Synthesis, anti-cancer evaluation of benzenesulfonamide derivatives as potent tubulin-targeting agents

Article abstract of DOI:10.1016/j.ejmech.2016.07.002

A series of benzenesulfonamide derivatives were synthesized and evaluated for their anti-proliferative activity and interaction with tubulin. These new derivatives showed significant activities against cellular proliferative and tubulin polymerization. Compound BA-3b proved to be the most potent compound with IC₅₀value ranging from 0.007 to 0.036 μM against seven cancer cell lines, and three drug-resistant cancer cell lines, which indicated a promising anti-cancer agent. The target tubulin was also verified by dynamic tubulin polymerization assay and tubulin intensity assay.

Full text of DOI:10.1016/j.ejmech.2016.07.002

Accepted Manuscript
Synthesis, anti-cancer evaluation of benzenesulfonamide derivatives as potent
tubulin-targeting agents
Jun Yang, Simin Yang, Shanshan Zhou, Dongbo Lu, Liyan Ji, Zhongjun Li, Siwang
Yu, Xiangbao Meng
PII:
S0223-5234(16)30549-9
10.1016/j.ejmech.2016.07.002
EJMECH 8720
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 April 2016
Revised Date: 20 June 2016
Accepted Date: 2 July 2016
Please cite this article as: J. Yang, S. Yang, S. Zhou, D. Lu, L. Ji, Z. Li, S. Yu, X. Meng, Synthesis, anti-
cancer evaluation of benzenesulfonamide derivatives as potent tubulin-targeting agents, European
Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.002.
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Graphical abstract
BA-3b, X=O, Y=CH2, R=7-F
IC50 7~36 nM against 7 cancer cell lines

ACCEPTED MANUSCRIPT
Synthesis, anti-cancer evaluation of benzenesulfonamide derivatives as potent
tubulin-targeting agents
Jun Yang, Simin Yang, Shanshan Zhou, Dongbo Lu, Liyan Ji, Zhongjun Li, Siwang Yu*, and
Xiangbao Meng*
The State Key Laboratory of Natural and Biomimetic Drugs; Department of Chemical Biology,
School of Pharmaceutical Sciences, Peking University, Beijing 100191, China
*Corresponding authors, Email: swang_yu@bjmu.edu.cn, or xbmeng@bjmu.edu.cn
Abstract:
A series of benzenesulfonamide derivatives were synthesized and evaluated for their
anti-proliferative activity and interaction with tubulin. These new derivatives showed significant
activities against cellular proliferative and tubulin polymerization. Compound BA-3b proved to be
the most potent compound with IC50 value ranging from 0.007 to 0.036 ꢀM against seven cancer
cell lines, and three drug-resistant cancer cell lines, which indicated a promising anti-cancer agent.
The target tubulin was also verified by dynamic tubulin polymerization assay and tubulin intensity
assay.
Keywords
Tubulin-targeting agents, sulfonamides, benzoxazepine, benzodiazepine, benzothiazepine.
1. Introduction:
Microtubules are hollow tubes formed by the polymerization of α, β-tubulin heterodimers,
which are essential in a diverse array of eukaryotic cell functions, like intracellular organelle
transport, cell motility and mitosis[1-4]. Numbers of clinically used compounds such as vinca
alkaloids, colchicines, paclitaxel, and epothilone attack microtubules by interfering with the
dynamics of the tubulin polymerization and depolymerization, resulting in mitotic arrest[5-8]
.
Undoubtedly, targeting tubulin is a successful strategy for cancer chemotherapy, However, there
are still many problems existing in clinical use of these anti-tubulin agents, like toxicity, poor
water solubility, poor bioavailability and multi-drug-resistant (MDR)[9-11]. Therefore, it is essential
to develop small molecular agents which can be effective not only in treating MDR tumors but
also in inhibiting tubulin polymerization.
Figure 1: New tubulin-targeting drug design
Stockwell et al identified compound 1 (Fig.1) as a highly potent tubulin-targeting agent after
analyzing more than 1 million simple synthetic compounds[11]. The lead compound 1 was proved
to be a highly potent structure by our previous work. Our group has actively engaged in searching
novel anticancer agents that target tubulin and have synthesized a series of 4-azaheterocycle
benzenesulfonamide derivatives (2, the ring contraction series), which show excellent activities
against a panel of cancer cell lines[12]. Our precious work revealed that cyclopropyl-oxazole
moiety was crucial for cytotoxicity. Moreover, benzodiazepine, benzoxazepine and
benzothiazepine skeleton as crucial pharmacophore cores have attracted much attention in the past
years owning to its broad spectrum of biological activities especially anticancer[13-17]
,
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anticonvulsant[18], CNS activities[19,20] and others[21]. To continue our earlier work[12], we designed
the ring expansion series (3, Fig. 1) of the lead compound 1, expecting an improvement in drug
potency and water solubility. We herein describe the rationale for the design, concise synthesis,
and structure-activity relationships (SAR) of a series of benzenesulfonamide derivatives as potent
antitubulin agents.
2. Results and discussion
2.1 Chemisty
The reference compound 1 was synthesized following the pathway depicted in Scheme 1. The
general syntheses of benzenesulfonamide derivatives are shown in Scheme 2. The
2,5-disubstituted oxazole was prepared from acetophenone and cyclopropylnitrile using
PhI(OAc)2 as an oxidant, which was subsequently treated with chlorosulfonic acid to yield
compound BA-1[22]. Compounds BA-2 were synthesized from 2-aminophenol, 2-aminothiophenol
or 2-aminobenzylalcohol with 1,3-dibromopropane or 1,2-dibromoehane in DMF, and then reacted
with BA-1 in presence of pridine to give the target compounds BA-3(a-l), BA-4(a-b),
respectively.
Scheme 1. Reagents and conditions: a) PhI(OAc)2, TfOH, cyclopropanecarbonitrile, DCE, 80°C; b)
Chlorosulfonic acid, DCM, 50°C; c) Pyridine, DCM, rt, 2h.
