Discovery of 2-[5-(4-fluorophenylcarbamoyl)pyridin-2-ylsulfanylmethyl]phenylboronic acid (SX-517): Noncompetitive boronic acid antagonist of CXCR1 and CXCR2

Article

pubs.acs.org/jmc

Discovery of 2‑[5-(4-Fluorophenylcarbamoyl)pyridin-2-

ylsulfanylmethyl]phenylboronic Acid (SX-517): Noncompetitive

Boronic Acid Antagonist of CXCR1 and CXCR2

Dean Y. Maeda,*^,†Angela M. Peck,^†Aaron D. Schuler,^†Mark T. Quinn,^‡Liliya N. Kirpotina,^‡

Winston N. Wicomb,^§Guo-Huang Fan,^∥and John A. Zebala^†

^†Syntrix Biosystems, 215 Clay Street, Auburn, Washington 98001, United States

^‡Department of Microbiology and Immunology, Montana State University, 960 Technology Boulevard, Bozeman, Montana 59717,

United States

^§Infectious Disease Research Institute, 1616 Eastlake Avenue East, Seattle, Washington 98102, United States

^∥Department of Pharmacology, Meharry Medical College, 1005 Dr. DB Todd Boulevard, Nashville, Tennessee 37208, United States

ABSTRACT: The G protein-coupled chemokine receptors CXCR1 and

CXCR2 play key roles in inﬂammatory diseases and carcinogenesis. In

inﬂammation, they activate and recruit polymorphonuclear cells (PMNs)

through binding of the chemokines CXCL1 (CXCR1) and CXCL8 (CXCR1

and CXCR2). Structure−activity studies that examined the eﬀect of a novel

series of S-substituted 6-mercapto-N-phenyl-nicotinamides on CXCL1-

stimulated Ca²⁺ﬂux in whole human PMNs led to the discovery of 2-[5-(4-

ﬂuorophenylcarbamoyl)pyridin-2-ylsulfanylmethyl]phenylboronic acid (SX-

517), a potent noncompetitive boronic acid CXCR1/2 antagonist. SX-517

inhibited CXCL1-induced Ca²⁺ﬂux (IC₅₀= 38 nM) in human PMNs but had

no eﬀect on the Ca²⁺ﬂux induced by C5a, fMLF, or PAF. In recombinant

HEK293 cells that stably expressed CXCR2, SX-517 antagonized CXCL8-

induced [³⁵S]GTPγS binding (IC₅₀= 60 nM) and ERK1/2 phosphorylation.

Inhibition was noncompetitive, with SX-517 unable to compete the binding of [¹²⁵I]-CXCL8 to CXCR2 membranes. SX-517

(0.2 mg/kg iv) signiﬁcantly inhibited inﬂammation in an in vivo murine model. SX-517 is the ﬁrst reported boronic acid

chemokine antagonist and represents a novel pharmacophore for CXCR1/2 antagonism.

therefore thought to play an important role in inﬂammatory

diseases characterized by a signiﬁcant neutrophil component.

Due to the involvement of these receptors in a wide range of

inﬂammatory diseases and carcinogenesis, CXCR1 and CXCR2

have attracted attention as targets for small-molecule drug

discovery (Figure 1).²¹Reparixin 1 is a ketoprofen derivative

being investigated in trials for the prevention and treatment of

delayed graft function and pancreatic islet transplantation.^22,23

Diarylureas exempliﬁed by 2 (SB225002) have been disclosed

as either selective CXCR2 antagonists^24,25or dual CXCR1/2

antagonists.²⁶The central urea motif in the diarylureas was later

replaced with the cyclic urea bioisostere 3,4-diaminocyclobut-3-

ene-1,2-dione to provide potent CXCR2-selective analogues as

represented by SCH527123 (3).²⁷The diarylurea SB656933

has advanced into clinical trials for chronic obstructive

pulmonary disease (COPD)^28,29and cystic ﬁbrosis,³⁰and

SCH527123 inhibited ozone inhalation-induced sputum

neutrophil recruitment in healthy subjects.³¹AZD-8309 (4) is

representative of the bicyclic thiazolopyrimidine class of

CXCR2 antagonists,³²and this antagonist eﬀectively inhibited

INTRODUCTION

■

The chemokine receptors CXCR1 and CXCR2 are closely

related members of the class A (rhodopsin-like) family of seven

transmembrane G-protein-coupled receptors.¹The chemokine

CXCL8 (Interleukin-8, IL8) activates the receptors CXCR1

and CXCR2, whereas the chemokine CXCL1 (growth related

oncogene α, GROα) is a selective agonist for CXCR2.²

CXCR1/2 signaling is sensitive to the pertussis toxin, indicating

involvement of the Gαi subunit in the heterotrimeric G-

protein.^3,4Agonist-induced changes in receptor conformation

uncouple the Gβγ subunit from the heterotrimeric G-protein

complex, activating signaling pathways that include phospho-

lipase Cβ, phosphatidylinositol-3-kinase, and mitogen-activated

protein kinases. Phospholipase Cβ in turn generates inositol-

1,4,5-triphosphate, which binds to the endoplasmic reticulum

and leads to a release of Ca²⁺into the cytoplasm.⁵CXCR1/2

signaling is involved in inﬂammation, wound healing, and

angiogenesis, and their dysregulation has been implicated in a

myriad of diseases involving acute and chronic inﬂamma-

tion,⁶⁻¹⁴as well as tumorigenesis.¹⁵⁻²⁰In particular, CXCR1/2

signaling mediates agonist-induced neutrophil activation and

recruitment to sites of inﬂammation (i.e., chemotaxis) and is

Received: June 4, 2014

Published: September 25, 2014

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Figure 1. CXCR1 and CXCR2 receptor antagonists.

the increase of LPS-mediated neutrophil recruitment in the

nasal lavage of healthy subjects.³³

Table 1. CXCL1-Inhibitory Activity of Carboxylate and

Carboxylate Isostere Substituted Thionicotinamides

As disclosed by Cutshall and co-workers, nicotinamide

glycolate esters exempliﬁed by the methyl ester 5 (Figure 1)

are antagonists of CXCR2-mediated human neutrophil chemo-

taxis and are distinct from the diarylurea and related

cyclobutane classes.³⁴We have previously shown that

CXCR2 antagonism by nicotinamide glycolate esters proceeded

through a novel intracellular mechanism that required hydro-

lytic cleavage of the ester within the neutrophil for activity.³⁵

However, the unique pharmacology of this class also led to

rapid degradation in plasma, making it untenable as a

therapeutic. The mechanistic insights gleaned from these

studies inspired us to search for new nicotinamide templates

that would directly antagonize CXCR2 without requiring

intracellular hydrolytic activation for their activity. Herein, we

report the discovery, structure−activity relationship (SAR), in

vitro pharmacology, and in vivo biologic activity of 7 (SX-517).

Compound 7 is a potent noncompetitive CXCR1/2 antagonist

resulting from a novel series of S-substituted 6-mercapto-N-

phenyl-nicotinamides active in their native form, and is the ﬁrst

reported boronic acid chemokine antagonist.

a

Each IC₅₀determination was performed in triplicate with ≥6

concentrations, and reported as the mean standard error.

Scheme 1. Synthesis of S-Substituted N-(4-Fluoro-phenyl)-6-

mercapto-nicotinamides 9−45, 47−48, 52, and 55

a

RESULTS AND DISCUSSION

■

The compounds described in this study are shown in Tables

1−4, and their synthetic methods are outlined in Schemes 1−4.

Synthetic Strategies and Focused Parallel Combina-

torial Synthesis. The synthesis of S-substituted N-(4-

ﬂuorophenyl)-6-mercapto-nicotinamides (7−53) was carried

out as described in Scheme 1 by ﬁrst condensing 6-thio-

nicotinic acid with 4-ﬂuoroaniline using the coupling reagent 2-

ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ). The

resulting N-(4-ﬂuorophenyl)-6-mercapto-nicotinamide 64 was

then S-alkylated with a commercially available alkyl halide,

typically an alkyl bromide, using one of three methods that

diﬀered primarily in their solvent and base. Method A utilized

the thio-nicotinamide intermediate 64 and bromomethyl

building blocks (Maybridge Chemical Co., Cornwall, U.K.)

dissolved in anhydrous DMF in the presence of resin-bound

tertiary amine (4-methylmorpholino polystyrene resin, Nova-

Biochem, La Jolla, CA). The reaction mixture was heated at 60

°C for 1 h, followed by thiol scavenger resin (mercaptomethyl

polystyrene resin, NovaBiochem, La Jolla, CA) addition. The

suspension was heated to 60 °C for 2 h, and then the reaction

mixture was ﬁltered and the ﬁltrate diluted with water. The

resulting precipitate was collected by centrifugation. Purity of

the synthesized compounds was assessed by HPLC, and the

a

Conditions: (i) 4-ﬂuoroaniline, EEDQ, DMF, rt; (ii) method A: Br-

CH₂−R, solid-phase base (N-methylmorpholino-substituted resin),

DMF, 60 °C; method B: Br-CH₂−R or Cl-CH₂−R, DMF, base (TEA,

DIPEA, or), rt; method C: Br-CH₂−R, EtOH, 1 N NaOH, reﬂux.

identiﬁcation of compounds was done by electrospray

ionization mass spectroscopy (ESI-MS). In all cases, the

major reaction product was the derivatized thionicotinamide.

Method A was used to initially synthesize compounds 13−43

in moderate to excellent purity by facile ﬁltration. The

synthesized compounds were screened at initial test concen-

trations of 5 and 10 μM for antagonism of CXCL1-mediated

intracellular Ca²⁺release in isolated human neutrophils

(hPMNs). Compounds that exhibited greater than 50%

inhibition at 5 μM were then resynthesized by either methods

B or C and re-evaluated in the assay to obtain IC₅₀values of

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a

Scheme 2. Synthesis of S-Substituted N-(4-Fluoro-phenyl)-6-mercapto-nicotinamides 8, 44, 47, 49, 51−52

a

Conditions: (i) TMS-N₃, Bu₂SnO, toluene, reﬂux; (ii) 1 N NaOH, MeOH, rt; and (iii) oxone, aq NaHCO₃, acetone, 4 °C

these selected compounds at a higher tested purity. Method B

utilized an alkyl halide and a tertiary amine [typically

triethylamine (TEA), diisopropylethylamine (DIPEA), or N-

methylmorpholine (NMM)] to give the ﬁnal compounds 8−

13, 15, 17, 22, 43, 46, 48, and 50. Method C utilized an alkyl

halide and aqueous NaOH in EtOH at reﬂux to give the ﬁnal

compounds 7, 30, and 53 in good yield.

Scheme 3. Synthesis of Anilide Derivatives of 6-(Thiobenzyl-

2-borono)-nicotinamides 54−59

a

Further chemical modiﬁcation of intermediates to ﬁnal test

compounds is shown in Scheme 2. Treatment of 7 by

potassium peroxymonosulfate (Oxone), according to the

procedure of Webb and Levy for the hydroxylation of aryl

boronic acids,³⁶aﬀorded compound 44. Saponiﬁcation of the

methyl esters 46, 48, and 50 with NaOH aﬀorded carboxylic

acids 47, 49, and 51, respectively. The tetrazoles 8 and 52 were

prepared via the cycloaddition of the cyano intermediates with

trimethylsilyl azide and dibutyltin oxide³⁷in toluene under

reﬂux.

The synthesis of N-substituted 6-(thiobenzyl-2-borono)-

nicotinamides 54−59 was performed as in Scheme 3. 6-Thio-

nicotinic acid was coupled to 2-bromomethyl-phenylboronic

acid using a modiﬁed method B employing TEA, DMF, and

elevated temperature (60 °C) to give intermediate 67.

Intermediate 67 was treated with neopentyl glycol in toluene

under reﬂux to aﬀord the protected neopentyl boronate ester.

After cooling to 0 °C, pivaloyl chloride was added with

triethylamine (TEA), and the reaction proceeded for an hour

and then with gradual warming to room temperature for

another hour to give the pivaloyl mixed anhydride intermediate

68, which was used without puriﬁcation. Intermediate 68 was

then coupled immediately to either 4-triﬂuoromethoxyaniline,

4-aminobenzoic acid, 5-(4-aminophenyl)-1H-tetrazole, 4-

amino-pyridine, 4-hydroxy-aniline, or 2-amino-5-ﬂuoropyridine

under heating to give the corresponding 6-(thiobenzyl-2-

neopentyl boronate ester)-nicotinamides. Exposure to water

liberated the boronic acid moiety from the neopentyl boronate

a

Conditions: (i) 2-bromomethyl-phenylboronic acid, DMF, TEA, 60

°C; (ii) neopentyl glycol, toluene, reﬂux; (iii) pivaloyl chloride, TEA,

toluene, 0 °C to rt; and (iv) aniline derivative, TEA, DMF, 60 °C.

ester, which after puriﬁcation by preparative HPLC aﬀorded

54−59.

The synthesis of N,N-disubstituted 6-(thiobenzyl-2-borono)-

nicotinamides 60−63 was performed through two alternative

routes as described in Scheme 4. In one route, 6-

chloronicotinoyl chloride was amidated with the secondary

amines 70a and 70b, using DBU as the base to give the N,N-

disubstituted 6-chloronicotinamides 71a and 71b, respectively.

The secondary amine 70a was derived from reductive

amination of N-Boc-2-piperidinecarbaldehyde (Chem-Impex,

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a

Scheme 4. Synthesis of N-Substituted 6-(Thiobenzyl-2-borono)-nicotinamides 60−63

a

Conditions: (i) 4-ﬂuoroaniline, K₂CO₃, THF, rt; (ii) N-Boc-2-piperidinecarbaldehyde, NaBH(OAc)₃, DCE, glacial AcOH, rt; (iii) tert-butyl

bromoacetate, DIPEA, DMF, 80 °C; (iv) phase-transfer reaction: bromomethylpyridine, toluene, 50% aq NaOH, TBAH, rt; (v) 6-chloronicotinoyl

chloride, DBU, DMF, heat; (vi) NaHS, DMF, heat; (vii) 2-bromomethyl-phenylboronic acid, EtOH, 1 N aq NaOH, reﬂux; (viii) 2-bromomethyl-

phenylboronic acid, DMF, TEA; (ix) 4 M HCl, dioxane, rt; and (x) 90% aq TFA, rt.

Wood Dale, IL) with 4-ﬂuoroaniline and sodium triacetox-

yborohydride. The secondary amine 70b was derived from the

coupling of 4-ﬂuoroaniline with tert-butyl bromoacetate in

DMF with DIPEA as base. In the alternate route, 6-

chloronicotinoyl chloride was amidated with 4-ﬂuoroaniline in

THF in the presence of potassium carbonate to form

intermediate 69. Compounds 72a−72b were synthesized

from intermediate 69 under phase transfer catalyzed conditions,

utilizing either 2-bromomethylpyridine or 4-bromomethylpyr-

idine, respectively. The tertiary amides 71a−71b and 72a−72b

were then subjected to the same treatment with sodium

hydrogen sulﬁde (NaHS) in DMF to displace the pyridinyl

chloride and form the corresponding thio-nicotinamide

intermediates 73a−73d, respectively. The resultant thio-

nicotinamide intermediates 73a and 73b were alkylated with

2-bromomethyl-phenylboronic acid using method C to aﬀord

60 and 61, respectively. Method C applied to 73c followed by

treatment with 4 M HCl in dioxane deprotected the Boc-

piperidyl moiety to yield 62. Method B applied to 73d followed

by acid hydrolysis of the tert-butyl protected carboxyl group

with 90% TFA yielded 63.

stimulated Ca²⁺ﬂux in whole hPMNs. Full dose−response

curves were determined for select compounds using at least

seven concentrations, and the resulting IC₅₀values listed in

Tables 1−4 as part of these SAR studies are the mean of at least

three determinations.

Structure−Activity Studies. The nicotinamide glycolate

methyl ester 5 antagonized CXCL1-stimulated Ca²⁺ﬂux by an

intracellular mechanism that required hydrolytic cleavage of the

ester within the hPMN to liberate the active species.³⁵The

required hydrolytic cleavage of the ester led to instability in

plasma that was inseparable from the intrinsic activation

mechanism of this pharmacophore class. Since in vivo stability

of a potential therapeutic is essential, novel CXCR2 antagonists

were sought that would directly antagonize the receptor

without the need for hydrolytic activation and, thus, resist in

vivo esterase activity. To this end and based on the above

activation mechanism involving the glycolate ester moiety, new

S-substituted nicotinamides and related congeners were

evaluated for antagonism of CXCL1-stimulated Ca²⁺ﬂux in

whole hPMNs, as summarized in Tables 1−4.

Our SAR eﬀorts ﬁrst involved the bioisosteric replacement of

the ester/acid moiety of lead nicotinamides 5 and 6 (Table 1).

Replacement of the carboxylate moiety with either a cyclic ester

(9), a sulfone (10), a nitro (11), or a phosphite (12) resulted in

loss of activity in our assay. We previously demonstrated that

the negatively charged nicotinamide carboxylate 6 could not

enter hPMNs, and its activity was only observed after hPMNs

were electropermeabilized.³⁵On the basis of this observation,

we hypothesized that the S-methyl-tetrazole 8 might serve as a

more lipophilic bioisostere of the thioglycolate moiety that

CXCL1-Stimulated Ca²⁺Flux in Human Neutrophils.

An increased ﬂux of intracellular Ca²⁺represents a key signaling

event in CXCL1-induced neutrophil activation through

CXCR2.²Since nicotinamide-based chemokine antagonists

act intracellularly,³⁵the activity of a compound will depend

not only on its target aﬃnity but also on its ability to transit

into the intracellular compartment. To simultaneously capture

both properties in initial SAR evaluations, all compounds

prepared were evaluated for their ability to inhibit CXCL1-

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Table 2. CXCL1-Inhibitory Activity of Heterocyclyl- and Heteroalkyl-Substituted Thionicotinamides

a

Select IC₅₀determinations were performed in triplicate with ≥6 concentrations, and reported as the mean standard error.

could passively enter hPMNs. Its activity (IC₅₀= 3900 nM)

supported this hypothesis, but its potency was relatively weak in

this capacity.

permeabilized hPMNs.³⁵Removal of the methylene between

the sulfur and the aryl ring resulted in loss of activity (45). The

results from this focused combinatorial chemistry eﬀort

indicated that the S-benzyl nicotinamide nucleus held potential

as a new CXCR2 inhibitor template.

