Lead identification of benzimidazolone and azabenzimidazolone arylsulfonamides as CC-chemokine receptor 4 (CCR4) antagonists

Article abstract of DOI:10.1016/j.bmc.2014.05.021

A knowledge-based library of 2,3-dichlorophenylsulfonyl derivatives of commercially available aryl amines was synthesised and screened as human CCR4 antagonists, in order to identify a suitable hit for the start of a lead-optimisation programme. Hits were required to be more potent than an existing indazole series, have better physicochemical properties (c log P <3.5, chrom log D_7.4 <5.3 and CLND solubility >116 μg/mL), and be stable to acid and light. The benzimidazol-2-one core was identified as a hit suitable for further investigation. Substitution at N1 with small alkyl groups was tolerated; however, these analogues were inactive in the whole blood assay (pA₂ <5). Azabenzimidazolone analogues were all found to be active, with compound 38 exhibiting whole blood activity of 6.1, low molecular weight (389) and chrom log D_7.4 (2.4), high LE (0.43), and solubility (152 μg/mL). In addition, 38 had human serum albumin binding of around 93% and met all the criteria for progression to lead optimisation.

Full text of DOI:10.1016/j.bmc.2014.05.021

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Lead identification of benzimidazolone and azabenzimidazolone
arylsulfonamides as CC-chemokine receptor 4 (CCR4) antagonists
Afjal H. Miah a, Hossay Abas a, Malcolm Begg b, Benjamin J. Marsh a, Daniel E. O’Flynn a, Alison J. Ford b,
Jonathan M. Percy c, Panayiotis A. Procopiou a, , Steve A. Richards d, Sally-Anne Rumley b
⇑
a Department of Medicinal Chemistry, Respiratory TAU, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
b Department of Respiratory Biology, 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
d UK Analytical Chemistry, Platform Technology & Science, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
A knowledge-based library of 2,3-dichlorophenylsulfonyl derivatives of commercially available aryl
amines was synthesised and screened as human CCR4 antagonists, in order to identify a suitable hit
for the start of a lead-optimisation programme. Hits were required to be more potent than an existing
indazole series, have better physicochemical properties (clogP <3.5, chrom logD7.4 <5.3 and CLND solu-
Received 13 February 2014
Revised 7 May 2014
Accepted 12 May 2014
Available online xxxx
bility >116 lg/mL), and be stable to acid and light. The benzimidazol-2-one core was identified as a hit
suitable for further investigation. Substitution at N1 with small alkyl groups was tolerated; however,
these analogues were inactive in the whole blood assay (pA2 <5). Azabenzimidazolone analogues were
all found to be active, with compound 38 exhibiting whole blood activity of 6.1, low molecular weight
(389) and chrom logD7.4 (2.4), high LE (0.43), and solubility (152 lg/mL). In addition, 38 had human
serum albumin binding of around 93% and met all the criteria for progression to lead optimisation.
Keywords:
CCR4 antagonist
Benzimidazolone
Azabenzimidazolone
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
allergic rhinitis.12 In addition, CCR4 antagonists were used as molec-
ular adjuvants in vaccines.13–16 Finally, CCR4 monoclonal antibod-
T helper 2 (Th2) cytokines in inflamed tissues lead to eosino-
philia, high levels of serum IgE and mast cell activation, all of which
contribute to the pathogenesis of allergic diseases.1 Upon exposure
to allergen, dendritic cells within tissue secrete macrophage-
derived chemokine (MDC), and thymus activation-regulated che-
mokine (TARC) which can recruit Th2 cells from the circulation.
The T cells can then migrate along this chemokine gradient to the
dendritic cells. The latter migrate from the inflamed tissue to local
lymph nodes where MDC and TARC may recruit further T cells.
Elevated levels of TARC and MDC as well as accumulation of
CCR4-positive cells were observed in lung biopsy samples from
patients with atopic asthma following allergen challenge.2 CCR4 is
also expressed by immune suppressive regulatory T cells,3,4 and a
minor subset of Th17 cells.5,6 Hence CCR4 antagonists represent a
novel therapeutic intervention in diseases where CCR4 is involved,
such as asthma,7 lung disease,3 atopic dermatitis,8 allergic broncho-
pulmonary aspergillosis,9 cancer,3,10 inflammatory bowel disease,4
the mosquito-borne tropical diseases, such as Dengue fever,11 and
ies were recently explored for CCR4+ T cell leukaemia and one
such antibody, Mogamulizumab, was approved in Japan for the
treatment of relapsed or refractory adult
T cell leukaemia/
lymphoma.17,18 The approval of the humanised monoclonal anti-
body Mogamulizumab underlines the value of generating small
molecule CCR4 antagonists in this area. The AstraZeneca pyrazine
sulfonamide 1,19 the Ono sulfonamide 2,20 and the GlaxoSmithKline
indazole 321,22 are recently published sulfonamide CCR4 receptor
antagonists (Fig. 1). Sulfonamides 1–3 bind at an intracellular
allosteric binding site, which is different from the extracellular
binding site where lipophilic amine antagonists such as 4–6 bind.
The two allosteric binding sites are distinct from each other and
from the orthosteric binding site where TARC and MDC bind.23 Inda-
zole 3 was the first candidate to be progressed to human studies;
however, the compound suffered from low solubility and weak
potency.24 A back-up programme was therefore initiated to identify
novel templates for lead optimisation studies. Our group has
recently reported the identification of bicyclic heteroarenes as novel
lead series of sulfonamide CCR4 antagonists.25 Herein we disclose
the identification of a second lead series, the benzimidazol-2-one
sulfonamides.
⇑
Corresponding author. Tel.: +44 1438 762883; fax: +44 1438 768302.
E-mail address: pan.a.procopiou@gsk.com (P.A. Procopiou).
http://dx.doi.org/10.1016/j.bmc.2014.05.021
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Miah, A. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.021

2
A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Cl
S
O
S
Cl
H
N
H
N
O
S
O
Cl
O
S
O
O
N
N
H
N
O
N
N
N
Br
O
N
Cl
O
O
O
N
O
HO
OH
N
H
1
2
3
Cl
Cl
F
HN
HN
N
HN
F
Cl
O
F
N
N
O
O
N
N
N
N
N
N
N
N
N
H
O
H
N
4
5
6
Figure 1. Structures for some recently published CCR4 antagonists.
Cl
2. Chemistry
Cl
Cl
The initial hit came from a focused library of 2,3-dichloroben-
zenesulfonamides designed on a knowledge driven approach. A
virtual set of 1600 commercially available aryl or heteroaryl pri-
mary amines were enumerated as 2,3-dichlorobenzenesulfona-
mides, ensuring that the set contained novel starting points with
desired physicochemical properties (MW <400, clogP <4, polar sur-
face area <100, H-bond acceptor <10, H-bond donor <5). A library
of about 100 sulfonamides was synthesised and assayed in the
Cl
HN
O
HN
S
O
O
S
O
O
O
N
N
HN
NH
8
7
O
GTP
of the starting point of the indazole series (7) in the GTP
and also better physicochemical properties than 7 (clogP <3.5,
chrom logD7.4 <5.3 and CLND solubility >116 g/mL). A subset of
c
S assay. Hits were required to have a pIC50 greater than that
Figure 2. Initial indazole (7) and benzimidazolone (8) hits.
cS assay,
l
regioselectively as the acidity of the nitrogen atoms of the imidazo-
lone ring was substantially different. The calculated pKa values
(ACD software, version 11) for N1 (para to the nitro-group) was
10.9 and for N3 (para to methoxy-group) was 17.1. Hence,
treatment of 9 with one equivalent of sodium hydride in DMF
and one equivalent of electrophile was expected to give the
N1-alkylation product as the major component. Indeed when
iodomethane was used as the electrophile 10a was obtained
in 25% yield. The other mono-methyl product 11a was present in
the reaction mixture only in trace amounts. The dimethyl product
12a was however also isolated in 14% yield, which suggested that
product 10a reacted further with iodomethane. The structure of
the monomethyl regioisomer 10a was confirmed by NOE experi-
ments. The HSQC spectrum confirmed the chemical shifts of the
aromatic protons at the C4 (7.51 ppm) and C7-position
(7.12 ppm). Irradiating the N-methyl protons (3.34 ppm) and
O-methyl protons (3.94 ppm), resulted in both sets of protons giv-
ing an NOE to the proton at the C7-position (7.12 ppm), confirming
the regiochemistry of the alkylation (Fig. 3).
Treatment of the sodium salt of 9 with 2-bromoacetamide gave
a 9:1 mixture of 10b and 11b (Scheme 1). Similarly treatment of 9
with iso-propyl bromide or with 3-iodo-oxetane gave 10c and 10d,
respectively. The nitro-group of 10a–d, 11b and 12a,b was
reduced with hydrogen over 10% palladium on carbon to give the
corresponding aromatic amines, which were sulfonylated with
2,3-dichlorobenzenesulfonyl chloride in pyridine to give 13a–d,
14b and 15a.
14 of the most active compounds was tested for chemical stability
in simulated gastric fluid and for photo-stability, and the unstable
compounds were removed from the set to leave 10 hits. We have
already reported for our indazole series,22 and more recently for
the bicyclic heteroarene series25 that introduction of a hydrogen
bond acceptor (HBA), suitably orientated to hydrogen-bond to
the sulfonamide NH, increased the potency of these compounds
approximately ten-fold. A methoxy group was then introduced to
each of the 10 stable hits of our sulfonamide library to act as an
HBA for the sulfonamide NH. Following screening of these methoxy
analogues in the GTPcS assay the benzimidazol-2-one sulfonamide
8 was identified as one of five hits suitable for further investiga-
tion. In this publication we disclose initial structure–activity rela-
tionships in this series, and the identification of a lead compound
(see Fig. 2).
Substitution with large benzyl groups was tolerated at the N1
position of the indazole series (for example compound 3).21,22 It
was therefore important to find out whether substitution in the
heterocyclic ring of the benzimidazolone scaffold was tolerated.
Alkylation of 8 would produce three potential products, which
would require separation and extensive characterisation. It was
also known from previous studies22 that alkylation of the
sulfonamide nitrogen provided compounds devoid of any CCR4
activity. It was considered that treatment of 5-methoxy-6-nitro-
benzimidazol-2-one 9 with one equivalent of electrophile in
the presence of
a base might produce one major product
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A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
The method of Nebra,30 utilising commercially available ruthe-
nium catalyst 24, and stoichiometric potassium periodate in water
gave 23 in 40% yield. Finally reduction of 23 using catalytic
hydrogenation over 10% Pd/C catalyst, followed by reaction with
2,3-dichlorobenzenesulfonyl chloride in pyridine gave the desired
N3-methylated sulfonamide 14a in 7% yield. The N1-propyl ana-
logue 13e was also obtained from this route by catalytic hydroge-
nation of 21 followed by sulfonylation.
H
4
3H
N
O2N
O
O
1N
7
H
Figure 3. NOE confirmation of the structure of N1-methyl regioisomer 10a. The
numbering of the N1-position in relation to the MeO- and nitro-groups shown
above could change depending on the substituent used, so throughout the report,
the N1-position as shown above is used as an arbitrary number for ease of
discussion.
A
regioselective synthetic route for the preparation of
N1-substituted benzimidazolones was developed and is outlined
in Scheme 3. Reaction of 16 with a range of primary amines, such
as N,N-dimethylaminoethylamine, 2-aminopropane-1,3-diol, and
3-amino-1-BOC-azetidine in the presence of diisopropylamine
(DIPEA) in DMF at room temperature gave 25 in 83–93% yields.
