Synthesis and pharmacological evaluation of novel N-aryl-3,4-dihydro- 1′H-spiro[chromene-2,4′-piperidine]-1′-carboxamides as TRPM8 antagonists

Article abstract of DOI:10.1016/j.bmc.2013.08.031

A novel series of N-aryl-3,4-dihydro-1′H-spiro[chromene-2,4′- piperidine]-1′-carboxamides was identified as transient receptor potential melastatin 8 (TRPM8) channel blockers through analogue-based rational design, synthesis and screening. Details of the synthesis, effect of aryl groups and their substituents on in-vitro potency were studied. The effects of selected functional groups on the 4-position of the chromene ring were also studied, which showed interesting results. The 4-hydroxy derivatives showed excellent potency and selectivity. Optical resolution and screening of alcohols revealed that (R)-(-)-isomers were in general more potent than the corresponding (S)-(+)-isomers. The isomer (R)-(-)-10e (IC₅₀: 8.9 nM) showed a good pharmacokinetic profile upon oral dosing at 10 mg/kg in Sprague-Dawley (SD) rats. The compound (R)-(-)-10e also showed excellent efficacy in relevant rodent models of neuropathic pain.

Full text of DOI:10.1016/j.bmc.2013.08.031

Bioorganic & Medicinal Chemistry 21 (2013) 6542–6553
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and pharmacological evaluation of novel N-aryl-3,
4-dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxamides
as TRPM8 antagonists
Sachin S. Chaudhari, Ashok B. Kadam, Neelima Khairatkar-Joshi, Indranil Mukhopadhyay, Pallavi V. Karnik,
Anupindi Raghuram, Shobha S. Rao, Thamil Selvan Vaiyapuri, Dinesh P. Wale, Vikram M. Bhosale,
⇑
Girish S. Gudi, Ramchandra R. Sangana, Abraham Thomas
Glenmark Research Centre, Glenmark Pharmaceuticals Ltd., A-607, TTC Industrial Area, MIDC Mahape, Navi Mumbai 400 709, India
a r t i c l e i n f o
a b s t r a c t
A novel series of N-aryl-3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxamides was identi-
fied as transient receptor potential melastatin 8 (TRPM8) channel blockers through analogue-based
rational design, synthesis and screening. Details of the synthesis, effect of aryl groups and their substit-
uents on in-vitro potency were studied. The effects of selected functional groups on the 4-position of the
chromene ring were also studied, which showed interesting results. The 4-hydroxy derivatives showed
excellent potency and selectivity. Optical resolution and screening of alcohols revealed that (R)-(–)-iso-
mers were in general more potent than the corresponding (S)-(+)-isomers. The isomer (R)-(–)-10e
(IC50: 8.9 nM) showed a good pharmacokinetic profile upon oral dosing at 10 mg/kg in Sprague–Dawley
(SD) rats. The compound (R)-(–)-10e also showed excellent efficacy in relevant rodent models of neuro-
pathic pain.
Article history:
Received 15 June 2013
Revised 14 August 2013
Accepted 16 August 2013
Available online 29 August 2013
Keywords:
Spiro[chromene-2,40-piperidines]
Optical resolution of alcohols
Cold allodynia
Wet-dog shake
Ó 2013 Elsevier Ltd. All rights reserved.
Neuropathic pain
1. Introduction
receptor potential ankyrin 1 (TRPA1) have been implicated in cold
sensation, while the heat-sensitive transient receptor potential
Transient receptor potential (TRP) ion channels comprise of an
extensive family of nonselective cation channels that are activated
by a wide variety of stimuli.1,2 More than 30 members of the TRP
channels have been identified thus far in mammals with a wide
range of expression profiles. Based on their amino acid sequence
and structural similarity, they are categorized into seven groups,
namely TRP canonical (C), TRP vanilloid (V), TRP melastatin (M),
TRP polycystin (P), TRP mucolipin (ML), TRP ankyrin (A), and TRP
nompc (N).3,4 All these channels were first discovered in Drosophila
melanogaster as light activated ion channels. TRP channels are
known to be activated by a wide range of physical, mechanical
and chemical stimuli.5 A growing body of evidence suggests that
certain members of the TRP family play critical roles in the trans-
duction of temperature and pain sensation.6 The melastatin sub-
group consists of eight channels namely TRPM 1–8. Among the
super-family of TRP channels, TRPM8, TRPA1 and TRPV1 share sig-
nificant sequence homology and appear to reproduce the broad
spectrum of temperature sensation, though they differ in their ana-
tomical distribution and the stimuli to which they respond.7 The
transient receptor potential melastatin 8 (TRPM8) and transient
vanilloid 1 (TRPV1) is activated by noxious heat above 42 °C.8
TRPM8 is expressed in the small dorsal root ganglia (DRG) neu-
rons and in neurons from trigeminal ganglia in the periphery. It is a
calcium (Ca2+)-permeable, nonselective cation channel and is of
special interest as this polymodal receptor responds to both tem-
perature changes and to several chemicals. TRPM8 is activated at
temperatures ranging from 8 to 26 °C.9–11 It is also activated by
several exogenous ligands, such as menthol, icilin, eucalyptol, gera-
niol and linalool. The therapeutic cooling sensation elicited by
these chemicals is due to the activation of TRPM8 channels.9,11
TRPM8 can sense temperature changes in the range of both innoc-
uous cold (15–28 °C) and noxious cold (<15 °C) and the role of
TRPM8 in skin cold sensation has been demonstrated using TRMP8
null mice.8,12,13 Recent studies revealed that intravenous adminis-
tration of certain known TRPM8 antagonists induces hypothermia
and decrease in deep body temperature.14
Cold allodynia is a pathological pain state induced by innocuous
cold. The pain is intractable and often occurs in patients with com-
plex regional pain syndrome.15 Approximately 80% of these pa-
tients suffer from cold allodynia. Cold allodynia is also present in
other disease conditions such as diabetic neuropathy and fibromy-
algia.16,17 Thus, the presence of cold allodynia in a wide range of
clinical disorders provides a strong rationale for the development
⇑
Corresponding author. Tel.: +91 22 6772 0000; fax: +91 22 2778 1206.
E-mail address: abraham_thomas@glenmarkpharma.com (A. Thomas).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.08.031

S. S. Chaudhari et al. / Bioorg. Med. Chem. 21 (2013) 6542–6553
6543
R2
of TRPM8 antagonists as a novel antihyperalgesic or antiallodynic
R2
agent.18,19 Several patents and publications have recently been re-
ported in the literature exploring the potential therapeutic use of
TRPM8 antagonists.20–26
O
N BOC
R1
O
OH
N BOC
5a-f
4
R1
b
COCH3
a
O
3
The two well-studied urea derivatives N-(4-tert-butylphenyl)-
4-(3-chloropyridine-2-yl)tetrahydropyrazine-1(2H)-carboxamide
(BCTC 1) and capsazepine 2 (Fig. 1) are known to inhibit rodent
TRPM8 with low potency and are nonselective.27,28 BCTC is also a
potent inhibitor of TRPV1 (IC50: 34.9 19 nM), thus limiting its po-
tential therapeutic use as a selective TRPM8 antagonist. Certain
other urea derivatives are also reported in the literature as TRPM8
antagonists. However, their TRPV1 activity had not been dis-
cussed.24 Based on the above literature, we designed urea deriva-
tives of spiro[chromene-2,40-piperidine] as potential TRPM8
antagonists.
R2
R2
R1
O
R1
O
c
N
BOC
NH2Cl
HO
7a-f
6a-f
3 5 7a
, :
-
R1 = R2 = H
R2
b
:
R1 = F, R2 = H
H
d
R1
O
N Ar
1
2
c
: R = H, R = F
N
d
e
:
R1 = H, R2 = Cl
O
1
2
: R = H, R = Br
R1 = F, R2 = Cl
:
f
8a-m
In this paper, we describe design, synthesis and structure–activ-
ity relationship (SAR) analysis of a novel series of spiro[chromene-
2,40-piperidine]-10-carboxamides as TRPM8 antagonists. The study
resulted in identification of (4R)-(–)-8-chloro-4-hydroxy-N-[4-(tri-
fluoromethoxy)phenyl]-3,4-di-hydro-10H-spiro[chromene-2,40-
piperidine]-10-carboxamide (–)-10e, as a potent, highly selective
and orally available TRPM8 antagonist which shows efficacy in
various rodent models of neuropathic pain.
Scheme 1. Reagents and conditions: (a) pyrrolidine, MeOH, RT, 24 h; (b) NaBH4,
EtOH, RT, 1 h.; (c) (i) triethylsilane, TFA, reflux, 18 h; (ii) ꢀ10% (w/v) HCl in EtOAc,
0 °C–RT, 2 h; (d) ArNHCO2Ph, Et3N, DMSO, RT, 2 h.
R2
R2
H
O
O
N
R3
a
N BOC
N
O
O
O
2
5c:
d:
e:
R2
R = F
9a-f
2. Results and discussion
2.1. Chemistry
2
R = Cl
2
R = Br
2
3
9 10a
-
: R = F, R = CF3
b R2 = Cl, R3 = CF
:
H
3
b
2
3
R3
c
: R = Br, R = CF3
O
N
The synthesis of urea derivatives of 3,4-dihydrospiro[chro-
mene-2,40-piperidine] 8a–m is described in Scheme 1. Thus, appro-
priately substituted 2-hydroxy-acetophenone 3 was reacted with
N-BOC-4-piperidone 4 in the presence of pyrrolidine in methanol
to give the spiro derivatives 5a–f in good to excellent yield.29–32
This well-known reaction proceeds via an enamine intermediate
of 3 with pyrrolidine, which adds to the carbonyl group of piperi-
done 4 and cyclizes through the phenolic oxygen to form the spi-
rocyclic compound.33 Sodium borohydride reduction of 5 gave
alcohol 6 which on treatment with triethylsilane in refluxing triflu-
oroacetic acid (TFA) resulted in reductive deoxygenation and con-
comitant deprotection of the N-BOC group.34 The crude amine,
isolated after basification of the TFA salt, was treated with ꢀ10%
(w/v) HCl in EtOAc to give 7 as its hydrochloride salt. The amine
hydrochloride 7 was coupled with various phenyl aryl carbamates
in the presence of excess triethylamine in dimethyl sulfoxide to af-
ford urea derivatives 8a–m in excellent yield.35,36
2
3
N
d
: R = F, R = OCF3
: R = Cl, R = OCF3
: R = Br, R = OCF3
O
2
3
e
f
HO
2
3
( )-10a-f
Scheme 2. Reagents and conditions: (a) (i) ꢀ10% (w/v) HCl in EtOAc; (ii) R3-
C6H4NHCO2Ph, Et3N, DMSO, RT, 2 h. (b) NaBH4, EtOH, 0 °C–RT, 1 h.
It was considered important to resolve the alcohol ( )-10 into
individual isomers as a part of this study. Initial attempts to pre-
pare the chiral alcohols via asymmetric reduction of ketone 9 using
(R)-2-methyl-CBS-oxazaborolidine–borane complex failed to give
the expected alcohol. Attempted asymmetric reduction using (–)-
DIP-chloride also failed to give the alcohol 10. This was probably
due to the presence of urea moiety in the ketone substrate. A con-
ventional approach of optical resolution via the diasteromeric es-
ters worked extremely well and is depicted in Scheme 3. The
The synthesis of 4-oxo derivatives 9a–f and 4-hydroxy deriva-
tives ( )-10a–f is given in Scheme 2. The ketones 9a–f were pre-
pared by direct coupling of free amine derived from 5c to 5e
with appropriately substituted aryl phenylcarbamate in the pres-
ence of triethylamine in dimethyl sulfoxide at room temperature.
The racemic alcohols 10a–f were prepared by carbonyl group
reduction of the corresponding 4-oxochromene 9a–f using sodium
borohydride in ethanol.29
R2
H
O
N
OCF3
N
O
b
(4S)-(+)-10d-f
O
O
NHCbz
NH
11a-c
R2
a
( )-10d-f
H
Cl
CH
3
H
N
O
TRPM8 IC50: 0.8 0.1 µM
TRPV1 IC50 : 34.9 19 nM
CH
3
N
N
O
N
OCF3
CH
3
N
N
O
b
BCTC 1
(4R)-(-)-10d-f
O
O
NHCbz
NH
HO
HO
H
11a, 12a, 10d: R2 = F
11b, 12b, 10e: R2 = Cl
11c, 12c, 10f: R2 = Br
Cl
N
TRPM8 IC50: 18 1.1 µM
TRPV1 IC50 : 2.6 1.2 µM
N
12a-c
S
Capsazepine 2
Scheme 3. Reagents and conditions: (a) Cbz-
18 h. (b) LiOHꢁH2O, THF–MeOH, water, RT, 1 h.
L
-Trp-OH, EDCI, DMAP, CH2Cl2, RT,
Figure 1. Structures of BCTC 1 and capsazepine 2.

