Lead optimisation of the N1 substituent of a novel series of indazole arylsulfonamides as CCR4 antagonists and identification of a candidate for clinical investigation

Article abstract of DOI:10.1016/j.bmcl.2012.02.104

Synthesis and preliminary SAR of the N1 substituent of a novel series of indazole sulfonamide chemokine receptor 4 (CCR4) antagonist is reported. Compound 7r was identified for further development.

Full text of DOI:10.1016/j.bmcl.2012.02.104

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2730–2733
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Lead optimisation of the N1 substituent of a novel series of indazole
arylsulfonamides as CCR4 antagonists and identification of a candidate
for clinical investigation
Panayiotis A. Procopiou a, , Alison J. Ford b, Rebecca H. Graves c, David A. Hall b, Simon T. Hodgson a,
⇑
Yannick M.L. Lacroix a, Deborah Needham a, Robert J. Slack b
a Department of Medicinal Chemistry, Respiratory CEDD, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
b Department of Respiratory Biology, Respiratory CEDD, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
c Department of Drug Metabolism and Pharmacokinetics, Respiratory CEDD, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1
2NY, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and preliminary SAR of the N1 substituent of a novel series of indazole sulfonamide chemokine
receptor 4 (CCR4) antagonist is reported. Compound 7r was identified for further development.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 13 January 2012
Revised 27 February 2012
Accepted 28 February 2012
Available online 7 March 2012
Keywords:
Chemokine receptor 4
CCR4
MDC
TARC
Antagonist
Chemokines are a group of small chemotactic proteins of 8–
10 kDa, which possess four conserved cysteines.1 More than 50
chemokines have been identified, and 17 chemokine receptors
have been described in humans. Chemokines are classified into
four groups designated CC, CXC, C and CX3C based on the arrange-
ment of the first two conserved cysteines located at or near the
N-terminus of each protein.2 Chemokines exert their effects by
binding to cell surface receptors, which belong to the seven trans-
membrane G-protein-coupled receptor family, and play an impor-
tant role in immune responses by directing leucocytes to inflamed
tissues. Ten CC chemokine receptors have been identified so far
and named as CC-chemokine receptor 1, 2 etc. Most chemokine
receptors recognise more than one chemokine and several chemo-
kines bind to more than one receptor.3 CCL17, also known as thy-
mus activation-regulated chemokine (TARC) and CCL22, also
known as macrophage-derived chemokine (MDC), are a pair of
CC chemokines that bind to CC-chemokine receptor 4 (CCR4).2
CCR4 is mainly expressed in T helper 2 (Th2) cells that produce
interleukin (IL)-4, IL-5 and IL-13.4 Th2 cytokines in inflamed tis-
sues lead to eosinophilia, high levels of serum IgE and mast cell
activation, all of which are believed to contribute to the patholog-
ical consequences of allergic diseases.5 Elevated levels of TARC and
MDC as well as accumulation of CCR4-positive cells have been ob-
served in lung biopsy samples from patients with atopic asthma
following allergen challenge.6,7 It has therefore been suggested
that interaction between CCR4 and its ligands plays an important
role in asthma. CCR4 antagonists represent a novel therapeutic
intervention in diseases where CCR4 has a central role in patho-
genesis. Progress in the discovery of small-molecule CCR4 antago-
nists as immunomodulatory agents was reviewed by Purandare
and Somerville in 2006.8 A number of publications on CCR4 antag-
onists have appeared in the literature since the last review9–18 and
these antagonists are classed into two broad chemo types (Fig. 1).
The first chemo type includes lipophilic amines such as Bristol
Myers Squibb compound 1,10 Pfizer compound 2,11 Astellas com-
pound 3,13 and the Daiichi Sankyo compound 4.17 The second che-
mo type is a series of pyrazine arylsulfonamides, such as the
AstraZeneca compound 519 and Ono compound 6.20 In this com-
munication we are disclosing our own studies on the identification
and the lead optimisation of the N1 substituent of a novel class of
indazole arylsulfonamides 7.
The assays used in this study were a [35S]-GTP
assay and/or a 125I-TARC radioligand binding assay both using
cS competition
⇑
Corresponding author.
E-mail address: pan.a.procopiou@gsk.com (P.A. Procopiou).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.104

