Discovery of (1R,2R)-N-(4-(6-isopropylpyridin-2-yl)-3-(2-methyl-2H-indazol- 5-yl)isothiazol-5-yl)-2-methylcyclopropanecarboxamide, a potent and orally efficacious mGlu5 receptor negative allosteric modulator

Article abstract of DOI:10.1016/j.bmcl.2013.01.009

A novel series of selective negative allosteric modulators (NAMs) for metabotropic glutamate receptor 5 (mGlu5) was discovered from an isothiazole scaffold. One compound of this series, (1R,2R)-N-(4-(6-isopropylpyridin-2-yl)-3- (2-methyl-2H-indazol-5-yl)isothiazol-5-yl)-2-methylcyclopropanecarboxamide (24), demonstrated satisfactory pharmacokinetic properties and, following oral dosing in rats, produced dose-dependent and long-lasting mGlu5 receptor occupancy. Consistent with the hypothesis that blockade of mGlu5 receptors will produce analgesic effects in mammals, compound 24 produced a dose-dependent reduction in paw licking responses in the formalin model of persistent pain.

Full text of DOI:10.1016/j.bmcl.2013.01.009

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1249–1252
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of (1R,2R)-N-(4-(6-isopropylpyridin-2-yl)-3-(2-methyl-2H-
indazol-5-yl)isothiazol-5-yl)-2-methylcyclopropanecarboxamide,
a potent and orally efficacious mGlu5 receptor negative allosteric
modulator
b
a
b
a,
Junliang Hao a, , Veronique Dehlinger , Adam M. Fivush , Helene C. E. Rudyk , Thomas C. Britton
,
⇑
Sean P. Hollinshead a, Benjamin P. Vokits a, , Barry P. Clark b, , Steven S. Henry a, Steven M. Massey a,
Langu Peng a, Bruce A. Dressman a, Beverly A. Heinz a, Edda F. Roberts a, Mallorie R. Bracey-Walker a,
Steven Swanson a, John T. Catlow a, Patrick L. Love a, , Anita D. Tepool a, , Steven C. Peters a,
Rosa Maria A. Simmons a, Smriti Iyengar a, David L. McKinzie a, James A. Monn a
,
a Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
b Lilly Research Center, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of selective negative allosteric modulators (NAMs) for metabotropic glutamate receptor 5
(mGlu5) was discovered from an isothiazole scaffold. One compound of this series, (1R,2R)-N-(4-(6-iso-
propylpyridin-2-yl)-3-(2-methyl-2H-indazol-5-yl)isothiazol-5-yl)-2-methylcyclopropanecarboxamide
(24), demonstrated satisfactory pharmacokinetic properties and, following oral dosing in rats, produced
dose-dependent and long-lasting mGlu5 receptor occupancy. Consistent with the hypothesis that
blockade of mGlu5 receptors will produce analgesic effects in mammals, compound 24 produced a
dose-dependent reduction in paw licking responses in the formalin model of persistent pain.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 7 November 2012
Revised 28 December 2012
Accepted 2 January 2013
Available online 12 January 2013
Keywords:
mGlu5 receptor
Negative allosteric modulator
Receptor occupancy
Formalin model
Rotorod test
Glutamate is the major excitatory neurotransmitter in the
mammalian central nervous system. The glutamate receptors are
classified into two types: ionotropic glutamate (iGlu) receptors
and metabotropic glutamate (mGlu) receptors. While the iGlu
receptors are ligand-gated ion channels, the mGlu receptors belong
to class C G-protein coupled receptor (GPCR) family.1 Eight mGlu
receptors (mGlu1-8) have been identified to date, and are further
classified into three groups (I–III) based on sequence homology,
signaling mechanisms, and pharmacology. Briefly, group I mGlu
receptors (mGlu1 and mGlu5) are predominantly localized in the
tic localization and are coupled to G i/o. Agonist activation of these
a
