Fused thiazolyl alkynes as potent mGlu5 receptor positive allosteric modulators

Article abstract of DOI:10.1016/j.bmcl.2013.05.070

A new series of potent fused thiazole mGlu₅ receptor positive allosteric modulators (PAMs) (10, 11 and 27-31) are disclosed and details of the SAR and optimization are described. Optimization of alkynyl thiazole 9 (Lu AF11205) led to the identification of potent fused thiazole analogs 10b, 27a, 28j and 31d. In general, substituted cycloalkyl, aryl and heteroaryl carboxamides, and carbamate analogs are mGlu₅ PAMs, whereas smaller alkyl carboxamide, sulfonamide and sulfamide analogs tend to be mGlu₅ negative allosteric modulators (NAMs).

Full text of DOI:10.1016/j.bmcl.2013.05.070

Bioorganic & Medicinal Chemistry Letters xxx (2013) xxx–xxx
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fused thiazolyl alkynes as potent mGlu5 receptor positive allosteric
modulators
⇑
Mathivanan Packiarajan , Michel Grenon, Samuel Zorn, Allen T. Hopper, Andrew D. White,
Gamini Chandrasena, Xiaosui Pu, Robbin M. Brodbeck, Albert J. Robichaud
Lundbeck Research USA, 215 College Road, Paramus, NJ 07652, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of potent fused thiazole mGlu5 receptor positive allosteric modulators (PAMs) (10, 11 and
27–31) are disclosed and details of the SAR and optimization are described. Optimization of alkynyl thi-
azole 9 (Lu AF11205) led to the identification of potent fused thiazole analogs 10b, 27a, 28j and 31d. In
general, substituted cycloalkyl, aryl and heteroaryl carboxamides, and carbamate analogs are mGlu5
PAMs, whereas smaller alkyl carboxamide, sulfonamide and sulfamide analogs tend to be mGlu5 negative
allosteric modulators (NAMs).
Received 18 April 2013
Revised 17 May 2013
Accepted 20 May 2013
Available online xxxx
Keywords:
mGlu5 Positive allosteric modulators
Fused thiazolyl aryl
Ó 2013 Elsevier Ltd. All rights reserved.
Cycloalkyl carboxamides and carbamates—
PAMs
Fused thiazolyl sulfonamides and
sulfamides—NAMs
PAM/NAM switching
Glutathione adducts
Glutamate is the major excitatory neurotransmitter in the
mammalian central nervous system, where it mediates its effects
through three ionotropic (AMPA, kinate and NMDA) and eight
metabotropic receptors (mGlu1 to mGlu8).1 The metabotropic glu-
tamate receptors (mGlus) are class C GPCRs and contain two dis-
tinct binding domains; a large extracellular domain that binds
glutamate and a heptahelical transmembrane (7TM) domain that
binds a variety of ligands at one or more allosteric binding sites.2
The mGlu5 receptor structurally and pharmacologically associates
with the NMDA receptor to modulate roles in synaptic plasticity,
cognition, and the learning and memory process.3 mGlu5 receptors
are primarily located post synaptically and are highly expressed in
the limbic brain regions,4a,4b also expressed by astrocytes.4c,4d Acti-
vation of the mGlu5 receptor is reported to potentiate NMDA
receptor function5b and thus positive allosteric activation of mGlu5
could indirectly correct NMDA hypo-function. It has been postu-
lated that positive modulation of mGlu5 receptor could treat the
positive symptoms and cognitive deficits in schizophrenia,5–9 and
also improve spatial learning10,11 and cognition.12
distinct chemotypes reported from various groups.14–19 Com-
pounds CDPPB (1)15 and ADX47273 (2)16 have been extensively
used as tools to probe the utility of mGlu5 positive allosteric mod-
ulation (Fig. 1). Compounds 317 and 418 have been shown to re-
verse amphetamine-induced hyperlocomotion and conditioned
avoidance responding. In addition, we have reported on several no-
vel classes of mGlu5 PAMs including nicotinamides 5,19b lactams
6,19c N-aryl pyrrolidinonyl oxadiazoles 7,19d azetidinyl carboxam-
ides 819e and alkynylthiazole carboxamides 9.19f
Early lead generation efforts led to the identification of alkynyl-
thiazole carboxamide compound 9 (Lu AF11205), a potent mGlu5
PAM (EC50 6.1 nM; Emax 167%). In addition, compound 9 is effica-
cious in the 24 h novel object recognition (NOR) and phencyclidine
(PCP) disrupted novel object recognition behavioral models in rats.
