Identification and structure-activity relationships of 1-aryl-3-piperidin-4-yl-urea derivatives as CXCR3 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2006.10.088

The synthesis and biological evaluation of a series of 1-aryl-3-piperidin-4-yl-urea derivatives as small-molecule CXCR3 antagonists is described. SAR studies resulted in significant improvement of potency and physicochemical properties and established the key pharmacophore of the series, and led to the identification of 9t, which exhibits an IC₅₀ of 16 nM in the GTPγS³⁵ functional assay.

Full text of DOI:10.1016/j.bmcl.2006.10.088

Bioorganic & Medicinal Chemistry Letters 17 (2007) 697–701
Identification and structure–activity relationships of 1-aryl-3-
piperidin-4-yl-urea derivatives as CXCR3 receptor antagonists
Daniel R. Allen, Amanda Bolt, Gayle A. Chapman, Roland L. Knight,
Johannes W. G. Meissner,* David A. Owen and Robert J. Watson
UCB, Inflammation Discovery, Granta Park, Great Abington, Cambridge CB21 6GS, UK
Received 22 September 2006; revised 26 October 2006; accepted 27 October 2006
Available online 2 November 2006
Abstract—The synthesis and biological evaluation of a series of 1-aryl-3-piperidin-4-yl-urea derivatives as small-molecule CXCR3
antagonists is described. SAR studies resulted in significant improvement of potency and physicochemical properties and established
the key pharmacophore of the series, and led to the identification of 9t, which exhibits an IC50 of 16 nM in the GTPcS35 functional
assay.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The chemokine receptor CXCR3 has been of interest for
a number of years as a potential target for treatment of
inflammatory diseases such as multiple sclerosis,1
asthma2 and rheumatoid arthritis.3 CXCR3 belongs to
the superfamily of seven transmembrane spanning
G-protein-coupled receptors (GPCRs) and is predomi-
nantly expressed by activated T cells, NK cells and B
cells. It binds to three members of the CXC chemokine
family: MIG (CXCL9), IP-10 (CXCL10) and I-TAC
(CXCL11) resulting in receptor activation and cellular
chemotaxis. Antagonising CXCR3 may result in de-
creased trafficking of activated Th1 effector cells and
memory T cells to sites of inflammation. CXCR3 may
also be involved in the retention of activated lympho-
cytes within inflamed tissues following activation with
MIG, IP-10 or I-TAC. Blocking strategies have demon-
strated efficacy in several murine disease models includ-
ing arthritis,4 transplant rejection,5 diabetes6 and
inflammatory bowel disease.7 Small-molecule CXCR3
antagonists have been reported in the literature,8 includ-
ing T487 18a and TAK779 28b (which blocks CXCR3 as
well as CCR2 and CCR59) (see Fig. 1).
selected for their hit-like molecular properties and simi-
larities to known GPCR ligands. From this screen, sev-
eral hit compounds were identified with potency in the
micromolar range, including the piperidine urea 3,
which was chosen as a starting point for a hit to lead
medicinal chemistry programme. Importantly this com-
pound provided excellent potency against both the hu-
man and murine receptors (Table 1), demonstrating
that cross-species activity is considerably more achiev-
able in this system than has been reported in develop-
ment of antagonists of CCR1–310 (see Fig. 2).
In order to support the medicinal chemistry project, a
radiolabelled GTPcS35 assay was developed,11 which
reconfirmed the HTS hits.
As shown in Table 1, there remained significant chal-
lenges in developing 3 into a lead-like antagonist series.
The compounds showed poor aqueous solubility, high
logD and potent CYP2D6 inhibitory activity. Neverthe-
less, the structure was ideally suited to rapid analogue
generation using solution-phase parallel synthesis, and
detailed investigation of the template was carried out
with the goal of identifying potent and drug-like mole-
cules for lead optimisation.
In order to identify hit compounds for our CXCR3 pro-
ject, a screen of 15,000 compounds was carried out using
a FLIPr-based calcium flux assay. The compounds were
Initial variation of the aryl urea moiety was carried out
on 0.1 mmol scale, coupling the aminopiperidine 412
with a set of 48 commercially available isocyanates to
give ureas 5 (Scheme 1). The final compounds were
obtained by adding the crude reaction mixtures to Oasis
Keywords: Chemokine; CXCR3; SAR; Antagonist.
