Synthesis and Biological Investigation of Coumarin Piperazine

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Synthesis and biological investigation of coumarin piperazine (piperidine) derivatives as potential multi-receptor atypical antipsychotics Yin Chen, Songlin Wang, Xiangqing Xu, Xin Liu, Minquan Yu, Song Zhao, Shicheng Liu, Yinli Qiu, Tan Zhang, Bifeng Liu, and Guisen Zhang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400408r • Publication Date (Web): 15 May 2013 Downloaded from http://pubs.acs.org on May 16, 2013

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Journal of Medicinal Chemistry

Synthesis and biological investigation of coumarin piperazine (piperidine) derivatives as potential multi-receptor atypical antipsychotics Yin Chen,†,‡ Songlin Wang,† Xiangqing Xu,‡ Xin Liu,† Minquan Yu,‡ Song Zhao,‡ Shicheng Liu,‡ Yinli Qiu, ‡ Tan Zhang,‡ 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

ACS Paragon Plus Environment


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Abstract The discovery and synthesis of potential and novel antipsychotic coumarin derivatives, associated with potent dopamine D2, D3 and serotonin 5-HT1A, 5-HT2A receptor properties, were

the

focus

of

the

present

paper.

The

most-promising

derivative

was

7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl)-piperidin-1-yl)butoxy)-4-methyl-8-chloro-2H-chrom en-2-one 17m. This derivative possesses unique pharmacological features, including high affinity for dopamine D2, D3 and serotonin 5-HT1A, 5-HT2A receptors. Moreover it possesses low affinity for 5-HT2C and H1 receptors (to reduce the risk of obesity associated with chronic treatment) and hERG channels (to reduce the incidence of torsade des pointes). In animal models, compound 17m inhibited apomorphine-induced climbing behavior, MK-801-induced hyperactivity and the conditioned avoidance response without observable catalepsy at the highest dose tested. Further, fewer preclinical adverse events were noted with 17m compared with risperidone in assays that measured prolactin secretion and weight gain. Acceptable pharmacokinetic properties were also noted with 17m. Taken together, 17m may constitute a novel class of drugs for the treatment of schizophrenia.


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Introduction Schizophrenia is a chronic and severe mental illness that affects 1% of the population.1 Typical antipsychotics (e.g., chlorpromazine and haloperidol, Figure 1) were effective in reducing positive symptoms, but failed to manage the negative symptoms and cognitive impairment associated with the disease.2 Moreover, typical antipsychotics are associated with major side effects, including extrapyramidal symptoms (EPS) and hyperprolactinemia.3-5 A breakthrough in pharmacotherapy for patients with schizophrenia occurred following the development of atypical antipsychotics (e.g., clozapine, ziprasidone and risperidone, Figure 1). Atypical antipsychotics possess potent antagonism for serotonin 5-HT2A and dopamine D2 receptors.6 These drugs are more effective than haloperidol against negative symptoms.7-10 However, a major issue with many atypical antipsychotics is their association with numerous side effects, including substantial weight gain and QT interval prolongation.11-14 Therefore, the development of innovative drugs that possess improved efficacy and favorable side-effect profiles is warranted. Research over the past decade has shown that the clinical efficacy of antipsychotic drugs is linked to a complex binding profile. Indeed, many previous studies have demonstrated the importance of multi-target G-protein-coupled receptors that are involved in schizophrenia. 15-17

The serotoninergic system plays is important in the regulation of the prefrontal cortex (PFC) and is highly associated with emotional control, cognitive behavior and working memory.18,19 The pyramidal neurons of the PFC possess numerous serotoninergic receptors, including 5-HT1A and 5-HT2A receptors.20 A growing number of studies have shown that 5-HT1A receptor is important biological target of antipsychotic medications. Activation of 5-HT1A receptor increases dopamine release in the frontal cortex, which may reduce negative symptoms and cognitive deficits in patients with schizophrenia.21 Serotonin acting at 5-HT2A receptor, inhibits neuronal activity in the substantia nigra and ventral tegmental areas. Several studies have reported that 5-HT2A receptor antagonists increase the firing rate of midbrain dopaminergic neurons in a state-dependent manner.22 Moreover, 5-HT2A receptor antagonists increase the activity of nigrostriatal DA-containing neurons following moderate D2 receptor blockade associated with antipsychotic drugs.23 Therefore, 5-HT2A receptor antagonism is



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believed to contribute to the atypical antipsychotic profile.24 Dopamine is a major neurotransmitter in the central nervous system (CNS) that plays important roles in behavior and cognition.25 Molecular genetic studies of G-protein coupled receptors have defined two families of dopamine receptors based upon structural and pharmacological similarities, the D1-like (D1 and D5 receptor subtypes) and the D2-like (D2, D3 and D4 receptor subtypes) receptors.26,27 Blockade of mesolimbic D2 receptor increases the efficacy

of

atypical

antipsychotics

against

positive

symptoms

associated

with

schizophrenia.28 Dopamine, through D3 receptor, modulates cholinergic systems in the prefrontal cortex. The role of D3 receptor in antipsychotic therapy is currently unknown; however, D3 antagonists may enhance acetylcholine release in the frontal cortex, thereby improving cognitive deficits. A growing number of preclinical studies suggest that the D3 receptor may be a useful target for amelioration of the negative and cognitive symptoms associated with schizophrenia and substance abuse disorders.29-33 Compound S33138 is a potent and selective dopamine D3 receptor antagonist that is currently being tested in patients with schizophrenia in Phase IIb clinical trials.34 Furthermore, two or more receptors may be involved in the weight gain associated with the treatment of schizophrenia via atypical antipsychotic drugs. Blockade of H1 receptor by antipsychotics is more likely to be the primary cause of these adverse reactions.35,36 Although 5-HT2C receptor blockade has been reported to counteract dopamine D2-mediated extrapyramidal side-effects (EPS)

37

and may

also confer anxiolytic/antidepressant properties38, 5-HT2C receptor may be involved in the risk of obesity under chronic treatment22,39,40. Thus, the aim of our work was to develop novel antipsychotics that act on dopaminergic and serotonergic receptors with a low affinity for 5-HT2C and H1 receptors. Our goal was to develop a pharmacological agent that would

effectively cure the positive symptoms, the negative symptoms and the cognitive impairment associated with schizophrenia, without producing weight gain. Several previous studies have reported on the development of novel antipsychotic drugs that preferentially bind to serotonin and dopamine receptors.22,41-44 Coumarin derivatives have in recent years been shown to increase CNS activity. Specifically, compound 1a displays a haloperidol-like profile at D2 and 5-HT2A receptors (pKi values of 7.93 and 6.76, respectively), and higher affinity for α1A receptors (pA2=9.07).45 Compound lb shows the


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strongest affinity for 5-HT1A receptors (Ki = 0.79 nM), and displays moderate selectivity for D2 and D3 receptors (Ki = 10.8 and 18.9 nM, respectively).46 To validate this multi-receptor affinity profile approach to antipsychotics and to achieve an optimum interaction with dopamine and serotonin receptors, the present study focused on the synthesis and pharmacological evaluation of a new class of antipsychotic agents with a coumarin system linked to the arylpiperazine (piperidine) group (Figure 2). We report here the synthesis and behavioral investigation of a set of coumarin derivatives as novel and potent antipsychotics, characterized by high affinity for D2, D3, 5-HT1A and 5-HT2A receptors. Their structure–activity relationships (SARs) for dopamine and serotonin receptors were associated with variation in the arylpiperazine (piperidine) moieties and the substituents on the coumarin system. Among the derivatives prepared, compound 17m, a selective antagonist at D2, D3, 5-HT1A and 5-HT2A receptors, was chosen for further biological investigation. Despite its structural similarity to risperidone, 17m shows a unique pharmacological profile, and was used to validate our novel approach to atypical antipsychotics based on D2, D3, 5-HT1A and 5-HT2A multi-receptor affinity profile. Chemistry The synthesis of the novel coumarin derivatives was performed according to the reaction pathways

illustrated

in

Schemes

1-5.

7-hydroxy-4-methylcoumarin

reacted

with

1,4-dibromobutane in acetone to give compound 2. Compound 2 reacted with an arylpiperazine (piperidine) in acetonitrile, in the presence of K2CO3 and KI, to give compounds 3a-m (Scheme 1, Table 1). Compounds 6a-b could be synthesized using the same approach (Scheme 1, Table 2). The starting 7-hydroxycoumarin intermediates 9 and 15 were obtained through the well-known von Pechmann reaction with slight modifications depending on the stability and reactivity of the β-dicarbonyl reagent used (Scheme 2 and Scheme 3).47 Subsequently, the 4-(chloromethyl)-7-hydroxy-2H-chromen-2-one

9

was

converted

to

the

4-(hydroxymethyl)-7-hydroxy-2H-chromen-2-one 10, which was then subjected to compound 11.48 Compound 11 reacted with an arylpiperazine (piperidine) to yield compounds 12a-b (Scheme 2, Table 2). 7-hydroxycoumarin intermediates 15 reacted with 1,4-dibromobutane to give compounds 16 in acetone. Compounds 16 reacted with an arylpiperazine (piperidine)



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in acetonitrile to give compounds 17a-t (Scheme 3, Table 2). 7-hydroxy-4-methylcoumarin reacted with 1,5-dibromopentane (1,3-dibromopropane or (E)-1,4-Dibromobut-2-ene), in acetone to give compounds 18 and 20 (Scheme 4). Compound 18 or 20 reacted with an arylpiperazine (piperidine) in acetonitrile to give compounds 19a-b and 21 (Scheme 4, Table 3). Epichlorohydrin reacted with 7-hydroxy-4-methylcoumarin or 7-hydroxy-4-phenyl-2H-chromen-2-one to give compounds 23a-b. Subsequently, compounds 23a-b reacted with arylpiperazine (piperidine) in MeOH to yield compounds 24a-b (Scheme 5, Table 3). Finally, (±)-7-hydroxy-4-phenyl-3,4-dihydrocoumarin (26) has been synthesized by the condensation of cinnamic acid (25) with resorcinol in the presence of concentrated HCl–HCl gas, resulting in a good yield.49 Subsequently, compound 26 reacted with 1,4-dibromobutane, in acetone to give compound 27. Compound 27 reacted with arylpiperazine (piperidine) in acetonitrile to give compound 28 (Scheme 5, Table 3). Results and Dissusion Structure–activity relationships Effect of coumarin moiety for different amine moieties As previously noted, ligands for D2, 5-HT1A and 5-HT2A receptors are of increasing therapeutic interest, and compounds from different chemical groups have been extensively studied in search of potentially novel antipsychotics. In the present study, we investigated the affinities of different amine moieties for D2, 5-HT1A and 5-HT2A receptors (Table 1, compounds

3a-m).

