Discovery of AZD-2098 and AZD-1678, Two Potent and Bioavailable CCR4 Receptor Antagonists

Article abstract of DOI:10.1021/acsmedchemlett.7b00315

N-(5-Bromo-3-methoxypyrazin-2-yl)-5-chlorothiophene-2-sulfonamide 1 was identified as a hit in a CCR4 receptor antagonist high-throughput screen (HTS) of a subset of the AstraZeneca compound bank. As a hit with a lead-like profile, it was an excellent sta

Full text of DOI:10.1021/acsmedchemlett.7b00315

Subscriber access provided by - Access paid by the | University Library, UC Santa Cruz
Letter
The discovery of AZD-2098 and AZD-1678, two
potent and bioavailable CCR4 receptor antagonists.
Nicholas D. Kindon, Glen Andrews, Andrew Baxter, David Cheshire, Paul Hemsley, Timothy Johnson,
Yu-Zhen Liu, Dermot McGinnity, Mark McHale, Antonio Mete, James Reuberson, Bryan Roberts,
John Steele, Barry Teobald, John Unitt, Deborah Vaughan, Iain Walters, and Michael John Stocks
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00315 • Publication Date (Web): 01 Sep 2017
Downloaded from http://pubs.acs.org on September 2, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Medicinal Chemistry Letters is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
The discovery of AZD-2098 and AZD-1678, two potent and bioavail-
able CCR4 receptor antagonists.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Nicholas Kindon,a, b Glen Andrews,a Andrew Baxter,a David Cheshire,a Paul Hemsley,a, c Timothy
Johnson,a Yu-Zhen Liu,a, Dermot McGinnity,a, d Mark McHale,a Antonio Mete,a James Reu-
berson,a Bryan Roberts,a John Steele, a, e Barry Teobald,a John Unitt,a Deborah Vaughan,a Iain
Walters,a and Michael J. Stocks* a, b
a AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH, UK.
b Current address School of Pharmacy, Centre for Biomolecular Sciences, The University of Nottingham, University
Park, Nottingham, NG7 2RD, UK.
c Current address Discovery Sciences, Innovative Medicines & Early Development, AstraZeneca, Darwin Building,
Cambridge Science Park, Cambridge, CB4 0FZ UK.
d Current address IMED Oncology, Innovative Medicines & Early Development, AstraZeneca, Chesterford Research
Park,, Cambridge, CB10 1XL. UK
e
Respiratory, Inflammation and Autoimmunity, Innovative Medicines and Early Development, AstraZeneca Gothenburg,
Pepparedsleden 1, SE-431 83 Mölndal, Sweden
KEYWORDS Chemokine receptor 4; MDC; TARC; CCR4; Antagonist
ABSTRACT: N-(5-Bromo-3-methoxypyrazin-2-yl)-5-chlorothiophene-2-sulfonamide 1 was identified as a hit in a CCR4
receptor antagonist high throughput screen (HTS) of a sub-set of the AstraZeneca compound bank. As a hit with a lead-
like profile, it was an excellent starting point for a CCR4 receptor antagonist program and enabled the rapid progression
through the Lead Identification and Lead Optimization phases resulting in the discovery of two bioavailable CCR4 recep-
tor antagonist candidate drugs.
The chemokine receptor family was originally identified
in the 1990s and has since grown in number, complexity
and range of biological functions. Originally thought to
be simple cell chemo attractants, chemokines have since
been shown to exhibit a broader range of functions cover-
ing involvement in HIV-1 infection to hematopoiesis and
control of cell growth.1,2
Figure 1: Structures of a selection of recently published CCR4 recep-
CCR4 is a G protein-coupled receptor (GPCR) and is ac-
tor antagonists.
