Structure-activity relationship and antitumor activity of thio-benzodiazepines as p53-MDM2 protein-protein interaction inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.08.003

In order to discuss the structure-activity relationship (SAR) of the thio-benzodiazepine compounds which showed excellent activity against p53-MDM2 protein-protein interaction, we designed and synthesized twenty compounds with electrophilic and nucleophil

Full text of DOI:10.1016/j.ejmech.2012.08.003

European Journal of Medicinal Chemistry 56 (2012) 10e16
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Structureeactivity relationship and antitumor activity of thio-benzodiazepines
as p53eMDM2 proteineprotein interaction inhibitors
Zizhao Guo 1, Chunlin Zhuang 1, Lingjian Zhu, Yongqiang Zhang, Jianzhong Yao, Guoqiang Dong,
***
*
**
Shengzheng Wang, Yang Liu, Hai Chen, Chunquan Sheng , Zhenyuan Miao , 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:
In order to discuss the structureeactivity relationship (SAR) of the thio-benzodiazepine compounds
which showed excellent activity against p53eMDM2 proteineprotein interaction, we designed and
synthesized twenty compounds with electrophilic and nucleophilic groups on the benzene ring. Among
them, compounds 8i (Ki ¼ 91 nM) and 8n (Ki ¼ 89 nM) showed better binding activity than that of the
reference drug Nutlin-3a (Ki ¼ 121 nM). In addition, in vitro antitumor activity against Saos-2, U-2 OS,
A549 and NCI-H1299 cell-lines were assayed by the MTT method. Especially, compounds 8i and 8n
possessed excellent biological activity and good selectivity comparable to Nutlin-3a, which were
promising candidates for further evaluation.
Received 22 April 2012
Received in revised form
19 July 2012
Accepted 1 August 2012
Available online 10 August 2012
Keywords:
p53eMDM2
Thio-benzodiazepine
SAR
Ó 2012 Elsevier Masson SAS. All rights reserved.
Antitumor activity
1. Introduction
many series of small-molecule inhibitors were described, including
benzodiazepinediones, nutlins, spiro-oxindoles, quinolinols, iso-
The tumor suppressor p53 is one of the most important proteins
in human cancers [1,2]. Its main functions are cell-cycle arrest, DNA
repair, and apoptosis [3]. The p53 mutations are very common in
human tumors, however it remains wild-type in approximately
50% of human cancers [4]. Although 50% of all human tumors
express wild-type p53, many are thought to have inadequate p53
function due to abnormalities in p53 regulation or defective
signaling in the p53 pathway [2]. Murine double minute 2 (MDM2)
is a protein which can inhibit p53’s ability to bind to DNA, activate
transcription and promote rapid degradation of p53. In turn, p53
activates the expression of the MDM2 protein in an autoregulatory
negative feedback loop [5].
indolinones, chlorofusin, norbornanes, sulfonamides, chalcones,
terphenyls, and piperazine-4-phenyl derivatives [3,7e15], many of
which showed relatively weak bioactivity, only the nutlins, the
spiro-oxindoles and the benzodiazepinediones are particularly
valuable [3,7]. Nultins were the first potent and selective
p53eMDM2 interaction inhibitors developed by Roche, and
promising candidates RG7112 [16] and RO5503781 [17] entered
phase I clinical trials, but their detailed structures were not re-
ported [18]. The spiro-oxindoles developed by Wang’s group
possessed good pharmacokinetic properties as well as a high
binding affinity to MDM2. Among them, one representative
candidate MI-219 with a Ki value of 5 nM was a potent and orally
active small-molecule inhibitor [19].
In 2005, Grasberger and his co-workers firstly reported 1,4-
benzodiazepine-2,5-dione compounds as the small molecule
inhibitors of the p53eMDM2 proteineprotein interaction [7]. The
benzodiazepine was thought to be a “privileged structure” in
medicinal chemistry [20]. In our previous work, we first reported
and synthesized a series of thio-benzodiazepines with the principle
of bioisosterism and found that many compounds had nanomolar
affinity toward MDM2 and exhibited potent antitumor activities
against the U-2 OS human osteosarcoma cell line (Fig.1) [21], which
is worthy of further structural optimization. Herein, we designed
and synthesized a series of thio-benzodiazepines with electrophilic
Due to the crucial role of p53 in tumor suppression, reactivation
of the p53 function by disruption of the p53eMDM2 interaction
using non-peptide small-molecule inhibitors is now recognized as
a new and promising strategy for anticancer drug design [6]. So far,
* Corresponding author. Tel./fax: þ86 21 81871241.
** Corresponding author. Tel./fax: þ86 21 81871243.
*** Corresponding author. Tel./fax: þ86 21 81871239.
E-mail addresses: shengcq@hotmail.com (C. Sheng), miaozhenyuan@
hotmail.com (Z. Miao), zhangwnk@hotmail.com (W. Zhang).
1
These two authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.08.003

Z. Guo et al. / European Journal of Medicinal Chemistry 56 (2012) 10e16
11
Fig. 1. The structures of TDP222669, Nutlin-3a and 1.
and nucleophilic groups on the benzene ring to extend the SAR and
find promising lead compounds with excellent biological
properties.
nucleophilic activity of sulfur atom, the substitution reaction took
place on sulfur instead of nitrogen atom.
