Discovery of a novel series of indolinylpyrimidine-based GPR119 agonists: Elimination of ether-a-go-go-related gene liability using a hydrogen bond acceptor-focused approach

Bioorg. Med. Chem. 34 (2021) 116034

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Bioorganic & Medicinal Chemistry

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Discovery of a novel series of indolinylpyrimidine-based GPR119 agonists:

Elimination of ether-a-go-go-related gene liability using a hydrogen bond

acceptor-focused approach

,

Masahiro Kamaura^a, Osamu Kubo^{a *}, Hiromichi Sugimoto^a, Naoyoshi Noguchi^a,

Hirohisa Miyashita^a, Shinichi Abe^a, Kae Matsuda^a, Yoshiyuki Tsujihata^a, Tomoyuki Odani^b,

Shinji Iwasaki^c, Toshiki Murata^a, Kenjiro Sato^a

^aCardiovascular & Metabolic Drug Discovery Unit, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan

^bBiomolecular Research Laboratories, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan

^cDMPK Research Laboratories, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan

A R T I C L E I N F O

A B S T R A C T

Keywords:

We previously identiﬁed a novel series of indolinylpyrimidine derivatives exempliﬁed by 2 in Figure 1, which is

an indoline based derivative, as potent GPR119 agonists. Despite the attractive potency of 2, this compound

inhibited the human ether-a-go-go-related gene (hERG) K⁺channel. We elucidated crucial roles of the methyl-

sulfonyl group of 2 in its interaction with the hERG channel and the GPR119 receptor, presumably as a hydrogen

bond acceptor (HBA). To remove the undesirable hERG inhibitory activity, a strategy was implemented to

arrange an HBA on a less conformationally ﬂexible framework at the indoline 5-position instead of the meth-

ylsulfonyl group. This successfully led to the discovery of a piperidinone ring as a desirable motif at the indoline

5-position, which could minimize hERG liability as shown by 24b. Further optimization focused on the reduction

of lipophilicity in terms of more favorable drug-like properties. Consequently, the introduction of a hydroxy

group at the 3-position of the piperidinone ring effectively reduced lipophilicity without compromising GPR119

potency, resulting in the identiﬁcation of (3S)-3-hydroxy-1-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]

piperidin-4-yl}oxy)pyrimidin-4-yl]- 2,3-dihydro-1H-indol-5-yl}piperidin-2-one ((S)-29) as a novel, potent, and

orally bioavailable GPR119 agonist with a well-balanced proﬁle. The pharmacological effects of this compound

were also conﬁrmed after single and chronic oral administration in diabetic animal models.

GPR119 agonist

GPCR

Type 2 diabetes mellitus

Indolinylpyrimidine

hERG

Hydrogen bond acceptor

1. Introduction

novel anti-diabetic agents without risk of hypoglycemia and are

considered to have potential therapeutic beneﬁts for obesity.^6,11

The GPR119 receptor has emerged as an attractive target for the

development of novel therapeutics against type 2 diabetes mellitus

(T2DM).^1–5The receptor is a member of class A GPCR, which is pre-

dominantly expressed on the pancreatic β-cells and enteroendocrine

cells within the gastrointestinal tract. Although several potential

endogenous ligands of GPR119, including oleoylethanolamide, have

been reported,^6–10their physiological importance remains unclear due

to their relatively low afﬁnity and/or insufﬁcient selectivity over other

targets. GPR119 receptor activation leads to insulin secretion from the

pancreatic β-cells in a glucose concentration-dependent manner and also

promotes secretion of incretins, such as GLP-1 and GIP, from the

enteroendocrine cells. As such, GPR119 agonists are expected to act as

Our previous report described the identiﬁcation of two novel series

of indoline-based GPR119 agonists with potent agonist activity, i.e.,

indoline carbamate derivatives and indolinylpyrimidine derivatives, as

exempliﬁed by 1 and 2, respectively.¹²Of the two series, indolinylpyr-

imidine derivatives were found to possess favorable metabolic stability

and low in vivo clearance. Despite its attractive in vitro potency and

pharmacokinetic proﬁle, however, further assessment revealed that

compound 2 potently blocked the human ether-a-go-go-related gene

(hERG) K⁺channel current, prohibiting further development of this

compound. Accordingly, our subsequent optimization effort was mainly

directed at addressing the remaining undesirable hERG issue of indoli-

nylpyrimidine derivatives. We identiﬁed the critical roles of the

* Corresponding author.

E-mail address: osamu.kubo@takeda.com (O. Kubo).

https://doi.org/10.1016/j.bmc.2021.116034

Received 25 November 2020; Received in revised form 13 January 2021; Accepted 16 January 2021

Available online 23 January 2021

Published by Elsevier Ltd.

This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

hydrogen bond acceptor (HBA) at the indoline 5-position in terms of

interaction with the hERG channel and activating the GPR119 receptor.

To remove the hERG inhibitory activity, a series of compounds with HBA

arranged in a conformationally restricted manner at the indoline 5-posi-

tion was designed. This led to the successful identiﬁcation of (S)-29,

characterized by potent GPR119 agonist activity, safe proﬁles, and

favorable pharmacokinetic properties. Herein, we report the design,

synthesis, and biological activity of indolinylpyrimidine derivatives as

novel GPR119 agonists.

(RS)-29 was synthesized similar to piperidinone 24b (Scheme 5).

The requisite carboxylic acid (RS)-26 was prepared from commercially

available tetrahydrofuran-2-carboxylic acid ((RS)-25) in three steps.¹⁵

Following acylation of aniline 21 with the acyl chloride prepared from

(RS)-26, intramolecular cyclization proceeded cleanly upon treatment

with potassium carbonate, followed by deacetylation to provide the

lactam (RS)-28 in high yield. Protecting group manipulation and con-

struction of a 1,2,4-oxadiazole ring using the same method (described in

Scheme 4) furnished the ﬁnal product, (RS)-29. To access the enantio-

meric pure isomers of (RS)-29, an identical synthetic procedure for (RS)-

29 was followed using chiral starting material (R)-25 or (S)-25. Conse-

quently, both enantiomers (R)-29 and (S)-29 were obtained without loss

of enantiomeric purity.

2. Chemistry

Scheme 1 describes the synthetic steps to N-thiocarbamate derivative

6. Compound 4, which was reported in our previous report, was treated

with HCl to remove the tert-butyloxycarbonyl (BOC) protecting group,

followed by reaction with isopropyl chlorothioformate to give 6 in good

yield.

The synthetic schemes of derivatization via 5-bromoindoline inter-

mediate 10 are summarized in Scheme 6. Syntheses of racemic 4- and 5-

hydroxypiperidinones (RS)-32a and 32b were accomplished by the

–

Ullmann–Goldberg type C N coupling of 5-bromoindoline 10 with

hydroxypiperidinones (RS)-30a or 30b and subsequent 1,2,4-oxadiazole

ring construction at the piperidine nitrogen. The palladium-catalyzed C-

arylation^16,17of cyclohexanone with 10 provided (RS)–33, a C-linked

analog of lactam 23b, which was converted to the ﬁnal product (RS)-34

as previously described.

Syntheses of 5-[(2-hydroxyethyl)sulfonyl]indolines and 5-[(2-

hydroxyethyl)sulﬁnyl]indolines were accomplished using the method

outlined in Scheme 2. The 5-bromoindoline 10, which was prepared in a

two-step sequence from 7 and 8 in the same manner as 4, was converted

to sulﬁde 11 by palladium-catalyzed coupling with 2-mercaptoethanol.

Oxidation of sulﬁde 11 by mCPBA delivered sulfone 12 and sulfoxide

(RS)-14. After the removal of the BOC group of 12 and (RS)-14, 1,2,4-

oxadiazole ring construction or thiocarbamate formation on the piper-

idine nitrogen afforded 13, (RS)-15a, or (RS)-15b, respectively. To

obtain single enantiomers of (RS)-15b, chiral preparative HPLC reso-

lution of racemic (RS)-15b was conducted to yield both enantiomers

15b-ent1 and 15b-ent2.

3. Results and discussion

The newly synthesized compounds were tested for functional

GPR119 agonism using a cAMP reporter assay in Chinese hamster ovary

(CHO) cells stably expressing human GPR119. In vitro metabolic sta-

bility was assessed by monitoring the disappearance of a parent com-

pound after incubation with human or rat liver microsomes and is

The 5-H-indoline derivative 17 was synthesized via the reductive

debromination of 10 (Scheme 3)

expressed as CL_int

hERG channel was measured in vitro using an automated patch-clamp

assay system at 10 or 30 M compound concentrations.

(μL/min/mg). The inhibitory activity against the

Scheme 4 describes in detail the syntheses of compounds bearing

various heterocycles at the 5-position on the indoline ring via aniline

intermediate 21. Lactam rings 23a and 23b and morpholinone ring 23c

were constructed by intramolecular cyclization of 22a–c, which were

prepared by acylation of 21 with appropriate acyl chlorides. Similarly,

the sulfonamidation of 21 with 3-chloropropanesulfonyl chloride and

subsequent intramolecular cyclization gave isothiazolidine 23d in fair

yield. To access piperazinone derivatives, the N-(2-hydroxyethyl)glyci-

namide derivative 22e was prepared by the chloroacetylation of 21,

followed by treatment with 2-aminoethanol. Alcohol 22e underwent

intramolecular cyclization upon Mitsunobu reaction, using the condi-

tion ⁿBu₃P/ADDP to afford the desired piperazinone 23e, after pro-

tecting piperazine nitrogen with a triﬂuoroacetyl group.

μ

To obtain in-depth proﬁles of indoline carbamate derivative 1 and

indolinylpyrimidine derivative 2 for the development of novel GPR119

agonists, we conducted further evaluation of these compounds in terms

of DMPKTox proﬁles (Table 1). As a result, indolinylpyrimidine deriv-

ative 2 was found to exhibit favorable metabolic stability, whereas

indoline carbamate derivative 1 was rapidly cleared by microsomes.

These in vitro metabolic stabilities were reﬂected through in vivo clear-

ance. That is, compound 2 exhibited much lowered clearance compared

with 1 after intravenous administration in rats (CL_total= 230 mL/h/kg

for 2; 2166 mL/h/kg for 1). The same trend of metabolic stability be-

tween indolinylpyrimidine and indoline carbamate derivatives was

observed for compounds with various piperidine N-substituents (data

not shown). We assumed that the high microsomal clearance of indoline

carbamate derivatives had been caused by metabolic vulnerability of the

central aliphatic hydrocarbon chains and/or relatively high ﬂexibility of

the central spacer, which may have allowed molecules to easily adopt

conformations ﬁt for binding to metabolism enzymes, such as CYPs.

In addition, further assessment revealed that compounds 1 and 2

strongly blocked the hERG channel current by automated patch-clamp

Synthesis of 1,2,6-thiadiazinane derivative 23f included a prepara-

tion of sulfamide 22f using a mild sulfamoylating reagent^{SPS:reﬁd::bib1313}

adopted by Montero et al. The sulfamide 22f was subjected to reaction

with 1,3-dibromopropane in the presence of potassium carbonate to

furnish BOC-protected 23f. The obtained 23a–f were processed to pro-

duce 1,2,4-oxadiazole derivatives 24a–f.

During our research program, Augustine et al. reported an efﬁcient

one-pot synthesis of C-linked 1,2,4-oxadiazoles from nitrile compounds

using a PTSA/ZnCl₂system as a catalyst.¹⁴We applied this methodology

to selected N-cyano compounds derived from 23, which afforded the

desired N-linked 1,2,4-oxadiazoles 24 in moderate to good yields.

Acyclic analog 24 g was obtained using similar synthetic methods.

assay (95% and 106% inhibition at 10 μM, respectively). Accordingly,

our optimization effort was directed at minimizing undesirable hERG

activity by focusing on indolinylpyrimidine derivatives, which exhibited

better metabolic stability. (See Figure 1).

Scheme 1. Synthesis of indolinylpyrimidine derivative 6^a. ^aReagents and conditions: (a) HCl, AcOEt, MeOH, rt, 94%; (b) ⁱPrSCOCl, TEA, THF, rt, 94%.

2

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

Scheme 2. Synthesis of 2-hydroxyethylsulfone 13 and 2-hydroxyethylsulfoxides 15a–b^a. ^aReagents and conditions: (a) EtOH, reﬂux, 83%; (b) tert-butyl 4-hydrox-

ypiperidine-1-carboxylate, NaH, THF, rt, 92%; (c) 2-mercaptoethanol, Pd₂(dba)₃, Xantphos, DIEA, toluene, 80 ^◦C, 62%; (d) mCPBA (2 eq.), AcOEt, rt, 89%; (e)

mCPBA (1 eq.), AcOEt, rt, 64%; (f) (1) HCl, AcOEt, rt; (2) BrCN, NaHCO₃, THF, H₂O, 0 ^◦C to rt; (3) ⁱPrCNH(NHOH), ZnCl₂, AcOEt, THF, DMSO, 80 ^◦C; (4) conc. HCl,

EtOH, 70 ^◦C, 67% (for 13) and 28% (for (RS)-15a); (g) (1) HCl, AcOEt, rt; (2) ⁱPrSCOCl, TEA, THF, rt, 92% (for (RS)-15b); (h) chiral HPLC separation.

Scheme 3. Synthesis of 5-H-indoline 17^a. ^aReagents and conditions: (a) H₂(1 atm), Pd on carbon, THF, MeOH, rt, 48%; (b) (1) TFA, rt; (2) ⁱPrSCOCl, TEA, THF,

rt, 90%.

A high degree of hERG inhibitory activities was also observed for N-

carbamate 3 and N-thiocarbamate 6 (Table 2), regardless of the acyclic

or cyclic structures of the piperidine N-substituent. Interestingly, the

deletion of methylsulfonyl group 6 resulted in a complete loss of hERG

inhibitory activity, accompanied by a rapid drop in GPR119 potency,

despite the substantially increased lipophilicity of compound 17

compared with 6, with a change in LogD_7.4from 3.6 to 5.3. This high-

lighted the critical roles of the methylsulfonyl group in the interaction

with the hERG channel and in the activation of GPR119. Hence, struc-

tural modiﬁcation to address the hERG issue was focused on the sub-

stituent at the indoline 5-position. We presumed that the sulfonyl group

would make crucial contact with the hERG channel as an HBA since the

role of HBA became more prominent in the case of uncharged hERG

ligands.¹⁸It was also assumed that the sulfonyl group functioned as an

HBA during GPR119 activation.¹⁹

on hERG inhibitory activity from the comparison of 2 and 6. These re-

sults indicated that the oxygen atom of the sulfonyl or sulﬁnyl group

would be involved in the interaction with the hERG channel, presum-

ably as HBA, and implied that structural modiﬁcation of a substituent at

the indoline 5-position with an appropriate arrangement of HBA could

control GPR119/hERG selectivity. Since a sulfoxide group can poten-

tially be susceptible to interconversion to the antipodal sulfoxide in

vivo,²²sulfoxide-containing derivatives were not further pursued.

5-position would change the GPR119/hERG selectivity by the com-

bination of the restricted orientation of HBA and steric hindrance of the

framework. Accordingly, a series of derivatives with an HBA-containing

heterocycle or carbocycle, rather than the alkylsulfonyl group, was

designed to identify novel indoline-based GPR119 agonists devoid of

hERG liability (Figure 2). We planned to incorporate a carbonyl group

and a sulfonyl group as HBA since one oxygen atom at the indoline 5-po-

sition was found to be sufﬁcient for activating the GPR119 receptor.

