Discovery of 4-benzoylpiperidine and 3-(piperidin-4-yl)benzo[d]isoxazole derivatives as potential and selective GlyT1 inhibitors

Abstract

Regulation of glycine transporter 1 (GlyT1) activity is a currently investigated strategy in drug discovery for schizophrenia. This study developed a series of new 4-benzoylpiperidine derivatives as GlyT1 inhibitors by bioisosteric replacement and mimicking of the pyridine ring of RG1678. Among the 4-benzoylpiperidine derivatives, 23q showed an IC₅₀ of 30 nM. Preliminary optimization of the blood–brain barrier penetration led to the discovery of 3-(piperidin-4-yl)benzo[d]isoxazole derivatives. Both series showed good selectivity over GlyT2, D₁, D₂, D₃, 5-HT_1A and 5-HT_2A receptors. Moreover, behavioral testing showed 23q (40 mg kg⁻¹, intragastric) can inhibit the hyperlocomotion induced by acute treatment of phencyclidine, and improve the impaired negative and cognitive symptoms in chronic phencyclidine-induced C57BL/6J mice. An interesting finding showed that 3-(piperidin-4-yl)benzo[d]isoxazole was a privileged scaffold of atypical antipsychotic agents but exhibited high selectivity and potency as a GlyT1 inhibitor.

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Introduction

Schizophrenia is a severe and chronic psychiatric illness, characterized by positive, negative and cognitive symptoms. Currently available drugs are effective in relief of the positive symptoms, but elicit weak or no improvement in the negative symptoms and cognitive impairments.¹ It is generally believed that the N-methyl-D-aspartate (NMDA) receptor hypofunction plays a critical role in the pathophysiology of schizophrenia and may associate with the development of negative symptoms and cognitive deficits in the illness.² Activating the NMDA receptor and restoring function of glutamatergic neurons may be an alternative therapeutic strategy for schizophrenia treatment.³ Because the NMDA receptor is widely distributed in the brain and direct activation of the NMDA receptor would cause seizures and serious neurotoxic side effects,⁴ efforts have turned to the auxiliary glycine binding site. Glycine is an obligatory co-agonist of the NMDA receptor, and elevated glycine levels in the synaptic cleft can promote activation of the NMDA receptor.⁵ Glycine in the synaptic cleft can be uptaken into neurons or surrounding glia by the glycine transporter (GlyT). There are two types of glycine transporters in the brain: GlyT1, which is localized to the NMDA receptor area, and GlyT2, which is distributed around the glycinergic neurons.⁶ Therefore, selective inhibition of GlyT1 could elevate glycine concentrations in the glutamatergic synapse and enhance NMDA receptor activity, thereby may be of potential therapeutic effects in schizophrenia.

So far, at least two series of GlyT1 inhibitors have been identified (Fig. 1).⁷ One is the sarcosine-derived GlyT1 inhibitor, such as 1 ⁸ and 2.⁹ However, some compounds of this series were discontinued in clinical trials because of undesired adverse effects. Another series is the non-sarcosine-based GlyT1 inhibitor, such as 3,¹⁰ 4,¹¹ and 5.¹² Among them, RG1678 demonstrated a beneficial effect in patients with schizophrenia characterized with predominant negative symptoms in a phase II proof-of-concept study¹³ and it is currently in phase III clinical trial.

Fig. 1 Structures of GlyT1 inhibitors.

In our effort to find new GlyT1 inhibitors, we considered RG1678 as a potential starting point for further structural modification. A patent from Roche revealed some bioisosteric replacements of piperazine ring, among them pyrrolidine series was the most potent and well investigated (compound 6 with an IC₅₀ = 90 nM) while piperidine ring did not exhibit good potency.¹⁴ In this case, we considered that piperidine ring was an appropriate bioisostere of piperazine and when the piperazine ring was replaced with piperidine, some heteroatoms should be incorporated to compensate for the loss of binding energy like hydrogen bond. Based on this consideration, we first introduced a carbonyl to 4-benzylpiperidine of compound 7 and a series of 4-benzoylpiperidine derivatives were synthesized, and tested (Fig. 2).

Fig. 2 Design of new GlyT1 inhibitors.

Results and discussion

Chemistry

All described target compounds were prepared by condensation of 2,5-disubstituted benzoic acids and corresponding piperidine moieties. The 2,5-disubstituted benzoic acids were performed as shown in Schemes 1 and 2. 8a–c were commercially available or synthesized as listed in ref. 10. The R² group preferred an electron-withdrawing groups (EWGs). So it was selected from nitro, methylsulfonyl, and N-methylsulfamoyl. First, 2-alkoxy-5-substituted benzoates 9a–h were prepared by substitution reactions with alkyl halides or Mitsunobu reactions with alkyl alcohols in good yields. Then the substituted esters were hydrolyzed by sodium hydroxide to afford the corresponding acids 10a–h. By reacting with triflic anhydride, the phenolic hydroxyl group of 8a–b was converted into trifluoromethylsulfonyloxy group in 72–79% yields and then coupled with 4-fluorophenylboronic acid or substituted by some alkyl amines to yield 12a–b. Further hydrolysis was performed to get 13a–b. Alternatively, considering the electron-withdrawing groups of the phenyl ring, the alkoxy and alkyl amine group at position 2 was also introduced by 2-fluorobenzoic acid analogues following the synthetic route described in Scheme 2.

Scheme 1 Reagents and conditions: (a) alkyl bromide, K₂CO₃, DMF, 40–60 °C, overnight; (b) alkyl alcohol, DIAD, PPh₃, THF, rt, 24 h; (c) NaOH, MeOH–THF–H₂O (1 : 1 : 1), 40 °C, 2 h; (d) triflic anhydride, Et₃N, CH₂Cl₂, 0 °C-rt, 20 min; (e) alkylamine, NMP, microwave, 210 °C, 5 min; (f) NaOH, MeOH–THF–H₂O (1 : 1 : 1), 60 °C, 2 h.

Scheme 2 Reagents and conditions: (a) alkylamine, THF, 100 °C, microwave, 40 min; (b) NaOH (2 N), THF, 60 °C, 2 h; (c) alkyl alcohol, t-BuOK, dry THF, rt-60 °C, 2 h; (d) propane-2-thiol, Cs₂CO₃, DMA, 100 °C, 3 h.

The synthesis of piperidine moieties is outlined in Scheme 3. Substituted bromobenzene was converted to a Grignard reagent and then reacted with Weinreb amide, resulting in 20a–b. Following reflux in HCl (6 N), 21a–b were prepared. 21c was synthesized according to a previous study¹⁵ and 21d was commercially available. Finally, the target compounds were prepared in a single step by HATU-mediated condensation yields ranging from 57% to 85%.

Scheme 3 Reagents and conditions: (a) Mg, I₂, dry THF, 1-acetyl-N-methoxy-N-methylpiperidine-4-carboxamide, reflux to rt, overnight; (b) HCl (6 N), reflux, 4 h; (c) HATU, Et₃N, CH₂Cl₂, rt, overnight.

In vitro biological evaluation and discussion

All final compounds were initially evaluated for their ability to inhibit GlyT1, and the results are summarized in Tables 1 and 2. Considering the convenience of synthesis, we introduced a nitro group to R² position first and then explored the R³ and R⁴ group. The structure–activity relationship study (SAR) of RG1678 revealed that the aromatic ring of the left side preferred hydrophobic substituents, so we incorporated trifluoromethyl and fluoro to R³ and R⁴ positions. As shown in Table 1, for the R⁴ group, fluoro was more potent than hydrogen (23a vs. 23b, 23c vs. 23d, and 23e vs. 23f), while on the R³ position, trifluoromethyl was less suitable than fluoro (23a vs. 23c and 23b vs. 23d). For the R¹ group, introduction of some alkylaminos, such as 23g and 23h, led to great loss in GlyT1 activity. Though 23e showed a slight increase of activity against 7, the others did not meet our satisfaction. This result prompted us to optimize the substituents of the benzoic moiety.

Table 1 In vitro biological evaluation of 23a–h

Compd	R^1	R^2	R^3	R^4	GlyT1 IC50 (nM)^a
a IC50 values were determined using [^3H] glycine and rat glioma C6 cell lines. Data are the mean ± SEM of at least three independent determinations.
23a		Nitro	F	F	471 ± 54
23b		Nitro	F	H	2790 ± 40
23c		Nitro	CF3	F	1855 ± 45
23d		Nitro	CF3	H	77.87%@10 μM
23e		Nitro	F	F	310 ± 94
23f		Nitro	F	H	461 ± 165
23g		Nitro	F	F	78.53%@10 μM
23h		Nitro	F	F	82.45%@10 μM

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Table 2 In vitro biological evaluation of 23i–t

Compd	R^1	R^2	GlyT1 IC50^a (nM)
a IC50 values were determined using [^3H] glycine and rat glioma C6 cell lines. Data are the mean ± SEM of at least three independent determinations.
23i		N-Methylsulfamoyl	172 ± 36
23j		N-Methylsulfamoyl	143 ± 7.25
23k		N-Methylsulfamoyl	117 ± 5.37
23l		N-Methylsulfamoyl	184 ± 14
23m		N-Methylsulfamoyl	80 ± 10.47
23n		N-Methylsulfamoyl	115 ± 32.9
23o		Methylsulfonyl	58.02%@10 μM
23p		Methylsulfonyl	501 ± 84.9
23q		Methylsulfonyl	30 ± 5
23r		Methylsulfonyl	572 ± 67
23s		Methylsulfonyl	44.73%@10 μM
23t		Methylsulfonyl	955 ± 101

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As shown in Table 2, nitro was first replaced with another electron withdrawing N-methylsulfamoyl group while R³ and R⁴ were kept as the most potent fluoro. To our delight, the N-methylsulfamoyl series exhibited well tolerance on the R¹ group, and all alkoxy and alkylamino groups on R¹ position showed moderate activities. However, none of the compounds had a better performance with an IC₅₀ value below 50 nM. Finally, we tested methylsulfonyl on R² position of benzoic moiety. The result was consistent with nitro series and alkoxy groups were more potent than alkylamino on R¹ position. Surprisingly, 23q gave the most satisfactory result (IC₅₀ = 30 nM). The various substituents at R¹ position of benzoic that were tolerated indicated that a lipophilic bulk pocket might exist at this position for the GlyT1 receptor.