Reduction of the nitro group of 25 with catalytic hydrogenation
over 10% Pd/C in ethanol gave 26 in 70–97% yields. In the case of
25f the hydroxyls were protected as TBDMS ethers before proceed-
ing to the hydrogenation step. The aromatic diamines 26 were then
cyclised with carbonyl diimidazole (CDI) in THF at 50 °C to give 27
in 69–83% yield. Benzimidazolones 27 were nitrated using 4 M
nitric acid to give the desired mono nitro products 10 in 33–53%
yield, accompanied by a small amount of the di-nitration product.
In the case of azetidine 10g the BOC protecting group was removed
under the nitration conditions and it was re-installed using BOC2O
in THF in the presence of NaHCO3. The nitro group of 10 was
reduced by catalytic hydrogenation over 10% Pd/C to give 28 in
high yields. The synthesis was completed by sulfonylation of 28
with 2,3-dichlorobenzenesulfonyl chloride in pyridine to give the
desired sulfonamide 13 in moderate yields. In the case of
BOC-13g the protecting group was removed using 4 M HCl in diox-
ane. Finally, 13g was converted to 13h using one equivalent of ace-
tic anhydride in pyridine.
The general method shown in Scheme 1 was unsuccessful for
the preparation of the N3-methylated regioisomer 11a, so an alter-
native route to explore the N3-substitution using allyl protection
was investigated (Scheme 2). The allyl group was chosen as a
protecting group as it is stable to both acidic (nitric acid) and basic
(sodium hydride) conditions, key requirements in the proposed
synthetic route. Reaction of commercially available 2-fluoro-4-
methoxy-1-nitrobenzene 16 with neat allylamine at room temper-
ature afforded the nitroaniline 17 in 97% yield. The nitro group of
17 was reduced with iron and ammonium chloride to provide
the phenylenediamine 18, which was then treated with CDI to give
the methoxy benzimidazolone 19 in 45% yield. Nitration of 19
using fuming nitric acid and concentrated sulfuric acid or neat
fuming nitric acid gave the undesired dinitro product 20. Investiga-
tion of various other concentrations of nitric acid and sulfuric acid,
identified 4 M nitric acid as the best reagent for the preparation of
the desired mono nitro product, 21 (23% yield). A small amount of
the dinitro product, 20 and starting material were also present in
the reaction mixture. Alkylation of 21 with methyl iodide was fac-
ile providing the methylated benzimidazolone 22 in excellent
yield. Removal of the allyl group proved to be problematic.
Attempts to use palladium catalysed conditions, involving the
Introduction of nitrogen at the C4-position of the benzimidazo-
lone series is outlined in Scheme 4. Commercially available
3-hydroxy pyridine 29 was reacted with bromine in aqueous
sodium hydroxide to give the dibromide 30 according to the
method of Clark and Deady.31 The latter was methylated with iodo-
methane in the presence of potassium carbonate in DMSO to give
31 in 80% yield. Nitration of 31 with concd HNO3 and concd
formation of p
–allyl complex,26 and those based on isomerisation
of the allylamine double bond using RuCl3 catalyst, followed by
hydrolysis of the resulting enamine, were all found to be
unsuccessful.27–29
R2
N
R2
N
H
N
H
N
O2N
b
O2N
O2N
O2N
O
O
O
O
N
H
N
R1
MeO
N
R1
N
H
MeO
MeO
MeO
9
12
11
10
c, d
c, d
c, d
a
O
S
Cl HN
O
S O
Cl HN
O
S
Cl HN
Cl
O
O
Cl
O
Cl
O
R2
N
R2
N
H
N
H
N
O
O
N
H
MeO
N
H
N
R1
MeO
N
R1
MeO
MeO
14
15
13
a =
-Me
b = -CH2CONH2
c = -
CHMe2
O
d =
Scheme 1. Reagents and conditions: (a) 17.5% HNO3, 0 °C 1 h, 20 °C, 3 h, 36%; (b) NaH, DMF, 20 °C then MeI (10a), or NH2COCH2Br (10b), or iso-PrBr (10c), or 3-iodo-oxetane
(10d); (c) H2, 10% Pd/C, EtOH, EtOAc, 18 h; (d) 2,3-dichlorobenzenesulfonyl chloride, pyridine, 20 °C.
Please cite this article in press as: Miah, A. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.021

4
A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
H
N
NO2
F
NO2
NH
NH2
NH
b
c
a
O
MeO
N
MeO
MeO
MeO
18
19
16
17
d
H
N
O2N
MeO
f
O2N
O2N
MeO
N
e
N
N
O
O
O
N
H
N
MeO
R
Ru
11a
g,h
22
Cl
Cl
24
20 R = NO2
21 R = H
O
S O
g,h
Cl
O
S O
Cl HN
Cl HN
N
Cl
O
H
N
N
H
MeO
O
N
14a
MeO
13e
Scheme 2. Reagents and conditions: (a) allylamine, 20 °C, 1.5 h, 97%; (b) Fe, NH4Cl, EtOH, H2O, 100 °C, 22 h, 83%; (c) CDI, THF, 50 °C, 2 h, 45%; (d) 4 M HNO3, 0–20 °C, 4 h, 23%;
(e) NaH, MeI, DMF, 0–20 °C, 2.5 h, 99%; (f) dichloro(2,6,10-dodecatriene-1,12-diyl)ruthenium(IV) (24), KIO4, H2O, 100 °C, 72 h, 39%; (g) H2, 10% Pd/C, EtOH–EtOAc (1:1), 5 h,
99%; (h) 2,3-dichlorobenzenesulfonyl chloride, pyridine, 20 °C, 1 h, 22%.
H
N
NO2
F
NO2
NH2
b
c
a
O
MeO
N
R
MeO
NH
R
MeO
NH
R
MeO
26
27
16
25
d
O
Cl
S O
Cl HN
H
H
N
H
e
H2N
O2N
b
N
N
O
O
O
N
R
N
R
N
R
MeO
MeO
MeO
28
13
10
NH
N
N
R
=
h
f
O
OH
OH
g
i
Scheme 3. Reagents and conditions: (a) RNH2, DIPEA, DMF, 20 °C; (b) H2, 10% Pd/C, EtOH; (c) CDI, THF, 50 °C; (d) 4 M HNO3, 0–20 °C; (e) 2,3-dichlorobenzenesulfonyl
chloride, pyridine, 20 °C.
H2SO4 at 65 °C gave 32 in 28% yield. The two bromine atoms of 32
were readily displaced by aqueous ammonia using microwave irra-
diation using a modification of the Barraclough procedure to give
diamine 33 in 78% yield.32 The latter was reacted with BOC2O to
give the bis-protected diamine 34 in 66% yield, followed by reduc-
tion of the nitro group using iron in acetic acid to provide amine 35
in 78% yield. This compound was then heated in pyridine in a
microwave oven at 100 °C to give the cyclised azabenzimidazolone
36 in 74% yield. Cleavage of the BOC group with TFA gave 37 in
93%, and reaction of this product with 2,3-dichlorobenzenesulfonyl
chloride in pyridine afforded the desired sulfonamide 38 in moder-
ate yield (30%).
Commercially available 6-methoxypyridine-2,3-diamine (39)
was used for the synthesis of the other regioisomeric azabenzimi-
dazolone series (Scheme 5). Reaction of 39 with CDI was slow giv-
ing the benzimidazolone 40 in only 25% yield after 48 h. Nitration
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A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Br
N
Br
N
Br
N
Br
Br
N
Br
a
d
b
c
HO
HO
MeO
MeO
NO2
29
30
31
32
BOC
HN
BOC
NH
BOC
BOC
NH
N
H2N
N
NH2
HN
MeO
35
N
g
e
f
MeO
NO2
MeO
NO2
NH2
33
34
H
N
H
N
H
N
H2N
MeO
N
O
S
HN
BOC
N
h
i
O
Cl
O
O
N
H
H
N
MeO
N
H
N
Cl
O
36
N
H
37
MeO
38
Scheme 4. Reagents and conditions: (a) Br2, 10% NaOH, 0 °C, 1 h, 20 °C, 4 h, 46%; (b) MeI, K2CO3, DMSO, reflux, 2 h, 80%; (c) concd HNO3, concd H2SO4, 65 °C, 36 h, 28%; (d) aq
NH3, 90 °C, 1 h, wave, 78%; (e) BOC2O, K2CO3, DMF, 20 °C, 18 h, 66%; (f) Fe, AcOH, 20 °C, 2 h, 78%; (g) pyridine, 100 °C, 1 h, wave, 74%; (h) TFA, 20 °C, 30 min, 93%; (i) 2,3-
l
l
dichlorobenzenesulfonyl chloride, pyridine, 20 °C, 2 h, 30%.
of 40, using triflic anhydride and fuming nitric acid gave the
desired nitro-benzimidazolone 41, which was reduced by catalytic
hydrogenation over 10% Pd/C to give the amino benzimidazolone
42. Finally, reaction with 2,3-dichlorobenzenesulfonyl chloride
afforded the target sulfonamide 43.
The preparation of the pyrazine analogue is shown in Scheme 6.
Reaction of commercially available 5-bromopyrazine-2,3-diamine
(44) with CDI gave the diazabenzimidazolone 45 in 85% yield.
Nitration, using nitronium tetrafluoroborate gave the nitro
compound 46 in 50% yield. The bromine of 46 was displaced by
methoxide to give the methoxy diazabenzimidazolone 47 in 49%
yield. Reduction of 47 using catalytic hydrogenation conditions
over 10% Pd/C gave 48 (80% yield), which was then sulfonylated
with 2,3-dichlorobenzenesulfonyl chloride in pyridine to afford
the desired sulfonamide 49.
determine potency against the native receptor for the more potent
compounds in the primary assay.22 The assay quantified cytoskel-
etal reorganization (formation of filamentous (F-) actin) which
occurs in a variety of cells in response to chemoattractants and is
a prelude to chemotaxis. This was achieved by staining the F-actin
with a fluorescent derivative of phalloidin, which binds with high
affinity and specificity to the interface between actin monomers
in F-actin. The response was measured as an increase in the fluo-
rescence intensity of the target cell population in a flow cytometer
and was expressed as a pA2. In this assay, human CD4+ CCR4+
lymphocytes were identified by staining with antibodies to CD4
and CCR4. Calculated partition coefficient (clogP), chromato-
graphic logD7.4 (chrom logD at pH 7.4) and ChemiLuminescent
Nitrogen Detection (CLND) kinetic solubility 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 precipi-
tated. Ligand efficiency (LE) was obtained from the equation
LE = À1.36 Â 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.
3. Results and discussion
All compounds in Table 1 were tested as human CCR4 antago-
nists in vitro. Antagonist potency was determined by a [35S]-GTP
cS
radioligand competition functional assay using recombinant
CCR4-expressing CHO cell membranes adhered to WGA-coated
Leadseeker SPA beads in assay buffer at pH 7.4.33 Another assay
using human whole blood was used as a secondary screen to
Compounds 2 and 4 were used as standards in the GTPcS assay
and included in Table 1. The data generated for 7, the initial lead
that lead to indazole candidate 3, are also included in Table 1 for
H
H
N
H
N
O2N
a
N
H2N
NH2
NH2
b
c
O
O
O
N
H
N
H
N
H
MeO
O
N
MeO
N
MeO
N
MeO
N
39
40
41
42
Cl
S O
Cl HN
d
H
N
O
N
H
MeO
N
43
Scheme 5. Reagents and conditions: (a) CDI, Et3N, THF, 20 °C, 48 h, 31%; (b) Tf2O, fuming HNO3, 20 °C, 3 h, 47%; (c) H2, 10% Pd/C, EtOH, 2 h, 76%; (d) 2,3-
dichlorobenzenesulfonyl chloride, pyridine, 20 °C, 1 h, 48%.
