Second-generation antibacterial benzimidazole ureas: Discovery of a preclinical candidate with reduced metabolic liability

Article

pubs.acs.org/jmc

Second-Generation Antibacterial Benzimidazole Ureas: Discovery

of a Preclinical Candidate with Reduced Metabolic Liability

Anne-Laure Grillot,* Arnaud Le Tiran,^†Dean Shannon,^‡Elaine Krueger, Yusheng Liao,

Hardwin O’Dowd, Qing Tang, Steve Ronkin, Tiansheng Wang, Nathan Waal, Pan Li, David Lauﬀer,

Emmanuelle Sizensky, Jerry Tanoury, Emanuele Perola, Trudy H. Grossman,^§Tim Doyle, Brian Hanzelka,

Steven Jones, Vaishali Dixit, Nigel Ewing, Shengkai Liao, Brian Boucher, Marc Jacobs, Youssef Bennani,

and Paul S. Charifson

Vertex Pharmaceuticals Incorporated, 50 Northern Avenue, Boston, Massachusetts 02210, United States

S

* Supporting Information

ABSTRACT: Compound 3 is a potent aminobenzimidazole urea with broad-spectrum Gram-positive antibacterial activity

resulting from dual inhibition of bacterial gyrase (GyrB) and topoisomerase IV (ParE), and it demonstrates eﬃcacy in rodent

models of bacterial infection. Preclinical in vitro and in vivo studies showed that compound 3 covalently labels liver proteins,

presumably via formation of a reactive metabolite, and hence presented a potential safety liability. The urea moiety in compound

3 was identiﬁed as being potentially responsible for reactive metabolite formation, but its replacement resulted in loss of anti-

bacterial activity and/or oral exposure due to poor physicochemical parameters. To identify second-generation amino-

benzimidazole ureas devoid of reactive metabolite formation potential, we implemented a metabolic shift strategy, which focused

on shifting metabolism away from the urea moiety by introducing metabolic soft spots elsewhere in the molecule.

Aminobenzimidazole urea 34, identiﬁed through this strategy, exhibits similar antibacterial activity as that of 3 and did not label

liver proteins in vivo, indicating reduced/no potential for reactive metabolite formation.

topoisomerase IV and have potent antibacterial activity.⁶This

work and subsequent studies led to the identiﬁcation of several

INTRODUCTION

■

Over the past decade, the introduction of a handful of new

potential preclinical candidates (Figure 1 and Table 1).

antibacterial agents as well as the implementation of programs

Compound 1 was extensively characterized and demonstrated

aimed at minimizing the spread of antibiotic-resistant bacterial

excellent activity in rodent models of infection,⁶but it was shown

strains and limiting the overuse of novel antibiotics has produced

some short-term successes in the battle against drug resistance.¹

However, resistance to antibiotics remains a global human threat

with the potential for catastrophic consequences in the future.

Infections caused by drug resistant Gram-positive organisms,

such as methicillin-resistant Staphylococcus aureus (MRSA) and

vancomycin-resistant enterococci (VRE), remain a critical issue

to be a potent CYP3A4 inhibitor and suﬀered from a short half-

life and poor physicochemical properties.⁷Compound 2 had

similar antibacterial properties as those of compound 1, with a

lower serum shift and lack of CYP inhibition. Its solubility proﬁle

was improved, but its oral bioavailability was suboptimal.⁷

Compound 3 resolved the above issues, exhibited potent activity

against a broad range of Gram-positive organisms, and showed

not only because of the morbidity and mortality caused by these

infections but also because of the associated economic cost.¹⁻³

eﬃcacy in rodent models of infection as well as an ADME proﬁle

compatible with oral and intravenous administration. The ability

Thus, an unmet medical need remains for a safe antibiotic

suitable for extended use to treat serious bacterial infections.^4,5

We previously reported the discovery of a novel class of

dual-targeting benzimidazole ureas that inhibit DNA gyrase and

Received: April 10, 2014

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Figure 1. Structures of lead aminobenzimidazole ureas 1−3.

Table 1. Enzyme Inhibition and Antibacterial Proﬁle of

Figure 2. In vitro labeling of human, monkey, dog, rat, and mouse

microsomal liver proteins by ¹⁴C-labeled 3 incubated at 30 μM for 1 h in

the presence or absence of NADPH. Covalent binding was observed

only in the presence of NADPH.

a

Aminobenzimidazole Ureas 1−3

compound

1

2

3

E. coli Gyrase K_i(nM)

6

<4

<6

5

cohort of rats received a single dose of the radiolabeled

compound. Four hours after the radiolabeled compound was

dosed, all animals were euthanized, and the amount of radiolabel

associated with liver proteins was determined (Figure 3). A

S. aureus TopoIV K_i(nM)

S. aureus MIC (μg/mL)

<6

0.031

1

0.031

0.25

0.016

0.25

S. aureus MIC + HS (μg/mL)

S. pneumoniae MIC (μg/mL)

E. faecalis MIC (μg/mL)

H. inﬂuenzae MIC (μg/mL)

≤0.008

0.016

1

≤0.008

0.032

1

≤0.008

<0.008

1

a

HS, 50% human serum.

to dose a Gram-positive agent intravenously would allow use in

the hospital for the treatment of infections caused by methicillin-

resistant S. aureus as well as vancomycin-resistant enterococci.

We deemed the option for oral dosing essential for a novel Gram-

positive agent because, in addition to providing the possibility

of step-down use outside of the hospital setting, there is a need

for new drugs active against methicillin-resistant S. aureus and

ﬂuoroquinolone-resistant Streptococcus pneumoniae in the

community setting, where intravenous drugs are not easily

administered. Compound 3, when dosed BID orally in a 72 h

thigh infection model (S. aureus), produced substantial

reductions in bacterial CFUs (colony forming units; ≥4 log),

demonstrating clear in vivo bactericidality at doses ≥25 mg/kg.⁸

However, in vitro and in vivo studies showed that compound 3

presented a potential safety liability, as it was shown to covalently

label liver proteins, presumably via the formation of a reactive

metabolite. We report herein a second-generation amino-

benzimidazole urea with an improved metabolic proﬁle,

identiﬁed via the implementation of a metabolic shift strategy.

Figure 3. Covalent labeling of liver proteins in vivo following 0, 6, or

13 days of dosing with compounds 3, followed by one dose of

¹⁴C-labeled 3, resulting in a time-dependent increase in amount of

radiolabel incorporation.

time-dependent increase in the amount of radiolabel bound

to liver proteins was observed, with levels above 50 pmol/mg at

14 days. The observed time-dependent increase in amount of

radiolabel incorporated suggests potential induction of the CYP

enzyme(s) responsible for the oxidation of 3 that led to reactive

metabolite formation.

Compound 3 Metabolites. To identify the putative reactive

metabolite of 3, metabolite identiﬁcation studies were conducted

in vitro in rat, dog, monkey, and human liver microsomes and S9

liver fractions. In vivo plasma samples from rat, dog, and monkey

were also examined, leading to a proposed route of metabolism

of 3 via dehydrogenation of the ethyl urea moiety (M − 2, 4)

and subsequent epoxidation (M + 14, 5) (Figure 4). A M + 14

metabolite, shown to be present in vitro in all species in liver

microsomes and S9 fractions, was thought to be epoxide 5, the

reactive metabolite responsible for protein covalent labeling.

Interestingly, this metabolite, while present in monkey and dog

plasma, was not present in rat plasma samples. A metabolite of

mass M − 2 was also present in plasma at low levels in all species.

RESULTS AND DISCUSSION

■

As part of our preclinical evaluation of compound 3, ¹⁴C-labeled

3 was synthesized and shown to covalently label liver proteins in

vitro in mouse, rat, dog, monkey, and human liver microsomes

(Figure 2) following methods published in the literature.⁹

Labeling occurred in the presence of NADPH but not in its

absence, suggesting that the formation of the reactive species was

mediated by CYP oxidation of compound 3.

To evaluate the potential of compound 3 to form a reactive

metabolite in vivo, covalent binding of 3 to rat liver proteins was

studied.⁸Male rats were dosed with 90 mg/kg of unlabeled 3

for 6 or 13 days. On day 7 or 14, the animals received 100 μCi of

¹⁴C-labeled 3 (speciﬁc activity of 1.43 mCi/mmol). A separate

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Figure 4. Initial proposed route of metabolism of 3.

Figure 5. Structures of tool compounds 6−8. In vivo metabolism according to Figure 3 was envisioned to yield depicted metabolites M − 3 for 6, M − 4

for 7, and M − 20 for 8. Instead, M − 2, M − 3, and M − 2 metabolites were observed, respectively.

Three follow-up compounds were prepared to investigate the

metabolism further: trideuterated analogue 6, penta-deuterated

analogue 7, and triﬂuoroethyl analogue 8 (Figure 5).

formed via α-hydroxylation of the ethyl substituent in 3, followed

by water elimination. This route of metabolism also explained the

ﬁndings for compounds 6−8 shown in Figure 5. Attempted de

novo synthesis of imine 9 did not result in isolation of the desired

compound; rather, compound 10, also of mass M − 2, resulting

from cyclization of imine 9 was isolated. The structure of

compound 10 was conﬁrmed by 2D NMR experiments (see

Supporting Information). Synthesized compound 10 was shown

to be identical to the metabolite obtained from compound 3 in

vivo by coelution experiments, and it showed the same frag-

mentation pattern in mass spectrometry (see Supporting

Information). Additional MS/MS studies of the observed M +

14 metabolites suggested that these metabolites arise from oxida-

tion of compound 10 (Figure 6, see Supporting Information).

These compounds were each dosed orally in rat at 30 mg/kg,

and plasma, bile, urine, and feces samples were analyzed to

identify their metabolites. In the case of deuterated analogues 6

and 7, the expected metabolites (of mass M − 3 and M − 4,

respectively) resulting from dehydrogenation of the urea’s ethyl

group were not observed. Instead, species of mass M − 2 (for

compound 6) and M − 3 (for compound 7) were found as major

metabolites in plasma, bile, urine, and feces. Triﬂuoroethyl urea 8

also yielded a metabolite of mass M − 2. These ﬁndings led us to

hypothesize that metabolism of 3 resulted in the formation of

imine 9 (Figure 6), another potential metabolite of mass M − 2,

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Figure 6. M − 2 metabolite formation. Initial oxidation takes place on the aliphatic carbon attached to the urea moiety in 3. Subsequent loss of water and

cyclization leads to 10. The asterisk-labeled carbon atoms in structures 9 and 10 denote activated centers susceptible to nucleophilic attack. Three M +

14 metabolites are formed by oxidation of compound 10.

Compound 6 was also shown to produce corresponding

M + 13 metabolites with structures similar to those obtained

from compound 3. Our conclusion from these studies is that

compound 10 is the M − 2 metabolite obtained from compound

3. We also hypothesize that the initial observed M + 14 metab-

olite is not the proposed structure 5 (Figure 4) but instead results

from further oxidation of compound 10 (Figure 6), although this

was not unequivocally determined through chemical synthesis.

Incubation of compound 3 in individual recombinant CYP

supersomes at 1 and 10 μM was shown to produce compound 10

(Figure 7). Incubations containing CYP3A4, CYP1A2, and

CYP2D6 showed formation of compound 10 at 10 μM, con-

ﬁrming that compound 10 is formed via oxidative metabolism

mediated by P450 enzymes.

Figure 7. Incubations of compound 3 in supersomes. Compound 10

Our conclusion from these studies is that cyclized intermediate

10, formed via CYP-mediated oxidation of 3 followed by loss of

water and intramolecular cyclization, is likely to be the putative

reactive metabolite formed upon dosing of compound 3. It is,

however, also possible that transient imine 9 itself acts as the

reactive metabolite.

Addressing Reactive Metabolite Formation: Attempts

To Replace the Urea Moiety. Our ﬁrst approach toward

identifying an aminobenzimidazole analogue with antibacterial

activity comparable to that of 3 and reduced potential for reactive

metabolite formation focused on identifying a replacement of the

presumed site for reactive metabolite formation in compound 3,

was not formed in the experiments without NADPH. Incubations

containing recombinant CYP2B6, CYP2C9, CYP2C8, and CYP2C19

did not show the formation of compound 10. Incubations with CYP3A4,

CYP1A2, and CYP2D6 showed formation of compound 10. Incubations

containing 1 μM compound 3 did not show formation of the imine

metabolite. The amount of metabolite formed increased signiﬁcantly at

10 μM at 30 min, and the highest amount was seen in incubations

containing CYP3A4 supersomes.

namely, the ethyl urea moiety. As observed for previously

reported aminobenzimidazole analogues,⁶the crystal structure

of 3 bound to bacterial S. aureus GyrB highlights several key

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interactions contributing to its aﬃnity: a hydrogen bond between

the C5-ﬂuoropyridine nitrogen and Arg136, a cation−π stacking

interaction between the C5-ﬂuoropyridine and Arg76, and a

bidentate hydrogen bond between the urea moiety and Asp73

(Figure 8). Additionally, an intramolecular hydrogen bond

constraints in this portion of the ATP binding site, with the

potential to maintain at least one hydrogen bond with Asp73.

Also essential to the design was the lack of potential for these

compounds to aﬀord M − 2 imines upon metabolism. These

analogues were synthesized, and their MIC’s against S. aureus

were measured (Figure 9).

In most instances, the MIC’s of these analogues against

S. aureus were greater than 8 μg/mL. Very small changes to the

ethyl urea moiety in compound 3 resulted in signiﬁcant loss

of antibacterial activity, as exempliﬁed by amides 12 and 13

(Table 2, S. aureus MICs > 8 μg/mL). Only two analogues,

carbamate 14 and amide 15, maintained MICs against S. aureus

below 0.1 μg/mL (Table 2). However, both compounds

exhibited poor properties, including poor solubility, large shifts

in MICs when tested in the presence of 50% human serum,

and/or lack of oral exposure; hence, these compounds were not

pursued further.

Addressing Compound 3 Reactive Metabolite For-

mation: Metabolic Shift Strategy. Having been unsuccessful

in identifying a suitable replacement for the urea moiety in

compound 3, we devised what we termed a metabolic shift

strategy, in which we attempted to shift metabolism away from

the urea by introducing a diverse set of metabolically labile

groups in our lead compounds.¹⁰Utilizing the SAR knowledge

gained in the work leading to the identiﬁcation of our preclinical

candidates,⁶we identiﬁed two C7 substituents as new starting

points toward aminobenzimidazole ureas with diﬀerent meta-

bolic proﬁles: 1-pyrazole and 2-tetrahydrofuran. Both of these

C7 moieties were attractive for several reasons: (a) they maintain

planarity between the benzimidazole core and the C7 sub-

stituent, and an intramolecular hydrogen bond between the C7

substituent and the aminobenzimidazole NH helps achieve

ligand preorganization (Figure 10); (b) they are structurally

diverse from previously investigated C7 substituents and

Figure 8. Crystal structure of compound 3 in complex with S. aureus

gyrase B (PDB ID: 4P8O). The depicted interactions are (a) a bidentate

hydrogen bond between the urea NH’s of 3 and Asp73, (b) a hydrogen

bond between the pyridine nitrogen of 3 and Arg136, (c) cation−π

stacking interaction between the pyridine ring of 3 and Arg76. Also

depicted is a hydrogen bond between one of the benzimidazole

nitrogens and a conserved water molecule.

helps to maintain coplanarity between the pyrimidine and the

benzimidazole rings, a feature previously shown to be optimal for

dual activity against gyrase B and topoisomerase IV for the

aminobenzimidazoles.⁶

With these elements in mind, we designed over 40 analogues

of compound 3 incorporating small urea mimics, due to the steric

Figure 9. Urea mimics of compound 3. All compounds were designed to eliminate the potential for formation of M − 2 imines (see 4 in Figure 3).

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a

Table 2. Antibacterial Proﬁles of Selected Urea Replacements

a

HS, 50% human serum.

oral exposure in rat after administration of a 30 mg/kg dose of

compound in an appropriate vehicle. Finally, our optimization

approach focused on exploring solubilizing substituents in order

to maintain potential for IV dosing.

1-Pyrazole at C7. In the C7 pyrazole series, the substituent at

C5 was investigated (Table 3). Compound 16 with a 3-pyridine

moiety at C5, which maintains a hydrogen bond with Arg136,

showed a reasonable MIC against S. aureus, but it showed a

16-fold serum shift. Introduction of a ﬂuorine atom at C6

(pyrazole 17), which improved antibacterial potency as well as

aﬃnity for gyrase B and topoisomerase IV in earlier series,⁶did

not result in a signiﬁcant improvement in S. aureus MIC.

However, oral exposure was improved 2-fold and therefore we

focused exclusively on C6-ﬂuorobenzimidazole pyrazoles. Next,

introduction of polarity at C5 was investigated in an eﬀort to

improve MICs and solubility. Solubilizing groups on the pyridine

moiety were tolerated, as shown by the activity of compound 18.

