Synthesis and structure-activity relationship of 4-substituted 2-(2-acetyloxyethyl)-8-(morpholine-4-sulfonyl)pyrrolo[3,4-c]quinoline-1, 3-diones as potent caspase-3 inhibitors

Article abstract of DOI:10.1021/jm048987t

Synthesis, biological evaluation, and SAR dependencies for a series of novel 1,3-dioxo-2,3-dihydro-1H-pyrrolo-[3,4-c]quinoline inhibitors of caspase-3 are described. The inhibitory activity of the synthesized compounds is highly dependent on the nature of

Full text of DOI:10.1021/jm048987t

3680
J. Med. Chem. 2005, 48, 3680-3683
Letters
of novel nonpeptide small-molecule inhibitors of caspase-3
having general formula I.
Synthesis and Structure-Activity
Relationship of 4-Substituted
2-(2-Acetyloxyethyl)-8-(morpholine-
4-sulfonyl)pyrrolo[3,4-c]quinoline-
1,3-diones as Potent Caspase-3 Inhibitors
Dmitri V. Kravchenko,† Yulia A. Kuzovkova,†
Volodymyr M. Kysil,‡ Sergey E. Tkachenko,‡,§
Sergey Maliarchouk,‡,# Ilya M. Okun,‡,#
Konstantin V. Balakin,‡,§ and
The synthesis of the 4-substituted pyrrolo[3,4-c]-
quinoline-1,3-dione ring system was accomplished ac-
cording to a sequence of reactions shown in Scheme 1.
Commercially available chloride 1 was reacted with
morpholine in 1,4-dioxane to give sulfonyl amide 2. The
latter was refluxed in a AcOH/water (1:1 v/v) mixture
to afford 8-sulfamoyl isatin 3. Dicarboxylic acids 5 were
prepared via a Pfitzinger reaction9 of 3 with keto esters
4a,b under strong alkali conditions. Acids 5 were then
converted into furandiones 6a,b upon the reaction with
acetic anhydride. Reactions of anhydrides 6a,b with
2-aminoethanol smoothly led to imides 7a,b.
Alexandre V. Ivachtchenko*,‡
Departments of Organic Chemistry, Medicinal Chemistry,
and Molecular Biology and HTS, Chemical Diversity
Research Institute, Khimki, Moscow Reg., Russia, and
ChemDiv, Inc., 11558 Sorrento Valley Road, Suite 5, San
Diego, California 92121
Received December 14, 2004
Abstract: Synthesis, biological evaluation, and SAR depend-
encies for a series of novel 1,3-dioxo-2,3-dihydro-1H-pyrrolo-
[3,4-c]quinoline inhibitors of caspase-3 are described. The
inhibitory activity of the synthesized compounds is highly
dependent on the nature of 4-substituents on the core scaffold.
4-Methyl- and 4-phenyl-substituted derivatives, which were
the most active compounds within this series, inhibited
caspase-3 with IC50 of 23 and 27 nM, respectively.
Scheme 1a
Caspases, a family of cysteine-dependent aspartate-
directed proteases, comprise highly homologous en-
zymes that play an important role in apoptotic cell
death.1 Caspase-3 (apopain) is situated at a key junction
in the apoptosis, mediating apoptotic cascade from the
intrinsic and extrinsic activation pathways.2 Therefore,
caspase-3 is an attractive target for therapeutic inter-
vention. For instance, inhibitors of caspase-3 were
described as promising cardioprotectants,3 neuropro-
tectants,4 and antiarthritic agents.5
1,3-Dioxo-2,3-dihydro-1H-pyrrolo[3,4-c]quinolines rep-
resent a relatively little explored class of heterocyclic
structures with promising physiological activities. Thus,
they were reported as cytotoxic agents6 and central
nervous system active agents, selective agonists, an-
tagonists, or inverse agonists for GABAA (γ-aminobu-
tyric acid A) brain receptors.7 Recently, we described
an efficient synthesis and biological activity of 8-sulf-
amide derivatives of 1,3-dioxo-2,3-dihydro-1H-pyrrolo-
[3,4-c]quinolines.8 Synthesis of 5H-pyrrolo[3,4-c]quino-
line-1,3,4-triones was also reported.7 In this paper, we
describe synthesis and biological evaluation for a series
a Reagents and conditions: (a) morpholine, 1,4-dioxane, room
temp, 1 h, 92%; (b) AcOH/H2O (1:1), reflux, 12 h, 89%; (c) KOH in
H2O, room temp, 12 h, 60-87%; (d) Ac2O, 100 °C, 3 h, 80-88%;
(e) 2-aminoethanol, pyridine, room temp, 1 h, then Ac2O, 80 °C,
30 min, 60-65%.
