Journal Pre-proof

Design, synthesis, and biological evaluation of novel benzodiazepine derivatives as

anticancer agents through inhibition of tubulin polymerization in vitro and in vivo

Yanqing Pang, Haibiao Lin, Caiwen Ou, Yingying Cao, Baijiao An, Jun Yan, Xingshu
Li

PII: S0223-5234(19)30814-1
DOI: https://doi.org/10.1016/j.ejmech.2019.111670
Reference: EJMECH 111670

To appear in: European Journal of Medicinal Chemistry

Received Date: 11 July 2019

Revised Date: 20 August 2019
Accepted Date: 30 August 2019

Please cite this article as: Y. Pang, H. Lin, C. Ou, Y. Cao, B. An, J. Yan, X. Li, Design, synthesis, and
biological evaluation of novel benzodiazepine derivatives as anticancer agents through inhibition of
tubulin polymerization in vitro and in vivo, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111670.

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© 2019 Published by Elsevier Masson SAS.

Design, synthesis, and biological evaluation of novel

benzodiazepine derivatives as anticancer agents through

inhibition of tubulin polymerization in vitro and in vivo

Yanqing Panga, Haibiao Linb, Caiwen Oub, Yingying Caoa, Baijiao Anc, Jun Yanb*, Xingshu Lic*
a
Department of Phase I Clinical Research Center, The Second Affiliated Hospital of Guangzhou
University of Chinese Medicine, Guangzhou 510006, China
b
Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou University of
Chinese Medicine, Guangzhou 510120, China
c
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China

ABSTRACT:

A series of novel structurally-related tubulin polymerization inhibitors based on

benzodiazepine were designed, synthesized, and evaluated for anticancer activity.

Extensive structure modifications were performed to investigate the detailed structure

and activity relationships (SARs). Most compounds exhibited potent antiproliferative

activity against a panel of cancer cell lines. Among these compounds, the optimal

compound, 9a, possessed the most superior activity, including cytotoxicity against

five cancer cell lines (IC50 = 6 ~ 15 nM) and inhibition of tubulin polymerization

(IC50 = 1.65 ± 0.11 µM). Mechanistic studies revealed that 9a could disrupt

intracellular microtubule organization, arrest cell cycle at the G2/M phase and

eventually induce cell apoptosis. Compound 9a exhibited good metabolic stability with

a t1/2 of 161.2 min, which was much better than the reference compound CA-4.

1
Moreover, the disodium salt of 9a, 9a-P, exhibited excellent in vivo antitumor activity

in xenograft mice model with inhibitory rate of 89.3%, which was better than the

reference compounds CA-4P (inhibitory rate: 52.8%) and Y-01P (inhibitory rate:

77.7%). Altogether, 9a could serve as a promising lead compound for the

development of highly efficient anticancer agents.

Keywords: tubulin polymerization inhibitors, benzodiazepine derivatives,

antiproliferative activity, cell cycle arrest, apoptosis, in vivo antitumor activity.

INTRODUCTION

Microtubules are key components of the cytoskeleton and play a pivotal role in

essential cellular processes including intracellular transportation, organelle

movements, and cell shape formation and maintenance.[1-3] Microtubules mainly

consist of α- and β-tubulin, which are involved in the tubulin dynamics and have

become a well-validated target for the development of clinically effective anticancer

drugs.[4-7] Some anticancer agents derived from natural products are well-known

tubulin polymerization disrupters via promoting or inhibiting tubulin polymerization.

For instance, Taxol, promotes microtubule over-aggregation to exert its anticancer

activity, and it is currently used in the treatment of ovarian and breast cancer, while

colchicine, dolastatins, and vinca alkaloids exert their drug effects via inhibiting

microtubule assembly.[8, 9] However, these natural products and their derivatives

2
have limited clinical application due to the complicated and difficult synthesis routes,

substantial cytotoxicity, and rapidly acquired drug resistance.[7]

Among the large class of anti-tubulin agents derived from natural products,

combretastatin A-4 (CA-4), isolated from the African willow tree Combretum caffrum,

is one of the most active small molecule compounds that has been well-studied in the

past decades. Through acting on the intracellular tubulin-microtubule system, CA-4

exerted the outstanding antitumor activity in vitro and in vivo. Its disodium phosphate

salt, CA-4P, is approved as an orphan drug for the treatment of anaplastic thyroid

cancer.[10, 11] However, some defects, such as the isomerization from Z-isomer to

more thermodynamically stable but inactive E-isomer, and poor solubility have

largely limited its clinical application.[12] Therefore, a drug discovery campaign

aimed at improving the intrinsic stability and retaining the superior anticancer activity

of CA-4 has been extensively carried out in the last few decades.[13]

Isocombretastatin A-4 (isoCA-4), replacing the olefinic bridge of CA-4 with 1,1-

diarylethylene, turned out to the promising anti-tubulin agent with comparable

activity and more stable structure to that of CA-4.[14] Thus, in previous work, our

group fused the anticancer selenium element with isoCA-4 to obtain the optimal

compound 1, which exhibited better antitumor activity than isoCA-4 in vivo.[15] On

the other hand, through introducing Millepachine group, a natural product founded to

3
display the anticancer activity, into CA-4, our group also designed and synthetized the

tubulin polymerization inhibitors 2 and 3, which possessed good anticancer activity

against a panel of cancer cell lines.[16-18] Meantime, our group designed a series of

cycle CA-4 derivatives to maintain its Z-configuration. As shown in Figure 1,

although all of these compounds could maintain Z-configuration, the benzodiazepine

derivative 6 exhibited best anticancer activity.[19-21] On basis of our previous

research work, it can be concluded that the 3-hydroxy-4-methoxy group is most

beneficial for activity in B ring (Figure 2). Inspired by previous works mentioned

above, we fused the benzodiazepine moiety of Y-01 with compound 1, 2, 3, and

extensive structure modifications have also been made to yield a novel series of

tubulin polymerization inhibitors. Detailed structure and activity relationships (SARs)

were also obtained to provide instructive guidance for further research work. Further

biological evaluation and mechanism studies were also performed in vitro and in vivo.

Figure 1. Structure of cycle CA-4 derivatives

4
Figure 2. Design strategy of novel tubulin polymerization inhibitors

RESULTS AND DISCUSSION

Chemistry

The synthetic route of the selenium-containing hybrids 9a-b is summarized in Scheme

1. Commercially available compound 5-bromo-2,3-dimethoxyaniline (1) was

diazotized by hydrochloric acid and sodium nitrite, followed by a reaction with

potassium selenocyanante to obtain 2, which was subsequently reacted with sodium

borohydride and methyl iodide to yield intermediate 3.[15] Then 3 was reacted with

4-methoxy-3-(methoxymethoxy)-2-nitrobenzaldehyde to afford diarylmethanol

derivative 4 in the presence of n-BuLi.[22] The oxidation reaction of 4 with 2-

iodoxybenzoic acid provided intermediate 5, which was subsequently reduced by

Na2S2O4 to produce intermediate 6. Intermediate 6 was reacted with BrCH2COBr or

CH3COBr to obtain compounds 7a or 7b, following cyclization to produce the ring

−b were
intermediates 8a or 8b, respectively.[19] Finally, target compounds 9a−

−b in good yields.
obtained by the deprotection reaction of 8a−

Scheme 1. Synthesis of compounds 9a−ba．

5
a
Reagents and reaction conditions: (a) NaNO2, 10% HCl, KSeCN; (b) NaBH4, CH3I, EtOH; (c) n-BuLi, anhydrous

THF; (d) IBX, THF; (e) Na2S2O4, CH3COCH3, H2O; (f) BrCH2COBr or CH3COBr, Et3N, anhydrous CH2Cl2 (g)

NH3.H2O/CH3CH2OH or CH3ONa/DMF; (h) HCl, CH3OH.

