Synthesis and antiproliferative evaluation of novel benzoimidazole- contained oxazole-bridged analogs of combretastatin A-4

Article abstract of DOI:10.1016/j.ejmech.2013.08.006

A series of novel oxazole-bridged analogs of combretastatin A-4 bearing a benzo[d]-imidazole as B ring were synthesized and evaluated for antiproliferative activities against five human cancer cell lines. Among all the synthesized compounds, the N-unsubstituted benzoimidazole analog 5 and the analogs 6b, 7a and 7b with a small hydrophobic group on nitrogen atom of benzoimidazole ring were identified as the most potent inhibitors of tumor cell growth with IC₅₀ values at nanomolar levels (5, IC₅₀ = 8.4 nM, HT29; 6b, 7a, 7b, IC₅₀ = 9.6 nM, 3.8 nM, 3.0 nM, A549). In a murine H22 tumor xenograft model, compound 5 exhibited significant antitumor activity. The binding mode of compound 5 in the colchicine binding site of tubulin was probed.

Full text of DOI:10.1016/j.ejmech.2013.08.006

Accepted Manuscript
Synthesis and antiproliferative evaluation of novel benzoimidazole-contained oxazole-
bridged analogs of combretastatin A-4
Jie Zhou, Jing Jin, Yi Zhang, Yuwen Yin, Xiaoguang Chen, Bailing Xu
PII:
S0223-5234(13)00507-2
10.1016/j.ejmech.2013.08.006
EJMECH 6346
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 February 2013
Revised Date: 31 July 2013
Accepted Date: 2 August 2013
Please cite this article as: J. Zhou, J. Jin, Y. Zhang, Y. Yin, X. Chen, B. Xu, Synthesis and
antiproliferative evaluation of novel benzoimidazole-contained oxazole-bridged analogs
of combretastatin A-4, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.08.006.
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ACCEPTED MANUSCRIPT
Graphical Abstract:
Synthesis and antiproliferative evaluation of novel benzoimidazole-contained
oxazole-bridged analogs of combretastatin A-4
Jie Zhou, Jing Jin, Yi Zhang, Yuwen Yin, Xiaoguang Chen, Bailing Xu
A series of novel oxazole-bridged analogs of combretastin A-4 bearing a benzo[d]-imidazole as B
ring were synthesized and evaluated for their antiproliferative activities in vitro and in vivo.
6a-13a
6b-14b

