Design, synthesis and biological studies of novel tubulin inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.04.078

A series of compounds originally derived from the vascular endothelial growth factor receptor tyrosine kinase inhibitor, SU5416, were synthesized and evaluated. The most potent compound in this series, compound 3, which structurally resembles the potent a

Full text of DOI:10.1016/j.bmcl.2013.04.078

Accepted Manuscript
Design, synthesis and biological studies of novel tubulin inhibitors
Yanjun Sun, Bulbul Pandit, Somsundaram N. Chettiar, Jonathan P. Etter,
Andrew Lewis, Jayasekar Johnsamuel, Pui-Kai Li
PII:
S0960-894X(13)00568-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.04.078
BMCL 20443
Reference:
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Bioorganic & Medicinal Chemistry Letters
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Accepted Date:
13 February 2013
23 April 2013
29 April 2013
Please cite this article as: Sun, Y., Pandit, B., Chettiar, S.N., Etter, J.P., Lewis, A., Johnsamuel, J., Li, P-K., Design,
synthesis and biological studies of novel tubulin inhibitors, Bioorganic & Medicinal Chemistry Letters (2013), doi:
http://dx.doi.org/10.1016/j.bmcl.2013.04.078
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Design, synthesis and biological studies of novel tubulin inhibitors
Yanjun Sun, Bulbul Pandit, Somsundaram N. Chettiar, Jonathan P. Etter, Andrew Lewis,
Jayasekar Johnsamuel and Pui-Kai Li*
Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State
University, Columbus Ohio 43210
Corresponence to:
Pui-Kai Li, Ph.D.,
College of Pharmacy, The Ohio State University,
338 Parks Hall, 500 West 12th Avenue,
Columbus, OH 43210-1291
Tel: (614) 688-0253
Fax: (614) 688-8556
E-mail: li.27@osu.edu
1

ABSTRACT: A series of compounds originally derived from the vascular endothelial growth
factor receptor tyrosine kinase inhibitor, SU5416, were synthesized and evaluated. The most
potent compound in this series, compound 3, structurally resemble the potent anti-microtubule
agent Combretastatin A-4, and inhibited tubulin polymerization and showed potent growth
inhibitory activities on both prostate and breast cancer lines with IC50 values in the low
nanomolar range.
2

Anti-microtubule agents such as Taxol and Vinca alkaloids have been used for the
treatment of cancer for many years. However, among the 3 classes of tubulin binding agents (i.
microtubule stabilizing agents, ii. vinca site binding agents and iii. colchicine site binding
agents), colchicine site binding agents are the only class of agents that do not have a
representative drug in clinical use for cancer. This class includes many natural products as well
as synthetic small molecules.1,2 Among all the colchicine site agents, Combretastatin A-4 (CA-4,
Fig. 1) has received special attention in the last decade or so.3 In addition to its potent
cytotoxicity and inhibitory activity on tubulin polymerization, CA-4 is one of the few anti-
microtubule agents reported to have selective vascular targeting activity, and it is an agent that
selectively destroyed tumor blood vessels and resulted in tumor cell death.4,5 The anti-tumor and
anti-vascular activities of CA-4 and its water-soluble prodrug, Combretastatin A-4 phosphate
(CA-4P), have been demonstrated in both animal and human studies.6-9
Many analogs of CA-4 have been designed to study the structure-activity-relationship of
the molecule in order to enhance both its cytotoxic and selective vascular targeting activity.3,10
We have been using SU5416,11,12 a potent ATP competitive VEGF receptor tyrosine kinase
inhibitor, as a template for the design of inhibitors for other growth factor receptors. Previously,
we discovered and reported a 2-indolinone containing compound (compound 1, Fig. 1) with
potent growth inhibitory activities on hormone-independent prostate (PC-3) and breast (MDA-
MB-231) cancer cell lines with IC50 values in low to subnanomolar range.13 The anti-
proliferative profile of compound 1 was also evaluated at the National Cancer Institute (NCI)
against 53 human cancer cell lines. Compound 1 was extremely potent with a GI50 below 10 nM
towards 46 out of 53 human cancer cell lines tested. The compound is active in colon, CNS,
prostate and renal cancer cell lines.14 Structure-activity-relationship studies showed that
compound 1 isomerized to compound 2 which has no tubulin polymerization inhibitory activity.
In our continuous search for potent inhibitors of tubulin polymerization and vascular disrupting
agents, we here report the synthesis and biological studies of seven analogs of compound 1 (Fig.
2). One of our analogs 3, exhibits potent cytotoxicity against prostate (PC-3) and breast cancer
cells (MDA-MB-231) in vitro, as well as inhibition of tubulin polymerization.
3

