Synthesis and in vitro biological evaluation of carbonyl group-containing analogues for σ₁ receptors

Abstract

To identify the ligands for σ₁ receptors that are potent and selective, the analogues of prezamicol and trozamicol scaffolds of carbonyl-containing vesicular acetylcholine transporter (VAChT) inhibitors were explored. Of the 23 analogues synthesized and tested, 5 displayed very high affinity for σ₁ (K_i = 0.48 - 4.05 nM) and high selectivity for σ₁ relative to σ₂ receptors (σ₁/σ₂ selectivity >749-fold). Four of the five compounds (14a, 14b, 14c and 14e) showed very low affinity for VAChT (K_i >290 nM) and the fifth compound (14g) showed moderate affinity for VAChT (K_i = 44.2 nM). The compound [1'-(4-fluorobenzyl)-3'-hydroxy[1,4']bipiperidinyl-4-yl]-(4-fluorophenyl)-methanone (14a) displayed very high affinity and selectivity for σ₁ receptor (K_i = 0.48 nM, σ₁/σ₂ >3600). All four of these most promising compounds (14a, 14b, 14c and 14e) can be radiosynthesized with fluorine-18 or carbon-11, which will allow further evaluation of their properties as PET probes for imaging σ₁ receptor in vivo.

1. Introduction

Sigma (σ) receptors were discovered in the 1970s,¹ and they were found to exhibit σ₁ and σ₂ subtypes.^{2, 3} Instead of opioid receptors as originally thought, they now are widely accepted as unique and independent.⁴ The σ₁ receptor shares no homology with other mammalian proteins⁵ and consists of 223 amino acids. It has been cloned and functionally expressed.⁶ The σ₂ receptor has not been cloned, and its structure has not been identified yet.⁷

Studies discovered that σ₁ receptors are highly expressed in the brain and several peripheral organs that include heart, spleen, kidney, liver, lung, ovary, testes, and placenta.^{8, 9} The σ₁ receptors are located in cytoplasmic, endoplasmic reticulum, and mitochondrial membranes.⁹ During development, elevated levels of σ₁ receptors were found in the embryonic stem cells at all stages of embryogenesis.⁹ In the central nervous system (CNS), the highest level of σ₁ transcripts is expressed in various cranial nerve nuclei, followed by mesencephalic structures that include red nucleus, periaqueductal gray matter and substantia nigra, and in some diencephalic structures that include habenula and arcuate, and paraventricular and ventromedial hypothalamic nuclei. Moderate levels of σ₁ transcripts were found in superficial and deeper cortical laminar layers, and mRNA of σ₁ receptor is found in the pyramidal cell layer and the dentate gyrus of the hippocampus formation.¹⁰ Also in the brain, thalamus and amygdaloid body express σ₁ receptor.¹⁰

Investigations have revealed that σ₁ receptor is involved in different functions associated with the central nervous, endocrine, motor and immune systems.^{11, 12} In the central nervous system, σ₁ receptor regulates neurotransmitter release, modulation of neurotransmitter receptor functions, learning and memory, and movement and posture control. Based on its broad modulatory effects,¹³ the σ₁ receptor is believed to play an important role in neuropsychiatric and neurodegenerative diseases.^14-16 Recent studies have shown that several σ₁ receptor agonists have positive therapeutic effects on diseases such as schizophrenia,¹⁷ major depression,¹⁶ Alzheimer's disease (AD),¹⁸ and substance abuse.¹⁹ Thus, σ₁ receptors are now considered as a therapeutic target for the treatment of depression, anxiety, schizophrenia and Alzheimer's disease.^20-22

Studies of cancer have revealed that σ₁ receptors are overexpressed in breast cancer, small cell and non-small cell lung carcinoma, renal carcinoma, colon carcinoma, sarcoma, brain tumors, melanoma, glioblastoma, neuroblastoma, and prostate cancers.^23-26 Based on the expression pattern of σ₁ receptors in numerous tumors, ligands for these receptors have potential for both imaging and therapy.²⁷ Administration of σ ligands inhibits ex vivo growth of tumor cells derived from human mammary adenocarcinoma, colon carcinoma, and melanoma.^{28, 29} For example, the σ₁ receptor ligand rimcazole inhibits cellular proliferation and induces cell death, although the mechanism is not clear.³⁰

The σ₁ receptor regulates the activity of diverse ion channels via protein-protein interactions, exhibits stereo-selective drug binding, and binds different types of drugs that exhibit structural diversity.^31-33 The activation of σ₁ receptors promotes both neuronal differentiation and anti-apoptotic action, potentially leading to cancer. The σ₁ receptor is a ligand-regulated mitochondrial membrane-associated endoplasmic reticulum (ER) protein receptor. σ₁ receptor ligands as potential therapeutic drugs may lead to decreased oxidative stress in mammalian cells, and may have antitumorigenic activity.³⁰ Specific σ₁ ligands that have high specific binding and suitable pharmaceutical properties may be potential therapeutic agents for the treatment of cancer.^{29, 34} Although the importance of σ₁ receptor in CNS disorders and cancer is recognized, details of the pathophysiological functions of σ₁ receptor in brain are still not clear.

Positron emission tomography (PET) is an elegant non-invasive imaging modality that can provide functional information about cellular processes. It has been used to quantify the density of σ₁ receptors in brain in vivo. A suitable clinical PET probe targeting the σ₁ receptors will advance our understanding of the functions of σ₁ receptors in the CNS and allow monitoring of the efficacy of disease treatment with σ₁ ligands.³⁵ Investigators have put tremendous efforts to identify PET probes for imaging σ₁ in vivo.^36-39 To date, the ¹¹C-labeled PET radiotracer [¹¹C]SA4503 is the only PET probe for imaging σ₁ receptor in neurological applications.^{40, 41} Compared to ¹¹C (t_1/2 = 20.4 min), ¹⁸F has a longer half-life (t_1/2 = 109.8 min) that places fewer time constraints on radiotracer synthesis and permits longer scan sessions usually resulting in higher target/non-target ratios. However, there is no report of an ¹⁸F-labeled probe for the σ₁ receptor that is clinically suitable.¹⁷ Identifying such a clinically suitable probe is imperative for diagnostic and therapeutic applications in CNS disorders and cancer.

In the search of highly selective and clinically suitable σ₁ ligands, investigations have revealed that high affinity σ₁ ligands must contain a basic amine having two hydrophobic appendages.^{7, 42} Structure-activity relationship analysis of vesamicol ligands (Figure 1), which bind to the vesicular acetylcholine transporter (VAChT) in presynaptic cholinergic nerve terminals, showed that many vesamicol ligands contain a pharmacophore that also have high affinity for σ₁ receptors.⁴³ In fact, some of the VAChT ligands show moderate to high binding affinities for σ₁ receptor. As shown in Figure 1, (-)-vasamicol (1) has moderate σ₁ receptor binding affinity value of 73.8 nM,⁴⁴ and its methyl substituted derivative (-)-p-methylvesamicol (2) has high σ₁ receptor binding affinity value of 8.10 nM;⁴⁴ the trozamicol analogues meta-iodobenzyltrozamicol (3) and-(+)-4-fluorobenzyltrozamicol (4) have σ₁ receptor binding affinity values of 92 and 21.6 nM respectively.^{43, 45} Several carbonyl containing VAChT analogues also bind to σ₁ very well; for example, the binding affinity of (1S,2S)-2-(4-(5-iodothiophen-2-yl)piperidin-1-yl)cyclohexanol (5) was high with a Ki value of 9.39 nM for σ₁ receptors.⁴⁶ To identify new, highly selective ligands for σ₁ receptors, our strategies are to (1) replace the 4-phenylpiperidinyl group in prezamicol structure with a 4-substituted benzoylpiperidinyl group which is known to favor σ receptors,⁴⁶ (2) alkylate or acylate the secondary amine in (3'-hydroxy-1,4'-bipiperidin-4-yl)(4-substituted phenyl)methanone structurally similar to the prezamicol scaffold, and (3) alkylate the secondary amine in (4'-hydroxy-1,3'-bipiperidin-4-yl)(4-substituted phenyl)methanone, which is structurally similar to the trozamicol scaffold. In the current paper, we detail our exploration of these new compounds to identify the ligands that have high affinity for σ₁ receptors and selectivity for σ₁ versus σ₂ receptors. This investigation was inspired by 1) the observation that σ₁ receptor ligands may be potential therapeutic drugs for the treatment of neurological disorders⁴ and cancer,³⁴ and 2) the need for highly selective and potent σ₁ receptor ligands that can be labeled with F-18 or C-11. Moreover, a novel clinical PET probe for imaging the σ₁ receptor will provide a unique tool to assess the relationship between changes in σ₁ receptors in the brain during the progression of CNS disorders and provide a useful tool to monitor the treatment efficacy of the CNS disorders and cancer.