Please cite this article in press as: Miah, A. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.021

6
A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
H
N
H
N
H
N
N
NH2
NH2
N
N
O2N
Br
N
N
a
O2N
N
N
b
c
O
O
O
Br
N
H
N
H
N
Br
N
N
H
MeO
44
45
46
47
O
Cl
S O
H
d
e
H2N
H
N
Cl HN
N
N
N
O
O
N
H
MeO
N
N
H
MeO
48
49
Scheme 6. Reagents and conditions: (a) CDI, THF, 50 °C, 48 h, 85%; (b) NO2BF4, CH3CN, 20 °C, 1 h, 50%; (c) NaOMe, MeOH, 20–50 °C, 20 h, 49%; (d) H2, 10% Pd/C, EtOH–EtOAc
(1:1), 1 h, 80%; (e) 2,3-dichlorobenzenesulfonyl chloride, pyridine, 20 °C, 1 h, 7%.
Table 1
In vitro data pIC50 for humanCCR4 GTPcS binding assay, calculated logP, ligand efficiency, measured chrom logD at pH 7.4 and CLND solubility
Compd
GTP
c
S pIC50 SEM
cLogP
LE
Chrom logD
CLND Solub. (
l
g/mL)
hWB pA2
(n)
2
4
8.41 0.02
(147)
8.26 0.01
(150)
6.22 0.03
(2)
6.5 0.1
(4)
6.7 0.1
(4)
4.7 0.2
(2)
5.7 0.1
(4)
6.34 0.03
(3)
<4.5
(3)
6.0 0.2
(3)
6.95 0.06
(3)
5.4 0.2
(2)
5.7 0.3
(2)
5.8 0.3
(2)
6.4 0.1
(2)
4.4
4.9
3.5
3.1
3.5
3.5
3.3
2.3
2.3
4.6
4.4
3.7
2.5
3.0
2.6
3.0
3.6
4.0
3.5
0.34
0.33
0.35
0.38
0.36
0.26
0.30
0.31
2.9
2.9
5.3
3.2
3.8
3.7
4.4
2.5
2.4
4.7
4.6
3.2
2.6
2.1
3.0
3.7
2.4
3.2
ND
193
145
116
32
6.6 0.1
(24)
7.3 0.3
(4)
<5.0
(6)
5.3
(4)
<5
7
8
13a
14a
15a
13b
14b
13c
13d
13e
13f
13g
13h
13i
38
12
66
ND
ND
ND
ND
ND
<5
58
11
167
3
0.30
0.35
0.25
0.27
0.28
0.28
0.33
0.43
0.40
0.41
180
176
82
ND
ND
ND
ND
<5
194
8
6.7 0.2
(5)
7.6 0.1
(2)
7.1 0.2
(3)
7.3 0.1
(2)
152
126
130
6.1
5.6
5.2
43
49
ND: not determined.
comparison. The initial benzimidazolone hit 8 was slightly more
potent than 7 (pIC50 = 6.5), with higher LE (0.38), and lower
lipophilicity (chrom logD7.4 3.2), but with lower solubility. Substi-
tution at the benzimidazolone N1-position with methyl or acetam-
ide groups (13a and 13b) was tolerated, however, substitution at
N3 (14a and 14b) reduced potency by 100-fold. The N1,
N3-dimethyl analogue 15a was ten-fold less potent than 13a. It
was encouraging to observe that removal of a hydrogen bond
donor (HBD) from the benzimidazolone core was tolerated. In
general reducing the number of HBD’s in a compound increases
permeability, although the solubility might be reduced.34,35 The
N1-position was further explored with small lipophilic, polar and
weakly basic groups in an attempt to improve the potency, solubil-
ity and physicochemical property of the series. Replacing the
methyl group at the N1-position for n-propyl group (13c) reduced
both the potency and the solubility. Replacing methyl for isopropyl
group (13d) caused a marginal increase in potency, accompanied
by increased lipophilicity (clogP = 4.4). The two basic analogues
Please cite this article in press as: Miah, A. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.021

A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
13e and 13g, the diol 13f, and the amide 13h were made to
increase the solubility of the molecules. However, despite the
observed increases in solubilities, their potencies were lower than
the previous compounds. The oxetane group was utilised to
improve the solubility and permeability of other scaffolds.36 The
oxetanyl analogue 13i was equipotent to the methyl analogue
13a, and it was also less lipophilic, however, its solubility remained
low. Furthermore, 13i was found to be unstable, and no further
substitutions were investigated.
and human serum albumin binding. Azabenzimidazolone 38 was
equipotent with our indazole candidate 3 in the whole blood assay
with a pA2 of 6.1, making 38 a very attractive starting point for lead
optimisation. The CLND solubility of 38 was 152 lg/mL, which is
on the lower limit for an oral candidate. Introduction of small
water-solubilising groups, either as substituents at N1, or in the
aromatic ring of the core would be expected to overcome this prob-
lem. Further investigation of this template will not be undertaken,
however, due to closure of the project.
It was noted that all three sulfonamide CCR4 antagonist candi-
dates 1, 2 and 3 contain a nitrogen heterocycle (pyrazine or inda-
zole), and that the nitrogen atom of the heterocyclic ring was
ortho- to the sulfonamide nitrogen. Similarly, a 10-fold increase
of potency was observed in our lead generation work for a
back-up series to compound 3 after the introduction of an ortho-
nitrogen atom to phenylpyrazole to form pyridylpyrazole sulfona-
mides. Furthermore, the introduction of nitrogen into the phenyl
ring of the benzimidazolone 8 was expected to enhance its solubil-
ity and physicochemical properties. This strategy was more
successful than the removal of an HBD, or the introduction of sol-
ubilising groups. Thus, replacement of the C4-methine with nitro-
gen (compound 38) resulted in more than a log unit increase in
5. Experimental section
Organic solutions were dried over anhydrous Na2SO4 or MgSO4.
TLC was performed on Merck 0.25 mm Kieselgel 60 F254 plates.
Products were visualised under UV light and/or by staining with
aqueous KMnO4 solution. LCMS analysis was conducted on either
System A or System B. System A: Acquity UPLC BEH C18 column
(2.1 mm  50 mm i.d. 1.7
lm packing diameter) eluting with
0.1% formic acid in water (solvent A), and 0.1% formic acid in
acetonitrile (solvent B), using the following elution gradient
0.0–1.5 min 3–100% B, 1.5–1.9 min 100% B, 1.9–2.0 min 3% B, at a
flow rate of 1 mL minÀ1 at 40 °C. The UV detection was an averaged
signal from wavelength of 210 nm to 350 nm, and mass spectra
were recorded on a mass spectrometer using alternate-scan elec-
trospray positive and negative mode ionization (ES+ve and ESÀve).
System B: Acquity UPLC BEH C18 column (50 mm  2.1 mm ID,
GTPcS potency (pIC50 = 7.6), and a substantial increase in ligand
efficiency (LE = 0.43). Furthermore, its lipophilicity was decreased
(clogP = 3.6 and chrom logD7.4 = 2.4), and its solubility increased
by more than three-fold compared to 8. Introducing nitrogen at
C7 (compound 43) also increased potency in the GTPcS assay,
but only by half a log unit while it increased its LE to 0.40. The lipo-
philicity was also decreased, but not by as much as in the case of
38. Its solubility was increased by three-fold to 126 lg/mL. Intro-
ducing two nitrogen atoms to the benzimidazolone phenyl ring
provided pyrazine 49, which increased its pIC50 to 7.3, LE to 0.41,
and its solubility by three-fold higher than 8.
1.7 lm packing diameter) eluting with 10 mM ammonium
bicarbonate in water adjusted to pH 10 with ammonia solution
(solvent A), and MeCN (solvent B) using the following elution
gradient 0–1.5 min 1–97% B, 1.5–1.9 min 97% B, 1.9–2.0 min
100% B at a flow rate of 1 mL minÀ1 at 40 °C. Column chromatogra-
phy was performed on Flashmaster II. Mass-directed auto-prepara-
tive HPLC (MDAP) was conducted on a Sunfire C18 column
Human whole blood F-actin polymerisation assay and physico-
chemical data were obtained for the most potent compounds of the
benzimidazolone series, and the results are also summarised in
Table 1. The parent benzimidazolone 8 was weakly active in this
assay and had a pA2 value of 5.3. Although the N1 isopropyl
(150 mm  30 mm i.d. 5
lm packing diameter) at ambient temper-
ature eluting with 0.1% formic acid in water (solvent A) and 0.1%
formic acid in acetonitrile (solvent B), using an appropriate elution
gradient over 15 min at a flow rate of 40 mL minÀ1 and detecting at
210–350 nm at room temperature. Mass spectra were recorded on
Micromass ZMD mass spectrometer using electro spray positive
and negative mode, alternate scans. 1H NMR spectra were recorded
at 400 MHz, unless otherwise stated. The chemical shifts are
expressed in ppm relative to tetramethylsilane. High resolution
positive ion mass spectra were acquired on a Micromass Q-Tof 2
hybrid quadrupole time-of-flight mass spectrometer. The purity
of all compounds screened in the biological assays was examined
by LCMS analysis and was found to be P95%, unless otherwise
specified. All animal studies were ethically reviewed and carried
out in accordance with Animals (Scientific Procedures) Act 1986
and the GSK Policy on the Care, Welfare and Treatment of
Laboratory Animals. The human biological samples were sourced
ethically and their research use was in accord with the terms of
the informed consents.
analogue 13d was slightly more potent than 8 in the GTPcS assay,
it was inactive in the whole blood assay (pA2 <5). The three
azabenzimidazolone analogues 38, 43 and 49 were all found to
be active, with compound 38 exhibiting whole blood activity sim-
ilar to that of the indazole candidate 3. The whole blood activity
data for 38 (pA2 = 6.1) is very encouraging as its molecular weight
is lower than 3, (389 vs 549), it displays a higher LE of 0.43 (cf. 3
LE = 0.29), and it is more soluble than 3. It is also much less
lipophilic than 3 (chrom logD7.4 = 2.4 vs 4.3). In addition, the
azabenzimidazolones 38, 43 and 49 have human serum albumin
binding around 93%, which is substantially lower than 3 (96.5%).
This translates to twice the unbound fraction of 38 available for
distribution to the desired tissues.