To investigate the effects of different substituting groups on the A ring to activity, a

series of compounds with different substituents were synthesized using similar

−b.
procedures as 9a−

Firstly, sulfur-containing analogues were synthesized. The total synthetic route was

similar to the selenium-containing analogues, except for the introduction way of

sulfur atom. 5-Bromo-2,3-dimethoxyaniline (1) was chosen as the starting material,

diazotized by hydrochloric acid and sodium nitrite, then reacted with sodium

thiomethoxide to produce the sulfur-containing intermediate 10.[23] Target

−d were subsequently synthesized using the same procedures as

compounds 16a−

described above in Scheme 1 (Scheme 2).

Scheme 2. Synthesis of compounds 16a−da

6
a
Reagents and reaction conditions: (a) NaNO2, 10% HCl, CH3SNa; (b) n-BuLi, anhydrous THF; (c) IBX, THF; (d)

Na2S2O4, CH3COCH3, H2O; (e) BrCH2COBr, BrCH3CHCOBr, CH3COBr or CH3CH2COBr, Et3N, anhydrous

CH2Cl2; (f) NH3.H2O/CH3CH2OH or CH3ONa/DMF; (g) HCl, CH3OH.

Secondly, the N,N-dimethyl group was introduced into the A ring. The total synthetic

route was similar to the selenium-containing analogues, except for the introduction

way of the N,N-dimethyl group. The synthesis started with 5-bromo-2,3-

dimethoxyaniline (1), which was reacted with sodium hydride and methyl iodide to

afford N,N-dimethyl-containing intermediate 17.[24] Target compound 23 was

subsequently synthesized using the same procedures as described above in Scheme 1

(Scheme 3).

Scheme 3. Synthesis of compound 23a

7
a
Reagents and reaction conditions: (a) CH3I, NaH, DMF; (b) n-BuLi, anhydrous THF; (c) IBX, THF; (d) Na2S2O4,

CH3COCH3, H2O; (e) BrCH2COBr, Et3N, anhydrous CH2Cl2; (f) NH3.H2O, CH3CH2OH; (g) HCl, CH3OH.

In addition, we also introduced a 3,6-dihydro-2H-pyran group into the A ring. The

total synthetic route was similar to the selenium-containing analogues, except for the

introduction way of the 3,6-dihydro-2H-pyran. 4-Bromo-2-methoxyphenol (24) was

chosen as the starting material and reacted with 3-chloro-3-methylbut-1-yne to afford

intermediate 25. In the presence of pyridine, 25 was converted to 3,6-dihydro-2H-

pyran-containing intermediate 26 via intramolecular cyclization. Subsequently, target

compound 32 was synthesized using the same procedures as described above in

Scheme 1 (Scheme 4).

Scheme 4. Synthesis of compound 32a

8
a
Reagents and reaction conditions: (a) 3-chloro-3-methylbut-1-yne, DBU, CuCl2, CH3CN; (b) Pyridine; (c) n-BuLi,

anhydrous THF; (d) IBX, THF; (e) Na2S2O4, CH3COCH3, H2O; (f) BrCH2COBr, Et3N, anhydrous CH2Cl2; (g)

NH3.H2O, CH3CH2OH; (h) HCl, CH3OH..

Finally, we also moved the 3,6-dihydro-2H-pyran in the A ring from 4,5-position to

5,6-position. As shown in Scheme 5, compound 41 was synthesized using the same

procedures as described above in Scheme 4, except for the starting material, which

was changed from 4-bromo-2-methoxyphenol to 2-bromo-5-methoxyphenol.

Scheme 5. Synthesis of compound 41a

9
a
Reagents and reaction conditions: (a) 3-chloro-3-methylbut-1-yne, DBU, CuCl2, CH3CN; (b) Pyridine; (c) n-BuLi,

anhydrous THF; (d) IBX, THF; (e) Na2S2O4, CH3COCH3, H2O; (f) BrCH2COBr, Et3N, anhydrous CH2Cl2; (g)

NH3.H2O, CH3CH2OH; (h) HCl, CH3OH.

Considering the bioavailability of drugs largely depends on their water solubility, 9a-

P and 16a-P, the disodium phosphate salts of 9a and 16a, respectively, were

synthesized for the in vivo tests. As shown in Scheme 6, 9a and 16a were esterified

with diethyl phosphite to afford intermediates 9aa and 16aa, which were subsequently

deprotected in trimethylsilyl bromide and neutralized with NaOH to give the target

compounds 9a-P and 16a-P.

Scheme 6. Synthesis of compounds 9a-P and 16a-Pa

a
Reagents and reaction conditions: (a) Diethyl phosphite, Et3N, CCl4, CH2Cl2; (b) TMSBr, anhydrous CH2Cl2; (c)

NaOH, MeOH.

10
BIOLOGY

Antiproliferative activities and SARs

The in vitro antiproliferative activities of these novel compounds were first evaluated

against a panel of five different cancer cell lines: A549 (human non-small cell lung

carcinoma); MDA-MB231 (human breast carcinoma); HepG2 (human hepatoma

carcinoma); Hela (human cervical carcinoma) and HCT116 (human colorectal

carcinoma). As shown in Table 1, 9a, with a methylselanyl group that replaced the 3-

methoxyl group of Y-01, exhibited pronounced activity with IC50 values ranging from

6 to 15 nM, which was more potent than the prototype compound Y-01 (IC50 = 9 ~ 54

nM). To examine the more detailed structure-activity relationships (SARs),

modifications toward A ring and benzodiazepine ring were performed. Firstly,

substituting the methylselanyl group with thiol group (16a, IC50 = 10 ~ 24 nM) or

N,N-dimethyl group (23, IC50 = 52 ~ 91 nM) resulted in a decrease to the activity.

Secondly, replacing the 3,4,5-trimethoxybenzene group with different substituted

rings has also played a vital role to the activity. Specifically, when the 3-methoxyl

group in A ring of Y-01 was kept unchanged and the 4,5-dimethoxy group was

substituted by 3,6-dihydro-2H-pyran, the obtained compound 32 still retained potent

antiproliferative activity with IC50 values ranging from 13 to 69 nM. However, the

activity decreased drastically (41, IC50 = 433 ~ 4588 nM) by moving the 3,6-dihydro-

11
2H-pyran ring from 4,5-position to 5,6-position, which demonstrated the importance

of the substituent position in A ring. Otherwise, the activities decreased when the

seven-membered benzodiazepine ring was reduced to six-membered amide ring (9b

vs 9a, 16c vs 16a). Meantime, introducing a methyl group into the methylene of the

benzodiazepine ring was unfavorable for the activity (16b vs 16a). Moreover, the

consistent effect on the antiproliferative activity was displayed by introducing the

methyl group in the amide ring (16d vs 16c). The detailed SARs were summarized in

Figure 3. Concluded from the results of structure and activity relationships above, 9a

turned out to be the most active compound toward five cancer cell lines.

Table 1. Antiproliferative Activities of Compounds Against Human Cancer Cell Linesa

IC50 (nM)b
Compd.
A549 Hela HepG2 HCT116 MDA-MB231
9a 5.82 ± 0.3 8.3 ± 0.5 12.6 ± 0.5 8.9 ± 0.3 15.5 ± 0.3

9b 60.7 ± 7.4 51.7 ± 1.4 22 ± 6.1 124 ± 16.9 69 ± 5.9

16a 18.7 ± 4.3 16.5 ± 1.8 14.2 ± 1.4 10.8 ± 0.3 24.7 ± 0.2

16b 93 ± 7.0 1068 ± 16.0 295 ± 23.0 89 ± 9.0 295 ± 14

16c 61.8 ± 1.4 35.5 ± 2.2 51.7 ± 7.7 28.8 ± 4.1 70 ± 1.6

16d 86 ± 2.6 158 ± 3.4 303 ± 19 229 ± 5.1 133 ± 3.1

12
 23 66.2 ± 6.1 54.9 ± 1.2 52 ± 5.0 53 ± 4.0 91 ± 5.0

32 56.2 ± 1.8 34.7 ± 6 30.5 ± 5.4 13.9 ± 1.5 69 ± 1.6

41 433 ± 6.7 1874 ± 8.1 4588 ± 5.3 207 ± 9.7 3709 ± 93.3

Y-01 9.15 ± 0.6 18.1 ± 0.2 15.7 ± 1.3 16.6 ± 0.3 54.6 ± 4.7

a
Cell lines were treated with compounds for 48 h. Cell viability was measured using the MTT assay as described in

the Supporting Information. bIC50 values are indicated as the means ± SD (standard error) of at least three

independent experiments.