tivated by the CC chemokines, CCL22 (MDC: macro-
In this communication we are disclosing our studies on
phage-derived chemokine) and CCL17 (TARC: T-cell and
the optimization of N-(5-bromo-3-methoxypyrazin-2-yl)-
5-chlorothiophene-2-sulfonamide 1 to afford two clinical
candidates AZD-2098 (47) and AZD-1678 (49). High
throughput screening of the AstraZeneca Mixed Chemo-
activation-related chemokine),3 leading to cell activation
and chemotaxis; it is expressed mainly by Th2 lympho-
cytes.4,5 In keeping with a role for CCR4 in the orches-
trated movement of Th2 cells into the allergic lung, both
kine Receptor Antagonist Compound library (~29,000
CCL17 and CCL22 have been shown to be elevated in the
compounds) using a hCCR4 Fluorescent Microvolume
human lung following allergen challenge.6 This potential
Assay Technology (FMAT) cell binding assay identified
compound 118,19 as an inhibitor of labeled CCL22 binding
role of CCR4 in driving human allergic lung disease led to
to CCR4 (pIC50 7.2).20 Subsequently functional antago-
many pharmaceutical companies attempting to identify
CCR4 receptor antagonists for the treatment of allergic
nism of 1 was shown using an hCCR4 Fluorescence Imag-
ing Plate Reader (FLIPR) intracellular calcium mobiliza-
tion assay (pIC50 7.0) and a human primary Th2 cell
rhinitis and asthma,7–14,15 but as yet no drug has been dis-
covered (Figure 1).16,17
chemotaxis assay (pIC50 6.7).
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 8
Table 1 Hit profile of pyrazine sulfonamide 1
4.1) and the lipophilic nature of the molecule (measured
logP 4.4).
1
2
3
4
5
6
7
8
9
The structure activity relationship (SAR) generated
with respect to modification of the 5-chlorothiophene
group is shown in Table 2. Removal of the 5-chloro sub-
stituent 2 gave over a 10-fold drop in affinity, as did the
isomeric unsubstituted thiophene 3. Addition of a chlo-
rine to the 4-position of the thienyl group also gave a re-
duction in affinity (compare 4 with 1) whilst a large sub-
stituent (bromine or phenyl) in the 3-position was toler-
ated (compare 5 and 6 with 1 and 2 respectively). The
phenyl analogue 7 was similar to the two unsubstituted
thiophenes in potency and introducing chlorine into the
2-, 3- or 4-positions of the phenyl sulfonamide ring (8, 9
and 10) gave an increase in binding relative to 7. The 6-
substituted dichlorophenyl sulfonamides (11-17) had a
potency range of almost 3 log units, with the 3,5-
dichlorophenylsulfonamide analogue 14 having only weak
Biological Assay / test procedure
Compound 1
hCCR4 Binding (pIC50)
hPPB (%)
7.2 ± 0.2
>99.9
<4.2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Calc. WBP (pIC50)*
logD
1.1
Solubility (mg/mL)
human hepatocytes (µL/mL/106cells)
rat / mouse in vivo PK
Cl (mL/min/kg)
0.34
<0.2
0.1 / 0.1
0.1 / 0.1
16 / 10
Vss (L/kg)
activity
(pIC50
5.3)
and
the
2,3-
T½ (h)
dichlorophenylsulfonamide 16 being optimal (pIC50 8.0),
almost a log unit more potent than 1. Similar affinity was
achieved with 3, 4-dichloro-2-thienyl (compare 18 and 17).
A number of other di-substituted phenyl sulfonamides
were screened, however no improvement in binding was
seen (19-24 compare with 17). Introduction of a nitrogen
into the aromatic ring lead to dramatic reduction in po-
tency (25) as did replacement of the aromatic ring with an
alkyl group as illustrated with 26.
Bioavailability (%)
Rat / mouse CCR4 binding (pIC50)
Rat / mouse PPB (%)
45 / 86
8.3 / 8.3
99.85 / 96.7
*Calculated from the hCCR4 binding and %age PPB
Compound 1 exhibited a good lead-like profile21 and was
shown to be selective for the CCR4 receptor, through
screening against an internal panel of chemokine recep-
tors (e.g. CXCR1 & CXCR2, CCR1, CCR2b, CCR5, CCR7 &
CCR8; inactive at 10 µM). When screened against a large
panel of receptors and enzymes (~120 screens at MDS-
Pharma), weak or no activity was observed and therefore 1
was chosen as a starting point for the CCR4 antagonist
program. Compound 1 had good solubility (0.34 mg/mL),
rat bioavailability (F = 45%) and half-life (T½ ~16 h) when
dosed in rat and a similar profile when dose in vivo in
mouse. This long half-life was accounted for by a very
low clearance (0.1 mL/min/kg), which counteracts the
small volume of distribution (Vss 0.1 L/kg).
