1,3-Dioxo-4-methyl-2,3-dihydro-1H-pyrrolo[3,4-c]quinolines as potent caspase-3 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.02.027

Synthesis, biological evaluation and structure-activity relationships for a series of novel nonpeptide small molecule inhibitors of caspase-3 are described. Among the studied compounds, 8-sulfamide derivatives of 1,3-dioxo-4-methyl-2,3-dihydro-1H-pyrrolo[3,4-c]quinolines have been identified as potent inhibitors of caspases-3. The most active compound within this series (8f) inhibited caspase-3 with IC₅₀ = 4 nM.

Full text of DOI:10.1016/j.bmcl.2005.02.027

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1841–1845
1,3-Dioxo-4-methyl-2,3-dihydro-1H-pyrrolo[3,4-c]quinolines
as potent caspase-3 inhibitors
Dmitri V. Kravchenko,a Vladimir V. Kysil,b Alexey P. Ilyn,b Sergey E. Tkachenko,b,c
Sergey Maliarchouk,b,d Ilya M. Okunb,d and Alexandre V. Ivachtchenkob,*
aDepartment of Organic Chemistry, Chemical Diversity Research Institute, 114401 Khimki, Moscow Region, Russia
bChemDiv, Inc., 92121 San Diego, CA, USA
cDepartment of Medicinal Chemistry, Chemical Diversity Research Institute, 114401 Khimki, Moscow Region, Russia
dDepartment of Molecular Biology and High-Throughput Screening, Chemical Diversity Research Institute,
114401 Khimki, Moscow Region, Russia
Received 27 October 2004; revised 7 February 2005; accepted 8 February 2005
Abstract—Synthesis, biological evaluation and structure–activity relationships for a series of novel nonpeptide small molecule inhibi-
tors of caspase-3 are described. Among the studied compounds, 8-sulfamide derivatives of 1,3-dioxo-4-methyl-2,3-dihydro-1H-pyr-
rolo[3,4-c]quinolines have been identified as potent inhibitors of caspases-3. The most active compound within this series (8f)
inhibited caspase-3 with IC50 = 4 nM.
Ó 2005 Elsevier Ltd. All rights reserved.
The caspase family comprises a family of highly homolo-
gous cysteine proteases that play key roles in inflamma-
tion and apoptosis.1 Among several different groups of
caspase enzymes, caspases-3 play a key role in apopto-
sis.2 Therefore, they are attractive targets for therapeutic
intervention in several diseases because of the central role
played by apoptosis in those conditions. For instance,
inhibitors of caspase-3 were described as promising car-
dioprotectants,3 neuroprotectants4 and hepatoprotec-
tants.5 Recently, we reported the discovery of a novel
class of potent small molecule inhibitors of caspase-3.6
In this paper, we describe synthesis, biological evaluation
and structure–activity relationships for this series of
novel nonpeptide small molecule inhibitors of caspase-3
having general formula I. The target 4-methyl-1,3-di-
oxo-2,3-dihydro-1H-pyrrolo[3,4-c]quinolines of general
formula I were synthesized using a previously reported
synthetic method based on Pfitzinger reaction.7
According to this approach depicted in Scheme 1, isatins
1a–e were suspended in water to an approximate con-
centration of 0.5 M and hydrolyzed with NaOH to give
oxoacetates 2a–e; the latter were then treated in situ with
methyl acetoacetate (2 mol. equiv) to afford the corre-
sponding dicarboxylic acids 3a–e. The acids 3a–e were
converted into furan-2,5-diones 4a–e upon the reaction
with an excess of acetic anhydride in dry pyridine. Reac-
tions of 1 M solutions of anhydrides 4a–e in pyridine
with equimolar amounts of different primary amines
5a–f smoothly led to imides 6a–t.
2
R
O
N
1
R
O
Using the described synthetic scheme, we have obtained
a series of novel compounds, which have not been previ-
ously reported in the literature. Thus, compound 6s was
synthesized from 1e as outlined in Scheme 1 in 67% yield
and used to synthesize an additional compound series as
outlined in Scheme 2.
N
CH
3
I
Keywords: Caspase-3; Inhibitor; 1H-pyrrolo[3,4-c]quinoline.
*
Chlorosulfonate 6w was synthesized using reaction of
6s with POCl3 at a temperature of 100 °C. Pyridinium
Corresponding author. Tel.: +1 858 794 4860; fax: +1 858 794 4931;
e-mail: av@chemdiv.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.027

