Synthesis and biological evaluation of new 6-hydroxypyridazinone benzisoxazoles: Potential multi-receptor-targeting atypical antipsychotics

Article abstract of DOI:10.1016/j.ejmech.2016.09.008

In recent years, multi-targeting directed ligands have attracted great interest as possible new atypical antipsychotics. Combinations of dopamine and serotonin receptor ligands within single molecules might afford new therapeutic opportunities. Herein, we describe the synthesis of a novel series of 6-hydroxypyridazinone benzisoxazoles and their binding behaviors to different receptors in terms of atypical antipsychotic behaviors. The most potent compound (46) exhibited excellent affinities for certain receptors (D₂, K_i?=?0.5?±?0.07?nM; 5-HT_1A, K_i?=?5.9?±?0.8?nM; 5-HT_2A, K_i?=?0.3?±?0.01?nM; 5-HT₆, K_i?=?0.5?±?0.04?nM) and combined with low affinities for the H₁, 5-HT_2C, and adrenergic α₁receptors. In contrast to risperidone, compound 46 exhibited a high cataleptic threshold; this may be useful in the development of a novel class of drugs treating schizophrenia.

Full text of DOI:10.1016/j.ejmech.2016.09.008

Accepted Manuscript
Synthesis and biological evaluation of new 6-hydroxypyridazinone benzisoxazoles:
Potential multi-receptor-targeting atypical antipsychotics
Xudong Cao, Yin Chen, Yifang Zhang, Yinli Qiu, Minquan Yu, Xiangqing Xu, Xin Liu,
Bi-Feng Liu, Guisen Zhang
PII:
S0223-5234(16)30736-X
10.1016/j.ejmech.2016.09.008
EJMECH 8874
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 July 2016
Revised Date: 2 September 2016
Accepted Date: 3 September 2016
Please cite this article as: X. Cao, Y. Chen, Y. Zhang, Y. Qiu, M. Yu, X. Xu, X. Liu, B.-F. Liu, G. Zhang,
Synthesis and biological evaluation of new 6-hydroxypyridazinone benzisoxazoles: Potential multi-
receptor-targeting atypical antipsychotics, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.09.008.
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Graphical Abstract
A series of new 6‑hydroxypyridazinone benzisoxazoles have been synthesized and the
target compounds evaluated for antipsychotic activity in in vitro and vivo.

ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of New 6‑Hydroxypyridazinone
Benzisoxazoles: Potential Multi-receptor-targeting Atypical Antipsychotics
Xudong Cao,†,§ Yin Chen,‡,§ Yifang Zhang,† Yinli Qiu,‡ Minquan Yu, ‡ Xiangqing
Xu,‡ 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
Author Contributions
§Xudong Cao and Yin Chen contributed equally to this work.
Abstract
In recent years, multi-targeting directed ligands have attracted great interest as
possible new atypical antipsychotics. Combinations of dopamine and serotonin
receptor ligands within single molecules might afford new therapeutic opportunities.
Herein, we describe the synthesis of a novel series of 6‑hydroxypyridazinone
benzisoxazoles and their binding behaviors to different receptors in terms of atypical
antipsychotic behaviors. The most potent compound (46) exhibited excellent affinities
for certain receptors (D2, Ki = 0.5 ± 0.07 nM; 5-HT1A, Ki = 5.9 ± 0.8 nM; 5-HT2A, Ki =
0.3 ± 0.01 nM; 5-HT6, Ki = 0.5 ± 0.04 nM) and combined with low affinities for the
H1, 5-HT2C, and adrenergic α1 receptors. In contrast to risperidone, compound 46
exhibited a high cataleptic threshold; this may be useful in the development of a novel
class of drugs treating schizophrenia.
Keywords: 6‑Hydroxypyridazinone; Multi-receptor-target; Atypical antipsychotics
1. Introduction
Schizophrenia is a chronic and disabling disease afflicting nearly 1% of the general
population. [1] The illness per se is characterized by heterogeneous groupings of
clinical features that can be subgrouped into different symptom domains: positive
(delusions and hallucinations), negative (apathy, amotivation, and blunted affect), and
cognitive symptoms (impaired memory, attention, and executive functions). [2]

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Without treatment, patients may undergo prolonged institutionalization and suffer
from neglect, poor health, and substance abuse. These events obviously burden
patients and impose financial costs on society. [3] The mainstay medications for
schizophrenia are antipsychotic drugs, but these have significant side effects. [4 - 6]
Antipsychotics fall into two classes, typical and atypical; these differ in terms of their
side effects and receptor-binding profiles. [7] All antipsychotics antagonize the
dopamine D2 receptor, but atypical antipsychotics act on a wider spectrum of
receptors, including all of the dopamine, serotonergic, cholinergic, and adrenergic
receptors.[7] Although typical antipsychotics such as haloperidol and chlorpromazine
effectively treat the positive symptoms of schizophrenia, they trigger a variety of
undesirable adverse extrapyramidal side-effects (EPS) that can be either acute
(Parkinsonism, akathisia, dystonia) or of later onset (tardive dyskinesia).[8] Compared
to the typical agents, atypical antipsychotics such as clozapine, risperidone,
iloperidone, olanzapine, and aripiprazole (Fig. 1) induced fewer EPS than did
haloperidol (even at low doses).[9] However, atypical antipsychotics trigger many
other adverse events, clozapine and olanzapine are the most likely to lead to weight
gain and glucose and lipid abnormalities, Sertindole and ziprasidone carry the
strongest risk of QTc prolongation among second-generation antipsychotics. [10]
Therefore, the discovery of safer and more effective antipsychotic medications that
effectively treat all aspects of the disease is tremendously important.
Several studies have sought to develop novel atypical antipsychotic drugs that
preferentially bind to the dopamine and serotonin receptors. [11] The dopamine
receptors are archetypal examples of G-protein-coupled receptors (GPCRs), which
mediate neurotransmission. Defects in dopaminergic neurotransmission trigger
several neurological and psychiatric disorders including Huntington’s disease,
attention-deficit hyperactivity disorder (ADHD), mood disorders, Parkinson’s disease,
and schizophrenia. [7]
Over the last 30 years, drugs targeting serotonin (5-HT) receptors have been
intensively investigated in the context of schizophrenia; many articles and reviews
suggest that drugs simultaneously targeting dopamine D2 and 5-HT receptors,

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especially the 5-HT1A and 5-HT2A receptors, may be of pharmacotherapeutic benefit
to schizophrenia patients. [4], [12] The 5-HT1A receptor is a major biological target of
antipsychotic medications. Activation of the postsynaptic 5-HT1A receptor increases
dopamine release in the frontal cortex, which may in turn reduce negative symptoms
and cognitive deficits in patients with schizophrenia. [7] The 5-HT2A receptor is
strategically located on dopaminergic neurons (which are generally excitatory) and
thus acts on brain dopaminergic neurotransmission. [13]. Blockade of the 5-HT2A
receptor with second-generation antipsychotics counteracts excessive D2 receptor
blockade and the EPS associated with typical antipsychotics. [12] An increasing body
of evidence supports the notion that modulation of 5-HT receptors improves treatment
of disorders such as schizophrenia. Moreover, one aim in development of
antipsychotic drugs is elimination or diminution of side effects. The 5-HT2C and
histamine H1 receptors may be involved in weight gain, and the adrenergic α1 receptor
is associated with the orthostatic hypotension that develops when schizophrenia is
treated using atypical antipsychotic drugs. [14] Thus, the aim of our present work was
to develop novel antipsychotics active on the dopamine D2, the serotonin 5-HT1A, and
the 5-HT2A receptors, but with low affinities for the histamine H1 receptor and
adrenergic α1 receptor. Such drugs might effectively reduce positive and negative
symptoms as well as cognitive impairment without inducing weight gain or
orthostatic hypotension.
Pyridazines and their oxygenated derivatives, pyridazinones, constitute a significant
group of six-carbon heterocyclic compounds that exhibit various biological activities.
[15] The central nervous system activities have attracted considerable interest;
compounds based on the pyridazine or pyridazinone scaffold have recently been
widely studied in terms of treatment of chronic pain and cognitive disorders [15b],
schizophrenia [16], and anxiety and depression. [17] Recently, novel σ1 receptor
ligands (based on 6‑hydroxypyridazinones) have been developed to treat neuropathic
pain. [18]
Benzoxazole and benzisoxazole are isomeric compounds, which are present in many

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natural products and often incorporated in drug design.[19a] Benzisoxazoles is an
important structure and has been widely used to prepared the biologically active
compounds, including atypical antipsychotics and anticonvulsant.[19b-e] The
arylpiperazine (piperidine) privileged core is recognized by the monoaminergic
receptors (e.g., 5-HT1A, 5-HT2A, D2, D3, a1, H1). [19f] We earlier explored the
structure–activity relationships (SARs) of arylpiperazine (piperidine) derivatives; we
developed
novel
multi-receptor
atypical
antipsychotics
containing
6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole. [19c-e] The optimal compound 1 of these
1,2-benzisoxazole derivatives exhibited obvious atypical antipsychotic potency; the
compound had high affinities for the dopamine D2, D3, and serotonin 5-HT1A and
5-HT2A receptors. [19d, 19e] We continued this work in efforts to better extend the
pharmacological activities of 6‑hydroxypyridazinones; we designed a new series of
1,2-benzisoxazole derivatives using molecular hybridization methods. [20] (Fig. 2)
The basic amino moiety of compound
2
was replaced with
6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole, compound 24 show moderate affinities to
dopamine D2 and serotonin 5-HT1A and 5-HT2A receptors. To further explore the SAR,
other arylpiperazine (piperidine) derivatives were introduced by biososteric
replacement; the new multi-targeting-ligand series 3 was prepared. The best
compound (46), which contained 6-fluoro-3-(4-piperidyl)-1, 2-benzisoxazole,
exhibited high affinity for the dopamine D2 and the serotonin 5-HT1A, 5-HT2A, and
5-HT6 receptors. Herein, we performed basic behavioral screening of atypical
antipsychotic potencies.

