Discovery of 3-Chloro-N-{(S)-[3-(1-ethyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidine-2-yl]methyl}-4-(trifluoromethyl)pyridine-2-carboxamide as a Potent Glycine Transporter 1 Inhibitor

Abstract

A novel glycine transporter 1 (GlyT1) inhibitor was designed by the superposition of different chemotypes to enhance its inhibitory activity. Starting from 2-chloro-N-{(S)-phenyl[(2S)-piperidin-2-yl]methyl}-3-(trifluoromethyl)benzamide (2, SSR504734), the introduction of heteroaromatic rings enabled an increase in the GlyT1 inhibitory activity. Subsequent optimization led to the identification of 3-chloro-N-{(S)-[3-(1-ethyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidine-2-yl]methyl}-4-(trifluoromethyl)pyridine-2-carboxamide (7w), which showed a powerful GlyT1 inhibitory activity (IC₅₀=1.8 nM), good plasma exposure and a plasma to brain penetration in rats that was sufficient to evaluate the compound’s pharmacological properties. Compound 7w showed significant effects in several rodent models for schizophrenia without causing any undesirable central nervous system side effects.

N-Methyl-D-aspartate (NMDA) receptor hypofunction is thought to be involved in the pathophysiology of schizophrenia.^1,2) This NMDA hypofunction hypothesis is based on the observation that NMDA receptor antagonists mimic the positive, negative, and cognitive symptoms of schizophrenia.^3,4) Thus enhancing NMDA function may improve the symptoms of schizophrenia, but excess activation of the glutamate binding site is considered to induce neurotoxicity.⁵⁾ Elevating the levels of glycine, which is an important coagonist of the NMDA receptor, is an alternative approach. In fact, the coadministration of glycine or the glycine site agonist D-serine with atypical antipsychotics helps to improve schizophrenia.⁶⁾

Levels of glycine are controlled by the glycine transporter, which consist of two subunits, glycine transporter 1 (GlyT1) and GlyT2. GlyT1 is widely expressed in forebrain areas such as cortex and hippocampus, where it might be co-localized with strychnine-insensitive glycine regulatory sites on NMDA receptors. In contrast, the expression of GlyT2 is limited to the brain stem, spinal cord, and cerebellum, and GlyT2 is known to be co-localized with strychnine-sensitive glycine receptors. Thus increasing NMDA receptor activity by inhibiting GlyT1 is an attractive drug discovery target for schizophrenia.^7,8)

Most effort has been focused on the development of GlyT1 inhibitors.⁹⁾ Early inhibitors were designed based on sarcosine (methylglycine) structure, a weak GlyT1 inhibitor, like 1¹⁰⁾ (ALX-5407, Fig. 1). Reported sarcosine-based inhibitors have suffered from poor pharmacokinetics (PK) profiles, including low brain penetration. Thus, recent development has focused on non-sarcosine type inhibitors, and several structurally diverse inhibitors have been reported from many groups. N-[Phenyl(piperidin-2-yl)methyl]benzamide derivatives, represented by 2 (SSR504734), are one type of non-sarcosine type inhibitor.¹¹⁾ The reported IC₅₀ values of 2 were 18 and 15 nM in in vitro human and rat glycine uptake inhibitory assays. Merck’s researchers have reported a sulfone based chemical class, represented by 3 (DCCCyB),¹²⁾ as well as sulfonamide class inhibitors, represented by 4.^13–16) At present, many other chemical classes, such as 5 (PF-0346275)¹⁷⁾ and 6 (RG1678),¹⁸⁾ have been reported. Among them, 6 showed a significant improvement in the negative symptoms of patients with schizophrenia in phase 2 clinical trial.¹⁹⁾

Fig. 1. Structures of GlyT1 Inhibitors

Recently, we reported the pharmacological profiles of 7w (TP0439150), which showed a potent in vitro glycine uptake inhibitory activity in rats (IC₅₀=1.5 nM) and exhibited significant effects in a rodent model of negative symptoms and cognitive impairment associated with schizophrenia.²⁰⁾ In the present article, we describe the medicinal chemistry efforts that resulted in the potent inhibitor 7w.

To discover a novel GlyT1 inhibitor with a strong in vitro potency and in vivo efficacy at low doses, we selected a ligand based drug design (LBDD) approach using known GlyT1 inhibitors. Among the reported chemotypes, we focused on the N-[phenyl(piperidin-2-yl)methyl]benzamide derivatives represented by 2 (scaffold A, Fig. 2) and the sulfonamide derivatives represented by 4 (scaffold B, Fig. 2). Both of these chemotypes have a secondary benzamide with ortho substituent as a similar substructure, and we supposed that these two different chemotypes inhibit GlyT1 by binding at the same site and have similar pharmacophore.

Fig. 2. Comparison of N-[Phenyl(piperidin-2-yl)]benzamide Derivative (Scaffold A) and Sulfonamide Derivative (Scaffold B)

Compound 4 (scaffold B) has been reported to have a potent GlyT1 inhibitory activity (IC₅₀=3 nM),¹⁵⁾ and we thought that the structurally characteristic SO₂ group likely played a key role as a hydrogen bond acceptor (HBA) in increasing potency. On the other hand, corresponding HBAs are absent in scaffold A; therefore, we presumed that the introduction of suitable HBAs to scaffold A would enhance the inhibitory activity. According to this hypothesis, the superposition of the two chemotypes was performed to explore suitable positions for introducing HBAs to scaffold A. Firstly, a pharmacophore model for scaffold B was generated from known sulfonamide derivatives described in the literature^13–16) by applying the “Common Feature Pharmacophore Generation” protocol in the BIOVIA Discovery Studio²¹⁾ (Fig. 3(a)). Secondly, compound 2, which represents a typical compound of scaffold A, was superposed onto the obtained pharmacophore model (Fig. 3(b)). The result clearly showed that the characteristic HBAs of scaffold B were missing in scaffold A and were placed over the outer space of the phenyl or piperidine moiety of 2.

Fig. 3. (a) A Pharmacophore Model of Sulfonamide Derivatives; (b) Superposition of 2 on the Pharmacophore Model; (c) Schematic Representation of Compounds of the Scaffold A and B, and the Designed Compounds in This Study

The green arrow and the associated spheres show a hydrogen bond acceptor, the purple ones show a hydrogen bond donor, the blue sphere shows a hydrophobic site, and the orange arrow and the spheres show an aromatic ring.

Based on this model, we decided to introduce HBAs around the phenyl moiety of scaffold A in consideration of the synthetic accessibility (Fig. 3(c)). For the HBAs, heteroaromatic rings were selected because of their size and the ease at which the heteroatom positions can be adjusted.

Chemistry

The compounds described in this work were prepared as shown in Charts 1–5. All the final compounds were formed from a hydrochloric acid salt. Chart 1 describes the synthesis of the reference biphenyl derivatives 7a, b, and intermediate 14c. The reaction of Grignard reagent with Weinreb amide 8 prepared from (S)-pipecolinic acid provided ketone (9a–c), which was reduced diastereoselectively by L-Selectride to yield an alcohol (10a–c). After the exchange of the protective group from a tert-butoxycarbonyl (Boc) to an allyl group, the mesylation of the hydroxyl group and the subsequent reaction with ammonia provided a benzylamine intermediate (13a–c). In this amination step, the configuration of the benzylic position was retained, since the aziridine intermediate was firstly formed by the intramolecular reaction of an allyl amine moiety with a methansulfonyloxy group. Condensation with 2-chloro-3-(trifluoromethyl)benzoic acid provided 14a–c, which was then converted to 7a and b by the removal of an allyl group.

Chart 1

Reagents and conditions: (a) 4-Biphenylmagnesium bromide (for 9a) or 3-biphenylmagnesium bromide (for 9b) or 3-methoxyphenylmagnesium bromide (for 9c), THF, 0°C; (b) L-Selectride, THF, −78°C; (c) KOH, MeOH, H₂O, reflux; (d) allyl bromide, K₂CO₃, DMF, 80°C; (e) (1) MsCl, Et₃N, CHCl₃; (2) ammonia, MeOH; (f) 2-Cl–3-CF₃-benzoic acid, HOBt–H₂O, WSC–HCl, DMF; (g) Pd(PPh₃)₄, 1,3-dimethylbarbituric acid, CHCl₃.

Intermediate 14c was used for the introduction of heteroaromatic rings (Chart 2). The removal of a methyl group by boran tribromide and the subsequent triflation led to 16. Then, the introduction of an Ar¹ group by Suzuki coupling and the removal of an allyl group yielded 7f, h, and i.

Chart 2

Reagents and conditions: (a) BBr₃, CHCl₃; (b) Tf₂O, pyridine, CHCl₃; (c) (1) Ar¹B(OH)₂ or pinacolester, Pd(PPh₃)₄, K₂CO₃, DMF, EtOH, 80°C; (2) Pd(PPh₃)₄, 1,3-dimethylbarbituric acid, CHCl₃.

An alternative route is shown in Chart 3. Compounds 17a and b, which were prepared in the same way as 9c, were hydrogenated, followed by triflation to yield 18a and b, respectively. Compounds 18a and b were reduced by L-Selectride followed by coupling with the desired boronic acid or its pinacol ester to yield 20a–g, which were converted to 21a–g using the same way as that shown in Charts 1(c)–(e). Subsequent condensation with desired carboxylic acid and deprotection provided 7c–e and k–y, respectively.

Chart 3

Reagents and conditions: (a) 4-Benzyloxyphenylmagnesium bromide or 3-benzyloxyphenylmagnesium bromide, THF, 0°C; (b) (1) H₂, Pd–C, EtOH; (2) Tf₂O, pyridine, CHCl₃, 0°C; (c) L-Selectride, THF, −78°C; (d) Ar¹B(OH)₂ or pinacol ester, Pd(PPh₃)₄, K₂CO₃, DMF, EtOH, 80°C; (e) Ar²–CO₂H, HOBt–H₂O, WSC–HCl, DMF; (f) Pd(PPh₃)₄, 1,3-dimethylbarbituric acid, CHCl₃; (g) 3-chloro-4-(trifluoromethyl)pyridine-2-carboxylic acid, HOBt–H₂O, WSC–HCl, DMF.

Compounds 7g and j were prepared according to Chart 4. The coupling reaction of 19b with bis(pinacolato)diboron provided 22. A subsequent Suzuki-coupling reaction with aryl halide provided 23a and b, which were converted to 7g and j using the same method as that shown in Charts 1(c)–(g).

Chart 4

Reagents and conditions: (a) Bis(pinacolato)diboron, Pd(dppf)Cl₂, dppf, KOAc, DMSO, 80°C; (b) Ar¹–Cl, Pd(PPh₃)₄, K₂CO₃, DMF, EtOH, 80°C.

An improved synthetic route for 7w is shown in Chart 5. In this scheme, we aimed to reduce the number of synthetic steps. Mono lithiation of 1,3-dibromobenzene and a subsequent reaction with Weinreb amide 8 provided ketone 25, which was converted to hydroxyimine 26 (mixture of cis and trans isomers) by reaction with hydroxylamine hydrochloride under reflux condition in ethanol. The Boc group was removed in this step; thus, re-protecion was needed. A Suzuki coupling reaction of N-hydroxyimine 26 using [1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride (PEPPSI-IPR) as a palladium catalyst yielded 27,²²⁾ which was reduced diastereoselectively by catalytic hydrogenation in a methanol solvent containing ammonia to yield amine 28. Racemization was not observed in any of steps. Subsequent condensation and deprotection yielded 7w. In this matter, 7w was prepared from Weinreb amide 8 in seven steps, compared with the eleven steps that were required in Chart 4.

Chart 5

Reagents and conditions: (a) 1,3-Dibromobenzene, n-BuLi, THF, −78°C; (b) (1) NH₂OH–HCl, EtOH, reflux; (2) Boc₂O, CHCl₃; (c) 1-ethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole, PEPPSI–IPR, K₂CO₃, toluene, EtOH, H₂O, 110°C; (d) H₂, Pd–C, NH₃/MeOH; (e) (1) 3-chloro-4-(trifluoromethyl)pyridine-2-carboxylic acid, WSC–HCl, HOBt–H₂O, DMF; (2) HCl, EtOAc.

Results and Discussion

Introduction of Heteroaromatic Rings to Improve Activity

The glycine uptake inhibitory activity was evaluated according to reported methods utilizing glioma T98G cells expressing human GlyT1.²³⁾ The investigation of optimal Ar¹ groups is described in Table 1. As a reference, we introduced phenyl group with the same size as the heteroaromatic rings that we intended to introduce. The biphenyl derivatives 7a and b showed reduced inhibitory activities (7a: IC₅₀=0.42 µM, 7b: 0.52 µM), compared with the literature value for 2, suggesting that the introduction of bulkiness without HBA at this position had a negative effect on the activity. According to our initial synthetic plan, several heteroaromatic rings were introduced. The introduction of a 3-pyridine group to the para and meta positions led to increased activities (7c: 0.037 µM, 7d: 0.014 µM), compared with those of the biphenyl derivatives. For the 5-pyrimidine derivatives, a meta-substituted derivative had a higher activity (7f: 0.0063 µM) than the corresponding 3-pyridine derivative 7d and the literature value for 2 (0.018 µM), while the para-substitution led to a reduced activity (7e: 0.093 µM). We thought that the increased activity of 7f was obtained through the introduction of a suitable HBA, as per our initial hypothesis.

