Lead identification and structure-activity relationships of heteroarylpyrazole arylsulfonamides as allosteric CC-chemokine receptor 4 (CCR4) antagonists

Article abstract of DOI:10.1039/c3ob42443j

A knowledge-based library of aryl 2,3-dichlorophenylsulfonamides was synthesised and screened as human CCR4 antagonists, in order to identify a suitable hit for the start of a lead-optimisation programme. X-ray diffraction studies were used to identify the pyrazole ring as a moiety that could bring about intramolecular hydrogen bonding with the sulfonamide NH and provide a clip or orthogonal conformation that was believed to be the preferred active conformation. Replacement of the core phenyl ring with a pyridine, and replacement of the 2,3-dichlorobenzenesulfonamide with 5- chlorothiophenesulfonamide provided compound 33 which has excellent physicochemical properties and represents a good starting point for a lead optimisation programme. Electronic structure calculations indicated that the preference for the clip or orthogonal conformation found in the small molecule crystal structures of 7 and 14 was in agreement with the order of potency in the biological assay.

Full text of DOI:10.1039/c3ob42443j

Organic &
Biomolecular Chemistry
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PAPER
Lead identification and structure–activity
relationships of heteroarylpyrazole
arylsulfonamides as allosteric CC-chemokine
receptor 4 (CCR4) antagonists†
Cite this: Org. Biomol. Chem., 2014,
12, 1779
Afjal H. Miah,a Royston C. B. Copley,b Daniel O’Flynn,a Jonathan M. Percyc and
Panayiotis A. Procopiou*a
A knowledge-based library of aryl 2,3-dichlorophenylsulfonamides was synthesised and screened as
human CCR4 antagonists, in order to identify a suitable hit for the start of a lead-optimisation pro-
gramme. X-ray diffraction studies were used to identify the pyrazole ring as a moiety that could bring
about intramolecular hydrogen bonding with the sulfonamide NH and provide a clip or orthogonal con-
formation that was believed to be the preferred active conformation. Replacement of the core phenyl ring
with a pyridine, and replacement of the 2,3-dichlorobenzenesulfonamide with 5-chlorothiophenesulfon-
amide provided compound 33 which has excellent physicochemical properties and represents a good
starting point for a lead optimisation programme. Electronic structure calculations indicated that the pre-
ference for the clip or orthogonal conformation found in the small molecule crystal structures of 7 and
14 was in agreement with the order of potency in the biological assay.
Received 6th December 2013,
Accepted 3rd February 2014
DOI: 10.1039/c3ob42443j
www.rsc.org/obc
endothelial cells) which can recruit Th2 cells from the circula-
tion. The T cells can then migrate along this chemokine gradi-
Introduction
Chemokines are a group of small basic proteins, which ent to the dendritic cells. Upon maturation, the dendritic cells
together with their receptors mainly regulate the trafficking of migrate from the inflamed tissue to local lymph nodes where
leucocytes down a chemoattractant gradient.1,2 Ten CC chemo- the MDC and TARC which they produce may recruit further
kine receptors have been identified so far and named as T cells to the inflammatory response. Elevated levels of TARC
CCR1–CCR10. CCR4 belongs to the 7-TM domain G-protein- and MDC as well as accumulation of CCR4-positive cells have
coupled receptor family and is mainly expressed in T helper been observed in lung biopsy samples from patients with
2 (Th2) cells. Th2 cytokines in inflamed tissues lead to eosino- atopic asthma following allergen challenge.5,6 Thus CCR4
philia, high levels of serum IgE and mast cell activation, all of antagonists represent a novel therapeutic intervention in dis-
which contribute to the pathogenesis of allergic diseases.3 eases where CCR4 has a central role in pathogenesis, such as
Thymus activation-regulated chemokine (TARC) and macro- asthma, atopic dermatitis,7 allergic bronchopulmonary asper-
phage-derived chemokine (MDC), bind to the orthosteric gillosis,8 cancer,9 the mosquito-borne tropical diseases, such
binding site of CCR4.4 Upon exposure to allergen, dendritic as Dengue fever,10 and allergic rhinitis.11 Progress in the dis-
cells within tissue secrete MDC and TARC (also produced by covery of small-molecule CCR4 antagonists as immunomodu-
latory agents was reviewed by Purandare and Somerville in
2006.12 Since then a number of additional CCR4 antagonists
have been published.13–22 Among these antagonists are two
pyrazine arylsulfonamides, the AstraZeneca 2,3-dichlorobenzene-
sulfonamide 123 and Ono’s 4-methylbenzenesulfonamide 2
(Fig. 1).24 Recently we have reported a class of indazole arylsul-
fonamides including 3, which we believe is the first small-
molecule clinical candidate targeting the CCR4 receptor.25,26
Furthermore, the pyrazine and indazole arylsulfonamide CCR4
antagonists 1–3 bind at an intracellular allosteric binding site,
which is different from the extracellular binding site where lipo-
philic amine antagonists such as 4 to 6 bind.27 The two allosteric
aAllergy & Inflammation DPU, Respiratory TAU, GlaxoSmithKline Medicines
Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK.
E-mail: pan.a.procopiou@gsk.com; Fax: +44 (0)1438 768302;
Tel: +44 (0)1438 762883
bUK Analytical Chemistry, Platform Technology & Science, GlaxoSmithKline
Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY,
UK
cWestCHEM Department of Pure and Applied Chemistry, University of Strathclyde,
295 Cathedral Street, Glasgow, G1 1XL, UK
†Electronic supplementary information (ESI) available. CCDC 975458–975461.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c3ob42443j
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Fig. 1 Structures for some recently published CCR4 antagonists.
binding sites are distinct from each other and from the orthos- Intramolecular hydrogen bonding has been used as a means
teric site where TARC and MDC bind. to reduce Sildenafil’s polar group interaction (pyrimidine NH)
Indazole 3 was progressed to human pharmacokinetic and with water and hence increase its oral absorption.29 Further
pharmacodynamic studies and the results were reported very uses of intramolecular hydrogen bonding to increase mem-
recently.28 The compound was generally safe and well tolerated brane permeability, water solubility, and lipophilicity were
by healthy male subjects. Following intravenous dosing, 3 dis- recently reported by Kuhn for a variety of motifs based on
played rapid, biphasic distribution and slow elimination (t1/2
=
X-ray crystal structure analysis.30
13.5 h). Following oral dosing, blood levels reached Cmax
rapidly (1.0–1.5 h), but bioavailability was low (16%). At a dose
of 1.5 g 3 inhibited TARC-induced actin polymerisation reach-
ing a mean CCR4 occupancy of 74%. Based on the low oral
bioavailability of 3 its further progression was halted. Herein
we present our attempts to identify an alternative lead series of
sulfonamide CCR4 antagonists, suitable for an optimisation
programme to develop a potential back-up compound to inda-
zole 3. We sought a low molecular weight and higher potency
compound that would bind to the intracellular sulfonamide
binding site. In our recent publication we reported a low
energy conformation based on small molecule X-ray diffraction
studies, where the plane normals of the indazole core and the
sulfonamide ring were positioned at right angles, with an
intramolecular hydrogen bond between the sulfonamide NH
and the OMe group.26 Furthermore a range of groups includ-
ing –F, –Cl, –OH, –CN, –CHF2, –COMe, –CH2OH, –CH(OH)Me
and –CMe2OH were investigated as alternative hydrogen bond
acceptors, and it was found that the four hydroxyl containing
compounds had increased potency in the human CCR4 GTPγS
assay. However, this increase did not translate in the human
whole blood assay, or to their pharmacokinetic properties. The
group which conferred optimal properties was found to be the
methoxy group, which was retained in the subsequent lead
Results and discussion
Antagonist potency was determined by a [35S]-GTPγS radio-
ligand competition functional assay using recombinant CCR4-
expressing CHO cell membranes adhered to WGA-coated Lead-
seeker SPA beads in pH 7.4 buffer.31 A secondary assay using
human whole blood was used as a screen to determine
potency against the native receptor for the more potent com-
pounds in the primary assay. The assay quantified cytoskeletal
reorganisation (formation of filamentous (F-) actin) which
occurs in a variety of cells in response to chemoattractants and
is a prelude to chemotaxis. Calculated partition coefficient
(clog P), chromatographic log D (chrom log D at pH 7.4) and
ChemiLuminescent Nitrogen Detection (CLND) kinetic solubi-
lity are included for all test compounds in this study. The high
throughput CLND solubility assay involved addition of
aqueous buffer to a test compound DMSO solution over a
period of time until the compound precipitated. Ligand
efficiency (LE) was obtained from the equation LE = −1.36 ×
pIC50/HAC, where HAC is the heavy atom count (number of
non-hydrogen atoms present in the molecule), with a desirable
figure of LE being >0.3.
optimisation that provided
3 as the clinical candidate.
