Synthesis and biological evaluation of thio-benzodiazepines as novel small molecule inhibitors of the p53-MDM2 protein-protein interaction

Article abstract of DOI:10.1016/j.ejmech.2011.09.043

A series of thio-benzodiazepine p53-MDM2 inhibitors were designed and synthesized based on the principle of bioisosterism. Most of the thio-benzodiazepines had nanomolar to micromolar affinity toward MDM2. Particularly, compounds 8a (K_i = 0.52

Full text of DOI:10.1016/j.ejmech.2011.09.043

European Journal of Medicinal Chemistry 46 (2011) 5654e5661
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of thio-benzodiazepines as novel small
molecule inhibitors of the p53eMDM2 proteineprotein interaction
Chunlin Zhuang 1, Zhenyuan Miao 1, Lingjian Zhu, Yongqiang Zhang, Zizhao Guo, Jianzhong Yao,
*
**
Guoqiang Dong, Shengzheng Wang, Yang Liu, Hai Chen, Chunquan Sheng , Wannian Zhang
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of thio-benzodiazepine p53eMDM2 inhibitors were designed and synthesized based on the
principle of bioisosterism. Most of the thio-benzodiazepines had nanomolar to micromolar affinity
Received 12 July 2011
Received in revised form
25 August 2011
Accepted 25 September 2011
Available online 2 October 2011
toward MDM2. Particularly, compounds 8a (Ki ¼ 0.52
m
m
M) and 8f (Ki ¼ 0.32
M). Meanwhile, compound 8j exhibited excellent
M, which
mM) showed binding activity
comparable to the positive drug nutlin-3a (Ki ¼ 0.23
antitumor activity against the U-2 OS human osteosarcoma cell line with an IC50 value of 1.06
m
was about 23 times higher than that of nutlin-3a. The docking model also successfully predicted that this
class of compounds mimicked three p53 critical residues binding to MDM2. The thio-benzodiazepines
represent a promising class of non-peptide inhibitors of the p53eMDM2 interaction.
Ó 2011 Published by Elsevier Masson SAS.
Keywords:
p53eMDM2
Small molecule inhibitors
Thio-benzodiazepine
Antitumor activity
1. Introduction
identified from a library of 300,000 compounds by high-
throughput screening (HTS) using the temperature-dependent
protein-unfolding assay ThermoFluor [13]. The strongest MDM2-
binding compound from this family was further optimized to
TDP222669 (Fig. 1), which has a Ki value of 80 nM and was confir-
med to be active in vitro [14,15].
However, this compound suffered from low bioavailability and
rapid in vivo clearance. Herein, we reported a series of thio-
benzodiazepines as small molecule inhibitors of p53eMDM2
interaction. In view of the sulfur atom’s wide application in drug
design [16e18], the thio-benzodiazepines were designed based on
the principle of bioisosterism. Biological assay showed that the
thio-benzodiazepines possessed both p53eMDM2 inhibitory
activity and in vitro antitumor activity.
The significance of the p53 tumor suppressor is not only from its
pivotal role in DNA-repair and cell-cycle control but also from its
overarching regulating role in carcinogenesis [1,2]. In approxi-
mately 50% of all human cancers, the function of p53 has been
disannulled by deletions or mutations [3]. While in the remaining
half of human cancers, p53 retains its wild-type form but its activity
is effectively inhibited through direct interaction with the human
murine double minute 2 (MDM2) oncoprotein [4].
Using a non-peptide small molecule inhibitor to restore the
impaired function of the p53 protein by disrupting the p53eMDM2
interaction offers an innovative avenue for the treatment of a broad
spectrum of cancers [4e6]. In recent years, a number of non-
peptide small molecule inhibitors of the p53eMDM2 interaction
have been reported [6e12]. These inhibitors can mainly be classi-
fied into nutlin derivatives, spiro-oxindole derivatives and benzo-
diazepine derivatives [6,9,10]. The benzodiazepine-based inhibitor
was reported by Grasberger et al. in 2005 [9]. The scaffold was
2. Chemistry
As depicted in Scheme 1, thio-benzodiazepines were synthe-
sized utilizing the highly efficient and versatile Ugi four-component
condensation (Ugi 4CC) reaction, which is a multi-component
reaction involving a ketone (or aldehyde), an amine, an isocya-
nide and a carboxylic acid to form a bis-amide [19]. In this study, we
used p-chlorobenzaldehyde (2) as the aldehyde, methyl amino(4-
chlorophenyl)acetate hydrochloride (1) as the amine, substituted
nitrobenzoic acid (4a,b) as the carboxylic acid and 1-isocyanocyclo-
hexene (3) as the isocyanide to perform the Ugi reaction and the
* Corresponding author. Tel./fax: þ86 21 81871239.
** Corresponding author. Tel./fax: þ86 21 81871243.
E-mail addresses: shengcq@hotmail.com (C. Sheng), zhangwnk@hotmail.com
(W. Zhang).
1
These two authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2011.09.043

C. Zhuang et al. / European Journal of Medicinal Chemistry 46 (2011) 5654e5661
5655
Table 1
Cl
Binding constants (Ki) of the MDM2 ligands and IC50 values of in vitro antitumor
activity.
Compounds R1
R2
R3
Kia
(m
M) IC50b
Saos-2
(mM)
U-2 OS
(p53 null) (wt-p53)
O
COOH
6a
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
H
H
H
H
e
e
10.6
0.52
1.02
2.26
>100
>100
0.32
15
5.58
6.55
12.41
17.76
13.3
13.94
13.68
91.17
>100
20.9
17.98
>100
>100
>100
>100
1.06
>100
>100
>100
>100
14.73
30.58
48.53
24.61
I
N
eNO2 eNH2 -CH2CH3
eNO2 eNH2 -Ph
Cl
eNO2 eNH2 -4-Cl-Ph
eNO2 eNH2 -C(CH3)3
eNO2 eNH2 -CH(CH3)2
eNO2 eNH2 -(CH2)3CH3
eNO2 eNH2 -(CH2)2CH3
N
H
2.19
O
15.74
20.66
27.85
19.04
2.87
72.23
91.74
88.12
3.37
13.8
9.81
11.28
54.38
Fig. 1. Structure of TDP222669.
49.6
eNO2 eNH2 -cyclopropyl 3.43
eNO2 eNH2 -4eCF3-Ph
eNO2 eNH2 -cyclohexyl
eNO2 eNH2 -2-naphthyl
5.34
>100
>100
conditions were similar to those in the literature [20]. Followed by
treating with iron powder, the key intermediates 6a,b were ob-
tained. Reductive amination reactions were performed between
compound 6b and commercially available aldehydes to give the
compounds 7aep. Finally, treatment of compounds 7aep with the
Lawesson’s Reagent afforded the target compounds 8aeq with the
yields of 54.9e86.1%.
eNO2 eNH2 -4eOCH3-Ph >100
eNO2 eNH2 -3-thienyl
eNO2 eNH2 -4-Br-Ph
eNO2 eNH2 -4-F-Ph
eNO2 eNH2 -2-furyl
___
>100
>100
1.71
43.1
0.23
8p
8q
Nutlin-3a
a
Values were determined by fluorescence polarization assay.
Values were measured with MTT method.
b
3. Results and discussion
replacement of the oxygen atom of the benzodiazepine scaffold
(compound 6a, Ki ¼ 10.6 M) by the sulfur atom (compound 8a,
M) led to the increase of the activity toward MDM2.
