Synthesis and Biological Evaluation of Novel σ1 Receptor Ligands for Treating Neuropathic Pain: 6-Hydroxypyridazinones

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Synthesis and Biological Evaluation of Novel σ₁Receptor Ligands for

Treating Neuropathic Pain: 6‑Hydroxypyridazinones

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

Song Zhao,^‡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: By use of the 6-hydroxypyridazinone frame-

work, a new series of potent σ₁receptor ligands associated with

pharmacological antineuropathic pain activity was synthesized

and is described in this article. In vitro receptor binding studies

revealed high σ₁receptor aﬃnity (K_iσ₁= 1.4 nM) and

excellent selectivity over not only σ₂receptor (1366-fold) but

also other CNS targets (adrenergic, μ-opioid, sertonerigic

receptors, etc.) for 2-(3,4-dichlorophenyl)-6-(3-(piperidin-1-

yl)propoxy)pyridazin-3(2H)-one (compound 54). Compound

54 exhibited dose-dependent antiallodynic properties in mouse

formalin model and rats chronic constriction injury (CCI)

model of neuropathic pain. In addition, functional activity of compound 54 was evaluated using phenytoin and indicated that the

compound was a σ₁receptor antagonist. Moreover, no motor impairments were found in rotarod tests at antiallodynic doses and

no sedative side eﬀect was evident in locomotor activity tests. Last but not least, good safety and favorable pharmacokinetic

properties were also noted. These proﬁles suggest that compound 54 may be a member of a novel class of candidate drugs for

treatment of neuropathic pain.

carry signiﬁcant risk of addiction.^10,11Thus, the new

antineuropathic pain agents with novel mechanisms of action

that improved the eﬃcacy of existing therapies and reduced the

adverse eﬀects are wanted.

INTRODUCTION

■

Neuropathic pain is serious and chronic and thus greatly

impacts quality of life, which can be relieved but cannot be

cured.¹Its exact incidence and prevalence are unknown, but

Pharmacological studies and biochemical analyses have

overall prevalence is conservatively estimated at 3−8% of the

shown that σ receptors represent a unique, non-opioid, non-

general population in developed countries.^2,3Unfortunately, all

phencyclidine, haloperidol-sensitive receptor family.^12,13Two

currently available treatments aﬀord only modest alleviation of

pain and minimal improvements in physical and emotional

distinct subtypes, termed the σ₁and σ₂receptors, have been

characterized. The σ₁receptor has been cloned from several

functioning.⁴Although very intensive research eﬀorts have

species and various tissues; the protein is 223 amino acids long

improved our understanding of the mechanisms that cause

and has a molecular weight of 25.3 kDa.^14,15Functionally, σ₁

pain, those eﬀorts have not yet yielded new antineuropathic

pain drugs.⁵In the absence of any real breakthrough in

receptor interacts physically with several receptors and ion

channels, or elements of their transduction machineries, to

antiallodynic drugs development, current guidelines for the

modulate receptor/ion channel activities.¹⁶The receptor

management of neuropathic pain feature only incremental

behaves as a unique ligand-regulated molecular chaperone

improvements in existing therapies, including combination

modulating the activities of various proteins and ion channels,

treatments, new formulations of existing drugs, me-too drugs,

including the K⁺and Ca²⁺channels, and the N-methyl-D-

and reﬁnements based on supposed drug mechanisms of

action.⁶⁻⁹Among these, tricyclic antidepressants, dual

aspartate (NMDAR) and opioid receptors, all of which are

involved in pain.¹⁷⁻²¹σ₁receptor is expressed in regions

serotonin−norepinephrine reuptake inhibitors (duloxetine),

important in terms of pain control; these include dorsal root

calcium channel α₂-δ ligands (gabapentin and prebagalin),

ganglion neurons, the dorsal spinal cord, the periaqueductal

and topical lidocaine are recommended as ﬁrst-line therapy for

gray matter, and the rostroventral medulla.¹⁶Receptor

expression in the spinal cord is upregulated during the

neuropathic pain.⁷⁻⁹Opioid antiallodynic drugs are typically

recommended for patients who have not responded to ﬁrst-line

medications.^10,11Although these medications are eﬀective in

reducing pain in neuropathic pain disorders, they may cause

serious side eﬀects such as weight gain and hyperalgesia and

Received: September 12, 2015

A

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induction phase of neuropathic pain.^22,23The pharmacological

proﬁles of σ₁receptor and σ₂receptor diﬀer, and both proteins

may serve as biomarkers of tumor cell proliferation and

proapoptotic tendencies.²⁴The proteins may thus play roles in

cancer imaging and treatment.^25,26

To date, mounting evidence indicates that targeting σ

receptors may be particularly beneﬁcial in a number of

neurodegenerative conditions including Alzheimer’s disease,

Parkinson’s disease, stroke, methamphetamine neurotoxicity,

Huntington’s disease, amyotrophic lateral sclerosis, and retinal

degeneration.²⁷The putative σ₁receptor antagonist, SN79 (6-

acetyl-3-(4-(4-(4-ﬂuorophenyl)piperazin-1-yl)butyl)benzo[d]-

oxazol-2(3H)-one), can attenuate psychostimulant eﬀects.²⁸

The selective σ receptor ligand, AC927 (1-(2-phenethyl)-

piperidine oxalate), can block several cocaine eﬀects that are

related to its abuse and excessive intake.²⁹Various 2(3H)-

benzoxazolones and 2(3H)-benzothiazolones have been

reported as highly selective σ₁receptor ligands, and ¹⁸F-labeled

2(3H)-benzothiazolones were developed as potential positron

emission tomography (PET) candidate radioligands for

imaging σ receptors in the central nervous system (CNS).³⁰

In addition, many selective σ₁receptor ligands have been tested

as treatments for CNS disorders in clinical studies.³¹⁻³⁵

Moreover, based on the recent preclinical data, selective σ₁

receptor antagonists may be both eﬃcacious and safe when

used to counter diﬃcult-to-treat chronic pain (including

neuropathic pain) and to enhance (or maintain) antiallodynic

eﬃcacy and increase the safety margins of opioid doses.³⁶BD-

1063 (1) is a typical σ₁receptor antagonist, inhibiting

mechanical allodynia induced by capsaicin.^37,38The recently

described σ₁receptor antagonists, S1RA (2), have good safety,

tolerability, and pharmacokinetic proﬁles and are currently in

phase II clinical trials for treatment of neuropathic pain.^39,40

Recently, a series of 4-aminotriazoles (3) exhibiting nanomolar

potency for σ₁receptor and good selectivity (compared to σ₂

receptor) has been reported⁴¹(Figure 1).

Figure 2. Glennon’s pharmacophoric σ₁receptor model (A) and

general structure of the new pyridazinone derivative (B).

exhibited a good pharmacophoric proﬁle, binding to σ₁

receptor, implicated in pain.⁴³

Pyridazine and pyrimidine are isomeric compounds in which

two nitrogen atoms diﬀer in position. Pyridazine and its

oxygenated derivative pyridazinone contain well-studied six-

membered heterocyclic rings. These compounds exhibit various

pharmacological activities and are present in many natural

sources.^46,47The compounds based on pyridazine or

pyridazinone scaﬀold have recently been shown to increase

CNS activity. Speciﬁcally, selective histamine H₃receptor

ligands contained the pyridazinones were reported as lead

candidates for potential use in the treatment of chronic pain

and cognitive disorders.⁴⁷A novel series of phosphodiesterase

10A inhibitors based on pyridazine or pyridazinone rings were

reported as clinical candidate for the treatment of schizo-

phrenia.⁴⁸To further explore novel σ₁ligands, we focused on

ring bioisosteres of the pyrimidine moiety.⁴⁹We introduced the

pyridazinone ring 6-hydroxy-2-phenylpyridazinone, combined

with the basic amino moiety, by molecular hybridization⁵⁰to

form a new series of ligands (5) with high aﬃnity to the σ₁

receptor and selectivity over the σ₂receptor subtype (Figure 3).

Figure 1. Representative σ₁receptor ligands.

Figure 3. Design of new pyridazinone derivatives.

Our interest has focused on the development of novel ligands

with high aﬃnity to the σ₁receptor and selectivity over the σ₂

receptor subtype.^42,43Recently, the pharmacophoric model of

Glennon et al.,^44,45(Figure 2A) was used to build a 3D

pharmacophore model allowing virtual drug screening. Hit

analysis showed that a pyrimidine scaﬀold was important, and

we prepared a series of such compounds. Compound 4

This strategy yielded a series of new compounds (Tables

1−5), which were used in SAR (structure−activity relationship)

studies to evaluate their pharmacological eﬃcacy and in

competitive receptor-binding assays to determine relative

aﬃnity for σ₁and σ₂receptors. Among the derivatives prepared,

compound 54 exhibited high aﬃnity for the σ₁receptor and

low aﬃnity for the σ₂receptor. Further, compound 54 exerted

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Table 1. Binding Aﬃnities for the σ₁and σ₂Receptor of Compounds 12−23

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 σ₁receptors. The values are the mean SEM of three experiments performed in duplicate.

dose-dependent antiallodynic eﬀects in the mouse formalin-

induced pain model and the rats CCI model. In addition,

function activity of compound 54 was evaluated using

phenytoin and indicated that the compound was a σ₁receptor

antagonist. Moreover, no motor impairments were found in

rotarod tests at antiallodynic doses and no sedative side eﬀects

were evident in locomotor activity tests, and the pharmacoki-

netic properties were favorable. Thus, compound 54 is a novel

compound with great potential for the treatment of neuropathic

pain.

using two alternative procedures. The key intermediate 6-

hydroxy-2-phenylpyridazin-3(2H)-one 8 was obtained via one-

step cyclization with phenylhydrazine hydrochloride 6 and

maleic anhydride 7. According to the substitution pattern, the

ﬁnal compounds 9 and 12−26 were prepared using method A

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

method A, 8 was reacted with commercially available 1-(2-

chloroethyl)piperidine under basic conditions to obtain

compound 9 in high yield. In method B, the intermediates

10 and 11 were produced by the alkylation with 8 and 1, n-

dibromoalkanes, and then reacted with the corresponding

amines under mild basic conditions to aﬀord the ﬁnal

compounds 12−26.

CHEMISTRY

■

Compounds were synthesized according to the reaction

pathways illustrated in Schemes 1−3. As depicted in Scheme

1, phenylaminoalkoxypyridazinone derivatives were prepared

The route depicted in Scheme 2 was used to prepare

substituted pyridazinone scaﬀold compounds 50−60 and 62−

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a

Scheme 1

a

Reagents and conditions: (I) H₂O, conc HCl, reﬂux; (II) Br(CH₂)_n+1Br, K₂CO₃, acetone, reﬂux; (III) HNRR, K₂CO₃, KI, acetonitrile, reﬂux; (IV)

K₂CO₃, KI, acetonitrile, reﬂux.

Table 2. Binding Aﬃnities for the σ₁and σ₂Receptor of Compounds 9, 13, 14, and 24−26

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 σ₁receptors. The values are the mean SEM of three experiments performed in duplicate.

72. The intermediates 27−38 were obtained using the

cyclization procedure with substituted phenylhydrazine hydro-

chloride, which was reacted with maleic anhydride 7, depending

on the substitution pattern of the ﬁnal pyridazinone. Next,

standard alkylation with 1,3-dibromopropane was conducted to

produce 39−49, and then reaction with the piperidine or 4-

methylpiperidine aﬀorded the ﬁnal compounds 50−60 and

62−72, respectively, in moderate yields (Tables 3 and 4).

Compound 61 was obtained by reaction with commercially

available 1-(3-chloropropyl)piperidine, and intermediate 38,

under basic conditions (Table 3).

The route depicted in Scheme 3 was used to prepare the

substituted pyridazinone scaﬀold compounds 79−81. Inter-

mediates 73−75 were obtained by cyclization of 3,4-

dichlorophenylhydrazine hydrochloride with various substi-

tuted maleic anhydrides, depending on the substitution patterns

desired in the ﬁnal pyridazinones. Next, standard alkylation

with 1,3-dibromopropane produced compounds 76−78, and

reaction with piperidine aﬀorded the ﬁnal compounds 79−81,

respectively, in moderate yields (Table 5).

Indeed, the 1-phenyl-1,2-dihydropyridazine-3,6-dione and 6-

hydroxy-2-phenylpyridazin-3(2H)one are ambident tautomeric

D

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a

Scheme 2

a

Reagents and conditions: (I) H₂O, conc HCl, reﬂux; (II) Br(CH₂)₃Br, K₂CO₃, acetone, reﬂux; (III) piperidine, K₂CO₃, KI, acetonitrile, reﬂux; (IV)

4-methylpiperidine, K₂CO₃, KI, acetonitrile, reﬂux; (V) K₂CO₃, KI, acetonitrile, reﬂux.