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Scheme 1 Reagents and conditions: (a) 1,2-phenylenedimethanol (5 equiv.), tert-BuOK (7.5 equiv.), THF, NMP, 5 h, 17%.
Sulfonamide 7, the starting point for the indazole lead on the conformation of these intramolecular interactions
optimisation process, was used as a reference compound for involving five-membered rings is uncertain given the classical
the analogues made in this investigation. Compounds of inter- intermolecular hydrogen bonds from the sulfonamide hydro-
est had to have higher potency than 7 in the GTPγS assay gen atoms that are also present in the crystal structure (see
(pIC50 > 6.2), and better physicochemical properties (clog P < ESI† for details).
3.48; chrom log D < 5.3; CLND solubility > 116 μg mL−1). The
The indazole sulfonamide 7 was also crystallised and suit-
novel pyrazine 8, a regioisomer of the AstraZeneca CCR4 antag- able crystals were obtained for an X-ray diffraction study. The
onist 1, was used as a standard in the GTPγS assay. Compound structure contained two crystallographically independent
8 was prepared from 9 and 1,2-phenylenedimethanol by regio- molecules, both of which possessed an intramolecular hydro-
selective displacement of the 3-chloro substituent (Scheme 1), gen bond between the sulfonamide NH and the methoxy ether
and its structure was unambiguously confirmed by an X-ray oxygen atom, as we reported previously in the case of the
diffraction study (Fig. 2). In both independent molecules 5-chlorothiophenesulfonamide analogue of 7.26 The confor-
present in the crystal structure, the sulfonamide hydrogen mations of the two independent molecules are related by a
atom was located and refined approximately in the plane of pseudo-inversion centre. The metric details of the intramole-
the pyrazine ring, allowing a favourable electrostatic inter- cular hydrogen bonds are as follows: for the first molecule
action to the ether oxygen in the same molecule. The influence (shown in Fig. 3), N–H 0.80(2) Å, H⋯O 2.49(2) Å; N⋯O 3.011(2)
Å; and ∠N–H⋯O 124(2)°; and for the second molecule, N–H
0.79(2) Å, H⋯O 2.47(2) Å; N⋯O 3.0238(19) Å; and ∠N–H⋯O
129(2)°. A consequence of the hydrogen bonds is to allow the
plane normals to the 2,3-dichlorobenzene rings and the
indazole rings to be approximately orthogonal [81.15(5)° and
Fig. 3 A view of one of the independent molecules from the X-ray
Fig. 2 A view of one of the independent molecules from the X-ray
crystal structure of 8. 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.
crystal structure of 7. 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.
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75.92(5)° for the above two molecules respectively]. This con-
formation was not previously reported 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 prior to our previous disclosure.26 It is postu-
lated that this conformation might be a preferred one for
increased binding activity.
Investigations to identify other hit series began with the
introduction of an ortho substituent into the simplest 2,3-
dichlorobenzenesulfonamide 10 (pIC50 = 5.3) (Scheme 2 and
Table 2 Structure of substituent R in Scheme 2
Compound number
R
Compound number
R
10
–H
15
11
12
–OMe
16
17
18
Table 2). Three groups were introduced, MeO–, MeCO–, 13
MeCH(OH)– and the respective analogues 11–13 were screened
in the GTPγS assay, which confirmed that the MeO– group
14
increased the potency of 10 by ten-fold (Table 1). Prototypes 7
and 11 were equipotent (pIC50 6.2), with 11 being more
lipophilic (clog P 3.93 vs. 3.48 and chrom log D 6.1 vs. 5.3),
and with a correspondingly lower CLND solubility (20 μg vs.
116 μg mL−1). The ketone 12 and alcohol 13 were less potent
than the methoxy analogue 11 confirming our earlier findings.
In our search for alternative groups to the MeO– group we
envisaged that a 5-membered heteroaryl ring containing two
or more heteroatoms, and placed ortho to the sulfonamide
group might be a good hydrogen bond acceptor for the sulfon-
amide NH, thus driving the 2,3-dichlorophenyl and phenyl
ring to which the sulfonamide group is attached, to be orthog-
onal as in the case of 7. Five compounds possessing 5-mem-
bered rings, including pyrazole, triazole, tetrazole, oxazole and
oxadiazole were synthesised from commercially available
(a) Compound 13 was obtained by reduction of 12 with sodium
borohydride in methanol.
substituted anilines and 2,3-dichlorobenzenesulfonyl chloride
to give analogues 14–18. These analogues were screened
in vitro and the data are presented in Table 1. Pyrazole 14 was
the most potent of the six heterocyclic analogues, with a pIC50
of 6.6 and LE of 0.39. In addition it was weakly active in the
human whole blood assay (pA2 5.3), whereas the other hetero-
cyclic analogues were not active. Its lipophilicity however was
unacceptably high (clog P 4.08 and chrom log D 6.4) as was its
low solubility (48 μg mL−1). A small molecule X-ray crystal
structure of the phenyl pyrazole 14 was obtained (Fig. 4) which
confirmed the existence of an intramolecular hydrogen bond
between the sulfonamide NH and a nitrogen in the pyrazole
Scheme 2 Reagents and conditions: (a) 2,3-dichlorobenzenesulfonyl
chloride, pyridine.
Table 1 In vitro data pIC50 for human CCR4 GTPγS binding assay, cal-
culated log P, ligand efficiency, measured chrom log D at pH 7.4 and
CLND solubility
GTPγS pIC50
SEM (n)
clog
P
Chrom
log D
CLND solub.
Cmpd
LE
(μg mL−1
)
7
8
6.22 0.03 (2)
8.2 0.0 (252)
5.3 0.2 (2)
6.2 0.0 (2)
<4.5 (2)
5.24 0.03 (3)
6.63 0.08 (6)
5.8 0.2 (4)
5.9 0.5 (4)
5.1 0.1 (2)
<4.5 (2)
5.8 0.1 (2)
5.46 0.07 (2)
7.2 0.1 (4)
6.6 0.4 (2)
6.83 0.01 (2)
7.2 0.2 (2)
3.48
3.97
3.99
3.93
3.95
2.70
4.08
3.0
3.23
3.79
2.84
3.26
3.26
3.26
3.26
2.48
2.66
0.35
0.38
0.40
0.42
0.29
0.34
0.39
0.34
0.35
0.30
0.25
0.34
0.33
0.43
0.39
0.40
0.47
5.3
4.0
5.8
6.1
6.45
5.0
6.4
3.3
2.9
4.4
6.3
6.7
2.85
3.72
3.23
3.0
116
153
3
20
5
132
48
180
233
123
8
10
11
12
13
14
15
16
17
18
19
20
23
26
30
33
34
Fig. 4 X-ray crystal structure of phenyl pyrazole 14. 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.
164
203
76
181
133
2.84
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Fig. 5 Least-squares fit (RMS = 0.05 Å) of the 12 non-hydrogen atoms
in the sulfonamide side-chain of indazole 7 (magenta) and pyrazole 14
(black). The intramolecular hydrogen bonds are depicted as dotted lines.
Fig. 6 X-ray crystal structure of phenyl tetrazole 16 showing the
ring [N–H 0.81(2) Å, H⋯N 1.95(2) Å; N⋯N 2.638(3) Å; and ∠N– absence of an intramolecular hydrogen bond between the NH of the
sulfonamide and the tetrazole ring. 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.
H⋯N 143(2)°]. Plane normals to the two phenyl rings were
inclined at 76.65(7)°.
The structures of 7 (the independent molecule shown in
Fig. 3) and 14 were overlaid and are shown in Fig. 5. The
overlay was done on the basis of a least-squares fit for the non-
hydrogen atoms in the sulfonamide side-chains. The RMS for
this fit was just 0.05 Å, indicating how close the conformations
are for this moiety. The remainder of the molecules, although
clearly different, occupy a similar region of space relative to
the atoms fitted, potentially driven by the presence of the
intramolecular bonds.
An X-ray crystal structure of the phenyl tetrazole 16, was
obtained (Fig. 6), which showed that the sulfonamide NH did
not take part in an intramolecular hydrogen bond but instead
was associated with an intermolecular hydrogen bond to N17
[N–H 0.80(2) Å, H⋯N 2.34(2) Å; N⋯N 3.107(2) Å; and ∠N–
H⋯N 161(2)°]. With this arrangement, the rings at either end
of the sulfonamide group are somewhat less orthogonal,
having their normals inclined at 65.65(7)°.
