norandrodiol select 300

Transcription

B E R N S L A W O F F I C E , P.C.
MICHAELA. BERNS
P H O N E21 7-367-9000
F A X 217-367-9005
107 WESTGOOSEA L L E Y
URBANA,IL 61801
mberns @shout.net
November 07,2000
U.S. International Trade Commission
500 E. Street S.W.
Washington, D.C. 20436
RE:337 COMPLAINT
On behalf of my client, LPJ Research, Inc., I enclose an original complaint with 14 copies, along
with a certified copy of the prosecution history for the patent in question with three copies, and
four copies of the technical references for the patent in question.
I also enclose a Small Business Application for Technical Assistance. LPJ, Inc. is a small business
and would like any assistance to help protect their product from the flood of infringing goods.
If you have any questions or need additional inforniation, please contact me.
Sincerely,
Michael Berm
,
REGISTERED
ATTORNEY #38,378
UNITED STATES PATENT AND TRADEMARK OFFICE
'.
SMALL BUSINESS APPLICATION
FOR TECHNICAL ASSISTANCE
Certifications of Applicant
The undersigned certifies that LPJ Research, Inc.
(hereinafter referred to as
“Applicant”) is an independently owned and operated company and that it qualifies as a small
business under the Small Business Administration’s Size Standards (hereinafter referred to as
“SBA Size Standards”) set forth in 13 C F R 5 121 The undersigned further certifies that
(1) the Standard Industrial Classification (“SIC”)codes (up to the 4 digit codes, if
known) for each of the Applicant’s lines of business are I J m w n
,
(2) the Applicant employs 1 5 - 3 0
number of employees at the time of
submitting this application (The method of calculating the number of employees is set
forth in the SBA Size Standards at 13 C F R 121 106.), and
(3) the Applicant’s annual receipts as calculated at the time of submitting this application
are < $5M
. (The method of calculating annual receipts is set forth in the
SBA Size Standards at 13 C F R.4 121 104 )
Acknowledgments of Applicant
Applicant acknowledges that it has received copies of the relevant statutory provisions (19 U.S.C.
$ 1339) and International Trade Commission’s (ITC) Rules (19 C F R. 0 213) concerning trade
remedy assistance Applicant acknowledges that it has reviewed these statutes and rules and
understands that
( 1 ) in accordance with the relevant statute and ITC rules, ITC personnel, in coordination
with other agencies responsible for administering U.S.trade laws, will provide technical
assistance to eligible small businesses seeking benefits and relief under U.S. trade laws. (A
definition of “technical assistance” is set forth in 19 U.S.C.5 1339 (b) and 19 C.F.R.
3 213.2 (d); a definition of “U.S. trade laws” is set forth in 19 U.S.C. 5 1339 (c) (2) and
19 C.F R. 4 213 2 (b); and a definition of the “agencies responsible for administering
the U . S .trade laws” is set forth in 19 C.F.R. 9 213.2 (c));
(2) the ITC’s determination of eligibility for technical assistance is not reviewable by any
other agency or by any court;
(3) technical assistance does not include legal representation or advocacy on behalf of an
applicant and, therefore, is not a substitute for the advice and/or retention of private legal
counsel; and
(4) technical assistance does not ensure that the recipient will prevail in any trade
remedy proceeding.
For questions concerning the required certifications and acknowledgments, applicants should
contact the ITC’s Trade Remedy Assistance Office (TRAO)at 1-800-343-9822 (toll free) or
(202) 205-2200. Applicant may also want to consult with private counsel about these matters.
(Print name of offrcer or principal signing under
oath on behalf of Applicant)
(Print name, address and telephone number of Applicant)
Sworn to before me t h i s a d a y of
My Commission expires on
d o vembe,r
5 /do! 06,
,1 9 3
(Place notary seal above.)
Public reporting burden for this collection of information is estimated to average one (1) hour per
response, including the time for reviewing instructions, searching existing data sources, gathering
and maintaining the data needed, and completing and reviewing the collection of information.
Send comments regarding this burden estimate or any other aspect of this collection of
information, including suggestions for streamlining the application process, to Trade Remedy
Assistance Office, U.S. International Trade Commission, 500 E Street, S.W., Washington, D.C.
20436, and to the OEce of Information and Regulatory Affairs, Office of Management and
Budget, Washington, D.C. 20503.
UNITED STATES INTERNATIONAL TRADE COMMISSION
Washington, D.C.
1
In the Matter of
4-ANDROSTENEDIOL
Inv. No. 337-TA-
COMPLAINT UNDER SECTION 337
OF THE TARIFF ACT OF 1930, AS AMENDED
Filed on Behalf of Complainant:
LPJ Research, Inc.
205 South Main Street
P.O. Box 160
Seymour, IL 6 1875
Shanghai Freemen International Trading Co., Ltd.
Shanghai Industrial Investment Bldg. 18, Rm.22D
Cao Xi Rd. (N)
Shanghai 200030, China
Counsel for Complainant:
21218 Vanowen St.
Canoga Park, CA 9 1303
(217) 687-4038
Michael Berns, Esq.
Berns Law Office, P.C.
107 West Goose Alley
Urbana, IL 61801
(217) 367-9000
(217) 367-9005(fax)
Proposed Respondents:
Asymchem, Inc.
2 Davis Dr.
P.O. Box 12076
Research Triangle, NC 27709
Changzhou Huabang Pharmaceutical
Group, Ltd.
22E, International Building ,
Changzhou, Jiangsu, China
Kingchem, Inc.
296 Kinderkamack Road
Oradell, NJ 07649
Purechem Co., Ltd.
71North Zhujiang Rd.
P.O. Box 528
Kunshan, JS, 215301, China
Stryka Botanics Company, Inc.
Taizhou Xingye
Baita
Linhai City, Zhejiang 217000, China
TUTT China - Sinochem Yangzhou
3710 E. Ovid Ave.
Des Moines, IA 50317
Uking Team, China
Wenzhou M&C Foreign Trade Corporation
307 Xueyaun Road East
Wenzhou Zhejiang, China
Wujin Jiaerke Group Corp., Ltd.
Hengshan Bridge Town
Zhejiang Provincial Light & Textile Industry Group
Corporation
No. 8 Mei Hua Bei
Hangzhou, China
DOCUMENTARY EXHIBITS
Exhibit No.
Description
Parawaph & Pape Nos.
1
Asymchem, Inc. webpage
par. 3,
pp. 4-5
2
e-mail correspondence with Changzhou
par. 4,
P. 5
3
advertisement for 4-Androstenediol from 1999
Spring-Summer Buyer's Guide of the Natural
Products Industry Insider
par. 5,
P. 5
4
Kingchem, Inc. webpage
par. 5,
P. 5
5
Purechem webpage
par. 6,
pp. 5-6
6
e-mail correspondence with Purechem
par. 6,
pp. 5-6
7
Shanghai Freemen webpage
par. 7,
P. 6
8
e-mail correspondence with Shanghai Freemen
par. 7,
P- 6
9
Stryka Botanics Company, Inc. webpage
par. 8,
pp. 6-7
10
Taizhou factory product list
par. 9,
P.7
11
TUTT China webpage
par. 10,
P. 7
12
Uking Team webpage
par. 11,
P. 7
13
e-mail correspondence with Uking Team
par. 11,
P. 7
14
Wujin Jiaerke Group webpage
par. 12,
pp. 7-8
15
e-mail correspondence with Jiaerke Group
par. 12,
pp. 7-8
16
claim charts
par. 15,
P. 9
17
U.S. Letters Patent 5,880,117
par. 17,
pp. 9-10
18
Assignment of ' 117 Patent to LPJ
par. 17,
pp. 9-10
19
Letters from counsel for LPJ to Respondents
par. 23,
p. 11
20
current LPJ price list
par. 30,
p. 13
21
LPJ Research, Inc. webpage
par. 31,
p. 13
22
Physical exhibit of a bottle of 4-Androstenediol
distributed by Syntrax Innovations, Inc. and sold
under the trademark TETRABOL.
par. 32,
p. 13
23
Physical exhibit of a sample of 4-Androstenediol
manufactured by LPJ Research, Inc.
par. 33,
p. 13
24
Certificate of Analysis of 4-Androstenediol
manufactured by LPJ Research, Inc.
par. 33,
p. 13
25
Invoice and packing list of shipment from
Zhejiang
par. 13,
26
Physical exhibit of a sample of 4-Androstenediol
from Zhejiang.
par. 13,
27
Laboratory analysis performed by LPJ of physical par. 13,
sample of 4-Androstenediol from Zhejiang.
28
Affidavit of Patrick Arnold
par. 29
Appendices
A
File Wrapper of the ‘1 17 Patent
par. 17,
pp. 9-10
B
References Cited in the ‘117 Patent File Wrapper
par. 17,
pp. 9-10
COMPLAINT
I. Introduction
1. This complaint is filed by LPJ Research, Inc. (“LPJ”) pursuant to section 337 of the
Tariff Act of 1930, as amended, based upon the importation into the United States, the sale for
importation, and the sale within the United States after importation by Proposed Respondents of
4-Androstenediol which infringes U.S. Letters Patent 5,880,117 (“the ‘117 patent”). LPJ
Research, Inc. is the owner by assignment, the inventor being Patrick Arnold, President of LPJ.
LPJ now seeks, as relief, an order excluding proposed respondents’ infringing 4-Androstenediol
from entry into the United States and a cease and desist order or orders halting the sale by
respondents of infringing, imported 4-Androstenediol.
11. Complainant
2. LPJ is a business entity that is incorporated under the laws of the State of Illinois. LPJ
has its principal place of business at 205 South Main Street, Seymour, Illinois 61875. At this
facility, LPJ has its main office, manufacturing facilities, laboratory facilities, and warehouse.
LPJ manufactures 4-Androstenediol at this facility.
111. ProDosed Respondents
3. Upon information and belief, Asymchem, Inc. (“Asymchem”) has its main office
located at 2 Davis Dr., Research Triangle, NC 27709. Upon information and belief, Asymchem
is involved in importing 4-Androstenediol and selling it through a distribution network that
includes the Internet. Attached to this complaint as Exhibit 1 is a copy of Asymchem’s website
located at www.asvmchem.com. This includes an advertisement for 4-Androstenediol, as well
4
as a list of Asymchem’s suppliers as: Zhejian Shou & Fu Chemical Co., Zhejiang, China and
Tianjing Fuxingda Pharmaceutical Intermediates Co., Ltd., Tianjing, China.
4. Upon information and belief, Changzhou Huabang Pharmaceutical Group, Ltd.
(“Changzhou”) has its main office located at 22/F, International Building, Changzhou, Jiangsu,
China. Upon information and belief, Changzhou is involved in the manufacture and export to
the United States of 4-Androstenediol. Attached to this complaint as Exhibit 2 is a copy of
recent e-mail correspondence between Jiang Xuejun, Assistant Manager of Marketing at
Changzhou and counsel for Complainant, Changzhou has offered 4-Androstenediol for sale in
the U.S. for $400/Kg.
5. Upon information and belief, Kingchem, Inc. (“Kingchem”) has its main office
located at 296 Kinderkamack Road, Oradell, NJ 07649. Upon information and belief, Kingchem
is an independent sales and marketing organization representing overseas manufacturers in
Europe and in Asia. Upon information and belief, Kingchem is involved in importing 4Androstenediol and selling it through a distribution network that it has established. Attached to
this complaint as Exhibit 4 is a copy of Kingchem’s webpage located at www.kingchem.com as
well as an advertisement for 4-Androstenediol from the webpage. Kingchem represents that it
has offices in Shanghai and Qingdao, China and direct contact with most Chinese manufacturers.
Kingchem was listed as a distributor of 4-Androstenediol in the 1999 Spring-Summer Buyer’s
Guide of the National Products Industry Insider, a copy of which is attached to this complaint as
Exhibit 3. Kingchem offered to sell 4-Androstenediol in a telephone call to its office.
6. Upon information and belief, Purechem Co., Ltd. (“Purechem”) has its main offices
located at 7 1 North Zhujiang Rd., Kunshan, JS,2 15301, China. Upon information and belief,
Purechem is involved in the manufacture and export to the United States of 4-Androstenediol.
5
Attached to this complaint as Exhibit 5 is a copy of a webpage located at
www.tradezone.com/tradesites/uurechem.html
that includes an advertisement for 4Androstenediol. Attached to this complaint as Exhibit 6 is a copy of recent e-mail
correspondence between Zhou wei dong of Purechem and counsel for Complainant. Purechem
has offered 4-Androstenediol for sale in the U.S. for $380/Kg.
7. Upon information and belief, Shanghai Freemen International Trading Co., Ltd.
(“Freemen”) has offices located at Shanghai Industrial Investment Bldg. 18, Rm. 22D, Cao Xi
Rd. (N), Shanghai 200030, China. Upon information and belief, Freemen is involved in the
manufacture and export to the United States of 4-Androstenediol. Attached to this complaint as
Exhibit 7 is a copy of Freemen’s webpage located at freemen.cOrn.cn that includes an
advertisement for 4-Androstenediol. Attached to this complaint as Exhibit 8 is a copy of recent
e-mail correspondence between David Zhang, President and CEO of Freemen, Hu Changchun of
Freemen, and counsel for Complainant that includes an offer to sell 4-Androstenediol in the U.S.
for $360/Kg.
8. Upon information and belief, Stryka Botanics Company, Inc. (“Stryka”) has offices
located at 2 16 Route 206, Somerville, NJ 08876 and 2 1218 Vanowen Street, Canoga Park, CA
9 1303. Upon information and belief, Stryka is involved in importing 4-Androstenediol and
selling it through a distribution network that it has established. Attached to this complaint as
Exhibit 9 is a copy of Stryka’s webpage located at www.stryka.com. Stryka was listed as a
distributor of 4-Androstenediol in the 1999 Spring-Summer Buyer’s Guide of the National
Products Industry Insider, a copy of which is attached to this complaint as Exhibit 3. Stryka
offered to sell 4-Androstenediol in a telephone call to its office.
9. Upon information and belief, Taizhou Xingye (“Taizhou”) has offices located at
6
Baita, Linhai City, Zhejiang 2 17000, China. Upon information and belief, Taizhou is involved
in the manufacture and export to the United States of 4-Androstenediol. Attached to this
complaint as Exhibit 10 is a copy of Taizhou’s factory product list e-mailed to Counsel for
Complainant. Taizhou is offering 4-Androstenediol for sale for $3 16/Kg.
10. Upon information and belief, TUTT China - Sinochem Yangzhou (“TUTT”) has
offices located at 3710 E. Ovid Ave., Des Moines, IA 50317. Upon information and belief,
TUTT is involved in importing 4-Androstenediol and selling it through a distribution network
that it has established. Attached to this complaint as Exhibit 11 is a copy of TUTT’s webpage
located at www.tuttg;roup.comwhich includes an advertisement for 4-Androstenediol.
1 1. Upon information and belief, Uking Team, Chma, Wenzhou M&C Foreign Trade
Corporation (“Uking”) has offices located at 307 Xueyaun Road East, Wnzhou Zhejiang, China.
Upon information and belief, Uking is involved in the manufacture and export to the United
States of 4-Androstenediol. Attached to this complaint as Exhibit 12 is a copy of Uking’s
webpage located at www.ukingteam.com. Attached to this complaint as Exhibit 13 is a copy of
recent e-mail correspondence between Kevin Ren, the Main Manager of Uking and counsel for
Complainant that includes an offer to sell 4-Androstenediol in the U.S. for $360/Kg.
12. Upon information and belief, Wujin Jiaerke Group Corp., Ltd. (“Jiaerke”) has offices
located at Hengshan Bridge Town, Changzhou, Jiangsu, China. Upon information and belief,
Jiaerke is involved in the manufacture and export to the United States of 4-Androstenediol.
Attached as Exhibit 14 is a copy of Jiaerke’s webpage located at www.iiaerlte.com which
includes an advertisement for 4-Androstenediol. Attached as Exhibit 15 is a copy of recent email correspondence between Angel Huang of Jiaerke and counsel for Complainant that includes
an offer to sell 4-Androstenediol in the U.S. for $350/Kg.
7
13. Upon information and belief, Zhejiang Provincial Light & Textile Industry Group
Corporation (“Zhejiang”) has offices located at No. 8 Mei Hua Bei, Hangzhou, China. Upon
information and belief, Zhejiang is involved in the manufacture and export to the United States
of 4-Androstenediol. Attached as Exhibit 25 is a copy of a Commercial Invoice and Packing
List dated May 18,2000, shipping 4-Androstenediol from China to the United States. Attached
as Exhibit 26 is a physical sample of the 4-Androstenediol imported by Zhejiang. Attached as
Exhibit 27 is a laboratory test analysis result of the 4-Androstenediol imported by Zhejiang.
IV. The Product at Issue
14. The ‘ 117 patent covers the use of 4-Androstenediol to increase testosterone levels.
The chemical term 4-Androstenediol refers to two isomers: 4-androstene-3beta, 17betadiol and
4-androstene-3alpha, 17beta-diol. It acts as a very effective precursor to testosterone, making
the human body produce more testosterone. Testosterone is considered to be the male virilizing
hormone. Its effects include maintenance of muscle and bone mass, sexual function, and
psychological well being among others. As males grow older, especially after the age of 35, a
slow decline in testosterone levels is observed which is accompanied by symptoms that have
been associated with the condition known as “andropause”. Symptoms of andropause include
lethargy, depression, lack of sexual desire and function, and loss of muscle mass and strength.
15. The use of 4-Androstenediol is a major breakthrough in health supplements. It is far
superior to popular androstenedione and 5-Androstenediol, which are also used for similar
purposes. Attached to this complaint as Exhibit 16 are claim charts showing practice of the
patent by LPJ and infringement by Respondents.
16. LPJ Research, Inc. is recognized as a leader in the health supplement industry. They
8
brought androstenedione to the United States and have worked on developing new, safe products
for health supplements. LPJ manufactures many of the supplements that are sold by other
brands across the country. LPJ manufactures 4-Androstenediol and sells to distributors for
encapsulation and retail sale. LPJ currently manufactures 4-Androstenediol,
Norandrodstenediol, Cyclodextrin complexed 4-Androstenediol, and Cyclodextrin complexed
Norandrostenediol. LPJ believes that 4-Androstenediol is assigned Harmonized Tariff Schedule
of the U.S. (HTSUS) Item No. 2937.99.9510, although it is not an anabolic steroid.
V. The Patent in Issue
17. LPJ is the owner of U.S. Letters Patent 5,880,117 entitled “USE OF 4Androstenediol TO INCREASE TESTOSTERONE LEVELS IN HUMANS” (“the ‘117
patent”). A copy of this patent is attached to this complaint as Exhibit 17. The patent was issued
on March 9, 1999, based on an application (Application No. 09/114,114) filed by Patrick Arnold
on July 13, 1998. Mr. Arnold is an employee of LPJ. and has assigned all rights in the invention
and all patents related to it to LPJ. A copy of this assignment, as duly filed with the United
States Patent and Trademark Office, is attached to this complaint as Exhibit 18. The assignment
is recorded in the PTO at Reel 009526 and Frame 0043. The ‘117 patent itself was issued to LPJ
as assignee. Accompanying this complaint as Appendix A are a certified copy and three other
copies of the Patent and Trademark Office file wrapper of the ‘ 117 patent. Appendix B consists
of four copies of each patent and appropriate pages from each technical reference mentioned in
the file wrapper of the ‘ 117 patent.
18. The ‘ 117 patent has four claims. All of the claims are at issue in this complaint.
Claim 1 is an independent claim and claims 2-4 are dependent claims that depend from claim 1.
9
The claims relate to the administration of 4-Androstenediol to humans to increase testosterone
levels.
19. No foreign patent applications corresponding to the ' 117 patent have been filed,
abandoned, withdrawn, or rejected.
20. LPJ has sold bulk quantities of 4-Androstenediol to distributors, thus granting an
implied license to sell the product. LPJ has not granted any express licenses covered by the
patent. No licenses have been granted for the manufacture of the product.
VI. Litigation
2 1. The ' 117 patent was the subject of a patent infringement lawsuit against Sports One,
Inc.: LPJ Research, Inc. v. Sports One, Inc., 99-2175, filed Aug. 10, 1999 in the Central District
of Illinois. That action has been settled by the parties and the validity of the patent was not
challenged. No litigation has brought into question the validity and enforceability of the ' 117
patent.
VII. Infringement of the ' 117 Patent
22. Soon after the release of 4-Androstenediol onto the United States market by LPJ,
foreign companies began selling products claiming to contain 4-Androstenediol. These products
have been advertised primarily in magazines, and sold through catalogs. Sales of the product
consist of actively inducing infringement and contributory infringement of all 4 claims of the
'1 17 patent. Upon information and belief, Respondents sell 4-Androstenediol in bulk powder
form.
10
A. Induced Infringement
23. Upon information and belief, Respondents have induced the infringement of the ‘117
patent under 35 U.S.C. 0 271(b) by supplying the elements of the patent. Sale of bulk product,
and sale of encapsulated product, both actively induce infringement by consumers. Counsel for
LPJ has sent letters to all Respondents informing them that LPJ is the owner of the ‘117 patent;
advising them that the products that they manufacture, sell for importation into the United States,
import, and sell after importation violate claims of the ‘ 117 patent; and demanding that the sale
of these products in the United States cease at once. Respondents have continued their activities
notwithstanding receipt of these letters. Copies of these letters, which were sent by registered
mail, return receipt requested, accompany this complaint as Exhibit 19. Appended to these
letters are copies of signed return receipts establishing that these letters were received by
Respondents.
B. Contributory Infringement
24. Upon information and belief, Respondents have contributed to the infringement of
the ‘1 17 patent under 35 U.S.C. 0 271(c). Respondents sale of the product is for use in
practicing the claims of the ‘1 17 patent. The product constitutes a material part of the invention.
Respondents knew the product was especially made for use in an infringement of the ‘117
patent. The product is not a staple article and not a commodity of commerce suitable for
substantial non-infringing use. The sale of 4-Androstenediol has no commercial noninfringing
use.
11
VIII. Importation
25. As set forth above, upon information and belief, Respondents’ products are imported
into the United States. Upon information and belief, there is no production of 4-Androstenediol
in the United States other than that by the Complainant. Upon information and belief, 4Androstenediol products are manufactured in China and imported to United States distributors.
LPJ does not sell 4-Androstenediol to any of the Respondents. Price quotes from Respondents,
who are willing to provide price quotes, are lower than the price LPJ charges for 4Androstenediol.
26. Upon information and belief, Respondents are mostly active in the bulk sales of 4Androstenediol. Their customers often encapsulate the product and resell to the consumer
market.
IX. Domestic Industry
27 LPJ maintains a laboratory with computers and research equipment that cost
thousands of dollars. LPJ has also performed clinical testing of the product to verify its
performance by measuring testosterone levels in subjects.
28. LPJ operates a 20,000 square foot production facility capable of producing 5,000
kilograms of 4-Androstenediol per month. The cost of the equipment used in the facility is
approximately $900,000. LPJ currently produces approximately 600 kilograms per month of 4Androstenediol which makes up approximately 80% of LPJ’s sales.
29. Patrick Arnold, President of LPJ Research, and inventor of the ‘117 patent, performs
a large amount of research and development in the field of prohormones for the bodybuilding
market. He is considered an expert in the field. He has a B.S. in Chemistry and has also worked
12
toward his Master’s degree. Mr. Arnold also uses 4-Androstenediol for increasing his
testosterone levels, as claimed in the patent. Attached to this complaint as Exhibit 28 is an
Affidavit of Patrick Arnold.
30. LPJ currently sells 4-Androstenediol for S515IKg for orders of 1-50 Kg. This is
higher than the quoted prices from the Respondents providing information. Attached to this
complaint as Exhibit 20 is the current price list from LPJ.
3 1. Attached to this complaint as Exhibit 2 1 is a copy of LPJ’s webpage located at
www.lpjresearch.com, showing its capabilities and products, including 4-Androstenediol.
32. Included with this complaint as Exhibit 22 is a physical sample of a bottle of 4Androstenediol manufactured by LPJ and distributed by Syntrax Innovations, Inc. and sold under
the trademark TETRABOL.
33. Included with this complaint as Exhibit 23 is a physical sample of 4-Androstenediol
manufactured by LPJ. A Certificate of Analysis of 4-Androstenediol manufactured by LPJ is
attached as Exhibit 24.
X. Relief
WHEREFORE, by reason of the foregoing, LPJ Research, Inc. requests that the United
States International Trade Commission:
(a) institute an immediate investigation pursuant to section 337 of the Tariff Act of 1930,
as amended, with respect to violations of that section based upon the importation into the United
States, the sale for importation, or the sale within the United States after importation by
Respondents of 4-Androstenediol which infringes valid and enforceable U.S. Letters Patent
13
5,880,117;
(b) schedule and conduct a hearing on said unlawful acts and, following said hearing:
(c) issue a permanent exclusion order excluding entry into the United States of 4Androstenediol through a general exclusion order;
(d) issue permanent cease and desist orders prohibiting respondents from selling in the
United States any 4-Androstenediol;
(e) issue such other and further relief as the Commission deems just and proper based on
the facts determined by the investigation and the authority of the Commission.
Respectfully submitted,
Michael Berns
14
VERIFICATION
I, Patrick Arnold, am President of LPJ Research, Inc., and inventor of United States Letters
Patent 5,880,117, and am duly authorized to sign this complaint on behalf of LPJ Research, Inc.
I have read the complaint and am aware of its contents. To the best of my knowledge,
information, and belief, formed after an inquiry reasonable under the circumstances, I hereby
certify as follows:
1. The complaint is not being presented for any improper purpose, such as to harass or to
cause unnecessary delay or needless increase in the cost of the investigation;
1
2. The claims and other legal connections in the complaint are warranted by existing law
or by a nonfrivolous argument for the extension, modification, or reversal of existing law or the
establishment of new law; and
3. The allegations and other factual contentions in the complaint have evidentiary
support or, if specifically so identified, are likely to have evidentiary support after a reasonable
opportunity for further investigation or discovery.
I declare under penalty of perjury that the foregoing is true and correct.
Patrick Arnold, President
LPJ Research, Inc.
15
Company Overview
Asymchem is a custom synthesis producer of advanced fine organic
chemical and pharmaceutical intermediates, based in the Research
Triangle Park, North Carolina, USA.
As a custom synthesis producer driven by inquiries, our edge over other
competitors is this: with lower operation and labor costs that come with
having production facilities in China, Asymchem can provide the most
competitive prices available on the market today.
Based on our vision of providing high quality products at competitive costs,
Asymchem is continually developing and refining innovative chemical
technology to meet customer’s demanding custom synthesis needs.
Contact Information
Telephone: (1)919-460-5179
FAX: (1)919-319-9888
Postal address:
2 Davis Dr., PO Box 12076, Research Triangle Park, NC 27709, USA
General Information and Sales: jnfo@asymchem.com
Webmaster: webmaster@asymchem.com
Send mad to snfo@asvmchem.com with questions or comments about this web site.
Last modified: July 20,1.999
E
l
EXHIBIT
Asymchem’s representation of Chinese fine organic chemical and bulk pharmaceutical
intermediates producers was compiled through rigorous background checks and on-site audits
by Asymchem’s American office.
From start to finish, Asymchem’s own technical team is there overseeing our client’s orders.
Quality is guaranteed, backed by Asymchem’s own highly experienced chemists and
purification facilities.
With our well-established network and database of prescreened Chinese producers, a supplier
can be found upon request with the assurance of guaranteed purity from Asymchem.
1. Zhejiang Shou 8, Fu Chemical Co., Zhejiang, China: Mercaptans
2. Tianjing Fuxingda Pharmaceutical Intermediates Co., Ltd., Tianjing, China:
Pharmaceutical Intermediates and Steroid Derivatives
Steroid Intermed iates
7%
4-And rost enedio1
# 4-Androstenedione
# 3b-Hydroxy-pregna-5,16-diene-2O-one-3-acetate
# 19-Norandrostenediol
$( 17-Methyltestosterone
$( 17-Methylandrost-5-en-3,17-dioI
I( Prasterone
# Prasterone Acetate
Pharmaceutical Intermediates
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2-Amino-4-chloro-2’-fluorobenzophenone
6-Benzylade nine
5-Bromoacetylsalicylamide
3-Bromoanisole
4-Bromo-2-fluorobiphenyl
6-Chloro-2,4-diaminopyrimidine
2-(2-Chloromethyl)-3,5-dimethoxypyridine
2-Chloronicotinic acid
Cytosine
4,4’-Difluorobenzophenone
5-Difluoromethoxy-2-mercapto-benzimidazole
Indole-3-butyric acid
Indole-3-carboxylic acid
2-Picolinic acid
Trifluoromethyl phenyl acetone
Send mail to info@asvmclwm.comwith questions or comments about this web site.
Last modified: May 27,1999
Subject : 4-andros tenediol
Date: Sun, 13 Feb 2000 18:53:31 +0800
From: czhbpc@public.cz.js.cn
To: <mberns@shout.net>
Attn:Mr mike berns of Blo
From:Changzhou Huabang Pharmaceutical Group Ltd.
Add: 22/F,International Building,Changzhou,
Jiangsu,China
Tel:86-5 19-8170087
Fax:86-5 19-8104456
Date:Fcb. 13,2000
Dear Mr.Mike Berns,
We are very indebted for Chinapharm-chemnet.com that they inform us you
are in market for above product. As for this item,you actually find a
right nianufacturer.It is the strongest for us and has been exported to
Europe,USA,India etc.The quality has been approved by ail of our customers.
Now our capacity is 500kg/month.
Following to your inquiry,please find herewith our best price:
Payment Term; At USD400/kg CIF Air USA by T/T in advance
Packing : In 25kg net cardboard drum
Delivcry Time:Prompt shipment €or 25 kg lot after your payment received
If you have anything unclear and further request,please feel free to let us
know.We shall try our best to cooperate with you.
Looking forward to your early reply.
Best Regards
Jiang xuejun
Asst. Manager of Marketing
1-
EXHIBIT
4-androstenediol
Subject: 4-androstenediol
Date: Thu, 24 Feb 2000 18: 18:15 +OS00
From: czhbpc@public.cz.js.cn
Jiangsu,China
Tel:86-5 19-8170087
Fax:86-5 19-8104456
Date:Feb.24,2000
Dear Mr.Mike Bems,
Good Morning! This is Jiang xuejun from Changzhou Huabang Pharmaceutical
Group Ltd. Very sorry for bothering you.
Have you already received our offer? Please kindly confirm it. Today
our President inquiry me this matter,and also our Manu.Dept.need make
a monthly plan. So please send us your definite reply,so that I hand
a reported to President and Manu.Dept.check with coopcrating possibility.
If you have no purchasing plan,there is no problem.We only hope that we
can keep a good understanding and close relation by this email.
Waiting for your favourable reply.
Best Regards
Jiang xuejun
lofl
2t24100 9:31 A M
coopera tion
Subject: cooperation
Date: Fri, 25 Feb 2000 13:17:40 +0800
From: czhbpc@public .cz.js.cn
Jiangsu,China
Tel:86-5 19-8I70087
Fax:86-519-8104456
Date:Feb.24,2000
Dear Mr.Mike Berns
Many thanks for your email. We shall awaiting for your decision.
But we hope that you decide this matter asap.,because the price
is advancing.
In addition,we are strongest on following items:
D-C 1i t cosani itic S uI fa t e 2 KC1,
D-GI iico sa in iri c M C1,
Chi tos a n
Chito-Oligosaccharides
N -acel y I gl 11 c o sam ie
I A X i t a mine
progestcrone
16-dcliydroprogcstcrone
pregnenolone
16-dehydro prcgncnolone
prednisolone
prednisolone acetate
methylprednisolone
megcsrerol acetate
rnedroxyprogesterone acetate
17α-hydroxy progesterone
chlormadinone
16αl7α-dihydroxy progesterone
1o f 2
2/25/00 12:22 P M
Plant Extract(P1case see attachment)
Are you interested in a.m.items? Please fell free to let
us know.We can provide you more competitive price with
good quality.
Waiting for your favourable reply.
Best regards
Jiang xuejun
Asst Manager of Marketing
1
I
Name: Products List Extract.doc
Type: Microsoft Word Document (applicatiodrnsword)
DProducts List Extract.doc
I
Encoding: base64
of 2
1
2/25/00 12:22 PM
Raw materials for the
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amino acids:
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1-lsoleudne
L-Glutamine
Bllberry 25%
Guarana 20%
Horse Chestnut 20%
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Olive l e a f 10%
specla(ty supplements:
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Co Enzyme Q 10
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CARDBNA
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Rochem Internaliil Ina. .h.i
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Source Connectha LLC
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Falcon Trading
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Unh-arJAl Preaervschem
IN,
Irma Corp.
hem Inc.
Wntgon Imluslrtes Inc.
MT NulrlenlS I ~ c .
westco Fine Inpcdisnlr
MW lnlernallonal
AMINO ACIDS
Maypro Indunlrle9 Inc
A.S I.lnlernnllanal 1%.
MinlStnr Inlernetional Inc.
N a h d logredienls Yb
ABCO C n ~ l o r l e a
AT2 Naliiral (cfiv. d AT2
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Cheinicel Inc.)
ACRIOCorpornllm
Pnul SmtwHer
Ani Chein 11%.
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Agumm 1%
Phefmllne Inc.
AllaChem
.
RIA Inletnellonal LLC
Ainerknn Ingrcrdlenls Inc.
RNN In(erna1imal
Amerlcnn InlernsNonal '
Markellng InC.
Chemtcsl Im.
Rochem lnlernallonal Inc.
Aatiland ChnmtnlS
S C ~ l X O l h R I Inc.
I
hzhlninl Niilrillnnnl
e Strykn Bolenlce Cmpeny
fJnrrlnplonClwmhxl Cwp.
Bioslnl USA Inc
CPR Inlnrnnlbnnl Inc.
Cliarlna Barrmnn b Co
ChRmOK Isborntorim I
N
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C m p o ~ Solutions
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Inc.
ONP IntnrnellocMll Go. 1%.
[)nglech lnlrrnellonnl Inc.
Esaentlnl NulrlenlJ Ine.
Ffllcorr Tfadlng
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Cmp.
F~IIJW,erldga CO. tnc.
Freemanlndiislrles LLC
Fuxtionnl F d s Cwp
Qnflnrd.Sc(ilmingsr
'T
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H.Rebmeri
Hnrlen Cwpnrtlon
Helm New Vork Chem
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InterChsrn Corp
,lo Mer Cehornlofies
KRC Irk?.
a HAkko USA Im.
I C Niitrmnln Inc.
MW Iiilnrrirrlimfll
KT
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Mnypro lnrluslrlor Im.
MiniSIar Inleii~llonatInC.
NX GsHtraticn lld.
I NalureMml I.RborslwbB
Nnl Chnitiicnl Cofp.
NutrnIwti Inc
PRlli wwntrnr
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peritr Mninrl~c(urIng
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Pliclrmliiu liw
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RIA Intorflalionnl1LC
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nwnra Ainercon Corp
kchem InlarnaliooalInc
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Seltznr Cheinlcals iw
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Circle No. 56
pring-bummer '99 Buyer's Guide
S
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!
F
The New Alternative
...
[ Pharmaceutical Intermediates and Fine Chemicals 1 Nutritional Products I Dves
Bnd Pigment Intermediates I Custom Chemica1 Manufacturing Capabilities I
Inquiry Sheet ]
Kingchem Inc.
Oradell, N.J. 07649
Telephone:
201-261-6002
Fax:
201-262-9436
Toll Free: 1-800-211-4330
E-Mail: kinpchem@,worldnet.att.net
EXHIBIT
~
Kingchem InC. is an independent sales and marketing organization
representing overseas manufacturers in Europe and Asia and serving the global
market.
O u r commitment to our customers is to provide products and service that meet or
exceed our
clien t's expectations.
Kinmhem's three divisions are headed by Stephen Wang-President and managed
by highly
trained individuals who are experts in their field and
account for over
100 years of accumulated experience.
))
))
))
Pharmaceutical Intermediates & Fine Chemicals
contact: Austin Bishop-Vice President
Nutritional Products & OTC Pharmaceuticals
contacts: (Mr.) Carmine Covino - Vice President
Neil C. Sullivan-Sales Manager, East
Patrick Sivolella-Sales Manager, West
Dvestuff & PiEments
contact: Frank Fortuna-Marketing Manager
Produe$ J:ikegt'\ ilud .%crviccs:
*
"
*
Pharmaceutical Intermediates (including some bulk actives)
Custom Chemical Manufacturing (cGMP and FDA inspected facilities)
Nutritional Supplements Bulk Ingredients
Agrochemical Intermediates
Fine and Specialty Chemicals
. Dyes & Pigments
'
. Dyestuff & Pigment Intermediates
We exclusively represent W h im S.A. in the North American Market. Norchim is a
Fine Chemical and Pharmaceutical Manufacturer located in France, whose facility has
been inspected by the Food and Drug Administration and fully operates under cGMP
conditions. With Norchim's strong R & D and manufacturing capability, as well as
Kingchem's efficient worldwide network, we are capable of providing a wide range of
products from commodity chemicals to advanced intermediates which include
pharmaceuticals & pharmaceutical intermediates and a wide range of nutritional
supplement ingredients.
In our Fine Chemical and Pharmaceutical Intermediates business, we offer service from
process development to full scale production, as well as custom chemical
synthesis.
Kingchem has a well established network in
China and direct contact with most of the Chinese
manufacturers. We maintain two (2) offices in
China (Shanghai & Qingdao), which provide us
with the advantage in serving those customers
who are working on cost saving projects or
offering the products in which China has either a
production or resource advantage.
The essence of our business is commitment to
customer service by meeting and/or exceeding our
client's expectations of cost, quality, and efficient
service. We are confident that our competitive
edge will provide the foundation required for our
customers to be successful in their own market
areas.
Put Kingchem Inc. to work at becoming your
new alternative as the source for quality
products.
We are positive that you will enjoy the
difference!
Kingchem Inc. I296 Kinderkamack Road I Oradell, NJ 07649
Phone 201-261-6002 Fax 201-262-9436
Email: kinackem@worldnet.att.net
[ Pharmaceutical Intermediates and Fine Chemicals I Nutrional Products 1 Dves
and Pigment Intermediates I Custom Chemical Manufacturing Cagabilities
hauiry Sheet 1-1
I
Nutritional Supplement lnpredients:
Kingchem is a leading supplier of bulk nutritional supplement ingredients to the
Nutritional and Health Food industry. Our Nutritional Product Division has been
serving the U.S. Nutritional and Health Food Industry for many years. We have
been working very closely with many Nutritional Supplement Manufacturers by
providing them quality bulk ingredients with a consistent source of supply at
competitive pricing and timely delivery. Our well established global source,
from the herbal growing farms in China to our exclusive cGMP manufacturer in
France, provides us the unique position in offering quality nutritional supplements
and herbal extracts competitively. In addition, our R&D department will continue
their focus on new product development.
We maintain inventory for many key ingredients,which allows Kingchem to make
prompt delivery (usually within 24-48hrs. after receipt of an order). Please call
our sales department 20 1-261-6002 or toll free 1-800-2 1 1-4330 for a price quote
and the latest product information.
Special. Invredients
Adenosine Triphosphate Na2
* Alpha Lipoic Acid
- Calcium D-Saccharate
Chondroitin Sulfate
Chrysin
- Colostrum
Daidzein
DHA
DHEA
* Gamma Oryzanol
Genistein
L-5-Hydroxytryptophane (5-HTP)
Idebenone
Indole3-Carbinol
- Ipriflavone
* Kava Kava
Lecithin
Lutein
*
*
*
*
*
-
Lycopene
Magnesium Oxide
Manganese Ascorbate
n-Methyl Glycine (Sercosine)
Pacl it axe1
Phosphatidylserine
Pyruvate Calcium & Sodium
. Red Yeast Rice
- RNA (Ribonucleic Acid)
* Royal Jelly Powder
SAMe (S-Adenosyl-L-Methionine Disulfate Tosylate)
Spirulina
- Beta-Sitosterol
Vitamin B I 2 Coenzyme (Dibencozide)
Zeaxanthin
Zinc Gluconate USP23
*
I
-
-
Vitamins
Beta Carotene
Biotin
* FolicAcid
PABA
. Vitamin B 12 (Cyanocobalamin)
* Vitamin E, Natural
Vitamin E, Synthetic
Vitamin K
Inositol Hexanicotinate
Inositol Hexaphosphate 70%
* Inositol Hexaphosphate 50%
4
Herb;ii/Botanicals/Extracts
* Astraglus
. Andrographolide
* Baicalin
Bilberry
' Barley Grass Juice
BlackCohosh
Caffeinematural)
CarrotJuice
Citrus Aurantium
Clausenae Leaf
* Codonopsis
Coptidis
Corydalis Yanhusuo
- Dandelion
- Dong Quai (Angelica)
Devil's Claw
Elderberry
Ephedra 6 %
Ephedra 8 YO
Epimedium Brericornum
e
-
- Epimedium Root Powder
Fangfeng
Feverfew
Garlic
Gentian Root
* Giant Knotweed Rhizome
Gingko Biloba Leaf
Ginseng
Glucomanan
GrapePeal
- Grape Seed
GreenTea
Griffonia Simplicifolia Seed
* Golden Seal
GotuKola
. Gynostemma
Hesperidin
Horsetail
Honeysuckle Flower
Huperzine A, (HupapureTM)
98 YOminimum
5 % blend
1 % blend
Custom blends manufactured to customer specifications
* Ivy DryAqueous
Jingjie
Kava Kava
* Kudzu 30 %
Kudzll40 %
Kudzu 80 Yo
- Ligustrum Berries
Momordica Grosvenoti Fruit
* Pueraria Root
Quercetin
Reishi Mushroom
Resveratrol
. Senna
Schisandra
. Siberian Ginseng
. Silyniarin (Milk Thistle)
Shitalte Mushroom
* St. JO~III'S
Wort
Stevia
Tribulus Terrestris
* Troxeru tin
Tumeric 95% (standard extract)
- Valerian
- Vanadyl sulfate (tech. grade)
- Vinpocetine
Whitc Willow Bark
WildYam
*
*
6
*
4
-
4
+
-
a
Yarrow
Yohimbe
* Zingiber Officinale
- Zingiber Officinale Fluid
Ma ri rk e Prod u ct s
Chitosan
. Glucosamine Hcl/Sulfate
Oyster Powder
Sea Cucumber Powder
Shark Cartilage Powder
Amino Acids
N-Acetyl-L-Carnitine HCI
0-Acetyl-L-Carnitine HCl
- N-Acetyl-L-Cysteine
Acetyl Glucosamine
L-Alanine
* L-Arginine
L-Arginine Hcl
- L-Aspartic Acid
- L-Cysteine
* L-Cysteine Hcl (AnhyIMono)
* L-Cystine
L-Glutamine
L-Isoleucine
L-Leucine
- L-Lysine Mono Hcl
L-Methionine
DL-Phenylalanine
L-Proline
. L-Pyroglutamic Acid
L-Tyrosine
*
*
*
*
*
Wild Yam Related Products
Please note well: The following products are derived from Wild Yam. Some o f the products have regulatory
implications and should be used responsibly. It IS the purchasers' responsibility to clarify regulatory issues upon
purchasc and use of the material.
4(5)-Androstene-3*, 17-Diol
----3 4(5)-Androstene-3,17-Dione
*
5(6)-Androstene-3*,17*-Diol
5(6)-Androstene-3,17-Dione
19-Nor-4(5)-Androstene-3*, 17*-Diol
19-Nor-4(5)Androstene-3,17-Dione
19-Nor-5(6)-Androstene-3*,17*-DioI
. 19-Nor-5(6)-Androstene-3,17-Dione
* 19-Nor-5( 10)-Androstene-3*, 17*-Diol
19-Nor-5( lO)-Androstene-3,17-Dione
* DHEA
* Diosgcnin
Estradiol
*
*
-
*
- Estriol (USP grade, micronized)
Estrone
Pregnenolone
Progesterone (USP grade, micronized)
Wild Yam
*Denotes alpha (a)or beta ( p )
Kingchem Inc. I 296 Kinderkamack Road I Oradell, NJ 07649
Phone 201-261-6002 Fax 201-262-9436
Email: ~achembworldnet.att.net
PURECHEM CO.,LTD
Our company serves the human nutrition and health market based on China, we niainly deal in
nutrients raw materials and herbal extracts, we are rcprcsentatives of some main factories in
China. Besides the above products, we are also responsible for sales of all pharmaceuticals &
internicdiates of CSP(ChangShu Pharmaceutical Material Factory) in the market out of China.
For prices & further information of our listed products below, please feel free to contact us!
e Nutrients raw materials
1,4-Androstadien-3,17-dione,
4-Androstene-3,17-dione 99% rnin,5-Androstene-3,17-dione,
4 4-Androsteiie-3,17-diol, 5-Androstene-3,17-diol,Androstcrone,
Allopregnan, DL-Carnitine,
L-Carnitine, Acetyl-L-Carnitine, Chondroitine Sulfate 90%,Chrysin, Dehydroepiandrosterone
(DHEP)99?Lomin, Epiandrosterone, Hydroxyprogesterone, L-5-Hydroxytryptophane (5-HTP) 99%
niin,alpha-Lipoic acid (Thioctic acid) 99%, Melatonin 99.5%, 19-Nor-4-androstene-3,17-dione
99%
min, 19-Nor-5-androstene-3,17-dione,
19-Nor-4-andorstene-3,17-diol,
19-Nor-5-androstene-3,17-diol,
Progesterone BP93, Sildenafil citrate 99%, Testosterone.
