Synthesis and optimization of novel α-phenylglycinamides as selective TRPM8 antagonists

Article abstract of DOI:10.1016/j.bmc.2016.11.049

Transient receptor potential melastatin 8 (TRPM8) is activated by innocuous cold and chemical substances, and antagonists of this channel have been considered to be effective for pain and urinary diseases. N-(3-aminopropyl)-2-{[(3-methylphenyl)methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride (AMTB), a TRPM8 antagonist, was proposed to be effective for overactive bladder and painful bladder syndrome; however, there is a potential risk of low blood pressure. We report herein the synthesis and structure–activity relationships of novel phenylglycine derivatives that led to the identification of KPR-2579 (20l), a TRPM8 selective antagonist. KPR-2579 reduced the number of icilin-induced wet-dog shakes and rhythmic bladder contraction in rats, with no negative cardiovascular effects at the effective dose.

Full text of DOI:10.1016/j.bmc.2016.11.049

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and optimization of novel
a-phenylglycinamides as selective
TRPM8 antagonists
a
⇑
Jun-ichi Kobayashi , Hideaki Hirasawa, Tetsuji Ozawa , Tomonaga Ozawa, Hiroo Takeda,
Yoshikazu Fujimori, Osamu Nakanishi, Noboru Kamada, Tetsuya Ikeda b
Discovery Research, R&D, Kissei Pharmaceutical Co., Ltd., 4365-1 Hotakakashiwabara, Azumino, Nagano 399-8304, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Transient receptor potential melastatin 8 (TRPM8) is activated by innocuous cold and chemical sub-
stances, and antagonists of this channel have been considered to be effective for pain and urinary dis-
eases. N-(3-aminopropyl)-2-{[(3-methylphenyl)methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride
(AMTB), a TRPM8 antagonist, was proposed to be effective for overactive bladder and painful bladder
syndrome; however, there is a potential risk of low blood pressure. We report herein the synthesis
and structure–activity relationships of novel phenylglycine derivatives that led to the identification of
Received 8 November 2016
Revised 23 November 2016
Accepted 25 November 2016
Available online xxxx
Keywords:
TRPM8
TRPM8 antagonist
Phenylglycinamide
KPR-2579
KPR-2579 (20l),
a TRPM8 selective antagonist. KPR-2579 reduced the number of icilin-induced
wet-dog shakes and rhythmic bladder contraction in rats, with no negative cardiovascular effects at
the effective dose.
Ó 2016 Elsevier Ltd. All rights reserved.
Ugi reaction
OAB
1. Introduction
sub-urothelial nerve fibers, and an increase of TRPM8 expression
has been observed in patients with idiopathic detrusor overactivity
Transient receptor potential (TRP) channels are non-selective
cation channels that are activated by environmental and endoge-
nous stimuli such as temperature, osmotic pressure, redox poten-
tial, chemical substances, and mechanical stimulus.1 TRPM8 is a
temperature-sensing TRP channel that can be activated by innocu-
ous cold stimuli and chemical substances such as menthol and
icilin.2 Expression of TRPM8 is observed mainly in the prostate,
dorsal root ganglion, and trigeminal ganglion, and activation of
these sensory neurons via TRPM8 are thought to induce the
sensation of cold.3 Cooling sensation and pain appear to be related,
and therefore TRPM8 has been regarded as a potential target for
analgesia.4 A number of TRPM8 antagonists have been reported
(Fig. 1), some of which have gone through clinical trials, for
treatment of pain and migraines (PF-05105679 1, AMG 333).5,6
Treatment of urinary diseases have been proposed as another
and painful bladder syndromes.8 Also, researchers of GlaxoSmithK-
line have reported that N-(3-aminopropyl)-2-{[(3-methylphenyl)
methyl]oxy}-N-(2-thienylmethyl)benzamide
hydrochloride
(AMTB, 2) reduced the frequency of rhythmic bladder contractions
and nociceptive reflex responses to noxious bladder distension in
rats.9 Intravesical instillation of TRPM8 stimulants, such as cold-
saline and menthol, decrease bladder threshold volume in guinea
pigs, and this effect was reduced by PBMC (3), a TRPM8 antago-
nist.10,11 In addition, application of menthol or similar stimuli to
the skin that induces the sensation of cold to the body, increased
detrusor overactivity in rats, and this response was prevented by
treatment of TRPM8 antagonists (BCTC 4, AMTB 2, DFL23448
6).12–14 Furthermore, similar cold-induced bladder responses have
been reported in mice; the responses were reduced in
Trpm8ꢀ/ꢀ mice compared with Trpm8+/+ mice.13
potential
therapeutic
use
of
TRPM8
antagonists.7
Recently, ex vivo single-unit mechanosensitive bladder afferent
TRPM8-immunoreactive staining has been observed in human
activities induced by intravesical L-menthol administration in rats
have been recorded, and the activation of C-fibers was suppressed
by pretreatment of RQ-00203078 (5), a selective TRPM8 antago-
nist.15 In clinical treatment of overactive bladder (OAB), anticholin-
ergic agents and b-adrenergic antagonist have been widely used,
but these drugs are considered to have potential risks such as
dry mouth and cardiovascular effects, respectively,16 and therefore
⇑
Corresponding author.
E-mail address: jun-ichi_kobayashi@pharm.kissei.co.jp (J.-i. Kobayashi).
Present address: Intellectual Property, Kissei Pharmaceutical Co., Ltd., 19-48
a
Yoshino, Matsumoto, Nagano 399-8710, Japan.
b
Present address: Medical Research, Kissei Pharmaceutical Co., Ltd., 1-8-9 Nihon-
bashimuromachi, Chuo-ku, Tokyo 103-0022, Japan.
http://dx.doi.org/10.1016/j.bmc.2016.11.049
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049

2
J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
NH2
O
N
N
O
O
CO2H
N
O
O
O
N
NH2
O
S
F
PF-05105679 (1)
AM TB (2)
PBMC (3)
CF3
Cl
N
N
N
N
O
N
O
S O
S
N
N
OCF3
N
N
H
N
N
OH
F
Cl
CO2H
RQ-00203078 (5)
Fig. 1. Reported TRPM8 antagonists. (1) A clinical trial was performed; (2–6) effects on urinary functions were investigated; (4) non-selective TRPM8 antagonist.
BCTC (4)
DFL23448 (6)
novel pharmacological targets are needed. For these reasons, we
selected TRPM8 as a therapeutic target for treatment of OAB.
When our research began, the effect of AMTB on distension-
induced bladder contraction, mentioned above, had already been
reported. However, administration of this compound also induces
low blood pressure at a high effective dose in rats.9 Our off-target
binding study of AMTB revealed that the compound has affinity for
b-adrenoceptor (rat, non-selective, 66%), and muscarinic receptor
by basic hydrolyzation, but racemization occurred during the
hydrolysis. To obtain a 1:1 mixture of diastereomers (rac-10), the
racemic ester (rac-7h) was used as starting material.
The preparation of N-phenethyl derivatives was problematic. As
is the case with N-benzyl derivatives, we attempted acylation of
secondary amine of N-phenethyl amino acid amide (13) which
was synthesized from chiral phenethylamine (16c) with mande-
lamide derivative (11). But the acylation did not proceed, presum-
ably because of steric hindrance around a secondary amine, like
diisopropylamine. Fortunately, acylation of benzyl ester derivative
(14) reacted; however, a long reaction time was needed for several
days at room temperature. The desired product (20c) was obtained
by catalytic hydrogenation of the benzyl ester (15) followed by
amidation. However, because of the low pKa of the alpha proton
of phenylglycine, epimerization occurred in all reactions, except
for the hydrogenation step. The total yield was only 17%, and sep-
aration of diastereomers in each step was complicated. To avoid
this problem, a reduction in the number of reaction steps was
needed after construction of the chiral phenylglycine.
(rat, non-selective, 69%) at 10 l
M.17 Moreover, binding to other
receptors with natural ligands that have primary amines (e.g. sero-
tonin receptor) was observed. From these findings, we hypothe-
sized that the reduction of basicity of primary amine would be
effective in avoiding such off-target binding affinity and cardiovas-
cular effects. In addition, intravesical administration of transient
receptor potential ankyrin 1 (TRPA1) agonists has been reported
to increase voiding frequency,18 and therefore the ideal compound
also needs to have selectivity against TRPA1 agonistic activity. Allyl
isothiocyanate and cinnamaldehyde have been reported as TRPA1
agonists, and the electrophilic structures such as thiocyanate and
a
,b-unsaturated aldehyde are considered to react with cysteines
Hence, we decided to use the Ugi reaction.21 The Ugi reaction
can create an N-acyl-N-alkylamino acid amide skeleton from a cor-
responding amine, aldehyde, carboxylic acid and isocyanide. In the
field of medicinal chemistry, the reaction was employed to build a
chemical library to analyze SAR,21b and some applications to
process chemistry have also been reported.22 Pfizer researchers
have disclosed racemic arylglycinamide derivatives as oxytocin
antagonists,23 with structures that are related to the compounds
in this paper, however, the use of chiral phenethylamine was not
described. Asymmetric Ugi reactions have been attempted with
phenethylamine as a chiral component in the synthesis of Fura-
nomycin24 and other compounds,25 but to the best of our knowl-
edge, most of the cases resulted in poor diastereoselectivity. The
results of these reports suggested to us that using the Ugi reaction
might yield the desired diastereomer easily. The imine intermedi-
ates (17a–c, i, k, l) were prepared from chiral amine (16a–c, k, l)
and aldehyde, and then reacted with carboxylic acid and iso-
cyanide to afford a mixture of four diastereomers (19a–l) in mod-
erate yield (Scheme 2). The enamine that formed was hydrolyzed
under acidic conditions to afford a mixture of 20a–l and 21a–l.
Although the steric hindrance around the secondary amine of
intermediate (18a–l) is similar to that of 13 in Scheme 1, the Ugi
reaction proceeded smoothly. The N-acylation step of the Ugi reac-
tion proceeds via an acyl rearrangement from iminoester (18a–l),
and the intramolecular assistance may contribute greatly to the
in the N-terminal of TRPA1 for channel opening.19 For this reason,
we also planned to avoid electrophilic structures during molecular
designing.
Our goal was to discover an orally available TRPM8 antagonist
with a sufficient safety margin for cardiovascular effects as well
as selectivity against other TRP channels. In this article, we
describe synthesis and the structure–activity relationships (SARs)
of novel arylglycinamides as TRPM8 antagonists. The selected
compound, N-((R)-carbamoylphenylmethyl)-N-[(R)-1-(3,5-difluo-
rophenyl)ethyl]benzamide (KPR-2579, 20l) has good bioavailabil-
ity and potency; moreover, no cardiovascular effect was observed
at the effective dose.
2. Chemistry
To prepare N-benzyl derivatives (9a–r), the amino group of
amino acid amides (7a, e, f, h–j) were functionalized by reductive
alkylation followed by acylation (Scheme 1). Although some ana-
logues of imine intermediates, prepared from phenylglycinamide
and benzaldehyde, have been reported to racemize under basic
conditions in solvent,20 the optical purity of our products was up
to 90% ee, with the exception of the OCF3 derivative (9p). The car-
boxylic acid derivative was prepared from the corresponding ester
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J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
3
R4
R4
R4
R5
a, b
c, (d)
R5
N
N
H
R2
R1
R2
R1
HCl · H2N
R5
R6
O
8a-c, e, f, h-r
9a-r, rac-10
7a, b, e, f, h-j
N
R5a
e
Ph
f
NH2
N
H
R5a
O
p-NsO
R5a
Cl
Cl
Cl
11R5a = CONH2
12 R5a = CO2Bn
16c
13 R5a = CONH2
14 R5a = CO2Bn
15 R5a = CO2Bn
20cR5a = CONH2
g, h
Scheme 1. Synthesis of N-benzyl and phenethyl derivatives via stepwise modifications. Reagents and conditions: (a) R1R2PhCHO, Et3N, MeOH, rt; (b) NaBH4, 0 °C; (c) R6COCl,
Et3N, CH2Cl2, rt; (d) 2 mol/L NaOH aq., MeOH, 40 °C (if applicable); (e) KHCO3, MeCN, 50 °C; (f) PhCOCl, Et3N, DMAP, CH2Cl2, 5 days, rt; (g) 10% Pd–C, H2, THF, 1 h, rt; (h) NH4Cl,
HATU, DIPEA, CH2Cl2, 12 h, rt, 46% for 3 steps from 14; p-NsO: p-nitrobenzenesulfonyloxy.
R3
R4
R3 R4
N
R3
N
a
b
N
H
R2
R1
NH2
R2
R1
17a-c, i, k, l
R2
R1
O
R6
Ph
O
18a-l
16a-c, k, l
R3 R4
N
R3 R4
N
R3 R4
N
H
N
NH2
NH2
c
R2
R1
R2
R1
R2
R1
+
O
O
O
R6
O
R6
O
R6
O
Ph
19a-l
20a-l
21a-l
20g R6 = 2-Acetoxyphenyl
21g R6 = 2-Acetoxyphenyl
d
e
d
20m R6 = 2-Hydroxyphenyl
21m R6 = 2-Hydroxyphenyl
20h R6 = 2-Nitrophenyl
20n R6 = 2-Aminophenyl
Scheme 2. Synthesis of N-phenethyl and N-phenylpropyl derivatives via the Ugi reaction. Reagents and conditions: (a) R4CHO, Et3N, MeOH, 50 °C; (b) R6CO2H, 4-
Phenylcyclohexenyl isocyanide, 50 °C; (c) HCl, dioxane, H2O, rt; (d) K2CO3 aq., MeOH, rt; (e) 10% Pd–C, H2, THF, rt, 64%.
acylation. Regarding diastereoselectivity, the undesired S-configu-
ration was dominantly formed, with the exception of 20b, how-
ever, a 2-fold improvement in the total yield was obtained
compared with the previous method. Both diastereomers could
be separated by column chromatography. For the synthesis of the
salicylamide derivative (20m, 21m), acetylsalicylic acid was used
as the carboxylic acid reagent, and the acetyl group was partially
hydrolyzed during the Ugi reaction and the following hydrolysis
of imine. The residual acetyl group was hydrolyzed with K2CO3
aq./MeOH at room temperature. The amino derivative (20n) was
prepared from o-nitrobenzoic acid, and the nitro group was
reduced during the last step of synthesis. Absolute configuration
was confirmed by single-crystal X-ray diffraction of KPR-2579
(20l).26
amine might be necessary for TRPM8 inhibition, however, com-
pound 9a exhibited moderate TRPM8 inhibitory activity. From this,
we focused on the N-substituted amino acid amide scaffold, and
performed a screening campaign to afford compound 9b. Surpris-
ingly, compound 9c, which has a BMAC substructure, was inactive.
Because of the function of TRPM8 as a thermosensor, the protein
tetramer should be highly flexible in lipid bilayers; therefore com-
pound 9b may have more binding modes with TRPM8 compared
with BMAC. From this hypothesis, we synthesized some deriva-
tives of compound 9b to reveal SAR of this scaffold (Table 2).
Comparison of the enantiomers showed that the R-configura-
tion (9b) was favored. Since it is known that electron-rich aromatic
rings such as thiophene and furan were susceptible to CYP metab-
olization,29 we replaced thiophene with phenyl to improve meta-
bolic stability (9d), which maintained inhibitory activity.
Homologation of the phenyl of R4 and R6 to a benzyl group resulted
in decrease of potency (9e, g), and removal of the aromatic ring led
to a loss of activity (9f). The results show that the phenyl ring of R4
is necessary for TRPM8 inhibition, and that the activity was greatly
affected by the distance from the central tertiary amide to the phe-
nyl ring of R4 and R6. Although the ester analogue (9h) showed
enhanced activity, the methyl ester was readily hydrolyzed to car-
boxylic acid by incubation in rat plasma (data not shown). Alkyla-
tion of carbamoyl nitrogen (9i, j) was also unfavorable, therefore
we concluded that the unsubstituted carbamoyl was the optimal
structure. Another arylglycine scaffold has been independently
reported as an TRPM8 antagonist,30 but all of the compounds
reported have a pyrrolidine amide in the C-terminus. This SAR
3. Results and discussion
TRPM8 inhibition was measured by monitoring a menthol-
induced Ca2+ increase in HEK293T cells expressing hTRPM8. The
inhibitory activity of AMTB (menthol IC50 = 0.414 0.027
lin IC50 = 0.448 0.048 M, pIC50 = 6.35 0.05) was consistent
with the value reported by GlaxoSmithKline (icilin
lM; ici-
l
pIC50 = 6.23 0.02).9 As mentioned above, our primary objective
was to reduce the basicity of the primary amine of AMTB. We syn-
thesized an amide analogue of BMAC,27 one of the AMTB deriva-
tives (Table 1, 9a). From claims and examples from the patent
application for BMAC,28 we speculated that the terminal primary
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4
J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Table 1
Table 3
Inhibitory activities of BMAC-related compounds.
Substituent effects of the N-benzyl group of compound 9d.
R4
N
R5
R3
O
R2
O
NH2
N
O
S
O
R1
Compounds
R2
R4
R5
hTRPM8 IC50 (l
M)a
b
4-Fluoro-BMAC
4-Florobenzyloxy
4-Florobenzyloxy
H
H
H
Ph
Ph
CH2NH2
CONH2
CONH2
CONH2
–
9a
9b
9c
9.1
1.7
>100
Compounds
R1
R3
CLint
(
lL/min/mg protein)
hTRPM8
IC50 (l
M)a
4-Florobenzyloxy
9d
9k
9l
9m
9n
9o
9p
9q
9r
3-OMe
H
3-F
H
H
H
H
H
H
H
H
>150
12
10
12
>150
4
8
12
4
0.90
11
a
Each IC50 value represents an average of at least two independent experiments.
b
We could not evaluate pure 4-Fluoro-BMAC28 because
a rearrangement
8.1
3.0
0.87
5.0
4.7
5.6
28
reaction of the thienyl group to the terminal amine occurred during handling.27
3-Cl
3-Me
3-CF3
3-OCF3
2-Cl
Table 2
Derivatization of amino acid and benzoyl moiety of compound 9b.
4-Cl
H
20a
ent-21a
20b
20c
3-OMe
3-OMe
H
(R)-Me
(S)-Me
(R)-Et
(R)-Me
>150
>150
8
0.24
43
19
R4
R5
N
3-Cl
11
0.40
O
a
R6
O
Each IC50 value represents an average of at least two independent experiments.
O
Compounds
Config.a
R4
R5
R6
hTRPM8 IC50 (l
M)b
stability in vivo. The calculated logD7.4 of 20c was 4.62, which indi-
cated that further improvement was required to avoid oxidative
metabolism. Therefore, we attempted to introduce a hydrophilic
substituent to 20c to decrease the clearance value.
ent-9b
9d
9e
9f
9g
9h
rac-10
9i
S
R
R
R
R
R
Ph
Ph
Bn
Me
Ph
Ph
Ph
Ph
Ph
NH2
NH2
NH2
NH2
NH2
OMe
OH
2-Thienyl
Ph
2-Thienyl
Ph
Bn
Ph
Ph
Ph
Ph
54
0.90
37
>100
19
0.21
59
Because introduction of substituents on the phenyl ring of R4
decreased activity (data not shown), we aimed to modify the phe-
nyl ring of R6 (Table 4). According to the screening results of
methyl substitutions of R6 (20d–f), only the 2-substitutied com-
pound (20d) was acceptable. Changing to a hydrophilic substituent
at 2-position maintained efficacy (20m, n), but there was no
improvement in clearance. As another approach to reduce
lipophilicity, we replaced the phenyl rings of R4 and R6 with pyri-
dyl, which led to a marked improvement of clearance but a drop in
potency (20i, j). These results seemed to suggest that simultaneous
improvement of metabolic stability and activity were difficult to
achieve solely focusing on lowering logD7.4. According to the pre-
sent metabolite study, hydroxylation of chlorobenzene was
assumed to be one of the metabolic pathways of 20c, so we next
attempted to introduce additional electron-withdrawing sub-
stituents on the ring. As shown in Table 3, substitution to the ortho
and para positions were considered to be unfavorable for activity;
we therefore introduced more substituents to the meta position. As
expected, the 3,5-difluoro derivative showed low clearance in
RLM; we selected the compound 20l (KPR-2579) as a candidate.
The pharmacokinetic profiles of KPR-2579 in rats are shown in
Fig. 2 and Table 5. Oral administration of the compound at 10 mg/
kg showed good bioavailability and sufficient concentration in
plasma to evaluate TRPM8 antagonism in vivo. To confirm the effi-
cacy of TRPM8 antagonism in vivo, icilin-induced wet-dog shake
was used. A suspension of KPR-2579 (1, 3, 10 mg/kg) was orally
administered to rats 1 h prior to intraperitoneal injection of icilin.
At 5 min after the injection, the number of induced wet-dog shakes
was counted for 5 min. The number of wet-dog shakes was
reduced dose-dependently (31% inhibition at 1 mg/kg, 73% inhibi-
tion at 3 mg/kg), and the behavior was completely inhibited at
10 mg/kg. Because we had confirmed general TRPM8 antagonism
in vivo, an effect on bladder contraction was evaluated. As is the
Racemic
R
R
NHMe
NMe2
10
86
9j
a
Absolute configuration of amino acid moiety.
Each IC50 value represents an average of at least two independent experiments.
b
inconsistency implies that these derivatives also might have other
binding mode against TRPM8 compared with our compounds.
We selected 9d as a lead compound for further modification as
this compound exhibited poor metabolic stability in rat liver
microsome (RLM). Metabolite analysis indicated that demethyla-
tion of the methoxy group and N-demethoxybenzylation predom-
inately occurred. Replacing the 3-methoxy group with halogen (9l,
m), haloalkyl (9o), and haloalkyloxy (9p) groups improved meta-
bolic stability, however, all of them exhibited weak inhibition com-
pared with 9d (Table 3). Meta-substitution proved to optimal for
the position of the substituent in terms of the balance of activity
and stability (9m, q, r). To prevent oxidative cleavage of the C-N
bond, another major metabolic reaction, introduction of alkyl
group at benzylic position was attempted. About a 4-fold enhance-
ment of activity was observed for R-phenethyl derivative (20a); in
contrast, the S-phenethyl derivative (ent-21a) showed a 40-fold
decrease of activity. The results show that the R,R-configuration
was optimal for this scaffold. Replacement with an ethyl group
(20b) did not improve activity compared with 9k. Therefore, fur-
ther elongation of the side alkyl chain proved to be ineffective.
From these observations, we synthesized compound 20c which
showed good activity and moderate metabolic stability.
Although we chose compound 20c as the next lead, the clear-
ance value was still insufficient to achieve several hours duration
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J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
5
Table 4
Optimization of compound 20c for lowering metabolic clearance.
R4
R6
NH2
N
R2
R1
O
O
R1
R2
R4
R6
logD7.4
CLint
(lL/min/mg protein)
hTRPM8 IC50 (l
M)a
b
Compounds
20d
20e
20f
20m
20n
20i
20j
20k
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
3-F
H
H
H
H
H
H
H
5-F
5-F
Ph
Ph
Ph
Ph
Ph
3-Pyridyl
Ph
Ph
Ph
2-MePh
3-MePh
4-MePh
2-OHPh
2-NH2Ph
Ph
3-Pyridyl
Ph
Ph
5.14
5.14
5.14
4.90
4.44
3.41
3.41
4.77
4.31
83
0.39
2.1
0.85
0.10
0.42
4.1
2.5
0.18
>150
>150
102
77
7
8
13
2
20l (KPR-2579)
0.080 0.005
a
Each IC50 value represents an average of at least two independent experiments.
LogD7.4 values were calculated using Marvin 6.2.0 (ChemAxon).
b
Fig. 3. Effect of KPR-2579 for rhythmic bladder contractions (RBCs). n = 4–5,
Fig. 2. Plasma concentrations of KPR-2579 in rats (n = 3).
**p < 0.01.
case with previous study of AMTB,9 we compared the frequencies
of distension-induced rhythmic contractions for each 10 min
before and after administration. Intravenous administration of
KPR-2579 reduced the number of rhythmic bladder contractions
at 0.1 and 0.3 mg/kg (Fig. 3), but the amplitude of the contractions
was unchanged. This observation is consistent with the result
found with AMTB. After 15 min of 1 mg/kg intravenous administra-
Table 5
Pharmacokinetic parameters of KPR-2579 in rats (n = 3).
Parameters
Dose
KPR-2579
t1/2
Vss
Cmax
Tmax
BA
1 mg/kg i.v.
1 mg/kg i.v.
10 mg/kg p.o.
10 mg/kg p.o.
10 mg/kg p.o.
34 min
1.2 L/kg
3.23 lM
50 min
tion, the concentration of KPR-2579 in plasma was 1.12
therefore the effective plasma concentration for suppressing rhyth-
mic bladder contraction was estimated to be below 1 M. As
described in Fig. 2, the plasma concentration of KPR-2579 was
kept over 1 M for 3.5 h after oral administration at 10 mg/kg.
lM, and
59%
l
l
Table 6
From these observations, we concluded that oral administration
of KPR-2579 at 10 mg/kg can be effective for more than 4 h in ani-
mal models with frequent urination.
To confirm cardiovascular effects of compound KPR-2579, off-
target binding affinity and effects to blood pressure and heart rate
were evaluated. As expected, the binding affinity to b-adrenergic,
muscarinic, and serotonergic receptors were independently sup-
pressed below 20% at 10 lM. We assume that this is because the
initial structural conversion from primary amine to amide was
effective. As a change in blood pressure and heart rate were not
In vitro activity of KPR-2579 with TRP channels.
a
TRP Channels
IC50
hTRPM8
rTRPM8
hTRPA1
hTRPV1
hTRPV4
hTRPA1
0.080 0.005
0.089 0.004
l
l
M
M
>30
>30
>30
lM
lM
lM
EC50 > 30 lM
a
Each IC50 value represents an average of three
independent experiments SEM.
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049

