Synthesis and evaluation of a new generation of orally efficacious benzimidazole-based poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors as anticancer agents

Article abstract of DOI:10.1021/jm900697r

Small molecule inhibitors of PARP-1 have been pursued by various organizations as potential therapeutic agents either capable of sensitizing cytotoxic treatments or acting as stand-alone agents to combat cancer. As one of the strategies to expand our portfolio of PARP-1 inhibitors, we pursued unsaturated heterocycles to replace the saturated cyclic amine derivatives appended to the benzimidazole core. Not only did a variety of these new generation compounds maintain high enzymatic potency, many of them also displayed robust cellular activity. For example, the enzymatic IC₅₀ and cellular EC₅₀ values were as low as 1 nM or below. Compounds 24 (EC₅₀ = 3.7 nM) and 44 (EC₅₀ = 7.8 nM), featuring an oxadiazole and a pyridine moiety, respectively, demonstrated balanced potency and PK profiles. In addition, these two molecules exhibited potent oral in vivo efficacy in potentiating the cytotoxic agent temozolomide in a B16F10 murine melanoma model.

Full text of DOI:10.1021/jm900697r

J. Med. Chem. 2009, 52, 6803–6813 6803
DOI: 10.1021/jm900697r
Synthesis and Evaluation of a New Generation of Orally Efficacious Benzimidazole-Based
Poly(ADP-ribose) Polymerase-1 (PARP-1) Inhibitors as Anticancer Agents
Yunsong Tong,* Jennifer J. Bouska, Paul A. Ellis, Eric F. Johnson, Joel Leverson, Xuesong Liu, Patrick A. Marcotte,
Amanda M. Olson, Donald J. Osterling, Magdalena Przytulinska, Luis E. Rodriguez, Yan Shi, Nirupama Soni, Jason Stavropoulos,
Sheela Thomas, Cherrie K. Donawho, David J. Frost, Yan Luo, Vincent L. Giranda, and Thomas D. Penning
Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064
Received May 22, 2009
Small molecule inhibitors of PARP-1 have been pursued by various organizations as potential
therapeutic agents either capable of sensitizing cytotoxic treatments or acting as stand-alone agents
to combat cancer. As one of the strategies to expand our portfolio of PARP-1 inhibitors, we pursued
unsaturated heterocycles to replace the saturated cyclic amine derivatives appended to the benzimida-
zole core. Not only did a variety of these new generation compounds maintain high enzymatic potency,
many of them also displayed robust cellular activity. For example, the enzymatic IC50 and cellular EC50
values were as low as 1 nM or below. Compounds 24 (EC50 = 3.7 nM) and 44 (EC50 = 7.8 nM),
featuring an oxadiazole and a pyridine moiety, respectively, demonstrated balanced potency and PK
profiles. In addition, these two molecules exhibited potent oral in vivo efficacy in potentiating the
cytotoxic agent temozolomide in a B16F10 murine melanoma model.
Introduction
repair caused bygenotoxic treatments.7 Morerecently, several
reports have suggested that PARP inhibition could have
cytotoxic activities against BRCA1/2 deficient cells in the
absence of any DNA damaging agent.8,9 Meanwhile, small
molecule inhibitors of PARP-1 have been pursued by various
organizations as potential therapeutic agents either capable of
sensitizing cytotoxic treatments or acting as stand-alone
agents.10-15 Some of them, including 4-(4-(4-(cyclopropanecar-
bonyl)piperazine-1-carbonyl)-3-fluorobenzyl)phthalazin-1(2H)-
one (AZD2281),11 8-fluoro-5-(4-((methylamino)methyl)phenyl)-
2,3,4,6-tetrahydro-1H-azepino[5,4,3-cd]indol-1-one (AG014699),16
and (2-[(R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carbox-
amide (4, ABT-888),17 have demonstrated efficacy in various
cancer xenograft models and are currently in human clinical
trials.
Poly(ADP-ribose) polymerase-1 (PARP-1), also known as
poly(ADP-ribose) synthetase or poly(ADP-ribose) transfer-
ase, is a nuclear enzyme that is part of a larger family of
PARPa enzymes. DNA damage activates PARP-1, causing it
to cleave its substrate nicotinamide adenine dinucleotide
(NADþ) and transfer ADP-ribose units to nuclear target
proteins that can facilitate DNA repair. This mechanism
allows cancer cells to escape apoptosis induced by DNA
damaging treatments such as chemotherapy and radiation
and enables cancer cells to repair the drug-induced DNA
lesions.1-3 The outcome represents one of the major compo-
nents of acquired drug resistance. Overexpression of PARP-1
in cancer compared to normal cells thus makes the former
more resistant to genotoxic stress.4-6 Since DNA damaging
agents (cytotoxic chemotherapy and radiation) remain a
primary regimen to treat most cancer patients, maximizing
their therapeutic benefit through inhibition of PARP-1 has
become an attractive strategy. Experiments involving PARP-
1 knockout have beenshown to be effectiveinimpairing DNA
More than a decade ago, scientists from the University of
Newcastle identified a class of benzoxazole-4-carboxamide
compounds as exemplified by 1 as weak PARP inhibitors.18
*To whom correspondence should be addressed. Phone: 1-847-935-
1252. Fax: 1-847-935-5165. E-mail: yunsong.tong@abbott.com.
a Abbreviations: PARP, poly(ADP-ribose) polymerase; TMZ, temo-
zolomide; PK, pharmacokinetic; TosMIC, toluenesulfonylmethyl iso-
cyanide; CDI, 1,10-carbonyldiimidazole; NBS, N-bromosuccinimide;
BPO, benzoyl peroxide; TFA, trifluoroacetic acid; DME, 1,2-dimethoxy-
ethane; DMF, dimethylformamide; TBTU, O-(benzotriazol-1-yl)-N,N,
N0,N0-tetramethyluronium tetrafluoroborate; HOBT, 1-hydroxybenzo-
triazole; TFFH, tetramethylfluoroformamidinium hexafluoropho-
sphate; TCDI, thiocarbonyldiimidazole; DBU, 1,8-diazabicyclo[5.4.0]-
undec-7-ene; b.i.d., twice daily; q.d., every day; TGI, tumor growth inhibi-
tion; SEM, standard error of measurement; PBS, phosphate buffered saline;
They proposed that the nitrogen on benzoxazole formed an
intramolecular hydrogen bond to the amide moiety, result-
ing in a desirable conformation for the latter to interact with
the ribose nucleoside-binding domain of PARP. Later, they
discovered structurally related benzimidazole-4-carboxa-
mides as more potent PARP inhibitors.19,20 The above-
mentioned interaction in the nicotinamide binding site was
confirmed by X-ray crystallography.20 These compounds, as
F, oral bioavailability; AUC, pharmacokinetic area under curve; Cmax,
pharmacokinetic maximum concentration; Vd, volume of distribution; CL,
pharmacokinetic clearance; brd, broad.
r
2009 American Chemical Society
Published on Web 10/21/2009
pubs.acs.org/jmc

6804 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tong et al.
represented by 2-(4-hydroxyphenyl)benzimidazole-4-carboxa-
mide (NU1085), showed weak to modest cellular activities.20,21
We have recently reported a series of benzimidazole-based
inhibitors for PARP-1 as anticancer agents.22,23 Our earlier
PARP-1 inhibitors (2) featured a piperidyl or pyrrolidinyl moiety,
Scheme 1a
a Reagents and conditions; (i) for 8, NaOMe, TosMIC, MeOH; (i) for
9, (a) TosMIC, NaCN, (b) NH3; (i) for 10, glyoxal, NH4OH.