In vivo pharmacokinetic study

With the identification of 23q as the most potent GlyT1 inhibitor in vitro, the pharmacokinetic parameters of 23q were evaluated in ICR mice. After oral administration of 23q (10 mg kg⁻¹), blood samples and brain tissues were collected and analyzed at different time points. As shown in Table 3, 23q had a short half-life (T_1/2 = 0.7 h), moderate plasma exposure (AUC_0–∞ = 737 h ng mL⁻¹) with a low brain exposure (C_max in brain = 26.3 ng g⁻¹), indicating a poor B/P ratio. However, the concentration in the brain (C_max in brain = 26.3 ng g⁻¹) was just above the IC₅₀ value. This result promoted us to make structure modifications to improve the brain exposure.

Table 3 Pharmacokinetics parameters of 23q in ICR mice

Compd	T1/2 (h)	Tmax (h)	Cmax,p^a (ng mL^−1)	AUC0–t (h ng mL^−1)	AUC0–∞ (h ng mL^−1)	MRT0–∞ (h)	Cmax,b^b (ng g^−1)	B/P^c
a Cmax,p = Cmax in plasma.b Cmax,b = Cmax in brain.c B/P = Cmax,b/Cmax,p. n = 3 mice per group; male ICR mice (18–20 g); 10 mg kg^−1, intragastric administration, formulated in 0.5% HMPC. PK parameters were calculated by non-compartmental model analysis on Phoenix WinNonlin 6.0 (Pharsight, Mountain View, CA).
23q	0.7	0.5	955	728	737	0.91	26.3	0.03

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Initial attempts to improve brain exposure

The low B/P ratio of 23q indicated that it may be difficult to penetrate the Blood–Brain Barrier (BBB). Strategies to improve BBB permeability have been reviewed and the structure optimization methods include increasing lipophilicity, reducing hydrogen bond donors, increasing rigidity and lowering polar surface area (PSA).¹⁶ Herein we used two parameters, C log P and tPSA (topological PSA) calculated by ChembioDraw, to represent the lipophilicity and PSA.

As shown in Table 4, 23q had a similar tPSA value with RG1678 while a slightly lower C log P, which indicating that higher lipophilicity may be helpful to improve the B/P ratio. At the same time, we made a confirmation restriction of 4-benzoylpiperidine moiety, leading to the 3-(piperidin-4-yl)benzo[d]isoxazole derivatives. By this transformation, we kept the heteroatom in proper position, increased the rigidity and lipophilicity of molecule, but caused a slight adverse effect to tPSA. When 23q was transformed to 24a, a great loss in activity was also observed (IC₅₀ = 185 nM). A scale of optimization was performed and the selected compounds summarized in Table 4 demonstrated that 3-(piperidin-4-yl)benzo[d]isoxazole derivatives had moderate to acceptable GlyT1 inhibiting activities (24g with an IC₅₀ = 38 nM). But the initial optimization failed to reach a balance among the activity, C log P and tPSA. As we expected, with a higher tPSA value, 24g did not have better performance than 23q in vivo pharmacokinetic study (data not shown).

Table 4 In vitro biological evaluation of 3-(piperidin-4-yl)benzo[d]isoxazole derivatives

Compd	R^1	R^2	GlyT1 IC50^a (nM)	C log P^b	tPSA^c
a IC50 values were determined using [^3H] glycine and rat glioma C6 cell lines. Data are the mean ± SEM of at least three independent determinations.b Data were calculated by ChemBioDraw.c Data were calculated by ChemBioDraw.d Data was selected from published reference.
RG1678	—	—	30^d	2.99	79.28
23q	—	—	30 ± 5	2.67	80.75
24a		Methylsulfonyl	185 ± 56	3.28	85.27
24b		Methylsulfonyl	159 ± 43	2.93	85.27
24c		Methylsulfonyl	496 ± 58.9	2.58	85.27
24d		Nitro	89 ± 7.8	4.19	102.94
24e		Nitro	85 ± 5.15	2.46	79.28
24f		N-Methylsulfamoyl	61 ± 6.83	2.92	97.3
24g		N-Methylsulfamoyl	38 ± 7.63	3.54	97.3

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Selectivity evaluation of 23 and 24 series

Though 24 series was unsuccessful to improve the B/P ratio, we were surprised to find that the 3-(piperidin-4-yl)benzo[d]isoxazole moiety was a privileged scaffold that appears frequently in atypical antipsychotic agents¹⁷ such as risperidone¹⁸ and iloperidone,¹⁹ which were dopamine and serotonin receptors dual antagonists. 4-Benzoylpiperidine moiety was also incorporated to discover multitarget ligands of aminergic receptors.²⁰ With this in mind, we assessed the selectivity of some selected compounds. Data were shown in Table 5. Both 23 and 24 series exhibited high selectivity over GlyT2, dopamine and serotonin receptors (IC₅₀ > 10 μM), indicating that 23 and 24 series were selective GlyT1 inhibitors. On the other hand, the result demonstrated that 3-(piperidin-4-yl)benzo[d]isoxazole moiety was not only a privileged scaffold in dopamine hypothesis, but also well tolerated in NMDA hypofunction hypothesis.

Table 5 Selectivity evaluation of 23 and 24 series

Compd	GlyT1 IC50 (nM)	GlyT2^a @10 μM	D1^b @10 μM	D2^b @10 μM	D3^b @10 μM	5-HT1A^b@10 μM	5-HT2A^b @10 μM	hERG^c IC50 (μM)
a GlyT2 inhibiting activity was examined with the cultured primary brain stem neurons.b D1, D2, D3, 5-HT1A, and 5-HT2A were obtained from HEK293 stable-transfecting cell lines.c hERG channels were obtained from HEK293 stable-transfecting cell lines.d Not tested.
23i	172 ± 36	25.67%	19.73%	5.87%	25.14%	2.90%	0.66%	—^d
23k	117 ± 5.37	11.94%	7.62%	18.45%	14.43%	33.06%	8.84%	—
23m	80 ± 10.47	14.92%	14.87%	6.20%	10.80%	5.80%	4.86%	—
23q	30 ± 5	14.62%	16.33%	10.31%	15.29%	11.22%	5.21%	8.1
24b	159 ± 43	8.65%	22.25%	24.24%	17.49%	3.91%	4.05%	—
24d	89 ± 7.8	18.95%	23.37%	5.20%	21.51%	1.75%	31.55%	—
24f	61 ± 6.83	19.70%	6.21%	3.94%	30.30%	24.01%	6.39%	—
24g	38 ± 7.63	17.61%	8.02%	22.48%	25.81%	35.67%	16.51%	—

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In vivo behavioral tests on C57BL/6J mice

With the identification of 23q as a potent and selective GlyT1 inhibitor, we assessed in vivo anti-psychotic activity using phencyclidine (PCP)-induced schizophrenia-like animal models. Considering the low B/P ratio, we assessed using higher doses (20, 40 mg kg⁻¹, intragastric).

Firstly, the effect of 23q on the acute PCP-induced hyperlocomotor activity was examined as described previously,²¹ 23q alone did not change the mice's locomotor activity. Pretreatment of 23q (40 mg kg⁻¹) attenuated PCP (5 mg kg⁻¹)-induced hyperlocomotion of mice, indicating that 23q is a potential anti-psychotic drug in vivo (Fig. 3).

Fig. 3 Effects of 23q on PCP-induced hyperlomotor activity in male C57BL/6J mice. The locomotor activity was indicated by the total travelling distance during 1 hour recording period after the injection of PCP (5 mg kg⁻¹). Vehicle: treated with saline without PCP or 23q. PCP + vehicle: treated with PCP (5 mg kg⁻¹, i.p.). PCP + 23q: treated with 23q (20 mg kg⁻¹ or 40 mg kg⁻¹, i.g.) for 60 min, then followed by the injection of PCP (5 mg kg⁻¹, i.p.). Data were expressed as mean ± SEM and analyzed using one-way analysis of variance (ANOVA) and Dunnett's post-hoc test. *: p < 0.05, vs. vehicle; #: p < 0.05, vs. PCP + vehicle. n = 10.