Moreover, the compounds with aliphatic substituents on the
benzene ring displayed moderate to high affinity against
3.1. p53eMDM2 binding assay and structureeactivity relationships
m
Ki ¼ 0.52
m
The binding Ki constants of small molecule ligands were
measured by fluorescence polarization (FP) binding assay. Nutlin-
3a, one of the most active small molecule p53eMDM2 inhibitor,
was used as the reference drug. The results were presented in
Table 1. Most of the thio-benzodiazepines had nanomolar to
micromolar affinity towards MDM2. SAR analysis showed that the
p53eMDM2. Particularly, compounds 8a (Ki ¼ 0.52
(Ki ¼ 0.32 M) showed comparable binding activity to nutlin-3a
(Ki ¼ 0.23 M). However, for the compounds with aromatic
mM) and 8f
m
m
Scheme 1. Synthetic Route. Reagents and conditions: (a) CH3OH, KOH, r.t., 3 days; (b) Fe powder, CH3COOH, 65e70 ꢀC, 1 h; (c) R3CHO, NaBH3CN, r.t., 16 h; (d) Lawesson’s Reagent,
toluene, 70 ꢀC, 4 h.

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C. Zhuang et al. / European Journal of Medicinal Chemistry 46 (2011) 5654e5661
substituents, only the fluorine-containing compounds (8j and 8p)
revealed moderate affinity. The crystal structure of MDM2 and p53
peptide (residues 15e29) provided insight into p53eMDM2 inter-
action [21]. The model revealed that p53 formed an amphipathic a-
helix interaction on the MDM2 protein surface containing three key
amino acid (Phe19, Trp23 and Leu26). Our docking models (Fig. 2)
demonstrated that thio-benzodiazepines could interact with
MDM2 by filling its Trp23 subpocket with p-chlorobenzyl group.
The substituted phenyl ring and 1-(4-chlorobenzyl) group are
located in the Phe19 and Leu26 pockets, respectively. Based on the
previously reported structures of the antagonists binding to MDM2
[8] and our docking model, we proposed a binding model for the
thio-benzodiazepine-MDM2 complex (Fig. 3). The binding inter-
action involves three lyophobic pockets that are filled by the three
aromatic rings of the thio-benzodiazepine. This binding model
predicts that the ester group is well positioned as hydrogen bond
acceptor with Gly16 or Ser17, while the secondary amine functions
as a donator to form a hydrogen bond with Gly58 (Fig. 2). This
prediction was further validated by the inhibitory activity of
compounds 8a and 8f (Fig. 2A and C). Furthermore, with regard to
thio-benzodiazepines with aromatic substituents, the affinity with
the MDM2 decreased significantly, which may be accounted for the
changed binding mode to the hydrophobic pocket Phe19 due to
their relatively larger steric volumes (Fig. 2B). In addition, the
docking models also illustrated that the sulfur atom with better
lipid solubility was easier to be exposed to the solvents, which may
explain the fact that the affinity of compound 8a is 20 times higher
than that of 6a.
Fig. 3. 3D schematic binding model for the complex of thio-benzodiazepines and
MDM2.
to the results (Table 1), compounds 6a, 8a, 8d, 8e, 8j and 8o showed
better biological activity against the U-2 OS cell line with wild-type
p53 than nutlin-3a. Especially, compound 8j showed the best
antitumor activity (IC50 ¼ 1.06
mM). However, compounds 8k, 8l,
8m and 8n displayed weak in vitro antitumor activity, which were
consistent with the results of the p53eMDM2 binding assays.
Interestingly, compound 8j was more active against the U-2 OS cell
line with wild-type p53 than the Saos-2 cell line with p53 deficient.
Therefore, compound 8j can be used as a lead compound and
further structural modification on solvent exposed position is
necessary for enhancing its antitumor activity. Further pharmaco-
logical and toxicological evaluation of this promising compound is
in progress.
3.2. In vitro antitumor activity
All the compounds were further evaluated in MTT assays for
ascertaining whether their in vitro antitumor activity was consis-
tent with the inhibitory activity of p53eMDM2 binding. According
Fig. 2. The docking models of thio-benzodiazepines inhibitors with MDM2. The inhibitors are shown in stick models with carbon atoms colored yellow, nitrogen blue, oxygen red
and sulfur deep yellow. Hydrogen bonds are depicted as green dashed lines. The amino acid residues which were formatted H-bonds are labeled. (A) Compound 8a; (B) Compound
8b; (C) Compound 8f. The figures were prepared from PDB entry: 1T4E, Using PyMol (http://pymol.souceforge.net/). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

C. Zhuang et al. / European Journal of Medicinal Chemistry 46 (2011) 5654e5661
5657
4. Conclusion
separated, dried (Na2SO4), and evaporated to dryness. The residue
was purified by column chromatography (n-hexaneeethyl acetate,
5:1) to give 6a,b, yields: 37.5e41.3%.
A series of thio-benzodiazepines were designed and synthesized
via Ugi reaction with the purpose to find more potent p53eMDM2
inhibitors. In the p53eMDM2 binding assay, the affinity of thio-
benzodiazepines with MDM2 had been largely increased compared
with the benzodiazepine derivative. Moreover, compounds 8a and 8f
showed good binding activity in nanomolar range, which were
comparable to the positive drug nutlin-3a. In addition, all the
compounds were evaluated for invitro antitumor activityagainst two
human osteosarcoma cell lines. For the U-2 OS cell line, compounds
8a, 8d, 8e, 8j and 8o showed higher in vitro inhibitory activity than
nutlin-3a. Furthermore, compound 8j showed potent antitumor
5.2.2. General procedure B: reductive amination
Aldehyde (10 mmol) and 6b (10 mmol) were mixed in CH3OH
(10 mL), adjusted to pH 6e7 with acetic acid. After stirring at room
temperature for 30 min, NaBH3CN (15e20 mmol) was then added to
the solvent, stirred for another 16 h. Removal of the solvent under
reduced pressure and the residue was purified by column chroma-
tography (CH2Cl2eCH3OH, 100:1) to give 7aeq, yields: 25.0e74.3%.
5.2.3. General procedure C: synthesis of thio-benzodiazepines
A dry toluene solution of 7aeq (10 mmol) under an atmosphere
of nitrogen was treated with Lawesson’s Reagent (2,4-bis(4-
methoxyphenyl)-2,4-disulfide, 5.5 mmol) under 70 ꢀC for 4 h,
cooled to room temperature, concentrated under vacuum and
purified by column chromatography(n-hexaneeethyl acetate, 10:1)
to give 8aeq, yields: 54.9e86.1%.
activity with an IC50 value of 1.06 mM. Several highly potent comp-
ounds are being further evaluated utilizing in vivo models. In
conclusion, this class of thio-benzodiazepines can be used as prom-
ising lead structures for further optimization.