3. Results and discussion
2. Chemistry
3.1. p53eMDM2 binding assays
A general pathway for the synthesis of target compounds 8aet
(Table 2) was outlined in Scheme 1. The skeleton of thio-
benzodiazepines was synthesized utilizing the highly efficient
and versatile Ugi four-component condensation (Ugi 4CC reaction)
[22]. We used substituted benzaldehydes (5) as the aldehyde,
methyl amino(4-chlorophenyl)acetate hydrochloride or methyl 2-
amino-2-(4-fluorophenyl) acetate hydrochloride (3) as the amine,
substituted nitrobenzoic acids (2) as the carboxylic acid and 1-
isocyanocyclohexene (4) as the isocyanide to perform the Ugi
reaction. In this synthetic process, the purity of the starting mate-
rial 4 had significant influence on the yield of Ugi reaction. The
obtained key intermediate 7 was treated with the Lawesson’s
reagent to afford the compounds 8aer with the yield of 50e87%. In
order to increase the structural diversity, two compounds 8s and 8t
with substituents on sulfur atom were synthesized by the nucleo-
philic substitution reaction in presence of 1,8-diazabicyclo[5.4.0]
undec-7-ene (DBU) at room temperature. Owing to the stronger
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 inhibitors,
was used as the reference drug. The results were listed in Table 1.
Most of the targeted thio-benzodiazepines had nanomolar to
micromolar affinity toward MDM2. The detailed SAR analysis
demonstrated that the proper substituents on the position 7 and 8
were beneficial to the binding activity. In particular, most fluorine-
containing compounds on the position 7 or 8 exhibited moderate to
excellent affinity. For example, compounds 8i (Ki ¼ 91 nM) and 8n
(Ki ¼ 89 nM) with trifluoromethyl group and fluorine atom on
position 8 showed excellent binding activity superior to the refer-
ence compound (Ki ¼ 121 nM). In addition, compounds 8d, 8f, 8g,
8j, 8o and 8q also displayed good binding affinity comparable to
Nutlin-3a. However, the difluorinated compound 8e had relatively
weak activity. Interestingly, compound 8c with three chlorine
atoms also showed good binding affinity with a Ki value of 721 nM.
The results also revealed that the substituents on the position 9
generated negative influence on the activity. For instance,
compounds 8b, 8h and 8l exhibited weak binding activity.
Furthermore, we found that the substituents on sulfur atom led to
significant decrease of activity, such as compounds 8s and 8t.
According to the previously reported structures of the inhibitors
binding to MDM2 and our docking model, we presented
Table 1
Binding constants (Ki) of the MDM2 ligands and IC50 values of in vitro antitumor
activity.
b
Compounds
Kia
(
m
M)
IC50
(m
M)
Saos-2
U-2 OS
A549
NCI-H1299
(p53 null)
(p53 null)
(wt-p53)
(wt-p53)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
1.37
11.1
61.8
8.91
6.59
3.84
12.7
6.48
>100
6.78
13.7
20.0
19.1
25.5
11.9
32.1
11.0
9.23
>100
>100
>100
12.1
25.3
12.1
88.5
3.50
12.0
2.61
44.3
7.23
>100
5.69
11.8
35.3
27.8
32.7
7.53
28.0
35.7
27.4
33.9
67.6
>100
19.6
15.9
37.2
>100
18.7
8.44
11.3
25.6
19.7
>100
26.9
9.44
>100
>100
>100
12.9
19.4
>100
23.7
>100
>100
>100
15.0
16.5
>100
24.7
16.5
5.73
>100
29.7
>100
11.1
14.0
>100
73.3
73.6
14.5
25.0
35.4
39.5
>100
>100
>100
20.4
42.6
>100
0.721
0.707
>100
1.72
0.315
8.18
0.0910
3.02
>100
>100
37.7
0.0890
1.05
3.98
0.518
11.9
Table 2
The structures of thio-benzodiazepines compounds 8ae8r.
compounds
R1
R2
R3
R4
R
R0
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
F
H
Cl
H
H
H
H
H
CH3
F
Cl
H
H
F
F
Cl
H
H
CF3
CF3
OCH3
H
H
H
F
Br
Cl
Cl
H
Cl
H
H
H
H
H
OCH3
H
H
H
CH3
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
F
Cl
Cl
Cl
Cl
Cl
F
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
Cl
Cl
F
Cl
Cl
Cl
Cl
F
8s
8t
>100
21.1
0.121
6.70
H
H
H
H
Nutlin-3a
1
Cl
Cl
Cl
25.7
CF3
OCH3
a
Values were determined by fluorescence polarization assay.
Values were measured with MTT method.
b

12
Z. Guo et al. / European Journal of Medicinal Chemistry 56 (2012) 10e16
Scheme 1. Reagents and conditions: (i) MeOH, KOH, r.t., 24 h; (ii) Fe powder, AcOH, 70 ꢀC, 1 h; (iii) Lawesson’s reagent, toluene, 70 ꢀC, 4 h (iv) MeI or BrCH2COOEt, DBU, DCM, r.t.,
0.5e1 h.
Fig. 2. The docking models of thio-benzodiazepines inhibitors with MDM2. The inhibitors are shown in stick models with carbon atoms light purple, nitrogen blue, oxygen red,
fluorine light green and sulfur yellow. Hydrogen bonds are depicted as green dashed lines. The amino acid residues which were formatted H-bonds are labeled. (A) Compound 8i;
(B) Compound 8n. The figures were prepared from PDB ID: 1T4E, using PyMol (http://pymol.souceforge.net/).
a hypothetical binding model for the thio-benzodiazepine-MDM2
complex (Fig. 2). The binding interactions involved three hydro-
phobic pockets that were filled by three aromatic rings of the thio-
benzodiazepine. This binding model also predicted that the ester
group was well positioned as hydrogen bond acceptor with Gly16,
which may account for the enhancement of binding activity.
better antitumor activity than the most potent compound 1.
Meaningfully, most compounds revealed good selectivity against
the U-2 OS cell line with wild type p53 and the Saos-2 cell line with
p53 deficiency. Several compounds also had good inhibitory
selectivity against the A549 cell line (wild type p53) and the NCI-
H1299 cell line (p53 null). Importantly, based on the data in
Table 1, we found that most compounds showed good in vitro
antitumor activity, which were consistent with the results in the
p53eMDM2 binding assays. Two promising compounds, 8i and 8n
showed excellent p53eMDM2 binding affinity and excellent in vitro
antitumor activity, which were promising candidates for further
evaluation.
