tfk building block for organic synthesis

The Three ‚Clicking‘ Amigos!
Valery Fokin
Hartmuth Kolb
fokin@scripps.edu
hckolb@scripps.edu
M.G. Finn
mgfinn@scripps.edu
H.C. Kolb, M.G. Finn, K.B.Sharpless Angew. Chem. 2001, 40, 2004.
H.C. Kolb, K.B.Sharpless Drug Discovery Today 2003, 8, 1128.
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The Dendrimer Team
QuickTime?and a TIFF (Uncompressed) decompressor are needed to see this picture.
Alina Feldman
Peng Wu
alinaf@scripps.edu
pengwu@scripps.edu
“The Trinity”
C N O
Just three simple letters,
but getting them assembled
in the ‘right’ order takes
more than a good typesetter, even
one of Benjamin Franklin’s skill.
Life’s Spartan Construction Plan
Nature is full of surprises, but few are
more striking than the contrast between
the irreducibly-complex web of functions
believed to enable ‘life’ and the
simple synthetic strategy from which
the phenomenon apparently springs.
Life, like the petrochemical industry, is based on
modular construction of oligomers
•
•
biopolymers of staggering diversity and function from < 36 modules
modules are attached one at a time, via long sequences of
INTERMOLECULAR reactions
HN H O
N
N
N H N
N
PO2 O
O
N
O 2P
O
O
O
Polynucleotides
4 D-nucleotides
H
N
N
O H NH
O
HS
H
O
O
Polypeptides
20 L-amino acids
HO
O
OH HO
O
O HO
OH
O
OH
Polysaccharides
8 D-pyranoses
Click Chemistry:
Diverse Chemical Function from a Few Good Reactions?
The time from discovery to implementation of useful
new ‘compounds’ is far too long.
Discovery
• Short,
Optimization
Delivery
modular sequences of near-perfect reactions
• Going ‘back to the future’: peeling off the layers of chemical
complexity that straight-jackets contemporary discovery efforts
• Embracing efficiency and simplicity
• Losing the losers & pursuing the winners, daily
H.C. Kolb, M.G. Finn, K.B.Sharpless Angew. Chem. 2001, 40, 2004.
H.C. Kolb, K.B.Sharpless Drug Discovery Today 2003, 8, 1128.
Why
Back to the Future?
Polaroid is gone,
and Kodak is…?
Journals and
Newspapers?
Click Chemistry’s Surprise Gifts:
1. ‘In situ’ click chemistry: hijacks enzymes to create
their own best inhibitors
2. Cu(I)-catalyzed azide-alkyne cycloaddition: a new
reaction with the alien property of being
‘unstoppable’ under the conditions found on Earth?
3. phenomenon of enhanced reactivity ‘floating on
water’
4. Ru(II)-catalyzed azide-alkyne cycloaddition
5. ✓
6. ?
Huisgen Dipolar Cycloaddition of
Azides and Alkynes
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1
N
N
N
N
1
N
N
4
"anti"
+
N
N
N
5
"syn"
R. Huisgen, in 1,3-Dipolar Cycloaddition Chemistry;
A. Padwa, Ed.; Wiley: New York, 1984; pp 1 – 176.
Cream of the Crop:
Best Blocks for Making Stable
Inter-molecular Connections
X
R
O
N
R
S
N
R
R
R
OH
Ar
H
R
R
N
N
R
N
N
H
N
R1
NH2
NH 2
N
R2
H
SH
R
R2
N
H
R1
R
O
R2
R
R
1
NH2
O
O
O
S
C
O
N
R
O
R
R
R
H
NH 2
O
R
X
C
R
X
Het
S
N
X
O
Azide and Alkyne Groups:
as good as it gets?
• built-in high energy content
• ‘invisible’ in all terrestrial environments
• but, when properly introduced, they ‘click’
forming an indestructible triazole link
Target-Guided Synthesis
Polyvalent interactions can be collectively much stronger
than the corresponding monovalent interactions.