5. Experimental protocols
5.1. General methods
5.2.4. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-2, 5-dioxo-
2, 3-dihydro -1H-benzo [e] [1,4] diazepin-4 (5H)-yl) acetate (6a)
Following General Procedure A, 2-nitrobenzoic acid (4a)
All reagents and solvents were purchased from commercial
suppliers and used as received unless otherwise stated. Melting
points were measured on an uncorrected X-5 digital melting point
apparatus (Gongyi City Yuhua Instrument Co., Ltd.; China). 1H NMR
and 13C NMR spectra were recorded on a BRUKER AVANCE 500
spectrometer (Bruker Company, Germany), using TMS as an internal
(10 mmol), compound
1 (10 mmol), p-chlorobenzaldehyde (2)
(10 mmol) and 1-isocyanocyclohexene (3) (11 mmol) got compound
6a as a white solid (1.755 g, yield: 37.5%). 1H NMR (500 MHz, DMSO-
d6)
d
: 10.78 (s,1H), 6.84e7.53 (m,12H, Ar-H), 6.29 (s,1H), 5.32 (s,1H),
3.77 (s, 3H). 13C NMR (125 MHz, DMSO-d6)
d: 169.97, 169.65, 167.15,
standard and CDCl3 or DMSO-d6 as solvents. Chemical shifts (
d
135.77, 134.12, 133.57, 133.48, 132.84, 132.52, 131.96, 130.60, 129.21,
128.78,126.78,126.70,124.36,120.56, 65.35, 64.73, 52.92. ESI-MS (m/
z): 467.57 [M ꢃ 1]-. Anal. calcd. for C24H18Cl2N2O4: C, 61.42; H, 3.87; N,
5.97. Found: C, 61.53; H, 3.86; N, 5.95.
values) and coupling constants (J values) are given in ppm and Hz,
respectively. Elemental analyses were performed with a MOD-1106
instrument and were consistent with theoretical values within
ꢁ0.4%. TLC analysis was carried out on silica gel plates GF254
(Qindao Haiyang Chemical, China). Flash column chromatography
was carried out on silica gel 300e400 mesh. Anhydrous solvent and
reagents were all analytical pure and dried through routine
protocols.
5.2.5. Methyl 2-(8-amino-3-(4-chlorophenyl)-2, 5-dioxo-2, 3-
dihydro-1H-benzo [e] [1,4] diazepin-4(5H)-yl)-2-(4-chlorophenyl)
acetate (6b)
Following General Procedure A, 2, 4-dinitrobenzoic acid (4b)
(10 mmol), compound 1 (10 mmol), p-chlorobenzaldehyde (2)
(10 mmol) and 1-isocyanocyclohexene (3) (11 mmol) gave
compound 6b as a light-yellow solid (2.0 g, yield: 41.3%). 1H NMR
5.2. Synthesis of target compounds
5.2.1. General procedure A: synthesis of benzodiazepines via Ugi 4CC
Powdered 2-nitrobenzoic acid or 2, 4-dinitrobenzoic acid (4a,b)
(10 mmol) was added to a well stirred solution of KOH (10 mmol) in
CH3OH (10 mL). The resulting suspension was stirred at room
temperature for 10 min and then cooled to 0 ꢀC and treated with
methyl 2-amino-2-(4-chlorophenyl)acetate hydrochloride (1)
(10 mmol), a solution of 1-isocyanocyclohexene (3) (11 mmol) in
(500 MHz, DMSO-d6) d: 10.44 (s, 1H), 6.18e7.48 (m, 11H, Ar-H), 5.90
(s, 1H), 5.72 (s, 2H), 5.20 (s, 1H), 3.72 (s, 3H). ESI-MS (m/z): 485.29
[M þ 1]þ. Anal. calcd. for C24H19Cl2N3O4: C, 59.52; H, 3.95; N, 8.68.
Found: C, 59.40; H, 3.96; N, 8.70.
5.2.6. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-2, 5-
dioxo-8-(propyl amino)-2, 3-dihydro-1H-benzo [e] [1,4] diazepin-4
(5H)-yl) acetate (7a)
CH3OH (2 mL) and
a solution of p-chlorobenzaldehyde (2)
(10 mmol) in CH3OH (2 mL), in the order given. The cooling bath
was removed and the reaction mixture was stirred at room
temperature for 3 days. Removal of the solvent under reduced
pressure left a residue which was stirred with H2O (10 mL) and
CH2Cl2 (80 mL). The organic layer was dried (Na2SO4) and evapo-
rated to dryness. The residue was stirred with boiling hexanes
(50 mL) for 5 min. The supernatant was discarded while still warm.
An analogous treatment was performed with boiling H2O
(50 mL ꢂ 2). The resulting solid product was stirred with AcOH
(60 mL).The resulting solution was heated at 45 ꢀC and treated
under vigorous stirring with iron powder (50 mmol) in one portion.
When the exothermic reaction had subsided, the reaction mixture
was heated at 65e70 ꢀC for 1 h and then allowed to cool and stirred
with CH2Cl2 (50 mL) and H2O (50 mL). The resulting suspension
was filtered to remove the unreacted iron and the filtrate trans-
ferred to a separating funnel. The organic layer was washed with
H2O (50 mL), NaHCO3 (aq, 2%; 50 mL), H2O (50 mL) and then
Following General Procedure B, propaldehyde (28 mg) and 6b
(240 mg) gave 7a (200 mg) as a white solid, yield: 76.5%. 1H NMR
(500 MHz, DMSO-d6) d: 10.44 (s, 1H), 6.19e7.48 (m, 11H, Ar-H), 6.19
(s, 1H), 5.90 (s, 1H), 5.21 (s, 1H), 3.72 (s, 3H), 2.84 (t, 2H, J ¼ 5.5 Hz),
1.46 (m, 2H), 0.85 (t, 3H, J ¼ 7.3 Hz). ESI-MS (m/z): 526.80 [M þ 1] þ
,
524.43 [M ꢃ 1]-. Anal. calcd. for C27H25Cl2N3O4: C, 61.60; H, 4.79; N,
7.98; Found: C, 61.45; H, 4.80; N, 7.99.
5.2.7. Methyl 2-(8-(benzylamino)-3-(4-chlorophenyl)-2, 5-dioxo-2,
3-dihydro-1H -benzo [e] [1,4] diazepin-4 (5H)-yl)-2-(4-chlorophenyl)
acetate (7b)
Following by General Procedure B, benzaldehyde (53.6 mg) and
6b (240 mg) gave 7b (170 mg) as a white solid, yield: 58.1%. 1H NMR
(500 MHz, DMSO-d6) d: 10.45 (s, 1H), 6.21e7.45 (m, 16H, Ar-H), 6.20
(s, 1H), 5.96 (s, 1H), 5.20 (s, 1H), 4.17 (d, 2H, J ¼ 5.9 Hz), 3.72 (s, 3H).
ESI-MS (m/z): 572.59 [M ꢃ 1]ꢃ. Anal. calcd. for C31H25Cl2N3O4: C,
64.81; H, 4.39; N, 7.31; Found: C, 64.69; H, 4.40; N, 7.33.

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C. Zhuang et al. / European Journal of Medicinal Chemistry 46 (2011) 5654e5661
5.2.8. Methyl 2-(8-(4-chlorobenzylamino)-3-(4-chlorophenyl)-2, 5-
dioxo-2, 3 -dihydro-1H-benzo [e] [1,4] diazepin-4 (5H)-yl)-2-(4-
chlorophenyl) acetate (7c)
5.2.14. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-2, 5-
dioxo-8-(4 -(trifluoromethyl) benzylamino)-2, 3-dihydro-1H-benzo
[e] [1,4] diazepin-4 (5H)-yl) acetate (7i)
Following General Procedure B, 4-chlorobenzaldehyde (30 mg)
Following General Procedure B, 4-(trifluoromethyl)benzaldehyde
(37 mg) and6b(100 mg) gave 7i(82 mg) asawhite solid, yield:61.8%.
and 6b (100 mg) gave 7c (31.3 mg) as a white solid, yield: 25.0%. 1H
NMR (500 MHz, DMSO-d6)
d
: 10.45 (s, 1H), 6.20e7.46 (m, 15H, Ar-
1H NMR (500 MHz, DMSO-d6)
d: 10.44 (s, 1H), 6.19e7.66 (m, 15H, Ar-
H), 6.17 (s, 1H), 5.94 (s, 1H), 5.20 (s, 1H), 4.17 (d, 2H, J ¼ 6.0 Hz),
3.71 (s, 3H). ESI-MS (m/z): 606.62 [M ꢃ 1]ꢃ. Anal. calcd. for
C31H24Cl3N3O4: C, 61.15; H, 3.97; N, 6.90; Found: C, 61.05; H, 3.98; N,
6.92.
