Synthesis and monoamine transporter binding properties of 2β-[3’-(substituted benzyl)isoxazol-5-yl]- and 2β-[3’-methyl-4’-(substituted phenyl)isoxazol-5-yl]-3β-(substituted phenyl)tropanes

Abstract

A series of 2β-[3’-(substituted benzyl)isoxazol-5-yl]- and 2β-[3’-methyl-4’-(substituted phenyl)isoxazol-5-yl]-3β-(substituted phenyl)tropanes were prepared and evaluated for affinities at dopamine, serotonin, and norepinephrine transporters using competitive radioligand binding assays. The 2β-[3’-(substituted benzyl)isoxazol-5-yl]-3β-(substituted phenyl)tropanes (3a–h) showed high binding affinities for the dopamine transporter (DAT). The IC₅₀ values ranged from 5.9 to 22 nM. On the other hand, the 2β-[3’-methyl-4’-(substituted phenyl)isoxazol-5-yl]-3β-(substituted phenyl)tropanes (4a–h), with IC₅₀ values ranging from 65 to 173 nM, were approximately 3- to 25-fold less potent than the corresponding 2β-[3’-(substituted benzyl)isoxazol]tropanes. All tested compounds were selective for the DAT relative to the norepinephrine transporter (NET) and serotonin transporter (5-HTT). 3β-(4-Methylphenyl)-2β-[3’-(4-fluorobenzyl)isoxazol-5-yl]tropane (3b) with IC₅₀ of 5.9 nM at the DAT and K_is of 454 nM and 113 nM at the NET and 5-HTT, respectively, was the most potent and DAT selective analog. Molecular modeling study suggested that the rigid conformation of the isoxazole side chain in 4a–h might play an important effect on their low DAT binding affinities.

1. Introduction

Cocaine abuse continues to be a significant medical problem in the United States. An estimated 2 million Americans currently use cocaine (past-month) according to the National Survey on Drug Use and Health (NSDUH).¹ In addition to its direct effects, cocaine abuse has also contributed to the increase of the spread of Human Immunodeficiency Virus (HIV) infection and drug-resistant tuberculosis.² At present, no effective medication is in clinical use for the treatment of cocaine abuse. In the central nervous system (CNS), cocaine (1a, Fig. 1) binds to the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (5-HTT), and inhibits presynaptic reuptake of the respective neurotransmitters. It also has effects on the cholinergic, muscarinic, and σ receptors and sodium channels.^3,4 It is believed that the DAT is the key recognition site for cocaine, mediating the behavioural and reinforcing effects that contribute to its abuse liability.^4–8 The dopamine hypothesis of cocaine addiction has received further support from molecular biological studies involving DAT knockout mice and Positron Emission Tomography (PET).^9,10 Accordingly, the discovery and development of potent and selective DAT inhibitors represents one of the promising approaches in the treatment of cocaine abuse.

Figure 1.

Structures of cocaine (1a) and 3-phenyltropanes

Many research groups have sought to develop the 3-phenyltropane class of DAT inhibitors, with 3β-phenyltropane-2β-carboxylic acid methyl ester (WIN 35,065-2, 1b) as the lead compound.^11–19 A large part of our structure-activity relationships (SAR) studies was directed towards modification of the 4’-chloro and 4’-methyl analogs 1c,d.¹⁶ Over the last several years, we have synthesized a large number of 3-phenyltropane analogs and evaluated them for binding at monoamine transporters. We have shown that a variety of 2β-esters, amides, and heterocyclic groups possessing either a 4’-chloro or 4’-methylphenyl group at the 3β-position had high-affinity for the DAT, in some cases, with considerably reduced affinity at the NET and 5-HTT.¹⁶ One of the more interesting classes of compounds in the heterocyclic series is the 2β-[3’-(substituted phenyl)isoxazol-5-yl]-3β-(substituted phenyl)tropanes (2a–h).²⁰ The DAT selective inhibitor RTI-336 (2e) is currently in advanced preclinical development.^21,22 The present study was undertaken to further explore the effect of substituents on the isoxazole ring of the 3β-phenyltropanes. In this paper, we describe the synthesis of several 2β-[3’-(substituted benzyl)isoxazol-5-yl]-3β-(substituted phenyl)tropanes (3a–h) and 2β-[3’-methyl-4’-(substituted phenyl)isoxazol-5-yl]-3β-(substituted phenyl)tropanes (4a–h), and report their monoamine transporter binding properties.

2. Chemistry

The synthesis of 2β-[3’-(substituted benzyl)isoxazol-5-yl]-3β-(substituted phenyl)tropanes (3a–h) and 2β-[3’-methyl-4’-(substituted phenyl)isoxazol-5-yl]-3β-(substituted phenyl)tropanes (4a–h) starting with anhydroecgonine methyl ester (5), is outlined in Scheme 1. Briefly, conjugate addition of 5 with the appropriate Grignard reagent at −45 °C in ethyl ether followed by trifluroacetic acid (TFA) yielded 3β-aryltropanes 6a,b.²³ Following our previously reported procedure,^20,24 a solution of 6a was added at 0 °C to the oxime dilithium salt, generated from the oxime of 1-(4-fluorophenyl)propan-2-one with n-butyllithium in tetrahydrofuran (THF) at room temperature, and the reaction was warmed to room temperature. After 20 hours, the reaction mixture was added to a 1:1 mixture of 3N hydrochloric acid and THF, and was refluxed for 5 hours. Column chromatography purification of the crude product afforded 2β-[3’-(4-fluorobenzyl)isoxazol-5-yl]-3β-(4-chlorophenyl)tropane (3a) in 41% yield. Under these conditions, none of the desired 2β-[3’-methyl-4’-(4-fluorophenyl)isoxazol-5-yl]-3β-(4-chlorophenyl)tropane (4a) was obtained. Alternatively, treatment of the oxime of 1-(4-fluorophenyl)propan-2-one with n-butyllithium in THF at 70 °C for 30 minutes followed by addition of 6a and refluxing for 3 hours afforded the corresponding mixture of addition intermediates. Subsequent cyclization in refluxing 3N hydrochloric acid and THF furnished a mixture of 3a and 4a, which were obtained in 4% and 7% yield, respectively, after careful column chromatography. The low yields of 3a and 4a were due to the formation of multiple by-products under the reaction conditions. However, none of the by-products was formed in quantities justifying isolation and identification. The stereochemistry of 3a and 4a was determined by ¹H NMR spectral analysis, particularly with the aid of coupling constants of C(2)-H and C(3)-H. The vicinal couplings of J_2eq,3ax = 5.9 Hz and J _3ax,4ax = 13.0 Hz for 3a, and J_2eq,3ax = 6.0 Hz and J _3ax,4ax = 12.9 Hz for 4a, respectively, are in good agreement with stereochemical assignments. By employing the modified reaction conditions, 2β-(substituted isoxazol)tropane analogs 3b–h and 4b–h were prepared in the range of 3–43% and 4–18% yield, respectively. The yields obtained along with the analytical data of target compounds are given in Table 1.