Figure 1.

Representative structures which are potent for σ₁ receptors

2. Results and Discussion

2.1. Chemistry

The target compounds were synthesized as depicted in Schemes 1–3. The synthesis started with 1,2,3,6-tetrahydropyridine, in which the secondary amine was first protected by reacting with benzoyl chloride to form benzamide 10. The alkenyl double bond of compound 10 was oxidized by meta-chloroperoxybenzoic acid (m-CPBA) to form epoxide 11. The epoxide 11 was refluxed with substituted 4-benzoylpiperidine hydrochloride salts, namely, either 4-(4'-fluorobenzoyl)piperidine hydrochloride or 4-(4'-methoxybenzoyl)piperidine hydrochloride, in ethanol with triethylamine as the base to afford tertiary amino alcohol intermediates. To improve the yield, commercially available 4'-substituted 4-benzoylpiperidine hydrochloride salts were used in excess and the reaction temperature was kept below 75 °C. This reaction condition afforded (3'-hydroxy-1,4'-bipiperidin-4-yl)(4-substituted phenyl)methanone intermediates and their regioisomers, (4'-hydroxy-1,3'-bipiperidin-4-yl)(4-substituted phenyl)methanone intermediates. The separation of these regioisomers by silica gel chromatography was difficult. To overcome the difficulty, the mixture of regioisomers was reacted with acetic anhydride to convert the free hydroxyl group to the corresponding acetates 12a–d. Subsequently, 12a and 12b were separated easily by silica gel chromatography.

Scheme 1.

Synthesis of compounds 13a-d

Scheme 3.

Synthesis of compounds 16a-h

Under strongly acidic conditions, hydrolysis of 12a and 12b to remove both the acetyl and benzoyl groups afforded key intermediates 13a and 13b. A similar procedure was used to make corresponding regioisomers 13c and 13d. Intermediates 13a and 13c are similar in structure to prezamicol, and 13b and 13d are similar in structure to trozamicol (Scheme 1). Starting with key intermediates 13a and 13c, the target compounds 14a–h were obtained via N-alkylation with various substituted benzyl halides in yields ranging from 34% to 84%. To assign the regio- and relative stereochemistry of the compounds obtained in the oxirane ring opening reaction, X-ray crystal structure of compound, 14c was carried out. The regiochemistry (the nitrogen of piperidine ring is on C13 and hydroxyl group on C17, Figure 2) was assigned based on the x-ray structure of 14c. The oxirane ring opening reaction was performed under the basic conditions. As predicted, the relative stereochemistry of the ring opening resulted in the trans stereochemistry (C13 and C17 in Figure 2). All the substituted benzyl halides were obtained commercially and were used in slight excess (Scheme 2). Target compounds 15a–g were obtained by coupling various substituted benzoic acids with piperidine intermediates 13a and c using bis(2-oxo-3-oxazolidinyl)phosphonic chloride (BOP-Cl) in yields ranging from 22% to 90%. Compounds 16a–h were synthesized by following the similar procedure to synthesize compounds 14a–h, except that the trozamicolanalogues 13b and 13d were utilized (Scheme 3). All the products were converted into oxalates for determining the bioactivity affinities.

Figure 2.

Chemical structure and x-ray crystal structure of 14c

Scheme 2.

Synthesis of compounds 14a-h and 15a-g

2.2. Biological binding affinity studies

The σ₁ and ₂σ binding affinities (K_i nM) of the new compounds were determined by using the competitive inhibition method with tritiated σ ligands according to reported procedures.^{7, 42} The σ₁ binding sites were assayed by using guinea pig brain membranes with the selective radioligand (+)-[³H]pentazocine. The σ₂ binding sites were assayed in rat liver membranes, a rich source of these sites, with [³H]DTG in the presence of (+)-pentazocine (100 nM) to mask σ₁ sites. VAChT binding was assayed using highly expressed human VAChT assayed with homogenized and partially clarified PC12^123.7 cells by displacement of bound 5 nM (-)-[³H]vesamicol. Apparent dissociation constants for binding of the novel compounds in vitro are shown in Table 1.

Table 1.

Affinities of new analogues for σ₁ receptor, σ₂ receptor, and VAChT^a

Compounds	σ1 (Ki, nM)b	σ2 (Ki, nM)c	VAChT (Ki, nM)d	Selective ratio of σ1 vs. σ2	Selective ratio of σ1 vs. VAChT	LogPe
1f	73.8 ± 11.2	346 ± 37	4.4 ± 0.6	4.6	16.7	1.40
14a	0.48 ± 0.14	1741 ± 286	1360 ± 295	3627	2833	2.83
14b	4.03 ± 0.47	5521 ± 1352	3310 ± 907	1370	821	2.61
14c	1.36 ± 0.28	2301 ± 249	401 ± 42.0	1692	295	2.73
14d	22.8 ± 2.32	4208 ± 115	2030 ± 385	184	89	1.48
14e	2.51 ± 0.34	2788 ± 718	294 ± 16.1	1111	117	2.82
14f	25.9 ± 0.96	5157 ± 202	14800 ± 3460	199	569	2.61
14g	4.05 ± 0.88	3033 ± 248	44.2 ± 3.03	749	11	2.72
14h	59.64 ± 2.22	4540 ± 1606	137 ± 14.3	76	2.2	1.46
15a	3144 ± 140	8642 ± 812	3600 ± 499	2.7	1.1	1.92
15b	2238 ± 271	>10000	30900 ± 6400	> 4.5	14	2.25
15c	2833 ± 374	20450 ± 4887	683 ± 90	7.2	0.24	1.67
15d	7739 ± 662	13565 ± 1435	3340 ± 706	1.7	0.43	0.57
15e	2088 ± 154	32850 ± 443	3460 ± 476	15.7	1.66	1.90
15f	2061 ± 113	15862 ± 3045	555 ± 65.4	7.7	0.27	1.65
15g	26646 ± 8738	17041 ± 8626	372 ± 65.7	0.64	0.01	0.56
16a	50.0 ± 7.9	3443 ± 928	136 ± 13.8	69	2.72	1.98
16b	91.1 ± 19.9	4979 ± 507	1970 ± 196	54	22	1.73
16c	106 ± 28	832 ± 147	149 ± 22.5	7.8	1.4	1.88
16d	1159 ± 128	13018 ± 2626	237 ± 41.4	11	0.20	0.79
16e	137 ± 21	5598 ± 1033	48.6 ± 8.37	41	0.35	2.00
16f	297 ± 27	4208 ± 439	1080 ± 296	14	3.6	1.75
16g	208 ± 53	8539 ± 1900	35.5 ± 11.1	41	0.17	1.89
16h	2225 ± 168	17135 ± 3863	107 ± 11	7.7	0.40	0.80

^aK_i values (mean ± SEM) were determined in at least three experiments.

^bThe σ₁ binding assay used membrane preparations of guinea pig brain.

^cThe σ₂ binding assay used homogenates of rat liver.

^dThe VAChT binding assay used expressed human VAChT.

^eCalculated value at pH 7.4 by ACD/I-Lab, version 7.0. (Advanced Chemistry Development, Inc., Canada).

^fValues from ref 44.

