Structure-based design of rhodanine-based acylsulfonamide derivatives as antagonists of the anti-apoptotic Bcl-2 protein

Article abstract of DOI:10.1016/j.bmc.2012.05.079

A series of novel rhodanine-based acylsulfonamide derivatives were designed, synthesized, and evaluated as small-molecule inhibitors of anti-apoptotic Bcl-2 protein. These compounds exhibit potent antiproliferative activity in three human tumor cell lines

Full text of DOI:10.1016/j.bmc.2012.05.079

Bioorganic & Medicinal Chemistry 20 (2012) 4194–4200
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure-based design of rhodanine-based acylsulfonamide derivatives
as antagonists of the anti-apoptotic Bcl-2 protein
Huan-qiu Li a, , Jing Yang a, , Shuhua Ma b, Chunhua Qiao a,
⇑
a College of Pharmaceutical Science, Soochow University, PR China
b Department of Chemistry, Towson University, 8000 York Road, Towson, MD 21252, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel rhodanine-based acylsulfonamide derivatives were designed, synthesized, and evaluated
as small-molecule inhibitors of anti-apoptotic Bcl-2 protein. These compounds exhibit potent antiprolif-
erative activity in three human tumor cell lines (Hep G2, PC-3 and B16-F10). Among them, the most
potent compounds 10 and 11 bind to Bcl-2 with a Ki of 20 and 25 nM, respectively. Docking studies dem-
onstrated that these two compounds orient similarly at the binding site of Bcl-2, and the calculated bind-
ing affinities (Glide XP score) of compound 10 is more negative than that of compound 11. The binding
interactions of compounds with high binding affinity to Bcl-2 protein were analyzed.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 25 April 2012
Revised 31 May 2012
Accepted 31 May 2012
Available online 7 June 2012
Keywords:
Bcl-2
Apoptosis
Antagonist
Structure-based design
Acylsulfonamide
1. Introduction
of inhibitors from Abbott Laboratories, exemplified by A (ABT-
737), was reported to inhibit Bcl-xL, Bcl-2, and Bcl-w with an
Apoptosis is an evolutionarily conserved process that regulates
normal development and tissue homeostasis by eliminating un-
wanted and damaged cells. Inhibition of apoptosis is implicated
in virtually every known human malignancy.1,2
IC50 less than 1.0 nM, greater than 3 orders of magnitude more po-
tently than previously described small-molecule inhibitors.9 A re-
lated compound, B (ABT-263), was subsequently prepared and
demonstrated oral availability across multiple species, and has
now entered phase II clinical trials.10 A hallmark of this class of
drugs is the presence of an acylsulfonamide core. This moiety rep-
resents a potential metabolic liability and indeed has been used in
prodrug approaches.11 In recent years, the structure-based ap-
proach has become very powerful for the discovery of novel lead
compounds and for lead optimization, and new rhodanine-based
derivatives as potent Bcl-2 inhibitors are reflected in recent publi-
cations (Fig. 1).12,13 The reported anti-apoptotic/prosurvival pan-
Bcl-2 inhibitors are some biarylrhodanine and pyridylrhodanine
derivatives. Thus, we postulated that a rhodanine-based sulfon-
amide may serve such a purpose while being topographically
appropriate to maintain high affinity binding to Bcl-2 family
proteins (Fig. 1). Herein, we report the synthesis of a series of rho-
danine-based acylsulfonamide derivatives, their in vitro antitumor
activity, and their binding affinities to Bcl-2 protein.
Proteins of the Bcl-2 (B-cell lymphocyte/leukemia-2) family are
the key inhibitors of apoptosis in the mitochondria-mediated path-
way and comprise both pro- and anti-apoptotic proteins.3–5
Several homologues, as defined by sequence similarity to some
or all of the four Bcl-2 homology (BH) domains in Bcl-2, are found
in humans and function as either blockers or inducers of apoptosis.
The most structurally disparate of the Bcl-2 family members are
those that contain a single BH domain. As molecular sentinels, in
response to cellular damage, the proapoptotic BH3-only proteins
(e.g., Bad, Bim, Noxa) are activated to propagate death signaling.
Antiapoptotic Bcl-2 proteins are over-expressed in many human
cancers. Bcl-2 is over-expressed in 80% of B-cell lymphomas, 30–
60% of prostate cancers, 90% of colorectal adenocarcinomas, and
a wide variety of other cancers; and Bcl-xL is over-expressed in
breast and lung cancers.6–8 Therefore, the antiapoptotic Bcl-2 fam-
ily protein represents an attractive target for the development of a
novel therapy for the treatment of many forms of cancer.