6
J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
observed with oral administration at 10 mg/kg, the dose at which
wet-dog shake behavior was completely inhibited in rats, we con-
cluded that KPR-2579 had a sufficient safety margin between sup-
pression of rhythmic bladder contraction and cardiovascular
effects. Moreover, KPR-2579 showed excellent selectivity over
other TRP channels including TRPA1 agonistic activity (Table 6);
methanol. The mixture was stirred for 1–5 h at room temperature.
Sodium borohydride (5.0 equiv) was added carefully to the mix-
ture cooled with ice. The mixture was stirred for 1 h at 0 °C and
then partitioned between water and EtOAc. The organic layer
was washed with brine, and dried over anhydrous MgSO4. The
solution was concentrated in vacuo, and the residue was purified
using either silica gel column chromatography or crystallization.
therefore, this compound proved to be
antagonist.
a selective TRPM8
5.1.2. 2-[4-(4-Fluorobenzyloxy)-3-methoxybenzylamino]acetamide
(8a)
4. Conclusion
The title compound was prepared from glycinamide hydrochlo-
ride (7a, 166 mg, 1.5 mmol), 4-(4-fluorobenzyloxy)-3-methoxy-
In summary, our screening campaign around AMTB to avoid
cardiovascular effect afforded a novel phenylglycinamide scaffold,
and the optimization of lead compound 9b led to the potent and
selective TRPM8 antagonist KPR-2579 (20l). The compound
showed good efficacy against wet-dog shakes and rhythmic blad-
der contractions in rats, a moderate pharmacokinetic profile, and
no cardiovascular effects at the effective dose. We propose KPR-
2579 as a tool for analyzing how TRPM8 plays a role in bladder
function, and suggest further modification of it to identify a clinical
candidate.
benzaldehyde (390 mg, 1.5 mmol),28 triethylamine (209
lL,
1.5 mmol), methanol (12 mL), and sodium borohydride (284 mg,
7.5 mmol) according to the general procedure of N-benzyl amino
acid derivatives. The crude product was suspended in a mixture
of EtOAc and hexane (1:1, 10 mL) to afford 8a (395 mg, 83%) as a
colorless solid. 1H NMR (CDCl3) d 3.31 (s, 2H), 3.73 (s, 2H), 3.89
(s, 3H), 5.09 (2H, s), 5.43 (br s, 1H), 6.78 (dd, J = 8.2, 1.9 Hz, 1H),
6.81–6.86 (m, 2H), 6.98 (br s, 1H), 7.02–7.09 (m, 2H), 7.38–7.44
(m, 2H); 13C NMR (DMSO-d6) d 51.1, 52.4, 55.5, 69.3, 112.1,
113.5, 115.2 (d, J = 21.5 Hz), 119.9, 130.0 (d, J = 8.4 Hz), 133.4,
133.6 (d, J = 3.2 Hz), 146.5, 149.1, 161.8 (d, J = 243.7 Hz), 173.4;
HRMS calcd for C17H19FN2O3 (M+H)+ 319.1452, found 319.1451;
mp 133 °C.
5. Experimental
5.1. Chemistry
5.1.3. (R)-2-(3-Methoxybenzylamino)-2-phenylacetamide (8b)
The title compound was prepared from (R)-phenylglycinamide
hydrochloride (7b, 373 mg, 2.0 mmol), 3-methoxybenzaldehyde
All reagents and solvents were purchased from commercial
sources and used without further purification. Reactions were
monitored by thin layer chromatography (TLC) using Merck Kiesel-
gel 60 F254 plates or Fuji Silysia Chemical Ltd. Chromatorex NH-TLC
plates. Flash column chromatography was performed on Biotage
prepacked columns. Melting points were determined on a Yanaco
micro melting point apparatus (MP-J3). 1H NMR spectra were
recorded on a Bruker AV400M spectrometer at 400 MHz, and
chemical shifts (d) are reported in parts per million (ppm) units
using tetramethylsilane as an internal standard. Data are presented
as follows; chemical shift, multiplicity (s, singlet; d, doublet; t, tri-
plet; q, quartet; dd, doublet of doublets; dt, doublet of triplets; td,
triplet of doublets; tt, triplet of triplets; m, multiplet; br, broad; br
s, broad singlet; br d, broad doublet), coupling constant, and inte-
gration. 13C NMR spectra were recorded on a Bruker AV400M spec-
trometer at 100 MHz with complete proton decoupling, and
chemical shifts (d) are reported in parts per million (ppm) with
the solvent as the internal reference (d 77.16 in CDCl3; d 39.52 in
DMSO-d6; d 67.21 in THF-d8).31 High resolution mass spectra
(HRMS) were recorded on an Agilent Technologies 6520 Accu-
rate-Mass Q-TOF instrument using electrospray ionization (ESI,
positive or negative ion mode). HPLC analysis for tested com-
pounds was performed on a Shimadzu LC-VP or Prominence series
instrument. Chemical purity was determined as following analyti-
cal condition; L-column2 (Chemicals Evaluation and Research
(272 mg, 2.0 mmol), triethylamine (279 lL, 2.0 mmol), methanol
(12 mL), and sodium borohydride (378 mg, 10.0 mmol) according
to the general procedure of N-benzyl amino acid derivatives. The
crude product was suspended in Et2O (10 mL) to afford 8b
(427 mg, 79%) as a colorless solid. 1H NMR (CDCl3) d 2.12 (br s,
1H), 3.78 (s, 2H), 3.81 (s, 3H), 4.24 (s, 1H), 5.44 (br s, 1H),
6.78–6.97 (m, 4H), 7.22–7.45 (m, 6H); 13C NMR (DMSO-d6) d
50.5, 54.9, 64.8, 112.2, 113.4, 120.2, 127.2, 127.3, 128.2, 129.2,
140.2, 142.0, 159.3, 174.0; HRMS calcd for C16H18N2O2 (M+H)+
271.1441, found 271.1441; mp 133 °C.
5.1.4. (S)-2-(3-Methoxybenzylamino)-2-phenylacetamide (ent-8b)
The title compound was prepared from (S)-phenylglycinamide
hydrochloride (ent-7b, 373 mg, 2.0 mmol) according to the prepa-
ration of 8b. The crude product was suspended in Et2O (10 mL) to
afford ent-8b (350 mg, 65%) as a colorless solid. 1H NMR (CDCl3) d
2.12 (br s, 1H), 3.78 (s, 2H), 3.81 (s, 3H), 4.25 (s, 1H), 5.42 (br s, 1H),
6.80–6.97 (m, 4H), 7.22–7.45 (m, 6H); 13C NMR (DMSO-d6) d 50.5,
54.9, 64.8, 112.2, 113.4, 120.2, 127.2, 127.3, 128.2, 129.2, 140.2,
142.0, 159.3, 174.0; HRMS calcd for
C
16H18N2O2 (M+H)+
271.1441, found 271.1439; mp 136 °C.
5.1.5. (R)-2-[4-(4-Fluorobenzyloxy)-3-methoxybenzylamino]-2-
phenylacetamide (8c)
Institute, Japan),
3
lm, 4.6 ꢁ 150 mm, flow rate 1.0 mL/min,
T = 40 °C, 220 nm, mobile phase (MeCN in water): 0–0.1 min
(10%), 0.1–13.0 min (10–85%), 13.0–20.0 min (85%). Optiical purity
was determined as following analytical conditions; Chiralcel OD,
Chiralpak IA-3 or IB-3 (DAICEL), 4.6 ꢁ 250 mm, flow rate 1.0 mL/
min, T = 40 °C, 220 nm. Optical rotations were recorded on a HOR-
IBA SEPA-300 using a 3.5 mm ꢁ 50 mm cell. Data are presented as
follows; specific rotation, concentration (c = g/100 mL), and
solvent.
The title compound was prepared from (R)-phenylglycinamide
hydrochloride (7b, 373 mg, 2.0 mmol), 4-(4-fluorobenzyloxy)-3-
methoxybenzaldehyde (520 mg, 2.0 mmol),28 triethylamine
(279 lL, 2.0 mmol), methanol (12 mL), and sodium borohydride
(378 mg, 10.0 mmol) according to the general procedure of N-ben-
zyl amino acid derivatives. The crude product was suspended in a
mixture of EtOAc and hexane (1:1, 6 mL) to afford 8c (300 mg, 38%)
as a colorless solid. The filtrate was concentrated in vacuo, and the
residue was suspended in a mixture of EtOAc and hexane (1:1,
6 mL) to afford 8c (170 mg, 22%) as a second crop. 1H NMR (CDCl3)
d 2.11 (br s, 1H), 3.68–3.78 (m, 2H), 3.87 (s, 3H), 4.22 (s, 1H), 5.09
(s, 2H), 5.42 (br s, 1H), 6.75–6.90 (m, 4H), 7.02–7.09 (m, 2H), 7.28–
7.45 (m, 7H); 13C NMR (CDCl3) d 52.2, 56.1, 66.6, 70.6, 112.0, 114.3,
5.1.1. General procedure for preparation of N-benzyl amino acid
derivatives
Triethylamine (1.0 equiv) was added to a mixture of amino acid
derivative hydrochloride (1.0 equiv) and aldehyde (1.0 equiv) in
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J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
7
115.6 (d, J = 21.3 Hz), 120.4, 127.6, 128.4, 129.0, 129.2 (d,
5.1.10. (R)-2-(3-Methoxybenzylamino)-N-methyl-2-phenylacetamide
(8i)
J = 8.3 Hz), 132.8, 133.0 (d, J = 3.0 Hz), 139.1, 147.3, 149.9, 162.6
(d, J = 246.1 Hz), 175.1; HRMS calcd for C23H23FN2O3 (M+H)+
395.1765, found 395.1762; mp 129 °C.
The imine intermediate was prepared from (R)-2-amino-N-
methyl-2-phenylacetamide (7i, 329 mg, 2.0 mmol), 3-methoxy-
benzaldehyde (272 mg, 2.0 mmol) in methanol (12 mL), and fol-
lowing reduction was performed by sodium borohydride
(378 mg, 10.0 mmol) according to the general procedure of N-ben-
zyl amino acid derivatives. The crude product was purified using
silica gel column chromatography (eluent EtOAc:Hexane = 1:1–
2:1) to afford 8i (286 mg, 50%) as a colorless solid. 1H NMR (CDCl3)
d 2.07 (br s, 1H), 2.83 (d, J = 5.0 Hz, 3H), 3.75 (s, 2H), 3.81 (s, 3H),
4.24 (s, 1H), 6.79–6.91 (m, 3H), 6.98–7.08 (m, 1H), 7.23–7.40 (m,
6H); 13C NMR (CDCl3) d 26.2, 52.4, 55.3, 66.9, 112.6, 113.9, 120.5,
127.5, 128.2, 128.9, 129.7, 139.3, 141.0, 159.9, 172.7; HRMS calcd
for C17H20N2O2 (M+H)+ 285.1598, found 285.1595; mp 69 °C.
5.1.6. (R)-2-(3-Methoxybenzylamino)-3-phenylpropionamide (8e)
The title compound was prepared from (R)-phenylalaninamide
hydrochloride (7e, 401 mg, 2.0 mmol), 3-methoxybenzaldehyde
(272 mg, 2.0 mmol), triethylamine (279 lL, 2.0 mmol), methanol
(12 mL), and sodium borohydride (378 mg, 10.0 mmol) according
to the general procedure of N-benzyl amino acid derivatives. The
crude product was suspended in a mixture of Et2O and hexane
(1:2, 10 mL) to afford 8e (412 mg, 72%) as a colorless solid. 1H
NMR (CDCl3) d 2.78 (dd, J = 13.9, 9.7 Hz, 1H), 3.22 (dd, J = 13.9,
4.2 Hz, 1H), 3.38 (dd, J = 9.7, 4.2 Hz, 1H), 3.54 (d, J = 13.5 Hz, 1H),
3.70–3.78 (m, 4H), 5.41 (br s, 1H), 6.61–6.69 (m, 2H), 6.74–6.80
(m, 1H), 7.09–7.21 (m, 4H), 7.22–7.35 (m, 3H); 13C NMR (DMSO-
d6) d 50.8, 54.8, 62.5, 112.3, 112.8, 119.9, 126.0, 128.0, 129.1,
129.4, 138.9, 142.2, 159.2, 175.6; HRMS calcd for C17H20N2O2 (M
+H)+ 285.1598, found 285.1596; mp 103 °C.
5.1.11. (R)-2-(3-Methoxybenzylamino)-N,N-dimethyl-2-
phenylacetamide (8j)
The title compound was prepared from (R)-2-amino-N,N-
dimethyl-2-phenylacetamide
1.5 mmol), 3-methoxybenzaldehyde (204 mg, 1.5 mmol), triethy-
lamine (209 L, 1.5 mmol), methanol (12 mL), and sodium borohy-
hydrochloride
(7j,
322 mg,
l
dride (284 mg, 7.5 mmol) according to the general procedure of N-
benzyl amino acid derivatives. The crude product was purified
using silica gel column chromatography (eluent EtOAc:Hex-
ane = 1:1–5:1) to afford 8j (262 mg, 59%) as a pale yellow oil. 1H
NMR (CDCl3) d 2.82 (s, 3H), 2.98 (s, 3H), 3.63 (d, J = 13.3 Hz, 1H),
3.74 (d, J = 13.3 Hz, 1H), 3.80 (s, 3H), 4.48 (s, 1H), 6.77–6.82 (m,
1H), 6.90–6.95 (m, 2H), 7.20–7.37 (m, 6H); 13C NMR (CDCl3) d
36.0, 36.8, 51.4, 55.3, 61.6, 112.9, 113.7, 120.8, 127.9, 128.0,
129.0, 129.4, 138.7, 141.7, 159.8, 172.2; HRMS calcd for
5.1.7. (R)-2-(3-Methoxybenzylamino)propionamide (8f)
The title compound was prepared from (R)-alaninamide
hydrochloride (7f, 249 mg, 2.0 mmol), 3-methoxybenzaldehyde
(272 mg, 2.0 mmol), triethylamine (279 lL, 2.0 mmol), methanol
(12 mL), and sodium borohydride (378 mg, 10.0 mmol) according
to the general procedure of N-benzyl amino acid derivatives. The
crude product was suspended in a mixture of Et2O and hexane
(1:1, 10 mL) to afford 8f (280 mg, 67%) as a colorless solid. 1H
NMR (CDCl3) d 1.36 (d, J = 7.0 Hz, 3H), 3.27 (q, J = 7.0 Hz, 1H),
3.76 (dd, J = 25.6, 13.3 Hz, 2H), 3.82 (s, 3H), 5.38 (br s, 1H), 6.79–
6.91 (m, 3H), 7.09 (br s, 1H), 7.23–7.29 (m, 1H); 13C NMR
(DMSO-d6) d 19.4, 51.0, 54.9, 56.4, 112.1, 113.3, 120.1, 129.2,
C
18H22N2O2 (M+H)+ 299.1754, found 299.1754.
5.1.12. (R)-2-Benzylamino-2-phenylacetamide (8k)
The title compound was prepared from (R)-phenylglycinamide
hydrochloride (7b, 373 mg, 2.0 mmol), benzaldehyde (212 mg,
142.2, 159.3, 176.9; HRMS calcd for
C
11H16N2O2 (M+H)+
209.1285, found 209.1275; mp 108 °C.
2.0 mmol), triethylamine (279 lL, 2.0 mmol), methanol (12 mL),
and sodium borohydride (378 mg, 10.0 mmol) according to the
general procedure of N-benzyl amino acid derivatives. The crude
product was suspended in a mixture of Et2O and hexane (1:1,
10 mL) to afford 8k (398 mg, 83%) as a colorless solid. 1H NMR
(DMSO-d6) d 2.90 (br s, 1H), 3.62 (dd, J = 20.5, 13.7 Hz, 2H), 4.08
(br s, 1H), 7.11 (br s, 1H), 7.20–7.35 (m, 8H), 7.37–7.42 (m, 2H),
7.53 (br s, 1H); 13C NMR (DMSO-d6) d 50.6, 64.8, 126.7, 127.2,
127.3, 128.0, 128.1, 128.2, 140.2, 140.3, 174.0; HRMS calcd for
5.1.8. Methyl (R)-2-(3-methoxybenzylamino)-2-phenylacetate (8h)
The title compound was prepared from (R)-phenylglycine
methyl ester hydrochloride (7h, 403 mg, 2.0 mmol), 3-methoxy-
benzaldehyde (272 mg, 2.0 mmol), triethylamine (279 lL,
2.0 mmol), methanol (12 mL), and sodium borohydride (378 mg,
10.0 mmol) according to the general procedure of N-benzyl amino
acid derivatives. The crude product was purified using silica gel
column chromatography (eluent EtOAc:Hexane = 1:10–1:5) to
afford 8h (290 mg, 51%) as a pale yellow oil. 1H NMR (CDCl3) d
2.37 (br s, 1H), 3.66–3.75 (m, 5H), 3.80 (s, 3H), 4.39 (s, 1H), 6.78–
6.83 (m, 1H), 6.87–6.91 (m, 2H), 7.21–7.27 (m, 1H), 7.28–7.40
(m, 5H); 13C NMR (CDCl3) d 51.3, 52.4, 55.3, 64.3, 112.9, 113.8,
120.7, 127.7, 128.3, 128.9, 129.6, 138.1, 141.2, 159.9, 173.6; HRMS
calcd for C17H19NO3 (M+H)+ 286.1438, found 286.1433.
C
15H16N2O (M+H)+ 241.1335, found 241.1334; mp 132 °C.
5.1.13. (R)-2-(3-Fluorobenzylamino)-2-phenylacetamide (8l)
The title compound was prepared from (R)-phenylglycinamide
hydrochloride (7b, 373 mg, 2.0 mmol), 3-fluorobenzaldehyde
(248 mg, 2.0 mmol), triethylamine (279 lL, 2.0 mmol), methanol
(12 mL), and sodium borohydride (378 mg, 10.0 mmol) according
to the general procedure of N-benzyl amino acid derivatives. The
crude product was suspended in a mixture of Et2O and hexane
(1:1, 10 mL) to afford 8l (472 mg, 91%) as a colorless solid. 1H
NMR (CDCl3) d 2.17 (br s, 1H), 3.79 (s, 2H), 4.23 (s, 1H), 5.56 (br
s, 1H), 6.78 (br s, 1H), 6.97 (td, J = 8.6, 2.5 Hz, 1H), 7.01–7.10 (m,
2H), 7.25–7.43 (m, 6H); 13C NMR (DMSO-d6) d 50.0 (d, J = 1.4 Hz),
64.9, 113.4 (d, J = 21.1 Hz), 114.5 (d, J = 21.2 Hz), 123.9 (d,
J = 2.3 Hz), 127.27, 127.30, 128.2, 130.0 (d, J = 8.4 Hz), 140.1,
143.6 (d, J = 7.1 Hz), 162.3 (d, J = 243.0 Hz), 173.9; HRMS calcd for
5.1.9. Methyl 2-(3-methoxybenzylamino)-2-phenylacetate (rac-8h)
The title compound was prepared from phenylglycine methyl
ester hydrochloride (rac-7h, 403 mg, 2.0 mmol) according to the
preparation of 8h. The crude product was purified using silica gel
column chromatography (eluent EtOAc:Hexane = 1:10–1:5) to
afford rac-8h (307 mg, 54%) as a pale yellow oil. 1H NMR (CDCl3)
d 2.37 (br s, 1H), 3.66–3.75 (m, 5H), 3.80 (s, 3H), 4.39 (s, 1H),
6.78–6.83 (m, 1H), 6.87–6.91 (m, 2H), 7.21–7.27 (m, 1H), 7.28–
7.40 (m, 5H); 13C NMR (CDCl3) d 51.4, 52.4, 55.3, 64.3, 112.9,
113.8, 120.7, 127.7, 128.3, 128.9, 129.6, 138.1, 141.2, 159.9,
C
15H15FN2O (M+H)+ 259.1241, found 259.1240; mp 126 °C.
5.1.14. (R)-2-(3-Chlorobenzylamino)-2-phenylacetamide (8m)
The title compound was prepared from (R)-phenylglycinamide
hydrochloride (7b, 373 mg, 2.0 mmol), 3-chlorobenzaldehyde
173.6; HRMS calcd for
286.1434.
C
17H19NO3 (M+H)+ 286.1438, found
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049