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2. Chemistry
The new compounds were synthesized according to Schemes 1-3. The starting 6‑
hydroxypyridazinone derivatives were prepared as described in our previous work.
[18] As shown in Scheme 1, our initial investigation focused on
2-(3,4-dichlorophenyl)-6-hydroxypyridazine-3(2H)-one (4); intermediate 5 was
produced by alkylation with 4 and 1, n-dibromoalkanes and then reacted with the
corresponding arylpiperazines (piperidines) under mildly basic conditions to yield the
final compounds 6-26 (Table 1).
The routes depicted in Schemes 2 and 3 were used to prepare the substituted 6‑
hydroxypyridazinone scaffold compounds 29-31 and 34-50. Intermediates 28 and 33
were obtained via standard alkylation of the substituted pyridazinone scaffold
compounds 27 and 32 with 1, 3-dibromopropane and then followed by reaction with
6-fluoro-3-(4-piperidyl)-1, 2-benzisoxazole under basic conditions yielded the final
compounds 29-31 and 34-50 (Tables 2 and 3).
To explore configurations, the most active compound 46 (Table 3) was subjected to
single-crystal X-ray diffraction (SCXRD). As no suitable crystals were obtained from
the free base, limited salt screening was performed. The hydrochloride yielded
crystals suitable for SCXRD, and a relative configuration was reliably determined. As
shown in Figure 3, this confirmed the configuration assigned above.
3. Pharmacology
3.1. In vitro studies
All of the new compounds were dissolved to 5% (w/v) in DMSO (initial
concentration). The following specific radioligands and tissue sources were used: (a)
the serotonin 5-HT1A receptor, [3H]8-OH-DPAT, from rat brain cortex; (b) the
serotonin 5-HT2A receptor, [3H]ketanserin in the present of 4-dione hydrochloride
hydrate (35 nM) , from rat brain cortex; (c) the serotonin 5-HT2C receptor,
[3H]mesulergine in the present of spiperone (40 nM), from rat brain cortex; (d) the
serotonin 5-HT6 receptor, [3H]lysergic acid diethylamide, from 5-HT6-C3 cells
(CHO-K1); (e) the dopamine D2 receptor, [3H]spiperone, from rat striatum; (f) the
histamine H1 receptor, [3H]mepyramine, from guinea pig cerebellum; and (g) the

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adrenergic α1 receptor, [3H]prazosin, from rat cerebral cortex.
In 5-HT1A receptor binding assays, total binding (TB) was determined in the
presence of the radioligand [3H]8-OH-DPAT. Nonspecific binding (NB) was
determined in the presence of the radioligand [3H]8-OH-DPAT and serotonin,
whereas compound binding (CB) was determined in the presence of the radioligand
[3H]8-OH-DPAT and the compound of interest. Each specific binding (SB) was
calculated as the total binding (TB) minus the nonspecific binding (NB) at a particular
concentration of radioligand. Each percentage of inhibition (%) was calculated as
follows: percentage of inhibition (%) = [(TB − CB)/(TB − NB)] × 100.
Blank binding experiments (5% [v/v] DMSO only) were performed; DMSO had no
effect. All compounds were tested at least three times over a six-fold concentration
range (10-5 M to 10-12 M). IC50 values were determined by nonlinear regression
analysis with fitting to the Hill equation curve. Ki values were calculated using the
Cheng and Prussoff equation, Ki = IC50/(1 + C/Kd), where C represents the
concentration of the hot ligand used, and Kd the receptor dissociation constant of each
labeled ligand. The mean Ki values and SEs were derived in at least three independent
experiments. Binding affinities are expressed as Ki values in Tables 1-3.
3.2. In vivo studies
Selected compounds were further evaluated using in vivo animal models featuring
apomorphine-induced climbing, MK801-induced hyperactivity, and catalepsy.
4. Results and Discussion
4.1. In vitro studies of the new compounds
As mentioned above, ligands for the D2, 5-HT1A, and 5-HT2A receptors are of
increasing therapeutic interest, and compounds of various chemical groupings have
been extensively studied in searches for potentially novel antipsychotics. In the
present work, our initial focus was on the effects of various arylpiperazine (piperidine)
moieties in terms of affinities for the D2, 5-HT1A, and 5-HT2A receptors (Table 1,
compounds 6-26). Table 1 shows that when electron-donating groups (methoxy
groups in the ortho- or parapositions) were introduced into the phenylpiperazine and
phenyl rings, compounds 6-8 exhibited good affinities for the 5-HT1A receptor, but

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weak affinities for the D2 and 5-HT2A receptors, compared to those of risperidone
(5-HT1A, Ki = 182 ± 15 nM). The 5-HT1A receptor is important in terms of the
therapeutic efficacy of atypical antipsychotic drugs. Specifically, 5-HT1A receptor
activity reduces negative symptoms and the incidence of EPS in patients with
schizophrenia. [19] Upon replacement of the 2,3-dimethylphenylpiperazine,
compound 9 exhibited moderate affinity for the 5-HT2A receptor. Substitution of the
N-phenyl group with a pyridine or pyrimidine group (compounds 10 and 11) reduced
the affinities for all three receptors. Compounds 12-16 contained halogen substituents
of the phenyl groups, and differed in terms of binding affinities to the three receptors.
Substitution of the phenyl ring with fluorine in the ortho- or paraposition (compounds
12 and 13) increased the affinities for 5-HT1A and 5-HT2A; compounds 14-16 with a
trifluoromethyl group or a chlorine atom on the phenyl ring exhibited reduced binding
affinities. Compound 17, in which a benzyl group replaced the N-phenyl group,
exhibited reduced affinities for all three receptors. When the phenyl ring was replaced
with a heterocyclic group (compounds 18 and 19), good affinities for 5-HT2A (18, Ki =
81.2 ± 10.5 nM; 19, Ki = 75.1 ± 9.0 nM) were apparent. Compound 20, with a
benzofuran group, exhibited good affinity for 5-HT1A (Ki = 64.8 ± 7.8 nM). When the
piperazine was replaced by piperidine, the resulting compounds 21 and 22 exhibited
moderate affinities for 5-HT2A (21, Ki = 107 ± 13.2 nM; 22, Ki = 74.5 ± 11.3 nM).
When the amine moiety was changed to (4-fluorophenyl)(piperidin-4-yl)methanone
(23), the affinities for 5-HT1A and 5-HT2A increased (Ki: 5-HT1A, 0.02 ± 0.01 nM;
5-HT2A, 13.8 ± 1.2 nM), accompanied by a moderate affinity for D2 (Ki =179 ± 15.3
nM). Compound 24, containing 6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole, exhibited
remarkably increased binding affinities (Ki: D2, 7.8 ± 0.8 nM; 5-HT1A, 25.0 ± 3.2 nM;
5-HT2A, 21.7 ± 2.4 nM).
We explored the effect of the distance between the 6‑hydroxypyridazinone scaffold
and the 6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole moiety. Compared to compound
24, interposing chain lengths of four (25) or five (26) carbons significantly reduced
affinity for the 5-HT1A receptor (25, 5-HT1A, Ki = 117 ± 15.8 nM; 26, 5-HT1A, Ki =326
± 29.4 nM), but activities against the D2 and 5-HT2A receptors were retained (albeit

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less than those of compound 24). Taken together, the data indicate that the three
carbon chain length (compound 24) was the most active.
To further investigate the SAR, we replaced groups on the 4- and 5-positions of the
pyridazinone ring. We first replaced the hydrogen atom with a methyl group (Table 2,
compound 29). Compared to compound 24, compound 29 had less affinity for the D2,
5-HT1A, and 5-HT2A receptors, due to either the size or the hydrophobicity of the
substituting group. Substitutions of cyclohexyl and phenyl groups yielded compounds
30 and 31, which behaved as did analogs containing methyl groups (29), confirming
that large substitutions may hinder receptor binding.
We finally explored the effects of substitution on the 2-position of the pyridazinone
ring on SAR. When the 3,4-dichlorophenyl ring was replaced with a phenyl group,
compound 34 retained moderate affinity for the three receptors. Compared to
compound 34, compounds 35 and 36 (containing electron-donating groups;
4-methylphenyl and 2-naphthyl ring) on the 2-position of the pyridazinone ring
exhibited improved binding affinities, albeit less than those of compound 24. When
halogens were introduced into the phenyl group, compound 37 (with fluorine on the
phenyl ring) exhibited similar affinities for the D2 and 5-HT2A receptors, but only a
weak affinity for the 5-HT1A receptor (37, Ki, D2, 10.8 ± 1.2 nM; 5-HT1A, 52.8 ± 5.8
nM; 5-HT2A, 25.9 ± 2.6 nM), compared to compound 24. When phenyl rings
substituted with chlorine in different positions were studied, compounds 38-40 had
high affinities for the 5-HT2A receptor, but weak affinities for the D2 and 5-HT1A
receptors (38, 5-HT2A, Ki = 12.3 ± 1.2 nM; 39, 5-HT2A, Ki = 10.3 ± 1.3 nM; 40,
5-HT2A, Ki = 6.0 ± 1.1 nM). Substitution of other dichlorophenyls for the
3,4-dichlorophenyl group yielded compounds 41-44; the 2,3-dichlorophenyl (41) and
2,5-dichlorophenyl (42) groups were associated with excellent affinities for the three
receptors (41, D2, Ki = 0.2 ± 0.01 nM; 5-HT1A, Ki = 9.3 ± 0.8 nM; 5-HT2A, Ki = 0.5 ±
0.04 nM; 42, D2, Ki = 2.7 ± 0.2 nM; 5-HT1A, Ki = 0.01 ± 0.002 nM; 5-HT2A, Ki = 0.7 ±
0.03 nM). Substitution with the 2,4-dichlorophenyl group (43) maintained the D2 and
5-HT2A receptor-binding affinities; with the exception of the 3,5-dichlorophenyl
group (44), only 5-HT2A receptor-binding was maintained (43, D2, Ki = 0.4 ± 0.02 nM;

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5-HT2A, Ki = 0.4 ± 0.03 nM; 44, 5-HT2A, Ki = 9.3± 1.0 nM). This indicates that
affinities for the D2, 5-HT1A, and 5-HT2A receptors depend on the location of
substituents on the phenyl ring. Some activities (albeit less than those of compounds
41 and 42) were retained upon replacement of the 3,4-dichloro moiety with
2,5-difluorophenyl or 2,6-difluorophenyl substituents (45, 46). Substitution of other
difluorophenyls or chlorofluorophenyl for the 3,4-dichlorophenyl group yielded
compounds 47-50, which exhibited excellent affinities only for the 5-HT2A receptor
(47, 5-HT2A, Ki = 2.3± 0.1 nM; 48, 5-HT2A, Ki = 3.2± 0.2 nM; 49, 5-HT2A, Ki = 0.5 ±
0.04 nM; 50, 5-HT2A, Ki = 12.3± 1.2 nM).
Treatment of schizophrenia with atypical antipsychotic drugs has been associated
with weight gain and/or orthostatic hypotension. Two receptors, the histamine H1 and
5-HT2C receptor, have been suggested to be involved in weight gain. [14], [21]
Blockade of the adrenergic α1 receptor triggered orthostatic hypotension; many
antipsychotic agents have this effect. [22] Expression of the rat 5-HT6 receptor is
almost exclusively central; thus, it is found in limbic and memory-related areas, the
olfactory tubercle, the striatum, the nucleus accumbens, the hippocampus, and some
cortical regions.[23] 5-HT6 receptor antagonists improved cognitive functional
performance in various animal models. [24]
In line with approaches toward multiple-receptor targeting by new antipsychotic
agents, compounds 41, 42, 45, and 46, with high affinities for the D2, 5-HT1A, and
5-HT2A receptors, were selected for further tests of binding to the H1, 5-HT2C,
adrenergic α1, and 5-HT6 receptors. We found that compounds 45 and 46 had strong
affinities for the 5-HT6 receptor (45, 5-HT6, Ki = 3.8 ± 0.4 nM; 46, 5-HT6, Ki = 0.5 ±
0.04 nM) compared to those of risperidone (Ki = 1260 ± 115 nM). Such high affinity
is an indication that compounds 45 and 46 may improve cognitive function, if they
blocked the receptor.
Schizophrenia treatment using atypical antipsychotic drugs has been associated
with weight gain and orthostatic hypotension. Three receptors (the histamine H1,
5-HT2C, and adrenergic α1 receptors) have been suggested to be involved in such
adverse events. Hence, it is relevant that compounds 42 and 45 exhibited high