Introduction of a methyl substituent at the 4-position of the

pyrazole in 18 (compound 19) resulted in a 4-fold improvement

in the S. aureus MIC. However, this modiﬁcation resulted in

reduced oral exposure. As in our earlier report,⁶pyridones

continued to show excellent antimicrobial activity, as exempliﬁed

by compound 20 (S. aureus MIC = 0.016 μg/mL), but failed to

show acceptable oral exposure. Solubilizing substituents other

than amines, particularly alcohols, were also investigated. Com-

pound 21 showed a MIC against S. aureus of 0.125 μg/mL and

could be improved slightly by the addition of a methyl group on

the C5 substituent, as in 22. Resolution of 22 yielded compounds

23 and 24. The (S)-isomer 23 was 4-fold more potent than the

(R)-isomer 24 against S. aureus and showed acceptable oral

exposure. However, compound 23 exhibited a high serum shift

(16-fold) and was not investigated further.

Figure 10. Intramolecular hydrogen bond in the C7-pyrazoles and C7-

tetrahydrofurans resulting in ligand preorganization.

therefore are likely to have a diﬀerent metabolic proﬁle; and

(c) both of these substituents present lower cLogP’s, which is

advantageous, as higher LogP values have been correlated with

increased toxicity.¹¹We have demonstrated previously that for

the aminobenzimidazoles, maintaining planarity between the

aminobenzimidazole core and the substituent at C7 is key for

dual inhibition of gyrase and topoisomerase IV.⁶Other dual

inhibitors of bacterial gyrase and topoisomerase IV that bind to

the ATP-binding site have been reported in the literature.¹²⁻¹⁷

Series that are isosteric to the aminobenzimidazoles exist,^13−15,17

and conformational analysis of representative compounds

from these series suggest that their lowest-energy conformation

is planar as well, independently of whether the analysis is per-

formed on the bound or unbound state. To drive our op-

timization eﬀort, we focused primarily on MICs against S. aureus

because, historically, this Gram-positive organism had been the

most diﬃcult to inhibit eﬀectively with the aminobenzimidazole

series. MIC assays were run under Clinical and Laboratory

Standards Institute (CLSI) conditions as well as in the presence

of 50% human serum to assess the eﬀect of protein binding

on compound potency (serum-shifted MIC). A serum-shifted

MIC against S. aureus of 0.5 μg/mL or lower was deemed to be

acceptable to justify compound progression. Additionally, we

evaluated potential for acceptable pharmacokinetics by assessing

2-Tetrahydrofuranyl Substituent at C7. The potency of

the 2-tetrahydrofuranyl C7 series was ﬁrst evaluated with com-

pounds 25 (3-pyridyl at C5) and 26 (5-pyrimidinyl substituent

at C5) (Table 4). Both showed moderate to good activity against

F

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a

Table 3. C7 Pyrazole Series SAR

a

HS, 50% human serum; ND, not determined.

S. aureus and produced low serum shifts when tested in

the presence of 50% human serum. As in the case of the C7

pyrazoles, introduction of a pyridone substituent at C5 resulted

in an improvement in antibacterial activity and a minimal serum

shift. However, neither of the two pyridones (27 and 28) showed

good exposure when dosed orally in rat. On the basis of previous

SAR, C5-azine alcohols were also investigated. Introduction of

a gem-dimethyl hydroxymethyl substituent at the 6-position of

the pyridine ring (compound 29) resulted in a S. aureus MIC of

0.5 and a 4-fold serum shift. The corresponding pyrimidine

(compound 30) showed a slightly improved S. aureus MIC and a

serum shift of only 2, probably due to this compound’s higher

polar surface area. Addition of a C6 ﬂuorine atom (compound

33) produced the expected further improvement in antibacterial

activity, along with a higher but still acceptable serum shift of 8.

Resolution of compounds 30 and 33 yielded 31 and 32 and 34

and 35, respectively. In both cases, the (R)-isomers were more potent

than the (S)-isomers (the R stereochemistry for both of these

compounds was conﬁrmed through small molecule X-ray crystallog-

raphy; see Supporting Information for the small molecule X-ray

structure of compound 34). When dosed at 1 mg/kg in rat (in 25%

dimethylacetamide/30% propylene glycol/5% polysorbate-80/40%

water as the vehicle), the IV clearance for 34 was 49 mL/min/kg,

and the half-life was 1.9 h. Oral bioavailability in rat was 48%

following a 3 mg/kg PO dose (vehicle: 0.5% methylcellulose in

water). The rat IV PK parameters of compound 34 were similar to

those of compound 3 (clearance = 31 mL/min/kg; half-life = 1.1 h

when dosed at 1 mg/kg in 5% dextrose in water at pH 3).

Compound 34 was selected for further evaluation because of its

overall antibacterial proﬁle and its adequate oral exposure in rat.

In Vitro and in Vivo Antibacterial Proﬁle of Compound

34. Compound 34 was shown to be a potent dual inhibitor of

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a

Table 4. C7 THF Series SAR

a

HS: 50% human serum; BQL: below quantitation levels; ND, not determined.

S. aureus gyrase and S. aureus topoisomerase IV with K_i’s of <12

and 9 nM, respectively. In a panel of Gram-positive organisms

and selected Gram-negative organisms (Table 5), 34 exhibited

antibacterial potencies generally below 0.25 μg/mL; this proﬁle

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mouse, rat, dog, monkey, and human liver microsomes and

hepatocytes and in vivo through studies in bile duct-cannulated

rats that received a single oral dose of the compound. Metabolites

were analyzed on multiple MS platforms, including high-resolu-

tion LC-MS/MS. As summarized in Figure 12, compound 34

was metabolized primarily via oxidation followed by glucur-

onidation (see Supporting Information). Only a very low amount

of the M − 2 cyclized metabolite (<2%) was observed.

To study the potential of compound 34 to form reactive

metabolites, covalent binding of 34 to rat liver protein was

evaluated in vivo.⁹Male rats were dosed with 90 mg/kg of

unlabeled 34 for 6 or 13 days. On day 7 or 14, the animals

received 100 μCi of ¹⁴C-labeled 34 (speciﬁc activity of 55 mCi/

mmol). A separate cohort of rats received a single dose of the

radiolabeled compound. Four hours after the radiolabeled

compound was dosed, all animals were euthanized, and the

amount of radiolabel associated with liver proteins was

determined. The amount of radiolabel bound to liver protein

in all samples was less than 5 pmol/mg of protein, suggesting that

34 has very low potential to covalently bind to rat liver proteins in

vivo (Figure 13). Taken in isolation, the lack of a time-dependent

increase in radiolabel incorporation with compound 34 could be

due to a reduction of potential CYP induction with compound 3.

However, the lack of liver labeling after a single dose of com-

pound 34 supports a metabolic shift from the ethyl group of

compound 3 to another position in compound 34.

Table 5. Antibacterial Activity of Compound 34 against

Selected Gram-Positive and -Negative Microorganisms

species

strain/assay conditions

MIC (μg/mL)

Gram-Positive

Staphylococcus aureus

+50% human serum

S. epidermidis

ATCC 29213

0.125

0.016

ATCC 12228

ATCC 29212

ATCC 49624

ATCC 10015

≤0.008

0.016

Enterococcus faecalis

E. faecium

0.063

Streptococcus pneumoniae

Gram-Negative

≤0.008

Haemophilus inﬂuenzae

Moraxella catarrhalis

ATCC 49247

ATCC 25238

0.5

≤0.016

was well-aligned with our target indications, which included

bacteremia, endocarditis, and osteomyelitis. In addition,

compound 34 showed bactericidal activity against key organisms,

exhibited low resistance frequencies, and maintained activity

against multidrug resistant organisms.⁷

The potential of 34 to treat skin and skin structure infec-

tions was evaluated in a neutropenic rat thigh infection model

(S. aureus ATCC 29213).^6,18In untreated animals, the bacterial

loads in thigh tissue increased by 1.41 and 1.62 log₁₀colony

forming units (CFUs) at 8 and 24 h (LC, late controls),

respectively, as compared to the bacterial load at the start of dosing

(EC, early controls). Administration of 34 to infected animals

resulted in a dose-related and time-dependent antibacterial eﬀect

(Figure 11). All doses of compound 34 provided statistically

signiﬁcant decreases versus the 24 h late controls, indicating

bacteriostatic activity for all doses. Compared with initial burden

prior to treatment (EC), median bacterial density in the thigh after

24 h of treatment with the two higher doses (30 and 60 mg/kg) of

compound 34 was signiﬁcantly reduced (at least −1 log₁₀

diﬀerence versus EC), indicating modest bactericidal compound

activity. Bacterial killing was time-dependent; it was less pro-

nounced after the shorter 8 h treatment with 30 and 60 mg/kg of

compound 34 (approximately −0.6 log₁₀in median bacterial

density versus EC). The q12h doses of 30 and 60 mg/kg of

compound 34 provided similar decreases in S. aureus thigh

burdens as that of 30 mg/kg q12h of the comparator moxiﬂoxacin.

Metabolism of Compound 34. The metabolism of com-

pound 34 was investigated in vitro through incubations with

Taken together, these studies suggest that metabolism of

34 occurs away from the urea moiety. Studies in bile duct-

cannulated rats have shown metabolism of compounds 3 and 34

to be mainly hepatic. Because of their similar rat IV clearance

numbers, 3 and 34 are cleared at the same rate but through

diﬀerent pathways, supporting a metabolic shift from compound

3 to compound 34.

CONCLUSIONS

■

Aminobenzimidazole ureas are a novel class of antibacterial

agents that exert their potent activity via dual inhibition of both

bacterial gyrase B and topoisomerase IV. The development of an

early preclinical candidate (3) from this series was halted due

to evidence for the formation of a reactive metabolite in vivo. By

implementing a metabolic shift strategy to circumvent the

metabolic path responsible for reactive metabolite formation, we

have identiﬁed a second-generation aminobenzimidazole urea

Figure 11. Eﬃcacy of compound 34 in a neutropenic S. aureus thigh infection model. Compound 34 was dosed q12h at 10, 30, and 60 mg/kg, and

moxiﬂoxacin was dosed q12h at 30 mg/kg. Compound 34 at 30 and 60 mg/kg provided similar reduction in bacterial burden as that of moxiﬂoxacin at

30 mg/kg at both 8 and 24 h post-treatment.

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Figure 12. Putative metabolites of compound 34 from in vitro and in vivo experiments.

to the methods reported in refs 6, 19, and 20. Ortho-phenylenediamines

were synthesized by reduction of the corresponding ortho-nitroanilines.

C7 substituents were generally introduced via a Suzuki coupling

(pyridine and tetrahydrofuran) or via S_NAr of an appropriately

substituted phenyl halide (pyrazole). C5 substituents were installed

via Suzuki couplings.

The preparation of compound 2 started with conversion of bromo-

iodopyrimidine 36 to alcohol 37 (Scheme 1). Coupling of 37 to 38⁶

under Miyaura−Suzuki conditions aﬀorded nitroaniline 39. Reductive

hydrogenation followed by condensation with reagent 41^6,19aﬀorded

compound 2.

The synthesis of 6 and 7 required the preparation of deuterated

versions of the urea forming reagents 43a and 43b.²⁰Condensation with

ortho-phenylenediamine 44²⁰at pH 3.5 aﬀorded 6 and 7, respectively

(Scheme 2).

The synthesis of compounds 8, 10, and 11 proceeded through

intermediate 45 obtained via condensation of amine 44 with com-

mercially available methyl N-(N-methoxycarbonyl-C-methylsulfanyl-

carbonimidoyl)carbamate. Compounds 8 and 11 were synthesized via

aminolysis of 45 with triﬂuoroethylamine or ammonia, respectively.

Treatment of 11 with acetaldehyde under dehydrating conditions

yielded tricycle 10 (Scheme 3).

The synthesis of compounds 12−15 required the introduction of

amide or carbamate moieties in the last step. For compound 12, this was

achieved by preparing the amide transfer reagent 46 and condensing

it with diamine 44. Carbamate 14 was prepared in the same manner as

that for compound 45, using the commercially available 1,3-

bis(ethoxycarbonyl)-S-methylisothiourea (Scheme 4). Another con-

venient way to prepare amides was via aminobenzimidazole

intermediate 47, prepared eﬃciently by hydrolysis of compound 3

(Scheme 5). Coupling of 47 with the desired carboxylic acid under

standard amide coupling conditions yielded compounds 13 and 15.

Figure 13. Covalent labeling of liver proteins in vivo following 0, 6, or

13 days of dosing with compounds 3 or 34, followed by one dose of

¹⁴C-labeled 3 or 34. Experiments with compound 34 did not result in

incorporation of radiolabel, whereas studies with compound 3 resulted

in a time-dependent increase in the amount of radiolabel incorporation.

(34) devoid of the metabolic liability of compound 3. Com-

pound 34 has been selected as a preclinical candidate, and studies

are ongoing toward ﬁrst-in-human clinical trials. Results will be

reported in due course.

EXPERIMENTAL SECTION

■

Syntheses of Benzimidazole Ureas. Benzimidazole ureas were

generally synthesized according to the methods published in ref 6. All

syntheses proceeded to an appropriately substituted ortho-phenylenedi-

amine intermediate that was then converted to the ethyl urea according

J

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a

Scheme 1.

a

(a) n-BuLi, CH₂Cl₂, −65 °C; (b) acetone, CH₂Cl₂, −65 °C to rt; (c) Pd(PPh₃)₄, Na₂CO₃, DMF, reﬂux; (d) 45 psi H₂, Raney Ni, methanol; (e) pH

3.5 buﬀer, 1,4-dioxane, reﬂux.

a

Scheme 2.

a

(a) DIPEA, CDI, DMF; (b) d₃-diethylamine hydrochloride or d₅-diethylamine hydrochloride; (c) 43a or 43b, pH 3.5 buﬀer, DME, 80 °C.

The synthesis of compound 17 (Scheme 6) started from 2,3,6-

triﬂuoroaniline, which was brominated and then oxidized to aﬀord 50.

Treatment of 50 with the anion of pyrazole resulted in the exclusive

displacement of the ﬂuorine at the 2-position in 50, to aﬀord 51.

Treatment of 51 with ammonia yielded bromonitroaniline 52a, which

was converted to nitroaniline 53 by Suzuki coupling with the desired

boronic acid. Subsequent reduction of 53 and condensation of the

intermediate ortho-phenylenediamine completed the synthesis of 17.

Ureas 18−22 were prepared in a similar fashion (Schemes 7−9).

Coupling of boronate 55 with 52a or 52b aﬀorded the desired

K

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a

Scheme 3.

a

(a) Methyl N-(N-methoxycarbonyl-C-methylsulfanyl-carbonimidoyl)carbamate, pH 3.5 buﬀer, 1,4-dioxane, reﬂux; (b) 2,2,2-triﬂuoroethanamine,

NMP; (c) ammonia, NMP, 120 °C; (d) acetaldehyde, TFA, 4 Å sieves, rt.

a

Scheme 4.

a

(a) Butyric anhydride, 2 N NaOH(aq), DME, rt; (b) 46, pH 3.5 buﬀer, DME, rt; (c) pH 3.5 buﬀer, 1,4-dioxane, reﬂux.

nitroanilines, which were converted to 18 and 19, respectively, using the

same two-step process described for 17. Coupling of triﬂate 57 with

bromonitroaniline 52b aﬀorded 58, which was converted to 20. Com-

pound 21 was prepared via coupling of 1-(5-bromo-2-pyridyl)ethanol to

the boronate of 52a. For the synthesis of 22, boronate 60 was coupled

to bromide 52a. Ureas 23 and 24 were obtained via SFC separation

of 22.

The synthesis of the C7 tetrahydrofuranyl derivatives proceeded

through intermediates 61 (for C6 = H) or 62 (where C6 = F) (Scheme 10).