Figure 3. Summarized SARs of synthesized compounds

Inhibition of tubulin polymerization

To elucidate whether 9a could inhibit tubulin polymerization in vitro, 9a was

incubated with unpolymerization tubulin using the method originally described by D.

Bonne et al. with some modifications.[25, 26] As presented in the Figure 4, after 9a

was incubated with tubulin at various concentrations (0.75~7.5 µM), the increased

tendency of the fluorescence intensity was obviously slowed down as compared with

the control, which indicated that 9a could inhibit the tubulin polymerization. In

addition, as the concentration increases, the fluorescence intensity decreases gradually.

13
Therefore, 9a could inhibit microtubule polymerization in a dose-dependent manner,

with an IC50 value of 1.65 ± 0.11 µM.

Figure 4. Compound 9a inhibited microtubule polymerization. Purified tubulin protein at 10 mM in a reaction

buffer was incubated at 37°C in the absence (control) or presence of 9a at the indicated concentrations (ranging

from 0.75 to 7.5 µM). The experiments were performed three times, and the results of representative experiments

are shown.

Disruption of microtubule organization

The inhibition effect of 9a toward microtubule organization in living cells was

measured using immunofluorescence assay. As shown in Figure 5, the microtubule

network in A549 cells exhibited normal arrangement with slim and fibrous

microtubules wrapped around the cell nucleus in control group. When treated with 9a

at indicated concentration after 24 h, the microtubule spindle shrunk and was heavily

disrupted. Especially when the concentration was increased to 10 nM, the microtubule

organization was completely disrupted and a number of dotted disorder formations

14
were easily observed. These phenomena indicated 9a could induce the disruption of

microtubule, which might eventually lead to cell cycle disorder.

Figure 5. Compound 9a disrupted the organization of the cellular microtubule network at indicated concentrations.

A549 cells were plated in confocal dishes and incubated with DMSO or 9a at 1, 5, 10 nM for 24 h, followed by

direct microscopy. The detection of the fixed and stained cells was performed with an LSM 570 laser confocal

microscope (Carl Zeiss, Germany). The experiments were performed three times, and the results of representative

experiments are shown.

Cell cycle arrest

As 9a could disrupt the normal organization of spindle, the arrest effect of 9a on the

cell cycle distribution was measured using flow cytometry analysis. As shown in

Figure 6A, most cells were in G1 phase in vehicle group. However, when treated with

9a at indicated concentration after 24 h, the population of cells in G2/M phase

dramatically increased. Especially when the concentration was increased to 10 nM,

almost all cells were arrested in G2/M phase. After the incubation time was increased

to 48 h, these phenomena were more obvious (Figure 6B). These results indicated that

9a could effectively arrest cells in the G2/M phase in a dose- and time-dependent

manner. Moreover, the characteristic hypodiploid DNA content peak (Sub-G1 phase)

15
could been obviously observed after cells incubated with 9a after 48 h, which

indicated 9a might eventually lead to cell apoptosis.

Figure 6. Cell cycle arrest effect of 9a. A549 cells were treated with compound 9a at 1, 5, or 10 nM for 24 h (A)

or 48 h (B), trypsinized and harvested for PI-stained DNA content using flow cytometry. The experiments were

performed three times, and the results of representative experiments are shown.

Cell apoptosis

As the appearance of Sub-G1 phase was observed in cell cycle assay, it was

hypothesized that 9a might induce apoptosis of A549 cells. To test this hypothesis,

A549 cells were incubated with 9a at different concentration and different time. As

shown in Figure 7A, when the cells were incubated with 9a at 1, 5, and 10 nM for 24

h, the percentages of early and late apoptosis cells were 5.88%, 13.47%, and 23.19%,

respectively, being elevated from the control group (0.21%). After 48 h incubation,

the percentages of early and late apoptosis cells were strikingly increased to 16.89%,

40.39%, and 84.55% with the incubated concentrations of 1, 5, and 10 nM,

16
respectively (Figure 7B). These results indicated that 9a could profoundly induce cells

apoptosis in a dose- and time-dependent manner.

Figure 7. Compound 9a induced A549 cell apoptosis. A549 cells were treated with compound 9a at 1, 5, or 10 nM

for 24 h (A) or 48 h (B). The percentages of cells in each stage of cell apoptosis were quantified using flow

cytometry: (upper left quadrant) necrotic cells; (upper right quadrant) late-apoptotic cells;(bottom left quadrant)

live cells; and (bottom right quadrant) early apoptotic cells. Total apoptosis cell percentages were obtained using

EXPO32 ADC analysis software. The experiments were performed three times, and the results of representative

experiments are shown.

Metabolic stability

Considering metabolic stability is an important part of drug-like properties, the assay

of 9a incubated with rat liver microsomes in buffer solution containing NADPH

regenerating system was performed. CA-4 and Y-01 were used as reference

compounds. As shown in Table 2, the remaining percentages of 9a at each time point

were higher than that of CA-4 and approximate to that of Y-01. After 180 min

incubation, After 180 min incubation, 50.4% and 49.9% of 9a and Y-01, respectively,

still remained, while CA-4 was almost undetectable due to its fast metabolic rate. A

17
short half-life of drug might lead to low bioavailability in vivo and require frequent

administration at a high dose, resulting in the drug accumulation and related toxicity.

The half-lives of 9a (t1/2 = 161.2 min), Y-01 (t1/2 = 157.5 min), and CA-4 (t1/2 < 60

min) indicated that 9a possessed a comparable metabolic stability to that of Y-01,

being greater than CA-4, which encouraged us to carry out further studies in vivo.

Table 2. Metabolic Stability of 9a, CA-4, and Y-01.

Remains %
Compd
Time (min) 30 60 90 120 150 180 t1/2

9a 95.6% 86.3% 75.9% 66.1% 57.4% 50.4% 161.2

CA-4 66.3% 36.4% 21.6% 17.9% NTa NTa NDb

Y-01 94.3% 85.5% 76.2% 65.3.% 55.2% 49.9% 157.5

a
NT: not tested because of the fast metabolic rate of CA-4. bND: not determined.

In vivo antitumor effect

In light of the potent antitumor activity of 9a and 16a in vitro, the in vivo antitumor

effects were evaluated in nude mouse A549 xenograft models established via the

subcutaneously injecting A549 cells in the logarithmic phase into the right armpit of

the mice. Considering the bioavailability of drugs largely depends on their water

solubility, 9a-P and 16a-P, the disodium phosphate salts of 9a and 16a, respectively,

were synthesized as prodrugs for the in vivo test. After nude mouse A549 xenograft

models were established, mice were randomly divided to five groups with six of each

(vehicle-treated, CA-4P-treated, Y-01P-treated, 9a-P-treated, and 16a-P-treated

groups). When the tumor volume grew to 100-200 mm3 in each group, 9a-P, 16a-P,

18
Y-01P, and CA-4P were intraperitoneally injected at a dose of 30 mg/kg to the mice

every other day for the entire observation period. As shown in Figure 8, 9a-P, 16a-P,

Y-01P, CA-4P and vehicle groups gave 215.2, 369.25, 408.17, 984.86 and 2006.37

mm3 of the mean final tumor volumes at the end of 26 days, respectively. The average

tumor weights of the 9a-P-treated and 16a-P-treated groups were 0.201 g (inhibitory

rate: 89.3%) and 0.373 g (inhibitory rate: 79.3%), respectively, comparing with 1.894

g in the vehicle group, which were less than that of CA-4P-treated group (0.879 g,

inhibitory rate: 52.8%) and Y-01-treated group (0.415 g, inhibitory rate: 77.7%).