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affinities for the 5-HT2C receptor (42, 5-HT2C, Ki = 1.0 ± 0.1 nM; 45, 5-HT2C, Ki = 9.1
± 1.5 nM), and that compound 41 exhibited good affinity for the histamine H1
receptor (Ki = 16.6 ± 1.8 nM). Furthermore, compound 46 had weak affinities for the
histamine H1, 5-HT2C, and adrenergic α1 receptors (Table 4), suggesting that
compound 46 may minimally elicit treatment-associated weight gain and orthostatic
hypotension.
4.2. Acute toxicities
We found that compound 46 exhibited high affinities for the D2, 5-HT1A, 5-HT2A,
and 5-HT6 receptors, and low affinities for the histamine H1, 5-HT2C, and adrenergic
α1 receptors. The acute toxicity of compound 46 was evaluated in terms of LD50
values. Compound 46 exhibited a good safety profile, even at the highest dose tested
(LD50 > 2,000 mg/kg).
4.3. Behavioral studies
Because of its excellent affinities for serotonergic and dopaminergic receptors and
its good safety profile, compound 46 was considered a promising atypical
antipsychotic agent and was subjected to in vivo pharmacological characterization.
Endogenous dysfunction of NMDA receptor-mediated neurotransmission may
contribute to the pathogenesis of schizophrenia. [25] MK-801-induced hyperactivity
has been used to indirectly evaluate the ability of a compound to oppose cortical
dopaminergic hypofunction induced by NMDA receptor blockade. [26] In this test,
compound 46 significantly inhibited MK-801 induced hyperactivity (Fig. 4), with an
ED50 value of 0.30 mg/kg (Table 5). By comparison, risperidone and haloperidol had
ED50 values of 0.018 and 0.19 mg/kg, respectively.
The apomorphine-induced climbing model has been classically linked to motor
agitation, one of the positive symptoms of schizophrenia. Inhibition or reversal of
apomorphine-induced cage climbing by mice indicates that mesolimbic dopaminergic
D2 receptor antagonism is in play. [26] As shown in Table 5, compound 46 had an
ED50 of 0.47 mg/kg in this test (Fig. 5); by comparison, risperidone and haloperidol
reversed apomorphine-induced climbing with ED50 values of 0.020 and 0.18 mg/kg,
respectively.

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We performed catalepsy testing to predict the incidence of extrapyramidal motor
disorders. [27] As shown in Table 5, haloperidol was most likely to induce catalepsy
(ED50, 0.38 mg/kg), consistent with high-level blockade of the D2 receptor. The
therapeutic indices of compound 46 based on both efficacy (using the apomorphine or
MK-801 models) and side effects (catalepsy) were in the range 50.68–79.40; the
therapeutic indices of risperidone were approximately 19.00–21.11. In contrast to
risperidone, compound 46 had a high catalepsy threshold.
Overall, compound 46 significantly inhibited apomorphine-induced climbing
behavior, and MK-801-induced hyperactivity. Compound 46 also demonstrated a low
propensity to induce unwanted extrapyramidal motor disturbances at the tested dose.
Conclusion
In summary, we have described the syntheses and pharmacological evaluation of a
series of 6‑hydroxypyridazinone benzisoxazoles (potential multi-targeting
antipsychotics). Of the derivatives synthesized, compound 46 exhibited high affinities
for the dopamine D2 and for the serotonin5-HT1A, 5-HT2A, and 5-HT6 receptors, with
low affinities for the H1 and 5-HT2C and adrenergic α1 receptors. Work with in vivo
animal models showed that compound 46 was potentially very useful for treating
schizophrenia without causing catalepsy. Compound 46 exhibited a higher threshold
of catalepsy induction compared to the currently favored atypical antipsychotic,
risperidone. Compound 46 may be a useful progenitor of novel drugs for effectively
treating the positive symptoms of schizophrenia.
5. Experimental section
6.1. Chemistry experimental
Melting points were determined in open capillary tubes and are uncorrected. 1H NMR
spectra were recorded at 600 MHz on a Varian Inova Unity 200 spectrometer in
CDCl3 solution. Chemical shifts were given in d 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). Reagents were all of analytical grade or of chemical
purity. Analytical TLC was performed on silica gel GF254. Column chromatographic

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purification was carried out using silica gel. Purity analysis was carried out by
LC-Mass analysis using an Agilent 1100 system. The HPLC conditions were as
follows: column, Shim-pack ODS 5.0 mm × 150 mm × 2.0 mm I.D (SHIMADZU,
Japanese); mobile phase, 0.018% HCOOH (TEDIA Company, USA)/acetonitrile
(Merck Company, Germany) 65/35; flow rate, 0.2 mL/min; column temperature, 40
oC. Compound purity is determined by high performance liquid chromatography
(HPLC), and all final test compounds were >95% purity. HRMS was measured on
Agilent 6530, Q-TOF.
6.1.1. General procedure for the preparation of compounds 5
To a solution of 3,4-Dichlorophenylhydrazine hydrochloride (55 mmol) and maleic
anhydride (50 mmol) in 400 mL of water, 40 mL of concentrated hydrochloric acid
solution was added. 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 to yield 4. [18] Pale yellow solid; yield 75.8%;
+
mp 291−292 °C;MS (ESI) m/z 257.2 (calcd 257.0 for C10H7Cl2N2O2 [M + H]+).
To a solution of 4 (10 mmol) and 1,3-dibromopropane (1,4-Dibromobutane or
1,5-dibromopentane) (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 5.
6.1.1.1 6-(3-Bromopropoxy)-2-(3,4-dichlorophenyl)pyridazin-3(2H)-one (5a). Pale
yellow solid; yield 71.8%; mp 82-85 °C; MS (ESI) m/z 376.9 (calcd 376.9 for
+
C13H12BrCl2N2O2 [M + H]+).
6.1.1.2 6-(4-Bromobutoxy)-2-(3,4-dichlorophenyl)pyridazin-3(2H)-one (5b). Pale
white solid; yield 72.1%; mp 86-87 °C; 1H NMR (600 MHz, CDCl3) δ 7.89 (d, J = 2.5
Hz, 1H), 7.65 (dd, J = 8.7, 2.5 Hz, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.00 (s, 2H), 4.22 (t,
J = 6.2 Hz, 2H), 3.48 (t, J = 6.5 Hz, 2H), 2.13 – 2.00 (m, 2H), 2.00 – 1.89 (m, 2H).

ACCEPTED MANUSCRIPT
6.1.1.3 6-((5-Bromopentyl)oxy)-2-(3,4-dichlorophenyl)pyridazin-3(2H)-one (5c). Pale
white solid; yield 73.2%; mp 83-84 °C; 1H NMR (600 MHz, CDCl3) δ 7.89 (t, J = 2.8
Hz, 1H), 7.65 (dt, J = 6.1, 3.0 Hz, 1H), 7.52 (t, J = 5.9 Hz, 1H), 7.07 – 6.95 (m, 2H),
4.20 (t, J = 6.4 Hz, 2H), 3.46 (t, J = 6.7 Hz, 2H), 2.09 – 1.89 (m, 2H), 1.89 – 1.75 (m,
2H), 1.69 – 1.58 (m, 2H).
6.1.2. General procedure for the preparation of compounds 6-26
To a suspension of compounds 5 (0.32 mmol) and K2CO3 (1.22 mmol) in acetonitrile
(5.0 mL), arylpiperazine (piperidine) (0.32 mmol) and a catalytic amount of KI were
added and the resulting mixture was refluxed for 7-9 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 (25 mL×3). The combined
organic layers were evaporated under reduced pressure, and the crude product was
purified by means of chromatography (10% MeOH/CHCl3) to yield compounds 6-26.
6.1.2.1. 2-(3,4-dichlorophenyl)-6-(3-(4-phenylpiperazin-1-yl)propoxy)Pyridazin
-3(2H)-one (6). Yield: 80.2%, oil. 1H NMR (600 MHz, CDCl3) δ 7.91 (d, J = 2.4 Hz,
1H), 7.67 (dd, J = 8.7, 2.5 Hz, 1H), 7.55 – 7.50 (m, 1H), 7.29 (dd, J = 8.7, 7.3 Hz, 2H),
7.05 – 6.98 (m, 2H), 6.95 (d, J = 7.9 Hz, 2H), 6.88 (t, J = 7.3 Hz, 1H), 4.28 (t, J = 6.4
Hz, 2H), 3.27 – 3.19 (m, 4H), 2.70 – 2.51 (m, 6H), 2.09 – 1.98 (m, 2H). HRMS (ESI)
m/z 459.1357 (calcd 459.1349 for C23H25Cl2N4O2 [M + H]+).
+
6.1.2.2. 2-(3,4-dichlorophenyl)-6-(3-(4-(2-methoxyphenyl)piperazin-1-yl)propoxy)
pyridazin-3(2H)-one (7). Yield: 81.0%, oil.1H NMR (600 MHz, CDCl3) δ 7.89 (d, J =
2.5 Hz, 1H), 7.65 (dd, J = 8.7, 2.5 Hz, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.06 – 6.98 (m,
3H), 6.98 – 6.89 (m, 2H), 6.87 (dd, J = 8.1, 1.1 Hz, 1H), 4.26 (t, J = 6.4 Hz, 2H), 3.87
(s, 3H), 3.12 (s, 4H), 2.70 (s, 4H), 2.63 – 2.51 (m, 2H), 2.09 – 1.94 (m, 2H). HRMS
+
(ESI) m/z 489.1451 (calcd 489.1455 for C24H27Cl2N4O3 [M + H]+).
6.1.2.3. 2-(3,4-dichlorophenyl)-6-(3-(4-(4-methoxyphenyl)piperazin-1-yl)propoxy)
pyridazin-3(2H)-one (8). Yield: 79.0%, oil. 1H NMR (600 MHz, CDCl3) δ 7.89 (d, J
= 2.5 Hz, 1H), 7.66 (dd, J = 8.7, 2.5 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.06 – 6.96 (m,
2H), 6.92 – 6.90 (m, 2H), 6.89 – 6.82 (m, 2H), 4.27 (t, J = 6.4 Hz, 2H), 3.78 (s, 3H),