Binding affinity data identified a number of potentially useful structure-activity trends. First, these new analogues generally have very low affinity for σ₂ receptors (K_i > 1000 nM). For VAChT, only compounds 14g, 16e, and 16g display moderate affinity of 44.2 ± 3.03, 48.6 ± 8.37, and 35.5 ± 11.1 nM. For the other ligands, the K_i values are greater than 100 nM. On the other hand, compounds 14a, 14b, 14c, 14e and 14g displayed very high affinity for σ₁ receptor (K_i <5 nM) and very good selectivity for σ₁ versus σ₂ receptors (>1000 fold). Among these latter compounds, 14a displayed the highest affinity for σ₁ receptors (K_i = 0.48 ± 0.14 nM), very high selectivity for σ₁ versus σ₂ receptors (>3000 fold), and for σ₁ receptors vs VAChT (2800 fold). These results suggest that 14a has high potency and high selectivity for σ₁ receptor.

More importantly, 14a has two fragments that contain a fluorine atom. One is in the para-position of the carbonyl group in 4-fluorobenzoylpiperidine fragment, which is easy to label by displacement of a nitro group with ¹⁸F^-. The other fluorine atom is in the para-position of the 1-(4-fluorobenzyl)piperidin-3-ol fragment. The position of this fluorine atom allows incorporation of fluorine-18 in two steps. The first step is to make 4-[¹⁸F]fluorobenzyl iodide,⁴⁷ and the second step is to make the final labeled radiotracer by N-alkylation of 13a with 4-[¹⁸F]fluorobenzyl iodide. Labeling 14a with fluorine-18 at different positions also provides a unique way to investigate the metabolic stability of 14a in vivo by determining which fragment of the structure contains the radioactivity from fluorine-18. This information will be very useful for guiding further structural optimization of 14a to identify a metabolically stable PET tracer favorable for clinical use. In addition, the moderate lipophilicity of 14a (Log P value = 2.83) suggests that compound 14a has good ability to enter into the brain and has high potential to be a suitable PET imaging probe or therapeutic agent for CNS disorders.

Comparing compounds 14a–h with 15a–g, N-benzyl substituted ligands displayed significantly higher binding affinities for σ receptors, particularly for σ₁ receptor sites. Whereas, N-benzoyl substituted ligands displayed low binding affinities for σ receptor. For example, when the 4-fluorobenzoyl group in 15a was replaced by the 4-fluorobenzyl group in 14a, the σ₁ receptor affinity had increased dramatically, changing the K_i value from 3144 nM for 15a to 0.48 nM for 14a. The same trend was observed in all other corresponding compounds. In general, compounds containing substituted benzyl groups preferred to bind to σ₁ receptor sites; 14a–h, displayed very high σ₁ binding affinities in which the K_i values ranged from 0.48 nM for 14a to 59.6 nM for 14h. However, compound 14g showed moderate VAChT binding affinity (44.2 ± 3.03 nM), whereas the other compounds 14a–f and 14h displayed lower VAChT binding affinities. The observation that compounds containing the substituted benzoyl groups 15a–g led to dramatic decrease in σ₁ receptor binding affinities (K_i >1000 nM) is consistent with results reported in the literature.⁴² This confirms that a carbonyl group between the substituted aromatic ring and the piperidine moiety plays an important role by affecting σ₁ receptor binding affinities. Incorporating the carbonyl group leads to more rigidity in the structure and might prevent the interaction of ligands with the σ₁ receptor binding site.

When the electron-withdrawing group fluoride (14a–d) was replaced with the electron-donating group methoxy (14e–h) at the para-position of the 4-substitued benzoylpiperidine fragment, the σ₁ binding affinities (nM) were decreased. For compounds 14a–d, σ₁ binding affinities (nM) were 0.48 ± 0.14, 4.03 ± 0.47, 1.36 ± 0.28 and 22.8 ± 2.32 respectively; for compounds 14e–h, the σ₁ binding affinities (nM) were 2.51 ± 0.34, 25.9 ± 0.96, 4.05 ± 0.88 and 59.64 ± 2.22 respectively. The decrease in affinity ranged from 2.6 fold for 14h vs. 14d to 6.4 fold for 14b vs. 14f.

Comparing the structures of 14a, 14b, and 14c, the only difference among them is the substitution at the para-position of the benzyl moiety on the terminal piperidine ring of (3'-hydroxy-1,4'-bipiperidin-4-yl)(4-fluorophenyl)methanone. The σ₁ K_i-values (nM) are displayed in the order of -F > -H > -OCH₃ with the dissociation constant values being 0.48 ± 0.14 for 14a, 1.36 ± 0.28 for 14c and 4.03 ± 0.47 for 14b. Compared with 14b, incorporating an electron-withdrawing fluorine atom at the 4-position of the benzyl group (14a) diminished the basicity of the nitrogen atom in the terminal pipeidine ring and the ligand affinity for σ₁ receptor was increased by 2.8 fold; on the contrary, incorporating an electron-donating methoxyl group (14b) enhanced the basicity of the nitrogen atom in the terminal pipeidine ring and the ligand affinity for σ₁ receptor was decreased by approximately 3 fold. A similar trend was observed in 4-methoxybenzoyl containing compounds 14e, 14f and 14g. To further test the effect of the electron density on the benzyl ring, compounds 14d and 14h containing the relatively high electron-density pyridine ring, with the N-atom in the meta-position, were synthesized and screened. However, decrease in σ₁ binding affinities was observed. For 14d, the σ₁ binding affinity was decreased more than 17 fold as expressed by K_i value changing from 1.36 ± 0.28 nM for 14c to 22.8 ± 2.32 nM for 14d. For 14h, the σ₁ binding affinity was decreased 15 fold as expressed by K_i value changing from 4.05 ± 0.88 nM for 14g to 59.64 ± 2.22 nM for 14h. To clearly understand how the electron density of the benzyl ring affects basicity of nitrogen atom in the piperidine rings resulted in the changes of σ₁ binding affinity, further structure-affinity investigation of these analogues is necessary.

The compounds with prezamicol scaffold (14a–h) displayed substantially higher affinities for σ₁ receptors than that of trozamicol scaffold (16a–h). The only structural difference between prezamicol and trozamicol is the position of the nitrogen atom in the terminal piperidine ring. Some trozamicol scaffold analogues displayed moderate σ₁ receptor binding affinities, for example 16a (50.0 ± 7.9 nM) and 16b (91.1 ± 19.9 nM). The difference in binding affinities for prezamicol and trozamicol analogues is 11 fold for 14f vs. 16f (25.9 ± 0.96 nM vs. 297 ± 27 nM) and 104 fold for the most potent 14a vs. 16a (0.48 ± 0.14 nM vs. 50.0 ± 7.9 nM).

Since our initial in vitro screening showed compound 14a (K_i-_σ1 = 0.48 ± 0.14 nM, 3627-fold for σ₁ vs. σ₂,-2833 fold for σ₁ vs. VAChT) and 14e (K_i-_σ1 = 2.51 ± 0.34 nM, 1111-fold for σ₁ vs. σ_2, 117-fold for σ₁ vs. VAChT) had high binding specificity for σ₁ receptors; both the compounds 14a and 14e will be easy to label with carbon-11 or fluorine-18 to test the feasibility of imaging σ₁ receptors in animals. We also determined the D₂ and D₃ affinities for both 14a and 14e and 5-HT_1A affinity for 14a. The in vitro binding data suggested that 14a and 14e had very low affinities for D₂, D₃ and 5HT_1A as shown in Table 2. This observation eliminates the concern that D₂, D₃ and 5-HT_1A receptor binding might interfere with the imaging signal when a C-11 or F-18 labeled ligands of 14a or 14e were used for PET imaging studies of the σ₁ receptors in vivo.

Table 2.