6544
S. S. Chaudhari et al. / Bioorg. Med. Chem. 21 (2013) 6542–6553
esterification of racemic spirocyclic alcohol ( )-10 with (2S)-2-
{[(benzyloxy)-carbonyl]amino}-3-(1H-indol-3-yl)propanoic acid
of potency. Di-substitution on the left-hand side of the molecule
as in the case of 8k also resulted in a loss of potency. The thiazole
derivative 8l and benzothiazole derivative 8m showed only moder-
ate potency.
The in-vitro screening results of keto compounds 9a–f and the
corresponding alcohols ( )-10a–f are given in Table 2. The keto
compounds 9a–c with trifluoromethyl group at the 4-position of
the phenyl ring showed moderate potency. Introduction of a triflu-
oromethoxy group at the 4-position of the phenyl ring as in the
case of 9d–f resulted in improved potency. The conversion of the
hydrogen bond acceptor group (carbonyl) at 4-position to a donor
group (hydroxyl) significantly and consistently improved potency
(compounds ( )-10a–f).
(Cbz-L
-Trp-OH) using N-ethyl-N0-(3-dimethylaminopropyl)carbo-
diimide hydrochloride (EDCI) as a coupling agent in the presence
of 4-(dimethylamino)pyridine (DMAP) in dichloromethane gave a
diastereomeric mixture of esters 11 and 12.37 The mixture of esters
was separated by flash silica gel column chromatography to yield
the less polar isomer 11 followed by the more polar isomer 12.
Compounds 11a–c and 12a–c were found to be enantiomerically
and diasteriomerically pure (>98%) based on chiral HPLC analysis.
Hydrolysis of the less polar diastereomers 11a–c with aqueous
lithium hydroxide yielded corresponding optically pure alcohols
(+)-10d–f.
Similarly, hydrolysis of the more polar diastereomers 12a–c
yielded corresponding optically pure alcohols (–)-10d–f in good
yields. Stereochemical descriptors (R or S) of (+)-10d–f and (–)-
10d–f were assigned by correlating the sign of optical rotation of
these compounds with that of known chromene alcohol deriva-
tives prepared through enantoselective reduction of the corre-
The effect of absolute stereochemistry of the hydroxyl group on
the hTRPM8 in-vitro potency of 10d–f is depicted in Table 3. The
inhibitory potency of individual isomers (+)-10–f and (–)-10d–f
on related channels such as TRPV1, TRPV3, TRPV4 and TRPA1
was also studied and the results are shown in the Table 3. Interest-
ingly, the optical antipodes showed significant differences in their
inhibitory potency on TRPM8 channel and (R)-isomers of 10d–f
were more potent than the corresponding (S)-isomers. A similar
but opposite trend was observed in the case of TRPV1 and the
(S)-isomers of 10d–f were more potent than the corresponding
(R)-isomers. Both (R)-(–)-10d–f and (S)-(+)-10d–f showed very
good selectivity over TRPV3, TRPV4 and TRPA1 and absolute ste-
reochemistry did not play any significant role on selectivity.
sponding ketones using
a
chiral oxazaborolidine–borane
complex.38
2.2. In-vitro pharmacology
The urea derivatives 8a–m (Scheme 1) and 9a–f, ( )-10a–f
(Scheme 2), were prepared as part of a structure–activity relation-
ship (SAR) analysis and were subjected to in-vitro hTRPM8 assay
using recombinant Chinese hamster ovary (CHO) cells. Tables 1–
2 show SAR of these series of compounds. The compound 8a (Ta-
ble 1) bearing no substitution on both aryl rings showed poor
TRPM8 antagonistic activity (IC50: 2423 nM). Introduction of an
appropriate substituent on the aryl ring seemed to be essential
to achieve good potency. A trifluoromethyl group at 4-position of
the aryl ring as in the case of 8b resulted in nearly nine-fold
improvement in potency. An additional fluorine substituent at 6-
position of the chromene ring as in the case of 8c resulted in signif-
icant loss of potency. A fluorine, chlorine or a bromine substituent
at the 8-position of the chromene ring in addition to the trifluoro-
methyl group at 4-position of the phenyl ring resulted in excellent
potency (compounds 8d–f). The 8-chloro derivative 8g with a tri-
fluoromethoxy group at 4-position of the phenyl ring also retained
good potency. Compound 8h with an electron-withdrawing cyano
group on the phenyl ring showed significant loss in potency. A
methyl substitution at 4-position of the phenyl ring as in the case
of 8i showed six-fold loss in potency compared to the correspond-
ing trifluoromethyl derivative 8e. An additional chlorine substitu-
ent on the phenyl ring as in the case of 8j resulted in partial loss
2.3. Metabolic stability and PK profile of (R)-(–)-10e
The in-vitro metabolic stability of (–)-10e was determined by
measuring the disappearance of (–)-10e in liver microsomal incu-
bations at 37 °C for 60 min. Compound (–)-10e showed moderate
to high stability across the species with 81%, 79%, 42%, 72% and
67% remaining in the male CD1 mouse, male Wistar rat, male Bea-
gle dog, male Cynomolgus monkey and pooled human liver micro-
somes, respectively.
A single dose pharmacokinetic (PK) profile of (–)-10e was
examined in male Sprague Dawley (SD) rats at 10 mg/kg dose
(Fig. 2). Following an oral administration, quantifiable levels of
(–)-10e were found up to 8 h post dose in all the animals. The com-
pound (–)-10e showed rapid absorption with a median Tmax of 1 h
post dose. A mean Cmax of 1088 ng/mL and mean AUC0–t of
4978 ng/h/mL were observed at 10 mg/kg oral dose.
2.4. In-vivo pharmacology
To establish correlation between in-vitro potency, pharmacoki-
netics (PK) and pharmacodynamics (PD), (–)-10e was also screened
Table 1
In-vitro hTRPM8 activity of 8a–ma
Table 2
In-vitro hTRPM8 activity of 9a–f and ( )-10a–fa
Compd.
R1
R2
Ar
hIC50 (nM)
Compd.
R2
R3
hIC50 (nM)
8a
8b
8c
8d
8e
8f
8g
8h
8i
H
H
F
H
H
H
F
Cl
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-OCF3Ph
4-CNPh
4-CH3Ph
2-Cl, 4-CF3Ph
4-CF3Ph
4-CF3, 1,3-thiazol-2-yl
6-F, 1,3-benzothiazol-2-yl
2423.0
280.7
666.9
97.3
48.5
18.9
9a
9b
9c
9d
9e
9f
( )-10a
( )-10b
( )-10c
( )-10d
( )-10e
( )-10f
F
CF3
CF3
CF3
OCF3
OCF3
OCF3
CF3
295.2
230.4
656.9
182.1
111.0
107.3
90.1
Cl
Br
F
Cl
Br
F
Cl
Br
F
Cl
Br
H
H
H
H
H
H
H
F
41.1
543.7
289.6
177.8
457.2
265.8
113.1
CF3
65.9
8j
8k
8l
CF3
44.5
OCF3
OCF3
OCF3
22.5
13.3
19.5
H
H
8m
a
a
IC50 values were determined by radiometric 45Ca+2 uptake assay from at least
IC50 values were determined by radiometric 45Ca+2 uptake assay from at least
two independent experiments using six point concentration dose–response curves.
two independent experiments using six point concentration dose–response curves.

S. S. Chaudhari et al. / Bioorg. Med. Chem. 21 (2013) 6542–6553
6545
Table 3
hTRPM8 activity and selectivity of optical isomers (S)-(+)-10d–f and (R)-(ꢂ)-10d–f
4
Compd.
IC50 (nM)
% Inhibition at 1.0 lM
3
2
1
0
39%
**
hTRPM8a
hTRPV1a
hTRPV3
hTRPV4
hTRPA1
41%
**
18%
(S)-(+)-10d
(R)-(ꢂ)-10d
(S)-(+)-10e
(R)-(ꢂ)-10e
(S)-(+)-10f
(R)-(ꢂ)-10f
94.0
9.5
46.3
8.9
60.0
16.6
266.3
841.6
217.8
>1000
412.9
>1000
23.2
26.4
17.7
27.7
8.0
6.6
12.5
12.8
38.8
3.3
2.8
7.8
15.9
23.5
4.3
24%
44%
**
66%
***
74%
***
65%
***
16.8
16.3
1.6
TRPM8 and TRPV1 IC50 values were determined by radiometric 45Ca+2 uptake
assay from at least two independent experiments using six point concentration
dose–response curves. TRPV3, TRPV4 and TRPA1 assays were run in duplicate and
a
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Time (h)
the results are expressed as percentage inhibition at 1.0
compounds.
lM concentration of the
Vehicle
Capsazepine 10 mg/kg, i.p.
(-)-10e, 10 mg/kg, p.o.
Figure 3. Effect of (ꢂ)-10e on acetone spray induced cold allodynia in rats with CCI.
Clinical scoring pattern—0: no response; 1: brisk withdrawal or flicking of paw; 2:
repeated flicking of paw; 3: repeated flicking and licking of paw (<5 s); 4: repeated
flicking and licking of paw (>5 s). ⁄⁄p < 0.01, ⁄⁄⁄p < 0.001, treatment groups vs.
vehicle group, two-way ANOVA, followed by Bonferroni’s test.
40
30
###
20
57%
***
72%
***
10
Figure 2. Pharmacokinetic profile of (R)-(ꢂ)-10e in male SD rats (mean SD of
three animals per time point).
0
Icilin
in rat TRPM8 channels using 45Ca2+ uptake assay. It showed an IC50
of 16.5 nM, which is close to the human potency of 8.9 nM (Ta-
ble 3). TRPM8 is considered as the predominant mammalian cold
sensor in the induction of hypersensitivity to cold stimuli.12,13,39
The in-vivo efficacy of compound (–)-10e was evaluated in three
rodent models of neuropathic pain and the results are given below.
First, the compound (–)-10e was evaluated in acetone spray in-
duced cold allodynia model in SD rats that had undergone chronic
constriction injury (CCI) neuropathic surgical procedure, using
Naive
(-)-10e , 30 mg/kg, p.o.
Pregabalin 30 mg/kg, p.o.
Vehicle
(-)-10e, 3 mg/kg, p.o.
(-)-10e , 10 mg/kg, p.o.
value is "0"
Figure 4. Effect of (ꢂ)-10e on icilin-induced WDS in C57BL/6J mice. Percent
inhibition is given above the respective bar. ###p < 0.001, vehicle group versus naïve
group, ⁄⁄⁄p < 0.001 vehicle group versus treated groups, one-way ANOVA, followed
by Bonferroni’s test.
capsazepine as
a
reference compound.28 Compound (ꢂ)-10e
(10 mg/kg, po) significantly inhibited the cold allodynia induced
by acetone spray in rats, with CCI nerve injury, with a maximal effi-
cacy of 74% reversal observed at 2 h post administration. Capsaze-
pine (10 mg/kg, ip) also produced a moderate efficacy of 44%
inhibition of cold-allodynia (Fig. 3).
7
6
###
5
4
The compound (ꢂ)-10e was then evaluated in a behavioral as-
say of icilin-induced wet dog shakes (WDS) in C57/BL6 mice to
measure responses to sensation of intense cold.40 Icilin a TRPM8
agonist, was used in the experiment to induce WDS in mice.41,42
The compound (ꢂ)-10e (3, 10 and 30 mg/kg, po) was evaluated
in this study. The top dose, 30 mg/kg, significantly inhibited WDS
like cold hypersensitivity in mice with a maximal of 72% efficacy.
The standard comparator drug pregabalin (30 mg/kg, po) produced
a significant 57% inhibition of icilin-induced WDS behavior (Fig. 4).
Finally, the compound (ꢂ)-10e was evaluated in oxaliplatin-in-
duced peripheral neuropathic pain in C57/BL6 mice. Oxaliplatin, a
platinum-based chemotherapeutic drug, is known to induce signif-
icant cold hypersensitivity presumably due to increase in TRPM8
expression.43–45 Compound (ꢂ)-10e (3, 10 and 30 mg/kg, po) pro-
duced a dose-dependent inhibition of nocifensive paw licking in
oxaliplatin-induced cold allodynia in mice with a maximal of
100% effect observed at 30 mg/kg dose. Pregabalin (30 mg/kg, po)
also produced a significant 69% efficacy (Fig. 5).
69%
73%
***
3
***
2
1
0
100%
***
Oxaliplatin
Naive
(-)-10e, 10 mg/kg, p.o.
Vehicle
(-)-10e, 30 mg/kg, p.o.
(-)-10e, 3 mg/kg, p.o.
Pregabalin 30 mg/kg, p.o.
Figure 5. Effect of (ꢂ)-10e on oxaliplatin-induced cold allodynia in male C57BL/6J
mice. Percent inhibition is given above the respective bar. ###p < 0.001, vehicle
group versus naïve group, ⁄⁄⁄p < 0.001 vehicle group versus treated groups, one-way
ANOVA, followed by Bonferroni’s test.
Thus, the compound (ꢂ)-10e produced significant efficacy
in various preclinical rodent models of cold allodynia/
hypersensitivity.