The syntheses of 61 and 62 are reported in ref 14. Similar to the C7

L

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a

Scheme 5.

a

(a) 12 M HCl, 110 °C; (b) N-BOC-sarcosine, HBTU, HOBt, DIPEA, DMF, rt; (c) HCl, Et₂O, CH₂Cl₂, MeOH, rt; (d) 2-methoxyacetic acid,

HBTU, HOBt, DIPEA, DMF, rt.

a

Scheme 6.

a

(a) NBS, DMF, rt; (b) NaBO₃, AcOH, 55 °C; (c) pyrazole or 4-methyl-1H-pyrazole, NaH, THF, 0 °C to rt; (d) NH₃, EtOH, reﬂux; (e) 3-pyridineboronic

acid 1,3-propanediol ester, Pd(PPh₃)₄, NaHCO₃, DME, reﬂux; (f) H₂, 10% Pd/C, EtOAc, 55 psi; (g) 41, pH 3.5 buﬀer, 1,4-dioxane, 90 °C.

pyrazole analogues, coupling of 61 or 62 to the desired coupling

partner aﬀorded compounds 25−30 and 33. SFC separation

of 30 and 33 yielded enantiomers 31 and 32 and enantiomers

34 and 35, respectively (Scheme 11). The stereochemistry of

M

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a

Scheme 7.

a

(a) NaB(OAc)₃H, DCE; (b) bis(pinacolato)diboron, PdCl₂(Cy₃P)₂, KOAc, 1,4-dioxane, 130 °C; (c) Na₂CO₃, Pd(PPh₃)₄, DME, 90 °C; (d) Ra Ni,

48 psi H₂, MeOH; (e) 41, pH 3.5 buﬀer, 1,4-dioxane, 90 °C.

a

Scheme 8.

a

(a) 1,1,1-Triﬂuoro-N-phenyl-N-(triﬂuoromethylsulfonyl)methanesulfonamide, Et₃N, DMF, rt; (b) bis(pinacolato)diboron, KOAc, PdCl₂(Cy₃P)₂,

2-Me THF, 130 °C; (c) 57, Pd(PPh₃)₄, Na₂CO₃, LiCl, DME, 100 °C; (d) Ra Ni, 45 psi H₂, MeOH; (e) 41, pH 3.5 buﬀer, dioxane, 90 °C.

31 and 34 was determined by obtaining small molecule X-ray

structures.

LC-MS analyses were performed on a Waters Acquity UPLC system

with a binary pump, a photodiode array detector, and an SQD Mass

Spectrometer using the following methods: Waters CSH C₁₈column

with water/acetonitrile/0.1% TFA; Waters Xbridge C₈column with

water/acetonitrile/0.1% TFA; Waters Xbridge C₈column with water/

acetonitrile/50 mM NH4OAc/pH 9; Waters Xselect perﬂuorophenyl

column with water/acetonitrile/0.1% TFA. The purity of the ﬁnal com-

pounds was determined by HPLC and/or LC-MS and was 95% or

greater unless speciﬁed otherwise. High-resolution mass measurements

Chemistry. All commercial reagents and anhydrous solvents were

obtained from commercial sources and were used without further

puriﬁcation, unless otherwise speciﬁed. Proton and carbon NMR

spectra (¹H NMR and ¹³C NMR) were recorded on a Bruker Avance

instrument with a QNP probe using TMS as the internal standard in

the indicated deuterated solvent. Preparative HPLC isolations were

performed using Agilent 1100 mass-directed puriﬁcation chromatog-

raphy system with 0.1% TFA in acetonitrile/water mobile phase.

N

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a

Scheme 9.

a

(a) Bis(pinacolato)diboron, KOAc, PdCl₂(Cy₃P)₂, 2-Me-THF, microwave, 130 °C; (b) 1-(5-bromo-2-pyridyl)ethanol, Pd(PPh₃)₄, Na₂CO₃, DME,

100 °C; (c) Ra Ni, MeOH, 40 psi H₂; (d) 41, pH 3.5 buﬀer, dioxane, 90 °C; (e) 60, PdCl₂(dppf), NaHCO₃, 2-Me-THF, microwave, 130 °C; (f)

SFC separation.

were performed on a Thermo QExactive mass spectrometer with a

heated electrospray source operated in positive ion mode.

was stirred at 100 °C for 1.5 h. The reaction was partitioned between

ethyl acetate and water and ﬁltered through Celite. The aqueous layer

was extracted with ethyl acetate. The combined organic phases were

washed with water and brine, dried over sodium sulfate, and con-

centrated to a slurry under reduced pressure. The slurry was triturated

with water and then with isopropyl alcohol. The resulting solid was

ﬁltered and dried under vacuum to aﬀord 27.8 g (91%) of 39 as an

orange solid. ¹H NMR (300 MHz, CDCl₃) δ 9.10 (s, 2H), 8.57 (d, 1H),

8.52 (s, 1H), 8.00 (s, 2H), 7.96 (m, 1H), 7.68 (t, 1H), 7.43 (m, 1H), 4.61

(s,1H), 1.64 (s, 6H) ppm. MS, m/z: 370 (M + H)⁺.

2-(5-Bromopyrimidin-2-yl)propan-2-ol (37). A 5 L ﬂask

containing 2.0 L of CH₂Cl₂was cooled to −65 °C under nitrogen,

and n-butyllithium (294.8 mL of 2.5 M, 0.737 mol) was added drop-

wise. 5-Bromo-2-iodo-pyrimidine (36, 200 g, 0.702 mol) in 500 mL

of CH₂Cl₂was added dropwise over 20 min. The solution was stirred for

15 min, and a precipitate formed. Acetone (257.8 mL, 3.510 mol) was

added at once, and the precipitate dissolved. The reaction mixture was

stirred for 30 min at −65 °C, warmed to room temperature, and then

quenched with 700 mL of aqueous 2 M NaHPO₄. The aqueous phase

was extracted with CH₂Cl₂, and the combined organic phases were

washed with brine and concentrated under reduced pressure. The

residue was puriﬁed by column chromatography over silica gel eluted

with 30% ethyl acetate/hexanes to yield 38 g (25%) of 37 as a yellow

solid. ¹H NMR (300 MHz, CDCl₃) δ 8.76 (s, 2H), 4.33 (s, 1H), 1.58 (s,

6H) ppm.

2-(5-(3,4-Diamino-5-(3-ﬂuoropyridin-2-yl)phenyl)pyrimidin-2-

yl)propan-2-ol (40). To a solution of 2-(5-(3-amino-5-(3-ﬂuoropyr-

idin-2-yl)-4-nitrophenyl)pyrimidin-2-yl)propan-2-ol (39, 5.05 g, 13.67

mmol) in methanol (750 mL) was added Raney nickel (4.0 mL of

suspension in water). The mixture was placed under 45 psi of hydrogen

on a Parr apparatus and shaken for 4 h. The catalyst was removed, and

the reaction was ﬁltered and concentrated under vacuum to aﬀord 4.64 g

1

(quant.) of 40 as a tan solid. H NMR (300 MHz, CDCl₃) δ 9.05 (s,

2-(5-(4-Amino-3-(3-ﬂuoropyridin-2-yl)-5-nitrophenyl)-

pyrimidin-2-yl)propan-2-ol (39). A mixture of 2-(5-bromopyrimidin-

2-yl)propan-2-ol (37, 17.84 g, 0.0822 mol), 2-(3-ﬂuoropyridin-2-yl)-6-

nitro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline (38,⁶29.52 g,

0.822 mol), tetrakis(triphenylphosphine) palladium(0) (3.79 g, 0.0033

mol), and sodium carbonate (2 M, 0.822 L, 0.1644 mol) in DMF (0.55 L)

2H), 8.53 (d, 1H), 7.58 (t, 1H), 7.31 (m, 2H), 6.96 (s, 1H), 4.79 (s, 2H),

4.71 (s, 1H), 3.44 (s, 2H), 1.64 (s, 6H) ppm. MS, m/z: 340 (M + H)⁺.

1-Ethyl-3-(7-(3-ﬂuoropyridin-2-yl)-5-(2-(2-hydroxypropan-2-yl)-

pyrimidin-5-yl)-1H-benzo[d]imidazol-2-yl)urea (2). 2-(5-(3,4-Dia-

mino-5-(3-ﬂuoropyridin-2-yl)phenyl)pyrimidin-2-yl)propan-2-ol (40, 4.6 g,

O

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a

Scheme 10.

a

(a) ArBR₂or ArB(OR)₂, Pd catalyst, base; (b) reduction catalyst, H₂; (c) 41, pH 3.5 buﬀer, dioxane, 90 °C; (d) bis(pinacolato)diboron,

PdCl₂(dppf), KOAc, DME, reﬂux; (e) ArOTf, Pd catalyst, base.

Scheme 11

P

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13.55 mmol) was combined with a 1 M solution of 1-ethyl-3-(N-

(ethylcarbamoyl)-C-methylsulfanyl-carbonimidoyl)urea (41)^6,19in dioxane

(14.9 mL, 14.9 mmol) and 300 mL of pH 3.5 buﬀer (prepared with 57.5 g

NaOAc·3H₂O and 400 mL of 1 N H₂SO₄), and the mixture was stirred at

105 °C for 8 h. The reaction was cooled to room temperature, diluted with

water, and brought to pH 7.5 by adding solid NaHCO₃. The mixture was

allowed to stir for 20 min, and the resulting solid was collected by ﬁltration,

washed successively with water and ethyl acetate, and dried in vacuo to aﬀord

2.67 g of 2 as an oﬀ-white solid. This solid was suspended in EtOH (25 mL),

and 2 equiv of methanesulfonyl chloride(1.41g, 12.3mmol)wasadded. The

mixture was warmed to 40 °C, stirred until homogeneous, ﬁltered, and

concentrated in vacuo. The resulting solid was dissolved in dichloro-

methane and added dropwise to a stirred solution of diethyl ether (200 mL)

to aﬀord an oﬀ-white suspension. The solid was collected by ﬁltration and

dried in vacuo to aﬀord 3.93 g (46%) of 2 as the bis-mesylate salt. ¹H NMR

(DMSO-d₆, 500 MHz) δ 11.3 (br, 1H), 9.18 (s, 2H), 8.70 (br d, J = 4.5 Hz,

1H), 8.24 (s, 1H), 8.04 (d, J = 1.5 Hz, 1H), 8.03 (m, 1H), 7.72 (br t, J = 5

Hz, 1H), 7.67 (quint, J = 4.0 Hz, 1H), 3.27 (m, 2H), 2.48 (s, 6H, 2 MsOH),

1.58 (s, 6H), 1.16 (t, J = 7.0 Hz, 3H) ppm. HRMS calcd. for (M + H)⁺, m/z:

436.1897, found m/z: 436.1888.

1-d₅-Ethyl-3-(5-(5-ﬂuoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-

benzo[d]imidazol-2-yl)urea (7). To a round-bottomed ﬂask was

added 44 (1.91 g, 6.7 mmol) and pH 3.5 aqueous phosphate buﬀer

(23 mL). A solution of 43b (3.68 g, 10.1 mmol) in DME (17 mL) was

added, and the solution was warmed to 80 °C for 3 h. The reaction

mixture was cooled to ambient temperature, and the mixture was

ﬁltered. The solids were rinsed with water and dried to aﬀord 2.31 g

(90% yield) of 7. ¹H NMR (500 MHz, DMSO-d₆) δ 12.09 (s, 1H), 10.03

(s, 1H), 9.04 (dd, J = 4.9, 1.0 Hz, 2H), 8.86 (t, J = 1.8 Hz, 1H), 8.57 (d,

J = 2.6 Hz, 1H), 8.49 (d, J = 1.7 Hz, 1H), 8.12 (dt, J = 10.4, 2.3 Hz, 1H),

7.98 (d, J = 1.7 Hz, 1H), 7.59−7.48 (m, 1H), 7.34 (s, 1H) ppm. HRMS

calcd. for (M + H)⁺, m/z: 383.1792, found m/z: 383.1791.

Methyl (5-(5-Fluoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-benzo-

[d]imidazol-2-yl)carbamate (45). A suspension of 44 (4 g, 14.2 mmol)

and methyl N-(N-methoxycarbonyl-C-methylsulfanyl-carbonimidoyl)-

carbamate (3.5 g, 17.1 mmol) in 1,4-dioxane (70 mL) and buﬀer pH 3.5

(20 mL, stock solution made from 1 N H₂SO₄and NaOAc) was stirred at

reﬂux for 4 h. The crude reaction mixture was ﬁltered while warm, and

the beige solid was washed successively with water (100 mL), acetone

(15 mL), EtOAc (15 mL), and Et₂O (100 mL) and dried in vacuo to

aﬀord 45 (5.33 g, quant.) as a white solid. ¹H NMR (300 MHz, TFA) δ

9.19 (dd, J = 1.2, 5.5 Hz, 2H), 8.95 (s, 1H), 8.62 (s, 1H), 8.52 (m, 2H),

8.27 (s, 1H), 7.79 (t, J = 5.5 Hz, 1H), 3.74 (s, 3H) ppm. An aliquot of

227 mg was then salted as the monoesylate salt. ¹H NMR (300 MHz,

CDCl₃) δ 9.00 (d, J = 4.9 Hz, 2H), 8.91 (d, J = 0.8 Hz, 1H), 8.83 (d, J =

1.6 Hz, 1H), 8.64 (d, J = 2.5 Hz, 1H), 8.01−7.97 (m, 2H), 7.40 (t, J = 4.9

Hz, 1H), 4.01 (s, 3H), 3.07 (q, J = 7.4 Hz, 2H), 1.45 (t, J = 7.4 Hz,

3H) ppm. MS, m/z: 365.25 (M + H)⁺.

1-(5-(5-Fluoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-benzo[d]-

imidazol-2-yl)-3-(2,2,2-triﬂuoroethyl)urea (8). To a suspension of

45 (330 mg, 0.89 mmol) in NMP (20 mL) was added 2,2,2-

triﬂuoroethylamine (2.64 g, 26.6 mmol), and the mixture was heated

at 120 °C in a sealed Parr ﬂask for 5 h. The resulting clear yellow solution

was cooled to room temperature and poured into water (70 mL). The

resulting suspension was stirred for 1 h and ﬁltered, and the oﬀ-white

solid was washed with water (2 × 50 mL) and then MeOH (10 mL),

which partially redissolved the product, explaining the low yield.

The resulting white solid (89.7 mg, 23%) was then converted to the

monoesylate salt. ¹H NMR (300 MHz, CDCl₃) δ 8.98 (d, J = 4.9 Hz,

2H), 8.80 (m, 1H), 8.79 (dd, J = 1.6, 13.6 Hz, 1H), 8.55 (d, J = 2.6 Hz,

1H), 7.94 (d, J = 1.6 Hz, 1H), 7.79−7.74 (m, 1H), 7.37 (t, J = 4.9 Hz,

1H), 7.09 (t, J = 6.2 Hz, 1H), 4.05−3.94 (m, 2H), 3.08 (q, J = 7.4 Hz,

2H),1.45 (t, J = 7.4 Hz, 2H) ppm. HRMS calcd. for (M + H)⁺, m/z:

432.1196, found m/z: 432.1190.

1-(5-(5-Fluoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-benzo[d]-

imidazol-2-yl)urea (11). To a suspension of 45 (280 mg, 0.75 mmol)

in NMP (20 mL) was bubbled ammonia (384.9 mg, 22.60 mmol) for

10 min. The mixture was heated at 120 °C in a sealed Parr ﬂask for 5 h.

The resulting clear yellow solution was then cooled to rt and poured into

water (70 mL). The resulting suspension was stirred for 1 h and ﬁltered,

and the oﬀ-white solid was washed with water (2 × 50 mL), triturated in

hot MeOH for 2 h, and then triturated with hot DMF (70 °C). The

insoluble solid was collected to yield 19.7 mg of 11 (5.7%) as the free

base, which was converted to its monoesylate salt. ¹H NMR (300 MHz,

MeOD) δ 9.03 (d, J = 4.8 Hz, 2H), 8.83 (s, 1H), 8.74 (s, 1H), 8.58 (d, J =

2.2 Hz, 1H), 8.06 (m, 2H), 7.53 (t, J = 4.8 Hz, 1H), 2.73 (q, J = 7.4 Hz,

2H), 1.25 (t, J = 7.4 Hz, 3H) ppm. MS, m/z: 350.14 (M + H)⁺.

7-(5-Fluoropyridin-3-yl)-4-methyl-9-(pyrimidin-2-yl)-3,4-

dihydrobenzo[4,5]imidazo[1,2-a][1,3,5]triazin-2(1H)-one (10).

To a solution of 11 (9.8 mg, 0.028 mmol) in TFA (1.5 mL) were added

in succession excess acetaldehyde (6.176 mg, 7.868 μL, 0.140 mmol) and 4Å

molecular sieves. The reaction mixture was stirred at rt for 3 days, concen-

trated in vacuo, and puriﬁed by reverse-phase HPLC to aﬀord 10 (2.4 mg,

23%) as a white solid. ¹H NMR (800 MHz, DMSO-d₆) δ 9.00 (d, 2H), 8.93

(s, 1H), 8.61(Hb, 1H), 8.40(Hd, 1H), 8.22(m, 1H), 8.20(m, 1H), 7.54(m,

1H), 6.08 (m, 1H), 1.65 (d, 3H) ppm. ¹³C NMR (125 MHz, DMSO-d₆) δ

162.50, 159.44 (d, J = 254.98 Hz), 158.75, 157.58, 148.96, 147.56, 143.96,

137.06, 136.30 (d, J = 23.75 Hz), 131.05, 130.00, 125.26, 122.34, 121.10

(d, J = 18.75 Hz), 120.05, 111.16, 61.79, 22.84 ppm. HRMS calcd. for

(M + H)⁺, m/z: 376.1322, found m/z: 376.1317.