D₂, D₃ and 5-HT_1A Affinities (Ki ± SD, nM) of compound 14a and 14e^a

Compounds	σ1 (Ki, nM)	D2 (Ki, nM)b	D3 (Ki, nM)b	5-HT1A (Ki, nM)c
14a	0.48 ± 0.14	1350 ± 53	>20,000	2540 ± 5.6
14e	2.51 ± 0.34	1489 ± 114	>15,000	ND

^aK_i values (mean ± SEM) were determined in at least three experiments

^bK_i values (mean ± SEM) for D₂, D₃ binding assay were determined according our published procedure.⁴⁸

^cK_i values (mean ± SEM) for 5-HT_1A binding assay were determined according the protocol with minor modification.⁴⁹

Overall, (1) N-alkylation with benzyl groups of (3'-hydroxy-1,4'-bipiperidin-4-yl)(4-substituted phenyl)methanone to form tertiary amines generally gives very high σ₁ receptor binding affinities and high selectivity for σ₁ receptor vs. σ₂ receptor and VAChT sites, (2) N-acylation with benzoyl groups of (3'-hydroxy-1,4'-bipiperidin-4-yl)(4-substituted phenyl)methanone to form tertiary amides diminishes the σ₁ receptor binding affinity, and (3) prezamicol analogues display high σ₁ receptor binding affinities, whereas, trozamicol analogues display moderate to low σ₁ receptor binding affinities. Although it is well known that σ₁ receptor distinguishes between enantiomers,^{31, 32} only racemic mixtures are used in this study. Further study will be focused on resolving the racemic mixtures to obtain enantiomeric isomers and conduct the binding affinity screening for the enantiomeric isomers. It is expected that enantiomers of compounds 14a–c and 14e will provide compounds with improved σ₁ binding affinity and selectivity. In addition, the in vitro affinities of 14a and 14a-c and 14e binding to other neurotransmitter receptors, transporters, enzymes and ion channels will be performed to determine if 14a-c and 14e bind to σ₁ receptor specifically.

3. Conclusion

In the present study, we synthesized a new class of compounds targeting σ₁ receptors. In an effort to identify compounds that have high affinity and selectivity for σ₁ receptors, we explored carbonyl containing VAChT inhibitors by introducing substituted benzyl or benzoyl groups on the nitrogen of the terminal piperidine ring. From this study, we have identified five racemic compounds, 14a (0.48 nM), 14b (4.03 nM), 14c (1.36 nM), 14e (2.51 nM), and 14g (4.05 nM), that have very high binding affinities for σ₁ receptors. The first four compounds also displayed high selectivity for σ₁ vs. σ₂ receptors and do not bind well to VAChT. They have the potential to be useful pharmacological agents targeting σ₁ receptors. More importantly, introducing fluorine-18 or carbon-11 isotopes into these compounds can be achieved easily. This accomplishment should permit further evaluation of these ligands to identify clinical PET probes for imaging σ₁ receptors in human beings. Particularly for 14a and 14c, the σ₁/σ₂ ratios are greater than 3600 and 1600 fold and the σ₁/VAChT ratios are greater than 2800 and 290 fold, respectively. After screening the in vitro binding affinities of 14a-c and 14e with other neurotransmitter receptors, transporters, enzymes and ion channels to confirm the σ₁ receptor binding specificity, the fluorine-18 or carbon-11 analogues of 14a-c and 14e will be synthesized to test their feasibility as useful PET probes to quantify the density of σ₁ receptors in human beings. In addition, the SAR information from the current study provides new insight into σ₁ receptor ligands.

4. Experimental Section

General

All reagents and chemicals were purchased from commercial suppliers and used without further purification unless otherwise stated. All anhydrous reactions were carried out in an oven-dried and nitrogen purged glassware unless otherwise stated. Flash column chromatography was conducted using silica gel 60A, “40 Micron Flash” [32-63 μm] (Scientific Absorbents, Inc.); the mobile phase used is reported in the experimental procedure for each compound. Melting points were determined using the MEL-TEMP 3.0 apparatus and are uncorrected. ¹H NMR spectra were recorded at 300 MHz on a Varian Mercury-VX spectrometer with CDCl₃ as solvent and tetramethylsilane (TMS) as the internal standard unless otherwise stated. All chemical shift values are reported in parts per million (PPM) (δ). Peak multiplicities are singlet, s; doublet, d; triplet t; multiplet, m; broad, br. Elemental analyses (C, H, N) were determined by Atlantic Microlab, Inc. Elemental analysis and HPLC were used to determine the purity of the target compounds that were used for binding assay. All the final compounds reported the biologic activity in the manuscript have a purity of ≥ 95%.

(5,6-Dihydropyridin-1(2H)-yl)(phenyl)methanone (10)

Into a solution of 1,2,3,6-tetrahydropyridine (1.00 g, 12.0 mmol) and triethylamine (3 mL) in CH₂Cl₂ (30 mL), benzoyl chloride (1.69 g, 12.0 mmol) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 2 hrs and washed with saturated Na₂CO₃ (20 mL × 3) and brine solution (20 mL), dried over Na₂SO₄ and concentrated under vacuum to afford crude product. The crude product was purified by silica gel column chromatography using hexane/ethyl acetate (10/3, v/v) to afford target product 10 as a colorless oil (2.27 g, 99.5%). ¹H NMR (CDCl₃): δ 2.16–2.25 (m, 2H), 3.46–4.20 (m, 4H), 5.53–5.91 (m, 2H), 7.29–7.53 (m, 5H).

7-Oxa-3-azabicyclo[4.1.0]heptan-3-yl(phenyl)methanone (11)

Into a solution of 10 (2.27 g, 12.1 mmol) in CH₂Cl₂ (20 mL), a solution of 3-chloroperoxybenzoic acid (5.43 g, 77% pure, 24.3 mmol) in CH₂Cl₂ (40 mL) was added dropwise at 0 °C. The reaction mixture was stirred at room temperature for 5 hrs. After the reaction was complete as determined by TLC (hexane/ethyl acetate, 1/1, v/v), saturated Na₂CO₃ solution (50 mL) was added into the reaction vial slowly with stirring. The reaction mixture was stirred for 30 min. The organic layer was washed with saturated Na₂CO₃ solution (50 mL × 3), brine solution (50 mL), dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using hexane/ethyl acetate (3/1, v/v) as the mobile phase to afford the product as a sticky oil (1.54 g, 62%). ¹H NMR (CDCl₃): δ 2.04–2.18 (m, 2H), 3.09–4.40 (m, 6H), 7.28–7.47 (m, 5H).

Procedure A: General method for preparation of 1'-substituted benzoyl-4-acyl-[1,4'-bipiperidin]-3'-yl acetates (12a and 12c) and 1'-substituted benzoyl-4-acyl-[1,3'-bipiperidin]-4'-yl acetates (12b and 12d)

A mixture of 11 (0.50 g, 1.97 mmol), 4-(4-fluorobenzoyl)piperidine hydrochloride (1.20 g, 4.92 mmol), and triethylamine (1 mL) in ethanol (50 mL) was heated to 70 °C with stirring for over 24 hrs. The reaction mixture was cooled, filtered and concentrated under reduced pressure. The crude product was dissolved in CH₂Cl₂ (50 mL) and washed with water (50 mL), saturated Na₂CO₃ (50 mL) and brine solution. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure to afford crude product as an oil. Acetic anhydride (1.11 g, 10.92 mmol) was added into a solution of this crude product in CH₂Cl₂ (30 mL). The reaction mixture was stirred at room temperature overnight. The mixture was washed with saturated Na₂CO₃ (30 mL × 3) and brine solution. The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using ethyl acetate/hexane (2/1, v/v) as the mobile phase to afford 12a (0.41 g, 37%), the first eluted compound as a colorless oil. ¹H NMR (CDCl₃): δ : 1.40–1.74 (m, 3H), 2.04 (s, 3H), 2.30–2.37 (m, 1H), 2.51–2.69 (m, 2H, ), 2.86–3.21 (m, 6H), 4.30–4.70 (m, 1H), 4.80–5.10 (br s, 1H), 7.13 (t, J = 5.7 Hz, 2H), 7.40–7.41 (m, 5H), 7.93–7.97 (m, 2H). At the same time, 1'-benzoyl-4-(4-fluorobenzoyl)-[1,3'-bipiperidin]-4'-yl acetate (12b) (0.50 g, 45%) was eluted as the second compound and 12b was a colorless oil. ¹H NMR (CDCl₃): δ 1.50–1.90 (m, 3H), 2.07 (s, 3H), 2.81–3.29 (m, 6H), 2.20–2.80 (m, 4H), 3.50–4.00 (m, 2H), 4.64 (br s, 1H), 5.10–5.18 (m, 1H), 7.12 (t, J = 5.7 Hz, 2H), 7.41–7.42 (m, 5H), 7.93 (m, 2H).