Table 3. Binding Aﬃnities for the σ₁and σ₂Receptor of Compounds 13 and 50−61

a

b

compd

R₅

K_iσ₁(nM)

K_iσ₂(nM)

selectivity (σ₂/σ₁)

c

13

50

51

52

53

54

55

56

57

58

59

60

61

phenyl

21.4 2.5

69.0 7.2

74.1 8.9

17.0 2.0

9.8 1.4

1.4 0.1

48 2.1

30 1.2

25 2.1

45 2.3

10 1.3

1074 96

50.2

18.6

12.1

−

160.5

1365.7

33.6

43.3

32.3

20.0

158.1

238.5

−

4-methylphenyl

2-naphthyl

1280 120

895 95

>2000

4-ﬂuorophenyl

4-chlorophenyl

1573 127

1912 210

1617 184

1301 89

809 15

902 10

1581 120

1908 130

>2000

3,4-dichlorophenyl

2,3-dichlorophenyl

3,5-dichlorophenyl

2,4-dichlorophenyl

2,5-dichlorophenyl

3-chloro-4-ﬂuorophenyl

3,4-diﬂuorophenyl

cyclopentyl

8

1.9

>2000

a

b

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

presence of (+)-SKF-10047 to block σ₁receptors. The values are the mean SEM of three experiments performed in duplicate.

c

heterocyclic. They are produced via a cyclization of the

phenylhydrazine hydrochloride 6 and maleic anhydride 7 in the

presence of acetic acid as solvent.⁵¹In this paper, 6-hydroxy-2-

phenylpyridazin-3(2H)-one (8) was prepared in solution of

water and hydrochloric acid. When the tautomeric pyridazi-

nones were reacted with compound 1, n-dibromoalkane,

chemoselectivity may be in play. The N-alkylation products

were obtained when 1-phenyl-1,2-dihydropyridazine-3,6-dione

was reacted with 1, n-dibromoalkane in the presence of

acetonitrile with K₂CO₃.^52,53While acetonitrile was replaced

with acetone, 6-hydroxy-2-phenylpyridazin-3(2H)one was

treated with 1, n-dibromoalkane, and the O-alkylation product

may be obtained mainly. To determine the absolute

conﬁguration, the most active compound 54 (Table 3) was

subjected to single crystal X-ray diﬀraction (SCXRD). As no

suitable crystals were obtained using the free base, limited salt

screening was performed. The hydrochloride yielded crystals

suitable for SCXRD, by which a relative conﬁguration was

reliably determined. As shown in Figure 4, this conﬁrmed the

selectivity assigned above. The result indicates indirectly that 6-

hydroxy-2-phenylpyridazin-3(2H)-one was prepared in solution

of water and hydrochloric acid and the O-alkylation product

was obtained mainly during the alkylation.

RESULTS AND DISCUSSION

■

Structure−Activity Relationships. The new series of

pyridazinone derivatives was designed to exploit the known

pharmacophoric features of σ₁receptor ligands. As recently

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Table 4. Binding Aﬃnities for the σ₁and σ₂Receptor of Compounds 14 and 62−72

a

b

compd

R₅

K_iσ₁(nM)

K_iσ₂(nM)

selectivity (σ₂/σ₁)

c

14

62

63

64

65

66

67

68

69

70

71

72

phenyl

28.0 3.4

61.6 65

84.1 9.1

19.8 2.4

9.5 1.2

5.0 0.6

55 2.3

43 1.4

35 2.6

60 3.4

10 1.4

1464 101

1549 135

913 10.4

1619 147

1783 210

1742 198

1201 34

1321 57

1100 23

870 15

52.3

25.1

4-methylphenyl

2-naphthyl

10.8

4-ﬂuorophenyl

81.7

4-chlorophenyl

187.7

348.4

21.8

3,4-dichlorophenyl

2,3-dichlorophenyl

3,5-dichlorophenyl

2,4-dichlorophenyl

2,5-dichlorophenyl

3-chloro-4-ﬂuorophenyl

3,4-diﬂuorophenyl

30.7

31.4

14.5

1627 36

1701 45

162.7

15 1.2

113.4

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 σ₁receptors. The values are the mean SEM of three experiments performed in duplicate.

a

Scheme 3

a

Reagents and conditions: (I) H₂O, conc HCl, reﬂux; (II) Br(CH₂)₃Br, K₂CO₃, acetone, reﬂux; (III) piperidine, K₂CO₃, KI, acetonitrile, reﬂux.

summarized by Glennon et al., σ₁receptor ligands should

contain a basic amino group and no fewer than two

hydrophobic regions at speciﬁed distances from the amino

group, thus 2.5−3.9 and 6−10 Å.^44,45(Figure 2A). Compounds

of this general structure (Figure 2B) were designed in which

the pyridazinones served as scaﬀolds bearing various groups

meeting the pharmacophoric requirements; certain variations

were also included. In detail, the chemical structures of these

pyridazinone derivatives were designed according to the

following rationale: we tested various basic amino moieties to

ﬁt the amine site and secondary hydrophobic region (NR1R2),

changed the distance between the pyridazinone ring and basic

amino moiety (carbon chain linker) to meet the pharmaco-

phoric requirements, replaced the 2-phenyl goup of pyridazi-

none by an aromatic ring or aliphatic ring (R5), and took

diﬀerent substitution on the 4- and 5-positions of the

F

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Figure 4. ORTEP showing the molecular structure of the hydrochloride salt of 54.

pyridazinone ring (R3 and R4) to ﬁnd the best conformational

ﬁtting to the receptor.

nM). Open-chain amines (21−23) did not enhance binding

aﬃnity or σ₁receptor selectivity.

The new compounds were ﬁrst evaluated in vitro by

determining their binding aﬃnities for the σ₁and σ₂receptors.

[³H](+)-Pentazocine was used determining binding at σ₁

receptor. Aﬃnity at σ₂receptor was determined using [³H]di-

o-tolylguanidine in the presence of (+)-SKF-10047 (to block

the σ₁receptor), respectively.^36,54The results of these primary

assays are shown in Tables 1−5.

It is consistent with the previous work that the nature of the

surrounding of the tertiary amine nitrogen had a profound

impact on aﬃnity.^30c

Eﬀect of the Distance between the Pyridazinone Ring and

the Basic Amino Moiety. The eﬀect of the length of the linker

between the pyridazinone and the basic amino moiety was

determined. As shown in Table 2, changes in linker length to

two (9) or four (24−26) carbon atoms signiﬁcantly reduced

binding to both the σ₁and σ₂receptors. Therefore, the binding

aﬃnities for both receptors were dependent upon chain length.

Compared to compound 13, the use of a linker containing two

carbon atoms to connect to the piperidine ring (9) inactivated

binding to both receptors; replacement of the linker with four

carbon atoms (24) reduced the binding aﬃnity for σ₁. When

substituted piperidine rings were used, compounds 25 and 26,

with linkers of four carbon atoms, had low σ₁receptor and σ₂

receptor aﬃnities. Thus, the alkyl chain length appeared to

directly aﬀect the aﬃnities for the two receptors. Together, the

data show that a linker of three carbon atoms (compounds 13

and 14) was optimal. It is consistent with the previous work

that three carbons showed high aﬃnity to the σ₁receptor and

excellent selectivity to σ₂receptor.^42,43

Eﬀect of Substitution on the 2-Position of the

Pyridazinone Ring. Compounds exhibiting good binding

aﬃnities and selectivities (13 and 14), containing piperidinyl

or 4-methylpiperidinyl groups as amino moieties with three-

carbon linker, were selected for further SAR investigation. The

eﬀects of replacing the phenyl group at the 2-position of the

pyridazinone ring with other aromatic or aliphatic rings were

explored (Tables 3 and 4).

When the amine moiety was piperidine (Table 3),

compound 50 containing electron-donating group (methyl)

on the phenyl group retained moderate activity (albeit less than

compound 13) and exhibited lower selectivity. Compound 51

was produced by replacing the phenyl with a naphthyl ring,

which did not improve the binding aﬃnity or selectivity.

Compounds 52−54 contained halogens substituent on the

phenyl group, remarkably increasing binding aﬃnity and

selectivity; the ﬂuorine-containing compound’s (52) constants

were K_iσ₁= 17.0 2.0 nM and K_iσ₂> 2000 nM, while those

for the chlorine-bearing molecule (53) were K_iσ₁= 9.8 1.4

nM and K_iσ₂= 1573 127 nM. Next, the 3,4-dichlorophenyl

Eﬀect of the Basic Amino Moiety for Diﬀerent Amine

Moieties. In our previous work, the σ₁receptor ligands

(pyrimidines derivatives and 3,4-dihydroxy-2(1H)-quinolinone

derivatives) designed were consistent with the known

pharmacophoric features summarized by Glennon et al.

Along with full explorations of SAR, the optimal chain length

between the pyrimidine ring (or 3,4-dihydroxy-2(1H)-quinoli-

none scaﬀold) and the basic amino moiety for high aﬃnity to

the σ₁receptor was determined to be three carbons.^42,43

In this work, our initial investigation focused on the eﬀect of

various basic amino moieties (Table 1, compounds 12−23).

First, the morpholine group (12) was used because of its

frequent appearance in σ₁receptor ligands, such as 2, yielding

moderate aﬃnity (K_iσ₁= 66.3 6.0 nM and K_iσ₂= 502

49.2 nM) to both receptors. Next, the improvements of

compounds with regard to hydrophobicity over a morpholino

group, piperidine, 4-methylpiperidine, 3,5-dimethylpiperidine

(13−15) were selected, resulting in compound 13, which

showed signiﬁcantly improved σ₁receptor binding aﬃnity and

selectivity (K_iσ₁= 21.4 2.5 nM and K_iσ₂= 1074 96 nM).

Increasing the hydrophobicity of piperidinyl by adding a methyl

group on the ring (14) led to similar binding aﬃnity (K_iσ₁=

28.0 3.4 nM and K_iσ₂= 1464 101 nM) to compound 13,

as was observed for compound (15), bearing 3,5-dimethylpi-

peridine (K_iσ₁= 46.0 4.1 nM and K_iσ₂= 1822 148 nM).

Substituting the oxygen atom of the morpholine with a

carbonyl group (16) greatly decreased aﬃnity to the σ₁

receptor.

When piperazine was substituted with small alkyl groups

(methyl or ethyl), the resulting compounds (17, 18) bound

with high aﬃnity to the σ₁receptor but showed weak selectivity

over the σ₂receptor. The same activity was observed in the

larger diazepane or smaller pyrolidine derivative, compound 19

(K_iσ₁= 30.3

2.8 nM and K_iσ₂= 508

54 nM), and

compound 20 (K_iσ₁= 35.5 3.8 nM and K_iσ₂= 458 54

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Table 5. Binding Aﬃnities for the σ₁and σ₂Receptor of Compounds 54 and 79−81

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 σ₁receptors. The values are the mean SEM of three experiments performed in duplicate.

group [present in the known σ₁receptor ligands 1 and 3] was

introduced, and compound 54 had even higher aﬃnity to σ₁

and excellent selectivity over σ₂(K_i= 1.4 0.1 nM and K_iσ₂=

1912 210 nM). Substituting other dichlorophenyls for the

3,4-dichlorophenyl group yielded compounds 55−58, which

showed lower activity than 54 but with moderate selectivities.

Some activity (albeit less than that of compound 54) was

retained upon replacement of the 3,4-dichloro moiety with 3-

chloro-4-ﬂuoro or 3,4-diﬂuoro substituents (59, 60); this

improved selectivity about 4- to 10-fold compared to

compounds 55−58. Replacing the aromatic rings at the 2-

position with cyclopentyl 61 resulted in a marked reduction in

the aﬃnities for the receptors.

Compounds bearing the amine moiety 4-methylpiperidine

(Table 4), 62 with a methyl on the phenyl group and 63 with a

naphthyl ring, displayed low aﬃnities for the σ₁receptor. Again,

introduction of halogens on the phenyl group remarkably

increased binding aﬃnity and selectivity: for ﬂuorine (64),

constants were K_iσ₁= 19.8 2.4 nM and K_iσ₂= 1619 147

nM, for chlorine (65), K_iσ₁= 9.5 1.2 nM and K_iσ₂= 1783

210 nM, and for the 3,4-dichloro (66), K_iσ₁= 5.0 0.6 nM

linker optimally matched the pharmacophoric requirement.

Thus, compound 54 was selected for further investigation of

the SAR.

We ﬁrst explored the eﬀects of replacing the hydrogen atom

with a methyl group (Table 5, compound 79). Compound 79

(compared to 54) for σ₁and σ₂receptors had a detrimental

eﬀect on aﬃnities, attributable either to the size of the group or

to its hydrophobicity. Substitution of the methyl group with a

cyclohexyl (80) or phenyl (81) group signiﬁcantly decreased

receptor aﬃnities even further; large group sizes may have

hindered receptor binding.

Overall, compounds 53, 54, 59, 60, 65, 66, 71, and 72

exhibited aﬃnity for the σ₁receptor (K_i< 15 nM) with

selectivity (compared to σ₂receptor; >100-fold). Therefore,

these compounds were selected for further studies.

Human Ether-a-Go-Go-Related Gene (hERG) Channel

Blockade. Recent studies have shown that many medications

can prolong the time between the start of the Q wave and the

end of the T wave as shown on an electrocardiogram (the QT

interval).⁵⁵Indeed, eﬀorts to predict the risk of long-QT

syndrome have focused on assays that explore hERG channel

activity.^55aThis is because hERG channel blockade is an

important indicator of potential proarrhythmic risk.^55bTo date,

work on hERG channel blockade has been a signiﬁcant

component of drug discovery within the pharmaceutical

industry. The eight compounds with K_iσ₁values of <15 nM

and selectivities of >100-fold were evaluated the ability to block

hERG through electrophysiological patch-clamp measurements.

As shown in Table 6, compounds 54, 71, and 72 exhibited

and K_iσ₂= 1742

198 nM. Introduction of other

dihalophenyl groups yielded compounds 67−72, which

behaved as did the analogs that contained piperidinyl ring

(55−60), conﬁrming the important contribution of the 3,4-

dichlorophenyl group to σ receptor aﬃnity.^36,39,41

Eﬀect of Replacing Groups on the 4- and 5-Positions of

the Pyridazinone Ring. The presence of a 3,4-dichlorophenyl

group on the 2-position of the pyridazinone ring aﬀorded the

best receptor-binding aﬃnity and selectivity, and a three-carbon

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lower aﬃnities for hERG than did the others (IC₅₀> 2000

nM).