H), 6.17 (s,1H), 5.94 (s,1H), 5.20 (s,1H), 4.29 (d, 2H, J ¼ 5.8 Hz), 3.71 (s,
3H). ESI-MS (m/z): 642.92[M þ 1]þ. Anal. calcd. for C32H24Cl2F3N3O4:
C, 59.82; H, 3.77; N, 6.54; Found: C, 59.72; H, 3.78; N, 6.55.
5.2.15. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
(cyclohexyl methylamino)-2, 5-dioxo-2, 3-dihydro-1H-benzo [e]
[1,4] diazepin-4 (5H)-yl) acetate (7j)
5.2.9. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
(neopentylamino) ꢃ2, 5-dioxo-2, 3-dihydro-1H-benzo[e] [1,4]
diazepin-4 (5H)-yl) acetate (7d)
Following General Procedure B, cyclohexanecarbaldehyde
(23.2 mg) and 6b (100 mg) gave 7j (72 mg) as a white solid, yield:
Following General Procedure B, pivalaldehyde (18 mg) and 6b
(100 mg) gave 7d (51.4 mg) as a white solid, yield: 45.0%. 1H NMR
60.2%. 1H NMR (500 MHz, DMSO-d6)
d: 10.41 (s, 1H), 6.19e7.47 (m,
(500 MHz, DMSO-d6)
d
: 10.40 (s, 1H), 6.22e7.47 (m, 11H, Ar-H), 6.19
11H, Ar-H), 6.18 (s,1H), 5.90 (s,1H), 5.21 (s,1H), 3.72 (s, 3H), 2.74 (m,
2H), 1.98 (m, 1H), 1.65e0.85 (m, 10H). ESI-MS (m/z): 1184.27
[2M þ Na]þ. Anal. calcd. for C31H31Cl2N3O4: C, 64.14; H, 5.38; N,
7.24; Found: C, 64.24; H, 5.37; N, 7.21.
(s, 1H), 5.98 (s, 1H), 5.21 (s, 1H), 3.72 (s, 3H), 2.75 (m, 2H), 0.85 (s,
9H). ESI-MS (m/z): 554.82 [M ꢃ 1]ꢃ. Anal. calcd. for C29H29Cl2N3O4:
C, 62.82; H, 5.27; N, 7.58; Found: C, 62.72; H, 5.28; N, 7.60.
5.2.10. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
(isobutylamino)-2, 5-dioxo-2, 3-dihydro-1H-benzo[e] [1,4]
diazepin-4 (5H)-yl) acetate (7e)
5.2.16. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
(naphthalen-2-yl methylamino)-2, 5-dioxo-2, 3-dihydro-1H-benzo
[e] [1,4] diazepin-4 (5H)-yl) acetate (7k)
Following General Procedure B, isobutyraldehyde (13 mg) and
Following General Procedure B, 2-naphthaldehyde (32.3 mg)
6b (87 mg) gave 7e (41.6 mg) as a white solid, yield: 41.8%. 1H NMR
and 6b (100 mg) gave 7 k (93 mg) as a white solid, yield: 72.2%. 1H
(500 MHz, DMSO-d6)
d: 10.42 (s, 1H), 6.20e7.47 (m, 11H, Ar-H), 6.19
NMR (500 MHz, DMSO-d6) d: 10.44 (s, 1H), 7.86 (m, 2H), 7.84
(s, 1H), 5.92 (s, 1H), 5.22 (s, 1H), 3.72 (s, 3H), 2.72 (m, 2H), 1.74 (m,
1H), 0.85 (d, 6H, J ¼ 6.6 Hz). ESI-MS (m/z): 1103.59 [2M þ Na]þ.
Anal. calcd. for C28H27Cl2N3O4: C, 62.23; H, 5.04; N, 7.78; Found: C,
62.10; H, 5.05; N, 7.80.
(m,1H), 7.79 (s, 1H), 7.37e7.67 (m, 7H), 7.14 (m, 1H), 7.03 (m, 2H),
7.00 (m, 3H), 6.25 (d, 1H, Ar-H, J ¼ 8.6 Hz), 6.18 (s, 1H), 6.01 (s, 1H),
5.19 (s, 1H), 4.34 (d, 2H, J ¼ 5.9 Hz), 3.71 (s, 3H). ESI-MS (m/z):
624.41 [M þ 1]þ. Anal. calcd. for C35H27Cl2N3O4: C, 67.31; H, 4.36; N,
6.73; Found: C, 67.42; H, 4.35; N, 6.71.
5.2.11. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-2, 5-
dioxo-8-(pentyl amino)-2, 3-dihydro-1H-benzo [e] [1,4] diazepin-4
(5H)-yl) acetate (7f)
5.2.17. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-(4-
methoxy benzylamino)-2, 5-dioxo-2, 3-dihydro-1H-benzo [e] [1,4]
diazepin-4 (5H)-yl) acetate (7l)
Following General Procedure B, pentanal (21.5 mg) and 6b
(120 mg) gave 7f (90 mg) as a white solid, yield: 65.6%. 1H NMR
Following General Procedure B, 4-methoxybenzaldehyde
(28.1 mg) and 6b (100 mg) gave 7l (78 mg) as a white solid, yield:
(500 MHz, DMSO-d6) d: 10.44 (s, 1H), 6.18e7.47 (m, 11H, Ar-H), 6.17
(s, 1H), 5.90 (s, 1H), 5.21 (s, 1H), 3.72 (s, 3H), 2.87 (t, 2H, J ¼ 5.8 Hz),
1.44 (m, 2H), 1.25 (m, 4H), 0.85 (t, 3H, J ¼ 6.4 Hz). ESI-MS (m/z):
554.95 [M þ 1]þ. Anal. calcd. for C29H29Cl2N3O4: C, 62.82; H, 5.27; N,
7.58; Found: C, 62.69; H, 5.28; N, 7.60.
62.6%. 1H NMR (500 MHz, DMSO-d6)
d: 10.44 (s, 1H), 6.21e7.46 (m,
15H, Ar-H), 6.18 (s, 1H), 5.97(s, 1H), 5.20 (s, 1H), 4.09 (d, 2H,
J ¼ 5.8 Hz), 3.72 (s, 3H), 3.71 (s, 3H). ESI-MS (m/z): 1231.41
[2M þ Na]þ. Anal. calcd. for C32H27Cl2N3O5: C, 63.58; H, 4.50; N,
6.95; Found: C, 63.68; H, 4.49; N, 6.94.
