A novel class of small-molecule caspase-3 inhibitors prepared by multicomponent reactions

Article abstract of DOI:10.1016/j.ejmech.2012.05.001

A series of tetra- and pentasubstituted polyfunctional dihydropyrroles 5 and 6 were synthesized via practical multicomponent reactions (MCRs) for research on their structure-activity relationship as caspase-3 inhibitors. Among 39 compounds evaluated, 14 of them exhibited inhibition against caspase-3 with IC₅₀ ranging from 5 to 20 μM. The inhibitory activities of 5 and 6 depend on the nature of substituents on different positions. 5 and 6 possess a different scaffold from those previously reported and are the first caspase-3 inhibitors prepared via MCRs. The most active compounds 5k (IC₅₀ = 5.27 μM) could therefore be used as a lead for the development of highly potent caspase-3 inhibitors as drug candidates for therapeutic agents by taking advantage of MCRs.

Full text of DOI:10.1016/j.ejmech.2012.05.001

European Journal of Medicinal Chemistry 54 (2012) 232e238
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A novel class of small-molecule caspase-3 inhibitors prepared
by multicomponent reactions
,
,
b,
**
Qiuhua Zhu a b, Lixin Gao c, Zhipeng Chen a, Sichao Zheng a, Huafei Shu a, Jia Li c , Huanfeng Jiang
,
***
,
Shuwen Liu a
*
a School of Pharmaceutical Sciences, Southern Medical University, 1838 Northern Guangzhou Road, Guangzhou, Guangdong 510515, China
b School of Chemistry and Chemical Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510640, China
c Chinese National Center for Drug Screening, Shanghai Institute of Materia Medica, Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, 189 Guo Shou Jing Road,
Zhangjiang Hi-Tech Park, Shanghai 201203, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
< The MCR scope for the synthesis of
4-BrC6H4 > cyclohexyl > PhCH2 >> Ph > CH3C2H4
pentasubstituted
polyfunctional
R4CHO
R3NH2
R2NH2
R2
HN
4
3
dihydropyrroles 6 were expanded.
< Tetra- and pentasubstituted dihy-
O
4-BrC6H4
MCRs
> 3,4-(Me)2C6H3
> 3-ClC6H4
........
N
R3
dropyrroles
5 and 6 are a novel
O
series of caspase-3 inhibitors.
one-pot
2
R1
R4
>> cyclohexyl
< The inhibitory activity of 5 and 6
depend on the nature of substitu-
ents on different positions.
R1O2C
CO2R1
O
Et > Me
H, Ph > Me
1
5a-y: R4 = H; 6a-n: R4 = alkyl / aryl
(14 examples with IC50 of 5 - 20 µM)
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tetra- and pentasubstituted polyfunctional dihydropyrroles 5 and 6 were synthesized via
practical multicomponent reactions (MCRs) for research on their structureeactivity relationship as
caspase-3 inhibitors. Among 39 compounds evaluated, 14 of them exhibited inhibition against caspase-3
Received 26 January 2012
Received in revised form
30 April 2012
Accepted 1 May 2012
Available online 11 May 2012
with IC50 ranging from 5 to 20
substituents on different positions. 5 and 6 possess a different scaffold from those previously reported
M)
mM. The inhibitory activities of 5 and 6 depend on the nature of
and are the first caspase-3 inhibitors prepared via MCRs. The most active compounds 5k (IC50 ¼ 5.27
m
could therefore be used as a lead for the development of highly potent caspase-3 inhibitors as drug
candidates for therapeutic agents by taking advantage of MCRs.
Keywords:
Caspase-3 inhibitor
Dihydropyrrole
Multicomponent reaction
Synthesis
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
pathogenesis of many diseases is closely connected with aberrantly
regulated apoptosis [1e6]. For example, cancer and Alzheimer’s
Apoptosis, or programmed cell death, is an essential physio-
logical process of tissue development and homeostasis. The
disease are characterized by insufficient and excessive apoptosis,
respectively [4,5]. Caspases, a family of cysteinyl aspartate-specific
proteases, have been proved to be critical in mediating the signal
transduction and execution of apoptosis [7]. Evidence from
caspase-deficient cells and cell lines, as well as immunodepleted
cell-free extracts, have indicated that caspase-3 is a key executioner
caspase and the inhibition of caspase-3 activity can significantly
prevent apoptosis in vitro and in vivo [8e10]. Consequently, great
* Corresponding author. Tel.: þ86 20 61648538; fax: þ86 20 61648655.
** Corresponding author. Tel.: þ86 20 22236518; fax: þ86 20 87112906.
*** Corresponding author. Tel./fax: þ86 21 50801552.
E-mail addresses: jli@mail.shcnc.ac.cn (J. Li), jianghf@scut.edu.cn (H. Jiang),
liusw@smu.edu.cn (S. Liu).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.05.001

Q. Zhu et al. / European Journal of Medicinal Chemistry 54 (2012) 232e238
233
interest has emerged in developing caspase-3 inhibitors for use as
therapeutic drugs in the treatment of excessive apoptosis-related
diseases such as Alzheimer’s and Parkinson’s diseases.
dihydropyrroles 6 with R2 ¼ aryl or alkyl and R3 ¼ alkyl (all of 6 with
the exception of 6f, 6g and 6n) and 6n with R2 ¼ R3 ¼ aryl (Ph) were
prepared by the MCRs under conditions C and D, respectively [24].
When 6f and 6g (R2 ¼ 4-HOOCC6H4) were prepared under condition
C or D, complex products were obtained. We found that triethyl-
amine (N(Et)3, 0.5 equiv) was needed for the synthesis of 6f as well
as 6g and that the ratios of starting materials were related to the
nature of groups R4. Conditions E and F were suitable for the
synthesis of 6f with R4 ¼ aryl and 6g with R4 ¼ alkyl, respectively.
A total of 39 tetra- and pentasubstituted polyfunctional dihy-
dropyrroles 5 and 6 were synthesized in 60e98% yields (19
compounds of which were not previously reported). All the new
compounds were characterized by 1H and 13C NMR, MS (GCeMS or
ESIeMS) and elemental analysis. Satisfactory analytical data
consistent with the shown molecular structures and a purity of at
least 95% (determined by elemental analysis) were obtained for all
compounds.
Until now, two kinds of inhibitors of caspase-3, peptidic and
nonpeptidic inhibitors, have been reported. Most of peptidic
inhibitors exhibit high inhibitory activities against caspase-3 with
IC50 in the nanomolar range [11] and even with Ki in the picomolar
range [12]. However, they have limited clinical utility due to poor
cell permeability and metabolic stability. Thus, considerable efforts
have been devoted to the development of nonpeptidic caspase-3
inhibitors.