Table 1. In Vitro GlyT1 Inhibitory Activity of Compounds 7a–j

Compound	Position	Ar1	hGlyT1 IC50 (µM)
2 (SSR504734)	—	—	0.018
7a	4		0.42
7b	3		0.52
7c	4		0.037
7d	3		0.014
7e	4		0.093
7f	3		0.0063
7g	3		0.010
7h	3		0.0025
7i	3		0.042
7j	3		0.0036

A comparison between para-substituted derivatives (7c, e) and meta-substituted derivatives (7d, f) suggested that the meta-position was suitable for modification. Since the 5-pyrimidine derivative 7f had a higher activity than the 3-pyridine derivative 7d, we further explored rings that included two nitrogen atoms. The introduction of pyrazine resulted in an intermediate activity between that of 3-pyridine 7d and that of 5-pyrimidine 7f, while the 1-methylpyrazol-4-yl derivative 7h had a greater inhibitory activity (7h: 0.0025 µM). The introduction of the 1-methylpyrazol-5-yl group as a regioisomer of the pyrazole derivative 7h proved to decrease the activity by more than 10 times (7i: 0.042 µM). The 1-methylimidazol-5-yl derivative 7j, which was a nitrogen atom transferred analog of 7i, had a better IC₅₀ value (7j: 0.0036 µM) than that of 7i and was equal or slightly inferior to that of 7h. Based on these modifications of the Ar¹ moiety, the two nitrogen atoms of the 1-methylpyrazol-4-yl group were thought to be suitable for interaction with GlyT1.

Modification of Substituents on Benzamide and Pyrazole Moieties

The influence of substituents on the benzamide moiety of scaffold A has not been reported until now. The results of modification of the benzamide moiety (Ar²) of 7h are shown in Table 2. We synthesized the 2,4-dichlorobenzamide derivative 7k, the substituents of which were often used in the derivatization of scaffold B; however, only weak inhibitory activity was observed (Table 2, 7k: 0.20 µM). We thought that the suitable substituents might differ depending on the scaffold, and further structure–activity relationships were studied to confirm the optimum substituents on Ar².

Table 2. In Vitro GlyT1 Inhibitory Activity of Compounds 7h and k–v

Compound	Ar2	hGlyT1IC50 (µM)	Compound	Ar2	hGlyT1IC50 (µM)
7h		0.0025	7q		0.025
7k		0.20	7r		0.036
7l		0.068	7s		0.21
7m		0.22	7t		0.0097
7n		0.012	7u		0.0049
7o		0.021	7v		0.0015
7p		0.042

The replacement of a chlorine atom of 7h with a fluorine atom led to a decrease in activity (7l: 0.068 µM), as did the removal of 2-chlorine atom (7m: 0.22 µM). Replacement with a methyl group also produced a 5-fold decrease in activity (7n: 0.012 µM), compared with the chlorine substituent. These results indicated that substituents at the ortho-position had a considerable effect on the GlyT1 inhibitory activity. To increase the potency, we examined 2,6-disubstitution, but both the dichloro derivative 7o and the dimethyl derivative 7p showed moderate activities (7o: 0.021 µM, 7p: 0.042 µM). We thought that these reductions in activity were caused by the removal of the meta-substituent.

The replacement of the trifluoromethyl group of 7h with chlorine, methyl, or a methoxy group each reduced the activity; in particular, the methoxy group had a detrimental effect on the activity (7s: 0.21 µM). We considered that electron-withdrawing substituents at the meta-position might be important for potent inhibitory activity. The 2-chloro-5-(trifluoromethyl)benzamide derivative 7t also exhibited a reduced activity (7t: 0.0097 µM). A comparison of 7k, o, and q suggested that 2,3-disubstitution was the best pattern for higher activity.

Pyridinecarboxamide derivatives were also examined in an attempt to reduce the lipophilicity of 7h (7h: C log P=4.56).²⁴⁾ The 3-chloro-2-(trifluoromethyl)pyridine-4-carboxamide derivative 7u (C log P=3.52) and the 3-chloro-4-(trifluoromethyl)pyridine-2-carboxamide derivative 7v (C log P=3.87) were synthesized, and both analogs showed favorable inhibitory activities (7u: 0.0049 µM, 7v: 0.0015 µM). In particular, the 2-pyridine derivative 7v showed an equipotent or slightly higher activity than 7h.

Regarding drug-likeness properties, the reduction in lipophilicity provided an opportunity for another derivatization. As shown in Table 3, the elongation of the alkyl group on the pyrazole moiety was investigated as a final fine tuning. The ethyl pyrazole derivative 7w showed almost the same inhibitory activity (7w: 0.0018 µM), although isopropyl and normal-propyl substitution showed slightly reduced activities (7x: 0.0024 µM, 7y: 0.0039 µM). These results indicated that the modification of the alkyl substituent of the pyrazole moiety was unlikely to increase the activity.

Table 3. In Vitro GlyT1 Inhibitory Activity of Compounds 7v–y

Compound	Ar1	hGlyT1 IC50 (µM)	C log P
7v		0.0015	3.87
7w		0.0018	4.40
7x		0.0024	4.71
7y		0.0039	4.93

Results of Metabolism, Pharmacokinetics, and Pharmacological Studies

Among the synthetic derivatives, we selected the most potent compounds 7v and w for further evaluation. Both compounds had low metabolism rates in human liver microsomes (7v: 13.8%, 7w: 16.4%),²⁵⁾ and did not significantly inhibit the major CYP450 enzymes (3A4, 2D6, 2C9, 2C19, 1A2) at a concentration of 10 µM. As shown in Table 4, both compounds showed acceptable plasma exposure and brain/plasma concentration ratios after oral administration in rats. Since the pharmacokinetics properties of 7w were more favorable, we selected 7w for further in vivo evaluation studies. The detailed pharmacological properties of 7w have already been reported.²⁰⁾ Compound 7w significantly improved MK-801-impaired cognition in social recognition tests performed in rats at a dose of 0.1 mg/kg orally and also significantly reversed the phencyclidine-induced reduction in the social interaction of paired mice at a dose of 0.3 mg/kg orally. These studies may reflect the cognitive dysfunction and negative symptom of schizophrenia, respectively. Moreover, compound 7w did not affect spontaneous locomotor activity or rotarod performance when administered 10 mg/kg orally. These results suggest that 7w may be useful for the treatment of cognitive dysfunction and the negative symptom associated with schizophrenia without any undesirable side effects in patients.

Table 4. Pharmacokinetics Profiles of 7v and w after Oral Administration in Rats

Compound	7v	7w
Dose (mg/kg)	10	10
Tmax (h)	3.0±1.73	4.0±0.0
Cmax (ng/mL)	49.3±12.4	117±15.4
B/P ratio	0.69 (4 h)	0.52 (4 h)
AUC (ng·h/mL)	235±53.7	661±42.2

Each value represents the mean±S.D. of three animals.

Conclusion

In this article, we have described the identification and efficient synthetic route of 7w, which showed a potent GlyT1 uptake inhibitory activity. To create a novel GlyT1 inhibitor with a strong in vitro potency and an in vivo efficacy when administered at low doses, we selected a ligand based drug design (LBDD) approach using known GlyT1 inhibitors. We added HBAs to 2 as a novel interactive structure, which was designed by superposition with different known chemotype like sulfonamide-based GlyT1 inhibitors. After reviewing several heteroaromatic rings as HBA, the introduction of a 1-methylpyrazol-4-yl group led to an increased inhibitory activity. Through optimization of the benzamide and pyrazole moieties, we successfully achieved 7w. Compound 7w exhibited an inhibitory activity that was about 10 times higher than the reference value of 2 and showed good plasma exposure and a plasma-to-brain penetration sufficient to evaluate its pharmacological properties. The results of in vivo pharmacological evaluations showed significant effects in several rodent models for schizophrenia, suggesting that 7w may be useful for the treatment of cognitive dysfunction and the negative symptoms associated with schizophrenia without causing any undesirable side effects.

Experimental

¹H- and ¹³C-NMR spectra were recorded on a JEOL JNM-ECA600, JEOL JNM-ECA500, Varian UNITYNOVA300, or Varian GEMINI2000/200, and the chemical shifts were expressed in δ (ppm) values with trimethylsilane as an internal reference (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, and br=broad peak). Mass spectra (MS) were recorded on a micromass Platform LC or Shimadzu LCMS-2010EV. High resolution (HR) mass spectral data were acquired using a Shimadzu LCMS-IT-TOF equipped with an electrospray ionization (ESI)/atmospheric pressure chemical ionization (APCI) dual ion source. The purities of the final compounds were confirmed using LC-MS on an Agilent instrument with electrospray ionization. The LC-MS conditions were as follows: Agilent 1290 infinity and Agilent 6150; column Waters Acquity CSH C18, 1.7 µm, 2.1×50 mm; eluent A, water+0.1% formic acid; eluent B, acetonitrile+0.1% formic acid; 20–99% B for 1.2 min, 99% B for 0.2 min; flow rate 0.8 mL/min; UV detection, λ=254 nm. Optical rotations were measured on a Rudolph Research Analytical AUTOPOL V.

tert-Butyl (2S)-2-(3-Methoxybenzoyl)piperidine-1-carboxylate (9c)

Under a nitrogen gas atmosphere, 3-bromoanisole (35 mL, 277 mmol) was added dropwise to a mixture of magnesium (7.1 g, 292 mmol) and a catalytic amount of iodine in tetrahydrofuran (THF) (270 mL), and the mixture was stirred for 2 h. This reaction solution was added dropwise to a solution of tert-butyl (2S)-2-{[methoxy(methyl)amino]carbonyl}piperidine-1-carboxylate (8, 50.0 g, 184 mmol) in THF (300 mL) under ice cooling, and the mixture was stirred at room temperature for 2 h. Ten percent aqueous NH₄Cl solution was added, followed by extraction with EtOAc. The organic layer was washed with water and brine, dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (11% EtOAc in hexane) to yield 9c (32.7 g, 56%) as a pale yellow oil. ¹H-NMR (600 MHz, CDCl₃) δ: 1.29–1.53 (m, 11H), 1.53–1.88 (m, 3H), 1.99–2.22 (m, 1H), 3.07–3.29 (m, 1H), 3.85 (s, 3H), 3.88–4.05 (m, 1H), 5.41–5.69 (m, 1H), 7.05–7.15 (m, 1H), 7.30–7.57 (m, 3H); MS (ESI): m/z 342 [M+Na]⁺.

tert-Butyl (2S)-2-([1,1′-Biphenyl]-4-carbonyl)piperidine-1-carboxylate (9a)

Compound 9a (55%) was obtained in a manner similar to that described for 9c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.37–1.52 (m, 12H), 1.59–1.74 (m, 1H), 1.77–1.95 (m, 1H), 2.06–2.22 (m, 1H), 3.11–3.29 (m, 1H), 3.88–4.05 (m, 1H), 5.47–5.78 (m, 1H), 7.35–7.49 (m, 3H), 7.62 (br d, J=7.4 Hz, 2H), 7.64–7.71 (m, 2H), 7.94–8.05 (m, 2H); MS (ESI): m/z 266 [M−Boc+H]⁺.

tert-Butyl (2S)-2-([1,1′-Biphenyl]-3-carbonyl)piperidine-1-carboxylate (9b)

Compound 9b (55%) was obtained in a manner similar to that described for 9c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.33–1.51 (m, 11H), 1.57–1.73 (m, 2H), 1.76–1.89 (m, 1H), 2.04–2.22 (m, 1H), 3.08–3.25 (m, 1H), 3.87–4.05 (m, 1H), 5.49–5.78 (m, 1H), 7.34–7.41 (m, 1H), 7.42–7.56 (m, 3H), 7.61 (br d, J=7.4 Hz, 2H), 7.78 (br d, J=7.4 Hz, 1H), 7.82–7.96 (m, 1H), 8.06–8.20 (m, 1H); MS (ESI): m/z 266 [M−Boc+H]⁺.

tert-Butyl (2S)-{(S)-Hydroxy(3-methoxyphenyl)methyl}piperidine-1-carboxylate (10c)

Under a nitrogen gas atmosphere, lithium tri-sec-butylborohydride (L-Selectride, 1 mol/L solution in THF, 200 mL, 200 mmol) was added dropwise to a solution of 9c (32.7 g, 103 mmol) in THF (300 mL) cooled with a dryice–acetone bath, and the mixture was stirred for 3 h. The reaction was quenched with 15% aqueous H₂O₂ solution (200 mL) and extracted with EtOAc. The organic layer was dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was suspended in 10% EtOAc in hexane, and the precipitate was filtered to yield 10c (22.0 g, 67%) as a colorless powder. ¹H-NMR (600 MHz, CDCl₃) δ: 1.26–1.34 (m, 1H), 1.35–1.75 (m, 5H), 1.51 (s, 9H), 2.84–3.15 (m, 1H), 3.82 (s, 3H), 3.95–4.46 (m, 2H), 4.83–4.88 (m, 1H), 6.81–6.87 (m, 1H), 6.90–7.00 (m, 2H), 7.22–7.31 (m, 1H); MS (ESI): m/z 344 [M+Na]⁺.

tert-Butyl (2S)-2-[(S)-([1,1′-Biphenyl]-4-yl)(hydroxy)methyl]piperidine-1-carboxylate (10a)

Compound 10a (96%) was obtained in a manner similar to that described for 10c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.32–1.38 (m, 1H), 1.38–1.65 (m, 4H), 1.52 (s, 9H), 1.66–1.74 (m, 1H), 2.89–3.17 (m, 1H), 3.98–4.55 (m, 2H), 4.93 (d, J=10.5 Hz, 1H), 7.32–7.37 (m, 1H), 7.40–7.49 (m, 4H), 7.54–7.64 (m, 4H); MS (ESI): m/z 390 [M+Na]⁺.

tert-Butyl (2S)-2-[(S)-([1,1′-Biphenyl]-3-yl)(hydroxy)methyl]piperidine-1-carboxylate (10b)

Compound 10b (81%) was obtained in a manner similar to that described for 10c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.30–1.37 (m, 1H), 1.37–1.65 (m, 4H), 1.52 (s, 9H), 1.65–1.74 (m, 1H), 2.87–3.18 (m, 1H), 3.97–4.54 (m, 2H), 4.95 (d, J=10.1 Hz, 1H), 7.31–7.40 (m, 2H), 7.39–7.49 (m, 3H), 7.50–7.65 (m, 4H); MS (ESI): m/z 368 [M+H]⁺.