We then examined whether 23q could improve the social interaction and cognitive function. C57BL/6J mice received daily injections of PCP (10 mg kg⁻¹) for 2 weeks prior to administration of 23q for an additional 2 weeks. Then social interaction and novel object recognition tests were performed. Methods are summarized in the Experimental section.^21–23

As shown in Fig. 4, mice treated with PCP exhibited a social behavioral deficit in the social interaction test. Two weeks of treatment with 23q (40 mg kg⁻¹) significantly restored the impaired social interaction in PCP-treated mice. Similarly, administration of 23q (40 mg kg⁻¹) significantly improved the cognitive deficit in the chronic PCP-treated mice (Fig. 5).

Fig. 4 Effects of 23q on PCP-induced C57BL/6J mice in the social interaction test. Social interaction time was defined as sniffing at any part of the partner's body, grooming, following, or crawling over/under. Vehicle: treated with saline without PCP or 23q. PCP + vehicle: treated with PCP (10 mg kg⁻¹, i.p.) once daily for 2 weeks and then saline for an additional 2 weeks. PCP + 23q: treated with PCP (10 mg kg⁻¹, i.p.) once daily for 2 weeks and then 23q (20 mg kg⁻¹ or 40 mg kg⁻¹, i.g.) for another 2 weeks. Data were expressed as mean ± SEM and analyzed using one-way analysis of variance (ANOVA) and Dunnett's post-hoc test. *: p < 0.05, vs. vehicle; #: p < 0.05, vs. PCP + vehicle. n = 10.

Fig. 5 Effects of 23q on PCP-induced C57BL/6J mice in novel object recognition test. Exploratory preference was a ratio of the amount of time spent exploring the novel object over the total time spent exploring both familiar and novel objects. Vehicle: treated with saline without PCP or 23q. PCP + vehicle: treated with PCP (10 mg kg⁻¹, i.p.) once daily for 2 weeks and then saline for an additional 2 weeks. PCP + 23q: treated with PCP (10 mg kg⁻¹, i.p.) once daily for 2 weeks and then 23q (20 mg kg⁻¹ or 40 mg kg⁻¹, i.g.) for another 2 weeks. Data were expressed as mean ± SEM and analyzed using one-way analysis of variance (ANOVA) and Dunnett's post-hoc test. *: p < 0.05, vs. vehicle; #: p < 0.05, vs. PCP + vehicle. n = 10.

Conclusions

In summary, based on the bioisosteric replacement and mimic of nitrogen atom of pyridine ring of RG1678, we synthesized and evaluated a series of 4-benzoylpiperidine derivatives (23a–t). Among them 23q was the most potent GlyT1 inhibitor in vitro with an IC₅₀ value of 30 nM and showed high selectivity against GlyT2 and dopamine and serotonin receptors. Further in vivo study demonstrated that 23q was effective on the chronic PCP-treated schizophrenia-like behavioral models with 40 mg kg⁻¹. The relatively high dose of 23q was needed to achieve the anti-psychotic efficacy may attribute to the poor pharmacokinetic parameters and low B/P ratio. Preliminary optimization did not improve the pharmacokinetic parameters but led to the discovery of another potential and selective GlyT1 inhibitors: the 3-(piperidin-4-yl)benzo[d]isoxazole derivatives. Next we will make more structure modifications to improve the brain exposure. 4-Benzoylpiperidine and 3-(piperidin-4-yl)benzo[d]isoxazole, which were privileged scaffolds in atypical antipsychotic agents, exhibited high potency in GlyT1 inhibition. We suspect whether they may be potential templates to build multitarget ligands of GlyT1, dopamine and serotonin receptors.

Experimental section

General

All reagents were purchased from commercial suppliers and used without further purification unless otherwise stated. Yields were not optimized. Microwave reactions were performed in a Biotage Initiator. Column chromatography was performed using pre-packed silica cartridges (from 4 to 40 g) from Bonna-Agela Technologies Inc. (Tianjin, China) and eluted with a CombiFlash® Rf 200 from Teledyne Isco. ¹H NMR and ¹³C NMR spectra was recorded on a Bruker AC300 or a Bruker AC400 NMR spectrometer, using tetramethylsilane as an internal reference. Low-resolution mass spectra were determined on Agilent liquid-chromatography mass spectrometer system that consisted of an Agilent 1260 infinity LC coupled to Agilent 6120 Quadrupole mass spectrometer (electrospray positive ionization; ESI) using an Agilent ZORBAX 1.8 mm SB-C18 column (2.1 × 50 mm) with aqueous CH₃CN (30–90%) containing 0.05% formic acid monitored at 240 nm. High-resolution mass spectra were recorded on a Q-Tof Ultima Globe mass spectrometer (Micromass, Manchester, UK). HPLC analysis for all compounds tested in biological systems was performed on an Agilent 1200 series LC system (Agilent ChemStation, Agilent Eclipse XDB-C18, 5 μm, 4.6 × 150 mm, 30 °C, UV240 nm, 1.0 mL min⁻¹) with aqueous CH₃CN (35–90%) containing formic acid (0.05%) for 25 min. Experiments in pharmacokinetics study and behavioral tests with live animals were performed in compliance with the Guidelines for the Care and Use of Laboratory Animals (National Research Council, People's Republic of China, 1996). The animal protocols were approved by the Institutional Animal Care and Use Committees of Shanghai Institute of Materia Medica (SIMM) and Soochow University.

General synthetic procedure for benzoic acids and piperidine moieties

Preparation of 10a–h, 13a–b, 16a–e, 18a–d and 21a–b was summarized in ESI.†

General synthetic procedure for 23a–t, 24a–g

To a solution of benzoic acid moiety (1 equiv.) in CH₂Cl₂ (15 mL) was added HATU (1.3 equiv.) and the mixture was stirred at room temperature for 30 min. Then Et₃N (3 equiv.) and piperidine moiety (1.2 equiv.) were added subsequently and the reaction mixture was stirred at room temperature overnight. The mixture was diluted with CH₂Cl₂ (10 mL) and washed with water. The organic layer was dried over MgSO₄ and concentrated. The residue was purified by flash column chromatography to give the final compounds.

(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(2-isopropoxy-5-nitrophenyl)methanone (23a). 23a was prepared from 10a (0.1 g, 0.44 mmol) and 21d (0.14 g, 0.53 mmol). Yellow solid (0.14 g, 76%). HPLC purity: 96.66%; ¹H NMR (500 MHz, CDCl₃): δ 8.28–8.08 (m, 2H), 7.95–7.81 (m, 1H), 7.04–6.94 (m, 2H), 6.93–6.83 (m, 1H), 4.79–4.62 (m, 2H), 3.51 (tt, J = 13.3, 4.1 Hz, 1H), 3.44–3.30 (m, 1H), 3.23–2.91 (m, 2H), 2.13–2.02 (m, 1H), 1.94–1.53 (m, 3H), 1.48–1.35 ppm (m, 6H); ¹³C NMR (126 MHz, CDCl₃) 198.54, 166.83–164.88 (d, J = 245.7 Hz), 165.29, 163.05–161.02 (d, J = 255.8 Hz), 158.75, 140.92, 133.19–133.00 (m), 127.60, 126.26, 124.22, 121.39–121.29 (d, J = 12.6 Hz), 112.70, 112.37–112.20 (d, J = 21.4 Hz), 105.07–104.64 (d, J = 27.1 Hz), 72.19, 47.66, 46.64 (2C), 28.14 (2C), 21.85 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₂₃O₅N₂F₂: 433.1575, found: 433.1564.

(4-(4-Fluorobenzoyl)piperidin-1-yl)(2-isopropoxy-5-nitrophenyl)methanone (23b). 23b was prepared from 10a (0.1 g, 0.44 mmol) and 21a (0.11 g, 0.53 mmol). Light yellow solid (0.14 g, 77%). HPLC purity: 99.86%; ¹H NMR (600 MHz, CDCl₃): δ 8.28–8.13 (m, 2H), 8.06–7.93 (m, 2H), 7.22–7.12 (m, 2H), 6.99 (t, J = 8.7 Hz, 1H), 4.83–4.67 (m, 2H), 3.61–3.45 (m, 2H), 3.26–2.98 (m, 2H), 2.10–1.66 (m, 4H), 1.52–1.35 ppm (m, 6H); ¹³C NMR (151 MHz, CDCl₃) 199.40, 166.20–164.92 (d, J = 193.3 Hz), 164.76, 158.31, 140.43, 131.48, 130.45–130.39 (d, J = 9.1 Hz, 2C), 127.14, 125.78, 123.75, 115.50, 111.90–111.72 (d, J = 27.2 Hz, 2C), 71.76, 46.04 (2C), 40.86, 27.97 (2C), 21.36 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₂₄O₅N₂F: 415.1669, found:415.1658.

