Synthesis and biological evaluation of novel sigma-1 receptor antagonists based on pyrimidine scaffold as agents for treating neuropathic pain

Article

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Synthesis and Biological Evaluation of Novel Sigma‑1 Receptor

Antagonists Based on Pyrimidine Scaﬀold As Agents for Treating

Neuropathic Pain

Yu Lan,^†,§Yin Chen,^‡,§Xudong Cao,^†Juecheng Zhang,^†Jie Wang,^†Xiangqing Xu,^‡Yinli Qiu,^‡

Tan Zhang,^‡Xin Liu,^†Bi-Feng Liu,^†and Guisen Zhang*^,†,‡

^†Systems Biology Theme, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of

Science and Technology, Wuhan 430074, China

^‡Jiangsu Nhwa Pharmaceutical Co., Ltd., 69 Democratic South Road, Xuzhou, Jiangsu 221116, China

S

* Supporting Information

ABSTRACT: The discovery and synthesis of a new series of pyrimidines as

potent sigma-1 receptor (σ₁R) antagonists, associated with pharmacological

antineuropathic pain activity, are the focus of this article. The new compounds

were evaluated in vitro in σ-1 and σ-2 receptor binding assays. The nature of

the pyrimidine scaﬀold was crucial for activity, and a basic amine was shown to

be necessary according to the known pharmacophoric model. The most

promising derivative was 5-chloro-2-(4-chlorophenyl)-4-methyl-6-(3-(piper-

idin-1-yl)propoxy)pyrimidine (137), which exhibited a high binding aﬃnity to

σ₁R receptor (K_iσ₁= 1.06 nM) and good σ-1/2 selectivity (1344-fold). In in

vivo tests, compound 137 exerted dose-dependent antinociceptive eﬀects in

mice formalin model and rats CCI models of neuropathic pain. In addition, no

motor impairments were found in rotarod tests; acceptable pharmacokinetic

properties were also noted. These data suggest compound 137 may constitute

a novel class of drugs for the treatment of neuropathic pain.

of addiction relative to the ﬁrst-line medications, opioid

analgesics are recommended typically for patients who have

not responded to ﬁrst-line medications. Several medications are

generally used as third-line treatments because of weak eﬃcacy,

discrepant results, or safety concerns, including anticonvulsants,

NMDA receptor antagonists, and topical capsaicin. Thus,

neuropathic pain analgesics with novel mechanisms of action

that improve the eﬃcacy of existing therapies and reduce

unwanted eﬀects are needed.⁸⁻¹⁰

INTRODUCTION

■

Neuropathic pain is widely recognized as one of the most

diﬃcult clinical syndromes, which aﬀects 8% of the population

and is considerably costly for the health care system and

devastating to the people who experience it.^1,2Although

progress has been made from basic science and clinical research

and preclinical neuropathic pain models have facilitated

identiﬁcation of several promising targets, innovations in the

ﬁeld of pain in recent years are lacking. Available therapies

often provide incomplete pain relief and treatment-related side

eﬀects are common; thus neuropathic pain management

remains a challenge.³

Various guidelines have been established for the pharmaco-

logical management of neuropathic pain.⁴⁻⁶Although diﬀering

in detail, the guidelines are generally consistent.⁷Medications

recommended as ﬁrst-line treatments for neuropathic pain

include antidepressants (such as tricyclic antidepressants, TCA,

and dual reuptake inhibitors of both serotonin and

norepinephrine), calcium channel α₂-δ ligands (gabapentin

and prebagalin), and topical lidocaine. These medications are

very eﬀective in reducing pain in several neuropathic pain

disorders, but treatment may be compromised and outweighed

by its side eﬀects. Opioid analgesics can alleviate nociceptive

and neuropathic pain with a mean decrease in pain intensity,

comparable with TCA and gabapentin/pregabalin. Regarding

their long-term safety, opioid-induced hyperalgesia and the risk

The sigma receptors (σR) were ﬁrst discovered in 1976¹¹

and mischaracterized originally as a new class of opioid

receptors. Later, sigma receptors were confused with the PCP/

NMDA glutamate receptor complex because of nonselective

ligands.^12,13Currently, sigma receptors are considered unique

proteins without homology to opioid receptors or other

mammalian proteins.¹⁴Pharmacological studies and biochem-

ical analyses have identiﬁed two distinct subtypes of sigma

receptor, termed the sigma-1 (σ₁) and sigma-2 (σ₂)

receptors.^15,16The σ₁R has been puriﬁed and cloned and

encodes a protein of 223 amino acids with a molecular weight

of ∼24 kDa.¹⁷Investigations have suggested that the σ₁R plays

an important role in central nervous system (CNS) function.¹⁸

This protein acts as a unique ligand-regulated molecular

Received: August 6, 2014

A

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chaperone that modulates the activity of diﬀerent proteins such

as N-methyl-D-aspartic (NMDA)¹⁹receptors and several ion

channels.²⁰⁻²²The σ₂R has not been characterized as well as

the σ₁R, and cloning of the σ₂R has not been reported.

Recently, the σ₂R was shown to be involved in cellular

proliferation and cell death, and radiolabeled ligands acting on

σ₂R have been used for tumor imaging techniques (i.e., positron

emission tomography, PET). Numerous studies suggest that

the σ₂R is an excellent biomarker and a promising therapeutic

target for the treatment of cancer.^23,24Due to their diﬀerent

pharmacological functions, selectivity between the σ₁R and σ₂R

is desirable.

subtype.⁴⁴Using a three-dimensional (3D) pharmacophore

model of σ₁R antagonists and virtual screening program, we

identiﬁed a new series of compounds based on pyrimidine

scaﬀold. Herein, we report the synthesis and investigated the

behavior of pyrimidine derivatives as novel and potent

antineuropathic analgesics. The compounds were subjected to

preliminary pharmacological evaluation to determine their

aﬃnities to the σ₁R and σ₂R. Their structure−activity

relationships (SARs) for the σ₁R and σ₂R were associated

with variation in the basic amino moieties and substituents on

the pyrimidine scaﬀold. Among the derivatives prepared,

compound 137 exhibited a high binding aﬃnity to σ₁R (K_iσ₁

= 1.06 nM), good σ₁R/σ₂R selectivity (1344-fold), and was

identiﬁed as a σ₁R antagonist. Weak aﬃnity to hERG channels

indicated that compound 137 had a low incidence of cardiac

toxicity. In addition, compound 137 inhibited dose-dependent

antinociceptive eﬀects in the mouse formalin-induced pain

model and rat chronic constriction injury (CCI) model. Motor

impairments were not found in rotarod tests at analgesic doses,

and acceptable pharmacokinetic properties were observed. On

the basis of these properties, compound 137 may be considered

a novel class of drugs for the treatment of neuropathic pain.

To date, selective σ₁R ligands have not been introduced to

the market, but many of the compounds have been tested in

clinical studies as antidepressants,²⁵antipsychotics,²⁶treat-

ments for drug abuse,²⁷and learning/memory enhancers.²⁸

Moreover, numerous groups have studied the potential role of

the σ₁R in pain management, especially neuropathic pain.²⁹

Since the 1990s, the σ₁R has been known to modulate opioid

analgesia,³⁰⁻³²and recently, the relationship between the μ-

opioid and σ₁R was shown to involve a direct physical

interaction.³³Treatment with σ₁R antagonists or σ₁R antisense

oligodeoxynucleotides enhanced the antinociceptive eﬀect

induced by morphine and other μ-opioid receptor agonists in

the acute nociceptive test, while σ₁R agonists reduced the

analgesic response elicited by opioids.^34,35The σ₁R also plays a

nociceptive role in the absence of opioids, as shown in studies

using σ₁R knockout mice and certain behavioral models

involving pain sensitization pathways. Mice lacking σ₁R were

unable to develop completely the two phases of formalin-

induced paw licking/biting behavior³⁶and showed no

mechanical hypersensitivity following capsaicin sensitization.³⁷

In an animal model of neuropathic pain, cold and mechanical

hypersensitivity were strongly attenuated in σ₁R knockout mice

treated with paclitaxel³⁸or exposed to partial sciatic nerve

ligation.³⁹Pharmacological antagonism of the σ₁R produced

similar results. Haloperidol and its metabolites I and II, which

have an aﬃnity for the σ₁R, inhibited formalin-induced pain⁴⁰

and capsaicin-induced sensitization in wild-type mice.⁴¹The

recently described σ₁R antagonists, S1RA, with high aﬃnity to

σ-1 receptors (K_i= 17 nM) and excellent selectivity ratio (σ₂/

σ₁> 550), are currently undergoing phase II clinical trials for

treatment of neuropathic pain^42,43(Figure 1).

MOLECULAR DESIGN

■

As the lack of three-dimensional protein structure of a σ₁

receptor,¹⁸the main focus of current eﬀorts toward the

development of a new σ₁R ligand is concentrated on structure-

based ligand design. In this work, Discovery Studio 2.5 software

(DS) was utilized to build and validate pharmacophore model.

3D pharmacophore hypotheses were built based on a set of

known diverse σ₁R antagonists. The best pharmacophore

model, which identiﬁed compounds with an associated

correlation coeﬃcient of 0.94027 between the experimental

and estimated K_ivalue, consisted of four pharmacophoric

feature: HBA (green), Hydrophob_aromatic (blue), Hydro-

phobic (light blue), and PosIonizable (red), as shown in Figure

2A. Detailed information about 3D pharmacophore model

generation can be found in the Support Information.

On the basis of the preliminary founding of virtual screening

by this 3D pharmacophore model and analysis of hits

identiﬁcation, we got the pyrimidine scaﬀold and design a

series of compounds (Figure 2B). In detail, the chemical

structures of these pyrimidine derivatives were designed

according to the following rationale: we changed the distance

between the pyrimidine ring and basic amino moiety (carbon

chain linker) to validate the requirement of the pharmacophoric

model, tested various basic amino moieties to ﬁt the

PosIonizable and Hydrophobic feature (NR₁R₂), took diﬀerent

substitution on the 4- and 5-positions of the pyrimidine ring

(R₃and R₄), and replaced the 2-phenyl goup of pyrimidine by

an aromatic ring or small alkyl group (R₅) to ﬁnd the best

conformational ﬁtting to the receptor.

Our interest has focused on the development of novel ligands

with high aﬃnity to the σ₁R and selectivity over the σ₂R

CHEMISTRY

■

The synthesis of the new compounds was accomplished using

diﬀerent routes, as shown in Schemes 1−5.

As depicted in Scheme 1, phenyl aminoalkoxypyrimidine

derivatives 8 and 14−34 were obtained using two alternative

procedures. The key intermediate 6-methyl-2-phenylpyrimidin-

4-ol 7 was prepared using a one-step procedure from

benzimidamide 5, which was cyclized with a moderate yield

of ethyl 3-oxobutanoate 6. Depending on the substitution

Figure 1. Representative σ₁receptor ligands.

B

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Table 1. Binding Aﬃnities for the σ₁and σ₂Receptor of

Compounds 8, 14−18, 36, and 38

selectivity

(σ₂/σ₁)

a

b

compd

Z

K_iσ₁(nM)

K_iσ₂(nM)

8

(CH₂)₂

(CH₂)₃

(CH₂)₄

(CH₂)₅

(CH₂)₆

>2000

c

14

15

16

17

18

36.3 4.8

949 65

26.1

11.2

95.2 7.6

389 63

>2000

1072 82

>2000

CH₂CHC

116 9.4

1451 82

12.5

HCH₂

36

38

(CH₂)₂O(CH₂)₂

CH₂CHOHCH₂

422 73

486 85

1924 107

1644 124

4.6

3.4

a

Aﬃnities were determined in guinea pig brain using [³H]-

b

(+)-pentazocine. Aﬃnities were determined in guinea pig brain

Figure 2. (A) The 3D-QSAR pharmacophore model of σ-1R

antagonist. The model is constituted with four pharmacophore

features: HBA, green; Hydrophob_aromatic, blue; Hydrophobic,

light blue; and PosIonizable, red. (B) The virtual screening result

and molecular design of pyrimidine scaﬀold by this 3D-QSAR model.

using [³H]-DTG in the presence of (+)-SKF-10047 to block σ-1

c

receptors. The values are means

SEM of three experiments

performed in duplicate.

Elongated analogues 36 and 38 were obtained starting from

6-methyl-2-phenylpyrimidin-4-ol 7, as shown in Scheme 2.

Alkylation of 7 with 1-bromo-2-(bromoethoxy)ethane under

basic conditions produced the monosubstituted 35, which was

reacted with morpholine to aﬀorded compound 36. Hydroxyl

derivative 38 was prepared by treatment of 7 with 2-

(chloromethyl)oxirane under basic conditions, followed by

ring-opening with morpholine.

The route depicted in Scheme 3 was used for the preparation

of substituted pyrimidine scaﬀold compounds 69−78 and 79−

88. The intermediates 49−58 were obtained using the

cyclization procedure with benzimidamide 5, which was reacted

pattern, the ﬁnal compounds 8 and 14−34 were prepared using

method A or the two-step procedure, method B (Tables 1, 2).

In method A, 7 was reacted with commercially available

chloroalkylamine under a basic condition to obtain a good yield

of compound 8. Method B involved standard alkylation with

1,n-dibromoalkanes to produce 9−13, and reaction with the

corresponding amines under mild basic conditions resulted in

moderate yields of the ﬁnal compounds 14−34. The general

synthetic methods for phenyl aminoalkoxypyrimidine deriva-

tives 8 and 14−34 (methods A and B) are described in the

Experimental Section.

a

Scheme 1

a

Reagents and conditions: (I) t-BuOK, MeOH, reﬂux; (II) Br(CH₂)_nBr, K₂CO₃, acetone, reﬂux; (III) HNR₁R₂, Cs₂CO₃, acetonitrile, reﬂux; (IV)

K₂CO₃, KI, acetonitrile, reﬂux;

C

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produce 59−68, and then reaction with the piperidine or

pyrrodine aﬀorded the ﬁnal compounds 69−78 and 79−88,

respectively, with moderate yields (Table 3).

Table 2. Binding Aﬃnities for the σ₁and σ₂Receptors of

Compounds 14 and 19−34

6,7-Dihydro-5H-cyclopenta[d]pyrimidines 94−95 and

5,6,7,8-tetrahydroquinazoline 97−98 (Scheme 4 and Table 4)

were prepared by reaction of 5 with ethyl 2-oxocyclopentane-

carboxylate or 2-oxocyclohexanecarboxylate to obtain inter-

mediates 91 and 92, which were subsequently alkylated with

1,3-dibromopropane and reacted with piperidine or pyrrolidine,

under conditions similar to those used for substituted

pyrimidine derivatives.

The syntheses of the 2-substituted 5-chloro-4-methyl-6-

aminoalkoxy pyrimidine derivatives 132−142 were accom-

plished by the corresponding aryl- or alkyl- amidine 99−109

that were reacted with ethyl 2-chloro-3-oxobutanoate 48,

followed by alkylation with 1,3-dibromopropane and reaction

with piperidine (Scheme 5 and Table 5). The nonsubstituted 5-

chloro-4-methyl-6-aminoalkoxy pyrimidine, intermediate 118,

which is soluble in water, was puriﬁed using ﬂash chromatog-

raphy.

RESULTS AND DISCUSSION

■

Structure−Activity Relationships. The design of the new

series of pyrimidines derivatives was based on the known

pharmacophoric features of σ₁R ligands. As recently summar-

ized by Glennon et al., σ₁R inhibitors should contain a basic

amino group and no less than two hydrophobic regions at

speciﬁed distances, 2.5−3.9 and 6−10 Å from the amino

group^45,46(Figure 3A). Compounds of the general structure

(Figure 3B) were designed in which the pyrimidine acted as an

important scaﬀold and various groups added according to these

pharmacophoric requirements; some variations were also

considered.

Eﬀect of the Distance between the Pyrimidine Ring and

the Basic Amino Moiety. In this study, our initial focus was to

investigate the eﬀect of diﬀerent chain lengths on aﬃnities to

σ₁R and σ₂R (Table 1, compounds 8, 14−34, 36, and 38). The

morpholine group was used because of its frequent appearance

in σ₁R ligands, such as S1RA. At position 2 of the pyrimidine

ring, a simple nonsubstituted phenyl was selected to meet the

requirement of the pharmacophoric model.

Initially, the two-carbon linker compound 8 was prepared but

did not activate either receptor; apparently the linker was too

short. Increasing the linker with only one carbon atom (14)

demonstrated binding aﬃnity and selectivity for the σ₁R over

the σ₂R (K_iσ₁= 36.3 nM and K_iσ₂= 949 nM). Further

elongation of the straight carbon linker between the pyrimidine

scaﬀold and morpholine moiety provided derivatives 15−16,

which had lower σ₁R and σ₂R aﬃnities than their propylene

counterparts. Derivatives of a six-atom spacer, compound 17,

did not bind either receptor, suggesting the distance was too

large for the binding site. Compared to compound 15, replacing

the single bond with a trans double bond (18) did not promote

aﬃnity to the σ₁/σ₂receptor. A ﬁve-atom linker with an

intercalated oxygen atom (36) maintained the potency for both

receptors as compound 16. The introduction of a hydroxyl

group in the linker (38) resulted in decreased potency for both

receptors, suggesting that polar groups are not preferred in this

region. Taken together, the data indicated that the distance

between the pyrimidine and basic amino group aﬀected

signiﬁcantly the potency of both receptors; three-carbon

chain length (14) was the most preferred and so was further

investigated.

a

Aﬃnities were determined in guinea pig brain using [³H]-

b

(+)-pentazocine. Aﬃnities were determined in guinea pig brain

using [³H]-DTG in the presence of (+)-SKF-10047 to block σ-1

c

receptors. The values are means

performed in duplicate.