Intraplantar (ipl) injection of formalin elicits a biphasic pain

response: early (phase I) and delayed (phase II), characterized

by paw licking, biting, and other behaviors. Phase I pain is

predominantly caused by direct activation of C-ﬁbers, whereas

phase II pain appears to be dependent on a combination of

peripheral tissue inﬂammation and functional changes in the

spinal cord, involving both peripheral and central sensitiza-

tion.^60,61

In the formalin test, pretreatment with compound 54 (80

mg/kg, ig) inhibited formalin-induced pain responses to a

similar degree as 2, reducing licking and biting times in phase II

to 45.88 3.54 s and 41.16 5.21 s, respectively. In contrast,

the antiallodynic eﬀects of compounds 71 and 72 attained

statistical signiﬁcance only when much higher doses (160 mg/

kg, ig) were given, reducing licking and biting times to 59.65

6.32 s and 56.43 6.77 s, respectively.

Table 6. Additional Data for Compounds Complying with

K_iσ₁< 15 nM and Selectivity of >100-Fold

a

compd

LD₅₀(mg/kg)

hERG IC₅₀(μM)

53

54

59

60

65

66

71

72

754.5 (654.6−962.5)

>2000

1.45 0.29

6.34 0.60

0.98 0.1

1.89 0.3

1.61 0.28

1.46 0.18

2.16 0.24

2.68 0.32

900 (732.1−1762.5)

780 (424.6−1586.2)

717.3 (588.8−973.5)

574.8 (453.2−769.2)

1530 (1398.3−1809.2)

1300 (948.5−1723.4)

a

95% conﬁdence limits.

To better characterize the antiallodynic eﬀects of 54, a wide

range of doses was tested. Compound 54 (20−160 mg/kg)

produced dose-dependent antinociception in phase II of the

formalin test and was (slightly) more eﬃcacious against

delayed-phase pain (Figure 5). The ED₅₀value was 48.97

3.89 mg/kg for phase II pain.

Acute Toxicity. The eight compounds that exhibited high

aﬃnities for the σ₁receptor, with good selectivity compared to

σ₂receptor, were evaluated in terms of acute toxicity (LD₅₀)

(Table 6). Compound 54 was nontoxic even at the highest

dose tested (LD₅₀> 2000 mg/kg).

Functional Proﬁle of the σ₁Receptor. More recent

studies using molecular biological tools have clearly demon-

strated that σ₁receptors and associated ligands modulate

inositol 1,4,5-trisphosphate (IP3) receptors at the endoplasmic

reticulum (ER) membrane. For example, the IP3 receptor-

inhibiting protein, ankyrin, was found to be “removed” from

IP3 receptors when σ₁receptor agonists were applied to NG-

108 cells, thus enhancing Ca²⁺eﬄux from the ER into the

cytosol.⁵⁶The same results were observed by Wu and Bowen

who found that the C-terminus portion of σ₁receptors caused

the dissociation of ankyrin from IP3 receptors in MCF-7 tumor

cells.⁵⁷In a study using CHO cells, Hayashi and Su found that

σ₁receptor agonists may activate σ₁receptor by dissociating σ₁

receptors from binding-immunoglobulin protein (BiP), and σ₁

receptor antagonist did not aﬀect the σ₁receptor-BiP

association and stabilized this complex.⁵⁸

Recently, phenytoin-media shifts in σ₁receptor ligand

aﬃnities and FRET-based biosensor studies of σ₁receptor

ligands have been used to categorize compounds as agonists or

antagonists.⁵⁹The functional activity of σ₁receptor ligand was

evaluated using phenytoin,^59aa low potency allosteric

modulator of σ₁receptor that shifts known σ₁receptor agonists

to signiﬁcantly higher aﬃnities (K_iratios without phenytoin vs

with phenytoin of >1), while σ₁receptor antagonists show

small or no shift to lower aﬃnity values (K_iratios without

phenytoin vs with phenytoin of ≤1). In such work, the

functional activities of compound 54 with high-level σ₁

receptor-binding aﬃnities, exhibiting greater selectivity for σ₁

receptor than σ₂receptor, have been evaluated using phenytoin.

Compound 54 aﬀorded a small shift, lowering aﬃnity when

incubated in the presence of phenytoin (K_iratio without vs

with phenytoin, 0.73), thus acting antagonistically on the σ₁

receptor.

Figure 5. Antiallodynic eﬀect of 2 and compound 54 in phase II (15−

45 min) of the mice formalin test. Each column and vertical line

represent the mean

SEM of the values obtained in at least 10

animals. Statistically signiﬁcant diﬀerences are the following: (#) p <

0.05, (##) p < 0.01 vs vehicle; (∗) p < 0.05, (∗∗) p < 0.01 vs vehicle +

formalin (two-way ANOVA followed by Newman−Keuls test).

CCI (Chronic Constriction Injury) Model. Compound 54

was evaluated in a representative and frequently used model of

neuropathic pain, the rat CCI model. Chronic constriction

injury results in the development of allodynia- and spontaneous

pain-like behaviors and hyperalgesia, which are normally

maximal 10−14 days after surgery.^62,63CCI rats exhibited

signs of allodynia upon mechanical stimulation, and hyper-

algesia to thermal stimulation; these are measures of neuro-

pathic pain. Mechanical allodynia was quantiﬁed by measuring

the hind-paw withdrawal response to von Frey hair stimulation,

whereas thermal (heat) hyperalgesia was assessed using a

Formalin-Induced Nociceptive Behavior. Together, the

data indicated that compounds 54, 71, and 72 had high

aﬃnities for σ₁receptor, with good selectivities (compared to σ₂

receptor) together with low aﬃnities for hERG and good safety

proﬁles. These compounds were considered promising and

were subjected to in vivo activity testing.

The classical model of acute and chronic pain, the formalin

test, was performed to evaluate the antinociceptive eﬀects.

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Figure 6. Antiallodynic eﬀects of compound 54 on the expression of neuropathic pain in CCI and sham operated rats. Data are obtained from 10 rats

per group and expressed as the mean SEM pressure threshold evoking paw withdrawal or latency to paw withdrawal. Statistically signiﬁcant

diﬀerences between the rats before surgery (basal) and surgery groups are the following: (∗) p < 0.05, (∗∗) p < 0.01 vs basal; (#) p < 0.05; (##) p <

0.01 vs vehicle treatment on days 14, 15, and 18 (two-way ANOVA followed by Newman−Keuls test).

plantar test; this was the hind-paw withdrawal latency in

response to radiant heat.

As shown in Figure 6, compound 54 dose-dependently

inhibited both mechanical allodynia and thermal hyper-

sensitivity when given in a single dose (day 15) or in two

doses (days 15 and 18); sham-operated rats exhibited no

signiﬁcant changes in mechanical or thermal sensitivity

compared to those before surgery (the basal levels). The

ED₅₀values after single dose treatment in terms of mechanical

allodynia and thermal hypersensitivity were 51.88 5.85 and

18.57

2.32 mg/kg, respectively. For the double-dose

treatment, the ED₅₀values were 40.26

2.17 mg/kg, respectively.

3.52 and 16.93

Motor Coordination: Rotarod Test. To investigate the

possibility that the observed eﬃcacy of compound 54 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.^41,42

As shown in Figure 7, no signiﬁcant eﬀect of compound 54 at

an antiallodynic dose was observed and no treatment-related

adverse eﬀects were found. In contrast, 2 did a signiﬁcant

reduction in the permanence time on the rotating rod was

observed at 80 mg/kg, after single intragastric administration.

Figure 7. Dose−response eﬀect of compounds 54 and 2 on motor

coordination (rotarod test). Data are obtained from 8−10 mice per

group and expressed as the mean SEM latency to fall down from

rod. Statistically signiﬁcant diﬀerences are the following: (∗) p < 0.05,

(∗∗) p < 0.01 vs vehicle (two-way ANOVA followed by Newman−

Keuls test).

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Locomotor Activity Test. In the exploratory locomotor

activity test, haloperidol (a known sedative)^64,65served as a

positive control. Pretreatment with 40, 80, and 160 mg/kg of

compound 54 only mildly reduced exploratory locomotor

activity (Table 7); in comparison, haloperidol signiﬁcantly

reduced spontaneous locomotor activity.

the pyridazinone was essential for activity, and the introduction

of halogens on the aromatic ring led to moderate aﬃnities to σ₁

receptor and increased selectivity to σ₂receptor.

In this study, a series of pyridazinone derivatives were

synthesized via an eﬃcient method, several of which were

shown to be potent and selective σ₁receptor ligands.

Compound 54 appears safe in preliminary tests and exerts

clear dose-dependent antiallodynic eﬀects against formalin-

induced pain in mice and antinociception (both mechanical

allodynia and thermal hypersensitivity) in the rat CCI model.

Moreover, compound 54 exhibited fovorable pharmacokinetic

properties and a good selective proﬁle against other speciﬁc

targets implicated in pain. Thus, compound 54 may facilitate

the development of a novel class of candidate drugs for the

treatment of neuropathic pain. Further studies of compound 54

and this series of derivatives are currently underway in our

laboratory and will be reported in due course.

Table 7. Eﬀects of Compound 54 and Haloperidol on the

Spontaneous Locomotor Activity in Mice (Mean SD, n =

8)

compd

control

dose (mg/kg)

total distance (mm)

24881.4 9545.8

23540.4 7124.5

haloperidol

0.1

0.3

1

a

8539.4 5329.7*

a

1169.7 662.3**

control

24881.4 9545.8

22826.6 6293.7

54

40

a

EXPERIMENTAL SECTION

80

18282.4 7050.5*

■

a

160

16458.3 6260.3**

Chemistry. All commercially available chemicals and reagents were

used without further puriﬁcation. Reagents were all of analytical grade

or of chemical purity (>95%). Melting points were determined in open

capillary tubes and are uncorrected. ¹H NMR spectra was recorded on

a Bruker Avance III 600 spectrometer at 600 MHz (¹H) using CDCl₃

or DMSO-d₆as solvent. Chemical shifts were given in δ values (ppm),

using tetramethylsilane (TMS) as the internal standard; coupling

constants (J) were given in Hz. Signal multiplicities were characterized

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

(broad signal). Analytical thin layer chromatography (TLC) was

performed on silica gel GF254. Column chromatographic puriﬁcation

was carried out using silica gel. Compound purity is determined by

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

compounds display purity higher than 95%. HPLC methods used the

following: Agilent 1100 spectrometer; column, InertSustain C18 5.0

mm × 150 mm × 4.6 mm i.d. (SHIMADZU, Japan); mobile phase, 30

mmol NH₄COOH (ROE SCIENTIFIC INC, USA) aq/acetonitrile

(Merck Company, Germany) 45/55; ﬂow rate, 0.6 mL/min; column

temperature, 40 °C. UV detection was performed at 210 nm.

a

(∗∗) p < 0.01. (∗) p < 0.05 vs control.

Pharmacokinetic Properties of Compound 54. We

evaluated the in vitro and in vivo eﬃcacies and the safety of

compound 54 in rats (Table 8). Intravenous administration (5

mg/kg, n = 3) yielded detectable plasma levels, with a half-life

(t_1/2) of 1.39 0.18 h. Oral administration (80 mg/kg, n = 3)

was associated with a t_1/2of 2.88 0.36 h. The area under the

curve (AUC) was 956

administration vs 13390

131 ng·h/mL after intravenous

1568 ng·h/mL after oral

administration. After oral administration of 80 mg/mL, the

peak serum concentration was 2923 350 ng/mL and the T_max

value 0.33 0.04 h. The clearance was 49.3 6.9 mL min⁻¹

kg⁻¹in the intravenous administration. The bioavailability of

compound 54 was 87.5%. Such encouraging preclinical data

suggest that compound 54 has desirable drug-like human

pharmacokinetic properties.

Selectivity Proﬁle of Compound 54. To assess the

interactions of compound 54 with other receptors or ion

channels, a selectivity proﬁle was created using additional

receptors (including the μ-opioid, serotoninergic, H₃, cannabi-

noid, and NMDA receptors) and ion channels (voltage-gated

sodium channel Nav 1.7 and the TRPV-1 protein) implicated in

pain. Compound 54 showed no signiﬁcant aﬃnity (%

inhibition at 1 μM of <50%) to any putative target (Supporting

Information).

6-Hydroxy-2-phenylpyridazin-3(2H)-one (8).^51bPhenylhydra-

zine hydrochloride (6) (55 mmol) was added to maleic anhydride (7,

50 mmol), 400 mL of water, and 40 mL of concentrated hydrochloric

acid solution. The mixture was heated at reﬂux for 9 h. After it was

cooled, the resulting solid was collected by ﬁltration, washed with ice−

water. The solid was dissolved in saturated sodium bicarbonate

solution; after ﬁltration, the resulting ﬁltrate was neutralized with 1

mol/L hydrochloric acid. The precipitate thus formed was ﬁltered and

washed with water. The resulting 8 was a white solid. Yield 79.8%; mp

272−273 °C; ¹H NMR (600 MHz, CDCl₃) δ 7.62 (d, J = 7.5 Hz, 2H),

7.52−7.47 (m, 2H), 7.43−7.38 (m, 1H), 7.10 (q, J = 9.8 Hz, 2H). A

signal for the OH-proton is not visible.