High throughput and virtual screening of libraries of small
molecules already on hand have helped identify novel lead
compounds, such as lead compounds IeIII (Fig. 1) [13e15], which
have been used as starting points for further developments in the
field of caspase-3 inhibitors. New inhibitors with higher potency
can often be developed from lead compounds by further structure-
based design and optimization. For example, lead compound isatin
(I, Fig. 1) has led to a series of more potent analogs, such as IV in
Fig. 1 [16], with IC50 in the nanomolar range. Although rapid
advances on nonpeptidic caspase-3 inhibitors have been made,
none of these compounds have been developed as a drug for
treatment of caspase-3-related diseases. Therefore, diverse potent
and specific caspase-3 inhibitors are needed.
2.2. Biological activity
Compounds 5 and 6 were evaluated for their inhibitory activities
against caspase-3 by the method as described in our previous work
[14]. For all the compounds that exhibited more than 50% inhibition
at the concentration of 20
mg/mL, the concentration-dependent
Multicomponent reaction (MCRs) is an ideal reaction mode
which is consistent with the concept of green chemistry, such as
atom economy, high efficiency. In addition, MCRs could be used to
build structural diversity and complexity of compound libraries [17],
whichletthemhave playedimportantrole inmoderndrugdiscovery
[18,19]. In order to discover new lead compounds, we developed
practical MCRs for the synthesis of heterocyclic compounds with
biological activity [20e25]. It was found that the MCR products,
tetra- and pentasubstituted polyfunctional dihydropyrroles 5 and 6,
reported in Ref. [24] also acted as caspase-3 inhibitors [26]. To our
knowledge, 5 and 6 possess a different molecular scaffold from
existingnonpeptidiccascpase-3inhibitorsandarethefirstinhibitors
against caspase-3 prepared by MCRs. Therefore, new dihy-
dropyrroles 5 and 6 were synthesized for research on their
structureeactivity relationship as caspase-3 inhibitors.
caspase-3 inhibition curves were further conducted and the IC50
values were calculated by using PRISM 4 (GraphPad) software. Final
IC50 values were the average of three independent experimental
results. Peptidic caspase-3 inhibitor Ac-DEVD-CHO was used as
positive control compound. In the same experimental conditions,
Ac-DEVD-CHO inhibited the caspase-3 activity with IC50 value of
8.75 ꢀ 0.63 nM. The IC50 values against caspase-3 for 5 and 6 are
summarized in Table 1.
The activity results indicate that most of 5 displayed inhibitory
activities against caspase-3 (Table 1). Replacement of the methyl
group by the ethyl group (R1) caused a modest activity increase up to
2-fold (5b > 5a, 5d > 5c). Both of R2 and R3 could significantly
influence the inhibitory activities of 5. The orders of inhibitory
activities of 5 with different R2 and R3 against caspase-3 was 4-
BrC6H4
BrC6H4
MeC6H4
>
cyclohexyl > PhCH2 >> Ph
3,4-(Me)2C6H3 3-ClC6H4
Ph 4-NO2C6H4
> CH3C2H4; and 4-
>
>
z
3,4-(Cl)2C6H3
>
4-
4-
2. Results and discussion
>
>
>
4-MeOC6H4 z
ClC6H4 > Naphthalen-1-yl >> cyclohexyl > CH3C2H4, allyl and
2.1. Chemistry
Pyridin-2-yl, respectively. Generally, 4-BrC6H4 group was an opti-
mized group for both of R2 and R3 (5k, 5.27
mM) in this in vitro
As shown in Scheme 1, the reaction conditions for the synthesis
of 5 and 6 correlate with the nature of substituents. Tetrasubstituted
polyfunctional dihydropyrroles 5 with R2 ¼ alkyl and R3 ¼ aryl or
alkyl (all of 5 with the exception of 5g, 5ie5k) as well as 5 with
R2 ¼ R3 ¼ aryl (5g, 5ie5k) were synthesized by the MCRs of but-2-
ynedioates 1, amines 2 and 3, formaldehyde 4a under conditions A
and B, respectively [24]. Pentasubstituted polyfunctional
caspase-3 inhibition assay. Cyclohexyl as R2 increased compounds
activity, but as R3 afforded inactive compounds. In addition, 5y
showed no inhibition, which may indicate that the hydrogen on the
nitrogen in 5 is needed for their inhibitoryactivityagainst caspase-3.
As shown in Table 1, R1 showed similar influence on the inhib-
itory activity of 6 to that on 5 with Et > Me. All of R2, R3 and R4 could
significantly influence the inhibitory activity of 6. The activity order
O
O
O
S
O
O
O
N
O
O
O
N
O2N
O
NH
O
O
O
N
O
OH
N
O
II
III
I
Ki (app) = 0.5 µM
IV
3.9 nM
13.6 µM
0.150 µM
Fig. 1. Structures and IC50 values of several lead compounds with different scaffolds.

234
Q. Zhu et al. / European Journal of Medicinal Chemistry 54 (2012) 232e238
Scheme 1. Synthesis of tetra- and pentasubstituted polyfunctional dihydropyrroles 5 and 6.a.
of 6 with different R2 was Naphthalen-1-yl > 4-BrC6H4 > Ph > 4-
NO2C6H4 >> 4-HOOCC6H4. The most potent caspase-3 inhibitor
among this series was 6d with an IC50 of 7.92 mM. 6 with carboxyl or
hydroxyl groups show much lower inhibitory activity (6f, 6g,
6ie6k), which indicates active hydrogen on these substituted
positions could significantly decrease the activity of 6.
dihydropyrroles 5 and 6 were synthesized (19 compounds of which
were not previously reported) and evaluated for their inhibitory
activities against caspase-3. The activity results show that 5 and 6
are a series of caspase-3 inhibitors as well as that the activities of
these compounds depend on the nature of substituents on different
positions. These caspase-3 inhibitors possess a different scaffold
from those previously reported and are the first caspase-3 inhibi-
tors prepared via MCRs. The most active compounds 5k
Compared with the structures of most of existing peptide and
nonpeptide caspase-3 inhibitors, 5 and 6 have not high electro-
philic groups that react with the active site cysteine residue of
caspase-3, such as high electrophilic carbonyl groups in I and II
(Fig. 1) [11e14] as well as high electrophilic Michael addition
acceptors [27,28]. Because different binding modes are observed
with different kinds of inhibitors bound to the same enzyme
frequently [11,29], a different inhibitory mechanism might be
expected for the novel class of caspase-3 inhibitors 5 and 6.