5.2.12. Methyl 2-(8-(butylamino)-3-(4-chlorophenyl)-2, 5-dioxo-2,
3-dihydro -1H-benzo [e] [1,4] diazepin-4 (5H)-yl)-2-(4-chlorophenyl)
acetate (7g)
5.2.18. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-2, 5-
dioxo-8- (thiophen-3-ylmethylamino)-2, 3-dihydro-1H-benzo [e]
[1,4] diazepin-4 (5H)-yl) acetate (7m)
Following General Procedure B, n-butanal (17.9 mg) and 6b
(120 mg) gave 7g (70 mg) as a white solid, yield: 52.5%. 1H NMR
Following General Procedure B, thiophene-3-carbaldehyde
(23.2 mg) and 6b (100 mg) gave 7m (86 mg) as a white solid,
(500 MHz, DMSO-d6) d: 10.44 (s, 1H), 6.19e7.48 (m, 11H, Ar-H), 6.18
(s, 1H), 5.90 (s, 1H), 5.22 (s, 1H), 3.72 (s, 3H), 2.88 (t, 2H, J ¼ 6.1 Hz),
1.44 (m, 2H), 1.25 (m, 2H), 0.86 (t, 3H, J ¼ 7.3 Hz). ESI-MS (m/z):
540.90 [M þ 1] þ, 538.68 [M ꢃ 1]ꢃ. Anal. calcd. for C28H27Cl2N3O4:
C, 62.23; H, 5.04; N, 7.78; Found: C, 62.40; H, 5.03; N, 7.77.
yield: 71.9%. 1H NMR (500 MHz, DMSO-d6)
d: 10.46 (s, 1H),
6.23e7.47 (m, 14H, Ar-H), 6.19 (s, 1H), 5.99 (s, 1H), 5.22 (s, 1H), 4.13
(d, 2H, J ¼ 5.7 Hz), 3.72 (s, 3H). ESI-MS (m/z): 580.74 [M þ 1]þ. Anal.
calcd. for C29H23Cl2N3O4S: C, 60.00; H, 3.99; N, 7.24; Found: C,
60.11; H, 3.98; N, 7.22.
5.2.13. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
(cyclopropyl methylamino)-2, 5-dioxo-2, 3-dihydro-1H-benzo [e]
[1,4] diazepin-4 (5H)-yl) acetate (7h)
5.2.19. Methyl 2-(8-(4-bromobenzylamino)-3-(4-chlorophenyl)-2,
5-dioxo-2, 3-dihydro-1H-benzo [e] [1,4] diazepin-4(5H)-yl)-2-(4-
chlorophenyl) acetate (7n)
Following General Procedure B, cyclopropanecarbaldehyde
(14.5 mg) and 6b (100 mg) gave 7h (65 mg) as a white solid, yield:
Following General Procedure B, 4-bromobenzaldehyde
(38.2 mg) and 6b (100 mg) gave 7n (78 mg) as a white solid,
58.6%. 1H NMR (500 MHz, DMSO-d6)
d: 10.44 (s, 1H), 6.22e7.48 (m,
11H, Ar-H), 6.19 (s, 1H), 5.93 (s, 1H), 5.22 (s, 1H), 3.72 (s, 3H), 2.80 (t,
2H, J ¼ 6.1 Hz), 0.94 (m, 1H), 0.40 (t, 2H, J ¼ 4.0 Hz), 0.15 (t, 2H,
J ¼ 4.5 Hz). ESI-MS (m/z): 536.70 [M ꢃ 1]ꢃ. Anal. calcd. for
C28H25Cl2N3O4: C, 62.46; H, 4.68; N, 7.80; Found: C, 62.35; H, 4.69;
N, 7.82.
yield: 58.0%. 1H NMR (500 MHz, DMSO-d6)
d: 10.45 (s, 1H),
6.20e7.49 (m, 15H, Ar-H), 6.18 (s, 1H), 5.93 (s, 1H), 5.20 (s, 1H), 4.15
(d, 2H, J ¼ 5.9 Hz), 3.71(s, 3H). ESI-MS (m/z): 654.47 [M þ 1]þ. Anal.
calcd. for C31H24BrCl2N3O4: C, 56.99; H, 3.70; N, 6.43; Found: C,
56.87; H, 3.71; N, 6.41.

C. Zhuang et al. / European Journal of Medicinal Chemistry 46 (2011) 5654e5661
5659
5.2.20. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-(4-
fluorobenzyl amino)-2, 5-dioxo-2, 3-dihydro-1H-benzo [e] [1,4]
diazepin-4 (5H)-yl) acetate (7o)
6.08 (s,1H), 5.74 (s,1H), 4.18 (d, 2H, J ¼ 6.0 Hz), 3.70 (s, 3H). 13C NMR
(125 MHz, DMSO-d6)
d: 199.13, 169.73, 166.33, 151.67, 138.52,
138.18, 134.35, 133.80, 133.63, 132.26, 132.20, 132.08, 131.74, 129.30,
128.80, 128.64, 128.57, 127.13, 115.43, 110.89, 101.68, 70.97, 65.79,
52.61, 45.36. ESI-MS (m/z): 622.61 [M ꢃ 1]ꢃ. Anal. calcd. for
C31H24Cl3N3O3S: C, 59.58; H, 3.87; N, 6.72; Found: C, 59.66; H, 3.86;
N, 6.70.
Following General Procedure B, 4-fluorobenzaldehyde (25.6 mg)
and 6b (100 mg) gave 7o (90 mg) as a white solid, yield: 73.7%. 1H
NMR (500 MHz, DMSO-d6) d: 10.45 (s, 1H), 6.21e7.47 (m, 15H, Ar-
H), 6.18 (s, 1H), 5.93 (s, 1H), 5.20 (s, 1H), 4.15 (d, 2H, J ¼ 5.9 Hz),
3.71 (s, 3H). ESI-MS (m/z): 1207.96 [2M þ Na]þ. Anal. calcd. for
C31H24FCl2N3O4: C, 62.85; H, 4.08; N, 7.09; Found: C, 62.74; H, 4.09;
N, 7.11.
5.2.26. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
(neopentylamino)-5 -oxo-2-thioxo-2, 3-dihydro-1H-benzo [e] [1,4]
diazepin-4 (5H)-yl) acetate (8e)
5.2.21. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-(furan-
2-yl methylamino)-2, 5-dioxo-2, 3-dihydro-1H-benzo [e] [1,4]
diazepin-4 (5H)-yl) acetate (7p)
Yield: 86.0%, a light-yellow solid, mp: 150e151 ꢀC. 1H NMR
(500 MHz, DMSO-d6)
(s, 1H), 6.10 (s, 1H), 5.75 (s, 1H), 3.71 (s, 3H), 2.74 (m, 2H), 0.85 (s,
9H). 13C NMR (125 MHz, DMSO-d6)
: 199.01, 169.70, 166.36, 152.30,
d: 12.40 (s, 1H), 6.33e7.53 (m, 11H, Ar-H), 6.15
Following General Procedure B, furfural (19.9 mg) and 6b
d
(100 mg) gave 7o (89 mg) as a white solid, yield: 74.3%. 1H NMR
138.22, 134.38, 133.70, 133.65, 132.23, 132.18, 131.98, 128.73, 128.52,
127.12, 114.40, 110.70, 100.95, 70.94, 65.73, 52.53, 48.59, 33.12,
(500 MHz, DMSO-d6) d: 10.48 (s, 1H), 6.18e7.54 (m, 14H, Ar-H), 6.17
(s, 1H), 6.01 (s, 1H), 5.21 (s, 1H), 4.14 (d, 2H, J ¼ 5.9 Hz), 3.72 (s, 3H).
ESI-MS (m/z): 1151.93 [2M þ Na]þ. Anal. calcd. for C29H23Cl2N3O5: C,
61.71; H, 4.11; N, 7.44; Found: C, 61.82; H, 4.10; N, 7.42.
27.19. ESI-MS (m/z): 570.70 [M
C29H29Cl2N3O3S: C, 61.05; H, 5.12; N, 7.37; Found: C, 61.20; H, 5.11;
N, 7.35.
