Resistance-modifying agents. 9. Synthesis and biological properties of benzimidazole inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase

Article abstract of DOI:10.1021/jm000950v

The nuclear enzyme poly(ADP-ribose) polymerase (PARP) facilitates the repair of DNA strand breaks and is implicated in the resistance of cancer cells to certain DNA-damaging agents. Inhibitors of PARP have clinical potential as resistance-modifying agents capable of potentiating radiotherapy and the cytotoxicity of some forms of cancer chemotherapy. The preclinical development of 2-aryl-1H-benzimidazole-4-carboxamides as resistance-modifying agents in cancer chemotherapy is described. 1H-Benzimidazole-4-carboxamides, particularly 2-aryl derivatives, are identified as a class of potent PARP inhibitors. Derivatives of 2-phenyl-1H-benzimidazole-4-carboxamide (23, K(i) = 15 nM), in which the phenyl ring contains substituents, have been synthesized. Many of these derivatives exhibit K(i) values for PARP inhibition < 10 nM, with 2-(4-hydroxymethylphenyl)-1H-benzimidazole-4-carboxamide (78, K(i) = 1.6 nM) being one of the most potent. Insight into structure-activity relationships (SAR) for 2-aryl-1H-benzimidazole-4-carboxamides has been enhanced by studying the complex formed between 2-(3-methoxyphenyl)-1H-benzimidazole-4-carboxamide (44, K(i) = 6 nM) and the catalytic domain of chicken PARP. Important hydrogen-bonding and hydrophobic interactions with the protein have been identified for this inhibitor. 2-(4-Hydroxyphenyl)-1H-benzimidazole-4-carboxamide (45, K(i) = 6 nM) potentiates the cytotoxicity of both temozolomide and topotecan against A2780 cells in vitro (by 2.8- and 2.9-fold, respectively).

Full text of DOI:10.1021/jm000950v

4084
J . Med. Chem. 2000, 43, 4084-4097
Resista n ce-Mod ifyin g Agen ts. 9.1 Syn th esis a n d Biologica l P r op er ties of
Ben zim id a zole In h ibitor s of th e DNA Rep a ir En zym e P oly(ADP -r ibose)
P olym er a se
Alex W. White,† Robert Almassy,‡ A. Hilary Calvert,§ Nicola J . Curtin,§ Roger J . Griffin,† Zdenek Hostomsky,‡
Karen Maegley,‡ David R. Newell,§ Sheila Srinivasan,† and Bernard T. Golding*,†
Department of Chemistry, Bedson Building, The University, Newcastle upon Tyne NE1 7RU, U.K.;
Cancer Research Unit, Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, U.K.; and
Agouron Pharmaceuticals Inc., San Diego, California 92121
Received March 22, 2000
The nuclear enzyme poly(ADP-ribose) polymerase (PARP) facilitates the repair of DNA strand
breaks and is implicated in the resistance of cancer cells to certain DNA-damaging agents.
Inhibitors of PARP have clinical potential as resistance-modifying agents capable of potentiating
radiotherapy and the cytotoxicity of some forms of cancer chemotherapy. The preclinical
development of 2-aryl-1H-benzimidazole-4-carboxamides as resistance-modifying agents in
cancer chemotherapy is described. 1H-Benzimidazole-4-carboxamides, particularly 2-aryl
derivatives, are identified as a class of potent PARP inhibitors. Derivatives of 2-phenyl-1H-
benzimidazole-4-carboxamide (23, Ki ) 15 nM), in which the phenyl ring contains substituents,
have been synthesized. Many of these derivatives exhibit Ki values for PARP inhibition < 10
nM, with 2-(4-hydroxymethylphenyl)-1H-benzimidazole-4-carboxamide (78, Ki ) 1.6 nM) being
one of the most potent. Insight into structure-activity relationships (SAR) for 2-aryl-1H-
benzimidazole-4-carboxamides has been enhanced by studying the complex formed between
2-(3-methoxyphenyl)-1H-benzimidazole-4-carboxamide (44, Ki ) 6 nM) and the catalytic domain
of chicken PARP. Important hydrogen-bonding and hydrophobic interactions with the protein
have been identified for this inhibitor. 2-(4-Hydroxyphenyl)-1H-benzimidazole-4-carboxamide
(45, Ki ) 6 nM) potentiates the cytotoxicity of both temozolomide and topotecan against A2780
cells in vitro (by 2.8- and 2.9-fold, respectively).
In tr od u ction
binding of PARP to a DNA nick may cause a transient
halt to cellular activity, allowing access of DNA repair
enzymes to the damage.10 Alternatively, formation
of poly(ADP-ribose) or the fragments from metabolism
of this polymer may signal to a cell that its DNA is
damaged.8
The ability of tumor cells to recognize and repair
damage to DNA inflicted by cancer therapy is an
important mechanism of resistance to treatment. How-
ever, in the absence of DNA damage, PARP does not
appear to be necessary for cell survival. PARP ‘knockout’
mice are viable and appear to be largely unaffected by
the lack of the enzyme.11 If PARP has no involvement
in critical physiological processes, then inhibiting the
enzyme is unlikely to induce major toxicity. PARP is
therefore an attractive target for drug design in the
context of cancer treatment.
Early studies identified nicotinamide (1) and various
3-substituted benzamides [e.g. 2, R ) NH2 (3AB)] as
inhibitors of PARP.12-14 However, these compounds lack
potency and specificity, and some are poorly soluble in
water. A significant advance was the synthesis of the
dihydroisoquinolines 3 and 4 in which the carboxamide
is constrained within a ring. Compound 3 is much more
active than 4 as an inhibitor of PARP (IC50 values of
0.41 and 8 µM, respectively).15 These results suggest
that the conformation of NAD+ that binds to PARP has
the carboxamide carbonyl anti to the C2-C3 bond. This
supposition was confirmed by ab initio molecular orbital
Poly(ADP-ribose) polymerase (PARP, EC 2.4.2.30) is
a nuclear enzyme with an important role in the process
of genomic repair.2,3 Inhibition of PARP in tumor cells
may potentiate radiotherapy and certain kinds of cancer
chemotherapy targeted to DNA.4 Three structural do-
mains control the activity of PARP: the N-terminal
DNA binding domain (containing two zinc fingers), a
central automodification domain, and the highly con-
served C-terminal catalytic region.5 The enzyme initially
recognizes and binds to a site of DNA damage. Binding
to DNA stimulates PARP to catalyze the synthesis of
linear and branched ADP-ribose polymers from sub-
strate nicotinamide adenine dinucleotide (NAD+), with
concomitant release of nicotinamide.6 The mechanism
of polymer formation has recently been probed using the
techniques of X-ray crystallography and site-directed
mutagenesis, which have shown the significance of the
strictly conserved active site residue Glu 988.7 The
polymer can be anchored either to the automodification
domain of PARP or to an adjacent histone. Poly(ADP-
ribose) is in a dynamic state, its rapid synthesis being
followed by a degradation catalyzed by the enzyme poly-
(ADP) glycohydrolase, before resynthesis occurs.8,9 The
* To whom correspondence should be addressed. Tel: +44 191 222
6647. Fax: +44 191 222 6929. E-mail: b.t.golding@newcastle.ac.uk.
† Department of Chemistry, Newcastle University.
§ Cancer Research Unit, Newcastle University.
‡ Agouron Pharmaceuticals Inc.
10.1021/jm000950v CCC: $19.00 © 2000 American Chemical Society
Published on Web 10/19/2000

Resistance-Modifying Agents. 9
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4085
Sch em e 1a
calculations on the nicotinamide moiety of NAD+, which
revealed that the conformation with the carbonyl posi-
tioned anti to the 2,3-bond (5) is preferred over that in
6.16 The carboxamide group can also be restricted via
an intramolecular hydrogen bond, as demonstrated for
benzoxazole-4-carboxamides 7 (X ) O, R ) Me)17a and
for benzimidazole-4-carboxamides17b (see also below).
In this paper, we report the synthesis of a series of
2-arylbenzimidazole-4-carboxamides 7 (X ) NH), which
are potent inhibitors of PARP and are able to act as
resistance-modifying agents18 for radiotherapy and
certain types of chemotherapy.19 These compounds
contain an intramolecular hydrogen bond, which helps
to impose the conformation of the molecule necessary
for inhibitory activity against PARP. We have previously
reported on the ability of quinazolinone inhibitors of
PARP to augment DNA-damaging drugs.18-20
a
Reagents: (i) NH3(aq); (ii) KOH, Br2, H2O; (iii) 10% Pd/C, H2,
MeOH; (iv) HCl, MeOH; (v) SOCl2/DMF, THF.
Sch em e 2a
Resu lts a n d Discu ssion
Ch em istr y. A variety of methods have been employed
to synthesize the benzimidazole-4-carboxamides de-
scribed herein. The 2-substituted benzimidazole-4-car-
boxylic acids 18, 20, and 22 were obtained by heating
2,3-diaminobenzoic acid (11) with a carboxylic acid in
the presence of hydrochloric or polyphosphoric acid
(Phillips synthesis; cf. Scheme 2).21,22 The required
2,3-diaminobenzoic acid (11) was readily obtained in
multigram quantities from 3-nitrophthalic anhydride
(8) by a literature procedure (Scheme 1).23 Thus,
regioselective ring opening of 8 with ammonia yielded
amide 9, a substrate for a Hofmann rearrangement that
afforded 2-amino-3-nitrobenzoic acid (10). Reduction of
10 gave 2,3-diaminobenzoic acid (11), while other stan-
dard reactions gave the useful intermediates 12-14 and
68 (Scheme 1). The benzimidazole-4-carboxylic acids 18,
20, and 22 were each converted into the corresponding
amides 19, 21, and 23, respectively, via the acid chloride
(Scheme 2).
Methyl 2,3-diaminobenzoate (12) was used to pre-
pare 2-arylbenzimidazole-4-carboxamides 24-30. The
more nucleophilic 3-amino group of 12 was selectively
acylated with an aryl acid chloride. The resulting
intermediate amide was cyclized under acidic conditions
to give the methyl ester of a 2-arylbenzimidazole-4-
carboxylic acid, which was converted to the required
amide by treatment with ammonia under pressure.
Both of the methods described above were inefficient
and not generally applicable. A modified version of a
procedure first reported by Weidenhagen24 was devel-
a
Reagents: (i) RCO2H, 4 M HCl or PPA, heat; (ii) SOCl2 then
NH3(aq); (iii) RCOCl, Et3N, DMAP, THF, 0 °C; (iv) AcOH, heat;
(v) NH3(l), 80 °C, 40 atm; (vi) RCHO, Cu(OAc)2, MeOH, heat, then
H2S.
oped for the direct preparation of numerous 2-aryl-
benzimidazole-4-carboxylic acids and amides. In this
method either 2,3-diaminobenzoic acid (11) or its amide
(13) was treated with an aryl aldehyde and copper(II)
acetate25 to give the desired 2-arylbenzimidazole-4-
carboxylic acid or amide (Tables 2 and 3). Of all the
methods utilized to synthesize the benzimidazole inhibi-
tors described herein, the route starting with amide 13
(Scheme 2) was the most efficient.
For the preparation of 2-trifluoromethylbenzimidazole
(17; Scheme 3), methyl 2-amino-3-nitrobenzoate (14)
was acylated with trifluoroacetic anhydride to yield 15.
Hydrogenation (see introduction to the Experimental
Section) of 15 yielded methyl 2-trifluoromethylbenzimi-
dazole-4-carboxylate (16) directly, the cyclization step
occurring in situ. The activating effect of the trifluoro-
methyl group enabled the conversion of 16 into amide