Scheme 2. a) m=1, 1,2-dibromoethane, K2CO3/DMF, 60°Cꢀm=2, 1,3-dibromopropane, K2CO3/DMF, 60°C; b)
BA-1, pyridine, DCM, rt, 2-3h.
The synthetic route for compound BA-2m was different from other precursor compounds BA-2,
which prepared from commercially available tetrahydronaphthalene following four steps with a 36%
total yield according to the reference methods[23], as shown in Scheme 3.
.
Scheme 3. aꢁFeCl3 6H2O, TBHP(70%), pyridine, 82°C; b) NaBH4/MeOH, 0°C; c) triethylamine, methanesulfonyl
.
chloride, CH2Cl2, then NaN3, DMF; d) BF3 OEt2, anhydrous CH2Cl2. -78°C, then NaBH4, NaOH(15% aqueous).
In order to improve the compounds’ water solubility, we designed three compounds, BA-3n,
BA-3o and BA-3p. Compounds BA-3n, BA-3o were expected to introduce amino and
tert-ammonia to form salt with suitable acid, which can largely increase water solubility.
Compound BA-3k was oxidized to sulfone[24], sulfoxide[25], which were expected to improve the
water solubility. These compounds’ general synthesis routes are shown respectively in Scheme
4-6.
Scheme 4. The route of introducing amine:a) Et3N, HCOOH, Pd/C, EtOH, 80°C.
Scheme 5. The route of introducing tert-amine: a) Sarcosine, DMSO, 180°C; b) LiAlH4, THF, 0°C; c) BA-1,
pyridine, DCM.
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Scheme 6. Synthesis of sulfone and sulfoxide from sulfide: a) 2.10 equiv mCPBA, DCM, r.t; b) 1.05 equiv
mCPBA, DCM, rt
2.2 Bilogical results
2.2.1 In vitro cell growth Inhibitory Activity
Preciously, we synthesized four lead compounds and evaluated for antiproliferative activities
against four types of human cancer cell lines, colorectal carcinoma HCT-116 cells, prostate
carcinoma PC3 cells, liver cancer HepG2 cells and ovarian cancer SK-OV-3 cells. As a result,
benzoxazepine derivative BA-3a showed highest potent than benzothiazepine, benzoxazine and
benzothiazine, and the lead compound 1 (see Table1). So we designed a series of benzoxazepine
derivatives and evaluated their cytotoxic potency, as shown in Table 2. All compounds exhibited
excellent cytotoxic activities.
Table 1. IC50 values of fist-run compounds over four cancer cell linesa.
Compared with compounds BA-3(g-j), BA-3(b-f) generally exhibited better anti-cancer activities,
respectively. The result indicated that substituent at the C-7 position is more potent than that at
C-8 position except nitro group (BA-3j vs BA-3e). Furthermore, electron-withdrawing group
substitutions, like -F, -Cl, -NO2, showed higher antiproliferative potency than those with
electron-donating groups, like methyl and methoxyl. More interesting, the 1,4-oxazepine
derivative BA-3a is more potent than its 1,3-oxazepine counterpart, BA-3l. Of all potent
compounds, BA-3b and BA-3f showed the best activities with IC50 values 0.015-0.036 and
0.018-0.039 μM, respectively.
Table 2. Antiproferliferative effects of benzoxazepane derivatives
Of the compounds designed to improve water solubility, only compound BA-3n exhibited
potent activity. The rest two compounds (BA-3p and BA-3q) showed a sharply decrease in activity
than the benzenothiozepine derivative BA-3K, which turned out to be a failure of their structural
modification, together with the N-methyl benzodiazepine derivative BA-3o (Table 3).
Table 3. Antiproferliferative effects of benzothiazepine and benzodiazepine derivatives
Clinical use of chemotherapeutics, including anti-tubulin agents, multi-drug-resistant problem
arose eventually. Compound BA-3b and BA-3g were chosen to test their potential against several
MDR cell lines. As shown in Table 4, compound BA-3b and BA-3g exhibited strong cytotoxicity
against both MDR cell lines K562/A02, KB/Vcr, MCF-7/Adr, and their drug-sensitive parental cell
lines.
Table 4. IC50 values of compounds in three MDR and drug-sensitive parental cell lines
2.2.2 Inhibition of tubulin polymerization.
To investigate whether the ring-expansion derivatives of lead compound 1 are tubulin-targeting
agents, BA-3b, BA-3g were chosen to undergo tubulin polymerization assay in vitro using
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purified porcine brain tubulin[26]. In this assay, tubulin monomer was self-polymerized to
microtubules, increasing light scattering at 340 nm. Microtubule-depolymerizing agents, like
vinblastine, colchicine, nacodazole, are known to inhibit self-polymerization activity of tubulin,
on the contrary, microtubule-stabilizing agent, like Taxol, accelerated polymerizationin this
assay[27]. Compounds BA-3b and BA-3g showed a similar dynamic curve with nacodazole, not
Taxol (Fig. 2). These results indicated that BA-3b, BA-3g directly binds to tubulin and induces
depolymerization of microtubule network.
Figure 2. Tubulin polymerization in the presence of BA-3b, BA-3g, nocodazole, and taxol
The effects of these compounds on tubulin polymerization were further examined by
fluorescent tubulin intensity assay. Treatment of SW480 cells with compounds BA-3b, BA-3g,
and vincristine, respectively, strong tubulin inhibitory activities were observed (Fig. 3).
Figure 3. Tubulin intensity assay (DMSO as control, Vin for vinblastine)
2.2.3Induction of SW480 cell apoptosis
Considering the potent antiproliferative activity, compound BA-3b was selected for flow
cytometry analysis. As shown in Fig. 4, compound BA-3b could induce early apoptosis in SW480
cells at very low concentration (50 nM) with a ratio of 7.74%, compared with 3.41% in the vehicle
control group, in 24 hours.
