Novel dual-targeting benzimidazole urea inhibitors of DNA gyrase and topoisomerase IV possessing potent antibacterial activity: Intelligent design and evolution through the judicious use of structure-guided design and stucture-activity relationships

Article abstract of DOI:10.1021/jm800318d

The discovery of new antibacterial agents with novel mechanisms of action is necessary to overcome the problem of bacterial resistance that affects all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV are well-characterized clinically validated targets of the fluoroquinolone antibiotics which exert their antibacterial activity through inhibition of the catalytic subunits. Inhibition of these targets through interaction with their ATP sites has been less clinically successful. The discovery and characterization of a new class of low molecular weight, synthetic inhibitors of gyrase and topoisomerase IV that bind to the ATP sites are presented. The benzimidazole ureas are dual targeting inhibitors of both enzymes and possess potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and community-acquired infections. The discovery and optimization of this novel class of antibacterials by the use of structure-guided design, modeling, and structure-activity relationships are described. Data are presented for enzyme inhibition, antibacterial activity, and in vivo efficacy by oral and intravenous administration in two rodent infection models.

Full text of DOI:10.1021/jm800318d

J. Med. Chem. 2008, 51, 5243–5263
5243
Novel Dual-Targeting Benzimidazole Urea Inhibitors of DNA Gyrase and Topoisomerase IV
Possessing Potent Antibacterial Activity: Intelligent Design and Evolution through the Judicious
Use of Structure-Guided Design and Stucture-Activity Relationships
Paul S. Charifson,*,‡ Anne-Laure Grillot,‡ Trudy H. Grossman,‡ Jonathan D. Parsons,‡ Michael Badia,‡ Steve Bellon,‡,§
David D. Deininger,‡ Joseph E. Drumm,‡ Christian H. Gross,‡ Arnaud LeTiran,‡ Yusheng Liao,‡ Nagraj Mani,‡
David P. Nicolau,† Emanuele Perola,‡ Steven Ronkin,‡ Dean Shannon,‡ Lora L. Swenson,‡ Qing Tang,‡ Pamela R. Tessier,†
Ski-Kai Tian,‡,# Martin Trudeau,‡ Tiansheng Wang,‡ Yunyi Wei,‡ Hong Zhang,‡ and Dean Stamos‡
Vertex Pharmaceuticals Incorporated, 130 WaVerly Street, Cambridge, Massachusetts 02139, and Center for Anti-InfectiVe Research and
DeVelopment, Hartford Hospital, 80 Seymour Street, Hartford, Connecticut 06102
ReceiVed March 20, 2008
The discovery of new antibacterial agents with novel mechanisms of action is necessary to overcome the
problem of bacterial resistance that affects all currently used classes of antibiotics. Bacterial DNA gyrase
and topoisomerase IV are well-characterized clinically validated targets of the fluoroquinolone antibiotics
which exert their antibacterial activity through inhibition of the catalytic subunits. Inhibition of these targets
through interaction with their ATP sites has been less clinically successful. The discovery and characterization
of a new class of low molecular weight, synthetic inhibitors of gyrase and topoisomerase IV that bind to the
ATP sites are presented. The benzimidazole ureas are dual targeting inhibitors of both enzymes and possess
potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and
community-acquired infections. The discovery and optimization of this novel class of antibacterials by the
use of structure-guided design, modeling, and structure-activity relationships are described. Data are presented
for enzyme inhibition, antibacterial activity, and in vivo efficacy by oral and intravenous administration in
two rodent infection models.
Introduction
the point of diminishing returns largely because resistance
mechanisms to earlier members of a given class of antibiotics
often impact, to some degree, the effectiveness of subsequent
generations of molecules from the same class. Identifying new
scaffold classes that hit new molecular targets is the obvious
approach to maintain a pipeline of effective antibiotics.10 In the
past 35 years, only two antibiotics representing new scaffold
classes linezolid and daptomycin have been approved in the
U.S. This creates a compelling need for new classes of
antibacterial agents with novel mechanisms of action. While
genomics has offered the potential to identify novel druggable
antibacterial targets, this has not yet proven successful.1
DNA gyrase and topoisomerase IV (topoIVa) have long been
recognized as attractive targets for antibiotics.11-13 Each enzyme
is a heterotetrameric type II topoisomerase characterized by its
ability to alter chromosome structure through breaking and
rejoining double stranded DNA; in addition, each enzyme is
independently essential for bacterial DNA replication. Gyrase
is primarily responsible for introducing negative supercoils into
conformationally constrained DNA, while topoIV primarily
resolves linked chromosome dimers at the conclusion of DNA
replication.12,14-16 The fluoroquinolones inhibit the catalytic
subunits of gyrase (GyrA) and/or topoIV (ParC).12 The associ-
ated subunits responsible for supplying the energy necessary
for catalytic turnover/resetting of the enzymes via ATP hy-
drolysis are GyrB (gyrase) and ParE (topoIV), respectively.17
The ATP binding sites in these subunits have been less
successfully exploited as antibacterial targets with the exception
of the natural product coumarins, e.g., novobiocin (Scheme 1)
and cyclothialadines.18 The benzimidazole ureas described in
Resistant Gram-positive organisms have emerged over the
past 2 decades and pose significant public health concerns
because of the serious types of infections they cause, the
vulnerable patient populations they infect, and their ability to
spread within the hospital environment and from the hospital
to the community. Clinically relevant organisms include me-
thicillin-resistant Staphylococcus aureus, vancomycin-resistant
Enterococcus, penicillin-resistant Streptococcus, macrolide-
resistant Streptococcus, and more recently, fluoroquinolone-
resistant Staphylococcus and Streptococcus.1-5 Data from the
National Nosocomial Infections Surveillance system6 suggest
that the prevalence of methicillin-resistant S. aureus infections
in hospital ICUs has increased steadily over the past decade to
a value exceeding 60% in 2004. Similarly, vancomycin-resistant
Enterococcus infections have reached a level exceeding 30%.
A recent troubling trend is the increase of clinical isolates that
are multidrug resistant.7,8
Bacterial resistance has been demonstrated against all of the
commonly prescribed classes of antibiotics.1 Antibiotic sales
are comprised primarily of multiple generations of ꢀ-lactams
(cephalosporins, penicillins, penems, and monobactams), fluo-
roquinolones, and macrolides.9 A major driver for continual
modification of these scaffolds has been to stay one step ahead
of resistance mechanisms, a strategy that is rapidly approaching
* To whom correspondence should be addressed. Phone: 617-444-6442.
Fax: 617-444-6566. E-mail: paul_charifson@vrtx.com.
‡
Vertex Pharmaceuticals Inc.
Present Address: Amgen, Inc., One Kendall Square, Cambridge, MA
§
0213.
†
a Abbreviations: topoIV, topoisomerase IV; MIC, minimum inhibitory
conventration; CFU, colony-forming unit; ADPNP, 5′-adenylyl-ꢀ,γ-imido-
diphosphate.
Hartford Hospital.
Present Address: Department of Chemistry, University of Science and
#
Technology of China, 96 Jinzhai Road, Hefei, Anhui, 230026, P. R. China.
10.1021/jm800318d CCC: $40.75
2008 American Chemical Society
Published on Web 08/09/2008

5244 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Charifson et al.
Scheme 1
the nonhydrolyzable ATP analogue, ADPNP (Figure 1C). It was
expected that this would reduce the selection of resistant mutants
in this region of the binding site, since they would affect the
viability of the organism if the binding of ATP were perturbed.
The second design principle to emerge from this initial
analysis was that the hydrogen bond interaction between Arg-
136 and the exocyclic carbonyl oxygen of the novobiocin
coumarin ring (Figure 1A) could potentially be mimicked by a
heterocyclic hydrogen bond acceptor directly attached to the
benzimidazole urea core (Figure 1D).
Compound 2 (Table 1) was the end result of this initial design
and provided an approximately 250-fold improvement in Ki
against E. coli gyrase (i.e., from 20 µM to 81 nM) and
approximately 15-fold in Ki against S. aureus gyrase (i.e., from
2 µM to 130 nM). This compound showed modest antibacterial
potency with an MIC of 16 µg/mL against S. aureus and 2 µg/
mL against S. pneumoniae (Table 1). Interestingly, 2 also
showed some weak inhibitory potency against E. coli topoIV
with a Ki of 2.3 µM. While compound 2 failed to show
antibacterial activity against the Gram-negative organism, E.
coli (MIC > 64 µg/mL), it did show an MIC of 16 µg/mL
against the respiratory Gram-negative pathogen, H. influenzae.
(For most compounds presented in this paper, there was a
general lack of antibacterial potency against E. coli. This has
been previously attributed to the effects of compound-mediated
efflux.)23
this study were designed and optimized to target these same
ATP binding sites in the GyrB and ParE subunits.
To maximize effectiveness and longevity of an antibiotic, as
well as to gain acceptance for use as an empiric monotherapy,
it is important that a new antibacterial agent suppress the
appearance of resistant strains. One way to accomplish this is
for a compound to work by interacting with more than one
essential target such that multiple mutations or other significant
geneticalterationsareneededforclinicallyrelevantresistance.19-21
The statistical likelihood of significant simultaneous mutations
in two independently essential targets is low (∼1 in 1014
bacteria).22 Dual inhibition of gyrase and topoIV is, therefore,
expected to minimize the frequency of resistant mutations.
Biochemical, genetic, and structural data show that the benz-
imidazole ureas are potent dual targeting agents of both
enzymes.23-25
Compound 1 (Scheme 1) was identified in a high-throughput
ATPase assay targeting the GyrB subunit.26 Approximately
30 000 compounds prefiltered for druglike properties27 were
used as the screening deck. Both publically available28 and in-
house crystal structures of novobiocin bound to the E. coli GyrB
subunit served as the initial guide for ligand optimization. While
the use of structure-guided design has proven itself with respect
to optimizing enzyme inhibitors,29 there are certainly additional
factors that contribute to antibacterial potency. Foremost among
these factors are permeability into the bacteria and avoidance
of bacterial efflux.
Selected SAR at the 5-, 6-, and 7-Positions of the Benzimid-
azole Urea Scaffold. Examination of the model of 2 bound in
the ATP binding site suggested that a variety of substitutions
might be tolerated at both the 5- and 6- positions of the
benzimidazole core. A limited number of examples of substit-
uents at each of these positions are presented to illustrate specific
points; however, it should be noted that many additional
analogues were synthesized and have contributed to the overall
SAR understanding.
5-Position SAR. As mentioned above, formation of a
hydrogen bond between the 3-pyridyl nitrogen of 2 and the side
chain of Arg-136 appeared to mimic an interaction observed
with novobiocin. Compound 3 lacks the ability to form such a
hydrogen bond and consequently loses 5-fold in gyrase Ki,
compared with 2, accompanied by a decrease in antibacterial
potency. However, compound 4 contains a 5-pyrimidinyl
substituent and shows modest improvements relative to 2 in
terms of Ki against both gyrase and topoIV that translates to an
improvement in overall MIC. Compound 5 represents several
imidazole amide analogues wherein the design intended the
amide carbonyl oxygen to act as a hydrogen bond acceptor with
Arg-136. This substitution resulted in approximately 10- to 20-
fold improvement in Ki against gyrase and a 30-fold improve-
ment against topoIV. These improvements in Ki relative to 2
resulted in a 4-fold improvement in antibacterial potency against
S. aureus and H. influenzae and a 30-fold improvement in MIC
against S. pneumoniae. This hydrogen bond was confirmed by
crystallography with later structures (Figure 2A, compound
19).30 It appeared that the exocyclic nature of the imidazole
amide carbonyl allowed for a more optimal hydrogen-bonding
pattern with the Arg-136 guanidino group. It is also interesting
that compounds 4 and 5 began to show improved antibacterial
activity against H. influenzae that was reflected by their
improvement in enzyme inhibitory potency against gyrase. In
addition to the modifications at the 5-position, the 6-fluoro
substituent of compounds 5 and 6 may have also contributed
to the improvement in both enzyme inhibition and antibacterial
potency relative to 2.
Results and Discussion
All compounds were evaluated for enzymatic inhibition and
antibacterial potency. Relative serum binding potential was
evaluated by S. aureus MIC assays containing 50% human
serum (Table 1).
The benzimidazole carbamate, 1, was identified as a micro-
molar GyrB inhibitor with MIC values greater than 16 µg/mL
against all organisms tested (Scheme 1, Table 1). Docking of 1
into the S. aureus X-ray structure from which novobiocin was
removed immediately suggested two types of structural modi-
fications (Figure 1). First, it appeared that modification of the
carbamate oxygen of 1 to a nitrogen (urea) might change a
seemingly repulsive interaction into an attractive one that would
further contribute to an extensive hydrogen bond network
involving Asp-73, Thr-165 (E. coli gyrase numbering), and a
highly conserved structural water (Figure 1B). Since the
benzimidazole portion of the scaffold also contributes to this
hydrogen bond network, it was thought that this would further
provide an optimization path to potent, low MW inhibitors. The
benzimidazole urea scaffold is positioned deep in the ATP
binding site and maximizes overlap with the adenine ring of

Benzimidazole Urea Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5245
Table 1. Substituent Effects on Gyrase and Topoisomerase IV Inhibition and Antibacterial Activitiesa
enzyme inhibiton data Ki (µM)
minimum inhibitory concentration (µg/mL)
S. aureus
S. aureus
gyrase
E. coli
gyrase
E. coli
topoIV - HS + HS S. pneumoniae H. influenzae
compd
5-position
6-position
7-position
novobiocin NA
NA
H
H
H
H
NA
H
H
H
H
0.01
2
0.13
0.47
0.049
0.010
0.014
20
0.081
0.4
0.02
<0.004
0.11
>60
2.3
9.5
1.1
0.125 >16
2
>16
2
>16
0.5
0.063
0.063
>16
16
>16
2
1
2
3
4
5
4-hydroxystyrylphenyl
3-pyridyl
phenyl
5-pyrimidinyl
1-imidazole-4-
cyclopropylamide
3-pyridyl
>16
16
>16
4
>16
>16
>16
4
F
H
0.078
4
8
4
6
F
H
H
H
H
0.038
0.085
0.19
0.033
0.061
0.13
0.68
1.4
3.5
1
16
0.25
0.5
2
16
16
>16
>16
0.5
16
1
2
>16
1
7
8
3-pyridyl
3-pyridyl
methyl
methoxy
piperidinyl
H
H
H
H
H
16
16
16
>16
>16
>16
9
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
3-pyridyl
2-methoxy-5-
pyrimidinyl
5-pyrimidinyl
1-imidazole-4-
cyclopropylamide
1-imidazole-4-
cyclopropylamide
4-(1-methyl-2-
pyridone)
0.081
0.008
0.026
0.015
0.007
0.017
0.014
0.035
0.044
0.12
3.3
2
10
11
12
13
14
15
16
17
methyl ester
methyl amide
1-pyrazole
2-pyridyl
<0.004
0.005
<0.004
<0.004
0.006
<0.004
0.018
0.015
0.035
0.15
0.046
0.014
1.3
0.023
0.39
0.68
0.063 0.5
0.016
0.063
0.008
0.004
0.5
0.004
1
0.125
16
>16
0.063
0.031
4
4
2
16
3-pyridyl
H
H
3-fluoropyridin-2-yl
benzyloxy
H
0.031 0.5
2
2
>16
8
>16
4
methoxy
18
19
H
H
1-pyrazole
1-pyrazole
0.016
0.01
<0.004
<0.004
0.058
0.012
0.031 0.25
0.5
<0.008
0.003
0.25
0.25
4
20
21
H
H
2-pyridyl
0.012
0.023
<0.004
<0.004
0.007
0.011
0.063 0.25
0.016 0.032
0.002
0.125
0.125
3-fluoropyridin-2-yl
<0.008
a S. aureus strain ATCC 29213; S. pnemoniae strain ATCC 10015; HS ) 50% human serum; H. influenzae strain ATCC 51907; compound 1 is a
carbamate (X ) O); compounds 2-21 are ureas (X ) NH).
6-Position SAR. Exploration at the 6-position showed that a
variety of substituents were well tolerated with respect to gyrase
and topoIV with minimal effect on either Ki or antibacterial
potency (Table 1). As mentioned above, the 6-fluoro analogue,
6, showed a 3-fold improvement in Ki relative to 2, for both
gyrase and topoIV, but showed a 16-fold improvement in MIC
against S. aureus and an 8-fold improvement against S.
pneumoniae. Presumably, the fluorine has either increased
bacterial permeability or decreased efflux potential for this
compound with respect to these two organisms.
A docking model of the 6-methyl analogue, 7, further
suggested that a variety of substituents might be tolerated at
both the 5- and 6-positions of the benzimidazole core (Figure
2B). This model also suggested that the preferred orientation
of the 5-aryl substituent (i.e., with the 5-aryl ∼40-50° relative
to benzimidazole core) can be maintained by any substituent at
the 6-position that does not perturb this “preorganized” con-
formation.31 The 6-piperidinyl (compound 9, as well as other
larger substituents, data not shown) did not cause any additional
loss in potency. Another important insight provided by this
structure is that it should be possible to fill the space adjacent
to the 7-position of the benzimidazole.
7-position because of the apparent unfilled space. The 7-car-
bomethoxy compound 10 showed a 15- to 20-fold improvement
in Ki against gyrase and an even greater than expected 65-fold
improvement against topoIV. This compound was the first
example of a benzimidazole urea with highly potent enzyme
inhibition against both gyrase and topoIV (Table 1). Dual
targeting further resulted in a dramatic improvement in overall
antibacterial potency with MICs of 0.063, 0.016, and 0.5 µg/
mL against S. aureus, S. pneumoniae, and H. influenzae,
respectively. Surprisingly, compound 11, the N-methylcarboxa-
mide analogue of 10, also showed potent gyrase inhibition;
however, it was less active against topoIV (Ki ) 150 nM)
relative to compound 10 (Ki ) 35 nM). One possible explanation
for this reduced topoIV inhibitory potency might be repulsion
between the amide proton with the 6-position proton causing
the methylamide moiety to rotate slightly out of plane (∼15°)
with respect to the benzimidazole core.
On the basis of the observed differences between the amide
and ester, the hypothesis began to emerge that coplanarity at
the 7-position provided optimal enzyme inhibitory potency
against topoIV. The 7-aryl analogues 12-14 were synthesized
to test this hypothesis and to further explore the steric limits of
this region. All of these compounds were equivalent to 10 with
respect to gyrase inhibitory potency; compounds 12 and 13 were
7-Position SAR. As suggested in the above section, it was
desirable to explore the region of the ATP site adjacent to the