1-Ethyl-3-(5-(5-ﬂuoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-benzo-

[d]imidazol-2-yl)urea (3). See refs 21 and 22.

1-Ethyl-3-(N-(ethylcarbamoyl)-C-(para-nitrobenzylsulfanyl-

carbonimidoyl)urea-d₆(43a). To a solution of CDI (959 mg,

5.9 mmol) and DIEA (1.82 g, 2.453 mL, 14.08 mmol) in DMF was

added at 0 °C commercially available 42 (823 mg, 2.8 mmol). The

reaction mixture was stirred at 0 °C for 1 h. To the dark purple solution

was added 2,2,2-d₃-diethylamine hydrochloride (500 mg, 5.913 mmol),

and the mixture was warmed to rt and stirred for 16 h. The reaction

mixture was diluted with water (50 mL), EtOAc (50 mL), and hexanes

(15 mL). The phases were separated, and the aqueous layer was

extracted twice with EtOAc. The combined organic extracts were

washed with saturated aqueous KHSO₄, water, and brine. The organic

phase was dried (MgSO₄), ﬁltered, and concentrated in vacuo. The

residue was diluted in EtOAc/Et₂O, and hexanes was added until

cloudiness was observed. The mixture was then cooled to ca. −15 °C

over 90 min, and the resulting precipitate was ﬁltered. The ﬁltrate was con-

centrated in vacuo to aﬀord 43b (0.34 g, 34%). MS, m/z: 360.22 (M + H)⁺.

1-Ethyl-3-(N-(ethylcarbamoyl)-C-(para-nitrobenzylsulfanyl-

carbonimidoyl)urea-d₁₀(43b). A solution of CDI (7.81 g, 48.2 mmol)

in DMF (75 mL) was cooled to −5 °C, and 41⁶(6.72 g, 22.9 mmol) was

added. The reaction mixture was stirred for 1 h. To the solution was added

d₅-diethylamine hydrochloride (4.17 g, 48.2 mmol). The reaction mixture

was warmed to room temperature and stirred for 16 h. Water (75 mL) and

isopropyl acetate (75 mL) were added. Heptane (10 mL) was added, and

the phases were separated. The lower aqueous phase was extracted with

isopropyl acetate (2 × 10 mL), and the combined organic phases were

washed with saturated aqueous NaHSO₄solution, then water, and then

saturated aqueous NaCl solution. The organic phase was dried (Na₂SO₄)

and ﬁltered, and the solvent was removed in vacuo to give 3.68 g (44%) of

the desired product 43b, which was carried to the next step without further

puriﬁcation.

1-d₃-Ethyl-3-(5-(5-ﬂuoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-

benzo[d]imidazol-2-yl)urea (6). To a suspension of 44²⁰(132 mg,

0.47 mmol) in DME (3 mL) was added a solution of 43a (337 mg,

0.9376 mmol) in DME (3 mL). To the mixture was added buﬀer (pH

3.5, NaOAc/1 N H₂SO₄). The mixture was stirred at 90 °C for 6 h. After

6 h, the beige reaction mixture was cooled to room temperature, diluted

with water, and ﬁltered. The solid was triturated in hot EtOAc, ﬁltered,

and washed with MeOH, water, and Et₂O in succession to aﬀord a 90%

pure solid, which was resolubilized in hot DMSO/2 N aq HCl and was

puriﬁed by reverse-phase HPLC Gilson to yield 6 (33 mg, 14%). The

ﬁnal product was converted to its esylate salt by dilution in MeOH and

1

addition of 1 equiv of ethanesulfonic acid. H NMR (300.0 MHz,

CDCl₃) δ 8.94 (d, J = 4.9 Hz, 2H), 8.77 (s, 1H), 8.71 (d, J = 1.6 Hz, 1H),

8.52 (d, J = 2.6 Hz, 1H), 7.83 (d, J = 1.6 Hz, 1H), 7.75−7.71 (m, 1H),

7.32 (t, J = 4.9 Hz, 1H), 6.15 (s, 1H), 3.35 (br d, J = 5.4 Hz, 2H), 3.03

(q, J = 7.4 Hz, 2H) and 1.42 (t, J = 7.4 Hz, 3H) ppm. HRMS calcd. for

(M + H)⁺, m/z: 381.1667, found m/z: 381.1665.

Q

dx.doi.org/10.1021/jm500563g | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

N-[N-Butanoyl-C-[4-nitrophenyl)methylsulfanyl]carbonimidoyl]-

butanamide (46). The hydrobromide salt of 42 (2.82 g, 9.65 mmol),

butanoyl butanoate (6.11 g, 4.33 mL, 38.6 mmol), water (15 mL), and

DME (24 mL) were charged in a 50 mL ﬂask. The pH of the mixture was

adjusted to 8 with a 2 N aqueous NaOH solution, and the resulting

biphasic reaction mixture was stirred at room temperature for 16 h. The

residue was diluted with EtOAc (50 mL), and the biphasic mixture was

stirred for 10 min and separated, and the organics were washed with

a saturated aqueous solution of NaHCO₃and brine, dried (MgSO₄),

and concentrated in vacuo to aﬀord an oﬀ-white solid. The solid was

triturated with Et₂O/hexanes, ﬁltered, and dried in vacuo to yield 46

and buﬀer pH 3.5 (25 mL, prepared from 1 N H₂SO₄and NaOAc) was

stirred at reﬂux overnight. The crude reaction mixture was ﬁltered while

warm, and the beige solid was successively washed with water (100 mL)

and methanol (75 mL) to aﬀord 1.19 g of 14 (52%) as free base, which

was converted to the monoesylate salt. ¹H NMR (300.0 MHz, CDCl₃)

δ 9.00 (d, J = 4.9 Hz, 2H), 8.88 (s, 1H), 8.81 (d, J = 1.6 Hz, 1H), 8.61 (d,

J = 2.6 Hz, 1H), 7.96−7.90 (m, 2H), 7.40 (t, J = 4.9 Hz, 1H), 4.45 (q, J =

7.1 Hz, 2H), 3.07 (q, J = 7.4 Hz, 2H), 1.48−1.42 (m, 6H) ppm. HRMS

calcd. for (M + H)⁺, m/z: 379.1319, found m/z: 379.1312.

N-(5-(5-Fluoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-benzo[d]-

imidazol-2-yl)-2-methoxyacetamide (15). To compound 47

(68.9 mg, 0.23 mmol) in DMF (3 mL) were added 2-methoxyacetic

acid (26.4 mg, 23 μL, 0.29 mmol), DIEA (66.9 mg, 90 μL, 0.52 mmol),

1-hydroxybenzotriazole (39.6 mg, 0.29 mmol), and HBTU (111.0 mg,

0.29 mmol). The reaction mixture was stirred at rt for 18 h. The resulting

solid was collected, washed with DMF (2 mL), MeOH (2 mL), and

CH₂Cl₂(3 mL), dried in vacuo, and salted as the monoesylate salt to

aﬀord 15 (70 mg, 64%). ¹H NMR (300 MHz, CD₃OD) δ 9.04 (d, J = 5.1

Hz, 2H), 8.92 (s, 1H), 8.86 (s, 1H), 8.59 (d, J = 2.1 Hz, 1H), 8.14 (m,

2H), 7.55 (t, J = 4.8 Hz, 1H), 4.34 (s, 2H), 3.62 (s, 3H), 2.80 (q, J = 7.5

Hz, 2 H), 1.30 (t, J = 7.5 Hz, 3 H) ppm. HRMS calcd. for (M + H)⁺, m/z:

379.1319, found m/z: 379.1318.

4-Bromo-2,3,6-triﬂuoro-aniline (49). To a solution of 2,3,6-

triﬂuoroaniline (20 g, 136.0 mmol) in DMF (300.0 mL) was added

N-bromosuccinimide (29.05 g, 163.2 mmol). The mixture was allowed

to stir at room temperature for 2 h, diluted with iPrOAc, washed with

water (3×) and brine, dried (Na₂SO₄), and concentrated in vacuo. The

residue was puriﬁed by column chromatography over silica gel (120 g)

and eluted with a 0 to 25% CH₂Cl₂/hexanes gradient to aﬀord 49

(22.36 g, 72%) as a brown crystalline solid. ¹H NMR (300 MHz, CDCl₃)

δ 7.02 (ddd, J = 9.8, 5.5, 2.5 Hz, 1H), 3.90 (s, 2H) ppm.

1

(2.61 g, 83%) as an oﬀ-white solid. H NMR (300 MHz, CDCl₃) δ

8.18−8.14 (m, 2H), 7.57−7.53 (m, 2H), 4.32 (s, 2H), 2.48 (t, J = 6.9 Hz,

2H), 2.37 (t, J = 7.0 Hz, 2H), 1.71−1.67 (m, 5H), 0.96 (t, J = 7.3 Hz,

6H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 186.8, 171.9, 167.8, 146.9,

144.9, 129.8, 123.2, 43.2, 39.9, 34.4, 18.3, 17.8, 13.6, 13.3 ppm. MS, m/z:

352.28 (M + H)⁺.

N-(5-(5-Fluoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-benzo[d]-

imidazol-2-l)butyramide (12). A suspension of 44 (400 mg, 1.42

mmol) and 46 (600 mg, 1.71 mmol), 1,4-dioxane (10 mL), and pH

3.5 buﬀer (10 mL, stock solution made from 1 N H₂SO₄and NaOAc)

was stirred for 8 h at reﬂux. The reaction mixture was cooled to room

temperature and ﬁltered, and the beige solid was washed successively

with water (50 mL) and MeOH (70 mL) and then dried in vacuo to

yield 12 (500 mg, 94%) as a beige solid, which was converted to its

monoesylate salt. ¹H NMR (300.0 MHz, CDCl₃) δ 9.00 (d, J = 4.9 Hz,

2H), 8.83−8.82 (m, 2H), 8.57 (d, J = 2.7 Hz, 1H), 7.94 (d, J = 1.6 Hz,

1H), 7.39 (t, J = 4.9 Hz, 1H), 3.07 (q, J = 7.4 Hz, 2H), 2.70 (t, J = 7.4 Hz,

2H), 1.85 (q, J = 7.4 Hz, 2H), 1.46 (t, J = 7.4 Hz, 3H), 1.07 (t, J = 7.4 Hz,

3H) ppm. HRMS calcd. for (M + H)⁺, m/z: 377.1526, found m/z:

377.1522.

1-Bromo-2,3,5-triﬂuoro-4-nitrobenzene (50). To a suspension

of sodium perborate tetrahydrate (26.02 g, 443 mmol) in glacial acetic

acid (450 mL) at 55 °C was added a solution of 4-bromo-2,3,6-triﬂuoro-

aniline (20 g, 88.5 mmol) in acetic acid (75 mL) dropwise. The mixture

was stirred at 55 °C for 2 h, and an additional 5 equiv of sodium

perborate tetrahydrate (25.5 g, 0.435 mol) was added. The reaction

mixture was stirred for 1.5 h and concentrated in vacuo. The residue was

diluted with water and extracted with diethyl ether (2×). The combined

organic extracts were washed with brine, dried (MgSO₄), and con-

centrated in vacuo. The residue was puriﬁed by column chromatography

over silica gel (120 g) and eluted with a 0 to 5% DCM/hexanes gradient

to aﬀord 8.53 g (38%) of 50 as an orange solid. ¹H NMR (300 MHz,

CDCl₃) δ 7.46−7.31 (m, 1H) ppm.

1-(3-Bromo-2,5-diﬂuoro-6-nitrophenyl)-1H-pyrazole (51a).

To an ice cold suspension of sodium hydride (1.68 g, 42.2 mmol) in

THF (90 mL) was added 1H-pyrazole (2.39 g, 35.2 mmol). The

reaction mixture was stirred for 20 min and then added to an ice cold

solution of 1-bromo-2,3,5-triﬂuoro-4-nitro-benzene (50, 9 g, 35.2

mmol) in THF (75 mL). The resulting mixture was stirred for 1.5 h,

quenched with water, and concentrated in vacuo. The residue was

extracted with diethyl ether, and the organic phase was dried (MgSO₄)

and concentrated in vacuo. The residue was chromatographed over

120 g of silica gel and eluted with a 0 to 25% ethyl acetate/petroleum

ether gradient to aﬀord 51 (8.25 g, 77%) as a light yellow solid. ¹H NMR

(300 MHz, CDCl₃) δ 7.93−7.84 (m, 1H), 7.79 (d, J = 1.5 Hz, 1H), 7.58

(dd, J = 8.2, 5.3 Hz, 1H), 6.62−6.54 (m, 1H) ppm.

1-(3-Bromo-2,5-diﬂuoro-6-nitrophenyl)-4-methyl-1H-pyra-

zole (51b). To an ice cold suspension of sodium hydride (1.58 g,

39.6 mmol) in THF (90 mL) was added 4-methyl-1H-pyrazole (2.71 g,

33.0 mmol). The mixture was stirred at 0 °C for 20 min and then added

to an ice cold solution of 1-bromo-2,3,5-triﬂuoro-4-nitrobenzene

(8.45 g, 33.0 mmol) in THF (75 mL). The reaction mixture was stirred

for 2 h, quenched with water, and concentrated under reduced pressure.

The residue was extracted with diethyl ether, and the organics were dried

(MgSO₄) and concentrated in vacuo. The residue was puriﬁed by

column chromatography over 120 g of silica gel and eluted with a 0 to

8% ethyl acetate/petroleum ether gradient to aﬀord 9.0 g (86%) of 51b

as a light orange solid. ¹H NMR (300 MHz, CDCl₃) δ = 7.63−7.57 (m,

1H), 7.54−7.44 (m, J = 8.2, 5.3, 1H), 2.16 (s, 2H) ppm.

5-(5-Fluoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-benzo[d]-

imidazol-2-amine (47). A mixture of 3 (15 g, 30.8 mmol) in aqueous

HCl (12 M, 150 mL) was heated in a sealed tube at 110 °C for 48 h.

The reaction mixture was cooled to 0 °C and brought to pH 8 by the

addition of 6 N aqueous NaOH. The white solid was collected by

ﬁltration, washed with water, and dried in vacuo to aﬀord 9 g (95%) of

47. ¹H NMR (300 MHz, DMSO-d₆) δ 11.12 (br s, 1H), 8.99 (d, J = 4.8

Hz, 2H), 8.81 (s, 1H), 8.53 (d, J = 2.7 Hz, 1H), 8.29 (d, J = 1.7 Hz, 1H),

8.07−8.02 (m, 1H), 7.69 (d, J = 1.7 Hz, 1H), 7.47 (t, J = 4.9 Hz, 1H),

6.56 (s, 2H) ppm. MS, m/z 307.04 (M + H)⁺.

tert-Butyl (2-((5-(5-Fluoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-

benzo[d]imidazol-2-yl)amino)-2-oxoethyl)(methyl)carbamate

(48). To a solution of 5-(5-ﬂuoro-3-pyridyl)-7-pyrimidin-2-yl-1H-

benzimidazol-2-amine (47, 60 mg, 0.20 mmol) in DMF were added

(benzotriazol-1-yloxy-dimethylamino-methylene)-dimethylammonium

hexaﬂuorophosphate (96.6 mg, 0.25 mmol), 1-hydroxybenzotriazole

(34.4 mg, 0.25 mmol), N-ethyl-N-isopropyl-propan-2-amine (58.2 mg,

78.5 μL, 0.45 mmol), and 2-(tert-butoxycarbonyl(methyl)amino)acetic

acid (48.2 mg, 0.25 mmol). The reaction mixture was stirred for 18 h

at room temperature. The solids were then ﬁltered, washed with DMF

(1 mL × 2), methanol (1 mL × 2), and dichloromethane (1 mL × 2),

and dried in vacuo to provide 48 (60 mg, 64%) as a white solid. MS, m/z:

478.40 (M + H)⁺.

N-(5-(5-Fluoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-benzo[d]-

imidazol-2-yl)-2-(methylamino)acetamide (13). To a solution of

48 (60 mg) in methanol (1 mL) and dichloromethane (1 mL) was

added 2 mL of 2.0 M hydrogen chloride in diethyl ether. The solution

was stirred at room temperature for 2 h. The resulting solid was collected

by ﬁltration, washed with DMF (1 mL × 2), methanol (1 mL × 2), and

dichloromethane (1 mL × 2), and dried in vacuo to aﬀord 13 (40 mg,

71%) as a white solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.19−8.14 (m,

3H), 8.03 (d, J = 1.7 Hz, 2H), 7.94 (dd, J = 1.4, 2.5 Hz, 1H), 7.28 (d,

J = 1.7 Hz, 1H), 6.64 (t, J = 4.9 Hz, 1H), 3.92 (d, J = 1.4 Hz, 2H), 3.40 (s,

H), 2.45−2.40 (m, H), 1.99 (d, J = 1.6 Hz, 3H) ppm. HRMS calcd. for

(M + H)⁺, m/z: 378.1479, found m/z: 378.1473.