*
Corresponding author. Tel.: +44 1223 896504; fax: +44 1223
896400; e-mail: Hans.Meissner@ucb-group.com
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.088

698
D. R. Allen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 697–701
O
O
O
O
N
N
H
N+
N
N
N
N
Cl
O
CF3O
Figure 1. CXCR3 antagonists.
Table 1. Properties of compound 3
Table 2. hCXCR3 Ki values for ureas 5 in GTPcS35 functional assay
hCXCR3 mCXCR3 CYP 2D6 Aq sol
(mg/ml)b
logD hMic
Compound
Ar
Kia (nM)
Ki (lM)a Ki (lM)a (lM)
(lg/ml/min)
3
2-Naphthyl
1-Naphthyl
110
IA
0.110
0.40
0.6
0.001
4.95
72
5a
5b
5c
5d
5e
5f
3-(Trifluoromethyl)phenyl
3-Chlorophenyl
88
a GTPcS35 assay.11
201
104
637
36
b At pH 6.5.
3-(Trifluoromethoxy)phenyl
3-Acetylphenyl
3,5-Bis(trifluoromethy)lphenyl
3-Fluoro-5-(trifluoromethyl)phenyl
3,5-Dichlorophenyl
4-Phenoxyphenyl
5g
5h
5i
47
41
O
N
N
H
N
306
IA
H
5j
3-Cyanophenyl
4-Cyanophenyl
3
5k
5l
IA
4-tert-Butylphenyl
4-(2,6-Dichloropyridyl)
Thien-3-yl
1040
3400
IA
Figure 2. CXCR3 hit structure.
5m
5n
5o
5p
3-Methoxycarbonylphenyl
2-(5-Phenylthienyl)
660
108
O
N
N
a IA <50% at 10 lM.
Ar
N
N
H2N
H
H
Investigation of the right-hand side cycloaliphatic group
was carried out by reductive amination of the piperidine
6 with a range of aldehydes chosen to explore the steric
requirements for CXCR3 potency (Scheme 2).
Scheme 1. Reagents: ArNCO, CH2Cl2, 60–85%.
MCX acidic ion exchange cartridges. Washing with eth-
yl acetate, methanol and water to remove unwanted by-
products; then elution with methanolic ammonia gave
the products as solids with purity greater than 95%.
As shown in Table 2, a broad range of aromatic groups
gave potent CXCR3 antagonists, the most active being
the 3,5-bis-trifluoromethyl urea 5f with a Ki of 36 nM.
However, the most potent compounds retained
logD > 4 and poor aqueous solubility, an exception
being 5e which provided improved properties at the
expense of affinity for the receptor.
As shown in Table 3, CXCR3 activity in this template is
highly dependent on the nature of the right-hand side
group, with only the myrtenyl derivative 7h giving
potency comparable to 3. It was striking that the cyc-
looctyl derivative 7d was inactive. A range of benzylic
groups such as 7f were included in the set, none of which
gave measurable antagonism of the receptor.
Compound 7h was further characterised and found,
unsurprisingly, to have a similarly high logD and poor
O
a, b
O
NH
N
O
+
N
N
H
H2N
NCO
H
6
O
N
R
c
N
N
H
H
Scheme 2. Reagents: (a) CH2Cl2; (b) 1 N HCl in Et2O, 86% over two steps; (c) RCHO, NaBH(OAc)3, trimethyl orthoformate/CH2Cl2 (1:1),
40–65%.

D. R. Allen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 697–701
Table 3. hCXCR3 Ki values for ureas 7 in GTPcS35 functional assay
699
N
OHC
a, b
NH
+
Compound
R
Kia (nM)
H2N
BocHN
8
3
Cyclooctene
Cyclohexyl
110
IA
IA
IA
IA
IA
IA
7a
7b
7c
7d
7e
7f
7g
7h
7i
1-Norbornene
1-Cyclohexene
Cyclooctane
NO2
O
NCO
F
c, d, e
( )n
O
2,4,4-Trimethylpentane
3,4-Dichlorophenyl
f
X
X
X = H, F
(2,6,6-Trimethylcyclohex-1-enyl)methyl IA
(À)Myrtenyl
X
290
IA
O
( )n
O
N
R(+)-4-Isopropylcyclohexen-1-yl
Phenylmethyl
Phenylethyl
7j
7k
1300
2200
O
N
N
H
H
a IA <50% at 10 lM.