Whilst an increase in affinity was achieved with com-
pounds 16 and 17, changing the 5-chlorothiophene group
for 2, 3-dichlorophenyl did not significantly change the
lipophilicity. Consequently, the hPPB figures for these
two compounds was also high (>99.9%).
Table 2 Exploration of the 5-position of the pyrazine
ring
Compound 1 had very good cross-over with rat and
mouse CCR4 receptors (both pIC50 8.3). Subsequently,
this species cross-over was shown to be a general feature
of the sulfonamide series (see Table 5), with the CCR4
receptor from all three species displaying very similar
structural activity relationships (SAR). When corrected
for plasma protein binding, the predicted rat whole blood
potency of 1 was pIC50 5.5 (rat ppb 99.85%) and for mouse
6.8 (mouse ppb 96.7%), giving 1 a suitable profile for use
as a target validation tool for in vivo hypothesis testing.
a
Example
R
X
CCR4 pIC50
7.2 ± 0.2
6.2 ± 0.2
5.9 ± 0.1
6.0 ± 0.1
7.4 ± 0.2
6.0 ± 0.1
6.1 ± 0.1
1
5-Chloro-2-thienyl
2-Thienyl
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
2
3
3-Thienyl
4
4,5-Dichloro-2-thienyl
3-Bromo-5-chloro-2-thienyl
3-Phenyl-2-thienyl
Phenyl
5
6
7
However, the calculated human whole blood potency
(WBP) was very poor with predicted activity (pIC50) < 4.2
(calculated from hCCR4 pIC50 7.2 and hPPB >99.9%).
8
2-Chlorophenyl
3-Chlorophenyl
4-Chlorophenyl
2,6-Dichlorophenyl
2,5-Dichlorophenyl
2,4-Dichlorophenyl
3,5-Dichlorophenyl
3,4-Dichlorophenyl
6.8 ± 0.2
7.0 ± 0.2
6.9 ± 0.2
6.2 ± 0.2
5.5 ± 0.1
6.9 ± 0.2
5.3 ± 0.1
6.7 ± 0.2
9
10
11
12
13
14
15
With a candidate drug requiring a WBP pIC50 ~ 6.0, the
key issue was to increase WBP by a combination of in-
creasing CCR4 receptor affinity and lowering plasma pro-
tein binding,22 whilst maintaining all the other good fea-
tures inherent in 1. The high plasma protein binding was
attributed to the acidic sulfonamide-NH (measured pKa
ACS Paragon Plus Environment

Page 3 of 8
ACS Medicinal Chemistry Letters
16
2,3-Dichlorophenyl
Br
8.0 ± 0.2
7.9 ± 0.2
8.1 ± 0.2
7.8 ± 0.2
7.1 ± 0.2
7.7 ± 0.2
thylthio, methylamino or ethyl (28, 29 and 30) gave a
1
2
3
4
5
6
7
8
9
10
11
12
large drop in potency, establishing oxygen as the optimal
atom with which to attach the 3-substituent to the pyra-
zine ring. Next, a small number of 3-alkoxy analogues
were synthesized (27, 31 and 32). In each case there was a
substantial drop in potency relative to the parent meth-
oxy-substituted compound (1 or 17). However, the 3-
allyloxy analogue (33) maintained activity and with the 3-
propargyloxy (34) analogue affording a slight increase in
activity. Further increases in activity was obtained with 3-
OCH2Aryl and 3-OCH2heteroaryl (35, 36, 37 and 38),
where substitution gave substantial increase in activity
(pIC50 up to 9.5). Although this SAR demonstrated how to
improve on CCR4 affinity, the increase in lipophilicity
associated with these changes did not address the plasma
protein binding issue with hPPB >99.9% in each case.
17
2,3-Dichlorophenyl
Cl
Cl
Cl
Cl
Cl
18
3,4-Dichloro-2-thienyl
2-Chloro-3-fluorophenyl
3-Chloro-2-fluorophenyl
3-Chloro-2-methylphenyl
19
20
21
2-Chloro-3-
(trifluoromethyl)phenyl
22
Cl
7.2 ± 0.1
23
3-Chloro-2-cyanophenyl
3-Chloro-2-methylthiophenyl
2,3-Dichloro-4-pyridyl
Butyl
Cl
Cl
Cl
Cl
7.2 ± 0.2
7.1 ± 0.2
5.0 ± 0.1
5.5 ± 0.2
24
25
26
13
a Potency is given as pIC50 values with n = ≥ 2 replicates.