Ethyl (5-(5-Fluoropyridin-3-yl)-7-(pyrimidin-2-yl)-1H-benzo-

[d]imidazol-2-yl)carbamate (14). A suspension of 44 (1.18 g,

4.20 mmol) and ethyl N-(N-ethoxycarbonyl-C-methylsulfanyl-

carbonimidoyl)carbamate (1.18 g, 5.04 mmol) in dioxane (25 mL)

R

dx.doi.org/10.1021/jm500563g | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

5-Bromo-4-ﬂuoro-2-nitro-3-(1H-pyrazol-1-yl)aniline (52a).

1-(3-Bromo-2,5-diﬂuoro-6-nitro-phenyl)pyrazole (51, 7.5 g, 24.67

mmol) was dissolved in 15 mL of ethanol and 7 M ammonia in

methanol (35.24 mL, 246.7 mmol). The reaction was stirred at 60 °C

overnight. An additional portion of 7 M ammonia in methanol

(35.24 mL, 246.7 mmol) was added, and stirring was continued for 24 h.

Another portion of ammonia in methanol was added, and stirring was

continued overnight. The reaction mixture was evaporated in vacuo to

aﬀord a bright yellow solid, which was recrystallized from dichloro-

methane/hexanes to aﬀord 5.25g of 52 as a bright yellow solid. A 1.31 g

second crop was collected to yield a total of 6.56 g (84%) of 52. ¹H NMR

(300 MHz, CDCl₃) δ 7.74−7.62 (m, 2H), 7.03 (d, J = 5.7 Hz, 1H),

6.48−6.42 (m, 1H), 4.99 (s, 2H) ppm.

5-Bromo-4-ﬂuoro-3-(4-methyl-1H-pyrazol-1-yl)-2-nitroaniline

(52b). A suspension of 1-(3-bromo-2,5-diﬂuoro-6-nitrophenyl)-4-

methyl-1H-pyrazole (9.0 g, 28.30 mmol) and ammonia in methanol

(40.4 mL of 7 M, 283.0 mmol) in ethanol (15 mL) was heated to 60 °C

and allowed to stir overnight. An additional 5 equiv of 7 M NH₃in

MeOH was added, and the mixture was heated to 80 °C for 48 h. The

reaction was cooled to rt and concentrated in vacuo. The residue was

diluted with water and extracted with diethyl ether (2×). The combined

organics were dried (MgSO₄) and concentrated in vacuo. The residue

was puriﬁed by column chromatography over 120 g of silica gel and

eluted with a 0 to 30% ethyl acetate−hexanes gradient to aﬀord 5.1 g

(56%) of 52b as a yellow solid. ¹H NMR (300 MHz, CDCl₃) δ = 7.61−

7.46 (m, 2H), 7.06 (d, J = 5.7, 1H), 4.97 (s, 2H), 2.17 (s, 3H) ppm.

4-Fluoro-2-nitro-3-(1H-pyrazol-1-yl)-5-(pyridin-3-yl)aniline (53).

A mixture of 52 (175 mg, 0.58 mmol), 3-pyridineboronic acid 1,3-

propanediol ester (114 mg, 0.70 mmol), and palladium tetrakis

triphenylphosphine (69 mg, 0.06 mmol) in 1.16 mL of 1 M NaHCO₃

and 4 mL of DME was stirred at 70 °C overnight. The mixture was

diluted with EtOAc, washed with water and brine, dried (MgSO₄), and

concentrated in vacuo. The residue was puriﬁed by column

chromatography over silica gel (30 g) and eluted with ethyl acetate−

hexanes, 3:1, then 4:1, then 5:1, to aﬀord 100 mg (57%) of 53 as an

orange solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.82 (m, 1H), 8.70 (m,

1H), 8.20 (m, 1H), 8.05 (m, 1H), 7.80 (m, 1H), 7.65 (m, 1H), 7.20 (m,

1H), 6.55, (s, 1H), 6.50 (br s, 2H) ppm.

(9.35 g, 95.3 mmol) was added dichlorobis(tricyclohexylphosphine)-

palladium(II) (5.0 mol %, 710 mg) in 1,4-dioxane (254 mL) . The mix-

ture was degassed for 15 min before and after addition of the catalyst,

heated at 130 °C for 18 h, cooled to ambient temperature, ﬁltered

through a pad of Celite, and washed three times with EtOAc. The

combined ﬁltrates were concentrated in vacuo to aﬀord 55 (19.3 g, 80%)

as green/brown solid. ¹H NMR (300 MHz, DMSO-d₆) δ 8.69 (s, 1H),

7.98 (dd, J = 7.7, 1.7 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 3.58 (d, J = 4.7

Hz, 4H), 2.46−2.35 (m, 4H), 1.31 (s, 12H) ppm.

1-Ethyl-3-(6-ﬂuoro-5-(6-(morpholinomethyl)pyridin-3-yl)-7-

(1H-pyrazol-1-yl)-1H-benzo[d]imidazol-2-yl)urea (18). A mixture

of 52a (300 mg, 1.0 mmol), 4-[[5-(4,4,5,5-tetramethyl-1,3,2-dioxabor-

olan-2-yl)-2-pyridyl]methyl]morpholine (568 mg, 1.5 mmol), Na₂CO₃

(1.5 mL of 2.0 M, 3.0 mmol), and Pd(PPh₃)₄(115 mg, 0.1 mmol) in

DME (5 mL) was degassed with N₂and heated to 80 °C for 14 h. The

reaction mixture was diluted with EtOAc, washed with water, ﬁltered

though a layer of Celite, and concentrated in vacuo. The crude product

was loaded onto a 12 g silica gel cartridge eluted with 0−10% MeOH/

DCM to aﬀord 4-ﬂuoro-5-[6-(morpholinomethyl)-3-pyridyl]-2-nitro-3-

pyrazol-1-yl-aniline (380 mg, 0.95 mmol, 96%). MS, m/z: 399.27 (M + H)⁺.

To a solution of 4-ﬂuoro-5-[6-(morpholinomethyl)-3-pyridyl]-2-nitro-

3-pyrazol-1-yl-aniline (380 mg, 0.95 mmol) in MeOH (30 mL) was

added Raney nickel (82 mg, 0.95 mmol). The reaction mixture was

shaken on a Parr apparatus for 2 h under 40 psi of hydrogen, ﬁltered

through a layer of Celite, and concentrated in vacuo to aﬀord 4-ﬂuoro-5-

[6-(morpholinomethyl)-3-pyridyl]-3-pyrazol-1-yl-benzene-1,2-diamine

(350 mg, 99%). MS, m/z: 369.3 (M + H)⁺. To a mixture of 4-ﬂuoro-5-

[6-(morpholinomethyl)-3-pyridyl]-3-pyrazol-1-yl-benzene-1,2-diamine

(350 mg, 0.97 mmol) and 1-ethyl-3-(N-(ethylcarbamoyl)-C-methyl-

sulfanyl-carbonimidoyl)urea (272 mg, 1.17 mmol) in dioxane (5 mL)

was added pH 3.5 buﬀer (12 mL). The mixture was heated at 90 °C

for 15 h. The reaction mixture was cooled to room temperature and

neutralized with saturated aqueous NaHCO₃. The resulting precipitate

was collected by ﬁltration, washed with H₂O, EtOAc, and Et₂O, and

dried in vacuo to aﬀord 18 as the free base, which was converted to the

bis-HCl salt (220 mg, 38%). ¹H NMR (300 MHz, DMSO-d₆) δ 8.89 (s,

1H), 8.34 (s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.93 (s, 1H), 7.82 (d, J = 8.0

Hz, 1H), 7.58 (t, J = 10.4 Hz, 2H), 6.65 (s, 1H), 4.58 (s, 2H), 3.90 (s,

3H), 3.26 (dt, J = 15.8, 10.9 Hz, 5H), 1.25−1.05 (m, 3H) ppm. HRMS

calcd. for (M + H)⁺, m/z: 465.2163, found m/z: 465.2157.

1-Ethyl-3-(6-ﬂuoro-7-(4-methyl-1H-pyrazol-1-yl)-5-(6-

(morpholinomethyl)pyridin-3-yl)-1H-benzo[d]imidazol-2-yl)urea

(19). To a suspension of 55 (2.41 g, 6.35 mmol), 52b (1.0 g, 3.17 mmol),

and Na₂CO₃(3.17 mL of 2.0 M, 6.35 mmol) was added Pd(PPh₃)₄

(183 mg, 0.159 mmol) in DME (10.6 mL). The mixture was degassed

for 10 min and then heated at 90 °C for 14 h. The mixture was cooled

to ambient temperature and partitioned between EtOAc and water. The

organic phase was dried (Na₂SO₄) and concentrated in vacuo. The

crude residue was puriﬁed by silica gel chromatography (0−10% 7 N

NH₃/MeOH/CH₂Cl₂gradient) to aﬀord the desired nitroaniline

adduct (1.09 g, 83%) as a dark brown oil. MS, m/z: 413.32 (M + H)⁺.

A suspension of the above nitroaniline (1.09 g, 2.643 mmol) and Raney

nickel (3.0 mL, suspension in water) in MeOH (18.8 mL) was placed

under 48 psi of hydrogen on a Parr shaker for 2 h. The reaction mixture

was ﬁltered through Celite, and the ﬁltrate was concentrated in vacuo to

aﬀord the diamine (0.83g, 82%) as a dark gray solid. MS, m/z: 383.38

(M + H)⁺. To a solution of the above diamine (0.83g, 2.168 mmol) and

1-ethyl-3-(N-(ethylcarbamoyl)-C-methylsulfanyl-carbonimidoyl)urea

(655 mg, 2.82 mmol) in 1,4-dioxane (12.0 mL) was added pH 3.5 buﬀer

(36 mL, stock solution prepared from 1 M H₂SO₄and NaOAc). The

resulting mixture was stirred at 90 °C for 16 h. Saturated sodium

bicarbonate was added to the reaction, and the mixture was stirred for

10 min. The resulting solid was collected by ﬁltration, washed twice with

water, and dried in vacuo to aﬀord 19 (0.64 g, 58%) as a beige solid. ¹H

NMR (300 MHz, DMSO-d₆) δ 8.68 (s, 1H), 8.11−7.89 (m, 2H), 7.65

(s, 1H), 7.56 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 6.2 Hz, 1H), 3.65 (s, 2H),

3.64−3.58 (m, 4H), 3.21 (dt, J = 13.9, 7.1 Hz, 2H), 2.49−2.43 (m, 4H),

2.14 (s, 3H), 1.11 (t, J = 7.2 Hz, 3H) ppm. HRMS calcd. for (M + H)⁺,

m/z: 479.2319, found m/z: 479.2316.

1-Ethyl-3-(6-ﬂuoro-7-(1H-pyrazol-1-yl)-5-(pyridin-3-yl)-1H-

benzo[d]imidazol-2-yl)urea (17). To a solution of 200 mg (0.67

mmol) of 53 in 20 mL of EtOAc was added 10% palladium on carbon

(50 mg). The mixture was shaken on a Parr apparatus under 45 psi of

hydrogen for 3h, ﬁltered over Celite, and concentrated in vacuo. To a

solution of the residue in 1.8 mL of 1,4-dioxane were added 1-ethyl-3-

(N-(ethylcarbamoyl)-C-methylsulfanyl-carbonimidoyl)urea (155 mg,

0.67 mmol) and 20 mL of pH 3.5 buﬀer. The mixture was heated at

95 °C overnight, cooled to room temperature, and neutralized by

addition of saturated aqueous NaHCO₃. The resulting solid was

collected by ﬁltration, washed with hot water (100 mL), and puriﬁed by

column chromatography over silica gel (5 to 100% of CH₂Cl₂/20%

1

MeOH/CH₂Cl₂) to aﬀord 17 (50 mg, 20%) as an oﬀ-white solid. H

NMR (300 MHz, CD₃OD) δ 8.79 (s, 1H), 8.57 (d, J = 4.7 Hz, 1H), 8.27

(br, 1H), 8.10 (dd, J = 7.7, 2.1 Hz, 1H), 7.89 (s, 1H), 7.65−7.45 (m,

2H), 6.62 (s, 1H), 4.84 (brs, 2H), 1.21 (t, J = 7.2 Hz, 3H) ppm. HRMS

calcd. for (M + H)⁺, m/z: 366.1479, found m/z: 366.1476.

4-[(5-Bromo-2-pyridyl)methyl]morpholine (54). To a solution

of 5-bromopyridine-2-carbaldehyde (25 g, 134.4 mmol) and morpho-

line (23.42 g, 23.4 mL, 268.8 mmol) in 1,2-dichloroethane (224 mL)

was added sodium triacetoxyborohydride (56.97 g, 268.8 mmol). The

resulting mixture was stirred at room temperature for 16 h and then

partitioned between EtOAc and saturated NaHCO₃. The organic phase

was washed with brine, dried (Na₂SO₄), and concentrated in vacuo. The

crude product was puriﬁed by silica gel chromatography (0−25%

EtOAc/hexanes gradient) to aﬀord 54 (21.7 g, 62%) as an oﬀ-white

crystalline solid. ¹H NMR (300 MHz, CD₃OD) δ 8.58 (d, J = 2.3 Hz,

1H), 7.97 (dd, J = 8.4, 2.3 Hz, 1H), 7.48 (d, J = 8.4 Hz, 1H), 3.76−3.65

(m, 4H), 3.61 (s, 2H), 2.54−2.41 (m, 4H) ppm.

4-((5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-

2-yl)methyl)morpholine (55). To a suspension of 54 (16.5 g,

63.5 mmol), bis(pinacolato)diboron (19.36 g, 76.2 mmol), and KOAc

S

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6-Methyl-2-oxo-1-(pyridin-2-ylmethyl)-1,2-dihydropyridin-

4-yl Triﬂuoromethanesulfonate (57). To a mixture of commercially

available 4-hydroxy-6-methyl-1-(2-pyridylmethyl)pyridin-2-one (56,

6.9 g, 31.91 mmol) and triethylamine (3.88 g, 5.34 mL, 38.3 mmol)

in DMF (50 mL) was added 1,1,1-triﬂuoro-N-phenyl-N-(triﬂuoro-

methylsulfonyl)methanesulfonamide (12.54 g, 35.1 mmol). The

reaction mixture was stirred at rt for 8 h and concentrated in vacuo.

The residue was puriﬁed by column chromatography over silica gel

(80 g) eluted with a 0 to 60% ethyl acetate/hexanes gradient to aﬀord

10.1 g (90%) of compound 57. MS, m/z: 348.84 (M + H)⁺.

crude yellow oil that was used without further puriﬁcation. MS, m/z:

182.16 (M + H)⁺(boronic acid).

1-Ethyl-3-(6-ﬂuoro-5-(6-(1-hydroxyethyl)pyridin-3-yl)-7-(1H-

pyrazol-1-yl)-1H-benzo[d]imidazol-2-yl)urea (21). 5-Bromo-4-

ﬂuoro-2-nitro-3-(1H-pyrazol-1-yl)aniline (52a, 3 g, 9.47 mmol),

4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

1,3,2-dioxaborolane (2.77 g, 10.9 mmol), dichloro-bis(tricyclo-

hexylphosphoranyl)palladium (350 mg, 0.47 mmol), and KOAc

(2.787 g, 28.40 mmol) were each split evenly among 3 microwave

tubes, suspended in 13 mL of solvent, and degassed for 10 min. The

reactions were heated for 45 min at 135 °C and cooled to room

temperature, ﬁltered, and concentrated in vacuo to aﬀord a dark brown

solid. This material was dissolved in minimal EtOAc and dropped into

an Erlenmeyer ﬂask containing hexanes stirring vigorously. The

resulting tan solid was ﬁltered and dried to aﬀord 550 mg of boronate.

