Synthesis and biological evaluation of benzo[d]imidazole derivatives as potential anti-cancer agents

Article abstract of DOI:10.1016/j.bmcl.2011.12.088

We herein report the synthesis, biological activity and structure-activity relationship of derivatives of 5,6-dichloro-1-β-d- ribofuranosylbenzimidazole and benzo[d]imidazole. A lead compound 6o demonstrates potent anti-proliferative activity and the ability to induce cancer cell apoptosis.

Full text of DOI:10.1016/j.bmcl.2011.12.088

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1317–1321
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of benzo[d]imidazole derivatives
as potential anti-cancer agents
⇑
Hamad M. Alkahtani, Abdullahi Y. Abbas, Shudong Wang
School of Pharmacy and Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
We herein report the synthesis, biological activity and structure–activity relationship of derivatives of
Received 17 October 2011
Revised 14 December 2011
Accepted 16 December 2011
Available online 23 December 2011
5,6-dichloro-1-b-
D-ribofuranosylbenzimidazole and benzo[d]imidazole. A lead compound 6o demon-
strates potent anti-proliferative activity and the ability to induce cancer cell apoptosis.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
RNA polymerase-II
Cyclin-dependent kinase inhibitor
Apoptosis
Anti-cancer therapy
Despite more than 13 members having been identified, the
cyclin-dependent kinases (CDKs) can generally be divided into
two groups based on roles in cell-cycle progression and transcrip-
tional regulation. Association of CDK1, CDK2, CDK4, and CDK6 with
A-, B-, E-, and D-type cyclins ensures an orderly and controlled pro-
gression of the cell proliferation cycle. CDK7 and CDK9, on the
other hand, are primarily involved in the regulation of transcrip-
tion through phosphorylation of the C-terminal domain (CTD) of
RNA polymerase-II (RNAP-II).1 De-regulation of various compo-
nents controlling the cell-cycle plays an essential role in tumor
pathogenesis. This has promoted the development of pharmaco-
logical small-molecule CDK inhibitors that can be used for cancer
therapy. However, the fact that transformed cells depleted of cyc-
lins and CDKs continue to proliferate2 indicates that that the spe-
cific targeting of individual cell-cycle CDKs may not be an
optimal therapeutic strategy due to functional redundancy.1
Flavopiridol, a pioneer pan-CDK inhibitor, has demonstrated
marked efficacy in refractory and relapsed chronic lymphocytic
leukemia (CLL).3 This has been attributed to flavopiridol inhibition
of CDK-mediated transcriptional regulation. CLL cells are mostly
accumulated mature B cells which are resistant to apoptosis.
Although strongly inhibiting most CDKs, the primary mechanism
of action of flavopiridol is believed to be through inhibiting CDK9
and altering the transcription of apoptosis-related proteins in
CLL. Flavopiridol is the most potent CDK9 inhibitor identified,
and it has been shown that this inhibition is associated with a de-
cline in short-lived anti-apoptotic proteins Mcl-1 and X-linked
Cl
Cl
N
N
DRB
OH
O
HO
OH
HO
R
R
N
N
R1
R
R
N
N
R1
O
HO
R2
HO
I
II
Figure 1. Structures of DRB and the designated derivatives.
inhibitor of apoptosis (XIAP), which results in reinstatement of
the ability of CLL cell to undergo apoptosis.4
5,6-Dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) is an
adenosine analog that has been shown to inhibit RNAPII transcrip-
tion both in vitro and in vivo.5,6 It acts by competing with ATP7 and
is believed to be the most selective CDK9 inhibitor available to
date.1 It inhibits CDK9 potently with an IC50 value of 600 nM by
biochemical assay and shows excellent selectivity against other
CDKs. However, this striking selectivity is not associated with the
level of cellular potency essential for anti-cancer agents.
Our interest in developing drugs that target transcriptional
CDKs has resulted in the discovery of several pre-clinical and clin-
ical candidates for cancer therapy.8–13 In order to identify more
selective CDK9 inhibitors we selected DRB as a model for chemical
⇑
Corresponding author. Tel.: +44 1158466863; fax: +44 01159513412.