Scheme 2a
with or without a simple alkyl group (R) on the nitrogen, off
the 2-position of a benzimidazole core. We have successfully
developed preclinical and clinical candidates, A-620223 (3)22
and 4.17,23,24 We also learned that R being a proton or an
alkyl group on 2 was generally required for good cellular
potency. In an attempt to diversify the benzimidazole-based
PARP-1 inhibitors, we opted to develop a newer generation
of this class of inhibitors by drastically changing the char-
acteristics of the non-benzimidazole portion of the mole-
cules. A more recent inhibitor (5) with a bis-phenyl moiety
exhibited encouraging enzymatic inhibition (Ki = 2 nM) but
suffered from relatively weak cellular activity (EC50 = 71
nM). In this paper, we disclose our development of a distinct
class of inhibitors (6) possessing unsaturated hetero-
cycles appended to the benzimidazole core. Not only did a
number of these inhibitors demonstrate high potency in
both enzymatic and cellular assays, some of them, such as
24 and 44, also exhibited good pharmacokinetic (PK) prop-
erties and potent oral in vivo efficacy in potentiating the
cytotoxic agent temozolomide (TMZ) in a mouse xenograft
model.
a Reagents and conditions: (i) 4-acetylbenzoic acid, CDI, pyridine,
DMF; (ii) AcOH; (iii) NBS, BPO, TFA; (iv) for 15, formamide, P2S5,
MgCO3; (iv) for 16, urea, AcOH; (iv) for 17, thiourea; (iv) for 18, 2-
aminopyridine; (iv) for 19, 4-(trifluoromethyl)pyridin-2-amine.
The isomeric imidazole analogue 10 was obtained by treating
7 with glyoxal and ammonium hydroxide.27
The synthesis of compounds 15-19 is outlined in Scheme 2.
Acylation of 2,3-diaminobenzamide (11)28 in the presence of
CDI and pyridine in DMF led to 12, which was in turn cyclized
under acidic (AcOH) conditions at 90 °C to furnish 13. This
ring closure strategy was shown in the early work by Griffin
et al.19 and later modified by White et al.20 and Barkalow
et al.28 Subsequent bromination using NBS and catalytic BPO
in TFA gave the versatile R-bromo ketone intermediate 14,
setting the stage for a number of different transformations. In
situ formation of thioformamide with P2S5 and formamide
followed by the addition of MgCO3 converted 14 to thiazole
15.29 The 2-methyloxazole derivative 16 was prepared when 14
was heated in urea and acetic acid. Reacting 14 with thiourea in
ethanol and water at 80 °C afforded 2-aminothiazole 17. The
R-bromo ketone moiety of 14 underwent cyclization in the
presence of 2-aminopyridine to give the imidazopyridine
analogue 18. A similar transformation involving 14 and
4-(trifluoromethyl)pyridin-2-amine provided 19.
Chemistry
Because of the vast variety of heterocycles in existence and
the diverse chemistry associated with their syntheses, the plan
for the structure-activity relationship studies discussed in this
paper was to strike a balance between the analogue diversity
and synthetic convergence and simplicity. As such, we gen-
erally chose to take advantage of the well-established chem-
istry previously reported by us22,28 and others19,21 to assemble
key intermediates with proper functional groups attached for
heterocycle formation.
As illustrated in Scheme 1, the known 2-(4-formylphenyl)-
1H-benzo[d]imidazole-4-carboxamide 721 was allowed to
react with TosMIC and NaOMe to give oxazole 8.25 Reaction
of 7 with TosMIC and NaCN followed by heating with NH3/
MeOH in a sealed tube at 110 °C afforded imidazole 9.26

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6805
Scheme 4a
Scheme 3a
a Reagents and conditions: (i) 4-cyanobenzoic acid, CDI, pyridine,
DMF; (ii) AcOH; (iii) NaN3, NH4Cl; (iv) HONH2 HCl, K2CO3; (v) for
3
29, CDI; (v) for 30, TCDI, silica gel; (v) for 31, TCDI, DBU.
Scheme 5a
a Reagents and conditions: (i) 4-(methoxycarbonyl)benzoic acid, CDI,
pyridine, DMF; (ii) AcOH; (iii) LiOH; (iv) acetamide oxime, TBTU,
HOBT, Et3N; (v) TFFH, NH2NH2, Et3N; (vi) HC(OMe)3; (vii) CDI.
The methods described in Scheme 3 were employed to
synthesize 22, 24, and 25. The benzoic acid intermediate 21
was produced after acylation of 11 followed by acid promoted
cyclization and ester saponification with LiOH. The synthesis
of 21 was reported by White et al.,20 although we chose a
different route to take advantage of an ample supply of the
intermediate 11 in our lab that allowed us to pursue our
targets moreefficiently. Coupling of the acid moiety of21 with
acetamide oxime using TBTU, HOBT, and triethylamine led
to cyclization to give the 3-methyl-1,2,4-oxadiazole 22.30 The
initial attempt to make 23 by reacting the methyl ester
precursor of 21 with hydrazine at an elevated temperature
led to very poor yield. Intermediate 21 was alternatively
treated with TFFH, hydrazine, and triethylamine to afford
the benzohydrazide 23. Subsequent condensation with methyl
orthoformate at 160 °C under microwave irradiation gave the
1,3,4-oxadiazole derivative 24. Treatment of 23 with CDI
yielded the 5-oxo-1,3,4-oxadiazole 25.
Preparation of compounds 29-31 is shown in Scheme 4.
Although benzonitrile derivative 26 has been reported ear-
lier,20 we carried out our synthesis using intermediate 11. The
cyano moietywas transformed into a tetrazoleby treatment of
26 with sodium azide and ammonium chloride in DMF at 80
°C to afford 27. Meanwhile, addition of hydroxylamine into
the cyano group produced the hydroxybenzamidine 28, a
convenient substrate labile to cyclization for analogue synth-
eses. Upon CDI treatment at 110 °C it was converted into the
1,2,4-oxadiazol-5-one 29.31 Treating 28 with 1,10-thiocarbo-
nyldiimidazole under either acidic (silica) or basic (DBU)
conditions yielded the 1,2,4-thiadiazol-5-one 30 and the
1,2,4-oxadiazole-5-thione 31, respectively.32
a Reagents and conditions: (i) hydroxylamine, MeOH, water; (ii)
LiOH; (iii) 11, CDI, pyridine, DMF; (iv) AcOH.
Treatment of 13 with DMF-dimethyl acetal failed to give
the desired enamino ketone derivative for convergent synth-
esesof 37and 39. Thus, the heterocyclic rings werebuilt before
the benzimidazole core was assembled. Compound 37 was
made according to Scheme 5. Following literature proce-
dures,33 enamino ketone 33 was obtained by the reaction of
methyl 4-acetylbenzoate (32) with DMF-dimethyl acetal.
Cyclization with hydroxylamine and subsequent saponifica-
tion with LiOH provided 35. The standard chemistry was
performed to give the isoxazole 37. As shown in Scheme 6,
methylpyrazolylbenzoic acid 38, derived from cyclization of
the enamino ketone 33 in the presence of methylhydrazine and
AcOH, was subjected to previously described conditions to
afford the methylpyrazole 39.
Scheme 7 provides the synthesis for compounds 41, 42, and
44. 4-Bromobenzaldehyde was treated with 11 in the presence
of Pd/C in refluxing methanol to yield the bromo derivative
40.34 An alternative method was to couple 11 with 4-bromo-
benzoic acid followed by an acidic ring closure. The

6806 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tong et al.
Scheme 6a
Table 1. Enzymatic and Cellular Activities
a Reagents and conditions: (i) methylhydrazine, AcOH; (ii) LiOH;
(iii) 11, CDI, pyridine, DMF; (iv) AcOH.
Scheme 7a
a Reagents and conditions: (i) 4-bromobenzaldehyde, Pd/C, MeOH;
(ii) for 41, 4-pyridineboronic acid, Pd (dppf)2Cl2, Na2CO3, dioxane;
for 42, 3-pyridineboronic acid, Pd (dppf)2Cl2, Na2CO3, dioxane; (iii) 11,
Pd/C, MeOH.
subsequent Suzuki reactions led to 41 and 42. Compound 44
was synthesized from 43 following similar procedures de-
scribed for 40.
Results and Discussion
The fact that our earlier PARP-1 inhibitors with a benzi-
midazole core, exemplified by 4, have demonstrated robust
efficacy in multiple tumor models17 prompted us to further
investigate and diversify the benzimidazole-based inhibitors,
one of the strategies we took to expand our portfolio of
PARP-1 inhibitors. According to X-ray cocrystal structures
we and others reported earlier,20,22,35,36 the benzimidazole
carboxamide scaffold is mainly responsible for enzyme in-
hibition through its network of hydrogen-bond and π-stack-
ing interactionswiththe nicotinamidebinding site of PARP-1,
leaving the area off the 2-position of the benzimidazole a
primary target for substantial modifications. Also, as disco-
vered in the prior studies, the basic piperidyl (e.g., 3) or
pyrrolidinyl (e.g., 4) moieties appeared necessary for good
cellular potencies. For example, when the alkyl group on the
nitrogen of the corresponding saturated rings was replaced by
an acyl or a sulfonyl group, the resulting inhibitors suffered
about 20- to 1000-fold decrease in cellular EC50 values.22 This
observation significantly limits the diversity of potential
a Values are the mean of two experiments. b Single measurement
except for 24 and 44.
analogues to be explored. Facing this limitation and also
knowing compound 5 to possess potent enzymatic inhibition,
we decided to make more profound changes to the molecules.