4086 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
White et al.
Sch em e 3a
(2, R ) NH2, Ki ) 3100 nM). However, placing a phenyl
group at the 2-position gave a significant increase
in activity (23, Ki ) 15 nM). A series of 2-arylbenzimi-
dazole-4-carboxamides was therefore synthesized to
enable the SAR for substituents on the phenyl ring to
be explored. A variety of substituents, with differing
electronic properties, were introduced at the 3- and
4-positions (38-46). Inhibition data showed that PARP
tolerates a variety of 3- and 4-substituents on the
2-phenyl group well and does not discriminate signifi-
cantly between electronically differing groups. 2-(4-
Cyanophenyl)benzimidazole-4-carboxamide (41, Ki ) 4
nM) was the most active compound among these inhibi-
tors. Comparison of 2-(3-methoxyphenyl)benzimidazole-
4-carboxamide (44) with 2-(3-hydroxyphenyl)benzimi-
dazole-4-carboxamide (60) showed little difference in
activity (Ki ) 6 nM for both compounds), indicating that
there is no favorable hydrogen-bonding interaction with
the protein for the 3-hydroxy group. Similarly, 2-(4-
methoxyphenyl)benzimidazole-4-carboxamide (38) and
2-(4-hydroxyphenyl)benzimidazole-4-carboxamide (45)
were equipotent (Ki ) 6.8 and 6 nM, respectively). The
position of the substituent (3 versus 4) is also relatively
unimportant for the aforementioned examples. How-
ever, 2-(4-hydroxymethylphenyl)benzimidazole-4-car-
boxamide (78) was significantly more active (Ki ) 1.6
nM) than its 3-hydroxymethyl isomer 79 (6.8 nM). As
expected, the precursor (75) of 78, containing a bulky
trialkylsilyl group, exhibited much reduced activity
(Ki ) 85 nM).
Both 2-(2-methoxyphenyl)benzimidazole-4-carbox-
amide (61, Ki ) 126 nM) and 2-(2-trifluoromethyl-
phenyl)benzimidazole-4-carboxamide (81, Ki ) 190 nM)
were considerably less active than their 3- and 4-sub-
stituted counterparts. However, comparison of the
data for the 2-methoxy group (61) with the isosteric
2-hydroxymethyl group (80, Ki ) 10.6 nM) suggests that
the hydroxymethyl group of 80 interacts with the
enzyme via a hydrogen bond or solvent molecule.
Furthermore, this compound is nearly as potent as its
3-isomer 79 (see above). It is important to note that 2-(2-
chlorophenyl)benzimidazole-4-carboxamide (82) and 2-(2-
fluorophenyl)benzimidazole-4-carboxamide (84) are both
well-tolerated by the enzyme (Ki ) 9.4 and 9 nM,
respectively) and are comparable in activity to their
3-analogues (e.g. 83). It appears that relatively small
groups and substituents containing a hydroxyl group
can be accommodated at the 2-position.
a
Reagents: (i) (CF3CO)2O, Et3N, THF; (ii) 10% Pd/C, H2,
MeOH; (iii) NH3(aq).
Sch em e 4a
a
Reagents: (i) NaH, TIPSCl, THF; (ii) MnO2, THF; TIPS )
trisopropylsilyl.
17 by treatment with aqueous ammonia. 2-(4-Hydroxy-
phenyl)benzimidazole-4-carboxamide (45) was prepared
from the corresponding 4-methoxy compound 38 by
treatment with BBr3 in DCM. 4-Carboxyphenylbenz-
imidazole (46) was obtained by basic hydrolysis of 2-(4-
cyanophenyl)benzimidazole-4-carboxamide (41). Benz-
imidazole N-alkylation and N-acylation (47-49) was
achieved by treatment of the appropriate benzimidazole
with an equivalent of potassium hydroxide in acetone
before addition of the alkylating (MeI) or acylating agent
(BzCl and CbzCl).26
2-Arylbenzimidazole-4-carboxamides containing a hy-
droxymethyl group at the 2-, 3-, and 4-positions (78-
80) were prepared by the Weidenhagen method from
the corresponding aldehyde precursors (70, 72, 74).
These were prepared by monosilylation of the appropri-
ate benzene-dimethanol with triisopropylsilyl chloride
(Scheme 4), with benzene-1,2-dimethanol giving the
highest yield (85%) because further silylation is inhib-
ited by the adjacent bulky ortho-silyl group. Oxidation
of each monosilylated benzene-dimethanol with man-
ganese dioxide afforded the required aldehyde.
Compounds expected to exhibit low activity against
PARP were synthesized for comparative biological stud-
ies. The N-methylamides 88 and 90 were obtained by
reaction of the corresponding methyl ester with methyl-
amine. In these amides a critical hydrogen-bonding site
is blocked by N-methylation. Likewise, 2-arylbenzimi-
dazole-4-carboxylic acids (e.g. 91 synthesized from 2,3-
diaminobenzoic acid (11) and 4-hydroxybenzaldehyde
via the Weidenhagen method) lack the amide function-
ally required for binding to PARP.
In h ibitor y Activities of 2-Su bstitu ted Ben zim i-
dazole-4-car boxam ides Again st P ARP . In this study,
structure-activity relationships (SAR) for a series of
2-substituted benzimidazole-4-carboxamides have been
probed by assaying for in vitro activity against recom-
binant human PARP. The simple benzimidazole-4-
carboxamides (17, 19, 21) showed sub-micromolar ac-
tivity against PARP and are considerably more active
than the classical PARP inhibitor 3-aminobenzamide
Benzimidazole-4-carboxamides containing a disub-
stituted (62, 63, 67, 86, 87) and trisubstituted (64)
2-phenyl group were synthesized to explore further the
SAR. These compounds showed improved aqueous
solubility compared to monosubstituted analogues. The
disubstituted inhibitors were generally well-accepted by
the enzyme, with the benzimidazole-4-carboxamide (86)
being particularly active (Ki ) 2.0 nM). The isomeric
benzimidazole 62 was 6-fold less active (Ki ) 13 nM).
The 2-(3,4,5-trimethoxyphenyl)benzimidazole-4-carbox-
amide (64) exhibited a sharp decrease in activity (Ki )
136 nM) indicating that while one meta-substituent is
well-tolerated, an additional meta-substituent is not.
For compounds 47 and 49 there is a decrease in
inhibitory activity as the size of the N(1) substituent
increases (Ki ) 32 and 530 nM for methyl and benzyl-

Resistance-Modifying Agents. 9
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4087
Ta ble 1. Physical and Biological Activity Data
compd
structure
X
Y
R
methoda
yield (%)
molecular formula
anal.b
Ki (nM)c
3100e
3-ABd
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
I
I
I
I
I
I
I
II
II
II
II
II
II
II
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
III
NH2
OH
NH2
OH
NH2
OH
NH2
CF3
H
H
Me
Me
Ph
Ph
75
77
13
72
14
20
62
62
14
45
37
92
26
23
75
70
65
72
91
96
58
80
48
74
73
25
72
46
57
47
30
15
61
C9H6F3N3O2
C8H6N2O2
C8H7N3O
C9H8N2O2
C9H9N3O
C, H, N
C, H, N
C, H, Ng
h
350
NT f
95 ( 11
NT
99 ( 6
NT
15
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
A
B
A
B
A
C
D
D
D
D
j
D
D
E
E
E
E
E
E
E
F
C, H, Ni
h
C14H10N2O2
C14H11N3O‚0.1EtOH
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
h
C, H, N
C, H, N
h
C, H, N
C, H, N
h
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
h
4′-OMe
4′-CF3
4′-NO2
4′-CN
4′-NH2
3′-CF3
3′-OMe
4′-OMe
4′-CF3
4′-NO2
4′-CN
4′-NH2
3′-CF3
3′-OMe
4′-OMe
4′-CF3
4′-NO2
4′-CN
4′-NH2
3′-CF3
3′-OMe
4′-OH
4′-CO2H
4′-OMe
4′-OMe
4′-OMe
C
16H16N2O4
C16H13F3N2O3
C15H13N3O5
C
16H13N3O3
C15H15N3O3
C16H13F3N2O3
C
C
16H16N2O4
18H18N2O5
OMe
OMe
OMe
OMe
OMe
OMe
OMe
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
C18H15F3N2O4
C15H11N3O4
C
16H11N2O5
C17H17N3O4
C18H15F3N2O4
NT
NT
NT
C
18H18N2O5
C15H13N3O2
C15H10F3N3O‚0.1H2O
C14H10N4O3
6.8 ( 3.9
F
F
F
F
F
F
F
F
1.2
8 ( 0e
4
C15H10N4O
C14H12N4O‚0.2MeOH
C15H10F3N3O
C15H13N3O2
C14H11N3O2
C15H11N3O3
C16H15N3O2
C22H17N3O3
C23H19N3O4
11
8 ( 0e
6
6
290
Me
Bz
Cbz
G
G
G
C, H, N
C, H, N
C, H, N
32e
NT (see text)
530
a
b
See Experimental Section. Elemental analyses are (0.4% of calculated values. c Assay against PARP full length protein unless
otherwise stated. 3-Aminobenzamide. e Assayed against PARP L713F protein. f Not tested. g Anal. C, H; N: found 24.59, expected 26.09.
Satisfactory analysis not obtained. Anal. C, H; N: found 23.39, expected 24.0. Prepared by hydrogenation of 26.
d
h
i
j
Ta ble 2. Physical and Biological Activity Data
compd
R1
R2
R3
methoda
yield (%)
molecular formula
anal.b
Ki (nM)c
59
60
61
62
63
64
65
66
67
4′-Cl
3′-OH
2′-OMe
3′-OMe
3′-OMe
3′-OMe
4′-NMe2
4′-CO2Et
3′,4′ (OCH2O)
H, I
H, I
H, I
H, I
H, I
H, I
H, I
H, I
H, I
38
62
38
20
14
12
17
20
15
C14H10ClN3O‚0.1MeOH
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, Nd
3 ( 0.4
6.3 ( 2.1
126 ( 3
13
C
C
C
C
C
14H11N3O2
15H13N3O2
4′-OH
4′-OMe
4′-OMe
15H13N3O3‚0.1MeOH
16H15N3O3‚0.5HCl
17H17N3O4
5.4 ( 1.2
136 ( 12
5.8 ( 0.9
8.1 ( 4
5′-OMe
C16H16N4O
C17H15N3O3‚0.1EtOH
C15H11N3O3
5.9 ( 0.4
a
b
See Experimental Section. Elemental analyses are (0.4% of calculated values. c Assay against PARP full length protein unless
d
otherwise stated. Anal. H, N; C: found 64.58, expected 64.05.
oxycarbonyl, respectively). An accurate value could not
be determined for 48 because of the instability of this
compound in the assay medium. These results indicate
a tight pocket in the active site region near the imida-
zole N(1)H. An unsubstituted imidazole is optimum,
either because the free N(1)H interacts with a water
molecule within the active site or because an N(1)
substituent interacts unfavorably with the protein.

4088 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
Ta ble 3. Physical and Biological Data
White et al.
compd
X
R1
R2
methoda
yield (%)
molecular formula
C24H33N3O2Si
C24H33N3O2Si
C24H33N3O2Si
C15H13N3O2
C15H13N3O2
C15H13N3O2
C15H10F3N3O‚0.2H2O
C14H10ClN3O
C14H10ClN3O‚0.33MeOH
C14H10FN3O
C15H13N3O2‚0.75H2O
C18H17N3O3
anal.b
NTd
NT
NT
Ki (nM)c
75
76
77
78
79
80
81
82
83
84
86
87
88
90
91
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NHMe
NHMe
OH
4′-CH2OTIPS
3′-CH2OTIPS
2′-CH2OTIPS
4′-CH2OH
3′-CH2OH
2′-CH2OH
2′-CF3
I
I
I
85
48
43
65
47
51
43
24
24
57
87
40
94
69
15
85
NT
NT
1.6 ( 0.4
6.8 ( 0.3
10.6 ( 0.5
190
9.4 ( 0.9
8.4 ( 0.5
9
C, H, Ne
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
f
I
I
I
I
2′-Cl
3′-Cl
2′-F
3′-OH
3′-O-allyl
4′-OH
4′-OMe
4′-OH
4′-OMe
4′-OMe
H, C
I
C, H, Ng
f
C, H, N
C, H, N
f
2.0 ( 0.2
9.4
C
15H13N3O2‚H2O
inactiveh
inactivei
104
C16H15N3O2‚0.6EtOH‚0.1H2O
C14H10N2O3
a
b
See Experimental Section. Elemental analyses are (0.4% of calculated values. c Assay against PARP full length protein unless
otherwise stated. Not tested. e Anal. C, H; N: found 14.42, expected 15.72. f Satisfactory analysis not obtained. Anal. C, H; N: found
13.62, expected 14.15. No inhibition at 100 µM. No inhibition at 10 µM.
d
g
h
i
As expected, replacement of the primary carboxamide
of 2-arylbenzimidazole-4-carboxamides with a secondary
amide (88, 90) or carboxylic acid (91) removed all
activity. Two amide protons are an absolute require-
ment for inhibitory activity: one proton being involved
in the essential intramolecular hydrogen bond, while
the other is required as a hydrogen-bonding donor to a
backbone amide carbonyl group of the enzyme, as
discussed below.
The data presented in this section indicate that a
variety of substituents at either the 3- or 4-position or
at both positions enhance the potency of 2-phenylbenz-
imidazole-4-carboxamide as an inhibitor of PARP. The
data can be understood with reference to the crystal
structure of 2-(3-methoxyphenyl)benzimidazole-4-car-
boxamide (44) (see below), which shows that the 3-meth-
oxyphenyl ring occupies a large, well-defined pocket in
the protein structure but with space around the ring
edge that permits a variety of further substituents.
F igu r e 1. Inhibition of A2780 cell growth by temozolomide:
potentiation by NU1085 (45). Cell growth was determined
by sulforhodamine B assay after 72 h of continuous exposure
to increasing concentrations of temozolomide with or without
co-exposure to 10 µM NU1085. Data are the mean of 5
replicates: temozolomide alone, b; temozolomide + 10 µM
NU1085, O.
P ot en t ia t ion of Top ot eca n a n d Tem ozolom id e
b y 2-(4-Hyd r oxyp h en yl)ben zim id a zole-4-ca r box-
a m id e (45). The ability of PARP inhibitors, including
2-methylbenzimidazole (21) to act as radio- and chemo-
potentiators in vitro has been demonstrated.20,27 Good
potentiation, as well as enzymatic inhibition, is vital if
these compounds are to function as therapeutically
useful resistance-modifying agents. Growth inhibition
assays demonstrated that 2-(4-hydroxyphenyl)benzimi-
dazole-4-carboxamide (NU1085, 45) potentiates topo-
tecan (TP), a topoisomerase I inhibitor, and temozolo-
mide (TM), a monofunctional alkylating agent recently
approved for the treatment of glioma.
A2780 human ovarian carcinoma cells were grown in
the presence of varying concentrations of TM with or
without 10 µM NU1085 (45). Cells exposed to TM alone
showed reduced cell survival compared to those treated
in combination with a PARP inhibitor (see Figure 1).
Likewise, cells exposed to TP and NU1085 (45) re-
sponded in a similar manner, the presence of the PARP
inhibitor leading to an increase in cell growth inhibition
(see Figure 2). Pooled data from two independent
experiments are given in Table 4. Calculation of the IC50
values for TM and TP with and without NU1085
allowed the determination of the potentiation factor,
PF50, for NU1085 against each drug. The benzimidazole
PARP inhibitor potentiated the cytotoxicity of temo-
zolomide and topotecan by 2-3-fold. A more detailed
discussion of related potentiation experiments has been
reported previously.20
Str u ctu r e Deter m in a tion of th e Com p lex Be-
tw een P ARP a n d 2-(3-Meth oxyp h en yl)ben zim id a -
zole-4-ca r boxa m id e (44) by X-r a y Cr ysta llogr a p h y.
The above conclusions from ‘traditional’ analysis of
the SAR data have been validated by an X-ray crys-
tallographic study of a complex between a representa-
tive inhibitor, 2-(3-methoxyphenyl)benzimidazole-4-car-
boxamide (44), and the catalytic domain of chicken
PARP (see Figure 3). The carboxamide group of 44 forms