As

shown

in

Table

1,

compound

3a

bearing

a

(benzo[d]isothiazol-3-yl)piperazine moiety showed moderate affinities for the D2, 5-HT1A and 5-HT2A receptors. It should be noted that the (6-fluorobenzo[d]isoxazol-3-yl)piperidine derivative 3b displayed high affinities for the D2, 5-HT1A and 5-HT2A receptors (Ki = 4.9, 3.5 and 8.4 nM, respectively). Moreover, compound 3b showed a higher affinity for 5-HT1A receptors than risperidone (Ki = 180 nM). 5-HT1A receptor is implicated in the therapeutic efficacy of atypical antipsychotic drugs. Specifically, 5-HT1A receptor reduce negative symptoms and reduce the incidence of EPS in patients with schizophrenia.21 These results indicate that compound 3b bearing a (6-fluorobenzo[d]isoxazol-3-yl)piperidine moiety showed higher affinities for three receptors compared with compound 3a with a


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(benzo[d]isothiazol-3-yl)piperazine fragment.44 The amine moieties 2-(piperidin-4-yl)benzo[d]oxazole, 2-(piperidin-4-yl)benzo[d]thiazole, (4-fluorophenyl)(piperidin-4-yl)-methanone and 1-tosylpiperazine (compounds 3c-f) , showed low affinities for D2, 5-HT1A and 5-HT2A receptors. When amine moieties were phenylpiperazines, compounds 3g-h, 3j-l exhibited good affinities for the 5-HT1A receptor. Specifically, the 2-methoxyphenylpiperazine derivative 3g (Ki = 0.012 nM) displayed the strongest affinity for 5-HT1A receptors. Moreover, compound 3g (D2, Ki = 1.4 nM; 5-HT2A, Ki = 8.0 nM) displayed the high affinity for D2 and 5-HT2A receptors. According to the results, compound 3g displayed a higher affinity for the D2 and 5-HT1A receptors compared with risperidone (D2, Ki = 3.7 nM; 5-HT1A, Ki = 180 nM). The phenyl ring substituted with methoxy in the ortho-position (compound 3g) increased the activity at all three receptors compared with in the para- and meta-positions (compounds 3h-i). Therefore, affinity for all three receptors is dependent upon the location of the substituent on the phenyl ring. However, replacement

of

the

2-methoxyphenylpiperazine

moiety

with

3-trifluoromethyl

phenylpiperazine, 2,3-dimethylphenylpiperazine and 2,3-dichlorophenylpiperazine moieties (compounds 3j-l) reduced the affinity for D2 and 5-HT2A receptors. Substitution of the N-phenyl with a pyridine group (compound 3m) reduced the affinity for all three receptors. These results indicate that compounds bearing (6-fluorobenzo[d]isoxazol-3-yl) piperidine and 2-methoxyphenylpiperazine moieties (compounds 3b and 3g) possess higher affinity for all three receptors compared with those with other amine moieties. Effect of Substitution on the 4-position (R1) We investigated the effects of replacing the methyl (R1) with other substituents (Table 2, compounds 6a-b, 12a-b and 17a-h). The affinities of compounds 6a-b and 12a-b vs 3b and 3g for D2, 5-HT1A and 5-HT2A receptors decreased as a result of the addition of H and CH2OH. 6-fluorobenzo[d]isoxazol-3-yl)piperidine derivative 17a (methyl is replaced with phenyl) showed good affinities for D2, 5-HT1A and 5-HT2A receptors (D2, Ki = 14.8 nM; 5-HT1A, Ki = 12.7 nM; 5-HT2A, Ki = 9.2 nM). However, bearing 2-methoxyphenylpiperazine moiety (compound 17b vs 3g) showed weak affinity for the three receptors. Compounds 17c and 17d (electron-withdrawing CF3) showed high affinity for the 5-HT1A receptor, but weak affinity for D2 and 5-HT2A receptors. Compound 17e is structurally identical to 3b except that



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the methyl is replaced with an ethyl group. This replacement resulted in reduced the affinity for D2 and 5-HT2A receptors. Compound 17f (replacement of the methyl with the n-propyl) showed high affinity for D2 receptor (Ki = 3.9 nM), moderate affinity for 5-HT2A receptor (Ki = 30.8 nM), and low affinity for 5-HT1A receptor. The substitution of the methyl (3b) group with isopropyl (17g) and cyclopropyl (17h) group produced a significant decrease in the affinity for the three receptors. Based on the above results, we observed that modifications of the alkyl group in the 4- position (R1) of the coumarin provided analogues with the following order of D2, 5-HT1A and 5-HT2A receptors: methyl > n-propyl > ethyl > isopropyl > cyclopropyl. These results confirmed the importance of the 4-methyl group of coumarin in the modulation of dopaminergic and serotonergic activity.45,46,50 Effect of substitution on the 3,5,6 or 8-position Introduction of methyl on the 3-position (R2) of coumarin (17i vs. 3b), led to increased potency at D2 and 5-HT2A receptors (D2, Ki = 4.0 nM; 5-HT2A, Ki = 0.3 nM) with no change in 5-HT1A receptor affinity. Compared with compound 3g, however, compound 17j with a 2-methoxyphenylpiperazine moiety resulted in weakly affinity for D2 and 5-HT1A receptors and a moderate affinity for 5-HT2A receptors. Encouraged by these results, we proceeded with our investigation by introducing different substituents at the eighth (17k-n), fifth (17p) or sixth (17o) position of coumarin (Table 2). Substituents with CH3 or Cl at the eighth position (R3) of compounds 17k-l bearing a 2-methoxyphenylpiperazine moiety showed moderate affinities for D2, 5-HT1A and 5-HT2A receptors.

Notably,

compounds

17m

(Cl)

and

17n

(CH3)

bearing

a

6-fluorobenzo[d]isoxazol-3-yl)piperidine showed significantly higher affinities for 5-HT2A (17m, 5-HT2A, Ki = 0.3 nM; 17n, 5-HT2A, Ki = 0.9 nM) receptor compared with compound 3b. Moreover, compounds 17m (Cl) and 17n (CH3) also displayed high affinities for D2 and 5-HT1A receptors. Specifically, compound 17m (Ki = 2.6 nM) showed higher affinity for the D2 receptor compared with compound 3b (Ki = 4.9 nM) and risperidone (Ki = 3.7 nM). Based on these results, introducing substituents at the eighth position (R3) of an amine moiety with 6-fluorobenzo[d]isoxazol-3-yl)piperidine appears to increase the affinity for D2, 5-HT1A and 5-HT2A receptors. The 6-chloro substitution (R4) derivative 17o displayed good affinity for 5-HT1A and


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5-HT2A receptors, but low affinity for D2 receptor. The 5-methyl substitution (R5) derivative 17p had a high affinity for 5-HT1A receptor (Ki = 18.3 nM), and moderate affinity for 5-HT2A and D2 receptors. Compound 17q with CH3 the third, fourth and eighth positions showed high affinity for D2 receptor, and weak affinity for 5-HT1A and 5-HT2A receptors. These results suggested that introduction of substituents at the fifth (R5) or sixth position (R4) could not increase the affinity to D2, 5-HT1A and 5-HT2A receptors. Effect of replacing the phenyl group (17a) with other aromatic ring Compound 17a had high affinity for the D2, 5-HT1A and 5-HT2A receptors. Therefore, we investigated the effects of introducing flouro- and methoxy- substituents on the phenyl group or replacing the phenyl ring with a heterocyclic (Table 2, compounds 17r-t). According to the results, compounds 17r-t displayed a dramatic decrease in affinity for all three receptors. These results also indicated the importance of the phenyl ring for affinity to the D2, 5-HT1A and 5-HT2A receptors. Effect of linker of coumarin and piperidine ring We determined the effect of the length of the linker between the coumarin and the piperidine ring. As shown in Table 3, chain lengths of three (19a) or five (19b) carbon atoms resulted in significantly reduced D2, 5-HT1A and 5-HT2A receptors binding. Therefore, the binding affinities for D2, 5-HT1A and 5-HT2A receptors are dependent upon chain length. Compared with compound 3b, replacing the single bond with a trans double bond (compound 21) reduced D2 and 5-HT1A affinity. Introducing OH to the carbon chain of compounds 24a-b resulted in inactivation of all three receptors. The length of the alkyl chain appeared to have a direct impact on affinity for the three receptors. Taken together, the data indicate that the four-carbon chain length (compound 3b) was the most active. Effect of double bond of coumarin As shown in Table 3, replacing the double bond with a single bond of coumarin reduced the affinity for D2, 5-HT1A and 5-HT2A receptors (compound 28, Ki: D2, 515.2 nM; 5-HT1A, >10000 nM; 5-HT2A, 558.9 nM). These results suggest the importance of the double bond for affinity to D2, 5-HT1A and 5-HT2A receptors. Overall, compounds 3b, 3g, 17a, 17i, 17m and 17n exhibited high affinity for the D2, 5-HT1A and 5-HT2A receptors. These eight compounds showed higher affinities for 5-HT1A



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receptors compared with clozapine (Ki = 141.6 nM) and risperidone (Ki = 180 nM). In particular, compound 3g (Ki = 0.012 nM) displayed strong affinities for 5-HT1A receptors. Moreover, compounds 3g (Ki = 1.4 nM) and 17m (Ki = 2.6 nM) had higher affinities for D2 receptors compared with clozapine (Ki = 128.7 nM) and risperidone (Ki = 3.7 nM). Furthermore, compounds 17i (Ki = 0.3 nM) and 17m (Ki = 0.3 nM) showed excellent affinities for 5-HT2A receptors, consistent with risperidone (Ki = 0.18 nM). Therefore, compounds 3b, 3g, 17a, 17i, 17m and 17n were selected for additional studies of binding to the D3, H1, 5-HT2C, a1 and a2 receptors due to their high affinities for D2, 5-HT1A and 5-HT2A receptors. D3 receptors are located presynaptically on dopamine terminals and antagonism of the D3 receptor enhances dopamine release. This is an important therapeutic aim in schizophrenia. The D3 receptor was proposed as a target for atypical antipsychotic drugs and various pharmacological studies have suggested that D3 antagonism might improve cognitive symptoms and reduce catalepsy.32,33 As shown in Table 4, compounds 3b, 3g, 17a, 17i, 17m and 17n exhibit higher affinity for D3 receptors compared with clozapine (Ki = 239.8 nM). Specifically, compounds 3g (Ki = 4.8 nM) and 17m (Ki = 4.3 nM) displayed higher affinity to the three receptors compared with risperidone (Ki = 9.7 nM). Thus, these results suggested that compounds 3g and 17m may improve cognitive symptoms and reduce catalepsy in patients with schizophrenia. Treatment of schizophrenia with atypical antipsychotic drugs has been associated with weight gain. Two receptors, histamine H1 and 5-HT2C have been suggested to be involved in this adverse event. 22,35,36, 39,40 As shown in Table 4, compounds 3g, 17a, 17i and 17m had lower affinities for H1 receptors compared with risperidone (Ki = 21.7 nM) and clozapine (Ki = 3.8 nM). In particular, compounds 3g, 17i and 17m showed significantly lower affinity for H1 receptors (Ki > 500 nM). Moreover, compound 17m had lower affinity to the 5-HT2C receptor (Ki > 1000 nM) in comparison to risperidone (Ki = 14.5 nM) and clozapine (Ki = 16.2 nM). Based on the above results, compound 17m exhibited low potential to elicit treatment-associated weight gain. Blockade of α1-adrenergic receptors resulted in orthostatic hypotension, an unwanted side effect associated with many antipsychotic agents.51,52 According to Table 4, compounds 17a,


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17i, 17m and 17n showed lower affinities for α1 receptors compared with risperdone (Ki = 2.9 nM) and clozapine (Ki = 32.5 nM). Compounds 17i and 17n had moderate affinities for α1 receptors. Compounds 17a and 17m exhibited very weak affinities for α1 receptors (Ki > 2000 nM). These results suggested that compounds 17a and 17m may not cause orthostatic hypotension. The α2 adrenergic occupancy might explain the marked increase in dopamine output in the prefrontal cortex induced by clozapine, which is associated with cognitive functioning.53 According to Table 4, compound 17m (Ki = 15.6 nM) exhibited higher affinity for α2 receptors compared with risperidone (Ki = 29.5 nM) and clozapine (Ki = 55.6 nM). These results suggested that compound 17m might exhibit enhanced clinical efficacy. Ether-a-gogo-Related Gene (hERG) KC channels Recent studies have shown that a wide range of medications can prolong the length of time between the start of the Q wave and the end of the T wave on an electrocardiogram (QT interval). Specific examples include sertindole, grepafloxacin and terfenadine.54 In extreme cases or in susceptible individuals, QT prolongation is associated with torsade de pointes (TdP), a polymorphic ventricular arrhythmia that can progress to ventricular fibrillation and sudden death. Indeed, efforts to predict an increased risk for long-QT syndrome have focused on assays that test hERG channel activity. This is because hERG channel blockade is an important indicator of potential pro-arrhythmic liability. To date, investigation of hERG channel blockade has been a significant step in the drug discovery trajectory in the pharmaceutical industry. As shown in Table 4, compounds 3b, 3g, 17i and 17n exhibited higher affinities for hERG compared with risperidone and clozapine. Interestingly, the low affinity of 17a (IC50 = 820 nM) and 17m (IC50 = 1591 nM) for hERG compared with risperidone and clozapine may decrease the propensity of these compounds to elicit treatment-induced QT interval prolongation. More importantly, when tested in vivo in the anesthetized rat model, compound 17m showed no QT prolongation or any cardiac liability despite at the highest dose (5, 15 and 60 mg/kg, po, supporting information). However, in comparison, risperidone could cause QT prolongation at the dose of 30 mg/kg (po). Intrinsic activity of 17m at selected receptors The intrinsic activity of compound 17m was selected for further investigation based on its