The second group of 4-substituted pyrrolo[3,4-c]quino-
line-1,3-diones was obtained utilizing the commercially
available 2,3-dioxo-2,3-dihydro-1H-indole-5-sulfonate 8
as starting reactant. According to this approach depicted
in Scheme 2, 8 was treated with keto esters 9a,b under
the conditions of Pfitzinger reaction to afford the cor-
responding dicarboxylic acids 10a,b. Acids 10a,b were
converted into furandiones 11a,b upon reaction with
acetic anhydride in dry pyridine. Compounds 11a,b
were successively treated with 2-aminoethanol and
acetic anhydride in pyridine to afford the corresponding
imides 12a,b. Sulfonyl chlorides 13a,b were then syn-
thesized from 12a,b by using POCl3 in tetramethylene
* To whom correspondence should be addressed. Phone: (858) 794-
4860. Fax: (858) 794-4931. E-mail: av@chemdiv.com.
† Department of Organic Chemistry, Chemical Diversity Research
Institute.
‡ ChemDiv, Inc.
§ Department of Medicinal Chemistry, Chemical Diversity Research
Institute.
# Department of Molecular Biology and HTS, Chemical Diversity
Research Institute.
10.1021/jm048987t CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/29/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3681
Scheme 2a
Scheme 4a
a Reagents and conditions: (a) CH2(CN)2, N-methylmorpholine,
MeOH, reflux, 30 min, 96%; (b) concentrated HCl/AcOH (1:1),
reflux, 12 h, 43%; (c) Ac2O, 100 °C, 30 min, 91%; (d) 2-amino-
ethanol, pyridine, 80 °C, 15 min, then Ac2O, 80 °C, 1 h, 86%; (e)
POCl3, reflux, 1 h, 96%.
Scheme 5a
a Reagents and conditions: (a) LiOH in H2O, room temp, 12 h,
60-85%; (b) Ac2O, pyridine, 100 °C, 30 min, 80-88%; (c) 2-ami-
noethanol, pyridine, room temp, 1 h, then Ac2O, 80 °C, 30 min,
75-97%; (d) POCl3, sulfolane, 80 °C, 2 h, 65-85%; (e) morpholine,
1,4-dioxane, 60 °C, 30 min, 65-95%.
Scheme 3a
a Reagents and conditions: (a) 4-(trifluoromethyl)benzaldehyde,
ZnCl2, Ac2O, 110 °C, 70 h, 20%.
a Reagents and conditions: (a) (for 21a) (NH2)2CS, i-PrOH,
reflux, 30 min, 75%; (b) (for 21b) HSCH2CO2Me, dimethoxyethane,
Cs2CO3, ultrasonic bath, room temp, 1 h, 65%; (c) morpholine (for
2a) or diethyl (2R)-2-amino-2-methylsuccinate (for 22b) or 3-chloro-
4-fluoroaniline (for 22c), i-PrOH, reflux, 20-60 min, 45-77%; (d)
m-CPBA, CH2Cl2, 0 °C, 1 h, 70%; (e) m-CPBA, CH2Cl2, room temp,
4 h, then 40 °C, 1.5 h, 56%.
sulfone (sulfolane). The reaction proceeded at 80 °C and
smoothly afforded the desired product. Finally, mor-
pholides 14a,b were obtained from the reaction of 13a,b
with morpholine in 1,4-dioxane at 60 °C.