SEM of three experiments

with various substituted ethyl 3-oxobutanoates 39−48, depend-

ing on the substitution pattern of the ﬁnal pyrimidine. Next,

standard alkylation with 1,3-dibromopropane was conducted to

D

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a

Scheme 2

a

Reagents and conditions: (I) Br(CH₂)₂O(CH₂)₂Br, K₂CO₃, KI, acetone, reﬂux; (II) morpholine, Cs₂CO₃, acetonitrile, reﬂux; (III) 2-

(cholomethyl)oxirane, K₂CO₃, KI, acetone, reﬂux; (IV) morpholine, MeOH, reﬂux.

a

Scheme 3

a

Reagents and conditions: (I) t-BuOK, MeOH, reﬂux; (II) Br(CH₂)₃Br, K₂CO₃, acetone, reﬂux; (III) piperidine, Cs₂CO₃, acetonitrile, reﬂux; (IV)

pyrrolidine, Cs₂CO₃, acetonitrile, reﬂux.

Eﬀect of the Basic Amino Moiety for Diﬀerent Amino

Groups. After determining the scaﬀold of the phenyl

pyrimidine derivatives, the eﬀect of various basic amino

moieties was investigated by synthesizing variations of

compound 14. As shown in Table 2, we selected piperidine,

4-methylpiperidine, 3,5-dimethylpiperidine, and 2,2,6,6-tetra-

methylpiperidine (compounds 19−22) to replace the morpho-

line moiety. The substitution of morpholine with piperidine

analogues resulted in compound 19, which showed signiﬁcantly

improved σ₁R binding aﬃnity and selectivity (K_iσ₁= 11.8 nM

and K_iσ₂= 1051 nM). The improvement was possibly due to

the greater hydrophobicity of the piperidine moiety, which was

closer in similarity to Glennon’s pharmacophoric σ₁R

model.^45,46Increasing the hydrophobic ability of piperidinyl

by adding a methyl group on the ring (20−21) did not enhance

the receptor’s binding aﬃnity, which suggested that the space in

this region was limited. The compound with 2,2,6,6-

tetramethylpiperidine (22) showed weak aﬃnity (K_iσ₁= 377

nM and K_iσ₂> 2000 nM) to both receptors, mainly because

the four methyls formed a strong steric hindrance around the N

atom, aﬀecting the ligand−receptor interaction. Substituting the

oxygen atom of the morpholino with a hydroxyl group or

carbonyl group (23−24) decreased greatly the aﬃnity to both

the σ₁R and σ₂R, which suggested the polar groups were not

conducive to σ₁R or σ₂R binding in this position.

Changing the oxygen atom in the morpholino group to a N

atom produced the piperazine derivative, compound 25, which

showed a weak receptor aﬃnity (K_iσ₁= 151 nM and K_iσ₂=

1212 nM) and may be due to the hydrophilic property of

piperazine. Substituting the hydrogen in piperazine with small

alkyl groups improved the hydrophobic property and resulted

in the potent and selective compounds 26−27 and in the larger

diazepane derivative, compound 28 (K_iσ₁= 18.7 nM and K_iσ₂

= 1295 nM). However, substituting with large groups, such as t-

butyloxycarboryl (29) or phenyl (30), was not tolerated by

either the σ₁R or σ₂R.

E

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Table 3. Binding Aﬃnities for the σ₁and σ₂Receptor of Compounds 19, 31, and 69−88

a

b

compd

n

R₃

R₄

K_iσ₁(nM)

K_iσ₂(nM)

selectivity (σ₂/σ₁)

c

19

69

70

71

72

73

74

75

76

77

78

31

79

80

81

82

83

84

85

86

87

88

2

1

CH₃

H

11.8 2.6

20.5 3.3

34.9 6.1

188 25

19.3 4.6

27.6 5.9

253 46

966 92

9.51 1.3

3.75 0.4

2.33 0.6

13.6 3.1

22.4 4.1

38.9 7.6

218 55

19.3 4.6

27.6 5.9

278 51

735 108

11.4 1.2

3.51 0.7

4.02 0.6

1051 87

1141 97

1456 127

1836 232

892 77

89.1

55.6

41.7

9.7

CH₂CH₃

(CH₂)₂CH₃

CH(CH₃)₂

OCH₃

H

46.2

48.0

CF₃

H

1327 101

>2000

cyclopropyl

phenyl

CH₃

H

>2000

CH₃

F

1221 104

1361 119

1649 119

1253 78

1429 133

1396 121

1092 172

892 77

128

CH₃

363

708

CH₃

Cl

H

CH₃

92.1

CH₂CH₃

(CH₂)₂CH₃

CH(CH₃)₂

OCH₃

H

63.8

35.8

5.0

H

46.2

48.0

CF₃

H

1327 101

>2000

cyclopropyl

phenyl

CH₃

H

>2000

CH3

F

1279 173

1194 97

1684 131

112

CH₃

340

419

CH₃

Cl

a

b

Aﬃnities were determined in guinea pig brain using [³H]-(+)-pentazocine. Aﬃnities were determined in guinea pig brain using [³H]-DTG in the

c

presence of (+)-SKF-10047 to block sigma-1 receptors. The values are means SEM of three experiments performed in duplicate.

a

Scheme 4

a

Reagents and conditions: (I) t-BuOK, MeOH, reﬂux; (II) Br(CH₂)₃Br, K₂CO₃, acetone, reﬂux; (III) piperidine, Cs₂CO₃, acetonitrile, reﬂux; (IV)

pyrrolidine, Cs₂CO₃, acetonitrile, reﬂux.

Smaller pyrrolidine analogues showed good potency and

maintained the selectivity (27, K_iσ₁= 13.6 nM and K_iσ₂=

1253 nM). Open-chain amines (32−34) retained some activity

(albeit less than compounds 19 and 31) and exhibited lower

selectivity.

emerged as the best two amino moieties in receptor binding

aﬃnity and selectivity, and the three-carbon linker was selected

as the proper distance to match the pharmacophoric require-

ment. Thus, compounds 19 and 31 were selected for further

investigation of the SAR.

Eﬀects of Substitution on the 4- and 5-Positions of the

Pyrimidine Ring. The piperidinyl and pyrrolidinyl groups

We ﬁrst explored the eﬀects of replacing the methyl (R₃)

with other substituents (Table 3, 69−75, 79−85). The aﬃnities

F

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aﬃnity to σ₁R (K_iσ₁= 2.33 nM) and σ₁/σ₂selectivity (708-

fold) compared with pyrrodinyl analogues (88). Fluorosub-

stituted analogue 77 showed less selectivity (363-fold), possibly

due to the hydrogen bonding eﬀect of the ﬂuorine atom, which

was not preferred in this position.

Table 4. Binding Aﬃnities for the σ₁and σ₂Receptor of

Compounds 94−95, 97, and 98

To better explain the SAR of substitution on the pyrimidine

ring, ring derivatives connecting the 4- or 5-positions were

prepared (Table 4) as 6,7-dihydro-5H-cyclopenta[d]-

pyrimidines (94−95) and 5,6,7,8-tetrahydroquinazolins (97−

98), respectively. The binding aﬃnities of these compounds

showed the ﬁve-membered ring on pyrimidine could maintain

the aﬃnity to the σ₁R and preferential selectivity with the

piperidinyl moiety (94, K_iσ₁= 44.2 nM, K_iσ₂= 1626 nM). A

six-membered ring in the same position resulted in loss of

activity, possibly due to the larger ring size hindering receptor

binding.

Eﬀect of Replacing the Phenyl Group on the 2-Position of

the Pyrimidine Ring (78) with Another Aromatic Ring.

Compound 78 exhibited signiﬁcant aﬃnity to the σ₁R and σ₁/

σ₂selectivity and was thus used for further investigation of the

eﬀect of various aromatic rings (Table 5, 132−142). The

nature of the aromatic group on the 2-position was important

for activity and selection. Either introducing an electron-

donating group (methyl) or electron-withdrawing group

(methoxyl and triﬂuoromethyl) would increase activity, but

halogens, such as ﬂuorine (135, K_iσ₁= 1.14 nM, K_iσ₂= 1329

nM) and chlorine (137, K_iσ₁= 1.06 nM, K_iσ₂= 1425 nM),

showed high aﬃnity to σ₁R and good selectivity to σ₂R. The

3,4-diﬂuorophenyl (136) group maintained the receptor-

binding proﬁle, but 3,4-dichlorophenyl (138) and 2-naphthyl

(139) were not conducive to σ₁or σ₂receptor binding in this

position. Replacing the aromatic group at the 2-position with

hydrogen (140) or a small alkyl such as methyl (141) resulted

in a marked reduction in potency. Cyclopropyl (142, K_iσ₁=

39.5 nM, K_iσ₂= 1328 nM) only maintained partial potency

and selectivity, thus suggesting the importance of an aromatic

ring in this position.

Human Ether-a-go-go-Related Gene (hERG) KC

Channel. Human ether-a-go-go-related gene (hERG) channel

blockade is an important predictor of cardiac toxicity. The

compounds with K_iσ₁< 5 nM and selectivity >400-fold were

subjected to a hERG patch-clamp assay⁴⁷as shown in Table 6.

In this experiment, a cutoﬀ value of IC₅₀> 5 μM was used.

Acute Toxicity. After considering the above-mentioned

results, the compounds that exhibited high aﬃnity to σ₁R and

good selectivity to σ₂R, with low aﬃnity for hERG, were

assayed for acute toxicity. The acute toxicities of compounds

were evaluated in terms of LD₅₀values (Table 6). Compound

137 showed a good proﬁle even at the highest dose tested

(LD₅₀> 2000 mg/kg).

a

Aﬃnities were determined in guinea pig brain using [³H]-

b

(+)-pentazocine. Aﬃnities were determined in guinea pig brain

using [³H]-DTG in the presence of (+)-SKF-10047 to block sigma-1

c

receptors. The values are means

performed in duplicate.

SEM of three experiments

of compounds 69−70 and 79−80 vs compounds 19 and 31 to

σ₁R and σ₂R decreased as a result of the additional group size

or hydrophobicity. The substitution of a methyl group with

isopropyl (71, 81), cyclopropyl (74, 84), and phenyl (75, 85)

groups produced a signiﬁcant decrease in the aﬃnities for the

receptors, which conﬁrmed the above-stated conclusion. On the

basis of these results, the modiﬁcation of the group in the 4-

position (R₃) of the pyrimidine produced analogues in the

following order of σ₁R aﬃnities: methyl > ethyl > isopropyl >

n-propyl > cyclopropyl > phenyl. The electron-withdrawing

groups, methyloxyl (72, 82) and triﬂuoromethyl (73, 83),

showed good potency for the σ₁R, but weaker selectivity than

the methyl group.

Introduction to the 5-position (R₄) in the pyrimidine scaﬀold

with a small group (76, K_iσ₁= 9.51 nM and K_iσ₂= 1221 nM;

86, K_iσ₁= 11.4 nM and K_iσ₂= 1279 nM) led to good aﬃnity

for the σ₁R and maintained selectivity to the σ₂R. Therefore, we

continued the investigation by introducing other substituents at

this position. Introducing halogens such as ﬂuorine (77, 87)

and chlorine (78, 88) showed moderate aﬃnities to the σ₁R

and increased selectivity to the σ₂R. Notably, compound 78,

with chlorine in the 5-position of the pyrimidine and

piperidinyl basic moiety, showed signiﬁcantly higher binding

a

Scheme 5

a

Reagents and conditions: (I) t-BuOK, MeOH, reﬂux; (II) Br(CH₂)₃Br, K₂CO₃, acetone, reﬂux; (III) piperidine, Cs₂CO₃, acetonitrile, reﬂux.

G

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Table 5. Binding Aﬃnities for the σ₁and σ₂Receptor of Compounds 78 and 132−142

a

b

compd

R₅

K_iσ₁(nM)

K_iσ₂(nM)

selectivity (σ₂/σ₁)

c

78

phenyl

2.33 0.6

1.96 0.7

3.88 1.0

4.42 1.4

1.14 0.2

0.97 0.l

1.06 0.2

89.8 13

102 19

187 14

58.8 7.2

39.5 6.8

1649 119

1554 92

1288 76

1776 135

1329 202

943 156

1425 139

>2000

708

792

132

133

134

135

136

137

138

139

140

141

142

4-methylphenyl

4-methoxyphenyl

4-(triﬂuoromethyl)phenyl

4-ﬂuorophenyl

3,4-diﬂuorophenyl

4-chlorophenyl

3,4-dichlorophenyl

2-naphthyl

332

402

1165

973

1344

>2000

H

1104 88

1081 63

1328 104

5.9

18.4

33.6

CH₃

cyclopropyl

a

b

Aﬃnities were determined in guinea pig brain using [³H]-(+)-pentazocine. Aﬃnities were determined in guinea pig brain using [³H]-DTG in the

c

presence of (+)-SKF-10047 to block σ-1 receptors. The values are means SEM of three experiments performed in duplicate.

selectivity to the σ₁R over the σ₂R was evaluated using

phenytoin, a low-potency allosteric modulator of the σ₁

receptor. Phenytoin shifts σ₁R agonists to signiﬁcantly higher

aﬃnities (K_iratios without phenytoin vs with phenytoin >1),

while σ₁R antagonists show no eﬀect or a very little eﬀect on

lowering the aﬃnity values (K_iratios without phenytoin vs with

phenytoin ≤1). Compound 137 produced a small shift,

lowering the aﬃnity when incubated in the presence of

phenytoin (K_iratios without phenytoin vs with phenytoin =

0.87), which exhibited antagonist properties on the σ₁receptor.

Formalin-Induced Nociceptive Behavior. Because of the

marked eﬀects on the σ₁R and σ₂R with its antagonist property

and good safety proﬁle, compound 137 was selected as a

promising candidate and subjected to further pharmacological

evaluation.

Recent research showed that σ₁R antagonists could reduce

both phases of formalin-induced paw licking/biting behavior in

mice.^44,49As a classic model of acute and chronic pain, the

formalin test was performed to evaluate the antinociceptive

eﬀect of selected compounds. Intraplantar injection of formalin

solution into the hind paw produces a biphasic pain response, a

brief, acute phase that indicates direct eﬀects on pain receptors

(phase I), followed by a longer-lasting tonic phase that reﬂects

Figure 3. Glennon’s pharmacophoric σ₁receptor model (A) and

general structure of the new pyrimidine derivative (B).

inﬂammation (phase II). The time spent licking or biting the

paw after injection was measured as an indicator of the pain

response in mice. As shown in Figure 4, pretreatment with

Table 6. Additional Data for Compounds Complying with K_i

σ₁< 5 nmol and Selectivity >400-Fold

compd

hERGIC₅₀(μmol)

LD₅₀(mg/kg)

compound 137 (80 mg/kg, ip) inhibited pain responses to a

similar degree as S1RA, reducing licking and biting time to

12.65 5.07 s in phase I and 37.41 8.89 s in phase II. S1RA

at the same dose reduced licking and biting time to 10.65

3.94 and 33.16 7.28 s during phase I and II, respectively; the

vehicle had no eﬀect relative to treatment with formalin alone.

To better characterize the antinociceptive eﬀects of 137, a wide

range of doses was tested (20−160 mg/kg). Compound 62

produced dose dependent antinociception in both phases; the

ED₅₀values were 48.36 5.11 and 42.15 3.96 mg/kg for

phases I and II, respectively.

78

0.28

3.76

0.95

2.61

1.54

1.40

6.98

439.5 (361.0−534.9)

>2000

88

132

134

135

136

137

1684.2 (1500.4−1892.1)

377.3 (298.8−476.5)

884.5 (755.7−1162.7)

584.8 (455.7−750.5)

>2000

Functional Proﬁle of σ₁Receptor. The phenytoin-media

shift on σ₁R ligand aﬃnity and FRET based biosensor of σ₁R

ligand were mainly used to categorize compounds into agonists

and antagonists recently.⁴⁸In these work, the functional activity

of compounds with high σ₁receptor binding aﬃnity and

CCI Model. Compound 137 was evaluated in a

representative and frequently used model of neuropathic pain,

the CCI model in rats. Chronic constriction injury results in the

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development of allodynia-like behavior, hyperalgesia, and

spontaneous pain-like behavior, which are normally maximal

10−14 days after surgery.^50,51CCI rats exhibited signs of

allodynia to mechanical stimulation and hyperalgesia to thermal

stimulation, which were used as outcome measures of

neuropathic pain. Mechanical allodynia was quantiﬁed by

measuring the hind paw withdrawal response to von Frey hair

stimulation, while thermal (heat) hyperalgesia was assessed

using a plantar test by measuring hind paw withdrawal latency

in response to radiant heat.

As shown in Figure 5, compound 137 dose-dependently

inhibited mechanical allodynia and thermal hypersensitivity,

both in single dose treatment (day 15) and repeated dose

treatment (day 18), while sham operated rats did not induce

signiﬁcant changes in mechanical and thermal sensitivity

respect to rats before surgery (basal). The ED₅₀values of

single dose treatment on mechanical allodynia and thermal

hypersensitivity were 58.25 6.04 and 47.23 3.87 mg/kg,

respectively. In repeated dose treatment, the ED₅₀values of

mechanical allodynia and thermal hypersensitivity were 61.82

6.15 and 50.21 5.07 mg/kg, respectively.

Figure 4. Antinociceptive eﬀect of S1RA and compound 137 in phase

I (0−5 min) and phase II (15−45 min) of the mice formalin test at the

dose of 80 mg/kg. Each column and vertical line represents mean

SEM of the values obtained in at least 10 animals. Statistically

signiﬁcant diﬀerences: p < 0.01 vs vehicle; ** p < 0.01 vs vehicle +

formalin (Two-Way ANOVA followed by Newman−Keuls test).

##

Figure 5. Antinociceptive eﬀects of compound 137 on the expression of neuropathic pain in CCI and sham operated rats. Date obtained from 10

rats per group and expressed as mean

SEM pressure threshold (g) evoking paw withdrawal or latency (s) to paw withdrawal. Statistically

signiﬁcant diﬀerences between the rats before surgery (basal) and surgery groups: * p < 0.05, ** p < 0.01 VS basal; ^#p < 0.05; ^##p < 0.01 vs vehicle

treatment on day 14 (Two-Way ANOVA followed by Newman−Keuls test).

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Motor Coordination: Rotarod Test. To investigate the

possibility that the observed eﬃcacy of compound 137 could

interfere with motor coordination and thus with the response of

mice in the nociceptive and neuropathic pain-related behavioral

tests, motor performance was measured in the rotarod test.⁴²

As shown in Figure 6, no signiﬁcant eﬀect of compound 137 at

1 μM < 50%) to these targets, as shown in part S2 in Support

Information.