E-mail address: shudong.wang@nottingham.ac.uk (S. Wang).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.088

1318
H. M. Alkahtani et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1317–1321
modification and expansion aimed at defining the structure activ-
ity relationship and improving cellular potency and drug-likeness
by replacing the metabolically-liable ribose of DRB with an aryl
or a carbocycle. Accordingly, we prepared a class of DRB and
benzo[d]imidazole derivatives (Type-I and Type-II, Fig. 1). In this
report, we describe the synthesis and biological evaluation of these
compounds. The initial structure–activity relationship analysis
provided invaluable guidance in progressing our CDK9-targeted
drug discovery programmes.
The synthetic chemistry employed to prepare Type-I and -II
compounds is outlined in Scheme 1. Condensation reactions be-
tween benzene-1,2-diamine 1 and the appropriate carboxylic acids
yielded benzo[d]imidazoles 2a–g14–16 where various substitutions,
including alkyls and halides, were introduced at 2C-position of the
imidazole ring system; that is, R1 to investigate their effects on bio-
logical activity. The preparation of o-acetylated nucleosides 3 in-
volved the formation of silylated benzimidazole by treating 2
with bis(trimethylsilyl)acetamide (BSA) in MeCN, followed by con-
densation with acylated sugar under Vorbrüggen’s conditions17 in
the presence of catalytic amounts of trimethylsilyl trifluorometh-
anesulfonate (TMSOTf). Subsequent hydrolysis of the latter in
methanolic ammonia resulted in benzo[d]imidazole nucleosides
4a–d in high yield.
was heated with carbon disulfide in ethanolic potassium hydrox-
ide, followed by methylation to give 8,19 which was in turn treated
with chlorocyclopentane to afford 5,6-dichloro-1-cyclopentyl-2-
(methylthio)-1H-benzo[d]imidazole 9. Methylsulfinyl- (10a) and
methylsulfonyl-1H-benzo[d]imidazole (10b) were obtained by oxi-
dation in the presence of m-CPBA.
Anti-proliferative activity of selected compounds was assessed
against HCT-116 colorectal carcinoma and MCF-7 breast carcinoma
cells using a standard 48-h MTT cytotoxicity assay.8 The results are
summarized in Table 1.
Replacement of the 5,6-dichloride group of the benzimidazole
nucleus with either fluoride (4a: R = F)20 or methoxide (4b:
R = OMe) in the context of 1-b-D-ribofuranosylbenzimidazole did
not offer any superiority over DRB in terms of cytotoxic effects
on the tumor cell lines tested. Similarly, introduction of a methyl
or ethyl group at the R1 in 4c21 or 4d failed to enhance activity,
irrespective of what substituent the benzene ring bears.
Surprisingly, compound 2a14 (R1 = CF3, R2 = H, R = Cl) demon-
strated good anti-proliferative activity in both HCT-116 and MCF-
7 cells with GI50 of 7 and 6 lM respectively, being 3.5- and 6-fold
more potent than DRB, suggesting that the ribose ring might be
dispensable. The structural modification of 2a by replacing 5,6-
dichlorides with 5,6-dimethyl groups, that is, 2c14
:
R1 = CF3,
Modification of the nucleoside moiety with several acyclic and
cyclic systems resulted in compound class Type-II (Fig. 1). Thus
treatment of benzene-1,2-diamines 1 with either 2-chlorobenzal-
dehyde or 2-florobenzaldehyde yielded the corresponding 5a18
and 5b. Diversity of 1N-substituted benzo[d]imidazoles 6a–k and
6n–r were obtained by alkylation reactions between 1H-
benzo[d]imidazoles 2a, 2d–g and the appropriate alkyl halides. Fi-
nal reduction of the nitrobenzene moieties of 6i–j in the presence
of SnCl4 under acidic conditions afforded the respective anilino
derivatives 6l–m.
R = Me, or depleting trifluoromethyl (2d15: R1 = H, R = Cl), however,
dramatically reduced the activity compared with 2a. Analogs with
different substitutions at R1, including alkyls (2e: R1 = Me, 2f:
R1 = Et, and 2g: R1 = i-Pr)15,16 and substituted phenyls (5a and
5b), in the context of 5,6-dichloro-1H-benzo[d]imidazole, had little
effect on cell viability. The trifluoromethyl substitution was there-
fore identified as the most effective residue at the position R1 of
5,6-dichlorid benzo[d]imidazole for cellular potency.