Scheme 1.

Reagents and conditions: (a) Grignard reagent, −45 °C, 2 h, then −78 °C, TFA; (b) 1-(4-substituted phenyl)propan-2-one oxime, BuLi, THF, 0 °C to 70 °C; (c) 3N HCl, THF, reflux.

Table 1.

2β-Isoxazol-3β-(substituted phenyl)tropanes, yields, and analytical data

Compounda	X	Y	Yield (%)	[α]20D (g/100mL, CH3OH)	mp (°C)	Molecular formula	Analysis
3a	Cl	F	4	−47.7° (0.34)	192 (dec)	C24H25Cl2FN2O·H2O	C, H, N
3b	CH3	F	7	−66.6° (0.29)	165 (dec)	C25H28ClFN2O·1.5H2O	C, H, N
3c	Cl	Cl	5	−88.3° (0.31)	192 (dec)	C24H25Cl3N2O·0.75H2O	C, H, N
3d	CH3	Cl	3	−75.4° (0.40)	180 (dec)	C25H28Cl2N2O·0.5H2O	C, H, N
3e	Cl	CH3	17	−70.0° (0.37)	108 (dec)	C25H28Cl2N2O·0.5H2O	C, H, N
3f	CH3	CH3	39	−61.9° (0.43)	193 (dec)	C26H31ClN2O·0.75H2O	C, H, N
3g	Cl	OCH3	33	−62.4° (0.34)	178 (dec)	C25H28Cl2N2O2·0.75H2O	C, H, N
3h	CH3	OCH3	43	−68.7° (0.53)	168 (dec)	C26H31ClN2O2·1.5H2O	C, H, N
4a	Cl	F	7	+22.2° (0.79)	231 (dec)	C24H25Cl2FN2O·H2O	C, H, N
4b	CH3	F	18	+18.0° (0.25)	208 (dec)	C25H28ClFN2O·1.75H2O	C, H, N
4c	Cl	Cl	12	+71.4° (0.37)	248 (dec)	C24H25Cl3N2O·0.25H2O	C, H, N
4d	CH3	Cl	11	+51.1° (0.33)	245 (dec)	C25H28Cl2N2O·1.25H2O	C, H, N
4e	Cl	CH3	9	+61.5° (0.35)	248 (dec)	C25H28Cl2N2O·0.5H2O	C, H, N
4f	CH3	CH3	7	+44.7° (0.34)	249 (dec)	C26H31ClN2O·1.25H2O	C, H, N
4g	Cl	OCH3	7	+69.4° (0.39)	243 (dec)	C25H28Cl2N2O2·1.25H2O	C, H, N
4h	CH3	OCH3	4	+50.7° (0.28)	238 (dec)	C26H31ClN2O2·1.25H2O	C, H, N

^aHCl salt used to characterize the compound.

3. Biology

The binding affinities for the target compounds at the DAT, NET, and 5-HTT were determined via competitive binding assays using the previously reported procedures.^25,26 The final concentration of radioligands in the assays were 0.5 nM [³H]WIN35,428 for the DAT, 0.5 nM [³H]nisoxetine for the NET and 0.2 nM [³H]paroxetine for the 5-HTT. The results of the binding studies, along with binding data of cocaine, WIN 35,065-2²¹ and 2a–h²⁰ for comparison are listed in Table 2. Since the DAT has two binding sites, IC₅₀ values are reported. The NET and 5-HTT have only one binding site, thus K_i values were calculated for inhibition of binding at these two transporters.

Table 2.

Monoamine transporter binding affinities of 2β-isoxazol-3β-(substituted phenyl)tropanes

Compounda	clog P	X	Y	DAT, IC50b (nM) [3H]WIN35,428	NET, Kib (nM) [3H]nisoxetine	5-HTT, Kib (nM) [3H]paroxetine
Cocainec				89.1	1990	95
WIN35,065-2c				23	556	182
2ad	4.48	Cl	F	1.86 ± 0.09	553 ± 65	660 ± 19
2bd	4.34	CH3	F	6.45 ± 1.6	552 ± 18	>2000
2cd	5.12	Cl	Cl	6.42 ± 0.46	>2000	>2000
2dd	4.98	CH3	Cl	8.74 ± 1.7	>2000	>2000
2ed	5.01	Cl	CH3	4.09 ± 0.44	1030 ± 24	522 ± 48
2fd	4.87	CH3	CH3	13 ± 2.1	>2000	>2000
2gd	4.36	Cl	OCH3	1.57 ± 0.1	454 ± 23	535 ± 16
2hd	4.23	CH3	OCH3	3.93 ± 0.49	455 ± 89	366 ± 2.9
3a	4.98	Cl	F	8.6 ± 3.0	435 ± 40	72 ± 8
3b	4.85	CH3	F	5.9 ± 1.5	454 ± 30	113 ± 8
3c	5.53	Cl	Cl	17.5 ± 1	677 ± 60	79 ± 36
3d	5.39	CH3	Cl	22.2 ± 5.5	840 ± 70	85 ± 12
3e	5.39	Cl	CH3	22.1 ± 2.8	1100 ± 140	42.9 ± 5
3f	5.26	CH3	CH3	6.9 ± 2.2	770 ± 40	89.7 ± 3.5
3g	4.85	Cl	OCH3	6.1 ± 1.4	347 ± 20	14.4 ± 1
3h	4.71	CH3	OCH3	9.6 ± 5	357 ± 50	41.1 ± 11
4a	5.09	Cl	F	111 ± 23	466 ± 40	908 ± 170
4b	4.96	CH3	F	76 ± 12	349 ± 70	>2000
4c	5.73	Cl	Cl	68.7 ± 4	788 ± 120	387 ± 130
4d	5.60	CH3	Cl	65.7 ± 22	519 ± 20	864 ± 250
4e	5.62	Cl	CH3	135 ± 34	>2000	297 ± 20
4f	5.49	CH3	CH3	173 ± 40	>2000	1000 ± 100
4g	4.98	Cl	OCH3	81.6 ± 22	>2000	1800 ± 300
4h	4.84	CH3	OCH3	119 ± 16	1300 ± 100	>2000