We thus turned our attention to investigating compounds
with unsaturated heterocycles extended off the 2-phenyl
group of the benzimidazole scaffold, replacing the previously
described piperidyl or pyrrolidinyl moieties.
The activity data for our next-generation inhibitors are
shown in Table 1. As expected, all the compounds synthesized
were potent against PARP-1, since the heterocycle portion of
the inhibitors was generally not involved in any key interac-
tions with the enzyme according to X-ray cocrystal structures
from early studies.22 The Ki values ranged from 0.76 nM (8) to

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6807
20 nM (27), in line with inhibitors (2) bearing piperidines or
pyrrolidines.22 The larger bicyclic moieties were also tolerated
as 18 and 19 exhibited Ki values of 2.9 and 6.8 nM, respec-
tively. More importantly, many of the these compounds also
possessed high cellular potency as measured by their ability to
inhibit H2O2-induced poly(ADP-ribosyl)ation in C-41 cervi-
cal carcinoma cells. Compounds 8, 9, 10, 15, 16, 18, 19, 22, 24,
39, 41, 42, and 44 all exhibited cellular EC50 values at20nM or
lower with the most active (8) being 0.99 nM. The size of the
heterocycles did not appear to be the main factor impacting
cellular activity. Some monocyclic derivatives possessed EC50
values below 4 nM (8, 9, 10, 15, and 24), while others were
above 300 nM (30, 31, and 37). Meantime, the values were 20
nM for the only two bicyclic inhibitors (18 and 19). It is
noteworthy that any substitution on the carbon of the ox-
adiazole on 24 was detrimental to the potencies (data not
shown). There was a general lack of correlation between
enzymatic and cellular activities, an outcome commonly
displayed by many enzyme inhibitors, since certain physico-
chemical properties such as cellular permeability is not re-
flected in an enzymatic assay. In contrast to our previous
observations, the finding associated with the cellular EC50
values from this new generation of compounds, however,
demonstrated that the more basic piperidyl or pyrrolidinyl
derivative is not required for good cellular potency.
The difference in molecular properties derived from the
unsaturated heterocycle portion appeared not to have a clear
impact on the cellular potency. For instance, compounds 8
and 37 shared similar polar surface area (PSA) with values
2
˚
around 102 A , yet 8 had the lowest EC50 value (0.99 nM)
while 37 was not active (EC50 > 1000 nM). Meanwhile,
compounds with a larger difference in PSA such as 15 and
Table 2. Mouse PK Data for Selected Inhibitors
mouse iva
mouse pob
2
24 (PSA = 87 and 118 A , respectively) could be equally
compd CLc
Vd
T1/2 (h) AUCe Cmax
AUCe F (%)
d
f
˚
potent (EC50 = 3.7 nM for both). Compounds withthe lowest
and highest clogP values (1.2 for 24; 5.0 for 19) possessed
cellular activity in the middle range of the spectrum. On the
other hand, 15, 17, 31, 37, 41, 42, and 44 had similar
lipophilicity (clogP value around 3) but their EC50 values
ranged from 3.7 nM (15) to greater than 1000 nM (37).
Relatively more basic heterocycles led to compounds with
8
18
32
26
26
0.97
0.56
0.36
0.40
0.58
0.62
0.54
0.31
3.0
0.07
0
0.11
0
6
0
10
24
41
42
44
3.3
10
8.8
7.4
1.7
2.7
0.76
2.2
1.8
4.0
1.4
2.7
6.1
41
44
73
141
5.9
7.3
6.6
0.94
1.1
1.3
a 3.0 mg/kg. b 10 mg/kg. c (L/h)/kg. d L/kg. e μM h. f μM.
3
Figure 1. In vivo efficacy of 24 in combination with temozolomide (TMZ) in the B16F10 murine melanoma model.

6808 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tong et al.
Figure 2. In vivo efficacy of 44 in combination with temozolomide (TMZ) in the B16F10 murine melanoma model.
EC50 values in the middle range between 2.2 and 20 nM (9, 10,
18, 19, 41, 42, and 44), while nonbasic species contributed to
the most and least potent compounds (EC50 values being 0.99
nMfor8 and >1000nM for 37). These observations indicated
that there was no correlation between physicochemical prop-
erties and cellular activity.
Six compounds with better overall enzymatic and cellular
activities were tested for pharmacokinetic (PK) properties in
CD-1 mice. As shown in Table 2, the oxazole and imidazole
derivatives 8 and 10 exhibited high clearance rates (at least
18 L/h/kg), high volumes of distribution (26 L/kg), and very
In this study, temozolomide was dosed orally, q.d., at 50 mg/
kg/day (mkd) for 5 consecutive days. Meanwhile, during the
sametime frame, 24 was administeredorally, b.i.d., atdoses of
10, 30, and 60 mkd. As shown in Figure 1, the effect of 24 in
combination with TMZ started to differentiate from temozo-
lomide monotherapy at day 12. At day 19, the relative tumor
growth inhibition (TGI) of 24 in combination with TMZ at
doses of 10, 30, and 60 mkd compared to TMZ monotherapy
was 53%, 72%, and 81%, respectively, showing significant
dose-dependent potentiation of TMZ by compound 24.
Compound 44 also demonstrated a clear TMZ-sensitizing
effect (Figure2) whendosed at10 or30mkd startingatday 12.
At day 16, TGI reached 83% and 72%, respectively.
However, its efficacy retreated much more quickly after day
16 compared to 24.
poor oral exposure (AUC = 0 or 0.11 μM h, respectively).
3
However, the oxadiazole compound 24 did possess greatly
improved PK properties with a moderated clearance rate (3.3
(L/h)/kg) and volume of distribution (1.7 L/kg) and much
higheroralAUC(4.0 μM h) andbioavailability(41%). When
3
dosedviaivadministration, the threepyridylanalogues41, 42,
and 44 displayed similar profiles. The clearance rates, volumes
of distribution, and AUC values ranged from 7.4 to 10 (L/h)/
Conclusion
In summary, we have replaced the piperidyl or pyrrolidinyl
moieties on the benzimidazole-based PARP-1 inhibitors with
unsaturated heterocycles extended off a 2-phenyl group in an
attempt to further expand the scope of this class of inhibitors.
Through diverse, yet often convergent synthetic schemes, a
wide range of different heterocycles were installed, resulting in
compounds not only with potent enzymatic potency but also
with high cellular activity (EC50 as low as 1 nM). This finding
broadened our earlier understanding regarding the necessity
of an appropriate piperidine or pyrrolidine derivative for
kg, 5.9-7.3 L/kg, and 0.94-1.3 μM h, respectively. On the
3
contrary, their oralexposures differed toa much largerdegree.
The oral AUC for 44 (6.1 μM h) was about twice as high as
3
for 42 (2.7 μM h) and 4 times as high as for 41 (1.4 μM h).
3
3
The two compounds with the best oral PK properties, 24
and 44, were further evaluated for their in vivo efficacy in
potentiating temozolomide (TMZ) in a B16F10 murine mela-
noma model. Temozolomide is a DNA-alkylating agent and
is used as a chemotherapy agent for melanoma and gliomas.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6809
cellular activity. Some compounds with balanced overall
activities were assessed for their PK properties. Among them,
compounds 24 and 44 showed reasonable oral exposure and
bioavailability. In addition, both inhibitors, 24 in particular,
demonstrated excellent oral in vivo efficacy in potentiating
TMZ in the B16F10 murine melanoma model. Therefore,
installation of an unsaturated heterocycle off the 2-phenyl
group of the benzimidazole core proved to be another viable
strategy for developing potent and orally efficacious PARP-1
inhibitors.