J. Kirby, Adv. Phys. Org. Chem. 1980, 37, 183;
W. P. Jencks, Proc. Natl. Acad. Sci. USA 1981, 78, 4046.
selective
reaction
binding
enzyme
monovalent
ligands
Examples:
-hydrazone formation -- [Darryle Rideout]
-disulfide bond formation
- epoxide ring-opening
- N-alkylation
- S-alkylation
inhibition of
the enzyme
“Fragment-Based Drug Discovery”,
J. Med. Chem. (Perspective), 2004,
D.A. Erlanson, et al., 3463-3482.
Acetylcholine Esterase
key role in the central and peripheral nervous system:
hydrolysis of the neurotransmitter acetylcholine to inactive choline
O
O
AChE
O
+
N
OH
N
HO
H2N
peripheral
binding site
narrow gorge
~ 20 Å depth
N
N
NH2
N
propidium
KD = 1.1 μM
N
N
NH2
active site
tacrine
KD = 18 nM
decamethonium
KD = 460 nM
P. Taylor, Z. Radic, Annu. Rev. Pharmacol. Toxicol. 1994, 34, 281;
Z. Radic, P. Taylor, J. Biol. Chem. 2001, 7, 4622.
Acetylcholinesterase
“Fishing” for novel PAS binders
H2N
Peripheral
Anionic Site
(PAS)
R2
R1
N
N
R3
NH2
R4
Gorge
N3
NH
Active
Center
N
AChE
Secret Life of Enzymes: An
Aggressive Strategy for Drug
Discovery
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mouse
M
o
u
s
e
fly
An Idea for Improving the Drug Discovery Process?
It is an unspoken assumption of medicinal chemistry that any
new molecule made for testing is a “finished” product, the
unique endpoint of an intentional synthetic sequence.
Only then is the candidate, with its locked-in structure,
exposed to the biological target, which of course
has a fixed structure of its own.
Arranging and observing such encounters is the essence of
medicinal chemistry. Despite the epic scale on which
these encounters have been conducted, positive
‘responses’ are exceptional. The majority of all these
arranged ‘meetings’ yield no response at all!
Clearly, known drug discovery methods need to be
improved dramatically, but how?
[continued]
The existing method seems flawed in the following
respect: it is really a ‘shoehorn’ approach, not only
lacking the subtlety to achieve Cinderella-like fits, but
even clumsy in much less demanding situations.
So, we thought of highjacking enzymes, to
catalyze the final step in their own ‘inhibitor’ syntheses.
Expecting at best, a ‘whisper’ from the target, we
were surprised to receive a ‘shout’!
[continued]
‘In situ’ inhibitor synthesis :
the final inhibitor structure remains nascent,
allowing the ‘best’ final bond constructions
to selectively ‘emerge’;
as an important corollary, it seems that
the latter bonding event occurs only when
it results in a final ligand-protein complex with
substantially improved non-covalent binding interactions
(i.e. lower Kd than the fragments) -- meaning, no false
positives to date!
