Identification of pyrazolopyrimidine arylsulfonamides as CC-chemokine receptor 4 (CCR4) antagonists

Article abstract of DOI:10.1016/j.bmc.2017.07.052

A novel 4-aminoindazole sulfonamide hit (13) was identified as a human CCR4 antagonists from testing a focussed library of compounds in the primary GTPγS assay. Replacing the indazole core with a pyrazolopyrimidine, and introduction of a methoxy group adjacent to the sulfonamide substituent, resulted in the identification of pyrazolopyrimidine 37a, which exhibited good binding affinity in the GTPγS assay (pIC₅₀ = 7.2), low lipophilicity (c log P = 2.2, chromlog D_7.4 = 2.4), high LE (0.41), high solubility (CLND solubility ≥581 μM), and an excellent PK profile in both the rat (F = 62%) and the dog (F = 100%). Further SAR investigation of the pyrazolopyrimidine suggested that substitution at N1 is tolerated, providing a suitable vector to modulate the properties, and increase the potency in a lead optimisation campaign.

Full text of DOI:10.1016/j.bmc.2017.07.052

Accepted Manuscript
Identification of pyrazolopyrimidine arylsulfonamides as CC-Chemokine Re-
ceptor 4 (CCR4) antagonists
Afjal H. Miah, Aurelie C. Champigny, Rebecca H. Graves, Simon T. Hodgson,
Jonathan M. Percy, Panayiotis A. Procopiou
PII:
S0968-0896(17)31171-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.052
BMC 13889
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
31 May 2017
20 July 2017
27 July 2017
Please cite this article as: Miah, A.H., Champigny, A.C., Graves, R.H., Hodgson, S.T., Percy, J.M., Procopiou, P.A.,
Identification of pyrazolopyrimidine arylsulfonamides as CC-Chemokine Receptor 4 (CCR4) antagonists,
Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.07.052
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Identification of pyrazolopyrimidine arylsulfonamides as CC-Chemokine Receptor 4
(
CCR4) antagonists.
a
Afjal H. Miah,* Aurelie C. Champigny, Rebecca H. Graves, Simon T. Hodgson, Jonathan
a
b
a
c
M. Percy, Panayiotis A. Procopiou.
a
Graphical abstract
1

Identification of pyrazolopyrimidine arylsulfonamides as CC-Chemokine Receptor 4
(
CCR4) antagonists.
a
Afjal H. Miah,* Aurelie C. Champigny, Rebecca H. Graves, Simon T. Hodgson, Jonathan
a
b
a
c
M. Percy, Panayiotis A. Procopiou.
a
a
b
Departments of Medicinal Chemistry, Drug Metabolism and Pharmacokinetics, Respiratory
TAU, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage,
Hertfordshire, SG1 2NY, United Kingdom,
c
WestCHEM Department of Pure and Applied Chemistry, University of Strathclyde, 295
Cathedral Street, Glasgow, G1 1XL, United Kingdom.
*
To whom correspondence should be addressed. Phone: (+44)1438 762037. E-mail:
afjal.h.miah@gsk.com
2

Abstract
A novel 4-aminoindazole sulfonamide hit (13) was identified as ahuman CCR4 antagonists
from testing a focussed library of compounds in the primary GTPγS assay. Replacing the
indazole core with a pyrazolopyrimidine, and introduction of a methoxy group adjacent to the
sulfonamide substituent, resulted in the identification of pyrazolopyrimidine 37a, which
exhibited good binding affinity in the GTPγS assay (pIC50 = 7.2), low lipophilicity (clogP =
2
.2, chromlogD7.4 = 2.4), high LE (0.41), high solubilty (CLND solubility ≥ 581 µM), and an
excellent PK profile in both the rat (F = 62%) and the dog (F = 100%). Further SAR
investigation of the pyrazolopyrimidine suggested that substitution at N1 is tolerated,
providing a suitable vector to modulate the properties, and increase the potency in a lead
optimisation campaign.
3

1
. Introduction
Chemokines are a group of small chemotactic cytokines (~ 8 – 16 Kda) that are involved in
directing leukocytes to the site of infection or inflammation and play an important role in host
defence. They exert their biological activity by binding with a subset of 7 transmembrane G-
protein-coupled chemokine receptors that cascade downstream intracellular signalling
through the activation of heterotrimeric G-proteins. To date, over 50 chemokines and 20
chemokine receptors have been identified in humans. In addition to the chemokines binding
to their own receptors with high affinity, most chemokines are promiscuous, binding and
activating a number of chemokine receptors, with each receptor often recognising more than
1
one chemokine. The chemokine ligands CCL17, also known as thymus activation-regulated
chemokine (TARC), and CCL22, also known as macrophage-derived chemokine (MDC)
solely bind to CC-chemokine receptor 4 (CCR4). CCR4 is predominantly expressed on the
membrane of T
H
2 cells that produce IL-4, IL-5 and IL-13, the cytokines responsible for the
recruitment of eosinophils, and production of high levels of serum IgE and activation of mast
2, 3
cells. There is clinical evidence suggesting that activated CCR4-positive cells and the
expression of CCR4 ligands (MDC and TARC) are upregulated in the airways after an
2
allergen challenge in mildly asthmatic patients. As the T
H
2 cells play an important role in the
allergic inflammatory response, it is suggested that inhibiting CCR4 with an antagonist will
provide an attractive new therapeutic intervention for allergic inflammatory diseases, such as
4
asthma and atopic dermatitis. CCR4 antagonists could also provide novel therapy for
5
6
allergic bronchopulmonary aspergillosis, inflammatory bowel disease and a variety of
7
8, 9
cancers. In addition, CCR4 is expressed on tumour cells from patients with adult T-cell
1
leukaemia, and a humanised monoclonal antibody, mogamulizumab, directed against CCR4
0
has been approved in Japan for demonstrating clinical anti-tumor activity in patients with
4