2. Results and discussions
2.1. Chemistry
Intense interest in small-molecule BH3 mimetics is reflected in
numerous recent publications (Fig. 1). The acylsulfonamide series
The synthesis of the rhodanine-based acylsulfonamide deriva-
tives is outlined in Scheme 1. The intermediates 2a–2l and 3a–3l
were prepared by the Knoevenagel reaction of various substitute
⇑
Corresponding author. Address: 199 Ren Ai Road, Suzhou 215123, China.
Fax: +86 512 65882089.
E-mail address: qiaochunhua@suda.edu.cn (C. Qiao).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.079

H. Li et al. / Bioorg. Med. Chem. 20 (2012) 4194–4200
4195
O
HO
HO
CHO
OHC
OH
OH
R1
COOH
R2
OH HO
N
S
R3
R4
S
N
Reported rhodanine-based compounds targeting Bcl-2 family protein
O
R-(-)-Gossypol
O
N
O
R
S
S
N
Rhodanine-based sulfonamide scaffold
O
HN
H
O
O
O
NH
NH
S O
O
S
O
N
N
N
N
F3CO2S
O2N
HN
S
HN
S
Cl
Cl
N
O
A (ABT-737)
N
B (ABT-263)
Figure 1. Structures of known BH3 mimetics with pro-apoptotic activity, acylsulfonamides A and B, R-(À)-Gossypol and the proposed rhodanine-based sulfonamide scaffold.
aldehydes with rhodanine, and methylated by CH3I. Then, the
rhodanine derivatives 4a–4l were synthesized from various -amino
ited remarkable antiproliferative activity comparable to the
positive control (R-)-Gossypol.
L
acids with 3a–3l and diisopropylethyl amine (DIEA). Finally, rhoda-
nine derivatives 4a–4l, 4-methylbenzenesulfonamide, dimethyl-
amino pyridine (DMAP) and 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDC) were dissolved in CH2Cl2
at room temperature to give the desired rhodanine-based acylsulf-
onamide derivatives 5–16. All compounds were purified by silica
gel column and identified by elemental H and C NMR and HRMS.
Generally, the results in Table 1 showed that those applied to
B16-F10 cells would perform better than that in Hep G2 and PC-3
cells. The substituents on the phenyl ring at R1 position didn’t input
substantial effects on the antiproliferative capability of the com-
pounds. Compound 9, 11, 12 exhibited single digit IC50value, which
is slightly more potent compared to other compounds, suggesting
substitute size or position on the phenyl ring would not exert signif-
icant effect on the compound anti-proliferative activity. Accord-
ingly, compound 6, 9, 11, 12 displayed moderate activity,
indicating relatively bulky isobutyl or isopropyl group at R2 position
would slightly favor the compound potency. Nonetheless, an aro-
matic benzyl group at R2 would not significantly tamper the com-
pound inhibitory activity compared to the aliphatic groups. Taken
together, the designed and synthesized rhodanine-based acylsulf-
onamide derivatives did demonstrate fairly potent antiproliferative
activity against all three tested cancer cell lines, although the struc-
tural-activity relationship study couldn’t reveal any defined struc-
tural feature at R1 and R2 position. Therefore, more structurally
diversified derivatives would be synthesized and evaluated to pro-
vide clues on the antagonist–protein interaction. Moreover, future
study on the sulfonamide moiety variation may afford remarkable
cell anti-proliferative candidates.
2.2. Antiproliferative assay
AlthoughBcl-2wasidentifiedas anoncogeneinitiallyin follicular
lymphoma, its expression has been identified in a wide array of can-
cers, including melanoma, myeloma, small cell lung cancer, prostate
and acute leukemias.14 Thus, all the synthesized rhodanine-based
acylsulfonamide derivatives 5–16 were evaluated for their antipro-
liferative activity against three human tumor cell lines Hep G2 (hu-
man liver cancer cell), B16-F10 (melanoma cancer cell) and PC-3
(prostatecancercell)byapplyingtheMTTcolorimetricassay. There-
sults were summarized in Table 1. (R-)-Gossypol (Fig. 1), reported to
bind to and antagonize the anti-apoptotic Bcl-2 family protein,15
was also screened under identical conditions for comparison. As ex-
pected, these rhodanine-based acylsulfonamide derivatives exhib-
O
NH2
OH
O
O
O
O
R1
H
R2
CH3I
R1
O
R1
NH
N
NH
S
NaOAc, HOAc
reflux, 78-86%
EtOH, DIEA
80-87%
S
S
EtOH, DIEA
S
SCH3
76-89%
S
3
1
2
O
S
O
O
H3C
NH2
O
S
R1
R1
N
O
N
O
S
CH3
S
EDC.HCl, DMAP,DCM
33-42%
NH
O
OH
HN
HN
R2
4
R2
5-16
Scheme 1. Synthesis of the desired rhodanine-based acylsulfonamide derivatives 5–16.