Herbal Extracts
Black Cohosh Extract 2.5%, Ginkgo Biloba Extract 24/6,Grape Seed Extract 95%, Horse Chestnut
18-22%,Horsetail Extract 7%, Kava Kava Extract 30%, Siberian Ginseng Extract 0.8%, St. John's
Wort Extract 0.3% & 0.5%, Valerian Extract 0.8%.
ePharm & Intermediates of CSP
Cimetidine BP93/JP 12,DL-Carnitine, L-Carnitine, Acetyl-L-Carnitine, alpha-Lipoic acid 99%,
Melatonin 99.5 %,Naproxen BP93/USP23,Naproxen sodium USP2 3, Raniti di ne
USP23,2-Acetyl-6-Methoxy-Naphthalene
98.5% (for naproxen), 6,8-Dichloro Ethyl Caprylate 95%
( for alpha-lipoic acid), 5-(Dimethylaminomethyl)furfuryl alcohol 95% ( for ranitidine),
2-[ [ [5-(Dimethylai~ine)methyl-2-furanyl]methyl]thio]ethanamin~
95% (for ranitidine), S-Methyl
monohydrate 97% (for cimetidine), N-Methyl-1 -(mcthylthio)-2-nitroethenamine 98% (for
ranitidine), Nitromethane 99-99.9%.
0 Contact us
Sales Dcpt. Tel: 0086-520-73058 1 1, Fax: 0086-520-73 10573,E-mail: purechem@publicl .sz.js.cn
ADD: 71 North Zhujiang Rd., P.O.BOX 528 Kunshan, Jiangsu 215301 China, FACTORY ADD:
Yaozhen Changshu, Jiangsu 21553 1 China.
p ur ec h em@@u blicl,sz,js.cn
PURECHEM CO., LTD
71 Nor
X 528
Telephone: 0086-520-7305811, Fax : 0086-520-7310573
Created by thc "Home Page Creator"
A free service o f T r a d e S h
Last modified: Thursday, 03-Dec-1998 17:4 1 :12 PST
Subject: Re: Pricc list
Date: Fri, 1 I Feb 2000 10:5 1 :06 +0800
From: purechem@public 1.sz.js.cn
To: "Michael Berns" <mberns@shout.net>
Dear sir,
Thanks for your email.
We list some products which we are selling to US as follows:
L-carnitine base USD82/kg cif air
alpha-Lipoic acid USD310/kg cif air
Chrysin
USD260/kg cif air
Melatonin USDGOO/kg cif air
and we also have 1,4-androstene-3,17-dione(diol),
please contact us if you need
further information.
Especiallly L-carnitine base and alpha-lipoic acid are our strong items, and US
market need large quantity of these two products.
Best regards,
Zhou wei dong
PURECHEM CO., LTD
71 North Zhujiang Road Kunshan, Jiangsu 215301 China
Tel: 0086-520-7305811; Fax: 0086-520-7310573
_-___
Original Message - - - - From: Michael Berns emberns@shout.net>
To: <purechem@publicl.sz.js.cn>
Sent: Friday, February 11, 2000 2:33 AM
Subject: Price list
> Please
send m e a price l i s t for y o u r " n u t r i e n t s r a w m a t e r i a l s f f in the u s
>
>
market
>
>
Thank you
> m b e r n s @ s h o ut . n e t
> FAX 2 1 7 - 3 6 7 - 9 0 0 5
>
1-
EXHIBIT
Subject: Re: Price list
Date: Mon, 14 Feb 2000 13:09:50 -to800
From: purechem@public 1.sz.js.cn
To: "Michael Berns" <mberns@shout.net>
Dear sirs,
Thanks for your email.
We are very interested in androstene series products, I offer as follows:
USD250/kg cif air
4-Androstene-3,17-dione99% rnin
USD598/kg cif air
5-Androstene-3,17-dione99% rnin
99% rnin
USD380/kg cif air
-4-Androstene-3,17-diol
5-Androstene-3,17-diol 99% min
USD258/kg cif air
19-Nor-4-Androstene-3,17-dione 99%min
USD788/kg cif air
19-Nor-5-Androstene-3,17-dione99%min
USD958/kg cif air
19-Nor-4-Androstene-3,17-diol 99%min
USD1580/kg cif air
19-Nor-5-Androstene-3,17-diol 99%min
USD1680/kg cif air
other products:
USD4500/kg cif a i r
Sildenafil citrate 99% MIN
Progesterone BP93/USP23 USD212/kg cif air
Chondroitin sulfate 90% min USD98/kg cif air
If you are interested in our above products, please let us know.
Best regards,
Zhou wei dong
PURECHEM CO. , LTD
71 North Zhujiang Road Kunshan, Jiangsu 215301 China
Tel: 0086-520-7305811;Fax: 0086-520-7310573
- - - - - Original Message - - - - From: Michael Berns <mberns@shout.netz
To: cpurechem@publicl.sz.js.cn>
Sent: Friday, February 11, 2000 11:44 PM
Subject: Re: Price list
> Please give me a price list of all your products. I am particularly interested in
androstenedione, androstenediol,
> norandrostenedione, and norandrostenediol. I would buy quantities of 25kg drums.
>
Thank you
Michael Berns
> 107 West Goose Alley
> Urbana, IL 61801 USA
>
z FAX 2 1 7 - 3 6 7 - 9 0 0 5
>
>
purechem@publicl . sz.js.en wrote:
Z
>
> >
>
> >
z >
5
>
Dear sir,
Thanks f o r your email.
We list some products which we are selling to US as follows:
>
>
>
>
>
>
>
>
>
>
> z
L - c a r n i t i n e b a s e USD82/kg c i f a i r
a l p h a - L i p o i c a c i d USD31O/kg c i f a i r
Chrysin
USD26O/kg c i f a i r
M e l a t o n i n USD6OO/kg c i f a i r
and we a l s o h a v e 1,4-androstene-3,17-dione(diol),
p l e a s e c o n t a c t u s i f y o u need
further information.
> >
> >
E s p e c i a l l l y L - c a r n i t i n e b a s e and a l p h a - l i p o i c a c i d a r e o u r s t r o n g i t e m s , and US
m a r k e t n e e d l a r g e q u a n t i t y o f these t w o p r o d u c t s .
> >
> Best
regards,
> >
z > Zhou w e i dong
> > PURECHEM CO,, LTD
z > 7 1 North Z h u j i a n g Road
> > T e l : 0086-520-7305811;
,>
>
>
>
>
>
>
>
>
>
- - - - - O r i g i n a l Message - - - - F r o m : M i c h a e l Berns < m b e r n s @ s h o u t . n e t >
T o : < p u r e c h e m @ p u b l i c l. s z . j s . c n >
S e n t : F r i d a y , F e b r u a r y 11, 2000 2 : 3 3 AM
S u b j e c t : Price l i s t
> > > P l e a s e s e n d me a
> > z market
> > >
> > Thank y o u
> > >
5
z > mberns@shout.net
> > > FAX 2 1 7 - 3 6 7 - 9 0 0 5
> > >
>
Kunshan, J i a n g s u 215301 China
Fax: 0086-520-7310573
p r i c e l i s t f o r y o u r ! ! n u t r i e n t s raw m a t e r i a l s " i n the u s
The following materials are mainly used in Nutriceutical Industry
----------------
I
1
(1) Joint & Skin Health Products:
Chitosan (Industrial Grade)
__
_.
- - _..
Chondroitin Sulfate (Bovine & Shark)
Chitosan (Food Grade)
Chitin
__ - - -
-
I_
__
D-Glucosamine Hydrochloride
-.
-
I
____I
D-Glucosamine Sulfate
__--
-?
2KCI
p a r n i n e Sulfate ? 2NaCI
_______..__I____
_--I.__
F - o n y l Methane
-___--
1 (2) Nutritional Amino Acids:
F__l_l..__.__.^_.
Acety I-L-Cy steine
S-Carboxymethyl-L-Cysteine
Creatine Monohydrate
I__...
I
_
-
L-Cysteine
L-Cysteine Hydrochloride Anhydrous
L-Cystine
I DL-Phenylaianie
1 D-Ribose
L-Leucine
D-Phenylalanie
__-
L-Tyrosine
(3) Botanical Products:
__
Angelica Standerized Extract
_ _ _ _ I
~
-
-_____
[iamboo Leaf Extract 24%
.-.___I--_
I Black
Bitter Melon Extract
1 Echinacea Purpurea P.E 6:l
Echinacea Purpurea P.E. 4:l
Garcinia Cambogia Extract
.
.____
_I
--potu
-
-
- _--.
Hawthorn Standerized Extract 3%
_____
-~
-.
I Nettles Extract 5:l
Kava Kava Extract 30%
-I_-___.__.
---__
I
-
Rhodiola Rosea Extract
Saw Palmetto Extract
St. John's Wort Extract
I Tribulus Fruit Extract
Wild Yam Extract
-
_"
_-
______
.
(4)Steroids:
4-Androstenediol
--
--
1
----r
Lutein Powdered Extract 5%
5-Androstenediol
I 5-Androstenedione
I
4-Androstenedione
-.
.
I
~~
______.
...
1p r itla v o nc
.
__ __ - __ 19-Nor-Androstene-4-diol
DHEA
-
Inula Helenium Extract 4:l
Horse Chestnut Extract
~
__
Kola Extract
_
I
Green Tea Extract
-
Cohosh Extract 2.5%
I Citrus Aurantium Extract 6%
Black Cohosh Extract 4.0%
___ - -
-_-
.
1 19-Nor-Androstenedione
-
--/19-Nor-Androstene-5-diol
1 7-Keto-DHEA
3-Acetyl-7-Keto-DHEA
(5)Synthetic Nutritional Products:
I__--
-___
- __-
I
Alpha Lipoic Acid
1 5,7-Dihydroxy flavone (Chrysinl
I
1 Germanium Sesquioxide
Ca-HMB
(CE132)
----------------
Send mail to kmsli~public,sta.net.cn
with questions or comments about this web site.
Last modified: 99-0 1-25
I
Subject: Prohormes and other ingredient for Bodybuilding
Date: Fri, 11 Feb 2000 13:48:47 +OOOO
From: "David Zhang" <david.zhang@freemen.sh.cn>
To: mberns@shout.net
C C : MAOJIAN@duracef.shout.net, hu.changchun@freemen.sh.cn
David Zhang <david.zhangefreemen.sh,cn>
Michael Berns<mberns@shout.net>
Fri. 11 Feb, 2000
Prohormones
From :
To :
Date :
Subj ect :
Dear Mike.
We are a annual 30 million US Dollar company involved in manufacturing and
distributing various kinds of nutraceuticals, including fine ingredients for
bodybuilding purpose.
We have 22 people, and the persons for bodybuilding ingredients are: Mr. Mao
Jian (mao.j ian@f reemen.sh.cns and
Mr. James Hook <hu.changchun@freemen.sh.cn>.They will work our a list
with reference pricing for you today.
We do sell to the US market a lot of prohormones and other fine ingredients for
bodybuilding purpose. But we also sell a lot of other fine ingresdients to US as
nutraceuticals directed for various purposes as well, like D-Glucosamines,
Chondroitin Sulfate, Chitosan, MSM, D-Ribose and various herbal extracts. If
you have interest, we will send you a list as well.
Looking forward to working with you.
All the best
David Zhang
President & CEO
Shanghai Freemen International
-------
Mike Wrote Originally
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I
I am looking for nutritional supplements, especially prohormones for
bodybuilding, to be sold in the US market.
Michael Berns
Urbana, IL 61801
Fax 217-367-9005
E
l
EXHIBIT
Subject: Re: Fwd: Re: Price List
Date: Sat, 12 Feb 2000 13:32:45 +0800
From: Hu Changchun <heide@guomai.sh.cn>
Organization: Shanghai Greenmen International
To: Michael Berns <mberns@shout.net>
Dear Mr.Michae1 Berns:
Would you pls kindly introduce your company a bit more so that we are
able to understand your role in
this nutritional supplement market. Prohormone and Bodybuilding
Ingredients are just our strong items.
Do you have any specific idea on some products you are now working on or
some product you have intention
to be involved into. WE can provide All Sorts of Pro-Hormone and Sports
nutrients like Creatine,HMB,
Pyruvate,D-Ribose,LipoicAcid,Chrysin,Ipriflavone and Various Amino
Acid.
Pls let us know your requirement.
Yours truly
Hu Changchun
kmsh wrote:
>
>
>
>
>
>
>
>
This i s a forwarded message
From: Michael Berns <mberns@shout.net>
T o : kmsh <kmsh@publ
i c . st a .n e t . cn>
Date: Friday, February 1 1 , 2 0 0 0 , 1 2 : 0 5 : 0 4 A M
Subject: Price L i s t
===8<==============0riginal
message text===============
am looking f o r n u t r i t i o n a l supplements, e s p e c i a l l y prohormones f o r
bodybuilding, t o be sold i n the US market.
> I
>
>
Michael Berns
> 107 West Goose A l l e y
> Urbana, I L
61801
> Fax 2 1 7 - 3 6 7 - 9 0 0 5
>
> kmsh wrote:
>
>
gr
> >
z
> >
> >
> z
> >
> >
>
>
>
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>
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>
>
Dear s i r s ,
Thank you v e r y much f o r your email.
I n order t o o f f e r you p r i c e f o r our products, k i n d l y p l e a s e l e t us
know your business range.
Thanks w i t h b e s t regards/Zhang Dong
Thursday, February 1 0 , 2 0 0 0 , 7 : 2 6 : 4 6 A M , you wrote:
MB> Please send me
a price l i s t .
>
> MB> Thank you.
z MB> mberns@shout.net
>
> B e s t regards,
Subject: Price List
Date: Mon, 14 Feb 2000 15:53:10 +0800
From: Hu Changchun <heide@guomai.sh.cn>
Organization: Shanghai Grcenmen International
To: Michael Berns <mberns@shout.net>, mao.jian@freemen.sh.cn
Great! We Will Consider Stock More Andros Prohormone in our LA
Warehouse.
So that you are able to directly working with us with product handling
in USA. Since We are not very familiar with one another now,So we Hope
YOU
are able to Provide Bank Wire Payment in Advance,then WE can Release our
Product from our LA Nakano Warehouse to your Hand.However,By This Way,
We will Allow you Very Good Price and High Quality Ingredients,So that
YOU
will a l s o share benefit from this.
This is the Price WE Give you with FOB LA Price:
Androstenedione 25kg : US$260/kg FOB LA.
4-Androstenediol 25kg : US$360/kg FOB LA.
5-Androstenediol 25kg : US$240/kg FOB LA.
19-Norandrostenedione 2 5 k g : US$720/kg FOB LA.
19-Norandrostenediol 25kg : US$1650/kg FOB LA.
If you are going to work with us,pls kindly consider placing some order
soon.
So that We are able to start cooperation with you soonest as we can.
Other BodyBuilding Ingredients We are Handling Now Aggressively is:
D-Ribose
Acetyl-L-Carnitine
Creatine Monohydrate
Creatine Pyruvate
BCAA
Chrys in
Ipriflavone
Indole-3-Carbinol
Diindolynmethane
Phytin
HMB Calcium
Calcium Pyruvate
Phytosterol
Vanadyl Sulphate
Tribulus
Yours truly
HU Changchun
Michael Berns wrote:
>
>
>
>
>
5
I am looking for nutritional supplements for the bodybuilding and muscle
development market in t h e US. Particularly, I am interested in
androstenedione, androstenediol, norandrostenedione, and
norandrostenediol. I would probably purchase in 25kg drums. I need the
items shipped to me in Illinois, USA.
>
> The
>
company I am working w i t h is looking to get into the bodybuilding
market and sell product in gyms, on the Internet, and through
>
>
nutritional stores.
Please send m e a p r i c e l i s t €or a l l your n u t r i t i o n a l supplements.
>
>
Michael Berns
> 1 0 7 West Goose A l l e y
> Urbana, I L
6 1 8 0 1 USA
> TEL 2 1 7 - 3 6 7 - 9 0 0 0
> FAX
>
Changchun wrote:
> Hu
>
>
>
>
>
>
>
>
>
>
> >
> >
>
> >
> >
217-367-9005
Would you p l s k i n d l y introduce your company a b i t more so t h a t we are
able t o understand your r o l e i n
t h i s n u t r i t i o n a l supplement market. Prohormone and Bodybuilding
Ingredients are j u s t our strong i t e m s .
D o you have any s p e c i f i c idea on some products you are now working on ox
some product you have i n t e n t i o n
t o be involved i n t o . WE can provide A l l Sorts o f Pro-Hormone and Sports
n u t r i e n t s l i k e Creatine,HMB,
Pyruvate, D-Ribose, Lipoic Acid, Chrysin, I p r i f l a v o n e and Various Amino
Acid.
> >
> >
> > Pls
> >
>
> >
l e t us know your requirement.
Yours t r u l y
Hu Changchun
3 . 5
>
>
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>
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>
kmsh wrote:
>
>
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>
>
This i s a forwarded message
From: Michael Berns <mberns@shout.net>
To: kmsh ckmsh@public.sta.net.cn>
> > Date: Friday, February 1 1 , 2 0 0 0 , 1 2 : 0 5 : 0 4 AM
> > S u b j e c t : Price L i s t
z
>
>
> >
> >
>
>
>
>
===8<==============0riginal
message text===============
n u t r i t i o n a l supplements, e s p e c i a l l y prohormones f o r
be sold i n the U S market.
> I am looking f o r
> bodybuilding, t o
>
> Michael Berns
> > 1 0 7 West Goose A l l e y
> Urbana, I L
61801
)r
> > Fax 2 1 7 - 3 6 7 - 9 0 0 5
> > >
> > >
> > >
> > >
> > >
>
>
> > >
>
z
> >
> >
> >
> >
kmsh wrote:
>
>
Dear s i r s ,
>
>
>
Thank you v e r y much f o r your email.
> >
I n order t o o f f e r you p r i c e €or our products, k i n d l y p l e a s e l e t u s
know your business range.
> >
> >
> >
> > >
> > >
> > >
> > >
>
Thanks w i t h b e s t regards/Zhang Dong
Thursday, February 1 0 , 2 0 0 0 , 7 : 2 6 : 4 6 AM, you wrote:
>
> MB2
>
Please send m e a p r i c e list.
>
>
>
>
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MBs Thank you.
MB> mberns@shou t net
> Best regards,
>
kmsh
.
mai 1to :kmsh@public.sta.net.cn
---a<
_ _ - ===========E n d of original message t e x t = = = = = = = = = = =
Best regards,
kmsh
.
mail t o :kmsh@Dubl i c s ta.net.cn
Welcome to O u r Green Health World!
- _ - Shanghai Greenmen International
Your Reliable Source for Nutritional Ingredients,
Innovative Pharmaceutical and Special chemicals.
httD://www,nutrients.corn.cn
httw://www.greenheaI.th.corn.cn
MEMBER OF "FA,
-
-
-
HPA
-
BOTANICALS HERBS EXTRACTS SPICES FRUIT -VEGETABLES
VITAMINS AMINO ACIDS GLUCONATES MINERALS CUSTOM BLENDS
-
-
-
-
About Us
The Stryka Story
Eight years ago Mr. Tom Christensen
decided to create a leading raw
material supplier based on his vast
business experience and emphasis
on quality. As a result, Stryka
Botanics was born and began
supplying vitamins, minerals,
chelates, and amino acids. As the
marketplace changed, so did Stryka.
Through long and careful
investigation we determined that our
valued customers were in need of a
company that could supply all1 their
botanical raw material requirements.
Hence we changed out emphasis to botanicals and began
carrying both the highest quality herbal extracts and powders.
With locations on both coasts, we are always in position to
quickly deliver the quality product you require. As the company
has grown we have added many things that keep us the leader in
the raw material field. Large investments have been made in the
latest computer systems, warehousing, equipment, inventory,
quality control and personnel to serve our clients to their fullest
needs.
1111
EXHIBIT
Today's industry is facing growth challenges, regulatory changes, quality
issues and changing consumer demands. The Stryka team of professionals
combines research, manufacturlng resources, production methods and quality
assurance to accept these challenges and become your raw material supplier
of the future.
[ Home I Herbs I Extracts & Vitamins I Contact Us ]
Stryka Botanics Co., lnc.
216 Route 206, Somerville, NJ 08876 Tel: (908) 281-5577 /Fax: (908) 281-5392
21218 Vanowen Street Canoga Park, CA 91303 - Tel: (818) 227-0555 /Fax: (818)
227-0560
,
E-Mail: info@strYka.com
-
-
Developed by
TAIZHOU XINGYE CHEMICAL!
0
0
I no
PRODUCT
000
Pyruvic acid
11000 Ca=15-16%
Calcium pyruvate Ca216.5%
I
FOEUO
Shanghai
lOO0D
SPEC.
000
ASSAY
298%
298%
299%
299%
10.00
13.00
15.00
25 KG
52009-14-0
25 KG
41 51-33- 1
298%
14.00
25 KG
81686-75-1
299%
15.00
25 KG
113-24-6
298%
11.80
250 KG
617-35-6
nnnno
298%
12.90
250KG
nnnono
298%
23.60
25
298%
675.00
5KG/TIN
298%
1447.00
5KGRIN
19793-20-5
5-0000
299%
215.00
5k3X"
52 1 - 17-5
onuoo
299%
180.00
25KG
53-43-0
198%
770.00
5IU3lIIN
298%
32.00
25KG
299%
214.00
25KG
298%
1930.00
5 U 3 "
298%
316.00
25KG
299%
25.00
25 KG
0000
Potassium pyruvate
0000
Magnesium pyruvate
0OUU
Sodium pyruvate
00UOO
Creeatinc pyruvate
19-0
nuono n
19-nor-4-androstened1one
19-13 o -4-00no
19-nor-4-androstene-3U ,1 X
I -diol
5-androstene-30 , 1 7 0 -diol
Dehydroepiandrosterone
i 9 c i o n - 5 n iou-ounn
19-norandrost-5( 10)-ene-&one
nnnou
1,3,5-benzenecarboxylicacid
4-0000
4-androstenedione
000 19-00000
Nandrolone
4-clnno
4-androstcne-30 ,lm-diol
N U U 00000
N-acetylglucosaminc
I
I
I
KG
-
734-32-7
63 -05 -8
75 12-17-6
nnnnououooo~ouoooaonn~~on
Address: Baita , Linhai City Zhejiang 317000 ,China
nnuooonounono
nnuooonuouannuo
00Fax: tt86-576-53 183190 5222250 0Tel: +4-86-576-5300O58,522225Oy53O0O68,5
11 1224
Ll
n
0
0
Email:
xiiz~~c~,;,mail.t;zptE.zj.cn
00Website: l i t t m : / / W \ ~ ~ ~ . X i n ~ c . n c t
1999/11/10
I-]
EXHIBIT
Company Information
Contact name:
Telephone:0086-576-5 1I 1224
Fax:0086-576-53 183 19
Chinese Name:
English Name:Taizhou Xingye Chemicals Factory
Address:Chengguan Bata Linhai City Zhejiang China
City :Linhai
Count1y:China
Postal Code/Zip:
Company URL:http://www.xii~gyenet
Brief introduction
Email: TUTT China - Sinochcm YanFzhou
Sinochem Yangzhou
Introduction
Sinochem Jiangsu Yangzhou Inip/Exp Corp. is a subsidiary of China National Chemical Imp/Exp Corp,
which is the largest foreign trade company across China. Sinochem Yangzhou is situated in a 2,000
year-old ancient city blessed with a rich cultural heritage and in close proximity to 60% of China's fine
chemical industry, most of which is within two hour's drive. Over the past few years thanks to the
pains-taking efforts that our most dynamic team of 20rplus your professionals have demonstrated our
business volume has reachcd the annual targct of USD 50,000,000 for several years straight. We aim
higher each year. Our R&D program has worked well to provide the international market with the products
in most urgent need and at a competitive price. We understand there is always rooni for improvement and
that there are always customers there we need to look for. That is why we are redoubling our efforts now
to explore an even brighter future.
PHARMACEUTICAL MATERIALS & INTERMEDIATES
ACYCLOVIWATENOLOL USP23
P-HYDROXY PHENYL ACETAMIDE 99%MIN
4-(2,3-ETHOXY)PROPANOXY PHENYL ACETAMIDE 98YoMIN
GUANINE 99'YoMIN
DIACETYL GUANINE 98%MIN
ALBUTEROL
P-HYDROXY ACETOPHENONE 99%MIN
AMIKACIN SULPHATE
N,N-DICYCLOHEXYLCAIIBODlIMIDE98%MIN
ANALGIN DAB 10
AZLOCILLINE
1-CHLOROCARBONYL-I M I DAZOLLIDONE-(2) 98.5%MIN
BENSERAZIDE
2,3,4-TRIHYDROXY BENZALDEHYDE 98%MIN
BUFLOMEDIL
1,3,5-TRIMETHOXY BENZENE 99YoMIN.
CAFFEINI VB6
ETHYL CYANOACRTATE 99YoMIN
METIIYL CYANOACETATE 99YoMIN
CAWAMAZEPIN BP93
1MINODIBENZIL 99'YoMIN
5-CHLOROCARBON YL IMINODIBENZIL
5-CEILOROCARBONYL IMINOSTTLBENE
CIMETIDINE
EXHIBIT
CYSTEAMINE HCL 95%MIN. 98%MlN.
S-METHYL MONOHYDRATE
4-METHYLIMIDAZOLE 98YoMIN
DIMETH YL-N-CY ANODITHZOI MI NOCARBONATE 98%MIN,
CLOFIBRATE & AI SALT
P-CHLORO PHENOXY ISOBUTYRIC ACID (CLOFZBRIC ACID) 98.5YoMIN
CLONAZEPAM
2-AMINO-5-NITRO-2'-CHLORO BENZOPHENONE
DEXAMETHASONE BP93
DILTIAZEM
2-AMINO THIOPI {ENOL 98YoMIN 99%MIN
P-ANTSLDEHYDE 98%MIN
EPRISONE HCL
P-ETHYL ACETOPHENONE
P-ETHYL PROPYLPHENONE 98%MlN.
ERY THROMY SIN THIOCYNA TE BP9_1/USP23
ECONAZOLE NITRATE
2',2,4-TRICHLORO ACETOPHENONE
FELODIPIN
2,3-DICHLOROBENZALDEMY DE
FENOFIBRATE
4-HYDROXY-LC-'CHLORO BENZOPHENONE
FLUNARIZINE HCL
4,4'-DIFLUORO BEN 20 PH ENONE
4,4'-DIFLUORO BENHYDROL
N-MONO-(4,4'-DI FLUORO-BENZHYDR0L)-PIPERAZINE
FOLIC ACID USP23/BP93/PROCAINE HCL BP93
P- N I TROBEN ZO IC A C I D 99.5 YOM IN .
P-AMINOBENZOIC ACID 99.8%MIN.
P-AMINO BENZOYLAMlDE 99%MIN
1 ,1,3-TRICHLORO ACETONE
2,4,5-TRIAMIN O-G-I I YDROXY PYRIMlDINE SULPHATE 98%MIN.
FURAZOLIDONE USP23/BP93
5-NITRO FURFURYL DIACETATE 98%MIN.
FUROSEMIDE
2,4-DICHLORO TOLUENE 99%MlN.
2,4-DICIJLORO BENZOIC ACID 99%MIN.
2,4-DICHLORO-5-SULPHONYLAINO
BENZOIC ACID 98.5%MIN.
GLUCUROLACTONE
INDOMETHACIN
P-CHLOROBENZOY LCHLORIDE 99%MIN.
KETOCONAZOLE BP88/USP22
1-ACETYL-4-(4-HYDROXY PHENYL)PPIPERALINE 99%MIN.
1,3-DICHLORO BENZENE 99%MIN. 99.5YoMIN.
KETOPROFENUM BP93
LOMEFLOXACJN
2,3,4-TRIFLUOROANlLINE 99YoMIN.
2,3,4-TRTFLUORONTTROBENZENE 99%MIN.
2,3,4,5-TETRAFLUORO BENZOIC ACID 99%MIN
2,3,4,5-TETRAFLUORO-6-NlTRO
BENZOIC ACID 98%MIN
EHTYL 2,3,4,5-TETRAFLUORO BENZOATE 99%MIN
2,3,4,5-TETRAFLUOROBENZOY
L CHLORIDE !BYOMIN
METARAMINOL
M-HYDROXY ACETOPHENONE
METOPROLOL
4-(2-METHOXY ETHYL) PHENOL 99%MIN.
MEZLOCILLINE
1XHLOROCA RBONYL-3-METHYLSULPHONYL-IMIDAZOLLIDONE-(2)
98 .S%MIN.
MICONAZOLE NITRATE
2,4-DICHLOROBENZYL CHLORIDE
NALIDIXIC ACID
DIETHYL MALONATE 99YoMIN.
EMME 99YoMIN.
NAPROXEN BP93
DL-NAPROXEN
6-METHOXY-2-PROPIONAPHTHONE
98YoMIN.
6-METHOXY-2-ACETYL NAPHTHALENE 96%MIN.
6-METHOXY-2-BROMO NAPHTHALENE 98%MIN.
NICLOSAMIDE BP93/USP23
5-CHLORO SALICYLIC ACID 99YoMIN.
5-CHLORO SALICYALDEHYDE
5-CHLORO SALICYLIC ACETAMIDE 99%MIN.
NICARDIPINE /NITREDPINE 97%MIN.
M-NITRO BENZALDEHYDE 99%MIN.
NIFEDIPINE USP23
0-NITROBENZALDEHYDE 99.5%MIN.
NORFLOXACIN U SP23IBP93 / CIPROFLOXACIN HCL USP23
2,4-DICHLORO FLUOROBENZENE 99%MIN.
2,4-DICHLORO-5-FLUORO ACETOPHENONE 99%MIN.99.5%MIN.
TRIETHYL ORTHOFORMATE 98%MIN.
CYCLOPROPYL CARBOXY ACID 99%MIN
3-CHLORO-4-FLUOR0 ANlLIN E 99%MIN. 99.5YoMIN.
OMEPRAZOLE
5-METHOXY-2-MERCAPLOBENZINIDAZOLE
99YoMIN
2-CHEOROMETHYE-3,5-DIMETHYEPRIL)IME-4-METHOXY
HCL 98%MIN
PARACETAMOL BP93/USP23
P-AMINO PHENOL
PHENACETIN
P-PHENETIDINE 98%MIN. 99%MIN.
PIPERACILLINE
I -CHLOROCARBONYL-4-ETHY L-2,3-DIOXO-PIPERAZINE 95%MIN
PIPERMIDTC ACID JPlO
DIMETHYL MALONATE 99%MIN.
TRIMETHYL ORTHOFORMATE 99YoMIN.
PROPAFENONE
0-HYDROXY ACETOPHENONE 98YoMIN
0-IIYDROXY PROPTOPI IENONE 99%MJN
RANITIDINE
1,1-BIS-METHYLTHlO-2-NITROETHENE
98%MIN.
98YoMIN.
N-METHYL- 1-(METHYLTHIO)-2-NITROETHENAMINE
I -METHOXY - 1-METHY LAM INO-2-NITROETHYLENE 98%MIN.
SULFADIAZINE BP93
P-ACETYLAMINO BENZENE SULPHONYL CHLORIDE
TOLBUTAMIDE
P-METHYL BENZENE SULPI-IONYL AMIDE
P-METHYL BENZENE SULPHONYL CHLORIDE 99YoMIN.
TRIMETHOPRIM (TMP) BP93
P-CRESOL 99%MIN.
GALLIC AICD 98%M1N. 99xMIN.
3,4,5-TRIMETHOXY BENZALDEHYDE 99%MIN
3,4,5-TRIMETHOXY BENZOIC ACID 99%MIN
METHYL 3,4,5-TRIMETtiOXY BENZOATE 99%MIN.
VIT.B2/BIATRIZOIC ACID
3,s-DINITRO BENZOIC ACID 98%MIN.
3,5-DINITRO BENZOYL CHLORIDE 98%MIN.
3,s-DIAMINO BENZOIC ACID 99%MIN.
l14-BUTENEDIOL 98%MIN
HERBAL EXTRACTS
GINKGO BILOBA EXTRACT 24%/6% (ginkgo acid:
5PPm 1
ST JOHN'WORT EXTARCT 0.30/0UV
SPIRULINA
FOOD GRADE
HUPERZINE- A
98% MIN
SILYMARIN
APOCYUM VENETUM EXTRACT
GINSENG EXTRACT
SIBERIAN GINSENG EXTRACT
WATER-SOLUBLE TEA POLYPHENOLS
LIPID-SOLUBLE TEA POLYPHENOLS
TEA POLYSACCHARIDE
GRAPE SEED EXTRACT
ALLlCIN(300/KG)
PACLITAXEL
99%MIN.
OTHER BIO CHEMICALS AND PHARMACEUTICALS
AGAR AGAR
CHONDROILINE SULPHATE
THIOCYANO ACID
D-GLUCOSAMINE HCL 99% MIN
D-GLUCOSAMINE S04(2-) 99% MIN
NATURAL VITAMIN E
SO%MIN.
NATURAL BETA-CAROTENE
CHOLESTERIN
SODIUM CHOLATE
CAMPTOTHECIN
98%MIN.
TRYPSIN
BP93/USP23/EUP97
EGG LECITHIN (30,70,85,90)
CHITIN
RIBONUCLEIC ACID
5'-N UCLEOTIDE
D-RIBOSE
RlBOSE TETRACETATE (BETA-D-TETRAACETYL RIBOSE)
VITAMIN A
VITAMIN BI, B2, B6, B12
SODIUM ISOASCORBATE
IVERMECTIN & AVERMECTIN
SIMVASTATIN & LOVASTATIN
FOOD ADDITIVES & AMINO ACID
CITRIC ACID
BP93/USP23
SODIUM (CALCIUM) CITRATE
BP93
FOLIC ACID BP93
CREATINE MONOHYDRATE 99% MIN
SORBIC ACID FCCIV
POTASSIUM SORBATE FCCIV
VANlLLINE FCCIII
D-XYLOSE BP93
XYLITOL FCCIV
STEVIOSIDE
XANTHUM GUM
FOOD GRADE
SARCOSINE
99%MIN.
SODIUM SACCI-IARIN BP93
ASPARTAME
FCCIlI
COUMARIN
FCCIII
L-CARNITINE
BENZOIC ACID A N D SODIUM BENZOATE TECH.FOOD GRADE
CASEIN
IND. GRADE
ACESULFAME-K FOOD GRADE
FUMARIC ACID FOOD GRADE
DL-TARTARIC
ACID
FOOD,IND.&PHARM.GRADE
3.4-Difluoro bromobenzene 99 %min
METHYL (ETHYL) MALTOL FCCIV
INOSITOL NF- 12
GLYCINE Technical Grade 98.5% MIN
Food Grade FCCIll
Pharin Grade USP23
DL(D,L)-ASPARTIC ACID AJ190
DL(D,L)-GLUTAMIC ACID AJI9O
L-GLUTA MlNE FCCIV
N-ACETYL-D-GLUTAMINE 98% MIN
DL-PHENYLALANINE ACID FCCIV
L-CY STINE
L-CYSTEINE HCL (MONO, ANHY)
AJI90
N-ACETY L CYSTEINE
S-CARBOXYMETHY I ,-L-CY STEINE AJI 88
L-LEUCIN E
FCCIII & USP23
L-ISOLEUCINE
FCCIll& USP23
L-VALINE
FCCIII & USP23
DL-€4O M 0 CYSTEINE
L-ARGININE HCL
MONO FCCIII
L-LYSINE
HCL
AJI90
L-PROLINE
AJ190
L-HYDROXY
PROLINE AJI90
L-ALANINE
AJ190
L-THREONINE
AJ190
L-TRYPTOPHAN
AJ190
_._....
__
.
..
.
. .. ., ..
-
Fluoro Compounds
Fluoro benzene 99.5%ni jn
1,2-Difluorobenzcne
99%min.
I ,3-Difluorobenzenc 9C).5'l/oInili
3,4-Dicliloro fluorobenzene 99%miii
3-Chloro-4-fluoro broniobenzene 99Yomin
3,4-Difluoro broniobenzene 99%rnin
2,4-Difluoro broinobenzcne 99Y0miii
.-
-
___
-
4-Fluoro phenol 99%inin
2-Fluor0 phenol 99%min
4-Fluoro tolueiic 99.5%min
3-Fluor0 toluene !@.5%min
2-Fluor0 toluene 99%min
2-Chloro-6-fluorotol~tene98Yomin.
2,6-Difluoro aniline C)8%1niii
4-Fluoroanilinc
99%mlll.
2,3,4-Trifluoro ani1inc 99.S'%mi11
2,3,4-Trifluoro nitrobenzene 99.5'%nii11
2,4-Difluoro aniline 990/;,min
2,4-Difluoro nitrobenzene 99%niin
3,4-Difluoro nitrobenzene 99%rnin
3,4-Difluoro aniline 99%min
4-Fluoro benzylchloride 98%111in.
4-FllIOrO benzaldehyde 99%niin
2-Chloro-6-fluorobci1zaldehyde 98%min.
2-Fluor0 phenylacetic acid 99%min
Trifluoro toluene (Benzotrifluoride) 99%1nin
2-Chloro benzotrifluoride 98%min
3-Chloro benzotrifluoride 98%niin
3,4-Dichloro benzotrifluoridc 99Xmin
1,3-Bis(trifluoromcthyl)benzene 99%min
3,5-Bis(trifluorometliyl)aniline 99%min.
3-Trifluoro methyl phenol 98.5%min
3-Trifluoro methyl anisole 98.5%min
3-Amino benzotrifluoride C)8%111in
4-Chloro benzotrifluoridc 98%miii
HORMONE MATERIALS & lNTERMEDlATES
5-Androstene-3bela-01- I7-onc 99.5% min
4-Androstenediol 97% min
4-Androstenedione 97% min
5-Androstenediol 98% min
5-Androstenedione 97% min
Betamethasone BP93
Chlormadinone Acetate
Cortisone Acetate BP93
Cyproterone Acetate BP93
DehydroepiandrostcronciDWEA 99% mi11
Desoxycorticosterone Acetate BP93
1 6-Dehydro Progesterone 97% min
16-Dchydro Prognenolone 99% min
16alpha, 17alpha-Diliydroxy Progesterone 97% min
16-DPA 96% min
Estradiol USP22
Ethyloestrenol 95% niin
Hydrocortisone BP93/USP23
Hydrocortisone Acetate BP93
17alpha-Hydroxy Progesteronc Acetate
Hydroxy Progesterone Caproate BP93
7-Keto-DHEA 97% min
Levo Norgcstrel USP23
Medroxy Progesterone Acetate BP93
Megesterol Acetate BP93
Mestanolon 97% min
Methandienone 97% rnin
17-Methylandrost-5-e1ie-3,17-dioI
M.P.: 191- 198 DEG C
Methylprcdnisolone BP93
Methyltestostcronc USP23
Nandroloni M.P.: 120- 125 DEC C
Nandroloni Phenylpropionate 97% niin
19-Norandro-4-ene-3,17-diol
97% rnin
19-Norandr0-4-ene-3,17-dione
98.5% min
19-Norandro-5-ene-3,17-diol
M.P.: 161 DEG C niin
19-Norandro-5-cne-3,I7-dione
Norethisterone Acetate BP93
Oxandroni 97% rnin
Prasterone Acetate M.P.:
162 DEG C min
Prcdnisolone BP93/USP23
Prednisolone Acetate USP23
Prednisone BP93/USP23
Pregnenolone 99% rnin
Progesterone Acetatc
Progesterone B P931l.JSP2 3
Prosteronc Sodium Sulfate 98% niin
Stanolone 975 min
Testosterone 97% niin
Testosterone Propionatc M.P.:2 10-2 13 DEG C
___ . . . . . . . . . . . . . . . . . . . . . . . . . .
INTERMEDIATES
3-Bromonitrobenzcne 99% rnin
3-Bromoaniline 99% niin
3-Bromophenol 99% niin
Cyclopropyl Bromide 98% niin
2-Bromopyridinc 98% min
2-Amino-5-Nitropyridine 98% mil?
2-Hydroxy-5-Nitropyridine 98% niin
2-Chloro-5-Nitropyridine98% min
2-Vinylpyridine 98% rnin
2-Aminopyridine 98% min
3-Aminopyridine 99% mill
4-Aminopyridine 99% inin
2-Amino-5-Mcthylpyridinc 98% rnin
2-Amino-6-Metliylpyridinc 98% rnin
._.l...----.l
_____
..................
2,6-Dichloropyridine 98% niin
Phloroglucinol anhydrous 98% min
2-Aniino-4,6-Diaminopyrimidinc
2,4,6-Triamino-5-Nitrosopyriinidine
92% inin
2,4,6-Triaininopyrimidine 98% mi11
2,4,5,6-Tetraminopyriniidine
Sulfate 98% min
Melatonin 99.5% niin
2-Methyl lndoline 99% i n i n
Dibenzfuran 99% min
2,3-Dichloropyrazine
2,6-Dichloropyrazine
3,6-Dichloropyridazinc
2-Acetylpyrazine 99% inin
Indole 98% niin
Hypoxanthine 99% iiiin
5-Methoxytrptatnine
_
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.
_
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__
~
TUTT Group
3710 E. Ovid Ave.
Des Moines, Iowa 503 17
U.S.A.
Tel: 5 15-265-9500
Fax: 5 15-265-9502
Eriiai 1: sales@tu tttrroup. corn
t-Ittp://www.tuttgroup.com
.
-
..
.
h t t p / / w w w . u k i n g t e a m . c o m
e-mail: sales@ukinateam.com rls@hi2000.com,
TEL:+86-577-8332756,
8312424,83501 15,8336171
- EA
Season’s Greeting!
Wish you a Happy New Year and a Successful New Millennium!
Uking Team, a team of experienced and energetic people who were highly educated and with rich
experience in international trade. Because of our high credit in business, because of experience and
knowledge during the past years, we accomplished a strong cooperative team, that knows how to
handle the demand of the customers. In the field of light and electric industry, chemicals,
pharmaceuticals and healthy products, we have developed our services well and built up a network
of qualified and reliable suppliers to ensure each order. Our quality standard and punctual delivery
have won us fame among our customers worldwide, for that we keep our aim to develop long term
business relations and give them our full concentration.
Furthermore we offer you our services as a mediator because we have our own organization for
international business: WENZHOU M AND C FOREIGN TRADE CORPORATION. That can be as
a spokesman to establish a cooperation for you with a Chinese manufacturer, or to help find a
suitable producer for the goods you need, or even if you have been doing business with a Chinese
enterprise, we can help you control the orders, quality and delivery time. Our service is increasing by
50% each year. In 1999, the total value of our work has broken through USD50,000,000.00 and now
is still booming.
All the while, we are committing ourselves to exploit new commercial fields and establish long-term
business relations with new parnters worldwide. Please visit our website
http//www.ukingteam.com and you will find tots of fevourites.
Kevin Ren
Main Manager
Ii-1
EXHlBIT
Ukhg- Team, China
'Leadng your Life in High Quality'
WENZHOU M6C FOREIGN TRADE CORPORATION
htt p//www.
u k i ngtea m .com
e-mail: sales@ukingteam,com rh@hi2000.com
307 XUNUAN ROAD EAST
WENZHOU ZHEJIANG CHINA
TEL: 46577-8332756,
8312424,8350115,8336171
FAX:+86-577-8335844
1 TO.
1
ATTN:
Michael Berns
FAWTEL: 12173679005
DATE
Thursday, February 17,2000
OUR REF: 2k0271nd190
SUBJECT
Quotation:androstenedione,androstenediol
Dear Mr. Michael Berns
Thanks for your mail. We are on the position of supplying you androstenedione and androstenediol. But your
inquiry is not dear. You did not mention spec, quantity and other mmercial conditions. Here is our general
quotation. Please note your interest. The price always bases on client's target quality and annual demand. If you
can inform us more abut your demand, we can negotiate the price again. Please send your reply with your
company details and contact details.Wish we can establish a nice communication in future.
ANDROSTENEDIONE 99%
USD237.001KG
CANDROSTENEDIOL
(4-ANDROSTENE-3 B ,17 B -DIOL)
5-ANDROSTENEDIOL
USD360.001KG
(4-ANDROSTENE-3,17-DIONE)
APPEARANCE
~
~
MELTING POINT
SPECIFIC ROTATION
LOSS ON DRYING
ASSAY(HPLC)
Thanks & Best Regards.