These results indicated that the selenium-containing compound 9a possessed more

potent antitumor activity compared with the sulfur-containing compound 16a and

oxygen-containing compound Y-01. It also can be seen from Figure 8E that 9a-P-

treated and 16a-P-treated groups have no change in body weight at 26 days,

comparing with vehicle group.

19
Figure 8. Efficacy of 9a-P and 16a-P against A549 xenografts. (A, B) Images of sacrificed mice and excised
tumors in each group. (C) The tumor volumes of mice in each group during observation period. (D) The weights of

the excised tumors in each group. (E) The body weights of the mice in each group at the end of 26 days. The data
were presented as the means ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, significantly different compared with the
control using ANOVA, n = 6. Data were not in conformity with Mauchly’s test of sphericity, and

Greenhouse−Geisser was performed for correction.

CONCLUSIONS

In summary, we reported the design, synthesis, and biological evaluation of a series of

novel tubulin polymerization inhibitions based on benzodiazepine. Most compounds

20
exhibited potent antiproliferative activity against a panel of five cancel cell lines with

IC50 values below the submicromolar concentration range. Among them, 9a,

represented the most active compound with IC50 values of 6−15 nM against five

cancer cell lines and 1.65 ± 0.11 µM toward tubulin polymerization inhibition.

Moreover, 9a could disrupt microtubule network in living cancer cells, arrest cell

cycle at G2/M phase and induce apoptosis in a dose- and time-dependent manner.

Metabolic assay indicated that 9a possessed good metabolic stability with a t1/2 of

161.2 min, being greater than reference compound CA-4. Notably, its phosphate salt,

9a-P exhibited potent antitumor effect in A549 xenograft model in vivo, being better

than the reference compounds CA-4P and Y-01P. All of these results highlighted 9a as

a promising lead compound for further investigation in anticancer drug development.

EXPERIMENT SECTION
Chemistry

General Methods. Reagents used in the synthesis were obtained commercially and

used without further purification, unless otherwise specified. Flash column

chromatography was performed using silica gel (200−300 mesh). The purity of the

compounds was determined by high-performance liquid chromatography (HPLC),

which was carried out on an Agilent 1200 series system with a TC-C18 column (4.6

mm × 250 mm, 5 µm). The compounds were eluted with a CH3OH/H2O (60:40)

mixture at a flow rate of 1 mL/min, and were detected at a wavelength of 254 nm. The

21
purity of all biologically evaluated compounds is greater than 95%. Melting points

were measured by an SRS-Opti melting point apparatus. 1H NMR and 13

C NMR

spectra were recorded using TMS as the internal standard on Bruker BioSpin GmbH

spectrometer (400, 100 MHz and 500, 125 MHz). The high-resolution mass spectra

(HRMS) were obtained using a Shimadzu LCMS-IT-TOF mass spectrometer.

Preparation of (5-bromo-2,3-dimethoxyphenyl)(methyl)selane (3) Intermediate 3 was

synthesized as the method we reported in reference 15. Colorless oil, 89% yield. 1H

NMR (400 MHz, Chloroform-d) δ 6.88 (q, J = 2.2 Hz, 2H), 3.84 (s, 3H), 3.83 (s, 3H),

2.26 (s, 3H).

Preparation of (5-bromo-2,3-dimethoxyphenyl)(methyl)sulfane (10) To a stirred

solution of 1 (232 mg, 1mmol) in 10% HCl (2mL) at 0 °C, sodium nitrite (83 mg, 1.2

mmol) was added and the reaction was maintained below 5 °C. After completion of

the reaction, the mixture was added dropwise to sodium thiomethoxide aqueous

solution (1.5 mmol). The reaction mixture was stirred at 50 °C for 3 h, then extracted

with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, and

concentrated under vacuum. The residue was purified by flash column

chromatography (PE/EtOAc, 20:1) to give 10. Colorless oil, 69% yield. 1H NMR (400

MHz, Chloroform-d) δ 6.97 – 6.63 (m, 2H), 3.84 (s, 3H), 3.82 (s, 3H), 2.40 (s, 3H).

22
Preparation of 5-bromo-2,3-dimethoxy-N,N-dimethylaniline (17) To a stirred solution

of 1 (232 mg, 1mmol) in THF (10mL) at 0 °C, NaH was added slowly, followed by

addition of CH3I dropwise after 5 min. The reaction mixture was stirred at rt for 3 h,

then extracted with ethyl acetate. The organic phase was dried over anhydrous sodium

sulfate, and concentrated under vacuum. The residue was purified by flash column

chromatography (PE/EtOAc, 20:1) to give 17. Yellow oil, 68% yield. 1H NMR (400

MHz, Chloroform-d) δ 6.79 – 6.58 (m, 2H), 3.83 (s, 3H), 3.77 (s, 3H), 2.82 (s, 6H).

General Procedures for the Preparation of 25 and 34.

DBU (304 mg, 2 mmol), 3-chloro-3-methylbut-1-yne (124 mg, 1.2 mmol) and CuCl2

(14 mg, 0.1 mmol) were added successively to a stirred solution of 24 (203 mg, 1

mmol) or 33 (203 mg, 1 mmol) in CH3CN (10 mL), respectively. The reaction

mixture was stirred at rt for 3 h, acidified by 10% HCl to adjust pH to 2.0, then

extracted with ethyl acetate. The organic phase was dried over anhydrous sodium

sulfate, and concentrated under vacuum. The residue was purified by flash column

chromatography (PE/EtOAc, 20:1) to give the products.

4-bromo-2-methoxy-1-(2-methylbut-3-yn-2-yloxy)benzene (25) White solid, 82% yield.

1
H NMR (400 MHz, Chloroform-d) δ 7.18 (dd, J = 7.5, 2.0 Hz, 1H), 7.11 (d, J = 2.0

Hz, 1H), 6.94 (d, J = 7.5 Hz, 1H), 3.77 (s, 3H), 2.57 (s, 1H), 1.76 (s, 6H).

23
1-bromo-4-methoxy-2-(2-methylbut-3-yn-2-yloxy)benzene (34) White solid, 82% yield.

1
H NMR (400 MHz, Chloroform-d) δ 7.40 (d, J = 8.8 Hz, 1H), 7.27 (d, J = 2.9 Hz,

1H), 6.51 (dd, J = 8.8, 2.9 Hz, 1H), 3.78 (s, 3H), 2.63 (s, 1H), 1.70 (s, 6H).

General Procedures for the Preparation of 26 and 35.

43 (269 mg, 1 mmol) or 52 (269 mg, 1 mmol) were added slowly to pyridine (10 mL)

under stirring, respectively. The reaction mixture were stirred at 80 °C for 12 h, then

concentrated under vacuum and purified by flash column chromatography (PE/EtOAc,

10:1) to give the products.

6-bromo-8-methoxy-2,2-dimethyl-2H-chromene (26) White solid, 84% yield. 1H NMR

(500 MHz, Chloroform-d) δ 7.36 (dd, J = 2.0, 1.1 Hz, 1H), 7.02 (d, J = 2.0 Hz, 1H),

6.54 (dd, J = 10.9, 1.1 Hz, 1H), 5.43 (d, J = 11.0 Hz, 1H), 3.82 (s, 3H), 1.86 (s, 6H).

8-bromo-5-methoxy-2,2-dimethyl-2H-chromene (35) White solid, 78% yield. 1H NMR

(400 MHz, Chloroform-d) δ 7.25 (d, J = 8.8 Hz, 1H), 6.63 (d, J = 9.9 Hz, 1H), 6.32 (d,

J = 8.8 Hz, 1H), 5.59 (d, J = 9.9 Hz, 1H), 3.80 (s, 3H), 1.46 (s, 6H).

General Procedures for the Preparation of 4, 11, 18, 27, and 36.