In order to widen our SAR eﬀorts, a focused combinatorial

library screen was implemented. As we discussed above, this

successful parallel synthetic eﬀort was made possible through

the use of excess alkyl bromide reagent and heat to drive the

alkylation to completion, as well as the use of solid phase

scavenging resins to aid in product isolation. The compounds

(13−43) were ﬁrst screened for activity versus CXCL1-

mediated calcium ﬂux in hPMNs at test concentrations of 5

and 10 μM. In our initial screen, compounds 13, 15, 17, 20, 22,

30, and 43 exhibited greater than 50% inhibition of CXCL1-

mediated intracellular Ca²⁺release at a test concentration of 5

μM and were therefore identiﬁed as potential hits. These

compounds were then resynthesized and retested, and the

results are shown in Tables 2 and 3. Upon resynthesis and

retesting, compounds 13, 15, 17, and 22 exhibited IC₅₀values

greater than 5 μM. Inhibitory activity was conﬁrmed for the

tetrahydrofuran derivative 20 (IC₅₀= 540 nM), the S-benzyl

derivative 30 (IC₅₀= 390 nM), and the S-dichlorobenzyl

derivative 43 (IC₅₀= 1610 nM). The activity of S-benzyl

derivative 30 was similar to the intrinsic activity of the de-

esteriﬁed carboxylate species (IC₅₀= 100−500 nM) of

nicotinamide methyl ester 5 reported previously in electro-

Elaboration of the benzyl ring with a carboxyl group resulted

in activity, with the 2-position (51, IC₅₀= 1480 nM) favored

over both the 3-position (49, IC₅₀= 2120 nM) and the 4-

position (47, IC₅₀> 10000 nM). We hypothesized that the

reduced activity of 51 was the result of poor accumulation in

the hPMN as a result of the negatively charged carboxylate

adversely aﬀecting uptake, eﬄux, or both. To examine this

hypothesis further and because we have shown previously that

the nicotinamide methyl ester 5 can be de-esteriﬁed within

hPMNs to liberate high concentrations of the corresponding

acid,³⁵we examined the uncharged S-methyl-benzoyl methyl

esters 46, 48, and 50 as potential intracellular precursors to the

corresponding acids. There was a 2-fold improvement in

potency in the 2-methyl ester 50 relative to the corresponding

free acid 51, but the 3- and 4-methyl esters 48 and 46 were

inactive. It is possible that the limited improvement in potency

arose because benzoyl methyl esters are poorer substrates for

intracellular esterase activity than the thioglycolate methyl ester

5.

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Table 3. CXCL1-Inhibitory Activity of Aryl-Substituted Thionicotinamides

a

Select IC₅₀determinations were performed in triplicate with ≥6 concentrations and reported as the mean standard error.

To explore bioisosteric replacements for the carboxylate in

our intermediate lead compound, the tetrazole derivative was

CXCR2 activation with an IC₅₀of 38 nM. Phenylboronic acid

has a pK_aof 8.9,⁴⁰and at neutral pH, compound 7 is expected

to be mostly uncharged and readily transit into the cell interior.

However, to test the possibility that greater lipophilicity would

further increase potency, we prepared the corresponding

pinacol ester 53. Although acyclic and unhindered cyclic esters

of boronic acids are rapidly hydrolyzed in water, hydrolysis is

slowed considerably for hindered cyclic aliphatic esters such as

the pinacol ester.⁴⁰Remarkably, the pinacol ester derivative 53

retained the same order of activity as the parent boronic acid,

synthesized and evaluated for activity. Compound 52 (IC₅₀

=

170 nM) exhibited an almost 10-fold increase in activity as

compared to the 2-carboxylate derivative 51. Our SAR eﬀorts

then turned to carboxyl replacements at the 2-position. An

important functionality being explored in recent pharmaceutical

development is the boronic acid moiety.^38,39Inclusion of a

boronic acid at the 2-postion of the S-benzyl nicotinamide

scaﬀold resulted in compound 7 exhibiting potent inhibition of

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with its activity modestly reduced by approximately 7-fold (IC₅₀

= 275 nM). Whether the pinacol ester was active at the target

or served to liberate the boronic acid inside the cell is unknown,

but importantly the enhanced lipophilicity of the boronyl ester

oﬀered no advantage in potency over the underivatived boronyl

group.

Concluding that the S-benzyl-2-borono scaﬀold in N-(4-

ﬂuorophenyl)-6-(thiobenzyl-2-borono)-nicotinamide 7 pro-

vided optimal nanomolar potency against CXCL1-induced

Ca²⁺ﬂux, we undertook separate focused SAR studies of the

apical 4-ﬂuorophenyl-carboxamido domain (Table 4). The 4-

ﬂuoroanilide moiety was previously investigated and optimized

for a related class of CXCR2 antagonists,⁴¹but our focus was to

introduce polar and/or ionizable substitutions that could

potentially increase aqueous solubility while retaining potent

chemokine antagonism. To accomplish this, the SAR of the

apical 4-ﬂuorophenyl-carboxamido- moiety in 7 was explored in

compounds 54−59, a series of R2 N-monosubstituted 6-

(thiobenzyl-2-borono)-nicotinamides (Table 4). The results

clearly revealed the importance of the 4-ﬂuoroanilide. Its

replacement with carboxyl (55) or tetrazolyl (56) abrogated all

activity. Its replacement with triﬂuoromethoxy (54), a pyridinyl

nitrogen (57), hydroxyl (58), or inclusion of a ring nitrogen

(59) resulted in a 7-, 14-, 3-, and 8-fold reduction in activity,

respectively. The N,N-disubstituted 6-(thiobenzyl-2-borono)-

nicotinamides 60−63 were explored by leaving the N-4-

ﬂuorophenyl R2 moiety constant and elaborating the R1 N-

substitution. The N-methyl-2-pyridyl (60) was found to be

well-tolerated (IC₅₀= 78 nM), yielding an activity nearly

equivalent to 7 and providing evidence that the pyridyl group,

which would be expected to be uncharged at neutral pH, does

not preclude entry of the molecule into the cell. Surprisingly,

the potency of the closely related congener 61 employing N-

methyl-4-pyridyl was 11-fold lower than 60, suggesting a model

where the pyridyl moieties are engaged in a highly structured

and restricted environment. N-methyl-2-piperidyl 62 was

explored as a nonplanar and nonaromatic analog of 60. It

was completely devoid of activity, possibly because it was not

accommodated at the target or because its positive charge

prevented passive diﬀusion into the cell. A similar ﬁnding was

obtained for the N-2-acetic acid analog 63. None of the

compounds 60−63 exhibited signiﬁcantly improved aqueous

solubility relative to 7, despite attempts to prepare conjugate

salts (data not shown).

Table 4. Eﬀect of N-Substituted 6-(Thiobenzyl-2-borono)-

nicotinamides on CXCL1-Induced Ca²⁺Flux in Human

Neutrophils

On the basis of its optimal in vitro potency in inhibiting

CXCL1-stimulated Ca²⁺release in the above SAR studies,

compound 7 was further evaluated with respect to its in vitro

signaling pharmacology and in vivo biologic activity as

described below.

G-Protein Coupling to CXCR2. CXC receptors transduce

signals to the interior of the cell through activation of a coupled

heterotrimeric G-protein, the most proximal signaling event

after agonist binding to the receptor.^3,4CXCR2 is bound and

activated by both CXCL1 and CXCL8.²The eﬀect of

compound 7 on G-protein coupling to CXCR2 was evaluated

by [³⁵S]GTPγS binding in CXCR2 membranes prepared from

HEK293 cells that stably expressed the human receptor (Figure

2). Compound 7 potently inhibited [³⁵S]GTPγS binding in

response to 10 nM CXCL8 with an IC₅₀of 60 7 nM (mean +

S.E.).

CXCL8 Binding at CXCR2. We have shown that

nicotinamide glycolates act away from the orthosteric chemo-

kine binding site to antagonize CXCR2 through an intracellular

mechanism.³⁵Maximal inhibition of radioligand binding by an

allosteric antagonist can be observed in displacement experi-

ments with radioligand concentrations much lower than the K_d

value.⁴²We examined the ability of compound 7 to displace

binding of [¹²⁵I]-CXCL8 from CXCR2 membranes using a

radioligand concentration (25 pM) that was >10-fold below the

K_dfor CXCL8 binding to CXCR2.⁴³Although compound 7

potently inhibited functional CXCR2 signaling by CXCL1

(IC₅₀= 38 nM, CXCL1-stimulated Ca²⁺ﬂux) and CXCL8

(IC₅₀= 60 nM, CXCL8-stimulated [³⁵S]GTPγS binding), up to

10 μM failed to compete the binding of [¹²⁵I]-CXCL8 to

CXCR2 membranes (Figure 3). In parallel controls, unlabeled

CXCL8 isopotently displaced its homologous radioligand (IC₅₀

= 28 pM). Collectively, these data support a model where

compound 7 acts as a noncompetitive, intracellular allosteric

inhibitor of CXCR2. These ﬁndings mirrored those of the

a

Each IC₅₀determination was performed in triplicate with ≥6

concentrations and reported as the mean standard error.

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Figure 4. Eﬀect of compound 7 on the cell surface expression of

CXCR2. HEK293 cells stably expressing CXCR2 were pretreated with

1% DMSO (vehicle) or 10 μM compound (cpd. 7) for 60 min.

HEK293 cells not expressing CXCR2 served as a negative isotype

control (isotype). All cells were then incubated with R-phycoerythrin

(PE)-conjugated antihuman CXCR2 mouse monoclonal antibody at 4

°C for 60 min. Cells were washed, ﬁxed in 2% formaldehyde in PBS,

and subjected to ﬂow cytometric ﬂuorescence-activated cell sorting

(FACS) analysis of the PE signal. Results are representative of three

independent experiments.

Figure 2. Inhibition of chemokine-stimulated [³⁵S]GTPγS binding by

compound 7. HEK293 cells stably expressing CXCR2 were incubated

with diﬀerent concentrations of compound (from 10⁻¹⁰to 10⁻⁴M) at

37 °C for 60 min, and then the membranes were prepared by lysis and

centrifugation. The membranes were then incubated in buﬀer

containing 50 μM GDP, 8 nM [³⁵S]GTPγS, and 10 nM CXCL8 at

30 °C for another 60 min. Membranes were harvested by rapid

ﬁltration and membrane-bound [³⁵S]GTPγS quantitated. Data,

expressed as a percentage of basal [³⁵S]GTPγS bound in the absence

of CXCL8, are the mean SE from three independent experiments,

each done in triplicate.

10 μM compound 7 did not signiﬁcantly alter the cell surface

expression of CXCR2. These data together with the data

showing inhibition of CXCL8-stimulated [³⁵S]GTPγS binding

are most consistent with a mechanism of antagonism involving

direct blockade of receptor activation.

CXCR2 MAPK Signaling. CXCR1 and CXCR2 mediate

downstream signaling in part through MAPK activation.^4,45To

assess the ability of compound 7 to inhibit MAPK in HEK293

cells that stably expressed CXCR2, we measured ERK1/2

phosphorylation in response to CXCL8 in the presence of

vehicle or 10 μM compound 7. As shown in the vehicle arm in

Figure 5, CXCR2 induced a time-dependent phosphorylation

Figure 3. Competition binding assay with 7 and CXCL8 at human

CXCR2. Membranes from recombinant HEK293-CXCR2 cells were

incubated with the compound at the indicated concentrations and 25

pM [¹²⁵I]-CXCL8. Radioligand binding to the membranes was

Figure 5. Eﬀect of Compound 7 on CXCR2-induced ERK1/2

phosphorylation. HEK293 cells stably expressing CXCR2 were

stimulated with CXCL8 (100 ng/mL) for 0−30 min in the presence

of vehicle or 10 μM compound 7. Phosphorylated ERK1/2 and total

ERK were determined by Western blotting using antiphospho-ERK1/

2 (P-ERK1/2) and antitotal ERK1/2 (T-ERK1/2) antibodies,

respectively. Data are representative of three independent experiments

performed in triplicate.

measured by scintillation. Data show the mean

SD (n = 3)

radioligand binding, expressed as the percent of control speciﬁc

radioligand binding with vehicle.

noncompetitive allosteric inhibitor reparixin 1, which inhibited

CXCR1 responses with no eﬀect on CXCL8 binding to

CXCR1.^22,23

Cell Surface Expression of CXCR2. CXCR2 activation by

chemokines is followed by receptor phosphorylation and

subsequent down-regulation; events that are accompanied by

receptor internalization.⁴⁴We considered the possibility that

compound 7 may antagonize CXCR2 in whole cells at least

partially through a similar mechanism of sequestering receptor

away from the cell membrane signaling machinery. We

therefore evaluated the eﬀect of compound 7 on the CXCR2

surface expression in stably transfected HEK293 cells using a

ﬂuorescently labeled antibody to the receptor and ﬂuorescence-

activated cell sorting. As shown in Figure 4, 60 min exposure to

of ERK1/2 upon activation by CXCL8. Maximum response was

obtained at 15 min. In contrast, compound 7 completely

blocked CXCR2-mediated phosphorylation of ERK1/2 by 30

min. There was no eﬀect on the amount of ERK1/2 present in

the lysates (Figure 5, lower panel) as assessed by an anti-

ERK1/2 antibody recognizing ERK1/2 irrespective of its

phosphorylation state. These data are consistent with receptor

blockade by compound 7 as the common event that abrogates

signaling pathways downstream of CXCR2 involving G-

proteins, MAP kinases, and intracellular Ca²⁺.

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Selectivity of Antagonism. CXCR1 and CXCR2 are

expressed in equal numbers on the surface of hPMNs.⁴⁶

Whereas CXCL8 activates both CXCR1 and CXCR2, CXCL1

is a selective agonist for CXCR2.²Compound 7 inhibited both

CXCL1- and CXCL8-stimulated Ca²⁺ﬂux in hPMNs with IC₅₀

values of 38 and 36 nM, respectively (Table 5).

Table 5. Eﬀect of 7 on Agonist-Induced Ca²⁺Flux

a

IC₅₀(nM)

agonist

hPMN

HEK293-CXCR1

HEK293-CXCR2

b

CXCL1

CXCL8

C5a

38

3

36 11

>5000

ND

880 90

210 40

c

PAF

d

fMLF

Figure 6. An air-pouch was formed on the backs of 10−15 week old,

male CD1 Swiss mice in four cohorts (n = 5 animals per cohort).

Compound dissolved in vehicle (0.02 mg/kg and 0.20 mg/kg cohorts)

or vehicle alone (positive and negative cohorts) was administered

intravenously. After 3 h, each air pouch was injected with 1 mL of PBS

(negative cohort) or 2% carrageenan in PBS (positive, 0.02 mg/kg and

0.20 mg/kg cohorts). After 4 h, the pouch ﬂuid was collected and

combined with an additional 2 mL PBS wash of the pouch. The cells in

the combined ﬂuid were stained with trypan blue and manually

a

Each IC₅₀determination was performed in triplicate with ≥6

b

concentrations and reported as the mean standard error. ND = not

determined. Platelet activating factor. Formyl-Met-Leu-Phe.

c

d

This inhibition was not partial, since a concentration of 1 μM

compound 7 was suﬃcient to completely eliminate CXCL8-

stimulated Ca²⁺ﬂux in hPMNs. The total antagonism of

CXCL8-induced Ca²⁺ﬂux by compound 7 presumably

reﬂected a dual blockade of both CXCR1 and CXCR2

signaling. The antagonism of CXCR1 and CXCR2 by

compound 7 in hPMNs was found to be selective; however,

as concentrations up to 5 μM failed to inhibit Ca²⁺ﬂux induced

by optimal concentrations of the chemokines C5a, PAF, and

fMLF (Table 5).

counted on a hemocytometer. Data show the mean

SE of the

absolute pouch cell count per cohort. Student’s t-test: **p < 0.01 vs

positive cohort.

2% carrageenan, which causes an inﬂammatory inﬁltrate

consisting predominantly of neutrophils. The total cell count

in the inﬂammatory inﬁltrate from each pouch was quantitated.

At a dose of 0.2 mg/kg of compound 7, there was a signiﬁcant

reduction in cell count in the pouches of treated animals

compared to the positive control cohort (**p < 0.01, Student’s

t-test).

Although the inhibition of CXCL8-induced Ca²⁺ﬂux in

hPMNs indicated dual antagonism of CXCR1 and CXCR2, the

coexpression of these receptors in hPMNs precluded evaluating

the relative inhibitory potency at each receptor. Additional

experiments were therefore performed to separately evaluate

the potency of compound 7 at CXCR1 and CXCR2. For these

determinations, CXCL8-induced Ca²⁺ﬂux was measured in

recombinant HEK293 cells that stably expressed CXCR1

(HEK293-CXCR1) or CXCR2 (HEK293-CXCR2) in the

presence of vehicle and diﬀerent concentrations of compound

7 (Table 5). For CXCL8-induced Ca²⁺ﬂux, the data showed

that compound 7 exhibited a 4-fold selectivity in inhibition for

CXCR2 (IC₅₀= 210 nM) over CXCR1 (IC₅₀= 880 nM) in

these recombinant systems. As a positive control, the CXCR2-

selective antagonist SB225002 inhibited CXCL8-induced Ca²⁺

ﬂux in the HEK293-CXCR2 cells with an IC₅₀of 40 nM, a

value consistent with its previously reported IC₅₀value of 30

nM for CXCL1-induced Ca²⁺ﬂux in hPMNs.²⁴The observed

discrepancy in the potency of 7 when evaluated in isolated

hPMNs versus HEK cells is not fully understood but may be

attributed to intracellular diﬀerences between the native

(hPMN) and artiﬁcially engineered (HEK) systems.

Eﬀect on Inﬂammation in Vivo. To conﬁrm the in vitro

eﬀects on the neutrophil function in vivo, compound 7 was

evaluated in the murine air-pouch model of inﬂammation

(Figure 6). An air-pouch was induced on the backs of male

CD1 Swiss mice as described.⁴⁷Cohorts of ﬁve animals each

were given vehicle (negative and positive control cohorts) or

compound 7 dissolved in vehicle (0.02 mg/kg and 0.20 mg/kg

test cohorts) by intravenous injection. Three hours afterward,

the pouches in the negative control cohort were injected with

sterile phosphate-buﬀered saline. Inﬂammation was induced in

the pouches of the remaining three cohorts by injection with

CONCLUSION

■

The results reported here describe SAR studies that examined

the eﬀect of a novel series of S-substituted 6-mercapto-N-

phenyl-nicotinamides on CXCL1-stimulated Ca²⁺ﬂux in whole

hPMNs. The SAR data established that an ortho-modiﬁed S-

benzyl substituent played a critical role in determining activity,

with an ortho-boronyl group being optimal. The ﬂuorine in the

4-ﬂuorophenyl-carboxamido- moiety was also an important

requirement for optimal inhibitory activity. Among the

derivatives exhibiting the most potent antagonism of CXCL1

described here, 7 was selected for further evaluation of its in

vitro pharmacology and in vivo biologic activity.

Compound 7 was found to be a dual CXCR2/1 antagonist

with a modest preference for CXCR2. It inhibited CXCL1- and

CXCL8-induced Ca²⁺ﬂux (IC₅₀= 38 and 36 nM, respectively)

in hPMNs. In response to CXCL8 stimulation, compound 7

directly antagonized [³⁵S]GTPγS binding (IC₅₀= 60 nM) and

ERK1/2 phosphorylation in HEK293 cells that stably expressed

CXCR2. In an in vivo murine model of inﬂammation

characterized by an inﬂammatory inﬁltrate predominated by

neutrophils, compound 7 signiﬁcantly reduced total cell count

at an intravenous dose of 0.2 mg/kg.

Early work by others to develop small-molecule chemokine

inhibitors focused on evolving compounds that were highly

speciﬁc for either CXCR1^22,23or CXCR2.^24,25These eﬀorts

were later followed by compounds with dual-activity at CXCR1

and CXCR2^26,48in recognition that these homologous

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receptors mediate inﬂammation through unique and over-

lapping pathways. For example, whereas both CXCR1 and

CXCR2 mediate CXCL8-induced chemotaxis,^23,49myeloper-

oxidase release from hPMNs occurs mainly through CXCR1

activation.^50,51CXCL8-mediated neutrophil chemotaxis is most

eﬀectively inhibited by dual CXCR1 and CXCR2 blockade.⁵²

Compared to speciﬁc antagonists, dual CXCR2/1 inhibition by

compound 7 may therefore oﬀer a more complete therapeutic

strategy in a number of inﬂammatory diseases where the

CXCR2 pathway is involved speciﬁcally or in conjunction with

CXCR1 signaling.