10 mL/kg. Blood samples were collected with a heparinized
syringe by cardiac puncture following CO2 asphixiation at
specified times. Plasma samples were aliquoted into 96-well
plates and proteins precipitated using acidified methanol.
Supernatants were stored at -20 °C. Sample analyses were
performed by LC-MS using a Shimadzu 10A-VP chromatog-
raphy system with a Phenomenex Polar-RP column. The mobile
phase consisted of various concentrations of acetonitrile and
0.1% acetic acid in water at a flow rate of 0.4 mL/min. Mass
detection was accomplished with an ESI equipped LCQ-Duo by
ThermoFinnigan. External standards were prepared from
spiked control plasma and used to generate a response factor
for every study. Limits of detection were between 20 and 50 nM.
PK parameters were calculated using WinNonlin software
(Pharsight).
Experimental Section
All commercially available chemical reagents were used with-
out further purification unless otherwise noted. Flash chroma-
tography was carried out using an ISCO Companion flash
system. Proton NMR datawererecordedusing a Varian Mercury
300 spectrometer or a Varian UNITY 400/500 spectrometer.
Mass spectral data were collected using a Finnigan SSQ7000
GC/MS (ESI) or a Finnigan DCI/MS SSQ7000. Elemental
analyses were performed by Quantitative Technologies, Inc.
Preparative HPLC was performed on a Zorbax RX-C18 column
(250 mm ꢀ 21.2 mm, 7 μm particle size) using a gradient of
10-100% acetonitrile/0.1% aqueous TFA over 40 min at a flow
rate of 15 mL/min. LC/MS was run on an Agilent 1100/Finnigan
Navigator system with a YMC C18 column (50 mm ꢀ 4.6 mm).
Melting point was measured using a Mel-Temp II apparatus.
The purity of all final products was determined by HPLC (at
least 95% pure unless otherwise noted). In addition, combustion
analyses were conducted on eight of the final products (8, 18, 22,
24, 27, 29, 39, and 42).
B16F10 Tumor Model. For B16F10 syngeneic studies, 1.2 ꢀ
106 cells/mL were mixed with an equal volume of Matrigel (BD
Biosciences, Bedford, MA) for a final cell concentration of 6 ꢀ
105 cells/mL. Mice were inoculated with 0.1 mL (6 ꢀ 104 cells/
mouse) of cell suspension by sc injection into the flank of 6-8
week old female C57BL/6 mice, 18-20 g (Charles River La-
boratories, Wilmington, MA). Mice were allocated into treat-
ment groups by size-matching. PARP inhibitor therapy was
initiated on day 5 (or 6) following inoculation, with temozolo-
mide treatment also starting on day 5 (or 6).
2-(4-(Oxazol-5-yl)phenyl)-1H-benzo[d]imidazole-4-carboxa-
mide (8). To a suspension of 2-(4-formylphenyl)-1H-benzo-
imidazole-4-carboxylic acid amide 721 (70.0 mg, 0.264 mmol)
in MeOH (2.5 mL) was added sodium methoxide in MeOH
(0.5 M, 2.1 mL, 1.1 mmol) followed by tosylmethyl isocyanide
(61.8 mg, 0.317 mmol). The mixture was heated at reflux
overnight. After cooling, the mixture was concentrated. The
residue was treated with 50% NaHCO3 and extracted with
EtOAc (2ꢀ). The combined organic layers were dried over
MgSO4, filtered, and concentrated. The crude material was
triturated with a small amount of MeOH and the solid was
filtered, washed with MeOH and diethyl ether, and oven-dried
to give 30.5 mg (38%) of the desired product as a yellow solid.
1H NMR (300 MHz, DMSO-d6) δ ppm 7.36 (t, J = 7.8 Hz,
1 H), 7.71-7.83 (m, 2 H), 7.84-7.91 (m, 2 H), 7.96 (d, J = 8.1
Hz, 2 H), 8.36 (d, J = 8.5 Hz, 2 H), 8.54 (s, 1 H), 9.34 (s, brd,
1 H), 13.47 (s, brd, 1 H). MS (DCI/NH3) m/z 305.1 (M þ H)þ.
The sample was further purified by HPLC for combustion
analysis. Anal. (C17H12N4O2 2TFA 0.15H2O) C, H, N.
PARP-1 Enzyme Assay.23 The enzyme assay was conducted
in a buffer containing 50 mM Tris, pH 8.0, 1 mM DTT, and
4 mM MgCl2. PARP reactions contained 1.5 μM [3H]NADþ
(1.6 μCi/mmol), 200 nM biotinylated histone H1, 200 nM
slDNA, and 1 nM PARP-1 enzyme. Autoreactions utilizing
SPA bead-based detection were carried out in 100 μL volumes in
white 96-well plates. Reactions were initiated by adding 50 μL of
2ꢀ NADþ substrate mixture to 50 μL of 2ꢀ enzyme mixture
containing PARP-1 and DNA. These reactions were terminated
by the addition of 150 μL of 1.5 mM benzamide (∼1000-fold
over its IC50 value). The stopped reaction mixtures (170 μL)
were transferred to streptavidin-coated flash plates, incubated
for 1 h, and counted using a TopCount microplate scintillation
counter. Ki data were determined from inhibition curves at
various substrate concentrations.
3
3
2-(4-(1H-Imidazol-5-yl)phenyl)-1H-benzo[d]imidazole-4-car-
boxamide (TFA Salt, 9). To a suspension of 721 (90.0 mg,
0.339 mmol) and tosylmethyl isocyanide (66.2 mg, 0.339 mmol)
in ethanol (8 mL) was added sodium cyanide (1.7 mg,
0.034 mmol) at room temperature. The reaction mixture was
heated at 40 °C for 1 h and then concentrated. The residue was
treated with 6 mL of NH3/MeOH (7 N), and the suspension was
heated at 110 °C in a pressure tube overnight. The mixture was
cooled and concentrated until most of the solvent was evapo-
rated. The residue was treated with water and extracted with
EtOAc. The organic layer was dried over MgSO4, filtered, and
concentrated. The crude product was purified by HPLC to give
30.1 mg (yellow solid, 29%) of the desired product as the TFA
salt. 1H NMR (300 MHz, CD3OD) δ ppm 7.42 (t, J = 7.8 Hz,
1 H), 7.80 (d, J = 8.1 Hz, 1 H), 7.91-8.00 (m, 3 H), 8.06 (d, J =
1.4 Hz, 1 H), 8.37 (d, J = 8.8 Hz, 2 H), 9.04 (d, J = 1.4 Hz, 1 H).
MS (DCI/NH3) m/z 304.1 (M þ H)þ.
Cellular Assay.23 C41 cells were treated with test compounds
for 30 min in a 96-well plate. PARP was activated by damaging
DNA with 1 mM H2O2 for 10 min. Cells were washed with ice-
cold PBS once and fixed with prechilled methanol/acetone (7:3)
at -20 °C for 10 min. After air-drying, plates were rehydrated
with PBS and blocked using 5% nonfat dry milk in PBS-tween
(0.05%) (blocking solution) for 30 min at room temperature.
Cells were incubated with anti-PAR antibody 10H (1:50) in
blocking solution at room temperature for 60 min followed by
washing with PBS-Tween20 five times and incubation with goat
antimouse fluorescein 5(6)-isothiocyanate (FITC)-coupled anti-
body (1:50) and 1 μg/mL 40,6-diamidino-2-phenylindole (DAPI)
in blocking solution at room temperature for 60 min. After
washing with PBS-Tween20 five times, analysis was performed
using an fmax fluorescence microplate reader set at the excita-
tion and emission wavelengths for FITC or the excitation and
emission wavelengths for DAPI. PARP activity (FITC signal)
was normalized with cell numbers (DAPI).
2-(4-(1H-Imidazol-2-yl)phenyl)-1H-benzo[d]imidazole-4-car-
boxamide (TFA Salt, 10). To a suspension of 721 (80.0 mg,
0.302 mmol) in ethanol (7 mL) was added glyoxal (40% in
water, 0.15 mL, 3.3 mmol) and ammonium hydroxide
(0.24 mL, 6.2 mmol). The reaction mixture was stirred over-
night. Most of the solvent was evaporated. The residue was
treated with water and extracted with EtOAc. The organic
layer was dried over MgSO4, filtered, and concentrated. The
crude product was purified by HPLC to give 32.1 mg (off-white
Mouse PK Protocols. Male CD-1 mice weighing 26-30 g
(Charles River Laboratories) were dosed iv via the tail vein or po
by gavage with a metal feeder tube. Dosing solutions were
prepared in 2.5% ethanol, 2.5% DMSO (iv only), 5% Tween-
80, 25% PEG400, and pH 7.4 PBS for a dosing volume of

6810 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tong et al.