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H. Li et al. / Bioorg. Med. Chem. 20 (2012) 4194–4200
Table 1
Structure of 4,6-substituted-(diaphenylamino) quinazolines and the anti-proliferative effect from MTT assay
O
O
R1
N
O
S
S
CH3
NH
O
HN
R2
Compound
R1
R2
IC50(lg/mL)
Hep G2
PC-3
B16-F10
5
6
7
8
9
10
11
12
13
14
15
16
4-MeO-Ph
2-F-Ph
2-F-Ph
3-Cl-Ph
2-Cl-Ph
2-Cl-Ph
2-Cl-Ph
Ph
2-MeO-Ph
Ph
2-MeO-Ph
2-MeO-Ph
—
(CH3)2CH
(CH3)2CHCH2
PhCH2
(CH3)2CHCH2
(CH3)2CHCH2
PhCH2
(CH3)2CH
(CH3)2CH
PhCH2
(CH3)2CHCH2
PhCH2
(CH3)2CH
—
12.79 0.72
16.43 1.33
18.73 1.35
16.51 1.10
13.35 1.01
21.01 1.21
21.25 1.41
18.23 1.22
23.88 2.31
13.43 0.93
16.58 1.39
20.17 1.36
10.16 0.66
20.27 3.31
15.31 1.22
22.07 1.98
16.75 1.66
15.61 2.03
16.01 1.79
11.87 1.37
11.90 1.22
12.84 0.88
16.64 1.13
16.37 1.23
15.04 0.73
6.71 0.37
11.62 0.83
11.29 0.39
11.90 0.58
13.37 1.30
8.52 1.11
12.20 0.93
8.80 0.49
7.46 0.52
11.84 1.01
14.03 1.27
12.54 1.32
13.49 1.59
5.56 0.31
(R)-Gossypol
Table 2
The binding affinities of representative rhodanine-based acylsulfonamide derivatives to Bcl-2 protein
Compound
5
10
11
12
15
(R)-Gossypol
Ki SD (nM)
671.5 27
20 1.3
25 2.4
3010 72
310 1.3
334 17
2.3. Binding assay to Bcl-2 protein
5.7, Schrödinger, LLC, New York, NY, 2011) module of a computa-
tional program Schrödinger. To validate the performance of Glide
docking and scoring, compounds 5, 10, 11, 12 and 15 were docked
to the binding site of Bcl-2 using the Glide extra precision (XP)
mode.19 Fig. 2 shows the correlation between the computed Glide
XP score and the measured Ki values for the above mentioned 5
inhibitors of Bcl-2.
The Bcl-2 binding affinity of these rhodanine-based acylsulfona-
mide derivatives and positive control (R-)-Gossypol was examined
and the results are summarized in Table 2. Except for compound
5, 10, 11, 12, 15, the binding affinity of all other compounds are
greater than 1.0 mM, thus the exact values were not listed in the ta-
ble. We have used a sensitive and quantitative in vitro fluorescence
polarization (FP) based binding assay.16 The basic principle behind
this assay is competition: a fluorescent peptide tracer (Flu-Bak-
BH3) and the nonfluorescent small molecule inhibitor competes
for binding to the target protein Bcl-2. Using this method, a binding
affinity of 300 nM (reported value 270 nM)17 IC50 was obtained for
Bak-BH3 peptide binding to the Bcl-2, and the binding affinity of
(R)-gossypol was determined to be 334 17 nM (reported value
320 nM),18 both values are consistent with that reported in the lit-
erature. As shown in Table 2, compounds 10 and 11 are the most
tight binding compounds in the FP assay, with binding affinities of
20 and 25 nM, respectively. This result basically correlates well with
that from the cell based assay. Another compound 15, with the 4-
phenyl phenyl at R1 position and the benzyl at R2 position, demon-
strated comparable binding affinity to the positive control (R-)-Gos-
sypol. This biological assay indicated that rhodanine-based
acylsulfonamide derivatives 10 and 11 are potential small-molecule
Bcl-2 inhibitors as anticancer agents.
The Bcl-2—compound 10 complex structure obtained from dock-
ing calculations is shown in Figure 3 (a). The acyl-phenyl-sulfon-
amide group occupies the similar Bcl-2 location as indicated in the
NMRstructure, inwhichthebenzeneringofthephenyl-sulfonamide
group formed p–p
interaction with the side chain of Tyr199,20 And
the PhCH2-group at R2 site of compound 10 occupied the binding site
of –SPh group of inhibitor 43B in the NMR structure (PDB ID:2O21);
2.4. Molecular modeling
The binding affinity of these rhodanine-based acylsulfonamide
derivatives to Bcl-2 was also examined through docking studies.
All docking calculations were performed using the Glide (Version
Figure 2. Correlation between the computed Glide XP score and the measured
logKi values.