Figure 4. BA-3b induce apoptosis in SW480 cells
The water solubility is a crucial chemo-physical factor for a drug candidate, especially for a
tubulin-targeting agent. The solubility of the most potent molecule BA-3b is measured as ~10
mg/L in pure water at 25°C, which is 1000-fold higher than its average antiproliferative IC50
values.
3. Conclusion
In summary, a series of novel sulfonamide derivatives were synthesized and displayed better
potent cytotoxicities than the lead compound 1. Generally, 7-substituted derivatives showed better
activity than those 8-substituted. The most potent compound BA-3b displayed excellent
cytotoxicity against 7 human tumor cell lines, and 3 MDR cell lines, with an IC50 range
0.007-0.036 ꢀM, as well as an excellent antitubulin activity. Moreover, the water solubility of this
compound is superior than Docetaxel, which used as an antitumor drug targeting to microtubule in
clinical treatment. The above results indicate this new compound might be an orally antitubulin
candidate after further investigation.
4. Material and methods
4.1 Chemical synthesis
General
All solvents were of analytical grade.1H NMR and 13C NMR spectra were recorded with
Bruker AM-400 MHz spectrometer. The chemical shifts were reported in ppm using TMS as
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internal standard. High resolution mass spectrometry data were measured on Bruker Apex
IVFTMS. Melting points were measured on an X-5 micro-melting point apparatus and
wereuncorrected. Column chromatography was conducted on silica gel 60 (E. Merck,
0.063-0.200mm).
4.1.1 General procedure for synthesis and purification of precursor compound BA-2(a-l).
To a solution of o-aminophenol (1.0 g, 9.2 mmol) in 10mL DMF was added 1,
3-dibromopropane(1.4 ml, 13.8 mmol) and potassium carbonate (6.36 g, 4.6 mmol), the reaction
mixture was refluxed overnight. After the reaction was finished monitored by TLC, the suspension
was cooled down to room temperature and removed DMF in vacuum. Then extracted with ethyl
acetate (3×10 mL) and brine (3×10 mL), dried over MgSO4, filtered and concentrated under
vacuum. The crude product was future purified by column chromatography (petroleum ether/
ethyl acetate=15:1) to obtain precursors compounds BA-2(a-l), yields 45-92%.
4.1.2 General procedure for synthesis and purification of precursor compound BA-2m.
A solution of tetrahydronaphthalene (674 mg, 5.0 mmol) in pyridine (5 mL) was added
.
FeCl3 6H2O (27 mg, 0.1 mmol) and TBHP (70%) (2.06 mL, 15.0 mmol). The suspension was
refluxed for 24 hour, then treated with hydrochloric acid (1M, 10 mL) and exacted with ethyl
acetate, the crude product was purified via column chromatography (petroleum ether / ethyl
acetate=10:1. The gained compound subsequently was added sodium borohydride (0.34 g, 8.9
mmol) in 9 mL MeOH and stirred at 0°C for 2 hours. When finished, the alcohol (2.0 mmol) was
added triethylamine (420 ꢀL, 3.0 mmol) in anhydrous CH2Cl2 (5 mL) and followed by
methanesulfonyl chloride (2.4 mmol) over a period of 10min. After an additional 15 min at 0°C,
the reaction mixture was diluted with CH2Cl2(10 mL), and the organic layer was washed with
brine (3×5 mL), and then dried with anhydrous Na2SO4. The solvent was removed under reduced
pressure to give the mesylate as yellow liquid. The crude mesylate (1.0 mmol) was re-dissolved in
DMF (10 mL), treated with NaN3 (2 mmol) and stirred at room temperature for 3 hours. The
reaction mixture was poured into ice-cold water (10 mL), extracted with brine (3×10 mL), dried
and purified by column chromatography (petroleum ether / ethyl acetate=10:1) to give pure
benzylicazide. The gained azide was re-dissolved in anhydrous CH2Cl2 (5mL) at -78°C, and added
.
BF3 OEt2 (2.2 mmol) dropwise. The resultant mixture was stirred at -78°C for 45 min and then
warmed to room temperature. After an additional 45 min reaction at room temperature, the
mixture was cooled to 0°C and treated with NaBH4 (265 mg, 7.0 mmol) in 15% aqueous NaOH (3
mL). The reaction was then warmed to room temperature and stirred for 45 min. The mixture was
extracted with CH2Cl2(2 × 10 mL), brine(2 × 5 mL), dried and purified by column
chromatography ( petroleum ether / ether acetate =10:1) to give compound BA-2m.
4.1.3 General procedure for synthesis and purification of precursor compound BA-2o.
To a stirred solution of isatoic anhydride (333 mg, 2.0 mmol) in 4 mL DMSO was added
sarcosine (182 mg, 2.0 mmol), the reaction mixture was refluxed for 5 hours. After the reaction
was finished, the mixture was cooled down to 0°C and allowed to stand overnight. The solid
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product was collected by filtration, dried and recrystallized from ethanol. Then the solid (90 mg)
was dissolved in THF (4 mL) at 0°C and added LiAlH4 (79 mg, 2.0 mmol). The reaction mixture
was stirred for 6 hours and then warmed to room temperature. To the solution was added water (3
mL), extracted with CH2Cl2 (3×5 mL), dried and purified by column chromatography (CH2Cl2:
MeOH=30:1) to give compound BA-2o. 1H NMR (400 MHz, CDCl3) δ 7.16-7.04 (m, 2H), 6.84 (t,
J = 7.4 Hz, 1H), 6.74 (d, J = 7.7 Hz, 1H), 3.90 (s, 1H), 3.71 (s, 2H), 3.25-3.10 (m, 2H), 2.94-2.82
(m, 2H), 2.40 (s, 3H). 13C NMR (100MHz, CDCl3) δ 150.16, 131.09, 128.76, 128.02, 120.77,
118.62, 62.09, 59.93, 45.93, 43.53. ESI MS: Calcd for C10H14N2 [M+H]+: 163.12; found: 163.28.
4.1.4 General procedure for synthesis and purification of compounds BA-3(a-m), BA-3oand
BA-4(a-b).