(Velcade), and other boronic acids have been previously

disclosed as inhibitors of serine proteases, proteasomes,

arginase, nitric oxide synthase, and transpeptidases.³⁹The

eﬀectiveness of boronic acids as inhibitors of proteosomes^58,59

and serine proteases⁶⁰⁻⁶³has been ascribed to the ability of the

boronyl group to form a reversible tetrahedral adduct with an

active-site nucleophile that has most often been serine but has

also included histidine.⁶⁰On the basis of crystallographic and

NMR studies of boronic acid inhibitors in complex with their

target enzymes, this adduct closely mimics the putative

tetrahedral intermediate or transition-state formed between

the enzyme and its substrates. Although CXCR1 and CXCR2

are not thought to possess intracellular enzymatic activity, we

speculate that the marked increase in potency observed upon

incorporation of the boronyl group at the 2-position of the S-

benzyl scaﬀold in compound 7 may be due to an analogous

reversible boronyl-mediated adduct with a receptor-based

nucleophile in the putative intracellular binding pocket. The

potential beneﬁt of such a postulated adduct in prolonging

pharmacodynamic eﬀects in subjects remains to be investigated.

However, like other CXCR2/1 inhibitors with a low oﬀ-rate

(e.g., SCH527123),⁴⁹the beneﬁt will likely be limited by the

kinetics of new receptor biosynthesis and the 1−2 day tissue

lifespan of the neutrophil itself.

G-protein coupled receptors (GPCRs) are thought to exist in

multiple conformational states, and activation by an agonist

mediates signaling to the cell interior through poorly

understood conformational changes in the receptor. It has

been proposed that allosteric sites in the transmembrane

domain of GPCRs may represent high-value targets for

noncompetitive inhibitors that block agonist-induced G-protein

activation.⁵³Indeed, studies have found that the antagonist

reparixin (1),^22,23and the CXCR2-speciﬁc antagonists

SB265610, SB332235, and SCH527123 (3), act as allosteric

inhibitors.^{43,49,54−56}These inhibitors achieve receptor selectiv-

ity by exploiting amino acid diﬀerences between these

homologous receptors, with reparixin (1) diﬀerentially binding

the extracellular half of the seven-transmembrane core²³and

SB332235 diﬀerentially binding an intracellular pocket made up

of several transmembrane helices.⁵⁵We previously demon-

strated that the nicotinamide glycolate methyl ester 5 inhibited

CXCL1-induced eﬀects in hPMNs through an intracellular

mechanism.³⁵In the studies herein, potent antagonism of

CXCL8-stimulated [³⁵S]GTPγS binding by compound 7

localized inhibition to a proximal signaling event involving

the CXCR2 receptor and its G-protein. Further data

demonstrated compound 7 was CXCR2/1 speciﬁc, with no

inhibition of fMLF-, C5a-, or PAF-induced Ca²⁺ﬂux through

their corresponding GPCRs. We therefore speculate that

compound 7 acts at an intracellular pocket, involving at least

the CXCR2/1 receptor to lock the receptor in a conformation

unable to activate downstream signaling. Studies to explore this

postulated mechanism are underway in our group.

Consistent with being a noncompetitive inhibitor, compound

7 potently inhibited functional CXCR2 signaling by CXCL1

and CXCL8 but failed to compete with the binding of [¹²⁵I]-

CXCL8 to CXCR2 at concentrations ∼100-fold larger than its

IC₅₀for inhibiting CXCR2 ligand-triggered Ca²⁺ﬂux. Others

have earlier recognized the possibility of identifying allosteric

small-molecule chemokine inhibitors that disrupt receptor

function without displacing the chemokine from its orthosteric

binding site.⁵⁷The SAR studies herein demonstrate that our

whole-cell functional assay for CXCL1-induced Ca²⁺ﬂux in

hPMNs beneﬁcially allowed for the selection of a novel class of

potent noncompetitive inhibitors that would have otherwise

been missed by a competitive displacement screen. Our

ﬁndings mirror those of the noncompetitive allosteric inhibitor

reparixin, which likewise inhibited CXCR1 responses with no

eﬀect on CXCL8 binding to CXCR1.^22,23

Current eﬀorts are underway in our laboratory to increase

the oral bioavailability of compound 7 and to further investigate

the interaction of this class of compounds with CXCR1 and

CXCR2 receptors. Compound 7 (SX-517) represents a novel

boronic acid containing pharmacophore for the antagonism of

CXCR1/2 chemokine receptors and may prove useful in the

treatment of inﬂammatory diseases with a signiﬁcant neutrophil

component.

EXPERIMENTAL METHODS

■

A. Pharmacology and Biology. Materials and Reagents.

Chemicals and carrageenan were obtained from Sigma-Aldrich

Chemical Co. (St. Louis, MO). Chemokines were from PeproTech

(Rocky Hill, NJ) or from R&D Systems (Minneapolis, MN). Control

CXCR2 antagonists (SB265610 and SB225002) were from Tocris

Biosciences (Ellisville, MO). The HEK293 cell line was from ATCC

(Manassas, VA). Erk1/2 and phospho-Erk (p-Erk) antibodies were

from Santa Cruz Biotechnology (Santa Cruz, CA). Cellulose nitrate

membrane ﬁlters were from Whatman Inc. (Piscataway, NJ).

[³⁵S]GTPγS (1250 Ci/mmol), ¹²⁵I-CXCL8 (2200 Ci/mmol), and

Uniﬁlter GF/C 96-well ﬁlter plates were from PerkinElmer Life and

Analytical Sciences (Waltham, MA).

HEK293 Cell Stably Expressing Human CXCR1 and CXCR2.

HEK293 cells were cultured in DMEM (Dulbecco’s modiﬁed Eagle’s

medium) supplemented with 50 units/mL penicillin, 50 μg/mL

streptomycin, 3 mM glutamine, 10% heat-inactivated fetal bovine

serum (Atlanta Biologicals, Lawrenceville, GA) at 37 °C, 5% CO₂.

Transfection with a human CXCR1³or CXCR2 plasmid⁴⁴was

performed with Lipofectamine (Invitrogen Life Technology) based on

the manufacturer’s protocol. For selection of stable polyclonal cell

lines, 800 μg/mL G418 (Sigma, St Louis, MO) were added 24 h after

transfection and cells were maintained in DMEM medium containing

800 μg/mL G418 through subculture procedures until a pooled, stable

cell line was established. Surface expression of CXCR1 and CXCR2

was conﬁrmed using R-phycoerythrin (PE)-conjugated antihuman

CXCR1 or CXCR2 mouse monoclonal antibody (BD Pharmingen,

San Diego, CA) and ﬂuorescence-activated cell sorting as described

below.

Compound 7 is notable for being the ﬁrst reported boronic

acid chemokine antagonist. Due to the unique chemical

similarities and diﬀerences between boron and carbon, boronic

acid based inhibitors have been the subject of considerable

recent interest as a new source of small-molecule therapeu-

tics.³⁸The clinical utility of the boronyl moiety has been

validated with the U.S. and European approval of bortezomib

Isolation of Human Neutrophils. Blood was collected from healthy

donors in accordance with a protocol approved by the Institutional

Review Board at Montana State University. Human polymorphonu-

clear leukocytes (hPMNs) were puriﬁed from the blood using dextran

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sedimentation, followed by Histopaque 1077 gradient separation and

hypotonic lysis of red blood cells, as described previously.⁶⁴hPMN

preparations were routinely >95% pure, as determined by light

microscopy, and >98% viable, as determined by trypan blue exclusion.

Isolated hPMNs were washed twice and resuspended in RPMI

containing 10% fetal bovine serum (FBS).

Animals. Male CD1 mice were obtained from Charles River

Laboratories (Wilmington, MA). Animals were housed and acclimat-

ized for 1 week under controlled temperature (20 2 °C), humidity

(55 10%), and lighting (7 a.m. to 7 p.m.). Standard sterilized food

and water were supplied ad libitum during acclimatization and

experiments. All procedures and protocols were approved by the

Institutional Animal Care and Use Committee and were carried out in

accordance with NIH guidelines for the handling and use of laboratory

animals.

Calcium Flux Assay. hPMNs (or cells expressing either CXCR1 or

CXCR2) were suspended in HBSS⁻(Hank’s balanced salt solution

without Ca²⁺and Mg²⁺) containing 10 mM HEPES and FLIPR

Calcium 3 dye (3.1 × 10⁷cells in total volume 1.7 mL). Cells were

aliquoted (200 μL of the cell suspension per tube, 8 tubes total), and 2

μL of the designated compound (with appropriate dilutions) were

added to each of 6 tubes. As controls, 2 μL of DMSO (1% ﬁnal

concentration) were added to two other tubes. Cells were incubated at

37 °C for 30 min. After dye loading, tubes were centrifuged at 6000

rpm for 1 min, supernatant was removed, and the cell pellet was

resuspended in 200 μL of HBSS⁺(with Ca²⁺and Mg²⁺), containing 10

mM HEPES. The test compound or DMSO (control) were added

again at the same concentrations that were used during cell loading.

The cell suspension was aliquoted into a 96-well Reading Plate

(Corning) in a volume of 90 μL (105 cells/well). The Compound

Plate contained agonist in HBSS⁻) or HBSS⁻(control). After 15 s of

reading the basal level of ﬂuorescence by FlexStation II, 10 μL of

agonist or HBSS⁻were automatically transferred from the compound

plate into the reading plate. The agonists used and their ﬁnal

concentrations were 25 nM CXCL1, 1 nM CXCL8, 10 nM N-formyl-

L-methionyl-L-leucyl-L-phenylalanine (fMLF), and 50 nM C5a.

Changes in ﬂuorescence were monitored (λ_ex= 485 nm, λ_em= 525

nm) every 5 s for 240 to 500 s at room temperature. The maximum

change in ﬂuorescence, expressed in arbitrary units over baseline

(max−min), was used to determine the agonist response. The eﬀect of

each compound on the agonist response was normalized and

expressed as a percent of the DMSO control, which was designated

as “100% response.” Curve ﬁtting and calculation of the compound

inhibitory concentration that reduced the level of the agonist response

by 50% (IC₅₀) was determined by nonlinear regression analysis of the

dose−response curves generated using Prism 4 (GraphPad Software,

Inc., San Diego, CA).

determined by nonlinear regression analysis of the dose−response

curves generated using Prism 4 (GraphPad Software, Inc., San Diego,

CA).

Competition ¹²⁵I-CXCL8 Binding Assay. This was performed

according to White et al. using HEK293-hCXCR2 membranes.²⁴

Brieﬂy, assays were performed in 96-well microtiter plates where the

reaction mixture contained 1.0 μg/mL membrane protein in 20 mM

Bis-trispropane, pH 8.0, with 1.2 mM MgSO₄, 0.1 mM EDTA, 25 mM

NaCl, and 0.03% CHAPS and compound (100 μM stock in DMSO)

added at the indicated concentrations, the ﬁnal DMSO concentration

was <0.5% under standard binding conditions. Binding was initiated

with 25 pM [¹²⁵I]-CXCL8. Nonspeciﬁc binding was determined with

30 nM CXCL8. After 1 h incubation at room temperature, membranes

were harvested by rapid ﬁltration. The ﬁlter was dried and counted

with a liquid scintillation counter. Speciﬁc CXCL8 binding to the

receptors was deﬁned as the diﬀerence between the total binding and

the nonspeciﬁc binding determined in the presence of an excess of

unlabeled CXCL8. The results were expressed as a percent of control

speciﬁc binding: (speciﬁc binding with compound)/(control speciﬁc

binding) × 100. IC₅₀for unlabeled CXCL8 was determined by

nonlinear regression analysis of the concentration−response curve

generated with mean replicate values ﬁtted to the Hill equation.

CXCR2 Cell Surface Expression by Flow Cytometry. HEK293 cells

stably expressing CXCR2 were pretreated with 1% DMSO (vehicle) or

compound (10 μM) for 60 min. HEK293 cells not expressing CXCR2

served as a negative isotype control. All cells were then incubated with

a 1:100 dilution of R-phycoerythrin (PE)-conjugated antihuman

CXCR2 mouse monoclonal antibody (BD Pharmingen, San Diego,

CA) at 4 °C for 60 min. Cells were washed, ﬁxed in 2% formaldehyde

in phosphate-buﬀered saline, and quantitated by ﬂuorescence-activated

cell sorting (FACS) using a FACScan ﬂow cytometer equipped with

CellQuest software (BD Biosciences, Mountain View, CA).

ERK1/2 Phosphorylation. HEK293 cells expressing CXCR2 were

harvested and plated in equal number to 60 mm plates (5 × 10⁶cells/

plate). The cells were then incubated with PBS containing 1% DMSO

(vehicle) or 10 μM compound for 60 min at 37 °C, followed by the

addition of 100 ng/mL CXCL8. All cells in a plate were then lysed at

0, 5, 15, and 30 min after agonist-treatment by adding lysis buﬀer:

0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10 mM Tris

(pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl

ﬂuoride, 10 μg/mL aprotinin, and 10 μg/mL leupeptin. Lysates

containing equal amounts of protein (∼50 μg) were resolved by 10%

SDS−polyacrylamide gel electrophoresis, transferred to a nitro-

cellulose membrane, and probed with antibody against phospho-

ERK1/2 or ERK1/2. Visualization was carried out with a horseradish

peroxidase-conjugated secondary antibody.

Murine Air-Pouch Model of Inﬂammation. An air-pouch was

induced on the backs of 10−15 week old, male CD1 Swiss mice by

subcutaneous injection (2 mL) of air as described.⁴⁷An additional

subcutaneous injection of air (1.5 mL) was given to reinﬂate the pouch

the following day. The compound was solubilized in vehicle consisting

of a 20:20:80 mixture of PEG400:DMF:saline. Compound dissolved in

vehicle, or vehicle alone was administered via iv tail vein injection at

diﬀerent doses according to body weight. After 3 h, either sterile

phosphate-buﬀered saline (PBS, 1 mL) or 2% carrageenan in sterile

PBS (1 mL) was injected into the air pouch. After 4 h, the mice were

sedated with ketamine and sacriﬁced by cervical dislocation. The

pouch ﬂuid was then collected by syringe and combined with an

additional 2 mL PBS wash of the pouch. The cells in the combined

ﬂuid were stained with trypan blue and manually counted on a

hemocytometer under 20× magniﬁcation.

[³⁵S]GTPγS Assay. [³⁵S]GTPγS assays were performed as previously

described⁶⁵with the following modiﬁcations: HEK293 cells stably

expressing hCXCR2, pretreated with diﬀerent concentrations of

compound, were lysed in buﬀer containing 5 mM Tris-HCl, pH 7.5,

5 mM EDTA, and 5 mM EGTA, and the cell lysate was centrifuged at

30000g for 10 min. Protein concentration in membrane preparations

was determined using the BioRad Protein Determination assay 18

from Bio-Rad (Hercules, CA). Membranes containing 50 μg of protein

were incubated in 50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 1 mM

EGTA, 100 mM NaCl, 50 μM GDP, 8 nM [³⁵S]GTPγS, 10 nM

CXCL8 in a total volume of 0.1 mL at 30 °C for 1 h. The reaction was

terminated by dilution into phosphate-buﬀered saline and rapid

ﬁltration through Uniﬁlter GF/C 96-well ﬁlter plates pretreated with

0.3% polyethylenimine and washed three times with ice-cold wash

buﬀer (50 mM Na₂HPO₄and 50 mM KH₂PO₄, pH 7.4). Bound

radioactivity was determined using a MicroBeta counter (PerkinElmer

Life and Analytical Sciences). Basal binding was assessed in the

absence of CXCL8, and nonspeciﬁc binding was determined in the

presence of 10 μM GTPγS. The percentage of CXCL8-stimulated

[³⁵S]GTPγS binding was calculated as [cpm_CXCL8− cpm_nonspecific]/

[cpm_basal− cpm_nonspecific]. Curve ﬁtting and calculation of the

compound inhibitory concentration that reduced the percentage of

CXCL8-stimulated [³⁵S]GTPγS binding by 50% (IC₅₀) was

B. Chemistry. Materials and Reagents. General chemicals and

reagents for synthesis were purchased from Sigma-Aldrich (Milwau-

kee, WI), and solvents were purchased from VWR International (West

Chester, PA) and used without further puriﬁcation. Commercial

synthetic precursors and intermediates were from Acros Organics

(Pittsburgh, PA), Sigma-Adrich Chemical (Milwaukee, WI), Avocado

Research (Lancashire, U.K.), Bionet (Cornwall, U.K.), Boron

Molecular (Research Triangle Park, NC), Combi-Blocks (San Diego,

CA), Eastman Organic Chemicals, Eastman Kodak Company

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(Rochester, NY), Fisher Scientiﬁc Co. (Pittsburgh, PA), Frontier

Scientiﬁc (Logan, UT), ICN Biomedicals, Inc. (Costa Mesa, CA),

Lancaster Synthesis (Windham, NH), Maybridge Chemical Co.

(Cornwall, U.K.), Pierce Chemical Co. (Rockford, IL), Riedel de

Haen (Hannover, Germany), Santa Cruz Biotechnology (Dallas, TX),

Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America

(Portland, OR), and Wako Chemicals USA, Inc. (Richmond, VA).

Solid phase scavenger resins were from NovaBiochem (La Jolla, CA).

General Analytical Procedures. Synthetic reaction progression was

monitored by thin-layer chromatography (TLC) using precoated

aluminum-backed plates with silica gel with ﬂuorescent indicator

(precoated F254 Macherey-Nagel plates, EMD Chemicals); the spots

were examined with UV light. Chromatographic puriﬁcation was

performed with 230−400 mesh (32−63 μm) ﬂash silica gel (Dynamic

Adsorbents, Norcross, GA) or by preparative high-performance liquid

chromatography (HPLC) using a Waters Delta Prep 4000 HPLC

ﬁtted with a Phenomenex Gemini 250 × 21 mm, 10 μm, C₁₈column

and monitored at 254 nm. Retention time (RT) is reported in minutes

(min), and purity as measured by UV absorbance is reported as a

percentage of all peak areas. HPLC analyses were performed using the

following gradients and systems:

Gradient A. Water:acetonitrile:formic acid (95:5:0.1) to water:-

acetonitrile:formic acid (5:95:0.1) at 35 °C over 12 min, on a

Shimadzu HPLC system, with a Phenomenex Gemini 50 × 2 mm, 5

μm, C₁₈column, monitored at 254 nm.

Gradient B. Water:acetonitrile:formic acid (95:5:0.1) to water:-

acetonitrile:formic acid (5:95:0.1) at 30 °C over 30 min, on an Agilent

1100 Series HPLC system, with a Phenomenex Gemini 50 × 2 mm, 5

μm, C₁₈column, monitored at 254 nm.

Gradient C. Water:acetonitrile:triﬂuoroacetic acid (95:5:0.1) to

water:acetonitrile:triﬂuoroacetic acid (5:95:0.1) at 35 °C over 0.5 min,

held at 5:95:0.1 for 6.5 min, on an Agilent 1100 Series HPLC system,

with Phenomenex Gemini 50 × 2 mm, 5 μm, C₁₈column, monitored

at 254 nm.

(1−18 h). The crude product was then precipitated out of solution by

the addition of water (5−50 mL/mmol).

