Identification of N-acyl-N-indanyl-α-phenylglycinamides as selective TRPM8 antagonists designed to mitigate the risk of adverse effects

Bioorg. Med. Chem. 30 (2021) 115903

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Identiﬁcation of N-acyl-N-indanyl-α-phenylglycinamides as selective

TRPM8 antagonists designed to mitigate the risk of adverse effects

Jun-ichi Kobayashi^*, Hideaki Hirasawa, Yoshikazu Fujimori, Osamu Nakanishi,

Noboru Kamada, Tetsuya Ikeda, Akitoshi Yamamoto, Hiroki Kanbe

Discovery Research, R&D, Kissei Pharmaceutical Co., Ltd., 4365-1 Hotaka kashiwabara, Azumino, Nagano 399-8304, Japan

A R T I C L E I N F O

A B S T R A C T

Keywords:

Transient receptor potential melastatin 8 (TRPM8), a temperature-sensitive ion channel responsible for detecting

cold, is an attractive molecular target for the treatment of pain and other disorders. We have previously

discovered a selective TRPM8 antagonist, KPR-2579, which inhibited bladder afferent hyperactivity induced by

acetic acid instillation into the bladder. However, additional studies have revealed potential adverse effects with

KPR-2579, such as the formation of a reactive metabolite, CYP3A4 induction, and convulsions. In this report, we

TRPM8

TRPM8 antagonist

Phenylglycinamide

Reactive metabolite

CYP3A4 induction

OAB

describe the optimization of α-phenylglycinamide derivatives to mitigate the risk of these adverse effects. The

optimal compound 13x exhibited potent inhibition against icilin-induced wet-dog shakes and cold-induced

frequent voiding in rats, with a wide safety margin against the potential side effects.

1. Introduction

therefore, TRPM8 is regarded as a potential pharmacological target for

OAB. An increase in TRPM8 expression has been observed in the sub-

Transient receptor potential (TRP) channels are non-selective cation

channels, which can be activated by temperature, osmotic pressure,

oxidation–reduction potential, and chemical or mechanical stimulus.¹

Transient receptor potential melastatin 8 (TRPM8), a member of the

melastatin subfamily of TRP channels, is expressed in sensory neurons

and is activated by a cold environment or chemicals to induce a cold

sensation.²The well-known TRPM8 agonist, menthol, is widely used in

the food and pharmaceutical industries;³however, TRPM8 antagonists

are still under development.⁴Recently, both apo- and ligand-bound

structures of TRPM8 have been revealed by cryo-electron micro-

scopy,⁵and their application in virtual screening and molecular design

for identiﬁcation of TRPM8 antagonists has been reported.⁶Currently,

neuropathic pain, migraine, and urinary diseases are proposed as ther-

apeutic targets for TRPM8 antagonists,^4,7and several compounds are

being investigated in clinical trials.^8,9

urothelial nerve ﬁbers of patients with idiopathic detrusor overactivity

and painful bladder syndromes, and the density was correlated with the

voiding frequency and pain scores.¹²In animal models, the frequency of

distention-induced rhythmic bladder contractions in rats was reduced

by treatment with a TRPM8 antagonist,¹³and detrusor overactivity in

rodents induced by dermal or intravesical application of TRPM8 stim-

ulants was also able to be prevented by TRPM8 antagonists.¹⁴From

these ﬁndings, we investigated TRPM8 antagonists for the treatment of

OAB and identiﬁed the potent and selective TRPM8 antagonist, KPR-

2579,¹⁵which inhibited bladder afferent hyperactivity induced by

acetic acid instillation into the bladder.¹⁶

Encouraged by these positive biological results, we performed

additional safety assessments of KPR-2579. Dose-escalation toxicity

studies in rodents and in vitro experiments revealed the following po-

tential problems. First, a glutathione (GSH) adduct of KPR-2579 was

detected in rat bile and urine (Fig. 1). GSH adducts are thought to be

formed via the reaction of GSH with electrophilic metabolites, such as

epoxides; therefore, the formation of such reactive metabolites can

cause depletion of GSH, a major contributor to the metabolism of xe-

nobiotics and reactive oxygen species.¹⁷Furthermore, these reactive

metabolites might react with proteins to elicit unexpected immune

responses.^18aBecause some drugs that form similar metabolites have

Overactive bladder (OAB) is deﬁned as having symptoms that

include urinary urgency, usually with urinary frequency, with or

without urgency urinary incontinence.¹⁰Anticholinergic drugs and β3-

adrenoceptor agonists are currently used for the clinical treatment of

OAB, but some of these drugs are considered to have potential adverse

events, such as dry mouth, constipation, or cardiovascular effects.¹¹

A

relationship between TRPM8 and bladder function has been reported;

* Corresponding author.

E-mail address: jun-ichi_kobayashi@pharm.kissei.co.jp (J.-i. Kobayashi).

https://doi.org/10.1016/j.bmc.2020.115903

Received 26 October 2020; Accepted 26 November 2020

Available online 3 December 2020

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

been reported to induce hepatotoxicity in clinical use,¹⁸such metabolite

formation must be avoided in drugs for chronic diseases. Second, con-

vulsions were observed in mice and rats after a single oral administra-

tion of KPR-2579 at 300 mg/kg. To the best of our knowledge, no

relationship between convulsions and TRPM8 has previously been re-

ported; thus, we speculated that another molecular target in the central

nervous system (CNS) might be associated with this symptom. The ce-

rebral and plasma concentrations at 4 h after administration of KPR-

2579 to rats were 171 and 103 µM, respectively, and the brain/plasma

ratio was determined to be 1.66. Reducing the CNS distribution of a

compound is thought to be a possible method to avoid convulsions.

Finally, the induction of CYP3A4 gene expression was observed after

incubation of KPR-2579 with HepaRG®, a hepatic stem cell line.¹⁹

CYP3A4 is responsible for the metabolism of >30% of launched drugs,²⁰

and inhibition or induction of this enzyme may change the metabolic

clearance of, not only the administered drug, but also co-administered

drugs.²¹The structure of the oxidative metabolite of KPR-2579 sug-

gested that KPR-2579 might be a substrate of CYP3A4;²²therefore, the

upregulation of CYP3A4 was considered to be a potential risk for both

the loss of activity of this antagonist and pharmacokinetic drug–drug

interactions (DDI).

3. Results and discussion

The activity of the TRPM8 antagonists was determined by measuring

changes in the Ca²⁺inﬂux into HEK293T cells expressing hTRPM8.

Comparing the activity of the diastereomers (13 and 14), the R-isomers

(13) were found to be the eutomers (see supplementary ﬁle for the ac-

tivity of 14), and this result was consistent with our previously reported

compounds.¹⁵Therefore, the following investigations focused on the R-

isomers.

Initially, we attempted to avoid the formation of reactive metabo-

lites. The structure of the GSH adduct was identiﬁed by LC-MS/MS

analysis, and the mechanism of formation was proposed to be epoxi-

dation of the diﬂuorophenyl ring followed by GSH addition and HF

elimination (Fig. 1).^22,25We attempted to quantify the amount of the

epoxide metabolite in human liver microsomes by forming the dansyl

glutathione (dGSH) adduct, a stable molecule containing a ﬂuorescent

probe.²⁶The nucleophilic trapping with dGSH proceeded smoothly for

KPR-2579, and the amount of adduct was estimated to be 163%

compared with that of the diclofenac adduct, which has been reported to

induce idiosyncratic hepatotoxicity.²⁷To avoid the epoxide formation,

modiﬁcation of the diﬂuorophenethyl moiety was considered to be an

attractive strategy. Because we have previously demonstrated that the

halogen atoms at the 3- and 5-positions on the phenethyl substructure

greatly contributed to the activity and metabolic stability, we initially

attempted to introduce additional substituents on the diﬂuorinated ring

(Table 1). Contrary to our expectations, additional substitution with a

ﬂuorine atom (13a, 13b) or methyl group (13c) resulted in a reduction

in activity. Then, we synthesized a conformationally ﬁxed, ring-fused

analog of compound 13c to improve the activity and metabolic char-

acteristics simultaneously. Although no change in activity was observed

for the fused six-membered ring analog (13e) compared with the phe-

nethylamine derivative (13d), fused ﬁve-membered ring analogs, such

as indane (13f), improved the activity. The observed difference between

the o-methyl phenethyl derivative (13c) and the ring-fused analog

(13m) suggested that the dihedral angle between the phenyl ring and

the C(sp³)–C(sp³) bond of phenethyl group (13c) or the C1–C2 bond of

indane (13m), might affect the activity. Introduction of heteroatoms to

indane was unfavorable (13g, 13h), which implied that the binding

pocket is highly hydrophobic. Since the non-substituted indane deriva-

tive exhibited moderate activity, we synthesized 4- and 6-halo-

substituted derivatives that structurally resembled meta-substituted

phenethyl derivatives. Substitutions with a ﬂuorine or chlorine atom

at the 4- and 6-positions were favorable, except for those with a chlorine

atom at the 4-position (13i–13l), and 4,6-disubstituted derivatives also

exhibited good activity (13m, 13n). Since some of these indane de-

rivatives exhibited equipotent activity to KPR-2579, we evaluated the

formation of the reactive metabolites of these compounds. The results

showed that no dGSH adducts were detected from compounds 13j, 13m,

and 13n. Presumably, the ﬁve-membered ring of indane prevented the

epoxidation of the phenyl ring by steric repulsion of the metabolic

enzyme and/or by altering the electron density of the phenyl ring.

Formation of the reactive metabolite was avoided, and therefore we

next focused on suppressing the CYP3A4 induction and CNS distribu-

tion. Most CYP3A4 protein expression is triggered by the formation of a

Considering that OAB is often a chronic disorder and there is a

relatively high prevalence in elderly people, a wide safety margin and

low DDI are required for potential treatments. Herein, we report the

development of N-acyl-N-indanyl-α-phenylglycinamides as selective

TRPM8 antagonists with reduced risk proﬁles.

2. Chemistry

We employed Ellman’s procedure to synthesize optically active

phenethylamines and fused ring analogs (Scheme 1).²³Sulﬁnimines

were prepared by condensation of the corresponding benzaldehyde

(1a–1c) or indanone (5g, 5i–5n) with tert-butanesulﬁnamide. For the

synthesis of phenethylamine derivatives, sulﬁnimines (2a–2c) were

alkylated with MeMgBr. For the ring fused analogs, the sulﬁnimines (6g,

6i–6n) were reduced with NaBH₄. The residual tert-butanesulﬁnyl group

was removed under acidic conditions to afford optically active amine

derivatives (4a–4c, 8g, 8i–8n). The core structure was constructed in a

similar manner to the synthesis of KPR-2579; that is, a Ugi reaction was

employed with a cleavable isocyanide followed by deprotection and

functional modiﬁcation (Scheme 2).^15,24The stereoselectivity of the Ugi

reaction with ring-fused amines, such as indanylamine derivatives, was

still poor (e.g., 27% de for 13m and 14m), and the conﬁguration of the

major isomer was the opposite of the reaction with phenethylamine, that

is, R-isomers (e.g. 13m) were formed dominantly. Although we assumed

that the fused ring induced conformational changes in the imine that

would affect the face selectivity of the isocyanide attack, it was difﬁcult

to explain the slight difference in selectivity from the calculated stable

conformation of the imine (data not shown). The stereochemistry of the

1

compounds was determined by comparison of the H NMR data with

that of KPR-2579, the structure of which has been previously conﬁrmed

by X-ray crystallography.¹⁵

Figure 1. Proposed metabolic pathway of KPR-2579 in rats. GSH, glutathione; the reactive site of the epoxidation and GSH addition on the ring was not identiﬁed.

2

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

Scheme 1. General procedure for the preparation of optically active amine intermediates. Reagents and conditions: (a) (S)-tert-butanesulﬁnamide, Ti(OEt)₄, THF, rt;

(b) MeMgBr, CH₂Cl₂, 0 ^◦C–rt; (c) HCl, 1,4-dioxane, MeOH, rt; (d) (R)-tert-butanesulﬁnamide, Ti(OEt)₄, THF, reﬂux; (e) NaBH₄, THF, rt.

Scheme 2. Synthetic route of phenylglycinamide derivatives. Reagents and conditions: (a) benzaldehyde, (Et₃N), MeOH, 50 ^◦C; (b) R^bCO₂H, 4-phenylcyclohexenyl

isocyanide, 50–60 ^◦C; (c) HCl, 1,4-dioxane, H₂O, rt; (d) K₂CO₃aq., MeOH, rt; (e) 10% Pd–C, H₂, THF or EtOH, rt.

complex between the pregnane xenobiotic receptor (PXR) and a ligand

molecule, which binds to the promoter region of the CYP3A4 coded

gene, followed by transcription to produce mRNA.²⁸Incubation of

HepaRG® cells with 3 and 10 µM of KPR-2579 for 24 h induced 5.6 and

11.1-fold expression of CYP3A4 mRNA, respectively. We set a criterion

for acceptable mRNA induction based on the latest FDA drug interaction

guidance;²⁹that is, the fold change in the mRNA expression relative to

the vehicle control should not exceed 2-fold at 30-fold of the unbound

maximum steady-state plasma concentration at the therapeutic dose

(I_max,u). Because prediction of the I_max,uvalue for clinical use from

preclinical data can be inaccurate, we tentatively determined the cri-

terion from the maximum unbound plasma concentration (C_max,u) at the

dose that completely inhibited icilin-induced wet-dog shake (WDS)³⁰in

rats. Using the value for 30-fold of the C_max,uat the dose of KPR-2579

that completely inhibited WDS (10 mg/kg),³¹the tentative criterion

was set as being < 2-fold at 7 µM for an equipotent compound; hence,

the expression of CYP3A4 mRNA should be reduced to less than a

quarter. Initially, we employed a time-resolved ﬂuorescence energy

transfer (TR-FRET) PXR binding assay³²for ease of use to assess the

CYP3A4 induction potential.³³A correlation between high lipophilicity

of a ligand molecule and strong PXR activation has previously been

reported.³⁴As high lipophilicity also enhances the penetration of com-

pounds into the brain, we attempted to lower the logD_7.4value of the

molecule to simultaneously suppress the CYP3A4 induction and reduce

the CNS distribution. Initially, we introduced polar substituents, such as

OH or NH₂, to the ortho position of the benzoyl group, because com-

pounds with modiﬁcations at this position retained inhibitory activity in

our previous study.¹⁵Indeed, these compounds kept the activity (13p,

13r) but their afﬁnity to PXR and the logD_7.4value were almost un-

changed. Indane derivatives (13m, 13n) also maintained the afﬁnity.

These ﬁndings suggested that a reduction in the logD_7.4value is needed

to weaken the interaction of these molecules with the ﬂexible and hy-

drophobic binding pocket of PXR.³⁵In another modiﬁcation, when R^b

was pyridine (13s, 13t), the PXR binding was suppressed with a

decrease in the logD_7.4value. A similar modiﬁcation has been previously

reported to result in attenuation of the TRPM8 inhibitory activity of

phenethylamine derivatives;¹⁵however, in the current study, the indane

derivatives had maintained or improved activity. The most favorable

3

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

Table 1

Structure–activity relationships (SARs) for (R)-phenethyl and ring fused phenylglycinamide derivatives.

Compound

R^a

R^b

Ph

logD_7.4

4.31

hTRPM8

dGSH adduct (%)^c

PXR binding EC₅₀(µM)

a

IC₅₀(µM)^b

KPR-2579

13a

0.08

163

10.3

d

Ph

4.45

1.7

–

13b

2.2

–

13c

Ph

4.78

2.0

–

13d

13e

Ph

4.02

4.59

1.2

1.4

–

13f

13g

13h

13i

Ph

4.15

2.80

3.33

4.29

0.27

19.5

3.3

–

0.09

13j

Ph

4.75

0.12

0

–

13k

13l

Ph

4.29

4.75

4.43

0.12

0.72

0.10

–

0

–

13m

9.5

13n

13p

13r

13s

13t

Ph

4.89

4.58

4.13

3.22

3.68

0.30

0.05

0.11

0

–

0

4.7

12.0

4.8

47

2-OHPh

2-NH₂Ph

3-pyridyl

131

4

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

a

Calculated using ChamAxon Marvin calculator plugin.

b

Each IC₅₀value represents the mean of at least two independent experiments.

c

d

The value was quantiﬁed by the ﬂuorescence of the adducts compared to that of the diclofenac adduct.

Not tested.

compound 13t had one-tenth of the afﬁnity toward PXR compared with

Table 3

KPR-2579, and no dGSH adduct was detected. Furthermore, no con-

In vitro activity of compound 13x against TRP channels.

vulsions were observed in mice after oral administration of 13t at a dose

of 300 mg/kg. The logD_7.4value of compound 13t was calculated as 0.63

less than that of KPR-2579 (3.68 vs 4.31); this decrease was assumed to

sufﬁciently reduce the CNS distribution to prevent convulsions. Despite

these positive results, inhibition of CYP3A4 was unexpectedly observed,

65% at 10 µM; therefore, more structural optimization was required.