Resistance-Modifying Agents. 9
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4089
F igu r e 2. Inhibition of A2780 cell growth by topotecan:
potentiation by NU1085 (45). Cell growth was determined by
sulforhodamine B after 72 h of continuous exposure to increas-
ing concentrations of topotecan with or without co-exposure
to 10 µM NU1085 assay. Data are the mean of 5 replicates:
topotecan alone, b; topotecan + 10 µM NU1085, O.
F igu r e 3. Compound 44 bound to the PARP active site. The
inhibitor is shown with white bonds, green spheres for carbon,
red spheres for oxygen, and blue spheres for nitrogen. The
protein is shown with bonds, also color-coded by elemental
type. Important residues surrounding the inhibitor are labeled
and shown with thick bonds. Three important hydrogen bonds
between the inhibitor and protein are shown as yellow lines.
Water molecules are shown as red crosses except for the one
water molecule that is hydrogen-bonded to the inhibitor and
is shown as a red sphere. The protein molecular surface is
shown with white dots.
Ta ble 4. Summary of Potentiation of Temozolomide and
Topotecan Growth Inhibition by 10 µM NU1085 (45)a
IC50
temozolomide (µM) topotecan (nM)
drug alone
525, 368
188, 183
2.8, 2.0
22.6, 22
7.8, 8.7
2.9, 2.5
drug + 10 µM NU1085
b
PF50
a
Data are from two independent experiments of the type shown
b
in Figures 1 and 2. PF50 is the potentiation factor at 50% growth
inhibition, i.e., IC50 for temozolomide (or topotecan) alone/IC50 for
temozolomide (or topotecan) +PARP inhibitor.
3-, 4-, and 5-positions, the most available space is at
the exposed position where the 3-methoxy is found.
Taking into consideration the mobility of the Gln 763
side chain, significant space is also available at the
4-position, whereas less space for substituents is found
extending from the 5-position. The information from
the crystal structure is in accord with the findings
regarding inhibitory potencies of inhibitors (see above),
in particular for 2-(3,4,5-trimethoxyphenyl)benzimida-
zole-4-carboxamide (64, Ki ) 136 nM).
An ordered water molecule is located 3.0 Å from the
benzimidazole N(1) nitrogen and is a hydrogen bond
acceptor. This water molecule is also 2.8 Å from the
catalytically important carboxylate of Glu 988. Another
water is found 3.5 Å from the 4-carboxamide nitrogen,
which is also 3.1 Å from the Gly 863 carbonyl oxygen,
3.6 Å from the side chain oxygen of Ser 864, and 3.5 Å
from a third water molecule.
three important hydrogen bonds, similar to the hydro-
gen bonding observed with other inhibitors bound to
PARP.28,29 In this case the inhibitor carbonyl oxygen
accepts two hydrogen bonds: one from the side chain
oxygen of Ser 904 (3.3 Å) and the other from a Gly 863
polypeptide amide NH (2.9 Å). The amide NH of 44 syn
to the carbonyl group is a hydrogen bond donor to the
Gly 863 backbone carbonyl oxygen (3.2 Å). The confor-
mation observed for the carboxamide group of 44 was
also seen in the crystal structure (i.e. measured for the
protein-free compound) of the analogous benzimidazole
38 and may be that intrinsically preferred by all of
2-arylbenzimidazole-4-carboxamides described herein.17b
The benzimidazole of 44 lies in a deep pocket formed
by residues Phe 897, Ala 898, Lys 903, and Glu 988 and
is buried between two tyrosine residues (Tyr 896 and
907). The residue Tyr 907 is approximately coplanar
with the benzimidazole, while Tyr 896 is rotated ap-
proximately 37° from coplanarity. The 2-(3-methoxy-
phenyl) group is located above a large, solvent-contain-
ing cavity of the enzyme. The 2-phenyl ring is rotated
approximately 23° relative to the benzimidazole plane.
Both faces of the 2-phenyl group are exposed to solvent,
as is the 3-methoxy group, although it is partially
shielded from solvent by a mobile Gln 763 side chain.
The 3-methoxy group is on the same side of the inhibitor
as the 4-carboxamide and is centered over the large
enzyme cavity. The unsubstituted 4- and 5-edge, on the
other side of the inhibitor, is oriented toward the surface
of the enzyme cavity, which is composed of the Gln 763
side chain (near 4) and main chain atoms from the
carbonyl of 888 through the amide of 890 (near 5). This
edge of the 2-phenyl ring is shielded from solvent, being
from 3.7 to 4.9 Å away from the protein. Comparing the
Con clu sion s
Structural modifications around a benzimidazole-4-
carboxamide core have identified 2-phenyl-1H-benz-
imidazoles as potent PARP inhibitors with clearly
defined structure-activity requirements.30 2-(4-Hydroxy-
phenyl)benzimidazole-4-carboxamide (NU1085, 45) has
been adopted as a benchmark inhibitor of PARP due
to its potency, ease of synthesis, and relatively good
solubility in water. Potentiation experiments have
shown that NU1085 (45) can increase the potency of the
cytotoxic agents topotecan and temozolomide by up to
3-fold. Clinically, a drug combination that could result
in reduced dosage levels and a decreased possibility of
drug resistance is extremely attractive. Consideration
of Ki, PF50, and crystallographic data is guiding the

4090 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
White et al.
(3.6 g, 60%): mp 95-96 °C; IR 3452, 3317, 1702, 1254 cm-1
;
synthesis of potent, selective, and bioavailable com-
pounds, which may be clinically useful PARP inhibitors.
1H NMR δ 3.96 (3 H, s), 6.80-6.88 (1 H, t), 8.29-8.34 (1 H,
d), 8.41-8.46 (1 H, d), 8.46 (2 H, br s); 13C NMR δ 52.7, 114.1,
114.4, 132.3, 132.9, 139.6, 146.6, 167.1; HRMS (EI) m/z
196.0479 [M+ calcd 196.0484 for C8H8N2O4].
Exp er im en ta l Section
Melting points were obtained on a Kofler hot stage ap-
paratus and are uncorrected. Infrared spectra (IR) were
recorded as KBr disks on a Nicolet 20 PC Fourier transform
spectrometer. Proton and carbon-13 nuclear magnetic reso-
nance (NMR) spectra were recorded at 200 and 50 MHz,
respectively, on a Bruker WP 200 spectrometer employing the
deuterated solvent as internal standard. Unless indicated
otherwise, spectra were recorded in DMSO-d6 as solvent. NH
and OH signals appeared as broad singlets (br s) exchangeable
with D2O. Chemical shift values are quoted in parts per million
(ppm) and coupling constants (J ) in hertz (Hz). 1H NMR
spectra of para-disubstituted aromatic rings were observed as
an AA′BB′ pattern but for simplicity are quoted as doublets;
key: s ) singlet, d ) doublet, t ) triplet, q ) quartet, m )
multiplet. Mass spectra were determined on a Kratos MS80
spectrometer in electron impact (EI) mode or fast atom
bombardment (FAB mode using a m-nitrobenzyl alcohol
matrix). The TLC systems employed Merck 1.05554 aluminum
sheets precoated with Kieselgel 60F254 (0.2 mm) and were
visualized with UV light at 254 or 365 nm. Column chroma-
tography was conducted under medium pressure on silica
(Kieselgel 60, 240-400 mesh). Elemental analyses were
performed either in-house on a Carlo-Erba Instrumentazione
1106 analyzer or by Butterworth Laboratories, Middlesex,
U.K., and are within (0.4% of theoretical value unless
otherwise specified. Reagents for organic synthesis were
purchased from Aldrich Chemical Co. (Gillingham, U.K.) and
Lancaster Synthesis (Morecambe, U.K.) and were used as
received unless otherwise stated. Routine and tissue culture
chemicals for growth inhibition assays were obtained from
Sigma Chemical Co. (Poole, U.K.) unless stated otherwise.
Ethanol and methanol were dried using Mg/I2 and stored over
molecular sieves. Diethyl ether and tetrahydrofuran were
predried over CaCl2 and distilled from sodium/benzophenone.
Petroleum ether refers to that fraction in the boiling range
40-60 °C. Solvents were removed under reduced pressure
using a rotary evaporator. Concentrated ‘880’ ammonia (aque-
ous ammonia solution) was used for amide formation from acid
chlorides.
Unless otherwise stated, catalytic hydrogenations were
performed for solutions in methanol using 10% Pd/C catalyst
(10 mol % w/w) stirred under hydrogen at atmospheric
pressure until gas absorption ceased. After filtration through
Celite and removal of the solvent, the resulting material was
used directly or purification was performed as described below.
2-Am in o-3-Nitr oben zoic Acid (10).22 Potassium hydrox-
ide pellets (22.6 g, 0.40 mol) in water (110 mL) were cooled
to 0 °C. Bromine (2.3 mL, 0.045 mol), followed by 3-nitro-
phthalamic acid (9)22 (9.0 g, 0.047 mol), was added. The
reaction mixture was warmed at 60 °C for 3 h, allowed to cool
to room temperature, and stirred overnight. The orange
precipitate was filtered off and taken up in the minimum
quantity of water to give a red solution, which was adjusted
to pH 4 with concentrated HCl, precipitating a yellow solid.
Filtration, followed by recrystallization from water, yielded
a bright yellow solid (7.27 g, 93%): mp 208-209 °C (lit.22
208-209 °C).
Meth yl 2,3-Dia m in oben zoa te (12). Methyl 2-amino-3-
nitrobenzoate (14) (1.12 g, 5.73 mmol) was hydrogenated (see
introduction to the Experimental Section) to give a brown solid
that was used directly (0.9 g, 94%): mp 63-64 °C; IR 3428,
3368, 3347, 1693 cm-1; 1H NMR δ 3.86 (3 H, s), 4.8-5.0 (2 H,
br s), 6.2-6.4 (2 H, br s), 6.45-6.53 (1 H, t), 6.78-6.84 (1 H,
d), 7.17-7.23 (1 H, d); 13C NMR δ 51.6, 109.3, 115.7, 117.7,
119.1, 136.2, 134.0, 169.0; HRMS (EI) m/z 166.0744 [M+ calcd
166.0742 for C8H10N2O2].
Meth od A. Ben zim id a zole Syn th esis (P h illip s Meth -
od ).21,22 A solution of 2,3-diaminobenzoic acid (11) (0.66-3.29
mmol) in 4 M HCl or polyphosphoric acid (PPA) (approximately
3 mL/mmol) containing an appropriate carboxylic acid (3 equiv)
was heated at reflux for 1 h. After cooling the reaction mixture
to room temperature, the precipitate was collected and redis-
solved in hot methanol to which activated charcoal was added.
The suspension was filtered and the solvents removed to yield
the product.
1H-Ben zim id a zole-4-ca r boxylic Acid (18). Method A.
2,3-Diaminobenzoic acid 11 and formic acid in 4 M HCl yielded
the product, 77%: mp >300 °C; IR 3125, 1715 cm-1; 1H NMR
δ 7.7-7.8 (1H, t), 8.2-8.3 (2H, dd), 9.65 (1H, s); HRMS (EI)
m/z 162.0445 [M+ calcd 162.0429 for C8H6N2O2]. Anal.
(C8H6N2O2) C, H, N.
Meth od B. Am id e F or m a tion Usin g Th ion yl Ch lor id e.
A solution of a benzimidazole-4-carboxylic acid (400-500 mg)
in thionyl chloride (10 mL) was boiled under reflux for 2-3 h.
Removal of the excess of thionyl chloride yielded a solid that
was suspended in dry THF (10 mL). The suspension was added
to stirred concentrated ammonia solution (50 mL). The am-
monia solution was evaporated and the residue was taken up
in water (20 mL). The resulting solution was extracted with
EtOAc and the combined extracts were dried (MgSO4). After
removal of the organic solvent the residual solid was purified
as appropriate.
1H-Ben zim id a zole-4-ca r boxa m id e (19). Method B. The
residual solid was redissolved in 0.1 M HCl (20 mL). After
filtration and adjusting the pH of the solution to pH 7, it was
progressively basified to pH 12 (1 M NaOH). Extractions
(EtOAc) were taken at each pH unit change. The extracts were
combined and dried (MgSO4) and the solvent was removed.
Recrystallization (EtOAc) yielded off-white crystals (50 mg,
1
13%): mp 214-218 °C; IR 3322, 3150, 1680 cm-1; H NMR δ
7.34-7.42 (1 H, t), 7.75 (1 H, s), 7.81-7.85 (1 H, d), 7.89-7.93
(1 H, d), 8.46 (1 H, br s), 9.4 (1 H, br s), 13.1 (1 H, br s); HRMS
(EI) m/z 161.0590 [M+ calcd 161.0589 for C8H7N3O]. Anal.
(C8H7N3O) C, H; N: found 24.59, expected 26.09.
2-Met h yl-1H -b en zim id a zole-4-ca r b oxylic a cid (20).
Method A. 2,3-Diaminobenzoic acid (11) and acetic acid in
4 M HCl yielded the product (72%): mp 188-191 °C; IR
1
2853, 1720 cm-1; H NMR δ 2.95 (3 H, s), 7.66-7.74 (1 H, t),
8.10-8.14 (2 H, dd); HRMS (EI) m/z 176.0582 [M+ calcd
176.0586 for C9H8N2O2].
2-Meth yl-1H-ben zim idazole-4-car boxam ide (21). Method
B. Recrystallization (EtOAc) yielded a white solid (70 mg,
1
14%): mp 233-238 °C; IR 3297, 3071, 1684 cm-1; H NMR δ
2,3-Dia m in oben zoic Acid (11). 2-Amino-3-nitrobenzoic
acid (10) (2.44 g, 13 mmol) was hydrogenated (see introduction
to the Experimental Section). The crude product was purified
by column chromatography (DCM-15% MeOH) to give a
deep red solid that was used directly (1.34 g, 66%): mp 204-
206 °C (lit.24 201 °C).
Meth yl 2-Am in o-3-n itr oben zoa te (14). A solution of
2-amino-3-nitrobenzoic acid (10) (5.0 g, 0.027 mol) in methanol
(80 mL) was cooled to 0 °C. Dry hydrogen chloride gas was
bubbled through the solution for 20 min, after which it was
heated at reflux for 6 h. After cooling and standing the reaction
mixture overnight, the precipitate was collected and recrystal-
lized (petroleum ether-EtOAc) to yield bright yellow crystals
2.68 (3 H, s), 7.30-7.38 (1 H, t), 7.72-7.76 (1 H, d), 7.86,7.90
(1 H, d), 7.72-7.90 (1 H, br s), 9.4 (1 H, br s), 12.8 (1 H, br s);
HRMS (EI) m/z 175.0749 [M+ calcd 175.0746 for C9H9N3O].
Anal. (C9H9N3O) C, H; N: found 23.39, expected 24.00.
2-P h en yl-1H -b en zim id a zole-4-ca r b oxylic Acid (22).
Method A. 2,3-Diaminobenzoic acid (11) and benzoic acid were
heated to 150-160 °C in PPA. After cooling to room temper-
ature, ice was added and the resulting solution was neutralized
with sodium hydroxide (10 M) and extracted (EtOAc). The
crude product was purified by column chromatography (DCM-
15% MeOH), 20%: mp 287-288 °C; IR 3433, 3336, 1680 cm-1
;
1H NMR δ 7.35-7.43 (1 H, t), 7.62-7.68 (3 H, m), 7.87-7.91