The GPR119 agonist activities and hERG inhibitory activities of

compounds with various substituents at the indoline 5-position are

presented in Table 3 alongside their microsomal clearances. For the

piperidine N-substituent, the 3-isopropyl-1,2,4-oxadiazole was selected

owing to its balanced potency proﬁle and metabolic stability. Our results

indicated that most of the compounds bearing a cyclic substituent

exhibited dramatically improved hERG inhibition proﬁles while main-

taining potent GPR119 activity. Conversion of the methylsulfonyl group

to a lactam ring conferred an excellent hERG proﬁle, despite the rela-

tively high lipophilicity of 24a and 24b (LogD_7.4= 3.7 for 24a and 3.8

for 24b). Among these, piperidinone 24b exhibited the desired meta-

bolic stability against both human and rat microsomes. The cyclohexa-

none derivative (RS)-34 (with greater hydrophobicity) also exhibited a

loss of hERG inhibitory activity. However, this compound was

Our ﬁrst approach for mitigating hERG inhibitory activity included

the conversion of the sulfonyl group to a sulfoxide group and reduction

of the lipophilicity of compounds.^20,21Introduction of a polar substitu-

ent, such as a (2-hydroxyethyl)sulfonyl group (13), maintained strong

hERG inhibitory activity and potent GPR119 agonist activity. Trans-

formation of the sulfonyl group into a sulfoxide group afforded racemic

(RS)-15a and (RS)-15b, for which no beneﬁcial effects on hERG liability

were observed. However, one enantiomeric isomer, 15b-ent1, exhibited

improved hERG inhibitory activity compared with antipode 15b-ent2,

while both compounds showed sufﬁcient GPR119 agonist activity. In

addition, comparing 13 and 15b-ent1, the GPR119 agonist activity was

equivalent for these 2 compounds, but the hERG inhibitory activity of

15b-ent1 was improved over that of 13. Although the right terminal

moiety of the molecule is different between 13 and 15b-ent1, it is un-

likely that the right terminal moiety of 15b-ent1 had a positive impact

3

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

Scheme 4. Synthesis of indolinylpyrimidine derivatives 24a–g^a. ^aReagents and conditions: (a) EtOH, reﬂux, 83%; (b) tert-butyl 4-hydroxypiperidine-1-carboxylate,

NaH, THF, rt, 69%; (c) H₂(1 atm), Pd on carbon, THF, MeOH, rt, 100%; (d) 4-chlorobutyryl chloride, TEA, THF, rt (for 22a); (e) 5-chlorovaleryl chloride, DMA, 0 ^◦C

to rt, 86% (for 22b); (f) (2-chloroethoxy) acetyl chloride, TEA, THF, rt, 96% (for 22c); (g) NaH, DMF, 0 ^◦C to rt, 81%–97%; (h) (1) HCl/AcOEt or TFA, rt; (2) BrCN,

NaHCO₃, THF, H₂O, 0 ^◦C to rt, 37%–98% over two steps; (3) ⁱPrCNH(NHOH), ZnCl₂, p-TsOH, DMF, 80 ^◦C, 36%–65% (for 24b–c, 24e, 24f, and 24 g); (i) (1) HCl,

AcOEt, rt; (2) BrCN, NaHCO₃, THF, H₂O, 0 ^◦C to rt; (3) ⁱPrCNH(NHOH), ZnCl₂, AcOEt, THF, reﬂux; then conc. HCl, EtOH, 70 ^◦C, 31%–34% (for 24a and 24d); (j) 3-

chloropropanesulfonyl chloride, TEA, THF, rt; (k) (1) chloroacetyl chloride, DMA, rt, 99%; (2) 2-aminoethanol, THF, ⁱPrOAc, reﬂux, 98%, (l) (1) ADDP, ⁿBu₃P, THF,

rt to 60 ^◦C; (2) TFAA, TEA, AcOEt, rt, 46% over two steps; (m) (1) NaOH, H₂O, MeOH, THF, rt; (2) HCl, MeOH, rt, 95%; (n) (tert-butoxycarbonyl){[4-(dimethy-

liminio)pyridin-1(4H)-yl]sulfonyl}azanide, THF, 50 ^◦C, 97%; (o) 1,3-dibromopropane, K₂CO₃, acetone, DMA, 70 ^◦C, 100%; (p) AcCl, TEA, THF, 0 ^◦C to rt, 95%; (q)

NaH, MeI, DMF, rt, 78%.

Scheme 5. Synthesis of 3-hydroxylpiperidinones (RS)-29, (R)-29, and (S)-29^a. ^aReagents and conditions: (a) (1) (COCl)₂, BnOH, pyridine, Et₂O, rt, 63%–69%; (2)

ZnCl₂, AcCl, reﬂux, 64%–83%; (3) H₂(1 atm), Pd on carbon, AcOEt, rt, 99%–100%; (b) (COCl)₂, rt, then 21, DMA, rt, 81%–85%; (c) (1) K₂CO₃, DMF, 50 ^◦C; (2)

K₂CO₃, MeOH, THF, rt, 86%–90% over two steps; (d) (1) HCl, AcOEt, rt; (2) BrCN, NaHCO₃, THF, H₂O, rt, 70%–94% over two steps; (3) ⁱPrCNH(NHOH), ZnCl₂, p-

TsOH, DMF, 85 ^◦C, 29%–52%.

4

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

Scheme 6. Synthesis of 4- and 5-hydroxypiperidinones (RS)-32a–b and cyclohexanone (RS)-34^a. ^aReagents and conditions: (a) CuI, trans-N,N’-dimethylcyclohexane-

1,2-diamine, K₂CO₃, toluene, 120 ^◦C, 4%–10%; (b) (1) HCl, AcOEt, rt; (2) BrCN, NaHCO₃, THF, H₂O, 0 ^◦C to rt, 83%–87% over two steps; (3) ⁱPrCNH(NHOH), ZnCl₂,

p-TsOH, DMF, 80 ^◦C, 11%–67%; (c) cyclohexanone, Pd₂(dba)₃, Xantphos, Cs₂CO₃, DME, 80 ^◦C, 24%.

Table 1

Proﬁles of indoline-based GPR119 agonists 1 and 2.

GPR119^aEC₅₀(nM)

CL_int

(μ

L/min/mg)

RLM

CL_total^c(mL/h/kg)

hERG^d%inhibition at 10

μM

LogD_7.4

b

e

Compound

HLM

1

2

3.9

7.7

329

69

365

ND

2,861

230

75 ± 10

106 ± 9

3.6

3.1

a

Agonist activity against human GPR119; EC₅₀values are expressed as means (n = 2).

b

c

d

HLM/RLM: Human/rat liver microsomal clearance; ND: a decrease in compound concentration was not observed.

Rat, 0.1 mg/kg, iv.

Automated patch-clamp assay; percentages of inhibition are expressed as means ± standard deviation (n = 4). ^eLogD value at pH 7.4.

Fig. 1. Indoline-based GPR119 agonists.

metabolically more vulnerable than piperidinone 24b, presumably due

to its more lipophilic nature. Contrary to expectations based on lip-

ophilicity, embedding an additional heteroatom in the piperidinone ring

had a somewhat negative impact on hERG inhibition and/or metabolic

stability (24c and 24e).

crystallographic analysis was conducted (Figure 3). The piperidinone

ring of 24b adopted a nearly perpendicular orientation to the indoline

ring. Considering the conformational similarity to aryl–sulfonyl systems,

which prefer orthogonal conformations with torsion angles of the

C unit between 60 and 120 ,²³we assumed that the carbonyl

– –

C S

◦

–

C

–

Interestingly, isothiazolidine 24d and 1,2,6-thidiazinane 24f

exhibited signiﬁcantly reduced hERG liability, regardless of bearing a

sulfonyl group. These improvements in hERG proﬁles indicated that the

introduction of a cyclic structure at the indoline 5-position would have

effectively interfered with the interaction between the compound and

the hERG channel as expected. A direct comparison between 24b and

the corresponding acyclic analog 24 g underscored the advantageous

effect of cyclization on GPR119/hERG selectivity. Taken together, a

lactam ring, particularly a piperidinone ring, emerged as a favorable

motif at the indoline 5-position, which suggested a well-balanced proﬁle

with low hERG liability.

group of the lactam ring of 24b would be able to function as a good

surrogate (as an HBA) for the sulfonyl group in terms of GPR119

agonism.

We also assumed that the decrease in hERG inhibitory activity of 24b

may have been driven by an unfavorable orientation of the carbonyl

group for interaction with the hERG channel. In addition, it was pre-

sumed that the steric bulk of the alkylene moiety of the lactam ring

disrupted the interaction within the spatially limited hERG channel

binding site.

Although the high GPR119/hERG selectivity of piperidinone 24b

was attractive, the relatively high LogD value of this compound moti-

vated us to reduce lipophilicity from the perspective of providing a

To understand the conformational preference of 24b, X-ray

5

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

Table 2

The GPR119 agonist activity and hERG inhibitory activity of indolinylpyrimidine derivatives: the effect of piperidine N-substituents and substituents at the indoline 5-

position.

R¹

H

R²

GPR119^aEC₅₀(nM)

17

hERG^b%inhibition at 10

μM

LogD_7.4

3.1

c

Compound

3

84 ± 14

6

3.2

89 ± 17

3.6

5.3

2.7

17

13

> 10,000

41

14 ± 5(16 ± 6)^d

102 ± 12

(RS)-15a

48

107 ± 2

2.7

(RS)-15b

15b-ent1

15b-ent2

14

57

9.7

95 ± 7

3.3

44 ± 10

101 ± 5

a

Agonist activity against human GPR119; EC₅₀values are expressed as means (n = 2).

b

c

d

Automated patch-clamp assay; percentages of inhibition are expressed as means ± standard deviation (n = 4).

LogD value at pH 7.4.

Data at 30 µM.

Table 3

The GPR119 agonist activity, hERG inhibitory activity, and microsomal clear-

ance of indolinylpyrimidine derivatives: introduction of cyclic substituents at

the indoline 5-position.

Fig. 2. The design of a novel GPR119 agonists with a cyclic LHS motif.

better opportunity for successful development.²⁴

d

e

Compound

R

GPR119^a

EC₅₀

hERG^b

CL_int

(μ

L/

LogD_7.4

3.7

Furthermore, we predicted that reducing lipophilicity could further

improve metabolic stability in 24b rats. Based on our SAR data, it was

assumed that the introduction of a hydroxy group in the substituent at

the indoline 5-position would be tolerated for GPR119 activity. Thus, 3-,

4-, and 5-hydroxy piperidinones were designed and synthesized

(Table 4). As expected, the installation of a hydroxy group effectively

reduced the lipophilicity of compounds, and the lowered degree was

distinct, depending on the position of OH-substitution. The 3-OH de-

rivative (RS)-29 was of comparable potency to the parent 24b and was

superior to the 4-OH (RS)-32a and 5-OH isomer (RS)-32b. In terms of

lipophilic efﬁciency,²⁵(RS)-29 exhibited the highest LLE value (pEC₅₀

ꢀ LogD_7.4) among the three regioisomers (LLE = 4.0 for 24b, 4.5 for

(RS)-29, 4.3 for (RS)-32a, and 4.4 for (RS)-32b). All hydroxylated

compounds were also found to exhibit desirable hERG inhibition proﬁles

and good stability in both human and rat liver microsomes. To fully

investigate the 3-OH derivative (RS)-29 with a high LLE value, the

enantiomeric pure isomers of this compound, (R)-29 and (S)-29, were

prepared. These isomers exhibited almost the same GPR119 agonist

activity and a clean hERG proﬁle, and good clearance in human/rat liver

microsome. Both isomers also showed good in vivo clearance when dosed

intravenously in rats. Among them, the (S)-isomer demonstrated a lower

in vivo clearance (148 mL/h/kg) than the (R)-isomer (375 mL/h/kg),

which made this compound an attractive candidate for further

evaluation.

%inhibition

min/mg)

HLM

(nM)

at 10

μ

M

RLM

138

24a

33

20 ± 3

ND

24b

17

48

15

36

9.7

31 ± 4

ND

78

13

141

46

98

1

3.8

4.7

3.1

2.7

3.2

(21 ± 2)^c

13 ± 6

(RS)-34

24c

54 ± 7

15 ± 2

19 ± 2

27

24e

ND

18

24d

24f

24

75

20 ± 7

79

21

17

3.2

3.7

24 g

66 ± 15

173

(108 ± 3)^c

a

Agonist activity against human GPR119; EC₅₀values are expressed as means

(n = 2).

b

Automated patch-clamp assay; percentages of inhibition are expressed as

means ± standard deviation (n = 4).

c

Data at 30 µM.

d

HLM/RLM: Human/rat liver microsomal clearance; ND: decrease in com-

Prior to in vivo evaluation, we prepared CHO cells stably expressing

rat GPR119 and evaluated the agonist activity of selected compounds.

Compound (S)-29 effectively increased intracellular cAMP level in rat

GPR119, expressing cells with an EC₅₀value of 120 nM (Table 5). To

further assess functional agonism for GPR119, an insulin-secretion assay

using HIT-T15 cells and a GLP-1-secretion assay using GLUTag cells

were conducted. Compound (S)-29 exerted potent hormone secretion

pound concentration was not observed.

e

LogD value at pH 7.4.

activities with EC₅₀values of nearly 10 nM in both assays. We observed a

good correlation between cAMP-based GPR119 agonism and these

hormone secretion activities (data not shown), conﬁrming that our

indoline-based chemotype could secrete several hormones by activating

6

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

dependent manner. These results strongly supported the usefulness of

GPR119 agonists for the treatment of T2DM.

3

1

The results from the in vitro safety assessment of compound (S)-29,

including the inhibition of the CYP isoforms, induction of CYP3A4, ATP

as a marker for cell viability, phototoxicity, time-dependent CYP inhi-

bition, phospholipidosis, and mutagenic side effects were also prom-

ising. In addition to the potent in vivo pharmacological effects, these

safety proﬁles warrant further development of (S)-29 as a useful anti-

diabetic agent.

4. Conclusion

Fig. 3. Single-crystal X-ray structure of compound 24b: (A) frontal view to the

It important that a new anti-diabetic agent without side effects be

made available, particularly as it relates to cardiovascular concerns. To

develop a novel class of GPR119 agonists with desirable DMPKTox

proﬁles, we continued our optimization campaign of indolinylpyr-

imidine derivatives to primarily address potential hERG liability.

Structural modiﬁcation based on an HBA-focused approach proved

effective in removing undesirable hERG inhibitory activity; however,

reducing the lipophilicity of compounds had a marginal impact on the

hERG proﬁles for this class of derivatives. We designed a series of

compounds with an HBA-containing heterocycle or carbocycle to restrict

HBA orientation, together with the introduction of steric bulk. Using this

approach, we found that a lactam ring served as an excellent substituent

at the indoline 5-position, which may offer an improved hERG proﬁle

coupled with high GPR119 potency. Furthermore, we pursued more

desirable physicochemical properties for a successful drug development,

particularly with a focus on lipophilicity reduction. Consequently, the

appropriate installation of a hydroxy group on the lactam ring resulted

in favorable lipophilicity without compromising GPR119 potency. A

representative compound (S)-29 demonstrated sufﬁcient in vivo phar-

macological effects after single and chronic oral administration in dia-

betic animal models, alongside excellent DMPKTox proﬁles.

central pyrimidine ring; (B) side view to the central pyrimidine ring.

