Design, synthesis, biological evaluation and cocrystal structures with tubulin of chiral β-lactam bridged combretastatin A-4 analogues as potent antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2017.12.004

A diverse of chiral β-lactam bridged analogues of combretastatin A-4 (CA-4), 3-substituted 1,4-diaryl-2-azetidinones, were asymmetrically synthesized and biologically evaluated, leading to identify a number of potent anti-proliferative compounds represent

Full text of DOI:10.1016/j.ejmech.2017.12.004

Accepted Manuscript
Design, synthesis, biological evaluation and cocrystal structures with tubulin of chiral
β-lactam bridged combretastatin A-4 analogues as potent antitumor agents
Pengfei Zhou, Yuru Liang, Hao Zhang, Hao Jiang, Kechang Feng, Pan Xu, Jie Wang,
Xiaoming Wang, Kuiling Ding, Cheng Luo, Mingming Liu, Yang Wang
PII:
S0223-5234(17)31010-3
10.1016/j.ejmech.2017.12.004
EJMECH 9982
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 August 2017
Revised Date: 28 November 2017
Accepted Date: 2 December 2017
Please cite this article as: P. Zhou, Y. Liang, H. Zhang, H. Jiang, K. Feng, P. Xu, J. Wang, X. Wang,
K. Ding, C. Luo, M. Liu, Y. Wang, Design, synthesis, biological evaluation and cocrystal structures with
tubulin of chiral β-lactam bridged combretastatin A-4 analogues as potent antitumor agents, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.004.
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ACCEPTED MANUSCRIPT
Graphical Abstract
A series of 3-substituted-1,4-diaryl-2-azetidinones were asymmetrically synthesized
and SARs revealed that the stereochemistry were critically important for the
antiproliferative activity.

ACCEPTED MANUSCRIPT
Design, Synthesis, Biological Evaluation and
Cocrystal Structures with Tubulin of Chiral
β-Lactam Bridged Combretastatin A-4 Analogues
as Potent Antitumor Agents
†
,§,1
†,1
‡,1
‡
†
‡
Pengfei Zhou,
Yuru Liang, Hao Zhang, Hao Jiang, Kechang Feng, Pan Xu, Jie
†
§
,§
,‡,
⊥
,†
,†
Wang, Xiaoming Wang, Kuiling Ding,* Cheng Luo,* Mingming Liu,* Yang Wang*
†
School of Pharmacy, Fudan University, Shanghai 201203, China
‡
Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
⊥
CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China
§
State Key Laboratory of Organometallic Chemistry, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
These authors contributed equally to this work.
1
ABSTRACT
A diverse of chiral β-lactam bridged analogues of combretastatin A-4 (CA-4), 3-substituted
1
,4-diaryl-2-azetidinones, were asymmetrically synthesized and biologically evaluated,
leading to identify a number of potent anti-proliferative compounds represented by 14b and
4c with IC values of 0.001-0.021 µM, against four human cancer cell lines (A2780, Hela,
1
5
0
1

ACCEPTED MANUSCRIPT
SKOV-3 and MDA-MB-231). Structure-activity relationship (SAR) studies on all
stereoisomers of 14b and 14c revealed that the absolute configurations of the chiral centers at
3
- and 4-position were critically important for the activity and generally a trans configuration
between the “A” and “B” rings is optimal. In addition, 14b and 14c displayed less
cytotoxicity on normal human oviduct epithelial cells than malignant cells indicating good
selectivity in vitro. Further biochemical evaluation and cocrystal structures with tubulin
demonstrated that both compounds disrupted tubulin polymerization through interacting at
the colchicine-binding site, suppressed angiogenesis in vitro and in vivo, blocked cell cycle
progression at mitotic phase and induced cellular apoptosis. The in vivo assays verified that
both compounds inhibited xenograft tumor growth in nude mice with acceptable therapeutic
window, showing promising potentials for further clinical development.
Keywords: 1,4-diaryl-2-azetidinone; combretastatin A-4; tubulin polymerization; antitumor
2

ACCEPTED MANUSCRIPT
1. Introduction
Microtubule is an important component of cytoskeleton, which is polymerized by α- and
β-subunit. Microtubule plays a role of separating the daughter chromosomes to the opposite
poles during the mitosis. The disruption of microtubule will result in the interruption of
mitosis and lead to the apoptosis of cell [1]. After Colchicine is the first discovered
compound which binds to the dimer and destabilizes microtubule [2], Combretastatin A-4
(CA-4, 1, Figure 1) is a natural product isolated from Combretum caffrum which can bind to
the colchicine binding-site, and shows extremely strong depolymerization of microtubule in
vitro [3]. Its water soluble phosphate prodrug CA-4P (2), is now in phase III clinical trials
[
4]. Since the cis-double bond is critically important for the activity of 1 and the spontaneous
isomerization from cis-isomer to trans-isomer leads to its chemical instability [5], a large
number of conformationally restricted analogues of CA-4 have been reported, in which the
cis double bond is replaced by a heterocycle [6]. Among them, β-lactam bridged analogues of
CA-4 are the most attractive and a number of substituted 1,4-diaryl-2-azetidinones have been
reported with significant suppressions on tubulin polymerization and cellular proliferation
[
7]. Due to two chiral carbon atoms of azetidin-2-one scaffold and the existence of two pairs
of enantiomers, accumulating studies have been focused on the relationship between
stereo-chemical structure and anticancer activity. The trans-form of C-3 and C-4 turned out
to be favor by the work of Meegan and Coccetti groups [7d,7f]. But the absolute
configurations of chiral centers remained unclear until compounds 3/4 were synthesized
asymmetrically by our group and firstly identified the favorable orientation of phenyl at
4
-position on β-lactam ring which demonstrated critically important for the antiproliferative
3