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interaction with multiple receptors and low hERG. As shown in Table 5, 17m showed stimulatory activity in an agonist assay on D2, D3, 5-HT1A and 5-HT2A receptors displayed minimal agonist activity, in all cases displaying less than 10% of the efficacy of the reference endogenous agonist. In an antagonist assay, 17m inhibited four receptors (D2, D3, 5-HT1A and 5-HT2A) by over 95%. Compound 17m behaves as antagonist at D2 receptor (IC50 26.3 nM), D3 receptor (IC50 24.8 nM), 5-HT2A receptor (IC50 39.2 nM) and 5-HT1A receptor (IC50 11.8 nM). Acute toxicity Taken together, the data indicated that compound 17m exhibited high affinity for dopamine (D2 and D3) and serotonin (5-HT1A and 5-HT2A) receptors, as well as low affinity for the H1 and 5-HT2C receptors and hERG. The acute toxicity of compound 17m was assayed in terms of LD50 values. Compound 17m displayed a good safety profile, even at the highest dose tested (LD50 > 2000 mg/kg). Weight gain and serum prolactin Antipsychotics have side effects including weight gain and hyperprolactinemia.13,14 As shown in the Figure 3, 17m showed negligible weight gain in mice that experienced chronic dosing (28 days). Risperidone, used as the positive control, resulted in a significantly increased weight gain in mice that received medium and high doses (Figure 3). This was also consistent with the estimated Ki values for H1 (risperidone, 21.7 nM; 17m, 1125.3 nM) and 5-HT2C (risperidone, 14.5 nM; 17m, 1700.7 nM) receptors. Moreover, compound 17m did not significantly influence serum prolactin levels compared with risperidone (Figure 4). Behavioral studies Due to the marked effect of 17m on serotonergic, dopaminergic and adrenergic receptors, it was selected as a promising atypical antipsychotic agent and subjected to in vivo pharmacological characterization. Atypical antipsychotics are used to relieve positive symptoms without EPS.22 In the present study, the side-effect liability was evaluated using the horizontal bar test, a very sensitive measure of catalepsy induced by dopamine D2 receptor blockade.22 The antipsychotic potential of the compounds was assessed by means of apomorphine-induced

climbing

and

dizocilpine-induced

(MK-801)

hyperactivity.55

Apomorphine-induced climbing was significantly reduced by D2 receptor antagonists.


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Selective antagonism of the noncompetitive N-methyl-D-aspartate (NMDA) antagonist, MK-801, has been proposed as an animal model of the negative and cognitive symptoms associated with schizophrenia.59 Conditioned avoidance response (CAR) is one of most important preclinical animal models used to study antipsychotic drugs.56 The

apomorphine-induced climbing

model

is

based

on the

induction

of

a

hyperdopaminergic state. This model has been classically linked to motor agitation and positive symptoms.55 Compound 17m produced significant dose-dependent responses in this model (Figure 5), with an ED50 value of 0.14 mg/kg (Table 6). In comparison, risperidone, clozapine and haloperidol reversed apomorphine-induced climbing with ED50 values of 0.02, 7.99 and 0.09 mg/kg, respectively. These results suggest that compound 17m was slightly more potent in terms of blocking D2 receptors in vivo compared with clozapine. This was also consistent with the estimated Ki values for the D2 receptor. The MK-801-induced hyperactivity model has been used to indirectly evaluate the ability of a compound to oppose cortical dopaminergic hypofunction induced by NMDA receptor blockade. In this test, compound 17m showed significant dose-dependent responses (Figure 6) with an ED50 value of 0.32 mg/kg (Table 6). In comparison, risperidone, clozapine and haloperidol yielded ED50 values of 0.01, 5.06 and 0.19 mg/kg, respectively. These results indicate that compound 17m was more potent than clozapine. Catalepsy is often used to predict the incidence of extrapyramidal motor disorders.57 In this model (Table 6), haloperidol had the highest propensity to induce catalepsy (ED50 0.22 mg/kg), consistent with its marked ability to block D2 receptors. In contrast, compound 17m exhibited a low potential for catalepsy (ED50, 70.85 mg/kg), consistent with risperidone and clozapine (ED50 risperidone 0.3 mg/kg, clozapine 92.73 mg/kg). The therapeutic indices of compound 17m based on its efficacy (apomorphine or MK-801 models) and its side effects (catalepsy) were in the range 214–222, while the therapeutic indices of risperidone and clozapine are roughly 11–30. In contrast to risperidone and clozapine, compound 17m had a high threshold for catalepsy, which might, by analogy, translate into lower clinical EPS liability. Disruption of the “avoidance” response in CAR is another preclinical model that is used to predict antipsychotic activity. In the rat CAR model, compound 17m effectively inhibited the



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avoidance response in a dose-dependent manner (Figure 7). Nonlinear regression analysis calculated an ED50 equivalent of 6.50 mg/kg (Table 8). Therefore,

compound

17m

inhibited

apomorphine-induced

climbing

behavior,

MK-801-induced hyperactivity and the conditioned avoidance response. Compound 17m also demonstrated a low propensity to induce unwanted extrapyramidal motor disturbances at therapeutically useful doses. Pharmacokinetic properties of compound 17m The pharmacokinetic properties of 17m including its in vitro and in vivo efficacy and the degree of safety were explored in the rat (Table 7). Intravenous administration of compound 17m to rats (5 mg/kg, n = 6) resulted in detectable plasma levels (half-life (t1/2) = 5.0 h). Oral administration of compound 17m to rats (12 mg/kg, n = 6) resulted in a t1/2 of 6.7 h. The area under the curve (AUC) value of compound 17m was 914.4 ng×h/ml after intravenous administration vs 702.7 ng×h/mL after oral administration. The Cmax value after oral dosing was 72.1 ng/mL, and the Tmax value was 3.0 h. The bioavailability of compound 17m was 32.0%. These encouraging preclinical data suggest that 17m possesses desirable drug-like human pharmacokinetic properties. Conclusion

A detailed structure activity relationship investigation of structure A and B has shown that several factors influence the binding affinity for the D2, 5-HT1A and 5-HT2A receptors of these compounds. First, bearing a 6-fluorobenzo[d]isoxazol-3-yl)piperidine moiety showed higher affinities to three receptors than those with other amine moieties, (2) the substitution on 4-position (R1) with methyl as favored substituent, (3) the substitution on the 3-position (R2) with unsubstituted or substitution (methyl) preferred, (4) the substitution on the 8-position (R3) with electron-withdrawing (chloro) as favored substituent, (5) the substitution on the 5-position (R5) or 6-position (R4) did lower the affinity for the three receptors, (6) the importance of the double bond for affinity to D2, 5-HT1A and 5-HT2A receptors, (7) straight


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four-carbon chain alkyl as preferred over other alkyl linker. From this SAR study, the compound 17m, which has unique affinities for dopamine, serotonin and adrenergic receptors, was identified as a potential multi-receptor antipsychotic. The binding profile of 17m showed high affinities for dopaminergic (D2 and D3) and serotonergic (5-HT1A and 5-HT2A) receptors and low affinities for H1 receptors and hERG channels. Use of animal models revealed that 17m significantly inhibited apomorphine-induced climbing behavior, MK-801-induced hyperactivity and CAR without catalepsy at the highest dose tested. Moreover, the data suggest a therapeutic index of at least 200-fold between efficacy (apomorphine or MK-801 models) and side effects (catalepsy). Compound 17m had a higher threshold for catalepsy induction compared with the two currently marketed atypical antipsychotics, risperidone and clozapine. Compound 17m also displayed fewer preclinical adverse events in assays of prolactin secretion and weight gain compared with risperidone. Moreover, compound 17m exhibited acceptable pharmacokinetic properties. Thus compound 17m may facilitate development of a novel class of drugs for the treatment of schizophrenia. Experimental Chemistry experimental Melting points were determined in open capillary tubes and are uncorrected. 1H NMR and 13

C NMR spectra were recorded at 400 MHz on a Varian Inova Unity 200 spectrometer in

CDCl3 solution. 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). Reagents were all of analytical grade or of chemical purity. Analytical 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 were >95% purity. HPLC methods used the followung: Agilent 1100 spectrometer; column, Shim-pack ODS 5.0 µm × 150 mm×2.0 mm I.D (SHIMADZU, Japanese); mobile phase, 0.0167% HCOOH (TEDIA Company，USA)/acetonitrile (Merck Company，Germany）50/50; flow rate, 0.2 mL/min; column temperature, 40 °C; UV detection was performed at 254 nm. 7-(4-Bromobutoxy)-4-methyl-2H-chromen-2-one (2) [58]



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1,4-Dibromobutane (2 mmol) was added to a solution of 7-hydroxy-4-methylcoumarin (1 mmol) and potassium carbonate in acetone (50 mL), and the mixture was refluxed for 4 h. The progress of the reaction was monitored by TLC. After cooling to room temperature, the mixture was filtered, the solvent was evaporated and the residue was recrystallized from EtOH to yield compound 2. Yield: 65%; mp: 65-67 ºC. 1H-NMR (CDCl3) δ 1.98-2.10 (m, 4H), 2.40 (s, 3H), 3.51 (t, 2H, J =6.0 Hz), 4.06 (t, 2H, J = 6.6 Hz), 6.13 (s, 1H), 6.79-6.87 (m, 2H), 7.50 (d, 1H, J =8.8 Hz). MS (ESI) m/z 311.2 (M+). General procedure for the preparation of compounds 3a-m To a suspension of compound 2 (0.32 mmol) and K2CO3 (0.64 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 6-12 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 evaporated under reduced pressure, and the crude product was purified by means of chromatography (5-10% MeOH/CHCl3) to yield compounds 3a-m. 7-(4-(4-(Benzo[d]isothiazol-3-yl)piperazin-1-yl)butoxy)-4-methyl-2H-chromen-2-one (3a): Yield: 69.3%; mp: 110-112 ºC. 1H-NMR (CDCl3) δ 1.75-1.90 (m, 4H), 2.38 (s, 3H), 2.51 (t, 2H, J = 7.2 Hz), 2.68-2.71 (m, 4H), 3.56-3.58 (m, 4H), 4.06 (t, 2H, J = 6.4 Hz), 6.11 (s, 1H), 6.80-6.86 (m, 2H), 7.30-7.36 (m, 1H), 7.43-7.48 (m, 2H), 7.79 (d, 1H, J = 8.8 Hz), 7.90 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 450.2 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-methyl-2H-chromen-2one (3b): Yield: 79.2%; mp: 126-128 ºC. 1H-NMR (CDCl3) δ 1.71-1.92 (m, 4H), 2.09-2.19 (m, 6H), 2.09 (s, 3H), 2.50 (t, 2H, J = 7.6 Hz), 3.09-3.12 (m, 3H), 4.07 (t, 2H, J = 6.4 Hz), 6.13 (s, 1H), 6.81-6.88 (m, 2H), 7.03-7.08 (m, 1H), 7.23-7.25 (m, 1H), 7.49 (d, 1H, J = 8.8 Hz), 7.69-7.72 (m, 1H). MS (ESI) m/z 451.2 ([M+H]+). 7-(4-(4-(Benzo[d]oxazol-2-yl)piperidin-1-yl)butoxy)-4-methyl-2H-chromen-2-one

(3c):

Yield: 71.3%; mp: 108-110 ºC. 1H-NMR (CDCl3) δ 1.68-1.89 (m, 4H), 1.99-2.19 (m, 6H), 2.39 (s, 3H), 2.45 (t, 2H, J = 7.2 Hz), 2.93-3.02 (m, 3H), 4.06 (t, 2H, J = 6.2 Hz), 6.12 (s, 1H), 6.81-6.87 (m, 2H), 7.27-7.32 (m, 2H ), 7.46-7.50 (m, 2H ), 7.67 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 433.2 ([M+H]+).