Resistance-Modifying Agents. 9
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4091
(1 H, d), 7.95-7.99 (1 H, d), 8.38-8.44 (2 H, d), 12.5 (1 H, br
s); HRMS (EI) m/z 238.0750 [M+ calcd 238.0742 for C14H10N2O2].
130.1, 133.2, 147.4, 162.2, 165.7, 168.3; HRMS (EI) m/z
300.1118 [M+ calcd 300.1110 for C16H16N2O4]. Anal. (C16H16N2O4)
C, H, N.
Meth od C. Am id e F or m a tion Usin g Th ion yl Ch lor id e/
DMF . A benzimidazole-4-carboxylic acid (0.2-1.8 mmol) was
suspended in THF. Thionyl chloride (1.5 equiv) and DMF (2
drops) were added. The resulting mixture was stirred at room
temperature until the carboxylic acid had been consumed
(monitoring by TLC). The mixture was added dropwise to
concentrated ammonia solution. Removal of the ammonia
allowed recovery of the amide by filtration or extraction
(EtOAc). Further purification was performed as appropriate.
Meth yl 2-Am in o-3-N-(4-tr iflu or om eth ylben zoyl)a m in o-
ben zoa te (25). Method D. Recrystallization from aqueous
MeOH yielded a white solid, 14%: mp 180-181 °C; IR 3389,
1
3292, 1701, 1653, 1330 cm-1; H NMR δ 3.93 (3 H, s), 6.70-
6.76 (3 H, m), 7.46-7.49 (1 H, d), 7.81-7.85 (1 H, d), 7.99-
8.03 (2 H, d), 8.29-8.33 (2 H, d), 10.05 (1 H, br s); HRMS (EI)
m/z 338.0886 [M+ calcd 338.0878 for C16H13F3N2O3]. Anal.
(C16H13F3N2O3) C, H, N.
Met h yl 2-Am in o-3-N-(4-n it r ob en zoyl)a m in ob en zoa t e
(26). Method D. Column chromatography (DCM-1% MeOH)
and recrystallization (MeOH) yielded the product, 45%: mp
2-P h en yl-1H-ben zim idazole-4-car boxam ide (23). Method
C. A white precipitate was obtained (31 mg, 62%): mp 239-
241 °C; IR 3184, 1669 cm-1 1H NMR δ 7.43-7.51 (1 H, t),
;
196-197 °C; IR 3382, 3293, 3257, 1702, 1658, 1525 cm-1
;
7.69-7.72 (3 H, m), 7.86-7.90 (1 H, d), 7.97 (1 H, br s), 7.98-
8.01 (1 H, d), 8.37-8.41 (2 H, d), 9.5 (1 H, br s); 13C NMR δ
115.4, 122.7, 123.3, 127.2, 129.4, 130.9, 135.7, 141.8, 152.3,
166.6; HRMS (EI) m/z 237.0907 [M+ calcd 237.0902 for
1H NMR δ 3.94 (3 H, s), 6.70-6.78 (1 H, t), 6.66 (2H, br s),
7.48-7.51 (1 H, d), 7.83-7.87 (1 H, d), 8.33-8.38 (2 H, d, J )
8.8), 8.46-8.51 (2 H, d, J ) 8.8), 10.15 (1 H, br s); (EI) m/z
315.0844 [M+ calcd 315.0855 for C15H13N3O5]. Anal. (C15H13N3O5)
C, H, N.
C
14H11N3O]. Anal. (C14H11N3O‚0.1EtOH) C, H, N.
Met h yl 2-N-(Tr iflu or oa cet yl)a m in o-3-n it r ob en zoa t e
Meth yl 2-Am in o-3-N-(4-cya n oben zoyl)a m in oben zoa te
(27). Method D. Column chromatography (DCM-1% MeOH)
and recrystallization (MeOH) yielded the product, 37%: mp
(15). To methyl 2-amino-3-nitrobenzoate (14) (100 mg, 0.51
mmol) and triethylamine (234 µL, 1.68 mmol) in THF (10 mL)
was added a solution of trifluoroacetic anhydride (238 µL, 1.68
mmol) in THF (5 mL) dropwise, with stirring at room tem-
perature (12 h). Water (0.5 mL) was added and the solvents
were removed to yield a yellow oil. Ice water (0.5 mL) was
added to the oil to give a white crystalline precipitate, which
was collected, washed with ice water and dried (95 mg, 64%):
mp 126-129 °C; IR 3295, 1726 (br) cm-1; 1H NMR δ 3.94 (3H,
s), 7.82-7.90 (1 H, t), 8.28-8.33 (1 H, d), 8.36-8.42 (1 H, d),
11.8 (1 H, br s); HRMS (EI) m/z 292.0316 [M+ calcd 292.0307
for C10H7F3N2O5].
1
198-202 °C; IR 3486, 3374, 3246, 2231, 1688, 1647 cm-1; H
NMR δ 3.93 (3 H, s), 6.68-6.76 (1 H, t), 6.72 (2 H, br s), 7.45-
7.49 (1 H, d), 7.81-7.86 (1 H, d), 8.11-8.15 (2 H, d, J ) 8.4),
8.25-8.29 (2 H, d, J ) 8.4), 10.1 (1 H, br s); HRMS (EI) m/z
295.0963 [M+ calcd 295.0957 for C16H13N3O3]. Anal. (C16H13N3O3)
C, H, N.
Meth yl 2-Am in o-3-N-(4-a m in oben zoyl)a m in oben zoa te
(28). Methyl 2-amino-3-N-(4-nitrobenzoyl)aminobenzoate (26)
(246 mg, 0.78 mmol) was hydrogenated (see introduction to
the Experimental Section) to give a white solid (204 mg,
92%): mp 197-200 °C; IR 3473, 3375, 3349, 3283, 1695, 1635
cm-1; 1H NMR δ 3.94 (3 H, s), 5.87 (2 H, s), 6.54 (2 H, s), 6.68-
6.73 (2 H, d), 6.73-6.76 (1 H, t), 7.42-7.47 (1 H, d), 7.78-
7.82 (1 H, d), 7.83-7.87 (2 H, d), 9.4 (1 H, br s); HRMS (EI)
m/z 285.1104 [M+ calcd 285.1113 for C15H15N3O3]. Anal.
(C15H15N3O3) C, H, N.
Meth yl 2-Am in o-3-N-(3-tr iflu or om eth ylben zoyl)a m in o-
ben zoa te (29). Method D. Column chromatography (DCM-
10% MeOH) and recrystallization (MeOH) yielded a white
solid, 26%: mp 157-159 °C; IR 3368, 3284, 1706, 1651, 1250
cm-1; 1H NMR δ 3.93 (3 H, s), 6.69-6.77 (1 H, t), 6.73 (2 H, s),
7.45-7.49 (1 H, d), 7.82-7.92 (2 H, m), 8.06-8.10 (1 H, d),
8.40-8.44 (1 H, d), 8.48 (1 H, s), 10.1 (1 H, br s); HRMS (EI)
m/z 338.0877 [M+ calcd 338.0878 for C16H13F3N2O3]. Anal.
(C16H13F3N2O3) C, H, N.
Met h yl 2-Am in o-3-N-(3-m et h oxyb en zoyl)a m in ob en -
zoa te (30). Method D. Column chromatography (DCM-10%
MeOH) and recrystallization (petroleum ether-EtOAc) yielded
the product, 23%: mp 124-125 °C; IR 3386, 3292, 1698, 1646
cm-1; 1H NMR δ 3.92 (3 H, s), 3.93 (3 H, s), 6.61 (2 H, s), 6.68-
6.76 (1 H, t), 7.22-7.27 (1 H, d), 7.44-7.47 (1 H, d), 7.49-
7.57 (1 H, t), 7.66 (1 H, s), 7.67-7.71 (1 H, d), 7.79-7.84 (1 H,
d), 9.8 (1 H, br s); HRMS (EI) m/z 300.1117 [M+ calcd 300.1110
for C16H16N2O4]. Anal. (C16H16N2O4) C, H, N.
Meth od E. Ben zim id a zole P r ep a r a tion Ca ta lyzed by
Gla cia l Acetic Acid . The N-acyl starting material (24-30,
0.2-1.6 mmol) was dissolved in glacial acetic acid (10 mL/
mmol), and heated at 120 °C until the reaction was complete
(monitoring by TLC). The solvent was removed and the solid
residue purified as appropriate.
Meth yl 2-Tr iflu or om eth yl-1H-ben zim id a zole-4-ca r box-
yla te (16). Hydrogenation (see introduction to the Experi-
mental Section) of 15 (51 mg, 0.21 mmol) gave a clear oil, which
crystallized under petroleum ether (38 mg, 75%): mp 86-88
°C; IR 3586, 3337, 3247, 3015, 1712, 1200, 1148 cm-1; 1H NMR
δ 4.07 (3 H, s), 7.56-7.63 (1 H, t), 8.11-8.15 (1 H, d), 8.19-
8.23 (1 H, d), 13.8 (1 H, br s); HRMS (EI) m/z 244.0462 [M+
calcd 244.0460 for C10H7F3N3O2].
2-Tr iflu or om et h yl-1H -b en zim id a zole-4-ca r b oxa m id e
(17). A solution of methyl 2-trifluoromethyl-1H-benzimidazole-
4-carboxylate (16) (26 mg, 0.1 mmol) in aqueous ammonia
solution (5 mL) was stirred at room temperature for 12 h.
Ammonia was removed under reduced pressure and the
aqueous residue was washed with EtOAc (3 × 5 mL). The
combined organic extracts were dried (MgSO4) and the solvent
was removed, to give the product (18 mg, 75%): mp 228-230
1
°C; IR 3351, 3187, 1669, 1157, 1144 cm-1; H NMR δ 7.57-
7.65 (1 H, t), 7.97-8.01 (2 H, d), 8.06-8.10 (1 H, d), 8.70 (1 H,
br s), 14.4 (1 H, br s); HRMS (EI) m/z 229.0460 [M+ calcd
229.0463 for C9H6F3N3O]. Anal. (C9H6F3N3O) C, H, N.
Meth od D. Acyla tion of Meth yl 2,3-Dia m in oben zoa te.
A solution of methyl 2,3-diaminobenzoate (12) (0.78-4.04
mmol, 1 equiv), triethylamine (1.5 equiv) and 4-(dimethyl-
amino)pyridine (DMAP, 5 mol %) in half the required volume
of THF (approximately 10 mL/100 mg 12) was cooled to 0 °C.
The appropriate acid chloride (1 equiv) was dissolved in the
remaining THF and added to the cooled solution over 30 min.
The reaction mixture was allowed to warm to room temper-
ature. The precipitate was collected, redissolved in EtOAc and
the resulting solution was washed twice with water followed
by saturated brine. The organic layer was dried (MgSO4), and
the solvents removed to leave a solid residue that was purified
as appropriate.
Meth yl 2-(4-Meth oxyp h en yl)-1H-ben zim id a zole-4-ca r -
boxyla te Aceta te Sa lt (31). Method E. Recrystallization
(petroleum ether-EtOAc) yielded a white crystalline solid,
1
75%: mp 141-142 °C; IR 3375, 1718, 1697 cm-1; H NMR δ
Met h yl 2-Am in o-3-N-(4-m et h oxyb en zoyl)a m in ob en -
zoa te (24). Method D. Recrystallization from MeOH-water
yielded the product, 62%: mp 179-180 °C; IR 3426, 3278,
1699, 1632 cm-1; 1H NMR δ 3.92 (3 H, s), 3.94 (3 H, s), 6.59 (2
H, br s), 6.68-6.75 (1 H, t), 7.13-7.17 (2 H, d), 7.43-7.46 (1
H, d), 7.79-7.83 (1 H, d), 8.07-8.12 (2 H, d), 9.7 (1 H, br s);
13C NMR δ 52.0, 55.8, 110.6, 113.8, 114.7, 125.0, 126.8, 129.1,
2.02 (3 H, s), 3.97 (3H, s), 4.09 (3 H, s), 7.21-7.25 (2 H, d, J )
8.6), 7.39-7.46 (1 H, t), 7.90-7.93 (1 H, d), 8.00-8.04 (1 H,
d), 8.36-8.40 (2 H, d, J ) 8.6), 12.1 (1 H, br s), 12.3-12.4
(1 H, br s); 13C NMR δ 21.4, 52.4, 55.6, 114.4, 121.7, 122.4,
124.3, 129.6, 153.6, 161.3, 166.1, 172.4; HRMS (EI) m/z
282.0991 [M+ - AcOH, calcd 282.1004 for C16H14N2O3]. Anal.
(C18H18N2O5) C, H, N.