ACCEPTED MANUSCRIPT
activity [8]. In this study, we would like to report a series of optically pure β-lactam linked
CA-4 analogues to define their SARs systematically, especially the influence of the absolute
configurations of C-3 and C-4 on the activities.
Figure 1. Small molecules that interact with tubulin.
2
. Results and discussion
.1 Chemistry
A series of chiral 3-methylene-2-azetidinones were synthesized asymmetrically according
2
to literature method [9] (Scheme 1). Racemic MBH adducts 6 were obtained by
Morita-Baylis-Hillman reaction of corresponding aromatic aldehydes 5 with ethyl acrylate in
the presence of DABCO in 3-14 days, depending on the reactivity of the carbonyl group of
aldehydes. After protecting the hydroxyl groups of 6 with acetic anhydride, 7 were submitted
to the asymmetric allylic amination using aromatic amines 8 as the nucleophiles under the
catalysis of Pd (dba) and 9a, affording the corresponding optically active amination products
2
3
1
0 in high yields (up to 94%) and enantioselectivities (up to 99% ee). Finally 10 can be
readily transformed into their corresponding β-lactam derivatives 11a-r in good yields
4

ACCEPTED MANUSCRIPT
(
37-92%) without loss of enantioselectivity by reaction with Sn[N(TMS) ] in refluxing
2
2
toluene.
a
Scheme 1. Synthesis of (S)-1,4-Diaryl-3-methylene-2-azetidinones 3 and 11a-r
a
o
Reagents and conditions: (a) DABCO, ethyl acrylate, MeOH, 25 C, 3-14 days, 37-99%;
o
(
b) Ac O, Et N, CH Cl , 0 C, 1 h, 37-91%; (c) i) Zn, AcOH, MeOH, reflux, 30 min; ii)
2 3 2 2
Boc O, THF, reflux, 16 h, 41% for 2 steps; (d) Pd (dba) (1%), 8a~e (1.5 equiv), 9a (2.5%),
2
2
3
o
K CO (aq.), CH Cl , 25 C, 3 h, 52-94%; (e) Sn[N(TMS) ] , toluene, reflux, 3 h, 37-92%,
5-99% ee; (f) Et N, acryloyl chloride, CH Cl , 25 C, 1 h, 76%; (g) TBAF, THF, 0 C, 1 h,
3 2 2
1-90%.
2
3
2
2
2 2
o
o
9
8
5

ACCEPTED MANUSCRIPT
Previous research has indicated clearly that the C-3 substituent is relatively flexible
6b,7a,7c,7d,8,10]. With 3-methylene-2-azetidinones in hand, modifications of 3-methylene
[
group allowed us to further examine the role of 3-substituent as well as the cis/trans/absolute
configurations of azetidin-2-ones on their antiproliferative effects. Accordingly, a diversity of
derivatives of 3 were designed and synthesized, and these transformations could be readily
realized with 3-methylene as latent functional group (Schemes 2-4).
Various hydroxyl-protected compounds 12a-h were obtained by acylation or alkylation of
3
with corresponding acyl chlorides or alkyl halides in good yields (Scheme 2). Then olefin
cross-metathesis of 3 were performed under the catalysis of Grubbs II to give 3-benzylidene
substituted compounds 13a-b, and the newly formed C=C bonds were identified as Z
configurations as indicated by NOESY. The Michael additions of 3 or 11g with
corresponding nucleophiles afforded 3-hydroxymethyl or aminomethyl substituted target
compounds 14a-j. The trans-diastereoisomer of 4, 14c and 14g could be obtained by
bromination of 14b or 14e followed by hydrogenation under the catalysis of Pd/C in the
presence of AcONa.
a
Scheme 2. Synthesis of Azetidinones 12-14
6