5246 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Charifson et al.
synthetic candidates possessing direct linked 7-aryl substituents.
One compound that met the criteria for coplanarity was the
3-fluoropyridin-2-yl compound, 15 (Scheme 1). While com-
pound 15 was almost identical to the des-fluoro analogue, 13,
in terms of both dual enzyme inhibition and general antibacterial
potency, the MIC against S. aureus was less affected by the
addition of human serum (Table 1). Finally, compound 16 with
a benzyloxy substituent at the 7-position showed an ap-
proximately 5-fold increase in Ki against gyrase and ap-
proximately 10-fold increase against topoIV relative to 10.
Possible explanations for this observation include that the
benzyloxy group has exceeded the optimum size for this region
of the ATP binding site and/or was unable to maintain the
required planarity in this region of the binding site because of
the additional torsional degrees of freedom and lack of ability
to achieve a constraining intramolecular hydrogen bond as
described above. Some of the structural similarities and differ-
ences between GyrB and ParE in this region of the binding site
will be discussed in further detail in the next section.
Figure 1. (A) Crystallographic data showing key binding interactions
of novobiocin with selected E. coli gyrase ATP binding site residues.
(B) Model of compound 1 docked into the ATP site highlighting the
interactions of the benzimidazole carbamate with a highly conserved
water and possible improvements suggested by the model. Potential
repulsion between the carbamate oxygen and Asp-73 is shown in green.
(C) Superposition of the modeled benzimidazole core with the adenine
moiety of ADPNP (from X-ray structure 1EI133). (D) Model of
compound 2 docked into the ATP site highlighting the potential
interactions that might simultaneously mimic those of novobiocin while
improving the strength of the hydrogen bond network involving Asp-
73 and a highly conserved water molecule (Thr-165 is not shown but
also stabilizes the conserved water via a hydrogen bond).
Selected Mix and Match Examples. Once it was established
that direct-linked 7-aryl substituents allowed for potent dual
targeting of gyrase and topoIV that translated into significant
improvements in antibacterial potency, many analogues were
prepared with key examples highlighted below. However, one
compound that does not possess a 7-position substituent but is
still noteworthy is compound 17 that contains a 2-methoxy-5-
pyrimidinyl substituent at the 5-position and a methoxy group
at the 6-position. This compound showed a modest 3- to 5- fold
improvement in Ki vs 2. Most importantly, its overall antibacte-
rial potency (Table 1) combined with favorable pharmacokinetic
profile served to clearly link in vitro potency with exposure and
pharmacologic response (data not shown) and validated the
potential of the class.
Compound 18 is a hybrid of compounds 4 and 12 showing
almost identical enzyme inhibitory potency and antibacterial
activity as 12 with an improvement in the H. influenzae MIC
and a lower serum effect. Another interesting compound
containing a pyrazole at the 7-position is 19. It was rationalized
that since the imidazole amide imparted a greater degree of
enzyme inhibitory potency relative to a 3-pyridyl or 5-pyrim-
idinyl substituent (compare 5 with compounds 2 and 4, Table
1) this compound when combined with a 7-aryl moiety might
be very potent. Compound 19 proved to be quite potent and
showed a 5-fold improvement in Ki against topoIV relative to
18; however, this did not result in an improvement in antibacte-
rial activity. In an attempt to improve the antibacterial activity
against S. aureus, the 2-pyridyl, compound 20, was tried in place
of the pyrazole at the 7-position since compound 13 retained
excellent antibacterial potency against S. aureus. Compound 20
turned out to be one of the best compounds in this study in
terms of potent dual targeting of gyrase and topoIV as well as
excellent antibacterial potency. As mentioned earlier, it is
believed that the increase in enzyme inhibitory potency of the
imidazole amide analogues is largely due to the ability of the
amide carbonyl oxygen to make stronger hydrogen bonds with
Arg-136. In this context, the 2-pyridone analogue, 21 also
attempted to mimic this optimized interaction. Compound 21
showed almost identical dual targeting inhibition of gyrase and
topoIV as compared with 20 and also showed a slight improve-
ment in antibacterial potency against S. aureus.
Figure 2. (A) Amide carbonyl oxygen of compound 19 making a
bifurcated hydrogen bond with Arg-136. (B) Model of compound 7
bound to S. aureus gyrase; solvent exposure of 6-position and
conformational effect of 6-methyl substituent on orientation of 5-pyridyl.
also equivalent to 10 with respect to topoIV inhibitory potency.
However, the regioisomeric compound 14 showed a 35-fold loss
in topoIV Ki relative to 10. Quantum mechanical rotational
barrier calculations32 performed at 6-31G* (Figure 3A) lend
support for this topoIV coplanarity requirement. Compound 10
shows a clear energetic preference for the methyl ester to be
perfectly coplanar. Similarly, compound 12 also showed a global
energy minimum corresponding to a roughly coplanar pyrazole
ring. However, compound 14 with its 3-pyridyl substituent at
the 7-position shows preferred minima that are 60° and 120°
out of plane. Interestingly, compounds 10 and 12 also appear
to have this coplanar minimum stabilized by an intramolecular
hydrogen bond between the imidazole NH and either the
carbonyl oxygen of 10 or 2-pyrazole nitrogen of 12 (Figure 3B).
The 3-pyridyl nitrogen of 14 would not be able to make such
an intramolecular hydrogen bond, and coplanarity would be
further destabilized by van der Waals repulsion between the
imidazole NH and the 2H proton of the 3-pyridyl ring. If the
pyridine nitrogen of 14 is moved over one position (i.e., a
2-pyridyl) as with 13, the intramolecular hydrogen bond would
occur, thereby resulting in restored coplanarity and potent
topoIV inhibitory potency. These observations allowed the use
of rotational barrier calculations in a prospective manner to rank
Compounds 18 and 19 contributed to a better understanding
of the SAR surrounding dual targeting of gyrase and topoIV
and the translation into antibacterial potency. More specifically,
these two compounds helped elucidate the structural understand-

Benzimidazole Urea Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5247
Figure 3. (A) Rotational barrier for compounds 10, 12, and 14. The torsion described is defined by the atoms comprising C6 and C7 on the
benzimidazole, the attachment atom and adjacent atoms for the 7-substituent. All rotational barrier calculations were performed at the HF/6-31G*
level of theory with Gaussian 98. (B) Chemical structures of compounds 10 and 12 illustrating intramolecular hydrogen bond that contributes to
ligand preorganization required for optimal topoIV enzyme inhibitory potency. Compound 14 is not capable of making this intramolecular hydrogen
bond.
ing of 7-aryl substitution as it applies to dual targeting. Both
compounds contain a pyrazole at the 7-position of the benz-
imidazole core although they differ with respect to their
5-position substituents. The X-ray crystal structures were solved
for compound 18 complexed with GyrB and compound 19
complexed with ParE.30 It has been previously shown that in
GyrB, the Ile-78 and Ile-94 side chains form hydrophobic
packing interactions; in ParE, Ile-78 is replaced by a methionine
(Met-74).25 It is plausible that this difference combined with
the residue packing adjacent to these residues causes the ParE
“wall” to extend further into the binding site (Figure 4), thereby
narrowing the region that a direct-linked 7-aryl substituent can
occupy. The net result of this relatively narrowed portion of
the binding site in ParE is the requirement for a greater degree
of coplanarity of any 7-aryl substituent for optimal inhibitory
potency against topoIV. The ability of several of these com-
pounds (e.g., 10, 12, 13, 15, 18-21) to make intramolecular
Figure 4. Molecular surface of GyrB ATP binding site (gray) from
crystallographic complex with compound 18 (yellow). The correspond-
ing portion of the ParE ATP site from the crystallographic complex
with compound 19 is shown as a purple mesh.
hydrogen bonds further imparts ligand preorganization in terms
of this required coplanarity.
In Vivo Efficacy for Compound 15. In addition to the in
vivo data presented in this study, an extensive microbiological
characterization of compound 15 (Scheme 1) has been recently
reported.23,24 In these studies, 15 demonstrated dual targeting
activity in a variety of bacteria, exhibited in vitro bactericidality,
and showed low in vitro resistance frequencies. Most impor-
tantly, compound 15 maintained potent antibacterial activity
against key multidrug resistant Gram-positive organisms.
The potential for compound 15 to be effective against skin
and skin structure infections was demonstrated in a thigh
infection model (Figure 5A). A single intravenous bolus dose
of 50 mg/kg compound 15 reduced bacterial density in thigh
tissue by 1.5 mean log10 CFUs respectively against S. aureus
ATCC 29213 at 6 h after dosing compared to the same hour
vehicle-treated control value (both P < 0.01); a reduction of
0.5 mean log10 CFU was achieved when compared to the 0 h
vehicle-treated control value (both P < 0.01). At 6 h after
dosing, the effect of linezolid at 50 mg/kg was equivalent to
that observed with compound 15 relative to both 0 h or same
hour vehicle-treated controls (both P < 0.01). The data from

5248 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Charifson et al.
Figure 5. Results for vehicle controls are in gray, compound 15 (purple bars), and linezolid (green bars). (A) Thigh infection: bar graph showing
reduction in mean log10 CFU from thigh tissue at 2 and 6 h postdose. Compound 15 and linezolid were administered as a single intravenous bolus
at a dose of 50 mg/kg. (B) Lung (pneumococcal pneumonia) infection: bar graph showing reduction in mean log10 CFU from lung tissue at 72 h
postinfection. Compound 15 and linezolid were administered as a two oral doses at 18 and 26 h postinfection. Relevant pharmacokinetic parameters
in rat are as follows: Cl ) 14.4 mL/min/kg, T1/2 ) 0.7 h, Vss ) 1.6 L/kg, F ) 88.4%.
this single dose, short time course study indicate that compound
15 distributes to the thigh tissue and provides a reduction in
bacterial density comparable to a relevant clinical standard.
In a lung infection model of pneumonia, orally administered
compound 15 at doses of 25 and 50 mg/kg given at 18 and
26 h postinfection resulted in a significant reduction of mean
log10 CFU in lung tissue at 72 h postinfection by 4.8 and 5.6
log10 units, respectively, compared with vehicle-treated controls
(P < 0.01 for all treatment groups). The mean log10 values for
total CFU, measured in lung extracts at 72 h postinfection, are
summarized in Figure 5B. Overall, the trend analysis for the
mean log10 CFU reduction exhibited by compound 15 over the
two oral doses appeared to be dose related. At these same doses,
linezolid reduced mean log10 CFU in lung tissue at 72 h
postinfection by 2.5 and 5.9 Log10 units, respectively, compared
with vehicle-treated controls (P < 0.01 for all treatment groups).
At the 25 mg/kg dose, the superior antibacterial potency of
compound 15 against S. pneumoniae has clearly translated to a
greater than 2 log10 reduction in lung bacterial density relative
to linezolid, while at the 50 mg/kg dose, the effects were
comparable.
Substituent changes at the 5- and 6-positions of the benz-
imidazole core dramatically improved enzyme inhibitory and
antibacterial potencies relative to initial compounds in the series.
However, an appropriate 7-substituent capable of forming a
preorganized intramolecular hydrogen bond, while at the same
time making complementary hydrophobic interactions with the
enzyme, appeared to be the key structural requirement for potent
dual targeting. In the case of most Gram-negative organisms,
however, potent enzyme inhibition did not translate to very
potent antibacterial activity with the exception of the respiratory
pathogens, H. influenzae and M. catarrhalis (data not shown).
Increased susceptibilities of E. coli mutant strains with either
improved permeability (impA mutant) or decreased efflux (tolC
mutant), suggested that efflux and, to a lesser degree, poor
permeability are responsible for the relative lack of antibacterial
potency in E. coli and other Gram-negative organisms.23
The benzimidazole ureas are a novel class of dual targeting
agents exerting their antibacterial effect via simultaneous
inhibition of the ATPase function of gyrase and topoisomerase
IV. Several of the compounds presented in this study possess
potent antibacterial activity against clinically relevant, multidrug
resistant organisms.23 Compound 15 was shown in previous
studies to possess low in Vitro spontaneous resistance frequen-
cies and demonstrated bactericidality against all organisms
tested. In the current study, this compound was shown to be
effective in rodent models of skin and skin structure infection
and pneumonia when dosed by both intravenous and oral routes.
In summary, the benzimidazole ureas represent one of the most
exciting recent chemical advances in the antibacterial field. As
a novel chemical class, the benzimidazole ureas have the
potential to become clinically viable antibiotics, directly ad-
dressing the resistance problem by their novel dual-targeting
mechanism of action.
Conclusion
History has shown many times over that familiarity with a
well-accepted antibiotic class has helped usher successive
generations of that class into widespread use by the medical
community. This phenomenon spans all of the major classes of
antibiotics: ꢀ-lactams, fluoroquinolones, cephalosporins, and
macrolides. This study represents the prospective design of dual
targeting inhibitors of both gyrase and topoisomerase IV using
tightly coupled insights from structural information, modeling,
and SAR.
There is no de facto expectation that an improvement in
enzyme inhibitory potency will result in greater antibacterial
potency, since the properties governing permeability and efflux
may be different from those imparting target affinity. A clear
understanding of SAR can be further complicated by the
possibility that different compounds can exhibit differing degrees
of enzyme inhibition across different bacterial species, and in
the present work, Ki values have only been determined for two
species (i.e., S. aureus and E. coli). Nonetheless, in the case of
the benzimidazole ureas, improvements in enzyme inhibitory
potency (especially when dual targeting was introduced) gener-
ally led to enhancements in antibacterial potency against Gram-
positive organisms.
Experimental Section
Synthesis of Benzimidazole Ureas. The general synthetic plan
to support SAR exploration within the benzimidazole urea (BI-
urea) series is outlined in Scheme 2. Each approach involved initial
placement of the various C5, C6, and C7 functional groups followed
by manipulation of the masked o-phenylenediamine to yield the
desired BI-urea. While our initial stepwise approach to converting
functionalized o-phenylenediamines to the BI-urea core did yield
final compounds, reaction efficiencies occasionally suffered. In some
cases complex mixtures of mono- and bis-urea adducts were formed
(Scheme 2, eq 1). If this was the dominant outcome, reactions were
driven to the bis-urea intermediates and then selectively endo-

Benzimidazole Urea Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5249
Scheme 2
Scheme 3a
a (a) Bis(pinacolotto)diboron, KOAc, Pd(dppf)Cl2, DMSO, 70 °C; (b): Ar-Br, Pd(PPh3)4, K3PO4, DMSO, 100 °C; (c) pyridin-3-yl-3-boronic acid,
Pd(dppf)Cl2, K3PO4, DMF, 120 °C; (d) 5% Pd-C, H2, EtOH, EtOAc; (e) NaHSO3, aq EtOH, reflux; (f) BrCN, MeOH, room temp; (g) 25a,b, EtNCO, THF,
reflux; (h) 24c, reagent A, aq HCl, p-dioxane, reflux.
deacylated to yield desired product using ammonia. Further pursuit
of milder and more expeditious methods identified reagents A and
B (Scheme 2, eqs 2 and 3) that allowed for facile one-step
conversions of o-phenylenediamines directly to BI-ureas in high
yields.34
Functionalization of the C5-position was most often accom-
plished either by direct Suzuki couplings for C5-carbon linked
aromatic groups or by nucleophilic aromatic substitution of activated
C5-fluorobenzenes for C5-nitrogen linked aryls. The preparation
of compounds 2 and 3 started with conversion of 2-nitro-4-
bromoaniline 22 to the corresponding biaryls 23a and 23b by direct
cross-couplings with 3-pyridyl- and phenylboronates under standard
conditions (Scheme 3). Alternatively, 22 could be converted to its
C4-boronate under conditions described by Miyaura35 et al. and
used either directly in a one pot, two-step sequence or as an isolated
intermediate to prepare other biaryls, including 23c. Reduction of

