1-[4-(1H-Benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl] -urea derivatives as small molecule heparanase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.09.069

A novel class of 1-[4-(1H-benzoimidazol-2-yl)-phenyl]-3-[4-(1H- benzoimidazol-2-yl)-phenyl]-ureas are described as potent inhibitors of heparanase. Among them are 1,3-bis-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea (7a) and 1,3-bis-[4-(5,6-dimethyl-1H-benzoimidazol-2-yl)-phenyl]-urea (7d), which displayed good heparanase inhibitory activity (IC₅₀ 0.075-0.27 μM). Compound 7a showed good efficacy in a B16 metastasis model.

Full text of DOI:10.1016/j.bmcl.2005.09.069

Bioorganic & Medicinal Chemistry Letters 16 (2006) 409–412
1-[4-(1H-Benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-
yl)-phenyl]-urea derivatives as small molecule heparanase inhibitors
Weitao Pan,a Hua-Quan Miao,b,* Yong-Jiang Xu,a Elizabeth C. Navarro,b
James R. Tonra,c Erik Corcoran,c Armin Lahiji,c Paul Kussie,b Alexander S. Kiselyov,a,
Wai C. Wonga and Hu Liua,*
aDepartment of Chemistry, ImClone Systems Incorporated, 710 Parkside Avenue, Brooklyn, NY 11226, USA
bDepartment of Protein Science, ImClone Systems Incorporated, 180 Varick Street, NY 10014, USA
cDepartment of Experimental Therapeutics, ImClone Systems Incorporated, 180 Varick Street, NY 10014, USA
Received 25 June 2005; revised 11 September 2005; accepted 26 September 2005
Available online 21 October 2005
Abstract—A novel class of 1-[4-(1H-benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-ureas are described as
potent inhibitors of heparanase. Among them are 1,3-bis-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea (7a) and 1,3-bis-[4-(5,6-di-
methyl-1H-benzoimidazol-2-yl)-phenyl]-urea (7d), which displayed good heparanase inhibitory activity (IC50 0.075–0.27 lM).
Compound 7a showed good efficacy in a B16 metastasis model.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Heparanase degrades heparan sulfate glycosaminogly-
can in the extracellular matrix (ECM) and the basement
membrane, and is involved in tumor cell invasion, angi-
ogenesis, and other physiological and pathological pro-
cesses.1 Different from the large number of proteases
know to disassemble the ECM,2 there is only one hepa-
ranase cDNA sequence coding functional enzyme iden-
tified to date.1a,c These findings indicated heparanase as
a major enzyme that degrades heparan sulfate glycos-
aminoglycan in mammalian tissues.1b,3 Among the hep-
aranase inhibitors known4,5 are sulfated oligosaccharide
derivatives, and the most advanced of them is PI-88,
which is currently in phase II clinical trials.4 Only
recently, small molecule heparanase inhibitors, exempli-
fied by 1H-isoindole-5-carboxylic acid6a and ben-
zoxazol-5-yl-acetic acid,6b were reported.
(Scheme 1), as a potent heparanase inhibitor with an
IC50 (compound concentration that causes 50% inhibi-
tion of the enzyme activity) of 0.29 lM.7 Nevertheless,
the highest plasma exposure observed for 1 in mice
was 5.5 lM, and this observation deterred us from using
it as the Ôproof-of-conceptÕ candidate for evaluation in
animal models. Our early effort in this area has also
identified (benzimidazol-2-yl)-phenyl-phenyl-urea 27
and 4-(1H-benzoimidazol-2-yl)-N-[4-(1H-benzoimidazol-
2-yl)-phenyl]-benzamide 38 as heparanase inhibitors
with IC50s of 0.93 and 0.95 lM, respectively. We hence
envisioned that the symmetrical urea 7 (Scheme 2),
which has combined structural features of 2 and 3,
might lead to another series of heparanase inhibitors
with desired potencies.
The synthesis of 1,3-bis-[4-(1H-arylimidazol-2-yl)-phen-
yl]-urea 7 is straightforward (Scheme 2). Heating an
aryldiamine 4 with 4-amino-benzoic acid 5 in poly-
phosporic acid at 220 ꢁC for 4 h afforded 4-(1H-ben-
zoimidazol-2-yl)-phenylamine 6 in good yield. In the
second step, 6 was dimerized at room temperature for
overnight with CDI to afford the urea 7,9 which was test-
ed in the heparanase enzymatic assay (Table 1).10
Our effort aimed at the development of Ôproof-of-con-
ceptÕ small molecule heparanase inhibitors led to the
identification of N-(4-{[4-(1H-benzoimidazol-2-yl)-
arylamino]-methyl}-phenyl)-benzamides, such as
1
Keywords: Heparanase inhibitor; Benzimidazole; Urea.
*
Corresponding authors. Tel.: +1 646 638 5014; fax: +1 212 645 205
(H-Q.M.); tel.: +1 917 755 5856; fax: +1 718 633 0856 (H.L.); e-mail
addresses: Hua-quan.miao@imclone.com; huliu205@yahoo.com
Present address: Chemical Diversity, 1155 Sorrento Valley Road,
Suite 5, San Diego, CA 92121, USA.
Results in Table 1 revealed that 1,3-bis-[4-(1H-ben-
zoimidazol-2-yl)-phenyl]-urea 7a has an IC50 of
0.27 lM against heparanase. Substitution on the phenyl
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.069