1842
D. V. Kravchenko et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1841–1845
Na+
O
O
COOH
COOH
O
O
O
R 1
R 1
R 1
Ac2O / pyridine
55-60 °C / 3h
20% NaOH
rt / 1h
H3C
OMe
O
O
rt / 12h
N
CH3
N
NH2
H
1a: R1 = H
2a-e
3a-e, 75-85% from 1a-e
1b: R1 = F
1c: R1 = Br
R 2
N
O
O
O
O
R2-NH2 5a-f
pyridine / Ac2O
O
S
1d: R1 =
N
R 1
O
O
R 1
O
1e: R1 = -SO3Na
reflux / 30 min
N
CH3
N
CH3
4a-e, 90-95%
6a-t, 80-92%
CH3
NH2
CH3
O
H2N
O
NH2
N
NH3
H3C-NH2
H2N
CH3
CH3
O
N
O
H3C
CH3
5a
5b
5c
5d
5e
5f
Scheme 1.
CH3
N
CH3
N
CH3
N
H3C
O
N
H3C
O
N
H3C
O
N
4
R
N
CH3
CH3
CH3
N
N
R3R4NH 7a-f
N
O
O
S
O
S
H
Cl
3
O
N+
R
DMSO
S
O
O
O
O
POCl3
O
O
rt / 30 min
100 °C / 3.5h
N
CH3
N
CH3
N
CH3
6s
8a-f, 80-91%
6w, 95%
conc. HCl
NaCl / H2O
CH3
N
CH3
N
H3C
N
H3C
N
O
O
CH3
CH3
O
N
Na+
O
S
N
HO
O
O
S
O
O
O
N
CH3
N
CH3
6u, 92%
6v, 96%
CH3
N
CH3
N
H3C
N
H3C
O
N
CuI / KCN
N,N'-dimethylethylendiamine
dimethoxyethane
O
CH3
CH3
N
N
N
O
Br
O
microwaves
300 W / 190 °C / 20 min
N
CH3
N
CH3
6t
9, 84%
Scheme 2.
sulfonate 6s was also converted into acid 6u and so-
dium salt 6v upon treatment with concd HCl or aque-
ous NaCl, correspondingly; these compounds have
also been used in the following biological experiments.
Chlorosulfonate 6w appeared to be a convenient inter-
mediate for synthesis of a small combinatorial library
of 8-sulfamide derivatives. The reaction between equi-
molar amounts of 6w and six linear and cyclic aliphatic
amines 7a–f dissolved in DMSO to an approximate
concentration of 1 M in each component proceeded at
room temperature and afforded individual sulfonamides
8a–f in good yields. In addition, the 8-bromo substi-
tuted compound 6t was reacted with KCN in the pres-
ence of CuI, N,N0-dimethylethylendiamine and