5250 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Charifson et al.
Scheme 4a
a (a) Na2CO3, DMF, 125 °C; (b) 50 psi of H2, Raney Ni, MeOH; (c) reagent B, pH 3.5 buffer, dioxane, reflux; (d) 6 N HCl, reflux; (e) DIEA, HBTU,
cyclopropylamine, DMF, room temp.
Scheme 5a
aminobenzimidazole 36 was obtained. Preparation of the C6 fluoro
analogue 6 began with the commercially available bromodifluo-
ronitrobenzene 37. Initial o-fluoro SnAr reaction followed by
installation of the C5 aryl via Suzuki coupling provided o-
nitroaniline 39, which was similarly reduced and treated with
cyanogen bromide to yield the aminobenzimidazole 40 (Scheme
6). The aminobenzimidazoles 44a and 44b were similarly prepared
from the appropriately functionalized nitrobromoanilines 41a and
41b through a Suzuki/reduction/cyanogen bromide sequence (Scheme
7). The conversion of 44a and 44b to the desired BI-ureas 7 and 8
was effected with simple acylation using ethyl isocyanate in
refluxing THF. However, under nearly identical conditions the
conversions of 36 and 40 to the desired BI-ureas provided complex
mixtures of mono- and diacylated products. To circumvent this
issue, these reactions were driven to their fully diacylated inter-
mediates and then regioselectively endodeacylated with ammonia
to provide 9 and 6, respectively. The BI-urea analogue 17 was
prepared from 41c using the most expeditious Suzuki/reduction/
reagent A sequence.
a (a) KNO3, concd H2SO4, 0 °C to room temp; (b) 0.5 N NH3, dioxane,
sealed tube, 70 °C; (c) piperidine, MeCN, 80 °C; (d) pyridine-3- boronic
acid pinacol ester, aq NaHCO3, Pd(PPh3)4, DME, 90 °C; (e) 10% Pd-C,
40 psi of H2, MeOH, HOAc; (f) NCBr, MeCN, MeOH, room temp; (g)
excess EtNCO, MeCN, 70 °C; (h) 7 N NH3, MeOH.
Preparation of the C7-carbonyl substituted analogues began with
nitration of bromoanthranilate 45 to provide 46 (Scheme 8).
Introduction of the C5-aryl via Suzuki coupling provided 47, which
was then reduced and directly converted to the BI-urea 10 using
the cyanamide derived reagent A. Subsequent methyl ester hy-
drolysis of 10 and conversion to the primary carboxamide under
standard amide forming conditions provided 11.
Preparation of the C7-ether analogue 16 began with bromination
of the hydroxynitroaniline 49 followed by chemoselective ether
formation providing 50 (Scheme 9). From this point BI-urea 16
was obtained through the usual sequence of Suzuki coupling to
introduce the C5-aryl, reduction, and then cyanogen bromide
treatment. The resulting aminobenzimidazole was then exhaustively
acylated and then treated with ammonia to effect the regioselective
endourea cleavage.
the resulting o-nitroanilines to their corresponding o-phenylenedi-
amines (24a, 24b, 24c) was accomplished by several means
including catalytic hydrogenation and stoichiometric methods using
inorganic reducing agents. Following a two-step sequence, 24a and
24b were first converted to their corresponding aminobenzimida-
zoles using cyanogen bromide then acylated with ethyl isocyanate
to provide 2 and 3. Alternatively, 24c was directly converted to 4
in a single step using reagent A.
Preparation of 5 began with an SnAr reaction between 26 and
27 under alkaline conditions (Scheme 4) to provide adduct 28.
Subsequent reduction to the o-phenylenediamine 29 and direct
conversion to the BI-urea using reagent B provided 30. Ester
hydrolysis to 31 and conversion to the corresponding carboxamide
provided compound 5.
C6 exploration normally began with placement of the C6-
substituent from commercial starting materials followed by incor-
poration of the C5-aryl and subsequent conversion to the BI-urea
final products. Preparation of the 1-piperidinyl analogue 9 (Scheme
5) began with nitration of 32 followed by a regioselective o-fluoro
SnAr reaction with ammonia to give nitroaniline 33. Exchange of
the second fluorine with the more nucleophilic piperidine followed
by Suzuki coupling to introduce the C5 aryl provided o-nitroaniline
35. Following reduction and cyanogen bromide treatment the
The preparation of the C5 and C7 bis-N-linked heteroaromatic
analogue 19 began with the two-step sequence involving initial
regeoselective para-sulfonation36 followed by ortho-nitration using
potassium nitrate in trifluoroacetic anhydride as solvent37 of aniline
54 through an in situ generated trifluoroacetanilide to provide 56.
To our surprise, cleavage of the sulfate blocking group occurred
under the nitration reaction conditions (Scheme 10) rather than in
a subsequent step involving heat and strong aqueous acid. As the
expected nitrated sulfonic acid intermediate was never observed,

Benzimidazole Urea Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5251
Scheme 6a
a (a) NH3, MeOH, reflux; (b) pyridine-3-boronic acid pinacol ester, aq NaHCO3, Pd(PPh3)4, DME, 90 °C; (c) 10% Pd-C, 40 psi of H2, MeOH, room
temp; (d) NCBr, MeCN, MeOH, room temp; (e) excess EtNCO, MeCN, 70 °C; (f) 7 N NH3, MeOH.
Scheme 7a
a (a) 3-Pyridineboronic acid pinacol ester, aq NaHCO3, Pd(PPh3)4, DME, 90 °C; (b): 2-methoxy-pyrimidine-5-pinacol boronate, Pd(PPh3)2Cl2, aq NaHCO3,
DME, reflux; (c) 10% Pd-C, 40 psi of H2, MeOH, HOAc; (d) Et3N, HCO2H, Pd-C, MeOH, reflux; (e) NCBr, MeCN, room temp; (f) excess EtNCO,
MeCN, 70 °C; (h) 7 N NH3, MeOH; (h) 43c, reagent A, aq H2SO4, reflux.
Scheme 8a
a (a) TFAA, KNO3, 0 °C; (b) 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine, aq NaHCO3, Pd(PPh3)4, DME, 90 °C; (c) 10% Pd-C, 40 psi of H2,
MeOH, HOAc; (d) reagent A, aq H2SO4, reflux; (e) 6 N HCl, reflux; (f) PyBrOP, MeNH2, DMAP, NMP.
it is presumed that loss of SO3 may have been facilitated by using
the strongly electrophilic trifluoroacetic anhydride as solvent. Acid
catalyzed cleavage for the trifluoroacetyl group followed by
introduction of the 1-pyrazolyl substituent at C7 via a regioselective
anionic SnAr reaction yielded 58. Incorporation of the C5 imidazole
amide group was similarly effected under more forcing conditions
to provide nitroaniline 59.
The preparation of analogues 12 and 18 began with oxidation
of aniline 59 to the corresponding nitro analogue 60 (Scheme 11).
Sequential ortho SnAr displacements with ammonia then anionic

5252 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Charifson et al.
Scheme 9a
provided 71a,b in high yields. Reduction of the resulting nitroa-
nilines and conversion to the final BI-ureas using reagent A gave
analogues 15 and 21, respectively.
An alternative approach to incorporating the C7 aryl functionality
was also developed starting with introduction of the C5 aryl
substituent via a Suzuki coupling to 76 (Scheme 14). Regioselective
bromination of 77 introduced the C7-bromide that could participate
in a subsequent organometallic cross-coupling with either 2-py-
ridylzinc chloride or 3-pyridylboronic acid to provide 79a and 79b,
respectively. The resulting nitroanilines were then reduced and
directly converted to the desired BI-ureas 13 and 14 using
reagent A.
Finally, the synthesis of analogue 20 with C5-nitrogen linked
azaheteroaromatics and C7-carbon linked heteroaryls began with
the regioselective nitration of bromodifuorobenzene 80 (Scheme
15). Ortho-selective SnAr displacement of 81 using ammonia
provided 82, which was then cross-coupled with 2-pyridylzinc
chloride under Negishi conditions to give 83. Sequential introduc-
tion of the C5 imidazolylamide under anionic SnAr conditions using
8738 (Scheme 16) followed by acid catalyzed cleavage of the tert-
butyl group yielded 85. Final conversion to the BI-urea 20 was
then achieved via the two-step sequence of reduction and treatment
of the resulting phenylenediamine with reagent B.
a (a) Br2, HOAc, room temp; (b) K2CO3, benzyl bromide, DMF, room
temp; (c) pyridine-3-boronic acid 1,3-propanediol cyclic ester, Pd(PPh3)4,
NaHCO3, DME, 90 °C; (d) sodium hydrosulfite, EtOH, water, 70 °C; (e)
NCBr, CH3CN, MeOH, room temp; (f) EtNCO, CH3CN, room temp; (g)
NH3, MeOH, 60 °C.
Scheme 10a
Chemistry. Proton NMR spectra were recorded on a Bruker
Advance instrument with a QNP probe using TMS as the internal
standard in the indicated deuterated solvent. LC-MS analyses were
performed on a Waters ZQ or ZMD or QuatroII mass spectrometer
using the electrospray (ESI) ionization technique. Preparative HPLC
isolations performed using Agilent 1100 mass-directed purification
chromatography system with 0.1% TFA in acetonitrile/water mobile
phase and Waters Sunfire C18 stationary phases. All commercially
available reagents were used without further purification. Purity
assessment for final compounds based on two orthogonal HPLC
methods. Method A consisted of the following: column, 4.6 mm
× 50 mm Waters YMC Pro-C18 column, 5 µm, 120A. Mobile
phases are as follows: A, H2O with 0.2% formic acid; B, acetonitrile
with 0.2% formic acid; gradient, 10% B to 90% B in 3 min with
5 min run time. Flow rate is 1.5 mL/min. Method B consisted of
the following: column: 4.6 mm × 50 mm Waters Symmetry C18
column, 5 µm. Mobile phases are as follows: A, H2O with 10 mM
ammonium formate; B, ACN with 10 mM ammonium formate;
gradient, 10% B to 90% B in 3 min with 5 min run time. Flow rate
is 1.5 mL/min.
a (a) H2SO4, 190 °C; (b) trifluoroacetic anhydride, KNO3, room temp;
(c) 6 N HCl, 100 °C; (d) NaH, pyrazole, DMF, 50 °C; (e) 87, Na2CO3,
DMF, 95 °C; (f) Pd-C, 45 psi of H2, 2 N HCl; (g) A, aq H2SO4, 100 °C.
Scheme 11a
2-Nitro-4-(pyridin-3-yl)benzenamine (23b). To a solution of
22 (217 mg, 1 mmol) in 6 mL of DMF was added successively
3-pyridineboronic acid (148 mg, 1.2 mmol), potassium phosphate
(276 mg, 1.3 mmol), and palladium dichloro-dppf (83 mg, 0.1
mmol). The mixture was stirred at 95 °C for 24 h and allowed to
cool to ambient temperature. It was diluted with 50 mL of ethyl
acetate and filtered through a pad of Celite. The filtrate was washed
successively with saturated aqueous sodium bicarbonate, water,
brine, was dried over magnesium sulfate, and was concentrated in
vacuo. The residue was purified by column chromatography over
silica gel eluted with ethyl acetate-hexanes (gradient from 1:4 to
5:1) to afford 23b (117 mg, 54% yield) as an orange solid. 1H
NMR (500 MHz, CDCl3): δ 8.80 (d, 1H), 8.55 (m, 1H), 8.35 (d,
1H), 7.85 (dd, 2H), 7.65 (dd, 2H), 7.35 (m, 1H), 6.95 (d, 1H), 6.25
(broad s, 2H).
a (a) NaBO3, HOAc, 100 °C; (b) NaH, 1H-pyrazole, DMF; (c) NH3,
ethanol, sealed tube, 80 °C; (d) ArB(OR)2 or ArBEt2, Pd(PPh3)4, NaHCO3,
aq DME, 90 °C; (e) 40 psi of H2, Pd(OH)2-C, MeOH; (f) A, pH 3.5 buffer,
100 °C.
pyrazole provided bromide 63. Introduction of the C5 aryl sub-
stituent via Suzuki coupling yielded the nitroanline intermediates
64a,b. Reduction of nitroanilines 59, 64a, and 64b followed by
treatment with reagent A gave the final BI-urea analogues 19, 12,
and 18 respectively.
The preparation of C7-aryl substituted analogues 15 and 21 was
effected by initial Suzuki couplings between the requisite halo-
heteroaromatics and boronate 65 to provide the biaryl 66 (Schemes
12 and 13). Following a three-step sequence involving para-
bromination, then ortho-nitration, and finally amide cleavage,
intermediate 69 was obtained. While direct Suzuki coupling to this
bromide was possible, it was also advantageous to prepare the C5-
boronate35 analogue 70 to allow for more diversification at the C5
position of the BI core. Suzuki couplings with either 69 or 70
4-(Pyridin-3-yl)benzene-1,2-diamine (24b). To a solution of
23b (117 mg, 0.544 mmol) in EtOAc (13 mL) was added 10%
palladium on carbon (51 mg). The resulting mixture was shaken
in a Parr bottle under a 40 psi of hydrogen and then filtered through
Celite. The solids were washed with EtOAc and the combined
filtrates concentrated in vacuo to afford compound 24b which was
1
used without further purification. H NMR (500 MHz, CDCl3): δ
8.80 (d, 1H), 8.45 (m, 1H), 7.75 (m, 1H), 7.25 (m, 1H), 6.95 (m,
2H), 6.80 (m, 1H), 3.25 (br s, 2H).
5-(Pyridin-3-yl)-1H-benzo[d]imidazol-2-amine (25b). To a
solution of compound 24b (0.54 mmol) in THF (2.5 mL) was added
5 mL of methanol and 5 mL of water, followed by an acetonitrile
solution of cynaogen bromide (0.116 mL, 0.580 mmol) at ambient

Benzimidazole Urea Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5253
Scheme 12a
a (a) Ar-Br, Pd(PPh3)4, NaHCO3, aq DME, 100 °C; (b) Br2, HOAc, room temp; (c) HNO3, TFAA, TFA, 0 °C; (d) 6 N HCl, reflux; (e) bis(pinacoloto)diboron,
KOAc, Cl2Pd(dppf)2, dioxane, 100 °C; (f) 3-pyridyl-1,3-propanediol boronate ester, Pd(PPh3)4, Na2CO3, aq DME, reflux; (g) 75, LiCl, Pd(PPh3)4, Na2CO3,
DMF, 95 °C; (h) Pd-C, 45 psi of H2, MeOH, EtOAc; (i) A, aq H2SO4, 100 °C.
Scheme 13a
1H), 7.7 (dd, 1H), 7.55 (m, 2H), 7.45 (m, 2H), 7.35 (m, 1H), 6.9
(d, 1H), 6.1 (br s, 2H).
5-Phenyl-1H-benzo[d]imidazol-2-amine (25a). To a solution of
of 23a (200 mg, 0.93 mmol) in 9 mL of EtOH and 6 mL of water
was added 1.42 g of NaHSO3. The mixture was stirred at 80 °C
for 90 min, cooled to ambient temperature, and partitioned between
100 mL of ethyl acetate and 50 mL of water. The aqueous layer
was extracted with three 50 mL portions of ethyl acetate. The
combined organic extracts were dried over magnesium sulfate and
concentrated in vacuo to afford 24a (152 mg, 89%) as an oil, which
was used without further purification.
a (a) Na2CO3, MeI, acetone, room temp; (b) H2, Pd(OH)2-C, MeOH,
room temp; (c) Tf2NPh, TEA, MeCN, room temp.
A solution of 24a (0.83 mmol) in 5 mL of THF was treated
with 8 mL of methanol and 8 mL of water, followed by 178 µL of
a 5 M solution of cynaogen bromide in acetonitrile. The mixture
was stirred at ambient temperature overnight, diluted with 80 mL
of water and 8 mL of 2 N NaOH, extracted with three 50 mL
portions of ethyl acetate. The combined organic extracts were
washed with aqueous 1 N NaOH, brine, dried (MgSO4), and
concentrated in vacuo to afford 25a (118 mg, 68%) as an oil. H
NMR (500 MHz, DMSO-d6): δ 10.6 (broad s, 1H), 7.45 (m, 2H),
7.25 (m, 4H), 7.15 (m, 1H), 7.0 (m, 2H), 6.1 (m, 1H).
1-Ethyl-3-(5-phenyl-1H-benzo[d]imidazol-2-yl)urea (3). To a
solution of 25a (40 mg, 0.19 mmol) in 2 mL of THF was added
ethyl isocyanate (0.34 mmol, 27 µL). The mixture was heated at
80 °C overnight, allowed to cool at ambient temperature, diluted
with 20 mL of ethyl acetate, washed with aqueous 1 N sodium
hydroxide, brine, was dried (MgSO4), and was concentrated in
vacuo. The residue was purified by preparatory HPLC to afford 3
(15 mg, 28% yield) as a white solid. Purity, method A 90%; method
B 90%. 1H NMR (500 MHz, DMSO-d6): δ 9.0 (s, 1H), 8.65 (d, J
) 4.0 Hz, 1H), 8.35 (d, J ) 8.7 Hz, 1H), 7.85 (s, 1H), 7.7 (dd, J
) 8.7, 4.0 Hz, 1H), 7.65 (s, 1H), 7.6 (m, 1H), 7.55 (m, 1H), 3.25
(m, 2H), 1.15 (q, J ) 7.5 Hz, 3H). MS, m/z: 281 (M + H)+
2-Nitro-4-pyrimidin-5-ylphenylamine (23c). In a sealed tube, 22
(0.32 g, 1.5mmol) and bis(pinacolotto)diboron (0.46 g, 1.8 mmol)
were dissolved in DMSO (3 mL) and treated with potassium acetate
(0.44 g, 4.5 mmol) and (0.1 g, 0.12 mmol) before heating overnight
at 70 °C in an oil bath. 5-Bromopyrimidine (0.24 g, 1.5 mmol),
finely ground potassium phosphate (0.96 g, 4.5 mmol), Pd(dppf)Cl2
(0.1 g), and DMSO (3 mL) were added to the cooled mixture,
sealed, heated at 100 °C for 18 h, then returned to ambient
temperature. The cooled mixture was poured into water (100 mL)
and extracted thrice with EtOAc. The combined organic extracts
were washed thrice with water, dried over MgSO4, filtered, then
concentrated to a yellow-orange solid which was purified by silica
temperature. The mixture was stirred overnight and then diluted
with 50 mL of water and 5 mL of 2 N NaOH and extracted with
three 50 mL portions of ethyl acetate. The combined organic extracts
were washed sequentially with 1 N NaOH, brine, dried over MgSO4,
and concentrated in vacuo to provide 25b (50 mg, 41% crude yield)
as a white solid which was used without further purification. H
NMR (500 MHz, MeOD): δ 8.8 (m, 1H), 8.45 (m, 1H), 8.05 (m,
1H), 7.45 (m, 2H), 7.25 (m, 2H), 4.9 (broad s, 3H).
1-Ethyl-3-(5-(pyridin-3-yl)-1H-benzo[d]imidazol-2-yl)urea (2).
To a solution of 25b (50 mg, 0.24 mmol) in THF (2 mL) was added
ethyl isocyanate (0.022 mL, 0.29 mmol). The mixture was heated
at 80 °C overnight and then returned to ambient temperature. The
crude reaction was diluted with 20 mL of ethyl acetate, washed
sequentially with 1 N sodium hydroxide and brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue was
purified by preparatory HPLC to afford 2 (25 mg, 16% yield) as a
white solid. Purity, method A 98%; method B 98%. 1H NMR (500
MHz, DMSO-d6): δ 9.0 (s, 1H), 8.65 (d, J ) 4.0 Hz, 1H), 8.35 (d,
J ) 8.7 Hz, 1H), 7.85 (s, 1H), 7.7 (dd, J ) 8.7, 4.0 Hz, 1H), 7.65
(s, 1H), 7.6 (m, 1H), 7.55 (m, 1H), 3.25 (m, 2H), 1.15 (7, J ) 7.5
Hz, 3H). MS, m/z: 282 (M + H)+.
1
1
4-Phenyl-2-nitroaniline (23a). To a solution of 22 (6 mmol,
1.30 g) in 36 mL of DMF was added successively phenylboronic
acid (7.2 mmol, 878 mg), potassium phosphate (7.8 mmol, 1.66 g),
and palladium dichloro-dppf (0.6 mmol, 489 mg). The mixture was
stirred at 95 °C for 24 h and allowed to cool to ambient temperature.
It was diluted with 200 mL of ethyl acetate and filtered through a
pad of Celite. The filtrate was washed successively with saturated
aqueous sodium bicarbonate, water, brine, was dried over magne-
sium sulfate, and was concentrated in vacuo. The residue was
purified by column chromatography over silica gel eluted with ethyl
acetate-hexanes (gradient from 1:4 to 5:1) to afford 23a (635 mg,
1
50%) as an orange solid. H NMR (500 MHz, CDCl3): δ 8.4 (d,