Kevin Ren
I
USD240.001KG
WHITE CRYSTALLINE 1
POWDER
170C-173C
+189 -+202
0.5% MAX
99% MIN
162C- 166C
46.4
0.5%
99% MIN
179C-182C
-51 --57
Q.5%
99% MIN
Subject: quotation: 2k027
Date: Thu, 17 Feb 2000 13:56:25 +0800
From: Kevin Ren <rls@hi2000.com>
To: "rnberns@shout.net" <rnbcrns@shout.net>
Dear Mr.Michae1 Berns
Thanks for your e-mail. Please take care of the attached sheet named 2k027-quot.
Please note your interest. Please enjoy our main introduction in the other attachment
Please send your reply with full details of your company. Thank you in advance.
Should you require any further information then please do not hesitate to
contact me.
Best Regards
Kevin Ren
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Uking Team, China "Leading your Life in High Quality"
Please visit our Website http//www.ukingteam.com
ADD: 307 XUEYUAN ROAD E A S T WENZHOU ZHEJIANG CHINA
TEL:+86-577-8332756,83l2424-2ll835Oll5-2l,8336l7l-2l'
MOBILE: + 8 6 - 1 3 8 0 6 8 7 7 2 8 4 FAX:+86-577-8335844,8350114
e-mail: sales@ukingteam.com, rls@hi2000.com
_-_____-----__--------------
00-2-15 1 5 : 5 3 : 0 0
Original Sheet:
>--
>Victor Pan, Customer Service Dept.
.....................................................
>China Chemi ca 1 Net work
.....................................................
>Providing t h e b e s t information resources t o l i n k
>chemical buyers and s e l l e r s worldwide and b u i l d i n g
>global o n - l i n e trade i n t h e chemical i n d u s t r y .
c E '* - de E i E
+ x ~ iBOD
f 1 ij >>- 1 ~ fc f>>
0 s pffi2 c P ' A ii x
@
>
>
>------------------------------------------------------------
>From: Michael Berns cmberns@shout.net>
>To: l , n o . l , r ~sales@hi2000.coms
>Subject: Price L i s t
>Date: T h u l O Feb 2 0 0 0 1 3 : 3 9 : 2 3 - 0 6 0 0
>
> P l e a s e send me a p r i c e l i s t f o r prohormones, androstenedione,
>androstenediol i n US market
>
>mberns@shout.net
>FAX 2 1 7 - 3 6 7 - 9 0 0 5
>
>------------------------------------------------------------
. . _.
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Name: 2k027-quot.doc
Type: Microsoft Word Document (application/msword)
2k027-auot.doc
Encoding:
base64
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0main introduction.doc
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Name: main introduction.doc
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Type: Microsoft Word Document (applicatiordmsword)
Encoding: base64
Add: Zhenglu town, Changzhou city, Jiangsu prov.,P.R of China.
Tel: 0086-5 19-8736501,8731714, 873 1706-8020/8022
Fax: 0086-5 19-8736500
Postcode: 213 1 1 1
E-mail: iiaerke~public.cz.is.cn
President & General manager: Jiang Hclin
Chemical formula
ISpecific rotation
/Loss on drying
]Packing
l+45"*3O
10.5% Max.
125kgs n.w. in cardboard drum or l0kgs n.w. in
I.@$%@%
lephone:0086-519-8601614,860 1774
Fi1~:0086-519-8601505
E-mail:jiaerke@public.cz.is.cn
Chinese Name:
English Name:Wujin Jiaerke Group Corp. Ltd.
Address:Hengshan bridge town,Changzhou,Jiangsu,China
City :Changzhou
Country:China
Postal CodeIZiv:
Brief introduction
Subject: =?hz-gb-2312?B?fntXKjcifnMIA=?=
Date: Sat, 12 Feb 2000 13: 17:38 +0800
From: jiaerke@public. cz.js.cn
-Original
Message---(7 "+MK-) :jiaerke <jiaerkke@pblic. cz.i s.cn>
-{JU&HK-): mbcms@,shout.nct Kmbcrns(iij,shuu(.uet>
-(HUFZ-}: 2000-(D~-}2-(TB-} 12-(W-} 13:17
Dear sirs,
Thank you for your e-mail dated on 1Ith,Feb.
1just quote the FOB SHANGHAI prices for the following products:
1.BENZOCAINE:
USD6.5KG
2 .CYANOACETAMIDE
USDS.O/KG
3 .CHLOROACETAMIDE
USD2.8KG
4,HYDROCORTISONEBASE
USDS2S.00iKG
5.IIYDROCORTISONE ACETATE
USD535.00KG
6.CORTISONE ACETATE
USD285.OO/KG
7 .PROGESTERONE
USD220.00KG
8 . ~ R O X Y P R O G E S T E R CAPROATE
0~
USD2 25.OO/KG
9,HMIROXYPROGESTERONEACETATE USD2 IO.OOKG
10.PREGNENOLONE
USD 165.OO/KG
11.DHEA
USD 185.OO/KCi
12.4-ANDROSTENEDIONE
USD220.00KG
13.5-ANDROSIENEDIOL
USD230.00KG
14.4-ANDROSTENEDIOL
USD350.00KG
15.TESTOSERONE
USD435.00KG
16.z-IMB CA
USD3S.OOKG
Looking forward to entering into business relationships with you.
Best regards,
Angel.huang
Feb.,12,2000
&
1-
EXHIBIT
U.S. PATENT NO. 5,880,117 COMPARED TO THE LPJ 4-ANDROSTENEDIOL
Claim 1
LPJ 4-Androstenediol
A method for increasing
There are currently no other known uses of 4Androstenediol. LPJ products are generally sold to
distributors in the nutritional supplement industry. The
product is then encapsulated and bottled for retail sales,
generally in the body building market.
testosterone levels in humans
by administration of 4Androstenediol.
Claim 2 (independent form)
I
The LPJ product is 4-Androstenediol, The specification
includes administration bv oral consumDtion.
~~
~
~~~
__
~
by administration of 4Androstenediol,
The LPJ product is 4-Androstenediol.
wherein the mode of
administration is peroral.
The LPJ product is typically encapsulated by distributors
for oral administration,
wherein a peroral daily dosage of
25 mg to 500 mg is taken.
LPJ’s products for retail sales includes directions to take
a dosage of 50- 100 mg per day.
1-
EXHIBIT
wherein the 4-Androstenediol is 4androstene-3beta, 17betadioI.
The LPJ product is 4-androstene-3betq 17betadiol.
Claim 1
ImDorted 4-androstenediol
There are currently no other known uses of 4androstenediol. These products are sold in the nutritional
supplement industry for human consumption. The retail
sales are generally in the body building market.
by administration of 4androstenediol.
The imported product is 4-androstenediol. The
specification includes administration by oral. Imported
products are sold in bulk.
I
I
I
I Imported 4-androstenediol
1
I
I
by administration of 4androstenediol,
The imported product is 4-androstenediol.
wherein the mode of
administration is peroral.
The imported product is typically encapsulated in the
U S . for oral administration.
I
I ImDorted 4-androstenediol
I
wherein a peroral daily dosage of
25 mg to 500 mg is taken.
One of the imported products for retail sales includes
directions to take a dosage of 100 mg per day.
I
Imported 4-androstenediol
wherein the 4-androstenediol is 4androstene-3beta, 17betadiol.
The imported product is 4-androstene-3betaY17betadiol.
5-androstenediol has also been available in the market. It
provides inferior results in increasing testosterone levels.
Advertising in the industry describes the sales of 4androstenediol as being 4-androstene-3beta, 17betadiol.
I1111111111lIl lll1
lI1111Il11111111
llll1
llI111111HI11
US00588011 7A
United States Patent
1191
Arnold
[MI
USE 01' 4hNDROSTENEDIOL 1Y)
INCREASE TESTOSTERONE LEVELS IN
HUMANS
Inventor: Patrick Arnold, P.O. Box 1G0,
Seymour, 111. 61875
Appl. No.: 114,114
Filed:
Jul. 13, 1998
..................................................... A61K 31/56
US. CI. .............................................................. 5!4/178
Fleld of Search ...............................................
514/178
Int, (3.6
1111
1561
Patent Number:
Date of f'ntent:
5,880,117
Mor. 9, 1999
References Cltcd
U.S. PATENT DOCUMENTS
5,387.583 2/199S l n r i n .......................................
5,391,776
211995 lleno el nl. .............................
5,578,588 11/1W6 Mntlero el nl.
.514/171
552/51)7
I'n'rti,ror,v Exmziner-Kayrnontl llenley, Ill
1571
ABSI'IMC'I'
This invention relates to a niethotl of administering the
(estosterone precursor I-androstenediol RS a means o f
increasing testclsterone levels in hiininns.
4 Claims, 1 Drawing Sheet
U.S. Patent
5,880,117
Mar. 9, 1999
Figure 1. Total Testosterone Responses
ye
17
Basebe
1
I
30
1
90
60
4-sndrostenedlone
I
Time (min)
Figure 2. Free Testosterone Rwponses
130
1
+placebo
-&-
90
d
I
Baseline
I
30
1
60
Time (min)
90
4.androstenediol
5,880,117
2
1
USE OF 4-ANDROSTENEDIOI.TO
INCREASE 'I'ESTOSTERONI? LEVELS IN
HUMANS
of anrlrostenetlinneare far I e s and irrorc rnriahle Ilian what
is dcscribcd in U.S. Pat. No. S.578.588. It was thcrcfnrc a n
ohject of this invention 10 tliscwwr ;inother n a l i i r a l l y occurring testosterone precursor that provided a greatcr blond
FIELD OF THE INVEN'I'ION
.S testosterone level response t h a n androstenedione but
retained all the advanlages of being a nun-toxic, natural, and
This invention relates to a method of administering the
quickly metabolizable precursor. 'Ihis would thercfore pertcslostcronc precursor 4-androstcnediol as a means of
mit oral adniinistration at a reasonalilc close providing a
increasing testosterone levels in humans. The steroid hordcpcndablc therapeutic rcsponsc.
mone testosterone is considered to be thc malc virilizing
The chemical term 4-nndroslenediol refers to two isohormone. Its effects include maintenance of muscle arid 1"
mers: 4-androstene-3be1a, 17betadiol and 4-andrnstenehone mass, sexual function, and psychological well being
3alpha, 17beta-diol. 'Ibis invention conccrns primarily the
among others As males grow oltlcr, esqecially alter [he age
former isomer in the preferred emhotlinient
of 35, a slow decline it] testosterone level? is observed which
is accompanied by symptoms that have been associaled with
4-androstenediol is a naturally occiirring coinpound. I t
the condition known as "andropause". Symptoms of andro- Is has heen itlenlified as a nietaliolite of testosterone i n
pause include lethargy, depression, lack of sexual desire and
placental, ulerine, testicular. adrenal, and hypothalamic/
fiinction, and loss of muscle mass and strength.
pituitary tissues. It acts as a very effective precursor to
testosterone. 4-androstcnet1io1converts to teslislerone via
DESCRIPTION OF TIIB PRIOR ART
the 3beta-hyciroxysteroid dehydrogenase enzyme. 1:. Ungar,
There are several plrarmaccutical methock 10 restore tes- lo M. Gut, a n d R. Dorfnian (-I Rid. U ~ I 224.
. , 191-2m)
round that after 4-antlrnslcncdiol was inciihatcd in livcr
tosterone levels in humans with suboptimal levels. Many of
limiie it melabdkml very readily t ( i testosterone. J. Blaclilier,
these have tlisaclvnntitges however. 'Ikstosterone &ers in oil
depot form have been used as injections for decades, how8. Porchielli, and R. Ilorftnan [Acm Edncrirlologico, 55,
ever these injections can be inconvenient and often painful.
697-704) also revealed that the in vitro conversion ul
These depot injeclions also reslllt in inconsistent blood 25 tritialed 4-androslene-3beta, I 7betadioI to testosterone in
levels as a supraphysiological surge is seen % O n after
whole humaii blood was very eflicient (15.76%) and was in
injection Iiut by the time the next injection is due, the levels
[act considerably more efficienl that tritiated androstenedihavc oftcn droppcd down below standard physiological
onc (5.61%).
in contrast with testosterone levels untlcr 2o , After learning of the in-vitro cflicacy of 4-androstencdiol
ons. which are quite stable within mild
i n regards to testosterone convcrsion, it was thcri thc intenrelease pulses of approximately 90 minute duration. Supration of the inventor lo iiivesligale whetlier 4-andrnstenedinl
physiological surges that are Seen with injectable preparawould act as an cffcclivc in-vivo peroral tcslostcronc prclions may increase the incidence o f undesirable side efl'ccts
cursor i n Iiurnaiis. It was also the intention of the inventor to
(i.e. prostrafe hypertrophy) as well as cause an amplified 35 investigate whether or no1 4-antlrostenediol would act as a
shutdown of thc hypothalamic/pituitary testicular axis
superior peroral lesloslerme precursor t ~ ant1rc)stenetlione.
i
(1-1
PTA) .
A clinical study was therefore undertaken. Seven adult
Other pharmaccutical rncthods for androgcn rcplaccmcn~
male subjects were liwd Each suhjecl was on separate
therapy include synthetic oral androgen derivatives. .These
occasions given an oral dose o f 100 rng. placebo,
compoiinds (i.e. methyltestosterone and fluoxymeslerone)
4-androstenediol, or antlrostencdione. Blood samples were
are altered i n the I7alpha position of the stcroid molecule
collected at 0, 30, 60, and 90 minutes following ingestion
wilh an alkyl group. This alkyl group renders the steroid
and analyzcd for total leslostcrnnc (Tl? (SCC PIG. I ) and rrcc
lestoslerone
(see PIG. 2) rising enzynie-linked immuimpervious to oxidation of the 17 beta hydroxyl group in the
liver and therefore greatly improves its oral bioavailahility
nosorbent assay. Rclativc to placcl,o, antlrnstcncdionc ingcscompared lo the non-alkylated steroids. Ilawever, this struc. 45 ticin C R I I S C ~a 1.43% increrx: i n t o t ; l l lesloslcronc and a
tural modification also has been associalcd with a greatly
10,996 increase i n free lesloslcronc n l 9 0 rninilles.
4-androstenediol ingestion canscd greater rcspcinscs, proincrcascd risk of hepatotoxicity. 'rhcrcforc, thcsc synthetic
ducing a 47.7% increase in total ~es~os~crone
arid a 4Z.SQz
cornpouncls are far from a n ideal solution.
increase i n free testostcronC a t 90 minvtcs.
U.S. Pat. No. S,S78,588 10 Mattern. C I 21, discloses a
niethocl of increasing testosterone levels i n huiriaris by 50
Oral 4-androslenediol can be given in daily closes of 25
administering a lcstoxtcronc prccursor, namcly androstcncrng. to SO0 nlg., preferably 100 to Xl(1 mg. 'I'hese daily doses
can be divided into several subdoses with 3-5 Iieing most
dionc. Modes of'ntlminislration discussed include peroral
preferahle. In addition to peroral administration,
and intranasal, The pharmacofinelics of such an administralion of a precursor is such lhat a pe.ak in [?\nod levels is
4-aiidroslenediol call also bc e~eclivelyattniinisteret~ b y
seen at approximately 90minutes with a sul~sequen~
tlecline 5.5 several other routes including trnnsdcrntal, rectal
(suppcisiltrry), inlrnn:isal, a n d sirhIingu;iI A [iNtic~lhr1y
to baseline within 3 hours, This facl permits one to morc
advantageous method of subling\laladministration involvcs
cloxfy sirnlllale the natural endogenous pulsatile relcasc of
tcstostcronc through multiple daily dosing of a precursor.
cornpieXing 4-antlroslenediol with beta-hydroxy~irc~~~\~IThis shoultl resilli i n a more normal physiological response
bels-cyclodexlrin which is then imsscrl into tablets.
4-androstenediol can also he effectively combined with
with a minimization of side effects and FIP'rA Shu(&wn.
androstenedione to produce a product t h a t contains t w ~
E'urihermore,since Itiesc precursors are natur;lI steroid hormanes found i n the blood, and are no( 17alpha alkylated
prccursors that convcrt lo Icstnstcrnnc through IWO clistincl
enzyme ,systems,
compounds. the hepatoxicity is minimal.
'Thc forcgoing drawings and dcscription nf thc invcnlinrl
DESCRIP'IION OF: 'U!E INVENI'ION
6s are for illustratioii only. k4o(lificatinmi not includctl in tllc
description which are obvious to those skilled in the art are
In the course of our research, we havc found that the blood
inlentled tu he inclutletl in thv cccil~eof the following clainjs
teslostcrone level increases seen with the oral atlminislrnlion
(m
3
5,880,117
4
DL5,SCRIPTION OF THE: DRAWINGS
The present invention will be more fully understood by
rcfcrence 10 the following dctrilcd dcscription thcreof whcn
read in con,iunctio,l
the altachecl rlrawings, and. wherein:
FIG. 1 is a graph or 'I'otal 'l'estosterone level versiis lime
in a clinical test of the invenlion.
FIG, 2 is a graph of F~~~T~~~~~~~~~~~
lcvcla
versus
In
in a clinical test rif the invention.
1claim:
1. A method of increasing leslosterone levels in hurnaos
b y administration of 4-androstenediol.
~
2. 'The nielhod of increasing testosterone levels in liiimans
according lo chim 1, wherein the mode ol atlrninistretion is
peroral'
3. The method o f increasing tcsrostcrone Icvels in humans
according to claim 1. whcrcin a pcroral clnily dosagc ol 25
mg is lakcn,
mg to
4. The method of increasing lesto.steronelevels in humans
according 10 claim 1. wherein the 4-andros~enediol is
4-androstene-3bcta, 17hetatliol.
* * * * *
ASSIGNMENT OF INVENTION AND PATENT APPLICATION
For value received, Patrick Arnold of Seymour, Illinois (hereinafter Assignor), hereby sells,
assigns, transfers, and sets over unto LPJ Research, Inc of Seymour, Illinois and its successors or
assigns (hereinafter Assignee) One Hundred Percent ( 100%) of the followitig:
Assignor’s right, title and interest in and to the invention entitled “USE OF 4ANDROSTENEDIOLL TO INCREASE TESTOSTERONE LEVELS M HUMANS”,
invented by Assignor,
the application for United States patent therefor,
any patent or reissues of any patent that may be granted thexeoa
any applications which are continuations. continuations-in-part, substitutes, or divisions of
said application.
Assignor authorizes and requests the United States Patent and Trademark Office to issue any
resulting patent(s) to Assignee.
Assignor hereby hrther sells, assigns, transfers, and sets over unto Assignee, the above
percentage of Assignor’s entire right, title, and interest in and to said invention in each and every
country foreign to the United States; and Assignor hrther conveys to Assignee the above
percentage of all priority rights resulting from the above-identified application for United States
patent. Assignor agrees to execute all papers, give any required testimony and perform other
IawfUl acts, at Assignee’s expense, as Assignee may require to enable Assignee to perfect
Assignee’s interest in any resulting patent of the United States and countries foreign thereto, and
to acquire, hold, enforce. convey, and uphold the validity o f said patent acid reissues and
extensions thereof, and Assignee’s interest therein.
In testimony whereof Assignor has hereunto set his hand on the dare below
I-[
EXHlBIT
MICHAEL A. BERNS
107 WEST GOOSE
ALLEY
URBANA. I
L 61801
2 1 7-367-9000
FAX 2 17-367-9005
PHONE
mberns@shout.net
March 7,2000
Asymchem, Inc.
2 Davis Dr.
P.O. Box 12076
Research Triangle, NC 27709
RE: U.S. Patent 5,880,117 to Patrick Arnold
I enclose a copy of U.S. Patent 5,880,117 to Patrick Arnold which issued on March 9, 1999,
entitled “Use of 4-Androstenediol to Increase Testosterone Levels in Humans.” LPJ Research,
Inc., assignee of the patent, is willing to grant nonexclusive licenses in the patent with respect to
all or limited fields of use upon negotiation of appropriate terms and conditions. LPJ Research,
lnc. is also the owner of the registered trademark ANDRODIOLB.
The patent discloses the use of 4-Androstenediol in humans. Patrick Arnold developed this
compound and brought it to the market. It has proven to be far more effective than the popular
androstenedione.
Asymchem, Inc. is reportedly a distributor of 4-Androstenediol. You must now have a license to
make any further sales. Sales without a license of the patent constitutes infringement in violation
of federal law.
This letter constitutes notice under 35 U.S.C. 287 of infringement of the 5,880,117 patent.
Damages may be tripled under the statute for willful infringement, and attorney’s fees may also be
included. We demand that you immediately cease and desist from any and all sales of 4Androstenediol until a license can be negotiated. LPJ Research, Inc. demands that the sale of the
products in the United States cease at once.
I will contact you in a few days to confirm your plans to license, or to cease the sale of 4Androstenediol. Please call me if you have any questions or need additional information.
Sincerely,
Michael Berns
Attorney-at-Law
REG15
UNITED STATE
-~
~~~
~
MICHAEL A. BERNS
PHONE217-367-9000
FAX 2 17-367-9005
107 WESTGOOSEALLEY
URBANA,
IL 61801
mberns @shout.net
June 14,2000
Changzhou Huabang Pharmaceutical Group, Ltd.
22/F, International Building
RE:US. Patent 5,880,117 to Patrick Arnold
I enclose a copy of U S . Patent 5,880,117 to Patrick Arnold, which issued on March 9, 1999, and
is entitled “Use of 4-Androstenediol to Increase Testosterone Levels in Humans”. LPJ Research,
all or limited fields of use upon negotiation of appropriate terms and conditions. LPJ Research
Inc. is also the owner of the U.S. registered trademark ANDRODIOL@.
Basically, the patent discloses the use of 4-Androstenediol in humans. As you may know, Patrick
Arnold developed this compound and brought it to the market. It has proven to be far more
effective than the popular androstenedione.
Changzhou Huabang Pharmaceutical Group, Ltd. was reportedly a distributor of 4Androstenediol. You now must license any further sales in the United States. Failure to license
the patent constitutes infringement, in violation of federal law.
The letter constitutes notice under 35 U.S.C. 287 of infringement of the 5,880,I 13 patent.
Damages may be tripled under the statute, and attorney fees may also be included. We must
demand that you immediately cease and desist any and all sales of 4-Androstenediol until a license
can be negotiated. LPJ Research Inc. demands that the sale of these products in the United
States cease at once.
Please contact me if you have any questions or need additional information.
Sincerely,
Michael Berns
Attorney-at-Law
REGISTERED
ATTORNEY #38,379
MICHAELA. BERNS
PHONE2 17-367-9000
1 0 7 WEST GOOSEALLEY
UREANA.
JL 6 1801
F A X 2 17-367-9005
mberns @shout.net
April 19, 1999
Kingchem, IIIC.
Oradell, NJ 07649
1enclose a copy of U.S. Patent 5,880,1 17 to Patrick Arnold, which issued on March 9, 1999, and
is entitled “Use of 4-Androstenediol to Increase Testosterone Levels in Hutnans”. LPJ Research,
tnc., assignee of the patent, is willing to grant nonexclusive licenses in the patent with respect to
Inc. is also the owner of the registered trademark ANDRODIOLB.
Basically, the patent discloses the use of 4-Androstenediol in htitnans. As you niay know,
Patrick Arnold developed this compound and brought it to the market. I t has proven to be far
niore effective than the popular androstenedione.
Kingchern, Itic. was rcportedly a distributor of 4-Androstenediol. You now must license any
fiirther sales. Failure to license the patent constitutes infringement, in violation of federal law.
The letter constitutes notice under 35 U.S.’C.287 of infringement of the 5,880,l 13 patent.
Damages may be tripled under tlie statute, and attorney fees may also be included. We iiiust
demand that you irritnediately cease and desist any and all sales of 4-Androstenediol until a
license can be negotiated. LPJ Research Inc. demands that the-sale of these products in the
United States cease at once.
I will contact you in a few days to confirrii your plans for licensing, or ceasing, the sale of
4-Androstenediol. Please call if you have any questjons or need additional infortnation.
Sincerely,
Michael Beriis
Attorney-at-Law
REGISTERED
ATTORNEY ff 38,379
UNITED S T A T E S P A T E N T AND TRADEMARK OFFICE
MICHAEL
A. BERNS
107 WEST GOOSEALLEY
URBANA,
IL 6 180 1
PHONE21 7-367-9000
FAX 21 7-367-9005
mberns @shout.net
June 14,2000
Purechem Co., Ltd.
7 1 North Zhujiang Rd.
P.O. Box 528
Kunshan, JS, 215301, China
RE:U.S. Patent 5,880,117 to Patrick Arnold
I enclose a copy of U.S. Patent 5,880,117 to Patrick Arnold, which issued on March 9, 1999, and
Inc. is also the owner of the U.S.registered trademark ANDRODIOLO.
Purechem Co., Ltd. was reportedly a distributor of 4-Androstenediol. You now must license any
further sales in the United States. Failure to license the patent constitutes infringement, in
violation of federal law.
The letter constitutes notice under 35 U.S.C. 287 of infringement of the 5,880,113 patent.
Sincerely,
Michael Berns
Attorney-at-Law
REGISTERED ATTORNEY #-,a78
MICHAEL A. BERNS
107 WEST GOOSEALLEY
URBANA.
IL 6180 1
PHONE2 1 7-367-9000
FAX 21 7-367-9005
mberns @shout.net
June 14,2000
Shanghai Freemen International Trading Co., Ltd.
Shanghai Industrial Investment Bldg., Rm. 22D
Cao Xi Rd. (N)
Shanghai 200030, China
Inc. is also the owner of the U.S. registered trademark ANDRODIOLO.
Shanghai Freemen International Trading Co., Ltd. was reportedly a distributor of 4Androstenediol. You now must license any further sales in the United States. Failure to license
The letter constitutes notice under 35 U.S.C.287 of infringement of the 5,880,113 patent.
demand that you immediately cease and desist any and a11 sales of 4-Androstenediol until a license
Michael Berns
Attorney-at-Law
REGISTERED AlTORNEY #38,378
SERNS L A W OFFICE,
P.C.
MICHAELA . BERNS
PHONE2 1 7-367-9000
F A X 2 17-367-9005
107 W E S T GOOSEALLEY
U R R A NI
A
L.6 180 1
niberns @shout.net
April 19, 1999
Stryka Botanics Company, lnc.
21218 Vanowen St.
Canoga Park, CA 91 303
I enclose a copy of U.S. Patent 5,880,l 17 to Patrick Arnold, which issued on March 9, 1999, and
Itic., assignec of the patent, is willing to grant nonexclusive licenses in the patent with respect to
a l l or limited fields of use upon negotiation of appropriate terms and conditions. LPJ Research
Jnc. js also the owner of the registered trademark ANDRODIOLB.
Basically, the patent discloses the use of 4-Androstenediol in hurnans. As you may know,
Patrick Arnold deveJoped this compound and brought it to the market. I t has proven to be far
more effective than the popular androstenedione.
Stryka Botanics Company, Inc. was reportedly a distributor o f 4-Androstenediol. You now must
license any further sales. Failure to license the patent constitutes infringement, in violation of
federal law.
The letter constitutes notice under 35 U.S.C. 287 of infringement of the 5,880,l I3 patent.
Damages may be tripled under the statute, arid attorney fees may also he included. We must
demand that you immediately cease and desist any and all sales of 4-Androstenediol until a
license can be negotiated. LPJ Research Inc. demands that the sale of these products in the
Clnited States cease at once.
I will contact you in a few days to confirm your plans for licensing, or ceasing, the sale of
4 -Androstenediol. Please call if you have any questions or need additional itifortnatioii.
Sincerely,
Michael Berm
Attorney-at-Law
REGISTEREDATTORNEY #38,379
UNITED S T A T E S P A T E N T AND TRADEMARK OFFICE
MICHAEL A. B E R N S
107 WESTGOOSEALLEY
URBANA.
IL 61801
PHONE21 7-367-9000
F A X 2 1 7-367-9005
mberns 42 shout.net
June 14,2000
Taizhou Xingye
Baita
Linhai City, Zhejiang 217000, China
Inc. is also the owner of the U S . registered trademark ANDRODIOLO.
Taizhou Xingye was reportedly a distributor of 4-Androstenediol. You now must license any
Sincerely,
Michael Berns
Attorney -at-Law
-
REGISTERED
ATTORNEY 838,379
UNITED STATES PATENT AND TRADEMARK
OFFICE
MICHAEL A. BERNS
107 WEST GOOSE
ALLEY
U R B A N A . IL 61801
PHONE2 1 7-367-9000
F A X 2 1 7-367-9005
mberns @shout.net
March 7,2000
3710 E. Ovid Ave.
Des Moines, IA 503 17
I enclose a copy of U.S. Patent 5,880,117 to Patrick Arnold which issued on March 9, 1999,
entitled “Use of 4-Androstenediol to Increase Testosterone Levels in Humans.” LPJ Research,
all or limited fields of use upon negotiation of appropriate terms and conditions. LPJ Research,
Inc. is also the owner of the registered trademark ANDRODIOLB.
The patent discloses the use of 4-Androstenediol in humans. Patrick Arnold developed this
compound and brought it to the market. It has proven to be far more effective than the popular
androstenedione.
TUTT China - Sinochem Yangzhou is reportedly a distributor of 4-Androstenediol. You must
now have a license to make any further sales. Sales without a license of the patent constitutes
infringement in violation of federal law.
This letter constitutes notice under 35 U.S.C. 287 of infringement of the 5,880,117 patent.
Damages may be tripled under the statute for willful infringement, and attorney’s fees may also be
included. We demand that you immediately cease and desist from any and all sales of 4Androstenediol until a license can be negotiated. LPJ Research, Inc. demands that the sale of the
products in the United States cease at once.
I will contact you in a few days to confirm your plans to license, or to cease the sale of 4Androstenediol. Please call me if you have any questions or need additional information.
Sincerely,
YMichaelBerns
Attorney-at-Law
REGISTERED ATTORNEY 838,379
UNITED STATES PATENT AND TRADEMARK
OFFICE
107 WEsr GOOSEALLEY
URBANA,
IL 61801
PHONE21 7-367-9000
FAX 2 17-367-9005
mberns @shout.net
June 14,2000
Uking Team, China
Wenzhou M&C Foreign Trade Corporation
307 Xueyaun Road East
Wenzhou Zhejiang, China
I enclose a copy of US.Patent 5,880,117 to Patrick Arnold, which issued on March 9, 1999, and
Inc. is also the owner of the U.S.registered trademark ANDRODIOLO.
Uking Team, China was reportedly a distributor of 4-Androstenediol. You now must license any
can be negotiated, LPJ Research Inc. demands that the sale of these products in the United
Sincerely,
Michael Berns
Attorney-at-Law
REGISTERED ATTORNEY #38,378
MICHAEL A. BERNS
107 WEST GOOSEALLEY
URBANA,
IL 6 1801
PHONE2 1 7-367-9000
FAX 2 17-367-9005
mberns @shout.net
June 14,2000
Wujin Jiaerke Group Corp., Ltd.
Hengshan Bridge Town
Inc. is also the owner of the U S . registered trademark ANDRODIOLB.
Wujin Jiaerke Group Corp., Ltd. was reportedly a distributor of 4-Androstenediol. You now
must license any further sales in the United States. Failure to license the patent constitutes
infringement, in violation of federal law.
Sincerely, 1
Michael Berns
Attorney-at-Law
REGISTERED ATTORNEY #38,378
MICHAEL A. BERNS
107 WEST
GOOSE
ALLEY
U R B A N A . IL 61801
PHONE21 7-367-9000
F A X 2 17-367-9005
mberns @shout.net
June 14,2000
Zhejiang Provincial Light & Textile Industry Group Corporation
No. 8 Mei Hua Bei
Hangzhou, Zhejiang, 3 10009, China
Inc. is also the owner of the U.S. registered trademark ANDRODIOLB.
Zhejiang Provincial Light & Textile Industry Group Corporation was reportedly a distributor of
4-Androstenediol. You now must license any further sales in the United States. Failure to license
Sincerely,
Michael Berm
Attorney-at-Law
REGISTERED
ATTORNEY138,379
SENDER:
‘f
w Complete Homo 1 UKVor 2 lor addiUond urvlcw,
Complelr itmr 3 4a ond 4b.
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F e b 2 9 00 1 0 : 4 9 a
John
P. R r n o l d
714-673-4158
P.3
LPJ Research Inc.
Home Office
Western Sales Office
205 South Main Sc.
PO.Box I60
Seymour, IL 6 I875
Phone: 2 f 7-687-4038
Fax: 2 17-687-4I38
352 Prom ontory Drive West
Newpol t Beach, CA 92660
PI one: 949-566-984 I
Fax: 949-566-98 I 4
email: infog Zlpjresearch.com
~p Research, In,.
Price List
J
Effective - October 15,1999
Bulk Products
Quantity in Kg
Product
AndrodiolB
(4-Androstenediol)
Norandrodiol"
(19-Nor-4-Androstenediol)
1to 50 kg
51 to 100 kg
Price per kg. In JSD
FOB - Seymour. IL
515
500
1 to 50 kg
51to 100 kg
1980
1 to 50 kg
51 to 100 kg
910
905
1 to50 kg
51to 100 kg
270
240
I to 50 kg
Cyclo-N ordiolTM
(Norandrodiol WICyclodextrin) 5 1 to 100 kg
710
680
19 Norandrostenedione
Cyclo-DiolTM
(Androdiol W/ Cyclodextrin)
---
For quantities over 100 kg please contact office
plastic bottles
Packaging: 1- 2kg
Lined fiber drums used for larger orders
Delivery:
In stock items shipped within 48 hours
10 to 20 days for items to be manufactured
J1-
EXHIBIT
1955
--
__-
--
AMERICAN PROHORMONE
MANUFACTURER AND RESEARCH
COMPANY
LPJ Research, Inc. (LPJ) is an American
Prohormone manufacturer and research
company based in Seymour, Illinois. Patrick
Arnold, President of LPJ, is a gifted chemist
who is known as the pioneer of modern day
prohormones. Patrick was the first person to
introduce the prohormone Androstenedione to
the national market. In addition, LPJ is the first
USA manufacturer to offer the prohormones
Androdiol, and Norandrodiol, in production
form to the domestic market.
By maintaining the highest manufacturing
standards in the industry, we can ensure the
highest product quality and consistency
available. Frequent lab testing is done to
assure guaranteed purity of finished products.
Independent laboratories are also used for
confirmation. In addition to quality, you can
expect one o f the lowest pricing structures in
the industry, providing the highest value in
prohormone products available anywhere.
Ergopham has formed an alliance with LPJ
Research Inc., the leader in prohormone
technology, to offer a cutting edge brand of
nutritional supplements. Patrick Arnold o f LPJ
Research will serve as a consultant on the
LPJ Research Inc (LPJ) is an American
Prohormone manufacturer and reserach
company based in Seymour, Illinois. President
Patrick Arnold is a gifted chemist who is
known as the pioneer o f modern day
prohormones. Patrick was the first person to
introduce the prohormone Androstenedione to
the national market. In addition, LPJ is the first
USA manufacturer to offer the prohorniones
Androdiol@, NorandrodiolQ, and
hydroxypropyl-beta-cyclodextrinprohormone
compounds, in production form to the
domestic market.
As a result of his wdrk in the area of
prohormones, The Sporting News magazine
recently named Patrick Arnold to their
Power 100 list. Patrick was voted as one of
the top 100 most influential sports figures in
the world!
Choose from a complete line of quality
prohormones, with options such as low bulk
pricing, finished bottle pricing (labels to be
supplied by client), and substantial volume
discounts.
A complete line of quality prohormones from
one source guarantees that you will be able to
get the right prohormone in the right quantity,
form, and price to meet your needs as you
grow.
LPJ RESEARCH: THE PRESIDENT
Patrick Arnold, President of LPJ
A gifted chemist, and world renowned 'father
of modem day prohonnones', Patrick Arnold's
writings are in constant demand because of
his intelligent, truthful, and effective style.His
articles are frequently published in magazines,
newspapers, and various industry journals.
Reprints of articles will be made available free
of charge. Patrick is also available to provide
technical support and to answer your
questions.
PRODUCTS OFFERED:
Androstenedione:
The original testosterone precursor
19 Norandrostenedione:
The orginal nortestosterone precursor
5 Androstenediol:
DHEA metabolite with low androgenic
potency. Immunostimulating activity. Direct
testosterone precursor.
4 Androstenediol (Androdiol 0)
Call for details.
19 Norandrostenediol
(Norandrodiol).
Call for details.
Cyclo-Diol TM
Call for details.
Cyclo-Nordiol TM
Call for details.
C of A is provided with each order.
For quantities over 100 kg please contact the
office.
PACKAGING: 1 kg - plastic bottle
2 kg - plastic bottle
Lined fiber drums are used for larger orders
DEL1VERY:In stock items shipped within 48
hours. Allow 10 to 20 days for items to be
manufactured for order sizes up to 100 kg.
Larger orders to be scheduled - contact office.
LPJ Research
205 South Main P.O. Box 160
Seymour, Illinois 61875
(217) 687-4038
FAX:(217) 687-4138
info@lpjresearch.com
LPJ RESEARCH: CONTACT US
LPJ Research
205 South Main P.O. Box 160
Seymour, Illinois 6 1875
(2 17) 687-4038
FAX:(217) 687-4138
info@lpjresearch.com
-
LPJ Research Inc.
Telephone: 217-687-4038
Fax: 2 17-687-4 I38
email: info@ Ipj research .c o m
205 South Main St.
PO.Box I60
Seymour, IL 6 I875
CERTIFICATE OF ANALYSIS
AndrodioIO
[ 4-Androstenediol]
LOT # 4AD04000
TEST
SPECIFICATION
Appearance
White to off white Powder
RESULT
Conforms
Assay
90 to 100% by HPLC
Melting Point
155- 165°C
155.6-163.1
Loss on Drying
1% Maximum
0.9%
96.6%
Chemical Structure
AndrodioKB
[4
Packaging
- Androstenediol ]
Please consult LPJ Research, Inc.
A Certificate of Analysis is provided for each shipment.
Androdiolo is a reQisteredtrademark of LPJ Research Inc.
Subj:
Re: HPBCD (urgent)
Date: 5/9/2000 7:04:51AM Central Daylight Time
From: inestamail.hz.zj.cn (Stephen Hou)
Reply-to: inves_t&mair,kA?j p~
(Stephen Hou)
To: RBoodram&aol.com
Dear Boodram,
Nice to receie your email.
How about your trip?
4andms tene3beta,Ifbetadiol
Punty>+98%
Prlce:$375.31/kg FOB
1g-nor-l-andros tene-3bela, 17betadiol
Purlty>=98%
Pnce:$1634.38/kgFOB
Delimy: Shipped by EMS and shipped three days after receidng your payment.
As to postage, Iwill check with postoffice and let you know tomorrow.
Sincerely yours,
Stephen Hou
-Original Message -
From: <RBoodram@aol.corn>
To: <inmt@mail.hz.zj.cn>
Sent: Monday, May 08, 2000 10:59 PM
Subject: Re: HPBCD (urgent)
> Stephen,
>
> Thanks for your email. Good to know the money amied safely. We would like
> to purchase one kilo of 4 Androstenediol and one kilo of l9Nor 4> Andorstenediol. Please adwse on price and deliwy.
>
> Regards,
> R. Boodram.
>
Reply-To: “Stephen Hou“ 4 nmst@mail.hz.z].c n>
TumW,MA.)09.2
1 25
EXHIBIT
-
*.p: 1
IS75 USA
Mark & Nos.
I
19-NOR-4-ANDNOSTEM-3
P ,I7 B
DIOL
IKG
-
. ._
SAY U.S.DOLLARS TWO TIlOUSAND AND
EIGHTY TWO AND CEBTS SIXTY NINE
ONLY
USD408.78KG
USD408.78
USD1673.91KG
USD1673.91
n'L:
USD2082.69
I I I
P CIKP:
(8)
ZEIEJIANC PROVINCIAL LIGHT & TEXTILE I1yDlJSTRY GROW COdWATnnU
jjyr
$1+k$
$.$$3 &
$
Bj
h\ q
%)I EJIANG PROVINCIAL LlGHT & TEXTILE INUUS'I'RY GROUP CORPORATION
No.8 Mei Hua Bei, Hangzhou, P.R.Chisa
PACKING
LIST
TO: R. BOODRAM LPJ RESEARCH
mm:
M ~
YIS, 2000
INVOICE NO: CEO81
S/C NO: ZLTCO8lE
205 SOU'1" MAIN STREET
SCYMOUR 1L 61875 USA
-Measurement
ENE-3 I3 ,I7 B
1KG
KG/2KG
0.01YM3
1KG
m.
E?;
American Analytical Chemistry Laboratories Corp.
Value & Quality-OrientedAnalytical Testing Services
Illinois Technology Center
101 Tomaras Avenue
Savoy, Illinois 61874
Phone: 217-352-6060
Fax: 217-352-6052
REPORT OF ANALYSIS
LPJ Research, Inc.
Sample No: 4ADl5400C
205 S. Main Street
Se:.niour. IL 61875
A m . : Patrick Arnold
Lab Sample ID:01-1-206
Product Name: Androdiol
Date Received: 06/07/00
Analysis Requested: 4-Androstendiol, Cholesterol Date Analyzed: 06112,22/00
Date Reported: 06/23/00
Analytical Methods: HPLC, GC
I TEST
~
4-Androstendiol
Cholesterol
I RESULT
I
87.3
<0.01
I
%
%
* This sample was tested against a 4-androstendiol reference from Steroidal, Inc.
Date: 06/23/2000
UNITED STATES INTERNATIONAL TRADE COMMISSION
Washington, D.C.
In the Matter of
Inv. No. 337-TA-
4-ANDROSTENEDIOL
Affidavit
Complainant LPJ Research, Inc. and Patrick Arnold, in support of its Complaint, states the
following:
1. I am the President of LPJ Research, Inc. of Seymour, Illinois, manufacturer of nutritional
supplement compounds.
2. I received a Bachelor of Science degree in Chemistry and have worked toward a Master’s
degree in Chemistry.
3. I have been involved in professional practice as a chemist and researcher.
4. I have been awarded U.S. Patent 5,880,117 and U.S. Patent 6,011,027 for nutritional
supplements.
5. It is well known in the nutritional supplement industry that 4-androstenediol can be
purchased from China.
6. LPJ Research, Inc. has been forced to reduce their prices to compete with illegally imported
products from China.
7. The imported products infringe U.S. Patent 5,880,117, owned by LPJ Research, Inc.
8. There are no legitimate usages €or 4-androstenediol, other than those described in the patent
claims.
9. The proposed respondents are involved in making, using, selling, or offering to sell 4androstenediol, covered by U.S. Patent 5,880,117.
1
I hereby declare that all statements made herein of my own knowledge are true and that all
statements made on information and belief are believed to be true; and further that these
statements were made with the knowledge that willfkl false statements and the like so made are
punishable by fine or imprisonment, or both, under Section 1001 of Title 18 of the United States
Code and that such willful false statements may jeopardize the validity of this action.
Very Respectfully,
hfYP
LPJ Researc , Inc.
Patrick Arnold, President
LPJ Research Inc.
By: Michael Berns
Its Attorney
BERNS LAW OFFICE, P.C.
Urbana, IL 6 1801
2 17-367-9000
F A X 2 17-367-9005
Dated:
2
UNITED STATES DEPARTMENT OF COMMERCE
United States Patent and Trademark Office
April 08, 1999
THIS IS TO CERTIFY THAT ANNEXED IS A TRUE COPY FROM THE RECORDS
OF THIS OFFICE OF THE FILE WRAPPER AND CONTENTS OF:
APPLICATION NUMBER: 09/114,114
FILING DATE: JuZy 13, 1998
PATENT NUMBER: 5,880,117
ISSUE DATE: March 09, 1999
TITLE OF INVENTION:
*
USE OF 4-ANDROSTENEDIOL TO INCREASE TESTOSTERONE LEVELS IN HUMANS
I"TOR(S) :
ARNOLD, PATRICK
By Authority of the
COMMISSIONER OF PATENTS AND TRADEMARKS
dy$zde
.L.J C
Certifying Officer
I
PATENT APPLICATION SERIAL, NO.
U.S. DEPARTMENT OF COMMERCE
PATENT AND TRADEMARK OFFICE
FEE RECORD SHEET
7RU1998 SCWW 00000051 09314114
moo aP
1 FC1201
PTO- 1556
(5187)
..... ............ .. .
. ._..
.
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USE OF 4-ANDROSTENEDIOL TO INCREASE
TESTOSTERONE'LEVELSI N HUMANS
ABSTRACT
This invention relates to a method of administering the testosterone precursor 4-androstenediol as
a means of increasing testosterone levels in humans.
7
,
I
$
FIELD OF THE INVENTION
This invention relates to a method of administering the testosterone precursor 4-androstenediol as
a means of increasing testosterone levels in humans. The steroid hormone testosterone is
considered to be the male virilizing hormone. Its effects include maintenance of muscle and bone
mass, sexual function, and psychological well being among others. As males grow older,
especially after the age of 35, a slow decline in testosterone levels is observed which is
accompanied by symptoms that have been associated with the condition known as “andropause”.
Symptoms of andropause include lethargy, depression, lack of sexual desire and fhnction, and loss
of muscle mass and strength.