Coordination of a heteroatom to heme is considered to be a key factor

in the inhibition of CYP enzymes.³⁶We hypothesized that the CYP3A4

inhibition observed for compound 13t might be attributed to the newly

introduced pyridine substructure, and modiﬁcation of the ring was also

considered to be a possible method to suppress the inhibition while

maintaining weak CYP3A4 induction. In this optimization step, CYP3A4

induction was evaluated by the expression of mRNA in HepaRG® cells as

described above. Although the throughput of the HepaRG® assay is poor

compared with the PXR binding assay, this method has the advantage of

providing an indication of the membrane permeability and transcription

activity of the test compounds. As shown in Table 1, ortho-substitution

of R^bhad a limited effect on the activity; therefore, we introduced

substituents at the 2-position of pyridine to increase the steric hindrance

around the nitrogen atom (Table 2). As expected, the 2-substituted de-

rivatives attenuated CYP3A4 inhibition, with slightly reduced TRPM8

inhibition, compared with compound 13t. Although the amino and

methoxy derivatives (13v, 13w) still had off-target effects, such as

CYP3A4 induction or formation of reactive metabolites, substitution

with methyl (13x) satisﬁed our requirements.

a

TRP Channel

IC₅₀

hTRPM8

rTRPM8

hTRPA1

hTRPV1

hTRPV4

0.21 ± 0.05 µM

0.08 ± 0.01 µM

>30 µM

a

Each value is presented as the mean ± SEM (n = 3).

Table 4

Effects of compound 13x and KPR-2579 on icilin-induced WDS in rats.

Compound^a

Dose (mg/

kg)

Time (h) prior to icilin

administration

%

inhibition^b

13x

3

1

4

1

4

100

96

KPR-2579

10

100

97

a

Compounds were orally administered.

b

Each value is presented as the mean ± SEM (n = 5).

Table 5

Effects of compound 13x and KPR-2579 on the voiding behavior in rats exposed

to cold.^a

Temperature

Compound^b

Dose

(mg/

kg)

Mean

Voiding

Total

The pharmacological proﬁle of compound 13x is shown in Table 3–5

and Fig. 2. Potent inhibition of rat TRPM8 and excellent selectivity for

TRPM8 over other TRP channels were conﬁrmed by in vitro experiments

(Table 3). Icilin-induced WDS in rats, a widely used index for evaluating

TRPM8 antagonism in vivo, was suppressed by more than 95% with

compound 13x (3 mg/kg) administered 1 or 4 h prior to icilin treatment

(Table 4). Considering the effective dose, compound 13x was estimated

voided

volume

(mL)

frequency

voided

volume

(mL)

Room temp.

Vehicle

13x

1.05 ±

0.09

4.0 ± 0.6

3.89 ±

0.25

(23–26 ^◦C)

4 ^◦

C

0.57 ±

0.05^##

0.76 ±

0.07*

10.1 ±

5.57 ±

0.27^##

5.14 ±

0.25

0.6^##

3

7.4 ± 0.9*

KPR-2579

10

0.75 ±

0.05*

8.1 ± 0.7*

5.81 ±

0.25

Table 2

SARs for 2-substituted nicotinamide derivatives.

a

Each value is presented as the mean ± SEM (n = 10).

Compounds were orally administered. ^##P < 0.01 vs. vehicle-treated rats

b

under the condition of room temperature, *P < 0.05 vs. vehicle-treated rats

under the condition of 4℃.

Compound

to be three-fold more potent than KPR-2579. To investigate bladder

function, we evaluated the effect of the compounds on cold-induced

frequent voiding in rats. We have previously reported a suppressive ef-

fect of KPR-2579 on the frequency of distension-induced rhythmic

bladder contractions in anesthetized rats; however, additional in vivo

experiments under non-anesthetized and noninvasive conditions are

required to properly evaluate the potency of compounds. We have

recently reported that frequent urination induced by cold temperatures

in rats can be noninvasively recorded by a frequency volume chart³⁷and

this method was considered to be useful to evaluate cold-induced OAB

symptoms. As shown in Fig. 2A, 2B, and Table 5, rats exposed to cold

showed a signiﬁcant increase in voiding frequency, accompanied by a

decrease in the mean voided volume. Pretreatment with compound 13x

(3 mg/kg) suppressed the frequent voiding without affecting the total

voided volume, and the effect appeared to be maintained for 4 h as

indicated by the frequency volume chart (Fig. 2C). This effect was

comparable to a dose of 10 mg/kg of KPR-2579 (Fig. 2D), which was

consistent with the three-fold improvement in the potency of compound

13x observed in the WDS experiment.

a

R^c

logD_7.4

hTRPM8

IC₅₀

CYP3A4

inhibition

(%, 10

µM)

CYP3A4

induction

(fold

dGSH

adduct

(%)^c

(µM)^b

change of

mRNA, 3

µM)

KPR-2579

13t

n/a^d

H

4.31

3.68

4.09

4.11

3.81

0.08

0.11

0.17

0.38

0.21

27

65

18

22

17

5.6

163

0

e

–

13v

NH₂

OMe

Me

1.8

42

0

13w

10.1

1.1

13x

0

a

Calculated using ChamAxon Marvin calculator plugin.

b

Each IC₅₀value represents the mean of at least two independent

experiments.

c

The value was quantiﬁed by ﬂuorescence of the adducts compared to that of

the diclofenac adduct.

d

e

Not applicable.

Not tested.

5

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

Figure 2. Representative traces of the frequency volume chart in rats given vehicle under the condition of room temperature (A) and rats given vehicle (B),

compound 13x 3 mg/kg (C), and KPR-2579 10 mg/kg (D) under the condition of 4 ^◦C.

Compound 13x had a potent effect on the voiding behavior, and

therefore we investigated the safety margin for CYP3A4 induction. As

described above, the effective dose for frequent voiding with cold

exposure was 3 mg/kg, which was the same dose of 13x that inhibited

more than 95% of icilin-induced WDS at 4 h. Table 6 shows the phar-

macokinetic proﬁle of compound 13x. The C_max,uvalue for oral

administration of 3 mg/kg was calculated as 158 nM from the C_maxvalue

and the fraction unbound in rat plasma (0.149), and 30-fold of the

concentration was determined to be 4.7 µM. The change in CYP3A4

mRNA was 1.1-fold after incubation of 3 µM of compound 13x with

HepaRG® cells; that is, the increase in CYP3A4 mRNA was only 10% at

that concentration. Therefore, the mRNA expression at 4.7 µM was

estimated not to exceed the two-fold threshold for CYP3A4 induction.

With regard to the effect in the CNS, no convulsions were observed in

mice after oral administration of 300 mg/kg. The cerebral and plasma

concentrations at 4 h after administration were 25.4 and 53.5 µM,

respectively, and the brain/plasma ratio was determined to be 0.48.

Although these data cannot be directly compared with the data for KPR-

2579 in rats, compound 13x achieved a lower brain concentration and

lower brain/plasma distribution ratio compared with KPR-2579, which

might contribute to the avoidance of CNS-related side effects.

kg for 4 h, and the potency was estimated to be three-fold stronger than

for KPR-2579. From these ﬁndings, we believe that compound 13x is a

potential clinical candidate; however, further safety investigations are

needed before clinical trials.

5. Experimental

5.1. Chemistry

All reagents and solvents were purchased from commercial sources

and used without further puriﬁcation. Reactions were monitored by thin

layer chromatography (TLC) using Merck Kieselgel 60 F₂₅₄plates or Fuji

Silysia Chemical Ltd. Chromatorex NH-TLC plates. Flash column chro-

matography 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 (δ) are reported in parts per million

(ppm) units using tetramethylsilane as an internal standard. For the

experiments at elevated temperature, the spectra were recorded on a

JEOL JNM-ECX400 spectrometer at 400 MHz, and ethylene glycol-d₆

was used as the internal reference (δ 3.57). Data are presented as fol-

lows; chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; 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 integration. ¹³C NMR

spectra were recorded on a Bruker AV400M spectrometer at 100 MHz

with complete proton decoupling, and chemical shifts (δ) are reported in

ppm with the solvent as the internal reference (δ 77.16 in CDCl₃; δ 39.52

in DMSO‑d₆; δ 67.21 in THF-d₈).³⁸High resolution mass spectra (HRMS)

were recorded on an Agilent Technologies 6520 Accurate-Mass Q-TOF

instrument using electrospray ionization (ESI, positive or negative ion

mode). HPLC analysis for tested compounds was performed on a Shi-

madzu LC-VP or Prominence series instrument. Chemical purity was

determined under the following analytical condition; L-column2

(Chemicals Evaluation and Research Institute, Japan), 3 µm, 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%). Optical rotations were recorded on a HORIBA SEPA-300 using a

3.5 mm × 50 mm cell. Data are presented as follows; speciﬁc rotation,

concentration (c = g/100 mL), and solvent.

4. Conclusion

We identiﬁed compound 13x with a reduced potential for the risk of

adverse effects by structural modiﬁcation of KPR-2579. The formation of

the reactive metabolite was prevented by structural modiﬁcation of the

N-phenethyl group to an N-indanyl group. Lowering the logD_7.4value of

the molecule provided a wide safety margin for the prevention of con-

vulsions and CYP3A4 induction. The unexpected inhibition of CYP3A4

was regulated by steric hindrance around the nitrogen atom of the

pyridine moiety. With regard to the voiding function, compound 13x

signiﬁcantly suppressed cold-induced frequent urination in rats at 3 mg/

Table 6

Pharmacokinetic parameters of compound 13x in rats.^a

i.v.

p.o.

Dose (mg/kg)

C_max(ng/mL)

(µM)

1

3

n/a^b

466

1.06

40

T

_max(min)

n/a

5.1.1. General procedure for preparation of imine 2a–2c

AUC_inf(ng⋅min/mL)

_1/2(min)

CL_tot(mL/min/kg)

40,454

64

66,360

n/a

55

Titanium ethoxide (1.4 equiv) was added to a mixture of benzalde-

hyde and (S)-tert-butanesulﬁnamide (1.05 equiv) in THF. The mixture

was stirred for 10 h at room temperature, and then partitioned between

EtOAc and brine. The insoluble solid was removed by ﬁltration, and

organic layer of the ﬁltrate was washed with brine. The organic layer

was dried over anhydrous MgSO₄, and concentrated in vacuo. The res-

idue was puriﬁed by silica gel column chromatography.

t

25

V

_ss(mL/kg)

1,893

n/a

F (%)

a

Each parameter was calculated from the mean plasma concentration of three

animals.

b

Not applicable.

6

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

5.1.2. (S,E)-2-Methyl-N-(3,4,5-triﬂuorobenzylidene)propane-2-

sulﬁnamide (2a)

methylmagnesium bromide in Et₂O (1.59 mL), and CH₂Cl₂(10 mL) ac-

cording to the general procedure. The crude product was puriﬁed using

silica gel column chromatography (eluent EtOAc:Hexane = 1:2–1:1) to

afford 3b (730 mg, 82%) as a colorless solid. ¹H NMR (CDCl₃) δ 1.22 (s,

9H), 1.57 (d, J = 6.8 Hz, 3H), 3.33 (d, J = 4.0 Hz, 1H), 4.84–4.92 (m,

1H), 6.80–6.90 (m, 2H); ¹³C NMR (CDCl₃) δ 22.6, 23.8, 49.2, 56.1, 104.5

(dd, J = 27.9, 21.3 Hz), 109.3 (dt, J = 24.2, 3.0 Hz), 134.2 (dd, J = 11.7,

8.1 Hz), 145.1 (ddd, J = 245.0, 13.2, 3.7 Hz), 150.5 (ddd, J = 251.6,

14.7, 12.8 Hz), 157.8 (ddd, J = 245.8, 10.3, 2.9 Hz); HRMS calcd for

The title compound was prepared from 3,4,5-triﬂuorobenzaldehyde

(1a, 640 mg), (S)-tert-butanesulﬁnamide (509 mg), titanium ethoxide

(1.277 g), and THF (10 mL) according to the general procedure. The

crude product was puriﬁed using silica gel column chromatography

(eluent EtOAc:Hexane = 1:10) to afford 2a (910 mg, 87%) as a colorless

solid. ¹H NMR (CDCl₃) δ 1.27 (s, 9H), 7.45–7.55 (m, 2H), 8.45 (s, 1H);

¹³C NMR (CDCl₃) δ 22.7, 58.4, 113.4 (dd, J = 16.0, 6.1 Hz), 130.0 (td, J

= 7.1, 4.5 Hz), 142.6 (dt, J = 259.0, 15.4 Hz), 151.8 (ddd, J = 252.6,

10.6, 3.6 Hz), 159.9 (dd, J = 4.1, 2.9 Hz); mp 85 ^◦C.

C

₁₂H₁₇F₃NOS (M + H)⁺280.0977, found 280.0979; mp 99 ^◦C.

5.1.8. (S)-N-[(R)-1-(3,5-Diﬂuoro-2-methylphenyl)ethyl]-2-

5.1.3. (S,E)-2-Methyl-N-(2,3,5-triﬂuorobenzylidene)propane-2-

sulﬁnamide (2b)

methylpropane-2-sulﬁnamide (3c)

The title compound was prepared from 2c (608 mg), 3 mol/L

methylmagnesium bromide in Et₂O (1.17 mL), and CH₂Cl₂(10 mL) ac-

cording to the general procedure. The crude product was puriﬁed using

silica gel column chromatography (eluent EtOAc:Hexane = 1:2–1:1) to

afford 3c (509 mg, 79%) as a colorless oil. ¹H NMR (CDCl₃) δ 1.22 (s,

9H), 1.48 (d, J = 6.8 Hz, 3H), 2.22–2.24 (m, 3H), 3.27 (d, J = 2.5 Hz,

1H), 4.78–4.86 (m, 1H), 6.66–6.74 (m, 1H), 6.89–6.94 (m, 1H); ¹³C

NMR (CDCl₃) δ 9.9 (d, J = 5.9 Hz), 22.7, 24.1, 50.1–50.2 (m), 55.9,

102.4 (dd, J = 27.6, 25.2 Hz), 108.9 (dd, J = 22.3, 3.4 Hz), 118.6 (dd, J

= 16.6, 3.6 Hz), 145.3 (dd, J = 8.0, 5.1 Hz), 161.2 (dd, J = 245.0, 13.2

Hz), 161.2 (dd, J = 245.0, 11.7 Hz); HRMS calcd for C₁₃H₂₀F₂NOS (M +

H)⁺276.1228, found 276.1230.

The title compound was prepared from 2,3,5-triﬂuorobenzaldehyde

(1b, 640 mg), (S)-tert-butanesulﬁnamide (509 mg), titanium ethoxide

(1.277 g), and THF (10 mL) according to the general procedure. The

crude product was puriﬁed using silica gel column chromatography

(eluent EtOAc:Hexane = 1:10) to afford 2b (906 mg, 86%) as a colorless

solid. ¹H NMR (CDCl₃) δ 1.28 (s, 9H), 7.06–7.14 (m, 1H), 7.45–7.51 (m,

1H), 8.85 (d, J = 2.0 Hz, 1H); ¹³C NMR (CDCl₃) δ 22.8, 58.6, 109.3 (dd,

J = 24.5, 4.0 Hz), 109.4 (dd, J = 28.0, 20.5 Hz), 124.3 (td, J = 8.8, 1.4

Hz), 147.6 (ddd, J = 255.7, 13.5, 3.8 Hz), 150.9 (ddd, J = 253.1, 14.0,

12.1 Hz), 154.8–155.0 (m), 157.8 (ddd, J = 247.2, 10.0, 3.2 Hz); mp

95 ^◦C.

5.1.4. (S,E)-N-(3,5-Diﬂuoro-2-methylbenzylidene)-2-methylpropane-2-

sulﬁnamide (2c)

5.1.9. General procedure for preparation of 7 g and 7i–7m

Titanium ethoxide (2.74 g, 12 mmol) was added to a mixture of

ketone (6 mmol) and (R)-tert-butanesulﬁnamide (764 mg, 6.3 mmol) in

THF (15 mL). The mixture was stirred for 5 h under reﬂux, and then

cooled with ice. Sodium borohydride (454 mg, 12 mmol) was added to

the mixture, then stirred for 10 min at 0 ^◦C and for 12 h at room tem-

perature. Ethyl acetate and water were added to the mixture, and the

formed precipitate was removed by ﬁltration. The ﬁltrate was concen-

trated in vacuo, and the residue was puriﬁed using silica gel column

chromatography.

The title compound was prepared from 3,5-diﬂuoro-2-methylbenzal-

dehyde (1c, 468 mg), (S)-tert-butanesulﬁnamide (381 mg), titanium

ethoxide (957 mg), and THF (10 mL) according to the general proced-

ure. The crude product was puriﬁed using silica gel column chroma-

tography (eluent EtOAc:Hexane = 1:10) to afford 2c (718 mg, 92%) as a

colorless oil. ¹H NMR (CDCl₃) δ 1.28 (s, 9H), 2.45 (br s, 3H), 6.94 (ddd, J

= 9.4, 8.5, 2.7 Hz, 1H), 7.48 (ddd, J = 9.0, 2.5, 1.5 Hz, 1H), 8.81 (d, J =

2.0 Hz, 1H); ¹³C NMR (CDCl₃) δ 10.1 (d, J = 5.2 Hz), 22.7, 58.2, 107.0

(dd, J = 27.8, 25.7 Hz), 110.9 (dd, J = 12.0, 3.7 Hz), 122.7 (dd, J = 17.5,

4.0 Hz), 134.8 (dd, J = 8.7, 5.9 Hz), 159.6 (dd, J = 4.4, 3.0 Hz), 160.8

(dd, J = 247.2, 13.1 Hz), 161.6 (dd, J = 247.2, 11.5 Hz).