3.2. In vitro antitumor activity
All the compounds were further evaluated in MTT assays for
ascertaining whether their in vitro antitumor activity showed good
relevance with the inhibitory activity of p53eMDM2 binding. In
order to demonstrate the rationality of this class of thio-
benzodiazepines, they were compared with the most potent thio-
compound 1 reported by our group [21]. The results (Table 1)
revealed that most compounds showed moderate to excellent
in vitro antitumor activity. With regard to the human osteosarcoma
U-2 OS cell line (wild-type p53), many compounds, such as 8a, 8c,
8d, 8e, 8g, 8i, 8j and 8n, showed better biological activity than
Nutlin-3a and 1. For the lung cancer A549 cell line (wild-type p53),
compounds 8d, 8e, 8j and 8n displayed higher in vitro anti-
proliferative activity than Nutlin-3a. At the same time, eight
compounds, namely 8a, 8c, 8e, 8g, 8i, 8n, 8p and 8q, also exhibited
4. Conclusion
In conclusion, we designed and synthesized a series of novel
thio-benzodiazepines by Ugi reaction with the purpose to explore
its SAR. In the p53eMDM2 binding assay, most of targeted
compounds have good binding affinity comparable to the reference
drug. Moreover, many compounds displayed good activity in
in vitro antitumor activity assay compared with Nutlin-3a. Inter-
estingly, several compounds exhibited good correlation between
the binding activity and in vitro antitumor activity. In particular,

Z. Guo et al. / European Journal of Medicinal Chemistry 56 (2012) 10e16
13
two representative compounds, namely 8i and 8n, showed excel-
5.2.2. Methyl 2-(9-chloro-3-(4-chlorophenyl)-2,5-dioxo-2,3-
dihydro-1H-benzo[e] [1,4]diazepin-4(5H)-yl)-2-(4-chlorophenyl)
acetate (7b)
lent biological activity in both p53eMDM2 binding affinity and
in vitro antitumor activity. A better understanding of this SAR could
provide meaningful insights for further optimization.
1H NMR (DMSO-d6, 500 MHz):
d 10.39 (s, 1H, NeH), 7.55e7.53
(d, 2H, J ¼ 8.55 Hz, AreH), 7.48e7.46 (d, 2H, J ¼ 8.55 Hz, AreH),
7.43e7.38 (m, 2H, AreH), 7.20e7.04 (m, 5H, AreH), 6.19 (s, 1H,
CeH), 5.38 (s, 1H, CeH), 3.77 (s, 3H, CeH). 13C NMR (150 MHz,
5. Experimental protocols
5.1. General methods
CDCl3) d: 169.69, 169.46, 166.67, 135.78, 133.80, 132.45, 131.45,
131.32, 131.19, 131.17, 129.65, 129.61, 128.61, 128.49, 125.49, 125.23,
123.59, 62.80, 61.69, 52.91; HRMS (ESI): m/z [M þ H]þ calcd for
C24H17Cl3N2O4: 503.0332; found: 503.0333.
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 600
spectrometer (Bruker Company, Germany), using TMS as an
internal standard and CDCl3 or DMSO-d6 as solvents. Chemical
5.2.3. Methyl 2-(7-chloro-3-(4-chlorophenyl)-2,5-dioxo-2,3-
dihydro-1H-benzo [e][1,4]diazepin-4(5H)-yl)-2-(4-chlorophenyl)
acetate (7c)
1H NMR (DMSO-d6, 500 MHz):
d 10.91 (s, 1H, NeH), 7.51e7.35
shifts (
d
values) and coupling constants (J values) are given in ppm
(m, 6H, AreH), 7.24e7.22 (d, 2H, J ¼ 9.9 Hz, AreH), 7.10e7.09 (d,
2H, J ¼ 8.05 Hz, AreH), 6.88e6.86 (d, 2H, J ¼ 8.6 Hz, AreH), 6.28 (s,
1H, CeH), 5.33(s, 1H, CeH), 3.76 (s, 3H, CeH). 13C NMR (150 MHz,
and Hz, respectively. TLC analysis was carried out on silica gel plates
GF254 (Qindao Haiyang Chemical, China). Flash column chroma-
tography (Biotage, LCD 2073 A) was carried out on silica gel
300e400 mesh. Anhydrous solvent and reagents were all analytical
pure and dried through routine protocols.
CDCl3)
d: 170.92, 169.87, 166.38, 135.76, 133.93, 133.16, 132.77,
131.30, 131.21, 130.59, 130.26, 129.62, 128.70, 127.62, 125.71, 121.41,
62.66, 61.94, 52.88; HRMS (ESI): m/z [M
C24H17Cl3N2O4: 503.0332; found: 503.0336.
þ
H]þ calcd for
5.2. General procedure for the synthesis of benzodiazepines (7) [23]
5.3. General procedure for the synthesis of thio-benzodiazepines
Powdered substituted 2-nitrobenzoic acid (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 (10 mmol) or 2-amino-2-(4-
fluorophenyl)acetate hydrochloride (10 mmol) (3), a solution of
1-isocyanocyclohexene (4) (11 mmol) in CH3OH (2 mL) and a solu-
tion of p-chlorobenzaldehyde or 4-fluorobenzaldehyde (5)
(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 1 day. 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 liquid was discarded while still
warm. An analogous treatment was performed with boiling H2O
(50 mL ꢁ 2). The resulting solid product was stirred with AcOH
(60 mL). The resulting solution was heated at 50 ꢀC and treated
under vigorous stirring with iron powder (40 mmol) in one portion.
When the exothermic reaction had subsided, the reaction mixture
was heated at 70 ꢀ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 transferred to
a separating funnel. The organic layer was washed with H2O
(50 mL), NaHCO3 (aq. 2%, 50 mL), H2O (50 mL) and then separated,
dried (Na2SO4), and evaporated to dryness. The residue was puri-
fied by column chromatography (n-hexaneeethyl acetate, 5:1) to
give 7aer, yield: 45e52%.