Molecular-scale Reaction Vessel Shepherds
ONE out of 98 Offered Combinations
R1
H2N
N
R1
NN
R1
N
N N
N
R2
N
R2
NH2
R2
syn
anti
H2N
H2N
N
H2N
NH2
N
N3
N3
N
NH2
N3
N
H2N
NH2
NH2
NH
NH2
H2N
N
N3
NH2
H2N
N
N
N3
N
N3
N
N3
NH
NH
NH
N
N
N3
NH
NH
N
NH
NH
N
N
N
N
The Trojan Horse
Orthogonal
Orthogonal
Orthogonal Reactivity
Empowers
Stealth Chemistry
Orthogonal
OrthoGON
The Target-Protein as the “Reaction Vessel”
PA6
Kd - 10-100 μM
TZ2
Kd - 10-100 nM
the most potent,
non-covalent inhibitor
of AChE known
syn-TZ2PA6
Kd = 77 fM
In Situ Click Chemistry
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Syn and anti triazoles derived from TZ2+PA6
H2N
NH2
H2N
NH2
N
N
N
N N
N
NH
N
N N
H
N
N
anti-isomer, not made in situ
syn-isomer, made in situ
Kd (eel)
Kd (mouse)
Kd (eel)
Kd (mouse)
= 14,000 fM
= 8,900 fM
= 99 fM
= 410 fM
Kd (mouse) = <2 fM (Y337A)
W.G. Lewis, K.B. Sharpless, et al. Angew. Chem., Int. Ed. 2002, 41, 1053,
Y. Bourne, H. C. Kolb, Z. Radiç, K. B. Sharpless, P. Taylor, P. Marchot, PNAS 2004, 101, 1449
H.C. Kolb et al. JACS, 2004, in press
Regioselectivity Control
syn
anti
The thermal process generates a
1:1 mixture of syn- and anti- PY6TZ2
syn
Whereas, the enzyme-guided
cycloaddition strongly favors the
syn- PY6TZ2
H 2N
N
N
N
NH2
N
NH
N
syn-TZ2/PA6
k
(10
10
k
on
M – 1 min
–1 )
K
off
( min
anti-TZ2/PA6
d
Enzyme
(fM)
–1)
k
(10
10
on
M – 1 min
k
off
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K
d
–1 ) ( min – 1 )
(fM)
14,000
1.5
0. 0015
99
Eel AChE
1.8
0. 25
1.3
0.0011
77
Torpedo AChE
3.2
0.026
1.7
2.0
0.36
0.0071
0. 072
0. 0026
410
3,600
720
mouse AChE
2.4
3.4
0.69
0.30
0. 058
0. 0032
8,900
1,700
460
1.0
0.0050
500
Y124Q mouseAChE
1.6
0.063
3,900
0.56
0..040
7,100
W286A mouseAChE
0.65
1.5
230,000
0.059
0.12
0.
0.073
0. 40
550,000
1.3
210,000
<0.000015
< 1.2
Drosophila AChE
mouse BuChE
Y72N/Y124Q/W286A mAChE
Y337A mouseAChE
1.3
<0.00021
720
< 16
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syn-TZ2/PA6 Y337A
(TFK+- eelAChE)
*
mAChE
(Kd < 1.2 fM)
metalloenzymes
natural ligands
enzyme inhibitors
small anions
In Situ Click Chemistry:
The Target-Protein as a Reaction Vessel
Discovers a ‘Freeze Frame-Inhibitor’
N
HN
H2N
TZ-2
Ph
N+
N3
PA-6
NH2
N
NH2
NH
N+
N
Ph
N
N
KD ~ 80 fM
H2N
Finn, M. G., Sharpless, K.B. et al.,
Angew. Chem., Int. Ed. 41, 1053-1057 (2002).
Shape Modification at the Gorge Entrance:
‘Freeze Frame’ Inhibitor
anti-TZ2PA6/mAChE
syn-TZ2PA6/mAChE
Bourne, Kolb, Radiç, Sharpless,Taylor, Marchot, PNAS 2004, 101, 1449
Drosophila melanogaster AChE
mAChE
70% residue homology
eAChE
51% residue
homology
48% residue
homology
dmAChE
mAChE
dmAChE
Crystal Structures: Comparison WT and Y337A mAChE
W286
syn-TZ2PA6
WT mAChE
Kd=410 fM
W72
Y337A mAChE
Kd=1.2 fM
Y341
A337 / Y337
W86
In situ Click Chemistry with Acetylcholinesterase
Species Differences
binary mixtures
NH2
NH2
H2 N
H 2N
NH2
H2 N
N
N
NH2
N
H2N
(CH2)2-6
NH2
N
N
H2N
N
N
N
+
N
N
N
PA2-6
+
+
N3
NH
NH
N
N
anti-TZ6PA2
TZ3PA6
HN
N
N
(CH2)2-6
N
N
NH
NH
N
N
syn-TZ2PA6
syn-TZ2PA5
N
TZ2-6
N
N
Electric Eel
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TIFF
TIFF(Uncompressed)
(Uncompressed)decompressor
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picture.