410
W. Pan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 409–412
N
NH
NH
O
N
H
N
OMe
1
Br
IC50: 0.29 uM
OMe
Br
N
O
NH
O
N
NH
N
N
H
NH
N
H
N
H
3
2
IC50: 0.95 uM
IC50: 0.93 uM
Scheme 1.
HO2C
NH2
NH2
B
B
N
A
R
5
A
NH2
R
N
H
polyphosporic acid
220 o
NH2
C
4
6
O
A, B = CH or N
N
N
DMF, rt, 30-70%
N
N
R
5
A
B
N
4
NH
O
B
4
NH
N
A
NH
R
N
H
5
7
Scheme 2.
ring of 7a proved to have dramatic effects on the hepa-
ranase inhibitory activities. Improved activities were ob-
served for methyl substitution, such as 7b–d, with 7d, a
tetra-methyl-substituted urea, having an IC50 of
0.075 lM. On the other hand, fluoro (7e), or methoxy
(7f,g), substitutions diminished the activities. Pyridine
containing ureas, such as 7h,i, had IC50s of 0.90 and
3.42 lM, respectively.
Table 1. In vitro inhibition of heparanase activity of 7a–i
Compound
A
B
R
Heparanase
percent
inhibition
at 0.83 lMa (%)
Heparanase
inhibition
IC50 (lM)a
7a
7b
7c
7d
CH CH
H
0.27
0.15
0.15
0.075
CH CH 5-Me
CH CH 4-Me
CH CH 5-Me
6-Me
Encouraged by the results obtained from the symmetri-
cal urea series, we then turned our attention to the
unsymmetrical ureas 11. The synthesis of compound
11 is shown in Scheme 3. A phenyl diamine 8 was first
reacted with 4-formyl-benzoic acid 9 in nitrobenzene
to provide benzimidazole acid 10. Curtis rearrangement
of 10 in the presence of diphenylphosphoryl azide
(DPPA) and triethyl amine generated the isocyanate
intermediate, which upon reacting with 6 (R = H)
7e
7f
7g
7h
7i
CH CH 5-F
21
NTb
NTb
0.87
0.90
3.42
CH CH 5-OMe 16
CH CH 4-OMe
N
CH
CH
N
H
H
a Average of three experiments with SD < 15%.
b Not tested.

