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Article

Design, Synthesis and Biological Investigation of Flavone Derivatives as Potential Multi-Receptor Atypical Antipsychotics

by
Lanchang Gao
1,†,
Zhengge Yang
1,†,
Jiaying Xiong
1,
Chao Hao
1,
Ru Ma
2,
Xin Liu
1,
Bi-Feng Liu
1,
Jian Jin
2,
Guisen Zhang
1,2,* and
Yin Chen
2,*
1
Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
2
Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, School of Pharmacy, Jiangsu Ocean University, Lianyungang 222005, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2020, 25(18), 4107; https://doi.org/10.3390/molecules25184107
Submission received: 17 August 2020 / Revised: 26 August 2020 / Accepted: 28 August 2020 / Published: 8 September 2020
(This article belongs to the Section Medicinal Chemistry)
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Abstract

:
The design of a series of novel flavone derivatives was synthesized as potential broad-spectrum antipsychotics by using multi-receptor affinity strategy between dopamine receptors and serotonin receptors. Among them, 7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl) piperidin- 1-yl) butoxy)-2,2-dimethylchroman-4-one (6j) exhibited a promising preclinical profile. Compound 6j not only showed high affinity for dopamine D2, D3, and serotonin 5-HT1A, 5-HT2A receptors, but was also endowed with low to moderate activities on 5-HT2C, α1, and H1 receptors, indicating a low liability to induce side effects such as weight gain, orthostatic hypotension and QT prolongation. In vivo behavioral studies suggested that 6j has favorable effects in alleviating the schizophrenia-like symptoms without causing catalepsy. Taken together, compound 6j has the potential to be further developed as a novel atypical antipsychotic.

Graphical Abstract

1. Introduction

Schizophrenia is a severe neuropsychiatric disorder that affects over 1% of the world’s population [1]. The clinical symptoms of schizophrenia are categorized into three general types, including positive, negative, and cognitive symptoms [2]. Currently, antipsychotics are the mainstay in schizophrenia treatment. For example, the first-generation antipsychotics (FGAs), namely typical antipsychotics including chlorpromazine and haloperidol, are all dopamine D2 receptor antagonists and effective for the treatment of positive symptoms, but invalid for the negative symptoms and cognitive impairment [3,4]. However, the strong antagonism of D2 receptor results in serious side effects such as extrapyramidal symptoms (EPS), hyperprolactinemia, and cognitive impairments [5]. The second-generation antipsychotics (SGAs), also known as atypical antipsychotics, including clozapine [6], and risperidone [7], have antagonistic action on both dopamine and serotonin receptors, especially with high affinity for 5-HT1A and 5-HT2A receptors, which contributes to various kinds of therapeutic advantages, such as fewer EPSs than FGAs [8]. However, the SGAs also produce many adverse metabolic and cardiovascular effects [9], such as weight gain, hyperprolactinemia, constipation, QT prolongation, and also show limited effects on negative symptoms and cognitive impairments [10,11]. Fortunately, these defects could be avoided by developing a multi-receptor ligand with a “selective” multi-receptor profile which can precisely modulates several specific targets to get a better effect and safety in the therapy of schizophrenia [12,13].
To verify this multi-receptor affinity strategy, we have previously developed several novel potential antipsychotics, such as compounds 1 and 2 shown in Figure 1, both of which could significantly alleviate the positive symptoms of schizophrenia, are associated with lower weight gain and lower prolactin levels, and have obvious therapeutic effects. In order to obtain versatile molecules with better therapeutic effects and less side effects, in this paper, a series of new compounds was designed and synthesized by means of a polypharmacological strategy and molecular hybridization method on the basis of our previous research [14,15]. The design concept of new compounds as shown in Figure 1, where the general structure contains a flavone-like moiety, a heterocyclic or arylpiperazine (piperidine) moiety and a flexible linker. By optimizing the combination of the benzocaprolactam fragment of brexpiprazole and the coumarin derivative scaffolds of compounds 1 and 2, they were transformed into a flavone-like fragment. The choice of heterocyclic or arylpiperazine (piperidine) originates from the commercial atypical antipsychotics [16], such as risperidone, brexpiprazole, aripiprazole. The linker flexibility of the new compounds also be evaluated as our previous SAR studies have proved it to play an important role in modulating the receptor function profiles [14,15,17].
Among these derivatives, compound 6j [7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl) iperidin-1-yl) butoxy)-2,2-dimethylchroman-4-one] exhibited a favorable polypharmacological antipsychotic profile. In vitro, it showed much higher potency for the desired targets (D2, 5-HT1A, 5-HT2A, and D3) than other off-target receptors (5-HT2C, α1, and H1). Further in vivo behavioral studies suggested that it has favorable effects in alleviating schizophrenia-like symptoms without causing catalepsy and a low liability to induce side-effects. Thus, compound 6j has the potential to be developed as a novel atypical antipsychotic candidate to treat schizophrenia.

2. Results and Discussion

2.1. Chemistry

New compounds were synthesized as shown in Scheme 1, Scheme 2 and Scheme 3. In Scheme 1, 2,4-dihydroxyacetophenone (3) was converted into intermediate 4 by reaction with acetone in the presence of pyrrolidine and acetonitrile, and the intermediates 7a and 7b were prepared using a similar process. Compounds 5a–5b and 8a-8d were obtained through alkylation of 4 and 7a–7b with 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane. Compounds 6a–6j and 9a–9k were obtained by coupling 5a–5b and 8a-8d with different arylpiperazines or heterocyclic arylpiperidines in the presence of K2CO3 in acetonitrile. As shown in Scheme 2, the new compounds 11a–11e and 14a–14b were synthesized following similar approaches to those described in Scheme 1, that is treatment of resorcinol and its derivatives 10a-10e with trifluoromethanesulfonic acid in the presence of 3-chloropropionic acid, and then treatment with sodium hydroxide solution to afford 11a-11e. Compounds 14a and 14b were synthesized starting from resorcinol and its derivatives by treatment with crotonic acid in the presence of anhydrous zinc chloride. The preparation of 12a–12e and 15a–15b used the same procedure as the synthesis of 5a-5b. Subsequently the intermediates 12a–12e and 14a–14b were coupled with 6-fluoro-3-(piperidin-4-yl) benzo[d]isoxazole to yield compounds 13a-13d and 16a–16b. Compound 21 was synthesized in five steps as described in Scheme 3. The intermediate 17 was synthesized from 2,4-dihydroxypropiophenone. Hydrolysis of 17 under basic conditions (NaHCO3/MeOH) produced 18. Intermediate 19 was prepared by reduction with Pd/C, followed by reaction with 1-bromo-4-chlorobutane to afford 20. The target compound 21 was obtained by coupling 20 with 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole in the presence of K2CO3 in acetonitrile.