ACCEPTED MANUSCRIPT
Synthesis and antiproliferative evaluation of novel
benzoimidazole-contained oxazole-bridged analogs of combretastatin
A-4
Jie Zhoub,#, Jing Jina,#, Yi Zhanga, Yuwen, Yinb, Xiaoguang Chena,*,
Bailing Xub,*
aState Key Laboratory of Bioactive Substances and Functions of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences
Medical College, Beijing, 100050, China
＆Peking Union
bBeijing Key Laboratory of Active Substance Discovery and Druggability Evaluation,
Institute of Materia Medica, Chinese Academy of Medical Sciences
Medical College, Beijing, 100050, China
＆Peking Union
Abstract: A series of novel oxazole-bridged analogs of combretastin A-4 bearing a
benzo[d]-imidazole as B ring were synthesized and evaluated for antiproliferative activities
against five human cancer cell lines. Among all the synthesized compounds, the N-unsubstituted
benzoimidazole analog 5 and the analogs 6b, 7a and 7b with a small hydrophobic group on
nitrogen atom of benzoimidazole ring were identified as the most potent inhibitors of tumor cell
growth with IC50 values at nanomolar levels (5, IC50 = 8.4 nM, HT29; 6b, 7a, 7b, IC50 = 9.6 nM,
3.8 nM, 3.0 nM, A549). In a murine H22 tumor xenograft model, compound 5 exhibited
significant antitumor activity. The binding mode of compound 5 in the colchicine binding site of
tubulin was probed.
Keywords: Combretastatin; Oxazole; Benzo[d]-imidazole; Antiproliferative activity;
Structure-activity relationship
1. Introduction
Combretastatin A-4 (CA-4), as shown in Figure 1, is a natural product, and was first isolated by
Pettit et al. from the bark of the South African willow tree Combretum caffrum in 1989 [1,2]. It
was disclosed that CA-4 can act as vascular disrupting agent (VDAs), and preferentially cut off
the blood supply of immature tumors leading to their death [3-5]. Furthermore, it was found that
CA-4 binds to the colchicine site of tubulin and inhibit tubulin polymerization [6,7]. Importantly,
CA-4 displayed potent cytotoxicities against a variety of murine and human cancer cell lines with
IC50 values ranging from 10-8 to 10-10 M [8-10]. However, CA-4 does not show efficacy in vivo
because of the facts that it has low aqueous solubility and easily isomerizes into the inactive
# The first two authors contributed equally.
* Corresponding author. B. Xu, Tel: +86 10 63166764; X. Chen, Tel: +86 10 63165207
E-mail address: xubl@imm.ac.cn (B. Xu); chxg@imm.ac.cn (X. Chen)
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trans-isomer from the active cis-isomer [11,12]. As shown in Figure 1, up to now, only three
water-soluble prodrugs, such as CA-4 phosphate disodium (CA-4P) [13, 14], CA-1 phosphate
disodium (CA-1P) and AVE8062, a serine derivative of AC-7739, have been tested in Phase I, II
or III clinical trial, respectively [15-17].
To date, a great number of CA-4 analogs with diverse scaffolds were synthesized and their
anticancer activities have been extensively explored [18-23]. The structure-activity relationship
(SAR) studies indicated that the common key structural features of these analogs consist of two
cis-orienated aromatic A-ring and B-ring, and A-ring preferred to contain 3,4,5-trimethoxy
substituents and a 4-methoxy group was desired to be positioned on B ring [24,25].
In our efforts to search for novel anticancer agents, a class of novel CA-4 analogs have been
designed and synthesized with the use of 4-methoxy-1H-benzo[d]-imidazole as B ring and oxazole
ring as a linker (6a-13a, 6b-14b, Figure 1). In this work, 3,4,5-trimethoxybenzene was retained as
A ring, while benzo[d]-imidazole was utilized as B ring to mimic the 2- and 3-hydroxyl groups in
CA-1 and CA-4 with an aim to improve the metabolic stability and physicochemical property. In
conjunction with the known SAR studies on the B ring, a 4-methoxy group was installed on the
benzoimidazole ring [26,27]. In addition, the oxazole subunit which was employed in the known
CA-4 analogs [28] was incorporated to lock the cis-orientation of A ring and B ring. Herein, we
reported the chemical synthesis and cytotoxicities of these oxazole-bridged analogs of CA-4.
Meanwhile, the preliminary SAR of N-substitution on benzoimidazole ring was explored as well.
2. Chemistry
The target oxazole derivatives containing N-substituted benzoimidazole (6a-13a and 6b-14b) were
synthesized on the basis of Scheme 1 and their chemical structures were shown in Table 1.
Treatment of 2-amino-3-nitrophenol with methyl iodide and K2CO3 provided
2-methoxy-6-nitroaniline 1 in 89% yield. Compound 1 was hydrogenated to afford
3-methoxy-o-phenylenediamine (2) in 77% yield. Conversion of o-phenylenediamine 2 into
4-methoxy-1H-benzo[d]imidazole 3 was achieved in 83% yield in refluxing HCO2H. The
formylation of benzoimidazole 3 was accomplished with hexamethylenetetramine and TFA at
reflux, and thus furnishing 4-methoxy-1H-benzo[d]imidazole-7-carbaldeyde 4 in 47% yield.
Compound 5 was obtained in 78% yield via the van Leusen reaction of compound 4 with
3,4,5-trimethoxyphenyl substituted TosMIC reagent in the presence of K2CO3. The final alkyation,
acylation or sulfonylation on the nitrogen atom of benzoimidzole ring of compound 5 proceeded
smoothly, and provided the corresponding target molecules 6a-13a and 6b-14b in reasonable
yields, respectively.
Due to the tautomerization of benzoimidazole ring of compound 5, the alkylation or acylation on
the nitrogen atom resulted in the formation of a pair of regioisomers 6a-13a and 6b-13b.
Compounds 6b-13b were generated as major products (33% - 68%), and compounds 6a-13a as
minor ones (16%-37%). Noticeably, when the bulky 2-carbomethoxy-3-thiophenesulfonyl
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chloride was attempted as a sulfonylating reagent, compound 5 delivered the desired product 14b
in 81% yield as a single regioisomer.
In order to further confirm the chemical structures of a pair of regioisomers 6a and 6b, compound
6b was unambiguously prepared by following an alternative synthetic route as shown in Scheme 2.
Treatment of 2-amino-3-nitrophenol with methyl iodide and NaH provided
2-methoxy-N-methyl-6-nitroaniline 15 in 62% yield. By following a reaction sequence of
hydrogenation, condensation with HCOOH and formylation, compound 15 was readily
transformed into the key 7-methoxy-N-methyl-benzo[d]imidazole-4-carbaldeyde 18 in a
reasonable overall yield . In the presence of K2CO3, the condensation reaction of aldehyde 18 with
3,4,5-trimethoxyphenyl substituted TosMIC reagent furnished the desired compound 6b in 35%
yield.
1H NMR data of compounds 6a and 6b showed much differences in chemical shifts of N-methyl
on the benzoimidazole B-ring and 2,6-aromatic protons on the 3,4,5-trimethoxybenzene A-ring. In
compound 6a, the N-methyl and 2,6-aromatic protons resonated at upfield (δ: 3.46 ppm for
N-methyl; 6.79 ppm for ArH); in contrast, in compound 6b the chemical shifts of the N-methyl
and 2,6-aromatic protons signaled at downfield (δ: 3.98 ppm for N-methyl; 6.93 ppm for ArH).
1
Therefore, H NMR behaviors of both N-methyl and 2,6-aromatic protons of compounds 6a and
6b could be chosen as probes to differentiate series 8a-13a and 8b-13b. As indicated by 1H NMR
data, both N-methylene and 2,6-aromatic protons in the series 8a-13a tended to resonate at upfield
in comparison with those of series 8b-13b. In particular, the δ values of 2,6-aromatic protons on
3,4,5-trimethoxybenzene in compounds 6a-13a were in a range of 6.69-6.79 ppm, while the
corresponding chemical shifts ranged from 6.83 ppm to 6.93 ppm in compounds 6b-14b. The
chemical shift of these aromatic protons would be selected to characterize the regionisomers
without N-methylene substituent on benzoimidazole ring, such as compounds 7a, 7b and 14b (δ
value for 2,6-protons on A ring: 6.76 ppm for 7a, 6.86 ppm for 7b; 6.86 ppm for 14b).
3. Biological results and discussion
3.1 Antiproliferative activity in vitro
The in vitro antiproliferative activities of all the synthesized target molecules were evaluated by
MTT assay, wherein five human cancer cell lines were used: MCF-7, A549, HT29, HepG2 and
BxPC3. Vincristine (VCR) and CA-4 were chosen as reference molecules. The cytotoxicities were
expressed as IC50 values presented in Table 1.
As shown in Table 1, the N-unsubstituted benzoimidazole compound 5 displayed highly potent
cytotoxicities against cancer cell lines MCF-7, HT29 and BxPC-3 with IC50 values of 25 nM, 8.4
nM and 31 nM, respectively. In terms of the cytotoxic potency against cancer cell lines MCF-7
and HT-29, compound 5 was comparable to VCR, and it was 8 fold and 32 fold more active than
CA-4, respectively. In contrast, the antiproliferative activity of compound 5 against tumor cells
BxPC-3 was similar to that of CA-4, while it was 16 fold more potent than that of VCR.
Compound 5 was the most active derivative in this series against MCF-7, HT-29 and BxPC-3
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tumor cells. Obviously, the obtained bioactive data indicated that 4-methoxy-1H-benzo[d]-
imidazole is a favorable replacement of the B ring on CA-4 as we expected.
Further variations on nitrogen of benzoimidazole motif were performed and two series of
regioisomers 6a-13a and 6b-13b were obtained. Surprisingly, it was found that the substitution
pattern on the nitrogen atom did not result in consistent difference in the antiproliferative activities.
For example, N-methyl substituted regioisomer 6b produced more cytotoxic activity against all
five tested tumor cells than regioisomer 6a. The IC50s of compound 6b were in double-digit
nanomolar range against the screened tumor cells. In contrast, regioisomers 7a and 7b with
carbmethoxyl substituent had comparable cytotoxicities and they were highly active against all the
used cancer cell lines. Regarding the tumor cells A549, regioisomers 7a and 7b were the most
potent compounds which had IC50s with 3.8 nM and 3.0 nM, respectively. In the case of the
substitution with hydroxylpropyl group, regioisomer 11b was less active than 11a when tumor
cells MCF-7, HT-29 and HepG2 were tested.
In addition, in the series of 6a-13a and 6b-13b, the methyl and carbmethoxyl substitution
generated the most potent derivatives 6b, 7a and 7b, which exhibited antiproliferative activities
against all of the tested cancer cell lines with IC50 values in the range of 3 nM to 56 nM.
Furthermore, in terms of cytotoxicities against BxPC-3, compounds 6b, 7a and 7b generated the
comparable IC50s to that of CA-4, and they were much more potent than VCR.
By comparison with methyl and carbmethoxyl group, carbethoxymethyl and cyanomethyl
substitution on the nitrogen atom lowered the cytotoxicities against most of the used cancer cell
lines with IC50s at a micromolar level. When flexible alkyl group such as hydroxyethyl and
hydroxypropyl were incorporated, compounds 10a, 10b, 11a and 11b produced moderate
cytotoxic activities against MCF-7, HT29 and HepG2. It was noted that when the polar acidic
carboxymethyl was installed, the antiproliferative activities of compounds 12a and 12b were
completely lost. While a carbamoymethyl group, an isostere of a carboxymethyl was incorporated,
compounds 13a and 13b still showed very low activities. Compound 14b with a bulky
2-carbmethoxy-3-thiophenesulfonyl as a substituent exhibited antiproliferative activities against
four cancer cell lines at a submicromolar level. Consequently, the small hydrophobic group was
tolerated on nitrogen atom of benzoimidazole motif, while the introduction of bulky hydrophilic
substituent on the nitrogen atom was detrimental to the cytotoxicities.
The most potent compounds 5, 6b, 7a and 7b and moderately active compounds 11a and 11b
were chosen to be further evaluated by MTT assay against tumor cells KB, vincristine
resistant KB (KBV), MX-1 and taxol resistant MX-1(MX-1/T). The results were presented in
Table 2. In terms of tumor cells KB and KBV, compounds 5, 6b, 7a and 7b were more
cytotoxic than compounds 11a, 11b and VCR. In particular, compounds 5, 6b, 7a and 7b
greatly inhibited the vincristine resistant KB cells with IC50s in the double-digit nanomolar
range, although these compounds were less active than CA-4. Regarding the tumor cells
MX-1, compounds 5, 6b, 7a and 7b exhibited moderate growth inhibitory activities. As for
tumor cells MX-1/T, compounds 5 and 7a displayed moderate cytotoxicities, and they were
more active than compounds 6b and 7b. Compounds 11a and 11b were not active against
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both MX-1 and MX-1/T.
3.2 Inhibition of tubulin polymerization
It is well known that CA-4 interacts with tubulin and inhibits the tubulin assembly [6,7].
Therefore, compound 5, one of the most active compounds in this series of CA-4 analogs,
was evaluated for its inhibition of tubulin polymerization. It was observed that compound 5
displayed a significant inhibitory activity on tubulin with IC50 value of 0.39 ꢀM, which was
more active than CA-4 (IC50 = 2.7 ꢀM). It was indicated that compound 5 could bind to
tubulin and interfere with the dynamic process of tubulin polymerization to lead to cell death.
3.3 Effects on cell cycle
In general, G2/M cell cycle arrest is strongly associated with inhibition on tubulin
polymerization [29]. And it is well established that CA-4 causes G2/M arrest in cell cycle.
The effect of compound 5 on cell cycle progression was investigated in HT29 cells by flow
cytometry at various concentrations. The effect of CA-4 on cell cycle of HT29 cells was also
examined. As shown in Figure 2, the percentage of cells in the G2/M phase were 14.75%,
17.17%, 35.63% and 44.25% when the cells were treated with compound 5 for 24 h at
concentrations of 12.5 nM, 25 nM, 50 nM and 100 nM, respectively. In control (untreated
cells) 7.37% of accumulation in G2/M phase was observed. It was clearly demonstrated that
compound 5 caused a significant G2/M arrest in a concentration-dependent manner, and that
was consistent with the behavior of tubulin-binding agents. In addition, a concentration of 25
nM of CA4 increased the cells in G2/M phase to 25.02%, which was about 2 times higher
than that of control cells.
3.4 In vivo antitumor activity of compound 5
Due to the potent in vitro antiproliferative activity of compound 5, a preliminary in vivo antitumor
study on compound 5 was investigated using H22 xenograft model in KM mice. Vincristine (VCR)
was used as a positive control. The tumor growth inhibition of compound 5 on KM mice was
presented in Table 3. Four different doses were examined via intraperitoneal (i.p.) administration
of compound 5 for 8 days. When KM mice were dosed at 7.5 mg/kg on days 1-7 and 10 mg/kg on
days 1, 3, 5 and 7 after tumor implantation (day 0), the tumor growth inhibition were 55% and
52%, respectively. When the mice were treated at a dose of 15 mg/kg of compound 5 on days 1
and 4, the tumor growth was significantly reduced with an inhibition of 66%, which was
comparable to that of VCR at a dose of 0.5 mg/kg. It was also observed that an inhibition of 42%
on tumor growth was obtained upon treatment of the mice at a dose of 20 mg/kg only once and no
body weight was lost. Therefore, compound 5 possessed significant in vivo antitumor effect. It was
envisioned that the optimization of doses and administration schedules might further improve the
antitumor activity of compound 5.
4. Molecular Modeling
Since CA-4 was identified to bind with tubulin at colchicine site, compound 5, a highly potent
inhibitor of tubulin polymerization was chosen to investigate the binding mode of this set of
compounds using CDOCKER docking program (Accelrys Discovery Studio 2.5.5) [30]. For
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comparing the binding features of compound 5 with CA-4, both of them were docked into tubulin.
The coordinates of X-ray co-crystal structure of a colchicine derivative, DAMA-colchicine with
tubulin (pdb code: 1SA0) [31] was employed. The docked binding poses of CA-4 and compound 5
were shown in Figure 3. The binding orientation of compound 5 was very close to those of CA-4
and DAMA-colchicine. The trimethoxybenzene rings of these compounds were well overlapped
each other and the hydrogen bonds were formed between the oxygen of one methoxyl group and
the key residue Cys241 in the active site. The benzoimidazole ring was in the same disposition
with B ring of CA-4, and the nitrogen adjacent to the methoxyl group mimicked the hydroxyl
motif on CA-4. The oxazole bridge extended to a small hydrophobic region consisting of the side
chain Leu248 and Lys 254, indicating a small hydrophobic cyclic ring besides oxazole fragment
might also be chosen as a linker. Therefore, with the trimethoxybenzene ring A and
benzoimidazole ring B intact, the utilization of other cyclic rings as a bridge is taken into account
in our lab for the design of novel analogs of CA-4.
5. Conclusions
In summary, a series of novel oxazole analogs of CA-4 were synthesized, which involved
benzoimidazole as ring B and rigid oxazole as linker to fix cis-orientation of trimethoxybenzene
ring and benzoimidazole ring. Among which, compouds 5, 6b, 7a and 7b exhibited excellent
cytotoxic activities with IC50 values at nanomolar level. In addition, compounds 5, 6b, 7a and 7b
also displayed good potency against vincristine resistant KB tumor cells. Furthermore, the
significant in vivo antitumor activity was observed with compound 5 with H22 tumor growth
inhibition of up to 66%. It was demonstrated that compound 5 was a potent inhibitor of tubulin
polymerization and induced G2/M arrest in HT29 cells. Molecular modeling was performed and
offered some insights for further modification of the title compounds. Therefore, this work
provided a series of useful analogs of CA-4 showing high potency in cytotoxic activity. Further
optimization on these analogs is in progress and will be reported in due course.
6. Experimental
6.1 Chemistry
6.1.1 General
Melting points were measured on a Yanaco micro melting point apparatus and are
uncorrected. 1H NMR (300 MHz and 400 MHz) on a Varian Mercury spectrometer was recorded
in DMSO-d6 or CDCl3. Chemical shifts are reported in δ(ppm) units relative to the internal
standard tetramethylsilane (TMS). High resolution mass spectra (HRMS) were obtained on an
Agilent Technologies LC/MSD TOF spectrometer. All chemicals and solvents used were of
reagent grade without purified or dried before use. All the reactions were monitored by thin-layer
chromatography (TLC) on pre-coated silica gel G plates at 254 nm under a UV lamp. Column
chromatography separations were performed with silica gel (200-300 mesh).
6.1.2 2-Methoxy-6-nitroaniline (1)
The mixture of 2-amino-3-nitrophenol (308 mg, 2 mmol) and K2CO3 (552 mg, 4 mmol) in DMF
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(5 ml) were stirred at room temperature for 30 min before addition of MeI (0.15 ml, 2.5 mmol).
After stirred at room temperature for 1 h, the reaction mixture was diluted with water (30 ml) and
extracted with ethyl ether (15 mL × 3). The combined organic layer was washed with water and
brine and then dried over anhydrous MgSO4, filtered and concentrated to give the crude product,
which was purified by column chromatography on silica gel with petroleum ether/AcOEt (v/v =
12:1). The title compound (300 mg, 89% yield) was afforded as salmon pink powder. Mp 68 -
69 ; 1H-NMR (CDCl3, 300 MHz, δ ppm): 7.73 (1H, d, J = 9.3 Hz), 6.88 (1H, d, J = 7.8 Hz), 6.60
(1H, t, J = 7.5 Hz), 6.42 (2H, brs), 3.92 (3H, s); HRMS (ESI): m/z, calcd. For C7H9N2O3 [M+H+]:
169.0608, found 169.0607.
6.1.3 3-Methoxy-o-phenylenediamine (2)
The mixture of 2-methoxy-6-nitroaniline (1, 300 mg, 1.78 mmol) and 10% Pd/C (60 mg) in
ethanol (15 ml) was stirred under hydrogen atmosphere at room temperature and atmospheric
pressure for 2 h. The solid was removed by filtration and the filtrate was concentrated. The residue
was purified by column chromatography on silical gel with petroleum ether-AcOEt (v/v = 3/1).
The title compound 2 (190 mg, 77% yield) was obtained as off-white powder. Mp 77 - 78 ;
1H-NMR (CDCl3, 300 MHz, δ ppm): 6.68 (1H, t, J = 7.8 Hz), 6.41 (2H, td, J1 = 7.5 Hz, J2 = 1.2
Hz), 3.84 (3H, s), 3.31 (4H, brs); HRMS (ESI): m/z, calcd. For C7H11N2O [M + H+] 138.0866,
found 138.0870.
6.1.4 4-Methoxy-1H-benzo[d]imidazole (3)
The reaction mixture of 3-methoxy-o-phenylenediamine (2, 190 mg, 1.37 mmol)) and HCOOH (5
mL) was refluxed for 4 h, then cooled to room temperature and poured into ice/water (20 mL).
The mixture was treated with 10% NaOH aqueous solution to pH ≈ 8 and then extracted with
ethyl ether (15 mL × 3). The combined extracts were washed with water and brine, then dried over
anhydrous MgSO4. The organic phase was concentrated to give the crude product, which was
purified by column chromatography with CH2Cl2-MeOH (v/v = 60/1). The desired product
4-methoxy-1H-benzo[d]imidazole (162 mg) was afforded as white powder with 83.9% yield. Mp
165 - 167 ; 1H-NMR (acetone-d6, 300 MHz, δ ppm): 8.08 (1H, s), 7.21 (1H, d, J = 7.8 Hz), 7.11
(1H, t, J = 7.8 Hz), 6.73 (1H, d, J = 7.8 Hz), 3.97 (3H, s); HRMS (ESI): m/z, calcd. For
C8H9N2O [M + H+] 149.0709 found 149.0707
6.1.5 4-Methoxy-7-carbaldehyde-N1-1H-benzo[d]imidazole (4)
The mixture of 4-methoxy-1H-benzo[d]imidazole (3, 4.44 g, 30 mmol) and Urotropine (21 g, 150
mmol) in TFA (80 ml) were stirred at reflux for 36 h. The reaction mixture was cooled to room
temperature and poured into ice. The precipitation occurred when the mixture was treated with
10% NaOH aqueous solution to pH ≈ 8. The solid was filtered and washed with water, the title
compound was afforded as white powder (2.5 g, 47.3%). m.p.: 235 - 237 ; 1H-NMR (DMSO-d6,
300 MHz, δ ppm) 12.94 (brs, 1H), 9.96 (s, 1H), 8.15 (s, 1H), 7.82 (d, J = 7.8 Hz, 1H), 6.94 (d, J =
8.1 Hz, 1H), 4.06 (s, 3H); HRMS (ESI): m/z, calcd. For C9H9N2O2 [M + H+] 177.0658, found
177.0663..
6.1.6 4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxy-1H-benzo[d]imidazole-7yl) oxazole (5)
The mixture of aldehyde 4 (650 mg, 3.69 mmol), 5-(isocyano(tosyl)methyl)-1,2,3-trimethoxy-
Benzene [28] (1.33 g, 3.69 mmol) and K2CO3 (764 mg, 5.54 mmol) in CH3OH (40 mL) were
stirred at room temperature for 20 h and then refluxed for 5 h. After the reaction completed, the
reaction mixture was concentrated and the residue was purified by column chromatography on
silica gel with CH2Cl2-MeOH (v/v = 50:1 to 30:1). The desired product was afforded as white
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1
powder (1.1 g, 78.5% ). m.p. 213 - 214℃; H NMR (400 MHz, DMSO-d6): δ 13.02 (brs, 0.5H),
12.51 (brs, 0.5H), 8.52 (s, 1H), 8.17 (s, 1H), 7.35(d, J = 7.6 Hz, 1H), 6.86 (s, 3H), 3.98 (s, 3H),
3.63 (s, 3H), 3.51 (s, 6H); 13C NMR (100 MHz, DMSO-d6): δ 153.40, 151.79, 142.39, 137.71,
134.42, 127.74, 124.75, 104.41, 103.93, 60.71, 56.48, 56.09; HRMS (ESI): m/z, calcd. For
C20H20N3O5 [M + H+] 382.1397, found 382.1393.
6.1.7 4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxy-N1-methyl-benzo[d]imidazole-7yl) oxazole
(6a) and 4-(3,4,5-trimethoxyphenyl)-5-(7-methoxy-N1-methyl-benzo[d]imidazole-4yl) oxazole
(6b)
To the mixture of compound 5 (127 mg, 0.3 mmol) and K2CO3 (62 mg, 0.45 mmol) in acetone (10
mL), MeI (0.09 mL, 0.45 mmol) was added. After stirred at reflux for 24 h, the solution was
evaporated and purified by column chromatography on silica gel with CH2Cl2-MeOH (v/v = 60:1
to 45:1). The title compound 6a (20 mg) was afforded as light yellow solid with 16.8% yield, and
compound 6b (45 mg) was afforded as light yellow solid with 34.5% yield. 6a: m.p. 130 - 132℃;
1H NMR (600 MHz, CDCl3): δ 8.05 (s, 1H), 7.83 (s, 1H), 7.35 (d, J = 7.8 Hz, 1H), 6.82 (d, J = 8.4
Hz, 1H), 6.79 (s, 2H), 4.08 (s, 3H), 3.80 (s, 3H), 3.55 (s, 6H), 3.46 (s, 3H);13C NMR (150 MHz,
CDCl3): δ 153.29, 150.01, 143.77, 141.68, 137.72, 136.40, 134.38, 134.02, 127.99, 127.92, 126.42,
105.34, 103.15, 102.99, 60.85, 56.06, 55.72, 32.44; HRMS (ESI): m/z, calcd. For C21H22N3O5 [M
1
+ H+] 396.1554, found 396.1552; 6b: m.p.152 - 154℃, H NMR (600 MHz, CDCl3): δ 8.05 (s,
1H), 7.85 (s, 1H), 7.37 (d, J = 8.4 Hz, 1H), 6.93 (s, 2H), 6.70 (d, J = 8.4 Hz, 1H), 4.10 (s, 3H),
3.98 (s, 3H), 3.84 (s, 3H), 3.68 (s, 6H); 13C NMR (150 MHz, CDCl3): δ 153.01, 150.08, 148.70,
144.38, 143.94, 143.47, 137.50, 135.09, 127.67, 124.66, 124.27, 113.18, 104.43, 103.37, 60.86,
55.90, 55.78, 34.18. HRMS (ESI): m/z, calcd. For C21H22N3O5 [M + H+] 396.1554, found
396.1557.
6.1.8
7yl)-oxazole (7a)
4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxy-N1-methoxycarbonyl-benzo[d]imidazole-
and
4-(3,4,5-trimethoxyphenyl)-5-(7-methoxy-N1-methoxycarbonyl
-benzo[d]-imidazole-4yl) oxazole (7b).
The mixture of compound 5 (85 mg, 0.22 mmol) and DIEA (0.08 mL, 0.45 mmol) in dry CH2Cl2
(8 mL) were stirred at 0 before addition of methyl chloroformate (0.03 mL, 0.33 mmol). The
reaction mixture was stirred at room temperature for 16 h, and then CH2Cl2 (20 mL) was added to
quench the reaction. The organic phase was washed with water and brine, then dried over
anhydrous MgSO4. After concentration, the crude product was obtained and purified by column
chromatography on silica gel with CH2Cl2-MeOH (v/v = 100:1 to 75:1). The title compound 7a
(35 mg) was afforded as light yellow solid with 35.7% yield, and compound 7b (40 mg) was
afforded as off-white solid with 40.8% yield. 7a: m.p. 171 - 172℃; 1H NMR (400 MHz, CDCl3):
δ 8.37 (s, 1H), 7.95 (s, 1H), 7.48 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.76 (s, 2H), 4.08 (s,
3H), 3.80 (s, 3H), 3.71 (s, 3H), 3.58 (s, 6H); 13C NMR (100 MHz, CDCl3): δ 153.16, 153.03,
149.13, 148.68, 143.60, 142.06, 137.54, 135.14, 134.47, 130.76, 130.17, 126.90, 108.17, 105.90,
103.19, 60.80, 56.22, 55.75, 54.92; HRMS (ESI): m/z, calcd. For C22H22N3O7 [M + H+] 440.1457,
found 440.1456; 7b: m.p. 174 - 175℃; 1H NMR (400 MHz, CDCl3): δ 8.42 (s, 1H), 8.04 (s, 1H),
7.50 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.4 Hz, 1H), 6.86 (s, 2H), 4.09 (s, 3H), 4.00 (s, 3H), 3.82 (s,
3H), 3.65 (s, 6H); 13C NMR (100 MHz, CDCl3): δ 153.03, 150.23, 148.80, 148.74, 144.53, 143.63,
142.88, 137.63, 135.69, 127.25, 126.93, 121.24, 113.76, 107.70, 104.28, 60.80, 56.45, 55.83,
8

