N-(4-{[4-(1H-Benzoimidazol-2-yl)-arylamino]-methyl}-phenyl)-benzamide derivatives as small molecule heparanase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.09.070

A novel class of N-(4-{[4-(1H-benzoimidazol-2-yl)-arylamino]-methyl}- phenyl)-benzamides are described as inhibitors of the endo-β-glucuronidase heparanase. Among them are N-(4-{[4-(1H-benzoimidazol-2-yl)-phenylamino]-methyl} -phenyl)-3-bromo-4-methoxy-benzamide (15h), and N-(4-{[5-(1H-benzoimidazol-2-yl) -pyridin-2-ylamino]-methyl}- phenyl)-3-bromo-4-methoxy-benzamide (23) which displayed good heparanase inhibitory activity (IC₅₀ 0.23-0.29 μM), with the latter showing oral exposure in mice.

Full text of DOI:10.1016/j.bmcl.2005.09.070

Bioorganic & Medicinal Chemistry Letters 16 (2006) 404–408
N-(4-{[4-(1H-Benzoimidazol-2-yl)-arylamino]-methyl}-phenyl)-
benzamide derivatives as small molecule heparanase inhibitors
Yong-Jiang Xu,a Hua-Quan Miao,b,* Weitao Pan,a Elizabeth C. Navarro,b
James R. Tonra,c Stan Mitelman,c M. Margarita Camara,c Dhanvanthri S. Deevi,c
Alexander S. Kiselyov,a, Paul Kussie,b 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 N-(4-{[4-(1H-benzoimidazol-2-yl)-arylamino]-methyl}-phenyl)-benzamides are described as inhibitors of
the endo-b-glucuronidase heparanase. Among them are N-(4-{[4-(1H-benzoimidazol-2-yl)-phenylamino]-methyl}-phenyl)-3-bromo-
4-methoxy-benzamide (15h), and N-(4-{[5-(1H-benzoimidazol-2-yl)-pyridin-2-ylamino]-methyl}- phenyl)-3-bromo-4-methoxy-benz-
amide (23) which displayed good heparanase inhibitory activity (IC50 0.23–0.29 lM), with the latter showing oral exposure in mice.
Ó 2005 Elsevier Ltd. All rights reserved.
Heparanase, an endo-b-D-glucuronidase that degrades
heparan sulfate glycosaminoglycans in the extracellular
matrix (ECM) and the basement membrane, is involved
in tumor cell invasion, angiogenesis, and other physiolog-
ical and pathological processes.1 Experimental tumor cell
metastasis in an animal model is reduced by treatment
with heparanase inhibitors.2 Different from the large
number of proteases known to disassemble the ECM,3
there is only one heparanase cDNA sequence coding func-
tional enzyme identified to date.1a,c These findings indi-
cated heparanase as a major enzyme that degrades
heparan sulfate glycosaminoglycans in mammalian tis-
sues,1b,4 and made heparanase a promising target for
the development of antitumor agents, especially antimet-
astatic agents. Most of the heparanase inhibitors known
are sulfated oligosaccharide derivatives that resemble
the substrate heparan sulfate glycosaminoglycans.2,5
The most advanced of them is PI-88, a highly sulfated
pentasaccharide (1, Scheme 1), which is currently in Phase
II clinical trials.2 As a heparan sulfate analogue, PI-88 has
heterogeneous structure and multiple mechanisms of
action. Laminarin sulfate and synthetic phosphorothio-
ate oligodeoxynucleotides,6 suramin and its analogs,7
benzoic acid analogs,8 sulfated iminosugars,9 azapseud-
odisaccharides,10 and natural products such as trachyspic
acid and its derivatives11 also inhibit heparanase with
good to modest potency.
Recently, small molecules such as 2,3-dihydro-1,3-
dioxo-1H-isoindole-5-carboxylic acids (2)12 and ben-
zoxazol-5-yl acetic acids (3)13 have been reported to be
OPO3Na2
OR
RO
OR
RO
OR
O
RO
O
RO
RO
O
O
O
OR
O
O
O
O
RO
RO
RO
RO
OR
RO
RO
R = SO3Na or H
1
O
F
HO2C
N
O
N
NH
HO2C
NH
O
Br
O
O
N
Keywords: Heparanase inhibitor; Benzimidazole.
*
Corresponding authors. Tel.: +1 646 638 5014; fax: +1 212 645 2054
(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.
