Fragment-to-hit-to-lead discovery of a novel pyridylurea scaffold of ATP competitive dual targeting type II topoisomerase inhibiting antibacterial agents

Article

pubs.acs.org/jmc

Fragment-to-Hit-to-Lead Discovery of a Novel Pyridylurea Scaﬀold of

ATP Competitive Dual Targeting Type II Topoisomerase Inhibiting

Antibacterial Agents

Gregory S. Basarab,*^,†John I. Manchester,^†Shanta Bist,^†P. Ann Boriack-Sjodin,^†,§Brian Dangel,^†

Ruth Illingworth,^†Brian A. Sherer,^†,∥Shubha Sriram,^†Maria Uria-Nickelsen,^†,‡and Ann E. Eakin^†,⊥

^†Infection Innovative Medicines, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States

ABSTRACT: The discovery and optimization of a new class of bacterial topoisomerase (DNA gyrase and topoisomerase IV)

inhibitors binding in the ATP domain are described. A fragment molecule, 1-ethyl-3-(2-pyridyl)urea, provided suﬃciently potent

enzyme inhibition (32 μM) to prompt further analogue work. Acids and acid isosteres were incorporated at the 5-pyridyl position

of this fragment, bridging to a key asparagine residue, improving enzyme inhibition, and leading to measurable antibacterial

activity. A CF₃-thiazole substituent at the 4-pyridyl position improved inhibitory potency due to a favorable lipophilic interaction.

Promising antibacterial activity was seen versus the Gram-positive pathogens Staphylococcus aureus and Streptococcus pneumoniae

and the Gram-negative pathogens Haemophilus inﬂuenzae and Moraxella catarrhalis. Precursor metabolite incorporation and

mutant analysis studies support the mode-of-action, blockage of DNA synthesis by dual target topoisomerase inhibition.

Compound 35 was eﬃcacious in a mouse S. aureus disease model, where a 4.5-log reduction in colony forming units versus

control was demonstrated.

IV) contain the ATP binding and hydrolysis domain. Inhibitors

of DNA gyrase typically also inhibit bacterial topoisomerase IV,

although the relative importance for expression of antibacterial

activity can vary depending on the inhibitor and the bacterial

species.¹³⁻¹⁵The two most widely sold ﬂuoroquinolones,

ciproﬂoxacin and levoﬂoxacin, have encountered growing

resistance mediated by mutations in one or both of the targets

since their FDA approvals in 1987 and 1996, respectively.^16,17

While ﬂuoroquinolones bind to the DNA cleaved complex of

DNA gyrase and topoisomerase IV, the natural product

aminocoumarins, clorobiocin and novobiocin, instead inhibit

ATPase activity in the GyrB and ParE subunits.^18,19Amino-

coumarins have not received widespread usage due to poor

pharmacokinetic properties and issues of safety, and therefore

widespread clinical resistance has not materialized.^12,18

INTRODUCTION

■

The need for novel antibacterials has intensiﬁed due to the

continuing emergence of resistant bacteria limiting the utility of

many drug therapies.¹⁻⁵Since the start of the new millennium,

only seven new chemical entities have received FDA approval

as systemic antibacterials and, of these, only two, linezolid and

daptomycin, operate by novel modes-of-action unrelated to

previous drugs.⁶Many new antibiotics brought to the

marketplace are active against resistant clinical strains either

due to increased potency (as for tigecycline in the tetracycline

class of antibiotics)⁷or to decreased susceptibility to the

degradation mechanisms (as for the carbapenems).^8,9However,

increased resistance can be expected to develop in due time.

The ﬂuoroquinolone and aminocoumarin classes of anti-

bacterials demonstrate clinical utility via inhibition of the

topoisomerases DNA gyrase and topoisomerase IV necessary

for DNA replication.¹⁰⁻¹²DNA gyrase and topoisomerase IV

are tetrameric A₂B₂complexes wherein the A subunits

(designated GyrA for DNA gyrase, ParC for topoisomerase

IV) contain the DNA cleavage domain while the B subunits

(designated GyrB for DNA gyrase and ParE for topoisomerase

In addition to aminocoumarin antibiotics, a wide variety of

compound scaﬀolds exist from synthetic and biological sources

that bind in the ATP binding pocket of GyrB/ParE and result

Received: August 6, 2013

Published: October 7, 2013

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Figure 1. Hydrogen bonding array around urea and pyrrolamide inhibitors.

in antibacterial activity.²⁰This would include cyclothialidine,²¹

a natural product distinct from the aminocoumarins, a related

synthetic scaﬀold that aﬀorded in vivo eﬃcacy²²and a

rhodanine derivative that maintained a part of the cyclo-

thialidine pharmacophore.²³Eﬀorts to mimic the adenine of

ATP have led to an aminopyrazolopyrimidine replacement.²⁴

Recent substituted pyrrolopyrimidines,²⁵azaindoles,²⁶and

bithiazoles²⁷invoke scaﬀold hopping approaches to derive

novel structural motifs. An anilinopyrimidine class of GyrB/

ParE inhibitor was derived by ﬁnding a hit through screening a

corporate compound collection in a ParE ATPase assay.²⁸

There are at least four types of fused bicyclic scaﬀolds

incorporating a urea pharmacophore,²⁹⁻³³including the

benzimidazole urea class of antibacterials as in VRT-752586

(1, Figure 1).²⁹The use of fragments or low-molecular-weight

compounds to identify starting points for the design DNA

gyrase inhibiting antibacterials has proven useful³³⁻³⁵and has

led to the pyrrolamide class of antibacterials such as 2 (Figure

1).³⁶

X-ray crystallographic structures have shown that 1 and 2

each occupy the position of the adenine portion of ATP with

hydrogen bonds donating and receiving interactions with

Asp81 (Staphylococcus aureus DNA gyrase numbering) and a

conserved water molecule.^29,35Compared to the ATP binding

interactions determined from an Escherichia coli X-ray cocrystal

structure of a 43 kDa GyrB domain and a nonhydrolyzable

ATP analogue,³⁷both 1 and 2 extend outside of the ATP

pocket to form hydrogen bonds or a salt bridge to the side

chain of Arg144 (see Figure 1). With this information in hand,

another scaﬀold was desired that would bind similarly to the

adenine binding domain and form a hydrogen bond or salt

bridge to the guanidine moiety of Arg144. A novel scaﬀold

allows for the display of diverse functionality in the ATP

binding pocket, thereby diﬀerentiating the structure−activity

parameters that can be explored relative to other antibacterials

targeting topoisomerase ATPase domains.

Figure 2. Design features of pyridylureas.

with a properly positioned carboxylate, and substitution at the

pyridyl 4-position allows exploration into a more open region

that extends toward the binding site for the ATP ribose ring.

The pyridyl 6-position abuts a hydrophobic surface of the

enzyme and should not be substituted nor replaced with a

heteroatom. The urea substituent approaches a small hydro-

phobic space that allows for small complementary hydrophobic

substituents as with compound 1. Other monocyclic hetero-

cycles, in particular those with 5-membered rings such as

imidazoles and furans, were discounted as it was diﬃcult to

build a straightforward trajectory that would allow an

interaction with Arg144 and avoid steric clashes with the

enzyme. Here, we describe the progression of a set of fragment

compounds with modest biochemical inhibition and favorable

ligand eﬃciencies to a viable lead, which we denote as

demonstrating promising antibacterial activity and eﬃcacy in an

animal disease model.

CHEMISTRY

■

A set of monocyclic aryl urea fragments (Figure 3) was tested

for ATPase inhibitory activity of a hybrid DNA gyrase

containing two S. aureus GyrB units and two E. coli GyrA

units.³⁵The compounds were commercially available or

synthesized by reacting alkyl isocyanates with aminohetero-

cycles or alkylamines with the phenylcarbamate of amino-

pyridine. This survey led to ethyl-pyridylurea 6 being chosen as

the starting point for elaboration. Compounds were synthesized

(see Tables 1−3) to investigate the inﬂuence of 5-position

substitutions designed to extend toward Arg144 and 4-position

substitutions to ﬁll the allowable space abutting the ATP ribose

binding region. Scheme 1 shows the synthesis of 14, carried out

straightforwardly in four steps by formation of ether 43

followed by reaction with ethylisocyanate forming the urea and

ester hydrolysis.

A hybrid design was envisioned where a urea interaction with

Asp81 as with 1 and a salt bridge to Arg144 as with 2 would be

linked in the same molecule. To this end, previously

unexplored 2-pyridylureas were chosen for investigation

wherein the heterocycle nitrogen is positioned for a hydrogen

bond to the crystallographic water molecule similar to the

benzimidazole nitrogen of 1. Molecular modeling was carried

out after removing the ligand from a S. aureus DNA GyrB

structure (3U2K, the N-terminal domain including a loop

deletion residues 14−104 and 128−233)³⁵and docking a 2-

pyridylurea. The pose shows that substitution at the pyridyl 5-

position (see Figure 2 for numbering) reaches toward Arg144

For the synthesis of compounds 15−23, an aryl group was

attached directly to the pyridylurea core at the 5-position as

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Figure 3. IC₅₀s and ligand eﬃciencies (LE) of urea fragments versus S. aureus ATPase activity.

Scheme 1. Synthesis of Compound 14

Scheme 2. Synthesis of Compounds 15−23

Scheme 3. Synthesis of Compounds 24−25

outlined in Scheme 2. Addition of ethylisocyanate to the

pinacol ester of 2-aminopyridine-5-boronic acid aﬀorded 44;

subsequent treatment with arylhalides in palladium catalyzed

cross-coupling reactions aﬀorded 15 and esters 45a−f.

Hydrolysis of esters aﬀorded the corresponding acids 16−21

targeted to form the salt bridge to Arg144. In two instances, the

acids were converted to amides (compounds 22 and 23) by

conversion to the cumylamide and TFA removal of the cumyl

group. Overall, Suzuki cross-couplings constituted the primary

method for the assembly of such biaryl units that ultimately link

the urea pharmacophore with an acid or acid isoster

pharmacophore. The electrophilic component of the cross-

coupling lies on the pyridyl urea core, while the nucleophilic

component is on the appended aryl ring that extends toward

Arg144.

A series of analogues was assembled with substituents at the

pyridyl 5-position and phenyl or pyridyl acids at the 4-position

as diagrammed in Schemes 3−6. The electrophilic and

nucleophilic components were reversed relative to Scheme 2

with coupling of a bromo or iodo pyridylurea with aryl

boronates. Scheme 3 shows the synthesis of compounds 24 and

25 starting by preparation of 46a and 46b. The diﬀerential

halogen substitutions allowed for selective palladium catalyzed

cross-coupling of the commercially available pinacol ester of 3-

ethoxycarbonylpyridine-5-boronic acid with the requisite

dihalopyridyl ureas, leading to 47a and 47b. Subsequent

hydrolysis aﬀorded 24 and 25, where the halogen atoms are

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Scheme 4. Synthesis of Compound 26

Scheme 5. Synthesis of Compounds 27−29

directed toward the allowable space shown in Figure 2. For

these and most of the subsequent analogues, the 3,3-bipyridyl

scaﬀold was utilized as it proved to aﬀord overall higher enzyme

inhibitory potency and lower MIC values relative to other

bipyridyl scaﬀolds (data not shown).

The chemistries in Schemes 4−6 focus on building onto the

pyridyl urea 4-position where, in part, the ribose ring of ATP

resides. In Scheme 4, the nitrile serves as a synthetic handle for

incorporation of an oxadiazole ring. The aminoisopicolinoni-

trile was converted in two steps to bromide 48, setting up a

palladium catalyzed cross-coupling reaction with the pyridyl-

boronate to aﬀord 49. The nitrile was converted to the

isoxadiazole ring in 50, and the ester was hydrolyzed to the acid

of 26.

Scheme 5 outlines the synthesis of analogues with a variety of

aromatic groups at the pyridyl 4-position and a 3-

pyridylcarboxylic acid at the 5-position. Compound 54a was

made from 47a by Suzuki reaction with the pinacol ester of 4-

pyridylboronic acid. For the other two compounds in Scheme

5, 2-amino-4-bromopyridine was ﬁrst capped with ethyl-

isocyanate, allowing for palladium catalyzed arylation aﬀording

compounds 52b and 52c. Bromination proceeded selectively at

the 5-position of the pyridine setting the stage for a second

palladium catalyzed arylation aﬀording compounds 54b and

54c. The esters of 54a−c were subsequently hydrolyzed to the

acids 27−29.

Thiazole rings were introduced at the pyridylurea 4-position

starting by reaction of methyl 2-amino-5-bromopyridine-4-

carboxylic acid with ethylisocyanate and aminolysis of the ester

to aﬀord 55 as shown in Scheme 6. After converting the amide

of 55 to the thioamide, reaction with bromoketones and

dehydration led to compounds 57a and 57b. Suzuki arylation

and ester hydrolysis aﬀorded compounds 30 and 31. Analogues

with alternative functionality to the acid of 31 were targeted;

the ester of 58b was treated with hydrazine and carbonyl

diimidazole to aﬀord oxadiazolone 35. The acid of 31 was

coupled with acetylhydrazide followed by dehydration to aﬀord

oxadiazole 36. The acid of 31 was also converted to amide 32

by coupling with cumylamine followed by TFA solvolysis, and

the amide was converted to oxadiazole 37 by formation of the

intermediate acylamidine with Brederick’s reagent and

subsequent treatment with hydroxylamine.

To build compounds with thiazoles at the pyridylurea 4-

position and thiazoles/benzthiazoles at the 5-position, Suzuki

reactions required the electrophilic boronate partner to reside

on the pyridine while the nucleophilic halide resides on the

thiazole and benzthiazole (see Scheme 7). To this end, 57b was

converted to the boronate 59, allowing for cross-coupling with

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Scheme 6. Synthesis of Compounds 30−32, 35−37

Scheme 7. Synthesis of Compounds 33−34

the requisite chlorothiazole and chlorobenzthiazole, setting up

the subsequent hydrolysis to aﬀord acids 33 and 34.

Outlined in Scheme 8 is the synthesis of several additional

analogues with functionality alternative to the acid envisioned

to reach out to Arg144. Two sequential Suzuki cross-coupling

reactions starting from compound 57b allowed formation of

isoxazole 38. Cross-coupling of the pinacol ester of 5-cyano-3-

pyridyl boronic acid with 57b led to 39; the nitrile was

subsequently converted to the tetrazole of 40. Compound 39

was also treated with hydroxylamine to form hydroxyamidine

41; subsequent reaction with carbonyl diimidazole aﬀorded

oxadiazolone 42.

measured (Figure 3). The aim of this fragment approach was to

assess the constraints in the adenine binding region before

embarking on an extensive analogue campaign. Five of the

compounds showed IC₅₀values less than 100 μM and ligand

eﬃciencies (LE) greater than 0.38 kcal/mol/N.³⁸Benzimida-

zole urea 3, a positive control fragment of 1, displayed low

micromolar potency as expected. Phenylurea 4, lacking a

heterocyclic nitrogen deemed important for the hydrogen

bonding array with the conserved crystallographic water

molecule, served as a negative control, and accordingly, did

not inhibit activity at 1 mM, the highest concentration tested.

The ethylurea substituent of 6 produced the highest potency

and highest LE of the fragment set in line with the motif

reported for the benzimidazole ureas as in 1. The smaller

methylurea 5, although 2.5-fold less potent, nearly matched the

LE of 6. Substituents larger than ethyl on the urea were

disfavored for both potency and, consequently, LE. Notably, a

measure of branching with the cyclopropylurea substituent of 9

was not well-tolerated for activity. Compounds 11 and 12 were

evaluated to determine the eﬀect of eliminating the distal urea

hydrogen bond donors to Asp81, with the diethylurea 11

RESULTS AND DISCUSSION

■

The pyrrolamide antibacterial class of DNA gyrase inhibitors

(e.g., 2) was derived from NMR-based screening of a fragment

library and led to the identiﬁcation of a pyrrole carboxylic ester

with a K_D= 1.1 mM against an E. coli N-terminal 24 kDa ATP-

binding domain.^35,36In a similar approach, inhibitory potency

(IC₅₀, or the concentration at which ATPase activity was

reduced to 50%) for 11 readily available arylurea fragments was

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Scheme 8. Synthesis of Compounds 38−40

Table 1. Isozyme Inhibition and MIC Values for 5-Substitited Pyridylureas

a

b

Sau, Eco, and Spn indicate the enzymes from S. aureus, E. coli, and S. pneumoniae, respectively. Bacterial strains are described in experimental

c

d

procedures. Methicillin sensitive S. aureus. Methicillin resistant, quinolone resistant.

showing that elimination of one H-bond donor can be

compensated by increasing hydrophobicity. The lower potency

of carbamate 12 showed, however, the H-bond acceptor is not

tolerated. The isomeric carbamate of 12 where the side chain

oxygen and nitrogen atoms are transposed (the pyridyloxy N-

ethylcarbamate) was synthesized, but it was too unstable in

solution for evaluation. Finally, the pyrimidine urea 13 showed

2-fold lower potency than 6; presumably the lower lipophilicity

imparted by the additional heterocyclic nitrogen does not

contribute to a binding interaction with the enzyme. On the

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Table 2. Inﬂuence of Pyridylurea 4-Position on Isozyme Inhibition and MIC Values

a

b

Sau, Eco, and Spn indicate the enzymes from S. aureus, E. coli, and S. pneumoniae, respectively. Bacterial strains are described in experimental

c

d

procedures. Methicillin sensitive S. aureus. Methicillin resistant, quinolone resistant.

basis of this initial survey, ethyl-pyridylurea fragment 6 was

chosen as the most promising starting point for further

elaboration.

MIC data and, importantly, allowed exploration of structure−

activity hypotheses when combined with the crystal structures

of the ATPase domain of S. pneumoniae ParE. In general,

relative inhibition by the compounds against the various

isozymes showed good correlations, with the best inhibitors

versus the S. aureus GyrB isozyme displaying better IC₅₀values

against the other three isozymes. It is perhaps better to consider

relative rather than absolute IC₅₀against the isozymes as the

ATPase assays might not directly reﬂect the eﬀect of inhibition

of the biological topoisomerase function in cells.