GPR119.

Next, we investigated pharmacokinetic parameters when compounds

were dosed orally in rats at a dose of 1 mg/kg. Pharmacokinetic studies

revealed that compound (S)-29 had favorable bioavailability (F = 24%).

The low clearance of compound (S)-29 was reﬂected in a high area

under the curve (AUC) (1670 ng⋅h/mL) and a prolonged MRT (3.8 h).

This pharmacokinetic data promised potent in vivo pharmacological

effects and encouraged us to evaluate their efﬁcacy in animal models.

The anti-diabetic effects of compounds by single administration were

assessed by an oral glucose tolerance test in the N-STZ-1.5 rats. Candi-

date compounds and vehicle were administered 1 h before the oral

glucose load, and plasma glucose and insulin concentrations were

monitored over 2 h. As presented in Figure 4, (S)-29 effectively lowered

plasma glucose levels after oral administration accompanied by insulin

secretion in a glucose-dependent manner. The glucose-lowering effect of

compound (S)-29 was dose-dependent and signiﬁcant (p < 0.025) at a

dose of 1 mg/kg. To further evaluate in vivo pharmacological effects, the

effect on glycosylated hemoglobin (GHb) levels after chronic treatment

in the N-STZ-1.5 rats was examined (Figure 5). Compound (S)-29

signiﬁcantly lowered GHb levels after a 4- week treatment in a dose-

Table 4

Proﬁles of indolinylpyrimidine derivatives: the effect of a hydroxy group on the piperidinone ring.

c

d

e

Compound

OH

GPR119^a

hERG^b

CL_int

(μ

L/min/mg)

RLM

CL_total

LogD_7.4

EC₅₀(nM)

%inhibition at 10

μM

HLM

(mL/h/kg)

(RS)-29

(RS)-32a

(RS)-32b

(R)-29

3-OH (Rac)

4-OH (Rac)

5-OH (Rac)

3-OH (R)

17

89

90

25

16

20 ± 2

18 ± 4

23 ± 4

31 ± 4

ND

4

–

3.3

2.8

2.6

3.3

13

7

–

1

–

375

148

ND

9

ND

1

(S)-29

3-OH (S)

a

b

c

Agonist activity against human GPR119; EC₅₀values are expressed as means (n = 2).

Automated patch-clamp assay; percentages of inhibition are expressed as means ± standard deviation (n = 4).

HLM/RLM: Human/rat liver microsomal clearance; ND: a decrease in the compound concentration was not observed.

d

e

Rat, 0.1 mg/kg, iv.

LogD value at pH 7.4.

Table 5

Proﬁles of compound (S)-29.

Rat GPR119^a

EC₅₀(nM)

Insulin secretion^b

GLP-1 secretion^c

EC₅₀(nM)

Pharmacokinetic proﬁles in rats^{d, e}

Vdss^d

CL_total

AUC^e

MRT^e

F^e

d

EC₅₀(nM)

(mL/kg)

(mL/h/kg)

(ng⋅h/mL)

(h)

3.8

(%)

24

120

a

b

c

25

19

332

148

1670

Agonist activity against rat GPR119; EC₅₀values are expressed as means (n = 2).

Insulin secretion assay in HIT-T15 cells (n = 2).

GLP-1 secretion assay in GLUTag cells (n = 2).

Rat, 0.1 mg/kg, iv.

d

e

Rat, 1 mg/kg, po.

7

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

Fig. 4. Acute effects of (S)-29 on glucose excursion and insulin secretion during an oral glucose tolerance test in type 2 diabetic rats. Male N-STZ-1.5 rats were fasted

overnight and orally given vehicle or (S)-29 (1 and 3 mpk); (A) and (C) show time-dependent changes in plasma glucose and insulin levels after a 1.5 g/kg glucose

challenge, respectively. Data in (B) and (D) represents the AUC of plasma glucose shown in (A) and AUC of plasma insulin shown in (C). The data are expressed as

mean ± SD (n = 6) #, p ≤ 0.025 vs vehicle by one-tailed Williams’ test.

Fig. 5. The chronic effects of (S)-29 on GHb in male N-STZ-1.5 rats under time-restricted feeding conditions; (A) GHb levels before and after a 4-week treatment of

(S)-29; (B) the percentage of GHb change after a 4-week treatment of (S)-29. Data are expressed as mean ± SD (n = 10) #, p ≤ 0.025 versus vehicle by one-tailed

Williams’ test.

5. Experimental section

deuterated solvent and coupling constants (J) are in Hertz (Hz). Data are

reported as follows: chemical shift, integration, multiplicity (s = singlet,

d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of

doublets, dt = doublet of triplets, ddd = doublet of doublet of doublets,

bs = broad singlet), and coupling constants. All solvents and reagents

were obtained from commercial suppliers and used without further

puriﬁcation. Thin-layer chromatography (TLC) was performed on Merck

silica gel plates 60F254. Column chromatography was performed on

5.1. Chemistry

¹H NMR spectra were recorded on Bruker AVANCE III (300 MHz),

Bruker AVANCE 300 (300 MHz), or Bruker Advance III plus (400 MHz)

spectrometer. Chemical shifts for ¹H NMR are given in parts per million

(ppm) downﬁeld from tetramethysilane (δ) as the internal standard in

8

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

silica gel 60 (0.063–0.200 or 0.040–0.063 mm, E. Merck), basic silica gel

(Chromatorex NH, 100–200 mesh, Fuji Silysia Chemical Ltd.) or Purif-

Pack (Si or NH, Moritex Corporation). LC–MS analysis was performed

on a Waters, Agilent, or Shimadzu Liquid Chromatography–Mass Spec-

trometer System, operating in APCI (+or ꢀ ) or ESI (+or ꢀ ) ionization

mode. Analytes were eluted using a linear gradient of 0.05% TFA con-

taining water/acetonitrile or 5 mM ammonium acetate containing

water/acetonitrile mobile phase. Determination of chemical purity by

HPLC (detection at 220 nm) was conducted using a Shimadzu Liquid

Chromatography System with 0.05% TFA containing water/acetonitrile

mobile phase. Elemental analyses were performed by Takeda Analytical

Research Laboratories, Ltd. Yields are not optimized.

tert-Butyl 4-[(6-{5-[(2-hydroxyethyl)sulfanyl]-2,3-dihydro-1H-

indol-1-yl}pyrimidin-4-yl)oxy]piperidine-1-carboxylate (11).

A mixture of compound 10 (10.0 g, 21.0 mmol), 2-mercaptoethanol

(1.80 g, 23.0 mmol), Pd₂(dba)₃(1.83 g, 2.00 mmol), Xantphos (2.30 g,

4.00 mmol), and N,N-diisopropylethylamine (8.14 g, 63.0 mmol) in

toluene (100 mL) was stirred at 80 ^◦C under Ar atmosphere for 16 h. The

mixture was partitioned between with AcOEt and water. The organic

layer was washed with brine, dried over MgSO₄, and concentrated. The

residue was puriﬁed by silica gel column chromatography (hexane/

AcOEt = 80/20 to 50/50) to give the title compound as a solid (6.20 g,

62%). ¹H NMR (300 MHz, DMSO‑d₆) δ 1.41 (9H, s), 1.46–1.65 (2H, m),

1.87–2.05 (2H, m), 2.94 (2H, t, J = 7.0 Hz), 3.05–3.26 (4H, m),

3.46–3.59 (2H, m), 3.63–3.83 (2H, m), 3.97 (2H, t, J = 8.7 Hz), 4.86

(1H, t, J = 5.7 Hz), 5.17–5.31 (1H, m), 6.08 (1H, s), 7.19 (1H, dd, J =

8.3, 1.9 Hz), 7.27 (1H, s), 8.30 (1H, d, J = 8.3 Hz), 8.45 (1H, s).

tert-Butyl 4-[(6-{5-[(2-hydroxyethyl)sulfonyl]-2,3-dihydro-1H-

indol-1-yl}pyrimidin-4-yl)oxy]piperidine-1-carboxylate (12).

To a mixture of compound 11 (800 mg, 1.69 mmol) in AcOEt (100

mL) was added mCPBA (70%, 868 mg, 3.50 mmol), and the mixture was

stirred at room temperature for 2 h. The mixture was passed through

silica gel (NH, AcOEt) to give the title compound as a white solid (760

mg, 89%). MS (ESI/APCI) m/z 505 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.41 (9H, s), 1.49–1.66 (2H, m), 1.89–2.07 (2H, m),

3.08–3.31 (4H, m), 3.34–3.43 (2H, m), 3.58–3.84 (4H, m), 4.09 (2H, t, J

= 8.9 Hz), 4.86 (1H, t, J = 5.7 Hz), 5.16–5.37 (1H, m), 6.23 (1H, s),

7.64–7.76 (2H, m), 8.48–8.60 (2H, m). Anal. Calcd for C₂₄H₃₂N₄O₆S: C,

57.13; H, 6.39; N, 11.10. Found: C, 57.21; H, 6.39; N, 11.05.

2-({1-[6-({1-[3-(Propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-

yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}sulfonyl)

ethanol (13).

5-(Methylsulfonyl)-1-[6-(piperidin-4-yloxy)pyrimidin-4-yl]-

2,3-dihydro-1H-indole (5).

To a mixture of tert-butyl 4-({6-[5-(methylsulfonyl)-2,3-dihydro-1H-

indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate (4, 6.17 g, 13.0

mmol), AcOEt (100 mL), and MeOH (100 mL) was added 4 M HCl in

AcOEt (15 mL). The mixture was stirred at room temperature for 16 h.

After the mixture was concentrated under reduced pressure, the residue

was diluted with AcOEt and basiﬁed with 1 M aqueous NaOH solution.

The organic layer was separated, washed with water and brine, and

dried over MgSO₄. The solvent was removed by evaporation to give the

title compound as a white solid (4.56 g, 94%). This product was used for

the next step without further puriﬁcation. MS (ESI/APCI) m/z 375 [M +

H]⁺.

S-Propan-2-yl 4-({6-[5-(methylsulfonyl)-2,3-dihydro-1H-indol-

1-yl]pyrimidin-4-yl}oxy)piperidine-1-carbothioate (6).

To a mixture of compound 5 (150 mg, 0.401 mmol) and triethyl-

amine (0.139 mL, 0.997 mmol) in THF (10 mL) was added S-isopropyl

chlorothioformate (111 mg, 0.801 mmol). The mixture was stirred at

room temperature for 16 h. The reaction mixture was diluted with

AcOEt, washed with water and brine, and dried over MgSO₄. The solvent

was removed by evaporation to give the title compound as a white solid

(180 mg, 94%). MS (ESI/APCI) m/z 477 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.28 (6H, d, J = 6.8 Hz), 1.53–1.71 (2H, m), 1.94–2.10 (2H,

m), 3.15 (3H, s), 3.22–3.40 (4H, m), 3.48 (1H, q, J = 6.9 Hz), 3.61–3.98

(2H, m), 4.08 (2H, t, J = 8.9 Hz), 5.25–5.40 (1H, m), 6.23 (1H, s),

7.67–7.79 (2H, m), 8.54 (2H, t, J = 4.7 Hz). Anal. Calcd for

Step A. A mixture of compound 12 (2.10 g, 4.16 mmol), 4 M HCl in

AcOEt (10 mL), and AcOEt (250 mL) was stirred at room temperature for

16 h. The solvent was removed by evaporation and dried to give 2-({1-

[6-(piperidin-4-yloxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}sulfo-

nyl)ethanol hydrochloride as a solid (1.83 g, 100%). MS (ESI/APCI) m/z

405 [M + H]⁺.

Step B. To a mixture of 2-({1-[6-(piperidin-4-yloxy)pyrimidin-4-yl]-

2,3-dihydro-1H-indol-5-yl}sulfonyl)ethanol hydrochloride (1.83 g,

4.15 mmol), NaHCO₃(2.10 g, 25.0 mmol), THF (90 mL), and water (30

C

₂₂H₂₈N₄O₄S₂: C, 55.44; H, 5.92; N, 11.76. Found: C, 55.55; H, 5.97; N,

11.66.

5-Bromo-1-(6-chloropyrimidin-4-yl)-2,3-dihydro-1H-indole (9).

◦

mL) was added cyanogen bromide (595 mg, 5.62 mmol) at 0 C. The

mixture was stirred at room temperature for 16 h. Aqueous NaHCO₃

solution was added and the mixture was extracted with a mixed solvent

of AcOEt and THF. The organic layer was washed with brine, dried over

MgSO₄, and concentrated under reduced pressure to give 4-[(6-{5-[(2-

hydroxyethyl)sulfonyl]-2,3-dihydro-1H-indol-1-yl}pyrimidin-4-yl)oxy]

piperidine-1-carbonitrile as a solid. This product was used for the next

step without further puriﬁcation. MS (ESI/APCI) m/z 430 [M + H]⁺.

Step C. The solid obtained in step B was dissolved in AcOEt/THF/

DMSO (5:5:1, 220 mL), and then N-hydroxy-2-methylpropanimidamide

(623 mg, 6.10 mmol) and zinc chloride (1 M Et₂O solution, 6.1 mL, 6.1

mmol) were added. The mixture was stirred at 80 ^◦C for 2 h. After the

mixture was concentrated under reduced pressure, EtOH (100 mL) and

concentrated hydrochloric acid (5 mL) were added to the residue. The

resulting mixture was stirred at 70 ^◦C for 3 h. The reaction mixture was

concentrated under reduced pressure and partitioned between AcOEt

and aqueous NaOH solution. The organic layer was washed with brine

and dried over MgSO₄. After removal of the solvent, the residue was

puriﬁed by silica gel column chromatography (AcOEt 100%) to give the

title compound as a white solid (13, 1.42 g, 67% over 2 steps). MS (ESI/

APCI) m/z 515 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.19 (6H, d, J

= 6.8 Hz), 1.64–1.87 (2H, m), 2.02–2.19 (2H, m), 2.70–2.96 (1H, m),

3.28 (2H, t, J = 8.7 Hz), 3.34–3.42 (2H, m), 3.43–3.57 (2H, m), 3.66

(2H, q, J = 6.2 Hz), 3.75–3.91 (2H, m), 4.09 (2H, t, J = 8.9 Hz), 4.86

(1H, t, J = 5.5 Hz), 5.27–5.42 (1H, m), 6.25 (1H, s), 7.65–7.75 (2H, m),

8.51–8.62 (2H, m). Anal. Calcd for C₂₄H₃₀N₆O₅S: C, 56.02; H, 5.88; N,

16.33. Found: C, 56.04; H, 5.93; N, 16.07.

A mixture of 4,6-dichloropyrimidine (17.3 g, 116 mmol), 5-bromo-

2,3-dihydro-1H-indole (20.0 g, 101 mmol), and EtOH (500 mL) was

reﬂuxed for 16 h. After the mixture was concentrated under reduced

pressure, the residual solid was successively washed with 1 M aqueous

NaOH solution, H₂O, and Et₂O and then dried under reduced pressure to

give the title compound as a white solid (26.0 g, 83%). MS (ESI/APCI)

m/z 312 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 3.24 (2H, t, J = 8.1

Hz), 4.07 (2H, t, J = 8.3 Hz), 6.97 (1H, s), 7.30–7.58 (2H, m), 8.35 (1H,

d, J = 8.7 Hz), 8.60 (1H, s).

tert-Butyl 4-{[6-(5-bromo-2,3-dihydro-1H-indol-1-yl)pyrimidin-

4-yl]oxy}piperidine-1-carboxylate (10).