1
solid, 35%) of the desired product as the TFA salt. H NMR
with CH2Cl2/MeOH (97:3 to 90:10) to give 7.2 mg (14%) of the
desired product as an off-white solid. 1H NMR (400 MHz,
CD3OD) δ ppm 2.53 (s, 3 H), 7.36 (t, J = 7.8 Hz, 1 H), 7.73 (d,
J = 8.0 Hz, 1 H), 7.91 (d, J = 8.6 Hz, 2 H), 7.97 (d, J = 8.0 Hz,
1 H), 8.24 (d, J = 8.3 Hz, 2 H), 8.27 (s, 1 H). MS (DCI/NH3) m/z
319.1 (M þ H)þ.
(300 MHz, DMSO-d6) δ ppm 7.36-7.44 (m, 1 H), 7.80 (d, J =
8.1 Hz, 2 H), 7.85 (s, 2 H), 7.91 (d, J = 8.5 Hz, 1 H), 8.21 (d, J =
8.8 Hz, 2 H), 8.50 (d, J = 8.8 Hz, 2 H), 9.22 (s, brd, 1 H). MS
(DCI/NH3) m/z 304.1 (M þ H)þ.
2-(4-Acetylphenyl)-1H-benzo[d]imidazole-4-carboxamide (13).
A solution of 4-acetylbenzoic acid (4.98 g, 30.3 mmol) in a
mixture of pyridine (30 mL) and DMF (30 mL) was treated with
CDI (5.41 g, 33.4 mmol). The reaction mixture was heated at
2-(4-(2-Aminothiazol-4-yl)phenyl)-1H-benzo[d]imidazole-4-car-
boxamide (TFA Salt, 17). A mixture of 14 (60.0 mg, 0.168 mmol)
and thiourea (14.7 mg, 0.193 mmol) in EtOH (2.5 mL) and water
(1.0 mL) was heated at 80 °C for 1 h. The solids were filtered,
washed with EtOH and water, and purified by HPLC to give
35.0 mg (light-yellow solid, 46%) of the desired product as the
TFA salt. 1H NMR (500 MHz, DMSO-d6) δ ppm 7.27 (s, 1 H),
7.38 (t, J = 7.8 Hz, 1 H), 7.72-7.82 (m, 2 H), 7.88 (d, J = 7.6 Hz,
1 H), 8.01 (d, J = 8.5 Hz, 2 H), 8.27 (d, J = 8.5 Hz, 2 H), 9.24 (s,
brd, 1 H). MS (DCI/NH3) m/z 336.1 (M þ H)þ.
45 °C for 2 h. 2,3-Diaminobenzamide 2HCl (11,28 6.80 g,
3
30.3 mmol) was added, and the mixture was stirred at room
temperature overnight. After concentration, the crude intermedi-
ate (12) was treated with acetic acid (70 mL) and the mixture
heated at 90 °C for 6 h. After the mixture was cooled, the solvent
was evaporated. The resulting oil was dissolved in EtOAc and
treated with saturated NaHCO3 until pH 8 was obtained. The
solid that formed was filtered, washed with water, and oven-dried
to give 7.46 g of the desired product (89%, 2 steps) as a yellow
solid. 1H NMR (300 MHz, DMSO-d6) δ ppm 2.66 (s, 3 H), 7.40 (t,
J = 7.8 Hz, 1 H), 7.78 (d, J = 7.8 Hz, 1 H), 7.80-7.84 (m, 1 H),
7.91 (d, J = 7.5 Hz, 1 H), 8.17 (d, J = 8.5 Hz, 2 H), 8.39 (d, J =
8.5 Hz, 2H), 9.30(s, 1 H), 13.61(s, 1H). MS(DCI/NH3) m/z280.1
(M þ H)þ. Melting point: 285-286 °C.
2-(4-(Imidazo[1,2-a]pyridin-2-yl)phenyl)-1H-benzo[d]imidazole-
4-carboxamide (TFA Salt, 18). A suspension of 14 (0.0600 g,
0.168 mmol) and 2-aminopyridine (0.0170 g, 0.184 mmol) in
ethanol (1.5 mL) was heated at 70 °C for 8 h. The solids were
filtered, washed with EtOAc, and purified by HPLC to give 25 mg
(tan solid, 32%) of the desired product as the TFA salt. 1H NMR
(300MHz, DMSO-d6) δppm 7.28 (t, J =6.7Hz, 1 H), 7.38(t, J =
7.7 Hz, 1 H), 7.63-7.72 (m, 1 H), 7.75-7.93 (m, 4 H), 8.19 (d, J =
8.7 Hz, 2 H), 8.42 (d, J = 8.7 Hz, 2 H), 8.72-8.83 (m, 2 H), 9.28 (s,
brd, 1 H). MS (DCI/NH3) m/z 354.1 (M þ H)þ. Anal.
(C21H15N5O 1.4TFA 0.35H2O) C, H, N.
2-(4-(2-Bromoacetyl)phenyl)-1H-benzo[d]imidazole-4-carbox-
amide (14). To a solution of 13 (5.60 g, 20.1 mmol) in TFA
(500 mL) were added N-bromosuccinimide (4.28 g, 24.1 mmol)
and benzoyl peroxide (0.243 g, 1.00 mmol). After heating at
50 °C for 4 h, additional NBS (2 ꢀ 400 mg) was added at 1 h
intervals. The reaction was continued for 1 h after the final
addition of NBS. Water (10 mL) was added, and the mixture was
concentrated. The residue was treated with EtOAc and washed
with 50% NaHCO3. The organic layer was dried over MgSO4,
filtered, and concentrated. The residue was treated with warm
MeOH and EtOAc, and the suspension was concentrated until
most of the solvent was evaporated. The solid was filtered,
washed with EtOAc, and oven-dried to give 1.87 g of the desired
product as a yellow solid. The filtrate was loaded on silica and
purified on an 80 g column using the ISCO Companion flash
system eluting with CH2Cl2/EtOAc (6:4 to 4:6) to give an
additional 0.614 g product. The total yield was 2.49 g (35%).
1H NMR (300 MHz, DMSO-d6) δ ppm 5.01 (s, 2 H), 7.40 (t, J =
7.8 Hz, 1 H), 7.74-7.84 (m, 2 H), 7.87-7.98 (m, 1 H), 8.21 (d,
J = 8.1 Hz, 2 H), 8.41 (d, J = 8.1 Hz, 2 H), 9.27 (s, 1 H), 13.63 (s,
1 H). MS (DCI/NH3) m/z 358.0 (M þ H)þ. Melting point:
78-79 °C.
3
3
2-(4-(7-(Trifluoromethyl)imidazo[1,2-a]pyridin-2-yl)phenyl)-1H-
benzo[d]imidazole-4-carboxamide (TFA Salt, 19). A suspension of
14 (0.0600 g, 0.168 mmol) and 4-(trifluoromethyl)pyridin-2-amine
(0.0298 g, 0.184 mmol) in ethanol (1.5 mL) was heated at reflux for
24 h. The solids were filtered, washed with EtOAc, and purified by
HPLC to give 0.0317 g (orange solid, 29%) of the desired product
1
as the TFA salt (HPLC purity is 93%). H NMR (400 MHz,
DMSO-d6) δ ppm 7.24 (dd, J = 7.1, 1.8 Hz, 1 H), 7.39 (t, J = 7.8
Hz, 1 H), 7.74-7.82 (m, 2 H), 7.90 (d, J = 7.7 Hz, 1 H), 8.13 (s,
1 H), 8.24 (d, J = 8.3 Hz, 2 H), 8.37 (d, J = 8.3 Hz, 2 H), 8.78 (s,
1 H), 8.81 (d, J = 7.4 Hz, 1 H), 9.23 (s, brd, 1 H). MS (DCI/NH3)
m/z 422.1 (M þ H)þ.
4-(4-Carbamoyl-1H-benzo[d]imidazol-2-yl)benzoic Acid (21).