ACCEPTED MANUSCRIPT
OMe
N
OMe
OMe
N
MeO
MeO
MeO
MeO
MeO
MeO
O
O
O
a
N
b
R'
3
, R = OH
MeO
MeO
MeO
MeO
R
R
c
R
11g, R = NH2
1
1
1
1
1
1
1
1
2a, R = OMe
2b, R = OEt
2c, R = OPr-n
2d, R = OPr-i
2e, R = OBn
2f, R = OAc
2g, R = OTs
13a, R = OH, R' = Ph
13b, R = OH, R' = 4-t-BuPh
OMe
N
1
1
1
1
1
4a, R = R' = OH, cis-(3S,4R)
4b, R = R' = OH, trans-(3R,4R)
4c, R = OH, R' = H, trans-(3S,4R)
4d, R = NH , R' = OH, cis-(3S,4R)
4e, R = NH , R' = OH, trans-(3R,4R)
d
e
MeO
O
2h, R = OSO NH2
2
2
2
14f, R = NH , R' = H, cis-(3R,4R)
2
1
4g, R = NH , R' = H, trans-(3S,4R)
R'
2
1
4h, R = OMe, cis-(3S,4R)
MeO
R
14i, R = NMe , cis-(3R,4R)
2
14j, R = NHBn, cis-(3R,4R)
a
Reagents and conditions: (a) acyl chlorides, anhydrides or alkyl halides, Et N or K CO ,
3
2
3
CH Cl , 78-97%; (b) styrene or 1-(tert-butyl)-4-vinylbenzene (2.5 equiv), Grubbs II (15%
2
2
equiv), 1,2-dichloroethane, reflux, 48 h, 30-51%; (c) for 14a and 14b: i) B (pin) (1.3 equiv),
2
2
o
CuCl (5%), MeOH (1.5 equiv), PPh (15%), t-BuOLi (10%), THF, 25 C, 12 h; ii)
3
o
NaBO ·4H O, H O, THF, 25 C, 2 h, 46% and 21% for 2 steps; for 14d and 14e: i) B (pin)
3
2
2
2
2
o
(
1.3 equiv), CuCl (5%), MeOH (1.5 equiv), PPh (15%), t-BuOLi (10%), THF, 25 C, 12 h;
3
o
ii) NaBO ·4H O, H O, THF, 25 C, 2 h, 42% and 17% for 2 steps; for 14f: Pd/C, H , EtOH,
3
2
2
2
o
o
2
5 C, 12 h, 89%; for 14h: MeONa, MeOH, 25 C, 12 h, 66%; for 14i: Me NH·HCl, Et N,
2 3
MeOH, reflux, 6 h, 75%; for 14j: BnNH , EtOH, reflux, 12 h, 30%; (d) i) BnCl, K CO ,
2
2
3
o
o
MeCN, reflux, 12 h; ii) CBr , PPh , THF, 25 C, 6 h; iii) Pd/C, AcONa, H , EtOH, 25 C, 12
h, 21% for 3 steps; e) i) Boc O, Et N, THF, reflux, 24 h; ii) CBr , PPh , THF, 25 C, 6 h; iii)
Pd/C, AcONa, H , EtOH, 25 C, 12 h; iv) TFA, DCM, 25 C, 1 h, 18% for 4 steps.
4
3
2
o
2
3
4
3
o
o
2
In order to further diversify the substitution of C-3, the dihydroxylation of 12e proceeded
under the catalysis of K OsO ·2H O with 4-methylmorpholine N-oxide (NMO) as the
2
4
2
oxidant, yielding 3-hydroxy-3-hydroxymethyl substituted compound 15, whose configuration
was determined by NOESY (Scheme 3). The direct catalytic hydrogenolysis or methylation
followed by catalytic hydrogenolysis of 15 provided products 16a-b. Oxidation with NaIO4
and subsequent reduction with NaBH of 15 afforded cis-(3R,4S)-3-hydroxy substituted
4
7

ACCEPTED MANUSCRIPT
1
analogue 17, whose configuration was determined as indicated by H NMR. The alkylation or
esterification followed by the debenzylation of 17 produced 3-alkoxy or 3-acyloxy
substituted analogues 18a-b. The sulfonylation of 17 with mesyl chloride gave 3-mesyloxy
substituted product 19 in 86% yield.
a
Scheme 3. Synthesis of Azetidinones 15-19
a
o
Reagents and conditions: (a) K OsO ·2H O (3%), NMO (50%, aq.), acetone, H O, 25 C,
2
4
2
2
o
1
2 h, 92%; (b) for 16a: Pd/C, H , EtOH, 25 C, 12 h, 96%; for 16b: i) Me SO , K CO ,
2 2 4 2 3
acetone, reflux, 6 h; ii) Pd/C, H , EtOH, 25 C, 12 h, 73% for 2 steps; (c) i) NaIO , MeOH,
o
2
4
o
o
H O, 25 C, 4 h; ii) NaBH , MeOH, 0 C, 1 h, 85% for 2 steps; (d) for 18a: i) Me SO ,
2
4
2
4
o
K CO , acetone, reflux, 6 h; ii) Pd/C, H , EtOH, 25 C, 12 h, 82% for 2 steps; for 18b: i)
2
3
2
o
Succinic anhydride (1.2 equiv), DIPEA, DMAP, CH Cl , 25 C, 6 h; ii) Pd/C, H , EtOH, 25
C, 12 h, 45% for 2 steps; (e) MsCl, Et N, CH Cl , 0 C, 1 h, 86%.
2
2
2
o
o
3
2
2
Nucleophilic substitution of 19 with tetrabutylammonium bromide (TBAB) under the
condition of microwave yielded trans-(3S,4S)-3-bromo substituted product 20 and its
8

ACCEPTED MANUSCRIPT
1
configuration was identified as indicated by H NMR (Scheme 4). Debenzylation of 20 with
H catalyzed by Pd/C resulted in the expected product 21 and the further debrominated
2
product 22 in the yields of 62% and 28%, respectively. Nucleophilic substitution of 19 with
o
NaN at 120 C in DMF provided trans-(3S,4S)-3-azido substituted product 23, whose
3
1
configuration was confirmed by H NMR. Then 23 was submitted to azide-alkyne click
reaction with propargyl alcohol afforded 3-(1,2,3-triazol-1-yl) substituted analogue 24.
Reduction of 23 with SnCl produced trans-(3S,4S)-3-amino substituted compound 25.
2
Derivatization of the amino of 25 with acid anhydride or acyl chloride gave the 3-acylamino
or 3-sulphonylamino substituted analogues 26a-c.
a
Scheme 4. Synthesis of Azetidinones 20-26
OMe
OMe
OMe
N
OMe
N
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
O
N
O
O
O
R
N
d
c
a
b
N
19
N
N
N3
Br
MeO
MeO
MeO
MeO
OH
OBn
23
OBn
20
OH
HO
24
21, R = Br
2, R = H
2
e
OMe
OMe
N
MeO
MeO
MeO
MeO
O
O
f
N
NH2
NHR
MeO
MeO
OBn
OH
25
26a, R = Ac
26b, R = COPh
26c, R = Ms
a
o
Reagents and conditions: (a) TBAB (3.5 equiv), DMF, microwave, 170 C, 6 h, 42%; (b)
o
o
Pd/C, H , EtOH, 25 C, 8 h, 62% for 21 and 28% for 22; (c) NaN ( 1.5 equiv), DMF, 120 C,
2
3
2
4 h, 53%; (d) i) CuSO ·5H O (5%), L-ascorbic acid sodium salt (15%), propargyl alcohol
4 2
o
o
(1.1 equiv), EtOH, H O, 25 C, 8 h; ii) Pd/C, H , EtOH, 25 C, 12 h, 93%; (e) SnCl (3
2 2 2
9