H. Li et al. / Bioorg. Med. Chem. 20 (2012) 4194–4200
4197
Figure 3. Molecular surface of the complex structure of Bcl-2 and compound 10 (docked structure from Glide); (b) Zoom-in image of compounds 5, 10, 11, 12, and 15 binding
to Bcl-2 protein.
the 2-Cl-Ph-group at R1 site of compound 10 falls into the hydropho-
bic site that the benzothiazole group occupied in Bcl-2 and 43B com-
plex structure. Compounds 5, 11, 12 and 15 bind to Bcl-2 protein in a
similar pattern as compound 10.
10 h. The resulting mixture was cooled to room temperature, and
then 150 mL water was added. The yellow precipitate that formed
was filtered, washed with ethyl ether, and dried to give 2j (1.32 g,
4.43 mmol) (Yield: 80.9%).1H NMR (300 MHz, DMSO-d6) d 7.38–
7.58 (m, 3H), 7.64–7.80 (m, 5H), 7.82–7.92 (m, 2H).
3. Conclusions
4.3. General procedure for the preparation of compounds 3a–3l
(3j)
In summary, a series of rhodanine-based acylsulfonamide deriv-
atives were designed, synthesized, and evaluated as anti-apoptotic
Bcl-2 protein antagonist. Two of these compounds exhibit protein
binding affinity comparable to positive control (R-)-Gossypol. The
cell based MTT assay demonstrated that some compounds dis-
played fairly potent cytotoxicity. The docking experiment indicated
that all potent compounds bind tightly to Bcl-2 protein.
Compound 2j (1.00 g, 3.37 mmol), DIEA (0.65 g, 5.05 mmol)
were suspended in anhydrous ethanol (50 mL), then CH3I (0.72 g,
5.05 mmol) was slowly added within ten minutes, the mixture
was stirred at room temperature for 2 h. Then 150 mL water was
added, the yellow precipitate was filtered, and dried to give 3j
(0.85 g, 2.64 mmol) (Yield: 81.1%). 1H NMR (300 MHz, DMSO-d6)
d 2.85 (s, 3H), 7.38–7.56 (m, 3H), 7.72–7.81 (m, 4H), 7.84–7.92
(m, 3H).
4. Experimental section
4.1. General chemistry
4.4. General procedure for the preparation of compounds 4a–4l
(4j)
All chemicals (reagent grade) used were purchased from Sig-
ma–Aldrich (USA) and Sinopharm Chemical Reagent Co. Ltd. (Chi-
na). 1H NMR spectra were measured on Varian Unity Inova 300/
400 MHz NMR Spectrometer at 25 °C and referenced to TMS.
Chemical shifts are reported in ppm (d) using the residual solvent
line as internal standard. Splitting patterns are designed as s, sin-
glet; d, doublet; t, triplet; m, multiplet. HRMS spectra were ac-
quired on Bruker Esquire Liquid Chromatography-Ion Trap Mass
Spectrometer. Analytical thin-layer chromatography (TLC) was
performed on the glass-backed silica gel sheets (silica gel 60 Å
GF254). All compounds were detected using UV light (254 or
365 nm). Analytical HPLC was conducted on SHIMADZU LC-20AD.
Prior to biological evaluation, all compounds were determined to
be >95% pure using appropriate analytical methods (MeOH/H2O
80% v/v, MeOH/H2O 75% v/v, MeOH/H2O 60% v/v) based on the
peak area percentage.
Compound 3j (0.60 g, 1.93 mmol), DIEA (0.25 g, 1.93 mmol) and
-phenylalanine (0.32 g, 1.93 mmol) were suspended in ethanol
L
(25 mL) and heated at reflux at 70–80 °C for 7 h. The reaction mix-
ture was cooled to room temperature and concentrated under re-
duced pressure. The resulting solid was extracted with ethyl
acetate for column chromatography. Column chromatography
was performed using silica gel (200–300 mesh), eluted with ethyl
acetate and petroleum ether (1:1, v/v) to give 4j (0.66 g,
1.42 mmol) (Yield: 80.2%).1H NMR (400 MHz, DMSO-d6) d 3.07
(dd, J = 9.2 Hz, 13.6 Hz, 1H), 3.26 (dd, J = 4.4 Hz, 13.6 Hz, 1H),
4.83–4.94 (m, 1H), 7.18–7.34 (m, 5H), 7.38–7.42 (m, 1H), 7.46–
7.52 (m, 2H), 7.61–7.67 (m, 3H), 7.72–7.75 (m, 2H), 7.80–7.86
(m, 2H).