5254 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Charifson et al.
Scheme 14a
a (a) 3-Pyridine-1,3-propanediol boronic ester, aq NaHCO3, Pd(PPh3)4, DME, 90 °C; (b) Br2, HOAc, room temp; (c) 3-(1,3,2-dioxaborinan-2-yl)pyridine,
Pd(PPh3)4, NaHCO3, aq DME, 90 °C; (d) 2-pyridylzinc chloride, Pd(PPh3)4, THF, DMF, reflux; (e) H2, Pd-C, EtOAc; (f) reagent A, aq H2SO4, 100 °C.
Scheme 15a
t
a (a) HNO3, H2SO4, room temp; (b) BuNH2, dioxane, sealed tube, 80 °C; (c) 2-pyridylzinc chloride, Pd(PPh3)4, THF, DMF, 90 °C; (d) 87, Na2CO3,
DMF, 120 °C; (e) HCl, MeOH, reflux; (f) 40 psi of H2, Pd(OH)2-C, MeOH; (g) reagent B, pH 3.5 buffer, 100 °C.
Scheme 16a
(m, 3H), 7.8 (s, 1H), 7.4-7.5 (m, 2H), 7.2 (broad s, 1H), 3.2 (q,
2H), 1.1 (t, 3H). MS, m/z: 283 (M + H)+; 281 (M - H)-.
Methyl 1-(5-Amino-2-fluoro-4-nitrophenyl)-1H-imidazole-4-car-
boxylate (28). To a solution of 27 (54.31 g, 0.312 moles) and 26
(43.27 g, 0.343 moles) in DMF (1.3 L) was added solid sodium
carbonate (36.4 g, 0.343 moles), and the mixture was heated to
100 °C for 7 h under N2 atmosphere and then stirred overnight at
ambient temperature. The mixture was then poured into water/ice
(5 L), resulting in a precipitate. The solids were stirred vigorously
at 0 °C for 20 min, then filtered. Solids were washed with water
and dried in vacuo at 50 °C to give 28 (82.5 g, 97.4% yield) as a
yellow solid. 1H NMR (500 MHz, DMSO-d6): δ 8.28 (s, 1H), 8.20
(s, 1H), 8.05 (d, 1H), 7.51 (s, 2H), 7.29 (d, 1H), 3.81 (s, 3H). MS,
m/z: 281(M + H)+.
1-(2-(3-Ethylureido)-6-fluoro-1H-benzo[d]imidazol-5-yl)-1H-imi-
dazole-4-carboxylic Acid (31). A suspension of 28 (4.2 g, 0.015
moles) and Raney nickel (0.42 g) in methanol (150 mL) was placed
under 45 psi of hydrogen gas on a Parr shaker for 4 h. The mixture
was then filtered through Celite to give a clear solution. The solids
were washed with MeOH. The combined filtrates were concentrated
in vacuo to the intermediate phenylenediamine 29 (3.3 g, 88% yield
crude) which was then diluted with pH ∼3.5 buffer (1 N H2SO4
buffered with NaOAc) and treated with solid reagent B (4.60 g,
0.0198 mol). The resulting mixture was heated to 100 °C for 5 h,
then returned to ambient temperature. The reaction pH was adjusted
to ∼7 with addition of aqueous NaHCO3 resulting in the precipita-
tion of the methyl ester of crude 30. The solids were taken up in
6 N HCl (50 mL) and heated to reflux. After 2.5 h the ester
hydrolysis was complete. The mixture was cooled to ambient
a (a) Cyclopropylamine, dioxane, sealed tube, 80 °C.
gel chromatography (1:0 to 49:1 CH2Cl2/MeOH gradient) to afford
1
23c (0.21 g, 66% yield) as a yellow solid. H NMR: (500 MHz,
CDCl3): δ 9.2 (s, 1H), 8.9 (s, 2H), 8.4 (s, 1H), 7,6 (d, 1H), 6.9 (d,
1H), 6.2 (broad s, 2H). MS, m/z: 217 (M + H)+; 215 (M - H)-.
4-Pyrimidin-5-ylbenzene-1,2-diamine (24c). A solution of 23c
in EtOAc (30 mL) and EtOH (30 mL) in a Parr bottle was treated
with 5% Pd-C (0.1 g) before pressurizing to 45 psi with hydrogen.
After 3 h, the mixture was filtered through a syringe filter disk and
concentrated to give 24c (0.18 g, 100% yield) as yellow solid. MS,
m/z: 187 (M + H)+.
1-Ethyl-3-(5-pyrimidin-5-yl-1H-benzoimidazol-2-yl)urea (4). To
a solution of 24c (0.66 g, 0.35 mmol) in a mixture of water (5 mL)
and p-dioxane (5 mL) was added a solution of reagent A (0.5 mmol)
which had been preacidified to pH 3 with 3 M H2SO4. The mixture
was refluxed for 16 h, returned to ambient temperature, and diluted
with water producint a precipitate. The solids were filtered and
further washed with water, then concentrated to dryness to give 4
(0.05 g, 50% yield) as an off-white. Purity, method A 98%; method
1
B 98%. H NMR (500 MHz, DMSO-d6): 10.0 (broad s, 1H), 9.1

Benzimidazole Urea Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5255
temperature and concentrated in vacuo. The resulting product was
azeotroped thrice with ethanol and pumped dry to yield 31 (3.0 g,
60% yield) as an off-white solid. 1H NMR (500 MHz, DMSO-d6):
δ 11.9 (s, 1H), 10.08 (s, 1H), 8.20 (s, 1H), 8.05 (s, 1H), 7.55 (d,
1H), 7.42 (d, 1H), 7.03 (m, 1H), 3.8 (s, 3H), 3.21 (dt, 2H), 1.13 (t,
3H). MS, m/z: 333 (M + H)+.
5-Methyl-4-(pyridin-3-yl)-2-nitroaniline (42a). To a solution of
41a (347 mg, 1.5 mmol) in DMSO (10 mL) was added pyridine-
3-boronic acid pinacol ester (203 mg, 1.65 mmol), potassium
phosphate (1.59 g, 5 mmol), and tetrakis(triphenylphosphine)-
palladium (0.1 equiv). The resulting mixture was heated at 95 °C
for 72 h, then returned to ambient temperature and quenched with
water (10 mL). The resulting crude mixture was extracted thrice
with EtOAc, the combined organic extracts were then dried over
Na2SO4, filtered and concentrated in vacuo, and the residue was
purified by silica gel chromatography (1:1 hexanes/EtOAc) to afford
42a (140 mg, 41% yield) as a yellow solid. MS, m/z: 230 (M +
H)+.
5-Methyl-4-pyridin-3-yl-benzene-1,2-diamine (43a). To a solution
of compound 42a (140 mg, 0.61 mmol) in ethyl acetate (20 mL)
was added 10% palladium on carbon (100 mg). The resulting
suspension was placed in a Parr hydrogenation apparatus under 40
psi of hydrogen gas while shaking at ambient temperature for 2 h.
The catalyst was removed by filtration and the filtrate concentrated
in vacuo to afford compound 43a (83 mg, 62% yield) as a white
solid. MS, m/z: 200 (M + H)+.
6-Methyl-5-(pyridin-3-yl)-1H-benzo[d]imidazol-2-amine (44a).
To a solution compound 43a (83 mg, 0.42 mmol) in MeCN/MeOH/
water (5/4/1, 100 mL) was added BrCN (0.5 mmol from 5 M
solution in MeCN), and the resulting mixture was stirred at ambient
temperature for 18 h. The mixture was partitioned between EtOAc
and 1 N NaOH, and the organic phase was sequentially washed
with water and brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The crude product was purified by silica gel chromatog-
raphy (1:0 to 9:1 EtOAc/MeOH gradient) to afford compound 44a
(40 mg, 43% yield) as a light-brown solid.
1-(5-(4-(Cyclopropylcarbamoyl)-1H-imidazol-1-yl)-6-fluoro-1H-
benzo[d]imidazol-2-yl)-3-ethylurea (5). To a solution of 31 (400
mg, 1.2 mmol) and diisopropylethylamine (0.5 mL, 2.88 mmol) in
DMF (1.6 mL) were added cyclopropylamine (0.12 mL, 1.8 mmol)
and HBTU (680 mg, 1.8 mmol), and the resulting mixture was
stirred at ambient temperature for 15 h. The mixture was then
poured onto water (30 mL), resulting in a precipitate that was
vigorously stirred for 1 h. The heterogeneous mixture was then
filtered, and the solids were washed with copious amounts of water.
The solids were then dried in vacuo, then washed sequentially with
toluene, then hexanes providing 5 (200 mg, 45% yield) as a white
solid. Purity, method A 97%; method B 97%. 1H NMR (500 MHz,
DMSO-d6): δ 11.9 (m, 1H), 10.06 (s, 1H), 8.20 (s, 1H), 8.05 (s,
1H), 7.55 (m, 1H), 7.41 (d, J ) 10.8 Hz, 1H), 6.98 (m, 1H), 3.23
(dq, J ) 11.7, 9.0 Hz, 2H), 2.84 (m, 1H), 1.12 (dd, J ) 9.0 Hz,
3H), 0.65 (m, 2H), 0.60 (m, 2H). MS, m/z: 372 (M + H)+
5-Bromo-4-fluoro-2-nitroaniline (38). To a solution of 37 (1 g,
4.2 mmol) in DMF (10 mL) was added (NH4)2CO3 (2 g, 21 mmol).
The resulting mixture was heated at 90 °C for 18 h, then cooled to
ambient temperature and quenched with water (10 mL). The product
was extracted thrice with EtOAc. The combined organic extracts
were dried over Na2SO4, filtered, concentrated in vacuo and the
residue was purified by silica gel chromatography (7:3, hexanes/
1
EtOAc) to afford 38 (600 mg) as a yellow solid. H NMR (500
1-Ethyl-3-(6-methyl-5-pyridin-3-yl-1H-benzoimidazol-2-
yl)urea (7). To a solution compound 44a (40 mg, 0.4 mmol) in
THF (5 mL) was added ethyl isocyanate (28 µL, 0.3 mmol). The
resulting mixture was heated to reflux overnight then returned to
ambient temperature and concentrated in vacuo. The crude product
was directly purified by silica gel chromatography (1:0 to 19:1
EtOAc/MeOH gradient) to afford compound 7 (5 mg, 9% yield)
as a white solid. Purity, method A 92%; method B 92%. 1H NMR
(500 MHz, CD3CN): δ 8.65 (m, 2H), 8.08 (dd, J ) 8.0 Hz, 1H),
7.69 (dd, J ) 7.8, 5.3 Hz, 1H), 7.46 (s, 1H), 7.38 (s, 1H), 3.62 (m,
2H), 2.89 (m, 2H), 2.25 (s, 3H), 1.14 (t, J ) 7.4 Hz, 3H), 1.07 (t,
J ) 7.2 Hz, 3H). MS, m/z: 296 (M + H)+.
5-Methoxyl-4-(pyridin-3-yl)-2-nitroaniline (42b). To a solution
of 41b (495 mg, 2 mmol) in DMSO (10 mL) was added pyridine-
3-boronic acid 1,3-propanediol cyclic ester (342 mg, 2.1 mmol),
potassium phosphate (2.1 g, 10 mmol), and tetrakis(triphenylphos-
phine)palladium (0.1 equiv). The resulting mixture was heated at
95 °C for 48 h, then returned to ambient temperature and quenched
with water (10 mL). The crude mixture was extracted thrice with
EtOAc, the combined organic extracts were then dried over Na2SO4,
filtered, concentrated in vacuo, and the residue was purified by silica
gel chromatography (EtOAc) to afford 42b (450 mg, 46% yield)
as a yellow solid. MS, m/z: 246 (M + H)+.
MHz, CDCl3): δ 7.90 (d, 1H), 7.10 (d, 1H), 6.0 (s, 2H). MS, m/z:
245(M + H)+.
5-Methoxyl-4-(pyridin-3-yl)-2-nitroaniline (39). To a solution of
38 (286 mg, 1.2 mmol) in DMSO (10 mL) was added pyridine-
3-boronic acid (177 mg, 1.44 mmol), potassium phosphate (1.3 g,
6 mmol), and tetrakis(triphenylphosphine)palladium (0.1 equiv). The
resulting mixture was heated at 95 °C for 24 h, returned to ambient
temperature, then quenched with water (10 mL). The product was
extracted thrice with EtOAc, the combined organic extracts dried
over Na2SO4 and then concentrated in vacuo, and the residue was
purified by silica gel chromatography (7:3 to 1:1 hexanes/EtOAc
gradient) to afford 39 (120 mg, 41% yield) as a yellow solid. MS,
m/z: 234 (M + H)+.
6-Fluoro-5-pyridin-3-yl-1H-benzoimidazol-2-ylamine (40). To a
solution of compound 39 (120 mg, 0.52 mmol) in ethyl acetate
(20 mL) was added 10% palladium on carbon (catalytic amount).
The resulting suspension was placed in a Parr hydrogenation
apparatus under 40 psi of hydrogen gas while shaking at ambient
temperature for 2 h. The catalyst was removed by filtration and
the filtrate concentrated in vacuo to afford the reduced product (75
mg, 0.37 mmol) as an off-white solid. MS, m/z: 204 (M + H)+.
To the above reduced product (0.37 mmol) in MeCN/MeOH/water
solution (5/4/1, 10 mL) was added BrCN (0.44 mmol, 1.2 equiv),
and the resulting mixture was stirred at ambient temperature for
18 h. The mixture was then partitioned between EtOAc and 1 N
NaOH, and the organic phase was washed sequentially with water
and brine, dried over Na2SO4, filtered, and concentrated in vacuo.
The crude product was purified by silica gel chromatography (1:0
to 49:1 EtOAc/MeOH gradient) to afford compound 40 (25 mg,
90% yield) as a white solid. MS, m/z: 229 (M + H)+.
5-Methoxyl-4-pyridin-3-ylbenzene-1,2-diamine (43b). To a solu-
tion of compound 42b (450 mg, 1.84 mmol) in ethyl acetate (20
mL) was added 10% palladium on carbon (100 mg). The resulting
suspension was placed in a Parr hydrogenation apparatus under 40
psi of hydrogen gas while shaking at ambient temperature for 2 h.
The catalyst was removed by filtration, and the filtrate concentrated
in vacuo to afford compound 43b (290 mg, 73% yield) as a white
solid. MS, m/z: 216 (M + H)+.
1-Ethyl-3-(6-fluoro-5-pyridin-3-yl-1H-benzoimidazol-2-yl)urea
(6). To a solution compound 40 (25 mg, 0.1 mmol) in THF (5 mL)
was added ethyl isocyanate (0.13 mmol). The resulting mixture was
heated to reflux overnight, returned to ambient temperature, then
concentrated in vacuo. The crude product was purified by prepara-
tive HPLC to afford compound 6 (6 mg) as a white solid. Purity,
7-Methoxy-5-pyridin-3-yl-1H-benzoimidazol-2-ylamine (44b). To
a solution compound 43b (140 mg, 0.65 mmol) in MeCN/MeOH/
water (5/4/1, 100 mL) was added BrCN (0.78 mmol from 5 M
solution in MeCN), and the resulting mixture was stirred at ambient
temperature for 18 h. The mixture was partitioned between EtOAc
and NaOH (1 N) and the aqueous phase extracted thrice with
EtOAc. The combined organic extracts were sequentially washed
with water and brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The crude product were purified by silica gel chroma-
tography (1:0 to 9:1 EtOAc/MeOH gradient) to afford 44b (95 mg,
1
method A 97%; method B 97%. H NMR (500 MHz, CDCl3): δ
8.79 (s, 1H), 8.61 (d, J ) 5.1 Hz, 1H), 8.17 (d, J ) 7 Hz, 1H),
7.63 (m, 2H), 7.43 (d, J ) 10.1 Hz, 1H), 6.30 (s, 1H, NH), 3.20
(m, 2H), 1.07 (t, J ) 7.2 Hz, 3H). MS, m/z: 300 (M + H)+.