2.2. In Vitro Evaluation and Structure-Activity Relationship (SAR)

At beginning of this study, the 7-hydroxy-2,2-dimethylchroman-4-one group was selected as a privileged structure derived from coumarin and flavone. The influence of various heterocyclic and aryl piperazines(piperidine) on the activities of D2, 5-HT1A and 5-HT2A receptors were primarily evaluated and the results are summarized in Table 1. Among those compounds, 6a–6d bearing phenylpiperazine and substituted phenylpiperazines (-F, -CF3, -OCH3) showed moderate affinity for 5-HT1A and 5-HT2A receptors and lower affinity for D2 receptors. When the phenylpiperazine was replaced with a heterocyclic piperazine, compound 6e bearing a pyripiperazine devoid of affinity for D2 receptor resulted. Compound 6f with pyrimidine almost lost the affinity for all the three receptors. Compound 6g bearing a 1-(benzo[d][1,3] dioxol-5-ylmethyl) piperazine showed lower affinity for the D2 receptor. Interestingly, replacing the phenylpiperazine with a benzoheterocycle-piperazines(piperidine), such as benzothiazole-piperazine and benzisothiazole- piperazine, gave compounds 6h and 6i, which showed significantly increased for all three receptors. Especially, compound 6j bearing a 6-fluorobenzo[d]isoxazole-piperidine moiety displayed excellent D2 (Ki = 8.1 nM), 5-HT1A (Ki = 9.7 nM) and 5-HT2A (Ki = 3.2 nM) receptor potency.
According to the above results, the 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole moiety was selected as a privileged fragment, as our previous studies have proved that compounds bearing this pharmacophore show favorable activities for D2, 5-HT1A and 5-HT2A receptors [14,15,17]. Next the effect of substituents on the 7-hydroxy-2,2-dimethylchroman-4-one group (Table 2) was investigated, such as five-membered rings, six-membered rings, methyl and chlorine groups. First, when dimethyl was replaced with a cyclopentyl ring, the obtained compound 9d had reduce activities on the D2, 5-HT1A and 5-HT2A receptors (9d vs. 6j). Similarly, enlargement of the five-membered ring to a cyclohexyl ring (compound 9h) slightly lowered the affinity for the three receptors compared with 6j. Removing the dimethyl or replacing the dimethyl with a methyl (13a and 16a) showed a negative impact on all the three receptors compared with compound 6j. Meanwhile, when the two methyl substituents at the 5 and 6 positions (compounds 21) of 7-hydroxy-2,2-dimethylchroman-4-one group were changed simultaneously, it resulted in increased activities for the 5-HT2A receptor but decreased activity for the D2 and 5-HT1A receptors. We further investigated the influence of a methyl group substituted at different positions on the phenyl ring. The activities of the substitution of methyl at the 2- (13c), 3- (13b) and 8- (13d) positions of the phenyl ring were retained, but were slightly lower than without a substituent (13a). According the results, when the methyl was substituted at the 6 position of 7-hydroxy-2,2-dimethylchroman-4-one group (16a) and the 8-chloro substituted derivative 16b displayed decreased activity for the 5-HT1A receptor (16a vs. 16b).
As the compounds bearing a cyclopentyl ring (9c and 9d) and a cyclohexyl ring (9g and 9h) moiety showed higher potency for the three receptors (Table 1 and Table 2), we therefore attempted to expand the structural transformation of 6-fluoro-3-(piperidin-4-yl) benzo[d]isoxazole fragment. When the privileged structure 6-fluoro-3-(piperidin-4-yl) benzo[d]isoxazole was replaced with phenylpiperazine derivatives and heterocyclic piperazines, such as 1-(2-methoxyphenyl) piperazine (9a,9b,9e),1-(2-(trifluoromethyl)phenyl)piperazine (9f), 1-(2,3-dimethylphenyl)piperazine (9i), 1-(2,3-dichlorophenyl)piperazine (9j) and 3-(piperidin-4-yl)benzo[d]isothiazole (9k), all of the resulting compounds fail to improve the affinities for the D2, 5-HT1A and 5-HT2A receptors, indicating that the 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole was the optimum structure for this type of compound.
Our previous SAR studies reveal that the linker length and flexibility play an important role in the regulation of the receptor potency, and the lengths of the linker generally considered appropriate were three carbons and four carbons 1415. Therefore, investigation of chain length was focused on three carbons and four carbons. As shown in Table 1 and Table 2, most of the compounds with chain lengths of four carbons exhibited higher affinities for the three receptors than that of three carbon derivatives (6j vs. 6l; 9a vs. 9b; 9g vs. 9h), for example, elongation of the linker of 9c from three carbons to four carbons resulted in improved activities for D2 and 5-HT1A but had a negative effect on the 5-HT2A receptor (9c vs. 9d). This indicated that the appropriate length of the linker was four carbons.
Overall, compounds 6j, 9d, 9g and 9h demonstrated high affinity for the D2, 5-HT1A and 5-HT2A receptors (D2, Ki < 20 nM; 5-HT1A, Ki <10 nM; 5-HT2A, Ki < 10 nM) and the potency ratio between D2 and 5-HT1A, 5-HT2A was less than 5, which means the compounds have balanced receptor activity profiles. Therefore, compounds 6j, 9d, 9g and 9h were selected for further evaluation.
A vast number of clinical studies has demonstrated that atypical antipsychotics can induce a series of adverse effects in the treatment of schizophrenia, such as weight gain, hyperglycaemia and QTc prolongation [18,19,20]. These adverse side effects have been proved to be associated with several off-target receptors, mainly focused on histamine H1, 5-HT2C, and adrenergic α1 [21,22]. For instance, the synergistic effects of H1 and 5-HT2C antagonism may cause weight gain and hyperglycaemia while antagonism on the adrenergic α1 receptor may trigger orthostatic hypotension [23]. Therefore, the selected compounds were further evaluated for these receptors in the present study. As shown in Table 3, compounds 6j, 9d, 9g and 9h exhibited moderate activities for the α1 and H1 receptors and lower activities for 5-HT2c receptors compared with risperidone (α1, Ki = 2.8 ± 0.4 nM; H1, Ki = 26.1 ± 2.8 nM; 5-HT2c, Ki = 19.7 ± 2.7 nM), suggesting that 6j, 9d, 9g and 9h had low liability to elicit treatment-associated adverse effects. In addition, the selected compounds were subjected to additional studies of binding to the D3 receptor, as the effect of atypical antipsychotic drugs on the D3 receptor is related to cognitive and motivational behaviors [24], and several recent pharmacological studies strongly support the idea that antagonism on the D3 receptor might reduce catalepsy [25]. As shown in Table 3, compounds 6j, 9d, 9g and 9h had high affinities for the D3 receptor, which may be beneficial for the treatment of schizophrenia.
Cardiotoxicity is an important drug evaluation indicator, which is often caused by the blockade of the human ether-a-go-go-related gene (hERG) potassium channel [26,27]. To assess the cardiotoxicity of compounds 6j, 9d, 9g and 9h, their inhibitory actions on hERG were assessed in vitro. As shown in Table 3, all of the tested compounds exhibited lower levels of inhibition on hERG compared to clozapine and risperidone, indicating that they have low risks for QT interval prolongation.
To sum up, all the in vitro evaluations results have shown that compound 6j exhibited higher affinity for D2, D3, 5-HT1A and 5-HT2A receptors and weaker affinity for 5-HT2c, α1 and H1 receptors than other candidates, therefore, compound 6j was then subjected to further investigation.

2.3. Acute Toxicity

To assess the safety of compound 6j, its acute toxicity was assayed in vivo in terms of LD50 values. As shown in Table 4, compound 6j showed a high safety threshold, even exceeds the maximum dose (LD50 > 2000 mg/kg). The results indicated that compound 6j possesses high safety performance and low acute toxicity.

2.4. Intrinsic Activity of Compound 6j

As shown in Table 5, compound 6j stimulated the D2L, D3, 5-HT1A, 5-HT2A receptors in the agonist assay and showed weak agonist activity, the efficacy of the reference compounds less than 10%, respectively. In the antagonist assay, the efficacy of compound 6j blocked the four receptors more than 90%. Thus, 6j functioned as an antagonist at the D2L (IC50 = 8.9 nM), D3 (IC50 = 31.5 nM), 5-HT1A (IC50 = 201.4 nM), 5-HT2A (IC50 = 195.5 nM) receptors.

2.5. Behavioral Studies

As schizophrenic patients show many psychiatric and somatic symptoms in clinical manifestations, more intuitive and visual models to evaluate the antipsychotic-like activities of the new compounds are required. Nowadays, there are several models commonly used to assess the antipsychotic-like efficacy of potential compounds, such as apomorphine (APO)-induced hyperlocomotion [28,29] and MK-801-induced hyperactivity models [30]. In the APO-induced hyperlocomotion model, the D2 receptor agonist apomorphine is commonly used to induce behavioral agitation in mice to simulate the positive symptoms of schizophrenia, but this can be reversed by compounds with antipsychotic-like efficacies. In the MK-801-induced hyperactivity model, the non-competitive NMDA receptor antagonist MK-801 can induce the schizophrenia-like symptoms in healthy animals, but compounds with antipsychotic-like efficacies can significantly reverse these symptoms. In addition, the catalepsy test model is used to predict the propensity of antipsychotics to induce EPS in humans [31]. This model has become a common and important animal model for assessing the potential therapeutic effect in schizophrenia [31]. In this study, 6j was subjected to these models to verify its potential antipsychotic activity.

2.5.1. Apomorphine-Induced Hyperlocomotion

As shown in Figure 2, compound 6j induced a dose-dependent response in APO-induced hyperlocomotion model, with an ED50 value of 0.19 mg/kg (Table 4). Mice in the control groups received clozapine and risperidone inhibit the APO-induced climbing with ED50 values of 17.92 and 0.046 mg/kg, respectively. The comparison of results indicates that compound 6j has certain efficacy against the positive symptoms of psychosis.

2.5.2. MK-801-Induced Hyperactivity

In the MK-801-induced hyperactivity model, compound 6j significantly and dose dependently attenuated the increased locomotor activity (Figure 3) with an ED50 value of 0.16 mg/kg (Table 4). In the control group, mice treated with clozapine and risperidone with ED50 values of 2.28 and 0.011 mg/kg, respectively. The comparison of the results shows that compound 6j was more potent than clozapine.