^aAll compounds were tested as the HCl salt.

^bAll values are means ± standard error of three or four experiments performed in duplicate.

^cData taken from Ref. 21.

^dData taken from Ref. 20.

4. Results and Discussion

SAR studies from our laboratory as well as others have shown that a variety of functional groups and substituents are well tolerated at the C(2)-position of 3β-phenyltropanes without loss of high-affinity for the DAT.^16,27–29 Results from some of the analogs suggest an electrostatic interaction between the 2β-subsituent and the binding site, whereas results from other analogs are more consistent with a hydrophobic or steric interaction, which is important for the high binding potency at the DAT. A possible explanation for the wide range of substituents that can be accommodated at the C(2)-position is the existence of more than one binding mode. Although the DAT tolerates ligands having a broad variety of 2β-substituents with little change in the affinity of the ligand, the nature of the substituents has a profound effect on the monoamine transporter selectivity.¹⁶ To obtain analogs with increased metabolic stability, we have replaced the metabolically labile 2β-ester group of the 3β-phenyltropanes by stable bioisosteric heterocyclic groups, which led to several analogs with high-affinity and selectivity for the DAT.^20,24,30,31 Computational analyses of the electrostatic (molecular electrostatic potential), hydrophobic (calculated logP), and steric (substituted volume) properties of these 2β-heterocyclic analogs strongly suggested electrostatic interactions predominately contributed to their DAT binding affinities.²⁴

We previously reported that 2β-[3’-(substituted phenyl)isoxazol-5-yl]-3β-(substituted phenyl)tropanes (2a–h) possessed high binding affinities (IC₅₀ = 1.57 to 13 nM) at the DAT with lower affinities at the NET and 5-HTT.²⁴ One of the most potent and selective compounds in the series, RTI-336 (2e), had an IC₅₀ of 4.09 nM at the DAT and K_is of 1030 nM and 522 nM at the NET and 5-HTT, respectively, and is currently in advanced preclinical development.^21,22 In the present study, we expanded the 2β-(substituted isoxazol)tropanes including 2β-[3’-(substituted benzyl)isoxazol]tropanes 3a–h and 2β-[3’-methyl-4’-(substituted phenyl)isoxazol]tropanes 4a–h to further explore the effect of the substituents on the isoxazole ring. All of the analogs 3a–h exhibited high DAT binding affinities similar to the corresponding 2β-[3’-(substituted phenyl)isoxazol]tropanes 2a–h. The IC₅₀ values ranged from 5.9 to 22 nM. On the other hand, the 2β-[3’-methyl-4’-(substituted phenyl)isoxazol]tropanes 4a–h, with IC₅₀ values ranging from 65 to 173 nM, were approximately 8- to 60-fold less potent than 2a–h, and 3- to 25-fold less potent than 3a–h. The particular substituents (F, Cl, CH₃, OCH₃) on the phenyl group of the isoxazole moiety appeared to have only a subtle effect on the DAT affinities. No correlation was observed between the DAT binding affinities with the calculated logP (clogP) of the compounds. All tested compounds in this study were selective for the DAT relative to the NET and 5-HTT. However, none of the compounds was as selective as the corresponding 2β-[3’-(substituted phenyl)isoxazol]tropanes 2a–h.

A molecular modelling study was performed to determine whether conformational differences between 3a–h and 4a–h account for the observed DAT binding properties. The minimum-energy conformations (Fig. 2) of 3a and 4a were generated using the MMFF94 force field in Spartan’06 (Wavefunction, Inc., Irvine, CA). There were no significant differences in the relative conformations of the tropane ring and the benzene ring attached at C(3)-position. However, significant difference in the relative conformations of the isoxazole ring attached at C(2)-position was revealed by the alignment of minimum-energy conformations shown in Figure 2. It is worthy to note that the isoxazole oxygen atom in 4a is pointed in an almost opposite direction compared to that in 3a. The two benzene rings in 4a are proximal, and the resultant steric hindrance increases the rigidity of the isoxazole side chain and perhaps decreases the ability of the ligand to adopt a higher affinity conformation at the DAT.

Figure 2.

Overlay (center; green: 3a and purple: 4a) of the calculated minimum-energy conformations of 3a (left) and 4a (right) (black: carbon; blue: nitrogen; red: oxygen; orange: chlorine; and green: fluorine)

In summary, a new series of 3β-phenyltropanes with various 2β-[3’-(substituted benzyl)isoxazol] and 2β-[3’-methyl-4’-(substituted phenyl)isoxazol] substituents were synthesized and evaluated for their monoamine transporter binding affinities. The 2β-[3’-(substituted benzyl)isoxazol]tropanes 3a–h exhibited high affinities (IC₅₀ = 5.9 to 22 nM) at the DAT, which is similar to that of the corresponding 2β-[3’-(substituted phenyl)isoxazol]tropanes 2a–h. On the other hand, the 2β-[3’-methyl-4’-(substituted phenyl)isoxazol]tropanes 4a–h were approximately 3- to 25-fold less potent than the corresponding 3a–h. Molecular modelling studies suggested that the conformational properties of the isoxazole moiety of 3a–h and 4a–h might play an important effect on their DAT binding affinities.