receptors leads (via the G subunits) to the inhibition of adenylate
a
cyclase and a concomitant reduction of cAMP formation. In addi-
tion, agonist activation of these receptors can also lead (via Gb
c
subunit activation) to the inhibition of voltage-gated Ca2+ chan-
nels2 and the activation of inwardly rectifying K+ channels.3
The mGlu5 receptors have been widely studied since the first
report of selective mGlu5 negative allosteric modulators (NAMs)
(E)-6-methyl-2-(phenyldiazenyl)pyridin-3-ol (Fig. 1, 1, SIB-1757)
and (E)-2-methyl-6-styrylpyridine (2, SIB-1893)4 and subsequent
identification and characterization of important tool compounds
2-methyl-6-(phenylethynyl)pyridine (3, MPEP)5 and 3-((2-methyl-
4-thiazolyl)ethynyl)pyridine (4, MTEP).6 These tool compounds
have demonstrated efficacy in numerous preclinical models of
CNS-related diseases such as depression,7 anxiety,8,9 and pain.10,11
Because of robust preclinical activity and clinical therapeutic po-
tential, there has been an intense effort to identify potent mGlu5
receptor NAMs with novel scaffolds.12,13 Recently we reported an
mGlu1 receptor selective NAM, (1R,2R)-N-(3-(4-methoxyphenyl)-
4-methylisothiazol-5-yl)-2-methylcyclopropane-carboxamide (5,
hmGlu1 IC50 = 9 2 nM, hmGlu5 IC50 = 158 65 nM) based on a
postsynaptic density and are coupled to G q. Agonist activation
a
of these receptors leads to the activation of phospholipase C,
resulting in increased intracellular second messengers such as ino-
sitol triphosphate (IP3) and diacylglycerol (DAG) and subsequent
intracellular Ca2+ release. Group II (mGlu2 and mGlu3) and group
III (mGlu4, mGlu6, mGlu7, mGlu8) show a predominant presynap-
⇑
Corresponding author. Tel.: +1 317 277 5703; fax: +1 317 276 7600.
E-mail address: haoju@lilly.com (J. Hao).
T. C. Britton, B. P. Votkis, B. P. Clark, P. L. Love and A. D. Tepool were employees of
Eli Lilly and Company when this work was conducted.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.009

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J. Hao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1249–1252
novel isothiazole scaffold.14 Here, we report further optimization of
the isothiazole scaffold, which has resulted in mGlu5 receptor
NAMs with good selectivity profiles (>20-fold) against mGlu1
receptors. We have furthermore characterized an optimized analog
from this work, compound 24, with respect to its pharmacokinetic
properties, mGlu5 receptor occupancy, and antinociceptive effects
in rats.
The synthesis started with the commercially available 2-cya-
noethanethioamide 6 (Scheme 1). Condensation of 6 with acetyl
chloride afforded compound 7. Treatment of 7 in AcOH with Br2
gave the cyclized isothiazole 8,15 which was condensed with acyl
chloride 914 to provide 3-bromoisothiazole 10. Transformation of
C4 acetyl to bromide was achieved with 4.5 equiv of Br2 through
a tandem haloform/Hunsdiecker-type decarboxylative reaction16
to yield the essential intermediate dibromide 11. Coupling of 3,4-
dibromoisothiazole 11 with boronic ester 12 under Suzuki–Miya-
ura conditions17 occurred selectively at C3 bromide to provide
compound 13. Alternatively, compound 13 was obtained by the Su-
zuki coupling of 12 with bromide 10, followed by the tandem
decarboxylative reaction. This also confirmed the regio-selectivity
of the Suzuki reaction on dibromide 11. Finally, desired com-
pounds 15–32 were obtained when the bromide 13 was reacted
with a variety of aryl stannanes (14) with the highly active (t-
Bu3P)2Pd18 as a catalyst in hot 1,2-dimethoxyethane (DME).
