Discovery of a 4-Azetidinyl-1-thiazoyl-cyclohexane CCR2 antagonist as a development candidate

Article abstract of DOI:10.1021/ml300260s

We have discovered a novel series of 4-Azetidinyl-1-Aryl-cyclohexanes as CCR2 antagonists. Divergent SAR studies on hCCR2 and hERG activities led to the discovery of compound 8d, which displayed good hCCR2 binding affinity (IC ₅₀, 37 nM) and po

Full text of DOI:10.1021/ml300260s

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Letter
Discovery of a 4-Azetidinyl-1-thiazoyl-cyclohexane
CCR2 Antagonist as a Development Candidate
Xuqing Zhang, Cuifen Hou, Heather Hufnagel, Monica Singer,
Evan Opas, Sandra McKenney, Dana Johnson, and Zhihua Sui
ACS Med. Chem. Lett., Just Accepted Manuscript • Publication Date (Web): 08 Oct 2012
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Discovery of a 4-Azetidinyl-1-thiazoyl-cyclohexane CCR2 An-
tagonist as a Development Candidate
Xuqing Zhang*, Cuifen Hou, Heather Hufnagel, Monica Singer, Evan Opas, Sandra McKenney, Dana
Johnson and Zhihua Sui
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Janssen Research and Development, LLC, Welsh & McKean Roads, Box 776, Spring House, PA 19477
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KEYWORDS azetidine, CCR2, hERG, cardiovascular (CV) safety
ABSTRACT: We have discovered a novel series of 4-azetidinyl-1-aryl-cyclohexanes as CCR2 Antagonists. A divergent SAR
studies on hCCR2 and hERG activities led to the discovery of compound 8d , which displayed good hCCR2 binding affinity (IC50,
37 nM) and potent functional antagonism (chemotaxis IC50, 30 nM). It presented an IC50 of > 50 ꢀM in inhibition of the hERG
channel and had no effect on QTc interval up to 10 mg/kg (i.v.) in anesthetized guinea pig and dog CV studies. It also displayed
high selectivity over other chemokine receptors and GPCRs, and amendable oral bioavailability in dogs and primates. In a thiogly-
collate-induced inflammation model in hCCR2KI mice, it had ED50 of 3 mg/kg on inhibition of the influx of leukocytes, mono-
cytes/macrophages and T-lymphocytes.
Monocyte chemotactic protein-1 (MCP-1) is one of the pri-
mary molecules controlling the influx of mononuclear leuko-
cant dose dependent prolongation of the QTc interval (ꢁQTc).
At a dose of 10 mg/kg, a +20% ꢁQTc was observed, with drug
plasma level of 12 ꢀM being attained. Thus, 1 was abandoned
cytes into sites of inflammation.1-2 MCP-1 is a potent chemo-
tactic factor for monocytes and memory T lymphocytes, and
from further development due to its unacceptable CV safety
stimulates the movement of those cells along a chemotactic
blockade of the voltage-gated potassium (K+) channel encoded
gradient following binding to its cell-surface receptor, CC
profile. Since QTc prolongation has been linked to preferential
chemokine receptor-2 (CCR2). This ligand/receptor pair is
overexpressed in numerous inflammatory conditions wherein
by the hERG, optimization of a structural series for lack of in
vitro hERG affinity appears to be generally predictive of de-
excessive monocyte recruitment is observed. Indeed, CCR2-
and MCP-1-deficient mice and CCR2 or MCP-1 antibody-
creased potential to cause QTc prolongation in vivo. Herein,
we report our continuous SAR studies on identifying potent
treated rodents show decreased recruitment of monocytes and
and selective hCCR2 antagonists devoid of this ancillary car-
produce markedly attenuated inflammatory responses in ani-
diac activity. Owing to the correlation between the ꢁQTc
mal models of rheumatoid arthritis (RAs),3 multiple sclerosis,4
observed with 1 in guinea pigs and the potency of 1 in the
asthma,5 atherosclerosis,6 diabetes,7 allograft rejection,8 and
hERG binding assay, we defined hERG IC50’s >25 ꢀM must
be achieved to address QTc issue. As a critical follow-up, a
of CCR2 in the pathogenesis of several immune-based in-
hERG patch clamp assay was used as a second front-line
neuropathic pain.9 Clearly, these observations confirm the role
flammatory diseases and identify this chemokine receptor as a
potentially valuable therapeutic target. Thus, an antagonist of
the binding of MCP-1 to its receptor may be an effective
treatment for any inflammatory disease in which monocytes,
mast cells, or basophils play major roles.