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S. S. Chaudhari et al. / Bioorg. Med. Chem. 21 (2013) 6542–6553
were successively washed with water (2 ꢃ 250 mL), saturated
3. Conclusion
NaHCO3 solution (250 mL) and brine (200 mL). The residue ob-
tained after evaporation of the solvent was purified by flash silica
gel column chromatography using 20–30% EtOAc in petroleum
ether (PE) as eluent.
In summary, several novel, structurally diverse urea derivatives
of 3,4-dihydro-10H-spiro[chromene-2,40-piperidines] were synthe-
sized and evaluated as TRPM8 antagonists. A detailed SAR analysis
revealed that introduction of a hydroxyl group at the 4-position of
the dihydrochromene ring resulted in improved potency. Optical
resolution of the chiral alcohols and re-screening of the individual
isomers demonstrated that the (R)-(ꢂ)-isomers were three to four-
fold more potent than the corresponding (S)-(+)-isomers. The se-
lected compound (ꢂ)-10e showed excellent potency, metabolic
stability and favorable pharmacokinetics properties. From the ro-
dent models of neuropathic pain explored in the present work,
we believe that (ꢂ)-10e has potential utility in the management
of pain. Further work is in progress to understand the effect of
(ꢂ)-10e on body temperature in relevant preclinical models.
4.1.1.1. tert-Butyl 4-oxo-3,4-dihydro-10H-spiro[chromene-2,40-
piperidine]-10-carboxylate 5a.
White solid; yield 76%; mp
91–92 °C; 1H NMR (300 MHz, DMSO-d6) d 1.39 (s, 9H), 1.57–1.65
(m, 2H), 1.85–1.90 (m, 2H), 2.84 (s, 2H), 3.01–3.19 (m, 2H), 3.69–
3.73 (m, 2H), 7.02–7.09 (m, 2H), 7.58 (t, J = 7.5 Hz, 1H), 7.72 (d,
J = 7.8 Hz, 1H); APCI-MS (m/z) 218 [M+H–CO2, –(CH3)2C@CH].
4.1.1.2. tert-Butyl 6-fluoro-4-oxo-3,4-dihydro-10H-spiro[chro-
mene-2,40-piperidine]-10-carboxylate 5b.
White solid; yield
51%; mp 117–119 °C; 1H NMR (300 MHz, DMSO-d6) d 1.39 (s,
9H), 1.56–1.66 (m, 2H), 1.84–1.89 (m, 2H), 2.86 (s, 2H), 3.10 (br
s, 2H), 3.68–3.73 (m, 2H), 7.11–7.17 (m, 1H), 7.41–7.50 (m, 2H);
APCI-MS (m/z) 236 [(M+H)–CO2,–(CH3)2C@CH].
4. Experimental
4.1. Chemistry
4.1.1.3. tert-Butyl 8-fluoro-4-oxo-3,4-dihydro-10H-spiro[chro-
mene-2,40-piperidine]-10-carboxylate 5c.
Off-white solid;
Melting points are uncorrected. Infrared spectra were recorded
on Perkin Elmer spectrum one FT-IR spectrometer. 1H and 13C NMR
spectra were recorded on a Varian 300 MHz FT NMR spectrometer
in either CDCl3 or DMSO-d6 as specified using tetramethylsilane as
internal standard. Chemical shifts are quoted in ppm (d) relative to
TMS (1H) using residual protonated solvent as internal standard.
Routine mass spectra (MS) were recorded on a Thermo Finnigan
model LCQ Deca XP Max spectrometer using ESI or APCI source
at positive or negative polarity mode by direct infusion method.
High-resolution mass spectra (HRMS) of all test compounds were
measured on a Thermo Scientific LTQ Orbitrap Discovery MS sys-
tem coupled with a LQT Tune Plus (version 2.5.5 SPI) software
operating in a positive electron spray ionization (ESI) mode. Opti-
cal purity of all chiral test compounds was measured on a Chir-
alpak IA or Chiralpak AD-H Diacel column. Optical rotations were
measured on a Jasco P-1030 polarimeter. All test compounds used
in biological assay were greater than 98% pure as determined by
HPLC on a Shimadzu or Waters system using PDA detector.
yield 88%; mp 98–100 °C; 1H NMR (300 MHz, DMSO-d6) d 1.40 (s,
9H), 1.58–1.71 (m, 2H), 1.87–1.98 (m, 2H), 2.92 (s, 2H), 3.05–
3.13 (m, 2H), 3.69–3.76 (m, 2H), 7.00–7.08 (m, 1H), 7.52–7.60
(m, 2H); APCI-MS (m/z) 236 [(M+H)–CO2, –(CH3)2C@CH].
4.1.1.4. tert-Butyl 8-chloro-4-oxo-3,4-dihydro-10H-spiro[chro-
mene-2,40-piperidine]-10-carboxylate 5d.
Off-white solid;
yield 85%; mp 117–118 °C; 1H NMR (300 MHz, DMSO-d6) d 1.39
(s, 9H), 1.60–1.69 (m, 2H), 1.86–1.95 (m, 2H), 2.92 (s, 2H), 2.99–
3.18 (m, 2H), 3.79–3.85 (m, 2H), 7.07 (t, J = 7.8 Hz, 1H), 7.70 (d,
J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H); APCI-MS (m/z) 352 [M+H]+.
4.1.1.5. tert-Butyl 8-bromo-4-oxo-3,4-dihydro-10H-spiro[chro-
mene-2,40-piperidine]-10-carboxylate 5e.
Off-white solid;
yield 77%; mp 127–128 °C; 1H NMR (300 MHz, CDCl3) d 1.40 (s,
9H), 1.59–1.68 (m, 2H), 1.82–1.94 (m, 2H), 2.91 (s, 2H), 3.01–
3.21 (m, 2H), 3.80–3.86 (m, 2H), 7.01 (t, J = 7.8 Hz, 1H), 7.74 (d,
J = 7.5 Hz, 1H), 7.90 (d, J = 7.5 Hz, 1H); APCI-MS (m/z) 396 [M+H]+.
All chemicals, reagents and solvents were procured from com-
mercial sources and were used as received. Petroleum ether (PE)
refers to the fraction boiling in the 40–60 °C range. tert-Butyl-4-
oxo-3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxyl-
ate derivatives 5 were synthesized according to literature proce-
4.1.1.6. tert-Butyl 8-chloro-6-fluoro-4-oxo-3,4-dihydro-10H-
spiro[chromene-2,40-piperidine]-10-carboxylate
5f.
Off-
white solid; yield 78%; mp 131–133 °C; 1H NMR (300 MHz, CDCl3)
d 1.46 (s, 9H), 1.56–1.66 (m, 2H), 2.00–2.11 (m, 2H), 2.75 (s, 2H),
3.19–3.32 (m, 2H), 3.90–4.10 (m, 2H), 7.36 (dd, J = 7.5, 3.0 Hz,
1H), 7.47 (dd, J = 7.8, 3.0 Hz, 1H); APCI-MS (m/z) 370 [M+H]+.
dures
hydroxyacetophones 3 and N-BOC-4-piperidone 4.34 3,4-Dihydro-
spiro[chromene-2,40-piperidine]hydrochloride
was prepared
starting
from
commercially
available
2-
7
from 5 by carbonyl group reduction and reductive deoxygenation
by following a literature procedure.34 All aryl phenyl carbamates
used for the preparation of urea derivatives were prepared by
the reaction of appropriate aromatic or hetero-aromatic amines
with phenyl chloroformate in the presence of pyridine in tetrahy-
drofuran by the following literature procedure.35
4.1.2. General procedure for the synthesis of 4-hydroxy-3,4-
dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxylates
6a–f
To a stirred solution of appropriate spiroketone 5 (10 mmol) in
ethanol (30 mL) was added sodium borohydride (378 mg,
10 mmol) portion wise and the mixture was further stirred at RT
for 1 h. The reaction mixture was diluted with water (300 ml)
and extracted with EtOAc (3 ꢃ 200 mL). The combined extracts
were washed with water (200 mL) and brine (100 mL). The solvent
was evaporated under reduced pressure and the residue obtained
was dried under vacuum to give alcohols 6a–f.
4.1.1. General procedure for the synthesis of 4-oxo-3,4-dihydro-
10H-spiro[chromene-2,40-piperidines] 5a–f
A solution of appropriate 2-hydroxyacetophone derivative 3
(100 mmol), N-tert-butoxycarbonyl-4-piperidone
4
(19.92 g,
100 mmol) and pyrrolidine (8.21 mL, 100 mmol) in methanol
(150 mL) was stirred at room temperature for 18–24 h. Upon com-
pletion of the reaction (monitored by TLC), the mixture was con-
centrated under vacuum and the brown residue thus obtained
was partitioned between EtOAc (500 mL) and 1 N hydrochloric
acid (500 mL). The layers were separated. The aqueous layer was
extracted with EtOAc (2 ꢃ 250 mL). The combined organic extracts
4.1.2.1. tert-Butyl 4-hydroxy-3,4-dihydro-10H-spiro[chromene-
2,40-piperidine]-10-carboxylate 6a.
White semisolid; yield
91%; 1H NMR (300 MHz, DMSO-d6) d 1.39 (s, 9H), 1.48–1.52 (m,
1H), 1.55–1.63 (m, 2H), 1.69–1.77 (m, 2H), 2.05–2.11 (m, 1H),
3.10–3.20 (m, 2H), 3.66 (br s, 2H), 4.67 (q, J = 7.2 Hz, 1H), 5.36 (d,
J = 6.0 Hz, 1H), 6.76 (d, J = 7.8 Hz, 1H), 6.88 (t, J = 7.2 Hz, 1H), 7.12