5. Experimental

Melting points were determined using a MEL-TEMP II capillary melting point apparatus and are uncorrected. Nuclear magnetic resonance (¹H NMR and ¹³C NMR) spectra were obtained on a Bruker Avance DPX-300 MHz NMR spectrometer. Chemical shifts are reported in parts per million (ppm) with reference to internal solvent. Mass spectra (MS) were obtained by electron impact at 70 eV on a Hewlett Packard 5989A instrument. Elemental analyses were performed by Atlantic Microlab Inc., Atlanta, GA. Optical rotations were measured on an AutoPol III polarimeter, purchased from Rudolf Research. Analytical thin-layer chromatography (TLC) was carried out using EMD silica gel 60 F₂₅₄ TLC plates. TLC visualization was achieved with a UV lamp or in an iodine chamber. Flash column chromatography was done on a CombiFlash Companion system using Isco prepacked silica gel columns or using EM Science silica gel 60Å (230–400 mesh). Unless otherwise stated, reagent-grade chemicals were obtained from commercial sources and were used without further purification. All moisture- and air-sensitive reactions and reagent transfers were carried out under dry nitrogen. All compounds described herein were prepared from natural (−)-cocaine, and therefore, they all possess the same absolute configuration as (−)-cocaine.

5.1. 3β-(4-Chlorophenyl)-2β-[3’-(4-fluorobenzyl)isoxazol-5-yl]tropane (3a)

To a stirred solution of 1-(4-fluorophenyl)propan-2-one (5.00 g, 33.0 mmol) in EtOH (75 mL) at room temperature under nitrogen was added K₂CO₃ (12.4 g, 99.0 mmol) followed by hydroxylamine hydrochloride (4.17 g, 66.0 mmol). After stirring for 5 h, the reaction mixture was filtered through a plug of Celite and the filtrate was concentrated under reduced pressure. The resultant residue was dissolved in Et₂O (200 mL), washed with brine (3 × 100 mL), and dried (Na₂SO₄). Removal of the solvent under reduced pressure afforded 1-(4-fluorophenyl)propan-2-one oxime (5.40 g) as an oil, which was used in the next step without further purification.

To a stirred solution of 1-(4-fluorophenyl)propan-2-one oxime (1.84 g, 11.0 mmol) in anhydrous THF (20 mL) at 0 °C under nitrogen was added a solution of BuLi in hexanes (1.6 M, 13.7 mL, 22.0 mmol). The reaction mixture was warmed to room temperature and stirred for 1 h. A solution of 6a²³ (1.07 g, 3.66 mmol) in anhydrous THF (5 mL) was then added. After stirring at room temperature for 20 h, the reaction was quenched by addition of a 20% NH₄Cl aqueous solution (10 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine (3 × 50 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The resultant residue was then dissolved in a 1:1 mixture of 3 N HCl aqueous solution and THF (20 mL), and refluxed for 5 h. After cooling to room temperature, the mixture was washed with Et₂O (3 × 10 mL). The aqueous layer was made basic with NaHCO₃ and extracted with EtOAc (3 × 50 mL). The combined EtOAc extracts were dried (Na₂SO₄) and concentrated under reduced pressure. Flash column chromatography of the crude product on silica gel (40 g Isco prepacked column) using 0 → 10% Et₂O in hexanes with 5% Et₃N afforded 3a (610 mg, 41%) as an oil: ¹H NMR (CDCl₃) δ 7.12−6.90 (m, 6H), 6.88−6.78 (m, 2H), 6.11 (s, 1H), 3.89 (d, J = 15.6 Hz, 1H), 3.80 (d, J = 15.6 Hz, 1H), 3.32−3.12 (m, 4H), 2.30−2.02 (m, 6H), 1.81−1.52 (m, 3H); ¹H NMR (300 MHz; C₆D₆) δ 7.05 (d, J = 8.7 Hz, 2H), 6.80−6.72 (m, 4H), 6.58 (d, J = 8.7 Hz, 2H), 6.11 (s, 1H), 3.67 (d, J = 15.3 Hz, 1H), 3.53 (d, J = 15.3 Hz, 1H), 2.93 (dd, J = 5.9, 2.6 Hz, 1H), 2.88−2.80 (m, 2H), 2.74 (ddd, J = 13.0, 5.9, 5.6 Hz, 1H), 1.91 (ddd, J = 13.0, 12.9, 2.7 Hz, 1H), 1.86 (s, 3H), 1.80−1.60 (m, 2H), 1.30−1.13 (m, 3H); ¹³C NMR (CDCl₃) δ 173.2, 162.1, 161.8 (d, J_C,F = 243 Hz), 140.3, 133.6 (d, J_C,F = 3.2 Hz), 132.1, 130.2 (d, J_C,F = 7.9 Hz), 128.8, 128.2, 115.3 (d, J_C,F = 21.1 Hz), 103.6, 65.4, 61.6, 46.3, 42.0, 35.4, 34.9, 31.7, 26.4, 25.0; MS (EI) m/z 410 (M⁺). The free base was converted to the hydrochloride salt: mp 192 °C (dec); [α]²⁰_D −47.7° (c 0.34, CH₃OH); Anal. Calcd for C₂₄H₂₅Cl₂FN₂O·H₂O: C, 61.94; H, 5.85; N, 6.02. Found: C, 61.95; H, 5.74; N, 5.97.