5256 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Charifson et al.
61%) as a pale-yellow solid. 1H NMR (500 MHz, CD3CN): δ 8.77
(d, 1H), 8,54 (m, 1H), 7.95 (m, 1H), 7.43 (m, 1H), 7.23 (s, 1H),
7.07 (s, 1 H), 5.12 (m, 2H), 3.86 (t, 3H). MS, m/z: 241 (M + H)+.
1-Ethyl-3-(7-methoxyl-5-pyridin-3-yl-1H-benzoimidazol-2-
yl)urea (8). To a solution compound 44b (95 mg, 0.4 mmol) in
THF (1 mL) was added ethyl isocyanate (63 µL, 0.8 mmol). The
resulting mixture was heated to reflux overnight, returned to ambient
temperature, concentrated in vacuo, then treated with 7 N NH3 in
MeOH (3 mL) at 55 °C for 1 h. The ambient temperature reaction
mixture was concentrated in vacuo and purified by silica gel
chromatography (1:0 to 19:1 EtOAc/MeOH gradient) to afford
compound 8 (42 mg, 34% yield) as a white solid. Purity, method
followed by acetone, and then dried under high vacuum giving 17
(17.0 g, 90% yield) as a tan solid. Purity, method A 92%; method
B 92%. 1H NMR (500 MHz, DMSO-d6): δ 11.40-11.75 (very
broad s, 1H), 9.76-10.04 (very broad s, 1H), 8.68 (s, 2H), 7.33 (s,
1H), 7.23-7.31 (broad s, 1H), 7.14 (s, 1H), 3.95 (s, 3H), 3.77 (s,
3H), 3.21 (quintet, J ) 7 Hz, 2H), 1.11 (t, J ) 7 Hz, 3H). MS,
m/z: 343 (M + H)+; 341 (M - H)-.
1-Bromo-2,4-difluoro-5-nitrobenzene (33). A 0 °C solution of
32 (3.0 g, 15.5 mmol) in concentrated sulfuric acid (16 mL) was
treated with potassium nitrate (1.60 g, 15.7 mmol) portionwise and
the reddish mixture stirred at 0 °C for 5 min and then at ambient
temperature for 3 h. The mixture was carefully poured directly onto
ice, then extracted with ethyl acetate. The organic phase was washed
with brine, dried over anhydrous Na2SO4, filtered, and evaporated
in vacuo to give (3.66 g, 99%) of crude product as a brown oil
which was used directly in the next step. 1H NMR (500 MHz,
CDCl3): δ 8.40 (t, 1 H), 7.14 (dd, 1 H). The intermediate bromo-
2,4-difluoro-5-nitrobenzene (3.35 g, 14.0 mmol) was dissolved in
0.5 N ammonia in dioxane (30 mL, 15 mmol) and the mixture
heated in a sealed tube at 70 °C for 48 h. The mixture was cooled
to ambient temperature, then evaporated in vacuo to give crude
compound 33 as a yellow solid which was not purified further. 1H
NMR (500 MHz, CDCl3): δ 8.40 (d, 1H), 6.6 (d, 1H), 6.2 (broad
s, 2H).
4-Bromo-2-nitro-5-(piperidin-1-yl)benzenamine (34). A solution
of 33 (500 mg, 2.13 mmol) in CH3CN (5 mL) was treated with
excess piperidine (1.05 mL, 10.7 mmol) and the yellow mixture
heated at 80 °C for 2 h and then returned to ambient temperature.
The mixture was partitioned between EtOAc and saturated NaHCO3
and the organic layer washed with brine, dried over Na2SO4, filtered,
and evaporated in vacuo to give 583 mg of an orange oil. The crude
product was purified by silica gel chromatography (4:1 hexanes/
ethyl acetate) to give 34 (450 mg, 70%) as a yellow solid. 1H NMR
(500 MHz, DMSO-d6): δ 8.10 (s, 1H), 7.5 (broad s, 2H), 6.55 (s,
1H), 3.0 (m, 4H), 1.7 (m, 4H), 1.55 (m, 2H) ppm.
1
A 90%; method B 90%. H NMR (CD3CN): δ 8.70 (d, J ) 1.6
Hz, 1H), 8.48 (dd, J ) 4.8, 1.6 Hz, 1H), 7.87 (m, 1H), 7.36 (m,
1H), 7.13 (s, 1H), 3.80 (s, 3H), 3.28 (m, 2H), 1.15 (t, J ) 7.2 Hz,
3H). MS, m/z: 312 (M + H)+.
4-Methoxy-5-(2-methoxypyrimidin-5-yl)-2-nitroaniline (42c). To
a mechanically stirring suspension of 2-methoxy-5-(4,4,5,5-tetram-
ethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (119 g, 0.506 mol) and
41c (125 g, 0.506 mol) in 1,2-dimethoxyethane (506 mL) was added
trans-dichlorobis(triphenylphosphine)palladium(II) (36 g, 0.0506
mol) followed by saturated aqueous NaHCO3 (∼1 M, 759 mL,
0.759 mol). The reaction mixture was stirred at reflux for 6 h
(analyzed by HPLC), diluted with water (1.3 L), cooled using an
ice-water bath while stirring for 30 min and then allowed to stand
at 5 °C for 2 h, and collected the precipitate by filtration. The solid
was washed with water until neutral pH, washed with hexane, and
dried under high vacuum. The resulting solids were then slurried
in CHCl3, stirred at reflux for 1.5 h, allowed to cool to ambient
temperature, then collected by filtration, washed with CHCl3, and
dried under high vacuum to give 42c (101 g, 72% yield) as an
orange powdery solid. 1H NMR (500 MHz, CDCl3): δ 8.69 (s, 2H),
7.67 (s, 1H), 6.78 (s, 1H), 5.98 (broad s, 2H), 4.08 (s, 3H), 3.83 (s,
3H).
4-Methoxy-5-(2-methoxypyrimidin-5-yl)benzene-1,2-diamine (43c).
To a mechanically stirring orange suspension of 42c (6.15 g, 22.28
mmol) and methanol (22 mL) was added triethylamine (13.9 mL,
100.26 mmol) followed by 10% palladium on activated carbon (50%
wet, 0.22 g). Formic acid (96%, 3.8 mL, 95.80 mmol) was added
slowly via dropping funnel over 5-10 min while the mixture was
simultaneously slowly heated. After complete addition, stirring was
continued at reflux for 2 h (all of the orange color had disappeared,
leaving a greenish black solution, and TLC indicated that the
reaction was complete). It was allowed to cool, diluted with
methanol, filtered, and the filtrate was concentrated under reduced
pressure. The residue was dissolved in a minimal amount of aqueous
2 N HCl and washed thrice with CH2Cl2. The stirring aqueous phase
was then neutralized (pH 5-8) with slow addition of solid NaHCO3,
giving rise to a light-brown precipitate which was extracted five
times with CH2Cl2. The combined organic extracts were washed
with brine, dried over Na2SO4/MgSO4, filtered, and concentrated
in vacuo to give 43c (5.15 g, 94% yield) as a light greenish brown
solid. 1H NMR (500 MHz, DMSO-d6): δ 8.57 (s, 2H), 6.55 (s,
1H), 6.37 (s, 1H), 4.82 (broad s, 2H), 4.18 (broad s, 2H), 3.92 (s,
3H), 3.62 (s, 3H).
1-Ethyl-3-(6-methoxy-5-(2-methoxypyrimidin-5-yl)-1H-benzo[d]im-
idazol-2-yl)urea (17). To 43c (13.6 g, 55.3 mmol) in a three-neck
round-bottom flask (leaving plenty of head space for frothing) was
added a solution of concentrated H2SO4 (4.6 mL, 165.9 mmol) in
water (130 mL), giving rise to a slight exotherm. To this
mechanically stirring suspension was added a mixture of the
monosodium salt of 1-cyano-3-ethylurea reagent A (14.9 g, 110.6
mmol) in water (60 mL + 27 mL rinse). An immediate mild
frothing was observed which lasted only momentarily. The reaction
mixture was stirred vigorously at a gentle reflux overnight (it was
very important to adjust the pH to between 3 and 6). When the
mixture was heated, the solids went into solution and then an off-
white tan precipitate formed giving rise to substantial frothing.
When the reaction was complete (as analyzed by HPLC and
LCMS), it was allowed to cool to ambient temperature and the
precipitated solid collected by filtration, washed with water,
2-Nitro-5-(piperidin-1-yl)-4-(pyridin-3-yl)benzenamine (35). A
solution of 34 (450 mg, 1.5 mmol) and pyridine-3-boronic acid
pinacol ester (318 mg, 1.95 mmol) in DME (6 mL) and saturated
NaHCO3 (3 mL, 3.0 mmol) was treated with Pd(PPh3)4 (87 mg,
0.075 mmol) and the biphasic suspension heated at 80 °C for 12 h.
The mixture was partitioned between CH2Cl2 and saturated NaH-
CO3, the organic phase was washed with brine, dried over
anhydrous Na2SO4, and evaporated in vacuo to give 572 mg of a
brown oil. Crude product was chromatographed on silica gel (9:1
to 4:1 CH2Cl2/EtOAc gradient) to give 35 (373 mg, 83%) as a
yellow solid. 1H NMR (500 MHz, DMSO-d6): δ 8.80 (s, 1H), 8.55
(m, 1H), 7.95 (m, 1H), 7.85 (s, 1H), 7.6 (s, 2H), 7.4 (m, 1H), 6.5
(s, 1H), 2.75 (m, 4H), 1.4 (m, 6H).
6-(Piperidin-1-yl)-5-(pyridin-3-yl)-1H-benzo[d]imidazol-2-
amine (36). A suspension of 35 (273 mg, 0.92 mmol) in methanol
(4 mL) and acetic acid (2 drops) was treated with 10% Pd/C (70
mg) and the black suspension shaken on a Parr apparatus under 45
psi of hydrogen for 12 h. Catalyst was removed by filtration, and
the filtrate was reduced in volume to give a solution of crude
diamine which was used immediately in the next step. A solution
of the crude diamine (∼0.9 mmol) in methanol (4 mL) was treated
with cyanogen bromide (3.7 mmol from 5 M solution in MeCN)
and the red-brown mixture stirred at ambient temperature under
an N2 atmosphere for 25 min. The reaction mixture was added
directly, purified by silica gel and chromatography (95:5 CH2Cl2/
MeOH, to 93:7 CH2Cl2:7N NH3 in MeOH gradient) to give 36 (66
mg, 26% yield) as an oily brown solid. MS, m/z: 294 (M + H)+
1-Ethyl-3-(6-(piperidin-1-yl)-5-(pyridin-3-yl)-1H-benzo[d]imida-
zol-2-yl)urea (9). A solution 36 (66 mg, 0.24 mmol) in dry CH3CN
(0.5 mL) was treated with excess ethyl isocyanate (200 µL) and
the brown mixture heated at 70 °C for 3 h. The mixture was
evaporated in vacuo to give crude bis-urea as an oil which was
then immediately treated with 7 N NH3 in MeOH (2 mL) and heated
at 70 °C for 1 h. The mixture was returned to ambient temperature,
evaporated in vacuo and then purified by column chromatography
on silica gel (98:2 to 96:4 to 95:5 CH2Cl2/MeOH gradient) to give

Benzimidazole Urea Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5257
28 mg of product as an off-white solid. Crude product was
suspended in water, stirred for 20 min, then collected by filtration,
rinsed twice with water, and dried in vacuo to give 9 (22 mg, 26%)
as a white solid. Purity, method A 94%; method B 94%. 1H NMR
(500 MHz, DMSO-d6): δ 11.40 (broad s, 1H), 9.8 (broad s, 1H),
8.90 (1H, s), 8.50 (dd, J ) 1.8, 2.6 Hz, 1H), 8.0 (dt, J ) 1.6, 8.4
Hz, 1H), 7.4 (dd, J ) 2.2, 7.4 Hz, 1H), 7.3 (broad s, 1H), 7.2 (s,
1H), 7.1 (s, 1H), 3.2 (q, J ) 8.4 Hz, 2H), 2.7 (m, 4H), 1.3 (m,
6H), 1.1 (t, J ) 8.4 Hz, 3H). MS, m/z: 365 (M + H)+.
(0.0022 g, 21% yield) as a white solid. Purity, method A 98%;
method B 98%. 1H NMR (500 MHz, CD3OD): δ 9.16 (d, J ) 1.6
Hz, 1H), 8.79-8.85 (m, 2H), 8.19 (d, J ) 1.2 Hz, 1H,), 8.09-.10
(m, 1H), 8.02 (d, J ) 1.2 Hz, 1H), 3.33 (q, J ) 14.5 and 7.3 Hz,
2H), 3.31 (s, 3H), 1.22 (t, J ) 7.3 Hz, 3H). MS, m/z: 339 (M +
H)+; 337 (M - H)-.
2-Amino-5-bromo-3-nitrophenol (50). To a stirred solution of
49 (3.0 g, 19.5mmol) in acetic acid (45 mL) was added bromine
(3.1 g, 19.5mmol) in acetic acid (25 mL) over 1 h. The resulting
mixture was allowed to stir for 18 h under nitrogen at ambient
temperature. The mixture was made homogeneous with the addition
of ethanol (100 mL) followed by the addition of ethyl acetate (300
mL). The solution was then neutralized with the addition of 2 N
NaOH and then reacidified to pH 3 with the addition of 6 N HCl.
The organic phase was washed with brine, dried over Na2SO4,
filtered, and the resulting filtrate was concentrated to dryness under
Methyl-2-amino-5-bromo-3-nitrobenzoate (46). To a 0-5 °C
solution of trifluoroacetic anhydride (60 mL) was slowly added 45
(5 g, 21.73 mmol), resulting in a beige precipitate formed during
the addition. After the mixture was stirred for 15 min, solid
potassium nitrate was added in (2.64 g, 26.08 mmol) in one portion.
The ice bath was then removed, and the resulting mixture was
allowed to stir overnight at ambient temperature. The resulting crude
reaction was then concentrated to dryness, dissolved in ethyl acetate,
and carefully neutralized with excess sodium bicarbonate. The
organic layer was washed with brine, dried over Na2SO4, filtered,
then concentrated to dryness. The resulting crude solid (6.39 g)
was heated to 65 °C in a mixture of methanol (150 mL) and 6 N
hydrochloric acid (50 mL) for 3 h. The mixture was cooled to
ambient temperature and concentrated to dryness to provide 46 (3.5
g, 59% yield) as a light-yellow solid. 1H NMR (500 MHz, CDCl3):
δ 8.52 (s, 1H), 8.43 (s, 1H), 3.94 (s, 3H). MS, m/z: 275 (M + H)+.
Methyl-2-amino-3-nitro-5-(pyridine-3-yl)benzoate (47). To a
solution of 46 (0.4 g, 1.095mmol) in dimethoxyethane (8 mL) were
added pyridin-3-ylboronic acid (0.2 g, 1.64 mmol), tetrakis(tri-
phenylphospine)palladium (0.125 g, 0.109mmol), and 1 M aqueous
sodium bicarbonate (2.18 mL, 2.18mmol). The resulting mixture
was refluxed overnight, then diluted with saturated sodium bicar-
bonate and extracted thrice with ethyl acetate. The combined organic
extracts were subsequently washed with brine, dried over Na2SO4,
filtered, concentrated in vacuo, and purified by column chroma-
tography on silica gel (1:0 to 0: EtOAc/CH2Cl2 gradient) to provide
47 (0.169 g, 57% yield) as a pale-yellow solid. 1H NMR (500 MHz,
CDCl3): δ 8.81 (s, 1H), 8.67 (s, 1H), 8.63 (d, 1H), 8.52 (s, 1H),
7.88 (d, 1H), 7.41 (dd, 1H), 3.96 (s, 3H). MS, m/z: 274 (M + H)+.
Methyl-2-(3-ethylureido)-5-(pyridine-3-yl)-1H-benzo[d]imidazole-
7-carboxylate (10). A slurry of 10% palladium on carbon (0.035
g) and 47 (0.167 g, 0.611mmol) in ethanol was subjected to 40 psi
of H2 in a Parr shaker for 4 h. The depressurized mixture was then
filtered through Celite and concentrated to dryness to provide the
corresponding phenylenediamine (0.110 g) which was then sus-
pended in water buffered to pH 3 with 1 N sulfuric acid and treated
with reagent A. The mixture was refluxed overnight, cooled to
ambient temperature, and the resulting solids were collected via
filtration. Further purification by column chromatography on silica
gel (100% ethyl acetate, then switching to 9:1 CH2Cl2/7 N ammonia/
methanol) provided 0.055 g of material that was dissolved in
methanol and reprecipitated with diethyl ether to provide 10 (0.01
g, 5% yield) as a white solid. Purity, method A 98%; method B
98%. 1H NMR (400 MHz, DMSO-d6): δ 11.5 (broad s, 1H), 9.89
(broad. s, 1H), 8.91 (broad s, 1H), 8.56 (dd, J ) 4.7 and 1.5 Hz,
1H), 8.11 (d, J ) 8.1 Hz, 1H), 8.01 (s, 1H), 7.9 (d, J ) 1.7 Hz,
1H), 7.40-7.51 (m, 2H), 3.96 (s, 3H), 3.25 (q, J ) 7.0, 5.6 Hz,
2H), 1.13 (t, J ) 7.2 Hz, 3H). MS, m/z: 340 (M + H)+; 338 (M -
H)-.
1
vacuum to provide 50 (4.35 g, 82% yield) as a yellow solid. H
NMR (500 MHz, CDCl3): δ 5.6 (broad s, 1H), 6.35 (broad s, 2H),
7.00 (s, 1H), 7.91 (s, 1H).
2-(Benzyloxy)-4-bromo-6-nitrobenzenamine (51). To a stirred
mixture of 50 (0.33 g, 1.2mmol) and potassium carbonate (0.46 g,
3.1mmol) was added benzyl bromide (0.21 g, 1.2mmol). The
mixture was allowed to stir under nitrogen at ambient temperature
for 18 h. The mixture was partitioned between ethyl acetate and
water. The organic phase was washed 5 times with water, brine,
dried over Na2SO4, filtered, then concentrated to dryness under
vacuum. The resulting residue was purified by column chromatog-
raphy on silica gel using (19:1 hexanes/EtOAc) to give 51 (0.21 g,
53% yield) as a dark-yellow solid. 1H NMR (500 MHz, CDCl3): δ
5.07 (s, 2H), 6.44 (broad s, 2H), 7.02 (s, 1H), 7.42 (m, 5H), 7.89
(s, 1H).
2-(Benzyloxy)-6-nitro-4-(pyridin-3-yl)benzenamine (52). To a
suspension of 51 (0.21 g, 0.64mmol), pyridine-3-boronic acid-1,
3-propanediolcyclic ester (0.14 g, 0.84mmol), and 1 M sodium
bicarbonate (1.3 mL) was added tetrakistriphenylphosphine pal-
ladium (0.07 g, 0.06 mmol) in ethylene glycol dimethyl ether. The
mixture was heated to reflux and allowed to stir for 5 h, then cooled
to ambient temperature. The mixture was diluted with ethyl acetate
and filtered through Celite. The filtrate was sequentially washed
with saturated sodium bicarbonate, water, and brine, then dried over
Na2SO4, filtered, and concentrated in vacuo. The resulting residue
was purified by column chromatography on silica gel 95.5:0.5
CH2Cl2/MeOH) to give 52 (0.10 g, 50% yield) as a dark-yellow
solid. 1H NMR (500 MHz, MeOH-d4): δ 5.24 (s, 2H), 7.21 (s, 1H),
7.34-7.5 (m, 6H), 7.91 (d, 1H), 8.5 (d, 1H), 8.71 (s, 1H).
7-(Benzyloxy)-5-(pyridin-3-yl)-1H-benzo[d]imidazol-2-amine (53).
To a solution of 52 (0.10 g, 0.31mmol) in ethanol (15 mL) and
water (2 mL) at 70 °C was added sodium sulfite (0.43 g, 2.49mmol)
in water (2 mL). The mixture was allowed to stir for 30 min, then
cooled to ambient temperature. The solvent was evaporated under
reduced pressure, and the resulting residue was purified by column
chromatography on silica gel (0.5:5.0:94.5 NH4OH/MeOH/CH2Cl2).
The resulting diamine (0.07 g, 0.23mmol) was dissolved in
acetonitrile (4 mL) and methanol (1 mL), and to it was added
cyanogen bromide (0.46 mmol from 5 M solution in MeCN). The
resulting mixture was allowed to stir for 2 h. The solvent was
evaporated under reduced pressure and the resulting residue was
purified by column chromatography on silica gel (95:5 CH2Cl2/7
N ammonia in methanol) to provide 53 (0.05 g, 46% yield) as an
off-white solid. 1H NMR (500 MHz, MeOD): δ 5.28 (s, 2H), 6.84
(s, 1H), 7.13 (s, 1H), 7.32-7.45 (m, 4H), 7.50 (d, 2H), 7.91 (d,
1H), 8.48 (d, 1H), 8.76 (s, 1H).
2-(3-Ethylureido)-5-(pyridine-3-yl)-1H-benzo[d]imidazole-7-car-
boxylate (48). A solution of 10 (0.143 g, 0.421mmol) in 6 N
hydrochloric acid (5 mL) was refluxed for 16 h, then cooled to
ambient temperature and concentrated in vacuo to provide 48 (0.128
g, 94% yield) as a white solid.
2-(3-Ethylureido)-N-methyl-5-(pyridine-3-yl)-1H-benzo[d]imida-
zole-7-carboxamide (11). To an ambient temperature mixture of
48 (0.0125 g, 0.031mmol) in dimethylformamide (1 mL) was added
bromo tris-pyrolidino-hexafluorophosphate (0.029 g, 0.063mmol),
methyamine hydrochloride (0.263 g, 0.094 mmol), diisopropyl-
ethylamine (0.32 mL, 0.188mmol), and a catalytic amount of
dimethylaminopyridine. After being stirred overnight, the mixture
was directly submitted for reverse phase HPLC to provide 11
1-(7-(Benzyloxy)-5-(pyridin-3-yl)-1H-benzo[d]imidazol-2-yl)-3-
ethylurea (16). To a mixture of 53 (0.05 g, 1.5mmol) in acetonitrile
(5 mL) was added an excess of ethyl isocyanate (1 mL). The
mixture was heated to 70 °C and allowed to stir for 3 h. The mixture
was returned to ambient temperature, concentrated in vacuo, and
the resulting residue was suspended in 7 N ammonia in methanol
(5 mL). The mixture was heated to 60 °C and allowed to stir for
3 h, then cooled to ambient temperature and concentrated in vacuo.
The resulting residue was purified by column chromatography on