Mouse
Click Chemistry Selection In Situ
for mouse AChE
TZ2 Kd = 23 nM
PA6 Kd = 360 nM
Trp-286
Tyr-72
?
Gly-342
syn-TZ2PA6 Kd = 410 fM
Tyr-341
Phe-338
Trp-286
Tyr-124
Ser-125
Phe-297
Gly-121
Tyr-72
Tyr-124
Gly-342
Tyr-337
Trp-86
Tyr-341
Phe-338
Ser-125
Phe-297
His-447
Gly-121
anchor molecule
Tyr-337
Trp-86
His-447
in situ click chemistry with low
affinity building blocks possible!
Binding Constants of Building Blocks (mAChE)
NH2
O
O
N3
HN
N
H2N
N
O
( )6
( )n
N
O
( )n
N
CN
( )n
N
TZ2: Kd=23 nM
PA6: Kd=360 nM
IQN-A5: Kd=77 μM
IQN-A6: Kd=210 μM
PIQ-A5: Kd=34 μM
PIQ-A6: Kd=21 μM
C-A5: Kd>400 μM
C-A6: Kd>400 μM
N
N
N
( )n
N
( )n
N
( )n
N
P-A5: Kd>400 μM
P-A6: Kd>400 μM
N
( )n
( )n
O
DPA-A5: Kd=42 μM
DPA-A6: Kd=83 μM
DMB-A5: Kd=14 μM
DMB-A6: Kd=14 μM
HIQ-A5: Kd=99 μM
HIQ-A6: Kd=190 μM
PO-A5: Kd>400 μM
PO-A6: Kd>400 μM
Cl
N
N
N
N
( )3
PQH-A4: Kd=38 μM
O
O2N
N
N
H
N
()
3
N
O N
N
N
N
()
3
QH-A4: Kd>400 μM BOH-A4: : Kd>4.1 μM
O
N
( )n
IIQ-A5:Kd>400 μM
IIQ-A6:Kd>400 μM
O
( )n
PHN-A5: Kd n.d.*
PHN-A6: Kd n.d.*
* not determined due to low solubility
Dissociation Constants (eAChE)
NH2
NH2
H2N
H2N
N
NH2
NH2
N
N
N
H2N
N
N
H2N
N
N
N
N N
N
N
N N
N
NH
NH
NH
NH
N
N
N
N
syn-TZ2PA5
Kd=100 fM
syn-TA2PZ6
Kd=830 fM
syn-TA2PZ5
Kd=540 fM
syn-TZ2PA6
Kd=99 fM
MeO
MeO
OMe
OMe
N
OMe
N
OMe
N
N
N
N
N
OMe
OMe
N
N
N
N
N
N
N
N
N
NH
NH
NH
NH
N
N
N
N
syn-(S)-TZ2PIQA6
Kd=96 fM
syn-(S)-TZ2PIQA5
Kd=33 fM
syn-(R)-TZ2PIQA6
Kd=360 fM
syn-(R)-TZ2PIQA5
Kd=36 fM
Concerted Huisgen [3+2] cycloadditions
with and without enzymic assistance
~Half-life comparisons
at the concentrations (micromolar) used
3000 years
‘alone’ -directly over the
27K summit
of Mount Concerted
----->
days
a ‘perfect’ polypeptide
embrace
and Mount Concerted
“ is brought low”
Concerted Huisgen [3+2] cycloadditions
versus the Cu-catalyzed stepwise process
~Half-life comparisons
at the concentrations (micromolar) used
3000 years
‘alone’ -directly over the
27K summit
of Mount Concerted
----->
days
------>
a ‘perfect’ polypeptide
embrace
and Mount Concerted
“ is brought low”
hours
finally, with copper’s ‘guidance’,
the 27K summit of Mount Concerted
is avoided altogether -------the stepwise
path meanders, and
goes up and down several times,
but never exceeds 19K!
