1. Zujajah Malik
Supervisor: Dr. Lana Mikhaylichenko
Course Coordinator: Dr. Kagan Kerman
CHMD90 Fall 2013- Winter 2014
Department of Physical and
Environmental Sciences
25th April 2014

2. Outline
• Caspases and biological significance of inhibiting their function
• Structure of Caspase-3
• Caspase-3 inhibitor Lead structure
• Reaction Scheme
• Results and their discussion
• Next Steps & Conclusion
• Acknowledgement
• References
2

3. What are caspases?
Cysteine-dependent, aspartate specific proteases1
 Key role in Apoptosis1
 Programmed cell death
 Maintains cellular abundance
 Caspases inactivate cellular infrastructure1
 3 families of caspases1
 Inflammatory (caspase-1,-4 &-5)
 Initiator (caspase-2,-8,-9 & -10)
 Effector (caspase-3,-6 & -7)
3
Caspase-3 catalyzes
committing step
1. Friedlander, R. N. Engl. J. Med. 2003, 348, 1365-1375. 3

4. Why inhibit Caspase-3?
UNREGULATED APOPTOSIS CHARACTERIZES DISEASE CONDITIONS
 AIDS (autoimmune deficiency syndrome) 2
 Neurodegenerative disorders eg .Alzheimer’s and Huntington’s
Disease2
 AD affects 747,000 Canadian individuals 3
 > 1 million by 20303
 Total annual expenditure $33 billion3
4
4
2. Elmore, S. Toxicol. Pathol. 2007, 35, 495-516.
3. Canadian Institute of Health Research. 2012.

5. • Research into therapeutic
strategies that target abnormal
apoptosis i.e caspase-3
• Synthesis of non-peptide
inhibitors
• Peptide inhibitors have poor
metabolic stability and
membrane permeability4
5
5
Why inhibit Caspase-3?
4. Chu, W.; Zhang, J.; Zeng, C.; Rothfuss, J.; Tu, Z.; Chu, Y.; Reichert, D.; Welch, M.; Mach, R. J. Med. Chem.
2005, 48, 7637-7647.

6. Structure of Caspase-35
• Activated caspase-3 exists as a homodimer
• 2 active sites on each homodimer
• 4- binding pockets on each active site
 S1, S2, S3. S4
6
S2: tryptophan,
tyrosine,
phenylalanine-hydrophobic
S3: Serine and
tryptophan
S1 – cysteine
forms oxyanion
for aspartic acid
capture
S4: hydrophilic and
narrow
Determinant of
variability among
caspases
6
5. Sakai, J.; Yoshimori, A.; Nose, Y.; Mizoroki, A.; Okita, N.; Takasawa, R.; Tanuma, S. Bioorg.
Med. Chem. 2008, 16, 4854-4859.

7. Caspase-3 Inhibitor: Lead Structure5
ISATIN SULFONAMIDE ANALOGUE
7
5. Sakai, J.; Yoshimori, A.; Nose, Y.; Mizoroki, A.; Okita, N.; Takasawa, R.; Tanuma, S. Bioorg.

8. ISATIN SULFONAMIDE ANALOGUE: LEAD STRUCTURE
• One of the first reported non-peptide
inhibitors: 5-Nitrosulfonylisatins5
• Electron withdrawing group at position 55
 N,N-dialkylsulfonamide groups
 High levels of inhibition
• N- substituent off isatin nitrogen
 Used as a means to introduce radioisotope for PET
scanning and imaging5
 Traizole rings show improved potency and
lipophilicity6
[18F]ICMT-11
6. Krause-Heuer, A. M.; Howell, N. R.; Matesic, L.; Dhand, G.; Young, E. L.; Burgess, L.; Jiang, C. D.; Lengkeek, N. A.; Fookes, C. J. R.;
Pham, T. Q.; Sobrio, F.; Greguric, I.; Fraser, B. H. Med chem. Comm. 2013, 4, 347-352.
7. Lawrence, H. R.; Pireddu, R.; Chen, L.; Luo, Y.; Sung, S.; Szymanski, A. M.; Yip, M. L. R.; Guida, W. C.; Sebti, S. M.; Wu, J.;
Lawrence, N. J. J. Med. Chem. 2008, 51, 4948-4956.
8

9. 9
Reaction Scheme

10. IR
GC-MS
1H-NMR
Melting
point
Results
10
10

11. Isatin-5-sulfonyl Chloride
•Rationale: chloride is a
better leaving group than
OH, favors nucleophilic
attack of amine in
subsequent steps
• IR/cm-1: 1362 & 1128(S=O
str); 1611 & 1469 (sp2 C=C
phenyl), 3371 (NH str), 553
(S-Cl str)
11
Compound Yield (%), mp (°C)
and Rf on TLC
ESI (+) m/z
from GCMS
I
90%
189-191°C
(Lit. mp 188-190°C)
0.89
244.9 m/z