A
solution of 4-(methoxycarbonyl)benzoic acid (4.47 g,
24.8 mmol) in a mixture of pyridine (25 mL) and DMF
(25 mL) was treated with CDI (4.43 g, 27.3 mmol) at 45 °C for
2 h. Compound 11 (5.56 g, 24.8 mmol) was added to the mixture,
and the mixture was stirred at room temperature overnight.
After concentration, the crude product 20 in acetic acid (50 mL)
was heated at 90 °C for 2.5 h. After the mixture was cooled, the
solvent was evaporated and the resulting solids were treated
with 8 mL of EtOAc and 300 mL of saturated NaHCO3. The
suspension was filtered, washed with water and diethyl ether,
then oven-dried to give 6.98 g (95%, 2 steps) of the methyl 4-(4-
carbamoyl-1H-benzo[d]imidazol-2-yl)benzoate intermediate as
a white solid. To this intermediate (1.50 g, 5.08 mmol) in THF
(30 mL) and MeOH (9 mL) was added an emulsion of lithium
hydroxide (0.639 g, 15.2 mmol) in water (6 mL) at room
temperature. The mixture was stirred overnight at room tem-
perature. Most of the solvent was evaporated; the residue was
treated with 5% citirc acid (∼50 mL). The resulting precipitate
was filtered, washed with water and diethyl ether, then oven-
dried to give 1.56 g (quantitative yield) of the desired product as
a tan solid. 1H NMR (300 MHz, DMSO-d6) δ ppm 7.39 (t, J =
7.7 Hz, 1 H), 7.74-7.84 (m, 2 H), 7.90 (d, J = 7.5 Hz, 1 H), 8.14
(d, J = 8.7 Hz, 2 H), 8.36 (d, J = 8.3 Hz, 2 H), 9.30 (d, J =
3.2 Hz, 1 H), 13.60 (s, 1 H). MS (DCI/NH3) m/z 282.0 (M þ H)þ.
2-(4-(3-Methyl-1,2,4-oxadiazol-5-yl)phenyl)-1H-benzo[d]imid-
azole-4-carboxamide (TFA Salt, 22). To a solution of 21
(0.0700 g, 0.249 mmol), HOBT (1.9 mg, 0.012 mmol), and
triethylamine (0.17 mL, 1.2 mmol) in DMF (2 mL) were added
TBTU (0.0840 g, 0.261 mmol) and acetamide oxime (0.0200 g,
0.274 mmol). The reaction mixture was stirred overnight and
2-(4-(Thiazol-4-yl)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(TFA Salt, 15). To a suspension of phosphorus pentasulfide
(0.621 g, 2.79 mmol) in dioxane (7 mL) was added formamide
(0.67 mL, 17 mmol). The mixture was heated at reflux for 2 h.
The solution (thioformamide) was decanted. To 14 (0.100 g,
0.279 mmol) in 2.8 mL of dioxane were added MgCO3 (0.235 g,
2.79 mmol) and the above-mentioned thioformamide solution.
The mixture was heated at reflux for 2 h. After cooling, the
reaction mixture was treated with 5% NaOH and extracted with
EtOAc. The organic layer was dried over MgSO4, filtered,
concentrated, and purified by HPLC to give 45 mg (light-yellow
solid, 37%) of the desired product as the TFA salt (HPLC purity
was 91%). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.41 (t, J =
7.8 Hz, 1 H), 7.77 (s, 1 H), 7.80 (d, J = 7.1 Hz, 1 H), 7.91 (d, J =
7.4 Hz, 1 H), 8.24 (d, J = 8.3 Hz, 2 H), 8.32-8.36 (m, 2 H), 8.37
(d, J = 1.8 Hz, 1 H), 9.19 (s, brd, 1 H), 9.27 (d, J = 2.2 Hz, 1 H).
MS (DCI/NH3) m/z 321.0 (M þ H)þ.
2-(4-(2-Methyloxazol-4-yl)phenyl)-1H-benzo[d]imidazole-4-car-
boxamide (16). A mixture of 14 (60.0 mg, 0.168 mmol) and urea
(40.2 mg, 0.670 mmol) in acetic acid (2 mL) was heated in a CEM
microwave reactor at 160 °C for 15 min. The reaction mixture was
concentrated. The resulting residue was treated with saturated
NaHCO3 and extracted with CH2Cl2/MeOH (9:1). The organic
layer was dried over MgSO4, filtered, concentrated, and purified
on a 4 g column using the ISCO Companion flash system eluting

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6811
then diluted with 50% brine. The resulting suspension was
filtered, washed with water, and oven-dried to give 0.0320 g of
an off-white solid as the intermediate. This solid was dissolved in
DMF, and the solution was heated at 160 °C for 30 min in a
Biotage microwave reactor. The crude material was purified by
HPLC to give 0.0210 g (white solid, 19%) of the desired product
as the TFA salt. 1H NMR (300 MHz, DMSO-d6) δ ppm 2.46 (s, 3
H), 7.40 (t, J = 7.8 Hz, 1 H), 7.75-7.83 (m, 2 H), 7.91 (d, J = 7.8
Hz, 1 H), 8.31 (d, J = 8.5 Hz, 2 H), 8.49 (d, J = 8.8 Hz, 2 H), 9.21
(s, brd, 1 H). MS (DCI/NH3) m/z 320.2 (M þ H)þ. Anal.
(C17H13N5O2 0.65TFA 0.05H2O) C, H, N.
2-(4-(Hydrazinecarbonyl)phenyl)-1H-benzo[d]imidazole-4-car-
boxamide (23). To a suspension of 21 (0.700 g, 2.49 mmol) and
tetramethylfluoroformamidinium hexafluorophosphate (TFFH,
0.789 g, 2.99 mmol) in DMF (9 mL) at 0 °C were added
triethylamine (0.69 mL, 5.0 mmol) and hydrazine (0.16 mL,
5.0 mmol). The mixture was stirred at room temperature for
6 h. Water was added to the reaction mixture. The resulting
precipitate was filtered, washed with water, and oven-dried to
give 0.35 g (48%) of the desired product as an off-white solid. 1H
NMR (300 MHz, DMSO-d6) δ ppm 4.57 (s, brd, 2 H), 7.37 (t, J =
7.7 Hz, 1 H), 7.69-7.83 (m, 2 H), 7.89 (d, J = 7.5 Hz, 1 H), 8.02
(d, J = 8.3 Hz, 2 H), 8.32 (d, J = 8.3 Hz, 2 H), 9.31 (s, 1 H), 9.93 (s,
1 H), 13.49 (s, brd, 1 H). MS (DCI/NH3) m/z 296.1 (M þ H)þ.
Melting point: 248-249 °C.
filtered, washed with water, and oven-dried to give 29.4 mg
(58%) of the desired product as an off-white solid. H NMR
1
(300 MHz, DMSO-d6) δ ppm 7.39 (t, J = 7.8 Hz, 1 H),
7.71-7.85 (m, 2 H,) 7.91 (d, J = 7.1 Hz, 1 H), 8.26 (d, J =
8.1 Hz, 2 H), 8.47 (d, J = 8.1 Hz, 2 H), 9.31 (s, 1 H), 13.58 (s, 1
H). MS (DCI/NH3) m/z 306.1 (M
þ
H)þ. Anal.
(C15H11N7O 2H2O) C, H, N.
3
2-(4-(N0-Hydroxycarbamimidoyl)phenyl)-1H-benzo[d]imida-
zole-4-carboxamide (28). A mixture of 26 (0.500 g, 1.91 mmol),
potassium carbonate (1.317 g, 9.53 mmol), and hydroxylamine
hydrochloride (0.199 g, 2.86 mmol) in EtOH (30 mL) was
heated at reflux. After 5 h, more hydroxylamine hydrochloride
(0.199 g) was added and the reaction mixture was heated at
reflux overnight. The reaction mixture was cooled and the
suspension filtered. The solids were washed with EtOH, stirred
in water for 30 min, filtered, washed with water, and oven-dried
to give 0.209 g of the desired product as a white solid. The initial
filtrate was concentrated, and the residue was triturated with a
small amount of MeOH. The solids were filtered, washed with
MeOH, and dried to give 0.0800 g of the desired product. The
total yield was 0.289 g (51%). 1H NMR (300 MHz, DMSO-d6)
δ ppm 5.92 (s, 2 H), 7.35 (t, J = 7.8 Hz, 1 H), 7.75 (d, J = 7.1
Hz, 2 H), 7.83-7.94 (m, 3 H), 8.24 (d, J = 8.5 Hz, 2 H), 9.35 (s,
brd, 1H), 9.81 (s, 1 H). MS (DCI/NH3) m/z 296.1 (M þ H)þ.