5.1.10. (R)-N-((R)-6,7-Dihydro-5H-cyclopenta[b]pyridin-5-yl)-2-

methylpropane-2-sulﬁnamide (7g)

The title compound was prepared from 6,7-dihydro-5H-cyclopenta

[b]pyridin-5-one (5g, 799 mg) according to the general procedure. The

crude product was puriﬁed using silica gel column chromatography

(eluent EtOAc:Hexane = 2:1) to afford 7g (146 mg, 10%) as a pale

yellow solid. ¹H NMR (CDCl₃) δ 1.26 (s, 9H), 1.99–2.10 (m, 1H),

2.65–2.75 (m, 1H), 3.10–3.20 (m, 1H), 3.45 (ddd, J = 18.6, 9.3, 3.5 Hz,

1H), 3.52 (d, J = 9.0 Hz, 1H), 4.98 (q, J = 8.3 Hz, 1H), 7.30–7.35 (m,

1H), 8.30 (d, J = 7.5 Hz, 1H), 8.53 (d, J = 5.8 Hz, 1H); ¹³C NMR (CDCl₃)

δ 22.8, 30.8, 32.9, 56.4, 60.9, 123.6, 137.2, 141.2, 147.6, 162.1; HRMS

calcd for C₁₂H₁₉N₂OS (M + H)⁺239.1213, found 239.1211; mp

188–195 ^◦C (dec.)

5.1.5. General procedure for preparation of 3a–3c

A solution of methylmagnesium bromide in Et₂O (3 mol/L, 1.5

equiv) was added to a solution of imine in CH₂Cl₂at 0 ^◦C. The mixture

was stirred for 1 h at that temperature and for 2 h at room temperature.

A saturated aqueous solution of ammonium chloride and EtOAc were

added to the mixture cooled with ice, then the organic layer was washed

with brine, and dried over anhydrous MgSO₄. The solution was

concentrated in vacuo, and the residue was puriﬁed using silica gel

column chromatography.

5.1.6. (S)-2-Methyl-N-{(R)-1-(3,4,5-triﬂuorophenyl)ethyl}propane-2-

sulﬁnamide (3a)

5.1.11. (R)-N-((R)-6-Fluoroindan-1-yl)-2-methylpropane-2-sulﬁnamide

(7i)

The title compound was prepared from 2a (828 mg), 3 mol/L

methylmagnesium bromide in Et₂O (1.57 mL), and CH₂Cl₂(10 mL) ac-

cording to the general procedure. The crude product was puriﬁed using

silica gel column chromatography (eluent EtOAc:Hexane = 1:1) to

afford 3a (742 mg, 85%) as a colorless solid. ¹H NMR (CDCl₃) δ 1.22 (s,

9H), 1.49 (d, J = 6.8 Hz, 3H), 3.29 (d, J = 2.3 Hz, 1H), 4.52 (ddd, J =

13.3, 6.6, 2.9 Hz, 1H), 6.92–7.01 (m, 2H); ¹³C NMR (CDCl₃) δ 22.6, 25.1,

53.6, 55.9, 111.1 (dd, J = 15.4, 5.9 Hz), 139.1 (dt, J = 251.6, 15.4 Hz),

140.0 (td, J = 6.6, 4.4 Hz), 151.4 (ddd, J = 250.1, 10.3, 3.7 Hz); HRMS

calcd for C₁₂H₁₇F₃NOS (M + H)⁺280.0977, found 280.0978; mp 74 ^◦C.

The title compound was prepared from 6-ﬂuoroindan-1-one (5i, 901

mg) according to the general procedure. The crude product was puriﬁed

using silica gel column chromatography (eluent EtOAc:Hexane = 1:2) to

afford 7i (373 mg, 24%) as a colorless solid. ¹H NMR (CDCl₃) δ 1.24 (s,

9H), 1.97–2.08 (m, 1H), 2.47–2.57 (m, 1H), 2.73–2.83 (m, 1H), 2.94

(ddd, J = 15.6, 8.8, 4.0 Hz, 1H), 3.45 (d, J = 6.5 Hz, 1H), 4.88 (q, J = 7.0

Hz, 1H), 6.93 (td, J = 8.7, 2.5 Hz, 1H), 7.16 (dd, J = 8.4, 5.1 Hz, 1H),

7.29 (dd, J = 8.7, 2.4 Hz, 1H); ¹³C NMR (CDCl₃) δ 22.8, 29.4, 35.8, 56.0,

61.8 (d, J = 2.2 Hz), 112.1 (d, J = 22.7 Hz), 115.3 (d, J = 22.0 Hz), 125.8

(d, J = 8.8 Hz), 138.8 (d, J = 2.2 Hz), 145.8 (d, J = 7.3 Hz), 162.4 (d, J =

244.3 Hz); HRMS calcd for C₁₃H₁₉FNOS (M + H)⁺256.1166, found

256.1165; mp 172 ^◦C.

5.1.7. (S)-2-Methyl-N-{(R)-1-(2,3,5-triﬂuorophenyl)ethyl}propane-2-

sulﬁnamide (3b)

The title compound was prepared from 2b (837 mg), 3 mol/L

7

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

5.1.12. (R)-N-((R)-6-Chloroindan-1-yl)-2-methylpropane-2-sulﬁnamide

(7j)

= 1:2) to afford 7n (198 mg, 11%) as a pale yellow solid. ¹H NMR

(CDCl₃) δ 1.24 (s, 9H), 1.99–2.09 (m, 1H), 2.49–2.58 (m, 1H), 2.73–2.83

(m, 1H), 3.01 (ddd, J = 16.1, 8.6, 4.0 Hz, 1H), 3.45 (d, J = 7.0 Hz, 1H),

The title compound was prepared from 6-chloroindan-1-one (5j,

1000 mg) according to the general procedure. The crude product was

puriﬁed using silica gel column chromatography (eluent EtOAc:Hexane

= 1:2) to afford 7j (408 mg, 25%) as a colorless solid. ¹H NMR (CDCl₃) δ

1.24 (s, 9H), 1.97–2.07 (m, 1H), 2.45–2.55 (m, 1H), 2.73–2.83 (m, 1H),

2.95 (ddd, J = 16.1, 8.5, 4.3 Hz, 1H), 3.44 (d, J = 6.5 Hz, 1H), 4.88 (q, J

= 6.9 Hz, 1H), 7.15 (d, J = 8.0 Hz, 1H), 7.21 (dd, J = 8.0, 1.5 Hz, 1H),

7.54–7.56 (m, 1H); ¹³C NMR (CDCl₃) δ 22.8, 29.6, 35.5, 56.0, 61.6,

125.2, 126.0, 128.4, 132.8, 141.9, 145.7; HRMS calcd for C₁₃H₁₉ClNOS

(M + H)⁺272.0870, found 272.0871; mp 163 ^◦C.

4.89 (q, J = 7.2 Hz, 1H), 6.97 (dd, J = 8.5, 1.2 Hz, 1H), 7.39 (s, 1H); ¹³

C

NMR (CDCl₃) δ 22.8, 25.7, 35.1, 56.1, 61.9 (d, J = 2.2 Hz), 115.7 (d, J =

23.5 Hz), 121.3 (d, J = 3.7 Hz), 128.2 (d, J = 19.1 Hz), 133.7 (d, J = 8.8

Hz), 148.4 (d, J = 6.6 Hz), 158.8 (d, J = 250.9 Hz); HRMS calcd for

C

₁₃H₁₈ClFNOS (M + H)⁺290.0776, found 290.0779; mp 127 ^◦C.

5.1.17. General procedure for preparation of 4a–4c, 8g and 8i–8n

A solution of HCl in 1,4-dioxane (4 mol/L) was added to a solution of

sulﬁnamide in methanol at room temperature. The mixture was stirred

for 1 h, then concentrated in vacuo, and the residue was suspended in

Et₂O. The insoluble solid was collected by ﬁltration and dried under

reduced pressure to afford amine hydrochloride.

5.1.13. (R)-N-((R)-4-Fluoroindan-1-yl)-2-methylpropane-2-sulﬁnamide

(7k)

The title compound was prepared from 4-ﬂuoroindan-1-one (5k,

901 mg) according to the general procedure. The crude product was

puriﬁed using silica gel column chromatography (eluent EtOAc:Hexane

= 1:2) to afford 7k (386 mg, 25%) as a colorless solid. ¹H NMR (CDCl₃) δ

1.23 (s, 9H), 1.98–2.08 (m, 1H), 2.47–2.57 (m, 1H), 2.78–2.88 (m, 1H),

3.05 (ddd, J = 16.1, 8.6, 4.4 Hz, 1H), 3.43 (d, J = 6.4 Hz, 1H), 4.93 (q, J

= 6.9 Hz, 1H), 6.91–6.97 (m, 1H), 7.18–7.25 (m, 1H), 7.36 (d, J = 7.5

Hz, 1H); ¹³C NMR (CDCl₃) δ 22.8, 26.0, 35.0, 56.0, 61.8 (d, J = 2.2 Hz),

114.8 (d, J = 20.5 Hz), 120.7 (d, J = 2.9 Hz), 129.1 (d, J = 6.6 Hz), 129.6

(d, J = 18.3 Hz), 147.3 (d, J = 5.1 Hz), 159.4 (d, J = 247.2 Hz); HRMS

calcd for C₁₃H₁₉FNOS (M + H)⁺256.1166, found 256.1165; mp 148 ^◦C.

5.1.18. (R)-1-(3,4,5-Triﬂuorophenyl)ethylamine hydrochloride (4a)

The title compound was prepared from 3a (677 mg), 4 mol/L HCl in

1,4-dioxane (1.21 mL), and methanol (5 mL) according to the general

procedure. Compound 4a (390 mg, 76%); colorless solid; ¹H NMR

(DMSO‑d₆) δ 1.49 (d, J = 6.8 Hz, 3H), 4.44 (q, J = 6.8 Hz, 1H), 7.52–7.61

(m, 2H), 8.53 (br s, 3H); ¹³C NMR (DMSO‑d₆) δ 20.3, 49.0, 112.2 (dd, J

= 16.4, 5.6 Hz), 136.7 (td, J = 7.6, 4.4 Hz), 138.4 (dt, J = 249.4, 15.4

Hz), 150.1 (ddd, J = 247.2, 9.5, 3.7 Hz); HRMS calcd for C₈H₉F₃N (M +

H)⁺176.0682, found 176.0677; mp 213 ^◦C.

5.1.19. (R)-1-(2,3,5-Triﬂuorophenyl)ethylamine hydrochloride (4b)

The title compound was prepared from 3b (663 mg), 4 mol/L HCl in

1,4-dioxane (1.19 mL), and methanol (5 mL) according to the general

procedure. Compound 4b (350 mg, 70%); colorless solid; ¹H NMR

(DMSO‑d₆) δ 1.53 (d, J = 6.8 Hz, 3H), 4.67 (q, J = 6.8 Hz, 1H), 7.42–7.49

(m, 1H), 7.59–7.68 (m, 1H), 8.61 (br s, 3H); ¹³C NMR (DMSO‑d₆) δ 19.6,

43.3, 106.0 (dd, J = 28.6, 21.3 Hz), 110.2 (d, J = 24.9 Hz), 129.8 (dd, J

= 12.2, 9.5 Hz), 144.0 (ddd, J = 244.3, 13.2, 3.9 Hz), 149.5 (dt, J =

248.7, 13.9 Hz), 157.2 (ddd, J = 243.6, 11.2, 3.0 Hz); HRMS calcd for

C₈H₉F₃N (M + H)⁺176.0682, found 176.0677; mp 203 ^◦C.

5.1.14. (R)-N-((R)-4-Chloroindan-1-yl)-2-methylpropane-2-sulﬁnamide

(7l)

The title compound was prepared from 4-chloroindan-1-one (5l,

1000 mg) according to the general procedure. The crude product was

puriﬁed using silica gel column chromatography (eluent EtOAc:Hexane

= 1:2) to afford 7l (271 mg, 17%) as a colorless solid. ¹H NMR (CDCl₃) δ

1.23 (s, 9H), 1.96–2.06 (m, 1H), 2.47–2.56 (m, 1H), 2.80–2.89 (m, 1H),

3.06 (ddd, J = 16.5, 8.8, 4.3 Hz, 1H), 3.44 (d, J = 6.8 Hz, 1H), 4.95 (q, J

= 7.0 Hz, 1H), 7.16–7.26 (m, 2H), 7.48 (d, J = 7.3 Hz, 1H); ¹³C NMR

(CDCl₃) δ 22.8, 29.4, 34.4, 56.0, 62.4, 123.4, 128.3, 128.7, 131.1, 141.7,

145.9; HRMS calcd for C₁₃H₁₉ClNOS (M + H)⁺272.0870, found

272.0871; mp 183 ^◦C.

5.1.20. (R)-1-(3,5-Diﬂuoro-2-methylphenyl)ethylamine hydrochloride

(4c)

The title compound was prepared from 3c (472 mg), 4 mol/L HCl in

1,4-dioxane (0.86 mL), and methanol (4 mL) according to the general

procedure. Compound 4c (334 mg, 94%); colorless solid; ¹H NMR

(DMSO‑d₆) δ 1.46 (d, J = 6.8 Hz, 3H), 2.21 (s, 3H), 4.61 (q, J = 6.8 Hz,

1H), 7.22–7.29 (m, 1H), 7.32–7.38 (m, 1H), 8.47 (br s, 3H); ¹³C NMR

(DMSO‑d₆) δ 9.5 (d, J = 5.1 Hz), 20.1, 46.1, 103.2 (dd, J = 27.9, 25.7

Hz), 109.0 (dd, J = 22.7, 3.7 Hz), 118.7 (dd, J = 16.9, 3.7 Hz), 141.8

(dd, J = 8.6, 5.5 Hz), 160.3 (dd, J = 244.3, 12.5 Hz), 160.4 (dd, J =

243.6, 14.0 Hz); HRMS calcd for C₉H₁₂F₂N (M + H)⁺172.0932, found

172.0928; mp 245 ^◦C.

5.1.15. (R)-N-((R)-4,6-Diﬂuoroindan-1-yl)-2-methylpropane-2-

sulﬁnamide (7m)

The title compound was prepared from 4,6-diﬂuoroindan-1-one (5m,

1.01 g) according to the general procedure. The crude product was

puriﬁed using silica gel column chromatography (eluent EtOAc:Hexane

= 1:2) to afford 7m (462 mg, 28%) as a pale yellow solid. ¹H NMR

(CDCl₃) δ 1.24 (s, 9H), 1.98–2.08 (m, 1H), 2.50–2.60 (m, 1H), 2.72–2.82

(m, 1H), 3.00 (ddd, J = 16.0, 8.7, 3.8 Hz, 1H), 3.46 (d, J = 7.3 Hz, 1H),

4.88 (q, J = 7.3 Hz, 1H), 6.69 (td, J = 9.0, 2.2 Hz, 1H), 7.16 (dd, J = 8.0,

1.8 Hz, 1H); ¹³C NMR (CDCl₃) δ 22.8, 25.5, 35.3, 56.1, 62.2 (t, J = 2.2

Hz), 103.3 (dd, J = 26.4, 24.7 Hz), 108.2 (dd, J = 22.7, 3.7 Hz), 125.0

(dd, J = 19.0, 3.0 Hz), 148.2 (dd, J = 8.8, 6.6 Hz), 158.8 (dd, J = 250.2,

12.5 Hz), 162.7 (dd, J = 247.6, 10.3 Hz); HRMS calcd for C₁₃H₁₈F₂NOS

(M + H)⁺274.1072, found 274.1072; mp 141 ^◦C.

5.1.21. (R)-6,7-Dihydro-5H-cyclopenta[b]pyridin-5-ylamine

dihydrochloride (8g)

The title compound was prepared from 7g (125 mg), 4 mol/L HCl in

1,4-dioxane (0.39 mL), and methanol (6 mL) according to the general

1

procedure. Compound 8g (100 mg, 92%); pale yellow solid; H NMR

5.1.16. (R)-N-((R)-6-Chloro-4-ﬂuoroindan-1-yl)-2-methylpropane-2-

sulﬁnamide (7n)

(DMSO‑d₆) δ 2.07–2.17 (m, 1H), 2.52–2.63 (m, 1H), 3.03–3.13 (m, 1H),

3.28 (ddd, J = 17.3, 9.2, 5.5 Hz, 1H), 4.80–4.89 (m, 1H), 7.60 (dd, J =

7.6, 5.5 Hz, 1H), 8.36 (d, J = 7.6 Hz, 1H), 8.65 (dd, J = 3, 1.0 Hz, 1H),

8.75 (br s, 3H); ¹³C NMR (DMSO‑d₆) δ 28.3, 30.1, 52.5, 123.8, 136.6,

139.5, 144.7, 161.4; HRMS calcd for C₈H₁₁N₂(M + H)⁺135.0917,

found 135.0914; mp 208 ^◦C (dec.).

Titanium ethoxide (1.44 g) was added to a mixture of 6-chloro-4-ﬂu-

oroindan-1-one (5n, 1.11 g) and (R)-tert-butanesulﬁnamide (764 mg) in

THF (15 mL). The mixture was stirred for 4 h under reﬂux, and then

cooled with ice. An aqueous solution of sodium borohydride (227 mg in

0.6 mL) was added to the mixture, then stirred for 10 min at 0 ^◦C and for

5 h at room temperature. Methanol (1.2 mL) and water (1.2 mL) were

added to the mixture, and the formed precipitate was removed by

ﬁltration. The ﬁltrate was concentrated in vacuo, and the residue was

puriﬁed using silica gel column chromatography (eluent EtOAc:Hexane

5.1.22. (R)-6-Fluoroindan-1-ylamine hydrochloride (8i)

The title compound was prepared from 7i (313 mg), 4 mol/L HCl in

1,4-dioxane (0.61 mL), and methanol (3 mL) according to the general

procedure. Compound 8i (212 mg, 92%); colorless solid; ¹H NMR

8

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

(DMSO‑d₆) δ 1.96–2.06 (m, 1H), 2.45–2.55 (m, 1H), 2.79–2.89 (m, 1H),

2.97–3.07 (m, 1H), 4.72 (t, J = 7.0 Hz, 1H), 7.17 (td, J = 8.8, 2.5 Hz,

1H), 7.36 (dd, J = 8.3, 5.3 Hz, 1H), 7.43 (dd, J = 9.1, 2.5 Hz, 1H), 8.45

(br s, 3H); ¹³C NMR (DMSO‑d₆) δ 29.2, 30.9, 54.5 (d, J = 2.2 Hz), 112.0

(d, J = 22.8 Hz), 115.9 (d, J = 22.0 Hz), 126.4 (d, J = 8.8 Hz), 139.8 (d,

J = 2.2 Hz), 141.7 (d, J = 8.1 Hz), 161.2 (d, J = 241.4 Hz); HRMS calcd

for C₉H₁₁FN (M + H)⁺152.0870, found 152.0865; mp 264 ^◦C.

determined due to sublimation.