8
J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
(281 mg, 2.0 mmol), triethylamine (279
l
L, 2.0 mmol), methanol
NMR (CDCl3) d 2.20 (br s, 1H), 3.91 (dd, J = 26.6, 13.5 Hz, 2H),
4.22 (s, 1H), 5.56 (br s, 1H), 7.07 (br s, 1H), 7.22–7.44 (m, 9H);
13C NMR (DMSO-d6) d 47.9, 65.1, 127.1, 127.30, 127.32, 128.2,
(12 mL), and sodium borohydride (378 mg, 10.0 mmol) according
to the general procedure of N-benzyl amino acid derivatives. The
crude product was suspended in a mixture of Et2O and hexane
(1:1, 10 mL) to afford 8m (503 mg, 92%) as a colorless solid. 1H
NMR (CDCl3) d 2.16 (br s, 1H), 3.77 (s, 2H), 4.23 (s, 1H), 5.54 (br
s, 1H), 6.76 (br s, 1H), 7.14–7.21 (m, 1H), 7.23–7.45 (m, 8H); 13C
NMR (DMSO-d6) d 50.0, 64.9, 126.6, 127.3, 127.7, 128.2, 130.0,
133.0, 140.1, 143.2, 173.8; HRMS calcd for C15H15ClN2O (M+H)+
275.0946, found 275.0945; mp 129 °C.
128.4, 129.0, 129.7, 132.5, 137.6, 140.0, 173.8; HRMS calcd for C15
H
-
15ClN2O (M+H)+ 275.0946, found 275.0945; mp 141 °C.
5.1.19. (R)-2-(4-Chlorobenzylamino)-2-phenylacetamide (8r)
The title compound was prepared from (R)-phenylglycinamide
hydrochloride (7b, 373 mg, 2.0 mmol), 4-chlorobenzaldehyde
(281 mg, 2.0 mmol), triethylamine (279 lL, 2.0 mmol), methanol
(12 mL), and sodium borohydride (378 mg, 10.0 mmol) according
to the general procedure of N-benzyl amino acid derivatives. The
crude product was suspended in a mixture of EtOAc and hexane
(1:1, 20 mL) to afford 8r (396 mg, 72%) as a colorless solid. 1H
NMR (CDCl3) d 2.15 (br s, 1H), 3.76 (s, 2H), 4.22 (s, 1H), 5.43 (br
s, 1H), 6.75 (br s, 1H), 7.21–7.43 (m, 9H); 13C NMR (DMSO-d6) d
49.8, 64.7, 127.25, 127.29, 128.1, 128.2, 129.8, 139.4, 140.1,
173.9; HRMS calcd for C15H15ClN2O (M+H)+ 275.0946, found
275.0944; mp 160 °C.
5.1.15. (R)-2-(3-Methylbenzylamino)-2-phenylacetamide (8n)
The title compound was prepared from (R)-phenylglycinamide
hydrochloride (7b, 373 mg, 2.0 mmol), 3-methylbenzaldehyde
(240 mg, 2.0 mmol), triethylamine (279 lL, 2.0 mmol), methanol
(12 mL), and sodium borohydride (378 mg, 10.0 mmol) according
to the general procedure of N-benzyl amino acid derivatives. The
crude product was suspended in a mixture of Et2O, EtOAc, and hex-
ane (2:1:2, 12.5 mL) to afford 8n (430 mg, 85%) as a colorless solid.
1H NMR (CDCl3) d 2.07 (br s, 1H), 2.35 (s, 3H), 3.72–3.81 (m, 2H),
4.24 (s, 1H), 5.56 (br s, 1H), 7.00 (br s, 1H), 7.07–7.13 (m, 3H),
7.20–7.26 (m, 1H), 7.28–7.43 (m, 5H); 13C NMR (DMSO-d6) d
21.1, 50.6, 64.9, 125.1, 127.2, 127.3, 127.4, 128.1, 128.2, 128.7,
137.2, 140.16, 140.19, 174.0; HRMS calcd for C16H18N2O (M+H)+
255.1492, found 255.1489; mp 129 °C.
5.1.20. General procedure for preparation of N-acyl derivatives
Acyl chloride (1.1 equiv) was added to a mixture of N-benzyl
amino acid derivatives (1.0 equiv) and triethylamine (1.2 equiv)
in CH2Cl2. The mixture was stirred for 1 h at room temperature
and then partitioned between water and EtOAc. The organic layer
was washed with brine, and dried over anhydrous MgSO4. The
solution was concentrated in vacuo, and the residue was purified
using either silica gel column chromatography or crystallization.
5.1.16. (R)-2-(3-Trifluoromethylbenzylamino)-2-phenylacetamide
(8o)
The title compound was prepared from (R)-phenylglycinamide
hydrochloride (7b, 373 mg, 2.0 mmol), 3-trifluoromethylbenzalde-
5.1.21. N-Carbamoylmethyl-N-[4-(4-fluorobenzyloxy)-3-
methoxybenzyl]-2-thienamide (9a)
hyde (348 mg, 2.0 mmol), triethylamine (279 lL, 2.0 mmol),
methanol (12 mL), and sodium borohydride (378 mg, 10.0 mmol)
according to the general procedure of N-benzyl amino acid deriva-
tives. The crude product was suspended in a mixture of Et2O and
hexane (1:1, 10 mL) to afford 8o (502 mg, 81%) as a colorless solid.
1H NMR (DMSO-d6) d 3.10 (br s, 1H), 3.67 (dd, J = 20.3, 14.5 Hz, 2H),
4.06 (s, 1H), 7.12 (br s, 1H), 7.19–7.47 (m, 9H), 7.55 (br s, 1H); 13C
NMR (DMSO-d6) d 50.1, 65.1, 123.5 (q, J = 3.7 Hz), 124.4 (q,
J = 3.8 Hz), 124.5 (q, J = 272.8 Hz), 127.0, 127.4, 128.3, 129.0 (q,
The title compound was prepared from 8a (150 mg,
0.471 mmol), 2-thenoyl chloride (55
lL, 0.518 mmol), triethy-
lamine (79 L, 0.565 mmol), and CH2Cl2 (5 mL) according to the
l
general procedure of N-acyl derivatives. The crude product was
suspended in EtOAc and hexane (1:1) to afford 9a (179 mg, 89%)
as a colorless solid. 1H NMR (CDCl3) d 3.87 (s, 3H), 4.08 (s, 2H),
4.86 (br s, 2H), 5.11 (s, 2H), 5.42 (br s, 1H), 6.39 (br s, 1H), 6.75–
6.80 (m, 1H), 6.82 (br s, 1H), 6.88 (d, J = 8.3 Hz, 1H), 7.01 (dd,
J = 5.0, 3.8 Hz, 1H), 7.03–7.10 (m, 2H), 7.37 (dd, J = 3.8, 1.0 Hz,
1H), 7.39–7.45 (m, 2H), 7.51 (dd, J = 5.0, 1.0 Hz, 1H); 13C NMR
(CDCl3) d 50.6 (br s), 53.9 (br s), 56.1, 70.6, 110.7 (br s), 114.4,
115.6 (d, J = 21.5 Hz), 119.1 (br s), 127.4, 129.0, 129.3 (d,
J = 8.2 Hz), 129.6, 130.4, 132.8 (d, J = 3.0 Hz), 136.7, 147.8, 150.3,
162.6 (d, J = 246.4 Hz), 165.7, 171.1; HRMS calcd for C22H21FN2O4S
(MꢀH)ꢀ 427.1133, found 427.1126; mp 166 °C; chemical purity
99.5%, tR 12.4 min.
J = 31.1 Hz), 129.2, 132.2, 140.1, 142.0, 174.0; HRMS calcd for C16
-
H15F3N2O (M+H)+ 309.1209, found 309.1209; mp 112 °C.
5.1.17. (R)-2-(3-Trifluoromethoxybenzylamino)-2-phenylacetamide
(8p)
The title compound was prepared from (R)-phenylglycinamide
hydrochloride (7b, 373 mg, 2.0 mmol), 3-trifluoromethoxyben-
zaldehyde (380 mg, 2.0 mmol), triethylamine (279 lL, 2.0 mmol),
methanol (12 mL), and sodium borohydride (378 mg, 10.0 mmol)
according to the general procedure of N-benzyl amino acid deriva-
tives. The crude product was suspended in a mixture of Et2O and
hexane (1:1, 10 mL) to afford 8p (550 mg, 85%) as a colorless solid.
1H NMR (DMSO-d6) d 3.22 (br s, 1H), 3.71 (dd, J = 19.8, 14.3 Hz, 2H),
4.07 (s, 1H), 7.12 (br s, 1H), 7.23–7.36 (m, 3H), 7.38–7.44 (m, 2H),
7.52–7.65 (m, 4H), 7.69 (br s, 1H); 13C NMR (DMSO-d6) d 49.9, 65.0,
119.2, 120.17, 120.20 (q, J = 256.5 Hz), 127.0, 127.3, 128.2, 130.1,
140.1, 143.6, 148.6 (m), 173.9; HRMS calcd for C16H15F3N2O2 (M
+H)+ 325.1158, found 325.1157; mp 126 °C.
5.1.22. N-((R)-Carbamoylphenylmethyl)-N-(3-methoxybenzyl)-2-
thienamide (9b)
The title compound was prepared from 8b (100 mg,
0.370 mmol), 2-thenoyl chloride (43.5
lL, 0.407 mmol), triethy-
lamine (62 L, 0.444 mmol), and CH2Cl2 (5 mL) according to the
l
general procedure of N-acyl derivatives. The crude product was
purified using silica gel column chromatography (eluent EtOAc:
Hexane = 1:1–2:1) to afford 9b (127 mg, 90%) as a colorless amor-
phous. 1H NMR (CDCl3) d 3.76 (s, 3H), 4.62 (d, J = 17.7 Hz, 1H), 5.10
(d, J = 17.7 Hz, 1H), 5.36 (br s, 1H), 5.54 (br s, 1H), 5.71 (br s, 1H),
6.77 (dd, J = 8.0, 2.0 Hz, 1H), 6.82–6.86 (m, 1H), 6.94 (dd, J = 5.0,
3.8 Hz, 1H), 6.96 (br s, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.28–7.36 (m,
4H), 7.42–7.50 (m, 3H); 13C NMR (CDCl3) d 52.8 (br s), 55.3, 65.7,
111.7, 113.3, 118.9, 127.3, 128.9, 129.4, 129.7, 130.0, 130.4,
134.6, 137.7, 138.8, 160.0, 165.9, 171.6; HRMS calcd for C21H20N2-
O3S (MꢀH)ꢀ 379.1122, found 379.1129; chemical purity 97.9%, tR
13.0 min; optical purity 92.4% ee, tR 7.3 min (IA-3, 40% ethanol in
5.1.18. (R)-2-(2-Chlorobenzylamino)-2-phenylacetamide (8q)
The title compound was prepared from (R)-phenylglycinamide
hydrochloride (7b, 373 mg, 2.0 mmol), 2-chlorobenzaldehyde
(281 mg, 2.0 mmol), triethylamine (279 lL, 2.0 mmol), methanol
(12 mL), and sodium borohydride (378 mg, 10.0 mmol) according
to the general procedure of N-benzyl amino acid derivatives. The
crude product was suspended in a mixture of EtOAc and hexane
(1:1, 20 mL) to afford 8q (376 mg, 68%) as a colorless solid. 1H
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049

