Isatin 1,2,3-triazoles as potent inhibitors against caspase-3

Article abstract of DOI:10.1016/j.bmcl.2011.01.110

Sixteen disubstituted 1,2,3-triazoles were prepared using the Huisgen cycloaddition reaction and evaluated as inhibitors against caspase-3. The two most potent inhibitors were found to be (S)-1-((1-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)-1H-1,2,3-triazol-4-yl)methyl)-5-((2-(methoxymethyl)pyrrolidin-1-yl) sulfonyl)indoline-2,3-dione (7f) and (S)-1-((1-benzyl-1H-1,2,3-triazol-5-yl) methyl)-5-((2-(methoxymethyl)pyrrolidin-1-yl)sulfonyl)indoline-2,3-dione (8g) with IC₅₀-values of 17 and 9 nM, respectively. Lineweaver-Burk plots revealed that these two triazoles show competitive inhibitory mechanism against caspase-3.

Full text of DOI:10.1016/j.bmcl.2011.01.110

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1626–1629
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Isatin 1,2,3-triazoles as potent inhibitors against caspase-3
⇑
Yang Jiang , Trond Vidar Hansen
School of Pharmacy, Department of Pharmaceutical Chemistry, University of Oslo, PO Box 1068 Blindern, N-0316 Oslo, Norway
a r t i c l e i n f o
a b s t r a c t
Article history:
Sixteen disubstituted 1,2,3-triazoles were prepared using the Huisgen cycloaddition reaction and evaluated
as inhibitors against caspase-3. The two most potent inhibitors were found to be (S)-1-((1-(2,3-dihydro-
benzo[b][1,4]dioxin-6-yl)-1H-1,2,3-triazol-4-yl)methyl)-5-((2-(methoxymethyl)pyrrolidin-1-yl)sulfonyl)
indoline-2,3-dione (7f) and (S)-1-((1-benzyl-1H-1,2,3-triazol-5-yl)methyl)-5-((2-(methoxymethyl)pyrro-
lidin-1-yl)sulfonyl)indoline-2,3-dione (8g) with IC50-values of 17 and 9 nM, respectively. Lineweaver–
Burk plots revealed that these two triazoles show competitive inhibitory mechanism against caspase-3.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 24 December 2010
Revised 24 January 2011
Accepted 25 January 2011
Available online 31 January 2011
Keywords:
Caspase-3
Inhibitors
1,2,3-Triazole
Isatin
Apoptosis, or programmed cell death, plays an essential role in
all multi-cellular organisms. Dysregulation of apoptosis is related
to many human diseases, including cancer, diabetes and neurode-
generative diseases.1 Several members from the caspase family are
central in this process, among which caspase-3 is the key effector
caspase involved in both intrinsic and extrinsic apoptotic path-
ways.2 Caspase-3 inhibitors with high potency and selectivity
may be useful remedies against stroke, hepatitis, brain injury and
neurodegenerative diseases.
Caspases are cysteine proteases that cleave their substrate after
an aspartate residue. In convention, the N-terminal residue of the
scissile bond in the substrate is named P1, the one to the C-termi-
nal residue is named P01, and other residues are numbered consec-
utively (Fig. 1).
leading to blocked protease activity. The presence of alkylating
functionalities such as aldehydes or ketones results in reversible
inhibitors, while
a-haloketones and acyloxymethyl substituted
ketones give rise to irreversible inhibitors. Although being useful
in research, peptide based caspase inhibitors in general are less suit-
able for drug development because of low cell permeability and lack
of specificity among the different caspases, as well as non-specific
toxicity. Hence, developing non-peptide based caspase inhibitors
remains an interesting research topic in medicinal chemistry.4
Isatin derivatives, such as 5-nitroisatin (1) and (S)-5-pyrrolidi-
nyl-1-ylsulfonyl)isatins (2a, 2b), were first reported by Lee et al.