General Procedure for the Preparation of Compounds 9 and

12−26. Method A. 2-Phenyl-6-(2-(piperidin-1-yl)ethoxy)-

pyridazin-3(2H)-one(9). To a suspension of 8 (10 mmol) and 1-

(2-chloroethyl)piperidine (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

CONCLUSIONS

■

This detailed SAR investigation of pyridazinone scaﬀold

derivatives reveals that several factors inﬂuence the binding

aﬃnity of these compounds to σ₁and σ₂receptor. The

pyridazinone scaﬀold was crucial for activity, and a basic amine

was necessary, matching the known σ₁receptor pharmaco-

phoric model. Piperidine was the preferred amino moiety

(NRR), greatly improving σ₁receptor binding aﬃnity and

maintaining selectivity. The aromatic group on the 2-position of

Table 8. Plasma Pharmacokinetic Data after Administration of Compound 54 in Rats (n = 3/Group)

dose (mg/kg)

C_max(ng/mL)

T_max(h)

CL (mL min⁻¹kg⁻¹

)

t_1/2(h)

AUC_0−last(ng·h/mL)

AUC_0−inf(ng·h/mL)

F (%)

80 (po)

5 (iv)

2923 350

0.33 0.04

2.88 0.36

1.39 0.18

11698 1222

931 125

13390 1568

956 131

87.5

49.3 6.9

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ﬁltrate evaporated under reduced pressure, and the crude product was

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

9. Pale yellow oil; yield 81.9%; ¹H NMR (600 MHz, CDCl₃) δ 7.67−

7.65 (m, 2H), 7.43 (dd, J = 10.8, 5.1 Hz, 2H), 7.32 (t, J = 7.4 Hz, 1H),

7.02−6.95 (m, 2H), 4.28 (t, J = 5.9 Hz, 2H), 2.71 (t, J = 5.9 Hz, 2H),

2.46 (s, 4H), 1.63−1.53 (m, 4H), 1.45 (dd, J = 26.5, 5.0 Hz, 2H).

HRMS (ESI) m/z 300.1713 (calcd 300.1707 for C₁₇H₂₂N₃O₂[M +

H]⁺).

CDCl₃) δ 7.62 (dd, J = 8.5, 1.0 Hz, 2H), 7.43 (dd, J = 11.3, 4.6 Hz,

2H), 7.35−7.31 (m, 1H), 6.98 (q, J = 9.7 Hz, 2H), 4.19 (t, J = 6.3 Hz,

2H), 2.90−2.66 (m, 8H), 2.63−2.54 (m, 2H), 2.50 (s, 3H), 1.99−1.88

(m, 2H). HRMS (ESI) m/z 329.1980 (calcd 329.1972 for

C₁₈H₂₅N₄O₂[M + H]⁺).

6-(3-(4-Ethylpiperazin-1-yl)propoxy)-2-phenylpyridazin-

1

3(2H)-one (18). Pale yellow oil; yield 83.6%; H NMR (600 MHz,

CDCl₃) δ 7.61 (dd, J = 8.5, 1.0 Hz, 2H), 7.41 (t, J = 7.9 Hz, 2H),

7.33−7.29 (m, 1H), 7.01−6.93 (m, 2H), 4.39−4.10 (m, 2H), 2.81−

2.39 (m, 12H), 2.02−1.81 (m, 2H), 1.14 (t, J = 7.3 Hz, 3H). HRMS

(ESI) m/z 343.2135 (calcd 343.2129 for C₁₉H₂₇N₄O₂[M + H]⁺).

6-(3-(4-Methyl-1,4-diazepan-1-yl)propoxy)-2-phenylpyrida-

zin-3(2H)-one (19). Pale yellow oil; yield 83.0%; ¹H NMR (600

MHz, CDCl₃) δ 7.68−7.65 (m, 2H), 7.49 (t, J = 7.4 Hz, 2H), 7.38 (t, J

= 7.4 Hz, 1H), 7.05 (d, J = 9.9 Hz, 1H), 7.01 (d, J = 9.8 Hz, 1H), 4.25

(t, J = 6.2 Hz, 2H), 3.39−3.09 (m, 6H), 2.95 (t, J = 5.9 Hz, 2H), 2.82

(dd, J = 15.2, 8.0 Hz, 2H), 2.75 (s, 3H), 2.29 (s, 2H), 2.06−1.98 (m,

2H). HRMS (ESI) m/z 343.2131 (calcd 343.2129 for C₁₉H₂₇N₄O₂[M

+ H]⁺).

Method B. 6-(3-Bromopropoxy)-2-phenylpyridazin-3(2H)-

one (10). To a solution of 8 (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 = 15/1) to yield 10. Pale yellow oil; yield

61.7%; ¹H NMR (600 MHz, CDCl₃) δ 7.67 (dt, J = 8.7, 1.7 Hz, 2H),

7.49−7.44 (m, 2H), 7.38−7.33 (m, 1H), 7.08−6.99 (m, 2H), 4.32 (t, J

= 5.9 Hz, 2H), 3.55 (t, J = 6.5 Hz, 2H), 2.30 (quintet, J = 6.2 Hz, 2H).

6-(4-Bromobutoxy)-2-phenylpyridazin-3(2H)-one (11). Pale

2-Phenyl-6-(3-(pyrrolidin-1-yl)propoxy)pyridazin-3(2H)-one

1

(20). Pale yellow oil; yield 83.0%; H NMR (600 MHz, CDCl₃) δ

1

yellow oil; yield 66.1%; H NMR (600 MHz, CDCl₃) δ 7.67 (dt, J =

7.69−7.65 (m, 2H), 7.47 (t, J = 7.8 Hz, 2H), 7.36 (t, J = 7.4 Hz, 1H),

7.08−6.94 (m, 2H), 4.24 (t, J = 6.4 Hz, 2H), 2.63−2.58 (m, 2H), 2.54

(t, J = 6.0 Hz, 4H), 2.02−1.95 (m, 2H), 1.85−1.73 (m, 4H). HRMS

(ESI) m/z 300.1715 (calcd 300.1707 for C₁₇H₂₂N₃O₂[M + H]⁺).

6-(3-(Dimethylamino)propoxy)-2-phenylpyridazin-3(2H)-

one (21). Pale yellow oil; yield 63.2%; ¹H NMR (600 MHz, CDCl₃) δ

7.66−7.59 (m, 2H), 7.47−7.41 (m, 2H), 7.37−7.31 (m, 1H), 7.07−

6.98 (m, 2H), 4.32−4.20 (m, 2H), 3.24−3.17 (m, 2H), 2.80 (br, 6H),

2.35−2.21 (m, 2H). HRMS (ESI) m/z 274.1552 (calcd 274.1550 for

C₁₅H₂₀N₃O₂[M + H]⁺).

8.7, 1.7 Hz, 2H), 7.49−7.44 (m, 2H), 7.38−7.33 (m, 1H), 7.08−6.99

(m, 2H), 4.20 (t, J = 6.2 Hz, 2H), 3.47 (t, J = 6.6 Hz, 2H), 2.10−1.98

(m, 2H), 1.97−1.87 (m, 2H).

6-(3-Morpholinopropoxy)-2-phenylpyridazin-3(2H)-one

(12). To a suspension of 10 (2 mmol) and morpholine (2.2 mmol),

K₂CO₃(4 mmol) in acetonitrile (50 mL) and a catalytic amount of KI

(1% mol) were added, and the resulting mixture was reﬂuxed for 6−8

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

reduced pressure. The residue was suspended in water (10.0 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

6-(3-(Diethylamino)propoxy)-2-phenylpyridazin-3(2H)-one

1

(22). Pale yellow oil; yield 66.0%; H NMR (600 MHz, CDCl₃) δ

7.67−7.63 (m, 2H), 7.49−7.44 (m, 2H), 7.35 (dd, J = 10.7, 4.2 Hz,

1H), 7.04−6.98 (m, 2H), 4.25 (t, J = 6.1 Hz, 2H), 3.20−3.15 (m, 2H),

2.96−2.84 (m, 4H), 2.22−2.08 (m, 2H), 1.24 (t, J = 7.2 Hz, 6H).

HRMS (ESI) m/z 302.1870 (calcd 302.1863 for C₁₇H₂₄N₃O₂[M +

H]⁺).

6-(3-(Diisopropylamino)propoxy)-2-phenylpyridazin-3(2H)-

one (23). Pale yellow oil; yield 64.0%; ¹H NMR (600 MHz, CDCl₃) δ

7.63 (d, J = 7.6 Hz, 2H), 7.37 (t, J = 7.9 Hz, 2H), 7.25 (t, J = 7.4 Hz,

1H), 6.96−6.86 (m, 2H), 4.15 (t, J = 6.4 Hz, 2H), 2.49 (t, J = 7.1 Hz,

2H), 2.35−2.28 (m, 4H), 1.86−1.78 (m, 2H), 1.44−1.32 (m, 4H),

0.80 (t, J = 7.4 Hz, 6H). HRMS (ESI) m/z 330.2181 (calcd 330.2176

for C₁₉H₂₈N₃O₂[M + H]⁺).

6-(4-(4-Piperidin-1-yl)butoxy)-2-phenylpyridazin-3(2H)-one

(24). Pale yellow oil; yield 79.7%; ¹H NMR (600 MHz, CDCl₃) δ 7.65

(d, J = 7.6 Hz, 2H), 7.48 (t, J = 8.2 Hz, 2H), 7.33 (t, J = 7.3 Hz, 1H),

7.05−7.02 (m, 2H), 4.25−4.21 (m, 2H), 3.15−3.06 (m, 2H), 2.97−

2.88 (m, 2H), 2.78−2.70 (m, 2H), 1.82−1.75 (m, 2H), 1.71−1.57 (m,

4H), 1.38−1.6 (m, 2H), 1.30−1.22 (m, 2H). HRMS (ESI) m/z

328.2029 (calcd 328.2020 for C₁₉H₂₆N₃O₂[M + H]⁺).

1

compound 12. Pale yellow oil; yield 89.8%; H NMR (600 MHz,

CDCl₃) δ 7.65−7.63 (m, 2H), 7.45 (t, J = 7.9 Hz, 2H), 7.34 (t, J = 7.4

Hz, 1H), 7.02−6.95 (m, 2H), 4.32−4.17 (m, 2H), 3.77−3.57 (m, 4H),

2.80−2.62 (m, 1H), 2.53−2.42 (m, 5H), 2.00−1.88 (m, 2H). HRMS

(ESI) m/z 316.1657 (calcd 316.1656 for C₁₇H₂₂N₃O₃[M + H]⁺).

2-Phenyl-6-(3-(piperidin-1-yl)propoxy)pyridazin-3(2H)-one

(13). Pale yellow oil; yield 91.6%; ¹H NMR (600 MHz, CDCl₃) δ 7.65

(d, J = 1.1 Hz, 1H), 7.64 (s, 1H), 7.44 (dd, J = 10.8, 5.0 Hz, 2H),

7.35−7.30 (m, 1H), 7.00−6.94 (m, 2H), 4.19 (t, J = 6.4 Hz, 2H),

2.49−2.32 (m, 6H), 2.00−1.90 (m, 2H), 1.64−1.54 (m, 4H), 1.43 (s,

2H). HRMS (ESI) m/z 314.1872 (calcd 314.1863 for C₁₈H₂₄N₃O₂[M

+ H]⁺).

6-(3-(4-Methylpiperidin-1-yl)propoxy)-2-phenylpyridazin-

1

3(2H)-one (14). Pale yellow oil; yield 88.3%; H NMR (600 MHz,

CDCl₃) δ 7.66 (d, J = 7.7 Hz, 2H), 7.44 (t, J = 7.9 Hz, 2H), 7.33 (t, J =

7.4 Hz, 1H), 7.01−6.95 (m, 2H), 4.20 (t, J = 6.4 Hz, 2H), 2.90 (d, J =

11.4 Hz, 2H), 2.52−2.39 (m, 2H), 2.01−1.85 (m, 4H), 1.66−1.56 (m,

2H), 1.41−1.30 (m, 1H), 1.24 (qd, J = 12.5, 3.6 Hz, 2H), 0.92 (d, J =

6.5 Hz, 3H). HRMS (ESI) m/z 328.2021 (calcd 328.2020 for

C₁₉H₂₆N₃O₂[M + H]⁺).

6-(3-(3,5-Dimethylpiperidin-1-yl)propoxy)-2-phenylpyrida-

zin-3(2H)-one(15). Pale yellow oil; yield 83.5%; ¹H NMR (600 MHz,

CDCl₃) δ 7.68−7.60 (m, 2H), 7.42 (t, J = 7.0 Hz, 2H), 7.31 (t, J = 7.4

Hz, 1H), 7.00−6.93 (m, 2H), 4.23−4.15 (m, 2H), 2.97−2.87 (m, 2H),

2.59−2.51 (m, 2H), 2.01 (dd, J = 14.5, 7.1 Hz, 2H), 1.81−1.66 (m,

2H), 1.56 (t, J = 11.2 Hz, 2H), 0.83 (d, J = 6.6 Hz, 6H), 0.64−0.49 (m,

2H). HRMS (ESI) m/z 342.2180 (calcd 342.2176 for C₂₀H₂₈N₃O₂[M

+ H]⁺).