(S)-{(2S)-1-Allylpiperidin-2-yl}(3-methoxyphenyl)methanol (12c)

KOH (39.2 g, 699 mmol) in water (350 mL) was added to a solution of 10c (28.1 g, 87.4 mmol) in MeOH (400 mL), and the mixture was stirred under reflux condition for 16 h. The organic solvent was distilled off, followed by extraction with CHCl₃. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was suspended in 5% EtOAc in hexane, and precipitate was filtered to yield 11c (19.0 g, 98%) as a colorless powder. ¹H-NMR (600 MHz, CDCl₃) δ: 1.20–1.32 (m, 2H), 1.34–1.44 (m, 2H), 1.54–1.61 (m, 1H), 1.73–1.79 (m, 1H), 2.56–2.63 (m, 1H), 2.63–2.68 (m, 1H), 3.06–3.11 (m, 1H), 3.82 (s, 3H), 4.37 (d, J=6.9 Hz, 1H), 6.79–6.85 (m, 1H), 6.88–6.94 (m, 2H), 7.23–7.28 (m, 1H); MS (ESI): m/z 222 [M+H]⁺.

K₂CO₃ (15.9 g, 116 mmol) and allyl bromide (7.3 mL, 84.4 mmol) were added to a solution of 11c (17.0 g, 76.8 mmol) in N,N-dimethylformamide (DMF) (200 mL), and the mixture was stirred at 50°C for 2 h. The reaction mixture was partitioned between water and EtOAc, and the organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified using NH-silica gel column chromatography (5% EtOAc in hexane) to yield 12c (16.8 g, 84%) as a colorless oil. ¹H-NMR (200 MHz, CDCl₃) δ: 1.12–1.77 (m, 6H), 2.56–2.81 (m, 2H), 2.93–3.11 (m, 1H), 3.27–3.51 (m, 2H), 3.81 (s, 3H), 4.72 (d, J=9.7 Hz, 1H), 5.11–5.26 (m, 2H), 5.76–5.99 (m, 1H), 6.76–6.85 (m, 1H), 6.89–6.97 (m, 2H), 7.18–7.28 (m, 1H); MS (ESI): m/z 262 [M+H]⁺.

(S)-([1,1′-Biphenyl]-4-yl)[(2S)-1-(prop-2-en-1-yl)piperidin-2-yl]methanol (12a)

Compound 12a (33% in 2 steps) was obtained in a manner similar to that described for 12c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.20–1.31 (m, 1H), 1.32–1.40 (m, 1H), 1.49–1.66 (m, 3H), 1.68–1.78 (m, 1H), 2.64–2.74 (m, 1H), 2.74–2.81 (m, 1H), 3.00–3.12 (m, 1H), 3.34–3.41 (m, 1H), 3.43–3.51 (m, 1H), 4.80 (br d, J=9.9 Hz, 1H), 5.15–5.26 (m, 2H), 5.90 (ddt, J=17.0, 10.4, 6.3, 6.3 Hz, 1H), 7.30–7.36 (m, 1H), 7.40–7.45 (m, 4H), 7.54–7.61 (m, 4H); MS (ESI): m/z 308 [M+H]⁺.

(S)-([1,1′-Biphenyl]-3-yl)[(2S)-1-(prop-2-en-1-yl)piperidin-2-yl]methanol (12b)

Compound 12b (44% in 2 steps) was obtained in a manner similar to that described for 12c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.21–1.29 (m, 1H), 1.32–1.40 (m, 1H), 1.48–1.66 (m, 3H), 1.68–1.77 (m, 1H), 2.66–2.73 (m, 1H), 2.73–2.80 (m, 1H), 3.01–3.11 (m, 1H), 3.37 (br dd, J=13.6, 6.6 Hz, 1H), 3.43–3.51 (m, 1H), 4.82 (d, J=9.9 Hz, 1H), 5.15–5.25 (m, 2H), 5.84–5.94 (m, 1H), 7.32–7.37 (m, 2H), 7.38–7.47 (m, 3H), 7.47–7.53 (m, 1H), 7.57–7.62 (m, 3H); MS (ESI): m/z 308 [M+H]⁺.

(S)-1-{(2S)-1-Allylpiperidin-2-yl}-1-(3-methoxyphenyl)methanamine (13c)

Methanesulfonyl chloride (6.9 mL, 89.1 mmol) was added to an ice-cooled solution of 12c (16.6 g, 63.5 mmol) and triethylamine (15 mL, 108 mmol) in CHCl₃ (160 mL), and the mixture was stirred under cooling for 1.5 h and at room temperature for an additional 1 h. The reaction mixture was concentrated in vacuo and the residue was dissolved in 8 mol/L NH₃ solution in MeOH (150 mL). After stirring at 40°C for 1 h, the reaction mixture was concentrated in vacuo. The residue was purified using silica gel column chromatography (CHCl₃–MeOH–28% NH₃ in water=50 : 1 : 0.5) to yield 13c (12.9 g, 78%) as a yellow oil. ¹H-NMR (600 MHz, CDCl₃) δ: 1.01–1.07 (m, 1H), 1.29–1.54 (m, 4H), 1.59–1.69 (m, 1H), 2.62–2.71 (m, 2H), 3.01–3.08 (m, 1H), 3.32–3.42 (m, 2H), 3.81 (s, 3H), 4.17 (d, J=9.6 Hz, 1H), 5.10–5.15 (m, 1H), 5.18–5.24 (m, 1H), 5.84–5.93 (m, 1H), 6.76–6.80 (m, 1H), 6.91–6.96 (m, 2H), 7.19–7.25 (m, 1H); MS (ESI): m/z 261 [M+H]⁺.

(S)-1-([1,1′-Biphenyl]-4-yl)-1-[(2S)-1-(prop-2-en-1-yl)piperidin-2-yl]methanamine (13a)

Compound 13a (83% in 2 steps) was obtained in a manner similar to that described for 13c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.04–1.16 (m, 1H), 1.28–1.57 (m, 4H), 1.60–1.71 (m, 1H), 2.66–2.73 (m, 2H), 3.07 (ddd, J=13.8, 9.9, 3.4 Hz, 1H), 3.35–3.43 (m, 2H), 4.25 (d, J=9.6 Hz, 1H), 5.09–5.26 (m, 2H), 5.87–5.96 (m, 1H), 7.29–7.35 (m, 1H), 7.39–7.47 (m, 4H), 7.52–7.62 (m, 4 H); MS (ESI): m/z 307 [M+H]⁺.

(S)-1-([1,1′-Biphenyl]-3-yl)-1-[(2S)-1-(prop-2-en-1-yl)piperidin-2-yl]methanamine (13b)

Compound 13b (74% in 2 steps) was obtained in a manner similar to that described for 13c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.02–1.12 (m, 1H), 1.29–1.46 (m, 3H), 1.46–1.56 (m, 1H), 1.60–1.74 (m, 1H), 2.66–2.75 (m, 2H), 3.02–3.10 (m, 1H), 3.33–3.45 (m, 2H), 4.27 (d, J=9.6 Hz, 1H), 5.09–5.27 (m, 2H), 5.86–5.95 (m, 1H), 7.28–7.52 (m, 6H), 7.54–7.66 (m, 3H); MS (ESI): m/z 307 [M+H]⁺.

N-[(S)-{(2S)-1-Allylpiperidin-2-yl}(3-methoxyphenyl)methyl]-2-chloro-3-(trifluoromethyl)benzamide (14c)

1-Hydroxybenzotriazole monohydrate (2.18 g, 16.1 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.54 g, 13.2 mmol) were added to a solution of 2-chloro-3-(trifluoromethyl)benzoic acid (2.58 g, 11.5 mmol) in DMF (15 mL), and the mixture was stirred for 30 min. A solution of 13c (3.00 g, 11.5 mmol) was added to the reaction mixture, and the mixture was stirred for 3 h. The reaction was quenched with saturated NaHCO₃ aqueous solution and extracted with EtOAc. The organic layer was washed with water and brine, dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (2% MeOH in CHCl₃) to yield 14c (3.42 g, 64%) as a colorless powder. ¹H-NMR (200 MHz, CDCl₃) δ: 1.25–1.88 (m, 6H), 2.45–2.64 (m, 1H), 2.75–3.03 (m, 2H), 3.10–3.38 (m, 2H), 3.81 (s, 3H), 4.88–4.98 (m, 1H), 5.02–5.17 (m, 2H), 5.61–5.83 (m, 1H), 6.75–6.84 (m, 1H), 6.90–7.01 (m, 2H), 7.19–7.32 (m, 1H), 7.36–7.48 (m, 1H), 7.64–7.81 (m, 3H); MS (ESI): m/z 467 [M+H]⁺.

N-{(S)-([1,1′-Biphenyl]-4-yl)[(2S)-1-(prop-2-en-1-yl)piperidin-2-yl]methyl}-2-chloro-3-(trifluoromethyl)benzamide (14a)

Compound 14a (67%) was obtained in a manner similar to that described for 14c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.35–1.62 (m, 5H), 1.77–1.90 (m, 1H), 2.54–2.62 (m, 1H), 2.85–2.91 (m, 1H), 2.94–3.01 (m, 1H), 3.21–3.27 (m, 1H), 3.33 (br dd, J=13.8, 6.4 Hz, 1 H), 4.97–5.04 (m, 1H), 5.08–5.18 (m, 2H), 5.70–5.80 (m, 1H), 7.26 (s, 1H), 7.31–7.36 (m, 1H), 7.41–7.49 (m, 5H), 7.56–7.61 (m, 4H), 7.71–7.79 (m, 2H); MS (ESI): m/z 513 [M+H]⁺.

N-{(S)-([1,1′-Biphenyl]-3-yl)[(2S)-1-(prop-2-en-1-yl)piperidin-2-yl]methyl}-2-chloro-3-(trifluoromethyl)benzamide (14b)

Compound 14b (84%) was obtained in a manner similar to that described for 14c. ¹H-NMR (600 MHz, dimethyl sulfoxide (DMSO)-d₆) δ: 1.08–1.14 (m, 1H), 1.27–1.45 (m, 4H), 1.66–1.72 (m, 1H), 1.99 (s, 1H), 2.45–2.49 (m, 1H), 2.73 (s, 1H), 2.90–2.95 (m, 1H), 2.96–3.01 (m, 1H), 3.34–3.41 (m, 1H), 3.46 (br dd, J=14.2, 6.4 Hz, 1H), 5.11–5.15 (m, 1H), 5.21–5.26 (m, 1H), 5.43–5.48 (m, 1H), 5.88 (ddt, J=17.0, 10.4, 6.2, 6.2 Hz, 1H), 7.35–7.40 (m, 1H), 7.42–7.51 (m, 4H), 7.53–7.61 (m, 1H), 7.62–7.69 (m, 4H), 7.76–7.79 (m, 1H), 7.93 (dd, J=7.1, 2.5 Hz, 1H), 9.04–9.10 (m, 1H); MS (ESI): m/z 513 [M+H]⁺.

N-{(S)-([1,1′-Biphenyl]-4-yl)[(2S)-piperidin-2-yl]methyl}-2-chloro-3-(trifluoromethyl)benzamide Hydrochloride (7a)

1,3-Dimethylbarbituric acid (299 mg, 1.9 mmol) and tetrakis(triphenylphosphine)palladium(0) (7.4 mg, 0.0064 mmol) were added to a solution of 14a (328 mg, 0.64 mmol) in CHCl₃ (5 mL), and the mixture was stirred for 2 h. The reaction mixture was partitioned between CHCl₃ and saturated NaHCO₃ aqueous solution, and the organic layer was dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (2% MeOH in CHCl₃) to yield the free form of 7a (212 mg). 4 mol/L HCl in EtOAc solution (0.2 mL) was added to a solution of the free form of 7a in EtOAc (1 mL), and the precipitate was filtered to yield 7a (202 mg, 62%) as a colorless powder. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.35–1.51 (m, 3H), 1.56–1.67 (m, 1H), 1.68–1.78 (m, 2H), 2.79–2.92 (m, 1H), 3.27–3.41 (m, 1H), 3.50–3.60 (m, 1H), 5.20–5.26 (m, 1H), 7.35–7.41 (m, 1H), 7.45–7.57 (m, 4H), 7.65–7.76 (m, 5H), 7.93–8.05 (m, 2H), 8.73–8.85 (m, 1H), 8.99–9.16 (m, 1H), 9.63 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.3, 139.8, 139.5, 138.7, 137.0, 133.4, 129.0, 128.7, 128.4, 128.0, 127.7, 127.6, 127.2 (q, J=31 Hz), 126.9, 126.7, 122.7 (q, J=273 Hz), 58.2, 55.6, 44.8, 25.8, 21.6, 21.4; HR-MS: Calcd for C₂₆H₂₄ClF₃N₂O [M+H]⁺ 473.1602. Found 473.1590; [α]_D=+72 (c=0.50, MeOH).

N-{(S)-([1,1′-Biphenyl]-3-yl)[(2S)-piperidin-2-yl]methyl}-2-chloro-3-(trifluoromethyl)benzamide (7b)

Compound 7b (38%) was obtained from 14b in a manner similar to that described for 7a. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.36–1.45 (m, 1H), 1.45–1.52 (m, 2H), 1.57–1.67 (m, 1H), 1.68–1.77 (m, 2H), 2.80–2.90 (m, 1H), 3.31–3.38 (m, 1H), 3.56–3.65 (m, 1H), 5.25–5.32 (m, 1H), 7.36–7.46 (m, 2H), 7.47–7.54 (m, 3H), 7.63–7.69 (m, 2H), 7.69–7.73 (m, 2H), 7.79 (s, 1H), 7.92–7.97 (m, 1H), 8.01–8.07 (m, 1H), 8.81–8.99 (m, 1H), 9.05–9.19 (m, 1H), 9.69 (d, J=8.3 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.4, 140.5, 139.7, 138.7, 138.5, 133.4, 129.3, 128.9, 128.6, 127.7, 127.7, 127.6 (q, J=82 Hz), 127.1, 127.0, 126.8, 126.4, 126.0, 122.7 (q, J=274 Hz), 58.2, 55.8, 44.8, 25.7, 21.6, 21.3; HR-MS: Calcd for C₂₆H₂₄ClF₃N₂O [M+H]⁺ 473.1602. Found 473.1596; [α]_D=+76 (c=0.52, MeOH).