5.1.28. General procedure of the Ugi reaction and deprotection

Amine (1 equiv.) and aldehyde (1 equiv.) were dissolved in MeOH.

When amine hydrochloride was used, an equivalent amount of Et₃N was

added. The mixture was stirred for 2 h at 50 ^◦C. After being cooled to

room temperature, carboxylic acid (1 equiv.) and 4-phenylcyclohenen-

1-yl isocyanide (1 equiv.) were added to the solution. The mixture was

stirred 5–12 h at 50–60 ^◦C, and then concentrated in vacuo. The residue

was suspended in a mixture of 1,4-dioxane and water, then added a

solution of HCl in 1,4-dioxane (4 mol/L). The mixture was stirred for 1 h

at room temperature. A saturated aqueous solution of NaHCO₃and

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

silica gel column chromatography.

5.1.23. (R)-6-Chloroindan-1-ylamine hydrochloride (8j)

The title compound was prepared from 7j (350 mg), 4 mol/L HCl in

1,4-dioxane (0.64 mL), and methanol (3 mL) according to the general

procedure. Compound 8j (243 mg, 93%); colorless solid; ¹H NMR

(DMSO‑d₆) δ 1.96–2.06 (m, 1H), 2.43–2.55 (m, 1H), 2.80–2.90 (m, 1H),

3.04 (ddd, J = 16.3, 8.8, 5.0 Hz, 1H), 4.67–4.74 (m, 1H), 7.33–7.41 (m,

2H), 7.69–7.72 (m, 1H), 8.55 (br s, 3H); ¹³C NMR (DMSO‑d₆) δ 29.5,

30.6, 54.3, 125.2, 126.6, 128.8, 131.0, 141.8, 143.0; HRMS calcd for

C₉H₁₁ClN (M + H)⁺168.0575, found 168.0570; mp 262 ^◦C (dec.).

5.1.29. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3,4,5-triﬂuorophenyl)

ethyl]benzamide (13a), N-((S)-Carbamoylphenylmethyl)-N-[(R)-1-(3,4,5-

triﬂuorophenyl)ethyl]benzamide (14a)

5.1.24. (R)-4-Fluoroindan-1-ylamine hydrochloride (8k)

The title compound was prepared from 7k (328 mg), 4 mol/L HCl in

1,4-dioxane (0.64 mL), and methanol (3 mL) according to the general

procedure. Compound 8k (219 mg, 91%); colorless solid; ¹H NMR

(DMSO‑d₆) δ 2.00–2.10 (m, 1H), 2.47–2.57 (m, 1H), 2.85–2.95 (m, 1H),

3.09 (ddd, J = 16.4, 8.7, 5.2 Hz, 1H), 4.72–4.80 (m, 1H), 7.18 (t, J = 8.8

The title compounds were prepared from 4a (212 mg), benzaldehyde

(106 mg), triethylamine (139 µL), methanol (4 mL), benzoic acid (122

mg), 4-phenylcyclohenen-1-yl isocyanide (183 mg), 1,4-dioxane (3 mL),

water (0.75 mL) and 4 mol/L HCl in 1,4-dioxane (0.75 mL) according to

the general procedure. The crude product was puriﬁed using silica gel

column chromatography (eluent EtOAc:Hexane = 1:2–2:1) to afford 13a

(145 mg, 35%, colorless amorphous solid, low-polarity) and 14a (149

mg, 36%, colorless amorphous solid, high-polarity). 13a: ¹H NMR

(CDCl₃) δ 1.29 (d, J = 6.8 Hz, 3H), 4.73 (br s, 1H), 5.12 (br s, 1H), 5.36

Hz, 1H), 7.33–7.39 (m, 1H), 7.47 (d, J = 7.5 Hz, 1H), 8.56 (br s, 3H); ¹³

C

NMR (DMSO‑d₆) δ 26.0, 30.2, 54.6 (d, J = 2.2 Hz), 115.4 (d, J = 19.8

Hz), 121.4 (d, J = 3.7 Hz), 129.2 (d, J = 6.6 Hz), 130.0 (d, J = 19.1 Hz),

143.3 (d, J = 5.9 Hz), 158.6 (d, J = 245.0 Hz); HRMS calcd for C₉H₁₁FN

(M + H)⁺152.0870, found 152.0864; mp 263 ^◦C (dec.).

(br s, 1H), 5.75 (br s, 1H), 7.25–7.34 (m, 2H), 7.35–7.54 (m, 10H); ¹³

C

5.1.25. (R)-4-Chloroindan-1-ylamine hydrochloride (8l)

NMR (CDCl₃) δ 19.1 (br s), 56.8 (br s), 63.3 (br s), 112.1 (dd, J = 16.2,

5.9 Hz), 126.3, 128.7, 128.8, 129.0, 129.4, 130.2, 136.2, 136.4 (br s),

137.1 (br s), 139.1 (dt, J = 251.9, 15.3 Hz), 151.2 (ddd, J = 250.2, 9.5,

3.7 Hz), 171.3, 172.2; HRMS calcd for C₂₃H₁₉F₃N₂O₂(M ꢀ H)^ꢀ

411.1326, found 411.1325; chemical purity 99.2%, t_R14.9 min. 14a: ¹H

NMR (CDCl₃) δ 1.74 (br s, 3H), 4.52 (br s, 1H), 5.05 (br s, 1H), 5.45 (br s,

1H), 5.69 (br s, 1H), 6.62–6.74 (m, 2H), 7.18–7.28 (m, 5H), 7.45–7.55

(m, 5H); ¹³C NMR (CDCl₃) δ 17.4 (br s), 56.9, 63.3 (br s), 112.1 (dd, J =

16.1, 5.9 Hz), 126.0, 128.6, 128.8, 128.9, 129.1, 129.8, 135.6 (br s),

136.5 (br s), 138.9 (br d, J = 252.4 Hz), 151.2 (dd, J = 250.8, 8.5 Hz),

171.6, 172.2; HRMS calcd for C₂₃H₁₈F₃N₂O₂(M ꢀ H)^ꢀ411.1326, found

411.1326; chemical purity 96.6%, t_R14.4 min.

The title compound was prepared from 7l (239 mg), 4 mol/L HCl in

1,4-dioxane (0.44 mL), and methanol (6 mL) according to the general

procedure. Compound 8l (165 mg, 92%); colorless solid; ¹H NMR

(DMSO‑d₆) δ 1.98–2.08 (m, 1H), 2.46–2.56 (m, 1H), 2.86–2.95 (m, 1H),

3.09 (ddd, J = 16.6, 8.9, 5.3 Hz, 1H), 4.79 (dd, J = 8.0, 5.5 Hz, 1H), 7.35

(t, J = 7.8 Hz, 1H), 7.43 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H),

8.51 (br s, 3H); ¹³C NMR (DMSO‑d₆) δ 29.4, 55.1, 124.1, 128.8, 128.9,

129.9, 141.9, 142.1; HRMS calcd for C₉H₁₁ClN (M + H)⁺168.0575,

found 168.0574; The melting point could not be determined due to

sublimation.

5.1.26. (R)-4,6-Diﬂuoroindan-1-ylamine hydrochloride (8m)

The title compound was prepared from 7m (408 mg), 4 mol/L HCl in

1,4-dioxane (0.75 mL), and methanol (3 mL) according to the general

procedure. Compound 8m (290 mg, 95%); colorless solid; ¹H NMR

(DMSO‑d₆) δ 2.01–2.11 (m, 1H), 2.49–2.59 (m, 1H), 2.82–2.91 (m, 1H),

3.00–3.10 (m, 1H), 4.74–4.79 (m, 1H), 7.25 (td, J = 9.5, 2.2 Hz, 1H),

7.36 (dd, J = 8.5, 2.0 Hz, 1H), 8.54 (br s, 3H); ¹³C NMR (DMSO‑d₆) δ

25.6, 30.4, 54.6, 104.1 (dd, J = 27.1, 24.9 Hz), 108.7 (dd, J = 23.3, 3.8

Hz), 126.2 (dd, J = 19.1, 2.9 Hz), 144.2 (dd, J = 9.9, 7.7 Hz), 158.1 (dd,

J = 248.0, 13.2 Hz), 161.6 (dd, J = 244.3, 11.0 Hz); HRMS calcd for

C₉H₁₀F₂N (M + H)⁺170.0776, found 170.0772; The melting point could

not be determined due to sublimation.

5.1.30. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(2,3,5-triﬂuorophenyl)

ethyl]benzamide (13b), N-((S)-Carbamoylphenylmethyl)-N-[(R)-1-(2,3,5-

triﬂuorophenyl)ethyl]benzamide (14b)

The title compounds were prepared from 4b (212 mg), benzaldehyde

(106 mg), triethylamine (139 µL), methanol (4 mL), benzoic acid (122

mg), 4-phenylcyclohenen-1-yl isocyanide (183 mg), 1,4-dioxane (3 mL),

water (0.75 mL) and 4 mol/L HCl in 1,4-dioxane (0.75 mL) according to

the general procedure. The crude product was puriﬁed using silica gel

column chromatography (eluent EtOAc:Hexane = 1:2–2:1) to afford 13b

(112 mg, 27%, colorless solid, low-polarity) and 14b (118 mg, 29%,

colorless solid, high-polarity). 13b: ¹H NMR (CDCl₃) δ 1.61 (d, J = 7.0

Hz, 3H), 4.80 (br s, 1H), 5.28 (br s, 1H), 5.39 (q, J = 7.0 Hz, 1H), 6.73 (br

s, 1H), 6.89–6.97 (m, 1H), 7.05–7.12 (m, 1H), 7.30–7.42 (m, 3H),

7.43–7.52 (m, 5H), 7.54–7.59 (m, 2H); ¹³C NMR (CDCl₃) δ 17.8, 53.2,

63.6, 105.9 (dd, J = 27.3, 21.1 Hz), 110.2 (d, J = 24.2 Hz), 126.6, 127.5,

128.3, 128.8, 129.0, 129.8 (t, J = 10.3 Hz), 130.5, 136.1, 136.4, 146.3

(ddd, J = 246.5, 13.1, 3.9 Hz), 150.3 (ddd, J = 251.6, 14.7, 12.5 Hz),

157.7 (ddd, J = 246.5, 10.3, 3.0 Hz), 171.7, 173.0; HRMS calcd for

5.1.27. (R)-6-Chloro-4-ﬂuoroindan-1-ylamine hydrochloride (8n)

The title compound was prepared from 7n (131 mg), 4 mol/L HCl in

1,4-dioxane (0.23 mL), and methanol (3 mL) according to the general

procedure. Compound 8n (91 mg, 91%); colorless solid; ¹H NMR

(DMSO‑d₆) δ 2.00–2.11 (m, 1H), 2.45–2.58 (m, 1H), 2.83–2.92 (m, 1H),

3.06 (ddd, J = 16.3, 8.8, 5.0 Hz, 1H), 4.77 (dd, J = 7.5, 6.0 Hz, 1H), 7.43

(dd, J = 8.9, 1.6 Hz, 1H), 7.58–7.61 (m, 1H), 8.59 (br s, 3H); ¹³C NMR

(DMSO‑d₆) δ 25.8, 30.2, 54.4 (d, J = 2.2 Hz), 116.0 (d, J = 24.2 Hz),

121.8 (d, J = 2.9 Hz), 129.4 (d, J = 19.8 Hz), 132.1 (d, J = 9.8 Hz), 144.7

(d, J = 6.6 Hz), 158.2 (d, J = 248.7 Hz); HRMS calcd for C₉H₁₀ClFN (M

+ H)⁺186.0480, found 186.0476; The melting point could not be

C

₂₃H₁₈F₃N₂O₂(M ꢀ H)^ꢀ411.1326, found 411.1326; mp 222 ^◦C;

chemical purity 97.1%, t_R14.4 min. 14b: ¹H NMR (CDCl₃) δ 1.73 (d, J =

5.8 Hz, 3H), 4.49 (br s, 1H), 5.21–5.31 (m, 1H), 5.44 (br s, 1H), 5.52 (br

s, 1H), 6.54–6.63 (m, 1H), 6.79 (br s, 1H), 7.19 (s, 5H), 7.46–7.53 (m,

3H), 7.60–7.65 (m, 2H); ¹³C NMR (CDCl₃) δ 16.6 (br s), 52.1, 63.0 (br s),

9

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

104.7–105.6 (m), 109.6 (d, J = 25.7 Hz), 126.1, 128.3, 128.4, 128.6,

128.8, 129.7, 136.1 (br s), 136.4, 145.8 (br d, J = 245.0 Hz), 149.9 (dt, J

= 251.6, 14.0 Hz), 157.6 (ddd, J = 245.7, 10.3, 2.7 Hz), 171.4, 172.2;

HRMS calcd for C₂₃H₁₈F₃N₂O₂(M ꢀ H)^ꢀ411.1326, found 411.1325; mp

192 ^◦C; chemical purity 98.0%, t_R14.0 min.

methanol (4 mL), benzoic acid (244 mg), 4-phenylcyclohenen-1-yl iso-

cyanide (367 mg), 1,4-dioxane (6 mL), water (1.5 mL) and 4 mol/L HCl

in 1,4-dioxane (1.5 mL) according to the general procedure. The crude

product was puriﬁed using silica gel column chromatography (eluent

EtOAc:Hexane = 1:1–2:1) to afford 13e (253 mg, 33%, colorless

amorphous solid, low-polarity) and 14e (223 mg, 29%, colorless solid,

1

5.1.31. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3,5-diﬂuoro-2-

methylphenyl)ethyl]benzamide (13c), N-((S)-Carbamoylphenylmethyl)-N-

[(R)-1-(3,5-diﬂuoro-2-methylphenyl)ethyl]benzamide (14c)

high-polarity). 13e: H NMR (CDCl₃) δ 1.35–1.57 (m, 2H), 1.69–1.80

(m, 2H), 2.56–2.71 (m, 2H), 4.65 (s, 1H), 5.15–5.22 (m, 1H), 5.47 (br s,

1H), 5.86 (br s, 1H), 7.07 (d, J = 7.5 Hz, 1H), 7.22 (t, J = 7.4 Hz, 1H),

7.31–7.44 (m, 7H), 7.49–7.54 (m, 2H), 7.57–7.62 (m, 2H), 8.12 (d, J =

7.8 Hz, 1H); ¹³C NMR (CDCl₃) δ 22.5, 29.4, 31.1, 60.8, 63.3, 126.1,

126.7, 127.8, 128.4, 128.5, 128.81, 128.84, 129.2, 129.3, 129.8, 134.0,

136.6, 138.2, 138.5, 171.3, 172.9; HRMS calcd for C₂₅H₂₃N₂O₂(M ꢀ

H)^ꢀ383.1765, found 383.1761; chemical purity 97.6%, t_R14.8 min.

14e: ¹H NMR (CDCl₃) δ 1.44–1.56 (m, 1H), 1.92–2.02 (m, 1H),

2.13–2.36 (m, 2H), 2.61–2.70 (m, 1H), 2.78–2.89 (m, 1H), 4.51 (s, 1H),

5.20 (dd, J = 9.8, 6.8 Hz, 1H), 5.45 (br s, 1H), 6.07 (br s, 1H), 6.68–6.75

(m, 1H), 6.95–7.06 (m, 3H), 7.20–7.33 (m, 5H), 7.40–7.45 (m, 3H),

7.55–7.61 (m, 2H); ¹³C NMR (CDCl₃) δ 22.1, 29.6, 29.7, 60.7, 64.7,

125.6, 126.2, 127.4, 128.2, 128.7, 128.8, 129.0, 129.3, 129.4, 129.7,

133.3, 136.8, 137.2, 139.2, 172.6, 173.3; HRMS calcd for C₂₅H₂₃N₂O₂

(M ꢀ H)^ꢀ383.1765, found 383.1762; mp 212 ^◦C; chemical purity

99.3%, t_R14.7 min.