Fig. 7 shows an overlay of the non-hydrogen atoms of the
2,3-dichlorobenzenesulfonamide moiety in 14 and 16, indicat-
ing that as before, the conformations of these groups are
nearly identical. The RMS is again just 0.05 Å. However, rela-
tive to the fitted atoms, the conformations of the rest of the 14
Fig. 7 Least-squares fit (RMS = 0.05 Å) of the 12 non-hydrogen atoms
and 16 molecules are clearly different. In 14, the S21–N9–C10–
in the sulfonamide side-chain of pyrazole 14 (black) and phenyl tetra-
zole 16 (orange). The intramolecular hydrogen bond in the former is
depicted as a dotted line.
C11 torsion angle (that defines the orientation of the central
phenyl ring relative to the sulfonamide group) is −150.44(18)°,
whereas for 16, without the intramolecular hydrogen bond,
the equivalent torsion angle is 78.8(2)°. This represents a rela-
tive rotation of the phenyl ring of approximately 131°.
and we do not wish to suggest that the presence of the intra-
molecular interaction is the primary or only driver. Nonethe-
Clearly, in the solid state, the various intermolecular
packing forces play a large role in the conformations adopted
less, having shown that the pyrazole 14 can adopt
a
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Scheme 3 Reagents and conditions: (a) 2,3-dichlorobenzenesulfonyl chloride, pyridine, 20 °C, 18 h.
conformation in the solid state similar to those of the indazole NMP at 100 °C to give 28 in 64% yield. Reduction of the nitro
sulfonamides and pyrazine 8, it was decided to focus on group of 28 with tin(II) chloride gave 29 in 68% yield. Reaction
further analogues of 14, optimising the physicochemical pro- of the latter with 2,3-dichlorobenzenesulfonyl chloride in pyri-
perties (clog P, chrom log D and solubility). In addition, the dine afforded the desired sulfonamide 26 in 51%.
core ring of 14 is a substituted aniline, which is not very attrac-
Replacement of the phenyl core of 14 with a pyrazine ring
tive in a drug candidate, because of potential genotoxicity would provide an even more hydrophilic analogue than the
issues.32 One way of bypassing such a problem is to introduce corresponding pyridines. The synthesis of analogue 30 is
a nitrogen atom into the phenyl core, which will also lower the shown in Scheme 6 and it commenced with reaction of
lipophilicity, and increase its solubility. The synthesis of ana- 3-bromo-pyrazine-2-amine 31 with 2,3-dichlorobenzenesulfo-
logues containing a nitrogen atom at each one of the four nyl chloride to give sulfonamide 32, followed by displacement
available positions on the core ring of 14 was undertaken. The of bromine by pyrazole in the presence of sodium hydride in
two pyridyl analogues 19 and 20 were prepared from the NMP to give 30 in 71% yield.
corresponding commercially available amines 21 and 22 in
60% and 47% yield respectively (Scheme 3).
The four pyridine analogues 19, 20, 23, 26 and pyrazine 30
were tested in vitro and the data is shown in Table 1. Pyridines
The synthesis of pyrazolyl-5-pyridine sulfonamide 23 is out- 19 and 20 had disappointingly weak affinities for the CCR4
lined in Scheme 4. 3-Bromopyridin-2-amine (24) was reacted receptor, whereas 26 was equipotent to 14. Pyridine 23
with pyrazole, in the presence of copper iodide, potassium car- however, was significantly more potent (pIC50 7.2), its clog P
bonate and N,N′-dimethylethylenediamine in xylene at 142 °C was low at 3.26, and its chrom log D was 3.72. Furthermore, its
to afford 3-(1H-pyrazol-1-yl)pyridin-2-amine 25 in 50% yield.33 ligand efficiency was 0.43 making it one of the most attractive
Reaction with 2,3-dichlorobenzenesulfonyl chloride in pyri- compounds in this study. In addition its CLND solubility
dine afforded the desired sulfonamide 23 in 41% yield.
increased to 203 μg mL−1. In contrast the pyrazine 30, despite
The synthesis of the pyrazolyl-3-pyridine sulfonamide 26 is its even lower clog P and chrom log D, had a slightly lower
shown in Scheme 5 and involved reaction of 3-fluoro-4-nitro- affinity (pIC50 6.8) and a correspondingly lower LE (0.40). The
pyridine 27 with pyrazole in the presence of sodium hydride in reason for 23 being the most potent regioisomer of the four
pyridine analogues was not investigated any further. An expla-
nation for the higher potency of 23, might be a favourable
interaction between the pyridine nitrogen when it is situated
ortho to the sulfonamide group and the receptor, and this is
also true in the pyrazine 30, which was the second most potent
analogue in the series.
In a final iteration to improve the potency and ligand
efficiency of 23, we examined the replacement of the 2,3-
dichlorobenzenesulfonamide group. In our investigation of
the indazole sulfonamide series the 5-chlorothiophenesulfon-
amide group was identified as a superior group as it had a
Scheme 4 Reagents and conditions: (a) Pyrazole, K2CO3, CuI, N,N-
dimethylethylenediamine, p-xylene, 142 °C, 72 h, 50%; (b) 2,3-dichloro-
benzenesulfonyl chloride, pyridine, 20 °C, 3 h, 41%.
Scheme 5 Reagents and conditions: (a) NaH, pyrazole, NMP, 0–100 °C,
18 h, 64%; (b) SnCl2, conc. HCl, MeOH, 20 °C, 48 h, 68%; (c) 2,3-
dichlorobenzenesulfonyl chloride, pyridine, 20 °C, 8 h, 51%.
Scheme 6 Reagents and conditions: (a) NaH, DME, 0 °C, 30 min, 2,3-
dichlorobenzenesulfonyl chloride, 20 °C, 8 h, 73%; (b) NaH, pyrazole,
NMP, 0 °C, 5 min, 100 °C, 18 h, 71%.
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significant basis set effects on the rankings of conformer ener-
gies or on the geometries obtained.
Full frequency calculations were carried out with the
smaller 6-31G* basis (in vacuo and in water) to identify minima
unequivocally and show that electronic energies provide a
reliable guide to free energies (see the ESI† for more details). An
excellent correlation (R2 = 0.991) was obtained between relative
electronic energies, Erel and relative free energies Grel for all the
conformers of 7 and 14 (B3LYP/6-31G*, in vacuo and in water).
Electronic energies obtained with the larger basis set (B3LYP/
6-31+G* in vacuo and in water) are therefore used as the basis
for the subsequent discussion, avoiding the much higher cost
of the full frequency calculations.
The application of the solvation model affects both energies
and geometries; the calculated dipoles were bigger for the
structures in water, consistent with the much higher polarity
of the medium. All the energies and geometries referred to
subsequently were calculated in water.
Clip-shaped or orthogonal conformations (corresponding
to those revealed in the crystal structures of 7, 14 and to a
lesser extent, 16) were identified, though closely-related and
energetically-similar conformations were also found. The clip
is formed from the dichlorophenyl ring and the arene which
bears the hydrogen bond acceptor/donor array; the angles
between the planes measured from the optimised structures
are 77° for 7, 66° for 14 and 62° for 16 (Fig. 8).
Scheme 7 Reagents and conditions: (a) 5-chlorothiophene-2-sulfonyl
chloride, pyridine, 20 °C, 3 h, 57%.
lower molecular weight and was also less lipophilic than the
2,3-dichlorobenzenesulfonamide.
The sulfonamide 33 was prepared from 25 and 5-chloro-
thiophene-2-sulfonyl chloride in pyridine in 57% yield
(Scheme 7). The lipophilicity of 33 (clog P 2.66) was the lowest
in this study, as was its chrom log D (2.84). It was equipotent
to 23 (pIC50 7.2) and its LE increased to 0.47 making it the
most ligand-efficient compound of this study. In addition its
molecular weight was low (341), the number of hydrogen bond
acceptors (6) and number of hydrogen bond donors (1) are
well within the Lipinski guidelines. Its polar surface area was
76, well below the recommended limit of 140 Å2. Furthermore,
33 was tested in the human whole blood assay and it was
weakly active (pA2 5.2). These encouraging data make 33 a
good starting point for a lead-optimisation study.