[22]
The precursor compound BA-1 was synthesized according to the reference methods
,
illustrated above in Chemistry section. Then BA-1(1.0 mmol) was added with compounds
BA-2(1.2 mmol) and pyridine (2.0 mmol) in CH2Cl2 (5 mL), the reaction was stirred at room
temperature for 2h, and then purified by column chromatography (petroleum ether/ ethyl
acetate=5:1) to obtain compounds BA-3(a-m), BA-3o and BA-4(a-b).
4.1.5 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepine
(BA-3a)
White solid, yield 85%. M.p. 87-88°C. 1H NMR (400 MHz, CDCl3) δ 7.69-7.56 (m, 4H), 7.52
(d, J=8.0 Hz,1H), 7.29 (s, 1H), 7.23 (t, J=8.0Hz, 1H), 7.10 (t, J=8.0 Hz, 1H), 6.97 (d, J= 8.0
Hz,1H), 4.03-3.67 (m, 4H), 2.13 (tt, J=8.2, 4.4 Hz, 1H), 1.94-1.80 (m, 2H), 1.19-1.06(m, 4H). 13
C
NMR (100MHz, CDCl3) δ 166.90, 156.54, 148.74, 139.62, 132.22, 131.92, 131.20, 129.39,
128.01, 124.45, 124.03, 123.65, 122.39, 70.88, 48.84, 29.38, 9.09, 8.65. HR-ESI-MS: Calcd for
C21H20N2O4S [M+H]+: 397.12220; found: 397.12071.
4.1.6 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-7-fluoro-2,3,4,5-tetrahydrobenzo[b][1,4]
oxazepine (BA-3b)
White solid, yield: 72%. M.p. 117-118°C.1H NMR (400 MHz, CDCl3) δ 7.71-7.59 (m, 4H),
7.30 (s, 1H), 7.28-7.23(m 1H), 6.99-6.88 (m, 2H), 3.98-3.71(m, 4H), 2.20-2.08 (m, J=8.2, 5.2 Hz,
1H), 1.94-1.80 (m, 2H), 1.20-1.07 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 167.07, 156.87, 148.63,
139.23, 132.12, 128.00, 124.58, 123.74, 123.10, 123.01, 117.87, 117.63, 116.08, 115.85, 70.96,
48.81, 29.33, 9.10, 8.70. HR-ESI-MS: Calcd for C21H19FN2O4S [M+H]+: 415.11278; found:
415.11265.
4.1.7 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-7-methoxy-2,3,4,5-tetrahydrobenzo[b][1,
4]oxazepine (BA-3c)
Brown oil, yield: 83%. 1H NMR (400 MHz, CDCl3) δ 7.71-7.54(m, 4H), 7.29 (s, 1H), 7.05 (d,
J=3.1 Hz, 1H), 6.89 (d, J=8.8 Hz, 1H), 6.78 (dd, J=8.8, 3.1 Hz, 1H), 3.97-3.69 (m, 7H), 2.21-2.06
(m,1H), 1.91-1.76 (m, 2H), 1.18-1.05 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 166.94, 155.58,
150.37, 148.72, 139.58, 132.89, 131.89, 128.04, 124.41, 123.62, 122.73, 115.55, 115.33, 71.01,
55.74, 48.95, 29.47, 9.08, 8.65. HR-ESI-MS: Calcd for C22H22N2O5S [M+H]+: 427.13277; found:
427.13146 .
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4.1.8 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-7-methyl-2,3,4,5-tetrahydrobenzo[b][1,4]
oxazepine (BA-3d)
Yellow solid, yield: 85%. M.p. 66-67°C. 1H NMR (400 MHz, CDCl3) δ 7.61 (q, J=8.7 Hz, 4H),
7.34 (d, J=1.8 Hz, 1H), 7.29 (s, 1H), 7.03 (dd, J=8.2, 1.8 Hz, 1H), 6.86 (d, J=8.2 Hz, 1H),
4.02-3.58 (m, 4H), 2.33 (s, 3H), 2.17-2.10 (m, 1H), 1.91-1.78 (m, 2H), 1.18-1.06 (m, 4H). 13C
NMR (100MHz, CDCl3) δ 166.93, 154.34, 148.75, 139.69, 133.83, 131.90, 131.83, 131.65,
130.05, 128.03, 124.40, 123.61, 122.01, 70.97, 48.90, 29.37, 20.70, 9.10, 8.68. HR-ESI-MS:
Calcd for C22H22N2O4S [M+H]+: 411.13785; found: 411.13688.
4.1.9 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-7-nitro-2,3,4,5-tetrahydrobenzo[b][1,4]
oxazepine (BA-3e)
Yellow solid, yield: 68%. M.p. 80-81°C. 1H NMR (400 MHz, CDCl3) δ 8.45 (d, J=2.7 Hz, 1H),
7.99 (dd, J=9.0, 2.7 Hz, 1H), 7.86 (d, J= 8.6 Hz, 2H),7.65 (d, J=8.6 Hz, 2H), 7.31 (s, 1H), 6.88 (d,
J=9.0 Hz, 1H), 4.16 (t, J=6.0 Hz, 2H),3.48 (t, J=6.0 Hz, 2H), 2.33 (p, J=6.0 Hz, 2H), 2.21-2.05 (m,
1H), 1.20-1.04 (m,4H). 13C NMR (100 MHz, CDCl3) δ167.42, 152.84, 150.58, 141.82, 137.13,
132.94, 127.97, 126.45, 125.05, 124.04, 121.30, 115.80, 110.78, 67.36, 31.38, 28.99, 9.12, 8.87.
HR-ESI-MS: Calcd for C21H19N3O6S [M+H]+: 432.10728; found: 432.10678.