Inhibitory potency for compounds 14−41 was determined

using two DNA gyrase and two topoisomerase IV isozymes

(Table 1). The isozymes developed for assays in this

investigation included DNA gyrase and topoisomerase IV

from E. coli, DNA gyrase from S. aureus, and topoisomerase IV

from Streptococcus pneumoniae. All compounds were also tested

for antibacterial activities (deﬁned by MIC, the minimum

inhibitory concentration that aﬀorded no visible growth in

culture media) using seven bacterial strains from ﬁve bacterial

species (Tables 1, 2, and 3). Results from the two E. coli

topoisomerase assays allowed for correlation with the MIC

values from two E. coli strains, a wild-type and a tolC⁻eﬄux

pump deﬁcient strain.³⁹MIC values were generally above the

concentrations tested against the wild-type E. coli strain despite

reaching low nanomolar potencies against the E. coli DNA

gyrase. However, in the absence of eﬄux with the tolC⁻stain,

MIC values were improved, providing the hypothesis that low

cellular accumulation of the compound prevented engagement

of the target in the wild-type strains. In contrast, growth of the

fastidious Gram-negative pathogens Haemophilus inﬂuenzae and

Moraxella catarrhalis proved more susceptible to the series of

compounds, perhaps due to higher membrane permeability.

Activity against the S. aureus GyrB could be correlated with two

S. aureus strains, a methicillin susceptible (MSSA) strain and a

methicillin-resistant, quinolone-resistant (MRQR) strain. The

S. pneumoniae ParE isozyme assay enabled correlation with

All the compounds of Table 1 displayed improved potency

against S. aureus GyrB ATPase relative to 6. This includes the

unsubstituted pyridine ring of compound 15, which increased

potency 12-fold over 6. Modeling suggests that this pyridine

substituent is coplanar with the scaﬀold’s core pyridine ring and

forms a π-interaction with the protein surface created by a ﬂat

edge-to-edge salt bridge between the binding pocket carbox-

ylate of Glu58 and the guanidine of Arg84 (see below). The

pyridine ring could be positioned to receive a hydrogen bond to

Arg144 (see below) similar to the pyridine ring of 1. The side

chain of Arg144 is quite ﬂexible, lacking a clear anchoring

hydrogen bonding array or salt bridge association of the

guanidine within the enzyme, and is thus able to adjust to the

various inhibitors. Potency was improved versus all four

isozymes (as much as 3-fold) for 19 relative to 15, reﬂecting

the addition of a carboxylate substituent potentially in

proximity to Arg144. Other aromatic rings served well to

position a carboxylate that maintained or improved enzyme

inhibition, including the phenyl rings of 17 and 18, the

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Table 3. Inﬂuence of Bipyridyl Substituent on Isozyme Inhibition and MIC Values

a

b

Sau, Eco, and Spn indicate the enzymes from S. aureus, E. coli, and S. pneumoniae, respectively. Bacterial strains are described in experimental

c

d

procedures. Methicillin sensitive S. aureus. Methicillin resistant, quinolone resistant.

thiazoles of 16 and 20, and the benzthiazole of 21. Extension to

a carboxylate with the phenyl ether of 14 decreased potency,

perhaps due to increased conformational ﬂexibility relative to

the directly bonded aromatic rings and disfavored coplanarity

with the pyridine ring necessary to direct the carboxylate for the

interaction with the arginine. Nonetheless, the phenylcarbox-

ylate substituent of 14 improved potency 3-fold over 6.

Meshing with the apparent mobility of the Arg144 side chain,

there were preferences for potency depending on the position

of the carboxylate. The 1,3 orientation between a carboxylate

and the point of attachment to the scaﬀold for thiazole 20

shows a 10-fold higher potency against S. aureus GyrB relative

to the 1,3 orientation of the isomeric 16. Extending the

carboxylate further outward from the pyridylurea pharmaco-

phore with the benzthiazole of 21 improved potency 3−4-fold

over 20, depending on the isozyme. Many of the compounds in

Table 1 showed higher ligand eﬃciencies (the ligand

eﬃciencies ranged from 0.52 for 14 to 0.76 for 20 using the

S. aureus IC₅₀data) than 6. Furthermore, measurable MIC

values versus S. pneumoniae, M. catarrhalis, and H. inﬂuenzae

indicated that the higher inhibition translated into antibacterial

activity allowing for rational structure−activity correlations.

Considerable improvement in potency was further achieved

by positioning substituents at the pyridylurea 4-position (Table

2). Modeling showed that substituents at this 4-position extend

into the ATP binding pocket toward an open region where the

ATP ribose ring is situated. This region is bounded by a loop

that is ordered in the dimeric structures of the 43 kDa GyrB

domain bound to an ATP analogue³⁷and is disordered in

monomeric 24 kDa GyrB and ParE-inhibitor complexes. The

smaller chlorine and bromine substituents of compounds 24

and 25 improved potency 6−24-fold depending on the

isozyme. Compounds 27 and 28, with pyridine substituents

on the 4-position, also displayed improved potency relative to

hydrogen (compound 19), albeit the improvement was less

than that of the halogen substituents. Compound 29 with the

phenyl substituent increased potency 10-fold or more relative

to 19 versus three of the isozymes and 2-fold versus the fourth.

Notably, the thiazole substituents of 30−34 aﬀorded further

potency improvements that can be attributed to positioning a

lipophilic group (t-butyl for 30 and CF₃for 31−34) toward

complementary lipophilic regions of GyrB and ParE (see

below).

Higher solubility is a critical factor for the development of a

therapeutic molecule to enable formulation development and

maintain eﬃcacious levels of the drug in vivo. Compounds with

an ionizable carboxylic acid substituent consistently imparted

higher solubility; however, the carboxamide analogues dis-

played superior antibacterial activity compared to those with

acid substituents (e.g., compare 22 to 20, Table 1 and 32 to 31,

Table 3). Therefore, achieving suﬃcient solubility for this drug

scaﬀold was required while also improving antibacterial activity

either by increasing inhibitory potency or improving bacterial

membrane permeability. That carboxamides and carboxylic

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Figure 4. (A) 31 in binding site of S. pneumoniae ParE ATPase domain. Hydrogen bonds are shown as dotted lines. 2F_o− F_cdensity has been

contoured to 1.0σ. The urea pyridylurea core is oriented in the binding pocket to form a bifurcated hydrogen bond between the urea and Asp78. A

crystallographic water bridges Asp78 to the pyridine nitrogen. Another crystallographic water bridges Arg140 to the second pyridine nitrogen. The

CF₃-thiazole abuts a hydrophobic surface formed by Met83, Ile98, and Pro84. (B) Surface representation of the binding site of 31. The pyridine

substituent π-stacks with Arg81(blue surface, upper right), and the carboxylate is oriented away from Arg140 (blue surface, right).

acids show comparable enzyme inhibition presumably reﬂects a

trade-oﬀ for greater enthalpic contribution to binding with

ionized acids and greater entropic contribution to binding with

neutral amides. In essence, there is a greater desolvation penalty

for taking the carboxylate from the aqueous environment to the

enzyme binding region for the interaction with the Arg144

guanidine. The dichotomy arises when analyzing antibacterial

activity as the capability of charged acids to traverse the

bacterial lipid bilayer is lower than neutral, more lipophilic

compounds.^40,41Hence, compounds 32 and 34 with higher log

D displayed the better MIC values among compounds of

similar inhibitor potencies in Table 3. The comparison of

benzthiazoles 23 and 21 provides, perhaps, a counterexample to

this, as antibacterial activity does not track well with either

potency or lipophilicity in that the former performed worse

than the latter, in particular against the E. coli tolC⁻strain. This

is attributed to the low solubility of 23 (<1 μM), likely

undermining reliable MIC measurement under the assay

conditions. Higher lipophilicity generally favors membrane

permeability and thus, antibacterial activity, but disfavors

solubility crucial for achieving higher plasma concentrations

in vivo and, as in the case with 23, for achieving suﬃcient

concentrations for even in vitro MIC assessments.

Indeed, the fact that there is not a direct correlation between

GyrB/ParE enzyme inhibition and antibacterial activity can be

attributed to a variety of reasons beyond the vagaries of

compound membrane permeability and solubility. Considerable

extrapolations have been made among the isozymes surveyed

for correlations with MIC data from the diﬀerent bacterial

species. As mentioned, inhibitors of the GyrB ATPase activity

are generally also inhibitors of ParE ATPase activity, and the

relative role of each for inhibition in an enzyme assay and in the

bacterial cytoplasm cannot easily be teased apart and might be

better ascertained from resistance development studies and

genomic analysis.⁴²Enzyme inhibition is measured by ATPase

activity coupled through detection of phosphate release in a

Malachite Green assay, which is diﬀerent from the physiolog-

ically relevant catalytic cycle with DNA that occurs inside the

bacterial cell. Finally, the assays do not account for expression

levels and regulatory mechanisms within the bacterial cell that

are also important for translation to MIC.⁴³Nonetheless, the

enzyme assays serve well to ascertain structure−activity

relationships; for example, IC₅₀values less than 50 μM against

the S. aureus GyrB isozyme were necessary to aﬀord MIC values

less than 64 μg/mL versus the S. aureus strains.

The antibacterial mode-of-action of compound 32 was

assessed by monitoring its inﬂuence on the incorporation of

radioactive thymidine, uridine, leucine, and valine, acetic acid,

and N-acetylglucoseamine into bacterial cells as indicators for

DNA, RNA, protein, fatty acid, and peptidoglycan biosynthesis,

respectively (see Table 5).^28,44Two pathogens, S. aureus and S.

pneumoniae, were surveyed, and the low IC₅₀values recorded

for thymidine incorporation indicate that the primary mode of

action of these inhibitors is the preferential disruption of DNA

synthesis, consistent with topoisomerase (GyrB and ParE)

inhibition. The observed inhibition of RNA and protein

syntheses, as measured by reduced uridine and amino acid

incorporation, is believed to be a consequence of the inhibition

of DNA synthesis rather than a secondary mode of action and

was similarly observed for compound 1.⁴⁵However, the

ciproﬂoxacin IC₅₀values for metabolite incorporation for

DNA synthesis was considerably more pronounced relative to

the other metabolic processes despite the fact that this molecule

also acts through inhibition of bacterial topoisomerases. This

may be the consequence of faster killing kinetics observed for

ciproﬂoxacin versus 32 (data not shown), triggering cell death

before eﬀects are seen for RNA and protein synthesis and may

reﬂect the diﬀerent mechanisms of topoisomerase inhibition for

the two molecules. No eﬀects were seen at the concentrations

of 32 tested on peptidoglycan or fatty acid biosyntheses.

Positive controls rifampicin, erythromycin, triclosan, and

penicillin G showed the expected proﬁles for the inhibition of

radiolabel precursor incorporation into RNA, protein, fatty acid,

and cell wall biosyntheses, respectively (data not shown).

A 2.06 Å crystal structure of compound 31 with the 23 kDa

N-terminal ATPase domain of the S. pneumoniae ParE

topoisomerase IV isozyme was determined (Figure 4 and

Table 8). The particular isozyme had proven robust for

crystallography work, aﬀording high resolution data with a

variety of inhibitor complexes including 4EMV and 4EM7 in

the Protein Data Bank.²⁶The isozyme binding pocket further

reﬂects the high degree amino acid sequence conservation

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Table 4. PK Properties of Selected Compounds

across species and between DNA gyrase and topoisomerase

IV.⁴⁶Although the construct itself does not have ATPase

activity displayed by the full ParE used for activity assays, the

integrity of the inhibitor binding pocket was maintained. The

structure showed that the pyridylurea pharmacophore of the

molecule formed a bifurcated hydrogen bond with Asp78

(equivalent to Asp81 for S. aureus GyrB used for modeling

work), which was, in turn, hydrogen-bonded to a structurally

conserved water molecule similar to the diagram for 1 in Figure

1. The water molecule otherwise bridged the core pyridine

nitrogen of 31 and Thr172 through hydrogen bonds. From the

perspective of inhibitor design, this water molecule is tightly

coordinated in this and other published structures and

therefore would be diﬃcult to displace. A second water

molecule bridged the urea carbonyl to the side chain

carboxamide of Asn51 and the hydroxyl of Ser124. The key

hydrogen bond interactions with Asp78 and the two water

molecules seen for 31 mimicked those made by adenine with

ATP analogues bound to the enzyme.³⁷In contrast, an X-ray

structure determination of 2 with the S. aureus GyrB ATPase

domain did not show the second water molecule, with the

region being occupied by the dichloropyrrole moiety and a

direct interaction being seen to Asn54 (S. aureus GyrB

numbering, equivalent to Asn51 of S. pneumoniae ParE).³⁶

The triﬂuoromethylthiazole substituent of 31 was positioned in

the hydrophobic patch that forms part of the ribose binding

pocket with the thiazole ring contacting Met83 and the

triﬂuoromethyl group ﬁtting snugly in a small pocket formed by

the adjacent residues Pro84, Thr94, and Ile98, perhaps

accounting for the potency enhancements. The 5-position

pyridine substituent oﬀ the core pyridylurea formed a π-

stacking interaction with Arg81 in line with what was

considered in the compound design. Interestingly, the pyridine

nitrogen engages the Arg140 (equivalent to Arg144 for S.

aureus GyrB) through a bridging water molecule rather than

through a salt bridge with the acid, which is oriented away from

the arginine residue toward the solvent. This result is in

contrast to the design hypothesis and inhibition data that would

have predicted the carboxylate improving inhibitory potency. It

also contrasts the structure solved for 1, where the pyridine

nitrogen makes a direct hydrogen bond to Arg144. It should be

noted that the crystallization was carried out with the 226

residue N-terminal ATPase domain of the S. pneumonia ParE

rather the entire 648 residue GyrB protein or a larger

topoisomerase IV ParC−ParE complex, which could misrepre-

sent the physiologically relevant binding orientation. Nonethe-

less, among the structures that have been solved, although the

hydrogen bond interactions with arginine have been deemed

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important for improving inhibitor potencies, there appears to

be a measure of plasticity for the arginine side chain

conformation.

As suggested earlier, compounds should have suﬃciently

high solubility and high antibacterial activity as two important

components toward the design of a viable drug. To this end, the

compounds of Table 3 incorporated alternative functionality to

the acid of 31 that had low antibacterial activity despite high

topoisomerase inhibitory potency and the amide of 32 that had

high antibacterial activity but moderate solubility (see Table 4).

Inhibitory potencies of 35−40 versus the four topoisomerase

isozymes were among the highest reported herein, albeit the

weak antibacterial activity of 39 and 42 with the more acidic

acid isosteres paralleled the properties of 31. Indeed, a clear

correlation emerged in that R groups with higher acidity (Table

4) led to weaker antibacterial activities despite inhibitory

potencies that were comparably high. Compounds 31, 35, 40,

and 42 displayed potent inhibition across the four topoisomer-

ase isozymes but were varied in acidity displaying correspond-

ingly varied MIC values; for example, the least acidic

compound 35 having a measured pK_aof 6.6 aﬀorded the

highest antibacterial activity. Figure 5 illustrates this correlation

Figure 6. X-ray crystal structure of 35 in S. pneumoniae ParE. The

hydrogen bonding array around Asp78 is maintained relative to 32

(Figure 4). The pyridyl-oxadiazolone substituent π-stacks with Arg81;

the oxadiazolone forms a salt bridge with Arg140.

interactions between the 35 and the protein paralleled those

seen for 31. However, unlike the strong acid carboxylate of

compound 31, a clear interaction was seen between the weakly

acidic oxadiazolone moiety and the Arg140 guanidinium. It is

notable that Arg81 (which stacks over the π-cloud of the distal

pyridine rings of both 31and 35) and Arg140 lie outside the

ATP binding region but are well conserved due to their

contributions to protein−protein interactions critical to

stabilizing the GyrB and ParE dimers.^37,47Although these

residues are not important for substrate binding, they

nonetheless play important roles for the enzyme and therefore

should not be candidates for nonsimilar mutation leading to

drug resistance. Binding interactions with these arginine

residues have been exploited in other classes of GyrB

inhibitors.^{25−32,34−36,48−50}

For compounds active versus S. aureus in Tables 2 and 3, it is

noteworthy that the MRQR strain was nearly equally aﬀected as

the MSSA strain. This MRQR strain carries a SCCmec type II

resistant allele imparting methicillin resistance⁵¹and an S85P

GyrA and S80Y ParC mutation imparting reduced susceptibility

to ﬂuoroquinolone DNA gyrase inhibitors. In contrast, the

ﬂuoroquinolone levoﬂoxacin showed large diﬀerences between

the strains with MIC values of 0.13 and 4 μg/mL for the MSSA

and MRQR S. aureus strains, respectively. Similarly, clinical

strains of S. pneumoniae with reduced susceptibility to

levoﬂoxacin were equally susceptible to compound 35

compared with the wild-type strain (data not shown, however

see Table 6 for nonclinical strains). These results were expected

because the mode of DNA gyrase inhibition of ATP-

competitive compounds diﬀers from that of ﬂuoroquinolones,

with the latter binding to cleaved DNA at the interface between

GyrA and GyrB⁵²as well as ParC and ParE.⁵³As a positive

control, novobiocin behaved similarly to 35 in that MIC values

were not aﬀected by the ﬂuoroquinolone resistant strains nor

were they aﬀected by nontopoisomerase binding antibacterials

such as tetracycline.⁵⁴These data bode well for this series of

DNA gyrase inhibiting compounds in that susceptibility in the

Figure 5. Correlation between antibacterial activity and pK_a.

in a plot of S. pneumoniae pMIC versus pK_afor the four

compounds. Although higher acidity improved solubility,

multiple factors (physical properties, antibacterial activity, and

in vivo kinetics) must be balanced to design potent and

eﬀective drug candidates for clinical use. Compounds 36, 37,

and 39 showed similar properties to 32 in that antibacterial

activity was high but solubility was low. Compound 41 showed

considerably weaker intrinsic potency and antibacterial activity

relative to 35 but better antibacterial activity relative to the

more acidic compounds 36, 37, and 39. Overall, compounds 32

and 35, though of moderate solubility, had the balance of

properties necessary for demonstrating in vivo activity. Because

the concentration of unbound compound in blood must be

suﬃciently high and sustained to demonstrate eﬃcacy in animal

disease models, compound 35 with the lower clearance in the

rat (Table 4) was selected for eﬃcacy experiments in an in vivo

disease model (see below).