During the course of our continued optimization of the isothiaz-
ole scaffold, we discovered that replacement of the p-methoxyben-
zyl at C3 and the methyl at C4 on the isothiazole of compound 5
with the 2-methylindazol-5-yl at C3 and the 2-pyridyl at C4,
respectively, gave compound 15 that was modestly mGlu5 recep-
tor selective (8-fold), with an IC50 = 29 11 nM (Table 1).19 Adding
a methyl group at the C6 of the pyridine ring (16) did not signifi-
cantly change the mGlu5 potency, but increased the selectivity to
about 50-fold. Further increasing the size of the C6 group on the
pyridine ring from methyl to ethyl (19), n-propyl (23) or i-propyl
(24), increased the mGlu5 potency (vs 16) slightly, but decreased
the selectivity. Changing the alkyl groups at C6 of the pyridine ring
to cycloalkyls (25–27), or adding fluorines (17, 18) did not mean-
ingfully affect mGlu5 potency, but decreased selectivity when
compared to 16. Compound 20 with the methoxyl group at the
C6 position of the pyridine ring was the least selective compound
within this series. Replacement of the methoxyl group by methyl-
amino (21) increased selectivity, and dimethylamino substitution
(22) further increased the selectivity to 477-fold. Substituting the
methyl group on the C3 indazole with ethyl further decreased
mGlu1 potency and increased selectivity (28 vs 22). Removing
the methyl group on the cyclopropyl amide gave compounds (29,
30, 31) with significantly better selectivity over mGlu1 receptors,
with compound 31 having no measurable mGlu1 antagonist activ-
ity up to the highest tested concentration. Finally, removal of the
methyl group on the indazole (32) decreased selectivity (vs 24).
Compound 24 was chosen for additional pharmacological profiling
since it displayed high receptor occupancy at the single dose
screen (data not shown). When examined for functional activity
in cells expressing recombinant rat mGlu5 receptors, 24 demon-
strated an IC50 = 167 49 nM, about 10-fold less potent than what
was observed in cells expressing human mGlu5 receptors. A broad-
er assessment of the in vitro pharmacological selectivity of com-
pound 24 was also investigated against other mGlu receptors and
a panel of 23 other CNS targets at CEREP™. We found less than
N
N
N
N
N
OH
2 SIB-1893
1 SIB-1757
3 MPEP
N
O
N
S
N
N
H
H3CO
S
4 MTEP
5
Figure 1. Representative mGlu5 and mGlu1 receptor NAMs.
50% inhibition when tested at a concentration of 1 lM in all assays
except 5-HT2B (56% inhibition, binding affinity Ki = 3160 nM). Fur-
thermore, 5-HT2B functional activity of compound 24 was deter-
mined. It proved to have no measurable agonist activity (À8.2%
S
N
S
S
a
b
NC
NH2
Br
NH2
NC
Cl
NH2
at 10
at 10
lM), whereas it did demonstrate antagonist activity (45.3%
O
O
l
M).
6
7
8
The pharmacokinetic attributes of compound 24 were next
evaluated in male Harlan Sprague Dawley (HSD) rats. Plasma con-
centrations were determined after oral and IV administrations at
1.5 and 1 mg/kg, respectively.20 After oral dosing, the plasma Cmax
(255 32 ng/mL) was attained at 2 h, and the AUC0–24h was
1570 145 ng h/mL (Fig. 2). The IV and oral elimination half-lives
were comparable at 4.9 1.5 h and 4.6 0.7 h, respectively. The
observed clearance was 8.4 1.4 mL/min/kg and the volume of dis-
tribution was 2.7 0.4 L/kg. The bioavailability of 24 was calcu-
lated to be 56.5 2.0%.