5258 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Charifson et al.
silica gel (97:3 CH2Cl2/7 N ammonia-methanol). The chromato-
graphed material was the triturated with water, filtered, and dried
under high vacuum to give 16 (0.05 g, 86% yield) as an off-white
added dropwise until the pH was approximately 3, then heated to
reflux and allowed to stir for 18 h. The mixture was cooled to
ambient temperature, resulting in a heterogeneous mixture. The
solvent was decanted, and the remaining solids were triturated twice
with water. The resulting solid was further purified by preparative
HPLC (10-90% gradient of acetonitrile-water with 0.1% TFA),
then converted to the bis-HCl salt by dissolving purified product
in 2 N hydrochloric acid (1.5 mL) and methanol (5 mL) and stirring
the mixture for 5 min. The solvent was removed in vacuo and
residual water was removed azeotropically with methanol to give
19 (24 mg, 32% yield as bis-HCl salt) as an off-white solid. Purity,
method A 96%; method B 96%. 1H NMR (500 MHz, DMSO-d6):
δ 0.62 (m, 2H), 0.71 (m, 2H), 1.11 (t, J ) 7.2 Hz, 3H), 2.89 (m,
1H), 3.23 (q, J ) 6.7 Hz, 2H), 6.69 (t, J ) 2.3 Hz, 1H), 7.4 (broad
s, 1H), 7.67 (s, 1H), 7.95 (s, 1H), 8.07 (m, 1H), 8.52 (m, 2H), 8.89
(broad s, 1H), 9.07 (s, 1H), 10.66 (broad s, 1H).
5-Bromo-1,3-difluoro-2-nitrobenzene (61). To a suspension of
sodium perborate tetrahydrate (1.04 g, 5 mmol) in acetic acid (20
mL), stirred at 55 °C, was added a solution of 60 in acetic acid (10
mL) over 1 h in a dropwise fashion. After being stirred at 55 °C
for an additional 3 h, the solution was allowed to cool to ambient
temperature and filtered. The filtrate was poured into ice and
extracted twice with ethyl acetate. The combined organic extracts
were washed successively with water (5 times), brine, dried over
MgSO4, and concentrated in vacuo. The resulting residue was
purified by column chromatography on silica gel (20:1 hexanes/
EtOAc) to provide 61 (780 mg, 66% yield) as a tan solid. 1H NMR
(500 MHz, CDCl3): δ 7.32 (dt, 2H).
1
solid. Purity, method A 98%; method B 98%. H NMR (MeOH-
d4): δ 1.20 (t, J ) 7.2 Hz, 3H), 3.30 (q under H2O peak, 2H), 5.39
(s, 2H), 7.03 (s, 1H), 7.31 (m, 2H), 7.39 (t, J ) 2.3 Hz, 2H), 7.49
(m, 1H), 8.06 (d, J ) 4.7 Hz, 1H), 8.47 (m, 1H), 8.77 (s, 1H). MS,
m/z: 388 (M + H)+.
2,2,2-Trifluoro-N-(3,5-difluoro-2-nitrophenyl)acetamide (56). A
mixture of 54 (19.4 g, 150 mmol) in concentrate sulfuric acid (22
mL) was heated with stirring at 190 °C for 5 h and then returned
to ambient temperature. The mixture was poured onto ice and the
resulting precipitate was filtered and washed with cold water,
affording 24.7 g of the sulfated intermediate as a gray solid. A
small fraction of the resulting product (2.0 g, 13.6mmol) was
dissolved in trifluoroacetic anhydride (18 g, 85.0mmol), allowed
to stir for 1 h at ambient temperature, then treated with solid
potassium nitrate (1.8 g, 17.8mmol) and allowed to stir for an
additional 18 h. The resulting crude reaction was poured slowly
onto ice and extracted 4 times with CH2Cl2. The combined organics
were dried over Na2SO4, and the solvent was evaporated in vaccuo.
The crude sample was purified by column chromatography on silica
gel (19:1 hexanes/EtOAc) to provide 56 (2.94 g, 75% yield) as a
1
dark-yellow solid. H NMR (500 MHz, CDCl3): δ 6.91 (m, 1H),
8.23 (m, 1H), 10.55 (broad s, 1H).
3,5-Difluoro-2-nitrobenzenamine (57). A mixture of 56 (0.41 g,
1.5mmol) and 6 N HCl (15 mL) was heated to reflux and allowed
to stir for 5 h. The mixture was cooled to ambient temperature and
poured into ice-cold aqueous sodium carbonate and extracted 4
times with CH2Cl2 and then a mixture of ethyl acetate and ethanol
(90:10). The combined organics were dried over Na2SO4, filtered,
then concentrated in vacuo to give 57 (0.17 g, 64% yield) as a
1-(5-Bromo-3-fluoro-2-nitrophenyl)-1H-pyrazole (62). To a stir-
ring 0 °C suspension of sodium hydride (44 mg, 1.1 mmol, 60%
oil dispersion) in THF (4 mL) was added a solution of pyrazole
(72 mg, 1.05 mmol) in THF (1 mL). The resulting mixture was
stirred at 0 °C for 5 min, then treated with a THF solution of 61
(238 mg, 1 mmol) dropwise over 10 min. The resulting mixture
was warmed to ambient temperature and allowed to stir for 1 h.
The reaction was then quenched by addition of water (1 mL),
partitioned between water and ethyl acetate, and the phases were
separated. The organic layer was washed with brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue was
purified by column chromatography on silica gel (6:1 hexanes/
1
yellow solid. H NMR (500 MHz, CDCl3): δ 5.89 (broad s, 2H),
6.29 (m, 2H).
5-Fluoro-2-nitro-3-(1H-pyrazol-1-yl)benzenamine (58). To a
solution of 57 (0.20 g, 1.2mmol) and pyrazole (0.094 g, 1.38 mmol)
in dimethylformamide (5 mL) was added sodium hydride, 60%
dispersion in mineral oil (0.055 g, 1.38 mmol). The mixture was
heated to 50 °C and allowed to stir for 18 h, returned to ambient
temperature, then quenched by the addition of aqueous ammonium
chloride. The crude mixture was extracted with twice with EtOAc,
the combined organics were washed with brine, dried over Na2SO4,
and concentrated under vacuum. The resulting residue was purified
by column chromatography on silica gel (4:1 hexanes/EtOAc) to
1
EtOAc) to provide 62 (240 mg, 86% yield) as a yellow solid. H
NMR (500 MHz, CDCl3): δ 6.55 (t, 1H), 7.45 (d, 1H), 7.60 (s,
1H), 7.80 (m, 2H). MS, m/z: 289 (M + H)+; 287 (M - H)-.
5-Bromo-2-nitro-3-pyrazol-1-ylphenylamine (63). To a solution
of 62 (240 mg, 0.84 mmol) in ethanol (3 mL) was added ammonia
(3 mL, 2 N in methanol). The resulting mixture was heated in a
sealed tube at 80 °C for 16 h, returned to ambient temperature and
pressure, then concentrated in vacuo. The residue was purified by
column chromatography on silica gel (3:1 hexanes/EtOAc) to
1
provide 58 (0.064 g, 25% yield) as a dark-yellow solid. H NMR
(CDCl3): δ 5.3 (broad s, 2H) 6.45-6.68 (m, 3H) 7.65 (d, 1H) 7.71
(d, 1H).
1-(3-Amino-4-nitro-5-(1H-pyrazol-1-yl)phenyl)-N-cyclopropyl-
1H-imidazole-4-carboxamide (59). To a mixture of 58 (0.063 g,
0.28 mmol) and 87 (0.064 g, 0.43 mmol) in dimethyformamide (4
mL) was added sodium carbonate (0.045 g, 0.43 mmol). The
mixture was heated to 95 °C and allowed to stir for 18 h, then
returned to ambient temperature. Water was added to the mixture
and the heterogeneous reaction allowed to stir for 30 min. The
mixture was allowed to settle, and the aqueous solution was
decanted. The resulting solids were triturated with water (2 × 30
mL) and filtered with a methanol wash. The mother liquor was
concentrated in vacuo and the resulting solid was triturated with a
10 mL solution of ethyl acetate/hexanes (1:1), filtered, and combined
with the original filtered solid to give 59 (0.06 g, 96% yield) as a
1
provide 63 (205 mg, 86%yield) as a yellow solid. H NMR (500
MHz, CDCl3): δ 5.20 (broad s, 2H), 6.50 (t, 1H), 6.9 (d, 1H), 7.1
(d, 1H), 7.7 (d, 1H), 7.8 (d, 1H). MS, m/z: 285 (M + H)+; 283 (M
- H)-.
2-Nitro-3-pyrazol-1-yl-5-pyridin-3-ylphenylamine (64a). To a
solution of 63 (200 mg, 0.71 mmol) in THF (8 mL) was added
3-pyridyldiethyl borane (157 mg), (tetrakistriphenylphosphine)
palladium(0) (84 mg), and sodium carbonate (2.2 mmol from 2 M
aqueous). The resulting mixture was stirred at 70 °C overnight,
then cooled to ambient temperature. The reaction mixture was
diluted with ethyl acetate (100 mL) and sequentially washed with
water then brine, dried over MgSO4, filtered, then concentrated in
vacuo. The resulting residue was purified by column chromatog-
raphy on silica gel (3:1 to 1:8 hexanes/EtOAc slow gradient) to
afford 64a (120 mg, 60% yield) as a yellow solid. 1H NMR
(DMSO-d6): δ 6.45 (broad s, 2H), 6.55 (t, 1H), 7.1 (s, 1H), 7.25
(s, 1H), 7.55 (m, 1H), 7.7 (s, 1H), 8.1 (dt, 1H), 8.3 (d, 1H), 8.7 (d,
1H), 8.9 (s, 1H).
1
dark-yellow solid. H NMR (500 MHz, CDCl3): δ 0.64 (m, 4H,
2.84 (m, 1H), 6.52 (broad s, 2H), 6.55 (t, 1H), 7.21 (m, 2H), 7.70
(s, 1H), 8.10 (d, 1H), 8.23 (s, 1H), 8.33 (d, 1H), 8.38 (s, 1H).
1-(5-(4-(Cyclopropylcarbamoyl)-1H-imidazol-1-yl)-7-(1H-pyra-
zol-1-yl)-1H-benzo[d]imidazol-2-yl)-3-ethylurea (19). A mixture of
59 (0.06 g, 0.17 mmol) and 10% Pd-C (10 mg) in methanol (20
mL) was placed under an atmosphere of hydrogen (45 psi) using a
Parr apparatus. The mixture was shaken for 2 h, the catalyst was
filtered off, and the solvent was evaporated under vacuum. The
resulting solid mostly dissolved in 1 N sulfuric acid (1.3 mL), and
to it was added reagent A (0.5 mmol). Then 1 N sulfuric acid was
1-Ethyl-3-(7-pyrazol-1-yl-5-pyridin-3-yl-1H-benzoimidazol-2-
yl)urea (12). A suspension of 64a (120 mg, 0.40 mmol) and 10%
Pd (12 mg) in ethyl acetate (10 mL) was placed in a Parr
hydrogenator under a hydrogen pressure of 45 psi. The mixture