4092 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
White et al.
Meth yl 2-(4-Tr iflu or om eth ylph en yl)-1H-ben zim idazole-
4-ca r boxyla te Aceta te Sa lt (32). Method E. Recrystallization
from petroleum ether-EtOAc yielded the product, 70%: mp
2-(4-Nit r op h en yl)-1H -b en zim id a zole-4-ca r b oxa m id e
(40). Method F. Column chromatography (DCM-1% MeOH)
and recrystallization from MeOH yielded the product, 74%:
mp >310 °C; IR 3436, 3073, 1661 cm-1; 1H NMR δ 7.48-7.56
(1 H, t), 7.90-7.94 (1 H, d), 8.00 (1 H, s), 8.00-8.04 (1 H, d),
8.52-8.56 (2 H, d, J ) 8.8), 8.60-8.64 (2 H, d, J ) 8.8), 9.3-
9.4 (1 H, br s), 13.8-14.0 (1 H, br s); HRMS (EI) m/z 282.0750
[M+ calcd 282.0753 for C14H10N4O3]. Anal. (C14H10N4O3) C,
H, N.
138-140 °C; IR 3329, 1709, 1694 cm-1 1H NMR δ 2.01
;
(3 H, s), 7.44-7.52 (1H, t), 7.97-8.14 (4H, m), 8.62-8.66
(2H, d), 12.1 (br s), 12.7-12.8 (1 H, br s); HRMS (EI) m/z
320.0402 [M+ - AcOH, calcd 320.0773 for C16H11F3N2O2]. Anal.
(C18H15F3N2O4) C, H, N.
Meth yl 2-(4-Nitr op h en yl)-1H-ben zim id a zole-4-ca r box-
yla te (33). Method E. Recrystallization from MeOH yielded
2-(4-Cya n op h en yl)-1H-ben zim id a zole-4-ca r boxa m id e
(41). Method F. Recrystallization from MeOH yielded the
the product, 65%: mp 208-210 °C; IR 1720, 1513 cm-1 1H
;
product, 73%: mp >310 °C; IR 3275, 3178, 2231, 1659 cm-1
;
NMR δ 4.21 (3 H, s), 7.57-7.65 (1 H, t), 8.10-8.12 (1 H, d),
8.23-8.27 (1 H, d), 8.60-8.64 (2 H, d, J ) 8.8), 8.78-8.82
(2 H, d, J ) 8.8), 13.04 (1 H, br s); HRMS (EI) m/z 297.0743
[M+ calcd 297.0750 for C15H11N3O4]. Anal. (C15H11N3O4) C,
H, N.
1H NMR δ 7.45-7.49 (1 H, t); 7.87-7.91 (1 H, d), 7.91 (1 H, br
s), 7.98-8.02 (1 H, d); 8.13-8.17 (2 H, d, J ) 8.3), 8.50-8.54
(2 H, d, J ) 8.3), 9.2-9.4 (1 H, br s), 13.6-13.8 (1 H, br s); 13
C
NMR δ 112.8, 115.8, 118.8, 123.4, 123.8, 127.8, 133.3, 133.6,
141.6, 141.8, 150.3, 166.4; HRMS (EI) m/z 262.0849 [M+ calcd
262.0855 for C15H10N4O]. Anal. (C15H10N4O) C, H, N.
Met h yl 2-(4-Cya n op h en yl)-1H -b en zim id a zole-4-ca r -
boxyla te (34). Method E. Recrystallization from petroleum
ether-EtOAc yielded the product, 72%: mp 195-198 °C; IR
2-(4-Am in op h en yl)-1H-ben zim id a zole-4-ca r boxa m id e
(42). Method F. Column chromatography (DCM-10% MeOH)
yielded the product, 25%: mp 237-240 °C; IR 3338, 3214, 1651
1
3448, 2229, 1692 cm-1; H NMR δ 4.09 (3 H, s), 7.44-7.53 (1
H, t), 7.97-8.01 (1 H, d), 8.10-8.13 (1 H, d), 8.13-8.17 (2 H,
d, J ) 8.4), 8.58-8.62 (2 H, d, J ) 8.4), 12.8 (1 H, br s); HRMS
(EI) m/z 277.0851 [M+ calcd 277.0851 for C16H11N2O5].
Met h yl 2-(4-Am in op h en yl)-1H -b en zim id a zole-4-ca r -
boxyla te Aceta te Sa lt (35). Method E. Recrystallization from
petroleum ether-EtOAc yielded the product, 91%: mp 162-
164 °C; IR 3451, 3369, 1692, 1648 cm-1; 1H NMR δ 2.02 (3 H,
s), 4.08 (3 H, s), 5.81 (2 H, s), 6.75-6.80 (2 H, d, J ) 8.6),
7.32-7.40 (1 H, t), 7.83-7.86 (1 H, d), 7.93-7.97 (1 H, d), 8.08-
8.13 (2 H, d, J ) 8.6), 11.9 (1 H, br s), 12.1 (1 H, br s); HRMS
(EI) m/z 267.1016 [M+ - AcOH, calcd 267.1008 for C15H13N3O2].
Anal. (C17H17N3O4) C, H, N.
cm-1 1H NMR δ 5.90 (2 H, s), 6.79-6.83 (2 H, d, J ) 8.3),
;
7.31-7.39 (1 H, t), 7.71-7.75 (1 H, d), 7.84 (1 H, s), 7.88-7.92
(1 H, d), 8.00-8.04 (2 H, d, J ) 8.3), 9.55 (1 H, br s), 13.0 (1
H, br s); HRMS (EI) m/z 252.1002 [M+ calcd 252.1011 for
C
14H12N4O]. Anal. (C14H12N4O‚0.2MeOH) C, H, N.
2-(3-Tr iflu or om eth ylp h en yl)-1H-ben zim id a zole-4-ca r -
boxa m id e (43). Method F. Recrystallization from MeOH
yielded the product, 72%: mp 268-270 °C; IR 3349, 3176,
1
1668, 1330 cm-1; H NMR δ 7.44-7.52 (1 H, t), 7.88-8.04 (5
H, m), 8.66-8.70 (1 H, d), 8.70 (1 H, s), 9.3 (1 H, br s), 13.6 (1
H, br s); 13C NMR δ 48.9, 115.6, 121.9, 123.1, 123.6, 127.2,
129.9, 130.6, 130.7, 131.2, 135.7, 136.0, 150.7, 166.5; HRMS
(EI) m/z 305 [M+], 288 [-CH3OH for C15H7F3N2O]. Anal.
(C15H10F3N3O) C, H, N.
Meth yl 2-(3-Tr iflu or om eth ylph en yl)-1H-ben zim idazole-
4-ca r boxyla te Aceta te Sa lt (36). Method E. The product was
obtained as a white solid, 96%: mp 105-107 °C; IR 3339, 1708,
1328 cm-1; 1H NMR δ 2.01 (3 H, s), 4.09 (3 H, s), 7.44-7.51 (1
H, t), 7.79-8.13 (4 H, m), 8.71-8.75 (1 H, d), 8.82 (1 H, s),
11.8-12.2 (1 H, br s), 12.8-13.0 (1 H, br s); HRMS (EI) m/z
320 [M+ - AcOH], 288.0529 [-CH3OH, calcd 288.0510 for
2-(3-Met h oxyp h en yl)-1H -b en zim id a zole-4-ca r b oxa m -
id e (44). Method F. Recrystallization from MeOH yielded the
product, 46%: mp 223-225 °C; IR 3409, 3169, 1662 cm-1; 1H
NMR δ 3.99 (3 H, s), 7.22-7.27 (1 H, d), 7.43-7.51 (1 H, t),
7.58-7.66 (1 H, t). 7.85-8.01 (5 H, m), 9.4-9.5 (1 H, br s),
13.5 (1 H, br s); HRMS (EI) m/z 267.1005 [M+ calcd 267.1008
for C15H13N3O2]. Anal. (C15H13N3O2) C, H, N.
C
15H7F3N2O]. Anal. (C18H15F3N2O4) C, H, N.
Meth yl 2-(3-Meth oxyp h en yl)-1H-ben zim id a zole-4-ca r -
boxyla te Aceta te Sa lt (37). Method E. Recrystallization from
petroleum ether-EtOAc yielded the product, 58%: mp 93-
94 °C; IR 3453, 3375, 1707 cm-1; 1H NMR δ 1.99 (3 H, s), 3.96
(3 H, s), 4.06 (3 H, s), 7.15-7.21 (1 H, d), 7.38-7.46 (1 H, t),
7.51-7.59 (1 H, t), 7.91-8.00 (3 H, m), 8.04-8.08 (1 H, d),
12.0 (1 H, br s), 12.5 (1 H, br s); HRMS (EI) m/z 282.1003 [M+
- AcOH, calcd 282.1004 for C16H14N2O3]. Anal. (C18H18N2O5)
C, H, N.
2-(4-H yd r oxyp h en yl)-1H -b en zim id a zole-4-ca r b oxa m -
id e (45). Boron tribromide (1 M/DCM) (3.8 mL, 3.8 mmol) was
transferred to a flask containing 2-(4-methoxyphenyl)-1H-
benzimidazole-4-carboxamide (38) (202.4 mg, 0.76 mmol). The
mixture was refluxed for 24 h and distilled to dryness. The
solid residue was treated with 10% NaOH (10 mL), followed
by the dropwise addition of concentrated hydrochloric acid to
neutralization. The white precipitate was collected and dis-
solved in EtOAc (10 mL). The resulting solution was washed
with water (2 × 3 mL) and dried (MgSO4). Removal of the
solvent gave the product, 110 mg, 57%: mp 266-267 °C; IR
Meth od F . Am id e P r ep a r a tion Un d er High P r essu r e.
A benzimidazole methyl ester (38-44, 0.1-1.06 mmol) was
dissolved in an excess of liquid ammonia (20-40 mL), placed
in a sealed reaction vessel (a glass-lined stainless steel bomb),
and heated to 80 °C for 24 h generating an internal pressure
of ca. 40 atm. The ammonia was removed and the solid residue
washed with ice-cold water and purified appropriately.
2-(4-Met h oxyp h en yl)-1H -b en zim id a zole-4-ca r b oxa m -
id e (38). Method F. Recrystallization from MeOH-water
yielded the product, 80%: mp 261-263 °C; IR 3321, 3141, 1656
1
3424, 3309, 3156, 1642 cm-1; H NMR δ 7.03-7.07 (2 H, d, J
) 8.5), 7.34-7.42 (1 H, t), 7.75-7.79 (1 H, d), 7.85 (1 H, br s),
7.90-7.94 (1 H, d), 8.15-8.19 (2 H, d, J ) 8.5), 9.4-9.6 (1 H,
br s), 10.0-10.4 (1 H, br s), 13.0-13.4 (1 H, br s); HRMS (EI)
m/z 253.0859 [M+ calcd 253.0851 for C14H11N3O2]. Anal.
(C14H11N3O2) C, H, N.
cm-1
;
1H NMR δ 3.96 (3 H, s), 7.23-7.27 (2 H, d, J ) 8.6),
2-(4-Ca r b oxyp h en yl)-1H -b en zim id a zole-4-ca r b oxa m -
id e (46). A solution of 41 (150 mg, 0.57 mmol) in aqueous
sodium hydroxide (20%, 2 mL) and ethanol (5 mL) was boiled
under reflux overnight. The solvent was removed, to yield a
brown oil to which water (2 mL) was added followed by
concentrated HCl to pH 7. The precipitated white solid was
collected, washed with water and recrystallized from MeOH
to yield the product (75 mg, 47%): mp > 300 °C; IR 3342, 3152,
7.37-7.45 (1 H, t), 7.78-7.82 (1 H, d), 7.87 (1 H, br s), 7.93-
7.96 (1 H, d), 8.27-8.31 (2 H, d, J ) 8.6), 9.4-9.5 (1 H, br s),
13.3-13.4 (1 H, br s); 13C NMR δ 60.6, 119.8, 119.9, 126.8,
127.2, 127.4, 128.0, 133.8, 140.6, 146.9, 157.3, 166.4, 171.6;
HRMS (EI) m/z 267.1016 [M+ calcd 267.1008 for C15H13N3O2].
Anal. (C15H13N3O2) C, H, N.
2-(4-Tr iflu or om eth yl)-1H-ben zim id a zole-4-ca r boxa m -
id e (39). Method F. Recrystallization from MeOH-water
yielded the product, 48%: mp 301-305 °C; IR 3155, 1668, 1318
cm-1; 1H NMR δ 7.45 (1 H, t), 7.88-7,92 (1 H, d), 7.99 (1 H, br
s), 8.03 (1 H, d), 8.06-8.10 (2 H, d, J ) 8.1), 8.55-8.59 (2 H,
d, J ) 8.1), 9.3-9.4 (1 H, br s), 13.7-13.8 (1 H, br s); HRMS
(EI) m/z 288.0535 [M+ - NH3, calcd 288.0510 for C15H7F3N2O].
Anal. (C15H10F3N3O‚0.1H2O) C, H, N.
1
1711, 1649 cm-1; H NMR δ 7.45-7.52 (1 H, t), 7.87-7.91 (1
H, d), 7.95 (1 H, s), 7.98-8.02 (1 H, d), 8.22-8.26 (2 H, d, J )
8.2), 8.46-8.50 (2 H, d, J ) 8.2), 9.35 (1 H, br s), 13.39 (1 H,
br s); HRMS (EI) m/z 281.0793 [M+ calcd 281.0800 for
C
15H11N3O3].
Meth od G. Ben zim id a zole 1-N-Alk yla tion /Acyla tion .26
The benzimidazole amide was suspended in acetone (100 mg/4