Melting point: 255-256 °C.
3
3
2-(4-(1,3,4-Oxadiazol-2-yl)phenyl)-1H-benzo[d]imidazole-4-car-
boxamide (TFA Salt, 24). A mixture of 23 (75.0 mg, 0.254 mmol)
and trimethyl orthoformate (1 mL) was heated in a CEM micro-
wave reactor at 160 °C for 30 min. The reaction mixture was
concentrated and purified by HPLC to give 43.0 mg (off-white
2-(4-(5-Oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)phenyl)-1H-
benzo[d]imidazole-4-carboxamide (TFA Salt, 29). To a suspen-
sion of 28 (40.0 mg, 0.135 mmol) in dioxane (2 mL) was added
CDI (26.4 mg, 0.163 mmol). The mixture was heated at reflux
for 40 min. The suspension was filtered and washed with diethyl
ether. The solids were stirred in water for 20 min, filtered,
washed with water and diethyl ether, and dried. The crude
material was purified by HPLC to give 29.0 mg (white solid,
50%) of the desired product as the TFA salt. 1H NMR
(500 MHz, DMSO-d6) δ ppm 7.40 (t, J = 7.8 Hz, 1 H),
7.79-7.81 (m, 2 H), 7.91 (d, J = 6.7 Hz, 1 H), 8.03 (d, J =
8.5 Hz, 2 H), 8.44 (d, J = 8.2 Hz, 2 H), 9.20 (s, brd, 1 H), 13.12 (s,
brd, 1 H). MS (DCI/NH3) m/z 322.1 (M þ H)þ. Anal.
(C16H11N5O3 1.4TFA 1.25H2O) C, H, N.
2-(4-(5-Oxo-4,5-dihydro-1,2,4-thiadiazol-3-yl)phenyl)-1H-benzo[d]-
imidazole-4-carboxamide (TFA Salt, 30). A mixture of 28 (40.0 mg,
0.135 mmol), and 1,10-thiocarbonyldiimidazole (30.0 mg, 0.149
mmol) in THF (5 mL) was heated at 50 °C for 1 h. Silica gel (0.6 g)
in CH2Cl2 (5 mL) and MeOH (1 mL) was added to the reaction at
room temperature, and the reaction mixture was stirred overnight.
Silica gel was filtered and washed with MeOH. The filtrate was
purified by HPLC to give 10.0 mg (light-yellow solid, 16%) of the
desired product as the TFA salt. 1H NMR (400 MHz, DMSO-d6)
δ ppm 7.40 (t, J = 7.8 Hz, 1 H), 7.73-7.82 (m, 2 H), 7.91 (d, J =
8.6 Hz, 1 H), 8.16 (d, J = 8.6 Hz, 2 H), 8.40 (d, J = 8.6 Hz, 2 H),
9.20 (s, brd, 1 H), 13.54 (s, brd, 1 H). MS (DCI/NH3) m/z 338.1
(M þ H)þ.
1
solid, 41%) of the desired product as the TFA salt. H NMR
(400 MHz, DMSO-d6) δ ppm 7.40 (t, J = 7.8 Hz, 1 H), 7.74-7.84
(m, 2 H), 7.91 (d, J = 7.7 Hz, 1 H), 8.25 (d, J = 8.3 Hz, 2 H), 8.48
(d, J = 8.3 Hz, 2 H), 9.21 (s, brd, 1 H), 9.42 (s, 1 H). MS
(DCI/NH3) m/z 306.1 (M
0.95TFA 0.05H2O) C, H, N (error for N, 0.43%).
þ
H)þ. Anal. (C16H11N5O2
3
3
2-(4-(5-Oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl)-1H-benzo[d]-
imidazole-4-carboxamide (TFA Salt, 25). To a suspension of
23 (70.0 mg, 0.237 mmol) in dioxane (2.5 mL) was added CDI
(46.1 mg, 0.284 mmol). The mixture was heated at reflux for
45 min. The suspension was concentrated and purified by HPLC
to give 9.0 mg (off-white solid, 9%) of the desired product as the
TFA salt. 1H NMR (400 MHz, DMSO-d6) δ ppm 7.39 (t, J =
7.7 Hz, 1 H), 7.75-7.81 (m, 2 H), 7.90 (d, J = 7.7 Hz, 1 H), 8.01
(d, J = 8.6 Hz, 2 H), 8.41 (d, J = 8.3 Hz, 2 H), 9.19 (s, brd, 1 H),
12.71 (s, 1 H). MS (DCI/NH3) m/z 322.1 (M þ H)þ.
3
3
2-(4-Cyanophenyl)-1H-benzo[d]imidazole-4-carboxamide (26).
A solution of 4-cyanobenzoic acid (1.641 g, 11.16 mmol) in a
mixture of pyridine (12 mL) and DMF (12 mL) was treated with
CDI (1.990 g, 12.27 mmol) and stirred at 45 °C for 2 h. After the
mixture was cooled, compound 11 (2.500 g, 11.16 mmol) was
added and the mixture was stirred at room temperature overnight.
After concentration, the residue was suspended in AcOH (20 mL)
and the mixture was heated at 80 °C for 4 h. The reaction mixture
was cooled and concentrated. The resulting residue was treated
with saturated NaHCO3, and the mixture was stirred for 15 min.
The suspension was filtered. The solids were washed with water
and diethyl ether, then oven-dried to give 2.726 g (93%) of the
desired product as an off-white solid. 1H NMR (300 MHz,
DMSO-d6) δ ppm 7.40 (t, J = 7.7 Hz, 1 H), 7.75-7.83 (m,
2 H), 7.90 (d, J = 7.5 Hz, 1 H), 8.07 (d, J = 8.3 Hz, 2 H), 8.44 (d,
J = 8.3 Hz, 2 H), 9.23 (s, brd, 1 H). MS (DCI/NH3) m/z 263.0
(M þ H)þ. Melting point: 323-324 °C.
2-(4-(5-Thioxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)phenyl)-1H-
benzo[d]imidazole-4-carboxamide (31). A mixture of 28 (40.0 mg,
0.135 mmol), 1,10-thiocarbonyldiimidazole (36.0 mg, 0.203
mmol), and DBU (0.082 mL, 0.54 mmol) in acetonitrile
(4 mL) was stirred at 80 °C for 1.5 h. The reaction mixture
was cooled, diluted with 5% citric acid, and extracted with
EtOAc. The suspension staying between two clear layers was
filtered, washed with water and EtOAc, and dried to give 35.0
mg (76%) of the desired product. 1H NMR (400 MHz, DMSO-
d6) δ ppm 7.39 (t, J = 7.7 Hz, 1 H), 7.74-7.81 (m, 2 H), 7.90 (d,
J = 8.6 Hz, 1 H), 8.10 (d, J = 8.6 Hz, 2 H), 8.44 (d, J = 8.6 Hz, 2
H), 9.21 (s, brd, 1 H). MS (DCI/NH3) m/z 338.0 (M þ H)þ.
2-(4-(Isoxazol-5-yl)phenyl)-1H-benzo[d]imidazole-4-carboxa-
mide (TFA Salt, 37). A suspension of 36 (60.0 mg, 0.186 mmol)
in acetic acid (2 mL) was heated at 90 °C for 90 min. The solids
were filtered, washed with AcOH, diethyl ether, and oven-dried
to give 44.0 mg of a white solid. The material was further
purified by HPLC to give 23.5 mg (30%) of the desired product
2-(4-(1H-Tetrazol-5-yl)phenyl)-1H-benzo[d]imidazole-4-car-
boxamide (27). To a solution of 26 (43.3 mg, 0.165 mmol) in
DMF (1.5 mL) were added ammonium chloride (44.2 mg,
0.825 mmol) and sodium azide (53.7 mg, 0.825 mmol). The
mixture was heated at 60 °C overnight, then at 80 °C for 24 h.