as caspase-3 and -7 inhibitors.5 These compounds were discovered
after high-throughput screening. It is presumed that the thiol
group in Cys-285 in caspase-3 forms a reversible covalent bond
with the carbonyl group of the isatin moiety leading to inhibition
of enzyme activity. Podichetty et al. synthesized fluorinated deriv-
atives of isatin and evaluated their inhibition potency against
several caspases.6 Chu et al. have explored the possibility of chang-
ing the isatin 3-carbonyl group to a Michael acceptor that resulted
in several inhibitors with medium to low nM IC50-values.7
Compounds with either a pyrrolo[3,4-c]quinoline-1,3-dione or an
isoquinoline-1,3,4-trione moiety have also been reported to inhibit
The corresponding binding pockets of the enzyme for these res-
idues are named S1, S0 and so on. For all caspases, P1 to P4 and P0
1
1
residues contribute to substrate recognition. For caspase-3 the
sequence Asp-Glu-Ala/Val-Asp (DEAD/DEVD) in the substrate is
favored for P4 to P1 positions. For the P01 position, residues with
small, or polar but uncharged side chains are favored (Gly, Ala,
Thr, Ser and Asn), while residues with bulky or charged side chains
are not.3
Active caspase-3 is a homodimer of two heterodimer subunits,
with one active site on each subunit. Peptide based caspase-3
inhibitors have been designed by adding an alkylating functional
group to such enzyme-recognition sequences. These inhibitors
bind to the substrate recognition pocket of the caspase followed
by reaction with the catalytic cysteine residue in the enzyme
caspase-3 with IC50-values at low l
M to nM range.8
Lately the Huisgen 1,3-dipolar cycloaddition reaction between
terminal alkynes and azides has attracted interest in medicinal
chemistry.9 This reaction has been used in several aspects for lead
discovery. For example, 1,4-disubstituted 1,2,3-triazoles have been
employed as mimics for amides,10 in combinatorial chemistry
library syntheses,11 in modifications of natural products12 and
in situ library screening.13 Grimm’s bioisosteric rule14 suggests
substitution of five-membered heterocyclic rings with triazoles.15
Moreover, the 1,2,3-triazole moiety is also a suitable mimic of a
⇑
Corresponding author. Tel.: +47 22857451; fax: +47 228555947.
E-mail address: yang.jiang@farmasi.uio.no (Y. Jiang).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.110

Y. Jiang, T. V. Hansen / Bioorg. Med. Chem. Lett. 21 (2011) 1626–1629
1627
Table 1
Inhibition data expressed as IC50-values
a
Compd
Caspase-3 inhibition IC50, nM
7a
7b
7c
7d
7e
7f
21
23
21
18
44
17
7g
7h
8a
8b
8c
8d
8e
8f
30
243
103
136
213
267
70
136
9
207
8g
8h
a
Values are means of two independent experiments, each
carried out in duplicates.
co-workers have used this reaction to prepare 18F-labeled isatin
derivatives for positron emission tomography studies of apoptosis,
and two triazole derivatives showed high inhibition potency with
IC50-values of 16.7 and 0.5 nM, respectively (Fig. 1, compounds
2c and 2d).19 Hence, we became interested in preparing disubsti-
tuted 1,2,3-triazole derivatives of 2a as potentially more potent
inhibitors against capsase-3.
Figure 1. The sequence of caspase-3 substrates and examples of known caspase-3
inhibitors.