The clip-shaped or orthogonal conformations are shown in
the left-hand column of Fig. 9. For pyrazolyl species 14, the
lowest energy species corresponds to the clip conformation;
the next lowest conformer is also a clip or orthogonal at
+1.2 kcal mol−1. The dichlorophenyl ring has flipped through
180° via a rotation in the sulfonamido group. The next confor-
mation at +2.1 kcal mol−1 is more extended. The same behav-
iour was observed for indazole 7, with a pair of clip-shaped
conformers 1.4 kcal mol−1 apart; however, in this case a more
extended conformation is much more competitive at +0.2 kcal
Conformational analysis
Conformations identified from crystal structures may not be
representative of states occupied in solution due to the pres-
ence of packing forces, so electronic structure calculations
were used to explore this more fully. Structures corresponding
to 7, 14 and 16 were built in Spartan’0834 and Monte Carlo
conformational searching was carried out using the PM3 semi-
empirical method. Relatively small sets of conformers (10 typi-
cally) were obtained and geometry optimisation calculations
were carried out for all members of the sets using the B3LYP
functional35,36 and the 6-31G* and 6-31+G* basis sets, in vacuo
and in water (Truhlar’s SM8 model37,38 was applied). Single
point energy calculations were also carried out (in vacuo) with
the larger 6-311+G** basis set (using the dual basis set
approximation implemented in Spartan’08). There were no
mol−1
.
Tetrazole 16 behaved differently; the lowest energy confor-
mation is clip-shaped but the dichlorophenyl ring is flipped
over into a different orientation, presenting the chlorine atoms
away from the hydrogen bond acceptor/donor array. The clip-
shaped conformation corresponding to the minima for 7 and
14 lies 0.7 kcal mol−1 higher in energy.
Fig. 8 Stick diagrams of orthogonal conformers calculated (B3LYP/6-31+G* in water [SM8]) for 7, 14 and 16.
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Fig. 9 CPK models of low energy conformers (Erel in kcal mol−1) optimised (B3LYP/6-31+G* in water [SM8]) for 7, 14 and 16. The clip or orthogonal
conformers form the left-hand column.
The lowest energy conformation found for 16 still contains substituents capable of hydrogen bonding to a sulfonamide
an intramolecular hydrogen bond, unlike the arrangement NH. It was envisaged that a five-membered heterocyclic ring
found in the crystal structure; with an isolated molecule, with two or more heteroatoms would be capable of hydrogen
opening the intramolecular hydrogen bond (IHB) leaves the bonding to bring about an orthogonal or clip conformation,
hydrogen bond acceptor and donor sites unsatisfied, which which then binds to the receptor preferentially. X-ray crystal
will have an enthalpic cost. However, the lowest energy confor- structure determinations helped to identify the pyrazole ring
mation which lacks the IHB, lies only 1.7 kcal mol−1 above the as a moiety that could bring about the desired hydrogen
global minimum, whereas opening the IHB in 14 appears to bonding and conformation. Replacement of the core phenyl
cost 7.5 kcal mol−1. Though the geometries of 14 and 16 ring with a pyridine and replacement of the 2,3-dichlorobenzene-
should be broadly similar, there are significant differences sulfonamide with 5-chlorothiophenesulfonamide provided
between the two azoles. Pyrazole is significantly more basic compound 33 with excellent physicochemical properties, and
(ca. 5 pKa units) than tetrazole, and the IHB acceptor site in 16 suitable for further elaboration. Conformational analysis per-
is not the usual site of tetrazole protonation (see the ESI† for formed on compounds 7, 14 and 16 was in agreement with the
more details). The apparent difference in IHB strength there- order of preference for the clip or orthogonal conformation
fore appears reasonable. Searching also revealed the confor- found in the small molecule crystal structures, and the order
mation found in the crystal for 16, at 5.1 kcal mol−1 above the of potency in the GTPγS assay.
global minimum.
These energy differences are small, but even very modest
differences in binding energies can affect potency signifi-
cantly; the order of preference for the clip or orthogonal con-
Experimental
Organic solutions were dried over anhydrous Na2SO4, MgSO4
or using a hydrophobic frit. TLC was performed on Merck
0.25 mm Kieselgel 60 F254 plates. Products were visualised
under UV light and/or by staining with aqueous KMnO4 solu-
tion. LCMS analysis was conducted on an Acquity UPLC BEH
C18 column (50 mm × 2.1 mm ID, 1.7 μm packing diameter)
eluting with 0.1% formic acid in water (solvent A), and 0.1%
formation found in the small molecule crystal structures of
7 and 14 is the same (14 > 7 > 16) as the order of potency in
the GTPγS assay data.
Conclusion
The aim of this study was to identify a lead compound suitable formic acid in MeCN (solvent B), using the following elution
for the start of a lead-optimisation programme to deliver a gradient 0.0–1.5 min 3–100% B, 1.5–1.9 min 100% B,
CCR4 antagonist as a potential back-up to the clinical candi- 1.9–2.0 min 100–3% B, at a flow rate of 1 mL min−1 at 40 °C.
date 3. The study began by examining a number of ortho- The UV detection was an averaged signal from wavelength of
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210 nm to 350 nm, and mass spectra were recorded on a mass 2,3-dichlorobenzenesulfonyl chloride (3.36 g, 13.7 mmol) at
spectrometer using alternate-scan electrospray positive and 20 °C, and the reaction mixture was stirred for 18 h. An
negative mode ionization (ESI+ve and ESI−ve). Column chrom- additional portion of 2,3-dichlorobenzenesulfonyl chloride
atography was performed on a Flashmaster II system. Purifi- (1 g, 4 mmol) was added and the mixture was stirred at room
cations by mass-directed auto-preparative HPLC (MDAP) was temperature for 18 h. The reaction mixture was partitioned
conducted on a Sunfire C18 column (150 mm × 30 mm i.d. between dichloromethane (250 mL) and water (250 mL). The
5 μm packing diameter) at ambient temperature eluting with organic phase was washed with 2 M hydrochloric acid
0.1% formic acid in water (solvent A) and 0.1% formic acid in (100 mL), dried and evaporated under reduced pressure. The
MeCN (solvent B), using an appropriate elution gradient over residue was purified by chromatography on a silica (100 g) car-
15 or 25 min at a flow rate of 40 mL min−1 and detecting at tridge eluting with a gradient of 0–100% ethyl acetate–cyclo-
210–350 nm at room temperature. Mass spectra were recorded hexane over 60 min. The appropriate fractions were combined
on Micromass ZMD mass spectrometer using electrospray and evaporated in vacuo to give 7 (2.51 g, 48%) as an off-white
positive and negative mode, alternate scans. The software used solid: 1H NMR (500 MHz, DMSO-d6) δ = 10.43 (s, 1H), 7.91 (dd,
was MassLynx 3.5 with OpenLynx and FractionLynx options.
J = 8.0, 1.0 Hz, 1H), 7.85 (dd, J = 8.0, 1.0 Hz, 1H), 7.47 (t, J = 8.0
1H NMR spectra were recorded at 400, 500 or 600 MHz. Hz, 1H), 7.26 (t, J = 8.0 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 6.46
Chemical shifts (δ) are expressed in ppm relative to tetra- (d, J = 8.0 Hz, 1H), 3.86 (s, 3H), 3.64 (s, 3H); 13C NMR
methylsilane. 13C NMR spectra were recorded at 101, 126 or (126 MHz, DMSO-d6) δ = 153.5, 142.9, 141. 6, 134.8, 134.5,
150 MHz. 1H and 13C NMR spectra were recorded using the 134.3, 129.9, 129.6, 128.6, 128.5, 110.4, 102.8, 100.4, 55.5, 36.0;
deuterated solvent as the lock and the residual solvent as the LCMS (ESI) RT = 1.05 min, 100% pure, m/z 386, 388 [M + H]+.
internal reference. In the cases indicated solid NaHCO3 was Anal. Calcd for C15H14Cl2N3O3S = 386.0128. Found = 386.0125
added to the NMR solution to sharpen up the line-widths of the [M + H]+. Crystal data: C15H13Cl2N3O3S; M = 386.24; colourless
13C signals, as some signals were so broad that did not show up wedge; spontaneous crystallisation from methanol, 10% 2 M
ˉ
in the absence of base. It is assumed that the original samples HCl; 0.82 × 0.62 × 0.34 mm; triclinic; space group, P1 (no. 2);
had very broad signals due to inter-conversion between unit cell dimensions, a = 7.9294(12) Å, b = 12.763(2) Å, c =
different forms at an intermediate rate over the NMR timescale. 16.761(3) Å, α = 96.664(17)°, β = 99.981(17)°, γ = 97.378(14)°,
These different forms might be due to the zwitterionic nature of V = 1640.1(5) Å3; Z = 4; dcalc = 1.564 Mg m−3; and μ(Mo-Kα, λ =
the compounds. Adding the base generated the sodium salt 0.71073 Å) = 0.543 mm−1
and created a single form with a sharp set of lines.
.