4.1.10 7-chloro-5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-2,3,4,5-tetrahydrobenzo[b][1,4]
oxazepine (BA-3f)
1
Light yellow solid, yield: 86%. M.p. 122-123°C. H NMR (400 MHz, CDCl3) δ 7.68-7.59 (m,
4H), 7.53 (d, J=2.6 Hz, 1H), 7.31 (s, 1H), 7.19 (dd, J=8.6, 2.6 Hz, 1H), 6.91 (d, J=8.6 Hz, 1H),
4.15-3.58 (m, 4H), 2.20-2.08 (m, 1H), 2.00-1.80 (m, 2H), 1.21-1.07 (m, 4H). 13C NMR (100 MHz,
CDCl3) δ 167.09, 155.10, 148.63, 139.23, 133.15, 132.15, 130.88, 129.28, 128.51, 128.02, 124.59,
123.76, 123.38, 70.95, 48.78, 29.23, 9.10, 8.71. HR-ESI-MS: Calcd for C21H19ClN2O4S [M+H]+:
431.08323; found: 431.08182.
4.1.11 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-8-fluoro-2,3,4,5-tetrahydrobenzo[b][1,4]
oxazepine (BA-3g)
White solid, yield: 78%. M.p. 146-147°C. 1H NMR (400 MHz, CDCl3) δ 7.61 (s, 4H), 7.48 (dd,
J=8.9, 6.2 Hz, 1H), 7.30 (s, 1H), 6.81 (ddd, J=8.9, 6.2, 2.9Hz, 1H), 6.69 (dd, J=8.9, 2.9 Hz, 1H),
3.97-3.64 (m, 4H), 2.20-2.07 (m, 1H), 1.97-1.78 (m, 2H), 1.20-1.04 (m, 4H). 13C NMR (100 MHz,
CDCl3) δ 167.04, 148.66, 139.35, 135.36, 132.45, 132.34, 132.06, 128.00, 124.56, 123.71, 111.12,
110.90, 109.75, 109.52, 77.34, 77.02, 76.70, 71.18, 48.76, 29.35, 9.09, 8.66. HR-ESI-MS: Calcd
for C21H19FN2O4S [M+H]+: 415.11278; found: 415.11204.
4.1.12 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-8-methoxy-2,3,4,5-tetrahydrobenzo[b][1,
4]oxazepine (BA-3h)
1
Yellow solid, yield: 85%. M.p. 169-171°C. H NMR (400 MHz, CDCl3)δ 7.64-7.56 (m, 4H),
7.41 (d, J=8.8 Hz, 1H), 7.30 (s, 1H), 6.65 (dd, J=8.8, 2.8 Hz, 1H), 6.50 (d, J= 2.8 Hz, 1H), 3.79 (s,
7H), 2.17-2.10 (m, 1H), 1.84 (s, 2H), 1.20-1.07 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 170.50,
160.24, 157.93, 147.16, 139.66, 132.12, 131.81, 128.05, 124.86, 124.37, 123.62, 109.67, 107.41,
71.22, 55.55, 48.96, 29.55, 9.11, 8.69. HR-ESI-MS: Calcd for C22H22N2O5S [M+H]+: 427.13277;
found: 427.13143.
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4.1.13 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-8-methyl-2,3,4,5-tetrahydrobenzo[b][1,4]
oxazepine (BA-3i)
1
White solid, yield: 86%. M.p.146-147°C. H NMR (400 MHz, CDCl3) δ 7.68-7.56 (m, 4H),
7.37 (d, J=8.1 Hz, 1H), 7.27 (s, 1H), 6.90 (d, J=8.1, 1H), 6.79(s, 1H), 3.95-3.73 (m, 4H), 2.31 (s,
3H), 2.19-2.08 (m, 1H), 1.91-1.77 (m, 2H), 1.18-1.06 (m, 4H). 13C NMR (100 MHz, CDCl3) δ
166.91, 156.28, 148.76, 139.83, 139.76, 131.81, 130.84, 129.42, 128.01, 124.76, 124.39, 123.63,
122.82, 70.91, 48.90, 29.47, 21.03, 9.08, 8.65. HR-ESI-MS: Calcd for C22H22N2O4S [M+H]+:
411.13785; found: 411.13750.
4.1.14 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-8-nitro-2,3,4,5-tetrahydrobenzo[b][1,4]
oxazepine (BA-3j)
Yellow solid, yield: 65%. M.p. 191-193°C. 1H NMR (400 MHz, CDCl3) δ 7.94 (dd, J=8.8, 2.6
Hz, 1H), 7.83 (d, J=2.6 Hz, 1H), 7.76-7.61 (m, 5H), 7.33 (s, 1H), 4.09-3.80 (m, 4H), 2.25-2.11 (m,
1H), 2.04-1.88 (m, 2H), 1.22-1.05 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 169.92, 148.38, 147.13,
138.63, 138.10, 132.59, 130.86, 127.90, 124.95, 123.99, 118.58, 118.00, 70.91, 48.71, 28.72, 9.13,
8.82. HR-ESI-MS: Calcd for C21H19N3O6S [M+H]+: 442.10728; found: 442.10676 .
4.1.15 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-2,3,4,5-tetrahydrobenzo[b][1,4]
thiazepine (BA-3k)
1
White solid. yield: 78%. M.p. 129-131°C. H NMR (400 MHz, CDCl3) δ 7.71 (d, J=8.6 Hz,
2H), 7.60 (d, J=8.6 Hz, 2H), 7.56-7.47 (m, 2H), 7.32-7.26 (m,2H), 7.21 (dt, J=7.5, 1.4 Hz, 1H),
3.79 (s, 2H), 2.67-2.55 (m, 2H), 2.25-2.01 (m,3H), 1.23-1.01 (m, 4H).13C NMR (100 MHz, CDCl3)
δ 171.11, 148.87, 142.65,139.73, 136.40, 133.79, 131.89, 130.90, 128.64, 128.36, 128.31, 124.37,
123.59, 49.91, 31.38, 30.74, 9.10, 8.67. HR-ESI-MS: Calcd for C21H20N2O3S2 [M+H]+: 413.09936;
found: 413.09886.