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7-(4-(4-(Benzo[d]thiazol-2-yl)piperidin-1-yl)butoxy)-4-methyl-2H-chromen-2-one (3d): Yield: 78.3%; mp: 119-121 ºC. 1H-NMR (CDCl3) δ 1.72-1.74 (m, 2H), 1.85-1.87 (m, 4H), 2.11-2.20 (m, 4H), 2.38 (s, 3H), 2.46 (t, 2H, J = 7.0 Hz), 3.05-3.12 (m, 3H), 4.06 (t, 2H, J = 6.6 Hz), 6.12 (s, 1H), 6.80-6.86 (m, 2H), 7.28-7.36 (m, 3H), 7.85 (d, 1H, J = 8.8 Hz), 7.97 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 449.3 ([M+H]+). 7-(4-(4-(4-Fluorobenzoyl)piperidin-1-yl)butoxy)-4-methyl-2H-chromen-2-one (3e): Yield: 72.3%; mp: 101-103 ºC. 1H-NMR (CDCl3) δ 1.68-1.72 (m, 2H), 1.81-1.87 (m, 6H), 2.08-2.11 (m, 2H), 2.40 (s, 3H), 2.42 (t, 2H, J = 7.6 Hz), 3.00-3.03 (m, 2H), 3.18-3.21 (m, 1H), 4.05 (t, 2H, J = 6.0 Hz), 6.13 (s, 1H), 6.80-6.87 (m, 2H), 7.43-7.50 (m, 3H), 7.87-7.88 (m, 2H). MS (ESI) m/z 438.2 ([M+H]+). 7-(4-(4-Tosylpiperidin-1-yl)butoxy)-4-methyl-2H-chromen-2-one (3f): Yield: 70.2%; mp: 105-107 ºC. 1H-NMR (CDCl3) δ 1.60-1.64 (m, 2H), 1.77-1.81 (m, 2H), 2.39-2.53 (m, 12H), 3.01 (s, br, 4H), 4.00 (t, 2H, J = 6.0 Hz), 6.13 (s, 1H), 6.76-6.83 (m, 2H), 7.32 (d, 2H, J = 8.8 Hz), 7.47 (d, 1H, J = 8.8 Hz), 7.63 (d, 2H, J = 8.8 Hz). MS (ESI) m/z 471.2 ([M+H]+). 7-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butoxy)-4-methyl-2H-chromen-2-one

(3g):

Yield: 61.2%; mp: 99-101 ºC. 1H-NMR (CDCl3) δ 1.68-1.88 (m, 4H), 2.40 (s, 3H), 2.47 (t, 2H, J = 7.6 Hz), 2.68 (s, br, 4H), 3.11 (s, br, 4H), 3.87 (s, 3H), 4.06 (t, 2H, J = 6.4 Hz), 6.13 (s, 1H), 6.81-7.00 (m, 6H), 7.49 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 423.2 ([M+H]+). 7-(4-(4-(3-Methoxyphenyl)piperazin-1-yl)butoxy)-4-methyl-2H-chromen-2-one

(3h):

Yield: 68.2%; mp: 103-105 ºC. 1H-NMR (CDCl3) δ 1.71-1.78 (m, 2H), 1.86-1.89 (m, 2H), 2.41 (s, 3H), 2.49 (t, 2H, J = 7.2 Hz), 2.62-2.64 (m, 4H), 3.21-3.23 (m, 4H), 3.80 (s, 3H), 4.07 (t, 2H, J = 6.4 Hz), 6.14 (s, 1H), 6.43 (d, 1H, J = 8.8 Hz), 6.48 (d, 1H, J = 8.8 Hz), 6.55-6.58 (m, 1H), 6.82-6.85 (m, 1H), 6.89 (d, 1H, J = 8.8 Hz), 7.18 (t, 1H, J = 8.8 Hz), 7.50 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 423.2 ([M+H]+). 7-(4-(4-(4-Methoxyphenyl)piperazin-1-yl)butoxy)-4-methyl-2H-chromen-2-one

(3i):

Yield: 65.3%; mp: 109-111 ºC. 1H-NMR (CDCl3) δ 1.72-1.78 (m, 2H), 1.85-1.89 (m, 2H), 2.40 (s, 3H), 2.49 (t, 2H, J = 7.2 Hz), 2.63-2.65 (m, 4H), 3.10-3.13 (m, 4H), 3.78 (s, 3H), 4.07 (t, 2H, J = 6.4 Hz), 6.14 (s, 1H), 6.81-6.93 (m, 6H), 7.49 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 423.3 ([M+H]+). 7-(4-(4-(3-(Trifluoromethyl)phenyl)piperazin-1-yl)butoxy)4-methyl-2H-chromen-2-one



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(3j): Yield: 78.9%; mp: 86-88 ºC. 1H-NMR (CDCl3) δ 1.72-1.89 (m, 4H), 2.17 (s, 3H), 2.47 (t, 2H, J = 7.6 Hz), 2.62-2.64 (m, 4H), 3.24-3.26 (m, 4H), 4.07 (t, 2H, J = 6.2 Hz), 6.13 (s, 1H), 6.81-6.87 (m, 2H), 7.05-7.11 (m, 3H), 7.29-7.35 (m, 1H), 7.48 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 461.5 ([M+H]+). 7-(4-(4-(2,3-Dimethylphenyl)piperazin-1-yl)butoxy)-4-methyl-2H-chromen-2-one

(3k):

Yield: 81.2%; mp: 96-98 ºC. 1H-NMR (CDCl3) δ 1.74-1.84 (m, 4H), 2.22 (s, 3H), 2.26 (s, 3H), 2.40 (s, 3H), 2.49 (t, 2H, J = 7.2 Hz), 2. 64 (s, br, 4H), 2.91-2.93 (m, 4H), 4.06 (t, 2H, J = 6.0 Hz), 6.13 (s,1H), 6.81-6.93 (m, 4H), 7.06 (d, 1H, J = 8.8 Hz), 7.48 (d, 1H, J = 8.4 Hz). MS (ESI) m/z 421.2 ([M+H]+). 7-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butoxy)-4-methyl-2H-chromen-2-one (3l) : Yield: 81.2%; mp: 98-100 ºC. 1H-NMR (CDCl3) δ 1.67-1.71 (m, 2H), 1.81-1.86 (m, 2H), 2.34 (s, 3H), 2.46 (t, 2H, J = 7.2 Hz), 2.62 (s, br, 4H), 3.03 (s, br, 4H), 4.02 (t, 2H, J = 6.0 Hz), 6.06 (s, 1H), 6.74-6.82 (m, 2H), 6.90-6.94 (m, 1H), 7.07-7.12 (m, 2H), 7.43 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 461.2 ([M+H]+). 7-(4-(4-(Pyridin-2-yl)piperazin-1-yl)butoxy)-4-methyl-2H-chromen-2-one (3m): Yield: 78.1%; mp: 89-91 ºC. 1H-NMR (CDCl3) δ 1.72-1.78 (m, 2H), 1.84-1.89 (m, 2H), 2.38 (s, 3H), 2.47 (t, 2H, J = 7.2 Hz), 2.54-2.59 (m, 4H), 3.54-3.57 (m, 4H), 4.06 (t, 2H, J = 6.4 Hz), 6.11 (s, 1H), 6.60-6.65 (m, 2H), 6.79-6.86 (m, 2H), 7.44-7.51 (m, 2H), 8.18 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 394.1 ([M+H]+). 7-(4-Bromobutoxy)- 2H-chromen-2-one (5) 1,4-dibromobutane (2 mmol) was added to a solution of 7-hydroxycoumarin (1 mmol) and potassium carbonate in acetone (50 mL), and the mixture was refluxed for 5 h. The reaction was monitored by TLC. After cooling to room temperature, the mixture was filtered, the solvent was evaporated and the residue was recrystallized from EtOH to yield compound 5. Yield: 85.2%; mp: 66-68 ºC. 1H-NMR (CDCl3) δ 1.97-2.14 (m, 4H), 3.51 (t, 2H, J = 6.0 Hz), 4.08 (t, 2H, J = 6.0 Hz), 6.27 (d, 1H, J = 8.8 Hz), 6.81-6.86 (m, 2H), 7.39 (d, 1H, J = 8.8 Hz), 7.65 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 297.0 ([M+H]+). General procedure for the preparation of compounds 6a-b To a suspension of compound 5 (0.32 mmol) and K2CO3 (0.64 mmol) in acetonitrile (5.0 mL), arylpiperazine (piperidine) (0.32 mmol) and a catalytic amount of KI were added and


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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 evaporated under reduced pressure, and the crude product was purified by means of chromatography (2% MeOH/CHCl3) to yield compounds 6a-b. 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-2H-chromen-2-one

(6a):

Yield: 70.8%; mp: 116-118 ºC. 1H NMR (CDCl3) δ 1.73-1.88 (m, 4H), 2.06-2.16 (m, 6H), 2.48 (t, 2H, J = 7.2 Hz), 3.07-3.10 (m, 3H), 4.07 (t, 2H, J = 6.4 Hz), 6.24 (d, 1H, J = 6.4 Hz), 6.80-6.86 (m, 2H), 7.05 (t, 1H, J = 1.6 Hz), 7.22-7.24 (m, 1H), 7.37 (d, 1H, J = 8.4 Hz), 7.63-7.69 (m, 2H). MS (ESI) m/z 437.2 ([M+H]+). 7-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butoxy)-2H-chromen-2-one (6b): Yield: 80.8%; mp: 81-83 ºC. 1H NMR (CDCl3) δ 1.73-1.88 (m, 4H), 2.49 (t, 2H, J = 7.6 Hz), 2.68 (s, br, 4H), 3.11 (s, br, 4H), 3.86 (s, 3H), 4.06 (t, 2H, J = 6.0 Hz), 6.24 (d, 1H, J = 6.4 Hz), 6.81-7.00 (m, 6H), 7.36 (d, 1H, J = 6.4 Hz), 7.63 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 409.3 ([M+H]+). 4-(Chloromethyl)-7-hydroxy-2H-chromen-2-one (9) 43 Resorcinol (10 g, 91 mmol) was dissolved in 100 mL of conc sulfuric acid at 0 0C. Then ethyl 4-chloroacetoacetate (10 mL, 74 mmol) was slowly added and the mixture was stirred at 0-5 0C for 2 h. The reaction mixture was then poured onto ice-water and the solid was filtered and washed with water, the residue was recrystallized from EtOH to yield white solid 11.8 g, yield: 61.8%, mp: 182-184 °C. MS (ESI) m/z 211.7 ([M+H]+). 4-(Hydroxymethyl)-7-hydroxy-2H-chromen-2-one (10) 44 A suspension of 9 (2 g, 7.8 mmol) in 200 ml of water was heated under refluxed for 8h. After cooling to room temperature the reaction mixture was extracted three times with EtOAc. The combined extracts were dried over anhydrous MgSO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography (EtOAc) providing white solid 1.4 g, yield: 76.9%, mp: 186-188 °C. MS (ESI) m/z 193.3 ([M+H]+). 7-(4-Bromobutoxy)-4-(hydroxymethyl)-2H-chromen-2-one (11) 1,4-dibromobutane (2 mmol) was added to a solution of compound 10 (1 mmol) and potassium carbonate in acetone (50 mL), and the mixture was refluxed for 6 h. The progress of the reaction was monitored by TLC. After cooling to room temperature, the mixture was