In view of the influences of the logarithm of the partition
coefficient (log P) on hydrophileelipophile balance of drugs [30] as
well as their release [31] and transport [32] in vivo, the log P values
in octanolewater of compounds 5 and 6 were calculated (ClogP in
Table 1). The ClogP error codes indicate that the ClogP values of only
14 compounds are based on trusted fragment values, while the
other 25 compounds are based on approximated, priori fragment
values or even very high log P unrealistic in nature (note d in
Table 1). Therefore, experimental log P values are needed to eval-
uate the calculated ones. Considering structure, biological activity
and solubility, the log P values of 5k, 5w, 6c and 6d were deter-
mined by shake-flask method [30,33]. To our surprise, the experi-
mental log P values for these compounds are much lower than the
calculated ones (Table 1). Since other compounds in Table 1 are the
derivatives of these compounds, it is expected that the experi-
mental log P values of 5 and 6 may be lower than 3. According to
(IC50 ¼ 5.27
mM) could therefore be used as a lead for the devel-
opment of highly potent caspase-3 inhibitors as drug candidates for
therapeutic agents by taking advantage of MCRs. The scope of the
MCRs for the synthesis of 5 and 6 as well as their inhibitory
mechanism, permeability and further structureeactivity relation-
ship as caspase-3 inhibitors are under investigation in our groups.
4. Experimental protocols
4.1. Chemistry
All melting points were taken on an XT-4 micro melting point
apparatus and are uncorrected. 1H NMR (400 MHz) and 13C NMR
(100.6 MHz) spectra were recorded using a Bruker Avance 400 MHz
NMR spectrometer and respectively referenced to 7.24 and
77.0 ppm for chloroform-d with TMS as internal standard. Mass
spectra were recorded on an API 4000QTRAP. TLC was performed
using commercially prepared 100e400 mesh silica gel plates
(GF254), and visualization was effected at 254 and 365 nm. All the
other chemicals were purchased from Aldrich Chemicals.
4.1.1. General procedure for the preparation of tetrasubstituted
polyfunctional dihydropyrroles 5 with alkyl R2 and alky/aryl R3
(condition A) [24]
Lipinski’s Five of Rules (H-bond donors
< 5, molecular
weight < 500, Log P < 5 and H-bond acceptors < 10) [34], both of 5
and 6 are drug-like compounds and hence could be used as new
drug leads for the development of more potent analogs by taking
advantages of MCRs.
Primary amines 3 (2 mmol), 38% formaldehyde 4a (120 mg,
1.5 mmol) and HOAc (120 mg, 2 mmol) were dropwise added into
the mixture of EtOH (4 mL), but-2-ynedioates 1 (1 mmol) and
primary or secondary aliphatic amines 2 (1 mmol) kept at room
temperature for 10e30 min in sequence, followed with stirring at
70 ꢁC for desired time (monitored by TLC). After completion of the
reactions, the product mixture was purified by preparative TLC
with n-hexane/ethyl acetate (10:1e1:1) as eluent to afford the
desired products in 85e93% yields (Table 1, all of 5 with the
exception of 5g, 5iek). For the characterization data of 5ae5d, 5f,
5h and 5x see Ref. [24].
3. Conclusions
In conclusion, we have expanded the scope of the MCRs for the
synthesis of pentasubstituted dihydropyrroles
6 to include
aromatic amines with carboxyl by developing new reaction
conditions. 39 tetra- and pentasubstituted polyfunctional

Q. Zhu et al. / European Journal of Medicinal Chemistry 54 (2012) 232e238
235
Table 1
Inhibitory activity of polyfunctional dihydropyrroles 5 and 6 against caspase-3.
R2
HN
O
O
O
N
R3
N
N
Br
R1O2C
R4
EtO2C
5a-5x (R4=H)
5y
6a-6n (R4=aryl, alkyl)
.
Compound
R1
R2
R3
R4
Inhibition / %a
IC50
/
m
Mb
ClogPc
Errord
5a
5b
5c
5d
5e
5f
5g
5h
5i
Me
Et
Me
Et
Me
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Me
Et
Et
Et
Et
Et
Me
Et
Et
Et
Et
Et
Et
Et
Cyclohexyl
Cyclohexyl
PhCH2
Ph
Ph
Ph
Ph
Ph
Ph
Ph
PhCH2
4-MeC6H4
4-FC6H4
4-BrC6H4
CH3C2H4
Cyclohexyl
allyl
4-MeC6H4
3,4-MeC6H3
4-MeOC6H4
Naphthalen-1-yl
Pyridin-2-yl
3-ClC6H4
4-ClC6H4
3,4-ClC6H3
4-BrC6H4
4-NO2C6H4
4-BrC6H4
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
HOC2H4
HOC2H4
HOC2H4
PhCH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
95.03 ꢀ 3.25
100.5 ꢀ 2.13
98.00 ꢀ 2.97
98.36 ꢀ 2.58
62.54 ꢀ 15.86
28.21 ꢀ 1.46
74.10 ꢀ 10.94
97.88 ꢀ 2.95
96.38 ꢀ 1.17
67.86 ꢀ 6.83
97.44 ꢀ 2.52
17.57 ꢀ 3.24
91.69 ꢀ 7.97
12.75 ꢀ 4.39
99.88 ꢀ 3.85
105.6 ꢀ 2.92
97.04 ꢀ 9.14
94.52 ꢀ 1.59
29.29 ꢀ 10.27
99.25 ꢀ 3.88
97.14 ꢀ 3.28
97.72 ꢀ 1.80
100.4 ꢀ 0.84
95.11 ꢀ 2.26
48.71 ꢀ 14.47
69.29 ꢀ 9.57
90.30 ꢀ 1.19
85.81 ꢀ 14.88
85.29 ꢀ 2.78
98.72 ꢀ 3.38
38.37 ꢀ 2.73
24.21 ꢀ 8.25
40.29 ꢀ 8.99
24.66 ꢀ 8.95
35.81 ꢀ 2.89
33.27 ꢀ 14.33
36.31 ꢀ 25.22
89.66 ꢀ 5.40
91.70 ꢀ 4.18
28.18 ꢀ 1.28
13.25 ꢀ 0.46
19.67 ꢀ 0.97
19.14 ꢀ 1.05
>45
4.35
4.88
4.03
4.56
3.38
3.91
4.70
4.50
10
10
40
40
10
10
30
40
30
30
30
30
30
30
10
10
10
10
10
10
10
10
10
10
30
40
40
51
51
40
40
30
30
40
40
40
42
40
51
PhCH2
CH3C2H4
CH3C2H4
Ph
PhCH2
4-MeC6H4
4-FC6H4
>45
16.58 ꢀ 0.86
11.33 ꢀ 0.46
>45
5.27 ꢀ 0.40
5.70
5.29
5j
5k
5l
4-BrC6H4
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Cyclohexyl
Morpholino
Ph
6.73/1.75e
4.43
5m
5n
5o
5p
5q
5r
5s
5t
5u
5v
5w
5x
5y
6a
6b
6c
6d
6e
6f
>45
5.31
4.15
5.38
5.83
4.80
6.06
3.38
5.59
11.32 ꢀ 0.73
9.76 ꢀ 0.44
23.74 ꢀ 2.18
25.23 ꢀ 6.43
10.44 ꢀ 1.02
24.23 ꢀ 2.66
10.96 ꢀ 1.06
8.35 ꢀ 1.07
15.99 ꢀ 1.60
5.59
6.19
5.74/1.80e
4.62
H
3.81
5.76
6.29
Ph
Ph
Ph
Ph
Ph
Ph
Me
Me
4-FC6H4
Ph
Ph
Ph
Ph
32.32 ꢀ 1.19
27.66 ꢀ 1.53
9.98 ꢀ 1.69
7.92 ꢀ 1.02
40.96 ꢀ 6.15
Ph
4-BrC6H4
Naphthalen-1-yl
4-NO2C6H4
4-HOOCC6H4
4-HOOCC6H4
Ph
Ph
Ph
4-MeC6H4
4-FC6H4
4-BrC6H4
4-MeC6H4
7.45/2.25e
7.46/2.00e
6.68
6.37
4.91
5.35
4.07
3.93
4.43
6.56
6.57
6g
6h
6i
6j
6k
6l
6m
6n
CH3C2H4
4-MeC6H4
>45
26.80 ꢀ 2.66
Ph
7.41
a
Inhibition in a initial screening at the concentration of 20 mg/mL.