Method C. Thionicotinamide 64 (1 equiv) and the corresponding

bromomethyl derivative (1 equiv) were suspended in EtOH (5 mL/

mmol). To the suspension, 1 N NaOH (1 equiv) was added, and the

reaction mixture was brought to gentle reﬂux and monitored by either

TLC or LC−MS until complete (0.5−2 h). The crude product was

then precipitated out of solution by the addition of water (5−20 mL/

mmol).

2-((5-(4-Fluorophenylcarbamoyl)pyridin-2-ylthio)methyl)-

phenylboronic acid (7, SX-517). Prepared via method C using

thionicotinamide 64 (1.27 g, 5.10 mmol) and 2-bromomethyl-

phenylboronic acid (1.09 g, 5.10 mmol) suspended in EtOH (50

mL). To the suspension, 1 N NaOH (5.1 mL, 5.10 mmol) was added

and the reaction mixture heated to gentle reﬂux for 2 h. Then water

(50 mL) was added to the reaction mixture while still hot. Upon

cooling, a white precipitate formed and this was ﬁltered, washed with

50% aqueous EtOH, and then water and dried in an oven to yield 1.53

g (78%) of 7 (SX-517) as an oﬀ-white solid. ESI-MS m/z = 383.1 [M

+ H]⁺. ¹H NMR (500 MHz, DMSO-d₆): δ 10.39 (s, 1H), 8.99 (s, 1H),

8.23 (s, 2H), 8.13−8.11 (m, 1H), 7.80−7.77 (m, 2H), 7.55 (d, J = 7.0

Hz, 1H), 7.45 (d, J = 8.0 Hz, 2H), 7.30 (t, J = 7.0 Hz, 1H), 7.22 (t, J =

8.8 Hz, 3H), 4.69 (s, 2H). ¹³C NMR (100.6 MHz, DMSO-d₆): d

163.6, 162.7, 159.6, 157.2, 148.6, 141.5, 136.2, 135.5, 135.3, 134.0,

129.3, 129.0, 126.2, 122.2, 122.1, 120.7, 115.4, 115.1. Anal. Calcd for

C₁₉H₁₆BFN₂O₃S: C, 59.71%; H, 4.22%; N, 7.33%; S, 8.39%. Found: C,

59.54%; H, 4.38%; N, 7.48%; S, 8.49%. HPLC (gradient B): RT =

18.45 min, purity 96.1%.

6-((2H-Tetrazol-5-yl)methylthio)-N-(4-ﬂuorophenyl)nicotinamide

(8). The cyano intermediate was prepared via method B using

thionicotinamide 64 and chloroacetonitrile. This intermediate was

used without further puriﬁcation. The cyano-intermediate (190 mg,

0.66 mmol), dibutyltin oxide (33 mg, 0.14 mmol) and trimethylsilyl

azide (174 μL, 1.32 mmol) were suspended in toluene (50 mL) and

reﬂuxed for 24 h. The mixture was allowed to cool to room

temperature, and the resulting precipitate was ﬁltered and washed with

toluene to yield 160 mg (73%) of 8 as a light yellow solid. TLC (1%

Gradient D. Water:acetonitrile:formic acid (95:5:0.1) to water:-

acetonitrile:formic acid (5:95:0.1) at 35 °C over 4 min, held at

5:95:0.1 for 5 min, on a Shimadzu HPLC system, with Phenomenex

Gemini 50 × 2 mm, 5 μm, C₁₈column, monitored at 254 nm.

Electrospray ionization mass spectrometric analysis (ESI-MS) was

performed using a Micromass Quattro II mass spectrometer with

1

AcOH/ethyl acetate): R_f= 0.32. ESI-MS m/z = 331.3 [M + H]⁺. H

NMR (500 MHz, DMSO-d₆): δ 10.41 (s, 1H), 8.97 (s, 1H), 8.18 (d, J

= 8.0 Hz, 1H), 7.77 (t, J = 6.0 Hz, 6.6 Hz, 2H), 7.58 (d, J = 8.2 Hz,

1H), 7.21 (t, J = 8.2 Hz, 2 H), 4.81 (s, 2H). HPLC (gradient B): RT =

14.52 min, purity 96.4%.

1

MassLynx 4.0. H NMR spectra were obtained on a Bruker AVance

(300 or 500 MHz, ¹H) and are reported as parts per million downﬁeld

from tetramethylsilane with number of protons, multiplicities, and

coupling constants in Hertz indicated parenthetically. Elemental

analyses were performed by Atlantic Microlab (Norcross, GA).

General Procedures for the Synthesis of S-Substituted N-(4-

Fluoro-phenyl)-6-mercapto-nicotinamides (Scheme 1). Accom-

plished by one of the following three methods. If required, the

crude was puriﬁed by either ﬂash silica gel chromatography or

preparative HPLC. Final product was characterized by HPLC, ESI-MS,

N-(4-Fluorophenyl)-6-(2-oxotetrahydrofuran-3-ylthio)-

nicotinamide (9). Prepared via method B using thionicotinamide 64

and α-bromo-γ-butyrolactone and puriﬁed by ﬂash silica gel

chromatography (2:3 ethyl acetate:hexanes) to yield 135 mg (41%)

of 9 as a white waxy solid. ESI-MS: m/z = 333.3 [M + H]⁺. ¹H NMR

(500 MHz, DMSO-d₆): δ 10.43 (s, 1H), 8.96 (s, 1H), 8.19 (dd, J = 8.3

Hz, 2.3 Hz, 1H), 7.80−7.77 (m, 2H), 7.57 (d, J = 8.4 Hz, 1H), 7.22 (t,

J = 8.8 Hz, 2H), 4.77 (t, J = 9.7 Hz, 1H), 4.50−4.46 (m, 1H), 4.40−

4.35 (m, 1H), 2.79−2.74 (m, 1H), 2.47−2.39 (m, 1H). Anal. Calcd for

C₁₆H₁₃FN₂O₃S: C, 57.82%; H, 3.94%; N, 8.43%. Found: C, 57.61%;

H, 4.04%; N, 8.14%. HPLC (gradient B): RT = 16.23 min, purity

99.8%.

1

and H NMR where indicated.

Method A. A solution of the bromomethyl derivative (2.5 equiv) in

anhydrous DMF (5 mL/mmol) was added to thionicotinamide 64 (1

equiv) and N-methylmorpholino-substituted polystyrene resin (5

equiv) and heated at 60 °C for 2 h in a screw cap glass vial.

Sulfhydryl-bearing scavenger resin (5 equiv) was then added to the

reaction mixture and heated at 60 °C for a further 4 h. After cooling,

the organic reaction solution was ﬁltered, and then diluted into water

(100 mL/mmol) to precipitate the product. The resulting suspension

was then centrifuged at 5000 rpm for 15 min, the aqueous supernatant

was decanted, and the product dried in a vacuum oven overnight at 50

°C. Compounds prepared by this method were used without further

puriﬁcation.

Method B. Thionicotinamide 64 (1 equiv) and the corresponding

bromomethyl or chloromethyl derivative (1 equiv) was dissolved in

anhydrous DMF (2 mL/mmol). To the solution, a tertiary amine base

(diisopropylethylamine, triethylamine, or N-methylmorpholine, 1

equiv) was added. The reaction was allowed to proceed at room

temperature and monitored by either TLC or LC−MS until complete

N-(4-Fluorophenyl)-6-(phenylsulfonylmethylthio)nicotinamide

(10). Prepared via method B using thionicotinamide 64 and

bromomethyl phenyl sulfone and puriﬁed by ﬂash silica gel

chromatography (step gradient of 1:2 to 2:3 ethyl acetate:hexanes)

to yield 159 mg (40%) of 10 as a white waxy solid. ESI-MS: m/z =

1

403.3 [M + H]⁺. H NMR (500 MHz, DMSO-d₆): δ 10.41 (s, 1H),

8.86 (s, 1H), 8.15 (dd, J = 8.2 Hz, 2.0 Hz, 1H), 7.92 (s, 1H), 7.90 (d, J

= 1.1 Hz, 1H), 7.80−7.77 (m, 2H), 7.69 (t, J = 7.3 Hz, 1H), 7.60 (t, J

= 7.6 Hz, 2H), 7.57 (d, J = 8.6 Hz, 1H), 7.22 (t, J = 8.8 Hz, 2H), 5.33

(s, 2H). Anal. Calcd for C₁₉H₁₅FN₂O₃S₂: C, 56.70%; H, 3.76%; N,

6.96%. Found: C, 56.20%; H, 3.97%; N, 6.51%. HPLC (gradient B):

RT = 19.06 min, purity 97.9%.

N-(4-Fluorophenyl)-6-(nitromethylthio)nicotinamide (11). Pre-

pared via method B using thionicotinamide 64 and bromonitro-

methane to yield 197 mg (51%) of 11 as an oﬀ-white solid. ESI-MS:

1

m/z = 308.1 [M + H]⁺. H NMR (500 MHz, DMSO-d₆): δ 10.44 (s,

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1H), 8.97 (s, 1H), 8.24 (d, J = 8.3 Hz, 7.78 (t, J = 7.1 Hz, 5.3 Hz, 2H),

7.71 (d, J = 8.5 Hz, 1H), 7.22 (t, J = 8.0 Hz, 8.4 Hz, 2H), 6.32 (s, 2H).

Purity determined by HPLC (gradient B): RT = 17.78 min, purity

88.4%.

(q, J = 7.0 Hz, 1H), 3.43−3.35 (m, 2H), 2.04−1.97 (m, 1H), 1.94−

1.78 (m, 2H), 1.67−1.60 (m, 1H). Anal. Calcd for C₁₇H₁₇FN₂O₂S: C,

61.43%; H, 5.16%; 8.43%. Found: C, 61.34%; H, 5.07%; N, 8.47%.

HPLC (gradient B): RT = 18.14 min, purity 95.6%.

Diethyl (5-(4-ﬂuorophenylcarbamoyl)pyridin-2-ylthio)-

methylphosphonate (12). The screened compound was prepared

via method B using thionicotinamide 64 and diethyl-(bromomethyl)-

phosphonate and puriﬁed by ﬂash silica gel chromatography (step

gradient of 1:1, 2:1, and 1:0 ethyl acetate:hexanes) to yield 138 mg

(34.7%) of 12 as a white waxy solid. ESI-MS: m/z = 399.4 [M + H]⁺.

¹H NMR (500 MHz, DMSO-d₆): δ 10.42 (s, 1H), 9.00 (s, 1H), 8.20

(dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.80−7.77 (m, 2H), 7.60 (d, J = 8.3 Hz,

1H), 7.22 (t, J = 8.8 Hz, 2H), 4.08−4.03 (m, 4H), 3.78 (d, J = 13.1 Hz,

2H), 1.21 (t, J = 6.7 Hz, 7.2 Hz, 6H). Anal. Calcd for C₁₇H₂₀FN₂O₄PS:

C, 51.25%; H, 5.06%; N, 7.03%. Found: C, 51.52%; H, 5.09%; N,

7.19%. HPLC (gradient B): RT = 17.56 min, purity 98.5%.

6-(2-(3,5-Dimethyl-1H-pyrazol-4-yl)ethylthio)-N-(4-ﬂuorophenyl)-

nicotinamide (21). Prepared via method A using thionicotinamide 64

and 4-(2-bromoethyl)-3,5-dimethyl-1H-pyrazole. ESI-MS: m/z =

371.1 [M + H]⁺. HPLC (gradient D): RT = 5.47 min, purity 81.0%.

N-(4-Fluorophenyl)-6-((5-nitrofuran-2-yl)methylthio)-

nicotinamide (22). The screened compound was prepared via method

B using thionicotinamide 64 and 2-(bromomethyl)-5-(nitro)furan and

puriﬁed by ﬂash silica gel chromatography (1:2 ethyl acetate:hexanes)

to yield 91 mg (24%) of 22 as an orange solid. ESI-MS: m/z = 374.1

[M + H]⁺. ¹H NMR (500 MHz, DMSO-d₆): δ 10.42 (s, 1H), 9.02 (d,

J = 2.0 Hz, 1H), 8.20 (dd, J = 8.3 Hz, 2.2 Hz, 1H), 7.80−7.77 (m, 2H),

7.64 (d, J = 3.6 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.23−7.20 (m, 2H),

6.79 (d, J = 3.8 Hz, 1H), 4.71 (s, 2H). Anal. Calcd for C₁₇H₁₂FN₃O₄S:

C, 54.69%; H, 3.24%; N, 11.25%. Found: C, 55.20%; H, 3.24%; N,

11.01%. HPLC (gradient B): RT = 20.13 min, purity 91.1%.

N-(4-Fluorophenyl)-6-((5-methylisoxazol-3-yl)methylthio)-

nicotinamide (23). Prepared via method A using thionicotinamide 64

and 3-(bromomethyl)-5-methylisoxazole. ESI-MS: m/z = 344.1 [M +

H]⁺. HPLC (gradient C): RT = 6.21 min, purity 92.9%.

N-(4-Fluorophenyl)-6-((5-methyl-2-phenyl-2H-1,2,3-triazol-4-yl)-

methylthio)nicotinamide (24). Prepared via method A using

thionicotinamide 64 and 4-(bromomethyl)-5-methyl-2-phenyl-2H-

1,2,3-triazole. ESI-MS: m/z = 420.1 [M + H]⁺. HPLC (gradient C):

RT = 7.03 min, purity 91.7%.

N-(4-Fluorophenyl)-6-((3-methyl-5-phenylisoxazol-4-yl)-

methylthio)nicotinamide (25). Prepared via method A using

thionicotinamide 64 and 4-(bromomethyl)-3-methyl-5-phenylisoxa-

zole. ESI-MS: m/z = 420.1 [M + H]⁺. HPLC (gradient C): RT = 6.81

min, purity 85.8%.

N-(4-Fluorophenyl)-6-((5-methyl-3-phenylisoxazol-4-yl)-

methylthio)nicotinamide (26). Prepared via method A using

thionicotinamide 64 and 4-(bromomethyl)-5-methyl-3-phenylisoxa-

zole. ESI-MS: m/z = 420.1 [M + H]⁺. HPLC (gradient B): RT = 24.01

min, purity 72.0%.

N-(4-ﬂuorophenyl)-6-((4-methyl-2-(4-(triﬂuoromethyl)phenyl)-

thiazol-5-yl)methylthio)nicotinamide (27). Prepared via method A

using thionicotinamide 64 and 5-(bromomethyl)-4-methyl-2-[4-

(triﬂuoromethyl)phenyl]-1,3-thiazole. ESI-MS: m/z = 504.1 [M +

H]⁺. HPLC (gradient D): RT = 6.41 min, purity 86.9%.

N-(4-Fluorophenyl)-6-((4-methyl-2-phenylthiazol-5-yl)-

methylthio)nicotinamide (28). Prepared via method A using

thionicotinamide 64 and 5-(bromomethyl)-4-methyl-2-[4-phenyl]-

1,3-thiazole. ESI-MS: m/z = 436.1 [M + H]⁺. HPLC (gradient C):

RT = 6.85 min, purity 70.7%.

N-(4-Fluorophenyl)-6-((tetrahydro-2H-pyran-2-yl)methylthio)-

nicotinamide (13). Prepared via method B using thionicotinamide 64

and 2-(bromomethyl)-tetrahydropyran to yield 143 mg (41%) of 13 as

1

an oﬀ-white solid. ESI-MS: m/z = 346.9 [M + H]⁺. H NMR (500

MHz, DMSO-d₆): δ 10.37 (s, 1H), 8.97 (s, 1H), 8.13 (d, J = 8.2 Hz,

1H), 7.80−7.77 (m, 2H), 7.47 (d, J = 8.1 Hz, 1H), 7.21 (t, J = 7.8 Hz,

8.4 Hz, 2H), 3.90 (d, J = 11.1 Hz, 1H), 3.49−3.46 (m, 1H), 3.40−3.35

(m, 2H), 3.28−3.24 (m, 1H), 1.78−1.72 (m, 2H), 1.45 (broad m,

3H), 1.33−1.27 (m, 1H). Anal. Calcd for C₁₈H₁₉FN₂O₂S: C, 62.41%;

H, 5.53%; N, 8.09%. Found: C, 62.37%; H, 5.49%; N, 8.20%. HPLC

(gradient B): RT = 19.80 min, purity 95.5%.

N-(4-Fluorophenyl)-6-(4-(2-methylthiazol-4-yl)benzylthio)-

nicotinamide (14). Prepared via method A using thionicotinamide 64

and 4-[4-(bromomethyl)phenyl]-2-methyl-1,3-thiazole. ESI-MS: m/z

= 436.1 [M + H]⁺. HPLC (gradient C): RT = 6.66 min, purity 71.1%.

N-(4-Fluorophenyl)-6-(pyridin-4-ylmethylthio)nicotinamide (15).

Prepared via method B using thionicotinamide 64 and 4-

(bromomethyl)pyridine to yield 146 mg (43%) of 15 as a white

1

solid. ESI-MS: m/z = 340.4 [M + H]⁺. H NMR (500 MHz, DMSO-

d₆): δ 10.39 (s, 1H), 8.98 (s, 1H), 8.53 (d, J = 3.9 Hz, 1H), 8.16 (d, J =

8.3 Hz, 1H), 7.79−7.75 (m, 3H), 7.54−7.53 (m, 2H), 7.29 (t, J = 2.9

Hz, 5.5 Hz, 1H), 7.21 (t, J = 8.2 Hz, 8.8 Hz, 2H), 4.61 (s, 2H). Anal.

Calcd for C₁₈H₁₄FN₃OS: C, 63.70%; H, 4.16%; N, 12.38%. Found: C,

63.28%; H, 3.90%; N, 12.11%. HPLC (gradient B): RT = 12.63 min,

purity 93.7%.

Benzyl 4-((5-(4-Fluorophenylcarbamoyl)pyridin-2-ylthio)methyl)-

piperidine-1-carboxylate (16). Prepared via method A using

thionicotinamide 64 and benzyl 4-(bromomethyl)tetrahydro-1-(2H)-

pyridinecarboxylate. ESI-MS: m/z = 495.0 [M + H]⁺. HPLC (gradient

D): RT = 5.90 min, purity 89.1%.

N-(4-Fluorophenyl)-6-((tetrahydro-2H-pyran-4-yl)methylthio)-

nicotinamide (17). Prepared via method B using thionicotinamide 64

and 4-(bromomethyl)-tetrahydropyran to yield 286 mg (83%) of 16 as

6-(2-(3,5-Dimethyl-1H-pyrazol-4-yl)ethylthio)-N-(4-ﬂuorophenyl)-

nicotinamide (29). Prepared via method A using thionicotinamide 64

and 4-(2-bromoethyl)-3,5-dimethyl-1H-pyrazole. ESI-MS: m/z =

371.1 [M + H]⁺. HPLC (gradient D): RT = 5.47 min, purity 81.0%.

6-(Benzylthio)-N-(4-ﬂuorophenyl)nicotinamide (30). Prepared via

method C using thionicotinamide 64 and benzyl bromide to yield 108

mg (64%) of 30 as an oﬀ-white powder. ESI-MS: m/z = 338.9 [M +

1

a white solid. ESI-MS: m/z = 347.1 [M + H]⁺. H NMR (500 MHz,

DMSO-d₆): δ 10.38 (s, 1H), 8.98 (s, 1H), 8.13 (d, J = 8.5 Hz, 1H),

7.80−7.77 (m, 2H), 7.48 (d, J = 8.3 Hz, 1H), 7.21 (t, J = 8.7 Hz, 9.1

Hz, 2H), 3.86−3.84 (m, 2H), 3.26 (t, J = 11.4 Hz, 2H), 3.20 (d, J = 6.7

Hz, 2H), 1.83−1.80 (m, 1H), 1.73 (d, J = 12.3 Hz, 2H), 1.33−1.23

(m, 2H). HPLC (gradient B): RT = 18.64 min, purity 89.1%.