3
2
Cl
Scheme 1.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.070

Y.-J. Xu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 404–408
Table 1. In vitro inhibition of heparanase activity of 10a–h
405
potent heparanase inhibitors. The best compounds from
these series have IC50s (compound concentrations that
cause 50% inhibition of the enzyme activity) of around
200 nM. These reports prompted us to reveal in this
and the following letter our effort aimed at the identifi-
cation of Ôproof-of-conceptÕ small molecule heparanase
inhibitors with efficacy in animal models.
Compound Ring A
Heparanase
inhibition IC50 (lM)a
10a
10b
10c
10d
10e
3-Fluoro-4-methoxy
>15
>15
4.0
3-Chloro-4-methoxy
3-Bromo-4-methoxy
3-Bromo-4-ethoxy
1.2
2.2
3-Bromo-4-n-propoxy
From the hits identified by high throughput screening, 4
(Scheme 2) was selected as the structure–activity relation-
ship (SAR) template mainly based on synthetic feasibility.
Early SAR investigation demonstrated that a 1,3-disposi-
tion of the benzamide moiety and the pendant benzimid-
azole ring system in 4 resulted in reduced inhibiting
activity. Furthermore, the central phenyl ring in 4 toler-
ates little change in terms of substitution. Hence, more de-
tailed SAR strategies were devised (5, Scheme 2) focusing
on: (a) modifications of the aromatic ring A and ring B
mainly through substitution changes; and (b) modifica-
tion of the amide linkage. In most cases, the 1,4-substitut-
ed central phenyl ring was kept intact.
OH
10f
11
O
10g
10h
3-(30-Pyridyl)-4-methoxy
3-(1H-Indazol-5-yl)-4-methoxy
5.7
6.1
a Average of three experiments with SD < 15%.
the more preferred substituents for ring A are 3-bro-
mine-4-alkoxy. When the 3-bromine in 10c was replaced
with either fluorine (10a) or chlorine (10b), the activity
against heparanase decreased dramatically. On the other
hand, the 4-alkoxy moiety of ring A tolerates more
changes, with the 4-methoxy (10c), 4-propoxy (10e)
giving comparable results, and the 4-ethoxy (10d) a slight-
ly better IC50 of 1.2 lM. This result indicated potential
room for extension to the right-hand side of the molecule.
We also found that the 3-bromine of compound 10c could
be replaced with certain heteroaryl moieties via Suzuki
coupling with boronic acids (10f–h), and the activity
against heparanase was partially retained.
In order to examine the effect of modification of ring A
on heparanase inhibitory activity, a typical procedure
was developed for the preparation of the N-[4-(1H-ben-
zoimidazol-2-yl)-phenyl]-amides 10 (Scheme 3). Heat-
ing the phenyl diamine 6 with 4-amino-benzoic acid 7
in polyphosphoric acid at 220 °C for 4 h afforded
4-(1H-benzoimidazol-2-yl)-phenylamine 8 in 55% yield.
In the second step, 8 was allowed to react with a
substituted benzoyl chloride in pyridine at 80 °C over-
night to produce 10,14 which was tested for heparanase
inhibitory activity in a radio-labeled enzymatic assay15
(Table 1).
The results in Table 1 led to the selection of 10d as a tem-
plate for further optimization of ring B (Table 2). A con-
vergent synthesis of compounds 14 was developed to
allow modification of ring B in the last step of the synthe-
sis (Scheme 4). First, 4-aminobenzyl alcohol 11 was
reacted with 3-bromo-4-ethoxy-benzoyl chloride to form
alcohol 12. A Dess Martin oxidation of 12 yielded alde-
hyde 13, which was allowed to condense with a substitut-
Early SAR investigation on ring A indicated that a 3,4-di-
substitution pattern on ring A of 10 seemed to be pre-
ferred for heparanase inhibiting activity. Furthermore,
Ring A
Ring B
modification
modification
O
R
N
4
1
NH
O
1 NH
Table 2. In vitro inhibition of heparanase activity of 14a–i
N
H
A
N
4
B
Compound
R
Heparanase percent Heparanase inhibition
inhibition at 25 lMa IC50 (lM)a
N
H
4
Amide Displacemet
5
10d
14a
14b
H
1.2
Scheme 2.