Synthesis of the alkynes was achieved using the literature pro-
cedures with minor modifications.5b,20 In brief, compound 3 was
treated with chlorosulfonic acid to yield the gem-dichloro deriva-
tive 4. Reacting 4 with (S)-2-(methoxymethyl)pyrrolidine followed
by acidic hydrolysis of the gem-dichloro moiety afforded 5-(S)-(2-
methoxymethyl)pyrrolidinylsulfonyl isatin 2a. Alkylation of the
amide nitrogen using 3-bromopropyne or 4-bromobutyne in the
peptide bond due to topological and electronic similarities.16 This
feature becomes especially attractive when developing inhibitors
of different proteases.17 Ng et al. reported in 2008 several 1,4-
disubstituted 1,2,3-triazoles as caspase-3 and -7 inhibitors with
activity in the low micromolar range.18 Recently Aboagye and
Scheme 1. Synthesis of triazole compounds. Reagents and conditions: (i) ClSO3H, 10 equiv (86%, crude); (ii) (S)-2-methoxylmethyl-pyrrolidine; (iii) AcOH–H2O 1:1 (46% yield
over two steps); (iv) Cs2CO3, acetonitrile, HCC(CH2)nBr (6a, n = 1, 76%; 6b, n = 2, 60%); (v) R-N3, MeOH, ; (vi) Ph-N3, CuSO4, sodium ascorbate, MeOH, rt.
D

1628
Y. Jiang, T. V. Hansen / Bioorg. Med. Chem. Lett. 21 (2011) 1626–1629
presence of Cs2CO3 yielded alkynes 6a and 6b in 30% and 24% over-
all yields, respectively. Azides were prepared using a literature pro-
cedure.21 The disubstituted triazoles 7a–7g or 8a–8g were
obtained either by thermal9a or by copper(I)-catalyzed9b,c cycload-
dition reactions (Scheme 1). The 1,4- and 1,5-disubstituted triazole
isomers obtained from the thermal reaction were separated by
chromatography. The products were characterized by spectral data
(see Supplementary data).
higher potency than the 1,4-isomer was observed. Moreover, for
compounds 7h and 8h with two methylene groups between the tri-
azole ring and the isatin moiety, 10- to 12-fold lower potency as the
compound 7a was observed. In addition, the potencies were lower
than most other new inhibitors reported herein (Table 1). These re-
sults indicate that the 1,2,3-triazole ring provides additional affinity
to the enzyme when it is connected to the isatin nitrogen via one
methylene group, not with two.
The enzyme inhibition assay was carried out as previously re-
For the most potent triazoles 7f and 8g, the inhibition kinetics
was measured (Fig. 2).23 These compounds were determined to
be competitive inhibitors, the same mechanism as that has been
observed for other isatin inhibitors, including 2a.5b Thus the Ki-val-
ues for 7f and 8g were determined to be 14.5 0.4 nM and
10.5 3.4 nM respectively, as calculated from inhibition kinetics
data.24
ported with fluorogenic substrate Ac-DEVD-AFC22 and the IC50
-
values of all synthesized triazoles were determined and compiled
in Table 1.
Compared to lead compound 2a (IC50 = 120 nM)5b the 1,4-disub-
stituted triazole 7a was approximately 5-fold more potent
(IC50 = 21 nM). Changing the phenyl group to a bulkier biphenyl
group had no effect on the potency (7b, IC50 = 23 nM). Adding an
electron-withdrawing nitro (7c, IC50 = 21 nM) or trifluoromethyl
(7d, IC50 = 18 nM) group in the para-position of the phenyl C-ring
gave little effect on the potency, either. However, introducing an
electron-donating methoxy group in the para-position of the C-ring
decreased the potency 2-fold (7e, IC50 = 44 nM). Replacing the phe-
nyl C-ring with a benzodioxin moiety as in 7f, the inhibition po-
tency (IC50 = 17 nM) was observed at the same level as 7a
(IC50 = 21 nM). The 1,5-disubstituted 1,2,3-triazoles 8a–8f were
all less potent than the corresponding 1,4-regioisomers 7a–7f
(Table 1). Interestingly, the 1,5-disubstituted triazole 8g derived
from benzyl azide exhibited more than 2-fold higher potency
Various substitutions at P01 position have been explored for sev-
eral peptide based caspase inhibitors,25 and structure–activity rela-
tionship studies on isatinsulfonamide compounds have been
carried out by several groups also.5b,7a Substitution groups contain-
ing aromatic rings were found to be preferable in many of these
explorations. Our work has shown that introduction of a 1,2,3-
triazole ring at the nitrogen atom of the isatin moiety results in sig-
nificantly enhanced inhibitory activity. Aulabaugh et al. reported
that sulfonyl isatin inhibitors showed a different mode of interac-
tion with caspase-3 compared to peptide based inhibitors.26 In the
report on isoquinolin-1,3,4-trione inhibitors by Du et al., it was
proposed that caspase-3 was inactivated by reactive oxygen spe-
cies generated by the inhibitor.8d All these results indicate that
the inhibition of caspase-3 by isatin sulfonamide derivatives or
analogs may involve different modes or mechanisms. Our results
would provide the basis for further development of other inhibi-
tors against caspase-3, as well as experimental basis for further
molecular modeling studies. These efforts will be reported in due
time.