ACCEPTED MANUSCRIPT
equiv), 9% HCl (aq.), MeOH, reflux, 4 h, 64%; (f) i) acid anhydride or acyl chloride, DIPEA,
o
o
CH Cl , 0 C, 1 h; ii) Pd/C, H , EtOH, 25 C, 12 h, 58-99%.
2
2
2
The synthesis of (R)-1,4-diaryl-3-methylene-2-azetidinone 29 was achieved by the similar
method of 3, with 9b rather than 9a as the ligand (Scheme 5). The C-3 modified analogues
3
0a-d were synthesized just as the corresponding enantiomers 14a-c and 4, respectively.
a
Scheme 5. Synthesis of Azetidinones 28-30
a
o
Reagents and conditions: (a) Pd (dba) , 9b, K CO (aq.), CH Cl , 25 C, 3 h, 96%; (b)
2
3
2
3
2
2
o
Sn[N(TMS) ] , toluene, reflux, 6 h, 91%, 99% ee; (c) TBAF, THF, 0 C, 1 h, 71%; (d) for
0a and 30b: i) B (pin) , CuCl, MeOH, PPh , t-BuOLi, THF, 25 C, 12 h; ii) NaBO 4H O,
2 2 3 3 2
2
2
o
.
3
o
H O, THF, 25 C, 2 h, 52% and 19% for 30a and 30b respectively; for 30d: 10% Pd/C,
2
o
EtOH, H , 25 C, 12 h, 92%; (e) i) BnCl, K CO , MeCN, reflux, 12 h; ii) CBr , PPh , THF,
5 C, 6 h; iii) 10% Pd/C, AcONa, EtOH, H , 25 C, 12 h, 20% for 3 steps.
2
2
3
o
4
3
o
2
2
2
.2 Biological results and discussion
2.2.1 Activities of Anti-proliferation in Vitro. The newly synthesized chiral β-lactam
analogues of CA-4 were screened for anti-proliferative activities against four human cancer
cell lines A2780, Hela, SKOV-3, and MDA-MB-231 with CA-4 (1) and paclitaxel （PTX）
as positive control. As summarized in Table 1, most of target compounds display moderate to
1
0

ACCEPTED MANUSCRIPT
potent inhibition on cell growth. Among them, compounds 11g, 11h, 12f, 14b, 14c, 14e, 14g,
2
1, 22, 24, 26a and 26b exhibited considerable activities with IC values less than 100 nM,
50
therein compound 14c exhibited similar activity with the referenced agents CA-4 (1) and
PTX. In addition, we also tested the cytotoxicity of 14b and 14c in a normal ovarian
epithelial cell line HOSE, and the result shows that both compounds achieve less 50%
inhibition ratio at 1 µM, indicating the low toxicity of 14b and 14c against normal cells.
Table 1. Antiproliferative Activities on Different Cell Lines
a
IC (µM)
5
c
0
Compound
b
d
e
A2780
> 20
> 20
0.127
> 20
0.121
0.316
0.031
0.065
MDA-MB-231
> 20
SKOV-3
> 20
> 20
0.265
> 20
0.136
0.341
0.036
0.063
14.490
0.365
0.414
1.388
> 20
Hela
1
1a
> 20
> 20
0.209
> 20
0.131
0.371
0.032
0.125
15.021
0.547
0.657
2.172
> 20
1
1
1
1
1
1
1
1
1b
1c
1d
1e
1f
1g
1h
1j
1k
> 20
0.193
> 20
0.112
0.512
0.040
0.034
6.643
0.784
0.560
1.368
> 20
10.472
0.215
0.287
0.677
> 20
1
11l
1
1
1n
1p
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1r
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
4a
4b
4c
0.112
0.854
0.854
3.417
11.330
> 20
0.081
> 20
0.133
2.545
> 20
0.125
1.018
0.958
5.623
15.930
> 20
0.133
12.845
0.240
3.165
> 20
0.175
2.260
1.135
5.257
18.030
> 20
0.104
12.080
0.177
2.988
> 20
0.295
3.157
2.032
6.888
> 20
> 20
0.116
17.785
0.224
4.732
> 20
0.358
0.021
0.001
0.220
0.014
0.002
0.182
0.016
0.003
0.408
0.021
0.003
1
1