Scheme 3. Reagents and conditions: (a) NaBH(OAc)3, trimethyl
orthoformate/CH2Cl2 (1:1), 76%; (b) 1 N HCl in Et2O; (c) ROH,
NaH, DMF, 55–70%; (d) H2 Pd/C, EtOH, 95%; (e) triphosgene,
DiPEA, CH2Cl2, 0 ꢁC, quant.; (f) CH2Cl2, quant.
solubility to compound 3, with the cycloaliphatic group
having a strong contribution to the lipophilicity of the
template.
O
a, b, c
NCO
Alteration of the central 4-aminopiperidine core was
investigated and it was found that changes such as
replacement with 3-aminopyrrolidine, 3-aminoazetidine,
3-aminomethylmorpholine, piperazine and homopiper-
azine (Fig. 3) all resulted in significant loss of potency.
Subsequent work therefore continued to focus on the
4-aminopiperidine template.
S
S
Br
OH
Br
d, e
O
N
R
S
N
N
H
H
Scheme 4. Reagents and conditions: (a) (COCl)2, cat. DMF, CH2Cl2;
(b) NaN3, acetone/water, 0 ꢁC; (c) D, PhMe, 82% over three steps; (d)
8, CH2Cl2, 78%; (e) RB(OH)2, Pd(PPh3)4, aq Na2CO3, THF, reflux,
35–62%.
In order to further investigate the SAR of the left-hand
side aromatic group, particularly towards improving the
drug-like properties, the myrtenylpiperidine 8 was pre-
pared and reacted with a series of commercially avail-
able arylisocyanates. The synthetic approach to the
more elaborated ureas was carried out either through
the coupling of a constructed isocyanate with amine 8
(Scheme 3; 9o, 9p, and 9q) or through palladium cross-
couplings with a common arylbromide (Scheme 4; 9i,
9j, and 9k).
Table 4. hCXCR3 Ki values for ureas 9 in GTPcS35 functional assay
O
N
Ar
N
N
H
H
9
Compound Ar
Kia (nM)
9a
9b
9c
9d
9e
9f
Phenyl
2000
73
In exploring the aryl SAR for the myrtenyl template, the
3,5-bis(trifluoromethyl)phenyl (9b) was again found to
give potent inhibitory activity as was the 3-fluoro-5-tri-
fluoromethylphenyl derivative 9t, which was found to
have a Ki against murine CXCR3 of 227 nM. The regio-
isomeric derivatives 9s and 9u were significantly less ac-
tive. Polar substituents were included with the aim of
identifying potent antagonists with tractable physico-
chemical properties. The 3-methoxycarbonyl phenyl
compound 9v was moderately active (376 nM), but
hydrolysis to the carboxylic acid 9w or reduction
to the hydroxymethyl derivative 9x gave inactive
3,5-Bis(trifluoromethyl)phenyl
3-Acetylphenyl
3,5-Difluorophenyl
258
171
429
31
3-Methoxyphenyl
3-Trifluoromethoxyphenyl
4-Trifluoromethoxyphenyl
5-Phenylthien-2-yl
9g
9h
9i
175
191
39
5-(4-Trifluoromethylphenyl)thien-2-yl
5-(3-Methoxyphenyl)thien-2-yl
5-(2-Chlorophenyl)thien-2-yl
5-Bromopyridin-3-yl
9j
29
46
9k
9l
171
70
9m
9n
9o
9p
9q
9r
9s
9t
5-(Thien-2-yl)pyridin-3-yl
5-(Thien-3-yl)pyridin-3-yl
3-(Tetrahydrofuran-3-yloxy)phenyl
107
141
3-(Tetrahydrofuran-3-yloxy)-5-fluorophenyl 83
O
O
O
O
N
R
N
R
3-(Tetrahydropyran-4-yloxy)phenyl
3-(Morpholin-4-ylmethyl)phenyl
2-Fluoro-5-trifluoromethylphenyl
3-Fluoro-5-trifluoromethylphenyl
4-Fluoro-5-trifluoromethylphenyl
3-Methoxycarbonylphenyl
191
Ar
Ar
N
R
Ar
Ar
N
N
N
N
IA
229
16
N
H
N
H
H
H
H
H
O
9u
9v
9w
9x
81
O
Ar
Ar
376
IA
IA
R
N
N
N
H
N
H
N
R
N
N
H
N
H
3-Hydroxycarbonylphenyl
3-Hydroxymethylphenyl
O
N
R
a IA <50% at 10 lM.