5-OMe 73
5-OMe 35
6-OMe
NTb
NTb
14c
14d
5-Cl
6-Cl
30
NTb
NTb
HO2C
NH2
NH2
NH2
N
N
7
5-Cl
30
NH2
6-Me
4-Me
5-Me
4-Me
5-Me
5-Me
6-Me
4-Me
7-Me
polyphosporic acid
220 o
(55%)
H
14e
14f
14g
2.3
1.6
1.3
C
8
6
O
Cl
pyridine, 80 oC
A
9
14h
14i
1.1
2.7
3
O
4
A
N
NH
N
H
a Average of three experiments with SD < 15%.
b Not tested.
10
Scheme 3.

406
Y.-J. Xu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 404–408
Br
OEt
Compound 15a was obtained by treating 10c with
O
sodium hydride, followed by methylation with methyl
iodide at room temperature. Compounds 15c, 15d,
and 15f were synthesized from 8 via standard sulfon-
amide formation with the corresponding sulfonyl chlo-
ride, reductive amination with the corresponding
aldehyde using NaBH(OAc)3 in dichloroethane, and
urea formation with an isocyanate. Compounds 15e,g
were prepared from 8 via standard amide formation
with the corresponding acid chlorides. Compounds
15b and 15h were prepared as shown in Scheme 6.
The phenyl diamine was first reacted with 4-formyl-
benzoic acid in nitrobenzene to provide acid 16.
Reacting 16 with CDI in DMF, followed by treatment
with aniline 17, provided 15b. Reacting 8 with alde-
hyde 18, which was prepared similarly as 13 (Scheme
4), under standard reductive amination condition pro-
vided 15h.
Br
Cl
NH
O
NH2
HO
OEt
HO
THF, DMAP
(80%)
11
12
Dess-Martin
CH2Cl2, rt
(64%)
NH2
R
Br
4
B
7
NH2
PhNO2
NH
5
Br
N
OEt
O
R
NH
O
N
H
OEt
130 0C, 12 h
6
O
13
14
Scheme 4.
ed phenyl diamine, followed by spontaneous oxidation in
nitrobenzene to yield 14 in good yields. Results in Table
2 revealed that methoxy (14a, 14b), or chlorine substitu-
ents on ring B (14c) resulted in a dramatically decreased
activity against heparanase when compared to 10d.
However, substitution of ring B with a methyl (14e,
14f), or a dimethyl (14g–i), largely retained the activity.
Results from Table 3 revealed that compound 15a, the
N methylated product of 10c, has a dramatically de-
creased activity against heparanase compared to 10c.
Replacement of the amide in 10c with a Ôreversed
amideÕ (15b), or a sulfonamide (15c), or an amino
methyl linkage (15d) all diminished the heparanase
inhibitory activity. Extending the amide linkage in
10c with an extra methylene unit was also met with dis-
appointment (15e). However, a urea replacement of the
amide (15f) improved the heparanase inhibitory activi-
ty by about 3-fold, and a similar improvement of activ-
ity was observed for 15g. Remarkably, the more
flexible compound 15h has an IC50 of 0.23 lM against
heparanase.
When dosed ip to mice (n = 3) at 50 mg/kg, 10d showed
a plasma concentration of 0.53 lM at 1 h and dropped
down to 0.38 lM by 4 h. Better exposure was observed
for the methoxy derivative 10c (plasma concentration
2.24 lM at 1 h and 0.67 lM by 4 h after ip dosing).
The observed sub-optimal plasma exposures, combined
with their modest activity against heparanase (IC50 of
1.2 and 4.0 lM, respectively), excluded 10d,c as Ôproof-
of-conceptÕ candidates for evaluation in animal models.
Nevertheless, these results prompted us to study the
effect of replacing the amide linkage of 10c with the
aim of improving anti-heparanase activity and phar-
macokinetic properties (Scheme 5 and Table 3).
Compound 15h was then selected as a template for fur-
ther modification, and the representative results from
this study are shown in Scheme 7. Compounds 19–23
were prepared similarly as 15h (Scheme 6) by using the
corresponding amines or aldehydes. Results in Scheme
7 indicated that a overall linear, yet slightly Ôbent,Õ dispo-
sition of the molecule is important for activity against
heparanase, as the more ÔbentÕ molecules, such as 19
and 20, have diminished activities. Similar reasons
may have also caused the loss of activity for compound
21, which has an additional methylene unit inserted be-
tween the benzimidazole and the central phenyl ring.