2.5.3. Catalepsy

In the catalepsy test model, compound 6j displayed a high threshold for catalepsy with an ED50 value of 56.84 mg/kg, but the ED50 values of clozapine and risperidone were 50.0 mg/kg and 0.92 mg/kg, the comparison of results indicates that 6j has a lower incidence of EPS (Table 4).
In addition, compound 6j displayed a wide therapeutic index (TI) range (299.16–352.5) based on its efficacy (APO and MK-801 models) and its side effects (catalepsy), while the TI of clozapine range from 5.58 to 21.93, and 20.0 to 83.63 of risperidone (Table 4). The result demonstrated that 6j has a higher safety margin than clozapine and risperidone.

3. Materials and Methods

3.1. General Information

All solvents were from commercial sources and used without further purification. The purity of all the reagents and the test compounds was more than 95%. Melting points were determined in open capillary tubes and are uncorrected. 1H-NMR spectra were recorded on an Avance III 400 spectrometer (Bruker, Karlsruhe, Germany) at 400 MHz (1H) using CDCl3 and DMSO-d6 as solvents. Chemical shifts recorded as δ values (ppm), using tetramethylsilane (TMS) as the internal reference; Coupling constants (J) are given in Hz. Proton multiplicities are labeled as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br (broad signal). Thin layer chromatography (TLC) was performed using silica gel GF254. Flash column chromatography was carried out using silica gel.

3.2. Instrumentation

High performance liquid chromatography (HPLC) methods: LC-20AD spectrometer (Shimadzu, Kyoto, Japan); column, Shimadzu VP-ODS (4.6 mm × 250 mm, 5 μm) C18-253; mobile phase, A: acetonitrile 0.01 mol/L NH4H2PO4 (0.2% Et3N, pH = 3.0) (10:90); B: acetonitrile 0.01 mol/L NH4H2PO4 (0.1% Et3N, pH = 3.0)(70:30); flow rate, 1.0 mL/min; sample size, 10 μL; column temperature, 40 °C; UV detection condition, 210 nm; High resolution mass spectra (HRMS) spectrometer, Agilent 6530 Q-TOF LC/MS and Agilent 1290 Infinity (G4212A) spectrometer (Agilent, Santa Carla, CA, USA). The HPLC conditions as following: column X BridgeR Shield RP18 (4.6 × 150 mm, 3.5 μm, Waters, Milford MA USA), C18-330; mobile phase: A, 5mmol/L NH4OAc (pH = 6.0)(10:90); B, MeOH; eluent, 30% A and 70% B (V:V); flow rate, 1.0 mL/min; column temperature, 35 °C; UV detection, 254 nm. Mass spectrometry conditions: Dual AJS ESI, positive; Gas temperature, 350 °C; Fragmentor, 80 KV; Drying gas, 7 mL/min; Nebulizer, 45 psi; Sheath gas temperature, 350 °C; Capillary Voltage, 4000 V; Sheath gas flow, 11 L/min; Nozzle Voltage, 500 V. Low resolution mass spectrometry (LRMS) and high performance liquid chromatography-mass spectrometry (HPLC-MS) were obtained using an Agilent MS/1200 HPLC liquid chromatograph/mass spectrometer (JXZX-FXS-064).Agilent ZORBAX SB-C18 (4.6 × 150 mm, 5 µm); ESI source: scan mode, positive/negative; Rage, 50–1000; Nitrogen gas flow, 11 L/min; Column pressure: −3000–3000 eV. Chromatographic parameter: Mobile phase, 50% acetonitrile, 50% water; UV detection, 210 nm, 245 nm; injection volume, 1.0 µL; flow rate, 0.3 mL/min; column temperature, 30 °C.

3.3. Synthesis

3.3.1. General Procedures for the Preparation of Intermediates 4 and 7

Compound 4, 7a and 7b were synthesized by the following reaction: 2,4-dihydroxyacetophenone and pyrrolidine were added to a suitable amount of acetonitrile and then corresponding ketone was added and the mixture was stirred at 50 °C for 12 h. Then, it was cooled down and poured into an ice-cold 2 M solution of hydrochloric acid and water, stirred for another 30 min, filtered and the obtained solid was recrystallized from EtOH (95%) to give 4 and 7a–7b.

3.3.2. General Procedures for the Preparation of Intermediates 5 and 8

To a suspension of intermediate 4 and 7 in DMF, 1.5 equivalents of K2CO3 and 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane were added and the suspension was stirred for 24 h at room temperature and then filtered. The filtrate was evaporated in vacuo followed by addition of dichloromethane (DCM), washed with water and dried over anhydrous Na2SO4, the solvent was evaporated in vacuo and the residue was separated by silica gel column chromatography to afford 5a, 5b and 8a–8d.

3.3.3. General Procedures for the Preparation of Target Compounds 6 and 9

A mixture of intermediates 5 or 8, anhydrous potassium carbonate, CH3CN and a catalytic amount of potassium iodide (KI) was stirred at 70 °C for 10 h. The mixture was filtered and the filtrate was concentrated under reduced pressure, the residue was dissolved in DCM, washed with water and dried with anhydrous MgSO4, the solvent was removed under reduced pressure and the residue was separated by silica gel column chromatography to afford 6a-6l and 9a-9k.

3.3.4. General Procedures for the Preparation of Intermediates 11, 12, 14, and 15

A mixture of resorcinol or a derivative, 3-chloropropionic acid and trifluoromethanesulfonic acid was stirred at 80 °C for 1 h. The reaction mixtures were cooled to room temperature and poured into water, extracted with ethyl acetate (EA), The combined organic phase was washed with water and dried over anhydrous MgSO4, the solvent was removed under reduced pressure and the residue was added to a certain amount of sodium hydroxide solution (2N) at 0–5 °C, then stirred for 2 h. The pH was adjusted to above 2 with hydrochloric acid solution and stand for 30 min, the formed intermediates 11a–11d were collected by filtration, washed with water and dried in vacuo.
A mixture of resorcinol or 2-chlororesorcinol, crotonic acid and anhydrous zinc chloride was stirred at 180 °C for 30 min. The reaction mixtures were cooled to room temperature and poured into water, extracted with EA, the combined organic layer was washed with water, brine, and dried over MgSO4, the solvents were removed under reduced pressure and the crude products was purified via column chromatography to give pure products 14a–14b.
Intermediates 12 and 15 were prepared as described for compound 5 using 11 or 14, 1-bromo-4-chlorobutane and anhydrous K2CO3 in DMF; the crude products were purified via column chromatography.

3.3.5. General Procedures for the Preparation of Target Intermediates 13 and 16

The target compounds 13a–13d and 16a–16b were prepared as described for compound 6 using 12a–12e, 15a–15b and K2CO3, KI, 6-fluoro-3-(piperidin-4-yl) benzo[d]isoxazole in acetonitrile, the crude products were purified via column chromatography.

3.3.6. General Procedures for the Preparation of Intermediates 17, 18 and 19

1-(2,4-Dihydroxyphenyl)propan-1-one (16.6 g) and sodium acetate anhydrous (8.3 g) were added to acetic anhydride (70 mL), the reaction mixture was stirred at reflux for 14 h and cooled to room temperature, diluted with water and extracted with DCM, the organic layer was separated and washed with water, dried over MgSO4, concentrated in vacuo to give intermediate 17 as a yellow solid. The intermediate 17 was dissolved in a solution (50 mL) of saturated sodium bicarbonate and MeOH (V:V = 1:1), the suspension was stirred at rt for 3 h, the product intermediate 18 obtained by filtration. Intermediate 18 was dissolved in MeOH, and then Pd/C added and stirred at rt for 10 h under hydrogen atmosphere, filtered and the filtrate concentrated in vacuo to give intermediate 19.

3.3.7. The Preparation of Intermediate 20 and Target Compound 21

The intermediate 20 and target compound 21 were prepared as described for intermediate 15 and target compound 16.