6-(4-(4-Methylpiperidin-1-yl)butoxy)-2-phenylpyridazin-

1

3(2H)-one (25). Pale yellow oil; yield 82.3%; H NMR (600 MHz,

CDCl₃) δ 7.63 (d, J = 7.6 Hz, 2H), 7.37 (t, J = 7.9 Hz, 2H), 7.25 (t, J =

7.4 Hz, 1H), 6.96−6.86 (m, 2H), 4.19 (t, J = 6.4 Hz, 2H), 2.91 (d, J =

11.4 Hz, 2H), 2.41−2.32 (m, 2H), 1.93 (dd, J = 20.6, 8.6 Hz, 2H),

1.82−1.75 (m, 2H), 1.71−1.57 (m, 4H), 1.38−1.6 (m, 1H), 1.30−1.22

(m, 2H), 0.93 (d, J = 6.5 Hz, 3H). HRMS (ESI) m/z 342.2179 (calcd

342.2176 for C₂₀H₂₈N₃O₂[M + H]⁺).

6-(4-(3,5-Dimethylpiperidin-1-yl)butoxy)-2-phenylpyrida-

zin-3(2H)-one (26). Pale yellow oil; yield 83.0%; ¹H NMR (600

MHz, CDCl₃) δ 7.64 (d, J = 7.6 Hz, 2H), 7.38 (t, J = 7.9 Hz, 2H), 7.25

(t, J = 7.4 Hz, 1H), 6.97−6.88 (m, 2H),4.29−4.16 (m, 2H), 4.16−4.10

(m, 2H), 3.10 (t, J = 20.3 Hz, 2H), 2.74−2.64 (m, 4H), 2.07−1.86 (m,

4H), 1.85−1.61 (m, 2H), 0.97 (d, J = 8.9 Hz, 3H), 0.83 (d, J = 6.6 Hz,

3H). HRMS (ESI) m/z 356.2340 (calcd 356.2333 for C₂₁H₃₀N₃O₂[M

+ H]⁺).

6-(3-(4-Oxopiperidin-1-yl)propoxy)-2-phenylpyridazin-

1

3(2H)-one (16). Pale yellow oil; yield 84.1%; H NMR (600 MHz,

CDCl₃) δ 7.68−7.63 (m, 2H), 7.49−7.42 (m, 2H), 7.36−7.32 (m,

1H), 7.05−6.93 (m, 2H), 4.46−4.20 (m, 4H), 3.76−3.72 (m, 4H),

2.54−2.37 (m, 2H), 2.26−2.09 (m, 2H), 2.02−1.91 (m, 2H). HRMS

(ESI) m/z 328.1663 (calcd 328.1656 for C₁₈H₂₂N₃O₃[M + H]⁺).

6-(3-(4-Methylpiperazin-1-yl)propoxy)-2-phenylpyridazin-

General Procedures for the Preparation of Intermediates

27−38. 6-Hydroxy-2-(p-tolyl)pyridazin-3(2H)-one (27). p-Tolyl-

hydrazine hydrochloride (55 mmol) was added to maleic anhydride

1

3(2H)-one (17). Pale yellow oil; yield 82.9%; H NMR (600 MHz,

L

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(7, 50 mmol), 400 mL of water, and 40 mL of concentrated

hydrochloric acid solution. The mixture was heated at reﬂux for 9 h.

After it was cooled, the resulting solid was collected by ﬁltration,

washed with ice−water. The solid was dissolved in saturated sodium

bicarbonate solution. After ﬁltration, the resulting ﬁltrate was

neutralized with 1 mol/L hydrochloric acid. The precipitate thus

formed was ﬁltered and washed with water. The resulting 27 was a

white solid. Yield 80.4%; mp 242−243 °C; ¹H NMR (600 MHz,

CDCl₃) δ 7.47 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 8.2 Hz, 2H), 7.13−7.04

(m, 2H), 2.42 (s, 3H). A signal for the OH-proton is not visible.

6-Hydroxy-2-(naphthalen-2-yl)pyridazin-3(2H)-one (28).

2H), 3.55 (t, J = 6.5 Hz, 2H), 2.36 (s, 3H), 2.29 (quintet, J = 6.2 Hz,

2H).

6-(3-Bromopropoxy)-2-(naphthalen-2-yl)pyridazin-3(2H)-

one (40). Pale yellow oil; yield 69.8%; ¹H NMR (600 MHz, CDCl₃) δ

8.23 (s, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.93−7.91 (m, 1H), 7.89−7.87

(m, 1H). 7.81 (dd, J = 8.8, 2.0 Hz, 1H), 7.56−7.49 (m, 2H), 7.07 (d, J

= 9.7 Hz, 1H), 7.00 (d, J = 9.7 Hz, 1H), 4.35 (t, J = 5.9 Hz, 2H), 3.56

(t, J = 6.5 Hz, 2H), 2.36−2.24 (m, 2H).

6-(3-Bromopropoxy)-2-(4-ﬂuorophenyl)pyridazin-3(2H)-one

1

(41). Pale yellow oil; yield 65.3%; H NMR (600 MHz, CDCl₃) δ

7.69−7.64 (m, 2H), 7.15−7.11 (m, 2H), 7.02−6.98 (m, 2H), 4.31 (t, J

= 5.9 Hz, 2H), 3.55 (t, J = 6.5 Hz, 2H), 2.30 (quintet, J = 6.2 Hz, 2H).

6-(3-Bromopropoxy)-2-(4-chlorophenyl)pyridazin-3(2H)-one

1

White solid; yield 78.1%; mp 268−269 °C; H NMR (600 MHz,

CDCl₃) δ 8.14 (s, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.89 (s, 1H), 7.73 (d,

J = 6.9 Hz, 1H), 7.57−7.52 (m, 2H), 7.46 (s, 1H), 7.17−7.09 (m, 2H).

A signal for the OH-proton is not visible.

1

(42). Pale yellow solid; yield 71.0%; mp 79−81 °C; H NMR (600

MHz, CDCl₃) δ 7.67 (d, J = 8.7 Hz, 2H), 7.42 (d, J = 8.7 Hz, 2H),

7.04−6.97 (m, 2H), 4.32 (t, J = 5.9 Hz, 2H), 3.55 (t, J = 6.4 Hz, 2H),

2.30 (quintet, J = 6.1 Hz, 2H).

2-(4-Fluorophenyl)-6-hydroxypyridazin-3(2H)-one (29).

1

White solid; yield 80.5%; mp 279−281 °C; H NMR (600 MHz,

6-(3-Bromopropoxy)-2-(3,4-dichlorophenyl)pyridazin-3(2H)-

CDCl₃) δ 7.69−7.64 (m, 2H), 7.12−7.09 (m, 2H), 7.03−6.99 (q, J =

9.7 Hz, 2H). A signal for the OH-proton is not visible.

1

one (43). Pale yellow solid; yield 71.8%; mp 83−85 °C; H NMR

(600 MHz, CDCl₃) δ 7.90 (d, J = 2.5 Hz, 1H), 7.65 (dd, J = 8.7, 2.5

Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.01(s, 2H), 4.34 (t, J = 5.9 Hz,

2H), 3.57 (t, J = 6.4 Hz, 2H), 2.32 (quintet, J = 6.2 Hz, 2H).

6-(3-Bromopropoxy)-2-(2,3-dichlorophenyl)pyridazin-3(2H)-

2-(4-Chlorophenyl)-6-hydroxypyridazin-3(2H)-one (30).

1

White solid; yield 83.0%; mp 281−282 °C; H NMR (600 MHz,

CDCl₃) δ 7.62 (d, J = 8.8 Hz, 2H), 7.47−7.43 (m, 2H), 7.10 (d, J = 8.5

Hz, 2H). A signal for the OH-proton is not visible.

1

one (44). Pale yellow solid; yield 71.3%; mp 53−55 °C; H NMR

2-(3,4-Dichlorophenyl)-6-hydroxypyridazin-3(2H)-one (31).

Pale yellow solid; yield 76.2%; mp 291−292 °C; ¹H NMR (600

MHz, CDCl₃) δ 7.90 (d, J = 2.5 Hz, 1H), 7.65 (dd, J = 8.7, 2.5 Hz,

1H), 7.52 (d, J = 8.7 Hz, 1H), 7.01(br, 2H). A signal for the OH-

proton is not visible.

2-(2,3-Dichlorophenyl)-6-hydroxypyridazin-3(2H)-one (32).

Pale yellow solid; yield 77.9%; mp 289−292 °C; ¹H NMR (600

MHz, DMSO-d₆) δ 11.50 (s, 1H), 7.77 (dd, J = 8.1, 1.6 Hz, 1H), 7.58

(dd, J = 7.9, 1.6 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.25 (d, J = 9.9 Hz,

1H), 7.08 (d, J = 9.9 Hz, 1H).

2-(3,5-Dichlorophenyl)-6-hydroxypyridazin-3(2H)-one (33).

Pale yellow solid; yield 74.8%; mp 288−289 °C; ¹H NMR (600

MHz, DMSO-d₆) δ 11.54 (s, 1H), 7.79 (d, J = 1.9 Hz, 2H), 7.62 (t, J =

1.9 Hz, 1H), 7.21 (d, J = 9.8 Hz, 1H), 7.05 (d, J = 9.8 Hz, 1H).

2-(2,4-Dichlorophenyl)-6-hydroxypyridazin-3(2H)-one (34).

Pale yellow solid; yield 76.9%; mp 279−281 °C; ¹H NMR (600

MHz, DMSO-d₆) δ 11.49 (s, 1H), 7.85 (d, J = 2.0 Hz, 1H), 7.65−7.55

(m, 2H), 7.24 (d, J = 9.9 Hz, 1H), 7.06 (d, J = 9.9 Hz, 1H).

2-(2,5-Dichlorophenyl)-6-hydroxypyridazin-3(2H)-one (35).

Pale yellow solid; yield 75.3%; mp 282−284 °C; ¹H NMR (600

MHz, DMSO) δ 11.48 (s, 1H), 7.78 (d, J = 2.5 Hz, 1H), 7.69 (d, J =

8.7 Hz, 1H), 7.60 (dd, J = 8.7, 2.5 Hz, 1H), 7.24 (d, J = 9.9 Hz, 1H),

7.07 (d, J = 9.9 Hz, 1H).

(600 MHz, CDCl₃) δ 7.57 (dd, J = 6.5, 3.1 Hz, 1H), 7.40−7.32 (m,

2H), 7.09−7.03 (m, 2H), 4.25 (t, J = 5.9 Hz, 2H), 3.55 (t, J = 6.5 Hz,

2H), 2.60−1.94 (m, 2H).

6-(3-Bromopropoxy)-2-(3,5-dichlorophenyl)pyridazin-3(2H)-

1

one (45). Pale yellow solid; yield 71.3%; mp 63−64 °C; H NMR

(600 MHz, CDCl₃) δ 7.72 (d, J = 1.9 Hz, 2H), 7.35 (t, J = 1.9 Hz,

1H), 7.11−6.96 (m, 2H), 4.35 (t, J = 5.9 Hz, 2H), 3.58 (t, J = 6.4 Hz,

2H), 2.36−2.31 (m, 2H).

6-(3-Bromopropoxy)-2-(2,4-dichlorophenyl)pyridazin-3(2H)-

one (46). Pale yellow oil; yield 70.1%; ¹H NMR (600 MHz, CDCl₃) δ

7.55 (dd, J = 1.5, 1.0 Hz, 1H), 7.42−7.35 (m, 2H), 7.08−7.01 (m,

2H), 4.24 (t, J = 5.9 Hz, 2H), 3.53 (t, J = 6.5 Hz, 2H), 2.28 (p, J = 6.2

Hz, 2H).

6-(3-Bromopropoxy)-2-(2,5-dichlorophenyl)pyridazin-3(2H)-

one (47). Pale yellow oil; yield 71.8%; ¹H NMR (600 MHz, CDCl₃) δ

7.49 (d, J = 8.6 Hz, 1H), 7.46 (d, J = 2.4 Hz, 1H), 7.39 (dd, J = 8.6, 2.4

Hz, 1H), 7.09−7.03 (m, 2H), 4.27 (t, J = 5.9 Hz, 2H), 3.55 (t, J = 6.5

Hz, 2H), 2.31−2.28 (m, 2H).

6-(3-Bromopropoxy)-2-(3-chloro-4-ﬂuorophenyl)pyridazin-

1

3(2H)-one (48). Pale yellow solid; yield 74.8%; mp 73−75 °C; H

NMR (600 MHz, CDCl₃) δ 7.53−7.37 (m, 2H), 7.07−6.96 (m, 2H),

6.81 (tt, J = 8.7, 2.3 Hz, 1H), 4.36 (t, J = 5.9 Hz, 2H), 3.58 (t, J = 6.4

Hz, 2H), 2.37−2.31 (m, 2H).

2-(3-Chloro-4-ﬂuorophenyl)-6-hydroxypyridazin-3(2H)-one

6-(3-Bromopropoxy)-2-(3,4-diﬂuorophenyl)pyridazin-3(2H)-

1

(36). Pale yellow solid; yield 76.8%; mp 291−292 °C; H NMR (600

one (49). Pale yellow solid; yield 69.9%; mp 43−45 °C; H NMR

MHz, DMSO-d₆) δ 11.54 (s, 1H), 7.54−7.44 (m, 2H), 7.29 (tt, J =

9.2, 2.4 Hz, 1H), 7.20 (d, J = 9.8 Hz, 1H), 7.09−7.03 (m, 1H).

2-(3,4-Diﬂuorophenyl)-6-hydroxypyridazin-3(2H)-one (37).

Pale yellow solid; yield 78.6%; mp 285−287 °C; ¹H NMR (600

MHz, DMSO-d₆) δ 11.48 (s, 1H), 7.78−7.75 (m, 1H), 7.59−7.49 (m,

2H), 7.22 (d, J = 9.8 Hz, 1H), 7.05 (d, J = 9.8 Hz, 1H).