14
15
16
The exploration of the 3-position is shown in Table 3.
Initially the atom linking the 3-substituent to the pyra-
zine ring was investigated. Changing methoxy for me-
Table 3 Exploration of the 3-position of the pyrazine ring.
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Example
R2
R3
Br
Br
Br
Br
Cl
Cl
Cl
Cl
R4
5-Chloro-2-thienyl
CCR4 receptor antagonism (pIC50)a
27
28
29
30
31
OEt
SMe
NHMe
Et
6.7 ± 0.2
5.5 ± 0.2
<5.0*
5-Chloro-2-thienyl
5-Chloro-2-thienyl
5-Chloro-2-thienyl
2,3-Dichlorophenyl
2,3-Dichlorophenyl
2,3-Dichlorophenyl
2,3-Dichlorophenyl
5.3 ± 0.2
6.8 ± 0.1
5.1 ± 0.2
7.8 ± 0.1
8.6 ± 0.2
OBu
t
32
33
34
OCH2 Bu
OCH2CHCH2
OCH2CCH
Cl
Cl
Cl
Cl
2,3-Dichlorophenyl
2,3-Dichlorophenyl
2,3-Dichlorophenyl
2,3-Dichlorophenyl
35
36
9.2± 0.3
8.4 ± 0.2
37
38
9.5 ± 0.2
7.9 ± 0.3
a Potency is given as pIC50 values with n = ≥ 2 replicates. *44% inhibition at 10 µM
In contrast to the 3-position, the exploration of the 5- and 6-positions on the pyrazine ring allowed for the discovery of
compounds with increased activity combined with lower lipophilicity relative to 17 (Table 4).
Table 4 Exploration of the 5- and 6- position of the pyrazine ring.
Example
17
X
R5
Cl
R6
H
CCR4 receptor antagonism (pIC50)a
7.9 ± 0.2
LogP
4.1
hPPB (%)
>99.9
Calc. WBP (pIC50)
-
CH=CH
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 8
39
40
41
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
CH=CH
S
H
Cl
H
H
H
Me
H
Cl
Me
H
H
H
F
8.1 ± 0.3
7.9 ± 0.2
5.3 ± 0.2
7.4 ± 0.2
7.3 ± 0.3
7.6 ± 0.2
8.4± 0.2
7.4 ± 0.1
7.8 ± 0.05
7.7 ± 0.2
8.6 ± 0.1
8.4 ± 0.2
9.0 ± 0.3
4.5
4.1
-
>99.9
>99.9
-
-
1
2
3
4
5
6
7
8
OEt
-
SO2Me
-
42
43
44
45
46
47
48
49
50
51
Me
3.0
-
99.65
>99.8
96.2
99.6
98.7
98.9
97.5
4.9
-
H
CH2OH
2.1
3.2
2.4
2.5
2.8
3.1
3.3
-
6.2
6.0
5.5
5.8
6.1
6.4
-
CH2OH
CH2OH
9
H
H
F
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CH=CH
CH=CH
CH=CH
99.4
>99.8
>99.8
H
F
F
-
a Potency is given as pIC50 values with n = ≥ 2 replicates.
F (%)
100/100
100/100
0.1
A wide variety of substituents were tolerated in the 5- and
6-positions and it was discovered that the percentage
plasma protein binding was reduced by lowering the lipo-
philicity of the compounds, affording plasma protein
binding below 99% for compounds of modest lipophilici-
ty (logP < 3.0). For 44, 45, 47, 48 and 49, the calculated
WBP (pIC50) of 5.8-6.4 was achieved. These met the can-
didate drug target profile set of WBP pIC50 ~ 6.0. Of these
four compounds, 47 and 49 had the best overall profile for
progression (Table 5). Compounds 47 and 49 demonstrat-
ed good selectivity for the CCR4 receptor when screened
in-house against a range of other chemokine receptors
(CXCR1 & CXCR2, CCR1, CCR2b, CCR5, CCR7 & CCR8; all
inactive at 10 µM) and little or no activity when screened
against a large panel of receptors and enzymes (~120
screens at MDS-Pharma). A pharmacokinetic profile
commensurate with once-daily oral administration ena-
bled both compounds to progress as candidate drugs.23
dose-to-man prediction
(mg/kg) UID
1
*PK studies conducted using 1% sodium bicarbonate solu-
tion at 9.0 µMol/kg (p.o.) or 3.0 µMol/kg (i.v.)