CONCLUSIONS

■

A detailed structure−activity relationship investigation of

pyrimidine scaﬀold derivatives has shown that several factors

inﬂuence the binding aﬃnity of these compounds to σ₁and σ₂

receptors.

Pyrimidine scaﬀold was crucial for activity, and a basic amine

was necessary matching the known σ₁R pharmacophoric model.

A straight three-carbon chain alkyl between the pyrimidine ring

and the amino moiety was preferred over other linkers. The

substitution on the 4-position (R₃) with a small alkyl group was

preferred, and methyl was the favored substituent. The

introduction of a halogen (chloro) on the 5-position (R₄)

showed moderate aﬃnities to σ₁R and increased selectivity to

σ₂R. An aromatic group on the 2-position of the pyrimidine

ring was essential for activity, and 4-chlorophenyl was favored.

From the SAR study, a series of 2-arylpyrimides were

synthesized, several of which were shown to be potent and

selective σ₁R ligands. The most promising compound, 137,

showed high aﬃnity to σ₁receptor, low aﬃnity to σ₂and hERG

channels, and was identiﬁed as a σ₁R antagonist. Compound

137 exerted clear dose-dependent analgesic eﬀects on the mice

formalin-induced pain model and antinociception on both

mechanical allodynia and thermal hypersensitivity in the rat

CCI model. Moreover, compound 137 exhibited good safety,

acceptable pharmacokinetic properties, and good selective

proﬁle to other speciﬁc targets that have been implicated in

pain. Thus, compound 137 may facilitate the development of a

novel class of drugs for the treatment of neuropathic pain.

Further studies of compound 137 and evaluation of these series

of derivatives are currently underway in our laboratory and will

be reported in due course.

Figure 6. Dose−response eﬀect of compound 137 and pregabalin on

motor coordination (rotarod test). Date obtained from 8−10 mice per

group and expressed as mean SEM latency (s) to fall down from

rod. Statistically signiﬁcant diﬀerences: * p < 0.05, ** p < 0.01 vs

vehicle (Two-Way ANOVA followed by Newman−Keuls test).

an analgesic dose was observed and no treatment-related

adverse eﬀects were found. In contrast, an analgesic dose of

pregabalin induced obvious motor impairment.⁴²

Pharmacokinetic Properties of Compound 137. The

evaluated pharmacokinetic properties of compound 137

included the in vitro and in vivo eﬃcacy and the degree of

safety, as characterized in rats (Table 7).

EXPERIMENTAL SECTION

■

Chemistry. Melting points were determined in open capillary tubes

1

and are uncorrected. H and ¹³C NMR spectra were recorded on a

Intravenous administration of compound 137 to rats (16

mg/kg, n = 8) resulted in detectable plasma levels, with a half-

life (t_1/2) of 10.57 h. Oral administration of compound 137 to

rats (320 mg/kg, n = 8) resulted in a t_1/2of 3.81 h. The area

under the curve (AUC) value of compound 137 was 3295.31

ng × h/mL after intravenous administration vs 54672.61 ng ×

h/mL after oral administration. After oral administration of 320

mg/mL, compound 137 achieved a peak concentration of

17394.90 ng/mL; the T_maxvalue was 0.5 h. The bioavailability

of compound 137 was 75.1%. These encouraging preclinical

data suggested that compound 137 possessed desirable drug-

like human pharmacokinetic properties.

Selectivity Proﬁle of Compound 137. To assess the

interaction of compound 137 with other receptors or ion

channels, a selectivity proﬁle for additional receptors (such as

μ-opioid, serotoninergic, H₃, cannabinoid, NMDA receptors, et.

al) and ion channels (voltage-gated sodium channel Nav 1.7

and TRPV-1 protein) that implicated in pain was performed.

Compound 137 showed no signiﬁcant aﬃnity (% inhibition at

Bruker Avance III 600 spectrometer at 600 MHz (¹H) and 150 MHz

(¹³C) using CDCl₃as solvent. Chemical shifts are given in δ values

(ppm), using tetramethylsilane (TMS) as the internal standard;

coupling constants (J) are given in Hz. Signal multiplicities are

characterized as s (singlet), d (doublet), t (triplet), q (quartet), m

(multiplet), br (broad signal). Commercial reagents and solvents were

all of analytical grade or of chemical purity (>99%). Analytical thin

layer chromatography (TLC) was performed on silica gel GF254.

Column chromatographic puriﬁcation was carried out using silica gel.

Purity analysis was carried out by LC-MS analysis using an Agilent

1200 system. The HPLC conditions are as follows: column, ZORBAX

BP-C18 5.0 μm × 150 mm × 4.6 mm I.D. (Agilent, USA); mobile

phase, 30 mmol of NH₄COOH (Roe Scientiﬁc Inc., USA) aq/

acetonitrile (Merck Company, Germany), 30/70; ﬂow rate, 1.0 mL/

min; column temperature, 40 °C. Compound purity is determined by

high performance liquid chromatography (HPLC), and all ﬁnal test

compounds display purity higher than 95% as determined by this

method.

6-Methyl-2-phenylpyrimidin-4-ol (7).⁵²To a suspension of

benzimidamide hydrochloride (5, 50 mmol) and ethyl 3-oxobutanoate

Table 7. Plasma Pharmacokinetic Data Following the Administration of Compound 137 in Rats (n = 8/Group)

dose (mg/kg)

C_max(ng/kg)

T_max(h)

t_1/2(h)

AUC_0−24h(ng × h/mL)

AUC_0−inf(ng × h/mL)

F (%)

iv 16

7261.45

10.57

3.81

3295.31

3686.17

po 320

17394.90

0.5

54672.61

55393.45

75.1

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(6, 55 mmol) in MeOH (200 mL) was added t-BuOK (10 mmol)

portionwise with stirring at 0 °C for 30 min. The resulting mixture was

heated to reﬂux for 4 h and then cooled down to room temperature.

After that, ice−water (200 mL) was added and neutralized with citric

acid. The precipitate thus formed was ﬁltered and washed with water.

The crude residue obtained was recrystallized from i-PrOH to give 6-

methyl-2-phenylpyrimidin-4-ol, 7, as a white solid (6.8 g, 73.0%), mp

dried with anhydrous magnesium sulfate, the ﬁltrate evaporated under

reduced pressure, and the crude product was puriﬁed by means of

chromatography (10% MeOH/CHCl₃) to yield 4-(3-((6-methyl-2-

phenylpyrimidin-4-yl)oxy)propyl)morpholine, 14, as a pale-yellow oil

1

(1.43 g, 91.6%). H NMR (600 MHz, CDCl₃) δ 8.45−8.41 (m, 2H),

7.45−7.40 (m, 3H), 6.44 (s, 1H), 4.52 (t, J = 6.5 Hz, 2H), 3.71 (t, J =

4.6 Hz, 4H), 2.57−2.39 (m, 9H), 2.05−1.94 (m, 2H). MS (ESI) m/z

314.6 (calcd 314.4 for C₁₈H₂₃N₃O₂[M + H]⁺).

1

220−222 °C. H NMR (600 MHz, CDCl₃) δ 12.65 (s, 1H), 8.22−

8.10 (m, 2H), 7.67−7.50 (m, 3H), 6.30 (s, 1H), 2.40 (s, 3H).

General Procedures for the Synthesis of 6-Methyl-2-phenyl-

pyrimidinyl Derivatives (8 and 14−34). Method A: 4-(2-((6-Methyl-

2-phenylpyrimidin-4-yl)oxy)ethyl)morpholine (8).⁶⁴To a suspension

of 7 (10 mmol) and 4-(2-chloroethyl) morpholine (10 mmol) in

acetonitrile (100 mL), potassium carbonate (20 mmol) and a catalytic

amount of potassium iodide (1% mol) were added. The resulting

mixture was heated and reﬂuxed for 6−8 h. The progress of the

reaction was monitored by TLC. After ﬁltering, the resulting ﬁltrate

was evaporated to dryness under reduced pressure. The residue was

suspended in water (50 mL) and extracted with dichloromethane (3 ×

25 mL). The combined organic layers were dried with anhydrous

magnesium sulfate, the ﬁltrate evaporated under reduced pressure, and

the crude product was puriﬁed by means of chromatography (10%

MeOH/CHCl₃) to yield 4-(2-((6-methyl-2-phenylpyrimidin-4-yl)-

oxy)ethyl)morpholine, 8, as a pale-yellow oil (2.42 g, 80.9%). ¹H

NMR (600 MHz, CDCl₃) δ 8.44−8.40 (m, 2H), 7.48−7.41 (m, 3H),

6.48 (s, 1H), 4.61 (t, J = 5.8 Hz, 2H), 3.71 (t, J = 4.6 Hz, 4H), 2.81 (t,

J = 5.8 Hz, 2H), 2.57−2.51 (m, 4H), 2.48 (s, 3H). MS (ESI) m/z

300.3 (calcd 300.2 for C₁₇H₂₁N₃O₂[M + H]⁺).

4-(4-((6-Methyl-2-phenylpyrimidin-4-yl)oxy)butyl)morpholine

(15). A pale-yellow oil (1.48 g, 90.5%). ¹H NMR (600 MHz, CDCl₃) δ

8.45−8.41 (m, 2H), 7.46−7.41 (m, 3H), 6.44 (s, 1H), 4.49 (t, J = 6.5

Hz, 2H), 3.70 (t, J = 4.6 Hz, 4H), 2.48 (s, 3H), 2.45−2.38 (m, 6H),

1.88−1.80 (m, 2H), 1.71−1.63 (m, 2H). MS (ESI) m/z 328.8 (calcd

328.4 for C₁₉H₂₅N₃O₂[M + H]⁺).

4-(5-((6-Methyl-2-phenylpyrimidin-4-yl)oxy)pentyl)morpholine

(16). A pale-yellow oil (1.50 g, 88.2%). ¹H NMR (600 MHz, CDCl₃) δ

8.46−8.38 (m, 2H), 7.46−7.40 (m, 3H), 6.45 (s, 1H), 4.48 (t, J = 6.6

Hz, 2H), 3.71 (t, J = 4.5 Hz, 4H), 2.49 (s, 3H), 2.40−2.33 (m, 6H),

1.86−1.82 (m, 2H), 1.63−1.54 (m, 2H), 1.53−1.46 (m, 2H). MS

(ESI) m/z 342.7 (calcd 342.4 for C₂₀H₂₇N₃O₂[M + H]⁺).

4-(6-((6-Methyl-2-phenylpyrimidin-4-yl)oxy)hexyl)morpholine

(17). A pale-yellow oil (1.58 g, 89.4%). ¹H NMR (600 MHz, CDCl₃) δ

8.47−8.41 (m, 2H), 7.48−7.42 (m, 3H), 6.44 (s, 1H), 4.46 (t, J = 6.6

Hz, 2H), 3.71 (t, J = 4.6 Hz, 4H), 2.49 (s, 3H), 2.42−2.32 (m, 6H),

1.85−1.77 (m, 2H), 1.53−1.49 (m, 4H), 1.44−1.32 (m, 2H). MS

(ESI) m/z 356.8 (calcd 356.5 for C₂₁H₂₉N₃O₂[M + H]⁺).

(E)-4-(4-((6-Methyl-2-phenylpyrimidin-4-yl)oxy)but-2-en-1-yl)-

1

morpholine (18). A pale-yellow oil (1.41 g, 86.7%). H NMR (600

Method B: 4-(3-Bromopropoxy)-6-methyl-2-phenylpyrimidine

(9). To a solution of 7 (10 mmol) and 1,3-dibromopropane (20

mmol) in acetone (100 mL), potassium carbonate (20 mmol) was

added and the mixture was reﬂuxed for 4−6 h. The progress of the

reaction was monitored by TLC. After cooling to room temperature,

the mixture was ﬁltered and the solvent was evaporated under reduced

pressure. The crude product was puriﬁed by means of chromatography

(petroleum ether/EtOAc = 50/1) to yield 4-(3-bromopropoxy)-6-

MHz, CDCl₃) δ 8.46−8.39 (m, 2H), 7.49−7.42 (m, 3H), 6.46 (s, 1H),

5.97−5.83 (m, 2H), 4.98 (d, J = 4.2 Hz, 2H), 3.65 (t, J = 4.6 Hz, 4H),

3.01 (d, J = 4.9 Hz, 2H), 2.48 (s, 3H), 2.45−2.38 (m, 4H). MS (ESI)

m/z 326.5 (calcd 326.4 for C₁₉H₂₃N₃O₂[M + H]⁺).

4-Methyl-2-phenyl-6-(3-(piperidin-1-yl)propoxy)pyrimidine

(19).⁶⁴A yellow oil (1.42 g, 91.4%). H NMR (600 MHz, CDCl₃) δ

1

8.48−8.37 (m, 2H), 7.47−7.41 (m, 3H), 6.44 (s, 1H), 4.50 (t, J = 6.5

Hz, 2H), 2.53−2.34 (m, 9H), 2.04−1.96 (m, 2H), 1.64−1.55 (m, 4H),

1.46−1.40 (m, 2H). MS (ESI) m/z 312.6 (calcd 312.4 for C₁₉H₂₅N₃O

[M + H]⁺).

4-Methyl-6-(3-(4-methylpiperidin-1-yl)propoxy)-2-phenylpyrimi-

dine (20). A yellow oil (1.40 g, 86.3%). ¹H NMR (600 MHz, CDCl₃)

δ 8.50−8.36 (m, 2H), 7.54−7.41 (m, 3H), 6.43 (s, 1H), 4.50 (t, J = 6.5

Hz, 2H), 2.93−2.85 (m, 2H), 2.51−2.45 (m, 5H), 2.05−1.96 (m, 2H),

1.95−1.87 (m, 2H), 1.62−1.58 (m, 2H), 1.39−1.30 (m, 1H), 1.28−

1.21 (m, 2H), 0.92 (d, J = 6.4 Hz, 3H). MS (ESI) m/z 326.6 (calcd

326.4 for C₂₀H₂₇N₃O [M + H]⁺).

4-(3-(3,5-Dimethylpiperidin-1-yl)propoxy)-6-methyl-2-phenylpyr-

imidine (21). A yellow oil (1.42 g, 83.5%). ¹H NMR (600 MHz,

CDCl₃) δ 8.48−8.38 (m, 2H), 7.50−7.40 (m, 3H), 6.43 (s, 1H), 4.50

(t, J = 6.5 Hz, 2H), 2.89−2.83 (m, 2H), 2.53−2.42 (m, 5H), 2.07−

1.96 (m, 2H), 1.75−1.61 (m, 4H), 1.42 (t, J = 10.9 Hz, 2H), 0.84 (d, J

= 6.5 Hz, 6H). MS (ESI) m/z 340.8 (calcd 340.5 for C₂₁H₂₉N₃O [M +

H]⁺).

1

methyl-2-phenylpyrimidine, 9, as a pale-yellow oil (1.92 g, 62.5%). H

NMR (600 MHz, CDCl₃) δ 8.50−8.38 (m, 2H), 7.48−7.43 (m, 3H),

6.45 (s, 1H), 4.61 (t, J = 6.0 Hz, 2H), 3.57 (t, J = 6.5 Hz, 2H), 2.49 (s,

3H), 2.38−2.32 (m, 2H). MS (ESI) m/z 308.3 (calcd 308.2 for

C₁₄H₁₅BrN₂O [M + H]⁺).

4-(4-Bromobutoxy)-6-methyl-2-phenylpyrimidine (10). A pale-

1

yellow oil (2.03 g, 63.3%). H NMR (600 MHz, CDCl₃) δ 8.46−

8.39 (m, 2H), 7.49−7.41 (m, 3H), 6.42 (s, 1H), 4.47 (t, J = 6.2 Hz,

2H), 3.46 (t, J = 6.6 Hz, 2H), 2.47 (s, 3H), 2.09−1.86 (m, 4H). MS

(ESI) m/z 322.6 (calcd 322.2 for C₁₅H₁₇BrN₂O [M + H]⁺).

4-((5-Bromopentyl)oxy)-6-methyl-2-phenylpyrimidine (11). A

pale-yellow oil (1.96 g, 58.7%). ¹H NMR (600 MHz, CDCl₃) δ

8.46−8.37 (m, 2H), 7.49−7.38 (m, 3H), 6.41 (s, 1H), 4.43 (t, J = 6.6

Hz, 2H), 3.39 (t, J = 6.8 Hz, 2H), 2.46 (s, 3H), 1.95−1.73 (m, 4H),

1.67−1.51 (m, 2H). MS (ESI) m/z 336.4 (calcd 336.2 for

C₁₆H₁₉BrN₂O [M + H]⁺).

4-Methyl-2-phenyl-6-(3-(2,2,6,6-tetramethylpiperidin-1-yl)-

propoxy)pyrimidine (22). A yellow oil (1.63 g, 88.6%). ¹H NMR (600

MHz, CDCl₃) δ 8.50−8.41 (m, 2H), 7.53−7.42 (m, 3H), 6.44 (s, 1H),

4.51 (t, J = 6.5 Hz, 2H), 2.53−2.42 (m, 5H), 2.07−1.96 (m, 2H),

1.66−1.48 (m, 4H), 1.49−1.33 (m, 2H), 0.76 (s, 12H). MS (ESI) m/z

368.3 (calcd 368.5 for C₂₃H₃₃N₃O [M + H]⁺).

4-((6-Bromohexyl)oxy)-6-methyl-2-phenylpyrimidine (12). A pale-

yellow oil (1.98 g, 56.6%). ¹H NMR (600 MHz, CDCl₃) δ 8.50−8.36

(m, 2H), 7.53−7.40 (m, 3H), 6.45 (s, 1H), 4.47 (t, J = 6.6 Hz, 2H),

3.42 (t, J = 6.8 Hz, 2H), 2.49 (s, 3H), 1.95−1.76 (m, 4H), 1.60−1.43

(m, 4H). MS (ESI) m/z 350.1 (calcd 350.3 for C₁₇H₂₁BrN₂O [M +

H]⁺).