(600 MHz, CDCl₃) δ 7.66−7.63 (m, 1H), 7.55−7.52 (m, 1H), 7.24

(dd, J = 18.5, 8.9 Hz, 1H), 7.05−6.97 (m, 2H), 4.34 (t, J = 5.9 Hz,

2H), 3.57 (t, J = 6.4 Hz, 2H), 2.36−2.28 (m, 2H).

General Procedures for the Synthesis of Compounds 50−

61. Method B. 6-(3-(Piperidin-1-yl)propoxy)-2-(p-tolyl)-

pyridazin-3(2H)-one (50). To a suspension of 39 (2 mmol) and

piperidine (2.2 mmol), K₂CO₃(4 mmol) in acetonitrile (50 mL) and a

catalytic amount of KI (1% mol) were added, and the resulting mixture

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

evaporated to dryness under reduced pressure. The residue was

suspended in water (10.0 mL) and extracted with dichloromethane (3

× 25 mL). 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 (10%

MeOH/CHCl₃) to yield compound 50. Pale yellow oil; yield 81.5%;

¹H NMR (600 MHz, CDCl₃) δ 7.48 (d, J = 8.3 Hz, 2H), 7.22 (d, J =

2-Cyclopentyl-6-hydroxypyridazin-3(2H)-one (38). Pale white

1

solid; yield 55.9%; mp 231−232 °C; H NMR (600 MHz, CDCl₃) δ

7.06 (d, J = 9.5 Hz, 2H), 5.44−5.25 (m, 1H), 1.93 (t, J = 31.1 Hz, 2H),

1.85−1.70 (m, 4H), 1.58−1.54 (m, 2H). A signal for the OH-proton is

not visible.

General Procedures for the Preparation of Intermediates

39−49. 6-(3-Bromopropoxy)-2-(p-tolyl)pyridazin-3(2H)-one

(39). To a solution of 27 (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 = 15/1) to yield compound 39. Pale yellow

8.1 Hz, 2H), 6.98−6.93 (m, 2H), 4.17 (t, J = 6.2 Hz, 2H), 2.59 (dd, J =

18.2, 10.4 Hz, 6H), 2.35 (s, 3H), 2.09−1.97 (m, 2H), 1.71−1.61 (m,

4H), 1.46 (s, 2H). HRMS (ESI) m/z 328.2027(calcd 328.2020 for

C₁₉H₂₆N₃O₂[M + H]⁺).

1

oil; yield 75.4%; H NMR (600 MHz, CDCl₃) δ 7.54 (d, J = 8.4 Hz,

2-(Naphthalen-2-yl)-6-(3-(piperidin-1-yl)propoxy)pyridazin-

1

2H), 7.27 (d, J = 8.2 Hz, 2H), 7.06−6.96 (m, 2H), 4.31 (t, J = 5.9 Hz,

3(2H)-one (51). Pale yellow oil; yield 87.2%; H NMR (600 MHz,

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CDCl₃) δ 8.20 (d, J = 1.7 Hz, 1H), 7.95−7.85 (m, 3H), 7.79 (dd, J =

8.8, 2.1 Hz, 1H), 7.54−7.49 (m, 2H), 7.03 (dd, J = 22.4, 9.7 Hz, 2H),

4.25 (t, J = 6.4 Hz, 2H), 2.45 (dd, J = 24.5, 16.7 Hz, 6H), 2.05−1.92

(m, 2H), 1.66−1.55 (m, 4H), 1.45 (s, 2H). HRMS (ESI) m/z

364.2026(calcd 364.2020 for C₂₂H₂₆N₃O₂[M + H]⁺).

layers were dried with anhydrous magnesium sulfate, the ﬁltrate was

evaporated under reduced pressure, and the crude product was

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

61. Pale yellow oil; yield 64.6%; ¹H NMR (600 MHz, CDCl₃) δ 6.67−

6.50 (m, 2H), 5.19−5.00 (m, 1H), 3.93 (dd, J = 14.8, 8.5 Hz, 2H),

2.19 (dd, J = 22.9, 15.5 Hz, 6H), 1.82−1.51 (m, 8H), 1.35 (dd, J =

23.9, 18.4 Hz, 6H), 1.18 (s, 2H). HRMS (ESI) m/z 306.2183 (calcd

306.2176 for C₁₇H₂₈N₃O₂[M + H]⁺).

General Procedures for the Synthesis of Compounds 62−

72. Method B. 6-(3-(4-Methylpiperidin-1-yl)propoxy)-2-(p-

tolyl)pyridazin-3(2H)-one (62). To a suspension of 39 (2 mmol)

and 4-methylpiperidine (2.2 mmol), K₂CO₃(4 mmol) in acetonitrile

(50 mL) and a catalytic amount of KI (1% mol) were added, and the

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

ﬁltrate was evaporated to dryness under reduced pressure. The residue

was suspended in water (10.0 mL) and extracted with dichloro-

methane (3 × 25 mL). 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 (10% MeOH/CHCl₃) to yield compound 62. Pale

yellow oil; yield 82.9%; ¹H NMR (600 MHz, CDCl₃) δ 7.52 (d, J = 8.3

Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 6.95 (q, J = 9.7 Hz, 2H), 4.18 (t, J =

6.4 Hz, 2H), 2.88 (d, J = 11.3 Hz, 2H), 2.45−2.42 (m, 2H), 2.37 (s,

3H), 1.97−1.86 (m, 4H), 1.60 (d, J = 12.8 Hz, 2H), 1.39−1.31 (m,

1H), 1.28−1.18 (m, 2H), 0.91 (d, J = 6.5 Hz, 3H). HRMS (ESI) m/z

342.2185(calcd 342.2176 for C₂₀H₂₈N₃O₂[M + H]⁺).

2-(4-Fluorophenyl)-6-(3-(piperidin-1-yl)propoxy)pyridazin-

1

3(2H)-one (52). Pale yellow oil; yield 80.6%; H NMR (600 MHz,

CDCl₃) δ 7.65−7.61 (m, 2H), 7.14−7.07 (m, 2H), 6.99 (d, J = 9.7 Hz,

2H), 4.20 (t, J = 6.2 Hz, 2H), 2.66 (dd, J = 17.3, 9.3 Hz, 6H), 2.15−

2.00 (m, 2H), 1.75−1.68 (m, 4H), 1.50 (s, 2H). HRMS (ESI) m/z

332.1775 (calcd 332.1769 for C₁₈H₂₃FN₃O₂[M + H]⁺).

2-(4-Chlorophenyl)-6-(3-(piperidin-1-yl)propoxy)pyridazin-

1

3(2H)-one (53). Pale yellow solid; yield 86.7%; mp 91−92 °C; H

NMR (600 MHz, CDCl₃) δ 7.68−7.64 (m, 2H), 7.42−7.38 (m, 2H),

6.97 (s, 2H), 4.20 (t, J = 6.4 Hz, 2H), 2.42 (dd, J = 22.1, 14.4 Hz, 6H),

2.04−1.86 (m, 2H), 1.65−1.53 (m, 4H), 1.43 (s, 2H). HRMS (ESI)

m/z 348.1477(calcd 348.1473 for C₁₈H₂₃ClN₃O₂[M + H]⁺).

2-(3,4-Dichlorophenyl)-6-(3-(piperidin-1-yl)propoxy)-

pyridazin-3(2H)-one (54). Pale yellow solid; yield 88.4%; mp 61−62

°C; ¹H NMR (600 MHz, CDCl₃) δ 7.88 (d, J = 2.4 Hz, 1H), 7.65 (dd,

J = 8.7, 2.5 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 7.04−6.96 (m, 2H), 4.22

(t, J = 6.4 Hz, 2H), 2.53−2.38 (m, 6H), 2.02−1.92 (m, 2H), 1.66−

1.58 (m, 4H), 1.45 (br, 2H). HRMS (ESI) m/z 382.1087 (calcd

382.1084 for C₁₈H₂₂Cl₂N₃O₂[M + H]⁺).

2-(2,3-Dichlorophenyl)-6-(3-(piperidin-1-yl)propoxy)-

1

pyridazin-3(2H)-one (55). Pale yellow oil; yield 81.5%; H NMR

(600 MHz, CDCl₃) δ 7.59−7.52 (m, 1H), 7.37−7.32 (m, 2H), 7.07−

6.98 (m, 2H), 4.13 (t, J = 6.3 Hz, 2H), 2.43−2.38 (m, 6H), 1.99−1.87

(m, 2H), 1.65−1.49 (m, 4H), 1.43 (s, 2H). HRMS (ESI) m/z

382.1088 (calcd 382.1084 for C₁₈H₂₂Cl₂N₃O₂[M + H]⁺).

6-(3-(4-Methylpiperidin-1-yl)propoxy)-2-(naphthalen-2-yl)-

1

pyridazin-3(2H)-one (63). Pale yellow oil; yield 81.3%; H NMR

(600 MHz, CDCl₃) δ 8.20 (d, J = 1.8 Hz, 1H), 7.89 (ddd, J = 16.8,

11.9, 6.1 Hz, 3H), 7.79 (dd, J = 8.8, 2.1 Hz, 1H), 7.54−7.47 (m, 2H),

7.03 (dd, J = 23.3, 9.7 Hz, 2H), 4.25 (t, J = 6.4 Hz, 2H), 2.90 (d, J =

11.4 Hz, 2H), 2.53−2.43 (m, 2H), 2.01−1.89 (m, 4H), 1.62 (d, J =

12.8 Hz, 2H), 1.43−1.18 (m, 3H), 0.93 (d, J = 6.5 Hz, 3H). HRMS

(ESI) m/z 378.2180(calcd 378.2176 for C₂₃H₂₈N₃O₂[M + H]⁺).

2-(4-Fluorophenyl)-6-(3-(4-methylpiperidin-1-yl)propoxy)-

2-(3,5-Dichlorophenyl)-6-(3-(piperidin-1-yl)propoxy)-

1

pyridazin-3(2H)-one (56). Pale yellow oil; yield 82.1%; H NMR

(600 MHz, CDCl₃) δ 7.70 (d, J = 1.9 Hz, 2H), 7.32 (t, J = 1.9 Hz,

1H), 6.99 (d, J = 1.1 Hz, 2H), 4.22 (t, J = 6.4 Hz, 2H), 2.47−2.41 (m,

6H), 2.03−1.93 (m, 2H), 1.63−1.56 (m, 4H), 1.45 (br, 2H). HRMS

(ESI) m/z 382.1079 (calcd 382.1084 for C₁₈H₂₂Cl₂N₃O₂[M + H]⁺).

2-(2,4-Dichlorophenyl)-6-(3-(piperidin-1-yl)propoxy)-

1

pyridazin-3(2H)-one (64). Pale yellow oil; yield 81.9%; H NMR

(600 MHz, CDCl₃) δ 7.68−7.63 (m, 2H), 7.15−7.09 (m, 2H), 7.01−

6.95 (m, 2H), 4.19 (t, J = 6.4 Hz, 2H), 2.88 (d, J = 11.1 Hz, 2H),

2.48−2.40 (m, 2H), 1.98−1.84 (m, 4H), 1.61 (d, J = 12.8 Hz, 2H),

1.35 (d, J = 6.0 Hz, 1H), 1.23 (q, J = 11.4 Hz, 2H), 0.91 (d, J = 6.5 Hz,

3H). HRMS (ESI) m/z 346.1933(calcd 346.1925 for C₁₉H₂₅FN₃O₂

[M + H]⁺).

1

pyridazin-3(2H)-one (57). Pale yellow oil; yield 83.2%; H NMR

(600 MHz, CDCl₃) δ 7.55 (d, J = 1.6 Hz, 1H), 7.41−7.34 (m, 2H),

7.02 (q, J = 9.9 Hz, 2H), 4.13 (t, J = 6.5 Hz, 2H), 2.44−2.39 (m, 6H),

2.00−1.87 (m, 2H), 1.66−1.52 (m, 4H), 1.44 (br, 2H). HRMS (ESI)

m/z 382.1086 (calcd 382.1084 for C₁₈H₂₂Cl₂N₃O₂[M + H]⁺).

2-(2,5-Dichlorophenyl)-6-(3-(piperidin-1-yl)propoxy)-

1

2-(4-Chlorophenyl)-6-(3-(4-methylpiperidin-1-yl)propoxy)-

pyridazin-3(2H)-one (58). Pale yellow oil; yield 85.3%; H NMR

pyridazin-3(2H)-one (65). Pale yellow solid; yield 82.2%; mp 95−96

(600 MHz, CDCl₃) δ 7.47 (d, J = 8.6 Hz, 1H), 7.44 (d, J = 2.4 Hz,

1H), 7.37 (dd, J = 8.6, 2.5 Hz, 1H), 7.03 (q, J = 9.9 Hz, 2H), 4.15 (t, J

= 6.5 Hz, 2H), 2.44−2.39 (m, 6H), 2.01−1.88 (m, 2H), 1.63−1.52 (m,

4H), 1.44 (br, 2H). HRMS (ESI) m/z 382.1088 (calcd 382.1084 for

C₁₈H₂₂Cl₂N₃O₂[M + H]⁺).

1

°C; H NMR (600 MHz, CDCl₃) δ 7.68−7.65 (m, 2H), 7.43−7.39

(m, 2H), 7.01−6.96 (m, 2H), 4.21 (t, J = 6.4 Hz, 2H), 2.89 (d, J = 11.4

Hz, 2H), 2.49−2.43 (m, 2H), 1.99−1.87 (m, 4H), 1.62 (d, J = 12.9

Hz, 2H), 1.42−1.33 (m, 1H), 1.28−1.21 (m, 3.6 Hz, 2H), 0.92 (d, J =

6.5 Hz, 3H). HRMS (ESI) m/z 362.1633 (calcd 362.1630 for

C₁₉H₂₅ClN₃O₂[M + H]⁺).

