Identification and initial evaluation of 4-N-aryl-[1,4]diazepane ureas as potent CXCR3 antagonists

Article abstract of DOI:10.1016/j.bmcl.2005.09.020

The identification and evaluation of aryl-[1,4]diazepane ureas as functional antagonists of the chemokine receptor CXCR3 are described. Specific examples exhibit IC₅₀ values of ～60 nM in a calcium mobilization functional assay, and dose-dependently inhibit CXCR3 functional response to CXCL11 (interferon-inducible T-cell α chemoattractant/I-TAC) as measured by T-cell chemotaxis, with a potency of approximately 100 nM.

Full text of DOI:10.1016/j.bmcl.2005.09.020

Bioorganic & Medicinal Chemistry Letters 16 (2006) 200–203
Identification and initial evaluation of 4-N-aryl-[1,4]diazepane
ureas as potent CXCR3 antagonists
Andrew G. Cole,a,* Ilana L. Stroke,a Marc-Raleigh Brescia,a Srilatha Simhadri,a
Joan J. Zhang,a Zahid Hussain,a Michael Snider,b Christopher Haskell,b Sofia Ribeiro,b
Kenneth C. Appell,a Ian Hendersona and Maria L. Webba
aPharmacopeia Drug Discovery, Inc., PO Box 5350, Princeton, NJ 08543, USA
bBerlex Biosciences, 2600 Hilltop Drive, PO Box 4099, Richmond, CA 94804, USA
Received 15 August 2005; revised 2 September 2005; accepted 7 September 2005
Available online 5 October 2005
Abstract—The identification and evaluation of aryl-[1,4]diazepane ureas as functional antagonists of the chemokine receptor
CXCR3 are described. Specific examples exhibit IC50 values of ꢀ60 nM in a calcium mobilization functional assay, and dose-depen-
dently inhibit CXCR3 functional response to CXCL11 (interferon-inducible T-cell a chemoattractant/I-TAC) as measured by T-cell
chemotaxis, with a potency of approximately 100 nM.
Ó 2005 Elsevier Ltd. All rights reserved.
Chemokines are a class of chemotactic cytokines, be-
tween 70 and 90 amino acids in length, that play an
important role in inflammatory and immune responses.
They are subdivided into different classes based on the
structural separation of conserved cysteine residues in
the chemokine sequence. The CXC subtype of chemo-
kines displays a single amino acid residue between the
first two conserved cysteines.1,2 CXCR3 is a chemokine
receptor belonging to the superfamily of seven trans-
membrane spanning G-protein-coupled receptors
(GPCRs) and is highly expressed in activated T cells.
Its natural chemokine ligands, CXCL9 (monokine in-
duced by interferon-c/Mig), CXCL10 (interferon-induc-
ible protein 10/IP-10), and CXCL11 (interferon-
inducible T-cell a-chemoattractant/I-TAC),1,2 are
thought to play a key role in directing activated T cells
to sites of inflammation.3 Consequently, inhibition of
CXCR3 activation provides potential therapeutic uses
for the treatment of a number of inflammatory disorders
including inflammatory bowel disease, multiple sclero-
sis, rheumatoid arthritis, and diabetes.4 As such, there
is significant interest in the identification of small-mole-
cule antagonists of CXCR3.5
High-throughput screening of an extensive series of
Encoded Combinatorial Libraries on Polymeric Support
(ECLiPSTM
)
libraries (90 Libraries, >4 million com-
6
pounds) employing a FLIPR7-based calcium mobiliza-
tion assay resulted in the identification of 4-aryl-
[1,4]diazepane ethyl ureas (6) as functional antagonists
of CXCR3. Distinct structural features were observed
in the screening hits, particularly the [1,4]diazepane ring
system and urea functionality, and further investigation
was initiated to define the SAR of this class of com-
pounds. Initial benzamide analogs 6 were prepared in
six steps as shown in Scheme 1. Thus, 4-fluoro-3-nitro
benzoic acid (1a) was converted to acid chloride 1b
and reacted with a primary amine to provide amide 2.
N-Arylation of homopiperazine with 2 and subsequent
urea formation with ethyl isocyanate was followed by
nitro-reduction to the aniline 5 using sodium hydrosul-
fite8 under mildly basic conditions. Final functionaliza-
tion of 5 with a range of acid chlorides provided the
target compounds 6 in good overall yield. A similar syn-
thetic approach was used to generate carboxylic acid 11
(Scheme 2) to allow direct modification of the carbox-
amide substituent on the central aromatic ring via
EDC-mediated amide formation.
A range of urea and carbamate analogs were also effi-
ciently constructed through utilization of intermediates
3 and 8 to investigate alternative functionalizations of
the diazepane ring.
Keywords: CXCR3; Chemokine; GPCR; Antagonist; Diazepane;
Urea.
*
Corresponding author. Tel.: +1 609 452 3653; fax: +1 609 655
4187; e-mail: acole@pcop.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.020