Keeping the portion of 5,6-dichloro-2-(trifluoromethyl)-1H-
benzo[d]imidazole while introducing a number of acyclic or cyclic
systems at 1N-position led to 6a–e. As shown in Table 1, although
6a–d, where R1 is alkyl; that is, Me (6a), Et (6b), i-Pr (6c) or Allyl
(6d), caused a loss in anti-proliferative activity, 5,6-dichloro-1-
To further probe the electron donating and withdrawing effects
thioether, sulfinyl and sulfonyl substituent were introduced to 2C-
position of benzo[d]imidazole. 4,5-Dichlorobenzene-1,2-diamine 1
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
N
N
N
N
N
N
g
h
d
R1
SH
S
S
N
H
N
H
R2
R2
10a-b
8
9
7
R2 = cyclopentyl
10a: R1 = SOMe
10b: R1 = SO2Me
f
R
R
R
N
N
R
R
N
R
R
NH2
NH2
R1
a
c
b
R1
R1
N
N
R
N
H
O
2a-g
O
1
2a: R = Cl, R1= CF3
HO
AcO
AcO
2b: R = Me, R1 = H
4a-d
HO
: R = Me, R1 = CF3
AcO
2c
2d
HO
: R = Cl, R1 = H
e
3
4a: R = F, R1= H
2e: R = Cl, R1 = Me
2f: R = Cl, R1 = Et
2g: R = Cl, R1 = i-Pr
d
4b: R = OMe, R1 = H
X
: R = Cl, R1 = Me
: R = H, R1 = Et
4c
4d
Cl
Cl
N
N
H
5a-b
Cl
Cl
5a: X = Cl
5b
N
N
R2
R1
: X = F
6a-r
Scheme 1. Reagents and conditions: (a) R1COOH, rf; 4 h, 70–98%, or R1COOH, Discovery Microwave, 145 °C; 20 min; (b) (i) BSA, MeCN, reflux, 1 h, (ii) (2S,3R,4R,5R)-5-
(acetoxymethyl)tetrahydrofuran-2,3,4-triyl triacetate in MeCN, TMSOTf, reflux, 24 h, 19–50%; (c) 7 M NH3 in MeOH, rt, 24 h, 100%; (d) 2a, 2d, 2e, 2f, 2 g, or 8; R2X, NaOH,
MeCN, 24 h, 1–70%; (e) ArCHO, Na2S2O5, EtOH, reflux, 38–86%; (f) CS2, KOH, EtOH, reflux, 4 hr, 87%; (g) MeI, K2CO3, reflux, 4 h, 80%; (h) DCM, m-CPBA, À40 °C–rt, 70–100%.

H. M. Alkahtani et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1317–1321
1319
Table 1
Structure and growth inhibitory activity of selected compounds against human tumor cancer cells
R
N
N
R1
R
R2
Code
Structure
R2
48 h MTT Cytotoxicity GI50
HCT-116
l
Ma
R1
R
MCF-7
DRB
4a
4b
4c
4d
2a
2b
2c
2d
2e
2f
2g
5a
5b
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
9
10a
10b
H
H
H
Me
Et
CF3
H
CF3
H
Me
Et
i-Pr
o-Cl-phenyl
o-F-phenyl
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
H
Me
Ethyl
CF3
i-Pr
SMe
SOMe
SO2Me
Ribose
Ribose
Ribose
Ribose
Ribose
H
H
H
H
H
H
H
H
H
Me
Et
i-Pr
Allyl
Cl
F
OMe
Cl
H
25.0
67.0
>100
nt
70.6
7.0
51.0
56.0
66.0
51.0
46.0
52.0
23.0
56.5
56.0
95.3
81.9
89.2
61.3
8.6
36.0
57.9
>100
nt
71.3
6.0
20.0
17.0
73.0
28.0
48.0
57.0
22.0
47.5
39.0
72.9
70.5
87.0
60.2
8.7
Cl
Me
Me
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
cyclopropylmethyl
Benzyl
m-CF3-benzyl
p-CF3-benzyl
o-NO2-benzyl
m-NO2-benzyl
p-NO2-benzyl
o-NH2-benzyl
m-NH2-benzyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
Cyclopentyl
50.5
19.0
5.9
68.0
17.0
1.5
22.8
81.6
26.1
22.6
37.0
13.0
38.0
22.0
9.0
60.0
97.3
55.6
77.5
42.0
40.0
52.0
21.0
43.0
3.9
2.9
0.5
0.6
0.5
0.6
aValues are means of at least two independent determinations; nt: not tested.