Compound 15 was obtained according to a modifica-
tion of the reported procedure10 by condensation of
4-methyl derivative 7a with 4-(trifluoromethyl)benzal-
dehyde in acetic anhydride in the presence of ZnCl2
(Scheme 3).
Scheme 4 shows the synthesis of 4-chloride interme-
diate 20, which was used for the synthesis of 4S- and
4N-substituted pyrrolo[3,4-c]quinoline-1,3-diones. Con-
densation of 3 with malononitrile led to intermediate
16, which was converted into dicarboxylic acid 17 by
reaction with a mixture of concentrated HCl and AcOH.
Acid 17 was converted into furandione 18 upon reaction
with acetic anhydride. Compound 18 was treated with
2-aminoethanol and acetic anhydride in pyridine at 80
°C, and the resulting imide 19 was reacted with POCl3
to afford the desired chloride 20.
Chloride 20 easily reacted with nucleophilic agents
(Scheme 5) such as thiourea, methyl mercaptoacetate,
morpholine, diethyl (2R)-2-amino-2-methylsuccinate,
and 3-chloro-4-fluoroaniline to smoothly afford the cor-
responding 4S- and 4N-substituted products 21a,b and
22a-c. Compound 21a was prone to rapid spontaneous
hydrolysis leading to the corresponding thiol; the latter
was also unstable and underwent slow oxidation, which
led to a disulfide dimer. Oxidation of sulfide 21b with
m-chloroperbenzoic acid (m-CPBA) in dichloromethane
at 0 °C afforded sulfinate 23. When the same reaction
was performed at higher temperature (∼40 °C), sul-
fonate 24 was formed.
Finally, Suzuki coupling11 of 20 with 3-chloro-4-
fluorophenylboronic acid, 4-(N,N-dimethylaminocarbo-
nyl)phenylboronic acid, or 3-pyridylboronic acid in the
presence of PdCl2(PPh3)2 afforded the corresponding
4-aryl substituted compounds 25a-c (Scheme 6).
Compounds 7a,b, 14a,b, 15, and 19-25 have been
tested for their ability to inhibit caspase-3 catalyzed
proteolytic breakdown of its fluorogenic substrate, Ac-
DEVD-AMC. The synthesized 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
(Table 1). The most active compounds within this series
have alkyl, aryl, and heteroaryl 4-substituents. They
demonstrated inhibitory activity (IC50) in the 20-60 nM
range. 4-Hydroxy- and 4-chloro-substituted derivatives
(19 and 20) and 4-sulfanyl derivatives with their
oxidized analogues (21a,b, 23, and 24) are significantly

3682 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11
Letters
Scheme 6a
a Reagents and conditions: (a) 3-chloro-4-fluorophenylboronic
acid (for 25a) or 4-[(dimethylamino)carbonyl]phenylboronic acid
(for 25b) or 3-pyridylboronic acid (for 25c), dimethoxyethane,
PdCl2(PPh3)2, Na2CO3, H2O, reflux, 4 h, 19-25%.
Figure 1. Caspase-3 inhibition curves in the presence of
different concentrations of the fluorogenic substrate.
Table 1. In Vitro Caspase-3 Inhibitory Potency of
4-Substituted 2-(2-Acetyloxyethyl)-8-(morpholine-4-
sulfonyl)pyrrolo[3,4-c]quinoline-1,3-diones of General Formula I
compd
R
IC50, nM
7a
Me
i-Pr
Ph
23 ( 2
7b
40 ( 3
14a
14b
15
19
20
27 ( 3
2-furyl
36 ( 4
4-CF3-C6H4-CHCH
OH
33 ( 3
10900 ( 3800
294 ( 16
255 ( 20
1290 ( 30
15350 ( 3500
5190 ( 2700
27900 ( 3300
352 ( 21
362 ( 26
58 ( 6
Cl
21a
21b
22a
22b
22c
23
S-C(NH)NH2
S-CH2CO2Me
1-morpholyl
Figure 2. Studies on mechanism of recombinant human
caspase-3 inhibition by 7a.