A. G. Cole et al. / Bioorg. Med. Chem. Lett. 16 (2006) 200–203
201
O
O
O
NO2
N
NO2
F
NO2
F
HN
HN
X
(ii)
(iv)
Ar
(iii)
NH
3
1a X=OH
Cl
(i)
Cl
2
1b X=Cl
Cl
Cl
O
O
O
O
NO2
NH
NH2
N
HN
HN
HN
(v)
(vi)
O
O
O
N
N
N
N
N
Cl
Cl
Cl
4
5
6a-o
NHEt
NHEt
NHEt
Cl
Cl
Cl
Scheme 1. Reagents and conditions: (i) (COCl)2, CH2Cl2, cat. DMF, 0–25 °C; (ii) 2,4-dichlorophenethylamine, CH2Cl2, 0 °C, 98% from 1a; (iii)
homopiperazine (3.0 equiv), DMF, 0 °C; (iv) EtNCO, CH2Cl2, 25 °C, 80% from 2; (v) Na2S2O4, NaHCO3, dioxane/MeOH, H2O, 0–25 °C, 68%; (vi)
ArCOCl, Et3N, cat. DMAP, CH2Cl2, 25 °C, 57–86%.
O
O
O
O
NO2
F
(ii)
NO2
N
(iv), (v)
NH2
N
NO2 (iii)
F
X
MeO
MeO
MeO
O
NH
N
7
8
9
1a X=OH
1b X=Cl
(i)
NHEt
O
O
O
O
Cl
O
Cl
O
O
Cl
O
NH
N
NH
(vi)
(vii)
NH
N
(viii)
HN
R
HO
MeO
O
N
N
N
N
11
6p-6bb
10
NHEt
NHEt
NHEt
Scheme 2. Reagents and conditions: (i) (COCl)2, CH2Cl2, cat. DMF, 0–25 °C; (ii) MeOH, 0 °C, 99% from 1a; (iii) homopiperazine (3.0 equiv), DMF,
0 °C; (iv) EtNCO, CH2Cl2, 25 °C, 95% from 7; (v) Na2S2O4, NaHCO3, dioxane/MeOH, H2O, 0–25 °C, 78%; (vi) 3-ClPhCOCl, Et3N, cat. DMAP,
CH2Cl2, 25 °C, 54%; (vii) LiOH, H2O/dioxane, 85%; (viii) EDC, HOBt, CH2Cl2, R-NH2, 61–89%.
Functional antagonism was measured by monitoring the
reduction in CXCL11-stimulated calcium release for a
cell line (HEK293) over-expressing recombinant human
CXCR3 and the chimeric G protein Gqi5,9 following
stimulation with 40 nM CXCL11 in a FLIPR-based cal-
cium mobilization assay10 (no agonist activity was ob-
served for the compounds described). As a specificity
control, antagonists were tested at 25 lM for effect on
activation of endogenous muscarinic receptors in the
same cell line by carbachol. Inhibition was found to be
specific for CXCR3. Furthermore, no cytotoxicity was
observed in CXCR3 HEK293 cells at 100 lM com-
pound concentration using a WST-1 readout (Roche)
after a 24 h incubation.11
ethyl derivative (6v). Incorporation of the racemic
trans-cyclopropylphenethyl derivative (6w) also dis-
played a moderate increase in potency in comparison
to the parent phenethyl system. The unsubstituted ben-
zylic analog (6y) resulted in only moderate activity.
However, as observed for the phenethyl-based analogs,
a ꢀ5-fold increase in potency was realized on inclusion
of a 4-chloro substituent (6z). Small aliphatic compo-
nents such as isopropyl (6aa) and cyclopropylmethyl
(6bb) resulted in a significant reduction in activity, dis-
playing IC50 values in the micromolar range. Com-
pounds involving removal of the amide carbonyl
acceptor (not shown) displayed no activity versus
CXCR3.
An investigation of the secondary carboxamide substitu-
ent (R) and the benzamide substituent (Ar) was initially
conducted to probe the steric and electronic require-
ments at these two positions for CXCR3 activity. A
strong preference for phenethyl-based functionality
was observed for the secondary amide, with 2,4-dichloro
substitution (6a–o) being optimal based on the analogs
synthesized (Table 1). Phenethyl-based systems incorpo-
rating mono halo substitution (6p–u) also maintained
reasonable activity superior to the unsubstituted phen-
Evaluation of the benzamide substituent (Ar) showed
good activity for the unsubstituted system (6a) and
electron-deficient systems substituted in the 3-position
(6c, 6e, and 6g), achieving potencies of 60–80 nM.
The analogous 2-substituted derivatives (6b and 6f)
resulted in a ꢀ10-fold reduction in potency. Inclusion
of simple heterocycles was tolerated (6k–o); however,
the 4-pyridyl analog (6m) displayed significantly better
potency than the corresponding 3-pyridyl derivative
(6l) (Table 1).