N-(4-Fluorophenyl)-6-(4-(5-methyl-1,2,4-oxadiazol-3-yl)-

benzylthio)nicotinamide (18). Prepared via method A using

thionicotinamide 64 and 3-[3-(bromomethyl)phenyl]-5-methyl-1,2,4-

oxadiazole. ESI-MS: m/z = 421.1 [M + H]⁺. HPLC (gradient C): RT

= 6.63 min, purity 80.7%.

1

H]⁺. H NMR (500 MHz, DMSO-d₆): δ 10.39 (s, 1H), 9.00 (s, 1H),

8.15 (d, J = 8.0 Hz, 1H), 7.80−7.77 (m, 2H), 7.49−7.45 (m, 3 H),

7.34 (t, J = 7.7 Hz, 6.9 Hz, 2H), 7.27−7.20 (m, 3H), 4.51 (s, 2H).

Anal. Calcd for C₁₉H₁₅FN₂OS: C, 67.44%; H, 4.47%; N, 8.28%.

Found: C, 67.17%; H, 4.37%; N, 8.13%. HPLC (gradient B): RT =

21.66 min, purity 96.5%.

N-(4-Fluorophenyl)-6-(4-nitrobenzylthio)nicotinamide (31). Pre-

pared via method A using thionicotinamide 64 and 1-(bromomethyl)-

4-nitrobenzene. ESI-MS: m/z = 384.1 [M + H]⁺. HPLC (gradient C):

RT = 6.63 min, purity 66.3%.

N-(4-Fluorophenyl)-6-(4-(triﬂuoromethoxy)benzylthio)-

nicotinamide (32). Prepared via method A using thionicotinamide 64

and 1-(bromomethyl)-4-(triﬂuoromethoxy)benzene. ESI-MS: m/z =

423.1 [M + H]⁺. HPLC (gradient C): RT = 7.12 min, purity 83.6%.

N-(4-Fluorophenyl)-6-(3-(triﬂuoromethoxy)benzylthio)-

nicotinamide (33). Prepared via method A using thionicotinamide 64

N-(4-Fluorophenyl)-6-((5-(triﬂuoromethyl)furan-2-yl)methylthio)-

nicotinamide (19). Prepared via method A using thionicotinamide 64

and 2-(bromomethyl)-5-(triﬂuoromethyl)furan. ESI-MS: m/z = 397.1

[M + H]⁺. HPLC (gradient C): RT = 6.82 min, purity 82.0%.

N-(4-Fluorophenyl)-6-((tetrahydrofuran-2-yl)methylthio)-

nicotinamide (20). Prepared via method C using thionicotinamide 64

and 2-(bromomethyl)tetrahydrofuran to yield 140 mg (41%) of 20 as

1

a white solid. ESI-MS: m/z = 333.1 [M + H]⁺. H NMR (500 MHz,

DMSO-d₆): δ 10.38 (s, 1H), 8.97 (s, 1H), 8.13 (d, J = 8.0 Hz, 1H),

7.78 (t, J = 6.1 Hz, 6.8 Hz, 2H), 7.49 (d, J = 8.3 Hz, 1H), 7.21 (t, J =

8.3 Hz, 8.6 Hz, 2H), 4.08−4.04 (m, 1H), 3.81 (q, J = 7.0 Hz, 1H), 3.66

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and 1-(bromomethyl)-3-(triﬂuoromethoxy)benzene. ESI-MS: m/z =

423.1 [M + H]⁺. HPLC (gradient C): RT = 7.12 min, purity 86.3%.

N-(4-Fluorophenyl)-6-(2-(thiophen-2-yl)benzylthio)nicotinamide

(34). Prepared via method A using thionicotinamide 64 and 2-[2-

(bromomethyl)phenyl]thiophene and screened without puriﬁcation.

ESI-MS: m/z = 421.1 [M + H]⁺. HPLC (gradient C): RT = 7.31 min,

purity 73.7%.

N-(4-Fluorophenyl)-6-(4-(2-methylthiazol-4-yl)benzylthio)-

nicotinamide (35). Prepared via method A using thionicotinamide 64

and 4-[4-(bromomethyl)phenyl]-2-methyl-1,3-thiazole. ESI-MS: m/z

= 436.1 [M + H]⁺. HPLC (gradient C): RT = 6.66 min, purity 71.1%.

6-(4-(1H-1,2,4-Triazol-1-yl)benzylthio)-N-(4-ﬂuorophenyl)-

nicotinamide (36). Prepared via method A using thionicotinamide 64

and 1-[4-(bromomethyl)phenyl]-1H-1,2,4-triazole. ESI-MS: m/z =

406.1 [M + H]⁺. HPLC (gradient C): RT = 6.06 min, purity 85.3%.

N-(4-Fluorophenyl)-6-(3-(5-methyl-1,2,4-oxadiazol-3-yl)-

benzylthio)nicotinamide (37). Prepared via method A using

thionicotinamide 64 and 3-[3-(bromomethyl)phenyl]-5-methyl-1,2,4-

oxadiazole. ESI-MS: m/z = 421.1 [M + H]⁺. HPLC (gradient C): RT

= 6.63 min, purity 80.7%.

6-(4-(1H-Pyrazol-1-yl)benzylthio)-N-(4-ﬂuorophenyl)-

nicotinamide (38). Prepared via method A using thionicotinamide 64

and 1-[4-(bromomethyl)phenyl]-1H-pyrazole. ESI-MS: m/z = 405.1

[M + H]⁺. HPLC (gradient C): RT = 6.54 min, purity 92.6%.

N-(4-Fluorophenyl)-6-(3-(2-methylthiazol-4-yl)benzylthio)-

nicotinamide (39). Prepared via method A using thionicotinamide 64

and 4-[3-(bromomethyl)phenyl]-2-methyl-1,3-thiazole. ESI-MS: m/z

= 436.1 [M + H]⁺. HPLC (gradient C): RT = 6.68 min, purity 69.2%.

6-(4-(1H-Pyrrol-1-yl)benzylthio)-N-(4-ﬂuorophenyl)nicotinamide

(40). Prepared via method A using thionicotinamide 64 and 1-[4-

(bromomethyl)phenyl]-1H-pyrrole. ESI-MS: m/z = 404.1 [M + H]⁺.

HPLC (gradient C): RT = 7.03 min, purity 70.7%.

(m, 2H), 7.11−7.07 (m, 1H), 6.85 (dd, J = 8.1 Hz, 0.8 Hz, 1H), 6.75−

6.72 (m, 1H), 4.42 (s, 2H). Anal. Calcd for C₁₉H₁₅FN₂O₂S: C,

64.39%; H, 4.27%; N, 7.90%. Found: C, 64.50%; H, 4.47%; N, 7.72%.

HRMS: calcd for C₁₉H₁₆FN₂O₂S [M + 1]⁺355.0911, found 355.0901;

calcd for C₁₉H₁₅FN₂NaO₂S [M + Na]⁺377.0730, found 377.0718.

HPLC (gradient B): RT = 20.18 min, purity 97.4%.

N-(4-Fluorophenyl)-6-(phenylthio)nicotinamide (45). Chloronico-

tinamide 69 (200 mg, 0.75 mmol), thiophenol (77 μL, 0.75 mmol),

potassium tert-butoxide (112 mg, 1.5 mmol) and 18-crown-6 (10 mg),

was dissolved in NMP (3 mL) and heated to 160 °C for 3 h. The

reaction mixture was diluted with water and extracted with ethyl

acetate. The organic layer was dried over Na₂SO₄and puriﬁed using

ﬂash chromatography (7:3 hexanes/ethyl acetate) to yield 99 mg

(31%) of 45 as an oﬀ-white powder. TLC (ethyl acetate/hexane, 1:1):

1

R_f= 0.47. ESI-MS: m/z = 325.1 [M + H]⁺. H NMR (500 MHz,

DMSO-d₆): δ 10.35 (s, 1H), 8.88 (d, J = 2.3 Hz, 1H), 8.08 (dd, J = 8.4

Hz, 2.3 Hz, 1H), 7.74−7.71 (m, 2H), 7.62−7.60 (m, 2H), 7.53−7.50

(m, 3H), 7.18 (t, J = 2.3 Hz, 2H), 7.02 (d, J = 8.5 Hz, 1H).

Methyl 4-((5-(4-Fluorophenylcarbamoyl)pyridin-2-ylthio)-

methyl)benzoate (46). Prepared via method B using thionicotinamide

64 and methyl 4-bromomethylbenzoate to yield 343 mg (87%) of 46

1

as an oﬀ-white solid. ESI-MS: m/z = 397.1 [M + H]⁺. H NMR (500

MHz, DMSO-d₆): δ 10.38 (s, 1H), 9.00 (d, J = 1.6 Hz, 1H), 8.15 (dd,

J = 8.6 Hz, 2.5 Hz, 1H), 7.92 (d, J = 8.3 Hz, 2H), 7.79−7.76 (m, 2H),

7.61 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.2 Hz, 1H), 7.21 (t, J = 9.0 Hz,

2H), 4.59 (s, 2H), 3.84 (s, 3H). Anal. Calcd for C₂₁H₁₇FN₂O₃S: C,

63.63%; H, 4.32%; N, 7.07%. Found: C, 63.38%; H, 4.22%; N, 7.23%.

Purity determined by HPLC (gradient B): RT = 21.59 min, purity

97.5%.

4-((5-(4-Fluorophenylcarbamoyl)pyridin-2-ylthio)methyl)benzoic

acid (47). The thionicotinamide methyl ester 46 (140 mg, 0.35 mmol)

was saponiﬁed using 1 N NaOH (1.1 equiv) in MeOH (5 mL) at

reﬂux for 1 h. The mixture was acidiﬁed using 1 N HCl and extracted

into ethyl acetate. The organic layer was washed with saturated NaCl,

dried over Na₂SO₄, ﬁltered, and evaporated to yield 81 mg (83%) of

compound 47 as a white solid. TLC (ethyl acetate/hexane 1:1): R_f=

6-(3-(1H-pyrrol-1-yl)benzylthio)-N-(4-ﬂuorophenyl)nicotinamide

(41). Prepared via method A using thionicotinamide 64 and 1-[3-

(bromomethyl)phenyl]-1H-pyrrole. ESI-MS: m/z = 404.1 [M + H]⁺.

HPLC (gradient C): RT = 6.98 min, purity 91.8%.

6-(2-Fluoro-3-methylbenzylthio)-N-(4-ﬂuorophenyl)nicotinamide

(42). Prepared via method A using thionicotinamide 64 and 1-

(bromomethyl)-2-ﬂuoro-3-methylbenzene. ESI-MS: m/z = 371.1 [M

+ H]⁺. HPLC (gradient C): RT = 6.97 min, purity 83.5%.

1

0.23. ESI-MS: m/z = 383.0 [M + H]⁺. H NMR (500 MHz, DMSO-

d₆): δ 10.55 (s, 1H), 9.01 (d, J = 1.6 Hz, 1H), 8.15 (dd, J = 8.6 Hz, 2.5

Hz, 1H), 7.92 (d, J = 8.3 Hz, 2H), 7.79−7.76 (m, 2H), 7.61 (d, J = 8.3

Hz, 2H), 7.50 (d, J = 8.2 Hz, 1H), 7.21 (t, J = 9.0 Hz, 2H), 4.59 (s,

2H), 3.84 (s, 3H).

6-(3,4-Dichlorobenzylthio)-N-(4-ﬂuorophenyl)nicotinamide (43).

Prepared via method B using thionicotinamide 64 and 1-

(bromomethyl)-3,4-dichlorobenzene. The crude material was puriﬁed

by ﬂash chromatography using a gradient elution of 10% ethyl acetate/

hexanes to 95% ethyl acetate/hexanes over 30 min to yield 355 mg

(83%) of compound 43 as a white solid. TLC (ethyl acetate/hexane,

1:4): R_f= 0.27. ESI-MS: m/z = 407.0, 409.0 [M + H]⁺. ¹H NMR (400

MHz, DMSO-d₆): δ 10.35 (s, 1H), 8.96 (s, 1H), 8.12−8.10 (m, 1H),

7.75−7.72 (m, 2H), 7.69 (s, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.50 (d, J =

12.9 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 7.17 (t, J = 8.9 Hz, 2H), 4.47

(s, 2H). Purity determined by HPLC (gradient B): RT = 26.18 min,

purity 71.2%.

N-(4-Fluorophenyl)-6-(2-hydroxybenzylthio)nicotinamide (44).

The boronic acid 6 (200 mg) was dissolved in 18 mL of THF/

acetone/0.1 N NaOH (1:1:1) and cooled to −3 °C. OXONE (1.0 eq,

311 mg) dissolved in 0.4 mM EDTA (2.1 mL) was added, according

to the procedure of Webb and Levy for the hydroxylation of aryl

boronic esters or boronic acids.³⁶The reaction was then stirred for 1 h

and quenched with sodium bisulﬁte (3 mL, 5 M aqueous), while being

stirred for 10 min. The solvent was removed by reduced pressure

rotary evaporation, leaving an aqueous suspension. The suspension

was extracted 3× with ethyl acetate, and the combined ethyl acetate

layers were washed with 0.01 N HCl, water, 0.01 N NaOH, and brine.

They were dried over MgSO₄, ﬁltered, and dried under vacuum. The

residue was dissolved in THF/MeOH and adhered to 2 g of silica gel.

The adhered silica was loaded onto 20 g of silica gel, eluting the

product with 3:1 hexanes:ethyl acetate, which was dried under vacuum

to provide 67 mg of compound 44 (36% yield). ESI-MS: m/z = 355.4

[M + H]⁺. ¹H NMR (500 MHz, DMSO-d₆): δ 9.75 (s, 1H), 9.01 (d, J

= 2.1 Hz, 1H), 8.14 (dd, J = 8.7 Hz, 2.6 Hz, 1H), 7.80−7.77 (m, 3H),

7.48 (d, J = 8.3 Hz, 1H), 7.33 (dd, J = 7.4 Hz, 1.3 Hz, 1H), 7.23−7.20

Methyl 3-((5-(4-Fluorophenylcarbamoyl)pyridin-2-ylthio)-

methyl)benzoate (48). Prepared via method B using thionicotinamide

64 and methyl 3-bromomethylbenzoate to yield 180 mg (45%) of 48

as a crystalline material. An analytical sample was recrystallized from

1

hot ethyl acetate. ESI-MS: m/z = 397.1 [M + H]⁺. H NMR (500

MHz, DMSO-d₆): δ 10.40 (s, 1H), 8.99 (d, J = 1.4 Hz, 1H), 8.15 (dd,

J = 8.6 Hz, 2.2 Hz, 1H), 8.07 (s, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.79−

7.74 (m, 3H), 7.51−7.47 (m, 2H), 7.21 (t, J = 8.8 Hz, 2H), 4.59 (s,

2H), 3.85 (s, 3H). Anal. Calcd for C₂₁H₁₇FN₂O₃S: C, 63.63%; H,

4.32%; N, 7.07%. Found: C, 63.53%; H, 4.18%; N, 7.13%. Purity

determined by HPLC (gradient B): RT = 21.57 min, purity 82.8%.

3-((5-(4-Fluorophenylcarbamoyl)pyridin-2-ylthio)methyl)benzoic

acid (49). The thionicotinamide methyl ester 48 (100 mg, 0.25 mmol)

was saponiﬁed using 1 N NaOH (1.1 equiv) in MeOH at room

temperature for 1 h. The mixture was acidiﬁed using 1 N HCl and

extracted into ethyl acetate. The organic layer was washed with

saturated NaCl, dried over Na₂SO₄, ﬁltered and evaporated to yield 81

mg (83%) of compound 49 as a white solid. ESI-MS: m/z = 382.9 [M

+ H]⁺. ¹H NMR (500 MHz, DMSO-d₆): δ 13.00 (br s, 1H), 10.39 (s,

1H), 9.00 (s, 1H), 8.16 (d, J = 8.6 Hz, 1H), 8.05 (s, 1H), 7.84 (d, J =

7.9 Hz, 1H), 7.78 (t, J = 7.3 Hz, 4.8 Hz, 2H), 7.71 (d, J = 7.5 Hz, 1H),

7.51 (d, J = 8.5 Hz, 1 H), 7.46 (t, J = 7.7 Hz, 7.5 Hz, 1H), 7.21 (t, J =

7.5 Hz, 8.8 Hz, 2H), 4.59 (s, 2H). Anal. Calcd for C₂₀H₁₅FN₂O₃S: C,

62.82%; H, 3.95%; N, 7.33%. Found: C, 62.53%; H, 3.82%; N, 7.28%.

Purity determined by HPLC (gradient B): RT = 19.11 min, purity

97.6%.

Methyl 2-((5-(4-Fluorophenylcarbamoyl)pyridin-2-ylthio)-

methyl)benzoate (50). Prepared via method B using thionicotinamide

64 and methyl 2-bromomethylbenzoate to yield 300 mg (76%) of 50

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as a white solid. ESI-MS: m/z = 397.1 [M + H]⁺. ¹H NMR (500 MHz,

DMSO-d₆): δ 10.40 (s, 1H), 9.00 (s, 1H), 8.13 (dd, J = 8.5 Hz, 1.8 Hz,

1H), 7.88 (d, J = 7.4 Hz, 1H), 7.80−7.77 (m, 2H), 7.66 (d, J = 7.9 Hz,

1H), 7.54 (t, J = 7.5 Hz, 1H), 7.46 (d, J = 8.6 Hz, 1H), 7.41 (t, J = 7.6

Hz, 7.1 Hz, 1H), 7.22 (t, J = 8.7 Hz, 9.0 Hz, 2H), 4.84 (s, 2H), 3.87 (s,

3H). HRMS: calcd for C₂₁H₁₈FN₂O₃S [M + 1]+ 397.1017, found

397.1006; calcd for C₂₁H₁₇FN₂NaO₃S [M + Na]+ 419.0836, found

419.0822. Purity determined by HPLC (gradient A): RT = 10.86 min,

purity 95.2%.

2-((5-(4-Fluorophenylcarbamoyl)pyridin-2-ylthio)methyl)benzoic

acid (51). The thionicotinamide methyl ester 50 (100 mg, 0.25 mmol)

was saponiﬁed using 1 N NaOH (1.55 mmol, 6.2 equiv) in MeOH at

room temperature for 1 h. The mixture was acidiﬁed using 1 N HCl

and extracted into ethyl acetate. The organic layer was washed with

saturated NaCl, dried over Na₂SO₄, ﬁltered and evaporated to yield

37.7 mg (40%) of compound 51 as a white solid. ESI-MS: m/z = 382.9

[M + H]⁺. ¹H NMR (500 MHz, DMSO-d₆): δ 13.14 (s, 1H), 10.40 (s,

1H), 9.01 (s, 1H), 8.13 (dd, J = 8.7 Hz, 2.1 Hz, 1H), 7.90 (d, J = 7.5

Hz, 1H), 7.80−7.77 (m, 2H), 7.64 (d, J = 7.5 Hz, 1H), 7.50 (t, J = 6.9

Hz, 7.3 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.38 (t, J = 7.3 Hz, 1H),

7.22 (t, J = 8.8 Hz, 2H), 4.87 (s, 2H). HRMS: calcd for C₂₀H₁₆FN₂O₃S

[M + 1]+ 383.0860, found 383.0852; calcd for C₂₀H₁₅FN₂NaO₃S [M

+ Na]+ 405.0680, found 405.0667. Purity determined by HPLC

(gradient B): RT = 19.28 min, purity 98.3%.