(8aer)
A dry toluene solution of 7aer (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 compounds 8aer, yield: 50e80%.
5.3.1. Methyl 2-(8-chloro-3-(4-chlorophenyl)-5-oxo-2-thioxo-2,3-
dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)-2-(4-chlorophenyl)
acetate (8a)
According to the general procedure, compound 7a (5 mmol),
Lawesson’s reagent (3 mmol) gave compound 8a as a light-yellow
solid (1.619 g, yield: 62%), m.p. 156e158 ꢀC. 1H NMR (DMSO-d6,
600 MHz): d 12.79 (s, 1H, NeH), 7.56e7.46 (m, 5H, CeH), 7.26e7.22
(m, 4H, AreH), 7.16 (dd, 1H, J ¼ 10.1, 1.7 Hz, AreH), 7.07 (d, 1H,
J ¼ 1.4 Hz, AreH), 6.21 (s, 1H, CeH), 5.85 (s, 1H, CeH), 3.74 (s, 3H,
CeH). 13C NMR (150 MHz, DMSO-d6)
d: 200.03, 169.35, 165.45,
137.49, 136.75, 134.17, 133.52, 132.90, 132.70, 132.61, 132.37, 128.98,
128.95, 127.26, 126.64, 125.96, 120.10, 70.63, 66.03, 52.86; ESI-MS
(m/z): 517.25 (M ꢂ H).
5.3.2. Methyl 2-(9-chloro-3-(4-chlorophenyl)-5-oxo-2-thioxo-2,3-
dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)-2-(4-chlorophenyl)
acetate (8b)
A light-yellow solid. Yield: 70%. M.p. 151e153 ꢀC. 1H NMR
(DMSO-d6, 600 MHz):
d
12.17 (s, 1H, NeH), 7.59 (d, 2H, J ¼ 8.55 Hz,
The 1H NMR data of the partly representative benzodiazepines.
AreH), 7.47e7.42 (m, 4H, AreH), 7.23e7.12 (m, 5H, AreH), 6.16 (s,
1H, CeH), 5.92 (s, 1H, CeH), 3.77 (s, 3H, CeH). 13C NMR (150 MHz,
5.2.1. Methyl 2-(8-chloro-3-(4-chlorophenyl)-2,5-dioxo-2,3-
dihydro-1H-benzo [e][1,4]diazepin-4(5H)-yl)-2-(4-chlorophenyl)
acetate (7a)
DMSO-d6) d: 204.20, 173.19, 169.29, 138.06, 137.55, 137.34, 137.19,
136.97, 136.80, 136.50, 135.67, 132.94, 132.82, 132.77, 131.86, 130.66,
129.48, 75.48, 70.41, 56.85; ESI-MS (m/z): 517.26 (M ꢂ H).
1H NMR (DMSO-d6, 600 MHz):
d 10.89 (s, 1H, NeH), 7.52e7.47
(m, 5H, CeH), 7.23e7.06 (m, 5H, AreH), 6.92 (s, 1H, AreH), 6.29
5.3.3. Methyl 2-(7-chloro-3-(4-chlorophenyl)-5-oxo-2-thioxo-2,3-
dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)-2-(4-chlorophenyl)
acetate (8c)
(s,1H, CeH), 5.33 (s,1H, CeH), 3.76 (s, 3H, CeH). 13C NMR (150 MHz,
CDCl3)
d: 171.35, 170.02, 166.94, 138.40, 135.76, 135.69, 133.87,
132.46, 131.38, 131.24, 129.66, 128.68, 125.77, 125.15, 124.90, 119.82,
A yellow solid. Yield: 59%. M.p. 260e262 ꢀC. 1H NMR (DMSO-d6,
62.62, 61.77, 52.91; HRMS (ESI): m/z [M
C24H17Cl3N2O4: 503.0332; found: 503.0327.
þ
H]þ calcd for
600 MHz):
d
12.84 (s, 1H, NeH), 7.55 (d, 2H, J ¼ 8.55 Hz, AreH),
7.48e7.45 (m, 3H, AreH), 7.42 (dd, 1H, J ¼ 8.7, 2.6 Hz, AreH),

14
Z. Guo et al. / European Journal of Medicinal Chemistry 56 (2012) 10e16
7.26e7.22 (m, 4H, AreH), 7.02 (d, 1H, J ¼ 8.7 Hz, AreH), 6.22 (s, 1H,
CeH), 5.87 (s, 1H, CeH), 3.76 (s, 3H, CeH). 13C NMR (150 MHz,
5.86 (s, 1H, CH), 3.73 (s, 6H, CH3). 13C NMR (150 MHz, DMSO-d6)
d:
198.88, 169.33, 165.91, 149.60, 133.96, 133.86, 133.37, 132.45, 132.39,
129.63, 128.79, 128.66, 127.26, 126.81, 126.27, 121.33, 114.93, 71.42,
66.33, 57.09, 52.78; ESI-MS (m/z): 515.52 (M þ H).
CDCl3)
d: 199.93, 169.21, 165.67, 135.92, 133.92, 133.82, 132.68,
131.84, 131.59, 130.91, 130.44, 129.50, 128.88, 128.72, 126.21, 120.59,
67.13, 64.27, 52.77; ESI-MS (m/z): 517.36 (M ꢂ H).