ACCEPTED MANUSCRIPT
3.29 – 3.09 (m, 4H), 2.77 – 2.62 (m, 4H), 2.64 – 2.51 (m, 2H), 2.19 – 1.86 (m, 2H).
+
HRMS (ESI) m/z489.1458 (calcd 489.1455 for C24H27Cl2N4O3 [M + H]+).
6.1.2.4. 2-(3,4-dichlorophenyl)-6-(3-(4-(2,3-dimethylphenyl)piperazin-1-yl)propoxy)
pyridazin-3(2H)-one (9). Yield: 78.3%, oil. 1H NMR (600 MHz, CDCl3) δ 7.91 (d, J
= 2.4 Hz, 1H), 7.68 (dd, J = 8.7, 2.5 Hz, 1H), 7.53 (d, J = 8.7 Hz, 1H), 7.10 (t, J = 7.7
Hz, 1H), 7.06 – 6.99 (m, 2H), 6.94 (t, J = 8.2 Hz, 2H), 4.29 (t, J = 6.4 Hz, 2H), 2.94 (t,
J = 4.2 Hz, 4H), 2.62 (dd, J = 26.6, 19.3 Hz, 6H), 2.29 (s, 3H), 2.25 (s, 3H), 2.14 –
+
1.99 (m, 2H). HRMS (ESI) m/z 487.1647 (calcd 487.1662 for C25H29Cl2N4O2 [M +
H]+).
6.1.2.5. 2-(3,4-dichlorophenyl)-6-(3-(4-(pyridin-2-yl)piperazin-1-yl)propoxy)
pyridazin-3(2H)-one (10). Yield: 76.8%, oil. 1H NMR (600 MHz, CDCl3) δ 8.24 –
8.14 (m, 1H), 7.88 (d, J = 2.5 Hz, 1H), 7.64 (dd, J = 8.7, 2.5 Hz, 1H), 7.56 – 7.45 (m,
2H), 7.05 – 6.96 (m, 2H), 6.69 – 6.60 (m, 2H), 4.26 (t, J = 6.4 Hz, 2H), 3.64 – 3.43 (m,
4H), 2.67 – 2.32 (m, 6H), 2.23 – 1.90 (m, 2H). HRMS (ESI) m/z 460.1310 (calcd
+
460.1302 for C22H24Cl2N5O2 [M + H]+).
6.1.2.6. 2-(3,4-dichlorophenyl)-6-(3-(4-(pyrimidin-2-yl)piperazin-1-yl)propoxy)
pyridazin-3(2H)-one (11). Yield: 77.2%, oil. 1H NMR (600 MHz, CDCl3) δ 8.30 (d, J
= 4.7 Hz, 2H), 7.88 (d, J = 2.5 Hz, 1H), 7.64 (dd, J = 8.7, 2.5 Hz, 1H), 7.50 (d, J = 8.7
Hz, 1H), 7.08 – 6.89 (m, 2H), 6.48 (t, J = 4.7 Hz, 1H), 4.25 (t, J = 6.3 Hz, 2H), 3.90 –
3.80 (m, 4H), 2.54 (dt, J = 7.2, 6.2 Hz, 6H), 2.04– 1.98 (m, 2H). HRMS (ESI) m/z
+
461.1267 (calcd 461.1254 for C21H23Cl2N6O2 [M + H]+).
6.1.2.7. 2-(3,4-dichlorophenyl)-6-(3-(4-(4-fluorophenyl)piperazin-1-yl)propoxy)
pyridazin-3(2H)-one (12). Yield: 78.9%, oil. 1H NMR (600 MHz, CDCl3) δ 7.89 (d, J
= 2.5 Hz, 1H), 7.66 (dd, J = 8.7, 2.5 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.06 – 6.99 (m,
2H), 6.99 – 6.95 (m, 2H), 6.90 – 6.88 (m, 2H), 4.27 (t, J = 6.4 Hz, 2H), 3.20 – 3.07 (m,
4H), 2.74 – 2.63 (m, 4H), 2.61 – 2.52 (m, 2H), 2.02 (dd, J = 14.2, 6.7 Hz, 2H). HRMS
+
(ESI) m/z 477.1249 (calcd 477.1255 for C23H24Cl2FN4O2 [M + H]+).
6.1.2.8. 2-(3,4-dichlorophenyl)-6-(3-(4-(2-fluorophenyl)piperazin-1-yl)propoxy)
pyridazin-3(2H)-one (13). Yield: 79.1%, oil. 1H NMR (600 MHz, CDCl3) δ 7.89 (d, J
= 2.5 Hz, 1H), 7.66 (dd, J = 8.7, 2.5 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.11 – 7.01 (m,

ACCEPTED MANUSCRIPT
4H), 6.99 – 6.92 (m, 2H), 4.27 (t, J = 6.4 Hz, 2H), 3.13 (s, 4H), 2.68 (s, 4H), 2.62 –
2.52 (m, 2H), 2.20 – 1.94 (m, 2H). HRMS (ESI) m/z 477.1258 (calcd 477.1255 for
+
C23H24Cl2FN4O2 [M + H]+).
6.1.2.9. 2-(3,4-dichlorophenyl)-6-(3-(4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)
propoxy)pyridazin-3(2H)-one (14). Yield: 82.5%, oil. 1H NMR (600 MHz, CDCl3) δ
7.89 (d, J = 2.5 Hz, 1H), 7.66 (dd, J = 8.7, 2.5 Hz, 1H), 7.57 – 7.51 (m, 1H), 7.36 (t, J
= 8.0 Hz, 1H), 7.13 (s, 1H), 7.11 – 7.05 (m, 2H), 7.04 – 6.99 (m, 2H), 4.28 (t, J = 6.4
Hz, 2H), 3.30 – 3.18 (m, 4H), 2.64 (dd, J = 20.9, 15.9 Hz, 4H), 2.63 – 2.55 (m, 2H),
2.17 – 1.92 (m, 2H). HRMS (ESI) m/z 527.1223 (calcd 527.1223 for
+
C24H24Cl2F3N4O2 [M + H]+).
6.1.2.10. 6-(3-(4-(2-chlorophenyl)piperazin-1-yl)propoxy)-2-(3,4-dichlorophenyl)
pyridazin-3(2H)-one (15). Yield: 80.2%, oil. 1H NMR (600 MHz, CDCl3) δ 7.90 (d, J
= 2.5 Hz, 1H), 7.66 (dd, J = 8.7, 2.5 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.37 (dd, J =
7.9, 1.5 Hz, 1H), 7.23 (ddd, J = 8.9, 7.7, 1.5 Hz, 1H), 7.07 – 7.03 (m, 1H), 7.03 – 6.96
(m, 3H), 4.27 (t, J = 6.4 Hz, 2H), 3.10 (s, 4H), 2.62 (dd, J = 32.5, 25.2 Hz, 6H), 2.11 –
+
1.91 (m, 2H). HRMS (ESI) m/z 493.0962 (calcd 493.0959 for C23H24Cl3N4O2 [M +
H]+).
6.1.2.11. 2-(3,4-dichlorophenyl)-6-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)
1
propoxy)pyridazin-3(2H)-one (16). Yield: 82.1%, oil. H NMR (600 MHz, CDCl3) δ
7.90 (d, J = 2.4 Hz, 1H), 7.67 (dd, J = 8.7, 2.5 Hz, 1H), 7.53 (d, J = 8.7 Hz, 1H), 7.22
– 7.12 (m, 2H), 7.07 – 7.00 (m, 2H), 7.01 – 6.94 (m, 1H), 4.28 (t, J = 6.4 Hz, 2H),
3.09 (s, 4H), 2.87 – 2.44 (m, 6H), 2.18 – 1.95 (m, 2H). HRMS (ESI) m/z
+
527.0581(calcd 527.0570 for C23H23Cl4N4O2 [M + H]+).
6.1.2.12. 6-(3-(4-benzylpiperazin-1-yl)propoxy)-2-(3,4-dichlorophenyl)
pyridazin-3(2H)-one (17). Yield: 81.4%, oil. 1H NMR (600 MHz, CDCl3) δ 7.88 (d,
J = 2.5 Hz, 1H), 7.65 (dd, J = 8.7, 2.5 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.33 (d, J =
4.4 Hz, 4H), 7.27 (dd, J = 8.6, 4.5 Hz, 1H), 7.00 (s, 2H), 4.23 (t, J = 6.4 Hz, 2H), 3.53
(d, J = 6.1 Hz, 2H), 2.66 – 2.37 (m, 8H), 2.10 – 1.78 (m, 3H). HRMS (ESI) m/z
+
473.1521(calcd 473.1506 for C24H27Cl2N4O2 [M + H]+).
6.1.2.13. 6-(3-(4-(benzo[b]thiophen-3-yl)piperazin-1-yl)propoxy)-2-(3,4-dichloro

