Synthesis and structure-activity relationships of indazole arylsulfonamides as allosteric CC-chemokine receptor 4 (CCR4) antagonists

Article abstract of DOI:10.1021/jm301572h

A series of indazole arylsulfonamides were synthesized and examined as human CCR4 antagonists. Methoxy- or hydroxyl- containing groups were the more potent indazole C4 substituents. Only small groups were tolerated at C5, C6, or C7, with the C6 analogues being preferred. The most potent N3-substituent was 5-chlorothiophene-2-sulfonamide. N1 meta-substituted benzyl groups possessing an α-amino-3-[(methylamino)acyl]- group were the most potent N1-substituents. Strongly basic amino groups had low oral absorption in vivo. Less basic analogues, such as morpholines, had good oral absorption; however, they also had high clearance. The most potent compound with high absorption in two species was analogue 6 (GSK2239633A), which was selected for further development. Aryl sulfonamide antagonists bind to CCR4 at an intracellular allosteric site denoted site II. X-ray diffraction studies on two indazole sulfonamide fragments suggested the presence of an important intramolecular interaction in the active conformation.

Full text of DOI:10.1021/jm301572h

Article
pubs.acs.org/jmc
Synthesis and Structure−Activity Relationships of Indazole
Arylsulfonamides as Allosteric CC-Chemokine Receptor 4 (CCR4)
Antagonists
Panayiotis A. Procopiou,*,† John W. Barrett,§ Nicholas P. Barton,∥ Malcolm Begg,‡ David Clapham,⊥
Royston C. B. Copley,∥ Alison J. Ford,‡ Rebecca H. Graves,§ David A. Hall,‡ Ashley P. Hancock,†
Alan P. Hill,∥ Heather Hobbs,† Simon T. Hodgson,† Coline Jumeaux,† Yannick M. L. Lacroix,†
Afjal H. Miah,† Karen M. L. Morriss,† Deborah Needham,† Emma B. Sheriff,§ Robert J. Slack,‡
Claire E. Smith,§ Steven L. Sollis,† and Hugo Staton†
‡
§
†Departments of Medicinal Chemistry, Respiratory Biology, Drug Metabolism and Pharmacokinetics, Respiratory CEDD,
GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
∥Computational and Structural Chemistry Department, Platform Technology & Science, GlaxoSmithKline Medicines Research
Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
⊥Exploratory Development Sciences, Platform Technology & Science, Research & Development, GlaxoSmithKline, Park Road, Ware,
Hertfordshire SG12 ODP, United Kingdom
S
* Supporting Information
ABSTRACT: A series of indazole arylsulfonamides were synthesized and examined as human CCR4
antagonists. Methoxy- or hydroxyl- containing groups were the more potent indazole C4 substituents.
Only small groups were tolerated at C5, C6, or C7, with the C6 analogues being preferred. The most
potent N3-substituent was 5-chlorothiophene-2-sulfonamide. N1 meta-substituted benzyl groups
possessing an α-amino-3-[(methylamino)acyl]− group were the most potent N1-substituents. Strongly
basic amino groups had low oral absorption in vivo. Less basic analogues, such as morpholines, had good
oral absorption; however, they also had high clearance. The most potent compound with high absorption
in two species was analogue 6 (GSK2239633A), which was selected for further development. Aryl
sulfonamide antagonists bind to CCR4 at an intracellular allosteric site denoted site II. X-ray diffraction
studies on two indazole sulfonamide fragments suggested the presence of an important intramolecular
interaction in the active conformation.
as macrophage-derived chemokine (MDC), bind to the
orthosteric binding site of CC-chemokine receptor 4
(CCR4).3 Upon exposure to allergen, dendritic cells within
the lung (or other tissue) secrete MDC and TARC (also
produced by endothelial cells) which can recruit Th2 cells from
the circulation. The T cells can then migrate along this
chemokine gradient to the dendritic cells. Upon maturation, the
dendritic cells migrate from the inflamed tissue to local lymph
nodes where the MDC and TARC which they produce may
recruit further T cells to the inflammatory response. Elevated
levels of TARC and MDC as well as accumulation of CCR4-
positive cells have been observed in lung biopsy samples from
patients with atopic asthma following allergen challenge.6,7
Thus CCR4 antagonists represent a novel therapeutic
intervention in diseases where CCR4 has a central role in
pathogenesis, such as asthma, atopic dermatitis,8 allergic
bronchopulmonary aspergillosis,9 cancer,10 the mosquito-
borne tropical diseases, such as Dengue fever,11 and allergic
INTRODUCTION
■
Chemokines are a group of small, basic proteins of 8−10 kDa,
which together with their receptors mainly regulate the
trafficking of leucocytes down a chemoattractant gradient.1,2
Chemokines possess four conserved cystein residues, and they
are classified into four groups designated CC, CXC, C, and
CX3C based on the arrangement of the first two conserved
cysteine residues located at or near the N-terminus of each
protein.3 Ten CC chemokine receptors have been identified so
far and named as CC-chemokine receptor 1, 2, 3, etc. Most
chemokine receptors recognize more than one chemokine and
several chemokines bind to more than one receptor.1 CCR4
belongs to the 7-TM domain G-protein-coupled receptor
family and is mainly expressed in T helper 2 (Th2) cells. The
latter are a subset of CD4-positive T helper cells that produce
interleukin (IL)-4, IL-5, and IL-13.4 Th2 cytokines in inflamed
tissues lead to eosinophilia, high levels of serum IgE, and mast
cell activation, all of which contribute to the pathogenesis of
allergic diseases.5 CC Chemokine ligand 17 (CCL17),
previously known as thymus activation-regulated chemokine
(TARC), and CC chemokine ligand 22 (CCL22), also known
Received: October 24, 2012
© XXXX American Chemical Society
A
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Figure 1. Structures for some recently published CCR4 antagonists.
a
Scheme 1
a
Reagents and conditions: (a) NH2NH2·H2O, 1-butanol, reflux, 80−92%; (b) KOH, DMSO, 3-cyanobenzyl chloride, 52−84%; (c) 5-chloro-2-
thiophenesulfonyl chloride, pyridine, 29−87%; (d) 1 M LiAlH4 solution in ether, THF, 55−77%; (e) Ac2O, Et3N, DCM, 38−89%; (f) BBr3, DCM,
33%.
rhinitis.12 Progress in the discovery of small-molecule CCR4
antagonists as immunomodulatory agents was reviewed by
Purandare and Somerville in 2006.13 A number of publications
on CCR4 antagonists have appeared in the literature since the
last review,14−23 and these antagonists appear to belong to two
chemical categories (Figure 1). The first category includes
lipophilic heteroarenes possessing basic amino groups such as
Bristol Myers Squibb (BMS) compound 1,15 Astellas
compound 2,18 and the Daiichi Sankyo compound 3.22 The
AstraZeneca (AZ) 2,3-dichlorobenzenesulfonamide 424 and
Ono 4-methylbenzenesulfonamide 525 belong to the second
category of pyrazine arylsulfonamides. More recently, we have
disclosed our own preliminary studies on a novel class of
indazole arylsulfonamides including 6, which we believe is the
first small-molecule clinical candidate targeting the CCR4
receptor.26
The AstraZeneca group have reported the discovery of a
novel mechanism for antagonism of CCR4 and CCR5
receptors with a series of small-molecule pyrazine sulfonamides,
which interact with an intracellular allosteric site on the
receptor.27 The precise location of this allosteric binding site
was not determined, however, it was suggested that it might be
a generic site for chemokine receptors, or even more broadly
for class A G-protein-coupled receptors. In our recent
communication disclosing sulfonamide 6, we focused our
investigations entirely on the variation of the indazole N1
substituent.26 In this publication, we present a fuller account of
the structure−activity relationships (SAR) of the indazole
arylsulfonamides covering the C4, C5, C6, C7, N3-sulfonamide,
and additional N1-substitutions, together with extensive in vivo
pharmacokinetic studies on 10 analogues, and shed light on the
spatial requirement of the sulfonamide antagonists based on
small-molecule X-ray diffraction studies.
B
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a
Scheme 2
a
Reagents and conditions: (a) NH2NH2·H2O, EtOH, 70 °C, 87%; (b) BOC2O, DMAP, Et3N, MeCN, 56%; (c) 5-chloro-2-thiophenesulfonyl
chloride, DCM, pyridine, 56%; (d) TFA, DCM, 43%; (e) N-BOC-3-(hydroxymethyl)benzylamine (16), PPh3, PrO2CNNCO2Pri, THF, 60 °C,
i
96%; (f) 2 M NaOH, MeOH, THF, 45 °C, 1 h; (g) TFA, DCM; (h) Ac2O, Et3N, DCM; (i) K2CO3, MeOH, 19%.
a
Scheme 3
a
Reagents and conditions: (a) DIBAL-H, PhMe; (b) NaBH4, MeOH, 17−48%; (c) TFA, DCM, 66%; (d) Ac2O, Et3N, DCM; (e) 2 M aq NaOH,
MeOH, 20 °C, 43%; (f) MeMgBr, THF, 30−77%.
chloro-2-thiophenesulfonyl chloride in pyridine gave the
sulfonamides 11a−d, the cyano group of which was reduced
with LiAlH4 to give the benzylamines 12a−d. Acetylation of
12a−d with acetic anhydride gave the required amides 7a−d.