1
1, 12
relapsed or refractory adult T-cell leukaemia/lymphoma.
More recently, CCR4
1
3, 14
antagonists were used as molecular adjuvants in vaccines.
Over the past decade, numerous small-molecule CCR4 allosteric antagonists have also been
15-21
reported in the literature,
and these are divided between two different chemotypes (Fig.1).
The first chemotype includes basic small lipophilic antagonists such as compounds 1 and 2
1
8, 19
22, 23
from Bristol Myers Squibb,
compound 3 from Astellas,
compounds 4 and 5 from
1
6, 24
25
and compound 6 from GlaxoSmithKline (GSK). The second chemotype
Daiichi Sankyo
2
6
includes aryl sulfonamide compounds such as compound 7 from AstraZeneca, compound 8
2
from Ono, and compounds 9, 10 and 11 from GSK. Recent advances in pharmacology,
7
28
29
30
3
1
based on competitive ligand binding studies from two recent papers published by our group
3
2
and AstraZeneca, has confirmed that CCR4 antagonism could be achieved through three
distinct binding sites. The endogenous ligands, MDC and TARC, bind to an extracellular
region called the orthosteric site. Basic small molecule antagonists 1, 2 and 3 were shown to
bind to an extracellular allosteric region, whereas the aryl sulfonamide compounds such as
pyrazine sulfonamide 7, 8 and indazole 9 bind to an intracellular allosteric region of the 7-
transmembrane CCR4 receptor. It is noteworthy that the compounds that bind to the
intracellular region need to have properties such as good membrane permeability to access
this site. At GSK, the allosteric extracellular site is termed Site 1, and the intracellular site is
termed Site 2.
Pharmaceutical companies have invested significant effort into progressing small molecule
CCR4 antagonists towards human studies, but so far only one candidate molecule, indazole
sulfonamide 9 from GSK, was reported to have progressed to phase I clinical trials for
3
asthma. Although indazole 9 demonstrated good pharmacokinetic data in preclinical animal
3
2
studies (bioavailability in rats and beagle dogs 85% and 97% respectively), it suffered from
8
low solubility, poor bioavailability in humans (16%) and weak ex vivo potency preventing it
5

3
3
from being progressed further, despite the fact that at the highest dose of 1500 mg/kg,
indazole 9 was found to be safe and demonstrated 74% CCR4 receptor occupancy. The
approval of mogamulizumab and the lack of small molecule candidates clearly highlight the
needs for identifying efficacious CCR4 antagonists, with improved potency, solubility and
good pharmacokinetic properties that are capable of engaging CCR4 at lower doses.
Figure 1. Examples of recently published small molecule CCR4 antagonists.
2
9
Our group has recently reported the identification of bicyclic heteroarene sulfonamide 10
30
and azabenzimidazolone sulfonamide 11 as novel lead series of Site 2 antagonists. In this
6

article, we disclose the identification and evaluation of the pyrazolopyrimidines, a lead series
with good oral bioavailability.
2
. Chemistry
We have already reported the design of a focused library of 2,3-dichlorobenzene
sulfonamides, where we synthesised 100 sulfonamides, and identified the
3
0
azabenzimidazolone series (as exemplified by compound 11 in Fig. 1). In this focused
library, hits were required to have both a pIC50 greater than that of the starting point of the
indazole series, 12, in the GTPγS assay, and improved physicochemical properties (Fig. 2). In
the same set, a novel 4-aminoindazole sulfonamide hit (compound 13) was identified that
showed good physicochemical properties and reasonable potency in the primary GTPγS
assay (Fig. 2).
Figure 2. Initial indazole hits
It was known from previous in house work on the candidate indazole series that introduction
of a hydrogen bond acceptor (HBA), such as a methoxy group, adjacent to the sulfonamide
substituent on the core, enhances the binding potency for the receptor. As we have already
2
reported with the indazole series and bicyclic heteroarene series, a suitably orientated
8
29
7

HBA to the sulfonamide NH is thought to bring about intramolecular hydrogen bonding,
locking the conformation of the molecule in a preferred active form. The sulfonamide 13 has
the 3 and 5-positions available for substitution and the focus of the chemistry was to
introduce methoxy groups on both of these positions to achieve the required hydrogen
bonding with the NH of the sulfonamide substituent on the 4-position. The chemistry used to
introduce a methoxy group at the 5-position is outlined in Scheme 1.
Scheme 1. Preparation of indazole 22. Reagents & Conditions: (a) 70% fuming HNO
3
, conc.
, 18, 110
2 3 2
CO , DMF, 98%; (e) SnCl , conc. HCl, MeOH, 52%; (f)
o
H
2
SO
4
, 0 C, 1 h, 90%; (b) NaH, MeI, DMF, 16 23% and 17 46%; (c) KOH, Pd
2
dba
3
o
C, 0.5 h, µwave, 80%; (d) MeI, K
2
,3-dichlorobenzenesulfonyl chloride, pyridine, 43%.
Nitration of commercially available 5-bromoindazole 14 at 0 °C occurred exclusively at the
1
-position affording 4-nitroindazole 15 in excellent yield. The H NMR spectrum of 15
4
contained a pair of doublets corresponding to the H-6 (7.77 ppm) and H-7 (7.87 ppm) protons
3
with a J coupling constant of 8.8 Hz, confirming that the nitration occurred at the 4-position.
8

The reaction of 15 with methyl iodide, using sodium hydride as base, gave a mixture of
regioisomers, 16 and 17, with the desired 1-methylindazole 17 predominating (ratio 1:2). The
1
regiochemistry of the two isomers was determined using a combination of C HMBC and
3
1
ROESY experiments (Fig. 3). In the C HMBC experiment for regioisomer 16, a correlation
3
was observed between the N-methyl protons (4.24 ppm) and the carbon at the 3-position
(
125.6 ppm), and for regioisomer 17 a cross peak was observed between the N-methyl
protons (4.14 ppm) and the tertiary carbon at the 8-position (141.4 ppm). In the ROESY
experiment an NOE correlation was observed between the N-methyl protons (4.24 ppm) and
the aromatic hydrogen at the 3-position (8.70 ppm) for regioisomer 16, whereas regioisomer
1
7 showed an NOE correlation between the N-methyl protons (3.93 ppm) to the aromatic
hydrogen at the 7-position (8.02 ppm).
13
Figure 3. C HMBC (red) and ROESY (blue) confirmation of the regioisomers 16 and 17.
3
Palladium-catalysed hydroxylation of 17, using Buchwald’s method (Scheme 1), employing
4
bulky monodentate phosphine ligand 18 with KOH, and Pd dba as the pre-catalyst, was
2
3
found to be very effective, affording the hydroxylated product 19 in 80% yield. The 5-
hydroxyindazole 19 was alkylated with methyl iodide under basic conditions, and then the
resulting nitroarene 20 was reduced with tin(II) chloride to give 4-amino-5-methoxyindazole
2
1. Finally, reaction with 2,3-dichlorobenzenesulfonyl chloride in pyridine afforded the
desired 5-methoxyindazole sulfonamide 22 in 43% yield.
9