(4-(2-Fluoro-4-(trifluoromethyl)benzoyl)piperidin-1-yl)(2-isopropoxy-5-nitrophenyl)methanone (23c). 23c was prepared from 10a (0.1 g, 0.44 mmol) and 21c (0.14 g, 0.53 mmol). Yellow solid (0.12 g, 57%). HPLC purity: 99.20%; ¹H NMR (400 MHz, CDCl₃): δ 8.23 (dd, J = 9.2, 2.8 Hz, 1H), 8.15 (dd, J = 16.1, 2.8 Hz, 1H), 7.94–7.84 (m, 1H), 7.52 (d, J = 8.1 Hz, 1H), 7.43 (dd, J = 10.8, 6.4 Hz, 1H), 6.97 (dd, J = 9.2, 5.6 Hz, 1H), 4.80–4.61 (m, 2H), 3.57–3.45 (m, 1H), 3.45–3.33 (m, 1H), 3.22–2.94 (m, 2H), 2.12–2.02 (m, 1H), 1.92–1.81 (m, 1H), 1.79–1.59 (m, 2H), 1.48–1.33 ppm (m, 6H); ¹³C NMR (101 MHz, CDCl₃) 199.29, 165.23, 161.82–159.29 (d, J = 255.5 Hz), 158.71, 140.99, 136.50–135.50 (m), 131.96–131.78 (m), 127.62, 126.21, 124.48–124.22 (d, J = 26.3 Hz), 123.95, 121.71, 128.10–121.23 (m), 114.50, 112.41–112.23 (m), 72.19, 47.92, 46.46 (2C), 27.88 (2C), 21.94 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₃O₅N₂F₄: 483.1543, found: 483.1533.

(1-(2-Isopropoxy-5-nitrobenzoyl)piperidin-4-yl)(4-(trifluoromethyl)phenyl)methanone (23d). 23d was prepared from 10a (0.1 g, 0.44 mmol) and 21b (0.14 g, 0.53 mmol). Off-white solid (0.14 g, 69%). HPLC purity: 98.61%; ¹H NMR (600 MHz, CDCl₃): δ 8.28–8.14 (m, 2H), 8.11–8.00 (m, 2H), 7.81–7.70 (m, 2H), 6.99 (t, J = 8.6 Hz, 1H), 4.82–4.67 (m, 2H), 3.62–3.51 (m, 2H), 3.27–3.01 (m, 2H), 2.12–1.69 (m, 4H), 1.52–1.36 ppm (m, 6H); ¹³C NMR (151 MHz, CDCl₃) 200.08, 164.81, 158.29, 140.44, 137.83, 134.24–134.03 (m), 128.11 (2C), 127.03, 126.78–122.09 (m), 125.95–125.82 (m, 2C), 125.46, 123.75, 111.92, 71.78, 45.95, 42.92 (2C), 27.76 (2C), 21.37 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₄O₅N₂F₃: 465.1637, found: 465.1625.

(1-(2-(Cyclopropylmethoxy)-5-nitrobenzoyl)piperidin-4-yl)(2,4-difluorophenyl)methanone (23e). 23e was prepared from 10c (0.1 g, 0.42 mmol) and 21d (0.13 g, 0.51 mmol). Light yellow solid (0.15 g, 80%). HPLC purity: 98.09%; ¹H NMR (400 MHz, CDCl₃): δ 8.36–8.14 (m, 2H), 7.94–7.78 (m, 1H), 7.04–6.84 (m, 3H), 4.87–4.59 (m, 1H), 4.05–3.85 (m, 2H), 3.53 (t, J = 13.7 Hz, 1H), 3.44–3.30 (m, 1H), 3.27–2.91 (m, 2H), 2.14–1.91 (m, 2H), 1.79–1.19 (m, 3H), 0.78–0.61 (m, 2H), 0.46–0.26 ppm (m, 2H); ¹³C NMR (101 MHz, CDCl₃) 198.42, 167.13–164.57 (d, J = 258.6 Hz), 165.02, 163.19–160.75 (d, J = 246 Hz), 159.64, 141.27, 132.97, 127.10, 126.30, 124.16, 121.48–121.36 (d, J = 12.1 Hz), 112.66–112.45 (d, J = 21.2 Hz), 111.49, 104.79 (m), 74.34, 47.82, 46.78 (2C), 28.00 (2C), 9.86, 3.93 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₃O₅N₂F₂: 445.1575, found: 445.1568.

(1-(2-(Cyclopropylmethoxy)-5-nitrobenzoyl)piperidin-4-yl)(4-fluorophenyl)methanone (23f). 23f was prepared from 10c (0.1 g, 0.42 mmol) and 21a (0.10 g, 0.51 mmol). Light yellow solid (0.13 g, 77%). HPLC purity: 99.71%; ¹H NMR (600 MHz, CDCl₃): δ 8.28–8.14 (m, 2H), 8.03–7.94 (m, 2H), 7.20–7.13 (m, 2H), 6.96 (d, J = 8.0 Hz, 1H), 4.86–4.65 (m, 1H), 4.07–3.92 (m, 2H), 3.63–3.45 (m, 2H), 3.29–2.98 (m, 2H), 2.10–1.99 (m, 2H), 1.87–1.76 (m, 2H), 1.40–1.20 (m, 1H), 0.81–0.60 (m, 2H), 0.46–0.31 ppm (m, 2H); ¹³C NMR (151 MHz, CDCl₃) 199.33, 166.16–164.47 (d, J = 255.2 Hz), 164.59, 159.21, 140.74, 131.51, 130.42–130.36 (d, J = 9.1 Hz, 2C), 126.54, 125.91, 123.74, 115.56–115.42 (d, J = 21.1 Hz, 2C), 111.02, 73.94, 46.23, 42.82 (2C), 28.10 (2C), 9.40, 3.52 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₄O₅N₂F: 427.1669, found: 427.1665.

(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(2-(isopropylamino)-5-nitrophenyl)methanone (23g). 23g was prepared from 13a (0.1 g, 0.45 mmol) and 21d (0.14 g, 0.54 mmol). Yellow solid (0.14 g, 73%). HPLC purity: 97.30%; ¹H NMR (600 MHz, CDCl₃): δ 8.14 (dd, J = 9.3, 2.6 Hz, 1H), 8.03 (d, J = 2.7 Hz, 1H), 7.89 (td, J = 8.6, 6.5 Hz, 1H), 7.00 (ddd, J = 9.2, 7.6, 2.4 Hz, 1H), 6.90 (ddd, J = 11.1, 8.6, 2.4 Hz, 1H), 6.66 (d, J = 9.3 Hz, 1H), 6.08 (d, J = 7.3 Hz, 1H), 4.25 (s, 1H), 3.79–3.69 (m, J = 6.5 Hz, 1H), 3.47–3.37 (m, 1H), 3.18 (t, J = 12.9 Hz, 2H), 2.07–1.95 (m, 2H), 1.83–1.62 (m, 3H), 1.29 ppm (d, J = 6.4 Hz, 6H); ¹³C NMR (151 MHz, CDCl₃) 197.87–197.83 (d, J = 6.04 Hz), 167.80, 166.28–164.49 (dd, J = 12.08, 258.2 Hz), 162.40–160.63 (dd, J = 12.08, 255.2 Hz), 150.93, 135.29, 132.65 (m), 127.08, 124.77, 120.84–120.77 (d J = 10.57 Hz), 115.96, 112.25–112.09 (d, J = 21.14 Hz), 109.94, 104.48–104.12 (t, J = 27.18 Hz), 47.13–47.08 (d, J = 7.55 Hz), 43.80 (3C), 27.74 (2C), 21.95 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₂₄O₄N₃F₂: 432.1735, found: 432.1722.

(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(5-nitro-2-(piperidin-1-yl)phenyl)methanone (23h). 23h was prepared from 13b (0.1 g, 0.40 mmol) and 21d (0.12 g, 0.48 mmol). Yellow solid (0.12 g, 66%). HPLC purity: 98.99%; ¹H NMR (600 MHz, CDCl₃): δ 8.20–8.05 (m, 2H), 7.95–7.83 (m, 1H), 7.05–6.85 (m, 3H), 4.85–4.56 (m, 1H), 3.56–3.46 (m, 1H), 3.44–3.28 (m, 3H), 3.26–2.91 (m, 4H), 2.14–2.05 (m, 1H), 1.96–1.51 ppm (m, 9H); ¹³C NMR (151 MHz, CDCl₃) 198.22, 167.72, 166.23–164.61 (d, J = 244.6 Hz), 162.52–160.60 (d, J = 255.2 Hz), 154.24, 140.03, 132.78–132.52 (m), 127.54, 125.53, 125.01, 124.77 (m), 120.89, 116.58, 112.27–112.07 (d, J = 7.55 Hz), 104.58–104.17 (m), 51.85 (2C), 47.41, 46.03 (2C), 27.36 (2C), 25.27 (2C), 23.43. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₂₆O₄N₃F₂: 458.1891, found: 458.1881.

4-(Cyclopropylmethoxy)-3-(4-(2,4-difluorobenzoyl)piperidine-1-carbonyl)-N-methylbenzenesulfonamide (23i). 23i was prepared from 10g (0.1 g, 0.35 mmol) and 21d (0.11 g, 0.42 mmol). White solid (0.13 g, 80%). HPLC purity: 95.16%; ¹H NMR (400 MHz, CDCl₃): δ 7.94–7.79 (m, 2H), 7.78–7.69 (m, 1H), 7.07–6.75 (m, 3H), 5.05–4.88 (m, 1H), 4.85–4.52 (m, 1H), 4.05–3.77 (m, 2H), 3.55 (d, J = 13.2 Hz, 1H), 3.37 (tt, J = 10.7, 3.8 Hz, 1H), 3.27–2.87 (m, 2H), 2.61 (d, J = 5.3 Hz, 3H), 2.06 (d, J = 13.5 Hz, 2H), 1.75 (h, J = 9.8, 8.6 Hz, 1H), 1.39–1.19 (m, 2H), 0.76–0.58 (m, 2H), 0.48–0.23 ppm (m, 2H); ¹³C NMR (101 MHz, CDCl₃) 198.58, 167.12–164.44 (d, J = 257.5 Hz), 166.14, 163.20–160.70 (d, J = 252.5 Hz), 157.94, 133.09 (m), 130.96, 130.08, 127.37, 127.05, 121.44, 112.66–112.45 (d, J = 21.2 Hz), 111.67, 105.10–104.57 (t, J = 26.8 Hz), 73.86, 47.94, 46.90 (2C), 29.32 (2C), 28.21, 9.95, 3.14 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₂₇O₅N₂F₂S: 493.1609, found: 493.1595.