P. A. Procopiou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2730–2733
2731
Cl
Cl
N
HN
N
HN
N
O
N
N
Cl
O
N
O
O
O
N
N
N
N
H
N
N
H
N
N
H
1
N
O
H
N
3
2
F
Cl
Cl
Cl
O
S
O
H
O
S
O
S
N
O
S
O
N
N
H
N
H
N
N
N
O
F
F
N
Br
N
O
Cl
O
N
N
O
O
N
N
N
7
O
4
6
5
HO
R
Figure 1. Structures for some recently published CCR4 antagonists.
recombinant human CCR4-expressing CHO cell membranes bound
to SPA beads with output measured on a Wallac Microbeta Trilux
scintillation counter. Our lead molecule 7 (R = H) was identified
from a focused pharmacophore array approach and had a pIC50
position on the benzyl group of 7a which tolerated a substituent.
These analogues were synthesised by the routes shown in Schemes
1 and 2, depending on the commercial availability of the benzyl
substituent.21 The synthetic route outlined in Scheme 1 started
with reaction of 2-fluoro-6-methoxybenzonitrile (8) with hydra-
zine hydrate in 1-butanol at reflux. The resulting 3-amino-indazole
9 was protected at the N1 position by reacting with di-tert-butyl
dicarbonate in the presence of triethylamine and DMAP to give
10. Reaction of 10 with one equivalent of 5-chloro-2-thi-
ophenesulfonyl chloride gave a mixture of mono-sulfonylated
of 6.4 in the GTP
the BMS compound 1 and Ono compound 6, which were used as
standards, had pIC50 of 8.3 and 8.4, respectively, in the GTP
cS assay and 6.6 in the TARC assay. By comparison
cS
assay, and 9.1 and 8.5, respectively, in the TARC assay (Table 1).
Lead optimisation started by investigating substitution with Cl-,
F-, Me-, CN- and MeO-groups, and attempting to identify the
Table 1
In vitro data pIC50 for GTP
polymerisation pA2
c
S and pKi for I125 TARC SPA binding assays, CLND solubility, lipophilicity (daylight clogP), and human whole blood TARC induced F-actin
Cl
S
O
H
O
S
O
N
N
N
R
Compd
R
GTP
cS pIC50
I125 TARC SPA pKi (n)
CLND solub. (lg/mL)
clogP
hWB actin polym. pA2 (n)
1
6
NA
NA
H–
8.26 0.01 (131)
8.41 0.02 (127)
6.41 0.01 (2)
ND
6.8 0.1 (2)
ND
7.64 0.02 (5)
6.49 0.02 (2)
7.49 0.13 (2)
7.32 0.03 (2)
7.04 0.09 (2)
7.26 0.03 (5)
7.1 0.1 (2)
6.38 0.08 (2)
7.50 0.04 (10)
6.89 0.03 (2)
ND
9.1 0.1 (11)
8.5 0.1 (8)
6.6 0.1 (10)
6.8 0.1 (2)
6.9 0.1 (2)
6.6 0.2 (2)
7.7 0.1 (5)
6.6 0.0 (2)
7.4 0.0 (2)
7.3 0.0 (2)
7.0 0.1 (2)
7.4 0.1 (5)
6.5 0.1 (2)
5.9 0.1 (2)
7.9 0.0 (2)
ND
145
170
1
12
12
7
46
74
4.9
4.4
4.6
4.5
4.5
4.5
4.3
3.5
3.6
3.6
3.6
3.1
3.4
3.1
3.4
3.5
3.4
3.1
3.4
3.7
7.3 0.3 (4)
6.6 0.1 (22)
5.2 0.2 (4)
ND
ND
ND
5.6 0.1 (2)
ND
5.2 0.2 (2)
ND
6.0 0.1 (3)
5.8 0.2 (6)
ND
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
2-MeO–
3-MeO–
4-MeO–
3,4-Di-MeO–
2-CH2OH
3-CH2OH
4-CH2OH
3-CH2NH2
3-CONH2
127
123
17
140
167
160
124
151
104
167
146
162
3-CONHMe
3-CONMe2
ND
6.0 0.1 (7)
ND
3-CH2NHCOCH3
3-CH2NMeCOCH3
4-CH2NHCOCH3
3-CH2NHCOCH2OH
3-CH2NHCOCH(Me)OH
3-CH2NHCOCMe2OH
6.8 0.1 (2)
7.8 0.2 (2)
8.1 0.1 (2)
8.0 0.0 (2)
ND
7.56 0.06 (2)
7.9 0.1 (3)
7.83 0.02 (95)
5.7 0.1 (2)
6.2 0.0 (2)
6.2 0.1 (11)
NA not applicable. ND not determined. When n <3 SEM is the SD.