Under an argon atmosphere, 3 (403 mg, 1.3 mmol), 10 (341 mg, 1.3 mmol), 17 (338

mg, 1.3 mmol), 26 (350 mg, 1.3 mmol), or 35 (350 mg, 1.3 mmol) was dissolved in

anhydrous THF (20 mL), respectively, and then the solutions were cooled to -78 °C.

N-BuLi (1.3 mmol) was added to the mixture in dropwise and stirred for 0.5 h. Then

4-methoxy-3-(methoxymethoxy)-2-nitrobenzaldehyde (241 mg, 1 mmol) in THF (2.5

24
ml) was added in dropwise. After stirred for 12 h, the reaction was quenched by

saturated ammonium chloride solution, and extracted with ethyl acetate. The organic

phase was dried over anhydrous sodium sulfate, and concentrated under vacuum. The

residue was purified by flash column chromatography (PE/EtOAc, 5:1) to give the

products.

(3,4-dimethoxy-5-(methylselanyl)phenyl)(4-methoxy-3-(methoxymethoxy)-2-

nitrophenyl)methanol (4) White solid, 60% yield. 1H NMR (400 MHz, Chloroform-d)

δ 7.23 (d, J = 1.5 Hz, 2H), 6.92 (ddd, J = 20.1, 2.0, 1.1 Hz, 2H), 6.62 (d, J = 1.1 Hz,

2H), 5.95 (dd, J = 5.0, 1.1 Hz, 1H), 5.23 (s, 2H), 3.90 (s, 6H), 3.85 (s, 3H), 3.50 (s,

3H), 2.22 (s, 3H).

(3,4-dimethoxy-5-(methylthio)phenyl)(4-methoxy-3-(methoxymethoxy)-2-

nitrophenyl)methanol (11) Yellow solid, 60% yield. 1H NMR (400 MHz, Chloroform-

d) δ 7.06 (d, J = 8.7 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 6.74 (q, J = 1.9 Hz, 2H), 5.83

(d, J = 3.7 Hz, 1H), 5.23 (s, 2H), 3.87 (s, 3H), 3.84 (d, J = 3.2 Hz, 6H), 3.50 (s, 3H),

2.39 (s, 3H).

(3-(dimethylamino)-4,5-dimethoxyphenyl)(4-methoxy-3-(methoxymethoxy)-2-

nitrophenyl)methanol (18) Light yellow solid, 61% yield. 1H NMR (400 MHz,

Chloroform-d) δ 7.10 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 8.8 Hz, 1H), 6.63 – 6.46 (m,

25
2H), 5.81 (d, J = 3.6 Hz, 1H), 5.18 (d, J = 2.3 Hz, 2H), 3.87 (s, 3H), 3.81 (d, J = 9.2

Hz, 6H), 3.50 (s, 3H), 2.81 (s, 6H).

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-3-(methoxymethoxy)-2-

nitrophenyl)methanol (27) White solid, 62% yield. 1H NMR (400 MHz, Chloroform-d)

δ 7.25 (dd, J = 2.1, 1.0 Hz, 1H), 7.12 (dd, J = 7.5, 1.1 Hz, 1H), 6.87 – 6.77 (m, 2H),

6.53 (dd, J = 10.9, 1.1 Hz, 1H), 5.91 (dd, J = 5.0, 1.1 Hz, 1H), 5.75 (d, J = 12.4 Hz,

1H), 5.44 (d, J = 10.8 Hz, 1H), 5.21 (s, 2H), 3.90 (s, 6H), 3.23 (s, 3H), 1.48 (s, 6H).

(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)(4-methoxy-3-(methoxymethoxy)-2-

nitrophenyl)methanol (36) White solid, 61% yield. 1H NMR (400 MHz, Chloroform-d)

δ 7.02 (ddd, J = 7.5, 2.2, 1.0 Hz, 2H), 6.87 (d, J = 7.5 Hz, 1H), 6.50 (d, J = 11.0 Hz,

1H), 6.39 (d, J = 7.3 Hz, 1H), 5.59 (d, J = 12.5 Hz, 1H), 5.13 (s, 2H), 3.90 (d, J = 5.0

Hz, 6H), 3.20 (s, 3H), 1.46 (s, 6H).

General Procedures for the Preparation of 5, 12, 19, 28, and 37.

To a stirred solution of 4 (472 mg, 1 mmol), 11 (425 mg, 1 mmol), 18 (422 mg, 1

mmol), 27 (431 mg, 1 mmol), or 36 (431 mg, 1 mmol) in THF (10 mL), respectively,

2-iodoxybenzoic acid (560 mg, 2 mmol) was added in portions. After the reaction

completed, the resulting precipitate was filtered off and the filtrate was concentrated,

following purified by flash column chromatography (PE/EtOAc, 5:1) to give the

products.

26
(3,4-dimethoxy-5-(methylselanyl)phenyl)(4-methoxy-3-(methoxymethoxy)-2-

nitrophenyl)methanone (5) Light yellow solid, 72% yield. 1H NMR (400 MHz,

Chloroform-d) δ 7.28 (m, 2H), 6.95 – 6.90 (m, 1H), 6.86 (d, J = 8.8 Hz, 1H), 5.20 (s,

2H), 3.95 (s, 3H), 3.84 (d, J = 2.1 Hz, 6H), 3.51 (s, 3H), 2.23 (s, 3H).

(3,4-dimethoxy-5-(methylthio)phenyl)(4-methoxy-3-(methoxymethoxy)-2-

nitrophenyl)methanone (12) Light yellow solid, 75% yield. 1H NMR (400 MHz,

Chloroform-d) δ 7.80 (d, J = 7.5 Hz, 1H), 7.61 – 7.25 (m, 3H), 5.19 (s, 2H), 3.85 (s,

3H), 3.81 (d, J = 3.2 Hz, 6H), 3.48 (s, 3H), 2.39 (s, 3H).

(3-(dimethylamino)-4,5-dimethoxyphenyl)(4-methoxy-3-(methoxymethoxy)-2-

nitrophenyl)methanone (19) Light yellow solid, 74% yield. 1H NMR (400 MHz,

Chloroform-d) δ 7.77 (d, J = 7.5 Hz, 1H), 7.36 (d, J = 7.5 Hz, 1H), 7.03 – 6.89 (m,

2H), 5.18 (d, J = 2.3 Hz, 2H), 3.90 (s, 6H), 3.85 (s, 3H), 3.50 (s, 3H), 2.81 (s, 6H).

(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)(4-methoxy-3-(methoxymethoxy)-2-

1
nitrophenyl)methanone (28) White solid, 77% yield. H NMR (400 MHz,

Chloroform-d) δ 7.39 – 7.27 (m, 2H), 7.09 – 7.00 (m, 2H), 6.27 (d, J = 9.9 Hz, 1H),

5.66 (d, J = 9.9 Hz, 1H), 5.23 (s, 2H), 3.97 (s, 3H), 3.89 (s, 3H), 3.52 (s, 3H), 1.52 (s,

6H).

(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)(4-methoxy-3-(methoxymethoxy)-2-

1
nitrophenyl)methanone (37) White solid, 77% yield. H NMR (400 MHz,

27
Chloroform-d) δ 7.39 (dd, J = 7.5, 4.2 Hz, 2H), 6.99 (d, J = 7.5 Hz, 1H), 6.56 – 6.50

(m, 2H), 5.66 (d, J = 10.8 Hz, 1H), 5.13 (s, 2H), 3.90 (d, J = 4.9 Hz, 6H), 3.27 (s, 3H),

1.50 (s, 6H).

General Procedures for the Preparation of 6, 13, 20, 29, and 38.

To a stirred solution of 5 (470 mg, 1 mmol), 12 (423 mg, 1 mmol), 19 (420 mg, 1

mmol), 28 (429 mg, 1 mmol), or 37 (429 mg, 1 mmol) in 10 mL acetone and H2O

(1:1), respectively, Na2S2O4 (870 mg, 5 mmol) was added slowly. The reaction

mixture was stirred at 50 °C for 3 h, then quenched by the addition of saturated

NaHCO3 solution and extracted with ethyl acetate. The organic phase was

concentrated under vacuum and purified by flash column chromatography (PE/EtOAc,

5:1) to give the products.