ACCEPTED MANUSCRIPT
1
1
1
1
4d
4e
4f
4g
0.327
0.035
0.081
0.021
1.772
1.753
1.021
1.545
17.723
1.221
0.079
0.008
0.022
0.015
0.033
0.027
0.164
>20
0.107
0.027
0.063
0.017
4.466
2.648
1.791
2.323
> 20
1.956
0.102
0.010
0.028
0.028
0.047
0.047
0.254
>20
0.054
0.328
0.044
0.215
0.035
3.419
2.559
2.269
2.099
> 20
1.794
0.178
0.013
0.030
0.033
0.035
0.024
0.192
>20
0.008
0.059
0.008
2.995
2.472
1.666
2.074
> 20
2.212
0.210
0.009
0.024
0.083
0.032
0.045
0.231
>20
1
4h
1
1
4i
4j
1
1
1
1
6a
6b
8a
8b
2
2
2
1
2
4
2
2
2
3
3
3
6a
6b
6c
0a
0b
0c
0.364
0.771
5.752
0.006
0.001
0.383
1.082
5.137
0.008
0.003
0.206
0.650
3.270
0.012
0.002
0.332
0.807
3.918
0.010
0.003
3
0d
1
PTX
a
The anti-proliferation activities of individual compound to tumor cells were determined by
b, d
the MTT assay. The data are the mean of triplicate determinations. A2780 and SKOV-3 are
human ovarian cancer cell lines. MDA-MB-231 is a human breast cancer cell line. Hela is a
human cervical carcinoma cell line.
c
e
2.2.2 Structure−Activity Relationships Study. According to the activities mentioned
above, the preliminary SARs of these β-lactam analogues can be summarized.
Comparing the data of 4 and 14a-g with 30a-d (Scheme 6), S-configuration of C-4 is
critically important for the activity, which showed the same conclusion as the reported
literature [8]. 3-Hydroxymethyl substituted products 14b and 14e with trans-(3R,4R)
configuration displayed about 4-20 fold more potent than cis-(3S,4R) diastereoisomers 14a
and 14d. 3-Methyl substituted compounds 14c and 14g with trans-(3S,4R) configuration also
displayed about 4-63 fold more potent than cis-(3R,4R) diastereoisomers 4 and 14f.
1
2