screening for eliminating compounds that displayed significant
blockade of the potassium current, which is another in vitro
indicator for causing QTc prolongation.
While a leading CCR2 antagonist MK-0812 failed to show
significant improvement on RAs in clinical trials, there has
been continuously intensive research interest in many distinct
chemical series as CCR2 antagonists for other indications.10-17
We have recently reported a novel series of 4-azetidinyl-1-
aryl-cyclohexanes as CCR2 antagonists.16 This scaffold is
characterized by a general hERG liable structure consisting of
a central basic amine flanked by two hydrophobes at the ends.
Divergent SARs on hCCR2 and hERG activities enabled us to
dial out hERG affinity and generated highly selective hCCR2
antagonists in the scaffold. The lead compound 1 (Figure 1)
possessed good hCCR2 binding affinity (IC50, 15 nM), potent
Figure 1. Early leads
functional activity (chemotaxis, IC50, 22 nM) and amendable
separation over hERG activities. Disappointingly, i.v. treat-
ment of anesthetized guinea pigs with 1 resulted in a signifi-
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Table 1. SAR of nitrogen containing six membered heterocyclic analogues
1
2
3
4
Het
N
NH
HO
O
NH
O
5
CF3
6
7
8
9
ID
hCCR2 binding
IC50 (nM)a
CTX
IC50 (nM)b
hERG
IC50 (ꢀM)c
Patch Clamp
%@3 ꢀM (control)d
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11
12
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58
59
60
190
650
nte
nt
35.0
43.8
28 7
19 7
2a
2b
190
170
45
nt
10.0
12.5
35.0
37.0
54 4
61 6
12 6
29 3
2c
2d
2e
2f
nt
74
56
44
310
220
nt
nt
22.5
21.6
61 10
55 5
2g
2h
71
nt
nt
17.5
25.0
43 3
30 3
2i
2j
250
18
19
50
49
27
30
33
44
17
43
14.5
7.0
68 2
nt
2k
2l
11.2
5.5
49 9
nt
2m
2n
2o
a
9.8
nt
b
MCP-1 Receptor Binding Assay in THP-1 Cells, for IC50>100 nM (n=1), for IC50<100 nM (n>2, average values, SEM< 25%); MCP-1 Induced
Chemotaxis in THP-1 Cells; c hERG 3H-astemizole binding activity on HEK-293 cell ; d The membrane K+ current IKr in hERG-transfected HEK293 cells
(n=3); e nt: not tested
In the discovery of lead compound 1, it was established that
the cyclohexyl azetidine core as well as the right-end bis-
amide functionality were critical for hCCR2 activity and might
be too sensitive for modifications.16 The left-end phenyl ring
was somehow tolerated for moderate to good hCCR2 activity.
For this reason, we focused further optimization on the left-
end aromatic group. Replacement of the phenyl group at the 1-
position on the cyclohexyl ring with the 3-pyridinyl group
resulted in a significant loss of hCCR2 activity but also further
attenuated hERG binding affinity (2a: hCCR2 IC50, 190 nM;
hERG IC50, 35 ꢀM).15-17 These data prompted us to take nitro-
gen containing six membered aromatic rings as the starting
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point for subsequent optimization (Table 1). Compound 2b
bearing a more polar pyrimidinyl group displayed even weaker
Table 2. SAR of five membered heterocyclic analogues
1
2
3
4
5
6
7
8
9
hCCR2 binding affinity (IC50, 650 nM) compared with 2a. We
then examined the substituent effect on both hCCR2 and
hERG inhibition among the pyridinyl derivatives. Electron
withdrawing groups such as fluoro or cyano did not improve
hCCR2 binding as indicated with 2c and 2d with IC50 of 190
nM and 170 nM respectively. In contrast, electron donating
group, exemplified by methoxy and methyl groups, had bene-
ficial effect on hCCR2 activity as evidenced by an approxi-
mate 3-5 fold increase of binding affinity (2e and 2f vs. 2a).