ACCEPTED MANUSCRIPT
54.97; HRMS (ESI): m/z, calcd. For C22H22N3O7 [M + H+] 440.1457, found 440.1458.
6.1.9 4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxy-N1-carbethoxymethyl-benzo[d]imidazole-7yl)
oxazole
(8a)
and
4-(3,4,5-trimethoxyphenyl)-5-(7-methoxy-N1--carbethoxymethyl
-benzo[d]imidazole-4yl) oxazole (8b).
The mixture of compound 5 (127 mg, 0.3 mmol), K2CO3 (62 mg, 0.45 mmol) and ethyl
bromoacetate (0.08 mL, 0.78 mmol) in acetone (10 mL) was stirred at room temperature for 20 h.
The reaction mixture was concentrated and the residue was purified by column chromatography
on silica gel with CH2Cl2-MeOH (v/v = 60:1 to 45:1). The desired product 8a (25 mg) was
afforded as light yellow solid with 16.2% yield, and product 8b (100 mg) was afforded as
1
off-white solid with 64.9% yield. 8a: m.p. 149 - 152℃; H NMR (600 MHz, CDCl3): δ 8.01 (s,
1H), 7.83 (s, 1H), 7.27 (d, J = 7.2 Hz, 1H), 6.79 (d, J = 7.8 Hz, 1H), 6.76 (s, 2H), 4.62 (s, 2H),
4.08 (s, 3H), 3.83 (q, J = 7.2 Hz, 2H), 3.81 (s, 3H), 3.57 (s, 6H), 1.06 (t, J = 7.2 Hz, 3H);13C NMR
(150 MHz, CDCl3): δ 166.69, 153.43, 153.15, 150.13, 143.89, 141.46, 137.70, 136.64, 134.69,
133.51, 128.32, 126.11, 105.06, 103.46, 103.41, 61.97, 60.83, 56.06, 55.74, 46.95, 13.81; HRMS
(ESI): m/z, calcd. For C24H26N3O7 [M + H+] 486.1765, found 486.1756; 8b: m.p. 196 - 197℃; 1H
NMR (400 MHz, CDCl3): δ 8.03 (s, 1H), 7.83 (s, 1H), 7.37 (d, J = 8.4 Hz, 1H), 6.91 (s, 2H), 6.71
(d, J = 8.4 Hz, 1H), 5.12 (s, 2H), 4.23 (q, J = 7.2 Hz, 2H), 3.91 (s, 3H), 3.82 (s, 3H), 3.65 (s, 6H),
1.26 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 167.73, 152.94, 149.99, 148.15, 144.15,
143.59, 143.36, 137.41, 135.18, 127.49, 124.47, 124.12, 113.57, 104.30, 103.91, 61.74, 60.76,
55.82, 55.75, 48.18, 14.11; HRMS (ESI): m/z, calcd. For C24H26N3O7 [M + H+] 486.1765, found
486.1767.
6.1.10
4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxy-N1-cyanomethyl-benzo[d]imidazole-7yl)
(9a) and
4-(3,4,5-trimethoxyphenyl)-5-(7-methoxy-N1-cyanomethyl-
oxazole
benzo[d]imidazole-4yl)oxazole (9b).
The mixture of compound 5 (200 mg, 0.52 mmol), K2CO3 (145 mg, 1.05 mmol) bromoacetonitrile
(0.05 mL, 0.68 mmol) in acetone (10 mL) were refluxed for 6 h. The mixture was concentrated
and the residue was purified by column chromatography on silica gel with CH2Cl2-MeOH (v/v =
60:1 to 40:1). The desired product 9a (40 mg) was afforded as light yellow solid with 18.1% yield,
and product 9b (150 mg) was afforded as off-white solid with 68% yield. 9a: m.p. 165 - 167℃;1H
NMR (400 MHz, CDCl3): δ 8.10 (s, 1H), 7.88 (s, 1H), 7.45 (d, J = 7.2 Hz, 1H), 6.90 (d, J = 7.6 Hz,
1H), 6.76 (s, 2H), 4.72 (s, 2H), 4.09 (s, 3H), 3.79 (s, 3H), 3.53 (s, 6H); 13C NMR (100 MHz,
CDCl3): δ 153.49, 150.75, 142.47, 140.59, 137.97, 136.71, 134.62, 131.94, 128.88, 126.03, 112.53,
105.18, 104.48, 103.10, 60.84, 56.19, 55.73, 33.55; HRMS (ESI): m/z, calcd. For C22H21N4O5 [M
1
+ H+] 421.1506, found 421.1501; 9b: m.p. 176 - 177℃; H NMR (400 MHz, CDCl3): δ 8.04 (s,
1H), 7.91 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H), 6.89 (s, 2H), 6.80 (d, J = 8.4 Hz, 1H), 5.36 (s, 2H),
4.02 (s, 3H), 3.82 (s, 3H), 3.66 (s, 6H); 13C NMR (100 MHz, CDCl3): δ 153.01, 150.16, 147.93,
143.35, 143.13, 142.90, 137.58, 135.46, 127.36, 125.32, 123.08, 114.21, 113.96, 104.78, 104.38,
60.80, 56.15, 55.87, 34.75. HRMS (ESI): m/z, calcd. For C22H21N4O5 [M + H+] 421.1506, found
421.1509.
6.1.11
4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxy-N1-hydroxyethyl-benzo[d]imidazole-7yl)
oxazole (10a) and 4-(3,4,5-trimethoxyphenyl)-5-(7-methoxy-N1-hydroxyethyl-benzo[d]-
9