Resistance-Modifying Agents. 9
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4093
mL), powdered potasssium hydroxide (1 equiv) was added and
the reaction stirred for 1 h when all solids had dissolved. The
alkylating or acylating agent (1 equiv) was added and the
reaction mixture stirred for a further 4 h. The solvent was
removed to give a solid residue that was washed with water
and purified appropriately.
MeOH) and recrystallization from MeOH yielded the product,
1
50%: mp 278-279 °C; IR 3419, 2838, 1677 cm-1; H NMR δ
4.19 (3 H, s), 7.30-7.33 (1 H, t), 7.42-7.50 (2 H, m), 7.63-
7.66 (1 H, m), 7.91-7.95 (1 H, d), 8.05-8.09 (1 H, d), 8.48-
8.52 (1 H, d), 11.9 (1 H, br s), 13.6 (1 H, br s); HRMS (EI) m/z
268.0847 [M+ calcd 268.0848 for C15H12N2O3]. Anal. (C15H12N2O3)
C, H, N.
2-(4-Meth oxyp h en yl)-1-N-m eth ylben zim id a zole-4-ca r -
boxa m id e (47). Method G. Benzimidazole 38 (105 mg, 0.4
mmol) and KOH (22 mg, 0.4 mmol) were treated with methyl
iodide (25 µL, 0.40 mmol). Column chromatography (DCM-
5% MeOH) gave fine white crystals (33 mg, 30%): mp 289-
2-(3-Meth oxy-4′-h yd r oxyp h en yl)-1H-ben zim id a zole-4-
ca r boxylic Acid (53). Method H. Product obtained in 44%
yield: mp 299-302 °C; IR 3519, 3075, 2847, 1600 cm-1 1H
;
NMR δ 4.00 (3 H, s), 6.99-7.03 (1 H, d), 7.34-7.42 (1 H, t),
7.85-7.97 (5 H, m,), 9.8 (1 H, br s); HRMS (EI) m/z 284.0800
[M+ calcd 284.0797 for C15H12N2O4]. Anal. (C15H12N2O4) C, H, N.
2-(3,4-Dim eth oxyp h en yl)-1H-ben zim id a zole-4-ca r box-
ylic Acid (54). Method H. Product obtained in 46% yield: IR
292 °C; IR 3309, 3141, 1671 cm-1 1H NMR δ 3.95 (3 H, s),
;
4.02 (3 H, s), 7.22-7.27 (2 H, d), 7.44-7.52 (1 H, t), 7.86-8.00
(5 H, m), 9.4 (1 H, br s); 13C NMR δ 37.3, 60.7, 119.5, 119.5,
126.6, 127.1, 127.5, 128.3, 136.3, 142.2, 145.4, 158.9, 166.0,
171.3; HRMS (EI) m/z 281.1158 [M+ calcd 281.1164 for
1
3079, 2839, 1604 cm-1; H NMR δ 3.95-4.01 (6H, d), 7.21-
C
16H15N3O2]. Anal. (C16H15N3O2) C, H, N.
7.25 (1 H, d), 7.41 (1 H, t), 7.88-8.02 (4 H, m); HRMS (EI)
m/z 298.0946 [M+ calcd 298.0954 for C16H14N2O4]. Anal.
(C16H14N2O4‚HCl) C, H, N.
2-(4-Meth oxyp h en yl)-1-N-ben zoylben zim id a zole-4-ca r -
boxa m id e (48). Method G. Benzimidazole 38 (366 mg, 1.37
mmol) and KOH (77 mg, 1.37 mmol) were treated with benzoyl
chloride (175 µL, 1.51 mmol). Column chromatography (DCM-
2% MeOH) and recrystallization (acetone-petroleum ether)
yielded white prisms (163 mg, 32%): mp 207-210 °C; IR 3446,
2-(3,4,5-Tr im et h oxyp h en yl)-1H -b en zim id a zole-4-ca r -
boxylic Acid (55). Method H. Product obtained in 40% yield:
1
mp 293-296 °C; IR 3260, 2937, 2833, 1593 cm-1; H NMR δ
3.84 (3 H, s), 4.02 (6 H, s), 7.50 (1 H, t), 7.84 (2 H, s), 7.95-
7.99 (1 H, d), 8.04-8.08 (1 H, d); HRMS (EI) m/z 328.1064
[M+ calcd 328.1059 for C17H16N2O5]. Anal. (C17H16N2O5‚
0.1MeOH) C, H, N.
1
1690, 1666 cm-1; H NMR δ 3.86 (3 H, s), 7.02-7.06 (2 H, d),
7.50-7.65 (4 H, m), 7.72-7.82 (3 H, m), 7.88-7.92 (2 H, d),
8.08 (1 H, s), 8.10-8.14 (1 H, d), 9.1-9.2 (1 H, br s); HRMS
(EI) m/z 371.1279 [M+ calcd 371.1270 for C22H17N3O3]. Anal.
(C22H17N3O3‚0.1H2O) C, H, N.
2-(4-N,N-Dim eth yla m in op h en yl)-1H-ben zim id a zole-4-
ca r boxylic Acid (56). Method H. Product obtained in 48%
yield: mp > 360 °C; IR 3436, 3258, 3179, 2927, 2843, 1646,
2-(4-Meth oxyp h en yl)-1-N-ben zyloxyca r bon ylben zim i-
d a zole-4-ca r b oxa m id e (49). Method G. Benzimidazole 38
(437 mg, 1.64 mmol) and KOH (92 mg, 1.64 mmol) were
treated with benzyl chloroformate (234 µL, 1.64 mmol).
Column chromatography (DCM-4% MeOH) and recrystalli-
zation (MeOH) afforded a white solid (398 mg, 61%): mp 202-
204 °C; IR 3379, 3164, 1746, 1687 cm-1; 1H NMR δ 3.92 (3 H,
s), 5.51 (2 H, s), 7.05-7.10 (2 H, d, J ) 8.8), 7.37-7.48 (5 H,
m), 7.57-7.65 (1 H, t), 7.68-7.86 (2 H, d, J ) 8.8), 8.01 (1 H,
br s), 8.08-8.13 (1 H, d), 8.23-8.27 (1 H, d), 9.02 (1 H, br s);
HRMS (EI) m/z 401.1394 [M+ calcd 401.1376 for C23H19N3O4].
Anal. (C23H19N3O4) C, H, N.
1603 cm-1 1H NMR δ 3.12 (6 H, s), 6.91-6.96 (2 H, d, J )
;
8.9), 7.37-7.41 (1 H, t), 7.82-7.86 (1 H, d), 7.88-7.92 (1 H,
d), 8.21-8.25 (2 H, d, J ) 8.8); HRMS (EI) m/z 281.1154 [M+
calcd 281.1164 for C16H15N3O2]. Anal. (C16H15N3O2‚0.65MeOH)
C, H, N.
2-(4-Eth oxyca r bon ylp h en yl)-1H-ben zim id a zole-4-ca r -
boxylic Acid (57). Method H. Product obtained in 31% yield:
mp 278 °C; IR 3297, 1721 cm-1; 1H NMR δ 1.41-1.48 (3 H, t),
4.40-4.50 (2 H, q), 5.49-7.58 (2 H, m), 7.93-7.96 (1 H, d),
8.02-8.06 (1 H, d), 8.17-8.24 (2 H, d, J ) 8.2), 8.55-8.59
(2 H, d, J ) 8.2); HRMS (EI) m/z 310.0955 [M+ calcd 310.0954
for C17H14N2O4].
Meth od H. Ben zim id a zole P r ep a r a tion fr om 2-Am in o-
3-n itr oben zoic Acid (10).24 A solution of 2-amino-3-nitroben-
zoic acid (10; 4.1-5.5 mmol) and sodium hydroxide (1.1 equiv)
in water was hydrogenated (see introduction to the Experi-
mental Section). Following acidification to pH 3-4 (glacial
acetic acid), a solution of the appropriate aldehyde (1.4 equiv)
in methanol and copper(II) acetate (1.4 equiv) in water was
added. The mixture was stirred vigorously, heated briefly to
boiling and filtered hot. The precipitate was washed with water
and dissolved in ethanol containing concentrated HCl (1 mL
HCl, 20 mL EtOH). A solution of sodium sulfide (1.4 equiv) in
water was added. The resulting solution was warmed and
filtered through Celite. After adjusting the pH of the filtrate
to 5-6 (concentrated HCl) it was diluted with water. The
ethanol was removed and the pH of the remaining aqueous
solution adjusted to 4-5 (concentrated HCl). The crude product
was collected by filtration, washed with water, and dissolved
in 0.5 M sodium hydroxide. The aqueous layer was washed
with EtOAc and the pH adjusted to 4-5 (concentrated HCl).
The carboxylic acid was recovered by filtration and purified
further as necessary.
2-(3,4-Meth ylen edioxyph en yl)-1H-ben zim idazole-4-car -
boxylic Acid (58). Method H. Column chromatography
(DCM-10% MeOH) yielded the product, 33%: mp 304-306
°C; IR 3270, 2786, 1503, 1480, 1249 cm-1 1H NMR δ 6.22
;
(2 H, s), 7.15-7.19 (1 H, d), 7.34-7.41 (1 H, t), 7.86-7.99
(4 H, m); HRMS (EI) m/z 282.0628 [M+ calcd 282.0641 for
C
15H10N2O4]. Anal. (C15H10N2O4‚0.5H2O) C, H, N.
2-(4-Ch lor op h e n yl)-1H -b e n zim id a zole -4-ca r b oxa m -
id e (59). Method C. Recrystallization from MeOH yielded the
product, 47%: mp 262-264 °C; IR 3389, 3173, 1657, 1596, 758
1
cm-1; H NMR δ 7.43-7.50 (1 H, t), 7.76-7.80 (2 H, d, J )
8.4), 7.84-7.88 (1 H, d), 7.93 (1 H, br s), 7.96-8.00 (1 H, d),
8.35-8.40 (2 H, d, J ) 8.5), 9.6 (1 H, br s), 13.6 (1 H, br s);
HRMS (EI) m/z 271.0648 [M+ calcd 271.0512 for C14H10ClN3O].
Anal. (C14H10ClN3O‚0.1MeOH) C, H, N.
2-(3-H yd r oxyp h en yl)-1H -b en zim id a zole-4-ca r b oxa m -
id e (60). Method C. Column chromatography (DCM-10%
MeOH) and recrystallization from MeOH yielded the product,
92%: mp 294-296 °C; IR 3411, 3332, 3190, 1661, 1600 cm-1
;
1H NMR δ 7.02-7.07 (1 H, dd), 7.40-7.53 (2 H, m), 7.73-
7.84 (3 H, m), 7.95-7.99 (2 H, d), 9.50 (1 H, s), 9.95 (1 H, br
s), 13.45 (1 H, br s); HRMS (EI) m/z 253.0863 [M+ calcd
253.0851 for C14H11N3O2]. Anal. (C14H11N3O2) C, H, N.
2-(2-Met h oxyp h en yl)-1H -b en zim id a zole-4-ca r b oxa m -
id e (61). Method C. Recrystallization from MeOH-water
yielded the product, 75%: mp 224-226 °C; IR 3421, 3321,
2-(4-Ch lor oph en yl)-1H-ben zim idazole-4-car boxylic Acid
(50). Method H. Product obtained in 80% yield: mp 306-308
1
°C;24 IR 3395, 3092, 1715, 755 cm-1; H NMR δ 7.53-7.60 (1
H, t), 7.77-7.81 (2 H, d), 8.01-8.05 (1 H, d), 8.09-8.13 (1 H,
d), 8.44-8.48 (2 H, d); HRMS (EI) m/z 272.0364 [M+ calcd
272.0353 for C14H9ClN2O2]. Anal. (C14H9ClN2O2) C, H, N.
2-(3-H yd r oxyp h en yl)-1H -b en zim id a zole-4-ca r b oxylic
Acid (51). Method H. Product obtained in 67% yield: mp 329-
330 °C; IR 3369, 1632 1596, 1491 cm-1; 1H NMR δ 7.01-7.04
(1 H, d), 7.42-7.44 (2 H, m), 7.81-8.01 (4 H, m); HRMS (EI)
m/z 236.0583 [M+ - H2O, calcd 236.0586 for C14H8N2O2].
2-(2-Met h oxyp h en yl)-1H -b en zim id a zole-4-ca r b oxylic
Acid (52). Method H. Column chromatography (DCM-10%
1
3154, 2849, 1673 cm-1; H NMR δ 4.15 (3 H, s), 7.23-7.31 (1
H, t), 7.36-7.47 (2 H, m), 7.61-7.69 (1 H, t), 7.89-7.97 (3 H,
m), 8.47-8.51 (1 H, d), 9.5 (1 H, br s), 12.6 (1 H, br s); HRMS
(EI) m/z 267.1003 [M+ calcd 267.1008 for C15H13N3O2]. Anal.
(C15H13N3O2) C, H, N.
2-(3-Meth oxy-4′-h yd r oxyp h en yl)-1H-ben zim id a zole-4-
ca r boxa m id e (62). Method C. Recrystallization from MeOH