On some occasions, 7 was accompanied by formation of a small
amount of diacetylated products, which were converted back to
7 by trans-esterification with methanol over anhydrous
potassium carbonate prior to workup. The phenolic analogue
7e was prepared from 12a by treatment with boron tribromide
to give 12e (33%), followed by acetylation with acetic acid in
CHEMISTRY
■
The SAR investigations commenced with examination of the
C4 substituted indazoles 7 outlined in Scheme 1. Reaction of
the appropriately substituted 2-fluoro-benzonitrile 8a−d with
hydrazine hydrate in n-butanol gave the amino indazoles 9a−d,
which were selectively alkylated with 3-cyanobenzyl chloride in
DMSO in the presence of powdered KOH to provide 10a−d.
This procedure to introduce the N1 substituent was an
improvement over our previous protocol which required the
use of protecting group chemistry.26 Reaction of 10a−d with 5-
C
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a
Scheme 4
a
Reagents and conditions: (a) DAST, DCM, 5 h, 15%; (b) TFA, DCM, 54%; (c) Ac2O, Et3N, DCM; (d) 2 M aq NaOH, MeOH, 60 °C, 3 h, 36%.
Table 1
the presence of HATU and diisopropylethylamine in DMF
(69%).
Mitsunobu reaction of 15 with N-BOC-3-(hydroxymethyl)-
benzylamine28 (16) gave 17 (96%), which was first hydrolyzed
with NaOH to give the monosulfonamide 18, then deprotected
with TFA, acetylated with Ac2O, and finally treated with
potassium carbonate in MeOH to cleave overacetylated
product, and provide 7f.
The C4-hydroxymethyl analogue 7g was obtained by
DIBAL-H reduction of the nitrile 17, followed by NaBH4
reduction of the resulting aldehyde 19 to give 20, cleavage of
the N-BOC protecting group with TFA to give the benzylamine
The 4-cyano analogue 7f was prepared by the route outlined
in Scheme 2. Reaction of 3-fluoro-1,2-benzenedicarbonitrile
(8f) with hydrazine hydrate in ethanol gave the 3-amino-
indazole 9f (87%), which was then protected with di-tert-butyl
dicarbonate in the presence of DMAP and Et3N in MeCN to
give 13 in 56% yield. Treatment of 13 with excess 5-chloro-2-
thiophenesulfonyl chloride gave the bis-sulfonamide 14 in 56%
yield, which was deprotected with TFA to give 15 in 43% yield.
D
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21, followed by acetylation with Ac2O, and finally hydrolysis
with NaOH (Scheme 3). The C4-acetyl analogue 7h was
prepared by methyl magnesium bromide addition to the nitrile
7f (77%), and the secondary alcohol 7i was prepared by NaBH4
reduction of 7h (48%). Addition of MeMgBr to ketone 7h gave
the tertiary alcohol 7j (30%).
Scheme 4 outlines the preparation of the C4-difluoromethyl
analogue 7k which was prepared from the aldehyde 19 using
diethylamino-sulfur trifluoride (DAST) in DCM to give 22 in
15% yield. The latter was deprotected with TFA to give the
amine 23 (54%), which was then acetylated and deprotected to
give 7k in 36% yield for the two steps.
Further amide analogues of 6 containing either an ether (a−
d) or amino (e−k) group were prepared by acylation of 12a
with the appropriate carboxylic acid in DMF in the presence of
N-[3-(dimethylamino)propyl]-N′-ethylcarbodiimide hydro-
chloride, N-hydroxybenztriazole hydrate (HOBT), and N-
methylmorpholine as base to give amides 24a−k (Table 1).
The amino-acids (e−i) used in the amide coupling reactions
were N-BOC protected and were subsequently deprotected
with either 4 M HCl in dioxane or with TFA in DCM. The N-
methyl-morpholine enantiomers 24j and 24k were made from
the racemic amino acid and separated by preparative chiral
HPLC. The two enantiomers were labeled isomer A and B as
their absolute configuration was not determined. Compound
24l was prepared from 12a and 3-oxetanone in the presence of
sodium triacetoxyborohydride in THF in 94% yield.
EtOH gave 35 in 25%. The latter was reacted with 3-
chloromethyl benzonitrile in the presence of powdered KOH in
DMSO to give 36, which was reduced with sodium
borohydride in the presence of nickel(II) chloride and di-tert-
butyl dicarbonate in THF and MeOH to give 37. Sulfonylation
of 37 gave 38, which was then deprotected with TFA in DCM
to give the amine 39. Acylation of 39 with AcOH or 2-acetoxy-
isobutyryl chloride and hydrolysis gave the two amides 33a and
33b, respectively.
Scheme 8 outlines the synthesis of the 6-fluoro analogues
40a and 40b. Treatment of 3,5-difluoroanisole with α,α-
dichloromethyl methyl ether in the presence of TiCl4 gave the
aldehyde 42, which was converted to the nitrile 43 with
hydroxylamine-O-sulfonic acid and then cyclized to the
indazole 44. Alkylation with 3-chloromethylbenzonitrile gave
45, which was converted to the sulfonamide 46 and then
reduced with LiAlH4 to give the amine 47. The latter was
acetylated to 40a and also converted to the amide 40b as
previously.
The 7-fluoro analogue 48 was obtained by the route outlined
in Scheme 9. Treatment of 2,3-difluoro-6-methoxybenzonitrile
49 with hydrazine hydrate in N-methylpyrrolidinone (NMP)
gave the indazole 50, which was alkylated with 3-chlorome-
thylbenzonitrile as before to provide 51. The latter was reacted
with 5-chloro-2-thiophenesulfonyl chloride to give 52, which
was then reduced with LiAlH4 to 53 and subsequently
acetylated with Ac2O to give 48.
Scheme 10 shows the synthetic route by which analogues of
6 possessing different sulfonamide groups were synthesized.
Benzonitrile 10a was reduced with LiAlH4 to give the diamine
54, which was selectively reacted with 2-acetoxy-isobutyryl
chloride at the benzylamine group to give 55. The latter was
reacted with the appropriate arylsulfonyl chloride and then
trans-esterified with MeOH in anhydrous potassium carbonate
to give the target sulfonamides 56a−d.
Compound 6 was prepared on a larger scale by reaction of
12a with 2-acetoxy-isobutyryl chloride in DCM and Et3N to
give 25 in 77% yield, followed by trans-esterification of the
acetate ester with methanol over anhydrous K2CO3 (71%)
(Scheme 5).
a
Scheme 5
RESULTS AND DISCUSSION
■
All compounds in Table 2 were tested as human CCR4
antagonists in vitro. Antagonist potency was determined by a
[35S]-GTPγS radioligand competition functional assay using
recombinant CCR4-expressing CHO cell membranes adhered
to WGA-coated Leadseeker SPA beads in assay buffer (20 mM
HEPES, 10 mM MgCl2, 100 mM NaCl, 0.05% BSA, 40 μg/mL
saponin at pH 7.4) with output measured on a Wallac
Microbeta Trilux scintillation counter.30 Another assay using
human whole blood was used as a secondary screen to
determine potency against the native receptor for the more
potent compounds in the primary assay. The assay quantified
cytoskeletal reorganization (formation of filamentous (F-)
actin) which occurs in a variety of cells in response to
chemoattractants and is a prelude to chemotaxis. This was
achieved by staining the F-actin with a fluorescent derivative of
phalloidin, which binds with high affinity and specificity to the
interface between actin monomers in F-actin. The response was
measured as an increase in the fluorescence intensity of the
target cell population in a flow cytometer and was expressed as
a pA2. In this assay, human CD4+ CCR4+ lymphocytes were
identified by staining with antibodies to CD4 and CCR4.
Ligand lipophilicity efficiency index (LLE) is a useful metric
introduced by Leeson and Springthorpe for assessing the
potency and lipophilicity of drug-like molecules during lead
optimization.31 We have used the more recently introduced
LLEAT, which combines lipophilicity, potency, and size and
a
Reagents and conditions: (a) 2-acetoxy-isobutyryl chloride, Et3N,
DCM, 77%; (b) K2CO3 (3 equiv), MeOH, 2 h, 20 °C, 71%.
The three pyridyl analogues of 7a, regioisomers 26, 27, and
28, were prepared from the bis-sulfonamide 2928 according to
Scheme 6, which outlines only the synthesis of regioisomer 26.