benzyl-2-(trifluoromethyl)-1H-benzo[d]imidazole 6f (R1 = benzyl)
exhibited comparable potency to 2a, and greater than threefold in-
crease in anti-proliferative effect compared with DRB, indicating
the tolerance of a large ring at 1N-position of the benzo[d]imidaz-
ole skeleton. An even more positive trend was displayed by 6i,
where an o-nitrobenzyl moiety at the 1N-position resulted in fur-
chemical assays.10 6o and 6r inhibited CDK9T1 with Ki values of
1.51 M and 3.33 respectively, with no activity against CDK2A. De-
spite their excellent cellular potency, 9, 10a and 10b showed low
inhibitory activity against CDK9T1, with Ki values >5 M, suggest-
ing that their cellular cytotoxicity might not be associated with
CDK9 kinase inhibition.
l
l
ther enhancement of activity, with GI50 values of 5.9
l
M and 1.5 in
In order to assess whether the lead compounds were capable of
inducing apoptosis in cancer cells, caspase-3 activation assay10 was
performed on 6o, 10a and 10b. As shown in Figure 2. 6o, 10a, 10b
and DRB activated significant caspase-3 activity at their respective
GI50 compared with DMSO, with enhanced activity at higher
concentrations.
respective HCT-116 and MCF-7 cells. Further investigating the R1
with different substituted benzyl functionalities giving 6g–h and
6j–n resulted in the loss of cellular activity.
Replacement of the ribose moiety in DRB with a cyclopentyl
ring was tolerated. 5,6-Dichloro-1-cyclopentyl-1H-benzo[d]imid-
azole (6n,22 R1 = H) slightly reduced anti-proliferative activity
compared with DRB. Keeping the 5,6-dichloro-1-cyclopentyl-1H-
benzo[d]imidazole portion, but structurally modifying R1 with a
methyl or isopropyl afforded 6o or 6r, with P2-fold increased cel-
Induction of apoptosis was further analysed by AnnexinV/PI
double staining in HCT-116 cells following treatment with each
compounds (Fig 3). The treatment with 6o or DRB resulted in sig-
nificant numbers of early apoptotic cells (>17% and 20% respec-
tively), being consistent with CDK9 inhibitory mechanism.10 10a
and 10b were much less effective when compared with 6o and
DRB, <7% Annexin V-positive cells were detected, indicating that
their anti-proliferative property might be the major contributor
to cytotoxicty in the cells.
lular potency in HCT-116 cells (GI50 = 13 and 9 lM respectively).
However MCF-7 cells appeared to be resistant to both compounds.
6p or 6q, containing a slightly bulkier group at R1, did not offer cel-
lular potency. The most potent anti-proliferative compounds, 10a
(GI50 = 0.5 lM) and 10b (GI50 = 0.6 lM), were obtained when
methylsulfinyl or methylsulfonyl groups respectively were intro-
We next examined the cell-cycle effects10 and analysis by flow
cytometry showed no effect on cell-cycle progression following
duced to R1 position.
In light of the potent anti-proliferative activity, the CDK kinase
inhibitory activities of 6o, 6r, 9, and 10a–b were evaluated by bio-
treatment of HCT-116 cells with 5xGI50
(Fig. 4). However DRB-treatment led to an observed accumulation
lM of 6o, 10a or 10b

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H. M. Alkahtani et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1317–1321
Figure 5. Cellular mode of action of 6o by Western blotting analysis: HCT-116 cells
were treated with 6o for 24 h at the concentrations shown. The reduced levels of
RNAPII CTD pSer-2 at GI50
as internal control.
lM indicated cellular CDK9 inhibition. b-Actin was used
Figure 2. Induction of caspase-3 activity in HCT-116 cells after treatment with each
compound at the respective GI50 or 5ÂGI50
lM for 24 h. Vertical bars represent the
mean s.d. of two independent experiments. Values significantly (p <0.05) different
from DMSO control are marked with an asterisk (Ã).