Benzimidazole Urea Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5259
was shaken for 16 h, filtered, and the filtrate was concentrated in
vacuo. The resulting residue was diluted with H2SO4 (1.6 mL of 1
N), combined with reagent A (0.8 mL from 1 M solution), and
heated at 95 °C for 4 h. The mixture was then cooled to ambient
temperature, concentrated in vacuo, and purified by preparative
HPLC to afford 12 as a bis-TFA salt. This resulting salt was
suspended in aqueous NaHCO3, stirred for 10 min, and filtered.
The resulting solids were washed with water and dried under high
vacuum to afford freebase 12 (69 mg, 50% yield) as a white solid.
Purity, method A 98%; method B 98%. 1H NMR (300 MHz,
DMSO-d6): δ 10.76 (broad s, 1H), 9.28 (d, J ) 1.5 Hz, 1 H), 9.06
(d, J ) 2.5 Hz, 1H), 8.83 (dd, J ) 5.5 and 1.3 Hz, 1H), 8.78 (d, J
) 7.9 Hz, 1H), 8.18 (d, J ) 1.3 Hz, 1H), 8.02 (dd, J ) 8.1 and 5.5
Hz, 1H), 7.94 (d, J ) 1.5 Hz, 1H), 7.82 (d, J ) 1.5 Hz, 1H), 7.59
(broad s, 1H), 6.69 (dd, J ) 2.6 and 1.9 Hz, 1H), 3.25 (m, 2H),
1.14 (t, J ) 7.2 Hz, 3H). MS, m/z: 348 (M + H)+; 346 (M - H)-.
2-Nitro-3-(1H-pyrazol-1-yl)-5-(pyrimidin-5-yl)benzenamine (64b).
To a solution of 63 (100 mg, 0.43 mmol) in DME (5 mL) was
added successively (1.3 mL of 1 M aqueous NaHCO3), 5-pyrimidine
boronic acid (65 mg, 0.52 mmol), and palladium tetrakistriph-
enylphosphine (50 mg, 0.04 mmol). The resulting mixture was
stirred at 80 °C overnight, cooled to ambient temperature, and
partitioned between ethyl acetate and water. The organic layer was
washed with brine, dried over MgSO4, filtered, and concentrated
in vacuo. The residue was purified by column chromatography on
silica gel (3:1 hexanes/EtOAc) to afford 64b (90 mg, 85%) as an
orange solid. 1H NMR (500 MHz, DMSO-d6): δ 9.25 (s, 1H), 9.1
(s, 2H), 7.2 (broad s, 2H), 7.1 (s, 1H), 7.05 (d, 1H).
1-(7-(1H-Pyrazol-1-yl)-5-(pyrimidin-5-yl)-1H-benzo[d]imidazol-
2-yl)-3-ethylurea (18). To a solution of 64b (100 mg, 0.35 mmol)
in ethyl acetate (15 mL) was added of 10% Pd(OH)2-C (15 mg,
catalytic). The mixture was shaken under a 45 psi hydrogen
atmosphere for 16 h, filtered over Celite, and concentrated in vacuo.
To a solution of the residual oil in 1 N sulfuric acid (1.4 mL) and
water (3 mL) was added reagent A (0.7 mL of a 1 M solution).
The mixture was stirred at reflux for 3.5 h, cooled to ambient
temperature, and concentrated in vacuo. The residue was purified
by preparative HPLC to afford 18 (42 mg, 20%) as an off white
solid. Purity, method A 98%; method B 98%. 1H NMR (500 MHz,
DMSO-d6): δ 10.25 (broad s, 1H), 9.2 (broad s, 1H), 9.15 (s, 2H),
9.05 (broad s, 1H), 8.05 (s, 1H), 7.85 (broad s, 1H), 7.75 (s, 1H),
7.25 (broad s, 1H), 6.65 (s, 1H), 3.25 (m, 2H), 1.15 (t, J ) 7.2 Hz,
3H). MS, m/z: 349 (M + H)+; 347 (M - H)-.
N-[2-(3-Fluoropyridin-2-yl)phenyl]-2,2-dimethylpropionamide
(66). A 3 L flask was charged with boronic acid 65 as a tetrahydrate
(92.1 g, 314 mmol), chlorofluoropyridine (37.6 g, 286 mmol),
NaHCO3 (48.0 g, 572 mmol), and Pd(PPh3)4 (3.3 g, 2.86 mmol).
Water (300 mL) and dimethoxyethane (300 mL) were added, and
the mixture was heated slowly to 83 °C (internal temperature) over
a 1 h period with overhead stirring. After ∼2 h all solids had
dissolved. The mixture was allowed to stir for an additional 10 h.
The mixture was cooled to ambient temperature and stirred
overnight, after which time a thick gum had formed. The crude
mixture was diluted with water (2 L) and stirred for an additional
2 h. The mixture was then allowed to rest without stirring until the
gum had settled to the bottom of the flask. The liquid phase was
removed via vacuum, then replaced with 0.1 N NaOH and stirred
for 15 min. The gum was allowed to settle and the liquid removed
via vacuum. The gum was then similarly washed three times with
water, then transferred to a 1 neck flask as an acetone solution.
The mixture was concentrated in vacuo and azeotroped five times
with ethyl acetate. No further purification was performed on
intermediate 66. 1H NMR (DMSO-d6, 300 MHz): δ 10.21 (s, 1H),
7.99 (dd, J ) 0.9, 8.1 Hz, 1H), 7.89 (m, 1H), 7.60 (ddd, J ) 1.5,
3.3, 7.8 Hz, 1H), 7.54 (quintet, J ) 3.9 Hz, 1H), 7.46 (dt, J ) 1.8,
7.8 Hz, 1H), 7.25 (dt, J ) 1.2, 7.8 Hz, 1H), 1.10 (s, 9H). MS, m/z:
273.14 (M + H)+.
heterogeneous mixture was stirred at ambient temperature for 5 h,
over which time a thick precipitate formed. The mixture was then
poured over ice, diluted with 1 N Na2S2O3 (500 mL), and stirred
for 1 h. The solids were filtered, resuspended in water (2 L), stirred
for 1 h, then filtered and washed with water again. The resulting
solids were pumped to dryness at 50 °C, resuspended in HOAc
(400 mL), and treated with bromine (4 mL, 76 mmol) in acetic
acid solution (20 mL) over a 20 min period. The resulting
heterogeneous mixture was stirred for 5 h, then quenched and treated
as described above. The resulting solids were vaccuum-dried at 50
°C to afford 67 (19.1 g, 72% yield) as a tan powder. 1H NMR
(DMSO-d6, 300 MHz): δ 10.13 (s, 1H), 8.58 (d, J ) 1.5 Hz, 1H),
7.90 (m, 2H), 7.56 (t, J ) 2.4 Hz, 1H), 7.65 (dd, J ) 2.4, 8.7 Hz,
1H), 7.57 (quintet, J ) 4.2 Hz, 1H), 1.10 (s, 9H). MS, m/z: 351.03
and 353.08 (M + H)+
N-[4-Bromo-2-(3-fluoropyridin-2-yl)-6-nitrophenyl]-2,2-dimeth-
ylpropionamide (68). To a suspension of 67 (6.45 g, 18.4 mmol)
in TFA (100 mL) and TFAA (25.5 mL, 183.6 mmol) at 0 °C was
added a TFA solution (30 mL) of 90% fuming nitric acid (2.46
mL, 55.1 mmol) over a 45 min period. The mixture was then stirred
at 0 °C for a total of 4 h. The crude mixture (now homogeneous)
was poured into ice, producing a pasty mass. The mixture was
diluted to 500 mL total volume with water, treated with 50 mL of
methanol, and vigorously stirred for 12 h. The resulting solids were
filtered, washed with copious amounts of water, then dried in vacuo
at 50 °C to afford 68 (6.1 g, 82% yield) as a tan powder. 1H NMR
(DMSO-d6, 300 MHz): δ 9.59 (s, 1H), 8.57 (d, J ) 1.5 Hz, 1H),
8.28 (d, J ) 2.4 Hz, 1H), 8.05 (dd, J ) 0.9, 2.4 Hz, 1H), 7.85 (dt,
J (q, J ) 1.2, 8.4 Hz, 1H), 7.58 (quintet, J ) 4.2 Hz) 1.10 (s, 9H).
MS, m/z: 396.09 (M + H)+
4-Bromo-2-(3-fluoropyridin-2-yl)-6-nitrophenylamine (69). A
suspension of 68 (522 g, 1.32 mol) was suspended in 6 M
hydrochloric acid (5 L), and the reaction mixture was heated at
reflux for 5 h. Pivalic acid and water were collected using a
Dean-Stark trap. After 5 h the LCMS results still indicated the
presence of starting material. Concentrated hydrochloric acid and
water (1 L each) were added, and the mixture was heated at reflux
until the starting material was consumed (additional 3 h). The
mixture was cooled overnight, and the solid was collected by
vacuum filtration and rinsed with water (5 L). The filter cake was
suspended in 10% sodium carbonate (5 L) and stirred for 30 min.
The solid was collected by vacuum filtration and rinsed with water
(5 L). The solid was dried in a convection oven at 50 °C for 48 h
to provide the product 8 (378.2, 92% yield) as an orange-yellow
1
solid. H NMR (CDCl3, 500 MHz): δ 8.53 (m, 1H), 8.40 (d, J )
3.0 Hz, 1H), 7.81 (t, J ) 3.0 Hz, 1H), 7.79 (br s, 2H), 7.63 (m,
1H), 7.41 (quintet, J ) 4.0 Hz, 1H). MS, m/z: 311.76 and 313.76
(M + H)+
2-(3-Fluoropyridin-2-yl)-6-nitro-4-(pyridin-3-yl)benzenamine (71a).
To a heterogeneous mixture of 69 (4.0 g, 12.82 mmol) and 3-(1,3,2-
dioxaborinan-2-yl)pyridine (2.7 g, 16.66 mmol) in DME/water (50:
25 mL) was added solid Na2CO3 (2.7 g, 25.63 mmol) and Pd(PPh3)4
(0.75 g, 0.64 mmol). The resulting mixture was stirred at reflux
for 6 h and then returned to ambient temperature, diluted with
EtOAc, then slowly acidified with 6 N HCl with stirring. The phases
were separated, and the aqueous phase was washed thrice with
EtOAc. The resulting aqueous extract was filtered to separate out
insoluble matter and then slowly basified with ammonium hydroxide
to pH ∼10. The resulting heterogeneous mixture was stirred at room
temperature for 5 h and solids were isolated via filtration, washed
with copious amounts of water, and dried under high vacuum to
afford 71a (3.65 g, 92% yield) as a yellow solid. 1H NMR (CDCl3,
500 MHz): major rotomer δ 8.91 (d, J ) 2.5 Hz, 1H), 8.61 (dt, J
) 1.5, 4.5 Hz, 1H), 8.54 (dd, J ) 2.5 Hz, 1H), 8.11 (m, 1H), 8.03
(t, J ) 2.5 Hz, 1H), 7.95 (m, 1H), 7.62 (quintet, J ) 4.0 Hz, 1H),
7.50 (m, 2H), 7.46 (m, 1H). MS, m/z: 310.9 (M + H)+; 309.37 (M
- H)-.
N-[4-Bromo-2-(3-fluoropyridin-2-yl)phenyl]-2,2-dimethylpropi-
onamide (67). To an ambient temperature suspension of 66 (∼77
mmol) in acetic acid (300 mL) was added bromine (12 mL, 228
mmol) as a solution in 50 mL of acetic acid over a 1 h period. The
1-Ethyl-3-(7-(3-fluoropyridin-2-yl)-5-(pyridin-3-yl)-1H-benzo[d]im-
idazol-2-yl)urea (15). To a suspension of 71a (350 mg, 1.13 mmol)
in EtOAc (10 mL) was added Pd-C (∼50 mg). The suspension
shaken in a Parr bottle under 45 psi of hydrogen for 1 h. The

5260 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Charifson et al.
reaction mixture was diluted with EtOAc (25 mL) and filtered on
2.7 µm glass microfiber filter. The filtrate was concentrated in vacuo
to give the crude intermediate diamine (245 mg), which was used
without further purification for the next step. MS, m/z: 281 (M +
H)+. A 50 mL round-bottom flask equipped with a reflux condenser
and a magnetic stirrer was charged with 1 N aqueous H2SO4 (3.5
mL), crude diamine (245.0 mg), and reagent A (4.52 mmol from 1
M solution). The pH of the reaction mixture was then adjusted to
3 with additional 1 N aqueous H2SO4 (3.5 mL). The mixture was
heated to 100 °C and stirred at that temperature for 10 h. The
resulting mixture was cooled to ambient temperature and filtered
to give an off-white solid, which was washed with water, 1 N
ammonium hydroxide, water, EtOAc, and MeOH in succession to
afford 15 (125 mg, 29% yield) as an off-white solid. Purity, method
A 98%; method B 98%. 1H NMR (500 MHz, CD3OD): δ 8.86 (d,
J ) 2.0 Hz, 2H), 8.69 (m, 1H), 8.52 (d, J ) 5.0 Hz, 1H), 8.16 (m,
2H), 7.79 (m, 2H), 7.56 (m, 1H), 7.50 (m, 1H), 3.55 (m, 2H), 1.23,
(t, J ) 7.2 Hz, 3H). MS, m/z: 377 (M + H)+.
2-(3-Fluoropyridin-2-yl)-6-nitro-4-(4,4,5,5-tetramethyl-1,3,2-di-
oxaborolan-2-yl)aniline (70). To a solution of compound 69 (430
mg, 1.4 mmol) in dioxane (8 mL) was added bis(pinacoloto)diboron
(462 mg, 1.82 mmol), KOAc (274 mg, 2.8 mmol), and PdCl2(dppf)4
(0.1 equiv). The resulting mixture was heated at 160 °C (micro-
wave) for 12 min. The crude reaction was concentrated in vacuo
and the resulting residue was purified by column chromatography
on silica gel (1:1 to 0:1 hexanes/EtOAc gradient) to afford 70 (360
mg, 71% yield) as a yellow solid, which was directly used for the
next step. MS, m/z: 360 (M + H)+.
then cooled to ambient temperature. The solids were collected,
washed sequentially with hot water and then 1:1 hexanes/EtOAc,
and dried to afford the biaryl product (200 mg, 59% yield) as a
yellow solid. To a solution of this adduct (200 mg, 0.59 mmol) in
ethyl acetate (20 mL) was added 10% palladium on carbon (catalytic
amount). The resulting suspension was placed in a Parr hydrogena-
tion apparatus under 40 psi of hydrogen gas while shaking at
ambient temperature for 1 h. The catalyst was removed by filtration
and the filtrate concentrated in vacuo to afford 71b (130 mg, 0.42
mmol) as an off-white solid. MS, m/z: 311 (M + H)+.
1-Ethyl-3-(7-(3-fluoropyridin-2-yl)-5-(1-methyl-2-oxo-1,2-dihy-
dropyridin-4-yl)-1H-benzo[d]imidazol-2-yl)urea (21). To an aqueous
solution of 71b (130 mg, 0.42 mmol) in sulfuric acid (0.84 mL of
1 N solution) was added solid reagent A (113 mg, 0.84 mmol) and
enough sulfuric acid to adjust the mixture to pH 3. The resulting
mixture was heated at 100 °C for 8 h and then cooled to ambient
temperature, resulting in a precipitate. The solids were collected
via filtration and washed with water, dried in vacuo, further washed
with EtOAc followed by 9:1 EtOAc/MeOH to afford compound
21 (100 mg, 59% yield) as a white solid. Purity, method A 98%;
method B 98%. 1H NMR (500 MHz, CDCl3): δ 8.72 (m, 1H), 8.43
(d, J ) 1.4 Hz, 1H), 8.00 (d, J ) 1.4 Hz, 1H), 7.91 (m, 1H), 7.88
(m, 1H), 7.60 (m, 1H), 6.99 (d, J ) 2 Hz, 1H), 6.96 (dd, J ) 7.0,
2.1 Hz, 1H), 3.36 (m, 2H), 3.72 (s, 3H), 1.24 (t, J ) 7.3 Hz, 3H).
MS, m/z: 407 (M + H)+.
4-(Pyridin-3-yl)-2-nitroaniline (77). To a solution of 76 (4.8 g,
22 mmol) in DME (100 mL) was added pyridine-3-boronic acid
1,3-propanediol cyclic ester (4.0 g, 24 mmol), sodium bicarbonate
(45 mmol from 1 M solution), and tetrakis(triphenylphosphine)-
palladium (1.27 g, 1.1 mmol). The resulting mixture was heated at
90 °C for 8 h and then cooled to ambient temperature. The solids
were collected, washed sequentially with water and then 19:1
hexanes/EtOAc, and dried to afford 77 (4.7 g, 100% yield) as a
yellow solid. 1H NMR (500 MHz, CDCl3):δ 8.8 (d, 1H), 8.55 (m,
1H), 8.35 (d, 1H), 7.85 (dd, 1H), 7.65 (dd, 1H), 7.35 (m, 1H), 6.95
(d, 1H), 6.25 (broad s, 2H).
2-Bromo-6-nitro-4-pyridin-3-ylphenylamine (78). To a solution
of 77 (1.3 g, 9 mmol) in acetic acid (25 mL) was added bromine
(1.58 g, 9.9 mmol) as a solution in acetic acid (5 mL). The resulting
mixture was stirred at ambient temperature for 1 h and then poured
into ice. The resulting solids were collected via filtration and washed
with water, then dissolved in EtOAc and sequentially washed with
NaOH (2 N, 20 mL), water, and brine. The organic phase was then
dried over MgSO4, filtered, and concentrated in vacuo. The
concentrate was purified by column chromatography on silica gel
(1:1 hexanes/EtOAc)] to afford 78 (0.8 g, 30% yield) as a yellow
solid. 1H NMR (500 MHz, CDCl3): δ 8.83 (d, 1H), 8.55 (m, 1H),
8.41 (d, 1H), 8.15 (d, 1H), 7.96 (m, 1H), 7.41 (m, 1H), 6.80 (broad
s, 2H). MS, m/z: 294 (M + H)+.
4-(Benzyloxy)-1-methylpyridin-2(1H)-one (73). To a 500 mL Parr
bottle with Teflon cap was added 72 (8.04 g, 0.04 mol) which was
partially dissolved in 50 mL of acetone. Then 11.6 g of potassium
carbonate (0.082 mol) was added followed by 3.0 mL of io-
domethane (0.048 mol) and the mixture was sealed and heated to
50 °C for 16 h. After the mixture was cooled to ambient
temperature, the solids were filtered, washed twice with acetone,
and the filtrate was treated with 1 g of activated charcoal. After
filtration through a 1 in. plug of Celite and after being washed with
acetone, the resulting residue was purified by column chromatog-
raphy on silica gel (1:1 to 0:1 hexanes/EtOAc gradient) to provide
73 (7.06 g, 82%) as an off-white solid. IR (cm-1): CdO (1652),
1
C(O)dN (1592), CH3 (1378), C-O (1226, 1210). H NMR (500
MHz, acetone-d6): δ 7.4-7.5 (m, 3H), 7.35 (t, 2H), 7.3 (m, 1H),
5.9 (d, 1H), 5.85 (s, 1H), 3.3 (s, 3H).
4-Hydroxy-1-methylpyridin-2(1H)-one (74). In a 500 mL Parr
bottle, 6.52 g of 73 (0.03 mol) was dissolved in 100 mL of
methanol. After charging with 0.5 g of 5% Pd/C, the mixture was
pressurized to 48 psi with hydrogen and shaken for 1.25 h, at which
time no further hydrogen uptake occurred. Filtration through Celite
and concentration gave 74 (2.75 g, 73% yield) as a white solid.
MS, m/z: 126 (M + H)+; 124 (M - H)-.
2-Nitro-6-pyridin-2-yl-4-pyridin-3-ylphenylamine (79a). A mix-
ture of 78 (100 mg, 0.34 mmol), 2-pyridylznic bromide (2.05 mmol,
from 0.5 M solution in THF) and tetrakis(triphenylphosphine)-
palladium (40 mg, 0.034 mmol) in THF (10 mL) was heated at
100 °C for 18 h. The mixture was returned to ambient temperature
and quenched with water. The product was extracted thrice with
EtOAc, the combined organic extracts were then dried over Na2SO4,
filtered, and concentrated in vacuo, and the residue was purified
by column chromatography on silica gel (EtOAc) to provide 79a
(75 mg, 75% yield) as a yellow solid. MS, m/z: 293 (M + H)+.
1-Ethyl-3-(7-pyridin-2-yl-5-pyridin-3-yl-1H-benzoimidazol-2-
yl)urea (13). To a solution of 79a (75 mg, 0.26 mmol) in ethyl
acetate (20 mL) was added 10% Pd-C (50 mg). The resulting
suspension was placed in a Parr hydrogenation apparatus under 40
psi of hydrogen gas while shaking at ambient temperature for 1 h.
The catalyst was removed by filtration through Celite and the filtrate
concentrated in vacuo to give the corresponding phenylenediamine
(50 mg, 0.19 mmol) which was directly combined with solid reagent
A (100 mg, 0.76 mmol) and sulfuric acid (0.76 mL, 1 N) in water
(1 mL). The resulting mixture was treated with enough sulfuric
acid to adjust the mixture to pH 3 and was heated at 100 °C for
8 h. The reaction mixture was then cooled to ambient temperature,
1-Methyl-2-oxo-1,2-dihydropyridin-4-yl Trifluoromethanesulfonate
(75). A partial solution of 74 (4.46 g, 0.036 mol) in a mixture of
DMF (30 mL) and dichloromethane (30 mL) was stirred for 30
min before 6.0 mL of triethylamine was added all at once. Complete
solution occurred rapidly. The resulting solution was treated with
solid N-phenyl-bis(trifluoromethanesulfonimide) (13.99 g, 0.0392
mol) added in 3 portions over 5 min. Some warming of the flask
was noted. The solution was stirred at ambient temperature for 3.5 h
before dilution with EtOAc. The solution was washed thrice with
water, brine and dried over MgSO4. Filtration and concentration
in vacuo gave an oil (∼19 g) which was purified by column
chromatography on silica gel (3:1 to 1:1 hexanes/EtOAc gradient)
1
to provide 75 (9.0 g, 98% yield) as an oil. H NMR (500 MHz,
acetone-d6): δ 7.9 (d, 1H), 6.4 (s, 1H), 6.3 (d, 1H), 3.5 (s, 3H).
MS, m/z: 258 (M + H)+; 255 (M - H)-.
4-(4-Amino-3-(3-fluoropyridin-2-yl)-5-nitrophenyl)-1-methylpy-
ridin-2(1H)-one (71b). To a solution of compound 75 (386 mg, 1.5
mmol) in DME (20 mL) was added boronate ester 70 (359 mg, 1.0
mmol), sodium carbonate (6 mmol from 2 M solution), LiCl (126
mg, 3 mmol), and tetrakis(triphenylphosphine)palladium (174 mg,
0.15mmol). The resulting mixture was heated at 90 °C for 4 h,