2732
P. A. Procopiou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2730–2733
Cl
S
O
S
O
NH2
N
H
N
O
O
O
NH2
N
b
CN
F
a
O
c
d
N
N
N
N
O
H
O
8
9
10
11
O
O
Cl
Cl
Cl
Cl
S
Cl
S
S
Cl
S
O
S
O
S
O
N
S
O
S
S
O
O
S
O
O
O
S
O
N
O
O
S
O
N
O
e
f
O
g
N
N
7
N
N
N
N
O
H
O
12
13
14
R
Scheme 1. Reagents and conditions: (a) NH2NH2ÁH2O, nBuOH, 92%; (b) BOC2O, Et3N, DMAP, DCM, 67%; (c) 5-chloro-2-thiophenesulfonyl chloride (1 equiv), pyridine, DCM,
22%; (d) 5-chloro-2-thiophenesulfonyl chloride (3 equiv), pyridine, DCM, 45 °C, 3 days, 80%; (e) TFA, DCM, 100%; (f) substituted benzyl alcohol, PPh3, iPrO2CN@NCO2Pri, THF,
70 °C, 20–80%; (g) 2 M NaOH, H2O, MeOH, 50 °C, 52–85%.
Cl
O
S
O
S
SEM
Cl
c
O
S
O
Cl
d
O
S
O
S
SEM
S
SEM
O
N
O
O
N
N
a
b
N
11
N
N
N
7
N
N
H
O
O
16
17
15
R
Scheme 2. Reagents and conditions: (a) SEM-Cl, DIPEA, DCM, 100%; (b) Na2CO3, H2O, DMF, 85 °C, 74%; (c) ArCH2Br, K2CO3, DMF, 44–88%; (d) nBu4NF, THF, 90–95%.
product 11, and bis-sulfonylated product 12, which were separable
by chromatography. Treatment of 10 or 11 with excess sulfonyl
chloride gave 12, which was then de-protected with TFA to give
13. Mitsunobu reaction of 13 with a variety of benzyl alcohols gave
the N1 alkylated product 14, accompanied by a small amount of N2
alkylation, which was readily removed by chromatography. Hydro-
lysis of 14 with aqueous sodium hydroxide in methanol gave target
test compounds 7. The synthetic route outlined in Scheme 2 com-
menced with reaction of 11 with SEM-Cl to give 15, removal of the
BOC protecting group to give 16, alkylation of 16 with an appropri-
ate benzyl halide to give 17, and finally cleavage of the SEM pro-
tecting group to give 7. It was possible to use benzyl bromides
instead of benzyl alcohols to produce compounds 14 by the meth-
od outlined in Scheme 1, however, it was not possible to use benzyl
alcohols to produce compounds 17 by the method outlined in
Scheme 2. Analogues bearing Cl-, F-, Me-, CN-, and MeO-groups
were very lipophilic (clogP 4.6–5) and had very low solubility as
measured by ChemiLuminescent Nitrogen Detection (CLND) solu-
The three regioisomeric analogues bearing a hydroxymethyl
substituent (7f, 7g and 7h) were prepared by the method shown
in Scheme 1, and for all three analogues their solubility increased
to >70 lg/mL. The meta-isomer (7g) and para-isomer (7h) were
more potent than the ortho-isomer (7f). Alcohol 7g had similar
activity to 7a on TARC-induced increases in the F-actin content
(a cytoskeletal reorganisation event of chemotaxis) of human
CD4+ CCR4+ lymphocytes measured in human whole blood (pA2
5.2). Introduction of the meta-aminomethyl moiety (7i) increased
the whole blood potency even further to 6.0, despite a drop in its
GTPcS potency to 7.0. The solubility of 7i was also reduced to
17
lg/mL and this might be due to the zwitterionic character of
the compound. The primary benzamide 7j was more potent than
the secondary amide 7k, which in turn was more potent than the
tertiary amide 7l in the TARC assay, which is consistent with the
requirement for a hydrogen-bond donor. In the GTPcS the tertiary
amide 7l was again less potent, however, the primary and second-
ary amides were equipotent in this assay. The solubility of all three
bility assay (<10
lg/mL). This high throughput kinetic solubility as-
benzamides increased to 140–167 lg/mL, and furthermore the
say involves addition of aqueous buffer to a test compound DMSO
solution over a period of time until the compound precipitates. The
potency of these lipophilic compounds (not shown in Table 1) was
not significantly different from that of 7a, apart from the MeO-
substituted analogues (Table 1), which highlighted a small prefer-
ence for meta-substitution (7c pIC50 6.9), and in the case of the 3,4-
disubstituted analogue 7e a significant increase in potency (pIC50
7.7). Since the solubility of all of the above analogues was very
whole blood activity of 7j was 5.8, similar to that of 7i. The acetam-
ide 7m was prepared by acetylation of 7i with acetic anhydride,
and was one of the more potent analogues in vitro (7.9 in the TARC
assay, and 7.5 in the GTPcS assay). In addition 7m was potent in
the whole blood assay (pA2 6.0) making it a candidate for further
investigation in pharmacokinetic studies. The N-methyl acetamide
7n was significantly less potent, further supporting the hypothesis
that a hydrogen-bond donor was preferred for an interaction with
the receptor. The para-acetamide analogue 7o was about ten-fold
less potent than 7m (6.8 vs 7.9 in the TARC assay confirming the
low (7–46
lg/mL), lead optimisation focused on the introduction
of solubilising groups on the N1 benzylic moiety.