An X-ray crystal structure of 35 bound to the S. pneumoniae

ParE was determined (Figure 6). As expected, most of the

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Table 5. Inhibition of Radioactive Precursor Incorporation into Bacterial Cells over 1 h

S. aureus IC₅₀(μM)

S. pneumoniae IC₅₀(μM)

radiolabel precursor

32

ciproﬂoxacin

radiolabel precursor

32

ciproﬂoxacin

³H-thymidine

³H-uridine

¹⁴C-leucine

¹⁴C-valine

¹⁴C-acetic acid

¹⁴C-N-acetyl glucosamine

1.6

6.9

3.9

³H- thymidine

³H-uridine

¹⁴C-leucine

¹⁴C-valine

¹⁴C-acetic acid

¹⁴C-N-acet-glucosamine

0.46

0.83

0.86

1.2

28

>195

>174

>195

>200

110

8.8

31

>200

>20

Table 6. MICs (μg/mL) versus S. pneumoniae Strains with Mutations in DNA Gyrase and Topoisomerase IV

wild-

type

GyrB T172A

ParE none

GyrB K143I

ParE none

GyrB none

GyrB T172A

ParE T172A

GyrB K143A

ParE T172A

GyrA S81F

ParC S79F

GyrA S81Y

ParC S79F

GyrA S81F

compd

35

ParE T172A

ParC S79Y

0.0025

0.5

0.01 (4×)

2 (8×)

0.5

0.01 (4×)

1 (4×)

1

0.01 (4×)

0.25 (1×)

1

0.02 (8×)

4 (16×)

0.5

0.04 (16×)

4 (16×)

0.5

0.0025

0.25

0.0025

0.25

0.0025

0.25

novobiocin

levoﬂoxacin

tetracycline

1

16 (16×)

0.13

16 (16×)

0.25

32 (32×)

0.13

0.25

clinic should be maintained versus bacteria with pre-existing

drug resistance due to DNA gyrase mutations.

Table 7. Frequency of Spontaneous Resistance for 35 against

S. aureus and MICs for Resulting Mutants

The importance of several interactions observed in the

cocrystal structure was investigated by creating point mutations

in the gyrB and parE genes of S. pneumoniae. The amino acids

selected for this study (see Table 6) were derived from those

investigated for other ATP competitive topoisomerase

inhibitors.^28,42,45,55The two threonine residues, Thr172 from

each of GyrB and ParE, lie in hydrogen bonding distance to the

water molecule otherwise coordinated with Asp78 in the

adenine binding pocket. The GyrB residue Lys143, analogous

to the ParE Arg140, is presumed to interact similarly with the

acidic oxadiazolone moiety of 35 as seen in the ParE complex

structure. Single point mutations T172A in GyrB and ParE and

K143I in GyrB resulted in a 4-fold elevation of MIC for 35

when compared to the MIC for the parent strain, whereas

combinations of these mutations in both genes resulted in an

8−16-fold elevation in MIC. In contrast, results for novobiocin

against these mutants were somewhat diﬀerent, where only the

GyrB mutants displayed debilitated MIC (4−16-fold) while the

ParE single mutant T172A showed no increase in MIC. These

results parallel those reported for aminobenzimidazole class of

GyrB inhibitors such as 1⁴²and similarly indicate that the dual

mode-of-action for 35 is more balanced than that for

novobiocin and that the interactions inﬂuencing ATPase

inhibition for each of the two topoisomerases can vary

depending on the scaﬀold.

S. pneumoniae strains were also made in which mutations in

gyrA and parC were constructed to confer resistance to

ﬂuoroquinolone drugs such as levoﬂoxacin (Table 6).⁵⁶

Compound 35 and novobiocin maintained potent MIC values

in line with data for the wild-type parent strain, while the MIC

values were elevated, appropriately, for levoﬂoxacin. None of

the GyrB/ParE mutations in Table 6 are necessarily relevant to

what might be seen clinically, but they do conﬁrm that the

binding site delineated by X-ray crystallography as being

relevant to antibacterial activity and that the mode-of-action

involving both DNA gyrase and topoisomerase IV for S.

pneumoniae that is distinct from that for ﬂuoroquinolones.

As a means to assess the risk of future resistance

development, the frequency of spontaneous resistance was

measured with increasing concentrations of 35 in S. aureus

(MSSA) cultures (Table 7). The frequency of spontaneous

a

[compound] μg/mL

fold MIC

resistance frequency

MIC shift

0.05

0.1

1×

2×

4×

1.1 × 10⁻⁹

1.3 × 10⁻⁹

3.0 × 10⁻¹⁰

16×

0.2

a

Elevation of MIC for selected mutants relative to parent strain.

resistance of approximately 1 × 10⁻⁹for 35 is low, suggesting

that development of resistance should not be rapid. MIC values

for the resulting mutant strains were elevated 16-fold relative to

the wild-type parent strain. DNA sequencing of the genes

encoding the topoisomerase subunits (gyrA, gyrB, parC, and

parE) from two of these resistant mutant strains revealed the

same point mutation in the gyrB gene when compared to

sequences obtained from the sensitive parent strain. This

mutation resulted in a single amino acid change in GyrB,

T173N, which is the same amino acid as Thr172 mutated in the

engineered S. pneumoniae strains described above. Similar

mutations, generated in vitro, have been reported to be

associated with resistance to several other chemical series that

target the ATP binding pocket of DNA gyrase.^28,45,55Although

these mutants can be generated in the laboratory, similar

mutations in gyrB have not been observed in any clinical

isolates of S. aureus or S. pneumoniae sequenced in house (data

not shown) or reported in the literature to date, presumably

due to the lack of ATPase inhibitors currently in clinical use.

Compound 35 was evaluated in vivo against S. aureus in a

neutropenic mouse thigh infection model.⁵⁷Thigh infection

was established with S. aureus at 1 × 10⁵CFU (colony-forming

units)/thigh in mice previously rendered neutropenic by

treatment with cyclophosphamide, as described previously.⁵⁸

Dosing of 35, or vehicle control, by intraperitoneal (ip)

injection commenced 2 h post infection and a second, equal

dose was administered 12 hours after the ﬁrst resulting in a

total daily doses of 80, 120, 160, 200, and 300 mg/kg. Activity

was quantiﬁed by viable counts of thigh homogenates 24 h after

start of the treatment and is presented in Figure 7 as mean log

CFU/g thigh tissue ( standard error) for each group of mice.

Compound 35 was found to be eﬃcacious against S. aureus in

this model, causing a dose-dependent decrease in viable

bacterial counts in the thigh. A dose of 300 mg/kg resulted

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strated with ip administration of 35. For translation to utility in

the clinic, further analogue optimization work from the

promising lead compounds shown here is needed to aﬀord

more soluble compounds that maintain potent antibacterial

activity and improve in vivo PK properties. The structural

features correlating with activity for these pyridylureas

diﬀerentiates from those for compounds 1 or 2 and oﬀer

alternative motifs to design and develop antibacterial agents

targeting bacterial topoisomerase binding targets.

EXPERIMENTAL PROCEDURES

■

General Considerations. All of the solvents and reagents used

were obtained commercially and used as such unless noted otherwise.

¹H NMR spectra were recorded in CDCl₃or DMSO-d₆solutions at

300 K using a Bruker 300 Ultrashield 300 MHz instrument. Chemical

shifts are reported as parts per million relative to the reference TMS

(0.00). ¹³C NMR spectra were recorded in DMSO-d₆solutions at 300

K and 126 MHz using a Bruker DRX-500 500 MHz instrument with a

QNP cryoprobe or at 101 MHz using a Bruker 300 Ultrashield 300

MHz instrument. Chemical shifts are reported as parts per million

relative to the reference TMS (0.00). High-resolution mass spectra

(HRMS) were obtained using a hybrid quadrupole time-of-ﬂight mass

spectrometer (microTOFq II, Bruker Daltonics) in ESI⁺mode. Silica

gel chromatographies were performed on an ISCO Combiﬂash

Companion instrument using ISCO RediSep ﬂash cartridges (particle

size: 35−70 μm) or Silacycle SiliaSep ﬂash cartridges (particle size:

40−63 μm). Preparative reverse phase HPLC was carried out using

YMC Pack ODS-AQ (100 mm × 20 mm ID, S-5 μ particle size, 12 nm

pore size) on Agilent instruments. Mass spectroscopy was performed

using a Waters ZQ mass spectrometer (for ESP) connected to a

Agilent 1100 HPLC instrument. All compounds tested possessed a

purity of ≥95%. Compounds 4 and 12 were purchased from chemical

supply houses. Compounds for oral and iv dosing in mice were

formulated in 25% PEG/water and 4:4:2 dimethylacetamide−

PEG400−water, respectively. Compounds for iv and ip dosing in

rats were formulated in 70% PEG/30% saline (13% HP cyclodextrin).

Wistar Han rats were used for pharmacokinetic studies and were

obtained from Charles River Laboratories (Raleigh, NC). CD-1 mice

were obtained from Charles River Laboratories (Kingston, NY) for

eﬃcacy studies. All animals were housed and acclimated in the animal

facility on site before each study. All experimental procedures were

conducted in accordance with protocols approved by the Institutional

Animal Care and Use Committee.

Figure 7. Eﬃcacy of compound 35 in a 24 h S. aureus thigh infection

model in neutropenic mice. Bars from left to right show the number of

S. aureus colony forming units (CFU) recovered from thighs of mice

treated with 0−300 mg/kg of compound 35 administered by ip

injection at 0 and 12 h. Total doses are shown. Error bars represent

standard error in the CFU measurements.

in the maximum response, a 3000-fold reduction in bacterial

burden relative to the vehicle control. The ED₅₀for 35 in this

model was estimated to be 200 mg/kg.

CONCLUSIONS

■

In summary, a series of urea pyridine fragments designed based

on the crystal structure of topoisomerase ATPase domains with

known inhibitors generated a valuable starting point for

elaboration into compounds with antibacterial activity. This

fragment-to-hit-to-lead sequence has spawned a robust

optimization program that will be reported in subsequent

publications and has been exempliﬁed in the public domain as

patent applications.⁵⁹⁻⁶⁵Lead compounds were developed to

engineer a higher aqueous solubility to facilitate further

development, hence, the emphasis on incorporating the

ionizable carboxylic acid and acid isoster moieties designed to

serve the dual roles of increasing solubility and interacting with

the binding domain arginine residue (Arg140, S. pneumoniae

ParE). The cocrystal structures of compounds 31 and 35 oﬀer

support for many of the design hypotheses including the

incorporation of binding interactions with an aspartate residue

associated with binding to the adenine portion of ATP and with

the arginine that lines the inhibitor pocket distal to the ATP

binding domain. That said, there remains a measure of

ambiguity for the importance of the carboxylate of 31 in the

interaction with Arg140 in that the X-ray derived crystallo-

graphic model showed the distal pyridine nitrogen oriented

toward the Arg140 with the S. pneumoniae ParE isozyme rather

than the carboxylate. By contrast, the oxadiazolone of 35 did

indeed bridge to Arg140 as anticipated by design. Building

substituents on the urea-pyridine 4-postion was central for a

notable improvement in inhibitory potency by occupying a

hydrophilic region of the enzyme with a lipophilic CF₃group.

Overall, potent inhibition across four topoisomerase isozymes

from three bacterial species was realized. Importantly,

inhibition of bacterial growth was demonstrated via a mode-

of-action that involves both DNA gyrase and topoisomerase IV,

which serves as the key criterion to continue optimization work

and to improve properties (e.g., antibacterial potency,

pharmacokinetics, solubility) that would support the demon-

stration of eﬃcacy in animal models and utility in the clinic.

Ultimately, eﬃcacy in a murine disease model was demon-

1-(1H-Benzimidazol-2-yl)-3-ethyl-urea (3). A mixture of 1H-

benzo[d]imidazol-2-amine (300 mg, 2.25 mmol) and ethylisocyanate

(0.36 mL, 4.5 mmol) in chloroform (4 mL) was heated at 100 °C in a

microwave reactor for 2 h. LC-MS shows two materials suggesting a

mixture of acylation products. Solvent was removed from the mixture,

and the residue was puriﬁed by reverse phase HPLC (5−79% 0.1%

formic acid in water and acetonitrile). The ﬁrst eluting component was

consistent with the title compound isolated as a solid (137 mg, 30%

1

yield). H NMR (DMSO-d₆) δ 11.5 (s, br, 1H), 9.6 (s, br, 1H), 7.3−

7.4 (m,3H), 6.9−7.0 (m, 2H), 3.2 (pentet, 2H), 1.1 (s, 3H). ¹³C NMR

(DMSO-d₆, 126 MHz) δ 154.1, 148.3, 120.4, 34.1, 15.3. LC-MS purity

100%. MS (ESI) m/z 275 [M + H]⁺. HRMS [M + H]⁺= 205.1090

(theoretical 205.1084).

Compounds 8 and 13 were synthesized similarly as compound 3

from the starting aminoheterocycle and the appropriate isocyanate.

1-Propyl-3-(2-pyridyl)urea (8). Solid, 500 mg (87%) using 2-

aminopyridine (300 mg) and n-propylisocyanate (0.45 mL). ¹H NMR

(DMSO-d₆) δ 9.6 (s, 1H), 9.0 (s, 1H), 8.6 (d, 2H), 7.0 (t, 1H), 3.25

(pentet, 2H), 1.1 (t, 3H). ¹³C NMR (DMSO-d₆, 126 MHz) δ 154.7,

153.5, 146.6, 138.1, 116.6, 111.5, 40.7, 22.9, 11.4. LC-MS purity 100%.

MS (ESI) m/z 167 [M + H]⁺. HRMS [M + H]⁺= 167.0921

(theoretical 167.0927).

1-Ethyl-3-(2-pyrimidin-2-yl)urea (13). Solid, 120 mg (32%) using

2-aminopyrimidine (320 mg) and ethylisocyanate (0.36 mL). ¹H

NMR (CDCl₃) δ 9.35 (s, br, 1H), 8.1−8.3(m, 1H), 7.6 (ddd, 1H), 6.9

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(m, 1H), 6.8 (d, 1H), 3.4 (dt, 2H), 1.7 (m, 2H), 1.0 (t, 3H). ¹³C NMR

isocyanate (0.082 mL) in chloroform (5 mL) was heated in a

microwave reactor at 110 C for 1 h. Solvent was removed, and the

ο

(DMSO-d₆, 126 MHz) δ 158.2, 158.0, 153.8, 114.4, 34.0, 15.3. LC-MS

purity 100%. MS (ESI) m/z 180 [M + H]⁺. HRMS [M + H]⁺

=

residue was chromatographed on silica gel (gradient elution with 2%

MeOH in CH₂Cl₂to 10% MeOH in CH₂Cl₂) to give 0.12 g (42%) of

180.1136 (theoretical 180.1131).

1

1-Ethyl-3-(2-pyridyl)urea (6). A mixture of 2-(phenoxycarbonyla-

mino)-pyridine⁶⁶(100 mg, 0.47 mmol) and 3 mL (6 mmol) of 2N

ethylamine in THF was heated at 80 °C for 30 min in a microwave

reactor. Solvent was removed, and the residue was puriﬁed by silica gel

chromatography (100% CH₂Cl₂followed by gradient elution to 80%

EtOAc in CH₂Cl₂) to aﬀord 70 mg (90%) of the title compound as a

the title compound as a white solid. H NMR (DMSO-d₆) δ 1.07 (t,

3H), 3.16−3.22 (m, 2H), 3.82 (s, 3H), 7.25−7.28 (m, 1H), 7.36 (s,

1H), 7.45−7.55 (m, 3H), 7.60−7.71 (m, 2H), 8.06 (s, 1H), 9.20 (s,

1H). MS (ESI) m/z 316 [M + H]⁺.

N-Ethyl-N′-[5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

pyridin-2-yl]urea (44). Ethylisocyanate (0.33 mL, 4.5 mmol) was

added to a solution of 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)pyridin-2-amine (1g, 4.5 mmol) in CHCl₃(5 mL). The resulting

mixture was heated in a microwave reactor at 110 °C for 1 h. Solvent

was removed to give 1 g of the title product as a white solid. ¹H NMR

(CDCl₃) δ 1.22 (t, 3H), 1.32 (s, 12H), 3.41 (m, 2H), 6.82 (d, 1H),

7.90 (dd, 1H), 8.52 (s, 1H), 8.62 (s, 1H), 9.40 (s, 1H). MS (ESI) m/z

292 [M + H]⁺.

Methyl 2-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)-1,3-thia-

zole-5-carboxylate (45e). N-Ethyl-N′-[5-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)pyridin-2-yl]urea (0.20 g, 0.69 mmol), methyl 2-

bromo-1,3-thiazole-5-carboxylate (0.152 g, 0.69 mmol), tetrakistriphe-

nylphosphine palladium (0.08 g, 0.069 mmol), and CsCO₃(0.245 mg,

0.754 mmol) were taken together in a microwave vial and degassed

with argon. Dioxane−water (4:1, 3 mL) was added, and the mixture

was heated in a microwave reactor at 110 °C for 30 min. The reaction

mixture was partitioned between water and EtOAc, and the layers were

separated. The organic layer was washed with saturated aqueous

NaHCO₃, water, and brine before being dried over MgSO₄. The

solvent was removed, and the residue was puriﬁed by chromatography

on silica gel (gradient eluting with 2% MeOH in CH₂Cl₂to 3% MeOH

in CH₂Cl₂to give 0.123 g of material consistent with the title

compound. ¹H NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.16−3.22 (m, 2H),

3.86 (s, 3 H), 7.60 (d, 1H), 7.79 (t, 1H), 8.27 (dd, 1H), 8.49 (s, 1H),

8.83 (s, 1H), 9.56 (s, 1H). MS (ESI) m/z 307 [M + H]⁺.