3-{(S)-{[2-Chloro-3-(trifluoromethyl)benzoyl]amino}[(2S)-piperidin-2-yl]methyl}phenyl Trifluoromethanesulfonate (16)

Boran tribromide (2.0 mL, 21.2 mmol) was added to an ice cooled solution of 14c (3.29 g, 7.05 mmol) in CHCl₃ (30 mL), and the mixture was stirred under ice cooling for 1.5 h. The reaction was quenched with saturated NaHCO₃ aqueous solution and extracted with EtOAc. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (0–12% MeOH in CHCl₃) to yield 15 (3.15 g, 98%) as a colorless amorphous. ¹H-NMR (300 MHz, CDCl₃) δ: 1.28–1.63 (m, 5H), 1.70–1.86 (m, 1H), 2.51–2.65 (m, 1H), 2.74–3.03 (m, 2H), 3.16–3.37 (m, 2H), 4.90 (d, J=9.2 Hz, 1H), 5.04–5.20 (m, 2H), 5.62–5.82 (m, 1H), 6.59–6.67 (m, 1H), 6.78–6.93 (m, 2H), 7.10–7.20 (m, 1H), 7.37–7.45 (m, 1H), 7.65–7.93 (m, 3H); MS (ESI): m/z 453 [M+H]⁺.

Trifluoromethanesulfonic anhydride (1.3 mL, 7.74 mmol) was added to an ice cooled solution of 15 (2.97 g, 6.56 mmol) and pyridine (2.6 mL, 32.2 mmol) in CHCl₃ (20 mL), and the mixture was stirred under ice cooling for 30 min and at room temperature for an additional 1.5 h. The reaction was quenched with saturated NaHCO₃ aqueous solution and extracted with CHCl₃. The organic layer was washed with water and brine, dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (0–3% MeOH in CHCl₃) to yield 16 (3.39 g, 88%) as a brown oil. ¹H-NMR (200 MHz, CDCl₃) δ: 1.22–1.91 (m, 6H), 2.49–2.70 (m, 1H), 2.71–3.03 (m, 2H), 3.12–3.38 (m, 2H), 4.92–5.03 (m, 1H), 5.05–5.20 (m, 2H), 5.61–5.84 (m, 1H), 7.12–7.34 (m, 2H), 7.37–7.50 (m, 3H), 7.65–7.85 (m, 3H); MS (ESI): m/z 585 [M+H]⁺.

2-Chloro-N-{(S)-(2S)-piperidin-2-yl[3-(pyrimidin-5-yl)phenyl]methyl}-3-(trifluoromethyl)benzamide Hydrochloride (7f)

Pyrimidine-5-boronic acid (212 mg, 1.71 mmol), potassium carbonate (236 mg, 1.71 mmol), and tetrakis(triphenylphosphine)palladium(0) (40 mg, 0.035 mmol) were added to a solution of 16 (657 mg, 1.12 mmol) in a mixed solvent of DMF–EtOH (2 : 1, 10 mL), and the mixture was stirred at 90°C for 4 h. The reaction mixture was partitioned between saturated NaHCO₃ aqueous solution and EtOAc, and the organic layer was washed with water and brine, dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using NH-silica gel column chromatography (20% EtOAc in hexane) to yield a colorless amorphous solid (237 mg). 1,3-Dimethylbarbituric acid (215 mg, 1.38 mmol) and tetrakis(triphenylphosphine)palladium(0) (5.3 mg, 0.0046 mmol) were added to a solution of the amorphous solid in CHCl₃ (4 mL), and the mixture was stirred for 3 h. The reaction mixture was partitioned between saturated NaHCO₃ aqueous solution and CHCl₃, and the organic layer was dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (5% MeOH in CHCl₃) and NH-silica gel column chromatography (100% EtOAc) to yield the free form of 7f (70 mg). 2 mol/L HCl in isopropanol solution (1.0 mL) was added to an ice-cooled solution of the free form of 7f in EtOH (1.0 mL). After concentration in vacuo, the residue was solidified with EtOAc to yield 7f (59 mg, 10% in 2 steps from 16) as a colorless powder. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.37–1.54 (m, 3H), 1.54–1.66 (m, 1H), 1.66–1.78 (m, 2H), 2.81–2.91 (m, 1H), 3.31–3.39 (m, 1H), 3.56–3.64 (m, 1H), 5.26–5.33 (m, 1H), 7.51–7.57 (m, 1H), 7.60 (t, J=7.8 Hz, 1H), 7.67 (t, J=7.8 Hz, 1H), 7.81–7.86 (m, 1H), 7.92–7.97 (m, 2H), 7.98–8.02 (m, 1H), 8.73–8.80 (m, 1H), 8.88–8.97 (m, 1H), 9.20 (s, 2H), 9.23 (s, 1H), 9.55 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.4, 157.5, 154.8, 138.9, 138.6, 134.1, 133.4, 132.8, 129.6, 128.6, 128.5, 128.0, 127.7, 127.2 (q, J=30 Hz), 126.6, 126.2, 122.7 (q, J=274 Hz), 58.2, 55.6, 44.8, 25.7, 21.6, 21.3; HR-MS: Calcd for C₂₄H₂₂ClF₃N₄O [M+H]⁺ 475.1507. Found 475.1493; [α]_D=+72 (c=0.48, MeOH).

2-Chloro-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}-3-(trifluoromethyl)benzamide Hydrochloride (7h)

Compound 7h (44% in 2 steps) was obtained from 16 in a manner similar to that described for 7f. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.34–1.49 (m, 3H), 1.56–1.65 (m, 1H), 1.68–1.77 (m, 2H), 2.80–2.90 (m, 1H), 3.33–3.38 (m, 1H), 3.48–3.55 (m, 1H), 3.88 (s, 3H), 5.15–5.19 (m, 1H), 7.24–7.27 (m, 1H), 7.39 (t, J=7.8 Hz, 1H), 7.52–7.55 (m, 1H), 7.63–7.69 (m, 2H), 7.86 (s, 1H), 7.93–7.97 (m, 1H), 7.97–8.02 (m, 1H), 8.14 (s, 1H), 8.68–8.81 (m, 1H), 8.90–9.02 (m, 1H), 9.51 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.3, 138.7, 138.4, 136.0, 133.5, 132.9, 129.2, 128.6, 128.0, 127.9, 127.7, 127.2 (q, J=31 Hz), 125.2, 124.6, 124.4, 122.7 (q, J=274 Hz), 121.6, 58.2, 55.8, 44.8, 38.7, 25.7, 21.6, 21.3; HR-MS: Calcd for C₂₄H₂₄ClF₃N₄O [M+H]⁺ 477.1664. Found 477.1663; [α]_D=+74 (c=1.05, MeOH).

2-Chloro-N-{(S)-[3-(1-methyl-1H-pyrazol-5-yl)phenyl][(2S)-piperidin-2-yl]methyl}-3-(trifluoromethyl)benzamide Hydrochloride (7i)

Compound 7i (21% in 2 steps) was obtained from 16 in a manner similar to that described for 7f. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.37–1.51 (m, 3H), 1.54–1.64 (m, 1H), 1.69–1.77 (m, 2H), 2.82–2.90 (m, 1H), 3.33–3.39 (m, 1H), 3.51–3.60 (m, 1H), 3.88 (s, 3H), 5.22–5.27 (m, 1H), 6.44 (d, J=1.8 Hz, 1H), 7.47–7.58 (m, 4H), 7.61 (s, 1H), 7.68 (t, J=7.8 Hz, 1H), 7.95 (s, 1H), 7.97 (s, 1H), 8.63–8.77 (m, 1H), 8.83–8.99 (m, 1H), 9.54 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.3, 142.3, 138.6, 138.5, 137.9, 133.4, 130.5, 129.2, 128.6, 128.1, 127.9, 127.9, 127.7, 127.6, 127.2 (q, J=31 Hz), 122.7 (q, J=274 Hz), 105.9, 58.1, 55.6, 44.8, 37.6, 25.7, 21.6, 21.3; HR-MS: Calcd for C₂₄H₂₄ClF₃N₄O [M+H]⁺ 477.1664. Found 477.1662; [α]_D=+66 (c=0.50, MeOH).

tert-Butyl (2S)-2-[4-(Benzyloxy)benzoyl]piperidine-1-carboxylate (17a)

Compound 17a (53%) was obtained in a manner similar to that described for 9c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.31–1.72 (m, 13H), 1.74–1.90 (m, 1H), 1.98–2.18 (m, 1H), 3.10–3.29 (m, 1H), 3.84–4.02 (m, 1H), 5.12 (s, 2H), 5.38–5.69 (m, 1H), 6.96–7.05 (m, 2H), 7.31–7.46 (m, 5H), 7.86–7.96 (m, 2H); MS (ESI): m/z 418 [M+Na]⁺.

tert-Butyl (2S)-2-[3-(Benzyloxy)benzoyl]piperidine-1-carboxylate (17b)

Compound 17b (60%) was obtained in a manner similar to that described for 9c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.30–1.50 (m, 11H), 1.52–1.73 (m, 2H), 1.74–1.85 (m, 1H), 1.98–2.16 (m, 1H), 3.07–3.28 (m, 1H), 3.86–4.03 (m, 1H), 5.10 (s, 2H), 5.39–5.70 (m, 1H), 7.13–7.20 (m, 1H), 7.30–7.41 (m, 4H), 7.41–7.45 (m, 2H), 7.46–7.58 (m, 2H); MS (ESI): m/z 418 [M+Na]⁺.

tert-Butyl (2S)-2-(4-{[(Trifluoromethyl)sulfonyl]oxy}benzoyl)piperidine-1-carboxylate (18a)

5% Pd/C (1.0 g) was added to a solution of 17a (9.90 g, 25 mmol) in MeOH (120 mL), and the mixture was stirred overnight under a hydrogen gas atmosphere. After filtration, the filtrate was concentrated in vacuo. The residue was solidified with 33% EtOAc in hexane to yield a colorless powder (4.94 g), which was converted to 18a (6.82 g, 62% in 2 steps from 17a, pale yellow powder) in a manner similar to that described for 16. ¹H-NMR (600 MHz, CDCl₃) δ: 1.30–2.16 (m, 15H), 2.96–3.26 (m, 1H), 3.83–4.05 (m, 1H), 5.35–5.65 (m, 1H), 7.32–7.43 (m, 2H), 7.99–8.11 (m, 2H); MS (ESI): m/z 438 [M+H]⁺, m/z 460 [M+Na]⁺.

tert-Butyl (2S)-2-{3-[(Trifluoromethanesulfonyl)oxy]benzoyl}piperidine-1-carboxylate (18b)

Compound 18b (66% in 2 steps) was obtained from 17b in a manner similar to that described for 18a. ¹H-NMR (600 MHz, CDCl₃) δ: 1.29–2.16 (m, 15H), 2.96–3.25 (m, 1H), 3.80–4.00 (m, 1H), 5.29–5.61 (m, 1H), 7.40–7.62 (m, 2H), 7.75–8.02 (m, 2H); MS (ESI): m/z 438 [M+H]⁺.

tert-Butyl (2S)-2-[(S)-Hydroxy(4-{[(trifluoromethyl)sulfonyl]oxy}phenyl)methyl]piperidine-1-carboxylate (19a)

Compound 19a (6.3 g, 94%) was obtained from 18a (6.70 g) in a manner similar to that described for 10c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.21–1.32 (m, 1H), 1.37–1.76 (m, 14H), 2.85–3.17 (m, 1H), 3.96–4.50 (m, 2H), 4.87–5.00 (m, 1H), 7.26–7.37 (m, 2H), 7.44–7.55 (m, 2H); MS (ESI): m/z 440 [M+H]⁺.

tert-Butyl (2S)-2-[(S)-Hydroxy{3-[(trifluoromethanesulfonyl)oxy]phenyl}methyl]piperidine-1-carboxylate (19b)

Compound 19b (96%) was obtained from 18a in a manner similar to that described for 10c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.22–1.29 (m, 1H), 1.38–1.73 (m, 5H), 1.51 (s, 9H), 2.93–3.11 (m, 1H), 3.99–4.36 (m, 2H), 4.92 (br d, J=10.3 Hz, 1H), 7.20–7.24 (m, 1H), 7.29 (s, 1H), 7.41–7.48 (m, 2H); MS (ESI): m/z 462 [M+Na]⁺.

tert-Butyl (2S)-2-{(S)-Hydroxy[4-(pyridin-3-yl)phenyl]methyl}piperidine-1-carboxylate (20a)

3-Pyridineboronic acid (2.50 g, 20.3 mmol), potassium carbonate (2.84 g, 20.5 mmol), and tetrakis(triphenylphosphine)palladium(0) (0.47 g, 0.41 mmol) were added to a solution of 19a (6.00 g, 13.7 mmol) in a mixed solvent of DMF–EtOH (2 : 1, 135 mL) were added, and the mixture was stirred at 90°C for 1.5 h. The reaction mixture was partitioned between water and EtOAc, and the organic layer was washed with water and brine, dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using NH-silica gel column chromatography (33–50% EtOAc in hexane) to yield 20a (4.40 g, 87%) as a pale yellow powder. ¹H-NMR (600 MHz, CDCl₃) δ: 1.31–1.38 (m, 1H), 1.40–1.55 (m, 2H), 1.52 (s, 9H), 1.56–1.84 (m, 3H), 2.97–3.16 (m, 1H), 3.99–4.50 (m, 2H), 4.89–5.01 (m, 1H), 7.35–7.39 (m, 1H), 7.45–7.62 (m, 4H), 7.85–7.90 (m, 1H), 8.56–8.61 (m, 1H), 8.82–8.86 (m, 1H); MS (ESI): m/z 369 [M+H]⁺.

tert-Butyl (2S)-2-{(S)-Hydroxy[3-(pyridin-3-yl)phenyl]methyl}piperidine-1-carboxylate (20b)