S. S. Chaudhari et al. / Bioorg. Med. Chem. 21 (2013) 6542–6553
6547
(t, J = 7.8 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H); APCI-MS (m/z) 220
[(M+H)–CO2, –(CH3)2C@CH].
4.1.3.2.
dine]hydrochloride 7b.
6-Fluoro-3,4-dihydrospiro[chromene-2,40-piperi-
Off-white solid; yield 88%; mp
231–233 °C; 1H NMR (300 MHz, DMSO-d6) d 1.78–1.85 (m, 6H),
2.72–2.77 (m, 2H), 3.04 (br s, 2H), 3.13 (br s, 2H), 6.80–6.86 (m,
1H), 6.90–6.97 (m, 2H), 9.21 (br s, 2H); ESI-MS (m/z) 222 [M+H]+.
4.1.2.2.
tert-Butyl
6-fluoro-4-hydroxy-3,4-dihydro-10H-
Color-
spiro[chromene-2,40-piperidine]-10-carboxylate 6b.
less liquid; yield 90%; 1H NMR (300 MHz, DMSO-d6) d 1.39 (s,
9H), 1.47–1.58 (m, 1H), 1.62–1.69 (m, 2H), 1.70–1.77 (m, 2H),
2.06–2.12 (m, 1H), 3.06 (br s, 1H), 3.18 (br s, 1H), 3.70 (br s, 2H),
4.62–4.69 (m, 1H), 5.50 (d, J = 6.3 Hz, 1H), 6.76–6.81 (m, 1H),
6.93–7.00 (m, 1H), 7.12–7.18 (m, 1H); APCI-MS (m/z) 238
[(M+H)–CO2, –(CH3)2C@CH].
4.1.3.3.
dine]hydrochloride 7c.
8-Fluoro-3,4-dihydrospiro[chromene-2,40-piperi-
Off-white solid; yield 83%; mp 209–
211 °C; 1H NMR (300 MHz, DMSO-d6) d 1.85–1.95 (m, 6H), 2.78
(t, J = 6.9 Hz, 2H), 3.04 (br s, 2H), 3.17–3.23 (m, 2H), 6.78–6.85
(m, 1H), 6.93 (d, J = 7.2 Hz, 1H), 7.00–7.06 (m, 1H), 9.06 (br s,
2H); APCI-MS (m/z) 222 [M+H]+.
4.1.2.3. tert-Butyl 8-fluoro-4-hydroxy-3,4-dihydro-10H-spiro
[chromene-2,40-piperidine]-10-carboxylate 6c.
White semi-
4.1.3.4.
dine]hydrochloride 7d.
8-Chloro-3,4-dihydrospiro[chromene-2,40-piperi-
Off-white solid; yield 85%; mp
solid; yield 89%; 1H NMR (300 MHz, DMSO-d6) d 1.40 (s, 9H),
1.54–1.62 (m, 1H), 1.67–1.74 (m, 2H), 1.79–1.84 (m, 2H), 2.10–
2.15 (m, 1H), 3.17 (br s, 2H), 3.68 (br s, 2H), 4.70 (q, J = 7.8 Hz,
1H), 5.51 (d, J = 6.0 Hz, 1H), 6.82–6.89 (m, 1H), 7.03–7.10 (m,
1H), 7.21 (d, J = 7.8 Hz, 1H); APCI-MS (m/z) 238 [(M+H)–CO2, –
(CH3)2C@CH].
221–223 °C; 1H NMR (300 MHz, DMSO-d6) d 1.84–1.90 (m, 6H),
2.77–2.81 (m, 2H), 2.98–3.06 (m, 2H), 3.19–3.25 (m, 2H), 6.85 (t,
J = 7.8 Hz, 1H), 7.09 (d, J = 7.8 Hz, 1H), 7.25 (d, J = 7.8 Hz, 1H),
9.30 (br s, 2H); APCI-MS (m/z) 238 [M+H]+.
4.1.3.5.
dine]hydrochloride 7e.
8-Bromo-3,4-dihydrospiro[chromene-2,40-piperi-
Off-white solid; yield 86%; mp 231–
4.1.2.4. tert-Butyl 8-chloro-4-hydroxy-3,4-dihydro-10H-spiro
[chromene-2,40-piperidine]-10-carboxylate 6d.
White semi-
232 °C; 1H NMR (300 MHz, DMSO-d6) d 1.80–1.88 (m, 6H), 2.78–
2.82 (m, 2H), 3.01–3.12 (m, 2H), 3.20–3.29 (m, 2H), 6.81 (t,
J = 7.8 Hz, 1H), 7.13 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H),
8.99 (br s, 2H); APCI-MS (m/z) 282 [M+H]+.
solid; yield 84%; 1H NMR (300 MHz, DMSO-d6) d 1.40 (s, 9H),
1.51–1.71 (m, 3H), 1.77–1.81 (m, 2H), 2.09–2.16 (m, 1H), 3.02–
3.16 (m, 2H), 3.69–3.78 (m, 2H), 4.68–4.75 (m, 1H), 5.53 (d,
J = 6.8 Hz, 1H), 6.90 (t, J = 7.8 Hz, 1H), 7.28 (d, J = 6.9 Hz, 1H), 7.37
(d, J = 6.9 Hz, 1H); APCI-MS (m/z) 254 [(M+H)–CO2, –(CH3)2C@CH].
4.1.3.6.
piperidine]hydrochloride 7f.
8-Chloro-6-fluoro-3,4-dihydrospiro[chromene-2,40-
Off-white solid; yield 71%; mp
4.1.2.5. tert-Butyl 8-bromo-4-hydroxy-3,4-dihydro-10H-spiro
>250 °C; 1H NMR (300 MHz, DMSO-d6) d 1.44–1.51 (m, 2H),
1.58–1.64 (m, 2H), 1.70–1.78 (m, 2H), 2.68–2.86 (m, 6H), 7.01
(dd, J = 8.4, 2.4 Hz, 1H), 7.20 (dd, J = 8.4, 2.4 Hz, 1H), 8.35 (br s,
2H); APCI-MS (m/z) 256 [M+H]+.
[chromene-2,40-piperidine]-10-carboxylate
6e.
Off-white
semisolid; yield 82%; 1H NMR (300 MHz, DMSO-d6) d 1.40 (s,
9H), 1.62–1.66 (m, 4H), 1.82–1.88 (m, 2H), 2.88–2.96 (m, 2H),
3.77–3.83 (m, 2H), 4.70–4.76 (m, 1H), 5.53 (d, J = 5.4 Hz, 1H,
exchangeable with D2O), 6.85 (t, J = 7.2 Hz, 1H), 7.42 (t, J = 7.8 Hz,
2H); APCI-MS (m/z) 298 [(M+H)–CO2, –(CH3)2C@CH].
4.1.4. General procedure for the synthesis of urea derivatives
8a–m
To a stirred solution of 3,4-dihydrospiro[chromene-2,40-piperi-
dine]hydrochloride 7a–f (0.50 mmol) in anhydrous DMSO (4 mL)
was added appropriate phenyl aryl carbamate (0.55 mmol) fol-
lowed by triethylamine (0.35 mL, 2.50 mmol). The resulting mix-
ture was stirred for 2–4 h (monitored by TLC) at room
temperature. On completion of the reaction, the mixture was di-
luted with EtOAc (200 mL) and washed with water (2 ꢃ 50 mL),
followed by brine (50 mL). The residue obtained after evaporation
of the solvent was purified by silica gel column chromatography
using 10–20% EtOAc in PE as eluent to obtain the product as white
to off-white solid.
4.1.2.6. tert-Butyl 8-chloro-6-fluoro-4-hydroxy-3,4-dihydro-
10H-spiro[chromene-2,40-piperidine]-10-carboxylate
6f.
d 1.40 (s, 9H), 1.59–1.67 (m, 2H), 1.71–1.83 (m, 2H), 2.11–2.17 (m,
2H), 3.00–3.15 (m, 2H), 3.76 (br s, 2H), 4.70 (t, J = 7.2 Hz, 1H), 5.68
(d, J = 6.3 Hz, 1H), 7.16–7.22 (m, 1H), 7.29–7.33 (m, 1H); APCI-MS
(m/z) 272 [(M+H)–CO2, –(CH3)2C@CH].
White semisolid; yield 87%; 1H NMR (300 MHz, DMSO-d6)
4.1.3. General procedure for the synthesis of 3,4-dihydro-10H-
spiro[chromene-2,40-piperidine]hydrochlorides (7a–f)
To a stirred solution of alcohol 6a–f (9.0 mmol) in TFA (30 mL)
was added triethylsilane (5.75 mL, 36 mmol) and the mixture was
refluxed for 18 h. The reaction mixture was concentrated under re-
duced pressure to result a viscous residue. The residue was dis-
solved in water (100 mL) and neutralized with saturated NaHCO3
solution. The mixture was extracted with EtOAc (2 ꢃ 200 mL).
The combined extracts were washed with water (100 mL) and
brine (100 mL). The residue obtained after evaporation of the sol-
vent was dissolved in minimum quantity of EtOAc. Excess of
ꢀ10% (w/v) HCl in EtOAc (300 mL) was added under stirring. The
white solid separated out was collected by filtration.
4.1.4.1. N-Phenyl-3,4-dihydro-10H-spiro[chromene-2,40-piperi-
dine]-10-carboxamide (8a).
Reaction of 7a (150 mg,
0.625 mmol) with phenyl phenylcarbamate (147 mg, 0.688 mmol)
gave 151 mg (75%) of the product as a white solid. Mp 167–168 °C;
1H NMR (300 MHz, DMSO-d6) d 1.55–1.62 (m, 2H), 1.69–1.74 (m,
2H), 1.78–1.83 (m, 2H), 2.72–2.76 (m, 2H), 3.17–3.27 (m, 2H),
3.83–3.90 (m, 2H), 6.78–6.83 (m, 2H), 6.91 (t, J = 7.5 Hz, 1H),
7.05–7.09 (m, 2H), 7.21 (t, J = 7.5 Hz, 2H), 7.45 (d, J = 7.8 Hz, 2H),
8.52 (s, 1H); HRMS (ESI) calcd for C20H22N2O2, [M+H]+, 323.1754;
found, 323.1752.
4.1.3.1. 3,4-Dihydrospiro[chromene-2,40-piperidine]hydro-chlo-
ride 7a.
(300 MHz, DMSO-d6) d 1.80–1.88 (m, 6H), 2.72–2.76 (m, 2H),
3.00–3.08 (m, 2H), 3.11–3.15 (m, 2H), 6.80–6.86 (m, 2H), 7.02–
7.11 (m, 2H), 9.11 (br s, 2H); ESI-MS (m/z) 204 [M+H]+.
4.1.4.2.
spiro[chromene-2,40-piperidine]-10-carboxamide
(8b). Reaction of 7a (50 mg, 0.208 mmol) with phenyl [4-(tri-
N-[4-(Trifluoromethyl)phenyl]-3,4-dihydro-10H-
White solid; yield 82%; mp 220–222 °C; 1H NMR
fluoromethyl)-phenyl]carbamate (64 mg, 0.229 mmol) gave 63 mg
(77%) of the product as a white solid. Mp 158–160 °C; 1H NMR