Acknowledgements
Acetylcholinesterase
TSRI
Hartmuth Kolb
Roman Manetsch
Antoni Krasinski
Jon Loren
Université de la Meditaranée (France)
UCSD
Palmer Taylor
Jessica Raushel
Zoran Radic
Pascale Marchot
Carbonic Anhydrase
HIV Protease
Vani Mocharla
John Cappiello
Benoit Colasson
Mat Whiting
Stefanie Röper
John Muldoon
Yves Bourne
Valery Fokin
M.G. 41
Finn
University of South Florida
Roman Manetsch
Prasanna Pullanikat
Lisa Malmgren
Richard Cross (no picture available)
Orthogonal
Orthogonal
Orthogonal Reactivity
Empowers
Stealth Chemistry
Orthogonal
OrthoGON
Siemens Biomarker Solutions:
Scientific Advisors
Hartmuth Kolb, Ph.D.
Associate Professor of
Molecular and Medical
Pharmacology, UCLA
VP, Siemens Biomarker Sol’ns
Chemistry and Molecular
Imaging
Leroy Hood, M.D.,
Ph.D. President and
Founder, The Institute for
Systems Biology
Systems Biology &
Molecular Medicine
Owen Witte, M.D., Ph.D.
Director, UCLA Institute for Stem
Cell Biology and Medicine
Distinguished Professor,
Microbiology, Immunology, and
Molecular Genetics
Oncology and Immunology
Slide 44
H-R Tseng, Ph.D.
Roy Doumani
Assistant Professor of
Molecular and Medical
Pharmacology, UCLA
Chemistry and Microfluidics
Professor, Molecular & Medical
Pharmacology, UCLA
Technology, Business, and
Investment Banking
Jim Heath, Ph.D.
Elizabeth W. Gilloon Professor
and Professor of Chemistry,
Caltech;
Pharmacology, UCLA
Nanotechnology, Chemistry
and Biotechnology
Johannes Czernin, M.D.
Director, Nuclear Medicine
Clinic, UCLA
PET/CT Molecular Imaging
Stephen Quake, Ph.D.
Professor of Bioengineering,
Stanford
Microfluidics and
Biotechnology
Michael Phelps, Ph.D.
Norton Simon Professor and
Chair, UCLA Molecular &
Medical Pharmacology
PET Molecular Imaging and
Molecular Medicine
September 2005
PET Molecular Imaging
Bridging Biology & Structure and Enabling Molecular Medicine
2-[F-18]Fluoro-2-Deoxy-D-Glucose
(FDG)
CH2OH
H
H
HO
511 keV photon
O
H
H
H
F
‹13N
‹11C
O
H
‹15O
‹18F
O
H
+ -
2 min
10 min
20 min
120 min
E = mc2
180o
511 keV photon
UCLA
The RDS 111 Cyclotron
Lung Carcinoma
Ovarian Carcinoma
Slide 45
September 2005
Discovery of Novel PET Tracers:
High-Affinity Protein Ligands through In Situ Click Chemistry
Imaging Probe
Extremely High Affinity
Anchor Molecule
~ 10–8 x 10–5 = 10–13
Secondary Ligand
Drug-like Affinity
Click
Medium Affinity
~10–8
~ 10–5
AM
18/19F
Biological Target
• Extremely high affinity for disease-related biological targets:
⇒ Affinities in the range of 10-9 to 10-14
• High specificity for disease-related biological targets:
⇒ Imaging probes designed by the target for the target
• Small molecules:
⇒ Imaging probes with access to surface receptors, cells & nucleus
• Predictable imaging probe generation:
⇒ The linking unit and the radionuclide are part of the design from the beginning
Indian Wells, California • October 2004
Click Chemistry PET Tracer Development:
Example: Carbonic Anhydrase Ligands
Biological Relevance
• Catalyze the interconversion of HCO3– and CO2
• Involved in key biological processes
– respiration and transport of CO2/HCO3–
– acid secretion and pH control
– bone resorption and calcification
– tumorigenicity and many others
• Inhibitors: Ar-SO2NH2
QuickTime?and a
a
Sorenson QuickTime?and
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Sorenson
decompressor
are
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to see3 this
picture.