Scheme 2 outlines the chemistry for introducing the 3-methoxy group in 13. 3-
Bromoindazole sulfonamide 24 was prepared from reaction of commercially available 4-
amino-3-bromoindazole 23 with 2,3-dichlorobenzenesulfonyl chloride in pyridine in 67%
yield. Reaction between 3-bromoindazole sulfonamide 24 and sodium methoxide in
methanol, using copper powder as a catalyst, afforded 3-methoxyindazole sulfonamide 25 in
3
9% yield.
Scheme 2. Preparation of 4-sulfonamidoindazole 25. Reagents & Conditions: (a) 2,3-
t
dichlorobenzenesulfonyl chloride, pyridine, rt, 18 h, 67%; (b) NaO Bu, MeOH, Cu powder,
o
sealed tube, 150 C, 18 h, 39%.
In order to improve the physicochemical properties for the series (higher solubility, lower
lipophilicity), it was decided to introduce nitrogen atoms into the phenyl ring of scafold 13.
2
Based on precedent from our work on the bicyclic heteroarene and azabenzimidazolone
9
3
0
sulfonamide series (10 and 11 ), replacing the carbon atom ortho- or para- to the
sulfonamide substituent with nitrogen results in improvement of binding affinity. In addition,
the generation of 4-aminoindazole as a possible metabolite following potential cleavage of
the sulfonamide moiety was considered a liability due to its electron-richness and potential
3
5
genotoxicity issues. Two analogues of 13, a pyrazolopyridine and a pyrazolopyrimidine
were prepared. The synthesis of the pyrazolopyridine analogue of 13, with replacement of
C7 with nitrogen is outlined in Scheme 3.
1
0

Scheme 3. Preparation of pyrazolopyridine analogue 31. Reagents & Conditions: (a) i) 110
o
o
, 110 C, 3.5 h, 54%; (b) 2,3-dichlorobenzenesulfonamide, Pd(OAc)
C, 1.5 h, ii) POCl
3
2
,
o
Xantphos, Cs
2
CO
3
, Dioxane, 150 C, 30 min, µwave, 63%; (c) NaOH, EtOH, 18 h, 82%; (d)
o
DMF, 190 C, µwave, 28%.
Condensation of aminopyrazole 26 with diethylethoxymethylenemalonate 27 using a
3
6
published procedure, followed by treatment with phosphoryl chloride gave 4-
chloropyrazolopyridine ester 28 in good yield. Buchwald coupling reaction of 28 with 2,3-
dichlorobenzenesulfonamide, using Xantphos as a ligand gave sulfonamide 29 in 63%
isolated yield. Saponification of the ester group of sulfonamide 29, followed by
o
decarboxylation of the resulting carboxylic acid 30 upon microwave irradiation at 190 C in
DMF, provided the desired target sulfonamide 31.
The synthesis of the analogue of 13 in which nitrogen was introduced to both the ortho- and
para positions to the sulfonamide substituent is outlined in scheme 4. The pyrazolopyrimidin-
1
1

4
-amine 32 was commercially available and reaction with 2,3-dichlorobenzenesulfonyl
chloride, in the presence of sodium hydride, afforded the sulfonamide product 33 in 21%
yield (Scheme 4).
Scheme 4. Synthesis of pyrazolopyrimidine 33. Reagents & Conditions: (a) 2,3-
dichlorobenzenesulfonyl chloride, NaH, DMF, 21%.
Variation of the 3-position of the pyrazolopyrimidine 33 was investigated next. This included
replacing hydrogen with hydroxy-, methoxy-, ethoxy- and 2-methoxyethoxy- groups using
the synthetic routes outlined in Scheme 5.
Starting materials 4-amino-pyrazolopyrimidine 36a-c, were prepared using conditions similar
3
7-39
to those described in the literature.
Reaction of tetracyanoethylene (34) with either MeOH
or 2-methoxyethanol in the presence of urea, and subsequent reactions with either
methylhydrazine or tert-butylhydrazine, followed by condensation with formamide afforded
3
6a-c in moderate to good yields. Reaction with 2,3-dichlorobenzenesulfonyl chloride in the
presence of sodium hydride afforded the desired methoxy- (37a), 2-methoxyethoxy- (37b)
and tert-butyl protected methoxy- (37c) pyrazolopyrimidine analogues. The 3-hydroxy
analogue 39 was obtained from 37c by removal of the tert-butyl group using concentrated
sulfuric acid, followed by cleavage of the 3-methoxy group with TMSI, generated in situ
from TMSCl in the presence of sodium iodide in refluxing acetonitrile.
1
2

Scheme 5. C3 variation of the pyrazolopyrimidine series. Reagents & Conditions: (a) i)
o
MeOH or 2-methoxyethanol, urea, 50 - 80 C, 0.5 h; ii) methylhydrazine or tert-
butylhydrazine hydrochloride, MeOH, reflux, 2.5 h, 35a (17%), 35b (17%), 35c (19%) (yield
o
over two steps); (b) formamide, 180-185 C, 2-4 h, 36a (39%), 36b (91%), 36c (46%) (c) 2,3-
o
dichlorobenzenesulfonyl chloride, NaH or LIHMDS, DMF, 0 C - rt, 37a (39%), 37b (10%),
3
4
7c (41%); (d) conc. H
2 4 3
SO , rt, 2 h, (80%); (e) TMSCl, NaI, CH CN, reflux, 18 h, 39 (90%),
o
0 (72%); (f) K CO , DMSO, 130 C, 4 h, 41 (50%), 42 (6%); (g) i) NaH, 2,3-
2
3
1
3