However, in vitro metabolic studies demonstrated that compound
9 forms significant amounts of glutathione adducts, thus hindering
further development. We envisioned two strategies to further opti-
mize this series and potentially minimize glutathione adducts for-
mation. The first one, is to expand SAR knowledge by fusing the
thiazole ring with the alkyl group of the carboxamide to form a
conformationally constrained analog as shown in Figure 2 (path
a). The second one was based on transposing the carbonyl group
to a position exocyclic to the ring as shown (Fig. 2, path b). To en-
able this strategy we synthesized a series of fused thiazole analogs
There has been considerable recent interest in the identification
of mGlu5 PAMs,13 as evidenced by the variety of structurally
⇑
Corresponding author. Tel.: +1 201 820 1971; fax: +1 201 261 0623.
E-mail address: packiara@yahoo.com (M. Packiarajan).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.070
Please cite this article in press as: Packiarajan, M.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.05.070

2
M. Packiarajan et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
N
N
O
Cl
N
F
F
H
N
N
N
N
N
O
CN
O
O
O
N
3
2: ADX47273
1: CDPPB
F
O
N
O
N
HN
HN
N
N
N
4
5
6
O
N
O
O
O
N
Cl
N
N
N
N
Cl
N
N
F
S
7
8
F
O
9: LuAF11205
F
Figure 1. mGlu5 Receptor positive allosteric modulators.
a
F
N
S
N
a
N
S
b
F
N
N
N
S
O
O
O
9: Lu AF11205
EC50 6.1 nM (167%)
28j: EC50 18 nM (89%)
10b: EC50 2.4 nM (180%)
b
Figure 2. Fused thiazoles as mGlu5 PAMs. The mGlu5 PAM EC50 and Emax data are shown.
O
O
O
O
O
O
R
S
S
BOC
Br
O
S
N
d
S
e
BOC
b
N
c
N
N
N
a
N
N
Br
Ar
Ar
d
N
g
N
N
N
O
12
13
14
15
10a, c-i
10b
O
O
O
O
O
O
S
N
S
S
S
N
S
S
N
O
i
OEt
OEt
Br
f
OEt
Br
h
Br
R
N
Br
Br
Br
N R
NHR
N
N
NHR
N
17
18
19a-b
20a-b
21a-b
11a-b
Scheme 1. Reagents and conditions: (a) NBS, THF, rt, 1 h, 87%; (b) Thiourea, K2CO3, DMF, 150 °C, 12 h, 68%; (c) CuBr2, Isoamyl nitrite, DMF, rt, 1 h, 39%; (d) (Ph3P)2PdCl2, CuI,
TEA, microwave, 140 °C, 15 min, 10–45%; (e) NaH, THF, 0 °C then MeI, 0 °C to rt, 65%; (f) NBS, AIBN, CCl4, >95%; (g) RNH2, DMF, K2CO3, heat, 58–76%; (h) LiOH, THF/H2O, rt,
15 h, >99% and (i) PyBOP, CH2Cl2, Et3N, reflux, 20 h, 35–55%.