J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
9
hexane); ½a 2D5
þ 91:2 (c = 1.18, CHCl3).
ꢂ
general procedure of N-acyl derivatives. The crude product was
purified using silica gel column chromatography (eluent EtOAc:
Hexane = 1:1–2:1) to afford 9e (242 mg, 87%) as a colorless gum.
1H NMR (CDCl3) d 3.25–3.38 (m, 2H), 3.76 (s, 3H), 4.52 (d,
J = 17.0 Hz, 1H), 4.77 (t, J = 7.7 Hz, 1H), 4.84 (d, J = 17.0 Hz, 1H),
5.29 (br s, 1H), 6.67–6.86 (m, 4H), 6.94 (dd, J = 5.0, 3.8 Hz, 1H),
7.14–7.32 (m, 7H), 7.47 (dd, J = 5.0, 1.0 Hz, 1H); 13C NMR (CDCl3)
d 34.8, 53.1 (br s), 55.2, 62.8, 112.8, 113.1, 119.3, 126.9, 127.3,
128.7, 129.3, 130.0, 130.3, 137.4, 137.5, 138.4, 159.9, 166.5,
172.4; HRMS calcd for C22H22N2O3S (MꢀH)ꢀ 393.1278, found
393.1272; chemical purity > 99.5%, tR 13.0 min; optical pur-
ity > 99.5% ee, tR 10.3 min (IA-3, 40% ethanol in hexane);
5.1.23. N-((S)-Carbamoylphenylmethyl)-N-(3-methoxybenzyl)-2-
thienamide (ent-9b)
The title compound was prepared from ent-8b (150 mg,
0.555 mmol), 2-thenoyl chloride (65
lL, 0.610 mmol), triethy-
lamine (93 L, 0.666 mmol), and CH2Cl2 (5 mL) according to the
l
general procedure of N-acyl derivatives. The crude product was
purified using silica gel column chromatography (eluent EtOAc:
Hexane = 1:1–2:1) to afford ent-9b (203 mg, 96%) as a colorless
amorphous. 1H NMR (CDCl3) d 3.76 (s, 3H), 4.62 (d, J = 17.7 Hz,
1H), 5.10 (d, J = 17.7 Hz, 1H), 5.35 (br s, 1H), 5.49 (br s, 1H), 5.68
(br s, 1H), 6.77 (dd, J = 8.2, 2.0 Hz, 1H), 6.84 (d, J = 7.6 Hz, 1H),
6.94 (dd, J = 5.0, 3.8 Hz, 1H), 6.97 (br s, 1H), 7.20 (t, J = 8.0 Hz,
1H), 7.28–7.36 (m, 4H), 7.42–7.50 (m, 3H); 13C NMR (CDCl3) d
52.7 (br s), 55.3, 65.6, 111.7, 113.3, 118.9, 127.3, 128.9, 129.4,
129.7, 130.0, 130.4, 134.6, 137.7, 138.8, 159.9, 165.9, 171.6; HRMS
calcd for C21H20N2O3S (MꢀH)ꢀ 379.1122, found 379.1128; chemi-
cal purity 97.2%, tR 12.4 min; optical purity > 99.0% ee, tR 9.9 min
½
a 2D5
ꢂ
þ 112:8 (c = 1.01, CHCl3).
5.1.27. N-((R)-1-Carbamoylethyl)-N-(3-methoxybenzyl)benzamide
(9f)
The title compound was prepared from 8f (150 mg,
0.720 mmol), benzoyl chloride (92
lL, 0.793 mmol), triethylamine
(120 L, 0.864 mmol), and CH2Cl2 (5 mL) according to the general
l
(IA-3, 40% ethanol in hexane); ½a D25
ꢀ 98:1 (c = 1.17, CHCl3).
ꢂ
procedure of N-acyl derivatives. The crude product was purified
using silica gel column chromatography (eluent EtOAc:Hex-
ane = 1:1–2:1) to afford 9f (213 mg, 95%) as a colorless solid. 1H
NMR (CDCl3) d 1.43–1.52 (m, 3H), 3.78 (s, 3H), 4.40–4.88 (m,
3H), 5.31 (br s, 1H), 6.50–6.95 (m, 4H), 7.23 (t, J = 7.9 Hz, 1H),
7.32–7.50 (m, 5H); 13C NMR (CDCl3) d 15.0 (br s), 51.9 (br s),
55.2 (br s), 55.3, 112.8, 119.3, 126.7, 128.7, 129.8, 130.2, 135.9,
139.1 (br s), 159.9, 173.7; HRMS calcd for C18H20N2O3 (MꢀH)ꢀ
311.1401, found 311.1393; mp 131 °C; chemical purity 99.2%, tR
11.0 min; optical purity 98.9% ee, tR 10.2 min (OD, 15% ethanol in
5.1.24. N-((R)-Carbamoylphenylmethyl)-N-[4-(4-fluorobenzyloxy)-3-
methoxybenzyl]-2-thienamide (9c)
The title compound was prepared from 8c (150 mg,
0.380 mmol), 2-thenoyl chloride (45
lL, 0.418 mmol), triethy-
lamine (64 L, 0.457 mmol), and CH2Cl2 (5 mL) according to the
l
general procedure of N-acyl derivatives. The crude product was
purified using silica gel column chromatography (eluent EtOAc:
Hexane = 1:1–2:1) to afford 9c (191 mg, 99%) as a colorless amor-
phous. 1H NMR (CDCl3) d 3.82 (s, 3H), 4.56 (d, J = 17.4 Hz, 1H), 5.04
(d, J = 17.4 Hz, 1H), 5.08 (s, 2H), 5.27 (br s, 1H), 5.45 (br s, 1H), 5.64
(br s, 1H), 6.71–6.76 (m, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.95 (dd,
J = 5.0, 3.8 Hz, 1H), 6.99–7.10 (m, 3H), 7.29–7.36 (m, 4H), 7.38–
7.49 (m, 5H); 13C NMR (CDCl3) d 52.7 (br s), 56.1, 65.9, 70.4,
110.5, 114.4, 115.6 (d, J = 21.6 Hz), 118.8, 127.3, 128.98, 129.0,
129.35 (d, J = 8.3 Hz), 129.41, 129.9, 130.3, 130.4, 133.0 (d,
J = 3.3 Hz), 134.8, 137.7, 147.0, 150.0, 162.6 (d, J = 246.3 Hz),
165.8, 171.4; HRMS calcd for C28H25FN2O4S (MꢀH)ꢀ 503.1446,
found 503.1450; chemical purity 99.1%, tR 13.6 min; optical purity
hexane); ½a 2D5
þ 94:4 (c = 1.07, CHCl3).
ꢂ
5.1.28. N-((R)-Carbamoylphenylmethyl)-N-(3-methoxybenzyl)
phenylacetamide (9g)
The title compound was prepared from 8b (100 mg,
0.370 mmol), phenylacetyl chloride (54
lL, 0.407 mmol), triethy-
lamine (62 L, 0.444 mmol), and CH2Cl2 (5 mL) according to the
l
general procedure of N-acyl derivatives. The crude product was
purified using silica gel column chromatography (eluent EtOAc:
Hexane = 1:1–2:1) to afford 9g (97 mg, 68%) as a colorless amor-
phous. 1H NMR (CDCl3) d 3.63–3.74 (m, 5H), 4.49 (d, J = 17.8 Hz,
1H), 4.73 (d, J = 17.8 Hz, 1H), 5.42 (br s, 1H), 5.64 (br s, 1H), 5.84
(s, 1H), 6.57 (br s, 1H), 6.62 (d, J = 7.6 Hz, 1H), 6.68–6.74 (m, 1H),
7.12 (t, J = 7.9 Hz, 1H), 7.17–7.43 (m, 10H); 13C NMR (CDCl3) d
41.2, 50.3, 55.3, 63.1, 111.4, 113.2, 118.5, 127.0, 128.7, 128.86,
128.94, 129.2, 129.6, 130.0, 134.7, 135.0, 139.1, 159.9, 171.9,
173.1; HRMS calcd for C24H24N2O3 (MꢀH)ꢀ 387.1714, found
387.1721; chemical purity 98.2%, tR 12.5 min; optical purity
93.2% ee, tR 9.7 min (IA-3, 40% ethanol in hexane); ½a D25
þ 85:0
ꢂ
(c = 1.04, CHCl3).
5.1.25. N-((R)-Carbamoylphenylmethyl)-N-(3-methoxybenzyl)
benzamide (9d)
The title compound was prepared from 8b (100 mg,
0.370 mmol), benzoyl chloride (47 L, 0.407 mmol), triethylamine
(62 L, 0.444 mmol), and CH2Cl2 (5 mL) according to the general
l
l
90.5% ee, tR 11.7 min (OD, 15% ethanol in hexane); ½a D25
ꢀ 46:1
ꢂ
procedure of N-acyl derivatives. The crude product was purified
using silica gel column chromatography (eluent EtOAc:Hex-
ane = 1:1–2:1) to afford 9d (131 mg, 95%) as a colorless amor-
phous. 1H NMR (CDCl3) d 3.72 (s, 3H), 4.42 (d, J = 16.8 Hz, 1H),
4.77 (d, J = 16.8 Hz, 1H), 5.41 (br s, 1H), 5.54 (br s, 1H), 5.73 (br s,
1H), 6.65 (d, J = 7.6 Hz, 1H), 6.68–6.78 (m, 2H), 7.11 (t, J = 7.8 Hz,
1H), 7.29–7.55 (m, 10H); 13C NMR (CDCl3) d 53.0 (br s), 55.3,
64.5 (br s), 112.1, 113.2, 119.3, 126.9, 128.6, 128.9, 129.0, 129.5,
129.9, 130.1, 135.0, 136.0, 138.9 (br s), 159.8, 171.7, 173.2; HRMS
calcd for C23H22N2O3 (MꢀH)ꢀ 373.1558, found 373.1560; chemical
purity 97.0%, tR 12.3 min; optical purity 91.8% ee, tR 9.7 min (IB-3,
(c = 0.99, CHCl3).
5.1.29. Methyl (R)-2-[N-benzoyl-N-(3-methoxybenzyl)amino]-2-
phenylacetate (9h)
The title compound was prepared from 8h (225 mg,
0.789 mmol), benzoyl chloride (101
lL, 0.868 mmol), triethy-
lamine (132 L, 0.946 mmol), and CH2Cl2 (7 mL) according to the
l
general procedure of N-acyl derivatives. The crude product was
purified using silica gel column chromatography (eluent EtOAc:
Hexane = 1:5–1:3) to afford 9h (303 mg, 99%) as a colorless oil.
1H NMR (CDCl3) d 3.72 (s, 3H), 3.78 (br s, 3H), 4.28 (br d,
J = 16.8 Hz, 1H), 4.74 (d, J = 16.8 Hz, 1H), 5.48 (br s, 1H), 6.55–
6.62 (m, 2H), 6.71 (d, J = 8.0 Hz, 1H), 7.10 (t, J = 8.0 Hz, 1H), 7.26–
7.43 (m, 8H), 7.48–7.53 (m, 2H); 13C NMR (CDCl3) d 52.1 (br s),
52.7, 55.3, 63.2 (br s), 112.0 (br s), 113.2 (br s), 119.2 (br s),
127.0, 128.7, 128.8, 129.6, 130.1, 130.2, 134.0, 135.7 (br s), 138.8
(br s), 159.8 (br s), 170.5, 173.2; HRMS calcd for C24H23NO4 (M
20% ethanol in hexane); ½a D25
þ 53:7 (c = 1.22, CHCl3).
ꢂ
5.1.26. N-((R)-1-Carbamoyl-2-phenylethyl)-N-(3-methoxybenzyl)-2-
thienamide (9e)
The title compound was prepared from 8e (200 mg,
0.703 mmol), 2-thenoyl chloride (83
lL, 0.773 mmol), triethy-
lamine (118 L, 0.844 mmol), and CH2Cl2 (5 mL) according to the
l
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049