ꢃ
1]ꢃ. Anal. calcd. for
5.2.22. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-5-oxo-2-
thioxo-2, 3-dihydro-1H-benzo [e] [1,4] diazepin-4(5H)-yl) acetate
(8a)
5.2.27. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
(isobutylamino)-5- oxo-2-thioxo-2, 3-dihydro-1H-benzo [e] [1,4]
diazepin-4 (5H)-yl) acetate (8f)
Following General Procedure C, 6a (100 mg) and Lawesson’s
Reagent (47.5 mg) gave 8a (80.9 mg) as a yellow solid, yield: 78.3%,
Yield: 76.6%, a yellow solid, mp: 206e208 ꢀC. 1H NMR (500 MHz,
DMSO-d6) d: 12.43 (s, 1H), 6.30e7.53 (m, 11H, Ar-H), 6.07 (s, 1H),
mp: 249e252 ꢀC. 1H NMR (500 MHz, DMSO-d6)
d
: 10.77 (s, 1H),
7.08e7.50 (m, 12H, Ar-H), 6.26 (s, 1H), 5.31 (s, 1H), 3.77 (s, 3H). 13C
NMR (125 MHz, DMSO-d6) : 199.63, 169.48, 166.33, 136.54, 134.12,
6.07 (s, 1H), 5.75 (s, 1H), 3.71 (s, 3H), 2.72 (t, 2H, J ¼ 6.3 Hz), 1.74 (m,
1H), 0.85 (d, 6H, J ¼ 6.6 Hz). 13C NMR (125 MHz, DMSO-d6)
d:
d
198.99, 169.71, 166.35, 152.34, 138.23, 134.38, 133.75, 133.65, 132.22,
132.17, 131.98, 128.74, 128.54, 127.12, 114.49, 110.72, 100.86, 70.93,
65.72, 52.53, 50.44, 27.57, 20.60, 20.56. ESI-MS (m/z): 1103.59
[2M þ Na]þ. Anal. calcd. for C28H27Cl2N3O3S: C, 60.43; H, 4.89; N,
7.55; Found: C, 60.33; H, 4.90; N, 7.57.
133.79, 133.17, 132.80, 132.52, 132.42, 129.30, 128.96, 128.80, 127.91,
127.27, 126.03, 120.82, 70.86, 66.11, 52.83. ESI-MS (m/z): 483.56
[M ꢃ 1]ꢃ. Anal. calcd. for C24H18Cl2N2O3S: C, 59.39; H, 3.74; N, 5.77;
Found: C, 59.29; H, 3.75; N, 5.78.
5.2.23. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-5-oxo-8-
(propyl amino)-2-thioxo-2, 3-dihydro-1H-benzo [e] [1,4] diazepin-4
(5H)-yl) acetate (8b)
5.2.28. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-5-oxo-8-
(pentyl amino)-2-thioxo-2, 3-dihydro-1H-benzo [e] [1,4] diazepin-4
(5H)-yl) acetate (8g)
Yield: 75.4%, a light-yellow solid, mp: 210e211 ꢀC. 1H NMR
Yield: 78.1%, a light-yellow solid, mp: 150e151 ꢀC. 1H NMR
(500 MHz, DMSO-d6)
d: 12.43 (s, 1H), 6.26e7.53 (m, 11H, Ar-H), 6.11
(500 MHz, DMSO-d6) d: 12.44 (s, 1H), 6.25e7.52 (m, 11H, Ar-H), 6.10
(s, 1H), 6.09 (s, 1H), 5.75 (s, 1H), 3.70 (s, 3H), 2.86 (t, 2H, J ¼ 5.6 Hz),
(s, 1H), 6.05 (s, 1H), 5.75 (s, 1H), 3.71 (s, 3H), 2.89 (t, 2H, J ¼ 4.5 Hz),
1.48 (m, 2H), 0.86 (t, 3H, J ¼ 7.3 Hz). 13C NMR (125 MHz, DMSO-d6)
1.44 (m, 2H),1.25 (m, 4H), 0.85 (t, 3H, J ¼ 6.8 Hz).13C NMR (125 MHz,
d: 199.01, 169.76, 166.41, 152.22, 138.26, 134.41, 133.80, 133.65,
DMSO-d6) d: 199.02, 169.83, 166.41, 152.19, 138.27, 134.48, 133.81,
132.26, 132.08, 128.79, 128.59, 127.15, 114.59, 110.61, 100.95, 70.94,
65.75, 52.57, 44.49, 21.92, 11.85. ESI-MS (m/z): 542.24 [M þ 1]þ.
Anal. calcd. for C27H25Cl2N3O3S: C, 59.78; H, 4.65; N, 7.75; Found: C,
59.68; H, 4.66; N, 7.77.
133.75, 132.29, 132.17, 132.11, 128.83, 128.67, 127.19, 114.57, 110.19,
100.92, 71.05, 65.84, 52.66, 42.65, 29.17, 28.34, 22.38, 14.38. ESI-MS
(m/z): 572.56 [M þ 1]þ. Anal. calcd. for C29H29Cl2N3O3S: C, 61.05;
H, 5.12; N, 7.37; Found: C, 61.16; H, 5.11; N, 7.35.
5.2.24. Methyl 2-(8-(benzylamino)-3-(4-chlorophenyl)-5-oxo-2-
thioxo-2, 3-dihydro-1H-benzo[e] [1,4] diazepin-4 (5H)-yl)-2-(4-
chlorophenyl) acetate (8c)
5.2.29. Methyl 2-(8-(butylamino)-3-(4-chlorophenyl)-5-oxo-2-
thioxo-2, 3-dihydro-1H-benzo [e] [1,4] diazepin-4 (5H)-yl)-2-(4-
chlorophenyl) acetate (8h)
Yield: 72.1%, a light-yellow solid, mp: 150e151 ꢀC. 1H NMR
Yield: 80.8%, a light-yellow solid, mp: 205e206 ꢀC. 1H NMR
(500 MHz, DMSO-d6)
(s, 1H), 6.06 (s, 1H), 5.75 (s, 1H), 4.18 (d, 2H, J ¼ 6.0 Hz), 3.73 (s, 3H).
13C NMR (125 MHz, DMSO-d6)
: 199.10, 169.67, 166.34, 151.88,
139.36, 138.17, 134.32, 133.77, 133.60, 132.23, 132.17, 132.01, 128.75,
128.66, 128.52, 127.44, 127.18, 127.09, 115.23, 110.79, 101.58, 70.92,
65.73, 52.55, 46.06. ESI-MS (m/z): 590.15 [M þ 1]þ, 588.42 [M ꢃ 1]ꢃ.
Anal. calcd. for C31H25Cl2N3O3S: C, 63.05; H, 4.27; N, 7.12; Found: C,
63.15; H, 4.26; N, 7.11.
d
: 12.45 (s, 1H), 6.12e7.52 (m, 16H, Ar-H), 6.10
(500 MHz, DMSO-d6) d: 12.44 (s, 1H), 6.25e7.53 (m, 11H, Ar-H), 6.10
(s, 1H), 6.06 (s, 1H), 5.75 (s, 1H), 3.71 (s, 3H), 2.88 (t, 2H, J ¼ 4.6 Hz),
d
1.44 (m, 2H), 1.25 (m, 2H), 0.86 (t, 3H, J ¼ 6.7 Hz). 13C NMR
(125 MHz, DMSO-d6) d: 199.02, 169.82, 166.41, 152.21, 138.26,
134.47, 133.80, 133.74, 132.28, 132.18, 132.10, 128.82, 128.66, 127.19,
114.57, 110.60, 100.95, 71.04, 65.83, 52.65, 42.38, 30.80, 20.13, 14.18.
ESI-MS (m/z): 554.81 [M ꢃ 1]ꢃ. Anal. calcd. for C28H27Cl2N3O3S: C,
60.43; H, 4.89; N, 7.55; Found: C, 60.31; H, 4.90; N, 7.57.