Thus Mitsunobu coupling of 29 with 2-(hydroxymethyl)-4-
pyridinecarbonitrile 3029 gave 31, which was reduced with
LiAlH4 and hydrolyzed with aqueous NaOH in MeOH to give
the benzylamine 32. The latter was acetylated with AcOH in
the presence of HATU and Et3N to give 26. The other two
regioisomers 27 and 28 were synthesized by an analogous
method starting from 29 and the appropriate hydroxymethyl-
pyridinecarbonitrile.
The 5-fluoro-analogues 33a and 33b were prepared by the
route shown in Scheme 7. Reaction of 3,6-difluoro-2-
methoxybenzonitrile (34) with hydrazine monohydrate in
E
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a
Scheme 6
a
Reagents and conditions: (a) PPh3, tBuO2CNNCO2But, THF, 80 °C, 88%; (b) LiAlH4, THF, 1 h; (c) 2 M NaOH, H2O, MeOH, 70 °C, 53%;
(d) AcOH, HATU, Et3N, DCM, 59%.
a
Scheme 7
a
Reagents and conditions (a) NH2NH2·H2O, EtOH, 80 °C, 25%; (b) 3-chloromethylbenzonitrile, KOH, DMSO, −5 °C, 48%; (c) NaBH4, NiCl2,
BOC2O, THF, MeOH, 65%; (d) 5-chloro-2-thiophenesulfonyl chloride, pyridine, DCM, 55%; (e) TFA, DCM, 45%; (f) (for 33a) AcOH, HATU,
DIPEA, DMF, 27%; (g) (for 33b) 2-acetoxyisobutyryl chloride, pyridine, DCM; (h) KOH, H2O, MeOH, 49%.
makes comparisons with conventional ligand efficiency (LE)
easier at the commonly used threshold value of 0.3 kcal/mol.32
In Table 2 the LLEAT, the chromatographic log D (chrom log D
at pH 7.4) and the chemiluminescent nitrogen detection
(CLND) kinetic solubility are included for all test compounds
in this study.33 The high throughput CLND solubility assay
involves addition of aqueous buffer to a test compound DMSO
solution over a period of time until the compound precipitates.
Finally, the pKa of the sulfonamide moiety for selected
compounds was measured and presented in Table 2. The test
compounds were compared with two standards, compounds 1
and 5, together with our own sulfonamides 6 and 7a.26
We started our SAR investigations by focusing our attention
on C4 substitutions with compounds 7b−7k. The compounds
with LLEAT higher than 7a were the three hydroxyl compounds
7e, 7g, and 7i. All three compounds had higher affinity than 7a,
ranging from 7.7 to 8.2, and lower log D by more than a log
unit. In addition, the CLND solubilities of 7e and 7i were
F
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a
Scheme 8
a
Reagents and conditions: (a) Cl2CHOMe, TiCl4, DCM, 57%; (b) NH2OSO3H, H2O, 110 °C, 3 h, 95%; (c) NH2NH2·H2O, n-BuOH, 110 °C, 21%;
(d) 3-chloromethylbenzonitrile, KOH, DMSO, 20%; (e) 5-chloro-2-thiophenesulfonyl chloride, pyridine, CHCl3, 45%; (f) LiAlH4, THF, 18%; (g)
(for 40a) Ac2O, Et3N, CHCl3, 52%; (h) (for 40b) 2-acetoxyisobutyryl chloride, pyridine, DCM; (i) K2CO3, MeOH, 51%.
a
Scheme 9
a
Reagents and conditions. (a) NH2NH2·H2O, NMP, 150 °C; (b) 3-chloromethylbenzonitrile, KOH, DMSO, 56%; (c) 5-chloro-2-thiophenesulfonyl
chloride, pyridine, DCM, 30%; (d) LiAlH4, THF, 36%; (e) Ac2O, Et3N, DCM, 34%.
higher than 7a. The higher affinity did not translate to the
whole blood actin polymerization assay but remained at
approximately the same level as 7a, with the highest potency
of 6.3 for alcohol 7i. On the basis of these data, compounds 7e,
7g, and 7i were selected for further investigation. It was
interesting to note that both the AZ and Ono compounds, 4
and 5, possessed an ortho-alkoxy substituent capable of
hydrogen bonding with the sulfonamide NH forming a five-
membered ring. Our own candidate 6 had a C4 methoxy
substituent capable of hydrogen bonding with the sulfonamide
NH, forming a six-membered ring. The effect of hydrogen
bonding to the acidic sulfonamide NH would be to alter its
acidity, leading us to hypothesize that there might be a
relationship between acidity and potency. The effects of ortho
substituents in the dissociation of substituted N-phenyl
benzenesulfonamides were reported by Ludwig, who demon-
strated that the pKa increased in the case of an ortho-methoxy
group in the aniline ring.34 The pKa of all the C4 substituted
analogues (7a−k) was measured and is shown in Table 2.
Comparing 7a with 7b the MeO- group increased the pKa in
line with Ludwig’s observations. Despite measurable differences
in the acidity of the various analogues’ sulfonamide NH, there
was no correlation between sulfonamide pKa and GTPγS
affinity. Interestingly, the affinity of the two analogues that
cannot form a hydrogen bond, namely the des-methoxy
analogue 7b and the cyano analogue 7f, have the lowest
affinity (6.6 and 6.5, respectively), whereas the three hydroxyl
analogues 7e, 7g, and 7i, which are capable of hydrogen
bonding, have the highest affinity (7.9, 8.2, and 7.7,
respectively). We have therefore hypothesized that the effect
of hydrogen-bonding would be to hold the analogues in a
locked active conformation. In the absence of suitable crystals
G
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a
Scheme 10
a
Reagents and conditions: (a) LiAlH4, THF, 50%; (b) 2-acetoxy-isobutyryl chloride, Et3N, DCM, 87%; (c) arylsulfonyl chloride, pyridine, DCM;
(d) K2CO3, MeOH, 39−55%.
of 6 for an X-ray diffraction study, we have examined smaller
fragments of 6 such as the N1-methyl analogues 57 and 58.
Fragment 57 gave suitable crystals for a small-molecule X-ray
diffraction study (Figure 2). In this structure, an intramolecular
hydrogen bond between the sulfonamide and the C4-OMe
group is clearly observed [N−H, 0.80(3) Å; H···O, 2.52(3) Å;
N···O, 3.036(3) Å; and ∠N−H···O, 124(3)°]. A consequence
of this hydrogen bond is to allow the plane normals to the
thiophene ring and the indazole core to be approximately
orthogonal [83.73(7)°]. Fragment 58 possesses a 3-thiophene
substituent at C4, which removes the possibility of this
intramolecular hydrogen bond. Figure 3 shows that the above
two plane normals are now closer to being parallel [17.2(2)°].
A search of the Cambridge Structural Database (v 5.33, Nov
2011) for [5,6] bicyclic cores containing any combination of
carbon, nitrogen, and oxygen atoms, and being substituted with
a sulfonamide moiety and an oxygen atom at the equivalent 3-
and 4-position of the indazole ring system, yielded no hits,
confirming the novel arrangement reported herein. Moreover,
the GTPγS affinity of fragment 57 was 6.4, whereas that of 58
was 5.8. Replacement of the N1-Me substituent of 57 with a
benzyl group leads to analogues with increased potency,
whereas in the case of 58 lead to nonadditive effects. These data
are consistent with the theory that disruption of the
intramolecular hydrogen bond leads to an alternative
conformation and a different binding mode, which in turn
leads to reduced potency.
Anisotropic atomic displacement ellipsoids for the non-
hydrogen atoms are shown at the 50% probability level.
Hydrogen atoms are displayed with an arbitrarily small radius.
The intramolecular hydrogen bond is indicated by a dashed
line.
Anisotropic atomic displacement ellipsoids for the non-
hydrogen atoms are shown at the 50% probability level.
Hydrogen atoms are displayed with an arbitrarily small radius.
In our preliminary communication, we have reported that
amide and hydroxyl-amide groups at the N1 benzyl group
increased both the potency and solubility.26 Recently, oxetanes
were reported as groups that increase the solubility of
compounds,35 therefore, oxetane amides 24a and 24b, and
related cyclic ether amides 24c and 24d, were investigated. The
LLEAT of all four compounds was inferior to 7a, whereas the
chrom log D was similar or even higher than 7a. Interestingly,
the CLND solubility of 24a, 24c, and 24d was slightly increased
compared to 7a, whereas 24b was similar. In comparing the 3-
oxetane with the 2-oxetane analogues (24a vs 24b), the former
had higher solubility and lower chrom log D, which might be
due to the greater exposure of the oxygen atom.