4.5. General procedure for the preparation of compounds 5–16
4.2. General procedure for the preparation of compounds 2a–2l
(2j)
Compound 4j (0.60 g, 1.40 mmol), p-TSA (0.36 g, 2.10 mmol),
EDCÁHCl (0.40 g, 2.10 mmol), DMAP (0.14 g, 1.12 mmol) were sus-
pended in CH2Cl2 (25 mL) and stirred at room temperature for
24 h. The mixture was cooled to 5 °C, and acidified to pH 1.0 with
addition of HCl aqueous solution (10%), followed by extraction
with CH2Cl2/MeOH (9:1, 3 Â 100 mL). The combined organic layers
Rhodanine
1 (0.73 g, 5.48 mmol), biphenyl-4-carbaldehyde
(1.00 g, 5.48 mmol) and sodium acetate (1.57 g, 19.18 mmol) were
suspended in acetic acid (50 mL) and the mixture was refluxed for

4198
H. Li et al. / Bioorg. Med. Chem. 20 (2012) 4194–4200
were washed with H2O and brine, dried over Na2SO4, and concen-
trated in vacuo. The residue was subjected to silica gel chromatog-
raphy or crystallization if necessary to afford the compounds 5–16.
Yield: 33–42%.
J = 8.4 Hz, 2H), 10.08 (d, J = 7.5 Hz, 1H); 13C NMR (100 MHz,
DMSO-d6) d 21.2, 36.7, 59.5, 125.3, 127.0, 127.6 (2C), 128.1,
128.4 (2C), 128.7, 129.2 (2C), 129.7 (2C), 130.3, 131.3, 132.0,
132.1, 134.2, 135.8, 136.1, 144.6, 169.1, 174.1, 178.7. ESI-MS: Cacld
for C26H22ClN3O4S2 [M+H]+: 540.0813, Found 540.0824 (error
2.0 ppm).
4.5.1. (Z)-2-(5-(4-Methoxybenzylidene)-4-oxo-4,5-
dihydrothiazol-2-ylamino)-3-methyl-N-tosylbutanamide 5
1H NMR (400 MHz, DMSO-d6) d 0.80 (d, J = 6.8 Hz, 3H), 0.83 (d,
J = 6.7 Hz, 3H), 2.05–2.13 (m, 1H), 2.37 (s, 3H), 3.78–3.86 (m, 4H),
7.08 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.7 Hz,
2H), 7.57 (s, 1H), 7.82 (d, J = 8.2 Hz, 2H), 9.69 (d, J = 7.8 Hz, 1H);
13C NMR (75 MHz, DMSO-d6) d 17.6, 18.7, 21.1, 30.5, 55.4, 63.0,
114.8 (2C), 125.6, 126.3, 127.5 (2C), 129.5 (2C), 129.9, 131.3 (2C),
136.2, 144.4, 160.4, 169.3, 174.3, 179.5. ESI-MS: Calcd for
4.5.7. (Z)-2-(5-(2-Chlorobenzylidene)-4-oxo-4,5-dihydrothiazol-
2-ylamino)-3-methyl-N-tosylbutanamide 11
1H NMR (400 MHz, DMSO-d6) d 0.78 (d, J = 6.8 Hz, 3H), 0.82 (d,
J = 6.8 Hz, 3H), 2.04–2.13 (m, 1H), 2.37 (s, 3H), 4.57–4.63 (m, 1H),
7.39–7.62 (m, 6H), 7.78 (s, 1H), 7.82 (d, J = 8.3 Hz, 2H), 9.93 (d,
J = 7.9 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d 17.6, 18.7, 21.1,
30.6, 63.1, 125.1, 127.6 (2C), 128.1, 128.6, 129.6 (2C), 130.2,
131.2, 132.1, 132.2, 134.2, 136.1, 144.5, 169.2, 174.5, 178.8. ESI-
MS: Cacld for C22H22ClN3O4S2 [M+H]+: 492.0813, Found 492.0819
(error 1.2 ppm).
C
23H25N3O5S2 [M+H]+ 488.1308, Found 488.1317 (error 1.8 ppm).
4.5.2. (Z)-2-(5-(2-Fluorobenzylidene)-4-oxo-4,5-dihydrothiazol-
2-ylamino)-4-methyl-N-tosylpentanamide 6
1H NMR (400 MHz, DMSO-d6) d 0.85 (d, J = 6.1 Hz, 3H), 0.89 (d,
J = 6.1 Hz, 3H), 1.48–1.60 (m, 3H), 2.37 (s, 3H), 4.67–4.74 (m, 1H),
7.34–7.43 (m, 4H), 7.48–7.55 (m, 2H), 7.66 (s, 1H), 7.81 (d,
J = 8.3 Hz, 2H), 9.96 (d, J = 7.9 Hz, 1H); 13C NMR (75 MHz, DMSO-
d6) d 21.1 (2C), 22.8, 24.5, 56.7, 116.2 (d, J = 21.3 Hz), 120.9 (d,
J = 6.7 Hz), 121.9 (d, J = 12.0 Hz), 125.4, 127.5 (2C), 128.5, 129.6
(2C), 131.1, 132.0 (d, J = 8.4 Hz), 136.2, 144.4, 158.8, 162.2, 170.2,
173.8, 178.9. ESI-MS: Cacld for C23H24FN3O4S2 [M+H]+: 490.1265,
Found 490.1274 (error 1.8 ppm).