Compound 20b (quant.) was obtained from 19b in a manner similar to that described for 20a. ¹H-NMR (600 MHz, CDCl₃) δ: 1.32–1.37 (m, 1H), 1.39–1.81 (m, 5H), 1.51 (s, 9H), 2.93–3.18 (m, 1H), 3.98–4.48 (m, 2H), 4.97 (br d, J=9.9 Hz, 1H), 7.37 (dd, J=7.6, 4.7 Hz, 1H), 7.39–7.54 (m, 3H), 7.59 (br s, 1H), 7.88–7.91 (m, 1H), 8.60 (dd, J=4.7, 1.4 Hz, 1H), 8.85 (d, J=2.1 Hz, 1H); MS (ESI): m/z 369 [M+H]⁺.

tert-Butyl (2S)-2-{(S)-Hydroxy[4-(pyrimidin-5-yl)phenyl]methyl}piperidine-1-carboxylate (20c)

Compound 20c (97%) was obtained from 19a in a manner similar to that described for 20a. ¹H-NMR (500 MHz, DMSO-d₆) δ: 1.20–1.39 (m, 3H), 1.40 (s, 9H), 1.43–1.49 (m, 1H), 1.59–1.82 (m, 2H), 2.85–3.02 (m, 1H), 3.86–3.96 (m, 1H), 4.07–4.23 (m, 1H), 4.89 (br dd, J=8.9, 4.1 Hz, 1H), 5.33 (br s, 1H), 7.51 (d, J=8.2 Hz, 2H), 7.80 (d, J=8.3 Hz, 2H), 9.15 (s, 2H), 9.18 (s, 1H); MS (ESI): m/z 370 [M+H]⁺.

tert-Butyl (2S)-2-{(S)-Hydroxy[3-(1-methyl-1H-pyrazol-4-yl)phenyl]methyl}piperidine-1-carboxylate (20d)

Compound 20d (quant.) was obtained from 19b in a manner similar to that described for 20a. ¹H-NMR (600 MHz, CDCl₃) δ: 1.31–1.75 (m, 6H), 1.52 (s, 9H), 2.85–3.19 (m, 1H), 3.95 (s, 3H), 3.97–4.50 (m, 2H), 4.90 (d, J=10.1 Hz, 1H), 7.21 (br d, J=7.3 Hz, 1H), 7.32–7.36 (m, 1H), 7.39–7.43 (m, 1H), 7.48 (br s, 1H), 7.63 (br s, 1H), 7.76 (s, 1H); MS (ESI): m/z 372 [M+H]⁺.

tert-Butyl (2S)-2-[(S)-[3-(1-Ethyl-1H-pyrazol-4-yl)phenyl](hydroxy)methyl]piperidine-1-carboxylate (20e)

Compound 20e (80%) was obtained from 19b in a manner similar to that described for 20a. ¹H-NMR (600 MHz, CDCl₃) δ: 1.29–1.72 (m, 6H), 1.52 (s, 9H), 1.54 (t, J=7.3 Hz, 3H), 2.86–3.17 (m, 1H), 3.97–4.47 (m, 2H), 4.21 (q, J=7.3 Hz, 2H), 4.90 (dd, J=10.5, 5.0 Hz, 1H), 7.21 (d, J=7.3 Hz, 1H), 7.32–7.36 (m, 1H), 7.39–7.44 (m, 1H), 7.50 (br s, 1H), 7.68 (br s, 1H), 7.78 (s, 1H); MS (ESI): m/z 386 [M+H]⁺.

tert-Butyl (2S)-2-[(S)-Hydroxy{3-[1-(propan-2-yl)-1H-pyrazol-4-yl]phenyl}methyl]piperidine-1-carboxylate (20f)

Compound 20f (quant.) was obtained from 19b in a manner similar to that described for 20a. ¹H-NMR (600 MHz, CDCl₃) δ: 1.27–1.77 (m, 15H), 1.53 (d, J=6.9 Hz, 3H), 1.56 (d, J=6.9 Hz, 3H), 2.86–3.15 (m, 1H), 3.99–4.47 (m, 2H), 4.48–4.57 (m, 1H), 4.91 (dd, J=10.3, 4.4 Hz, 1H), 7.20 (d, J=7.3 Hz, 1H), 7.32–7.36 (m, 1H), 7.40–7.45 (m, 1H), 7.51 (br s, 1H), 7.71 (br s, 1H), 7.78 (s, 1H); MS (ESI): m/z 400 [M+H]⁺.

tert-Butyl (2S)-2-{(S)-Hydroxy[3-(1-propyl-1H-pyrazol-4-yl)phenyl]methyl}piperidine-1-carboxylate (20g)

Compound 20g (78%) was obtained from 19b in a manner similar to that described for 20a. ¹H-NMR (600 MHz, CDCl₃) δ: 0.91–0.98 (m, 3H), 1.29–1.73 (m, 6H), 1.52 (s, 9H), 1.87–1.96 (m, 2H), 2.90–3.17 (m, 1H), 3.98–4.52 (m, 4H), 4.90 (br dd, J=10.1, 4.1 Hz, 1H), 7.20 (br d, J=7.8 Hz, 1H), 7.32–7.36 (m, 1H), 7.39–7.44 (m, 1H), 7.50 (br s, 1H), 7.66 (br s, 1H), 7.78 (s, 1H); MS (ESI): m/z 400 [M+H]⁺.

(S)-1-[(2S)-1-(Prop-2-en-1-yl)piperidin-2-yl]-1-[4-(pyridin-3-yl)phenyl]methanamine (21a)

Compound 21a (33% in 4 steps) was obtained from 20a in a manner smilar to that described for the synthesis of 13c from 10c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.04–1.12 (m, 1H), 1.23–1.56 (m, 4H), 1.62–1.72 (m, 1H), 2.67–2.74 (m, 2H), 3.03–3.11 (m, 1H), 3.36–3.42 (m, 2H), 4.28 (d, J=10.1 Hz, 1H), 5.12–5.16 (m, 1H), 5.20–5.25 (m, 1H), 5.87–5.95 (m, 1H), 7.33–7.37 (m, 1H), 7.47–7.50 (m, 2H), 7.52–7.56 (m, 2H), 7.85–7.89 (m, 1H), 8.57–8.59 (m, 1H), 8.85 (d, J=2.3 Hz, 1H); MS (ESI): m/z 308 [M+H]⁺.

(S)-1-[(2S)-1-(Prop-2-en-1-yl)piperidin-2-yl]-1-[3-(pyridin-3-yl)phenyl]methanamine (21b)

Compound 21b (43% in 4 steps) was obtained from 20b in a manner similar to that described for 21a. ¹H-NMR (600 MHz, CDCl₃) δ: 1.03–1.11 (m, 1H), 1.31–1.57 (m, 4H), 1.63–1.71 (m, 1H), 2.66–2.76 (m, 2H), 3.03–3.11 (m, 1H), 3.35–3.44 (m, 2H), 4.29 (d, J=10.1 Hz, 1H), 5.12–5.17 (m, 1H), 5.20–5.25 (m, 1H), 5.86–5.95 (m, 1H), 7.34–7.38 (m, 1H), 7.39–7.52 (m, 3H), 7.58–7.61 (m, 1H), 7.87–7.91 (m, 1H), 8.57–8.60 (m, 1H), 8.85–8.86 (m, 1H); MS (ESI): m/z 308 [M+H]⁺.

(S)-1-[(2S)-1-(Prop-2-en-1-yl)piperidin-2-yl]-1-[4-(pyrimidin-5-yl)phenyl]methanamine (21c)

Compound 21c (99% in 4 steps) was obtained from 20c in a manner similar to that described for 21a. ¹H-NMR (600 MHz, CDCl₃) δ: 1.31–4.44 (m, 12H), 5.46 (d, J=11.0 Hz, 2H), 6.05–6.19 (m, 1H), 7.49–7.72 (m, 4H), 8.89–8.92 (m, 2H), 9.20 (s, 1H); MS (ESI): m/z 309 [M+H]⁺.

(S)-1-[3-(1-Methyl-1H-pyrazol-4-yl)phenyl]-1-[(2S)-1-(prop-2-en-1-yl)piperidin-2-yl]methanamine (21d)

Compound 21d (17% in 4 steps) was obtained from 20d in a manner similar to that described for 21a. ¹H-NMR (600 MHz, CDCl₃) δ: 1.01–1.09 (m, 1H), 1.29–1.55 (m, 4H), 1.60–1.89 (m, 1H), 2.65–2.72 (m, 2H), 3.02–3.08 (m, 1H), 3.33–3.43 (m, 2H), 3.94 (s, 3H), 4.22 (d, J=10.1 Hz, 1H), 5.13 (dd, J=10.1, 1.8 Hz, 1H), 5.19–5.24 (m, 1H), 5.86–5.94 (m, 1H), 7.18–7.21 (m, 1H), 7.30 (t, J=7.8 Hz, 1H), 7.34–7.37 (m, 1H), 7.46–7.50 (m, 1H), 7.64 (s, 1H), 7.77 (s, 1H); MS (ESI): m/z 311 [M+H]⁺.

(S)-1-[3-(1-Ethyl-1H-pyrazol-4-yl)phenyl]-1-[(2S)-1-(prop-2-en-1-yl)piperidin-2-yl]methanamine (21e)

Compound 21e (48% in 4 steps) was obtained from 20e in a manner similar to that described for 21a. ¹H-NMR (600 MHz, CDCl₃) δ: 1.02–1.10 (m, 1H), 1.30–1.57 (m, 4H), 1.53 (t, J=7.3 Hz, 3H), 1.58–1.84 (m, 1H), 2.67–2.73 (m, 2H), 3.03–3.09 (m, lH), 3.34–3.44 (m, 2H), 4.18–4.24 (m, 3H), 5.11–5.16 (m, 1H), 5.18–5.26 (m, 1H), 5.85–5.95 (m, 1H), 7.19 (d, J=7.8 Hz, 1H), 7.30 (t, J=7.8 Hz, 1H), 7.35–7.38 (m, 1H), 7.50 (br s, 1H), 7.68 (s, 1H), 7.78 (s, 1H); MS (ESI): m/z 325 [M+H]⁺.

(S)-1-{3-[1-(Propan-2-yl)-1H-pyrazol-4-yl]phenyl}-1-[(2S)-1-(prop-2-en-1-yl)piperidin-2-yl]methanamine (21f)

Compound 21f (39% in 4 steps) was obtained from 20f in a manner similar to that described for 21a. ¹H-NMR (600 MHz, CDCl₃) δ: 1.02–1.09 (m, 1H), 1.30–1.59 (m, 4H), 1.55 (d, J=6.9 Hz, 6H), 1.61–1.85 (m, 1H), 2.67–2.73 (m, 2H), 3.03–3.09 (m, 1H), 3.34–3.43 (m, 2H), 4.23 (d, J=9.6 Hz, 1H), 4.50–4.56 (m, 1H), 5.11–5.15 (m, 1H), 5.19–5.25 (m, 1H), 5.86–5.95 (m, 1H), 7.16–7.19 (m, 1H), 7.29 (t, J=7.6 Hz, 1H), 7.36–7.39 (m, 1H), 7.50–7.52 (m, 1H), 7.71 (s, 1H), 7.79 (s, 1H); MS (ESI): m/z 339 [M+H]⁺.

(S)-1-[(2S)-1-(Prop-2-en-1-yl)piperidin-2-yl]-1-[3-(1-propyl-1H-pyrazol-4-yl)phenyl]methanamine (21g)

Compound 21g (33% in 4 steps) was obtained from 20g in a manner similar to that described for 21a. ¹H-NMR (600 MHz, CDCl₃) δ: 0.95 (t, J=7.3 Hz, 3H), 1.03–1.11 (m, 1H), 1.29–1.73 (m, 5H), 1.89–1.96 (m, 2H), 2.67–2.73 (m, 2H), 3.02–3.10 (m, 1H), 3.33–3.44 (m, 2H), 4.11 (t, J=7.1 Hz, 2H), 4.22 (d, J=9.6 Hz, 1H), 5.10–5.16 (m, 1H), 5.19–5.25 (m, 1H), 5.85–5.96 (m, 1H), 7.17–7.20 (m, 1H), 7.30 (t, J=7.6 Hz, 1H), 7.35–7.39 (m, 1H), 7.49–7.52 (m, 1H), 7.66 (s, 1H), 7.79 (s, 1H); MS (ESI): m/z 339 [M+H]⁺.

2-Chloro-N-{(S)-(2S)-piperidin-2-yl[4-(pyridin-3-yl)phenyl]methyl}-3-(trifluoromethyl)benzamide Dihydrochloride (7c)

Compound 7c (42% in 2 steps) was obtained from 21a in a manner similar to that described for the synthesis of 14c and 7a. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.35–1.52 (m, 3H), 1.59–1.68 (m, 1H), 1.70–1.77 (m, 2H), 2.80–2.89 (m, 1H), 3.34–3.40 (m, 1H), 3.54–3.63 (m, 1H), 5.23–5.28 (m, 1H), 7.63–7.65 (m, 2H), 7.67 (t, J=8.0 Hz, 1H), 7.90–7.97 (m, 4H), 8.04 (dd, J=7.8, 1.4 Hz, 1H), 8.64–8.69 (m, 1H), 8.81 (dd, J=5.5, 1.4 Hz, 1H), 8.85–8.90 (m, 1H), 9.16–9.23 (m, 2H), 9.76 (d, J=8.3 Hz, 1H); ¹³C-NMR (126 MHz, DMSO- d₆) δ: 165.3, 143.7, 143.0, 139.6, 139.1, 138.6, 136.9, 134.7, 133.5, 128.7, 128.6, 128.0, 127.7, 127.5, 127.2 (q, J=32 Hz), 125.9, 122.8 (q, J=274 Hz), 58.0, 55.6, 44.7, 25.8, 21.6, 21.3; HR-MS: Calcd for C₂₅H₂₃ClF₃N₃O [M+H]⁺ 474.1555. Found 474.1547; [α]_D=+71 (c=0.52, MeOH).