ACCEPTED MANUSCRIPT
imidazole-4yl)oxazole (10b).
The mixture of compound 5 (285 mg, 0.75 mmol) and NaH (90 mg, 2.25 mmol) in dry DMF (10
mL) was stirred at room temperature for 30 min before addition of
(2-bromoethoxy)(tert-butyl)dimethylsilane (375 mg, 1.5 mmol). The mixture was stirred at 60 for
6 h and then diluted with water (20 mL). The resulting mixture was extracted with CH2Cl2 (15 mL
× 3), and the combined organic phase was washed with brine (15 mL × 2) and then dried over
anhydrous MgSO4. After concentration, the crude product was obtained and purified by column
chromatography on silica gel with CH2Cl2-MeOH (v/v = 100:1 to 70:1) to give a mixture of the
protected compound 10a and 10b whose hydroxyl group was protected with
tert-butyldimethylsilane. Then the above compounds were dissolved in dry THF (15 mL) and the
solution of tetrabutyl ammonium fluoride (365 mg, 2.0 mmol) in dry THF (4 mL) was added. The
resulting reaction mixture was stirred at room temperature for 30 min and then poured into
ice/water (20 g). The aqueous phase was extracted with CH2Cl2/MeOH (15 mL × 3, 10:1 ratio),
and the combined organic phase was washed with brine (15 mL × 2) and then dried over
anhydrous MgSO4. After filtration and concentration, the crude product was obtained and purified
by column chromatography on silica gel with CH2Cl2-MeOH (v/v = 30:1 to 25:1 to 18:1). The
desired product 10a (120 mg) was afforded as white solid with 37.6% yield, and product 10b (130
1
mg) was afforded as white solid with 40.7% yield. 10a: m.p. 179 - 180℃; H NMR (400 MHz,
CDCl3): δ 8.05 (s, 1H), 7.80 (s, 1H), 7.21 (d, J = 8.0 Hz, 1H), 6.65 (s, 2H), 6.56 (d, J = 8.0 Hz,
1H), 3.94 (s, 3H), 3.85 (brs, 2H), 3.78 (s, 3H), 3.69 (brs, 2H), 3.46 (s, 6H); 13C NMR (100 MHz,
CDCl3): δ 153.21, 152.49, 149.93, 144.33, 141.54, 137.70, 136.58, 133.85, 132.01, 127.49, 126.03,
105.11, 102.83, 102.70, 60.77, 59.81, 55.87, 55.58, 49.30; HRMS (ESI): m/z, calcd. For
1
C22H24N3O6 [M + H+] 426.1660, found 426.1662; 10b: m.p. 240 - 241℃; H NMR (400 MHz,
DMSO-d6): δ 8.49 (s, 1H), 8.07 (s, 1H), 7.31 (d, J = 5.2 Hz, 1H), 6.93 (d, J = 5.6 Hz, 1H), 6.85 (s,
2H), 4.89 (s, 1H), 4.44 (brs, 2H), 3.96 (s, 3H), 3.72 (brs, 2H), 3.61 (s, 3H), 3.49 (s, 6H); 13C NMR
(100 MHz, DMSO-d6): δ 152.56, 151.05, 148.36, 145.56, 144.06, 143.37, 136.86, 134.09, 127.18,
124.22, 123.26, 112.51, 103.81, 103.76, 60.50, 59.98, 56.06, 55.39, 49.10; HRMS (ESI): m/z,
calcd. For C22H24N3O6 [M + H+] 426.1660, found 426.1659.
6.1.12 4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxy-N1-hydroxypropyl-benzo[d]imidazole-7yl)-
oxazole
(11a)
and
4-(3,4,5-trimethoxyphenyl)-5-(7-methoxy-N1-hydroxypropyl
-benzo[d]imidazole-4yl)oxazole (11b)
Following the same procedure as the compounds of 10a and 10b, the mixture of compound 5 (285
mg, 0.75 mmol), NaH (90 mg, 2.25 mmol) and (3-bromopropoxy)(tert-butyl)dimethylsilane (378
mg, 1.5 mmol) in dry DMF (10 mL) to give the desired product 11a (50 mg) as off-white solid
with 15.2% yield, and product 11b (110 mg) as off-white solid with 33.4% yield. 11a: m.p. 149 -
151℃; 1H NMR (400 MHz, CDCl3): δ 8.02 (s, 1H), 7.89 (s, 1H), 7.29 (d, J = 8.0 Hz, 1H), 6.78 (d,
J = 8.0 Hz, 1H), 6.76 (s, 2H), 4.04 (s, 3H), 3.99 (t, J = 6.8 Hz, 2H), 3.78 (s, 3H), 3.52 (s, 6H), 3.35
(t, J = 5.6 Hz, 2H), 2.55 (brs, 1H), 1.60 (m, 2H); 13C NMR (100 MHz, CDCl3): δ 153.33, 153.23,
150.05, 143.99, 142.06, 137.69, 136.16, 134.79, 133.00, 128.06, 126.30, 105.18, 102.99, 60.79,
58.15, 55.95, 55.66, 42.54, 32.18; HRMS (ESI): m/z, calcd. For C23H26N3O6 [M + H+] 440.1816,
found 440.1817; 11b: m.p. 185 - 186℃; 1H NMR (400 MHz, CDCl3): δ 8.03 (s, 1H), 7.90 (s, 1H),
7.35 (d, J = 8.4 Hz, 1H), 6.91 (s, 2H), 6.70 (d, J = 8.0 Hz, 1H), 4.53 (t, J = 6.4 Hz, 2H), 3.97 (s,
3H), 3.81 (s, 3H), 3.64 (s, 6H), 3.54 (t, J = 5.6 Hz, 2H), 2.64 (brs, 1H), 2.06 (m, 2H); 13C NMR
10