5.2. 3β-(4-Chlorophenyl)-2β-[3’-(4-fluorobenzyl)isoxazol-5-yl]tropane (3a) and 3β-(4-chlorophenyl)-2β-[3’-methyl-4’-(4-fluorophenyl)isoxazol-5-yl]tropane (4a)

To a stirred solution of 1-(4-fluorophenyl)propan-2-one oxime (1.84 g, 11.0 mmol) in anhydrous THF (20 mL) at 0 °C under nitrogen was added a solution of BuLi in hexanes (1.6 M, 13.7 mL, 22.0 mmol). The reaction mixture was warmed to room temperature over 30 min, and then heated to 70 °C for another 30 min. A solution of 6a (1.07 g, 3.66 mmol) in anhydrous THF (5 mL) was added. The resultant mixture was refluxed for 3 h. After cooling to room temperature, the reaction was quenched by addition of a 20% NH₄Cl aqueous solution (10 mL). The organic layer was separated and the aqueous layer was extracted with EtOAc (3 × 50 mL). The combined organic phases were washed with brine (3 × 50 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The residue was partitioned between Et₂O (100 mL) and 2 N HCl (50 mL). The aqueous layer was separated, made basic with NaHCO₃, and extracted with EtOAc (3 × 50 mL). The combined EtOAc extracts were washed with brine (3 × 30 mL), dried (Na₂SO₄), and concentrated under reduced pressure. The resultant residue was then dissolved in a 1:1 mixture of 3 N HCl aqueous solution and THF (10 mL), and refluxed for 5 h. After cooling to room temperature, the mixture was made basic with NaHCO₃ and extracted with EtOAc (3 × 50 mL). The combined EtOAc extracts were dried (Na₂SO₄) and concentrated under reduced pressure. Flash column chromatography of the crude product on silica gel (40 g Isco prepacked column) using 0 → 10% Et₂O in hexanes with 10% Et₃N afforded 3a (55.0 mg, 4%) and 4a (101 mg, 7%).

Compound 4a

oil; ¹H NMR (CDCl₃) δ 7.17−6.88 (m, 6H), 6.77−6.67 (m, 2H), 3.54−3.44 (m, 1H), 3.27−3.08 (m, 2H), 3.04 (dd, J = 6.0, 2.1 Hz, 1H), 2.65 (ddd, J = 12.9, 12.9, 2.7 Hz, 1H), 2.30−2.08 (m, 5H), 2.02 (s, 3H), 1.78−1.62 (m, 3H); ¹³C NMR (CDCl₃) δ 168.9, 164.1, 159.3 (d, J_C,F = 221 Hz), 140.8, 132.1, 131.4 (d, J_C,F = 8.0 Hz), 129.2, 128.3, 126.6 (d, J_C,F = 3.5 Hz), 117.6, 115.8 (d, J_C,F = 21.4 Hz), 66.3, 62.0, 45.6, 42.1, 36.4, 35.8, 26.9, 25.2, 10.5; MS (EI) m/z 410 (M⁺). The free base was converted to the hydrochloride salt: mp 231 °C (dec); [α]²⁰_D +22.2° (c 0.79, CH₃OH); Anal. Calcd for C₂₄H₂₅Cl₂FN₂O·H₂O: C, 61.94; H, 5.85; N, 6.02. Found: C, 61.69; H, 5.85; N, 5.80.

5.3. 3β-(4-Methylphenyl)-2β-[3’-(4-fluorobenzyl)isoxazol-5-yl]tropane (3b) and 3β-(4-methylphenyl)-2β-[3’-methyl-4’-(4-fluorophenyl)isoxazol-5-yl]tropane (4b)

The procedure for 3a and 4a was followed using 1.00 g (3.66 mmol) of 6b²³ and 1.84 g (11.0 mmol) of 1-(4-fluorophenyl)propan-2-one oxime to give 105 mg (7%) of 3b and 250 mg (18%) of 4b.

Compound 3b

oil; ¹H NMR (CDCl₃) δ 7.12−6.90 (m, 6H), 6.86−6.74 (m, 2H), 6.10 (s, 1H), 3.90 (d, J = 15.6 Hz, 1H), 3.80 (d, J = 15.6 Hz, 1H), 3.35−3.12 (m, 4H), 2.30−2.02 (m, 9H), 1.82−1.52 (m, 3H); ¹³C NMR (CDCl₃) δ 173.6, 161.9, 161.7 (d, J_C,F = 244 Hz), 138.6, 135.8, 133.8 (d, J_C,F = 3.2 Hz), 130.2 (d, J_C,F = 7.9 Hz), 128.8, 127.3, 115.3 (d, J_C,F = 21.1 Hz), 103.6, 65.5, 61.8, 46.5, 42.0, 35.5, 35.0, 31.7, 26.4, 25.1, 21.1; MS (EI) m/z 390 (M⁺). The free base was converted to the hydrochloride salt: mp 165 °C (dec); [α]²⁰_D −66.6° (c 0.29, CH₃OH); Anal. Calcd for C₂₅H₂₈ClFN₂O·1.5H₂O: C, 66.14; H, 6.88; N, 6.17. Found: C, 65.74; H, 6.51; N, 6.07.

Compound 4b

oil; ¹H NMR (CDCl₃) δ 7.05−6.84 (m, 6H), 6.69−6.59 (m, 2H), 3.51−3.43 (m, 1H), 3.23−3.08 (m, 2H), 3.03 (dd, J = 6.0, 2.1 Hz, 1H), 2.65 (ddd, J = 12.6, 12.6, 2.4 Hz, 1H), 2.35−2.08 (m, 8H), 2.01 (s, 3H), 1.75−1.62 (m, 3H); ¹³C NMR (75 MHz; CDCl₃) δ 169.4, 164.0, 159.2 (d, J_C,F = 226 Hz), 139.1, 135.8, 131.5 (d, J_C,F = 8.0 Hz), 128.8, 127.8, 126.8 (d, J_C,F = 3.4 Hz), 117.4, 115.5 (d, J_C,F = 21.3 Hz), 66.3, 62.1, 45.7, 42.1, 36.6, 36.1, 26.8, 25.2, 21.1, 10.5; MS (EI) m/z 390 (M⁺). The free base was converted to the hydrochloride salt: mp 208 °C (dec); [α]²⁰_D +18.0° (c 0.25, CH₃OH); Anal. Calcd for C₂₅H₂₈ClFN₂O·1.75H₂O: C, 65.49; H, 6.92; N, 6.11. Found: C, 65.24; H, 6.53; N, 6.03.