(E)-4-((4-Bromobut-2-en-1-yl)oxy)-6-methyl-2-phenylpyrimidine

(13). A pale-yellow oil (2.13 g, 67.0%). ¹H NMR (600 MHz, CDCl₃) δ

8.44−8.35 (m, 2H), 7.50−7.40 (m, 3H), 6.45 (s, 1H), 6.16−5.94 (m,

2H), 4.98 (d, J = 5.3 Hz, 2H), 3.95 (d, J = 7.1 Hz, 2H), 2.47 (s, 3H).

MS (ESI) m/z 320.3 (calcd 320.2 for C₁₅H₁₅BrN₂O [M + H]⁺).

4-(3-((6-Methyl-2-phenylpyrimidin-4-yl)oxy)propyl)morpholine

(14). To a suspension of 9 (5 mmol) and morpholine (5.5 mmol) in

acetonitrile (100 mL), cesium carbonate (10 mmol) was added, and

the resulting mixture was heated and reﬂuxed for 6−8 h. After ﬁltering,

the resulting ﬁltrate was evaporated to dryness under reduced pressure.

The residue was suspended in water (50 mL) and extracted with

dichloromethane (3 × 25 mL). The combined organic layers were

1-(3-((6-Methyl-2-phenylpyrimidin-4-yl)oxy)propyl)piperidin-4-ol

1

(23). A yellow oil (1.35 g, 82.7%). H NMR (600 MHz, CDCl₃) δ

8.47−8.38 (m, 2H), 7.48−7.43 (m, 3H), 6.45 (s, 1H), 4.50 (t, J = 6.4

Hz, 2H), 3.66 (s, 1H), 2.84−2.74 (m, 2H), 2.67−2.43 (m, 6H), 2.18−

2.07 (m, 2H), 2.03−1.96 (m, 2H), 1.92−1.83 (m, 2H), 1.63−1.54 (m,

2H). MS (ESI) m/z 328.8 (calcd 328.4 for C₁₉H₂₅N₃O₂[M + H]⁺).

1-(3-((6-Methyl-2-phenylpyrimidin-4-yl)oxy)propyl)piperidin-4-

one (24). A yellow oil (1.30 g, 80.3%). ¹H NMR (600 MHz, CDCl₃) δ

8.46−8.38 (m, 2H), 7.47−7.43 (m, 3H), 6.46 (s, 1H), 4.56 (t, J = 6.4

Hz, 2H), 2.76 (t, J = 6.1 Hz, 4H), 2.63 (t, J = 7.2 Hz, 2H), 2.51−2.43

(m, 7H), 2.06−1.97 (m, 2H). MS (ESI) m/z 326.7 (calcd 326.4 for

C₁₉H₂₃N₃O₂[M + H]⁺).

K

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Article

4-Methyl-2-phenyl-6-(3-(piperazin-1-yl)propoxy)pyrimidine (25).

4-(2-(2-((6-Methyl-2-phenylpyrimidin-4-yl)oxy)ethoxy)ethyl)-

morpholine (36). To a suspension of 35 (5 mmol) and morpholine

(5.5 mmol) in acetonitrile (100 mL), cesium carbonate (10 mmol)

was added and the resulting mixture was heated and reﬂuxed for 6−8

h. After ﬁltering, the resulting ﬁltrate was evaporated to dryness under

reduced pressure. The residue was suspended in water (50 mL) and

extracted with dichloromethane (3 × 25 mL). The combined organic

layers were dried with anhydrous magnesium sulfate, the ﬁltrate

evaporated under reduced pressure, and the crude product puriﬁed by

means of chromatography (10% MeOH/CHCl₃) to yield 4-(2-(2-((6-

methyl-2-phenylpyrimidin-4-yl)oxy)ethoxy)ethyl)morpholine, 36, as a

pale-yellow oil (1.49 g, 86.7%). ¹H NMR (600 MHz, CDCl₃) δ 8.47−

8.38 (m, 2H), 7.50−7.40 (m, 3H), 6.50 (s, 1H), 4.65 (t, J = 6.6 Hz,

2H), 3.88−3.79 (m, 2H), 3.72−3.66 (m, 6H), 2.62−2.56 (m, 2H),

2.49−2.39 (m, 7H). MS (ESI) m/z 344.7 (calcd 344.4 for C₁₉H₂₅N₃O₃

[M + H]⁺).

1

A yellow oil (1.22 g, 78.4%). H NMR (600 MHz, CDCl₃) δ 8.48−

8.38 (m, 2H), 7.50−7.42 (m, 3H), 6.44 (s, 1H), 4.51 (t, J = 6.5 Hz,

2H), 2.94−2.08 (m, 14H), 2.03−1.97 (m, 2H). MS (ESI) m/z 313.6

(calcd 313.4 for C₁₈H₂₄N₄O [M + H]⁺).

4-Methyl-6-(3-(4-methylpiperazin-1-yl)propoxy)-2-phenylpyrimi-

dine (26). A yellow oil (1.47 g, 90.1%). ¹H NMR (600 MHz, CDCl₃)

δ 8.46−8.39 (m, 2H), 7.47−7.42 (m, 3H), 6.44 (s, 1H), 4.51 (t, J = 6.5

Hz, 2H), 2.94−2.10 (m, 16H), 2.03−1.97 (m, 2H). MS (ESI) m/z

327.6 (calcd 327.4 for C₁₉H₂₆N₄O [M + H]⁺).

4-(3-(4-Ethylpiperazin-1-yl)propoxy)-6-methyl-2-phenylpyrimi-

dine (27). A yellow oil (1.48 g, 87.1%). ¹H NMR (600 MHz, CDCl₃)

δ 8.48−8.40 (m, 2H), 7.46−7.41 (m, 3H), 6.44 (s, 1H), 4.51 (t, J = 6.5

Hz, 2H), 2.95−2.17 (m, 15H), 2.02−1.96 (m, 2H), 1.08 (t, J = 7.2 Hz,

3H). MS (ESI) m/z 341.4 (calcd 341.5 for C₂₀H₂₈N₄O [M + H]⁺).

1-Methyl-4-(3-((6-methyl-2-phenylpyrimidin-4-yl)oxy)propyl)-1,4-

1

diazepane (28). A yellow oil (1.49 g, 87.6%). H NMR (600 MHz,

4-Methyl-6-(oxiran-2-ylmethoxy)-2-phenylpyrimidine (37). To a

suspension of 7 (10 mmol) and 2-(chloromethyl)oxirane (11 mmol)

in acetonitrile (100 mL), potassium carbonate (20 mmol) and a

catalytic amount of potassium iodide (1% mol) were added. The

resulting mixture was heated and reﬂuxed for 4−6 h, and the progress

of the reaction was monitored by TLC. After cooling to room

temperature, the mixture was ﬁltered and the solvent was evaporated

under reduced pressure. The crude product was puriﬁed by means of

chromatography (petroleum ether/EtOAc = 40/1) to yield 4-methyl-

6-(oxiran-2-ylmethoxy)-2-phenylpyrimidine, 37, as a pale-yellow oil

CDCl₃) δ 8.46−8.40 (m, 2H), 7.48−7.43 (m, 3H), 6.45 (s, 1H), 4.52

(t, J = 6.5 Hz, 2H), 2.85−2.28 (m, 15H), 2.02−1.94 (m, 2H), 1.86−

1.80 (m, 2H). MS (ESI) m/z 341.7 (calcd 341.5 for C₂₀H₂₈N₄O [M +

H]⁺).

4-(3-(4-Boc-piperazin-1-yl)propoxy)-6-methyl-2-phenylpyrimi-

dine (29). A yellow oil (1.75 g, 85.2%). ¹H NMR (600 MHz, CDCl₃)

δ 8.48−8.38 (m, 2H), 7.48−7.42 (m, 3H), 6.44 (s, 1H), 4.52 (t, J = 6.4

Hz, 2H), 3.44 (s, 4H), 2.57−2.46 (m, 5H), 2.46−2.24 (m, 4H), 2.02−

1.97 (m, 2H), 1.46 (s, 9H). MS (ESI) m/z 413.7 (calcd 413.5 for

C₂₃H₃₂N₄O₃[M + H]⁺).

1

(1.77 g, 71.1%). H NMR (600 MHz, CDCl₃) δ 8.44−8.24 (m, 2H),

4-Methyl-2-phenyl-6-(3-(4-phenylpiperazin-1-yl)propoxy)-

1

pyrimidine (30). A yellow oil (1.71 g, 88.1%). H NMR (600 MHz,

7.43−7.33 (m, 3H), 6.44 (s, 1H), 4.82−4.14 (m, 2H), 3.40−3.22 (m,

1H), 2.91−2.60 (m, 2H), 2.42 (s, 3H). MS (ESI) m/z 243.5 (calcd

243.3 for C₁₄H₁₄N₂O₂[M + H]⁺).

CDCl₃) δ 8.46−8.41 (m, 2H), 7.48−7.44 (m, 3H), 7.29−7.24 (m,

2H), 6.96−6.91 (m, 2H), 6.88−6.82 (m, 1H), 6.46 (s, 1H), 4.56 (t, J =

6.4 Hz, 2H), 3.28−3.15 (m, 4H), 2.68−2.62 (m, 4H), 2.61−2.56 (m,

2H), 2.49 (s, 3H), 2.08−2.02 (m, 2H). MS (ESI) m/z 389.2 (calcd

389.5 for C₂₄H₂₈N₄O [M + H]⁺).

1-((6-Methyl-2-phenylpyrimidin-4-yl)oxy)-3-morpholinopropan-

2-ol (38). A mixture of 37 and (5 mmol) and morpholine (5.5 mmol)

in MeOH was stirred and heated to reﬂux for 6−8 h. The solvent was

evaporated under reduced pressure, and the crude product was

puriﬁed by means of chromatography (petroleum ether/EtOAc = 10/

1 to EtOAc) to yield 1-((6-methyl-2-phenylpyrimidin-4-yl)oxy)-3-

4-Methyl-2-phenyl-6-(3-(pyrrolidin-1-yl)propoxy)pyrimidine (31).

1

A pale-red oil (1.36 g, 91.5%). H NMR (600 MHz, CDCl₃) δ 8.44−

8.40 (m, 2H), 7.46−7.43 (m, 3H), 6.45 (s, 1H), 4.53 (t, J = 6.5 Hz,

2H), 2.66−2.60 (m, 2H), 2.57−2.51 (m, 4H), 2.48 (s, 3H), 2.07−2.01

(m, 2H), 1.82−1.76 (m, 4H). MS (ESI) m/z 298.8 (calcd 289.4 for

C₁₈H₂₃N₃O [M + H]⁺).

1

morpholinopropan-2-ol, 38, as a yellow oil (0.83 g, 50.6%). H NMR

(600 MHz, CDCl₃) δ 8.44−8.35 (m, 2H), 7.51−7.46 (m, 3H), 6.53 (s,

1H), 4.35−4.26 (m, 2H), 4.12−4.08 (m, 2H), 3.85−3.64 (m, 6H),

2.57−2.54 (m, 4H), 2.51 (s, 3H). MS (ESI) m/z 330.4 (calcd 330.4

for C₁₈H₂₃N₃O₃[M + H]⁺).

N,N-Dimethyl-3-((6-methyl-2-phenylpyrimidin-4-yl)oxy)propan-

1

1-amine (32). A yellow oil (1.05 g, 77.4%). H NMR (600 MHz,

CDCl₃) δ 8.46−8.41 (m, 2H), 7.44−7.41 (m, 3H), 6.41 (s, 1H), 4.48

(t, J = 6.5 Hz, 2H), 2.46−2.40 (m, 5H), 2.23 (s, 6H), 1.99−1.91 (m,

2H). MS (ESI) m/z 272.6 (calcd 272.4 for C₁₆H₂₁N₃O [M + H]⁺).

N,N-Diethyl-3-((6-methyl-2-phenylpyrimidin-4-yl)oxy)propan-1-

amine (33). A yellow oil (1.13 g, 76.1%). ¹H NMR (600 MHz,

CDCl₃) δ 8.47−8.41 (m, 2H), 7.50−7.43 (m, 3H), 6.45 (s, 1H), 4.51

(t, J = 6.5 Hz, 2H), 2.64−2.59 (m, 2H), 2.58−2.52 (m, 4H), 2.48 (s,

3H), 2.00−1.92 (m, 2H), 1.03 (t, J = 7.2 Hz, 6H). MS (ESI) m/z

300.2 (calcd 300.4 for C₁₈H₂₅N₃O [M + H]⁺).

General Procedures for the Synthesis of 2-Phenylpyrimidinyl

Derivatives (49−58)..⁵³⁻⁵⁹6-Ethyl-2-phenylpyrimidin-4-ol (49). To

a suspension of benzimidamide hydrochloride (5, 50 mmol) and ethyl

3-oxopentanoate (39, 55 mmol) in MeOH (200 mL) was added t-

BuOK (10 mmol) portionwise, with stirring, at 0 °C for 30 min. The

resulting mixture was heated to reﬂux for 4 h and then cooled down to

room temperature. After that, ice−water (200 mL) was added and

neutralized with citric acid. The precipitate thus formed was ﬁltered

and washed with water. The crude residue obtained was recrystallized

from i-PrOH to give 6-ethyl-2-phenylpyrimidin-4-ol, 49, as a white

solid (7.60 g, 75.9%), mp 166−168 °C. ¹H NMR (600 MHz, CDCl₃)

δ 13.15 (s, 1H), 8.33−8.12 (m, 2H), 7.63−7.45 (m, 3H), 6.31 (s, 1H),

2.67 (q, J = 7.5 Hz, 2H), 1.30 (t, J = 7.5 Hz, 3H).

3-((6-Methyl-2-phenylpyrimidin-4-yl)oxy)-N,N-dipropylpropan-1-

amine (34). A yellow oil (1.31 g, 80.2%). ¹H NMR (600 MHz,

CDCl₃) δ 8.48−8.38 (m, 2H), 7.48−7.43 (m, 3H), 6.45 (s, 1H), 4.51

(t, J = 6.5 Hz, 2H), 2.63−2.57 (m, 2H), 2.49 (s, 3H), 2.42−2.36 (m,

4H), 1.99−1.91 (m, 2H), 1.51−1.41 (m, 4H), 0.87 (t, J = 7.4 Hz, 6H).

MS (ESI) m/z 328.5 (calcd 328.5 for C₂₀H₂₉N₃O [M + H]⁺).

4-(2-(2-Bromoethoxy)ethoxy)-6-methyl-2-phenylpyrimidine (35).

To a solution of 7 (10 mmol) and 1-bromo-2-(2-bromoethoxy)ethane

(20 mmol) in acetone (100 mL), potassium carbonate (20 mmol), and

a catalytic amount of potassium iodide (1% mol) were added. The

mixture was reﬂuxed for 4−6 h, and the progress was monitored by

TLC. After cooling to room temperature, the mixture was ﬁltered and

the solvent was evaporated under reduced pressure. The crude product

was puriﬁed by means of chromatography (petroleum ether/EtOAc =

100/1 to 50/1) to yield 4-(2-(2-bromoethoxy)ethoxy)-6-methyl-2-

2-Phenyl-6-propylpyrimidin-4-ol (50). A white solid (8.20 g,

1

76.7%), mp 145−147 °C. H NMR (600 MHz, CDCl₃) δ 12.84 (s,

1H), 8.26−8.15 (m, 2H), 7.66−7.48 (m, 3H), 6.29 (s, 1H), 2.61 (t, J =

7.6 Hz, 2H), 1.86−1.73 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H).

6-Isopropyl-2-phenylpyrimidin-4-ol (51). A white solid (8.37 g,

1

78.2%), mp 133−136 °C. H NMR (600 MHz, CDCl₃) δ 13.16 (s,

1H), 8.36−8.16 (m, 2H), 7.67−7.43 (m, 3H), 6.31 (s, 1H), 3.00−2.71

(m, 1H), 1.30 (d, J = 6.9 Hz, 6H).

6-Methoxy-2-phenylpyrimidin-4-ol (52). A white solid (7.31 g,

1

72.3%), mp 112−115 °C. H NMR (600 MHz, CDCl₃) δ 13.24 (s,

1

1H), 8.38−8.12 (m, 2H), 7.67−7.49 (m, 3H), 6.59 (s, 1H), 3.52 (s,

3H).

phenylpyrimidine, 35, as a pale-yellow oil (1.85 g, 54.8%). H NMR

(600 MHz, CDCl₃) δ 8.47−8.36 (m, 2H), 7.50−7.40 (m, 3H), 6.50 (s,

1H), 4.64 (t, J = 6.4 Hz, 2H), 3.88 (t, J = 6.3 Hz, 2H), 3.84 (t, J = 6.3

Hz, 2H), 3.47 (t, J = 6.3 Hz, 2H), 2.48 (s, 3H). MS (ESI) m/z 338.1

(calcd 338.2 for C₁₅H₁₇BrN₂O₂[M + H]⁺).

2-Phenyl-6-(triﬂuoromethyl)pyrimidin-4-ol (53). A white solid

1

(9.46 g, 78.8%), mp 107−110 °C. H NMR (600 MHz, CDCl₃) δ

12.78 (s, 1H), 8.45−8.13 (m, 2H), 7.89−7.47 (m, 3H), 6.84 (s, 1H).

L

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6-Cyclopropyl-2-phenylpyrimidin-4-ol (54). A white solid (8.41 g,

4-(3-Bromopropoxy)-5-ﬂuoro-6-methyl-2-phenylpyrimidine (67).

A pale-yellow oil (1.95 g, 60.1%). H NMR (600 MHz, CDCl₃) δ

8.42−8.26 (m, 2H), 7.51−7.40 (m, 3H), 4.70 (t, J = 6.0 Hz, 2H), 3.61

(t, J = 6.4 Hz, 2H), 2.52 (s, 3H), 2.45−2.36 (m, 2H). MS (ESI) m/z

326.1 (calcd 326.2 for C₁₄H₁₄BrFN₂O [M + H]⁺).