202
A. G. Cole et al. / Bioorg. Med. Chem. Lett. 16 (2006) 200–203
Table 1. IC50 values for 4-N-aryl-[1,4]diazepane ethyl ureas
Ar
Table 2. IC50 values for 1-substituted-4-N-aryl-[1,4]diazepanes
O
O
O
NH
O
Cl
HN
R
NH
N
HN
O
N
N
O
R
6
HN
N
Cl
12
IC50 SEMa
Cl
Compound R
Ar
(lM)
Compound
R
IC50 SEMa (lM)
6a
6b
6c
6d
6e
6f
2,4-Dichlorophenethyl Ph
2,4-Dichlorophenethyl 2-Cl Ph
2,4-Dichlorophenethyl 3-Cl Ph
0.07 0.00
0.70 0.14
0.06 0.01
6c
–NHEt
–NHnPr
–NHiPr
–NHnBu
–NHMe
–NMe2
–OMe
0.06 0.01
0.06 0.01
0.06 0.01
0.05 0.00
0.80 0.12
0.89 0.21
9.8 1.9
12a
12b
12c
12d
12e
12f
12g
2,4-Dichlorophenethyl 3-MeO Ph 0.17 0.03
2,4-Dichlorophenethyl 3-CN Ph
2,4-Dichlorophenethyl 2-F Ph
2,4-Dichlorophenethyl 3-F Ph
2,4-Dichlorophenethyl 4-F Ph
2,4-Dichlorophenethyl 3,4-F2 Ph
2,4-Dichlorophenethyl 3,5-F2 Ph
0.08 0.03
0.59 0.11
0.06 0.01
0.20 0.04
0.29 0.07
0.24 0.12
6g
6h
6i
–OEt
13.0 1.7
a IC50 standard error mean in response to stimulation with 40 nM
CXCL11.
6j
6k
6l
2,4-Dichlorophenethyl 2-Thiophene 0.13 0.02
2,4-Dichlorophenethyl 3-Pyridyl
2,4-Dichlorophenethyl 4-Pyridyl
2,4-Dichlorophenethyl 3-Thiophene 0.49 0.01
3.10 0.40
0.25 0.05
in the same HEK293 cell line.9 Stimulation with 100 nM
CXCL10 in the FLIPR-based calcium mobilization as-
say resulted in an IC50 value of 20 nM for 6c, demon-
strating that antagonism is not limited to receptor
activation by CXCL11.
6m
6n
6o
6p
6q
6r
2,4-Dichlorophenethyl 2-Furan
0.64 0.11
0.19 0.04
0.30 0.08
0.12 0.02
0.34 0.10
0.34 0.10
0.32 0.06
0.51 0.08
0.24 0.06
0.42 0.06
1.25 0.41
0.25 0.04
2.16 0.44
2.15 0.12
2-Chlorophenethyl
3-Chlorophenethyl
4-Chlorophenethyl
2-Fluorophenethyl
3-Fluorophenethyl
4-Fluorophenethyl
Phenethyl
3-Cl Ph
3-Cl Ph
3-Cl Ph
3-Cl Ph
3-Cl Ph
3-Cl Ph
3-Cl Ph
6s
The functional activity of 6c was further investigated
with respect to its ability to inhibit CXCL11 induced
T-cell chemotaxis in vitro. Cultured human T cells ex-
press both CXCR3 and CCR7, which respond to the li-
gands CXCL11 and CCL21 (secondary lymphoid-tissue
chemokine/SLC), respectively. Compound 6c successful-
ly inhibited the CXCR3 (CXCL11)-mediated migration,
whereas migration through CCR7 (CCL21) was unaf-
fected (Fig. 1).12 In other experiments 6c was able to
dose-dependently inhibit CXCR3-mediated migration
with an IC50 of approximately 100 nM.
6t
6u
6v
6w
6x
6y
6z
6aa
6bb
Cyclopropylphenethylb 3-Cl Ph
3,4-Dimethoxyphenethyl 3-Cl Ph
Benzyl
3-Cl Ph
3-Cl Ph
3-Cl Ph
3-Cl Ph
4-Chlorobenzyl
iPr
Cyclopropylmethyl
a IC50 standard error mean in response to stimulation with 40 nM
CXCL11.
b trans-Racemic.
The specificity of 6c was also evaluated against a panel
of 14 human GPCRs in radioligand binding assays.13
The GPCRs included adrenergic receptors a2B and
b2; chemokine receptors CCR1, CCR2B, CCR4,
CXCR1 (IL-8A), and CXCR1 (IL-8B); dopamine recep-
tors D1, D2S, D3, and D4.2; muscarinic receptors M1 and
M2; and the serotonin receptor 5-HT1A. For each of
Modification of the ethyl urea functionality (Table 2) to
generate the n-propyl (12a), i-propyl (12b), and n-butyl
(12c) analogs resulted in maintenance of 660 nM activ-
ity. Carbon deletion to generate the methyl urea (12d)
and incorporation of the N,N-dimethyl tertiary urea
(12e) resulted in >10-fold reduction in potency. Devia-
tion from the urea functionality to generate the methyl
carbamate (12f) and ethyl carbamate (12g) derivatives
resulted in the loss of activity to P10 lM.
250
DMSO
500 nM 6c
200
Alteration of the central core of 6 to investigate the
importance of the disubstituted [1,4]-diazepane ring sys-
tem was also conducted (not shown). Reduction in ring
size to generate the piperazinyl analog, and incorpora-
tion of isosteric acyclic variants exhibit no activity
against CXCR3. This suggests that the geometry adopt-
ed by the [1,4]-diazepane is a preferred conformation of
the 1- and 4-substituents for activity.
150
100
50
0
CXCL11 (100 nM) CCL21 (300 nM)
Chemokine Attractant
buffer
The activity of 6c was also measured by monitoring the
reduction in CXCL10 (IP-10)-stimulated calcium release
Figure 1. Inhibition of CXCL11 induced T-cell chemotaxis by 6c.
aArbitrary fluorescence units.