2,3-Dichloro-N-(3,6-dichloropyrazin-2-yl)benzenesulfonamide
High resolution mass spectra were acquired on a Micromass
Q-Tof 2 hybrid quadrupole time-of-flight mass spectrometer
(9)
coupled to HPLC. LC analysis for HRMS was conducted on a A solution of 3,6-dichloropyrazin-2-amine40 (6.1 g, 37 mmol)
Phenomenex Luna C18 (2) column (100 mm × 2.1 mm, 3 µm in DME (300 mL) was treated with solid NaH (60% oil dis-
particle size), eluting with 0.1% formic acid water (solvent A), persion, 7.5 g, 190 mmol) over 45 min at room temperature.
and 0.1% formic acid in MeCN (solvent B), using the following After the addition was complete the mixture was warmed to
elution gradient 0–2 min 5% B, 2–8 min 5–100% B, 8–10.5 min 50 °C for 1 h. At this temperature, 2,3-dichlorobenzenesulfonyl
100% B, 10.5–11.5 min 100–5% B, 11.5–14 min 5% B, at a flow chloride (11.9 g, 49 mmol) was added over 1 h and the mixture
rate of 0.5 mL min−1, at 35 °C. The elemental composition was stirred for a further 30 min at 50 °C. The solution was cooled
calculated using MassLynx v4.1 for the [M + H]+. The purity of all in an ice-bath and treated with 5% aqueous citric acid solu-
compounds screened in the biological assays was ≥95%, unless tion. The resulting mixture was partitioned between ethyl
otherwise specified. Melting points were determined on a Stuart acetate and brine, the organic layer separated, dried (sodium
SMP40 automatic melting point apparatus. Starting materials sulfate) and concentrated to an orange solid (16 g). The
were commercially available unless otherwise specified.
residue was triturated with diethyl ether to give 11 g of the
The four single crystal X-ray diffraction studies on 7, 8, 14 crude product, which was purified by chromatography in a gra-
and 16 were carried out at 150 K using Mo-Kα X-radiation (λ = dient of ethyl acetate–petrol to MeOH–EtOAc (starting with
0.71073 Å). Crystal data for each compound are given in the 30% EtOAc in petrol), and re-purified by chromatography
sections below. A description of the refinement and the full using a gradient of 0–30% MeOH–dichloromethane to give two
tables associated with each crystal structure are given in the fractions of varying purity. The first was triturated with diethyl
ESI.† Crystallographic information files have been deposited ether to give pure 9 (3.2 g, 23%). The second fraction was
with the Cambridge Crystallographic Data Centre (CCDC). further purified by chromatography using a gradient of 10%
CCDC 975458–975461 contain the supplementary crystallo- MeOH–dichloromethane to give pure 9 (2 g, 14%) and some
graphic data for this paper.
impure fractions. The impure fractions were concentrated and
triturated with diethyl ether to give a further 1.2 g of product.
All three product batches were combined to give 9 (6.4 g, 46%)
2,3-Dichloro-N-[1-methyl-4-(methyloxy)-1H-indazol-3-yl]-
benzenesulfonamide (7)
1
as a white solid: H NMR δ (400 MHz, DMSO-d6) 8.05 (dd, J =
A
solution of 4-methoxy-1H-indazol-3-amine39 (2.38 g, 8.0, 1.5 Hz, 1H), 7.68 (dd, J = 8.0, 1.5 Hz, 1H), 7.45–7.40 (m,
13.4 mmol) in pyridine (50 mL) was treated portionwise with 2H); 13C NMR δ (101 MHz, DMSO-d6) 152.2, 144.7, 143.2,
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137.5, 132.7, 131.9, 130.4, 128.4, 127.0, 126.6; LCMS (ESI) RT = 1H); 13C NMR (126 MHz, DMSO-d6) δ = 139.2, 137.2, 135.5,
1.06 min, 97% pure, m/z 372/374/376 [M + H]+, and RT = 134.7, 130.9, 129.8 (2C), 129.3, 129.1, 124.7, 119.9 (2C); LCMS
1.13 min, 3% (another isomer). Anal. Calcd for C10H6Cl4N3O2S = (ESI) RT = 1.12 min, 100% pure, m/z = 300, 302 [M − H]−. Anal.
371.8929. Found = 371.8933 [M + H]+.
Calcd for C12H10Cl2NO2S = 301.9804. Found = 301.9800
[M + H]+.
2,3-Dichloro-N-(6-chloro-3-((2-(hydroxymethyl)benzyl)oxy)-
pyrazin-2-yl)benzenesulfonamide (8)
2,3-Dichloro-N-(2-methoxyphenyl)benzenesulfonamide (11)41
A solution of 2,3-dichloro-N-(3,6-dichloropyrazin-2-yl)benzene-
sulfonamide (2.4 g, 6.5 mmol), 1,2-phenylenedimethanol
(4.5 g, 32 mmol) in NMP (40 mL) was treated with potassium
tert-butoxide (5.47 g, 48.7 mmol) in THF (60 mL) and the
mixture was stirred at room temperature for 5 h. The solution
was cooled to 0 °C and 5% aqueous citric acid added
(100 mL). The mixture was extracted with ethyl acetate
(200 mL) and the organic solution washed with water (100 mL)
and brine (100 mL) then dried over sodium sulfate and con-
centrated under reduced pressure. The crude material was
1
(105 mg, 79%): H NMR (500 MHz, DMSO-d6) δ = 9.79 (s, 1H),
7.89 (dd, J = 8.0, 1.5 Hz, 1H), 7.76 (dd, J = 8.0, 1.5 Hz, 1H), 7.44
(t, J = 8.0 Hz, 1H), 7.23–7.10 (m, 2H), 6.95–6.78 (m, 2H), 3.44
(s, 3H); 13C NMR (126 MHz, DMSO-d6) δ = 153.3, 140.1, 134.1,
133.7, 129.3, 129.2, 127.8, 127.6, 127.2, 124.0, 120.3, 111.6,
54.9; LCMS (ESI) RT = 1.18 min, 98% pure, m/z 332, 334
[M + H]+. Anal. Calcd for C13H12Cl2NO3S = 331.9910. Found =
331.9905 [M + H]+.
purified by chromatography eluting with a gradient of ethyl N-(2-Acetylphenyl)-2,3-dichlorobenzenesulfonamide (12)
acetate–petroleum ether (5–20%). The material recovered
(1.5 g) was still impure so it was further purified by chromato-
graphy eluting with a gradient of MeOH–DCM (1–2%). The
2,3-Dichlorobenzenesulfonyl chloride (100 mg, 0.41 mmol)
was added to a stirring solution of 2-aminoacetophenone
(50 mg, 0.37 mmol) in anhydrous pyridine (2 mL) and the reac-
appropriate fractions were evaporated under reduced pressure
tion mixture was stirred at ambient temperature for 4 h.
and the residue was triturated with diethyl ether to give the
Additional 2,3-dichlorobenzenesulfonyl chloride (75 mg,
desired material (390 mg). The filtrates from the trituration
0.30 mmol) was added and the reaction mixture was stirred for
were evaporated and triturated again with petroleum ether in
a further 18 h. The reaction mixture was evaporated under
DCM to give a further batch of product (130 mg). The batches
reduced pressure, and the solid was dissolved in DMSO (2 mL)
1
were combined to afford 8 (520 mg, 17%) as a solid: H NMR
and purified by MDAP. Appropriate fractions were combined
(400 MHz, CD3OD) 8.23 (dd, J = 8.0, 1.5 Hz, 1H), 7.78 (dd, J =
and evaporated under reduced pressure to afford 12 (46 mg,
8.0, 1.5 Hz, 1H), 7.66 (br s, 1H), 7.49 (t, J = 8.0 Hz, 1H),
7.46–7.42 (m, 2H), 7.38–7.27 (m, 2H), 5.54 (s, 2H), 4.74 (s, 2H)
(the NH and OH exchangeable protons were not observed);
LCMS (ESI) RT = 1.22 min, 100% pure, m/z 474, 476 [M + H]+.
Crystal data: C18H14Cl3N3O4S; M = 474.73; colourless needle;
slow cooling from ethanol; 0.64 × 0.10 × 0.10 mm; monoclinic;
space group, P21/n (alt. P21/c, no. 14); unit cell dimensions, a =
9.0361(16) Å, b = 20.725(2) Å, c = 21.381(6) Å, β = 96.612(16)°,
V = 3977.5(14) Å3; Z = 8; dcalc = 1.586 Mg m−3; and μ(Mo-Kα, λ =
1
36%) as a white solid: H NMR (500 MHz, DMSO-d6) δ = 12.09
(s, 1H), 8.20 (dd, J = 8.0, 1.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H),
7.97 (dd, J = 8.0, 1.0 Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.52 (t, J =
8.0 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H),
2.67 (s, 3H); 13C NMR (126 MHz, DMSO-d6) δ = 203.8, 138.0,
138.0, 136.1, 135.5, 134.8, 133.41, 131.2, 129.2, 129.0, 123.7,
122.9, 117.4, 28.9; LCMS (ESI) RT = 1.16 min, 100% pure, m/z
344, 346 [M + H]+. Anal. Calcd for C14H12Cl2NO3S = 343.9910.