W. Pan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 409–412
411
R
HO2C
CHO
R
O
N
H2N
H2N
9
R
O
R'
O
R'
NH
N
H
PhNO2, 130 o
C
HO
NH
NH
R
NH
8
10
13
12
R
1) DPPA, Et3N
2)
B
N
A
R'
5
NH2
N
O
R =
N
H
NH
4
N
H
6
N
R
NH
14
NH
O
B
N
NH
Scheme 4.
A
NH
N
H
11
against heparanase (less than 10% inhibition at
0.83 lM).
Scheme 3.
Urea 7a showed less than 50% proliferation inhibition
on B16-BL6 melanoma cells in vitro at 100 lM, and
hence was selected for further biological evaluation.
When dosed intraperitoneally (ip) to mice (n = 3) as a
suspension in cremophore/5% DMSO/CMC, compound
7a showed a plasma concentration of 31 lM at 1 h, and
23 lM by 4 h, after dosing. These levels were consider-
ably higher than its IC50 of 0.27 lM against heparanase.
In the B16 metastasis model5e (Fig. 1), compound 7a
was dosed ip to C57 mice at 30 mg/kg as a suspension
in cremophore/5% DMSO/CMC. About 30–60 min
after the injection of the compound, B16-BL6 melano-
ma cells (2 · 105/mouse) were injected into the tail vein
of the mice. Twenty-one days later, the mice were sacri-
ficed and their lungs were examined for nodules. Results
in Figure 1 demonstrate that the group of mice treated
with compound 7a (n = 10) showed around 50% inhibi-
tion of lung metastasis compared to the vehicle-treated
group (n = 8) or the untreated group (n = 10). This
result is comparable to that obtained from PSS (single
ip dose of 16 mg/kg, n = 10), which is a sulfated poly-
provided urea 11. The activities of 11 against heparanase
are summarized in Table 2.
Results from Table 2 indicate that, not surprisingly, the
unsymmetrical ureas showed structure–activity relation-
ship (SAR) patterns similar to those of the symmetrical
ones, such that methyl substitution (11a) improved the
heparanase inhibitory activity, and a methoxy substitu-
tion (11b,c) diminished the activity compared to 7a.
In the latter case, again a 4-methoxy (11c) is preferred
over the 5-methoxy (11b). On the other hand, pyridine
containing ureas, such as 11e–g, showed decreased
heparanase inhibitory activities.
The SAR on the ureas showed a preference for an over-
all linear, yet ÔbentÕ disposition of the molecule. In order
to examine effects of the overall shape and length of the
molecule on the heparanase inhibitory activity, several
of the more ÔbentÕ molecules, such as 12–14 (Scheme
4), were prepared similarly using the synthetic route
described in Scheme 3. Consistent with the SAR trends
observed in the N-(4-{[4-(1H-benzoimidazol-2-yl)-aryl-
amino]-methyl}-phenyl)-benzamides 1,7 the more ÔbentÕ
ureas 12–14 all have dramatically diminished activities
100
80
60
40
20
0
Table 2. In vitro inhibition of heparanase activity of 11a–g
Compound
A
B
R0
Heparanase
percent
Heparanase
inhibition
IC50 (lM)a
inhibition at
0.83 lMa (%)
11a
11b
11c
11d
11e
11f
11g
CH CH 4-Me
0.15
NTb
1.15
NTb
NTb
NTb
1.0
CH CH 5-OMe 17
CH CH 4-OMe
CH CH 4-NO2 20
N
CH
H
CH 5-OMe
17
8
N
CH
Vehicle Compound 7a Untreated
PSS
N
H
n=8
n=10
n=10
n=10
a Average of three experiments with SD < 15%.
b Not tested.
Figure 1.

412
W. Pan et al. / Bioorg. Med. Chem. Lett. 16 (2006) 409–412
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saccharide heparanase inhibitor with a heparanase
inhibitory IC50 of 15 nM in the enzymatic assay.
In summary, a novel class of 1-[4-(1H-benzoimidazol-
2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-
ureas are described as potent inhibitors of heparanase.
Among them are 1,3-bis-[4-(1H-benzoimidazol-2-yl)-
phenyl]-urea (7a), and 1,3-bis-[4-(5,6-dimethyl-1H-ben-
zoimidazol-2-yl)-phenyl]-urea (7d), which displayed
good heparanase inhibitory activity (IC50 0.075–
0.27 lM), with 7a showing good efficacy in the B16
metastasis model. Active effort is in progress to further
prove the link between the inhibition of heparanase
activity and the efficacy observed in the B-16 study,
and the results will be reported in due course.
6. (a) Courtney, S. M.; Hay, P. A.; Buck, R. T.; Colville, C.
S.; Porter, D. W.; Scopes, D. C.; Pollard, F. C.; Page, M.
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Acknowledgments
We are grateful to Dr. Peter Bohlen for his support and
Dr. Marc Labelle for his helpful advice and discussion.
7. Xu, Y-J.; Miao, H-Q.; Pan, W.; Navarro, E. C.; Tonra, J.
R.; Mitelman, S.; Camara, M. M.; Deevi, D. S.; Kiselyov,
A. S.; Kussie, P.; Wong, W. C.; Liu, H. Bioorg. Med.
Chem. Lett., in press.
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