3.3.8. The Characteristics and Spectroscopic Data of the Target Compounds

2,2-Dimethyl-7-(4-(4-phenylpiperazin-1-yl)butoxy) chroman-4-one (6a).Pale-white solid; m. p. 131–133 °C; yield 80.3%; 1H-NMR (CDCl3) δ 7.84 (d, J = 8.8 Hz, 1H), 7.32 (dd, J = 8.6, 7.4 Hz, 2H), 6.98 (dd, J = 8.8, 0.9 Hz, 2H), 6.91 (t, J = 7.3 Hz, 1H), 6.58 (dd, J = 8.8, 2.4 Hz, 1H), 6.42 (d, J = 2.3 Hz, 1H), 4.07 (t, J = 6.3 Hz, 2H), 3.31–3.21 (m, 4H), 2.71 (s, 2H), 2.70–2.65 (m, 4H), 2.56–2.43 (m, 2H), 1.89 (dd, J = 14.4, 6.3 Hz, 2H), 1.83–1.69 (m, 2H), 1.50 (d, J = 2.0 Hz, 6H). HRMS (ESI) calculated for C25H33N2O3 [M + H]+, 409.2486; found, 409.2479.
7-(4-(4-(2-Fluorophenyl) piperazin-1-yl) butoxy)-2,2-dimethylchroman-4-one (6b). Pale-white solid; m. p. 135–137 °C; yield 78.8%; 1H-NMR (CDCl3) δ 7.84 (d, J = 8.8 Hz, 1H), 7.19–6.87 (m, 4H), 6.57 (dd, J = 8.8, 2.3 Hz, 1H), 6.41 (d, J = 2.3 Hz, 1H), 4.06 (t, J = 6.3 Hz, 2H), 3.27–3.06 (m, 4H), 2.71–2.97 (m, 6H), 2.58–2.40 (m, 2H), 1.93–1.71 (m, 4H), 1.49 (s, 6H). HRMS (ESI) calculated for C25H32FN2O3 [M + H]+, 427.2391; found, 427.2390.
2,2-Dimethyl-7-(4-(4-(3-(trifluoromethyl) phenyl) piperazin-1-yl) butoxy) chroman-4-one (6c). Pale-white solid; m. p. 139–140 °C; yield 76.2%; 1H-NMR (CDCl3) δ 7.84 (d, J = 8.8 Hz, 1H), 7.40 (t, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.20 - 7.02 (m, 2H), 6.58 (dd, J = 8.8, 2.4 Hz, 1H), 6.41 (dd, J = 5.0, 2.7 Hz, 1H), 4.07 (dd, J = 8.0, 4.4 Hz, 2H), 3.33 (s, 4H), 2.68 (d, J = 24.9 Hz, 6H), 2.56 (s, 2H), 1.96–1.85 (m, 2H), 1.83–1.71 (m, 2H), 1.50 (d, J = 2.7 Hz, 6H). HRMS (ESI) calculated for C26H32F3N2O3 [M + H]+, 477.2360; found, 477.2358.
7-(4-(4-(2-Methoxyphenyl) piperazin-1-yl) butoxy)-2,2-dimethylchroman-4-one (6d).Off-white solid; m. p. 128–130 °C; yield 75.5%; 1H-NMR (CDCl3) δ 7.80 (t, J = 4.0 Hz, 1H), 7.02–6.87 (m, 4H), 6.57 (m, 1H), 6.41 (d, J = 4.0 Hz, 1H), 4.09 (d, J = 4.0 Hz, 2H), 3.87 (s, 2H), 2.70-2.60 (m, 4H), 2.05 (d, J = 8.0 Hz, 2H), 2.68 (d, J = 22.9 Hz, 2H), 2.67–2.58 (m, 4H), 2.57–2.42 (m, 2H), 1.89 (dd, J = 14.4, 6.3 Hz, 2H), 1.45 (s, 6H). HRMS (ESI) calculated for C26H34N2O4 [M + H]+, 438.2519; found, 438.2511.
2,2-Dimethyl-7-(4-(4-(pyridin-2-yl) piperazin-1-yl) butoxy) chroman-4-one (6e).Pale-white solid; m. p. 127–129 °C; yield 73.2%; 1H-NMR (CDCl3) δ 8.33 - 8.17 (m, 1H), 7.83 (t, J = 8.1 Hz, 1H), 7.53 (ddd, J = 8.9, 7.1, 2.0 Hz, 1H), 6.79–6.62 (m, 2H), 6.58 (dd, J = 8.8, 2.4 Hz, 1H), 6.42 (d, J = 2.3 Hz, 1H), 4.07 (t, J = 6.3 Hz, 2H), 3.67–3.53 (m, 4H), 2.68 (d, J = 22.9 Hz, 2H), 2.67–2.58 (m, 4H), 2.57–2.42 (m, 2H), 1.89 (dd, J = 14.4, 6.3 Hz, 2H), 1.78 (dd, J = 14.9, 8.1 Hz, 2H), 1.49 (s, 6H). HRMS (ESI) calculated for C24H32N3O3 [M + H]+, 410.2438; found, 410.2438.
2,2-Dimethyl-7-(4-(4-(pyrimidin-2-yl) piperazin-1-yl) butoxy) chroman-4-one (6f).Pale-white solid; m. p. 128–131 °C; yield 69.7%; 1H-NMR (CDCl3) δ 8.35 (d, J = 4.7 Hz, 2H), 7.83 (d, J = 8.8 Hz, 1H), 6.64–6.52 (m, 2H), 6.42 (d, J = 2.3 Hz, 1H), 4.06 (t, J = 6.3 Hz, 2H), 3.90 (s, 4H), 2.71 (s, 2H), 2.56 (d, J = 20.1 Hz, 4H), 2.51 (d, J = 7.4 Hz, 2H), 1.94 - 1.82 (m, 2H), 1.82 - 1.70 (m, 2H), 1.49 (s, 6H). HRMS (ESI) calculated for C23H31N4O3 [M + H] +, 411.2391; found, 411.2390.
7-(4-(4-(Benzo[d] [1,3] dioxol-5-ylmethyl) piperazin-1-yl) butoxy)-2,2-dimethylchroman-4-one(6g).White solid; m. p. 112–114 °C; yield 69.4%; 1H-NMR (CDCl3) δ 7.82 (d, J = 8.8 Hz, 1H), 6.89 (s, 1H), 6.78 (d, J = 0.8 Hz, 2H), 6.55 (dd, J = 8.8, 2.3 Hz, 1H), 6.39 (d, J = 2.3 Hz, 1H), 5.97 (s, 2H), 4.16 (q, J = 7.1 Hz, 2H), 4.03 (t, J = 6.4 Hz, 2H), 3.45 (s, 2H), 2.70 (s, 2H), 2.46 (m, 6H), 1.94–1.60 (m, 4H), 1.49 (s, 6H), 1.30 (t, J = 7.1 Hz, 2H). HRMS (ESI) calculated for C27H35N2O5 [M + H]+, 467.2540; found, 467.2529.
7-(4-(4-(Benzo[b]thiophen-4-yl) piperazin-1-yl) butoxy)-2,2-dimethylchroman-4-one(6h).Pale white solid; m. p. 117119 °C; yield 67.7%; 1H-NMR (CDCl3) δ 7.85 (d, J = 8.8 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 7.45 (q, J = 5.6 Hz, 2H), 7.32 (dd, J = 9.6, 6.1 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 6.59 (dd, J = 8.8, 2.3 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 4.27 - 3.97 (m, 2H), 3.26 (s, 4H), 2.75 (d, J = 30.9 Hz, 4H), 2.64–2.41 (m, 2H), 2.02–1.70 (m, 4H), 1.50 (s, 6H), 1.31 (t, J = 7.1 Hz, 2H). HRMS (ESI) calculated for C27H33N2O3S [M + H]+, 465.2206; found, 465.2201.
7-(4-(4-(Benzo[d]isothiazol-3-yl) piperazin-1-yl) butoxy)-2,2-dimethylchroman-4-one (6i). Pale white solid; m. p. 119–120 °C; yield 65.8%; 1H-NMR (CDCl3) δ 7.96 (d, J = 8.2 Hz, 1H), 7.85 (t, J = 8.1 Hz, 2H), 7.52 (ddd, J = 8.1, 7.0, 1.0 Hz, 1H), 7.41 (ddd, J = 8.0, 7.0, 0.9 Hz, 1H), 6.58 (dd, J = 8.8, 2.4 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 4.08 (t, J = 6.3 Hz, 2H), 3.71–3.50 (m, 4H), 2.86–2.66 (m, 4H), 2.64–2.44 (m, 2H), 1.98–1.68 (m, 4H), 1.50 (s, 6H), 1.31 (t, J = 7.1 Hz, 2H). HRMS (ESI) calculated for C26H32N3O3S [M + H]+, 466.2195; found, 466.2189.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl) piperidin-1-yl) butoxy)-2,2-dimethylchroman- 4-one (6j). Pale white solid; m. p. 116–117 °C; yield 70.2%; 1H-NMR (CDCl3) δ 7.80 (d, J = 8.8 Hz, 1H), 7.70 (dd, J = 8.7, 5.0 Hz, 1H), 7.25 (dd, J = 8.5, 1.9 Hz, 1H), 7.06 (td, J = 8.9, 2.1 Hz, 1H), 6.57 (dd, J = 8.8, 2.4 Hz, 1H), 6.40 (d, J = 2.4 Hz, 1H), 4.23–4.13 (m, 2H), 3.86 (dd, J = 5.4, 4.2 Hz, 2H), 3.74 (d, J = 5.8 Hz, 2H), 3.15 – 3.01 (m, 3H), 2.69 (dd, J = 11.2, 5.4 Hz, 4H), 2.26 (dd, J = 11.3, 3.0 Hz, 2H), 2.15 – 2.02 (m, 4H), 1.45 (s, 6H). HRMS (ESI) calculated for C27H32FN2O4 [M + H]+, 467.2341; found, 467.2335.