1'-Benzoyl-4-(4-methoxybenzoyl)-[1,4'-bipiperidin]-3'-yl acetate (12c) and 1'-benzoyl-4-(4-methoxybenzoyl)-1,3'-bipiperidin-4'-yl acetate (12d)

Procedure A was followed with 4-(4-methoxybenzoyl)piperidine hydrochloride, 11, triethylamine and acetic anhydride to afford 12c (0.42 g, 37%) as a colorless oil that was eluted first and 12d (0.34 g, 37%) as a colorless oil that was eluted second. The ¹H NMR of 12c (CDCl₃): δ 1.40–1.90 (m, 8H), 2.04 (s, 3H), 2.22–2.40 (m, 1H), 2.24–2.70 (m, 2), 2.80–3.22 (m, 4H), 3.87 (s, 3H), 4.00–4.60 (m, 1H), 6.94 (d, J = 8.7 Hz, 2H), 7.41 (s, 5H), 7.91 (d, J = 8.7 Hz, 2H). The ¹H NMR of 12d (CDCl₃): δ 1.45–1.90 (m, 7H), 2.04 (s, 3H), 2.22–2.75 (m, 2H), 2.80–3.30 (m, 5H), 3.64 (br s, 1H), 3.86 (s, 3H), 4.64 (br s, 1H), 5.10–5.20 (m, 1H), 6.92 (d, J = 8.1 Hz, 2H), 7.64 (br s, 5H), 7.89 (m, 2H).

Procedure B. General method for preparation of (3'-hydroxy-[1,4'-bipiperidin]-4-yl)(4-substituted-phenyl)methanones (13a and 13c) and (4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-substituted-phenyl)methanones (13b and 13d)

To a solution of 12a (0.66 mmol) in ethanol (10 mL), 6N HCl (4 mL, 2.40 mmol) was added. The reaction mixture was heated to reflux for 14 hours. The mixture was cooled and concentrated under vacuum. 1N NaOH solution (20 mL) was added to the residue, the aqueous phase was extracted with CH₂Cl₂ (20 mL × 3).The combined organic phases were washed with saturated sodium carbonate (20 mL × 3) and brine solution, dried over Na₂SO₄ and concentrated under vacuum. The crude product was purified by silica gel column chromatography using methanol/ethyl acetate/triethylamine (50/50/2, v/v/v) as the mobile phase to afford 13a as a colorless oil (132 mg, 66%).¹H NMR (CDCl₃): δ 1.37–1.89 (m, 8H), 2.24–2.77 (m, 6H), 2.98–3.01 (m, 1H), 3.12–3.24 (m, 2H), 3.35–3.51 (m, 2H), 7.11–7.17 (t, J = 5.7 Hz, 2H), 7.94–7.99 (m, 2H).

(3'-Hydroxy-[1,4'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (13b)

Procedure B was followed with 12b and 6N HCl to afford 13b as a colorless oil (173 mg, 76%). ¹H NMR (CDCl₃ + CD₃OD): δ 1.56–1.92 (m, 7H), 2.41–3.47 (m, 11H), 3.62–3.70 (m, 1H), 3.87 (s, 3H), 6.99–7.02 (m, 2H), 7.94–7.98 (m, 2H).

(1'-Benzoyl-4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-fluorophenyl)methanone (13c)

Procedure B was followed with 12c to afford 13c as a colorless oil (0.22 g, 67%). ¹H NMR (CDCl₃ + CD₃OD): δ 1.43–1.51 (m, 1H), 1.74–1.90 (m, 5H), 2.10–2.12 (m, 1H), 2.29–2.40 (m, 2H), 2.49–2.61 (m, 2H), 2.77–2.88 (m, 2H), 3.03–3.06 (m, 2H), 3.17–3.26 (m, 2H), 3.51–3.59 (m, 1H), 7.14 (t, J = 5.4 Hz, 2H), 7.94–7.98 (m, 2H).

(1'-Benzoyl-4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (13d)

Procedure B was followed with 12d to afford 13d as a colorless oil (0.18 g, 82%). ¹HNMR (CDCl₃ + CD₃OD): δ 1.56–1.92 (m, 6H), 2.07–2.11 (m, 1H), 2.41–2.80 (m, 5H), 2.80–3.00 (m, 1H), 3.00–3.18 (m, 2H), 3.20–3.40 (m, 2H), 3.62–3.70 (m, 1H), 3.87 (s, 3H), 6.99–7.02 (m, 2H), 7.94–7.98 (m, 2H).

Procedure C. General method for preparation of benzyl compounds (14a-h) (1'-(4-Fluorobenzyl)-3'-hydroxy-[1,4'-bipiperidin]-4-yl)(4-fluorophenyl)methanone (14a)

Into a solution of 13a (132 mg, 0.42 mmol) and triethylamine (17.1 mg, 1.69 mmol) in CH₂Cl₂ (15 mL), a solution of 4-fluorobenzyl bromide (80 mg, 0.42 mmol) in CH₂Cl₂ (5 mL) was slowly added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was washed with water (20 mL × 2) and brine solution (20 mL). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by silica gel column chromatograph using triethylamine/ethyl acetate (1/50, v/v) as mobile phase to afford 14a as a pale yellow oil (78.9 mg, 45%). ¹H NMR (CDCl₃): δ 1.53–2.04 (m, 9H), 2.20–2.32 (m, 2H), 2.71–2.76 (m, 2H), 2.91–3.01 (m, 2H), 3.17–3.22 (m, 2H), 3.51–3.63 (m, 3H), 6.96–7.02 (m, 2H), 7.10–7.17 (m, 2H), 7.23–7.27 (m, 2H), 7.93–7.98 (m, 2H). The free base was converted to the corresponding oxalate salt by dissolving it in acetone and mixing with 1 equivalent of oxalic acid in acetone. mp (oxalate salt): 234 °C (decomposed). Anal. (C₂₄H₂₈F₂N₂O₂•H₂C₂O₄•0.25H₂O) C, H, N.

(4-Fluorophenyl)(3'-hydroxy-1'-(4-methoxybenzyl)-[1,4'-bipiperidin]-4-yl)methanone (14b)

Procedure C was followed with 13a, triethylamine and 4-methoxybenzyl bromide to afford 14b (54 mg, 78%). ¹H NMR(CDCl₃): δ 1.55–1.87 (m, 7H), 2.23–2.27 (m, 2H), 2.70–2.75 (m, 2H), 2.92–3.00 (m, 2H), 3.19–3.24 (m, 2H), 3.48–3.60 (m, 4H), 3.80 (s, 3H), 6.83–6.86 (m, 2H), 7.10–7.21 (m, 4H), 7.93–7.98 (m, 2H). mp (oxalate salt): 237 °C (decomposed). Anal. (C₂₅H₃₁FN₂O₃•H₂C₂O₄•0. 5H₂O) C, H, N.

(1'-Benzyl-3'-hydroxy-[1,4'-bipiperidin]-4-yl)(4-fluorophenyl)methanone (14c)

Procedure C was followed with 13a, triethylamine and benzyl bromide to afford 14c (54 mg, 84%). ¹H NMR (CDCl₃): δ 1.57–2.03 (m, 8H), 2.17–2.28 (m, 2H), 2.71–2.76 (m, 2H), 2.93–3.01 (m, 2H), 3.20–3.25 (m, 2H), 3.50–3.63 (m, 4H), 7.10–7.16 (m, 2H), 7.24–7.31 (m, 5H), 7.93 –7.98 (m, 2H). mp (oxalate salt): 243 °C (decomposed). Anal. (C₂₄H₂₉FN₂O₂•H₂C₂O₄•0.25H₂O) C, H, N.

(4-Fluorophenyl)(3'-hydroxy-1'-(pyridin-3-ylmethyl)-[1,4'-bipiperidin]-4-yl)methanone (14d)

Procedure C was followed with 13a, triethylamine and pyridin-4-methyl bromide to yield 14d (19 mg, 50%). The mobile phase used for column chromatographic separation was triethylamine/ethyl acetate/methanol (1/9/1, v/v/v). ¹H NMR (CDCl₃): δ 1.71–2.02 (m, 8H), 2.24–2.28 (m, 2H), 2.74–2.76 (m, 2H), 2.91–3.00 (m, 2H), 3.17–3.21 (m, 2H), 3.50–3.60 (m, 4H), 7.10–7.17 ( m, 2H), 7.23–7.27 (m, 1H), 7.62–7.65 (m, 1H), 7.94–7.98 (m, 2H), 8.50–8.52 (m, 2H). mp (oxalate salt): 178 °C (decomposed). Anal. (C₂₃H₂₈FN₃O₂•1.5H₂C₂O₄•H₂O) C, H, N.