DESCRIPTION OF THE PRIOR ART
p
i-
There are several pharmaceutical methods to restore testosterone levels in humans with suboptimal levels. Many of these have disadvantages however. Testosterone esters in oil depot form
ti=
I!
have been used as injections for decades, however these injections can be inconvenient and oRen
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painful. These depot injections also result in inconsistent blood levels as a supraphysiological
$e
-
surge is seen soon after injection but by the time the next injection is due, the levels have often
{s
..-
dropped down below standard physiological levels. This is in contrast with testosterone levels
i
g
‘
under normal conditions, which are quite stable within mild release pulses of approximately 90
-
minute duration. Supraphysiological surges that are seen with injectable preparations may
increase the incidence of undesirable side effects @.e.prostrate hypertrophy) as well as cause an
amplified shutdown of the hypothalamic/pituitary testicular axis (”TA).
Other pharmaceutical methods for androgen replacement therapy include synthetic oral androgen
derivatives. These compounds (i.e. methyltestosterone and fluoxymesterone) are altered in the
17alpha position of the steroid molecule with an alkyl group. This alkyl group renders the steroid
impervious to oxidation of the 17beta hydroxyl group in the liver and therefore greatly improves
its oral bioavailability compared to the non-alkylated steroids. However, this structural
2
....~
.._... ..
c
modification also has been associated with a greatly increased risk of hepatotoxicity. Therefore,
these synthetic compounds are far from an ideal solution.
U.S.patent 5,578,588 to Mattern, et. al, discloses a method of increasing testosterone levels in
humans by administering a testosterone precursor, namely androstenedione. Modes of
administration discussed include peroral and intranasal. The pharmacokinetics of such an
administration of a precursor is such that a peak in blood levels is seen at approximately 90
minutes with a subsequent decline to baseline within 3 hours. This fact permits one to more
closely simulate the natural eXdogenous pulsatile release of testosterone through multiple daily
dosing of a precursor. This should result in a more normal physiological response with a
minimization of side effects and HPTA shutdown. Furthermore, since these precursors are
natural steroid hormones found in the blood, and are not 17alpha alkylated compounds, the
hepatoxicity is minimal.
PESCRIPTION OF THE INVENTION
In the course of our research, we have found that the blood testosterone level increases seen with
the oral administration of androstenedione are far less and more variable than what is described in
U.S. patent 5,578,588. It was therefore an object of this invention to discover another naturally
-
occurring testosterone precursor that provided a greater blood testosterone level response than
androstenedione but retained all the advantages of being a non-toxic, natural, and quickly
metabolizable precursor. This would therefore permit oral administration at a reasonable dose
providing a dependable therapeutic response.
The chemical term 4-androstenediol refers to two isomers: 4-androstene-3beta, 17betadiol and 4androstene3alpha, 17beta-diol. This invention concerns primarily the former isomer in the
preferred embodiment.
4-androstenediol is a naturally occurring compound. It has been identified as a metabolite of
3
c
c
testosterone in placental, uterine, testicular, adrenal, and hypothalamidpituitary tissues. It acts as
a very effective precursor to testosterone. 4-androstenediol converts to testosterone via the
d
/I.
I
3beta-hydroxysteroid dehydrogenase enzyme. F. Ungar, M.Gut, and R. Dorfinan (J Biol.
I
Chent.,224, 191-200) found that after 4-androstenediol was incubated in h e r tissue it
11
metabolized very readily to testosterone. J. Blaquier, E. Forchielli, and R. Dorfman (Acta
Endocrinologica, 55, 697-704) also revealed that the in vitro conversion of tritiated 4!
7.I , ..
I
androstene-3beta, 17betadiol to testosterone in whole human blood was very efficient(15.76%)
I
and was in fact considerably more efficient that tritiated androstenedione (5.61%).
!
I
i
I
i
i
After learning of the in-vitro efficacyof 4-androstenediol in regards to testosterone conversion, it
was then the intention of the inventor to investigate whether 4-androstenediol would act as an
effective in-vivo peroral testosterone precursor in humans. It was also the intention of the
inventor to investigate whether or not 4-androstenediol would act as a superior peroral
testosterone precursor to androstenedione.
1
A clinical study was therefore undertaken. Seven adult male subjects were used. Each subject
was on separate occasions given an oral dose of 100 mg. placebo, 4-androstenediol, or
i
androstenedione. Blood samples were collected at 0, 30, 60, and 90 minutes following ingestion
i
and analyzed for total testosterone (TT) (see Fig. 1) and free testosterone (FT) (see Fig. 2) using
I
i
i
enzyme-linked immunosorbent assay. Relative to placebo, androstenedione ingestion caused a
I
androstenediol ingestion caused greater responses, producing a 47.7% increase in total
t
testosterone and a 4.5% increase in fiee testosterone at 90 minutes.
I
t
I
I
I
14.8% increase in total testosterone and a 10.9% increase in free testosterone at 90 &Utes.
4-
Oral 4-androstenediol can be given in daily doses of 25 mg. to 500 mg., preferably 100 to 300
mg. These daily doses can be divided into several subdoses with 3-5being most preferable. In
addition to peroral administration, 4-androstenediol can also be effectively administered by several
other routes including transdermal, rectal (suppository), intranasal, and sublingual. A particularly
I
advantageous method of sublingual administration involves cornplexing 4-androstenediol with
I
beta-hydroxypropyl-beta-cyclodextrinwhich is then pressed into tabiets. 4-androstenediol can also
4
!
c
be effectivelycombined with androstenedione to produce a product that contains two precursors
that convert to testosterone through two distinct enzyme systems.
The foregoing drawings and description ofthe invention are for illustration only. Modifications
not included in the description which are obvious to those skilled in the art are intended to be
included in the scope of the following claims.
f
DE
l
s
W
The present invention w
ill be more hlly understood by reference to the following detailed
description thereof when read in conjunction with the attached drawings, and wherein:
FIG. 1is a graph of Total Testosterone level versus time in a clinical test of the invention.
FIG. 2 is a graph of Free Testosterone levels versus time in a clinical test of the invention.
5
,'
CI
.
I
,
P.
.
CLARMS
I claim:
I
'I
1. A method o f increasing testosterone levels in humans by administration of 4-androstenediol.
2. The method of increasing testosterone levels in humans according to claim 1, wherein the
mode of administration is peroral.
1;1
peroral daily dosage of 25 mg to 500 mg is taken.
4. The method of increasing testosterone levels in humans according to claim 1, wherein the 4-
i
2
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I
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3. The method of increasing testosterone levels in humans according to claim 1, wherein a
1
-3
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. .
androstenediol is 4-androstene-3betq 17betadiol.
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POWER OF ATTORNEY OR
AUTHORIZATION OF AGENT,
NOT ACCOMPANYING
APPLICATION
APprrutkn
H-
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arnnl d
GroupArtUnp
EmdnUNyM
1036
/
RegistrationNumber
Name
Mi-
38,379
Rerns
as my/our attomey(s) or agent@)to prosecute the apptlcatlon Mentitled above, and to transad all
business in the Patent and Trademark Office connected therewith.
aEz%~al
Address
Michael. B e r n s
Berns Tlaw Office. P.C.
4 0 5 East Main S t r e e t
citv
llrhnnn
Country
USA
Name
Addrest
Telephone
.
21 7 3 6 7 9 0 0 0
I am the:
Signature
Date
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State
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2173843355
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Name
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der the Paperwork Reduction Act of 1995. no persons are requld to repond
STATEMENT UNDER 37 CFR 3.73!b)
Applicant:
Patrick Arnold .
.
Apptiition NO.:
Filed:
-
7
Entitled: Use Of 4-Androstenediol to Increase Testosterone Levels
LPJ Research, Inc.
...
corporation
1 a
(Name of Assignee)
I
of Auignee. e.g., m u o n . m n h i p . univetslty,governmentagency, etc)
states that it is:
1. @ the assignee of the entire right, title, and interest or
2.
an assignee of an undivided part interest
in the patent application fdentifiedabove by virtue of either:
A.[.&An assignment from the inventor(s) of the patent applicationidentified above. The assignment was recordedin the Patent
and Trademark Office at Reel
Frame
,or for which a copy thereof Is attached.
OR
B. [ ] A chain of title from the inventor(s), of the patent application Identified above, to the current assignee a s shown below
1. From:
To:
The document was recorded in the Patent and Tradernark Office at
, Frame
,or for which a copy thereof is attached.
Reel
2. From:
To:
The document was recorded in the Patent and Trademark Officeat
,Frame
,or for which a copy thereof is attached.
Reel
3. From:
To:
The document was recorded in the Patent and Trademark Offickat
,Frame
,or for whlch a copy thereof isattache&Reel
c
[ ] Additional documents in the chain of title are listed on a supplemental sheet.
[ *pies
of asslgnments or other documents In the chain of title are attached.
The undersigned (whose title Is supplied below) is em
7-7 -48
Date
Typed or printedname
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IfcrAf
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UT
Burden Hour S t a t m n t This form is estimated to take 0.2 houn to complete. T i e War MW dependtng
h e needs of h e individual case. An .comments
on h e amount of tlme you are required to a m lete this form should be sent lo the Chief Infonnalton 0 cer, Patent and Trademark offce. WasIington, DC
20231. DO NOT SEND FEES OR COMPLETE6 FORMS TO MIS ADDRESS. SWD TO: AsslStant Commissicner for Patents. Washington. DC 20231.
..
.
-
L
STATEMENT CLAIMING SMALL ENTITY STATUS
(37 CFR 1.9(f) 8 1.27(c))-SMALL BUSINESSCONCERN
ApplicanfPatwnee,orldentifiec:
ApplicationorPatentNo.:
Filedorlssued:
Tic rT!z.p nf 4
..
Docket Number ( O m
1036
Arnold
-A n d r n q + m . i n t
.*.
frizz T-,st- Levelb
'LPJ Research I Inc
N4hlEOFSMALLBUSlNESSCONCEFtN
ADDRESS O F S W BUSINESSCONCERN
SeVmplu. TT, 61875
P - 0 . B o x 160
.
&usires~ ~ c qwrmeS
e m
as a small businessconcern as defined in
I hereby state that the above identified
13 CFR Part 121for potpwes o f paying red&
fetu the United States Patent and Trademark Offiw. in that the number
of employees ofthe mxcem, includii those o f its a m i . dws rid exceed 500 perrons.For pwposes of this statement.
(1) the n m k r of employees o f t h bcffiw
tonternisthe average 0verthepwiou.s fiscal year of theof the penons
empbyed M a wm,
part-time. 01 t v r y basis duting each of the pay periods of the fiscal year, and (2) concern
are sfiiktes of each other when either, dkectiyor i d i i , om cmxm conttds or has the power to cordrot the othcr. or
athirdpartyorpartiescantrdsorhasthe powcrtoconbdkdh
I hereby state that rights under oonbact 01 taw have been a w e y e d t o and remain wrth thb small business concern
.dentifid a b o r s w i t h r ~ t o t h e i n v e l r t i o ndesuikdbc
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a the~~medhereMlwithtitleaolistedakwe.
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0 theapplicationidentifiabove.
0 thepatentkhtifidabove.
.
.
If the dghk held by the above identified yndl bdness ~ x ~ c eare
m not exdusive. each individual. con~em.or
organbation having rights in the invention must file -ate
statements as to theii status as small entities, and no rights
to the invention are heU by any p s m , o+herthan the inventor, who would not quarry as an independent inventor under
37 CFR 19(c) 8 that penon made the invention. or by any concern which would not qualify as a y M P business concern
under 37 CFR 1.9(d). OT a nonprofd organbation under 37 CFR 1.9(e).
bch person, concern. or ofganha!nn having any rights m the hvenh is bled below:
such pwson. =em, OT organization exists.
5;k"ach
w,c o ~ ~ c e mor. orgabtion is listed below. .
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e
Separate sta!ements are required awn each ~ m e perron,
d
concern or orprimtion having rightsto the invention
stating their status as Mlan entilie.(37 CFR 1.27)
-
I advlowledge the duty to file. in this application OT patent, n o t i f i of any change In d a b resulting in 1&;t d
entitlement to -1
entity a t u s prior to paying, or st the time, ufpaying, the e a f i ofthe issos fee or any maintenance
lee due after the date on which status as a small entity is no bnger appropriate. (37 CFR 12Wb))
M
E OF PERSON SIGNING
P a t r i c k Arnold
r m OF
~ PERSON IF OTHER THAN OWNER
Box 160 Seymour, IL 61875
.
.
I
.
.
.
..
..,... , :..
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Docket Number (Optional)
STATEMENT CLAIMING SMALL ENTITY STATUS
(37 CFR l.Q(f) & ‘1,27(b))-INDEPENDENT INVENTOR
Applicant,Patentee,orldentifier;
1036
Arnold
Applicationor Patent No.:
Filedorlssued:
Tie:
.
.
-
rrsp nf 4 ~ n ~ r n s t+- - P ~ l-
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Is.
As a below named inventor, I hereby state that Iqualify as an independentinventoras defined in 37 CFR 1.9(c)
for purposes of paying reducedfees tothe Patentand Trademarkofficedescribed in:
a x t h e specification filed herewithwith title as listed above.
c]the application identified above.
0 the patent identified above.
I have not assigned,granted,conveyed,orlicensed, and am under no obligationunder contract or lawto assign,
grant,convey,or license,anyrightsin theinvention toany personwhowouldnotqualifyas an independent inventor
under 37 CFR 1.9(c) ifthat person had made the invention, or to any concern which would not qualify as a small
business concern under37CFR 1.9(d) oranonprofitorganization under37CFR 1.9(e).
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LPJ Research, Inc.
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..
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P a t r i c k Arnold
NAMEOFINVENTOR
ignature of inventor
7-7
Date
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Application NO.
Notice of Ail0 wabilit y
Applicantk)
09/114,114
Examiner
Patrick Arnold
Group Art Unit
Ray Henley
1614
~
0 received inthis national stage application from the International Bureau (PCT Rule 17.2(a)).
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Notice of References Cited, PTO-892
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UTILITY
PATENT APPLICATION
TRANSMITTAL
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nlytornew nonpmvisionalapplicatim under37&F.R
Tibe
Use of 4-Androstenediol to Incre
5 f.53(b); Express Mail Label No.
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48 MPEP chapter 600 concerning utility patent application conlents.
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UNITED STATES ~EPARTMENTOF COMMERCE
patent and Trademark Office
Address: COMMISSIONER OF PATENTS AND TRADEMARKS
Washmaton. DC 20231
Please find below andlor attached an Office communication concerning this application or
proceeding.
Commissioner of Patents and Trademarks
i
,
Serial Number: 09/114,114
Page 2
Artunit: 1614
REASONS FOR ALLOWANCE
The following is an examiner's statement of reasons for allowance:
The present invention is directed to a method for increasing testosterone levels through
-
the administration of 4-androstenediol.
Mattern et al. (U.S. Patent No. 5,578,588 includes the general concept of increasing
testosterone levels though the administration of a testosterone precursor (abstract and claim 1 at
column 4) and applicant has apparently acknowledged at page 4, lines 2-8 that in vifro,4androstenediol was known to be a testosterone precursor.
Applicant at page 4, second full paragraph, shows that the present compounds provides
for a significantly greater testosterone level than the preferred compound of Mattern et al. This
would not have been expected and thus, the present claims are believed to be allowable.
Ueno et al. (U.S.Patent No. 5,391,776) and Loria (U.S. Patent No. 5,387,583) are cited
to show the general state of the art.
Any comments considered necessary by applicant must be submitted no later than the
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SMALL ENTITY
TYPE
CLAIMS AS FILED PART I
(Column 1)
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FOR
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37 CF.R I 1.18 a Ihr hl.W
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Fee Fee FM Fee FeeDescripUon
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1oB 330
107 540
108 790
114 150
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208 165 DesignfEifee
207 270 Plantfilingfee
208 395 Reistuefillngfee
214 75 Prcvlslcnalfilingfee
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120 310
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Petitionto r d v e -unintentional
140 110 240
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NollceclAppeal .
Filing a brief in suppat cd an appeal
142 1.320 242 660
143 450 243 225
144 670 2 U 335
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Deslgn issue fee
Plant Issue fee
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US005880117A
[19]
Arnold
[54] USE OF 4ANJlROSTENEDIOL TO
INCREASE TESTOSTERONE LEVELS IN
"S
p76] Inventor: Patrick Arnold, P.O.Box 160.
Seymour. Ill. 61875
[21]
AppL No.: 114,114
1221 Filed:
Jul. l3,1998
...................................
-.-.-... -.....---l....-lll.....l...-
[511 ht.CL6
[52] U.S. (3. ......
[581 Field of Search
A61K 31/56
5141178
....__........-_._._...--.__
514178
-
1111
Patent Number:
~451
Date of Patent:
[5563
5,880,117
Mar. 9,1999
References Cited
us.P " T
DOCUMENTS
.----.....
5987,583 2/1995 Loria
.--...-..... 5I41171
5,391,776 W1995 Ueno et al. -........._-....... 552/507
5,578,588 11/19% Mattem et al..
primary Examiner-Raymond Henley, IiI
AESTRACT
[57l
This invention relates to a method of administering the
testosterone precursor 4-androstenediol as a m a s of
increasing testosterone levels i n humans.
4 Claims, 1 Drawing Sheet
J
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3,UUU,ll~
MX.Y, l Y Y Y
Figure 1. Total Testosterone Responses
Time (min)
1/
-4
Figure 2. Free TestosteroneResponses
:I
130
si! 120
+plaCebo
1
I
8a 110
100
90
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Baseline
I
30
I
60
Time (min)
90
I
J:
5,880,117
1
USE OF ~ANDROSTENEDIOLTO
INCREASE TESTOSTERONE LEVELS
HUMANS
FIELD OF THE ]["TION
This invention relates to a method of
tfie
testosterone precursor Candrostenediol a a means of
increasing testosterone levels in humans. The steroid hormone testosterone is considered to be the a e m
g
hormone. Its dfects include maintenma of muscle and
bone mass. sexual fundon. and p~chologicalwell being
among others. A s males grow older, especuy d e r the age
of 35. a slow decline in testosterone levels is
which
is accompaniedby symptoms that have been associated with
the condition known as "andropause". Symptoms of andre
pause include lethargy, depression. lack of sexual desire and
function. and loss of muscle mass and strength.
.. .
..
...:.-
5
2
of androstenedione are far less and more variable than what
is desaibed in U.S.Pat NO.5,578588. It was therefore an
object of this invention to discover another naturally occurnhg testosterone precursor that provided a great& blood
testosterone level response than androstenedione but
retained a l l the advantages of being a non-toxic. ~ t ~ r and
a l ,
quickly metabolizable precursor. This would therefore permit oral administration at a reasonable dose providing a
The chemical term dandrostenediol refers to two isomers: 4-andrOStene-3be-W 17betadiol and Candrostenethe
3alpha 17bekdOl. This invention concerns
former isomer in the prefaed embodiment.
4-androstenediol is a naturally ocauring compound. It
has been identified as a metabolite of testosterone in
placental. uterine, testicular, adrenal, and hypothalamic/
pituitary tissues. It acts as a very effective precursor to
testosterone. 4androstenediol convats to testosterone via
DES(3RIpTION OFTHE PRIOR AFtT
the 3beta-hydroxysteroid dehydrogenase enzyme. F. Ungar,
M. Gut, and R D o r f m ~(J
~ Bid C k , 224, 191-200)
There an: s e v d phannaceutid me&& to restore tesfound
that
after
CandroStenedioI
Was incubated in fiver
tosterone levels in humans with suboptimal levels. Many of
~ S ~ e i t m ~ b O to~testoskmne.
v ~ r ~J . B~h qYU k
these have disadvantageshowever. Testosterone esters in oil
E Forchielli, and R Dorftnan (Ac& ~ocrinologlca,55,
depot form have been used as injections for &xades, how697-704) ah0 revealed that the
Vitro conversion O f
ever these injections
be inconvenient and often painfut
resut
in
inconsistent
blood
25
hitiatad
4-andrOStene3beta,
17betadiOl
to kstOSkrOne in
depot injections
whole human blood Was Very tfident (15.76%) and Was in
levels as a supraphysiological surge is seen soon after
fad considerably more effident that tritiated andKOStenediinjection but by the h e tfie next injection is due, the levels
one (5.61%).
have often dropped down below standard physiological
levels. This is in contrast with testosterone levels under
After learning of the in-vitro efficacyof 4androstenediol
normal conditions. which are quite stable within mild 3o i n regards to testosterone conversion, it was then the intenrelease pulses of approximately 90 minute duration. supration of the inventor to investigate whether Candrostenediol
physiological surges that are seen with injectable -awould act an d f d v e h-vivo proral testosterone pretions may increase the incidence of undesirable side &ects
cursor in humans. It was also the intention of the inventor to
(ia prostrate hypertrophy) as well as cause an amplified 35 investigate whether or not Candrostenediol would act as a
shutdown of the hypothalamidpituitary testicular axis
Superior peroral tcstostaone prearrsor to androstenedione.
A clinical study was therefore undertaken. Seven adult
(Hpw.
~erpharmaceuticalmethodsforandrogenreplacement male subjects were used. Each subject was on separate
therapy include synthetic oral androgen derivatives. These
occasions given an oral dose of 100 mg. placebo,
compounds (Le. methyltestosterone and fluoxymestcrone) 4o 4-androstencdiol, or androstenedione. Blood samples were
are altered in the 17alpha position of the steroid molecule
collected at 0,30,60, and 90 minutes following ingestion
and analyzedfor total testosteroneerr) (seeFIG. 1) and free
with an alkyl group. This alkyl group renders the stezoid
impervious to oxidation of the 17 beta hydroxyl group in the
testosterone 0(see FIG, 2) using enzymdiakcd M u liver and therefore greatly improves its oral bimvailability
nosorbtnt assay. Relative to placebo, androstenedioneingescompared to the non-alkylatedsteroids,However, this s t ~ ~ c45- tion caused a 14.8% increase in total testosterone and a
a greatly
10.9% increase in free testosterone at 90 minutes.
turalmodification also has been associated
increased risk of hepatotoxicity. Therefore, these synthetic
Candrostencdiol ingestion caused gnater responses, producing a 47.7% hcrease in total testosterone and a 4 2 5 %
compounds are far from an ideal solution.
U.S. Pat. No. 5,578588 to Mattern, et. al, discloses a
increase in fiet e s t & ~ ~ n eat 90
method of increasing testosterone levels in humans by u) Oral 4-androstenediol can be given in d a y doses of 25
mg.to 500mg.,preferably 100to 300 mg.These daily doses
administering a testosterone precursor, namely androstene
dione. M d t s of administration discussed indude
can be divided into S C V subdoses
~
with 3-5 g i g most
adminispreferable. In addition to peroral administration,
and intranasal. The pharmacokinetics of su&
tration of a precursor is such that a peak in blood levels is
Candrostenediol CUI also be effectively administered by
seen at approximately 90 minutes with a subsequent decline 55 several other routes including transdermal, rectal
to baseline within 3 hours. This fact pennits one to more
(supposito~), intranasal, and SubEiagual. A particularly
closely simulate the natural endogenous pulsatile release of
advantageous method of sublingual administration involves
complwing Candrostendol with beta-hydroxypropyltestosterone though multiple daily dosing of a precursor.
This should result in a more normal physiological response
beta-cyclodextrin which is- then pressed into tablets.
with a minidzation of side effeds and I-IFl'A shutdown. 60 Candrostenediol CUI ab0 be effectively combined with
Furthamore, since these precursors are naturaI steroid horandrostenedione to prduce a product that contains two
mones found in the blood, and are not 17alpha alkylated
precursm that convert to testosterone through two distinct
enzyme systems.
compounds, the hepatoxicity is minimal.
The foregoing drawings aod dcsaiption of the invention
DESCRFIION OF THE INVENTION
65 are for illustration only. Modifications not included in the
description which are obvious to those slcilled in the art arc
In the course of our research. we have found that the blood
testosterone level increases seen with the oral adminimtion
intended to be induded in the scope of the following claims.
10
5,880,117
3
4
~ ~ C R J P T I OOF
N THE DRAWINGS
The present invention will be more fuUy understood by
reference to the following detailed description thereof when
read in conjunction
the attached hwings,and
wherein:
FIG. 1 is a graph of Total Testosterone level versus time
in a clinical test of the invention. :
FIG. 2 is a graph of Free Testosterone levels versus time
in a clinical test of the invention.
I*
1. A method of increasing testosterone levels in humans
by administration of Candrostenediol.
I
r
2.The method ofinc.seaSingtestosterone levels in humans
according to daim 1, wherein the mode of administration is
md
3.Themethodofinu~ingtestosterone levels io humans
according to claim 1, wherein a peroral daily dosage of 25
mg to 500
is taken*
4. The method of increasing testosterone levels in humans
according to claim
wherein the Candrostenodiol is
dandrostene9beta. 17betadiol.
* * * * *
-.
'
Notice of References Cited
Notice of References Cited
Part of Paper No.
2
..
'PTOSEbBA (6.35)
9130/98,OMB0651-0031
DEPARTMENT OF COMMERCE
c
1449pJpFo
Rev. tms
-.
U.S. Ckpartment of commerce
PalentandTrabe&m
LIST OF PRIOR ART CITED BY
APPLICANT
(use as many sheets as n e c e s q )
c
1
of
..""_....I.......-,..".
~
Complete if Known
Application Number
,
Filing
First N a m d Inventor
Group Art U d
Examher Name
Attorney Docket Number
.
I
"
.
-I..-".-"""".."-
..
ForelgnPatent Document
FOREIGN PATENT DOCUMENTS
a
1 03 6
....
--
+
-.
USE OF 4-ANDROSTENEDIOL TO INCREASE
TESTOSTERONE LEVELS IN HUMANS
Patrick Arnold
Inventor:
P.O. Box 160
Seymour, IL 61875
Attorney:
Michael Berns
RegistrationNumber 38,379
Urbana, Illinois 6 1802
Assignee:
LPJ Research Inc.
P.O. Box 160
Seymour, IL 61875
1
.. .
.
.
.
c
~iiiiiiniiiuilnaltlni~niiir;i~inuiuianiimii~
US005387583A
[I91
[ill
Loria
[S4]
[451
COMPOSITIONS CONTAINING
CORTI~~OIDSOBANAU)~
THERMlFAND CORTICOSTEROID
B~GEFFHXWEAMOU"3OP
5-ANDMBl"E 3B, 17B OR
S-ANDROSJXNE3B,7B, 178 TRIOL OR
ANALOGWTHEREDF
Inveutoc
Roorr M. bria, 3819 Brook Rd.,
Richmond, Va 23227
[21] AppLNo.: 50,579
P
I
Filed:
[Sl] Int.CI.6
[SZ] US. a
APr.mm
..-.-..-....-.....-.-........-....-.
A61g 3!X4/l71;514/178;
S1#182; 5W63155W636; 4W3.71
514/171, 178,
[SS] FiddafsearCa
le
552/634,636
A.imcyphhmht?do86a.Dets
C Jons
Asdstant Rxwnine+Dwaaync
,
Patent Number:
Date of Patent:
5,387,583
Feb. 7,1995
r'
.
5,387,583
f
'
c
.
or patients suffering
e
e
5 .'
i'
5,387,583
6
5,3 87,583
B
8
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5,387,583
14
c
15
5,387,583
16
c
5,387,583
17''
12
hGL2
iacr. 1
ina. lt
k.
6
49
41
41
31
15
55
46
5s
54
44
16
25
2)
.
18
cr
5,387,583
.l!3
A series of experiments was do=
20
daily or twice a day to achieve a daily dosage of 15 mg
and 5 mg dexamethasone Per day.
&kr&e.
-
whether BAED and PAET would cause a change in
Example 6:
the IeveI of the cytokine L 3 . The cultares wece preA preparation for application to the skin or muma
pared in accordance with tht g e n d rne t oa
fouo*g
manner:
above After 30 hours the level of I L 3 in & supuaa- 5 m y he prepared in
tantp of the cultures was measured using the I L 3
ELISA kit ma~~ttfactured
by EndoGen Inc, h t a n ,
46 w/w
Mass The findings are shown m Tabla 4and 5 below.
AED
03%
TABLE 4
Example 7:
A formuladon for admInisndtidnas a reteu&n enema
may k:formulated in the following mannec
TABLE 5
EFFJ3XOFBAEDAND
BAETON--3
PRODUCTION IN TRE ABSENCE OF COrA
30
r
21
5,387,583
"
1. A pharmm~~tical
compoSition comprising an antiinflammstory d e c t i v e amount of at ktut one cortic0stcroid or analogue thenof and a cmtiaxteroid bulTering effective amount of at least one immUno pgulating
mmpottnd selectad firom compouods of the formolp: ' 5
md
15
50
55
60
65
.". . . ....
_ ..
. .*a
..C
22
lone, ffupsedddene and its derivatives,flnrandrenolide.,
clobetasol sad its derivatiues, clobstsonc and its derivatives, aldomctasone, flumethasoneand its derivatives,
-andffuoc~rtolone
and its derivatives.
3. A compositjon of c l h 1 contabin$ BAET.
4. A composition of claim 1 containing BAET and
*,
US005391776A
[19~
Ueno et al.
[54] SXXROIDDERKVATIVIB
Inventors: Hiroald Ueno, San Diego? CaliE;
Patent Number:
1451 Date of Patent:
2-104593 (A990 Japan
W-187
W992 WrPo
sY=f*-aw=#
[73] Assignee: MfbsrtbfatdgnsdcOrpatntion,
Tokyo, Jaw?
-Ap-npriorityJapan
Feb. 1% I992
[58]
[561
..--.-._....
*
mmqu Extnt~im-Paul J. Kilto~
Agent. ~rFImt-WenderOth,
M
Lind & POWk
ABSrRA!x
4428497
."..".-........-."...
....
51/00
....".-.-.".....".552/506
EIeld ofsevch ...
5W507,M6; SlVlQL
514/107
[5l] 5th U . 6
[S2]
[m
.
Asterokiderivative of the gcnasl formula:
Pl] Appl.No: 15,800
[U]FiM
Fsb.lO.1993
POI
Feb. 21, 1995
0
PUBLICATIONS
et at., Sdencc, vol. 241, pp. 84-86 Jut 1988.
Japaq M t o Kmfmno, Tokyo,
Japin;MasaWio MorioLn, Tokyo,
Jep4n; Akibisa Mod, TokyoIJapan
5,391,776
[ii]
cQ7J
us.a.
Bdereaoes Cited
FOREIGN PATENT
0496520 7/1992 learopernpdo&.
0548884 6/1993 Humpm~Prt.OFf..
10 Cldms, No D r d r i s
,
.
.
1
5,391,776
'~
2
c
5,391,776
3'
4
X*
cornpi
-A-
0
I
B
H
0
n
M
-c-~ac-N-
HO
e
2
0'
I
0
0
a
*NH(cH&c-NH-
a
HO
3
'0
-
0
0
W
R
U
-c-"(~NH-
HO
4
*
0'
0
II
0
II
H
--DNHCEe-NH-
I
-3
v
-
.e
5,391,776
6
TABLE l-continucd
0
n
X--Q--A--CH[P(ORhb
X
*
-A-
R
?'
H
m
0
I
0
U
m
&
H
-C-NH$SIc-NH-
H
0
'
0
U
-C-"CHC-"-
0
n
R
R
&
HO
c
5,339 1,776
7
8
X*
ccmpd Na
12
-A-
?0
R
0
0
II
H
0
n
U
-C-NHfHC-NH-
0
H
0
0
H
I
fl
HO
13
14
O0
II
n
O0
0
0
II
n
H
-c-"ac-mI
HO
16
0
I
0
I1
CH
t
e5,391,776
9
10
-A-
X-O-
CcwPdNo.
R
0
0
11
'
n
It
-C-"C-N"
H
H
18
0
'
I
19
0
&
E
I
O0
*m-
Em
'0
I
,
0
0
U
n
-w--*~-
H
H
21
O0
H
22
O0
--&
HO
.. .
c
5,391,776
12
fl
'
TABLE lcantinutd
0
U
'x-O-A-~OR)~~
0 0
24
1 II
?'
If
-mi-
H
25
H
26
0
'
H
00
I
€30
'
?'
&
HO
,'
..
.
I.
c
13
5,39 1,776
..
'
14
TABLE Isontinned
0
X-C?-A-U@(OR)&
Wpd.No.
U
*X
-A0
29
0
I1
n
-c-(cmrc-"-
0
30
I
-C-a3*"--
31
R
0I
0
II
H
0
I
H
0
H
I
n
H
33
H
34
..
'
I
E
c
5,391,776
is
16
TABLE l a n t i n u e d
0
II
X-O-A--cII[p(OR)&
campd Na
36
X-0-
-A-
R
H
?-
37
0
1
em-
R
-@
iqj
38
0
\
39
41
42
H
0-
0
'& 45
H
H
.
c
c
17
5,39 1,776
,,
18
TABLE lcontinued
0
X--O-A-ON&
Cornpa No.
ll
-A-
X*
R
43
44
t
I:
0I
46
0
0
U
H
a
-c-c&"c+NIf-
Q-
41
48
0-
0
0
~a
I
-
II
m
H
~
-
-
,
49
JGL"EIn.
H
c
c
5,391,776
19
20
TABLE Iumtinued
0
X-O-A--ctqp(OR)&
n
cornpa No.
-A-
X-0-
50
0-
R
H
$ ,
53
49
H
c
H
55
0
a
--o
€I
-
f
5391,776
21
22
TABLE loontinued
0
B
X-O-A--CH[P(OR)ZI2
hpd.
-A-
X-0-
Na
n
0
R
0
u
II
-cNEic&c-NEi-
4.
0
I
0
n
n
~~a.2C-m0
“0
59
‘0
a,
@
0
0
0
0
U
n
-=wc=hm-
tl
U
‘
0
61
0
0
n
o
-aHcFic!NR-
I
* ‘
‘0
F
62
,
63
‘0
7
0
0
W
It
0
J
-cNn-
ii
5,391,776
23
ccmpd.
24
x-0-
-A-
64
R
0
0
n
I
H
-cw=wk~-
\
0
PH
6S
'0
66
0 0
@
&
H
IIU
-c+c-"-
0
0
H
I I H
-c-m-
\0
0
61
0
61
U
H
-c-KwZ*~-
68
H
\
0
69
H
?n
'0
L
0
u
m
,
.=: Y.
4
-,
.
....
Q..
.. .
L.^t
' .,
.: .
;,u.-
... .
0
I1
e
5,391,776
25.
26
TABLE l-continned
0
n
X--O-ATOR)&
x-0-
campah
71
-A0
OH
ar,
1 1
m
-
I
a
0
a
R
H
3
'0
PF P
72
H
7"-
I
ws
'0
n
e
e
-e
s c ) g -"-
H
-c
'0
74
R
.
'0
75
H
'0
76
P
OH
'0
n
PH
'0
@
H
--c
H
.
c
c
5,391,776
2?
28
TABLE X-&ntinaed
x*A--ctqp(OR&
campd Na
0
n
X
*
78
-A-
R
OH
@
‘0
?9
n
\
0
-c
-6-
83
‘0
81
H
@
W
8
H
‘0
83
H
84
c
c
5,391,776
29 ..
30
TABLE lcontinued
0
n
x--O-A--cLqp(OR)&
X*
R
-A0
I
-q---
0
II
H
‘0
‘e
‘0
‘0
0
---II
‘0
&
8
H
A
c
5,391,776
31
93
P
94
?H
32
0
H
U
-c-Qb-
0
n
H
-c-t~h-
95
?H
0
a
*fl
H
--o-(--
‘0
97
?”
‘0
98
OH
‘0
&
0
n
--o(cEI36
H
c
33
5,391,776
..
34
TABLE I a n t i n d
0
I
. ,X - - O - A m O R ) z k
Gmpd.No.
---A-
X-Q-
0
99
0
It
u
0
0
0
n
u
-c-=s-"-
0
lor
I
-+m&-+rn-
-a-
0'
0'
E
8
0
0
ICn
B
El
N
*-X
I-
H
0
0
0
W
H
0
u
0
a
0
U
0
H
n
-c+cE*m-
U
0
0'
&
0
*WkP-*
D
..
r*
.C
5,391,776
35
Cornpa No.
36
-A-
X-O-
106
R
0
'0
2
-
H
I
0
107
0
0
n
-C-"g!-NH-
ll
H
0
0
U
0
'
-m-
&
0
I
H
0
H
0
0
i
I
-c-mAs-"-
'0
I
0
I10
0
&
&
&
-11
0
111
e s r
H
-
H
-c-NF€c€l*N€F
\0
0
tU
\0
.
..
..
..
C
..
'
.
- - .-
H
5,3 9 1,776
37 * '
38
TABLE lcontinued
0
X-O-A-ORfi
Cornpa
No.
B
-A-
X-0-
1u
e
-e
R
0
'0
0
0
$14
II
H
-1-
\
I IS
H
'0
I16
0
0
II
I
E
~ ~ - C - N n -
117
0
0
H
I
U
* ( ~ - -
-
'0
I18
0
H
II
-C-c&--
119
0
I
0
II
-c-NIic&c-"-,
,
E
P
121
P
A
-cwCR&+rn-
HO
--P
E
HO
0
0
I
I
-c-NHC&c-m-
'0
w
y
o
. .
\
0
M
v
- .. . .. . .
'0
... , .
.
I
~
. .
.1'
t
c
f:
5,391,776
42
TABLE l~ontinua3
-A-
X-0-
C-PaNa
I26
'0
in
&
&
R
0
H
I
--c--cHr
0
0
U
-PNH=N€l-
II
H
0
H
0
128
0
n
-ern-
0
&
n
0
H
-c+ww2-c-N=-
-.
n
tl
0
130
B
H
-l-
I31
r
..-
c
5,391,776
43
44
TABLE lcontiaucd
X-0-
Cornpa No.
m
R
H
-A-
0
I
0
0
0
II
-c-ai2-c-m-
HO
u3
H
II
a
-c-wWs-~-
0
l34
H
D
-c-cHa-
0
0
US
tl
+NEa&€+"-
0
136
. _...
0
U
-c-cH+NE-
137
H
U
0
I
H
iI
-
0
H
H
-cwzC-m-
138
P
--
n
--c-Q[I
\
0
*-.
c
5,391,776
45.
46
TABLE l-continned
0
n
X--O-A-UiROR)&
aDmpaNa
-A-
x-0-
139
0
R
0
- c U+ N i f ~ - " -I1
0
n
0
'
*"ac-Nx-
a
3
0
D
0)
&
HO
e
142
0
'
FIO
143
?'
O0
0 .
.-
HO
,e-
,.
di
.
.
.(.I.
..
.
5,391,776
47
48
TABLE l-continued
0
?'
?'
.
-&NFiC€FlE-NH-
0
U
0
ti
-c-m~c-"-
H
148
149
H
0
P
m
-
0
U
H
-
I
c
49
5,391,776
r .
iii Where p is 1 and Y is -NH-
(m=l):
..
c
50
f
'
ii
5,391,776
6s
C3
c
c
53
5,391,776
54
'
'55
5,391,776
56
c
!j7.
5,391,776
58
I
C'
'
'59
5,391,776
60
Preparation 12
Preparation 8
0
10
H
O
W U o
II
0
IS
m
.4
c
5,39 1,776
62
Preparation 18
45
Yidd: 6.3% N M R (CDCIs 6). 7.62 (d, IH,
J=lO.O&),
7.19 (d, lH, J=8.!Zk), 6.83 (dd, 1
9 50
J=2,J;IIzI&5H& 6.77 (d, lH, J = m ) , 5.14 (s,2H),
5.05 (JX, lH, Js21.932, lO.OHz), 4.75 (t, lEI, 8.Wz),
4254.16 (tu, 8% 3.47 (s, 3m3.40(a, ZEI) 284 (t, 2H,
J=4.2Hz), 229-220 (w 3 m 1.91-1.31 (m22H), 0.83
(a 3 m
ss
Preparation 17
0
-AT
0
I
-C+(CI&i+NE-
tl
60
y]eM:33% NMR ( m a s a), 8.11,7.86 (each d,WI,
8.4Hz). 7.18 (d, 1 s 8.6Hz), 6.9-6.7 (m, 3Hj, 5.23 (dt,
1?€, 10- 21.8&), 5.13 (E+ 2H), 4.93 ($ lF& 7.8EL),
4.404.10 (m,1% 3.45 (4 3H), 230-2.m (Q w),
21-22 (m.3 q , 20-11 (m, 22€Q0.95 (a 3H).
preparatioaz
0
I
c-m-
36
Yid& 97% NMR(CDCh,6),731)(d, lH,J=S.QiZ),
.
-AT
r
6.83 (dd, IH,Jd.S€k, am),678 (4 lH,J=2Sh),
6.36 (4 1% J=l(L(3Hz), 5.15 (s, 2€Q 5.03 (4 lH,
J=lO.O€h, 21.W) 469 (t, lH, J=7.8&), 4.244.14 65
YA& S3% NMR (CLX%, a), 7.95-7.90 (a W
(m, 8H) 3.47 (s, 3H) 280-283(m, w),271-2.59 (m,
4H), 231-216 (a 1.90-1.09 (Q lOH),,1.34(t, lW,
7.s7.45 (% 3H), 7.18 (4 1% 8.6Hz), 7.80 (d4 1%
J=7.1Hz), 0.82 (s,3EI)
zs)Iz. 8.6H.z). 6.76 (d, IH, 25Hz),664 (d, I& IO=),
i*- F - * . r ' f c w 7 . F Y.k *
.. -. *
. G *,.,ti ?
p
5
5
.
!
?'
7 .
..-
c
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IN THE UNITED STATES PATENT AND
Serial Number:
Appl. Filed:
Applicants:
Patrick Arnold
Appl. Title:
Use of+&drostenedioI to Increase Testosterone Levels in Humans
Examiner:
PETITION TO MAKE SPECIAL
Assistant Commissioner of Patents
Applicant hereby respectfirlly petitions that the above application be made special under MPEP
Section 708.02 for the following reason; attached is a declaration in support thereof:
L
Manufacturer Available
VI.
Energy Savings Will Result
Ih,
Irkiingement Exists
VII.
Recombinant DNA is Involved
111.
Applicant’s Health is Poor
VIII. Special Procedure: Search was made
IV.
Applicant’s Age is 65 or Greater
E. Superconductivity is Advanced
V.
Environmental Quality will be Enhanced
-
Also attached, since reason I, 11, VII, or VIII has been checked, is the $130.00 Petition Fee
pursuant to Rules 102 and 17(i).
Very respecthvy submitted,
.....-.......
,.
. . . . .. ....
,
c
4
.-
IN THE UNITEDSTATES PATENT AND TRADEMARK OFFICE
Serial Number:
Appl. Filed:
Applicants:
Patrick Amold
Appl. Title:
Use of 4-Androstenediol to Increase Testosterone Levels in Humans
Examiner:
Declaration in Support of Accompanyidg Petition to Make Special
Reason I - Manufacturer Available
-
Reason II Infringement Exists
-
Reason v1[I Search was Made
In support of the accompanying Petition to Make Special, applicant declares as follows:
1. I am the applicant in the above-identified patent application.
2. This patent application was assigned to LPJResearch Inc., who is prepared to manufacture the
product.
3. Foreign manufacturers have begun importing the product to infhge on this patent application.
-
4. Axarch of the prior art was made by my myself and additional search was made by my patent
attorney, Michael Berns.
5. An Information Disclosure Statement is enclosed with the patent application.
c
a
6. I fbrther declare that all statements herein of my own knowledge are true and that all
statements made upon information and belief are believed to be true, and firther that these
statements were made with the knowledge that willfil false statements and the like so made are
punishable by fine or imprisonment, or both, under Section 1001 of Title 18 of the United States
Code, and that such willfil false statements may jeopardize the validity of the application and any
patent issuing therefrom.
Very respectfully,
!
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Patrick Arnold, Applicant
..
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In the United States Patent and Trademark Office
,
Serial Number:
Appn. Filed:
Applicant(s):
'Appn. Title:
Examiner:
Group Art Unit:
..
Patrick Arnold
Use of 4-Androstenediol to Increase Testosterone Levels in Humans
. ..
Mailed:
At:
-
Information Disclosure Statement
Assistant Commissioner of Patents
Washington, District of Columbia 20231
.
Attached is a completed Form PTO-1449 and copies of the pertinent parts of the
references listed on this form. The comments on the relevance of any non-English
references, pursuant to Rule 98 are contained in the Prior Art section of the specification.
B
Patent #5,578,588to Mattern et al. shows the use of Androstenedione as a testosterone
precursor.
This reference does not show the use of 4-Androstenediol.
Very respectfully,
...
.
Michael Berns
Re istration Number 38,379
40 East Main Street
Urbana, IL 61801
2 17-384-1144
!
...
!
. . . .
I
1I11111111111111It41ll1111US005578588A
11111ll1l11111111111111111111111I1111111
(191
Mattern et al.
[541 MEDICAMENT FOR INCREASING THE
TESTOSTERONE LEVEL
[75] Inventors: Claudia Matiern. Stamberg: Radiger
. mcker.Hcrnching, both of Germany
[73] Assigncc: Arrowdean Ltd., Ireland
[21], Appl. No.:
[22] PCT Filed:
%
Fordga Application Priority Data
Nov. 26,1996
References Cited
[561
U.S.PATENT DOCUMENTS
..........................................