To a stirred solution of tert-butyl 4-hydroxypiperidine-1-carboxylate

(75.0 g, 373 mmol) in THF (1 L) was added sodium hydride (60% oil

dispersion, 14.9 g, 373 mmol) at 0 ^◦C. After the mixture was stirred at

room temperature for 1.5 h, compound 9 (37.0 g, 119 mmol) was added

to the mixture. The resulting mixture was stirred at room temperature

for 16 h. The reaction mixture was quenched with water and extracted

with AcOEt. The organic layer was washed with brine, dried over

MgSO₄, and concentrated under reduced pressure. The residue was

triturated with ⁱPr₂O and the precipitated solid was collected to give the

title compound as a white solid (52.0 g, 92%). MS (ESI/APCI) m/z 475

[M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.41 (9H, s), 1.47–1.65 (2H,

m), 1.88–2.05 (2H, m), 3.05–3.27 (4H, m), 3.64–3.80 (2H, m), 3.98 (2H,

t, J = 8.7 Hz), 5.16–5.30 (1H, m), 6.09 (1H, s), 7.33 (1H, dd, J = 8.7, 2.3

Hz), 7.40 (1H, s), 8.32 (1H, d, J = 8.7 Hz), 8.46 (1H, s).

9

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

tert-Butyl 4-[(6-{5-[(2-hydroxyethyl)sulﬁnyl]-2,3-dihydro-1H-

indol-1-yl}pyrimidin-4-yl)oxy]piperidine-1-carboxylate ((RS)-14).

To a mixture of compound 11 (800 mg, 1.69 mmol) in AcOEt (100

mL) was added mCPBA (70%, 419 mg, 1.69 mmol), and the mixture was

stirred at room temperature for 1 h. The mixture was passed through

silica gel (NH, AcOEt) to give the title compound as a white solid (530

mg, 64%). MS (ESI/APCI) m/z 489 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.41 (9H, s), 1.48–1.69 (2H, m), 1.87–2.06 (2H, m),

2.78–3.02 (2H, m), 3.07–3.31 (4H, m), 3.54–3.89 (4H, m), 4.05 (2H, t, J

= 8.7 Hz), 5.02 (1H, t, J = 5.1 Hz), 5.17–5.36 (1H, m), 6.16 (1H, s), 7.45

(1H, dd, J = 8.7, 1.9 Hz), 7.52 (1H, s), 8.44–8.62 (2H, m). Anal. Calcd for

temperature under H₂atmosphere (1 atm, balloon) for 3 days. The

catalyst was removed by ﬁltration through a PTFE membrane ﬁlter and

the ﬁltrate was concentrated under reduced pressure. The residue was

partitioned between AcOEt and aqueous NaHCO₃solution. The organic

layer was washed with brine and dried over Na₂SO₄. After removal of

the solvent, the residue was puriﬁed by silica gel column chromatog-

raphy (hexane/AcOEt = 80/20 to 65/35) to give the title compound as a

white solid (400 mg, 48%). MS (ESI/APCI) m/z 397 [M + H]⁺. ¹H NMR

(300 MHz, DMSO‑d₆) δ 1.41 (9H, s), 1.48–1.63 (2H, m), 1.89–2.02 (2H,

m), 3.09–3.24 (4H, m), 3.71 (2H, dt, J = 13.5, 4.8 Hz), 3.97 (2H, t, J =

8.7 Hz), 5.18–5.29 (1H, m), 6.08 (1H, s), 6.88–6.96 (1H, m), 7.16 (1H, t,

J = 7.8 Hz), 7.23 (1H, d, J = 7.2 Hz), 8.37 (1H, d, J = 8.0 Hz), 8.45 (1H,

s).

C

₂₄H₃₂N₄O₅S: C, 59.00; H, 6.60; N, 11.47. Found: C, 58.72; H, 6.60; N,

11.36.

2-({1-[6-({1-[3-(Propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-

S-Propan-2-yl 4-{[6-(2,3-dihydro-1H-indol-1-yl)pyrimidin-4-yl]

oxy}piperidine-1-carbothioate (17).

yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}sulﬁnyl)

ethanol ((RS)-15a).

Compound 17 was prepared from compound 16 in a manner similar

to that described for compounds 5 and 6. White solid. Yield 90% over 2

steps. MS (ESI/APCI) m/z 399 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆)

δ 1.28 (6H, d, J = 6.8 Hz), 1.54–1.69 (2H, m), 1.93–2.08 (2H, m),

3.14–3.26 (2H, m), 3.28–3.41 (2H, m), 3.42–3.55 (1H, m), 3.64–3.89

(2H, m), 3.97 (2H, t, J = 8.7 Hz), 5.25–5.36 (1H, m), 6.10 (1H, s),

6.89–6.98 (1H, m), 7.12–7.29 (2H, m), 8.37 (1H, d, J = 8.3 Hz), 8.45

(1H, s). Anal. Calcd for C₂₁H₂₆N₄O₂S: C, 63.29; H, 6.58; N, 14.06. Found:

C, 63.31; H, 6.71; N, 13.94.

Compound (RS)-15a was prepared from (RS)-14 in a manner similar

to that described for compounds 13. White solid. Yield 28% over 3 steps.

MS (ESI/APCI) m/z 499 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.19

(6H, d, J = 6.8 Hz), 1.65–1.88 (2H, m), 2.00–2.20 (2H, m), 2.72–3.04

(3H, m), 3.26 (2H, t, J = 8.5 Hz), 3.42–3.93 (6H, m), 4.05 (2H, t, J = 8.7

Hz), 4.79–5.09 (1H, m), 5.25–5.43 (1H, m), 6.06–6.31 (1H, m),

7.14–7.61 (2H, m), 8.43–8.62 (2H, m). Anal. Calcd for

C

₂₄H₃₀N₆O₄S⋅0.2H₂O: C, 57.40; H, 6.10; N, 16.73. Found: C, 57.28; H,

6.15; N, 16.53.

1-(6-Chloropyrimidin-4-yl)-5-nitro-2,3-dihydro-1H-indole (19).

Compound 19 was prepared from 5-nitro-2,3-dihydro-1H-indole in a

manner similar to that described for compound 9. Pale yellow solid.

Yield 83%. MS (ESI/APCI) m/z 277 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 3.33 (2H, t, J = 8.5 Hz), 3.96–4.34 (2H, m), 7.16 (1H, s),

8.09–8.28 (2H, m), 8.56 (1H, d, J = 9.0 Hz), 8.72 (1H, s).

S-Propan-2-yl 4-[(6-{5-[(2-hydroxyethyl)sulﬁnyl]-2,3-dihydro-

1H-indol-1-yl}pyrimidin-4-yl)oxy]piperidine-1-carbothioate

((RS)-15b).

Compound (RS)-15b was prepared from (RS)-14 in a manner similar

to that described for compounds 5 and 6. White solid. Yield 92% over 2

steps. MS (ESI/APCI) m/z 491 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆)

δ 1.28 (6H, d, J = 7.2 Hz), 1.50–1.71 (2H, m), 1.91–2.09 (2H, m),

2.78–3.00 (2H, m), 3.15–3.40 (5H, m), 3.41–3.56 (1H, m), 3.56–3.93

(3H, m), 3.98–4.15 (2H, m), 5.03 (1H, t, J = 5.1 Hz), 5.23–5.40 (1H, m),

6.17 (1H, s), 7.46 (1H, dd, J = 8.3, 1.9 Hz), 7.52 (1H, s), 8.47–8.59 (2H,

m). Anal. Calcd for C₂₃H₃₀N₄O₄S₂⋅0.1H₂O: C, 56.10; H, 6.18; N, 11.38.

Found: C, 55.85; H, 6.20; N, 11.16.

tert-Butyl 4-{[6-(5-nitro-2,3-dihydro-1H-indol-1-yl)pyrimidin-

4-yl]oxy}piperidine-1-carboxylate (20).

Compound 20 was prepared from compound 19 in a manner similar

to that described for compound 10. Pale yellow solid. Yield 69%. MS

1

(ESI/APCI) m/z 442 [M + H]⁺. H NMR (300 MHz, DMSO‑d₆) δ 1.41

(9H, s), 1.49–1.66 (2H, m), 1.90–2.06 (2H, m), 3.08–3.24 (2H, m),

3.24–3.36 (2H, m), 3.64–3.82 (2H, m), 4.12 (2H, t, J = 8.7 Hz),

5.21–5.37 (1H, m), 6.28 (1H, s), 8.04–8.10 (1H, m), 8.10–8.19 (1H, m),

8.51 (1H, d, J = 9.1 Hz), 8.56 (1H, s). Anal. Calcd for C₂₂H₂₇N₅O₅: C,

59.85; H, 6.16; N, 15.86. Found: C, 59.83; H, 6.12; N, 15.90.

tert-Butyl 4-{[6-(5-amino-2,3-dihydro-1H-indol-1-yl)pyrimidin-

4-yl]oxy}piperidine-1-carboxylate (21).

S-Propan-2-yl 4-[(6-{5-[(2-hydroxyethyl)sulﬁnyl]-2,3-dihydro-

1H-indol-1-yl}pyrimidin-4-yl)oxy]piperidine-1-carbothioate (15b-

ent1 and 15b-ent2).

Compound (RS)-15b (racemate, 97.7 mg) was subjected to chiral

HPLC separation (CHIRALPAK AD LF001 (50 mmID × 500 mmL),

eluting with EtOH at 60 mL/min ﬂow rate) to afford two enantiomers,

15-ent1 (the ﬁrst eluted enantiomer, white solid, 40.7 mg) and 15-ent2

(the second eluted enantiomer, white solid, 44.6 mg). 15b-ent1:

Retention time 20.1 min (CHIRALPAK AD-3 NC002 (4.6 mmID × 250

mmL), eluting with EtOH at 0.7 mL/min ﬂow rate). > 99.9%ee. MS (ESI)

m/z 491 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.29 (6H, d, J = 6.9

Hz), 1.54–1.70 (2H, m), 1.95–2.07 (2H, m), 2.81–2.99 (2H, m),

3.19–3.39 (5H, m), 3.42–3.54 (1H, m), 3.57–3.88 (3H, m), 3.99–4.11

(2H, m), 4.97–5.05 (1H, m), 5.26–5.37 (1H, m), 6.18 (1H, s), 7.46 (1H,

dd, J = 8.5, 1.9 Hz), 7.53 (1H, s), 8.48–8.56 (2H, m). Anal. Calcd for

A mixture of compound 20 (8.80 g, 19.9 mmol), 10% Pd on carbon

(500 mg), THF (150 mL), and MeOH (300 mL) was stirred at room

temperature under H₂atmosphere (1 atm, balloon) for 16 h. The catalyst

was removed by ﬁltration through a PTFE membrane ﬁlter and the

ﬁltrate was concentrated under reduced pressure to give the title com-

pound as a white solid (8.44 g, quant.). MS (ESI/APCI) m/z 412 [M +

H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.41 (9H, s), 1.46–1.65 (2H, m),

1.84–2.03 (2H, m), 2.98–3.25 (4H, m), 3.63–3.78 (2H, m), 3.86 (2H, t, J

= 8.5 Hz), 4.79 (2H, s), 5.11–5.31 (1H, m), 5.91 (1H, s), 6.38 (1H, dd, J

= 8.5, 2.5 Hz), 6.49 (1H, d, J = 2.3 Hz), 8.03 (1H, d, J = 8.3 Hz), 8.34

(1H, s). Anal. Calcd for C₂₂H₂₉N₅O₃: C, 64.21; H, 7.10; N, 17.02. Found:

C, 64.09; H, 7.23; N, 16.73.

C

₂₃H₃₀N₄O₄S₂⋅0.2H₂O: C, 55.89; H, 6.20; N, 11.34. Found: C, 55.96; H,

6.24; N, 11.10. 15b-ent2: Retention time 24.5 min (CHIRALPAK AD-3

NC002 (4.6 mmID × 250 mmL), eluting with EtOH at 0.7 mL/min

ﬂow rate). 96.8%ee. MS (ESI) m/z 491 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.29 (6H, d, J = 6.9 Hz), 1.54–1.68 (2H, m), 1.95–2.06 (2H,

m), 2.83–2.98 (2H, m), 3.22–3.39 (5H, m), 3.43–3.54 (1H, m),

3.56–3.87 (3H, m), 4.01–4.10 (2H, m), 4.98–5.03 (1H, m), 5.26–5.36

(1H, m), 6.18 (1H, s), 7.46 (1H, dd, J = 8.5, 1.9 Hz), 7.53 (1H, s),

8.49–8.55 (2H, m). Anal. Calcd for C₂₃H₃₀N₄O₄S₂: C, 56.30; H, 6.16; N,

11.42. Found: C, 56.12; H, 6.24; N, 11.24.

tert-Butyl 4-({6-[5-(2-oxopyrrolidin-1-yl)-2,3-dihydro-1H-indol-

1-yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate (23a).

Compound 23a was prepared from compound 21 in a manner similar

to that described for compounds 22b and 23b. Pale yellow solid. Yield

90% over 2 steps. MS (ESI/APCI) m/z 494 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.41 (9H, s), 1.46–1.67 (2H, m), 1.89–2.13 (4H, m),

2.42–2.50 (2H, m), 3.07–3.26 (4H, m), 3.65–3.76 (2H, m), 3.80 (2H, t, J

= 7.0 Hz), 3.97 (2H, t, J = 8.5 Hz), 5.16–5.33 (1H, m), 6.06 (1H, s), 7.35

(1H, dd, J = 9.1, 2.3 Hz), 7.61 (1H, d, J = 1.9 Hz), 8.32 (1H, d, J = 9.1

Hz), 8.44 (1H, s).

tert-Butyl

4-{[6-(2,3-dihydro-1H-indol-1-yl)pyrimidin-4-yl]

oxy}piperidine-1-carboxylate (16).

A mixture of compound 10 (1.00 g, 2.10 mmol), 10% Pd on carbon

(200 mg), THF (20 mL), and MeOH (10 mL) was stirred at room

1-{1-[6-({1-[3-(Propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-

yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}pyrrolidin-2-

10

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

one (24a).

stirred at 80 ^◦C for 8 h. The reaction mixture was diluted with AcOEt and

THF, washed with 0.03 N hydrochloric acid, 0.1 N aqueous NaOH so-

lution, water and brine, and dried over Na₂SO₄. The ﬁltrate was passed

through a pad of silica gel (NH, AcOEt) and concentrated under reduced

pressure to give a pale yellow solid. The solid was suspended in AcOEt-

EtOH (10:1 v/v, 165 mL) and stirred at 80 ^◦C for 1 h. After the mixture

was cooled to rt, the precipitated solid was collected to give the title

compound as a white powder (24b, 25.5 g, 62%). MS (ESI/APCI) m/z

504 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.19 (6H, d, J = 6.8 Hz),

1.67–1.92 (6H, m), 2.02–2.15 (2H, m), 2.36 (2H, t, J = 6.0 Hz),

2.74–2.91 (1H, m), 3.19 (2H, t, J = 8.5 Hz), 3.42–3.59 (4H, m),

3.76–3.88 (2H, m), 4.00 (2H, t, J = 8.7 Hz), 5.26–5.38 (1H, m), 6.12

(1H, s), 7.03 (1H, dd, J = 8.7, 2.3 Hz), 7.12 (1H, d, J = 1.9 Hz), 8.33 (1H,

d, J = 8.7 Hz), 8.47 (1H, s). Anal. Calcd for C₂₇H₃₃N₇O₃: C, 64.39; H,

6.60; N, 19.47. Found: C, 64.27; H, 6.63; N, 19.41.