ACCEPTED MANUSCRIPT
1
phenyl)pyridazin-3(2H)-one (18). Yield: 79.4%, oil. H NMR (600 MHz, CDCl3) δ
8.00 – 7.87 (m, 1H), 7.67 (ddd, J = 8.8, 2.4, 0.6 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H),
7.53 (d, J = 8.7 Hz, 1H), 7.42 (q, J = 5.5 Hz, 2H), 7.33 – 7.25 (m, 1H), 7.07 – 7.00 (m,
2H), 6.91 (d, J = 7.7 Hz, 1H), 4.29 (t, J = 6.3 Hz, 2H), 3.21 (s, 4H), 2.92 – 2.53 (m,
6H), 2.15 – 1.97 (m, 2H). HRMS (ESI) m/z 516.1043(calcd 515.1070 for
C25H25Cl2N4O2S+ [M + H]+).
6.1.2.14. 6-(3-(4-(benzo[d]isothiazol-3-yl)piperazin-1-yl)propoxy)-2-(3,4-dichloro
1
phenyl)pyridazin-3(2H)-one (19). Yield: 81.5%, oil. H NMR (600 MHz, CDCl3) δ
7.96 – 7.88 (m, 2H), 7.82 (d, J = 8.1 Hz, 1H), 7.70 – 7.64 (m, 1H), 7.52 (dd, J = 9.1,
3.5 Hz, 1H), 7.50 – 7.45 (m, 1H), 7.41 – 7.35 (m, 1H), 7.07 – 6.92 (m, 2H), 4.28 (t, J
= 6.4 Hz, 2H), 3.75 – 3.54 (m, 4H), 2.76 – 2.69 (m, 4H), 2.65 – 2.53 (m, 2H), 2.10 –
1.95 (m, 2H). HRMS (ESI) m/z 516.1030 (calcd 516.1022 for C25H24Cl2N5O2S+ [M +
H]+).
6.1.2.15. 3-(4-(3-((1-(3,4-dichlorophenyl)-6-oxo-1,6-dihydropyridazin-3-yl)oxy)
1
propyl)piperazin-1-yl)benzofuran-5-carboxamide (20). Yield: 78.5%, oil. H NMR
(600 MHz, CDCl3) δ 7.89 (d, J = 2.4 Hz, 1H), 7.71 – 7.64 (m, 1H), 7.56 – 7.50 (m,
1H), 7.45 (s, 1H), 7.42 (d, J = 9.0 Hz, 1H), 7.18 – 7.13 (m, 2H), 7.07 – 6.98 (m, 2H),
4.29 (t, J = 6.3 Hz, 2H), 3.24 (d, J = 4.3 Hz, 4H), 2.66 (dd, J = 35.5, 28.3 Hz, 6H),
2.07 (t, J = 6.9 Hz, 2H), 1.90 – 1.54 (m, 2H). HRMS (ESI) m/z 542.1341(calcd
+
542.1356 for C26H26Cl2N5O4 [M + H]+).
6.1.2.16. 6-(3-(4-(benzo[d]thiazol-2-yl)piperidin-1-yl)propoxy)-2-(3,4-dichloro
phenyl)pyridazin-3(2H)-one (21). Yield: 77.9%, oil. 1H NMR (600 MHz, CDCl3) δ
7.98 (d, J = 8.1 Hz, 1H), 7.89 (d, J = 2.5 Hz, 1H), 7.88 – 7.84 (m, 1H), 7.65 (dd, J =
8.7, 2.5 Hz, 1H), 7.51 (d, J = 8.7 Hz, 1H), 7.49 – 7.42 (m, 1H), 7.38 – 7.32 (m, 1H),
7.06 – 6.89 (m, 2H), 4.25 (t, J = 6.4 Hz, 2H), 3.16 – 3.09 (m, 3H), 2.62 – 2.51 (m, 2H),
2.18 (dd, J = 20.0, 11.1 Hz, 4H), 2.02 – 1.93 (m, 4H). HRMS (ESI) m/z 515.1059
(calcd 515.1070 for C25H25Cl2N4O2S+ [M + H]+).
6.1.2.17. 6-(3-(4-(1H-indol-3-yl)piperidin-1-yl)propoxy)-2-(3,4-dichloroPhenyl)
1
pyridazin-3(2H)-one (22). Yield: 81.3%, oil. H NMR (600 MHz, CDCl3) δ 8.22 (s,
1H), 7.91 (t, J = 5.4 Hz, 2H), 7.66 (dd, J = 8.7, 2.5 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H),

ACCEPTED MANUSCRIPT
7.40 (d, J = 8.1 Hz, 1H), 7.26 – 7.13 (m, 3H), 7.08 – 6.97 (m, 2H), 4.31 (t, J = 6.4 Hz,
2H), 3.29 (d, J = 2.8 Hz, 2H), 2.80 (t, J = 5.7 Hz, 2H), 2.74 – 2.59 (m, 3H), 2.22 –
1.99 (m, 2H), 1.70 (s, 4H). HRMS (ESI) m/z 497.1523 (calcd 497.1506 for
+
C26H27Cl2N4O2 [M + H]+).
6.1.2.18. 2-(3,4-dichlorophenyl)-6-(3-(4-(4-fluorobenzoyl)piperidin-1-yl)propoxy)
1
pyridazin-3(2H)-one (23). Yield: 82.4%, oil. H NMR (600 MHz, CDCl3) δ 7.98 (dd,
J = 8.7, 5.5 Hz, 2H), 7.89 (d, J = 2.4 Hz, 1H), 7.65 (dd, J = 8.7, 2.4 Hz, 1H), 7.53 (d,
J = 8.7 Hz, 1H), 7.16 (t, J = 8.6 Hz, 2H), 7.07 – 6.91 (m, 2H), 4.25 (t, J = 6.3 Hz, 2H),
3.25 – 3.22 (m, 1H), 3.04 (d, J = 11.5 Hz, 2H), 2.55 (t, J = 7.2 Hz, 2H), 2.17 (s, 2H),
2.03 – 1.98 (m, 2H), 1.90 – 1.81 (m, 4H). HRMS (ESI) m/z 504.1266 (calcd 504.1252
+
for C25H25Cl2FN3O3 [M + H]+).
6.1.2.19. 2-(3,4-dichlorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
1
-1-yl)propoxy)pyridazin-3(2H)-one (24). Yield: 83.2%, oil. H NMR (600 MHz,
CDCl3) δ 7.88 (d, J = 2.4 Hz, 1H), 7.73 – 7.60 (m, 2H), 7.56 – 7.41 (m, 1H), 7.23 (dd,
J = 8.5, 1.6 Hz, 1H), 7.10 – 6.93 (m, 3H), 4.25 (t, J = 6.3 Hz, 2H), 3.08 (d, J = 11.8
Hz, 3H), 2.57 (t, J = 7.3 Hz, 2H), 2.25 – 1.89 (m, 6H), 1.25 (d, J = 0.6 Hz, 2H).
+
HRMS (ESI) m/z 517.1231(calcd 517.1204 for C25H24Cl2FN4O3 [M + H]+).
6.1.2.20. 2-(3,4-dichlorophenyl)-6-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)butoxy)pyridazin-3(2H)-one (25). Yield: 79.8%, oil. 1H NMR (600 MHz, CDCl3)
δ 7.89 (d, J = 2.5 Hz, 1H), 7.71 (dd, J = 8.7, 5.1 Hz, 1H), 7.67 (dd, J = 8.7, 2.5 Hz,
1H), 7.52 (d, J = 8.7 Hz, 1H), 7.26 (dd, J = 8.5, 2.1 Hz, 1H), 7.07 (td, J = 8.8, 2.1 Hz,
1H), 7.01 (d, J = 2.9 Hz, 2H), 4.23 (t, J = 6.4 Hz, 2H), 3.11 (dd, J = 12.9, 10.4 Hz,
3H), 2.58 – 2.43 (m, 2H), 2.23 – 2.04 (m, 6H), 1.88 – 1.78 (m, 2H), 1.78 – 1.63 (m,
+
2H). HRMS (ESI) m/z 531.1345 (calcd 531.1361 for C26H26Cl2FN4O3 [M + H]+).
6.1.2.21. 2-(3,4-dichlorophenyl)-6-((5-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
1
-1-yl)pentyl)oxy)pyridazin-3(2H)-one (26). Yield: 78.9%, oil. H NMR (600 MHz,
CDCl3) δ 7.89 (d, J = 2.1 Hz, 1H), 7.73 (dd, J = 8.6, 5.1 Hz, 1H), 7.66 (dd, J = 8.7,
2.1 Hz, 1H), 7.52 (d, J = 8.7 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.07 (t, J = 8.8 Hz,
1H), 7.04 – 6.99 (m, 2H), 4.20 (t, J = 6.5 Hz, 2H), 3.11 (d, J = 10.4 Hz, 3H), 2.57 –
2.42 (m, 2H), 2.14 (dt, J = 47.0, 27.2 Hz, 4H), 1.89 – 1.73 (m, 2H), 1.77 – 1.58 (m,

ACCEPTED MANUSCRIPT
2H), 1.54 – 1.47 (m, 2H), 1.34 – 1.20 (m, 2H). HRMS (ESI) m/z 545.1510 (calcd
+
545.1517 for C27H28Cl2FN4O3 [M + H]+).
6.1.3. General procedure for the preparation of compounds 28
To a solution of 3,4-Dichlorophenylhydrazine hydrochloride (55 mmol) and
3,4-dimethylfuran-2,5-dione (4,5,6,7-tetrahydroisobenzofuran-1,3-dione) (50 mmol)
in 400 mL of water, 40 mL of concentrated hydrochloric acid solution was added. 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 to yield 27. [18]
2-(3,4-Dichlorophenyl)-4-hydroxy-5,6,7,8-tetrahydrophthalazin-1(2H)-one
(27a).
Pale yellow solid; yield 79.1%; mp 261−263°C;MS (ESI) m/z 311.1 (calcd 311.0 for
C14H13Cl2N2O2 [M + H]+).
+
2-(3,4-Dichlorophenyl)-4-hydroxyphthalazin-1(2H)-one (27b). Pale yellow solid;
+
yield 78.2%; mp 271−272 °C;MS (ESI) m/z 307.2 (calcd 307.0 for C14H9Cl2N2O2
[M + H]+).
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 28.
6.1.3.1. 6-(3-Bromopropoxy)-2-(3,4-dichlorophenyl)-4,5-dimethylpyridazin-3
(2H)-one (28a). oil; yield 69.3%; MS (ESI) m/z 405.0 (calcd 405.0 for
+
C15H16BrCl2N2O2 [M + H]+).
6.1.3.2. 4-(3-Bromopropoxy)-2-(3,4-dichlorophenyl)-5,6,7,8-tetrahydrophthalazin-
1(2H)-one (28b). Pale yellow solid; yield 72.4%; mp 45−46 °C; MS (ESI) m/z 430.9
+
(calcd 431.0 for C17H18BrCl2N2O2 [M + H]+).
6.1.3.3. 4-(3-Bromopropoxy)-2-(3,4-dichlorophenyl)phthalazin-1(2H)-one (28c). Pale