Table 1
SAR of 2-(arylethynyl)-6,7-dihydrothiazolo[5,4-c]pyridin-4(5H)-one analogs 10
O
R1
S
N
N
X
n
10
R
Compound
X
R
R1
n
EC50 (nM)
EMax (%)
IC50 (nM)
Inh. (%)
10a
10b
10c
10d
10e
10f
10g
10h
10i
CH
CH
CH
CH
CH
CH
CH
CH
N
H
H
H
CH3
H
H
H
H
H
H
H
1
1
1
1
1
1
1
1
1
112
2.4
290
2500
1500
5200
>10,000
>10,000
51
243
180
310
120
320
310
44
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
À260
À170
À340
À180
À390
À350
À34
3-F
3-Cl
2-F
4-F
2-OMe
3-Me
H
50
120
À22
À48
11a
11b
CH
CH
H
H
0
0
2.5
150
11
>10,000
23
À16
>10,000
59
aThe mGlu5 EC50 and IC50 FLIPR data were generated using a human mGlu5 cell line.21
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M. Packiarajan et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
3
Boc
Boc
Boc
S
S
Boc
N
a
b
N
c
S
N
N
d
S
N
H2N
Br
NH
Ar
Ar
N
N
O
N
22
23
24
25a - c
26a - c
Scheme 2. Reagents and conditions: (a) Pyrrolidine, cat. PTSA, Azeotropic condition, 2 h, then Sulfur, cyanamide, rt, 16 h, 70%; (b) t-BuONO, CuBr2, DMF, 0 °C to rt, 1 h, 43%;
(c) Aryl alkynes, (Ph3P)2PdCl2, CuI, Et3N, reflux, 1 h, 48–53% and (d) 4 M HCl in dioxane, CH2Cl2, >99%.
O
O
R
R
S
N
S
N
S
N
Ar
N
R
Ar
Ar
a
b
c
27: R = alkyl, cycloalkyl
30
31
S
NH
O
Ar
O
N
S
N
R
S
N
N
N
R
d
Ar
26a - c
N
28: R = alkyl, cycloalkyl
29: R = Aryl and hetAr
Scheme 3. Reagents and conditions: (a) RCHO, NaBH(OAc)3, DCE, rt, 23–58%; (b) RCOCl, Et3N, CH2Cl2, rt or RCO2H, HBTU, Et3N, CH2Cl2, rt, overnight, 40–63%; (c) RSO2Cl, Et3N,
CH2Cl2, rt, 30–60% and (d) (i) 4-NO2C6H4OCOCl, DIEA, rt, 84%, and (ii) R1R1NH, CH2Cl2, Et3N, rt, 25–65%.
based on 10b and 28j. In this letter we report our efforts towards
the synthesis and optimization of such fused thiazole based mGlu5
receptor PAMs.
sumed that the extensive conjugation within these molecules
facilitates Michael type thiol addition to both the alkyne and thia-
zole moieties. We envisioned that reducing conjugation by moving
the carbonyl group exocyclic to the ring would minimize glutathi-
one adduct formation. To test this hypothesis we synthesized ana-
logs 27–31 utilizing the key precursor 26 in a manner similar to a
literature method as shown in Scheme 2.23
N-Boc-piperidin-4-one 22 was heated with pyrrolidine in the
presence of a catalytic amount of p-TsOH for 2 h to yield the enam-
ine, followed by treatment with elemental sulfur and cyanamide to
provide N-Boc-protected thiazolo[5,4-c]piperidine 23 in 70% yield.
Diazotization with t-BuONO in the presence of CuBr2 yielded 2-
bromo-thiazole analog 24 in 43% yield. Subsequent Sonogashira
coupling with aryl alkynes under microwave conditions yielded
N-Boc protected aryl alkynes 25a–c in 48–53% yield.20c N-Boc
deprotection under acidic conditions (4 M HCl in dioxane at 0 °C)
afforded desired 2-(arylethynyl)-4,5,6,7-tetrahydrothiazolo[5,4-
c]pyridine HCl salts 26a–c in near quantitative yield. Reductive
Compounds 10 and 11 were synthesized as outlined in
Scheme 1. Bromination of commercially available N-Boc 2,4-diox-
opiperidine 1220 with NBS in THF afforded bromo derivative 13
in 87% yield. Treatment of compound 13 with thiourea in DMF in
the presence of K2CO3 at 150 °C for 12 h afforded aminothiazole
14 in 68% yield.20 Diazotization using isoamyl nitrite in the pres-
ence of CuBr2 in DMF at 0 °C and warming to room temperature
over a period of 1 h afforded bromothiazole 15 in 39% yield. Sono-
gashira coupling of 2-bromothiazole 15 with aryl alkynes in the
presence of Et3N at 140 °C under microwave conditions yielded
fused thiazoles 10a, c–i in 10–45% yield.20 Base-mediated alkyl-
ation of arylalkynyl thiazole 10a with NaH in THF at 0 °C followed
by addition of methyl iodide afforded compound 10b in 65% yield.