b
c
Mean value ꢀ standard deviation (at least three independent assays).
Calculated log P value using ClogP program in Sybyl 7.3.
d
ClogP error codes: 10 (trusted fragment value used), 30 (approximated fragment value used), 40 (a priori fragment value used), 42 (vinyl approximation based on priori
value) and 51 (very high log P unrealistic in nature).
e
Experimental log P.
4.1.1.1. Methyl
2,5-dihydro-5-oxo-1-phenyl-4-(propylamino)-1H-
(m, 2H), 1.73e1.67 (m, 11H), 0.92 (t, J ¼ 7.2 Hz, 3H) ppm; 13C NMR
pyrrole-3-carboxylate
(5e). 91%
yield,
White
solid,
(150 MHz, CDCl3):
d
¼ 165.4, 100.0, 59.4, 44.5, 34.9, 25.5, 24.7, 21.5,
mp ¼ 87.0e88.0 ꢁC; 1H NMR (400 MHz, DMSO):
d
¼ 7.80e7.77 (m,
14.6, 11.3 ppm; GCeMS: m/z
C16H26N2O3; C, 65.28; H, 8.90; N, 9.52; O, 16.30; Found: C, 65.41; H,
8.79; N, 9.38.
¼
294 (Mþ); Anal. Calcd for
2H), 7.47e7.38 (m, 2H), 7.26e7.17 (m, 1H), 4.44 (s, 2H), 3.91e3.80
(m, 5H), 1.70e1.64 (m, 2H), 1.01 (t, J ¼ 7.2 Hz, 3H) ppm; 13C NMR
(101 MHz, CDCl3):
d
¼ 164.80, 164.77, 139.06, 129.35, 125.27, 119.71,
96.33, 51.14, 48.31, 29.92, 24.72, 11.31 ppm; GCeMS: m/z ¼ 274
(Mþ); Anal. Calcd for C15H18N2O3: C, 65.68; H, 6.61; N, 10.21; O,
17.50; Found: C, 65.71; H, 6.53; N, 10.02.
4.1.1.3. Ethyl 1-cyclohexyl-4-(cyclohexylamino)-2,5-dihydro-5-oxo-
1H-pyrrole-3-carboxylate
(5m). 80%
yield,
White
solid,
mp ¼ 101.5e104.5 ꢁC; 1H NMR (600 MHz, CDCl3):
d
¼ 6.62 (b, 1H),
4.48 (b, 1H), 4.09e4.08 (m, 2H), 3.89 (b, 1H), 3.80 (b, 2H), 1.87e1.28
4.1.1.2. Ethyl 4-(cyclohexylamino)-2,5-dihydro-5-oxo-1-propyl-1H-
(m, 10H), 1.19e1.05 (m, 13H) ppm; 13C NMR (150 MHz, CDCl3):
pyrrole-3-carboxylate (5l). 86% yield, yellow solid, mp ¼ 64ꢂ67 ꢁC;
d
¼ 165.6,164.7, 96.4, 59.2, 51.1, 50.3, 43.4, 34.8, 31.0, 25.5, 25.4, 24.7,
1H NMR (600 MHz, CDCl3):
d
¼ 6.75 (b, 1H), 4.595e4.591 (m, 1H),
14.5 ppm; GCeMS: m/z ¼ 334 (Mþ); Anal. Calcd for C19H30N2O3: C,
4.20 (q, J ¼ 7.2 Hz, 2H), 3.93 (s, 2H), 3.41 (t, J ¼ 7.2 Hz, 2H),1.98e1.96
68.23; H, 9.04; N, 8.38; O, 14.35; Found: C, 68.10; H, 9.31; N, 8.17.

236
Q. Zhu et al. / European Journal of Medicinal Chemistry 54 (2012) 232e238
4.1.1.4. Ethyl 1-allyl-4-(cyclohexylamino)-2,5-dihydro-5-oxo-1H-pyr
4H), 4.59 (b, 1H), 4.38 (s, 2H), 4.26 (q, J ¼ 7.2 Hz, 2H), 2.01e1.99 (m,
2H), 1.76e1.61 (m, 3H), 1.45e1.19 (m, 8H) ppm; 13C NMR (150 MHz,
role-3-carboxylate (5n). 54% yield, yellow solid, mp ¼ 94.5e97.0 ꢁC;
1H NMR (400 MHz, CDCl3):
d
¼ 5.80e5.71 (m, 1H), 5.20e5.16 (m,
CDCl3):
d
¼ 164.6, 140.0, 134.9, 130.1, 124,9, 119.3, 117.1, 59.8, 50.6,
2H), 4.60e4.54 (m, 1H), 4.18 (q, J ¼ 7.2 Hz, 2H), 4.04 (d, J ¼ 6.0 Hz,
2H), 3.89 (s, 2H), 1.97e1.93 (m, 2H), 1.72e1.67 (m, 2H), 1.38e1.35
(m, 1H), 1.38e1.14 (m, 8H) ppm. 13C NMR (101 MHz, CDCl3):
47.9, 34.9, 34.8, 25.5, 24.7, 14.6 ppm; GCeMS: m/z ¼ 362 (Mþ); Anal.