3-(4-(2,4-Difluorobenzoyl)piperidine-1-carbonyl)-N-methyl-4-(2,2,3,3,3-pentafluoropropoxy)benzenesulfonamide (23j). 23j was prepared from 18c (0.1 g, 0.27 mmol) and 21d (0.086 g, 0.33 mmol). Off-white solid (0.13 g, 71%). HPLC purity: 95.93%; ¹H NMR (600 MHz, CDCl₃): δ 7.95–7.74 (m, 3H), 7.11–7.03 (m, 1H), 7.03–6.96 (m, 1H), 6.95–6.83 (m, 1H), 5.17–5.00 (m, 1H), 4.74–4.47 (m, 3H), 3.48 (dt, J = 13.9, 4.1 Hz, 1H), 3.37 (dtd, J = 14.4, 10.5, 9.9, 4.7 Hz, 1H), 3.20–2.98 (m, 2H), 2.63 (dd, J = 5.4, 2.6 Hz, 3H), 2.07 (d, J = 13.7 Hz, 1H), 1.94–1.56 ppm (m, 3H); ¹³C NMR (151 MHz, CDCl₃) 197.89–197.76 (d, J = 19.6 Hz), 166.25–164.54 (d, J = 258.2 Hz), 164.46, 162.35–160.66 (d, J = 255.2 Hz), 155.35, 133.48, 132.67 (m), 129.68, 127.28, 127.00, 120.87, 116.98 (m), 112.20, 112.09 (m), 111.65, 104.43–104.07 (t, J = 27.2 Hz), 65.01–64.64 (m), 47.00, 45.60 (2C), 28.79 (2C), 27.35. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₂O₅N₂F₇S: 571.1138, found: 571.1121.

3-(4-(2,4-Difluorobenzoyl)piperidine-1-carbonyl)-4-(isopropylamino)-N-methylbenzenesulfonamide (23k). 23k was prepared from 16c (0.1 g, 0.37 mmol) and 21d (0.11 g, 0.44 mmol). White solid (0.11 g, 63%). HPLC purity: 98.07%; ¹H NMR (600 MHz, CDCl₃): δ 7.86 (td, J = 8.6, 6.5 Hz, 1H), 7.67 (dd, J = 8.8, 2.3 Hz, 1H), 7.55 (d, J = 2.3 Hz, 1H), 6.98 (ddd, J = 9.5, 7.5, 2.4 Hz, 1H), 6.88 (ddd, J = 11.1, 8.5, 2.4 Hz, 1H), 6.70 (d, J = 8.9 Hz, 1H), 5.49 (d, J = 7.3 Hz, 1H), 4.83 (q, J = 5.4 Hz, 1H), 3.68 (h, J = 6.4 Hz, 1H), 3.43–3.33 (m, 1H), 3.13 (t, J = 12.7 Hz, 2H), 2.57 (d, J = 5.4 Hz, 3H), 2.07–1.89 (m, 3H), 1.77–1.60 (m, 2H), 1.24 ppm (d, J = 6.4 Hz, 6H); ¹³C NMR (151 MHz, CDCl₃) 198.02–197.99 (d, J = 4.53 Hz), 168.32, 166.21–164.51 (d, J = 256.7 Hz), 162.37–160.67 (d, J = 256.7 Hz), 148.85, 132.64–132.55 (m), 129.96, 127.64, 123.00, 120.89, 117.13, 112.19–112.03 (d, J = 21.14 Hz), 110.58, 104.47–104.11 (t, J = 27.18 Hz), 47.11–47.06 (d, J = 7.55 Hz), 43.44 (2C), 38.10, 28.75 (2C), 27.71, 22.01 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₈O₄N₃F₂S: 480.1769, found: 480.1757.

3-(4-(2,4-Difluorobenzoyl)piperidine-1-carbonyl)-N-methyl-4-(piperidin-1-yl)benzenesulfonamide (23l). 23l was prepared from 16d (0.1 g, 0.34 mmol) and 21d (0.10 g, 0.40 mmol). Off-white solid (0.12 g, 69%). HPLC purity: 97.67%; ¹H NMR (400 MHz, CDCl₃): δ 7.98–7.82 (m, 1H), 7.81–7.63 (m, 2H), 7.09–6.96 (m, 2H), 6.89 (q, J = 9.6 Hz, 1H), 4.93–4.54 (m, 2H), 3.55–3.15 (m, 4H), 3.13–2.85 (m, 4H), 2.63 (d, J = 5.4 Hz, 3H), 2.18–1.65 (m, 8H), 1.29 ppm (d, J = 20.1 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) 198.84, 168.81, 167.02–164.5 (d, J = 254.5 Hz), 153.51, 133.16–132.95 (m), 130.71, 130.07, 129.62, 129.43, 127.98, 121.36, 117.77, 112.69–112.52 (d, J = 17.2 Hz), 104.88–104.62 (m), 52.76 (2C), 47.82, 46.49 (2C), 29.68 (2C), 27.82, 26.35 (2C), 24.00. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₅H₃₀O₄N₃F₂S: 506.1925, found: 506.1911.

3-(4-(2,4-Difluorobenzoyl)piperidine-1-carbonyl)-N-methyl-4-morpholinobenzenesulfonamide (23m). 23m was prepared from 16e (0.1 g, 0.33 mmol) and 21d (0.10 g, 0.40 mmol). White solid (0.13 g, 77%). HPLC purity: 98.45%; ¹H NMR (400 MHz, CDCl₃): δ 7.96–7.85 (m, 1H), 7.82 (dd, J = 8.6, 2.3 Hz, 1H), 7.73 (dd, J = 21.7, 2.3 Hz, 1H), 7.11–6.97 (m, 2H), 6.95–6.85 (m, 1H), 4.88 (p, J = 5.4 Hz, 1H), 4.80–4.57 (m, 1H), 3.94–3.73 (m, 4H), 3.50–3.18 (m, 4H), 3.13–2.91 (m, 4H), 2.63 (d, J = 5.3 Hz, 3H), 2.16–1.90 (m, 2H), 1.80–1.48 ppm (m, 2H); ¹³C NMR (101 MHz, CDCl₃) 198.60–198.20 (d, J = 40.4 Hz), 168.38, 167.26–164.6 (d, J = 268.6 Hz), 163.45–160.78 (d, J = 268.7 Hz), 152.42, 133.24–133.02 (m), 132.41, 130.95, 129.51, 127.70, 121.38–121.25 (d, J = 13.1 Hz), 117.98, 112.78–112.57 (d, J = 21.2 Hz), 104.88–104.60 (m), 66.70 (2C), 51.88 (2C), 47.70, 46.55 (2C), 29.35 (2C), 28.04. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₂₈O₅N₃F₂S: 508.1718, found: 508.1704.

3-(4-(2,4-Difluorobenzoyl)piperidine-1-carbonyl)-4-(isopropylthio)-N-methylbenzenesulfonamide (23n). 23n was prepared from 18d (0.1 g, 0.35 mmol) and 21d (0.11 g, 0.42 mmol). Off-white solid (0.10 g, 58%). HPLC purity: 96.27%; ¹H NMR (400 MHz, CDCl₃): δ 7.88 (q, J = 8.0 Hz, 1H), 7.81–7.74 (m, 1H), 7.66 (s, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.06–6.95 (m, 1H), 6.95–6.80 (m, 1H), 5.11–4.95 (m, 1H), 4.67 (s, 1H), 3.62 (hept, J = 6.8 Hz, 1H), 3.52–3.29 (m, 2H), 3.27–3.03 (m, 2H), 2.64 (d, J = 5.2 Hz, 3H), 2.16–1.57 (m, 4H), 1.37 ppm (d, J = 6.6 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) 198.45, 167.16–164.61 (d, J = 257.5 Hz), 167.03, 163.18–160.75 (d, J = 245.3 Hz), 140.10, 137.76, 136.10, 133.11, 128.85, 127.64, 125.26, 121.43–121.26 (d, J = 13.1 Hz), 112.72–112.51 (d, J = 21.2 Hz), 105.03–104.49 (t, J = 27.3 Hz), 47.63, 46.75 (2C), 37.24, 29.34 (2C), 27.62, 22.69 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₇O₄N₂F₂S₂: 497.1380, found: 497.1367.