10
J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
+H)+ 390.1700, found 390.1693; chemical purity 97.8%, tR
14.8 min; optical purity 96.8% ee, tR 5.3 min (OD, 15% ethanol in
5.1.33. N-((R)-Carbamoylphenylmethyl)-N-benzylbenzamide (9k)
The title compound was prepared from 8k (150 mg,
hexane); ½a 2D5
ꢂ
þ 63:2 (c = 1.00, CHCl3).
0.624 mmol), benzoyl chloride (79
lL, 0.687 mmol), triethylamine
(105 L, 0.749 mmol), and CH2Cl2 (5 mL) according to the general
l
procedure of N-acyl derivatives. The crude product was purified
using silica gel column chromatography (eluent Et2O:Hex-
ane = 3:1–5:1) to afford 9k (145 mg, 67%) as a colorless amor-
phous. 1H NMR (CDCl3) d 4.45 (d, J = 16.7 Hz, 1H), 4.79 (d,
J = 16.7 Hz, 1H), 5.45 (br s, 1H), 5.51 (br s, 1H), 5.75 (br s, 1H),
7.05–7.10 (m, 2H), 7.14–7.22 (m, 3H), 7.27–7.54 (m, 10H); 13C
NMR (CDCl3) d 53.0 (br s), 64.4, (br s), 126.9, 127.1, 128.4, 128.6,
128.9, 129.0, 129.8, 130.0, 134.9, 136.1, 137.1 (br s), 171.7,
173.3; HRMS calcd for C22H20N2O2 (MꢀH)ꢀ 343.1452, found
343.1452; chemical purity 98.5%, tR 12.2 min; optical purity
5.1.30. Methyl 2-[N-benzoyl-N-(3-methoxybenzyl)amino]-2-
phenylacetate (rac-9h)
The title compound was prepared from rac-8h (242 mg,
0.848 mmol), benzoyl chloride (108
lL, 0.933 mmol), triethy-
lamine (142 L, 1.017 mmol), and CH2Cl2 (8 mL) according to the
l
general procedure of N-acyl derivatives. The crude product was
purified using silica gel column chromatography (eluent EtOAc:
Hexane = 1:5–1:3) to afford rac-9h (322 mg, 98%) as a colorless
oil. 1H NMR (CDCl3) d 3.72 (s, 3H), 3.78 (br s, 3H), 4.29 (br d,
J = 16.8 Hz, 1H), 4.75 (d, J = 16.8 Hz, 1H), 5.48 (br s, 1H), 6.55–
6.62 (m, 2H), 6.68–6.75 (m, 1H), 7.10 (t, J = 8.0 Hz, 1H), 7.26–7.43
(m, 8H), 7.48–7.53 (m, 2H); 13C NMR (CDCl3) d 52.1 (br s), 52.7,
55.3, 63.2 (br s), 112.0 (br s), 113.2 (br s), 119.2 (br s), 127.0,
128.7, 128.8, 129.6, 130.1, 130.2, 134.0, 135.7 (br s), 138.7 (br s),
159.8 (br s), 170.5, 173.2; HRMS calcd for C24H23NO4 (M+H)+
390.1700, found 390.1697; chemical purity > 99.5%, tR 14.6 min.
95.8% ee, tR 10.7 min (OD, 15% ethanol in hexane); ½a D25
þ 39:3
ꢂ
(c = 1.13, CHCl3).
5.1.34. N-((R)-Carbamoylphenylmethyl)-N-(3-fluorobenzyl)
benzamide (9l)
The title compound was prepared from 8l (150 mg,
0.581 mmol), benzoyl chloride (74 L, 0.639 mmol), triethylamine
(97 L, 0.697 mmol), and CH2Cl2 (5 mL) according to the general
l
l
procedure of N-acyl derivatives. The crude product was purified
using silica gel column chromatography (eluent EtOAc:Hex-
ane = 1:1) to afford 9l (182 mg, 87%) as a colorless amorphous.
1H NMR (CDCl3) d 4.42 (d, J = 16.9 Hz, 1H), 4.78 (br s, 1H), 5.42–
5.86 (m, 3H), 6.65–6.72 (m, 1H), 6.76–6.85 (m, 2H), 7.06–7.15
(m, 1H), 7.25–7.55 (m, 10H); 13C NMR (CDCl3) d 52.1 (br s), 63.8
(br s), 113.9 (d, J = 21.9 Hz), 122.5 (br s), 126.7, 128.6, 128.99,
129.01, 129.9, 130.1, 134.5, 135.9, 140.3 (br s), 162.7 (d,
J = 245.8 Hz), 171.8, 173.3; HRMS calcd for C22H19FN2O2 (MꢀH)ꢀ
361.1358, found 361.1359; chemical purity 98.8%, tR 12.4 min;
optical purity 95.2% ee, tR 11.4 min (OD, 15% ethanol in hexane);
5.1.31. N-(3-Methoxybenzyl)-N-[(R)-(N-methylcarbamoyl)
phenylmethyl]benzamide (9i)
The title compound was prepared from 8i (150 mg,
0.528 mmol), benzoyl chloride (67
lL, 0.581 mmol), triethylamine
(88 L, 0.633 mmol), and CH2Cl2 (5 mL) according to the general
l
procedure of N-acyl derivatives. The crude product was purified
using silica gel column chromatography (eluent EtOAc:Hex-
ane = 1:1–2:1) to afford 9i (204 mg, 99%) as a colorless gum. 1H
NMR (CDCl3) d 2.82 (d, J = 4.4 Hz, 3H), 3.73 (s, 3H), 4.41 (br d,
J = 16.8 Hz, 1H), 4.75 (d, J = 16.8 Hz, 1H), 5.34 (br s, 1H), 5.72 (br
s, 1H), 6.70–6.75 (m, 2H), 6.77 (br s, 1H), 7.13 (t, J = 7.8 Hz, 1H),
7.29–7.53 (m, 10H); 13C NMR (CDCl3) d 26.5, 53.0 (br s), 55.2,
64.7 (br s), 112.0, 113.1, 119.2, 126.8, 128.5, 128.7, 128.9, 129.4,
129.7, 130.0, 135.1, 136.0, 138.8 (br s), 159.7, 169.9, 173.2; HRMS
calcd for C24H24N2O3 (MꢀH)ꢀ 387.1714, found 387.1713; chemical
purity 97.5%, tR 12.6 min; optical purity 98.3% ee, tR 6.1 min (IA-3,
½
a 2D5
ꢂ
þ 17:3 (c = 1.12, CHCl3).
5.1.35. N-((R)-Carbamoylphenylmethyl)-N-(3-chlorobenzyl)
benzamide (9m)
The title compound was prepared from 8m (150 mg,
30% ethanol in hexane); ½a D25
ꢂ
þ 80:8 (c = 1.10, CHCl3).
0.546 mmol), benzoyl chloride (70
lL, 0.601 mmol), triethylamine
(91 L, 0.655 mmol), and CH2Cl2 (5 mL) according to the general
l
procedure of N-acyl derivatives. The crude product was purified
using silica gel column chromatography (eluent EtOAc:Hex-
ane = 1:1) to afford 9m (189 mg, 91%) as a colorless amorphous.
1H NMR (CDCl3) d 4.40 (d, J = 16.8 Hz, 1H), 4.75 (br s, 1H), 5.46–
5.84 (m, 3H), 6.84–6.87 (m, 1H), 6.89–6.95 (m, 1H), 7.03–7.10
(m, 2H), 7.25–7.53 (m, 10H); 13C NMR (CDCl3) d 52.0 (br s), 63.7
(br s), 125.0 (br s), 126.7, 127.1, 128.7, 129.0, 129.1, 129.5, 130.0,
130.1, 134.1 (br s), 134.5, 135.9, 139.7 (br s), 171.7, 173.3; HRMS
calcd for C22H19ClN2O2 (MꢀH)ꢀ 377.1062, found 377.1065; chem-
ical purity 97.1%, tR 12.9 min; optical purity 99.3% ee, tR 11.9 min
5.1.32. N-(3-Methoxybenzyl)-N-[(R)-(N,N-dimethylcarbamoyl)
phenylmethyl]benzamide (9j)
The title compound was prepared from 8j (150 mg,
0.503 mmol), benzoyl chloride (64
lL, 0.554 mmol), triethylamine
(84 L, 0.603 mmol), and CH2Cl2 (5 mL) according to the general
l
procedure of N-acyl derivatives. The crude product was purified
using silica gel column chromatography (eluent EtOAc:Hex-
ane = 1:1–2:1) to afford 9j (204 mg, quant.) as a colorless gum. This
compound exists as a mixture of rotamers in CDCl3 (4:1). 1H NMR
(CDCl3) d 2.26 (br s, minor 3H), 2.89 (s, major 3H), 2.94 (br s, minor
3H), 3.03 (s, major 3H), 3.61 (s, major 3H), 3.68 (br s, minor 3H),
4.37 (br d, J = 14.5 Hz, minor 1H), 4.49 (d, J = 17.2 Hz, major 1H),
4.78 (d, J = 17.2 Hz, major 1H), 5.24 (br d, J = 14.5 Hz, minor 1H),
5.67 (br s, minor 1H), 6.09 (s, major 1H), 6.19 (d, J = 7.0 Hz, major
1H), 6.45–7.52 (m, major 13H and minor 14H); 13C NMR (CDCl3)
d 35.9 (br s, minor), 36.1, 36.3 (br s, minor), 37.1, 48.4 (br s, minor),
51.2, 55.1, 59.9, 63.9 (br s, minor), 111.8, 112.2, 113.0 (br s, minor),
119.0, 120.2 (br s, minor), 126.1 (br s, minor), 126.7, 128.2, 128.7,
128.8, 129.3, 130.5, 133.6, 136.8, 137.1 (br s, minor), 140.4, 140.7
(br s, minor), 159.1, 170.0 (br s, minor), 170.3, 172.5 (br s, minor),
(OD, 15% ethanol in hexane); ½a D25
þ 12:6 (c = 1.20, CHCl3).
ꢂ
5.1.36. N-((R)-Carbamoylphenylmethyl)-N-(3-methylbenzyl)
benzamide (9n)
The title compound was prepared from 8n (150 mg,
0.590 mmol), benzoyl chloride (75
lL, 0.649 mmol), triethylamine
(99 L, 0.708 mmol), and CH2Cl2 (5 mL) according to the general
l
procedure of N-acyl derivatives. The crude product was purified
using silica gel column chromatography (eluent EtOAc:Hex-
ane = 1:1) to afford 9n (182 mg, 86%) as a colorless amorphous.
1H NMR (CDCl3) d 2.22 (s, 3H), 4.43 (d, J = 16.6 Hz, 1H), 4.75 (d,
J = 16.6 Hz, 1H), 5.43 (br s, 1H), 5.54 (br s, 1H), 5.77 (br s, 1H),
6.78 (s, 1H), 6.90 (d, J = 7.6 Hz, 1H), 6.96 (d, J = 7.6 Hz, 1H), 7.08
(t, J = 7.6 Hz, 1H), 7.27–7.54 (m, 10H); 13C NMR (CDCl3) d 21.4,
53.2 (br s), 64.4 (br s), 124.1, 126.9, 127.9, 128.3, 128.6, 128.8,
173.8; HRMS calcd for
C
25H26N2O3 (M+H)+ 403.2016, found
403.2011; chemical purity > 99.5%, tR 14.3 min; optical pur-
ity > 99.0% ee, tR 5.5 min (IA-3, 40% ethanol in hexane);
½
a 2D5
ꢂ
ꢀ 112:8 (c = 1.10, CHCl3).
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049

J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
11
128.9, 129.9, 130.0, 135.0, 136.1, 137.0 (br s), 138.0, 171.8, 173.3;
HRMS calcd for C23H22N2O2 (MꢀH)ꢀ 357.1609, found 357.1607;
chemical purity 98.8%, tR 13.8 min; optical purity 96.8% ee, tR
in a mixture of EtOAc and hexane (1:2, 15 mL) to afford 9r (164 mg,
79%) as a colorless solid. 1H NMR (CDCl3) d 4.39 (d, J = 16.7 Hz, 1H),
4.76 (br s, 1H), 5.34–5.80 (m, 3H), 6.96 (d, J = 8.4 Hz, 2H), 7.12 (d,
J = 8.1 Hz, 2H), 7.25–7.54 (m, 10H); 13C NMR (CDCl3) d 52.1 (br s),
63.9 (br s), 126.7, 128.4 (br s), 128.6 (br s), 129.02, 129.05, 129.9,
130.1, 132.7 (br s), 134.6, 135.9 (br s), 171.7, 173.3; HRMS calcd
for C22H19ClN2O2 (MꢀH)ꢀ 377.1062, found 377.1069; mp 157 °C;
chemical purity 96.9%, tR 13.0 min; optical purity > 99.0% ee, tR
10.0 min (OD, 15% ethanol in hexane);
CHCl3).
½
a 2D5
ꢂ
þ 40:1 (c = 1.02,
5.1.37. N-((R)-Carbamoylphenylmethyl)-N-(3-trifluoromethylbenzyl)
benzamide (9o)
11.4 min (OD, 15% ethanol in hexane); ½a D25
þ 22:9 (c = 1.07,
ꢂ
The title compound was prepared from 8o (150 mg,
CHCl3).
0.487 mmol), benzoyl chloride (62
lL, 0.535 mmol), triethylamine
(81 L, 0.584 mmol), and CH2Cl2 (5 mL) according to the general
l
procedure of N-acyl derivatives. The crude product was purified
using silica gel column chromatography (eluent EtOAc:Hex-
ane = 1:1–2:1) to afford 9o (177 mg, 88%) as a colorless amor-
phous. 1H NMR (CDCl3) d 4.32–4.60 (m, 1H), 4.82 (br s, 1H),
5.49–5.93 (m, 3H), 7.02 (br s, 1H), 7.10–7.60 (m, 13H); 13C NMR
5.1.41. 2-[N-Benzoyl-N-(3-methoxybenzyl)amino]-2-phenylacetic
acid (rac-10)
To a suspension of rac-9h (207 mg, 0.532 mmol) in methanol
(10 mL) was added NaOH aq. (2 mol/L, 0.53 mL, 1.06 mmol), and
the mixture was stirred for 4 h at 40 °C. To the reaction mixture
was added HCl aq. (1 mol/L, 1.06 mL), and the mixture was parti-
tioned between EtOAc and water. The organic layer was washed
with brine, and then dried over anhydrous MgSO4. The solution
was concentrated in vacuo to afford rac-10 (191 mg, 96%) as a col-
orless solid. 1H NMR (CDCl3) d 3.72 (s, 3H), 4.33 (d, J = 16.6 Hz, 1H),
4.79 (br d, J = 16.6 Hz, 1H), 5.35 (br s, 1H), 6.62–6.68 (m, 2H), 6.75
(br d, J = 7.4 Hz, 1H), 7.10–7.20 (m, 1H), 7.28–7.45 (m, 8H), 7.50–
7.55 (m, 2H); 13C NMR (CDCl3) d 52.9 (br s), 55.3, 63.8 (br s),
112.1, 113.5, 119.3, 127.1, 128.7, 128.8, 129.9, 130.4, 133.7, 135.2
(br s), 138.1 (br s), 159.9 (br s), 173.6, 173.9 (br s); HRMS calcd
for C23H21NO4 (M+H)+ 376.1543, found 376.1539; mp 142 °C.
(CDCl3)
d 51.9 (br s), 63.3 (br s), 123.6, 123.8, 124.0 (q,
J = 272.6 Hz), 126.6, 128.6 (br s), 129.0, 129.2, 130.0, 134.3, 136.0,
138.8 (br s), 171.8, 173.4 (br s); HRMS calcd for C23H19F3N2O2
(MꢀH)ꢀ 411.1326, found 411.1331; chemical purity 97.9%, tR
13.2 min; optical purity 90.3% ee, tR 10.8 min (OD, 15% ethanol in
hexane); ½a 2D5
ꢀ 3:14 (c = 1.09, CHCl3).
ꢂ
5.1.38. N-((R)-Carbamoylphenylmethyl)-N-(3-
trifluoromethoxybenzyl)benzamide (9p)
The title compound was prepared from 8p (150 mg,
0.463 mmol), benzoyl chloride (59
lL, 0.509 mmol), triethylamine
(77 L, 0.554 mmol), and CH2Cl2 (5 mL) according to the general
l
5.1.42. (S)-Carbamoylphenylmethyl 4-nitrobenzenesulfonate (11)
procedure of N-acyl derivatives. The crude product was purified
using silica gel column chromatography (eluent Et2O:Hex-
ane = 3:1–5:1) to afford 9p (157 mg, 79%) as a colorless amor-
phous. 1H NMR (CDCl3) d 4.45 (br d, J = 15.8 Hz, 1H), 4.77 (br s,
1H), 5.53–5.93 (m, 3H), 6.76 (br s, 1H), 6.90 (d, J = 7.3 Hz, 1H),
6.93 (d, J = 8.3 Hz, 1H), 7.12 (t, J = 8.0 Hz, 1H), 7.20–7.58 (m,
10H); 13C NMR (CDCl3) d 51.8 (br s), 63.4 (br s), 119.4 (br s),
120.5 (q, J = 257.4 Hz), 125.2 (br s), 126.6, 128.6 (br s), 129.0,
129.1, 129.5 (br s), 130.0, 134.3, 135.9, 140.2 (br s), 149.1, 171.8,
173.4 (br s); HRMS calcd for C23H19F3N2O3 (MꢀH)ꢀ 427.1275,
found 427.1279; chemical purity 97.9%, tR 13.4 min; optical purity
A
solution of 4-nitrobenzenesulfonyl chloride (665 mg,
3.00 mmol) in CH2Cl2 (10 mL) was added dropwise to a mixture
of l-mandelamide (454 mg, 3.00 mmol), triethylamine (501 L,
l
3.60 mmol) and N,N-dimethylaminopyridine (37 mg, 0.30 mmol)
in CH2Cl2 (10 mL) at 0 °C. The mixture was stirred for 1 h and
washed with water and brine. The organic layer was dried over
anhydrous MgSO4, and concentrated in vacuo. The residue was sus-
pended in a mixture of EtOH and hexane (5:1, 6 mL) to afford 11
(527 mg, 52%) as a colorless solid. 1H NMR (CDCl3) d 5.72 (br s,
1H), 5.87 (s, 1H), 6.40 (br s, 1H), 7.19–7.33 (m, 5H), 7.85–7.90
(m, 2H), 8.18–8.22 (m, 2H); 13C NMR (CDCl3) d 82.4, 124.3, 128.0,
129.1, 129.3, 130.2, 132.8, 142.1, 150.7, 168.6; mp 120 °C dec;
88.0% ee, tR 10.2 min (OD, 15% ethanol in hexane); ½a D25
ꢀ 62:6
ꢂ
(c = 0.62, THF).
½
a 2D5
ꢂ
þ 22:5 (c = 0.87, DMF).
5.1.39. N-((R)-Carbamoylphenylmethyl)-N-(2-chlorobenzyl)
5.1.43. Benzyl (S)-2-(4-nitrobenzenesulfonyloxy)-2-phenylacetate
(12)
benzamide (9q)
The title compound was prepared from 8q (150 mg,
0.546 mmol), benzoyl chloride (70 L, 0.601 mmol), triethylamine
(91 L, 0.655 mmol), and CH2Cl2 (5 mL) according to the general
The title compound was prepared from 4-nitrobenzenesulfonyl
chloride (2.22 g, 10.0 mmol), l-mandelic acid benzyl ester (2.42 g,
10.0 mmol), triethylamine (1.67 mL, 12.0 mmol), and N,N-
dimethylaminopyridine (122 mg, 1.00 mmol) according to the
preparation of 11. The crude product was purified using silica gel
column chromatography (eluent CH2Cl2:Hexane = 2:1) to afford
12 (2.74 g, 64%) as a colorless solid. 1H NMR (CDCl3) d 5.07 (d,
J = 12.2 Hz, 1H), 5.16 (d, J = 12.2 Hz, 1H), 5.96 (s, 1H), 7.16–7.20
(m, 2H), 7.28–7.40 (m, 8H), 7.96–8.00 (m, 2H), 8.19–8.23 (m,
2H); 13C NMR (CDCl3) d 68.1, 80.1, 124.3, 127.9, 128.4, 128.7,
128.9, 129.1, 129.4, 130.3, 132.1, 134.6, 142.3, 150.7, 166.8; mp
l
l
procedure of N-acyl derivatives. The crude product was purified
using silica gel column chromatography (eluent EtOAc:Hex-
ane = 1:1) to afford 9q (191 mg, 92%) as a colorless amorphous.
1H NMR (CDCl3) d 4.59 (d, J = 17.6 Hz, 1H), 4.76 (br s, 1H), 5.40
(br s, 1H), 5.51 (br s, 1H), 5.65 (br s, 1H), 7.05–7.78 (m, 14H); 13C
NMR (CDCl3) d 50.9 (br s), 64.8 (br s), 126.8, 128.3 (br s), 128.6,
129.0, 129.3, 129.8, 130.3, 132.3, 134.5 (br s), 134.6, 135.6 (br s),
171.5, 173.3; HRMS calcd for C22H19ClN2O2 (MꢀH)ꢀ 377.1062,
found 377.1059; chemical purity 98.3%, tR 12.7 min; optical purity
109 °C; ½a 2D5
þ 26:5 (c = 1.23, CHCl3).
ꢂ
99.0% ee, tR 10.9 min (OD, 15% ethanol in hexane); ½a D25
ꢂ
+71.3
(c = 1.05, CHCl3).
5.1.44. (R)-2-[(R)-1-(3-Chlorophenyl)ethylamino]-2-phenylacetamide
5.1.40. N-((R)-Carbamoylphenylmethyl)-N-(4-chlorobenzyl)
(13)
benzamide (9r)
To
a
mixture of (R)-1-(3-chlorophenyl)ethylamine (16c,
The title compound was prepared from 8r (150 mg,
0.546 mmol), benzoyl chloride (70 L, 0.601 mmol), triethylamine
(91 L, 0.655 mmol), and CH2Cl2 (5 mL) according to the general
procedure of N-acyl derivatives. The crude product was suspended
231 mg, 1.487 mmol), KHCO3 (149 mg, 1.487 mmol), acetonitrile
(5 mL), and THF (5 mL) was added 11 (250 mg, 0.743 mmol), and
the mixture was stirred for 6 h at 50 °C. After being cooled to room
temperature, the mixture was partitioned between EtOAc and
l
l
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049