The improved potency of 2e and 2f was also translated into
good levels of activity in the chemotaxis assay with IC50 of 74
nM and 56 nM, respectively. To our delight, installation of
both substituents maintained good selectivity between hCCR2
and hERG binding affinities (hERG binding IC50 of 35 and 37
ꢀM). The weak hERG affinities of both compounds were fur-
ther confirmed with only 12% and 29% of inhibition at 3 ꢀM
in the patch clamp assay. The dimethylamino analogue 2g was
~ 8-fold weaker for hCCR2 binding than 2f. Addition of a
methyl group ortho to the methoxy group also resulted in sig-
nificant loss of hCCR2 affinity as shown by 2h (IC50, 220
nM). The two methoxy-pyridinyl regioisomers 2i and 2j dis-
played weaker hCCR2 binding affinities (IC50, 71 and 250
nM) compared with 2f indicating electronic density of the
pyridinyl ring had great impact on hCCR2 activity. As ex-
pected, installation of more lipophilic 4-alkoxy groups main-
tained or improved both hCCR2 binding affinity and chemo-
taxis activity as illustrated by 2k to 2o. Steric bulkiness on the
alkoxy group was well tolerated for good hCCR2 activity (2m,
2n, 2o). Unfortunately, the unwanted hERG signals among
these derivatives reappeared to be problematic for further
evaluations.
hCCR2 Binding
IC50 (nM)
hERG Binding
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ID
3
IC50 (ꢀM)
2,820
5,300
2,700
> 50
15.0
> 50
4
5
6
790
> 50
100
62
27.0
32.0
24.0
7
8a
9
36
Given the good in vitro selectivity between hCCR2 and
hERG activities, 2e and 2f were selected for evaluation in an
anesthetized guinea pig CV (GPCV) study. Disappointingly,
both 2e and 2f still displayed electro-cardiographic effects
consisting of dose dependent increase in ꢁQTc. With a dosage
of 10 mg/kg (i.v.), 2e and 2f exhibited +13% and +6% of the
ꢁQTc respectively. These results led us to re-define our crite-
ria on in vitro hERG profile for advancing compounds into
GPCV study. Our efforts then focused on identifying com-
pounds with decreased hERG affinity to our detection limit
(IC50, 50 ꢀM) while maintaining or improving hCCR2 activi-
ties. However, attempt on determining the direct correlation
between hERG binding and QTc prolongation could be chal-
lenging due to the impact on QTc from other factors, for an
example, serum protein binding. Therefore, the final selection
on the compounds for further development should be solely
determined by comprehensively in vivo CV safety evaluation.
abolished hERG affinity, it also significantly reduced hCCR2
activity. To our satisfaction, installation of a 2-thiophene (7),
2-thiazole (8a) or 2-isothiazole (9) at the 1-postion of the cy-
clohexyl ring was tolerated for good hCCR2 binding affinity
(IC50, 100, 62, 36 nM). Among them, 8a displayed relatively
weak hERG binding affinity with IC50 of 32 ꢀM and promis-
ing separation between hCCR2 and hERG affinities, which
promoted us to investigate this substitution pattern in detail.