5.3.9. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-5-oxo-2-
thioxo-8-(trifluoromethyl)-2,3-dihydro-1H-benzo[e][1,4]diazepin-
4(5H)-yl)acetate (8i)
5.3.4. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-fluoro-
5-oxo-2- thioxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)
acetate (8d)
A light-yellow solid. Yield: 53%. M.p. 151e152 ꢀC. 1H NMR
A light-yellow solid. Yield: 58%. M.p. 140e142 ꢀC. 1H NMR
(DMSO-d6, 600 MHz):
d
12.89 (s, 1H, NeH), 7.68 (d, 1H, J ¼ 8.15 Hz,
(DMSO-d6, 600 MHz):
d
12.79 (s, 1H, NH), 7.57e7.55 (m, 3H, AreH),
AreH), 7.56 (d, 2H, J ¼ 8.5 Hz, AreH), 7.48e7.43 (m, 3H, AreH), 7.36
(s, 1H, AreH), 7.26e7.21 (m, 4H, AreH), 6.24 (s, 1H, CeH), 5.90 (s,
7.46 (d, 2H, J ¼ 8.52 Hz, AreH), 7.23 (s, 4H, AreH), 6.98e6.95 (m,1H,
AreH), 6.82 (dd, 1H, J ¼ 9.9, 2.4 Hz, AreH), 6.21 (s, 1H, CH), 5.85 (s,
1H, CeH), 3.76 (s, 3H, CeH). 13C NMR (150 MHz, DMSO-d6)
d:
1H, CH), 3.74 (s, 3H, CH3). 13C NMR (150 MHz, CDCl3)
d
: 200.48,
200.19, 169.26, 165.23, 136.84, 134.21, 133.31, 132.76, 132.73, 132.26,
132.20, 131.08, 130.54, 129.70, 129.00, 128.92, 127.28, 126.61, 124.25,
122.44, 122.14, 117.60, 70.58, 66.08, 52.92; ESI-MS (m/z): 553.24
(M þ H).
169.51, 166.40, 165.04, 163.35, 137.20, 137.13, 135.93, 133.89, 133.82,
133.78, 131.99, 131.88, 130.40, 129.53, 128.64, 126.23, 124.14, 124.12,
113.75, 113.60, 106.43, 106.26, 67.15, 64.30, 52.79; ESI-MS (m/z):
501.35 (M ꢂ H).
5.3.10. Methyl 2-(3-(4-chlorophenyl)-5-oxo-2-thioxo-8-
(trifluoromethyl)-2,3-dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)-
2-(4-fluorophenyl)acetate (8j)
5.3.5. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-7,8-difluoro-
5-oxo-2- thioxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)
acetate (8e)
A light-yellow solid. Yield: 59%. M.p. 132e135 ꢀC. 1H NMR
A yellow solid. Yield: 69%. M.p. 94e95 ꢀC. 1H NMR (DMSO-d6,
(DMSO-d6, 600 MHz):
d
12.89 (s, 1H, NeH), 7.68 (d, 1H, J ¼ 8.25 Hz,
600 MHz):
d
12.77 (s, 1H, NH), 7.52 (d, 2H, J ¼ 8.52 Hz, AreH), 7.48
AreH), 7.62e7.60 (m, 2H, AreH), 7.44 (d, 1H, J ¼ 7.45 Hz, AreH),
7.36 (s, 1H, AreH), 7.26e7.21 (m, 6H, AreH), 6.27 (s, 1H, CeH),
(dd, 1H, J ¼ 8.58, 10.62 Hz, AreH), 7.44 (d, 2H, J ¼ 8.64 Hz, AreH),
7.25 (d, 2H, J ¼ 8.82 Hz, AreH), 7.21 (d, 2H, J ¼ 7.98 Hz, AreH),
7.04 (dd, 1H, J ¼ 11.2, 7.00 Hz, AreH), 6.20 (s, 1H, CH), 5.86 (s, 1H,
5.84 (s, 1H, CeH), 3.75 (s, 3H, CeH). 13C NMR (150 MHz, CDCl3)
d:
200.50,169.69,166.14,164.20,162.54,135.76,134.23,134.00,133.95,
132.56, 132.51, 132.25, 131.91, 130.28, 128.73, 127.46, 126.22, 122.16,
116.56, 116.42, 66.91, 64.40, 52.82; ESI-MS (m/z): 537.13 (M þ H).
CH), 3.73 (s, 3H, CH3). 13C NMR (150 MHz, CDCl3)
d: 200.05, 169.42,
165.29, 164.28, 164.26, 164.14, 136.06, 134.53, 134.42, 134.07, 134.02,
133.96, 131.87, 131.80, 130.14, 129.58, 128.84, 126.13, 67.08, 64.43,
52.89; ESI-MS (m/z): 519.53 (M ꢂ H).
5.3.11. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-8-
methoxy-5-oxo-2-thioxo- 2,3-dihydro-1H-benzo[e][1,4]diazepin-
4(5H)-yl)acetate (8k)
5.3.6. Methyl 2-(8-chloro-3-(4-fluorophenyl)-5-oxo-2-thioxo-2,3-
dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)-2-(4-fluorophenyl)
acetate (8f)
A light-yellow solid. Yield: 56%. M.p. 135e136 ꢀC. 1H NMR
(DMSO-d6, 600 MHz):
d
12.64 (s, 1H, NeH), 7.54 (d, 2H, J ¼ 8.40 Hz,
A yellow solid. Yield: 70%. M.p. 143e144 ꢀC. 1H NMR (DMSO-d6,
AreH), 7.54e7.45 (m, 3H, AreH), 7.23 (s, 4H, AreH), 6.69 (dd, 1H,
J ¼ 2.00, 6.65 Hz, AreH), 6.54 (d, 1H, J ¼ 1.6 Hz, AreH), 6.18 (s, 1H,
CeH), 5.82 (s, 1H, CeH), 3.73 (s, 3H, CeH), 3.69 (s, 3H, CeH); 13C
600 MHz):
d
12.75 (s, 1H, NH), 7.59 (d, 1H, J ¼ 5.52 Hz, AreH), 7.58
(d, 1H, J ¼ 5.46 Hz, AreH), 7.47 (d, 1H, J ¼ 8.58 Hz, AreH), 7.24e7.19
(m, 4H, AreH), 7.13 (dd, 1H, J ¼ 2.10, 8.52 Hz, AreH), 7.06 (d, 1H,
J ¼ 1.98 Hz, AreH), 6.98 (t, 2H, J ¼ 8.82 Hz, AreH), 6.23 (s, 1H, CH),
NMR (150 MHz, DMSO-d6) d: 199.57, 169.57, 166.00, 162.11, 138.05,
134.03, 133.93, 133.24, 132.75, 132.47, 132.33, 128.91, 128.78, 127.20,
120.48, 112.23, 105.25, 70.75, 65.92, 55.94, 52.74; ESI-MS (m/z): 513
(M ꢂ H).