Compound 24a was prepared from compound 23a in a manner

similar to that described for compounds 13. White solid. Yield 34% over

3 steps. MS (ESI/APCI) m/z 490 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.19 (6H, d, J = 6.8 Hz), 1.65–1.84 (2H, m), 1.96–2.18 (4H,

m), 2.41–2.50 (2H, m), 2.82 (1H, q, J = 6.9 Hz), 3.20 (2H, t, J = 8.5 Hz),

3.41–3.59 (2H, m), 3.73–3.90 (4H, m), 3.98 (2H, t, J = 8.7 Hz),

5.23–5.39 (1H, m), 6.09 (1H, s), 7.35 (1H, dd, J = 8.7, 2.3 Hz), 7.61 (1H,

d, J = 2.3 Hz), 8.32 (1H, d, J = 9.0 Hz), 8.46 (1H, s). Anal. Calcd for

C

₂₆H₃₁N₇O₃⋅0.1H₂O: C, 63.55; H, 6.40; N, 19.95. Found: C, 63.39; H,

6.52; N, 19.70.

tert-Butyl 4-[(6-{5-[(5-chloropentanoyl)amino]-2,3-dihydro-

1H-indol-1-yl}pyrimidin-4-yl)oxy]piperidine-1-carboxylate (22b).

To a solution of compound 21 (20.0 g, 48.6 mmol) in DMA (200 mL)

was added dropwise 5-chloropentanoyl chloride (7.76 g, 50.1 mmol) at

0

^◦C, and the mixture was stirred at room temperature for 1 h. The

tert-Butyl 4-{[6-(5-{[(2-chloroethoxy)acetyl]amino}-2,3-dihy-

dro-1H-indol-1-yl)pyrimidin-4-yl]oxy}piperidine-1-carboxylate

(22c).

mixture was diluted with AcOEt, successively washed with water,

saturated aqueous NaHCO₃solution and brine, and dried over Na₂SO₄.

The solvent was evaporated to give the title compound as a gray powder

(22.2 g, 86%). MS (ESI/APCI) m/z 530 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.41 (9H, s), 1.47–1.63 (2H, m), 1.64–1.82 (4H, m),

1.89–2.02 (2H, m), 2.31 (2H, t, J = 7.0 Hz), 3.07–3.24 (4H, m),

3.62–3.78 (4H, m), 3.95 (2H, t, J = 8.5 Hz), 5.17–5.30 (1H, m), 6.04

(1H, s), 7.27 (1H, dd, J = 8.9, 2.1 Hz), 7.58 (1H, d, J = 1.5 Hz), 8.25 (1H,

d, J = 8.7 Hz), 8.42 (1H, s), 9.80 (1H, s).

Compound 22c was prepared from compound 21 in a manner similar

to that described for compound 22b. Brown oil. Yield 96%. MS (ESI/

APCI) m/z 532 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.41 (9H, s),

1.46–1.63 (2H, m), 1.88–2.02 (2H, m), 3.11–3.23 (4H, m), 3.62–3.77

(2H, m), 3.81(4H, s), 3.96 (2H, t, J = 8.7 Hz), 4.11 (2H, s), 5.15–5.29

(1H, m), 6.05 (1H, s), 7.35 (1H, dd, J = 8.7, 1.9 Hz), 7.60 (1H, s), 8.28

(1H, d, J = 8.7 Hz), 8.43 (1H,s), 9.58 (1H, s).

tert-Butyl 4-({6-[5-(2-oxopiperidin-1-yl)-2,3-dihydro-1H-indol-

1-yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate (23b).

tert-Butyl

4-({6-[5-(3-oxomorpholin-4-yl)-2,3-dihydro-1H-

indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate (23c).

Compound 23c was prepared from compound 22c in a manner

similar to that described for compound 23b. Brown solid. Yield 97%. MS

To a solution of compound 22b (22.1 g, 41.7 mmol) in anhydrous

DMF (300 mL) was added sodium hydride (60% oil dispersion, 2.00 g,

50.0 mmol) at 0 ^◦C, and the mixture was stirred at room temperature for

3 h. After the mixture was cooled to at 0 ^◦C again, water (300 mL) was

added dropwise to the reaction mixture. The precipitated solid was

collected, washed with water and Et₂O, and dried to give the title

compound as a pale yellow powder (19.0 g, 92%). MS (ESI/APCI) m/z

494 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.41 (9H, s), 1.47–1.63

(2H, m), 1.77–2.02 (6H, m), 2.36 (2H, t, J = 6.0 Hz), 3.09–3.23 (4H, m),

3.55 (2H, t, J = 5.3 Hz), 3.65–3.78 (2H, m), 3.99 (2H, t, J = 8.7 Hz),

5.19–5.30 (1H, m), 6.09 (1H, s), 7.03 (1H, dd, J = 8.7, 2.3 Hz), 7.12 (1H,

d, J = 1.9 Hz), 8.33 (1H, d, J = 8.7 Hz), 8.46 (1H, s).

(ESI/APCI) m/z 496 [M + H]⁺. H NMR (300 MHz, DMSO‑d₆) δ 1.41

1

(9H, s), 1.47–1.64 (2H, m), 1.86–2.03 (2H, m), 3.08–3.26 (4H, m),

3.63–3.78 (4H, m), 3.89–4.05 (4H, m), 4.18 (2H, s), 5.17–5.32 (1H, m),

6.10 (1H, s), 7.15 (1H, dd, J = 8.5, 2.1 Hz), 7.24 (1H, d, J = 2.3 Hz), 8.36

(1H, d, J = 8.7 Hz), 8.46 (1H, s).

4-{1-[6-({1-[3-(Propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-

yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}morpholin-3-

one (24c).

Compound 24c was prepared from compound 23c in a manner

similar to that described for compound 24b. White solid. Yield 24% over

3 steps. MS (ESI/APCI) m/z 506 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.19 (6H, d, J = 7.2 Hz), 1.66–1.83 (2H, m, J = 12.9, 8.7,

8.7, 4.0 Hz), 2.01–2.15 (2H, m), 2.82 (1H, q, J = 6.9 Hz), 3.20 (2H, t, J

= 8.5 Hz), 3.49 (2H, ddd, J = 13.2, 9.2, 3.6 Hz), 3.64–3.73 (2H, m),

3.76–3.88 (2H, m), 3.91–4.07 (4H, m), 4.18 (2H, s), 5.32 (1H, dt, J =

7.9, 4.3 Hz), 6.12 (1H, s), 7.15 (1H, dd, J = 8.7, 2.3 Hz), 7.24 (1H, d, J =

1.9 Hz), 8.36 (1H, d, J = 8.7 Hz), 8.48 (1H, s). Anal. Calcd for

1-{1-[6-({1-[3-(Propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-

yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}piperidin-2-one

(24b).

Step A. A mixture of compound 23b (18.9 g, 38.3 mmol), 4 N HCl in

AcOEt (77 mL), AcOEt (200 mL), and MeOH (100 mL) was stirred at

room temperature for 15 h. After the mixture was concentrated under

reduced pressure, the residue was suspended with AcOEt (100 mL). The

precipitated solid was collected and washed with AcOEt. The solid was

dissolved in a mixed solvent of THF (270 mL) and water (126 mL), and

then sodium bicarbonate (16.1 g, 191 mmol) and cyanogen bromide

(5.27 g, 49.8 mmol) were added to the mixture. The resulting mixture

was stirred at room temperature for 3 h. The reaction mixture was

diluted with AcOEt, washed with brine, and dried over Na₂SO₄. The

solvent was removed by evaporation to give 4-({6-[5-(2-oxopiperidin-1-

yl)-2,3-dihydro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1-carbon-

itrile as a pale yellow solid (15.7 g, 98%). MS (ESI/APCI) m/z 419 [M +

H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.66–1.90 (6H, m), 1.98–2.11 (2H,

m), 2.36 (2H, t, J = 5.9 Hz), 3.13–3.47 (6H, m), 3.55 (2H, t, J = 5.3 Hz),

4.00 (2H, t, J = 8.5 Hz), 5.15–5.28 (1H, m), 6.11 (1H, s), 7.03 (1H, dd, J

= 8.5, 2.1 Hz), 7.12 (1H, d, J = 1.9 Hz), 8.33 (1H, d, J = 8.7 Hz), 8.45

(1H, s).

C

₂₆H₃₁N₇O₄: C, 61.77; H, 6.18; N, 19.39. Found: C, 61.69; H, 6.21; N,

19.32.

tert-Butyl 4-({6-[5-(1,1-dioxido-1,2-thiazolidin-2-yl)-2,3-dihy-

dro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate

(23d).

To a solution of compound 21 (5.00 g, 12.2 mmol) and triethylamine

(2.79 mL, 20.0 mmol) in THF (220 mL) was added dropwise 3-chloro-

propanesulfonyl chloride (2.58 g, 14.6 mmol), and the mixture was

stirred at room temperature for 16 h. The mixture was diluted with

AcOEt, successively washed with water and brine, dried over MgSO₄,

and concentrated under reduced pressure. The residual solid was dis-

solved in DMF (100 mL). Sodium hydride (60% oil dispersion, 583 mg,

14.6 mmol) was added to the mixture at 0 ^◦C, and the mixture was

stirred at room temperature for 3 h. The reaction was quenched by

addition of water and extracted with AcOEt. The organic layer was

washed with brine and dried over MgSO₄. The solvent was removed by

evaporation to give the title compound as a brown solid (5.10 g, 81%

Step B. To a mixture of 4-({6-[5-(2-oxopiperidin-1-yl)-2,3-dihydro-

1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1-carbonitrile (34.0 g,

81.2 mmol) and N-hydroxy-2-methylpropanimidamide (10.8 g, 106

mmol) in anhydrous DMF (510 mL) were added zinc chloride (1.0 M

Et₂O solution, 24.4 mL, 24.4 mmol) and toluenesulfonic acid mono-

hydrate (4.64 g, 24.4 mmol) at room temperature, and the mixture was

1

over 2 steps). MS (ESI/APCI) m/z 516 [M + H]⁺. H NMR (300 MHz,

DMSO‑d₆) δ 1.41 (9H, s), 1.47–1.69 (2H, m), 1.87–2.06 (2H, m),

2.30–2.45 (2H, m), 3.06–3.27 (4H, m), 3.46 (2H, t, J = 7.4 Hz),

11

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

3.62–3.80 (4H, m), 3.98 (2H, t, J = 8.7 Hz), 5.13–5.35 (1H, m), 6.07

(1H, s), 7.02 (1H, dd, J = 8.9, 2.5 Hz), 7.17 (1H, d, J = 2.3 Hz), 8.33 (1H,

d, J = 8.7 Hz), 8.45 (1H, s). Anal. Calcd for C₂₅H₃₃N₅O₅S: C, 58.23; H,

6.45; N, 13.58. Found: C, 58.12; H, 6.43; N, 13.68.

7.09–7.18 (1H, m), 7.23 (1H, s), 8.37 (1H, d, J = 8.7 Hz), 8.47 (1H, s).

1-{1-[6-({1-[3-(Propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-

yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}piperazin-2-one

hydrochloride (24e).

5-(1,1-Dioxido-1,2-thiazolidin-2-yl)-1-[6-({1-[3-(propan-2-yl)-

1,2,4-oxadiazol-5-yl]piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihy-

dro-1H-indole (24d).

Step A. 1-{1-[6-({1-[3-(Propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-

4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}-4-(tri-

ﬂuoroacetyl)piperazin-2-one was prepared from compound 23e in a

manner similar to that described for compound 24b. White solid. Yield

63% over 3 steps. MS (ESI/APCI) m/z 601 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.19 (6H, d), 1.67–1.83 (2H, m), 2.02–2.15 (2H, m),

2.74–2.91 (1H, m), 3.20 (2H, t, J = 8.3 Hz), 3.43–3.56 (2H, m),

3.73–3.88 (4H, m), 3.90–4.07 (4H, m), 4.23–4.38 (2H, m), 5.26–5.38

(1H, m), 6.14 (1H, s), 7.13 (1H, dd, J = 8.7, 2.3 Hz), 7.23 (1H, s), 8.37

(1H, d, J = 8.7 Hz), 8.48 (1H, s).

Compound 24d was prepared from compound 23d in a manner

similar to that described for compound 13. White solid. Yield 31% over

3 steps. MS (ESI/APCI) m/z 526 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.19 (6H, d, J = 6.8 Hz), 1.66–1.84 (2H, m), 1.99–2.18 (2H,

m), 2.30–2.45 (2H, m), 2.68–2.93 (1H, m), 3.21 (2H, t, J = 8.5 Hz),

3.40–3.59 (4H, m), 3.69 (2H, t, J = 6.6 Hz), 3.76–3.90 (2H, m), 3.98

(2H, t, J = 8.7 Hz), 5.22–5.40 (1H, m), 6.10 (1H, s), 7.02 (1H, dd, J =

8.9, 2.5 Hz), 7.17 (1H, d, J = 2.3 Hz), 8.34 (1H, d, J = 9.1 Hz), 8.46 (1H,

s). Anal. Calcd for C₂₅H₃₁N₇O₄S: C, 57.13; H, 5.94; N, 18.65. Found: C,

56.89; H, 5.84; N, 18.47.

Step B. A mixture of 1-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-

yl]piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}-4-

(triﬂuoroacetyl)piperazin-2-one (225 mg, 0.37 mmol), 1 M aqueous

NaOH solution (1.12 mL, 1.12 mmol), MeOH (4 mL) and THF (4 mL) was

stirred at room temperature for 1 h. The reaction mixture was diluted

with AcOEt, washed with brine, and dried over Na₂SO₄. The solvent was

removed by evaporation to give a pale yellow solid. The solid was sus-

pended in MeOH (4 mL) and a solution of HCl in MeOH (0.37 mmol,

prepared from acetyl chloride (0.027 mL, 0.37 mmol) and MeOH (4 mL)

was added. The mixture was concentrated under reduced pressure and

triturated with Et₂O. The precipitated solid was collected to give the title

compound as a pale yellow powder (24e, 193 mg, 95%). MS (ESI/APCI)

m/z 505 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.19 (6H, d, J = 6.8

Hz), 1.65–1.85 (2H, m, J = 12.9, 8.6, 8.6, 3.8 Hz), 2.00–2.17 (2H, m),

2.74–2.91 (1H, m), 3.22 (2H, t, J = 8.7 Hz), 3.40–3.57 (4H, m),

3.75–3.91 (6H, m), 4.02 (2H, t, J = 8.7 Hz), 5.25–5.40 (1H, m), 6.15

(1H, s), 7.10 (1H, dd, J = 8.7, 2.3 Hz), 7.17 (1H, s), 8.40 (1H, d, J = 8.7

Hz), 8.49 (1H, s), 9.70 (2H, bs). Anal. Calcd for C₂₆H₃₃ClN₈O₃⋅0.9H₂O:

C, 56.04; H, 6.29; N, 20.11. Found: C, 56.21; H, 6.25; N, 19.81.

tert-Butyl 4-{[6-(5-{[(tert-butoxycarbonyl)sulfamoyl]amino}-

2,3-dihydro-1H-indol-1-yl)pyrimidin-4-yl]oxy}piperidine-1-

carboxylate (22f).

tert-Butyl

4-{[6-(5-{[N-(2-hydroxyethyl)glycyl]amino}-2,3-

dihydro-1H-indol-1-yl)pyrimidin-4-yl]oxy}piperidine-1-carbox-

ylate (22e).