The title compounds were prepared from 4c (208 mg), benzaldehyde

(106 mg), triethylamine (139 µL), methanol (4 mL), benzoic acid (122

mg), 4-phenylcyclohenen-1-yl isocyanide (183 mg), 1,4-dioxane (3 mL),

water (0.75 mL) and 4 mol/L HCl in 1,4-dioxane (0.75 mL) according to

the general procedure. The crude product was puriﬁed using silica gel

column chromatography (eluent EtOAc:Hexane = 1:2–2:1) to afford 13c

(151 mg, 37%, colorless solid, low-polarity) and 14c (149 mg, 37%,

colorless solid, high-polarity). 13c: ¹H NMR (CDCl₃) δ 1.55 (d, J = 6.8

Hz, 3H), 2.00–2.03 (m, 3H), 4.75 (s, 1H), 5.16 (q, J = 6.8 Hz, 1H), 5.25

(br s, 1H), 6.21 (br s, 1H), 6.76–6.82 (m, 1H), 7.12–7.17 (m, 1H),

7.30–7.52 (m, 8H), 7.54–7.58 (m, 2H); ¹³C NMR (CDCl₃) δ 10.5 (d, J =

5.9 Hz), 20.0, 55.8, 63.4, 103.6 (dd, J = 27.9, 25.0 Hz), 110.4 (dd, J =

22.7, 3.7 Hz), 120.9 (dd, J = 16.5, 3.8 Hz), 126.8, 127.9, 128.5, 128.7,

129.2, 130.4, 136.3, 137.3, 141.3 (dd, J = 8.1, 5.1 Hz), 161.0 (dd, J =

245.8, 13.2 Hz), 161.2 (dd, J = 246.5, 12.2 Hz), 171.3, 172.5; HRMS

calcd for C₂₄H₂₁F₂N₂O₂(M ꢀ H)^ꢀ407.1577,₁found 407.1576; mp

177 ^◦C; chemical purity 98.4%, t_R14.9 min. 14c: H NMR (CDCl₃) δ 1.71

(s, 3H), 1.80 (d, J = 6.8 Hz, 3H), 4.51 (br s, 1H), 5.00 (q, J = 6.8 Hz, 1H),

5.39 (br s, 2H), 6.50–6.57 (m, 1H), 6.92–6.97 (m, 1H), 7.09–7.20 (m,

5H), 7.45–7.49 (m, 3H), 7.59–7.64 (m, 2H); ¹³C NMR (CDCl₃) δ 10.3 (d,

J = 5.9 Hz), 18.3, 55.0, 63.4 (br s), 103.0 (t, J = 26.4 Hz), 110.2 (dd, J =

22.3, 3.2 Hz), 120.8 (br s), 126.3, 128.4, 128.59, 128.64, 129.0, 129.8,

136.5 (br s), 136.6 (br s), 141.0 (br s), 160.9 (dd, J = 246.5, 12.2 Hz),

161.0 (dd, J = 245.8, 13.6 Hz), 171.4, 172.2; HRMS calcd for

5.1.34. N-((R)-Carbamoylphenylmethyl)-N-((R)-indan-1-yl)benzamide

(13f), N-((S)-Carbamoylphenylmethyl)-N-((R)-indan-1-yl)benzamide

(14f)

The title compounds were prepared from (R)-indan-1-ylamine (8f,

266 mg), benzaldehyde (212 mg), methanol (4 mL), benzoic acid (244

mg), 4-phenylcyclohenen-1-yl isocyanide (367 mg), 1,4-dioxane (6 mL),

water (1.5 mL) and 4 mol/L HCl in 1,4-dioxane (1.5 mL) according to the

general procedure. The crude product was puriﬁed using silica gel col-

umn chromatography (eluent EtOAc:Hexane = 1:1–2:1) to afford 13f

(253 mg, 34%, colorless solid, low-polarity) and 14f (169 mg, 23%,

colorless solid, high-polarity). 13f: ¹H NMR (CDCl₃) δ 1.68–1.82 (m,

1H), 2.00–2.11 (m, 1H), 2.55–2.66 (m, 1H), 2.70–2.80 (m, 1H), 4.67 (s,

1H), 5.36 (br s, 1H), 5.54 (t, J = 8.5 Hz, 1H), 5.82 (br s, 1H), 7.19 (d, J =

7.5 Hz, 1H), 7.26–7.53 (m, 10H), 7.59–7.64 (m, 2H), 7.96 (d, J = 7.3 Hz,

1H); ¹³C NMR (CDCl₃) δ 29.7, 31.4, 62.8, 66.4, 125.0, 125.7, 126.6,

127.2, 128.3, 128.4, 128.5, 128.8, 129.2, 129.8, 136.6, 137.8, 140.0,

143.0, 171.4, 172.6; HRMS calcd for C₂₄H₂₁N₂O₂(M ꢀ H)^ꢀ369.1609,

found 369.1605; mp 172 ^◦C; chemical purity 98.7%, t_R14.23 min. 14f:

¹H NMR (CDCl₃) δ 2.34–2.46 (m, 1H), 2.58–2.70 (m, 1H), 2.72–2.83 (m,

1H), 3.05–3.15 (m, 1H), 4.49 (s, 1H), 5.46 (br s, 1H), 5.56 (t, J = 7.2 Hz,

1H), 6.06 (br s, 1H), 6.78 (d, J = 7.3 Hz, 1H), 6.85 (t, J = 7.1 Hz, 1H),

7.13 (t, J = 7.5 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 7.25–7.32 (m, 5H),

7.42–7.47 (m, 3H), 7.56–7.61 (m, 2H); ¹³C NMR (CDCl₃) δ 30.1, 30.2,

63.7, 66.5, 125.1, 125.3, 126.1, 126.6, 128.1, 128.3, 128.6, 128.7,

128.8, 129.7, 136.7, 137.1, 139.3, 143.9, 172.5, 172.6; HRMS calcd for

C

₂₄H₂₁F₂N₂O₂(M ꢀ H)^ꢀ407.1577, found 407.1577; mp 214 ^◦C;

chemical purity 91.2%, t_R14.6 min.

5.1.32. N-((R)-Carbamoylphenylmethyl)-N-((R)-1-phenylethyl)

benzamide (13d), N-((S)-Carbamoylphenylmethyl)-N-((R)-1-phenylethyl)

benzamide (14d)

The title compounds were prepared from (R)-1-phenylethylamine

(4d, 242 mg), benzaldehyde (212 mg), methanol (4 mL), benzoic acid

(244 mg), 4-phenylcyclohenen-1-yl isocyanide (367 mg), 1,4-dioxane

(6 mL), water (1.5 mL) and 4 mol/L HCl in 1,4-dioxane (1.5 mL) ac-

cording to the general procedure. The crude product was puriﬁed using

silica gel column chromatography (eluent EtOAc:Hexane = 1:1–2:1) to

afford 13d (225 mg, 31%, colorless amorphous solid, low-polarity) and

14d (243 mg, 34%, colorless solid, high-polarity). 13d: ¹H NMR (CDCl₃)

δ 1.42 (d, J = 7.0 Hz, 3H), 4.72 (s, 1H), 5.21 (br s, 1H), 5.28 (q, J = 7.0

Hz, 1H), 6.06 (br s, 1H), 7.31–7.54 (m, 13H), 7.56–7.62 (m, 2H); ¹³C

NMR (CDCl₃) δ 18.9, 58.2, 62.8, 126.3, 127.7, 128.2, 128.3, 128.8,

128.9, 129.1, 129.9, 136.7, 137.7, 139.0, 171.4, 172.3; HRMS calcd for

C

₂₄H₂₁N₂O₂(M ꢀ H)^ꢀ369.1609, found 369.1604; mp 198 ^◦C; chemical

purity > 99.5%, t_R14.16 min.

C

₂₃H₂₁N₂O₂(M ꢀ H)^ꢀ357.1609, found 357.1606; chemical purity

94.5%, t_R13.9 min. 14d: ¹H NMR (CDCl₃) δ 1.73 (d, J = 7.0 Hz, 3H),

4.65 (s, 1H), 5.22 (q, J = 7.0 Hz, 1H), 5.46 (br s, 1H), 6.20 (br s, 1H),

7.06–7.21 (m, 10H), 7.42–7.51 (m, 3H), 7.54–7.59 (m, 2H); ¹³C NMR

(CDCl₃) δ 17.4, 58.2, 63.3, 126.1, 127.8, 128.0, 128.4, 128.9, 129.5,

136.7, 137.0, 138.7, 172.3, 172.7; HRMS calcd for C₂₃H₂₁N₂O₂(M ꢀ

H)^ꢀ357.1609, found 357.1602; mp 149 ^◦C; chemical purity 97.2%, t_R

13.7 min.

5.1.35. N-((R)-Carbamoylphenylmethyl)-N-((R)-6,7-dihydro-5H-

cyclopenta[b]pyridin-5-yl)benzamide (13g), N-((S)-

Carbamoylphenylmethyl)-N-((R)-6,7-dihydro-5H-cyclopenta[b]pyridin-5-

yl)benzamide (14g)

The title compounds were prepared from 8g (155 mg), benzaldehyde

(80 mg), triethylamine (209 µL), methanol (2 mL), benzoic acid (92 mg),

4-phenylcyclohenen-1-yl isocyanide (137 mg), 1,4-dioxane (2 mL),

water (0.5 mL) and 4 mol/L HCl in 1,4-dioxane (0.56 mL) according to

the general procedure. The crude product was puriﬁed using silica gel

column chromatography (eluent EtOAc:MeOH = 10:1) to afford 13g

(25 mg, 9%, colorless amorphous solid, low-polarity) and 14g (15 mg,

5.1.33. N-((R)-Carbamoylphenylmethyl)-N-((R)-1,2,3,4-

tetrahydronaphthalen-1-yl)benzamide (13e), N-((S)-

Carbamoylphenylmethyl)-N-((R)-1,2,3,4-tetrahydronaphthalen-1-yl)

benzamide (14e)

1

5%, colorless solid, high-polarity). 13g: H NMR (CDCl₃) δ 1.50–2.18

The title compounds were prepared from (R)-1,2,3,4-tetrahy-

dronaphthalen-1-amine (9e, 294 mg), benzaldehyde (212 mg),

(m, 2H), 2.66–3.25 (m, 2H), 4.60 (br s, major 1H), 5.00 (br s, minor 1H),

5.44–5.87 (m, 3H), 7.01–7.62 (m, 11H), 8.08–8.52 (m, 2H); ¹³C NMR

10

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

(CDCl₃) δ 29.8, 31.7, 62.7, 64.5, 122.1, 126.4, 128.5, 128.8, 128.9,

129.4, 130.1, 133.6, 134.2, 136.3, 137.4, 150.1, 163.8, 171.3, 172.6;

HRMS calcd for C₂₃H₂₀N₃O₂(M ꢀ H)^ꢀ370.1561, found 370.1559;

chemical purity 99.1%, t_R10.73 min. 14g: ¹H NMR (CDCl₃) δ 2.43–2.61

(m, 1H), 2.66–2.79 (m, 1H), 2.83–2.96 (m, 1H), 3.12–3.27 (m, 1H), 4.45

(br s, 1H), 5.52 (br s, 1H), 5.68 (br s, 2H), 6.68 (dd, J = 7.7, 4.9 Hz, 1H),

6.84–6.94 (m, 1H), 7.24–7.34 (m, 5H), 7.43–7.48 (m, 3H), 7.54 (br s,

2H), 8.32 (d, J = 4.0 Hz, 1H); ¹³C NMR (CDCl₃) δ 28.3, 32.3, 63.7, 64.3,

120.9, 126.5, 128.6, 128.9, 129.1, 129.9, 132.8, 133.4, 136.5, 137.3,

150.1, 164.5, 171.7, 172.6; HRMS calcd for C₂₃H₂₀N₃O₂(M ꢀ H)^ꢀ

370.1561, found 370.1558; mp 217 ^◦C; chemical purity 98.8%, t_R10.66

min.

(d, J = 244.3 Hz), 172.2, 172.6; HRMS calcd for C₂₄H₂₀FN₂O₂(M ꢀ H)^ꢀ

387.1514, found 387.1513; chemical purity 94.5%, t_R14.0 min.

5.1.38. N-((R)-Carbamoylphenylmethyl)-N-((R)-6-chloroindan-1-yl)

benzamide (13j), N-((S)-Carbamoylphenylmethyl)-N-((R)-6-chloroindan-

1-yl)benzamide (14j)

The title compounds were prepared from 8j (153 mg), benzaldehyde

(80 mg), triethylamine (105 µL), methanol (2 mL), benzoic acid (92 mg),

4-phenylcyclohenen-1-yl isocyanide (137 mg), 1,4-dioxane (2 mL),

water (0.5 mL) and 4 mol/L HCl in 1,4-dioxane (0.56 mL) according to

the general procedure. The crude product was puriﬁed using silica gel

column chromatography (eluent EtOAc:Hexane = 1:2–2:1) to afford 13j

(105 mg, 35%, colorless solid, low-polarity) and 14j (84 mg, 28%,

colorless solid, high-polarity). 13j: ¹H NMR (CDCl₃) δ 1.72–1.86 (m,

1H), 2.01–2.15 (m, 1H), 2.50–2.62 (m, 1H), 2.65–2.77 (m, 1H), 4.66 (s,

1H), 5.29–5.90 (m, 3H), 7.08 (d, J = 8.0 Hz, 1H), 7.20–7.27 (m, 1H),

7.33–7.52 (m, 8H), 7.55–7.61 (m, 2H), 7.96 (s, 1H); ¹³C NMR (CDCl₃) δ

29.2, 31.8, 62.9, 66.1, 125.9, 126.0, 126.6, 128.4, 128.6, 128.8, 128.9,

129.3, 130.0, 133.1, 136.4, 137.6, 141.4, 142.2, 171.2, 172.6; HRMS

calcd for C₂₄H₂₀ClN₂O₂(M ꢀ H)^ꢀ403.1219, found 403.1217; mp

174 ^◦C; chemical purity 98.2%, t_R15.0 min. 14j: ¹H NMR (CDCl₃) δ

2.38–2.51 (m, 1H), 2.62–2.79 (m, 2H), 2.98–3.10 (m, 1H), 4.41 (s, 1H),

5.38–5.55 (m, 2H), 5.78 (br s, 1H), 6.57 (s, 1H), 7.05–7.11 (m, 2H),

7.26–7.35 (m, 5H), 7.43–7.47 (m, 3H), 7.53–7.58 (m, 2H); ¹³C NMR

(CDCl₃) δ 29.8, 30.5, 63.8, 66.3, 126.0, 126.1, 126.6, 128.66, 128.70,

128.9, 129.0, 129.8, 132.1, 136.6, 137.3, 141.4, 142.3, 172.0, 172.6;

HRMS calcd for C₂₄H₂₀ClN₂O₂(M ꢀ H)^ꢀ403.1219, found 403.1218; mp

189 ^◦C; chemical purity 92.0%, t_R14.9 min.

5.1.36. N-((R)-Carbamoylphenylmethyl)-N-((S)-2,3-dihydrobenzofuran-

3-yl)benzamide (13h), N-((S)-Carbamoylphenylmethyl)-N-((S)-2,3-

dihydrobenzofuran-3-yl)benzamide (14h)

The title compounds were prepared from (S)-2,3-dihy-

drobenzofuran-3-ylamine hydrochloride (10h, 172 mg), benzaldehyde

(106 mg), triethylamine (139 µL), methanol (2 mL), benzoic acid (122

mg), 4-phenylcyclohenen-1-yl isocyanide (183 mg), 1,4-dioxane (3 mL),

water (0.75 mL) and 4 mol/L HCl in 1,4-dioxane (0.75 mL) according to

the general procedure. The crude product was puriﬁed using silica gel

column chromatography (eluent EtOAc:Hexane = 2:1–3:1) to afford

13h (55 mg, 15%, colorless amorphous solid, low-polarity) and 14h (38

1

mg, 10%, colorless solid, high-polarity). 13h: H NMR (CDCl₃) δ 4.10

(dd, J = 10.1, 5.2 Hz, 1H), 4.23 (br s, 1H), 4.68 (br s, 1H), 5.30–6.10 (m,

3H), 6.76 (d, J = 7.8 Hz, 1H), 6.97 (br s, 1H), 7.22 (br s, 1H), 7.34–7.72

(m, 11H); ¹³C NMR (CDCl₃) δ 62.4, 62.5, 74.0, 110.3 (br s), 121.5 (br s),

123.2 (br s), 126.8, 128.9, 129.1, 129.5, 130.1, 131.0 (br s), 136.2,

137.2 (br s), 160.7, 170.8, 171.9 (br s); HRMS calcd for C₂₃H₁₉N₂O₃(M

ꢀ H)^ꢀ371.1401, found 371.1400; chemical purity 97.1%, t_R12.9 min.

14h: ¹H NMR (CDCl₃) δ 4.45–4.68 (m, 2H), 5.10–5.21 (m, 1H),

5.42–5.74 (m, 3H), 6.51 (t, J = 7.4 Hz, 1H), 6.67 (br s, 1H), 6.80 (d, J =

8.0 Hz, 1H), 7.07–7.30 (m, 6H), 7.41–7.53 (m, 5H); ¹³C NMR (CDCl₃) δ

62.0 (br s), 63.7 (br s), 74.8, 110.6, 120.7, 123.2 (br s), 126.3 (br s),

126.8, 128.5 (br s), 128.9, 129.0, 129.2, 129.9, 130.7 (br s), 136.3,

137.2 (br s), 160.9, 171.47, 171.54 (br s); HRMS calcd for C₂₃H₁₉N₂O₃

(M ꢀ H)^ꢀ371.1401, found 371.1398; mp 178 ^◦C; chemical purity

95.9%, t_R13.1 min.