4.1.16 1-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-1,2,3,5-tetrahydrobenzo[e][1,4]oxazepine
(BA-3l)
1
Colorless solid, yield: 79%. M.p. 66-67°C. H NMR (400 MHz, CDCl3) δ 7.75-7.57 (m, 5H),
7.41 (t, J=7.8 Hz, 1H), 7.36 (s, 1H), 7.19 (t, J=7.8 Hz, 1H), 6.51(d, J=7.8 Hz, 1H), 5.00 (d, J=12.5
Hz, 1H), 4.67 (d, J=12.5 Hz, 1H), 4.28-4.07(m, 1H), 3.71-3.52 (m, 1H), 3.48-3.27 (m, 2H),
2.20-2.12 (m, 1H), 1.20-1.08 (m,4H). 13C NMR (100 MHz, CDCl3) δ 167.43, 148.40, 142.49,
136.67, 135.67, 132.72, 131.31, 129.57, 128.78, 128.72, 127.24, 124.95, 123.86, 60.91, 53.78,
28.42, 9.15, 8.88. HR-ESI-MS: Calcd for C21H20N2O4S [M+H]+: 397.12220; found: 397.12176.
4.1.17 2-cyclopropyl-5-(4-(2,3,4,5-tetrahydro-1H-benzo[b]azepin-1-ylsulfonyl)phenyl)oxazole
(BA-3m)
White solid, yield: 81%. M.p.112-113°C. 1H NMR (400 MHz, CDCl3) δ 7.72 (d, J=8.6Hz, 2H),
7.63 (d, J=8.6 Hz, 2H), 7.32-7.28 (m, 2H), 7.21-7.16(m, 2H), 7.14-7.09 (m, 3.2 Hz, 1H), 3.99-3.39
(m, 2H), 2.49-2.29 (m, 2H), 2.23-2.08 (m, 1H), 1.88-1.75 (m, 2H), 1.65-1.48 (m, 2H), 1.20-1.07
(m, 4H). 13CNMR (100 MHz, CDCl3) δ 167.03, 148.69, 141.86, 140.61, 139.73, 131.79, 130.20,
129.53, 128.15, 127.79, 126.99, 124.42, 123.83, 50.96, 34.15, 29.69, 25.66, 9.09, 8.68.
HR-ESI-MS: Calcd for C22H22N2O3S [M+H]+: 395.142394; found: 395.14239.
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4.1.18 2-cyclopropyl-5-(4-(4-methyl-2,3,4,5-tetrahydro-1H-benzo[e][1,4]diazepin-1-ylsulfonyl)
phenyl)oxazole (BA-3o)
1
Yellow solid, yield: 46%. M.p. 108-110°C. H NMR (400 MHz, CDCl3)δ 7.76-7.67 (m, 4H),
7.37 (d, J=7.2 Hz, 1H), 7.32 (s, 1H), 7.27-7.20 (m, 2H), 7.19-7.11 (m, 1H), 3.26 (s, 2H), 3.13-2.68
(m, 4H), 2.22 (s, 3H), 2.16-2.10 (m, 1H), 1.23-1.06 (m, 4H). 13C NMR (100 MHz, CDCl3) δ
167.14, 148.55, 140.27, 139.36, 132.06, 130.88, 129.12, 128.33, 128.02, 127.78, 124.59, 123.91,
60.34, 57.46, 47.86, 42.70, 9.11, 8.76. HR-ESI-MS: Calcd for C22H23N3O3S [M+H]+: 410.15384;
found: 410.15406.
4.1.19 4-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine
(BA-4a)
Yellow solid, yield: 88%. M.p. 114-115°C. 1H NMR (400 MHz, CDCl3)δ7.86 (dd, J=8.2, 1.5 Hz,
1H), 7.67-7.57 (m, 4H), 7.30 (s, 1H), 7.08 (ddd, J=8.2, 7.4, 1.5 Hz, 1H), 6.95 (ddd, J=8.2, 7.4, 1.5
Hz, 1H), 6.80 (dd, J=8.2, 1.5 Hz, 1H),4.01-3.83 (m, 2H), 3.83-3.62 (m, 2H), 2.16-2.06 (m, J=8.1,
5.3 Hz, 1H), 1.18-1.07 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 146.85, 137.25, 132.68, 127.89,
126.52, 124.90, 124.87, 124.07, 123.67, 120.98, 117.58, 62.63, 44.39, 9.09, 8.75. HR-ESI-MS:
Calcd C20H18N2O4S [M+H]+: 383.10655; found: 383.10545.
4.1.20 5-(4-(2H-benzo[b][1,4]thiazin-4(3H)-ylsulfonyl)phenyl)-2-cyclopropyloxazole (BA-4b)
Yellow solid, yield: 86%. M.p. 147-148°C. 1H NMR (400 MHz, CDCl3) δ 7.69 (dd, J= 6.0, 3.6
Hz, 1H), 7.58 (s, 4H), 7.30 (s, 1H), 7.11 (dt, J=7.2, 3.6 Hz, 2H), 7.08-7.03 (m, 1H), 4.08-3.96 (m,
2H), 2.96-2.85 (m, 2H), 2.20-2.08 (m, 1H),1.19-1.07 (m, 4H).13C NMR (100 MHz, CDCl3) δ
167.16, 148.28, 138.86, 134.15,132.38, 128.34, 128.21, 127.78, 126.92, 126.77, 124.75, 123.91,
44.82, 25.92, 9.09, 8.73. HR-ESI-MS: Calcd for C20H18N2O3S2 [M+H]+: 399.08371; found:
399.08235.
4.1.21 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-
8-amine (BA-3n)
To a solution of BA-3j (32 mg, 0.07 mmol) in 3 mL ethanol was added Et3N (0.31 mmol),
formic acid (0.3 mmol) and palladium on activated carbon (5 mg) in sequence. The reaction
mixture was refluxed for 2h, then cooled down to room temperature, then filtered and collected
the filtrate. After removal of the solvent in vacuum, the crude product was purified by column
chromatography (CH2Cl2: MeOH=20:1) to obtain compound BA-3n as light yellow solid, yield:
90%. M.p. 91-92°C. 1H NMR (400 MHz, CDCl3) δ7.72-7.54 (m, 4H), 7.36-7.21 (m,2H), 6.39 (dd,
J=8.5, 2.6 Hz, 1H), 6.25 (d, J=2.6 Hz, 1H), 4.23-3.59 (m, 5H), 2.23-2.04 (m, 1H), 2.03-1.71 (m,
2H), 1.18-1.04 (m, 4H).13C NMR (100 MHz,CDCl3) δ 166.85, 157.86, 148.86, 147.82, 139.84,
132.27, 131.68, 128.08, 124.28,123.54, 122.43, 110.54, 107.88, 71.13, 49.05, 29.63, 9.07, 8.60.