Step A. To a mixture of compound 21 (2.00 g, 4.86 mmol) in DMA

(20 mL) was added chloracetyl chloride (0.426 mL, 5.35 mmol) at 0 ^◦C,

and the mixture was stirred at room temperature for 3 h. The reaction

mixture was diluted with AcOEt, washed with water and brine, and

dried over Na₂SO₄. The solvent was removed by evaporation to give tert-

butyl 4-[(6-{5-[(chloroacetyl)amino]-2,3-dihydro-1H-indol-1-yl}pyr-

imidin-4-yl)oxy]piperidine-1-carboxylate as a white powder (2.35,

99%). MS (ESI/APCI) m/z 488 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆)

δ 1.41 (9H, s), 1.47–1.64 (2H, m), 1.89–2.02 (2H, m), 3.08–3.26 (4H,

m), 3.65–3.77 (2H, m), 3.97 (2H, t, J = 8.5 Hz), 4.22 (2H, s), 5.16–5.30

(1H, m), 6.06 (1H, s), 7.29 (1H, dd, J = 8.9, 2.1 Hz), 7.57 (1H, d, J = 1.5

Hz), 8.30 (1H, d, J = 8.7 Hz), 8.44 (1H, s), 10.21 (1H, s).

Step B. A mixture of tert-butyl 4-[(6-{5-[(chloroacetyl)amino]-2,3-

dihydro-1H-indol-1-yl}pyrimidin-4-yl)oxy]piperidine-1-carboxylate

(1.00 g, 2.05 mmol) and 2-aminoethanol (0.618 mL, 10.3 mmol) in a

mixed solvent of THF (20 mL) and AcOⁱPr (20 mL) was stirred at 70 ^◦C

for 8 h and then reﬂuxed for further 3 h. The reaction mixture was

diluted with AcOEt, washed with saturated aqueous NaHCO₃solution

and brine, and dried over Na₂SO₄. The solvent was removed by evapo-

ration to give the title compound as a pale yellow powder (22e, 1.03 g,

98%). MS (ESI/APCI) m/z 513 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆)

δ 1.41 (9H, s), 1.47–1.64 (2H, m), 1.89–2.03 (2H, m), 2.44 (1H, bs), 2.61

(2H, t, J = 5.5 Hz), 3.08–3.23 (4H, m), 3.26 (2H, s), 3.42–3.52 (2H, m),

3.66–3.77 (2H, m), 3.96 (2H, t, J = 8.5 Hz), 4.63 (1H, t, J = 5.3 Hz),

5.17–5.29 (1H, m), 6.04 (1H, s), 7.34 (1H, dd, J = 8.7, 1.9 Hz), 7.61 (1H,

d, J = 1.9 Hz), 8.27 (1H, d, J = 8.7 Hz), 8.43 (1H, s), 9.80 (1H, bs).

A mixture of compound 21 (2.18 g, 5.30 mmol) and (tert-butox-

ycarbonyl){[4-(dimethyliminio)pyridin-1(4H)-yl]sulfonyl}azanide

(1.92 g, 6.36 mmol) in THF (20 mL) was stirred at 50 ^◦C for 5 h. The

reaction mixture was diluted with AcOEt, successively washed with 0.1

M hydrochloric acid, water and brine, and dried over Na₂SO₄. The sol-

vent was removed by evaporation to give the title compound as a pale

yellow amorphous solid (3.05 g, 97%) MS (ESI/APCI) m/z 591 [M +

H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.38 (9H, s), 1.41 (9H, s),

1.47–1.63 (2H, m), 1.88–2.05 (2H, m), 3.06–3.26 (4H, m), 3.65–3.78

(2H, m), 3.88–4.10 (2H, m), 5.17–5.31 (1H, m), 6.07 (1H, s), 6.92–7.00

(1H, m), 7.05 (1H, s), 8.28 (1H, d, J = 8.7 Hz), 8.44 (1H, s), 9.95 (1H, s),

11.01 (1H, bs).

tert-Butyl

4-[(6-{5-[2-oxo-4-(triﬂuoroacetyl)piperazin-1-yl]-

2,3-dihydro-1H-indol-1-yl}pyrimidin-4-yl)oxy]piperidine-1-

carboxylate (23e).

tert-Butyl 6-[1-(6-{[1-(tert-butoxycarbonyl)piperidin-4-yl]oxy}

pyrimidin-4-yl)-2,3-dihydro-1H-indol-5-yl]-1,2,6-thiadiazinane-2-

carboxylate 1,1-dioxide (23f).

To a mixture of compound 22e (1.00 g, 1.95 mmol) and tri-n-

butylphosphine (0.729 mL, 2.93 mmol) in THF (20 mL) was added 1,1^′-

(azodicarbonyl)dipiperidine (0.738 g, 2.93 mmol) at 0 ^◦C, and the

mixture was stirred at room temperature for 15 h. Tributylphosphine

(0.729 mL, 2.93 mmol) and 1,1^′-(azodicarbonyl)dipiperidine (0.738 g,

2.93 mmol) were added again and the resulting mixture was stirred at

60 ^◦C for 4 h. After the mixture was cooled to room temperature, AcOEt

(20 mL) was added and the insoluble materials were ﬁltered off. To the

ﬁltrate were added triethylamine (1.63 mL, 11.7 mmol) and triﬂuoro-

acetic anhydride (0.542 mL, 3.91 mmol). After being stirred for 6 h, the

reaction mixture was diluted with AcOEt, washed with water, aqueous

NaHCO₃solution and brine, and dried over Na₂SO₄. After removal of the

solvent, the residue was puriﬁed by crystallization from toluene to give

the title compound as a white powder (0.530 g, 46%). MS (ESI/APCI) m/

z 591 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.41 (9H, s), 1.48–1.64

(2H, m), 1.89–2.04 (2H, m), 3.09–3.26 (4H, m), 3.65–3.83 (4H, m),

3.90–4.08 (4H, m), 4.24–4.39 (2H, m), 5.17–5.32 (1H, m), 6.11 (1H, s),

A mixture of compound 22f (460 mg, 0.779 mmol), 1,3-dibromopro-

pane (0.120 mL, 1.17 mmol) and potassium carbonate (323 mg, 2.34

mmol) in a mixed solvent of acetone (15 mL) and DMA (5 mL) was

stirred at 70 ^◦C for 6 h. The reaction mixture was diluted with AcOEt,

washed with water and brine, and dried over Na₂SO₄. The solvent was

removed by evaporation to give the title compound as a pale yellow

amorphous solid (490 mg, 100%). MS (ESI/APCI) m/z 631 [M + H]⁺. ¹H

NMR (300 MHz, DMSO‑d₆) δ 1.41 (9H, s), 1.43 (9H, s), 1.48–1.63 (2H,

m), 1.88–2.01 (4H, m), 3.09–3.26 (4H, m), 3.66–3.76 (2H, m), 3.81 (2H,

t, J = 5.8 Hz), 3.95–4.06 (4H, m), 5.18–5.31 (1H, m), 6.12 (1H, s), 7.15

(1H, dd, J = 8.9, 2.4 Hz), 7.22 (1H, d, J = 1.9 Hz), 8.36 (1H, d, J = 8.7

Hz), 8.47 (1H, s).

5-(1,1-Dioxido-1,2,6-thiadiazinan-2-yl)-1-[6-({1-[3-(propan-2-

yl)-1,2,4-oxadiazol-5-yl]piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-

dihydro-1H-indole (24f).

12

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

Compound 24f was prepared from compound 23f in a manner

similar to that described for compound 24b. Pale yellow amorphous

to that described for compound (S)-27. White amorphous solid. Yield

81%. MS (ESI/APCI) m/z 588 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ

1.41 (9H, s), 1.47–1.63 (2H, m), 1.76–2.00 (6H, m), 2.10 (3H, s),

3.10–3.23 (4H, m), 3.66–3.76 (4H, m), 3.92–4.00 (2H, m), 4.94–4.99

(1H, m), 5.19–5.28 (1H, m), 6.05 (1H, s), 7.28 (1H, dd, J = 8.7, 2.3 Hz),

7.56 (1H, d, J = 1.9 Hz), 8.29 (1H, d, J = 9.1 Hz), 8.44 (1H, d, J = 0.8

Hz), 10.01 (1H, s).

1

solid. Yield 47% over 3 steps. MS (ESI/APCI) m/z 541 [M + H]⁺. H

NMR (300 MHz, DMSO‑d₆) δ 1.19 (6H, d, J = 7.2 Hz), 1.67–1.88 (4H,

m), 2.01–2.15 (2H, m), 2.73–2.90 (1H, m), 3.20 (2H, t, J = 8.5 Hz),

3.31–3.40 (2H, m), 3.42–3.61 (4H, m), 3.76–3.87 (2H, m), 4.00 (2H, t, J

= 8.7 Hz), 5.25–5.37 (1H, m), 6.13 (1H, s), 7.13 (1H, dd, J = 8.7, 2.3

Hz), 7.17–7.25 (2H, m), 8.34 (1H, d, J = 8.7 Hz), 8.48 (1H, s). Anal.

Calcd for C₂₅H₃₂N₈O₄S⋅0.1AcOEt: C, 55.52; H, 6.02; N, 20.39. Found: C,

55.26; H, 5.91; N, 20.20.

tert-Butyl

4-({6-[5-(3-hydroxy-2-oxopiperidin-1-yl)-2,3-dihy-

dro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate

((RS)-28).

tert-Butyl

4-({6-[5-(acetylamino)-2,3-dihydro-1H-indol-1-yl]

Compound (RS)-28 was prepared from (RS)-27 in a manner similar

to that described for compound (S)-28. Pale brown solid. Yield 86% over

2 steps. MS (ESI/APCI) m/z 510 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.41 (9H, s), 1.49–1.63 (2H, m), 1.67–2.02 (5H, m),

2.04–2.15 (1H, m), 3.10–3.23 (4H, m), 3.45–3.55 (1H, m), 3.56–3.66

(1H, m), 3.67–3.76 (2H, m), 3.95–4.08 (3H, m), 5.18 (1H, d, J = 3.8 Hz),

5.21–5.30 (1H, m), 6.09 (1H, s), 7.05 (1H, dd, J = 8.7, 1.9 Hz), 7.13 (1H,

d, J = 1.9 Hz), 8.34 (1H, d, J = 8.7 Hz), 8.46 (1H, s).

pyrimidin-4-yl}oxy)piperidine-1-carboxylate (22 g).

To a mixture of compound 21 (1.00 g, 2.43 mmol) and triethylamine

(1.01 mL, 7.29 mmol) in THF (20 mL) was added acetyl chloride (0.225

mL, 3.16 mmol) dropwise at 0 ^◦C. The mixture was stirred at room

temperature for 30 min. The reaction mixture was partitioned between

AcOEt and water. The organic layer was successively washed with water

and brine, and dried over Na₂SO₄. After removal of the solvent, the

residue was puriﬁed by silica gel column chromatography (hexane/

AcOEt = 40/60 to 0/100) to give the title compound as a brown

amorphous solid (1.05 g, 95%). MS (ESI/APCI) m/z 454 [M + H]⁺. ¹H

NMR (300 MHz, DMSO‑d₆) δ 1.41 (9H, s), 1.47–1.63 (2H, m), 1.90–2.04

(5H, m), 3.07–3.23 (4H, m), 3.62–3.77 (2H, m), 3.95 (2H, t, J = 8.5 Hz),

5.17–5.29 (1H, m), 6.03 (1H, s), 7.21–7.29 (1H, m), 7.57 (1H, d, J = 1.5

Hz), 8.25 (1H, d, J = 9.1 Hz), 8.42 (1H, d, J = 0.8 Hz), 9.81 (1H, s).

N-Methyl-N-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]

piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}

acetamide (24 g).

3-Hydroxy-1-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]

piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}

piperidin-2-one ((RS)-29).

Compound (RS)-29 was prepared from (RS)-28 in a manner similar

to that described for compound 24b. White solid. Yield 20% over 3

steps. MS (ESI/APCI) m/z 520 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆)

δ 1.19 (6H, d), 1.67–1.81 (3H, m), 1.82–2.00 (2H, m), 2.03–2.14 (3H,

m), 2.77–2.87 (1H, m), 3.19 (2H, t, J = 8.5 Hz), 3.43–3.55 (3H, m),

3.57–3.67 (1H, m), 3.77–3.88 (2H, m), 3.96–4.09 (3H, m), 5.19 (1H, d,

J = 3.4 Hz), 5.27–5.37 (1H, m), 6.12 (1H, s), 7.05 (1H, d, J = 8.7 Hz),

7.13 (1H, s), 8.35 (1H, d, J = 8.3 Hz), 8.48 (1H, s). Anal. Calcd for

Step A. N-{1-[6-({1-[3-(Propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-

4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}acetamide

was

C

₂₇H₃₃N₇O₄: C, 62.41; H, 6.40; N, 18.87. Found: C, 62.24; H, 6.42; N,

18.59.

(2R)-2-(Acetyloxy)-5-chloropentanoic acid ((R)-26).

prepared from compound 22 g in a manner similar to that described for

compound 24b. White solid. Yield 37% over 3 steps. MS (ESI/APCI) m/z

464 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆) δ 1.19 (6H, d, J = 6.8 Hz),

1.66–1.82 (2H, m), 1.97–2.14 (5H, m), 2.76–2.90 (1H, m), 3.17 (2H, t, J

= 8.3 Hz), 3.49 (2H, ddd, J = 13.2, 9.3, 3.6 Hz), 3.76–3.87 (2H, m), 3.96

(2H, t, J = 8.5 Hz), 5.25–5.35 (1H, m), 6.06 (1H, s), 7.26 (1H, dd, J =

8.9, 2.1 Hz), 7.57 (1H, d, J = 1.9 Hz), 8.25 (1H, d, J = 8.7 Hz), 8.44 (1H,

d, J = 0.8 Hz), 9.81 (1H, s).

Compound (R)-26 was prepared from (2R)-tetrahydrofuran-2-car-

boxylic acid in a manner similar to that described for compound (S)-26.

Colorless oil. Yield 39% over 3 steps. ¹H NMR (300 MHz, CDCl₃) δ

1.86–2.13 (4H, m), 2.15 (3H, s), 3.49–3.74 (2H, m), 4.86–5.18 (1H, m),

6.51 (1H, bs).

tert-Butyl

4-{[6-(5-{[(2R)-2-(acetyloxy)-5-chloropentanoyl]

Step B. To a mixture of N-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadia-

zol-5-yl]piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}

acetamide (300 mg, 0.65 mmol) in anhydrous DMF (15 mL) was added

sodium hydride (60% oil dispersion, 38.8 mg, 0.970 mmol) at room

temperature, and the mixture was stirred at room temperature for 1 h.