The antagonist potency of 47 and 49 were assessed in a
number of cell systems in which a response mediated by
the human CCR4 receptor can be evoked by MDC or
TARC and quantified using changes in intracellular calci-
um concentration or chemotaxis. The cell systems used
were a cell line (CHO) transfected with the human CCR4
receptor and human CD4+, CD45RA+ Th2 cells in 0.3%
HSA. Findings are summarized in Table 6. The reduced
potency in the presence of added protein is consistent
with the drop-off predicted from plasma protein binding
measurements. Both 47 and 49 demonstrated no agonist
activity at concentrations up to 10 µM.
Table 5 Compound profiles of 47 and 49
Table 6 Potency of 47 and 49 for inhibition of cellular
responses mediated by the human CCR4 receptor
Cell system
Assay
readout
47 (pIC50
mean ± SEM) mean ± SEM)
49 (pIC50
Inhibition
of MDC-
8.4
±
0.1
7.5
(n=3)
±
0.04
Screen
47 (R7 = H)
49 (R7 = F)
CCL22 Ca2+ re- induced
(n=4)
sponse
Ca2+flux
(HEK
expressing
hCCR4)
cells
(CHO cells
expressing
hCCR4)
hCCR4 pIC50
hPPB (%)
7.8 ± 0.05 (n = 11)
8.6 ± 0.1 (n = 10)
98.9
5.9
99.4
6.4
0.6
1.3
Calc. WBP (pIC50)
logD
Inhibition of Th2 MDC-
cell CCL22 driven induced
chemotaxis in 0.3% chemotaxis
HSA
6.3
(n=3)
±
0.2 6.8
(n=3)
±
0.2
0.35
1.5
Solubility (mg/mL)
Human
hepatocytes <0.2
<1.0
(µL/mL/106cells)
Inhibition of Th2 TARC-
cell CCL17 driven induced
chemotaxis in 0.3% chemotaxis
HSA
6.3
(n=3)
±
0.1 6.5 (n=1)
Species cross over
Rat CCR4 pIC50
Mouse CCR4 pIC50
Dog CCR4 pIC50
Rat/dog in vivo PK*
Cl (mL/min/kg)
Vss (L/kg)
8.0 ± 0.27 (n = 3)
8.0 ± 0.04 (n = 3)
7.6 ± 0.06 (n = 7)
9.0 ± 0.1 (n = 3)
9.0 ± 0.1 (n = 3)
8.5 ± 0.2 (n = 3)
The anti-inflammatory effects of 47 were investigated in
Brown-Norway rats sensitized to ovalbumin. Sensitized
rats were dosed orally (bid) with 47 at 0.22, 0.75, 2.2, 3.0,
7.5 and 15 µmol/kg 1 hour prior to antigen challenge and
every 12 hours thereafter prior to termination 96 hours
2.0/1.5
0.2/0.1
1.7/1.0
2.3/3.7
0.2/0.2
2.5/0.5
T½ (h)
ACS Paragon Plus Environment

Page 5 of 8
ACS Medicinal Chemistry Letters
Scheme 1. i) Br2, dichloromethane, 2,6-lutidine (85-95%)
post challenge. Histopathological examination of the lung
1
2
3
4
5
6
7
8
ii) NaOMe (3 eq.), MeOH, reflux (90-95%) iii) H2, Pd/C, eth-
anol, rt (95%) iv) ArSO2Cl, KOtBu, 0-25°C, THF (80-95%) v)
HNO3, AcOH, 70-85oC (75-82%) vi) H2, Pd/C, AcOH, 60oC
(75-92%) vii) HBF4, CH3CN, 0-5oC, NaNO2 (40%) or HF /
Pyridine / NaNO2 (85%). Range of percentage yields across
multiple reactions and scale (~100 mg to >50g).
tissue showed a marked, dose-dependent reduction in a
range of histological correlates with reduced alveolitis and
leukocyte trafficking in the microvasculature being the
most diagnostic features of efficacy. The changes were
first visible at a dose of 0.22 µmol/kg and maximal at 7.5
µmol/kg. Plasma samples were taken 12 hours after the
last dose of compound for measurement of terminal
trough concentrations of 47 to demonstrate a strong
PKPD correlation (Table 7).