7-(3-(4-(2-Methoxyphenyl)piperazin-1-yl)propoxy)-2,2-dimethylchroman-4-one (6k). Pale-white solid; m. p. 103–105 °C; yield 71.5%; 1H-NMR (CDCl3) δ 7.80 (d, J = 8.0 Hz, 1H), 7.02–6.87(m, 4 H), 6.54–6.57 (m, 1H), 6.40 (d, J =4 Hz, 1H), 3.88 (s, 3H), 3.13 (s, br, 4H), 2.70–2.67 (m, 8H), 2.60 (t, J = 8.0 Hz, 2H), 2.05-2.03 (m, 2H), 1.46 (s, 6H). HRMS (ESI) calculated for C25H33FN2O4 [M + H]+, 425.2435; found, 425.2433.
7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propoxy)-2,2-dimethylchroman-4-one (6l). Pale-white solid; m. p. 116–118 °C; yield 73.4%; 1H-NMR (CDCl3) δ 7.80 (d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 1H), 7.27–7.25 (m, 1H), 7.09-7.05 (m, 1H), 6.57-6.54 (m, 1H), 6.40 (d, J = 4.0 Hz, 1H), 4.10 (t, J = 8.0 Hz, 2H), 3.13-3.09 (m, 3H), 2.68 (s, 2H), 2.59 (t, J = 8.0 Hz, 2H), 2.21-2.00 (m, 8H), 1.46 (s, 6H).HRMS (ESI) calculated for C26H30FN2O4 [M + H]+, 453.2184; found, 453.2182.
7-(3-(4-(2-Methoxyphenyl) piperazin-1-yl) propoxy) spiro[chromane-2,1′-cyclopentan]-4-one (9a). Off-white solid; m. p. 128–130 °C; yield 68.5%; 1H-NMR (CDCl3) δ 7.81 (d, J = 8.0 Hz, 1H), 7.04–6.88(m, 4H), 6.58–6.55 (m, 1H), 6.40 (d, J = 4.0 Hz, 1H), 4.09 (t, J = 8.0 Hz, 2H), 3.89 (s, 3H), 3.13 (s, br, 4H), 2.79 (s, 2H), 2.71 (s, br, 4H), 2.61 (t, J = 8.0 Hz, 2H), 2.03–2.12 (m, 4H), 1.87-1.91 (m, 2H), 1.74-1.65 (m, 4H). HRMS (ESI) calculated for C27H35FN2O4 [M + H]+, 451.2519; found, 451.2511.
7-(4-(4-(2-Methoxyphenyl) piperazin-1-yl) butoxy) spiro[chromane-2,1′-cyclopentan]- 4-one (9b). Off-white solid; m. p. 96–98 °C; yield 76.4%; 1H-NMR (CDCl3) δ 7.80 (d, J = 8.0 Hz, 1H), 7.03–6.86 (m, 4H), 6.56–6.53 (m, 1H), 6.37 (d, J = 4.0 Hz, 1H), 4.03 (t, J = 8.0 Hz, 2H), 3.88 (s, 3H), 3.12 (s, br, 4H), 2.78 (s, 2H), 2.68 (s, br, 4H), 2.49 (t, J = 8.0 Hz, 2H), 2.10–2.05 (m, 2H), 1.87–1.84 (m, 4H), 1.75–1.69 (m, 6H). HRMS (ESI) calculated for C27H35FN2O4 [M + H]+, 451.2519; found, 451.2511. HRMS (ESI) calculated for C28H37N2O4 [M+H]+, 465.2748; found, 465.2745.
7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propoxy)spiro[chromane-2,1′-cyclopentan]-4-one (9c). White solid; m. p. 91–93 °C; yield 70.2%; 1H-NMR (CDCl3) δ 7.80 (d, J = 8.0 Hz, 1H), 7.72-7.69(m, 1H), 7.26–7.24 (m, 1H), 7.09-7.04 (m, 1H), 6.57–6.54(m, 1H), 6.41 (d, J = 4.0 Hz, 1H), 4.09 (t, J = 8.0 Hz, 2H), 3.11-3.08 (m, 3H), 2.78 (s, 2H), 2.58 (t, J = 8.0 Hz, 2H), 2.23-1.86 (m, 12H), 1.74-1.64 (m, 4H). HRMS (ESI) calculated for C28H32FN2O4 [M + H]+, 479.2341; found, 479.2338.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)spiro[chromane-2,1′-cyclopentan]-4-one (9d). Pale-white solid; m. p. 103–105 °C; yield 68.6%; 1H-NMR (CDCl3) δ 7.80 (d, J = 8.0 Hz, 1H), 7.69–7.73(m, 1H), 7.26–7.24 (m, 1H), 7.09–7.04 (m, 1H), 6.56–6.54 (m, 1H), 6.38 (d, J = 4.0 Hz, 1H), 4.04 (t, J = 8.0 Hz, 2H), 3.12-3.08 (m, 3H), 2.78 (s, 2H), 2.48 (t, J = 8.0 Hz, 2H), 2.19-2.07 (m, 8H), 1.90-1.63 (s, 10H). HRMS (ESI) calculated for C29H34FN2O4 [M + H]+, 493.2497; found, 493.2496.
7-(3-(4-(2-Methoxyphenyl)piperazin-1-yl)propoxy)spiro[chromane-2,1′-cyclohexan]-4-one (9e). Pale-white solid; m. p. 105–107 °C; yield 71.4%; 1H-NMR (CDCl3) δ 7.79 (d, J = 8.0 Hz, 1H), 6.85–7.00 (m, 4H), 6.56–6.53 (m, 1H), 6.44 (d, J = 4.0 Hz, 1H), 4.09 (t, J = 8.0 Hz, 2H), 3.87 (s, 3H), 3.12 (m, 4H), 2.70 (s, br, 4H), 2.64 (s, 2H), 2.60 (t, J = 8.0 Hz, 2H), 2.07-1.97 (m, 4H), 1.72-1.66 (m, 2H), 1.52-1.45 (m, 4H), 1.34–1.30 (s, 2H). HRMS (ESI) calculated for C28H37N2O4 [M + H]+, 465.2748; found, 465.2740.
7-(3-(4-(2-(Trifluoromethyl)phenyl)piperazin-1-yl)propoxy)spiro[chromane-2,1′-cyclohexan]-4-one (9f). Off-white solid; m. p. 138–140 °C; yield 74.7%; 1H-NMR (CDCl3) δ 7.80–7.73 (m, 1H), 7.42–7.34 (m, 1H), 7.19–7.06 (m, 3H), 6.56–6.52 (m, 1H), 6.41 (d, J = 4.0 Hz, 1H), 4.05 (t, J = 8.0 Hz, 2H), 3.31-3.28 (m, 2H), 2.70–2.52 (m, 6H), 2.30–2.05 (m, 4H), 1.99–1.48 (m, 10H), 1.36–1.32 (m, 2H). HRMS (ESI) calculated for C28H34F3N2O3 [M + H]+, 503.2516; found, 503.2511.
7-(3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)propoxy)spiro[chromane-2,1′-cyclohexan]-4-one (9g). Pale-white solid; m. p. 96–98 °C; yield 67.1%; 1H-NMR (CDCl3) δ 7.79(d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 1H), 7.26–7.24 (m, 1H), 7.10–7.05 (m, 1H), 6.58-6.55 (m, 1H), 6.45 (d, J = 4.0 Hz, 1H), 4.11 (t, J = 8.0 Hz, 2H), 3.12–3.10 (m, 3H), 2.66 (s, 2H), 2.61 (t, J = 8.0 Hz, 2H), 2.2–1.98 (m, 12H), 1.74–1.48 (m, 4H), 1.36-1.33 (m, 2H). HRMS (ESI) calculated for C29H34FN2O4 [M + H]+, 493.2497; found, 493.2495.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)spiro[chromane-2,1′-cyclohexan]-4-one (9h). White solid; m. p. 126–128 °C; yield 72.9%; 1H-NMR (CDCl3) δ 7.78(d, J = 8.0 Hz, 1H), 7.70–7.73 (m, 1H), 7.26–7.24 (m, 1H), 7.09–7.04 (m, 1H), 6.55–6.52 (m, 1H), 6.42 (d, J = 4.0 Hz, 1H), 4.05 (t, J = 8.0 Hz, 2H), 3.13-3.06 (m, 3H), 2.65 (s, 2H), 2.49(t, J = 8.0 Hz, 2H), 2.18-1.98 (m, 8H), 1.86-1.84 (m, 2H), 1.75-1.69 (m, 4H), 1.53-1.50 (m, 4H), 1.37-1.31 (m, 2H). HRMS (ESI) calculated for C30H36FN2O4 [M + H]+, 507.2654; found, 507.6259.