(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(2-isopropoxy-5-(methylsulfonyl)phenyl)methanone (23o). 23o was prepared from 10d (0.1 g, 0.38 mmol) and 21d (0.12 g, 0.46 mmol). White solid (0.14 g, 83%). HPLC purity: 98.06%; ¹H NMR (600 MHz, CDCl₃): δ 7.91–7.82 (m, 2H), 7.82–7.71 (m, 1H), 7.02 (t, J = 8.7 Hz, 1H), 6.98 (t, J = 8.3 Hz, 1H), 6.92–6.83 (m, 1H), 4.75–4.57 (m, 2H), 3.52–3.43 (m, 1H), 3.42–3.31 (m, 1H), 3.18–3.07 (m, 1H), 3.04 (d, J = 3.5 Hz, 3H), 3.00–2.91 (m, 1H), 2.08–1.99 (m, 1H), 1.91–1.52 (m, 3H), 1.44–1.31 ppm (m, 6H); ¹³C NMR (151 MHz, CDCl₃) 198.21–197.97 (d, J = 36.2 Hz), 166.25–164.54 (d, J = 258.2 Hz), 165.57, 162.38–160.77 (d, J = 243.1 Hz), 157.35, 132.61–132.41 (m), 131.79–131.60 (m), 129.77, 129.61, 127.47, 120.88–120.79 (d, J = 13.60 Hz), 112.50, 112.23–112.01 (m), 104.53–104.18 (t, J = 26.43 Hz), 71.26, 47.11, 46.22, 44.17 (2C), 27.62 (2C), 21.11 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₆O₅NF₂S: 466.1500, found: 466.1494.

(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(2-isobutoxy-5-(methylsulfonyl)phenyl)methanone (23p). 23p was prepared from 10e (0.1 g, 0.37 mmol) and 21d (0.11 g, 0.44 mmol). White solid (0.13 g, 73%). HPLC purity: 98.82%; ¹H NMR (400 MHz, CDCl₃): δ 7.97–7.74 (m, 3H), 7.09–6.96 (m, 2H), 6.95–6.82 (m, 1H), 4.78–4.62 (m, 1H), 4.02–3.73 (m, 2H), 3.57–3.28 (m, 2H), 3.22–3.08 (m, 1H), 3.04 (d, J = 2.8 Hz, 3H), 2.25–2.11 (m, 1H), 2.10–1.66 (m, 5H), 1.04 ppm (d, J = 6.6 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) 198.52–198.27 (d, J = 25.3 Hz), 167.15–164.47 (d, J = 270.6 Hz), 165.71, 163.21–160.67 (d, J = 256.5 Hz), 159.15, 133.23–132.93 (m), 132.79, 130.35, 130.20, 127.27, 121.48–121.33(d, J = 15.1 Hz), 112.77–112.47 (m), 112.26, 105.09–104.56 (m), 75.52, 47.60 (m), 46.66, 44.76 (2C), 28.18 (2C), 27.71, 19.09 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₂₈O₅NF₂S: 480.1656, found: 480.1644.

(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(5-(methylsulfonyl)-2-(2,2,3,3,3-pentafluoropropoxy)phenyl)methanone (23q). 23q was prepared from 18b (0.2 g, 0.57 mmol) and 21d (0.18 g, 0.69 mmol). White solid (0.26 g, 84%). HPLC purity: 98.78%; ¹H NMR (400 MHz, CDCl₃): δ 7.98 (dt, J = 8.7, 2.6 Hz, 1H), 7.93–7.83 (m, 2H), 7.09 (t, J = 9.0 Hz, 1H), 7.04–6.96 (m, 1H), 6.94–6.84 (m, 1H), 4.74–4.45 (m, 3H), 3.52–3.41 (m, 1H), 3.42–3.29 (m, 1H), 3.21–2.96 (m, 2H), 3.06 (d, J = 3.2 Hz, 3H), 2.12–2.00 (m, 1H), 1.90–1.80 (m, 1H), 1.79–1.62 ppm (m, 2H); ¹³C NMR (101 MHz, CDCl₃) 198.25, 167.10–164.65 (d, J = 247.5 Hz), 164.42, 163.27–160.75 (d, J = 254.5 Hz), 156.37, 135.49, 133.12 (m), 130.40, 128.23, 127.92, 121.42–121.29 (d, J = 13.1 Hz), 121.42 (m), 112.77(m), 112.99, 112.43, 104.71–104.46 (t, J = 12.6 Hz), 65.38 (m), 47.50 (m), 46.63, 44.65 (2C), 26.65 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₁O₅NF₇S: 556.1029, found: 556.1013.

(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(5-(methylsulfonyl)-2-((1,1,1-trifluoropropan-2-yl)oxy)phenyl)methanone (23r). 23r was prepared from 18a (0.10 g, 0.32 mmol) and 21d (0.10 g, 0.38 mmol). White solid (0.13 g, 79%). HPLC purity: 98.73%; ¹H NMR (600 MHz, CDCl₃): δ 7.99–7.94 (m, 1H), 7.94–7.83 (m, 2H), 7.18–7.07 (m, 1H), 7.00 (td, J = 9.2, 8.5, 2.5 Hz, 1H), 6.93–6.84 (m, 1H), 4.90–4.78 (m, 1H), 4.75–4.59 (m, 1H), 3.52–3.44 (m, 1H), 3.42–3.32 (m, 1H), 3.22–3.00 (m, 2H), 3.07 (d, J = 2.8 Hz, 3H), 2.07 (dd, J = 13.7, 3.8 Hz, 1H), 1.91–1.82 (m, 1H), 1.80–1.49 ppm (m, 5H); ¹³C NMR (151 MHz, CDCl₃) 197.82, 166.10–164.19 (d, J = 286.5 Hz), 164.37, 162.23–160.54 (d, J = 255.2 Hz), 156.02–155.70 (m), 134.55, 132.71–132.61 (m), 129.73, 129.57, 127.35, 124.47 (m), 120.88–120.80 (d, J = 12.08 Hz), 114.21, 112.21–112.06 (d, J = 22.65 Hz), 104.42–104.06 (t, J = 27.18 Hz), 73.37–72.47 (m), 47.05, 46.31, 44.16 (2C), 27.31 (2C), 13.33–13.21 (m). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₃O₅NF₅S: 520.1217, found: 520.1205.

(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(2-(isopropylamino)-5-(methylsulfonyl)phenyl)methanone (23s). 23s was prepared from 16a (0.1 g, 0.39 mmol) and 21d (0.12 g, 0.47 mmol). Off-white solid (0.11 g, 61%). HPLC purity: 98.63%; ¹H NMR (400 MHz, CDCl₃): δ 7.93–7.82 (m, 1H), 7.74 (dd, J = 8.9, 2.1 Hz, 1H), 7.64–7.58 (m, 1H), 7.04–6.94 (m, 1H), 6.93–6.83 (m, 1H), 6.74 (d, J = 8.9 Hz, 1H), 5.65 (d, J = 7.3 Hz, 1H), 3.71 (h, J = 6.6 Hz, 1H), 3.46–3.34 (m, 1H), 3.15 (t, J = 12.6 Hz, 2H), 2.99 (d, J = 0.9 Hz, 3H), 2.00 (d, J = 13.9 Hz, 2H), 1.73 (q, J = 12.7, 12.1 Hz, 3H), 1.27 ppm (d, J = 6.3 Hz, 6H); ¹³C NMR (101 MHz, CDCl₃) 198.34–198.30 (d, J = 4.04 Hz), 168.59, 167.16–164.47 (d, J = 258.5 Hz), 163.29–160.62 (d, J = 256.5 Hz), 150.11, 133.11–133.04 (m), 130.47, 128.17, 125.42, 121.37–121.26 (d, J = 11.1 Hz), 117.64, 112.70–112.49 (d, J = 21.2 Hz), 111.14, 105.01–104.48 (t, J = 26.7 Hz), 47.59–47.52 (d, J = 7.07 Hz), 45.00, 43.97 (2C), 38.56, 28.19 (2C), 22.44 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₇O₄N₂F₂S: 465.1660, found: 465.1651.

(4-(2,4-Difluorobenzoyl)piperidin-1-yl)(5-(methylsulfonyl)-2-(piperidin-1-yl)phenyl)methanone (23t). 23t was prepared from 16b (0.1 g, 0.35 mmol) and 21d (0.11 g, 0.42 mmol). Off-white solid (0.12 g, 72%). HPLC purity: 98.28%; ¹H NMR (400 MHz, CDCl₃): δ 7.92–7.66 (m, 3H), 7.09–6.93 (m, 2H), 6.87 (t, J = 10.0 Hz, 1H), 4.62 (d, J = 12.6 Hz, 1H), 3.44–2.87 (m, 8H), 3.01 (s, 3H) 2.10–1.99 (m, 1H), 1.96–1.55 ppm (m, 9H); ¹³C NMR (101 MHz, CDCl₃) 198.73, 168.48, 167.16–164.48 (d, J = 257.6 Hz), 163.15–160.65 (d, J = 252.5 Hz), 154.10, 133.09, 132.11 (m), 129.54, 128.43, 121.43–121.27 (d, J = 13.1 Hz), 117.94 (2C), 112.73–112.52 (d, J = 21.2 Hz), 104.80 (m), 52.54 (2C), 47.81, 46.49 (2C), 38.61, 28.33 (2C), 26.21 (2C), 23.95. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₅H₂₉O₄N₂F₂S: 491.1816, found: 491.1806.