5.4. 3β-(4-Chlorophenyl)-2β-[3’-(4-chlorobenzyl)isoxazol-5-yl]tropane (3c) and 3β-(4-chlorophenyl)-2β-[3’-methyl-4’-(4-chlorophenyl)isoxazol-5-yl]tropane (4c)

The procedure for 3a and 4a was followed using 1.07 g (3.66 mmol) of 6a and 2.02 g (11.0 mmol) of 1-(4-chlorophenyl)propan-2-one oxime to give 85.0 mg (5%) of 3c and 180 mg (12%) of 4c.

Compound 3c

oil; ¹H NMR (CDCl₃) δ 7.31−7.22 (m, 2H), 7.14−7.07 (m, 2H), 7.03 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 6.11 (s, 1H), 3.90 (d, J = 15.6 Hz, 1H), 3.81 (d, J = 15.6 Hz, 1H), 3.36−3.14 (m, 4H), 2.30−2.04 (m, 6H), 1.82−1.53 (m, 3H); ¹³C NMR (CDCl₃) δ 173.3, 161.8, 140.3, 136.5, 132.6, 132.2, 130.1, 128.9, 128.7, 128.3, 103.7, 65.5, 61.7, 46.4, 42.1, 35.5, 34.9, 31.9, 26.5, 25.1; MS (EI) m/z 426 (M⁺). The free base was converted to the hydrochloride salt: mp 192 °C (dec); [α]²⁰_D −88.3° (c 0.31, CH₃OH); Anal. Calcd for C₂₄H₂₅Cl₃N₂O·0.75H₂O: C, 60.39; H, 5.60; N, 5.87. Found: C, 60.70; H, 5.85; N, 5.62.

Compound 4c

oil; ¹H NMR (CDCl₃) δ 7.35−7.27 (m, 2H), 7.14−7.05 (m, 2H), 6.91 (d, J = 8.7 Hz, 2H), 6.67 (d, J = 8.7 Hz, 2H), 3.54−3.42 (m, 1H), 3.22−3.08 (m, 2H), 3.04 (dd, J = 6.0, 2.1 Hz, 1H), 2.64 (ddd, J = 12.9, 12.9, 2.7 Hz, 1H), 2.30−2.06 (m, 5H), 2.03 (s, 3H), 1.77−1.63 (m, 3H); ¹³C NMR (CDCl₃) δ 169.0, 157.7, 140.7, 133.9, 132.2, 131.0, 129.2, 129.0, 128.3, 117.5, 66.3, 62.0, 45.7, 42.1, 36.4, 35.8, 26.8, 25.2, 10.5; MS (EI) m/z 426 (M⁺). The free base was converted to the hydrochloride salt: mp 248 °C (dec); [α]²⁰_D +71.4° (c 0.37, CH₃OH); Anal. Calcd for C₂₄H₂₅Cl₃N₂O·0.25H₂O: C, 61.55; H, 5.49; N, 5.89. Found: C, 61.49; H, 5.75; N, 5.78.

5.5. 3β-(4-Methylphenyl)-2β-[3’-(4-chlorobenzyl)isoxazol-5-yl]tropane (3d) and 3β-(4-methylphenyl)-2β-[3’-methyl-4’-(4-chlorophenyl)isoxazol-5-yl]tropane (4d)

The procedure for 3a and 4a was followed using 1.00 g (3.66 mmol) of 6b and 2.02 g (11.0 mmol) of 1-(4-chlorophenyl)propan-2-one oxime to give 40.0 mg (3%) of 3d and 160 mg (11%) of 4d.

Compound 3d

oil; ¹H NMR (CDCl₃) δ 7.31−7.20 (m, 2H), 7.10−6.77 (m, 6H), 6.10 (s, 1H), 3.91 (d, J = 15.6 Hz, 1H), 3.81 (d, J = 15.6 Hz, 1H), 3.40−3.17 (m, 4H), 2.40−2.03 (m, 9H), 1.88−1.57 (m, 3H); ¹³C NMR (CDCl₃) δ 161.8, 138.6, 136.6, 136.1, 132.5, 131.1, 130.2, 129.0, 128.7, 127.4, 103.8, 65.6, 62.0, 46.5, 42.1, 35.5, 34.9, 31.9, 26.6, 25.1, 21.2; MS (EI) m/z 406 (M⁺). The free base was converted to the hydrochloride salt: mp 180 °C (dec); [α]²⁰_D −75.4° (c 0.40, CH₃OH); Anal. Calcd for C₂₅H₂₈Cl₂N₂O·0.5H₂O: C, 66.37; H, 6.46; N, 6.19. Found: C, 66.38; H, 6.61; N, 5.87.

Compound 4d

oil; ¹H NMR (CDCl₃) δ 7.30−7.23 (m, 2H), 6.95 (d, J = 8.1 Hz, 2H), 6.86 (d, J = 8.1 Hz, 2H), 6.67−7.57 (m, 2H), 3.53−3.44 (m, 1H), 3.23−3.09 (m, 2H), 3.04 (dd, J = 6.0, 2.0 Hz, 1H), 2.66 (ddd, J = 12.9, 12.9, 2.7 Hz, 1H), 2.26 (s, 3H), 2.25 (s, 3H), 2.24−2.04 (m, 2H), 2.02 (s, 3H), 1.78−1.63 (m, 3H); ¹³C NMR (CDCl₃) δ 169.4, 157.6, 139.0, 136.0, 133.7, 131.2, 129.4, 128.9, 128.8, 127.8, 117.4, 66.3, 62.2, 45.7, 42.1, 36.6, 36.0, 26.9, 25.2, 21.1, 10.6; MS (EI) m/z 406 (M⁺). The free base was converted to the hydrochloride salt: mp 245 °C (dec); [α]²⁰_D +51.1° (c 0.33, CH₃OH); Anal. Calcd for C₂₅H₂₈Cl₂N₂O·1.25H₂O: C, 64.45; H, 6.60; N, 6.01. Found: C, 64.50; H, 6.42; N, 5.92.