6548
S. S. Chaudhari et al. / Bioorg. Med. Chem. 21 (2013) 6542–6553
(300 MHz, DMSO-d6) d 1.57–1.64 (m, 2H), 1.70–1.83 (m, 4H), 2.74
(t, J = 5.7 Hz, 2H), 3.20–3.26 (m, 2H), 3.86–3.82 (m, 2H), 6.78–6.84
(m, 2H), 7.04–7.10 (m, 2H), 7.57 (d, J = 8.4 Hz, 2H), 7.68 (d,
J = 8.4 Hz, 2H), 8.93 (br s, 1H); HRMS (ESI) calcd for C21H21F3N2O2,
[M+H]+ 391.1627; found, 391.1625.
mate (96 mg, 0.401 mmol) gave 107 mg (77%) of the product as a
white solid. Mp >250 °C; 1H NMR (300 MHz, DMSO-d6) d 1.60–
1.67 (m, 2H), 1.73–1.86 (m, 4H), 2.75–2.84 (m, 2H), 3.15–3.26
(m, 2H), 3.95–4.03 (m, 2H), 6.83 (t, J = 7.2 Hz, 1H), 7.07 (d,
J = 7.0 Hz, 1H), 7.23 (d, J = 7.5 Hz, 1H), 7.64–7.70 (m, 4H), 9.05 (br
s, 1H); HRMS (ESI) calcd for C21H20ClN3O2, [M+H]+, 382.1316;
found, 382.1316.
4.1.4.3.
10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(8c). Reaction of 7b (50 mg, 0.194 mmol) with phenyl[4-(tri-
6-Fluoro-N-[4-(trifluoromethyl)phenyl]-3,4-dihydro-
4.1.4.9.
[chromene-2,40-piperidine]-10-carboxamide (8i).
8-Chloro-N-(4-methylphenyl)-3,4-dihydro-10H-spiro
Reaction
fluoromethyl)phenyl]carbamate (60 mg, 0.213 mmol) gave 51 mg
(65%) of the product as a white solid. Mp 162–164 °C; 1H NMR
(300 MHz, DMSO-d6) d 1.56–1.63 (m, 2H), 1.69–1.81 (m, 4H),
2.74 (t, J = 6.6 Hz, 2H), 3.21–3.28 (m, 2H), 3.86–3.91 (m, 2H),
6.78–6.82 (m, 1H), 6.89–6.95 (m, 2H), 7.57 (d, J = 8.1 Hz, 2H),
7.68 (d, J = 8.7 Hz, 2H), 8.93 (br s, 1H); HRMS (ESI) calcd for C21H20-
F4N2O2, [M+H]+, 409.1533; found, 409.1530.
of 7d (100 mg, 0.364 mmol) with phenyl(4-methylphenyl) carba-
mate (91 mg, 0.401 mmol) gave 111 mg (82%) of the product as a
white solid. Mp 194–196 °C; 1H NMR (300 MHz, DMSO-d6) d
1.56–1.64 (m, 2H), 1.70–1.86 (m, 4H), 2.22 (s, 3H), 2.79 (t,
J = 6.3 Hz, 2H), 3.14–3.22 (m, 2H), 3.92–3.97 (m, 2H), 6.83 (t,
J = 7.8 Hz, 1H), 7.01–7.08 (m, 3H), 7.23 (d, J = 7.8 Hz, 1H), 7.68 (d,
J = 8.1 Hz, 2H), 8.45 (br s, 1H); HRMS (ESI) calcd for C21H23Cl1N2O2,
[M+H]+, 371.1520; found, 371.1519.
4.1.4.4.
10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(8d). Reaction of 7c (50 mg, 0.194 mmol) with phenyl[4-(tri-
8-Fluoro-N-[4-(trifluoromethyl)phenyl]-3,4-dihydro-
4.1.4.10. 8-Chloro-N-[2-chloro-4-(trifluoromethyl)phenyl]-3,4-
dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxamide
fluoromethyl)phenyl]carbamate (60 mg, 0.213 mmol) gave 53 mg
(67%) of the product as a white solid. Mp 163–165 °C; 1H NMR
(300 MHz, DMSO-d6) d 1.57–1.68 (m, 2H), 1.74–1.85 (m, 4H),
2.78 (br s, 2H), 3.20–3.26 (m, 2H), 3.87–3.93 (m, 2H), 6.78–6.83
(m, 1H), 6.92 (d, J = 7.8 Hz, 1H), 6.97–7.05 (m, 1H), 7.58 (d,
J = 8.7 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 8.95 (br s, 1H); HRMS (ESI)
calcd for C21H20F4N2O2, [M+H]+, 409.1533; found, 409.1530.
(8j).
Reaction of 7d (75 mg, 0.273 mmol) with phenyl[2-
chloro-4-(trifluoromethyl)phenyl]carbamate (95 mg, 0.301 mmol)
gave 102 mg (81%) of the product as a white solid. Mp 157–
158 °C; 1H NMR (300 MHz, CDCl3) d 1.60–1.79 (m, 4H), 1.80–1.89
(m, 2H), 2.77–2.82 (m, 2H), 3.20–3.31 (m, 2H), 3.93–3.99 (m,
2H), 6.84 (t, J = 7.8 Hz, 1H), 7.08 (d, J = 7.2 Hz, 1H), 7.24 (d,
J = 7.8 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.79–7.84 (m, 2H), 8.49 (br
s, 1H); HRMS (ESI) calcd for C21H19Cl2F3N2O2, [M+H]+, 459.0848;
found, 459.0849.
4.1.4.5.
10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(8e). Reaction of 7d (500 mg, 1.823 mmol) with phenyl[4-(tri-
8-Chloro-N-[4-(trifluoromethyl)phenyl]-3,4-dihydro-
fluoromethyl)phenyl]carbamate (564 mg, 2.006 mmol) gave
627 mg (81%) of the product as a white solid. Mp 158–159 °C; 1H
NMR (300 MHz, CDCl3) d 1.61–1.69 (m, 2H), 1.84–1.94 (m, 4H),
2.80–2.86 (m, 2H), 3.42–3.50 (m, 2H), 3.90–3.97 (m, 2H), 6.60 (br
s, 1H), 6.80 (t, J = 7.8 Hz, 1H), 6.98 (d, J = 7.5 Hz, 1H), 7.20 (d,
J = 7.8 Hz, 1H), 7.47–7.55 (m, 4H); HRMS (ESI) calcd for C21H20ClF3-
N2O2, [M+H]+, 425.1238; found, 425.1230.
4.1.4.11. 8-Chloro-6-fluoro-N-[4-(trifluoromethyl)phenyl]-3,4-
dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(8k).
Reaction of 7f (100 mg, 0.342 mmol) with phenyl[4-(tri-
fluoromethyl)phenyl]carbamate (106 mg, 0.376 mmol) gave
103 mg (68%) of the product as a white solid. Mp 159–160 °C; 1H
NMR (300 MHz, DMSO-d6) d 1.59–1.68 (m, 2H), 1.71–1.77 (m,
2H), 1.82–1.87 (m, 2H), 2.75–2.82 (m, 2H), 3.16–3.24 (m, 2H),
3.94–4.03 (m, 2H), 7.01 (dd, J = 5.3, 2.4 Hz, 1H), 7.25 (dd, J = 5.4,
2.4 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 8.97
4.1.4.6.
10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(8f). Reaction of 7e (200 mg, 0.628 mmol) with phenyl[4-(tri-
8-Bromo-N-[4-(trifluoromethyl)phenyl]-3,4-dihydro-
(br s, 1H); HRMS (ESI) calcd for
443.1143; found, 443.1144.
C
21H19ClF4N2O2, [M+H]+,
fluoromethyl)phenyl]carbamate (194 mg, 0.690 mmol) gave
224 mg (76%) of the product as a white solid. Mp 159–160 °C; 1H
NMR (300 MHz, DMSO-d6) d 1.59–1.66 (m, 2H), 1.73–1.86 (m,
4H), 2.74–2.81 (m, 2H), 3.18–3.28 (m, 2H), 3.98–4.05 (m, 2H),
6.79 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 7.2 Hz, 1H), 7.38 (d, J = 7.8 Hz,
1H), 7.58 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz, 2H), 8.96 (br s,
1H); HRMS (ESI) calcd for C21H20BrF3N2O2, [M+H]+, 469.0733;
found, 469.0729.
4.1.4.12. 8-Chloro-N-[4-(trifluoromethyl)-1,3-thiazol-2-yl]-3,4-
dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(8l).
Reaction of 7d (250 mg, 0.911 mmol) with phenyl[4-(tri-
fluoromethyl)-1,3-thiazol-2-yl]carbamate (289 mg, 1.002 mmol)
gave 197 mg (50%) of the product as a white solid. Mp 96–97 °C;
1H NMR (300 MHz, DMSO-d6) d 1.63–1.76 (m, 4H), 1.80–1.86 (m,
2H), 2.75–2.81 (m, 2H), 3.19–3.26 (m, 2H), 4.03–4.10 (m, 2H),
6.84 (t, J = 7.8 Hz, 1H), 7.07 (d, J = 7.5 Hz, 1H), 7.24 (d, J = 7.2 Hz,
1H), 7.80 (s, 1H), 11.42 (br s, 1H); HRMS (ESI) calcd for C18H17ClF3-
N3O2S, [M+H]+, 432.0754; found, 432.0753.
4.1.4.7. 8-Chloro-N-[4-(trifluoromethoxy)phenyl]-3,4-dihydro-
10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(8g).
Reaction of 7d (100 mg, 0.364 mmol) with phenyl[4-(tri-
fluoromethoxy)phenyl]carbamate (119 mg, 0.401 mmol) as de-
scribed gave 140 mg (87%) of the product as a white solid. Mp
139–140 °C; 1H NMR (300 MHz, DMSO-d6) d 1.62–1.70 (m, 2H),
1.72–1.80 (m, 2H), 1.82–1.87 (m, 2H), 2.75–2.81 (m, 2H), 3.17–
3.24 (m, 2H), 3.94–4.00 (m, 2H), 6.83 (t, J = 7.8 Hz, 1H), 7.07 (d,
J = 7.5 Hz, 1H), 7.20–7.29 (m, 3H), 7.56 (d, J = 8.9 Hz, 2H), 8.76 (br
s, 1H); HRMS (ESI) calcd for C21H20ClF3N2O3, [M+H]+, 441.1187;
found, 441.1187.
4.1.4.13. 8-Chloro-N-(6-fluoro-1,3-benzothiazol-2-yl)-3,4-dihy-
dro-10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(8m).
Reaction of 7d (75 mg, 0.273 mmol) with phenyl(6-
fluoro-1,3-benzothiazol-2-yl)carbamate (87 mg, 0.301 mmol) gave
105 mg (89%) of the product as a white solid. Mp 188–189 °C; 1H
NMR (300 MHz, DMSO-d6) d 1.64–1.78 (m, 4H), 1.80–1.87 (m,
2H), 2.72–2.80 (m, 2H), 3.22–3.28 (m, 2H), 4.05–4.11 (m, 2H),
6.84 (t, J = 7.5 Hz, 1H), 7.08 (d, J = 7.5 Hz, 1H), 7.21–7.26 (m,
2H), 7.60–7.66 (m, 1H), 7.78–7.84 (m, 1H), 11.30 (br s, 1H); HRMS
4.1.4.8.
[chromene-2,40-piperidine]-10-carboxamide (8h).
of 7d (100 mg, 0.364 mmol) with phenyl(4-cyanophenyl)carba-
8-Chloro-N-(4-cyanophenyl)-3,4-dihydro-10H-spiro
Reaction
(ESI) calcd for
432.0934.
C
21H19ClFN3O2S, [M+H]+, 432.0943; found,