o
dichlorobenzenesulfonyl chloride, DMF, rt, 2 h; ii) conc. H
2 4
SO , 0 C, 1 h, 43 (34%), 44
(
13%) (yield over two steps).
For the the preparation of the ethoxy compound 43, an alternative route was developed in
which the variation of the C3 substituent was carried out at a later stage (Scheme 5).
Pyrazolopyrimidine amine 36c was reacted with TMSCl, in the presence of sodium iodide, to
give the hydroxyl pyrazolopyrimidine 40 in 72% yield. Alkylation of 40 with ethyl iodide
afforded 41 as the major, and 42 as the minor product. The structures of the regioisomers
1
3
15
were confirmed by C and N HMBC experiments. The key correlation in an HMBC
experiment from the methylene protons (4.33 ppm) of the ethoxy group to C3 (152.2 ppm) on
the pyrazolopyrimidine core confirmed the structure of regioisomer 41 (Fig. 4). For
1
5
3
regioisomer 42, N HMBC experiment showed a key JN-H correlation from N1 to both the
tert-butyl protons and the methylene proton of the ethyl group, and N2 showed correlation to
all the protons of the ethyl group (Fig. 4). Sulfonylation reactions of both 41 and 42 with 2,3-
dichlorobenzenesulfonyl chloride, followed by hydrolysis using concentrated sulfuric acid
afforded the desired target sulfonamides 43 and 44 respectively.
Figure 4. HMBC confirmation of the regioisomers 41 and 42.
Substitution at N1 and C6 of the promising lead of 36c was then explored, starting first with
N1. The pyrazolopyrimidine series already contains three aromatic rings, and to maintain the
desired physicochemical properties by keeping the clogP and MW low, it was decided that
incorporation of a third substituent on the core template should be limited to small non-
aromatic groups in order to maintain or increase solubility, permeability and absorption.
1
4

The most direct approach to investigate the SAR at the N1-position involved reaction of
pyrazolopyrimidine 38 with 2 equivalents of LiHMDS, followed by addition of one
equivalent of a range of alkylating agents (Scheme 6). The first equivalent of base
deprotonates the more acidic sulfonamide proton (measured pK
a
= 5.71) and then the second
of the
equivalent deprotonates the N1-proton. The more basic anion (the measured pK
a
conjugate acid is 11.95) at the N1-position reacted first with the alkylating agents, affording
the desired N1-alkylated products (45a – 45g) in moderate to good yield. A small amount of
di-alkylation products (both N1 and sulfonamide NH alkylated) were also observed by
LCMS, but these were not isolated. The carboxylic acid analogue 45h was obtained by
saponification of ester 45g (Scheme 6).
Scheme 6. Preparation of analogues of 45 substituted at N1. Reagents & Conditions: (a)
o
o
LiHMDS (2.0 equiv.), THF, RBr or RI (1.0 equiv.) -78 C – 20 C, 45a 37%, 45b 17%, 45c
5
3%, 45d 70%, 45e 56%, 45f 19%; (b) NaOH, rt, 54% yield over two steps.
Finally, the C6-position of the pyrazolopyrimidine template was investigated. The approach
taken involved preparation of a key intermediate, 48 following a modified version of the
4
0
published method described by Cheng and Robins (Scheme 7). The pyrazole nitrile, 35a
was hydrolysed using concentrated sulfuric acid to give the pyrazole carboxamide, 46. This
was then reacted with CDI to give the pyrazolopyrimidone, 47, which was reacted with 5 mol
1
5

equivalents of phosphorus pentachloride in phosphoryl chloride to afford the
dichloropyrimidine 48.
N
The selective displacement of the 4-chloro group of dichloropyrazolopyrimidine by S Ar
4
1-44
reaction is well documented.
Thus the chlorine atom on the 4-position was displaced with
o
,3-dichlorobenzenesulfonamide in THF, in the presence of NaH at 60 C to afford the
2
substituted intermediate, 49 in 57% yield.
Scheme 7. Synthetic route to prepare pyrazolopyrimidine analogues with changes at the 6-
o
, rt, 18 h, 94%; (b) CDI, DMF, 85 C, 1 h, 68%;
position. Reagents & Conditions: (a) H
2
SO
4
1
6

o
(
c) PCl
5 3
(5 equiv.), POCl (22 equiv.), 100 C, 18 h, 44%; (d) 2,3-
o
dichlorobenzenesulfonamide, NaH, THF, 0 C - 60 C, 57%; (e) trimethylboroxine,
o
o
o
PdCl
2
(dppf), Cs
2
CO
3
, 1,4-dioxane, 100 C, µwave, 2 h, 22%; (f) NaOMe, 130 C, µwave, 1 h,
o
5
2%; (g) amine, neat or in IPA, 100 - 130 C, µwave, 1 - 3 h, 26-56%.
Finally, the SAR at the 6-position of 49 was investigated following displacement of chloride
with sodium methoxide, or a variety of amine nucleophiles, to give the corresponding
pyrazolopyrimidine analogues 51, 52a – h. As expected, the displacement of chloride from
o
C6 required higher temperatures, typically about 130 C. The methoxy group was introduced
by heating 49 with sodium methoxide in methanol to afford 51 in 52% yield. Reactions of 49
with the corresponding amine nucleophiles were carried out either in neat amine or excess
amine in isopropanol as solvent. The reactions were heated using microwave irradiation at
o
temperatures between 100 - 130 C for 1 - 3 h to give the corresponding amino analogues.
The methyl group at C6 was introduced by a palladium-catalysed reaction of chloro
pyrazolopyrimidine 49 with trimethylboroxine to give the methyl analogue 50 in 22% yield.
2
6, 28, 32
SAR of previous Site 2 series,
indicated that aryl sulfonamides, such as 2,3-
dichlorophenyl- or 5-chlorothienyl- were optimal for activity, whereas alkyl, cycloalkyl,
naphthyl or other heteroaryl sulfonamides were inferior. The 5-chlorothienyl group was found
to have better physicochemical properties in comparison to the 2,3-dichlorophenyl- group, as
it had lower molecular weight, and was less lipophilic. In order to improve the properties of
the series, the 5-chlorothienyl analogue of 37a (pyrazolopyrimidine 53) was obtained by
reacting 4-amino-3-methoxypyrazolopyrimidine 36a with 5-chloro-thiophenesulfonyl
chloride in the presence of sodium hydride (Scheme 8).
1
7