4.5.8. (Z)-2-(5-Benzylidene-4-oxo-4,5-dihydrothiazol-2-
ylamino)-3-methyl-N-tosylbutanamide 12
1H NMR (400 MHz, DMSO-d6) d 0.80 (d, J = 6.5 Hz, 3H), 0.83 (d,
J = 6.7 Hz, 3H), 2.05–2.13 (m, 1H), 2.37 (s, 3H), 4.58–4.68 (m, 1H),
7.34–7.45 (m, 3H), 7.49–7.58 (m, 4H), 7.61 (s, 1H), 7.82 (d,
J = 7.2 Hz, 2H), 9.79 (d, J = 7.1 Hz, 1H) ; 13C NMR (75 MHz, DMSO-
d6) d 17.7, 18.8, 21.2, 30.6, 63.1, 125.7, 127.6, 128.5, 129.3 (2C),
129.6 (2C), 129.8, 130.0, 134.0, 136.2, 141.5, 141.9, 144.5, 169.4,
174.5, 179.4. ESI-MS: Cacld for C22H23N3O4S2 [M+H]+: 458.1203,
Found 458.1190 (error 2.8 ppm).
4.5.3. (Z)-2-(5-(2-Fluorobenzylidene)-4-oxo-4,5-dihydrothiazol-
2-ylamino)-3-phenyl-N-tosylpropanamide 7
1H NMR (300 MHz, DMSO-d6) d 2.40 (s, 3H), 2.82 (dd, J = 9.9,
13.5 Hz, 1H), 3.13 (dd, J = 3.6, 13.8 Hz, 1H), 4.82–4.99 (m, 1H),
7.16–7.24 (m, 5H), 7.33–7.44 (m, 4H), 7.46–7.54 (m, 2H), 7.61 (s,
1H), 7.82 (d, J = 8.1 Hz, 2H), 10.08 (d, J = 7.4 Hz, 1H); 13C NMR
4.5.9. (Z)-2-(5-Benzylidene-4-oxo-4,5-dihydrothiazol-2-
ylamino)-4-methyl-N-tosylpentanamide 13
1H NMR (400 MHz, DMSO-d6) d 0.85 (d, J = 6.1 Hz, 3H), 0.88 (d,
J = 6.1 Hz, 3H), 1.45–1.65 (m, 3H), 2.37 (s, 3H), 4.64–4.77 (m, 1H),
7.38–7.45 (m, 3H), 7.49–7.57 (m, 4H), 7.63 (s, 1H), 7.77–7.82 (m,
2H) , 9.93 (d, J = 7.3 Hz, 1H); 13CNMR (75 MHz, DMSO-d6) d 21.2
(2C), 23.0, 24.6, 29.2, 56.8, 127.6 (2C), 128.4, 129.4 (2C), 129.6
(2C), 129.7 (2C), 130.0, 130.2, 134.0, 136.4, 144.5, 170.5, 174.1,
179.5. ESI-MS: Cacld for C23H25N3O4S2 [M+H]+: 472.1359, Found
472.1359 (error 0 ppm).
(75 MHz, DMSO-d6)
d 21.1 (2C), 22.8, 24.5, 56.7, 116.2 (d,
J = 21.3 Hz), 120.9 (d, J = 6.7 Hz), 121.9 (d, J = 12.0 Hz), 125.4,
127.5 (2C), 128.5, 129.6 (2C), 131.1, 132.0 (d, J = 8.4 Hz), 136.2,
144.4, 158.8, 162.2, 170.2. ESI-MS: Cacld for
C23H24FN3O4S2
[M+H]+: 490.1265, Found 490.1274 (error 1.8 ppm).