1

79.2%), mp 181−183 °C. H NMR (600 MHz, CDCl₃) δ 12.93 (s,

1H), 8.39−8.02 (m, 2H), 7.65−7.42 (m, 3H), 6.33 (s, 1H), 2.00−1.74

(m, 1H), 1.35−1.13 (m, 2H), 1.08−0.86 (m, 2H).

2,6-Diphenylpyrimidin-4-ol (55). A white solid (9.98 g, 80.4%), mp

1

285−288 °C. H NMR (600 MHz, CDCl₃) δ 11.50 (s, 1H), 8.28−

4-(3-Bromopropoxy)-5-chloro-6-methyl-2-phenylpyrimidine (68).

1

8.20 (m, 2H), 8.17−8.09 (m, 2H), 7.68−7.45 (m, 6H), 6.89 (s, 1H).

5,6-Dimethyl-2-phenylpyrimidin-4-ol (56). A white solid (7.44 g,

A pale-yellow oil (2.12 g, 62.2%). H NMR (600 MHz, CDCl₃) δ

8.46−8.33 (m, 2H), 7.51−7.41 (m, 3H), 4.69 (t, J = 6.0 Hz, 2H), 3.62

(t, J = 6.5 Hz, 2H), 2.62 (s, 3H), 2.47−2.35 (m, 2H). MS (ESI) m/z

342.8 (calcd 342.6 for C₁₄H₁₄BrClN₂O [M + H]⁺).

1

74.3%), mp 157−160 °C. H NMR (600 MHz, CDCl₃) δ 13.36 (s,

1H), 8.40−8.14 (m, 2H), 7.65−7.40 (m, 3H), 2.41 (s, 3H), 2.13 (s,

3H).

4-Ethyl-2-phenyl-6-(3-(piperidin-1-yl)propoxy)pyrimidine (69). A

mixture of 59 (5 mmol) and piperidine (5.5 mmol) in acetonitrile

(100 mL) and cesium carbonate (10 mmol) was heated and reﬂuxed

for 6−8 h. After ﬁltering, the resulting ﬁltrate was evaporated to

dryness under reduced pressure. The residue was suspended in water

(50 mL) and extracted with dichloromethane (3 × 25 mL). The

combined organic layers were dried with anhydrous magnesium

sulfate, the ﬁltrate evaporated under reduced pressure, and the crude

product puriﬁed by means of chromatography (10% MeOH/CHCl₃)

to yield 4-ethyl-2-phenyl-6-(3-(piperidin-1-yl)propoxy)pyrimidine, 69,

as a yellow oil (1.41 g, 86.4%). ¹H NMR (600 MHz, CDCl₃) δ 8.49−

8.41 (m, 2H), 7.49−7.43 (m, 3H), 6.45 (s, 1H), 4.52 (t, J = 6.5 Hz,

2H), 2.77 (q, J = 7.6 Hz, 2H), 2.52−2.33 (m, 6H), 2.06−1.97 (m,

2H), 1.64−1.57 (m, 4H), 1.50−1.38 (m, 2H), 1.33 (t, J = 7.6 Hz, 3H).

MS (ESI) m/z 326.5 (calcd 326.4 for C₂₀H₂₇N₃O [M + H]⁺).

2-Phenyl-4-(3-(piperidin-1-yl)propoxy)-6-propylpyrimidine (70).

5-Fluoro-6-methyl-2-phenylpyrimidin-4-ol (57). A white solid

1

(7.18 g, 70.3%), mp 148−151 °C. H NMR (600 MHz, CDCl₃) δ

13.13 (s, 1H), 8.34−7.97 (m, 2H), 7.67−7.44 (m, 3H), 2.43 (s, 3H).

5-Chloro-6-methyl-2-phenylpyrimidin-4-ol (58). A white solid

1

(7.88 g, 71.4%), mp 166−169 °C. H NMR (600 MHz, CDCl₃) δ

12.86 (s, 1H), 8.27−7.95 (m, 2H), 7.70−7.37 (m, 2H), 2.55 (s, 3H).

General Procedures for the Synthesis of 2-Phenyl-6-(3-(piperidin-

1-yl)propoxy)pyrimidine Derivatives (59−78). 4-(3-Bromopropoxy)-

6-ethyl-2-phenylpyrimidine (59). To a solution of 49 (10 mmol) and

1,3-dibromopropane (20 mmol) in acetone (100 mL), potassium

carbonate (20 mmol) was added and the mixture was reﬂuxed for 4−6

h. The progress of the reaction was monitored by TLC. After cooling

to room temperature, the mixture was ﬁltered and the solvent was

evaporated under reduced pressure. The crude product was puriﬁed by

means of chromatography (petroleum ether/EtOAc = 50/1) to yield

4-(3-bromopropoxy)-6-ethyl-2-phenylpyrimidine, 59, as a pale-yellow

1

A yellow oil (1.50 g, 88.3%). H NMR (600 MHz, CDCl₃) δ 8.51−

oil (1.95 g, 60.8%). H NMR (600 MHz, CDCl₃) δ 8.49−8.40 (m,

8.44 (m, 2H), 7.50−7.42 (m, 3H), 6.45 (s, 1H), 4.53 (t, J = 6.5 Hz,

2H), 2.75−2.69 (m, 2H), 2.55−2.29 (m, 6H), 2.08−1.99 (m, 2H),

1.87−1.77 (m, 2H), 1.65−1.58 (m, 4H), 1.51−1.40 (m, 2H), 1.01 (t, J

= 7.4 Hz, 3H). MS (ESI) m/z 340.8 (calcd 340.5 for C₂₁H₂₉N₃O [M +

H]⁺).

2H), 7.49−7.43 (m, 3H), 6.46 (s, 1H), 4.62 (t, J = 6.0 Hz, 2H), 3.58

(t, J = 6.5 Hz, 2H), 2.77 (q, J = 7.6 Hz, 2H), 2.42−2.31 (m, 2H), 1.33

(t, J = 7.6 Hz, 3H). MS (ESI) m/z 322.4 (calcd 322.2 for

C₁₅H₁₇BrN₂O [M + H]⁺).

4-(3-Bromopropoxy)-2-phenyl-6-propylpyrimidine (60). A pale-

yellow oil (2.08 g, 62.2%). ¹H NMR (600 MHz, CDCl₃) δ 8.49−8.40

(m, 2H), 7.49−7.43 (m, 3H), 6.46 (s, 1H), 4.62 (t, J = 6.0 Hz, 2H),

3.58 (t, J = 6.5 Hz, 2H), 2.77 (q, J = 7.6 Hz, 2H), 2.42−2.31 (m, 2H),

1.33 (t, J = 7.6 Hz, 3H). MS (ESI) m/z 336.4 (calcd 336.2 for

C₁₆H₁₉BrN₂O [M + H]⁺).

4-(3-Bromopropoxy)-6-isopropyl-2-phenylpyrimidine (61). A

pale-yellow oil (2.03 g, 60.7%). ¹H NMR (600 MHz, CDCl₃) δ

8.53−8.36 (m, 2H), 7.49−7.42 (m, 3H), 6.46 (s, 1H), 4.61 (t, J = 6.0

Hz, 2H), 3.57 (t, J = 6.5 Hz, 2H), 3.05−2.89 (m, 1H), 2.43−2.27 (m,

2H), 1.32 (d, J = 6.9 Hz, 6H). MS (ESI) m/z 336.7 (calcd 336.2 for

C₁₆H₁₉BrN₂O [M + H]⁺).

4-(3-Bromopropoxy)-6-methoxy-2-phenylpyrimidine (62). A pale-

yellow oil (1.88 g, 58.2%). ¹H NMR (600 MHz, CDCl₃) δ 8.47−8.36

(m, 2H), 7.48−7.42 (m, 3H), 6.75 (s, 1H), 4.62 (t, J = 6.0 Hz, 2H),

3.57 (t, J = 6.5 Hz, 2H), 3.50 (s, 3H), 2.39−2.31 (m, 2H). MS (ESI)

m/z 324.5 (calcd 324.2 for C₁₄H₁₅BrN₂O₂[M + H]⁺).

4-(3-Bromopropoxy)-2-phenyl-6-(triﬂuoromethyl)pyrimidine

(63). A pale-yellow oil (2.22 g, 61.4%). ¹H NMR (600 MHz, CDCl₃) δ

8.50−8.40 (m, 2H), 7.55−7.38 (m, 3H), 6.88 (s, 1H), 4.66 (t, J = 6.0

Hz, 2H), 3.56 (t, J = 6.4 Hz, 2H), 2.42−2.31 (m, 2H). MS (ESI) m/z

362.6 (calcd 362.2 for C₁₄H₁₂BrF₃N₂O [M + H]⁺).

4-Isopropyl-2-phenyl-6-(3-(piperidin-1-yl)propoxy)pyrimidine

1

(71). A yellow oil (1.51 g, 89.2%). H NMR (600 MHz, CDCl₃) δ

8.52−8.46 (m, 2H), 7.50−7.44 (m, 3H), 6.48 (s, 1H), 4.54 (t, J = 6.5

Hz, 2H), 3.00 (dt, J = 13.8, 6.9 Hz, 1H), 2.54−2.36 (m, 6H), 2.09−

2.01 (m, 2H), 1.66−1.59 (m, 4H), 1.51−1.42 (m, 2H), 1.35 (d, J = 6.9

Hz, 6H). MS (ESI) m/z 340.2 (calcd 340.5 for C₂₁H₂₉N₃O [M +

H]⁺).

4-Methoxy-2-phenyl-6-(3-(piperidin-1-yl)propoxy)pyrimidine

1

(72). A yellow oil (1.37 g, 84.1%). H NMR (600 MHz, CDCl₃) δ

8.45−8.39 (m, 2H), 7.46−7.40 (m, 3H), 6.74 (s, 1H), 4.52 (t, J = 6.5

Hz, 2H), 3.50 (s, 3H), 2.51−2.38 (m, 6H), 2.07−1.97 (m, 2H), 1.65−

1.55 (m, 4H), 1.45−1.38 (m, 2H). MS (ESI) m/z 328.5 (calcd 328.4

for C₁₉H₂₅N₃O₂[M + H]⁺).

2-Phenyl-4-(3-(piperidin-1-yl)propoxy)-6-(triﬂuoromethyl)-

1

pyrimidine (73). A yellow oil (1.57 g, 86.2%). H NMR (600 MHz,

CDCl₃) δ 8.52−8.42 (m, 2H), 7.52−7.44 (m, 3H), 6.90 (s, 1H), 4.59

(t, J = 6.5 Hz, 2H), 2.52−2.36 (m, 6H), 2.09−2.00 (m, 2H), 1.66−

1.55 (m, 4H), 1.48−1.42 (m, 2H). MS (ESI) m/z 366.4 (calcd 366.4

for C₁₉H₂₂F₃N₃O [M + H]⁺).

4-Cyclopropyl-2-phenyl-6-(3-(piperidin-1-yl)propoxy)pyrimidine

1

(74). A yellow oil (1.53 g, 90.5%). H NMR (600 MHz, CDCl₃) δ

8.45−8.37 (m, 2H), 7.61−7.48 (m, 3H), 6.39 (s, 1H), 4.51 (t, J = 6.5

Hz, 2H), 2.53−2.34 (m, 6H), 2.04−1.84 (m, 3H), 1.66−1.53 (m, 4H),

1.46−1.41 (m, 2H), 1.29−1.14 (m, 2H), 1.08−0.95 (m, 2H). MS

(ESI) m/z 338.2 (calcd 338.5 for C₂₁H₂₇N₃O [M + H]⁺).

2,4-Diphenyl-6-(3-(piperidin-1-yl)propoxy)pyrimidine (75). A yel-

low oil (1.71 g, 91.4%). ¹H NMR (600 MHz, CDCl₃) δ 8.62−8.54 (m,

2H), 8.22−8.15 (m, 2H), 7.54−7.46 (m, 6H), 7.02 (s, 1H), 4.58 (t, J =

6.5 Hz, 2H), 2.56−2.37 (m, 6H), 2.06−1.96 (m, 2H), 1.66−1.58 (m,

4H), 1.45−1.38 (m, 2H). MS (ESI) m/z 374.8 (calcd 374.5 for

C₂₄H₂₇N₃O [M + H]⁺).

4-(3-Bromopropoxy)-6-cyclopropyl-2-phenylpyrimidine (64). A

pale-yellow oil (2.13 g, 63.8%). ¹H NMR (600 MHz, CDCl₃) δ

8.47−8.31 (m, 2H), 7.48−7.41 (m, 3H), 6.46 (s, 1H), 4.61 (t, J = 6.0

Hz, 2H), 3.59 (t, J = 6.5 Hz, 2H), 2.43−2.30 (m, 2H), 2.02−1.91 (m,

1H), 1.24−1.21 (m, 2H), 1.05−1.01 (m, 2H). MS (ESI) m/z 334.1

(calcd 334.2 for C₁₆H₁₇BrN₂O [M + H]⁺).

4-(3-Bromopropoxy)-2,6-diphenylpyrimidine (65). A pale-yellow

1

oil (2.44 g, 66.1%). H NMR (600 MHz, CDCl₃) δ 8.64−8.51 (m,

2H), 8.22−8.14 (m, 2H), 7.53−7.47 (m, 6H), 7.02 (s, 1H), 4.70 (t, J =

6.0 Hz, 2H), 3.62 (t, J = 6.5 Hz, 2H), 2.47−2.37 (m, 2H). MS (ESI)

m/z 370.2 (calcd 370.3 for C₁₉H₁₇BrN₂O [M + H]⁺).

4,5-Dimethyl-2-phenyl-6-(3-(piperidin-1-yl)propoxy)pyrimidine

1

(76). A yellow oil (1.42 g, 87.1%). H NMR (600 MHz, CDCl₃) δ

4-(3-Bromopropoxy)-5,6-dimethyl-2-phenylpyrimidine (66). A

pale-yellow oil (1.90 g, 59.3%). ¹H NMR (600 MHz, CDCl₃) δ

8.44−8.34 (m, 2H), 7.52−7.36 (m, 3H), 4.62 (t, J = 6.0 Hz, 2H), 3.59

(t, J = 6.6 Hz, 2H), 2.49 (s, 3H), 2.43−2.34 (m, 2H), 2.13 (s, 3H). MS

(ESI) m/z 322.6 (calcd 322.2 for C₁₅H₁₇BrN₂O [M + H]⁺).

8.44−8.36 (m, 2H), 7.48−7.37 (m, 3H), 4.51 (t, J = 6.4 Hz, 2H),

2.56−2.30 (m, 9H), 2.12 (s, 3H), 2.06−1.97 (m, 2H), 1.64−1.55 (m,

4H), 1.47−1.41 (m, 2H). MS (ESI) m/z 326.6 (calcd 326.4 for

C₂₀H₂₇N₃O [M + H]⁺).

M

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5-Fluoro-4-methyl-2-phenyl-6-(3-(piperidin-1-yl)propoxy)-

5-Fluoro-4-methyl-2-phenyl-6-(3-(pyrrolidin-1-yl)propoxy)-

1

pyrimidine (77). A yellow oil (1.36 g, 82.6%). H NMR (600 MHz,

pyrimidine (87). A pale-red oil (1.31 g, 83.1%). H NMR (600 MHz,

CDCl₃) δ 8.38−8.28 (m, 2H), 7.48−7.39 (m, 3H), 4.58 (t, J = 6.6 Hz,

2H), 2.62−2.26 (m, 9H), 2.12−1.98 (m, 2H), 1.66−1.52 (m, 4H),

1.46−1.41 (m, 2H). MS (ESI) m/z 330.2 (calcd 330.4 for

C₁₉H₂₄FN₃O [M + H]⁺).

CDCl₃) δ 8.39−8.29 (m, 2H), 7.49−7.40 (m, 3H), 4.61 (t, J = 6.5 Hz,

2H), 2.69−2.63 (m, 2H), 2.59−2.47 (m, 7H), 2.13−2.03 (m, 2H),

1.85−1.75 (m, 4H). MS (ESI) m/z 316.7 (calcd 316.4 for

C₁₈H₂₂FN₃O [M + H]⁺).

5-Chloro-4-methyl-2-phenyl-6-(3-(piperidin-1-yl)propoxy)-

5-Chloro-4-methyl-2-phenyl-6-(3-(pyrrolidin-1-yl)propoxy)-

1

pyrimidine (78). A yellow oil (1.41 g, 81.5%). H NMR (600 MHz,

pyrimidine (88). A pale-red oil (1.37 g, 82.5%). H NMR (600 MHz,

CDCl₃) δ 8.44−8.33 (m, 2H), 7.49−7.40 (m, 3H), 4.61 (t, J = 6.5 Hz,

2H), 2.72−2.65 (m, 2H), 2.64−2.51 (m, 7H), 2.14−2.06 (m, 2H),

1.85−1.75 (m, 4H), 1.44−1.39 (m, 2H). MS (ESI) m/z 346.9 (calcd

346.9 for C₁₉H₂₄ClN₃O [M + H]⁺).

CDCl₃) δ 8.42−8.33 (m, 2H), 7.49−7.42 (m, 3H), 4.61 (t, J = 6.5 Hz,

2H), 2.71−2.65 (m, 2H), 2.63−2.52 (m, 7H), 2.14−2.06 (m, 2H),

1.86−1.75 (m, 4H). MS (ESI) m/z 332.9 (calcd 332.8 for

C₁₈H₂₂ClN₃O [M + H]⁺).