Scheme 8. Synthetic step to prepare pyrazolopyrimidine analogue 53. Reagents &
Conditions: (a) 5-chlorothiophene-2-sulfonyl chloride, NaH, DMF, rt, 47%.
3
. Results and Discussion
The analogues prepared above were screened for human CCR4 antagonist activity in vitro in
3
5
28, 45
a [ S]-GTPγS radioligand competition functional assay.
The affinity was expressed as
the negative logarithm of the concentration of the test molecule that produced 50% inhibition
35
of the target protein (pIC50). For all test compounds, [ S]-GTPγS activity, ligand efficiency
LE), calculated partition coefficient (clogP - ACD version 12), chromatographic logD at pH
.4 (chromlogD7.4) and ChemiLuminescent Nitrogen Detection (CLND) kinetic solubility are
(
7
presented in Table 1. LE estimates the efficiency of binding interaction with respect to the
size of the ligand, it was obtained from the equation LE = – 1.36 × pIC50 / HAC, where HAC
4
6
is the heavy atom count (number of non-hydrogen atoms present in the molecule). Based on
a typical drug profile, an LE of greater than 0.3 is accepted as a good value representing an
effective binding ligand. The high throughput CLND solubility assay involves the addition of
aqueous buffer to a test compound in DMSO solution over a period of time until the
4
7
compound precipitates.
Compounds 2 and 8 were used as standards in the GTPγS assay and included in Table 1. The
data generated for 12 and 54 (see Fig. 5), the initial ‘hit’ and ‘lead’ that provided our indazole
candidate 9, are also included in Table 1 for comparison.
1
8

Figure 5. Structure of the initial hit, 12 and lead, 54 that led to the indazole candidate 9.
The new 4-aminoindazole hit 13 had a pIC50 = 5.5 in the GTPγS assay with good LE (0.34),
and a reasonable physicochemical profile (chromlogD7.4 = 3.2 & CLND solubility = 170
µM). Introduction of methoxy group at C3 on the indazole ring (25) resulted in half a log unit
improvement in affinity (pIC50 = 6.1), but no change in LE. In contrast introduction of
methoxy group at the 5-position (22) lowered affinity (pIC50 = 5.0). Although, indazole 25
was more potent than 13, it was also more lipophilic (chromlogD7.4 = 6.5) and this was
reflected in its lower solubility (80 µM). As discussed before, the 4-aminoindazole is not very
3
5
attractive as a drug candidate because of potential genotoxicity issues. We sought to resolve
both these problems by introducing nitrogen atoms into the phenyl ring of the indazole
moiety. The 7-aza analogue of 13 (pyrazolopyridine 31) was found to reduce potency and LE
to pIC50: 4.9 and 0.3 respectively. However, the pyrimidine analogue of 13
(
pyrazolopyrimidine 33) resulted in a 0.5 log unit improvement in potency (pIC50 = 5.9), with
compound 33 demonstrating much improved solubility (476 µM) and much more acceptable
level of lipophilicity (chromlogD7.4 = 1.7). Furthermore, introduction of a methoxy group
adjacent to the sulfonamide substituent of compound 33 provided pyrazolopyrimidine 37a,
which had a one order of magnitude higher affinity in the GTPγS assay (pIC50 = 7.2)
compared to 54 (pIC50 = 6.2).
1
9

Investigation of substitution at C3 of the pyrazolopyrimidine template with H-bond acceptor
groups, such as methoxy- (37a), hydroxy- (39), ethoxy- (43) or 2-methoxyethoxy- (37b)
revealed that the methoxy- group was the optimal substituent and for this reason this group
was retained in all further modifications.
The pyrazolopyrimidine 44 with an ethyl group on N2 was found to have much reduced
binding affinity (pIC50 < 4.5), so no further N2 substituted analogues were synthesised.
The pyrazolopyrimidine series already contained three aromatic rings, and in order to
maintain the clogP and MW at a low level and the solubility high, it was necessary not to add
another aromatic ring to improve affinity. Therefore, further variation at N1 was limited to
small aliphatic substituents (38, 45a – h). The data showed that either small groups, such as a
methyl (37a), or no substitution at all (38) were preferred, and that all other modifications at
the N1-position reduced potency. However, it does appear that polar groups such as 2-
methoxyethyl- (45d, pIC50 = 6.5), 3-oxetanyl- (45e, pIC50 = 6.6), and a carboxylic acid group
(
45h, pIC50 = 6.7) were tolerated and these groups could be used to improve further the
physicochemical properties of the molecule, such as solubility and permeability or oral
absorption.
Intoduction of a methyl group at the 6-position of 37a (50), a methoxy- (51) or amino group
(
52a) resulted in reduced affinity by 0.5-1 log unit. Introduction of an NHMe group (52b)
was tolerated, with pyrazolopyrimidine 52b demonstrating similar potency (pIC50 = 7.1), but
lower LE (0.37), higher clogP (3.1) and reduced solubility (375 µM) in comparison to 37a
(
pIC50 = 7.2, LE = 0.41, clogP = 2.2 and solubility ≥ 581 µM). Replacing the NHMe (52b)
with NHEt (52c) resulted in a loss in potency of 0.5 log units, and all other modifications
52d-h) were found to be detrimental for activity.
(
2
0

Finally, exchanging the 2,3-dichlorophenyl group of compound 37a with 5-chlorothienyl
provided pyrazolopyrimidine 53; as expected, this exchange not only maintained the potency,
but also increased the ligand efficiency (LE = 0.44), and reduced the molecular weight and
clogP (1.6) but preserved the solubility (in comparison to 37a).
Pyrazolopyrimidine 37a possessed high in vitro passive membrane permeability (Pexact =
4
22 nm/s) across Madin Darby canine kidney – multidrug resistance 1 (MDCKII-MDR1)
cells in the presence of a potent P-glycoprotein (PGP) inhibitor. The compound also had low
microsomal clearance in rat, dog and human (0.58, 0.65 and <0.53 mL/min/g respectively).
Following the encouraging in vitro data, pyrazolopyrimidine 37a was submitted for rat and
dog pharmacokinetic studies in the male Wistar Han rat and Beagle dog, and the results are
summarised in Table 2. The pharmacokinetic data for indazole 54 in male Sprague Dawley
(
crl:CD(SD)) rat is also included in Table 2 for comparison. Following an IV dose (1 mg/kg),
the blood clearance rate of pyrazolopyrimidine 37a was very low in the rat (2 mL/min/kg),
with a half life of 1.9 h, and a low volume of distribution (0.3 L/kg). After an oral dose (1
mg/kg), the oral bioavailability was moderate to high at 62%. In contrast, indazole 54 showed
a higher plasma clearance (17 mL/min/kg), with 24% bioavailability in the rat at equivalent
doses. The blood clearance of 37a in the Beagle dog was also low (4 mL/min/kg) following
an IV dose (0.5 mg/kg) , and following an oral dose (1 mg/kg), the compound demonstrated
complete oral bioavailability in the dog. The phamacokinetic profile of 37a is very
encouraging, and as it has higher affinity than 54, (the starting lead compound that provided
the indazole candidate 9), is much less lipophilic, and has much higher solubilty, presents
itself as a suitable candidate for further lead optimisation.
Compounds 37a and 53 were screened in the secondary human actin polymerisation whole
2
8, 45
blood assay
2
and both compounds were found to be inactive (pA < 5.2). They have high
2
1