12
J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
water. The organic layer was washed with water and brine, and
dried over anhydrous MgSO4. The solution was concentrated in
vacuo, and the residue was purified using silica gel column chro-
matography (eluent EtOAc:Hexane = 1:5–1:1) to afford 13
(144 mg, 67%) as a pale yellow solid. 1H NMR (CDCl3) d 1.40 (d,
J = 6.7 Hz, 3H), 2.13 (br s, 1H), 3.81 (q, J = 6.7 Hz, 1H), 4.02 (s,
1H), 5.57 (br s, 1H), 6.88 (br s, 1H), 7.12 (dt, J = 6.2, 2.1 Hz, 1H),
7.21–7.35 (m, 8H); 13C NMR (CDCl3) d 24.2, 57.0, 65.0, 124.9,
126.8, 127.3, 127.6, 128.4, 129.1, 130.1, 134.7, 139.3, 146.9,
175.5; HRMS calcd for C16H17ClN2O (M+H)+ 289.1102, found
0.698 mmol) in CH2Cl2 (5 mL) was added diisopropylethylamine
(332 L, 1.90 mmol). The mixture was stirred for 12 h at room tem-
l
perature, and partitioned with EtOAc and water. The organic layer
was washed with water and brine, and dried over anhydrous
MgSO4. The solution was concentrated in vacuo, and the residue
was purified using silica gel column chromatography (eluent
EtOAc:Hexane = 1:2–1:1) to afford 20c (159 mg, 64%, colorless
solid, low-polarity) and 21c (79 mg, 32%, colorless solid, high-
polarity).
289.1101; mp 114 °C; ½a D25
ꢀ 36:9 (c = 1.16, CHCl3).
ꢂ
5.1.48. General procedure of the Ugi reaction and deprotection
A mixture of amine (2.00 mmol) and aldehyde (2.00 mmol) in
MeOH (4 ml) was stirred for 2 h at 50 °C. After being cooled to
room temperature, carboxylic acid (2.00 mmol) and 4-phenylcy-
clohenen-1-yl isocyanide (367 mg, 2.00 mmol) were added to the
solution. The mixture was stirred 5 h at 50 °C, and then concen-
trated in vacuo. The residue was suspended in 1,4-dioxane (6 mL)
and water (1.5 mL). To the mixture was added HCl in 1,4-dioxane
(4 mol/L, 1.5 mL), and the mixture was stirred for 1 h at room tem-
perature. A saturated aqueous solution of NaHCO3 and EtOAc were
added to the reaction mixture. The organic layer was washed with
water and brine, and dried over anhydrous MgSO4. The solution
was concentrated in vacuo, and the residue was purified using sil-
ica gel column chromatography.
5.1.45. Benzyl (R)-2-[(R)-1-(3-chlorophenyl)ethylamino]-2-
phenylacetate (14)
To a mixture of (R)-1-(3-chlorophenyl)ethylamine (16c, 1.37 g,
8.77 mmol), KHCO3 (1.17 g, 11.70 mmol) in acetonitrile (15 mL)
was added 12 (2.50 g, 5.85 mmol), and the mixture was stirred
for 4 h at 50 °C. After being cooled to room temperature, the mix-
ture was partitioned between EtOAc and water. The organic layer
was washed with water and brine, and dried over anhydrous
MgSO4. The solution was concentrated in vacuo, and the residue
was purified using silica gel column chromatography (eluent
Et2O:Hexane = 1:20) to afford 14 (1.31 g, 59%, colorless oil, low-
polarity) and benzyl (S)-2-[(R)-1-(3-chlorophenyl)ethylamino]-2-
phenylacetate 140 (425 mg, 19%, colorless oil, high-polarity). 14:
1H NMR (CDCl3) d 1.34 (d, J = 6.5 Hz, 3H), 2.33 (br s, 1H), 3.75 (q,
J = 6.5 Hz, 1H), 4.22 (s, 1H), 5.10 (d, J = 12.3 Hz, 1H), 5.22 (d,
J = 12.3 Hz, 1H), 7.14–7.36 (m, 14H); 13C NMR (CDCl3) d 24.8,
56.4, 63.1, 66.9, 125.2, 127.2, 127.3, 127.5, 128.2, 128.4, 128.7,
5.1.49. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3-methoxyphenyl)
ethyl]benzamide (20a), N-((S)-Carbamoylphenylmethyl)-N-[(R)-1-(3-
methoxyphenyl)ethyl]benzamide (21a)
The title compounds were prepared from (R)-1-(3-methoxyphe-
nyl)ethylamine (16a, 302 mg, 2.00 mmol), benzaldehyde (212 mg,
2.00 mmol), benzoic acid (244 mg, 2.00 mmol) according to the
general procedure of the Ugi reaction and deprotection. The crude
product was purified using silica gel column chromatography (elu-
ent Et2O:Hexane:MeOH = 10:10:1) to afford 20a (230 mg, 30%, col-
orless amorphous, low-polarity) and 21a (262 mg, 34%, colorless
solid, high-polarity). 20a: 1H NMR (CDCl3) d 1.36 (d, J = 7.0 Hz,
3H), 3.87 (s, 3H), 4.73 (br s, 1H), 5.24 (q, J = 7.0 Hz, 1H), 5.29 (br
s, 1H), 5.91 (br s, 1H), 6.87 (dd, J = 8.1, 2.5 Hz, 1H), 7.00–7.04 (m,
1H), 7.21–7.25 (m, 1H), 7.27–7.47 (m, 7H), 7.50–7.60 (m, 4H);
13C NMR (CDCl3) d 19.3, 55.5, 58.1, 62.8, 113.4, 113.7, 119.7,
126.3, 128.35, 128.43, 128.8, 129.1, 129.8, 129.9, 136.6, 137.9,
140.9, 159.9, 171.3, 172.3; HRMS calcd for C24H24N2O3 (MꢀH)ꢀ
387.1714, found 387.1715; chemical purity 98.9%, tR 13.2 min;
128.9, 130.0, 134.5, 135.7, 138.1, 147.1, 173.6; HRMS calcd for C23
H22ClN2O2 (M+H)+ 380.1412, found 380.1411;
-
½
a 2D5
ꢂ
þ 23:8
(c = 1.35, CHCl3). 140: 1H NMR (CDCl3) d 1.32 (d, J = 6.6 Hz, 3H),
2.44 (br s, 1H), 3.54 (q, J = 6.6 Hz, 1H), 4.23 (s, 1H), 5.03 (d,
J = 12.5 Hz, 1H), 5.08 (d, J = 12.5 Hz, 1H), 7.08–7.16 (m, 3H), 7.20–
7.38 (m, 11H); 13C NMR (CDCl3) d 24.4, 54.5, 62.8, 66.8, 125.2,
127.1, 127.5, 127.8, 127.9, 128.26, 128.28, 128.6, 128.9, 129.9,
134.5, 135.6, 138.0, 146.9, 172.6; HRMS calcd for C23H22ClN2O2
(M+H)+ 380.1412, found 380.1412; ½a 2D5
þ 126:4 (c = 1.42, CHCl3).
ꢂ
5.1.46. Benzyl (R)-2-{N-benzoyl-N-[(R)-1-(3-chlorophenyl)ethyl]
amino}-2-phenylacetate (15)
Benzoyl chloride (917 lL, 7.90 mmol) was added to a mixture of
14 (1.00 g, 2.63 mmol), triethylamine (2.20 mL, 15.8 mmol), N,N-
dimethylaminopyridine (32 mg, 0.263 mmol) in CH2Cl2 (20 mL).
The mixture was stirred for 5 days at room temperature, and par-
titioned between EtOAc and water. The organic layer was washed
with water and brine, and dried over anhydrous MgSO4. The solu-
tion was concentrated in vacuo, and the residue was purified using
silica gel column chromatography (eluent EtOAc:Hexane = 1:10–
1:5) to afford a mixture of 15 and corresponding diastereomer
(912 mg, 72%, R,R:S,R = 5:1). 1H NMR (CDCl3) d 1.27 (d, J = 7.0 Hz,
3H), 4.60 (br s, 1H), 5.02 (d, J = 12.0 Hz, 1H), 5.12–5.23 (m, 2H),
½
a 2D5
ꢂ
þ 142:5 (c = 1.00, CHCl3). 21a: 1H NMR (CDCl3) d 1.71 (d,
J = 6.8 Hz, 3H), 3.59 (s, 3H), 4.64 (br s, 1H), 5.18 (q, J = 6.8 Hz,
1H), 5.45 (br s, 1H), 6.13 (br s, 1H), 6.64–6.70 (m, 2H), 6.77–6.81
(m, 1H), 7.05–7.11 (m, 1H), 7.12–7.20 (m, 5H), 7.42–7.50 (m,
3H), 7.52–7.58 (m, 2H); 13C NMR (CDCl3) d 17.6, 55.2, 58.2, 63.4,
113.5, 113.9, 120.1, 126.1, 127.9, 128.5, 128.6, 128.9, 129.5,
129.6, 136.9, 140.3, 159.6, 172.3, 172.5; HRMS calcd for
C
24H24N2O3 (MꢀH)ꢀ 387.1714, found 387.1707; mp 162 °C; chem-
ical purity > 99.5%, tR 12.8 min; ½a D25
þ 84:2 (c = 1.07, CHCl3).
ꢂ
6.92–7.52 (m, 19H); HRMS calcd for
484.1674, found 484.1673.
C
30H26ClNO3 (M+H)+
5.1.50. N-((S)-Carbamoylphenylmethyl)-N-[(S)-1-(3-methoxyphenyl)
ethyl]benzamide (ent-20a), N-((R)-Carbamoylphenylmethyl)-N-[(S)-
1-(3-methoxyphenyl)ethyl]benzamide (ent-21a)
5.1.47. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3-chlorophenyl)
ethyl]benzamide (20c), N-((S)-Carbamoylphenylmethyl)-N-[(R)-1-(3-
chlorophenyl)ethyl]benzamide (21c)
To a mixture of 15 and corresponding diastereomer (5:1,
650 mg, 1.34 mmol) in THF (20 mL) was added 10% Pd–C (evonik
degussa, E101 NE/W, 65 mg), and the mixture was stirred for 1 h
under H2 atmosphere. The catalyst was removed by filtration,
and the filtrate was concentrated in vacuo to afford carboxylic acid
intermediate (531 mg, quant). To a mixture of the carboxylic acid
(250 mg, 0.635 mmol), NH4Cl (85 mg, 1.59 mmol), HATU (266 mg,
The title compounds were prepared from (S)-1-(3-methoxyphe-
nyl)ethylamine (ent-16a, 302 mg, 2.00 mmol), benzaldehyde
(212 mg, 2.00 mmol), benzoic acid (244 mg, 2.00 mmol) according
to the general procedure of the Ugi reaction and deprotection. The
crude product was purified using silica gel column chromatogra-
phy (eluent Et2O:Hexane:MeOH = 10:10:1) to afford ent-20a
(216 mg, 28%, colorless amorphous, low-polarity) and ent-21a
(285 mg, 37%, colorless solid, high-polarity). ent-20a: 1H NMR
(CDCl3) d 1.36 (d, J = 7.0 Hz, 3H), 3.88 (s, 3H), 4.73 (br s, 1H),
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049