5.1.39. N-((R)-Carbamoylphenylmethyl)-N-((R)-4-ﬂuoroindan-1-yl)

benzamide (13k), N-((S)-Carbamoylphenylmethyl)-N-((R)-4-ﬂuoroindan-

1-yl)benzamide (14k)

The title compounds were prepared from 8k (141 mg), benzaldehyde

(80 mg), triethylamine (105 µL), methanol (2 mL), benzoic acid (92 mg),

4-phenylcyclohenen-1-yl isocyanide (137 mg), 1,4-dioxane (2 mL),

water (0.5 mL) and 4 mol/L HCl in 1,4-dioxane (0.56 mL) according to

the general procedure. The crude product was puriﬁed using silica gel

column chromatography (eluent EtOAc:Hexane = 1:2–1:1) to afford 13k

(105 mg, 36%, colorless amorphous solid, low-polarity) and 14k (78 mg,

27%, colorless solid, high-polarity). 13k: ¹H NMR (CDCl₃) δ 1.70–1.85

(m, 1H), 2.02–2.15 (m, 1H), 2.50–2.62 (m, 1H), 2.77–2.89 (m, 1H), 4.65

(s, 1H), 5.37 (br s, 1H), 5.50–5.60 (m, 1H), 5.76 (br s, 1H), 6.92–7.00

(m, 1H), 7.26–7.53 (m, 9H), 7.58–7.61 (m, 2H), 7.76 (d, J = 7.3 Hz, 1H);

¹³C NMR (CDCl₃) δ 25.5, 31.3, 62.9, 66.4, 115.0 (d, J = 19.8 Hz), 121.4,

126.5, 128.4, 128.6, 128.9, 129.1 (d, J = 19.1 Hz), 129.3, 130.0, 136.4,

137.6, 143.6, 159.4 (d, J = 247.2 Hz), 171.3, 172.6; HRMS calcd for

5.1.37. N-((R)-Carbamoylphenylmethyl)-N-((R)-6-ﬂuoroindan-1-yl)

benzamide (13i), N-((S)-Carbamoylphenylmethyl)-N-((R)-6-ﬂuoroindan-

1-yl)benzamide (14i)

The title compounds were prepared from 8i (141 mg), benzaldehyde

(80 mg), triethylamine (105 µL), methanol (2 mL), benzoic acid (92 mg),

4-phenylcyclohenen-1-yl isocyanide (137 mg), 1,4-dioxane (2 mL),

water (0.5 mL) and 4 mol/L HCl in 1,4-dioxane (0.56 mL) according to

the general procedure. The crude product was puriﬁed using silica gel

column chromatography (eluent EtOAc:Hexane = 1:2–2:1) to afford 13i

(83 mg, 29%, colorless solid, low-polarity) and 14i (67 mg, 23%,

colorless amorphous solid, high-polarity). 13i: ¹H NMR (CDCl₃) δ

1.73–1.86 (m, 1H), 2.02–2.14 (m, 1H), 2.49–2.61 (m, 1H), 2.65–2.76

(m, 1H), 4.67 (s, 1H), 5.30–5.55 (m, 2H), 5.78 (br s, 1H), 6.91–7.00 (m,

1H), 7.07–7.13 (m, 1H), 7.34–7.53 (m, 8H), 7.55–7.61 (m, 2H),

7.67–7.74 (m, 1H); ¹³C NMR (CDCl₃) δ 29.0, 32.1, 62.9, 66.2, 112.8 (d,

J = 22.0 Hz), 115.7 (d, J = 22.0 Hz), 125.9 (d, J = 8.1 Hz), 126.5, 128.4,

128.6, 128.9, 129.3, 130.0, 136.4, 137.6, 138.3 (d, J = 2.9 Hz), 142.3 (d,

J = 7.3 Hz), 162.5 (d, J = 244.3 Hz), 171.3, 172.6; HRMS calcd for

C

₂₄H₂₀FN₂O₂(M ꢀ H)^ꢀ387.1514, found 387.1513; chemical purity

97.8%, t_R14.4 min. 14k: ¹H NMR (CDCl₃) δ 2.40–2.58 (m, 1H),

2.65–2.82 (m, 2H), 3.08–3.20 (m, 1H), 4.47 (s, 1H), 5.40–5.62 (m, 2H),

5.91 (br s, 1H), 6.52 (br s, 1H), 6.78–6.84 (m, 2H), 7.26–7.34 (m, 5H),

7.42–7.47 (m, 3H), 7.53–7.59 (m, 2H); ¹³C NMR (CDCl₃) δ 26.2, 30.1,

63.9, 66.5, 114.8 (d, J = 20.5 Hz), 121.1, 126.6, 128.2 (d, J = 6.6 Hz),

128.4, 128.86, 128.93, 129.9, 130.3 (d, J = 19.1 Hz), 136.6, 137.2,

143.1, 159.5 (d, J = 248.0 Hz), 172.2, 172.7; HRMS calcd for

C

₂₄H₂₀FN₂O₂(M ꢀ H)^ꢀ387.1514, found 387.1513; mp 215 ^◦C; chem-

ical purity 99.1%, t_R14.2 min.

5.1.40. N-((R)-Carbamoylphenylmethyl)-N-((R)-4-chloroindan-1-yl)

benzamide (13l), N-((S)-Carbamoylphenylmethyl)-N-((R)-4-chloroindan-

1-yl)benzamide (14l)

C

₂₄H₂₀FN₂O₂(M ꢀ H)^ꢀ387.1514, found 387.1514; mp 186 ^◦C; chem-

ical purity > 99.5%, t_R14.4 min. 14i: ¹H NMR (CDCl₃) δ 2.39–2.52 (m,

1H), 2.64–2.79 (m, 2H), 2.99–3.10 (m, 1H), 4.46 (br s, 1H), 5.37–5.55

(m, 2H), 5.89 (br s, 1H), 6.35 (br s, 1H), 6.78–6.85 (m, 1H), 7.11 (dd, J

= 8.4, 5.0 Hz, 1H), 7.29–7.35 (m, 5H), 7.43–7.47 (m, 3H), 7.53–7.58 (m,

2H); ¹³C NMR (CDCl₃) δ 29.6, 30.8, 63.8, 66.4, 112.3 (d, J = 23.5 Hz),

115.7 (d, J = 23.5 Hz), 126.1 (d, J = 7.3 Hz), 126.6, 128.6, 128.9, 129.0,

129.9, 136.6, 137.3, 139.3 (d, J = 2.2 Hz), 141.6 (d, J = 6.6 Hz), 161.8

The title compounds were prepared from 8l (122 mg), benzaldehyde

(64 mg), triethylamine (84 µL), methanol (2 mL), benzoic acid (73 mg),

4-phenylcyclohenen-1-yl isocyanide (110 mg), 1,4-dioxane (2.5 mL),

water (0.45 mL) and 4 mol/L HCl in 1,4-dioxane (0.45 mL) according to

the general procedure. The crude product was puriﬁed using silica gel

column chromatography (eluent EtOAc:Hexane = 1:1–3:1) to afford 13l

11

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

(46 mg, 19%, colorless amorphous solid, low-polarity) and 14l (20 mg,

8%, colorless solid, high-polarity). 13l: ¹H NMR (CDCl₃) δ 1.69–1.82 (m,

1H), 1.98–2.12 (m, 1H), 2.51–2.63 (m, 1H), 2.76–2.87 (m, 1H), 4.66 (br

s, 1H), 5.48 (br s, 1H), 5.56 (t, J = 8.2 Hz, 1H), 5.73 (br s, 1H), 7.20–7.60

(m, 12H), 7.89 (d, J = 6.2 Hz, 1H); ¹³C NMR (CDCl₃) δ 28.8, 30.7, 62.8,

66.8, 124.1, 126.5, 128.4, 128.58, 128.64, 128.9, 129.3, 130.0, 131.2,

136.3, 137.6, 141.1, 142.3, 171.3, 172.6; HRMS calcd for C₂₄H₂₀ClN₂O₂

(M ꢀ H)^ꢀ403.1219, found 403.1217; chemical purity 97.5%, t_R15.3

min. 14l: ¹H NMR (CDCl₃) δ 2.38–2.52 (m, 1H), 2.63–2.83 (m, 2H),

3.08–3.19 (m, 1H), 4.47 (br s, 1H), 5.42–5.66 (m, 2H), 5.86 (br s, 1H),

6.55–6.65 (m, 1H), 6.77 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 8.0 Hz, 1H),

7.24–7.34 (m, 5H), 7.42–7.48 (m, 3H), 7.52–7.60 (m, 2H); ¹³C NMR

(CDCl₃) δ 29.5, 29.7, 63.8, 67.0, 123.7, 126.6, 127.8, 128.4, 128.9,

129.0, 129.9, 131.4, 136.6, 137.2 (br s), 141.7 (br s), 142.2, 172.1,

172.7; HRMS calcd for C₂₄H₂₀ClN₂O₂(M ꢀ H)^ꢀ403.1219, found

403.1217; mp 188 ^◦C; chemical purity 90.1%, t_R15.1 min.

HRMS calcd for C₂₄H₁₉ClFN₂O₂(M ꢀ H)^ꢀ421.1125, found 421.1124;

mp 168 ^◦C; chemical purity 97.5%, t_R15.4 min. 14n: ¹H NMR (CDCl₃) δ

2.41–2.58 (m, 1H), 2.65–2.81 (m, 2H), 3.02–3.17 (m, 1H), 4.40 (br s,

1H), 5.49 (br s, 2H), 5.70 (br s, 1H), 6.35 (br s, 1H), 6.83 (d, J = 8.3 Hz,

1H), 7.29–7.41 (m, 5H), 7.43–7.58 (m, 5H); ¹³C NMR (CDCl₃) δ 25.9,

30.1, 63.8, 66.2, 115.6 (d, J = 23.5 Hz), 121.9, 126.5, 128.87, 128.87 (d,

J = 19.1 Hz), 129.1, 129.9, 133.0 (br s), 136.4, 137.3, 144.2 (br s), 158.8

(d, J = 250.9 Hz), 171.7, 172.5; HRMS calcd for C₂₄H₁₉ClFN₂O₂(M ꢀ

H)^ꢀ421.1125, found 421.1125; chemical purity 97.5%, t_R15.2 min.

5.1.43. N-((R)-Carbamoylphenylmethyl)-N-[(R)-1-(3,5-diﬂuorophenyl)

ethyl]-2-hydroxybenzamide (13p), N-((S)-Carbamoylphenylmethyl)-N-

[(R)-1-(3,5-diﬂuorophenyl)ethyl]-2-hydroxybenzamide (14p)

A mixture of the title compounds, 2-acethyloxy-N-((R)-Carbamoyl-

phenylmethyl)-N-[(R)-1-(3,5-diﬂuorophenyl)ethyl]benzamide (13o),

and

2-acethyloxy-N-((S)-Carbamoylphenylmethyl)-N-[(R)-1-(3,5-

diﬂuorophenyl)ethyl]benzamide (14o) was prepared from (R)-1-(3,5-

diﬂuorophenyl)ethylamine (314 mg), benzaldehyde (212 mg), meth-

anol (4 mL), acetylsalicylic acid (360 mg), 4-phenylcyclohenen-1-yl

isocyanide (367 mg), 1,4-dioxane (6 mL), water (1.5 mL) and 4 mol/L

HCl in 1,4-dioxane (1.5 mL) according to the general procedure. The

obtained mixture was dissolved in methanol (10 mL), and K₂CO₃(276

mg) was added. The mixture was stirred for 1 h at room temperature,

then 1 mol/L HCl (2 mL) was added, and 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

EtOAc:Hexane = 1:1–2:1) to afford 13p (247 mg, 30%, colorless solid,

low-polarity) and 14p (325 mg, 40%, colorless solid, high-polarity).

13p: ¹H NMR (CDCl₃) δ 1.28 (d, J = 7.0 Hz, 3H), 4.74 (br s, 1H), 5.28

(q, J = 7.0 Hz, 1H), 5.52 (br s, 1H), 5.56 (br s, 1H), 6.78 (tt, J = 8.7, 2.3

Hz, 1H), 6.90 (td, J = 7.5, 1.0 Hz, 1H), 7.06 (dd, J = 8.3, 0.9 Hz, 1H),

7.10–7.17 (m, 2H), 7.27–7.46 (m, 5H), 7.51–7.56 (m, 2H), 8.60 (s, 1H);

¹³C NMR (THF-d₈) δ 18.1, 56.5 (br s), 64.8, 102.2 (t, J = 25.0 Hz), 111.6

(dd, J = 19.1, 6.6 Hz), 116.9, 120.0, 124.7, 128.5, 128.6, 129.1, 129.9,

131.2, 138.4, 147.3 (br s), 155.5, 163.5 (dd, J = 245.0, 12.5 Hz), 170.4,

171.9; HRMS calcd for C₂₃H₁₉F₂N₂O₃(M ꢀ H)^ꢀ409.1369, found

409.1368; mp 212 ^◦C; chemical purity 94.6%, t_R13.3 min. 14p: ¹H NMR

(CDCl₃) δ 1.73 (d, J = 6.8 Hz, 3H), 4.67 (br s, 1H), 5.18 (q, J = 6.8 Hz,

1H), 5.65 (br s, 1H), 5.72 (br s, 1H), 6.49 (tt, J = 8.8, 2.3 Hz, 1H),

6.53–6.60 (m, 2H), 6.94 (td, J = 7.5, 1.0 Hz, 1H), 7.03 (dd, J = 8.3, 0.8

Hz, 1H), 7.18–7.36 (m, 7H), 8.28 (br s, 1H); ¹³C NMR (THF-d₈) δ 17.2,

57.6, 64.8 (br s), 101.8–102.8 (m), 111.4–111.9 (m), 116.9, 120.1,

125.8, 127.8, 128.2, 128.5, 130.57, 130.62, 137.8 (br s), 146.7 (br s),

154.4, 163.3 (dd, J = 246.4, 13.0 Hz), 170.3, 172.5; HRMS calcd for

5.1.41. N-((R)-Carbamoylphenylmethyl)-N-((R)-4,6-diﬂuoroindan-1-yl)

benzamide (13m), N-((S)-Carbamoylphenylmethyl)-N-((R)-4,6-

diﬂuoroindan-1-yl)benzamide (14m)

The title compounds were prepared from 8m (206 mg), benzalde-

hyde (106 mg), triethylamine (139 µL), methanol (2 mL), benzoic acid

(122 mg), 4-phenylcyclohenen-1-yl isocyanide (183 mg), 1,4-dioxane

(3 mL), water (0.75 mL) and 4 mol/L HCl in 1,4-dioxane (0.75 mL)

according to the general procedure. The crude product was puriﬁed

using silica gel column chromatography (eluent EtOAc:Hexane =

1:2–3:1) to afford 13m (100 mg, 25%, colorless solid, low-polarity) and

14m (65 mg, 16%, pale yellow amorphous solid, high-polarity). 13m:

This compound exists as a mixture of rotamers in CDCl₃(3:1); ¹H NMR

(CDCl₃) δ 1.20–2.15 (m, 2H), 2.42–3.10 (m, 2H), 4.66 (br s, major 1H),

4.96 (br s, minor 1H), 5.41–6.16 (m, 3H), 6.50–6.77 (m, 1H), 7.23–7.62

(m, 11H); ¹³C NMR (CDCl₃) δ 25.0, 26.4 (br s, minor), 30.0 (br s, minor),

31.5, 62.9, 66.2, 66.8 (br s, minor), 103.7 (t, J = 25.3 Hz), 108.8 (d, J =

23.5 Hz), 124.6 (dd, J = 19.1, 2.9 Hz), 126.4, 127.1 (minor), 128.4,

128.7, 128.9, 129.4, 130.0, 136.2, 137.4, 144.8 (br s), 158.8 (dd, J =

251.0, 13.0 Hz), 162.8 (dd, J = 246.6, 10.0 Hz), 171.2, 172.6; HRMS

calcd for C₂₄H₁₉F₂N₂O₂(M ꢀ H)^ꢀ405.1420, found 405.1420; mp

183 ^◦C; chemical purity > 99.5%, t_R14.7 min. 14m: ¹H NMR (CDCl₃) δ

2.40–2.58 (m, 1H), 2.64–2.84 (m, 2H), 3.00–3.16 (m, 1H), 4.45 (br s,

1H), 5.50 (br s, 1H), 5.67 (br s, 1H), 5.78 (br s, 1H), 6.10–6.20 (m, 1H),

6.56 (t, J = 8.5 Hz, 1H), 7.20–7.59 (m, 10H); ¹³C NMR (CDCl₃) δ 25.8,

30.2, 63.8, 66.4, 103.5 (t, J = 25.5 Hz), 108.4 (d, J = 22.3 Hz), 125.7 (d,

J = 19.8 Hz), 126.5, 128.7, 128.9, 129.1, 130.0, 136.4, 137.2, 144.0 (br

s), 158.9 (dd, J = 250.4, 12.6 Hz), 162.1 (dd, J = 247.2, 10.3 Hz), 171.8,

172.6; HRMS calcd for C₂₄H₁₉F₂N₂O₂(M ꢀ H)^ꢀ405.1420, found

405.1420; mp 183 ^◦C; chemical purity 97.9%, t_R14.4 min.

C

₂₃H₁₉F₂N₂O₃(M ꢀ H)^ꢀ409.1369, found 409.1368; mp 204 ^◦C;

chemical purity 98.8%, t_R13.0 min.