ACCEPTED MANUSCRIPT
Analogues (21, 24, 26a-b) with trans-orientation also showed excellent antiproliferative
activities. In conclusion, trans-orientation of substituents at 3,4-positions of β-lactam scaffold
benefit the activity, which coincide with the results reported by Coccetti and Meegan groups
[
7d,7f].
Scheme 6. The anti-proliferative activities of 3-hydroxymethyl and 3-methyl substituted
stereoisomers.
OMe
N
OMe
N
OMe
N
OMe
N
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
O
O
O
O
Me
Me
HO
HO
MeO
MeO
MeO
MeO
R
OH
R
OH
cis-(3S,4R), R = OH, 14a: 182-408 nM cis-(3S,4S), 30a: >20000 nM
trans-(3R,4R), R = OH, 14b: 14-21 nM trans-(3R,4S), 30b: 206-383 nM trans-(3S,4R), R = OH, 14c: 1-3 nM trans-(3S,4S), 30c: 650-1082 nM
cis-(3S,4R), R = NH , 14d: 54-328 nM cis-(3R,4R), R = NH , 14f: 59-215 nM
cis-(3R,4R), R = OH, 4: 31-63 nM
cis-(3R,4S), 30d: 3270-5752 nM
2
2
trans-(3R,4R), R = NH
2
, 14e: 8-44 nM
trans-(3S,4R), R = NH
2
, 14g: 8-35 nM
Many of 3-methylene substituted analogues (11e, 11g, 11h) showed good activities, but
-(Z)-benzylidene analogues (13a-b) were inactive, indicating that 3-methylene is tolerant
3
but bulky steric hindrance in the same plane is forbidden. The 3,4,5-trimethoxyphenyl turned
out to be the best choice for N-1 substitution, which is consistent with CA-4 and previous
research results. The analogue with 3,5-dimethoxy substituted N-phenyl ring (11r) exhibited
a little loss of activity, whereas 11n and 11p with only one MeO- group on their N-phenyl
rings showed considerable loss in potency. The 4-methoxy of the phenyl ring at C-4 was very
important for the activity and the lack of 4-methoxy (11a, 11b, 11j) resulted in loss of
activity, whereas the 3-hydroxy of 4-phenyl ring can tolerate various arrangements. Removal
or replacement of 3-hydroxy of 4-phenyl ring with other bioisosteres, such as F, Cl and NH2,
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as well as the acylation of 3-hydroxy can maintain the activity (11e, 11f, 11g, 11h, 12f, 12h).
In the cases of the alkylated derivatives (12a-e) of 3-hydroxy at 4-phenyl ring, the bulkier
steric hindrance of alkyl group the less potent the activity is.
2.2.3 Disruption of Tubulin Assembly. Representative compounds 14b and 14c were
tested for inhibitory effects on tubulin assembly, using isolated pig brain tubulin. As shown in
Figure 2A, 14b and 14c suppressed in vitro tubulin polymerization in a dose-dependent
manner, compared to the solvent (DMSO) control. When incubated with 6 µM of the two
compounds, tubulin polymerization was almost completely inhibited. The IC values were
5
0
listed in Table 2. Both compounds exhibited stronger effects than positive control Colchicine.
Figure 2. (A) Effects of compounds 14b and 14c on tubulin polymerization in vitro. (B)
1
4b (500 nM) and 14c (500 nM) induce depolymerization of tubulin in HeLa cells. The
supernatant faction (S) contains unpolymerized tubulin and the pellet fraction (P) contains
polymerized tubulin. DMSO (0.2%) was used as negative control. (C) Immunofluorescence
assay of SKOV-3 cells shows the effect of compounds 14b and 14c on the organizations of
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cellular microtubule network. CA-4 (1) served as tubulin depolymerized control, whereas
PTX was used as tubulin stabilized control. DMSO (0.2%) was used as negative control.
Scale bars: 20 µm.
Table 2. IC Values of 14b and 14c on Inhibition of Tubulin Polymerization
5
0
Compounds
14b
3.5 ± 0.1
14c
1.7 ± 0.0
Colchicine
8.6 ± 0.9
a,b
IC (µM)
5
0
a
b
IC50 values were calculated by inhibition ratio of 9 concentrations of each compound.
The values present as mean ± SD from at least two independent experiments.
Next, the effects of 14b and 14c on the inhibition of cellular tubulin assembly were also
determined. After treatment with 14b, 14c, 1 or PTX respectively, the cells were lysed with
detergents, and centrifuged. The pellet (P, polymerized tubulin) and supernatant (S,
depolymerized tubulin) fraction were collected and analyzed by western blotting (Figure 2B
and S1). Treatment of 1 significantly decreased polymerized microtubules while treatment of
PTX increased polymerized tubulin in Hela cells. As expected, cells treated with the two
tested compounds showed dramatically reduction of polymerized tubulin.
Furthermore, we employed cellular immunofluorescence assay to visualize the effects of
β-lactam analogues 14b and 14c on microtubule structure. As shown in Figure 2C, when
treated with solvent (DMSO) control, the microtubule network (green) of SKOV-3 cells were
slim and hair-like, distributing around nucleus (blue). However, when treated cells with 1,
1
4b or 14c, the fibrous microtubule structures were disorganized and their density was also
significantly reduced. In contrast, cells treated with PTX exhibited a spindle shaped
microtubule network and the density of microtubule was significantly increased [11]. Taken
together, these results clearly demonstrated that our β-lactam bridged CA-4 analogues
strongly inhibit tubulin assembly in both purified protein and cancer cells.
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2.2.4 Anti-angiogenic Effects in Vitro and in Vivo. Targeting angiogenesis has been one
of the strategies for increasing the efficacy of cancer therapeutics. Since many tubulin
binding agents, including CA-4, have been reported to interfere with vascular formation [12],
1
4b and 14c were evaluated for their effects against angiogenesis in vitro and in vivo in this
study. In capillary-like tube formation assay, vascular endothelial cells could form
capillary-like structures on matrigel [13], an extracellular matrix rich in proangiogenic
factors. As shown in Figure 3A, the HUVEC cells only treated with diluent (DMSO)
produced a complete network structure. However, both 14b and 14c could effectively alter
the tubule-like structures after 12 h incubation. At the concentration of 50 nM, 14c almost
completely disrupted the capillary-like tube formation.
Furthermore, the in vivo anti-vascular activities of 14b and 14c were also tested using
matrigel plug assay [14]. In this assay, matrigel supplemented with human VEGF (100
ng/mL) was mixed with 14b (100 nM), 14c (100 nM) or diluent (DMSO), followed by
injection into nude mice subcutaneously. One week later, the growth of capillary blood vessel
in the plug was massively induced by VEGF, when matrigel was only mixed with diluent
(Figure 3B upper line). However, the existence of compound 14b or 14c attenuated the
VEGF-induced capillary blood vessel in the matrigel plug. The relative level of angiogenesis
was also presented by hemoglobin content in the matrigel plug. As shown in Figure 3B lower
line, the levels of hemoglobin in matrigel were reduced by 14b and 14c treatment compared
with diluent treated control. Collectively, these results demonstrated that 14b and 14c
suppressed VEGF-induced angiogenesis in vitro and in vivo.
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Figure 3. Effects of compounds 14b and 14c on angiogenesis in vitro and in vivo. (A) 14b
and 14c disrupted capillary-like tubule formed by HUVECs. CA-4 (1) served as positive
control. (B) Matrigel plug assay showed that 14b and 14c inhibited growth of capillary
vascular in nude mice.
2.2.5 Inhibition of Colony Formation and Cell Cycle Arrest. In the following
experiments, we further confirmed the cytotoxic effects of 14b and 14c by colony formation
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assay. As shown in Figure 4A and 4B, the colonies formed by MDA-MB-231 cells were
dose-dependently reduced by the exposure to 14b and 14c.
To further characterize the inhibitory effects of 14b and 14c on cell growth, cell cycle
progression was analyzed by quantitating DNA content, since chemical agents generally
decrease growth and proliferation of cancer cells through altering the regulation of cell cycle
[
15]. As expected, results showed that, similar to the positive compound 1, both compounds
4b and 14c arrest Hela cells in the G2/M phase (Figure 4C and S2). In the DMSO treated
1
group, only about 24% of cells were distributed in the G2/M phase. However, after being
treated with 14c or 1 at the dose of 50 nM, nearly 100% of cells were arrested in G2/M phase.
Moreover, immunoblotting showed that 14b and 14c markedly up-regulated the expression of