S. S. Chaudhari et al. / Bioorg. Med. Chem. 21 (2013) 6542–6553
6549
4.1.5. General procedure for the synthesis of urea derivatives
9a–f
pared
by
deprotection
of
5c,
with
phenyl[4-
(trifluoromethoxy)phenyl]carbamate (361 mg, 1.214 mmol) gave
To a stirred suspension of tert-butyl 4-oxo-3,4-dihydro-10H-
spiro[chromene-2,40-piperidine]-10-carboxylate 5c–e (10 mmol)
in ethyl acetate (10 mL) was added excess of ꢀ10% (w/v) HCl in
EtOAc (300 mL) and the mixture was stirred at room temperature
for 2 h. Ethyl acetate was evaporated under reduced pressure to af-
ford the amine hydrochloride (yield ꢀ100%) as a white solid, which
was used as such for the coupling reaction.
407 mg (84%) of the product as a white solid. Mp 168–170 °C; IR
(KBr) 3299, 1697, 1640, 1489, 1427, 1254, 1153 cm–1 1H NMR
;
(300 MHz, DMSO-d6) d 1.72–1.81 (m, 2H), 1.93–2.00 (m, 2H),
2.96 (s, 2H), 3.15–3.25 (m, 2H), 3.88–3.95 (m, 2H), 7.01–7.09 (m,
1H), 7.23 (d, J = 8.4 Hz, 2H), 7.53–7.63 (m, 4H), 8.78 (s, 1H); HRMS
(ESI) calcd for C21H18F4N2O4, [M+H]+, 439.1275; found, 439.1271.
To a stirred solution of above amine hydrochloride (10 mmol) in
anhydrous DMSO (30 mL), was added appropriate phenyl aryl car-
bamate (11 mmol) followed by triethylamine (6.97 mL, 50 mmol).
The resulting mixture was stirred at room temperature until com-
pletion of reaction (2–4 h). The mixture was diluted with EtOAc
(300 mL) and washed with water (2 ꢃ 100 mL), followed by brine
(100 mL). The residue obtained after evaporation of the solvent
was purified by silica gel column chromatography using 10–20%
EtOAc in PE as eluent to afford the products 9a–f as white to off-
white solid.
4.1.5.5.
dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(9e).
Reaction of 8-chloro-4-oxo-3,4-dihydro-10H-spiro[chro-
8-Chloro-4-oxo-N-[4-(trifluoromethoxy)phenyl]-3,4-
mene-2,40-piperidine]hydrochloride (2.0 g, 6.941 mmol), prepared
by the deprotection of 5d, with phenyl[4-(trifluorometh-
oxy)phenyl]carbamate (2.27 g, 7.635 mmol) gave 2.75 g (87%) of
the product as a white solid. Mp 152–153 °C; IR (KBr) 3336,
1638, 1534, 1417, 1282, 1159 cm–1 1H NMR (300 MHz, DMSO-
;
d6) d 1.73–1.77 (m, 2H), 1.94–1.98 (m, 2H), 2.95 (s, 2H), 3.14–
3.22 (m, 2H), 3.97–4.01 (m, 2H), 7.11 (t, J = 7.8 Hz, 1H), 7.23 (d,
J = 8.7 Hz, 2H), 7.55 (d, J = 8.7 Hz, 2H), 7.72 (d, J = 7.2 Hz, 1H),
7.78 (d, J = 7.8 Hz, 1H), 8.78 (s, 1H); HRMS (ESI) calcd for C21H18Cl1-
F3N2O4, [M+H]+, 455.098; found, 455.0977.
4.1.5.1. 8-Fluoro-4-oxo-N-[4-(trifluoromethyl)phenyl]-3,4-dihy-
dro-10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(9a).
mene-2,40-piperidine]hydrochloride (300 mg, 1.104 mmol), pre-
pared by deprotection of 5c, with phenyl[4-
Reaction of 8-fluoro-4-oxo-3,4-dihydro-10H-spiro[chro-
4.1.5.6.
dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(9f).
Coupling reaction of 8-bromo-4-oxo-3,4-dihydro-10H-
spiro[chromene-2,40-piperidine]hydrochloride
(600 mg,
8-Bromo-4-oxo-N-[4-(trifluoromethoxy)phenyl]-3,4-
(trifluoromethyl)phenyl]carbamate (342 mg, 1.214 mmol) gave
443 mg (95%) of the product as a white solid. Mp 186–188 °C; IR
(KBr) 3370, 1691, 1655, 1492, 1338, 1223, 1100 cm–1
;
1H NMR
1.804 mmol), prepared by the deprotection of 5e, with phenyl[4-
(trifluoromethoxy)phenyl]carbamate (590 mg, 1.984 mmol) gave
847 mg (94%) of the product as a white solid. Mp 176–178 °C; IR
(300 MHz, DMSO-d6) d 1.74–1.81 (m, 2H), 1.93–2.01 (m, 2H),
2.96 (s, 2H), 3.15–3.26 (m, 2H), 3.90–3.98 (m, 2H), 7.02–7.10 (m,
1H), 7.52–7.60 (m, 4H), 7.64–7.70 (m, 2H), 8.97 (br s, 1H); HRMS
(ESI) calcd for C21H18F4N2O3, [M+H]+, 423.1326; found, 423.1322.
(KBr) 3421, 1638, 1534, 1438, 1281, 1160 cm–1
;
1H NMR
(300 MHz, DMSO-d6) d 1.68–1.76 (m, 2H), 1.92–2.00 (m, 2H),
2.95 (s, 2H), 3.12–3.26 (m, 2H), 3.98–4.06 (m, 2H), 7.03 (t,
J = 7.5 Hz, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.55 (d, J = 8.7 Hz, 2H),
7.75 (d, J = 7.8 Hz, 1H), 7.91 (d, J = 7.8 Hz, 1H), 8.78 (br s, 1H);
HRMS (ESI) calcd for C21H18BrF3N2O4, [M+H]+, 499.0474; found,
499.0472.
4.1.5.2. 8-Chloro-4-oxo-N-[4-(trifluoromethyl)phenyl]-3,4-dihy-
dro-10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(9b).
mene-2,40-piperidine]hydrochloride (500 mg, 1.735 mmol), pre-
pared by deprotection of 5d, with phenyl[4-
Reaction of 8-chloro-4-oxo-3,4-dihydro-10H-spiro[chro-
(trifluoromethyl)phenyl]carbamate (537 mg, 1.908 mmol) gave
4.1.6. General procedure for the synthesis of alcohols ( )-10a–f
To a stirred solution of appropriate 4-oxo derivative 9a–f
(2.0 mmol) in ethanol (10 mL) was added NaBH4 (76 mg,
2.0 mmol) portion-wise at room temperature. After 1 h, the mix-
ture was diluted with water (100 mL) and extracted with EtOAc
(2 ꢃ 200 mL). The combined organic extracts were washed with
water (100 mL) and brine (100 mL). The solvent was evaporated
and the residue obtained was purified on a short length silica gel
column using 40–50% EtOAc in PE to yield the product as a white
solid.
639 mg (84%) of the product as a white solid. Mp 203–204 °C; IR
(KBr) 3449, 3373, 1690, 1654, 1597, 1337, 1100, 840 cm–1 1H
;
NMR (300 MHz, DMSO-d6) d 1.70–1.80 (m, 2H), 1.92–2.02 (m,
2H), 2.96 (s, 2H), 3.15–3.25 (m, 2H), 3.97–4.05 (m, 2H), 7.09 (t,
J = 7.8 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.66–7.80 (m, 4H), 8.98 (br
s, 1H); HRMS (ESI) calcd for C21H18ClF3N2O3, [M+H]+, 439.1030;
found, 439.1028.
4.1.5.3. 8-Bromo-4-oxo-N-[4-(trifluoromethyl)phenyl]-3,4-dihy-
dro-10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(9c).
mene-2,40-piperidine]hydrochloride (500 mg, 1.503 mmol), pre-
pared by deprotection of 5e, with phenyl[4-
Reaction of 8-bromo-4-oxo-3,4-dihydro-10H-spiro[chro-
4.1.6.1. ( )-8-Fluoro-4-hydroxy-N-[4-(trifluoromethyl)phenyl]-
3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxam-
ide (10a).
White solid; yield 94%; mp 140–141 °C; IR (KBr)
(trifluoromethyl)phenyl]carbamate (465 mg, 1.654 mmol) gave
3336, 1650, 1482, 1327, 1223, 1114, cm–1
;
1H NMR (300 MHz,
603 mg (83%) of the product as a white solid. Mp 233–235 °C; IR
DMSO-d6) d 1.66–1.79 (m, 3H), 1.86–1.92 (m, 2H), 2.13–2.20 (m,
1H), 3.12–3.23 (m, 2H), 3.86–3.92 (m, 2H), 4.70–4.76 (m, 1H),
5.50–5.56 (m, 1H), 6.85–6.91 (m, 1H), 7.04–7.12 (m, 1H), 7.21–
7.26 (m, 1H), 7.58 (d, J = 7.8 Hz, 2H), 7.68 (d, J = 7.8 Hz, 2H), 8.94
(br s, 1H); HRMS (ESI) calcd for C21H20F4N2O3, [M+H]+, 425.1482;
found, 425.1479.
(KBr) 3321, 1693, 1647, 1596, 1542, 1315, 1226, 1120, 835 cm–1
;
1H NMR (300 MHz, DMSO-d6) d 1.68–1.77 (m, 2H), 1.92–2.00 (m,
2H), 2.94 (s, 2H), 3.15–3.26 (m, 2H), 3.98–4.06 (m, 2H), 7.02 (t,
J = 7.8 Hz, 1H), 7.58 (d, J = 8.7 Hz, 2H), 7.67 (d, J = 8.7 Hz, 2H),
7.75 (d, J = 7.2 Hz, 1H), 7.91 (d, J = 7.2 Hz, 1H), 8.98 (br s, 1H);
HRMS (ESI) calcd for C21H18BrF3N2O3, [M+H]+, 483.0525; found,
483.0525.
4.1.6.2. ( )-8-Chloro-4-hydroxy-N-[4-(trifluoromethyl)phenyl]-
3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxam-
4.1.5.4.
dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxamide
(9d).
Reaction of 8-fluoro-4-oxo-3,4-dihydro-10H-spiro[chro-
mene-2,40-piperidine]hydrochloride (300 mg, 1.104 mmol), pre-
8-Fluoro-4-oxo-N-[4-(trifluoromethoxy)phenyl]-3,4-
ide (10b).
White solid; yield 80%; mp 152–154 °C; IR (KBr)
3402, 1650, 1453, 1326, 1235, 1114, cm–1
;
1H NMR (300 MHz,
DMSO-d6) d 1.61–1.78 (m, 3H), 1.81–1.93 (m, 2H), 2.12–2.19 (m,
1H), 3.10–3.17 (m, 2H), 3.93–3.99 (m, 2H), 4.70–4.76 (m, 1H),

6550
S. S. Chaudhari et al. / Bioorg. Med. Chem. 21 (2013) 6542–6553
5.55 (d, J = 5.7 Hz, 1H), 6.92 (t, J = 7.8 Hz, 1H), 7.31 (d, J = 7.8 Hz,
1H), 7.39 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 8.4 Hz, 2H), 7.68 (d,
J = 8.4 Hz, 2H), 8.95 (br s, 1H); HRMS (ESI) calcd for C21H20ClF3N2-
O3, [M+H]+, 441.1187; found, 441.1184.
{[4-(trifluoromethoxy)phenyl]carbamoyl}-3,4-dihydrospi-
ro[chromene-2,40-piperidin]-4-yl N-[(benzyloxy) carbonyl]-
L-
tryptophanate (12a).
EDCI mediated esterification of ( )-
10d (320 mg, 0.726 mmol) with Cbz-L-Trp-OH (295 mg,
0.871 mmol) afforded 575 mg of a mixture of 11a and 12a. Flash
4.1.6.3. ( )-8-Bromo-4-hydroxy-N-[4-(trifluoromethyl)phenyl]-
3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-10-carboxam-
silica gel chromatography (30% EtOAc in PE) afforded 205 mg
(37%) of 11a as
a
white solid; mp 105–106 °C; 1H NMR
ide (10c).
White solid; yield 87%; mp 159–161 °C; IR (KBr)
(300 MHz, DMSO-d6) d 1.63–1.76 (m, 3H), 1.85–2.00 (m, 2H),
2.20–2.29 (m, 1H), 3.06–3.27 (m, 5H), 3.79–3.91 (m, 2H), 4.25–
4.36 (m, 1H), 4.97 (s, 2H), 5.80–5.86 (m, 1H), 6.47 (d, J = 7.8 Hz,
1H), 6.70–6.80 (m, 1H), 6.95 (t, J = 7.2 Hz, 1H), 7.08 (t, J = 7.8 Hz,
1H), 7.13–7.30 (m, 8H), 7.37 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 7.8 Hz,
1H), 7.53 (d, J = 8.7 Hz, 2H), 7.88 (d, J = 7.8 Hz, 1H), 8.77 (br s,
1H), 10.89 (br s, 1H); HRMS (ESI) calcd for C40H36F4N4O7, [M+H]+,
761.2592; found, 761.2599. HPLC: Chiralpak IA (Daicel),
3397, 1651, 1559, 1326, 1235, 1114, cm–1
;
1H NMR (300 MHz,
DMSO-d6) d 1.64–1.78 (m, 3H), 1.80–1.93 (m, 2H), 2.11–2.18 (m,
1H), 3.10–3.17 (m, 1H), 3.27–3.35 (m, 1H), 3.93–4.01 (m, 2H),
4.70–4.77 (m, 1H), 5.55 (d, J = 6.6 Hz, 1H), 6.86 (t, J = 7.8 Hz, 1H),
7.43 (t, J = 6.9 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4 Hz,
2H), 8.94 (br s, 1H); HRMS (ESI) calcd for
C21H20BrF3N2O3,
[M+H]+, 485.0682; found, 485.0680.
250 ꢃ 4.6 mm, 5
lm, eluting with hexane/EtOH, 70:30, 99.17%;
4.1.6.4.
oxy)phenyl]-3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-
10-carboxamide (10d).
White solid; yield 93%; mp 143–
144 °C; IR (KBr) 3348, 1644, 1511, 1260, 1200, 735, cm–1 1H
NMR (300 MHz, DMSO-d6) d 1.65–1.78 (m, 3H), 1.82–1.90 (m,
2H), 2.10–2.20 (m, 1H), 3.10–3.25 (m, 2H), 3.84–3.90 (m, 2H),
4.70–4.75 (m, 1H), 5.52 (d, J = 6.0 Hz, 1H), 6.85–6.91 (m, 1H),
7.04–7.11 (m, 1H), 7.20–7.26 (m, 3H), 7.56 (d, J = 8.4 Hz, 2H),
8.74 (br s, 1H); HRMS (ESI) calcd for C21H20F4N2O4, [M+H]+,
441.1432; found, 441.1428.
( )-8-Fluoro-4-hydroxy-N-[4-(trifluorometh-
retention time: 14.5 min. Further elution with increasing amounts
of EtOAc in PE gave 166 mg (30%) of 12a as a white solid, mp 102–
103 °C; 1H NMR (300 MHz, DMSO-d6) d 1.17–1.60 (m, 3H), 1.68–
1.73 (m, 2H), 1.97–2.03 (m, 1H), 3.00–3.23 (m, 5H), 3.72–3.80
(m, 1H), 3.84–3.91 (m, 1H), 4.23–4.32 (m, 1H), 5.01 (s, 2H), 5.84–
5.90 (m, 1H), 6.79–7.02 (m, 4H), 7.16–7.33 (m, 9H), 7.49 (d,
J = 7.2 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H), 7.95 (d, J = 7.2 Hz, 1H),
8.75 (br s, 1H), 10.90 (br s, 1H); HRMS (ESI) calcd for C40H36F4N4O7,
[M+H]+, 761.2592; found, 761.2598. HPLC: Chiralpak IA (Daicel),
;
250 ꢃ 4.6 mm, 5
lm, eluted with hexane/EtOH, 70:30, 99.69%,
retention time: 10.4 min.
4.1.6.5.
oxy)phenyl]-3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-
10-carboxamide (10e).
White solid; yield 87%; mp 150–
152 °C; IR (KBr) 3333, 1644, 1510, 1453, 1238, 1159, 964 cm–1
( )-8-Chloro-4-hydroxy-N-[4-(trifluorometh-
4.1.7.2. (4S)-8-Chloro-10-{[4-(trifluoromethoxy)phenyl]carbam-
oyl}-3,4-dihydrospiro[chromene-2,40-piperidin]-4-yl N-[(ben-
;
zyloxy)carbonyl]-
{[4-(trifluoromethoxy)phenyl]carbamoyl}-3,4-dihydrospi-
ro[chromene-2,40-piperidin]-4-yl
N-[(benzyloxy)carbonyl]-
tryptophanate (12b). EDCI mediated esterification of ( )-
10e (2.10 g, 4.596 mmol) with Cbz- -Trp-OH (1.86 g, 5.515 mmol)
L
-tryptophanate (11b) and (4R)-8-chloro-10-
1H NMR (300 MHz, DMSO-d6) d 1.60–1.77 (m, 3H), 1.81–1.92 (m,
2H), 2.12–2.19 (m, 1H), 3.09–3.16 (m, 1H), 3.21–3.29 (m, 1H),
3.88–3.96 (m, 2H), 4.71–4.77 (m, 1H), 5.54 (d, J = 6.3 Hz, 1H),
6.91 (t, J = 7.8 Hz, 1H), 7.23 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 7.2 Hz,
1H), 7.39 (d, J = 7.2 Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 8.75 (br s,
1H); HRMS (ESI) calcd for C21H20ClF3N2O4, [M+H]+, 457.1136;
found, 457.1133.
L-
L
yielded 3.51 g of a mixture of 11b and 12b. Flash silica gel chroma-
tography (eluent, 30% EtOAc in PE) afforded 1.54 g (43%) of 11b as a
white solid. Mp 114–116 °C; 1H NMR (300 MHz, DMSO-d6) d 1.58–
1.80 (m, 3H), 1.92–2.05 (m, 2H), 2.21–2.30 (m, 1H), 3.04–3.34 (m,
5H), 3.85–4.01 (m, 2H), 4.24–4.31 (m, 1H), 4.97 (s, 2H), 5.80–5.86
(m, 1H), 6.59 (d, J = 7.8 Hz, 1H), 6.78 (t, J = 7.8 Hz, 1H), 6.96 (t,
J = 7.5 Hz, 1H), 7.06 (t, J = 7.8 Hz, 1H), 7.17–7.39 (m, 9H), 7.47 (d,
J = 7.8 Hz, 1H), 7.55 (d, J = 8.7 Hz, 2H), 7.89 (d, J = 6.9 Hz, 1H),
8.77 (s, 1H), 10.89 (br s, 1H); HRMS (ESI) calcd for C40H36ClF3N4O7,
[M+H]+, 777.2297; found, 777.2303. HPLC: Chiralpak IA (Daicel),
4.1.6.6.
oxy)phenyl]-3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-
10-carboxamide (10f).
White solid; yield 81%; mp 159–
( )-8-Bromo-4-hydroxy-N-[4-(trifluorometh-
161 °C; IR (KBr) 3340, 1643, 1510, 1447, 1238, 1160, 964 cm–1
;
1H NMR (300 MHz, DMSO-d6) d 1.63–1.77 (m, 3H), 1.82–1.91 (m,
2H), 2.10–2.17 (m, 1H), 3.09–3.17 (m, 2H), 3.95–4.04 (m, 2H),
4.70–4.78 (m, 1H), 5.54 (d, J = 6.0 Hz, 1H), 6.86 (t, J = 7.2 Hz, 1H),
7.23 (d, J = 8.4 Hz, 2H), 7.39–7.47 (m, 2H), 7.56 (d, J = 8.4 Hz, 2H),
8.75 (br s, 1H); HRMS (ESI) calcd for C21H20BrF3N2O4, [M+H]+,
501.0631; found, 501.0630.
250 ꢃ 4.6 mm, 5
lm, eluting with hexane/EtOH, 70:30, 98.97%;
retention time: 14.6 min. Further elution with increasing amounts
of EtOAc in PE gave 1.14 g (32%) of 12b as a white solid. Mp 113–
115 °C; 1H NMR (300 MHz, DMSO-d6) d 1.00–1.23 (m, 2H), 1.38–
1.44 (m, 1H), 1.52–1.59 (m, 1H), 1.66–1.79 (m, 2H), 1.89–1.99
(m, 1H), 2.98–3.23 (m, 4H), 3.78–3.85 (m, 1H), 3.93–4.01 (m,
1H), 4.23–4.32 (m, 1H), 5.00 (s, 2H), 5.85–5.90 (m, 1H), 6.89–7.03
(m, 4H), 7.10 (d, J = 7.8 Hz, 1H), 7.17–7.31 (m, 7H), 7.40 (d,
J = 7.8 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 7.59 (d, J = 8.7 Hz, 2H),
7.94 (d, J = 6.9 Hz, 1H), 8.75 (br s, 1H), 10.90 (br s, 1H); HRMS
4.1.7. General procedure for synthesis of
11a–c and 12a–c
L
-tryptophan esters
-Trp-OH
To a stirred solution of ( )-10a–f (1.5 mmol) and Cbz-
L
(609 mg, 1.8 mmol) in dichloromethane (20 ml) were added EDCI
(345 mg, 1.8 mmol) and DMAP (220 mg, 1.8 mmol) at room tem-
perature. The mixture was stirred at room temperature for 18 h.
The solvent was evaporated under vacuum and the residue thus
obtained was diluted with water (250 mL) and EtOAc (250 mL).
The layers were separated and the aqueous layer was extracted
with EtOAc (2 ꢃ 100 mL). The combined EtOAc extracts were
washed with water (100 mL), dried (Na2SO4) and evaporated to af-
ford a ꢀ1:1 mixture of diastereomers 11a–c and 12a–c.
(ESI) calcd for
C
40H36ClF3N4O7, [M+H]+, 777.2297; found,
m, eluting
777.2302. HPLC: Chiralpak IA (Daicel), 250 ꢃ 4.6 mm, 5
l
with hexane/EtOH, 70:30, 99.75%; retention time: 11.1 min.
4.1.7.3. (4S)-8-Bromo-10-{[4-(trifluoromethoxy)phenyl]carbam-
oyl}-3,4-dihydrospiro[chromene-2,40-piperidin]-4-yl N-[(ben-
zyloxy)carbonyl]-L-tryptophanate (11c) and (4R)-8-bromo-
10-{[4-(trifluoromethoxy)phenyl]carbamoyl}-3,4-dihydrospi-
4.1.7.1. (4S)-8-Fluoro-10-{[4-(trifluoromethoxy)phenyl]carbam-
oyl}-3,4-dihydrospiro[chromene-2,40-piperidin]-4-yl N-[(ben-
ro[chromene-2,40-piperidin]-4-yl N-[(benzyloxy) carbonyl]-
L-
tryptophanate (12c).
EDCI mediated esterification of ( )-10f
zyloxy)carbonyl]-
L
-tryptophanate (11a) and (4R)-8-fluoro-10-
(660 mg, 1.316 mmol) with Cbz-L-Trp-OH (534 mg, 1.579 mmol)