plasma protein binding (Human Serum Albumin (HSA) binding 98.5 and 97.7% respectively)
suggesting that there is a low free fraction available to elicit an effect in the whole blood
assay. It should be noted that the starting lead compound for 9, indazole 54 (HSA binding:
9
7.7%) was also inactive. In a lead optimisation programme, reducing the plasma protein
binding of the pyrazolopyrimidine template could therefore be an initial strategy to obtain
whole blood activity.
4
. Conclusion
The aim of this project was to identify a novel template with improved potency,
physicochemical and pharmacokinetic properties compared to the starting point of the
indazole candidate 9. From testing a focussed library of compounds in the primary GTPγS
assay, a novel 4-aminoindazole sulfonamide hit (13) was identified that had similar potency
and physicochemical properties to that of the indazole lead 54. Replacing the indazole core of
1
3 with a pyrazolopyrimidine core, and introducing a methoxy group adjacent to the
sulfonamide substituent, resulted in the identification of 37a which had very good binding
affinity in the GTPγS assay, lower lipophilicity, better solubilty, and acceptable PK profile in
both the rat and the dog. SAR investigation of the pyrazolopyrimidine suggested that
substitution with small alkyl substituents at N1 is tolerated, providing a suitable vector to
modulate the properties, and increase the potency in a lead optimisation campaign. Based on
SAR from our azabenzimidazolone sulfonamide 11 in Fig. 1), one obvious change which
might improve the properties and potency is to replace the nitrogen at the 7-position of
pyrazolopyrimidine 37a with carbon to give a 5-aza pyrazolopyridine analogue. This
template will also provide an additional position to exploit substitution. Further investigation
of Site 2 CCR4 templates was halted because of recent findings that binding of Site 1 only
antagonists, such as the basic lipophilic amine 2, 3 and 6, were capable of invoking
2
2

internalisation of the CCR4 receptor from the cell surface into the cell cavity, whereas Site 2
4
antagonists showed no effect. In addition, Site 1 antagonists have been reported to show
5
efficacy in ovalbumin-induced airway inflammation and murine allergic inflammation
1
6, 18, 19, 48
models.
In contrast, no Site 2 antagonists have so far been reported to show any in
vivo activity. This has led us to speculate that in vivo activity is a consequence of receptor
internalisation which is observed with Site 1 antagonists. The possibility that internalisation
of the CCR4 receptor may contribute to a biological response was also highlighted by the
Kirin group with their CCR4 inhibitor, K777, which has a chemotype for a Site 1
4
8
antagonist.
Although, it is not yet clear why binding to Site 1 causes internalisation of the receptor, it is
widely appreciated that reducing the number of receptors expressed on the cell membrane will
result in reduction of accessible binding sites for the natural agonist to bind. Ultimately, this will
result in loss of functional responsiveness of the receptor towards chemokines.
5
5
. Experimental
.1 Synthetic Chemistry
Organic solutions were dried over anhydrous Na
TLC was performed on Merck 0.25 mm Kieselgel 60 F254 plates. Products were visualised
under UV light and/or by staining with aqueous KMnO solution.
2 4 4
SO , MgSO or using a hydrophobic frit.
4
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 MeCN (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 100 – 3% B, at a flow rate of 1
–
mLmin at 40 °C. The UV detection was an averaged signal from wavelength of 210 nm to
1
2
3

3
50 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 an
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 0.0 – 1.5 min 1 –
–
7% B, 1.5 – 1.9 min 97% B, 1.9 – 2.0 min 100% B at a flow rate of 1 mL min at 40 °C; or
1
9
System C an Acquity UPLC BEH C18 column (50 mm × 2.1 mm ID, 1.7 µm packing
diameter) eluting with 0.1% trifluoroacetic acid in water (solvent A), and 0.1% trifluoroacetic
acid in MeCN (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 100 – 3 %B at a flow rate of 1 mL min at 40 °C; or
1
–
System D an Agilent Sunfire C18 column (30 mm × 4.6 mm ID, 3.5 µm packing diameter)
eluting with 0.1% trifluoroacetic acid in water (solvent A), and 0.1% trifluoroacetic acid in
MeCN (solvent B) using the following elution gradient 0.0 – 4.2 min 3 – 100% B, 4.2 – 4.8
–
min 100% B, 4.8 – 5.0 min 100 – 3 % B at a flow rate of 3 mL min at 30 °C.
1
Column chromatography was performed on a Flashmaster II system. The Flashmaster II is an
automated multi-user flash chromatography system, available from Argonaut Technologies
Ltd, which utilizes disposable, normal phase, SPE cartridges (2 g to 100 g).
Purifications by mass-directed auto-preparative HPLC (MDAP) was conducted on either
System A a Sunfire C18 column (150 mm × 30 mm i.d. 5 µm packing diameter) at ambient
temperature eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in
MeCN (solvent B), using an appropriate elution gradient over 15 or 25 min at a flow rate of
–
0 mL min and detecting at 210 - 350 nm at room temperature. Mass spectra were recorded
1
4
on Micromass ZMD mass spectrometer using electro spray positive and negative mode,
alternate scans; or System B an XBridge C18 column (150 mm × 30 mm i.d. 5 µm packing
2
4