Found = 343.9909 [M + H]+.
0.71073 Å) = 0.597 mm−1
.
2,3-Dichloro-N-phenylbenzenesulfonamide (10)
2,3-Dichloro-N-(2-(1-hydroxyethyl)phenyl)benzenesulfonamide
(13)
Aniline (37.2 mg, 0.4 mmol) was dissolved in dichloromethane
(1 mL) and treated with a solution of 2,3-dichlorobenzenesul- Sodium borohydride (4 mg, 0.11 mmol) was added to a stirring
fonyl chloride in dichloromethane (0.44 M, 1 mL, 1.1 equiv.), solution of 12 (25 mg, 0.07 mmol) in methanol (0.5 mL) and
followed by a stock solution of pyridine in dichloromethane the reaction mixture was stirred at ambient temperature for
(10 M, 0.12 mL, 3 equiv.). The reaction mixture was stirred at 2 h. Additional sodium borohydride (4 mg, 0.11 mmol) was
room temperature for 2.5 h, and then treated with saturated added and the reaction was stirred for a further 4 h. The reac-
aqueous sodium bicarbonate solution (2 mL). After stirring for tion mixture was treated with 2 M HCl solution (0.1 mL), dis-
15 min the reaction mixture was transferred to a hydrophobic solved in DMSO (1 mL) and purified by MDAP. The solvent was
frit and the organic phase isolated. The aqueous phase was evaporated under reduced pressure to give 13 (14 mg, 53%) as
1
further extracted with dichloromethane (2 mL) and the com- a colourless oil: H NMR (500 MHz, DMSO-d6) δ = 10.16 (br s,
bined extracts were evaporated under nitrogen in a blow-down 1H), 8.02–7.91 (m, 2H), 7.56 (t, J = 8.0 Hz, 1H), 7.39 (dd, J =
apparatus. The product was purified by mass-directed auto- 7.5, 1.0 Hz, 1H), 7.22–7.06 (m, 2H), 6.88 (d, J = 8.0 Hz, 1H),
preparative HPLC (MDAP) to give 10 (84 mg, 69%): 1H NMR 5.60 (br s, 1H), 5.05 (q, J = 6.3 Hz, 1H), 1.26 (d, J = 6.3 Hz, 3H);
(500 MHz, DMSO-d6) δ = 10.77 (s, 1H), 8.03 (dd, J = 8.0, 1.5 Hz, 13C NMR (126 MHz, DMSO-d6) δ = 141.2, 140.3, 135.3, 134.8,
1H), 7.91 (dd, J = 8.0, 1.5 Hz, 1H), 7.53 (t, J = 8.0 Hz, 1H), 133.4, 130.2, 129.4, 129.2, 127.8, 127.3, 126.5, 123.9, 65.8, 25.0;
7.31–7.18 (m, 2H), 7.09 (d, J = 7.5 Hz, 2H), 7.02 (t, J = 7.5 Hz, LCMS (ESI) RT = 1.03 min, 100% pure, m/z 344, 346 [M + H]+.
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N-(2-(1H-Pyrazol-1-yl)phenyl)-2,3-dichlorobenzenesulfonamide
(14)
88.763(11)°, β = 84.177(10)°, γ = 64.880(13)°, V = 741.50(16) Å3;
Z = 2; dcalc = 1.658 Mg m−3; and μ(Mo-Kα, λ = 0.71073 Å) =
0.595 mm−1
.
General procedure for the preparation of 14–20. 2,3-Dichloro-
benzenesulfonyl chloride (120 mg, 0.48 mmol) was added to a
stirring solution of 2-(1H-pyrazol-1-yl)aniline (70 mg,
0.44 mmol) in anhydrous pyridine (2 mL) and the reaction was
stirred at ambient temperature for 18 h. The reaction mixture
was evaporated under reduced pressure, and the solid was dis-
solved in DMSO (1 mL) and purified by MDAP. The solvent was
evaporated under reduced pressure to 14 (118 mg, 71%) as a
white solid: mp = 152–154 °C (from MeOH); 1H NMR
(400 MHz, DMSO-d6) δ = 10.9 (s, 1H), 8.23 (d, J = 2.0 Hz, 1H),
7.94–7.78 (m, 3H), 7.55 (dd, J = 8.0, 2.0 Hz, 1H), 7.51–7.43 (m,
2H), 7.35–7.25 (m, 2H), 6.55 (t, J = 2.0 Hz, 1H); 13C NMR
(101 MHz, DMSO-d6) δ = 141.1, 138.2, 135.1, 134.3, 131.4,
130.9, 130.0, 128.8, 128.4, 128.2, 127.9, 126.3, 123.6, 123.5,
107.5; LCMS (ESI) RT = 1.23 min, 100% pure, m/z 368, 370
[M + H]+. Anal. Calcd for C15H12Cl2N3O2S = 368.0027. Found =
368.0028 [M + H]+. Crystal data: C15H11Cl2N3O2S; M = 368.23;
colourless block; slow evaporation of methanol solution; 0.44
× 0.19 × 0.11 mm; monoclinic; space group, P21/n (alt. P21/c,
no. 14); unit cell dimensions, a = 7.5758(9) Å, b = 8.7573(9) Å,
2,3-Dichloro-N-(2-(oxazol-5-yl)phenyl)benzenesulfonamide (17)
From 2-(oxazol-5-yl)aniline (54 mg, 0.34 mmol) and 2,3-
dichlorobenzenesulfonyl chloride (91 mg, 0.37 mmol). (77 mg,
1
61%) as a pale yellow solid; H NMR (400 MHz, DMSO-d6) δ =
10.29 (s, 1H), 8.39 (s, 1H), 7.92 (dd, J = 8.0, 1.5 Hz, 1H), 7.78
(dd, J = 8.0, 1.5 Hz, 1H), 7.68 (dd, J = 7.7, 1.5 Hz, 1H), 7.60 (s,
1H), 7.49 (t, J = 8.0 Hz, 1H), 7.40 (t, J = 7.7 Hz, 1H), 7.30 (td, J =
7.7, 1.5 Hz, 1H), 6.91 (dd, J = 7.7, 1.5 Hz, 1H); 13C NMR
(126 MHz, DMSO-d6 + NaHCO3) δ = 150.5, 149.7, 147.5, 147.1,
133.6, 131.5, 129.3, 128.9, 128.1, 127.7, 125.3, 125.0, 119.6,
119.4, 116.7; LCMS (ESI) RT = 1.00 min, 100% pure, m/z 369,
371 [M + H]+. Anal. Calcd for C15H11Cl2N2O3S = 368.9862.
Found = 368.9860 [M + H]+.
2,3-Dichloro-N-(2-(5-methyl-1,3,4-oxadiazol-2-yl)phenyl)-
benzenesulfonamide (18)
From
2-(5-methyl-1,3,4-oxadiazol-2-yl)aniline
(59
mg,
0.34 mmol) and 2,3-dichlorobenzenesulfonyl chloride (91 mg,
0.37 mmol). (46 mg, 35%) as a white solid: 1H NMR (400 MHz,
DMSO-d6) δ = 11.21 (s, 1H), 8.19 (dd, J = 8.0, 1.5 Hz, 1H), 7.97
(dd, J = 8.0, 1.5 Hz, 1H), 7.88 (d, J = 7.5 Hz, 1H), 7.61 (t, J =
8.0 Hz, 1H), 7.56–7.41 (m, 2H), 7.28 (t, J = 7.5 Hz, 1H), 2.60 (s,
3H); 13C NMR (126 MHz, DMSO-d6) δ = 164.1, 163.3, 138.3,
136.2, 135.6, 135.0, 133.4, 131.2, 129.4, 129.2, 129.1, 124.9,
118.7, 112.4, 11.1; LCMS (ESI) RT = 1.17 min, 100% pure, m/z
384, 386 [M + H]+. Anal. Calcd for C15H12Cl2N3O3S = 383.9971.
Found = 383.9967 [M + H]+.
c = 23.065(2) Å, β = 91.905(8)°, V = 1529.4(3) Å3; Z = 4; dcalc
=
1.599 Mg m−3; and μ(Mo-Kα, λ = 0.71073 Å) = 0.573 mm−1
.