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filtered, the solvent was evaporated and the residue was recrystallized from hexane /EtOH to yield compound 11. Yield: 55.6%; mp: 80-82 ºC. 1H-NMR (CDCl3) δ 1.98-2.14 (m, 4H), 3.52 (t, 2H, J = 6.4 Hz), 4.08 (t, 2H, J = 6.0 Hz), 4.91 (s, 2H), 6.49 (s, 1H), 6.84-6.87 (m, 2H), 7.44 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 327.2 ([M+H]+). General procedure for the preparation of compounds 12a-b To a suspension of compound 11 (0.32 mmol) and K2CO3 (0.64 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 10-12 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 evaporated under reduced pressure, and the crude product was purified by means of chromatography (10% MeOH/CHCl3) to yield compounds 12a-b. 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-(hydroxymethyl)-2Hchromen-2-one (12a): Yield: 65.9%; mp: 160-162 ºC. 1H-NMR (CDCl3) δ 1.71-1.75 (m, 2H), 1.84-1.89 (m, 2H), 2.08-2.17 (m, 6H), 2.49 (t, 2H, J = 7.2 Hz), 3.08-3.11 (m, 3H), 4.05 (t, 2H, J = 6.0 Hz), 4.88 (s, 2H), 6.46 (s, 1H), 6.83-6.85 (m, 2H), 7.03-7.08 (m, 1H), 7.24 (d, 1H, J = 8.8 Hz), 7.42 (d, 1H, J = 8.8 Hz), 7.68-7.72 (m, 1H). MS (ESI) m/z 467.2 ([M+H]+). 7-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butoxy)-4-(hydroxymethyl)-2H-chromen-2-on e (12b): Yield: 60.1%; mp: 94-96 ºC. 1H-NMR (CDCl3) δ 1.73-1.75 (m, 2H), 1.83-1.87 (m, 2H), 2.51 (t, 2H, J = 7.2 Hz), 2.72 (s, br, 5H), 3.12 (s, br, 5H), 3.86 (s, 3H), 4.01 (t, 2H, J = 6.4 Hz), 4.83 (s, 2H), 6.45 (s, 1H), 6.77-7.00 (m, 8H), 7.39 (d, 1H, J = 8.4 Hz). MS (ESI) m/z 439.1 ([M+H]+). General procedure for the synthesis of 7-hydroxy-4-methylcoumarins (15)43 To a mixture of resorcinol (1.0 g, 9 mmol) in alkylated ethyl acetoacetate was slowly added concentrated sulfuric acid (5 mL, dropwise) at 0 oC. The mixture was stirred at room temperature for 3-4 h. The progress of reaction was monitored on TLC (5% MeOH/CHCl3). On completion of the reaction 100 mL ice/water was added. The crude solid so obtained was then filtered, washed with water, dried and crystallized from ethanol to give the coumarins 15. General procedure for the synthesis of compounds (16)


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1,4-Dibromobutane (2 mmol) was added to a solution of compounds 15 (1 mmol) and potassium carbonate in acetone (50 mL), and the mixture was refluxed for 5-8 h. The reaction was monitored by TLC. After cooling to room temperature, the mixture was filtered, the solvent was evaporated and the residue was recrystallized from hexane /EtOH to yield compounds 16. 7-(4-Bromobutoxy)-4-phenyl-2H-chromen-2-one (16a): Yield: 70.1%; mp: 64-66 ºC. 1

H-NMR (CDCl3) δ 1.94-2.14 (m, 4H), 3.51 (t, 2H, J = 6.0 Hz), 4.14 (t, 2H, J = 6.0 Hz), 6.22

(s, 1H), 6.78-6.89 (m, 2H), 7.38-7.56 (m, 6H). MS (ESI) m/z 372.1 ([M+H]+). 7-(4-Bromobutoxy)-4-(trifluoromethyl)-2H-chromen-2-one (16b): Yield: 50.5%; mp: 84-86 ºC. 1H-NMR (CDCl3) δ 1.99-2.14 (m, 4H), 3.52 (t, 2H, J = 6.0 Hz), 4.11 (t, 2H, J = 6.6 Hz), 6.63 (s, 1H), 6.88-6.94 (m, 2H), 7.62-7.65 (m, 1H). MS (ESI) m/z 365.0 ([M+H]+). 7-(4-Bromobutoxy)-4-ethyl-2H-chromen-2-one (16c): Yield: 70.8%; mp: 94-96 ºC. 1

H-NMR (CDCl3) δ 1.33 (t, 3H, J = 7.2 Hz), 1.99-2.11 (m, 4H), 2.75-2.81 (m, 2H), 3.51 (t,

2H, J = 6.4 Hz), 4.07 (t, 2H, J = 6.0 Hz), 6.15 (s, 1H), 6.80-6.87 (m, 2H), 7.53 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 325.2 ([M+H]+). 7-(4-Bromobutoxy)-4-propyl-2H-chromen-2-one (16d): Yield: 65.8%; mp: 88-90 ºC. 1

H-NMR (CDCl3) δ 1.06 (t, 3H, J = 7.2 Hz), 1.99-2.12 (m, 6H), 2.70-2.74 (m, 2H), 3.51(t,

2H, J = 6.4 Hz), 4.07 (t, 2H, J = 6.0 Hz), 6.14 (s, 1H), 6.81-6.87 (m, 2H), 7.54 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 339.2 ([M+H]+). 7-(4-Bromobutoxy)-4-isopropyl-2H-chromen-2-one (16e): Yield: 75.8%; mp: 58-60 ºC. 1

H-NMR (CDCl3) δ 1.30 (s, 3H), 1.32 (s, 3H), 1.96 -2.12 (m, 4H), 3.22-3.29 (m, 1H), 3.50 (t,

2H, J = 6.4 Hz), 4.07 (t, 2H, J = 6.0 Hz), 6.17 (s, 1H), 6.81-6.87 (m, 2H), 7.57 (d, 1H, J = 8 Hz). MS (ESI) m/z 339.2 ([M+H]+). 7-(4-Bromobutoxy)-4-cyclopropyl-2H-chromen-2-one (16f): Yield: 62.9%; 65-67 ºC. 1

H-NMR (CDCl3) δ 0.83-0.85 (m, 2H), 1.13-1.16 (m, 2H), 1.99-2.12 (m, 5H), 3.51 (t, 2H, J =

6.2 Hz), 4.07 (t, 2H, J = 6.0 Hz), 5.89 (s, 1H) 6.80-6.82 (m, 1H), 6.85-6.89 (m, 1H), 7.81 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 337.1 ([M+H]+). 7-(4-Bromobutoxy)-3,4-dimethyl-2H-chromen-2-one (16g): Yield: 78.9%; 95-97 ºC. 1

H-NMR (CDCl3) δ 1.97-2.14 (m, 4H), 2.22 (s, 3H), 2.38 (s, 3H), 3.51 (t, 2H, J = 6.4 Hz),

4.06 (t, 2H, J = 6.0 Hz), 6.79-6.85 (m, 2H), 7.48-7.52 (m, 1H). MS (ESI) m/z 325.3 ([M+H]+).



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7-(4-Bromobutoxy)-8-chloro-4-methyl-2H-chromen-2-one (16h): Yield: 71.2%; 105-107 ºC. 1H-NMR (CDCl3) δ 2.05-2.17 (m, 4H), 2.49 (s, 3H), 3.55 (t, 2H, J = 5.8 Hz), 4.18 (t, 2H, J = 6.4 Hz), 6.16 (d, 1H, J = 1.2 Hz), 6.89-6.91 (m, 1H), 7.46-7.48 (m, 1H). MS (ESI) m/z 345.1 ([M+H]+). 7-(4-Bromobutoxy)-4,8-dimethyl-2H-chromen-2-one (16i): Yield: 78.3%; 108-110 ºC. 1


4.12 (t, 2H, J = 6.4 Hz), 6.15 (s, 1H), 6.83-6.85 (m, 1H), 7.42-7.44 (m, 1H). MS (ESI) m/z 325.2 ([M+H]+). 7-(4-Bromobutoxy)-6-chloro-4-methyl-2H-chromen-2-one (16j): Yield: 50.9%; 118-120 ºC. 1H-NMR (CDCl3) δ 2.04-2.18 (m, 4H), 2.43 (s, 3H), 3.55 (t, 2H, J = 6.4 Hz), 4.13 (t, 2H, J = 6.0 Hz), 6.18 (s, 1H), 6.84 (s, 1H), 7.58 (s, 1H). MS (ESI) m/z 345.0 ([M+H]+). 7-(4-Bromobutoxy)-4,5-dimethyl-2H-chromen-2-one (16k): Yield: 60.6%; 88-90 ºC. 1


4.08 (t, 2H, J = 6.0 Hz), 6.05 (s, 1H), 6.53 (s, 1H), 6.75 (s, 1H). MS (ESI) m/z 325.1 ([M+H]+). 7-(4-Bromobutoxy)-4-(4-fluorophenyl)-2H-chromen-2-one (16l): Yield: 68.1%; mp: 72-74 ºC. 1H-NMR (CDCl3) δ 2.01-2.11 (m, 4H), 3.53 (t, 2H, J = 6.4 Hz), 4.09 (t, 2H, J = 6.0 Hz), 6.21 (s, 1H), 6.80-6.83 (m, 1H), 6.89 (d, 1H, J = 4 Hz), 7.21-7.25 (m, 2H), 7.36 (d, 1H, J = 8 Hz), 7.43-7.47 (m, 2H). MS (ESI) m/z 391.1 ([M+H]+). 7-(4-Bromobutoxy)-4-(3-methoxyphenyl)-2H-chromen-2-one (16m): Yield: 72.2%; mp: 78-80 ºC. 1H-NMR (CDCl3) δ 1.98-2.13 (m, 4H), 3.51 (t, 2H, J = 6.4 Hz), 3.88 (s, 3H), 4.09 (t, 2H, J = 6.0 Hz), 6.24 (s, 1H), 6.78-6.81 (m, 1H), 6.89 (d, 1H, J = 4 Hz), 6.97 (d, 1H, J = 4 Hz), 7.01-7.06 (m, 2H), 7.43-7.46 (m, 2H). MS (ESI) m/z 402.2 ([M+H]+). 7-(4-Bromobutoxy)-4-(thiophen-2-yl)-2H-chromen-2-one (16n): Yield: 70.1%; mp: 58-60 ºC. 1H-NMR (CDCl3) δ 1.98-2.15 (m, 4H), 3.52 (t, 2H, J = 6.6 Hz), 4.10 (t, 2H, J = 6.4 Hz), 6.35 (s, 1H), 6.85-6.88 (m, 2H), 7.22-7.24 (m, 1H), 7.42 (d, 1H, J = 8 Hz), 7.56 (d, 1H, J = 4 Hz), 7.84 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 379.0 ([M+H]+). General procedure for the synthesis of compounds (17) To a suspension of compounds 16 (0.32 mmol) and K2CO3 (0.64 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 8-12 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


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evaporated under reduced pressure, and the crude product was purified by means of chromatography (5-10% MeOH/CHCl3) to yield compounds 17. 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-phenyl-2H-chromen-2one (17a): Yield: 75.1%; mp: 97-99 ºC. 1H-NMR (CDCl3) δ 1.72-1.76 (m, 2H), 1.87-1.91 (m, 2H), 2.07-2.18 (m, 6H), 2.48 (t, 2H, J = 7.2 Hz), 3.07-3.10 (m, 3H), 4.08 (t, 2H, J = 6.4 Hz), 6.21 (s, 1H), 6.80-6.80 (m, 1H), 6.88-6.89 (m, 1H), 7.03-7.07 (m, 1H), 7.21-7.24 (m, 1H), 7.37-7.52 (m, 6H), 7.68-7.71(m, 1H). MS (ESI) m/z 513.3 ([M+H]+). 7-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butoxy)-4-phenyl-2H-chromen-2-one

(17b):