In summary, a new series of potent and bioavailable
amino-pyrazine sulfonamide CCR4 receptor antagonists
have been developed from a lead optimization program
initiated from a high-throughput screening / synthesis
campaign. The initial hit series proved to have reasonable
activity but was hindered by having very high human
plasma protein binding resulting in a low free fraction of
compound. Through chemical modification, both the
potency of the series and the free fraction was enhanced
to ultimately deliver two clinical candidates. Further stud-
ies on these compounds will be reported in due course.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 7 Terminal plasma concentrations of 47
Dose
Total plasma
Free plasma
(µmol/kg)
concentration/pIC50
concentration (µM)
0.22
0.75
2.2
3.0
7.5
15
<LOQ*
-
<LOQ*
-
<LOQ*
-
0.033 ± 0.018
0.14 ± 0.069
0.49 ± 0.18
0.6
3
Supporting Information
Representative synthetic procedures and analytical data for
47 and 49. Description of biological assay (CCR4 receptor
antagonism using FMAT whole cell binding).
9
*LOQ was 0.03 µM for the 0.22, 0.75 and 2.2 µmol/kg dos-
es
The Supporting Information is available free of charge on the
ACS Publications website.
The synthesis of 47 and 49 is shown (Scheme 1). Dibro-
mination of 2-aminopyrazine followed by selective dis-
placement of the bromine in the 3-position with sodium
methoxide gave 2-amino-5-bromo-3-methoxypyrazine.
The bromine in the 5-position was then removed by hy-
AUTHOR INFORMATION
Corresponding Author
* michael.stocks@nottingham.ac.uk
drogenation
dichlorophenylsulphonyl chloride with 2-amino-3-
methoxypyrazine afforded 47. highly-selective 5-
and
coupling
of
the
2,
3-
Author Contributions
A
All authors have given approval to the final version of the
manuscript.
position nitration of 47 followed by reduction of the nitro
group 52 gave an amino-pyrazine 53 that was diazotized
in the presence of hydrofluoric acid to give 49.
ACKNOWLEDGMENT
The author’s thank Drs Michael Bernstein, Sara Duncan and
Eva Lenz for NMR spectroscopy support and Mrs Hemma
Pancholi for detailed analysis work.
ABBREVIATIONS
CCR4, CC chemokine receptor 4; CCL12, CC chemokine lig-
and 12; F (bioavailability), FLIPR (Fluorescence Imaging Plate
Reader), FMAT (Fluorescent Microvolume Assay Technolo-
gy), GPCR G protein-coupled receptor, MDC (macrophage-
derived chemokine ), PPB (plasma protein binding), TARC
(T-cell and activation-related chemokine ), UID (once-a-day),
Vss (volume of distribution at steady state).
(3)
(4)
Proudfoot, A. E. I. Chemokine receptors: multifaceted
therapeutic targets. Nat. Rev. Immunol. 2002, 2 (2), 106–115.
Bonecchi, R.; Bianchi, G.; Bordignon, P. P.; D’Ambrosio, D.;
Lang, R.; Borsatti, A.; Sozzani, S.; Allavena, P.; Gray, P. A.;
Mantovani, A.; Sinigaglia, F. Differential Expression of
Chemokine Receptors and Chemotactic Responsiveness of
Type 1 T Helper Cells (Th1s) and Th2s. J. Exp. Med. 1998, 187
REFERENCES
(1)
Pease, J.; Horuk, R. Chemokine Receptor Antagonists. J.
Med. Chem. 2012, 55 (22), 9363–9392.
(2)
Charo, I. F.; Ransohoff, R. M. The Many Roles of
Chemokines and Chemokine Receptors in Inflammation. N.
Engl. J. Med. 2006, 354 (6), 610–621.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 8
(1), 129–134.