ACCEPTED MANUSCRIPT
yellow solid; yield 72.4%; mp 48−50 °C; MS (ESI) m/z 427.2 (calcd 427.0 for
+
C17H14BrCl2N2O2 [M + H]+).
6.1.4. General procedure for the preparation of compounds 29-31
To a suspension of compounds 28 (0.32 mmol) and K2CO3 (1.22 mmol) in acetonitrile
(5.0 mL), 6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole (0.32 mmol) and a catalytic
amount of KI were added and the resulting mixture was refluxed for 7-9 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 (25
mL×3). The combined organic layers were evaporated under reduced pressure, and
the crude product was purified by means of chromatography (10% MeOH/CHCl3) to
yield compounds 30-32.
6.1.4.1. 2-(3,4-dichlorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
1
-1-yl)propoxy)-4,5-dimethylpyridazin-3(2H)-one (29). Yield: 75.2%, oil. H NMR
(600 MHz, CDCl3) δ 7.92 (d, J = 2.5 Hz, 1H), 7.71 (dd, J = 8.7, 5.1 Hz, 1H), 7.67 (dd,
J = 8.7, 2.5 Hz, 1H), 7.54 – 7.46 (m, 1H), 7.25 (dd, J = 8.3, 2.1 Hz, 1H), 7.06 (td, J =
8.8, 2.1 Hz, 1H), 4.26 (t, J = 6.3 Hz, 2H), 3.18 – 2.93 (m, 3H), 2.65 – 2.52 (m, 2H),
2.26 – 2.16 (m, 8H), 2.16 – 1.97 (m, 6H). HRMS (ESI) m/z 545.1529 (calcd 545.1517
for C27H28Cl2FN4O3 [M + H]+).
+
6.1.4.2. 2-(3,4-dichlorophenyl)-4-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
1
-1-yl)propoxy)-5,6,7,8-tetrahydrophthalazin-1(2H)-one (30). Yield: 77.3%, oil. H
NMR (600 MHz, CDCl3) δ 7.93 (d, J = 2.4 Hz, 1H), 7.70 (ddd, J = 11.2, 8.7, 3.8 Hz,
2H), 7.53 – 7.48 (m, 1H), 7.26 (dd, J = 8.4, 2.0 Hz, 1H), 7.07 (td, J = 8.8, 2.1 Hz, 1H),
4.26 (t, J = 6.3 Hz, 2H), 3.10 (t, J = 15.2 Hz, 3H), 2.68 – 2.48 (m, 6H), 2.34 – 1.94 (m,
6H), 1.93 – 1.71 (m, 6H). HRMS (ESI) m/z 571.1687 (calcd 571.1674 for
+
C29H30Cl2FN4O3 [M + H]+).
6.1.4.3. 2-(3,4-dichlorophenyl)-4-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)phthalazin-1(2H)-one (31). Yield: 76.8%, oil. 1H NMR (600 MHz,
CDCl3) δ 8.54 – 8.46 (m, 1H), 8.08 – 8.01 (m, 1H), 8.00 (d, J = 2.5 Hz, 1H), 7.91 –
7.81 (m, 2H), 7.77– 7.54 (m, 2H), 7.55 (d, J = 8.7 Hz, 1H), 7.27 (dd, J = 8.5, 2.1 Hz,
1H), 7.08 (td, J = 8.8, 2.1 Hz, 1H), 4.46 (t, J = 6.3 Hz, 2H), 3.16 (d, J = 11.1 Hz, 3H),

ACCEPTED MANUSCRIPT
2.69 (t, J = 6.9 Hz, 2H), 2.43 – 1.94 (m, 8H). HRMS (ESI) m/z 567.1342 (calcd
+
567.1361 for C29H26Cl2FN4O3 [M + H]+).
6.1.5. General procedure for the preparation of compounds 33
Phenylhydrazine hydrochloride (55 mmol) was added to maleic anhydride ( 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 32a was a white solid. [18] Yield 79.8%; mp 272−273 °C;MS (ESI)
+
m/z189.0 (calcd 189.1 for C10H9N2O2 [M + H]+).
6-Hydroxy-2-(p-tolyl)pyridazin-3(2H)-one (32b). White solid. Yield 80.2%; mp
242−243 °C; MS (ESI) m/z 203.2 (calcd 203.1 for C11H11N2O2 [M + H]+).
+
6-Hydroxy-2-(naphthalen-2-yl)pyridazin-3(2H)-one (32c). White solid; yield 78.4%;
+
mp 268−269 °C; MS (ESI) m/z 239.3 (calcd 239.1 for C14H11N2O2 [M + H]+).
2-(4-Fluorophenyl)-6-hydroxypyridazin-3(2H)-one (32d). White solid; yield 77.8%;
+
mp 279−281 °C; MS (ESI) m/z 207.2 (calcd 207.1 for C10H8FN2O2 [M + H]+).
2-(4-Chlorophenyl)-6-hydroxypyridazin-3(2H)-one (32e). White solid; yield 81.2%;
mp 281−282 °C; MS (ESI) m/z 223.0 (calcd 223.0 for C11H8ClN2O2 [M + H]+).
+
2-(2-Chlorophenyl)-6-hydroxypyridazin-3(2H)-one (32f). White solid; yield 80.4%;
mp 282−284 °C; 1H NMR (600 MHz, DMSO) δ 11.44 (s, 1H), 7.72 (t, J = 2.0 Hz,
1H), 7.62 (ddd, J = 8.1, 1.9, 1.0 Hz, 1H), 7.50 (t, J = 8.1 Hz, 1H), 7.44 (ddd, J = 8.0,
2.0, 1.0 Hz, 1H), 7.23 – 7.14 (m, 1H), 7.09 – 6.99 (m, 1H).
2-(3-Chlorophenyl)-6-hydroxypyridazin-3(2H)-one (32g). White solid; yield 79.2%;
mp 285−286 °C; 1H NMR (600 MHz, DMSO) δ 11.41 (s, 1H), 7.69 – 7.61 (m, 1H),
7.57 – 7.52 (m, 1H), 7.52 – 7.46 (m, 2H), 7.23 (d, J = 9.9 Hz, 1H), 7.06 (d, J = 9.9 Hz,
1H).
2-(2,3-Dichlorophenyl)-6-hydroxypyridazin-3(2H)-one (32h). Pale yellow solid; yield
+
78.9%; mp 289−292 °C; MS (ESI) m/z 257.2 (calcd 257.0 for C10H7Cl2N2O2 [M +
H]+).

ACCEPTED MANUSCRIPT
2-(2,5-Dichlorophenyl)-6-hydroxypyridazin-3(2H)-one (32i). Pale yellow solid; yield
+
76.1%; mp 282−284 °C; MS (ESI) m/z 257.1 (calcd257.0 for C10H7Cl2N2O2 [M +
H]+).
2-(2,4-Dichlorophenyl)-6-hydroxypyridazin-3(2H)-one (32j). Pale yellow solid; yield
+
76.9%; mp 279−281 °C; MS (ESI) m/z 257.2 (calcd 257.0 for C10H7Cl2N2O2 [M +
H]+).
2-(3,5-Dichlorophenyl)-6-hydroxypyridazin-3(2H)-one (32k). Pale yellow solid; yield
+
74.8%; mp 288−289 °C; MS (ESI) m/z 257.0 (calcd 257.0 for C10H7Cl2N2O2 [M +
H]+).
2-(2,5-Difluorophenyl)-6-hydroxypyridazin-3(2H)-one (32l). Pale yellow solid; yield
73.3%; mp 287−289 °C; 1H NMR (600 MHz, DMSO) δ 11.52 (s, 1H), 7.53 (ddd, J =
8.8, 5.8, 3.2 Hz, 1H), 7.46 (td, J = 9.3, 4.8 Hz, 1H), 7.39 (ddd, J = 9.0, 6.8, 3.6 Hz,
1H), 7.23 (d, J = 9.9 Hz, 1H), 7.06 (d, J = 9.9 Hz, 1H).
2-(2,6-Difluorophenyl)-6-hydroxypyridazin-3(2H)-one (32m). Pale yellow solid;
yield 74.2%; mp 286−287 °C; 1H NMR (600 MHz, DMSO) δ 11.46 (s, 1H), 7.88 (dd,
J = 6.8, 2.6 Hz, 1H), 7.66 (ddd, J = 8.9, 4.4, 2.6 Hz, 1H), 7.53 (t, J = 9.0 Hz, 1H),
7.19 (d, J = 9.8 Hz, 1H), 7.04 (d, J = 9.8 Hz, 1H).
2-(3,5-Difluorophenyl)-6-hydroxypyridazin-3(2H)-one (32n). Pale yellow solid; yield
73.5%; mp 285−288 °C; 1H NMR (600 MHz, DMSO) δ 11.59 (s, 1H), 7.60 (tt, J =
8.5, 6.4 Hz, 1H), 7.33 (t, J = 8.3 Hz, 2H), 7.27 (d, J = 9.9 Hz, 1H), 7.11 (d, J = 9.9 Hz,
1H).
2-(3,4-Difluorophenyl)-6-hydroxypyridazin-3(2H)-one (32o). Pale yellow solid; yield
+
77.8%; mp 285−287 °C; MS (ESI) m/z 225.1 (calcd 225.0 for C10H7F2N2O2 [M +
H]+).
2-(2,4-Difluorophenyl)-6-hydroxypyridazin-3(2H)-one (32p). Pale yellow solid; yield
75.7%; mp 281−283 °C; 1H NMR (600 MHz, DMSO) δ 11.48 (s, 1H), 7.62 (td, J =
8.7, 6.1 Hz, 1H), 7.47 (ddd, J = 10.4, 9.2, 2.8 Hz, 1H), 7.27 – 7.19 (m, 2H), 7.05 (d, J
= 9.9 Hz, 1H).
2-(3-Chloro-4-fluorophenyl)-6-hydroxypyridazin-3(2H)-one (32q). Pale yellow solid;
+
yield 76.8%; mp 291−292 °C; MS (ESI) m/z 240.9 (calcd 241.0 for C10H7ClFN2O2

ACCEPTED MANUSCRIPT
[M + H]+).
To a solution of 32 (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 33.
6.1.5.1. 6-(3-Bromopropoxy)-2-phenylpyridazin-3(2H)-one (33a). Pale yellow oil;
+
yield 62.1%; MS (ESI) m/z 309.0 (calcd 309.0 for C13H14BrN2O2 [M + H]+).
6.1.5.2. 6-(3-Bromopropoxy)-2-(p-tolyl)pyridazin-3(2H)-one (33b). Pale yellow oil;
+
yield 72.4%; MS (ESI) m/z 323.1 (calcd 323.0 for C14H16BrN2O2 [M + H]+).
6.1.5.3. 6-(3-Bromopropoxy)-2-(naphthalen-2-yl)pyridazin-3(2H)-one (33c). Pale
+
yellow oil; yield 70.2%; MS (ESI) m/z 359.0 (calcd 359.0 for C17H16BrN2O2 [M +
H]+).
6.1.5.4. 6-(3-Bromopropoxy)-2-(4-fluorophenyl)pyridazin-3(2H)-one (33d). Pale
+
yellow oil; yield 64.6%; MS (ESI) m/z 327.2 (calcd 327.0 for C13H13BrFN2O2 [M +
H]+).
6.1.5.5. 6-(3-Bromopropoxy)-2-(4-chlorophenyl)pyridazin-3(2H)-one (33e). Pale
yellow solid; yield 71.6%; mp 79−81 °C; MS (ESI) m/z 343.0 (calcd 343.0 for
+
C13H13BrClN2O2 [M + H]+).
6.1.5.6. 6-(3-Bromopropoxy)-2-(2-chlorophenyl)pyridazin-3(2H)-one (33f). Pale
yellow solid; yield 71.6%; mp 72−73 °C; 1H NMR (600 MHz, DMSO) δ 11.41 (s,
1H), 7.69 – 7.61 (m, 1H), 7.57 – 7.52 (m, 1H), 7.52 – 7.46 (m, 2H), 7.23 (d, J = 9.9
Hz, 1H), 7.06 (d, J = 9.9 Hz, 1H).
6.1.5.7. 6-(3-Bromopropoxy)-2-(3-chlorophenyl)pyridazin-3(2H)-one (33g). Pale
yellow solid; yield 71.6%; mp 75−77 °C; 1H NMR (600 MHz, CDCl3) δ 7.74 (t, J =
1.9 Hz, 1H), 7.68 – 7.61 (m, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.36 – 7.30 (m, 1H), 7.08 –
6.93 (m, 2H), 4.34 (t, J = 5.9 Hz, 2H), 3.57 (t, J = 6.5 Hz, 2H), 2.32 (p, J = 6.2 Hz,
2H).
6.1.5.8. 6-(3-Bromopropoxy)-2-(2,3-dichlorophenyl)pyridazin-3(2H)-one (33h). Pale