2-Phenyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-ol (91).⁶⁰To

a suspension of benzimidamide hydrochloride (5, 50 mmol) and ethyl

2-oxocyclopentanecarboxylate (89, 55 mmol) in MeOH (200 mL) was

added t-BuOK (10 mmol) portionwise, with stirring, at 0 °C for 30

min. The resulting mixture was heated to reﬂux for 4 h and then

cooled down to room temperature. After that, ice−water (200 mL)

was added and neutralized with citric acid. The precipitate thus formed

was ﬁltered and washed with water. The crude residue obtained was

recrystallized from i-PrOH to give 2-phenyl-6,7-dihydro-5H-

cyclopenta[d]pyrimidin-4-ol, 91, as a white solid (8.6 g, 81.0%), mp

General Procedures for the Synthesis of 2-Phenyl-6-(3-(pyrroli-

din-1-yl)propoxy)pyrimidine Derivatives (79−88). 4-Ethyl-2-phenyl-

6-(3-(pyrrolidin-1-yl)propoxy)pyrimidine (79). A mixture of 59 (5

mmol) and pyrrolidine (5.5 mmol) in acetonitrile (100 mL) and

cesium carbonate (10 mmol) was heated and reﬂuxed for 6−8 h. After

ﬁltering, the resulting ﬁltrate was evaporated to dryness under reduced

pressure. The residue was suspended in water (50 mL) and extracted

with dichloromethane (3 × 25 mL). The combined organic layers

were dried with anhydrous magnesium sulfate, the ﬁltrate evaporated

under reduced pressure, and the crude product puriﬁed by means of

chromatography (10% MeOH/CHCl₃) to yield 4-ethyl-2-phenyl-6-(3-

(pyrrolidin-1-yl)propoxy)pyrimidine, 79, as a pale-red oil (1.37 g,

1

232−235 °C. H NMR (600 MHz, CDCl₃) δ 12.61 (s, 1H), 8.26−

8.07 (m, 2H), 7.60−7.44 (m, 3H), 2.97 (t, J = 7.8 Hz, 2H), 2.88 (t, J =

7.5 Hz, 2H), 2.24−2.05 (m, 2H).

1

88.2%). H NMR (600 MHz, CDCl₃) δ 8.48−8.41 (m, 2H), 7.48−

2-Phenyl-5,6,7,8-tetrahydroquinazolin-4-ol (92).⁶⁰To a suspen-

sion of benzimidamide hydrochloride (5, 50 mmol) and ethyl 2-

oxocyclohexanecarboxylate (90, 55 mmol) in MeOH (200 mL) was

added t-BuOK (10 mmol) portionwise, with stirring, at 0 °C for 30

min. The resulting mixture was heated to reﬂux for 4 h and then

cooled down to room temperature. After that, ice−water (200 mL)

was added and neutralized with citric acid. The precipitate thus formed

was ﬁltered and washed with water. The crude residue obtained was

recrystallized from i-PrOH to give 2-phenyl-5,6,7,8-tetrahydroquina-

7.43 (m, 3H), 6.46 (s, 1H), 4.54 (t, J = 6.5 Hz, 2H), 2.77 (q, J = 7.6

Hz, 2H), 2.67−2.62 (m, 2H), 2.58−2.50 (m, 4H), 2.09−2.01 (m, 2H),

1.84−1.75 (m, 4H), 1.33 (t, J = 7.6 Hz, 3H). MS (ESI) m/z 312.8

(calcd 312.4 for C₁₉H₂₅N₃O [M + H]⁺).

2-Phenyl-4-(3-(pyrrolidin-1-yl)propoxy)-6-propylpyrimidine (80).

A pale-red oil (1.40 g, 86.2%). ¹H NMR (600 MHz, CDCl₃) δ

8.47−8.40 (m, 2H), 7.47−7.43 (m, 3H), 6.44 (s, 1H), 4.53 (t, J = 6.5

Hz, 2H), 2.73−2.68 (m, 2H), 2.67−2.61 (m, 2H), 2.58−2.51 (m, 4H),

2.08−2.02 (m, 2H), 1.83−1.77 (m, 6H), 1.00 (t, J = 7.4 Hz, 3H). MS

(ESI) m/z 326.6 (calcd 326.4 for C₂₀H₂₇N₃O [M + H]⁺).

1

zolin-4-ol, 92, as a white solid (8.9 g, 78.7%), mp 246−249 °C. H

4-Isopropyl-2-phenyl-6-(3-(pyrrolidin-1-yl)propoxy)pyrimidine

NMR (600 MHz, CDCl₃) δ 12.90 (s, 1H), 8.32−8.09 (m, 2H), 7.70−

7.36 (m, 3H), 2.74 (t, J = 6.1 Hz, 2H), 2.59 (t, J = 6.0 Hz, 2H), 1.93−

1.71 (m, 4H).

1

(81). A pale-red oil (1.38 g, 84.5%). H NMR (600 MHz, CDCl₃) δ

8.50−8.44 (m, 2H), 7.50−7.41 (m, 3H), 6.46 (s, 1H), 4.54 (t, J = 6.5

Hz, 2H), 3.03−2.93 (m, 1H), 2.69−2.61 (m, 2H), 2.60−2.57 (m, 4H),

2.09−2.02 (m, 2H), 1.85−1.76 (m, 4H), 1.32 (d, J = 6.9 Hz, 6H). MS

(ESI) m/z 326.3 (calcd 326.4 for C₂₀H₂₇N₃O [M + H]⁺).

2-Phenyl-4-(3-(piperidin-1-yl)propoxy)-6,7-dihydro-5H-

cyclopenta[d]pyrimidine (94). To a solution of 91 (10 mmol) and

1,3-dibromopropane (20 mmol) in acetone (100 mL), potassium

carbonate (20 mmol) was added and the mixture was reﬂuxed for 4−6

h. The progress of the reaction was monitored by TLC. After cooling

to room temperature, the mixture was ﬁltered and the solvent was

evaporated under reduced pressure. The crude product was puriﬁed by

means of chromatography (petroleum ether/EtOAc = 100/1 to 50/1)

to yield 4-(3-bromopropoxy)-2-phenyl-6,7-dihydro-5H-cyclopenta[d]-

4-Methoxy-2-phenyl-6-(3-(pyrrolidin-1-yl)propoxy)pyrimidine

1

(82). A pale-red oil (1.28 g, 81.7%). H NMR (600 MHz, CDCl₃) δ

8.45−8.38 (m, 2H), 7.48−7.44 (m, 3H), 6.75 (s, 1H), 4.56 (t, J = 6.5

Hz, 2H), 3.51 (s, 3H), 2.70−2.62 (m, 2H), 2.60−2.53 (m, 4H), 2.12−

2.02 (m, 2H), 1.84−1.76 (m, 4H). MS (ESI) m/z 314.8 (calcd 314.4

for C₁₈H₂₃N₃O₂[M + H]⁺).

2-Phenyl-4-(3-(pyrrolidin-1-yl)propoxy)-6-(triﬂuoromethyl)-

1

pyrimidine, 93, as a pale-yellow oil (2.17 g, 65.3%). H NMR (600

pyrimidine (83). A pale-red oil (1.57 g, 89.6%). H NMR (600 MHz,

MHz, CDCl₃) δ 8.47−8.36 (m, 2H), 7.52−7.40 (m, 3H), 4.67 (t, J =

6.0 Hz, 2H), 3.59 (t, J = 6.6 Hz, 2H), 3.03 (t, J = 7.8 Hz, 2H), 2.87 (t,

J = 7.5 Hz, 2H), 2.45−2.35 (m, 2H), 2.20−2.10 (m, 2H). MS (ESI)

m/z 334.3 (calcd 334.2 for C₁₆H₁₇BrN₂O [M + H]⁺).

CDCl₃) δ 8.50−8.42 (m, 2H), 7.53−7.43 (m, 3H), 6.91 (s, 1H), 4.62

(t, J = 6.5 Hz, 2H), 2.69−2.62 (m, 2H), 2.59−2.52 (m, 4H), 2.12−

2.04 (m, 2H), 1.84−1.77 (m, 4H). MS (ESI) m/z 352.5 (calcd 352.4

for C₁₈H₂₀F₃N₃O [M + H]⁺).

A mixture of 93 (5 mmol) and piperidine (5.5 mmol) in acetonitrile

(100 mL) and cesium carbonate (10 mmol) was heated and reﬂuxed

for 6−8 h. After ﬁltering, the resulting ﬁltrate was evaporated to

dryness under reduced pressure. The residue was suspended in water

(50 mL) and extracted with dichloromethane (3 × 25 mL). The

combined organic layers were dried with anhydrous magnesium

sulfate, the ﬁltrate evaporated under reduced pressure, and the crude

product puriﬁed by means of chromatography (10% MeOH/CHCl₃)

to yield 2-phenyl-4-(3-(piperidin-1-yl)propoxy)-6,7-dihydro-5H-

cyclopenta[d]pyrimidine, 94, as a yellow oil (1.27 g, 75.6%). ¹H

NMR (600 MHz, CDCl₃) δ 8.44−8.31 (m, 2H), 7.53−7.40 (m, 3H),

4.56 (t, J = 6.5 Hz, 2H), 2.87 (t, J = 6.2 Hz, 2H), 2.68−2.63 (m, 2H),

2.61−2.48 (m, 6H), 2.41−2.36 (m, 2H), 2.08−2.00 (m, 2H), 1.67−

1.54 (m, 4H), 1.47−1.41 (m, 2H). MS (ESI) m/z 338.6 (calcd 338.5

for C₂₁H₂₇N₃O [M + H]⁺).

4-Cyclopropyl-2-phenyl-6-(3-(pyrrolidin-1-yl)propoxy)pyrimidine

1

(84). A pale-red oil (1.42 g, 88.1%). H NMR (600 MHz, CDCl₃) δ

8.44−8.37 (m, 2H), 7.59−7.47 (m, 3H), 6.41 (s, 1H), 4.52 (t, J = 6.5

Hz, 2H), 2.54−2.34 (m, 6H), 2.03−1.82 (m, 3H), 1.86−1.73 (m, 4H),

1.30−1.14 (m, 2H), 1.12−1.02 (m, 2H). MS (ESI) m/z 324.6 (calcd

324.4 for C₂₀H₂₅N₃O [M + H]⁺).

2,4-Diphenyl-6-(3-(pyrrolidin-1-yl)propoxy)pyrimidine (85). A

1

pale-red oil (1.66 g, 92.2%). H NMR (600 MHz, CDCl₃) δ 8.64−

8.55 (m, 2H), 8.23−8.18 (m, 2H), 7.58−7.47 (m, 6H), 7.03 (s, 1H),

4.56 (t, J = 6.5 Hz, 2H), 2.56−2.37 (m, 6H), 2.08−1.99 (m, 2H),

1.82−1.70 (m, 4H). MS (ESI) m/z 360.3 (calcd 360.5 for C₂₃H₂₅N₃O

[M + H]⁺).

4,5-Dimethyl-2-phenyl-6-(3-(pyrrolidin-1-yl)propoxy)pyrimidine

1

(86). A pale-red oil (1.33 g, 85.3%). H NMR (600 MHz, CDCl₃) δ

8.43−8.37 (m, 2H), 7.48−7.39 (m, 3H), 4.53 (t, J = 6.4 Hz, 2H),

2.66−2.60 (m, 2H), 2.57−2.50 (m, 4H), 2.47 (s, 3H), 2.13 (s, 3H),

2.08−2.01 (m, 2H), 1.85−1.73 (m, 4H). MS (ESI) m/z 312.8 (calcd

312.4 for C₂₀H₂₅N₃O [M + H]⁺).

2-Phenyl-4-(3-(pyrrolidin-1-yl)propoxy)-6,7-dihydro-5H-

cyclopenta[d]pyrimidine (95). A mixture of 93 (5 mmol) and

pyrrolidine (5.5 mmol) in acetonitrile (100 mL) and cesium carbonate

N

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Journal of Medicinal Chemistry

Article

(10 mmol) was heated and reﬂuxed for 6−8 h. After ﬁltering, the

resulting ﬁltrate was evaporated to dryness under reduced pressure.

The residue was suspended in water (50 mL) and extracted with

dichloromethane (3 × 25 mL). The combined organic layers were

dried with anhydrous magnesium sulfate, the ﬁltrate evaporated under

reduced pressure, and the crude product puriﬁed by means of

chromatography (10% MeOH/CHCl₃) to yield 2-phenyl-4-(3-

(pyrrolidin-1-yl)propoxy)-6,7-dihydro-5H-cyclopenta[d]pyrimidine,

(d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 2.58 (s, 3H), 2.40 (s,

3H).

5-Chloro-2-(4-methoxyphenyl)-6-methylpyrimidin-4-ol (111). A

1

white solid (10.12 g, 80.8%), mp 143−146 °C. H NMR (600 MHz,

CDCl₃) δ 11.58 (s, 1H), 8.33 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 8.9 Hz,

2H), 3.87 (s, 3H), 2.58 (s, 3H).

5-Chloro-6-methyl-2-(4-(triﬂuoromethyl)phenyl)pyrimidin-4-ol

(112).⁶¹A white solid (12.87 g, 89.2%), mp 157−161 °C. H NMR

1

(600 MHz, CDCl₃) δ 11.60 (s, 1H), 8.50 (d, J = 8.2 Hz, 2H), 7.69 (d,

J = 8.3 Hz, 2H), 2.60 (s, 3H).

95, as a pale-red oil (1.13 g, 70.2%). H NMR (600 MHz, CDCl₃) δ

8.45−8.29 (m, 2H), 7.52−7.40 (m, 3H), 4.57 (t, J = 6.5 Hz, 2H), 2.88

(t, J = 6.2 Hz, 2H), 2.69−2.64 (m, 2H), 2.63−2.51 (m, 6H), 2.42−

2.35 (m, 2H), 2.06−2.00 (m, 2H), 1.85−1.73 (m, 4H). MS (ESI) m/z

324.5 (calcd 324.4 for C₂₀H₂₅N₃O [M + H]⁺).

5-Chloro-2-(4-ﬂuorophenyl)-6-methylpyrimidin-4-ol (113). A

white solid (10.07 g, 84.4%), mp 164−167 °C. ¹H NMR (600

MHz, CDCl₃) δ 12.05 (s, 1H), 8.25−7.98 (m, 2H), 7.24−7.12 (m,

2H), 2.51 (s, 3H).

2-Phenyl-4-(3-(piperidin-1-yl)propoxy)-5,6,7,8-tetrahydroquina-

zoline (97). To a solution of 92 (10 mmol) and 1,3-dibromopropane

(20 mmol) in acetone (100 mL), potassium carbonate (20 mmol) was

added and the mixture was reﬂuxed for 4−6 h. The progress of the

reaction was monitored by TLC. After cooling to room temperature,

the mixture was ﬁltered and the solvent was evaporated under reduced

pressure. The crude product was puriﬁed by means of chromatography

(petroleum ether/EtOAc = 100/1 to 50/1) to yield 4-(3-

bromopropoxy)-2-phenyl-5,6,7,8-tetrahydroquinazoline, 96, as a pale-

yellow oil (1.90 g, 54.9%). ¹H NMR (600 MHz, CDCl₃) δ 8.42−8.34

(m, 2H), 7.47−7.41 (m, 3H), 4.64 (t, J = 5.9 Hz, 2H), 3.59 (t, J = 6.6

Hz, 2H), 2.86 (t, J = 6.2 Hz, 2H), 2.60 (t, J = 6.2 Hz, 2H), 2.43−2.34

(m, 2H), 1.94−1.77 (m, 4H). MS (ESI) m/z 348.8 (calcd 348.2 for

C₁₇H₁₉BrN₂O [M + H]⁺).

5-Chloro-2-(3,4-diﬂuorophenyl)-6-methylpyrimidin-4-ol (114). A

1

white solid (11.04 g, 86.1%), mp 170−173 °C. H NMR (600 MHz,

CDCl₃) δ 12.78 (s, 1H), 8.25−8.20 (m, 1H), 8.19−8.13 (m, 1H),

7.30−7.15 (m, 1H), 2.58 (s, 3H).

5-Chloro-2-(4-chlorophenyl)-6-methylpyrimidin-4-ol (115). A

white solid (10.66 g, 83.6%), mp 171−174 °C. ¹H NMR (600

MHz, CDCl₃) δ 11.84 (s, 1H), 8.44−8.30 (m, 2H), 7.48−7.35 (m,

2H), 2.60 (s, 3H).

5-Chloro-2-(3,4-dichlorophenyl)-6-methylpyrimidin-4-ol (116). A

1

white solid (12.87 g, 88.9%), mp 177−180 °C. H NMR (600 MHz,

CDCl₃) δ 11.92 (s, 1H), 8.50−8.45 (m, 1H), 8.27−8.20 (m, 1H),

7.56−7.48 (m, 1H), 2.59 (s, 3H).

5-Chloro-6-methyl-2-(naphthalen-2-yl)pyrimidin-4-ol (117). A

white solid (12.41 g, 91.7%), mp 204−207 °C. ¹H NMR (600

MHz, CDCl₃) δ 12.08 (s, 1H), 8.92 (s, 1H), 8.52−8.42 (m, 1H),

8.05−7.95 (m, 1H), 7.93−7.88 (m, 1H), 7.86−7.82 (m, 1H), 7.57−

7.49 (m, 2H), 2.63 (s, 3H).

A mixture of 96 (5 mmol) and piperidine (5.5 mmol) in acetonitrile

(100 mL) and cesium carbonate (10 mmol) was heated and reﬂuxed

for 6−8 h. After ﬁltering, the resulting ﬁltrate was evaporated to

dryness under reduced pressure. The residue was suspended in water

(50 mL) and extracted with dichloromethane (3 × 25 mL). The

combined organic layers were dried with anhydrous magnesium

sulfate, the ﬁltrate evaporated under reduced pressure, and the crude

product puriﬁed by means of chromatography (10% MeOH/CHCl₃)

to yield 2-phenyl-4-(3-(piperidin-1-yl)propoxy)-5,6,7,8-tetrahydroqui-

5-Chloro-6-methylpyrimidin-4-ol (118).⁶²To a suspension of

formimidamide hydrochloride (107, 100 mmol) and ethyl 2-chloro-3-

oxobutanoate (48, 110 mmol) in MeOH (300 mL) was added t-BuOK

(20 mmol) portionwise, with stirring, at 0 °C for 30 min. The resulting

mixture was heated to reﬂux for and then cooled down to room

temperature. After that, ice−water (500 mL) was added and

neutralized with citric acid. The mixture was extracted with EtOH

(3 × 150 mL), and the combined organic layers were dried with

anhydrous magnesium sulfate. The ﬁltrate was evaporated under

reduced pressure, and the crude product was puriﬁed by means of

chromatography (petroleum ether/EtOAc = 20/1 to 2/1) to yield 5-

chloro-6-methylpyrimidin-4-ol, 118, as a pale-yellow solid (7.56 g,

1

nazoline, 97, as a yellow oil (1.47 g, 84.0%). H NMR (600 MHz,

CDCl₃) δ 8.46−8.32 (m, 2H), 7.51−7.38 (m, 3H), 4.57 (t, J = 6.5 Hz,

2H), 2.86 (t, J = 6.2 Hz, 2H), 2.68−2.64 (m, 2H), 2.63−2.50 (m, 6H),

2.08−2.00 (m, 2H), 1.92−1.84 (m, 4H), 1.65−1.52 (m, 4H), 1.46−

1.40 (m, 2H). MS (ESI) m/z 352.7 (calcd 352.5 for C₂₂H₂₉N₃O [M +

H]⁺).