5.6. 3β-(4-Chlorophenyl)-2β-[3’-(4-methylbenzyl)isoxazol-5-yl]tropane (3e) and 3β-(4-chlorophenyl)-2β-[3’-methyl-4’-(4-methylphenyl)isoxazol-5-yl]tropane (4e)

The procedure for 3a and 4a was followed using 1.07 g (3.66 mmol) of 6a and 1.79 g (11.0 mmol) of 1-(4-methylphenyl)propan-2-one oxime to give 250 mg (17%) of 3e and 130 mg (9%) of 4e.

Compound 3e

oil; ¹H NMR (CDCl₃) δ 7.16−6.96 (m, 6H), 6.88−6.79 (m, 2H), 6.13 (s, 1H), 3.91 (d, J = 16.5 Hz, 1H), 3.80 (d, J = 16.5 Hz, 1H), 3.33−3.13 (m, 4H), 2.34 (s, 3H), 2.30−2.03 (m, 6H), 1.82−1.53 (m, 3H); ¹³C NMR (CDCl₃) δ 172.8, 162.3, 140.3, 136.0, 134.8, 132.1, 129.2, 128.9, 128.5, 128.2, 103.8, 65.4, 61.6, 46.3, 41.9, 35.5, 34.9, 32.0, 26.4, 25.0, 21.1; MS (EI) m/z 406 (M⁺). The free base was converted to the hydrochloride salt: mp 108 °C (dec); [α]²⁰_D −70.0° (c 0.37, CH₃OH); Anal. Calcd for C₂₅H₂₈Cl₂N₂O·0.5H₂O: C, 66.37; H, 6.46; N, 6.19. Found: C, 66.26; H, 6.64; N, 6.04.

Compound 4e

oil; ¹H NMR (CDCl₃) δ 7.20−7.03 (m, 4H), 6.93 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 8.4 Hz, 2H), 3.52−3.44 (m, 1H), 3.27−2.96 (m, 3H), 2.67 (ddd, J = 12.0, 12.0, 2.1 Hz, 1H), 2.36 (s, 3H), 2.32 (s, 3H), 2.26−2.07 (m, 2H), 2.03 (s, 3H), 1.76−1.62 (m, 3H); ¹³C NMR (CDCl₃) δ 168.5, 158.0, 140.8, 137.4, 132.0, 129.5, 129.4, 129.2, 128.2, 127.6, 118.3, 66.4, 62.0, 45.4, 42.1, 36.2, 35.8, 26.8, 25.1, 21.3, 10.6; MS (EI) m/z 406 (M⁺). The free base was converted to the hydrochloride salt: mp 248 °C (dec); [α]²⁰_D +61.5° (c 0.35, CH₃OH); Anal. Calcd for C₂₅H₂₈Cl₂N₂O·0.5H₂O: C, 66.37; H, 6.46; N, 6.19. Found: C, 66.62; H, 6.42; N, 6.07.

5.7. 3β-(4-Methylphenyl)-2β-[3’-(4-methylbenzyl)isoxazol-5-yl]tropane (3f) and 3β-(4-methylphenyl)-2β-[3’-methyl-4’-(4-methylphenyl)isoxazol-5-yl]tropane (4f)

The procedure for 3a and 4a was followed using 1.00 g (3.66 mmol) of 6b and 1.79 g (11.0 mmol) of 1-(4-methylphenyl)propan-2-one oxime to give 550 mg (39%) of 3f and 105 mg (7%) of 4f.

Compound 3f

oil; ¹H NMR (CDCl₃) δ 7.10 (d, J = 8.1 Hz, 2H), 7.01 (d, J = 8.1 Hz, 2H), 6.95 (d, J = 7.8 Hz, 2H), 6.81 (d, J = 7.8 Hz, 2H), 6.15 (s, 1H), 3.92 (d, J = 15.0 Hz, 1H), 3.81 (d, J = 15.0 Hz, 1H), 3.35−3.13 (m, 4H), 2.33 (s, 3H), 2.30−2.02 (m, 9H), 1.85−1.50 (m, 3H); ¹³C NMR (CDCl₃) δ 173.1, 161.9, 138.1, 135.6, 135.4, 134.8, 128.9, 128.6, 128.4, 127.2, 103.4, 65.2, 61.5, 46.3, 41.8, 35.4, 34.8, 31.7, 26.2, 24.8, 20.9, 20.8; MS (EI) m/z 386 (M⁺). The free base was converted to the hydrochloride salt: mp 193 °C (dec); [α]²⁰_D −61.9° (c 0.43, CH₃OH); Anal. Calcd for C₂₆H₃₁ClN₂O·0.75H₂O: C, 71.54; H, 7.50; N, 6.42. Found: C, 71.61; H, 7.37; N, 6.34.

Compound 4f

oil; ¹H NMR (CDCl₃) δ 7.11 (d, J = 7.8 Hz, 2H), 6.94 (d, J = 7.8 Hz, 2H), 6.89 (d, J = 8.1 Hz, 2H), 6.61 (d, J = 8.1 Hz, 2H), 3.50−3.41 (m, 1H), 3.22−3.08 (m, 3H), 2.68 (ddd, J = 12.3, 12.3, 2.1 Hz, 1H), 2.40 (s, 3H), 2.24 (s, 6H), 2.21−2.06 (m, 2H), 2.01 (s, 3H), 1.72−1.60 (m, 3H); ¹³C NMR (CDCl₃) δ 169.1, 157.8, 139.2, 137.2, 135.7, 129.6, 129.2, 128.8, 127.9, 127.7, 118.1, 66.5, 62.1, 45.6, 42.1, 36.4, 36.1, 26.8, 25.2, 21.3, 21.1, 10.6; MS (EI) m/z 386 (M⁺). The free base was converted to the hydrochloride salt: mp 249 °C (dec); [α]²⁰_D +44.7° (c 0.34, CH₃OH); Anal. Calcd for C₂₆H₃₁ClN₂O·1.25H₂O: C, 70.10; H, 7.58; N, 6.29. Found: C, 70.22; H, 7.36; N, 6.35.