MS, m/z: 267.20 (M + H)⁺(boronic acid). A mixture of 4-ﬂuoro-2-

nitro-3-pyrazol-1-yl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

aniline (200 mg, 0.57 mmol), 1-(5-bromo-2-pyridyl)ethanol (116 mg,

0.57 mmol), sodium hydrogen carbonate (960 μL of 1.2 M, 1.15 mmol),

and PdCl₂(Cy₃P)₂(50 mg, 0.06 mmol) in DMF (2 mL) was degassed

with a stream of nitrogen for 10 min. The reaction mixture was reﬂuxed

for 10 h, diluted with EtOAc, washed with brine, dried (Na₂SO₄), and

concentrated in vacuo. The residue was puriﬁed by column

chromatography over a 12 g silica gel cartridge and eluted with a 0 to

100% EtOAc/hexanes gradient to aﬀord the desired nitroaniline (70 mg,

36%). MS, m/z: 344.20 (M + H)⁺. A mixture of nitroaniline (70 mg,

0.204 mmol) and Raney nickel (20 mg, 0.20 mmol) in MeOH (20 mL)

was shaken for 1 h under 40 psi of hydrogen. The catalyst was removed

by ﬁltration though a layer of Celite. The ﬁltrate was concentrated

in vacuo the give the desired diamine (60 mg, 94%). MS, m/z: 314.20

(M + H)⁺. To a mixture of the above diamine (60 mg, 0.19 mmol) and

1-ethyl-3-(N-(ethylcarbamoyl)-C-methylsulfanyl-carbonimidoyl)urea

(53 mg, 0.23 mmol) in dioxane (1 mL) was added pH 3.5 buﬀer

(10 mL). The mixture was stirred for 15 h at 90 °C and neutralized with

saturated aqueous NaHCO₃. The resulting precipitate was collected by

ﬁltration, washed with H₂O and EtOAc, and dried in vacuo. This free

base of 21 was converted to the bis-HCl salt (56 mg, 0.11 mmol, 58%).

¹H NMR (300 MHz, CD₃OD) δ 9.04 (s, 1H), 8.86 (d, J = 8.3 Hz, 1H),

4-(5-Amino-2-ﬂuoro-3-(4-methyl-1H-pyrazol-1-yl)-4-nitro-

phenyl)-1-(pyridin-2-ylmethyl)pyridin-2(1H)-one (58). A mixture

of 5-bromo-4-ﬂuoro-3-(4-methyl-1H-pyrazol-1-yl)-2-nitroaniline (52b,

250 mg, 0.7934 mmol), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (302 mg, 1.190 mmol),

potassium acetate (233 mg, 2.38 mmol), and dichlorobis(tricyclo-

hexylphosphine)palladium(II) (30 mg, 0.04 mmol) in 2-Me THF

(3.2 mL) was degassed with a nitrogen stream in a 5 mL high-pressure

vial for 20 min. The tube was sealed and heated to 130 °C for 7 h. The

reaction mixture was then cooled to rt, ﬁltered through a pad of Florisil,

eluted with ethyl acetate and 20%MeOH/DCM, and concentrated in

vacuo. The resulting oil was dissolved in ethyl acetate, washed with

saturated NaHCO₃(Na₂SO₄), and concentrated in vacuo. The residue

was combined with [2-methyl-6-oxo-1-(2-pyridylmethyl)-4-pyridyl]

triﬂuoromethanesulfonate (276 mg, 0.793 mmol), LiCl (134 mg,

3.170 mmol), tetrakis(triphenylphosphine)palladium(0) (137 mg,

0.119 mmol), and sodium carbonate (595 μL of 2 M, 1.189 mmol) in

dimethoxyethane (4.7 mL) and degassed with a nitrogen stream for

20 min, and was then heated to 90 °C overnight. The mixture was cooled

to rt and partitioned between ethyl acetate and water. The organic phase

was washed with brine, dried (Na₂SO₄), and concentrated in vacuo. The

residue was puriﬁed by column chromatography over 12 g of silica gel

eluted with ethyl acetate to aﬀord 128 mg (37%) of 58. ¹H NMR (300

MHz, CDCl₃) δ 8.54 (d, J = 4.8 Hz, 1H), 7.66 (td, J = 7.7, 1.7 Hz, 1H),

7.54 (d, J = 4.2 Hz, 2H), 7.31 (d, J = 7.7 Hz, 1H), 7.24−7.10 (m, 1H),

6.86 (d, J = 6.1 Hz, 1H), 6.64 (s, 1H), 6.21 (s, 1H), 5.44 (s, 2H), 5.00 (s,

1H), 2.47 (s, 1H), 2.17 (s, 1H) ppm.

1-Ethyl-3-(5-ﬂuoro-4-(4-methyl-1H-pyrazol-1-yl)-6-(2-oxo-1-

(pyridin-2-ylmethyl)-1,2-dihydropyridin-4-yl)-1H-benzo[d]-

imidazol-2-yl)urea (20). To a solution of 58 (128 mg, 0.295 mmol) in

methanol (20 mL) was added Raney nickel (1.0 mL of suspension in

water). The mixture was placed under 45 psi of hydrogen in a Parr

apparatus and allowed to shake for 3 h. The catalyst was removed, and

the reaction mixture was ﬁltered and concentrated under vacuum. The

residue was combined with 1-ethyl-3-(N-(ethylcarbamoyl)-C-methyl-

sulfanyl-carbonimidoyl)urea (75.2 mg, 0.324 mmol), pH 3.5 buﬀer

(prepared with 57.5 g NaOAc·3H₂O and 400 mL of 1 N H₂SO₄), and

1,4-dioxane (1 mL) and was stirred at 105 °C for 8 h. The reaction

mixture was cooled to rt, diluted with water,and neutralized by adding

solid NaHCO₃. The mixture was allowed to stir for 20 min, and the

resulting precipitate was collected by ﬁltration and washed with water

(2×) and ethyl acetate to yield 91 mg (61%) of 20 as free base, which

was converted to the bis-HCl salt by dilution in CH₂Cl₂/MeOH,

8.48 (s, 1H), 8.22 (d, J = 8.4 Hz, 1H), 8.03 (s, 1H), 7.79 (d, J = 5.7 Hz,

1H), 6.74 (s, 1H), 5.32 (dd, J = 13.4, 6.7 Hz, 1H), 3.3−3.4 (q, 2H), 1.25

(t, 3H) ppm. HRMS calcd. for (M+H)⁺, m/z: 410.1741, found m/z:

410.1739.

1-Ethyl-3-(6-ﬂuoro-5-(6-(1-hydroxypropyl)pyridin-3-yl)-7-

(1H-pyrazol-1-yl)-1H-benzo[d]imidazol-2-yl)urea (22). A mixture

of crude 1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)-

propan-1-ol (60, 270 mg, 1.03 mmol), 5-bromo-4-ﬂuoro-2-nitro-3-(1H-

pyrazol-1-yl)aniline (52a, 325 mg, 1.03 mmol), sodium bicarbonate

(170 mg, 2.05 mmol), and PdCl₂(dppf) (84 mg, 0.10 mmol) was

microwaved for 20 min at 120 °C. The crude mixture was ﬁltered over

Celite, and the Celite pad was washed with ethyl acetate. The ﬁltrate was

concentrated to dryness and puriﬁed by column chromatography over

silica gel eluted with a 25 to 100% gradient of ethyl acetate in hexanes to

aﬀord 220 mg (60%) of the desired nitroaniline as a brown oil. MS, m/z:

358.36 (M + H)⁺. To a solution of the above nitroaniline (220 mg, 0.62

mmol) in methanol (15 mL) was added Raney nickel. The mixture was

shaken on a Parr apparatus under 40 psi of hydrogen for 90 min. The

reaction mixture was ﬁltered, concentrated to dryness, puriﬁed by

column chromatography over silica gel, and eluted with 10% methanol

in dichloromethane to aﬀord 81 mg (40%) of the diamine. MS, m/z:

328.28 (M + H)⁺. To the above diamine (81 mg, 0.247 mmol) in 4 mL

of a 2:1 mixture of pH 3.5 buﬀer (NaOAc/H₂SO₄/water) and dioxane

was added 3-(N-(ethylcarbamoyl)-C-methylsulfanyl-carbonimidoyl)-

urea (75 mg, 0.322 mmol) . The reaction was stirred at 90 °C for 2 h.

The mixture was cooled to room temperature and diluted with water.

The resulting brown precipitate was ﬁltered and dried to give 33 mg

(32%) of 22 as the free base. This free base was dissolved in a minimal

amount of methanol and treated with aqueous hydrochloric acid

(1.2 equiv of 6 N, 15 uL, 0.09 mmol). The resulting mixture was diluted

with water and lyophilized to aﬀord 36 mg (32%) of desired product

as the mono hydrochloride salt. ¹H NMR (300 MHz, DMSO-d₆) δ 8.93

1

addition of 4 N HCl/dioxane (0.5 mL), and lyophilization. H NMR

(300 MHz, DMSO-d₆) δ 10.49 (s,1H), 8.57 (d, J = 4.2, 1H), 8.10 (t, J =

6.7, 1H) 7.90 (s, 1H) 7.53 (d, J = 6.0, 1H), 7.43−7.25 (m, 4H), 6.48 (d,

J = 9.6, 2H), 5.40 (s, 2H), 3.30−3.12 (m, 2H), 2.43 (s, 3H), 2.15 (s, 3H),

1.11 (t, J = 7.2, 3H) ppm. HRMS calcd. for (M + H)⁺, m/z: 501.2163,

found m/z: 501.2159.

1-(5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)-

propan-1-ol (60). A mixture of commercially available 1-(5-bromopyridin-

2-yl)propan-1-ol (59, 400 mg, 1.85 mmol), bis(pinacolato)diboron

(541 mg, 2.13 mmol), potassium acetate (545 mg, 5.55 mmol), and

dichloro-bis (tricyclohexylphosphoranyl)palladium (69 mg, 0.09 mmol)

in 2-methyltetrahydrofuran (7.4 mL) was degassed under a nitrogen

stream for 10 min. The mixture was microwaved for 45 min at 135 °C,

cooled to rt, and ﬁltered over Celite. The ﬁltrate was concentrated to

dryness, dissolved in minimal ethyl acetate, and dropped into cold

hexanes while stirring vigorously. The resulting precipitate was removed

by ﬁltration, and the ﬁltrate was concentrated to aﬀord 620 mg of 60 as a

T

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(s, 1H), 8.62 (d, J = 8.2 Hz, 1H), 8.35 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H),

7.94 (d, J = 1.5 Hz, 1H), 7.73−7.54 (m, 2H), 6.60 (s, 1H), 4.96 (dd, J =

7.2, 4.8 Hz, 1H), 3.31−3.11 (m, 2H), 1.85 (dtd, J = 28.4, 13.7, 7.2 Hz,

2H), 1.22−1.04 (m, 3H), 0.94 (t, J = 7.3 Hz, 3H) ppm. HRMS calcd. for

(M + H)⁺, m/z: 424.1897, found m/z: 424.1896.

chromatography over silica gel eluted with a gradient of 20 to 100%

ethyl acetate in hexanes to aﬀord the intermediate nitroaniline (250 mg,

50.2%). ¹H NMR (300 MHz, CDCl₃) δ 9.20 (s, 1H), 8.39 (d, J = 2.2 Hz,

1H), 7.58 (d, J = 2.0 Hz, 1H), 7.28 (s, 1H), 7.13 (s, 2H), 4.93 (t, J = 7.2

Hz, 1H), 4.17 (dt, J = 8.2, 7.0 Hz, 1H), 4.00 (dt, J = 8.5, 6.9 Hz, 1H),

2.43−2.28 (m, 1H), 2.25−2.07 (m, 3H) ppm. MS, m/z: 287.23 (M +

H)⁺. To a solution of the above Suzuki adduct in MeOH and EtOAc

(∼100 mL; 1:2) was added 10% Pd/C (0.087 mmol). The reaction

mixture was stirred under H₂(45 psi) for 2 h. The depressurized mixture

was ﬁltered through Celite and concentrated to dryness to provide the

intermediate diamine (170 mg, 76%), which was used as is. MS, m/z:

257.18 (M + H)⁺. The above diamine and 3-(N-(ethylcarbamoyl)-C-

methylsulfanyl-carbonimidoyl)urea (154.1 mg, 0.66 mmol) were

dissolved in dioxane (3 mL) and pH 3.5 buﬀer (6 mL, stock solution

made from 1 N H₂SO₄and NaOAc). The mixture was reﬂuxed for 3 h,

cooled to ambient temperature, and neutralized by addition of 1 M

aqueous NaHCO₃. The resulting solids were collected by ﬁltration,

washed with hot water (200 mL), dried in vacuo, and salted as the

(+)-(S)-1-Ethyl-3-(6-ﬂuoro-5-(6-(1-hydroxypropyl)pyridin-3-

yl)-7-(1H-pyrazol-1-yl)-1H-benzo[d]imidazol-2-yl)urea (23) and

(−)-(R)-1-Ethyl-3-(6-ﬂuoro-5-(6-(1-hydroxypropyl)pyridin-3-yl)-

7-(1H-pyrazol-1-yl)-1H-benzo[d]imidazol-2-yl)urea (24). 150 mg

of 22 was separated by chiral SFC using 40% MeOH + 0.2% TEA @

170 mL/min on a Chiralpak IC (30 × 150); detection: 220 nm. 180 mg

of 23 and 84 mg of 24 were obtained as oﬀ-white solids. The purity of 23

was assessed at 99.4% using a Chiralpak IC (4.6 × 100), eluted with

40% MeOH + 0.2% TEA, detection: 220 nm, and ee calculated to be

99.9%, and the purity of 24 was 98.8%, with ee of 99.7%, using the same

analytical method. Both compounds were converted to their

monomesylate salt. Analytical data for 23: ¹H NMR (300 MHz,

CD₃OD) δ 8.85 (s, 1H), 8.50 (d, J = 8.4, 1H), 8.29 (t, J = 2.3, 1H), 7.95

(d, J = 8.4, 1H), 7.87 (d, J = 1.7, 1H), 7.54 (d, J = 5.9, 1H), 6.66−6.56 (m,

1H), 4.95 (dd, J = 7.5, 4.6, 2H), 3.32−3.28 (m, 2H), 2.74 (s, 3H), 2.11−

1.92 (m, 1H), 1.92−1.76 (m, 1H), 1.20 (t, J = 7.2, 3H), 1.04 (t, J = 7.4,

3H) ppm. HRMS calcd. for (M + H)⁺, m/z: 424.1897, found m/z:

424.1895. [α]_D²⁶= 16.0 (c 0.1, MeOH). Analytical data for 24: ¹H NMR

(300 MHz, TFA) δ 8.74 (s, 1H), 8.50 (d, J = 8.6, 1H), 8.13 (d, J = 2.5,

1H), 8.08 (d, J = 2.5, 1H), 7.97 (d, J = 5.6, 1H), 7.80 (d, J = 8.5, 1H), 6.75

(d, J = 2.5, 1H), 5.02 (dd, J = 8.1, 4.4, 1H), 3.09 (q, J = 7.2, 2H), 1.90−

1

monomesylate salt to aﬀord 26 (144 mg, 45%). H NMR (300 MHz,

CD₃OD) δ 9.19 (s, 1H), 9.13 (s, 2H), 7.81 (d, J = 1.5 Hz, 1H), 7.69−

7.59 (m, 1H), 5.28 (t, J = 7.3 Hz, 1H), 4.25 (dd, J = 14.4, 7.5 Hz, 1H),

4.03 (dd, J = 14.9, 7.5 Hz, 1H), 3.36 (dd, J = 9.6, 4.9 Hz, 2H), 2.75 (s,

3H), 2.58 (tt, J = 12.4, 6.3 Hz, 1H), 2.13 (dt, J = 14.9, 5.6 Hz, 2H), 1.95

(ddd, J = 16.5, 12.0, 8.1 Hz, 1H), 1.23 (t, J = 7.2 Hz, 3H) ppm. HRMS

calcd. for (M + H)⁺, m/z: 353.1726, found m/z: 353.1724.

2-Nitro-6-(tetrahydrofuran-2-yl)-4-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)aniline (63). A solution of 61²¹(3.91 g, 13.6

mmol), bis(pinacolato)diboron (3.80 g, 14.98 mmol), and potassium

acetate (4.01 g, 14.86 mmol) in 39 mL of DME was degassed with a stream

of nitrogen for 1 h. 1,1′-Bis(diphenylphosphino)ferrocenepalladium(II)

dichloride (556 mg, 0.68 mmol) was added, and the reaction mixture was

stirred at reﬂux for 1.5 h. The mixture was then allowed to cool to rt, diluted

with hexanes, ﬁltered through a plug of Florisil, eluted with EtOAc, and

concentrated to dryness in vacuo to aﬀord 6.47 g of a dark amber oil. This oil

was puriﬁed by ISCO silica gel chromatography eluting with 0 to 35%

EtOAc/hexanes to aﬀord 4.24 g (93%) of 63 as an orange glass. ¹H NMR

(300 MHz, CDCl₃) δ 8.6 (m, 1H), 7.7 (m, 1H), 7.1 (br s, 2H), 4.8 (m, 1H),

4.1 (m, 1H), 3.9 (m, 1H), 2.3−2.0 (m, 4H), 1.3 (s, 12H) ppm. MS, m/z:

335.45 (M + H)⁺.