Iodomethane (0.081 mL, 1.3 mmol) was added followed by stirring at

room temperature for 3 h. The mixture was diluted with AcOEt, suc-

cessively washed with water and brine, and dried over Na₂SO₄. After

removal of the solvent, the residue was suspended in Et₂O. The

precipitated solid was collected to give the title compound as a white

solid (24 g, 240 mg, 78%). MS (ESI/APCI) m/z 478 [M + H]⁺. ¹H NMR

(300 MHz, DMSO‑d₆) δ 1.19 (6H, d, J = 7.2 Hz), 1.67–1.84 (5H, m),

2.02–2.14 (2H, m), 2.74–2.91 (1H, m), 3.11 (3H, s), 3.22 (2H, t, J = 8.5

Hz), 3.49 (2H, ddd, J = 13.2, 9.2, 3.6 Hz), 3.76–3.88 (2H, m), 4.02 (2H,

t, J = 8.7 Hz), 5.32 (1H, dt, J = 7.9, 4.3 Hz), 6.13 (1H, s), 7.11 (1H, d, J

= 8.3 Hz), 7.20 (1H, s), 8.40 (1H, d, J = 8.7 Hz), 8.48 (1H, s). Anal. Calcd

for C₂₅H₃₁N₇O₃: C, 62.88; H, 6.54; N, 20.53. Found: C, 62.88; H, 6.59; N,

20.29.

amino}-2,3-dihydro-1H-indol-1-yl)pyrimidin-4-yl]oxy}piperidine-

1-carboxylate ((R)-27).

Compound (R)-27 was prepared from (R)-26 in a manner similar to

that described for compound (S)-27. White amorphous solid. Yield 83%.

MS (ESI/APCI) m/z 588 [M + H]⁺. ¹H NMR (300 MHz, CDCl₃) δ 1.47

(9H, s), 1.65–1.73 (2H, m), 1.86–2.00 (4H, m), 2.08–2.16 (2H, m), 2.23

(3H, s), 3.16–3.42 (4H, m), 3.53–3.65 (2H, m), 3.72–3.88 (2H, m), 3.96

(2H, t, J = 8.7 Hz), 5.21–5.42 (2H, m), 5.89 (1H, s), 7.00–7.18 (1H, m),

7.64 (1H, s), 7.76 (1H, s), 8.31 (1H, d, J = 8.7 Hz), 8.44 (1H, s).

tert-Butyl 4-[(6-{5-[(3R)-3-hydroxy-2-oxopiperidin-1-yl]-2,3-

dihydro-1H-indol-1-yl}pyrimidin-4-yl)oxy]piperidine-1-carbox-

ylate ((R)-28).

Compound (R)-28 was prepared from (R)-27 in a manner similar to

that described for compound (S)-28. White solid. Yield 86% over 2 steps.

MS (ESI/APCI) m/z 510 [M + H]⁺. ¹H NMR (300 MHz, CDCl₃) δ 1.47

(9H, s), 1.65–1.91 (3H, m), 1.93–2.10 (4H, m), 2.36–2.47 (1H, m),

3.19–3.34 (4H, m), 3.56–3.86 (5H, m), 3.99 (2H, t, J = 8.5 Hz),

4.15–4.27 (1H, m), 5.22–5.34 (1H, m), 5.91 (1H, s), 7.04 (1H, dd, J =

8.7, 2.3 Hz), 7.12 (1H, s), 8.38 (1H, d, J = 8.7 Hz), 8.45 (1H, s).

(3R)-3-Hydroxy-1-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-

yl]piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}

piperidin-2-one ((R)-29).

2-(Acetyloxy)-5-chloropentanoic acid ((RS)-26).

Compound (RS)-26 was prepared from tetrahydrofuran-2-carboxylic

acid in a manner similar to that described for compound (S)-26. Pale

yellow oil. Yield 47% over 3 steps. ¹H NMR (300 MHz, CDCl₃) δ

1.88–2.12 (4H, m), 2.15 (3H, s), 3.58 (2H, t, J = 6.2 Hz), 5.06 (1H, dd, J

= 7.3, 4.7 Hz), 6.52 (1H, bs).

Compound (R)-29 was prepared from (R)-28 in a manner similar to

that described for compound 24b. White solid. Yield 43% over 3 steps.

tert-Butyl 4-{[6-(5-{[2-(acetyloxy)-5-chloropentanoyl]amino}-

2,3-dihydro-1H-indol-1-yl)pyrimidin-4-yl]oxy}piperidine-1-

carboxylate ((RS)-27).

Retention time 15.5 min (CHIRALCEL OD3 (4.6 mmID × 250 mmL),

25

eluting with MeOH at 1 mL/min ﬂow rate). 99.9%ee. [

α]

+ 17.1 (c

D

1.014, CHCl₃). MS (ESI/APCI) m/z 520 [M + H]⁺. ¹H NMR (300 MHz,

Compound (RS)-27 was prepared from (RS)-26 in a manner similar

DMSO‑d₆) δ 1.19 (6H, d, J = 6.8 Hz), 1.68–2.01 (5H, m), 2.03–2.15 (3H,

13

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

m), 2.76–2.89 (1H, m), 3.19 (2H, t, J = 8.3 Hz), 3.43–3.67 (4H, m),

3.76–3.88 (2H, m), 3.96–4.08 (3H, m), 5.16 (1H, d, J = 3.0 Hz),

5.27–5.37 (1H, m), 6.12 (1H, s), 7.05 (1H, dd, J = 8.7, 1.9 Hz), 7.14 (1H,

d, J = 1.9 Hz), 8.34 (1H, d, J = 8.7 Hz), 8.48 (1H, s). Anal. Calcd for

[(3S)-3-(acetyloxy)-2-oxopiperidin-1-yl]-2,3-dihydro-1H-indol-1-yl}

pyrimidin-4-yl)oxy]piperidine-1-carboxylate as a white powder (1.19 g,

90%). MS (ESI/APCI) m/z 552 [M + H]⁺. ¹H NMR (300 MHz, CDCl₃) δ

1.47 (9H, s), 1.96–2.12 (6H, m), 3.17–3.35 (7H, m), 3.56–3.85 (6H, m),

3.94–4.04 (2H, m), 5.23–5.46 (2H, m), 5.91 (1H, s), 7.04 (1H, dd, J =

8.5, 2.1 Hz), 7.14 (1H, s), 8.34 (1H, d, J = 8.7 Hz), 8.44 (1H, s).

Step B. A mixture of tert-butyl 4-[(6-{5-[(3S)-3-(acetyloxy)-2-oxopi-

peridin-1-yl]-2,3-dihydro-1H-indol-1-yl}pyrimidin-4-yl)oxy]piperidine-

1-carboxylate (16.3 g, 29.6 mmol), potassium carbonate (8.17 g, 59.1

mmol), THF (100 mL) and MeOH (100 mL) was stirred at room tem-

perature for 3 h. After cooling to 0 ^◦C, 0.45 M hydrochloric acid (250

mL) was added to the reaction mixture followed by stirring at room

temperature for 1 h. The precipitated solid was collected by ﬁltration

and washed with water to give a pale yellow solid. The solid was dis-

solved in THF and dried over Na₂SO₄. The solvent was removed by

evaporation to give the title compound as a pale yellow solid ((S)-28,

15.0 g, 100%). MS (ESI/APCI) m/z 510 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.41 (9H, s), 1.48–1.63 (2H, m), 1.65–2.15 (6H, m),

3.10–3.24 (4H, m), 3.46–3.77 (4H, m), 3.95–4.09 (3H, m), 5.15 (1H, d,

J = 3.8 Hz), 5.20–5.31 (1H, m), 6.09 (1H, s), 7.05 (1H, dd, J = 8.7, 2.3

Hz), 7.13 (1H, d, J = 1.9 Hz), 8.34 (1H, d, J = 8.7 Hz), 8.46 (1H, s).

(3S)-3-Hydroxy-1-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-

yl]piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}

piperidin-2-one ((S)-29).

C

₂₇H₃₃N₇O₄: C, 62.41; H, 6.40; N, 18.87. Found: C, 62.40; H, 6.51; N,

18.81.

(2S)-2-(Acetyloxy)-5-chloropentanoic acid ((S)-26).

Step A. A mixture of (2S)-tetrahydrofuran-2-carboxylic acid (1.16 g,

9.99 mmol) and oxalyl dichloride (3 mL, 34.4 mmol) was stirred at room

temperature for 20 h. After the mixture was concentrated under reduced

pressure, the residual oil was added to a solution of phenylmethanol

(0.973 g, 9.00 mmol) and pyridine (2.14 g, 27.0 mmol) in Et₂O (₂0 mL)

at 0 ^◦C. The resulting mixture was allowed to warm to room temperature

followed by stirring for 2 h. The reaction mixture was quenched with

water and concentrated under reduced pressure. The residue was puri-

ﬁed by silica gel column chromatography (hexane/AcOEt = 100/0 to

95/5) to give benzyl (2S)-tetrahydrofuran-2-carboxylate as colorless oil

(1.17 g, 63%). ¹H NMR (300 MHz, CDCl₃) δ 1.82–2.11 (3H, m),

2.14–2.35 (1H, m), 3.82–4.08 (2H, m), 4.44–4.55 (1H, m), 5.09–5.26

(2H, m), 7.29–7.44 (5H, m).

Step B. A mixture of benzyl (2S)-tetrahydrofuran-2-carboxylate

(1.15 g, 5.58 mmol), acetyl chloride (2.63 g, 33.5 mmol) and zinc

chloride (3.8 mg, 0.028 mmol) was stirred under reﬂux overnight. The

reaction mixture was concentrated under reduced pressure, diluted with

Et₂O and saturated aqueous NaHCO₃solution, and extracted with Et₂O.

The organic layer was washed with brine, dried over MgSO₄and

concentrated under reduced pressure. The residue was puriﬁed by silica

gel column chromatography (hexane/AcOEt = 100/0 to 85/15) to give

benzyl (2S)-2-(acetyloxy)-5-chloropentanoate as colorless oil (1.01 g,

64%). ¹H NMR (300 MHz, CDCl₃) δ 1.76–1.91 (2H, m), 1.96–2.08 (2H,

m), 2.14 (3H, s), 3.53 (2H, t, J = 6.2 Hz), 5.01–5.11 (1H, m), 5.12–5.28

(2H, m), 7.28–7.49 (5H, m).

Compound (S)-29 was prepared from (S)-28 in a manner similar to

that described for compound 24b. White solid. Yield 49% over 3 steps.

Retention time 13.6 min (CHIRALCEL OD3 (4.6 mmID × 250 mmL),

25

eluting with MeOH at 1 mL/min ﬂow rate). 99.9%ee. [

α]

ꢀ 16.2 (c

D

1.027, CHCl₃). MS (ESI/APCI) m/z 520 [M + H]⁺. ¹H NMR (300 MHz,

DMSO‑d₆) δ 1.19 (6H, d, J = 6.8 Hz), 1.67–2.01 (5H, m), 2.03–2.16 (3H,

m), 2.74–2.91 (1H, m), 3.14–3.25 (2H, m), 3.43–3.68 (4H, m),

3.76–3.90 (2H, m), 3.96–4.10 (3H, m), 5.16 (1H, d, J = 3.8 Hz),

5.26–5.39 (1H, m), 6.12 (1H, s), 7.05 (1H, d, J = 8.7 Hz), 7.14 (1H, s),

8.34 (1H, d, J = 8.7 Hz), 8.47 (1H, s). Anal. Calcd for C₂₇H₃₃N₇O₄: C,

62.41; H, 6.40; N, 18.87. Found: C, 62.41; H, 6.55; N, 18.79.

Step C. A mixture of benzyl (2S)-2-(acetyloxy)-5-chloropentanoate

(1.01 g, 3.55 mmol) and 10% Pd on carbon (80 mg) in AcOEt was stirred

at room temperature under H₂atmosphere (1 atm, baloon) overnight.

After the catalyst was removed by ﬁltration, the ﬁltrate was concen-

trated under reduced pressure to give the title compound as a colorless

oil ((S)-26, 0.690 g, 100%). ¹H NMR (300 MHz, CDCl₃) δ 1.87–2.11 (4H,

m), 2.13–2.24 (3H, m), 3.51–3.73 (2H, m), 4.93–5.19 (1H, m), 6.40 (1H,

bs).

tert-Butyl

4-({6-[5-(4-hydroxy-2-oxopiperidin-1-yl)-2,3-dihy-

dro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate

((RS)-31a).

A mixture of compound 10 (240 mg, 0.505 mmol), (RS)-4-hydrox-

ypiperidin-2-one (174 mg, 1.51 mmol), trans-N,N’-dimethylcyclohex-

ane-1,2-diamine (57.4 mg, 0.404 mmol), copper(I) iodide (38.5 mg,

0.202 mmol) and potassium carbonate (349 mg, 2.52 mmol) in toluene

tert-Butyl

4-{[6-(5-{[(2S)-2-(acetyloxy)-5-chloropentanoyl]

amino}-2,3-dihydro-1H-indol-1-yl)pyrimidin-4-yl]oxy}piperidine-

1-carboxylate ((S)-27).

◦

(10 mL) was stirred at 120 C for 2 days. The reaction was quenched

A mixture of compound (S)-26 (680 mg, 3.49 mmol) and oxalyl

dichloride (1.50 mL, 17.2 mmol) was stirred at room temperature for 20

h and concentrated under reduced pressure. The residual oil was added

to a mixture of compound 21 (1.25 g, 3.04 mmol) in DMA at room

temperature. The resulting mixture was stirred for 4 h, quenched with

water, and extracted with AcOEt. The organic layer was washed with

brine, dried over MgSO₄and concentrated under reduced pressure. The

residue was puriﬁed by silica gel column chromatography (hexane/

AcOEt = 100/0 to 50/50) to give the title compound as a pale yellow

amorphous solid (1.74 g, 85%). MS (ESI/APCI) m/z 588 [M + H]⁺. ¹H

NMR (300 MHz, CDCl₃) δ 1.47 (9H, s), 1.69–1.79 (2H, m), 1.85–2.02

(4H, m), 2.07–2.18 (2H, m), 2.23 (3H, s), 3.15–3.36 (4H, m), 3.57 (2H, t,

J = 6.4 Hz), 3.69–3.87 (2H, m), 3.96 (2H, t, J = 8.7 Hz), 5.16–5.38 (2H,

m), 5.89 (1H, s), 7.09 (1H, dd, J = 8.7, 2.3 Hz), 7.64 (1H, s), 7.75 (1H, s),

8.31 (1H, d, J = 8.7 Hz), 8.44 (1H, s).

with water and extracted with AcOEt. The organic layer was washed

with brine, dried over MgSO₄and concentrated under reduced pressure.

The residue was puriﬁed by silica gel column chromatography (AcOEt/

MeOH = 100/0 to 90/10) gave the title compound as a white solid (27.0

mg, 10%). MS (ESI/APCI) m/z 510 [M + H]⁺. ¹H NMR (300 MHz,

CDCl₃) δ 1.47 (9H, s), 1.84–1.87 (2H, m), 1.95–2.00 (2H, m), 2.09–2.23

(2H, m), 2.53–2.64 (1H, m), 2.80–2.92 (1H, m), 3.19–3.34 (4H, m),

3.51–3.61 (1H, m), 3.71–3.87 (4H, m), 3.99 (2H, t, J = 8.7 Hz),

4.29–4.42 (1H, m), 5.24–5.36 (1H, m), 5.92 (1H, s), 7.00–7.14 (2H, m),

8.35 (1H, d, J = 8.7 Hz), 8.45 (1H, s).

tert-Butyl

4-({6-[5-(5-hydroxy-2-oxopiperidin-1-yl)-2,3-dihy-

dro-1H-indol-1-yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate

((RS)-31b).