D. V. Kravchenko et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1841–1845
1843
Table 1. In vitro caspase-3 inhibition assay results for compounds
6a–v and 9
dimethoxyethane under microwave irradiation condi-
tions to afford the 8-cyano derivative 9. The microwave
instrument used was a commercial household microwave
oven (Moulinex FM1935G, frequency: 2450 MHz).
Compd
R1
R2
IC50 (lM)
6a
6b
6c
H
F
H
H
H
>100
62.80
37.10
All the synthesized compounds were characterized by
1H NMR; LCMS and HRMS spectral data. Satisfactory
analytical data consistent with the shown molecular
structures were obtained for all compounds.
Br
O
S
6d
H
0.21
N
N
O
O
*
O
6e
6f
H
Br
CH3
CH3
6.36
1.58
Compounds 6a–v, 8a–f and 9 have been tested on their
ability to inhibit caspase-3 catalyzed proteolytic break-
down of its fluorogenic substrate, Ac-DEVD-AMC.
The caspase-3 activity with and without inhibitors was
measured in accordance with the reported protocol8
using VICTOR2V (Perkin–Elmer) multimode 96/384-
well plate reader by the rate of fluorescence increase
(kex 360 nm, kem 460 nm) due to the liberation of a meth-
ylcoumarin moiety with concomitant increase in its
quantum yield. For all the compounds that exhibited
more than 50% inhibition at a concentration of
100 lM, the dose-dependent caspase-3 inhibition curves
were registered and the IC50 values were calculated using
PRISM 4 (GraphPad) software. The most active com-
pounds displayed dose–response curves with a Hill slope
close to unity, which indicates a high probability of the
compounds being real inhibitors and not promiscuous
ones.
O
S
6g
CH3
0.044
*
O
6h
6i
6j
H
F
CH2–CO2CH3
CH2–CO2CH3
CH2–CO2CH3
4.65
2.50
0.46
Br
O
S
6k
CH2–CO2CH3
0.016
N
N
O
O
*
O
6l
H
F
CH2CH2–CO2CH3
CH2CH2–CO2CH3
CH2CH2–CO2CH3
23.3
5.5
6m
6n
Br
1.08
O
S
6o
CH2CH2–CO2CH3
0.037
*
O
H3C
6p
6q
6r
H
Br
8.11
2.54
0.015
*
H3C
The synthesized 4-methyl-1,3-dioxo-2,3-dihydro-1H-
pyrrolo[3,4-c]quinolines of general formula I displayed
high activity in this in vitro caspase-3 inhibition assay
(Tables 1 and 2). The activity strongly depends on the
nature of substituents in position 2 and especially in
the position 8 of this heterocyclic system. In the general
case, the activity increases with the increase of electron-
withdrawing capacity of the 8-substitutent. Thus, for a
group of 2-unsubstituted compounds 6a–d, compound
6a with R1 = H has IC50 > 100 lM; 8-fluoro derivative
6b and 8-bromo derivative 6c have IC50 equal to 62.8
and 37.1 lM, correspondingly; and 8-(morpholine-4-sul-
fonyl)-substituted compound 6d has IC50 = 0.21 lM.
In this group, the activity changed by three orders of
magnitude. Similar dependencies were observed within
all other congeneric series with identical 2-substituents.
*
O
S
H3C
N
O
*
*
O
CH3
N
6s
6t
SOꢀ3 PyHþ
0.1
H3C
N
CH3
*
*
CH3
N
H3C
N
Br
0.36
CH3
CH3
N
H3C
N
6u
SO3H
0.09
CH3
*
CH3
N
The observed correlations between the electron-with-
drawing ability of the 8-substituent (R1) and the potency
of inhibition are shown in Figure 1. These data suggest
that electrophilicity of the imide carbonyls plays a defi-
nite role in activity of the studied compounds. The
mechanism of inhibition has been studied for a large
variety of compounds possessing the electrophilic carbo-
nyls, such as peptidealdehydes,9 isatins,10 homop-
hthalimides,11 quinazolinones,12 etc. In the reported
cases, the mechanism involved addition of the enz-
ymeÕs catalytic cysteine residue to carbonyl moiety
(Fig. 2).
6v
9
SOꢀ3 Naþ
0.14
H3C
H3C
N
CH3
*
*
CH3
N
N
CN
0.016
CH3
eral formula I given appropriate substituents R1 and
R2 are present.
The nature of the 2-substituents also influences the
activity of the synthesized compounds against caspase-
3. Thus, in all the studied congeneric series with identical
8-substituents, minimal activity was observed for 2-
unsubstituted compounds. The most active compounds
The ability of thiols to reversibly interact with phthal-
imide-like compounds13 in a similar manner suggests
that the caspase enzyme could also be reversibly inacti-
vated by electrophilic carbonyls of compounds of gen-