.................
...........
W 9 6 6 Meli
4.8'37.114 1011989 pib et al.
5.053.403 1W1991 Orenucich et al.
3984,333
16lll4
514n6
514/170
OTHER PUBUCATIONS
'
Qermany ..........................42 14 953.3
............... .........-".......... A61K 31/56
............................................. 514/177;5144182
...................................... 514177, 182
May 6, 1992 [DE]
1511 Int. Cl?
[52] US. CI.
[SEI Field of Search
[451 Date of Patent:
034909181 91992 European Pat. Off..
[87] FCI' Pub. No.: W093/21924
PCT P x D a t e : Nov. 11,1993
(301
5,578,588
_..
335,729
Apr. 30,1993
PcT/DE93/00397
[86]. PCTNo.:
8 371 Date:
Nov. 7,1994
8 102(c) Date! Nov. 7, 1994
Patent Number:
[ill
...
Vmder et al.. Human Physiology, 4th Ed.. 1985.
Primary Eurminerc-Kimberly Jordan
Attorney. Agent. or Firm--Merchant,Oould. Smith. Edcll,
Welter & Schmidt, P.A.
P71
ABSTRACT
'
The inventicm concerns a drug for increasing the level of
testosterone in the human body, the,drug containing at least
one testosmne precursor.
3 Claims, No Drawings
-.'
.
I
i
5,578,588
.
.1
MEDICAMENT FOR INCREASING THE
TESTOSTERONE LEVEL
This application is a 371 of pcT/DE93/00397 filed Apr.
30,1993. The invention relates to a medicament for inmas- s
ing the testosterone level in humans.
The main action o f the steroid hormone testosterone is
the intensifying of the primary and secondary sex characters
o f man, as well as the mafntaining of the functions associated therewith. Apart from this main eITect testosterone has IO
a number of secondary eKects. which are of great irnportance for the stressability and performance characteristics of
the human organism. These include the maintaining of m
anabolic metabolic sihiation. thc restoration of thc pcxformancc o f man following exhausting exercise and Incrensing 15
the psychophysiological stressability and stress resistance.
The action mechanisms o f testosterone have been investigatcd in dctaU. The secondary effects on the psychophysiological state have, according to the latest resmch, been
attributed to iko presence o f testosterone receptors ‘in the u)
central nervous system.
Over 90% of the testosterone in the blood is bound to
protein and the biologically active component is htestostcronc rcprcsenting 4 to 8% o f the total concentration in
thc blood. Thc testostcrone concenuation in the blood is z
subjcct to a physiological daily cycle (maximum concentration in the morning) a seasonal cycle (lowest concentration
in May) and influences by living circumstances and ageing
processes.
The overall testostergne concentration in the blood i.3 30
individually very stable undcr normal conditions. High
physical cffort, long-lasting stress situations and unfavourable diet lower the blood level. With increasing age and in
pardcular from about 35 in man there is a reduction o f the
free testosterone concentration. Thcsc changes lcad to a 35
reduced, general performance, to higher time requirements
for restodng the organism after exhaustive exercise and to a
reduction o f the psychophysiological strcssability and stras
resistance. Research on physically and cyclically highly
stressed persons have revealed that a risc in thc tcstostcmnc 40
level in the upper part of the individual physiological
fluctuation range leads to a cancelling out of this negative
situation and to an increase in the general performance
characteristics. However, a concentration rise above the
individual, upper standard limit leads to no b e t t a therapcu- 4s
tic effect and instead causes side effects.
The increase in the testosterone level in humans in the
sense o f a substitution has consequently become part o f
preventative and therapeutic concepts in old-age medicine,
particularly for man.The supply conventionally lakes place 50
perorally or in an oily solution in ffltramuscular form and in
part as a depot preparation.
However, the following disadvantages are associated
with these administration forms:
the influencing of the blood level is overall difficult to 55
control;
be
the individual starling situation and svess
, adequately taken into account Tor the medication;
Peroral and i~tramuscular
lead Lo a metabolkalion 60
via the circulatory system liver-bile-intestine-liver
(“flrst-pass effect”);
.
this effect reduces the bioavailabilily and requires the
supply of higher doses with the resulting higher stressing o f the metabolism;
65
the supply of higher doses can lead to an undesired rise in
the overall concentration, which via the physiological
2
control mechanism reduces the endogenic testosterone
production.
The problem of the present invention is therefore to
providc a mcdicamcnt for raising the testosterone level in
humans, whosc application is cquivalcnt in its effcct to the
inlramuscular supply o f tcstosteronc. which avoids thc
aforementioned disadvantages. requires a much lower dose
and permits a stressing o f the s e c o n w action on the central
nervous system
According to the invention this problem is solved by a
medicament having a content of at least one precursor o f
teatostcrone and which is preferably androstendione. progesterone or 17-a-hydroxy progesterone.
A pdcularly advantageous embodiment o f the invention
is charactmized by a galcnic preparation, which allows the
supply by per nasal application using a dosing spray and
having a preferred content o f 3.5 to 15 mg of active
substance per pump thrust.
Alternatively thereto the medicament according to the
invention can also be in the form o f a sustained relea.% drag
&,depot form or buccal tnblet for peroral administration. In
this case the preferred content per ingestion unit is 50 to 100
mg of active substance.
It has been shown that through thc wc of a precursor o f
testosterone, which is only uansformcd into thc active
substance in the organism. thcrc is a rnorc complcx rcaction
of the steroid metabolism, which is more balanced and better
corresponds to the physiological conditions. so ihar overall
an optimum action can be obtnined whilst avoiding side
effects.
Animal tcsts canied out on the guinea pig havc fundamcntally pmvcd thc rapid transfornation of radioactively
labelled androstendione, progesterone or 17-a-hydroxy
progesterone into testosterone.
In humans 50 to 100 mg o f perorally supplicd androstendione. progeswrone or 17-a-hydroxy progesterone are also
lapidly transformed into testosterone. In the case of androstendione supply e.g. aftcr only I5 minutes in the blood thcrc
is a rise in the overall testosterone concentration from 40 to
83% (50 mg) or Ill to 3 7 % (100 mg). There is an increase
in the proportion of free, biologically active testosterone, the
appearance o f the concentration maximum and the path of
the blood level in the case of a positive basic reaction reveal
clear, repeating. individual differences.
In the case of the preferred pernasal administration by
mcans o f a dosing spray a single supply of 3.5 to 15 mg of
androstendione,progesterone or 17-a-hydroxy progesterone
led to testosterone level rises in the blood of 34 to 97%. The
extent and time sequence thereof are comparable with the
results which were obtainable in the case of the peroral
supply of much higher doses or the intramuscular supply o f
teStosLerone propionate. Unlike in the case of peroral and
intramuscular administralion, with pernasal administration
there was no significant “first-pass” melabolization of the
precursor molecule.
This led to the good‘ controllability o f the influencing,
which could be proved by multiple adminismions. The
Individual reaction position is taken into account by the
regulating mechanisms of h e metabolism. An adapted
of ,he free testosErone was obtained, whose
and
are
with the values oblained wilh
peroral adminismion of a ten Limes higher dose.
A significant long-term efwt was detected with multiple.
pernasal Pdmjnistration. Three to four days following the
final adminislralion lhere was a lurther testosterone level
increase or 48 to 97% in the blood and this was maintained
lor a M e r 6 to 7 days. This reaction is probably attribut-
t'
. s,57a,588
3
able U, an inflUcnchg Of the control cycle for endogenic
testosterone production.
In addition, pernasal administration facilitatcs Lc transfcr
into the cerebrospinal fluid and also into othcr tissucs and
organs o f the human organism. As thc overcoming o f thc s
b]ood-5& b&er fs a WJor problem for d l phmaccuticds =ting on the central nCrvauS sysrcm, bc f a c i l i t m
X C C to
. ~mc
~ ccrcbrospjnal fluid via t h pemmd
~
adminisvation rcprcscnts a particular advantage of thc medicament
according IO thc invcntion. Thus, for the drat time it is io
possiblc to influcncc tho testosterone receptors in the brain,
which represents a novel thernpeutic npproach for testosler.one. The subsequently described fmprovernent of the plychophydological perfomlance ChSaCteriStiCS is probably
due to the influencing of the central nervous system.
There is an increase in the lutos~rone/epileutoslerone
quotient in the meldbolile profile of urine. However. it is not
as -ked
in he
or pemmd administration(3.8 to
14.3) and &comes n o m on the day following the supply,
20
whilst the leslosterone level in the blood remains high.
The use o n e nasal spray for 6 days (daily dose 5 to 7
mg) in physically and cyclically highly stressed persons in
-
4
mlddlc agc Icd IO a shomning o f the regeneration following
exhausting exercise and a &Iter balanced metabolic situation. It mut in particular be smssd
thab umequesled, d l
Lest penonr
a
psychophysiological
suasability and
improved
resislnnCe.
The features of the invcnlion disclosed in the description
and claims can be essential to the different embodiments Of
the invention. either singly or in random subcomblnations.
We cldm:
1. A method for increasing the level of testosterone in a
human comprising nasal adminismtion of at least one
teStOSteronc pncunor.
2. me method according to
1,
the bsrostcronc prccursor is androstenedione, progesterone, 17-aIhereof*
hydroxyprogesteronc* Or
3. Thc method according 10 clnirn 1. wherein the nasd
adminislration COmpnSCS administration Of 3.5 10 about 15
mg of tcstostcronc precllrsor pcr pump thrust.
-..
* * . * *
. ..
. .
-..
c-
f
UNITED STAT&, ~EPARTMENT
OF COMMERCE
Patent and Trademark Office
NOTICE OF ALLOWANCE AND ISSUE FEE DUE
..... .
.
.
.
.
..
TION IDENTIFIED ABOVE HAS BEENEXAMINED AND IS ALLOWED FOR ISSUANCE AS A PATENT.
SUE FEEMUST BE PAID WITHIN THREE MONTHS FROM THE MAILING DATE OF THIS NOTICE OR THIS
TION SHALL BE REGARDEDAS ABANDONED, TU1S STATUTORY PERIOD CANNOT BE EXTENDED.
,
.
I
.)
TO RESPOND TO TH/S NOTICE:
MALL ENTITY status shown above.
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If the SMALL ENTITY is shown as NO:
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I
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;
I
i
PATENT AND TRADEMARK OFFICE COPY
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' U S GPO 1998437639/80023
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TRANSMIT THIS FORM WITH FEE
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Patent and Tmdemark Office; U.S. MPAFITMENTOF ColPLlERcE
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Patent ana I raaemarK uftice
Address: COMMISSIONER OF PATENTS AND TRADEMARKS
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DATE MAILED:
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111f: / 2 [I / '3:3
Please find below and/or attached an Office communication concerning this application or
proceedina.
Commissioner of Patents and Trademarks
c IWV. 21851
1 - F 5 COPY
W.S.aOyERNMENT PRlNTlNO OFFICE 1995-319-826
"pp,,"O.w.'
'sc+y~C#u.~W
-
Notice of Aflowability
mpp,,L,a,,,,*,
I.".
091114,114
.
Patrick Arnold
Examiner
Group Art Unit
1614
Ray Henley
laims being allowable, PROSECUTION ON THE MERITS IS (ORREMAINS) CLOSED in this application. If not included
with (or previously mailed), a Notice of Allowance and Issue Fee Due or other appropriate communication will be
Ed in due course.
his communication is responsive to the Notice of Allowability dated August 13, 1998
he allowed clalmIs) islare 7-4
he drawings filed on
-
are acceptable.
,cknowledgementis made of a claim for foreign priority under 3 5 U.S.C. § 119(a)-ld).
I
All
0 Some' 0 None
of the CERTIFIED copies of the priority documents have been
0 received.
0 received in Application No. (Series Code/Serial Number)
0 received in this national stage application from the International Bureau (PCT Rule 17.2(a)).
Certified copies not received:
cknowledgement is made of a claim for domestic priority under 35 U.S.C. § 1191e).
IORTENED STATUTORY PERIOD FOR RESPONSE to comply with the requirements noted below is set to EXPIRE
EE MONTHS FROM THE "DATE MAILED" of this Office action. Failure to timely comply will result in
NDONMENT of this application. Extensions of time may be obtained under the provisions of 3 7 CFR 1.136(a).
ote the attached EXAMINER'S AMENDMENT or NOTICE
OF INFORMAL APPLICATION, PTO-152, which discloses
iat the oath or declaration is deficient. A SUBSTITUTE OATH OR DECLARATION IS REQUIRED.
pplicant MUST submit NEW FORMAL DRAWINGS
1 because the orlglnally filed drawings were declared by applicant to be informal.
3 including changes required by the Notice of Draftsperson's Patent Drawing Review, PTO-948, attached hereto or
to Paper No.
.
r including changes required by the proposed drawing correction filed on
approved by the examiner.
, which
has been
I including changes required by the attached Examiner's Amendment/Comment.
entifying indicia such as the application number (see 37 CFR 1.84(c)) should be written on the r e v e m side of the
awings. The drawings should be filed as a separate paper with a transmittal lettter addressed to the Officlal
raftsperson.
ote the attached Examiner's comment regarding REOUIREMENT FOR THE DEPOSIT OF BIOLOGICAL MATERIAL.
response to this letter should include, in the upper right hand corner, the APPLICATION NUMBER (SERIES
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I Notice of Informal Patent Application, PTO-152
I Interview Summary, PTO-413
I Examiner's AmendmentKomment
4
&T&
MYMOM3 ;:j..;;:.;y, fj!
PRIM$? \/ E
:.$;> ;:;1
Q R ' j : p JyCJ
-0 Examiner's Comment Regarding Requirement for Deposit of Biological Material
10 Examiner's Statement of Reasons for Allowance
mi and Trademark Office
17 (Rev. 9-95)
Notice of Allowability
Part of Paper No.
6
I ULGIIC
aiiu
I iaultriiarn
L
ASSISTANT SECRETARY AND C O ~ I M I S S I ~ J N E R
OF PATENTS AND TRADEMARKS
.
.
MPW
Paper Number 7
In re Application of:
Patrick Arnold
Serial No. 09/114114
Filed: July 13, 1998
For: USE OF 4-ANDROSTENEDIOL
TO INCREASE TESTOSTERONE
LEVELS M HUMANS
DECISION ON PETITION
This is a decision on the petition under 37 CFR 0 1.102, filed July 13, 1998, to make the
application special for examination purposes.
Petitioner asserts that a manufacturer is available for the claimed invention, that foreign
manufacturers are importing the claimed product and that a search of the prior art was made by
the inventor and his patent attorney.
I
However, petitioner has not met the requirements necessary to establish each of the above
assertions. No evidence has been presented demonstrating (1)that the manufacturerhas the
requisite capital and facilities, (2) that the manufacturer will not proceed until a patent is granted,
(3)that the manufacturer has obligated itself to the manufacture of the invention upon allowance
and (4) that a careful and thorough search of the prior art has been made. With respect to an
iniiinging product no rigid comparison of the alleged infringing product ant that of the claims of
the application has been presented nor that a careful and thorough search of the prior art has been
made. With respect to the search of the prior art no details have been provided so as to determine
whether or not the search was careful and thorough.
*
The petition is DISMISSED.
-
If petitioner desires to file a renewed petition it must be filed within TWO (2) MONTHS o f the
mailing date o f this decision.
I
Michael Berns
Berns Law Office
Urbana, IL 61802
i
/o- 2.1-w
L A W O F F I C E , P.C.
MICHAEL
A. BERNS
107 WEST GOOSEALLEY
URBANA.
IL 6 1 80 1
October 5, 1998
I
Re:09/113,1 14
Batch H72
Daed 08/13/98
.
.
PHONE2 1 7-367-9000
FAX 2 1 7-367-9005
mberns @shout.net
.
Kajniond Henley 111
Primary Examiner Group 1200
U.S. Patent and Trademark Oifice
Waahlngtou D C. 20231
..
Sincerely,
Michael Berns
Attorney-at-Laiv
*
I
.!
...
REGISTEREDATTORNEY 138,379
UN~TEDSTATES PATENT AND TRADEMARK OFFICE
Y
I
Pigure.1. Total Testosterone Responses
27
25
23
I
21
-A-
4-androstenedione
4-androstenediol
I
.
.. .
I9
!
17
I
Baseline
60
30
I
I
I
90
Time (min)
Figure 2. Free Testosterone Responses
140
130
--
2 120
ipc 110
+placebo
.
4-androstenedione
-A-
4-androstenediol
100.
90
A
I
Baseline
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US005387583A
[I91
Loria
1541 COMeOSITIONS CONTAINING
CORTICOSFEROIDS OR ANALOGUES
THEREOF AND CORTICOfXEROID
BUFFERING EFFECTIVEAMOUNTS OF
5-ANDROSTENE 3B, 17B OR
5-ANDROSTENE 3B, 7B, 17B TRIOL OR
ANALOGUES THEREOF
[76] Inventor:
Roger M. ]Loria, 3819 Brook Rd.,
Richmond, Va. 23227
[21] Appl. NO.: 50,579
[22] Filed:
[51] Int. CIP
[52] U.S. CI.
[58]
Apr. 20, 1993
............................................
A61K 31/565
.................................... 514/171; 514/178;
514/182; 552/634; 552/636; 424/93.71
Field of Search ............
514/171, 178, 182;
Primary Examiner-Josk a. Dees
Assistant Examine-Dwayne C. Jones
552/634, 636
[11]
Patent Number:
5,387,583
[451
Date of Patent:
Feb. 7, 1995
Attorney, Agent or Firm-GIenna Hendricks; Stephen
Gates
t571
ABSWCX
3/3,17&androstenediol (“PAED) and 313,713, 17pandrosteaetriol (“#MET”) may be used to counteract
the aatiproliferative and immunosuppressive effects of
hydrocortisone and other corticosteroids (i.e., to act as
buffers to counteract the lymphosuppressive response
to such steroids). @AED and PAET are steroids which
mediate immuae response to provide the body protection against b u a e down-regulation. A method for
testing analogues of PAED and PAET to compare the
effectiveness of such analogues as buffers of certain
effects of hydrocortisone and other corticosteroids,
including immune response and proliferative effects is
described. Cytokiaes, including most particularly IL-3,
arc produced by addition of PAET and BAED and
their analogues to the growth media of cell cultures of
lymphatic cells.
5,387,583
COMPOSlTIONS C 0 “ I N G
CORTICOSTEROIDS O R ANALOGUES THEREOF
AND CORTICOSTEROm BUFFERING
EFFECMVE AMOUNTS OF 5-ANDRO3B, 5
17B OR 5 - A ” E
3B,7B, 17B TRIOL O R
ANALOGUES THEREOF
mELD O F THE INVENTION
This invention relates to 3&17j3-androstenedioI
10
(“BAED”) and 3/3,7p,17/3-androstenetriol (“&4ET”)
and their use to counteract the antiproliferative and
immunosuppressive effects of hydrocortisone and other
corticosteroids (Le., to act as buffers to counteract the 15
lymphosupprwive response to such steroids). BAED
andflAETactasimmuneregulatingsteroidshthebody.
By “immune regulatingsteroids” is meant steroids which
mediate immune response to provide the body protection
against immune down-regulation. The invention also 20
relates to means for testing analogues of BAED and
P m T to compare the effectivenessof such analoguesas
buffers of certain effects of hydrocortisone and other
corticosteroids,includingimmune response and proliferative effects. Finally the invention relates to production 25
of cytokines, including most particularly IL-3, by addigion of PAET and BAED and their analogues to the
growth media of cell cultures of lymphatic cells.
BACKGROUND OF THE INVENTION
30
In vertebrates the development of host protection
against pathogens requires a selective host immune
response that involves the mobilization of the humoral
and/or cellular mediated immune responses. Several
factors adversely affect the body’s protective response, 35
cap&itity by
prolmgd- u n ~ s u p p r ~ i o n or
l~dowrr-re&ulation#~ hune
system. is,in real~s~-regulation,.
or
iw,
appropriate to speaL
“dmegdationSs of the immune system than of downis a failure to protect the body 40
regulation since the
from
lmmuno-suppression provides an Opportuni@for Pathogens to @ow in the
It
not matter what
the primw insult to immunity’ The
inability to muster the appropriate immune 45
Among the
different
response has the&ame
causes Of immuoO-su~~ressio~ viral, bacterial9funYeast and PataSitiC infectionsi chemothmpy, hadid
ation, seveze stress, chronic fatigue syndrome, diabetes
mellitus, autoimmune disease, rheumatoid arthritis and u)
some forms of steroid therapy.
It has long been known that patients receiving steroid
hormones of adrenocor&ical origin at pharmacologically
doses show increa!xd incidence Of
infectious disease. AS. Fauci, Immunolo. RH., 65, 55
133-155 (1982); and J.E. Parillo and A.S. Fauci, Annual
Review of Pharmacology and T o x i d ~ 19,
~ n 179-201
(1979). Dehydroepiandrosterone, also known 89 3Bhydroxyandrost-5-en-17a1e or dehydrobandrosterone (referred to hereinafter as DHEA),is a 17-ket0s- 60
teroid which is quantitatively one of the major adrene
cortical steroid hormones found in mammals. M.E.
Windholz, The Merck Index, N h f h Edition (1976); K.
Diem and C. Lentner, &igv Scienhpc Tubles (1979).
(Although DHEA appears to serve as an intermediary 65
in gonadd steroid synthesis, the primary physiological
function of DHEA has not been fully understood. It has
been known, however, that levels of this hormone begin
2
to decline in the second decade of life, reaching 5% of
the original level in the elderly.)
Clinically, DHEA has been used systemically and/or
topically for treating patients suffering from psoriasis,
gout, hyperlipemia, and it has been administered to
post-coronary patients. (W. Regelson et al., New York
Academy of Sciences, 518, 260-273 (1988)). In mammals
DHEA has been shown to have weight optimizing and
anticarcinogenic effects.
DHEA has been used clinicallyin Europe in conjunction with estrogen as an agent to reverse menopausal
symptoms and also has been used in the treatment of
manic depression, schizophrenia, and Alzheimer’s disease. DHEA has also been used clinically at 40
mg/lcg/day in the treatment of advanced cancer and
multiple sclerosis. Mild androgenic effects, hirsutism,
and increased libido were the side effects observed.
These side effects can be overcome by monitoring the
dose and/or by wing analogues.
US.Pat. No.5,077,284 entitled “Use of Dehydroepiandrosterone to Improve Immune Response” describes
the subcutaneous or oral administration of DHEA to
improve the host’s response to infections. U.S. Pat. No.
4,978,532 describes use of patch technology to deliver
DHEA.
It is now disclosed that DHEA is a precursor in a
metabolic pathway which ultimately leads to more
powerful agents that increase immune response in mammals. That is, DHEA acts as a biphasic compound: it
acts as art immuno-modulator when converted to androstenediol (androst-5-ene3/3,17/3-diol,BAED) or androstenetriol (androst-5-ene-3/3,7&17B-triol, PAET).
However, in vitro DHEA has certain lymphotoxic and
suppressive effects on cell proliferationprior to its conversion to BAED and/or PAET. It is, therefore, postulated that the superior immunity enhancing properties
obtained by administration of DfWA result from its
conversion to more active metabolites.
An agent that would advance the protective regulawithout giving &? to Ilndetion of the immune
suable side effects seen with DHEA administration
would
p & c ~ ~ l advanbgeous
y
improvement
of host resistance against i&ption. Protetive regulation of the immune system could then be effected using
lower doses of the chemotherapeutic agent, and would
provide more
response wjth a wjdm range of
protection. It has previously been disclosed in US.Pat.
No. 5,206,008, incorporated herein in its entirety, that
BAED and p m (asteroid
~
found in the skin, other
tissue) enhmw immune
hue
response and lymphocytic cell proliferation.
DESCRIPTION OF THE INVENTION
In U.S. Fat. No. 5,206,008 a new class of steroids
which act as b m e regulators has been identified.
3~,17fi-~&o~tene&ol
(IS-D)
and 3P,7/3,17B-androst e n e h l (PAWhavebeen shown to enhance immune
response in the presence of stress, including infection
and exposure to chemotherapy. It is believed that
3fl,17@-androstenediol @PLED)is converted in the skin
to 3/3,7p,l7P-androstenetriol (PAET) and 38,7a,17/3andrastenetriol ( M T ) .Both the BAED and BAET
have been shown to be potent immune regulators, with
the PAET being about 100 times as potent as BAED.
The present invention provides methods of t a h g
the effect of immune-regulating steroids by measuring
the counteracting effect of BAED and PAET and their
analogues against the antiproliferativeeffects of corti-
‘
3
5,387,583
4
costeroids in vitro. The method is particularly useful for
hypoplastic or aplastic anemias, or diabetes are such
evaluating the effectiveness of analogues of BAED and
susceptible patients who would benefit from prophylacPAET and other immune-regulating steroids as buffers
tic administrationofPAED and/or PAET. Also among
of corticosteroid activities. Uses of PAED and PAET
the causesof immuno-suppressiontreatable with BAED
have been disclosed in U.S. patent application 5 and PAET are viral, bacterial, fungal, yeast and para07/739,809. These compounds have use 89 immune
sitic infections, chemotherapy,irradiation,severe stress,
regulating steroids for several purposes, as described
chronic fatigue syndrome and untoward effects of steroid therapy. The compositions of the invention are
below. A second aspect of the invention is the use of
BAED and PAET in conjunction with corticosteroids
particularly useful for treating patients suffering from
to ameliorate some of the undesirable effects Of COrtiCO- 10 infections caused by viruses that destroy the body’s
steroid therapy. It has been found in vivo that the spleen
immune response, such 89 human immunodeficiency
and thymus O f d f d s under Stress decrease in She. It
virus (Hrv) and hepatitis. m e protective value of
Was found that B U D and PAET do not effect a
BAED and PAET is particularly important to patients
change in the size of the spleen Of ~ O r f n d ,
aniundergoing
procedures such BS bowel surgery
mds, but protected the spleen and
from ~ J O ~ U15- or repair of gunshot wounds of the abdomen where
presents a sedous threat. patients with a
tion in animals during infection or other conditions of
stress. In spleen cell cultures exposed to hydrocortisone
history of conditions such as rheumatic fever would
a decrease proliferation is seen. spleen lymphocfles
benefit from prophylactic
maintenance use of
in culture, when exposed to mitogem such as concanavthe regulatory
p m and PAET or in condin A (con Ah liPoPolYsaccharide A (Lps)JOr Some 2o junction with other steroids as disclosed herein,
undergo a marked ProlifeatiVe inCmSe. Adtreating any condition wherein both inflammationand
dition of hydrocortisone to such a culture prevents such
of infection
present, treatment with a combiproliferative response. The addition of BAED and
nationof BAED or PAET or their analogues in conPAET to cultures containingproliferatin@wbi%
junction with corticosteroids (both natural and syntheOf hydrocortisone has
found to
counteract or buffer the antioroliferative effects
of 25 sized analogues) can provide benefit of anti-inflammatory action and modification of allergenic response
corticosteroids such as hydr6cortisone. BAED or
coupled
with avoidance of increased susceptibility to
BAET may be given concurrently with corticosteroids
infection.
or may be administered in a composition containing a
Both PAED and BAET are steroids and possess
combination of BAED or PAET with corticosteroids.
30
lipophilic
characteristics. Solvents for lipophilic steIn addition to their use as immune regulators, BAED
roids are known in the art and would be used as carriers
and BAET, their esters and other analogues are useful
for these compounds. Examples of such carriers are
for production of interleukin-3 (IL-3) in cell culture,
pharmaceutically acceptableglycols, and cyclodextrins,
including autogenous It3, as well as other cytokines
which are effective in regulation of the body’s re- 35 especially the intrinsically amorphous cyclodextrins.
Other vehicles that should be considered include fatty
sponses. IL-3 is an important cytokine capable of stimuacid esters such as esters of polyoxyethylene sorbitan
lating granulopoiesis, erythropoiesis and thrombopoie(Tweens) or sorbitan (Spans) to prepare oil-in-water
sis. It is a major factor governing differentiation and
emulsions. For subcutaneous administration to animals
proliferation of hematopoietic cells. Presently several
forms of IL-3 are available. Recombinaat human IL-3 40 used in the examples, the agents were dissolved 1:l
dimethyl sulfoxide @MSO)/ethanol, then diluted for
has been produced in several organisms. (See U.S. Pat.
No. 5,128,450, which is incorporated herein by refer- subcutaneous administration to the animals. When the
compositions were administered by mouth, BAED and
ence.) However, E - 3 obtained from non-human
BAET were added (without being dissolved) to the diet
sources has caused unacceptable side effects.
It has b a n disclosed in U.S.Pat. No. 5,206,008 that 45 to provide a composition containing 0.4% BAED. The
compositions may be provided in capsules formulated
BAED and @Am,their esters and analogues, provide
with the usual fillers. For application to the skin, PAED
means for regulating the immune response, for amelioor PAET may, for example, be dissolved in carrier
rating effects of stress, and for avoiding untoward efmaterial containing DMSO and alcohol, then applied to
fects of chemotherapy or exposure to irradiation by
administration of androstenediol (PAED) and andros- 59 a patch or directly to epidermal tissues. For vaginal or
rectal administration, BAED or PAET may be administenetnol (PAET). The improved means of regulating
tered by suppository, enema, or by application of
immune response to viral, bacterial, and other infections
creams, etc. Cornpositions of the invention may be adcan be utilized in treating not only infectious diseases,
ministered by any method that will result in contact of
but also in treating other immune disturbances such as
diabetes and chronic fatigue syndrome (both diseases 55 the active agent with tissue of ectodermal origin.
BAED and BAET have been found to inhiiit the
now considered to be immune response related synadherence properties of body cells. The antiadherence
dromes), rheumatoid arthritis and autoimmone reproperties of the active agents of the invention may be
sponses.In the case of virus-induced heart or pancreatic
infection where no other antiviral chemotherapeutic delivered directly to epithelialtissue during surgery. An
modality exists, BAED and PAET and their analogues, 60 example of such use would involve the application of
compositions containing the active agents of the invenincluding esters thereof, have value as prophylactic
tion to the omentum in conditions such as infection,
protective agents.
endometritis and malignancies of the bowel and ovary
In clinical medicine, treatment with BAED and
wherein adherence of foreign cells or particles to norP A P can lower morbidity in patients exposed to
pathogenic organisms.These agents can be effectively 65 malcells of the peritoneal lining is a problem. Carnposiused prophylactically in patients known to be particutions of the invention may, for example, be administered
as mists or sprays. Compositions can also be adminislarly susceptible to infection. Patients nndergoing surgery or chemotherapy or patients suffering from burns,
tered intrathecally.
5,387,583
7
-continued
ppm
37.49 32.12 72.01 4291
12
30.65
9
10
11
43.58 38.55 21.49
pprn
Carbon 16
I1
18
ppm
42.51 8130 11.41
Carbon
19
146.75
13
93.65
124.88
14
45.35
63.46 39.09
IS
38.13
19.53
DISCUSSION
5
8
alkylated derivatives are useful as materials which act as
precursors to PAED and PAET. Suitable derivatives
include but are not limited to compounds of the general
formula:
Stereochemistry was assigned to 3&7fl,t7&trihy
droxyandrost-Sene 0 and 3fl,7&17/3-t&ydroxyan- 10
drost-5ene (n) by comparison of their proton nmr with
cholest-5-ene-3/3,7/3-dioland cholest-5cne-3/3,7~-diol
proton nmr [Smith et al., J. Org. Chem., 38, 119-123
RiO
(1973) 1.
Preparatory method #2
15
and
A second method of preparing PAET has been developed which also used 3/3,17/3 diacetoxyandrost-Sene 8s
a starting material as before. This compound was oxidized with t-butylhydroperoxide in the presence of
chromium hexacarbonyl (Cr(c0)a) to form 38,1713- 20
diacetoxyandrost-5-en-7sneas described by Pearson
[Pearson, et al., J. Chem Soc. Perkin Trans. X, 267
(1985)l. The enone formed then was reduced with triRIO
isobutylaluminum (TIBA) to give the acetylated 7Phydroxy 0 product.
25
wherein each R1 individually may be H,alkenyl of 2-8
PREPARATION OF 3/3,7fl,l7/3-TRIHYDROXcarbons, alkyl of 1-8 carbons, phenylalkyl of 1-4 carYANDROST-SENE
A solution of 3/3,17fl diacetoxyandrost-5-en-7-one bons, phenyl, or COR2 (acyl), wherein Rz is H,alkyl of
1-8 carbons, alkenyl of 2-8 carbons, phenylalkyl
(0.9581 g, 2,466 mmol).and pentane (30 ml), dried over
MgSO4) were mixed under a nitrogen atmosphere. 30 wherein the alkyl has 1-4 carbons (including benzyl) or
phenyl. Any phenyl moiety may have up to three subTZBA (9.5 ml.9.5 mmol, 1M in toluene) was then added
drop& by syringe. The solution w& stirred at room
stituents chosen from among hydroxy, &boxy of 1-4
temperature for about 1 hour. The reaction was termicarbons, halo, alkoxy of 1-4 carbons, alkyl of 1-4 carnated by the addition of diluted hydrochloric acid (ap
bons, or alkenyl of 2-4 carbons and wherein any alkyl
proximately 5 ml). This solution was added to water 35 may be a straight chain, branched chain, or the alkyl
(100 ml)and extracted with ethyl acetate (3 times with
may be wholly or partially cyclized.
50 ml). The organic layers were combined and then
The following compounds also may be evaluated for
washed several times with saturated sodium bicarbonuseful “AED-activity” and “AET-activity” using the
ate solution (50 ad), saturated sodium chloride solution
process of the present invention. “ A E D is 3fl,17&
and “ A E T is 3p,70,17P(two times with 50 ml,)and Water (50ml). The organic 40 djy&oxymdrost-5-ene (W)
layer was dried over magnesium Sulfate and the solvent
t&ydroxym&ost+ene 0. me compounds and the
n x i ~ ~ ebY
d r o w evaporation to geld 86% Crude
methods for their preparation have been chosen to inproduct. 1H N m indicated that the crude Product
troduce substituents on the a-face or in equatorial posicontained
tions ofthe molecule in order to avoid presenting ad&45 tiom1
7-one (86%) and 38,17P-diacetoxy-7a-h~droxdroxyandrostsubstituents on the fi-face which is the face
xne.
most biologically active steroid molecules have been
The final product I was recovered from another
found to present to receptor sites.
preparation by saponification of the mixed acetate by
Certain generalized processes which are well-underadding a solution of K2CQ in methanol/water to the
stood in the c811be used as indicated in the following
crude product. The solution was stirred at room tem- 50
descriptions of synthesis of the selected compounds to
perature and monitored by TLC every hour (TLC conprotect @lock) sensitive functional groups during conditions 0.25 mm silica gel on glass, solvent system
versions of other functionaIities and then may be easily
60% ether, 20% hexane, 15% methanol, 5% water,
removed using methods well-known in the art.
developed with 12 chamber). The reaction was stopped
when only one spot was detected (approximately 5 hr, 55 1. Keto groups may be protected as ketals. Conversion of keto groups to ketaIs may be accomplished by
depending on the K 2 C O 3 concentration). Dilute acid
reaction of the ketone with ethylene glycol in the preswas added to the reaction mixture until it was slightly
ence of p-toluene sulfonic acid (PTSA). The ketal
acidic. The reaction mixhue was worked up by extracgroup may be removed subsequently by reaction with
tion with ethyl acetate (3x50 ml), and the combined
organic extracts were washed with saturated NaCl solu- 60 aqueous acetone in the presence of PTSA.
2. Hydroxyl groups may be blocked by acetylation.
tion (2 x 50ml), and then water (1 X 50 ml).
The organic
Acetylation is accomplished by reaction with acetic
solution was dried over MgzS04 and the solvent reanhydride. Deacetylation is accomplished by hydrolymoved by evaporation. Recrystallization from 95%
sis with sodium carbonate, or i f a base-sensitive funcethanovethyl acetate mixture gave cr), m.p. 232’-234‘
65 tionality is present, with an acidic medium.
C.(lit. 236’ C., Butenandt, op.cit)
3. Hydroxyl groups may also be blocked by converPAED and BAET may be substituted with protecsion to tetrahydropyranyl ethers by reaction with dihytive groups which yield BAED or PAET on hydrolysis
in order to prolong their activity. Hence, acylated and
dropyran in the presence of PTSA. The ether may be
-
-
9
5,381,583
10
d. the trimethylsilyl ether o f (XWI) is reacted with
hydrolyzed back to a hydroxyl group using HCl in
lithium aluminum hydride which removes the broaqueous acetone.
mine and reduces the 3-keto group causing the
4. Hydroxyl groups atso may be protected by converdouble bond to isomerize to Cs to form 3psion to tximethylsilyl ethers by reaction with trimethylhydroxy-17~-trimethylsiloxy-~drost-J-ene
(Xnr)
silyl chloride in DMF in the presence of an HC1 accep- 5
e. the trimethylsifyl group is removed to fonn
tor such as imidazole. The trimethylsily1 groups may be
Other 4u-alkyl compounds may be made by substiremoved later by reaction with tetrabutylammonium
tuting the appropriate alkyl iodide for methyl iofluoride.
dide in step a.
The products of ea& reaction step are recovered by
10 5. 6-methyl-3j3,17&dihydroxymdm~t-5-ene
mIX)
standard methods weI1-known to the art.
a. testosterone (XII) is converted to its trimethylsilyl
1. 3~,17P~ydroxy-5a-androstane
(V)
ether (XXI)
an alcohol solution of /3AEI)(IV) is hydrogenated
b. the keto group of XXI is converted to its enol
with molecular hydrogen in the presenceof a pallaacetate (a 3,5-diene) (XXII) by reaction with acetic
dium on charcoal catalyst to yield a solution of 0.
anhydride in the presence of PTSA
2. la-alkyl-substituted BAED compounds may be pre- 15
c. the enol ester (XXII) is reduced with sodium boropared by the method for la-methyl-3P,17&dihyhydride to form 3P-hydroxy-17/3-trimethybiloxydroxyandrost-S-ene (XI)which follows:
androst-5-ene (XXIII)
a. dehydrotestosteroneacetate (VI)(Florey, U.S. Pat.
d. XXIII is reacted with p-toluene sulfonyl chloride
No.2,875,196) is dylically brominated by reaction
to form its 3-tosylate 04Uv)
with N-bromosuccinimide and then treated with 20
e. XXJY is solvolyzed in aqkous alcohol and underzinc dust to isomerize the Q-CS double bond to
goes the i-steroid rearrangement to form 1713form 17~-acetoxyandrosta-1,5dien-3-one
(VI9
trimethylsiloxy-6-hydroxy-3,5-cyclo-androstane
b. the dienone (VI0 is reacted with lithiadimethyl
(XXV)
cuprate to form la-methyl-l7/3-acetoxyandrost4
25
f
.
the 6-hydroxyl of XXV is oxidized to a keto group
en-3-one (VIII) by 1,eaddition
with chromium oxide to form 1713-trimethylsiloxyc. the 3-keto group is acetylated with acetic anhy3,5-cycloandrostan-6one (XXVJJ
dride to form enol acetate lk-methyl 3,17Pg. XXVI is converted to the &methylene compound
diacetoxyandrost-3,4e (EQ
XXVXX using the Wittig reaction (Stache and
d. reduction of the enol acetate (rx) with sodium 30
Fritsch, Liebig's Annalen 1966, 697, 204)
borohydride in pyridine forms la-methyl-17/3h XXVII is hydrolyzed with aqueous PTSA and
acetoxy-3~-hydroxy~~ost-5ene
(x)
isomerizes to produce XXVIII
e. (X) is deacetylated by reaction with alcoholic soi. reaction ofXXVIII with tetrabutylammonium fluodium carbonate to obtain (XI)
ride removes the trimethylsiilyl group to form 6la-alkenyl-3~,17~~ihydroxyandrost-5-enes
may be 35
methyl-3~,17/3-dihydroxyandrost-5-me
(XXIX)
made by substituting appropriatedialkenyltithiocu- Other 6a-alkyl compounds may be made by using the
prates for lithidimethyl cuprate in step (b).
appropriate Wittig reagent in step g.
3. 2a-alkyl-3/3,17P-dihydroxyandtost-5~nes
may be
6.
16u-methyl-3~,17~-dihydroxyandrost-S-ene
prepared using the method given below for 2a-methm m
yI-3&17p&yhXYdaibudroxyaadrost-5cne (XVI)bY uskg 40
a'.&&&ph&ost,xone
(DHEAXXXX) is reacted
the appropriate alkyl iodide in step a.
with dihydropyran to form the tetrahydropyranyl
a. testosterone Qtr) is reacted at -30' C. with diisoether (XXXI)
PrOPYldOfit~m
in tetrahydrofuranto form the
b. the 17-keto group is converted to the dimethylhydrazone (XXXII)
2-lithio salt followed by reaction with methyl iodide to form amixture of 2u-methyl-17gdihydrox- 45 C.
is reacted b t with butyl
a d then
y a n d r o s t ~ - 3 - o n e(XIII) and its 2fl-isomer
methyl iodide to introduce the 16a-methyl group
b. the mixed Za,2/3-isomers are reacted with sodium
forming XXXIII
methoxide to epimerize the 2B-iSOm~f to form
d. reaction of M I 1 1 with cuprous chloride causes
cleavage of the hydrazone and also cleaves the
IxW
c. (xm) is PeraCetyIated by reaction with an excess $0
ether groups to produce 14a-xriethyl-3P,17&dihyof acetic anhydride in the presence of PTSA to
droxyandrost-5-ene (?LXXlv) Other 16a-alkyl
fonn 2a-methyl-3,17~-diacetoxyandrosta-3,5-diene
compounds may be made by using the appropriate
alkyl iodide instead of methyl iodide in step c.
0
d. oav) is reacted with sodium borohydride in etha17a-methyl-3&17~-trihydroxyandrost-5-ene
7.
no1 to product ~-meayl-3P,17P-dihydroxyan- 55 (XxXvrI)
drosten-Eene-17-acetare0
a. DHEA (XXXJ is reacted with trimethylsilyl chloe. XV is then deacetylated using sodium carbonate to
ride to form the trimethylsilyl ether XXXV
2a-methyl-3fi,17P-dihydroxyandiosc
b. XXXV is reactad with methyl magnesiumiodide to
produce
5vxvr)
form 17a-methyl-3~-trimethylsiioxy)-17@-hydrox4.4a-methyI-3p, 17&lihydroxyandrost-5-cne 0
60
ymdrost-5-ene ( X X X V r )
a. testosterone (YCII)is reacted with potassium butoxc. the trimethylsilyl group of XXXVI is removed
ide and methyl iodide to form 4-methyl-17bwith fluoride ion to form 17a-methyl-3P,17P-dihyhydroxyandrat4en-3-0ne
droxyandrost-Sene (XXXVIr)
b. XVII is reacted with N-bromosuccinimide in carOther 17a-alkyl compounds may be made by use of the
bon tetrachloride to form 61)-bromo4methyl-17/3- 65 appropriate alkyl magneium halide in step b.
hydroxyandrost4en-3-one (XVIII)
8. 7a-methyl-3/3,17~-dihydroxyandrost-5-ene
(XLIII)
a. aadrosta-4,6.dien-3,17~0nc17ketal (XXXVIII) is
c. the hydroxyl of (XVlII) is converted to a trimethreacted with methyl magnesium bromide in the
ylsilyl ether group
-
11
5,387,583
presence of cuprous chloride to produce 7amethylandrost4ene-3,17-dione17 ketal QCXXlX)
b. allylic bromination of XXXIX with N-bromosuccinimide produces 6&bromo-7a-methylandrosten3.17-dione 17 ketal (XL)
5
c. reduction of XJ.,with lithium aluminum hydride
produces the ketal of 36-hydroxyandrost-5-cnen17-one (XLI)
d. the ketal group of XLI is removed to form XLII
e. the keto group of XLII is reduced by the M e r - 10
wein-Pondorf-Verley reaction to yield 7a-methyl3&17&dihydroxyandrost-5-ene (XLIII)
Other 7a-alkyl compounds may be made by using an
appropriate alkyl magnesium halide in step a
9. 3a-methyl-3/3,17fl-tdhydroxyandrost-5-ene
QCLVI) 15
a.
3~-hydroxy-17~-trimethy~oxy-androst-5-ene
(JCXIII) is subjected to an Oppenauer oxidation to
form the corresponding androst-5-en-3-one
WIV)
12
above for making PAED-type compounds, in all cases
below where the 7P-hydroxyl group is introduced it is
accomplished by oxidizing a Abndrostene compound
with chromium hexactubonyl/tert-butylhydroperoxide
reagent to the 7-keto compound and then reducing the
7-keto group to a 7&hydroxyl using triisobutylaluminun in pentane or hexme.
Products of each step are recovered by standard
methods which are well-understood to those skilled in
the art.