5.78 (s, 1H, CH), 3.29 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6)
d:
200.43, 169.64, 165.49, (163.71, 162.60), (161.75, 160.66), 137.52,
136.63, 132.91, 132.84, 130.64, 130.03, 127.50, 126.77, 125.83, 120.02,
116.01, 115.89, 70.01, 65.72, 52.80; ESI-MS (m/z): 487.43 (M þ H).
5.3.12. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-9-
methyl-5-oxo-2-thioxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-
4(5H)-yl)acetate (8l)
5.3.7. Methyl 2-(7-chloro-3-(4-chlorophenyl)-5-oxo-2-thioxo-2,3-
dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)-2-(4-fluorophenyl)
acetate (8g)
A light-yellow solid. Yield: 50%. M.p. 240e241 ꢀC. 1H NMR
(DMSO-d6, 600 MHz):
d
11.97 (s, 1H, CeH), 7.58 (d, 2H, J ¼ 8.5 Hz,
A light-yellow solid. Yield: 73%. M.p. 120e123 ꢀC. 1H NMR
AreH), 7.44 (d, 2H, J ¼ 8.55 Hz, AreH), 7.30 (d, 1H, J ¼ 7.65 Hz,
AreH), 7.21e7.12 (m, 5H, AreH), 7.01 (t, 1H, J ¼ 7.7 Hz, AreH), 6.12
(s, 1H, CeH), 5.88 (s, 1H, CeH), 3.76 (s, 3H, CeH), 2.18 (s, 3H, CeH);
(DMSO-d6, 600 MHz):
d
12.70 (s, 1H, NH), 7.53 (d, 1H, J ¼ 5.52 Hz,
AreH), 7.52 (d, 1H, J ¼ 5.28 Hz, AreH), 7.49 (d, 1H, J ¼ 2.52 Hz,
AreH), 7.38 (dd, 1H, J ¼ 2.58, 8.70 Hz, AreH), 7.25e7.17 (m, 6H,
AreH), 6.97 (d, 1H, J ¼ 8.7 Hz, AreH), 6.16 (s, 1H, CH), 5.76 (s, 1H,
13C NMR (150 MHz, DMSO-d6)
d: 199.92, 169.38, 166.52, 135.09,
133.93, 133.83, 133.47, 132.50, 132.28, 130.11, 128.77, 128.65, 127.86,
126.71, 126.64, 71.51, 66.42, 52.81, 17.94; ESI-MS (m/z): 499.28
(M þ H).
CH), 3.71 (s, 3H, CH3). 13C NMR (150 MHz, CDCl3)
d: 199.70, 169.59,
165.92, 164.13, 162.47, 139.19, 134.17, 133.84, 132.61, 132.55, 132.50,
131.97, 131.45, 130.73, 129.84, 128.80, 128.69, 126.23, 124.45, 123.95,
120.98, 67.10, 64.33, 52.78; ESI-MS (m/z): 503.64 (M þ H).
5.3.13. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-7-
methyl-5-oxo-2-thioxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-
4(5H)-yl)acetate (8m)
5.3.8. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-9-
methoxy-5-oxo-2-thioxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-
4(5H)-yl)acetate (8h)
A light-yellow solid. Yield: 71%. M.p. 220e222 ꢀC. 1H NMR
(DMSO-d6, 600 MHz):
d
12.67 (s, 1H, NeH), 7.54 (d, 2H, J ¼ 8.5 Hz,
A light-yellow solid. Yield: 62%. M.p. 123e125 ꢀC. 1H NMR
AreH), 7.45 (d, 2H, J ¼ 8.55 Hz, AreH), 7.32 (d, 1H, J ¼ 1.25 Hz,
AreH), 7.24e7.20 (m, 4H, AreH), 7.14 (dd, 1H, J ¼ 1.55, 8.35 Hz,
AreH), 6.88 (d, 1H, J ¼ 8.3 Hz, AreH), 6.17 (s, 1H, CeH), 5.87 (s, 1H,
CeH), 3.74 (s, 3H, CeH), 2.16 (s, 3H, CeH); 13C NMR (150 MHz,
(DMSO-d6, 600 MHz):
d
11.70 (s, 1H, NH), 7.59 (d, 2H, J ¼ 5.52 Hz,
AreH), 7.42 (d, 2H, J ¼ 8.58 Hz, AreH), 7.17 (s, 4H, AreH), 7.06e7.01
(m, 2H, AreH), 6.98 (dd,1H, J ¼ 1.50, 7.92 Hz, AreH), 6.12 (s,1H, CH),

Z. Guo et al. / European Journal of Medicinal Chemistry 56 (2012) 10e16
15
DMSO-d6)
d
: 198.84, 169.41, 166.23, 135.38, 134.39, 133.96, 133.81,
125.69, 119.93, 114.26, 70.54, 65.87, 55.42, 52.77; ESI-MS (m/z):
515.83 (M þ H).
133.57, 133.31, 132.46, 132.31, 130.44, 128.85, 128.78, 127.48, 127.21,
120.81, 71.10, 66.20, 52.78, 20.59; ESI-MS (m/z): 499.54 (M þ H).