Another way of increasing the solubility would be the
introduction of an ionizable group such as an amino group. All
three amines, 24e, 24f, and 24g, had identical affinity, lower log
D, and slightly increased CLND solubility. Furthermore, all
three analogues had high whole blood potency, and indeed they
were the most potent compounds in this assay, making them
suitable for further investigation. It was interesting to
investigate the reason that these amines were uniformly more
potent in the whole blood assay, and we hypothesized that this
might be due to lower plasma protein binding. The rapid
equilibrium dialysis human plasma protein binding for a range
of analogues is presented in Table 3. The three strongly basic
analogues 24e, 24f, and 24g had the lowest plasma protein
binding, indicating that the higher potency in the whole blood
actin polymerization assay was related to the higher levels of
unbound compound. The four less basic analogues 24h−k
containing a morpholine moiety had very high affinity in the
GTPγS assay. Compounds 24j and 24k, which are enantiomers,
were found to have identical activity with a pIC50 value of 8.2,
one of the highest values in this series. However, due to their
high molecular weight, their LLEAT value was slightly reduced
at 0.26. The solubility of analogues 24h−k was higher as
expected, making these compounds worthy of further
investigation. The basic analogue containing the 3-oxetane
24l was less potent with a LLEAT of 0.2 and low solubility, and
it was therefore rejected.
Another approach to increase the solubility of analogues of
7a was replacement of the phenyl ring of the benzylic
substituent by a heterocyclic ring, such as pyridine, in analogues
26−28. These analogues had reduced lipophilicity and very
good LLEAT, however, their potency was lower than 7a and
they were not examined any further. Similarly introduction of
H
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Table 2. In Vitro Data pIC50 for GTPγS Binding Assay, Human Whole Blood F-Actin Polymerization pA2, Ligand Efficiency,
Chrom log D, CLND Solubility, and pKa
a
compd
GTPγS pIC50
LLE(AT)
chrom log D
CLND solubility (μg/mL)
hWB actin polymerization pA2 (n)
pKa
1
8.26 0.01 (150)
8.41 0.02 (147)
7.83 0.02 (113)
7.50 0.04 (10)
6.6 0.0 (4)
7.5 0.1 (2)
7.1 0.1 (2)
7.9 0.2 (2)
6.5 0.1 (4)
8.2 0.1 (2)
7.0 0.1 (2)
7.7 0.1 (4)
7.0 0.1 (2)
7.0 0.1 (2)
7.7 0.2 (4)
7.5 0.1 (4)
7.4 0.2 (4)
7.5 0.2 (4)
7.8 0.1 (2)
7.8 0.2 (6)
7.8 0.1 (10)
7.6 0.2 (4)
7.8 0.1 (6)
8.2 0.1 (3)
8.2 0.0 (3)
6.6 0.2 (4)
6.8 0.1 (4)
7.0 0.1 (6)
6.9 0.2 (2)
7.9 0.1 (3)
7.4 0.1 (2)
7.4 0.2 (4)
7.8 0.0 (14)
7.1 0.1 (4)
6.4 0.3 (4)
7.3 0.2 (4)
6.4 0.2 (4)
7.8 0.2 (3)
0.25
0.27
0.27
0.28
0.24
0.25
0.26
0.32
0.26
0.35
0.27
0.31
0.26
0.24
0.25
0.27
0.23
0.26
0.27
0.28
0.24
0.27
0.28
0.26
0.26
0.20
0.32
0.33
0.32
0.29
0.25
0.26
0.26
0.25
0.19
0.24
0.19
0.25
2.9
2.9
4.3
4.1
3.3
3.8
3.0
2.9
2.5
2.6
4.8
2.7
2.9
3.7
4.0
4.5
4.9
4.4
3.1
3.3
3.3
3.9
3.9
4.4
4.4
4.3
2.6
2.5
3.0
4.1
4.1
4.1
4.2
4.0
5.1
4.5
4.7
4.5
≥145
193
7.3 0.3 (4)
6.6 0.1 (24)
6.2 0.1 (11)
6.0 0.1 (7)
ND
ND
ND
6.54
6.60
5.93
6.03
5.62
6.12
5.40
5.66
ND
5.62
5.46
5.60
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
6.76
7.06
6.82
7.26
5
6
≥162
≥124
≥117
≥226
≥139
≥166
≥155
≥113
57
7a
7b
7c
5.2 0.2 (2)
5.3 0.2 (2)
6.1 0.1 (2)
ND
7d
7e
7f
7g
6.0 0.3 (2)
ND
7h
7i
≥208
≥159
≥188
≥173
124
6.3 0.1 (3)
ND
7j
7k
5.1 0.1 (2)
6.0 0.2 (3)
ND
24a
24b
24c
24d
24e
24f
24g
24h
24i
24j
24k
24l
26
≥153
≥193
191
6.1 0.1 (2)
6.3 0.1 (3)
6.7 0.0 (5)
6.85 0.04 (4)
6.9 0.1 (4)
6.25 0.15 (4)
7.1 0.3 (4)
6.6 0.2 (6)
6.6 0.2 (6)
ND
≥180
≥220
≥250
≥212
≥203
≥180
122
≥141
≥161
≥182
≥199
≥174
≥161
≥198
≥177
76
<5.0 (1)
27
ND
28
ND
33a
33b
40a
40b
48
5.5 0.2 (3)
6.2 0.0 (2)
6.0 0.2 (5)
6.2 0.1 (4)
ND
56a
56b
56c
56d
ND
≥198
157
ND
ND
36
5.9 0.2 (3)
a
CLND solubility values that are within 85% of maximum possible concentration (as determined from DMSO stock concentration) are reported as
≥. ND: not determined. When n < 3, SEM is the SD.
nitrogen in the indazole benzene ring to provide aza-indazole
analogues gave compounds with reduced potency (data not
shown).
fluoro analogue of 7a, 48, was of lower potency and lower
solubility and was rejected.
Finally, variation of the sulfonamide moiety of 6 was
investigated with a large number of analogues, including alkyl,
cycloalkyl, heterocyclic, heteroaryl, and aryl analogues; four of
the latter are shown in Table 2. None of these analogues
however demonstrated any improvement over compound 6.
The passive membrane permeability (Pex) of the more potent
compounds across Madin−Darby canine kidney−multidrug
resistance 1 (MDCKII-MDR1) cells in the presence of a potent
P-glycoprotein inhibitor is shown in Table 3. The permeability
of 6 and its 6-fluoro analogue 40b was good but lower than 7a,
and this might be a reflection of the additional hydrogen bond
donor in these two analogues. The compound with the highest
cell membrane permeability was the morpholine 24k, which
was less basic and did not have any additional hydrogen bond
donors, whereas the morpholine 24i with an additional
hydrogen bond donor was lower than 24k but still at the
Substitution in the aromatic ring of the indazole core at C5,
C6, and C7 was not tolerated, apart from very small groups. Of
particular interest was substitution with fluorine, as this might
reduce metabolism of the phenyl ring. The C5 fluoro analogue
33a was more potent than 7a, however, it had a lower whole
blood potency, whereas analogue 33b was less potent in the
GTPγS assay than 6 but equipotent in the whole blood assay.
Since neither compound had any additional advantage over 7a
nor 6, these analogues were not progressed any further.
Substitution with fluorine at C6 gave 40a and 40b. Both
compounds were equipotent in both assays with their
respective analogues 7a and 6. The LLEAT was slightly reduced
as a result of the higher molecular weight, and the CLND
solubility for both 40a and 40b was marginally improved.
Compound 40b was chosen for further investigation. The C7
I
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amino analogue of 6. It is interesting to speculate whether this
was due to an active transport mechanism; however, we have
no evidence for this. The compounds shown in Table 3 were
progressed to preliminary CD rat pharmacokinetic studies in
vivo and are compared with the previously reported data for
compounds 7a and 6.26 Each compound was dosed iv and po at
1 mg/kg, n = 2 for each leg of the study, except for the iv leg of
24g, which had n = 1. The compounds were dosed as solutions
in DMSO−PEG200−H2O (5:45:50), except for 24k, which
was dosed in the ratio of 10:40:50. The bioavailability of the
phenolic analogue 7e was 12%, whereas that of the secondary
alcohol 7i was 35%, which was greatly diminished by
comparison with 7a, and consequently their progression was
terminated. The strongly basic analogues 24e and 24g had low
bioavailability, whereas the less basic morpholines 24i and 24k
had bioavailability of 32% and 68%, respectively. Compound
24f, the strongly basic amino analogue of 6, also had reduced
oral bioavailability compared to 6 although it was slightly higher
at 29% than the other strongly basic compounds, which may be
a reflection of its higher permeability, and further highlighting
the possibility of an active transport mechanism for this
analogue. The morpholines were considered worthy of further
investigation in the dog. The 6-fluoro analogue 40b had high
bioavailability similar to that of 6. The five compounds shown
in Table 4 were progressed to pharmacokinetic studies in the
Figure 2. The 2D connectivity and 3D conformation taken from the
crystal structure of 57.