Calcd for C19H23ClN2O3: C, 62.89; H, 6.39; Cl, 9.77; N, 7.72; O, 13.23;
Found: C, 62.91; H, 6.29; N, 7.51.
d
¼ 165.67, 165.30, 132.46, 120.05, 118.14, 96.75, 59.42, 50.40, 47.17,
45.45, 34.88, 25.55, 24.69, 14.55 ppm; GCeMS: m/z ¼ 292 (Mþ);
Anal. Calcd for C16H24N2O3: C, 65.73; H, 8.27; N, 9.58; O, 16.42;
Found: C, 65.61; H, 8.12; N, 9.37.
4.1.1.11. Ethyl 1-(4-chlorophenyl)-4-(cyclohexylamino)-2,5-dihydro-
5-oxo-1H-pyrrole-3-carboxylate (5u). 94% yield, White solid,
mp ¼ 91.5e92.5 ꢁC; 1H NMR (400 MHz, CDCl3):
d
¼ 7.72e7.30 (m,
4H), 4.58e4.53 (m, 1H), 4.35 (s, 2H), 4.23 (q, J ¼ 7.2 Hz, 2H),
4.1.1.5. Ethyl 4-(cyclohexylamino)-2,5-dihydro-5-oxo-1-p-tolyl-1H-
2.00e1.96 (m, 2H), 1.74e1.69 (m, 3H), 1.40e1.15 (m, 8H) ppm; 13C
pyrrole-3-carboxylate
(5o). 87%
yield,
White
solid,
NMR (100 MHz, CDCl3):
d
¼ 165.3, 164.4, 137.3, 129.9, 129.0, 120.2,
mp ¼ 106.4e107.4 ꢁC; 1H NMR (600 MHz, CDCl3):
d
¼ 7.63e7.18 (m,
96.2, 59.7, 50.5, 47.8, 34.7, 25.4, 24.6, 14.5 ppm; GCeMS: m/z ¼ 362
(Mþ); Anal. Calcd for C19H23ClN2O3: C, 62.89; H, 6.39; Cl, 9.77; N,
7.72; O, 13.23; Found: C, 62.81; H, 6.32; N, 7.61.
4H), 4.63e4.62 (m, 1H), 4.38 (s, 2H), 4.25 (q, J ¼ 7.2 Hz, 2H), 2.34 (s,
3H), 2.03e2.00 (m, 2H), 1.76e1.19 (m, 11H) ppm; 13C NMR
(150 MHz, CDCl3):
d
¼ 164.3, 136.4, 134.7, 129.6, 119.6, 59.6, 50.6,
48.1, 34.8, 25.6, 24.7, 20.9, 14.6 ppm; GCeMS: m/z ¼ 342 (Mþ); Anal.
Calcd for C20H26N2O3: C, 70.15; H, 7.65; N, 8.18; O, 14.02; Found: C,
70.01; H, 7.49; N, 8.32.
4.1.1.12. Ethyl 1-(3,4-dichlorophenyl)-4-(cyclohexylamino)-2,5-dihydro-
5-oxo-1H-pyrrole-3-carboxylate (5v). 91% yield, White solid,
mp ¼ 114.5e115.0 ꢁC; 1H NMR (600 MHz, CDCl3):
d
¼ 7.99e7.42 (m,
3H), 4.58e4.56 (m, 1H), 4.36 (s, 2H), 4.26 (q, J ¼ 7.2 Hz, 2H), 2.01e1.99
4.1.1.6. Ethyl4-(cyclohexylamino)-2,5-dihydro-1-(3,4-dimethylphenyl)-
(m, 2H), 1.76e1.19 (m, 11H) ppm; 13C NMR (150 MHz, CDCl3):
5-oxo-1H-pyrrole-3-carboxylate (5p). 91% yield, White solid,
d
¼ 164.6, 133.1, 130.6, 120.7, 118.1, 100.0, 59.9, 50.7, 47.8, 34.8, 25.6,
mp ¼ 113.9e114.5 ꢁC; 1H NMR (400 MHz, CDCl3):
d
¼ 7.53e7.10 (m,
25.5, 24.7, 14.6 ppm; GCeMS: m/z ¼ 397 (Mþ); Anal. Calcd for
C19H22Cl2N2O3: C, 57.44; H, 5.58; Cl, 17.85; N, 7.05; O, 12.08; Found: C,
57.18; H, 5.69; N, 7.31.
3H), 4.63e4.58 (m, 1H), 4.35 (s, 2H), 4.23 (q, J ¼ 7.2 Hz, 2H), 2.26 (s,
3H), 2.22 (s, 3H), 2.01e1.97 (m, 2H), 1.74e1.58 (m, 3H), 1.41e1.15 (m,
8H) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 165.4, 164.2, 137.3, 136.5,
133.4, 130.0, 120.8, 119.5, 117.0, 94.3, 59.5, 50.5, 48.1, 34.8, 25.5, 24.6,
20.0, 19.2, 14.5 ppm; GCeMS: m/z ¼ 356 (Mþ); Anal. Calcd for
C21H28N2O3: C, 70.76; H, 7.92; N, 7.86; O, 13.47; Found: C, 70.93; H,
7.71; N, 7.69.
4.1.1.13. Ethyl 1-(4-bromophenyl)-4-(cyclohexylamino)-2,5-dihydro-
5-oxo-1H-pyrrole-3-carboxylate (5w). 91% yield, White solid,
mp ¼ 107.0e108.0 ꢁC; 1H NMR (400 MHz, CDCl3):
¼ 7.67e7.46 (m,
d
4H), 4.56e4.55 (m, 2H), 4.36 (s, 2H), 4.24e4.22 (m, 2H), 1.99e1.96
(m, 2H), 1.74e1.15 (m, 11H) ppm; 13C NMR (100 MHz, CDCl3):
4.1.1.7. Ethyl 4-(cyclohexylamino)-2,5-dihydro-1-(4-methoxyphenyl)-
d
¼ 165.4, 164.4, 137.9, 132.0, 120.6, 119.5, 117.7, 96.2, 59.7, 50.5, 47.8,
5-oxo-1H-pyrrole-3-carboxylate (5q). 90% yield, White solid,
34.7, 25.4, 24.6, 14.5 ppm; GCeMS: m/z ¼ 407 (Mþ); Anal. Calcd for
C19H23BrN2O3: C, 56.03; H, 5.69; Br, 19.62; N, 6.88; O, 11.78; Found:
C, 56.23; H, 5.48; N, 6.69.
mp ¼ 101.8e102.8 ꢁC; 1H NMR (600 MHz, CDCl3):
¼ 7.64e6.91 (m,
d
4H), 4.62 (b, 1H), 4.37 (s, 2H), 4.25 (q, J ¼ 7.2 Hz, 2H), 3.82 (s, 3H),
2.03e2.00 (m, 2H), 1.76e1.61 (m, 3H), 1.45e1.19 (m, 8H) ppm; 13C
NMR (150 MHz, CDCl3):
d
¼ 164.2,156.9,132.0,121.5,114.2, 59.6, 55.5,
4.1.1.14. Ethyl 1-(4-bromophenyl)-2,5-dihydro-4-morpholino-5-oxo-
50.5, 48.4, 34.8, 25.5, 24.7, 14.6 ppm; GCeMS: m/z ¼ 358 (Mþ); Anal.