5.1.42. N-((R)-Carbamoylphenylmethyl)-N-((R)-6-chloro-4-ﬂuoroindan-

1-yl)benzamide (13n), N-((S)-Carbamoylphenylmethyl)-N-((R)-6-chloro-

4-ﬂuoroindan-1-yl)benzamide (14n)

5.1.44. N-((R)-Carbamoylphenylmethyl)-N-((R)-1-(3,5-diﬂuorophenyl)

ethyl)-2-nitrobenzamide (13q), N-((S)-Carbamoylphenylmethyl)-N-((R)-1-

(3,5-diﬂuorophenyl)ethyl)-2-nitrobenzamide (14q)

The title compounds were prepared from 8n (444 mg), benzaldehyde

(212 mg), triethylamine (279 µL), methanol (4 mL), benzoic acid (244

mg), 4-phenylcyclohenen-1-yl isocyanide (367 mg), 1,4-dioxane (6 mL),

water (1.5 mL) and 4 mol/L HCl in 1,4-dioxane (1.5 mL) according to the

general procedure. The crude product was puriﬁed using silica gel col-

umn chromatography (eluent EtOAc:Hexane = 1:2–2:1) to afford 13n

(313 mg, 37%, colorless solid, low-polarity) and 14n (205 mg, 24%,

colorless amorphous solid, high-polarity). 13n: This compound exists as

a mixture of rotamers in CDCl₃(3:1); ¹H NMR (CDCl₃) δ 1.20–2.16 (m,

2H), 2.44–3.16 (m, 2H), 4.65 (br s, major 1H), 4.99 (br s, minor 1H),

5.24–5.85 (m, 3H), 6.81–7.06 (m, 1H), 7.31–7.88 (m, 11H); ¹³C NMR

(CDCl₃) δ 25.2, 26.6 (br s, minor), 29.9 (br s, minor), 31.4, 62.9, 66.1,

66.8 (br s, minor), 114.2–114.8 (m, minor), 116.0 (d, J = 24.2 Hz),

120.3 (br s, minor), 121.9, 126.5, 127.2 (br s, minor), 127.8 (d, J = 19.8

Hz), 128.5, 128.7, 128.9, 129.4, 130.1, 134.0 (d, J = 9.5 Hz), 136.2,

137.4, 145.0, 147.9 (br s, minor), 158.8 (d, J = 251.6 Hz), 171.1, 172.6;

The title compounds were prepared from (R)-1-(3,5-diﬂuorophenyl)

ethylamine (314 mg), benzaldehyde (212 mg), methanol (4 mL), 2-

nitrobenzoic acid (334 mg), 4-phenylcyclohenen-1-yl isocyanide (367

mg), 1,4-dioxane (6 mL), water (1.5 mL) and 4 mol/L HCl in 1,4-dioxane

(1.5 mL) according to the general procedure. The crude product was

puriﬁed using silica gel column chromatography (eluent EtOAc:Hexane

= 1:1–2:1) to afford 13q (237 mg, 27%, colorless solid, low-polarity)

and 14q (346 mg, 39%, colorless solid, high-polarity). 13q: ¹H NMR

(CDCl₃) δ 1.30 (d, J = 7.0 Hz, 3H), 4.73 (s, 1H), 4.83 (q, J = 7.0 Hz, 1H),

5.31–5.48 (m, 2H), 6.72–6.82 (m, 1H), 7.08–7.75 (m, 10H), 8.23 (d, J =

8.3 Hz, 1H); ¹³C NMR (CDCl₃) δ 20.8, 57.9, 63.0, 103.4 (t, J = 25.3 Hz),

110.3 (dd, J = 19.1, 7.3 Hz), 124.9, 128.2, 129.0, 129.4, 129.6, 130.3,

132.9, 134.9, 137.9, 144.1 (t, J = 8.4 Hz), 145.1, 163.5 (dd, J = 248.7,

12.5 Hz), 168.6, 170.9; HRMS calcd for C₂₃H₁₈F₂N₃O₄(M ꢀ H)^ꢀ

438.1271, found 438.1278; mp 205 ^◦C; chemical purity > 99.5%, t_R

12

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

14.3 min. 14q: This compound exists as a mixture of rotamers in CDCl₃

(6:1); ¹H NMR (CDCl₃) δ 1.69 (d, J = 6.8 Hz, major 3H), 1.80 (d, J = 6.8

Hz, minor 3H), 4.45 (s, 1H), 4.74 (q, J = 6.8 Hz, 1H), 5.42 (br s, 2H),

6.42–6.57 (m, 1H), 6.73–6.81 (m, 2H), 7.08–7.86 (m, 8H), 8.31 (d, J =

8.0 Hz, 1H); ¹³C NMR (CDCl₃) δ 16.3, 57.2, 63.6, 103.4 (t, J = 25.3 Hz),

111.8 (dd, J = 18.7, 7.2 Hz), 125.1, 128.5, 128.8, 128.9, 129.2, 130.4,

132.8, 134.8, 136.5, 141.6 (t, J = 8.4 Hz), 144.9, 162.8 (dd, J = 249.0,

12.6 Hz), 168.3, 171.3; HRMS calcd for C₂₃H₁₈F₂N₃O₄(M ꢀ H)^ꢀ

438.1271, found 438.1276; mp 216 ^◦C; chemical purity 97.7%, t_R13.8

min.

min. 14t: ¹H NMR (CDCl₃) δ 2.46–2.61 (m, 1H), 2.69–2.82 (m, 2H),

3.02–3.17 (m, 1H), 4.44 (br s, 1H), 5.34–5.66 (m, 3H), 6.30 (br s, 1H),

6.84 (d, J = 8.0 Hz, 1H), 7.26–7.45 (m, 6H), 7.86 (d, J = 7.3 Hz, 1H),

8.71 (dd, J = 4.9, 1.6 Hz, 1H), 8.80 (s, 1H); ¹³C NMR (CDCl₃) δ 26.0,

30.2, 63.9, 66.3, 115.9 (d, J = 22.0 Hz), 122.0 (br s), 123.7, 128.9 (d, J

= 19.8 Hz), 129.3, 132.4, 133.1–133.4 (m), 134.5, 137.0, 143.7 (br s),

147.4, 151.0, 158.9 (d, J = 251.6 Hz), 170.0 (br s), 171.1; HRMS calcd

for C₂₃H₁₈ClFN₃O₂(M ꢀ H)^ꢀ422.1077, found 422.1077; mp 185 C;

◦

chemical purity 97.8%, t_R13.1 min.

5.1.47. N-((R)-Carbamoylphenylmethyl)-N-((R)-6-chloro-4-ﬂuoroindan-

1-yl)-2-nitronicotinamide (13u), N-((S)-Carbamoylphenylmethyl)-N-((R)-

6-chloro-4-ﬂuoroindan-1-yl)-2-nitronicotinamide (14u)

5.1.45. N-((R)-Carbamoylphenylmethyl)-N-((R)-4,6-diﬂuoroindan-1-yl)

nicotinamide (13s), N-((S)-Carbamoylphenylmethyl)-N-((R)-4,6-

diﬂuoroindan-1-yl)nicotinamide (14s)

The title compounds were prepared from 8n (444 mg), benzaldehyde

(212 mg), triethylamine (279 µL), methanol (4 mL), 2-nitronicotinic

acid (336 mg), 4-phenylcyclohenen-1-yl isocyanide (367 mg), 1,4-

dioxane (6 mL), water (1.5 mL) and 4 mol/L HCl in 1,4-dioxane (1.5

mL) according to the general procedure. The crude product was puriﬁed

using silica gel column chromatography (eluent EtOAc:Hexane =

2:1–3:1) to afford 13u (454 mg, 48%, colorless amorphous solid, low-

polarity) and 14u (271 mg, 29%, colorless amorphous solid, high-

polarity). 13u: This compound exists as a mixture of rotamers in

CDCl₃(6:1); ¹H NMR (CDCl₃) δ 1.35–1.48 (m, minor 1H), 1.72–1.86 (m,

major 1H), 2.12–2.25 (m, 1H), 2.41–2.63 (m, 1H), 2.70–2.83 (m, major

1H), 2.99–3.09 (m, minor 1H), 4.64 (s, major 1H), 4.86 (br s, minor 1H),

5.05–5.60 (m, 3H), 6.88 (d, J = 8.5 Hz, minor 1H), 7.02 (d, J = 8.5 Hz,

major 1H), 7.38–7.62 (m, 5H), 7.70–7.85 (m, 2H), 8.07 (dd, J = 7.7, 1.5

Hz, minor 1H), 8.28 (d, J = 7.7 Hz, major 1H), 8.65 (dd, J = 4.6, 1.5 Hz,

minor 1H), 8.68–8.72 (m, major 1H); ¹³C NMR (CDCl₃) δ 25.0, 26.3

(minor), 29.5 (minor), 31.5, 62.6 (minor), 62.7, 66.0, 66.6 (minor),

114.5 (d, J = 24.2 Hz, minor), 116.3 (d, J = 24.2 Hz), 120.1 (minor),

121.0, 127.8 (minor), 128.0, 128.16, 128.20, 128.8, 129.0, 129.4,

129.5, 129.6 (minor), 130.3 (minor), 132.6 (d, J = 9.5 Hz, minor), 133.6

(minor), 134.1 (d, J = 8.8 Hz), 136.6, 138.2 (minor), 138.4, 143.9 (d, J

= 5.1 Hz), 146.9–147.2 (m, minor), 149.1 (minor), 149.4, 152.8, 154.2

(minor), 158.7 (d, J = 249.4 Hz, minor), 158.9 (d, J = 251.6 Hz), 164.6

(minor), 166.7, 170.4, 171.3 (minor); HRMS calcd for C₂₃H₁₇ClFN₄O₄

(M ꢀ H)^ꢀ467.0928, found 467.0935; chemical purity 97.4%, t_R14.6

The title compounds were prepared from 8m (206 mg), benzalde-

hyde (106 mg), triethylamine (139 µL), methanol (2 mL), nicotinic acid

(123 mg), 4-phenylcyclohenen-1-yl isocyanide (183 mg), 1,4-dioxane

(3 mL), water (0.75 mL) and 4 mol/L HCl in 1,4-dioxane (0.75 mL)

according to the general procedure. The crude product was puriﬁed

using silica gel column chromatography (eluent EtOAc:Hexane = 10:1 –

EtOAc:MeOH = 20:1) to afford 13s (103 mg, 25%, colorless solid, low-

polarity) and 14s (60 mg, 15%, colorless solid, high-polarity). 13s: This

compound exists as a mixture of rotamers in CDCl₃(2.5:1); ¹H NMR

(CDCl₃) δ 1.20–2.13 (m, 2H), 2.43–3.14 (m, 2H), 4.74 (br s, major 1H),

5.01 (br s, minor 1H), 5.32–6.29 (m, 3H), 6.50–6.82 (m, 1H), 7.23–7.67

(m, 7H), 7.84 (br s, 1H), 8.63–8.72 (m, 1H), 8.78 (br s, 1H); ¹³C NMR

(CDCl₃) δ 25.0, 26.3 (br s, minor), 30.1 (br s, minor), 31.8, 62.9, 66.2,

66.9 (br s, minor), 102.0–103.0 (m, minor), 103.9 (t, J = 25.1 Hz),

106.9–107.7 (m, minor), 108.8 (d, J = 22.0 Hz), 123.8, 124.7 (dd, J =

19.4, 2.3 Hz), 128.7, 129.0, 129.5, 132.2, 134.4, 135.1 (br s, minor),

137.1, 144.4 (br s), 147.3, 148.0 (br s, minor), 151.1, 158.9 (dd, J =

250.6, 11.2 Hz), 162.9 (dd, J = 248.7, 9.0 Hz), 170.1, 170.8; HRMS

calcd for C₂₃H₁₈F₂N₃O₂(M ꢀ H)^ꢀ406.1373, found 406.1372; mp

199 C; chemical purity 99.3%, t_R12.8 min. 14s: ¹H NMR (CDCl₃) δ

◦

2.45–2.63 (m, 1H), 2.68–2.82 (m, 2H), 2.99–3.16 (m, 1H), 4.50 (br s,

1H), 5.42 (br s, 1H), 5.66 (br s, 1H), 5.76 (br s, 1H), 6.08 (br s, 1H), 6.58

(t, J = 8.5 Hz, 1H), 7.28–7.46 (m, 6H), 7.84 (d, J = 7.0 Hz, 1H), 8.69 (dd,

J = 4.9, 1.5 Hz, 1H), 8.77 (br s, 1H); ¹³C NMR (CDCl₃) δ 25.8, 30.3, 63.8,

66.3, 103.8 (t, J = 23.5 Hz), 108.4 (d, J = 19.8 Hz), 123.7, 125.8 (dd, J

= 19.8, 2.9 Hz), 129.0, 129.2, 132.4, 134.5, 136.9, 143.5 (br s), 147.4,

151.0, 158.9 (dd, J = 250.9, 12.5 Hz), 162.1 (dd, J = 248.0, 10.3 Hz),

170.0, 171.2; HRMS calcd for C₂₃H₁₈F₂N₃O₂(M ꢀ H)^ꢀ406.1373, found

406.1373; mp 147 ^◦C; chemical purity 97.4%, t_R12.3 min.

1

min. 14u: H NMR (CDCl₃) δ 2.31–2.45 (m, 1H), 2.65–2.84 (m, 2H),

3.07–3.19 (m, 1H), 4.35 (s, 1H), 5.12–5.20 (m, 1H), 5.42–5.57 (m, 2H),

6.53 (s, 1H), 6.82 (d, J = 8.3 Hz, 1H), 7.30–7.40 (m, 6H), 7.81 (dd, J =

7.7, 4.6 Hz, 1H), 8.19 (d, J = 6.8 Hz, 1H), 8.72 (dd, J = 4.5, 1.8 Hz, 1H);

¹³C NMR (CDCl₃) δ 26.1, 29.0, 64.1, 66.0, 116.0 (d, J = 23.5 Hz), 123.2,

128.5 (d, J = 14.7 Hz), 128.7, 129.1, 129.18, 129.23, 129.3, 133.4 (d, J

= 8.8 Hz), 136.4, 138.8, 142.6 (d, J = 5.1 Hz), 149.4, 152.8, 158.6 (d, J

= 250.9 Hz), 166.5, 171.1; HRMS calcd for C₂₃H₁₇ClFN₄O₄(M ꢀ H)^ꢀ

467.0928, found 467.0934; chemical purity 84.4%, t_R14.0 min.

5.1.46. N-((R)-Carbamoylphenylmethyl)-N-((R)-6-chloro-4-ﬂuoroindan-

1-yl)nicotinamide (13t), N-((S)-Carbamoylphenylmethyl)-N-((R)-6-

chloro-4-ﬂuoroindan-1-yl)nicotinamide (14t)

The title compounds were prepared from 8n (444 mg), benzaldehyde

(212 mg), triethylamine (279 µL), methanol (4 mL), nicotinic acid (246

mg), 4-phenylcyclohenen-1-yl isocyanide (367 mg), 1,4-dioxane (6 mL),

water (1.5 mL) and 4 mol/L HCl in 1,4-dioxane (1.5 mL) according to the

general procedure. The crude product was puriﬁed using silica gel col-

umn chromatography (eluent EtOAc:Hexane = 2:1–5:1) to afford 13t

(415 mg, 49%, colorless solid, low-polarity) and 14t (225 mg, 27%,

colorless solid, high-polarity). 13t: This compound exists as a mixture of

rotamers in CDCl₃(4:1); ¹H NMR (CDCl₃) δ 1.73–2.15 (m, 2H),

2.45–2.63 (m, 1H), 2.70–3.15 (m, 1H), 4.75 (br s, major 1H), 5.00 (br s,

minor 1H), 5.35–5.76 (m, 3H), 7.00 (br s, 1H), 7.35–7.54 (m, 6H),

7.77–7.92 (m, 2H), 8.69 (d, J = 4.0 Hz, 1H), 8.81 (s, 1H); ¹³C NMR

(CDCl₃) δ 25.1, 26.6 (br s, minor), 29.9 (br s, minor), 31.7, 62.9, 66.0,

66.9 (br s, minor), 114.0–115.0 (m, minor), 116.2 (d, J = 24.2 Hz),

120.1–120.7 (m, minor), 121.8 (d, J = 4.4 Hz), 123.8, 127.9 (d, J = 20.5

Hz), 128.7, 129.0, 129.6, 132.3, 134.2, 134.5, 135.0 (br s, minor),

137.0, 144.6 (br s), 147.3, 148.1 (br s, minor), 151.1, 158.8 (d, J =

250.9 Hz), 170.1, 170.7; HRMS calcd for C₂₃H₁₈ClFN₃O₂(M ꢀ H)^ꢀ

422.1077, found 422.1078; mp 204 ^◦C; chemical purity 98.8%, t_R13.5

5.1.48. N-((R)-Carbamoylphenylmethyl)-N-((R)-6-chloro-4-ﬂuoroindan-

1-yl)-2-methoxynicotinamide (13w), N-((S)-Carbamoylphenylmethyl)-N-

((R)-6-chloro-4-ﬂuoroindan-1-yl)-2-methoxynicotinamide (14w)

The title compounds were prepared from 8n (444 mg), benzaldehyde

(212 mg), triethylamine (279 µL), methanol (4 mL), 2-methoxynicotinic

acid (306 mg), 4-phenylcyclohenen-1-yl isocyanide (367 mg), 1,4-

dioxane (6 mL), water (1.5 mL) and 4 mol/L HCl in 1,4-dioxane (1.5

mL) according to the general procedure. The crude product was puriﬁed

using silica gel column chromatography (eluent EtOAc:Hexane =

1:1–3:1) to afford 13w (388 mg, 43%, colorless amorphous solid, low-

polarity) and 14w (240 mg, 26%, colorless amorphous solid, high-

polarity). 13w: This compound exists as a mixture of rotamers in

CDCl₃; ¹H NMR (CDCl₃) δ 1.70–2.25 (m, 2H), 2.44–3.10 (m, 2H),

3.97–4.10 (m, 3H), 4.52–4.96 (m, 1H), 5.20–5.30 (m, 1H), 5.31–6.03

(m, 2H), 6.83–7.04 (m, 2H), 7.34–7.89 (m, 7H), 8.20–8.30 (m, 1H); ¹³

C

NMR (CDCl₃) δ 25.3, 26.4 (minor), 29.8 (br s, minor), 30.6 (br s, minor),

30.8, 53.4 (minor), 53.8, 62.5, 62.7 (minor), 64.5 (br s, minor), 65.7 (br

s), 114.1 (d, J = 22.0 Hz, minor), 115.5 (d, J = 24.2 Hz, minor), 116.0

13

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

(d, J = 24.2 Hz), 116.9 (br s, minor), 117.4, 119.9, 120.3 (minor), 120.7

(br s, minor), 121.8, 122.6 (br s, minor), 127.8 (d, J = 19.8 Hz), 128.4,

128.6, 128.9, 129.16, 129.24, 132.6 (d, J = 7.3 Hz, minor), 133.8 (d, J

= 8.8 Hz), 135.9 (minor), 136.5, 136.7, 137.2 (minor), 138.8 (minor),

144.7 (d, J = 6.6 Hz), 148.1, 148.7 (minor), 158.68 (d, J = 248.0 Hz,

minor), 158.75 (d, J = 250.9 Hz), 159.4 (br s, minor), 159.6, 161.1

(minor), 166.8 (br s, minor), 168.7, 169.0 (br s, minor), 171.0, 171.2

(minor); HRMS calcd for C₂₄H₂₀ClFN₃O₃(M ꢀ H)^ꢀ452.1183, found

452.1183; chemical purity 93.8%, t_R14.6 min. 14w: ¹H NMR (CDCl₃) δ

2.29–2.90 (m, 3H), 3.01–3.28 (m, 1H), 4.00 (s, 3H), 4.39 (br s, 1H), 5.26

(t, J = 7.9 Hz, 1H), 5.36–6.20 (m, 2H), 6.31–6.50 (m, 1H), 6.76–6.85 (m,

1H), 7.01 (dd, J = 7.0, 5.2 Hz, 1H), 7.23–7.49 (m, 5H), 7.73 (d, J = 6.8

Hz, 1H), 8.25 (dd, J = 4.9, 1.6 Hz, 1H); ¹³C NMR (CDCl₃) δ 25.9, 29.7,

53.9, 63.6, 66.1, 115.6 (d, J = 23.5 Hz), 117.3, 120.1, 122.5 (br s),

128.7, 128.9, 129.6, 133.0 (d, J = 8.8 Hz), 136.6, 137.4, 144.0 (d, J =

5.9 Hz), 148.3, 158.8 (d, J = 250.9 Hz), 159.2, 168.7, 171.7; HRMS

calcd for C₂₄H₂₀ClFN₃O₃(M ꢀ H)^ꢀ452.1183, found 452.1184; chemical

purity > 99.5%, t_R14.4 min.