7-(4-(4-(2,3-Dimethylphenyl)piperazin-1-yl)butoxy)spiro[chromane-2,1′-cyclohexan]-4-one (9i). White solid; m. p. 138–140 °C; yield 74.3%; 1H-NMR (CDCl3) δ 7.81–7.76 (m, 1H), 7.10–6.92(m, 3H), 6.56–6.52 (m, 1H), 6.42 (d, J = 4.0 Hz, 1H), 4.06(t, J = 8.0 Hz, 2H), 3.06 (s, br, 4H), 2.66 (s, 2H), 2.60 (t, J = 8.0 Hz, 2H), 2.29 (s, 3H), 2.23 (s, 3H), 2.07–1.48 (m, 16H), 1.37–1.31 (m, 2H).HRMS (ESI) calculated for C30H41N2O3 [M + H]+,477.3112; found, 477.3110.
7-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butoxy)spiro[chromane-2,1′-cyclohexan]-4-one(9j). White solid; m. p. 119–121 °C; yield 70.7%; 1H-NMR (CDCl3) δ 7.81–7.76(m, 1H), 7.10–6.92 (m, 3H), 6.56–6.52 (m, 1H), 6.42 (d, J = 4.0 Hz, 1H), 4.06 (t, J = 8.0 Hz, 2H), 3.00 (s, br, 4H), 2.66 (s, 2H), 2.54(t, J = 8.0 Hz, 3H), 2.29 (s, 3H), 2.01-1.48 (m, 12H), 1.37–1.33 (m, 2H). HRMS (ESI) calculated for C28H35Cl2N2O3 [M + H]+, 517.2019; found, 517.2013.
7-(4-(4-(Benzo[d]isothiazol-3-yl)piperidin-1-yl)butoxy)spiro[chromane-2,1′-cyclohexan]-4-one (9k). Pale-white solid; m. p. 129–130 °C; yield 62.8%; 1H-NMR (CDCl3) δ 7.79 (d, J = 8.0 Hz, 2H), 7.19–7.16 (m, 2H), 7.49–7.45 (m, 1H), 6.99–6.96 (m, 1H), 6.56–6.53 (m, 1H), 6.42 (d, J = 4.0 Hz, 1H), 4.05 (t, J = 8.0 Hz, 2H), 3.11(s, br, 4H), 2.70 (s, br, 4H), 2.66(s, 2H), 2.52 (t, J = 8.0 Hz, 2H), 2.00-1.48 (m, 12H), 1.34-1.31 (m, 2H). HRMS (ESI) calculated for C30H37N2O3S [M + H]+, 505.2519; found, 505.2514.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)chroman-4-one (13a). Colorless oily liquid; yield 58.1%; 1H-NMR (CDCl3) δ 7.80(d, J = 8.0 Hz, 1H), 7.75–7.71 (m, 1H), 7.26–7.24 (m, 1H), 7.11–7.06 (m, 1H), 6.56–6.53 (m, 1H), 6.38 (d, J = 4.0 Hz, 1H), 4.42 (t, J = 8.0 Hz, 2H), 4.06 (t, J = 8.0 Hz, 2H),3.06–2.95 (m, 4H), 2.81–2.78 (m, 1H), 2.52–2.43 (m, 4H), 1.92–1.69 (m, 6H), 1.41-1.35 (m, 2H). HRMS (ESI) calculated for C25H28FN2O4 [M + H]+, 439.2018; found, 439.2017.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-5-methylchroman-4-one (13b). Pale-white solid; m. p. 85–87 °C; yield 63.6%; 1H-NMR (CDCl3) δ 7.75–7.71 (m, 1H), 7.25–7.23 (m, 1H), 7.06–7.11 (m, 1H), 6.54–6.57 (m, 1H), 6.39 (d, J = 4.0 Hz, 1H), 4.45 (t, J = 8.0 Hz, 2H), 4.07 (t, J = 8.0 Hz, 2H), 3.07–2.96 (m, 4H), 2.80–2.77 (m, 1H), 2.52–2.43 (m, 4H), 2.39 (s, 3H), 1.91–1.67 (m, 6H), 1.40–1.33 (m, 2H). HRMS (ESI) calculated for C26H30FN2O4 [M + H]+, 453.2184; found, 453.2182.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-6-methylchroman-4-one (13c). Pale-white solid; m. p. 91–93 °C; yield 60.3%; 1H-NMR (CDCl3) δ 7.80 (d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 1H), 7.26-7.24 (m, 1H), 7.12–7.07 (m, 1H), 6.38 (d, J = 4.0 Hz, 1H), 4.46 (t, J = 8.0 Hz, 2H), 4.06 (t, J = 8.0 Hz, 2H), 3.08–2.99 (m, 4H), 2.80–2.77 (m, 1H), 2.52–2.43 (m, 4H), 2.38 (s, 3H), 1.92–1.68 (m, 6H), 1.41–1.35 (m, 2H). HRMS (ESI) calculated for C26H30FN2O4 [M + H]+, 453.2184; found, 453.2182.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-8-methylchroman-4-one (13d). Pale-white solid; m.p. 88–90 °C; yield 67.0%; 1H-NMR (CDCl3) δ 7.80 (d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 1H), 7.26–7.24 (m, 1H), 7.12–7.07 (m, 1H), 6.57–6.54 (m, 1H), 4.45 (t, J = 8.0 Hz, 2H), 4.06 (t, J = 8.0 Hz, 2H), 3.08–3.01 (m, 4H), 2.82–2.79 (m, 1H), 2.55–2.44 (m, 4H), 2.40 (s, 3H), 1.91–1.69 (m, 6H), 1.41–1.36 (m, 2H). HRMS (ESI) calculated for C26H30FN2O4 [M + H]+, 453.2184; found, 453.2179.
7-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-2-methylchroman-4–one (16a). Pale-white solid; m. p. 95–97 °C; yield 63.4%; 1H-NMR (CDCl3) δ 7.80(d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 1H), 7.27–7.25 (m, 1H), 7.10–7.05 (m, 1H), 6.57–6.54 (m,1H), 6.39 (d, J = 4.0 Hz, 1H), 4.41–4.38 (m, 1H), 4.05 (t, J = 8.0 Hz, 2H), 3.06–3.01 (m, 3H), 2.81–2.78 (m, 2H), 2.51–2.41 (m, 4H), 1.93–1.67 (m, 6H), 1.50–1.47 (m, 5H). HRMS (ESI) calculated for C26H30FN2O4 [M + H]+, 453.2184; found, 453.2180.
8-Chloro-7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-2-methylchroman-4-one (16b). Pale-white solid; m. p. 99–101 °C; yield 64.7%; 1H-NMR (CDCl3) δ 7.81(d, J = 8.0 Hz, 1H), 7.74–7.71 (m, 1H), 7.26–7.23 (m, 1H), 7.10–7.05 (m, 1H), 6.56–6.54 (m,1H), 4.40–4.38 (m, 1H), 4.05 (t, J = 8.0 Hz, 2H), 3.05–3.01 (m, 3H), 2.81–2.78 (m, 2H), 2.51–2.41 (m, 4H), 1.92–1.67 (m, 6H), 1.50–1.46 (m, 5H). HRMS (ESI) calculated for C26H29ClFN2O4 [M + H]+, 487.1794; found, 487.1785.
7-(4-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butoxy)-2,3-dimethylchroman-4-one (21). Pale-white solid; m. p. 82–84 °C; yield 86.1%; 1H-NMR (CDCl3) δ 7.80 (d, J = 8.0 Hz, 1H), 7.75–7.71 (m, 1H), 7.26–7.24 (m, 1H), 7.13–7.08 (m, 1H), 6.58–6.54 (m, 1H), 6.38 (d, J = 4.0 Hz, 1H), 4.45 (m, 1H), 4.05 (t, J = 8.0 Hz, 2H), 3.69 (m, 1H), 3.03-3.01(m, 2H), 2.82-2.78 (m, 1H), 2.55–2.44 (m, 4H), 1.92–1.69 (m, 6H), 1.41–1.28 (m, 8H). HRMS (ESI) calculated for C27H32FN2O4 [M + H]+, 467.2341; found, 467.2335.