Calcd for C20H26N2O4: C, 67.02; H, 7.31; N, 7.82; O, 17.85; Found: C,
66.89; H, 7.50; N, 7.69.
1H-pyrrole-3-carboxylate
(5y). 85%
yield,
White
¼ 7.65e7.47 (m,
solid,
mp ¼ 151.0e152.0 ꢁC; 1H NMR (400 MHz, CDCl3):
d
4H), 4.39 (s, 2H), 4.22 (q, J ¼ 7.2 Hz, 2H), 3.80e3.75 (m, 8H), 1.31 (t,
J ¼ 7.2 Hz, 3H) ppm; 13C NMR (100 MHz, CDCl3):
d
¼ 165.6, 162.7,
4.1.1.8. Ethyl 4-(cyclohexylamino)-2,5-dihydro-1-(naphthalen-1-yl)-
146.5, 137.7, 132.1, 120.8, 117.9, 104.1, 67.5, 60.3, 50.2, 48.7, 14.5 ppm;
GCeMS: m/z ¼ 394 (Mþ); Anal. Calcd for C17H19BrN2O4: C, 51.66; H,
4.85; Br, 20.22; N, 7.09; O, 16.19; Found: C, 51.39; H, 4.94; N, 7.15.
5-oxo-1H-pyrrole-3-carboxylate (5r). 45% yield, White solid,
mp ¼ 152.5e153.5 ꢁC; 1H NMR (600 MHz, CDCl3):
¼ 7.92e7.40 (m,
d
7H), 4.66e4.62 (s, 1H), 4.44 (s, 2H), 4.24 (q, J ¼ 7.1 Hz, 2H),
2.08e1.25 (m, 13H) ppm; 13C NMR (150 MHz, CDCl3):
d
¼ 165.6,
4.1.2. General procedure for the preparation of tetrasubstituted
polyfunctional dihydropyrroles 5 with aryl R2 and R3 (condition B)
[24]
134.6, 134.5, 130.1, 128.9, 128.6, 127.1, 126.5, 125.5, 125.3, 122.7, 97.8,
59.6, 51.4, 50.6, 45.1, 35.0, 25.5, 24.8, 14.6 ppm; ESI-MS: m/z ¼ 379
(M þ Hþ); Anal. Calcd for C23H26N2O3: C, 72.99; H, 6.92; N, 7.40; O,
12.68; Found: C, 72.71; H, 6.99; N, 7.35.
Primary aromatic amines 3 (4 mmol), 38% formaldehyde 4a
(120 mg, 1.5 mmol) and HOAc (120 mg, 2 mmol) were dropwise
added into the mixture of EtOH (4 mL), but-2-ynedioates 1
(1 mmol) and primary aromatic amines 2 (1 mmol) kept at room
temperature for 30e60 min in sequence, followed the same steps
described in general procedure A to afford the desired products in
85e89% yields (Table 1, 5g, 5iek). For the characterization data of
5g, 5iek see Ref. [24].
4.1.1.9. Ethyl 4-(cyclohexylamino)-2,5-dihydro-5-oxo-1-(pyridin-2-
yl)-1H-pyrrole-3-carboxylate (5s). 84% yield, White solid,
mp ¼ 94.5e96.5 ꢁC; 1H NMR (600 MHz, CDCl3):
d
¼ 8.47e7.07 (m,
4H), 4.64 (s, 2H), 4.58 (b, 1H), 4.25 (q, J ¼ 7.2 Hz, 2H), 2.03e2.00 (m,
2H), 1.77e1.74 (m, 3H), 1.44e1.22 (m, 8H) ppm; 13C NMR (150 MHz,
CDCl3):
d
¼ 165.9, 165.0, 151.2, 147.9, 137.9, 120.0, 114.2, 98.3, 59.7,
50.6, 46.7, 34.9, 31.1, 25.5, 24.7, 14.5 ppm; GCeMS: m/z ¼ 329 (Mþ);
Anal. Calcd for C18H23N3O3: C, 65.63; H, 7.04; N, 12.76; O, 14.57;
Found: C, 65.43; H, 7.21; N, 12.58.
4.1.3. General procedure for the preparation of pentasubstituted
polyfunctional dihydropyrroles 6 with aryl/alky R2 and alkyl R3
(condition C) [24]
Aldehydes 4bed (3 mmol) and primary aliphatic amines 3
(2 mmol) were dropwise added into the mixture of EtOH (4 mL),
but-2-ynedioates 1 (1 mmol) and primary amines 2 (1 mmol) kept
at room temperature for 30e60 min in sequence, followed with
4.1.1.10. Ethyl 1-(3-chlorophenyl)-4-(cyclohexylamino)-2,5-dihydro-
5-oxo-1H-pyrrole-3-carboxylate (5t). 91% yield, White solid,
mp ¼ 102.5e103.5 ꢁC; 1H NMR (600 MHz, CDCl3):
¼ 7.86e7.14 (m,
d

Q. Zhu et al. / European Journal of Medicinal Chemistry 54 (2012) 232e238
237
stirring at rt or 70 ꢁC for desired time (monitored by TLC). After
completion of the reactions, the product mixture was purified by
preparative TLC with n-hexane/ethyl acetate (10:1e1:1) as eluent
to afford the desired products in 60e98% yields (Table 1, all of 6
with the exception of 6f, 6g and 6n). For the characterization data of
6a, 6b, 6e, 6he6l see Ref. [24].