cyclin B1, which is synthesized at G2-phase and reaches maximum level at metaphase. In
addition, 14b and 14c treatment also induced hyper-phosphorylation of the mitotic
checkpoint kinase BubR1 and mitotic trigger histone H3 in a dose-dependent manner (Figure
4
D). These results clearly demonstrate that, consistent with other reported microtubule
inhibitors [16], β-lactam analogues 14b and 14c significantly induce cellular mitotic arrest in
Hela cells.
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Figure 4. (A) Treatment with 14b and 14c does-dependently suppressed cell colony
formation of MDA-MB-231 cells. (B) Qualification of the numbers of colonies. (C) Cell
cycle distribution of HeLa cells after treatment with indicated concentrations of 14b and 14c
for 24 h. (D) Western blot analysis of mitosis regulated proteins. ** P<0.01; *** P<0.001.
2.2.6 Induction of Cellular Apoptosis. Numerous studies have demonstrated that tubulin
polymerization inhibitors possess the abilities to induce cellular apoptosis [17]. For this
reason, we assessed whether the active analogues 14b and 14c could induce apoptosis of Hela
cells by double staining with Annexin V-FITC and propidium iodide (PI). The flow
cytometric data (Figure 5A) showed the high activities of 14b and 14c on the induction of
cellular apoptosis. As qualified in Figure S3, the total proportions of apoptotic cells,
including Annexin V+/PI- (the right lower quadrant representing early apoptosis) and
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Annexin V+/PI+ (the right upper quadrant representing late apoptosis or necrosis), were
3
8.7%, 57.5% and 70.7% after treatment with 50, 100 and 200 nM of 14b, and were 52.7%,
5.1% and 71.3% after treatment with 50, 100 and 200 nM of 14c, respectively. Exposure to
6
positive control 1 (100 nM) leaded to totally 62.5% cell apoptosis, however, only 2.7%
apoptotic cells were detected in solvent (DMSO) treated group. Subsequently, we further
examined apoptosis associated proteins by immunoblotting analysis. As indicated in Figure
5
B, pro-apoptotic protein Bax and p53 was up-regulated by both 14b and 14c treatment in a
dose-dependent manner. In addition, both compounds also dose-dependently promoted
cleavage of poly(ADP-ribose) polymerase-1 (PARP-1), a marker of cells undergoing
apoptosis. These results implied that the β-lactam analogues exhibited their anti-tumor effects
through, at least partly, induction of cellular apoptosis.
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Figure 5. (A) Representative flow cytometry profiles showed that 14b and 14c
does-dependently induced apoptosis of Hela cells. (B) Western blot analysis of
apoptosis-related proteins. Cells were treated with indicated concentrations of 14b, 14c, 1 or
diluent (0.2% DMSO) for 48 h.
2.2.7 Anti-tumor Activities in Vivo. Firstly, we performed single-dose acute toxicity
assay to assess the safety of compounds 14b and 14c. ICR mice (n=10, half male and half
female) were injected intraperitoneally with various dosages of 14b (95, 70, 50, 35 and 25
mg/kg) or 14c (275, 200, 150, 125 and 100 mg/kg), or vehicle control. As summarized in
Table 3, none of the mice died in the two days after treatment with 14b, whereas the death of
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mice mainly occurred in the second day after treatment with 14c. With LD values of 136.5
5
0
kg/mg, 14c exhibited less toxicity than 14b, whose LD value was calculated as 61.5 mg/kg.
5
0
Table 3. Acute Toxicity of 14b and 14c in Mice
No. of dead mice
Dose
No. of
Total
death
Survival (%)
on day 14
Compd.
1
2
3
4
5-14
days
1
1
0
0
0
0
0
0
0
0
0
(mg/kg) mice
day days days days
9
7
50
3
2
5
0
10
10
10
10
10
10
10
10
10
10
10
0
0
0
0
0
2
1
2
0
0
0
0
0
0
0
0
4
4
4
2
0
0
6
4
1
1
0
2
2
0
1
1
0
1
2
0
0
0
1
0
0
0
0
0
8
7
1
1
0
9
7
6
3
1
0
20
30
90
90
100
10
30
40
70
90
14b
5
5
2
2
75
00
1
4c
150
1
1
25
00
vehicle
100
1
4b: LD = 61.5 mg/kg; 14c: LD = 136.5 mg/kg.
50 50
Next, we evaluated the in vivo anti-tumor effects of compounds 14b and 14c using human
ovarian cancer xenograft mice model, which is established by subcutaneous inoculation of
A2780 cells in the female Balb/C nude mice. After the mean value of tumor volumes reached
3
approximately 100 mm , the mice were randomly divided into 4 groups and administrated
with 7.5 mg/kg of 14b, 8 mg/kg of 14c, 10 mg/kg of PTX (as positive control) or vehicle
(negative control) by intraperitoneal injection once every two days. As shown in Figure 6A,
treatment of both 14b and 14c significantly suppressed the tumor growth, achieving 78.9%
and 76.3% reduction in tumor size at the end of the observation period, respectively. When
the observation time ended, the mice were sacrificed and the tumors were excised and
weighed. The results were illustrated in Figures 6B and 6D, and again confirmed the
anti-tumor activities of the two compounds. Slight weight loss was observed in 14b treated
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animals, but was not statistically significant (Figure 6C). Furthermore, H&E staining of
tumor sections showed extensive areas of necrosis or cell death (indicated by black arrow in
Figure 6E) in both 14b and 14c treated groups, however, none of abnormal areas were
observed in the vehicle treatment group. In addition, we also examined organs of agents
treated and vehicle treated mice, including liver, kidney and spleen, using H&E staining
(Figure 6F). No detectable abnormalities were observed in the organs examined, indicating
the safety of 14b and 14c at the therapeutic dosage. Taken together, considering its LD50
value, it could be proposed that compound 14c possesses a satisfied therapeutic window and
is worth for further validation.
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Figure 6. Effects of compounds 14b and 14c on xenografts tumor growth in nude mice. Nude
mice bearing tumors formed by A2780 cells were administered with 14b, 14c, PTX, or
vehicle (n = 10). Figures show the average tumor volumes (A), weights (B), the isolated
tumors (C) and body weights (D) of mice at the end of observation. (E) Representative
images of H&E-stained tumor sections show that administration of 14b and 14c induced
extensive areas of necrosis in xenografts tumors. (F) Representative images of H&E-stained
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organic sections. No obvious alternation was observed in the organs examined, including
liver, kidney and spleen after treatment with 14b and 14c. ** P<0.01; *** P<0.001.
2.2.8 The Cocrystal Structure of Tubulin in Complex with Compounds 14b and 14c.
To reveal the specific details of binding, as well as to provide solid basis for further structural
optimization, compounds 14b and 14c were soaked into the crystals of a protein complex
(T2R-TTL) consisting of two α,β-tubulin heterodimers, the stathmin-like protein RB3, and
the tubulin tyrosine ligase. Finally the co-crystal structures of these two compounds with
tubulin were determined at 2.56 Å for 14b (PDB code 5XAG) and 2.55 Å for 14c (PDB code
5
XAF), respectively (Figure 7). The data collection and refinement statistics was
summarized in Table S1. As expected, 14b and 14c occupied the binding site of colchicine
with satisfactory electron density. To determine the impact of ligand binding against the
overall conformation of tubulin, these two complex structures were subsequently
superimposed to the tubulin structure in the absence of ligand (PDB code 5JQG). The RMSD
values for the superimposed structures of 14b and 14c were 0.603 Å over 1892 Cα atoms and
0
.608 Å over 1917 Cα atoms, respectively, indicating that these two compounds did not
affect the global conformation of tubulin. However, the major conformational changes come
from two loops of tubulin near the binding site after ligand binding. Similar conformational
changes were also observed in the structure of tubulin with colchicine, indicating that 14b
and 14c may possess the same inhibitory mechanism as colchicine. In addition, as shown in
Figure S4, the conformations of these two loops were not the same in three structures,
suggesting that the flexibility of this binding site might be further exploited. Both
hydrophobic contacts and hydrogen bonds were observed in the structures of tubulin with
1
4b and 14c. As shown in Figure 7G, 14b formed a direct hydrogen bond with β-Ala250, as
2
5