ACCEPTED MANUSCRIPT
yellow solid; yield 70.3%; mp 53−55 °C; MS (ESI) m/z 376.9 (calcd 376.9 for
+
C13H12BrCl2N2O2 [M + H]+).
6.1.5.9. 6-(3-Bromopropoxy)-2-(2,5-dichlorophenyl)pyridazin-3(2H)-one (33i). Pale
+
yellow oil; yield 70.9%; MS (ESI) m/z 377.1 (calcd 376.9 for C13H12BrCl2N2O2 [M
+ H]+).
6.1.5.10. 6-(3-Bromopropoxy)-2-(2,4-dichlorophenyl)pyridazin-3(2H)-one (33j). Pale
+
yellow oil; yield 71.2%; MS (ESI) m/z 376.8 (calcd 376.9 for C13H12BrCl2N2O2 [M
+ H]+).
6.1.5.11. 6-(3-Bromopropoxy)-2-(3,5-dichlorophenyl)pyridazin-3(2H)-one (33k). Pale
yellow solid; yield 72.5%; mp 63−64 °C; MS (ESI) m/z 376.9 (calcd 376.9 for
+
C13H12BrCl2N2O2 [M + H]+).
6.1.5.12. 6-(3-Bromopropoxy)-2-(2,5-difluorophenyl)pyridazin-3(2H)-one (33l). Pale
yellow solid; yield 68.9%; mp 41−42 °C; 1H NMR (600 MHz, CDCl3) δ 7.24 – 7.17
(m, 2H), 7.12 (ddt, J = 9.2, 7.1, 3.4 Hz, 1H), 7.07 – 7.00 (m, 2H), 4.29 (t, J = 5.9 Hz,
2H), 3.56 (t, J = 6.5 Hz, 2H), 2.30 (p, J = 6.2 Hz, 2H).
6.1.5.13. 6-(3-Bromopropoxy)-2-(2,6-difluorophenyl)pyridazin-3(2H)-one (33m).
Pale yellow oil; yield 71.2%; 1H NMR (600 MHz, CDCl3) δ 7.41 (tt, J = 8.5, 6.1 Hz,
1H), 7.11 – 6.98 (m, 4H), 4.26 (t, J = 5.9 Hz, 2H), 3.54 (t, J = 6.5 Hz, 2H), 2.28 (p, J
= 6.3 Hz, 2H).
6.1.5.14. 6-(3-Bromopropoxy)-2-(3,5-difluorophenyl)pyridazin-3(2H)-one (33n). Pale
yellow oil; yield 72.3%; 1H NMR (600 MHz, CDCl3) δ 7.92-7.90 (m, 1H), 7.65-7.60
(m, 1H), 7.25 – 7.19 (m, 1H), 7.03 – 6.99 (m, 2H), 4.33 (t, J = 5.9 Hz, 2H), 3.57 (t, J
= 6.4 Hz, 2H), 2.32 (p, J = 6.2 Hz, 2H).
6.1.5.15. 6-(3-Bromopropoxy)-2-(3,4-difluorophenyl)pyridazin-3(2H)-one (33o). Pale
yellow solid; yield 68.9%; mp 43−45 °C; MS (ESI) m/z 345.2 (calcd 345.0 for
+
C13H12BrF2N2O2 [M + H]+).
6.1.5.16. 6-(3-Bromopropoxy)-2-(2,4-difluorophenyl)pyridazin-3(2H)-one (33p). Pale
yellow solid; yield 72.9%; mp 46−47 °C; 1H NMR (600 MHz, CDCl3) δ 7.51 – 7.35
(m, 1H), 7.10 – 6.85 (m, 4H), 4.27 (t, J = 5.9 Hz, 2H), 3.55 (t, J = 6.5 Hz, 2H), 2.29 (p,
J = 6.2 Hz, 2H).

ACCEPTED MANUSCRIPT
6.1.5.17. 6-(3-Bromopropoxy)-2-(3-chloro-4-fluorophenyl)pyridazin-3(2H)-one (33q).
Pale yellow solid; yield 71.8%; mp 73−75 °C; MS (ESI) m/z 360.9 (calcd 361.0 for
+
C13H12BrClFN2O2 [M + H]+).
6.1.6. General procedure for the preparation of compounds 34-50
To a suspension of compounds 34 (0.32 mmol) and K2CO3 (1.22 mmol) in acetonitrile
(5.0 mL), 6-fluoro-3-(4-piperidyl)-1,2-benzisoxazole (0.32 mmol) and a catalytic
amount of KI were added and the resulting mixture was refluxed for 7-9 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 (25
mL×3). The combined organic layers were evaporated under reduced pressure, and
the crude product was purified by means of chromatography (10% MeOH/CHCl3) to
yield compounds 34-50.
6.1.6.1. 6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propoxy)-2-phenyl
1
pyridazin-3(2H)-one (34). Yield: 77.8%, oil. H NMR (600 MHz, CDCl3) δ 7.75 –
7.66 (m, 3H), 7.51 – 7.44 (m, 2H), 7.40 – 7.33 (m, 1H), 7.26 (dd, J = 8.5, 2.1 Hz, 1H),
7.11 – 6.97 (m, 3H), 4.26 (t, J = 6.4 Hz, 2H), 3.09 (dd, J = 13.4, 5.3 Hz, 3H), 2.61 –
2.47 (m, 2H), 2.24 – 1.94 (m, 8H). HRMS (ESI) m/z 449.1989 (calcd 449.1983 for
C25H26FN4O3 [M + H]+).
+
6.1.6.2. 6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propoxy)-2-(p-tolyl)
pyridazin-3(2H)-one (35). Yield: 81.5%, oil. 1H NMR (600 MHz, CDCl3) δ 7.71 (dd,
J = 8.7, 5.1 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H), 7.31 – 7.19 (m, 3H), 7.07 (td, J = 8.8,
2.1 Hz, 1H), 7.04 – 6.98 (m, 2H), 4.25 (t, J = 6.4 Hz, 2H), 3.09 (d, J = 11.2 Hz, 3H),
2.65 – 2.53 (m, 2H), 2.40 (s, 3H), 2.18 (t, J = 12.3 Hz, 2H), 2.12 – 2.04 (m, 4H), 2.04
+
– 1.97 (m, 2H). HRMS (ESI) m/z 463.2151 (calcd 463.2140 for C26H28FN4O3 [M +
H]+).
6.1.6.3. 6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propoxy)-2-(naph
thalene-2-yl)pyridazin-3(2H)-one (36). Yield: 80.1%, oil. 1H NMR (600 MHz, CDCl3)
δ 8.20 (d, J = 1.9 Hz, 1H), 7.95 – 7.84 (m, 3H), 7.79 (dd, J = 8.8, 2.1 Hz, 1H), 7.68
(dd, J = 8.7, 5.1 Hz, 1H), 7.54 – 7.48 (m, 2H), 7.25 (dd, J = 8.5, 2.1 Hz, 1H), 7.11 –
6.99 (m, 3H), 4.30 (t, J = 6.4 Hz, 2H), 3.23 – 3.03 (m, 3H), 2.66 – 2.50 (m, 2H), 2.23

ACCEPTED MANUSCRIPT
+
– 1.92 (m, 8H). HRMS (ESI) m/z 499.2122 (calcd 499.2140 for C29H28FN4O3 [M +
H]+).
6.1.6.4. 6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propoxy)-2-(4-fluoro
phenyl)pyridazin-3(2H)-one (37). Yield: 79.5%, oil. 1H NMR (600 MHz, CDCl3) δ
7.70 (dd, J = 8.7, 5.1 Hz, 1H), 7.68 – 7.65 (m, 2H), 7.27 – 7.23 (m, 1H), 7.18 – 7.12
(m, 2H), 7.09 – 7.04 (m, 1H), 7.03 – 6.97 (m, 2H), 4.25 (t, J = 6.4 Hz, 2H), 3.16 –
2.99 (m, 3H), 2.76 – 2.48 (m, 2H), 2.29 – 1.93 (m, 6H), 1.26 (s, 2H). HRMS (ESI)
+
m/z 467.1897 (calcd 467.1889 for C25H25F2N4O3 [M + H]+).
6.1.6.5. 2-(4-chlorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (38). Yield: 81.2%, oil. 1H NMR (600 MHz,
CDCl3) δ 7.74 – 7.64 (m, 3H), 7.46 – 7.40 (m, 2H), 7.26 (dd, J = 8.5, 2.0 Hz, 1H),
7.07 (td, J = 8.9, 2.2 Hz, 1H), 7.04 – 6.97 (m, 2H), 4.26 (t, J = 6.4 Hz, 2H), 3.09 (d, J
= 10.7 Hz, 3H), 2.57 (t, J = 7.4 Hz, 2H), 2.34 – 1.91 (m,6H), 1.27 (s, 2H). HRMS
(ESI) m/z 483.1579 (calcd 483.1594 for C25H25ClFN4O3 [M + H]+).
+
6.1.6.6. 2-(2-chlorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (39). Yield: 82.1%, oil. 1H NMR (600 MHz,
CDCl3) δ 7.75 – 7.69 (m, 2H), 7.68 – 7.63 (m, 1H), 7.40 (t, J = 8.1 Hz, 1H), 7.38 –
7.30 (m, 1H), 7.26 (dd, J = 8.5, 2.1 Hz, 1H), 7.07 (td, J = 8.8, 2.1 Hz, 1H), 7.05 – 6.99
(m, 2H), 4.28 (t, J = 6.4 Hz, 2H), 3.20 – 3.06 (m, 3H), 2.58 (t, J = 7.3 Hz, 2H), 2.38 –
+
1.86 (m, 8H). HRMS (ESI) m/z 483.1612 (calcd 483.1594 for C25H25ClFN4O3 [M +
H]+).
6.1.6.7. 2-(3-chlorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (40). Yield: 80.7%, oil. 1H NMR (600 MHz,
CDCl3) δ 7.71 (dd, J = 8.7, 5.1 Hz, 1H), 7.57 – 7.53 (m, 1H), 7.47 – 7.43 (m, 1H),
7.43 – 7.36 (m, 2H), 7.26 (dd, J = 8.5, 2.1 Hz, 1H), 7.09 – 7.01 (m, 3H), 4.20 (t, J =
6.4 Hz, 2H), 3.08 (d, J = 11.6 Hz, 3H), 2.56 (t, J = 7.3 Hz, 2H), 2.32 – 1.88 (m, 8H).
+
HRMS (ESI) m/z 483.1582 (calcd 483.1594 for C25H25ClFN4O3 [M + H]+).
6.1.6.8. 2-(2,3-dichlorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (41). Yield: 78.9%, oil. 1H NMR (600 MHz,
CDCl3) δ 7.69 (dd, J = 8.7, 5.1 Hz, 1H), 7.53 (dd, J = 7.7, 1.9 Hz, 1H), 7.39 – 7.31 (m,