4.1.5.1. 4-(4-(Ethoxycarbonyl)-1-cyclohexyl-2,5-dihydro-2-oxo-5-
phenyl-1H-pyrrol-3-ylamino)benzoic acid (6f). 76% yield, white
solid, mp ¼ 218ꢂ222 ꢁC; 1H NMR (400 MHz, MeOH):
d
¼ 7.92 (d,
J ¼ 8.7 Hz, 2H), 7.41e7.26 (m, 5H), 7.12 (d, J ¼ 8.7 Hz, 2H), 5.40 (s,
1H), 3.87 (q, J ¼ 7.2 Hz, 2H), 3.62 (m, 1H), 1.91e1.46 (m, 6H),
1.25e1.11 (m, 3H), 0.96 (m, 1H), 0.86 (t, J ¼ 7.2 Hz, 3H) ppm; 13C
NMR (101 MHz, MeOH):
d
¼ 145.87, 138.70, 131.30, 129.53, 129.33,
4.1.3.1. Ethyl 4-(4-bromophenylamino)-1-cyclohexyl-2,5-dihydro-5-
121.11, 63.81, 61.26, 56.00, 49.63, 49.42, 31.87, 31.50, 27.06, 26.91,
26.36, 14.03 ppm; MS (ESI): m/z ¼ 449 (M þ Hþ); Anal. Calcd for
C26H28N2O5: C, 69.63; H, 6.29; N, 6.25; O, 17.84; Found: C, 69.83; H,
6.32; N, 6.18.
oxo-2-phenyl-1H-pyrrole-3-carboxylate (6c). 83% yield, Yellow
solid, mp ¼ 161.5e163.5 ꢁC; 1H NMR (600 MHz, CDCl3):
¼ 8.14 (s,
d
1H), 7.43e7.04 (m, 9H), 5.21 (s, 1H), 3.97 (t, J ¼ 7.1 Hz, 2H), 3.67 (m,
1H), 1.84e1.58 (m, 7H), 1.21e1.06 (m, 3H), 0.97 (d, J ¼ 7.1 Hz, 3H)
ppm; 13C NMR (150 MHz, CDCl3):
d
¼ 164.6, 164.4, 142.7, 137.9,
4.1.6. Procedure for the preparation of pentasubstituted
polyfunctional dihydropyrroles 6g (condition F)
137.6, 131.3, 128.3, 128.2, 128.0, 124.2, 117.3, 110.5, 61.8, 60.0, 54.4,
30.9, 30.6, 26.0, 25.8, 25.2, 13.8 ppm; MS (ESI): m/z ¼ 484 (M þ Hþ);
Anal. Calcd for C25H27BrN2O3: C, 62.12; H, 5.63; Br, 16.53; N, 5.80; O,
9.93; Found: C, 62.35; H, 5.25; N, 5.69.
The reaction mixture of diethyl but-2-ynedioate 1a (142 mg,
1 mmol), 4-aminobenzoic acid 2r (205 mg, 1.5 mmol) and N(Et)3
(55 mg, 0.5 mmol) in MeOH (0.5 mL) kept at room for 1 h was added
dropwise to the mixture of EtOH (5 mL), acetaldehyde 4c (318 mg,
3 mmol) and cyclohexanamine 3a (198 mg, 2 mmol), followed with
stirring at room temperature for desired time (about 24 h, moni-
tored by TLC). After completion of the reactions, the product
mixture was purified by preparative TLC with n-hexane/ethyl
acetate/HOAc (48:12:1) as eluent to afford the desired products in
79% yield (Table 1, 6g).
4.1.3.2. Ethyl 1-cyclohexyl-2,5-dihydro-4-(naphthalen-1-ylamino)-5-
oxo-2-phenyl-1H-pyrrole-3-carboxylate (6d). 83% yield, Yellow
solid, mp ¼ 190.5e191.5 ꢁC; 1H NMR (600 MHz, CDCl3) :
¼ 8.40 (s,
d
1H), 8.11 (d, J ¼ 8.3 Hz,1H), 7.89 (d, J ¼ 7.7 Hz,1H), 7.73 (d, J ¼ 8.1 Hz,
1H), 7.49 (m, 3H), 7.40e7.30 (m, 6H), 5.26 (s, 1H), 3.89 (t, J ¼ 7.1 Hz,
2H), 3.75e3.66 (m, 1H), 1.84e0.84 (m, 13H) ppm; 13C NMR
(150 MHz, CDCl3):
d
¼ 164.7, 144.2, 138.7, 134.7, 134.1, 129.1, 128.4,
128.2, 128.1, 128.0, 126.2, 126.0, 125.7, 125.1, 121.4, 109.3, 100.0, 61.7,
59.8, 54.2, 31.0, 30.7, 16.0, 25.8, 25.2, 13.7 ppm; MS (ESI): m/z ¼ 455
(M þ Hþ). Anal. Calcd for C29H30N2O3: C, 76.63; H, 6.65; N, 6.16; O,
10.56; Found: C, 76.32; H, 6.71; N, 6.02.
4.1.6.1. 4-(4-(Methoxycarbonyl)-1-cyclohexyl-2,5-dihydro-5-methyl-
2-oxo-1H-pyrrol-3-ylamino)benzoic acid (6g). 79% yield, white
solid, mp ¼ 223ꢂ225 ꢁC; 1H NMR (400 MHz, MeOH):
d
¼ 7.90 (d,
J ¼ 8.5 Hz, 2H), 7.06 (d, J ¼ 8.4 Hz, 1H), 4.41 (q, J ¼ 6.4 Hz, 0H),
3.71e3.60 (m, 2H), 2.06e1.62 (m, 8H), 1.51 (d, J ¼ 6.4 Hz, 2H), 1.33
(ddd, J ¼ 56.8, 28.5, 8.1 Hz, 6H) ppm; 13C NMR (101 MHz, MeOH):
4.1.3.3. Ethyl 4-(4-bromophenylamino)-2,5-dihydro-5-oxo-2-phenyl-
1-propyl-1H-pyrrole-3-carboxylate (6m). 89% yield, White solid,
d
¼ 165.99, 165.64, 145.82, 142.51, 131.23, 121.11, 120.99, 115.91,
mp ¼ 134.0e135.0 ꢁC; 1H NMR (600 MHz, CDCl3):
d
¼ 8.23 (s, 1H),
56.41, 55.76, 51.69, 31.83, 31.29, 27.15, 27.03, 26.49, 19.90 ppm; MS
(ESI): m/z ¼ 373 (M þ Hþ); Anal. Calcd for C20H24N2O5 : C, 64.50; H,
6.50; N, 7.52; O, 21.48; Found: C, 64.12; H, 6.30; N, 7.43.