To confirm the cellular CDK9 inhibitory activity, HCT-116 cells
were treated with 6o for 24 h at the concentrations shown (Fig
5). Western blot analysis showed that the level of phosphorylation
at Ser-2 of RNAPII CTD was reduced, starting from GI50 lM in a
dose-dependent manner, indicating cellular CDK9 inhibition. The
same treatment reduced the levels of Mcl-1 and MDM2 proteins
and the cellular mechanism was consistent with the CDK9-medi-
ated RNAPII transcriptional inhibition in cancer cells.10
In conclusion, a series of analogs of DRB and benzo[d]imidazole25
were prepared and their SAR was studied. 5,6-Dichloro-1-cyclopen-
tyl-1H-benzo[d]imidazoles 6o, 10a and 10b possessed potent anti-
proliferative activity in cancer cell lines. 6o inhibited cellular CDK9
activity and was capable of inducing apoptosis via down-regulation
of anti-apoptotic proteins Mcl-1 at the concentration used to elicit
its cytotoxic GI50 response.
Acknowledgment
Figure 3. HCT-116 cells were exposed to 5ÂGI50
lM each compounds or DMSO for
24 h and the cells were analysed by annexinV/PI strained DNA content. The
percentage of cells undergoing apoptosis was defined as the sum of early apoptosis
cells (AnnV+/PIÀ) and late apoptosis (AnnV+/PI+). Vertical bars represent the
mean s.d. of two independent experiments. Values significantly (p <0.05) different
from DMSO control are marked with an asterisk (Ã).
Hamad M. Alkahtani and Abdullahi Y. Abbas thank King Saud
University and Islamic Development Bank (IDB) for their respective
studentships.
References and notes
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25. 6o: 1H NMR (DMSO-d6) d 1.71 (m, 2H, CH2), 1.9–2.13 (m, 6H, CH2), 2.58 (s, 3H,
CH3), 4.86 (q, J = 8.8 Hz, 1H, CH), 7.75 (s, 1H, ArH), 7.79 (s, 1H, ArH); 13C NMR
(DMSO-d6) d 15.0, 24.8, 30.0, 56.7, 112.9, 120.1, 124.1, 133.2, 143.0, 155.4;
HRMS (EI+) m/z: 269.0647 [M+H]+; C13H14Cl2N2 requires 268.0534. 10a: 1H
NMR (DMSO-d6) d 1.73 (m, 2H, CH2), 1.99 (m, 2H, CH2), 2.15 (m, 6H, CH2), 3.18
(s, 3H, CH3), 5.35 (q, J = 8.9 Hz, 1H, CH), 8.04 (s, 1H, ArH), 8.14 (s, 1H, ArH). 13
C
NMR (DMSO-d6) d 24.69, 30.91, 31.34, 38.91, 57.80, 114.60, 122.47, 127.46,
133.83, 141.70, 156.87. HRMS (EI+) m/z: 318.0174 [M+H]+; C13H14Cl2N2OS
requires 317.2341. 10b: 1H NMR (DMSO-d6) d 1.73 (m, 2H, CH2), 2 (m, 2H, CH2),
2.15 (m, 4H, 2 Â CH2), 3.69 (s, 3H, CH3), 5.43 (q, J = 9 Hz, 1H, CH), 8.05 (s, 1H,
ArH), 8.20 (s, 1H, ArH). 13C NMR (DMSO-d6) d 24.6, 30.4, 42.9, 58.6, 115.3,
123.1, 127.2, 128.6, 133.0, 140.6, 151.6; HRMS (EI+) m/z: 333.0318;
20. Moore, C. L.; Zivkovic, A.; Engels, J. W.; Kuchta, R. D. Biochemistry 2004, 43,
12367.
21. Meggio, F.; Shugar, D.; Pinna, L. A. Eur. J. Biochem. 1990, 187, 89.
22. Tuncbilek, M.; Kiper, T.; Altanlar, N. Eur. J. Med. Chem. 2009, 44, 1024.
C13H14Cl2N2O2S requires 333.2335.