Nicotinanilides as inhibitors of neutrophil chemotaxis

Article abstract of DOI:10.1016/S0960-894X(02)00188-9

A series of 4-fluoronicotinanilides was synthesized and shown to be novel, potent, and selective inhibitors against GRO-α-driven human neutrophil chemotaxis. Compounds of this class may be useful for the treatment of inflammatory, autoimmune, and allergic

Full text of DOI:10.1016/S0960-894X(02)00188-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1517–1520
Nicotinanilides as Inhibitors of Neutrophil Chemotaxis
Neil S. Cutshall,a,* Kristin A. Kucera,b Rocky Ursino,b
John Lathamb and Nathan C. Ihlea
aDepartment of Chemistry, Celltech R&D Inc., 1631 220th Street SE, Bothell, WA98021, USA
bDepartment of Gene Function & Target Validation, Celltech R&D Inc., 1631 220th Street SE, Bothell, WA98021, USA
Received 5January 2002; accepted 27 February 2002
Abstract—A series of 4-fluoronicotinanilides was synthesized and shown to be novel, potent, and selective inhibitors against GRO-
a-driven human neutrophil chemotaxis. Compounds of this class may be useful for the treatment of inflammatory, autoimmune,
and allergic disorders. # 2002 Elsevier Science Ltd. All rights reserved.
Activated neutrophils (polymorphonuclear neutrophils:
PMN) have been implicated in the pathogenesis of
many inflammatory conditions.1 Neutrophils are acti-
vated and attracted by interleukin-8 (IL-8) and other
Glu-Leu-Arg (ELR) containing CXC chemokines,
including epithelial cell-derived neutrophil-activating
peptide-78 (ENA-78), and growth-related oncogene
(GRO)-a, GRO-b, and GRO-g. In response to trau-
matic injury or infection, human neutrophils are direc-
Figure 1. Inhibitors of neutrophil chemotaxis.
ted to the site of injury or infection by CXC chemokines
that signal via two distinct seven-transmembrane
G-protein coupled receptors (GPCRs), CXCR1 and
CXCR2. Both CXCR1 and CXCR2 bind IL-8 with
high affinity; however, only CXCR2 binds the other
neutrophil-activating and ELR motif-bearing chemo-
kines with high affinity. Leukocyte recruitment to these
inflammatory sites is multi-faceted involving chemo-
attractants ranging from formyl-methionyl-leucyl-
phenylalanine (FMLP), platelet-activating factor
(PAF), leukotriene B4 (LTB4) and C5a anaphylotoxin.
a class of nicotinanilide N-oxides containing heteroaryl
4-fluoroanilides were antagonists of the CXCR2 recep-
tor and inhibited neutrophil chemotaxis.4 Further work
in this area revealed that the related class of com-
pounds, 6-substituted nicotinanilides, are also potent
and selective inhibitors of GRO-a driven human neu-
trophil chemotaxis. Here we report these results, includ-
ing the design and synthesis of 5b, a functionally active
and selective inhibitor of chemotaxis (IC50=42 nM).
Two routes were explored toward the synthesis of
6-alkylsulfanyl-nicotinanilides (Scheme 1). In route A,
6-mercaptonicotinic acid 3 was first converted to the
corresponding nicotinanilide 4 then S-alkylated to pro-
vide thioether 5a in good yield. Because of the limited
commercial availability of diverse haloacetates, a sec-
ond synthetic strategy was developed (route B). Com-
mercially available 6-chloronicotinoyl chloride 6 was
converted to anilide 7.4 Treatment of 7 with various
thioalkoxides led to the thioether analogues 5b–g.
Compound 5h was readily obtained from a mild sapo-
nification of 5b following neutralization.
Several nonpeptide small molecules have recently been
reported to inhibit neutrophil chemotaxis (Fig. 1).