Compounds 11a and b were synthesized from commercially avail-
able bromothiazole carboxylate 17 in five steps. Bromination with
NBS in the presence of AIBN in CCl4 afforded bromomethyl thiazole
18,20d which was treated with an excess of cyclopentylamine or
cyclopropylamine with heating to provide aminoalkyl thiazole es-
ter derivatives 19a and 19b in moderate yield. Saponification using
LiOH resulted in carboxylic acids 20a and 20b and ring closure, by
warming to reflux with HBTU or PyBOP, afforded desired 2-bro-
mothiazolyldihydropyrrolidinones 21a and b in 35–55% yield.
Sonogashira coupling of 2-bromothiazoles 21 with phenylacety-
lene in the presence of Et3N at 140 °C under microwave conditions
yielded fused thiazoles 11a and 11b in 18% and 24% yield
resepectively.20
Table 2
SAR of alkylamine analogs 27
R1
S
N
N
R
27
Compound
R
R1
EC50 (nM) EMax (%) IC50 (nM) Inh. (%)
27a
27b
H
CH3
34
>10,000
110
À8
>10,000
>10,000
À38
3-F CH3
6
Results for conformationally constrained analogs 10 and 11 are
summarized in Table 1. Unsubstituted lactam 10a possesses moder-
ate mGlu5 PAM potency (EC50 112 nM; Emax 243%), while N-methyl-
ation, to provide the corresponding N-methyl analog 10b,
remarkably improves potency (EC50 2.4 nM; Emax 150%). Aryl group
modifications of the NH-lactams had variable effects on potency. For
example, analogs 10d–f possess weak PAM potency while 3-fluoro-
phenyl analog 10c and 3-pyridyl analog 10i (EC50 51 nM; Emax 120%)
maintain similar activity as 10a. Both the 2-methoxy (10g) and 3-
methyl (10h) substituted aryl analogs result in complete loss of po-
tency. Interestingly, cyclopentyl analog 11a exhibits potent PAM
activity (EC50 2.5 nM; Emax 150%) while the corresponding cyclopro-
pyl analog 11b is a fairly potent NAM (IC50 23 nM; Inh. 59%).
Incubation of compound 10b with human microsomes in the
presence of reduced glutathione and NADPH results in the produc-
tion of substantial amounts of glutathione adducts.22 We pre-
27c
27d
27e
H
>10,000
>10,000
110
À6
À2
32
1200
72
51
87
28
3-F
H
>10,000
27f
H
100
180
>10,000
À30
27g
27h
27i
H
>10,000
>10,000
>10,000
À48
À40
À44
650
200
640
94
91
94
3-F
3-F
27j
3-F
H
>10,000
>10,000
À47
400
91
1
O
27k
19
>10,000
aThe mGlu5 EC50 and IC50 FLIPR data were generated using a human mGlu5 cell
line.21
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4
M. Packiarajan et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
Table 4
amination of tetrahydrothiazolo[5,4-c]pyridine HCl salts 26 with
aldehydes or ketones using NaBH(OAc)3 in the presence of DIEA
at room temperature afforded N-alkyl amine analogs (27a–27k)
in 23–58% yield (Scheme 3).
SAR of aryl and heteroaryl carboxamide analogs 29
O
S
N
R
Treatment of substituted aryl alkynyl tetrahydrothiazolo[5,4-
c]pyridine HCl salts 26a–26c with alkyl, cycloalkyl, aryl and het-
eroaryl carbonyl chlorides or carboxylic acids under standard
N
29
EC50 (nM) EMax (%) IC50 (nM) Inh. (%)
Compound
R
CH3
Cl
29a
99
170
170
160
150
170
150
120
110
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
À140
26
Table 3
SAR of alkyl carboxamide analogs 28
29b
29c
29d
29e
29f
8.7
30
O
F
S
N
R1
À64
16
N
R
CN
28
71
Compound
R
R1
EC50
(nM)
EMax
(%)
IC50
(nM)
Inh.