(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)(5-(methylsulfonyl)-2-(2,2,3,3,3-pentafluoropropoxy)phenyl)methanone (24a). 24a was prepared from 18b (0.1 g; 0.29 mmol) and 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride (0.09 g, 0.34 mmol, commercial available). White solid (0.11 g, 71%). HPLC purity: 96.45%; ¹H NMR (600 MHz, CDCl₃): δ 8.03–7.86 (m, 2H), 7.70–7.61 (m, 1H), 7.27 (dq, J = 8.6, 2.1 Hz, 1H), 7.18–7.04 (m, 2H), 4.81–4.50 (m, 3H), 3.58 (td, J = 13.3, 3.7 Hz, 1H), 3.38 (td, J = 10.7, 5.0 Hz, 1H), 3.30–3.10 (m, 2H), 3.08 (dd, J = 4.5, 1.5 Hz, 3H), 2.29–1.97 ppm (m, 4H). ¹³C NMR (151 MHz, CDCl₃) 164.55–162.89 (d, J = 250.6 Hz), 164.20, 163.46–163.37 (d, J = 13.6 Hz), 159.42 (m), 155.86, 134.82, 130.07, 127.82, 127.43, 121.73–121.67 (t, J = 4.53 Hz), 118.60 (m), 116.48, 112.60, 112.17 (m), 111.92, 97.15–96.98 (d, J = 25.67 Hz), 65.01 (m), 45.80 (2C), 44.07, 33.56, 29.81 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₁O₅N₂F₆S: 551.1075, found: 551.1060.

(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)(2-isobutoxy-5-(methylsulfonyl)phenyl)methanone (24b). 24b was prepared from 10f (0.1 g, 0.37 mmol) and 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride (0.11 g, 0.44 mmol). White solid (0.13 g, 76%). HPLC purity: 96.76%; ¹H NMR (400 MHz, CDCl₃): δ 7.97–7.91 (m, 1H), 7.86 (dd, J = 32.0, 2.4 Hz, 1H), 7.70–7.59 (m, 1H), 7.28–7.24 (m, 1H), 7.13–7.02 (m, 2H), 4.80 (dd, J = 33.0, 13.5 Hz, 1H), 3.99–3.74 (m, 2H), 3.67–3.54 (m, 1H), 3.43–3.08 (m, 3H), 3.05 (d, J = 3.6 Hz, 3H), 2.32–1.94 (m, 5H), 1.11–0.96 ppm (m, 6H); ¹³C NMR (101 MHz, CDCl₃) 165.82, 165.42–162.92 (d, J = 252.5 Hz), 163.88, 159.99, 159.13, 132.85, 130.40, 127.62, 127.32, 122.05 (m), 117.14, 112.60 (m), 112.15, 97.68–97.41 (d, J = 27.3 Hz), 75.54, 46.82 (2C), 44.75, 34.35, 30.08 (2C), 28.18, 19.09 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₂₈O₅N₂FS: 475.1703, found: 475.1690.

(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)(5-(methylsulfonyl)-2-((1,1,1-trifluoropropan-2-yl)oxy)phenyl)methanone (24c). 24c was prepared from 18a (0.1 g, 0.32 mmol) and 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride (0.10 g, 0.38 mmol). White solid (0.13 g, 81%). HPLC purity: 98.52%; ¹H NMR (400 MHz, CDCl₃): δ 7.98 (d, J = 8.4 Hz, 1H), 7.96–7.84 (m, 1H), 7.65 (td, J = 8.2, 4.7 Hz, 1H), 7.28–7.24 (m, 1H), 7.22–7.04 (m, 2H), 4.95–4.69 (m, 2H), 3.63–3.53 (m, 1H), 3.44–3.12 (m, 3H), 3.08 (s, 3H), 2.25 (dd, J = 13.6, 4.0 Hz, 1H), 2.14–1.89 (m, 3H), 1.66–1.52 ppm (m, 3H); ¹³C NMR (101 MHz, CDCl₃) 165.53–163.03 (d, J = 252.5 Hz), 165.02, 164.08–163.94 (d, J = 14.1 Hz), 160.02, 156.59, 135.09, 130.43, 128.52, 127.95, 122.34 (m), 122.32–122.21 (d, J = 11.1 Hz), 117.08, 113.64, 112.82 (m), 97.82–97.55 (d, J = 27.3 Hz), 73.64 (m), 47.12 (2C), 44.76, 34.25, 30.17 (2C), 13.89. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₃O₅N₂F₄S: 515.1264, found: 515.1251.

(2-(Cyclopentyloxy)-5-nitrophenyl)(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)methanone (24d). 24d was prepared from 10b (0.1 g, 0.40 mmol) and 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride (0.12 g, 0.48 mmol). Light yellow solid (0.12 g, 65%). HPLC purity: 97.55%; ¹H NMR (400 MHz, CDCl₃): δ 8.29–8.12 (m, 2H), 7.69–7.58 (m, 1H), 7.28 (d, J = 2.1 Hz, 1H), 7.09 (tt, J = 8.8, 2.1 Hz, 1H), 7.04–6.94 (m, 1H), 4.97–4.86 (m, 1H), 4.84–4.70 (m, 1H), 3.68–3.51 (m, 1H), 3.43–3.04 (m, 3H), 2.33–2.15 (m, 1H), 2.12–1.50 ppm (m, 11H); ¹³C NMR (101 MHz, CDCl₃) 165.43–162.95 (d, J = 250.5 Hz), 165.26, 163.90, 160.02, 159.05, 141.03, 127.51, 126.18, 124.44, 124.01, 121.93 (m), 117.13, 112.80–112.49 (d, J = 31.3 Hz), 97.72–97.45 (d, J = 27.3 Hz), 81.64, 46.84 (2C), 33.97, 33.01 (2C), 30.29 (2C), 23.96 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₂₅O₅N₃F: 454.1778, found 454.1770.

(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)(5-nitro-2-(piperidin-1-yl)phenyl)methanone (24e). 24e was prepared from 13b (0.1 g, 0.40 mmol) and 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride (0.12 g, 0.48 mmol). Yellow solid (0.12 g, 67%). HPLC purity: 97.32%; ¹H NMR (600 MHz, CDCl₃): δ 8.24–8.08 (m, 2H), 7.70–7.60 (m, 1H), 7.31–7.24 (m, 1H), 7.10 (tdd, J = 8.8, 4.8, 2.1 Hz, 1H), 6.97 (dd, J = 11.2, 9.1 Hz, 1H), 4.95–4.59 (m, 1H), 3.67–3.53 (m, 1H), 3.43–3.25 (m, 4H), 3.23–3.02 (m, 3H), 2.35–2.21 (m, 1H), 2.19–1.82 (m, 3H), 1.80–1.59 ppm (m, 6H); ¹³C NMR (151 MHz, CDCl₃) 167.81, 164.60–162.94 (d, J = 250.7 Hz), 163.54–163.34 (d, J = 30.2 Hz), 159.63, 154.25, 140.06, 127.44, 125.62, 125.08, 121.72–121.45 (d, J = 40.7 Hz), 116.64, 116.74, 112.33–112.16 (d, J = 25.6 Hz), 97.22–97.04 (d, J = 27.2 Hz), 51.86 (2C), 46.30 (2C), 33.98, 29.68 (2C), 25.71 (2C), 23.42. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₂₆O₄N₄F: 453.1938, found: 453.1927.

3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-carbonyl)-4-isopropoxy-N-methylbenzenesulfonamide (24f). 24f was prepared from 10h (0.1 g, 0.37 mmol) and 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride (0.11 g, 0.44 mmol). Off-white solid (0.10 g, 60%). HPLC purity: 97.72%; ¹H NMR (400 MHz, CDCl₃): δ 7.85–7.68 (m, 2H), 7.68–7.59 (m, 1H), 7.25–7.18 (m, 1H), 7.06 (td, J = 8.8, 2.1 Hz, 1H), 6.98 (dd, J = 8.8, 2.9 Hz, 1H), 4.82–4.60 (m, 2H), 3.58 (d, J = 13.8 Hz, 1H), 3.42–3.01 (m, 3H), 2.58 (s, 3H), 2.27–1.82 (m, 5H), 1.42–1.31 ppm (m, 6H); ¹³C NMR (101 MHz, CDCl₃) 166.07, 165.02–162.52 (d, J = 252.5 Hz), 163.40–163.27 (d, J = 13.1 Hz), 159.77, 156.41, 130.38, 129.83, 129.61, 127.25, 126.96, 126.53, 121.96–121.68 (d, J = 28.3 Hz), 116.67–116.43 (d, J = 24.2 Hz), 112.19 (m), 71.11, 46.56 (2C), 33.77, 30.02, 28.78 (2C), 21.48 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₃H₂₇O₅N₃FS: 476.1655, found: 476.1645.

3-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidine-1-carbonyl)-4-isobutoxy-N-methylbenzenesulfonamide (24g). 24g was prepared from 10f (0.1 g, 0.35 mmol) and 6-fluoro-3-(4-piperidinyl)-1,2-benzisoxazole hydrochloride (0.11 g, 0.42 mmol). White solid (0.13 g, 67%). HPLC purity: 96.83%; ¹H NMR (500 MHz, CDCl₃): δ 7.90–7.71 (m, 2H), 7.71–7.60 (m, 1H), 7.27–7.23 (m, 1H), 7.08 (tdd, J = 8.8, 3.9, 2.1 Hz, 1H), 7.01 (dd, J = 8.8, 5.2 Hz, 1H), 4.99–4.89 (m, 1H), 4.87–4.70 (m, 1H), 3.96–3.73 (m, 2H), 3.67–3.57 (m, 1H), 3.41–3.31 (m, 1H), 3.30–3.04 (m, 2H), 2.62 (d, J = 6.6 Hz, 3H), 2.31–1.88 (m, 5H), 1.07–1.00 ppm (m, 6H); ¹³C NMR (126 MHz, CDCl₃) 166.25, 165.19–163.20 (d, J = 250.7 Hz), 163.87, 160.09, 158.27, 131.14, 130.35, 127.36, 126.92, 122.39–122.10 (d, J = 36.5 Hz), 117.18, 112.70 (m), 112.00–111.71 (d, J = 36.5 Hz), 97.68–97.46 (d, J = 27.7 Hz), 75.39, 46.90 (2C), 34.39, 30.36, 29.34 (2C), 28.25, 19.12 (2C). HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₂₉O₅N₃FS: 490.1812, found: 490.1798.