Compounds 15 and 45a−d and 45f were made similarly to the

synthesis of ethyl 2-(6-{[(ethylamino)carbonyl]amino}pyridin-3-yl)-

1,3-thiazole-5-carboxylate from compound 44 and the indicated aryl

halide.

1

solid. H NMR (DMSO-d₆) δ 9.15 (s, 1H), 8.2 (m, 2H), 7.6 (t, 1H),

7.3 (d, 1H), 6.9 (m, 1H), 3.2 (q, 3H), 1.1 (t, 3H). ¹³C NMR (DMSO-

d₆, 126 MHz) δ 154.6, 153.5, 146.6, 136.5, 138.1, 116.6, 111.5, 33.8,

15.4. LC-MS purity 99%; MS (ESI) m/z 166 [M + H]⁺. HRMS [M +

H]⁺= 166.0982 (theoretical 166.0982).

Compounds 5, 7, and 9−11 were synthesized similarly as

compound 6 from 75 mg of 2-(phenoxycarbonylamino)-pyridine.

1-Methyl-3-(2-pyridyl)urea (5). Solid, 46 mg (65%) using 2N

1

methylamine in THF. H NMR (DMSO-d₆) δ 9.2 (s, 1H), 8.1 (m,

2H), 7.7 (t, 1H), 7.3 (d, 1H), 6.9 (t, 1H), 2.7 (s, 3H). LC-MS purity

100%. ¹³C NMR (DMSO-d₆, 126 MHz) δ 155.3, 153.5, 146.6, 138.1,

116.6, 111.4, 25.9. MS (ESI) m/z 152 [M + H]⁺. HRMS [M + H]⁺=

152.0823 (theoretical 152.0826).

1-Prop-2-ynyl-3-(2-pyridyl)urea (7). Solid, 48 mg (78%) using

diethylamine. ¹H NMR (DMSO-d₆) δ 9.35 (s, 1H), 8.5 (s, 1H), 8.2 (d,

1H), 7.7 (t, 1H), 7.3 (d, 1H), 6.95 (t, 1H) 4.0 (dd, 1H), 3.1 (t, 1H).

¹³C NMR (DMSO-d₆, 126 MHz) δ 154.4, 153.1, 146.7, 138.3, 117.0,

111.6, 81.7, 73.0, 28.5. LC-MS purity 100%. MS (ESI) m/z 176 [M +

H]⁺. HRMS [M + H]⁺= 176.0823 (theoretical 176.0826).

1-Cyclopropyl-3-(2-pyridyl)urea (9). Solid, 41 mg (66%) using

1

cyclopropylamine,. H NMR (DMSO-d₆) δ 9.1 (s, 1H), 8.1 (s, 2H),

7.7 (m, 1H), 7.3 (m, 1H), 6.8 (m, 1H), 2.6 (m, 1H), 0.7 (m, 2H), 0.4

(m, 2H). LC-MS purity 100% MS (ESI) m/z 178 [M + H]⁺. HRMS

[M + H]⁺= 178.0984 (theoretical 178.0982).

1-(2-Pyridyl)-3-(2,2,2-triﬂuoroethyl)urea (10). Solid, 45 mg (59%)

using triﬂuoroethylamine. ¹H NMR (DMSO- d₆) δ 9.5 (s, 1H), 8.7 (s,

1H), 8.2 (d, 1H), 7.7 (m, 1H), 7.4 (m, 1H), 7.0 (m, 1H), 4.0 (m, 2H).

¹³C NMR (DMSO-d₆, 126 MHz) δ 154.6, 152.9, 146.7, 138.5, 125.0,

117.4, 111.8, 39.5 (overlaps DMSO resonace). LC-MS purity 100%.

MS (ESI) m/z 220 [M + H]⁺. HRMS [M + H]⁺= 220.0702,

(theoretical 220.0699).

1,1-Diethyl-3-(2-pyridyl)urea (11). Oil, 40 mg (59%) using

diethylamine. ¹H NMR (DMSO- d₆) δ 8.6 (s, 1H), 8.2 (d, 1H),

7.85 (d, 1H), 7.7 (t, 1H), 7.0 (t, 1H), 3.4 (q, 4H), 1.1 (t, 6H). ¹³C

NMR (DMSO-d₆, 126 MHz) δ 155.5, 152.0, 147.3, 137.4, 117.6,

113.3, 40.6, 13.8. LC-MS purity 100%. MS (ESI) m/z 194 [M + H]⁺.

HRMS = 194.1296 (theoretical 194.1295).

1-(3,3′-Bipyridine-6-yl)-3-ethylurea (15). Isolated as a solid (25

mg, 25%) from 84 mg of 3-iodopyridine. ¹H NMR (DMSO-d₆)δ 1.10

(t, 3H), 3.19−3.22 (m, 2H), 7.45−7.53 (m, 2H), 8.00 (brs, 1H),

8.07−8.10 (m, 2H), 8.57 (brs, 2H), 8.90 (s, 1H), 9.33 (s, 1H), 3.54

(brs, 1H). MS (ESI) m/z 243 [M + H]⁺. HRMS [M + H]⁺243.1246

(theoretical 243.1240).

Ethyl 2-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)-1,3-thia-

zole-4-carboxylate (45a). Isolated as a solid (162 mg, 72%) from

1

165 mg ethyl 2-bromo-1,3-thiazole-4-carboxylate. H NMR (DMSO-

Methyl 3-(6-Nitropyridin-3-yloxy)benzoate. NaH (0.095 g, 2.36

mmol, 60% dispersion in oil) was added to a solution of the methyl 3-

hydroxybenzoate (0.36 mg, 2.36 mmol) in DMF (4 mL) at room

temperature with gas evolution and formation of a light-yellow

solution. 5-Bromo-2-nitropyridine (0.40 mg, 1.97 mmol) was added,

turning the reaction mixture into a brown solution. The reaction

mixture was heated at 60 °C for 1 h before being partitioned between

water and EtOAc. The layers were separated and the organic layer was

washed with water and dried over MgSO₄. The EtOAc was ﬁltered and

concentrated to give a brown oil that was puriﬁed by chromatography

on silica gel (hexanes/EtOAc) to give 0.32 g of the title compound,

which was used subsequently without further puriﬁcation. MS (ESI)

m/z 275 [M + H]⁺.

Methyl 3-(6-Aminopyridin-3-yloxy)benzoate (43). A mixture of

10% palladium on carbon (30 mg) in methanol (5 mL) and methyl 3-

(6-nitropyridin-3-yloxy)benzoate (320 mg, 1.17 mmol) was degassed

with nitrogen and left under a hydrogen atmosphere overnight. The

reaction mixture was ﬁltered, and the ﬁltrate was concentrated to give

230 mg of a clear oil consistent with the title compound, which was

used subsequently without further puriﬁcation. MS (ESI) m/z 245 [M

+ H]⁺.

d₆) δ 1.09 (t, 3H), 1.31 (t, 3H), 3.14−3.22 (m, 2H), 4.32 (q, 2 H),

7.60 (d, 1H), 7.80 (brs, 1H), 8.21 (dd, 1H), 8.54 (s, 1H), 8.77 (s, 1H),

9.52 (s, 1H). MS (ESI) m/z 321 [M + H]⁺.

Methyl 3-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)benzoate

(45b). Isolated as a solid (110 mg, 54%) from 147 mg of methyl 3-

1

bromobenzoate. H NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.15−3.24 (m,

2H), 3.88 (s, 3 H), 7.49 (d, 1H), 7.61 (t, 1H), 7.92−8.03 (m, 3H),

8.05 (dd, 1H), 8.16 (s, 1H), 8.53 (d, 1H) 9.32 (s, 1H). MS (ESI) m/z

300 [M + H]⁺.

Methyl 4-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)benzoate

(45c). Isolated as a solid (89 mg, 43%) from 148 mg of methyl 4-

1

bromobenzoate. H NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.15−3.21 (m,

2H), 3.86 (s, 3 H), 7.50 (d, 1H), 7.83 (d, 2H), 7.96 (t, 1H), 8.01 (m,

2H), 8.09 (dd, 1H), 8.59 (d, 1H) 9.34 (s, 1H). MS (ESI) m/z 300 [M

+ H]⁺.

Ethyl 6′-{[(Ethylamino)carbonyl]amino}-3,3′-bipyridine-5-car-

boxylate (45d). Isolated as a solid (78 mg, 48%) from 118 mg of

ethyl 5-bromonicotinate. ¹H NMR (DMSO-d₆) δ 1.09 (t, 3H), 1.35 (t,

3H), 3.14−3.25 (m, 2H), 4.37 (q, 2 H), 7.53 (d, 1H), 7.96 (t, 1H),

8.15 (dd, 1H), 8.47 (s, 1H), 8.64 (s, 1H), 9.04 (s, 1H), 9.13 (s, 1H),

9.37 (s, 1H). MS (ESI) m/z 315 [M + H]⁺.

3-(6-(3-Ethylureido)pyridin-3-yloxy)benzoate. A solution of meth-

yl 3-(6-aminopyridin-3-yloxy)benzoate 42 (0.23 mg) and ethyl-

Ethyl 2-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)-1,3-ben-

zothiazole-7-carboxylate (45f). Isolated as a solid (101 mg, 53%)

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from 147 mg of ethyl 2-bromo-1,3-benzothiazole-7-carboxylate.⁶⁷¹H

NMR (DMSO-d₆) δ 1.10 (t, 3H), 1.40 (t, 3H), 3.20 (m, 2H), 4.44 (q,

2 H), 7.68 (q, 2H), 7.82 (t, 1H), 8.10 (dd, 1H), 8.30 (d, 1H), 8.39

(dd, 1H), 8.94 (d, 1H), 9.59 (s, 1H). MS (ESI) m/z 371 [M + H]⁺.

1-(4-Chloropyridin-2-yl)-3-ethylurea. A suspension of 4-chloropyr-

idin-2-amine (2.186 g, 17 mmol), ethyl isocyanate (2.69 mL, 34.00

mmol), and chloroform (8 mL) was heated in the microwave at 100

°C for 1 h. The resultant solution was concentrated in vacuo to give

the title compound in quantitative yield. No further puriﬁcation was

N-(4-Bromopyridin-2-yl)-N′-ethylurea (51). A mixture of 4-

bromopyridin-2-amine (2 g, 11.6 mmol) and ethylisocyanate (0.91

mL, 11.6 mmol) in chloroform (10 mL) was heated at 110 °C for 2 h.

The reaction mixture was concentrated under reduced pressure, and

the residue was triturated with acetonitrile before being collected by

ﬁltration to give the title compound as a white solid (2.15 g). ¹H NMR

(DMSO-d₆) δ 1.08 (t, 3H), 3.12−3.18 (m, 2H), 7.16 (dd, 1H), 7.65

(br s, 1H), 7.74 (s, 1H), 8.07 (d, 1H), 9.29 (s, 1H). MS (ESI) m/z

244, 246 [M + H]⁺.

1-(4-Cyanopyridin-2-yl)-3-ethylurea. In a microwave reactor vessel

ﬁtted with a stir bar, 2-aminoisonicotinonitrile (2.0 g, 16.79 mmol)

and ethylisocyanate (2.66 mL, 33.58 mmol) were suspended in CHCl₃

(8 mL). The vessel was sealed and heated at 100 °C for 1 h in a

microwave reactor. The reaction mixture was concentrated under

reduced pressure to provide 3.1 g of the title compound as a cream-

colored solid. ¹H NMR (DMSO-d₆) δ 1.07 (t, 3H), 3.17 (m, 2H), 7.34

(m, 2H), 7.91 (s, 1H), 8.40 (d, 1H), 9.47 (s, 1H). LC MS (ESI) m/z

191, 193 [M + H].

1

performed. H NMR (DMSO-d₆) δ 9.31 (s, 1H), 8.16 (d, 1H), 7.63

(m, 1H), 7.59 (m, 1H), 7.03 (m, 1H), 3.16 (m, 2H), 1.07 (t, 3H). MS

(ESI) m/z 200, 202 [M + H]⁺.

1-(5-Bromo-4-chloropyridin-2-yl)-3-ethylurea (46a). A solution of

1-(4-chloropyridin-2-yl)-3-ethylurea (3.39 g, 16.98 mmol), N-

bromosuccinimide (3.02 g, 16.98 mmol), acetonitrile (32 mL), and

DMF (10 mL) were combined and heated at 80 °C for 2 h. Upon

cooling to RT, a precipitate formed. Water was added, and the solid

was collected and washed with water to give 2.78 g (59%) of the title

compound which was used without further puriﬁcation. ¹H NMR

(DMSO-d₆) δ 9.37 (s, 1H), 8.44 (s, 1H), 7.92 (s, 1H), 7.17 (m, 1H),

3.15 (m, 2H), 1.06 (t, 3H). MS (ESI) m/z 278, 280 [M + H]⁺.

4-Bromo-5-iodopyridin-2-amine. 4-Bromopyridin-2-amine (2.5g,

14.45 mmol) was dissolved in DMF (6 mL)/CHCl₃(20 mL), 1-

iodopyrrolidine-2,5-dione (6.50 g, 28.90 mmol) was added, and the

mixture was stirred at 45 °C for 2 days. CHCl₃was evaporated, and

the remaining solution was poured into water (15 mL) and extracted

with EtOAc (15 mL × 3). The organic phase was concentrated and

puriﬁed by chromatography on silica gel, and elution with a hexanes−

EtOAc gradiant to give the title compound (3.2 g). ¹H NMR (DMSO-

d₆) 4.51 (br, 2H), 6.80 (s, 1H), 8.35 (s, 1H). MS (ESI) m/z 299.9 [M

+ H]⁺.

N-Ethyl-N′-(4-phenylpyridin-2-yl)urea (52c). 1-(4-Bromopyridin-

2-yl)-3-ethylurea 47 (500 mg, 2.05 mmol), phenylboronic acid (749

mg, 6.15 mmol), tetrakis(triphenylphosphine)palladium(0) (237 mg,

0.20 mmol), and CsCO₃(2 g, 6.15 mmol) were suspended in a

solution of dioxane−water (4:1). The reaction mixture was degassed

and then heated to 110 °C in a microwave reactor for 30 min. The

reaction mixture was ﬁltered through a pad of Celite and then

partitioned between EtOAc and water. The organic layer was dried

over Na₂SO₄, ﬁltered, and concentrated. The residue was puriﬁed by

silica gel column chromatography (95:5 CH₂Cl₂/MeOH) to give 550

1

mg of the title compound as a white solid (95% yield). H NMR

(DMSO-d₆) δ 1.3 (t, 3H), 3.45 (m, 2H), 7.0 (s, 1H), 7.1 (d, 1H), 7.3−

7.9 (m, 6H), 8.2 (d, 1H), 9.3 (br s, 1H). MS (ESI) m/z 242 [M + H]⁺.

N-(3,4′-Bipyridin-2′-yl)-N′-ethylurea (52b). Prepared from 750 mg

47 and 630 mg of 3-pyridyl boronic acid as described for 48d to aﬀord

570 mg of the title compound. ¹H NMR (DMSO-d₆) δ 1.3 (t, 3H), 3.2

(m, 2H), 7.3 (d, 1H), 7.5 (m, 1H), 7.7 (s, 1H), 7.9−8.1 (m, 2H), 8.3

(d, 1H), 8.65 (d, 1H), 8.9 (s, 1H), 9.3 (s, 1H). MS (ESI) m/z 243 [M

+ H]⁺.

1-(4-Bromo-5-iodopyridin-2-yl)-3-ethylurea (46b). 4-Bromo-5-io-

dopyridin-2-amine (3.2g, 10.71 mmol) was dissolved in dry chloro-

form (15 mL). Ethylisocyanate (2.52 mL, 32.12 mmol) was added, and

the reaction mixture was heated at reﬂux for 24 h. The reaction was

cooled to room temperature, and hexanes were added. The resulting

precipitate was collected by ﬁltration to give the desired product (3.14

1

1-(5-Bromo-4-cyanopyridin-2-yl)-3-ethylurea (47). A suspension

of 1-(4-cyanopyridin-2-yl)-3-ethylurea (3.50 g, 18.40 mmol) and NBS

(3.93 g, 22.08 mmol) in acetonitrile (100 mL) was heated at 60 °C.

Upon heating, the suspension became homogeneous and turned dark-

brown in color. After heating for 12 h, a precipitate formed. The

mixture was cooled to room temperature, and the solids were ﬁltered

and washed ﬁrst with water, and then with hexanes. Drying of the

collected solids in vacuo gave 2.61 g of the title compound in a 52%

g). H NMR (DMSO-d₆) 1.06 (t, 3H), 3.32 (q, 2H), 7.24 (br, 1H),

8.05 (s, 1H), 8.52 (s, 1H), 9.31 (s, 1H). MS (ESI) m/z 372 [M + H]⁺.

Ethyl 4′-Chloro-6′-(3-ethylureido)-3,3′-bipyridine-5-carboxylate

(47a). The 1-(5-bromo-4-chloropyridin-2-yl)-3-ethylurea 46a (0.404

g, 1.45 mmol), ethyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

nicotinate (0.482 g, 1.74 mmol), and CsCO₃(0.945 g, 2.90 mmol)

were added to a microwave vessel. The vessel was degassed and

purged with N₂. Tetrakis (triphenylphosphine)palladium(0) (0.168 g,

0.15 mmol) was added, and the vessel was degassed and purged with

N₂. Dioxane (10 mL) and water (2.5 mL) were added, and the vessel

was degassed and purged with N₂three more times. The vessel was

placed in the microwave and heated at 100 °C for 2 h. The organic

layer was separated and concentrated in vacuo. After puriﬁcation by

silica gel chromatography (0−10% MeOH/CH₂Cl₂), the resultant

solid was triturated with hot acetonitrile to give 0.339 g (67%) of the

1

yield. H NMR (DMSO-d₆) δ 1.07 (t, 3H), 3.16 (m, 2H), 7.04 (m,

1H), 8.12 (s, 1H), 8.60 (s, 1H), 9.56 (s, 1H). MS (ESI) m/z 269, 271

[M + H]⁺.