Figure 1. Structures of Combretastatin A-4 (CA-4), compounds 1 and 2.
Figure 2. Proposed compound 1 analogs.
Compound 1 has potent anti-proliferative and tubulin polymerization inhibitory activity,
but tends to isomerize to compound 2 in solution which has no tubulin polymerization inhibitory
activity. This phenomenon is also observed in the KDR inhibitor SU5416.15 In our continuous
effort to design potent anti-microtubule agents; we have designed, synthesized and biologically
evaluated analogs of compound 1 that do not have the ability to be deactivated through
4

isomerization. To design these types of analogs, the indolin-2-one in compound 1 has been
replaced with a benzimidazol-2-one (3, 4 and 5), benzoxazol-2-one (6 and 7), or 1H-indole-2,3-
dione (8). For compound 1, the 6-methoxy group in the indolin-one and the trimethoxy group on
the benzylidene ring are essential for potent cytotoxicity and inhibition of tubulin
polymerization. Compounds 3 – 9 were designed with a methoxy in the position corresponding
to the 6-methoxy group of compound 1. In addition, the compounds also have 3,4,5-trimethoxy
phenyl ring that mimic the same group in compound 1.
The synthetic route used for the synthesis of 3 and 4 are shown in Fig. 3. In brief,
benzylation of 4-methoxy-2-nitroaniline yielded 10. The nitro group of 10 was reduced using
using Zn/HOAc to afford 11. Compound 3 was prepared by cyclization of 11 with N,N-
carbonyldiimidazole (CDI). Methylation of 3 with methyl iodide provided 4.
Figure 3. Synthesis of 3 and 4: a. K2CO3, KI, DMF, rt, 4h; b. Zn, HOAc, 110°C, 2h; c. CDI,
THF, rt, overnight; d. MeI, K2CO3, KI, DMF, rt, 24h.
The conversion of 10 to 11 was carried out using two different reaction conditions;
SnCl2/HCl (reaction condition a or c in Fig 4) and Zn/HOAc (condition b in Fig. 4). Compound
11 appeared to be formed under both reaction conditions, when monitored by TLC. The reactions
were worked up and the product was extracted with ethyl acetate. Surprisingly only compound
5

12 was isolated (Fig 4). The structure of 12 was confirmed by 1D and 2D NMR spectra as well
as high resolution mass spectrometry. Interestingly, compound 11 was recovered instead of 12,
when dichloromethane was used for extraction instead of ethyl acetate (Fig. 4).
Figure 4. Formation of compound 12: a. SnCl2·2H2O, HCl, EtOH, 80°C-90°C, 30 min (workup
with EtOAc); b. Zn, HOAc, 110°C, 2h (workup with EtOAc); c. SnCl2·2H2O, HCl, EtOH, 80°C-
90°C, 30 min (workup with CH2Cl2).
We postulated that compound 12 was formed from the reaction between 11 and ethyl
acetate (Fig. 5). The reaction is proposed to start with the acetylation of 11 to form the
intermediate, N-acetamide, which went through intramolecular cyclization to form the
intermediate, benzoimidazol-2-ol. Compound 12 was then formed by dehydration of the
benzoimidazol-2-ol intermediate.
6

Figure 5. Proposed mechanism for the formation of 12 from 11.
Preparation of compound 5 was achieved by using a procedure similar to the one described
in Fig. 3, where the corresponding commercially available 3,4,5-trimethoxy benzoyl chloride
was used instead of 3,4,5-trimethoxy benzyl bromide (Fig. 6).
Figure 6. Synthesis of compound 5: a. Et3N, CH2Cl2, rt, 2h ; b. SnCl2·2H2O, HCl, EtOH, 80°C-
90°C, 30 min; c. CDI, THF, rt, overnight.
7