5.2.25. Methyl 2-(8e((4-chlorobenzyl) amino)-3-(4-chlorophenyl)-
5-oxo-2-thioxo-2, 3-dihydro-1H-benzo [e] [1,4] diazepin-4 (5H)-
yl)-2-(4-chlorophenyl) acetate (8d)
5.2.30. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
((cyclopropyl methyl) amino)-5-oxo-2-thioxo-2, 3-dihydro-1H-
benzo [e] [1,4] diazepin-4 (5H)-yl) acetate (8i)
Yield: 62.9%, a yellow solid, mp: 150e151 ꢀC. 1H NMR (500 MHz,
Yield: 77.0%, a light-yellow solid, mp: 210e211 ꢀC. 1H NMR
DMSO-d6)
d
: 12.45 (s, 1H), 6.10e7.52 (m, 15H, Ar-H), 6.10 (s, 1H),
(500 MHz, DMSO-d6) d: 12.44 (s, 1H), 6.28e7.53 (m, 11H, Ar-H), 6.10

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C. Zhuang et al. / European Journal of Medicinal Chemistry 46 (2011) 5654e5661
(s, 1H), 6.09 (s, 1H), 5.75 (s, 1H), 3.71 (s, 3H), 2.80 (t, 2H, J ¼ 6.1 Hz),
(s,1H), 6.11 (s,1H), 5.75 (s,1H), 4.16 (d, 2H, J ¼ 5.9 Hz), 3.71 (s, 3H).13
C
0.94 (m, 1H), 0.41 (m, 2H), 0.15 (m, 2H). 13C NMR (125 MHz, DMSO-
NMR (125 MHz, DMSO-d6) d: 199.11, 169.70, 166.36, 151.78, 140.30,
d6)
d
: 198.99, 169.72, 166.37, 152.18, 138.20, 134.75, 133.82, 133.68,
138.17, 134.34, 133.77, 133.63, 132.25, 132.20, 132.00, 128.77, 128.56,
127.75,127.12,126.60,115.28,110.90,101.54, 70.95, 65.75, 52.57, 41.78.
ESI-MS (m/z): 594.64 [M ꢃ 1]ꢃ. Anal. calcd. for C29H23Cl2N3O3S2: C,
58.39; H, 3.89; N, 7.04; Found: C, 58.49; H, 3.88; N, 7.02.
132.23, 132.20, 132.06, 128.76, 128.58, 127.13, 114.65, 110.59, 101.11,
70.94, 65.73, 52.55, 47.00, 10.59, 3.75. ESI-MS (m/z): 554.87
[M þ 1]þ. Anal. calcd. for C28H25Cl2N3O3S: C, 60.65; H, 4.54; N, 7.58;
Found: C, 60.75; H, 4.53; N, 7.57.
5.2.36. Methyl 2-(8e((4-bromobenzyl) amino)-3-(4-chlorophenyl)-
5-oxo-2-thioxo-2, 3-dihydro-1H-benzo [e] [1,4] diazepin-4 (5H)-
yl)-2-(4-chlorophenyl) acetate (8o)
5.2.31. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-5-oxo-2-
thioxo-8e((4- (trifluoromethyl) benzyl) amino)-2, 3-dihydro-1H-
benzo [e] [1,4] diazepin-4 (5H)-yl) acetate (8j)
Yield: 70.8%, a yellow solid, mp: 236e237 ꢀC. 1H NMR (500 MHz,
Yield: 82.0%, a yellow solid, mp: 151e152 ꢀC. 1H NMR (500 MHz,
DMSO-d6) d: 12.44 (s, 1H), 6.27e7.95 (m, 15H, Ar-H), 6.10 (s, 1H),
DMSO-d6)
d
: 12.44 (s, 1H), 6.30e7.67 (m, 15H, Ar-H), 6.10 (s, 1H),
6.08 (s, 1H), 5.73 (s, 1H), 4.17 (d, 2H, J ¼ 6.1 Hz), 3.71(s, 3H). 13C NMR
6.09 (s, 1H), 5.74 (s, 1H), 4.30 (d, 2H, J ¼ 6.1 Hz), 3.70 (s, 3H). 13C
(125 MHz, DMSO-d6) d: 199.11,169.67, 166.30, 151.62,138.93, 138.16,
NMR (125 MHz, DMSO-d6)
d
: 199.15, 169.67, 166.30, 151.58, 144.54,
134.31, 133.77, 132.23, 132.17, 132.04, 131.51, 129.62, 128.75, 128.51,
127.09, 120.15, 115.44, 110.87, 101.67, 70.92, 65.74, 52.55, 45.40. ESI-
MS (m/z): 668.79 [M þ 1]þ. Anal. calcd. for C31H24BrCl2N3O3S: C,
55.62; H, 3.61; N, 6.28; Found: C, 55.51; H, 3.62; N, 6.30.
138.19, 134.31, 133.79, 133.58, 132.24, 132.16, 132.11, 128.76, 128.50,
128.03, 127.11, 125.53, 125.50, 115.59, 110.85, 101.68, 70.92, 65.75,
52.55, 45.58. ESI-MS (m/z): 656.60 [M ꢃ 1]ꢃ. Anal. calcd. for
C32H24Cl2F3N3O3S: C, 58.36; H, 3.67; N, 6.38; Found: C, 58.50; H,
3.66; N, 6.36.
5.2.37. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8e((4-
fluorobenzyl) amino)-5-oxo-2-thioxo-2, 3-dihydro-1H-benzo [e]
[1,4] diazepin-4 (5H)-yl) acetate (8p)
5.2.32. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
((cyclohexyl methyl) amino)-5-oxo-2-thioxo-2, 3-dihydro-1H-
benzo [e] [1,4] diazepin-4 (5H)-yl) acetate (8k)
Yield: 75.2%, a yellow solid, mp: 147e148 ꢀC. 1H NMR (500 MHz,
DMSO-d6) d: 12.45 (s, 1H), 6.30e7.52 (m, 15H, Ar-H), 6.10 (s, 1H),
Yield: 86.1%, a yellow solid, mp: 151e152 ꢀC. 1H NMR (500 MHz,
6.10 (s, 1H), 5.74 (s, 1H), 4.17 (d, 2H, J ¼ 5.9 Hz), 3.70 (s, 3H). 13C NMR
DMSO-d6)
6.05 (s, 1H), 5.74 (s, 1H), 3.71 (s, 3H), 2.74 (m, 2H), 1.98 (m, 1H),
1.65e0.85 (m, 10H). 13C NMR (125 MHz, DMSO-d6)
: 198.99,
d: 12.42 (s, 1H), 6.25e7.53 (m, 11H, Ar-H), 6.10 (s, 1H),
(125 MHz, DMSO-d6) d: 199.12, 169.73, 166.34, 151.74, 138.18,
135.52, 134.36,133.80,133.64,132.26,132.20,132.08,129.47,129.40,
128.79, 128.58, 127.13, 115.50, 115.33, 110.88, 101.64, 70.98, 65.78,
52.61, 45.36. ESI-MS (m/z): 1239.59 [2M þ Na]þ. Anal. calcd. for
C31H24FCl2N3O3S: C, 61.19; H, 3.98; N, 6.91; Found: C, 61.29; H, 3.97;
N, 6.89.
d
169.72, 166.36, 152.37, 138.24, 134.66, 133.78, 133.68, 132.22, 132.17,
131.96, 128.74, 128.52, 127.12, 114.40, 110.76, 100.76, 70.92, 65.71,
52.52, 49.08, 37.07, 30.92, 26.45, 25.84. ESI-MS (m/z): 594.66
[M ꢃ 1]ꢃ. Anal. calcd. for C31H31Cl2N3O3S: C, 62.41; H, 5.24; N, 7.04;
Found: C, 62.52; H, 5.23; N, 7.02.