4. Conclusion
5.1. 2,3-Dichloro-N-(6-methoxy-2-oxo-2,3-dihydro-1H-
benzo[d]imidazol-5-yl)benzenesulfonamide (8)
The aim of this work was to obtain human CCR4 antagonists
which were more potent than the starting point of our indazole
candidate, had better physicochemical properties (clogP <3.5,
A solution of 5-amino-6-methoxy-1,3-dihydro-2H-benzimidazol-
2-one (55.6 mg, 0.31 mmol) and 2,3-dichlorobenzenesulfonyl
chloride (76 mg, 0.31 mmol) in pyridine (0.5 mL) was stirred over-
night. The reaction mixture was concentrated under a stream of
nitrogen in a blow-down apparatus at 40 °C. The residue was purified
by Mass-Directed Auto-Preparative HPLC (MDAP) to give 8 (65 mg,
54%) as a solid: 1H NMR (400 MHz, DMSO-d6) d = 10.55 (s, 1H),
10.36 (s, 1H), 9.56 (s, 1H), 7.87 (dd, J = 8.0, 2.0 Hz, 1H), 7.70 (dd,
J = 8.0, 2.0 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 6.75 (s, 1H), 6.44 (s, 1H),
chrom logD7.4 <5.3 and CLND solubility >116 lg/mL), and were
stable to acid and light. A novel series of 2,3-dichlorophenylsulf-
onamide derivatives of benzimidazol-2-one were synthesised and
examined as CCR4 antagonists. Substitution with small alkyl
groups at N1 was tolerated; however, substitution at N3 or di-sub-
stitution at both N1 and N3 was not tolerated. Introduction of
nitrogen in the benzimidazolone phenyl ring increased potency,
ligand efficiency and solubility, whilst reducing the lipophilicity
Please cite this article in press as: Miah, A. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.021

8
A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3.33 (s, 3H); 13C NMR (101 MHz, DMSO-d6) d = 155.5, 149.6, 140.3,
134.0, 133.8, 129.7, 129.4, 129.2, 127.7, 122.6, 116.4, 109.4, 93.6,
55.5; HRMS (ESI) calcd for C14H12Cl2N3O4S (M+H)+ 387.9920; found:
387.9927; LCMS (System A) RT = 0.85 min, 95%, ES+ve m/z 388, 390,
392 (M+H)+, 429, 431, 433 (M+H+MeCN)+.
a white solid (20 mg, 38%): 1H NMR (400 MHz, DMSO-d6) d = 9.67
(br s, 1H), 7.87 (dd, J = 8.0, 1.5 Hz, 1H), 7.70 (dd, J = 8.0, 1.5 Hz,
1H), 7.38 (t, J = 8.0 Hz, 1H), 7.01 (s, 1H), 6.77 (s, 1H), 3.35 (s, 3H),
3.27 (s, 3H), 3.26 (s, 3H); LCMS (System A) RT = 0.95 min, 93%,
ES+ve m/z 416, 418,420 (M+H)+, 457, 459, 461 (M+H+MeCN)+.
5.2. 5-Methoxy-6-nitro-1H-benzo[d]imidazol-2(3H)-one (9)
5.6. 2-(6-Methoxy-5-nitro-2-oxo-2,3-dihydro-1H-
benzo[d]imidazol-1-yl)acetamide (10b) and 2-(5-methoxy-6-
nitro-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)acetamide
(11b)
A solution of 17.5% fuming nitric acid (12.5 mL, 48.9 mmol) was
slowly added to 5-methoxy-1H-benzo[d]imidazol-2(3H)-one
(0.25 g, 1.5 mmol) at 0 °C, and the reaction was stirred for 1 h at
0 °C, and at 20 °C for 3 h. The reaction mixture was cooled to 0 °C
and neutralised with saturated aqueous NaHCO3 solution. The
product was extracted with EtOAc (2 Â 50 mL), washed with
water, dried, and concentrated to give 9 (113 mg, 36%) as a yellow
solid: 1H NMR (400 MHz, DMSO-d6) d = 11.21 (br s, 1H), 10.81 (br s,
1H), 7.47 (s, 1H), 6.80 (s, 1H), 3.89 (s, 3H); MS ES+ve m/z 210
(M+H)+, 251 (M+H+MeCN)+.
Were prepared from 9 (200 mg, 0.956 mmol) and 2-bromoacet-
amide (132 mg, 0.956 mmol) according to the procedure described
for the preparation of 13a. A 9:1 mixture of 10b and 11b (90 mg,
32%) was obtained as a yellow solid: 1H NMR (400 MHz, DMSO-d6)
major d = 11.11 (br s, 1H), 7.62 (br s, 1H), 7.52 (s, 1H), 7.22 (br s,
1H), 7.11 (s, 1H), 4.48 (s, 2H), 3.89 (s, 3H); MS ES+ve m/z 267 (M+H)+.
5.7. 2-(5-(2,3-Dichlorophenylsulfonamido)-6-methoxy-2-oxo-
2,3-dihydro-1H-benzo[d]imidazol-1-yl)acetamide (13b)
N19663-84-3 and 2-(6-(2,3-dichlorophenylsulfonamido)-5-
methoxy-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)acetamide (14b)
5.3. 6-Methoxy-1-methyl-5-nitro-1H-benzo[d]imidazol-2(3H)-
one (10a) and 5-methoxy-1,3-dimethyl-6-nitro-1H-
benzo[d]imidazol-2(3H)-one (12a)
Sodium hydride (15 mg of a 60% w/w dispersion in mineral oil,
0.38 mmol) was added to a stirring solution of 9 (75 mg, 0.36 mmol)
in anhydrous DMF (1 mL) at 0 °C. Methyl iodide (0.02 mL,
0.36 mmol) was added and the reaction mixture was stirred at
ambient temperature for 4 h. The reaction mixture was diluted with
water (15 mL) and ethyl acetate (15 mL). The organic layer was sep-
arated, washed with water (10 mL), brine (10 mL), dried and evap-
orated under reduced pressure. The residue was taken up in
DMSO (2 mL) and purified by MDAP. The appropriate fractions were
combined and the solvent was evaporated under reduced pressure
to give 10a (20 mg, 25%); 1H NMR (400 MHz, DMSO-d6) d = 11.06
(br s, 1H), 7.51 (s, 1H), 7.12 (s, 1H), 3.94 (s, 3H), 3.34 (s, 3H); LCMS
(System A) RT = 0.60 min, 95%, ES+ve m/z 265 (M+H+MeCN)+, ESÀve
m/z 222 (MÀH)À, and 12a (12 mg, 14%): 1H NMR (400 MHz, DMSO-
d6) d = 7.80 (s, 1H), 7.19 (s, 1H), 3.95 (s, 3H), 3.39 (s, 3H), 3.34 (s, 3H);
1H NMR (400 MHz, MeOD-d4) d = 7.74 (s, 1H), 7.06 (s, 1H), 3.99 (s,
3H), 3.45 (s, 3H), 3.41 (s, 3H); LCMS (System A) RT = 0.69 min,
99%, ES+ve m/z 238 (M+H)+, 279 (M+H+MeCN)+.
Were prepared from a mixture of 10b and 11b (9:1) (60 mg,
0.22 mmol) according to the procedure described for the prepara-
tion of 13a. Obtained 13b (18 mg, 18%) as a white solid: mp = 301–
304 °C; 1H NMR (400 MHz, DMSO-d6) d = 10.59 (br s, 1H), 9.61 (br
s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.72 (dd, J = 8.0, 1.5 Hz, 1H), 7.52 (br
s, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.11 (br s, 1H), 6.79 (s, 1H), 6.66 (s,
1H), 4.31 (s, 2H), 3.36 (s, 3H); 1H NMR (400 MHz, MeOD-d4)
d = 7.79–7.66 (m, 2H), 7.27 (t, J = 8.0 Hz, 1H), 7.13 (s, 1H), 6.59 (s,
1H), 4.47 (s, 2H), 3.52 (s, 3H); HRMS (ESI) calcd for C16H15Cl2N4O5S
(M+H)+ 445.0140; found = 445.0152; LCMS (System A)
RT = 0.80 min, 95%, ES+ve m/z 445, 447, 449 (M+H)+; and 14b
(4 mg, 4%) as
a
white solid: 1H NMR (400 MHz, MeOD-d4)
d = 7.86–7.60 (m, 2H), 7.27 (t, J = 8.0 Hz, 1H), 7.04 (s, 1H), 6.59 (s,
1H), 4.48 (s, 2H), 3.51 (s, 3H); LCMS (System A) RT = 0.79 min,
95%, ES+ve m/z 445, 447, 449 (M+H)+.
5.8. 1-Isopropyl-5-methoxy-6-nitro-1H-benzo[d]imidazol-
2(3H)-one (10c)
5.4. 2,3-Dichloro-N-(6-methoxy-1-methyl-2-oxo-2,3-dihydro-
1H-benzo[d]imidazol-5-yl)benzenesulfonamide (13a)
From 9 according to the procedure described for the prepara-
tion of 10a: 1H NMR (400 MHz, DMSO-d6) d = 11.10 (br s, 1H),
7.50 (s, 1H), 7.13 (s, 1H), 4.62 (spt, J = 7.0 Hz, 1H), 3.95 (s, 3H),
1.47 (d, J = 7.0 Hz, 6H); MS ES+ve m/z 252 (M+H)+, 293
(M+H+MeCN)+.
A solution of 10a (20 mg, 0.09 mmol) in ethanol (2 mL) and
ethyl acetate (2 mL) was hydrogenated over 10% palladium on car-
bon (5 mg). The catalyst was collected by filtration through celite,
and washed with ethanol. The filtrate and washings were evapo-
rated under reduced pressure and the residue was dissolved in
anhydrous pyridine (0.6 mL). 2,3-Dichlorobenzenesulfonyl chlo-
ride (20 mg, 0.09 mmol) was added and stirred for 1 h. The reaction
mixture was evaporated under reduced pressure and the residue
was purified by MDAP. The solvent was evaporated under reduced
pressure to afford 13a (9 mg, 25%) as a white solid: 1H NMR
(400 MHz, DMSO-d6) d = 10.57 (s, 1H), 9.61 (br s, 1H), 7.86 (dd,
J = 8.0, 1.5 Hz, 1H), 7.69 (dd, J = 8.0, 1.5 Hz, 1H), 7.39 (t, J = 8.0 Hz,
1H), 6.80 (s, 1H), 6.72 (s, 1H), 3.39 (s, 3H), 3.21 (s, 3H); LCMS (Sys-
tem A) RT = 0.89 min, 95%, ES+ve m/z 402, 404 (M+H)+, 443, 445,
447 (M+H+MeCN)+.
5.9. 2,3-Dichloro-N-(1-isopropyl-6-methoxy-2-oxo-2,3-dihydro-
1H-benzo[d]imidazol-5-yl)benzenesulfonamide (13c)
From 10c according to the procedure described for the prepara-
tion of 13a afforded 13c (13 mg, 70%) as a white solid: 1H NMR
(DMSO-d6, 600 MHz) d = 10.54 (s, 1H), 9.63 (br s, 1H), 7.92–7.82
(m, 1H), 7.73 (dd, J = 8.1, 1.5 Hz, 1H), 7.41 (t, J = 8.1 Hz, 1H), 6.78
(s, 1H), 6.76 (s, 1H), 4.48 (spt, J=6.9 Hz, 1H), 3.41 (s, 3H), 1.39 (d,
J=6.8 Hz, 6H); LCMS (System A) RT = 1.00 min, 100%, ES+ve m/z
430, 432, 434 (M+H)+.
5.10. 2,3-Dichloro-N-(6-methoxy-1-(oxetan-3-yl)-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol-5-yl)benzenesulfonamide (13d)
5.5. 2,3-Dichloro-N-(6-methoxy-1,3-dimethyl-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol-5-yl)benzenesulfonamide (15a)
as a white solid: 1H NMR (400 MHz, DMSO-d6) d = 10.70 (br s,
1H), 9.69 (br s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.73 (dd, J = 8.0, 1.5 Hz,
1H), 7.41 (t, J = 8.0 Hz, 1H), 6.96 (s, 1H), 6.84 (s, 1H), 5.46–5.33 (m,
Was prepared from 12a (30 mg, 0.13 mmol) according to the
procedure described for the preparation of 13a. Obtained 15a as
Please cite this article in press as: Miah, A. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.021

A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
1H), 5.05 (t, J = 6.5 Hz, 2H), 4.86 (m, 2H), 3.44 (s, 3H); LCMS (System
B) RT = 0.93 min, 100%, ES+ve m/z 444, 446, 448 (M+H)+.
of 0–100% ethyl acetate–cyclohexane over 60 min. The appropriate
fractions were combined and evaporated under reduced pressure
to afford 21 (0.53 g, 23%) as a brown solid: 1H NMR (400 MHz,
DMSO-d6) d = 11.17 (br s, 1H), 7.54 (s, 1H), 7.06 (s, 1H), 6.27–
5.62 (m, 1H), 5.41–4.85 (m, 2H), 4.49 (m, 2H), 3.91 (s, 3H); MS
ES+ve m/z 250 (M+H)+, 291 (M+H+MeCN)+.