1-Ethyl-3-(5-(1-methyl-2-oxo-1,2-dihydropyridin-4-yl)-7-

(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea (27). A

mixture of (1-methyl-2-oxo-4-pyridyl) triﬂuoromethanesulfonate (923 mg,

3.59 mmol), 2-nitro-6-tetrahydrofuran-2-yl-4-(4,4,5,5-tetramethyl-1,3,2-di-

oxaborolan-2-yl)aniline (63, 1000 mg, 2.99 mmol), LiCl (508 mg, 11.97

mmol), Pd(PPh₃)₄(519 mg, 0.45 mmol), and 2 M aqueous Na₂CO₃

(3 mL) in DME (25 mL) was purged with N₂for 2 min and then heated

at 100 °C for 48 h. The reaction mixture was then cooled to ambient

temperature, diluted with ethyl acetate, and ﬁltered through Celite. The

ﬁltrate was sequentially washed with saturated sodium bicarbonate,

water, and brine, dried (Na₂SO₄), and concentrated in vacuo. The

residue was puriﬁed by column chromatography over silica gel eluted

with a 1 to 10% gradient of MeOH in CH₂Cl₂to aﬀord the intermediate

nitroaniline (650 mg, 69%). MS, m/z: 315.32 (M + H)⁺. To the above

Suzuki adduct in MeOH and EtOAc (100 mL; 1:2) was added 10%

Pd/C (219 mg, 0.21 mmol). The reaction mixture was stirred under

45 psi of H₂for 4 h. The depressurized mixture was ﬁltered through

Celite and concentrated to dryness to provide the intermediate diamine

(400 mg, 68% yield). MS, m/z: 286.26 (M + H)⁺. The above diamine

and 3-(N-(ethylcarbamoyl)-C-methylsulfanyl-carbonimidoyl)urea (326

mg, 1.4 mmol) were dissolved in dioxane (6.5 mL) and pH 3.5 buﬀer

(13 mL, stock solution made from 1 N H₂SO₄and NaOAc). The

mixture was reﬂuxed for 4 h, cooled to ambient temperature, and

neutralized with 1 M aqueous NaHCO₃. The resulting solids were

collected by ﬁltration and washed with hot water (200 mL). The solids

were dried and salted as mono-HCl salt to aﬀord 27 (450 mg, 76%). ¹H

NMR (300 MHz, CD₃OD) δ 8.08 (d, J = 7.0 Hz, 1H), 7.87 (s, 1H), 7.68

(s, 1H), 7.22 (dd, J = 6.9, 1.8 Hz, 1H), 7.15 (s, 1H), 5.29 (t, J = 7.4 Hz,

1H), 4.26 (dd, J = 14.6, 7.3 Hz, 1H), 4.03 (dd, J = 14.7, 7.7 Hz, 1H), 3.82

(s, 2H), 3.42−3.33 (m, 2H), 2.60 (td, J = 12.1, 6.8 Hz, 1H), 2.24−2.04

1.57 (m, 2H), 0.94 (t, J = 7.3, 3H), 0.80 (t, J = 7.3, 3H) ppm. HRMS

26

calcd. for (M + H)⁺, m/z: 424.1897, found m/z: 424.1890. [α]_D

−15.0 (c 0.1, MeOH).

=

1-Ethyl-3-(5-(pyridin-3-yl)-7-(tetrahydrofuran-2-yl)-1H-

benzo[d]imidazol-2-yl)urea (25). A mixture of 4-bromo-2-nitro-6-

tetrahydrofuran-2-yl-aniline (61,²¹138 mg, 0.48 mmol), PdCl₂(PPh₃)₂

(10 mg, 0.024 mmol), 2 M sodium carbonate (0.43 mL), and 3-pyridyl-

diethylborane (92 mg, 0.62 mmol) in DME (4 mL) was heated at

140 °C in the microwave for 15 min. The reaction mixture was cooled to

ambient temperature, diluted with ethyl acetate, and ﬁltered through

Celite. The ﬁltrate was sequentially washed with saturated sodium

bicarbonate, water, and brine, dried (Na₂SO₄), and concentrated in

vacuo. The residue was puriﬁed by column chromatography over silica

gel and eluted with a gradient of 40 to 80% ethyl acetate in hexanes to

aﬀord the intermediate nitroaniline (100 mg, 73%). To a solution of the

nitroaniline (99 mg, 0.35 mmol) in MeOH (20 mL) was added a

catalytic amount of PtO₂. The reaction mixture was stirred under 1 atm

of H₂for 1 h, ﬁltered through Celite, and concentrated to dryness to

provide the intermediate diamine (70 mg, 78%), which was used as is.

The above diamine (70 mg, 0.27 mmol) and 3-(N-(ethylcarbamoyl)-C-

methylsulfanyl-carbonimidoyl)urea (135 mg, 0.54 mmol) were

dissolved in dioxane (3 mL) and pH 3.5 buﬀer (6 mL, stock solution

made from 1 N H₂SO₄and NaOAc). The mixture was reﬂuxed for 4 h,

cooled to ambient temperature, neutralized by addition of 1 M aqueous

NaHCO₃, and extracted with ethyl acetate 3 times. The combined

organic extracts were washed with water and brine, dried (MgSO₄), and

concentrated in vacuo. The residue was puriﬁed by column

chromatography over silica gel and eluted with a 10 to 20% gradient

of methanol in methylene chloride to aﬀord a brown oil. Crystallization

of this oil using acetonitrile/ethyl acetate aﬀorded 25 (33 mg, 35%) as a

yellow solid. ¹H NMR (CD₃OD, 500 MHz) δ 8.80 (s, 1H), 8.48 (dd,

1H), 8.10 (m, 1H), 7.58 (d, 1H), 7.50 (dd, 1H), 7.34 (br s, 1H), 5.30 (m,

1H), 4.23 (m, 1H), 4.01 (m, 1H), 3.34 (q, 2H), 2.52 (m, 1H), 2.13 (m,

2H), 1.98 (m, 1H), 1.22 (t, 3H) ppm. MS, m/z 352.2 (M + H)⁺.

1-Ethyl-3-(5-(pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-1H-

benzo[d]imidazol-2-yl)urea (26). A mixture of 4-bromo-2-nitro-6-

tetrahydrofuran-2-yl-aniline (61,²¹500 mg, 1.74 mmol), Pd(PPh₃)₄

(201.1 mg, 0.17 mmol), 2 M sodium carbonate (2.6 mL), and 5-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidine (538.2 mg, 2.61 mmol)

in DME (10.00 mL) was heated at 100 °C for 18 h. The reaction mixture

was cooled to ambient temperature, diluted with ethyl acetate, and

ﬁltered through Celite. The ﬁltrate was sequentially washed with

saturated sodium bicarbonate, water, and brine, dried (Na₂SO₄), and

concentrated in vacuo. The residue was puriﬁed by column

U

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(m, 2H), 1.94 (ddd, J = 16.5, 12.0, 8.0 Hz, 1H), 1.23 (t, J = 7.2 Hz, 3H)

ppm. HRMS calcd. for (M + H)⁺, m/z: 382.1879, found m/z: 382.1879.

1-Ethyl-3-(5-(6-methyl-2-oxo-1-(pyridin-2-ylmethyl)-1,2-di-

hydropyridin-4-yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]-

imidazol-2-yl)urea (28). A mixture of [2-methyl-6-oxo-1-(2-pyridyl-

methyl)-4-pyridyl] triﬂuoromethanesulfonate (57, 1.60 g, 4.55 mmol),

2-nitro-6-tetrahydrofuran-2-yl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-

lan-2-yl)aniline (63, 1.22 g, 3.64 mmol), LiCl (617 mg, 14.6 mmol),

Pd(PPh₃)₄(630 mg, 0.55 mmol), and 2 M aqueous Na₂CO₃(3.6 mL) in

DME (30 mL) was purged with N₂for 2 min and then heated at 100 °C

for 48 h. The reaction mixture was then cooled to ambient temperature,

diluted with ethyl acetate, and ﬁltered through Celite. The ﬁltrate was

sequentially washed with saturated sodium bicarbonate, water, and

brine, dried (Na₂SO₄), and concentrated in vacuo. The residue was

puriﬁed by column chromatography over silica gel eluted with a 1 to 10%

gradient of MeOH in CH₂Cl₂to aﬀord the intermediate nitroaniline

(1.2 g, 81%). MS, m/z: 407.25 (M + H)⁺. To the above Suzuki adduct in

MeOH and EtOAc (100 mL; 1:2) was added 10% Pd/C (314 mg,

0.30 mmol). The reaction mixture was stirred under H₂(45 psi) for 4 h.

The depressurized mixture was ﬁltered through Celite and concentrated

to dryness to provide the intermediate diamine (910 mg, 82%). MS,

m/z: 377.27 (M + H)⁺. The above diamine 3-(N-(ethylcarbamoyl)-C-

methylsulfanyl-carbonimidoyl)urea (3.20 g, 1.38 mmol) were dissolved

in 1,4-dioxane (6.5 mL) and pH 3.5 buﬀer (13 mL, stock solution made

from 1 N H₂SO₄and NaOAc). The mixture was reﬂuxed for 4 h, cooled

to ambient temperature, and neutralized with 1 M aqueous NaHCO₃.

The resulting solids were collected by ﬁltration, washed with hot water

(200 mL), and dried to aﬀord 28 (450 mg, 76%). ¹H NMR (300 MHz,

CDCl₃) δ 8.59 (d, J = 5.1 Hz, 1H), 7.67 (td, J = 7.9, 1.3 Hz, 2H), 7.21(dd,

J = 14.1, 5.8 Hz, 4H), 6.63 (d, J = 1.3 Hz, 1H), 5.51 (s, 2H), 5.20 (d, J =

7.5 Hz, 1H), 4.31−4.13 (m, 1H), 4.06−3.87 (m, 1H), 3.42−2.99 (m,

2H), 2.59−2.31 (m, 4H), 2.04 (dd, J = 19.4, 13.0 Hz, 3H), 1.04 (t, J = 7.2

Hz, 3H) ppm. MS, m/z: 473.32 (M + H)⁺.

2-(5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-

yl)propan-2-ol (65). A microwave tube was charged with commer-

cially available 2-(5-bromo-2-pyridyl)propan-2-ol (796 mg, 3.684

mmol), bis(dipinacolato)diboron (982 mg, 3.87 mmol), dichloro-bis

(tricyclohexylphosphoranyl) palladium (136 mg, 0.18 mmol), KOAc

(1.08 g, 11.1 mmol), and 2-methyltetrahydrofuran (14.9 mL). The

mixture was degassed for 10 min and then heated in a microwave for

45 min at 135 °C. The crude mixture was allowed to cool and was

ﬁltered on Florisil (eluted with CH₂Cl₂/MeOH). The ﬁltrate was

washed with saturated aqueous NaHCO₃, dried (MgSO₄), and con-

centrated in vacuo. The residue was then used without further

puriﬁcation. MS, m/z: 181.93 (M + 1)⁺(boronic acid).

1-Ethyl-3-(5-(6-(2-hydroxypropan-2-yl)pyridin-3-yl)-7-(tetra-

hydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea (29). A mixture

of crude 2-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]-

propan-2-ol (65, 500 mg, 1.9 mmol), 4-bromo-2-nitro-6-tetrahydrofur-

an-2-yl-aniline (61, 469 mg, 1.58 mmol), Pd(PPh₃)₄(183 mg, 0.158

mmol), and sodium carbonate (2.37 mL of 2 M, 4.75 mmol) in 1,2-

dimethoxyethane (10 mL) was heated at reﬂux for 8 h. The dark yellow

reaction mixture was cooled to rt and diluted with water, saturated

aqueous NaHCO₃, and CH₂Cl₂. The phases were separated and dried

(MgSO₄), and the organics were concentrated in vacuo. The residue was

puriﬁed by ISCO (40 g SiO₂, CH₂Cl₂to 60% EtOAc in CH₂Cl₂) to yield

259 mg of the desired product (257 mg, 48%) as a yellow oil that

solidiﬁed upon standing. MS, m/z: 344.23 (M + 1)⁺. A solution of the

above nitroaniline (259 mg, 0.75 mmol) in MeOH (20 mL)/EtOAc

(30 mL) was placed ion a Parr ﬂask, and 10% Pd/C (wet, Degussa,

200 mg, 1.88 mmol) was added. The mixture was shaken in a Parr apparatus

under 45 psi of hydrogen for 4 h. The reaction mixture was ﬁltered and

concentrated in vacuo to aﬀord the desired diamine (230 mg, 97%), which

was used as is. MS, m/z: 314.25 (M + 1)⁺. To a solution of crude diamine

(230 mg, 0.734 mmol) in 1,4-dioxane (4.4 mL) was added 1-ethyl-3-(N-

(ethylcarbamoyl)-C-methylsulfanylcarbonimidoyl)urea (222 mg, 0.954

mmol) and pH 3.5 buﬀer (10 mL). The reaction solution was stirred at

90 °C for 10 h, cooled to rt, diluted with water (∼20 mL), and

neutralized with saturated aqueous NaHCO₃(∼15 mL). The resulting

turbid reaction mixture was extracted twice with CH₂Cl₂/MeOH (9:1).

The organics were dried (MgSO₄) and concentrated in vacuo. The

residue was dissolved in CH₂Cl₂and puriﬁed by ISCO (24 g SiO₂, DCM

to 4% 7 N NH₃/MeOH in DCM) to aﬀord a beige solid, which was

triturated in hot Et₂O to yield the free base of 29 (140 mg, 47%) as an

1

oﬀ-white solid. This was converted to the bis mesylate salt. H NMR

(300 MHz, CD₃OD) δ 8.95 (d, J = 1.6 Hz, 1H), 8.90 (dd, J = 8.6, 2.2 Hz,

1H), 8.23 (d, J = 8.6 Hz, 1H), 7.92 (d, J = 1.5 Hz, 1H), 7.72 (d, J = 0.8

Hz, 1H), 5.31 (t, J = 7.3 Hz, 1H), 4.26 (dd, J = 14.4, 7.5 Hz, 1H), 4.03

(dd, J = 14.9, 7.6 Hz, 1H), 3.36 (dd, J = 12.0, 4.8 Hz, 2H), 2.73 (s, 6H),

2.61 (dd, J = 12.0, 5.1 Hz, 1H), 2.12 (ddd, J = 9.7, 7.3, 4.3 Hz, 2H), 2.04−

1.88 (m, 1H), 1.73 (s, 6H), 1.22 (dd, J = 11.4, 4.1 Hz, 3H) ppm. HRMS

calcd. for (M + H)⁺, m/z: 410.2192, found m/z: 410.2188.

1-Ethyl-3-(5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-

(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea (30). Amix-

ture of 4-bromo-2-nitro-6-tetrahydrofuran-2-yl-aniline (61, 500 mg,

1.74 mmol), Pd(PPh₃)₄(201 mg, 0.17 mmol), 2 M aqueous sodium

carbonate (2.6 mL), and commercially available 2-(5-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-yl)propan-2-ol (608 mg,

2.30 mmol) in DME (12.50 mL) was heated at 100 °C for 18 h. The

reaction mixture was then cooled to ambient temperature, diluted with ethyl

acetate, and ﬁltered through Celite. The ﬁltrate was sequentially washed

with saturated sodium bicarbonate, water, and brine, dried (Na₂SO₄), and

concentrated in vacuo. The residue was puriﬁed by column chromatog-

raphy over silica gel eluted with a 20 to 100% gradient of EtOAc in hexanes

to aﬀord the intermediate nitroaniline (900 mg, 75%). ¹H NMR (300 MHz,

CDCl₃) δ 8.89 (s, 2H), 8.36 (d, J = 2.2 Hz, 1H), 7.57 (d, J = 2.1 Hz, 1H),

7.11 (s, 2H), 4.93 (t, J = 7.2 Hz, 1H), 4.68 (s, 1H), 4.15 (ddd, J = 10.2, 7.7,

5.4 Hz, 1H), 4.04−3.94 (m, 1H), 2.43−2.26(m, 1H), 2.16(ddd, J = 9.4, 6.5,

1.9 Hz, 3H), 1.64 (s, 6H) ppm. MS, m/z: 345.18 (M + H)⁺. To the above

Suzuki adduct in MeOH and EtOAc (∼100 mL; 1:2) was added 10% Pd/C

(278 mg, 0.26 mmol). The reaction mixture was stirred under H₂(45 psi)

for 2 h. The depressurized mixture was then ﬁltered through Celite and

concentrated to dryness to provide the intermediate diamine (565 mg,

69%). MS, m/z: 315.26 (M + 1)⁺. The above diamine and 1-ethyl-3-(N-

(ethylcarbamoyl)-C-methylsulfanylcarbonimidoyl)urea (417 mg, 1.80

mmol) were dissolved in dioxane (6 mL) and buﬀer pH 3.5 (18 mL,

stock solution made from 1 N H₂SO₄and NaOAc). The resulting mixture

was reﬂuxed for 3 h, cooled to ambient temperature, and neutralized with

1 M aqueous NaHCO₃. The resulting beige solids were collected by

ﬁltration, washed with hot water (200 mL), dried in vacuo, and salted as the

monomesylate salt to aﬀord 30 (576 mg, 62% yield). ¹H NMR (300 MHz,

CD₃OD) δ 9.07 (s, 2H), 7.79 (d, J = 1.5 Hz, 1H), 7.62 (s, 1H), 5.41−5.21

(m, 1H), 4.32−4.17 (m, 1H), 4.04 (dd, J = 14.7, 8.0 Hz, 1H), 3.38 (d, J = 7.2

Hz, 2H), 2.72 (s, 3H), 2.66−2.52 (m, 1H), 2.12 (d, J = 7.6 Hz, 2H), 2.03−

1.89 (m, 1H), 1.64 (s, 6H), 1.23 (t, J = 7.2 Hz, 3H) ppm. HRMS calcd. for

(M + H)⁺, m/z: 411.2145, found m/z: 411.2140.