Included is SB-265601 (1), a compound that has
demonstrated potent inhibition of CXCR2 ligand bind-
ing and neutrophil chemotaxis (IC50=10 nM) in vitro
and was efficacious in vivo in an LPS-induced rabbit
model of neutrophilia.2 PD 0220245( 2) was recently
claimed as a selective antagonist of IL-8 binding
(IC50=110 nM) and potent inhibitor of neutrophil
chemotaxis (IC50=170 nM).3 We recently disclosed that
Scheme 2 outlines the chemical routes used to prepare
the inhibitors shown in Table 2. Treatment of the
6-bromonicotinanilide5 8 in the presence of excess of
*Corresponding author. Tel.: +1-425-489-4911; fax: +1-425-485-
9026; e-mail: ncutshall@ceptyr.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00188-9

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N. S. Cutshall et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1517–1520
Table 1. Representative activities of nicotinanilides
Compd
n
R1
GRO-a
Chemotaxis
IC50 (mM)a
FMLP
Chemotaxis
IC50 (mM)a
5a
5b
5c
5d
5e
1
1
1
1
2
0.046
0.042
0.043
0.083
0.76
>40
ntb
nt
Scheme 1. Reactions and conditions: (a) 4-FC6H4NH2, EEDQ, DMF,
rt, 90%; (b) propyl bromoacetate, Cs2CO3, DMF, rt, 76%; (c) 4-
FC6H4NH2, i-Pr2NEt, CH2Cl2, 0 ꢀC to rt, 92%; (d) HS(CH2)nCO2R1,
t-BuOK, THF, rt; (e) 5b, NaOH THF-H2O, 86%.
nt
>40
5f
5g
5h
3
2
1
H
H
H
5.1
>20
>20
nt
nt
nt
aValues were calculated from the geometric mean of at least two
experiments.
bNot tested.
Scheme 2. Reactions and conditions: (a) methyl glycolate, t-BuOK,
THF, 65%; (b) R1OH, H2SO4 (cat.), 80 ꢀC.
accomplished by exposing the crude reaction mixture to
reduced pressure. Alternatively, 9a could be heated to
reflux in toluene under acidic conditions with a small
excess of the higher molecular weight alcohols to pro-
vide analogues 9d–h after purification by flash chroma-
tography or trituration.
Scheme 3 outlines the route used to prepare 6-sub-
stituted-carbamoylmethylsulfanyl-N-(4-fluorophenyl)-
nicotinanilides. Treatment of the acid 5h with PyBrOP
in the presence of excess amine provided the desired
amides 10a–f. Efforts to improve the chemical yields of
this class were limited due to their lower potencies.
Scheme 3. Reactions and conditions: (a) R1R2NH, PyBrOP, DMF,
<35%.
It was of interest to design a synthetic route toward
nicotinanilide analogues where SAR diversity could be
explored around the 4-fluoroanilide moiety. Scheme 4
describes a route toward the synthesis of analogues
where the amide functionality at the 3 position of the
nicotinanilide core was reversed. 2-Chloro-5-nitro-pyri-
dine 11 was converted to 12 by treatment with the
potassium alkoxide of methyl glycolate. Reduction of
the nitro functionality followed by acylation with
4-fluorobenzoyl chloride readily provided 13.
Scheme 4. Reactions and conditions: (a) methyl glycolate, t-BuOK,
THF, 20%; (b) 10% Pd/C, H2, MeOH; (c) 4-FC6H4COCl, i-Pr2NEt,
CH2Cl2, 55% from 12.
methyl glycolate with potassium tert-butoxide provided
the methyl ester 9a. The less reactive 6-chloro-N-(4-
fluoro-phenyl)-nicotinamide could also be converted to
9a under similar conditions but in lower yield. Trans-
esterification of methyl ester 9a into esters 9b–c was
readily accomplished by heating 9a with the appropriate
alcohol neat in a sealed tube in the presence of a cata-
lytic amount of sulfuric acid or triethylamine. Removal
of the excess low molecular weight alcohol was easily
As part of a lead optimization strategy, nicotinanilides
were screened for both their ability to block GRO-a and
FMLP driven human neutrophil chemotaxis.6
SAR studies initially focused on changes around the
6-position of the nicotinanilide core (Table 1). It was

N. S. Cutshall et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1517–1520
Table 2. Representative activities of nicotinanilides Table 3. Representative activities of nicotinanilides
1519
R1
GRO-a
Chemotaxis
IC50 (mM)a
FMLP
Chemotaxis
IC50 (mM)a
Compd
R1
R2
GRO-a
Chemotaxis
IC50 (mM)a
FMLP
Chemotaxis
IC50 (mM)a
Compd
10a
10b
10c
10d
10e
10f
H
H
H
>20
>20
1.0
ntb
nt
9a
0.11
>40
9b
9c
0.16
0.20
>40
>40
9d
9e
9f
0.22
>40
ntb
>40
>40
>40
>40
0.41
12.5
2.7
0.094
0.050
0.68
>40
>40
>40
H
H
9g
9h
0.22
aValues were calculated from the geometric mean of at least two
experiments.