Tao, J.-C. Synthesis, Cytotoxic Activity Evaluation and
(5)
(6)
(7)
(8)
Bochner, B. S.; Hudson, S. A.; Xiao, H. Q.; Liu, M. C.
Release of Both CCR4-Active and CXCR3-Active
Chemokines during Human Allergic Pulmonary Late-Phase
Reactions. J. Allergy Clin. Immunol. 2003, 112 (5), 930–934.
Pilette, C.; Francis, J. N.; Till, S. J.; Durham, S. R. CCR4
Ligands Are up-Regulated in the Airways of Atopic
Asthmatics after Segmental Allergen Challenge. Eur. Respir.
J. 2004, 23 (6), 876–884.
Hall, D.; Ford, A.; Hodgson, S. Therapeutic Potential of
CCR4 Antagonistste. In New Drugs and Targets for Asthma
and COPD; Karger Medical and Scientific Publishers, 2010;
Vol. 39, pp 161–165.
Newhouse, B.; Allen, S.; Fauber, B.; Anderson, A. S.; Eary, C.
T.; Hansen, J. D.; Schiro, J.; Gaudino, J. J.; Laird, E.; Chantry,
D.; Eberhardt, C.; Burgess, L. E. Racemic and Chiral
Lactams as Potent, Selective and Functionally Active CCR4
Antagonists. Bioorg. Med. Chem. Lett. 2004, 14 (22), 5537–
5542.
Yokoyama, K.; Ishikawa, N.; Igarashi, S.; Kawano, N.;
Masuda, N.; Hattori, K.; Miyazaki, T.; Ogino, S. ichi; Orita,
M.; Matsumoto, Y.; Takeuchi, M.; Ohta, M. Potent CCR4
Antagonists: Synthesis, Evaluation, and Docking Study of
2,4-Diaminoquinazolines. Bioorganic Med. Chem. 2008, 16
(17), 7968–7974.
Yokoyama, K.; Ishikawa, N.; Igarashi, S.; Kawano, N.;
Hattori, K.; Miyazaki, T.; Ogino, S.; Matsumoto, Y.;
Takeuchi, M.; Ohta, M. Discovery of Potent CCR4
Antagonists: Synthesis and Structure–activity Relationship
Study of 2,4-Diaminoquinazolines. Bioorg. Med. Chem.
2008, 16 (14), 7021–7032.
Burdi, D. F.; Chi, S.; Mattia, K.; Harrington, C.; Shi, Z.;
Chen, S.; Jacutin-Porte, S.; Bennett, R.; Carson, K.; Yin, W.;
Kansra, V.; Gonzalo, J.-A.; Coyle, A.; Jaffee, B.; Ocain, T.;
Hodge, M.; LaRosa, G.; Harriman, G. Small Molecule
Antagonists of the CC Chemokine Receptor 4 (CCR4).
Bioorg. Med. Chem. Lett. 2007, 17 (11), 3141–3145.
Purandare, A. V.; Wan, H.; Somerville, J. E.; Burke, C.;
Vaccaro, W.; Yang, X.; McIntyre, K. W.; Poss, M. A. Core
Exploration in Optimization of Chemokine Receptor CCR4
Antagonists. Bioorganic Med. Chem. Lett. 2007, 17 (3), 679–
682.
Allen, S.; Newhouse, B.; Anderson, A. S.; Fauber, B.; Allen,
A.; Chantry, D.; Eberhardt, C.; Odingo, J.; Burgess, L. E.
Discovery and SAR of Trisubstituted Thiazolidinones as
CCR4 Antagonists. Bioorganic Med. Chem. Lett. 2004, 14
(7), 1619–1624.
HQSAR Study of Novel Isosteviol Derivatives as Potential
Anticancer Agents. Eur. J. Med. Chem. 2016, 115, 26–40.
Procopiou, P. A.; Barrett, J. W.; Barton, N. P.; Begg, M.;
Clapham, D.; Copley, R. C. B.; Ford, A. J.; Graves, R. H.;
Hall, D. A.; Hancock, A. P.; Hill, A. P.; Hobbs, H.; Hodgson,
S. T.; Jumeaux, C.; Lacroix, Y. M. L.; Miah, A. H.; Morriss, K.
M. L.; Needham, D.; Sheriff, E. B.; Slack, R. J.; Smith, C. E.;
Sollis, S. L.; Staton, H. Synthesis and Structure–Activity
Relationships of Indazole Arylsulfonamides as Allosteric
CC-Chemokine Receptor 4 (CCR4) Antagonists. J. Med.