J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
13
5.19–5.35 (m, 2H), 5.91 (br s, 1H), 6.87 (dd, J = 8.2, 2.5 Hz, 1H),
7.00–7.04 (m, 1H), 7.21–7.25 (m, 1H), 7.27–7.47 (m, 7H), 7.50–
7.60 (m, 4H); 13C NMR (CDCl3) d 19.3, 55.5, 58.1, 62.8, 113.4,
113.7, 119.7, 126.3, 128.37, 128.45, 128.9, 129.1, 129.8, 129.9,
136.6, 138.0, 140.9, 160.0, 171.3, 172.3; HRMS calcd for
63.4 (br s), 126.1, 128.0, 128.2, 128.3, 128.7, 129.0, 129.6, 129.7,
134.3, 136.7 (br s), 136.8, 140.9, 172.0, 172.2; HRMS calcd for C23
-
H
21ClN2O2 (MꢀH)ꢀ 391.1219, found 391.1220; mp 151 °C; chemi-
cal purity > 99.5%, tR 13.5 min; ½a D25
þ 58:7 (c = 0.96, CHCl3).
ꢂ
C
24H24N2O3 (MꢀH)ꢀ 387.1714, found 387.1711; chemical pur-
5.1.53. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3-chlorophenyl)
ethyl]-2-methylbenzamide (20d), N-((S)-Carbamoylphenylmethyl)-N-
[(R)-1-(3-chlorophenyl)ethyl]-2-methylbenzamide (21d)
ity > 99.5%, tR 13.2 min; ½a D25
ꢀ 147:4 (c = 1.04, CHCl3). ent-21a:
ꢂ
1H NMR (CDCl3) d 1.71 (d, J = 6.8 Hz, 3H), 3.59 (s, 3H), 4.64 (br s,
1H), 5.18 (q, J = 6.8 Hz, 1H), 5.49 (br s, 1H), 6.13 (br s, 1H), 6.64–
6.70 (m, 2H), 6.77–6.81 (m, 1H), 7.05–7.11 (m, 1H), 7.12–7.20
(m, 5H), 7.42–7.50 (m, 3H), 7.52–7.58 (m, 2H); 13C NMR (CDCl3)
d 17.6, 55.2, 58.2, 63.4, 113.5, 113.9, 120.1, 126.1, 127.8, 128.5,
128.6, 128.9, 129.5, 129.6, 136.9, 140.3, 159.6, 172.3, 172.5; HRMS
calcd for C24H24N2O3 (MꢀH)ꢀ 387.1714, found 387.1710; mp
The title compounds were prepared from (R)-1-(3-chlorophe-
nyl)ethylamine (16c, 311 mg, 2.00 mmol), benzaldehyde (212 mg,
2.00 mmol), 2-methylbenzoic acid (272 mg, 2.00 mmol) according
to the general procedure of the Ugi reaction and deprotection.
The crude product was purified using silica gel column chromatog-
raphy (eluent EtOAc:Hexane = 1:2–1:1) to afford 20d (203 mg,
25%, colorless solid, low-polarity) and 21d (307 mg, 38%, colorless
solid, high-polarity). 20d: This compound exists as a mixture of
rotamers in CDCl3 (3:2). 1H NMR (CDCl3) d 1.27 (d, J = 6.8 Hz, minor
3H), 1.37 (d, J = 6.6 Hz, major 3H), 2.45 (br s, minor 3H), 2.53 (br s,
major 3H), 4.64 (br s, 1H), 4.95–5.12 (m, 1H), 5.20–5.95 (m, 2H),
7.16–7.71 (m, 13H). In 13C NMR, some peaks of the rotamers could
not be distinguished. 13C NMR (CDCl3) d 19.1, 19.3, 19.6, 20.6, 57.5
(minor), 58.0, 62.4, 62.8 (minor), 125.0, 125.7, 125.8, 126.0, 126.1,
126.3, 127.6, 128.1, 128.2, 128.3, 128.6, 128.8, 129.25, 129.31,
130.0, 130.7, 131.4, 134.4, 134.57, 134.61, 135.61, 135.66, 136.0,
137.9, 138.7 (minor), 141.0, 141.6 (minor), 171.0 (minor), 171.2,
172.2, 172.4 (minor); HRMS calcd for C24H23ClN2O2 (MꢀH)ꢀ
405.1375, found 405.1375; mp 129 °C; chemical purity > 99.5%, tR
161 °C; chemical purity > 99.5%, tR 12.8 min; ½a D25
ꢀ 81:3 (c = 1.17,
ꢂ
CHCl3).
5.1.51. N-((R)-Carbamoylphenylmethyl)-N-((R)-1-phenylpropyl)
benzamide (20b), N-((S)-Carbamoylphenylmethyl)-N-((R)-1-
phenylpropyl)benzamide (21b)
The title compounds were prepared from (R)-1-phenylpropy-
lamine (16b, 270 mg, 2.00 mmol), benzaldehyde (212 mg,
2.00 mmol), benzoic acid (244 mg, 2.00 mmol) according to the
general procedure of the Ugi reaction and deprotection. The crude
product was purified using silica gel column chromatography (elu-
ent Et2O:Hexane:MeOH = 20:20:1) to afford 20b (284 mg, 38%, col-
orless amorphous, low-polarity) and 21b (211 mg, 28%, colorless
solid, high-polarity). 20b: 1H NMR (CDCl3) d 0.93 (t, J = 7.3 Hz,
3H), 2.16–2.33 (m, 2H), 4.86 (s, 1H), 4.94 (t, J = 7.5 Hz, 1H), 5.55
(br s, 1H), 6.83 (br s, 1H), 6.97–7.03 (m, 2H), 7.07–7.27 (m, 8H),
7.44–7.58 (m, 5H); 13C NMR (CDCl3) d 11.4, 25.0, 63.5, 64.6,
126.6, 127.46, 127.52, 128.27, 128.31, 128.5, 128.7, 128.8, 129.6,
136.3, 137.0, 137.5, 172.6, 173.1; HRMS calcd for C24H24N2O2
(MꢀH)ꢀ 371.1765, found 371.1761; chemical purity > 99.5%, tR
15.6 min; ½a 2D5
þ 153:5 (c = 1.02, CHCl3). 21d: This compound
ꢂ
exists as a mixture of rotamers in CDCl3 (3:2). 1H NMR (CDCl3) d
1.69 (d, J = 7.0 Hz, major 3H), 1.73 (d, J = 6.7 Hz, minor 3H), 2.47
(br s, minor 3H), 2.53 (br s, major 3H), 4.54 (br s, 1H), 4.85–4.95
(m, minor 1H), 5.09 (q, J = 7.0 Hz, major 1H), 5.46 (br s, 1H), 5.79
(br s, minor 1H), 5.93 (br s, major 1H), 6.92–7.57 (m, 13H); In
13C NMR, some peaks of the rotamers could not be distinguished.
13C NMR (CDCl3) d 17.0 (minor), 17.6, 19.2, 19.6 (minor), 57.6,
57.8 (minor), 63.0, 63.4 (minor), 125.6, 125.7, 125.9, 126.0, 126.3,
128.1, 128.2, 128.3, 128.59, 128.66, 128.73, 128.9, 129.2, 129.3,
129.6, 130.9, 131.4, 133.9, 134.4, 135.7, 135.8, 136.1, 136.9, 137.1
(minor), 140.8 (minor), 140.9, 171.9, 172.1, 172.4; HRMS calcd
for C24H23ClN2O2 (MꢀH)ꢀ 405.1375, found 405.1371; mp 141 °C;
14.5 min; ½a 2D5
ꢂ
þ 161:9 (c = 1.13, CHCl3). 21b: 1H NMR (CDCl3) d
0.69 (t, J = 7.3 Hz, 3H), 1.68–1.81 (m, 1H), 2.00–2.13 (m, 1H), 4.73
(s, 1H), 4.97 (t, J = 7.5 Hz, 1H), 5.14 (br s, 1H), 5.82 (br s, 1H),
7.31–7.51 (m, 11H), 7.55–7.62 (m, 4H); 13C NMR (CDCl3) d 11.3,
25.4, 62.9, 64.5, 126.8, 128.4, 128.5, 128.8, 128.93, 128.95, 129.0,
129.8, 136.9, 137.58, 137.64, 171.2, 172.5; HRMS calcd for
chemical purity 96.7%, tR 15.3 min; ½a D25
þ 69:5 (c = 1.03, CHCl3).
ꢂ
C
24H24N2O2 (MꢀH)ꢀ 371.1765, found 371.1759; mp 129 °C; chem-
ical purity > 99.5%, tR 14.8 min; ½a D25
þ 156:2 (c = 1.11, CHCl3).
ꢂ
5.1.54. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3-chlorophenyl)
ethyl]-3-methylbenzamide (20e), N-((S)-Carbamoylphenylmethyl)-N-
[(R)-1-(3-chlorophenyl)ethyl]-3-methylbenzamide (21e)
5.1.52. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3-chlorophenyl)
ethyl]benzamide (20c), N-((S)-Carbamoylphenylmethyl)-N-[(R)-1-(3-
chlorophenyl)ethyl]benzamide (21c)
The title compounds were prepared from (R)-1-(3-chlorophe-
nyl)ethylamine (16c, 311 mg, 2.00 mmol), benzaldehyde (212 mg,
2.00 mmol), 3-methylbenzoic acid (272 mg, 2.00 mmol) according
to the general procedure of the Ugi reaction and deprotection.
The crude product was purified using silica gel column chromatog-
raphy (eluent EtOAc:Hexane = 1:2–1:1) to afford 20e (250 mg, 31%,
colorless amorphous, low-polarity) and 21e (303 mg, 37%, colorless
solid, high-polarity). 20e: 1H NMR (CDCl3) d 1.38 (d, J = 7.0 Hz, 3H),
2.39 (s, 3H), 4.69 (br s, 1H), 5.23 (q, J = 7.0 Hz, 1H), 5.29 (br s, 1H),
5.97 (br s, 1H), 7.23–7.47 (m, 10H), 7.48–7.57 (m, 3H); 13C NMR
(CDCl3) d 19.0, 21.5, 57.6, 63.0, 123.1, 126.2, 126.9, 127.8, 128.2,
128.4, 128.5, 128.7, 129.2, 130.0, 130.7, 134.5, 136.4, 137.5,
138.9, 141.6, 171.3, 172.5; HRMS calcd for C24H23ClN2O2 (MꢀH)ꢀ
405.1375, found 405.1377; chemical purity 96.9%, tR 15.7 min;
The title compounds were prepared from (R)-1-(3-chlorophe-
nyl)ethylamine (16c, 311 mg, 2.00 mmol), benzaldehyde (212 mg,
2.00 mmol), benzoic acid (244 mg, 2.00 mmol) according to the
general procedure of the Ugi reaction and deprotection. The crude
product was purified using silica gel column chromatography (elu-
ent EtOAc:Hexane = 1:1–3:1) to afford 20c (236 mg, 30%, colorless
solid, low-polarity) and 21c (356 mg, 45%, colorless solid, high-
polarity). 20c: 1H NMR (CDCl3) d 1.38 (d, J = 7.0 Hz, 3H), 4.70 (br
s, 1H), 5.22 (q, J = 7.0 Hz, 1H), 5.30 (br s, 1H), 5.94 (br s, 1H),
7.28–7.48 (m, 9H), 7.49–7.59 (m, 5H); 13C NMR (CDCl3) d 19.1,
57.7, 63.1, 126.1, 126.3, 127.8, 128.3, 128.4, 128.5, 128.9, 129.2,
130.0, 134.6, 136.4, 137.6, 141.5, 171.3, 172.4; HRMS calcd for C23
-
21ClN2O2 (MꢀH)ꢀ 391.1219, found 391.1223; mp 136 °C; chemi-
½ ꢂ
a 2D5
þ 120:4 (c = 1.14, CHCl3). 21e: 1H NMR (CDCl3) d 1.73 (d,
H
cal purity 99.0%, tR 13.8 min; ½a D25
ꢂ
þ 128:6 (c = 1.11, CHCl3). 21c: 1H
J = 6.6 Hz, 3H), 2.41 (s, 3H), 4.55 (br s, 1H), 5.11–5.22 (m, 1H),
5.45 (br s, 1H), 5.94 (br s, 1H), 6.99–7.23 (m, 9H), 7.24–7.30 (m,
1H), 7.32–7.40 (m, 3H); 13C NMR (CDCl3) d 17.3 (br s), 21.6, 57.6,
63.3 (br s), 123.0, 126.1, 126.6, 128.0, 128.1, 128.3, 128.6, 128.8,
NMR (CDCl3) d 1.73 (d, J = 6.8 Hz, 3H), 4.56 (br s, 1H), 5.10–5.19 (m,
1H), 5.45 (br s, 1H), 5.90 (br s, 1H), 6.99–7.22 (m, 9H), 7.43–7.51
(m, 3H), 7.53–7.58 (m, 2H); 13C NMR (CDCl3) d 17.3 (br s), 57.6,
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049

14
J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
129.5, 130.4, 134.3, 136.7, 138.9, 141.0 (br s), 172.1, 172.4; HRMS
5.1.57. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3-chlorophenyl)
ethyl]-2-nitrobenzamide (20h), N-((S)-Carbamoylphenylmethyl)-N-
[(R)-1-(3-chlorophenyl)ethyl]-2-nitrobenzamide (21h)
calcd for C24H23ClN2O2 (MꢀH)ꢀ 405.1375, found 405.1374; mp
160 °C; chemical purity 98.4%, tR 15.4 min; ½a D25
þ 70:8 (c = 1.10,
ꢂ
The title compounds were prepared from (R)-1-(3-chlorophe-
nyl)ethylamine (16c, 311 mg, 2.00 mmol), benzaldehyde (212 mg,
2.00 mmol), 2-nitrobenzoic acid (334 mg, 2.00 mmol) according
to the general procedure of the Ugi reaction and deprotection.
The crude product was purified using silica gel column chromatog-
raphy (eluent EtOAc:Hexane = 1:2–1:1) to afford 20h (326 mg,
37%, pale yellow amorphous, low-polarity) and 21h (437 mg,
50%, pale yellow amorphous, high-polarity). 20h: This compound
exists as a mixture of rotamers in CDCl3 (5:1). 1H NMR (CDCl3) d
1.31 (d, J = 7.0 Hz, major 3H), 1.59 (d, J = 6.3 Hz, minor 3H), 4.73
(s, major 1H), 4.85 (q, J = 7.0 Hz, major 1H), 4.90–4.98 (m, minor
2H), 5.25–5.71 (m, 2H), 6.72–7.90 (m, 12H), 8.23 (d, J = 8.3 Hz,
major 1H), 8.28 (d, J = 8.0 Hz, minor 1H). 13C NMR (CDCl3) d 18.3
(minor), 20.6, 58.1, 58.2 (minor), 62.8, 63.4 (minor), 124.9, 125.5,
125.7 (minor), 127.4, 127.6 (minor), 128.1, 128.3, 128.8 (minor),
128.9, 129.1 (minor), 129.4, 129.5, 130.18, 130.21, 130.7 (minor),
132.5 (minor), 133.0, 134.4 (minor), 134.5 (minor), 134.82,
134.84, 138.0, 139.6 (minor), 141.8, 145.1, 168.6, 171.0; HRMS
CHCl3).
5.1.55. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3-chlorophenyl)
ethyl]-4-methylbenzamide (20f), N-((S)-Carbamoylphenylmethyl)-N-
[(R)-1-(3-chlorophenyl)ethyl]-4-methylbenzamide (21f)
The title compounds were prepared from (R)-1-(3-chlorophe-
nyl)ethylamine (16c, 311 mg, 2.00 mmol), benzaldehyde (212 mg,
2.00 mmol), 4-methylbenzoic acid (272 mg, 2.00 mmol) according
to the general procedure of the Ugi reaction and deprotection.
The crude product was purified using silica gel column chromatog-
raphy (eluent EtOAc:Hexane = 1:2–1:1) to afford 20f (241 mg, 30%,
colorless amorphous, low-polarity) and 21f (322 mg, 40%, colorless
solid, high-polarity). 20f: 1H NMR (CDCl3) d 1.38 (d, J = 7.0 Hz, 3H),
2.39 (s, 3H), 4.70 (br s, 1H), 5.18–5.38 (m, 2H), 6.02 (br s, 1H), 7.21–
7.58 (m, 13H); 13C NMR (CDCl3) d 19.0, 21.5, 57.6, 63.1, 126.1,
126.4, 127.8, 128.2, 128.3, 128.4, 129.1, 129.5, 130.0, 133.5,
134.5, 137.5, 140.2, 141.6, 171.4, 172.6; HRMS calcd for C24H23
-
calcd for
C
23H20ClN3O4 (MꢀH)ꢀ 436.1070, found 436.1063;
ClN2O2 (MꢀH)ꢀ 405.1375, found 405.1372; chemical purity
½
a 2D5
ꢂ
þ 136:0 (c = 0.89, CHCl3). 21h: This compound exists as a mix-
95.8%, tR 15.8 min; ½a D25
ꢂ
þ 118:3 (c = 1.13, CHCl3). 21f: 1H NMR
ture of rotamers in CDCl3 (4:1). 1H NMR (CDCl3) d 1.70 (d,
J = 6.7 Hz, major 3H), 1.80 (d, J = 6.7 Hz, minor 3H), 4.47 (s, major
1H), 4.75 (q, J = 6.7 Hz, major 1H), 4.87–4.96 (m, minor 1H), 5.03
(br s, minor 1H), 5.44 (br s, major 2H and minor 1H), 5.76 (br s,
minor 1H), 6.89 (t, J = 7.8 Hz, major 1H), 6.96–7.10 (m, major 2H
and minor 3H), 7.13–7.46 (m, 6H), 7.61–7.72 (m, major 1H and
minor 2H), 7.79–7.88 (m, major 2H and minor 1H), 8.32 (d,
J = 8.3 Hz, 1H); 13C NMR (CDCl3) d 16.3, 17.7 (minor), 57.4, 58.5
(minor), 63.5, 125.0, 125.5 (minor), 125.9 (minor), 127.1, 127.5
(minor), 127.9 (minor), 128.0, 128.2, 128.4 (minor), 128.6, 128.9,
129.2, 129.3, 130.2, 130.6 (minor), 132.9, 134.1, 134.8, 136.4,
139.5, 140.6 (minor), 144.7, 145.1 (minor), 168.2, 171.6, 172.7
(minor); HRMS calcd for C23H20ClN3O4 (MꢀH)ꢀ 436.1070, found
(CDCl3) d 1.73 (d, J = 6.9 Hz, 3H), 2.40 (s, 3H), 4.56 (br s, 1H),
5.14–5.24 (m, 1H), 5.45 (br s, 1H), 5.97 (br s, 1H), 6.99–7.22 (m,
9H), 7.25–7.31 (m, 2H), 7.45 (d, J = 8.0 Hz, 2H); 13C NMR (CDCl3)
d 17.4 (br s), 21.5, 57.7, 63.5 (br s), 126.0, 126.1, 127.9, 128.1,
128.3, 128.6, 129.6, 133.8, 134.3, 136.7 (br s), 139.8, 141.1 (br s),
172.2, 172.5; HRMS calcd for C24H23ClN2O2 (MꢀH)ꢀ 405.1375,
found 405.1377; mp 185 °C; chemical purity 95.6%, tR 15.5 min;
½
a 2D5
ꢂ
þ 77:8 (c = 1.05, CHCl3).
5.1.56. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3-chlorophenyl)
ethyl]-2-hydroxybenzamide (20m), N-((S)-Carbamoylphenylmethyl)-
N-[(R)-1-(3-chlorophenyl)ethyl]-2-hydroxybenzamide (21m)
436.1064; ½a 2D5
þ 110:8 (c = 1.21, CHCl3).
ꢂ
A mixture of the title compounds, 2-acetyloxy-N-((R)-Car-
bamoylphenylmethyl)-N-[(R)-1-(3-chlorophenyl)ethyl]benzamide
(20g) and 2-acetyloxy-N-((S)-Carbamoylphenylmethyl)-N-[(R)-1-
(3-chlorophenyl)ethyl]benzamide (21g) was prepared from (R)-1-
(3-chlorophenyl)ethylamine (16c, 311 mg, 2.00 mmol), benzalde-
hyde (212 mg, 2.00 mmol), acetylsalicylic acid (360 mg,
2.00 mmol) according to the general procedure of the Ugi reaction
and deprotection. To a solution of the mixture in MeOH (10 mL)
was added K2CO3 (276 mg, 2.00 mmol), and the mixture was stir-
red for 1 h at room temperature. The crude product was purified
using silica gel column chromatography (eluent EtOAc:Hex-
ane = 1:1) to afford 20m (197 mg, 24%, colorless solid, low-polar-
ity) and 21m (262 mg, 32%, colorless solid, high-polarity). 20m:
1H NMR (CDCl3) d 1.31 (d, J = 7.0 Hz, 3H), 4.67 (br s, 1H), 5.33 (q,
J = 7.0 Hz, 1H), 5.48 (br s, 1H), 5.59 (br s, 1H), 6.90 (td, J = 7.5,
1.0 Hz, 1H), 7.04–7.09 (m, 1H), 7.29–7.58 (m, 11H), 8.65 (br s,
1H); 13C NMR (THF-d8) d 18.6, 57.3 (br s), 64.5, 117.0, 119.9,
124.8, 127.3, 127.6, 128.1, 128.4, 128.9, 129.0, 129.9, 130.0,
131.1, 134.4, 138.8, 143.9 (br s), 155.4, 170.6, 171.9; HRMS calcd
for C23H21ClN2O3 (MꢀH)ꢀ 407.1168, found 407.1170; mp 224 °C;
5.1.58. N-[(R)-Carbamoyl(prirdin-3-yl)methyl]-N-[(R)-1-(3-
chlorophenyl)ethyl]benzamide (20i), N-[(S)-Carbamoyl(prirdin-3-yl)
methyl]-N-[(R)-1-(3-chlorophenyl)ethyl]benzamide (21i)
The title compounds were prepared from (R)-1-(3-chlorophe-
nyl)ethylamine (16c, 311 mg, 2.00 mmol), 3-formylpyridine
(214 mg, 2.00 mmol), benzoic acid (244 mg, 2.00 mmol) according
to the general procedure of the Ugi reaction and deprotection. The
crude product was purified using silica gel column chromatogra-
phy (eluent EtOAc:Hexane:MeOH = 10:4:1) to afford 20i (158 mg,
20%, colorless amorphous, low-polarity) and 21i (342 mg, 43%, pale
yellow amorphous, high-polarity). 20i: 1H NMR (CDCl3) d 1.52 (d,
J = 7.0 Hz, 3H), 4.77 (s, 1H), 5.27 (q, J = 7.0 Hz, 1H), 5.37 (br s,
1H), 6.32 (br s, 1H), 7.30–7.43 (m, 5H), 7.46–7.58 (m, 5H), 7.93–
7.98 (m, 1H), 8.59 (dd, J = 4.8, 1.5 Hz, 1H), 8.69 (d, J = 2.3 Hz, 1H);
13C NMR (CDCl3) d 18.7, 57.8, 61.1, 123.8, 126.0, 126.3, 127.9,
128.7, 129.1, 130.2, 130.4, 133.0, 134.8, 135.9, 136.1, 140.8,
149.5, 149.6, 170.5, 172.6; HRMS calcd for C22H20ClN3O2 (MꢀH)ꢀ
392.1171, found 392.1172; chemical purity 98.9%, tR 12.6 min;
chemical purity 97.4%, tR 13.8 min; ½a D25
ꢂ
þ 31:0 (c = 1.07, DMF).
½
a 2D5
ꢂ
þ 52:9 (c = 1.05, CHCl3). 21i: 1H NMR (CDCl3) d 1.74 (d,
21m: 1H NMR (CDCl3) d 1.73 (d, J = 6.9 Hz, 3H), 4.66 (br s, 1H),
5.23 (q, J = 6.9 Hz, 1H), 5.67 (br s, 2H), 6.91–7.06 (m, 6H), 7.14–
7.24 (m, 5H), 7.29–7.37 (m, 2H), 8.29 (s, 1H); 13C NMR (CDCl3) d
16.3, 57.7, 63.5, 117.9, 120.0, 122.1, 125.8, 126.5, 127.9, 128.1,
128.6, 129.6, 129.8, 131.5, 134.4, 136.0, 141.3, 154.4, 171.3,
172.8; HRMS calcd for C23H21ClN2O3 (MꢀH)ꢀ 407.1168, found
407.1169; mp 202 °C; chemical purity > 99.5%, tR 13.5 min;
J = 7.0 Hz, 3H), 4.60 (br s, 1H), 5.18 (q, J = 7.0 Hz, 1H), 5.60 (br s,
1H), 6.00 (br s, 1H), 7.04–7.20 (m, 5H), 7.46–7.57 (m, 5H), 7.72–
7.77 (m, 1H), 8.07 (d, J = 2.4 Hz, 1H), 8.44 (dd, J = 4.8, 1.6 Hz, 1H);
13C NMR (CDCl3) d 17.0, 57.7, 61.1, 123.4, 125.9, 126.0, 128.2,
128.5, 129.1, 129.88, 129.92, 132.5, 134.7, 136.2, 136.5, 140.6,
149.3, 149.9, 171.1, 172.4; HRMS calcd for C22H20ClN3O2 (MꢀH)ꢀ
392.1171, found 392.1174; chemical purity 97.7%, tR 12.2 min;
½
a 2D5
ꢂ
þ 115:5 (c = 1.09, DMF).
½
a 2D5
ꢂ
þ 92:4 (c = 0.95, CHCl3).
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049