ACCEPTED MANUSCRIPT
(100 MHz, CDCl3): δ 152.95, 150.01, 148.23, 144.46, 143.84, 143.69, 137.47, 135.04, 127.56,
124.21, 123.81, 113.19, 104.38, 103.46, 60.79, 58.30, 55.84, 55.78, 43.71, 33.64; HRMS (ESI):
m/z, calcd. For C23H26N3O6 [M + H+] 440.1816, found 440.1817.
6.1.13
4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxy-N1-carboxymethyl-benzo[d]imidazole-7yl)
oxazole (12a).
The solution of LiOH (21 mg, 0.52 mmol) in water (2 mL) was added to the solution of compound
8a (65 mg, 0.14 mmol) in THF (2 mL) and MeOH (2 mL). The resulting mixture was stirred at
room temperature for 40 min, and then concentrated under reduced pressure. The residue was
dissolved in water (5 mL) and treated with 1N HCl aqueous solution to pH ≈ 4~5. The precipitate
was filtered and washed with water. The title compound 12a (20 mg) was obtained as white solid
with 32% yield. m.p.196 - 198℃; 1H NMR (400 MHz, DMSO-d6): δ 12.90 (brs, 1H), 8.53 (s, 1H),
8.11 (s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.69 (s, 2H), 4.66 (s, 2H), 3.99 (s,
3H), 3.60 (s, 3H), 3.45 (s, 6H) 13C NMR (100 MHz, DMSO-d6): δ 168.80, 152.88, 152.71, 151.59,
144.99, 141.11, 137.10, 135.62, 134.15, 133.68, 127.43, 126.33, 104.82, 103.73, 103.44, 59.99,
55.93, 55.29, 46.19; HRMS (ESI): m/z, calcd. For C22H22N3O7 [M + H+] 440.1452, found
440.1455.
6.1.14 4-(3,4,5-Trimethoxyphenyl)-5-(7-methoxy-N1-carboxymethyl-benzo[d]imidazole-4yl)-
oxazole (12b).
Following the same procedure as the compound of 12a, the mixture of compound 8b (65 mg, 0.14
mmol) THF/MeOH (4 mL) and LiOH (21 mg, 0.52 mmol) in water (2 mL) to give the title
1
compound 12b (40 mg) as white solid with 67.5% yield. m.p.126 - 128℃; H NMR (400 MHz,
DMSO-d6): δ 13.11 (brs, 1H), 8.50 (s, 1H), 8.13 (s, 1H), 7.32 (d, J = 8.4 Hz, 1H), 6.94 (d, J = 8.0
Hz, 1H), 6.83 (s, 2H), 5.19 (s, 2H), 3.91 (s, 3H), 3.61 (s, 3H), 3.48 (s, 6H); 13C NMR (100 MHz,
DMSO-d6): δ 169.97, 152.57, 151.11, 148.47, 145.34, 143.51, 143.09, 136.82, 134.22, 127.13,
124.50, 123.91, 112.62, 104.29, 103.67, 59.98, 56.16, 55.41, 47.80; HRMS (ESI): m/z, calcd. For
C22H22N3O7 [M + H+] 440.1452, found 440.1452.
6.1.15 4-(3,4,5-Trimethoxyphenyl)-5-(4-methoxy-N1-carbamoymethyl-benzo[d]imidazole-
7yl)oxazole (13a).
The mixture of compound 9a (35 mg, 0.083 mmol) and KOH (14 mg, 0.25 mmol) in t-BuOH (5
mL) was refluxed for 1 h. The mixture was cooled to room temperature and diluted with water (15
mL). The aqueous phase was extracted with CH2Cl2/MeOH (15 mL × 6, v/v = 10:1), and the
combined organic phase was washed with water and then dried over anhydrous MgSO4. The
organic phase was concentrated to give the crude product, which was purified by column
chromatography on silica gel with CH2Cl2-MeOH (v/v = 30:1 to 15:1 to 10:1). The desired
product compound 13a (8 mg) was afforded as light yellow solid with 22% yield. m.p.225 - 227℃;
1H NMR (600 MHz, CDCl3): δ 8.02 (s, 1H), 7.94 (brs, 1H), 7.35 (d, J = 7.8 Hz, 1H), 6.84 (d, J =
8.4 Hz, 1H), 6.76 (s, 2H), 5.40 (brs, 1H), 5.33 (brs, 1H), 4.54 (s, 2H), 4.08 (s, 3H), 3.80 (s, 3H),
1
3.56 (s, 6H); H NMR (150 MHz, CDCl3): δ 167.58, 153.33, 153.27, 150.52, 143.88, 140.82,
137.83, 136.87, 134.18, 132.94, 128.82, 126.22, 105.36, 103.89, 103.33, 60.85, 56.16, 55.79,
48.31; HRMS (ESI): m/z, calcd. For C22H23N4O6 [M + H+] 439.1612, found 439.1613.
11