(1'-(4-Fluorobenzyl)-3'-hydroxy-[1,4'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (14e)

Procedure C was followed with 13c, triethylamine and 4-fluorobenzyl bromide to afford 14e (24.5 mg, 63%). ¹H NMR (CDCl₃): δ 1.56–2.28 (m, 8H), 2.18–2.30 (m, 2H), 2.70–2.74 (m, 2H), 2.92–2.99 (m, 2H), 3.19–3.23 (m, 2H), 3.48–3.62 (m, 4H), 3.87 (s, 3H), 6.83–6.95 (m, 4H), 7.18–7.21 (m, 2H), 7.90–7.94 (m, 2H). mp (oxalate salt): 231 °C (decomposed). Anal. (C₂₅H₃₁FN₂O₃•H₂C₂O₄•0.5H₂O) C, H, N.

(3'-Hydroxy-1'-(4-methoxybenzyl)-[1,4'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (14f)

Procedure C was followed with 13c, triethylamine and 4-methoxybenzyl bromide to afford 14f (30 mg, 73%). ¹H NMR (CDCl₃): δ 1.51–2.00 (m, 8H), 2.20–2.32 (m, 2H), 2.71–2.76 (m, 2H), 2.91–3.01 (m, 2H), 3.17–3.22 (m, 2H), 3.51–3.63 (m, 4H), 3.79 (s, 3H), 3.86 (s, 3H), 6.96–7.02 (m, 2H), 7.10–7.17 (m, 2H), 7.23–7.27 (m, 2H), 7.93–7.98 (m, 2H). mp (oxalate salt): 225 °C (decomposed). Anal. (C₂₅H₃₁FN₂O₃•H₂C₂O₄•0.5H₂O) C, H, N.

(1'-Benzyl-3'-hydroxy-[1,4'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (14g)

Procedure C was followed with 13c, triethylamine and benzyl bromide to afford 14g (32 mg, 34%). ¹H NMR (CDCl₃): δ 1.54–2.04 (m, 8H), 2.22–2.27 (m, 2H), 2.70–2.75 (m, 2H), 2.93–3.00 (m, 2H), 3.20–3.25 (m, 2H), 3.54–3.62 (m, 4H), 3.86 (s, 3H), 6.94 (d, J = 9 Hz, 2H), 7.26–7.31 (m, 5H), 7.93 (d, J = 9 Hz, 2H).. mp (oxalate salt): 234 °C (decomposed). Anal. (C₂₅H₃₂N₂O₃•2H₂C₂O₄).

(3'-Hydroxy-1'-(pyridin-3-ylmethyl)-[1,4'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (14h)

Procedure C was followed with 13c, triethylamine and pyridin-4-methyl bromide to prepare 14h (20 mg, 52%). The mobile phase used for column chromatographic separation was triethylamine/ethyl acetate/methanol (1/9/1, v/v/v). ¹H NMR (CDCl₃): δ 1.72–2.03 (m, 8H), 2.25–2.29 (m, 2H), 2.74–2.76 (m, 2H), 2.91–3.00 (m, 2H), 3.19–3.21 (m, 2H), 3.55–3.61 (m, 4H), 3.87 (s, 3H), 6.95 (d, J = 9 Hz, 2H), 7.23–7.27 (m, 1H), 7.62–7.65 (m, 1H), 7.93 (d, J = 9 Hz, 2H), 8.50–8.52 (m, 2H). The free base was converted to the oxalate salt. mp: 188 °C (decomposed). Anal (C₂₄H₃₁N₃O₃ •2H₂C₂O₄•0.5H₂O) C, H, N.

Procedure D. General method for preparation of benzamides (15a-g) (3'-Hydroxy-[1,4'-bipiperidine]-1',4-diyl)bis(4-fluorophenyl)methanone (15a)

Into a solution of 13a (50 mg, 0.163 mmol), BOP-Cl (100 mg, 0.40 mmol), and triethylamine (1 mL) in methylene chloride (30 mL) was added 4-fluorobenzoic acid (40 mg, 0.28 mmol). The reaction mixture was stirred overnight at room temperature. The reaction mixture was washed with saturated sodium carbonate (20 mL × 5) and brine solution (20 mL). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using triethylamine/ethyl acetate (1/50, v/v) as mobile phase to afford 15a (62 mg, 89%). ¹H NMR (CDCl₃): δ 1.54–1.93 (m, 7H), 2.27–2.34 (m, 1H), 2.44–2.52 (m, 1H), 2.80–2.98 (m, 5H), 3.23–3.26 (m, 1H), 3.30–3.60 (m, 1H), 4.80 (br s, 1H), 7.07–7.18 (m, 4H), 7.40–7.44 (m, 2H), 7.94–7.99 (m, 2H). The free base was converted to the oxalate salt. mp: 213 °C (decomposed). Anal. (C₂₄H₂₆F₂N₂O₅•H₂C₂O₄) C, H, N.

(4-(4-Fluorobenzoyl)-3'-hydroxy-[1,4'-bipiperidin]-1'-yl)(4-methoxyphenyl)methanone (15b)

Procedure D was followed with 13a, BOP-Cl, triethylamine and 4-methoxybenzoic acid to afford 15b (48 mg, 22%). ¹H NMR (CDCl₃) : δ 1.54–1.93 (m, 7H), 2.31–2.34 (m, 1H), 2.44 –2.51 (m, 1H), 2.79 –2.98 (m, 5H), 3.23–3.26 (m, 1H), 3.47 (m, 2H), 3.82 (s, 3H), 6.91 (d, J = 9 Hz, 2H), 7.14 (t, J = 6 Hz, 2H), 7.38 (d, J = 9 Hz, 2H), 7.94–7.99 (m, 2H). The free base was converted to the oxalate salt. mp: 206 °C (decomposed). Anal. (C₂₄H₂₇FN₂O₃•H₂C₂O₄•0.5H₂O) C, H, N.

(1'-Benzoyl-3'-hydroxy-[1,4'-bipiperidin]-4-yl)(4-fluorophenyl)methanone (15c)

Procedure D was followed with 13a, BOP-Cl, triethylamine and benzoic acid to afford 15c (0.34 g, 78%). ¹H NMR (CDCl₃): δ 1.75–1.80 (m, 2H), 1.80–1.95 (m, 4H), 2.20–2.40 (m, 1H), 2.40–2.60 (m, 1H), 2.60–3.00 (m, 5H), 3.10–3.30 (m, 1H), 4.10 (s br s, 1H), 4.86 (br s, 1H), 7.12–7.18 (m, 2H), 7.28–7.42 (m, 5H), 7.95–7.99 (m, 2H). The free base was converted to the oxalate salt. mp: 152 °C (decomposed). Anal. (C₂₄H₂₇FN₂O₃•H₂C₂O₄•H₂O) C, H, N.

(4-(4-Fluorobenzoyl)-3'-hydroxy-[1,4'-bipiperidin]-1'-yl)(pyridin-3-yl)methanone (15d)

Procedure D was followed with 13a, BOP Cl, triethylamine and pyridine-3-carboxylic acid to afford15d (33 mg, 83%). The mobile phase used for column chromatographic separation was triethylamine/ethyl acetate/methanol (1/9/1, v/v/v). ¹H NMR (CDCl₃): δ 1.44–2.11 (m, 6H), 2.20–2.40 (m, 1H), 2.40–2.60 (m, 1H), 2.60–3.10 (m, 5H), 3.10–3.30 (m, 1H), 3.38–3.85 (m, 2H), 3.90–4.30 (m, 1H), 4.80–5.10 (m, 1H), 7.12–7.18 (m, 2H), 7.34–7.38 (m, 1H), 7.73–7.75 (m, 1H), 7.95–7.99 (m, 2H), 8.66–8.68 (m, 2H). The free base was converted to the oxalate salt. mp: 215 °C (decomposed). Anal. (C₂₃H₂₆FN₃O₃•H₂C₂O₄•0.25H₂O) C, H, N.