Benzimidazole Urea Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5261
resulting in a precipitate. The solids were collected, washed with
water, and dried in vacuo. The solids were then purified by column
chromatography on silica gel (1:0 to 9:1 EtOAc/MeOH gradient)
to afford 13 (27 mg, 29% yield) as a white solid. Purity, method A
95%; method B 95%. 1H NMR (500 MHz, CDCl3): δ 8.92 (d, J )
1.8 Hz, 1H), 8,80 (m, 1H), 8.52 (m, 1H), 8,30 (m, 1H), 8.21 (d, J
) 8 Hz, 1H), 8.04 (s, 1 H), 7.94 (m, 1H), 7.75 (s, 1H), 7.56 (d, J
) 7.9 Hz, 1H), 7.37 (m, 2H), 3.36 (q, 7.3 Hz, 2H), 1.24 (t, J ) 7.3
Hz, 3H). (M + 1). MS, m/z: 359 (M + H)+.
2-Nitro-6-pyridin-3-yl-4-pyridin-3-ylphenylamine (79b). To a
solution of 78 (50 mg, 0.17 mmol) in DME (10 mL) was added
pyridine-3-boronic acid 1,3-propanediol cyclic ester (42 mg, 0.25
mmol), potassium phosphate (2.1 g, 10 mmol), and tetrakis(triph-
enylphosphine)palladium (20 mg, 0.017 mmol). The resulting
mixture was heated at 90 °C for 18 h, then cooled to ambient
temperature. The reaction was quenched with water and extracted
thrice with EtOAc. The combined organic extracts were then dried
over Na2SO4, filtered, concentrated in vacuo and the residue was
purified by silica gel chromatography eluting with ethyl acetate to
afford 79b (50 mg, 100% yield) as a yellow solid. MS, m/z: 293
(M + H)+.
1-Ethyl-3-(7-pyridin-3-yl-5-pyridin-3-yl-1H-benzoimidazol-2-
yl)urea (14). To a solution of 79b (50 mg, 0.17 mmol) in ethyl
acetate (20 mL) was added 10% Pd-C (catalytic amount). The
resulting suspension was placed in a Parr hydrogenation apparatus
under 40 psi of hydrogen gas while shaking at ambient temperature
for one hour. The catalyst was removed by filtration and the filtrate
concentrated in vacuo to afford the intermediate phenylelediamine
(42 mg, 0.16 mmol). MS, m/z: (M + H)+ 263. To a solution of
above product (42 mg, 0.16 mmol) and sulfuric acid (0.76 mL, 1
N) in water (1 mL) was added solid reagent A (102 mg, 0.76 mmol).
Enough sulfuric acid was added dropwise to achieve pH 3. The
resulting mixture was heated at 100 °C for 8 h and then cooled to
ambient temperature, resulting in a precipitate. The solids were
collected, washed with water, and dried in vacuo. The solids were
purified by column chromatography on silica gel (1:0 to 9:1 EtOAc/
MeOH gradient) to afford compound 14 (10 mg, 16% yield) as a
white solid. Purity, method A 90%; method B 90%. 1H NMR (500
MHz, DMSO-d6): δ 9.32 (s, 1H), 8.95 (d, J ) 2.1 Hz, 1H), 8.53
(m, 3H), 8.12 (m, 1H), 7.74 (d, J ) 2.1 Hz, 1H), 7.68 (s, 1H), 7.49
(m, 2H), 3.22 (m, 2H), 1.13 (t, J ) 7.2 Hz, 3H). MS, m/z: 359 (M
+ H)+.
N-tert-Butyl-5-fluoro-2-nitro-3-(pyridin-2-yl)benzenamine (83).
To an ambient temperature solution of 82 (1.0 g, 3.44 mmol) in
DMF (5 mL) was added Pd(PPh3)4 (200 mg, 0.172 mmol) and then
2-pyridylzinc bromide (5.15 mmol as 0.5 M solution in THF). The
resulting mixture was heated to reflux for 8 h and then returned to
ambient temperature. The crude reaction was then diluted with ethyl
acetate and saturated NaHCO3, and the resulting slurry was
vigorously stirred for 30 min. Celite was added, and stirring was
continued for an additional 30 min. The heterogeneous mixture was
filtered through a pad of fresh Celite, and the solids were washed
sequentially with ethyl acetate, water, then ethyl acetate again. The
combined filtrates were separated, and the organic phase was dried
over Na2SO4, filtered through a short pad of silica gel with ethyl
acetate, and concentrated in vacuo. The resulting crude product was
then purified by column chromatography on silica gel (30:1 to 9:1
CH2Cl2/ethyl acetate slow gradient) to provide 83 (600 mg, 60%
1
yield) as an orange oil. H NMR (500 MHz, DMSO-d6): δ 8.52
(d, 1H), 7.91 (ddd, 1H), 7.61 (dd, 1H), 7.40 (ddd, 1H), 7.01 (dd,
1H), 6.72 (dd, 1H), 6.33 (s, 1H), 1.40 (s, 9H).
1-(3-(tert-Butylamino)-4-nitro-5-(pyridin-2-yl)phenyl)-N-cyclo-
propyl-1H-imidazole-4-carboxamide (84). To a solution of 83 (300
mg, 1.03 mmol) and 87 (190 mg, 1.24 mmol) in DMF (3 mL) was
added solid Na2CO3 (130 mg. 1.24 mmol) at ambient temperature.
The resulting mixture was heated to 120 °C for 8 h and then
returned to ambient temperature. The crude mixture was diluted
with water (20 mL), the resulting heterogeneous mixture was stirred
for 20 min, and the solids were then filtered. The solids were washed
with copious amounts of water and concentrated in vacuo to provide
1
84 (400 mg, 92% yield) as a yellow solid. H NMR (500 MHz,
DMSO-d6): δ 8.57 (d. 1H), 8.48 (s, 1H), 8.40 (s, 1H), 8.12 (d,
1H), 7.96 (dd, 1H), 7.82 (d, 1H), 7.41 (dd, 1H), 7.22 (d, 1H), 7.21
(d, 1H), 6.20 (s, 1H), 2.85 (m, 1H), 1.48 (s, 9H), 0.65 (m, 2H),
0.61 (m, 2H).
1-(3-Amino-4-nitro-5-(pyridin-2-yl)phenyl)-N-cyclopropyl-1H-
imidazole-4-carboxamide (85). A solution of 84 (400 mg, 0.95
mmol) in MeOH (20 mL) and 2 N HCl (5 mL, 10 mmol) was
heated to reflux for 2 h and then returned to ambient temperature.
The resulting mixture was then concentrated in vacuo and diluted
with water (50 mL) and treated with enough solid NaHCO3 to raise
the pH to ∼7.0. The resulting heterogeneous mixture was then
stirred for 30 min and filtered. The solids were washed with copious
amounts of water and concentrated in vacuo to provide 85 (312
1
mg, 90% yield) as a yellow solid. H NMR (500 MHz, DMSO-
1-Bromo-3,5-difluoro-2-nitrobenzene (81). To an ambient tem-
perature, heterogeneous mixture of 80 (6.0 mL, 52.1 mmol) in neat
sulfuric acid (10 mL) was added fuming nitric acid (2.5 mL, 62.5
mmol) dropwise over a 1 h period. The resulting mixture was stirred
for an additional hour, resulting in a precipitate that progressively
inhibited stirring. The resulting solids were broken up with vigorous
stirring and the mixture was then poured onto ice, producing a sticky
solid. The mixture was allowed to warm to ambient temperature,
diluted with hexanes/ethyl acetate (1:2 v/v), and stirred until all
solids dissolved. The organic phase was washed with brine, dried
over Na2SO4, and filtered through a short pad of silica gel with 1:1
hexanes/ethyl acetate. The filtrate was then concentrated in vacuo
at 0 °C until solids formed providing 81 (11.2 Grams, 90% yield)
d6): δ 8.54 (d, 1H), 8.40 (s, 1H), 8.23 (s, 1H), 8.12 (d, 1H), 7.95
(dd, 1H), 7.80 (d, 1H), 7.41 (dd, 1H), 7.28 (d, 1H), 7.15 (d, 1H),
6.51 (s, 2H), 2.82 (m, 1H), 0.66 (m, 2H), 0.61 (m, 2H).
N-Cyclopropyl-1H-imidazole-4-carboxamide (87). A mixture of
86 (5.0 g, 26.6 mmol) and cyclopropylamine (3.9 mL, 55.8 mmol)
in dioxane was heated to 80 °C in a sealed tube for 4 h. The mixture
was cooled to ambient temperature and concentrated in vacuo,
providing a sticky solid. The crude product was azeotroped thrice
with ethanol and the resulting solids were triturated with toluene,
filtered, and dried under high vacuum to afford 87 (7.2 Grams,
90% yield) as an off-white solid. 1H NMR (500 MHz, MeOH-d4):
δ 7.7 (s, 1H), 7.6 (s, 1H), 2.8 (m, 1H) 0.8 (m, 2H) 0.6 (m, 2H).
1-(6-(4-(Cyclopropylcarbamoyl)-1H-imidazol-1-yl)-4-(pyridin-2-
yl)-1H-benzo[d]imidazol-2-yl)-3-ethylurea (20). To a partial suspen-
sion of 85 (100 mg, 0.275 mmol) in MeOH (20 mL) was added
Raney Ni (∼0.2 mL of slurry) in a Parr bottle. The mixture was
exposed to 45 psi of H2 for 2 h with shaking and then purged of
excess H2. The resulting black solution was filtered through a 0.5
µm polycarbonate membrane, and the solids were washed with
MeOH. The filtrate was concentrated in vacuo, and the resulting
waxy solid was diluted with dioxane (2 mL) and pH 3.5 buffer (5
mL), treated with reagent B (83 mg, 0.358 mmol), and heated to
reflux for 6 h. The crude mixture was then cooled to ambient
temperature, diluted with 1 M NaHCO3 (20 mL), and the
heterogeneous mixture was stirred for 20 min. The solids were then
collected via filtration, washed sequentially with water and 9:1
EtOH/water, then dried in vacuo to provide 20 (62 mg, 52% yield)
1
as a light-yellow solid. H NMR (500 MHz, DMSO-d6): δ 7.92
(ddd, 1H), 7.88 (ddd, 1H).
N-tert-Butyl-3-bromo-5-fluoro-2-nitrobenzenamine (82). To a
solution of 81 (3.0 g, 12.61 mmol) in dioxane (10 mL) was added
tert-butylamine (3.3 mL, 31.56 mmol), and the mixture was sealed
in a high-pressure tube. The mixture was then heated to 65 °C for
5 h and then returned to ambient temperature and pressure. The
mixture was then diluted with water and 1:1 hexanes/EtOAc and
stirred until all solids had dissolved. The resulting phases were then
separated, the organic phase was then washed with brine, dried
over Na2SO4, filtered through a short pad of silica gel with 1:1
hexanes/EtOAc, and concentrated in vacuo. The crude product was
then purified by column chromatography on silica gel (3:1 to 2:1
hexanes/CH2Cl2 gradient) to provide 82 (2.95 g, 81% yield) as an
orange oil. 1H NMR (500 MHz, DMSO-d6): δ 6.98 (dd, 1H), 6.93
(dd, 1H), 5.81 (s, 1H), 1.35 (s, 9H).
1
as an off-white solid. Purity, method A 91%; method B 91%. H

5262 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Charifson et al.
NMR (500 MHz, MeOH-d4): δ 8.96 (s, 1H), 8.89 (d, J ) 5.1 Hz,
1H), 8.47 (d, J ) 8.2, 1H), 8.37 (m, 2H), 8.20 (ddd, J ) 7.8, 7.8,
2.3 Hz, 1H), 7.93 (s, 1H), 7.63 (dd, J ) 7.0, 6.6 Hz, 1H), 3.37 (q,
J ) 7.1 Hz, 1H), 2.88 (m, 1H), 1.24 (t, J ) 6.3 Hz, 3H), 0.87 (m,
2H), 0.68 (m, 2H). MS, m/z: 431 (M + H)+; 429 (M - H)-.
Ki Determination. The details of the enzyme inhibition assays
can be found in ref 23.
Acknowledgment. The authors gratefully acknowledge
Hong Tsao and Betty Suh for developing and performing LC/
MS methods on the compounds included in this manuscript.
We also thank Mark Namchuk for a thorough reading and
critique of this manuscript.
Supporting Information Available: LC/MS data for compounds
2-21. This material is available free of charge via the Internet at
http://pubs.acs.org.
Susceptibility Testing. All bacteria were obtained from the
American Type Culture Collection (ATCC; Manassas, VA).
Minimal inhibitory concentration (MIC) determinations were
performed in liquid medium in 96-well microtiter plates according
to the methods described by the Clinical Laboratory and Standards
Institute (CLSI23) with modifications as described previously.23 To
measure the relative effects of serum on compound susceptibility,
human serum (U.S. Biological, Swampscott, MA) was added to
liquid medium to a final concentration of 50% in assays performed
with S. aureus ATCC 29213. All test methods met acceptable
standards based on recommended quality control ranges for all
comparator antibiotics and the appropriate ATCC strain.
Computational Methods. Docking calculations were performed
with the ICM program (Molsoft LLC).39 Compounds 1 and 2 were
docked using unconstrained docking with the docking region defined
by the crystallographic position of novobiocin. Subsequent docking
calculations employed harmonic constraints applied to the benz-
imidazole urea portion of the molecules to the crystallographic
position of related molecules.30 The docking and postprocessing
were performed according to previous methods.40 Final pose
selection was determined on the basis of both nonbond interaction
energies and strain energies relative to the closest local minimum,
as well as visual inspection. All quantum mechanical calculations
described were performed with Gaussian 98 at the HF/6-31G* level
of theory.32
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