After cooling, the reaction mixture was treated with 5% citric
acid and water until a suspension formed. The solids were

6812 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Tong et al.
as the TFA salt. 1H NMR (300 MHz, DMSO-d6) δ ppm 4.83 (s,
2 H), 7.40 (t, J = 7.8 Hz, 1 H), 7.72-7.86 (m, 2 H), 7.90 (t, J =
7.0 Hz, 1 H), 8.14 (d, J = 8.1 Hz, 2 H), 8.42 (d, J = 8.5 Hz, 2 H),
9.26 (s, brd, 1 H), 13.64 (s, 1 H). MS (DCI/NH3) m/z 305.1 (M þ
H)þ.
J = 7.9 Hz, 2 H), 7.91 (d, J = 7.6 Hz, 1 H), 8.24 (d, J = 8.6 Hz,
2 H), 8.36 (d, J = 6.7 Hz, 2 H), 8.48 (d, J = 8.9 Hz, 2 H), 8.96 (d,
J =6.7 Hz, 2 H), 9.23 (s, brd, 1 H). MS (ESI) m/z 315.2 (M þ H)þ.
2-(4-(Pyridin-3-yl)phenyl)-1H-benzo[d]imidazole-4-carboxamide
(TFA Salt, 42). A mixture of 40 (40.0 mg, 0.127 mmol), pyridin-3-
ylboronic acid (18.7 mg, 0.152 mmol), bis(triphenylphos-
phine)palladium(II) dichloride (4.4 mg, 6.3 μmol), and 1 M
Na2CO3 (0.15 mL, 0.152 mmol) in 1.5 mL of DME/EtOH/H2O
(7:2:3) was heated at 160 °C for 15 min in a Biotage microwave
reactor. The reaction mixture was diluted with brine and water and
extracted with EtOAc/MeOH (9:1). The organic layer was dried,
concentrated, and purified by HPLC to give 43.5 mg (white solid,
63%) of the desired product as the TFA salt. 1H NMR (400 MHz,
DMSO-d6) δ ppm 7.38 (t, J = 7.8 Hz, 1 H), 7.72-7.82 (m, 2 H),
7.83-7.92 (m, 2 H), 8.05 (d, J= 8.6 Hz, 2 H), 8.41 (d, J=8.6Hz, 2
H), 8.60 (d, J = 10.1 Hz, 1 H), 8.79 (dd, J = 5.2, 1.2 Hz, 1 H),
9.15-9.25 (m, 2 H). HR/MS (FAB) calculated for C19H15N4O:
315.1240; found, 315.1237.
4-(1-Methyl-1H-pyrazol-3-yl)benzoic Acid (38). A mixture of
methylhydrazine (carcinogen! 0.201 g, 4.37 mmol) and 33 (1.02
g, 4.37 mmol) in AcOH (12 mL) was heated at 70 °C for 1 h.
After concentration, the residue was treated with saturated
NaHCO3 and extracted with EtOAc. The organic layer was
dried, concentrated, and purified on a 40 g column using the
ISCO Companion flash system eluting with CH2Cl2/EtOAc
(97.5:2.5 to 90:10) to give the desired isomeric intermediate
(0.525 g, 56%). To a solution of this intermediate (0.500 g,
2.31 mmol) in THF (10 mL) and MeOH (3 mL) was added an
emulsion of lithium hydroxide (0.291 g, 6.94 mmol) in water (2
mL). After 2 h at room temperature, the mixture was heated at
40 °C for 3 h. Most of the solvent was evaporated. The
remaining material was treated with 5% citric acid. The result-
ing precipitate was filtered, washed with water, and oven-dried
to give 0.415 g (50%, two steps) of the desired product as a white
2-(4-(Pyridin-2-yl)phenyl)-1H-benzo[d]imidazole-4-carboxa-
mide (44). A mixture of 11 (2.00 g, 8.90 mmol), 4-(pyridin-2-
yl)benzaldehyde (43, 1.64 g, 8.96 mmol), and 10% Pd/C (0.600
g) in MeOH (60 mL) was heated at reflux overnight. After
cooling, the reaction mixture was filtered through Celite. The
filtrate was concentrated and purified by recrystallization from
MeOH to give 1.20 g (43%) of the desired product as a yellow
1
solid. H NMR (300 MHz, DMSO-d6) δ ppm 3.91 (s, 3 H),
6.80 (d, J = 2.0 Hz, 1 H), 7.78 (d, J = 2.4 Hz, 1 H), 7.93 (q, J =
8.8 Hz, 4 H), 12.87 (s, brd, 1 H). MS (DCI/NH3) m/z 203.0
(M þ H)þ. Melting point: 235-236 °C.
1
solid. H NMR (500 MHz, DMSO-d6) δ ppm 7.48 (t, J = 7.8
2-(4-(1-Methyl-1H-pyrazol-3-yl)phenyl)-1H-benzo[d]imidazole-
4-carboxamide (HCl Salt, 39). A solution of 38 (0.150 g,
0.742 mmol) in a mixture of pyridine (1.5 mL) and DMF
(1.5 mL) was treated with CDI (0.132 g, 0.816 mmol) at 45 °C
for 2 h. Compound 11 (0.166 g, 0.742 mmol) was added, and
the mixture was stirred at room temperature for 4.5 h. After
concentration, the residue was treated with 20% brine and
extracted with EtOAc (2ꢀ). The suspension in the aqueous
layer was filtered, washed with water, and oven-dried to give
0.161 g (65%) of clean intermediate as a yellow solid. A
suspension of this intermediate (0.110 g, 0.328 mmol) in acetic
acid (3 mL) was heated at 90 °C for 90 min. The reaction
mixture was concentrated. The residue was suspended in 3 mL
of MeOH and sonicated. To this suspension was added 1 N
HCl in ether. The precipitate was filtered, washed with diethyl
ether, and oven-dried to give 0.114 g (tan solid, 89%) of the
desired product as the HCl salt. 1H NMR (300 MHz, DMSO-
d6) δ ppm 3.93 (s, 3 H), 6.86 (d, J = 2.4 Hz, 1 H), 7.44 (t, J = 7.9
Hz, 1 H), 7.75-7.86 (m, 2 H), 7.91 (d, J = 7.5 Hz, 1 H), 8.04
(d, J = 8.3 Hz, 2 H), 8.30 (d, J = 8.7 Hz, 2 H), 9.07 (s, brd, 1 H).
MS (DCI/NH3) m/z 318.1 (M þ H)þ. Anal. (C18H15-
N5O 2HCl 1.75H2O) C, H, N.
2-(4-Bromophenyl)-1H-benzo[d]imidazole-4-carboxamide (40).
A mixture of 2,3-diaminobenzamide (0.500 g, 2.23 mmol),
4-bromobenzaldehyde (0.454 g, 2.45 mmol), and 10% Pd/C
(0.130 g) in MeOH (25 mL) was heated at reflux overnight. The
reaction mixture was filtered through Celite. The filtrate was
concentrated and purified on a 40 g silica column using the ISCO
Companion flash system eluting with CH2Cl2/MeOH (95:5 to
90:10) to give 0.185 g (26%) of the desired product as an off-white
solid. 1H NMR (300 MHz, DMSO-d6) δ ppm 7.39 (t, J = 7.8 Hz,
1 H), 7.74-7.86 (m, 4 H), 7.89 (dd, J = 7.5, 1.02 Hz, 1 H),
8.17-8.24 (m, 2 H), 9.14 (s, brd, 1 H). MS (ESI) m/z 316.1 (M þ
H)þ. Melting point: 219-220 °C.
Hz, 1 H), 7.62-7.68 (m, 1 H), 7.83 (s, 1 H), 7.87 (d, J = 7.9 Hz,
1 H), 7.94 (d, J = 7.6Hz, 1 H), 8.20 (t, J =7.6Hz, 1 H), 8.25-8.31
(m, 1 H), 8.36 (d, J = 8.5 Hz, 2 H), 8.49 (d, J = 8.5 Hz, 2 H),
8.82 (d, J = 5.2 Hz, 1 H), 9.04 (s, brd, 1 H). MS (ESI) m/z 315.1
(M þ H)þ.
Supporting Information Available: Combustion analysis data
for compounds 8, 18, 22, 24, 27, 29, 39, and 42 and experimental
procedures for 34-36. This material is available free of charge
via the Internet at http://pubs.acs.org.
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