HR-ESI-MS: Calcd for C21H21N3O4S [M+H]+: 412.13310; found: 412.13156.
4.1.22 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-2,3,4,5-tetrahydrobenzo[b][1,4]
thiazepine 1,1-dioxide (BA-3p)
To a solution of BA-3k (100 mg, 0.24 mmol) in 3 mL CH2Cl2 in an ice bath, was added
m-CPBA (86 mg, 0.50 mmol). The mixture was stirred at room temperature for 2h. The solvent
was removed under reduced pressure and purified by column chromatography (petroleum ether /
1
ether acetate =3:1) to give compound BA-3p as a white solid, yield: 86%. M.p. 203-205°C. H
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NMR (400 MHz, CDCl3) δ 8.09 (d, J=7.9, 1H), 7.94 (d, J= 8.6 Hz, 2H), 7.75 (d, J=7.9 Hz, 1H),
7.69 (d, J=8.6 Hz, 2H),7.64 (t, J=7.9, 1H), 7.53-7.47 (m, 1H), 7.33 (s, 1H), 3.26-3.04(m, 2H),
2.22-2.04(m, 3H), 1.21-1.04 (m, 4H). 13C NMR (100MHz, CDCl3) δ 148.57, 138.58,
138.36,137.61, 134.41, 132.73, 129.96, 129.28, 129.08, 128.29, 124.84, 123.72, 53.88,
49.70,24.55, 9.10, 8.71. HR-ESI-MS:Calcd for C21H20N2O5S2 [M+H]+: 445.08919; found:
445.08926.
4.1.23 5-(4-(2-cyclopropyloxazol-5-yl)phenylsulfonyl)-2,3,4,5-tetrahydrobenzo[b][1,4]
thiazepine 1-oxide (BA-3q)
To a solution of BA-3k (100 mg, 0.24 mmol) in 3 mL CH2Cl2 in an ice bath, was added
m-CPBA (43 mg, 0.25 mmol). The mixture was stirred at room temperature for 2h. After finished,
the solvent was removed under reduced pressure and purified by column chromatography
(petroleum ether / ether acetate =3:1) to give compound BA-3p as a white solid in 77% yield. M.p.
157-158°C. 1H NMR (400 MHz, CDCl3) δ7.78-7.80(m, 3H), 7.67-7.70(m, 2H), 7.59-7.63(m, 1H),
7.46-7.53(m, 2H), 7.35(s, 1H), 4.40-4.44(m, 1H), 3.18-3.23(m, 1H), 2.98(t, J=12.0 Hz, 1H),
2.83-2.90(m, 1H), 2.42-2.52(m, 1H), 2.13-2.20(m, 1H), 2.03-2.07(m, 1H),1.12—1.21(m, 4H). 13
C
NMR (100MHz, CDCl3) δ163.33, 148.38, 144.42, 138.61, 135.52, 132.70, 131.43, 129.73, 129.40,
128.18, 125.20, 124.94, 124.02, 53.43, 48.76, 25.96, 9.11, 8.78. HR-ESI-MS: Calcd for
C21H20N2O4S2 [M+H]+: 429.09427; found: 429.09253.
4.2 Pharmacological protocols
4.2.1 Cell culture
HCT116, PC-3, HepG2, SK-OV-3, MCF-7, KB, K562 cell lines were from ATCC (ATCC,
Rockville, MD). KB/VCR, K562/A02, MCF-7/ADR were from Institute of Hematology and
Blood Disease HospitalꢂTianjin, CAMS). The cells were maintained in RPMI 1640 medium
supplemented with 10% FBS (fetal bovine serum), 1 mmol/L sodium pyruvate, 2 mmol/L
L-Glutamine. FBS was heat inactivated for 30 min at 56°C before use. Cell cultures were grown at
37°C, in a humidified atmosphere of 5% CO2, in a CO2 incubator.
4.2.2 Cell viability analysis
All cells used in the research were prepared at 3.5×104 cells/mL concentration and each 100 mL
cells suspension was seeded in 96-well cell imcroplate for 24 h (37 ºC, 5% CO2). Then each
solution was added and incubated for another 72 h. For the control group, equivalent concentration
of DMSO (final concentration 0.5%) was added. MTT (3-[4,5-dimethylthiazol-2yl]-diphenyl
tetrazolium bromide) method was employed to measure the number of surviving cells and
recorded the OD value at 492nm/620 nm. The IC50 values were calculated using Prism Graphpad
software of the triplicate experiment.
4.2.3 Tubulin polymerization assay
Microtubule-associated protein-rich tubulin (2 mg/mL, bovine brain, Cytoskeleton) in buffer
containing 80 mM PIPES (pH 6.9), 2 mM MgCl2, 0.5 mM EGTA, and 5% glycerol was placed in
cuvettes, 200 ꢀL/assay, and incubated respectively with DMSO, 1 ꢀM compound BA-3b and
BA-3g, 3 ꢀM Nocodazole, and 10 ꢀM Taxol, respectively. Polymerization was started by adding 1
mM GTP and incubating at 37 °C, followed by absorption readings at 340 nm with a Varian Cary
50 series spectrophotometer.
4.2.4 Tubulin intensity assay
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A549 cells were treatment for 24h in the presence of 200 nM of BA-3b and BA-3g, 50 nM
vinblastine, and stained for α-tubulin (green) with tubulin primary antibody and dylight488
secondary antibody, and nucleus (blue) with Hoechst 33258, images were taken by a fluorescence
microscopy.