6-(2-(2H-Tetrazol-5-yl)benzylthio)-N-(4-ﬂuorophenyl)-

nicotinamide (52). The cyanobenzyl intermediate 66 (363 mg, 1

mmol) was suspended in anhydrous toluene (25 mL). Dibutyltin oxide

(25 mg, 0.1 mmol) was added, followed by trimethylsilyl azide (131

μL, 1 mmol). The mixture was then heated to reﬂux for 18 h. The dark

yellow brown solution was allowed to cool to room temperature, at

which time a brown precipitate was seen to form. The precipitate was

ﬁltered and washed with toluene to yield 190 mg (47%) of compound

52. ESI-MS: m/z = 407.2 [M + H]⁺. ¹H NMR (500 MHz, DMSO-d₆):

δ 10.39 (s, 1H), 8.93 (s, 1H), 8.11 (dd, J = 8.6 Hz, 1.6 Hz, 1H), 7.80−

7.75 (m, 4H), 7.55−7.49 (m, 2H), 7.42 (d, J = 8.3 Hz, 1H), 7.22 (t, J =

8.6 Hz, 2H), 4.90 (s, 2H). HRMS: calcd for C₂₀H₁₆FN₆OS [M + 1]+

407.1085, found 407.1078; calcd for C₂₀H₁₅FN₆NaOS [M + Na]⁺

429.0904, found 429.0896. Purity determined by HPLC (gradient B):

RT = 18.81 min, purity 94.6%.

N-(4-Fluorophenyl)-6-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)benzylthio)nicotinamide (53). Prepared via method C using

thionicotinamide 64 and 2-bromomethyl-phenylboronic acid pinacol

ester, and puriﬁed by ﬂash silica gel chromatography (step gradient of

1:6 and 1:3 ethyl acetate:hexanes) to yield 197 mg (42%) of 53 as an

oﬀ-white hygroscopic solid. ESI-MS: m/z = 465.4 [M + H]⁺. ¹H NMR

(500 MHz, DMSO-d₆): δ 10.39 (s, 1H), 9.01 (s, 1H), 8.14 (d, J = 8.7

Hz, 1H), 7.80−7.77 (m, 2H), 7.68 (d, J = 7.2 Hz, 1H), 7.51 (d, J = 7.6

Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.26 (t, J =

7.1 Hz, 7.5 Hz, 1H), 7.21 (t, J = 8.8 Hz, 2H), 4.73 (s, 2H), 1.29 (s,

12H). Anal. Calcd for C₂₅H₂₆BFN₂O₃S: C, 64.66%; H, 5.64%; N,

6.03%. Found: C, 64.45%; H, 5.60%; N, 5.83%. Purity determined by

HPLC (gradient B): RT = 24.93 min, purity 95.7%.

4-(6-(2-Boronobenzylthio)nicotinamido)benzoic acid (55). Fol-

lowing the same procedure described for 54 and starting from

intermediate 68 (0.5 mmol) and 4-aminobenzoic acid (70 mg, 0.5

mmol, 1 equiv), and puriﬁed using preparative HPLC (60:40:0.1

water:acetonitrile:formic acid isocratic) to yield 23 mg (11%) of

1

compound 55 as a white solid. ESI-MS: m/z = 409.2 [M + H]⁺. H

NMR (500 MHz, DMSO-d₆): δ 12.79 (s, 1H), 10.61 (s, 1H), 9.00 (s,

1H), 8.23 (s, 2H), 8.15 (dd, J = 8.2 Hz, 1.7 Hz, 1H), 7.97−7.90 (m,

4H), 7.54 (d, J = 6.8 Hz, 1H), 7.47−7.44 (m, 2H), 7.30 (t, J = 6.9 Hz,

7.2 Hz, 1H), 7.22 (t, J = 8.3 Hz, 7.1 Hz, 1H), 4.69 (s, 2H). HRMS:

calcd for C₂₀H₁₈BN₂O₅S [M + 1]+ 409.1024, found 409.1018; calcd

for C₂₀H₁₇BN₂NaO₅S [M + Na]+ 431.0843, found 431.0837. Purity

determined by HPLC (gradient B): RT = 16.61 min, purity 98.4%.

2-((5-(4-(2H-Tetrazol-5-yl)phenylcarbamoyl)pyridin-2-ylthio)-

methyl)phenylboronic acid (56). Following the same procedure

described for 54 and starting from intermediate 68 (0.5 mmol) and

the 4-tetrazole-aniline (80 mg, 0.5 mmol, Santa Cruz Biotechnology,

Dallas, TX), the crude material after workup was further puriﬁed using

preparative HPLC (60:40:0.1 water:acetonitrile:formic acid isocratic)

to yield 38 mg (18%) of compound 56 as a white solid. ESI-MS: m/z

1

= 433.2 [M + H]⁺. H NMR (500 MHz, DMSO-d₆) 10.63 (s, 1H),

9.02 (s, 1H), 8.23 (s, 1H), 8.16 (dd, J = 8.6 Hz, 1.8 Hz, 1H), 8.06−

8.00 (m, 4H), 7.55 (d, J = 6.9 Hz, 1H), 7.46 (t, J = 8.5 Hz, 7.7 Hz,

2H), 7.31 (t, J = 7.3 Hz, 1H), 7.22 (t, J = 7.2 Hz, 1H), 4.70 (s, 2H).

HRMS: calcd for C₂₀H₁₈BN₆O₃S [M + 1]⁺433.1249, found 433.1239.

HPLC (gradient B): RT = 16.29 min, purity 100.0%.

2-((5-(Pyridin-4-ylcarbamoyl)pyridin-2-ylthio)methyl)-

phenylboronic acid (57). Following the same procedure described for

54 and starting from intermediate 68 (0.33 mmol) and 4-amino-

pyridine (31 mg, 0.33 mmol), and puriﬁed by preparative HPLC

(95:5:0.1 water:acetonitrile:formic acid to 5:95:0.1 water:acetonitrile:-

formic acid over 30 min) to yield 18 mg (21%) of compound 57 as a

1

white solid. ESI-MS: m/z = 366.2 [M + H]⁺. H NMR (500 MHz,

DMSO-d₆): δ 10.68 (s, 1H), 9.01 (d, J = 1.7 Hz, 1H), 8.51 (d, J = 5.9

Hz, 2H), 8.24 (s, 2H), 8.15 (dd, J = 8.4 Hz, 2.4 Hz, 1H), 8.01−7.95

(m, 1H), 7.79 (dd, J = 5.0 Hz, 1.3 Hz, 2H), 7.48−7.43 (m, 2H), 7.32−

7.23 (m, 2H), 4.70 (s, 2H). HPLC (gradient B): RT = 10.79 min,

purity 95.7%.

2-((5-(4-Hydroxyphenylcarbamoyl)pyridin-2-ylthio)methyl)-

phenylboronic acid (58). Following the same procedure described for

54 and starting from intermediate 68 (0.33 mmol) and 4-hydroxy-

aniline (36 mg, 0.33 mmol), the crude material after workup was

further puriﬁed using preparative HPLC (60:40:0.1 water:acetoni-

trile:formic acid isocratic) to yield 21 mg (17%) of compound 58 as a

1

white solid. ESI-MS: m/z = 381.3 [M + H]⁺. H NMR (500 MHz,

DMSO-d₆): δ 10.11 (s, 1H), 9.30 (s, 1H), 8.96 (s, 1H), 8.23 (s, 1H),

8.11 (dd, J = 8.2 Hz, 1.6 Hz, 1H), 7.54−7.52 (m, 3H), 7.45−7.39 (m,

2H), 7.30 (t, J = 6.0 Hz, 7.5 Hz, 1H), 7.22 (t, J = 7.2 Hz, 1H), 6.76 (d,

J = 8.9 Hz, 2H), 4.68 (s, 2H). HRMS: calcd for C₁₉H₁₈BN₂O₄S [M +

1]⁺381.1075, found 381.1093; calcd for C₁₉H₁₇BN₂NaO₄S [M + Na]⁺

403.0894, found 403.0910. HPLC (gradient B): RT = 15.40 min,

purity 98.8%.

2-((5-(5-Fluoropyridin-2-ylcarbamoyl)pyridin-2-ylthio)methyl)-

phenylboronic acid (59). Following the same procedure described for

54 and starting from intermediate 68 (0.22 mmol) and 2-amino-5-

ﬂuoropyridine (25 mg, 0.22 mmol), the crude material after workup

was further puriﬁed using preparative HPLC (60:40:0.1 water:-

acetonitrile:formic acid isocratic) to yield 17 mg (20%) of compound

2-((5-(4-(Triﬂuoromethoxy)phenylcarbamoyl)pyridin-2-ylthio)-

methyl)phenylboronic acid (54). Intermediate 68 (0.5 mmol) was

reacted with 4-triﬂuoromethoxy aniline (177 mg, 0.5 mmol, 1 equiv)

and TEA (70 mL, 0.5 mmol, 1 equiv) in anhydrous DMF. The

reaction mixture was heated to 60 °C for 16 h. The crude material was

diluted with ethyl acetate and the organic layer washed with water and

saturated NaHCO₃. The organic layer was dried over Na₂SO₄, ﬁltered,

and evaporated. The crude was puriﬁed using ﬂash silica gel

chromatography (ethyl acetate/hexanes 1:2 as eluent) to provide 53

1

59 as a white solid. ESI-MS: m/z = 383.9 [M + H]⁺. H NMR (500

MHz, DMSO-d₆): δ 11.08 (s, 1H), 9.02 (s, 1H), 8.42 (s, 1H), 8.23−

8.16 (m, 4H), 7.82 (t, J = 8.8 Hz, 8.6 Hz, 1H), 7.54 (d, J = 7.3 Hz,

1H), 7.43 (t, J = 9.3 Hz, 10.5 Hz, 2H), 7.30 (t, J = 7.0 Hz, 7.8 Hz, 1H),

7.22 (t, J = 7.4 Hz, 7.2 Hz, 1H), 4.68 (s, 2H). HPLC (gradient B): RT

= 17.24 min, purity 97.8%.

2-((5-((4-Fluorophenyl)(pyridin-2-ylmethyl)carbamoyl)pyridin-2-

ylthio)methyl)phenylboronic acid (60). According to method C, N-

(4-ﬂuorophenyl)-N-pyridin-2-yl-methyl-6-mercapto-nicotinamide in-

termediate 73a (2.9 g, 8.7 mmol) and 2-bromomethyl-phenylboronic

acid (2.9 g, 8.7 mmol) were suspended in EtOH (100 mL). One N

NaOH (8.7 mL, 8.7 mmol) was added and the suspension brought to

1

mg (12%) of 54. ESI-MS: m/z = 449.1 [M + H]⁺. H NMR (500

MHz, DMSO-d₆): δ 10.52 (s, 1H), 9.00 (s, 1H), 8.23 (s, 2H), 8.14

(dd, J = 8.6 Hz, 1.8 Hz, 1H), 7.90−7.86 (m, 2H), 7.55 (d, J = 7.1 Hz,

1H), 7.46 (d, J = 3.8 Hz, 1H), 7.45 (d, J = 2.4 Hz, 1H), 7.40−7.37 (m,

2H), 7.30 (t, J = 7.1 Hz, 1H), 7.22 (t, J = 7.0 Hz, 1H), 4.69 (s, 2H).

HRMS: calcd for C₂₀H₁₇BF₃N₂O₄S [M + 1]⁺449.0949, found

449.0960. Purity determined by HPLC (gradient B): RT = 21.19 min,

purity 98.3%.

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reﬂux. After 2 h, the mixture was concentrated in vacuo and partioned

between ethyl acetate and water. The ethyl acetate was extracted 3×

with water and evaporated to yield 2.9 g (70%) of compound 60 as a

1H), 7.74−7.71 (m, 2H), 7.38 (d, J = 9.3 Hz, 1H), 7.19 (t, J = 8.7 Hz,

2H). TLC (ethyl acetate/hexanes/MeOH 1:1:0.1): R_f= 0.7.

6-(Cyanomethylthio)-N-(4-ﬂuorophenyl)nicotinamide (65). Thio-

nicotinamide 64 (248 mg. 1.00 mmol) dissolved in anhydrous DMF (3

mL) with chloroacetonitrile (63 μL, 1.0 mmol) was then added,

followed by triethylamine (140 μL, 1.00 mmol). The reaction was

heated to 100 °C for 30 min. After cooling to room temperature, the

solution was diluted into water (60 mL), and the resulting precipitate

was ﬁltered and washed with water to yield the cyano-intermediate 65

1

white solid. ESI-MS: m/z = 473.9 [M + H]⁺. H NMR (500 MHz,

DMSO-d₆): δ 8.52 (d, J = 4.4 Hz, 1H), 8.37 (s, 1H), 8.17 (s, 2H), 7.77

(t, J = 8.0 Hz, 7.4 Hz, 1H), 7.51−7.47 (m, 3H), 7.32−7.23 (m, 5H),

7.21−7.16 (m, 2H), 7.11 (t, J = 8.6 Hz, 2H), 5.15 (s, 2H), 4.56 (s,

2H). HPLC (gradient B): RT = 16.08 min, purity 95.9%.

2-((5-((4-Fluorophenyl)(pyridin-4-ylmethyl)carbamoyl)pyridin-2-

ylthio)methyl)phenylboronic acid (61). Following the same proce-

dure described for 60 and starting from the N-(4-ﬂuorophenyl)-N-

pyridin-4-yl-methyl-6-mercapto-nicotinamide intermediate 73b (2.8 g,

8.0 mmol) and 2-bromomethyl-phenylboronic acid (1.72 g, 8.0

mmol), workup yielded 2.88 g (76%) of compound 61 as a light

yellow foam. An analytical sample was puriﬁed by ﬂash silica gel

chromatography (97:3 ethyl acetate:MeOH). ESI-MS: m/z = 473.9

1

as a white solid (259 mg, 90%). ESI-MS: m/z = 288.2 [M + H]⁺. H

NMR (300 MHz, DMSO-d₆): δ 10.47 (s, 1H), 9.06 (dd, J = 2.4 Hz,

0.9 Hz, 1H), 8.22 (dd, J = 8.4, 2.4 Hz, Hz, 1H), 7.78 (dd, J = 9.0 Hz,

4.8 Hz, 2H), 7.64 (dd, J = 8.4 Hz, 0.6 Hz, 1H), 7.22 (t, J = 8.7 Hz, 2

H), 4.36 (s, 2H).

6-(2-Cyanobenzylthio)-N-(4-ﬂuorophenyl)nicotinamide (66). Pre-

pared via method B using thionicotinamide 64 (0.5 mmol) and α-

bromo-o-tolunitrile (0.5 mmol) to yield 137 mg (75%) of 66 as an oﬀ-

white solid. TLC (ethyl acetate/hexane, 1:1): R_f= 0.44. ESI-MS: m/z

= 364.1 [M + H]⁺. ¹H NMR (500 MHz, DMSO-d₆): δ 10.38 (s, 1H),

8.97 (d, J = 1.6 Hz, 1H), 8.11 (dd, J = 9.5 Hz, 2.1 Hz, 1H), 7.81 (d, J =

7.5 Hz, 1H), 7.75−7.68 (m, 3H), 7.62 (t, J = 7.3 Hz, 1H), 7.48 (d, J =

8.5 Hz, 1H), 7.42 (t, J = 8.3 Hz, 1H), 7.17 (t, J = 9.0 Hz, 2H), 4.64 (s,

2H).

1

[M + H]⁺. H NMR (500 MHz, DMSO-d₆): δ 8.52−8.49 (m, 2H),

8.40−8.37 (m, 1H), 8.18 (s, 2H), 7.52−7.51 (m, 2H), 7.37 (d, J = 4.1

Hz, 2H), 7.33−7.17 (m, 5H), 7.14−7.10 (m, 2H), 7.06−7.03 (m, 1H),

5.12 (s, 2H), 4.56 (s, 2H). Anal. Calcd for C₂₅H₂₁BFN₃O₃S: C,

63.44%; H, 4.47%; N, 8.88%. Found: C, 63.70%; H, 4.55%; N, 8.80%.

HPLC (gradient B): RT = 13.13 min, purity 96.8%.

2-((5-((4-Fluorophenyl)(piperidin-2-ylmethyl)carbamoyl)pyridin-

2-ylthio)methyl)phenylboronic acid (62). Thiol alkylation was

achieved according to method C by suspending the thionicotinamide

intermediate 73c (90 mg, 1 equiv) and 2-bromomethylphenyl boronic

acid (50 mg, 1.1 equiv) in EtOH (1 mL). Then 1 N NaOH (0.2 mL, 1

equiv) was added and the solution gently reﬂuxed for 2 h. Alkylated

product 74a was extracted with ethyl acetate (3 × 5 mL), washed with

water (3 × 5 mL), brine (3 × 5 mL), and dried over MgSO₄. The

organic layer was ﬁltered to remove MgSO₄, and then removed by

rotary evaporation and dried in vacuo to aﬀord 90 mg of the Boc-

protected piperidine boronic acid. The Boc group was then removed

by adding 4 M HCl in dioxane (1 mL) to the Boc-protected piperidine

boronic acid (20 mg). The resulting piperidine compound was then

puriﬁed using preparative HPLC (80:20:0.1 water:acetonitrile:formic

acid to 70:30:0.1 water:acetonitrile:formic acid over 30 min) to yield 5

mg (29%) of 62 as a white solid. ESI-MS: m/z = 480.1 [M + H]⁺. ¹H

NMR (500 MHz, D₂O): δ 8.35 (s, 1H), 8.08 (s, 1H), 7.41−7.37 (m,

2H), 7.28−7.15 (m, 4H), 7.05 (d, J = 7.6 Hz, 1H), 6.96−6.92 (m,

2H), 4.30 (s, 3H), 3.87 (dd, J = 14.8 Hz, 3.7 Hz, 1H), 3.38 (d, J = 10.8

Hz, 1H), 3.28 (br s, 1H), 2.88−2.84 (m, 1H), 1.78 (d, J = 7.7 Hz, 3H),

1.61−1.56 (m, 1H), 1.48−1.43 (m, 1H), 1.39−1.36 (m, 1H). HPLC

(gradient B): RT = 19.29 min, purity 90.1%.

6-(2-Boronobenzylthio)nicotinic acid (67). 6-Mercaptonicotinic

acid (1.55 g, 10 mmol) and 2-bromomethyl-phenylboronic acid (2.14

g, 10 mmol) were dissolved in anhydrous DMF (20 mL), and then

TEA (2.78 mL, 20 mmol) was added. The mixture was warmed to 60

°C for 1 h, and then removed from heat and let cool to room

temperature. The solution was acidiﬁed with 1 N HCl and extracted

with ethyl acetate. The ethyl acetate layer was washed with water,

saturated NaCl, dried over Na₂SO₄, and evaporated to yield

intermediate 67 as a yellow solid (1.54 g, 53%). TLC (AcOH/ethyl

acetate/EtOH, 0.1:80:20): R_f= 0.52; ESI-MS: m/z = 290.1 [M + H]⁺.