Table 4. Beagle Dog Pharmacokinetic Parameters
24e
24f
24g
24i
24k
7a
6
Cl (mL/min/kg)
Vdss (L/kg)
T1/2 (h)
17
6
18
5.0
2.4
32
11
41
3.0
2.6
39
22
5.7
2.6
NA
18
3.2
1.6
49
9
2.2
2.6
97
5.9
81
0.95
NA
a
F%
NA
a
NA: not applicable.
beagle dog and are compared with the previously reported data
for compounds 7a and 6.26 Each compound was dissolved in
DMSO−PEG200−H2O (5:45:50) and dosed iv at 0.5 mg/kg,
with n = 2 per compound, except for 24k (n = 1). Two
compounds, 24e and 24i, were progressed to the po leg of the
study at a dose of 1 mg/kg, n = 2 and had bioavailability of 81%
and 39%, respectively. The clearance of both 24e and 24i was
much higher than that of 6 and were therefore not progressed.
The remaining three compounds, 24f, 24g, and 24k, had at
least 2-fold higher clearance than 6 and were therefore of no
further interest. The only remaining compounds were 6 and
40b. Both compounds had a very similar rat pharmacokinetic
profile and very similar CLND solubility. The equilibrium
solubility of 6 and 40b in a range of physiologically relevant
media was measured using HPLC quantification and is
Figure 3. The 2D connectivity and 3D conformation taken from the
crystal structure of 58.
same levels as 6 and 40b. The compounds with the lowest
permeability were 7i, 24e, and 24g, which had extra hydrogen
bond donors and/or were strongly basic. The only odd
compound with good permeability but being strongly basic and
possessing two additional hydrogen bond donors was 24f, the
Table 3. Human Plasma Protein Binding (PPB) Measured by Equilibrium Dialysis, Membrane Permeability (Pex) across MDCK
Cells, and CD Rat Pharmacokinetic Parameters
7e
7i
24e
24f
24g
24i
24k
40b
7a
6
a
human PPB (%)
Pex (nm/s)
Cl (mL/min/kg)
Vdss (L/kg)
iv T1/2 (h)
100
ND
19
ND
94.6
50
96
121
15
92.2
45
29
2.6
3
98.2
97
98.6
300
24
ND
111
19
99.5
254
5
99
132
15
27.5
13
17
18
1.2
1.2
12
3.5
3.7
35
0.79
0.8
19
0.84
1.3
29
0.78
1.2
32
1
1.5
1.8
90
0.57
1.6
1.9
2.5
85
1.3
68
F%
<1
100
a
ND not determined.
J
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summarized in Table 5. The solubility of both compounds was
found to be low in all physiologically relevant media such as
dose, variability was high following the 1 mg/kg dose (range of
26−72%). These findings gave us confidence that despite the
low solubility of 6 high bioavailability could be achieved from a
solid dose and encouraged further progression of 6.
Table 5. Equilibrium Solubility after 24 h of 6 and 40b in
a
The selectivity of 6 for CCR4 over other chemokine
receptors was evaluated in a series of GTPγS binding assays
at Euroscreen. Compound 6 had a pIC50 of 7.4 for CCR4, and
the pIC50 for CCR1, CCR2, CCR8, CCR10, CXCR1, CXCR2,
CXCR3, and CXCR4 was <5, whereas for CCR5 was 5.2. The
selectivity of 6 against a panel of 7TM receptors, ion channels,
enzymes, transporters, and nuclear receptors was evaluated in
agonist or antagonist mode as appropriate. The only off-target
activity of note was weak antagonism (pXC50 5.1) at the α1
nicotinic ACh receptor, which potentially leads to neuro-
muscular blockade and generalized muscle relaxation. At this
level of activity, any physiological effect is unlikely. There was
also moderate inhibitory activity at the organic anion
transporting polypeptide OATP1B1 transporter (PXC50 6.4).
OATPs form a superfamily of sodium-independent transport
systems that mediate the trans-membrane transport of a wide
range of amphipathic endogenous and exogenous organic
compounds. OATPs are involved in the uptake of organic
anions from the blood to the liver and therefore may be
involved in drug distribution, metabolism, and elimination.
Compound 6 was negative in the Ames test and not toxic to
membranes and mitochondria in the HepG2 Cell Health assay.
Furthermore, there were no cardiovascular issues associated
with 6 in rabbit ventricular wedge (up to 10 μM), no cardiac
repolarization, conduction, contractility, nor any liability for
torsadogenic (TdP) arrhythmias. Similarly in the conscious rat,
there were no treatment related effects on body temperature,
cardiovascular, or electrocardiographic parameters at 30, 100,
and 300 mg/kg po. In addition, no toxic effects were observed
in a 7-day repeat oral study in the rat.36
Compound 6 was very highly bound (99% or greater) to
plasma proteins in rat, dog, and human plasma. The extent of
binding was determined in CD rat, beagle dog, and human
plasma using rapid equilibrium dialysis at a nominal
concentration of 1 μg/mL (n = 3). The modest half-life in
rat and dog are consistent with the low/moderate Cl and low/
moderate volume of distribution, suggesting that the majority
of compound was removed effectively from the body and that
the plasma protein binding does not appear to be restrictive
either for Cl or Vd. The in vitro clearance of 6 was very low in
three species: rat, dog, and human. There was no evidence of
tissue accumulation or toxicity on repeat oral administration in
rat or dog. This reinforces the lack of restrictive binding in vivo,
although it may also indicate that there is a nonmetabolic
clearance involved. It is possible that there was an underlying
longer terminal phase that was not detected due to being below
the limit of detection of the blood assay.
Various Physiologically Relevant Media and pHs
media
6 (μg/mL)
40b (μg/mL)
water
57
1
32
1
simulated gastric fluid (pH 1.6)
b
FeSSIF (pH 6.5)
23
10
73
3
33
36
c
FaSSIF (pH 6.5)
e
saline
ND
d
pH 2
ND
ND
ND
ND
d
pH 7
10
56
d
pH 8
d
pH 10
1324
a
b
Compounds were quantified by HPLC. FeSSIF = fed state
simulated intestinal fluid. FaSSIF = fasted state simulated intestinal
c
d
fluid. Britton−Robinson buffer was used for the pH 2−10
e
determinations. ND: not determined.
simulated gastric fluid (SGF), water, fed-state simulated
intestinal fluid (FeSSIF), and fasted-state simulated intestinal
fluid (FaSSIF). The 24 h water equilibrium solubility for 6 was
57 μg/mL, whereas for 40b was 32 μg/mL. These figures were
substantially lower than the values obtained from the high
throughput CLND kinetic solubility measurement of 162 and
198 μg/mL, respectively (Table 2). The difference in kinetic vs
equilibrium solubility of 6 obtained from either solid material
or from DMSO stock solutions was investigated using NMR
quantification. Solubility from solid in pH 7.4 phosphate
buffered saline (PBS) confirmed the data from the HPLC
quantified measurements, with the solubility increasing with
time. Kinetic solubility from 10 mM DMSO stock solution by
adding pH 7.4 buffer to give a 5% DMSO final concentration
(as with the CLND determination) provided a solubility of
about 270 μg/mL. This solution was stable even after 100 h.
Solubility from solid in the presence of a small amount of
MeOH (10 μL) to break up initial crystal lattice showed a
marked difference to the solubility obtained from solid alone.
The compound freely dissolved in MeOH and when diluted
down with PBS yielded an initial solution in excess of 270 μg/
mL. This solution, unlike the DMSO solution, was not stable;
the solubility decreased with time until equilibrium was reached
at the same final concentration as that obtained from solid
alone. These data are consistent with the formation of very
stable supersaturated solution of 6 in the case of the high
throughput CLND solubility assay, giving rise to the apparent
higher solubility figures. The stability of 6 in the solid state was
examined for 1 month at 50 °C, and at 40 °C−75% relative
humidity. No changes in the main peak, impurities, or physical
properties were observed, confirming that 6 was stable in the
solid state.