N-(5′-Bromo-3,4′-bipyridin-2′-yl)-N′-ethylurea (49c). Prepared

from 165 mg of 52b and 121 mg of NBS as described for 49 to

1

aﬀord 200 mg of the title compound. H NMR (DMSO-d₆) δ 1.1 (t,

3H), 3.2 (m, 2H), 7.4 (m, 1H), 7.5−7.6 (m, 1H), 7.7 (s, 1H), 7.9 (d,

1H), 8.5 (d, 1H), 8.6 (s, 1H), 8.7 (d, 1H), 9.4 (s, 1H). MS (ESI) m/z

321, 323 [M + H]⁺.

1

title compound. H NMR (DMSO-d₆) δ 9.47 (s, 1H), 9.12 (m, 1H),

8.92 (m, 1H), 8.37 (m, 1H), 8.33 (s, 1H), 7.85 (s, 1H), 7.46 (m, 1H),

4.38 (q, 2H), 3.18 (m, 2H), 1.35 (t, 3H), 1.09 (t, 3H). MS (ESI) m/z

349, 351 [M + H]⁺.

N-(5-Bromo-4-phenylpyridin-2-yl)-N′-ethylurea (53c). A mixture

of 1-ethyl-3-(4-phenylpyridin-2-yl)urea 52c (530 mg, 2.20 mmol) and

N-bromosuccinamide (391 mg, 2.20 mmol) in DMF (5 mL) was

heated at 80 °C for 2 h. Then the reaction mixture was partitioned

between water and EtOAc. The layers were separated, and the organic

layer was washed with 5% sodium thiosulfate solution, water, and brine

before being dried (MgSO₄) and concentrated. The solid obtained was

washed with acetonitrile and dried in vacuo to give oﬀ-white solid (250

mg). The acetonitrile wash was further concentrated and puriﬁed by

normal phase chromatography (silica gel, 2−5% MeOH in CH₂Cl₂) to

give additional 80 mg of the product as a white solid (330 mg total

Ethyl 4′-Bromo-6′-(3-ethylureido)-3,3′-bipyridine-5-carboxylate

(47b). 1-(4-Bromo-5-iodopyridin-2-yl)-3-ethylurea 45b (1.33g, 3.59

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

nate (1.049 g, 3.59 mmol), tetrakis(triphenylphosphine)palladium(0)

(0.415 g, 0.36 mmol), and K₂CO₃(0.745 g, 5.39 mmol) were

suspended in a mixture of DMF (10 mL) and water (1.0 mL). The

suspension was degassed, purged with nitrogen, and heated at 100 °C

for 1.5 h. The reaction mixture was cooled to room temperature and

ﬁltered; the ﬁltrate was concentrated and puriﬁed by column

chromatography on silica gel (0−10% MeOH/CH₂Cl₂) to give the

title compound as a solid (1.32 g). ¹H NMR (CDCl₃) δ 1.29 (t, 3H),

1.45 (t, 3H), 3.45 (q, 2H), 4.47 (q, 2H), 7.30 (br, 1H), 8.12 (s, 1H),

8.38 (t, 1H), 8.84 (2s, 2 × H), 9.29 (s, 1H). MS (ESI) m/z 393, 395

[M + H]⁺.

1

weight, 47%). H NMR (CDCl₃) δ 1.21 (t, 3H), 3.33−3.42 (m, 2H),

6.9 (s, 1H), 7.25 (s, 1H), 7.42−7.46 (m, 5 H), 8.34 (s, 1H), 8.84 (s,

1H). MS (ESI) m/z 320, 322 [M + H]⁺.

Ethyl 4′-Cyano-6′-(3-ethylureido)-3,3′-bipyridine-5-carboxylate

(49). 1-(5-Bromo-4-cyanopyridin-2-yl)-3-ethylurea 47 (1.1 g, 4.09

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

(1.35 g, 4.91 mmol), CsCO₃(1.59 g, 4.91 mmol), and tetrakis-

(triphenylphosphine)palladium(0) (422 mg, 0.41 mmol) were

combined in a microwave vial and suspended in a solution of

dioxane/water (4:1). The reaction mixture was degassed and then

heated at 100 °C in a microwave reactor for 1 h. The reaction mixture

was ﬁltered through a pad of Celite, and the ﬁltrate was partitioned

between water and EtOAc. The layers were separated, and the aqueous

was back extracted with EtOAc three times. The combined extract was

washed with water and brine, dried over MgSO₄, and concentrated.

The residue was puriﬁed by silica gel column chromatography

(EtOAc/hexanes). The fractions containing product were concen-

trated, and the residue was triturated with acetonitrile. Isolation gave

and EtOAc. The layers were separated, and the aqueous was back

extracted with EtOAc three times. The combined extracts were washed

with water and brine, dried over MgSO₄, and concentrated. The

residue was puriﬁed by silica gel chromatography (1% to 3% MeOH in

CH₂Cl₂) to obtain a oﬀ-white solid (155 mg, 55%) as the product. ¹H

NMR (DMSO-d₆) δ 1.09 (t, 3H), 1.27 (t, 3H), 3.18−3.22 (m, 2H),

4.27 (q, 2H), 7.12−7.15 (m, 2H), 7.32−7.34 (m, 3H), 7.54 (s, 1H),

7.85 (brs, 1H), 7.95 (s, 1H), 8.33 (s, 1H), 8.50 (d, 1H), 8.9 (d, 1H),

9.4 (s, 1H). MS (ESI) m/z 391 [M + H]⁺.

5-Bromo-2-(3-ethylureido)isonicotinamide (55). A mixture of

methyl 2-amino-5-bromoisonicotinate (3 g, 13 mmol) and ethyl-

isocyanate (1.12 mL, 14.3 mmol) in chloroform (12 mL) was heated

in a microwave reactor at 110 °C for 3 h. The reaction was

concentrated, and 50 mL of 7N NH₃in MeOH was added. The

resulting mixture was stirred at room temperature overnight. After

concentration of the solution, the residue was triturated with

1

970 mg of the title compound as a light-yellow solid (70% yield). H

NMR (DMSO-d₆) δ 1.1 (t, 3H), 1.35 (t, 3H), 3.2 (m, 2H), 4.4 (q,

2H), 7.25 (m, 1H), 8.1 (s, 1H), 8.5 (t, 1H), 8.6 (s, 1H), 9.05 (d, 1H),

9.2 (d, 1H), 9.6 (s, 1H). MS (ESI) m/z 340 [M + H]⁺.

1

acetonitrile to give a white solid (3.5 g). H NMR (DMSO-d₆) δ

1.06 (t, 3H), 3.1−3.3 (m, 2H), 7.1 (m, 1H), 7.6 (s, 1H), 8.0 (m, 1H),

Ethyl-6′-(3-ethylureido)-4′-(N′-hydroxycarbamimidoyl)-3,3′-bi-

pyridine-5-carboxylate. Hydroxylamine (0.099 mL, 1.62 mmol) was

added to a suspension of ethyl 4′-cyano-6′-(3-ethylureido)-3,3′-

bipyridine-5-carboxylate 49 (500 mg, 1.47 mmol) in ethanol (10

mL), and the reaction mixture was heated at 65 °C for 3 h. After

cooling, solvent was removed to give a white solid (457 mg). MS

(ESI) m/z 373 [M + H]⁺.

Ethyl-6′-(3-ethylureido)-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-3,3′-

bipyridine-5-carboxylate. Acetic anhydride (0.057 mL, 0.60 mmol)

was added to a solution of ethyl 6′-(3-ethylureido)-4′-(N′-hydrox-

ycarbamimidoyl)-3,3′-bipyridine-5-carboxylate (150 mg, 0.40 mmol)

in acetic acid (5 mL), and the reaction mixture was heated at 85 °C

overnight The reaction mixture was diluted with water and extracted

with EtOAc. The layers were separated, and the organic layer was

dried over Na₂SO₄, ﬁltered, and concentrated. The residue was

puriﬁed by silica gel column chromatography (EtOAc−hexanes) to

aﬀord 110 mg of the title compound as a white solid. MS (ESI) m/z

397 [M + H]⁺.

Ethyl 5-[6-(Ethylcarbamoylamino)-4-(4-pyridyl)-3-pyridyl]-

pyridine-3-carboxylate (54a). Dichlorobis(triphenylphosphine)-

palladium(II) (60 mg, 0.086 mmol) as added to a solution of ethyl

4′-chloro-6′-(3-ethylureido)-3,3′-bipyridine-5-carboxylate (46a) (300

mg, 0.86 mmol) in dioxane (4 mL) in a 10 mL microwave vial. The

mixture was stirred for 5−10 min before adding 4-pyridyl boronic acid

(1.29 mmol, 1.5 equiv), followed by a solution of NaHCO₃(0.774 g,

9.22 mmol) in water (2 mL). The vial was sealed and the mixture was

stirred for 10 min and then heated in a microwave reactor at 120 °C

for 4 h. EtOAc (3 mL) and brine (1 mL) were added to the reaction

mixture, and the organic layer was separated and dried over Na₂SO₄.

The volatiles were removed under vacuum, and the residue was

puriﬁed by ﬂash chromatography over silica gel, elution with isocratic

30% EtOAc in hexanes over 2 min, then gradient elution to 90%

EtOAc in hexanes to give the title compound as a white solid in 65%

yield. ¹H NMR (300 MHz, CD₃OD) δ 1.22 (t, 3H), 1.33 (t, 3H), 3.36

(q, 2H), 4.37 (q, 2H), 7.26 (d, 2H), 7.30 (s, 1H), 8.13 (s, 1H), 8.39 (s,

1H), 8.49 (d, 2H), 8.99 (s, 1H). MS (ESI) m/z 392 [M + H]⁺.

Ethyl 5-[6-(Ethylcarbamoylamino)-4-(3-pyridyl)-3-pyridyl]-

pyridine-3-carboxylate (54b). Prepared from 530 mg of 53b and

261 mg of ethyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

nicotinate as described for 49 to aﬀord 160 mg of the title compound.

¹H NMR (DMSO-d₆) δ 1.09 (t, 3H), 1.27 (t, 3H), 3.14−3.24 (m,

8.3 (s, 1H), 9.3 (s, 1H). MS (ESI) m/z 287, 289 [M + H]⁺.

5-Bromo-2-(3-ethylureido)pyridine-4-carbothioamide (56). A

mixture of 5-bromo-2-(3-ethylureido)isonicotinamide 55 (1.25 g,

4.35 mmol) and Lawesson’s Reagent (1.76 g, 4.35 mmol) in THF (20

mL) was heated at 70 °C overnight. Solid precipitates were ﬁltered and

washed with THF. The ﬁltrate was concentrated, and the residue was

triturated with THF to isolate additional solids. The solids were

1

combined to give 1 g of the title compound. H NMR (DMSO-d₆) δ

1.07 (t, 3H), 3.1−3.3 (m, 2H), 7.37 (m, 1H), 7.5 (s, 1H), 8.3 (s, 1H),

9.3 (s, 1H), 9.8 (s, 1H), 10.3 (s, 1H). MS (ESI) m/z 303, 305 [M +

H]⁺.

1-(5-Bromo-4-(4-tert-butylthiazol-2-yl)pyridin-2-yl)-3-ethylurea

(57a). A mixture of 5-bromo-2-(3-ethylureido)pyridine-4-carbothioa-

mide 56 (290 mg, 0.96 mmol) and 1-bromo-3,3-dimethylbutan-2-one

(0.387 mL, 2.87 mmol) in acetonitrile (8 mL) was heated at 80 °C for

3 h. The reaction mixture was concentrated and puriﬁed by silica gel

chromatography (1−3% MeOH in CH₂Cl₂) to give a white solid (230

1

mg, 63%). H NMR (DMSO-d₆) δ 1.08 (t, 3H), 1.37 (s, 9H), 3.16−

3.24 (m, 2H), 7.37 (brs, 1H), 7.64 (s, 1H), 8.35 (s, 1H), 8.50 (s, 1H),

9.36 (s, 1H). MS (ESI) m/z 383, 385 [M + H]⁺.

1-(5-Bromo-4-(4-hydroxy-4-(triﬂuoromethyl)-4,5-dihydrothiazol-

2-yl)pyridin-2-yl)-3-ethylurea. A mixture of 5-bromo-2-(3-

ethylureido)pyridine-4-carbothioamide 56 (1.1 g, 3.63 mmol) and 3-

bromo-1,1,1-triﬂuoropropan-2-one (2.26 mL, 21.8 mmol) in acetoni-

trile (25 mL) was heated at 80 °C for 4 h. A clear solution resulted

within an hour. The mixture was concentrated, and the residue was

partitioned between water and EtOAc. The layers were separated, and

the organic was washed with water and brine before being dried

(MgSO₄) and concentrated to give a light-yellow solid. Puriﬁcation by

silica gel chromatography (2−5% MeOH in CH₂Cl₂) gave the title

1

compound as a white solid (470 mg, 40%). H NMR (DMSO-d₆) δ

1.06 (t, 3H), 3.12−3.18 (m, 2H), 3.60 (dd, 1H), 3.90 (dd, 1H), 7.13

(brs, 1H), 7.98 (s, 1H), 8.47 (s, 1H), 9.41 (s, 1H). MS (ESI) m/z 414,

416 [M + H]⁺.

N-{5-Bromo-4-[4-(triﬂuoromethyl)-1,3-thiazol-2-yl]pyridin-2-yl}-

N′-ethylurea (57b). Triﬂuoroacetic anhydride (1.13 mL, 8.0 mmol)

and triethylamine (1.113 mL, 8.0 mmol) were added sequentially to a

mixture of 1-(5-bromo-4-(4-hydroxy-4-(triﬂuoromethyl)-4,5-dihydro-

thiazol-2-yl)pyridin-2-yl)-3-ethylurea (2.2 g, 5.32 mmol) in acetonitrile

(25 mL). The reaction mixture was allowed to stir overnight at room

temperature. Because the reaction was not complete as determined by

LC-MS, another 150 μL each of triﬂuoroacetic anhydride and

triethylamine were added and stirring was continued for additional 3

h. The reaction mixture was concentrated, and the residue was

partitioned between water and EtOAc. The layers were separated, and

the organic layer was washed with aqueous NaHSO₄, water, and brine

before being dried (MgSO₄) and concentrated. The residue was taken

up in CH₂Cl₂and some material precipitated. The solids were

collected by ﬁltration and dried (617 mg). The ﬁltrate was

concentrated, and the residue was puriﬁed by normal phase (silica

gel, 1−3% MeOH in CH₂Cl₂) to give 520 mg of additional solid. Both

solids were consistent with the title material for a total weight 1.14 g

2H), 4.28 (q, 2H), 7.35−7.39 (m, 1 H), 7.60 (s, 2H), 7.80 (s, 1H),

7.97 (s, 1H), 8.35 (s, 1H), 8.37 (s, 1H), 8.52 (d, 2H), 8.94 (s, 1H),

9.45 (s, 1H). MS (ESI) m/z 392 [M + H]⁺.

Ethyl 6′-(3-Ethylureido)-4′-phenyl-3,3′-bipyridine-5-carboxylate

(54c). 1-(5-Bromo-4-phenylpyridin-2-yl)-3-ethylurea 53c (230 mg,

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

nicotinate (398 mg, 1.44 mmol), CsCO₃(468 mg, 1.44 mmol), and

tetrakis(triphenylphosphine)palladium(0) (83 mg, 0.07 mmol) in a

sealed microwave reactor vial were degassed with N₂. Solvent (5 mL,

dioxane−water, 4:1) was added, and the mixture was heated in a

microwave reactor at 100 °C for 30 min. The reaction mixture was

ﬁltered through Celite, and the ﬁltrate was partitioned between water

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(52%). ¹H NMR (DMSO-d₆) δ 1.07 (t, 3H), 3.11−3.17 (m, 2H), 7.24

(t, 1H), 8.35 (s, 1H), 8.50 (s, 1H), 8.77 (s, 1H), 9.34 (s, 1H). MS

(ESI) m/z 420, 422 [M + H]⁺.

tetrakis(triphenylphosphine)palladium(0) (59 mg, 0.05 mmol) were

placed in a microwave reactor vial and degassed. Solvent (dioxane−

water, 4:1, 10 mL) was added, and the reaction mixture was heated at

100 °C in a microwave reactor for 30 min. The reaction was ﬁltered

through Celite and partitioned between water and EtOAc. The layers

were separated, and the aqueous was back extracted with EtOAc 3

times. The combined extracts were washed with water and brine, dried

(MgSO₄), and concentrated. The residue was taken up in CH₂Cl₂with

1% MeOH and solids precipitated. The solids were collected and dried

in vacuo to aﬀord 25 mg of material (MS (ESI) m/z 458 [M + H]⁺)

that was taken up in 5 mL MeOH. A 2N aqueous solution of LiOH

(0.5 mL) was added, and the solution was stirred at 40 °C for 2 h. The

solvent was removed, and the residue was dissolved in water. A 1N

HCl aqueous solution was added, and solids that precipitated were

collected and dried in vacuo. The material was consistent with title

compound (15 mg, 8% over 2 steps). ¹H NMR (DMSO-d₆) δ 1.09 (t,

3H), 3.16−3.24 (m, 2H), 7.50 (s, 1H), 8.09 (s, 1H), 8.25 (s, 1H), 8.68

(d, 2H), 9.67 (s, 1H). MS (ESI) m/z 444 [M + H]⁺. HRMS [M + H]⁺

= 444.0412 (theoretical 444.0406).