ACCEPTED MANUSCRIPT
6.1.16 4-(3,4,5-Trimethoxyphenyl)-5-(7-methoxy-N1-carbamoymethyl-benzo[d]imidazole-
4yl)oxazole (13b).
Following the procedure as the compound of 13a, the mixture of compound 9b (78 mg, 0.18 mmol)
and KOH (41 mg, 0.74 mmol) in t-BuOH (5 mL) to give the desired product 13b (35 mg) as light
1
yellow solid with 43% yield. m.p. 193 - 195℃; H NMR (400 MHz, DMSO-d6): δ 8.50 (s, 1H),
8.07 (s, 1H), 7.66 (s, 1H), 7.29 (d, J = 8.0 Hz, 1H), 7.20 (s, 1H), 6.90 (d, J = 8.4 Hz, 1H), 6.85 (s,
2H), 5.05 (s, 2H), 3.90 (s, 3H), 3.61 (s, 3H), 3.49 (s, 6H); 13C NMR (100 MHz, DMSO-d6): δ
169.08, 152.58, 151.07, 148.51, 145.90, 143.65, 143.31, 136.83, 134.13, 127.18, 124.24, 124.09,
112.50, 104.00, 103.73, 59.99, 56.05, 55.49, 48.58; HRMS (ESI): m/z, calcd. For C22H23N4O6 [M
+ H+] 439.1612, found 439.1615.
6.1.17 4-(3,4,5-Trimethoxyphenyl)-5-(7-methoxy-N1-(2-carbomethoxy-3-thiophenesulfonyl-
benzo[d]imidazole-4yl)oxazole (14b)
The mixture of compound 5 (200 mg, 0.53 mmol), pyride (0.085 mL, 1.05 mmol) and
2-carbomethoxy-3-thiophenesulfonyl chloride (151 mg, 0.63 mmol) in CH2Cl2 (10 mL) was
stirred at room temperature for 20 h and then diluted with CH2Cl2 (20 mL). The organic phase was
washed with brine (10 mL × 2) and then dried over anhydrous MgSO4, filtered and concentrated to
give the crude product, which was purified by column chromatography on silica gel with
CH2Cl2-MeOH (v/v = 120:1). The title compound 14b (250 mg) was afforded as off-white solid
with 81% yield; m.p. 201 - 202℃; 1H NMR (400 MHz, CDCl3): δ 8.73 (s, 1H), 8.05 (s, 1H), 7.85
(d, J = 5.2 Hz, 1H), 7.62 (d, J = 5.2 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 6.86 (s, 2H), 6.77 (d, J =
8.4 Hz, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 3.76 (s, 3H), 3.66 (s, 6H); 13C NMR (100 MHz, CDCl3): δ
158.63, 153.06, 150.27, 147.57, 145.43, 144.36, 142.61, 140.39, 137.75, 135.82, 135.24, 132.50,
128.95, 127.23, 126.66, 121.17, 114.11, 106.90, 104.48, 60.82, 55.93, 55.62, 53.21; HRMS (ESI):
m/z, calcd. For C26H24N3O9S2 [M + H+] 586.0948, found 586.0953.
6.1.18 2-Methoxy-N-methyl-6-nitroaniline (15)
The mixture of 2-methoxy-6-nitroaniline (504 mg, 3 mmol) and NaH (144 mg, 3.6 mmol) in THF
(10 ml) were stirred at 0℃ for 30 min before addition of MeI (0.2 ml, 3.3 mmol). After stirred at
room temperature for 6 h, the reaction mixture was diluted with water (30 ml) and extracted with
ethyl ether (20 mL × 3). The combined organic layer was washed with water and brine and then
dried over anhydrous MgSO4, filtered and concentrated to give the crude product, which was
purified by column chromatography on silica gel with petroleum ether/AcOEt (v/v = 45:1). The
title compound (340 mg, 62.2% yield) was afforded as salmon pink powder. m.p. 35-36 ;
1H-NMR (CDCl3, 400 MHz, δ ppm): 7.68 (d, J = 8.8 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 6.61 (t, J
= 8.4 Hz, 1H), 3.84 (s, 3H), 3.17 (s, 3H); HRMS (ESI): m/z, calcd. For C8H11N2O3 [M+H+]:
183.0764, found 183.0763.
6.1.19 3-Methoxy-N2-methyl-o-phenylenediamine (16)
The mixture of 2-methoxy-N-methyl-6-nitroaniline (15, 328 mg, 1.8 mmol) and 10% Pd/C (98 mg)
in ethanol (15 ml) was stirred under hydrogen atmosphere at room temperature and atmospheric
pressure for 2 h. The solid was removed by filtration and the filtrate was concentrated. The residue
was purified by column chromatography on silical gel with petroleum ether-AcOEt (v/v = 5:1 to
v/v = 1.5:1). The title compound 16 (188 mg, 68.8% yield) was obtained as off-white oil.
1H-NMR (CDCl3, 400 MHz, δ ppm): 6.83 (t, J = 8.0 Hz, 1H), 6.39 (d, J = 8.0 Hz, 1H), 6.34 (d, J
12