are needed to see this picture.
• CA-IX & XII overexpressed in tumors
Test Case for Validation Purposes:
R
• Carbonic Anhydrase-II
– Expressed in erythrocytes, lung,
stomach, kidneys
N N
N
R'
15 Å
O S
Š
O NH
Zn2+
Slide 47
V.P. Mocharla, H.C. Kolb, et al. Angew. Chem. Int. Ed. 2005, 44, 116-120.
September 2005
In situ Click Chemistry
Carbonic Anhydrase: Hit Discovery & Validation
H2N
S
O O
+
O
N3
N
H
N
N
N
H
N
O
H2N
S
O O
O
S
N
Slide 48
O
O Ethoxazolamide
S
Kd = 0.1 nM ± 0.02
NH2
September 2005
In situ Click Chemistry
Carbonic Anhydrase: Binding Affinities
N R
N
N
bovine Carbonic Anhydrase II
1 mg/mL (approx. 30µM)
+
H2N
S
O O
N3 R
60 µM
H2N
aq. pH7.4 buffer
37°C, 40 hrs
400 µM
S
O O
Kd = 37 nM ± 6 (bCA-II)
N3
• No ‘false positives’
R1
X
N3
R2
O
15
9
in situ hits
R
N3
N3
• Some ‘false negatives’
N3
N R
R1
S
N
H
R2
R
Ph
Ph
• In situ hits are the most potent compounds
R
4
1
1
2
1
1
in situ hit
1
in situ hit
No
in situ hits
No
in situ hits
No
in situ hits
inactive
1.3 & 9 nM
8 nM
28 & 4 x
4.6 x
Kd =
0.2 – 2.4 nM
5 nM
7 nM
185 – 15 x
7.4 x
5.2 x
Slide 49
September 2005
Probe Development with In Situ Click Chemistry:
Fast Development Cycle
Biological Target
• Disease related
• Overexpressed, overactivated or mutated
In situ click
5 - 6 weeks
High Affinity Ligand
• Small molecule, drug-like
• Easy to synthesize & label
• [F-19] containing
Copper click
MicroPET Study
High Affinity Probe
• Rodents
• Model of disease
• “Proof of concept”
• [F-18] labeled, w/o “retrofitting” of radionuclide
UCLA
Human PET Study
• Safety assessment with non-human primates
• eIND approved human study
Slide 50
September 2005
Probe Development by In Situ Click Chemistry:
Example: Carbonic Anhydrase-II
Biological Target
• Carbonic Anhydrase-II
• Erythrocytes, lung,
kidneys
In situ click
4 weeks
High Affinity Ligand
• Small molecule, drug-like
• Easy to synthesize & label
• [F-18] containing
R2
MicroPET Results
• Uptake in the blood, lung, kidneys
• 90% of tracer still bound after 2 hrs
• No metabolism that generates 18F–
Total: 5 weeks
Slide 51
N N
N
R1
18F
F18-PVBS
F-PPBS
CLogP: 0.95
CLogP: 3.1
M.W.: 432.1
M.W.: 493.2
O S
NH2
O
September 2005
Probe Development by In Situ Click Chemistry:
Example: Carbonic Anhydrase-II
R2
N N
N
18F
F18-PVBS
CLogP: 0.95
R1
M.W.: 432.1
O S
NH2
O
Results:
• F18-PVBS, shows a biodistribution that reflects the expression of CA-II
in the various tissues: Red Blood Cells, Lung, Kidneys
• Most of the tracer is still bound after 2 hours
CA-II References:
1 Hr Dynamic PET/CT
Annu. Rev. Biochem. 1995, 64, 375-401
Am. J. Respir. Cell Mol. Biol., 1994, 10 (5, 05), 499-505
J. Nephrol. 2002, 15 (suppl. 5), S61-S74
World J Gastroenterol 2005, 11(2), 155-163
Human Protein Reference Database: http://www.hprd.org/protein/02023
2 Hr Static PET/CT
2 Hr Static PET/CT
Lung
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Tumor
MTI/UCLA
Slide 52
September 2005
Probe Development by In Situ Click Chemistry:
Example: Carbonic Anhydrase-II
Biodistribution of F18- PVBS
# 5288 (155 min p.i.)
# 5289 (137 min p.i.)
# 5290 (81 min p.i.)
40
F18-PVBS shows a biodistribution, which is
consistent with the expression profile of CA-II
35
R2
N N
N
30
F18-PVBS
CLogP: 0.95
25
R1
20
M.W.: 432.1
O S
NH2
O
15
10
5
fe
m
ur
n
lo
co
sm
al
li
nt
es
t in
e
h
y
ne
st
om
ac
nc
pa
ki
d
re
as
ng
lu
t
ar
he
or
m
tu
us
cl
e
m
oo
d
0
bl
% ID/g
18F
Slide 53
September 2005
Future In Situ Click Chemistry Projects:
Cancer-Related Carbonic Anhydrases
Carbonic Anhydrase XII - Breast & Renal Cancer
• Expression is significantly upregulated by hypoxia in breast and renal cancer cell lines
• CA XII positive tumors are associated with a lower relapse rate and a better overall survival
Carbonic Anhydrase IX - Early Stage Non-Small Lung Cancer, Bladder Cancer
• Hypoxia marker
• CA IX expression of tumor cells plays an important role in the growth and survival of tumor cells
under normoxia and hypoxia
Develop Carbonic Anhydrase IX and XII selective
biomarkers through in situ click chemistry
Slide 54
September 2005
Cyclooxygenase-2:
Development of Inflammation & Cancer Markers
• COX enzymes are responsible for the conversion of
arachidonic acid into prostaglandins, prostacyclins &
thromboxanes.
• COX-1 is constitutively expressed in kidneys and GI.
Its inhibition can lead to GI toxicity (ulcer, bleeding,
perforation).
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• COX-2 is inducible in inflammatory cells (monocytes,
macrophages) and plays a role in the inflammatory
response. Selective COX-2 inhibitors can treat
inflammatory diseases w/o GI side effects
(Nonsteroidal Antiinflammatory Drugs, NSAIDs).
• COX-2 is overexpressed in various pathologies:
Inflammation, cancer, ischemia, neurodegenerative
disorders.
• COX-2 tracers are useful for measuring the in vivo
expression of COX-2 in connection with inflammatory
disease, ischemia (cardiac, cerebral), neuronal
degeneration (Parkinson, Alzheimer) and cancer
(colorectal, gastric, breast).