Ethyl 4′-(4-tert-Butylthiazol-2-yl)-6′-(3-ethylureido)-3,3′-bipyri-

dine-5-carboxylate (58a). A mixture of 57a (220 mg, 0.57 mmol),

ethyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinate (318

mg, 1.15 mmol), CsCO₃(374 mg, 1.15 mmol), and tetrakis-

(triphenylphospine)palladium(0) (131 mg, 0.113 mmol) were taken

in a microwave reactor vial and degassed with nitrogen. Dioxane (8

mL) and water (2 mL) were added, and the mixture was degassed

again. The mixture has heated in a microwave reactor at 100 °C for 30

min. After cooling to room temperature, the mixture was ﬁltered, and

the ﬁltrate was partitioned between water and EtOAc. The layers

separated, and the organic layer was washed with aqueous NaHCO₃,

water, and brine. The solution was dried (MgSO₄) and concentrated

to aﬀord a light-brown solid. The solid was triturated with acetonitrile

1

to give a white solid (395 mg). H NMR (DMSO-d₆) δ 1.05 (s, 9H),

1.09 (t, 3H), 1.30 (t, 3H), 3.16−3.24 (m, 2H), 4.33 (q, 2H), 7.41 (s,

1H), 7.66 (brs, 1H), 8.07 (d, 1H), 8.09 (s, 1H), 8.3 (s, 1H), 8.7 (d,

1H), 9.0 (s, 1H), 9.46 (s, 1H). MS (ESI) m/z 454 [M + H]⁺.

Ethyl 6′-{[(Ethylamino)carbonyl]amino}-4′-[4-(triﬂuoromethyl)-

1,3-thiazol-2-yl]-3,3′-bipyridine-5-carboxylate (58b). 1-(5-Bromo-4-

(4-(triﬂuoromethyl)thiazol-2-yl)pyridin-2-yl)-3-ethylurea 57b (2 g,

5.06 mmol), dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine

(0.724 g, 1.52 mmol), Pd₂(dba)₃(0.463 g, 0.51 mmol), ethyl 5-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)nicotinate (2.80 g, 10.12

mmol), and CsCO₃(3.63 g, 11.13 mmol) were placed in a round-

bottom ﬂask and degassed with nitrogen. Dioxane (30 mL) and water

(5 mL) were added, and the mixture was degassed by bubbling though

N₂gas. The reaction mixture was heated at 95 °C for 1 h. After cooling

to room temperature, the mixture was ﬁltered through Celite and

partitioned between water and EtOAc. The layers were separated, and

the aqueous was back extracted twice with EtOAc. The combined

extracts were washed with water and brine, dried (MgSO₄), and

concentrated. The residue was puriﬁed by silica gel chromatography

(hexanes/EtOAc) to give a light-brown solid that was triturated with

acetonitrile to give a white solid (2.2 g) consistent with the title

2-(6-(3-Ethylureido)-4-(4-(triﬂuoromethyl)thiazol-2-yl)pyridin-3-

yl)benzo[d]thiazole-7-carboxylate (55b). A mixture of 59 (300 mg,

0.68 mmol), ethyl 2-bromobenzo[d]thiazole-7-carboxylate (194 mg,

0.68 mmol), CsCO₃(221 mg, 0.68 mmol), and tetrakis-

(triphenylphosphine)palladium(0) (78 mg, 0.07 mmol) were placed

in a microwave reactor vial and degassed. Solvent (dioxane−water, 4:1,

10 mL) was added, and the reaction mixture was heated in a

microwave reactor at 100 °C for 30 min. The reaction was ﬁltered

through Celite and partitioned between water and EtOAc. The layers

were separated, and the aqueous was back extracted with EtOAc 3

times. The combined extracts were washed with water and brine, dried

(MgSO₄), and concentrated. The residue was puriﬁed by silica gel

chromatography (1−3% gradient of MeOH in CH₂Cl₂) to aﬀord 60

mg of a solid (MS (ESI) m/z 522 [M + H]⁺) that was taken up in 2

mL of 1:1 THF−water. A 2N aqueous solution of LiOH (0.5 mL) was

added, and the solution was stirred at 40 °C for 3 h. The solvent was

removed, and the residue was dissolved in water. A 1N HCl aqueous

solution was added, and solids that precipitated were collected and

dried in vacuo. The material was consistent with title compound (40

1

compound. H NMR (DMSO-d₆) δ 1.11 (t, 3H), 1.31(t, 3H), 3.18−

1

3.24 (m, 2H), 4.34 (q, 2H), 7.57 (brs, 1H), 8.16−8.18 (m, 1H), 8.21

(s, 1H), 8.39 (s, 1H), 8.58 (s, 1H), 8.75 (d, 1H), 9.10 (s, 1H), 9.52 (s,

1H). MS (ESI) m/z 466 [M + H]⁺.

mg, 12% over 2 steps). H NMR (DMSO-d₆) δ 1.1 (t, 3H), 3.2 (m,

2H), 7.5 (s, 1H), 7.7 (t, 1H), 8.1 (d, 1H), 8.18 (s, 1H), 8.20 (d, 1H),

8.6 (s, 1H), 8.75 (s, 1H), 9.7 (s, 1H), 13.8 (s, 1H). MS (ESI) m/z 494

[M + H]⁺. HRMS [M + H]⁺= 494.0558 (theoretical 494.0563).

2-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)-1,3-thiazole-5-

carboxylic Acid (20). Methyl 2-(6-{[(ethylamino)carbonyl]amino}-

pyridin-3-yl)-1,3-thiazole-5-carboxylate 44e (0.185 g, 0.60 mmol) was

taken in MeOH (6 mL), and 2N LiOH (1 mL) was added. The

resulting mixture was stirred at 45 °C for 2 h. The solvent was

removed, and the residue was dissolved in water before being acidiﬁed

with 1N HCl. The precipitated solid was collected by ﬁltration and

washed with water and dried in vacuo to aﬀord 0.17 g of material

6-(3-Ethylureido)-4-(4-(triﬂuoromethyl)thiazol-2-yl)pyridin-3-yl-

boronic Acid (59). A 2.0 M solution of i-PrMgCl in THF (800 mL,

1.60 mol, 2.53 equiv) was added to a suspension of 57b (250 g, 0.633

mol) in anhydrous THF (6.25 L) −50 °C over 45−60 min (keeping

the temperature below −35 °C). The reaction was stirred at −40 °C

for an hour and then cooled to −78 °C. A 2.5 M solution of n-BuLi in

hexane (1.42 L, 3.54 mol, 5.6 equiv) was added over 1 h (keeping the

temperature below −65 °C). The reaction was stirred at −78 °C for 1

h before adding trimethylborate (775 mL, 10.7 equiv) in one portion.

The mixture was allowed to warm to room temperature with stirring

for 2 h. HCl (6N, 1.5 L) was added carefully, and the mixture was

stirred for 45 min before being diluted with EtOAc (6.25 L) and water

(3.5 L). The layers were separated, and the aqueous layer was adjusted

to pH ∼7−8 with sodium hydroxide before extraction with EtOAc (3

× 2 L). All organic layers were combined and concentrated under

reduced pressure. The residue was suspended in water (3 L) and

MTBE (1.5 L) with stirring for 1 h. Solid material was collected by

vacuum ﬁltration to give the ﬁrst batch of material. The ﬁltrate (water

and MTBE) was concentrated under vacuum, and the residue was

triturated with MTBE to give a second batch of material. The two

batches were combined, triturated with water, and dried in a vacuum

oven to give compound the title compound as an oﬀ-white solid (183

g, 79%). ¹H NMR (DMSO-d₆) δ 1.10 (t, 3H), 3.18 (m, 2H), 7.75 (brt,

1H), 7.91 (s, 1H), 8.18 (br, 2H), 8.31 (s, 1H), 8.64 (s, 1H), 9.31 (s,

1H). MS (ESI) m/z 361 [M + H]⁺.

1

consistent with the title compound. H NMR (DMSO-d₆) δ 1.09 (t,

3H), 3.16−3.22 (m, 2H), 7.60 (d, 1H), 7.81 (t, 1H), 8.26 (dd, 1H),

8.38 (s, 1H), 8.81 (s, 1H), 9.54 (s, 1H), 13.54 (brs, 1H). MS (ESI) m/

z 293 [M + H]⁺. HRMS [M + H]⁺293.0716 (theoretical 293.0697).

Compounds 14, 16−19, 21, 24−26, and 25−34 were made

similarly to the synthesis of 20.

3-(6-(3-Ethylureido)pyridin-3-yloxy)benzoic Acid (14). Isolated as

a solid (63 mg, 62%) from 100 mg of 3-(6-(3-ethylureido)pyridin-3-

1

yloxy)benzoate. H NMR (DMSO-d₆) δ 1.07 (t, 3H), 3.16−3.22 (m,

2H), 7.25−7.28 (m, 1H), 7.36 (s, 1H), 7.45−7.55 (m, 3H), 7.60−7.71

(m, 2H), 8.05 (s, 1H), 9.21 (s, 1H), 13.15 (s, 1H). ¹³C NMR (DMSO-

d₆, 126 MHz) δ 166.6, 157.9, 154.5, 150.3, 146.2, 138.8, 132.6, 130.9,

130.4, 123.8, 121.7, 116.8, 112.6, 33.8, 15.4. MS (ESI) m/z 302 [M +

H]⁺. HRMS [M + H]⁺302.1139 (theoretical 302.1135).

2-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)-1,3-thiazole-4-

carboxylic Acid (16). Isolated as a solid (53 mg, 65%) from 85 mg of

1

44a. H NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.14−3.23 (m, 2H), 7.59

2-{6-{[(Ethylamino)carbonyl]amino}-4-[4-(triﬂuoromethyl)-1,3-

thiazol-2-yl]pyridin-3-yl}-1,3-thiazole-5-carboxylate (33). A mixture

of 59 (225 mg, 0.51 mmol), methyl 2-bromothiazole-5-carboxylate

(113 mg, 0.51 mmol), CsCO₃(166 mg, 0.51 mmol), and

(d, 1H), 7.80 (t, 1H), 8.21 (dd, 1H), 8.46(s, 1H), 8.77 (s, 1H), 9.51 (s,

1H), 13.13 (brs, 1H). ¹³C NMR (DMSO-d₆, 126 MHz) δ 164.7,

162.0, 154.8, 154.2, 147.9, 145.2, 136.0, 128.2, 122.1, 111.5, 33.9, 15.3.

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MS (ESI) m/z 293 [M + H]⁺. HRMS [M + H]⁺293.0697 (theoretical

293.0703).

111.9, 33.9, 15.3; .MS (ESI) m/z 363 [M + H]⁺. HRMS [M + H]⁺=

363.1468 (theoretical 363.1452).

3-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)benzoic Acid

(17). Isolated as a solid (43 mg, 56%) from 80 mg of 44b. ¹H

NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.16−3.22 (m, 2H), 7.48−7.56 (m,

2H), 7.84−7.91 (m, 2H), 8.02−8.05 (m, 2H), 8.15 (s, 1H), 8.53 (s,

1H), 9.33 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167.8, 154.6,

153.0, 144.6, 136.9, 136.3, 135.0, 129.0, 128.8, 128.2, 128.1, 126.6,

111.5, 33.8, 15.3. MS (ESI) m/z 286 [M + H]⁺. HRMS [M + H]⁺

286.1184 (theoretical 286.1186).

4′-(4-tert-Butylthiazol-2-yl)-6′-(3-ethylureido)-3,3′-bipyridine-5-

carboxylic Acid (30). Isolated as a solid (78 mg, 83%) from 100 mg of

58a. ¹H NMR (DMSO-d₆) δ 1.05 (s, 9H), 1.10 (t, 3H), 3.16- 3.27 (m,

2H), 7.39 (s, 1H), 7.66 (br s, 1H), 8.03 (s, 1H), 8.06 (s, 1H), 8.28 (s,

1H), 8.65 (d, 1H), 9.00 (s, 1H), 9.44 (s, 1H), 13.43 (s, 1H). MS (ESI)

m/z 426 [M + H]⁺. HRMS [M + H]⁺= 426.1596 (theoretical

426.1594).

6′-{[(Ethylamino)carbonyl]amino}-4′-[4-(triﬂuoromethyl)-1,3-

thiazol-2-yl]-3,3′-bipyridine-5-carboxylic Acid (31). Isolated as a

solid (60 mg, 88%) from 70 mg of 58b. ¹H NMR (DMSO-d₆) δ 1.11

(t, 3H), 3.18−3.24 (m, 2H), 7.57 (brs, 1H), 8.15−8.18 (m, 1H), 8.22

(s, 1H), 8.37 (s, 1H), 8.57 (s, 1H), 8.72 (s, 1H), 9.08 (s, 1H), 9.51 (s,

1H), 13.53 (s, 1H). LC MS (ESI) m/z 438.0848 [M + H]⁺; HRMS

[M + H]⁺= (theoretical 438.0842).

4-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)benzoic Acid

1

(18). Isolated as a solid (56 mg, 92%) from 64 mg of 44c. H NMR

(DMSO-d₆) δ 1.09 (t, 3H), 3.15−3.21 (m, 2H), 3.86 (s, 3 H), 7.50 (d,

1H), 7.80 (d, 2H), 7.98 (t, 1H), 7.99 (d 2H), 8.07 (dd, 1H), 8.59 (d,

1H), 9.33 (s, 1H), 12.98 (brs, 1H). ¹³C NMR (DMSO-d₆, 126 MHz)

δ 167.1, 154.4, 153.3, 144.7, 141.2, 136.5, 130.0, 129.4, 127.4, 126.0,

111.5, 33.9, 15.4. MS (ESI) m/z 286 [M + H]⁺. HRMS [M + H]⁺=

286.1189 (theoretical 286.1186).

6′-(3-Ethylureido)-4-(4-pyridyl)-3,3′-bipyridine-5-carboxylic Acid

(27). A 1N solution of LiOH (0.3 mL) was added dropwise to a

solution of 54a (50 mg, 0.13 mmol) in methanol. The reaction

progress was followed by LC/MS, and the reaction was stopped upon

reaching above 90−95% conversion to product. The methanol was

removed partially under reduced pressure, and EtOAc (5 mL) was

added. The mixture was cooled to 4 °C, acidiﬁed to pH ∼2 with 10%

solution of HCl, and extracted with EtOAc. The organic layer was

separated and dried with Na₂SO₄. Volatiles were removed under

reduced pressure to give a solid residue, which was dried to give the

6′-{[(Ethylamino)carbonyl]amino}-3,3′-bipyridine-5-carboxylic

1

Acid (19). Isolated as a solid (14 mg, 25%) from 63 mg of 44d. H

NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.15−3.23 (m, 2H), 7.53 (d, 1H),

7.95 (t, 1H), 8.14 (dd, 1H), 8.45 (t, 1H), 8.63 (s, 1H), 9.02 (s, 1H),

9.11 (s, 1H), 9.35 (s, 1H), 13.51 (brs, 1H). ¹³C NMR (DMSO-d₆, 126

MHz) δ 167.5, 166.6, 155.2, 154.4, 154.2, 152.6, 146.0, 136.9, 134.4,

127.1, 126.9, 126.7, 125.0, 122.1, 111.7, 33.9, 15.2. MS (ESI) m/z 287

[M + H]⁺. HRMS [M + H]⁺= 287.1134 (theoretical 287.1139).

2-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)-1,3-benzothia-

zole-7-carboxylic Acid (21). Isolated as a solid (38 mg, 92%) from 45

1

desired product. H NMR (CD₃OD) δ 1.22 (t, 3H), 3.36 (q, 2H),

7.68 (s, 1H), 8.05 (brs, 2H), 8.53 (brs, 2H), 8.88 (brs, 3H), 9.22 (brs,

1H). ¹³C NMR (DMSO-d₆, 126 MHz) δ 165.6, 153.8, 153.7, 154.4,

152.7, 148.6, 148.3, 145.2, 143.2, 138.4, 132.0, 126.5, 126.9, 123.8,

111.5, 33.9, 15.3. MS (ESI) m/z 369 [M + H]⁺. HRMS [M + H]⁺=

364.1407 (theoretical 364.1404).

1

mg of 44f. H NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.18−3.23 (m, 2H),

7.48 (t, 1H), 7.58 (d, 1H), 7.87−7.91 (m, 2H), 7.80 (d, 1H), 8.31 (dd,

1H), 8.86 (d, 1H), 9.52 (s, 1H). MS (ESI) m/z 343 [M + H]⁺. HRMS

[M + H]⁺= 343.0843 (theoretical 343.0859).

4′-Chloro-6′-(3-ethylureido)-3,3′-bipyridine-5-carboxylic Acid

2-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)-1,3-thiazole-5-

carboxamide (22). HATU (0.078 mg, 0.225 mmol) was added to a

solution of 2-(6-{[(ethylamino)carbonyl]amino}pyridin-3-yl)-1,3-thia-

zole-4-carboxylic acid 20 (60 mg, 0.205 mmol), Et₃N (84 μL, 0.41

mmol)), and cumylamine (28 mg, 0.205 mmol) in 3 mL of DMF.

After stirring at room temperature for 2 h, the mixture was diluted with

water and extracted with EtOAc. Solids, which precipitated during the

workup, were collected by ﬁltration, rinsed with water and EtOAc, and

dried in vacuo to aﬀord 20 mg of a solid (MS (ESI) m/z 410 [M +

H]⁺) that was dissolved 1 mL of TFA (1 mL) with stirring at room

temperature overnight. The TFA was removed under reduced

pressure, and the residue was taken up in aqueous NaHCO₃. Solids

that precipitated were collected by ﬁltration, washed with water, and

dried in vacuo to aﬀord 15 mg (25% over 2 steps) of material

1

(24). Isolated as a solid (126 mg, 91%) from 151 mg of 46a. H

NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.18 (q, 2H), 7.51 (s, 1H), 7.85 (s,

1H), 8.33 (s, 2H), 8.88 (s, 1H), 9.10 (s, 1H), 9.45 (s, 1H), 13.71 (s,

1H). ¹³C NMR (DMSO-d₆, 126 MHz) δ 166.0, 154.1, 153.2, 149.4,

148.9, 142.4, 137.5, 131.0, 126.2, 124.5, 111.2, 33.9, 15.2. MS (ESI)

m/z 321, 323 [M + H]⁺. HRMS [M + H]⁺= 321.0758 (theoretical

321.0748).