diameter) at ambient temperature eluting with 10 mM ammonium bicarbonate in water
adjusted to pH10 with ammonia solution (solvent A), and MeCN (solvent B), using an
–
1
appropriate elution gradient over 15 or 25 min at a flow rate of 40 mL min ; or System C a
Sunfire C18 column (150 mm × 30 mm i.d. 5 µm packing diameter) at ambient temperature
eluting with 0.1% trifluoroacetic acid in water (solvent A), and 0.1% trifluoroacetic acid in
MeCN (solvent B), using an appropriate elution gradient over 15 or 25 min at a flow rate of
–
0 mL min . The software used was MassLynx 3.5 with OpenLynx and FractionLynx
1
4
options.
1
Proton magnetic resonance spectra ( H NMR) were recorded at 400, 500 or 600 MHz, unless
otherwise stated. The chemical shifts (δ) are expressed in ppm relative to tetramethylsilane.
1
3
Carbon magnetic resonance spectra ( C NMR) were recorded at 100, 126 or 150 MHz. The
purity of all compounds screened in the biological assays was examined by LCMS analysis
and was found to be ≥ 95%, unless otherwise specified.
5
.1.1. 5-Bromo-4-nitro-1H-indazole (15)
A solution of 5-bromo-1H-indazole (1 g, 5.1 mmol) in concentrated sulfuric acid (20 mL of
8 wt% aqueous solution, 380 mmol) was cooled to 0 ºC. Fuming nitric acid (1.0 mL of a 70
wt% aqueous solution, 22.6 mmol) was added dropwise and the reaction was maintained for
h at 0 °C. The reaction mixture was poured onto ice water (100 mL) and the precipitate was
9
1
collected by filtration, washed with water (30 mL) and dried at 50 ºC under reduced pressure
1
to afford the title compound (1.1 g, 90%) as a yellow solid; H NMR (600 MHz, DMSO-d
6
,):
4
δ = 8.28 (d, J = 0.7 Hz, 1H), 7.87 (dd, J = 8.8, J = 0.9 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H); the
1
3
exchangeable NH proton was not observed; C NMR (151 MHz, DMSO-d
6
,): δ = 140.5,
+
40.3, 131.2, 131.2, 117.4, 117.0, 107.1; LCMS (UV, ES) RT = 0.88 min, [M+H] = 242,
1
2
5

+
2
44, 100% purity; LCMS (Method B, UV, ES) RT = 0.93 min, [M+H] = 242, 244, 100%
purity.
.1.2. 5-Bromo-2-methyl-4-nitro-2H-indazole (16) & 5-bromo-1-methyl-4-nitro-1H-
5
indazole (17)
Sodium hydride (43 mg of a 60% w/w dispersion in mineral oil, 1.07 mmol) was added to a
solution of 15 (200 mg, 0.83 mmol) in anhydrous DMF (2 mL). The mixture was stirred for
1
0 minutes, then methyl iodide (0.067 mL, 1.07 mmol) was added and the reaction mixture
was stirred at room temperature for 18 h. Several drops of HCl (2 M aqueous solution) were
added and the reaction mixture was concentrated under reduced pressure. The residue was
taken up in DMSO (2 mL) and purified by MDAP (Method A). The appropriate fractions
were combined and dried under reduced pressure to afford the two regioisomers as yellow
1
solids: 16 (49 mg, 23%); H NMR (400 MHz, DMSO-d
6
) δ = 8.70 (s, 1H), 7.95 (dd, J = 8.0,
) δ =
48.0, 140.4, 131.1, 125.6, 124.7, 116.7, 110.5, 41.1; LCMS (UV, ES) RT = 0.93 min,
1
3
1
1
.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 4.24 (s, 3H); C NMR (101 MHz, DMSO-d
6
+
M+H] = 256, 258 100% purity, and 17 (98 mg, 46%); H NMR (400 MHz, DMSO-d
1
[
6
) δ =
8
.24 (s, 1H), 8.02 (dd, J = 8.0, 1.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 4.14 (s, 3H); LCMS
+
(
Method A, UV, ES) RT = 0.98 min, [M+H] = 256, 258, 100% purity.
.1.3. 1-Methyl-4-nitro-1H-indazol-5-ol (19)
A mixture of 17 (30 mg, 0.12 mmol), 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl
2 (4 mg, 9.4 µmol), Pd (dba) (2 mg, 2.2 µmol), potassium hydroxide (20 mg, 0.35 mmol)
5
2
2
3
in 1,4-dioxane (0.25 mL) and water (0.25 mL) was heated in the microwave at 110 ºC for 30
min. The reaction mixture was concentrated under reduced pressure, HCl (10 mL of a 1 M
aqueous solution) was added and the product was extracted with ethyl acetate (2 x 15 mL).
The combined organic extracts were dried using a hydrophobic frit and concentrated under
reduced pressure. The crude sample was taken up in DMSO (2 mL) and purified by MDAP
2
6

(
Method A). The solvent was dried under reduced pressure to afford the title compound (18
1
mg, 80%) as a yellow solid; H NMR (400 MHz, DMSO-d
6
) δ = 11.26 (br.s, 1H), 8.25 (s,
1
H), 8.07 (d, J = 9.0 Hz, 1H), 7.25 (d, J = 9.0 Hz, 1H), 4.10 (s, 3H); LCMS (Method A, UV,
+
ES) RT = 0.78 min, [M+H] = 194, 98% purity.
5
.1.4. 5-Methoxy-1-methyl-4-nitro-1H-indazole (20)
Potassium carbonate (28 mg, 0.20 mmol) was added to a solution of 19 (30 mg, 0.16 mmol)
in anhydrous DMF (0.4 mL) and the mixture was stirred for 10 minutes. Methyl iodide (0.013
mL, 0.20 mmol) was then added and the mixture was stirred at ambient temperature for 18 h.
The reaction mixture was diluted with ethyl acetate (30 mL) and water (20 mL). The organic
phase was separated, and the aqueous layer was extracted further with ethyl acetate (2 x 30
mL). The organic extracts were combined, washed with brine (20 mL), dried using a
hydrophobic frit and evaporated under reduced pressure to afford the title compound (35 mg,
1
8%) as a pale green solid; H NMR (400 MHz, DMSO-d
9
6
) δ = 8.18 (s, 1H), 8.11 (d, J = 9.0
Hz, 1H), 7.58 (d, J = 9.0 Hz, 1H), 4.11 (s, 3H), 4.02 (s, 3H); LCMS (Method A, UV, ES) RT
+
0.75 min, [M+H] = 208, 90% purity.
=
5
.1.5. 5-Methoxy-1-methyl-1H-indazol-4-amine (21)
Tin(II) chloride (180 mg, 0.97 mmol) was added to a solution of 20 (40 mg, 0.19 mmol) in
MeOH (1 mL). Concentrated HCl (0.08 mL of a 37 wt% aqueous solution, 0.97 mmol) was
added and the reaction mixture was allowed to stir at ambient temperature for 4 h. The
reaction mixture was neutralised by the addition of solid sodium carbonate. The solution was
then diluted with ethyl acetate (20 mL), washed with sodium hydroxide (15 mL of a 1 M
aqueous solution), followed by sodium bicarbonate (20 mL of a saturated aqueous solution),
dried using a hydrophobic frit, and concentrated under reduced pressure to afford the title
1
compound (23 mg, 52%) as a brown gum. H NMR (400 MHz, MeOD-d
4
) δ = 7.98 (s, 1H),
7
.13 (d, J = 8.8 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H), 3.93 (s, 3H), 3.84 (s, 3H); the
2
7