(IC50 = 9 nM) than 7a. However, the 1,4-regioisomer 7g (IC50
=
30 nM) was less potent than 8g. Apparently, the substitution pat-
tern in the 1,2,3-triazole ring affects the binding affinity towards
caspase-3. The potency seems to be higher for 1,4-disubstiuted
1,2,3-triazoles prepared from phenyl azides, but for the 1,5-disub-
stituted 1,2,3-triazole 8g prepared from benzyl azide a 3-fold
Acknowledgments
The Research Council of Norway is gratefully acknowledged for
a post-doctoral fellowship to Y.J. Helpful discussions with profes-
sor Ragnhild Paulsen and technical assistance from professor
Harald Thideman Johansen are appreciated.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.110.
References and notes
1. Fischer, U.; Schulze-Osthoff, K. Pharmacol. Rev. 2005, 57, 187.
2. (a) Riedl, S. J.; Shi, Y. G. Nat. Rev. Mol. Cell Biol. 2004, 5, 897; (b) Fuentes-Prior, P.;
Salvesen, G. S. Biochem. J. 2004, 384, 201.
3. Timmer, J. C.; Salvesen, G. S. Cell Death Differ. 2007, 14, 66.
4. O’Brien, T.; Lee, D. Mini-Rev. Med. Chem. 2004, 4, 153.
5. (a) Lee, D.; Long, S. A.; Adams, J. L.; Chan, G.; Vaidya, K. S.; Francis, T. A.; Kikly,
K.; Winkler, J. D.; Sung, C. M.; Debouck, C.; Richardson, S.; Levy, M. A.; DeWolf,
W. E.; Keller, P. M.; Tomaszek, T.; Head, M. S.; Ryan, M. D.; Haltiwanger, R. C.;
Liang, P. H.; Janson, C. A.; McDevitt, P. J.; Johanson, K.; Concha, N. O.; Chan, W.;
Abdel-Meguid, S. S.; Badger, A. M.; Lark, M. W.; Nadeau, D. P.; Suva, L. J.;
Gowen, M.; Nuttall, M. E. J. Biol. Chem. 2000, 275, 16007; (b) Lee, D.; Long, S. A.;
Murray, J. H.; Adams, J. L.; Nuttall, M. E.; Nadeau, D. P.; Kikly, K.; Winkler, J. D.;
Sung, C. M.; Ryan, M. D.; Levy, M. A.; Keller, P. M.; DeWolf, W. E. J. Med. Chem.
2001, 44, 2015.
6. Podichetty, A. K.; Faust, A.; Kopka, K.; Wagner, S.; Schober, O.; Schafers, M.;
Haufe, G. Bioorg. Med. Chem. 2009, 17, 2680.
7. (a) Chu, W.; Zhang, J.; Zeng, C.; Rothfuss, J.; Tu, Z.; Chu, Y.; Reichert, D. E.;
Welch, M. J.; Mach, R. H. J. Med. Chem. 2005, 48, 7637; (b) Chu, W.; Rothfuss, J.;
d’Avignon, A.; Zeng, C.; Zhou, D.; Hotchkiss, R. S.; Mach, R. H. J. Med. Chem.