3.4. Biological Studies

Ethics Statement

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 light and temperature conditions and received standard rat chow and tap water at libitum. Animals were randomly assigned to different experimental groups and each group was kept in a separate cage. All the research involving animals in this study follows the guidelines of the bylaws on experiments on animals, and has been approved by the Ethics and Experimental Animal Committee of Jiangsu Ocean University (Project identification code:2020002, date of approval: 8 January 2020). For the procedural details of the biological studies see the Supplementary Materials.

4. Conclusions

In summary, a series of new flavone derivatives were synthesized based on heterocyclic and aromatic piperazines (piperidine), and their antipsychotic activities were evaluated. Of these compounds, 6j was favorable for binding to multiple acceptors with the KI value of D2 (12.1 nM), D3 (25.4 nM), 5-HT1A (9.7 nM) and 5-HT2A (3.2 nM), respectively. On the other hand, 6j showed low potency for 5-HT2C, α1, H1 receptors and human ether-a-go-go-related gene (hERG) channel (IC50 3769.8 nM), which are closely related to the adverse effects of existing antipsychotics. In addition, compound 6j exhibited significant inhibition of schizophrenia-like symptoms (APO and MK-801-induced motor behavior) without causing catalepsy. Furthermore, compound 6j with its high value of ED50 (58.4 mg/kg) in catalepsy and LD50 (>2000 mg/kg) acts at a low dose in apomorphine-induced climbing test (ED50 0.19 mg/kg), which results in a wider therapeutic index range compared with the typical antipsychotics clozapine and risperidone. Thus, compound 6j has great potential in the treatment of schizophrenia as a novel multi-target antipsychotic drug.

Supplementary Materials

Receptor Binding Studies; Intrinsic activity assessment; hERG Affinity; Acute toxicity; Behavioral studies; 1H-NMR, 13C-NMR and HR-MS of 6j.

Author Contributions

Data curation, L.G., Z.Y., and J.X.; Investigation, L.G., J.X., C.H., J.J. and Z.Y.; Methodology, L.G., Z.Y., Y.C., R.M., and G.Z.; writing—original draft preparation, L.G. and Y.C.; writing—review and editing, Y.C., and G.Z.; supervision, G.Z., Y.C., B.-F.L. and X.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was undertaken as part of the Six Talent Peak project in Jiangsu Province (grant 2019-SWYY-128) and the Key Research and Development Program of Lianyungang High-Tech District (ZD201916).

Acknowledgments

We are grateful for support Jiangsu Nhwa Pharmaceutical Co., Ltd. and the Priority Academic Program Development of Jiangsu Higher Education Institutions of China.