Because of the differences in immunology between animals
and human, the complexity of chemokine systems across
species, the presence of multiple chemokine ligands, and the
uncertain relevance of animal models to clinical disease, no in
vivo anti-inflammatory data has been generated for 6. Therefore
a microdose study administering radiolabeled 6 in man was
undertaken to provide preliminary information on the
distribution and clearance in man and hence allow accurate
modeling and dose predictions for the oral first-time-in-human
study.36 C3-[14C]-labeled 6 was synthesized by the same route
as 6 starting from [14CN]-8. The microdose study was
conducted in healthy male volunteers receiving an intravenous
On the basis of the interesting solubility findings of 6, we
speculated whether there was any difference between the rat
bioavailability of 6 following administration as a solution in
DMSO−PEG200−H2O (5:45:50) (F = 85% at 1 mg/kg, Table
3) and following administration of a suspension of wet bead
milled material (particle size <1 μm). Following administration
of the suspension at 1, 10, and 30 mg/kg to the rat, the oral
exposure of 6 increased with increasing dose and bioavailability
remained high (53%, 80%, and 101%, respectively). While the
interanimal variability was low following the 10 and 30 mg/kg
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infusion of 100 μg [14C]-6 per volunteer. The derived human
pharmacokinetic parameters confirmed that the compound had
a low to moderate clearance. These data, together with
absorption data from the rat and dog PK studies, were used
in simulations to predict blood exposure in man.36
EXPERIMENTAL SECTION
■
Organic solutions were dried over anhydrous Na2SO4 or MgSO4. TLC
was performed on Merck 0.25 mm Kieselgel 60 F254 plates. Products
were visualized under UV light and/or by staining with aqueous
KMnO4 solution. LCMS analysis was conducted on either System A,
an Acquity UPLC BEH C18 column (2.1 mm × 50 mm ID, 1.7 μm
packing diameter) eluting with 0.1% formic acid in water (solvent A)
and 0.1% formic acid in acetonitrile (solvent B), using the following
elution gradient 0.0−1.5 min 3−100% B, 1.5−1.9 min 100% B, 1.9−
2.0 min 3% B, at a flow rate of 1 mL min−1 at 40 °C. The UV detection
was an averaged signal from wavelength of 210 to 350 nm, and mass
spectra were recorded on a mass spectrometer using alternate-scan
electrospray positive and negative mode ionization (ES +ve and ES
−ve); or System B Sunfire C18 column (30 mm × 4.6 mm ID, 3.5 μm
packing diameter) eluting with 0.1% formic acid in water (solvent A),
and 0.1% formic acid in MeCN (solvent B), using the following
elution gradient 0−0.1 min 3% B, 0.1−4.2 min 100%, 4.2−4.9 min 3%
B, 4.9−5.0 min 3% B at a flow rate of 3 mL min−1 at 30 °C; or System
C Acquity UPLC BEH C18 column (50 mm × 2.1 mm ID, 1.7 μm
packing diameter) eluting with 10 mM ammonium bicarbonate in
water adjusted to pH10 with ammonia solution (solvent A) and
MeCN (solvent B) using the following elution gradient 0−1.5 min 1−
97% B, 1.5−1.9 min 97% B, 1.9−2.0 min 100% B at a flow rate of 1
mL min−1 at 40 °C. Column chromatography was performed on
Flashmaster II. The Flashmaster II is an automated multiuser flash
chromatography system, available from Argonaut Technologies Ltd,
which utilizes disposable, normal phase, SPE cartridges (2−100 g).
Mass-directed autopreparative HPLC (MDAP) was conducted on a
Sunfire C18 column (150 mm × 30 mm ID, 5 μm packing diameter)
at ambient temperature eluting with 0.1% formic acid in water (solvent
A) and 0.1% formic acid in acetonitrile (solvent B), using an
appropriate elution gradient over 15 min at a flow rate of 40 mL min−1
and detecting at 210−350 nm at room temperature. Mass spectra were
recorded on Micromass ZMD mass spectrometer using electro spray
positive and negative mode, alternate scans. The software used was
MassLynx 3.5 with OpenLynx and FractionLynx options. 1H NMR
spectra were recorded at 400 MHz unless otherwise stated. The
chemical shifts are expressed in ppm relative to tetramethylsilane. High
resolution positive ion mass spectra were acquired on a Micromass Q-
Tof 2 hybrid quadrupole time-of-flight mass spectrometer. Optical
rotations were measured with an Optical Activity AA100 digital
polarimeter. Analytical chiral HPLC was conducted on Chiralpak
column (250 mm × 4.6 mm) eluting with an appropriate ratio of
EtOH−heptane for 30 min at room temperature, flow rate 1 mL
min−1, injection volume 15 μL, detecting at 215 nm. The purity of all
compounds screened in the biological assays was examined by LCMS
analysis and was found to be ≥95% unless otherwise specified. 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. The human
biological samples were sourced ethically and their research use was in
accord with the terms of the informed consents.
As mentioned in our Introduction, the AstraZeneca group
have reported in 2008 the presence of an intracellular allosteric
site on CCR4 and CCR5 receptors to which small pyrazine
sulfonamides bind.27 Further pharmacological studies were
performed by our group to determine which binding site on
CCR4 mediated the effects of the indazole sulfonamide series.
These experiments used a combination of functional antagonist
interaction studies in isolated T cell actin polymerization assays
and radioligand binding with site-selective tritiated antagonists
1 and 4. The studies indicated that there are three distinct
binding sites on the CCR4 receptor, the orthosteric site to
which the macromolecular ligands MDC and TARC bind and
two additional allosteric sites to which small molecules bind.
Lipophilic amines such as compound 1 and 2 bind to an
allosteric site denoted arbitrarily as site I. Compound 6 and its
analogues bind to site II, the allosteric modulatory site, which is
accessible from the cytoplasmic face of the receptor and which
may also exist in other chemokine receptors.37 Site II overlaps
with the binding site for the pyrazine sulfonamides 4 and 5.
These experiments will be the subject of a future publication.38
The presence of multiple antagonist binding sites on CCR4, at
least one of which is allosteric, opens the potential for the
design of molecules which have selective effects on its two
chemokine agonists. Also, compounds which bind to more than
one of these sites would be predicted to have greater inhibitory
activity than the current site-selective compounds.
CONCLUSION
■
A novel series of indazole arylsulfonamides were synthesized
and examined as allosteric human CC chemokine receptor 4
antagonists. The preferred C4 substituents were methoxy- or
hydroxy-containing groups. Fluoro-groups at C5, C6, or C7
were tolerated, with the C6 analogues being preferred. The
most potent N3-substituent was 5-chlorothiophene-2-sulfona-
mide. A variety of potent N1 meta-substituted benzyl groups
were identified. The most potent substituents contained a 3-
methylaminoamide group possessing an α-amino moiety.
Strongly basic amino groups had low oral bioavailability in
rat and/or dog pharmacokinetic studies. Less basic analogues,
such as morpholines, had better apparent permeability and
moderate/high bioavailability in rat, however, they tended to
have high clearance in dog. The most potent compound with
high bioavailability in both rat and dog was the tertiary-hydroxy
analogue 6 (GSK2239633A). This compound was selective
against other chemokine receptors and a panel of 7TM
receptors, ion channels, enzymes, transporters, and nuclear
receptors. Compound 6 had high permeability in MDCK cells
and was highly bound to plasma proteins. It had low solubility
in all physiologically relevant media, with the solubility
increasing dramatically at pH 10. This suggested that 6 might
have low oral bioavailability at higher doses. However, the oral
exposure of 6 in the rat, following administration of a
suspension of wet bead milled material, increased with
increasing dose up to 30 mg/kg. Compound 6 was selected
for further development, and the clinical studies will be
reported shortly.36
4-(Methyloxy)-1H-indazol-3-amine (9a). A mixture of 2-fluoro-
6-(methyloxy)benzonitrile (8a) (10 g, 66 mmol) and hydrazine
hydrate (9.63 mL, 198 mmol) in n-butanol (100 mL) was heated to
reflux under nitrogen for 18 h. The reaction mixture was allowed to
cool, water (300 mL) was added, and the organic phase was removed.
The solid in the aqueous phase was collected by filtration and dried in
vacuo at 40 °C to give a white solid (0.6g). The butanol phase was
evaporated in vacuo, and the residue and the aqueous mother liquors
were combined and extracted using ethyl acetate (2 × 200 mL). The
combined ethyl acetate extractions were dried (MgSO4) and
evaporated in vacuo. The residue was dissolved in DCM and applied
to a 100 g silica cartridge. This was eluted with cyclohexane (500 mL),
cyclohexane−ethyl acetate (1:1, 500 mL), and ethyl acetate (500 mL).
The required fractions were combined and evaporated in vacuo to give
9a (9.92 g, 92%) as an off-white solid. MS ES + ve m/z 164 (M + H)+.
1H NMR δ (DMSO-d6) 11.35 (1H, s), 7.10 (1H, t, J = 8 Hz), 6.76
(1H, d, J = 8 Hz), 6.29 (1H, d, J = 8 Hz), 4.95 (2H, br s), 3.85 (3H, s).
L
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3-{[3-Amino-4-(methyloxy)-1H-indazol-1-yl]methyl}-
benzonitrile (10a). A solution of ground potassium hydroxide (6.75
g, 120 mmol) in DMSO (300 mL) was treated with 9a (7.85 g, 48.1
mmol) at room temperature under nitrogen, and this gave a deep-red
solution. After 5 min, the red solution was treated with 3-cyanobenzyl
chloride (8.84 g, 58.3 mmol) in one portion. The reaction mixture was
stirred for 20 min and then poured into water (500 mL), forming an
emulsion. The emulsion was extracted with chloroform (3 × 500 mL).