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well as water-bridged hydrogen bonds with β-Asn349. However, the main driving force of
binding was thought to be shape matching between the compound and surrounding
hydrophobic residues, including the residues Leu242, Leu248, Ala250, Leu255, Met259,
Val315, Ala316, Ala317, Ile318 of β-tubulin. Notably, as shown in Figure 7I, 14b and 14c
were more deeply buried than colchicine in the binding site, which is consistent with their
better potency against tubulin polymerization. However, the acetamido group of colchicine
was not overlaid in the superimposed structures, providing us a new possible direction for
further structural modification.
Figure 7. Crystal structures of the tubulin-14b and tubulin-14c complex.
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6

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7

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(A) Overall view of the complex formed between 14b and tubulin. α-tubulin is shown as
green cartoon, and β-tubulin is shown as cyan cartoon. 14b is shown in yellow sphere
representation (PDB code 5XAG). (B) Overall view of the complex formed between 14c and
tubulin. α-tubulin is shown as green cartoon, and β-tubulin is shown as cyan cartoon. 14c is
shown in yellow sphere representation (PDB code 5XAF). (C) 2Fo-Fc electron density (blue)
for 14b, and the contour level is set to 1.0 sigma. (D) 2Fo-Fc electron density (blue) for 14c,
and the contour level is set to 1.0 sigma. (E) 14b binds to the colchicine site of β-tubulin,
which is shown in surface representation. (F) 14c binds to the colchicine site of β-tubulin,
which is shown in surface representation. (G) Close-up views of the hydrogen bonds and
hydrophobic contacts formed between 14b (yellow sticks) and tubulin. Hydrogen bonds are
shown in yellow dashed lines, and corresponding distances were labeled in angstrom.
Interacting residues of tubulin are shown in white stick representation, and crystal waters are
shown in red sphere representation. (H) Close-up views of the hydrogen bonds and
hydrophobic contacts formed between 14c (yellow sticks) and tubulin. Hydrogen bonds are
shown in yellow dashed lines, and corresponding distances were labeled in angstrom.
Interacting residues of tubulin are shown in white stick representation, and crystal waters are
shown in red sphere representation. (I) Superimposition of 14b (cyan stick, PDB code
5
XAG) and 14c (green stick, PDB code 5XAF) with colchicine (salmon stick, PDB code
4
O2B).
3. Conclusion
In summary, a diverse of chiral β-lactam bridged combretastatin A-4 analogues have been
synthesized and biologically evaluated. Most of the target compounds displayed moderate to
potent anti-proliferative activities against four human cancer cell lines (A2780, Hela,
SKOV-3 and MDA-MB-231). The studies on SARs revealed that the absolute configurations
of the chiral C-4 were critically important for the activity, more specifically,
(S)-configuration for 3-methylene substituted series (11) and the same orientation for other
analogues. On this basis, trans-configuration of substituents at 3,4-positions of β-lactam
scaffold benefit the antiproliferative activity. Among all synthesized compounds, 14b and
1
4c turned out to be most potent and were selected for further pharmacological studies after
comprehensive consideration. The co-crystal structures of tubulin in complex with 14b and
4c were determined by X-ray crystallography, which showed that they bind to the same site
1
2
8