N-(2-(1H-1,2,4-Triazol-1-yl)phenyl)-2,3-dichlorobenzene-
sulfonamide (15)
From 2-(1H-1,2,4-triazol-1-yl)aniline (54 mg, 0.34 mmol) and
2,3-dichlorobenzenesulfonyl chloride (91 mg, 0.37 mmol).
(76 mg, 60%) as a pale yellow solid: 1H NMR (400 MHz,
DMSO-d6) δ = 10.32 (br s, 1H), 8.82 (s, 1H), 8.12 (s, 1H), 7.90
(dd, J = 8.0, 1.5 Hz, 1H), 7.77 (dd, J = 8.0, 1.5 Hz, 1H), 2,3-Dichloro-N-[2-(1H-pyrazol-1-yl)-3-pyridinyl]-
7.55–7.51 (m, 1H), 7.49–7.38 (m, 3H), 7.34–7.28 (m, 1H); 13C benzenesulfonamide (19)
NMR (126 MHz, DMSO-d6) δ = 152.5, 145.3, 139.5, 135.4, 134.8,
From 2-(1H-pyrazol-1-yl)-3-pyridinamine (59 mg, 0.37 mmol)
132.9, 130.2, 129.9, 129.6, 129.4, 129.0, 128.4, 128.2, 126.6;
LCMS (ESI) RT = 0.96 min, 99% purity, m/z 369, 371 [M + H]+.
Anal. Calcd for C14H11Cl2N4O2S = 368.9974. Found = 368.9974
[M + H]+.
and
2,3-dichlorobenzenesulfonyl
chloride
(100
mg,
0.41 mmol). (83 mg, 60%) as a white solid: mp = 135–138 °C;
1H NMR (400 MHz, DMSO-d6) δ = 12.08 (s, 1H), 8.62 (d, J =
2.0 Hz, 1H), 8.21 (d, J = 4.0 Hz, 1H), 8.10 (dd, J = 8.0, 1.0 Hz,
1H), 8.04 (s, 1H), 8.00–7.86 (m, 2H), 7.56 (t, J = 8.0 Hz, 1H),
7.34 (dd, J = 8.0, 4.0 Hz, 1H), 6.67 (d, J = 2.0 Hz, 1H); 13C NMR
(101 MHz, DMSO-d6) δ = 143.3, 141.7, 138.6, 137.4, 135.6,
N-(2-(1H-Tetrazol-1-yl)phenyl)-2,3-dichlorobenzene-
sulfonamide (16)
From 2-(1H-tetrazol-1-yl)aniline (84 mg, 0.34 mmol) and 2,3- 134.5, 130.5, 129.6, 128.9, 128.8, 128.7, 123.6, 122.8, 107.7;
dichlorobenzenesulfonyl chloride (91 mg, 0.37 mmol). (56 mg, LCMS (ESI) RT = 0.80 min, 98% pure, m/z 369, 371 [M + H]+.
47%) as a white solid; mp = 188–190 °C (from MeOH); 1H Anal. Calcd for C14H11Cl2N4O2S = 368.9980. Found = 368.9971
NMR (400 MHz, DMSO-d6) δ = 10.56 (br s, 1H), 9.60 (s, 1H), [M + H]+.
7.93 (dd, J = 8.0, 1.0 Hz, 1H), 7.74 (dd, J = 8.0, 1.0 Hz, 1H),
N-(4-(1H-Pyrazol-1-yl)pyridin-3-yl)-2,3-dichlorobenzene-
sulfonamide (20)
7.64–7.53 (m, 2H), 7.52–7.42 (m, 2H), 7.34–7.20 (m, 1H); 13C
NMR (101 MHz, DMSO-d6) δ = 144.6, 139.2, 134.8, 134.4, 131.4,
130.4, 130.2, 129.1, 128.9, 128.6, 128.5, 128.2, 127.5; LCMS From 4-(1H-pyrazol-1-yl)pyridin-3-amine (50 mg, 0.31 mmol)
(ESI) RT = 0.92 min, 95% purity, m/z 370, 372 [M + H]+. Anal. and 2,3-dichlorobenzenesulfonyl chloride (84 mg, 0.34 mmol).
Calcd for C13H10Cl2N5O2S = 369.9932. Found = 369.9930 (57 mg, 47%) as a pale brown solid: mp = 188–191 °C; 1H NMR
[M + H]+. Crystal data: C13H9Cl2N5O2S; M = 370.21; colourless (500 MHz, DMSO-d6 + NaHCO3) δ = 9.43 (d, J = 2.5 Hz, 1H),
rod; slow evaporation of methanol solution; 0.39 × 0.10 × 8.34 (s, 1H), 8.03 (dd, J = 8.0, 1.5 Hz, 1H), 7.79 (d, J = 8.0 Hz,
ˉ
0.06 mm; triclinic; space group, P1 (no. 2); unit cell dimen- 1H), 7.71–7.57 (m, 3H), 7.41 (t, J = 8.0 Hz, 1H), 6.44 (t, J =
sions, a = 7.4390(9) Å, b = 8.4770(12) Å, c = 13.0579(15) Å, α = 2.1 Hz, 1H); the exchangeable NH proton was not observed;
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13C NMR (126 MHz, DMSO-d6 + NaHCO3) δ = 146.6, 143.2, (126 MHz, DMSO-d6) δ = 153.8, 143.9, 140.3, 138. 6, 129.9,
139.8, 138.1, 135.9, 135.7, 133.5, 132.9, 131.7, 129.0, 128.8, 129.0, 120.3, 110.3; LCMS (ESI) RT = 0.70 min, 98% pure, m/z
127.7, 115.4, 106.0; LCMS (ESI) RT = 0.98 min, 95% pure, m/z 191 [M + H]+. Anal. Calcd for C8H7N4O2 = 191.0564. Found =
369, 371 [M + H]+. Anal. Calcd for C14H11Cl2N4O2S = 368.9974. 191.0560 [M + H]+.
Found = 368.9974 [M + H]+.
3-(1H-Pyrazol-1-yl)pyridin-4-amine (29)
3-(1H-Pyrazol-1-yl)pyridin-2-amine (25)33
Tin(II) chloride (449 mg, 2.37 mmol) was added to a solution
A
mixture of 3-bromopyridin-2-amine (24) (500 mg, of 28 (90 mg, 0.47 mmol) in MeOH (1.5 mL). Conc. HCl
2.89 mmol), potassium carbonate (839 mg, 6.07 mmol), N,N′- (0.2 mL of a 12 M solution, 2.4 mmol) was added and the reac-
dimethylethylenediamine (0.092 mL, 0.87 mmol), pyrazole tion mixture was allowed to stir at ambient temperature for
(236 mg, 3.47 mmol) and CuI (83 mg, 0.43 mmol) in p-xylene 48 h. The reaction mixture was neutralised by the addition of
(8 mL) was heated under nitrogen at 142 °C for 72 h. The solid sodium carbonate. The solution was diluted with ethyl
mixture was partitioned between ethyl acetate (100 mL) and acetate (100 mL), washed with sodium hydroxide (20 mL of a
water (50 mL). The organic layer was separated, washed with 1 M aqueous solution), followed by sodium bicarbonate
brine (50 mL), dried (MgSO4) and evaporated under reduced (40 mL of a saturated aqueous solution), dried (MgSO4), and
pressure. The sample was pre-absorbed on Florisil and puri- concentrated under reduced pressure to give 29 (55 mg, 68%)
1
fied by chromatography on a silica cartridge (100 g), using a as a white solid: H NMR (400 MHz, DMSO-d6) δ = 8.32 (d, J =
gradient of 0–50% ethyl acetate–cyclohexane over 40 min. The 3.0 Hz, 1H), 8.23 (d, J = 3.0 Hz, 1H), 7.77 (dd, J = 8.0, 2.0 Hz,
appropriate fractions were combined and the solvent was evap- 1H), 7.65 (d, J = 2.0 Hz, 1H), 6.54 (d, J = 8.0 Hz, 1H), 6.46 (t, J =
orated under reduced pressure to give 25 (230 mg, 50%) as a 2.0 Hz, 1H), 6.06 (br s, 2H); LCMS (ESI) RT = 0.33 min, 93%
beige coloured solid: 1H NMR (400 MHz, CDCl3) δ = 8.07 (d, J = pure, m/z 161 [M + H]+.