A. G. Cole et al. / Bioorg. Med. Chem. Lett. 16 (2006) 200–203
203
7. Fluorometric Imaging Plate Reader, Molecular Devices.
these GPCRs, less than 50% inhibition was observed at
10 lM.
8. Jones, T. R.; Smithers, M. J.; Taylor, M. A.; Jackman, A.
L.; Calvert, A. H.; Harland, S. J.; Harrap, K. R. J. Med.
Chem. 1986, 29, 468.
9. A CXCR3 cDNA clone (sequence as listed in GenBank
Accession No. BD195161) and the chimeric G protein
Gqi5, cotransfected into HEK293 cells, were used to
construct a stable cell line.
10. HEK293/CXCR3 Gqi5 cells were seeded at 10,000 cells
(25 lL) per well in poly (D-lysine)-treated 384-well plates
(Costar, black clear-bottomed cell culture-treated) 1–2
days prior to the assay. Culture medium was then
removed and replaced with 25 lL of 50% cell culture
medium/50% Calcium Plus Dye (Molecular Devices)/
2.5 mM probenecid. For dye loading, plates were incu-
bated for 30 min at 37 °C/5% CO2, followed by equili-
bration to room temperature for 30–90 min. Test
compounds were diluted in 20 lL HanksÕ balanced salt
solution (HBSS)/20 mM HEPES, pH 7.5/1% DMSO/
0.1% BSA/2.5 mM probenecid. 12.5 lL test compound
(or as controls, CXCL11 to 40 nM or buffer, also with
1% DMSO) was added in the FLIPR 384 to dye-loaded
cells. 12.5 lL CXCL11, in HBSS/20 mM HEPES, pH 7.5/
0.1% BSA, was then added to the cells/test compound, to
40 nM, and fluorescence measured over 3 min.
In conclusion, novel small-molecule functional antago-
nists of the chemokine receptor CXCR3 have been iden-
tified, as determined by inhibition of both calcium
mobilization and T-cell chemotaxis on stimulation with
CXCL11. In addition, compounds were shown to be
non-cytotoxic and display good general selectivity.
These initial findings provide a structural template for
the further optimization of potency and evaluation of
pharmacokinetic properties.
References and notes
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T cells were washed and resuspended to
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0.05% BSA). Multiscreen MIC chemotaxis chambers
(3 lm, Millipore) were used adding cells (100 lL,
106 cells/well) to the top chamber with 100 nM CXCL11
or 300 nM CCL21 antagonist (130 lL) added to the
bottom chamber, followed by incubation (90 min at
37 °C). Following migration, Calcein-AM (14 lL, 25 lg/
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10 lM.