5.11. N-Allyl-5-methoxy-2-nitroaniline (17)
Allylamine (2.19 mL, 29.2 mmol) was added to 2-fluoro-4-
methoxy-1-nitrobenzene (16) (2.5 g, 15 mmol) and the reaction
was stirred at ambient temperature for 1.5 h. The reaction mixture
was concentrated under reduced pressure to remove excess allyla-
mine. The residue was dissolved in ethyl acetate (300 mL) and
washed with HCl (1 M, 150 mL), followed by saturated aqueous
solution of sodium bicarbonate (100 mL), water (50 mL), dried,
and evaporated under reduced pressure to afford 17 (2.94 g, 97%)
as a yellow solid: 1H NMR (400 MHz, DMSO-d6) d = 8.47 (t,
J = 5.0 Hz, 1H), 8.04 (d, J = 9.0 Hz, 1H), 6.34–6.28 (m, 2H), 5.99–
5.88 (m, 1H), 5.27 (dd, J = 17.0, 1.5 Hz, 1H), 5.20 (dd, J = 10.3,
1.5 Hz, 1H), 4.08–4.03 (m, 2H), 3.83 (s, 3H); MS ES+ve m/z 209
(M+H)+, 250 (M+H+MeCN)+.
5.15. 1-Allyl-6-methoxy-3-methyl-5-nitro-1H-
benzo[d]imidazol-2(3H)-one (22)
Sodium hydride (18 mg of a 60% w/w dispersion in mineral oil,
0.46 mmol) was added to a stirring solution of 21 (105 mg,
0.42 mmol) in anhydrous DMF (1 mL) at 0 °C. The mixture was stir-
red for 20 min, then methyl iodide (0.03 mL, 0.46 mmol) was
added and the reaction mixture was allowed to warm to ambient
temperature over 2 h. The reaction mixture was quenched with
water (20 mL) and the product was extracted with ethyl acetate
(3 Â 20 mL). The combined organic extracts were washed with
water (20 mL), brine (2 Â 20 mL), dried and evaporated under
reduced pressure to afford 22 (110 mg, 99%) as an orange solid:
1H NMR (400 MHz, DMSO-d6) d = 7.83 (s, 1H), 7.13 (s, 1H), 5.97–
5.86 (m, 1H), 5.18 (dd, J = 10.5, 1.5 Hz, 1H), 5.13 (dd, J = 18.5,
1.5 Hz, 1H), 4.54 (m, 2H), 3.92 (s, 3H), 3.36 (s, 3H); MS ES+ve m/z
264 (M+H)+, 305 (M+H+MeCN)+.
5.12. N1-allyl-5-methoxybenzene-1,2-diamine (18)
Iron powder (3.5 g, 325 mesh, 62 mmol) was added to a solu-
tion of 17 (2.6 g, 12 mmol) in ethanol (70 mL) and water (35 mL).
Ammonium chloride (1.8 g, 33 mmol) was added in one portion,
and the reaction mixture was refluxed for 24 h. Ethanol was
removed under reduced pressure, and the residue was partitioned
between ethyl acetate (500 mL) and saturated aqueous sodium
bicarbonate solution (500 mL). The organic layer was separated
and the aqueous layer was extracted with more ethyl acetate
(2 Â 300 mL). The combined organic extracts were washed with
brine (2 Â 200 mL), dried, and concentrated under reduced pres-
sure to afford 18 (1.84 g, 83%) as a dark purple oil: LCMS (System
A) RT = 0.50 min, 80%, ES+ve m/z 179 (M+H)+. A portion (40 mg)
of the product was purified by MDAP to give 18 (22 mg) as an
orange oil: 1H NMR (400 MHz, CDCl3) d = 8.16 (br s, 1H), 6.72 (d,
J = 7.0 Hz, 1H), 6.32–6.16 (m, 2H), 6.05–5.92 (m, 1H), 5.47–5.24
(m, 5H), 5.19 (d, J = 10.0 Hz, 1H), 3.76 (s, 3H); LCMS (System A)
RT = 0.50 min, 91%, ES+ve m/z 179 (M+H)+.
5.16. 5-Methoxy-1-methyl-6-nitro-1H-benzo[d]imidazol-2(3H)-
one (11a)
Compound 22 (30 mg, 0.11 mmol), dichloro(2,6,10-dodecatri-
ene-1,12-diyl)ruthenium(IV) (24) (2.2 mg, 6.8 lmol) and potas-
sium periodate (52 mg, 0.23 mmol) were suspended in water
(1 mL) and stirred in a sealed vessel at 100 °C for 72 h. Ethyl ace-
tate (10 mL) was added and the organic layer was separated,
washed with brine (10 mL), dried and evaporated under reduced
pressure. The residue was purified by MDAP to give 11a (10 mg,
39%) as a yellow solid: 1H NMR (400 MHz, MeOD-d4) d = 7.70 (s,
1H), 6.93 (s, 1H), 3.94 (s, 3H), 3.38 (s, 3H), the NH was not observed
due to exchange; MS ES+ve m/z 224 (M+H)+, 265 (M+H+MeCN)+.
5.13. 1-Allyl-6-methoxy-1H-benzo[d]imidazol-2(3H)-one (19)
5.17. 2,3-Dichloro-N-(6-methoxy-3-methyl-2-oxo-2,3-dihydro-
1H-benzo[d]imidazol-5-yl)benzenesulfonamide (14a)
CDI (110 mg, 0.68 mmol) was added to a stirring solution of 18
(110 mg, 0.62 mmol) in THF (2 mL). The reaction mixture was
heated to 50 °C under nitrogen for 2 h, concentrated, and purified
by MDAP to give 19 (57 mg, 45%) as a pale purple solid: 1H NMR
(400 MHz, DMSO-d6) d = 10.63 (br s, 1H), 6.87 (d, J = 8.5 Hz, 1H),
6.67 (d, J = 2.5 Hz, 1H), 6.56 (dd, J = 8.5, 2.5 Hz, 1H), 5.92–5.81
(m, 1H), 5.15 (dd, J = 10.0, 1.5 Hz, 1H), 5.09 (dd, J = 17.0, 1.5 Hz,
1H), 4.38 (m, 2H), 3.72 (s, 3H); MS ES+ve m/z 205 (M+H)+, 246
(M+H+MeCN)+.
From 11a according to the procedure described for the prepara-
tion of 13a afforded 14a (4 mg, 22%) as a white solid: 1H NMR
(400 MHz, CDCl3) d = 9.03 (br, 1H), 7.82 (dd, J = 8.0, 1.5 Hz, 1H),
7.61 (dd, J = 8.0, 1.5 Hz, 1H), 7.47 (br s, 1H), 7.20 (t, J = 8.0 Hz,
1H), 7.18 (s, 1H), 6.52 (s, 1H), 3.61 (s, 3H), 3.38 (s, 3H); LCMS (Sys-
tem A) RT = 0.88 min, 96%, ES+ve m/z 443, 445, 447 (M+H+MeCN)+.
5.18. 2,3-Dichloro-N-(6-methoxy-2-oxo-1-propyl-2,3-dihydro-
1H-benzo[d]imidazol-5-yl)benzenesulfonamide (13e)
5.14. 1-Allyl-6-methoxy-5-nitro-1H-benzo[d]imidazol-2(3H)-
one (21)
From 21 according to the procedure described for the prepara-
tion of 13a afforded 13e (5 mg, 7%) as a beige coloured solid: 1H
NMR (400 MHz, DMSO-d6) d = 10.55 (br s, 1H), 9.60 (br, 1H), 7.87
(dd, J = 8.0, 1.5 Hz, 1H), 7.72 (dd, J = 8.0, 1.5 Hz, 1H), 7.40 (t,
J = 8.0 Hz, 1H), 6.79 (s, 1H), 6.74 (s, 1H), 3.67 (t, J = 7.0 Hz, 2H),
3.40 (s, 3H), 1.60 (sxt, J = 7.0 Hz, 2H), 0.83 (t, J = 7.0 Hz, 3H); LCMS
(System A) RT = 1.00 min, 100%, ES+ve m/z 430, 432, 434 (M+H)+;
471, 473, 475 (M+H+MeCN)+.
Nitric acid (4 M, 66.5 mL) was cooled to 0 °C and added slowly
to 19 (1.9 g, 9.3 mmol). The reaction mixture was stirred for 1 h at
0 °C and then allowed to warm to ambient temperature and stirred
for 3 h. Some of the starting material dropped out of solution and
formed a gum. Glacial acetic acid (5 mL) was added and the reac-
tion was stirred at ambient temperature for 2 h. The reaction mix-
ture was poured onto 30 mL of iced-water and was neutralised
with saturated sodium bicarbonate solution. The product was
extracted with ethyl acetate (2 Â 300 mL), and the combined
organic extracts were washed with brine (100 mL), dried and
evaporated under reduced pressure. The residue was purified by
chromatography on silica (100 g cartridge) eluting with a gradient
5.19. N1-(5-Methoxy-2-nitrophenyl)-N2,N2-dimethylethane-
1,2-diamine (25f)
N1,N1-Dimethylaminoethylamine (0.96 mL, 8.8 mmol) was
added to a solution of 16 (1.0 g, 5.8 mmol), in anhydrous DMF
Please cite this article in press as: Miah, A. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.021

10
A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
(2 mL). DIPEA (2.0 mL, 12 mmol) was added and the reaction mix-
ture was stirred under nitrogen at ambient temperature for 3 h.
The reaction mixture was diluted with ethyl acetate (50 mL) and
the organic layer was washed with water (2 Â 50 mL), brine
(50 mL), dried and evaporated under reduced pressure to afford
25f (1.45 g, 93%) as a yellow solid: 1H NMR (400 MHz, DMSO-d6)
d = 8.50 (br s, 1H), 8.02 (d, J = 9.5 Hz, 1H), 6.35–6.28 (m, 2H), 3.87
(s, 3H), 3.41–3.36 (m, 2H), 2.55 (t, J = 6.0 Hz, 2H), 2.22 (s, 6H);
MS ES+ve m/z 240 (M+H)+.
4H), 3.66 (s, 3H), 3.47–3.40 (m, 1H), 0.93 (s, 18H), 0.09 (s, 12H);
MS ES+ve m/z 441 (M+H)+.
5.25. tert-Butyl 3-((2-amino-5-methoxyphenyl)amino)
azetidine-1-carboxylate (N-BOC-26h)
as a dark solid (723 mg, 97%); 1H NMR (400 MHz, DMSO-d6)
d = 9.13 (br s, 2H), 7.05 (d, J = 8.5 Hz, 1H), 6.30 (dd, J = 8.5, 2.5 Hz,
1H), 6.20 (br s, 1H), 6.02 (d, J = 2.5 Hz, 1H), 4.30–4.12 (m, 3H),
3.74–3.64 (m, 2H), 3.71 (s, 3H), 1.39 (s, 9H); MS ES+ve m/z 294
(M+H)+.