Yield: 71.3%; mp: 86-88 ºC. 1H-NMR (CDCl3) δ 1.75-1.77 (m, 2H), 1.87-1.90 (m, 2H), 2.51 (t, 2H, J = 7.2 Hz), 2.69 (s, br, 4H), 3.12 (s, br, 4H), 3.86 (s, 3H), 4.08 (t, 2H, J = 6.4 Hz), 6.21 (s, 1H), 6.77-7.00 (m, 6H), 7.36-7.51 (m, 6H). MS (ESI) m/z 485.4 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-(trifluoromethyl)-2Hchromen-2-one (17c): Yield: 61.2%; mp: 122-124 ºC. 1H-NMR (CDCl3) δ 1.77-1.92 (m, 4H), 2.11-2.23 (m, 6H), 2.52 (t, 2H, J = 7.2 Hz), 3.10-3.13 (m, 3H), 4.10 (t, 2H, J = 6.4 Hz), 6.61 (s, 1H), 6.87-6.93 (m, 2H), 7.03-7.08 (m, 1H), 7.22-7.25 (m, 1H), 7.60-7.63 (m, 1H), 7.70-7.73 (m, 1H). MS (ESI) m/z 505.1 ([M+H]+). 7-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butoxy)-4-(trifluoromethyl)-2H-chromen-2one (17d): Yield: 69.8%; mp: 111-113 ºC. 1H-NMR (CDCl3) δ 1.77-1.90 (m, 4H), 2.54 (t, 2H, J = 7.2 Hz), 2.74 (s, br, 4H), 3.14 (s, br, 4H), 3.86 (s, 3H), 4.09 (t, 2H, J = 6.4 Hz), 6.61 (s, 1H), 6.85-7.00 (m, 6H), 7.61 (d, 1H, J = 4 Hz). MS (ESI) m/z 477.2 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-ethyl-2H-chromen-2-on e (17e): Yield: 79.1%; mp: 133-135 ºC. 1H-NMR (CDCl3) δ 1.32 (t, 3H, J = 7.6 Hz), 1.74-1.90 (m, 4H), 2.06-2.16 (m, 6H), 2.48 (t, 2H, J = 7.2 Hz), 2.75-2.81 (m, 2H), 3.05-3.10 (m, 3H), 4.07 (t, 2H, J = 6.4 Hz), 6.14 (s, 1H), 6.82-6.87 (m, 2H), 7.03-7.07 (m, 1H), 7.23-7.25 (m, 1H), 7.52 (d, 1H, J = 8 Hz), 7.68-7.71 (m, 1H). MS (ESI) m/z 465.3 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-propyl-2H-chromen-2one (17f) : Yield: 81.1%; mp: 136-138 ºC. 1H-NMR (CDCl3) δ 1.00 (t, 3H, J = 7.2 Hz), 1.64-1.69 (m, 2H), 1.93-2.22 (m, 6H), 2.64 (t, 2H, J = 7.2 Hz), 3.00-3.74 (m, 9H), 4.04 (t, 2H, J = 6.4 Hz), 6.05 (s, 1H), 6.71-6.78 (m, 2H), 7.05-7.23 (m, 2H), 7.80 (d, 1H, J = 8.8 Hz), 7.91 (d, 1H, J = 8 Hz). MS (ESI) m/z 479.2 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-isopropyl-2H-chromen2-one (17g) : Yield: 73.2%; mp: 112-114 ºC. 1H-NMR (CDCl3) δ 1.32 (d, 6H, J = 4 Hz),



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1.74-1.89 (m, 4H), 2.06-2.14 (m, 6H), 2.49 (t, 2H, J = 7.2 Hz), 3.08-3.11 (m, 3H), 3.24-3.28 (m, 1H), 4.08 (t, 2H, J = 6.2 Hz), 6.17 (s, 1H), 6.84-6.89 (m, 2H), 7.04-7.08 (m, 1H), 7.22-7.25 (m, 1 H), 7.58 (d, 1H, J = 4 Hz), 7.69-7.72 (m, 1H). MS (ESI) m/z 479.3 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-cyclopropyl-2Hchromen-2-one (17h): Yield: 72.3%; mp: 102-104 ºC. 1H-NMR (CDCl3) δ 0.81-0.86 (m, 2H), 1.13-1.16 (m, 2H), 1.73-1.91 (m, 4H), 2.05-2.19 (m, 7H), 2.49 (t, 2H, J = 7.2 Hz), 3.09-3.11 (m, 3H), 4.06 (t, 2H, J = 6.4 Hz), 5.89 (s, 1H), 6.83-6.91 (m, 2H), 7.04-7.09 (m, 1H), 7.22-7.24 (m, 1H), 7.70-7.74 (m, 1H), 7.79-7.83 (m, 1H). MS (ESI) m/z 477.6 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-3,4-dimethyl-2H-chromen -2-one (17i): Yield: 80.9%; mp: 119-121 ºC. 1H-NMR (CDCl3) δ 1.73-1.86 (m, 4H), 2.05-2.17 (m, 9H), 2.36 (s, 3H), 2.48 (t, 2H, J = 7.2 Hz), 3.07-3.10 (m, 3H), 4.05 (t, 2H, J = 6.4 Hz), 6.78-6.86 (m, 2H), 7.02-7.07 (m, 1H), 7.21-7.24 (m, 1H), 7.48 (d, 1H, J = 8.8 Hz), 7.68-7.71 (m, 1H). MS (ESI) m/z 465.3 ([M+H]+). 7-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butoxy)-3,4-dimethyl-2H-chromen-2-one (17j): Yield: 69.5%; mp: 129-131 ºC. 1H-NMR (CDCl3) δ 1.74-1.87 (m, 4H), 2.18 (s, 3H), 2.36 (s, 3H), 2.50 (t, 2H, J = 7.2 Hz), 2.68 (s, br, 4H), 3.11 (s, br, 4H), 3.87 (s, 3H), 4.04 (t, 2H, J = 6.6 Hz), 6.78-6.99 (m, 6H), 7.47 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 437.3 ([M+H]+). 7-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butoxy)-4-methyl-8-chloro-2H-chromen-2-one (17k): Yield: 70.1%; mp: 139-141 ºC. 1H-NMR (CDCl3) δ 1.75-1.95 (m, 4H), 2.40 (s, 3H), 2.52 (t, 2H, J = 7.2 Hz), 2.68 (s, br, 4H), 3.10 (s, br, 4H), 3.87 (s, 3H), 4.18 (t, 2H, J = 6.4 Hz), 6.17 (s, 1H), 6.86-6.93 (m, 5H), 7.45 (d, 1H, J = 8 Hz). MS (ESI) m/z 457.2 ([M+H]+). 7-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butoxy)-4,8-dimethyl-2H-chromen-2-one (17l): Yield: 68.3%; mp: 134-136 ºC. 1H-NMR (CDCl3) δ 1.76-1.93 (m, 4H), 2.31 (s, 3H), 2.40 (s, 3H), 2.51 (t, 2H, J = 7.6 Hz), 2.68 (s, br, 4H), 3.11 (s, br, 4H), 3.87 (s, 3H), 4.10 (t, 2H, J = 6.4 Hz), 6.12 (s, 1H), 6.82-7.00 (m, 5H), 7.40 (d, 1H, J = 8.8 Hz). MS (ESI) m/z 437.1 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-methyl-8-chloro-2Hchromen-2-one (17m): Yield: 78.1%; mp: 140-142 ºC. 1H-NMR (CDCl3) δ 1.78-1.80 (m, 2H), 1.93-2.15 (m, 8H), 2.41 (s, 3H), 2.50 (t, 2H, J = 7.2 Hz), 3.07-3.10 (m, 3H), 4.19 (t, 2H, J = 6.0 Hz), 6.15 (s, 1H), 6.92 (d, 1H, J = 8.8 Hz), 7.02-7.07 (m, 1H), 7.22-7.25 (m, 1H), 7.46


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(d, 1H, J = 8.8 Hz), 7.68-7.71 (m, 1H). MS (ESI) m/z 485.2 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4,8-dimethyl-2H-chromen -2-one (17n): Yield: 76.9%; mp: 155-157 ºC. 1H-NMR (CDCl3) δ 1.72-1.80 (m, 2H), 1.88-1.94 (m, 2H), 2.05-2.19 (m, 6H), 2.31 (s, 3H), 2.39 (s, 3H), 2.49 (t, 2H, J = 7.2 Hz), 3.08-3.10 (m, 3H) , 4.11 (t, 2H, J = 6.0 Hz), 6.11 (s, 1H), 6.84 (d, 1H, J = 8.8 Hz), 7.05 (t, 1H, J = 2.0 Hz), 7.22-7.24 (m, 1H), 7.40 (d, 1H, J = 8.8 Hz), 7.68-7.71 (m, 1H). MS (ESI) m/z 465.1 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-methyl-6-chloro-2Hchromen-2-one (17o): Yield: 70.9%; mp: 136-138 ºC. 1H-NMR (CDCl3) δ 1.70-1.79 (m, 2H), 1.93-1.95 (m, 2H), 2.38 (s, 3H), 2.51 (t, 2H, J = 7.2 Hz), 2.67 (s, br, 4H), 3.10 (s, br, 3H), 4.13 (t, 2H, J = 6.4 Hz), 6.17 (s, 1H), 6.85-7.00 (m, 4H), 7.57 (s, 1H). MS (ESI) m/z 485.1 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4,5-dimethyl-2H-chromen -2-one (17p): Yield: 60.8%; mp: 126-128 ºC. 1H-NMR (CDCl3) δ 1.74-1.76 (m, 2H), 1.91-1.93 (m, 2H), 2.06-2.17 (m, 6H), 2.39 (s, 3H), 2.49 (t, 2H, J = 7.2 Hz), 2.59 (s, 3H), 3.05-3.10 (m, 3H), 4.07 (t, 2H, J = 6.4 Hz), 6.04 (s, 1H), 6.53 (s, 1H), 6.73 (s, 1H), 7.03-7.08 (m, 1H), 7.22-7.25 (m, 1H), 7.68-7.71 (m, 1H). MS (ESI) m/z 465.3 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-3,4,8-trimethyl-2Hchromen-2-one (17q): Yield: 65.6%; mp: 109-111 ºC. 1H-NMR (CDCl3) δ

1.76-1.90 (m,

4H), 2.06-2.36 (m, 14H), 2.49(t, 2H, J = 7.6 Hz), 3.04-3.10 (m, 3H), 3.43-3.49 (m, 1H), 4.09 (t, 2H, J = 6.0 Hz), 6.81-6.85 (m, 1H), 7.05-7.08 (m, 1H), 7.22-7.25 (m, 1H), 7.39-7.41 (m, 1H), 7.68-7.70 (m, 1H). MS (ESI) m/z 479.2 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-(4-fluorophenyl)-2Hchromen-2-one (17r): Yield: 50.8%; mp: 87-89 ºC. 1H-NMR (CDCl3) δ 1.76-1.79 (m, 2H), 1.89-1.93 (m, 2H), 2.10-2.18 (m, 6H), 2.49 (t, 2H, J = 7.2 Hz), 3.09-3.12 (m, 3H), 4.11 (t, 2H, J = 6.4 Hz), 6.21 (s, 1H), 6.82-6.84 (m, 1H), 6.91-6.92 (m, 1H), 7.03-7.10 (m, 1H), 7.22-7.25 (m, 3H), 7.34-7.37 (m, 1H), 7.44-7.47 (m, 2H), 7.69-7.73 (m, 1H). MS (ESI) m/z 531.3 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-(3-methoxyphenyl)-2Hchromen-2-one (17s): Yield: 61.9%; mp: 93-95 ºC. 1H-NMR (CDCl3) δ 1.77-1.81 (m, 2H),