¹H NMR (500 MHz, DMSO-d₆): δ 13.23 (br s, 1H), 8.92 (s, 1H),

8.19 (br s, 2H), 8.05 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.53 (d, J = 7.3 Hz,

1H), 7.43−7.36 (m, 2H), 7.29 (t, J = 7.3 Hz, 1H), 7.21 (t, J = 7.4 Hz,

1H), 4.67 (s, 2H). HPLC (gradient B): RT = 14.03 min, purity 83.4%.

6-(2-(5,5-Dimethyl-1,3,2-dioxaborinan-2-yl)benzylthio)nicotinic

pivalic anhydride (68). Intermediate 67 (290 mg, 1 mmol) and

neopentyl glycol (1 mmol) were suspended in anhydrous toluene (5

mL) and set to reﬂux over 24 h. Without isolation, the resulting

mixture containing the boronic ester was cooled in an ice bath under

argon. Pivaloyl chloride (120 μL, 1 mmol) and triethylamine (140 μL,

1 mmol) were then added to the cooled solution. The reaction was

allowed to proceed at the lowered temperature for 1 h, and then

warmed to room temperature for an additional hour. The resulting

crystalline triethylammonium salt was ﬁltered away, and the reaction

mixture concentrated by rotary evaporation. The resulting oil

containing 70 was diluted with anhydrous DMF (3 mL). This

solution was used without further isolation.

2-(6-(2-Boronobenzylthio)-N-(4-ﬂuorophenyl)nicotinamido)-

acetic acid (63). The thionicotinamide 73d (110 mg, 0.3 mmol) and

2-bromomethyl-phenylboronic acid were coupled using method B to

yield the tert-butyl ester thionicotinamide intermediate 74b (138 mg,

93%). TLC (ethyl acetate): R_f= 0.6; ESI-MS: m/z = 496.9 [M + H]⁺.

The intermediate was dissolved in 90% aq TFA and incubated at room

temperature for 2 h. The TFA was removed by rotary evaporation to

and the crude material puriﬁed by preparative HPLC (65:35:01

water:acetonitrile:formic acid isocratic) to yield 74 mg (61%) of

compound 63 as a white hygroscopic solid. ESI-MS: m/z = 440.9 [M

N-(4-Fluoro-phenyl)-6-chloro-nicotinamide (69). 4-Fluoroaniline

(5.8 mL, 60 mmol) was stirred in THF (300 mL) and potassium

carbonate (16.6 g, 120 mmol) was added, followed by 6-

chloronicotinoyl chloride (10.6 g, 60 mmol) and stirred overnight.

The salts were removed by ﬁltration, and the organic solution was

cooled in an ice bath and diluted with water while stirring. The

resulting white precipitate was ﬁltered to yield 8.3 g (55%) of 69. TLC

(ethyl acetate/hexanes 1:1) R_f= 0.51. ESI-MS: m/z = 251.0 [M + H]⁺.

¹H NMR (500 MHz, DMSO-d₆): δ 10.53 (s, 1H), 8.95 (d, J = 2.5 Hz,

1

+ H]⁺. H NMR (500 MHz, DMSO-d₆): δ 12.95 (br s, 1H), 8.37 (s,

1H), 8.30 (s, 1H), 7.53 (d, J = 5.9 Hz, 1H), 7.41−6.92 (br m, 10H),

4.57 (s, 2H), 4.46 (s, 2H). HPLC (gradient B): RT = 16.75 min,

purity 96.6%.

N-(4-Fluoro-phenyl)-6-mercaptonicotinamide (64). To 150 mL

DMF was added and stirred 6-mercapto-nicotinic acid (50 mmol, 7.76

g). To the stirred solution, 4-ﬂuoroaniline (50 mmol, 4.8 mL) was

added followed by the addition of 2-ethoxy-1-ethoxycarbonyl-1,2-

dihydroquinoline (EEDQ, 50 mmol, 12.36 g). The dark brown

mixture was stirred for 12 h. The mixture was then diluted with water

(500 mL), and the precipitate collected by ﬁltration and washed

repeatedly with water. The oﬀ-white solid was dried in an oven (50

°C) for 72 h to aﬀord 6.42 g (52%) of the thionicotinamide product.

1H), 8.37 (dd, J = 8.4 Hz, 2.5 Hz, 1H), 7.81−7.70 (m, 3H), 7.26−7.18

(m, 2H). HPLC (gradient B): RT = 13.8 min, purity 94.3%.

tert-Butyl 2-((4-ﬂuorophenylamino)methyl)piperidine-1-carboxy-

late (70a). N-Boc-2-piperidinecarbaldehyde (1.1 g, 1 eq., Combi-

Blocks, San Diego, CA) and 4-ﬂuoroaniline (481 μL, 1 equiv) were

dissolved in DCE (20 mL). Under an inert atmosphere, NaBH(OAc)₃

(1.5 g, 1.4 equiv) and glacial acetic acid (294 μL, 1 equiv) were added,

and the reaction was monitored by TLC and LC−MS. The mixture

was diluted with ethyl acetate and washed with 10% citric acid (×3),

brine (×1) and dried over MgSO₄. The mixture was ﬁltered and the

solvent removed by rotary evaporation and dried in vacuo to aﬀord 1.4

1

ESI-MS: m/z = 248.9 [M + H]⁺. H NMR (500 MHz, DMSO-d₆): δ

13.87 (s, 1H), 10.23 (s, 1H), 8.32 (s, 1H), 7.89 (dd, J = 8.8 Hz, 2.2 Hz,

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Journal of Medicinal Chemistry

Article

g of the secondary aniline 70a (90% yield). The intermediate was

carried forward without further characterization. ESI-MS: m/z = 309.1

[M + H]⁺.

s, 1H), 7.47 (d, J = 7.9 Hz, 1H), 7.33−7.31 (m, 2H), 7.28 (t, J = 6.1

Hz, 1H), 7.10 (t, J = 8.1 Hz, 2H), 5.16 (s, 2H). HPLC (gradient B):

RT = 13.45 min, purity 99.1%.

tert-Butyl 2-(4-ﬂuorophenylamino)acetate (70b). A solution of 4-

ﬂuoroaniline (1.00 mL, 10 mmol) and DIPEA (1.74 mL, 10 mmol) in

anhydrous DMF (10 mL) was warmed to 80 °C, and then a solution

of tert-butyl bromoacetate (1.47 mL, 10 mmol) in anhydrous DMF

(10 mL) was added dropwise over 1 h. After addition, the mixture was

kept at 80 °C for 4 h. The mixture was then concentrated by rotary

evaporation and then partitioned between ethyl acetate and water. The

organic layer was washed with water and then evaporated to yield 70b

as a dark brown liquid (1.96 g, 87%). A sample was puriﬁed by ﬂash

silica gel chromatography (1:10 ethyl acetate/hexanes). TLC (ethyl

N-(4-Fluorophenyl)-6-mercapto-N-(pyridin-2-ylmethyl)-

nicotinamide (73a). N-(4-ﬂuorophenyl)-N-pyridin-2-ylmethyl-6-

chloro-nicotinamide intermediate 72a (3.4 g, 10 mmol) and

anhydrous sodium hydrogen sulﬁde (1.1 g, 20 mmol) were suspended

in anhydrous DMF (30 mL). The suspension was heated to reﬂux, and

the mixture turned a deep green color. After 2 h, the mixture was

diluted with ethyl acetate (150 mL) and extracted with water. The

aqueous layer was acidiﬁed with glacial AcOH to pH 6−7 and back

extracted with ethyl acetate (4 × 50 mL), and the organic layers were

combined, dried over Na₂SO₄, ﬁltered, and evaporated by rotary

evaporation to yield 2.9 g (87%) of N-(4-ﬂuorophenyl)-N-pyridin-2-

ylmethyl-6-mercapto-nicotinamide intermediate 73a as a dark yellow

oil. TLC (ethyl acetate/hexanes, 2:1): R_f= 0.1; ESI-MS: m/z = 339.9

[M + H]⁺.

1

acetate/hexanes, 1:4) R_f= 0.31. ESI-MS: m/z = 225.9 [M + H]⁺. H

NMR (500 MHz, DMSO-d₆): δ 6.94−6.90 (m, 2H), 6.55−6.52 (m,

2H), 5.85 (s, 1H), 3.75 (d, J = 4.3 Hz, 2H), 1.41 (s, 9H). HPLC

(gradient B): RT = 18.78 min, purity 86.5%.

tert-Butyl 2-((6-chloro-N-(4-ﬂuorophenyl)nicotinamido)methyl)-

piperidine-1-carboxylate (71a). The secondary aniline intermediate

70a (0.5 g, 1 equiv) was stirred in DMF (5 mL) at 80 °C followed by

the addition of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 240 μL, 1

equiv) and 6-chloronicotinoyl chloride (0.3 g, 1 equiv). The reaction

was monitored by TLC and LC−MS and was stirred overnight at 80

°C. The mixture was then diluted with ethyl acetate (25 mL), washed

with water, saturated NaHCO₃, water, 10% citric acid, water, saturated

NaCl, dried over Na₂SO₄, and ﬁltered. The organic solvent was then

removed by rotary evaporation and dried in vacuo to aﬀord 0.56 g of

the N-substituted N-(4-ﬂuorophenyl)-6-chloro-nicotinamide 71a (78%

yield). The intermediate was carried forward without further

puriﬁcation. ESI-MS: m/z = 448.2 [M + H]⁺.

N-(4-Fluorophenyl)-6-mercapto-N-(pyridin-4-ylmethyl)-

nicotinamide (73b). Following the same procedure described for 73a

and starting from the N-(4-ﬂuorophenyl)-N-pyridin-4-ylmethyl-6-

chloro-nicotinamide intermediate 72b (2.5 g, 7.4 mmol), workup

yielded 2.8 g (quant.) of compound crude 72b, which was carried

forward without further puriﬁcation. TLC (ethyl acetate/hexanes,

2:1): R_f= 0.1; ESI-MS: m/z = 339.9 [M + H]⁺.

tert-Butyl 2-((N-(4-ﬂuorophenyl)-6-mercaptonicotinamido)-

methyl)piperidine-1-carboxylate (73c). Thiolation of 71a was

achieved by suspending the tertiary amide (0.56 g, 1 equiv) and

sodium hydrogen sulﬁde (0.14 g, 2 equiv) in anhydrous DMF (2 mL)

under an inert atmosphere. The reaction was gently reﬂuxed for 2 h

and then diluted with ethyl acetate (30 mL) and extracted with water

(3 × 20 mL). The aqueous layer was then acidiﬁed with 10% citric acid

and stored at 2−8 °C. The ﬁne white precipitate was then collected by

ﬁltration to aﬀord 90 mg of thionicotinamide intermediate 73c (16%

yield). The intermediate was carried forward without further

puriﬁcation.

tert-Butyl 2-(N-(4-ﬂuorophenyl)-6-mercaptonicotinamido)-

acetate (73d). The 6-chloronicotinamide 71b (211 mg, 0.58 mmol)

and sodium hydrogen sulﬁde (74 mg) were dissolved in DMF (1 mL).

The mixture was heated to 85 °C for 15 min, and then allowed to cool

to room temperature. The mixture was diluted in ethyl acetate, washed

with water, saturated NaHCO₃, water, 10% citric acid, water, saturated

NaCl, and dried over Na₂SO₄. The crude material was ﬁltered and

evaporated to yield 123 mg (58%) of 73d as a yellow oil, which was

carried forward without further puriﬁcation. TLC (ethyl acetate): R_f=

0.53; ESI-MS: m/z = 362.9 [M + H]⁺.

tert-Butyl 2-(6-chloro-N-(4-ﬂuorophenyl)nicotinamido)acetate

(71b). The disubstituted aniline 70b (0.41 g, 2.31 mmol) was coupled

to 6-chloronicotinoyl chloride (0.52 g, 2.31 mmol) in anhydrous DMF

(2 mL) using DBU (344 mL, 2.31 mmol). The mixture was heated to

65 °C for 48 h, diluted with ethyl acetate, and then the organic layer

washed with water, saturated NaHCO₃, water, 10% citric acid, water,

saturated NaCl, dried over Na₂SO₄, and ﬁltered. The crude material

was concentrated by rotary evaporation, and puriﬁed by ﬂash

chromatography (ethyl acetate/hex 1:2) to yield 387 mg (46%) of

6-chloronicotinamide intermediate 71b as a clear oil. TLC (ethyl

1

acetate/hexanes, 1:2) R_f= 0.28; ESI-MS: m/z = 364.9 [M + H]⁺. H

NMR (300 MHz, DMSO-d₆): δ 8.25 (s, 1H), 7.69 (d, J = 7.0 Hz, 1H),

7.46 (d, J = 8.7 Hz, 1H), 7.30−7.26 (m, 2H), 7.17 (t, J = 8.8 Hz, 2H),

4.48 (s, 2H), 1.42 (s, 9H). HPLC (gradient B): RT = 19.88 min,

purity 83.5%.

6-Chloro-N-(4-ﬂuorophenyl)-N-(pyridin-2-ylmethyl)nicotinamide

(72a). N-(4-ﬂuorophenyl)-6-chloro-nicotinamide 69 (1.0 g, 4.0 mmol)

and 2-bromomethyl-pyridine hydrobromide (1.01 g, 4.0 mmol) were

suspended in toluene (5 mL). 50% aqueous NaOH (3.0 mL) was

added to the mixture, followed by tetra-n-butyl-ammonium hydroxide

(TBAH, 100 μL). The biphasic reaction mixture was vigorously stirred

overnight and the aqueous layer removed by pipet. The organic layer

was diluted with ethyl acetate and washed with water (3 × 25 mL) and

saturated aqueous NaCl (3 × 25 mL). The water washes were back-

extracted once with ethyl acetate (25 mL). The combined organic

layers were dried over Na₂SO₄, ﬁltered, and dried by rotary

evaporation to yield 1.3 g (96%) of N-(4-ﬂuorophenyl)-N-pyridin-2-

ylmethyl-6-chloro-nicotinamide intermediate 72a as an oﬀ-white solid.

ESI-MS: m/z = 341.9, 343.9 [M + H]⁺. ¹H NMR (500 MHz, DMSO-

d₆): δ 8.52 (d, J = 4.3 Hz, 1H), 8.35 (d, J = 2.3 Hz, 1H), 7.79−7.73 (m,

2H), 7.50 (d, J = 5.4 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.33−7.27 (m,

3H), 7.10 (t, J = 8.6 Hz, 2H), 5.16 (s, 2H).

AUTHOR INFORMATION

■

Corresponding Author

*E-mail: dmaeda@syntrixbio.com. Tel: (253) 833-8009. Fax:

(253) 833-8127.

Present Address

G.F.: Neuroinﬂammation Discovery Performance Unit, Glax-

oSmithKline R&D, Shanghai 201203, China.

Author Contributions

A.M.P. and A.D.S. contributed equally to this manuscript.

Notes

The authors declare no competing ﬁnancial interest.

6-Chloro-N-(4-ﬂuorophenyl)-N-(pyridin-4-ylmethyl)nicotinamide

(72b). Following the same procedure described for 72a and starting

from the N-(4-ﬂuorophenyl)-6-chloro-nicotinamide 69 (3.0 g, 12

mmol, 1.2 equiv) and 4-bromomethyl-pyridine hydrobromide (2.5 g,

10 mmol, 1.0 equiv), workup yielded 2.5 g (74%) of the N-(4-

ﬂuorophenyl)-N-pyridin-4-ylmethyl-6-chloro-nicotinamide intermedi-

ate 72b. ESI-MS: m/z = 342.1, 344.1 [M + H]⁺. ¹H NMR (500 MHz,

DMSO-d₆): δ 8.52 (s, 1H), 8.35 (s, 1H), 7.79−7.76 (m, 2H), 7.49 (br

ACKNOWLEDGMENTS

■

This work was supported by National Institutes of Health

Grant R44HL072614 (D.Y.M.) from the National Heart Lung

and Blood Institute. We thank both Dr. Joel Morgan and David

Nguyen (Syntrix Biosystems, Inc.) for careful review of the

manuscript.

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dx.doi.org/10.1021/jm500827t | J. Med. Chem. 2014, 57, 8378−8397

Journal of Medicinal Chemistry

Article

(15) Clarke, M.; Jamieson, T.; Steele, C.; Olson, M.; Samuel, M.;

Das, S.; Sansom, O.; Nibbs, R. Inhibition of neutrophil chemokine

receptor CXCR2 profoundly suppresses inflammation-driven and

spontaneous tumorigenesis. Immunology 2012, 137, 190−190.

(16) Gong, L.; Cumpian, A. M.; Caetano, M. S.; Ochoa, C. E.; De la

Garza, M. M.; Lapid, D. J.; Mirabolfathinejad, S. G.; Dickey, B. F.;

Zhou, Q. H.; Moghaddam, S. J. Promoting effect of neutrophils on

lung tumorigenesis is mediated by CXCR2 and neutrophil elastase.

Mol. Cancer 2013, 12, 14.

(17) Han, L.; Jiang, B.; Wu, H.; Wang, X. D.; Tang, X. J.; Huang, J.

F.; Zhu, J. High expression of CXCR2 is associated with tumorigenesis,

progression, and prognosis of laryngeal squamous cell carcinoma. Med.

Oncol. 2012, 29, 2466−2472.

(18) Jamieson, T.; Clarke, M.; Steele, C. W.; Samuel, M. S.;

Neumann, J.; Jung, A.; Huels, D.; Olson, M. F.; Das, S.; Nibbs, R. J. B.;

Sansom, O. J. Inhibition of CXCR2 profoundly suppresses

inflammation-driven and spontaneous tumorigenesis. J. Clin. Invest.

2012, 122, 3127−3144.

(19) Katoh, H.; Wang, D. Z.; Daikoku, T.; Sun, H. Y.; Dey, S. K.;

DuBois, R. N. CXCR2-Expressing Myeloid-derived suppressor cells

are essential to promote colitis-associated tumorigenesis. Cancer Cell

2013, 24, 631−644.

(20) Balkwill, F. R. The chemokine system and cancer. J. Pathol.

2012, 226, 148−157.

(21) Busch-Petersen, J. Small molecule antagonists of the CXCR2

and CXCR1 chemokine receptors as therapeutic agents for the

treatment of inflammatory diseases. Curr. Top. Med. Chem. 2006, 6,

1345−1352.

(22) Allegretti, M.; Bertini, R.; Cesta, M. C.; Bizzarri, C.; Di Bitondo,

R.; Di Cioccio, V.; Galliera, E.; Berdini, V.; Topai, A.; Zampella, G.;

Russo, V.; Di Bello, N.; Nano, G.; Nicolini, L.; Locati, M.; Fantucci, P.;

Florio, S.; Colotta, F. 2-Arylpropionic CXC chemokine receptor 1

(CXCR1) ligands as novel noncompetitive CXCL8 inhibitors. J. Med.

Chem. 2005, 48, 4312−4331.

(23) Bertini, R.; Allegretti, M.; Bizzarri, C.; Moriconi, A.; Locati, M.;

Zampella, G.; Cervellera, M. N.; Di Cioccio, V.; Cesta, M. C.; Galliera,

E.; Martinez, F. O.; Di Bitondo, R.; Troiani, G.; Sabbatini, V.;

D’Anniballe, G.; Anacardio, R.; Cutrin, J. C.; Cavalieri, B.; Mainiero,

F.; Strippoli, R.; Villa, P.; Di Girolamo, M.; Martin, F.; Gentile, M.;

Santoni, A.; Corda, D.; Poli, G.; Mantovani, A.; Ghezzi, P.; Colotta, F.