5.0 Hz, 1H), 7.85–7.65 (m, 2H), 7.44 (dd, J = 8.0, 1.5 Hz, 1H),
N-(3-(1H-Pyrazol-1-yl)pyridin-4-yl)-2,3-dichlorobenzene-
sulfonamide (26)
6.71 (dd, J = 8.0, 5.0 Hz, 1H), 6.47 (t, J = 2.0 Hz, 1 H), 5.75 (br s,
2H); LCMS (ESI) RT = 0.61 min, 100% pure, m/z 161 [M + H]+.
From 29 (35 mg, 0.22 mmol) and 2,3-dichlorobenzenesulfonyl
N-(3-(1H-Pyrazol-1-yl)pyridin-2-yl)-2,3-dichlorobenzene-
sulfonamide (23)
chloride (70 mg, 0.28 mmol) (42 mg, 51%) as a pale brown
solid: mp = 246–250 °C; 1H NMR (400 MHz, DMSO-d6) δ =
From 25 (15 mg, 0.09 mmol) and 2,3-dichlorobenzenesulfonyl 11.90 (br s, 1H), 8.52 (br s, 1H), 8.41 (d, J = 2.5 Hz, 1H), 8.16
chloride (25 mg, 0.10 mmol) (14 mg, 41%) as a white solid: (m, 2H), 7.91 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 1.5 Hz, 1H), 7.59
mp = 199–202 °C; 1H NMR (500 MHz, DMSO-d6 + NaHCO3) δ = (t, J = 8.0 Hz, 1H), 7.19 (br s, 1H), 6.52 (t, J = 2.5 Hz, 1H); 13C
9.10 (d, J = 2.0 Hz, 1H), 8.03 (dd, J = 8.0, 1.5 Hz, 1H), 7.77 (dd, NMR (126 MHz, DMSO-d6 + NaHCO3) δ = 160.2, 147.5, 140.5,
J = 8.0, 1.5 Hz, 1H), 7.66–7.52 (m, 3H), 7.36 (t, J = 8.0 Hz, 1H), 138.8, 133.2, 131.5, 129.7, 129.2, 128.4, 128.1, 127.8, 127.5,
6.52 (dd, J = 8.0, 5.0 Hz, 1H), 6.40 (t, J = 2.0 Hz, 1H); the 114.8, 107.4; LCMS (ESI) RT = 0.94 min, 98% pure, m/z 369,
exchangeable NH proton was not observed; 13C NMR 371 [M + H]+. Anal. Calcd for C14H11Cl2N4O2S = 368.9974.
(126 MHz, DMSO-d6 + NaHCO3) δ = 152.2, 147.7, 144.8, 139.3, Found = 368.9974 [M + H]+.
132.6, 131.9, 131.0, 130.1, 128.8, 128.5, 127.1, 126.0, 112.0,
N-(3-Bromopyrazin-2-yl)-2,3-dichlorobenzenesulfonamide (32)
105.3; LCMS (ESI) RT = 1.02 min, 100% pure, m/z 369, 371
[M + H]+. Anal. Calcd for C14H11Cl2N4O2S = 368.9974. Found = Sodium hydride (575 mg of a 60% w/w dispersion in mineral
368.9974 [M + H]+.
oil, 14.4 mmol) was added to a stirred solution of 3-bromo-
pyrazin-2-amine (31) (500 mg, 2.87 mmol) in anhydrous DME
(6 mL) at 0 °C. The mixture was stirred for 30 min and then
4-Nitro-3-(1H-pyrazol-1-yl)pyridine (28)
Sodium hydride (113 mg of a 60% w/w dispersion in mineral 2,3-dichlorobenzenesulfonyl chloride (776 mg, 3.16 mmol) was
oil, 2.82 mmol) was added to a stirred solution of 1H-pyrazole added. The reaction was allowed to warm to ambient tempera-
(287 mg, 4.22 mmol) in NMP (5 mL) at 0 °C. The mixture was ture and stirred for 18 h. The reaction mixture was quenched
stirred for 5 min, then 3-fluoro-4-nitropyridine (27) (200 mg, by the addition of HCl (20 ml of a 2 M aqueous solution) and
1.41 mmol) was added and the reaction was heated to 100 °C the product was extracted with ethyl acetate (3 × 50 mL). The
for 18 h. After cooling, ethyl acetate (50 mL) and water (50 mL) organic extracts were combined, dried (MgSO4), and evapor-
were poured into the mixture. The organic layer was separated, ated under reduced pressure to give the crude product. The
washed with water (50 mL), brine (30 mL), dried (MgSO4), and residue was adsorbed onto Florisil, and purified by chromato-
evaporated under reduced pressure. The sample was purified graphy on a silica cartridge (50 g) eluting with a gradient of
by chromatography on a silica cartridge (70 g) using a gradient 0–100% ethyl acetate–cyclohexane over 40 min. The appropri-
of 25–100% ethyl acetate–cyclohexane over 40 min. The appro- ate fractions were combined and evaporated under reduced
priate fractions were combined and evaporated under reduced pressure to give 32 (800 mg, 73%) as a beige coloured solid: 1H
pressure to give 28 (173 mg, 64%) as a white solid: 1H NMR NMR (600 MHz, DMSO-d6) δ = 8.15 (br s, 1H), 8.12 (br s, 1H),
(400 MHz, DMSO-d6) δ = 9.10 (d, J = 2.0 Hz, 1H), 8.57–8.41 (m, 8.06 (dd, J = 8.1, 1.5 Hz, 1H), 7.93 (dd, J = 8.1, 1.5 Hz, 1H), 7.58
3H), 7.86 (d, J = 2.0 Hz, 1H), 6.64 (t, J = 2.0 Hz, 1H); 13C NMR (t, J = 8.1 Hz, 1H); the exchangeable NH proton was not
1790 | Org. Biomol. Chem., 2014, 12, 1779–1792
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observed; 13C NMR (151 MHz, CD3OD + DCl) δ = 146.6, 141.4,
141.1, 139.2, 138.5, 136.1, 136.0, 132.3, 131.0, 128.9; LCMS
(ESI) RT = 0.98 min, 91% pure, m/z 382, 384, 386 [M + H]+.
References
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381.8819, [M + H]+.
C10H7BrCl2N3O2S = 381.8814. Found =
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Sodium hydride (60% oil dispersion, 52 mg, 1.3 mmol) was
added to a stirred solution of 1H-pyrazole (107 mg, 1.57 mmol)
in NMP (1.0 mL) at 0 °C. The mixture was stirred for 5 min,
then 32 (100 mg, 0.261 mmol) was added and the reaction was
heated to 100 °C for 18 h. After cooling, the mixture was
poured into water (20 mL), acidified to pH 5 with 2 M aqueous
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with MeOH to give 30 (68 mg, 71%) as a white solid: mp =
217–220 °C; 1H NMR (400 MHz, DMSO-d6) δ = 12.32 (br s, 1H),
8.77 (d, J = 2.5 Hz, 1H), 8.28 (dd, J = 8.0, 1.5 Hz, 1H), 8.19 (d,
J = 2.5 Hz, 1H), 8.15 (d, J = 1.5 Hz, 1H), 8.12 (d, J = 2.5 Hz, 1H),
7.98 (dd, J = 8.0, 1.5 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H), 6.80–6.75
(m, 1H); 13C NMR (126 MHz, DMSO-d6) δ = 142.9, 140.0, 139.1,
138.0, 136.8, 135.9, 134.5, 134.2, 131.9, 130.1, 129.2, 109.3 1C
missing; LCMS (ESI) RT = 1.18 min, 100% pure, m/z 370, 372
[M + H]+ = 370, 372. Anal. Calcd for C13H10Cl2N5O2S =
369.9927. Found = 369.9931 [M + H]+.
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1
NMR (500 MHz, DMSO-d6 + NaHCO3) δ = 8.91 (d, J = 2.0 Hz,
1H), 7.98 (dd, J = 5.0, 2.0 Hz, 1H), 7.82 (dd, J = 8.0, 2.0 Hz, 1H),
7.61 (d, J = 2.0 Hz, 1H), 7.30 (d, J = 4.0 Hz, 1H), 6.94 (d, J =
4.0 Hz, 1H), 6.65 (dd, J = 8.0, 5.0 Hz, 1H), 6.39 (t, J = 2.0 Hz,
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[M + H]+.
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Acknowledgements
We thank Mike Woodrow and Steve Sollis for the synthesis of
7, Viqui Vinader for crystallising 8, Sean Lynn and Steve 18 Y. Nakagami, K. Kawashima, K. Yonekubo, M. Etori,
Richards for some NMR spectra, Bill J. Leavens for collecting
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whole blood assay, and Professor W. Clegg, Dr N. R. Brooks,
Dr L. Russo and Dr R. W. Harrington for the GSK funded
crystal structures of 7, 8, 14 and 16.
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