2007, 50, 3751.
8. (a) Kravchenko, D. V.; Kysil, V. M.; Tkachenko, S. E.; Maliarchouk, S.; Okun, I. M.;
Ivachtchenko, A. V. Eur. J. Med. Chem. 2005, 40, 1377; (b) Kravchenko, D. V.;
Figure 2. Lineweaver–Burk plot of triazoles 7f and 8g.

Y. Jiang, T. V. Hansen / Bioorg. Med. Chem. Lett. 21 (2011) 1626–1629
1629
Kysil, V. V.; Ilyn, A. P.; Tkachenko, S. E.; Maliarchouk, S.; Okun, I. M.;
Ivachtchenko, A. V. Bioorg. Med. Chem. Lett. 2005, 15, 1841; (c) Chen, Y. H.;
Zhang, Y. H.; Zhang, H. J.; Liu, D. Z.; Gu, M.; Li, J. Y.; Wu, F.; Zhu, X. Z.; Li, J.; Nan,
F. J. Med. Chem. 2006, 49, 1613; (d) Du, J. Q.; Wu, J.; Zhang, H. J.; Zhang, Y. H.;
Qiu, B. Y.; Wu, F.; Chen, Y. H.; Li, J. Y.; Nan, F. J.; Ding, J. P.; Li, J. J. Biol. Chem.
2008, 283, 30205.
4829; (b) Odlo, K.; Fournier-Dit-Chabert, J.; Ducki, S.; Gani, A. B. S. M. O.; Sylte,
I.; Hansen, T. V. Bioorg. Med. Chem. 2010, 18, 6874.
22. Enzyme assay was performed at 25 °C on a PerkinElmer VICTOR plate reader
with the excitation and emission wavelengths at 405 and 530 nm, respectively.
The assay were run in 200
HEPES (pH 7.5), 50 mM KCl, 0.1% CHAPS, 5 mM b-mercaptoethanol, 1 mM
EDTA, 10% sucrose and 10 Ac-DEVD-AFC. The final concentration of
l
L volumes and contained the following: 25 mM K+
9. (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004;
(b) Kolb, H. C.; Sharpless, K. B. Drug Discovery Today 2003, 8, 1128; (c) Meldal,
M.; Tornøe, C. W. Chem. Rev. 2008, 108, 2952.
10. Kim, S.; Cho, M.; Lee, T.; Lee, S.; Min, H.-Y.; Lee, S. K. Bioorg. Med. Chem. Lett.
2007, 17, 4584.
lM
caspase-3 was 0.25 nM (calculated as heterodimer), and the final concentration
of DMSO was 5%. Compounds tested were diluted in DMSO prior to the assay.
23. The inhibition kinetics for compounds 7f and 8g against caspase-3 were
determined using buffer conditions described above. The concentration of Ac-
11. Li, J.; Zheng, M.; Tang, W.; He, P.-L.; Zhu, W.; Li, T.; Zuo, J.-P.; Liu, H.; Jiang, H.
Bioorg. Med. Chem. Lett. 2006, 16, 5009.
12. Suarez, P. L.; Gándara, Z.; Gómez, G.; Fall, Y. Tetrahedron Lett. 2004, 45, 4619.
13. Manetsch, R.; Krasinski, A.; Radic, Z.; Raushel, J.; Taylor, P.; Sharpless, K. B.;
Kolb, H. C. J. Am. Chem. Soc. 2004, 126, 12809.
14. Kier, L. B.; Hall, L. H. Chem. Biodivers. 2004, 1, 138.
15. Ciocoiu, C. C.; Nikolic´, N.; Nguyen, H. H.; Thoresen, G. H.; Aasen, A. J.; Hansen, T.
V. Eur. J. Med. Chem. 2010, 45, 3047.
16. Bock, V. D.; Perciaccante, R.; Jansen, T. P.; Hiemstra, H.; van Maarseveen, J. H.
Org. Lett. 2006, 8, 919.