6 h under H₂atmosphere. 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

= 3:1) to afford 13r as a colorless amorphous solid (95 mg, 68%). H

NMR (CDCl₃) δ 1.28 (d, J = 7.0 Hz, 3H), 4.49 (s, 2H), 4.57 (br s, 1H),

5.19 (q, J = 7.0 Hz, 1H), 5.32 (br s, 1H), 5.54 (br s, 1H), 6.72–6.79 (m,

3H), 7.15–7.23 (m, 4H), 7.35–7.45 (m, 3H), 7.51–7.56 (m, 2H); ¹³C

NMR (CDCl₃) δ 19.4, 57.7, 62.6, 103.6 (t, J = 25.3 Hz), 111.7 (dd, J =

18.8, 7.0 Hz), 116.6, 117.6, 120.8, 126.1, 128.9, 129.1, 129.4, 130.7,

137.6, 143.1 (br s), 144.4, 163.5 (dd, J = 248.7, 12.5 Hz), 171.3, 171.8;

HRMS calcd for C₂₃H₂₀F₂N₃O₂(M ꢀ H)^ꢀ408.1529, found 408.1530;

chemical purity 96.2%, t_R14.5 min.

5.1.51. 2-Amino-N-((R)-carbamoylphenylmethyl)-N-((R)-6-chloro-4-

ﬂuoroindan-1-yl)nicotinamide (13v)

To a solution of 13u (200 mg) in ethanol (10 mL) was added 10% Pd-

C (evonik degussa, E101 NE/W, 50 mg), and the mixture was stirred for

12 h under H₂atmosphere. 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

= 2:1–3:1) to afford 13v as a colorless solid (91 mg, 49%). ¹H NMR

(CDCl₃) δ 1.56–1.80 (m, 1H), 1.97–2.17 (m, 1H), 2.47–2.60 (m, 1H),

2.61–2.80 (m, 1H), 4.65 (br s, 1H), 5.25–5.66 (m, 5H), 6.68 (dd, J = 7.5,

5.0 Hz, 1H), 6.95–7.03 (m, 1H), 7.37–7.55 (m, 6H), 7.70 (br s, 1H), 8.13

(dd, J = 5.0, 1.8 Hz, 1H); ¹³C NMR (CDCl₃) δ 25.4 (br s), 31.8 (br s), 62.6

(br s), 66.0 (br s), 113.1, 115.2 (br s), 115.6–116.4 (m), 122.3 (br s),

128.2, 128.4, 129.2, 129.3, 129.5, 133.9 (br s), 135.0 (br s), 137.0 (br s),

144.3 (br s), 149.8, 155.5 (br s), 158.9 (d, J = 250.2 Hz), 170.2 (br s),

171.4; HRMS calcd for C₂₃H₁₉ClFN₄O₂(M ꢀ H)^ꢀ437.1186, found

5.1.49. N-((R)-Carbamoylphenylmethyl)-N-((R)-6-chloro-4-ﬂuoroindan-

1-yl)-2-methylnicotinamide (13x), N-((S)-Carbamoylphenylmethyl)-N-

((R)-6-chloro-4-ﬂuoroindan-1-yl)-2-methylnicotinamide (14x)

The title compounds were prepared from 8n (444 mg), benzaldehyde

(212 mg), triethylamine (279 µL), methanol (4 mL), 2-methylnicotinic

acid (274 mg), 4-phenylcyclohenen-1-yl isocyanide (367 mg), 1,4-

dioxane (6 mL), water (1.5 mL) and 4 mol/L HCl in 1,4-dioxane (1.5

mL) according to the general procedure. The crude product was puriﬁed

using silica gel column chromatography (eluent EtOAc:Hexane =

2:1–5:1) followed by recrystallization (EtOAc/Hexane) to afford 13x

(306 mg, 35%, colorless solid, low-polarity) and 14x (213 mg, 24%,

colorless solid, high-polarity). 13x: This compound exists as a mixture of

437.1185; mp 162–166 ^◦C; chemical purity 98.4%, t_R13.6 min; [α ²_D⁵

+

]

53.7 (c = 1.22, CHCl₃).

1

rotamers in DMSO‑d₆(6:5); H NMR (DMSO‑d₆) δ 1.22–1.45 (m, 1H),

1.98–2.11 (m, 1H), 2.36–2.65 (m, 4H), 2.83–2.95 (m, 1H), 4.93–5.23

(m, 1H), 5.27 (s, minor 1H), 5.37 (s, major 1H), 7.14 (d, J = 8.8 Hz, 1H),

7.24–7.95 (m, 10H), 8.55 (br s, 1H); ¹H NMR (ethylene glycol-d₆, 298 K)

δ 1.30–1.95 (m, 2H), 2.42–3.04 (m, 5H), 4.98–5.50 (m, 2H), 6.98–7.20

(m, 1H), 7.32–8.15 (m, 8H), 8.64 (br s, 1H); ¹H NMR (ethylene glycol-d₆,

383 K) δ 1.60–2.20 (m, 2H), 2.40–3.10 (m, 5H), 5.20 (t, J = 7.3 Hz, 1H),

5.30 (br s, 1H), 6.98 (d, J = 7.8 Hz, 1H), 7.32–7.49 (m, 6H), 7.60 (br s,

1H), 7.77–7.84 (m, 1H), 8.54 (d, J = 3.8 Hz, 1H); ¹³C NMR (DMSO‑d₆) δ

21.4, 22.3 (minor), 25.7 (minor), 26.0, 29.7, 61.8, 63.6, 65.2 (minor),

113.5 (d, J = 24.2 Hz), 120.2 (minor), 120.8, 121.3 (minor), 121.5,

128.5, 128.7, 128.9, 129.0, 129.2 131.1, 131.6 (minor), 133.0 (minor),

134.4, 135.7 (minor), 136.0, 149.15, 149.22, 149.4, 149.7, 153.1,

154.3, 158.1 (d, J = 246.5 Hz), 168.3, 171.0; HRMS calcd for

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 cal-

cium inﬂux assay, HEK293T cells (RIKEN Cell Bank, Tsukuba, 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 × 10⁴cells/well in

Dulbecco’s modiﬁed Eagle’s medium (Thermo Fisher Scientiﬁc, Wal-

tham, 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). EC₈₀concentration of menthol, AITC, capsaicin and RN-1747

were used as agonists for TRPM8, TRPA1, TRPV1 and TRPV4, respec-

tively. IC₅₀values were calculated from the concentration response

curve with nonlinear regression analysis using GraphPad Prims 4.0

(GraphPad Software Inc., La Jolla, CA).

C

₂₄H₂₀ClFN₃O₂(M ꢀ H)^ꢀ436.1234, found 436.1234; mp 204 ^◦C;

chemical purity 99.1%, t_R13.7 min; [α _D²⁰

]

+ 24.9 (c = 0.58, MeOH). 14x:

1

This compound exists as a mixture of rotamers in DMSO‑d₆(7:5); H

NMR (DMSO‑d₆) δ 2.40 (br s, 3H), 2.60–2.84 (m, 3H), 2.96–3.10 (m,

1H), 5.04–5.16 (m, 1H), 5.31 (s, minor 1H), 5.40 (s, major 1H), 5.70 (br

s, minor 1H), 5.81 (br s, major 1H), 7.02–7.13 (m, 1H), 7.21–7.92 (m,

10H), 8.46–8.60 (m, 1H); ¹³C NMR (DMSO‑d₆) δ 21.5, 21.9 (minor),

26.0, 26.1 (minor), 29.5, 29.8 (minor), 60.9 (minor), 61.1, 64.0, 65.7

(minor), 113.7 (d, J = 24.2 Hz), 118.1 (minor), 118.2, 121.3 (minor),

121.5, 128.3, 128.5, 128.6, 128.9, 129.0, 129.2, 129.7, 131.0, 131.1,

131.2, 131.3, 131.7, 133.0 (minor), 134.1, 136.4 (minor), 136.8, 148.5,

148.6, 149.4, 149.6 (minor), 153.5, 153.7 (minor), 158.1 (d, J = 247.2

Hz), 168.3 (minor), 168.4, 170.4 (minor), 170.5; HRMS calcd for

₂₄H₂₀ClFN₃O₂(M ꢀ H)^ꢀ436.1234, found 436.1233; mp 206 ^◦C;

5.2.2. dGSH trapping assay

C

chemical purity 98.5%, t_R13.3 min; [α _D²⁵

+ 85.3 (c = 1.18, MeOH).

]

Compounds (100 µM) were incubated with human liver microsomes

(Veritas Tokyo, Japan) at a protein concentration of 2 mg/mL in 0.06 M

Tris buffer pH 7.4 (Nacalai Tesque, Kyoto, Japan), containing 10 mM

MgCl₂(FUJIFILM Wako Pure Chemical, Osaka, Japan), and 2 mg/mL

NADPH (Nacalai Tesque) with or without 1 mM dansylated reduced

glutathione (dGSH). After incubation at 37 ^◦C for 0 or 30 min, the re-

5.1.50. 2-Amino-N-((R)-carbamoylphenylmethyl)-N-[(R)-1-(3,5-

diﬂuorophenyl)ethyl]benzamide (13r)

To a solution of 13q (150 mg) in THF (5 mL) was added 10% Pd-C

(evonik degussa, E101 NE/W, 50 mg), and the mixture was stirred for

actions were terminated with the addition of ice-cold

5 mM

14

J.-i. Kobayashi et al.

Bioorganic & Medicinal Chemistry 30 (2021) 115903

dithiothreitol/MeOH. After centrifugation (1800g, 20 min, 4 ^◦C), the

supernatants were dried under a nitrogen gas stream at 40 ^◦C. The res-

idue was dissolved in a mixture of acetonitrile and 0.5% formic acid

(2:8, v/v) and analyzed by HPLC (1100 series, Agilent Technologies,

Santa Clara, CA, USA) coupled with a ﬂuorescence detector (FP-920,

JASCO, Tokyo, Japan) and a mass spectrometer (ZQ, Waters, Milford,

MA, USA). The excitation and emission wavelengths for the ﬂuorescence

detector were set at 340 and 525 nm, respectively. The relative amount

(%) of dGSH adduct of the test compounds was determined by the

ﬂuorescence relative to the diclofenac dGSH adduct.

5.2.7. Icilin-induced wet-dog shake

Vehicle or test compound was orally administered to female rats.

After 1 or 4 h, icilin (1 mg/mL, in polyethylene glycol 400) was intra-

peritoneally administered and wet-dog shakes were induced. From 5

min after administration of icilin, wet-dog shakes were counted for 5

min. Results were calculated as percent inhibition of wet-dog shakes

compared with vehicle-treated wet-dog shake counts.

5.2.8. Voiding behavior measurements in rats exposed to cold

Vehicle or test compounds were orally administered after rats were

acclimated to a metabolic cage (Tecniplast Japan Co. Ltd., Tokyo,

Japan) mounted on an electrical balance (A&D Company, Ltd., Tokyo,

Japan) connected to a computer at room temperature (23–26 ^◦C). One

hour after drug administration, rats were orally loaded with 4 mL of

water and transferred to the metabolic cage placed in a low temperature

incubator (Fukushima Industries Corp., Osaka, Japan) regulated at 4 ^◦C.

Then, the cumulative voided volume was recorded on a computer at 2-

min intervals for 4 h and the voiding behavior of rats exposed to cold

was evaluated as follows: a change in the urine weight (>0.1 g) was

regarded as a voiding episode and the speciﬁc gravity of urine was

deﬁned as 1 g/mL. The mean voided volume, voiding frequency, and

total voided volume were calculated.

5.2.3. PXR binding assay

The LanthaScreen® TR-FRET PXR competitive binding assay was

conducted in accordance with the manufacturer’s (Thermo Fisher Sci-

entiﬁc, Waltham, MA, USA) protocols. The test compounds were diluted

in TR-FRET PXR assay buffer and 10 µL/well of these dilutions were

dispensed in quadruplicate to ﬁnal concentrations of 0.003–60 µM.

Fluormone PXR green (5 µL/well) at a ﬁnal concentration of 40 nM was

added. The following mixture (5 µL/well) was added: dithiothreitol,

terbium-labeled anti-glutathione S-transferase antibody, and hPXR-LBD

at ﬁnal concentrations of 50 µM, 10 nM, and 5 nM, respectively. The

plate was incubated for 1 h at room temperature in the dark. An Inﬁn-

ite® M1000 plate reader was used to measure the TR-FRET. For the TR-

FRET ratios, the emission signal at 520 nm was divided by the emission

signal at 495 nm. The EC₅₀values were calculated from the concen-

tration–response curve with nonlinear regression analysis using

GraphPad Prism 4.0 (GraphPad Software Inc., La Jolla, CA, USA).

5.2.9. Pharmacokinetic study in rats

Rat blood was collected from the jugular vein at 2, 15, and 30 min

and 1, 2, 4 and 6 h after single oral or intravenous administration of test

compound to rats. The plasma concentrations were measured using an

LC-MS/MS

(API-4000,

Applied

Biosystems/MDS

Analytical

5.2.4. CYP3A4 inhibition assay

Technologies).

Test compounds (100 µM) were pre-incubated at 37 ^◦C with human

liver microsomes (Veritas) at a protein concentration of 0.5 mg/mL in

0.1 M phosphate buffer pH 7.4 (Nacalai Tesque) containing 5 mM MgCl₂

(FUJUFILM Wako Pure Chemical) and 1 mM NADPH (Nacalai Tesque).

The mixture was diluted 10-fold with 25 µM midazolam solution con-

taining 0.1 M phosphate buffer pH 7.4, 5 mM MgCl₂, and 1 mM NADPH.

After incubation at 37 ^◦C for 10 min, the reactions were terminated by

the addition of ice-cold acetonitrile. After deproteinization by centrifu-

Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

◦

Acknowledgements

gation (1800g, 20 min, 4 C), the supernatants were prepared as the

measurement samples. Ketoconazole (1 µM) was used as a typical

CYP3A4 competitive inhibitor. The amount of midazolam metabolite,

1^′-hydroxymidazolam, was determined by HPLC (LC-10A, Simadzu,

Kyoto, Japan) coupled with MS/MS (API-4000, AB Sciex, Framingham,

MA, USA).

We thank Mr. Naohiro Kawamura for synthesis of the compounds,

and Ms. Akane Matsuzawa for the biological assay. We also thank Dr.

Tomonaga Ozawa for valuable discussions. This research received no

speciﬁc grant from any funding agency in the public, commercial, or

not-for-proﬁt sectors.

5.2.5. CYP3A4 mRNA induction assay

The process of preparing differentiated HepaRG® cell cultures was

performed according to the Biopredic protocol. HepaRG® cells were

cultured at 3.6 × 10⁴cells/well in Williams E medium supplemented

Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bmc.2020.115903.

´

with ADD670C (Biopredic International, Saint Gregoire, France) in a

type I collagen coated plate (Corning, Corning, NY, USA) for 72 h. After

removing the media, the HepaRG® cells were treated with rifampicin

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16

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Identification of N-acyl-N-indanyl-α-phenylglycinamides as selective TRPM8 antagonists designed to mitigate the risk of adverse effects

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