J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
15
5.1.59. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3-chlorophenyl)
ethyl]nicotinamide (20j), N-((S)-Carbamoylphenylmethyl)-N-[(R)-1-
(3-chlorophenyl)ethyl]nicotinamide (21j)
to the general procedure of the Ugi reaction and deprotection. The
crude product was purified using silica gel column chromatogra-
phy (eluent EtOAc:Hexane = 1:1–2:1) to afford 20l (243 mg, 31%,
colorless solid, low-polarity) and 21l (345 mg, 44%, colorless solid,
high-polarity). 20l: 1H NMR (CDCl3) d 1.33 (d, J = 6.8 Hz, 3H), 4.73
(br s, 1H), 5.12–5.22 (m, 1H), 5.36 (br s, 1H), 5.86 (br s, 1H), 6.76
(tt, J = 8.8, 2.1 Hz, 1H), 7.12–7.20 (m, 2H), 7.34–7.48 (m, 6H),
7.49–7.56 (m, 4H); 13C NMR (CDCl3) d 19.2 (br s), 57.3 (br s),
63.3 (br s), 103.4 (t, J = 25.8 Hz), 110.8 (dd, J = 19.0, 7.3 Hz),
126.3, 128.5, 128.7, 129.0, 129.3, 130.1, 136.2, 137.4 (br s), 143.9
(br s), 163.2 (dd, J = 248.9, 12.6 Hz), 171.2, 172.3; HRMS calcd for
The title compounds were prepared from (R)-1-(3-chlorophe-
nyl)ethylamine (16c, 311 mg, 2.00 mmol), benzaldehyde (212 mg,
2.00 mmol), nicotinic acid (246 mg, 2.00 mmol) according to the
general procedure of the Ugi reaction and deprotection. The crude
product was purified using silica gel column chromatography (elu-
ent EtOAc:Hexane:MeOH = 10:2:1) to afford 20j (236 mg, 30%, col-
orless amorphous, low-polarity) and 21j (356 mg, 45%, colorless
amorphous, high-polarity). 20j: 1H NMR (CDCl3)
d 1.36 (d,
J = 7.0 Hz, 3H), 4.74 (br s, 1H), 5.16 (q, J = 7.0 Hz, 1H), 5.37 (br s,
1H), 5.68 (br s, 1H), 7.29–7.45 (m, 6H), 7.47–7.58 (m, 4H), 7.85
(dt, J = 7.8, 2.0 Hz, 1H), 8.69 (dd, J = 4.9, 1.7 Hz, 1H), 8.80–8.82
(m, 1H); 13C NMR (CDCl3) d 19.7, 57.8, 63.1, 123.7, 126.0, 127.6,
128.4, 128.7, 128.9, 129.4, 130.2, 132.4, 134.2, 134.8, 137.4,
141.2, 147.2, 151.0, 169.9, 170.8; HRMS calcd for C22H20ClN3O2
(MꢀH)ꢀ 392.1171, found 392.1175; chemical purity 97.5%, tR
C
23H20F2N2O2 (MꢀH)ꢀ 393.1420, found 393.1425; mp 149 °C;
chemical purity 99.3%, tR 14.6 min; optical purity 98.9% ee, tR
6.2 min (IA-3, 40% ethanol in hexane); ½a D25
þ 135:7 (c = 1.12,
ꢂ
CHCl3). 21l: 1H NMR (CDCl3) d 1.66–1.80 (m, 3H), 4.54 (br s, 1H),
5.11 (br s, 1H), 5.46 (br s, 1H), 5.80 (br s, 1H), 6.49–6.57 (m, 1H),
6.62 (br d, J = 6.0 Hz, 2H), 7.16–7.25 (m, 5H), 7.43–7.58 (m, 5H);
13C NMR (CDCl3) d 17.3 (br s), 57.3, 63.4 (br s), 103.2 (t,
J = 25.1 Hz), 111.0 (dd, J = 19.0, 6.2 Hz), 126.0, 128.4, 128.8, 129.0,
129.8, 136.6 (br s), 143.1 (br s), 162.8 (dd, J = 249.6, 12.9 Hz),
171.7, 172.2; HRMS calcd for C23H20F2N2O2 (MꢀH)ꢀ 393.1420,
found 393.1423; mp 158 °C; chemical purity 99.2%, tR 14.2 min;
12.5 min; ½a 2D5
ꢂ
þ 154:4 (c = 1.14, CHCl3). 21j: 1H NMR (CDCl3) d
1.76 (d, J = 6.9 Hz, 3H), 4.58 (br s, 1H), 5.10 (q, J = 6.9 Hz, 1H),
5.48 (br s, 1H), 5.68 (br s, 1H), 6.96–7.25 (m, 9H), 7.42 (dd,
J = 7.5, 5.0 Hz, 1H), 7.85–7.91 (m, 1H), 8.72 (dd, J = 5.0, 1.5 Hz,
1H), 8.83 (br s, 1H); 13C NMR (CDCl3) d 17.3, 57.7, 63.5, 123.8,
126.0, 128.16, 128.20, 128.5, 128.8, 129.0, 129.6, 132.8, 134.0,
134.4, 136.3, 140.4, 147.0, 150.7, 169.7, 171.4; HRMS calcd for
½
a 2D5
ꢂ
þ 41:3 (c = 1.09, CHCl3).
5.1.62. 2-Amino-N-((R)-carbamoylphenylmethyl)-N-[(R)-1-(3-
chlorophenyl)ethyl]benzamide (20n)
C
22H20ClN3O2 (MꢀH)ꢀ 392.1171, found 392.1176; chemical pur-
ity > 99.5%, tR 12.4 min; ½a D25
þ 26:6 (c = 1.17, CHCl3).
ꢂ
To a solution of 20h (250 mg, 0.571 mmol) in THF (10 mL) was
added 10% Pd–C (evonik degussa, E101 NE/W, 50 mg), and the mix-
ture was stirred for 3 h under H2 atmosphere. The catalyst was
removed by filtration, and the filtrate was concentrated in vacuo.
The residue was purified using aminopropyl silica gel column
chromatography (eluent EtOAc:Hexane = 1:1–2:1) to afford 20n
as a colorless solid (149 mg, 64%). 1H NMR (CDCl3) d 1.31
(d, J = 7.0 Hz, 3H), 4.33–4.68 (m, 3H), 5.23 (q, J = 7.0 Hz, 1H), 5.28
(br s, 1H), 5.56 (br s, 1H), 6.72–6.80 (m, 2H), 7.16–7.24 (m, 2H),
7.28–7.45 (m, 5H), 7.51–7.56 (m, 4H); 13C NMR (CDCl3) d 19.4,
58.0, 62.3, 116.5, 117.5, 121.0, 126.2, 127.1, 128.4, 128.7, 128.8,
129.0, 129.4, 129.9, 130.6, 134.5, 137.8, 140.8, 144.4, 171.3,
171.8; HRMS calcd for C23H22ClN3O2 (MꢀH)ꢀ 406.1328, found
406.1329., mp 170 °C; chemical purity 99.3%, tR 15.0 min;
5.1.60. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3-chloro-5-fluoro-
phenyl)ethyl]benzamide (20k), N-((S)-Carbamoylphenylmethyl)-N-
[(R)-1-(3-chloro-5-fluorophenyl)ethyl]benzamide (21k)
Triethylamine (279
lL, 2.00 mmol) was added to a mixture
of
(R)-1-(3-chloro-5-fluorophenyl)ethylamine
hydrochloride
(420 mg, 2.00 mmol), benzaldehyde (212 mg, 2.00 mmol) in MeOH
(4 mL), and the mixture was stirred for 2 h at 50 °C to afford the
imine intermediate. The title compounds were prepared from the
imine and benzoic acid (244 mg, 2.00 mmol) according to the gen-
eral procedure of the Ugi reaction and deprotection. The crude pro-
duct was purified using silica gel column chromatography (eluent
EtOAc:Hexane = 1:2–1:1) to afford 20k (296 mg, 36%, colorless
amorphous, low-polarity) and 21k (312 mg, 38%, colorless amor-
phous, high-polarity). 20k: 1H NMR (CDCl3) d 1.33 (d, J = 7.0 Hz,
3H), 4.73 (br s, 1H), 5.12–5.22 (m, 1H), 5.37 (br s, 1H), 5.84 (br s,
1H), 7.02–7.07 (m, 1H), 7.25–7.57 (m, 12H); 13C NMR (CDCl3) d
19.1 (br s), 57.2 (br s), 63.3 (br s), 113.5 (d, J = 22.7 Hz), 115.7 (d,
J = 24.4 Hz), 123.8 (d, J = 2.9 Hz), 126.3, 128.6, 128.7, 129.0, 129.3,
130.1, 135.2 (d, J = 10.7 Hz), 136.2, 137.3 (br s), 143.7 (br s),
162.8 (d, J = 250.1 Hz), 171.2, 172.3; HRMS calcd for C23H20ClFN2O2
(MꢀH)ꢀ 409.1125, found 409.1128; chemical purity > 99.5%, tR
½
a 2D5
ꢂ
þ 21:3 (c = 1.07, DMF).
5.2. Biological methods
5.2.1. Calcium influx assay
The cDNA of human TRPM8 (NM_024080.4), TRPA1
(NM_007332.2), TRPV1 (NM_080704.3), TRPV4 (NM_021625.4)
and rat TRPM8 (NM_134371.2) were inserted into a pcDNA3.1
(TRPM8, TRPA1, TRPV4) or pCI-neo (TRPV1) expression plasmid.
For the calcium influx assay, HEK293T cells (RIKEN Cell Bank, Tsu-
15.3 min; ½a 2D5
ꢂ
þ 145:8 (c = 1.17, CHCl3). 21k: 1H NMR (CDCl3) d
1.74 (br s, 3H), 4.51 (br s, 1H), 5.10 (br s, 1H), 5.47 (br s, 1H),
5.75 (br s, 1H), 6.67–6.73 (m, 1H), 6.77–6.90 (m, 2H), 7.17–7.26
(m, 5H), 7.44–7.58 (m, 5H); 13C NMR (CDCl3) d 17.2, 57.2, 63.3,
113.5 (d, J = 22.1 Hz), 115.5 (d, J = 24.4 Hz), 124.1, 126.0, 128.5,
128.8, 129.0, 129.8, 135.0 (br s), 136.5 (br s), 142.8 (br s), 162.4
(d, J = 250.3 Hz), 171.7, 172.2; HRMS calcd for C23H20ClFN2O2
(MꢀH)ꢀ 409.1125, found 409.1127; chemical purity > 99.5%, tR
kuba, Japan) were seeded in poly-D-lysine-coated, 96-well culture
plates with black walls and a clear base (Corning, NY) at a density
of 5 ꢁ 104 cells/well in Dulbecco’s modified Eagle’s medium
(Thermo Fisher Scientific, Waltham, MA) containing 10% fetal
bovine serum. After 4 h, cells were transfected with each plasmid
using a Lipofectamine 2000 (Thermo Fisher Scientific) following
the manufacturer’s protocol. After 2 days of incubation, cells
expressing TRPM8, TRPA1 and TRPV1 were loaded with Fluo-4
AM (Dojindo, Kumamoto, Japan) and cells expressing TRPV4 were
loaded with Fura-2 AM (Dojindo), for 60 min at 37 °C. Cells were
then tested for an agonist-induced increase in intracellular calcium
level using an FDSS plate reader (Hamamatsu Photonics,
Hamamatsu, Japan). EC80 concentration of menthol, AITC, capsaicin
and RN-1747 were used as agonists for TRPM8, TRPA1, TRPV1 and
TRPV4, respectively. IC50 values were calculated from the
15.0 min; ½a 2D5
þ 41:9 (c = 1.02, CHCl3).
ꢂ
5.1.61. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3,5-difluoropheny-
l)ethyl]benzamide (20l), N-((S)-Carbamoylphenylmethyl)-N-[(R)-1-
(3,5-difluorophenyl)ethyl]benzamide (21l)
The title compounds were prepared from (R)-1-(3,5-difluo-
rophenyl)ethylamine (16l, 314 mg, 2.00 mmol), benzaldehyde
(212 mg, 2.00 mmol), benzoic acid (244 mg, 2.00 mmol) according
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049

16
J.-i. Kobayashi et al. / Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
concentration response curve with nonlinear regression analysis
using GraphPad Prims 4.0 (GraphPad Software Inc., La Jolla, CA).
biological assay. We also thank Dr. Hideyuki Muranaka and Mr.
Fumiya Tanada for HRMS and HPLC analysis.
5.2.2. Rat liver microsomal stability
A. Supplementary data
Test compounds (10 lmol/L) were incubated with rat liver
microsomes (Xenotech, LLC) at a protein concentration of 0.5 mg/
mL in Tris buffer (pH 7.4, 50 mmol/L) containing MgCl2
(10 mmol/L) and alamethicin (10 lg/mL). Incubations were initi-
ated by the addition of NADPH (1 mg/mL) and UDPGA (1 mmol/
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.11.049.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
L). The reactions were carried out for 60 min at 37 °C, and then
quenched with cold acetonitrile (125 lL). These prepared samples
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This research received no specific grant from any funding
agency in the public, commercial, or not-for-profit sectors.
Acknowledgments
We thank Dr. Hiroki Kanbe for measurement of metabolic sta-
bility and metabolite analysis, and Ms. Akane Matsuzawa for the
Please cite this article in press as: Kobayashi J.-i., et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.11.049