(2-amino-4-methoxy-3-(methoxymethoxy)phenyl)(3,4-dimethoxy-5-

(methylselanyl)phenyl)methanone (6) Yellow solid, 77% yield. 1H NMR (400 MHz,

Chloroform-d) δ 7.50 (d, J = 9.4 Hz, 1H), 7.44 – 7.31 (m, 2H), 7.05 – 6.89 (m, 1H),

5.51 (s, 2H), 4.00 (s, 3H), 3.96 (d, J = 5.4 Hz, 6H), 3.66 (s, 3H), 2.36 (s, 3H).

(2-amino-4-methoxy-3-(methoxymethoxy)phenyl)(3,4-dimethoxy-5-

(methylthio)phenyl)methanone (13) Yellow solid, 64% yield. 1H NMR (400 MHz,

Chloroform-d) δ 7.29 (d, J = 9.1 Hz, 1H), 7.06 – 6.96 (m, 2H), 6.24 (d, J = 9.1 Hz,

1H), 5.14 (s, 2H), 3.92 (s, 3H), 3.89 (d, J = 4.9 Hz, 6H), 3.63 (s, 3H), 2.41 (s, 3H).

28
(2-amino-4-methoxy-3-(methoxymethoxy)phenyl)(3-(dimethylamino)-4,5-

dimethoxyphenyl)methanone (20) Yellow solid, 67% yield. 1H NMR (400 MHz,

Chloroform-d) δ 7.73 (d, J = 7.5 Hz, 1H), 7.26 (d, J = 7.5 Hz, 1H), 7.01 – 6.79 (m,

2H), 5.18 (d, J = 2.3 Hz, 2H), 3.90 (s, 6H), 3.85 (s, 3H), 3.54 (s, 3H), 2.83 (s, 6H).

(2-amino-4-methoxy-3-(methoxymethoxy)phenyl)(5-methoxy-2,2-dimethyl-2H-

1
chromen-8-yl)methanone (38) Yellow solid, 77% yield. H NMR (400 MHz,

Chloroform-d) δ 7.41 (d, J = 7.5 Hz, 1H), 7.26 (d, J = 7.5 Hz, 1H), 6.68 (d, J = 7.5 Hz,

1H), 6.52 (d, J = 7.5 Hz, 1H), 6.39 (d, J = 11.0 Hz, 1H), 6.02 (s, 2H), 5.89 (d, J = 11.0

Hz, 1H), 4.35 (s, 2H), 3.90 (d, J = 4.9 Hz, 6H), 3.28 (s, 3H), 1.47 (s, 6H).

General Procedures for the Preparation of 7a, 14a, 21, 30, and 39.

Et3N (152 mg, 1.5 mmol) was added a stirred solution of 6 (440 mg, 1 mmol), 13 (393

mg, 1 mmol), 20 (390 mg, 1 mmol), 29 (399 mg, 1 mmol), or 38 (399 mg, 1 mmol) in

10 mL anhydrous CH2Cl2, respectively, following added BrCH2COBr (242 mg, 1.2

mmol) in dropwise at 0 °C. The reaction mixture was stirred at rt for 3 h, then

quenched by the addition of saturated NaHCO3 solution and extracted with CH2Cl2.

The organic phase was concentrated under vacuum and purified by flash column

chromatography (PE/EtOAc, 3:1) to give the products.

2-bromo-N-(6-(3,4-dimethoxy-5-(methylselanyl)benzoyl)-3-methoxy-2-

(methoxymethoxy)phenyl)acetamide (7a) White solid, 82% yield. 1H NMR (400 MHz,

Chloroform-d) δ 9.90 (s, 1H), 7.42 (d, J = 7.5 Hz, 1H), 7.40 (s, 2H), 6.76 (d, J = 7.5

29
Hz, 1H), 5.49 (s, 2H), 4.24 (s, 2H), 4.00 (s, 3H), 3.96 (d, J = 5.4 Hz, 6H), 3.46 (s, 3H),

2.36 (s, 3H).

2-bromo-N-(6-(3,4-dimethoxy-5-(methylthio)benzoyl)-3-methoxy-2-

(methoxymethoxy)phenyl)acetamide (14a) Light yellow solid, 80% yield. 1H NMR

(400 MHz, Chloroform-d) δ 9.15 (s, 1H), 7.25 – 7.17 (m, 3H), 6.82 (d, J = 8.7 Hz,

1H), 5.17 (s, 2H), 3.93 (d, J = 2.3 Hz, 6H), 3.88 (d, J = 4.8 Hz, 5H), 3.60 (s, 3H), 2.39

(s, 3H).

2-bromo-N-(6-(3-(dimethylamino)-4,5-dimethoxybenzoyl)-3-methoxy-2-

(methoxymethoxy)phenyl)acetamide (21) Light yellow solid, 85% yield. 1H NMR (400

MHz, Chloroform-d) δ 8.29 (s, 1H), 7.28 (s, 2H) 7.05 (d, J = 2.1 Hz, 2H), 6.82 (d, J =

8.7 Hz, 1H), 5.16 (s, 2H), 3.93 (s, 3H), 3.92 – 3.82 (m, 8H), 3.59 (s, 3H), 2.82 (s, 6H).

2-bromo-N-(3-methoxy-6-(8-methoxy-2,2-dimethyl-2H-chromene-6-carbonyl)-2-

(methoxymethoxy)phenyl)acetamide (30) White solid, 85% yield. 1H NMR (500 MHz,

Chloroform-d) δ 8.34 (s, 1H), 7.45 (dd, J = 2.0, 1.1 Hz, 1H), 7.28 (d, J = 7.5 Hz, 1H),

7.16 (d, J = 2.0 Hz, 1H), 6.79 (d, J = 7.5 Hz, 1H), 6.58 (dd, J = 10.9, 1.0 Hz, 1H),

6.02 (s, 2H), 5.50 (d, J = 11.0 Hz, 1H), 4.24 (s, 2H), 3.90 (s, 6H), 3.28 (s, 3H), 1.61 (s,

6H).

2-bromo-N-(3-methoxy-6-(5-methoxy-2,2-dimethyl-2H-chromene-8-carbonyl)-2-

(methoxymethoxy)phenyl)acetamide (39) White solid, 83% yield. 1H NMR (400 MHz,

30
Chloroform-d) δ 8.29 (s, 1H), 7.32 (d, J = 7.5 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 6.90

(d, J = 7.5 Hz, 1H), 6.53 (dd, J = 9.2, 1.7 Hz, 2H), 5.66 (d, J = 11.0 Hz, 1H), 5.13 (s,

2H), 4.12 (s, 2H) , 3.90 (d, J = 4.9 Hz, 6H), 3.29 (s, 3H), 1.46 (s, 6H).

General Procedures for the Preparation of 7b and 14c. The procedures were same

to that of preparation of 7a and 14a, except for the change of BrCH2COBr to

CH3COBr.

N-(6-(3,4-dimethoxy-5-(methylselanyl)benzoyl)-3-methoxy-2-

(methoxymethoxy)phenyl)acetamide (7b) White solid, 80% yield. 1H NMR (400 MHz,

Chloroform-d) δ 8.30 (s, 1H), 7.36 – 7.27 (m, 2H), 7.18 (d, J = 8.6 Hz, 1H), 6.77 (d, J

= 8.7 Hz, 1H), 5.14 (s, 2H), 3.93 (s, 3H), 3.91 (s, 3H), 3.87 (s, 3H), 3.58 (s, 3H), 2.24

(d, J = 4.4 Hz, 3H).