4.5.4. (Z)-2-(5-(3-Chlorobenzylidene)-4-oxo-4,5-dihydrothiazol-
2-ylamino)-4-methyl-N-tosylpentanamide 8
4.5.10. (Z)-2-(5-(Biphenyl-4-ylmethylene)-4-oxo-4,5-
1H NMR (400 MHz, DMSO-d6) d 0.85 (d, J = 6.1 Hz, 3H), 0.89 (d,
J = 6.2 Hz, 3H), 1.49–1.66 (m, 3H), 2.37 (s, 3H), 4.65–4.79 (m, 1H),
7.38–7.46 (m, 2H), 7.49–7.58 (m, 3H), 7.60–7.67 (m, 2H), 7.79–
7.90 (m, 2H), 9.91 (d, J = 7.0 Hz, 1H); 13C NMR (100 MHz, DMSO-
d6) d 17.6, 18.8, 21.2, 30.7, 63.3, 127.7 (2C), 127.8, 128.4, 129.2,
129.5, 129.7 (2C), 130.4, 131.2, 134.0, 136.2, 136.3, 144.6, 169.4,
dihydrothiazol-2-ylamino)-3-phenyl-N-tosylpropanamide 14
1H NMR (400 MHz, DMSO-d6)
d 2.40 (s, 3H), 2.84 (dd,
J = 10.0 Hz, 14.0 Hz, 1H), 3.14 (dd, J = 4.4 Hz, 14.0 Hz, 1H), 4.89–
4.97 (m, 1H), 7.17–7.26 (m, 5H), 7.39–7.43 (m, 3H), 7.46–7.52
(m, 2H), 7.62–7.66 (m, 3H), 7.72–7.76 (m, 2H), 7.80–7.85 (m, 4H)
, 9.94 (d, J = 10.1 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d 21.3,
36.8, 59.5, 126.9 (2C), 127.1, 127.5(2C), 127.7(2C), 128.2, 128.3,
128.5(2C), 129.2(3C), 129.3(2C), 129.8(2C), 130.3(2C), 133.0,
136.0, 136.2, 139.1, 141.3, 144.6, 169.3, 174.1, 179.5. ESI-MS: Cacld
174.4, 179.3. ESI-MS: Cacld for
C
22H22ClN3O4S2 [M+H]+:
492.0813, Found 492.0820 (error 1.4 ppm).
4.5.5. (Z)-2-(5-(2-Chlorobenzylidene)-4-oxo-4,5-dihydrothiazol-
2-ylamino)-4-methyl-N-tosylpentanamide 9
for
C
32H27N3O4S2 [M+H]+: 582.1521, Found 582.1525 (error
0.6 ppm).
1H NMR (400 MHz, DMSO-d6) d 0.86 (d, J = 5.8 Hz, 3H), 0.89 (d,
J = 5.8 Hz, 3H), 1.47–1.63 (m, 3H), 2.38 (s, 3H), 4.67–4.78 (m, 1H),
7.39–7.61 (m, 6H), 7.75–7.84 (m, 3H), 9.97 (d, J = 7.5 Hz, 1H); 13C
NMR (75 MHz, DMSO-d6) d 21.1 (2C), 22.7, 24.4, 40.1, 56.7, 125.2,
127.4 (2C), 127.9, 128.5, 129.5 (2C), 130.1, 131.1, 132.0, 132.1,
134.1, 136.2, 144.3, 170.1, 173.9, 178.6. ESI-MS: Cacld for
4.5.11. (Z)-2-(5-(2-Methoxybenzylidene)-4-oxo-4,5-
dihydrothiazol-2-ylamino)-3-phenyl-N-tosylpropanamide 15
1H NMR (300 MHz, DMSO-d6) d 2.40 (s, 3H), 2.80 (dd, J = 9.6,
13.5 Hz, 1H), 3.12 (dd, J = 4.2, 13.5 Hz, 1H), 3.86 (s, 3H), 4.86–
4.96 (m, 1H), 7.04–7.12 (m, 2H), 7.18–7.26 (m, 5H), 7.35–7.43
(m, 4H), 7.78–7.85 (m, 3H), 9.92 (d, J = 7.8 Hz, 1H); 13C NMR
(75 MHz, DMSO-d6) d 21.1, 36.8, 55.7, 59.5, 111.7, 120.8, 122.5,
124.5, 126.8, 127.5 (2C), 128.0, 128.3 (2C), 128.4, 129.2 (2C),
129.5 (2C), 131.5, 136.1, 136.7, 144.0, 157.8, 169.5, 174.1, 179.3.
C
23H24ClN3O4S2 [M+H]+: 506.0970, Found 506.0955 (error 2.9 ppm).
4.5.6. (Z)-2-(5-(2-Chlorobenzylidene)-4-oxo-4,5-dihydrothiazol-
2-ylamino)-3-phenyl-N-tosylpropanamide 10
1H NMR (300 MHz, DMSO-d6) d 2.40 (s, 3H), 2.82 (dd, J = 9.9,
13.8 Hz, 1H), 3.13 (dd, J = 4.5, 14.4 Hz, 1H), 4.89–4.99 (m, 1H),
7.15–7.27 (m, 5H), 7.40–7.62 (m, 6H), 7.75 (s, 1H), 7.82 (d,
ESI-MS: Cacld for
536.1310 (error 0.3 ppm).