EtOC(O)-CH2-CH(CO2Et)-NH
3-Cl-4-F-C6H3-NH
SO-CH2CO2Me
SO2-CH2CO2Me
4-(Me2NCO)-C6H4
3-Cl-4-F-C6H3
3-pyridyl
after dilution, the measured activity of the sample was
practically identical to that of inhibition in control
samples, which were preincubated at normal concentra-
tions of the compound and enzyme.
24
25a
25b
25c
33 ( 4
33 ( 5
The plots also demonstrate the noncompetitive char-
acter of inhibition because the inhibitor decreases Vmax
without effecting the apparent Km (Figure 2). The same
conclusion can be made from an analysis of Line-
weaver-Burk plots (see Supporting Information). The
dissociation constant Km for Ac-DEVD-AMC calculated
from these plots was estimated to be 6 ( 2 µM at 95%
confidence intervals. The selectivity of 7a was estimated
in the reactions with a panel of active recombinant
human caspases-1-9 and the specific substrates for
each individual enzyme. Compound 7a was the most
potent against caspase-3, but the level of selectivity
versus the remaining caspases was modest.
To assess the antiapoptotic activity of 7a in a cell-
based assay, we measured the viability of human Jurkat
T cells treated with 10 µM staurosporin. As a positive
control, we used a known cell-permeable apoptosis
inhibitor Z-VAD-FMK. Compound 7a demonstrated a
higher level of protection than the peptide inhibitor, as
can be seen from the cell viability values (Figure 3).
These data also suggest, though indirectly, good cell
permeability of 7a.
In summary, we synthesized a series of novel non-
peptide caspase-3 inhibitors based on a 1,3-dioxo-2,3-
dihydro-1H-pyrrolo[3,4-c]quinoline molecular scaffold.
The inhibitory activity depends on the nature of the
substituent in position 4 of this heterocyclic system.
Compounds 7a and 14a with 4-methyl and 4-phenyl
substituents are the most potent compounds (IC50 ) 23
and 27 nM, respectively). Our primary data suggest
noncompetitive and reversible character of caspase-3
less potent (IC50 ) 250-1300 nM). The less active
compounds within the synthesized set have alkyl- and
arylamino 4-substituents (IC50 ) 5-28 µM). It is sug-
gested that electrophilicity of imide carbonyls, which can
be affected by different types of 4-substituents, plays
an essential role in the activity of the studied com-
pounds. In accordance with this suggestion, we have
observed a clear correlation between the inhibitory
activity and nucleophilicity of the 4-substituents (see
Supporting Information).
The mechanism of inhibition has been assessed for
7a, which is the most active in this series. The noncom-
petitive character of the inhibition has been demon-
strated in the experiments, in which the inhibitory
potency was measured in the presence of increasing
concentration of the substrate Ac-DEVD-AMC (Figure
1). The incremental increase in the substrate concentra-
tion did not shift the inhibition curves to the right as
one would expect for competitive inhibition. An IC50 of
23 ( 6 nM was found for 7a from these curves. Parallel
experiments demonstrated that the IC50 value for Ac-
DEVD-CHO, a potent tetrapeptide inhibitor of caspase-
3, was equal to 3.1 ( 0.2 nM under the same experi-
mental conditions. Inhibition by 7a was reversible, as
demonstrated by an experiment in which a 10-fold
concentration of the compound was preincubated for 1
h with 10-fold caspase-3 concentration followed by 10-
fold dilution of the mixture into a standard assay buffer
to a final compound concentration of 30 nM. At 1.5 h

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 11 3683
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studies are continuing.
Acknowledgment. The authors thank Caroline T.
Williams (Department of Analytical Chemistry, Chem-
Div, Inc.) for NMR and LCMS data. The authors also
thank the colleagues of the Scripps Center for Mass
Spectrometry (La Jolla, CA) for HRMS data.
Supporting Information Available: Synthesis proce-
dures and analytical data for intermediates and final products
(1H NMR, 13C NMR, HRMS), experimental procedures for the
biological tests, inhibition kinetics, and selectivity data for 7a,
and data on the effect of 4-substituents on the caspase-3
inhibitory activity. This material is available free of charge
via the Internet at http://pubs.acs.org.
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