2-Phenyl-4-(3-(pyrrolidin-1-yl)propoxy)-5,6,7,8-tetrahydroquina-

zoline (98). A mixture of 96 (5 mmol) and pyrrolidine (5.5 mmol) in

acetonitrile (100 mL) and cesium carbonate (10 mmol) was heated

and reﬂuxed for 6−8 h. After ﬁltering, the resulting ﬁltrate was

evaporated to dryness under reduced pressure. The residue was

suspended in water (50 mL) and extracted with dichloromethane (3 ×

25 mL). The combined organic layers were dried with anhydrous

magnesium sulfate, the ﬁltrate evaporated under reduced pressure, and

the crude product puriﬁed by means of chromatography (10%

MeOH/CHCl₃) to yield 2-phenyl-4-(3-(pyrrolidin-1-yl)propoxy)-

5,6,7,8-tetrahydroquinazoline, 98, as a pale-red oil (1.30 g, 77.4%).

¹H NMR (600 MHz, CDCl₃) δ 8.44−8.32 (m, 2H), 7.49−7.36 (m,

1

53.2%), mp 211−213 °C. H NMR (600 MHz, CDCl₃) δ 13.34 (s,

1H), 8.11 (s, 1H), 2.52 (s, 3H).

5-Chloro-2,6-dimethylpyrimidin-4-ol (119).⁶³A white solid (9.32

g, 58.8%), mp 121−124 °C. ¹H NMR (600 MHz, CDCl₃) δ 13.31 (s,

1H), 2.50 (s, 3H), 2.45 (s, 3H).

5-Chloro-2-cyclopropyl-6-methylpyrimidin-4-ol (120). A white

solid (11.50 g, 62.3%), mp 133−136 °C. ¹H NMR (600 MHz,

CDCl₃) δ 13.45 (s, 1H), 2.39 (s, 3H), 2.00−1.91 (m, 1H), 1.24−1.18

(m, 2H), 1.14−1.09 (m, 2H).

4-(3-Bromopropoxy)-5-chloro-6-methyl-2-(p-tolyl)pyrimidine

(121). To a solution of 110 (10 mmol) and 1,3-dibromopropane (20

mmol) in acetone (100 mL), potassium carbonate (20 mmol) was

added and the mixture was reﬂuxed for 4−6 h. The progress of the

reaction was monitored by TLC. After cooling to room temperature,

the mixture was ﬁltered and the solvent was evaporated under reduced

pressure. The crude product was puriﬁed by means of chromatography

(petroleum ether/EtOAc = 100/1 to 50/1) to yield 4-(3-

bromopropoxy)-5-chloro-6-methyl-2-(p-tolyl)pyrimidine, 121, as a

3H), 4.55 (t, J = 6.4 Hz, 2H), 2.85 (t, J = 6.2 Hz, 2H), 2.68−2.62 (m,

2H), 2.60−2.48 (m, 6H), 2.10−2.01 (m, 2H), 1.92−1.73 (m, 8H). MS

(ESI) m/z 338.7 (calcd 338.5 for C₂₁H₂₇N₃O [M + H]⁺).

General Procedures for the Synthesis of 5-Chloro-6-methylpyr-

imidin-4-ol Derivatives (110−120). 5-Chloro-6-methyl-2-(p-tolyl)-

pyrimidin-4-ol (110). To a suspension of 4-methylbenzimidamide (99,

50 mmol) and ethyl 2-chloro-3-oxobutanoate (48, 55 mmol) in

MeOH (200 mL) was added t-BuOK (10 mmol) portionwise, with

stirring, at 0 °C for 30 min. The resulting mixture was heated to reﬂux

for 4 h and then cooled down to room temperature. After that, ice−

water (200 mL) was added and neutralized with citric acid. The

precipitate thus formed was ﬁltered and washed with water. The crude

residue obtained was recrystallized from i-PrOH to give 5-chloro-6-

methyl-2-(p-tolyl)pyrimidin-4-ol, 110, as a white solid (9.60 g, 81.8%),

1

pale-yellow oil (1.66 g, 46.8%). H NMR (600 MHz, CDCl₃) δ 8.26

(d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 4.65 (t, J = 6.0 Hz, 2H),

3.60 (t, J = 6.5 Hz, 2H), 2.58 (s, 3H), 2.46−2.33 (m, 5H). MS (ESI)

m/z 356.8 (calcd 356.7 for C₁₅H₁₆BrClN₂O [M + H]⁺).

4-(3-Bromopropoxy)-5-chloro-2-(4-methoxyphenyl)-6-methyl-

1

pyrimidine (122). A pale-yellow oil (1.64 g, 44.1%). H NMR (600

MHz, CDCl₃) δ 8.33 (d, J = 8.8 Hz, 2H), 6.95 (d, J = 8.9 Hz, 2H),

4.65 (t, J = 6.0 Hz, 2H), 3.86 (s, 3H), 3.61 (t, J = 6.5 Hz, 2H), 2.58 (s,

1

mp 175−178 °C. H NMR (600 MHz, CDCl₃) δ 11.60 (s, 1H), 8.26

O

dx.doi.org/10.1021/jm501207r | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

3H), 2.44−2.35 (m, 2H). MS (ESI) m/z 372.4 (calcd 372.7 for

C₁₅H₁₆BrClN₂O₂[M + H]⁺).

5-Chloro-2-(4-methoxyphenyl)-4-methyl-6-(3-(piperidin-1-yl)-

1

propoxy)pyrimidine (133). A yellow oil (1.54 g, 82.1%). H NMR

(600 MHz, CDCl₃) δ 8.33 (d, J = 8.9 Hz, 2H), 6.95 (d, J = 8.9 Hz,

2H), 4.57 (t, J = 6.5 Hz, 2H), 3.86 (s, 3H), 2.57 (s, 3H), 2.54−2.49

(m, 2H), 2.48−2.32 (m, 4H), 2.10−2.00 (m, 2H), 1.66−1.52 (m, 4H),

1.50−1.39 (m, 2H). MS (ESI) m/z 377.1 (calcd 375.9 for

C₂₀H₂₆ClN₃O₂[M + H]⁺).

4-(3-Bromopropoxy)-5-chloro-6-methyl-2-(4-(triﬂuoromethyl)-

phenyl)pyrimidine (123). A pale-yellow oil (2.15 g, 52.6%). ¹H NMR

(600 MHz, CDCl₃) δ 8.50 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.3 Hz,

2H), 4.69 (t, J = 6.0 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.62 (s, 3H),

2.50−2.35 (m, 2H). MS (ESI) m/z 410.7 (calcd 410.6 for

C₁₅H₁₃BrClF₃N₂O [M + H]⁺).

5-Chloro-4-methyl-6-(3-(piperidin-1-yl)propoxy)-2-(4-

(triﬂuoromethyl)phenyl)pyrimidine (134). A yellow oil (1.83 g,

4-(3-Bromopropoxy)-5-chloro-2-(4-ﬂuorophenyl)-6-methylpyri-

midine (124). A pale-yellow oil (1.75 g, 48.7%). ¹H NMR (600 MHz,

CDCl₃) δ 8.44−8.34 (m, 2H), 7.18−7.06 (m, 2H), 4.66 (t, J = 6.0 Hz,

2H), 3.62 (t, J = 6.4 Hz, 2H), 2.59 (s, 3H), 2.44−2.35 (m, 2H). MS

(ESI) m/z 360.6 (calcd 360.6 for C₁₄H₁₃BrClFN₂O [M + H]⁺).

4-(3-Bromopropoxy)-5-chloro-2-(3,4-diﬂuorophenyl)-6-methyl-

1

88.7%). H NMR (600 MHz, CDCl₃) δ 8.48 (d, J = 8.2 Hz, 2H),

7.68 (d, J = 8.3 Hz, 2H), 4.59 (t, J = 6.5 Hz, 2H), 2.60 (s, 3H), 2.55−

2.50 (m, 2H), 2.50−2.32 (m, 4H), 2.11−2.04 (m, 2H), 1.65−1.55 (m,

4H), 1.50−1.40 (m, 2H). MS (ESI) m/z 414.9 (calcd 414.9 for

C₂₀H₂₃ClF₃N₃O [M + H]⁺).

1

5-Chloro-2-(4-ﬂuorophenyl)-4-methyl-6-(3-(piperidin-1-yl)-

pyrimidine (125). A pale-yellow oil (1.95 g, 51.8%). H NMR (600

1

propoxy)pyrimidine (135). A yellow oil (1.46 g, 80.5%). H NMR

MHz, CDCl₃) δ 8.25−8.19 (m, 1H), 8.19−8.14 (m, 1H), 7.30−7.16

(m, 1H), 4.67 (t, J = 6.0 Hz, 2H), 3.62 (t, J = 6.4 Hz, 2H), 2.60 (s,

3H), 2.46−2.36 (m, 2H). MS (ESI) m/z 378.8 (calcd 378.6 for

C₁₄H₁₂BrClF₂N₂O [M + H]⁺).

5-Chloro-2-(4-chlorophenyl)-4-methyl-6-(3-(piperidin-1-yl)-

propoxy)pyrimidine (126). A pale-yellow oil (1.85 g, 49.2%). ¹H

NMR (600 MHz, CDCl₃) δ 8.42−8.29 (m, 2H), 7.50−7.36 (m, 2H),

4.69 (t, J = 6.0 Hz, 2H), 3.62 (t, J = 6.4 Hz, 2H), 2.61 (s, 3H), 2.47−

2.33 (m, 2H). MS (ESI) m/z 377.2 (calcd 377.1 for C₁₄H₁₃BrCl₂N₂O

[M + H]⁺).

(600 MHz, CDCl₃) δ 8.44−8.31 (m, 2H), 7.19−7.01 (m, 2H), 4.57 (t,

J = 6.5 Hz, 2H), 2.58 (s, 3H), 2.55−2.48 (m, 2H), 2.47−2.28 (m, 4H),

2.09−2.02 (m, 2H), 1.65−1.54 (m, 4H), 1.51−1.38 (m, 2H). MS

(ESI) m/z 365.1 (calcd 364.9 for C₁₉H₂₃ClFN₃O [M + H]⁺).

5-Chloro-2-(3,4-diﬂuorophenyl)-4-methyl-6-(3-(piperidin-1-yl)-

1

propoxy)pyrimidine (136). A yellow oil (1.65 g, 86.6%). H NMR

(600 MHz, CDCl₃) δ 8.21−8.16 (m, 1H), 8.15−8.10 (m, 1H), 7.23−

7.16 (m, 1H), 4.55 (t, J = 6.5 Hz, 2H), 2.57 (s, 3H), 2.53−2.48 (m,

2H), 2.47−2.36 (m, 4H), 2.11−1.99 (m, 2H), 1.64−1.55 (m, 4H),

1.49−1.38 (m, 2H). MS (ESI) m/z 383.3 (calcd 382.8 for

C₁₉H₂₂ClF₂N₃O [M + H]⁺).

4-(3-Bromopropoxy)-5-chloro-2-(3,4-dichlorophenyl)-6-methyl-

1

pyrimidine (127). A pale-yellow oil (2.19 g, 53.3%). H NMR (600

5-Chloro-2-(4-chlorophenyl)-4-methyl-6-(3-(piperidin-1-yl)-

MHz, CDCl₃) δ 8.51−8.45 (m, 1H), 8.26−8.19 (m, 1H), 7.55−7.48

(m, 1H), 4.68 (t, J = 6.0 Hz, 2H), 3.63 (t, J = 6.4 Hz, 2H), 2.61 (s,

3H), 2.46−2.39 (m, 2H). MS (ESI) m/z 411.7 (calcd 411.5 for

C₁₄H₁₂BrCl₃N₂O [M + H]⁺).

1

propoxy)pyrimidine (137). A yellow oil (1.55 g, 81.6%). H NMR

(600 MHz, CDCl₃) δ 8.38−8.26 (m, 2H), 7.45−7.36 (m, 2H), 4.56 (t,

J = 6.5 Hz, 2H), 2.57 (s, 3H), 2.52−2.47 (m, 2H), 2.46−2.34 (m, 4H),

2.09−1.99 (m, 2H), 1.64−1.53 (m, 4H), 1.48−1.40 (m, 2H). MS

(ESI) m/z 381.4 (calcd 381.3 for C₁₉H₂₃Cl₂N₃O [M + H]⁺).

5-Chloro-2-(3,4-dichlorophenyl)-4-methyl-6-(3-(piperidin-1-yl)-

4-(3-Bromopropoxy)-5-chloro-6-methyl-2-(naphthalen-2-yl)-

1

pyrimidine (128). A pale-yellow oil (2.20 g, 56.2%). H NMR (600

MHz, CDCl₃) δ 8.92 (s, 1H), 8.53−8.42 (m, 1H), 8.03−7.95 (m, 1H),

7.93−7.89 (m, 1H), 7.88−7.84 (m, 1H), 7.57−7.47 (m, 2H), 4.75 (t, J

= 6.0 Hz, 2H), 3.65 (t, J = 6.4 Hz, 2H), 2.65 (s, 3H), 2.51−2.39 (m,

2H). MS (ESI) m/z 392.9 (calcd 392.7 for C₁₈H₁₆BrClN₂O [M +

H]⁺).

1

propoxy)pyrimidine (138). A yellow oil (1.81 g, 87.5%). H NMR

(600 MHz, CDCl₃) δ 8.45−8.43 (m, 1H), 8.21−8.17 (m, 1H), 7.51−

7.46 (m, 1H), 4.56 (t, J = 6.5 Hz, 2H), 2.58 (s, 3H), 2.55−2.49 (m,

2H), 2.48−2.36 (m, 4H), 2.10−2.02 (m, 2H), 1.65−1.54 (m, 4H),

1.51−1.40 (m, 2H). MS (ESI) m/z 416.3 (calcd 414.8 for

C₁₉H₂₂Cl₃N₃O [M + H]⁺).

4-(3-Bromopropoxy)-5-chloro-6-methylpyrimidine (129). A pale-

1

yellow oil (1.07 g, 40.2%). H NMR (600 MHz, CDCl₃) δ 8.33 (s,

5-Chloro-4-methyl-2-(naphthalen-2-yl)-6-(3-(piperidin-1-yl)-

1H), 4.24 (t, J = 6.0 Hz, 2H), 3.62 (t, J = 6.4 Hz, 2H), 2.48 (s, 3H),

2.34−2.25 (m, 2H). MS (ESI) m/z 267.1 (calcd 266.5 for

C₈H₁₀BrClN₂O [M + H]⁺).

1

propoxy)pyrimidine (139). A yellow oil (1.71 g, 86.4%). H NMR

(600 MHz, CDCl₃) δ 8.89 (s, 1H), 8.54−8.41 (m, 1H), 8.02−7.93 (m,

1H), 7.95−7.90 (m, 1H), 7.86−7.82 (m, 1H), 7.56−7.45 (m, 2H),

4.62 (t, J = 6.5 Hz, 2H), 2.62 (s, 3H), 2.58−2.51 (m, 2H), 2.50−2.28

(m, 4H), 2.13−2.04 (m, 2H), 1.64−1.56 (m, 4H), 1.50−1.41 (m, 2H).

MS (ESI) m/z 397.2 (calcd 396.9 for C₂₃H₂₆ClN₃O [M + H]⁺).

5-Chloro-4-methyl-6-(3-(piperidin-1-yl)propoxy)pyrimidine (140).

4-(3-Bromopropoxy)-5-chloro-2,6-dimethylpyrimidine (130). A

1

pale-yellow oil (1.20 g, 42.9%). H NMR (600 MHz, CDCl₃) δ 4.18

(t, J = 6.0 Hz, 2H), 3.51 (t, J = 6.1 Hz, 2H), 2.60 (s, 3H), 2.39 (s, 3H),

2.34−2.25 (m, 2H). MS (ESI) m/z 280.8 (calcd 280.6 for

C₉H₁₂BrClN₂O [M + H]⁺).

4-(3-Bromopropoxy)-5-chloro-2-cyclopropyl-6-methylpyrimidine

(131). A pale-yellow oil (1.35 g, 44.5%). ¹H NMR (600 MHz, CDCl₃)

δ 4.20 (t, J = 6.0 Hz, 2H), 3.55 (t, J = 6.2 Hz, 2H), 2.46 (s, 3H), 2.43−

2.32 (m, 2H), 1.76−1.70 (m, 1H), 1.26−1.20 (m, 2H), 1.15−1.10 (m,

2H). MS (ESI) m/z 306.9 (calcd 306.6 for C₁₁H₁₄BrClN₂O [M +

H]⁺).

5-Chloro-4-methyl-6-(3-(piperidin-1-yl)propoxy)-2-(p-tolyl)-

pyrimidine (132). A mixture of 121 (5 mmol) and piperidine (5.5

mmol) in acetonitrile (100 mL) and cesium carbonate (10 mmol) was

heated and reﬂuxed for 6−8 h. After ﬁltering, the resulting ﬁltrate was

evaporated to dryness under reduced pressure. The residue was

suspended in water (50 mL) and extracted with dichloromethane (3 ×

25 mL). The combined organic layers were dried with anhydrous

magnesium sulfate, the ﬁltrate evaporated under reduced pressure, and

the crude product puriﬁed by means of chromatography (10%

MeOH/CHCl₃) to yield 5-chloro-4-methyl-6-(3-(piperidin-1-yl)-

propoxy)-2-(p-tolyl)pyrimidine, 132, as a yellow oil (1.52 g, 84.8%).