5.2.38. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
((furan-2-yl methyl) amino)-5-oxo-2-thioxo-2, 3-dihydro-1H-benzo
[e] [1,4] diazepin-4 (5H)-yl) acetate (8q)
5.2.33. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
((naphthalen-2-yl methyl) amino)-5-oxo-2-thioxo-2, 3-dihydro-
1H-benzo [e] [1,4] diazepin-4 (5H)-yl) acetate (8l)
Yield: 81.2%, a yellow solid, mp: 180e181 ꢀC. 1H NMR (500 MHz,
DMSO-d6) d: 12.43 (s,1H), 6.30e8.51 (m,14H, Ar-H), 6.09 (s,1H), 6.09
Yield: 54.9%, a yellow solid, mp: 259e260 ꢀC. 1H NMR (500 MHz,
(s, 1H), 5.74 (s, 1H), 4.28 (d, 2H, J ¼ 5.9 Hz), 3.70 (s, 3H). 13C NMR
DMSO-d6)
d: 12.44 (s, 1H), 7.87 (m, 2H), 7.68 (m, 1H), 7.51 (s, 1H),
(125 MHz, DMSO-d6) d: 199.12, 169.69, 166.33, 152.44, 151.51, 142.50,
7.43e7.50 (m, 7H), 7.24 (d, 1H, J ¼ 8.8 Hz), 7.14 (m, 5H), 6.35 (d, 1H,
138.07, 134.95, 133.87, 133.69, 132.24, 131.99, 128.77, 128.57, 127.11,
115.51, 110.79, 110.73, 107.58, 101.79, 70.95, 65.75, 52.56, 40.43. ESI-
MS (m/z): 1181.31 [2M þ Na]þ. Anal. calcd. for C29H23Cl2N3O4S: C,
60.00; H, 3.99; N, 7.24; Found: C, 60.11; H, 3.98; N, 7.23.
J ¼ 8.6 Hz), 6.17 (s,1H), 6.10 (s,1H), 5.72 (s,1H), 4.36 (d, 2H, J ¼ 5.7 Hz),
3.69 (s, 3H). 13C NMR (125 MHz, DMSO-d6)
d: 199.04, 169.68, 166.32,
151.89, 138.17, 136.98, 134.31, 133.76, 133.59, 133.28, 132.57, 132.22,
132.06, 128.75, 128.51, 128.31, 127.93, 127.89, 127.08, 126.55, 125.99,
125.56,115.26,110.85,101.90, 70.91, 65.73, 52.55, 46.30. ESI-MS(m/z):
642.75 [M þ 1]þ. Anal. calcd. for C35H27Cl2N3O3S: C, 65.62; H, 4.25; N,
6.36; Found: C, 65.75; H, 4.24; N, 6.34.
5.3. Computational protocol
Molecular docking was used to predict the binding mode of the
synthesized thio-benzodiazepine derivatives. The crystal structure
[9] of MDM2 (PDB code: 1T4E) was prepared by removing the
benzodiazepine and adding hydrogen atoms in GOLD 4.1.2. We
used two known potent inhibitors of the p53eMDM2 interaction
with different chemical structures (TDP222669 and nutlin-3a) as
positive controls. The docking parameters of GOLD were similar to
the literatures [15,22]. We also used MVD 4.3.0 for docking to
confirm the robustness of the docking pose. The active site was
defined to encompass all MDM2 atoms within a 10 Å radius sphere
from the center of the Trp23 of the p53 peptide ligand. Other
parameters were set by default. As a result, these two docking
programs obtained approximate binding poses.
5.2.34. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8e((4-
methoxy benzyl) amino)-5-oxo-2-thioxo-2, 3-dihydro-1H-benzo [e]
[1,4] diazepin-4 (5H)-yl) acetate (8m)
Yield: 78.6%, a yellow solid, mp: 222e223 ꢀC. 1H NMR (500 MHz,
DMSO-d6) d: 12.45 (s, 1H), 6.29e7.53 (m, 15H, Ar-H), 6.11 (s, 1H), 6.10
(s,1H), 5.74 (s,1H), 4.10 (d, 2H, J ¼ 5.7 Hz), 3.72 (s, 3H), 3.70 (s, 3H).13C
NMR (125 MHz, DMSO-d6) d: 199.07, 169.70, 166.34, 158.65, 151.88,
138.15, 134.34, 133.77, 133.62, 132.23, 132.16, 131.99, 131.15, 128.75,
128.53,127.11,115.12,114.12,110.81,101.56, 70.94, 65.73, 55.46, 52.53,
45.53. ESI-MS(m/z): 618.64 [Mꢃ 1]ꢃ. Anal. calcd. for C32H27Cl2N3O4S:
C, 61.94; H, 4.39; N, 6.77; Found: C, 61.81; H, 4.40; N, 6.79.
5.2.35. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-5-oxo-8-
((thiophen -3-ylmethyl) amino)-2-thioxo-2, 3-dihydro-1H-benzo
[e] [1,4] diazepin-4 (5H)-yl) acetate (8n)
5.4. p53eMDM2 binding assay
The dose-dependent binding experiments were carried out
with serial dilution in DMSO of compounds. A 5
Yield: 75.3%, a light-yellow solid, mp: 165e166 ꢀC. 1H NMR
mL sample of the
(500 MHz, DMSO-d6) d: 12.46 (s, 1H), 6.20e7.53 (m, 14H, Ar-H), 6.15
tested sample and preincubated (for 30 min) MDM2 binding

C. Zhuang et al. / European Journal of Medicinal Chemistry 46 (2011) 5654e5661
5661
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domain (1-118) (10 nM) and PMDM-F peptide (Anaspec) (10 nM) in
the assay buffer (100 mM potassium phosphate, pH 7.5; 100 g/mL
bovine gamma globulin; 0.02% sodium azide were added into black
96-well microplates with F-bottom and chimney wells (Corning) to
m
produce a final volume of 115
ml. For each assay, the controls
included the MDM2 binding domain and PMDM-F. The polarization
values were measured after 1 h of incubation at room temperature
using Biotek Synergy H4 with a 480 nm excitation filter, a 528-nm
static and polarized filter. The Ki values were determined from
a plot using nonlinear least-squares analysis. And curve fitting was
performed using GraphPad Prism software. Nutlin-3a (Sigma-
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assay in each plate.
5.5. In vitro antitumor activity
The cellular growth inhibitory activity was determined using
two human osteosarcoma cell lines [U-2 OS (wild-type p53), Saos-2
(p53 null)]. 5e6 ꢂ 104 cells per well were plated in 96-well plates.
After culturing for 24 h, test compounds were added onto triplicate
wells with different concentrations and 0.1% DMSO for control.
After 72 h of incubation, 20 mL of MTT (3-[4,5-dimethylthiazol-2-
yl]-2,5-di phenyltetrazoliumbromide) solution (5 mg/mL) was
added to each well, and after the samples were shaken for 1 min
the plate was incubated further for 4 h at 37 ꢀC. Thio-
benzodiazepines were dissolved with 100 mL of DMSO. The absor-
bance (OD) was quantitated with microplate using Biotek Synergy
H4 at 570 nm. Wells containing no drugs were used as blanks.
Concentration of the compounds that inhibited cell growth by 50%
(IC50) was calculated.
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Acknowledgement
This work was supported in part by the Key Project of
Science and Technology of Shanghai (No. 09431901700). We thank
Dr. Chuanxu Liu at Key Laboratory of Cell Differentiation and
Apoptosis of National Ministry of Education, Shanghai Jiao Tong
University School of Medicine for technical assistance about
p53eMDM2 binding assay.
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