S. S. Chaudhari et al. / Bioorg. Med. Chem. 21 (2013) 6542–6553
6551
yielded 722 mg of ꢀ1:1 mixture of 11c and 12c. Flash silica gel col-
98.28%, retention time: 17.2 min; Specific optical rotation: ½a D25
ꢄ
:
umn chromatography (eluent, 30% EtOAc in PE) afforded 389 mg
(36%) 11c as a white solid. Mp 109–110 °C; 1H NMR (300 MHz,
DMSO-d6) d 1.56–1.79 (m, 3H), 1.86–1.98 (m, 2H), 2.21–2.30 (m,
1H), 3.06–3.23 (m, 5H), 3.89–4.02 (m, 2H), 4.23–4.33 (m, 1H),
4.97 (s, 2H), 5.78–5.85 (m, 1H), 6.62 (d, J = 7.2 Hz, 1H), 6.73 (t,
J = 7.8 Hz, 1H), 6.96 (t, J = 7.2 Hz, 1H), 7.07 (t, J = 7.8 Hz, 1H),
7.17–7.38 (m, 8H), 7.46–7.57 (m, 4H), 7.88 (d, J = 6.9 Hz, 1H),
8.77 (br s, 1H), 10.89 (br s, 1H); HRMS (ESI) calcd for C40H36BrF3N4-
O7, [M+H]+, 821.1792; found, 821.1801. HPLC: Chiralpak IA (Dai-
+ 4.41° (c 0.1, CHCl3).
4.1.8.4.
(4R)-(ꢂ)-8-Fluoro-4-hydroxy-N-[4-(trifluorometh-
oxy)phenyl]-3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-
10-carboxamide (ꢂ)-(10d).
LiOH mediated hydrolysis of 12a
(145 mg, 0.190 mmol) as described above afforded 68 mg (81%)
of the product as a white solid. Mp 145–146 °C; 1H NMR and IR
spectra were identical to that of ( )-10d. HPLC: Chiralpak IA (Dai-
cel), 250 ꢃ 4.6 mm, 5
lm, eluting with hexane/isopropanol, 90:10,
cel), 250 ꢃ 4.6 mm, 5
lm, eluting with n-hexane/EtOH, 60:40,
98.20%, retention time: 35.7 min; Specific optical rotation: ½a D25
ꢄ
: –
98.46%; retention time: 9.8 min. Further elution of the column
gave 324 mg (30%) of 12c as a white solid. Mp 113–114 °C; 1H
NMR (300 MHz, DMSO-d6) d 1.07–1.15 (m, 1H), 1.39–1.44 (m,
1H), 1.51–1.59 (m, 1H), 1.65–1.75 (m, 2H), 2.98–3.25 (m, 5H),
3.79–3.86 (m, 1H), 3.95–4.02 (m, 1H), 4.24–4.31 (m, 1H), 5.00 (s,
2H), 5.84–5.90 (m, 1H), 6.86 (t, J = 7.8 Hz, 1H), 6.90–7.04 (m, 3H),
7.10–7.20 (m, 2H), 7.23–7.33 (m, 7H), 7.48 (d, J = 7.8 Hz, 1H),
7.53–7.63 (m, 3H), 7.94 (d, J = 6.6 Hz, 1H), 8.75 (br s, 1H), 10.89
7.79° (c 0.1, CHCl3).
4.1.8.5.
(4R)-(ꢂ)-8-Chloro-4-hydroxy-N-[4-(trifluorometh-
oxy)phenyl]-3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-
10-carboxamide (ꢂ)(ꢂ)-(10e).
LiOH mediated hydrolysis of
12b (975 mg, 1.254 mmol) as described afforded 499 mg (87%) of
the product as a white solid. Mp 149–150 °C; 1H NMR and IR spec-
tra were identical to that of ( )-10e. 13C NMR (75 MHz, DMSO-d6) d
13.9, 22.0, 30.9, 33.1, 35.6, 39.7, 40.3, 60.7, 75.4, 120.2 (q,
J = 253.0 Hz, OCF3), 120.5, 120.6 (2C), 121.1 (2C), 127.1, 128.5 (d,
J = 11.4 Hz, C–OCF3), 139.9, 142.5, 147.6, 154.7; HPLC: Chiralpak
(br s, 1H); HRMS (ESI) calcd for
821.1792; found, 821.1802. HPLC: Chiralpak IA (Daicel),
m, eluting with n-hexane/EtOH, 60:40, 100%;
C
40H36BrF3N4O7, [M+H]+,
250 ꢃ 4.6 mm, 5
l
retention time: 8.6 min.
AD-H (Daicel) 250 ꢃ 4.6 mm, 5
lm, eluting with hexane/isopropa-
nol, 80:20, 98.36%, retention time: 9.1 min; Specific optical rota-
tion: ½a 2D5
ꢄ
: ꢂ3.27° (c 0.1, CHCl3).
4.1.8. General procedure for the hydrolysis of esters: synthesis
of optically pure alcohols (+)-(10d–f) and (ꢂ)-(10d–f)
4.1.8.6.
(4R)-(ꢂ)-8-Bromo-4-hydroxy-N-[4-(trifluorometh-
To a solution of tryptophan ester 11a–c or 12a–c (0.5 mmol) in
mixture of THF (10 mL), MeOH (4 mL), and water (4 mL) was added
LiOHꢁH2O (42 mg, 1.0 mmol). The reaction mixture was stirred at
room temperature for 1 h. The solvents were evaporated under re-
duced pressure. The residue thus obtained was diluted with water
(100 mL) and acidified with 1.0 N hydrochloric acid to pH 4.0. The
mixture was extracted with EtOAc (2 ꢃ 200 mL) and the combined
extracts were washed with water (100 mL) and dried over anhy-
drous Na2SO4. The residue obtained after evaporation of the sol-
vent was purified by silica gel column chromatography using 40–
50% ethyl acetate in PE to give optically pure alcohols.
oxy)phenyl]-3,4-dihydro-10H-spiro-[chromene-2,40-piperidine]-
10-carboxamide (ꢂ)-(10f).
LiOH mediated hydrolysis of 12c
(300 mg, 0.365 mmol) as described afforded 130 mg (71%) of the
product as a white solid. Mp 161–162 °C; 1H NMR and IR spectra
were identical to that of ( )-10f. HPLC: Chiralpak AD-H (Daicel)
250 ꢃ 4.6 mm,
5 lm, eluting with hexane/isopropanol 80:20,
98.52%; retention time: 9.7 min; Specific optical rotation: ½a D25
ꢄ
:
ꢂ5.02° (c 0.1, CHCl3).
4.2. Biology
4.2.1. Screening of TRPM8 antagonists using 45calcium uptake
assay
4.1.8.1.
oxy)phenyl]-3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-
10-carboxamide (+)-(10d).
LiOH mediated hydrolysis of 11a
(170 mg, 0.223 mmol) as described gave 83 mg (84%) of the prod-
uct as a white solid. Mp 144–146 °C; 1H NMR and IR spectra were
identical to that of ( )-10d. HPLC: Chiralpak IA (Daicel),
(4S)-(+)-8-Fluoro-4-hydroxy-N-[4-(trifluorometh-
Radiometric calcium influx assay was performed according to
reported protocol.46,47 Chinese hamster ovary (CHO) cells stably
expressing human TRPM8 were generated in-house and main-
tained in F-12 Dulbecco’s Modified Eagle Medium (DMEM F-12)
supplemented with 10% FBS and G418. The cells were cultured at
37 °C in humidified air containing 5% CO2. A 10 mM stock of com-
pound (ꢂ)-10e was prepared in DMSO. Subsequent dilutions from
the stock solution were made in drug dilution buffer (DDB) (DMEM
F-12 containing 1.8 mM CaCl2). Cells were seeded in 96-well plates
at a density of 0.03 ꢃ 106 cells/well, and cultured overnight at
37 °C in the presence of 5% CO2. Before the assay, the cells were
washed twice with assay buffer (DMEM F12 with 0.1% BSA and
1.8 mM CaCl2). The assay was carried out at 25 °C in a total volume
250 ꢃ 4.6 mm,
5 lm, eluting with hexane/isopropanol, 90:10,
99.03%, retention time: 40.7 min; specific optical rotation: ½a D25
ꢄ
:
+8.11° (c 0.1, CHCl3).
4.1.8.2.
oxy)phenyl]-3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-
10-carboxamide (+)-(10e).
LiOH mediated hydrolysis of 11b
(4S)-(+)-8-Chloro-4-hydroxy-N-[4-(trifluorometh-
(1.50 g, 1.930 mmol) as described gave 785 mg (89%) of the prod-
uct as a white solid. Mp 154-156 °C; 1H NMR and IR spectra were
identical to that of ( )-10e. HPLC: Chiralpak AD-H (Daicel),
of 200
l
L. Cells were treated with test compounds for 10 min.
45Ca+2 solution was added at a final concentration of 5
lCi/mL fol-
250 ꢃ 4.6 mm,
5 lm, eluting with hexane/isopropanol, 80:20,
lowed by the addition of icilin as an agonist. After 4 min of agonist
treatment, the drug was washed out and the wells were rinsed
thrice with wash buffer (145 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2,
1.2 mM MgCl2, 10 mM HEPES, 10 mM glucose and 0.1% BSA). The
cells were lysed in lysis buffer (50 mM Tris–HCl (pH 7.5),
150 mM NaCl, 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS)
on a shaker. Radioactivity in cell lysates was measured as counts
per minute (cpm) using TopCount liquid scintillation counter (Per-
kin Elmer). IC50 values were calculated from concentration re-
sponse curve by nonlinear regression analysis using GraphPad
Prism 3.0 (GraphPad Software Inc.).
98.42%; retention time: 15.7 min; Specific optical rotation: ½a D25
ꢄ
:
+3.72° (c 0.1, CHCl3).
4.1.8.3.
oxy)phenyl]-3,4-dihydro-10H-spiro[chromene-2,40-piperidine]-
10-carboxamide (+)-(10f).
LiOH mediated hydrolysis of 11c
(4S)-(+)-8-Bromo-4-hydroxy-N-[4-(trifluorometh-
(360 mg, 0.438 mmol) as described gave 176 mg (80%) of the prod-
uct as a white solid. Mp 164–166 °C; 1H NMR and IR spectra were
identical to that of ( )-10f. HPLC: Chiralpak AD-H (Daicel)
250 ꢃ 4.6 mm,
5 lm, eluting with hexane/isopropanol, 80:20,