5.20. 2-((5-Methoxy-2-nitrophenyl)amino)propane-1,3-diol
(25g)
5.26. 1-(2-(Dimethylamino)ethyl)-6-methoxy-1H-benzo[d]
imidazol-2(3H)-one (27f)
Was similarly prepared to give 25g (1.3 g, 83%) as a yellow
solid: 1H NMR (400 MHz, DMSO-d6) d = 8.57 (d, J = 8.0 Hz, 1H),
8.02 (d, J = 9.5 Hz, 1H), 6.46 (d, J = 2.5 Hz, 1H), 6.30 (dd, J = 9.5,
2.5 Hz, 1H), 4.95 (t, J = 5.5 Hz, 2H), 3.86 (s, 3H), 3.77–3.68 (m,
1H), 3.67–3.48 (m, 4H); MS ES+ve m/z 243 (M+H)+.
A mixture of 26f (1.15 g, 5.49 mmol) and CDI (0.89 g, 5.49 mmol)
in anhydrous THF (20 mL) was heated to 50 °C for 2 h. The mixture
was concentrated under reduced pressure, and the residue was
taken up in ethyl acetate (40 mL). The organic layer was washed
with sodium bicarbonate solution (40 mL), brine (40 mL), dried,
and evaporated under reduced pressure to afford 27f (1.26 g, 83%)
as a dark purple solid: 1H NMR (400 MHz, DMSO-d6) d = 10.54 (br
s, 1H), 6.85 (d, J = 8.0 Hz, 1H), 6.76 (d, J = 2.5 Hz, 1H), 6.55 (dd,
J = 8.0, 2.5 Hz, 1H), 3.84 (t, J = 6.5 Hz, 2H), 3.74 (s, 3H), 2.53–2.47
(m, 2H), 2.18 (s, 6H); MS ES+ve m/z 236 (M+H)+.
5.21. N-(5-Methoxy-2-nitrophenyl)-2,2,3,3,9,9,10,10-
octamethyl-4,8-dioxa-3,9-disilaundecan-6-amine (25g-bis-
TBMS ether)
TBDMS-Cl (2.3 g, 15 mmol) and imidazole (1.0 g, 15 mmol) was
added to a solution of 25g (1.3 g, 6.8 mmol) in anhydrous DMF
(15 mL) and the reaction mixture was stirred under nitrogen at
ambient temperature for 3 h. The reaction mixture was partitioned
between water (50 mL) and ethyl acetate (3 Â 50 mL). The organic
extracts were washed with brine (50 mL), dried, and evaporated
under reduced pressure to afford 25g-bis-TBMS ether (3.38 g,
95%) as a yellow oil: 1H NMR (400 MHz, DMSO-d6) d = 8.60 (d,
J = 8.5 Hz, 1H), 8.02 (d, J = 9.5 Hz, 1H), 6.44 (d, J = 2.5 Hz, 1H),
6.30 (dd, J = 9.5, 2.5 Hz, 1H), 4.06–3.93 (m, 1H), 3.85 (s, 3H),
3.83–3.77 (m, 2H), 3.77–3.62 (m, 2H), 0.87 (s, 18H), 0.04 (s, 6H),
0.03 (s, 6H); MS ES+ve m/z 471 (M+H)+.
5.27. 6-Methoxy-1-(2,2,3,3,9,9,10,10-octamethyl-4,8-dioxa-3,9-
disilaundecan-6-yl)-1H-benzo[d]imidazol-2(3H)-one (27g-bis-
TBMS ether)
(2.04 g, 69%) as a dark brown solid: 1H NMR (400 MHz, DMSO-
d6) d = 10.56 (br s, 1H), 6.81 (d, J = 8.5 Hz, 1H), 6.79 (d, J = 2.5 Hz,
1H), 6.53 (dd, J = 8.5, 2.5 Hz, 1H), 4.40–4.31 (m, 1H), 4.06 (dd,
J = 10.5, 8 Hz, 2H), 3.97 (dd, J = 10.5, 6 Hz, 2H), 3.71 (s, 3H), 0.75
(s, 18H), -0.04 (s, 6H), -0.11 (s, 6H); MS ES+ve m/z 467 (M+H)+.
5.22. tert-Butyl 3-((5-methoxy-2-nitrophenyl)amino)azetidine-
1-carboxylate (N-BOC-25h)
5.28. tert-Butyl 3-(5-methoxy-2-oxo-1H-benzo[d]imidazol-
3(2H)-yl)azetidine-1-carboxylate (N-BOC-27h)
Was prepared according to the procedure described for the
preparation of 25f to give 25h (819 mg, 84%) as a yellow gum:
1H NMR (400 MHz, CDCl3) d = 8.37 (d, J = 4.5 Hz, 1H), 8.16 (d,
J = 9.5 Hz, 1H), 6.29 (dd, J = 9.5, 2.5 Hz, 1H), 5.83 (d, J = 2.5 Hz,
1H), 4.38–4.24 (m, 3H), 3.89–3.82 (m, 2H), 3.83 (s, 3H), 1.43 (s,
9H); MS ES+ve m/z 647 (2 M+H)+.
(620 mg, 79%) as a beige coloured solid: 1H NMR (400 MHz,
CDCl3) d = 8.61 (br s, 1H), 6.99 (d, J = 8.5 Hz, 1H), 6.98 (d,
J = 2.5 Hz, 1H), 6.69 (dd, J = 8.5, 2.5 Hz, 1H), 5.34–5.24 (m, 1H),
4.52–4.43 (m, 2H), 4.44–4.35 (m, 2H), 3.83 (s, 3H), 1.51 (s, 9H);
MS ES+ve m/z 320 (M+H)+, 639 (2 M+H)+.
5.29. 1-(2-(Dimethylamino)ethyl)-6-methoxy-5-nitro-1H-benzo
[d]imidazol-2(3H)-one (10f)
5.23. N1-(2-(Dimethylamino)ethyl)-5-methoxybenzene-1,2-
diamine (26f)
A suspension of 27f (1.26 g, 4.55 mmol) in nitric acid (4 M,
60 mL) was stirred at ambient temperature for 22 h. The reaction
mixture was poured over ice (50 mL), neutralised with solid
sodium bicarbonate, and extracted with ethyl acetate
(2 Â 150 mL). The combined organic extracts were then washed
with brine (100 mL), dried, and evaporated under reduced pressure
to afford 10f (750 mg, 53%) as a yellow solid: 1H NMR (400 MHz,
DMSO-d6) d = 11.05 (s, 1H), 7.51 (s, 1H), 7.15 (s, 1H), 4.00–3.88
(m, 2H), 3.94 (s, 3H), 2.55 (t, J = 6.5 Hz, 2H), 2.19 (s, 6H); MS ES+ve
m/z 281(M+H)+.
A solution of 25f (1.45 g, 6.06 mmol) in ethanol (100 mL) was
hydrogenated over 10% palladium on carbon (0.65 g) for 18 h.
The catalyst was removed by filtration through a pad of celite,
washed with ethanol (150 mL) and concentrated under reduced
pressure to give 26f (1.15 g, 91%) as a dark purple oil: 1H NMR
(400 MHz, DMSO-d6) d = 6.51 (d, J = 8.0 Hz, 1H), 6.08 (d,
J = 2.5 Hz, 1H), 6.03 (dd, J = 8.0, 2.5 Hz, 1H), 4.46 (m, 1H), 3.97 (br
s, 2H), 3.64 (s, 3H), 3.11–3.04 (m, 2H), 2.54–2.45 (m, 2H), 2.21 (s,
6H).
5.24. 5-Methoxy-N1-(2,2,3,3,9,9,10,10-octamethyl-4,8-dioxa-
3,9-disilaundecan-6-yl)benzene-1,2-diamine (26g-bis-TBMS
ether)
5.30. 1-(1,3-Dihydroxypropan-2-yl)-6-methoxy-5-nitro-1H-
benzo[d]imidazol-2(3H)-one (10g)
(40 mg, 33%) as a yellow solid: 1H NMR (400 MHz, DMSO-d6)
d = 11.03 (br s, 1H), 7.49 (s, 1H), 7.14 (s, 1H), 4.87 (t, J = 5.5 Hz,
2H), 4.42–4.34 (m, 1H), 3.95–3.88 (m, 2H), 3.91 (s, 3H), 3.81–
3.74 (m, 2H); MS ES+ve m/z 284 (M+H)+.
(2.53 g, 70%) as a dark purple oil: 1H NMR (400 MHz, DMSO-d6)
d = 6.58 (d, J = 8.0 Hz, 1H), 6.16 (d, J = 2.5 Hz, 1H), 6.08 (dd, J = 8.5,
2.5 Hz, 1H), 4.25 (d, J = 8.5 Hz, 1H), 3.92 (s, 2H), 3.77–3.67 (m,
Please cite this article in press as: Miah, A. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.021

A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
11
5.31. tert-Butyl 3-(6-methoxy-5-nitro-2-oxo-2,3-dihydro-1H-
benzo[d]imidazol-1-yl)azetidine-1-carboxylate (N-BOC-10h)
459.0661; found = 459.0654; LCMS (System A) RT = 0.69 min,
90%, ES+ve m/z 459, 461, 463 (M+H)+.
4 M Nitric acid (30 mL) was added to N-BOC-27h (620 mg,
1.94 mmol) and the suspension was shaken at ambient temperature
until the solid dissolved. After a couple of minutes a fine powder
started to precipitate, and the mixture was stirred for 4 h. The prod-
uct was very soluble in water and was thus converted to the N-BOC
protected analogue by treating the solution with solid sodium bicar-
bonate to pH 8, and then adding THF (20 mL) and Boc2O (0.76 mL,
3.27 mmol). The mixture was stirred at ambient temperature for
48 h, and then extracted with ethyl acetate (3 Â 50 mL). The com-
bined organic extracts were washed with HCl (2 M,40 mL), sodium
bicarbonate solution (40 mL), brine (40 mL), dried, and evaporated
under reduced pressure. The residue was purified by column chro-
matography on silica (100 g cartridge) eluting with 0–50% ethyl ace-
tate–cyclohexane for 30 min. The solvent was evaporated under
reduced pressure to afford N-BOC-10h (368 mg, 52%) as a dark yel-
low solid: 1H NMR (400 MHz, CDCl3) d = 9.01 (s, 1H), 7.77 (s, 1H),
7.14 (s, 1H), 5.28 (m, 1H), 4.54–4.36 (m, 4H), 3.99 (s, 3H), 1.51 (s,
9H); MS ES+ve m/z 365 (M+H)+.
5.36. 2,3-Dichloro-N-(1-(1,3-dihydroxypropan-2-yl)-6-methoxy-
2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-yl)
benzenesulfonamide (13g)
(21 mg, 48%) as a white solid; 1H NMR (400 MHz, DMSO-d6)
d = 10.55 (s, 1H), 9.59 (br s, 1H), 7.88 (dd, J = 8.0, 1.5 Hz, 1H), 7.73
(dd, J = 8.0, 1.5 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 6.76 (s, 2H), 4.77
(t, J = 5.5 Hz, 2H), 4.26–4.17 (m, 1H), 3.86–3.77 (m, 2H), 3.76–
3.67 (m, 2H), 3.38 (s, 3H). HRMS (ESI) calcd for C17H18Cl2N3O6S
(M+H)+ 462.0293; found 462.0295; LCMS (System A)
RT = 0.77 min, 100%, ES+ve m/z 462, 464, 466 (M+H)+.
5.37. tert-Butyl 3-(5-(2,3-dichlorophenylsulfonamido)-6-
methoxy-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-
yl)azetidine-1-carboxylate (N-BOC-13h)
From N-BOC-28h (321 mg, 0.96 mmol) and 2,3-dic-
hlorobenzenesulfonyl chloride (286 mg, 1.17 mmol) according to
the procedure described for the preparation of 13a. (300 mg,
57%) as a colourless oil: 1H NMR (400 MHz, CDCl3) d = 9.99 (br,
1H), 7.77 (d, J = 8.0 Hz, 1H), 7.58 (s, 1H), 7.56 (d, J = 8.0 Hz, 1H),
7.17 (t, J = 8.0 Hz, 1H), 6.77 (s, 1H), 5.25 (s, 1H), 5.23–5.16 (m,
1H), 4.39–4.28 (m, 4H), 3.60 (s, 3H), 1.43 (s, 9H); MS ES+ve m/z
543, 545, 547 (M+H)+.