4094 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
yielded the product, 46%: mp 244-245 °C; IR 3379, 3195 3001,
White et al.
added dropwise and the reaction mixture was stirred over-
night. Water (1 mL/5 mL reaction mixture) was added and
the product was extracted with diethyl ether (3 × 15 mL). The
combined organic extracts were dried (MgSO4) and the solvents
were removed to give the product which was purified as
appropriate.
1
2838, 1659, 1598 cm-1; H NMR δ 4.01 (3 H, s), 7.04-7.08 (1
H, d), 7.35-7.39 (1 H, t), 7.76-7.81 (4 H, m), 7.91-7.95 (1 H,
d), 9.5 (1 H, br s), 9.8 (1 H, br s), 13.2 (1 H, br s); HRMS (EI)
m/z 283.0961 [M+ calcd 283.0957 for C15H13N3O3]. Anal.
(C15H13N3O3‚0.1MeOH) C, H, N.
Meth od K. Ma n ga n ese Dioxid e Oxid a tion . Monosilyl-
protected benzene-dimethanol (5-17 mmol) was dissolved in
tetrahydrofuran (1 g/20 mL), activated manganese dioxide (5
equiv) was added, and the reaction mixture stirred overnight.
Filtration through Celite and removal of solvents yielded the
product that was purified as appropriate.
2-(3,4-Dim eth oxyp h en yl)-1H-ben zim id a zole-4-ca r box-
a m id e (63). Method C. Recrystallization from MeOH yielded
the product, 31%: mp 267-269 °C; IR 3422, 2834, 2777, 1648,
1603 cm-1; 1H NMR δ 3.96-4.03 (6 H, d), 7.26-7.30 (1 H, d),
7.39-7.46 (1 H, t), 7.80-7.98 (5 H, m), 9.5 (1 H, br s), 13.3 (1
H, br s); HRMS (EI) m/z 297.1110 [M+ calcd 297.1113 for
C
16H15N3O3]. Anal. (C16H15N3O3‚0.5HCl) C, H, N.
2-(3,4,5-Tr im et h oxyp h en yl)-1H -b en zim id a zole-4-ca r -
(4-Tr iisop r op ylsilyloxym eth ylp h en yl)m eth a n ol (69).
Method J . Column chromatography (petroleum ether-20%
EtOAc) gave a clear oil, 64%: 1H NMR (CDCl3) δ 1.06-1.09
(18 H, d), 1.09-1.14 (3 H, m), 1.77-1.83 (1 H, t), 4.63-4.66
(2 H, d), 4.82 (2 H, s), 7.32 (4 H, s).
boxa m id e (64). Method C. Recrystallization from MeOH-
water yielded the product, 30%: mp 275-276 °C; IR 3436,
3258, 3179, 2927, 2843, 1646 cm-1; H NMR δ 3.84 (3 H, s),
1
4.02 (6 H, s), 7.40-7.86 (1 H, t), 7.82 7.86 (2 H, s), 7.82-7.86
(2H, d), 7.95-7.91 (1 H, d), 9.6 (1 H, br s), 13.4 (1 H, br s);
HRMS (EI) m/z 327.1212 [M+ calcd 327.1219 for C17H17N3O4].
Anal. (C17H17N3O4) C, H, N.
4-(Tr iisopr opylsilyloxym eth yl)ben zaldeh yde (70). Meth-
od K. Alcohol 69 yielded an oil, purified by column chroma-
tography (petroleum ether-5% EtOAc), 78%: IR 2944, 2891,
2866, 2728, 1705, 1609, 1579 cm-1 1H NMR (CDCl3) δ
;
1.04-1.22 (21 H, m), 4.90 (2 H, s), 7.48-7.52 (2 H, d), 7.82-
7.86 (2 H, d), 10.0 (1 H, s); 13C NMR δ 12.0, 18.0, 64.7, 126.0,
129.8, 135.3, 148.9, 192.1; HRMS (EI) m/z 292.1873 [M+ calcd
292.1859 for C17H28O2Si].
(3-Tr iisop r op ylsilyloxym et h ylp h en yl)m et h a n ol (71).
Method J . Column chromatography (petroleum ether-20%
EtOAc) yielded a clear oil, 38%: 1H NMR (CDCl3) δ 1.07-
1.15 (21 H, m), 1.75 (1 H, t), 4.66-4.68 (2 H, d), 4.83 (2 H, s),
7.21-7.33 (4 H, m).
3-(Tr iisopr opylsilyloxym eth yl)benzaldehyde (72).Method
K. Alcohol 71 afforded a clear oil purified by column chroma-
tography (petroleum ether-5% EtOAc), 81%: IR 2944, 2866,
1707 cm-1; 1H NMR (CDCl3) δ 1.04-1.22 (21 H, m), 4.89 (2 H,
s), 7.45-7.53 (1 H, t), 7.62-7.66 (1 H, d), 7.73-7.77 (1 H, d),
7.85 (1 H, s), 10.0 (1 H, s); 13C NMR δ 12.0, 18.1, 64.4, 127.0,
128.2, 128.9, 131.8, 136.5, 142.9, 192.5.
(2-Tr iisop r op ylsilyloxym et h ylp h en yl)m et h a n ol (73).
Method J . Pure oil obtained directly, 85%: 1H NMR (CDCl3)
δ 1.03-1.22 (21 H, m), 4.67 (2 H, s), 4.86 (2 H, s), 7.24-7.38
(4 H, m).
2-(Tr iisopr opylsilyloxym eth yl)benzaldehyde (74).Method
K. Alcohol 73 afforded a clear oil, purified by column chroma-
tography (petroleum ether-5% EtOAc), 61%: IR 2943, 2866,
2729, 1696 cm-1; 1H NMR (CDCl3) δ 0.99-1.15 (21 H, m), 5.17
(2 H, s), 7.34-7.41 (1 H, t), 7.52-7.60 (1 H, t), 7.72-7.77
(1 H, d), 7.78-7.82 (1 H, d), 10.1 (1 H, s); HRMS (EI) m/z
249.1321 [M+ - isopropyl, calcd 249.1312 for C14H21O2Si].
2-(4-Tr iisop r op ylsilyloxym et h ylp h en yl)-1H -b en zim i-
d a zole-4-ca r b oxa m id e (75). Method I. Reaction with
aldehyde 70. Column chromatography (DCM-8% MeOH)
yielded a yellow solid, 85%: mp 171-176 °C; 1H NMR δ 1.15-
1.31 (21 H, m), 5.00 (2 H, s), 7.40-7.48 (1 H, t), 7.63-7.67 (2
H, d, J ) 8.2), 7.81-7.85 (1 H, d), 7.91 (1 H, br s), 7.96-7.99
(1 H, d), 8.30-8.34 (2 H, d, J ) 8.2), 9.5 (1 H, br s), 13.5 (1 H,
br s); HRMS (EI) m/z 423.2349 [M+ calcd 423.2342 for
2-(4-N,N-Dim eth yla m in op h en yl)-1H-ben zim id a zole-4-
ca r b oxa m id e (65). Method C. Column chromatography
(DCM-10% MeOH) and recrystallization from MeOH yielded
the product, 36%: mp 281-283 °C; IR 3302, 3167, 2817, 1660,
1610 cm-1; 1H NMR δ 3.12 (6 H, s), 6.94-6.98 (2 H, d), 7.31-
7.39 (1 H, t), 7.72-7.76 (1 H, d), 7.82-7.83 (1 H, br s), 7.88-
7.92 (1 H, d), 8.13-8.17 (2H, d); HRMS (EI) m/z 280.1326 [M+
calcd 280.1324 for C16H16N4O]. Anal. (C16H16N4O) C, H, N.
2-(4-Eth oxyca r bon ylp h en yl)-1H-ben zim id a zole-4-ca r -
boxa m id e (66). Method C. Column chromatography (DCM-
10% MeOH) and recrystallization from EtOH yielded the
product, 66%: mp 110-112 °C; IR 3380, 1691, 1654 cm-1; 1H
NMR δ 1.42-1.49 (3 H, t), 4.41-4.51 (2 H, q), 7.45-7.53 (1 H,
t), 7.86-8.02 (3 H, m), 8.23-8.27 (2 H, d, J ) 8.4), 8.47-8.51
(2 H, d, J ) 8.3), 9.40 (1 H, br s), 13.74 (1 H br s); HRMS (EI)
m/z 309.1114 [M+ calcd 309.1113 for C17H15N3O3]. Anal.
(C17H15N3O3‚0.1EtOH) C, H, N.
2-(3,4-Meth ylen edioxyph en yl)-1H-ben zim idazole-4-car -
boxa m id e (67). Method C. Column chromatography (DCM-
20% MeCN) yielded the product, 46%: mp 286-288 °C; IR
3345, 3144, 2788, 1650, 1602, 1245 cm-1
;
1H NMR δ 6.25
(2 H, s), 7.21-7.26 (1 H, d), 7.37-7.45 (1 H, t), 7.78-7.97
(5 H, m); HRMS (EI) m/z 281.0803 [M+ calcd 281.0800
for C15H11N3O3]. Anal. (C15H11N3O3) H, N; C: found 64.58,
expected 64.05.
Meth od I. F r om 2-Am in o-3-n itr oben za m id e (68).23,24
2-Amino-3-nitrobenzamide (1.35-5.56 mmol) in methanol
(20 mL/mmol) was hydrogenated (see introduction to the
Experimental Section). Following filtration through Celite, the
alcoholic filtrate was acidified with glacial acetic acid (5 equiv).
The resulting solution was stirred during the addition of the
appropriate aldehyde (1.05 equiv) dissolved in methanol
(10-20 mL) followed by copper acetate (1.2 equiv) dissolved
in water (10-20 mL). The colored suspension was briefly
heated to boiling, allowed to cool to room temperature and
stirred vigorously for 1 h. Hydrogen sulfide gas was bubbled
through the cooled (0 °C) solution for approximately 10 min,
and the resulting brown precipitate removed by filtration
through Celite. The filtrate was evaporated and purification
was performed as appropriate.
C
24H33N3O2Si].
2-(3-Tr iisop r op ylsilyloxym et h ylp h en yl)-1H -b en zim i-
d a zole-4-ca r boxa m id e (76). Method I. Reaction with alde-
hyde 72. Column chromatography (DCM-8% MeOH) yielded
an orange solid, 48%: 1H NMR δ 1.18-1.49 (21 H, m), 5.04 (2
H, s), 7.43-7.47 (1 H, t), 7.60-7.73 (2 H, m), 7.84-7.88 (1 H,
d), 7.98-8.02 (2 H, d), 8.18-8.22 (1 H, d), 8.43 (1 H, s), 9.45
(1 H, br s), 13.5 (1 H, br s).
2-Am in o-3-n itr oben za m id e (68). Method C. 2-Amino-3-
nitrobenzoic acid (5 g, 0.027 mol) yielded an orange precipitate
that was recovered by filtration. Recrystallization from MeOH
(500 mL) yielded the product (3.79 g, 76%): mp 234-235 °C;
2-(2-Tr iisop r op ylsilyloxym et h ylp h en yl)-1H -b en zim i-
d a zole-4-ca r boxa m id e (77). Method I. Reaction with alde-
hyde 74. Column chromatography (DCM-8% MeOH) yielded
1
IR 3430, 3297, 3206, 1695, 1686 cm-1; H NMR δ 6.74-6.82
(1 H, t), 7.74 (1 H, br s), 8.04-8.08 (1 H, d), 8.27 (1 H, br s),
8.27-8.31 (1 H, d), 8.6 (2 H, br s); 13C NMR δ 114.0, 119.2,
129.8, 132.5, 136.8, 146.4, 170.3; HRMS (EI) m/z 181.0495
[M+ calcd 181.0487 for C7H7N3O3].
1
an oily solid, 43%: IR 3186, 2943, 2865, 1661, 1598 cm-1; H
NMR δ 0.88-1.09 (21 H, m), 5.25 (2 H, s), 7.25-7.33 (1 H, t),
7.39-7.46 (1 H, t), 7.50-7.57 (1 H, t), 7.64-7.68 (1 H, d), 7.79-
7.83 (4 H, m), 9.06 (1 H, br s), 13.2 (1H, br s); HRMS (EI) m/z
423.2348 [M+ calcd 423.2342 for C24H33N3O2Si].
Meth od L. Silyl Dep r otection . Silyl-protected benzimi-
dazole (1.6-3.05 mmol) was dissolved in tetrahydrofuran (1
Meth od J . Silylation P r ocedu r e. The appropriate benzene-
dimethanol isomer (7.46-45 mmol) and sodium hydride (1
equiv) were suspended in THF (1 g/10 mL) under nitrogen and
stirred for 1 h. Triisopropylsilyl (TIPS) chloride (1 equiv) was