2-(3-Chloro-4-ﬂuorophenyl)-6-(3-(piperidin-1-yl)propoxy)-

1

pyridazin-3(2H)-one (59). Pale yellow oil; yield 81.6%; H NMR

2-(3,4-Dichlorophenyl)-6-(3-(4-methylpiperidin-1-yl)-

(600 MHz, CDCl₃) δ 7.50−7.38 (m, 2H), 7.07−6.93 (m, 2H), 6.80−

6.75 (m, 1H), 4.23 (t, J = 6.4 Hz, 2H), 2.47−2.40 (m, 6H), 2.03−1.90

(m, 2H), 1.67−1.54 (m, 4H), 1.44 (br, 2H). HRMS (ESI) m/z

366.1381 (calcd 366.1379 for C₁₈H₂₂ClFN₃O₂[M + H]⁺).

propoxy)pyridazin-3(2H)-one (66). Pale white solid; yield 83.7%;

1

mp 54−56 °C; H NMR (600 MHz, CDCl₃) δ 7.88 (d, J = 2.5 Hz,

1H), 7.65 (dd, J = 8.7, 2.5 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 6.99 (d, J

= 10.0 Hz, 2H), 4.22 (t, J = 6.4 Hz, 2H), 2.91 (d, J = 11.4 Hz, 2H),

2.49−2.45 (m, 2H), 2.02−1.90 (m, 4H), 1.63 (d, J = 12.8 Hz, 2H),

1.29−1.22 (m, 3H), 0.93 (d, J = 6.5 Hz, 3H). HRMS (ESI) m/z

396.1246 (calcd 396.1240 for C₁₉H₂₄Cl₂N₃O₂[M + H]⁺).

2-(3,4-Diﬂuorophenyl)-6-(3-(piperidin-1-yl)propoxy)-

1

pyridazin-3(2H)-one (60). Pale yellow oil; yield 82.4%; H NMR

(600 MHz, CDCl₃) δ 7.64−7.60 (m, 1H), 7.55−7.45 (m, 1H), 7.21

(dd, J = 18.5, 8.9 Hz, 1H), 7.03−6.93 (m, 2H), 4.20 (t, J = 6.4 Hz,

2H), 2.45−2.39 (m, 6H), 2.01−1.91 (m, 2H), 1.64−1.51 (m, 4H),

1.44 (d, J = 4.6 Hz, 2H). HRMS (ESI) m/z 350.1682 (calcd 350.1675

for C₁₈H₂₂F₂N₃O₂[M + H]⁺).

2-(2,3-Dichlorophenyl)-6-(3-(4-methylpiperidin-1-yl)-

propoxy)pyridazin-3(2H)-one (67). Pale yellow oil; yield 82.1%;

¹H NMR (600 MHz, CDCl₃) δ 7.61−7.52 (m, 1H), 7.38−7.30 (m,

2H), 7.04 (q, J = 9.9 Hz, 2H), 4.14 (t, J = 6.3 Hz, 2H), 2.88 (d, J =

11.5 Hz, 2H), 2.44 (t, J = 6.3 Hz, 2H), 2.00−1.85 (m, 4H), 1.61 (d, J =

13.0 Hz, 2H), 1.41−1.30 (m, 1H), 1.26−1.20 (m, 2H), 0.92 (d, J = 6.5

Hz, 3H). HRMS (ESI) m/z 396.1242 (calcd 396.1240 for

C₁₉H₂₄Cl₂N₃O₂[M + H]⁺).

2-(3,5-Dichlorophenyl)-6-(3-(4-methylpiperidin-1-yl)-

propoxy)pyridazin-3(2H)-one (68). Pale yellow oil; yield 81.3%;

¹H NMR (600 MHz, CDCl₃) δ 7.71 (d, J = 1.9 Hz, 2H), 7.34 (t, J =

Method A: 2-Cyclopentyl-6-(3-(piperidin-1-yl)propoxy)-

pyridazin-3(2H)-one (61). To a suspension of 38 (10 mmol) and

1-(3-chloropropyl)piperidine (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 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

1.9 Hz, 1H), 7.05−6.95 (m, 2H), 4.23 (t, J = 6.4 Hz, 2H), 2.91 (d, J =

N

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Article

11.5 Hz, 2H), 2.50−2.45 (m, 2H), 2.02−1.89 (m, 4H), 1.64 (d, J =

12.9 Hz, 2H), 1.44−1.32 (m, 1H), 1.30−1.20 (m, 2H), 0.94 (d, J = 6.5

Hz, 3H). HRMS (ESI) m/z 396.1243 (calcd 396.1240 for

C₁₉H₂₄Cl₂N₃O₂[M + H]⁺).

2-(2,4-Dichlorophenyl)-6-(3-(4-methylpiperidin-1-yl)-

propoxy)pyridazin-3(2H)-one (69). Pale yellow oil; yield 81.2%;

¹H NMR (600 MHz, CDCl₃) δ 7.56−7.54 (m, 1H), 7.40−7.33 (m,

2H), 7.02 (q, J = 9.9 Hz, 2H), 4.13 (t, J = 6.5 Hz, 2H), 2.88 (d, J =

11.5 Hz, 2H), 2.49−2.41 (m, 2H), 1.99−1.80 (m, 4H), 1.61 (d, J =

13.0 Hz, 2H), 1.43−1.29 (m, 1H), 1.29−1.15 (m, 2H), 0.92 (d, J = 6.5

Hz, 3H). HRMS (ESI) m/z 396.1248 (calcd 396.1240 for

C₁₉H₂₄Cl₂N₃O₂[M + H]⁺).

2-(2,5-Dichlorophenyl)-6-(3-(4-methylpiperidin-1-yl)-

propoxy)pyridazin-3(2H)-one (70). Pale yellow oil; yield 80.7%;

¹H NMR (600 MHz, CDCl₃) δ 7.47−7.44 (m, 1H), 7.43 (d, J = 2.4

CDCl₃) δ 7.93 (dd, J = 2.3, 1.0 Hz, 1H), 7.67 (ddd, J = 8.7, 2.4, 1.0 Hz,

1H), 7.51 (dd, J = 8.7, 0.9 Hz, 1H), 4.40−4.27 (m, 2H), 3.59 (td, J =

6.4, 0.7 Hz, 2H), 2.36 (p, J = 5.8 Hz, 2H), 2.23 (s, 3H), 2.17 (s, 3H).

4-(3-Bromopropoxy)-2-(3,4-dichlorophenyl)-5,6,7,8-tetrahy-

drophthalazin-1(2H)-one (77). Pale yellow solid; yield 72.4%; mp

1

45−46 °C; H NMR (600 MHz, CDCl₃) δ 7.93 (d, J = 2.5 Hz, 1H),

7.68 (dd, J = 8.7, 2.5 Hz, 1H), 7.50 (d, J = 8.7 Hz, 1H), 4.33 (t, J = 5.9

Hz, 2H), 3.58 (t, J = 6.5 Hz, 2H), 2.61 (t, J = 4.7 Hz, 2H), 2.53 (dd, J

= 5.4, 3.9 Hz, 2H), 2.34 (p, J = 6.2 Hz, 2H), 1.89−1.74 (m, 4H).

4-(3-Bromopropoxy)-2-(3,4-dichlorophenyl)phthalazin-

1

1(2H)-one (78). Pale yellow solid; yield 72.4%; mp 48−50 °C; H

NMR (600 MHz, CDCl₃) δ 8.51−8.44 (m, 1H), 8.06−8.01 (m, 1H),

7.99 (d, J = 2.5 Hz, 1H), 7.89−7.85 (m, 2H), 7.74 (dd, J = 8.7, 2.5 Hz,

1H), 7.54 (d, J = 8.7 Hz, 1H), 4.54 (t, J = 5.9 Hz, 2H), 3.66 (t, J = 6.5

Hz, 2H), 2.46 (p, J = 6.2 Hz, 2H).

General Procedures for the Synthesis of Compounds 79−

81. Method B. 2-(3,4-Dichlorophenyl)-4,5-dimethyl-6-(3-(pi-

peridin-1-yl)propoxy)pyridazin-3(2H)-one (79). To a suspension

of 73 (2 mmol) and piperidine (2.2 mmol), K₂CO₃(4 mmol) in

acetonitrile (50 mL) and a catalytic amount of KI (1% mol) were

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

the resulting ﬁltrate was evaporated to dryness under reduced pressure.

The residue was suspended in water (10.0 mL) and extracted with

dichloromethane (3 × 25 mL). 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 (10% MeOH/CHCl₃) to yield compounds 79.

Pale yellow oil; yield 82.1%; ¹H NMR (600 MHz, CDCl₃) δ 7.92 (d, J

= 2.5 Hz, 1H), 7.76−7.64 (m, 1H), 7.49 (dd, J = 7.7, 3.4 Hz, 1H), 4.21

(t, J = 6.3 Hz, 2H), 2.50−2.43 (m, 6H), 2.24−2.21 (m, 3H), 2.20−

2.13 (m, 3H), 2.04−1.94 (m, 2H), 1.68−1.58 (m, 4H), 1.46 (br, 2H).

HRMS (ESI) m/z 410.1405 (calcd 410.1397 for C₂₀H₂₆Cl₂N₃O₂[M +

H]⁺).

Hz, 1H), 7.36 (dd, J = 8.6, 2.5 Hz, 1H), 7.08−6.98 (m, 2H), 4.14 (t, J

= 6.5 Hz, 2H), 2.87 (d, J = 11.5 Hz, 2H), 2.47−2.37 (m, 2H), 1.98−

1.85 (m, 4H), 1.60 (d, J = 13.5 Hz, 2H), 1.34 (ddd, J = 11.2, 6.9, 4.0

Hz, 1H), 1.22 (ddd, J = 15.5, 12.3, 3.7 Hz, 2H), 0.91 (d, J = 6.5 Hz,

3H). HRMS (ESI) m/z 396.1244 (calcd 396.1240 for C₁₉H₂₄Cl₂N₃O₂

[M + H]⁺).

2-(3-Chloro-4-ﬂuorophenyl)-6-(3-(4-methylpiperidin-1-yl)-

propoxy)pyridazin-3(2H)-one (71). Pale yellow oil; yield 78.9%;

¹H NMR (600 MHz, CDCl₃) δ 7.55−7.39 (m, 2H), 7.00−6.97 (m,

2H), 6.80−6.75 (m, 1H), 4.23 (t, J = 6.4 Hz, 2H), 2.90 (d, J = 11.5 Hz,

2H), 2.56−2.41 (m, 2H), 2.02−1.86 (m, 4H), 1.71−1.57 (m, 2H),

1.39−1.33 (m, 1H), 1.27−1.21 (m, 2H), 0.92 (d, J = 6.5 Hz, 3H).

HRMS (ESI) m/z 380.1542 (calcd 380.1536 for C₁₉H₂₄ClFN₃O₂[M

+ H]⁺).

2-(3,4-Diﬂuorophenyl)-6-(3-(4-methylpiperidin-1-yl)-

propoxy)pyridazin-3(2H)-one (72). Pale yellow oil; yield 79.3%;¹H

NMR (600 MHz, CDCl₃) δ 7.65−7.61 (m, 1H), 7.54−7.51 (m, 1H),

7.23 (dd, J = 18.4, 9.0 Hz, 1H), 7.05−6.96 (m, 2H), 4.22 (t, J = 6.4 Hz,

2H), 2.90 (d, J = 11.5 Hz, 2H), 2.50−2.41 (m, 2H), 2.05−1.84 (m,

4H), 1.63 (d, J = 13.1 Hz, 2H), 1.44−1.30 (m, 1H), 1.28−1.21 (m,

2H), 0.93 (d, J = 6.5 Hz, 3H). HRMS (ESI) m/z 364.1838 (calcd

364.1831 for C₁₉H₂₄F₂N₃O₂[M + H]⁺).

General Procedures for the Preparation of Intermediates

73−75. 2-(3,4-Dichlorophenyl)-6-hydroxy-4,5-dimethylpyrida-

zin-3(2H)-one (73). 3,4-Dicholrophenylhydrazine hydrochloride (55

mmol) was added to 3,4-dimethylfuran-2,5-dione (50 mmol), 400 mL

of water, and 40 mL of 35% hydrochloric acid solution. The mixture

was heated at reﬂux for 9 h. After it was cooled, the resulting solid was

collected by ﬁltration, washed with ice−water. The solid was dissolved

in saturated sodium bicarbonate solution, and after ﬁltration, the

resulting ﬁltrate was neutralized with 1 mol/L hydrochloric acid. The

precipitate thus formed was ﬁltered and washed with water. The

resulting 73 was a white solid. Yield 77.4%; mp 255−257 °C; ¹H NMR

(600 MHz, DMSO-d₆) δ 11.42 (s, 1H), 7.97 (d, J = 2.4 Hz, 1H),

7.74−7.59 (m, 2H), 2.09 (s, 6H).

2-(3,4-Dichlorophenyl)-4-hydroxy-5,6,7,8-tetrahydrophtha-

lazin-1(2H)-one (74). Pale yellow solid; yield 79.1%; mp 261−263

°C; ¹H NMR (600 MHz, DMSO-d₆) δ 11.41 (s, 1H), 7.97 (d, J = 2.4

Hz, 1H), 7.71 (dt, J = 8.8, 5.6 Hz, 2H), 3.35 (s, 2H), 2.56−2.46 (m,

2H), 2.44 (s, 2H), 1.69 (s, 2H).