Noncompetitive allosteric inhibitors of the inflammatory chemokine

receptors CXCR1 and CXCR2: Prevention of reperfusion injury. Proc.

Natl. Acad. Sci. U.S.A. 2004, 101, 11791−11796.

(24) White, J. R.; Lee, J. M.; Young, P. R.; Hertzberg, R. P.; Jurewicz,

A. J.; Chaikin, M. A.; Widdowson, K.; Foley, J. J.; Martin, L. D.;

Griswold, D. E.; Sarau, H. M. Identification of a potent, selective non-

peptide CXCR2 antagonist that inhibits interleukin-8-induced

neutrophil migration. J. Biol. Chem. 1998, 273, 10095−10098.

(25) Podolin, P. L.; Bolognese, B. J.; Foley, J. J.; Schmidt, D. B.;

Buckley, P. T.; Widdowson, K. L.; Jin, Q.; White, J. R.; Lee, J. M.;

Goodman, R. B.; Hagen, T. R.; Kajikawa, O.; Marshall, L. A.; Hay, D.

W.; Sarau, H. M. A potent and selective nonpeptide antagonist of

CXCR2 inhibits acute and chronic models of arthritis in the rabbit. J.

Immunol. 2002, 169, 6435−6444.

ABBREVIATIONS USED

■

CXCR1, CXC receptor 1; CXCR2, CXC receptor 2; CXCL1,

CXC ligand 1; CXCL8, CXC ligand 8; DIPEA, diisopropyle-

thylamine; FACS, ﬂuorescence-activated cell sorting; C5a,

complement component 5a; fMLF, formyl-methionyl-leucyl-

phenylalanine; PAF, platelet-activating factor; iv, intravenous;

NMM, N-methylmorpholine; PMN, polymorphonuclear cells;

hPMN, human PMN; RT, retention time; SAR, structure

activity relationship; TEA, triethylamine

REFERENCES

■

(1) Murphy, P. M.; Baggiolini, M.; Charo, I. F.; Hebert, C. A.; Horuk,

R.; Matsushima, K.; Miller, L. H.; Oppenheim, J. J.; Power, C. A.

International union of pharmacology. XXII. Nomenclature for

chemokine receptors. Pharmacol. Rev. 2000, 52, 145−176.

(2) Petersen, F.; Flad, H. D.; Brandt, E. Neutrophil-activating

peptides NAP-2 and IL-8 bind to the same sites on neutrophils but

interact in different ways: Discrepancies in binding affinities, receptor

densities, and biologic effects. J. Immunol. 1994, 152, 2467−2478.

(3) Neptune, E. R.; Iiri, T.; Bourne, H. R. G alpha(i) is not required

for chemotaxis mediated by G(i)-coupled receptors. J. Biol. Chem.

1999, 274, 2824−2828.

(4) Jones, S. A.; Moser, B.; Thelen, M. A Comparison of postreceptor

signal-transduction events in Jurkat cells transfected with either IL-8R1

or IL-8R2 - chemokine mediated activation of P42/P44 MAP-Kinase

(ERK-2). Febs Lett. 1995, 364, 211−214.

(5) Schorr, W.; Swandulla, D.; Zeilhofer, H. U. Mechanisms of IL-8-

induced Ca2+ signaling in human neutrophil granulocytes. Eur. J.

Immunol. 1999, 29, 897−904.

(6) Kurdowska, A.; Noble, J. M.; Grant, I. S.; Robertson, C. R.;

Haslett, C.; Donnelly, S. C. Anti-interleukin-8 autoantibodies in

patients at risk for acute respiratory distress syndrome. Crit. Care Med.

2002, 30, 2335−2337.

(7) Erdem, H.; Pay, S.; Serdar, M.; Simsek, I.; Dinc, A.; Musabak, U.;

Pekel, A.; Turan, M. Different ELR(+) angiogenic CXC chemokine

profiles in synovial fluid of patients with Behcet’s disease, familial

Mediterranean fever, rheumatoid arthritis, and osteoarthritis. Rheuma-

tol. Int. 2005, 26, 162−167.

(8) Lally, F.; Smith, E.; Filer, A.; Stone, M. A.; Shaw, J. S.; Nash, G.

B.; Buckley, C. D.; Rainger, G. E. A novel mechanism of neutrophil

recruitment in a coculture model of the rheumatoid synovium. Arthritis

Rheum. 2005, 52, 3460−3469.

(9) Reich, K.; Garbe, C.; Blaschke, V.; Maurer, C.; Middel, P.;

Westphal, G.; Lippert, U.; Neumann, C. Response of psoriasis to

interleukin-10 is associated with suppression of cutaneous type 1

inflammation, downregulation of the epidermal interleukin-8/CXCR2

pathway and normalization of keratinocyte maturation. J. Invest.

Dermatol. 2001, 116, 319−329.

(10) Banks, C.; Bateman, A.; Payne, R.; Johnson, P.; Sheron, N.

Chemokine expression in IBD. Mucosal chemokine expression is

unselectively increased in both ulcerative colitis and Crohn’s disease. J.

Pathol. 2003, 199, 28−35.

(26) Traves, S. L.; Smith, S. J.; Barnes, P. J.; Donnelly, L. E. Specific

CXC but not CC chemokines cause elevated monocyte migration in

COPD: A role for CXCR2. J. Leukocyte Biol. 2004, 76, 441−450.

(27) Dwyer, M. P.; Yu, Y.; Chao, J.; Aki, C.; Biju, P.; Girijavallabhan,

V.; Rindgen, D.; Bond, R.; Mayer-Ezel, R.; Jakway, J.; Hipkin, R. W.;

Fossetta, J.; Gonsiorek, W.; Bian, H.; Fan, X.; Terminelli, C.; Fine, J.;

Lundell, D.; Merritt, J. R.; Rokosz, L. L.; Kaiser, B.; Li, G.; Wang, W.;

Stauffer, T.; Ozgur, L.; Baldwin, J.; Taveras, A. G. Discovery of 2-

hydroxy-N,N-dimethyl-3-{2-[[(R)-1-(5-methylfuran-2-yl)propyl]-

amino]-3,4-dioxocyclobut-1-enylamino}benzamide (SCH 527123): A

potent, orally bioavailable CXCR2/CXCR1 receptor antagonist. J.

Med. Chem. 2006, 49, 7603−7606.

(11) Beeh, K. M.; Kornmann, O.; Buhl, R.; Culpitt, S. V.; Giembycz,

M. A.; Barnes, P. J. Neutrophil chemotactic activity of sputum from

patients with COPD: Role of interleukin 8 and leukotriene B-4. Chest

2003, 123, 1240−1247.

(12) Keatings, V. M.; Collins, P. D.; Scott, D. M.; Barnes, P. J.

Differences in interleukin-8 and tumor necrosis factor-alpha in induced

sputum from patients with chronic obstructive pulmonary disease or

asthma. Am. J. Respir. Crit. Care Med. 1996, 153, 530−534.

(13) Chapman, R. W.; Phillips, J. E.; Hipkin, R. W.; Curran, A. K.;

Lundell, D.; Fine, J. S. CXCR2 antagonists for the treatment of

pulmonary disease. Pharmacol. Ther. 2009, 121, 55−68.

(14) Donnelly, L. E.; Barnes, P. J. Chemokine receptor CXCR2

antagonism to prevent airways inflammation. Drugs Future 2011, 36,

465−472.

(28) Aul, R.; Patel, S.; Summerhill, S.; Kilty, I.; Plumb, J.; Singh, D.

LPS challenge in healthy subjects: An investigation of neutrophil

8395

dx.doi.org/10.1021/jm500827t | J. Med. Chem. 2014, 57, 8378−8397

Journal of Medicinal Chemistry

Article

chemotaxis mechanisms involving CXCR1 and CXCR2. Int.

Immunopharmacol. 2012, 13, 225−231.

(29) Lazaar, A. L.; Sweeney, L. E.; MacDonald, A. J.; Alexis, N. E.;

Chen, C.; Tal-Singer, R. SB-656933, a novel CXCR2 selective

antagonist, inhibits ex vivo neutrophil activation and ozone-induced

airway inflammation in humans. Br. J. Clin. Pharmacol. 2011, 72, 282−

293.

(30) Moss, R. B.; Mistry, S. J.; Konstan, M. W.; Pilewski, J. M.;

Kerem, E.; Tal-Singer, R.; Lazaar, A. L.; Investigators, C. F. Safety and

early treatment effects of the CXCR2 antagonist SB-656933 in patients

with cystic fibrosis. J. Cystic Fibrosis 2013, 12, 241−248.

(31) Holz, O.; Khalilieh, S.; Ludwig-Sengpiel, A.; Watz, H.; Stryszak,

P.; Soni, P.; Tsai, M.; Sadeh, J.; Magnussen, H. SCH527123, a novel

CXCR2 antagonist, inhibits ozone-induced neutrophilia in healthy

subjects. Eur. Respir. J. 2010, 35, 564−570.

(32) Dwyer, M. P.; Yu, Y. N. CXCR2 modulators: a patent review

(2009−2013). Expert Opin. Ther. Pat. 2014, 24, 519−534.

(33) Virtala, R.; Ekman, A. K.; Jansson, L.; Westin, U.; Cardell, L. O.

Airway inflammation evaluated in a human nasal lipopolysaccharide

challenge model by investigating the effect of a CXCR2 inhibitor. Clin.

Exp. Allergy 2012, 42, 590−596.

Locati, M.; Galliera, E.; Di Benedetto, P. Vigilante, P.; Bertini, R.;

Allegretti, M. Design of noncompetitive interleukin-8 inhibitors acting

on CXCR1 and CXCR2. J. Med. Chem. 2007, 50, 3984−4002.

(49) Gonsiorek, W.; Fan, X.; Hesk, D.; Fossetta, J.; Qiu, H.; Jakway,

J.; Billah, M.; Dwyer, M.; Chao, J.; Deno, G.; Taveras, A.; Lundell, D.

J.; Hipkin, R. W. Pharmacological characterization of Sch527123, a

potent allosteric CXCR1/CXCR2 antagonist. J. Pharmacol. Exp. Ther.

2007, 322, 477−485.

(50) Jones, S. A.; Wolf, M.; Qin, S.; Mackay, C. R.; Baggiolini, M.

Different functions for the interleukin 8 receptors (IL-8R) of human

neutrophil leukocytes: NADPH oxidase and phospholipase D are

activated through IL-8R1 but not IL-8R2. Proc. Natl. Acad. Sci. U.S.A.

1996, 93, 6682−6686.

(51) Geiser, T.; Dewald, B.; Ehrengruber, M. U.; Clarklewis, I.;

Baggiolini, M. The interleukin-8-related chemotactic cytokines GRO-

alpha, GRO-beta, and GRO-gamma activate human neutrophil and

basophil leukocytes. J. Biol. Chem. 1993, 268, 15419−15424.

(52) Kaur, M.; Singh, D. Neutrophil chemotaxis caused by chronic

obstructive pulmonary disease alveolar macrophages: The role of

CXCL8 and the receptors CXCR1/CXCR2. J. Pharmacol. Exp. Ther.

2013, 347, 173−180.

(53) May, L. T.; Avlani, V. A.; Sexton, P. M.; Christopoulos, A.

Allosteric modulation of G protein-coupled receptors. Curr. Pharm.

Design 2004, 10, 2003−2013.

(54) Bradley, M. E.; Bond, M. E.; Manini, J.; Brown, Z.; Charlton, S.

J. SB265610 is an allosteric, inverse agonist at the human CXCR2

receptor. Br. J. Pharmacol. 2009, 158, 328−338.

(55) Nicholls, D. J.; Tomkinson, N. P.; Wiley, K. E.; Brammall, A.;

Bowers, L.; Grahames, C.; Gaw, A.; Meghani, P.; Shelton, P.; Wright,

T. J.; Mallinder, P. R. Identification of a putative intracellular allosteric

antagonist binding-site in the CXC chemokine receptors 1 and 2. Mol.

Pharmacol. 2008, 74, 1193−1202.

(56) de Kruijf, P.; Lim, H. D.; Roumen, L.; Renjaan, V. A.; Zhao, J.;

Webb, M. L.; Auld, D. S.; Wijkmans, J. C.; Zaman, G. J.; Smit, M. J.; de

Graaf, C.; Leurs, R. Identification of a novel allosteric binding site in

the CXCR2 chemokine receptor. Mol. Pharmacol. 2011, 80, 1108−

1118.

(57) Onuffer, J. J.; Horuk, R. Chemokines, chemokine receptors and

small-molecule antagonists: Recent developments. Trends Pharmacol.

Sci. 2002, 23, 459−467.

(58) Groll, M.; Berkers, C. R.; Ploegh, H. L.; Ovaa, H. Crystal

Structure of the Boronic Acid-Based Proteasome Inhibitor Bortezomib

in Complex with the Yeast 20S Proteasome. Structure 2006, 14, 451−

456.

(59) Hu, G. Q.; Lin, G.; Wang, M.; Dick, L.; Xu, R. M.; Nathan, C.;

Li, H. L. Structure of the Mycobacterium tuberculosis proteasome and

mechanism of inhibition by a peptidyl boronate. Mol. Microbiol. 2006,

59, 1417−1428.

(60) Tsilikounas, E.; Kettner, C. A.; Bachovchin, W. W. B-11 NMR-

spectroscopy of peptide boronic acid inhibitor complexes of alpha-lytic

protease: Direct evidence for tetrahedral boron in both boron histidine

and boron serine adduct complexes. Biochemistry 1993, 32, 12651−

12655.

(34) Cutshall, N. S.; Kucera, K. A.; Ursino, R.; Latham, J.; Ihle, N. C.

Nicotinanilides as inhibitors of neutrophil chemotaxis. Bioorg. Med.

Chem. Lett. 2002, 12, 1517−1520.

(35) Maeda, D. Y.; Quinn, M. T.; Schepetkin, I. A.; Kirpotina, L. N.;

Zebala, J. A. Nicotinamide glycolates antagonize CXCR2 activity

through an intracellular mechanism. J. Pharmacol. Exp. Ther. 2010,

332, 145−152.

(36) Webb, K. S.; Levy, D. A facile oxidation of boronic acids and

boronic esters. Tetrahedron Lett. 1995, 36, 5117−5118.

(37) Wittenberger, S. J.; Donner, B. G. Dialkyltin oxide mediated

addition of trimethylsilyl azide to nitriles. A novel preparation of 5-

substituted tetrazoles. J. Org. Chem. 1993, 58, 4139−4141.

(38) Hunter, P. Not boring at all Boron is the new carbon in the

quest for novel drug candidates. EMBO Rep. 2009, 10, 125−U110.

(39) Yang, W. Q.; Gao, X. M.; Wang, B. H. Boronic acid compounds

as potential pharmaceutical agents. Med. Res. Rev. 2003, 23, 346−368.

(40) Hall, D. G. Boronic Acids: Preparation and Applications in Organic

Synthesis and Medicine; WILEY-VCH: Weinheim, 2005.

(41) Cutshall, N. S.; Ursino, R.; Kucera, K. A.; Latham, J.; Ihle, N. C.

Nicotinamide N-oxides as CXCR2 antagonists. Bioorg. Med. Chem.

Lett. 2001, 11, 1951−1954.

(42) Christopoulos, A. Allosteric binding sites on cell-surface

receptors: Novel targets for drug discovery. Nat. Rev. Drug Discovery

2002, 1, 198−210.

(43) de Kruijf, P.; van Heteren, J.; Lim, H. D.; Conti, P. G.; van der

Lee, M. M.; Bosch, L.; Ho, K. K.; Auld, D.; Ohlmeyer, M.; Smit, M. J.;

Wijkmans, J. C.; Zaman, G. J.; Smit, M. J.; Leurs, R. Nonpeptidergic

allosteric antagonists differentially bind to the CXCR2 chemokine

receptor. J. Pharmacol. Exp. Ther. 2009, 329, 783−790.

(44) Mueller, S. G.; White, J. R.; Schraw, W. P.; Lam, V.; Richmond,

A. Ligand-induced desensitization of the human CXC chemokine

receptor-2 is modulated by multiple serine residues in the carboxyl-

terminal domain of the receptor. J. Biol. Chem. 1997, 272, 8207−8214.

(45) Shyamala, V.; Khoja, H. Interleukin-8 receptors R1 and R2

activate mitogen-activated protein kinases and induce c-fos,

independent of Ras and Raf-1 in Chinese hamster ovary cells.

Biochemistry 1998, 37, 15918−15924.

(61) Farrjones, S.; Smith, S. O.; Kettner, C. A.; Griffin, R. G.;

Bachovchin, W. W. Crystal versus solution structure of enzymes:

NMR-spectroscopy of a peptide boronic acid-serine protease complex

in the crystalline state. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 6922−

6924.

(46) Hammond, M. E. W.; Lapointe, G. R.; Feucht, P. H.; Hilt, S.;

Gallegos, C. A.; Gordon, C. A.; Giedlin, M. A.; Mullenbach, G.;

Tekampolson, P. IL-8 induces neutrophil chemotaxis predominantly

via type-I IL-8 receptors. J. Immunol. 1995, 155, 1428−1433.

(47) Posadas, I.; Terencio, M. C.; Guillen, I.; Ferrandiz, M. L.;

Coloma, J.; Paya, M.; Alcaraz, M. J. Co-regulation between cyclo-

oxygenase-2 and inducible nitric oxide synthase expression in the time-

course of murine inflammation. N-S Arch. Pharmacol. 2000, 361, 98−

106.

(62) Bachovchin, W. W.; Wong, W. Y. L.; Farrjones, S.; Shenvi, A. B.;

Kettner, C. A. N-15 NMR-spectroscopy of the catalytic-triad histidine

of a serine protease in peptide boronic acid inhibitor complexes.

Biochemistry 1988, 27, 7689−7697.

(63) Bone, R.; Shenvi, A. B.; Kettner, C. A.; Agard, D. A. Serine

protease mechanism: Structure of an inhibitory complex of alpha-lytic

protease and a tightly bound peptide boronic acid. Biochemistry 1987,

26, 7609−7614.

(64) Siemsen, D. W.; Schepetkin, I. A.; Kirpotina, L. N.; Lei, B.;

Quinn, M. T. Neutrophil isolation from nonhuman species. Methods

Mol. Biol. 2007, 412, 21−34.

(48) Moriconi, A.; Cesta, M. C.; Cervellera, M. N.; Aramini, A.;

Coniglio, S.; Colagioia, S.; Beccari, A. R.; Bizzarri, C.; Cavicchia, M. R.;

8396

dx.doi.org/10.1021/jm500827t | J. Med. Chem. 2014, 57, 8378−8397

Journal of Medicinal Chemistry

Article

(65) Hall, D. A.; Beresford, I. J.; Browning, C.; Giles, H. Signalling by

CXC-chemokine receptors 1 and 2 expressed in CHO cells: A

comparison of calcium mobilization, inhibition of adenylyl cyclase and

stimulation of GTPgammaS binding induced by IL-8 and GROalpha.

Br. J. Pharmacol. 1999, 126, 810−818.

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Discovery of 2-[5-(4-fluorophenylcarbamoyl)pyridin-2-ylsulfanylmethyl]phenylboronic acid (SX-517): Noncompetitive boronic acid antagonist of CXCR1 and CXCR2

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