(1'-(4-Fluorobenzoyl)-3'-hydroxy-[1,4'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (15e)

Procedure D was followed with 13c, BOP-Cl, triethylamine and 4-fluorobenzoic acid to prepare 15e (84 mg, 67%). ¹H NMR (CDCl₃): 1.30–1.98 (m, 5H), 2.16–2.33 (m, 1H), 2.34–2.41 (m, 1H), 2.45–3.65 (m, 8H), 3.77 (s, 3H), 4.03 (br s, 1H), 4.72 (br s, 1H), 6.83–6.87 (m, 2H), 6.97–7.03 (m, 2H), 7.29–7.35 (m, 2H), 7.81–7.85 (m, 2H). The free base was converted to the oxalate salt. mp: 216 °C (decomposed). Anal. (C₂₅H₂₉FN₂O₄•H₂C₂O₄) C, H, N.

(1'-Benzoyl-3'-hydroxy-[1,4'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (15f)

Procedure D was followed with 13c, BOP-Cl, triethylamine and benzoic acid to afford 15f (0.14 g. 80%). ¹H NMR (CDCl₃): δ 1.60–1.90 (m, 5H), 2.10–2.50 (m, 2H), 2.60–3.00 (m, 5H), 3.20–3.50 (m, 2H), 3.65 (s, 1H), 3.87 (s, 3H), 4.10 (s br, 1H), 5.30 (br s 1H), 6.94 (d, J = 8.7 Hz, 2H), 7.38–7.40 (m, 5H), 7.92 (d, J = 9 Hz, 2H). The free base was converted to the oxalate salt. mp: 188 °C (decomposed). Anal. (C₂₅H₃₀N₂O₈•H₂C₂O₄•1.5H₂O) C, H, N.

(3'-Hydroxy-1'-isonicotinoyl-[1,4'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (15g)

Procedure D was followed with 13c, BOP-Cl, triethylamine and pyridine-3-carboxylic acid to afford 15g (36 mg, 90%). The mobile phase used for column chromatographic separation was triethylamine/ethyl acetate/methanol (1/9/1, v/v/v). ¹H NMR (CDCl₃): δ 1.61–1.96 (m, 6H), 2.04–2.08 (m, 1H), 2.27–2.56 (m, 2H), 2.80–2.09 (m, 4H), 2.09–3.27 (m, 2H), 3.44–3.69 (m, 2H), 3.87 (s, 3H), 4.85–5.15 (m, 1H), 6.93–6.97 (m, 2H), 7.28–7.38 (m, 1H), 7.73–7.80 (m, 1H), 7.85–7.94 (m, 2H), 8.67–8.68 (m, 2H). The free base was converted to the oxalate salt. mp: 220°C (decomposed). EIMS: Calcd, 429.1843; Found, 429.1843. Anal. (C₂₄H₂₉N₃O₄•H₂C₂O₄) C, H, N.

(1'-(4-Fluorobenzyl)-4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-fluorophenyl)methanone (16a)

Procedure C was followed with 13b, triethylamine and 4-fluorobenzyl bromide to yield 16a as a pale yellow oil (28.7 mg, 43%). ¹H NMR (CDCl₃): δ 1.55–2.05 (m, 9H), 2.24–2.31 (m, 1H), 2.49–2.57 (m, 1H), 2.71–2.83 (m, 3H), 2.94–3.04 (m, 2H), 3.17–3.20 (m, 1H), 3.40–3.49 (m, 3H), 6.98–7.05 (m, 2H), 7.10–7.17 (m, 2H), 7.25–7.30 (m, 2H), 7.92–7.97 (m, 2H). The free base was converted to the oxalate salt. mp: 209 °C (decomposed). Anal. (C₂₄H₂₈F₂N₂O₂•H₂C₂O₄) C, H, N.

(4-Fluorophenyl)(4'-hydroxy-1'-(4-methoxybenzyl)-[1,3'-bipiperidin]-4-yl)methanone (16b)

Procedure C was followed with 13b, triethylamine and 4-methoxybenzyl bromide to afford 16b (28 mg, 45%). ¹H NMR (CDCl₃): δ 1.56–2.06 (m, 8H), 2.26–2.30 (m, 1H), 2.53– 2.56 (m, 1H), 2.72–2.86 (m, 3H), 3.00–3.03 (m, 2H), 3.17–3.20 (m, 1H), 3.40–3.50 (m, 4H), 3.81 (s, 3H), 6.86–6.89 (m, 2H), 7.10–7.27 (m, 4H), 7.92–7.97 (m, 2H). The free base was converted to the oxalate salt. mp: 204 °C (decomposed). Anal. (C₂₅H₃₁FN₂O₃•H₂C₂O₄) C, H, N.

(1'-Benzyl-4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-fluorophenyl)methanone (16c)

Procedure C was followed with 13b, triethylamine and benzyl bromide to afford 16c (40 mg, 62%). ¹H NMR (CDCl₃): δ 1.56–2.06 (m, 8H), 2.24–2.3 (m, 1H), 2.51–2.59 (m, 1H), 2.72–2.86 (m, 3H), 3.00–3.04 (m, 2H), 3.17–3.20 (m, 1H), 3.40–3.54 (m, 4H), 7.10 –7.15 (m, 2H), 7.26–7.33 (m, 5H), 7.92–7.97 (m, 2H). The free base was converted to the oxalate salt. mp: 200 °C (decomposed). Anal. (C₂₄H₂₉FN₂O₂•H₂C₂O₄) C, H, N.

(4-Fluorophenyl)(4'-hydroxy-1'-(pyridin-3-ylmethyl)-[1,3'-bipiperidin]-4-yl)methanone (16d)

Procedure C was followed with 13b, triethylamine and pyridine-4-methyl bromide to afford 16d (22 mg, 56%). ¹H NMR (CDCl₃): δ 1.56–2.08 (m, 8H), 2.24–2.32 (m, 1H), 2.48–2.53 (m, 1H), 2.70–3.04 (m, 5H), 3.19–3.59 (m, 5H), 7.13 (t, J = 5.7 Hz, 2H), 7.26–7.31 (m, 1H), 7.66–7.69 (m, 1H), 7.92–7.97 (m, 2H), 8.52–8.54 (m, 2H). The free base was converted to the oxalate salt. mp: 182 °C. Anal. (C₂₃H₂₈FN₃O₂•H₂C₂O₄•0.5H₂O) C, H, N.

(1'-(4-Fluorobenzyl)-4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (16e)

Procedure C was followed with 13d, triethylamine and 4-fluorobenzyl bromide to afford 16e (38 mg, 55%). ¹H NMR (CDCl₃): δ 1.55–2.06 (m, 8H), 2.28 (td, J = 2.1, 13.8 Hz, 1H), 2.52 (td, J = 3.3, 10.5 Hz, 1H), 2.71–2.84 (m, 3H), 2.90–3.10 (m, 2H), 3.04–3.20 (m, 1H), 3.40–3.49 (m, 4H), 3.86 (s, 3H), 6.92–7.04 (m, 4H), 7.25–7.30 (m, 2H), 7.89–7.92 (m, 2H). The free base was converted to the oxalate salt. mp: 221 °C (decomposed). Anal. (C₂₅H₃₁FN₂O₃•H₂C₂O₄) C, H, N.

(4'-Hydroxy-1'-(4-methoxybenzyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (16f)

Procedure C was followed with 13d, triethylamine and 4-methoxybenzyl bromide to afford 16f (32 mg, 78%). ¹H NMR (CDCl₃): δ 1.54–2.06 (m, 8H), 2.24–2.28 (m, 1H), 2.50–2.53 (m, 1H), 2.72–2.86 (m, 3H), 2.98–3.02 (m, 2H), 3.17–3.20 (m, 1H), 3.41–3.48 (m, 3H), 3.62 (br s, 1H), 3.81 (s, 3H), 3.86 (s, 3H), 6.85–6.95 (m, 4H), 7.22 (dd, J = 2.1, 6.9 Hz, 2H), 7.90 (dd, J = 2.1, 6.9 Hz, 2H). The free base was converted to the oxalate salt. mp: 182 °C (decomposed). Anal. (C₂₆H₃₄N₂O₄•H₂C₂O₄) C, H, N.