12. 3/3,7&lihydroxyandrosten-17-one (CIII)
a. the 17-keto group of DHEA (XXX)is protected by
ketalization and the 3-hydroxyl is acetylated to
form an acetoxy ketal (CI)
b. a 7P-hydroxyl is introduced into CI as set out
above to form the 17 ketal of 3&acetoxy-7&
hydroxyandrost-5-cn-17-one
c. CII is deacetylated and the ketal group is removed
to produce 3P,7&dihydroxyandrosten- 17-one
(CIII)
13. 7P,17/3-dihydroxyandrosth-en-3-one
(CVIII)
a. the ketal group of the 17 ketal of 3P-acetoxy-7Phydroxyandrost-5-en-17sne(CII above) is removed to form 3/3-acetoxy-7~-hydroxyandrost-5en-17-one (CIIa)
b. the keto group of CIIa is reduced with sodium
borohydride in pyridine to form 3o-acetoxy-
b. XLIV is reacted witb methyl magnesium iodide to 20
form 3a-methyl-3~-hydroxy-17P-trimethylsiloxyandrost-5-ene CXLW
c. b e trimethylsiloxy group is removed with fluoride
ion to form 3a-methyl-3&l7~-trihydroxyandrost5-ene (XLVI)
2s
Other 3a-alkyl compounds may be prepared by using an
appropriate alkyl magnesium halide in step b.
10. 6-halo-3a-methyl-3fi,17&dihydroxyandrost-5-enes
7/3,17~-djhydroxyandrost-5-ene
(CIV)
(LI,X=C4 Br, I, or F)
c. the 7 and 17 hydroxyls of CIV are blocked with
a. testosterone acetate (XLVII) is dylicdly bromi- 30
trimethylsilyl groups by reaction with trimethylnated to the &bromo derivative QLVIII, X=Br)
silyl chloride to form 3/3-acetoxy-7P,17/3-bis(trimeusing N-bromosuccinimide in the presence of a
thylsiloxy)androst-5-ene (CV)
freeradical initiator such as uv light, benzoyl perd. the 3-acetoxy group of CV is saponified with alcooxide or azobisisobutyonitrile,
holic sodium carbonate to form 3s-hydroxyor
3s
7P,17&bis-(trimethyfsioxy)androst-5-ene (CVI)
allylically chlorinated to the dchloro derivative
e. the 3-hydroxyl of CVI is oxidized to a keto group
(XLvIIr, xec1) using sulfuryl chloride with a
with chromium trioxide (the double bond will
free-radicalinitiator,
move to Q)to yield 3-keto-7P,17P-bis(trmethylor
siloxy)androst4ene (CVn)
allylically iodinated to the 6-iodo derivative 40 f. the trimethylsilyl groups are removed with tet(XLVIII, X=I) using mercuric iodide in the presrabutylamrnonium fluoride to produce 7/3,17&
ence of uv lieht
dihydroxyandrost4en-3-one(CWII)
c. the &halo co%pound (XLVIII) is acetylated with
14. 3P,7/3,17P-trihydroxy-5a-~~osndrostane
(CIX)
acetic anhydride h pyridine to form a &haloAET 0 is reduced by hydrogenation over a palla3,1713-diacetoxyandrosta-3,5-diene(XLIX)
45 dium on charcoal catalyst to yield 3/3,7/3,17P-trihydroxy-5a-androstane(CIX)
d. XLIX is reduced with sodium borohydride to form
a 6-halo-l7l3-acetoxy-3-hydroxyandrost-5-ene
Q
15. 7~,17&dihydroxyandrost-1,4-dien-3-one(CXI)
e. L is saponified to fonn the 6-halo-3a-methyla. 3-keto-7~,17/3-bis(trimethylsioxy)androst-4-ene
3P,17P-dihydroxyandrost-5-ene(LI)
(CVII above) is oxidized with SeOz to introduce a
double bond at CpC2 to form 7/3,17P-bis(trimeThe 6-bromo compound (LI, X=Br) may be con- 50
verted to the 6-fluor0 (LI,X=p) functionality by reacthy1siloxy)androst- 1,4-dien-3-one (CX)
tion with silver fluoride.
b. the trimethylsilyl groups of CX are removed by
11. 16a-halo-38,178-dihydroxyandrost-5-cnes (LV? reaction with tetrabutylammoniumfluoride to produce
X=Cl, Br or Fj
7/3,17fl-dihydroxyandrast-I
,edien-J-one (CXI)
8. DHEA acetate &11) is reacted with cuprous chlo- 55 16. JP,178-dihydro~yyandrosta-S,7d~e~e
(CXIv)
ride or cuprous bromide to form a 16a-hdo cama. a 7b-hydroxyl group is introduced into 3P,l7Pdiacetoxyandrost-5-ene(AEDdiacetate)(CXII)
pound (LXII, X-Cl or Br)(Glazier; J.0rg. Chem.,
1962,27,4397)
b. the 7-hydroxyl is removed by heating a solution of
b. the keto group of LID[ is reduced with sodium
CXIII with PTSA to form a 7,8-doublebond fonnborohydride to form 16a-haIo-3/3-acetoxy-l7/3- 60
ing CXIII
c. CXIII is saponified with alcoholic sodium carbonhydroxy-aodrost-5-en-17-one(LIV, X=Cl or Br)
ated to form 3fi,17/3-dihydroxyandrosta-5,7-diene
c. the acetate groups of LIV are hydrolyzed in acid
medium to yield (LV, X=CI or Br)
(CXIV)
17.
9,10-secoandrosta-5,7,10(19)-trime-3~,17~-diol
d. the bromo analog (LV, X-Br) is readily converted to the 16a-fluor0 compound (LV, X=F) by 69 (CXV)
reaction with silver acetate.
a solution of 3P,17/3-dihydroxyandrosta-5,7-diene
When preparing substituted aAET compounds, in
(CXIV above) is irradiated with ultra-violet light (ca
addition to using the generalized processes set out
280 nanometers) to form 9,1O-secoandrosta-5,7,10(19)-
5,387,583
13
14
tnen~3&17/3-diol (CXV)(analogous to the formation
a. the 7fl-hydroxyl of the 17 ketal of 3fi-acetoxy-7fi-
of Vitamin D3 from 7-dehydrocholesterol)
la-methyl-3F,7& 17B-trihydrox yandrost-5-ene
hydroxyandrost-5-en-17-one
(CII above) is con-
verted to the trimethylsiyl ether (CXXVIII) by
reaction with trimethylsilyl chloride
a 7~,17~-bis(trimethyls~oxy)androst-1,4-dien-3-one5 b. the 3-hydroxyl of CXXVIII is deacetylated by
(CX above) is reacted with N-bromosuccinimide
reaction with sodium carbonate
followed by treatment with zinc dust to isomerize
c. the 3-hydroxyl is reacted with p-toluene sulfonyl
the a-Csdouble bond is to form 7&17B-diacetoxchloride to form the 3-tosylate CXXIX
d. on solvolysis the CXXlX undergoes the i-steroid
yandrosta-1,5dien-3-one (CXVI)
rearrangement to form the 7,17-substituted 6Pb. the dienone (CXVI) is reacted with lithiodimethyl 10
cuprate to form la-methyl-7/3,17P-doxyanhydroxy-3,5-cycloandrostane(CXXX)
drostan-5en-hne (CXVII) by a 1,4-addition
e. the &hydroxyl of CXXX is oxidized to the ketone
c. the 3-keto group of CXVII is reduced with sodium
CXXXI with chromium oxide
borohydride in pyridine to form la-methylf. the Cketone CXYXI is converted to the 6-methylene compound CXXXII by the Wittig reaction
7P, 17~aiace~xy-3~-hydroxyandrost-5~ne 15
(CXVIII)
(Stache and Fritsch, Liebig's A d e n 1966, 697,
d. CXVIII is saponified with alcoholic sodium car204)
bonate to obtain la-methyl-3/3,7&17~-trihydroxg. the ti.methylene-3,5cycloandrostanecompound
yandrost-kene (CXIX)
CXXXII is hydrolyzed and isomerized with aquela-~keny1-3P,7/3,178-trihydroxyan~t20
ow PTSA to produce $methyl-3/3,7fl,17&trihyOther
5-enes may be made by using appropriatelithiocuprates
droxymdrostJ-ene (CXXXIIl)
Other 6-alkyl derivatives may be made by using the
in step (b)
19.
2a-methy1-3/3,7~,17~-~y~oxyandrost-5-ene
appropriate Wittig reagent in step f.
(c=I)
22.
16u-methyl-3/3,7&17fl-trihydroxyandrost-5-ene
a 7/3,17B-dihydroxyandro~~n-3-one (CVIII 25 (CXXXVI)
above) is reacted ut
30' C. with diisoa. 3~,7~dihydroxyandrosten-l7-one
(CIII)is reacted
propylaminolithium In tetrahydrofuranto form the
with dihydropyran to form the 3,7ditetrahy2-lithio salt followed by reaction with methyl iodropyranyl ether CXXXlV
b. the 17-keto group of CXXXIV is converted to its
dide to form a mixture of 2a-methyl-7@,17Pdihydroxyandmt4en-3-one (CXX)and its 2P-isomer 30
dimethylhydrazone by reaction with dimethylhyb. a solution of the mixed isomers is heated with sodrazine
dium methoxide to epimetize the 2B-hmer to
c. the dimethylhydrazone CXXXV is reacted first
cxx '
with butyl lithium and then methyl iodide to introc. CXX is peracetylated by reaction with acetic anhyduce the 16a-methyl group
dride in the presence of PTSA to form 2u-methyl- 35 d. cleavage of the hydrazone with cuprous chloride
3,7&17/3-triacctoxy~drosta-3,Sdiene (cxxz)
also cleaves the dihydropyranyl ether groups to
d. CXXI is reacted first with sodium borohydride in
produce
16a-methyl-3&7/3,17B-trihydrox yanethanol and then with alcoholic sodium carbonate
drostJ-tne (CXXXVl)
to produce 2a-methyl-38,7~,17S-rrihydroxyan- Other 16a-alkyl compounds may be made by using an
drost-5cne (Cxxn) Other 2u-alkyl compounds 40 appropriate alkyl halide i
n step c.
23.
may be made by using au appropriate alkyl hatide
17u~methyl-3~,7~,17/3-trlhydroxyandrost-5-ene
instead of methyl iodide in step a.
(C=w
20.
4a-methyl-3/3,7/3,17P-trlhydroxyandrost-S-crie a. 3fl,7&diiydroxyandmten- 17-one (CIIK above) is
acetylated with acetic anhydride to form the
(Cxxvn)
a. the hydroxyls of 7~,17/3dihydroxyandrost~n45
3p,7@diacetate (Cxxxvrt R=acetyl)or is reare converted to trimethylsiiylether
acted with trimethylsiyl chloride to form the bis3-e (CMI)
groups by reaction with trimethylsilyl chloride
trimethylsilyl ether (CXXXVII,Rdrhethylsilyl)
formingCXXrn
b. the diacetate (or disilyl ether) CXXXVII is reacted
b. CXIUII is reacted with potassium butoxide and
with methyl magnesium iodide to form 17/3-meth50
yl-3/3,7&diacetoxy (CXXXVIII, Raacetyl) or
methyl iodide to form 4-mahyl-7/3,17/3dihydroxyandrost4en-3sne (CVm)
bis-trimethylsiloxy)-l7fl-hydroxyandrost-5-ene
c. CXMN is reacted with N-bromosuccinimide in
(CXXXVIII,R-trimethylsilyl)
carbon tetrachloride to form 6/3-bromo4methylc. the diacetate is saponifed (or the trimethylsilyl
7fl,17/3-bis(trimethylsiloxy)androst-4-en-3-one
groups are removed with fluoride ion) to form
(cxxv)
55
17a-methyl- 3/3,7&17&trihydroxyandrost-5-ene
d. CXXV is reacted with lithium aluminum hydride
(CxxXrx)
to remove the bromine and reduce the >keto
Other 17a-akyl derivatives may be prepared by using
gronp, the double bond isomerizes to
forming
an appropriate alkyl magnesium halide in step b.
4cl-methy1-3~-hydrox~7~,17/3-bis(~ethyl~ox-24.
7a-methyl-3/3,7/3,17&trihydroxyandrost-5-ene
y)androst-S-ene (CXXVI)
60
(CXLII)
e. the trimethyldyl groups of CXXVI am removed
a. 3~,17~-dihydroxyandrost-5-en-7-one
(III) is reacted with trimethylsiiyl chloride to form the bisby reaction with tetrabutylammonium fluoride to
form
4a-methyl-3~,7~,17~-trihydroxyandrosttrimethylsilyl ether C X L
5 - m(CXXvn)
~
Other 4a-alkyl derivatives may be
b. C X L is reacted with methyl magnesium iodide to
prepared by substituthg an appropriate alkyl hal- 65
form 7u-methyl-7~-hydroxy-3~,17/3-bis(trimeth~lsiloxy)androst-5-ene (CXLI)
ide for methyl iodide in step b.
21.
6methyl-3~,7~,17/3-trihydroxyandrost-S-ene c. the trimethylsilyl groups are removed by reaction
of CXLI with tetrabutylammonium fluoride to
(C-I)
18.
(C=)
-
e,
5,387.5
, 83
--
1s
16
7a-methy1-3~,?/3,17/3-trihydroxyandrost- 28. Ia,3/3,7/3,17/3-tetrahydroxyandrost-5-ene
(CLX)
form
Sene (CXLID
Other 7a-dkyl derivatives may be prepared by using an
appropriate alkyl magnesium halide in step b.
a. DHEA ( X X X ) is fermented with penicillium asper-
gillus by the method of Dodson et al., JACS, 1957,
79, 3921 and 1960,82, 4026 to form la,f&dihy-
25.
3a-methyl-3/3,7~,17/3-trihydroxyandrost-5-ene
droxyandrostJ-en-17-one (CLVI)
(CXLV)
a. 3 ~ - h y ~ o ~ - 7 ~ , 1 7 ~ . b i s ( t ~ e t h y l s ~ o x y ) a n d r o sb.t - the keto group of CLVI is reduced to hydroxyl
with sodium borohydride to form la,3&17/3-trihy5-ene (CVI above) is subjected to an Oppenauer
droxyandrost-5-ene(CLVII)
oxidation to form 7/3,17fl-bis(trimethylsiloxy)an10
drostJ-en-3sne (CXLIII)
c. the hydroxyl groups of CLVII are acetylated with
b. the androstenone CXLIII is reacted with methyl
acetic anhydride to form la,3/3,17/3-triacetoxyan-
magnesium iodide to form 3u-methyl-3/3-hydroxy-
7/3,17/3-bis(trimethyIsiloxy)androst-5-ene
(CXLIV)
drost-5ene (CLVIK)
d. the 7fi-hydroxyl group is introduced by the stan-
dard method to form lq3/3,17/3-triacetoxy-7/3,s
hydroxyandrost-Sene (CLIX)
CXLIV by reaction with tctrabutylammonium
e. the acetate groups of CLIX are saponified with
fluoride to form 3a-methyl-3/3,7/3,17B-trihydroxsodium carbonate to yield la,3/3,7/3,17/3-tetrahyyandrost-5-ene (CXLV)
Other 3a-alkyl derivatives may be made by using an
droxyandrost-5-ene (CLX)
appropriate alkyl magnaim halide in step b.
20 29. 3/3,7/3,16a,17P-tetrahydr&yandrost-S-ene (CLXI)
26.
6-halo-3a-meth~l.3/3,7~,17P-trihydrox~~dro~t-16a-bromo-3/3,7/3,17P-trihydroxyandrost.s.ene
5-enes (CLI, X=Cl, Br, I or F)
(CLIII, X=Br) is reacted with sodium hydroxide
a. 7fl,17fl-dihydroxyandrost-4-en-3-one (CVIII) is
in dimethylformamide to form 3/3,7/3,16u,17/3-tetconverted to the 7P,17P-diacetate (CXLVI) by
reaction with acetic anhydride
25
rahydroxyandrost-%ne (CLXI)
Esters and ethers of the above ComPounb may also
b. the enone CXLVI is allylically brominated to the
&bromo derivative (CXLVTI, X=Br) using N- be evaluated by the methods of the present invention as
bromosucchhide in the presence of a free-radical
substitutes for PAED or PAET. Doubly (or more)
initiator such as uv light, benzoyl peroxide or azosubstituted BAED and pAET derivatives for such apbisisobutyronitrile,
30 plications may be made by performing appropriate
or
combinations of the above syntheses.
allylically chlorinated to khloro derivative
(CXLVII, X=Cl) using sulfuryl chloride with a
EXPERIMENTAL
free-radical initiator,
Cultures
of
spleen
ceUs used for testing properties of
or
35
allylically iodinated to the
compound
the products O f the invention Were prepared according
(CXLVII X=I) using mercuric iodide i
n the presto the fOhWing procedure:
ence of uv light
Lymphocytes were harvested from spleens of
c. the 6-halo compound is acetylated with acetic
BALB/c mice. The lymphocytes were set at a concenanhydride to the 6.halo-3-acetoxy-7P,l7~-bis(.40 tration of 5 . 0 106
~ cells/& in RPMI 1640 containing
trimeth~lsilox~)androsta-3,5.diene (CXLX
10% fetal calf Semm (FCS), 200 p~ L-glumine, 20
R=CI, Br or I)
mM Hepes, 2.5 U/ml penicillin, 2.5 p d m l streptomyd. reduction of the diene CXLIX with sodium borocin, and 5.0X 10-5 M 2-mercaptoethanol. The active
hydride foms the 6-halo~7~,17P-diacet0xy~3~
hydrowmdrost-5+ne (CXL, R=,-J, Br or I)
45 agents in amounts designated h the table Were added to
Cultures O f the cells in standard 96-well plates in the
e. C L is apoNfi& by reaction with alcoholic sodium
W M I 1640 media in 100 pl samples in triplicate. The
carbonate to form 6-halo-3a-rnethyl-3/3,7flIp,17/3steroids were dissolved in a 50:50 mixture of dimethyl
trihydroxyandrost-Sene(CLI, X=Q, Br, I or F)
The 6-brOmO CmpoWd (CLk X=Bd may be consulfoxide (DMSO)and ethanol before addition to the
verted to the &fluor0 functionality by PZaCtiOn with 50 culma, B~~
the 50. DMSO+&anol mixture
silver fluoride.
used to dissolve the steroids has a suppressive effect on
27.
16a-halo-3p,7P, 17/3-trihydroxyandrost-5-enes cell proliferation,all results were compared to standard
(CLV, X=CI, Br or F)
mixture.
cultures containing Oa2% Of the
a. 3/3,7/3-dihydroxyandrosten-17-one
(CIII) is reacted
with acetic anhydride to form the diacetate (CLII) 55
Effects Of Added BAED*
and Hyb. CLE is react& with cuprow chloride or cuprow
drocortisone on Proliferation in the Absence of a
bromide to form a l6a-halo compound (CLIII,
Mitogen
X=CI or Br) (Glazier; LOrg. Chem, 1962, 27,
A series of tests was run in triplicate using BALB/c
4397)
spleen cells to demonstrate the effect of the products of
C. the keto POUP Of CLIII is reduced with sodium 60 the invention and hy&opo&one (“Hycofi”) on celluto form 16u-hdo-3&7/3-diacetoxylar proliferationin the absence of a mitogen. Cultures of
17P-hydroxyandrost-5-ene (cLw*x=cl Or Br)
spleen cells were prepared by the procedure outlined
d. the acetate groups of CLW are hydrolyzed in acid
to field the 16a-halo.3~,7~,17/3.~y- above and steroids were added a$ shown in Table 1.
65 Twenty four houri after setup, 50 p a [3H]-thymidine
droxyandrast-5ene (CLV, X-Cl or Br)
was added to each cell. Four to six hours later, the cells
e. the bromo malog of CLV is readily converted to
were harvested and counted on a scintillation counter.
the 16a-fluor0 compound (CLV, R=F) by rmction with silver acetate.
The results are set forth in Table I.
c. the trimdhylsiloxy groups are removed from
oAET
5,387,583
17
TABLE 1
18
TABLE 24xntinued
l a n u a c t of BAED md Hydroconbone
on Splcen ccll Cultures fmm BALB/c Mice
Stimulated with 2 5 pa/mf Conclnrvdin A
MlTOC3EN INDUCED BALB/C
SPLEEN CELL PROt-TION
EFFECTS
OF BA€Z,BAED. AND HYDROCORTISONE
Avtmge
Stcroid
SL
5
AED
Concentration
0.1 phi
none, no mlvtnt
a o 02%
~ DMSOBOH
AET 3.0 ph!
A E S 0.5 #&M
A E D 5.0 pM
AE.D 0.5 JLM
0.MpM
Hycort
coo^
mtlon
0.1 pM
0.1 pM
Avcngs
count
(3 samples)
347,000
365,000
SuDdard
Q
Rtducdoa
Mtion
of Caatrol
38,M
44,ooO
18
14
10
A comparison of these results With those of Table 1
shows that addition of a mitogen such as ConA increases proliferationby nearly two orders ofmagnitude.
It can be seen that BAED slightly enhanced the prolif15
erative response of spleen cells to C o d , but it did not
AET 5.0 pM
AET 0.5 pM
AED 5.0 pM
AED 0.5 phi
AET 5.0 pM
AET 0.5 pM
significantly counteract the dcpressivc effect of 1.0 pM
hydrocortisone on proliferation. Although PAED
AED 5.0 JLM
counteracted the effect of 0.1 phl hydrocoirboae, the
AED 0.5 fl
since proliferation did
20 eflect was limited in magnitude,
not attain the control level. 'Example 3: Effects of Added PAEZ and HydrocortiThe solvent-oontaining control averaged about
sone on Proliferation in the Presence of a Mitogen
2800rt200 counts while the culturu to wbich steroids
Another s e i s of cultures was run in which PAET
had been added rang& from a low of about 1500k500
to about 6000fSOO counts. These roults do not allow 25 and/or hydrocortisone was added to cultures similar to
making a determination that there was a si@icmt
those of Example but to which 2.5 pg/d concanavalin
effect on proliferation from the addition of the steroids
A had been added. The results are set forth in Table 3.
to the culturq. Therefore it was concluded that BAED,
PAET and corticOsteroids have little effect on cellular
TABLE 3
proliferation in the absence of the stimulus created by 30
Influence
of
BAED
wd H y d r m h n e
mitogens and/or mitogens.
on Splean c d l CIll~lofmm BALB/c Mlce
Experiment 2 Effecuof Added BAED and HydrocorS h d a l e d with 2.5 ua/d Cbncu~valinA
tisone on Proliferation in the Presence of a Mitoga
70
Hymn
Avarge
Another series of cdtures was run addmg B U D
Sundud
Reduction
count
AET
Coocenand/or hydrocortisone to cultures to which concanava- 35 Corrccnmtion tration (3 umpln) Deviation of Control
lin A had b a n added in a manner $Warto those of
5.0 phi
502,000
zz.Oo0
hcr. 50
Example 1. Preliminary tests on cultures to which con1.0 F M
572,000
23,000
her. 71
29,000
incr. 56
canavalin A had been added at concentrationsof 10.0,
523,000
0.5 pM
23,ooO
incr. 45
0.1 fl
486,000
5.0, 2.5 and 1.0 pg/d showed that proliferation of
29,aoO
her.
62
541,000
pM
cultures was most sensitive to the effect of added hydro- 40
.- 0.0s
0.01
u M3S,000
incr. 65
953.000
. .. ,
cortisone when the concanavalin A concentration was
5.0 @
I
31
23OhO
9,000
2.5 pg/d,so all tests on the effects of BAED on cul34
21,000
222,003
1.0 pM
34
22.000
tures stimulated with concanavalin A were performed
0.5 phl
z21,Mxl
31
0.1 phf
19.Oo0
228,000
with cbncsoavslin A at 2.5 pg/dThe results are set
iacr. 23
24,000
0.05 pM
4ll,000
forth in Table 2.
45
bcr.
63
51,000
5.0 pM
547,000
1.0 fl
----
,
--
I
I
1.0 pM
0.5 pM
TABLE 2
Mumce of BAED md Hydroud~~m
011 S p k a Cell llJhutr frcm BALB/c Mice
Slimu(ited with 2.5 ra/d C a a ~ & A
Hyoort
AED
Coneentdoa
0.2%
DMSQE:OH
"Ow
I.Ofl
0.5 )LM
0.1 @vl
0.05 @vl
-
s.opM
I
I
LOpM
0.5 fl
0 I .uM
0.05 p M
5.0pM
1.0 )LM
0.5 p~
amW
o
n
---
5.0 pM
Avwgc
373,000
430,000
426,000
46969,600
U8.000
0.5
1.o)rM
361,000
191,000
0.1 pM
0.W pM
1.OM
LOpM
1.0pb4
1.0 pM
0.1 pM
0.1 fl
ai
pr+i
Dcvtrtioo
14,000
(3ramples)
424.000
216,000
2wJoo
Ulxxx)
r.opM
S h d d
cOWt
'
262,000
230,000
201,Oao
228.000
238,KU
3W,600
319,000
32~~00
18,000
31.000
21,000
29,ocO
23,ooO
26,000
13,Mxl
IS.000
22,W
II.Oo0
Z~,Ow
16ooO
15,000
6.000
18,,ooO
26,oaO
I0,ooO
zv~co
0.1 pM
0.0s pM
0.01 pM
%
50
1.0pM
W U C ~
0.9 phf
ofCoa:rol
0.1 pM
0.05 pM
0.2%
12
DMSO:EtOW
incr. 2
55
-
her. I
lao. 11
hm.6
49
41
41
38
15
s5
46
53
54
44
16
25
23
1.0 pM
1.0pM
1.0pM
1.0 p.M
1.0 pM
0.1 pM
0.1 pM
0.1 pM
0.1 pM
219,000
242,000
223,000
226,000
224,000
4
0
5
.
m
439.000
~os,oOo
419,000
3a.m
24,W
10,ooO
17,000
19,000
8,000
42lm
23,000
22,000
31,ooO
22.ooo
35
28
34
33
33
Incr. 21
incr. 31
Incr. 22
incr. 25
-
-
It can be s e n that PAET markedIy enhanced the
proliferative response of spleen cells to C o d . In the
prcscace of 1.0 p M hydrocortisone a relatively high
60 dose (5.0 pM) of BAET was required to counteract the
depressive effect of the hyydrocortisone, while lower
doses of BAED showed no s i d c a n t effect. When 0.1
pM hydromrtiSone was present PAET completety
counteracted the depressive effect of the hydrocorti65 sone, and mediated an increase to above the control
level.
Example 4 Effect of PAED and PAET on IL-3 production
5.387.583
-,- ,
19
A series of experiments was done to determine
whether BAED and BAET would cause a change in
the level of the cytokine xC3. The cultures were prepared in accordance with the general method set out
above. After 30 hours the level of IL-3 in the superna- 5
tants of the culture3 was measured using the I G 3
ELISA kit manufactured by EndoGen Inc., Boston,
Mass. The fmdings are shown in Tables 4 and 5 below.
_20_
daily or twice a day to achieve a daily dosage of 15 mg
and 5 mg dexamethasone per day.
Example 6:
A preparation for application to the skin or mucosa
may he prepared in the following manner:
Ingrdenl
AE!D
__
~
IO
TABLE 4
EFFECT OF BAED AND BAET ON
Petrolscum
THE PRODUCTION OF INTERLEUKIN-3
IN "EPRESENCE OF 5.0 M r n l c4nA
AED/A.ET
0.2%
DMSOEHIH
AED 5.0 pM
It3
Pg/ml
Hydrocortisone
168
AED 0.5 p M
AET 5.0 pM
AET 0.5 pM
AED 5.0 ph4
AED 0.5 pM
AET 5.0 pM
AET 0.5 pM
0.5 pM
0.5 pM
0.5 pM
0.5 pM
0.5 pM
.
184
I80
201
219
51
210
42
I18
102
TABLE 5
EFFECTOF BAED AND
BAET ON RJTERLEWN-3
PRODUCTION IN THE ABSENCE OF COnA
AED/AET
n 2%
RMS%OH
AED 5.0 pM
AET 5.0 pM
AET0.5
uM
AED 5.0 pM
AET 5.0 pM
AET 0.5 p M
struction and absorption in the upper gastro-htestind
tract. The active agents are most effective when the
20 period of e x p u r e to the mucosa of the intestinal tract
is increased. Hence use of capsules containing the active
agents in formulations that effect slow release in the
intestine are appropriate for treatment of intestinal disorders such as Crohn's disease and colitis. Use of reten25 tion enemas for treatment of inflammation of the large
bowel is also appropriate.
Example 7:
A formulation for administration as a retention enema
may be formulated in the following manner:
30
Ingredient
O S pM
0.5 pM
O S pM
35
deroxlmerasone
Propylene glycol
0.05%
99%
When the active agent is administered to the mucosa
100
1.2
36
s9
w/w %
0.05%
62
0.5 pM
13.0%
83.0%
When BAED or PAET or their analogues are admin-
Pg/d
30
19
0.5%
0.5%
3.0%
l 5 istered orally, the active agents may be utilized more
efficiently if the active agents are protected from de-
a-3
Hydrocortisone
crkmcinolont
elvcervl
-propyiene
_ monostearate
g~yco~
% w/w
of the oral cavity, it may be administered as a spray for
use in the oral-pharyngeal cavity and the nasal cavities.
Example 8:
C57BL/6J mice infected with a single dose of CB4
(9X106) and treated with BAED plus hydrocortisone
The results clearly show that SAET caused increased
or BAED alone. The dosage of BAED was 1 mg
secretionof IL-3both in the presence and inthe absence
BAED in DMS0:ETOH 1:l. The dosage of hydrocorof concanavalin-A. Further, they show that hydrocorti- 4, tisone was 2 mg administered as a 1% saline solution.
sone suppressed the production of E - 3 and that PAET
Groups of six mice were given the following:
counteracted the immunosuppressive effect of hydro(A) PAED and hydrocortisone (AED/HC) given concortisone to an extent that brought the level of IL-3
currently with the virus at the same site.
production to nearly the same level as that found in
(J3) BAED/HC given concurrently with the virus, but
cultures to which no hydrocortisone had been added. 50 at a different site.
Equally interesting was the increase in the le el of XL-3
(C)PAED/HC given four hours after administration of
production to higher than normal levels in t& cultures
virus.
to which hydrocortisone had not been added. fIence, it
(D) BAED only given concurrently with virus at the
can be said that BAET not only is useful for cqunteractsame site.
ing the effect of lowering IL-3 levels brought on by 55 The results were as follows:
corticosteroid therapy, but also that increased production of IL-3by cells can be induced in cultures by the
addition of BAET.
Mclbod
M o N l i y Results
The I t 3 expressed by a culture may be recovered
A
1/6
from the m& containing IL-3 by known methods 60
B
6/6
C
6/6
such as single or sequential reverse-phase HPLC steps
D
6/6
on a preparative HPLC column. (See Urdal, et al., J.
1%
Chromatog. 296171 (1984) and W.S. Pat. No.5,128,450).
Example 5:
It can be seen that treatment of the mice coacurrentIy
Capsules of a formulation of BAED and dexametha- 65 wi
with a mixture of BAED and hydrosone for oral administration arc prepared containing 15
co
greater protection than did the use of
rng BAED, 5 mg dexamethasone, 150 rng starch, a d 5
BAED alone.
mg magnesium stearate. The capsules are administwed
I claim:
76
40
5,387,583
21
22
lone, fluprednidene and its derivatives, flurandrenolide,
clobetasol and its derivatives,clobetasoneand its derivatives, alclometasone, flumethasone and its derivatives,
-andfluocortolone and its derivatives.
3. A composition of claim 1 c 0 n W . g PAET.
4. A composition of claim 1 containing BAET and
hydrocortisone.
5. A composition of claim 1 which is a patch.
6. A composition of claim 5 containing at least one
10 compound selected from compounds of the formula:
1. A pharmaceutical composition comprising an antiinflammatory effective amount of at least one conicosteroid or analogue thereof and a corticosteroid buffering effective amount of at least one immuno regulating
compound selected from compounds of the formula: ' 5
*
15
and
JdJ
RIO
20
and '-
25
wherein each R1 individually may be H,alkenyl of 2-8
carbons, alkyl of I-8carbons, phenylalkyl of 1-4 carbons, phenyl, or COR2 [(acyl)], wherein R2 is H,alkyl of
wherein the alkyl has 1-4 carbons -,
or phenyl
30
wherein each
phenyl moiety may have up to three
substituents chosen from among hydroxy, carboxy of
1-4 carbons, halo, alkoxy of 1-4 carbons, alkyl of 1-4
carbons, or a l h y l of 2-4 carbons and wherein any
alkyl may be a straight chain, branched chain, or the 35
alkyl may be wholly or partially cyclized.
2. A composition of claim 1 for topical application
wherein the corticosteroid is selected from among hydrocortisone,triamoinoloneand its derivatives, betamethasone and its derivatives, flunisolide, prednisone and 40
its derivatives, fluwinolone and its derivatives, diflorasone and its derivatives, halclnonide, dexamethasone
and its derivatives, desoximetasone, diflncortolone and
its derivatives, flucloronide, fluccinoaide, fluocorto-
-
-
-
45
55
65
Ri0
wherein each R1 indiidually may be H,alkenyl of 2-8
carbons, alkyl of 1-8 carbons, phenylalkyl of 1-4 car-
bons, phenyl, or COR2 [(acyl)], wherein R2is H,alkyl of
wherein the alkyl has 1-4 carbons, including benzyl , - or phenyl - wherein each-phenyl moiety may
have up to three substituents chosen from among hydroxy, carboxy of 1-4 carbons, halo, alkoxy of 1-4
carbons,alkyl of 1-4 carbons, or alkenyl of 2-4 carbons
and wherein any alkyl may be a straight chain,
branched chain, or the alkyl may be wholly or partially
cyclized; and triamcinolone.
7. A composition of claim 6 containing PAET.
* * * * *
llllll1l11111111111111111111l111l111111111111111111111111Ill1111111111
US005391776A
[191
Ueno et al.
1541 STEROID DERIVATIVES
p5] Inventors: Hiroaki Ueno, San Diego, Calif.;
Sdchiro Kadowaki, Kanagawa,
Jspaq W t o iCnmizono, Tokyo,
Japan; Masahiko Morioka, Tokyo,
Japan; Akihisa Mori, Tokyo, Japan
[73] Assignee:
Mitsubkhi Kasei Corporation,
Tokyo, Japan
[21] Appl. NO.: 15,800
[22] Filed:
[11]
Patent Number:
5,391,776
[45]
Date of Patent:
Feb. 21, 1995
2-104593 4/1990 Japan .
W092/05187 4/1992 WIPO .
OTHER PUBLICATIONS
Eriksen et al., Science, vol. 241, pp. 84-86 Jul. 1988.
Primoiy Examiner-Paul J. Killos
Attorney, Agent, or Firm-Wenderoth, Lind & Ponack
[571
ABSTRACT
Asteroid derivative of the general formula:
Feb. 10, 1993
Foreign Application Priority Data
Feb. 14, 1992 [JP] Japan ..................................
r
1
(0
[30]
Int. C L 6
..............................................
4-028497
cO7J 51/00
U.S. (3. .....................................
552/507; 552/506
Field of Search ................552/507, 506; 514/102,
5 14/107
References Cited
0496520 7/1992 European Pat. Off. .
0548884 6/1993 European Pat. Off. .
represents a residue of steroid comrepresents -CO[”(CHR1)rCI)pCO]mNH--,
-CO-(R~)X-(Z),+ZO-NH-,
or
-CO-(CH2)n-.
A therapeutic agent to osteopathy
comprising the above steroid derivative is also provided.
wherein X-Opound and -A-
5,391,776
1
STEROID DERIVATIVES
ro
1
The present invention relates to novel steroid derivaX-O-A-CHL!(OR)~]2
tives. More particularly, this invention relates to steroid 5
derivatives useful as therapeutic agents in osteopathy
X-0- represents a residue of steroid comosteoporo~or the like and p ~ e u t iwherein
~
such
pound,
A- represents
compositions containing at least one of them.
Steroids (e.g. estrogen, androgen) already known as
- C O ~ ( ~ ' ) ~ + l f n " having sexual hormone activity have been noted to be lo
useful as
agents in
treatment Of OSteoin which y representsan integer of from 1 to 3, p repreporosis, because they show osteogenesis accelerating
sents 0 or 1, m reprants m hteger of from 0 to 5, R1
activity and increase the bone
represents hydrogen atom, optionally substituted
Journal of M d c h t , 303,1195 (1980); J 0 w - d of chi- ~ 1 - c 4 & y l group or optionally substituted C S - C I ~ ~ ~ Y ~
cal and EndoCrinologicalMetabolism, 51, 1359 (1980)].
group, a d y represents & or -m-,
Further, it has recently been recognized that these ste--cO--(R1)~13 (Z)+NHroid compounds accelerate osteogenesis by direct action to bone, because receptors for estrogen or androgen were found in bone tissue [Science, 241,84 (1988); 2o which x and q each independently represent 0 or 1, R*
represents OPtionallY substituted
Proceedings of the National Academy of Sciences of
group,
the U.S.A., $6, 854 (1989)l. However, these steroid
-WW~C~O-(CHZ)Icom~oundsrauire careful administration when used as
a syketnic theiapeutic agent for osteoporosis, because
an integer Of 0-59
organs and induce xveral side 25 m which and
they &o act on
Cyclo represents CpC7 cycloalkylene, optionally subsuch s generation Of uterus cancer' abnormal
stituted phenylene group or optionally substituted
&Me
Prostatic hwrnophy, defemination
C,-C7 &ylcne group, represents -0- or -"-,
or the like.
or
On the other hand, bisphosphonic acid derivatives
are known easily to transfer to bone, and it is disclosed M
that a madidnal compound can be osteostlactively
taken in by binding said medicinal compound to a bisin which n represents an integer of From 0 to 10 with the
phosphonic acid derivative [Japanese Patent Publicaproviso that, when X-0is 17&(3-hydroxy-1,3,5tions (KOKAI) No.SHO 58-174393, SHO 62-26256and
estratrienenyloxy) group, n represents 0 or 1, and
HE1 2-1045931. These publicationsdisclose as a medici- 35 R represents hydrogen atom or C144 alkyl group, or
nal compound carbonic acid dehydrogenase inhibitors,
pbarmaceutically acceptable salts thereof. The present
invention will be explained in detail below.
antiinflammatory agents, anticancer agents and the like.
The present invention relates to steroid derivatives of
However, a compound consisting of asteroid compound
the general formula (I):
bound to a bisphosphonic acid derivative is not disclosed in the publications.
40
As the results of extensive studies seeking an effective
0
meaOS which allows asteroid compound to osteoselectively act, the present inventors have found that administration of a novel compound prepared by binding a
steroid compound to a bisphosphonic acid derivative 45
wherein X-O- represents a residue of steroid comthrough one of various spacers enables the steroid compounds having a cyclopentanohydrophenanthrenering,
pound to act more selectively on osseous tissue than
so-called "steroid nucleus", which is represented by the
other organs A conjugate of asteroid compound and a
following formula
bisphosphonic acid derivative exerts no steroid-like 5o
activity,since it does not bind to asteroid receptor. It is
considered, however, that, when said conjugate is administMcdto a living body, a considerable part of said
conjugate molecules would be adsorbed on bones
through the bisphosphonic acid, and the rest remained 5s
without being adsorbed would be smoothly excreted
out of the body. The coojugate bound to the bone is
gradually cleaved at the bonding site of the bisphosphonic acid and steroid compound to isolate the steroid
wherein the rings J, K,L, and M each independently
compound which will bind to the steroid receptor 60 represent a saturated, partially saturated, or unsaturated
within the bone, thereby osseoselectively showing osring, and they may be independently substituted by one
scogenesis accelerating activity. Further, the present
or more substituents selected from alkyl, alkenyl, akyinventors have found that the concentration of the stenyl, halogen, alkoxy, ester, acyl, hydroxy, and oxo
roid compound in bone c ~ be
n kept at a high level for a
groups. Specific examples are, for instance,those havlong time. The present invention has been established 65 ing hydroxy group (e.& estradiol, testosterone, dehybased on the above findings.
drotestosterone, pregnenolone, ethynylesrmdiol, esAccordingly, the present invention is directed to a
trone, estriol,dehydroepiandrmteronsterone,androstenediol,
steroid derivatim of the general formula (I):
17a-hydroxyproge&rone, norethandroolone, androster-
mew
.
2
3
one, norethidrone, nandrolone, etc.), and -Asents
5,391,776
4
repre-
in which k and 1 each represent an integer of 0 4 , and
Cyclo represents C347 cycloalkylene group which
-cot"(cHR'~ror)pcol,"includes divalent groups formed by elimination of two
5 hydrogen atoms from a single carbon atom to which
they attached (e.g. cyclopropylene group, cyclopentain Which Y represents an integer from 1 to 3, P represents 0 Or 1, represents an integer from 0 to 5, R1 lene group, cyclohcptalenegroup, ctc.)), phenyl group
represents hydrogen atom, c1-c4 alkyl F O U P optionoptionally substituted by a substituent (e.g. hydroxy
group, Carboxy group, CI-cs alkyl group, etc.) or
ally having a rnbstituent (e.& hydroxy group. mercapto
P U P , a Y l W 0 SOUPI -0
&roup, amido GOUP, 10 C~-C.~alkylene
group (e.g. methylene, methylene,proph x Y gtoup, OPtiOdlY Substituted Phenyl P U P ,
ylene, pentamethylene, heptamethylene, etc.) optionaryt P U P OPtiOd1Y hVing One or
ally substituted by a substituent (e.g. hydroxy group,
ek.) or C6-c~
cI-Cs alkyl group, CYCZOaralkyl group, wboxy
more substimm sclcctad from hydroxy group, wh x y group and CI-CS*Yl gr0W and y reprmnb
group, etc.), and 2 represents -0- or --NH--, or
-0- or NH-, or
1s
-CO-(CH&-
--co--(R3dZ)9--cQ--"-
in which n represents an integer from 0 to 10 with the
in which x and q each independently rePr=t 0 or 1,
proviso that, when X-0- is 1777-(3-hydroxy-1,3,5R'rePrents VinYlaC POUP optionally substituted bY a 20 estratrienenyloxy)group, n represents 0 or 1, and
substituent (e.& hydroxy S O U P , cI45 a Y 1 SOUP,
R represents hydrogen atom or Cl-C4 alkyl group
C7-Cm aralkyl group, carboxy group, etc.),
(e.g. methyl group, butyl group, etc.), or pharmaceutically acceptable salts thereof.
-(CHA-CYdO-(CHz)~Specific examples of the compounds of the present
inv&tion will be shown below ih Table 1.
TABLE I
0
It
X-O-A-CH[p(OR)&
2
'0
I
3
4
0'
0
ll
0
II
n
0
0
H
-C-NH(CH&C-NH-
II
II
-C-NH(CH&C-NH-
0
0
?'
I1
li
-c-NHcHc-NHI
CH3
H
5,391,776
5
Corn@.
6
X-O-
No.
-A-
5
R
0
0
II
II
?0
H
-C-NH~C-hTY-
@
HO
6
0
H
0
1
I
H
0
-0-NHCHC-NHII
1
I
0’
I
7
0’
&
0
II
-c-m?-m-
HO
8
9
10
0’
0’
0’
0
II
0
II
-C-NHCHC-N€+
n
n
0
II
H
0
II
-c-NHqHc-m-
I
CHzOH
H
5,391,776
7
8
TABLE l-continued
0
X--O--A--CH[P(ORkJ2
It
Compd. No.
x-o-
12
-A-
'0
R
0
0
-C-NHYHC-NHII
II
H
I
CHzSH
HO
13
&
0
0
-C-NHCHC-NHII
It
H
I
CHzCHzSCHj
HO
0
14
0
H
0
II
H
0
It
H
-c-"CHC-NHII
II
I
HO
IS
&
0
II
-c-NHaic-mI
HO
0
II
-c-
16
HO
NHpc-m-
5,39 1,776
9
10
TABLE l-continued
*
*
P
*
B
X-0- A-CHp(OR)&
Corn@. No.
17
x-o-
O0
I
-A-
0
It
-C-"CHC-Nx-
0
II
R
H
I
CW2CHzCOOH
HO
18
0
0
-C-I"
tCHC-"-
II
H
CH~CHICONH~
I
HO
19
?'
HO
m
?'
HO
21
P0
HO
22
e
'0
l
HO
0
I1
-C-NX-
H
5,39 1,776
11
12
TABLE I-continued
0
X--O-A-CHF(OR)2]2
II
Corn@. No.
x-o-
-A-
R
24
N
?’
2s
H
0.
26
H
HO
e
27
P’
0
II
0
II
H
0
0
H
-C-(C!HZ)~-C--NH-
HO
28
&
HO
I1
It
-C+(CHj)4-C-NH-
5,391,776
13
14
TABLE I-continued
0
II
X-O-A-CH~(OR)rl~
Compd. No.
-A-
x-o-
29
R
H
0
II
0
I1
-C-(CHds-C-NH-
€IO
30
0
II
&
&
&
0
-C-CH-C-"-
I1
I
CH3
€IO
31
R
32
0
H
0
II
II
-c-a-c-NHI
0 CH3 0
II I
II
-c-c-c-NHI
H
0 C2H5 0
II I
II
I
H
CH3
H
33
-c-c-c-"-
CzHs
H
34
&
8
H
fC)P
-C
C-"-
H
35
H
H
5,391,776
15
16
TABLE 1-continued
0
II
X-O-A-CH~(OR)d2
x-o-
Compd. No.