We next determined whether compound 24 would afford
meaningful mGlu5 receptor occupancy (RO) in fasted and drug na-
ive HSD rats after oral dosing.21,22 In this study, MPEPy was used as
the RO tracer.23 Within the same rats, mGlu1 RO was also deter-
mined using 1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-
phenylethanone24 as the mGlu1 RO tracer.25 As shown in Figure 3,
measured receptor occupancy was plotted against oral doses of 24
(0.3, 1, 3, 10 mg/kg). Compound 24 showed dose-dependent
mGlu5 receptor occupancy (ED50 = 2.1 mg/kg) without appreciable
receptor occupancy at mGlu1 receptors up to 10 mg/kg. Next, a
time-course study measured mGlu5 RO of compound 24 following
a single oral dose of 10 mg/kg. In this experiment, significant
mGlu5 RO was sustained over a 24 h period (Table 2). During these
O
R3
O
O
R3
N
S
R3
N
S
d
9
N
H
Br
N
Br
H
c
Br
O
10
11
O
B
O
O
R2
N
R3
N
S
R2
N
N
12
N
H
N
e
Br
13
SnBu3
N
O
R3
N
S
R2
N
R1
N
3
4
N
H
14
N
f
R1
15-32
Scheme 1. Reagents and conditions: (a) AcCl, pyridine, 0 °C to rt, 91%; (b) Br2,
AcOH, 40 °C, 72%; (c) Et3N, CH2Cl2, rt, 60–82%; (d) 5 N NaOH, Br2, 1,4-dioxane, À10
to 0 °C, 55–85%; (e) PdCl2(PPh3)2, 2 M aq Na2CO3, DME, 80 °C, 17–51%; (f) (t-
Bu3P)2Pd, DME, 100 °C; then 1 M HCl in Et2O, EtOAc, 24–65%.

J. Hao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1249–1252
1251
Table 1
mGlu5 and mGlu1 receptor functional activities (IC50) for compounds 15–32
Compound
R1
R2
R3
hmGlu5a (nM)
hmGlu1a (nM)
Selectivity
15b
16b
17
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH2CH3
CH3
CH3
CH3
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
29 11
45 11
50 21
81 49
238 55
2210 474
1300 219
1020 149
279 35
8
49
26
13
28
5
26
477
20
34
25
13
33
>357
86
102
>266
10
CH2F
CHF2
CH2CH3
OCH3
NHCH3
N(CH3)2
nPr
18
19b
20
10
2
36 15
62 14
190 48
21b
22
1590 273
9540 388
409 145
644 128
735 66
687 162
891 153
>12,500
20
20
4
3
23
24b
25b
26b
27
iPr
c-Pr
c-Butyl
c-Pentyl
N(CH3)2
CH2CH3
iPr
19 10
30
54 18
9
27
35
36
9
7
9
28b
29b
30b
31b
32b
3110 974
4570 953
>12,500
H
H
CH3
45 13
47
39 11
N(CH3)2
7
iPr
397 75
a
Represents the geometric mean ( SEM) of at least three separate determinations.
HCl salt.
b
Table 2
1000
IV administration; 1 mg/kg
Time course of mGlu5 RO and brain levels of compound 24 following a
PO administration; 1.5 mg/kg
10 mg/kg oral administration in rats
Time (h)
% mGlu5 RO
Brain levels (nM)
100
0.5
1
2
4
6
37.8 5.6
57.8 7.4
74.3 9.9
67.7 7.4
62.5 0.8
58.9 3.2
1245 232
2227 164
2851 135
2593 169
2975 263
2339 65
10
24
Data are represented as mean SEM.
0
2
4
6
8
10 12 14 16
Time (hr)
Figure 4, oral administration of compound 24 in fasted male HSD
rats resulted in dose dependent attenuation of late phase forma-
lin-induced paw-licking pain behavior. Statistically significant ef-
fects28 were noted between 10 and 60 mg/kg with a calculated
absolute ED50 of 25.5 mg/kg.
Figure 2. Plasma concentrations of compound 24 in rats following oral and
intravenous administration. Data are represented as mean SEM.
Performance on the rotorod29 was used as a measure of ataxia
or sedation to verify that the apparent analgesic effects of com-
pound 24 in the formalin model were not attributable to non-spe-
cific neurological consequences of the drug. The ability of
compound 24 to induce deficits in rotorod performance was exam-
ined using an automated accelerating rotorod at the highest dose
tested in the formalin model (60 mg/kg).30 No statistically signifi-
cant deficits28 were noted in the test for up to 4 h, indicating that
100
mGlu5
mGlu1
75
50
25
0
800
0.1
1
10
Vehicle
1 mg/kg
3 mg/kg
10 mg/kg
30 mg/kg
60 mg/kg
700
Dose (mg/kg)
600
*
Figure 3. Dose–response studies measuring mGlu5 and mGlu1 receptor occupancy
500
*
*
of compound 24 following
represented as mean SEM.
a 60 min oral administration in rats. Data are
400
300
200
100
0
studies, hippocampal concentrations of 24 were also measured for
each time point. Similar to the mGlu5 RO data, brain exposure was
high and was sustained out to 24 h after dose, and the ratio of brain
exposure to RO remained relatively constant throughout this time
period.