Compound (RS)-31b was prepared from compound 10 and (RS)-5-

hydroxypiperidin-2-one in a manner similar to that described for com-

pound (RS)-31a. White solid. Yield 4%. MS (ESI/APCI) m/z 510 [M +

H]⁺. ¹H NMR (300 MHz, CDCl₃) δ 1.47 (9H, s), 1.66–1.81 (3H, m),

1.92–2.22 (4H, m), 2.48–2.64 (1H, m), 2.70–2.84 (1H, m), 3.18–3.35

(4H, m), 3.51–3.64 (1H, m), 3.73–3.85 (3H, m), 3.98 (2H, t, J = 8.7 Hz),

4.26–4.36 (1H, m), 5.22–5.34 (1H, m), 5.91 (1H, s), 7.04 (1H, dd, J =

8.7, 2.3 Hz), 7.11 (1H, s), 8.36 (1H, d, J = 8.7 Hz), 8.45 (1H, s).

4-Hydroxy-1-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]

piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}

tert-Butyl 4-[(6-{5-[(3S)-3-hydroxy-2-oxopiperidin-1-yl]-2,3-

dihydro-1H-indol-1-yl}pyrimidin-4-yl)oxy]piperidine-1-carbox-

ylate ((S)-28).

Step A. A mixture of compound (S)-27 (1.41 g, 2.40 mmol) and po-

tassium carbonate (0.663 g, 4.80 mmol) in DMF (30 mL) was stirred at

50 ^◦C for 8 h. After the mixture was cooled to 0 ^◦C, 0.1 M hydrochloric

acid was added. The precipitated solid was collected by ﬁltration,

washed, and dried under reduced pressure to give tert-butyl 4-[(6-{5-

14

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

piperidin-2-one ((RS)-32a).

μ

g/mL streptomycin), and incubated with various concentrations of test

Compound (RS)-32a was prepared from (RS)-31a in a manner

similar to that described for compound 24b. Pale yellow solid. Yield

58% over 3 steps. MS (ESI/APCI) m/z 520 [M + H]⁺. ¹H NMR (300 MHz,

CDCl₃) δ 1.29 (6H, d, J = 6.8 Hz), 1.82–1.97 (2H, m), 1.98–2.09 (2H, m),

2.08–2.24 (2H, m), 2.52–2.65 (1H, m), 2.81–2.87 (2H, m), 3.24 (2H, t, J

= 8.5 Hz), 3.52–3.66 (3H, m), 3.77–4.05 (6H, m), 4.32–4.39 (1H, m),

5.32–5.43 (1H, m), 5.93 (1H, s), 7.05 (1H, dd, J = 8.5, 2.1 Hz), 7.11 (1H,

s), 8.36(1H, d, J = 8.3 Hz), 8.45 (1H, s). Anal. Calcd for C₂₇H₃₃N₇O₄: C,

62.41; H, 6.40; N, 18.87. Found: C, 62.31; H, 6.29; N, 18.72.

5-Hydroxy-1-{1-[6-({1-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]

piperidin-4-yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}

piperidin-2-one ((RS)-32b).

compounds diluted in assay buffer for 2 h. After removal of culture su-

pernatant, cAMP-induced luciferase activities were measured with

Steady-Glo reagent (Promega) and EnVision Multilabel Plate Reader

(PerkinElmer). Agonist activities of test compounds on GPR119 were

expressed as [(A ꢀ B)/(C ꢀ B)] × 100 (luciferase activities (A) in test

compounds-treated cells, (B) in vehicle-treated cells, and (C) in cells

treated with 10 μM N-[4-(methylsulfonyl)phenyl]-5-nitro-6-{4-[4-(tri-

ﬂuoromethoxy)phenoxy]piperidin-1-yl}pyrimidin-4-amine²⁶).

values were obtained with XLﬁt software (ID Business Solutions).

EC₅₀

5.3. hERG inhibition assay

Compound (RS)-32b was prepared from (RS)-31b in a manner

similar to that described for compound 24b. White solid. Yield 36% over

3 steps. MS (ESI/APCI) m/z 520 [M + H]⁺. ¹H NMR (300 MHz, CDCl₃) δ

1.29 (6H, d, J = 6.8 Hz), 1.86–1.97 (2H, m), 2.00–2.20 (5H, m),

2.47–2.61 (1H, m), 2.67–2.95 (2H, m), 3.15–3.30 (2H, m), 3.52–3.62

(3H, m), 3.75–3.82 (1H, m), 3.83–3.93 (2H, m), 3.98 (2H, t, J = 8.7 Hz),

4.25–4.37 (1H, m), 5.29–5.45 (1H, m), 5.93 (1H, s), 7.05 (1H, d, J = 8.7

Hz), 7.12 (1H, s), 8.36 (1H, d, J = 8.3 Hz), 8.45 (1H, s). Anal. Calcd for

hERG/CHO cells stably expressing hERG channel were purchased

from Millipore (UK) Ltd. (cat. # CYL3038). Cells were cultured at 32 ^◦C,

5% CO₂in Ham’s F-12 medium supplemented with 10% fetal bovine

serum, 500 μg/mL Geneticin (In vitrogen). The hERG inhibition assay

was performed on the IonWorks Quattro (Molecular Devices) system in

population patch clamp (PPC) mode. The extracellular solution was

phosphate-buffered salines (PBS) with calcium and magnesium (Cat.

#14040, In vitrogen). The intracellular solution contained 140 mM KCl,

2 mM MgCl₂, 1 mM EGTA and 20 mM HEPES, pH 7.3 with KOH. After

C

₂₇H₃₃N₇O₄: C, 62.41; H, 6.40; N, 18.87. Found: C, 62.26; H, 6.43; N,

18.59.

tert-Butyl 4-({6-[5-(2-oxocyclohexyl)-2,3-dihydro-1H-indol-1-

perforation using 100 μg/mL amphotericin B (Sigma-Aldrich), hERG

current was measured under the potential-clamp protocol (Holding

potential ꢀ 80 mV, the ﬁrst voltage 40 mV: 2 sec, the second voltage ꢀ

50 mV: 2 sec). The peaktail current before addition of the compounds

was measured as the pre hERG current. Test compounds were incubated

on the cells for a period of 5 min. The peaktail current after addition of

the compounds was measured as the post hERG current. %hERG inhi-

bition was calculated (n = 4) to the following.

yl]pyrimidin-4-yl}oxy)piperidine-1-carboxylate ((RS)–33).

To a solution of compound 10 (1.00 g, 2.10 mmol) and cyclohexa-

none (0.436 mL, 4.21 mmol) in DME (10 mL) were added Pd₂(dba)₃

(0.096 g, 0.11 mmol), Xantphos (0.122 g, 0.211 mmol), and cesium

carbonate (2.06 g, 6.31 mmol) at room temperature. The mixture was

stirred at 80 ^◦C under N₂atmosphere overnight. The reaction mixture

was diluted with water, and extracted with AcOEt. The organic layer was

washed with brine, dried over MgSO₄and concentrated under reduced

pressure. The residue was puriﬁed by silica gel column chromatography

(hexane/AcOEt = 85/15 to 50/50) to give a yellow solid. The obtained

solid was crystallized from AcOEt–hexane to give the title compound as

a white solid (0.244 g, 24%). MS (ESI/APCI) m/z 493 [M + H]⁺. ¹H NMR

(300 MHz, DMSO‑d₆) δ 1.41 (9H, s), 1.48–1.62 (2H, m), 1.68–2.00 (6H,

m), 2.02–2.15 (2H, m), 2.25–2.33 (1H, m), 2.45–2.58 (1H, m),

3.10–3.22 (4H, m), 3.65–3.76 (3H, m), 3.96 (2H, t, J = 8.7 Hz),

5.18–5.29 (1H, m), 6.06 (1H, s), 6.90 (1H, dd, J = 8.3, 1.5 Hz), 6.99 (1H,

s), 8.25 (1H, d, J = 8.3 Hz), 8.44 (1H, d, J = 0.8 Hz).

%hERGinhibition = 100 ꢀ (posthERGcurrent/prehERGcurrent) × 100

5.4. Estimation of LogD at pH 7.4

LogD7.4, which is a partion coefﬁcient between 1-octanol and

aqueous buffer pH 7.4, of the compounds was measured on the chro-

matographic procedure whose condition was developed based on a

published method.^20,21

2-{1-[6-({1-[3-(Propan-2-yl)-1,2,4-oxadiazol-5-yl]piperidin-4-

yl}oxy)pyrimidin-4-yl]-2,3-dihydro-1H-indol-5-yl}cyclohexanone

((RS)-34).

5.5. Solubility determination

Small volumes of the compound DMSO solutions were added to the

aqueous buffer solution (pH 6.8). After incubation, precipitates were

separated by ﬁltration. The solubility was determined by HPLC analysis

of each ﬁltrate.

Compound (RS)-34 was prepared from (RS)–33 in a manner similar

to that described for compound 24b. Pale yellow solid. Yield 9% over 3

steps. MS (ESI/APCI) m/z 503 [M + H]⁺. ¹H NMR (300 MHz, DMSO‑d₆)

δ 1.19 (6H, d, J = 7.2 Hz), 1.68–1.97 (6H, m), 2.02–2.14 (4H, m),

2.25–2.33 (1H, m), 2.44–2.59 (1H, m), 2.77–2.87 (1H, m), 3.17 (2H, t, J

= 8.7 Hz), 3.44–3.55 (2H, m), 3.69 (1H, dd, J = 12.3, 5.5 Hz), 3.77–3.87

(2H, m), 3.97 (2H, t, J = 8.5 Hz), 5.26–5.37 (1H, m), 6.09 (1H, s), 6.91

(1H, dd, J = 8.3, 1.5 Hz), 6.99 (1H, s), 8.25 (1H, d, J = 8.3 Hz), 8.46 (1H,

d, J = 0.8 Hz). Anal. Calcd for C₂₈H₃₄N₆O₃: C, 66.91; H, 6.82; N, 16.72.

Found: C, 66.75; H, 6.57; N, 16.44.

5.6. Single-crystal X-ray structure analysis

Crystal data for compound 24b: C27H33N7O3 MW = 503.60; crystal

size, 0.32 × 0.18 × 0.15 mm; colorless, block; triclinic, space group P-1,

a = 5.50960(10) Å, b = 12.9242(2) Å, c = 18.9707(3) Å,

α = 89.9541

(9)^◦, β = 87.6185(9)^◦, γ = 79.8701(9)^◦,V = 1328.61(4) Å3, Z = 2, Dx =

5.2. In vitro GPR119 agonist activity

1.259 g/cm3, T = 298 K, μ = 0.688 mm-1, λ = 1.54184 Å, R1 = 0.0902,

wR2 = 0.2665, S = 1.059.

All measurements were made on a Rigaku R-AXIS RAPID diffrac-

tometer using graphite monochromated Cu-K radiation. The structure

GPR119 agonist activities were evaluated in the reporter gene assay

using CHO cells stably co-expressing cyclic AMP response element

(CRE)–luciferase reporter gene (Promega) and GPR119. Cells were

α

was solved by direct methods with SHELXT-2018/21) and was reﬁned

using full-matrix least-squares on F2 with SHELXL-2018/3.2) All non-H

atoms were reﬁned with anisotropic displacement parameters.^27,28

CCDC 2,046,043 for compound 24b the supplementary crystallo-

graphic data for this paper. These data can be obtained free of charge

from The Cambridge Crystallographic Data Centre via http://www.ccdc.

cam.ac.uk/structures.

seeded at 10,000 cells/well in Minimum essential medium (MEM)

α

containing 10% fetal bovine serum, 100 U/mL penicillin, 100

streptomycin, and 500 g/mL Geneticin in 384-well white opaque

plates, and cultured at 37 C under 5% CO₂with saturated humidity

overnight. The cells were washed once with assay buffer (MEM , 20

mmol/L HEPES, 0.1% bovine serum albumin, 100 U/mL penicillin, 100

μg/mL

μ

◦

α

15

M. Kamaura et al.

Bioorganic & Medicinal Chemistry 34 (2021) 116034

5.7. In vitro metabolic clearance in human and rat hepatic microsomes

libitum. Male N-STZ1.5 rats (25–30 weeks of age) were fasted overnight

and orally given vehicle (0.5% methylcellulose) or compounds. Sixty

minutes later, all animals were received an oral glucose load (1.5 g/kg).

Blood samples were collected from tail vein before drug administration

(pre), and just before glucose load (time 0), and 10, 30, 60, and 120 min

after glucose load. Plasma glucose and plasma insulin levels were

measured by Autoanalyzer 7080 (Hitachi, Japan) and radioimmuno-

assay (Millipore, USA), respectively. Differences between two groups

were analyzed by one-tailed Williams test.

Human and mouse liver microsomes were purchased from Xenotech,

LLC (Lenexa, KS). An incubation mixture consisted of microsomal pro-

tein in 50 mM KH2PO4–K2HPO4 phosphate buffer (pH 7.4) and 1 μM

test compound. The concentration of microsomal protein was 0.2 mg/

mL. An NADPH-generating system containing 5 mM MgCl2, 5 mM

glucose-6-phosphate, 0.5 mM β-NADP⁺, and 1.5 units/mL glucose-6-

phosphate dehydrogenase was added to the incubation mixture to

initiate the enzyme reaction. The reaction was terminated 15 and 30 min

after the initiation of the reaction by mixing the reaction mixture with

acetonitrile, followed by centrifugation. The supernatant was subjected

to LC/MS/MS analysis. The metabolic velocity was calculated as the

slope of the concentration–time plot.

Declaration of Competing Interest

The authors declared that there is no conﬂict of interest.

Acknowledgments

5.8. In vitro GLP-1 secretion assay

We thank Tatsuru Tomokuni, Kana Furuyabu, and Chihiro Kawate

for conducting hERG inhibition assay. We also thank Mitsuyoshi Nish-

itani for X-ray crystallographic analysis, the members in charge of

determination of LogD value, and the members of the Takeda Analytical

Research Laboratories, Ltd. for elemental analyses. Finally, we

acknowledge Dr. Tsuyoshi Maekawa for supervision of the research,

careful reading of the manuscript, and valuable suggestions.

GLUTag cells, the murine L cell line, were cultured in Dulbecco’s

modiﬁed Eagle’s medium (DMEM, high glucose) containing 10% heat-

inactivated FBS, 100 IU/mL penicillin and 100 μg/mL streptomycin.

GLUTag cells were seeded at density of 1 × 104 cells/well in a 96 well

poly-^L-lysine coated plate. The following day, the medium was replaced

with DMEM (low glucose) containing 10% heat-inactivated FBS, 100 IU/

mL penicillin and 100 μg/mL streptomycin, and the cells were incubated

References

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acid free BSA, 10 mmol/L glucose, and compounds was added, and the

cells were incubated for 2 h at 37 ^◦C. After incubation, supernatants

from each well were collected, and secreted active GLP-1 concentration

was measured using active GLP-1 ELISA kit (Millipore, EGLP-35 K) ac-

cording to the manufacture’s instruction.

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◦

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17

Article Doi

Discovery of a novel series of indolinylpyrimidine-based GPR119 agonists: Elimination of ether-a-go-go-related gene liability using a hydrogen bond acceptor-focused approach

DOI: 10.1016/j.bmc.2021.116034

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