C
27H25N3O5S2 [M+H]+: 536.1308, Found

H. Li et al. / Bioorg. Med. Chem. 20 (2012) 4194–4200
4199
4.5.12. (Z)-2-(5-(2-Methoxybenzylidene)-4-oxo-4,5-
4.9. Molecular modeling procedure
dihydrothiazol-2-ylamino)-3-methyl-N-tosylbutanamide 16
1H NMR (300 MHz, DMSO-d6) d 0.78–0.84 (m, 6H), 2.02–2.11
(m, 1H), 2.38 (s, 3H), 3.88 (s, 3H), 4.59 (t, J = 6.9 Hz, 1H), 7.06–
7.14 (m, 2H), 7.39–7.42 (m, 4H), 7.81–7.84 (m, 3H), 9.72 (d,
J = 7.7 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) d 17.7, 18.8, 21.2,
30.7, 55.8, 63.1, 111.8, 121.0, 122.6, 124.7, 127.7 (2C), 128.1,
128.6, 129.7 (2C), 131.7, 136.3, 144.6, 158.0, 169.6, 174.8, 179.6.
Alldocking calculationswere performedusingthe Glide module of a
computational program Schrödinger. The NMR-derived structure of
Bcl-2 complexed with an acyl-sulfonamide based inhibitor 3-nitro-N-
{4-[2-(2-phenylethyl)-1,3-benzothiazol-5-yl]benzoyl}-4-{[2-(phenyls
ulfanyl)ethyl]amino}benzenesulfonamide (43B in PDB code: 2O21)
was used as the receptor for this docking experiment. The hydrogen
atoms from the original NMR structure were removed and then the
complex structure was processed with the Protein Preparation Wizard
of Schrödinger. In this step, hydrogen atoms were added to the protein
and ligand, the protein–ligand complex was then refined by Impact
minimization.
ESI-MS: Cacld for
488.1306 (error 0.4 ppm).
C
23H25N3O5S2 [M+H]+: 488.1308, Found
4.6. Cell proliferation assay
The antiproliferative activities of chalcone thiosemicarbazide
derivatives were determined using a standard (MTT)-based colori-
metric assay (Sigma). Briefly, cell lines were seeded at a density of
7 Â 103 cells/well in 96-well microtiter plates (Costar). After 24 h,
exponentially growing cells were exposed to the indicated
compounds at final concentrations ranging from 0.1 to 40 mg/
mL. After 48 h, cell survival was determined by the addition of
All the inhibitor structures used in the docking experiment were
modeled with Schrödinger and refined by its LigPrep module. The
docking calculations were performed using the Glide extra preci-
sion (XP) mode. Default input parameters were employed in dock-
ing calculations and a core of acyl-phenyl-sulfonamide group from
the prepared receptor structure was used as a reference. To com-
pensate for the rigid protein structure, Glide provides the options
to scale the van der Waals radii of nonpolar atoms of both the pro-
tein and ligand. In this work, the default scaling factors were used,
that is, the van der Waals radii for nonpolar atoms were scaled by a
factor of 0.8, and those of protein atoms were not scaled.
an MTT solution (20 lL of 5 mg/mL MTT in PBS). After 6 h,
100 mL of 10% SDS in 0.01 N HCl was added, and the plates were
incubated at 37 °C for a further 4 h; optical absorbance was mea-
sured at 570 nm on an LX300 Epson Diagnostic microplate reader.
Survival ratios are expressed in percentages with respect to un-
treated cells. IC50 values were determined from replicates of 6
wells from at least two independent experiments.
Acknowledgment
This work was financially supported by grants from Chinese
National Science & Technology Major Project on ‘‘Key New Drug
Creation and Manufacturing Program’’ (2009ZX09103-129) to
Professor Chunhua Qiao at Soochow University. Foundation from
the Priority Academic Program Development of Jiangsu Higher
Education Institutions is also acknowledged; The docking study
work was supported by Towson University start-up funds to
Professor Ma.
4.7. Protein expression and purification
The isoform 2 construct of the human Bcl-2 with an N-terminal
6xHis tag was used.21 Bcl-2 protein was produced in E. coli BL21
(DE3) cells. Cells were grown at 37 °C in 2xYT containing antibiot-
ics to an OD600 of 0.6. Protein expression was induced by 0.4 mM
IPTG at 20 °C for 20 h. Cells were lysed in 50 mM Tris pH 8.0 buffer
containing 500 mM NaCl, 0.1%bME and 40 lL of Leupectin/Aprotin.
The protein was purified from the soluble fraction using Ni-NTA
resin, following the manufacturer’s instructions. The protein was
further purified on a Superdex75 column (Amersham Biosciences)
in 25 mM Tris pH 8.0 buffer containing 150 mM NaCl and 2 mM
DTT.
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