Since mGlu5 NAMs have demonstrated robust efficacy in ani-
mal models for pain,10,11 compound 24 was evaluated in the for-
malin paw-licking model of persistent pain.26,27 As shown in
Early Phase
Late Phase
Figure 4. Effect of compound 24 on formalin-induced paw-licking behavior in rats.
Data are expressed as paw-licking pain behavior events (mean SEM) in early
phase (0–5 min) or late phase (11–40 min). ⁄p <0.05 significantly different from
vehicle group.

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J. Hao et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1249–1252
mGlu1 and mGlu5 receptors. The mGlu receptor-mediated responses were
determined by changes in intracellular calcium concentrations measured by a
fluorescent calcium sensitive dye Fluro-3 using a 96-channel fluorometric
imaging reader (FLIPR-Molecular Devices Corp.; La Jolla, CA, USA). The
antagonist effects of compounds were quantified using 10-point dose curves
by comparing the peak fluorescent responses to the agonist glutamate in the
presence and absence of compound. The assay window was defined as the
maximal response obtained by glutamate at its predetermined EC90%
concentration minus the response obtained by buffer alone. Antagonists
effects were calculated as a percent of the assay window. All data were
calculated as relative IC50 values using a four-parameter logistic curve fitting
program (ActivityBase v5.3.1.22).
the analgesic response of compound 24 in the formalin assay was
likely not caused by a general motor deficit (data not shown).
In summary, we have discovered a novel series of selective
mGlu5 receptor NAMs based on the isothiazole scaffold, repre-
sented by compound 24. We demonstrated that compound 24 dis-
played satisfactory pharmacokinetic properties in rats, and showed
dose responsive central mGlu5, but not mGlu1, receptor occu-
pancy. Furthermore, we determined that compound 24 was orally
efficacious in the formalin model of persistent pain at doses consis-
tent with selective mGlu5 receptor engagement.31
20. The oral doses were delivered as a suspension consisting of 1% carboxymethyl
cellulose (CMC), and 0.25% Tween 80, and the IV doses were delivered as a
solution in 10% Pharmasolve:10% PEG400:20% Captisol (10:10:80).
21. Chernet, E.; Martin, L. J.; Li, D.; Need, A. B.; Barth, V. N.; Rash, K. S.; Phebus, L. A.
Life Sci. 2005, 78, 340.
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22. Rats received compound 24 or vehicle (same as in PK study), and after 1 h, both
groups were intravenously administered (via lateral tail vein) the mGlu5
receptor RO tracer, MPEPy (10 lg/kg), and sacrificed 20 min later. Both the
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receptor density levels) were dissected and analyzed. Using LCMS
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27. In this assay, compound 24 or vehicle (same as in PK study) was dosed orally
(1, 3, 10, 30, or 60 mg/kg) in fasted male HSD rats. Formalin (50 lL; 5%) was
injected subcutaneously into the intraplantar surface of the right hindpaw
after 1 h. Paw-licking pain behavior was measured as number of events, from
0–60 min after formalin administration.
28. All data were analyzed with ANOVA followed by Dunnett’s test using
JMPv6.0.2 (SAS Institute, Cary, NC) statistical software.
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30. Rotorod testing was conducted in trained rats prior to administration of 24 and
again at 1, 2, 3 and 4 h after oral administration of 60 mg/kg. Rats that
maintained posture and did not fall off the rotorod were given a maximum
score of 40 s.
31. All the compounds disclosed in this paper were covered by this patent: Britton,
T. C.; Dehlinger, V.; Fivush, A. M.; Hollinshead, S. P.; Vokits, B. P. 3-Indazolyl-4-
Pyridylisothiazoles. WO2009/123855 A1, 2009.
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19. The relative potency and selectivity of the compounds in this Letter were
evaluated on stable AV12 clonal cell lines expressing recombinant human