The combined organic solutions were washed with water (400 mL)
and passed through a hydrophobic frit. The solvent was removed in
vacuo, and the residue was applied to a 340 g silica cartridge and eluted
with a gradient of 0−100% ethyl acetate in cyclohexane over 8 column
volumes (CV). The solvent was evaporated under reduced pressure to
give an orange solid, which was triturated in a mixture of ethyl acetate
(10 mL) and cyclohexane (90 mL). The solid was collected by
filtration and washed with cyclohexane (50 mL). The solid was dried
in vacuo to give 10a (7.89 g, 59%) as a pale-orange solid: MS ES +ve
cartridge. This was eluted with a gradient of 5−40% MeCN containing
0.1% 880 aqueous ammonia and 10 mM ammonium bicarbonate over
8 CV. The required fractions were combined, and the MeCN was
evaporated under reduced pressure. The residue was collected by
filtration and washed with water. The solid was dried at 40 °C in vacuo
for 3 d to give 7a (1.80 g, 89%) as a white solid: LCMS (system A) RT
1
= 0.99 min, 96%, ES +ve m/z 505/507 (M + H)+. H NMR δ (600
MHz, DMSO-d6) 10.41 (1H, s), 8.28 (1H, br t, J = 5.5 Hz), 7.38 (1H,
d, J = 4 Hz), 7.27 (1H, t, J = 8 Hz), 7.24 (1H, t, J = 8 Hz), 7.18 (1H, d,
J = 8 Hz), 7.17 (1H, d, J = 4 Hz), 7.16 (1H, br s), 7.14 (1H, d, J = 8
Hz), 6.99 (1H, d, J = 8 Hz), 6.51 (1H, d, J = 8 Hz), 5.49 (2H, s), 4.19
(2H, d, J = 5.5 Hz), 3.76 (3H, s), 1.84 (3H, s). 13C NMR δ (151 MHz,
DMSO-d6) 169.0, 153.3, 142.1, 140.1, 139.9,137.1, 135.7, 134.6, 131.9,
128.4, 127.4, 126.5, 126.4, 125.8, 109.5, 102.4, 100.1, 55.2, 51.8, 42.0,
22.5. HRMS ES +ve m/z 505.0770. C22H22ClN4O4S2 requires
505.0771.
2-{[(3-{[3-{[(5-Chloro-2-thienyl)sulfonyl]amino}-4-(methyl-
oxy)-1H-indazol-1-yl]methyl}phenyl)methyl]amino}-1,1-di-
methyl-2-oxoethyl acetate (25). A suspension of 12a (40.5 g, 87
mmol) in DCM (1 L) and triethylamine (36.4 mL, 262 mmol) was
cooled down and then treated slowly with 2-chloro-1,1-dimethyl-2-
oxoethyl acetate (12.52 mL, 87 mmol). The reaction mixture was
stirred at room temperature under nitrogen for 80 min. It was then
washed with 2 M HCl (300 mL), NaHCO3 (300 mL), and brine, dried
(MgSO4), and evaporated under reduced pressure to give a white
solid. The residue was purified by chromatography on a 1.5 kg silica
column, eluting with 40−100% ethyl acetate−cyclohexane over 8 CV.
The required fractions were combined and evaporated under reduced
pressure to give 25 (37.87 g, 73%) as a white foam: MS ES +ve m/z
591/593 (M + H)+. 1H NMR δ (CDCl3) 7.65 (1H, br s), 7.43 (1H, d,
J = 4 Hz), 7.29−7.18 (3H, m), 7.13 (1H, br s), 7.06 (1H, br d, J = 7.5
Hz), 6.85 (1H, d, J = 8 Hz), 6.77 (1H, d, J = 4 Hz), 6.37 (1H, d, J = 8
Hz), 6.33 (1H, br t, J = 6 Hz), 5.43 (2H, s), 4.43 (2H, d, J = 6 Hz),
3.92 (3H, s), 2.05 (3H, s), 1.62 (6H, s). Additional quantities of 25
(2.16 g, 4%) were obtained from mixed fractions (3.39 g), which were
further purified by chromatography on a 120 g silica column using a
gradient of 40−75% EtOAc−cyclohexane over 8 CV.
1
m/z 279 (M + H)+. H NMR δ (CDCl3) 7.61−7.56 (1H, m), 7.49−
7.43 (3H, m), 7.27 (1H, t, J = 8 Hz), 6.76 (1H, d, J = 8 Hz), 6.40 (1H,
d, J = 8 Hz), 5.36 (2H, s), 5.00−4.10 (2H, br), 4.00 (3H, s).
5-Chloro-N-[1-[(3-cyanophenyl)methyl]-4-(methyloxy)-1H-
indazol-3-yl]-2-thiophenesulfonamide (11a). To 10a (7.89 g,
28.3 mmol) was added a solution of 5-chloro-2-thiophenesulfonyl
chloride (6.15 g, 28.3 mmol) in pyridine (9.17 mL, 113 mmol) under
nitrogen at room temperature. An exothermic reaction occurred which
went deep-red colored. After 40 min, the reaction mixture was
partitioned between ethyl acetate (500 mL) and 2 M hydrochloric acid
(500 mL). The aqueous phase was extracted with ethyl acetate (400
mL). The combined organic solutions were dried (MgSO4) and
evaporated in vacuo. The deep-red residue was dissolved in DCM and
applied to a 340 g silica cartridge. The cartridge was eluted with a
gradient of 0−10% ethyl acetate in dichloromethane over 8 CV.
Evaporation of the appropriate fractions gave 11a (11.1 g, 85%) as an
1
off-white solid: MS ES +ve m/z 459/461 (M + H)+. H NMR δ
(CDCl3) 7.75 (1H, br s),7.60−7.56 (1H, m), 7.51 (1H, d, J = 4 Hz),
7.44−7.39 (2H, m), 7.35 (1H, br s), 7.27 (1H, t, J = 8 Hz), 6.82 (1H,
d, J = 4 Hz), 6.80 (1H, d, J = 8 Hz), 6.42 (1H, d, J = 8 Hz), 5.47 (2H,
s), 3.95 (3H, s).
N-[(3-{[3-{[(5-Chloro-2-thienyl)sulfonyl]amino}-4-(methyl-
oxy)-1H-indazol-1-yl]methyl}phenyl)methyl]-2-hydroxy-2-
methylpropanamide (6). A solution of 25 (1.09 g, 1.84 mmol) in
methanol (50 mL) was treated with potassium carbonate (0.765 g,
5.53 mmol), and the reaction mixture was stirred at room temperature.
After stirring for 30 min, the reaction mixture started to crystallize, and
it was allowed to stand for 2 h. It was then partitioned between water
(100 mL) and ethyl acetate (150 mL). The two phases were separated,
and the aqueous layer was extracted with ethyl acetate (100 mL). The
combined organic layers were washed with brine, dried (MgSO4), and
evaporated in vacuo. HPLC showed presence of product in the
aqueous phase, hence it was acidified with 2 M HCl and extracted with
ethyl acetate (100 mL). The organic layer was washed with brine,
dried (MgSO4), and evaporated under reduced pressure. The
combined residues were purified by chromatography on a 100 g silica
column, eluting with 0−100% EtOAc−cyclohexane over 40 min. The
required fractions were combined and evaporated under reduced
pressure to give 6 (0.72 g, 71%) as a white solid: LCMS (system A)
N-[1-{[3-(Aminomethyl)phenyl]methyl}-4-(methyloxy)-1H-
indazol-3-yl]-5-chloro-2-thiophenesulfonamide hydrochloride
(12a). A lithium aluminum hydride solution in ether (1M, 60.5 mL)
was added slowly under nitrogen to a cooled solution of 11a (11.1 g,
24.2 mmol) in THF (150 mL), maintaining temperature below 10 °C.
The suspension was stirred at room temperature for 1 h and then was
quenched by addition of water (7 mL), followed by a 2 M solution of
sodium hydroxide (42.5 mL). After stirring for 30 min, the solid was
removed by filtration and washed with THF. The combined filtrate
and washings were loaded on two 70 g SCX-2 cartridges. The
cartridges were washed with methanol (1 L) and then eluted with 10%
2 M hydrochloric acid in methanol (2 L). The required fractions were
combined and concentrated under reduced pressure. The resultant
solid was collected by filtration and washed with water. The solid was
dried in vacuo to give the hydrochloride salt of 12a (9.3 g, 77%) as a
white solid: MS ES +ve m/z 463/465 (M + H)+. 1H NMR δ (DMSO-
d6) 7.44−7.37 (4H, m), 7.37−7.34 (1H, d, J = 7.5 Hz), 7.28 (1H, t, J =
8 Hz), 7.20 (1H, d, J = 8 Hz), 7.19 (1H, d, J = 4 Hz), 7.14 (1H, br d, J
= 8 Hz), 6.51 (1H, d, J = 8 Hz), 5.52 (2H, s), 3.96 (2H, s), 3.76 (3H,
s).