(1'-Benzyl-4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (16g)

Procedure C was followed with 13d, triethylamine and benzyl bromide to afford 16g (90 mg, 71%). ¹HNMR (CDCl₃): δ 1.57–2.04 (m, 8H), 2.25–2.32 (t, J = 7.0 Hz, 1H), 2.51–2.60 (m, 1H), 2.70–2.86 (m, 3H), 3.00–3.04 (m, 2H), 3.17–3.20 (m, 1H), 3.40–3.59 (m, 4H), 3.86 (s, 3H), 6.91–6.94 (d, J = 9.0 Hz, 2H), 7.26–7.33 (m, 5H), 7.89–7.92 (d, J = 9.0 Hz, 2H). The free base was converted to the oxalate salt. mp: 220.3 °C (decomposed). Anal. (C₂₅H₃₂N₂O₃•H₂C₂O₄•0.5H₂O) C, H, N.

(4'-Hydroxy-1'-(pyridin-3-ylmethyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone (16h)

Procedure C was followed with 13d, triethylamine and pyridine-4-methyl bromide to afford 16h (13 mg, 34%). ¹H NMR (CDCl₃): δ 1.58–2.08 (m, 8H), 2.22 - 2.29 (m, 1H), 2.52–2.53 (m, 1H), 2.70–3.04 (m, 5H), 3.18–3.20 (m, 1H), 3.42–3.54 (m, 4H), 3.87 (s, 3H), 6.91–6.96 (m, 2H), 7.26–7.30 (m, 1H), 7.63–7.70 (m, 1H), 7.89–7.94 (m, 2H), 8.52–8.54 (m, 2H). The free base was converted to the oxalate salt. mp: 202 °C. Anal. (C₂₄H₃₁N₃O₃•H₂C₂O₄) C, H, N.

In vitro Biological Evaluation

Sigma receptor binding assays

The compounds were dissolved in DMF, DMSO, or ethanol, and then diluted in 50 mM Tris-HCl buffer containing 150 mM NaCl and 100 mM EDTA at pH 7.4, prior to performing the σ₁ and σ₂ receptor binding assays. The procedures for isolating the membrane homogenates and performing the σ₁ and σ₂ receptor binding assays have been previously described in detail.⁴²

Briefly, the σ₁ receptor binding assays were conducted in 96-well plates using guinea pig brain membrane homogenates (~300 Wg protein) and ~5 nM (+)-[³H]pentazocine (34.9 Ci/mmol, Perkin-Elmer, Boston, MA). The total incubation time was 90 min at room temperature. Nonspecific binding was determined from samples that contained 10 μM of cold haloperidol. After 90 min, the reaction was quenched by adding 150 μL of ice-cold wash buffer (10 mM Tris HCl, 150 mM NaCl, pH 7.4) using a 96 channel transfer pipet (Fisher Scientific, Pittsburgh, PA). The samples were harvested and filtered rapidly through a 96-well fiberglass filter plate (Millipore, Billerica, MA) that had been presoaked with 100 μL of 50 mM Tris-HCl buffer at pH 8.0 for 1 h. Each filter was washed 3 times with 200 μL of ice-cold wash buffer, and the filter counted in a Wallac 1450 MicroBeta liquid scintillation counter (Perkin-Elmer, Boston, MA).

The σ₂ receptor binding assays were conducted using rat liver membrane homogenates (~300 μg protein) and ~5 nM [³H]DTG (58.1 Ci/mmol, Perkin-Elmer, Boston, MA) in the presence of 1 μM of (+)-pentazocine to block σ₁ sites. The incubation time was 2 h at room temperature. Nonspecific binding was determined from samples that contained 10 μM of cold haloperidol. All other procedures were identical to those described for the σ₁ receptor binding assay above.

Data from the competitive inhibition experiments were modeled using nonlinear regression analysis to determine the concentration that inhibits 50% of the specific binding of the radioligand (IC₅₀ value). Competitive curves were best fit to a one-site fit and gave pseudo-Hill coefficients of 0.6-1.0. K_i values were calculated using the method of Cheng and Prusoff and are presented as the mean (± 1 SEM). For these calculations, we used a K_d value of 7.89 nM for (+) [³H]pentazocine and guinea pig brain and a K_d value of 30.73 nM for [³H]DTG and rat liver.

Vesicular acetylcholine transporter binding assays

In vitro VAChT binding assays of these novel compounds were performed on human vesicular acetylcholine transporter (VAChT) permanently expressed in PC12^A123.7 cells at about 50 pmol/mg of crude extract. The radioligand used was 5 nM (-)-[³H]vesamicol, and the assay was conducted as described at final concentrations of 10^-11 M to 10^-5 M novel compound (53). Unlabeled (-)-vesamicol was used as an external standard, for which K_i = 15 nM, and the mixture was allowed to equilibrate for 20 hrs. Duplicate data were averaged and fitted by regression with a rectangular hyperbola to estimate the K_i value of the novel compound. All compounds were independently assayed at least two times.

Dopamine Receptor Binding Assays

In vitro D₂, D₃ binding assays were performed on human D₂ and D₃ receptor expressed HEK 293 cells according our recently published protocol.⁴⁸

5-HT_1A receptor binding assays

The binding affinity at 5-HT_1A receptors was characterized by a filtration assay following the reported procedure⁴⁹ with minor modification. Human 5-HT_1A serotonin receptor membranes (~10 ug protein) (ChanTest, Cleveland, Ohio, USA) were diluted in 50 mM Tris–HCl buffer (10 mM MgCl₂, 0.1mM EDTA, pH 7.4) and incubated in a total volume of 150 uL with ~0.89 nM [³H] 8-OH-DPAT (Perkin Elmer, Boston, MA, USA) at 25°C in 96 well sample plates for 60 min. 10 μM WAY-100635 was used to define the non-specific binding. The radioligand concentration was equal to approximately twice the K_d value and the concentration of the competitive inhibitor ranged over 6 orders of magnitude for competition experiments (0.1 nM-10 μM or 0.01 nM-1 μM). The reactions were terminated by the addition of 150 μL of cold wash buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4, at 4°C) using a 96 channel transfer pipette (Fisher Scientific), and the samples harvested and filtered rapidly to Millipore MultiScreen_HTS 96-Well Filter Plates that had been presoaked with 100 μL of 50 mM Tris HCl buffer, pH 8.0 for 1 h. Each filter was washed with 200 μL of ice-cold wash buffer for a total of three washes. Filters were dissolved in 2.5 mL of scintillation fluid and a Wallac 1450 MicroBeta liquid scintillation counter (Perkin Elmer, Boston, MA, USA) was used to measure the radioactivity. The concentration of inhibitor that inhibits 50% of the specific binding of the radioligand (IC₅₀ value) was determined by using nonlinear regression analysis to analyze the data of competitive inhibition experiments. Competition curves were modeled for a single site and the IC₅₀ values were converted to equilibrium dissociation constants (K_i values) using the Cheng and Prusoff equation with equilibrium dissociation constant K_d = 0.35 nM.

Abbreviations List

Anal.

Analysis

BOP-Cl

bis(2-oxo-3-oxazolidinyl)phosphonic chloride

Calcd.

calculated

CIMS.

Chemical ionization mass spectrometry

CNS

central nervous system

SAR

structure-activity relationship

DCC

N,N'-dicyclohexylcarbodiimide

DMF

N,N-dimethylformamide

DMSO

dimethyl sulfoxide

DTG

1,3-ditolylguanidine

ND

not determined

PET

positron emission tomography

prezamicol

3-hydroxy-4-(4-phenylpiperidinyl)piperidine

THF

tetrahydrofuran

TLC

thin layer chromatography

trozamicol

4-hydroxy-3-(4-phenylpiperidinyl)piperidine

VAChT

vesicular acetylcholine transporter

vesamicol

(-)-trans-2-(4-phenylpiperidino)cyclohexanol