Urea 30 (576 mg) was dissolved in 23 mL of methanol (solution

concentration of 25 mg/mL) and separated into its two enantiomers

using chiral SFC (preparative conditions: 50% MeOH + 0.2% DEA @

170 mL/min on an IC (30*150), 100 bar, 35C, 220 nm, 3.0 mL

injections). The enantiomers were salted to aﬀord compound 32

(223 mg, 48.5%, ee 100%) and 31 (228 mg, 48.6%, ee 100%) as the

monomesylate salts (analytical conditions: 40% MeOH + 0.2% DEA @

5 mL/min on an IC (4.6*100), 100 bar, 35C, 220 nm; ee’s calculated

using analytical chromatogram peak areas).

(−)-(S)-1-Ethyl-3-(5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-

(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea (32). ¹H

NMR (300 MHz, CD₃OD) δ 9.08 (s, 2H), 7.80 (d, J = 1.2 Hz, 1H),

7.62 (s, 0H), 5.30 (t, J = 7.4 Hz, 1H), 4.24 (dd, J = 14.5, 7.5 Hz, 1H), 4.04

(dd, J = 14.7, 7.7 Hz, 1H), 3.36 (dd, J = 10.3, 4.1 Hz, 2H), 2.72 (s, 3H),

2.67−2.51 (m, 1H), 2.28−2.04 (m, 2H), 2.03−1.88 (m, 1H), 1.64 (s,

6H), 1.23 (t, J = 7.2 Hz, 3H) ppm. HRMS calcd. for (M + H)⁺, m/z:

411.2145, found m/z: 411.2143. [α]_D²⁵= −17.0 (c 0.1, MeOH).

(+)-(R)-1-Ethyl-3-(5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-

yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea

(31). ¹H NMR (300 MHz, CD₃OD) δ 9.08 (s, 2H), 7.80 (d, J = 1.3 Hz,

1H), 7.70−7.43 (m, 1H), 5.42−5.21 (m, 1H), 4.25 (d, J = 7.5 Hz, 1H),

4.03 (d, J = 6.8 Hz, 1H), 3.36 (dd, J = 10.2, 4.3 Hz, 2H), 2.73 (s, 3H),

2.59 (s, 1H), 2.29−2.05 (m, 2H), 1.96 (m, 1H), 1.64 (s, 3H), 1.23

V

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(t, J = 7.2 Hz, 3H) ppm. HRMS calcd. for (M + H)⁺, m/z: 411.2145,

found m/z: 411.2142. [α]_D²⁶= 17.0 (c 0.1, MeOH).

described by the Clinical Laboratory and Standards Institute (CLSI²³)

with modiﬁcations as described previously.²³To 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.

In Vitro Covalent Binding Experiments. Thirty micromolar

¹⁴C-radiolabeled 3 (speciﬁc activity: 0.43 mCi/mmole) was incubated

with microsomes from rat, dog, monkey, and mouse liver in the presence

of NADPH for 1 h. Incubation was quenched with acetonitrile, and the

precipitated protein was washed exhaustively until the supernatant from

the washes were devoid of unbound radioactivity. The remaining

protein pellet was dissolved in 1 N NaOH in a water bath at 60 °C for at

least 1 h or until completely dissolved. Ten microliters of the dissolved

sample was then analyzed on a liquid scintillation counter to determine

the amount of covalently bound radioactivity. An aliquot of the dissolved

protein was reserved for determination of protein concentration using

the Bradford assay. The amount of radioactivity was normalized to the

protein content in every sample and expressed as picomoles per

milligram of protein.

In Vivo Covalent Binding Experiments. Unlabeled 3 was dosed

to male rats at 90 mg/kg for 6 or 13 days. On day 7 or 14, the animals

received 100 μCi of ¹⁴C-labeled 3 (speciﬁc activity: 55mCi/mmol). A

separate cohort of rats received a single dose of the radiolabeled

compound. Four hours after the radiolabeled compound was dosed,

all animals were euthanized, and their livers were removed and weighed.

An aliquot of the livers was homogenized in 0.1 M phosphate buﬀer

using a Potter-Elvehjem tissue grinder. The liver homogenate was then

centrifuged at 9000g to separate the S9 fraction. An aliquot of the S9

fraction was then washed exhaustively with acetonitrile until the

supernatant was devoid of unbound radioactivity, as determined by

liquid scintillation counting. The remaining protein pellet was dissolved

in 1 N NaOH in a water bath at 60 °C for at least 1 h or until completely

dissolved. Ten microliters of the dissolved sample was then analyzed on

a liquid scintillation counter to determine the amount of covalently

bound radioactivity. An aliquot of the dissolved protein was reserved for

determination of protein concentration using the Bradford assay. The

amount of radioactivity was normalized to the protein content in every

sample and expressed as picomoles per milligram of protein. A similar

experiment was conducted with compound 34.

1-Ethyl-3-(6-ﬂuoro-5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-

yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)urea

(33). A suspension of 4-bromo-3-ﬂuoro-6-nitro-2-tetrahydrofuran-2-yl-

aniline (62,²¹9.41 g, 30.8 mmol), 2-[5-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)pyrimidin-2-yl]propan-2-ol (8.96 g, 33.9 mmol),

saturated aqueous NaHCO₃(77 mL, 93 mmol), and tetrakis-

(triphenylphosphine) palladium(0) (1.78 g, 1.54 mmol) in 1,4-dioxane

(94 mL) was stirred at 108 °C overnight, cooled to rt, and diluted with

ethyl acetate. The organics were washed with water and brine, dried

(MgSO₄), and concentrated in vacuo. To a stirred solution of the residue

in 20 mL of ethanol was added 100 mL of hexanes. After 1 h of stirring,

the precipitate was collected, washed with hexanes, and dried in vacuo

to aﬀord the intermediate nitroaniline (8.88 g, 79%). MS, m/z: 365.25

(M + 1)⁺. To a solution of nitroaniline (8.88 g, 24.5 mmol) in methanol

(44 mL) was added triethylamine (6.8 mL, 49 mmol) and 5% Pd/C

(50% wet, 1.78 g). The reaction mixture was placed on a Parr shaker

under 45 psi of hydrogen overnight. Filtration through Celite and

puriﬁcation by column chromatography (SiO₂, ethyl acetate) aﬀorded

the intermediate diamine (7.22 g, 89%). MS, m/z: 333.34 (M + 1)⁺. To

the diamine (7.22 g, 21.7 mmol) in 1,4-dioxane (36 mL) was added pH

3.5 buﬀer (72 mL). After addition of 1-ethyl-3-(N-(ethylcarbamoyl)-C-

methylsulfanyl-carbonimidoyl)urea (41, 6.05 g, 26.1 mmol), the

reaction was heated at 108 °C for 1.5 h. Following cooling to rt and

addition of aqueous sodium bicarbonate solution (144 mL), the mixture

was ﬁltered, and the collected solid was washed twice with water (2 × 10 mL)

and dried in vacuo to aﬀord 33 (7.90 g, 85%). MS, m/z: 429.45 (M + 1)⁺.

Urea 33 (7.50 g) was dissolved in 2330 mL of methanol/methylene

chloride 1:1 (solution concentration of 3 mg/mL) and, after ﬁltra-

tion through a 0.2 μm nylon membrane, was separated into its two

enantiomers using chiral SFC (preparative conditions: 70% MeOH +

0.2% DEA at 175 mL/min on an IC (30*150), 100 bar, 35C, 220 nm,

12 mL injections) to aﬀord 3.72 g (40%) of (+)-(R)-1-ethyl-3-(6-ﬂuoro-

5-(2-(2-hydroxypropan-2-yl)pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-

1H-benzo[d]imidazol-2-yl)urea (34), with an ee of 100%, and 3.70 g

(40%) of (−)-(S)-1-ethyl-3-(6-ﬂuoro-5-(2-(2-hydroxypropan-2-yl)-

pyrimidin-5-yl)-7-(tetrahydrofuran-2-yl)-1H-benzo[d]imidazol-2-yl)-

urea (35), with an ee of 100% (analytical conditions: 70% MeOH + 0.2%

DEA at 5 mL/min on an IC (4.6*100), 100 bar, 35C, 220 nm; ee’s

calculated using analytical chromatogram peak areas). Analytical data for

34: ¹H NMR (300 MHz, CD₃OD) δ 8.95 (d, J = 1.6 Hz, 2H), 7.45 (d, J =

6.4 Hz, 1H), 5.44−5.32 (m, 1H), 4.27 (q, J = 7.3 Hz, 1H), 4.01 (q, J = 7.3

Hz, 1H), 3.36−3.30 (m, 2H), 2.65−2.49 (m, 1H), 2.20−2.06 (m, 2H),

2.00−1.83 (s, 1H), 1.63 (s, 6H), 1.21 (t, J = 7.2 Hz, 3H) ppm. ¹³C NMR

(125 MHz, DMSO-d₆) δ 173.18, 156.28, 156.26, 152.61 (d, J = 240.87

Hz), 152.18, 146.99, 128.03, 126.69, 117.96 (d, J = 16.25 Hz), 115.72 (d,

J = 20 Hz), 112.80, 74.42, 72.64, 68.20, 34.46, 32.74, 29.59, 25.43, 14.74

ppm. HRMS calcd. for (M + H)⁺, m/z: 429.2050, found m/z: 429.2039.

[α]_D²⁶= 16 (c 0.1, MeOH). Analytical data for 35: ¹H NMR (300 MHz,

CD₃OD) δ 8.94 (d, J = 1.6 Hz, 2H), 7.44 (d, J = 6.4 Hz, 1H), 5.44−5.33

(m, 1H), 4.27 (q, J = 7.3 Hz, 1H), 4.01 (q, J = 7.3 Hz, 1H), 3.36−3.30

(m, 2H), 2.65−2.48 (m, 1H), 2.20−2.06 (m, 2H), 2.00−1.4 (s, 1H),

1.62 (s, 6H), 1.20 (t, J = 7.2 Hz, 3H) ppm. HRMS calcd. for (M + H)⁺,

m/z: 429.2050, found m/z: 429.2039. [α]_D²⁶= −14.0 (c 0.1, MeOH).

K_iDetermination. The details of the enzyme inhibition assays can

be found in ref 23.

Incubations in Supersomes. Compound 3 (1 and 10 μM) was

incubated with individual recombinant CYP supersomes in the presence

and absence of NADPH. The incubations were carried out for either 0,

15, and 30 min. Samples were analyzed by LC-MS/MS, and the forma-

tion of compound 10 was quantiﬁed.

Description of the Neutropenic Rat Thigh Infection Model.

The methicillin-susceptible S. aureus (MSSA) strain ATCC 29213 was

obtained from the American Type Culture Collection (ATCC). The

frozen bacterial test isolate was subcultured twice onto standard

microbiological agar media (trypticase soy agar with 5% sheep blood)

prior to inoculation. The second plating occurred less than 24 h before

use in the thigh infection model. Immediately prior to injection into

thighs, several bacterial colonies were suspended in 0.9% saline to

approximate a 0.5 McFarland turbidity standard and then diluted to

the target concentration of 10⁷colony forming units (CFU)/mL for

inoculation. Several dilutions of this inoculum were plated again to verify

the infection dose. Neutropenia was induced via an intraperitoneal

injection of 150 mg/kg of the immunosuppressant cyclophosphamide

(Baxter Healthcare Corp.) at a volume of 1 mL per animal 3 days prior to

bacterial infection. On day 0, the rats (121−159 g) were infected by an

intramuscular injection (0.2 mL) into both rear thighs using a sus-

pension of 10⁷CFU/mL S. aureus ATCC 29213 in normal saline.

Compound dosing commenced at the designated time 0 at 2 h post-

inoculation, when colonization was expected to be stable. Three rats

(n = 3) per thigh collection time point (8 and 24 h post treatment

initiation) were administered one of the following ﬁve test formulations:

vehicle (20% cavitron/1% HPMCAS-MG), 34 at 10, 30, or 60 mg/kg in

vehicle, or moxiﬂoxacin (Moxi) at 30 mg/kg in saline. Each treatment

Small Molecule X-ray Structure Determination. Colorless

crystals of 34 were obtained by crystallization from ethanol. A crystal

with dimensions of 0.15 × 0.15 × 0.10 mm³was mounted on a Bruker

APEX II CCD diﬀractometer with Cu Ka radiation. Diﬀraction data

were collected to a resolution of 0.85 Å using 0.5° steps and 30 s

exposure for each frame. Data were collected at 100 (2) K using a

nitrogen open-ﬂow system. Integration of intensities and reﬁnement of

cell parameters were accomplished using APEX software. The structure

was solved by direct methods and reﬁned by the SHELXTL package.

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

W

dx.doi.org/10.1021/jm500563g | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

was administered via oral gavage in a 10 mL/kg dosing volume at 0 and

12 h (q12h). Thigh tissues from untreated controls were harvested 2 h

after infection at time 0 (early control, EC). Thighs from vehicle- (late

controls, LC) and compound-treated animals were harvested after 8 or

24 h of treatment. Both rear thighs (n = 2) of each animal were

harvested, rinsed with sterile saline, weighed, placed in 50 mL sterile

normal saline, and homogenized. Approximately half of the unﬁltered

homogenate was frozen for subsequent drug concentration analysis. The

other half of the homogenized sample volume was passed through a

large pore ﬁlter to remove cartilage and large clumped pieces of tissue.

The ﬁltrate was then 10-fold serially diluted in saline, cultured onto agar

media plates (trypticase soy agar with 5% sheep blood), and incubated at

37 °C for 18−24 h. Colony forming units (CFU) were enumerated as

CFU/mL homogenate, and the median for each treatment group (n = 6,

i.e., two thighs from three animals each) was calculated. As a measure of

thigh burden, the median CFU/mL thigh homogenate for each

compound-treated group was compared to both the initial (time 0)

bacterial density (EC) and that of the harvest time-matched vehicle

controls (LC). To obtain statistics, the data were analyzed using a

Kruskal−Wallis nonparametric test followed by a Dunn’s post-test to

compare all data sets in an experiment (Prism Software). Signiﬁcant

diﬀerences were noted with their p values represented as follows: *,

p < 0.05; **, p < 0.01; ***, p < 0.001.

REFERENCES

■

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ASSOCIATED CONTENT

■

S

* Supporting Information

Two-dimensional NMR characterization of compound 10;

coelution data of synthesized compound 10 and M − 2

metabolite; small molecule X-ray structure of compound 34;

MS/MS data supporting the structure of the M + 14 metabolites

of compound 3, and of the metabolites of compound 34.

This material is available free of charge via the Internet at

http://pubs.acs.org.

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AUTHOR INFORMATION

■

Corresponding Author

*E-mail: Anne-Laure_Grillot@vrtx.com.

Present Addresses

^†Arnaud Le Tiran: Institut de Recherche Servier, 3 rue de la

́

Republique, 92150 Suresnes, France.

^‡Dean Shannon: DE Synthetics, 30 Dineen Drive, Fredericton,

NB, E3B 5A3, Canada.

^§Trudy H. Grossman: Tetraphase Pharmaceuticals, Inc., 480

Arsenal Street, Suite 110, Watertown, Massachusetts 02472,

United States.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

The authors gratefully acknowledge Jon Parsons for obtaining

K_i’s against gyrase and topoisomerase IV for compounds

included in this manuscript, Brian Krueger for developing the

SFC conditions allowing the separation of enantiomers reported

in this manuscript, John Pietryka for help with NMR spectra

analysis, and Mariusz Krawiec for solving the small molecule

X-ray structures of compounds 31 and 34. The authors also

gratefully acknowledge Sue Stokes for reviewing this manuscript.

X

dx.doi.org/10.1021/jm500563g | J. Med. Chem. XXXX, XXX, XXX−XXX

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dx.doi.org/10.1021/jm500563g | J. Med. Chem. XXXX, XXX, XXX−XXX

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Second-generation antibacterial benzimidazole ureas: Discovery of a preclinical candidate with reduced metabolic liability

DOI: 10.1021/jm500563g

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