37
*
0
0I
H
38
-C
0
H
\\
C-Mi-
H
?-
H
&
&
H
0
0-
41
H
\\c-NH-
0-
39
R
-A-
\\
Yf-
-C
H
0-
H
42
0
II
0
II
-C-CHXCH-C-NH-
H
5,391,776
17
18
TABLE I-continued
0
II
X--O-A-CT(OR)LJ~
compd. No.
x-o-
R
0-
43
44
-A-
e
?-
0
I1
0
II
-CNH-(cH~-O-C-"-
H
H
45
0
0-
I1
I
0
H
II
-C-C?INC-NH-
@
H
46
0
0
0-
H
II
II
-C-(CH~"C-NH-
47
e
01
0
II
0
-C-(CH&-N€I-C-NH-
I1
H
H
48
0
0-
I1
0
II
H
-C-CH-NHCNH-
49
H
0I
-C
NC-NH-
5,391,776
19
20
TABLE l-continued
0
I1
X-O-A-CH[P(OR~z
x-o-
Compd. No.
-A-
R
H
50
NC-NH-
H
51
0-
fi
0
II
0
II
H
-C-CHz-O-C-NH-
H
52
53
H
0-
H
H
54
H
56
0I
0
11
-c-mz-
H
5,391,776
21
22
TABLE l-continued
0
II
X-O--A-CH~(OR)212
Compd. No.
x-o-
-A-
OH
57
R
P
i
H
0
II
H
-CNHCH2C-NH-
'0
0
II
58
-C"(cH&C-NH-
'0
&
II
0
II
H
0
II
0
N
0
OH
59
-CNH(CH.&C"-
'0
II
-C-NHCHCNH-
I
a s
' 0
0
OH
OH
62
0
II
II
0
0
n
II
H
H
-C-N'HCHC-NH-
63
OH
I
'0
0
II
-C"-
a
5,391,776
23
24
TABLE lcontinued
h p d . No.
x-o-
R
-A-
64
0
II
0
II
H
-C(NI-ICHzC%NH-
\
0
65
0 0
I1 II
OH
I
@
0
'
e
e
66
OH
I
R
-C-C-"-
0
0
-C!-CHpZ-NHII
I1
H
'0
61
PH
0
0
-!-(CH*h-CNHIt
H
'0
68
OH
&
'0
69
0
H
II
-C-(CHd3-C-NH-
H
OH
' 0
70
\
8
&
0
0
1
I
0
II
-C-CHC-N€I-
I
H
5,39 1,776
25
26
TABLE lcontinued
Cornpa No.
x-o-
-A-
71
tl YH3 51
R
H
-c-c-c-"-
I
ma
0
'
12
P
?";1
H
-c-c-c-mI
CIHS
\
0
73
0
'
&
H
f C ) i C-"-
-C
74
H
75
16
H
O7"-
-.
H
71
'0
5,391,776
27
28
TABLE I-continued
0
II
X-O-A-CH~(ORh]z
H
19
H
OH
I
H
81
82
OH
I
@
0
0
II
II
H
-C-NH(CHdzNHCNH-
\0
03
\
0
0
II
II
-c-"(cH~)~-o-cNH-
H
0
84
OH
I
\
0
0
II
0
I1
-ccxzN€ic-N€i-
H
5,39 1,776
29
30
TABLE lcontinued
0
II
X-O-A-CH[P(ORhlz
Cmqxl. No.
-A-
x-o-
R
OH
85
I
86
U
OH
I
'0
&
OH
0
II
-C-CHz-O-CNH-
89
PH
90
PH
PH
91
'0
0
II
H
H
5,39 1,776
31
32
TABLE l-continued
0
II
X-O--A-CH[P(OR)&
x-o-
Compd. No.
-A-
R
0
H
92
II
-C-
\
0
93
0
II
H
0
I1
H
-C-CHz-
\
0
94
\
0
95
OH
H
PH
ti
I
96
e
YH
91
0
‘
98
&
-C-(CHh-
?H
0
II
H
0
II
H
--C-(CH?)6-
-C-(CH~)IO-
5,391,776
33
34
TABLE l-continued
0
W
X-O-A-CHF(OR)~]Z
-A-
x-o-
Corn@. No.
0
I1
0
99
0
II
-CN?ICH2CNH-
0
II
100
0
11
H
-c-mzc-N€I-
0
I1
H
0
II
-c--CHz-
H
0
H
0
101
II
-C-(cH&-C-”-
102
103
R
H
e
?‘
0
II
II
-c-”m~c-”-
0
104
&
0
II
0
II
H
0
H
-c-CH2-c-NH-
0
10s
P’
0
0
II
-C-(cH*C-”-
I1
5,391,776
35
36
TABLE ltontinued
0
II
X--O-A-CHP(OR)Zh
Compd.
-A-
x-o-
No.
R
H
0
11
106
-C-CHz-
0
0
107
I1
It
0
‘
H
-C-NHCH2C-NH-
0
0
0’
&
&
&
H
0
II
II
-c-cH2-c-NH-
0
109
0’
0
II
0
1
I
H
-C-(CHdzC-NH-
0
110
0
111
0
1
I
0’
e
0
‘0
H
-c-cn2-
P
P
-C-NHCHp2-M-
0
I1
0
II
-c-cH~-c--MI-
5,391,776
37
38
TABLE I-continued
0
X-O-A-CH~OR)&
II
Compd. No.
x-o-
-A-
113
0
0
It
R
H
0
I1
- C - ( c S r ~ ~ -
zc
0
0
1I4
0
H
II
-C-CHz-
\
0
11s
O
II
P
H
-C-NHCH2C-NIi-
116
OH
117
PH
0
0
It
II
-c-CH2-c-m-
H
0
H
P
1
I
-C-(CH&-Ch"-
0
H
II
-c-cHz-
'0
119
'0
0
0
-C-N€iCH$-N'€+
II
I1
H
5,391,776
39
40
TABLE l-continued
0
II
X--O-A-CH[p(ORhh
Compd. No.
-A-
x-o-
120
'0
I
121
&
&
R
II
0
II
H
0
II
0
II
H
0
-C-CHz-C-NH-
-C-(CH2hC-NH-
HO
122
H
0
II
'0
-C-CHz-
HO
123
0
'
&
0
II
0
I1
H
0
H
-c-"CH~c-NH-
0
II
II
-c-CH~-c-NH-
125
P
P
-C-(CH2)2C-"-
H
5,39 1,776
41
42
TABLE l-continued
0
II
X--O-A-CH[P(ORM1
-A-
X-O-
Compd. No.
R
0
126
H
II
-C-CH2-
0
\
I21
0
II
0
II
H
0
0
I1
H
-C-"2C-"-
I1
-C-CHyC-NH-
&
&'
&
Prn
0
129
0
0
It
-C-(CH~-C-"-
1
I
H
0
130
0
II
H
-C-CHz-
0
131
H
HO
5,391,776
43
44
TABLE l-continued
0
II
X-O-A-CH[P(OR)dz
Compd. No.
X-O-
132
-A-
0
It
0
1
I
0
II
0
II
R
H
-C-CHz-C-NH-
HO
133
H
-C-(CH~2c--"-
0
1
I
134
H
-C-CHz-
135
I1
0
II
H
0
II
0
1
I
H
0
0
ti
H
0
-C-NHCH2C-NH-
136
-C-C&-C-N€X-
137
I1
-C-(CHhC-NH-
I38
P
-C-CHz-
H
5,391,776
45
46
TABLE l-conthued
0
II
X-O-A-CH~(OR)&
x-o-
Compd. No.
139
-AO/
I
0
II
0
II
-C-NHCH2C-N"
0
141
R
I
0
II
-C--NHCH2C+NH-
HO
142
0'
143
HO
144
'P
a
3
5,391,776
47
48
TABLE l-continued
0
II
X-O-A--CH[P(OR&
x-o-
Compd. No.
R
-A-
II
0
0
II
0
0
145
ll
CH(CWCHzW
-c-mcH~c-"-
HO
146
'P
-C-NHCff$-NH-
I1
147
H
148
H
149
N
The compounds of the present invention can be for- 55
mulated into phan~ceuticalformulations suitable for
particular administration mute together with couvcntional carriers when used as a medicine. For example,
they can be prepared into formulations such as tablets,
capsules, granules, powders, solutions or the like for 60
oral route. In preparing solid formulations for oral
route. customary excipients, binders, lubricaats, coloring agents, disintegrators and &e lie may be wed.
Ehnplcs of the excipient are lactose, starch, talc,mag&urn stenrate, crystallint dulose, methy1 celluiose, 65
carboxymethyl cellulose, glycerin, sodium alginate,
arabic gum, and the like. Examples of the binder arc
polyvinyl alcohol, polyvinyl ether, ethyl cellulose,mabic gum, shellac, saccharose, and the like, and examplts
of the lubricant are magnesium stcarate, talc and the
like. In addition, customarycoloring agents and disinte-
grators already known may be used. Further, tablets
may be coated in a conventional manner. Liquid formulations include aqueous or oily suspensions. solutions,
syrups, elixirs and the like and these liquid formulations
may be prepared in a conventional manner. In preparing injcctiom, the compound of the p e n t invention
may be mixed With p H regulators, buffets, stabilizers,
isotonic agents, topical anesthetics, and the l&e, and
injections for subcutaneou~,intramuscular or htravenous administration may be prepared. As for the base
for preparing suppositorits, fatty and oily bases such as
49
5,39 1,776
cacao butter, polyethyleneglycol, Witepsol (Registered
trademark, Dynamite Novel Company) or the like may
50
011-h)
X-O-CO--NH(CHR~)~H
\1
be used.
Cuniu, rearrangement
Appropriate dosage of thus prepared formulations 5
varies, depending upon the symptoms, body weight, age
or the like of particular patients. Appropriate daily
dosage for adult of the compound of the present invention is, in general, about 0.01 to ZOO0 mg. The daily 10
dosage may preferably be administered after divided to
2-4 portions. Alternatively, a single dosage may be
X-O-CO-N?f(CHR~)~-CO-"CH
administered every other day or with longer time inter-
~H2NcH[!~0,1,
Val.
Salts of the steroid derivatives of the general formula
\1
15
(T) are those with non-toxic bases. Appropriate salts
include those of inorganic bases such as sodium,potasX-O-CO-NH(CHR~)~-CO-~CH
sium or the like,ammonium salt, those of organic bases 20
like triethylamine, and the like.
iu) Where p is 1 and Y is -0Production of the compounds of the present invention will be explained below under three sections, dex-0-co-w
pending upon the type of -A- in the general formula 25
0.(1) Compounds (III-g), (III-j), (n1-n) of the general
P - l ) @
formula (Iin
),
WMCh -Ais
X-O--CO-NH(CHR~)yOH
-WNM~'),-W+Irn~-
W-a)
]
pi
P(OR6h
[B ]
P ( O ~
on-9
W1-j)
(m- 1):
CnW
(n1-k)
on-0
30
wherein RI,y, Y , p and m have the same m d g s as
defined in the general formula 0,or their salts, can be
prepared, for example, according to the following synthetic ronte.
i) Where p is 0
40
In the above formulae, X
&,
R1, y and m have the
same meanings as defined in the general formula (I), W
represents halogen atom or imidazolyl group, and Rs
and R6 each independently represent c1-c4 lower alkyl
45 fF?UP*
Where p is 0:
Compound (m-b) can be prepared by reacting Compound (11) with Compound @La) in an appropriate
solvent and, ifnecessary, in the presence of an appropriate base. The solvent to be uscd illustratively includes
diethyl ether, tetrabydrofumn, dioxane, dimethoxyethme, sulfome, dimethyl sulfoxide, dhethylformamide
and the like, and the base to be used includestriethylamine, diisopropylamine, pyridine, collidhe, N-methylmorpholine, diazabicycloundwene and the l i e . The
reaction is preferably effected by reacting Compound
(
I
I
with
) 1 to 4 mol equivalent of Compouad @La) in
the presence of 1 to 4 mol equivalent of a base. The
reaction is effected usually at temperature from 0' to
150' C.,preferably fkom 20' to 120' C,over a period of
1 to 10 hours.
Compounds (IIId)can be prepared by reacting Compound (X-b) with Compound (II1-c) or their salts in an
appropriate solvat in the presence of an appropriate
base. The solvent illustratively includes diethyl ether,
tetrahydrofuran, dioxane, dimethoxyethane, sulforane,
dimethyl sulfoxide, dimethylformamide and the like,
and the base includes triethylamine, diisopropylamhe,
1)
50
55;
60
65
ii) Where p is 1and Y is -NH- (m=I)
51
5,391,776
-I
pyridine, collidine, N-methylmorpholine, diazabicynate. This reaction is carried out usually at temperature
doundecene and the like. The reaction is preferably
from -30" to 200' C., preferably 0' to 130' C.
effected by reacting Compound (XI-b) with 1 to 4 mol
The compound wherein m is an integer of 2-5 can be
equivalents of Compound (II1-c) or their salts in the
presence of 2 to 8 mol equivalents of a base. This reac- 5 prepared in ,accordance with the above-mentioned
preparation employed for the compound wherein m is
tion is carried out usually at temperaturefrom 0' to 150'
C., preferably 20' to 120' C.,over a period of 10 to 100
1.
hours.
iii) W e r e Y is -0-:
Compound (m-e) or its salt can be prepared by hyThe compound having the above general formula
drolyzing Compound (III-d)with an aqueous solution 10
or its salt ciin be prepared from Compound (III(IU-1)
of an inorganic base in an appropriate solvent or by
b) and (111-k) in the same manner as the preparation of
deprotecting Compound (md)(in which RS=benzyl
Compound (II1.d) dcscriid in the above item i).
group or the like) over palladium catalyst in an appropriate solvent. The solvent to be used includes convenCompound (III-m) can be prepared by reacting Comtional alkanols (e.g. methanol, ethanol, propanol, 2- IS pound (III-1) or its salt with Compound (IV-b) in the
propanol, butanol, hobutanol, 2-methoxyethanol, etc.),
presence of an appropriate base and an appropriate
dimethyl sulfoxide, dimethylformamide, and the like.
solvent.
The solvent to be uB6d illustratively includes
The inorganic base to be wed includa sxiium hydroxtoluene, dioxane, dichloromethane, dimcthoxyethaue,
ide, potassium hydroxide, Lithium hydroxide, calcium
hydroxide, barium hydroxide, sodium carbonate, potas- 20 and the like, and the b w inClUdS triethylamine, pyrisium carbonate and the &e. Preferable amount of the
dine, diisopropyiethy-le, N-methylmorpholine,
base used in the reaction is 0.1 to 10 mol equivalents.
and the like. The present reaction is preferably effected
This reaction is carried out usually at temperature from
by reacting Compound (111-1) with 1 to 2 mol equiva20' to loo' C over a period of 1to 50 hours.
Compound (X1-f) can be prepared by reacting Corn- 25 lents of Compound (IV-b) in the presence of 0 to 4 mol
equivalents of a base. This reaction is carried out ordipound (IIIc) or its salt with carboxylic acid activator
narily at temperature from -30' to 200' C., preferably
(e.g. dicyc1'*
'de, isobutyl chIoroformate,
N,N-cacbonyldiimidazole, thionyl chloride, oxalyl
0' to 130' C.
chloride, eto.) in the presence of an appropriatebase in
Compounds (IILg),(III-j)and (111-n) can be prepared
an appropriate solvent for activating the carboxyl 30 by respectively reacting Compounds (III-f), (III-i) and
group of Compound (In-e) and addiag tetraalkyl
(IIEm) with trimethylsilanehalide in the presence of an
ambmcthy1cnebispho6phonate w - a ) to the reaction
appropriate solvent, and then by hydrolyzing the resulmixture. The solvent to be used illustratively includes
tant product with water. The solvent to be used iuustradichloromethane, chloroform, diethyl ether, diisopropy1 ether, tetrahydrofuran, dioxane, dimethoxyethane, 35 tively includes tetrahydrofuran,dioxane, dimethoxyethand the like, and the base to be used includes pyridine,
ana acetonitrile and the like, and the trimethyldam
collidin~ triethylamine, diisopropylethylamine, nhalide includes trimethylsilane iodide, trimethylsilane
methylmorpholine, diazabicy-cloundcccnc, and the
bromide, trimethylsilane chloride and the like. In the
like. The piesent reaction is effected by activating carboxyl group of Compound (III-c) with 1to 2 mol cquiv- 40 present reaction preferably 4 to 8 mol equivalents of
tdmethylsilane halide with respect to Compounds (malents of a carboxylic acid activator in the preseace of 1
to 4 mol quivalents of a base and then adding thereto 1
f) (III-i) and (In-m) may be used. This reaction is carto 4 mol equivalents of tetraauryl aminomethylenebisried out at temperature from -50' to 20' C,preferably
phosphonate. This reaction is preferably cf€ectEd usu-20' to 0' C.. over a period of 0.5 to 5 hours.
ally at tempentore from -70' to 20. C., preferably 45
The compound wherein m is an integer of 2-5 can be
-20' to 0' C., over a period of 0.5 to 10 hours.
in accordance with the above-mentioned
prepared
The compound wherein n q u a l s 0 can be prepared
preparation employed for the compound wherein m is
from Compound (II1-b) and Compound (VI-a) under
conditions em~lovcdin the above-mentioned reaction
1.
50
betwan C0ml;or;nd p - b ) and ComPOund 0U-C)(2) Compounds
(V-g), and ('"-1). in which
ii)Whcrt pis 1 and Y is -NH-:
-A- in the general formula 0 represents
Several methods for convertina the carboxvl RTOUD
of Compound (III-h)or its salt to-ismyanate Goup
--cO-(R9dZ)+~kuown. For example, Compound (III-I) canbe prepared
by trrrting CoPnpound @I-h) with DPPA (Diphtnyl 55
as deWherein XDR2t and 9 have the -e
phosphonoazide) in the prescnce of an appropriatebase
fined in the general formula 0,or their salts can be
and an appropriatesolvent for Curtius rearrangementto
prepllre the hVm@ aod then adding W-1
prepared in the manner as shown In the following synamhomethyllenebisphosphonate(VI*) to the resultant
thetic
product. The solvent to be used illustratively includes 60
qio:
i)
toluene, dioxane, dichloromethane, dimethoxyethane
and the lilre,and the base includes hiethylamine, pyndine, diisopropylethy-lamhe, N-methyhnorphohe,
~0-co-m~~~-co-0~4
(v4
and the like, This reaction is prefetably effeoted by
X-0-H
(II)
reacting Compound (In-h) with 1 to 2 mol q~kdc~ts
65
of DPPA in the presence of 1to 4 mol equivalentsof a
(v4
x-O-CO-~%-CO-OR4
basc and adding 1 to 4 mol equivalents of tetraakyl
~omethyleaebisphosphonate
to the resulting isOcya-
we),
.
5,391,776
53
-continued
1
X-O-CO-(R3,-CO-OH
ii) Where 2 is -NH-
A
v
II
0
(Vc)
and qE1:
X-O-CB-(R3x-?-W-CQ-NH-CH
iii) Where Z is -0- and q-1:
i' J
P(0H)z
54
-continued
5
X-o-Co-~~),-o-Co-"-cX
[P ]
P(0m
WQ
In the above formulae, X-0-, x, Rz,Z and q have
the same meanings as defined in the general formula (I),
and R4 and R6 each independently represent C1-C4
10 lower alkyl poup or b a y 1 group.
i) Where q=O
Compounds (V-b) can be prepared by reacting respectively the carboxylic acids (V-a) with a carboxylic
acid activator (q.
dicyclocarbodiimide,isobutyl chloroformate, N,N'-carbonyldiimidamle, thionyl chloride,
oxalyl chloride, etc.) in an appropriate solvent for activating the carboxy group and then adding Compound
(
I
I
and
) an appropriate base to the reaction mixture.
The solvent to be used illustrativelyincludes dichloromethane, chloroform, diethyl ether, diisopropyl ether,
tetrahydrofuran, dioxane, dimethoxyethane and the
like,and the base to be used includes pyridine, collidine,
tsiethylamine, diisopropylethylamine, N-methylmorpholine, diazabicycloundcceneand the like. The present
reaction is preferably effected by treating respectively
Compound (V-a) with 1 to 2 mol equivalents of a carboxylic acid activator for activating the carboxy group
and then adding to the resulting mixture 1 to 2 mol
equivalents of Compound (11) and 1 to 4 mol equiva- , .
ients of a base. This reaction is carried out usually at
temperature from -20' to 100' C.,preferably 0" to 80'
C,over a period of 0.5 to 10 hours.
Cornpounds (V-c) or their salts can be prepared by
hydrolyzing Compound (V-b) with aqueous solution of
an inorganic base in an appropriate solvent or by hydrogenating Compounds (V-b) (R4-benzyl group) over
palladium catalyst in an appropriate solvent. Said solvent ordinarily includes alkanols (eg. methanol, ethanol, propanol, 2-propanol, butanol, isobutanol, 2methoxyethanol, etc.), dimethyl sulfoxide, dimethylfomamide and the like. The inorganic base to be used
includes sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, barium hydroxide,
sodium carbonate, potassium carbonate and the like.
preferred amount of the base is 0.1 to.10mol equivalents. The F c t i o n is effected usually at temperature
from 20' to 100' C. over a period of 1 to 100 hours.
Alternately, Compound (V-c) or its salt can be prepared
by treating Compound (
I
l
with
) 1to 6 mol equivalents
of acid anhydride (V-a') in the presence of a base. The
solvent to be used includes organic amines (e.g. pyridine, collidiue, etc.), toluene, dioxane and the lie. The
base includes triethylamine, pyridine, Mimethylamlnopyridine, and the like.
Compound (Vd)can be prepared by reacting Compound (V-c) or its salt with a carboxylic acid activator
(e.g. dicyclocarbodiimide, isobutyl chloroformate,
N,N-arbonyldiimidazolc, thionyl chloride, oxalyl
chloride, ctc.) in the presence of an appropriate base in
an appropriate solvent for activating the &oxy group
of Compound (V-c) and then adding to the resulting
mixture tetraallcyl aminomethylenebis-phosphosphonate
0.
The solvent to be used illustratively includes dichloromethane, chloroform, diethyl ether, dusopropyl
ether, tetrahydrofuran, dioxane, dimethoxyethaue and
the like, and the base to be used includes pyridine, collidine, triethylamine, diisopropylethylamine, N-methylmorpholine, diazabicyclound~ne, etc. "he present
5s
5,391,776
reaction is preferably effected by adding 1 to 2 mol
equivalents of a carboxylic acid activator and I to 4 mol
equivalents of a base to Compound (V-c) for activating
the carboxy group of Compound (v-c) and then adding
1 to 4 mol equivalents of tetraakyl aminomethylenebisphosphonate. This reaction is carried out usually at
temperature from -70' to 20' C.,preferably -20' to 0'
C..over a Deriod of 0.5 to 10 hours.
subjecting Compound (Vc) or its salt to Curtius rearrangement with DPPA (diphenyl phosphonoazide) in
the presence of an appropriate base and an appropriate
solvent and then reacting the resuiting isocyanate with 15
tetraalkyl aminomethylenebisphosphonatc (VI-a). The
SOlVCnt to be USCd inClUdeS tOlUen6 diOXanC, diChlOr0methane, dlmethoxyethane,and the like. The base to be
used includes triethylamine, pyridine, diisopropylethylamine, N-methylmorpholineand the like. The present reaction is preferably effected by adding 1to 2 mol
equivalents of DPPA and 1 to 4 mol quivalents of a
base to Compound (V-c) and then adding 1 to 4 mol
wuivalab ofk-1
aminamethy-lenebisphosphon-25
ate. his reaction is carried Out usu~uyat temperature
from -30' to 200' C.,preferably 0' tO 130' C
iii) Where Z=&
and q= 1:
fornula
compouad havine
above
w-BOr salt be
cornpod (v-h) 30
and Compound
via Compound (V-i) in the same
mannu as the preparation of Compound (V-c) described in the above item i).
Compound (V-k) cm be prepared by reacting Compound (V-j) or its salt with an appropriate base in an 3s
appropriate solvent. T h e solvent to bc used illustratively includes toluene, dioxane, dichloromethane, dimetlaoxyethane and the Ukc, and the base to be used
includes triethylamine, pyridlne, diisopropylethyla- 4o
mine, N-methylmorpholinc and the like In the present
reaction, 1 to 2 mol equivalents of Compound (IV-b)
and 0 to 4 mol equivalents of a base with respect to
Compound (V-j) arc preferably used. This reaction is
carried out usually at -30' to 200' C.,preferably 0' to 45
'
(Io
56
HO--cO-(CH2)n-CH
['
X-0-H
X--O--cO--(cEI2).--CH
P(OR6k
(n)
1
['
In the above formulae, X - 0 -
3
WI-4
and n have the m e
meanings as defined in the general formula (I)
and
, R6
represents C1-Q lower alkyl group.
Compound (VI-b) can be prepared by reacting the
carboxylic acid (VI-a) with a carboxylic acid activator
(e.g. dcyclocarbodiimide, isobutyl chloroformate,
N,N-carbonyIdhidaole, thimyl chloride, oxalyl
chloride, etc.) in an appropriate Sdvent for 8CtiVaring
the carboxyl PUP of Compound 0'1-4 and then adding to the resultant mixture Compound (?I) and an appropriatc base. T h e solvent to be used illustratively
includes dichloromethaae, chloroform, diethyl ether,
diisopropyl ether, tetrahydrofluon, dioxane, dimethoxyethane,.aad the like, and the basc to be used includes
py;idine, ~collidine, triethylamine, diisopropylethylamine, N-methylmorpholine, diazabicycloundecene, and
the like. In the present invention, preferably 1 to 2 mol
equivalents of a carboxylic acid activator is added to
Compound (VI-a) for activating the carboxy group, and
then 1 to 2 mol equivalents of Compound (11) and 1 to
4 mol equivalents of a base are added This reaction is
carried out w W y at tempuature from -20' to loo'
C.,preferably 0' to 80' C., over a period of 0.5 to 10
hours.
Compound (VIS)can be obtained by reacting Compound (VI-b) with a trimethylsilanehalide in an appropriate solvent and hydrolyzing the rcdtsnt product
with water. The Jolvmt to be used illustratively includes tetrahydrofuran, dioxane, dimethoxyethane,acc130' C.
tonitrile and the like, and the trimethylsilane halide
Compounds (V-e), (V-g) and (V-I) CBIL be prepared
includes trimethylshe iodide, trimethyldane broby reacting respectively Compounds (V-d), (V-f) and
mide, trimethylsilanechloride and the We. Xn the pres(V-k) with a trimethylshe halide in an appropriate
ent reaction, preferably 4 to 8 mol equivalents of trim&
solvent and then hydrolyzingthe resultant product with
thylDilnne halide is added to Compound (VI-b). This
water. The solvent to be used includes tetrahydrofuran,
reaction is carried out usually at temperature from
dioxane, dimethoxyethane,acetonitrileand the like, and
50' to 20' C.,preferably 20' to 0' C., over a period
the t r i m e t h y l s i l ~halide
~
includes trimethylsilane ioof 0.5 to 5 hours.
dide, trimethylsilane bromide, t r i m e t h y l s i chloride 55 The present invention willbe explained in more detail
by way of the followlng Preparations and Examples,
and the like. In the present reaction, 4 to 8 mol qUivabut the scope of the present invention should not be
lents of trimethylsilane halide is used for one mol of
construedto be limited to those Preparations and ExamCompounds (Vd),(V-f)
and (V-k). This r d o n is
plcs.
carried out usually at temperature from -50' to 20' C ,
preferably -20' to 0' C ,over a period of 0.5 to 5 horn. 60
Preparation 1
(3) Compound (VI<) in which -A- in the general
17~-Imidazolylcarbonyloxy-3-methoxymethy
formula 0 represents
1,3,5-estrabiae
-c%)#To a solution of 17~-hydroxy-3-methoxymethyloxy65 1,3,5-estratriene(3.34g) in dioxane (70ml) were added
N,N-carbonyldiimidazole (4.30 g) and triethylamine
wherein n has the same meaning as defiaed in the gen(3.3 d),
and the resultant mixture was refluxed under
eral forinula 0,or its salt can be prepared according to
heating and stirring for 3 h o w . After cooling, the reacthe following synthetic route:
-
-
57
5,391,776
58
tion mixture was mixed with water and extracted with
(2H, s), 4.45 (lH, t, J=$.5Hz), 3.33 (3% s), 3.22 (2H. d,
ethyl acetate. The organic layer was washed with satuJs4.3Hz). 2.84-2.74 (2H, m),
0.76 (3H, s)
rated saline, dried over anhydrous magnesium sulfate,
and the solvent was evaporated. The residue WBSchroPreparation 5
matographed on a silica gel column (120 E), eluting with 5
l7~-[(2-Carboxyethyl)aminocarbonyloxy]-3
-methoxy hexane-ethyl acetate to give 17&imidazolylcarmethyloxy-1,3,5-estene
bonyloxy-3-metho~ethyloxy-1,3,5cstratre (3.56
g, Yield: 82%).
l7j3-[(2-Methoxycarbonylethyl)~~~bonyrethyl)aminocarbonNMR (CDCl3,B) 8.14 (lH, Q, 7.42 (IH, 2), 7.20 (lH,
d, J=8.5HZ), 7.08 (IH,s), 6.84 (lH,dd, Js7.7LO 3-methoxymcthyloxy-l,3,5cstrabrieneobtained in Preparation 3 was treated in the Same manner as in Prepara7.7Hz), 3.47 (3H, s), 286 (2H, m), 0.94 (3H, s)
tion 4 to give 17~[(2-Carboxyethyi)amhocarbonyloxPreparation 2
y]-3-methoxymethyloxy-1,3,kstratriene. Yield: 85%
17/3-Methoxycarbonyhethylaminocarbonyloxy3N?dR@MSOd-6)7.16(1H,d, J=8.5&), 7.04 (lH, t,
methoxy-methyloxy 1,3,5.estratriene
1s J=S.OHz), 6.75 (lH, dd, J=2.5%, 8.5&), 6.69 (lH,
d,
To a solution of 17~-~dazolyIcarbonyloxy-3- J=2.5Hz), 5.10(W, s), 4.47(1H, t, J=8.2HZ), 3.33 (3H,
methoxymethyloxy-l,3,4.estratrienc(1.14 g) in dioxane
s), 3.16 (2H, dt, J=S.OHz, 7.6Hz), 2.83-2.73 (2H,m),
(25 ml)were added glycine methyl ester hydrochloride
2.37
(2H, t, J=7.6%), 0.75 (3H, S)
(1.05 g), triethylamine (1.16 ml), and diazabicycloundecene (1.25 d).The resultant mixture was refluxed 20
Preparation 6
under heating and stirring for 3 days. After cooling, the
reaction mixlure was mixed with water and extracted
17&[3-Methoxymethyloxy-l,
with ethyl acetate. The organic layer was washed with
3,5cstratriene]hemisuccinate
saturated saline, dried over anhydrous magnesium SUIfa& and the solvent was evaporated. The residue was 2s To a solution of 17&hydroxy-3-methoxymethytoxy1,3,5-~stratriene(3.65 g) and succinic anhydride (6.9 g)
chromatographedon 8 silica gel column (100 g), eluting
with hexane-ethyl acetate to give 17/3-methoxycar- in toluene (54 ml) were added pyridine (9.3 ml)and
bonylmethylamin~bonyloxy-3-mcthoxymethylo~ 4dimethyl-aminopyridhe (140 mg), and the resultant
1,3,5-atrat1icne (0.77 g, Yield: 64%).
mixture was refluxed under heating for 48 hours. The
Nh4R (CDCIo, 8) 7.20 (lH, d, J=8.5%), 6.83 (lH,30
reaction mixture was washed With saturated aqueous
dd, J-8.5 Hz, 2.5 Hz), 6.77 (lH,d, Js2.5 Hz), 5.14
potassium bisulfate, and the organic layer was concen(3H, s), 4.65 (lH, dd, J 3 7 . m 7.7H2), 3.98 (2H,d,
3H), 3.47 (38 s), 2.90-2.82 (ZH, trated to give a brown oil. This was chromatographed
J=5.5Hz), 3.77 (2H,
m), 0.81 (3H, s)
on a silica gel column (300 g), elnting with chloroform35
/methanol to give 17/3-[3-methoxy-methyloxy1,3,5Preparation 3
cstratriene)hemisuccinate
(3.0
g,
Yield:
63%)
as
a
color17&[(2-Methoxycarbonylethyl)
less oil.
~ocarbonyloxy]-3-methoxyme(hyloxy1,3,5-estraNMR (CDC13, 8) 7.19 (d, lH, 8.6Hz), 6.82 (33, lH,
triene
40 8.6Hz, 2.7Hz), 6.77 (d, lH,2.7Hz), 5.14 (s, 2H), 4.71 (6
The reaction was effected in the same manner as in
lH, 7.6Hz), 3.47 (s, 3H), 2.9-2.8 (m,2I9, 2.3-1.1 (a
Preparation 2 except that &alanine methyl ester hydro13H), 0.82 (s, 3H)
chloride was used in place of glycine methyl ester hydrochloride, whereby 17~-[(2-methoxycarbonylethylRreparationS 7-12
)aminocarbonyloxy]-3-methoxymethyloxy-l,3,5-cstra- 4s
triene WBS obtained. Yield 87%.
The following compounds were prepared according
NhfR (CDClh 6) 7.20 (lH, d, J=8.5HZ), 6.82 (lH,
to Preparation 6, using other anhydrides or steroids
dd, J=2.5Hz,
6.77 (lH,d, J=2.5&), 5.14 (lH,
than uscd in Prepamtion 6.
s), 4.62 (lH, t, J=8,2Htz), 3.71 (3H, s), 3.47 ( 3 8 s),
3.47-3.40 (W,
m), 2.84 (2H, t, J=4.3Hz), 256 (2% t, H)
Preparation 7
J=6.0Hz), 0.78 (3H, S)
Preparation 4
COOH
.
17~-Carboxymethylaminocarbonyloxy-3-metho~~
thy10w*l,3,hSW&he
5s
To a soludan of 17~-mthoxycarbOn~e-
thylaminocarbonyloxy-3-metho~thyloxy-l,3,5-
estratriae (0.77 g) in methanol (SO ml)was added 2 N
aqueous potassium hydroxide (6 ml), and the resultant
mixture was stirred at room temperature for 2 hours.
The reaction mixture was mixed with ion exchange
resin @OWEX, SW-XS), neutralized, and filtered to
.
remove the ion exchange resin. The filtrate was concentrated in vacuo to give 17l3-carboxymetbylamiaocarb o n y l o x y - 2 m ~ o x ~ e t h y l o ~ - l , 3 , S ~ ~(0.65
~ e n65
e
g, Yield 88%) as an amorphous solid.
NMR (DMSOd-6, 8) 7.16 ( 1 8 d, J~8.5Hz)s 6.75
(1H, dd,J=8.5=, 2.5HZ), 6.69 (lH, d, J-2.5%), 5.10
Yield 64% NMR (CDCl3, 8) 7.90-7.83 (m, lH),
7.75-7.70(m,H),7.65-7.50(m,2H),7.14(d, lH, 8.6Hz),
6.82-6.70 (I& ux),5.12 (s w),4.92 (t, 1% 7.4Hz), 3.45
(s, 3H), 2.90-2.75 (m,2H), 245-2.10 (m,3H), 2.05-1.62
(m.H), 1.55-1.20 (m, 6H),0.85 (s, 3H)
s9
5,39 1,776
Prepamtion 8
60
Preparation 12
Yield: 60% NMR (CDCl3, 8) 5.38 (d, lH, 4.8Hz),
(d* lH, 8.6m),
6*8115 4.7-4.5 (m,K), 2.70-2.50 (m, 4H), 2.50-2.22 (m,3H),
(dd, lH, 2.7% 8.6&), 6.76 (d, 1H,2 . 7 W 6.47 (4 1Ht
2.15-1.75 (m,6H), 1.70-0.95 (m,11H), 1.02,0.86 (each:
12.9Hz), 6.38 (d, lH, 12.9Hz), 5.13 (x, 2H), 4.82 (t, lH, s, 3 ~ )
7.4&), 3.45 (s, 3H), 2.90-2.78 (W,m), 2.88-2.78 (m,
Preparation 13
2H), 2.38-2.10 (m,3H), 1.98-1.25 (m,lOH), 0.85 (s,3H).
2o 17-0xo-3-(2'-carboxyethyl)aminocarbonyloxy-1,3,5Preparation 2
estratriene
To
a solution of succinic acid monobenzyl ester (571
0
mg) in toluene (5.7 ml) was added triethylamine (458
ml),and the resultant mixture was chilled at 0' C., g m d O
A
O
H
II
25 ually mixed with DPPA (620 pl), and stirred at 100' C.
0
for 30 minutes. After confirming that generation of gas
was finished, the reaction mixture was chilled at 0' C.,
mixed &@wise with a solution of 3-bydroxy-17-0~01,3,S-estratriene(740 mg)in toluene (7.4 ml), and stirred
30 at 100' C. for 1 hour. The reaction mixture was allowed
0
to cool to room temperature, diluted with chloroform,
washed with water, and then concentrated. The residue
Yield: 74% NMR (cm13,8) 5.71 (s, lH), 4.60 (t, lH, was chromatographed on a silica gel column, eluting
7.6W, 2.70-2.50 (mt 4H), 2.50-0.93 (m,19H). 1.16.0.80
with chloroform/methanol to give 343 mg of colorless
(each: s, 3H).
35 crystals.
To a solution of said product in ethanol (3.4 ml)/tetPreparation 10
rahydmfurau(3.4 ml)w89 added 10% palladium carbon
(34 mg), and the resultant mixture was hydrogenated
0
for 1 hour. The reaction mixture was filtered with CelII
ite, and the filtrate was concentrated to give 174x0-3H
O
(2'-carboxyethyl)aminocarbonyloxy-1,3,5-estratriene
I
0
(231 mg) as colorless crystals.Yield: 29%.
N M R (CDCl3) 7.66 @r-& IH)7.23 (d, lH,85Hz),
6.90-6.80 (m,3H), 5.62 (t, IH,6.2Hz), 3.W3.40 (m,
45 WT),2.95-2.80(m,2H),2.15-2.60(m,2H),2.50-1.90(m,
nr),1.70-1.30 (m,6H), 0.88 (s, 3H).
yield: 25q70M M R (cDcls
&
&
Preparation 14
[1713-(3-Methoxymethyloxy-e~yloxy-1,3,~cstratrieny~oxy)carYield: 36 % N M R (CDCl3, 6) 460 (t, 1H, 7.6=),
~nYl-ome~Yl~~~ruboxamidomethylenolbis2.70-2.58 (m,H),2.20-1.90 (m,6H), 1.75-0.70 (m,16H),
phonate)ktraethyl
0.99,0.78 (each: S, 3H).
To
a solution of 17B-carboxymethylaminocarPreparation 11
bonyloxy-3-methoxymcthylo1ty-l,3,5-cstrat1ie~(0.65
55 g) in tetrahydrofuran (20 ml) obtained in Preparation 4
was added N-methyhorpholine (0.19 ml), and the resultant mixturewas chllled at 10' C, g~aduallyadded
with isobutyl chloroformate (0.22 ml), and stirred at
-10' C. for 15 minutes. A solution of tetraethyl
60 amhomethylenebispbosphoaate (0.47 g) in tetrahydrofuran (5 ml) was added at 10' C.to the mixture, which
was stirred for 30 minutes. The reaction mixture was
.+
O
H 0
0
diluted with ethyl acetate, and washed with water and
saturated saline. The organic layer was dried over anhy65 drous magnesium suJfatc,and mncentratd to remove
the solvent. The residue was chromatographed on a
Yield 70% NMR (CDClk 8) 5.35 (d, lH, 4.3%).
silica gel column (30 g), eluting with chlorofordme4.66450 {m,H),2.70-2.44 (m,5H). 2.40-0.95 (m,19H),
than01 to give [17/3-(3-methoxymethyloxy-l,3,5-estra2.10,0.W, 0.60 (each: S, 3")
0
&
-
-
5,39 1,776
61
62
carbonyl-aminometbylcarboxamidomcPreparation 18
thylene]bis(ph~sphonate)tnraethyl (910 mg). Yield:
84%.
0
0
NhfR(CDCI~,8)7.19(1H,d, J=8.5HZ), 7.13(1H.d,
N
It
J=8.4&), 6.82 (IH,
dd, J=2.SHZ, 8.5&), 6.77 (IH,
d, 5
-A-:
-C-C(CH&-C-NHJ=2.5&), 5.46 (IH,J=4.9%), 5.14 (W,s), 5.04 (IH,
dt, JplOHZ, 22 &), 4.62 (IH, f J=;8.4*), 4.28 4.13
Yield: 1m%
(CDa3, a), 7.16 (d, 1H, 8.6%)
(8H,
m), 3.97
d*Jr4-9a),
3*47(%
2-84
7.13 (d, IH,IOHz), 6.79 (dd, lH,2.5Hz, 8.6Hz), 6.74 (d,
,t J=4.4%), 1.39-1.30 (ISH, m),0.81 (3H, s)
lH, 25Hz), 5.11 (5.2H), 5.00 (dt, lH, lo&, 21.8Hz),
lo 4.70 (t, lH, 7.8Hz), 4.30-4.25 (m, 8H), 3.44 (s, 3H),
Preparations 15-28
2.90-2.76 (m,2H) 2.40-2.05 (m, 3H), 1.95-1.10 (m,
=bWlic a d de*atiVCS already
usiag
low, 1.47, 1.6 .(at&s, each 3H), 1.32 (t, 12H,
known or the steroidal carbox~licacid derivatives ob5=7.1=), 0.83 (st 3 ~ )
tained above as starting materials, the reactions were
carried out in the same manner as in Preparation 14 to 15
Preparation 19
give the compounds having the following general fortrienyloxy)
3w~ -(m
m,
0
mulr.
['
P(%ZH5)2
O-A-CH
CH@CH20
]
-A-:
Yield: 13% NMR (CDCl3,S) 8.96 (d, lH, 9.8Hz) 7.14
(d, lH, 8.6)1[2), 6.79 (dd, lH, 8.6Hz, 2.6Hz), 6.73 (d, lH,
26Hz). 5.12 (at, lH, lo&, 21.8Hz). 5.10 (5.2H), 4.73 (t,
IH,7.8Hz), 4.30-4.10 (m,8H), 3.43 (s, 3H), 2.90-2.75
25 (m, H) 2.40-1.20 (m, 17)F), 1.32 (t, 12H, 7.1Hz),
0.90-0.75 (m, 9H).
Preparation 20
Preparation I5
0
-A-:
O
II
P
U
-C-C(CZHS)~-C-NH-
0
0
30
II
-C!-NIX(CH~~-C-hW-A-:
?T-
Yield 71% NMR (CDcl3,S) 7.19 (lH, d, J=B.SHz),
6.82 (16dd, Jp25NZ, 8.SHz), 6.77 (lH, d, J=25Hz), 3s
6.42 (1Hp 4 J=10*0=), 5-44 (1H, J 4 - 5 W v 5.14
Yield: 38% NMR (CDQ, a), 7.17 (4 1H, 8.6&),
(2H~
1', '*03 (lH* dt*
21-m)* 4*61(IH,
6.80 (dd, lH,2.5Hz, 8.6Hz), 6.52 (d, lH, lOHz), 5.12 (s,
J=8.3Hz)9
4*24413 (m 8*b 3454-3*40 (2H*m), 3.47
2H), 4.97 (dt, lH, 10% 21.8Hz), 4.71 (t, lH, 7.8Hz),
6 9 3% 2&? (2H, *,Jp4*3Hz)*
(%f Ja5.6Hz)9
4.15-4.05 (m, 8H), 3.45 (s, 3H), 2.90-2.78 (m, 2H),
1-35(at, J=7-1m), 1-34(6H, Ja7.1*),
0.77 (3K
2.40-2.05 (m,H), 2.00-1.20 (a 14H), 1.32 (t, 12H,
s).
J=7.1&), 0.85 (& 3H)
Prejmration 16
Preparation 21
Jplo-ow
-A-:
0
0
-C-CHz-~NHII
II
45
Yield: 6.3% NMR (CDCh 6), 7.62 (d, lH,
J=lO.OHz), 7.19 (4 1 6 J=S.SHz), 6.83 (dd, lH,
J=2.5"2, 8 . i 6.77 (d, lH, J=ZSHz), 5.14 (8, w),
5.05 @t, lH, J=21,5Hz, IO.OHz), 4.75 (t, lH, 8.8&),
4.25416 (m,8H), 3.47 (s,3H), 3.40 (s, 2H) 2.84 (t, 2%
J=4.5Hz), 2.29-2.20 (m,3H), 1.91-1.31 (m,22H), 0.83
55
6,3H)
Preparation 17
0
-A-:
.