N-(6-(3,4-dimethoxy-5-(methylthio)benzoyl)-3-methoxy-2-

(methoxymethoxy)phenyl)acetamide (14c) Light yellow solid, 84% yield. 1H NMR

(400 MHz, Chloroform-d) δ 8.29 (s, 1H), 7.24 – 7.18 (m, 3H), 6.91 (d, J = 8.7 Hz,

1H),5.14 (s, 2H), 3.92 (d, J = 3.0 Hz, 7H), 3.87 (s, 3H), 3.58 (s, 3H), 2.39 (s, 3H),

2.07 (s, 3H).

General Procedures for the Preparation of 14b and 14d.

BrCHCH3COBr (259 mg, 1.2 mmol) or CH3CH2COBr (164 mg, 1.2 mmol) were

added in dropwise to a stirred solution of 13 (393 mg, 1 mmol) and Et3N (152 mg, 1.5

31
mmol) in 10 mL anhydrous CH2Cl2 at 0 °C, respectively. The reaction mixture was

stirred at rt for 3 h, then quenched by the addition of saturated NaHCO3 solution and

extracted with CH2Cl2. The organic phase was concentrated under vacuum and

purified by flash column chromatography (PE/EtOAc, 3:1) to give the products.

2-bromo-N-(6-(3,4-dimethoxy-5-(methylthio)benzoyl)-3-methoxy-2-

(methoxymethoxy)phenyl)propenamide (14b). Light yellow solid, 80% yield. 1H NMR

(400 MHz, Chloroform-d) δ 8.29 (s, 1H), 7.58 (d, J = 2.0 Hz, 1H), 7.53 (d, J = 2.0 Hz,

1H), 7.47 (d, J = 7.5 Hz, 1H), 6.89 (d, J = 7.5 Hz, 1H), 5.42 (q, J = 6.8 Hz, 1H), 5.14

(s, 2H), 3.90 (s, 6H), 3.86 (s, 3H), 3.21 (s, 3H), 2.37 (s, 3H), 2.07 (d, J = 6.8 Hz, 3H).

N-(6-(3,4-dimethoxy-5-(methylthio)benzoyl)-3-methoxy-2-

(methoxymethoxy)phenyl)propionamide (14d). Light yellow solid, 81% yield. 1H

NMR (400 MHz, Chloroform-d) δ 8.33 (s, 1H), 7.27 – 7.21 (m, 3H), 6.80 (d, J = 8.7

Hz, 1H), 5.16 (s, 2H), 3.94 (d, J = 1.0 Hz, 6H), 3.90 (s, 3H), 3.59 (s, 3H), 2.41 (s, 3H),

2.32 (q, J = 7.6 Hz, 2H), 1.14 (t, J = 7.6 Hz, 3H).

General Procedures for the Preparation of 8a, 15a, 15b, 22, 31 and 40.

To a stirred solution of 7a (561 mg, 1 mmol), 14a (514 mg, 1 mmol), 14b (528 mg, 1

mmol), 21 (511 mg, 1 mmol), 30 (520 mg, 1 mmol) or 39 (520 mg, 1 mmol) in 10 mL

CH3CH2OH, respectively, NH3.H2O (5 mmol) was added dropwise. The reaction

32
mixture was stirred at 90 °C for 5 h, then concentrated under vacuum to obtain the

products without further purified.

General Procedures for the Preparation of 8b, 15c and 15d.

To a stirred solution of 7b (482 mg, 1 mmol), 14c (435 mg, 1 mmol) or 14d (449 mg,

1 mmol) in 10 mL DMF, respectively, CH3ONa (270 mg, 5 mmol) was added in

portions. The reaction mixture was stirred at 90 °C for 5 h, then quenched by addition

of large ice water. The resulting precipitate was filtered and dried under vacuum to

obtain the products without further purified.

General Procedures for the Preparation of 9a, 9b, 16a-16b, 23, 32, and 41.

To a stirred solution of 8a, 8b, 15a-15b, 22, 31, or 40 (1 mmol) in 10 mL CH3OH,

respectively, 37% HCl (5 mmol) was added dropwise. The reaction mixture was

stirred at rt for 5 h, then neutralized by addition of saturated NaHCO3 solution and

extracted with ethyl acetate. The organic phase was concentrated under vacuum and

purified by flash column chromatography (PE/EtOAc, 1:1) to give the products.

5-(3,4-dimethoxy-5-(methylselanyl)phenyl)-9-hydroxy-8-methoxy-1H-

benzo[e][1,4]diazepin-2(3H)-one (9a) White solid, 95% yield. m.p.: 178.2−179.3 °C.

1
H NMR (400 MHz, Chloroform-d) δ 7.89 (s, 1H), 7.04 (d, J = 1.8 Hz, 1H), 6.95 –

6.85 (m, 2H), 6.71 (d, J = 8.6 Hz, 1H), 4.31 (s, 2H), 3.98 (s, 3H), 3.90 (s, 3H), 3.85 (s,

13
3H), 2.19 (s, 3H). C NMR (100 MHz, Chloroform-d) δ 170.83, 170.02, 151.83,

33
148.30, 147.85, 136.37, 134.67, 127.14, 122.97, 122.14, 120.86, 119.80, 111.78,

105.69, 60.11, 56.81, 56.40, 56.02, 5.10. HRMS (ESI) (m/z) [M+H]+ calcd for

C19H20N2O5Se, 437.0611; found, 437.0616. Purity: 98.4% (by HPLC).

4-(3,4-dimethoxy-5-(methylselanyl)phenyl)-8-hydroxy-7-methoxyquinolin-2(1H)-one

(9b) White solid, 96% yield. M.p.: 193.4−195.1 °C 1H NMR (400 MHz, Chloroform-

d) δ 9.08 (s, 1H), 7.12 (d, J = 8.9 Hz, 1H), 6.86 – 6.76 (m, 3H), 6.49 (s, 1H), 3.97 (d,

J = 13.0 Hz, 6H), 3.88 (s, 3H), 2.25 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 161.67,

152.02, 151.53, 149.21, 144.99, 134.68, 133.65, 127.02, 124.48, 118.39, 117.67,

116.22, 114.60, 110.43, 107.81, 59.81, 56.64, 56.37, 4.91. HRMS (ESI) (m/z) [M+H]+

calcd for C19H19NO5Se, 422.0502; found, 422.0489. Purity: 98.4% (by HPLC).

5-(3,4-dimethoxy-5-(methylthio)phenyl)-9-hydroxy-8-methoxy-1H-

benzo[e][1,4]diazepin-2(3H)-one (16a) White solid, 93% yield. M.p.: 186.1−187.6 °C

1
H NMR (400 MHz, Chloroform-d) δ 7.82 (s, 1H), 6.99 (d, J = 1.9 Hz, 1H), 6.92 –

6.81 (m, 2H), 6.71 (d, J = 8.7 Hz, 1H), 4.31 (s, 2H), 3.99 (s, 3H), 3.87 (d, J = 14.3 Hz,

13
6H), 2.35 (s, 3H) C NMR (100 MHz, Chloroform-d) δ 170.80, 170.11, 152.00,

147.81, 147.43, 136.05, 134.65, 133.01, 127.11, 122.97, 120.87, 119.77, 111.07,

105.69, 60.13, 56.82, 56.40, 56.08, 14.74. HRMS (ESI) (m/z) [M+H]+ calcd for

C19H20N2O5S, 389.1166; found, 389.1161. Purity: 98.2% (by HPLC).

34
5-(3,4-dimethoxy-5-(methylthio)phenyl)-9-hydroxy-8-methoxy-3-methyl-1H-

benzo[e][1,4]diazepin-2(3H)-one (16b) White solid, 91% yield. M.p.:

193.6−194.7 °C 1H NMR (500 MHz, DMSO-d6) δ 9.44 (s, 1H), 6.98 (d, J = 1.9 Hz,

1H), 6.88 (d, J = 8.7 Hz, 1H), 6.84 (d, J = 1.9 Hz, 1H), 6.75 (d, J = 8.7 Hz, 1H), 3.88

(d, J = 3.6 Hz, 3H), 3.78 (s, 3H), 3.76 (s, 3H), 3.61 (q, J = 6.4 Hz, 1H), 2.29 (s, 3H),

1.50 (d, J = 6.4 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ 171.18, 167.16, 151.90,

149.49, 146.39, 136.11, 132.84, 128.50, 121.62, 118.67, 110.93, 107.39, 59.92, 58.83,

56.52, 56.28, 17.79, 14.02. HRMS (ESI) (m/z) [M+H]+ calcd for C20H22N2O5S,

403.1322; found, 403.1321. Purity: 97.6% (by HPLC).