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.
Molecules 25 04107 g001 550
Figure 1. Design of new flavone-linker-based heterocyclic arylpiperazine(piperidine) and arylpiperazine(piperidine) derivatives.
Figure 1. Design of new flavone-linker-based heterocyclic arylpiperazine(piperidine) and arylpiperazine(piperidine) derivatives.
Molecules 25 04107 g001
Molecules 25 04107 sch001 550
Scheme 1. Reagents and conditions: (i) (a) acetone, cyclopentanone or cyclohexanone, pyrrolidine, CH3CN, 50 °C, 12 h; (b) hydrochloric acid solution; (ii) 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane, K2CO3, DMF, rt, 24 h; (iii) 4-aromatic ring substituted piperazine (6a-6i,6k,9a,9b,9e,9f,9i,9l) or 4-aromatic ring substituted piperidine (6j,6l,9c,9d,9g,9h,9k) CH3CN, K2CO3, KI, 70 °C.
Scheme 1. Reagents and conditions: (i) (a) acetone, cyclopentanone or cyclohexanone, pyrrolidine, CH3CN, 50 °C, 12 h; (b) hydrochloric acid solution; (ii) 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane, K2CO3, DMF, rt, 24 h; (iii) 4-aromatic ring substituted piperazine (6a-6i,6k,9a,9b,9e,9f,9i,9l) or 4-aromatic ring substituted piperidine (6j,6l,9c,9d,9g,9h,9k) CH3CN, K2CO3, KI, 70 °C.
Molecules 25 04107 sch001
Molecules 25 04107 sch002 550
Scheme 2. Reagents and conditions: (i) (a) trifluoromethanesulfonic acid, 3-chloropropionic acid, 80 °C, 1 h; (b) sodium hydroxide solution(2 N); (ii) 1-bromo-4-chlorobutane, K2CO3, DMF, rt, 24 h; (iii) CH3CN, K2CO3, KI, 70 °C, 10 h; (iv) crotonic acid, anhydrous zinc chloride, 180 °C, 30 min.
Scheme 2. Reagents and conditions: (i) (a) trifluoromethanesulfonic acid, 3-chloropropionic acid, 80 °C, 1 h; (b) sodium hydroxide solution(2 N); (ii) 1-bromo-4-chlorobutane, K2CO3, DMF, rt, 24 h; (iii) CH3CN, K2CO3, KI, 70 °C, 10 h; (iv) crotonic acid, anhydrous zinc chloride, 180 °C, 30 min.
Molecules 25 04107 sch002
Molecules 25 04107 sch003 550
Scheme 3. Reagents and conditions: (i) acetic anhydride, sodium acetate anhydrous, reflux, 14 h; (ii) sodium bicarbonate solution, MeOH, rt, 3 h; (iii) Pd/C, H2, rt; (iv) 1-bromo-4-chlorobutane, K2CO3, DMF, rt, 24 h; (v) 6-fluoro-3-(piperidin-4-yl)-3a,7a-dihydrobenzo[d]isoxazole, CH3CN, K2CO3, KI, 70 °C.
Scheme 3. Reagents and conditions: (i) acetic anhydride, sodium acetate anhydrous, reflux, 14 h; (ii) sodium bicarbonate solution, MeOH, rt, 3 h; (iii) Pd/C, H2, rt; (iv) 1-bromo-4-chlorobutane, K2CO3, DMF, rt, 24 h; (v) 6-fluoro-3-(piperidin-4-yl)-3a,7a-dihydrobenzo[d]isoxazole, CH3CN, K2CO3, KI, 70 °C.
Molecules 25 04107 sch003
Molecules 25 04107 g002 550
Figure 2. Effect of compound 6j on APO-induced climbing in mice (10/group). The score was shown as means ± SEM. Statistical significances were performed by the nonparametric two-tailed Mann-Whitney U-test: # p < 0.05 vs. vehicle treatment; * p < 0.05 and ** p < 0.01 vs. apomorphine treatment.
Figure 2. Effect of compound 6j on APO-induced climbing in mice (10/group). The score was shown as means ± SEM. Statistical significances were performed by the nonparametric two-tailed Mann-Whitney U-test: # p < 0.05 vs. vehicle treatment; * p < 0.05 and ** p < 0.01 vs. apomorphine treatment.
Molecules 25 04107 g002
Molecules 25 04107 g003 550
Figure 3. Effect of compound 6j on MK-801-induced hyperactivity in mice (10/group). The distance expressed as the means ± SEM. Statistical evaluation was conducted by two-way ANOVA followed by Tukey’s test for multiple comparisons. # p < 0.05 vs. vehicle treatment; ** p < 0.01 and * p < 0.05 vs. MK-801 treatment.
Figure 3. Effect of compound 6j on MK-801-induced hyperactivity in mice (10/group). The distance expressed as the means ± SEM. Statistical evaluation was conducted by two-way ANOVA followed by Tukey’s test for multiple comparisons. # p < 0.05 vs. vehicle treatment; ** p < 0.01 and * p < 0.05 vs. MK-801 treatment.
Molecules 25 04107 g003
Table
Table 1. Binding Affinities for D2, 5-HT1A, and 5-HT2A Receptors of Compounds.
Table 1. Binding Affinities for D2, 5-HT1A, and 5-HT2A Receptors of Compounds.
Molecules 25 04107 i001
CompoundNR1R2Receptor Affinity Ki ± SEM (nM) a
D25-HT1A5-HT2A
6a Molecules 25 04107 i002553.5 ± 68.7118.9 ± 17.489.8 ± 11.3
6b Molecules 25 04107 i003776.8 ± 84.7155.6 ± 19.572.5 ± 8.4
6c Molecules 25 04107 i0041128.6 ± 108.4269.1 ± 30.3147.4 ± 16.9
6d Molecules 25 04107 i0052351.5 ± 317.4275.2 ± 25.286.8 ± 5.6
6e Molecules 25 04107 i0064587.3 ± 510.3627.4 ± 75.3722.1 ± 64.9
6f Molecules 25 04107 i007>10,000 b735.6 ± 82.71135.1 ± 151.7
6g Molecules 25 04107 i0081135.2 ± 140.4216.5 ± 20.8379.5 ± 39.1
6h Molecules 25 04107 i00943.1 ± 5.725.3 ± 2.855.2 ± 4.9
6i Molecules 25 04107 i01026.2 ± 2.781.7 ± 9.519.5 ± 2.2
6j Molecules 25 04107 i01112.1 ± 1.59.7 ± 1.33.2 ± 0.4
aKi values are taken from three experiments, expressed as means SEM. b The Ki values were not calculated because the inhibition percentages at 10 mM were too low.
Table
Table 2. Binding Affinities for D2, 5-HT1A, and 5-HT2A Receptors of Compounds and Reference Antipsychotics.
Table 2. Binding Affinities for D2, 5-HT1A, and 5-HT2A Receptors of Compounds and Reference Antipsychotics.
CompoundStructureReceptor Affinity Ki ± SEM (nM) a
D25-HT1A5-HT2A
6k Molecules 25 04107 i0123157.4 ± 466.4115.8 ± 15.833.1 ± 4.5
6l Molecules 25 04107 i01318.6 ± 2.311.5 ± 1.26.7 ± 0.7
9a Molecules 25 04107 i0143853.8 ± 327.9476.1 ± 57.572.0 ± 6.8
9b Molecules 25 04107 i0153793.4 ± 408.7384.9 ± 49.461.7 ± 8.6
9c Molecules 25 04107 i01618.9 ± 2.110.4 ± 1.48.3 ± 0.9
9d Molecules 25 04107 i01715.7 ± 1.98.2 ± 0.88.5 ± 0.7
9e Molecules 25 04107 i0182054.9 ± 229.5312.1 ± 41.493.5 ± 7.9
9f Molecules 25 04107 i019914.6 ± 90.348.3 ± 5.276.7 ± 9.4
9g Molecules 25 04107 i02018.4 ± 1.87.7 ± 0.99.1 ± 1.1
9h Molecules 25 04107 i02117.0 ± 2.35.6 ± 0.37.3 ± 0.4
9i Molecules 25 04107 i022154.2 ± 19.692.1 ± 8.867.5 ± 7.4
9j Molecules 25 04107 i02381.7 ± 9.776.3 ± 7.135.8 ± 5.2
9k Molecules 25 04107 i02435.1 ± 4.7213.3 ± 39.575.6 ± 8.6
13a Molecules 25 04107 i02522.5 ± 2.613.1 ± 1.79.6 ± 0.8
13b Molecules 25 04107 i02628.9 ± 3.424.6 ± 2.910.5 ± 1.3
13c Molecules 25 04107 i02725.8 ± 3.918.1 ± 1.315.9 ± 2.3
13d Molecules 25 04107 i02822.9 ± 3.117.0 ± 2.213.7 ± 1.9
16a Molecules 25 04107 i02919.7 ± 2.710.1 ± 1.37.9 ± 0.5
16b Molecules 25 04107 i03021.4 ± 2.478.8 ± 8.98.1 ± 1.1
21 Molecules 25 04107 i03128.4 ± 3.216.2 ± 1.52.4 ± 1.9
Clozapine 132.5 ± 13.7188.5 ± 18.312.7 ± 1.8
Risperidone 3.5 ± 0.4191.5 ± 16.90.17 ± 0.03
a Ki values obtained from three experiments, recorded as means ± SEM.
Table
Table 3. Activities on D3, 5-HT2C, α1 and H1 Receptors of compounds and reference antipsychotics.
Table 3. Activities on D3, 5-HT2C, α1 and H1 Receptors of compounds and reference antipsychotics.
CompoundReceptor Affinity Ki ± SEM (nM) ahERG
IC50 (nM)
D35-HT2Cα1H1
6j25.4 ± 3.1565.8 ± 75.4237.2 ± 86.4938.5 ± 101.33769.8 ± 297.5
9d42.6 ± 5.3425.6 ± 47.8161.7 ± 57.1784.2 ± 98.33380.3 ± 352.3
9g35.2 ± 2.8401.1 ± 55.6138.2 ± 39.8909.3 ± 78.53015.5 ± 290.8
9h38.6 ± 3.7528.2 ± 68.5101.5 ± 50.6819.5 ± 91.83982.6 ± 411.5
Risperidone10.9 ± 1.919.7 ± 2.72.8 ± 0.4.26.1 ± 2.8168.5 ± 23.1
Clozapine265.2 ± 18.516.1 ± 3.133.6 ± 3.94.3 ± 0.52750.4 ± 317.2
a Ki values obtained from three experiments, recorded as means ± SEM.
Table
Table 4. In Vivo Pharmacological Profile of Compound.
Table 4. In Vivo Pharmacological Profile of Compound.
CompoundAPO aMK-80 bCAT cCAT/APOCAT/MK-801LD50
6j0.190.1656.84299.16355.25>2000
Clozapine17.922.2850.021.935.58150.0
Risperidone0.0460.0110.922083.6382.1
a APO: apomorphine-induced climbing (ED50, mg/kg, po). b MK-801: MK-801-induced hyperactivity (ED50, mg/kg, po). c CAT: catalepsy (ED50, mg/kg, po).
Table
Table 5. Activities of Compound 6j and Reference Compounds to D2L, D3, 5-HT1A, 5-HT2A Receptors.
Table 5. Activities of Compound 6j and Reference Compounds to D2L, D3, 5-HT1A, 5-HT2A Receptors.
ReceptorCompdActivation (10 μM, %) (n = 3)EC50 (nM)Inhibition (10 μM, %) (n = 3)IC50
(nM)
D2Ldopamine101.3 ± 5.319.1
SCH23390 99.1 ± 3.231.6
6j4.5 ± 0.6 97.4 ± 4.18.9
D3dopamine98.4 ± 4.42.5
spiperone 98.8 ± 4.893.9
6j5.9 ± 0.7 97.5 ± 2.731.5
5-HT1A5-HT99.7 ± 3.82.3
WAY-100635 98.4 ± 3.711.2
6j0.8 ± 0.1 99.1 ± 4.5201.4
5-HT2A5-HT100.2 ± 2.620.4
ketanserin 98.2 ± 2.687.6
6j1.4 ± 0.2 99.8 ± 2.2195.5

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Gao, L.; Yang, Z.; Xiong, J.; Hao, C.; Ma, R.; Liu, X.; Liu, B.-F.; Jin, J.; Zhang, G.; Chen, Y. Design, Synthesis and Biological Investigation of Flavone Derivatives as Potential Multi-Receptor Atypical Antipsychotics. Molecules 2020, 25, 4107. https://doi.org/10.3390/molecules25184107

AMA Style

Gao L, Yang Z, Xiong J, Hao C, Ma R, Liu X, Liu B-F, Jin J, Zhang G, Chen Y. Design, Synthesis and Biological Investigation of Flavone Derivatives as Potential Multi-Receptor Atypical Antipsychotics. Molecules. 2020; 25(18):4107. https://doi.org/10.3390/molecules25184107

Chicago/Turabian Style

Gao, Lanchang, Zhengge Yang, Jiaying Xiong, Chao Hao, Ru Ma, Xin Liu, Bi-Feng Liu, Jian Jin, Guisen Zhang, and Yin Chen. 2020. "Design, Synthesis and Biological Investigation of Flavone Derivatives as Potential Multi-Receptor Atypical Antipsychotics" Molecules 25, no. 18: 4107. https://doi.org/10.3390/molecules25184107

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