2-Chloro-N-{(S)-(2S)-piperidin-2-yl[3-(pyridin-3-yl)phenyl]methyl}-3-(trifluoromethyl)benzamide Hydrochloride (7d)

Compound 7d (65% in 2 steps) was obtained from 21b in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.36–1.46 (m, 1H), 1.46–1.52 (m, 2H), 1.58–1.67 (m, 1H), 1.69–1.77 (m, 2H), 2.80–2.90 (m, 1H), 3.32–3.39 (m, 1H), 3.59–3.70 (m, 1H), 5.29–5.33 (m, 1H), 7.58–7.64 (m, 2H), 7.66 (t, J=7.8 Hz, 1H), 7.84–7.86 (m, 1H), 7.93–8.00 (m, 3H), 8.05 (dd, J=7.8, 1.4 Hz, 1H), 8.65–8.71 (m, 1H), 8.83 (dd, J=5.5, 1.4 Hz, 1H), 8.92–8.98 (m, 1H), 9.10–9.18 (m, 1H), 9.21 (d, J=2.3 Hz, 1H), 9.73 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.4, 144.1, 143.3, 139.3, 139.1, 138.6, 137.0, 135.4, 133.5, 129.7, 128.6, 128.6, 128.0, 127.7, 127.2 (q, J=30 Hz), 126.9, 126.6, 125.8, 122.7 (q, J=273 Hz), 58.1, 55.7, 44.8, 25.7, 21.6, 21.3; HR-MS: Calcd for C₂₅H₂₃ClF₃N₃O [M+H]⁺ 474.1555. Found 474.1552; [α]_D=+76 (c=0.60, MeOH).

2-Chloro-N-{(S)-(2S)-piperidin-2-yl[4-(pyrimidin-5-yl)phenyl]methyl}-3-(trifluoromethyl)benzamide Hydrochloride (7e)

Compound 7e (24% in 2 steps) was obtained from 21c in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.34–1.51 (m, 3H), 1.56–1.69 (m, 1H), 1.69–1.79 (m, 2H), 2.81–2.93 (m, 1H), 3.32–3.43 (m, 1H), 3.50–3.63 (m, 1H), 5.21–5.33 (m, 1H), 7.63 (d, J=8.3 Hz, 2H), 7.68 (t, J=7.8 Hz, 1H), 7.91 (d, J=8.3 Hz, 2H), 7.97 (d, J=7.8 Hz, 1H), 8.02 (d, J=7.3 Hz, 1H), 8.75–8.82 (m, 1H), 9.03–9.11 (m, 1H), 9.17–9.24 (m, 3H), 9.67 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.3, 157.4, 154.7, 138.7, 138.6, 133.5, 133.4, 132.6, 128.7, 128.6, 128.0, 127.7, 127.2, 127.2 (q, J=30 Hz), 122.7 (q, J=274 Hz), 58.1, 55.5, 44.8, 25.7, 21.6, 21.3; HR-MS: Calcd for C₂₄H₂₂ClF₃N₄O [M+H]⁺ 475.1507. Found 475.1498; [α]_D=+71 (c=0.53, MeOH).

2,4-Dichloro-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}benzamide Hydrochloride (7k)

Compound 7k (62% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.33–1.45 (m, 3H), 1.55–1.64 (m, 1H), 1.67–1.76 (m, 2H), 2.79–2.88 (m, 1H), 3.31–3.37 (m, 1H), 3.48–3.55 (m, 1H), 3.87 (s, 3H), 5.12–5.16 (m, 1H), 7.24 (d, J=7.8 Hz, 1H), 7.38 (t, J=7.6 Hz, 1H), 7.52 (d, J=7.8 Hz, 1H), 7.56 (dd, J=8.3, 2.3 Hz, 1H), 7.65 (br s, 1H), 7.67 (d, J=2.3 Hz, 1H), 7.74–7.77 (m, 1H), 7.86 (s, 1H), 8.13 (s, 1H), 8.63–8.76 (m, 1H), 8.94–9.08 (m, 1H), 9.42 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.3, 138.7, 136.0, 134.9, 134.6, 132.9, 131.5, 131.0, 129.2, 129.1, 127.9, 127.1, 125.2, 124.6, 124.3, 121.6, 58.2, 55.7, 44.7, 38.7, 25.8, 21.6, 21.3; HR-MS: Calcd for C₂₃H₂₄Cl₂N₄O [M+H]⁺ 443.1400. Found 443.1397; [α]_D=+66 (c=0.55, MeOH).

2-Fluoro-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}-3-(trifluoromethyl)benzamide Hydrochloride (7l)

Compound 7l (55% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.32–1.48 (m, 3H), 1.57–1.67 (m, 1H), 1.68–1.76 (m, 2H), 2.77–2.86 (m, 1H), 3.30–3.37 (m, 1H), 3.55–3.63 (m, 1H), 3.88 (s, 3H), 5.15–5.21 (m, 1H), 7.25–7.30 (m, 1H), 7.40 (t, J=7.8 Hz, 1H), 7.50–7.56 (m, 2H), 7.66–7.70 (m, 1H), 7.88 (s, 1H), 7.91–7.96 (m, 1H), 8.09–8.14 (m, 1H), 8.16 (s, 1H), 8.67–8.81 (m, 1H), 8.98–9.17 (m, 1H), 9.55 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 162.5, 156.3 (d, J=258 Hz), 139.0, 136.0, 135.2, 133.0, 129.3, 128.0, 125.4 (q, J=13 Hz), 125.1, 124.9, 124.8, 124.7, 124.3, 122.5 (q, J=272 Hz), 121.6, 116.9–117.4 (m), 58.2, 55.8, 44.6, 38.7, 25.9, 21.6, 21.3; HR-MS: Calcd for C₂₄H₂₄F₄N₄O [M+H]⁺ 461.1959. Found 461.1948; [α]_D=+56 (c=0.51, MeOH).

N-{(S)-[3-(1-Methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}-3-(trifluoromethyl)benzamide Hydrochloride (7m)

Compound 7m (58% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.34–1.48 (m, 3H), 1.57–1.68 (m, 1H), 1.68–1.79 (m, 2H), 2.80–2.89 (m, 1H), 3.32–3.38 (m, 1H), 3.67–3.76 (m, 1H), 3.87 (s, 3H), 5.19–5.26 (m, 1H), 7.28–7.32 (m, 1H), 7.36 (t, J=7.6 Hz, 1H), 7.48–7.53 (m, 1H), 7.71–7.77 (m, 2H), 7.87 (s, 1H), 7.94 (d, J=7.8 Hz, 1H), 8.14 (s, 1H), 8.33 (s, 1H), 8.38 (d, J=7.8 Hz, 1H), 8.63–8.80 (m, 1H), 9.07–9.21 (m, 1H), 9.71 (d, J=9.2 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.0, 139.5, 136.1, 134.8, 133.0, 132.0, 129.5, 129.3, 129.0 (q, J=32 Hz), 128.1, 127.9, 125.2, 124.5, 124.4, 124.4, 124.0 (q, J=273 Hz), 121.5, 58.0, 56.0, 44.6, 38.7, 26.0, 21.6, 21.3; HR-MS: Calcd for C₂₄H₂₅F₃N₄O [M+H]⁺ 443.2053. Found 443.2053; [α]_D=+22 (c=0.66, MeOH).

2-Methyl-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}-3-(trifluoromethyl)benzamide Hydrochloride (7n)

Compound 7n (59% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.34–1.44 (m, 3H), 1.55–1.65 (m, 1H), 1.67–1.77 (m, 2H), 2.26 (s, 3H), 2.78–2.90 (m, 1H), 3.33–3.40 (m, 1H), 3.48–3.55 (m, 1H), 3.88 (s, 3H), 5.13–5.18 (m, 1H), 7.24–7.27 (m, 1H), 7.40 (t, J=7.6 Hz, 1H), 7.48–7.55 (m, 2H), 7.64–7.67 (m, 1H), 7.75–7.81 (m, 2H), 7.87 (s, 1H), 8.15 (s, 1H), 8.61–8.73 (m, 1H), 8.89–9.08 (m, 1H), 9.37 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.1, 139.2, 139.2, 136.0, 133.9, 133.0, 131.5, 129.3, 128.0 (q, J=28 Hz), 127.9, 126.6, 126.1, 125.1, 124.6, 124.3, 124.3 (q, J=275 Hz), 121.6, 58.0, 55.8, 44.7, 38.7, 25.9, 21.6, 21.3, 15.5; HR-MS: Calcd for C₂₅H₂₇F₃N₄O [M+H]⁺ 457.2210. Found 457.2199; [α]_D=+53 (c=0.60, MeOH).

2,6-Dichloro-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}benzamide Hydrochloride (7o)

Compound 7o (30% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.38–1.61 (m, 3H), 1.65–1.72 (m, 1H), 1.73–1.82 (m, 2H), 2.86–2.95 (m, 1H), 3.23–3.58 (m, 2H), 3.88 (s, 3H), 5.27–5.33 (m, 1H), 7.27 (d, J=7.3 Hz, 1H), 7.39 (t, J=7.8 Hz, 1H), 7.45–7.49 (m, 1H), 7.51–7.56 (m, 3H), 7.66–7.69 (m, 1H), 7.85 (s, 1H), 8.11 (s, 1H), 8.52–8.80 (m, 2H), 9.58 (d, J=8.3 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 163.4, 137.3, 136.0, 135.7, 132.8, 131.3, 131.2, 129.0, 128.2, 127.8, 125.6, 124.6, 121.7, 58.6, 55.1, 45.1, 38.7, 25.0, 21.6, 21.4; HR-MS: Calcd for C₂₃H₂₄Cl₂N₄O [M+H]⁺ 443.1400. Found 443.1392; [α]_D=+79 (c=0.54, MeOH).

2,6-Dimethyl-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}benzamide Hydrochloride (7p)

Compound 7p (33% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆): δ1.32–1.44 (m, 2H), 1.50–1.62 (m, 2H), 1.68–1.77 (m, 2H), 2.12 (br s, 6H), 2.80–2.90 (m, 1H), 3.27–3.54 (m, 2H), 3.88 (s, 3H), 5.16–5.22 (m, 1H), 7.02 (s, 1H), 7.03 (s, 1H), 7.17 (t, J=7.6 Hz, 1H), 7.24 (d, J=7.3 Hz, 1H), 7.38 (t, J=7.6 Hz, 1H), 7.53 (d, J=7.8 Hz, 1H), 7.67 (br s, 1H), 7.85 (s, 1H), 8.12 (s, 1H), 8.37–8.56 (m, 1H), 8.67–8.84 (m, 1H), 8.95 (d, J=8.3 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆): δ169.2, 138.8, 137.5, 136.0, 133.9, 132.8, 129.0, 128.2, 127.8, 127.1, 125.6, 124.5, 121.7, 58.0, 55.5, 44.9, 38.7, 25.6, 21.6, 21.4, 19.0; HR-MS: Calcd for C₂₅H₃₀N₄O [M+H]⁺ 403.2492. Found 403.2499; [α]_D=+82 (c=0.54, MeOH).

2,3-Dichloro-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}benzamide Hydrochloride (7q)

Compound 7q (39% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.35–1.47 (m, 3H), 1.54–1.64 (m, 1H), 1.68–1.77 (m, 2H), 2.79–2.89 (m, 1H), 3.30–3.38 (m, 1H), 3.46–3.53 (m, 1H), 3.88 (s, 3H), 5.11–5.17 (m, 1H), 7.23–7.25 (m, 1H), 7.39 (t, J=7.6 Hz, 1H), 7.48 (t, J=7.8 Hz, 1H), 7.52–7.55 (m, 1H), 7.63–7.67 (m, 2H), 7.72–7.76 (m, 1H), 7.85–7.88 (m, 1H), 8.14 (s, 1H), 8.57–8.68 (m, 1H), 8.85–8.96 (m, 1H), 9.40 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.5, 138.6, 138.2, 136.0, 132.9, 132.0, 131.4, 129.1, 128.3, 128.0, 127.9, 125.3, 124.6, 124.3, 121.6, 58.2, 55.7, 44.8, 38.7, 25.7, 21.6, 21.3; HR-MS: Calcd for C₂₃H₂₄Cl₂N₄O [M+H]⁺ 443.1400. Found 443.1394; [α]_D=+86 (c=0.63, MeOH).

2-Chloro-3-methyl-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}benzamide Hydrochloride (7r)

Compound 7r (44% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.33–1.49 (m, 3H), 1.54–1.63 (m, 1H), 1.67–1.76 (m, 2H), 2.34 (s, 3H), 2.79–2.89 (m, 1H), 3.31–3.37 (m, 1H), 3.41–3.63 (m, 1H), 3.88 (s, 3H), 5.12–5.17 (m, 1H), 7.24 (d, J=7.3 Hz, 1H), 7.33 (t, J=7.6 Hz, 1H), 7.38 (t, J=7.6 Hz, 1H), 7.42–7.45 (m, 1H), 7.45–7.49 (m, 1H), 7.53 (d, J=7.8 Hz, 1H), 7.64 (br s, 1H), 7.84–7.87 (m, 1H), 8.13 (s, 1H), 8.46–8.61 (m, 1H), 8.84–8.99 (m, 1H), 9.19 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 166.7, 138.7, 136.5, 136.3, 136.0, 132.9, 131.9, 130.1, 129.1, 127.9, 126.9, 126.6, 125.3, 124.6, 124.4, 121.6, 58.3, 55.6, 44.8, 38.7, 25.7, 21.6, 21.3, 19.9; HR-MS: Calcd for C₂₄H₂₇ClN₄O [M+H]⁺ 423.1946. Found 423.1934; [α]_D=+85 (c=0.50, MeOH).