bNot tested.
aValues were calculated from the geometric mean of at least two
experiments.
bNot tested.
showed an inability to block ligand binding in a filter
mat assay using CXCR2 membranes (data not shown).8
5b and 5h were subsequently tested for GRO-a-depen-
dent GTP-g-S exchange with CXCR2 membranes.9 The
carboxylate 5h showed selective antagonism of GRO-a
mediated exchange (IC50=614 nm vs inactive against
CCR1 and CCR9) while ester 5b was inactive. Lastly,
based on this observation, the free acid 5h was
reevaluated and determined to have an IC50=1.2 mM
in a CXCR2 filter mat binding assay.8 A possible
explanation for these results is that the nicotinanilide
esters are membrane permeable and able to pass into the
interior of neutrophils. The esters are then hydrolyzed
by endogenous esterases to liberate the correspondingly
less permeable carboxylate salt, which accumulates
intracellularly and interferes with signaling of the
CXCR2 receptor. Additional studies to validate the
cellular targets of these compounds are ongoing.
found that the most potent analogues in this series
incorporated a thioglycolate ester pharmacophore as
illustrated by compounds 5a–d. All had potencies
<100 nM against GRO-a driven chemotaxis. Sub-
stantial structural diversity about the ester could be tol-
erated while maintaing acceptable biological activity,
however, efforts to extend the methylene unit of the
thioglycolate moiety significantly reduced the potency
as seen by 5e. Functional inhibitory activity against
chemotaxis was also lost upon conversion of the ester to
the corresponding acid as seen by 5f–h. Selectivity was
assessed in a neutrophil chemotaxis assay using FMLP
as an alternate chemotactic agonist. In all cases the
compounds were more potent GRO-a antagonists and
displayed little or no antagonist activity toward FMLP.
The restricted antagonism toward GRO-a infers inter-
ference is proximal to CXCR2 since these two receptors
extensively share their signal transduction cascade. To
further validate the selectivity of this class of com-
pounds for CXCR2, 5b and 5h were tested as repre-
sentative compounds in additional assays. Preliminary
tests indicated that 5b selectively interfered with
CXCR2 binding as it had an IC50=32 nM against
GRO-a induced calcium flux against human neutrophils
but was inactive at antagonizing CCR2 and CCR3 in
transfected CHO cells.7 Interestingly, the ester 5b
Because of concerns regarding the potential oxidation
of the thioether moiety at the 6-position of the nicoti-
nanilide core to the corresponding sulfoxide and sul-
fone, oxygen-based analogues were investigated (Table
2). As was observed with the thioglycolate-derived ana-
logues, a considerable amount of diversity was allowed
about the ester moiety while maintaining sub-
micromolar potencies. In general, the benzyl ester ana-
logues exemplified by 9f and 9g displayed the highest
functional potencies. This class retained the earlier

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N. S. Cutshall et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1517–1520
selectivity against GRO-a versus FMLP but removed
potential oxidative liabilities of the thioglycolate-based
analogues.
References and Notes
1. (a) Harada, A.; Mukaida, N.; Matsushima, K. Mol. Med.
Today 1996, 2, 482. (b) Murdoch, C.; Finn, A. Blood 2000, 95,
3032.
Potential replacements for the ester functional group
were investigated (Table 3). In general, aliphatic amides
displayed limited potency against GRO-a driven
chemotaxis while the aniline-based derivatives showed
modest potencies. Compound 10f was the only analogue
tested that showed activity <1 mM. Again, these ana-
logues have a similar selectivity profile as the series
previously described. Several explanations of the
attenuated overall potencies of this series is possible.
The compounds may display a reduced ability to pass
through the cell membrane or have increased enzymatic
stability toward hydrolases in the cell which could result
in lower intracellular concentrations of 5h. Alter-
natively, this series may simply have a reduced affinity
for the CXCR2 receptor.