Replacement of the phenyl group to the left hand side
of the molecule led to 22 that had an IC50 of 0.93 lM
against heparanase. Remarkably, the central phenyl
group could be replaced with a pyridine moiety and
the resultant 23 had an IC50 of 0.29 lM. When dosed
p.o. to mice (n = 3) as a suspension in ethanol/Tween
80/PEG 400 (E/T/P) at 30 mg/kg, compound 23 showed
a plasma concentration of 3.8 lM at 1 h, going up to
5.5 lM by 4 h. Interestingly, changing formulation or
dosing 23 intraperitoneally did not lead to improved
plasma exposure.
Br
Br
OMe
O
N
OMe
N
X
NH
N
H
N
H
10c
15
Scheme 5.
Table 3. In vitro inhibition of heparanase activity of 15a–h
Compound
X
Heparanase inhibition
IC50 (lM)a
10c
15a
15b
15c
15d
15e
15f
–NHCO–
4.0
>15
>15
>15
>15
>15
0.93
–N(Me)CO–
–CONH–
–NHSO2–
–NHCH2–
–NHCOCH2–
–NHCONH–
O
O
15g
15h
0.91
0.23
NH
NH
O
In summary, a novel class of N-(4-{[4-(1H-benzoimidazol-
2-yl)-arylamino]-methyl}-phenyl)-benzamides are de-
scribed as heparanase inhibitors. Among them are 15h
and 23 which displayed good heparanase inhibitory activ-
ity (IC50 0.23–0.29 lM), with the latter showing oral
NH
NH
a Average of three experiments with SD < 15%.

Y.-J. Xu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 404–408
407
O
1)
N
N
N
N
OHC
CO2H
N
O
O
N
NH2
NH2
N
H
HN
OMe
Br
PhNO2, 130 o
(80%)
C
H2N
N
H
OH
2)
16
6
15b
OMe
Br
17
(20% for 2 steps)
Br
OMe
NH
O
O
H
N
N
18
NH
NH
N
H
NH2
OMe
Br
N
NaBH(OAc)3, DMF
(47%)
O
8
15h
Scheme 6.
N
NH
R
N
H
N
R
N
NH
NH
N
H
HN
21
19
20
less than 10% inhibition
at 0.83 uM
less than 10% inhibition
at 0.83 uM
less than 10% inhibition
at 0.83 uM
R
N
N
N
NH
O
NH
R =
NH
OMe
R
N
H
R
N
H
N
Br
23
22
IC50 0.29 uM
IC50 0.93 uM
Scheme 7.
4. Kussie, P. H.; Hulmes, J. D.; Ludwig, D. L.; Patel, S.;
Navarro, E. C.; Seddon, A. P.; Giorgio, N. A.; Bohlen, P.
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exposure in mice. Furthermore, the SAR information
gathered during this study provided useful insights into
the design of potent small molecule heparanase inhibitors
suitable for animal model evaluation (see following
article).
Acknowledgments
We are grateful to Dr. Peter Bohlen for his support and
Dr. Marc Labelle for his helpful advice and discussion.
8. Ishida, K.; Wierzba, M. K.; Teruya, T.; Simizu, S.; Osada,
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Biotech). The specific activity was determined as
98.4 cpm/ng HS. The purified 3H-HS was then
immobilized on CNBr-activated Sepharose beads
(Pharmacia) according to manufacturerÕs instructions.
Heparanase activity was determined using 96-well
plates. Human platelet heparanase (2.67 nM) was
13. Courtney, S. M.; Hay, P. A.; Buck, R. T.; Colville, C. S.;
Phillips, D. J.; Scopes, D. C.; Pollard, F. C.; Page, M. J.;
Bennett, J. M.; Hircock, M. L.; McKenzie, E. A.;
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pre-mixed with
a compound of the present study
14. All synthetic intermediates were characterized by 1H
NMR and MS; all final products were characterized by
1H NMR and LCMS.
(various concentrations for IC50 studies) in a total
volume of 125 ll. 3H-HS–Sepharose slurry (25 ll,
4 nM) was then added into the mixture and
incubated overnight at 37 °C. The reaction buffer
was transferred to 96-well Luma plates (Perkin
Elmer). The plates were air dried and the radioac-
tivity was directly detected in a TopCounter (Perkin
Elmer).
15. Heparanase activity assays: human heparanase protein
was purified from human platelets using a modified
protocol (Freeman et al., Biochem. J. 1998, 330,
1341). Heparan sulfate (HS, Seikagaku) was labeled
with sodium boro[3H]hydride (Amersham-Pharmacia