5.4. General procedure for the synthesis of 8s and 8t
5.3.14. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-7-fluoro-
5-oxo-2-thioxo-2,3-dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)
acetate(8n)
To a dry CH2Cl2 solution of 8a or 8k (5 mmol) was added MeI
(5 mmol) or ethyl bromoacetate (5 mmol), then DBU (5 mmol) was
added to the resulting solution and stirred at room temperature for
30e60 min. After that, the reaction mixture was concentrated
under vacuum and purified by column chromatography (n-
hexaneeethyl acetate, 10:1) to give 8s or 8t, yield: 90e95%.
A light-yellow solid. Yield: 66%. M.p. 146e148 ꢀC. 1H NMR
(DMSO-d6, 600 MHz):
d
12.76 (s, 1H, NeH), 7.55 (d, 2H, J ¼ 8.55 Hz,
AreH), 7.45 (d, 2H, J ¼ 8.55 Hz, AreH), 7.24e7.20 (m, 6H, AreH),
7.06e7.02 (m, 1H, AreH), 6.20 (s, 1H, CeH), 5.85 (s, 1H, CeH),
3.74 (s, 3H, CeH); 13C NMR (150 MHz, DMSO-d6)
d: 199.19, 169.31,
165.07, (159.83, 158.21), 134.18, 133.54, 133.35, 132.89, 132.66,
132.40, (129.65, 129.60), 128.98, 128.93, 127.24, (123.52, 123.46),
(120.40, 120.25), (116.28, 116.11), 70.76, 66.12, 52.90; ESI-MS (m/z):
503.28 (M þ H).
5.4.1. Methyl 2-(8-chloro-3-(4-chlorophenyl)-2-(methylthio)-5-
oxo-3H-benzo[e] [1,4]diazepin-4(5H)-yl)-2-(4-chlorophenyl)acetate
(8s)
A light-yellow solid. Yield: 95%. M.p. 185e187 ꢀC. 1H NMR
(DMSO-d6, 600 MHz):
d
7.53 (d, 1H, J ¼ 8.46 Hz, AreH), 7.49 (d, 2H,
5.3.15. Methyl 2-(3-(4-chlorophenyl)-8-fluoro-5-oxo-2-thioxo-2,3-
dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)-2-(4-fluorophenyl)
acetate (8o)
J ¼ 8.58 Hz, AreH), 7.46 (d, 2H, J ¼ 8.76 Hz, AreH), 7.21 (d, 2H,
J ¼ 8.58 Hz, AreH), 7.17 (d, 2H, J ¼ 8.46 Hz, AreH), 7.11 (dd, 1H,
J ¼ 2.16, 8.58 Hz, AreH), 7.03 (d, 1H, J ¼ 2.10 Hz, AreH), 6.02 (s, 1H,
CH), 5.44 (s, 1H, CH), 3.75 (s, 3H, CH3), 2.52 (s, 3H, CH3). 13C NMR
A light-yellow solid. Yield: 80%. M.p. 106e107 ꢀC. 1H NMR
(DMSO-d6, 600 MHz):
d
12.79 (s, 1H, NeH), 7.56 (d, 2H, J ¼ 8.5 Hz,
(150 MHz, CDCl3) d: 170.10, 169.58, 167.38, 145.98, 137.63, 135.83,
AreH), 7.46 (d, 2H, J ¼ 8.5 Hz, AreH), 7.26e7.23 (m, 6H, AreH),
133.48, 132.97, 131.57, 131.50, 130.74, 129.52, 128.34, 126.25, 125.71,
125.33, 125.29, 64.31, 60.60, 52.76, 14.38; ESI-MS (m/z): 533.52
(M þ H).
7.06e7.04 (m, 1H, AreH), 6.22 (s, 1H, CeH), 5.87 (s, 1H, CeH),
3.75 (s, 3H, CeH); 13C NMR (150 MHz, DMSO-d6)
d: 199.18,
169.28, 165.04, (159.98, 158.04), 134.15, 133.52, 133.33, 132.89,
132.63, 132.38, (129.65, 129.59), 128.96, 128.90, 127.22, (123.51,
123.45), (120.39, 120.20), (116.27, 116.08), 70.77, 66.11, 52.87; ESI-
MS (m/z): 487.69 (M þ H).
5.4.2. Methyl 2-(4-chlorophenyl)-2-(3-(4-chlorophenyl)-2-((2-
ethoxy-2-oxoethyl) thio)-8-methoxy-5-oxo-3H-benzo[e][1,4]
diazepin-4(5H)-yl)acetate (8t)
A light-yellow solid. Yield: 90%. M.p. 130e131 ꢀC. 1H NMR
5.3.16. Methyl 2-(8-bromo-3-(4-chlorophenyl)-5-oxo-2-thioxo-2,3-
dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)-2-(4-chlorophenyl)
acetate (8p)
(DMSO-d6, 600 MHz): d 7.49e7.44 (m, 5H, AreH), 7.25e7.19 (m, 4H,
AreH), 6.66 (dd, 1H, J ¼ 2.45, 8.85 Hz, AreH), 6.28 (d, 1H, J ¼ 2.4 Hz,
AreH), 5.98 (s, 1H, CeH), 5.49 (s, 1H, CeH), 4.20e4.16 (m, 2H, CeH),
4.01 (s, 2H, CeH), 3.75 (s, 3H, CeH), 3.69 (s, 3H, CeH), 1.21 (t, 3H,
A yellow solid. Yield: 73%. M.p. 139e141 ꢀC. 1H NMR (DMSO-d6,
600 MHz):
d
12.76 (s, 1H, NeH), 7.55 (d, 2H, J ¼ 8.58 Hz, AreH), 7.46
J ¼ 7.1 Hz, CeH). 13C NMR (150 MHz, CDCl3)
d: 170.36,167.96,167.82,
(d, 2H, J ¼ 8.58 Hz, AreH), 7.42 (d, 1H, J ¼ 8.46 Hz, AreH), 7.29 (dd,
1H, J ¼ 1.86, 8.46 Hz, AreH), 7.26e7.24 (m, 4H, AreH), 7.21 (d, 1H,
J ¼ 1.86 Hz, AreH), 6.21 (s, 1H, CeH), 5.75 (s, 1H, CeH), 3.74 (s, 3H,
167.29, 162.16, 145.86, 135.72, 133.19, 133.07, 132.15, 131.92, 131.16,
129.54, 127.93, 127.21, 119.95, 112.53, 109.40, 62.09, 61.71, 60.01,
55.23, 52.54, 33.37, 14.21; ESI-MS (m/z): 601.59 (M þ H).