Table 3 highlighted the SAR within a series of thiazole sub-
stituted analogues. Installation of a thiazole moiety at the 1-
position of cyclohexyl ring was generally well tolerated for
good hCCR2 activity. However, modification on the thiazole
group had great impact on in vitro hERG profile and hence
dominated the QTc effect in GPCV study. There was a trend
that more lipophilic thiazoles led to stronger hERG in vitro
activity. Compared to 8a, simple methylation at the 3-position
of the thiazole caused the blockade of hERG potassium cur-
rent from 35% to 49% at 3 ꢀM (8b). It is therefore not surpris-
ing that intravenous infusion of 8b in anesthetized guinea pigs
triggered a dose dependent QTc prolongation (ꢁQTc, +15% at
10 mg/kg). Compound 8c bearing a hydrophobic benzothia-
zole moiety possessed enhanced hERG in vitro activity. In-
stallation of a 5-thaizole group onto the 1-position of the cy-
clohexyl ring (8d) remarkably attenuated the hERG binding
affinity to greater than 50 ꢀM. Consistent with its weak hERG
affinity, 8d also displayed low inhibition on the potassium
current in the patch clamp study (21% inhibition at 3 ꢀM). To
our delight, intravenous infusion of 8d in anesthetized guinea
Reviewing of recent literatures led us to postulate that the
weak basic pyridinyl group of 2e and 2f may participate in a
cation-π interaction with the Tyr-652 residue of the hERG
channel.18 Thus, elimination of the basic feature of the pyridi-
nyl group would disrupt this interaction and further attenuate
hERG binding. For chemistry effort, we decided to examine
the left-end aromatic ring with a series of five membered het-
erocyclic analogues. As illustrated in Table 2, substitution by
five membered heterocycle had great impact on hCCR2 affini-
ty. While incorporation of an oxazole (3), imidazole (4), pyr-
rozole (5) or thiadiazole (6) either efficiently attenuated or
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pigs had no effect on QTc up to 10 mg/kg. Plasma concentra- mg/kg (i.v.) in GPCV study, the plasma concentration was
1
2
3
4
5
6
7
8
tion reached at 26 ꢀM. Having identified 5-thiazole group as
the preferred left-end moiety, we extended our SAR studies to
several substituted 5-thiazole analogues. Compound 8e and 8f
with small alkyl substitutions displayed good hCCR2 activities
and desired in vitro hERG profile. Furthermore, 8f demon-
strated no effect on QTc up to 10 mg/kg (i.v.) in GPCV study
(plasma level, 10 ꢀM). It is noteworthy that the 5-thiazole is
not considered as a general pharmacophore for suppressing
hERG in vitro activities and eliminating QTc prolongation in
GPCV study. As illustrated by 8g and 8h, while maintaining
good hCCR2 activities, they both possessed enhanced inhibi-
tion on the potassium current (49% and 77% inhibition at 3
ꢀM). While compound 8g displayed no effect on QTc up to 10
only ~ 3 ꢀM which did not provide enough safety margin for
further development. Here, an extremely narrow line appeared
to determine the CV safety profile of this series.
Given its superior in vitro hERG and GPCV safety profiles,
8d was selected to be further evaluated in an anesthetized dog
CV safety study. Strikingly, it did not induce dose-dependent
or notable effects on most cardiohemodynamic, functional
respiratory and electrophysiological parameters up to 10
mg/kg (i.v.) with plasma level at 70 ꢀM. It also did not induce
changes in ECG morphology. Overall, 8d represented the first
compound with good CV safety profile as a development can-
didate.
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Table 3. SAR of thiazole substituted analogues
ID
Ar
hCCR2 binding CTX
IC50 (nM)
hERG Binding
Patch Clamp
GPCV
ꢁQTc@10 mg/kg (+%)a
IC50 (nM)
IC50 (ꢀM)
% @ 3 ꢀM (control)
62
13
60
32
28
30 4
49 3
nt
8a
18
16
+15
8b
8c
28
6.2
80 9
nt
37
78
30
65
> 50
> 50
21 1
21 5
no effect
nt
8d
8e
N
57
70
> 50
35 3
no effect
8f
Me
Me
S
5
4
28
49 6
77 5
no effect
nt
8g
8h
41
68
6.0
a
Cumulative doses (0.1, 0.3, 1, 3 and 10 mg/kg) were administered incrementally as five-minute i.v. infusions at 0, 20, 40, 60 and 80 minutes. QTc in-
tervals (ꢁQTc) were measured and recorded at each time point as a percent change from baseline for each animal. Values are mean SEM (n=3)
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Scheme 1. Synthesis of 8d
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7
8
9
10
11
12
(a) n-BuLi, -78oC. (b) TBAF, 2 steps, 55%. (C) HCl, acetone, room temperature, 90%. (d) NaBH(OAc)3, TEA, DCM, room temperature,
35% along with its isomer, 40%.