Slide 55
R
N
N
R
N
N3
O
O
S
S O
O
NH
NH22
R1
COX-2
September 2005
Probe Development by In Situ Click Chemistry:
COX-2: Inflammation & Cancer Markers
8 Azides
11
Azides
R
R
N
N3
IC50
> 500 nM
O
S O
NH2
N
30 µM anchor molecule,
100 µM azide, 6 µM enzyme
O
S O
NH2
1 day
R1
COX-2
N
R1
Hit 1: (non-F cmpd)
• M.W. 516.2
• clogP 2.7
• IC50 ~ 20 nM
(Valdecoxib: 6-8 nM)
• COX-2 specific
COX-2
COX-2
Cox-2 +
Inhibitor
BSA
Control
Hit 2: (non-F cmpd)
• M.W. 471.1
• clogP 3.3
Hit 3: (F analog of Hit 2)
• M.W. 475.5
• clogP 2.9
Hit 4: (F analog of Hit 1)
• M.W. 504.5
• clogP 2.5
• IC50 ~ 20 nM
MTI/UCLA
Slide 56
September 2005
SBS’s Current Biomarker Pipeline:
COX-2 Human Studies
>2mCi/nanomole
Time of Scan
(minutes)
Slide 57
5
28
71
SBS/UCLA
September 2005
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8 DISCOVERY TECHNIQUES
•
•
•
•
•
•
•
•
Look in dark places, e.g., those shielded by phobias
Find inspiration in anomalies
Accept uncertainty
The AntiScientific Method–—don’t prove, disprove
Have a game plan, but run with results
Won’t work? do it anyway
Always think weird
And, above all, respect the KISS Principle!?
We speak piously of taking measurements and
making small studies that will add another brick
to the temple of science. Most such bricks
just lie around the brickyard.
Platt, J.R. (1964) “Strong Inference”
Science. 146: 347-353.
HIV Protease: In Situ Approach
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Click Chemistry Reagents and Inhibitor
OH
OH
NH
N3
O
O
Ki ≈ 1mM
N
O
S
O
Ki ≈ 1 µM
OH
NH
O
O
N N
N
OH
N
O
S
O
Ki = 1.7nM
O
O
Triazoles as Connectors
“Pharmacophoric Linkers”
• Linker, like amides or other 5-membered heterocycles
• Capable of stacking with other aromatic groups
• Hydrogen bond acceptor (N3)
• Dipole similar to amides (N-methyl acetamide: 3.7 – 4.0 Debye )
ab initio results
(HF 6-311G**++)
Properties of 1,2,3-Triazoles, continued:
• Weakly basic. Hydrogen bond acceptors (N3 and N2):
N
NH2
N pKBH+ = 1.25
N
N
NH3
N
N
H
CH3
H
NO2
pKBH+ = 0
pKBH+ = 1
N
pKBH+ = 5.2
pKBH+ = 7.0
pKBH+ = 9.3
• Extremely stable, both thermally
and chemically:
H
N
N
N
Benzotriazole survives for I/2 second
at 600 degrees C! Hence more stable than
benzene or napthalene says Alan Katrizky
1,2,3-Triazoles:
Stable to Severe Reduction and Oxidation Conditions
N
Pd/C(10%)/ acetic acid
N
N
N
o
22 C, 2.7 bar H2
N
N
H2N
85%
H2 N
US 3,197,475, 1965
NH2
H2N
HOOC
KMnO4, NaOH
N
NH2
N
N
N
100 oC
N
H
N
H2N
Gazz. Chim. Ital. 1941, 71, 228
N
N
KMnO4
N
H
Jingxi Huagong 2003, 20, 628
80%
HOOC
NH
microwave
irradn.
H
N
N
HOOC
N
N
H
N
H
65%
Organic Azides:
late bloomers
In organic synthesis
Organic Azides: connectivity as good as it gets?
• Highly energetic species
• Yet, most are thermally stable and easily handled safely if simple
precautions are observed
• Virtually inert to most other functionalities, like tigers in a cage
• Form robust, aromatic heterocycles, such as 1,2,3-triazoles,
via 1,3-dipolar cycloaddition reactions
• However until recently, azides were seen ‘only’ as latent amines:
- 24,174 examples of reductions of an azide to an amine
- 809 examples of cycloadditions resulting in triazoles, and
421 of them are copper-catalyzed (i.e. post-2002)
(SciFinder, March 13, 2005)
1,2,3-Triazoles:
Permanent Connectors
with Pharmacophoric Properties
• Large dipoles:
4.7 Debye
5.2 Debye
1
N
1
N
N
N
4
N
N
5