P. A. Procopiou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2730–2733
2733
Table 2
comprehensive SAR report together with additional studies on 7r
will be published in due course.
CD rat and Beagle dog pharmacokinetic data for 7m and 7r
Species
CD Rat
7m
7r
Acknowledgments
iv Dose (mg/kg)
po Dose (mg/kg)
n Value (iv/po)
Cl (mL/min/kg)
Vdss (L/kg)
1.0
1.0
2/2
5
0.57
1.6
Complete
1.0
1.0
2/2
15
1.9
2.5
85
We thank Heather Barnett for technical assistance, and the
Screening and Compound Profiling Department at GlaxoSmithKline
for generating the GTPcS data.
T
½
(h)
F%
Supplementary data
Beagle dog
iv Dose (mg/kg)
po Dose (mg/kg)
n Value (iv/po)
Cl (mL/min/kg)
Vdss (L/kg)
0.54
1.1
4/4
18
3.2 1.1
1.6 0.2
0.55
1.1
4/4
9.0 1.9
2.2 0.8
2.6 0.5
97 27
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.02.104. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
5
T
½
(h)
F%
49
5
References and notes
meta-position as the preferred site for substitution of the benzylic
ring. The solubility of 7m was 124 g/mL, therefore, increasing the
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l
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groups became our next target. The analogues 7p, 7q and 7r bear-
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with the appropriate carboxylic acid.21 Analogues 7q and 7r pro-
vided increased potency, and whole blood activity. The tertiary
alcohol 7r had slightly lower potency than the two reference com-
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pounds 1 and 6 in the GTPcS assay, lower lipophilicity (clogP 3.7),
and whole blood activity of 6.2. The permeability of compounds
7m and 7r in MDCK cells was measured as 254 and 132 nm/s,
respectively, and was sufficiently high to progress the compounds
to pharmacokinetic studies. All animal studies were ethically
reviewed and carried out in accordance with Animals (Scientific
Procedures) Act 1986 and the GSK Policy on the Care, Welfare
and Treatment of Laboratory Animals. Compounds 7m and 7r were
studied in vivo in rat and dog, and the data are shown in Table 2. In
the rat the acetamide 7m had complete oral bioavailability, low
volume of distribution and low clearance [6% liver blood flow
(LBF)]. In the dog bioavailability was reduced to 49%, volume was
moderate, however, the clearance increased substantially to 58%
LBF. In the rat the a-hydroxy-isobutyramide 7r had high oral bio-
availability, moderate volume and low clearance (18% LBF), and a
half-life of 2.5 h which was longer than 7m. The bioavailability of
7r in the dog was 97% and was higher than 7m. The volume of
7r was moderate, however, its clearance (29% LBF) was lower than
7m, resulting in a longer half-life (2.6 h). In contrast the reference
compound 1 had negligible oral bioavailability in an in-house rat
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time in human clinical data will be reported shortly.22
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No in vivo pharmacodynamic data were generated for 7r due to
the differences in immunology between animals and human, the
complexity of chemokine systems across species, and the lack of
convincing evidence that CCR4 can drive inflammation in animal
models of asthma. Greater relevance was placed on in vitro studies
using target human CD4+ CCR4+ and CD45RO+ T cells and biopsies
taken from atopic asthmatics, supernatants from which were used
to induce chemotaxis of autologous memory T-cells. A more
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Aug 2010).
22. Cahn, A.; Hodgson, S. T.; Wilson, R.; Robertson, J.; Watson, J.; Beerahee, M.;
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tolerability, pharmacokinetics and pharmacodynamics of GSK2239633,
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a