5.32. 5-Amino-1-(2-(dimethylamino)ethyl)-6-methoxy-1H-
benzo[d]imidazol-2(3H)-one (28f)
A solution of 10f (0.75 g, 2.4 mmol) in a mixture of ethanol
(50 mL) and ethyl acetate (50 mL) was hydrogenated over 10% pal-
ladium on carbon (0.77 g) for 18 h. The catalyst was collected by
filtration through a pad of celite, washed with a mixture of ethanol
and ethyl acetate (1:1; 100 mL), and then the filtrate was concen-
trated under reduced pressure to afford 28f (0.63 g, 94%) as a
brown oil: 1H NMR (400 MHz, DMSO-d6) d = 10.21 (s, 1H), 6.70
(s, 1H), 6.38 (s, 1H), 4.40 (br s, 2H), 3.77 (t, J = 6.5 Hz, 2H), 3.75
(s, 3H), 2.48 (t, J = 6.5 Hz, 2H), 2.18 (s, 6H).
5.38. N-(1-(Azetidin-3-yl)-6-methoxy-2-oxo-2,3-dihydro-1H-
benzo[d]imidazol-5-yl)-2,3-dichlorobenzenesulfonamide
hydrochloride (13h)
4 M HCl solution in dioxane (4 mL, 16 mmol) was added to a
solution of N-BOC-13h (300 mg, 0.552 mmol) in chloroform
(1 mL), and the mixture was stirred at ambient temperature for
1.5 h. The white precipitate was collected by filtration to afford
13h (125 mg, 51%) as an off-white solid: 1H NMR (400 MHz,
CD3OD) d = 7.72 (br t, J = 8 Hz, 2H), 7.26 (t, J = 8.0 Hz, 1H), 7.14 (s,
1H), 6.75 (s, 1H), 5.31–5.22 (m, 1H), 4.85–4.79 (m, 2H), 4.45 (br
t, J = 9 Hz, 2H), 3.65 (s, 3H); LCMS (System A) RT = 0.68 min, 91%,
ES+ve m/z 443, 445, 447 (M+H)+.
5.33. 5-Amino-1-(1,3-dihydroxypropan-2-yl)-6-methoxy-1H-
benzo[d]imidazol-2(3H)-one (28g)
From 10g (40 mg, 0.14 mmol) obtained 28g (28 mg, 78%) as a
dark purple oil: 1H NMR (400 MHz, DMSO-d6) d = 10.24 (s, 1H),
6.74 (s, 1H), 6.37 (s, 1H), 4.80 (br t, J = 5.5 Hz, 2H), 4.34 (br s,
2H), 4.19 (quint, J = 6.5 Hz, 1H), 3.87–3.74 (m, 4H), 3.73 (s, 3H).
5.39. N-(1-(1-Acetylazetidin-3-yl)-6-methoxy-2-oxo-2,3-
dihydro-1H-benzo[d]imidazol-5-yl)-2,3-
dichlorobenzenesulfonamide (13i)
5.34. tert-Butyl 3-(5-amino-6-methoxy-2-oxo-2,3-dihydro-1H-
benzo[d]imidazol-1-yl)azetidine-1-carboxylate (N-BOC-28h)
From N-BOC-10h (367 mg, 1.00 mmol) obtained N-BOC-28h
(321 mg, 95%) as a light brown solid: 1H NMR (400 MHz, DMSO-
d6) d = 10.94 (s, 1H), 10.0–9.30 (br, 2H), 7.10 (s, 2H), 5.23–5.14
(m, 1H), 4.49–4.33 (m, 2H), 4.23 (t, J = 9 Hz, 2H), 3.87 (s, 3H),
1.43 (s, 9H); MS ES+ve m/z 335 (M+H)+, 376 (M+H+MeCN)+.
Acetic anhydride (50 lL, 0.53 mmol) was added to a solution of
13h (19 mg, 0.04 mmol) in pyridine (0.5 mL). The mixture was stir-
red at ambient temperature for 3 h; LCMS analysis showed that di-
acetylation had occurred. MeOH (7.5 mL) and K2CO3 (60 mg,
0.43 mmol) were added to the mixture, and after stirring for 24 h,
2 M aq HCl solution was added to pH 1. The mixture was extracted
using ethyl acetate (20 mL) and the organic layer was washed with
brine (20 mL), dried and concentrated under reduced pressure. The
solid was dissolved in DMSO (1 mL), and purified by MDAP. Appro-
priate fractions were evaporated under reduced pressure to afford
13i (5 mg, 25%) as a white solid: 1H NMR (400 MHz, CD3OD)
d = 7.73 (m, 2H), 7.28 (t, J = 8.0 Hz, 1H), 7.14 (s, 1H), 6.71 (s, 1H),
5.22–5.14 (m, 1H), 4.75–4.71 (m, 1H), 4.57 (t, J = 9.3 Hz, 1H), 4.53–
4.47 (m, 1H), 4.34 (t, J = 9.5 Hz, 1H), 3.54 (s, 3H), 1.93 (s, 3H); LCMS
(System A) RT = 0.82 min, 98%, ES+ve m/z 485, 487, 489 (M+H)+.
5.35. 2,3-Dichloro-N-(1-(2-(dimethylamino)ethyl)-6-methoxy-
2-oxo-2,3-dihydro-1H-benzo[d]imidazol-5-
yl)benzenesulfonamide (13f)
From 28f (100 mg, 0.40 mmol) and 2,3-dichlorobenzenesulfo-
nyl chloride (108 mg, 0.44 mmol) according to the procedure
described for the preparation of 13a afforded 13f (67 mg, 36%) as
a beige coloured solid: 1H NMR (400 MHz, DMSO-d6) d = 10.56 (s,
1H), 9.60 (br, 1H), 7.88 (dd, J = 8.0, 1.5 Hz, 1H), 7.72 (dd, J = 8.0,
1.5 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 6.78 (s, 1H), 6.74 (s, 1H), 3.80
(t, J = 6.5 Hz, 2H), 3.40 (s, 3H), 2.46 (t, J = 6.5 Hz, 2H), 2.15 (s, 6H);
13C NMR (101 MHz, DMSO-d6) d = 154.6, 150.0, 140.7, 134.2,
134.0, 130.0, 129.9, 129.6, 128.0, 121.3, 117.3, 109.5, 93.8, 56.7,
56.0, 45.4, 38.3. HRMS (ESI) calcd for C18H21Cl2N4O4S (M+H)+
5.40. 2,6-Dibromopyridin-3-ol (30)
An ice-cold solution of bromine (16.2 mL, 315 mmol) in sodium
hydroxide (2.5 M, 320 mL, 800 mmol) was added dropwise to a
Please cite this article in press as: Miah, A. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.021

12
A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
stirred solution of pyridin-3-ol (10.0 g, 105 mmol) in sodium
hydroxide (2.5 M, 110 mL, 275 mmol). The solution was stirred at
0 °C for 1 h and then at room temp for 4 h. A small amount of pre-
cipitate that formed was removed by filtration. The filtrate was
cooled, and concentrated hydrochloric acid was added until the
pH reached 1. The solid was collected by filtration, washed with
water, dried, and recrystallised from carbon tetrachloride to give
30 (12.1 g, 46%) as a beige coloured solid: 1H NMR (400 MHz,
DMSO-d6) d = 11.14 (br s, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.26 (d,
J = 8.0 Hz, 1H); MS ESÀve m/z 250, 252, 254 (MÀH)À.
pressure to give 34 (210 mg, 66%) as a yellow solid: 1H NMR
(400 MHz, DMSO-d6) d = 9.86 (s, 1H), 9.22 (s, 1H), 7.91 (s, 1H),
3.89 (s, 3H), 1.45 (s, 9H), 1.40 (s, 9H); MS ES+ve m/z 385 (M+H)+.
5.45. Di-tert-butyl (3-amino-5-methoxypyridine-2,6-
diyl)dicarbamate (35)
Iron powder (290 mg, 5.2 mmol) was added to a stirring solu-
tion of 34 (200 mg, 0.52 mmol) in glacial acetic acid (10 mL). The
reaction mixture was stirred at ambient temperature for 2 h, and
then concentrated under reduced pressure. The residue was parti-
tioned between sodium bicarbonate solution (10 mL) and ethyl
acetate (20 mL), the aqueous was extracted with further portion
of ethyl acetate (20 mL), and the organic extracts were washed
with brine (20 mL), dried, and concentrated under reduced pres-
sure to give 35 (170 mg, 78%) as a yellow oil: 1H NMR (400 MHz,
DMSO-d6) d = 8.58 (s, 1H), 8.23 (s, 1H), 6.81 (s, 1H), 4.81 (s, 2H),
3.70 (s, 3H), 1.43 (s, 9H), 1.39 (s, 9H); MS ES+ve m/z 355 (M+H)+.
5.41. 2,6-Dibromo-3-methoxypyridine (31)
A
suspension of 2,6-dibromopyridin-3-ol (12 g, 47 mmol),
potassium carbonate (6.0 g, 43 mmol), methyl iodide (10.1 mL,
162 mmol) and DMSO (20 mL) was refluxed for 2 h. The reaction
mixture was poured into water (60 mL) and was warmed gently
at 50 °C with stirring for 30 min. The reaction mixture was cooled
to room temperature and the precipitated solid was collected by
filtration, and dried under vacuum. The residue was crystallised
from cyclohexane to give 31 (10.1 g, 80%) as a pale orange solid:
1H NMR (400 MHz, DMSO-d6) d = 7.66 (d, J = 8.0 Hz, 1H), 7.52 (d,
J = 8.0 Hz, 1H), 3.90 (s, 3H); MS ES+ve m/z 266, 268, 270 (M+H)+.
5.46. tert-Butyl (6-methoxy-2-oxo-2,3-dihydro-1H-imidazo[4,5-
b]pyridin-5-yl)carbamate (36)
A solution of 35 (150 mg, 0.42 mmol) in dry pyridine (1.5 mL)
was heated a microwave oven for 1 h at 100 °C. The mixture was
concentrated under reduced pressure to give 36 (110 mg, 74%) as
a brown solid: 1H NMR (400 MHz, DMSO-d6) d = 11.01 (br s, 1H),
10.71 (br s, 1H), 8.44 (s, 1H), 7.04 (s, 1H), 3.75 (s, 3H), 1.41 (s,
9H); MS ES+ve m/z 281 (M+H)+.
5.42. 2,6-Dibromo-3-methoxy-5-nitropyridine (32)
Fuming nitric acid (16 M, 5 mL, 80 mmol) was added to a solu-
tion of 31 (2.0 g, 7.5 mmol) in sulfuric acid (18 M, 8 mL, 144 mmol)
at 0 °C, and then the mixture was heated at 65 °C for 18 h. Addi-
tional concd nitric acid (2.5 mL, 40 mmol) was added and the reac-
tion mixture was stirred at 65 °C for a further 18 h. The reaction
mixture was cooled and cautiously neutralised with solid sodium
carbonate. The resulting mixture was extracted with ethyl acetate
(3 Â 50 mL), and the combined organic extracts were dried and
concentrated under reduced pressure. The residue was purified
by chromatography on a silica cartridge (100 g) eluting with a gra-
dient of 0–25% ethyl acetate–cyclohexane over 60 min. The appro-
priate fractions were combined and evaporated under reduced
pressure to give 32 (0.69 g, 28%) as a white solid: 1H NMR
(400 MHz, DMSO-d6) d = 8.24 (s, 1H), 4.00 (s, 3H); LCMS (System
A) RT = 1.05 min, 98%, no mass ions observed in either +ve or
Àve mode.