4′-Bromo-6′-(3-ethylureido)-3,3′-bipyridine-5-carboxylic Acid

1

(25). Isolated as a solid (3.6 g, 68%) from 5.39 g of 46b. H NMR

(DMSO-d₆) δ 1.09 (t, 3H), 3.18 (q, 2H), 7.48 (s, 1H), 8.03 (s, 1H),

8.28 (s, 1H), 8.32 (s, 1H), 8.86 (s, 1H), 9.11 (s, 1H), 9.44 (s, 1H),

13.60 (s, 1H). MS (ESI) m/z 365, 367 [M + H]⁺. HRMS [M + H]⁺=

365.0248 (theoretical 365.0243).

6′-(3-Ethylureido)-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-3,3′-bipyri-

dine-5-carboxylic Acid (26). Isolated as a solid (75 mg, 73%) from

ethyl 6′-(3-ethylureido)-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-3,3′-bipyri-

1

consistent with the title compound. H NMR (DMSO-d₆) δ 1.1 (t,

3H), 3.2 (m, 2H), 7.6 (m, 2H), 8.15 (m, 1H), 8.3 (brs, 1H), 8.2 (d,

1H), 8.4 (s, 1H), 8.77 (d, 1H), 9.5 (s, 1H). MS (ESI) m/z 292 [M +

H]⁺. HRMS [M + H]⁺= 292.0875 (theoretical 292.0862).

1

dine-5-carboxylate 50 (110 mg, 0.28 mmol). H NMR (DMSO-d₆) δ

1.11 (t, 3H), 2.58 (s, 3H), 3.20 (m, 2H), 7.55 (t, 1H), 8.13 (s, 1H),

8.21 (s, 1H), 8.36 (s, 1H), 8.68 (s, 1H), 9.05 (s, 1H), 9.51 (s, 1H),

13.46 (brs, 1H). MS (ESI) m/z 369 [M + H]⁺. HRMS [M + H]⁺=

369.1324 (theoretical 369.1305).

6′-{[(Ethylamino)carbonyl]amino}-3,3′:4′,3″-terpyridine-5-car-

boxylic Acid (28). Isolated as a solid (57 mg, 45%) from 135 mg (0.34

mmol) 54b. ¹H NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.14−3.24 (m, 2H),

7.35−7.39 (m, 1 H), 7.57 (d, 1H), 7.66 (s, 1H), 7.94−7.95 (m, 2H),

8.31−8.33 (m, 3H), 8.51 (d, 1H), 8.88 (d, 1H), 9.54 (s, 1H). ¹³C

NMR (DMSO-d₆, 126 MHz) δ 166.2, 154.5, 153.7, 152.6, 149.2,

149.1, 148.6, 148.2, 146.3, 137.4, 136.6, 134.0, 132.4, 128.0, 124.9,

123.4, 111.9, 33.9, 15.3. MS (ESI) m/z 364 [M + H]⁺. HRMS [M +

H]⁺= 364.1421 (theoretical 364.1404).

2-(6-{[(Ethylamino)carbonyl]amino}pyridin-3-yl)-1,3-benzothia-

zole-7-carboxamide (23). Following the procedure for the synthesis

of compound 22, the title compound was isolated as a solid (43 mg,

1

61%) from 70 mg of compound 21. H NMR (DMSO-d₆) δ 1.10 (t,

3H), 3.16−3.23 (m, 2H), 7.60−7.66 (m, 2H), 7.82 (brs, 2H), 8.08 (d,

1H), 8.17 (d, 1H), 8.38 (dd, 1H), 8.41(s, 1H), 8.92 (d, 1H), 9.56 (s,

1H). MS (ESI) m/z 342 [M + H]⁺. HRMS [M + H]⁺= 342.1010

(theoretical 342.1019).

6′-(3-Ethylureido)-4′-(4-(triﬂuoromethyl)thiazol-2-yl)-3,3′-bipyri-

dine-5-carboxamide (32). Following the procedure for the synthesis

of compound 22, the title compound was isolated as a solid (9 mg,

14%) from 65 mg of compound 31. Isolated as a solid (9 mg, 54%).

¹H NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.18−3.24 (m, 2H), 7.45 (brs,

1H), 7.65 (s, 1H), 8.16 (s, 1H), 8.18 (s, 1H), 8.24 (s, 1H), 8.35 (s,

1H), 8.55 (d, 1H), 8.60 (d, 1H), 9.05 (s, 1H), 9.49 (s, 1H). ¹³C NMR

(DMSO-d₆, 126 MHz) δ 166.5, 166.1, 165.6, 155.0, 154.1, 153.7,

149.5, 142.8, 140.0, 135.7, 127.4, 127.3, 126.8, 126.7, 125.3, 120.7,

120.3, 111.1, 34.0, 15.2. MS (ESI) m/z 437 [M + H]⁺. HRMS [M +

H]⁺= 437.0994 (theoretical 437.1002).

6′-{[(Ethylamino)carbonyl]amino}-4′-phenyl-3,3′-bipyridine-5-

carboxylic Acid (29). Isolated as a solid (124 mg, 90%) from 140 mg

1

of 50c. H NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.14−3.22 (m, 2H),

7.11−7.15 (m, 2H), 7.32−7.34 (m, 3H), 7.53 (s, 1H), 7.90 (brs, 1H),

7.92 (s, 1H), 8.30 (s, 1H), 8.44 (d, 1H), 8.87 (s, 1H), 9.40 (s, 1H).

¹³C NMR (DMSO-d₆, 126 MHz) δ 165.9, 154.5, 153.6, 153.3, 149.7,

148.3, 147.9, 138.0, 137.3, 133.1, 129.0, 128.6, 128.2, 126.0, 124.5,

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1-Ethyl-3-(5′-(hydrazinecarbonyl)-4-(4-(triﬂuoromethyl)thiazol-

2-yl)-3,3′-bipyridin-6-yl)urea. A solution of 150 mg (0.32 mmol) of 1-

ethyl-3-(5′-(hydrazinecarbonyl)-4-(4-(triﬂuoromethyl)thiazol-2-yl)-

3,3′-bipyridin-6-yl)urea 58b and 0.30 mL (0.97 mmol) of hydrazine in

20 mL ethanol was heated at reﬂux for 5 h. Solvent was removed, and

the solid residue was triturated with 10% MeOH in CH₂Cl₂. The

solids were collected and dried in vacuo to aﬀord 101 mg (69%) of the

(m, 2H), 7.6 (m, 1H) 8.14 (s, 1H), 8.2 (s, 1H), 8.37 (s, 1H), 8.55 (s,

1H), 8.63 (s, 1H), 9.16 (d, 1H), 9.48 (s, 1H). MS (ESI) m/z 506 [M +

H]⁺.

1-Ethyl-3-(5′-(3-methyl-1,2,4-oxadiazol-5-yl)-4-(4-

(triﬂuoromethyl)thiazol-2-yl)-3,3′-bipyridin-6-yl)urea (37). A mix-

ture of N-(1-(dimethylamino)ethylidene)-6′-(3-ethylureido)-4′-(4-

(triﬂuoromethyl)thiazol-2-yl)-3,3′-bipyridine-5-carboxamide (80 mg,

0.16 mmol), hydroxylamine hydrochloride (13.20 mg, 0.19 mmol), 2N

NaOH (0.038 mL, 0.19 mmol), and 70% aqueous acetic acid (2 mL)

in 3 mL of dioxane was heated at 80 °C for 30 min. After cooling to

room temperature, solvent was removed and the residue was

partitioned between water and CH₂Cl₂. The layers separated, and

the aqueous was back extracted 3 times with CH₂Cl₂. Solids

precipitated from the CH₂Cl₂solution. The solids were ﬁltered and

dried in vacuo to give the title compound as a white solid (55 mg,

1

title compound. H NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.1−3.3 (m,

2H), 4.6 (brs, 2H), 7.55 (m, 1H), 8.13 (s, 1H), 8.23 (s, 1H), 8.34 (s,

1H), 8.55 (s, 1H), 8.59 (s, 1H), 8.99 (s, 1H), 9.48 (s, 1H), 9.97 (s,

1H). MS (ESI) m/z 452 [M + H]⁺.

1-Ethyl-3-(5′-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)-4-(4-

(triﬂuoromethyl)thiazol-2-yl)-3,3′-bipyridin-6-yl)urea (35). Carbon-

yldiimidazole (56.6 mg, 0.35 mmol) was added to a solution of 1-

ethyl-3-(5′-(hydrazinecarbonyl)-4-(4-(triﬂuoromethyl)thiazol-2-yl)-

3,3′-bipyridin-6-yl)urea (105 mg, 0.23 mmol) and diisopropylethyl-

amine (0.061 mL, 0.35 mmol) in DMF (1.5 mL), and the mixture was

stirred at room temperature for 1.5 h. The reaction mixture was

diluted with water and extracted with 5% methanol in CH₂Cl₂. The

combined extract was washed with water and brine and dried over

MgSO₄and concentrated. The residue was puriﬁed by silica gel

chromatography (2−8% MeOH gradient in CH₂Cl₂) to give the title

1

73%). H NMR (DMSO-d₆) δ 1.10 (t, 3H), 2.31 (s, 3H), 3.05−3.28

(m, 2H), 7.74 (brs, 1H), 8.24 (s, 1H), 8.40 (s, 2H), 8.56 (s, 1H), 8.77

(d, 1H), 9.25 (d, 1H), 9.60 (s, 1H). ¹³C NMR (DMSO-d₆, 126 MHz)

δ 166.3, 166.2, 158.2, 154.3, 153.9, 149.1, 148.8, 148.7, 142.8, 139.6,

137.7, 132.4, 126.4, 125.7, 124.6, 120.3, 112.6, 110.3, 33.9, 15.3, 11.2,

10.1. MS (ESI) m/z 476 [M + H]⁺. HRMS [M + H]⁺= 476.1107

(theoretical 476.1111).

1

compound as a white solid (65 mg). H NMR (DMSO-d₆) δ 1.10 (t,

1-(5′-(3,5-Dimethylisoxazol-4-yl)-4-(4-(triﬂuoromethyl)thiazol-2-

yl)-3,3′-bipyridin-6-yl)-3-ethylurea (38). 3-Bromo-5-(4,4,5,5-tetra-

methyl-1,3,2-dioxaborolan-2-yl)pyridine (596 mg, 2.10 mmol), 1-(5-

bromo-4-(4-(triﬂuoromethyl)thiazol-2-yl)pyridin-2-yl)-3-ethylurea

(53b) (830 mg, 2.10 mmol), tris(dibenzylideneacetone)-

dipalladium(0) (192 mg, 0.21 mmol), 2-dicyclohexylphosphino-

2′,4′,6′-tri-iso-propyl-1,1′-biphenyl (300 mg, 0.63 mmol), and

Na₂CO₃(223 mg, 2.10 mmol) were taken in a round bottomed

ﬂask, and the ﬂask was ﬂushed with nitrogen. Solvent (5:1;

acetonitrile−water, 10 mL) was added and degassed with nitrogen,

and the mixture was heated at 100 °C for 3 h. The reaction mixture

was ﬁltered, and the ﬁltrate was concentrated under reduced pressure.

The resulting crude residue was partitioned between water and EtOAc.

The layers were separated, and the aqueous was back extracted with

EtOAc three times. The combined organic layers were washed with

water and brine, dried over MgSO₄, and concentrated under reduced

pressure. The residue obtained was puriﬁed by normal phase

chromatography (gradient of MeOH in CH₂Cl₂) to give a white

solid (483 mg) consistent with 1-(5′-bromo-4-(4-(triﬂuoromethyl)-

thiazol-2-yl)-3,3′-bipyridin-6-yl)-3-ethylurea 57b (MS (ESI) m/z 472,

474 [M + H]⁺). The solid (100 mg, 0.21 mmol), 3,5-dimethyl-4-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isoxazole (70.9 mg, 0.32

mmol), dicyclohexyl(2′,4′,6′-triisopropylbiphenyl-2-yl)phosphine

(30.3 mg, 0.06 mmol), tris(dibenzylideneacetone)dipalladium(0)

(19.39 mg, 0.02 mmol), and Na₂CO₃(33.7 mg, 0.32 mmol) were

placed in a microwave reaction vial and degassed with nitrogen.

Acetonitrile (4 mL) and water (0.8 mL) were added, and the mixture

was heated in a microwave reactor at 100 °C for 40 min. After cooling

to room temperature, the mixture was partitioned between water and

EtOAc. The layers were separated, and the aqueous layer was back

extracted 3 times with EtOAc. Combined extracts were washed with

water and brine. Drying (MgSO₄) and removal of solvent was followed

by silica gel chromatography (1−3% gradient MeOH in CH₂Cl₂) of

the residue to give a white solid (63 mg) with some impurities. Further

puriﬁcation with a second silica gel chromatography (1−3% gradient

MeOH in CH₂Cl₂) aﬀorded a white solid (40 mg) consistent with the

3H), 3.16−3.28 (m, 2H), 7.55 (brs, 1H), 8.09 (s, 1H), 8.22 (s, 1H),

8.37 (s, 1H), 8.57 (s, 1H), 8.62 (s, 1H), 8.97 (s, 1H), 9.50 (s, 1H),

12.80 (s, 1H). ¹³C NMR (DMSO-d₆, 126 MHz) δ 166.2, 154.3, 154.2,

154.1, 152.4, 151.8, 149.3, 145.3, 142.8, 139.3, 134.0, 132.7, 126.3,

123.6, 120.1, 120.3, 110.3, 33.9, 15.3. MS (ESI) m/z 437 [M + H]⁺.

HRMS [M + H]⁺= 478.0918 (theoretical 478.0903).

1-(5′-(2-Acetylhydrazinecarbonyl)-4-(4-(triﬂuoromethyl)thiazol-

2-yl)-3,3′-bipyridin-6-yl)-3-ethylurea. HATU (89 mg, 0.23 mmol)

was added to a solution of 85 mg (0.19 mmol) of 6′-{[(ethylamino)-

carbonyl]amino}-4′-[4-(triﬂuoromethyl)-1,3-thiazol-2-yl]-3,3′-bipyri-

dine-5-carboxylic acid 31, triethylamine (54 μL, 0.39 mmol), and

acetohydrazide (14.1 mg, 0.19 mmol) in 2 mL of DMF, and the

mixture was stirred at room temperature for 1 h. The mixture was

diluted with EtOAc and washed 3 times with water and once with

brine. Drying (MgSO₄) and removal of solvent gave an oil (158 mg)

that was puriﬁed by reverse phase HPLC (acetonitrile−water gradient

with 0.1% TFA) to aﬀord 48 mg (52%) of the title compound as a

solid. ¹H NMR (DMSO-d₆) δ 1.10 (t, 3H), 2.58 (s, 3H), 3.1−3.3 (m,

2H), 4.6 (brs, 2H), 7.54 (brs, 2H), 7.5−7.6 (m, 1H), 8.23 (s, 1H),

8.29 (s, 1H), 8.4 (s, 1H), 8.56 (s, 1H), 8.69 (s, 1H), 9.15 (s, 1H), 9.51

(s, 1H). MS (ESI) m/z 494 [M + H]⁺.

1-Ethyl-3-(5′-(5-methyl-1,3,4-oxadiazol-2-yl)-4-(4-

(triﬂuoromethyl)thiazol-2-yl)-3,3′-bipyridin-6-yl)urea (36). Triphe-

nylphosphine (306 mg, 2.33 mmol), CCl₄(0.114 mL, 1.17 mmol),

and triethylamine (0.316 mL, 2.33 mmol) were added to a mixture of

1-(5′-(2-acetylhydrazinecarbonyl)-4-(4-(triﬂuoromethyl)thiazol-2-yl)-

3,3′-bipyridin-6-yl)-3-ethylurea (184 mg, 0.389 mmol) in CH₂Cl₂(5

mL). The resulting mixture was allowed to stir overnight at room

temperature. The mixture was partitioned between water and CH₂Cl₂.

The layers were separated, and the aqueous layer was back extracted 3

times with CH₂Cl₂. The combined extracts were washed with water,

dried (MgSO₄), and concentrated, and the residue was puriﬁed by

normal phase chromatography (1−3% gradient of MeOH in CH₂Cl₂)

1

to give the title compound as a white solid (48 mg, 26%). H NMR

(DMSO-d₆) δ 1.10 (t, 3H), 2.58 (s, 3H), 3.18−3.22 (m, 2H), 7.54

(brs, 1H) 8.23 (s, 1H), 8.28 (s, 1H), 8.40 (s, 1H), 8.56 (s, 1H), 8.69

(s, 1H), 9.15 (d, 1H), 9.51 (s, 1H). MS (ESI) m/z 476 [M + H]⁺.

HRMS [M + H]⁺= 476.1117 (theoretical 476.1111).

N-(1-(Dimethylamino)ethylidene)-6′-(3-ethylureido)-4′-(4-

(triﬂuoromethyl)thiazol-2-yl)-3,3′-bipyridine-5-carboxamide. A mix-

ture of 6′-(3-ethylureido)-4′-(4-(triﬂuoromethyl)thiazol-2-yl)-3,3′-bi-

pyridine-5-carboxamide 32 (270 mg, 0.62 mmol) in 1,1-dimethoxy-

N,N-dimethylethanamine (10 mL, 68.40 mmol) was heated to 120 °C

for 1 h. After cooling to room temperature, solids that precipitated

were ﬁltered, rinsed acetonitrile, and dried in vacuo. The solids were

consistent with the title compound, 178 mg (57%). ¹H NMR (DMSO-

d₆) δ 1.11 (t, 3H), 2.29 (s, 3H), 3.11 (s, 3H), 3.14 (s, 3H), 3.18−3.22

1

title compound. H NMR (DMSO-d₆) δ 1.10 (t, 3H), 2.15 (s, 3H),

2.36 (s, 3H), 3.08−3.29 (m, 2H), 7.58 (br s, 1H), 7.80 (s, 1H), 8.21

(s, 1H), 8.37 (s, 1H), 8.55 (d, 1H), 8.57 (s, 1H), 8.67 (d, 1H), 9.48 (s,

1H). MS (ESI) m/z 489 [M + H]⁺. HRMS [M + H]⁺= 489.1334

(theoretical 489.1315).