7.46e7.40 (m, 2H), 7.38e7.32 (m, 3H), 7.24e7.19 (m, 2H), 7.10e7.05
(m, 2H), 5.17 (s, 1H), 4.06e3.97 (m, 2H), 3.64 (s, 1H), 2.66 (s, 1H),
1.51 (m, 2H), 1.00 (t, J ¼ 7.1 Hz, 3H), 0.85 (t, J ¼ 7.4 Hz, 3H) ppm;
13C NMR (150 MHz, CDCl3):
d
¼ 164.5, 142.9, 137.7, 136.4, 131.4,
4.2. Caspase-3 assays
128.6, 128.4, 127.7, 124.2, 117.4, 110.1, 62.1, 60.1, 42.2, 21.5, 13.8,
11.3 ppm; MS (ESI): m/z ¼ 444 (M þ Hþ); Anal. Calcd for
C22H23BrN2O3: C, 59.60; H, 5.23; Br, 18.02; N, 6.32; O, 10.83; found:
C, 59.79; H, 5.12; N, 6.51.
Recombinant human caspase-3 catalytic domain was prepared
according to our previous work [14] with minor modification. The
typical assay of caspase-3 was carried out in a 100
including 50 mM HEPES pH 7.5, 150 mM NaCl, 2 mM dithiothreitol,
1 mM EDTA,100 M Ac-DEVD-pNA (pNA, p-nitroaniline) and 20 nM
caspase-3 in the presence or absence of 2 L of inhibitor in DMSO.
In screening, the enzyme was incubated with inhibitors for 30 min.
The rate of hydrolysis product pNA was monitored continuously by
change of absorbance at 405 nm for 3 min, and the initial rate of
hydrolysis was determined using the early linear region of the
enzymatic reaction curve. Compounds were tested in duplicate,
and IC50 curves were calculated for all inhibitors assayed. Final IC50
values were the average of three independent experiments.
mL system
4.1.4. Procedure for the preparation of pentasubstituted
polyfunctional dihydropyrroles 6 with R2 ¼ R3 ¼ aryl (condition D)
[24]
Benzaldehyde 4b (3 mmol), aniline 3b (2 mmol) and HOAc
(120 mg, 2 mmol) were dropwise added into the mixture of EtOH
(4 mL), ethyl but-2-ynedioate 1a (1 mmol) and aniline 2b (1 mmol)
kept at room temperature for 30 min in sequence, followed the
same steps described in general procedure C to afford the desired
product in 92% yield (Table 1, 6n). For the characterization data of
6n see Ref.[24].
m
m
4.3. Shake-flask determination of partition coefficients
4.1.5. Procedure for the preparation of pentasubstituted
polyfunctional dihydropyrroles 6f (condition E)
The octanolewater partition coefficients (log P) were measured
using the shake-flask technique as described in references [30,33]
with minor difference. Briefly, 0.10 M phosphate buffer (pH ¼ 7)
and sample in buffer-saturated octanol (1 mM) were prepared.
Then, 0.5 mL of sample in buffer-saturated octanol was added to
20 mL octanol-saturated buffer. The partitioning was carried out in
50 ml centrifuge tubes. The two phases were mutually saturated by
100 inversions in roughly 5 min at room temperature [30]. Then,
the phases were allowed to separate after centrifugation at
3000 rpm for 5 min (Eppendorf 5424 centrifuge). Sample concen-
trations in octanol phase were determined.
The reaction mixture of diethyl but-2-ynedioate 1a (255 mg,
1.5 mmol), 4-aminobenzoic acid 2j (137 mg, 1 mmol) and N(Et)3
(55 mg, 0.5 mmol) in MeOH (0.5 mL) kept at room for 1 h was added
dropwise to the mixture of EtOH (5 mL), benzaldehyde 4b (318 mg,
3 mmol) and cyclohexanamine 3a (198 mg, 2 mmol), followed with
stirring at room temperature for desired time (about 24 h, moni-
tored by TLC). After completion of the reactions, the product
mixture was purified by preparative TLC with n-hexane/ethyl
acetate/HOAc (48:12:1) as eluent to afford the desired products in
76% yield (Table 1, 6f).

238
Q. Zhu et al. / European Journal of Medicinal Chemistry 54 (2012) 232e238
[8] R.M. Siegel, Nat. Rev. Immunol. 6 (2006) 308e317.
Thirty mL of samples in octanol phase were diluted to 3 mL with
[9] C.H. Yi, J. Yuan, Dev. Cell 16 (2009) 21e34.
[10] S. Kumar, Cell Death Differ. 14 (2007) 32e43.
[11] J.W. Becker, J. Rotonda, S.M. Soisson, R. Aspiotis, C. Bayly, S. Francoeur,
M. Gallant, M. Garcia-Calvo, A. Giroux, E. Grimm, Y. Han, D. McKay,
D.W. Nicholson, E. Peterson, J. Renaud, S. Roy, N. Thornberry, R. Zamboni,
J. Med. Chem. 47 (2004) 2466e2474.
buffer-saturated octanol. The absorption spectra of samples were
determined by TU-1901 UVevis spectrophotometer. The concen-
tration measurements were performed by HPLC using a liquid
chromatograph Waters 2695 separation module equipped with
a Waters 2996 photodiode array detector. The column was a Phe-
nomenex P/NO.006-4337-E0 (4
phase was the mixture of 90% methanol and 10% water, and four
samples had retention times of 6.45e11.88 min.
[12] M.F. Schmidt, A. El-Dahshan, S. Keller, J. Rademann, Angew. Chem. Int. Ed. 48
(2009) 6346e6349.
m
m, 4.60 ꢃ 250 mm). The mobile
[13] D. Lee, S.A. Long, J.L. Adams, G. Chan, K.S. Vaidya, T.A. Francis, K. Kikly,
J.D. Winkler, C.-M. Sung, C. Debouck, S. Richardson, M.A. Levy, W.E. DeWolf Jr.,
P.M. Keller, T. Tomaszek, M.S. Head, M.D. Ryan, R.C. Haltiwanger, P.-H. Liang,
C.A. Janson, P.J. McDevitt, K. Johanson, N.O. Concha, W. Chan, S.S. Abdel-
Meguid, A.M. Badger, M.W. Lark, D.P. Nadeau, L.J. Suva, M. Gowen,
M.E. Nuttall, J. Biol. Chem. 275 (2000) 16007e16014.
Different concentrations of sample (10, 20, 40, 60, 80, 100 and
120 mM) were prepared by diluting 1 mM of sample solutions with
buffer-saturated octanol and determined by HPLC under the above
mentioned conditions. The correlation coefficients R2 of linear
equations for the stand curves of four samples are 0.9997e0.9999.
The concentration of sample in octanol phase was calculated using
the linear equation.
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Acknowledgments
We thank the National Natural Foundation of China (U0832001),
National Basic Research Program of China (973 Program
2011CB808603), Guangdong Natural Science Foundation
(10351086000000) and Guangdong Science and Technology
Project (2008B060600066) for financial support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2012.05.001. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
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