¹H NMR (600 MHz, CDCl₃) δ 8.26 (d, J = 8.2 Hz, 2H), 7.24 (d, J =

1

A yellow oil (1.04 g, 77.3%). H NMR (600 MHz, CDCl₃) δ 8.53 (s,

1H), 4.28 (t, J = 6.5 Hz, 2H), 2.58 (s, 3H), 2.51−2.45 (m, 2H), 2.47−

2.34 (m, 4H), 2.08−1.99 (m, 2H), 1.62−1.55 (m, 4H), 1.47−1.40 (m,

2H). MS (ESI) m/z 271.1 (calcd 270.8 for C₁₃H₂₀ClN₃O [M + H]⁺).

5-Chloro-2,4-dimethyl-6-(3-(piperidin-1-yl)propoxy)pyrimidine

1

(141). A yellow oil (1.12 g, 79.6%). H NMR (600 MHz, CDCl₃) δ

4.22 (t, J = 6.5 Hz, 2H), 2.54 (s, 3H), 2.47−2.39 (m, 2H), 2.36−2.28

(m, 7H), 2.03−1.95 (m, 2H), 1.61−1.53 (m, 4H), 1.45−1.38 (m, 2H).

MS (ESI) m/z 284.8 (calcd 284.8 for C₁₄H₂₂ClN₃O [M + H]⁺).

5-Chloro-2-cyclopropyl-4-methyl-6-(3-(piperidin-1-yl)propoxy)-

1

pyrimidine (142). A yellow oil (1.24 g, 80.1%). H NMR (600 MHz,

CDCl₃) δ 4.31 (t, J = 6.5 Hz, 2H), 2.56 (s, 3H), 2.48−2.38 (m, 2H),

2.38−2.27 (m, 7H), 2.02−1.94 (m, 2H), 1.75−1.69 (m, 1H), 1.60−

1.53 (m, 4H), 1.44−1.36 (m, 2H), 1.25−1.19 (m, 2H), 1.16−1.10 (m,

2H). MS (ESI) m/z 310.5 (calcd 310.8 for C₁₆H₂₄ClN₃O [M + H]⁺).

In Vitro Binding Assays. Materials. The following speciﬁc

radioligands and tissue sources were used: (a) σ₁receptor, [³H]-

(+)-pentazocine (250 μCi, PerkinElmer, NET-1056250UC), and male

Dunkin Hartley guinea pig brain membrane; (b) σ₂receptor, [³H]-di-

o-tolylguanidine ([³H]-DTG, 250 μCi, PerkinElmer, NET-

986250UC), and male Dunkin Hartley guinea pig brain membrane.

Chemicals and reagents were purchased from diﬀerent commercial

sources and of analytical grade.

8.0 Hz, 2H), 4.57 (t, J = 6.5 Hz, 2H), 2.58 (s, 3H), 2.54−2.49 (m,

2H), 2.49−2.32 (m, 7H), 2.09−2.01 (m, 2H), 1.63−1.54 (m, 2H),

1.49−1.38 (m, 2H). MS (ESI) m/z 361.2 (calcd 360.9 for

C₂₀H₂₆ClN₃O [M + H]⁺).

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Membrane Preparation. Membrane preparation was performed by

previously reported method.⁶⁵Crude P2 membranes were prepared

from frozen guinea pig brains minus cerebellum. Tissues were

homogenized in ice-cold 10 mM Tris-sucrose buﬀer (0.32 mol

sucrose in 10 mM Tris-HCl, pH 7.4) using an ULTRA TURAX

homogenizer. The homogenates were centrifuged at 4 °C at 1000g for

10 min, and the supernatant was saved. The resulting pellet was then

resuspended in the same buﬀer, incubated for 10 min at 4 °C, and

centrifuged at 1000g for 10 min. After that, the pellet was discarded

and the supernatants were combined and centrifuging at 31000g for 15

min. The pellets were resuspended in 10 mL of Tris-HCl, pH 7.4, in a

volume of 3 mL/g, and the suspension was allowed to incubate at 25

°C for 30 min. Following centrifuging at 31000g for 15 min, the pellet

was resuspended by gentle homogenization in 10 mM Tris-HCl, pH

7.4, in a ﬁnal volume of 1.53 mL/g tissue, and aliquots were stored at

−80 °C until used. The protein concentration of the suspension was

determined by the method of Bradford.⁶⁶Subsequently, the

preparation contained 4 mg protein/mL.

General Procedures for the Binding Assays. All the test

compounds were prepared by dissolving in 5% DMSO. The ﬁlter

mats were presoaked in 0.5% polyethylenimine solution for 2 h at

room temperature before use. The following speciﬁc radioligands and

tissue sources were used: (a) σ₁receptor, [³H]-(+)-pentazocine,

guinea pig brain membranes; (b) σ₂receptor, [³H]-DTG, guinea pig

brain membranes.

The total binding (TB) was determined in the absence of

nonspeciﬁc binding and compounds while speciﬁc binding (SB) was

determined in the presence of compounds. Nonspeciﬁc binding (NB)

was determined as the diﬀerence between total and speciﬁc binding.

Nonspeciﬁc binding was determined in the presence of 10 μmol of

haloperidol. Percentage of inhibition (%) was calculated as the

following equation:

Tris-HCl buﬀer, pH 8.0. For nonspeciﬁc binding, to each assay tube

was added 850 μL of the tissue suspension, 50 μL of [³H]-DTG, 50 μL

of 400 nM (+)-SKF10047, and 50 μL of 10 μM DTG. Each speciﬁc

binding assay tube was added 850 μL of the tissue suspension, 50 μL

of [³H]-DTG, 50 μL of 400 nM (+)-SKF10047, and 50 μL of

reference drug or test compounds solution in various concentrations

(10⁻⁵M to 10⁻¹⁰M). The tubes were incubated at 25 °C for 120 min.

The incubation was followed by a rapid vacuum ﬁltration through

Whatman GF/B glass ﬁlters, and the ﬁltrates were washed twice with 5

mL of cold buﬀer and transferred to scintillation vials. Scintillation

ﬂuid (2.0 mL) was added, and the radioactivity bound was measured

using a Beckman LS 6500 liquid scintillation counter.

Functional Proﬁle on σ₁Receptor. The functional activity of

compound 137 was evaluated by using guinea pig brain membranes

binding assays for σ₁receptor. The binding aﬃnities either in the

absence or presence of 1 mM phenytoin were measured to identify the

functional (agonistic or antagonistic) nature.⁴⁸

hERG Aﬃnity. Ability to block hERG potassium channels was

determined using the whole-cell patch clamp method and cloned

hERG potassium channels (expressed in HEK 293 cells) as biological

material.⁴⁷For this purpose, the patch clamp ampliﬁer (Axopatch

200B, Molecular Devices) and digital converter (Digidata 1440A,

Molecular Devices) were used. Recording electrodes were made from

borosilicate glass with ﬁlament (BF120-94-15, Sutter Instrument

Company). Creation of voltage-clamp command pulse protocols and

data acquisition were controlled by pCLAMP software (version 10.1,

Molecular Devices).

The bath solution consisted of 137 mM NaCl, 5.4 mM KCl, 10 mM

glucose, 10 mM HEPES, and 2 mM CaCl₂. The pH was adjusted to

7.5 by addition of NaOH. The pipet ﬁlling solution consisted of 140

mM KCl, 1 mM MgCl₂, 5 mM EGTA, 10 mM HEPES, and 5 mM

Na₂ATP. The pH was adjusted to 7.2 by addition of KOH.

To study voltage dependence of steady-state block of hERG

channels on diﬀerent drug concentrations (0.3, 1, 3, and 10 μM) in

HEK cells, the holding membrane potential was switched from −80 to

+50 mV for 2 s following return to −50 mV for 3s (sampling rate of 4

kHz, low-pass ﬁltered at 1 kHz) in intervals of 30s. Tail currents were

measured at −50 mV in control and in the presence of the drug at

concentrations determined empirically.

percentage of inhibition(%) = (TB − SB)/(TB − NB) × 100%

Blank experiments were carried out to determine the eﬀect of 5%

DMSO on the binding, and no eﬀects were observed. Compounds

were tested at least three times over a six concentration range (10⁻⁵M

to 10⁻¹⁰M), and IC₅₀values were determined by nonlinear regression

analysis using Hill equation curve ﬁtting. K_ivalues were calculated

based on the Cheng and Prusoﬀ equation: K_i= IC₅₀/(1 + C/K_d). In

the equation, C represents the concentration of the hot ligand used

and K_dits receptor dissociation constant calculated for each labeled

ligand. Mean K_ivalues and SEM are reported for at least three

independent experiments.

All raw measurements were performed using Clampﬁt (version

10.2), a part of pCLAMP software (version 10.1). Results were

transferred to the program Statistical Package for the Social Sciences

(SPSS) spreadsheets for further analysis.

In Vivo Test. Animals. Chinese Kun Ming (KM) mice (20 2.0

σ₁Receptor Binding Assays. Binding of [³H]-(+)-pentazocine at σ₁

receptor was performed according to D. L. Dehaven-Hudkins et al.,⁵²

with minor modiﬁcations. The binding properties of the test

compounds to guinea pig σ₁receptor were studied in guinea pig

brain membranes using [³H]-(+)-pentazocine as the radioligand.

To each total binding assay tube was added 900 μL of the tissue

suspension, 50 μL of 4.0 nM [³H]-(+)-pentazocine, 50 μL of Tris-HCl

buﬀer, pH 8.0. Nonspeciﬁc binding each assay tube was added 900 μL

of the tissue suspension, 50 μL of [³H]-(+)-pentazocine, and 50 μL of

10 μM haloperidol. To each speciﬁc binding assay tube was added 900

μL of the tissue suspension, 50 μL of [³H]-(+)-pentazocine, and 50 μL

of reference drug or test compounds solution in various concentrations

(10⁻⁵M to 10⁻¹⁰M). The tubes were incubated at 25 °C for 180 min.

The incubation was followed by a rapid vacuum ﬁltration through

Whatman GF/B glass ﬁlters, and the ﬁltrates were washed twice with 5

mL of cold buﬀer and transferred to scintillation vials. Scintillation

ﬂuid (2.0 mL) was added, and the radioactivity bound was measured

using a Beckman LS 6500 liquid scintillation counter.

σ₂Receptor Binding Assays. Binding assays were performed as

described by Ronsisvalle et al.⁶⁷with minor modiﬁcations.⁴²The

binding properties of the test compounds to guinea pig σ₂receptor

were studied in guinea pig brain membranes using [³H]-DTG.

The membranes were incubated with 3 nM [³H]-DTG in the

presence of 400 nM (+)-SKF10047 to block σ₁sites. To each total

binding assay tube was added 850 μL of the tissue suspension, 50 μL

of 3.0 nM [³H]-DTG, 50 μL of 400 nM (+)-SKF10047, and 50 μL of

g) and Sprague−Dawley (SD) rats (250

5.0 g) were used as

experimental animals in this study. All the animals were housed under

standardized conditions for light, temperature, and humidity and

received standard rat chow and tap water and libitum. Animals were

assigned to diﬀerent experimental groups randomly, each kept in a

separate cage. All research involving animals in this study follow the

guidelines of the Bylaws of Experiments on Animals and have been

approved by the Ethics and Experimental Animal Committee of

Jiangsu Nhwa Pharmaceutical Co., Ltd.

Acute Toxicity. Mice (10 mice for each group) were orally dosed

with po administration of a 10 mL/kg volume of vehicle 0.5%

methylcellulose (Sigma-Aldrich) or increasing dose of test compounds

(200, 500, 1000, 1500, and 2000 mg/kg). The number of surviving

animals was recorded after 24 h of drug administration. The percent

mortality in each group was calculated. The LD₅₀values were

calculated by using Statistical Package for Social Sciences (SPSS)

program.

Formalin Test.⁴⁴Mice were randomly separated into diﬀerent

groups: vehicle, vehicle + formalin, and drug-treated group, and each

group contained 10 mice. A diluted formalin solution (20 μL of a 2.5%

formalin solution, 0.92% formaldehyde) was injected into the dorsal

surface of the right hind paw of the mouse. Immediately thereafter, the

mouse was put into a glass cylinder and the observation period started.

A mirror was placed behind the glass cylinder to allow clear

observation of the paws. Nociceptive behavior induced by formalin

was quantiﬁed as the time spent licking or biting the injected paw

Q

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Journal of Medicinal Chemistry

Article

during two diﬀerent periods individually recorded: the ﬁrst period was

recorded 0−5 min after the injection of formalin and was considered

indicative of formalin-evoked nociception phase I; the second period

was recorded 15−45 min after formalin injection and was considered

indicative of formalin-evoked nociception phase II.⁴⁴The mice (n =

10−12 per group) received ip administration of a 10 mL/kg volume of

vehicle 30% PEG 400 (Sigma-Aldrich) or test compound 15 min

before intraplantar (ipl) formalin injection. The antinociceptive eﬀect

induced by the diﬀerent treatments in the formalin test was calculated

by the following formula: antinociceptive eﬀect (%) = (LT_V− LT_D)/

LT_V× 100%, where LT_Vrepresents the licking time in vehicle injected

animals and LT_Dmeans licking time in drug-injected animals.

CCI Model.^50,51The CCI of the sciatic nerve as an animal model

was used to study the eﬃcacy of treatments in neuropathic pain.

Rats were randomly separated into several groups: sham control and

vehicle- and drug-treated groups, and each group contained 10 rats.

Pain threshold base values of each group were measured 1−2 days

before surgery and those with the value of 2 days were picked. The

pain thresholds were measured again 14 days after surgery to check

whether the model was successful. Drugs were dose orally with po

administration of a 10 mL/kg volume of vehicle 0.5% methylcellulose

(Sigma-Aldrich) or increasing dose of test compounds twice a day for

4 days (15th, 16th, 17th, and 18th day). Each group was measured

after ﬁrst administration on the 15th day and the last administration on

the 18th day, which result in the single administration and repeated

administration, respectively.

(Agilent, USA); mobile phase, 30 mmol of NH₄COOH (Roe Scientiﬁc

Inc., USA) aq/acetonitrile (Merck Company, Germany) 30/70; ﬂow

rate, 1.0 mL/min; column temperature, 40 °C.

For routine compound 137 screening, rats (n = 8/group) were

dosed via the lateral tail vein at the indicated dose for iv administration

(16 mg/kg, 100% saline) or via oral gavage (320 mg/kg, suspension in

0.5% methylcellulose). At 30 min, 1, 2, 3, 4, 5, 6, 7, and 24 h after

administration, serial blood samples were collected from the lateral tail

vein into heparinized collection tubes (approximately 0.25 mL). The

plasma was separated by centrifugation, and the sample was prepared

for HPLC/MS analysis by protein precipitation with acetonitrile. The

plasma samples were analyzed for drug and internal standard via the

HPLC-MS/MS protocol.

ASSOCIATED CONTENT

* Supporting Information

■

S

Details of computational procedures, selectivity of compound

137 for additional receptors and ion channels, inhibition curves

ﬁgure of functional proﬁle of σ₁receptor, ¹³C NMR and HR-

MS of partially new compounds, and ¹H NMR, ¹³C NMR, HR-

MS, LC-MS, and elemental analyses of compound 137. This

material is available free of charge via the Internet at http://

pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Phone: +86-27-87792235. Fax: +86-27-87792170. E-mail:

gszhang@mail.hust.edu.cn.

■

The CCI of the sciatic nerve surgery was performed as described by

Bennett and Xie⁵⁰with minor modiﬁcation. Brieﬂy, the SD rats were

anesthetized with chloral hydrate (5%) and the right common sciatic

nerve was exposed at the level of the midthigh of the right hind paw.

At about 1 cm proximal to the nerve trifurcation, about 7 mm of the

nerve was freed and four ligatures (4−0 silk tread) were tight loosely

with a distance of ca. 1.0 mm. The nerve was only barely constricted.

The muscle was than stitched and the skin incision closed with wound

clips. The rats with sciatic nerve exposure without ligation served as

the sham control group.

Author Contributions

^§Y.L. and Y.C. contributed equally to this work;

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

Two tests were performed: the von Frey test and the plantar test.

Allodynia to mechanical stimuli and hyperalgesia to noxious thermal

stimulus were used as outcome measures of neuropathic pain by using

the von Frey and plantar tests, respectively.

■

We thank Prof. Liangren Zhang, Zhenming Liu, and Hongwei

Jin of the State Key Laboratory of Natural and Biomimetic

Drugs, School of Pharmaceutical Sciences, Peking University,

for guiding computational works of 3D Pharmacophore Model

Generation, molecular design, and performing HR-MS and

elemental analyses for partially new compounds.

In the von Frey test, brieﬂy, animals were placed in a transparent

test chamber with a wire-mesh grid ﬂoor through which von Frey

monoﬁlaments were applied. The monoﬁlaments were applied in

increasing force until the rats withdrew the ipsilateral, nerve injury paw

using an up−down paradigm. Clear paw withdrawal, sharking, or

licking was considered as nociceptive-lick response to determine the

mechanical withdrawal threshold (MWT). Animals were adapted to

the testing situation for at least 30 min before the sessions started. For

each measurement, the paw was sampled four times and a mean

calculated. At least 3 min elapsed between.

Thermal hyperalgesia was assessed through the plantar test by

measuring hind paw latency in response to radiant heat. Brieﬂy, rats

were placed in a clear plastic chamber with a glass ﬂoor and allowed to

acclimate to the environment for 30 min. A radiant heat source (BMC-

410A) was then positioned under the plantar surface of the hind paw.

The latency for the withdrawal reﬂex was recorded as thermal

withdraw latency (TWL). A cutoﬀ time of 30 s was imposed to

prevent tissue damage in the absence of response. For each

measurement, the paw was sampled four times and a mean calculated.

At least 3 min elapsed between.

ABBREVIATIONS USED

■

σ₁R, sigma-1 receptor; σ₂R, sigma-2 receptor; CNS, central

nervous system; NMDA, N-methyl-^D-aspartate; 3D, three-

dimensional; hERG, ether-a-go-go-related gene; CCI, chronic

constriction injury; ED₅₀, 50% eﬀective dose; LD₅₀, median

lethal dose

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