(%)
CF3
OCH3
CH3
11
12
28a
28b
H
H
CH3
CH2CH3
>10,000
>10,000
À18
À17
À13
90
38
79
79
29
6.7
19
28c
H
>10,000
290
61
N
N
28d
H
9.4
42
>10,000
6.3
29g
29h
160
220
28e
H
3.9
95
>10,000
10
3.7
S
28f
28g
28h
28i
H
H
>10,000
81
3
370
81
13
19
38
29i
1600
71
>10,000
À14
120
87
61
>10,000
>10,000
>10,000
N
O
3-
F
17
aThe mGlu5 EC50 and IC50 FLIPR data were generated using a human mGlu5 cell
line.21
F
H
85
F
28j
H
18
89
72
>10,000
>10,000
52
45
F
F
F
F
F
3-
F
28k
2.5
4-
F
28l
150
640
19
73
>10,000
>10,000
>10,000
>10,000
13
O
28m
28n
28o
H
H
H
43
À0.2
42
Table 5
280
96
SAR of sulfonamide and sulfamide analogs 30
O
N
O
R
130
45
S
S
N
O
28p
H
790
89
>10,000
À1
n
O
28q
28r
28s
H
H
H
167
70
153
190
80
>10,000
>10,000
>10,000
À57
10
0
Compound
30a
n
1
0
R
EC50 (nM)
>10,000
>10,000
EMax (%)
À4
IC50 (nM)
11
Inh. (%)
75
F
F
190
30b
À35
140
74
N
N
30c
30d
30e
1
1
1
>10,000
>10,000
>10,000
À6
5.7
34
36
71
87
78
28t
28u
28v
H
H
H
100
290
320
260
120
100
>10,000
>10,000
>10,000
20
À12
À9
O
À28
35
O
30f
30g
30h
30i
30j
1
0
1
0
1
>10,000
150
À17
110
6
480
81
O
28w
H
470
45
>10,000
À2
>10,000
780
À17
86
N
N
N
>10,000
250
28x
28y
H
H
18
280
70
>10,000
>10,000
8
89
>10,000
13
À74
89
H
O
>10,000
1.5
N
1200
À37
aThe mGlu5 EC50 and IC50 FLIPR data were generated using a human mGlu5 cell
line.21
aThe mGlu5 EC50 and IC50 FLIPR data were generated using a human mGlu5 cell
line.21
Please cite this article in press as: Packiarajan, M.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.05.070

M. Packiarajan et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
5
amide formation conditions afforded the respective alkyl and
cycloalkyl amide analogs (28a–28y) and aryl and heteroaryl car-
boxamide analogs (29a–29i) (Scheme 3). In an analogous manner,
tetrahydrothiazolo[5,4-c]pyridine HCl salts 26a–26c were treated
with sulfonyl/sulfamyl chlorides at room temperature to provide
the corresponding sulfonamide and sulfamide analogs (30a–30j)
in 30–60% yield.
Carbamate analogs 31a–31g were synthesized in moderate
yield in a two-step process by reaction of tetrahydrothiazolo[5,4-
c]pyridine HCl salts 26a–c with either triphosgene or 4-nitro-
phenyl chloroformate followed by treatment with cyclic amines
in the presence of DIEA (Scheme 3).
moderate to excellent NAM potency. Interestingly, cyclohexyl sul-
fonamide 30g and piperidine sulfamide 30i retained moderate
PAM potency.
Selected carbamate analogs 31a–31g were investigated as isos-
teric analogs of their corresponding carboxamides (Table 6). In
general, the carbamate analogs exhibit potent mGlu5 PAM activity,
similar to their carboxamide counterparts. The only exception to
this trend is compound 31g, which lacks activity in both PAM
and NAM mode.