6552
S. S. Chaudhari et al. / Bioorg. Med. Chem. 21 (2013) 6542–6553
4.2.2. Selectivity screening of TRPM8 antagonists using
45calcium uptake assay
tion versus time data was subjected to pharmacokinetic analysis
using Pharsight WinnonlinÒ version 5.2 Software for estimation
Radiometric calcium assay was performed as described above
with some minor modifications. CHO cells stably expressing hu-
man TRPA1, TRPV1, TRPV3 or TRPV4 were generated in-house
and maintained in DMEM F-12 supplemented with 10% FBS and
G418. All the cell types were cultured at 37 °C in humidified air
containing 5% CO2. Before the assay, the cells were washed twice
with assay buffer. The assay was carried out in total volume of
of pharmacokinetic parameters including Cmax, AUC0–t and Tmax.
4.2.5. Chronic constriction injury (CCI) acetone test for (4R)-(ꢂ)-
10e
Male SD rats weighing 160–200 g were used for the study.
Chronic constriction injury (CCI) of the sciatic nerve was performed
under aseptic conditions. The rats were anesthetized with an intra-
peritoneal injection of ketamine and xylazine (40 and 8 mg/kg,
respectively) combination. The sciatic nerve on the left side was
exposed through a mid-thigh incision and separation of the heads
of the biceps femoris muscle. Proximal to the sciatic trifurcation,
approximately 7 mm of nerve was freed, and four loose ligatures
of 4–0 chromic gut were placed around the sciatic nerve. The mus-
cle and skin were then closed in two layers and covered with io-
dine solution. A single experimenter performed all the sciatic
nerve ligations to maintain the uniformity. After seven days of
the ligation, the animals were taken for the experiment.48,49
On the day of the experiment, animals were acclimatized for
15 min in the experimental plexiglass chamber containing sieving
iron wire mesh (0.5 cm2 pore size) in the false bottom of the cham-
ber. Throughout the experimental period, ambient temperature
was maintained at 21 0.5 °C. After acclimatization period, 0.5%
MC (methyl cellulose), capsazepine (10 mg/kg, ip) and compound
200 lL at 30 °C for TRPV1, 32 °C for TRPV3 and 25 °C for TRPV4
and TRPA1. The cells were treated with Compound (ꢂ)-10e for
10 min. 45Ca+2 solution was added at a final concentration of
5
l
Ci/mL followed by the addition of respective agonist (allyl iso-
thiocyanate for TRPA1, capsaicin for TRPV1, 2-aminoethoxydiphe-
nyl borate (2-APB) for TRPV3 and 4 -phorbol 12,13-didecanoate
(4 PDD) for TRPV4). After 2–5 min of agonist treatment, the drug
a
a
was washed out and wells were rinsed thrice with wash buffer.
The cells were lyzed in lysis buffer on a shaker. Radioactivity in cell
lysates was measured as counts per minute (cpm) using TopCount
liquid scintillation counter (Perkin Elmer).
4.2.3. Metabolic stability test of (4R)-(ꢂ)-10e
The in-vitro metabolic stability of (ꢂ)-10e was determined
using liver microsomes from male CD1 mouse, male Wistar rat,
male Beagle dog, male Cynomolgus monkey and pooled human li-
ver microsomes obtained from Xenotech (USA). A 5.0 mM stock of
(ꢂ)-10e was prepared in DMSO from which a working solution of
(ꢂ)-10e (10 mg/kg, po) were administered and acetone (100
ll)
was applied as a spray on the ipsilateral paw and the response
was observed and recorded for 2 min. This procedure was repeated
3–5 times with a 5 min interval between each application. Cold
pain was measured by number of flinches and frequency of paw
licking measured in a 2 min interval at 0, 0.5, 1.0, 2.0 and 3.0 h post
treatment. Based on the flinching and licking, clinical scores were
assigned and correlated for the determination of cold pain
intensity.50
0.1 mM was prepared in DMSO. A 5.0
solution, 25
spiked into 445
tiated by the addition of a 25
nicotinamide adenine dinucleotide phosphate) in water to get a fi-
nal concentration of 2.0 mM of NADPH, 1.0 M of test item and
l
L aliquot of the working
L of 20 mg/mL of protein (liver microsomes) was
L of KH2PO4 buffer (pH 7.4). The reaction was ini-
L aliquot of 40 mM NADPH (reduced
l
l
l
l
1.0 mg/mL of protein. The reaction mixture was incubated at
37 °C for 60 min and terminated by the addition of 3 mL of TBME
(tert-butyl methyl ether). For the 0 min samples, the reaction was
terminated with 3 mL of TBME prior to the addition of NADPH. A
4.2.6. Icilin induced wet dog shake test for (4R)-(ꢂ)-10e
Healthy, male C57/BL6 mice (20–30 g) were used for the study.
Icilin was dissolved in 20% DMSO and 1% Tween 80 in distilled
water and injected ip in a volume of 10 ml/kg. The experiments
were performed during the light phase between 9 am and 3 pm.
Each animal was acclimatized in bell jar (22 ꢃ 18 ꢃ 25 cm;
L ꢃ W ꢃ H) for 10 min for three consecutive days before icilin
administration. The animals were weighed, grouped and icilin
(30 mg/kg, ip) was administered to all groups except the naïve
groups. Compound (ꢂ)-10e (3, 10 and 30 mg/kg, po) and pregaba-
lin (30 mg/kg, po) were administered 30 and 60 min prior to icillin
injection, respectively. The naïve and vehicle groups received 0.5%
MC as vehicle. Incillin-induced cold pain was estimated by the
number of WDS during a 30 min period post-icillin injection.
25 lL aliquot of a 10.0 lg/mL internal standard solution in metha-
nol was spiked in both 0 h and 60 min samples. The samples were
processed by vortex mixing for 5 min and centrifuging at 1000g for
5 min. The clear supernatant liquid was separated and dried under
nitrogen. The residue was reconstituted by adding a 500
reconstitution solvent (9:1 v/v mixture of acetonitrile and
L sample was in-
lL of
2.0 mM ammonium acetate in water) and ꢀ5
l
jected into the LC/MS/MS system. The peak area ratios of test item
to internal standard were determined.
Based on the comparison of area ratios of test item to internal
standard in the 60 min samples relative to the 0 min samples,
the percent metabolized and percent remaining of the test com-
pound were estimated.
4.2.7. Oxaliplatin-induced peripheral neuropathic pain test for
(4R)-(ꢂ)-10e
4.2.4. Single dose oral PK study of (4R)-(ꢂ)-10e in SD rats
Three male SD (Sprague Dawley) rats weighing between
180ꢂ220 g were used for the study. The animals were fasted over-
night before dosing and food was administered 4 h post dosing.
Water was removed just before dosing and was provided ad libitum
2 h post dosing. Compound (ꢂ)-10e was formulated using 1%
Tween 80Ò and 0.5% (w/v) methyl cellulose suspension in water
(qs). The suspension was administered by oral gavage and blood
samples were collected at 0.5, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0 and 24.0 h
post dose into tubes containing K2EDTA as anticoagulant. The
blood samples were centrifuged at 1000g for 10 min and plasma
was separated. The plasma samples, standard and quality control
samples were processed using liquid–liquid extraction after the
addition of an internal standard and the concentrations were esti-
mated using an achiral LC/MS/MS method. The plasma concentra-
Healthy, male C57/BL6 mice (20–30 g) were used for the study.
Oxaliplatin was dissolved in 5% glucose in distilled water and in-
jected ip in a volume of 10 ml/kg. Oxaliplatin is known to increase
TRPM8 mRNA expression in dorsal root ganglia that reaches the
peak on day 3. Hence, in the current study, animals were tested
for cold allodynia 3 days post oxaliplatin (3 mg/kg, ip) injection.
Acetone (50 ll) was sprayed on the sub plantar region of the right
paw with the help of a microsyringe sprayer and immediately the
animal was kept in a chamber to record licking time during a 60 s
observation period. This procedure was repeated three times with
a 15 min interval between each application. The naïve and vehicle
groups received 0.5% MC as vehicle. Compound (ꢂ)-10e (3, 10 and
30 mg/kg, po) and pregabalin (30 mg/kg, po) were administered 30
and 60 min prior to the acetone spray, respectively.

S. S. Chaudhari et al. / Bioorg. Med. Chem. 21 (2013) 6542–6553
6553
22. Chaudhari, S. S.; Thomas, A.; Kadam, A. B.; Adik, B. G.; Dhone, S. V.; Khairatkar-
Joshi, N.; Mukhopadhyay, I. WO 103,381, 2010.
Acknowledgments
23. Calvo, R. R.; Meegalla, S. K.; Parks, D. J.; Parsons, W. H.; Ballentine, S. K.; Lubin,
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Neeper, M.; Brandt, M. R.; Stone, D. J.; Flores, C. M. Bioorg. Med. Chem. Lett.
2012, 22, 2922.
We thank Dr. Srinivas Gullapalli for review of the manuscript
and for his valuable inputs on in-vivo pharmacology. We also
thank Dr. Rajesh Dwivedi and his team for analytical support espe-
cially for the optical purity measurements of compounds by chiral
HPLC. We extend our gratitude to Mr. Rambabu Pattem for the
HRMS analysis of compounds.
26. Zhu, B.; Xia, M.; Xu, X.; Ludovici, D. W.; Tennakoon, M.; Youngman, M. A.;
Matthews, J. M.; Dax, S. L.; Colburn, R. W.; Qin, N.; Hutchinson, T. L.; Lubin, M.
L.; Brandt, M. R.; Stone, D. J.; Flores, C. M.; Macielag, M. J. Bioorg. Med. Chem.
Lett. 2013, 23, 2234.
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Supplementary data
Supplementary data associated with this article can be found, in
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