Resistance-Modifying Agents. 9
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22 4095
g/20 mL), 1 M tetrabutylammonium fluoride (TBAF; 1.05
equiv) in THF was added, and the reaction mixture stirred
for 4 h at room temperature. Water (15 mL) was added and
the aqueous layer was extracted with EtOAc (3 × 15 mL). The
combined organic extracts were dried (Na2SO4) and purified
as appropriate.
3-O-Allyl-4-m et h oxyb en za ld eh yd e (85). To 3-hydroxy-
4-methoxybenzaldehyde (1.0 g, 6.6 mmol) and powdered
anhydrous potassium carbonate (1.0 g, 7.2 mmol) in aceto-
nitrile (30 mL) was added allyl bromide (0.63 mL, 7.2 mmol)
in acetonitrile (15 mL). The reaction was stirred for 16 h at
room temperature after which the solvent was removed. The
resulting yellow solid was suspended between water (20 mL)
and EtOAc (20 mL). The aqueous layer was washed with
EtOAc (2 × 20 mL). The combined organic extracts were dried
(MgSO4), and the solvent removed to yield an oil. This was
purified by column chromatography (petroleum ether-20%
EtOAc) to yield a clear oil, 1.12 g, 89%: IR 2935, 2841, 1685,
1595, 1585, 1510 cm-1; 1H NMR (CDCl3) δ 3.94 (3 H, s), 4.62-
4.66 (2 H, d), 5.27-5.33 (1 H, d, J ) 1.3, J ) 10.4), 5.37-5.46
(1 H, d, J ) 1.4, J ) 17.3), 6.05-6.16 (1 H, m), 6.94-6.98 (1
H, d), 7.38 (1 H, s), 7.41-7.46 (1 H, d), 9.8 (1 H, s); 13C NMR
δ (CDCl3) 56.1, 69.7, 110.7, 110.9, 118.5, 126.8, 130.0, 132.5,
148.5, 154.9, 190.8; HRMS (EI) m/z 192.0794 [M+ calcd
192.0786 for C11H12O3].
2-(3-Hyd r oxy-4-m eth oxyp h en yl)-1H-ben zim id a zole-4-
ca r boxa m id e (86). Methods H, C. 2-Amino-3-nitrobenzoic
acid (10) (1 g, 5.5 mmol) and isovanillin (1.2 g, 6 mmol) yielded
the product, 87%: mp 155-159 °C; IR 3379, 3169, 1648, 1600,
1490 cm-1; 1H NMR δ 3.96 (3 H, s), 7.20-7.24 (1 H, d), 7.36-
7.43 (1 H, t), 7.73-7.80 (3 H, m), 7.91-7.95 (2 H, d), 9.5 (2 H,
br s). 13.2 (1 H, br s); HRMS (EI) m/z 283.0949 [M+ calcd
283.0957 for C15H13N3O3]. Anal. (C15H13N3O3‚0.75H2O) C, H;
N: found 14.19, expected 14.73.
2-(4-Hyd r oxym eth ylp h en yl)-1H-ben zim id a zole-4-ca r -
boxa m id e (78). Method L. Column chromatography (DCM-
10% MeOH) yielded a white solid, 65%: mp 257-258 °C; IR
1
3332, 3295, 3170, 1658, 1600 cm-1; H NMR δ 4.69-4.72 (2
H, d), 5.47 (1 H, t), 7.40-7.48 (1 H, t), 7.61-7.65 (2 H, d, J )
8.1), 7.81-7.85 (1 H, d), 7.91 (1 H, s), 7.95-7.99 (1 H, d), 8.29-
8.33 (2 H, d, J ) 8.1), 9.5 (1 H, br s), 13.4 (1 H, br s); 13C NMR
δ 62.5, 114.9, 122.2, 122.3, 122.9, 126.7, 126.9, 127.5, 135.3,
141.5, 145.4, 152.0, 166.2; HRMS (EI) m/z 267.1006 [M+ calcd
267.1008 for C15H13N3O2]. Anal. (C15H13N3O2) C, H; N: found
14.42, expected 15.72.
2-(3-Hyd r oxym eth ylp h en yl)-1H-ben zim id a zole-4-ca r -
boxa m id e (79). Method L. Column chromatography (DCM-
10% MeOH) and recrystallization from EtOH yielded the
product, 47%: mp 249-250 °C; IR 3341, 3280, 3173, 1658,
1
1635, 1602 cm-1; H NMR δ 4.72-4.74 (2 H, d), 5.48-5.54 (1
H, t), 7.41-7.48 (1 H, t), 7.56-7.60 (1 H, d), 7.61-7.69 (1 H,
t), 7.81-7.85 (1 H, d), 7.92 (1 H, s), 7.96-7.99 (1 H, d), 8.19-
8.22 (1 H, d), 8.33 (1 H, s), 9.5 (1 H, br s), 13.55 (1 H, br s); 13
C
NMR δ 62.8, 115.2, 122.5, 123.1, 125.0, 125.4, 126.0, 127.5,
128.8, 129.0, 135.5, 141.6, 143.8, 152.2, 166.4; HRMS m/z (EI)
267.1012 [M+ calcd 267.1008 for C15H13N3O2]. Anal. (C15H13N3O2‚
0.2EtOH) C, H, N.
2-(2-Hyd r oxym eth ylp h en yl)-1H-ben zim id a zole-4-ca r -
boxa m id e (80). Method L. Column chromatography (DCM-
5% MeOH) and recrystallization from EtOH yielded the
product, 51%: mp 264-268 °C; IR 3402, 3179, 1650, 1604
cm-1; 1H NMR δ 5.05 (2 H, s), 5.65 (1 H, br s), 7.43-7.51 (1 H,
t), 7.56-7.71 (2 H. m), 7.87-8.01 (5 H, m), 9.4 (1 H, br s), 13.3
(1 H, br s); HRMS (EI) m/z 267.0998 [M+ calcd 267.1008 for
2-(3-O-Allyl-4-m et h oxyp h en yl)-1H -b en zim id a zole-4-
ca r boxa m id e (87). Method I. Reaction with aldehyde 85.
Column chromatography (DCM-10% MeOH) and recrystal-
lization from EtOH yielded the product, 40%: 1H NMR δ
3.79 (3 H, s), 4.79-4.82 (2 H, d), 5.40-5.45 (1 H, d, J ) 10.3),
5.54-5.63 (1 H, d, J ) 17.2), 6.14-6.33 (1 H, m), 7.27-7.31
(1 H, d), 7.38-7.46 (1 H, t), 7.79-7.83 (1 H, d), 7.87 (1 H, s),
7.94-7.97 (3 H, m), 9.45 (1 H, br s), 13.3 (1 H, br s); HRMS
(EI) m/z 323.1271 [M+ calcd 323.1270 for C18H17N3O3].
C
15H13N3O2]. Anal. (C15H13N3O2‚0.2EtOH) C, H, N.
2-(4-H yd r oxyp h en yl)-1H -b en zim id a zole-4-N-m et h yl-
ca r boxa m id e (88). Methyl 2-(4′-hydroxyphenyl)-1H-benzimi-
dazole-4-carboxylate (200 mg, 0.75 mmol) was dissolved in
ethanol containing 33% methylamine (10 mL) and stirred
overnight. Removal of solvents, followed by column chroma-
tography (DCM-7.5% MeOH) yielded an off-white solid, 187
mg, 94% (recrystallized from DCM-MeOH): mp 289-290 °C;
2-(2-Tr iflu or om eth ylp h en yl)-1H-ben zim id a zole-4-ca r -
boxa m id e (81). Method I. Column chromatography (DCM-
10% MeOH) and recrystallization from EtOH-petroleum ether
yielded the product (43%): mp 265-268 °C; IR 3309, 3163,
1670, 1658; 1H NMR δ 7.48-7.56 (1 H, t), 7.88-7.92 (3 H, m),
8.00-8.12 (4 H, m), 9.4 (1 H, br s), 13.6 (1 H, br s); 13C
NMR δ 115.5, 121.2, 123.0, 126.7, 127.2, 127.7, 128.3,
129.1, 130.9, 132.2, 132.8, 134.9, 141.3, 150.0, 166.2; HRMS
(EI) m/z 305.0764 [M+ calcd 305.0776 for C15H10F3N3O]. Anal.
(C15H10F3N3O‚0.2H2O) C, H, N.
1
IR 3108, 1614, 1479 cm-1; H NMR δ 3.11-3.13 (3 H, d, J )
4.65), 4.22 (1 H, br s), 7.04-7.08 (2 H, d, J ) 8.6), 7.35-7.43
(1 H, t), 7.75-7.79 (1 H, d), 7.93-7.97 (1 H, d), 8.21-8.26 (2
H, d, J ) 8.6), 9.95-9.97 (1 H, d, J ) 4.65), 10.22 (1 H, br s),
13.25 (1 H, br s); HRMS (EI) m/z 267.1001 [M+ calcd 267.1008
for C15H13N3O2]. Anal. (C15H13N3O2‚H2O) C, H, N.
Meth yl 2-(4-Meth oxyp h en yl)-1H-ben zim id a zole-4-ca r -
b oxyla t e (89). Method I. Methyl 2-amino-3-nitrobenzoate
(14) was used. Column chromatography (1:1 petroleum
ether-EtOAc) gave a brown solid, 62%: mp 142-144 °C; IR
3374, 1696 cm-1; 1H NMR δ 3.97 (3 H, s), 4.09 (3 H, s), 7.20-
7.24 (2 H, d, J ) 8.8), 7.38-7.46 (1 H, t), 7.90-7.94 (1 H, d),
8.02-8.06 (1 H, d), 8.37-8.41 (2 H, d, J ) 8.8), 12.38 (1 H, br
s); HRMS (EI) m/z 282.1018 [M+ calcd 282.1004 for C16H14N2O3].
2-(4-Met h oxyp h en yl)-1H -b en zim id a zole-4-N-m et h yl-
ca r boxa m id e (90). A solution of methyl 2-(4′-methoxyphenyl)-
1H-benzimidazole-4-carboxylate (89) (209 mg, 0.74 mmol) in
ethanol containing 33% methylamine (20 mL) was stirred
overnight. Removal of solvents and recrystallization from
EtOH yielded off-white needles, 144 mg, 69%: mp 252-256
°C; IR 3187, 1646 cm-1; 1H NMR δ 3.12 (3 H, d), 3.98 (3 H, s),
7.25-7.29 (2 H, d, J ) 8.4), 7.39-7.46 (1 H, t), 7.78-7.82 (1
H, d), 7.95-7.99 (1 H, d), 8.33-8.38 (2 H, d, J ) 8.4), 9.93 (1
H, br s), 13.3 (1 H, br s); 13C NMR δ 26.3, 55.7, 114.8, 121.8,
122.3, 122.7, 129.0, 135.5, 141.5, 152.4, 161.5, 165.7; HRMS
(EI) m/z 281.1152 [M+ calcd 281.1164 for C16H15N3O2]. Anal.
(C16H15N3O2‚0.6EtOH‚0.1H2O) C, H, N.
2-(2-Ch lor op h en yl)-1H-ben zim id a zole-4-ca r boxa m id e
(82). Method I. Column chromatography (1:1 petroleum ether-
EtOAc) and recrystallization from MeOH yielded the product,
24%: mp 234-235 °C; IR 3385, 3326, 3182, 1663 cm-1
;
1H NMR δ 7.47-7.54 (1 H, t), 7.67-7.92 (4 H, m), 7.99-8.12
(2 H, 2 d), 9.38 (1 H, br s), 13.39 (1 H, br s); HRMS (EI)
m/z 271.0521 [M+ calcd 271.0512 for C14H10ClN3O]. Anal.
(C14H10ClN3O) C, H, N.
2-(3-Ch lor op h en yl)-1H-ben zim id a zole-4-ca r boxa m id e
(83). Method I. Column chromatography (EtOAc) and re-
crystallization from MeOH yielded the product, 24%: mp
260-261 °C; IR 3349, 3175, 3156, 1657, 1635, 1602, 1578 cm-1
;
1H NMR δ 7.44-7.52 (1 H, t), 7.72-7.74 (2 H, d), 7.85-7.89
(1 H, d), 7.91 (1 H, s), 7.98-8.02 (1 H, d), 8.30-8.35 (1 H, t),
8.41 (1 H, s), 9.4 (1 H, br s), 13.6 (1 H, br s); HRMS (EI)
m/z 271.0507 [M+ calcd 271.0512 for C14H10ClN3O]. Anal.
(C14H10ClN3O‚0.33MeOH) C, H, N.
2-(2-F lu or op h en yl)-1H-ben zim id a zole-4-ca r boxa m id e
(84). Method I. Column chromatography (EtOAc-30% petro-
leum ether) and recrystallization from MeOH yielded the
product, 57%: mp 207-210 °C; IR 3436, 3354, 3189, 1664,
1603, 1585 cm-1; 1H NMR δ 7.36-7.54 (3 H, m), 7.64-7.69 (1
H, m), 7.80-7.85 (1 H, d), 7.87 (1 H, s), 7.91-7.97 (1 H, d),
8.34-8.38 (1 H, t), 9.35 (1 H, br s), 13.13 (1 H, br s); 13C NMR
δ 115.6, 116.6, 117.2, 122.7, 123.2, 125.3, 130.5, 132.7, 135.3,
140.7, 147.1, 158.6, 160.6, 166.1; HRMS (EI) m/z 255.0803
[M+ calcd 255.0808 for C14H10FN3O].
2-(4-H yd r oxyp h en yl)-1H -b en zim id a zole-4-ca r b oxylic
Acid (91). Method I. 2-Amino-3-nitrobenzoic acid (10) was
used. Column chromatography (DCM-MeOH 15%) gave an

4096 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 22
White et al.
orange solid that was recrystallized from EtOH, 15%: mp
5 replicate wells were used. A replicate plate was fixed as
described below to obtain an estimate of the cell density at
the start of the drug incubation. The plates were incubated
for 72 h at 37 °C before assaying (together with the preincu-
bation plate) for cell growth as previously described.40 The
optical density of the wells was read on a computer-interfaced
MR700 microtiter-plate reader (Dynex, Billingshurst, U.K.)
relative to an air blank using a 570-nm filter. Cell growth was
expressed as percent (%) of an appropriate control, i.e. 1%
DMSO or 10 µM NU1085 (45). The IC50 values (concentration
of drug required to reduce growth by 50%) were determined
by nonlinear regression curve-fitting using GraphPad PRISM
(San Diego, CA) software. The potentiation factor, PF50, was
calculated from IC50 of topotecan or temozolomide alone ÷ IC50
of topotecan or temozolomide + NU1085.
1
346-347 °C; IR 3429, 3217, 1615 cm-1; H NMR δ 4.47 (1 H,
br s) 6.99-7.03 (2 H, d, J ) 8.5), 7.34-7.42 (1 H, t), 7.84-
7.88 (1 H, d), 7.92-7.96 (1 H, d), 8.22-8.26 (2 H, d, J ) 8.5),
10.1-10.2 (1 H, br s); HRMS (EI) m/z 254.0687 [M+ calcd
254.0691 for C14H10N2O3].
Cr ysta llogr a p h y. Purified protein and cocrystals of the
C-terminal catalytic domain of chicken PARP were obtained
using procedures similar to those previously described.31 In
this study, 70 mM Tris, pH 8.5, 19% PEG-3400, and 8%
isopropyl alcohol were used as precipitant. X-ray diffraction
data were collected from this P212121 crystal form: a ) 59.4
Å, b ) 64.3 Å, c ) 97.0 Å, at SSRL beamline 7-1.32 Data
resolution is from 20.0-2.2 Å; 95006 observations were scaled
and merged into 19029 unique reflections using DENZO and
SCALEPACK.33 The overall R-merge is 6.1%, the ratio I/σ(I)
is 28.3, and the data is 97.9% complete. Corresponding values
for the high-resolution data shell (2.28-2.20 Å) are 22.2%, 9.8,
and 95.5%, respectively. The structure of 44 complexed to
PARP was solved and refined using this data, the program
X-PLOR,34 and the protein and solvent starting model 1PAX
from the Protein Data Bank.28 The conventional and free
R-factors after refinement are 19.4% and 27.4%. The rms
deviations between model and ideal bond distances, bond
angles, dihedral angles, and improper angles are 0.007 Å, 1.3°,
23.6°, and 1.1°. The model coordinates have been deposited in
the Protein Data Bank with the code 1EFY.
Ack n ow led gm en t . We thank Agouron Pharma-
ceuticals Inc., the Cancer Research Campaign, Cancer
Research Campaign Technology, and the Engineering
and Physical Sciences Research Council for grants
supporting this work. We gratefully acknowledge
Gilbert de Murcia for the gift of the baculovirus con-
structs used for the assays and the crystallography.
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Assa y of In h ibitor s w ith P ARP . The enzymatic assay was
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