Figure 3. Piperidine replacements investigated.

700
D. R. Allen et al. / Bioorg. Med. Chem. Lett. 17 (2007) 697–701
Table 5. hCXCR3 Ki values, physicochemical properties and PK data for selected compounds
b
c
Compound
Ki (nM)
Solubilitya
logD
CLint
(lL/min/mg protein)
CLp
(mL/min/kg)
(lg/ml)
3
110
70
0.1
4
4.95
3.47
3.87
3.00
4.21
2.35
226
324
287
97
nt
9m
9o
9p
9t
112
222
nt
141
83
927
874
23
16
28
67
66
nt
10
55
34.5
nt, not tested.
a At pH 6.5.
b Mouse microsomal clearance at 0.5 lM.
c Mouse clearance. Compounds dosed 1 mg/kg iv.
F
F
N+
N
O
O
CF3
N
N
CF3
N
N
H
H
H
H
I
Scheme 5. Reagents: MeI, Et2O.
compounds. We found that the 3-methoxy derivative 9e
gave modest potency (429 nM); however, modulation to
the 3-tetrahydrofuran ether 9o significantly improved
activity (141 nM). Addition of a 5-fluoro substituent
gave 9p which combined good potency (83 nM) with
much improved physicochemical properties (logD 3.0).
poor CaCO2 permeability and low oral absorption, qua-
ternisation was not pursued further.
In summary, a new series of small-molecule non-quater-
nary CXCR3 antagonists has been discovered. SAR
studies resulted in significant improvement in potency,
and established the key requirements for affinity in the
series. These findings provide a structural CXCR3
template for the further optimisation of potency and
pharmacokinetic properties. We will report on these
findings in due course.
Phenylthiophene 9h was found to be active and further
derivatives 9i, 9j and 9k gave excellent potency but poor
physicochemical properties. In order to improve this,
the meta-bromopyridine 9l was prepared from 5-bro-
monicotinic acid with identical reagents and conditions
as in Scheme 4 to give 9m. The logD of this compound
was found to be 3.47, which demonstrated that incorpo-
ration of more hydrophilic aromatic groups can signifi-
cantly improve the physicochemical properties whilst
retaining good potency (see Table 4).
Acknowledgment
The authors thank Dr. James Pease from Imperial
College London for performing the chemotaxis assay.
As this chemical series was providing a good range of
affinities and promising initial physicochemical proper-
ties, we evaluated selected compounds in preliminary
DMPK.
References and notes
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Although the compounds have moderate to high micro-
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2). For this reason, we investigated the effect of quater-
nisation in this series. Compound 9t was treated with
methyl iodide to give 10 as a mixture of cis/trans isomers
(see Scheme 5).
4. Salomon, I.; Netzer, N.; Wildbaum, G.; Schif-Zuck, S.;
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Healthcare; 5 mg/ml) in the presence or absence of GDP
(10 lM), saponin (50 lg/ml) and GTPcS35 (Amersham;
300 pM). Assays were carried out in 96-well plates where
membranes and ligand (ITAC/IP10; R&D Systems) were
incubated for 60 min (rt) in assay buffer (20 mM Hepes,
100 mM NaCl, 10 mM MgCl, 1 mM EDTA (pH 7.4), and
0.1% BSA) prior to addition of GTPcS35. Following a
further 2-h incubation at rt, plates were centrifuged and
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membrane was then put in place and 20 ll of
the appropriately treated cells was placed on top of
the filter, in duplicate (200,000 cells per well). The
chambers were then incubated at 37 ꢁC with 95% CO2
in a humid environment for 5 h. After incubation, the
upper surface of the filter was scraped to remove the un-
migrated cells, the filter removed and the cells migrating
into the lower chamber were counted on a haemocy-
tometer. Data are shown as the number of cells/high
power field and are means of three separate experiments.
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11. GTPcS35 binding was determined using CHO-hCXCR3
membranes (Euroscreen; 20 lg/ml), SPA beads (GE