4.2.5 SW480 cell apoptosis analysis
The effect of compound on apoptosis was assessed by flow cytometer. The human cancer cells
SW480 were cultured in 60 mm dishes to 70%-80% confluence. After treated with DMSO, BA-3b
for 24 hours, all the cells were harvested and fixed by 70% alcohol. Then the fixed samples were
washed twice by cold PBS buffer, incubated in staining solution (5μg/ml propidium iodide, 100
μg/ml RNase, 0.2% Triton X-100) at 37 °C for 30 min and analyzed by flow cytometry.
Acknowledgment
We gratefully acknowledge the National Natural Science Foundation of China (NSFC No.
81273370, 81573272) for generous financial support.
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X Y
R
this work
our previous work
R
N
N
N
O2S
O2S
O2S
O
O
N
O
N
N
1
target compounds
2
Figure 1. New tubulin-targeting drug design
Scheme 1. Reagents and conditions: a) PhI(OAc)2, TfOH, cyclopropanecarbonitrile, DCE, 80
Chlorosulfonic acid, DCM, 50 C; c) Pyridine, DCM, rt, 2h.
°C; b)
°
Scheme 2. a) m=1, 1,2-dibromoethane, K2CO3/DMF, 60
BA-1, pyridine, DCM, rt, 2-3h.
°Cꢀm=2, 1,3-dibromopropane, K2CO3/DMF, 60°C; b)
.
Scheme 3. aꢁFeCl3 6H2O, TBHP(70%), pyridine, 82
°C; b) NaBH4/MeOH, 0
°C; c) triethylamine,methanesulfonyl
.
chloride, CH2Cl2, then NaN3, DMF; d) BF3 OEt2, anhydrous CH2Cl2. -78
°C, then NaBH4, NaOH(15% aqueous).
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Scheme 4. The route of introduce amine: a) Et3N, HCOOH, Pd/C, EtOH, 80°C
Scheme 5. The route of introduce tert-amine: a) Sarcosine, DMSO, 180°C; b) LiAlH4, THF, 0°C; c) BA-1,
pyridine, DCM.
Scheme 6. Synthesis of sulfone and sulfoxide from sulfide: a) 2.10 equiv mCPBA, DCM, r.t; b) 1.05 equiv
mCPBA, DCM, r.t
Table 1. IC50 values of fist-run compounds over four cancer cell linesa.
IC50(µM)a
Comd No.
HCT116
0.040
0.088
0.071
0.045
0.049
PC3
HepG2
0.040
0.027
0.058
0.048
0.029
SK-OV-3
0.036
0.049
0.047
0.039
0.027
0.034
0.077
0.049
0.038
BA-3a
BA-3k
BA-4a
BA-4b
1
0.047
aMTT assays were used for evaluation, and values were expressed as mean IC50 of the triplicate experiment.
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Table 2. Antiproferliferative effects of benzoxazepane derivatives
IC50(μM)a
Compd No.
HCT116 PC3
HepG2 SK-OV-3
BA-3b
BA-3c
BA-3d
BA-3e
BA-3f
BA-3g
BA-3h
BA-3i
BA-3j
BA-3l
BA-3m
BA-3n
0.024
0.047
0.029
0.133
0.039
0.030
0.223
0.766
0.145
0.187
0.072
0.087
0.018 0.036 0.015
0.014 0.049
0.053
0.034
0.053
0.019
0.028
0.185
0.179
0.120
0.175
0.038
0.056
0.041 0.031
0.119 0.149
0.018 0.028
0.042 0.065
0.271 0.229
0.241 0.795
0.072 0.071
0.137 0.242
0.039 0.073
0.033 0.100
aMTT assays were used for evaluation, and values were expressed as mean IC50 of the triplicate experiment.
Table 3. Antiproferliferative effects of benzothiazepine and benzodiazepine derivatives
IC50(μM)a
Compd No.
HCT116 PC3
HepG2 SK-OV-3
BA-3k
BA-3p
BA-3q
BA-3o
0.088
0.263
>1.00
0.256
0.034 0.027 0.049
5.882 2.174
1.616
>1.00
0.199
>1.00 >1.00
0.347 0.525
a MTT assays were used for evaluation, and values were expressed as mean IC50 of the triplicate experiment.
Table 4. IC50 values of compounds in three MDR and drug-sensitive parental cell lines
IC50(ꢀM)± standard deviationa
Compd No.
K562
0.008
K562/A02 KB
0.013 0.007
±0.002 ±0.002 ±0.007 ±0.008
0.015 0.012 0.006 0.012
±0.004 ±0.003 ±0.002 ±0.007
0.141 1.289 0.140 2.940
±0.019 ±1.138 ±0.097 ±0.257
0.036 0.871 0.029 0.308
±0.034 ±0.366 ±0.003 ±0.037
KB/V MCF-7 MCF-7/A
0.007 0.021 0.020
BA-3b
BA-3g
Dox
±0.002 ±0.002
0.014 0.046
±0.004 ±0.007
0.006 5.961
±0.001 ±0.478
0.004 0.926
±0.002 ±0.132
VCR
a MTT assays were used for evaluation, and values were expressed as mean IC50 of the triplicate experiment.
Dox: doxorubicinꢃVCR: Vincristine.
K562/A02: K562 resistant to Dox; KB/V: KB resistant to VCR; MCF-7/A: MCF-7 resistant to Dox.
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Figure 2. Tubulin polymerization in the presence of BA-3b, BA-3g, nocodazole, and taxol
Figure 3. Tubulin intensity assay (DMSO as control, Vin for vinblastine)
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Figure 4. BA-3b induce apoptosis in SW480 cells
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Highlights
ꢀ
ꢀ
ꢀ
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A series of novel sulfonamide derivatives were synthesized.
The antiproliferative activities against four cancer cell lines were determined.
These compounds exert antitumor effect via tubulin depolymerization.
BA-3b was very potent against both drug-sensitive and MDR cancer cell lines.