Compounds 10b (Lu AF32105), 27a, 28j and 31d were selected
for additional profiling (Fig. 3, Table 7). Compound Lu AF32105
exhibits high potency at the mGlu5 receptor (EC50 2.4 nM) a cLogP
Removal of the carbonyl group from compounds such as 10b is
generally detrimental to mGlu5 PAM activity (Table 2). For exam-
ple, the des-carbonyl analog of 10b, 27a, is approximately 14-fold
less potent at mGlu5, and meta-fluorophenyl analog 27b has com-
plete loss of mGlu5 activity. While N-methyl (27a) to N-ethyl (27c)
modification results in weak mGlu5 NAM activity. PAM activity was
regained with N-n-butyl (27e) and N-2-methylbutyl (27f) analogs.
To our disappointment, however, all N-cycloalkyl analogs (27h–
27k) investigated were either moderate NAMs or inactive.
Results for the alkyl, cycloalkyl and aryl carboxamides of 2-
(arylethynyl)-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridines 28a–28y
and 29a–29i are summarized in Table 3 and Table 4. Small alkyl
amide analogs such as methyl (28a), ethyl (28b), cyclopropyl
(28c), and cyclobutyl (28f) carboxamides not only lack PAM activ-
ity, but rather are potent mGlu5 NAMs. Carboxamide analogs with
more lipophilic moieties such as methylcyclopropyl 28d and spiro-
cyclobutylcyclopropyl 28e result in potent PAMs with EC50’s of 9.4
and 3.9 nM, respectively. Unsubstituted cyclobutyl carboxamide
28f has modest NAM activity, but PAM activity is regained upon
cyclobutyl ring substitution with methyl, fluoro and methoxy
groups (see 28g–28m). Cycloalkyl carboxamides such as cyclopen-
tyl (28n), tetrahydrofuranyl (28o and 28p), substituted cyclohexyl
(28q–28t), tetrahydropyranyl (28u–28w) and bicycloheptane
(28x) results in varying degrees of mGlu5 PAM potency. Similarly,
3-substituted benzamide analogs (29a–29f) exhibit good to mod-
erate mGlu5 PAM potency, while heteroaryl carboxamide analogs
(29g, 29h and 29i) are 3 to 10-fold less potent, but retain PAM
activity.
of 2.2 and moderate solubility (20 lM). These analogs are highly
selective among other mGlu subtypes (mGlu1, mGlu2, mGlu4 and
mGlu7) in agonist and modulator (PAM and NAM) modes. Addition-
ally, these compounds do not show significant inhibition of cyto-
chrome P450 (CYP) isoforms 2D6 and 3A4 (<40% at 5 lM) and
have only moderately high rat and human plasma protein binding.
Table 6
SAR of Cyclic carbamate analogs 31
O
R
S
N
N
R
N
31
Compound
HNRR
HN
EC50 (nM)
75
EMax (%)
IC50 (nM)
>10,000
Inh. (%)
31a
120
À38
F
F
HN
31b
31c
31d
31e
72
74
9.8
53
100
100
180
170
>10,000
>10,000
>10,000
>10,000
3
HN
HN
HN
F
15
À53
26
F
F
F
31f
26
94
27
>10,000
>10,000
À9
HN
HN
F
Selected sulfonamide and sulfamide analogs were investigated
and data are summarized in Table 5. Disappointingly, many of
the sulfonamide analogs 30a, 30d and 30f, and sulfamide analogs
30c, 30e, 30h and 30j lacked PAM activity, with some exhibiting
F
31g
>10,000
9
aThe mGlu5 EC50 and IC50 FLIPR data were generated using a human mGlu5 cell
line.21
F
N
S
N
S
N
S
N
S
F
N
N
N
N
N
O
O
O
28j
27a
31d
10b
Figure 3. Selected key compounds.