II
-A-:
-E-@l-m-
Yield: 33% NMR (CDCh 6). 8.11,7.86 (each: d, 2H,
8.4Hz), 7.18 (d, lH, 8.6Hz), 6.9-6.7 (m,3H), 5.23 (dt,
lH, 10% 21,8HZ), 5.13 (s, 2H), 4.93 (f 1H, 7.8Hz),
4.40-4.10 (m,12H), 3.45 (s, 3H), 290-2.80 (m,2H),
2.5-2.2 (m,3H), 2.0-1.2 (m,22H), 0.95 (s, 3H).
Preparation 22
0
II
-C-(CH&-C-NH-
0
60
n
II
yNH-
Yield 97% NMR (CDCl3, a), 7.20 (4 la J=8.6Hz),
6.83 (dd, lH,J=2.5Hz, 8.6Hz), 6.78 (d, lH, J=2.5Hz),
6.36 (d, lH,J=IO.O=), 5.15 (s, w), 5.03 (dt, 1%
J=lO.OHZ, 21.8a) 4.69 (t, lH, J=7.8&), 4.24-4.14 65
(m,8H) 3.47 (s, 3H) 2.80-2.83 (m,2H), 2.71-2.59 (m,
Yield 53% N M R (CDClh a), 7.95-7.90 (m lH),
4H), 2.31-2.16 (m, H), 1.90-1.09 (m, lOH), 1.34 (t, lW,
7.80 (dd, lH,
7.60-7.45 (m,3H), 7.18 (d, lH, $.a),
J=7.1Hz), 0.82 (s, 3H)
2.5Hz, 8.6Hz), 6.76 (d, IH,2.5Hz), 6.64 (d, lH,lo=),
5,391,776
63
5.21 (dt, lH, IOHz, 21.8Hz), 5.12 (s, 3H), 4.86 (t, lH,
7.8Hz), 4.404.10 (m,SH), 3.45 (s, 3H). 2.90-2.80 (2H,
m), 2.40-2.10 (m, 3H), 2.0-1.7 (m, 22H), 0.91 (s, 3H)
Preparation 23
64
Preparation 26
e
5
x-0-:
-A-:
- E o i - m -
0
IO
Yield: 67 96 NMR (CDCl3, 6), 6.25 (d, IH, lo%),
4.99 (dt, lH, IO=, 21.8Hz), 4.58 (t, lH, 7.6Hz),
29% NMR (CDC13s 7*13 (" lH, 8'6Hz)' 15 4.25-4.10 (m,8H), 2.70-2.50 (m, 4H), 2.40-1.90 (m,6H),
6-7qdd*
lH>27*
8.6Hz), 6.33 (a, Om5H, 9.m)~
6-21
2.80-0.70 (m,16H), 0.99,0.78 (each s, 3H), 1.34 (t, 12H,
(d, lH, 9.2Hz), 5.08 (s, 2H), 4.95 (dt, lH, IOHz, 21.6Hz),
6.8Hz)
4.64 (t, lH, 7.6&), 4.3-4.0 (m, 8H), 3.41 (s, 3H), 2.9-2.7
Preparation 27
(m,2H), 2.6-2.4 (m, lH), 2.3-1.1 (m, 20H). 1.4-1.2 (m, 2o
12"). 0.76 (s, 3H)
Preparation 24
-A-:
25
0
0
II
II
-C-CH=CH-C-NH-
x-0-:
Yield 48% I W R ( c D c 1 3 , a), 9.55 (d, lH, 9.9Hz), 3o
7.17 (d, IH,8.6Hz), 6.81 (dd, IH,2.7Hz, 8.6HZ), 6.75 (d,
Yield: 74% NhfR (CDC13, 6), 5.35 (d, 1& 4.3Hz),
4.5-4.7 (m,lH), 2.70-2.20 (m,5H), 2.35-0.95 (m,19H),
lH, 2.7Hz), 6.32 (d, lH,13.2Hz), 6.22 (d, lH, 13.2&),
( each st
2*10p0'99*
5.12 (s, 2H), 5.09 (dt, lH,21.6Hz, lo&), 4.75 (t, lH,
Preparation 28
7.6Hz), 4.3-4.1 (xu,SH), 3.45 (s, 3H), 2.9-2.7 (m,2I-9, 35
2.4-1.2 (m, 13H), 1.4-1.2 (m, 12H). 0.82 (s, 3H)
3m
In the same m e r as h heparadons 16-26, the
compounds having the following general formula were
,,
x-0-:
obtained.
'0
0
II
0
II
X--O--C-(CH&-C-NHCH
['
P(0CzHs)z
e
0
Preparations 25-28
]
45
~
Yield: 65% NMR (CDC13, S), 5.38 (d, lN, 4.78Hz),
4.7-4.5 (m, H), 2.7-2.5 (m, 4H), 2.50-0.95 (m, 17H),
1.02, 0.86 (each s, 3H)
Preparation 29
I2-[3'-(17'13-Hydroxy-l',3',5'~etratrienen 1oxy)carbon~lamlno]ethy~~~~domethyl~e~~~~~~
phonate)tetraethyl
Using
!7~~~3-[2'-(Carb6xy)ethy~~~~nyl55 ]oxy-l,3,5cstratriene(231 mg), the reaction was carried
out in the same manner BS in Prmaration 8 to give light
yellow syrup (402 mg). TOa solition of this p;oductin
methanol (6.0 ml) was added sodfum borohydride (34
me) at 0' C under stirring, and the d t a n t mixture
was stirred for 30 minuttS. The mixture was poured into
x-0-:
0m
m saturated
aqueous ammonium chloride (50 ml) and
shaken with chloroform. T h e organic layer was concenYield: 84% NMR (CDCl3, a), 6.40 (d, 1% lo&),
trated, and the residue was chromatographedon a silica
5.68 (d, 1H,0.8Hz), 4.98 (dt, 1 8 lo&, 21.8&), 4.55
gel column, eluting with methanoUwatm to give the
( dd, IH, 7.6Hz, 9.OHz), 4.3-4.1 (m, 8H), 2.7-2.5 (m, 65 titled compound (242 mg). Yield 60%.
NTdR (CDc13, 6) 7.21 (d, lH,8.4HZ). 6.81 (dd, lH,
4H), 2.5-0.9 (m, 19H), 1.29 (t, 12% 7.1Hz), 1.15 (s, 3H),
2.0Hz, 10.4I-I~). 6.77 (d, lH, 2.0Hz), 6.02 (t, 1H,6.2Hz),
0.79 (s, 3H)
5.04 (dt, IH, lO.lHz, 21.8Hz), 4.3-4.1 (m,SH), 3.69 (t,
Preparation 25
K
,
H, S.lHZ), 3.62-3.50
5,391,776
65
2H), 2.85-2.70 (m, 2H),
2.60-2.50 (m,2K), 2.35-1.10 (m, 13H). f .31 (t, 12H),
0.73 (5.3H)
66
residue was chromatographedon a silica gel column to
give the titled compound (82.4 mg).
- "R,7.20 (d, lH,8.6HZ). 6.80-6.65 (m,2H), 4.97
(dt, IH,lOHz, 21.8Hz), 4.63 (t, lH, 8.3Hz), 4.3-4.0 (m.
Preparation 30-32
5 8H), 2.90-2.60 (m,4H), 2.3-2.1 (m,2H), 2.01 (s, 3H),
The reaction was carried out in the m e manner 119 in
1.90-1.20 (m,27H), 0.77 (s, 3H)
Prepnration 29 to give the compounds having the folPreparation 34
lowing general formula.
(DI,
{2-[3'-(17'~-Cyclohexylcarboxy-l',3',5'-estratrieny1ox-
O-A-CH
OH
.
h 1h
l P ( w ~ tJz
y)carbonyl]ethylcarboxamidomethylene}~~~h~ph0nate)tetraethyl
me reaction was canid out in the m e manner BS in
Preparation 33 to give the titled compound.
15
NMR, 7.22 (d, W , 8,6Hz), 6.79 (dd, IH, 2.4Hz,
8.3hz), 6.60-6.40 (m,IH), 5.02 (dt, 1% l o a , 21.8Hz),
4.66 (t, IH, 8.3Hz), 4.30-4.00 (m,8H), 2.90-2.75 (m,
4H), 2.70-2.60 (a, 2H), 2.40-1.20 (m,32H), 0.80 (s, 3II)
20
Preparation 35
[17~-(3-Hydroxy-1,3,s-eStratrienyloxy)~nylPreparation 30
m e t h y l u r e i a - m e ~ y l e n e ~ ~ ~ tetraethyl
~pho~~)
To
a
solution
of
I7~-[3-methoxymethyloxy-1,3,50
0
75 estratriene]hemisuccinate in toluene (2.5 ml) were
II
li
--A-:
-C-(CHdpC-NRadded triethylamine (127 pl) and DPPA (230 pl), and
the resultant mixture was stirred at loo'C. for 30 minfield: 52% NMR ( C ~ Za),, 7.24 (4 I& 8 . 6 W
utes and poured into chilled water (30 d).
Afw gener1@ 2.6Hz, 8 . 4 W 6-76(4
6-81(a,
2 . 5 W 6.28 (4
ation of gas was finished aminomcthylcaebispaosphon8.4&), 4-92 (at. 1
8 10I-h 21.8W, 4.25-4-05 (m, 8W, 30 ate in tetrahydrofuran (235 mg) were added and the
3.71 (t,la,8.3)Iz), 3.9-3.7 (m,
4H), 2.66 (t. W , 5.95Hz),
resultant mixture was stirred at 100' C. for 30 minutes.
2.40-1.10 (m, 25H), 0.75 (5.3")
The mixture was shaken with chloroform (10 ml x2),
and the organic layer was dried over magnesium sulfate
Preparation 31
and concentrated. The residue was chromatographed
35 on a silica gel column, eluting with metMoI/water to
0
0
&e the titled compound (145 mg).
II
II
-A-:
-C-NH-(C3X&-C--NHYield 33%. NMR,7.16 (d, IH,8.6Hz), 6.80(dd, lH,
2.5Hz, 8.6Hz), 6.75 (d, lH,2.5Hz), 5,954.70 (m,2H),
YWd: 60% NMR(CDC12,a), 7.90(d, 9.9&), 7.06(d,
5.12 (S, 2H), 4.90 (dt, 1& lo&, 21.8&), 4.66 (t, lH,
1H,8.6HZ), 6.78-6.60(m, w),6.29(t, lH,5.3IIz), 4.98
8.3Hz), 4.30-4.10 (m, SH), 3.45 (s, 3H), 2.90-2.80 (m,
(dt, lH, lOHz,22.1&),4.23.95(m, 8H), 3.50-3.30(ra,
ZH), 2.51 (t, 2H,6.2&), 2.40-2.10 (m, 3H), 1.90-1.10
2H) , 2.8-2.6 (m,2H), 2.50-1.10 (m,lSH), 1.21 (t-Ue,
(m,22H) 0.78 (s, 3H)
12H), 0.73 (s, 3")
Preparation 36
45
Preparation 32
Tetraethyl 2-carboxyethyl-1, 1-bisphosphonate
0
0
To a solution of tetramethylme diphosphonatc (500
pl) in tetrahydrofuran ( 5.0 ml ) was added sodium
II
II
-A-:
-C-C&-C-NHborohydride (161 mg) under ice cooliig, and the resul50 tant mixture was stirred for 20 minutes. M e r ~onfumYield: 25% NMR (-3,
a), 7.62 (d, lO.OHZ), 7.25 ing that generation of gas was finished, 1-brornoacetic
(d, 1K 8.m~
) 6.90-6.70 (m, 2% 5.05 (dt, 1K 9.8&
acid benzyl ester (319 pl) was added. The reaction mix21.6Hz), 4 . 3 4 1 (aSH), 3.70 (4 1% 8.3&), 3.62 (%
ture was allowed toreturn to room temperature, stirred
w),2.90-2.70 (m,1H), 2.40-1.0 (mt 25H), 0.75 (S, 3H)
for 30 minutes, and poured into sanuated aqueouS am55 monium chloride. The mixture was shaken with chloroPreparation 33
form (1 5.0 ml x2), and the organic layer was mncen{ 2 [ 3 ' - ( 1 7 ' ~ - A a t o x y - l ' ~ ~ ~ ~ ~ ~ y l o xw.
~ ~ ~The
n Yresidue
l - was chromatographodon a silica gel
]ethylcorbo~dometh~lene~~amiaomethylene)bis@hosphonat
(20 g), elutiag with doto-fodmethanol to
ethyl
give a colorless oil (660mg). To a solution of this prodTo a solution of ~2-[3'-(17'~-hydrox~l',3'J'-esrra. uct in ethanol (10 ml) was added 10% pauadiumcarbon
t r i e n y l o ~ ~ ~ ~ ~ y l ~ ~ ~ d (60
o mg),
m eandt the
h mhtwe
y
was hydtogenated at room
phosphonate)teW&hyl fn methylene chloride (2 ml)
temperatwe for 2 hours. The reaction mixture was fiwere added 4-dimethylaminopyriclhe (23.0 mg) and
tered with &lite to give the titled compound (503 mg)
acetic anhydride (16.7 ml), and the resultant mixture 65 asacolorlesssyrup. Yield: 72%.
was stirred at room temperature for 1hour and poured
N M R (CDCls, S), 7.1-6.0 @r-S,
4.224.05 (m,
intochilledwater. Themixturewssshakcnwithchloro- SH), 2.98-3.3 (m, IH), 2.79 (dt, ZH, 6.IHz, 16.1%).
form, and the organic layer was concentrated. The
k30 (t, 12H)
lw,
5,391,776
67
Preparation 37
3-Methoxymethyloxy-17&(3',3'-diphosphonopropiony-~oxy)-1,3,5-cstratriatrimetetraethyl
To a solution of 3-mcthoxymethyloxy- 17s-hydroxy-
68
EXAMPLE 2
{2-[ 17'&(3'-Hydroxy- 1',3',5'-estratrienyloxy)car-
5
bonylamino]cthylcatboxamidomethylene}bis~h~phonic acid) (Compound No. 2 in Table 1)
l,3,5-estratriene (212 mg), bisphosphonic a i d mtm
obtained in Preparation 35, aad &%iethyfiiopyri&e (107 mg) in methylme chloride (2.3 d)wm added
using {2-[17'B-(3'-methoxpethyloxy-l',3',5'-eslra~ ~ ~ ~ o x ~ ~ ~ ~ l ~ i n o ] ~ h y l ~ ~ ~ i d o ~
thYlme}bNkme-thYl P h o s h ~ ~ *Ob&&
)
in prepad ~ y c l o h e x y l - ~ ~ d e(lw me), and the resultant ,o ration 7, the reaction was carried out i
n the same manner as in Fhmple 1 to give {2-[17'B-(3'-hy&oxymixtute
stirred at room temptntuse '
or l2 hours' 1',3',5'-estratricn Ioxy) carbonylamino]-ethyIcsrboxThe
mixture was
into
and
amidomcthylwe~ba(phosphonic
acid). Yield: 45%.
shrlren with mtthylae chloride (lo dx2)s
N M R (DMSOd-6, 8) 8.09 (IH, d, J=lO.OHz), 7.02
Or@'
layer was concenhated to
The residue
(lH, d, J=8.5Hz), 6.90 ( lH, t, J15.0Hz ), 6.48 ( lH,
was dilutedwith
(lo ml)@ and so*utionwIu dd, J12.5&, 8.5Hz), 6.42 (lH, d, J=2.5Hz), 4.54 (lH,
with cotton and concmtmtcd to sivethe title
dt, J==IO.OHz, 21.8Hz), 4.74 (lH, t, J=8.3&),
compound (367 mg).
3.21-3.12 (2H, m), 2.75-2.60 (2H, rn), 2.38 (2H, t,
Yield: 94%.
7.17 (d, 1K 8.6Hz), 6.80 (dd, lH,
J16.9&),
0.74 (314, s)
2.6Hz, 8.6Hz), &74(d, lH, 26Hz), 5.12 (s, lH, 2H), 4.67 2o
EXAMPLE 3
(dd, lH, 7.5Hz, 9.1Hz), 4.25-4.05 (m,SH), 3.45 (s, 3H),
3.20-2.75 (m, SEI), 2.3-21 (81% 3H), 2.00-1.20 (m, lOH),
[17~~(3'-Hy&oxy-l',3',5'cstratrienyloxy)carbbnyl1.31 (t-like, 12H), 0.81 (s, 3H)
me~yl-catboxamiaomethylenelbis(phospho~c
acid)
(Compound No. 25 in Table 1)
Preparaiion 38
25
TO a solution of [17~~3-methoxymethyloxy-1.3,5~ 7 P - H ~ d r o x ~ - 3 ~ 3 ' , 3 ' d ~ h ~ ~ h o n o ~ r o p i o n ~ l o xestratrienyloxy)carbonylmethylcarboxamid.
~~-l,3,5artratrime tetraethyl
thylene]bie(tetraethyl-phosphonate) (350 mg) in acetonitrile (7 ml) chilled at -20' C. WIS added trimethylusing
17-oxo-3-(3',3'-diphropi~yloxy)
1,3,5-estntriene (270 mg), the reaction was carried out
say1 iodide (0.40 ml), and the resultant mixture was
stirred at -W C. for 30 minutes. me reaction mixture
in be
Bs h fiwmtion 36
dve17-0xe
3'aiphosphono.propionyloxy) 1,3,5+&&ime
was diluttd
methylene chbl'ide (m
A) and mixed
343:
(418 mg, Yield: 100%). To a solution of this product in
~
~
p
~
~
~
diethyl ether (4.3 ml) was added 0.425 M Solpdon of
driedto give the titled compo,,,,d (145ms) as
&(B&h in diethyl ether (2.8 @s md b e W W b I t 35 a co~or~ess
yidd: 54%.
mirhue was stirred at O' ' for
pourad
NMR (D20,8) 7.01 (d, 1H, J=8.3Hz), 6.48 (d, IH,
into chilled
wassha\rm. with chloroJ=4.3Hz), 6.42 (8, IH), 4.64 (t, 1% J=8.8Hz), 3.38 (s,
(15 mlx2)#
and
conaor
2H), 2.85-2.60 (m,2H), 232-1.09 (m, 13H), 0.76 (s, 3H)
mtcd.The residua was chromatographed on a silica gel
EXAMPLE 4
column, eluting WIth chlorofonn/methanol to give the 4
titled Compound (275 mg).
{l-[ 17'&(3'-Hydr0xy-l',3',S'~tratrienyloxy)carb0nyl]Yield 65%. N M R (CDCrs, a), 7.23 (d, lH,8.45HZ).
l-methylethyl~x~domethyl~ne~b~ho~h~ni~
6.82 (dd, IH,2.4Hz, 8.45-h
6.76 (d, lH, 2.4&),
acid) and its sodium salt (Compound No.32 in Table 1)
4.23-4.03 (m, SH), 3.67 (t, lH, 8.15Hz), 3.25-2.90 (m,
To a solution of {1-[17~-(3'-methoxythyloxy)3H), 2-85-2.70 (m, 2m9 2**1*m 6%13H), 1.30 ( t - l k 45 1 ' , 3 ' , 5 ' ~ ~ ~ ~ ~~ ~] n0 yx] J ~ 1 - m e t h y l & y l ~ 12H), 0.72 (a 3H)
b o M m M o . m t t h y l e ( t e ~ ~ t h yphoshponate)
l
(230
mg) in acetonitrile (23 ml) chilled at -20' C. was
EXAMPLE 1
added trimethylsilyl iodide (0.273 mi), and the resultant
[17s-(3-Hydroxy-l,3,5~~cn~loxy)car M mixture w89 stimd for 30 minutts. The reaction mixbon~lamiwmethylcarboxamido~th~~elbis@ ture was diluted with methylene chloride (3.0 ml),and
phonic acid) (Compound No. 1 in Table 1)
water (0.5 ml) was gradually dropwise added. The reA solution of [1719~methox~e~y(3-mclhoxymethyloxy-1,3,5-estras u l ~ PrbPitab
t
WM
washed
ethyl
tate to give the titled compound (100 mg) as colorless
trimy1oxy)cslbonylaminomethylcarboddome
thykne]bis(phollphonate)tetraethyl (Obtainsd in Prepa- 55
this product in methanol (3.0 dlwas
ration 6)(xromg) ia acetollItriIe(6 d,w1L8stirred
at
dropadded 8 mlution of^^ acetate (43 mg) in
-20' C. trkneth~1
d d dh p and
methanol (1.0
and the resultant precipitate was
*ed With W8tu (l ml)*
stirred for3o *utCS md
mkred a d washed wfth methanol/w.ter to give SOThe
@Mate
was
& @ dum salt ofthe titled compouod (59 mg).
dbdUed w*ter well, ad
dried to
[17&(3-hydroxYNMR @ 2 0 , 8), 7.04 (d, lH, 8.2Hz), 6.48 (d, lH,
1 , 3 , 5 c s t t a t r i e n y l o x y ~ n y ~ m ~ y I ~ ~ x -8.2€h), 6.45 (8, lH), 4.07 (t, lH, 18.6H.z). 2.70-2.50 (m,
~ d o m e t h y l ~ Ms(phOsPh0~C
e]
add) (170 mg) d- 2H), 2.10-1.O(m, 13H), 1.34,1.32,0.63 (each s, each 3H)
orless solid. Yield 73%.
EXAMPLE 5-12
NMR (DMsOa-6, 6) 7.01 (I€& d, Jr8.5Hz)s 6.48 65
(lH, dd, Y =2.5Hz, 8.5Hz), 6.41 (lH,
d, J=Z.nrZ), 4.48
The reaction was carried out in the same manner as in
(lH, t, J=4.9Hz), 3.93 (lH, dt, J=1.6Hz, 18.8Hz), 3.74
Example 4 to give sodium salts of the compounds of the
(2H, d, J-4.9&),
2.68 (W,d, J=4.4Kz), 0.77 (3H, S)
foUoWing general formula
m,
-
mg
:Eti;::;
a,
5,391,776
69
70
EXAMPLE 9
(Cornpod No.31'h Table 1)
Yield: 93% Nh4R @20, a), 7.80-7.50 (m, 3H),
7.50-7.30(m, H), 7.09(d, lH,8.2Hz), 7.60-7.40(m, 2H),
I5 4.73 (t, lH, 7.4Hz). 4.53 (t, l W , 19.8Hz), 2.70-2.50 (m,
2H), 2.30-1.10 (m, 13H), 0.71 (s, 3H)
EXAMPLE 5
-A-:
0
fl
EXAMPLE 10
1
I
-C-(CH2)2-C+NH-
(Compound No.26 in Table 1)
20
Yield 39% NMR 0 2 0 , a), 7.05 (d, lH, 8.2Hz), 6.52
(d, lH,-8.2Nz), 6.47 (s, IH), 4.23 (t, lH, 19.8Hz),
2.69-2.47 (m,6H), 2.11- 0.98 (m,13H), 0.63 (s, 3HJ
25
EXAMPLE 6
0
-A-:
0
II
It
-C-N€I-(CzH$h-C-N€I(Comm
.and No.33 in Tdlc 1)
EXAMPLE 7
40
I
(Compound No. 39 in Tabk 1)
Yield 90% NMR @20, a), 7.06 (d, lH, 7.6Hz).
6.70-6.50(m, 2H), 4.35 (d, lH,18.6Hz), 2.8-2.6(m, 2H),
2.40-1.00 (m, 17H), 0.67 (s, 3H)
EXAMPLE 11
-A-:
Fi
13H), 0.66 (s, 3H)
EXAMPLE 12
I1
45
(Compomd No. 46 in T&Ie 1)
(Compound No. 35 in Table I)
Yield 77% M M R @P,a), 6.91 (d, 1% 7.5&),
6.52-6.33 (m,w),4 , s (t, lH, 19.2Hz), 3.66 (1; 1H,
7.4Hz), 265-2.50 (m,2H), 2.10-0.90 (m,23H), 0.57 (s,
3H)
ii
-C-CH=CH-C-NH(Compmd No. 42 in Tablo I)
Yield: 46% "MR @20,8), 7.2-7.1 (m,2H), 6.7-6.4
(m,3H), 4.4-4.0 (m,lH), 2.7-2.5 (m,2H), 2.2-1.0 (m,
0
V '
--"-CJJi
30
Yield 78% NMR @zO, 6), 6.89 (d, lH, 7.5Hz),
6.526.44 (m,2H), 4.40 (t, lH, 19.5Hz), 2.70-2.50 (m,
2H), 2.10-0.90 (m,lnr), 0.85-0.62 (m,6H), 0.50 (s, 3H)
0
-A-:
Yield 82% NMR @20, 6), 7.18 (d, lH,7.6Hz),
6.74.4(m,ZW,4.23(t, lH,18.6&),3.52(t, lH,7,8Hz),
2*80-2.60 (m*6H)*
22-0*9 (a 13H),OS3 6~3H)
EXAMPLES 13-15
In the s ~ m cmanner as in Examples 5-12, the com-
EXAMPLE 8
-A-:
55 pounds of the following general formula were obtained.
-I@i-m---
0-A-CH
(Compotand Na 36 Ia T~bk1)
Yield 37% N M R ' W , 6) 7.89, 7.72 (each d, each
2H, each 8.4Hz), 6.94 (d, lW, 8.6Hz), 6.606..30 (m, w),65
4.50 (t-like), 1H, with bo), 3.90-3.68 (m, lH),
2.60-2.43 (m, w). 220-1.00 (m, 13H), 0.70 (s, 3H)
OH
[
!(0%l2
5,39 1,776
71
72
EXAMPLE 13
0
-A-:
EXAMPLE 17
0
II
II
-C--NH-(CHZ)I-C--NH(Compound No. 58 in Table 1)
5
Yield: 84% N M R (DzO, S), 7.24 (d, lN, 8.2Hz),
6.90-6.70 (m,2H), 4.33 (t, lH, 18.6Hz), 3.60 (t, lH, lo
7.4&), 3.43-3.30 (m, ZH), 2.80-2.70 (m, ZH), 2.60-2.40
(m,2H). 2.30-1.10 (m, 13H), 0.62 (s, 3H)
(t,
0
0
n
0
(Campound No. 109 in Table 1)
EXAMPLE 14
Yield: 95% NMR @zO, ti), 4.46 (t, IH,7.6&), 4.22
lH,lOHz), 2.6-0.6 (m,22H), 0.65 (s,6H)
It
-C-(CHh-C-NH(Compolmd No. 67 ia Table I)
-A-:
&
x-0-:
EXAMPLE 18
20
Yidd 87% N M R 0320, 8), 7.18 (d, IN, 8.4Hz),
6.70-6.60 (m,2H), 4.23 (t, IS,18.6Hz), 3.51 (t, 1%
7.SHz), 2.80-2.55 (m,6H), 2.20-0.95 (m,13H), 0.53 ( s , 25
o%c/Q1’
3w
EXAMPLE 15
30
0
t7
II
-C-Cli2-C-NH-
X-0--r
\
&
0
(Compound No. 125 In Table I)
-A-:
(Compouod No.66 in Table 1)
Yield: 93% N M R @20, a), 5.32 (s-like, IH), 4.5-4.3
Yidd: 88% N M R (DZO, S), 7.26 (d, IH,8.3%). 35 (m, lH), 4.25 (t, lR, 22.5Hz). 2.60.9 (m, 24H). 2.21,
6-85-6-75 (mv W),4-33 (t, 1K 18.6*),
3-58 (4 1H,
0.89, 0.45 (each s, 3H)
7.8Hz). 3.18 (s, H), 2.80-2.70 (m,2H), 2.30-1.00 (m,
EXAMPLE 19
UH), 0.59 (9, 3H)
40
EXAMPLES 16-19
In the same manner as in Examples 5-12, the compounds of the following general formula were obtained.
45
0
0
x--o-!-(cH*-c--MKcH
P1
por
EXAMPLE 16
x--0-:
&
x-0-:
50
‘0
(Compomrd No, 117 in Table I)
Yield: 72% N M R @20, a), 5.4-5.2 (m,I@, 4.5-4.3
(m, IH), 4.21 (t, lH, 18.6Hz), 3.45 (t, lH,7.”2),
55 2.6-0.95 (m,23H), 0.86,0.55 (each s, 3H)
EXAMPLES 20,21
In the same manner as in Examples 5-12, the com60 pounds of the following general formula were obtained.
0
(Compound Na 105 in Table 1)
Yield: 90% NMR 020,S), 5-75 (s-W,
lH), 4.48 (t, 65
lH, 7.6Hz). 4.20 (t, la,20Hz), 2.6-0.6 (m,18H), 0.75
(s-like, 6H)
0
II
x-0-c-CH2-cH
]
[Ip(0H)J
5,39 1,776
73
74
EXAMPLE 23
EXAMPLE 20
X--0-:
Yield 93%
(d,
1w8‘m)*
HO
(Ompound
No. 56 in Table 1)
x-0-:
NMR (D20,S), 7.05 (d, lH, 8.7Hz), 6.50
6*47(,‘
(m,13H), 0.59 (s,3H).
2’7G2’30(m* ’H), 2’20-**oo15
2.90-0.95 (m,30H). 0.60 (s, 3X-I)
20
25
‘0
(Can@
No. 93 h Table 1)
Yield 65% NMR (DZO, a), 7.21 (d, iH, 8.mZ),
6.80-6.70 (xu, H),
3.53 (t, lH,7.4Hz), 2.9-1.0 (m, 18I-9, 30
0.72 (s, 3H)
EXAMPLES 22,23
the same maMef in
5-12, h e
pounds of the following general formula were obtained. 35
[’ 3
0
0
II
li
x-o-c-(c3I~-c-”m
(Compound No. 148 in Table 1)
(DzO,a), 7.2047.00 (m, 1H). 6.80,
Yield: 83%
6.60 (m, ZH), 4.60-4.40 (m, lH), 4.33 (t, lH, 18&),
EXAMPLE 21
x-0-:
‘0
P(0n
EXAMPLE 22
45
Experiment 1 Transitional ability of the compound of
the invention to bones
Test Method
SD male rat (body weight about 220 g) subcutaneously received a vehicle (95% corn oil and 5% benzyl
alcohol) (Group A) or 17plStradiol (250 pgAcg)
(Group B) or equimolar amount of the compound of the
present
invention
[17&(3-hydroxy-l,3,Ssstra~~enyloxy)carbony~e~yl~~~domethyleneJbis(phosphonic acid) (Compound No. 25 in Table 1:
to as “hPOund
No. 25”)
pg/kg) (Group C). The blood and the tibia were collected after 2 hours, one day, and two days in each
group. l7P-Estradiol in the plasma was measured directly by RIA method. The tibia was pulverized after
the meat chip and marrow, and then Iy0philized. The pulverized bone (150 mg) was dissolved in 5
N hydrochloric acid (1.5 ml)at room temperature, and
the resultant solution (500 pl) was mixed with 0.5 M
EDTA (500 pl), water (1 ml) and 5 N sodium hydroxide (500 pl),and allowed to stand at room temperature
for 30 lninutes to isolate 171p-estradiolof Compound
No. 25. The isolated 17fl-estradiol was extracted with 5
ml of isoamyl alcohol,and the extract was concentrated
to dryness and dissolved in 500 pl of phosphate buffer
(PH7.4) for assaying by RIA method.
Test Result
Table 2 shows a mean value of the measurements for
one group consisting of five rats with standard error.
TABLE 2
Anourlt of I7kstmdi01 in P b m d b Bone
2
Im
y
2 Days
plrmu
Bone
Plrsmr
Bone
PL;lsm*
Bone
Wd) @s/IWmcr) W m l ) @8/1mmg)
@g/lWm&
nm
COmpMlnd
dmintMItd
vthidc
-&846
4 0
<m
<20
51 f 25
<20
<20
IM*30
<M
<m
<20
<20
359f 134
<20
<W
376+;92
<20
<m
The table shows that, in Group B, 17/3g-estradiolwas
detected in p h m a until the next day a h administration but it was below detectionlimitin bone throughout
the test period. On the other hand, in Group C in which
x-0-:
o.
(Campomhi No. 147 tP T d e 1)
Compound No.25 wns administered, 17fl-estradiolwas
65 below detection limit in plasma, and it was detected in
Yield 93% N M R 0,
S), 7.20-7.00 (m, lH), bone already 2 hours after administration. The amount
6.80-6.60 (m, ZH), 4.60-4.40 (rn, lH), 4.33 (t, lH, of 17fl-cstradiol in bone increased with the lapse of
time. Acwrdingly, it was concluded that Compound
lS.lHz), 2.90-0.95 (m,19H), 200,0.60(each s, 3H)
60
5,391,776
75
76
No. 25 of the present invention has an ability of transition into bone.
Experiment 2 Bone resorption inhiiitory action
observed in ovariectomy model
5
Test Method
SI) female rats of 12 wales age which had undergone
ovarcectomy (OVX)received subcutaueouslya vehicle
(95% corn oil and 5% bmzyl alcohol) (Group 2) or 10
i7flemdioi (XI p~/kg)(O~OUP3) or eq&~lrv
momt of Compound No.25 (40 pg/kg) (Group 4) for
28 days sinincc
the next
Of owration'The rats were
subjected to autopsy on 29th day, and the weight of wet 15
uterus and the amount of a &ne volume in tibia (Can-
cellous bone volume/Tissuc volumex 100) were meaSUrCd.
-CoI"(CHR')fl+lmrn-
Boris
I
2
3
4
in which x and q each independently represent 0 or 1,
R2 represents optionally substituted vinylene group,
volume
gsvm,
%)
Shrmopelnh
Adminwmtioa or Mhiols
0VX:Mminimubnorvvehlcle
O%AdmwrmbtoaO~I~Estndiol
O%A-Ot
c4mpMIDd No. 25
am^ I W, ~ i a r p P
a < aoi
a,
in which y representsan integer of from 1 to 3, p repre20 sents 0 or 1, m represents an integer of from 0 to 5, Rl
Test Result
oroup
wherein the rings J, M L and M each independently
represent a samted, partidy saturated,or unsaturated
be independently substituted by
and which
one or more subsdtuents selected from alkyl, alkenyl,
alkynyl, halogen, alkoxy ester, acyl, hydroxy and oxo
groups, -A- represents
26
*2
30
18 f 2
3Of2
30 f 3
35
-(~Z)&~O-(CWI-
in which k and 1each represent an integer of 0-5, and
Cycle representsC347 cycloalkylenegroup, optionally
substituted phenylene group or optionally substituted
C1-q alkylene group, and 2 represents -0or
-NH-, or
'
77
5,391,776
in which k and I each represent an integer of 0-5,and
Cyclo represents C3-Q cycloalkylene group, phenylme &roup or optiodly substituted C1-c~&ylene
group, and 2 represents -NH-, or
-cQ-(chh¶in which n represents an integer of from 0 to 10.
5. A compound according to claim 4, wherein R1 is
hydrogen atom or C1-a alkyl group optionally substituted by phenyl group.
6. A compound according to claim 4, wherein R2 is
vinylene group,
4~2)~cl0--(~2)1-
in which k and 1each represent an integer of 0-5, and
Cyclo represents C347 cycloalkylme group, phenylene POUP or C1-a alkylae POUP optionally substitutcd by one or two C1-Q alkyl groups.
7. A pharmaceutical composition comprising a steroid derivative described in claim 1 or a pharmaceuti-
78
cally acceptable salt thereof together with a p h m a e u t i d l y acceptable d e r .
in OS*OPathY COmPk8. A therapeutic agent for
ing a steroid derivative described in claim 1 or a phar5 maceutically acceptable salt as an effective ingredient.
9. A compound according to claim 4, wherein
X-0represents
3-(17/3-hydroxy-l,3,5:estratrienyloxy), -Arepresents
- C C ~ - [ N H ( ~ ) , , - O ~ C ~ ~ ~ -
in which y represents 2, p represents 0, m represents 1,
and R' represents h y h g e n atom and R represents
hydrogen.
10. A compound according to claim 4, wherein
15
X-0represents
17-fl(3-hydroxy-1,3,5-estratrienyloxy), -Arepresents
*'
-Co-(R9AZ)+"-
in which X represents l, q represents 0, R2 represents
methylene or phenylene, and R represents hydrogen
atom.
25
30
35
45
55
60
65
*
*
*
*
e
I Il1111111111Ill1111111111US005578588A
11111l11111IllilIIIII11111111111Ill111l111
1191
Mattern et a].
[45]
1541 MEDICAMENT FOR INCREASING THE
TESTOSTERONE LEVEL,
[75]
Inventors: Claudia Mattern, Stamberg; Riidiger
Hacker, Hewching, both of Germany
[73] Assignee: Arrowdean Ltd., Ireland
[21] Appl. No.:
335,729
[22] PCT Filed
Apr. 30, 1993
[86] PCT No.:
PCT/DE93/00397
.....................................................
Nov. 26, 1996
References Cited
U.S. PATENT DOCUMENTS
.....................
................
...........
3,284,303 W1966 Meli
4,877,774 10/1989 Pitha et al.
5,053,403 10/1991 Orentreich et al.
167fl4
514126
514/170
5/1992 European Pat. Off.
Primary Examiner-Kimberly Jordan
Attorney, Agent, or Firm-Merchant, Gould, Smith, Edell,
Welter & Schmidt, P.A.
r571
42 14 953.3
Int. Cl?
A61K 31/56
[52] U.S. C1. .............................................
5141177; 514/182
5141177, 182
[58] Field of Search ...................
[51]
5,578,588
OTHER PUBLICATIONS
Vandcr et al., Human Physiology, 4th Ed.,1985.
Foreign Application Priority Data
.............
~561
034909181
PCT Pub. Date: Nov. 11,1993
May 6, 1992 [DE] Germany
Patent Number:
Date of Patent:
5 371 Date:
Nov. 7,1994
8 102(e) Date: Nov. 7,1994
[87] PCT Pub. No.: W093/21924
[30]
[11]
ABSTRACT
The invention concerns a drug for increasing the level of
testosteronein the human body, the drug containing at least
one testosterone precursor.
3 Claims,No Drawings
5,578,588
1
2
MEDICAMENT FOR INCREASING THE
TESTOSTERONELEVEL
control mechanism reduces the endogenic testosterone
vroduction.
The problem of the present invention is therefore to
provide a medicament for raising the testosterone level in
humans, whosc application is equivalent in its effect to the
intramuscular supply of testosterone, which avoids the
aforementioned disadvantages,requires a much lower dose
and permits a stressing of the secondary action on the central
nervous system.
According to the invention this problem is solved by a
medicamcnt having a content of at least one precursor of
testosteroneand which is preferably androstendione, progesterone or 17-a-hydroxyprogesterone.
A particularly advantageousernbodimcnt of thc invention
is characterized by a galenic preparation, which allows the
supply by per nasal application using a dosing spray and
having a preferred content of 3.5 to 15 mg of active
substance per pump thrust.
Alternatively thereto the medicament according to the
!nvention can also be in the form of a sustainedrelease drag
ee, depot form or buccal tablet for peroral administration.In
this case the preferred content per ingestion unit is 50 to 100
mg of active substance.
It has been shown that through the use of a precursor of
testosterone, which is only transformcd into the active
substance in the organism, there is a more complex reaction
of the steroid metabolism, which is more balanced and better
corresponds to the physiological conditions, so that overall
an optimum action can be obtained whilst avoiding sidc
effects.
Animal tests carried out on the guinea pig have fundamentally proved thc rapid transformation of radioactively
labelled androstendione, progesterone or 17-a-hydroxy
progesterone into testosterone.
In humans 50 to 100 mg of perorally supplied androstendione, progesterone or 17-a-hydroxy progesterone are also
rapidly transformed into testosterone. In the case of androstendione supply e.g. after only 15 minutes in the blood there
is a rlse in the overall testosteroneconcentrationfrom 40 to
83% (50 mg) or 111 to 237% (100 mg). There is an increase
in the proportion of free, biologically active testosterone,the
appearance of the concentration maximum and the path of
thc blood level in the case of a positive basic reaction reveal
clear, repeating, individual differences.
In the case of the preferred pernasal administration by
means of a dosing spray a single supply of 3.5 to 15 mg of
androstendione,progesteroneor 17-a-hydroxyprogesterone
led to testosterone level riscs in the blood of 34 to 97%. The
extent and time sequence thereof are comparable with the
results which were obtainable in the case of the peroral
supply of much higher doses or the intramuscularsupply of
testosterone propionate. Unlike in the case of peroral and
intramuscular administration, with pernasal administration
there was no significant “first-pass” metabolization of the
precursor molecule.
This led to the good- controllability of the influencing,
which could be proved by multiple administrations. The
individual reaction position is taken into account by the
regulating mechanisms of the metab,olism. An adapted
increase of the free testosterone was obtained, whose extent
and kinetics are comparable with the values obtained with
peroral administration of a ten times higher dose.
A significantlong-term cffcct was detected with multiple,
pernasal administration. Three to four days following the
final administration there was a further tcstosterone level
increase of 48 to 97% in the blood and this was maintained
for a further 6 to 7 days. This reaction is probably attribut-
This application is a 371 of PCT/DE93/00397filed Apr.
30,1993. The invention relates to a medicament for increas- 5
ing the testosterone lcvel in humans.
The main action of the steroid hormone testosterone is
the intensifying of the primary and secondary sex characters
of man, as well as the maintaining of the functions associated therewith. Apart from this main effect iestosterone has 10
a number of secondary effects, which are of great importance for the stressability and performance characteristicsof
the human organism. These include the maintaining of an
anabolic metabolic situation, the restoration of the performance of man following exhausting exercise and increasing 15
the psychophysiological stressability and stress resistance.
The action mechanisms of testostcrone have been investigated in detail. The secondary effects on the psychophysiological state have, according to the latest research, been
attributed to the presence of testosterone receptors ‘in the 20
central nervous system.
Over 90% of the testosterone in the blood is bound to
protein and the biologically active component is free testosterone representing 4 to 8% of the total concentration in
thc blood. The testosterone concentration in the blood is 25
subject to a physiological daily cycle (maximum concentration in the morning) a seasonal cycle (lowest concentration
in May) and influences by living circumstances and ageing
processes.
The overall testosterone concentration in the blood is 30
individually very stable under normal conditions. High
physical effort, long-lasting stress situations and unfavourable diet lower the blood level. With increasing age and in
particular from about 35 in man there is a reduction of the
free testosterone concentration. These changes lead to a 35
reduced, general performance, to higher time requirements
for restoring the organism after exhaustive exercise and to a
reduction of the psychophysiologicalstressability and stress
resistance. Research on physically and cyclically highly
stressed persons have revealed that a rise in the testosterone 40
level in the upper part of the individual physiological
fluctuation range leads to a cancelling out of this negative
situation and to an increase in the general per€ormance
characteristics. However, a concentration rise above the
individual, upper standard limit leads to no better therapcu- 45
tic effect and instead causes side effects.
The increase in the testosterone level in humans in the
sense of a substitution has consequently become part of
preventative and therapeutic concepts in old-age’medicine,
particularly for man. The supply conventionallytakes place 50
perorally or in an oily solution in intramuscular form and in
part as a depot preparation.
However, the following disadvantagcs arc associated
with these administration forms:
the influencing of the blood level is overall difficult to 55
control;
the individual starting situation and stress cannot be
adequately taken into account for the medication;
peroral and intramuscular supply lead to a metabolization 60
via the circulatory system liver-bile-intestine-liver
(“first-pass effect”);
this effect reduces the bioavailability and requires the
supply of higher doses with the resulting higher stressing of the metabolism;
65
the supply of higher doses can lead to an undesired rise in
the overall concentration, which via the physiological
V
3
5,578,588
able to an influencing of the control cycle for endogenic
testosterone production.
In addition, pernasal administration facilitates the transfer
into the cerebrospinal fluid and also into other tissues and
organs of the human organism. As the overcoming of the 5
blood-brain barrier is a major problem for all phmaceuticals acting on f i e central nervous system, f i e facilitated
access to the cerebrospinal fluid via thc pernasal administration represents a particular advantage of the medicament
according to the invention. Thus, for the first time it is 10
possible to influence the testosterone receptors in the brain,
which represents a novel therapeutic approach for testosterone. The subsequently described improvement of the psychophysiological performance
is probably
due to the influencing.of the central nervous system.
I5
There is an increase in the testosterondepitestosterone
quotient in the metabolite profile of urine. However, it is not
as marked in the case of pcmasal administration (3.8 to m u
14.3) a d becomes normal on the day following the s~pply,
20
whilst the testosterone level in the blood remains high.
The use of the nasal spray for 6 days (daily dose 5 to 7
mg) in physically and cyclically highly stressed persons in
4
middle age led to a shortening of the regeneration following
exhausting exercise and a better balanced metabolic situain particular be stressed that, unrequcsted, dl
tion. lt
the test persons
a higher psychophysiological
strcssabilitY and an improvcd stress resistance*
The features of the invention disclosed in the dcscription
and chims Can be essentid tO the different embodiments Of
the invention, either singly or in random subcombinations.
We claim:
1.A method for increasing thc level of testosterone in a
human comprising nasal administration of at least one
precursor,
2. The method according to claim 1, wherein the testosterone precursor is androstencdione, progesterone, 17-ahy*oxyprogesterone3 Or
3. The method according to claim 1, wherein the nasal
administration COmpriStS administration of 3.5 to about 15
mg of testosterone precursor Per pump thrust.
* * * * *

norandrodiol select 300

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