N-[(3-{[3-{[(5-Chloro-2-thienyl)sulfonyl]amino}-4-(methyl-
oxy)-1H-indazol-1-yl]methyl}phenyl)methyl]acetamide (7a). A
suspension of 12a hydrochloride (2.0 g, 4.0 mmol) in DCM (10 mL)
was treated with triethylamine (1.67 mL, 12.0 mmol), and the mixture
was stirred for 20 min at room temperature before acetic anhydride
(0.397 mL, 4.20 mmol) was added. The reaction mixture was stirred at
room temperature for 2 h and then partitioned between DCM (50
mL) and saturated sodium bicarbonate solution (50 mL). The
aqueous phase was extracted with DCM (30 mL), and the combined
organic solutions were dried using a hydrophobic frit. The solution
was evaporated under reduced pressure, and the residue was dissolved
in DMSO−methanol (10 mL, 1:1) and applied to a 330 g C18
1
RT = 1.02 min, 100%, ES +ve m/z 549/551 (M + H)+. H NMR δ
(600 MHz, DMSO-d6) 10.40 (1H, s), 8.13 (1H, br t, J = 6 Hz), 7.37
(1H, d, J = 4 Hz), 7.26 (1H, t, J = 8 Hz), 7.23 (1H, t, J = 8 Hz), 7.17
(1H, d, J = 4 Hz), 7.17 (1H, d, J = 8 Hz), 7.14 (1H, br s), 7.13 (1H, d,
J = 8 Hz), 6.98 (1H, d, J = 8 Hz), 6.50 (1H, d, J = 8 Hz), 5.48 (2H, s),
5.34 (1H, s), 4.22 (2H, d, J = 6 Hz), 3.76 (3H, s), 1.24 (6H, s). 13C
NMR δ (151 MHz, DMSO-d6) 176.5, 153.3, 142.1, 140.2, 140.1,
137.0, 135.7, 134.6, 132.0, 128.4, 127.4, 126.1, 125.7, 109.5, 102.4,
100.1, 71.9, 55.2, 51.9, 41.7, 27.8. HRMS ES +ve m/z 549.1036.
C24H26ClN4O5S2 requires 549.1033.
CCR4 [35S]-GTPγS Binding Assay. Membranes derived from a
CHO cell line stably transfected with human CCR4 receptor (3 μg
membranes per well) were adhered to Wheat Germ Agglutinin
Polystyrene LEADSeeker scintillation proximity assay beads (250 μg/
well) in assay buffer containing N-2-hydroxyethylpiperazine-N′-2-
M
dx.doi.org/10.1021/jm301572h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
ethanesulfonic acid (20 mM), MgCl2 (10 mM), NaCl (100 mM),
saponin (40 μg/mL), bovine serum albumen (0.05% w/v), GDP (4.4
μM), and pH adjusted to 7.4 using 1 M KOH. After 1 h precoupling at
4 °C, the bead/membrane suspension was mixed in a 4:5 ratio with
[35S]-GTPγS (PerkinElmer LAS UK Ltd., radioactivity concentration
37 MBqmL−1; specific activity = 1250 Cimmol−1) made up in assay
buffer containing MDC (at a concentration that results in the final
assay concentration of MDC being EC80) to give a final radioligand
concentration of 0.6 nM. A 45 μL/well aliquot of this reagent mixture
was dispensed into white Greiner polypropylene 384-well plates
containing stock solution of the test compounds in DMSO (10 mM,
0.5 μL) or 0.5 μL of DMSO as a control, and the assay plates were
sealed and centrifuged at 1000 rpm for 20 s. The plates were left to
equilibrate in darkness for between 3 and 6 h before reading on a
ViewLux luminescence imager using a 613/55 filter for 5 min/plate.
Data were analyzed using a four-parameter logistical equation to
determine the antagonist IC50.
ASSOCIATED CONTENT
* Supporting Information
■
S
Preparative details and spectroscopic data for compounds: 10b,
11b, 12b, 7b, 9c, 10c, 11c, 7c, 9d, 10d, 11d, 12d, 7d, 12e, 7e,
9f, 13−18, 7f, 19−21, 7g, 7h, 7i, 7j, 22, 23, 7k, 24a−24l, 31,
32, 26−28, 35−39, 33a, 33b, 42−47, 40a, 40b, 50, 52, 53, 48,
54, 55, 56a, 56b, 56c, 56d, 57, 58, and solubility
determinations. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: (+44)1438 762883. Fax: (+44)1438 768302. E-mail:
pan.a.procopiou@gsk.com.
■
Notes
The authors declare no competing financial interest.
Human Whole Blood F-Actin Polymerization Assay. Volun-
teers gave informed consent for blood donation and denied taking any
medication in the 7 days prior to donation. Blood (9 volumes) was
taken from healthy human subjects into 3.8% sodium citrate solution
(1 volume). The blood was incubated at room temperature with
saturating concentrations of fluorescein isothiocyanate (FITC)-
conjugated mouse antihuman CD4 and a noninactivating phycoery-
thrin (PE)-conjugated mouse antihuman CCR4 (BD Biosciences) or
appropriate isotype control antibodies for 10 min. The blood was then
incubated with antagonists or vehicle (0.1% DMSO) at 37 °C for 30
min before addition of the agonist (MDC from ALMAC Sciences, UK;
TARC from PeproTech EC, UK) for 15 s. The assay was terminated
by adding 10 volumes of FACS Lysing solution (BD Biosciences).
After 30 min, the cells were centrifuged (1000g for 5 min),
resuspended in FACS Lysing solution (200 μL), and incubated at
room temperature for a further 15 min. The cell suspensions were then
centrifuged (1200g for 5 min), washed twice by resuspending in PBS
(150 μL), and centrifuging as above to recover the cells and incubated
for 20 min with lysophosphatidylcholine (100 μg.mL−1) and Alexa
fluor 647 phalloidin (0.075 units.mL−1). The cells were centrifuged at
1200g for 5 min and resuspended in PBS. The F-actin content of the
CD4+ CCR4+ lymphocytes in each sample was determined on a
FACSCalibur flow cytometer by measuring the mean Alexa fluor 647
(FL-4) fluorescence intensity of 500 cells. This was expressed as a
fraction of the Alexa fluor 647 fluorescence intensity of the CCR4−
lymphocytes in the same sample.
Single Crystal X-ray Diffraction Studies of 57 and 58. Crystal
structures of 57 and 58 were determined at 150K using Mo Kα X-
radiation (λ = 0.71073 Å).
Summary of the structure determination of 57: C13H12ClN3O3S2, M
= 357.83, monoclinic, space group P21/n (alt. P21/c, no. 14), a =
11.034(2) Å, b = 8.6297(17) Å, c = 16.101(3) Å, β = 94.54(3)°, V =
1528.3(5) Å3, Z = 4, Dcalc = 1.555 Mgm−3, F(000) = 736, μ(Mo Kα) =
0.538 mm−1; R1 [I > 2σ(I)] = 0.0358; wR2 (all data) = 0.0733; and S
= 1.061.
Summary of the structure determination of 58: C16H12ClN3O2S3, M
= 409.92, monoclinic, space group P21/c (no. 14), a = 10.360(2) Å, b
= 17.010(3) Å, c = 10.076(2) Å, β = 98.05(3)°, V = 1758.2(6) Å3, Z =
4, Dcalc = 1.549 Mgm−3, F(000) = 840, μ(Mo Kα) = 0.589 mm−1; R1
[I > 2σ(I)] = 0.0608; wR2 (all data) = 0.1071; and S = 1.056.
Full details of the structure determinations can be found in the
Supporting Information. In addition, crystallographic data (excluding
structural factors) for the structures in this paper have been deposited
with the Cambridge Crystallographic Data Centre as supplementary
publication numbers CCDC 906759 and 906760. Copies of the data
can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail:
deposit@CCDC.cam.ac.uk).”
ACKNOWLEDGMENTS
■
We thank Peter Eddershaw and Lisa Ranshaw for checking the
pharmacokinetic data, Hossay Abas, Heather Barnett, Stephania
Christou, Andrew Craven, and Benjamin Marsh for technical
assistance, Duncan Farrant and Shenaz Nunhuck for the NMR
solubility determinations, Bill J. Leavens for collecting the
HRMS data, and the Screening and Compound Profiling
Department at GlaxoSmithKline for generating the human
CCR4 GTPγS data. Professor William Clegg and Dr. Luca
Russo (University of Newcastle) are thanked for the
GlaxoSmithKline funded X-ray crystallographic analyses of 57
and 58.
ABBREVIATIONS USED
■
CCR4, CC-chemokine receptor 4; TARC, thymus activation-
regulated chemokine; MDC, macrophage-derived chemokine;
[35S]-GTPγS, guanosine-5′-[γ-thio]triphosphate; MDCK,
Madin Darby canine kidney; Cl, clearance; Vd, volume of
distribution; PK, pharmacokinetics
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