3-Methoxy-2-methyl-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}benzamide Hydrochloride (7s)

Compound 7s (67% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.31–1.46 (m, 3H), 1.53–1.64 (m, 1H), 1.66–1.77 (m, 2H), 2.01 (s, 3H), 2.77–2.86 (m, 1H), 3.30–3.37 (m, 1H), 3.42–3.60 (m, 1H), 3.78 (s, 3H), 3.88 (s, 3H), 5.11–5.16 (m, 1H), 7.04 (d, J=8.3 Hz, 1H), 7.10 (d, J=7.8 Hz, 1H), 7.22–7.27 (m, 2H), 7.38 (t, J=7.8 Hz, 1H), 7.50–7.53 (m, 1H), 7.65 (br s, 1H), 7.86 (s, 1H), 8.13 (s, 1H), 8.46–8.58 (m, 1H), 8.92–9.01 (m, 1H), 9.04 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 168.9, 157.3, 139.4, 137.7, 136.0, 132.9, 129.2, 127.9, 126.3, 125.1, 124.5, 124.3, 123.7, 121.6, 119.4, 111.6, 58.1, 55.6, 44.7, 38.7, 25.9, 21.6, 21.4, 12.4; HR-MS: Calcd for C₂₅H₃₀N₄O₂ [M+H]⁺ 419.2442. Found 419.2429; [α]_D=+59 (c=0.51, MeOH).

2-Chloro-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}-5-(trifluoromethyl)benzamide Hydrochloride (7t)

Compound 7t (55% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.34–1.45 (m, 3H), 1.58–1.68 (m, 1H), 1.68–1.78 (m, 2H), 2.82–2.92 (m, 1H), 3.35–3.40 (m, 1H), 3.47–3.56 (m, 1H), 3.87 (s, 3H), 5.15–5.20 (m, 1H), 7.22–7.25 (m, 1H), 7.38 (t, J=7.6 Hz, 1H), 7.52–7.55 (m, 1H), 7.66 (br s, 1H), 7.74 (d, J=8.3 Hz, 1H), 7.82–7.88 (m, 2H), 8.10–8.13 (m, 1H), 8.13 (s, 1H), 8.78–8.91 (m, 1H), 8.92–9.08 (m, 1H), 9.49 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.1, 138.4, 136.8, 136.0, 134.7, 132.9, 130.9, 129.2, 127.9, 127.8, 127.6 (q, J=34 Hz), 126.3, 125.3, 124.7, 124.3, 123.6 (q, J=270 Hz), 121.6, 58.3, 55.8, 44.7, 38.7, 25.8, 21.6, 21.2; HR-MS: Calcd for C₂₄H₂₄ClF₃N₄O [M+H]⁺ 477.1664. Found 477.1658; [α]_D=+68 (c=0.52, MeOH).

3-Chloro-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}-2-(trifluoromethyl)pyridine-4-carboxamide Hydrochloride (7u)

Compound 7u (43% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.33–1.49 (m, 3H), 1.55–1.66 (m, 1H), 1.67–1.77 (m, 2H), 2.81–2.91 (m, 1H), 3.34–3.39 (m, 1H), 3.48–3.58 (m, 1H), 3.87 (s, 3H), 5.11–5.24 (m, 1H), 7.25 (d, J=7.8 Hz, 1H), 7.39 (t, J=7.6 Hz, 1H), 7.54 (d, J=7.3 Hz, 1H), 7.65 (s, 1H), 7.87 (s, 1H), 8.07–8.21 (m, 2H), 8.82 (d, J=4.6 Hz, 1H), 8.89–9.10 (m, 2H), 9.78–9.86 (m, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 163.5, 147.7, 146.2, 143.3 (q, J=34 Hz), 138.1, 136.1, 133.0, 129.2, 127.9, 127.4, 126.2, 125.2, 124.8, 124.3, 121.6, 120.9 (q, J=277 Hz), 58.2, 55.8, 44.8, 38.7, 25.7, 21.6, 21.2; HR-MS: Calcd for C₂₃H₂₃ClF₃N₅O [M+H]⁺ 478.1616. Found 478.1605; [α]_D=+69 (c=0.53, MeOH).

3-Chloro-N-{(S)-[3-(1-methyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}-4-(trifluoromethyl)pyridine-2-carboxamide Hydrochloride (7v)

Compound 7v (43% in 2 steps) was obtained from 21d in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.33–1.52 (m, 3H), 1.53–1.63 (m, 1H), 1.66–1.77 (m, 2H), 2.77–2.86 (m, 1H), 3.25–3.36 (m, 1H), 3.55–3.64 (m, 1H), 3.88 (s, 3H), 5.12–5.21 (m, 1H), 7.25–7.30 (m, 1H), 7.41 (t, J=7.8 Hz, 1H), 7.52–7.57 (m, 1H), 7.65 (s, 1H), 7.87 (s, 1H), 8.07 (d, J=5.0 Hz, 1H), 8.15 (s, 1H), 8.46–8.60 (m, 1H), 8.75–8.86 (m, 1H), 8.88 (d, J=5.0 Hz, 1H), 9.60 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 163.6, 152.6, 148.5, 138.4, 136.1, 135.8 (q, J=32 Hz), 133.0, 129.3, 127.9, 125.7, 125.2, 124.7, 124.4, 123.1, 121.8 (q, J=275 Hz), 121.6, 58.1, 55.5, 44.7, 38.7, 25.6, 21.6, 21.4; HR-MS: Calcd for C₂₃H₂₃ClF₃N₅O [M+H]⁺ 478.1616. Found 478.1603; [α]_D=+71 (c=0.50, MeOH).

3-Chloro-N-{(S)-[3-(1-ethyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}-4-(trifluoromethyl)pyridine-2-carboxamide Hydrochloride (7w)

Compound 7w (71% in 2 steps) was obtained from 21e in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.35–1.52 (m, 3H), 1.41 (t, J=7.3 Hz, 3H), 1.54–1.65 (m, 1H), 1.68–1.77 (m, 2H), 2.78–2.87 (m, 1H), 3.27–3.34 (m, 1H), 3.57–3.64 (m, 1H), 4.17 (q, J=7.3 Hz, 2H), 5.16–5.21 (m, 1H), 7.26–7.29 (m, 1H), 7.41 (t, J=7.8 Hz, 1H), 7.53–7.57 (m, 1H), 7.66–7.68 (m, 1H), 7.88 (s, 1H), 8.06 (d, J=4.6 Hz, 1H), 8.20 (s, 1H), 8.52–8.65 (m, 1H), 8.81–9.00 (m, 1H), 8.87 (d, J=5.0 Hz, 1H), 9.65 (d, J=9.2 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 163.6, 152.7, 148.5, 138.5, 135.9, 135.8 (q, J=37 Hz), 133.1, 129.3, 126.5, 125.7, 125.1, 124.7, 124.5, 123.0, 121.7 (q, J=275 Hz), 121.4, 58.1, 55.5, 46.4, 44.6, 25.5, 21.6, 21.4, 15.5; HR-MS: Calcd for C₂₄H₂₅ClF₃N₅O [M+H]⁺ 492.1772. Found 492.1763; [α]_D=+68 (c=0.52, MeOH).

3-Chloro-N-[(S)-[(2S)-piperidin-2-yl]{3-[1-(propan-2-yl)-1H-pyrazol-4-yl]phenyl}methyl]-4-(trifluoromethyl)pyridine-2-carboxamide Hydrochloride (7x)

Compound 7x (58% in 2 steps) was obtained from 21f in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.32–1.52 (m, 3H), 1.46 (d, J=6.9 Hz, 6H), 1.52–1.65 (m, 1H), 1.67–1.78 (m, 2H), 2.77–2.87 (m, 1H), 3.27–3.35 (m, 1H), 3.56–3.65 (m, 1H), 4.49–4.56 (m, 1H), 5.15–5.20 (m, 1H), 7.27 (d, J=7.3 Hz, 1H), 7.41 (t, J=7.6 Hz, 1H), 7.56 (d, J=7.8 Hz, 1H), 7.67 (br s, 1H), 7.87 (s, 1H), 8.07 (d, J=5.0 Hz, 1H), 8.23 (s, 1H), 8.50–8.58 (m, 1H), 8.83–8.92 (m, 2H), 9.63 (d, J=9.2 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 163.5, 152.5, 148.4, 138.4, 135.8 (q, J=31 Hz), 135.6, 133.2, 129.2, 125.7, 125.1, 124.8, 124.7, 124.4, 123.1, 121.7 (q, J=275 Hz), 121.1, 58.1, 55.5, 53.1, 44.6, 25.6, 22.7, 21.6, 21.3; HR-MS: Calcd for C₂₅H₂₇ClF₃N₅O [M+H]⁺ 506.1929. Found 506.1917; [α]_D=+70 (c=0.49, MeOH).

3-Chloro-N-{(S)-(2S)-piperidin-2-yl[3-(1-propyl-1H-pyrazol-4-yl)phenyl]methyl}-4-(trifluoromethyl)pyridine-2-carboxamide Hydrochloride (7y)

Compound 7y (61% in 2 steps) was obtained from 21g in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 0.85 (t, J=7.3 Hz, 3H), 1.34–1.52 (m, 3H), 1.54–1.65 (m, 1H), 1.67–1.76 (m, 2H), 1.82 (tq, J=7.3, 7.2 Hz, 2H), 2.77–2.87 (m, 1H), 3.25–3.34 (m, 1H), 3.56–3.65 (m, 1H), 4.09 (t, J=6.9 Hz, 2H), 5.16–5.21 (m, 1H), 7.28 (d, J=7.8 Hz, 1H), 7.41 (t, J=7.8 Hz, 1H), 7.53–7.57 (m, 1H), 7.66–7.68 (m, 1H), 7.89 (s, 1H), 8.06 (d, J=5.0 Hz, 1H), 8.19 (s, 1H), 8.51–8.62 (m, 1H), 8.83–8.94 (m, 2H), 9.65 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 163.5, 152.5, 148.5, 138.4, 136.0, 135.8 (q, J=32 Hz), 133.1, 129.3, 127.2, 125.7, 125.1, 124.7, 124.4, 123.1, 121.7 (q, J=275 Hz), 121.2, 58.1, 55.5, 53.0, 44.7, 25.6, 23.2, 21.6, 21.3, 10.9; HR-MS: Calcd for C₂₅H₂₇ClF₃N₅O [M+H]⁺ 506.1929. Found 506.1916; [α]_D=+84 (c=0.41, MeOH).

tert-Butyl (2S)-2-{(S)-Hydroxy[3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methyl}piperidine-1-carboxylate (22)

Potassium acetate (5.27 g, 53.7 mmol), [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1.46 g, 1.79 mmol), 1,1′-bis(diphenylphosphino)ferrocene (1.19 g, 2.51 mmol), and bis(pinacolato)diboron (5.00 g, 19.7 mmol) were added to a solution of 19b (7.86 g, 17.9 mmol) in 1,4-dioxane (150 mL), and the mixture was stirred at 80°C for 3 h. The reaction solution was partitioned between water and EtOAc, and the organic layer was washed with water and brine, dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (33–50% EtOAc in hexane) to yield 22 (6.82 g, 91%) as a pale yellow amorphous. ¹H-NMR (600 MHz, CDCl₃) δ: 7.83 (br s, 1H), 7.74 (d, J=7.3 Hz, 1H), 7.44–7.49 (m, 1H), 7.33–7.40 (m, 1H), 4.89 (d, J=10.1 Hz, 1H), 3.98–4.50 (m, 2H), 2.87–3.41 (m, 1H), 1.52 (s, 9H), 1.34 (s, 12H), 1.25–1.73 (m, 6H); MS (ESI): m/z 418 [M+H]⁺.

tert-Butyl (2S)-2-{(S)-Hydroxy[3-(pyrazin-2-yl)phenyl]methyl}piperidine-1-carboxylate (23a)

2-Chloropyrazine (0.64 g, 5.56 mmol), tetrakis(triphenylphosphine)palladium(0) (96 mg, 0.083 mmol), and potassium carbonate (0.77 g, 5.56 mmol) were added to a solution of 22 (1.16 g, 2.78 mmol) in a mixed solvent of DMF–EtOH (2 : 1, 18 mL), and the mixture was stirred at 90°C for 2 h. The reaction solution was partitioned between water and EtOAc, and the organic layer was washed with water and brine, dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (33–67% EtOAc in hexane) to yield 23a (0.79 g, 77%) as a colorless amorphous. ¹H-NMR (200 MHz, CDCl₃) δ: 1.32–1.86 (m, 6H), 1.52 (s, 9H), 2.91–3.18 (m, 1H), 3.99–4.25 (m, 1H), 4.35–4.54 (m, 1H), 5.00 (d, J=10.5 Hz, 1H), 7.48–7.57 (m, 2H), 7.92–7.99 (m, 1H), 8.03–8.08 (m, 1H), 8.52 (d, J=2.6 Hz, 1H), 8.62–8.66 (m, 1H), 9.04 (d, J=1.3 Hz, 1H); MS (ESI): m/z 370 [M+H]⁺.

tert-Butyl (2S)-2-{(S)-Hydroxy[3-(1-methyl-1H-imidazol-5-yl)phenyl]methyl}piperidine-1-carboxylate (23b)

Compound 23b (74%) was prepared from 22 in a manner similar to that described for 23a. ¹H-NMR (600 MHz, CDCl₃) δ: 1.30–1.74 (m, 6H), 1.51 (s, 9H), 2.89–4.47 (m, 3H), 3.67 (s, 3H), 4.93 (br d, J=9.1 Hz, 1H), 7.11 (s, 1H), 7.31–7.35 (m, 1H), 7.36–7.47 (m, 3H), 7.52 (s, 1H); MS (ESI): m/z 372 [M+H]⁺.

(S)-1-[(2S)-1-(Prop-2-en-1-yl)piperidin-2-yl]-1-[3-(pyrazin-2-yl)phenyl]methanamine (24a)

Compound 24a (49% in 4 steps) was prepared from 23a in a manner similar to that described for the synthesis of 13c from 10c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.04–1.11 (m, 1H), 1.31–1.77 (m, 5H), 2.67–2.77 (m, 2H), 3.04–3.11 (m, 1H), 3.34–3.46 (m, 2H), 4.32 (d, J=10.1 Hz, 1H), 5.13–5.16 (m, 1H), 5.20–5.25 (m, 1H), 5.87–5.96 (m, 1H), 7.45–7.52 (m, 2H), 7.88–7.91 (m, 1H), 8.02–8.04 (m, 1H), 8.50–8.52 (m, 1H), 8.63–8.65 (m, 1H), 9.04–9.06 (m, 1H); MS (ESI): m/z 309 [M+H]⁺.