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Compound 8d was a selective hCCR2 inhibitor, showing no
significant inhibitory activity at concentration of 10 µM in
Cerep and Invitrogen protein kinase panel screens. It did not
significantly inhibit the binding of any of the relevant chemo-
kines tested including CCR1, CCR3, CCR4, CCR5, CXCR1,
CXCR2, CXCR4, and CX3CR1 at concentration of 25 ꢀM.
nists. Through divergent SARs of hCCR2 and hERG on the
left-end heterocyclic ring, 8d was identified as the lead com-
pound. It exhibits potent hCCR2 activity, high selectivity,
weak hERG affinity and good oral bioavailability. Evaluated
in guinea pigs and dogs, 8d possesses a clean CV safety pro-
file and provides good safety margin as a development candi-
date.
Compound 8d also exhibited amendable DMPK profiles for
further development. It was highly orally bioavailable and
exhibited oral bioavailability of 70.2% in dogs when dosed at
6.7 mg/kg using 0.5% methocel as a vehicle (Cmax = 1617
ng/mL, AUClast = 5887 h*ng/mL). In addition, it showed
25.4% oral bioavailability in non-human primates (7.2 mg/kg
p.o., Cmax = 740 ng/mL, AUClast = 3061 h*ng/mL), but only
19% of oral bioavailability in mice (10 mg/kg p.o., Cmax = 74
ng/mL, AUClast = 204 h*ng/mL), and 15.3% oral bioavailabil-
ity in rats (10 mg/kg p.o., Cmax = 100 ng/mL, AUClast = 416
h*ng/mL).
ASSOCIATED CONTENT
Supporting Information. Experimental procedures for the syn-
thesis of 8d and characterization data for 2a-2o, 3-7, 8a-h, 9 as
well as in vitro and in vivo biological protocols are available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 215-628-7877. Fax: 215-540-4612. E-mail:
xzhang5@its.jnj.com
Compound 8d only displayed a Ki of 9.6 ꢀM for mCCR2
binding. Human CCR2 knock-in/murine CCR2 knock-out
mice (hCCR2KI) have been adopted to overcome the species
issue and validate in vivo models. Compound 8d was as-
sessed in several pharmacological models using hCCR2KI
mice. In a thioglycollate-induced peritonitis (TG) model, it
dose-dependently inhibited the influx of leukocytes, mono-
cytes/macrophages and T-lymphocytes into the peritoneal
cavity with an ED50 of 3 mg/kg p.o. bid. In an OVA-induced
asthma model, 8d inhibited airway eosinophil infiltration by
89% at a dose of 10 mg/kg p.o. bid. The efficacy of 8d in
mouse model suggests that it may have potential as a single
agent in asthma to improve signs and symptoms and reduce
airway resistance. The detailed pharmacological profile of 8d
will be reported later.
Notes
The authors declare no competing financial interest..
ACKNOWLEDGMENT
We thank LG Biology, CoE for Cardiovascular Safety Research,
ADME/PK and HOPS at JRD for technical assistance.
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The synthesis of this series is exemplified by compound 8d
as illustrated in Scheme 1. Thiazole 10 was treated with n-
BuLi in THF at –78oC and the resulting anion was reacted
with commercially available ketone 11 followed by desilyla-
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gave the corresponding ketone 13 in 90% yield. Reductive
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