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1.88-1.93 (m, 2H), 2.02-2.10 (m, 6H), 2.52 (t, 2H, J = 7.6 Hz), 3.09-3.12 (m, 3H), 3.88 (s, 3H), 4.11 (t, 2H, J = 6.2 Hz), 6.24 (s, 1H), 6.80-6.83 (m, 1H), 6.91-6.93 (m, 1H), 6.98-7.08 (m, 4H), 7.23-7.25 (m, 1H), 7.42-7.44 (m, 2H), 7.70-7.74 (m, 1H). MS (ESI) m/z 543.7 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-(thiophen-2-yl)-2Hchromen-2-one (17t): Yield: 65.8%; mp: 101-103 ºC. 1H-NMR (CDCl3) δ 1.72-1.76 (m, 2H), 1.89-1.92 (m, 2H), 2.08-2.15 (m, 6H), 2.50 (t, 2H, J = 7.2 Hz), 3.06-3.12 (m, 3H), 4.11 (t, 2H, J = 6.4 Hz), 6.36 (s, 1H), 6.86-6.91 (m, 2H), 7.02-7.09 (m, 1H), 7.23-7.25 (m, 2H),7.42-7.44 (m, 1H), 7.56-7.58 (m, 1H), 7.71-7.73 (m, 1H), 7.82-7.84 (m, 1H). MS (ESI) m/z 519.3 ([M+H]+). General procedure for the preparation of compounds 18a-b, 20 1,4-Dibromopentane (1,3-Dibromopropane or (E)-1,4-Dibromobut-2-ene) (2 mmol) was added to a solution of 7-hydroxy-4-methylcoumarin (1 mmol) and potassium carbonate in acetone (50 mL), 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, the solvent was evaporated and the residue was recrystallized from hexane /EtOH to yield compounds 18a-b, 20. 7-(3-Bromopropoxy)-4-methyl-2H-chromen-2-one (18a): Yield: 55.1%; mp: 78-80 ºC. 1

H-NMR (CDCl3) δ 2.27-2.40 (m, 5H), 3.62 (t, 2H, J = 6.4 Hz), 4.16 (t, 2H, J = 6.0 Hz), 6.14

(s, 1H), 6.82-6.88 (m, 2H), 7.49-7.51 (m, 1H). MS (ESI) m/z 297.1 ([M+H]+). 7-((5-Bromopentyl)oxy)-4-methyl-2H-chromen-2-one (18b): Yield: 69.1%; mp: 86-88 ºC. 1

H-NMR (CDCl3) δ 1.65-1.68 (m, 2H), 1.85-1.99 (m, 4H), 2.43 (s, 3H), 3.47 (t, 2H, J = 6.8

Hz), 4.05 (t, 2H, J = 6.4 Hz), 6.14 (s, 1H), 6.81-6.88 (m, 2H), 7.49-7.52 (m, 1H). MS (ESI) m/z 325.0 ([M+H]+). (E)-7-((4-Bromobut-2-en-1-yl)oxy)-4-methyl-2H-chromen-2-one (20): Yield: 70.7%; mp: 90-92 ºC. 1H-NMR (CDCl3) δ 2.41 (s, 3H), 4.02 (d, 2H, J = 7.2 Hz), 4.63 (d, 2H, J = 4.4 Hz), 5.97-6.12 (m, 2H), 6.14 (s, 1H), 6.80-6.99 (m, 2H), 7.47-7.58 (m, 1H). MS (ESI) m/z 309.2 ([M+H]+). General procedure for the preparation of compounds 19a-b, 21 To a suspension of compound 18a (18b or 20) (0.32 mmol) and K2CO3 (0.64 mmol) in


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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 (3 × 25 mL). The combined organic layers were evaporated under reduced pressure, and the crude product was purified by means of chromatography (10% MeOH/CHCl3) to yield compound 19a-b, 21. 7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propoxy)-4-methyl-2H-chromen-2 -one (19a) : Yield: 75.6%; mp: 145-147 ºC. 1H-NMR (CDCl3) δ 2.02-2.23 (m, 8H), 2.40 (s, 3H), 2.60 (t, 2H, J = 7.2 Hz), 3.07-3.10 (m, 3H), 4.12 (t, 2H, J = 6.4 Hz), 6.13 (s, 1H), 6.84-6.89 (m, 2H), 7.06 (t, 1H, J = 2 Hz), 7.23-7.25 (m, 1H), 7.50 (d, 1H, J = 8.8 Hz), 7.71 (t, 1H, J = 5.2 Hz). MS (ESI) m/z 437.2 ([M+H]+). 7-((5-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)pentyl)oxy)-4-methyl-2H-chrome n-2-one (19b) : Yield: 79.8%; mp: 98-100 ºC. 1H-NMR (CDCl3) δ 1.53-1.63 (m, 4H), 1.85-1.89 (m, 2H), 2.05-2.14 (m, 6H), 2.40 (s, 3H), 2.43 (t, 2H, J = 7.2 Hz), 3.06-3.08 (m, 3H), 4.04 (t, 2H, J = 6.4 Hz), 6.12 (s, 1H), 6.80-6.87 (m, 2H), 7.02-7.07 (m, 1H), 7.22-7.24 (m, 1H), 7.49 (d, 1H, J = 4.8 Hz), 7.67-7.71 (m, 1H). MS (ESI) m/z 465.3 ([M+H]+). (E)-7-((4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)but-2-en-1-yl)oxy)-4-methyl2H-chromen-2-one (21): Yield: 80.8%; mp: 129-131 ºC. 1H-NMR (CDCl3) δ 2.05-2.17 (m, 6H), 2.39 (s, 3H), 3.05-3.13 (m, 5H), 4.61 (d, 2H, J = 5.2 Hz), 5.91-5.96 (m, 2H), 6.13 (s, 1H), 6.83-6.90 (m, 2H), 7.05-7.06 (m, 1H), 7.22-7.25 (m, 1H), 7.49 (d, 1H, J = 8.8 Hz), 7.68-7.70 (m, 1H). MS (ESI) m/z 449.3 ([M+H]+). General procedure for the preparation of compounds 23a-b Epichlorohydrin (1 mmol) was added to a solution of 7-hydroxy-4-methylcoumarin or 7-hydroxy-4-phenyl-2H-chromen-2-one (1 mmol) and potassium carbonate in acetone (50 mL), 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, the solvent was evaporated and the residue was recrystallized from EtOH to yield compounds 23a-b. 7-(Oxiran-2-ylmethoxy)-4-methyl-2H-chromen-2-one (23a): Yield: 52.8%; mp: 125-127 ºC. 1H-NMR (CDCl3) δ 2.81-2.83 (m, 1H), 2.96-2.98 (m, 1H), 3.39-3.43 (m, 1H), 3.99-4.03 (m, 1H), 4.35-4.39 (m, 1H), 6.26 (s, 1H), 6.84-6.94 (m, 2H), 7.51-7.53 (m, 1H). MS (ESI)



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m/z 233.1 ([M+H]+). 7-(Oxiran-2-ylmethoxy)-4-phenyl-2H-chromen-2-one (23b): Yield: 59.2%; mp: 137-139 ºC. 1H-NMR (CDCl3) δ 2.42 (s, 3H), 2.80-2.82 (m, 1H), 2.95-2.97 (m, 1H), 3.39-3.42 (m, 1H), 3.97-4.01 (m, 1H), 4.33-4.37 (m, 1H), 6.16 (s, 1H), 6.84-6.93 (m, 2H), 7.41-7.46 (m, 3H), 7.51-7.53 (m, 3H). MS (ESI) m/z 295.1 ([M+H]+). General procedure for the preparation of compounds 24a-b The compound 23 (0.70 mmol) and arylpiperazine (piperidine) (0.7 mmol) were dissolved in ethanol (15 mL) and refluxed for 5 h. After completion of the reaction excess ethanol was removed through high vacuo and the residue was diluted with water and extracted with ethyl acetate. The organic layer was washed with water, brine, and dried over anhydrous Na2SO4. Column chromatography over silica gel and elution with 50% chloroform in hexane furnished the final product compound 24a-b. 7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-hydroxypropoxy)-4-methyl-2Hchromen-2-one (24a): Yield: 60.5%; mp: 183-185 ºC. 1H-NMR (CDCl3) δ 2.08-2.14 (m, 4H), 2.26-2.27 (m, 1H), 2.40 (s, 3H), 2.59-2.65 (m, 3H), 3.02-3.20 (m, 3H), 3.63 (br, 1H), 4.08-4.18 (m, 3H), 6.15 (s, 1H), 6.85 (d, 1H, J = 2.8 Hz), 6.91-6.93 (m, 1H), 7.06-7.09 (m, 1H), 7.24-7.26 (m, 1H), 7.51 (d, 1H, J = 8.8 Hz), 7.66-7.69 (m, 1H). MS (ESI) m/z 453.2 ([M+H]+). 7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)-2-hydroxypropoxy)-4-phenyl-2Hchromen-2-one (24b): Yield: 65.4%; mp: 193-195 ºC. 1H-NMR (CDCl3) δ 2.08-2.14 (m, 4H), 2.24-2.27 (m, 1H), 2.52-2.68 (m, 3H), 3.02-3.21 (m, 3H), 3.63 (br, s, 1H), 4.08-4.18 (m, 3H), 6.23 (s, 1H), 6.84-6.87 (m, 1H), 6.93-6.94 (m, 1H), 7.05-7.10 (m, 1H), 7.24-7.26 (m, 1H), 7.39-7.51 (m, 6H), 7.66-7.69 (m, 1H). MS (ESI) m/z 515.2([M+H]+). 7-Hydroxy-4-phenyl-3,4-dihydrocoumarin (26) 45 A slow stream of hydrogen chloride gas was passed through a boiling mixture of cinnamic acid (5 mmol), resorcinol (5 mmol) and concentrated hydrochloric acid (20 mL) until a clear solution was obtained (6 h), the solution was cooled and the solid that separated out was filtered, washed repeatedly with cold water, dried and purified by column chromatography using acetone–chloroform (3:97) as eluent to afford 1.03g compound 26. Yield: 83.3%; mp: 140-141 oC. MS (ESI) m/z 241.2 ([M+H]+).


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7-(4-Bromobutoxy)-4-phenylchroman-2-one (27) 1,4-dibromobutane (2 mmol) was added to a solution of compound 26 (1 mmol) and potassium carbonate in acetone (50 mL), and the mixture was refluxed for 6 h. The progress of the reaction was monitored by TLC. After cooling to room temperature, the mixture was filtered, the solvent was evaporated and the residue was purified by means of chromatography to yield compound 27.Yield: 50.3%; oil. 1H-NMR (CDCl3) δ 1.86-1.98 (m, 4H), 3.03 (d, 2H, J = 8.8 Hz), 3.49 (t, 2H, J = 7.2 Hz), 3.57 (t, 1H, J = 6.4 Hz), 3.99 (t, 2H, J = 6.0 Hz), 6.62-6.67 (m, 1H), 6.85-6.87 (m, 1H), 7.14-7.16 (m, 1H), 7.22-7.25 (m, 3H), 7.32-7.36 (m, 2H). MS (ESI) m/z 375.2 ([M+H]+). 7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-4-phenylchroman-2-one (28) To a suspension of compound 27 (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 6 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 evaporated under reduced pressure, and the crude product was purified by means of chromatography (5% MeOH/CHCl3) to yield compound 28. Yield: 55.1%; mp：98-100 oC. 1H-NMR (CDCl3) δ 1.61-1.76 (m, 4H), 2.07-2.16 (m, 6H), 2.47 (t, 2H, J = 7.2 Hz), 3.08-3.10 (m, 5H), 3.57 (t, 1H, J = 6.4 Hz), 3.89 (t, 2H, J = 6.4 Hz), 6.67-6.69 (m, 1H), 6.91-6.93 (m, 1H), 7.01-7.05 (m, 2H), 7.22-7.26 (m, 6H), 7.65-7.70 (m, 1H). MS (ESI) m/z 515.3 ([M+H]+). 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. 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. In vitro binding assays