(S)-1-[3-(1-Methyl-1H-imidazol-5-yl)phenyl]-1-[(2S)-1-(prop-2-en-1-yl)piperidin-2-yl]methanamine (24b)

Compound 24b (20% in 4 steps) was prepared from 23b in a manner similar to that described for the synthesis of 13c from 10c. ¹H-NMR (600 MHz, CDCl₃) δ: 1.02–1.09 (m, 1H), 1.31–1.68 (m, 5H), 2.65–2.72 (m, 2H), 3.02–3.09 (m, 1H), 3.36–3.41 (m, 2H), 3.67 (s, 3H), 4.25 (d, J=10.1 Hz, 1H), 5.11–5.16 (m, 1H), 5.19–5.26 (m, 1H), 5.85–5.95 (m, 1H), 7.10–7.11 (m, 1H), 7.27–7.29 (m, 1H), 7.36–7.41 (m, 3H), 7.51 (s, 1H); MS (ESI): m/z 311 [M+H]⁺.

2-Chloro-N-{(S)-(2S)-piperidin-2-yl[3-(pyrazin-2-yl)phenyl]methyl}-3-(trifluoromethyl)benzamide Hydrochloride (7g)

Compound 7g (37% in 2 steps) was prepared from 24a in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.34–1.49 (m, 3H), 1.55–1.66 (m, 1H), 1.68–1.77 (m, 2H), 2.82–2.91 (m, 1H), 3.33–3.39 (m, 1H), 3.54–3.63 (m, 1H), 5.25–5.31 (m, 1H), 7.57–7.62 (m, 2H), 7.68 (t, J=7.8 Hz, 1H), 7.94–7.97 (m, 1H), 7.98–8.02 (m, 1H), 8.13–8.15 (m, 1H), 8.24–8.27 (m, 1H), 8.66 (d, J=2.8 Hz, 1H), 8.72–8.87 (m, 2H), 8.95–9.10 (m, 1H), 9.29–9.35 (m, 1H), 9.62 (d, J=8.7 Hz, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.4, 151.1, 144.3, 143.7, 142.2, 138.8, 138.6, 136.2, 133.4, 129.4, 128.7, 128.0, 127.7, 127.2 (q, J=31 Hz), 126.4, 126.1, 126.0, 122.7 (q, J=273 Hz), 58.1, 55.8, 44.8, 25.7, 21.6, 21.3; HR-MS: Calcd for C₂₄H₂₂ClF₃N₄O [M+H]⁺ 475.1507. Found 475.1498; [α]_D=+77 (c=0.55, MeOH).

2-Chloro-N-{(S)-[3-(1-methyl-1H-imidazol-5-yl)phenyl][(2S)-piperidin-2-yl]methyl}-3-(trifluoromethyl)benzamide Hydrochloride (7j)

Compound 7j (40% in 2 steps) was prepared from 24b in a manner similar to that described for 7c. ¹H-NMR (600 MHz, DMSO-d₆) δ: 1.34–1.51 (m, 3H), 1.56–1.65 (m, 1H), 1.69–1.77 (m, 2H), 2.79–2.88 (m, 1H), 3.29–3.43 (m, 1H), 3.57–3.66 (m, 1H), 3.88 (s, 3H), 5.24–5.29 (m, 1H), 7.57–7.64 (m, 3H), 7.66 (t, J=7.8 Hz, 1H), 7.72 (s, 1H), 7.88 (s, 1H), 7.93–7.97 (m, 1H), 7.98–8.04 (m, 1H), 8.77–8.92 (m, 1H), 9.07–9.22 (m, 2H), 9.72–9.77 (m, 1H); ¹³C-NMR (126 MHz, DMSO-d₆) δ: 165.3, 139.1, 138.6, 136.8, 133.8, 133.5, 129.5, 129.1, 128.8, 128.6, 128.3, 128.0, 127.7, 127.2 (q, J=30 Hz), 126.3, 122.8 (q, J=274 Hz), 118.2, 57.9, 55.5, 44.7, 34.5, 25.7, 21.5, 21.4; HR-MS: Calcd for C₂₄H₂₄ClF₃N₄O [M+H]⁺ 477.1664. Found 477.1662; [α]_D=+54 (c=0.59, MeOH).

tert-Butyl (2S)-2-(3-Bromobenzoyl)piperidine-1-carboxylate (25)

Under a nitrogen gas atmosphere, n-BuLi (2.64 mol/L hexane solution, 175 mL) was added dropwise to a solution of 1,3-dibromobenzene (148 g, 629 mmol) in THF (680 mL) cooled with a dry ice-acetone bath, and the mixture was stirred at a temperature below −70°C for 30 min. A solution of 8 (114 g, 419 mmol) in THF (230 mL) was added in a dropwise manner, and the reaction mixture was stirred for 2 h. The reaction was quenched with saturated NH₄Cl aqueous solution and extracted with EtOAc. The organic layer was dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (11% EtOAc in hexane) to yield 25 (71.2 g, 46%) as a pale yellow powder. ¹H-NMR (600 MHz, CDCl₃) δ: 1.28–1.88 (m, 14H), 1.96–2.20 (m, 1H), 2.98–3.17 (m, 1H), 3.82–4.06 (m, 1H), 5.33–5.64 (m, 1H), 7.29–7.37 (m, 1H), 7.63–7.72 (m, 1H), 7.76–7.89 (m, 1H), 8.00–8.09 (m, 1H); MS (ESI): m/z 368 [M+H]⁺.

tert-Butyl (2S)-2-[(3-Bromophenyl)(hydroxyimino)methyl]piperidine-1-carboxylate (26)

Hydroxylamine hydrochloride (1.92 g, 27.6 mol) was added to a solution of 26 (5.10 g, 13.8 mmol) in EtOH (50 mL), and the mixture was stirred for 5 h under reflux condition. After cooling with an ice bath, 4 mol/L HCl solution in EtOAc was added to the reaction mixture and the mixture was stirred overnight at room temperature. A mixed solvent of hexane–EtOAc (1 : 1, 50 mL) was added to the solution, and the precipitate was filtered to yield colorless solid (4.97 g). This solid was suspended in MeOH (50 mL), and triethylamine (5.8 mL, 41.6 mmol) and di-t-butyl dicarbonate (9.5 mL, 41.4 mmol) were added under ice cooling. The mixture was stirred for 3 h at rom temperature and then concentrated in vacuo. The residue was partitioned between EtOAc and 5% aqueous potassium bisulfate solution, and the organic layer was washed with saturated NaHCO₃ aqueous solution, dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (10–33% EtOAc in hexane) to yield 26 (4.18 g, 79%) as a pale yellow oil. ¹H-NMR (600 MHz, CDCl₃) δ: 1.37–1.96 (m, 14H), 2.15–2.24 (m, 1H), 2.28–2.94 (m, 1H), 3.66–3.75 (m, 1H), 5.49–5.54 (m, 1H), 7.18–7.52 (m, 4H), 8.27 (s, 1H); MS (ESI): m/z 383 [M+H]⁺.

tert-Butyl (2S)-2-{[3-(1-Ethyl-1H-pyrazol-4-yl)phenyl](hydroxyimino)methyl}piperidine-1-carboxylate (27)

A solution of potassium carbonate (117 g, 850 mmol) in water (1 L) and a solution of 1-ethyl-1H-pyrazole-4-boronic acid pinacol ester (163 g, 736 mmol) in EtOH (1 L) and [1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride (7.70 g, 11.4 mmol) were added to a solution of 26 (217 g, 566 mmol) in toluene (1 L), and the mixture was stirred for 2 h at 72°C. After the reaction mixture was cooled to room temperature, brine (400 mL) was added, followed by extraction with EtOAc (400 mL×2). The organic layer was dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (20–60% EtOAc in hexane) to yield 27 (174 g, 77%) as a pale yellow amorphous. ¹H-NMR (600 MHz, CDCl₃) δ: 1.23–1.39 (m, 9H), 1.40–2.26 (m, 9H), 2.28–3.03 (m, 1H), 3.67–3.77 (m, 1H), 4.16–4.24 (m, 2H), 5.59–5.65 (m, 1H), 7.12–7.19 (m, 1H), 7.30–7.48 (m, 3H), 7.65 (s, 1H), 7.74–7.76 (m, 1H), 8.01–8.53 (m, 1H); MS (ESI): m/z 399 [M+H]⁺.

tert-Butyl (2S)-2-{(S)-Amino[3-(1-ethyl-1H-pyrazol-4-yl)phenyl]methyl}piperidine-1-carboxylate (28)

Compound 27 (50.1 g, 126 mmol) and 10% Pd/C (5.0 g) were added to an 8 mol/L NH₃ solution in MeOH (300 mL), and the mixture was stirred under a hydrogen gas atmosphere for 20 h at room temperature. 8 mol/L NH₃ solution in MeOH (140 mL) and 10% Pd/C (5.0 g) were added, and the mixture was stirred for an additional 7.5 h at 45°C. After filtration, the solvent was removed in vacuo. The residue was purified using silica gel column chromatography (5% MeOH in CHCl₃) to yield 28 (37.7 g, 78%) as a colorless syrup. ¹H-NMR (600 MHz, CDCl₃) δ: 1.28–1.32 (m, 1H), 1.34–1.73 (m, 14H), 1.54 (t, J=7.3 Hz, 3H), 2.80–3.04 (m, 1H), 4.01–4.51 (m, 3H), 4.21 (q, J=7.3 Hz, 2H), 7.14–7.21 (m, 1H), 7.28–7.35 (m, 1H), 7.37–7.41 (m, 1H), 7.42–7.50 (m, 1H), 7.62–7.71 (m, 1H), 7.77 (s, 1H); MS (ESI): m/z 385 [M+H]⁺.

Synthesis of 3-Chloro-N-{(S)-[3-(1-ethyl-1H-pyrazol-4-yl)phenyl][(2S)-piperidin-2-yl]methyl}-4-(trifluoromethyl)pyridine-2-carboxamide (7w) from 28

1-Hydroxybenzotriazole monohydrate (63.8 g, 417 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (68.5 g, 357 mmol) were added to an ice-cooled solution of 3-chloro-4-(trifluoromethyl)pyridine-2-carboxylic acid (67.2 g, 298 mmol) in DMF (500 mL), and the mixture was stirred under ice cooling for 10 min. A solution of 28 (114.5 g, 298 mmol) in DMF (400 mL) was added dropwise to the reaction mixture, and the mixture was stirred at room temperature for 4 h. The reaction was quenched with saturated NaHCO₃ aqueous solution and extracted with a mixed solvent of hexane–EtOAc (1 : 1). The organic layer was dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (40–60% EtOAc in hexane) to yield a colorless amorphous. The amorphous solid was dissolved in EtOAc (1100 mL), and 4 mol/L HCl in EtOAc (280 mL) was then added under ice cooling. The mixture was stirred at room temperature for 3 h, and the reaction solution was concentrated in vacuo. The residue was suspended in CHCl₃, and 2 mol/L aqueous sodium hydroxide solution was added under ice cooling. The mixture was partitioned, and the organic layer was dried over anhydrous MgSO₄, filtered, and concentrated in vacuo. The residue was purified using silica gel column chromatography (3–7% MeOH in CHCl₃) to yield free form of 7w (96.2 g, 66% in 2 steps) as a colorless amorphous. ¹H-NMR (600 MHz, CDCl₃) δ: 1.31–1.59 (m, 4H), 1.53 (t, J=7.2 Hz, 3H), 1.74–1.80 (m, 1H), 1.81–1.86 (m, 1H), 2.46–2.54 (m, 1H), 2.94–3.03 (m, 2H), 4.20 (q, J=7.2 Hz, 2H), 5.11 (dd, J=8.1, 3.1 Hz, 1H), 7.20 (d, J=7.8 Hz, 1H), 7.33–7.41 (m, 2H), 7.45–7.48 (m, 1H), 7.65 (s, 1H), 7.73 (d, J=4.5 Hz, 1H), 7.76–7.78 (m, 1H), 8.35 (br d, J=7.8 Hz, 1H), 8.72 (d, J=4.5 Hz, 1H); MS (ESI): m/z 492 [M+H]⁺.

Biological Assay

Glioma T98G cells expressing human GlyT1 were used. The T98G cells were seeded at a density of 2.0×10⁴ cells/well onto a 96-well plate and cultured overnight in a carbon dioxide incubator. The test compound was dissolved in 100% DMSO and then dissolved in 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer solution (pH 7.4) containing 150 mM sodium chloride, 1 mM calcium chloride, 5 mM potassium chloride, 1 mM magnesium chloride, 10 mM glucose, and 0.2% bovine serum albumin. After the medium for the cell culture was removed, the test compound and [³H] glycine (final concentration, 250 nM) were added to the cells and reacted at room temperature for 15 min. After the completion of the reaction, the labeled glycine solution was aspirated with a manifold. The cells were then lysed with 0.5 mol/L sodium hydroxide solution. The amount of intracellular glycine was determined by measuring the radio activity in the cell lysate using a liquid scintillation counter. The quantity of glycine uptake in the presence of 10 µM ALX5407 was defined as nonspecific uptake, and the specific uptake amount was determined by subtracting the nonspecific uptake amount from the total uptake amount in the absence of 10 µM ALX5407. The glycine uptake inhibitory activity (IC₅₀ value) was calculated from an inhibition curve for test compound concentrations of 10⁻¹⁰ to 10⁻⁶.

Acknowledgments

The authors would like to thank Drs. Shigeyuki Chaki, Yoshinori Sekiguchi, Akito Yasuhara and Taketoshi Okubo for helpful discussions and Dr. Atsushi Okada and Mr. Akira Higuchi for the analytical and spectrum studies.

Conflict of Interest

All authors are employees of Taisho Pharmaceutical Co., Ltd.

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