ACCEPTED MANUSCRIPT
= 8.0 Hz, 1H), 3.81 (s, 3H), 3.70 (brs, 2H), 2.67 (s, 3H); HRMS (ESI): m/z, calcd. For C8H13N2O
[M + H+] 153.1022, found 153.1021.
6.1.20 7-Methoxy-1-methyl-1H-benzo[d]imidazole (17)
The reaction mixture of 3-methoxy-N2-methyl-o-phenylenediamine (16, 188 mg, 1.23 mmol)) and
HCOOH (3 mL) was refluxed for 2 h, then cooled to room temperature and poured into ice water
(20 mL). The mixture was treated with 10% NaOH aqueous solution to pH ≈ 8 and then extracted
with ethyl ether (15 mL × 3). The combined extracts were washed with water and brine, then dried
over anhydrous MgSO4. The organic phase was concentrated to give the crude product, which was
purified by column chromatography with CH2Cl2-MeOH (v/v = 100:1). The desired product
7-methoxy-N-methyl-1H-benzo[d]imidazole (100 mg) was afforded as waxy solid in 50% yield.
m.p. 62-63℃; 1H-NMR (CDCl3, 400 MHz, δ ppm): 7.71 (s, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.14 (t,
J = 8.0 Hz, 1H), 6.68 (d, J = 8.0 Hz, 1H), 4.04 (s, 3H), 3.93 (s, 3H); HRMS (ESI): m/z, calcd. For
C9H11N2O [M + H+] 163.0866 found 163.0864.
6.1.21 4-Methoxy-7-carbaldehyde-N1-1H-benzo[d]imidazole (18)
The mixture of 7-methoxy-N-methyl-1H-benzo[d]imidazole (17, 98 mg, 0.605 mmol) and
urotropine (677 mg, 4.84 mmol) in TFA (8 ml) were stirred at reflux for 72 h. The reaction
mixture was cooled to room temperature and poured into ice. The precipitation occurred when the
mixture was treated with 10% NaOH aqueous solution to pH ≈ 8 and then extracted with ethyl
ether (15 mL × 3). The combined extracts were washed with water and brine, then dried over
anhydrous MgSO4. The organic phase was concentrated to give the crude product, which was
purified by column chromatography with CH2Cl2-MeOH (v/v = 100:1 to v/v = 60:1). the title
compound was afforded as white powder (58 mg, 50.4%). m.p. 140 - 142℃; 1H-NMR (DMSO-d6,
400 MHz, δ ppm) 10.51 (s, 1H), 8.25 (s, 1H), 7.69 (d, J = 8.4 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H),
4.02 (s, 3H), 4.00 (s, 3H); HRMS (ESI): m/z, calcd. For C10H11N2O2 [M + H+] 191.0815, found
191.0816.
6.1.22
(6b)
4-(3,4,5-trimethoxyphenyl)-5-(7-methoxy-N1-methyl-benzo[d]imidazole-4yl)oxazole
The mixture of aldehyde 18 (28 mg, 0.145 mmol), 5-(isocyano(tosyl)methyl)-1,2,3-trimethoxy-
benzene (58 mg, 0.16 mmol) and K2CO3 (30 mg, 0.217 mmol) in CH3OH (3 mL) were stirred at
room temperature for 4 h and then refluxed for 5 h. After the reaction completed, the reaction
mixture was concentrated and the residue was purified by column chromatography on silica gel
with CH2Cl2-MeOH (v/v = 60:1 to 45:1). The desired product was afforded as white powder (20
1
mg, 35% ). H-NMR ( CDCl3, 400 MHz, δ ppm) 8.05 (s, 1H), 7.80 (s, 1H), 7.36 (d, J = 8.4 Hz,
1H), 6.93 (s, 2H), 6.70 (d, J = 8.4 Hz, 1H), 4.10 (s, 3H), 3.98 (s, 3H), 3.84 (s, 3H), 3.67 (s, 6H).
6.2 Biological evaluation
6.2.1 Cell culture
The human breast adenocarcinoma cell line MCF-7, the human colon carcinoma cell line
HT29, the human pancreas carcinoma cell line BxPC-3, the human lung adenocarcinoma cell line
A549, the human hepatocellular carcinoma cell line HepG2, human nasopharyngeal
carcinoma cell line KB, vincristine resistant KB cell line KBV, human breast cancer
cell line MX-1 and taxol resistant MX-1 cell line MX-1/T were cultured in RPMI-1640
medium supplemented with 10% FBS, and incubated at 37°C in a humidified atmosphere that
13

ACCEPTED MANUSCRIPT
contained 5% CO2. All the cells were purchased from the American Type Culture Collection
(ATCC) and the Cell Culture Center of Institute of Basic Medical Science, Chinese Academy of
Medical Sciences.
6.2.2 Cytotoxicity assay
The in vitro cytotoxic activities of tested compounds were determined by MTT (Sigma) assay.
Briefly, cells were seeded into 96-well plates at a density of 1~2×103/well (depended on the cell
growth rate). 24 hr later, triplicate wells were treated with media and agents. After 72 h incubated
at 37℃ in 5% CO2, the drug containing medium was removed and replaced by 100 ꢀl fresh
medium with 0.5 mg/ml MTT solution. After 4h incubation, the medium with MTT was removed
and 150 ꢀl DMSO was added to each well. The plates were gently agitated until the color reaction
was uniform and the OD570 was determined using a microplate reader (Wellscan MK3,
Labsystems Dragon). Microsoft® Excel 2003 was used for data analysis. Media-only treated cells
served as the indicator of 100% cell viability. The 50% inhibitory concentration (IC50) was defined
as the concentration that reduced the absorbance of the untreated wells by 50% of the vehicle in
the MTT assay.
6.2.3 Tubulin polymerization assay
The inhibitory activity of compounds 5 and CA-4 on the polymerization of tubulin was
evaluated using Cytoskeleton assay kit (Cat. #BK011P). According to the manufacture’s protocol,
the reaction mixture containing assay buffer 1, tubulin glycerol buffer, GTP stock and tubulin
stock was prepared immediately and stored on ice. The 96 well plate was warmed in the plate
reader at 37ºC for 10 min. Then, 5 ꢀL compound 5 or CA-4 was added at appropriate
concentrations into the well and re-warmed in the plate reader for 1 min. 50 ꢀL tubulin reaction
mixture was pipetted quickly into each well and the data were collected at 340 nm using
SpectraMax 190 spectrophotometer (Company of Molecular Devices). The readings were taken at
37ºC for 60 min at 1 min interval. IC50s, which were the compound concentrations required to
inhibit the polymerization of tubulin by 50%, were obtained using GraphPad Prism 4.00 software.
6.2.4 Flow cytometric analysis
Human colorectal cancer cell line HT29, preserved by our institute, was cultured in
DMEM/F12 medium supplemented with 10% fetal calf serum (Gibco BRL Life Technologies,
Grand Island, NY, USA), and incubated at 37°C in a humidified atmosphere that contained 5%
CO2. HT29 cells treated without or with compound 5 at various concentrations (12.5 nM, 25 nM,
50 nM and 100 nM) and CA4 at 25 nM concentration for 24 hour were collected by trypsinization,
washed twice with phosphate-buffered saline (PBS) and fixed in ice-cold 70% (v/v) ethanol at
-20°C for 24 h. Before testing, the fixed cells were washed with PBS and re-suspended in PBS
that contained 50 mg/ml propidium iodide and 0.5 mg/ml RNase A for 30 min at 37°C. The DNA
contents of HT29 cells were measured by flow cytometer (Partec cyflow ML, Germany). All data
were recorded and analyzed using FCS Express 4 software.
6.2.5 The in vivo antitumor activity
The male KM mice with the weight of 18-22 g were purchased from the Vital River
Laboratory Animal Technology Co. Ltd (Beijing, China). Mice stained with H22 liver cancer used
14

ACCEPTED MANUSCRIPT
in our study were housed under SPF breeding facility and maintained according to the guidelines
of the Committee on Animals of the Institute of Materia Medica, Chinese Academy of Medical
Sciences & Peking Union Medical College. Ascites was removed under sterile condition from
mice with H22 liver cancer and diluted with normal saline in a 1:3 ratio (v/v). The suspension of
tumor cells was inoculated subcutaneously into the oxter of KM mice. After inoculation for 24 h,
tumor-bearing mice were randomized to seven groups. The control group was treated with saline
by intraperitoneal (i.p.) injection; the positive control group was treated with VCR (0.25 mg/kg
and 0.5 mg/kg) on days 1 and 4; and other four groups were treated with compound 5 at different
doses via i.p. injection. The first group was administrated at a dose of 7.5 mg/kg on days 1-7. The
second group was administrated at a dose of 10 mg/kg on days 1, 3, 5 and 7. The third group was
administrated at a dose of 15 mg/kg on days 1 and 4. The fourth group was administrated at a dose
of 20 mg/kg on day 1. The mice were sacrificed on day 8 and the body weight and tumors were
weighed. The experimental results were expressed as mean ± standard deviation (SD). The
inhibition rate of tumor weight (%) was calculated using the equation [(Wcontrol
–
Wtreatment)/Wcontrol]×100%, where W is tumor weight. Independent t test was used to evaluate the
differences between experimental and control groups with SPSS version 13.0 (SPSS, Chicago, IL,
USA).
6.3 Computational studies
All molecular computation studies were performed on a Dell 2.83 GHz Core 2 running Windows
XP. The X-ray crystal structure of tubulin complexed with DAMA-colchicine was retrieved from
PDB (PDB code: 1SA0) [30]. DAMA-colchicine, CA-4 and compound 5 were drawn with
Chemdraw and fully minimized using the CHARMm force field. The CDOCKER software
package integrated in Accelrys Discovery Studio 2.5.5 was used to dock each of the molecules
into the colchicine site of tubulin. CDOCKER uses molecular dynamics (MD) with CHARMm
force field scheme to dock ligands into a binding site. The binding site was constructed within 5 Å
with DAMA-colchicine set as the center. DAMA-colchicine was then docked into the binding site
and 10 conformations were gained with the RMSD values between 0.8 Å and 0.9 Å to prove the
availability of the docking method. Then CA-4 and compound 5 were docked into the same
binding site, respectively. The 10 final docked conformations of each of ligands were ranked
according to the negative CDOCKER interaction energy. The docking mode was chosen on the
basis of the negative CDOCKER interaction energy.
Acknowledgments
This work is supported by the Special Funds for National Public Benefit Research Institutes (No.
2010CHX03) and National Program of New Drug Innovation and Production (No.
2009ZX09301-003-1-1).
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The collected figure captions:
Figure 1. Structures of CA-4, CA-4 analogs in clinical trial and designed oxazole derivatives.
Figure 2. Flow cytometry analysis of compound 5 in HT29 cells. (A) Cell cycle distribution of HT29 cells after
treatment with compound 5 at different concentrations and CA4 at 25 nM concentration for 24 h. An increased
proportion of cells at G2/M phase were observed in cells treated with compound 5. (B) The statistical graph of cell
cycle distribution.
Figure 3. Comparison of the docked poses of compound 5 (pink), CA-4 (orange) with the crystal structure of
DAMA-colchicine (green). H-Bonding interactions are presented with green line. The chemical structure of
DAMA-colchicine is presented. Molecular image was generated with UCSF Chimera [32].
18