Chem. 2013, 56 (5), 1946–1960.
1
2
3
4
5
6
7
8
(15)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(16)
(17)
Pease, J. E.; Horuk, R. Chemokine Receptor Antagonists:
Part 1. Expert Opin. Ther. Pat. 2009, 19 (1), 39–58.
Cahn, A.; Hodgson, S.; Wilson, R.; Robertson, J.; Watson, J.;
Beerahee, M.; Hughes, S. C.; Young, G.; Graves, R.; Hall, D.;
van Marle, S.; Solari, R. Safety, Tolerability,
Pharmacokinetics and Pharmacodynamics of GSK2239633,
a CC-Chemokine Receptor 4 Antagonist, in Healthy Male
(9)
(10)
(11)
Subjects: Results from an Open-Label and from a
Randomised Study. BMC Pharmacol. Toxicol. 2013, 14 (1), 14.
Andrews, G.; Jones, C.; Wreggett, K. A. An Intracellular
Allosteric Site for a Specific Class of Antagonists of the CC
Chemokine G Protein-Coupled Receptors CCR4 and CCR5.
Mol. Pharmacol. 2008, 73 (3), 855–867.
Slack, R. J.; Russell, L. J.; Barton, N. P.; Weston, C.; Nalesso,
G.; Thompson, S.-A.; Allen, M.; Chen, Y. H.; Barnes, A.;
Hodgson, S. T.; Hall, D. A. Antagonism of Human CC-
Chemokine Receptor 4 Can Be Achieved through Three
Distinct Binding Sites on the Receptor. Pharmacol. Res.
Perspect. 2013, 1 (2), e00019.
(18)
(19)
(20)
(21)
(22)
(23)
Baxter, A.; Timothy, J.; Kindon, N.; Roberts, B.; Steele, J.;
Stocks,
M.;
Nicholas,
T.
Preparation
of
N-
pyrazinylthiophenesulfonamides as Chemokine Receptor
Modulators. WO 2003051870, 2003.
Davis, A. M.; Teague, S. J.; Kleywegt, G. J. Application and
Limitations of X-Ray Crystallographic Data in Structure-
Based Ligand and Drug Design. Angew. Chem. Int. Ed. Engl.
2003, 42 (24), 2718–2736.
Luker, T.; Bonnert, R.; Paine, S. W.; Schmidt, J.; Sargent, C.;
Cook, A. R.; Cook, A.; Gardiner, P.; Hill, S.; Weyman-Jones,
C.; Patel, A.; Thom, S.; Thorne, P. Zwitterionic CRTh2
Antagonists. J. Med. Chem. 2011, 54 (6), 1779–1788.
Grime, K. H.; Barton, P.; McGinnity, D. F. Application of In
Silico, In Vitro and Preclinical Pharmacokinetic Data for
the Effective and Efficient Prediction of Human
Pharmacokinetics. Mol. Pharm. 2013, 10 (4), 1191–1206.
(12)
(13)
(14)
Liu, C.-J.; Yu, S.-L.; Liu, Y.-P.; Dai, X.-J.; Wu, Y.; Li, R.-J.;
6
ACS Paragon Plus Environment

Page 7 of 8
ACS Medicinal Chemistry Letters
1
2
3
4
For Table of Contents Use Only
5
6
7
8
The discovery of AZD-2098 and AZD-1678, two potent and bioa-vailable CCR4 receptor
antagonists.
9
Nicholas Kindon, Glen Andrews, Andrew Baxter, David Cheshire, Paul Hemsley, Timothy
Johnson, Yu-Zhen Liu, Dermot McGinnity, Mark McHale, Antonio Mete, James Reu-berson,
Bryan Roberts, John Steele, Barry Teobald, John Unitt, Deborah Vaughan, Iain Walters, and
Michael J. Stocks
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table of Contents graphic
88x34mm (151 x 151 DPI)
ACS Paragon Plus Environment