5.47. 5-Amino-6-methoxy-1H-imidazo[4,5-b]pyridin-2(3H)-one
(37)
A mixture of 36 (50 mg, 0.18 mmol) and TFA (0.25 mL) was stir-
red at ambient temperature for 30 min. The mixture was concen-
trated under reduced pressure, the residue was dissolved in
methanol and dichloromethane (1:1, 1 mL), and passed down an
aminopropyl ion-exchange cartridge (500 mg), eluting with meth-
anol and dichloromethane (1:1, 10 mL). The fractions were concen-
trated under reduced pressure to give 37 (30 mg, 93%) as a brown
solid: 1H NMR (400 MHz, DMSO-d6) d = 10.56 (br s, 1H), 10.12 (s,
1H), 6.82 (s, 1H), 5.12 (s, 2H), 3.73 (s, 3H); MS ES+ve m/z 181
(M+H)+.
5.43. 3-Methoxy-5-nitropyridine-2,6-diamine (33)
5.48. 2,3-Dichloro-N-(6-methoxy-2-oxo-2,3-dihydro-1H-
imidazo[4,5-b]pyridin-5-yl)benzenesulfonamide (38)
A solution of 32 (0.5 g, 1.6 mmol) in aqueous ammonia (15 M,
12 mL, 180 mmol) was heated at 90 °C for 1 h in a microwave oven.
The reaction mixture was cooled, the yellow solid was collected by
filtration, washed with a small amount of water, and dried under
vacuum to afford 33 (230 mg, 78%) as a yellow solid: 1H NMR
(400 MHz, DMSO-d6, 393 K) d = 7.48 (s, 1H), 7.35 (br, 2H), 6.81
(br, 2H), 3.80 (s, 3H); MS ES+ve m/z 185 (M+H)+.
From 37 (30 mg, 0.17 mmol) and 2,3-dichlorobenzenesulfonyl
chloride (45 mg, 0.18 mmol) gave 38 (20 mg, 29%) as a white solid:
1H NMR (400 MHz, DMSO-d6) d = 11.00 (s, 1H), 10.77 (s, 1H), 10.21
(s, 1H), 7.94 (dd, J = 8.0, 1.3 Hz, 1H), 7.90 (dd, J = 8.0, 1.3 Hz, 1H),
7.51 (t, J = 8.0 Hz, 1H), 7.02 (s, 1H), 3.58 (s, 3H); LCMS (System A)
RT = 0.76 min, 100%, ES+ve m/z 389, 391, 393 (M+H)+.
5.44. Di-tert-butyl (3-methoxy-5-nitropyridine-2,6-
5.49. 5-Methoxy-1H-imidazo[4,5-b]pyridin-2(3H)-one (40)
diyl)dicarbamate (34)
CDI (1.25 g, 7.73 mmol) was added to a solution of 6-methoxy-
pyridine-2,3-diamine (39) (1.0 g, 7.0 mmol) and triethylamine
(2.0 mL, 14 mmol) in THF (13 mL), and the reaction was stirred
for 2 h at ambient temperature under nitrogen. The reaction was
evaporated under reduced pressure to remove THF, and the residue
was triturated with ethyl acetate. The solid was collected by filtra-
tion, and further purified by chromatography on a silica cartridge
(100 g) eluting with a gradient of 0–25% methanol–dichlorometh-
ane over 40 min. Appropriate fractions were evaporated under
reduced pressure to give 40 (360 mg, 31%) as a white solid: 1H
NMR (400 MHz, DMSO-d6) d = 11.18 (br s, 1H), 10.54 (br s, 1H),
Di-tert-butyl dicarbonate (0.76 mL, 3.3 mmol) and K2CO3
(560 mg, 4.10 mmol) were added to a solution of 33 (150 mg,
0.82 mmol) in DMF (4 mL). The reaction mixture was stirred at
ambient temperature for 18 h, and then diluted with water
(20 mL) and ethyl acetate (40 mL). The organic layer was sepa-
rated, washed with water (2 Â 10 mL), brine (10 mL), dried, and
concentrated under reduced pressure. The residue was purified
by chromatography on a silica cartridge (50 g) eluting with a gra-
dient of 0–50% ethyl acetate–cyclohexane over 40 min. The appro-
priate fractions were combined and evaporated under reduced
Please cite this article in press as: Miah, A. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.021

A. H. Miah et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
13
7.21 (d, J = 8.5 Hz, 1H), 6.35 (d, J = 8.5 Hz, 1H), 3.77 (s, 3H); MS
ES+ve m/z 166 (M+H)+.
12.50 (br s, 1H); 13C NMR (101 MHz, DMSO-d6) 154.4, 146.2,
143.1, 138.1, 120.0; MS ESÀve m/z 258, 260 (MÀH)À.
5.50. 5-Methoxy-6-nitro-1H-imidazo[4,5-b]pyridin-2(3H)-one
5.55. 5-Methoxy-6-nitro-1H-imidazo[4,5-b]pyrazin-2(3H)-one
(41)
(47)
Compound 40 (266 mg, 1.61 mmol) was slowly added to triflu-
oroacetic anhydride (5.5 mL, 40 mmol) at 0 °C, and the reaction
was stirred at this temperature for 1 h. Fuming nitric acid
(0.14 mL of a 70 wt% aqueous solution, 1.63 mmol) was added
dropwise and the reaction was stirred at 0 °C for a further 2 h.
The reaction mixture was then slowly added to a solution of
sodium metabisulfite (304 mg, 1.6 mmol) in water (14 mL) at
0 °C, and the precipitate formed was collected by filtration to give
41 (160 mg, 47%) as a bright yellow solid: 1H NMR (400 MHz,
DMSO-d6) d = 12.09 (br s, 1H), 11.06 (br s, 1H), 7.85 (s, 1H), 3.98
(s, 3H); MS ES+ve m/z 211 (M+H)+; 228 (M+NH4)+.
Sodium methoxide solution in methanol (25% w/v, 0.4 mL,
100 mg, 1.8 mmol) was added in portions to a solution of 46
(150 mg, 0.58 mmol) dissolved in MeOH (1 mL). The reaction was
stirred at ambient temperature for 18 h and was then heated to
50 °C and stirred at this temperature for a further 24 h. Water
was added (1 mL) and the reaction mixture was diluted with DMSO
(4 mL). The precipitated solid was removed by filtration. The fil-
trate was diluted with more DMSO (2 mL), and purified by MDAP.
The appropriate fractions were combined and the solvent was
evaporated under reduced pressure to give 47 (60 mg, 49%) as a
yellow solid: 1H NMR (400 MHz, DMSO-d6) d = 12.5 (br, 1H), 12.0
(br, 1H), 4.00 (s, 3H); MS ESÀve m/z 210 (MÀH)À.
5.51. 6-Amino-5-methoxy-1H-imidazo[4,5-b]pyridin-2(3H)-one
(42)
5.56. 5-Amino-6-methoxy-1H-imidazo[4,5-b]pyrazin-2(3H)-one
(48)
A solution of 41 (130 mg, 0.62 mmol) in ethanol (2 mL) was
hydrogenated over 10% palladium on carbon (66 mg) for 2 h. The
catalyst was removed by filtration through celite, washed with eth-
anol (30 mL), and the filtrate was concentrated under reduced
pressure to give 42 (42 mg, 38%) as a yellow solid: 1H NMR
(400 MHz, DMSO-d6) d = 10.59 (br s, 1H), 10.15 (s, 1H), 6.69 (s,
1H), 4.38 (s, 2H), 3.80 (s, 3H).
A solution of 47 (60 mg, 0.28 mmol) in ethanol (2 mL) and ethyl
acetate (2 mL) was hydrogenated over 10% palladium on carbon
(91 mg) for 1 h. The catalyst was removed by filtration through cel-
ite, washed with a mixture of ethanol and ethyl acetate (1:1,
30 mL), and evaporated under reduced pressure to give 48
(41 mg, 80%) as a grey solid: 1H NMR (400 MHz, DMSO-d6)
d = 11.9 (br s, 1H), 8.98 (s, 1H), 7.27 (s, 1H), 6.46 (s, 1H), 3.73 (s,
3H); MS ES+ve m/z 182 (M+H)+.
5.52. 2,3-Dichloro-N-(5-methoxy-2-oxo-2,3-dihydro-1H-
imidazo[4,5-b]pyridin-6-yl)benzenesulfonamide (43)
5.57. 2,3-Dichloro-N-(6-methoxy-2-oxo-2,3-dihydro-1H-
imidazo[4,5-b]pyrazin-5-yl)benzenesulfonamide (49)
From 42 (23 mg, 0.13 mmol) and 2,3-dichlorobenzenesulfonyl
chloride (35 mg, 0.14 mmol) gave 43 (24 mg, 48%) as a white solid:
1H NMR (400 MHz, DMSO-d6) d = 11.23 (br s, 1H), 10.52 (s, 1H),
9.90 (br s, 1H), 7.88 (dd, J = 8.0, 1.5 Hz, 1H), 7.71 (dd, J = 8.0,
1.5 Hz, 1H), 7.41 (t, J = 8.0 Hz, 1H), 7.08 (s, 1H), 3.39 (s, 3H); 13C
NMR (101 MHz, DMSO-d6) d = 178.8, 154.4, 154.0, 140.4, 140.3,
134.1, 133.8, 129.7, 129.4, 127.8, 118.3, 117.1, 53.0; Anal. Calcd
for C13H11Cl2N4O4S = 388.9878. Found = 388.9881 (M+H)+; LCMS
(System A) RT = 0.83 min, 100%, ES+ve m/z 389, 391, 393 (M+H)+.
From 48 (41 mg, 0.23 mmol) and 2,3-dichlorobenzenesulfonyl
chloride (70 mg, 0.28 mmol) to give 49 (8 mg, 7%) as a pale orange
solid: 1H NMR (400 MHz, CD3OD) d = 8.06 (dd, J = 8.0, 1.5 Hz, 1H),
7.77 (dd, J = 8.0, 1.5 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 3.83 (s, 3H); LCMS
(System A) RT = 0.78 min, 88%; ES+ve m/z 390, 392, 394 (M+H)+.
Acknowledgements
We thank Bill J. Leavens for collecting the HRMS data, and the
Screening and Compound Profiling Department at GlaxoSmithK-
5.53. 5-Bromo-1H-imidazo[4,5-b]pyrazin-2(3H)-one (45)
line for generating the human CCR4 GTPcS data.
A
mixture of 5-bromopyrazine-2,3-diamine (44) (0.70 g,
3.7 mmol) and CDI (1.2 g, 7.4 mmol) in anhydrous THF (20 mL)
was heated at 50 °C for 22 h. More CDI (0.60 g, 3.7 mmol) was
added and the reaction was stirred at 50 °C for a further 24 h.
The reaction mixture was concentrated under reduced pressure,
and the residue was purified by chromatography on a silica car-
tridge (100 g), eluting with a gradient of 0–100% ethyl acetate–
dichloromethane over 40 min. The appropriate fractions were
combined and evaporated under reduced pressure to give 45
(0.68 g, 85%) as a white solid: 1H NMR (400 MHz, DMSO-d6)
d = 11.95 (br s, 2H), 7.97 (s, 1H); MS ES+ve m/z 215, 217 (M+H)+.
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Please cite this article in press as: Miah, A. H.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.05.021