2. Palovich, M. R.; Widdowson, K.; Elliott, J. D.; White, J.
R.; Sarau, H. M.; Rutledge, M. C.; Bi, J.; Dede, K. A.; Ben-
son, G. M.; Griswold, D. E.; Martin, L. D.; Nie, H.; Schmidt,
D. M.; Foley, J. J. Abstracts of Papers, Part 2, 219th National
Meeting of the American Chemical Society, San Francisco,
CA, March 26–30, 2000; American Chemical Society:
Washington, DC, 2000; MEDI 338.
3. Li, J. J.; Yue, W. S.; Trivedi, B. K.; Miller, S. R.; Connor, D.
T.; Roth, B. D.; Low, J. E.; Heilig, D. J.; Hunt, S.; Carson, K.
G.; Glynn, R. A.; Ye, Q.; Luly, J. R.; Yang, W.; Qin, S. Abstracts
of Papers, 220th National Meeting of the American Chemical
Society, Washington, D.C., August 20–24, 2000; American
Chemical Society: Washington, DC, 2000; MEDI 144.
4. Cutshall, N. S.; Ursino, R.; Kucera, K. A.; Latham, J.;
Ihle, N. C. Bioorg. Med. Chem. Lett. 2001, 11, 1951.
5. Windscheif, P.; Vogtle, F. Synthesis 1994, 87.
6. Neutrophil isolation and GRO-a or FMLP chemotaxis was
conducted according to Frevert, C. W.; Wong, V. A.; Good-
man, R. B.; Goodwin, R.; Martin, T. R. J. Immunol. Methods
1998, 213, 41. The following is a representative procedure: The
lower chambers of a ChemoTx plate (Neuro Probe, Gaithers-
burg, MD) were filled with 29 mL of 50 nM GRO-a (Pepro-
Tech, Rocky Hill, NJ) and test compound. The empty upper
chambers were affixed to the lower (plate), and 25 mL of PMN
suspension of calcein AM tagged cells (3E6 cells/mL), without
(control) or with 0.04–40 mM test compound, preincubated
30 min, was added to the upper wells. Test compounds were
dissolved in DMSO (100%) at 20 mM and diluted in assay
buffer to the desired concentrations; final DMSO concen-
tration was 0.2%. Neutrophil migration proceeded for 40 min at
37ꢀC in a humidified incubator with 5% CO2. After removing
nonmigrated cells from the top of the plate, migrated cells were
quantified by reading fluorescence on a Wallac Victor. Max-
imum chemotactic response was determined by cells to which no
compound was added (positive control), whereas the negative
control (unstimulated) was defined by the absence of chemokine
in the lower chamber. The ratio of the positive to negative con-
trols represents the chemotactic index of the cells.
Lastly, a limited amount of SAR was explored where
the amide functionality at the 3 position of the nicoti-
nanilide core was reversed (Scheme 4). Compound 13,
representative of this class of antagonists, showed an
IC50=360 nM against GRO-a driven neutrophil
chemotaxis. Since significant potency and selectivity of
this series has been retained it is expected that addi-
tional changes to this portion of the molecule may be
tolerated.
In conclusion, the synthesis and evaluation of a novel
series of potent and selective GRO-a driven neutrophil
chemotaxis inhibitors have been described. Initial data
indicates that this class of compounds may function as
ester prodrugs by which they penetrate the cell and are
then converted to the bioactive species via enzymatic
hydrolysis to the corresponding carboxylate salt.10
Additional studies are planned to further explore the
cellular signaling pathway by which these compounds
function.
7. Ng, H. P.; May, K.; Bauman, J. G.; Ghannam, A.; Islam,
I.; Liang, M.; Horuk, R.; Hesselgesser, J.; Snider, R. M.;
Perez, H. D.; Morrissey, M. M. J. Med. Chem. 1999, 42, 4680.
8. Binding assays were conducted at Panlabs (Bothell, WA)
and were based on the displacement of [125I]-IL-8 from CHO
cell membranes expressing human CXCR2 receptors.
Acknowledgements
The authors would like to thank Dr. Breda Twomey
for performing assays on these compounds and Mr.
Ted Baughman and Dr. Scott Jeffrey, for helpful
discussions.
9. Hall, D. A.; Beresford, I. J. M.; Browning, C.; Giles, H. Br.
J. Pharmacol. 1999, 126, 810.
10. Robbins, B. L.; Srinivas, R. V.; Kim, C.; Bischofberger,
N.; Fridland, A. Antimicrob. Agents Chemother. 1998, 42, 612.