ACCEPTED MANUSCRIPT
2H), 7.24 (dd, J = 8.5, 2.1 Hz, 1H), 7.10 – 6.99 (m, 3H), 4.30 – 3.99 (m, 2H), 3.16 –
2.98 (m, 3H), 2.54 – 2.44 (m, 2H), 2.20 – 1.85 (m, 8H). HRMS (ESI) m/z 517.1195
+
(calcd 517.1204 for C25H24Cl2FN4O3 [M + H]+).
6.1.6.9. 2-(2,5-dichlorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (42). Yield: 79.7%, oil. 1H NMR (600 MHz,
CDCl3) δ 7.69 (dd, J = 8.7, 5.1 Hz, 1H), 7.46 – 7.43 (m, 2H), 7.35 (dd, J = 8.6, 2.5 Hz,
1H), 7.24 (dd, J = 8.5, 2.1 Hz, 1H), 7.08 – 6.99 (m, 3H), 4.19 (t, J = 6.4 Hz, 2H), 3.17
– 3.00 (m, 3H), 2.58 – 2.47 (m, 2H), 2.23 – 1.87 (m, 8H). HRMS (ESI) m/z 517.1223
+
(calcd 517.1204 for C25H24Cl2FN4O3 [M + H]+).
6.1.6.10. 2-(2,4-dichlorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (43). Yield: 79.2%, oil. 1H NMR (600 MHz,
CDCl3) δ 7.70 (dd, J = 8.7, 5.1 Hz, 1H), 7.55 (s, 1H), 7.39 (d, J = 1.0 Hz, 2H), 7.26
(dd, J = 8.5, 2.0 Hz, 1H), 7.12 – 6.91 (m, 3H), 4.28 – 4.11 (m, 2H), 3.24 – 2.94 (m,
3H), 2.54 (t, J = 7.3 Hz, 2H), 2.28 – 1.91 (m, 8H). HRMS (ESI) m/z 517.1217 (calcd
+
517.1204 for C25H24Cl2FN4O3 [M + H]+).
6.1.6.11. 2-(3,5-dichlorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (44). Yield: 80.3%, oil. 1H NMR (600 MHz,
CDCl3) δ 7.76 – 7.63 (m, 3H), 7.33 (t, J = 1.8 Hz, 1H), 7.25 (dd, J = 8.5, 2.1 Hz, 1H),
7.06 (td, J = 8.8, 2.1 Hz, 1H), 7.04 – 6.99 (m, 2H), 4.28 (t, J = 6.4 Hz, 2H), 3.49 –
2.92 (m, 3H), 2.72 – 2.48 (m, 2H), 2.20 (dd, J = 16.2, 9.0 Hz, 2H), 2.12 – 1.94 (m,
+
6H). HRMS (ESI) m/z 517.1215 (calcd 517.1204 for C25H24Cl2FN4O3 [M + H]+).
6.1.6.12. 2-(2,5-difluorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (45). Yield: 77.8%, oil. 1H NMR (600 MHz,
CDCl3) δ 7.69 (dd, J = 8.7, 5.1 Hz, 1H), 7.26 – 7.16 (m, 3H), 7.13 – 7.08 (m, 1H),
7.07 – 6.97 (m, 3H), 4.20 (t, J = 6.4 Hz, 2H), 3.08 (dd, J = 17.3, 9.5 Hz, 3H), 2.58 –
2.47 (m, 2H), 2.25 – 2.10 (m, 2H), 2.13 – 1.93 (m, 6H). HRMS (ESI) m/z 485.1812
+
(calcd 485.1795 for C25H24F3N4O3 [M + H]+).
6.1.6.13. 2-(2,6-difluorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (46). Yield: 78.9%, oil. 1H NMR (400 MHz,
DMSO) δ 7.70 (dd, J = 8.7, 5.1 Hz, 1H), 7.39 (dq, J = 8.5, 6.1 Hz, 1H), 7.25 (dd, J =

ACCEPTED MANUSCRIPT
8.5, 2.0 Hz, 1H), 7.11 – 6.99 (m, 5H), 4.20 (t, J = 6.4 Hz, 2H), 3.08 (t, J = 12.7 Hz,
3H), 2.54 (t, J = 7.3 Hz, 2H), 2.20 – 1.95 (m, 8H). HRMS (ESI) m/z 485.1793 (calcd
485.1795 for C25H24F3N4O3+ [M + H]+).
6.1.6.14. 2-(3,5-difluorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (47). Yield: 79.1%, oil. 1H NMR (600 MHz,
CDCl3) δ 7.81 (dd, J = 6.6, 2.6 Hz, 1H), 7.70 (dd, J = 8.7, 5.1 Hz, 1H), 7.65 (ddd, J =
8.9, 4.2, 2.6 Hz, 1H), 7.27 – 7.19 (m, 2H), 7.07 (td, J = 8.8, 2.1 Hz, 1H), 7.04 – 6.97
(m, 2H), 4.26 (t, J = 6.4 Hz, 2H), 3.12 – 3.03 (m, 3H), 2.66 – 2.48 (m, 2H), 2.20 (dd,
J = 16.2, 8.9 Hz, 2H), 2.13 – 1.93 (m, 6H). HRMS (ESI) m/z 485.1805 (calcd
+
485.1795 for C25H24F3N4O3 [M + H]+).
6.1.6.15. 2-(3,4-difluorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (48). Yield: 79.9%, oil. 1H NMR (600 MHz,
CDCl3) δ 7.70 (dd, J = 8.7, 5.1 Hz, 1H), 7.64 (ddd, J = 11.5, 7.2, 2.6 Hz, 1H), 7.57 –
7.51 (m, 1H), 7.27 – 7.21 (m, 2H), 7.07 (td, J = 8.8, 2.1 Hz, 1H), 7.04 – 6.97 (m, 2H),
4.27 (t, J = 6.4 Hz, 2H), 3.27 – 3.03 (m, 3H), 2.71 – 2.45 (m, 2H), 2.20 (dd, J = 16.1,
9.3 Hz, 2H), 2.14 – 1.95 (m, 6H). HRMS (ESI) m/z 485.1790 (calcd 485.1795 for
+
C25H24F3N4O3 [M + H]+).
6.1.6.16. 2-(2,4-difluorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin
-1-yl)propoxy)pyridazin-3(2H)-one (49). Yield: 79.7%, oil. 1H NMR (600 MHz,
CDCl3) δ 7.69 (dd, J = 8.7, 5.1 Hz, 1H), 7.44 (td, J = 8.5, 5.9 Hz, 1H), 7.24 (dd, J =
8.5, 2.1 Hz, 1H), 7.09 – 6.93 (m, 5H), 4.19 (t, J = 6.4 Hz, 2H), 3.16 – 2.90 (m, 3H),
2.59 – 2.46 (m, 2H), 2.24 – 2.13 (m, 2H), 2.09 – 1.92 (m, 6H). HRMS (ESI) m/z
+
485.1781 (calcd 485.1795 for C25H24F3N4O3 [M + H]+).
6.1.6.17. 2-(3-chloro-4-fluorophenyl)-6-(3-(4-(6-fluorobenzo[d]isoxazol-3-yl)
piperidin-1-yl)propoxy)pyridazin-3(2H)-one (50). Yield: 81.0%, oil. 1H NMR (600
MHz, CDCl3) δ 7.70 (dd, J = 8.7, 5.1 Hz, 1H), 7.49 – 7.41 (m, 2H), 7.25 (dd, J = 8.5,
1.9 Hz, 1H), 7.07– 7.03 (m, 1H), 7.03 – 6.97 (m, 2H), 6.85 – 6.74 (m, 1H), 4.29 (t, J =
6.3 Hz, 2H), 3.20 – 2.98 (m, 3H), 2.57 (t, J = 7.3 Hz, 2H), 2.19 (dd, J = 16.3, 9.1 Hz,
2H), 2.14 – 1.98 (m, 6H). HRMS (ESI) m/z 501.1513 (calcd 501.1500 for
+
C25H24ClF2N4O3 [M + H]+).

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6.2. Pharmacological methods
6.2.1. Animals
Chinese Kun Ming (KM) Mice (20 ± 2.0 g) and Spraguee-Dawley (SD) rats (250 ±
5.0 g) were used as experimental animals in this study. Animals were housed under
standardized conditions for light and temperature and received standard rat chow and
tap water and libitum. Animals were randomly assigned to different experimental
groups, each kept in a separate cage. All research involving animals in this study
follow the guidelines of the byelaw of experiments on animals, and have been
approved by the Ethics and Experimental Animal Committee of Jiangsu Nhwa
Pharmaceutical Co., Ltd.
6.2.2. In vitro binding assays
6.2.2.1. 5-HT1A receptor [19]. Rat cerebral cortex was homogenized in 20 volumes of
ice-cold Tris-HCl buffer (50 mM, pH 7.7) using an ULTRA TURAX homogeniser,
and was then centrifuged at 32,000 g for 10 min. The resulting pellet was then
o
resuspended in the same buffer, incubated for 10 min at 37 C, and centrifuged at
32000 g for 10 min. The final pellet was resuspended in Tris-HCl buffer containing 10
mM Pargyline, 4 mM CaCl2 and 0.1% ascorbic acid. Total binding each assay tube
was added 900 ꢀL of the tissue suspension, 50 ꢀL of 0.5 nM [3H] 8-OH-DPAT (187.4
Ci/mmol, Perkin Elmer Life Sciences, Boston, MA, USA), 50 ꢀL TriseHCl buffer
containing 10 mM Pargyline, 4 mM CaCl2 and 0.1% ascorbic acid. Non-specific
binding each assay tube was added 900 ꢀL of the tissue suspension, 50 ꢀL of [3H]
8-OH-DPAT, 50 ꢀL of 10 mM serotonin. Compound binding each assay tube was
added 900 ꢀL of the tissue suspension, 50 ꢀL of [3H] 8-OH-DPAT, 50 ꢀL of new
o
compounds or reference drug. The tubes were incubated at 37 C for 30 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 cold buffer and transferred to
scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound
was measured using a PE Microbeta 2450 liquid scintillation counter.
6.2.2.2. 5-HT2A receptor [19]. Rat cerebral cortex was homogenized in 20 volumes of
ice-cold Tris-HCl buffer (50 mM, pH 7.7) using an ULTRA TURAX homogeniser,