Open Access

4-aroylpiperidines and 4-(α-hydroxyphenyl)piperidines as selective sigma-1 receptor ligands: synthesis, preliminary pharmacological evaluation and computational studies

Chemistry Central Journal201610:53

DOI: 10.1186/s13065-016-0200-1

Received: 4 April 2016

Accepted: 9 August 2016

Published: 23 August 2016

Abstract

Background

Sigma (σ) receptors are membrane-bound proteins characterised by an unusual promiscuous ability to bind a wide variety of drugs and their high affinity for typical neuroleptic drugs, such as haloperidol, and their potential as alternative targets for antipsychotic agents. Sigma receptors display diverse biological activities and represent potential fruitful targets for therapeutic development in combating many human diseases. Therefore, they present an interesting avenue for further exploration. It was our goal to evaluate the potential of ring opened spipethiane (1) analogues as functional ligands (agonists) for σ receptors by chemical modification.

Results

Chemical modification of the core structure of the lead compound, (1), by replacement of the sulphur atom with a carbonyl group, hydroxyl group and 3-bromobenzylamine with the simultaneous presence of 4-fluorobenzoyl replacing the spirofusion afforded novel potent sigma-1 receptor ligands 7a–f, 8a–f and 9d–e. The sigma-1 receptor affinities of 7e, 8a and 8f were slightly lower than that of 1 and their selectivities for this receptor two to threefold greater than that of 1.

Conclusions

It was found that these compounds have higher selectivities for sigma-1 receptors compared to 1. Quantitatitive structure–activity relationship studies revealed that sigma-1 binding is driven by hydrophobic interactions.

Keywords

Sigma-1 binding Piperidines QSAR Pharmacophore

Background

Sigma (σ) receptors are membrane-bound proteins that bind several psychotropic drugs with high affinity [1]. They were initially proposed to be related to opioid receptors [2] but were later found to be a distinct pharmacological entity distinguished by an unusual promiscuous ability to bind a wide variety of drugs [3]. Initial interest in σ receptors came mainly from their high affinity for typical neuroleptic drugs, such as haloperidol, and their potential as alternative targets for antipsychotic agents [4, 5]. However, no endogenous functional ligand (agonist) for σ receptors has been conclusively identified.

These receptors are classified into two subtypes: subtype 1 (σ1 receptor) and subtype 2 (σ2 receptor) which are differentiated by their pharmacological profiles, distribution in tissues, functions, and molecular sizes [6], with the σ1 being the most documented. Basically, the σ1 receptor is believed to have a ligand binding profile such that (+)-benzomorphans are at least fivefold to tenfold more potent than their corresponding (−)-isomers [7]. On the other hand, for the σ2 subtype, the (−)-benzomorphans are more potent than their corresponding (+)-isomers in the binding assay. The gene coding the σ1 receptor has been isolated and cloned from guinea pig [8], mouse [9], rat [10], and human [11]. The protein coded by the σ1 receptor gene in rat brain consists of a 223 amino acid sequence (23 kDa). In contrast, the σ2 receptor has not been cloned yet and is estimated to have a molecular weight of 19–21.5 kDa [12]. The presence of a σ3 subtype has not been confirmed yet, even though its existence was proposed in a few papers [1315].

The specific participation and character of σ receptors in the processes of the psychiatric and neurological disorders is still not clear [16]. Nevertheless, some of the ligands have drawn attention as potentially useful antipsychotics, antidepressants [17, 18], anxiolytics [19], anti-amnesics, for mental improvement [20], analgesics [21], anti-epileptics, anticonvulsants, and as seizure reducing neuroprotective agents [22]. Apart from their involvement in psychiatric disorders and nervous system diseases, σ receptors and their ligands offer a plethora of means for dealing with several cancer cell types through a variety of strategies [23]. A typical endogenous σ1 receptor regulator is the hallucinogen N,N-dimethyltryptamine [24]. Moreover, σ1 receptor ligands have recently been shown to be potent noncompetitive antagonists at the N-methyl-d-aspartate (NMDA) receptor with IC50 values similar to those of the dissociative anesthetic (S)-(+)-ketamine [25]. Ghandi et al. recently carried out a one pot synthesis of new spirocyclic-2,6-diketopiperazine derivatives, with benzylpiperidine and cycloalkane moieties, some of which showed up to a 95-fold σ12 selectivity ratio [26].

Over the years, a large number of compounds with unrelated chemical structures have been reported to display affinity for σ receptors (Fig. 1). To explain the binding of these structurally diverse compounds to sigma receptors, a number of models or pharmacophores have been proposed [13, 2733]. Generally, the pharmacophore (ph4) for σ1 receptor binding consists of three major sites: an amine site as an essential proton acceptor site, flanked by two hydrophobic domains, a primary hydrophobic site that binds phenyl group “B” from the central amine and a secondary binding site that binds phenyl group “A” from the central amine (Fig. 2). Gund and coworkers [13] suggested that the chains between the amine site and aromatic rings need not be simple alkyl chains. They could bear a polar substituent such as S or O, which could be considered as the second binding site (Fig. 3). Other functional groups can be piperidyl, guanidinyl, pyrrolidyl, piperazyl, thiochromanyl, and benzamidyl [7]. A pharmacophore for σ2 binding has also been proposed [24, 3033]. The latter is also characterized by a central amine site flanked by two hydrophobic sites. However, the two models (σ1 and σ2) differ in a number of respects, such as the distance between the central amine site and the hydrophobic sites [13, 32].
Fig. 1