Table 7
Data for selected key compounds
M)b
cLogPc
LLEd
hClint (L/min)
rClint (mL/min)
hPPB (%)
rPPB (%)
a
e
e
Compound
mGlu5 EC50 (nM)
Sol (
l
10b (Lu AF32105)
27a
28j
2.4
34
18
20
240
1
2.2
3.0
1.9
3.7
6.4
4.5
5.8
4.3
3.1
4.9
1.1
1.0
52.0
88.0
7.6
96.7
96.5
98.9
97.6
95.1
93.9
98.8
95.3
31d
9.8
0.2
49.0
a
b
c
For the mGlu5 EC50 FLIPR assay see Table 1.
The kinetic solubility (/mL) was measured using DMSO stock solutions.24
cLogP was calculated using ChemBioDraw Ultra version 12.0.
Lipophilic ligand efficiency (LLE).25 = pEC50ÀcLogP.
d
e
The hClint and rClint are human (L/min) and rat (mL/min) intrinsic clearances, respectively and the clearances were determined according to Obach et al.26 The human and
rat maximum liver blood flow corresponds to 1.5 L/min and 20 mL/min, respectively.
Please cite this article in press as: Packiarajan, M.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.05.070

6
M. Packiarajan et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
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Acknowledgments
We thank our colleagues Kimloan Nguyen and Zack Zou (meta-
bolic stability, CYP inhibition and glutathione study); Xu Zhang and
Chi Zhang (purification and physico-chemical property determina-
tion); Michelle B. Uberti and Christina L. Bonvicino (functional
data) for providing experimental data to support this work. We
also thank Dr. Diptendu Goswami (Chembiotek, India) for synthetic
support for this work.
References and notes
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D-
Lysine coated 384 well plates (Greiner). After overnight incubation in
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5%CO2, cells were loaded with calcium dye (Calcium 5 Assay Kit, Molecular
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glutamate in the presence of test compound. Positive modulator activity was
calculated from the fluorescence max to min data normalized to yield
responses for no modulation (EC20 response) and full stimulation (1 mM L-
glutamate) as 0% and 100% modulation, respectively. Concentration–response
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potency (EC50
activity was assessed by measuring a concentration dependent inhibition of
the EC80 response to -glutamate in the presence of test compound.
) and efficacy (Emax). Negative allosteric modulator (NAM)
L
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devoid of test compound or the positive control, clozapine. The glutathione
adducts were measured using LC–MS/MS method. The Waters UPLC and
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Please cite this article in press as: Packiarajan, M.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.05.070

M. Packiarajan et al. / Bioorg. Med. Chem. Lett. xxx (2013) xxx–xxx
7
Thermo TSQ Quantum systems in tandem were used with a 15 min gradient
run time using a C18 column (100 Â 2.1 mm, 1.7). Initial neutral loss (129 Da)
scans were performed for all reactions and the confirmatory negative precursor
(272 Da) scan was followed when the compound was positive in the neutral
loss scan. The major GSH adduct of clozapine metabolite was detected at m/z
632 with a retention time of 5.3 min under these assay conditions which has
been used to validate the assay.
in a Titer plate shaker at a speed of 1.8 for 4 h. The supernatant was analyzed
by Waters Acquity UPLC system (Column:Acquity UPLC BEH C-18, 1.7
2.1 Â 50 mm column and PDA detector at 254 nm for quantification) with a
gradient (mobile phase A: 2% (w/v) ammonium formate and 20% acetonitrile in
water, mobile phase B: 100% acetonitrile) for 2 min.
lm
25. Leeson, P. D.; Springthorpe, B. Nat. Rev. Drug Disc. 2007, 6, 881.
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Rance, D. J.; Wastall, P. J. Pharmacol. Exp. Ther. 1997, 283, 46. The rat and human
maximum liver blood flow corresponds to 20 mL/min and 1.5 L/min,
respectively.
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M. WO 2001074774. (b) Noguchi, S.; Koyama, T. WO 2008156159.
24. The solubility (
l
M) was measured from DMSO stock solution of the test
L, 25 mM potassium phosphate buffer
compound (300) diluted with 2 Â 200
l
Please cite this article in press as: Packiarajan, M.; et al. Bioorg. Med. Chem. Lett. (2013), http://dx.doi.org/10.1016/j.bmcl.2013.05.070