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Table 1.
The in vitro antiproliferative activities of oxazole derivatives (5, 6a-13a, 6b-14b), VCR and CA-4 against five
human cancer cell lines including MCF-7, A549, HT29, HepG2 and BxPC-3.
N
O
O
R1
N
O
N
O
O
6a-13a
6b-14b
Compound
R1
Cytotoxicity (IC50, nM) a
number
5
MCF – 7
25
A549
NDb
HT29
8.4
HepG2
BxPC-3
31
H
CH3
NDb
200
20
6a
41
37
52
220
7a
COOCH3
17
3.8
11
42
8a
CH2COOC2H5
CH2CN
419
23.4
>5000
NDb
NDb
>5000
>5000
9.6
350
500
870
130
310
>5000
870
29
>500
1390
NDb
NDb
>5000
1390
56
9a
2050
540
1600
180
10a
11a
12a
13a
6b
(CH2)2OH
(CH2)3OH
CH2COOH
CH2CONH2
CH3
500
290
>5000
2050
39
>5000
1600
14
7b
COOCH3
47
3.0
11
25
45
8b
CH2COOC2H5
CH2CN
>500
89
>500
2600
NDb
NDb
>5000
NDb
2510
>500
190
>500
77
>500
2190
NDb
NDb
>5000
NDb
941
9b
10b
11b
12b
13b
14b
(CH2)2OH
(CH2)3OH
CH2COOH
CH2CONH2
2-Carbomethoxy-3-
thiophenesulfonyl
VCR
510
170
250
410
>5000
990
134
2110
>5000
>10000
435
2220
>5000
>10000
163
13
53
NDb
12
210
NDb
>500
<50
CA-4
220
270
aCompound dose (nM) required to inhibit cell growth by 50%;
bND, not determined.
19

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Table 2.
The in vitro antiproliferative activities of oxazole derivatives, CA-4 and VCR against cancer cell lines KB, KBV,
MX-1 and MX-1/T.
Compound
Cytotoxicity (IC50, nM) a
KB
16
KBV
MX-1
397
MX-1/T
249
5
6b
65
27
41
334
12820
565
7a
27
70
393
7b
107
346
406
8.0
139
53
241
4860
>10000
21080
71
11a
11b
CA-4
VCR
>10000
>10000
6.0
3778
>10000
52
>100000
12
75
aCompound dose (nM) required to inhibit cell growth by 50%;
20

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Table 3.
The in vivo antitumor activity of compound 5 against H22 xenografts in KM mice.
Group
Dose
Animal No.
Body Weight(g)±SD
(initial/finish)
Tumor Weight
(g)±SD
Inhibition
(%)c
(mg/kg×t)
(initial/finish)
Control
VCR(i.p.)a,b
VCR(i.p.) a,b
5(i.p.)b
5(i.p.) b
5(i.p.) b
8/8
8/8
8/7
8/8
8/6
8/8
8/8
18.50±1.20/30.00±3.24
18.90±0.83/24.60±1.36
19.00±1.31/25.50±1.98
18.90±0.83/30.40±1.96
18.75±1.28/24.40±3.39
20.00±1.20/28.40±3.69
18.90±0.83/25.50±1.56
2.01±0.31
1.38±0.24
0.78±0.30
1.15±0.44
0.68±0.32
0.96±0.33***
0.90±0.35***
0.25×2
0.5×2
20×1
31***
61***
42***
66***
52***
55***
15×2
10×4
5(i.p.) b
7.5×7
Note: aVCR is an abbreviation of vincristine; bi.p. means intraperitoneally; c ***p<0.001
21

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R1 = H, R2 = OH (CA-4)
6a-13a
6b-14b
R1 = H, R2 = OPO3Na2 (CA4P)
R1 = H, R2 = NH2 (AC-7739)
R1 = H, R2 = NHCOCHNH2CH2OH (AVE-8062)
R1 = OPO3Na2, R2 = OPO3Na2 (CA1P)
Figure 1. Structures of CA-4, CA-4 analogs in clinical trial and designed oxazole derivatives.
22

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Figure 2. Flow cytometry analysis of compound 5 in HT29 cells. (A) Cell cycle distribution of HT29 cells after
treatment with compound 5 at different concentrations and CA4 at 25 nM concentration for 24 h. An increased
proportion of cells at G2/M phase were observed in cells treated with compound 5. (B) The statistical graph of cell
cycle distribution.
23

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O
H3CO
H3CO
CH2SH
NH
H3CO
O
OCH3
DAMA-colchicine
Figure 3. Comparison of the docked poses of compound 5 (pink), CA-4 (orange) with the crystal structure of
DAMA-colchicine (green). H-Bonding interactions are presented with green line. The chemical structure of
DAMA-colchicine is presented. Molecular image was generated with UCSF Chimera [32].
24

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O
O
O
2
O
1
OH
N
NH2
NH2
NH2
NO2
N
NH2
NO2
d
c
a
b
N
H
N
H
3
4
O
H
N
N
N
2
2
O
O
O
O
O
O
O
O
R1
N
f
e
A
A
O
N
N
+
6
6
O
B
B
N
NH
N
O
O
R1
O
O
O
5
6a-11a
6b-11b,14b
g
8a,8b
9a,9b
12a,12b
13a,13b
h
Scheme 1. Reagents and conditions: a) CH3I, K2CO3, DMF, rt; b) 10%Pd/C, EtOH, H2, rt; c) HCOOH, reflux; d)
hexamethylenetetramine, TFA, reflux; e) 3,4,5-trimethoxyphenyl TosMIC, K2CO3, MeOH, rt-reflux; f)
MeI/BrCH2CO2C2H5/BrCN, K2CO3, acetone, reflux or CH3OCOCl, DIEA, DCM, 0℃-rt or
(2-bromoethoxy)(tert-butyl)dimethylsilane/ (3-bromopropoxy)(tert-butyl)dimethylsilane, NaH, DMF, 40℃ or
2-carbomethoxy-3-thiophenesulfonyl chloride, pyridine, DCM, rt; g) LiOH, THF/H2O, rt; h) KOH, t-BuOH,
reflux
25

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NO2
NH2
NO2
NH2
N
N
a
b
c
N
H
N
H
OH
O
O
O
15
16
17
H
O
N
O
O
O
N
e
d
A
O
N
N
B
N
O
O
18
6b
Scheme 2. Reagents and conditions: a) CH3I/NaH/THF/0℃-rt; b) 10% Pd/C, EtOH, H2, rt; c) HCOOH, reflux; d)
hexamethylenetetramine, TFA, reflux; e) 3,4,5-trimethoxyphenyl TosMIC, K2CO3, MeOH, rt-reflux.
26

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Highlights:
► Novel benzoimidazole-contained analogs of combretastatin A-4 were synthesized.
► The chemical structures of regioisomers 6a-13a and 6b-14b were confirmed.
► Four compounds exhibited excellent antiproliferative activities in vitro.
► Compound 5 possessed significant in vivo antitumor activity.
► Benzoimidazole is a favorable replacement of the B ring on CA-4.

ACCEPTED MANUSCRIPT
Supporting Information
Synthesis and antiproliferative evaluation of novel
benzoimidazole-contained oxazole-bridged analogs of combretastatin
A-4
Jie Zhou, Jing Jin, Yi Zhang, Xiaoguang Chen, Bailing Xu*
Table of Contents
Page
S-3
1H NMR Spectrum of Compound 5
13C NMR Spectrum of Compound 5
1H NMR Spectrum of Compound 6a
13C NMR Spectrum of Compound 6a
1H NMR Spectrum of Compound 6b
13C NMR Spectrum of Compound 6b
1H NMR Spectrum of Compound 7a
13C NMR Spectrum of Compound 7a
1H NMR Spectrum of Compound 7b
13C NMR Spectrum of Compound 7b
1H NMR Spectrum of Compound 8a
13C NMR Spectrum of Compound 8a
1H NMR Spectrum of Compound 8b
13C NMR Spectrum of Compound 8b
1H NMR Spectrum of Compound 9a
13C NMR Spectrum of Compound 9a
1H NMR Spectrum of Compound 9b
13C NMR Spectrum of Compound 9b
1H NMR Spectrum of Compound 10a
13C NMR Spectrum of Compound 10a
1H NMR Spectrum of Compound 10b
13C NMR Spectrum of Compound 10b
1H NMR Spectrum of Compound 11a
13C NMR Spectrum of Compound 11a
1H NMR Spectrum of Compound 11b
13C NMR Spectrum of Compound 11b
1H NMR Spectrum of Compound 12a
13C NMR Spectrum of Compound 12a
1H NMR Spectrum of Compound 12b
13C NMR Spectrum of Compound 12b
S-3
S-4
S-4
S-5
S-5
S-6
S-6
S-7
S-7
S-8
S-8
S-9
S-9
S-10
S-10
S-11
S-11
S-12
S-12
S-13
S-13
S-14
S-14
S-15
S-15
S-16
S-16
S-17
S-17
S-1