4-(3,4-dimethoxy-5-(methylthio)phenyl)-8-hydroxy-7-methoxyquinolin-2(1H)-one

(16c) White solid, 93% yield. M.p.: 201.2−202.9 °C 1H NMR (400 MHz, DMSO-d6)

δ 9.56 (s, 1H), 6.98 (d, J = 9.0 Hz, 1H), 6.93 (dd, J = 5.5, 3.6 Hz, 2H), 6.78 (d, J = 1.9

Hz, 1H), 6.30 (s, 1H), 3.86 (d, J = 9.6 Hz, 6H), 3.79 (s, 3H), 2.39 (s, 3H).13C NMR

(100 MHz, DMSO-d6) δ 161.62, 152.23, 151.73, 148.38, 144.74, 134.14, 133.81,

132.14, 129.80, 119.16, 117.75, 116.91, 114.03, 110.44, 107.70, 59.84, 56.64, 56.43,

13.80. HRMS (ESI) (m/z) [M+H]+ calcd for C19H19NO5S, 374.1057; found, 374.1046.

Purity: 98.6% (by HPLC).

4-(3,4-dimethoxy-5-(methylthio)phenyl)-8-hydroxy-7-methoxy-3-methylquinolin-

2(1H)-one (16d) White solid, 94% yield. M.p.: 176.8−178.2 °C 1H NMR (400 MHz,

35
Chloroform-d) δ 9.39 (s, 1H), 6.78 – 6.65 (m, 2H), 6.58 (s, 2H), 3.97 (d, J = 4.1 Hz,

6H), 3.87 (s, 3H), 2.40 (s, 3H) 2.05 (s, 3H). 13C NMR (101 MHz, DMSO) δ 162.28,

152.46, 147.72, 147.61, 144.10, 134.15, 133.78, 133.35, 128.28, 124.68, 116.85,

116.52, 115.84, 110.39, 108.17, 59.90, 56.75, 56.48, 14.61, 13.96.HRMS (ESI) (m/z)

[M+H]+ calcd for C20H21NO5S, 388.1213; found, 388.1209. Purity: 98.1% (by HPLC).

5-(3-(dimethylamino)-4,5-dimethoxyphenyl)-9-hydroxy-8-methoxy-1H-

benzo[e][1,4]diazepin-2(3H)-one (23) White solid, 93% yield. M.p.: 181.6−183.4 °C

1
H NMR (400 MHz, Chloroform-d) δ 7.92 (s, 1H), 6.93 (d, J = 8.8 Hz, 1H), 6.76 (d, J

= 2.0 Hz, 1H), 6.73 – 6.64 (m, 2H), 4.30 (s, 2H), 3.98 (s, 3H), 3.83 (d, J = 10.4 Hz,

13
6H), 2.79 (s, 6H). C NMR (100 MHz, Chloroform-d) δ 170.79, 169.83, 152.11,

149.03, 147.67, 136.63, 135.04, 128.64, 122.75, 121.19, 120.46, 119.35, 109.83,

107.28, 60.14., 56.78, 56.35, 56.06, 42.80. Purity: 96.3% (by HPLC).

9-hydroxy-8-methoxy-5-(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)-1H-

benzo[e][1,4]diazepin-2(3H)-one (32) White solid, 94% yield. M.p.: 210.2−211.6 °C

1
H NMR (400 MHz, Chloroform-d) δ 7.82 (s, 1H), 7.09 (d, J = 1.9 Hz, 1H), 6.93 (d, J

= 8.8 Hz, 1H), 6.79 – 6.54 (m, 2H), 6.25 (d, J = 9.8 Hz, 1H), 5.63 (d, J = 9.8 Hz, 1H),

4.29 (s, 2H), 3.99 (s, 3H), 3.86 (s, 3H), 1.49 (s, 6H). 13C NMR (125 MHz, DMSO-d6)

δ 170.01, 162.93, 152.24, 148.77, 149.36, 135.30, 131.64, 130.82, 127.44, 125.38,

123.46, 121.28, 118.58, 111.37, 109.66, 105.78, 76.46, 57.80, 57.03, 56.77, 27.51.

36
HRMS (ESI) (m/z) [M+H]+ calcd for C22H22N2O5, 395.1601; found, 395.1606. Purity:

96.4% (by HPLC).

9-hydroxy-8-methoxy-5-(5-methoxy-2,2-dimethyl-2H-chromen-8-yl)-1H-

benzo[e][1,4]diazepin-2(3H)-one (41) White solid, 96% yield. M.p.: 208.5−209.9 °C

1
H NMR (500 MHz, DMSO-d6) δ 9.40 (s, 1H), 7.24 (d, J = 8.5 Hz, 1H), 6.74 (d, J =

8.7 Hz, 1H), 6.61 (d, J = 8.5 Hz, 1H), 6.52 (dd, J = 9.3, 5.8 Hz, 2H), 5.56 (d, J = 10.0

Hz, 1H), 3.99 (s, 2H), 3.82 (d, J = 6.5 Hz, 6H), 0.89 (s, 6H).13C NMR (125 MHz,

DMSO-d6) δ 170.12, 169.47, 156.12, 151.58, 148.95, 136.34, 130.52, 129.57, 127.62,

124.36, 122.82, 120.17, 116.33, 109.94, 107.53, 103.39, 75.74, 57.06, 56.62, 56.14,

27.51. HRMS (ESI) (m/z) [M+H]+ calcd for C22H22N2O5, 395.1601; found, 395.1605.

Purity: 97.1% (by HPLC).

Biological assays

The antiproliferative activity assay, tubulin polymerization inhibition assay,

microtubule organization disruption assay, cell cycle arrest assay, apoptosis assay,

metabolic stability assay and in vivo antitumor activity evaluation were performed in

accordance with the previously reported procedures and described in the Supporting

Information.

Supporting information

1 13
H and C NMR spectra, HPLC chromatograms, high resolution mass spectra,

37
methods of biological activity assay.

Abbreviations

SARs, structure and activity relationships; CA-4, combretastatin A-4; isoCA-4,

isocombretastatin A-4; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-

tetrazolium bromide; SD, standard error; PI, propidium iodide; HPLC, high-

performance liquid chromatography; HRMS, high-resolution mass spectra.

Corresponding author

*For Jun Yan, Phone number, +086-020-81887233-35264; Fax number, +086-020-

81887233-35267; e-mail, yanjun1989_happy@126.com

*For Xingshu Li, Phone number, +86-020-39943050; Fax number, +86-020-

39943050; E-mail address: lixsh@mail.sysu.edu.cn

Notes

The authors declare no competing financial interest.

Acknowledgments

We thank the natural science foundation of Guangdong province [2018A030313539],

the youth innovation project of Guangdong university [E1-KFD015181K35], and the

Science Foundation of Guangdong Provincial Hospital of Chinese Medicine

[2017KT1525] for financial support.

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38
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TOC:

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Highlights:

A series of novel structurally-related tubulin polymerization inhibitors were designed

and synthesized.

Most compounds exhibited potent antiproliferative activity against a panel of cancer

cell lines.

Compound 9a arrested cell cycle and induced apoptosis in A549 cells.

Compound 9a exhibited potent in vivo antitumor activity in xenograft mouse models

43
A series of novel structurally-related tubulin polymerization inhibitors were designed

and synthesized.

Most compounds exhibited potent antiproliferative activity against a panel of cancer

cell lines.

Compound 9a arrested cell cycle and induced apoptosis in A549 cells.

Compound 9a exhibited potent in vivo antitumor activity in xenograft mouse models.