CeH); 13C NMR (150 MHz, DMSO-d6)
d: 200.00, 169.35, 165.57,
137.47, 134.17, 133.51, 132.89, 132.71, 132.60, 132.36, 128.97, 128.83,
127.26, 126.93, 125.47, 122.98, 70.63, 66.03, 52.86; ESI-MS (m/z):
560.89 (M ꢂ H).
5.5. Computational protocol
Molecular docking was used to predict the binding mode of the
synthesized thio-benzodiazepine derivatives. The crystal structure
[7] of MDM2 (PDB code: 1T4E) was prepared by removing the
benzodiazepine and adding hydrogen atoms in MVD 4.3.0. We used
two known potent inhibitors of the p53eMDM2 interaction with
different chemical structures (TDP222669 and nutlin-3a) as posi-
tive controls. The docking parameters of MVD were similar to the
literatures [24]. The active site was defined to encompass all MDM2
5.3.17. Methyl 2-(8-chloro-5-oxo-2-thioxo-3-(4-(trifluoromethyl)
phenyl)-2,3-dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)-2-(4-
chlorophenyl)acetate (8q)
A light-yellow solid. Yield: 68%. M.p. 160e162 ꢀC. 1H NMR
(DMSO-d6, 600 MHz):
d
12.83 (s, 1H, NeH), 7.56 (d, 4H, J ¼ 8.52 Hz,
AreH), 7.50 (d, 1H, J ¼ 8.58 Hz, AreH), 7.46 (d, 4H, J ¼ 8.52 Hz,
AreH), 7.14 (dd, 1H, J ¼ 2.04, 7.38 Hz, AreH), 7.06 (d, 1H, J ¼ 2.04 Hz,
AreH), 6.23 (s, 1H, CeH), 5.97 (s, 1H, CeH), 3.76 (s, 3H, CeH). 13C
ꢀ
atoms within a 12 A radius sphere from the center of the Trp23 of
the p53 peptide ligand. Other parameters were set by default.
NMR (150 MHz, CDCl3) d: 199.84, 169.54, 166.46, 138.47, 137.58,
136.38, 136.09, 132.65, 131.91, 130.26, 130.14, 130.04, 129.63, 126.33,
125.91, 125.51, 125.48, 125.29, 124.37, 122.57, 119.37, 67.25, 64.45,
52.89; ESI-MS (m/z): 551.37 (M ꢂ H).
5.6. p53eMDM2 binding assay
The dose-dependent binding experiments were carried out with
serial dilution in DMSO of compounds. A 5 mL sample of the tested
5.3.18. Methyl 2-(8-chloro-3-(4-methoxyphenyl)-5-oxo-2-thioxo-
2,3-dihydro-1H-benzo[e][1,4]diazepin-4(5H)-yl)-2-(4-
chlorophenyl)acetate (8r)
sample and preincubated (for 30 min) MDM2 binding domain
(1e118) (10 nM) and PMDM-F peptide (Anaspec) (10 nM) in the assay
buffer (100 mM potassium phosphate, pH 7.5; 100 mg/mL bovine
gamma globulin; 0.02% sodium azide was added into black 96-well
microplates with F-bottom and chimney wells (Corning) to produce
A yellow solid. Yield: 75%. M.p. 137e138 ꢀC. 1H NMR (DMSO-d6,
600 MHz): d 12.72 (s, 1H, NeH), 7.57e7.47 (m, 5H, AreH), 7.15e7.08
(m, 4H, AreH), 6.70 (d, 2H, J ¼ 8.8 Hz, AreH), 6.23 (s, 1H, CeH), 5.78
a final volume of 115 ml. For each assay, the controls included the
(s, 1H, CeH), 3.74 (s, 3H, CeH), 3.61 (s, 3H, CeH); 13C NMR
MDM2 binding domain and PMDM-F. The polarization values were
measured after 1 h of incubation at room temperature using Biotek
Synergy H2 with a 485 nm excitation filter, a 528 nm static and
(150 MHz, DMSO-d6)
d: 201.03, 169.45, 165.60, 158.85, 137.54,
136.52, 134.12, 132.97, 132.56, 132.41, 128.95, 126.74, 126.61, 125.99,

16
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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 (SigmaeAldrich), the first
potent and specific non-peptide small-molecule MDM2 inhibitor was
used as reference compound for validating the assay in each plate.
5.7. In vitro antitumor activity
The cellular growth inhibitoryactivity was determined using two
human osteosarcoma cell lines [U-2 OS (wild type p53), Saos-2 (p53
null)]. 5e6 ꢁ 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 dis-
solved with 100 mL of DMSO. The absorbance (OD) was quantitated
with microplate using Biotek Synergy H2 at 570 nm. Wells con-
taining no drugs were used as blanks. Concentration of the
compounds that inhibited cell growth by 50% (IC50) was calculated.
Acknowledgments
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This research was supported in part by the Key Project of Science
and Technology of Shanghai (No. 09431901700), Major Special
Project for the Creation of New Drugs (Grant No. 2009ZX09301-011),
the 863 Hi-Tech Program of China (No. 2012AA020302), Shanghai
Rising-Star Program (No. 12QH1402600), Shanghai Municipal
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