[³H] glycine uptake assay (GlyT1)

Rat glioma C6 cells that stably expressed GlyT1 were plated into 24-well culture plates (1 × 10⁶ per well). After 18 h, the culture medium was discarded and HEPES buffer solution (160 μL) was added. Thereafter, tested compounds (20 μL) and ³H-glycine (20 μL; final concentration ranges: 10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, and 10⁻⁵ M) were added to a total volume of 200 μL. In the total uptake wells, the tested compounds were replaced with Hank's balanced salt solution (HBSS; 20 μL). NFPS²⁴ (10 μM) was used to determine nonspecific uptake. After a 30 min incubation, HBSS was discarded and the plate was washed twice with phosphate-buffered saline (500 μL). Then, NaOH (100 μL, 2 M) was added to lyse the cells. The lysate activity was examined, and the inhibiting ratio and IC₅₀ values were calculated.

[³H] glycine uptake assay (GlyT2)

GlyT2 inhibiting activity was examined using cultured primary brain stem neurons. In brief, brain stems were removed from brains of rat pups. The tissues were minced with a sterile razor blade and digested with 0.05% trypsin (Sigma) and 0.01% Dnase I (Sigma) at 37 °C for 10 min. Then, the tissues were collected by centrifugation for 5 min. After centrifugation, complete culture media Dulbecco's modified Eagle's medium/F12 (3 mL; 1 : 1; Gibco BRL, Gaithersburg, MD, USA) was added, which contained 10% fetal bovine serum (Hyclone, Logan, UT, USA), penicillin (100 U mL⁻¹), and streptomycin. Thereafter, this tissue was mechanically dissociated into a single-cell suspension. The dissociated cells were plated into 24 plates pre-coated with polylysine (Sigma). At 48 h later, cytosine arabinoside (5 μM) was added to kill the glial cells. Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO₂. On the 10th day of culture, the inhibiting activity of GlyT2 was tested. Sarcosine (10⁻³ M) was added to all wells to block GlyT1 activity, and glycine (10⁻⁴ M) was used to determine nonspecific uptake action. The remaining procedures were identical to those in the GlyT1 test.

Binding assay for dopamine and serotonin receptors

Dopamine receptors 1, 2, and 3 were obtained from HEK293 stable-transfecting cell lines. Binding assays were conducted as described before.^{25,26 3}H-SCH23390 (10 nM) was used for the binding test of D₁ receptor, and ³H-spiperone for the D₂ or D₃ receptor-binding assay. (+)-Butaclamol (final concentration: 10 μM) was used to determine nonspecific binding activity. After incubating 1 h at 30 °C, Tris–HCl (pre-cooled, 3 mL) was added to stop the binding reaction. The receptor-bound isotope was collected with a Whatman GF/B glass fiber filter. Radioactivity on the fiber filter was examined, and the inhibiting ratio was calculated. In the binding test of 5-HT receptor, ³H-8-OH-DPAT (10 nM) was used to test binding of 5-HT_1A, and nonspecific binding activity was determined with 5-HT (10 μM). ³H-ketanserin was used for the 5-HT_2A test, and spiperone was used for nonspecific binding activity. The remaining procedures were identical to those in the dopamine receptor tests.

hERG channel assay

The K⁺ current by the hERG was recorded using an Axopatch 200 A amplifier at 24–25 °C. HEK293 cells stably expressing hERG channels were held at a resting voltage of −80 mV. Voltage protocols were controlled by pClamp 9.0 software via the DigiData-1322A interface (Axon Instruments, USA). Electrodes (a tip resistance of 3–5 mΩ) were pulled from borosilicate grass pipettes (Sutter Instruments, USA) and filled with a pipette solution consisted of the following (in mM): 140 KCl, 2 MgCl₂, 1 CaCl₂, 10 HEPES, 10 EGTA, pH 7.4 adjusted with KOH. The test compound was diluted (1 nM–0.1 mM) in extracellular buffer (NaCl 150 mM, KCl 4 mM, CaCl₂ 1.2 mM, MgCl₂ 1 mM, HEPES 10 mM, pH 7.4 with NaOH, 300–310 mOsm), and was added directly using a RSC-100 rapid solution changer with a 9-tube head (BioLogic Co, France). After the cells were stabilized and the currents steady, the amplitude and kinetics of I_KhERG were recorded. Offline analysis of the peak tail current was performed using pClamp software 9.0. The amplitude and kinetics of I_KhERG were recorded for each drug concentration. Concentration-response curves were fitted by nonlinear regression analysis and IC₅₀ values were reported.

Pharmacokinetic study

Male ICR mice (body weight: 18–20 g) were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd (Shanghai, China). Animals were fasted for 12 h but free access to water before administration of the compound. All animals were fed in 3 h after administration. Compound was formulated in 0.5% HMPC and administered orally by gavage at a dose of 10 mg kg⁻¹. Blood samples (0.1 mL) were drawn from retrobulbar venous plexus of mice at 0.5, 1, 2, 4, 8 and 24 h postdose (n = 3 mice per group) and stored in heparinized tubes. After centrifugation for 5 min, plasma (50 μL) was added MeOH–ACN (1 : 1, 200 μL) and centrifuged for 10 min. Supernatant fluid was stored at −20 °C for analysis. Brain tissues were collected at the same time and stored at −20 °C until analysis. Brain tissues were added 10 volumes of MeOH–ACN (1 : 1). After vortex and centrifugation, supernatant fluid was used to analysis. A LC-MS/MS method was used for the quantification of the tested compound. The PK parameters were calculated by non-compartmental model analysis on Phoenix WinNonlin 6.0 (Pharsight, Mountain View, CA).

Locomotion test

Male C57BL/6J mice were pre-treated with 23q (20, 40 mg kg⁻¹, intragastric) 60 min before the injection of PCP (5 mg kg⁻¹, intraperitoneal). The mice were placed in a Plexiglas open field arena (40 × 40 × 45 cm, Jiliang Co. Ltd., Shanghai, China) connected with a video-based recording system. Automated activity was recorded, and the total travelling distance was calculated.

Social interaction test

In this test, C57BL/6J mice received daily injections of either saline or PCP (10 mg kg⁻¹, intraperitoneal) for 2 weeks prior to administration of vehicle control (saline) or compound 23q (20, 40 mg kg⁻¹, intragastric) for an additional 2 weeks. After treatment, the mice were individually placed into an unfamiliar arena (40 × 40 × 45 cm, Jiliang Co., Ltd., Shanghai, China) simultaneously with a weight-matched male mouse. Their behavior was video-recorded for 10 min. The time spent in interaction was defined as sniffing at any part of the partner's body (mainly the anogenital area), grooming, following, crawling over/under, or boxing/wrestling. Interactions were manually scored.

Novel object recognition test

Animal treatment was identical to that described in the social interaction test. On the first day, mice were placed into an open field box (40 × 40 × 45 cm) for 5 min and then immediately returned to their home cages. This was repeated on the second day. On the third day, two identical objects were placed into the open-field box and animals were allowed to explore for 5 min. After this training, the box and objects were cleaned with 75% ethanol to avoid possible instinctive odorant cues. On the fourth day, mice were placed back into the same box, a novel object replaced one of the objects used during training, and mice were allowed to explore freely for 5 min. The familiar object and the novel one were different in shape and color, but similar in size. An exploratory preference index, or the ratio of time spent exploring novel object over total time spent exploring both objects, was used to score recognition memory.

Abbreviations

THF	tetrahydrofuran
DMF	N,N-dimethylformamide
DMA	N,N-dimethylacetamide
HATU	2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
DIAD	diisopropyl azodicarboxylate
EWG	electron-withdrawing group
NFPS	N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine
PCP	phencyclidine
SAR	structure–activity relationship
HPLC	high performance liquid chromatography
EGTA	ethylene glycol tetraacetic acid
ACN	acetonitrile
HMPC	hydroxypropyl methyl cellulose
i.g.	intragastric
i.p.	intraperitoneal

Acknowledgements

This work was financially supported by grants from the National Nature Science Foundation of China (grant 81402786, 81130023), National Basic Research Plan (973) of the Ministry of Science and Technology of China (2011CB5C4403). Supports from Priority Academic Program Development of Jiangsu Higher Education Institutes (PAPD) and Grant from Jiangsu Science and Technology commission (BY2011131) are also appreciated.

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Footnote

† Electronic supplementary information (ESI) available: Preparation of 10a–h, 13a–b, 16a–e, 18a–d and 21a–b. See DOI: 10.1039/c5ra04714e

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