Both compounds 6 and 7 were synthesized via a one-step reaction (Fig 7). For the
synthesis of 6, 6-methoxy-3H-benzooxazol-2-one was stirred with 3,4,5-trimethoxybenzyl
bromide in DMF at 65oC for 3 hours. A milder reaction condition was used for the preparation of
7; 6-methoxy-3H-benzooxazol-2-one was stirred with triethylamine and methylene chloride
overnight at room temperature.
Figure 7. Synthesis of compound 6 and 7: a. K2CO3, DMF, 65°C, 3h; b. Et3N, CH2Cl2, rt,
overnight.
For the synthesis of 9, we first adopted a procedure similar to the synthesis of 6, the
reaction failed to produce the desired product (Fig. 8). Instead, compound 9 was obtained
through Wolff Kisner reduction of indole-2,3-dione (8), which was readily synthesized by
stirring 5-methoxy-1H-2,3-dione with 3,4,5-trimethyl benzyl bromide.
8

Figure 8. Synthesis of compounds 8 and 9: a. K2CO3, DMF, 65°C, 3h; b. Et3N, CH2Cl2, rt,
overnight. c. K2CO3, KI, DMF, rt, 4h; d. H2NNH2, reflux, overnight.
The anti-proliferative activities of compounds 3 – 9 were first examined in PC-3, and
MDA-MB-231 cancer cells via the standard MTS assay (Table 1). Compounds 3 – 5 all contain
the benimidazol-2-one moiety. Compound 3 exhibited potent anti-proliferative activities on both
PC-3 and MDA-MB-231 cells with IC50 of 44.25 and 52.75 nM respectively. Replacing the
trimethoxybenzyl group of 3 with a trimethoxybenzoyl group (5) caused a slight decrease in the
anti-proliferative activities in both cancer cell lines. In addition, replacing the NH group of 3
with a CH2 (9) also caused a slight decrease in anti-proliferative activities in both cancer cell
lines to the same extent as 5. However, the N-methylated version of compound 3, which is 4,
resulted in more than a 10 fold decrease in the anti-proliferative activities in PC-3 cells and more
than a 50 fold decrease in MDA-MB-231 cells when compared to 3. Lastly, replacement of the
indol-2-one moiety of 3 with a benzoxazol-2-one (compounds 6 and 7) proved to be detrimental
to the anti-proliferative activities. Both compounds 6 and 7 are at least 100 fold less active than
compound 3.
In addition to anti-proliferative activities, all of the compounds were also tested on their
abilities to inhibit tubulin polymerization in purified porcine brain tubulin. We observed a direct
9

correlation between anti-proliferative activities and inhibition of tubulin polymerization.
Compound 3 is the most potent inhibitor of tubulin polymerization whereas compounds 6 and 7
are inactive.
Compound #
PC-3
(IC50 nM)
MDA-MB-231
(IC50 nM)
Tubulin polymerization
inhibition
(IC50 M)
4.5
8.2 ± 1.1
44.25 ± 2.3
555.1 ± 10.1
125.5 ± 5.5
4486
0.88 ± 0.02
52.75 ± 4.20
3027.5
1
3
16.4
>40
19.14
>40
n.d
4
108.5 ± 4.7
4520
5
6
>20000
>20000
7
318.5 ± 9.8
165.2 ± 6.6
934.6 ± 22.9
193.3 ± 8.8
n.d
8*
9
31.2
* Solubility was poor in DMSO, IC50 values not accurate
Table 1. Inhibition of cancer cell proliferation and tubulin polymerization by compounds 3 - 9. Cells
(2,500 cells/well) were treated with varying concentration of compounds and cell associated protein was
determined using MTS assay. The IC50 values represent the means of 3 independent experiments that
were run in triplicate. Tubulin polymerization inhibition was determined as described in reference 13.
In conclusion, we have designed and synthesized several tubulin inhibitors derived from
our lead molecule - compound 1. The most potent compound in this series, compound 3,
exhibited potent anti-proliferative activities on both PC-3 and MDA-MB-231 cells as well as
potent inhibitory activities on tubulin polymerization.
Acknowledgment
This research is supported in part by the Department of Defense Prostate Cancer Research
Program Award W81XWH-08-1-0555
10

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11

*Graphical Abstract (for review)
Graphical Abstract
Design, synthesis and biological studies of novel
tubulin inhibitors
Leave this area blank for abstract info.
Yanjun Sun, Bulbul Pandit, Somsundaram N. Chettiar, Jonathan P. Etter, Andrew Lewis, Jayasekar Johnsamuel and
Pui-Kai Li*