5.8. 3β-(4-Chlorophenyl)-2β-[3’-(4-methoxybenzyl)isoxazol-5-yl]tropane (3g) and 3β-(4-chlorophenyl)-2β-[3’-methyl-4’-(4-methoxyphenyl)isoxazol-5-yl]tropane (4g)

The procedure for 3a and 4a was followed using 1.07 g (3.66 mmol) of 6a and 1.97 g (11.0 mmol) of 1-(4-methoxylphenyl)propan-2-one oxime to give 510 mg (33%) of 3g and 101 mg (7%) of 4g.

Compound 3g

oil; ¹H NMR (CDCl₃) δ 7.14−6.99 (m, 4H), 6.90−6.80 (m, 4H), 6.10 (s, 1H), 3.89 (d, J = 15.6 Hz, 1H), 3.81 (s, 3H), 3.77 (d, J = 15.6 Hz, 1H), 3.34−3.13 (m, 4H), 2.30−2.05 (m, 6H), 1.83−1.56 (m, 3H); ¹³C NMR (CDCl₃) δ 172.9, 162.6, 158.4, 140.4, 132.1, 130.0, 129.7, 128.9, 128.3, 114.0, 103.8, 65.4, 61.7, 55.4, 46.4, 42.0, 35.5, 35.0, 31.6, 26.5, 25.1; MS (EI) m/z 422 (M⁺). The free base was converted to the hydrochloride salt: mp 178 °C (dec); [α]²⁰_D −62.4° (c 0.34, CH₃OH); Anal. Calcd for C₂₅H₂₈Cl₂N₂O₂·0.75H₂O: C, 63.49; H, 6.29; N, 5.92. Found: C, 63.20; H, 6.38; N, 5.78.

Compound 4g

white solid; mp 58.0–59.0 °C; ¹H NMR (CDCl₃) δ 7.14−7.08 (m, 2H), 6.95−6.80 (m, 4H), 6.72−6.63 (m, 2H), 3.82 (s, 3H), 3.52−3.42 (m, 1H), 3.24−3.06 (m, 3H), 2.66 (ddd, J = 12.3, 12.3, 2.4 Hz, 1H), 2.25 (s, 3H), 2.24−2.05 (m, 2H), 2.02 (s, 3H), 1.75−1.62 (m, 3H); ¹³C NMR (CDCl₃) δ 168.6, 159.2, 158.1, 140.9, 132.0, 130.9, 129.2, 128.2, 122.9, 118.1, 114.2, 66.5, 62.0, 55.4, 45.5, 42.1, 36.3, 35.8, 26.9, 25.2, 10.6; MS (EI) m/z 422 (M⁺). The free base was converted to the hydrochloride salt: mp 243 °C (dec); [α]²⁰_D +69.4° (c 0.39, CH₃OH); Anal. Calcd for C₂₅H₂₈Cl₂N₂O₂·1.25H₂O: C, 62.31; H, 6.38; N, 5.81. Found: C, 62.34; H, 6.42; N, 5.77.

5.9. 3β-(4-Methylphenyl)-2β-[3’-(4-methoxybenzyl)isoxazol-5-yl]tropane (3h) and 3β-(4-methylphenyl)-2β-[3’-methyl-4’-(4-methoxyphenyl)isoxazol-5-yl]tropane (4h)

The procedure for 3a and 4a was followed using 1.00 g (3.66 mmol) of 6b and 1.97 g (11.0 mmol) of 1-(4-methoxylphenyl)propan-2-one oxime to give 631 mg (43%) 3h and 52.0 mg (4%) of 4h.

Compound 3h

oil; ¹H NMR (CDCl₃) δ 7.08−6.90 (m, 4H), 6.86−6.77 (m, 4H), 6.13 (s, 1H), 3.89 (d, J = 15.3 Hz, 1H), 3.80 (s, 3H), 3.79 (d, J = 15.3 Hz, 1H), 3.32−3.10 (m, 4H), 2.30−2.01 (m, 9H), 1.84−1.53 (m, 3H); ¹³C NMR (CDCl₃) δ 173.2, 162.2, 158.2, 138.6, 135.6, 130.0, 129.6, 128.7, 127.3, 113.8, 103.5, 65.3, 61.7, 55.1, 46.4, 41.9, 35.5, 34.9, 31.4, 26.3, 24.9, 21.0; MS (EI) m/z 402 (M⁺). The free base was converted to the hydrochloride salt: mp 168 °C (dec); [α]²⁰_D −68.7° (c 0.53, CH₃OH); Anal. Calcd for C₂₆H₃₁ClN₂O₂·1.5H₂O: C, 67.01; H, 7.35; N, 6.01. Found: C, 66.72; H, 7.33; N, 5.80.

Compound 4h

oil; ¹H NMR (CDCl₃) δ 7.00−6.76 (m, 6H), 6.66−6.55 (m, 2H), 3.82 (s, 3H), 3.50−3.40 (m, 1H), 3.20−3.02 (m, 3H), 2.67 (ddd, J = 12.3, 12.3, 2.1 Hz, 1H), 2.24 (s, 6H), 2.21−2.05 (m, 2H), 2.01 (s, 3H), 1.75−1.60 (m, 3H); ¹³C NMR (CDCl₃) δ 169.1, 159.1, 158.10, 139.3, 135.8, 131.0, 128.8, 127.8, 123.2, 117.9, 114.0, 66.5, 62.2, 55.4, 45.7, 42.2, 36.5, 36.2, 26.9, 25.2, 21.1, 10.6; MS (EI) m/z 402 (M⁺). The free base was converted to the hydrochloride salt: mp 238 ° C (dec); [α]²⁰_D +50.7° (c 0.28, CH₃OH); Anal. Calcd for C₂₆H₃₁ClN₂O₂·1.25H₂O: C, 67.67; H, 7.32; N, 6.07. Found: C, 67.56; H, 7.35; N, 5.96.