1844
D. V. Kravchenko et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1841–1845
Table 2. In vitro caspase-3 inhibition assay results for 8-(morpholin-4-
ylsulfonyl) and 1,3,5-trimethyl-1H-pyrazol-4-yl substituted compounds
Among the studied compounds, 8-sulfamide and 8-cya-
no derivatives of 1,3-dioxo-4-methyl-2,3-dihydro-1H-
pyrrolo[3,4-c]quinolines have been identified as potent
inhibitors of caspases-3. The most active compounds
within this series, such as 6k, 6r, 8b, 8f and 9, inhibited
caspase-3 in the 4–20 nM range. Compound 8f was the
most potent inhibitor with IC50 value equal to 4 nM.
Parallel experiments demonstrated that the IC50 values
for Ac-DEVD-CHO, a potent tetrapeptide inhibitor of
caspase-3, was equal to 3.1 nM under the same experi-
mental conditions.
Compd
R1
R2
IC50 (lM)
O
8a
S
O
O
NH2
0.033
*
*
*
H
N
8b
8c
0.020
0.021
S
OH
O
O
H
N
S
O CH3
O
In summary, here we have described the synthesis and
activity of a novel class of potent caspase-3 inhibitors
based on pyrrolo[3,4-c]quinoline-1,3-dione molecular
scaffold. Caspase-3 inhibitory activity of the synthesized
compounds is highly dependent on the substitutions on
the core scaffold, especially at the 8-position. Compound
8f with a morpholinesulfonyl moiety at the 8-position
and 1,3,5-trimethyl-1H-pyrazol-4-yl group at the 2-posi-
tion is the lead compound with potent inhibitory activity
(IC50 = 4 nM). Evaluation against other caspases in-
volved in apoptosis, as well as further SAR studies, is
continuing.
CH3
N
H3C
N
O
O
8d
0.023
O
S
CH3
N
H
CH3
*
*
O
O
O
S
8e
8f
0.055
0.004
N
N
*
*
O
O
S
O
O
Acknowledgements
The authors thank Caroline T. Williams (Department of
Analytical Chemistry, ChemDiv, Inc.) for LCMS and
NMR spectral data.
have methoxycarbonylmethyl (e.g., 6j and 6k with IC50
=
0.46 and 0.016 lM, correspondingly), 2-methylphenyl
(e.g., 6r with IC50 = 0.015 lM) and 1,3,5-trimethyl-1H-
pyrazol-4-yl (e.g., 8f with IC50 = 0.004 lM) substituents
in the position 2. Submicromolar activity was observed
for two 2-bromo substituted derivatives 6j and 6t (IC50
equal to 0.46 and 0.36 lM, correspondingly). Sulfonates
6s, 6u and 6v inhibited caspase-3 in the 0.09–0.14 lM
range.
References and notes
1. Stennicke, H. R.; Ryan, C. A.; Salvesen, G. S. Trends
Biochem. Sci. 2002, 27, 94.
2. Porter, A. G.; Janicke, R. U. Cell Death Differ 1999, 6, 99.
3. Chapman, J.; Magee, W.; Stukenbrok, H.; Beckius, G.;
Milici, A.; Tracey, W. Eur. J. Pharmacol. 2002, 456, 59.
4. Scott, C.; Sobotka-Briner, C.; Wilkins, D.; Jacobs, R.;
Folmer, J.; Frazee, W.; Bhat, R.; Ghanekar, S.; Aharony,
D. Pharmacol. Exp. Ther. 2003, 304, 433.
5. Anselmo, D.; Katori, M.; Kaldas, M.; Hoglen, N.;
Valentino, K.; Busuttil, R.; Kupiec-Weglinski, W.;
Farmer, D. Am. J. Transplant. 2002, 2, 920.
6. Ivachtchenko, A.; Khvat, A.; Kysil, V.; Maliartchuk, S.;
Tkachenko, S.; Okun, I. Drugs Future 2004, 29, 191.
7. (a) Pfitzinger, W. J. Prakt. Chem. 1886, 33, 100; (b)
Godard, A.; Queguiner, G. J. Heterocycl. Chem. 1980, 17,
465; (c) Campaigne, E.; Hutchinson, J. H. J. Heterocycl.
Chem. 1970, 7, 655; (d) Ried, W.; Weidemann, P. Chem.
Ber. 1971, 104, 3341; (e) Ganjian, I.; Khorshidi, M.;
Lalezari, I. J. Heterocycl. Chem. 1991, 28, 1173; (f)
Figure 1. Relationship between inhibitory activities (pIC50) and rp
Hammett constants for R1 substituents in the series 6.
Enz-SH
Enz
S
R2
R2
R2
Enz
O
O
HO
N
N
N
S
R1
R1
O
R1
O
OH
N
N
N
Figure 2. Possible modes of caspase nucleophilic attack on the ÔphtalimideÕ carbonyls.

D. V. Kravchenko et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1841–1845
1845
Ivachtchenko, A. V.; Kobak, V. V.; IlÕyin, A. P.; Trifilen-
kov, A. S.; Busel, A. A. J. Comb. Chem. 2003, 5,
645.
10. Webber, S. E.; Tikhe, J.; Worland, S. T.; Fuhrman, S. A.;
Hendrickson, T. F.; Matthews, D. A.; Love, R. A.; Patick,
A. K.; Meador, J. W.; Ferre, R. A.; Brown, E. L.; DeLisle, D.
A.; Ford, C. E.; Binford, S. L. J. Med. Chem. 1996, 39, 5072.
11. Wang, Q. M.; Johnson, R. B.; Jungheim, L. N.; Cohen, J.
D.; Villarreal, E. C. Antimicrob. Agents Chemother. 1998,
42, 916.
8. Durrieu, F.; Belloc, F.; Lacoste, L.; Dumain, P.; Chabrol,
J.; Dachary-Prigent, J.; Morjani, H.; Boisseau, M. R.;
Reiffers, J.; Bernard, P.; Lacombe, F. Exp. Cell Res. 1998,
240, 165.
9. Webber, S. E.; Okano, K.; Little, T. L.; Reich, S. H.; Xin,
Y.; Fuhrman, S. A.; Matthews, D. A.; Love, R. A.;
Hendrickson, T. F.; Patick, A. K.; Meador, J. W., III;
Ferre, R. A.; Brown, E. L.; Ford, C. E.; Binford, S. L.;
Worland, S. T. J. Med. Chem. 1998, 41, 2786.
12. Scott, C.; Sobotka-Briner, C.; Wilkins, D.; Jacobs, R.;
Folmer, J.; Frazee, W.; Bhat, R.; Ghanekar, S.; Aharony,
D. J. Pharmacol. Exp. Ther. 2003, 304, 433.
13. Oswaldo, N.; Jose, R.; Larry, A. J. Phys. Org. Chem.
1994, 7, 80.