exchangeable NH
2
protons were not observed; LCMS (Method A, UV, ES) RT = 0.57 min,
+
M+H] = 178, 80% purity.
[
5
.1.6. 2,3-Dichloro-N-(5-methoxy-1-methyl-1H-indazol-4-yl)benzenesulfonamide (22)
2
,3-Dichlorobenzenesulfonyl chloride (33 mg, 0.14 mmol) was added to a stirring solution of
aniline 21 (20 mg, 0.11 mmol) in anhydrous pyridine (0.8 mL) and the reaction mixture was
stirred at ambient temperature for 4 h. The reaction mixture was evaporated under reduced
pressure, and the solid was taken up in DMSO (2 mL) and purified by MDAP (Method A).
The appropriate fractions were combined and the solvent was evaporated under reduced
1
pressure to afford the title product (19 mg, 43%) as a white solid; H NMR (400 MHz,
MeOD-d
4
) δ = 8.01 (s, 1H), 7.73 (dd, J = 8.0 1.5 Hz, 1H), 7.63 (dd, J = 8.0, 1.5 Hz, 1H), 7.38
(
d, J = 9.0 Hz, 1H), 7.28 - 7.18 (m, 1H), 7.09 (d, J = 9.0 Hz, 1H), 3.99 (s, 3H), 3.46 (s, 3H);
the exchangeable NH proton was not observed; LCMS (Method A, UV, ES) RT = 1.02 min,
+
M+H] = 386, 388, 390, 100% purity.
[
5
.1.7. N-(3-Bromo-1-methyl-1H-indazol-4-yl)-2,3-dichlorobenzenesulfonamide (24)
The sulfonamide was prepared from 3-bromo-1-methyl-1H-indazol-4-amine 17 (150 mg,
.66 mmol) and 2,3-dichlorobenzenesulfonyl chloride (180 mg, 0.73 mmol) according to the
0
procedure described for the preparation of 22. The same work up, followed by purification on
a 20 g silica column, using a gradient of 0-100% ethyl acetate-cyclohexane over 30 min, and
evaporation of the solvent under reduced pressure afforded 24 (192 mg, 67%) as a yellow
1
solid; H NMR (400 MHz, MeOD-d
4
) δ = 8.00 (dd, J = 8.0, 1.5 Hz, 1H), 7.78 (dd, J = 8.0, 1.5
Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 7.5 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 6.85 (d, J
+
7.5 Hz, 1H), 3.99 (s, 3H); LCMS (Method A, UV, ES) RT = 1.17 min, [M+H] = 434, 436,
=
4
38, 440, 99% purity.
5
.1.8. 2,3-Dichloro-N-(3-methoxy-1-methyl-1H-indazol-4-yl)benzenesulfonamide (25)
2
8

A suspension of 24 (40 mg, 0.09 mmol), sodium tert-butoxide (27 mg, 0.28 mmol) and
copper powder (2 mg, 0.03 mmol) in anhydrous MeOH (1 mL) was sealed in a Reacti-Vial
and heated to 150 ºC for 18 h. The reaction was allowed to cool to ambient temperature.
Several drops of HCl (2 M aqueous solution) were added and the reaction mixture was
diluted with DMSO (1 mL) and filtered through a small pad of celite. The filtrate was
purified on the MDAP (Method B), and the appropriate fractions were combined and dried
TM
1
under reduced pressure to afford the sulfonamide (14 mg, 39%) as a brown gum; H NMR
(
400 MHz, CDCl
3
) δ = 8.32 (br.s, 1H), 8.14 (dd, J = 8.0, 1.0 Hz, 1H), 7.61 (dd, J = 8.0, 1.0
Hz, 1H), 7.32 (t, J = 8.0 Hz, 1 H), 7.15 (t, J = 8.0 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 6.82 (d, J
1
3
=
3
8.0 Hz, 1H), 4.13 (s, 3H), 3.80 (s, 3H); C NMR (101 MHz, CDCl ) δ = 154.9, 142.9,
1
38.4, 135.7, 134.8, 130.6, 130.5, 130.2, 128.6, 127.1, 105.1, 104.1, 102.9, 56.6, 35.2; LCMS
+
Method A, UV, ES) RT = 1.19 min, [M+H] = 386, 388, 390 100% purity.
(
5
.1.9. Ethyl 4-chloro-1-methyl-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (28)
1
-Methyl-1H-pyrazol-5-amine (4.85 g, 49.9 mmol) and diethyl ethoxymethylenemalonate (10
mL, 50 mmol) were heated at 110 ºC for 1.5 h. The ethanol by-product was removed by
evaporation under reduced pressure. Phosphoryl trichloride (12.1 mL, 130 mmol) was added
and the mixture was heated at 110 ºC for 3.5 h. The mixture was cooled to room temperature
and MeCN (5 mL) was added, and then the solution was further cooled down to 10 ºC. Water
(
80 mL) was added slowly maintaining the temperature below 10 ºC and the suspension was
stirred at ambient temperature overnight. The resultant solid was collected by filtration,
washed with minimum amount of water and dried under reduced pressure to afford the title
1
compound (6.7 g, 54%) as a white solid; H NMR (400 MHz, DMSO-d
6
) δ = 8.96 (s, 1H),
1
.40 (s, 1H), 4.38 (q, J = 7.0 Hz, 2H), 4.10 (s, 3H), 1.36 (t, J = 7.0 Hz, 3H); C NMR (101
3
8
MHz, DMSO-d ) δ = 163.6, 151.0, 150.7, 137.8, 132.2, 118.0, 115.1, 61.5, 34.2, 14.0. LCMS
6
2
9