1-(5′-Cyano-4-(4-(triﬂuoromethyl)thiazol-2-yl)-3,3′-bipyridin-6-

yl)-3-ethylurea (39). 1-(5-Bromo-4-(4-(triﬂuoromethyl)thiazol-2-yl)-

pyridin-2-yl)-3-ethylurea 53b (300 mg, 0.76 mmol), CsCO₃(495 mg,

1.52 mmol), tetrakis(triphenylphosphine)palladium (0) (88 mg, 0.08

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

nicotinonitrile (349 mg, 1.52 mmol) were placed in a microwave

reaction vial and degassed with nitrogen. Then dioxane−water (4:1, 6

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Journal of Medicinal Chemistry

Article

mL) was added, and the mixture has heated in a microwave reactor at

100 °C for 30 min. The reaction mixture was partitioned between

water and EtOAc, and layers were separated. The aqueous layer was

back extracted with EtOAc (3 times). The combined EtOAc layers

were washed with aqueous NaHCO₃, water, and brine before being

dried (MgSO₄). The solvent was removed, and the residue was

triturated with acetonitrile to give the title compound as a white solid

wells. Curve ﬁtting to determine IC₅₀values was performed using the

equation %I = 100/{1 + (IC₅₀/[I])ⁿ}.

Log D Determination. The partition coeﬃcient (log D) was

measured by shake ﬂask method, using 10 mM phosphate buﬀer at pH

7.4 and n-octanol. The samples were allowed to reach equilibrium by

shaking for 1 h at 1200 rpm, and sample analysis was done by LC/UV,

with MS for mass conﬁrmation.

pK_aDetermination. pK_avalues were determined at Sirius

Analytical Instruments Ltd. (Forest Row Business Park, Station

Road, Forest Row, East Sussex TH18 5DW, UK) by a Gold Standard

pH metric assay on a Sirius T3 automated system in triplicate. The

accuracy of the measurement was approximately 0.02 log units.

Plasma Protein Binding Determination. Human plasma protein

binding was determined from a 10 μM compound solution in a

Dianorm plasma well incubating at 37 °C for 16 h. Free fractions were

calculated from ratios of drug concentration in buﬀer and plasma wells

determined by LC-MS/MS.

Microsomal Clearance Determination. Metabolic stability was

determined by incubating a 2 μM compound solution in 0.5 mg/mL

human microsomes and adding 1 mM NADPH. Compound

concentration at diﬀerent time points was quantiﬁed by LC-MS/MS.

Cl_intwas calculated from disappearance of the compound and

correction factors.

1

(270 mg, 85%). H NMR (DMSO-d₆) δ 1.09 (t, 3H), 3.16−3.22 (m,

2H), 7.49 (t, 1 H), 8.22 (s, 1H), 8.36 (s, 1H), 8.38 (d, 1H), 8.60 (s,

1H), 8.76 (s, 1H), 9.04 (s, 1H), 9.52 (s, 1H). MS (ESI) m/z 419 [M +

H]⁺. HRMS [M + H]⁺= 419.0893 (theoretical 419.0896).

1-(5′-(2H-Tetrazol-5-yl)-4-(4-(triﬂuoromethyl)thiazol-2-yl)-3,3′-bi-

pyridin-6-yl)-3-ethylurea (40). A mixture of 1-(5′-cyano-4-(4-

(triﬂuoromethyl)thiazol-2-yl)-3,3′-bipyridin-6-yl)-3-ethylurea (60 mg,

0.14 mmol), NaN₃(18.65 mg, 0.29 mmol), and NH₄Cl (14.57 mg,

0.27 mmol) in DMF (1.5 mL) was heated at 100 °C for 6 h. Solvent

was removed, and the residue was puriﬁed by reverse phase HPLC

(acetonitrile−water gradient with 0.1% TFA) to aﬀord the title

compound as a white solid (23 mg, 36% yield). ¹H NMR (DMSO-d₆)

δ 1.09 (t, 3H), 3.17−3.22 (m, 2H), 7.53 (t, 1 H), 8.25 (s, 1H), 8.35 (s,

1H), 8.40 (s, 1H), 8.55 (s, 1H), 8.77 (d, 1H), 9.22 (s, 1H), 9.53 (s,

1H). MS (ESI) m/z 462 [M + H]⁺. HRMS [M + H]⁺= 462.1080

(theoretical 462.1066).

6′-(3-Ethylureido)-N′-hydroxy-4′-(4-(triﬂuoromethyl)thiazol-2-

yl)-3,3′-bipyridine-5-carboximidamide (41). A mixture of 1-(5′-

cyano-4-(4-(triﬂuoromethyl)thiazol-2-yl)-3,3′-bipyridin-6-yl)-3-ethyl-

urea 40 (70 mg, 0.17 mmol) and hydroxylamine (0.015 mL, 0.25

mmol, 50% in water) in ethanol (3 mL) was heated to 80 °C for 1 h in

a microwave reactor. Solvent was removed, and the residue was

slurried in acetonitrile. Filtration of the solids and drying in vacuo gave

the title compound as a white solid (52 mg, 69%). ¹H NMR (DMSO-

d₆) δ 1.11 (t, 3H), 3.1−3.3 (m, 2H), 6.0 (s, 2H), 7.57 (brs, 1H), 8.03

(s, 1H), 8.26 (s, 1H), 8.35 (s, 1H), 8.46 (s, 1H), 8.56 (s, 1H), 8.93 (s,

1H), 9.48 (s, 1H), 9.1 (s, 1H). ¹³C NMR (DMSO-d₆, 126 MHz) δ

166.0, 154.3, 153.9, 150.1, 149.2, 148.4, 146.0, 142.7, 139.3, 133.9,

131.6, 128.7, 126.4, 124.6, 120.4, 110.0, 33.9, 15.3. MS (ESI) m/z 452

[M + H]⁺. HRMS [M + H]⁺= 452.1117 (theoretical 452.1111).

1-Ethyl-3-(5′-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)-4-(4-

(triﬂuoromethyl)thiazol-2-yl)-3,3′-bipyridin-6-yl)urea (42). A mix-

ture of compound 41 (70 mg, 0.16 mmol), DBU (0.023 mL, 0.16

mmol), and carbonyl diimidazole (25.1 mg, 0.16 mmol) in dioxane (3

mL) was stirred overnight at room temperature. The solvent was

removed, and the residue was puriﬁed by reverse phase HPLC (Gilson,

25%ACN in water to 70% ACN with 0.1% TF) to give the title

Pharmacokinetic Studies. Pharmacokinetic properties of selected

compounds were studied in male rats. Plasma pharmacokinetics were

determined from 0 to 24 h following 15 min iv infusions at 10 mg/kg.

Serial 200 μL samples of whole blood were taken at time intervals.

Concentration of compound in plasma was determined by LC-MS/

MS, and pharmacokinetic parameters were estimated using a

noncompartmental model in WinNonLin (Pharsight). Mean results

were determined from experiments with three mice.

Minimum Inhibitory Concentration (MIC). MIC values were

determined by the broth microdilution method in accordance with the

Clinical and Laboratory Standards Institute (CLSI) guidelines. In brief,

bacterial suspensions were adjusted to a 0.5 McFarland standard to

yield a ﬁnal inoculum between 3 × l0⁵and 7 × l0⁵colony-forming

units (CFU)/mL. For S. aureus strains (MSSA, wild-type from the

AstraZeneca screening collection and MRQR, from the AstraZeneca

screening collection),⁵⁷bacterial inocula were made in sterile, cation

adjusted Mueller−Hinton broth (Beckton Dickinson), and for S.

pneumoniae (D39; NCTC 7466) in sterile, cation adjusted Mueller−

Hinton broth containing 2.5% lysed horse blood (Hema Resource &

Supply Inc.). For E. coli (W3110; ATCC 27325), E. coli tolC⁻(W3110

ΔtolC::Tn10 from the AstraZeneca screening collection),⁵⁷H.

inﬂuenzae (wild-type, Rd KW20; ATCC 51907), or M. catarrhalis

(wild-type, ATCC43617) bacterial inocula were made in sterile, cation

adjusted Mueller−Hinton broth (Beckton Dickinson) containing 0.5%

yeast extract (Beckton Dickinson) plus 30 mL of 15 μg/mL Bovine

Hematin stock (Sigma) and 3 mL of 15 μg/mL β-nicotinamide

adenine dinucleotide (Sigma). An inoculum volume of 100 μL was

added to wells (using a Tecan EVO robot) containing 2 μL of DMSO

containing 2-fold serial dilutions of drug. All inoculated microdilution

trays were incubated in ambient air at 35 °C for 18−24 h. Following

incubation, the lowest concentration of the drug that prevented visible

growth as read at OD600 nm was recorded as the MIC. Performance

of the assay was monitored by the use of laboratory quality-control

strains and commercially available control compounds with deﬁned

MIC spectra, in accordance with CLSI guidelines.

DNA Biosynthesis Inhibition. Incorporation of radiolabeled

precursors (³H-thymidine, ³H-uridine, ¹⁴C-leucine, ¹⁴C-valine, ¹⁴C-

acetic acid and ¹⁴C-N-acetyl glucosamine) of biological macro-

molecules was measured in S. aureus (MSSA) and S. pneumoniae

(D39) following previously described methods.³²The DNA gyrase

inhibitor, ciproﬂoxacin, that inhibits DNA biosynthesis, was included

as a reference.

Resistance Frequency and Isolation of Resistant Mutants.

Resistance studies were performed following methods previously

described.^28,45Suspensions of bacterial cultures of methicillin-

susceptible S. aureus (MSSA) were transferred onto Mueller−Hinton

agar (BD, Franklin Lakes, NJ) plates containing compound at

1

compound as a white solid (33 mg, 43%). H NMR (DMSO-d₆) δ

1.09 (t, 3H), 3.1−3.3 (m, 2H), 7.51 (brs, 1H), 8.15 (s, 1H), 8.24 (s,

1H), 8.37 (s, 1H), 8.59 (s, 1H), 8.70 (s, 1H), 8.99 (d, 1H), 9.52 (s,

1H), 13.14 (brs, 1H). ¹³C NMR (DMSO-d₆, 126 MHz) δ 166.1,

159.6, 155.5, 154.3, 154.2, 153.2, 149.4, 145.9, 142.9, 139.2, 134.9,

132.7, 126.3, 123.5, 119.5, 120.2, 110.4, 34.0, 15.3. MS (ESI) m/z 478

[M + H]⁺. HRMS [M + H]⁺= 478.0924 (theoretical 478.0903).

Inhibition of ATPase Activity of Topoisomerase Enzymes.

Assays for the ATPase activity of the various topoisomerase isozymes

were performed as described previously^35,68The ATPase activities of

E. coli DNA gyrase and topoisomerase IV were measured from

reconstituted tetramers of E. coli GyrA/GyrB and ParC/ParE,

respectively. The ATPase activity of S. aureus DNA gyrase was

measured from the reconstituted hybrid tetramer of the GyrB subunit

from S. aureus with the GyrA subunit from E. coli. The S. pneumoniae

topoisomerase IV ATPase assay was performed using the uncoupled

GyrB subunit. Inhibition of ATPase activity was monitored by reduced

production of inorganic phosphate, a product of the topoisomerase-

catalyzed ATP hydrolysis reaction. Inorganic phosphate was quantiﬁed

using the ammonium molybdate/malachite green-based detection

system, measuring absorbance at 650 (A₆₅₀) nm, and assays were

performed in microtiter plates containing of range concentrations of

compounds dissolved in DMSO. For determination of the IC₅₀values

reported, raw A₆₅₀nm was converted to percent inhibition calculated

from the means of 32 wells each per assay plate of 0% inhibition

(DMSO control) and 100% inhibition (2 μM novobiocin control)

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Table 8. X-ray Data Processing and Reﬁnement Statistics

31

35

PDB code

4LP0

4LPB

Space group:

C222(1)

cell constants

a, b, c (Å)

72.9, 94.3, 62.3

73.4, 94.7, 61.2

90.0, 90.0, 90.0

22.09−1.75

99.9 (100)

21859

α, β, γ (deg)

90.0, 90.0, 90.0

resolution range (Å)

completeness overall (%)

reﬂections, unique

multiplicity

62.27−1.95 (2.06−1.95)

99.8 (100.0)

15830

7.1 (7.3)

0.084 (0.386)

17.6

7.1 (7.2)

0.093 (0.577)

21.8

a

R_{merge overall}

b

R_{value overall}(%)

c

R_{value free}(%)

20.8

23.6

non-hydrogen protein atoms

non-hydrogen ligand atoms

solvent molecules

1511

1531

30

33

107

120

rmsd from ideal values

bond lengths (Å)

0.010

1.39

0.005

1.08

bond angles (deg)

d

Φ, Ψ angle distribution for residues

in most favored regions (%)

in additional allowed regions (%)

in generously regions (%)

in disallowed regions (%)

89.1

10.3

0.0

96.0

3.4

0.0

0.6

a

b

c

R_merge= ∑_hkl[(∑_i|I_i− ⟨I⟩|)/∑_iI_i]. R_value= ∑_hkl||F_obs| − |F_calc||/∑_hkl|F_obs|. R_freeis the cross-validation R factor computed for the test set of 5% of

d

unique reﬂections. Ramachandran statistics as deﬁned by PROCHECK.

multiples of the MIC (0.04 μg/mL) as well as plates without

compound. The frequencies of resistance were calculated from the

ratio of the number of colonies on the compound-containing plates

divided by the number of colonies on compound-free plates following

incubation for 7 days. Values from three separate plates were averaged

to yield the reported frequency of spontaneous resistance. To ensure

resistance stability, the recovered colonies were passaged twice on

compound-free agar plates. Resistant strains that showed a signiﬁcant

shift in MIC as compared to the parental strain were further

characterized by PCR ampliﬁcation and sequencing of gyrA, gyrB,

parC, and parE genes to identify mutations conferring resistance.

Generation of DNA Gyrase Mutations in S. pneumoniae and

Cross-Resistance Determination. Point mutations in gyrB and parE

genes in S. pneumoniae (D39; NCTC 7466) were generated as

described previously.^28,45Following similar methods, point mutations

in gyrA and parC genes in S. pneumoniae were generated to create

known ﬂuoroquinolone-resistance mutations. MIC values of com-

pounds against the parental strain and mutants of S. pneumoniae were

determined following standard CLSI methods, as described above.

S. pneumoniae ParE Crystallization, X-ray Diﬀraction Data

Collection and Reﬁnement. Crystals of the ATP binding domain of

S. pneumoniae ParE (residues 1−226) were grown using vapor

diﬀusion methodologies. A solution of 3 μL of 8 mg/mL protein in

buﬀer containing 20 mM Tris pH 8.0, 100 mM NaCl was added to 3

μL of well solution (18−25% Peg4K, 0.2 M ammonium acetate, 0.1 M

MIB, pH 7.0) and incubated at 16 °C. Crystals appeared within 3 days

and grew to full size within 1 week. To create cocomplexes, crystals

were transferred into a solution containing 25% Peg4000, 0.2 M

ammonium acetate, 0.1 M MIB pH 7, and a 10% DMSO solution of

26 in buﬀer with a ﬁnal compound concentration of 10 mM. Crystals

were incubated for 6−24 h at 16 °C before a quick transfer into a

cryosolution containing the soak solution and 2,3-butanediol (4.5:1

ratio) before freezing in liquid nitrogen. Data were collected at LRL-

CAT. Indexing and data processing were performed using the

autoPROC pipeline.⁶⁹The molecular replacement solution was

found using the program AMoRe⁷⁰as implemented in the CCP4

suite of programs.⁷¹Iterative cycles of reﬁnement and model building

were performed using Refmac⁷²and COOT.⁷³A summary of data

collection and reﬁnement statistics are listed in Table 8.

AUTHOR INFORMATION

Corresponding Author

*Phone: 781-839-4844. E-mail: greg.basarab@astrazeneca.com.

■

Present Addresses

^§Epizyme, Inc., 400 Technology Square, Cambridge, Massa-

chusetts 02139, United States.

^∥EMD Serono Research and Development Institute, 45A

Middlesex Turnpike, Billerica, Massachusetts 01821, United

States.

^‡Pﬁzer Worldwide Research and Development, 620 Memorial

Drive, Cambridge, Massachusetts 02139, United States.

^⊥Oﬃce of Biodefense, Research Resources & Translational

Research, Division of Microbiology & Infectious Diseases,

NIAID, NIH, 6610 Rockledge Drive, Bethesda, Maryland

20892, United States.

Notes

The authors declare the following competing ﬁnancial

interest(s): We are or have been employed by AstraZeneca

Pharmaceuticals.

ACKNOWLEDGMENTS

■

We thank the following individuals from AstraZeneca for their

valuable technical contributions and helpful advice: Barbara

Arsenault, Dr. Allison Choy, Nancy DeGrace, Elise Gorseth,

Lena Grosser, Sussie Hopkins, Dr. Ken Hull, Dr. Camil

Joubran, David McKinney, Ying Liu, and Linda Otterson.

ABBREVIATIONS USED

■

CFU, colony-forming unit; GyrA, A-subunit of DNA gyrase;

GyrB, B-subunit of DNA gyrase; LE, ligand eﬃciency; MSSA,

methicillin sensitive S. aureus; MRQR, methicillin resistant,

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dx.doi.org/10.1021/jm401208b | J. Med. Chem. 2013, 56, 8712−8735

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novel 5-(2-hydroxybenzylidene) rhodanine inhibitors of DNA gyrase

quinolone resistant S. aureus; ParC, the C-subunit of

topoisomerase IV; ParE, E-subunit of topoisomerase IV; tolC,

gene encoding outer membrane transport channel

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Design, synthesis, and structure−activity relationship studies of ATP

analogues as DNA gyrase inhibitors. Bioorg. Med. Chem. Lett. 2000, 10,

821−826.

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Fragment-to-hit-to-lead discovery of a novel pyridylurea scaffold of ATP competitive dual targeting type II topoisomerase inhibiting antibacterial agents

DOI: 10.1021/jm401208b

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