12. Ethyl-4-hydroxy benzoate & ethyl-4- amino benzoate
Corresponding sulfoxy ester and Sulfonamide derivative
•Multiple attempts with varying
reaction conditions
•Poor nucleophilicity
•Resonance stabilization
12
•THF/DMF & THF/DCM
•Triethylamine &
diisopropylethyl amine
•Reflux, 60-80°C
•This procedure was discontinued

13. Different Amines & Conditions
•4-(2-aminoethyl)morpholine &
1-(2-aminoethyl)pyrrolidine
•Isatin sulfonic acid instead of
isatin sulfonyl chloride
•Use of coupling reagents
EDC
2-hydroxy pyridine
13
•Coupling reagent not
removed
•Approach discontinued

14. 14
•Low yield
•Crude initial
•Loss during column
chromatography
•Distinguishing IR stretches
for S-N seen at 724 cm-1 and
778 cm-1 for II and III
respectively
14
Different Amines & Conditions
Compound Yield (%),
mp (°C) and
Rf on TLC
ESI (+) m/z
from GCMS
II
19%
88-91°C
0.33
339.1 m/z
III.
10%
110-113°C
0.18
323 m/z

15. •methanol in III
•OH shoulder in IR
•Bulky molecule-holds solvent
•Removal of solvent under
reduced pressures
N-(2-morpholinoethyl)-2,3-
dioxoindolin-5-sulfonamide
2,3-dioxo-N-(2-(pyrrolidin-1-
yl)ethyl) indolin-5-sulfonamide
15

16. Substituting Isatin nitrogen
•N-benzyl moiety will bind at the S1 binding pocket
16
Compound Yield (%),
mp (°C) and
Rf on TLC
ESI (+) m/z
from GCMS
IIa
14%
99-105°C
0.69
447.1 m/z
IIIa
51%
75-78°C
0.72
In Progress

17. 1-(4-flurobenzyl)-N-(2-
morpholinoethyl)-2,3-
dioxoindoline-5-
sulfonamide
1-(4-flurobenzyl)-2,3-dioxo-
N-(2-(pyrrolidin-1-
yl)ethyl)indoline-5-
sulfonamide
17

18. Enzyme
inhibition
Assay
Will these
compounds
inhibit Caspase-3
in-vivo?
Molecular
docking
studies
Prediction
of potential
bioactivities
Next Steps…
Determining biological activities
Pass Inet Predictions
18
Compound Pa Pi Activity
IIa 0.268 0.004 Caspase-3
IIIa 0.250 0.005 Caspase-3
•Pa < 0.5 = compounds are new
chemical entities
•Purpose: synthesis of NOVEL
coompounds
18

19. Isatin sulfonic acid
Add a better leaving group to
support nucleophilic attack
Link amines to sulfonyl group to
generate sulfnamide
Addf fuorobenzyl moitey to isatin
nitrogen
Conclusion
•Synthesized novel non-peptide
compounds
•Need to be analyzed for potential
bioactivity in-vivo and in-vitro
•Refine synthesis to obtain better
yields & modify linkages to enhance
inhibition
•Potential to replace peptide
inhibitors with enhanced
lipophilicity and potency
•Can save $$$$
19

20. Acknowledgements
•Sincere gratitude to my supervisor Dr. Lana Mikhaylichenko
•Course Coordinator: Dr. Kagan Kerman
•Lab Management Staff: Tony, Ronald, Michael, Kony, Vlad, Scott
•Fellow Labmates: Eva, Thayalan and Sagar
•Department of Physical and Environmental Sciences at UTSC (DPES)
20

21. References
1. Friedlander, R. N. Engl. J. Med. 2003, 348, 1365-1375.
2. Elmore, S. Toxicol. Pathol. 2007, 35, 495-516.
3. Canadian Institute of Health Research. 2012. http://www.cihr-irsc.gc.ca/e/41053.html
4. Sakai, J.; Yoshimori, A.; Nose, Y.; Mizoroki, A.; Okita, N.; Takasawa, R.; Tanuma, S. Bioorg. Med.
Chem. 2008, 16, 4854-4859.
5. Chu, W.; Zhang, J.; Zeng, C.; Rothfuss, J.; Tu, Z.; Chu, Y.; Reichert, D.; Welch, M.; Mach, R. J. Med.
Chem. 2005, 48, 7637-7647.
6. Krause-Heuer, A. M.; Howell, N. R.; Matesic, L.; Dhand, G.; Young, E. L.; Burgess, L.; Jiang, C. D.;
Lengkeek, N. A.; Fookes, C. J. R.; Pham, T. Q.; Sobrio, F.; Greguric, I.; Fraser, B. H. Med chem.
Comm. 2013, 4, 347-352.
7. Lawrence, H. R.; Pireddu, R.; Chen, L.; Luo, Y.; Sung, S.; Szymanski, A. M.; Yip, M. L. R.; Guida, W.
C.; Sebti, S. M.; Wu, J.; Lawrence, N. J. J. Med. Chem. 2008, 51, 4948-4956.
21

22. 22
Thank you …
Questions ?