DEVD-AFC was varied from 6.25 to 100 lM, and the concentration of the
inhibitor was varied from 0 to 120 nM and from 0 to 60 nM for compounds 7f
and 8g, respectively. The maximum slope of the curve of AFC fluorescence was
used as the initial rate.
24. Km (without inhibitor) and K0 (with inhibitor) values were calculated by fitting
the raw
v
versus [S] data imnto
v = Vmax[S]/(Km + S), using BioDataFit (http://
www.changbioscience.com/stat/ec50.html). Ki-values were calculated using
the equation for competitive inhibitors, K0m = Km(1 + [I]/Ki). We are grateful to
an anonymous referee for helpful suggestions on how to perform these
calculations.
17. Whiting, M.; Tripp, J. C.; Lin, Y.-C.; Lindstrom, W.; Olson, A. J.; Elder, J. H.;
Sharpless, K. B.; Fokin, V. V. J. Med. Chem. 2006, 49, 7697.
18. Ng, S. L.; Yang, P. Y.; Chen, K. Y. T.; Srinivasan, R.; Yao, S. Q. Org. Biomol. Chem.
2008, 6, 844.
19. (a) Smith, G.; Glaser, M.; Perumal, M.; Nguyen, Q. D.; Shan, B.; Arstad, E.;
Aboagye, E. O. J. Med. Chem. 2008, 51, 8057; (b) Nguyen, Q. D.; Smith, G.; Glaser,
M.; Perumal, M.; Arstad, E.; Aboagye, E. O. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
16375.
20. Kravchenko, D. V.; Kuzovkova, Y. A.; Kysil, V. M.; Tkachenko, S. E.; Maliarchouk,
S.; Okun, I. M.; Balakin, K. V.; Ivachtchenko, A. V. J. Med. Chem. 2005, 48, 3680.
21. (a) Odlo, K.; Hentzen, J.; Fournier-Dit-Chabert, J.; Ducki, S.; Gani, A. B. S. M. O.;
Sylte, I.; Skrede, M.; Flørenes, V. A.; Hansen, T. V. Bioorg. Med. Chem. 2008, 16,
25. (a) Han, Y. X.; Giroux, A.; Grimm, E. L.; Aspiotis, R.; Francoeur, S.; Bayly, C. I.;
Mckay, D. J.; Roy, S.; Xanthoudakis, S.; Vaillancourt, J. P.; Rasper, D. M.; Tam, J.;
Tawa, P.; Thornberry, N. A.; Paterson, E. P.; Garcia-Calvo, M.; Becker, J. W.;
Rotonda, J.; Nicholson, D. W.; Zamboni, R. J. Bioorg. Med. Chem. Lett. 2004, 14, 805;
(b) Ullman, B. R.; Aja, T.; Chen, N.; Diaz, J. L.; Gu, X.; Herrmann, J.; Kalish, V. J.;
Karanewsky, D. S.; Kodandapani, L.; Krebs, J. J.; Linton, S. D.; Meduna, S. P.; Nalley,
K.; Robinson, E. D.; Roggo, S. P.; Sayers, R. O.; Schmitz, A.; Ternansky, R. J.;
Tomaselli, K. J.; Wu, J. C. Bioorg. Med. Chem. Lett. 2005, 15, 3632; (c) Ekici, O. D.; Li,
Z. Z.; Campbell, A. J.; James, K. E.; Asgian, J. L.; Mikolajczyk, J.; Salvesen, G. S.;
Ganesan, R.; Jelakovic, S.; Grutter, M. G.; Powers, J. C. J. Med. Chem. 2006, 49, 5728.
26. Aulabaugh, A.; Kapoor, B.; Huang, X. Y.; Dollings, P.; Hum, W. T.; Banker, A.;
Wood, A.; Ellestad, G. Biochemistry 2007, 46, 9462.