2-(3,4-Dichlorophenyl)-4-hydroxyphthalazin-1(2H)-one (75).

Pale yellow solid; yield 78.2%; mp 271−272 °C; ¹H NMR (600 MHz,

DMSO-d₆) δ 11.45 (s, 1H), 8.51−8.44 (m, 1H), 8.06−8.01 (m, 1H),

7.99 (d, J = 2.5 Hz, 1H), 7.89−7.85 (m, 2H), 7.74 (dd, J = 8.7, 2.5 Hz,

1H), 7.54 (d, J = 8.7 Hz, 1H)

2-(3,4-Dichlorophenyl)-4-(3-(piperidin-1-yl)propoxy)-

5,6,7,8-tetrahydrophthalazin-1(2H)-one (80). Pale yellow oil;

1

yield 79.8%; H NMR (600 MHz, CDCl₃) δ 7.92 (d, J = 2.5 Hz,

1H), 7.67 (dd, J = 8.7, 2.5 Hz, 1H), 7.48 (d, J = 8.7 Hz, 1H), 4.21 (t, J

= 6.3 Hz, 2H), 2.64−2.29 (m, 10H), 2.05−1.92 (m, 2H), 1.83−1.71

(m, 4H), 1.66−1.53 (m, 4H), 1.46 (br, 2H). HRMS (ESI) m/z

463.1557 (calcd 436.1553 for C₂₂H₂₈Cl₂N₃O₂[M + H]⁺).

2-(3,4-Dichlorophenyl)-4-(3-(piperidin-1-yl)propoxy)-

1

phthalazin-1(2H)-one (81). Pale yellow oil; yield 80.3%; H NMR

(600 MHz, CDCl₃) δ 8.49−8.43 (m, 1H), 8.06−8.00 (m, 1H), 7.98

(d, J = 2.5 Hz, 1H), 7.86−7.73 (m, 2H), 7.74 (dd, J = 8.7, 2.5 Hz, 1H),

7.52 (d, J = 8.7 Hz, 1H), 4.41 (t, J = 6.3 Hz, 2H), 2.58−2.47 (m, 6H),

2.20−2.06 (m, 2H), 1.66−1.57 (m, 4H), 1.47 (br, 2H). HRMS (ESI)

m/z 432.1249 (calcd 432.1240 for C₂₂H₂₄Cl₂N₃O₂[M + H]⁺).

Single Crystal X-ray Structure Determination of 54.

Crystallization and Sample Preparation. Crystals of the hydro-

chloride salt of 54 were obtained by slow evaporation of cosolvent of

methanol and acetone solution of the compound.

Data Collection. Crystal structure determination for the hydro-

chloride salt of 54 was carried out using Bruker SMART APEX II.

Structure Solution and Reﬁnement. Crystal structure solution

was achieved using direct methods as implemented in SHELXTL⁶⁶

and visualized using the program XP. Missing atoms were

subsequently located from diﬀerence Fourier synthesis and added to

the atom list. Least-squares reﬁnement on F²using all measured

intensities was carried out using the program SHELXTL. All non-

hydrogen atoms were reﬁned, including anisotropic displacement

parameters. The chlorine anion corresponding to one cationic

molecule is distributed in two half-positions located on 2-fold rotation

axes and shared with the neighboring cationic molecules.

General Procedures for the Preparation of Intermediates

76−78. 6-(3-Bromopropoxy)-2-(3,4-dichlorophenyl)-4,5-dime-

thylpyridazin-3(2H)-one (76). To a solution of 73 (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 = 15/1) to yield

Crystal Data at sf20150715_zyc_0m. C₁₈H₂₆Cl₃N₃O₄, crystal

size 0.39 × 0.32 × 0.3 mm³, 454.77 g mol⁻¹, triclinic, P1, a =

8.7813(3) Å, b = 10.4026(3) Å, c = 12.2094(4) Å, α = 97.6772(14)°, β

= 91.8626(13)°, γ = 105.0210(12)°, V = 1064.92(6) Å³, Z = 2, ρ_calcd

=1.418 Mg/m³, R1 = 0.0364 (0.0427), wR2 = 0.1002 (0.1063),

absorption coeﬃcient is 0.459 mm⁻¹, F(000) = 476, goodness-of-ﬁt on

F²is 1.028, largest diﬀerence peak (hole) is 0.337 (−0.289) e Å⁻³.

1

compound 76. Pale yellow oil; yield 71.8%; H NMR (600 MHz,

O

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Article

Receptor Binding Studies. 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), (+)-SKF-10047 (Sigma-Aldrich), and male Dunkin

Hartley guinea pig brain membrane. Chemicals and reagents were

purchased from diﬀerent commercial sources and of analytical grade.

Membrane Preparation. Membrane preparation was performed

by previously reported method.³⁶Crude P2 membranes were prepared

from frozen guinea pig brains minus cerebellum. Tissues were

homogenized in ice-cold 10 mmol Tris-sucrose buﬀer (0.32 mol

sucrose in 10 mmol 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 centrifuged at 31 000g 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 31 000g for 15 min, the pellet

was resuspended by gentle homogenization in 10 mmol of 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.⁶⁷Subsequently, the

preparation contained 4 mg protein/mL.

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.³⁶with minor modiﬁcations.⁶⁸The

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 (+)-SKF-10047 to block σ₁sites. To each total binding assay

tube were added 850 μL of the tissue suspension, 50 μL of 3.0 nM

[³H]-DTG, 50 μL of 400 nM (+)-SKF-10047, 50 μL Tris-HCl buﬀer,

pH 8.0. To each nonspeciﬁc binding assay tube were added 850 μL of

the tissue suspension, 50 μL of [³H]-DTG, 50 μL of 400 nM (+)-SKF-

10047, 50 μL of 10 μM DTG. To each speciﬁc binding assay tube

were added 850 μL of the tissue suspension, 50 μL of [³H]-DTG, 50

μL of 400 nM (+)-SKF-10047, 50 μL of reference drug or test

compounds solution in various concentrations (10⁻⁵mol to 10⁻¹⁰

mol). 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 54 was evaluated by using guinea pig brain membranes

binding assays for σ₁receptor. The binding aﬃnities in either the

absence or presence of 1 mM phenytoin were measured to identify the

functional (agonistic or antagonistic) nature.⁵⁹

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, (+)-SKF-10047

(blocked the σ₁receptor) guinea pig brain membranes.

In Vivo Test. Animals. Chinese Kun Ming (KM) mice (20 2.0

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 studies involving animals in this research follow the

guidelines of the bylaw of experiments on animals and have been

approved by the Ethics and Experimental Animal Committee of

Jiangsu Nhwa Pharmaceutical Co., Ltd.

For the σ₁receptor binding assays, the total binding (TB) was

determined in the presence of the radioligand [³H]-(+)-pentazocine.

Nonspeciﬁc binding (NB) was determined in the presence of the

radioligand [³H]-(+)-pentazocine and haloperidol, while the com-

pound binding (CB) was determined in the presence of the

radioligand [³H]-(+)-pentazocine and the determining compound.

Speciﬁc binding (SB) is calculated by the total binding (TB) minus the

nonspeciﬁc binding (NB) at a particular concentration of radioligand.

Percentage of inhibition (%) was calculated as the following equation:

percentage of inhibition (%) = [(TB − CB)/(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 6 concentration range (10⁻⁵mol

to 10⁻¹⁰mol), 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_d, its receptor dissociation constant, was calculated

for each labeled ligand (for [³H]-(+)-pentazocine, K_dwas 1.53 0.30

nM (n = 3), and for [³H]-DTG, K_dwas 6.76 0.94 nM (n = 3)).

Mean K_ivalues and SEM are reported for at least three independent

experiments.

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.

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.⁵⁵For 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 3 s (sampling rate of 4

kHz, low-pass ﬁltered at 1 kHz) in intervals of 30 s. Tail currents were

measured at −50 mV in control and in the presence of the drug at

concentrations determined empirically. 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

σ₁Receptor Binding Assays. Binding of [³H]-(+)-pentazocine at

σ₁receptor was performed according to D. L. Dehaven-Hudkins et

al.⁵⁴with minor modiﬁcations.^42,43The 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 were added 900 μL of the tissue suspension,

50 μL of 4.0 nM [³H]-(+)-pentazocine, 50 μL Tris-HCl buﬀer, pH 8.0.

To nonspeciﬁc binding each assay tube were added 900 μL of the

tissue suspension, 50 μL of [³H]-(+)-pentazocine, 50 μL of 10 μM

haloperidol. To each speciﬁc binding assay tube were added 900 μL of

the tissue suspension, 50 μL of [³H]-(+)-pentazocine, 50 μL of

reference drug or test compounds solution in various concentrations

(10⁻⁵mol to 10⁻¹⁰mol). 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

P

DOI: 10.1021/acs.jmedchem.5b01416

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Package for the Social Sciences (SPSS) spreadsheets for further

analysis.

Motor Coordination (Rotarod Test).⁴²The motor performance

of mice was assessed by means of an automated rotarod (YLS-4C,

China). Mice were trained, and those that could not stay moving on

the rod for 300 s in 10 rpm were discarded from the study before drug

treatment. In the test, mice were required to walk against the motion

of an elevated rotating drum at 10 rpm and the latencies to fall-down

were recorded. With the selected animals, rotarod latencies were

measured 30, 60, 90, and 120 min after ig administration of drugs.

Locomotor Activity Test. Locomotor activity was assessed in 14

automated activity frames equipped with infrared photobeam emitters

and sensors. To assess drug eﬀect on exploratory locomotor activity,

the mice were transferred to new home cages immediately before test

start and activity was measured for 30 min. Test or reference

compounds were orally administered 30 min before test start at the

following doses of 40, 80, 160 mg/kg; haloperidol was at 0.1, 0.3, 1

mg/kg. The average speed was measured before and 30, 60, 90, and

120 min after treatment.

Pharmacokinetics Study in Rats. The HPLC conditions were as

follows: column, XSELECT CSH XP C18 (2.1 mm × 50 mm, 2.5

μm); mobile phase, 0.025% FA and 1 mM NH₄OAc (ROE

SCIENTIFIC INC, USA) in water/acetonitrile (Merck Company,

Germany) (v:v, 45:55); ﬂow rate, 0.6 mL/min; column temperature,

50 °C. UV detection was performed at 210 nm. For routine compound

54 screening, rats (n = 3/group) were dosed via the lateral tail vein at

the indicated dose for iv administration (5 mg/kg, 100% saline) or via

oral gavage (80 mg/kg, suspension in 0.5% methylcellulose). At 30

min and 1, 2, 3, 4, 5, 6, 8 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 LC/MS analysis by

protein precipitation with acetonitrile. The plasma samples were

analyzed for drug and internal standard via the LC−MS/MS protocol.

Statistics. To estimate the potency of test and reference

compounds, the ED₅₀values and their 95% conﬁdence limits were

calculated by using the program SPSS (Statistical Package for the

Social Science).

Formalin Test. Formalin tests were performed as described by

Cendan

́

et al.⁶¹with minor modiﬁcations. 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 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-voked nociception phase

II.⁴⁹The 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_Vrepresent the

licking time in vehicle injected animals, LT_Dmeans licking time in

drug-injected animals.

CCI Model.^62,63The 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 resulted in the single administration and repeated

administration, respectively.

The CCI of the sciatic nerve surgery was performed as described by

Bennett and Xie^62awith 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 ∼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. Two tests were performed: the von Frey test

(electronic von Frey rigid tips with 90 g range (IITC Life Science Inc.,

U.S.A, 2390)) 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. 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 withdrawal 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.

ASSOCIATED CONTENT

■

S

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.5b01416. CCDC 1423214 contains the supplementary

crystallographic data for this paper. These data can be obtained

free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html

(or from the Cambridge Crystallographic Data Centre, 12

Union Road, Cambridge CB21EZ, U.K. (fax (+44) 1223-336-

033, e-mail deposit@ccdc.cam.ac.uk).

Inhibition curves of functional proﬁle of σ₁receptor;

additional receptors binding aﬃnities of compound 54;

data of compound 54 in phase I of the mouse formalin

test; ¹H NMR, HR-MS, and HPLC of compound 54; X-

ray crystal data of compound 54 (PDF)

Molecular formula strings (CSV)

AUTHOR INFORMATION

■

Corresponding Author

*E-mail: gszhang@mail.hust.edu.cn. Phone: +86-27-87792235.

Fax: +86-27-87792170.

Author Contributions

^§X.C. and Y.C. contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

Q

DOI: 10.1021/acs.jmedchem.5b01416

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

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ACKNOWLEDGMENTS

■

We are grateful for ﬁnancial support from the NSFC (Grant

81573291) and the National Science and Technology Major

Project “Key New Drug Creation and Manufacturing Program”

(Grant 2012ZX09103-101-010). The authors thank Prof.

Liangren Zhang, Zhenming Liu, and Hongwei Jin of State

Key Laboratory of Natural and Biomimetic Drugs, School of

Pharmaceutical Sciences, Peking University, for performing

HRMS.

ABBREVIATIONS USED

■

CNS, central nervous system; NMDA, N-methyl-D-aspartate;

hERG, ether-a-go-go-related gene; ig, intragastric administra-

tion; CCI, chronic constriction injury; ED₅₀, 50% eﬀective

dose; LD₅₀, median lethal dose

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sigma receptor as a ligand-regulated auxiliary potassium channel

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