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General procedures All the new compounds were dissolved in 5% DMSO. The following specific radioligands and tissue sources were used: (a) serotonin 5-HT1A receptor, [3H]8-OH-DPAT, rat brain cortex; (b) serotonin 5-HT2A receptor, [3H]ketanserin, rat brain cortex; (c) serotonin 5-HT2C receptor, [3H]mesulergine, rat brain cortex; (d) dopamine D2 receptor, [3H]spiperone, rat striatum; (e) dopamine D3 receptor, [3H]7-OH-DPAT, rat olfactory tubercle; (f) histamine H1 receptor, [3H]mepyramine, guinea pig cerebellum; (g) adrenergic a1 receptor, [3H]prazosin, rat brain cortex; (h) adrenergic a2 receptor, [3H]rauwolscine, rat brain cortex; Total binding was determined in the absence of no-specific binding and compounds. Specific binding was determined in the presence of compounds. Non-specific binding was determined as the difference between total and specific binding. Percentage of inhibition (%) = (total binding − specific binding) × 100% / (total binding − nonspecific binding) 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 (10-5 M to 10-11 M), IC50 values were determined by nonlinear regression analysis using Hill equation curve fitting. Ki values were calculated based on the Cheng and Prussoff equation: Ki = IC50/(1 + C/Kd) where C represents the concentration of the hot ligand used and Kd its receptor dissociation constant were calculated for each labeled ligand. Mean Ki values and SEM are reported for at least three independent experiments. 5-HT1A receptor 22,59 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 32000 g for 10 min. The resulting pellet was then resuspended in the same buffer, incubated for 10 min at 37 ºC, and centrifuged at 32000 g for 10 min. The final pellet was resuspended in Tris-HCl buffer containing 10 µM Pargyline, 4 mM CaCl2 and 0.1% ascorbic acid. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 0.5 nM [3H]8-OH-DPAT (187.4 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL Tris–HCl buffer containing 10 µM Pargyline, 4 mM CaCl2 and 0.1% ascorbic acid. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]8-OH-DPAT, 50 µL of 10


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µM serotonin. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]8-OH-DPAT, 50 µL of new compounds or reference drug. The tubes were incubated at 37 ºC 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 5mL cold buffer and transferred to scintillation vials. Scintillation fluid (3.0 mL) was added and the radioactivity bound was measured using a Beckman LS 6500 liquid scintillation counter. 5-HT2A receptor 22,59 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 centrifuged at 32000 g for 20 min. The resulting pellet was resuspended in the same quantity of the buffer centrifuged for 20min. The final pellet was resuspended in 50 volumes of the Tris-HCl buffer. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 0.6 nM [3H]ketanserine (60.0 Ci/mmol, Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL Tris-HCl buffer. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]ketanserin, 50 µL of 10 µM methisergide. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]ketanserin, 150 µL of new compounds or reference drug. The tubes were incubated at 37 ºC for 15 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 Beckman LS 6500 liquid scintillation counter. 5-HT2C receptor 22,59 Rat cerebral cortex was homogenized in 20 volumes of ice-cold Tris-HCl buffer (50 mM, pH7.7) using ULTRA TURAX homogeniser, and centrifuged at 32000 g for 20 min. The resulting pellet was resuspended in the same quantity of the buffer centrifuged for 20min. The final pellet was resuspended in 50 volumes of the Tris-HCl buffer. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]mesulergine, 50 µL Tris-HCl buffer. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 1 nM [3H]mesulergine (85.4 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL of 10 µM mianserin. Specific binding each assay tube was added 900 µL of the tissue



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suspension, 50 µL of [3H]mesulergine, 50 µL of new compounds or reference drug. The tubes were incubated at 37 ºC for 15 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 Beckman LS 6500 liquid scintillation counter. Dopaminergic D2 receptor 22,59 Rat striatum was homogenized in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.7) using an ULTRA TURAX homogeniser, and centrifuged twice for 10 min at 48,000 g with resuspension of the pellet in fresh buffer. The final pellet was resuspended in 50 mM ice-cold Tris-HCl containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% ascorbic acid and 5µM pargyline. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 0.5 nM [3H]spiperone (16.2 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL Tris-HCl buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 0.1% ascorbic acid and 5µM pargyline. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]spiperone, 50 µL of 10 µM (+)-butaclamol. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]spiperone, 50 µL of new compounds or reference drug. The tubes were incubated at 37 ºC for 15 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 Beckman LS 6500 liquid scintillation counter. Dopaminergic D3 receptor 52 Rat olfactory tubercle was homogenized in 20 volumes of ice-cold 50 mM Hepes Na (pH 7.5) using an ULTRA TURAX homogeniser, and centrifuged twice for 10 min at 48,000 g with resuspension of the pellet in fresh buffer. The final pellet was resuspended in 50 mM Hepes Na, pH 7.5, containing 1 mM EDTA, 0.005% ascorbic acid, 0.1% albumin, and 200 nM eliprodil. Total binding each assay tube was added 900 µL of membranes，50 µL of 0.6 nM [3H] 7-OH-DPAT (50 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL of 50 mM Hepes Na, pH 7.5, containing 1 mM EDTA, 0.005% ascorbic acid, 0.1% albumin, 200 nM eliprodil. Non-specific binding each assay tube was added 900 µL of membranes, 50


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µL of [3H] 7-OH-DPAT (50 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL of 1 µM dopamine. Specific binding each assay tube was added 900 µL of Membranes, 50 µL of [3H] 7-OH-DPAT (50 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL of new compounds or reference drug. The tubes were incubated at 25 ºC for 60 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 Beckman LS 6500 liquid scintillation counter. Histamine H1 receptor 60 Guinea pig cerebellum was homogenized in 20 volumes of ice-cold 50 mM phosphate buffer (pH = 7.4) using an ULTRA TURAX homogeniser, and centrifuged twice for 10 min at 50,000 g with resuspension of the pellet in fresh buffer. The final pellet was resuspended in phosphate buffer. Total binding each assay tube was added 900 µL of membranes 50 µL of 1 nM [3H]mepyramine (20.0 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL phosphate buffer. Non-specific binding each assay tube was added 900 µL of membranes, 50 µL of [3H]mepyramine, 50 µL of 1 µM promethazine. Specific binding each assay tube was added 900 µL of Membranes, 50 µL of [3H]mepyramine, 50 µL of new compounds or reference drug. The tubes were incubated at 30 ºC for 60 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 Beckman LS 6500 liquid scintillation counter. Adrenergic a1 receptor61 Rat cerebral cortex was homogenized in 20 volumes of ice-cold Tris-HCl buffer containing 5 mM EDTA (50 mM, pH7.7) using ULTRA TURAX homogeniser, and centrifuged at 44000 g for 20 min at 4℃. The resulting pellet was resuspended in the same quantity of the buffer centrifuged for 20min. The final pellet was resuspended in 50 volumes of the Tris-HCl buffer. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 1 nM [3H]prazosin (85.4 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL Tris-HCl buffer. Non-specific binding each assay tube was added 900 µL of the tissue



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suspension, 50 µL of 1 nM [3H]prazosin, 50 µL of 10 µM prazosin. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H]prazosin, 50 µL of new compounds or reference drug. The tubes were incubated at 25 ℃ for 60 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 Beckman LS 6500 liquid scintillation counter. Adrenergic a2 receptor61 Rat cerebral cortex was homogenized in 20 volumes of ice-cold Tris-HCl buffer containing 5 mM EDTA (50 mM, pH7.7) using ULTRA TURAX homogeniser, and centrifuged at 44000 g for 20 min at 4℃. The resulting pellet was resuspended in the same quantity of the buffer centrifuged for 20min. The final pellet was resuspended in 50 volumes of the Tris-HCl buffer. Total binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 1 nM [3H] rauwolscine, 50 µL Tris-HCl buffer. Non-specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of 1 nM [3H] rauwolscine (73.0 Ci/mmol; Perkin Elmer Life Sciences, Boston, MA, USA), 50 µL of 10 µM rauwolscine. Specific binding each assay tube was added 900 µL of the tissue suspension, 50 µL of [3H] rauwolscine, 50 µL of new compounds or reference drug. The tubes were incubated at 25℃ for 60 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 Beckman LS 6500 liquid scintillation counter. In Vitro Assays for hERGAffinity Evaluation62 Cell: Ability to block hERG potassium channels was determined using the electrophysiological method and cloned hERG potassium channels (expressed in HEK 293 cells, Creacell Company, France) as biological material. Electrophysiology: The effects evaluated using Nanion Technologies (Patchliner® NPC-16, Germany). Currents were elicited using a voltage step from a holding potential of -80 mV to +40 mV for 500 ms, then to the test potential for 500 ms and back to holding. Pulses were elicited every 20 seconds.


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Data analysis: Data acquisition and analyses was performed using the Nanion Technologies. Standardization of current amplitude through the following equations is fitting: 1(1-(c(IC50)-1)h)-1. Equation for drug concentration in c, for the biggest effect of 50% IC50 drug concentration, h for hill coefficient. Each value is the mean SD of 3-5 determinations and represents nM values. Experimental in Vitro Pharmacology for Intrinsic Activity Assessment 63,64 CHO-K1 cells expressing four receptors (CHO-K1/D2/Gα15, CHO-K1/D3/Gα15, CHO-K1/5-HT1A/Gα15 and CHO-K1/5-HT2A) were seeded in a 384-well black-wall, clear-bottom plate at a density of 20,000 cell per well in 20 µL of growth medium,18 hours prior to the day of experiment and maintained at 37°C/5% CO2. For agonist assay, 20 µL of dye-loading solution (20 mM HEPES/HBSS with 2.5 mM Probenecid, pH 7.4) was added into the well. Then the plate was placed into a 37°C incubator for 60 minutes, followed by a 15 minutes at room temperature. At last, 10 µL compounds or control agonist were added into respective wells of the assay plate. For antagonist assay, 20 µL of dye-loading solution (20 mM HEPES/HBSS with 2.5 mM Probenecid, pH 7.4) and 10 µL of compound or control antagonist solution were added into the well. Then the plate was placed into a 37°C incubator for 60 minutes, followed by 15 minutes at room temperature. At last, 12.5 µL of control agonist was added into respective wells of the assay plate during reading in FLIPR. For agonist test, the compounds or agonist plate contained compounds or control agonist at 5×final test concentration, and was placed at Source 2. Compounds or control agonist were added to the reading plate at 20 sec and the change in cell fluorescence signal was measured in a FLIPR (FLIPR® Calcium 4 assay kit, Molecular Devices) for an additional 100 sec (21 sec to 120 sec.). For antagonist test, only compounds or control agonist solution at Source 2 plate was replaced with control agonist solution at 5× EC80 concentration. Data were recorded by ScreenWorks (version 3.1) as FMD files with FLIPR. Data acquisition and analyses was performed using ScreenWorks (version 3.1) program and exported to Excel. The average value of 20 (1s to 20s) seconds reading was calculated as the baseline reading and the relative fluorescent units (∆RFU) intensity values were calculated with the maximal fluorescent units (21s to 120s) subtracting the average value of baseline



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reading . The % activation of compound was calculated from the following equation: % activation = {(∆RFUCompound- ∆RFUBackground)/ (∆RFUAgonist

control

- ∆RFUBackground)}

×100 % activation was then plotted as a function of the log of the cumulative doses of compounds. The % inhibition of the test article was calculated from the following equation: % inhibition = {1- (∆RFUCompound- ∆RFUBackground)/ (∆RFUAgonist control - ∆RFUBackground)} ×100 % inhibition was then plotted as a function of the log of the cumulative doses of compounds. Behavioral tests Acute toxicity Mice (10 mice in each group) were orally dosed with increasing doses of the compound 17m (200, 500, 1000, 1500 and 2000 mg/kg). The number of surviving animals was recorded after 24h of drug administration, and the percent mortality in each group was calculated. The LD50 values were calculated by using the program SPSS (Statistical Package for the Social Science). MK-801- induced hyperactivity 52 Mice (10 mice in each group) were orally dosed with vehicle or increasing doses of the haloperidol (0.06, 0.2, 0.6, 2.0 and 6 mg/kg), clozapine (1, 2.5, 7, 20 and 60 mg/kg), risperidone (0.01, 0.03, 0.1, 0.3 and 1.0 mg/kg) and compound 17m (0.25, 0.5, 1, 2 and 10 mg/kg). Animals were placed in Plexiglas cages for evaluating locomotor activity. After 30 min, the animals were challenged with 0.3 mg/kg (sc) of MK-801 and the locomotor activity of each animal was recorded for 90 min. Statistical evaluation was performed by two-way ANOVA followed by Tukey test for multiple comparisons. # p

Synthesis and Biological Investigation of Coumarin Piperazine

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