Some sigma receptor ligands

Fig. 2

Gund’s pharmacophore model

Fig. 3

Design of 1,4-disubstituted piperidines from proposed pharmacophore model for sigma receptor ligands

Owing to the apparent involvement of σ receptors in a variety of biological processes, and the potential applications of σ ligands in pharmacology and medicine, interest in these receptors and their ligands has remained high, and there is a continuing search for new selective ligands that can serve either as agonists or antagonists at those biological processes that are mediated by σ receptors.

Among the compounds reported to bind σ receptors, a large number of benzylpiperidine and benzylpiperazine derivatives display remarkable affinity. In reviewing these collections of compounds, our attention was attracted to spipethiane (1), a spirocyclic compound that contains the elements of benzylpiperidine. Spipethiane is a very potent and selective ligand for σ1 receptors (K i : σ1, 0.5 nM; σ2, 416 nM) [34]. The design of this compound was inspired by the reported high affinity of the spirotetralins (2) for σ1 receptors. In contrast to the spirotetralins, spipethiane does not display high affinity for 5-HT2 receptors. Consequently, the compound was one of the most selective σ1 ligands reported at the time. Since this initial discovery, the work on this spirocyclic skeleton has been extended to include several compounds which are reported to display high affinity and selectivity for σ1 receptors, and potential antitumor activity [33, 35]. Spipethiane and it analogues therefore provide interesting targets for further investigation.

Deprived of conformational freedom, spirocyclic compounds such as spipethiane and the spirotetralins may derive their receptor selectivity from their ability to adopt only a restricted number of molecular conformations. The current study sought to investigate the role of spirofusion in the biological activity of the spipethiane/spirotetralin skeleton. The compounds obtained from the study were tested for binding to σ1 and σ2 receptors.

Results and discussion

Compounds 7a–f, 8a–f and 9d–e were synthesized according to methods A–C reported in Scheme 1. Reaction of 4-(4-fluorobenzoyl)piperidine with various substituted benzyl halides in the presence of sodium acetate, in aqueous ethanol afforded the methanone analogues 7a–f [36] (Scheme 1, method A). Reduction of the methanone analogues with LiBH4 in THF provided the corresponding alcohols 8a–f [33] (Scheme 1, method B). Reductive amination of the methanone analogues 7d and 7e with 3-bromobenzylamine afforded 9d and 9e (Scheme 1, method C) [37]. The synthesized 1,4-disubstituted piperidine derivatives were evaluated for their affinity at both σ1 and σ2 receptors.
Scheme 1

Reagents: a Substituted benzyl halide, EtOH, H2O, NaOAc reflux; b LiBH4, THF, reflux; c 3-bromobenzylamine hydrochloride, LiBH4, THF, HOAc, reflux

Sigma receptor binding

Overall, the majority of target compounds displayed significantly higher affinity for σ1 receptors than for σ2 receptors (Tables 1, 2, 3). K i values at σ1 receptors were below 15 nM for all the compounds except 7b, 7d, 8b and 8d. In contrast, all compounds except 7a and 7f were found to have K i values greater than 500 nM at the σ2 receptor. Among the ketones, 7e and 7a emerged as the most potent σ1 receptor ligands followed closely by 7c and 7f. All four compounds are para substituted, suggesting that this substitution pattern is favored. In contrast, substitution at the ortho or disubstituted at meta and para positions was disfavoured, as both the 2″-nitro compound (7d) and 3″, 4″-dichloro substituted analogue (7b) displayed equally poor affinity for the σ1 receptor. Reduction of the carbonyl compounds to the corresponding alcohols led to a significant increase in σ1 receptor affinity for the most potent ligands: 11-fold for 7f versus 8f; twofold for both 7a versus 8a and 7c versus 8c. In contrast, for compounds 7e versus 8e the σ1 binding affinity decreased fivefold upon reduction of the carbonyl to the corresponding alcohol. Compounds 7e, 8a and 8f exhibited the highest selectivity (ki-σ21 = 610, 606 and 589 respectively) for σ1 receptors among all compounds tested, with K i values between 1.00 to 2.00 nM and >500 nM for σ2 receptors; their affinities were slightly lower than that of spipethiane (K i : σ1, 0.50 nM; σ2, 416 nM; ki-σ21 = 208) [34] but greater than that of (+)-pentazocine (K i : σ1, 3.58 ± 0.20 nM; σ2, 1923 nM; ki-σ21 = 540) [38]. Therefore, these compounds are more selective than spipethiane. Compounds 7b, 7d, 8b, 8d and 9d interact non-selectively with both receptor subtypes but with mediocre binding affinities (K i : σ21 = 2). In particular, ortho substitution with the nitro group results in mediocre binding affinity for both receptor subtypes (σ1: 7d vs 8d vs 9d; σ2: 7d, 8d and 9d), 2″-N). As a result, the nitro substituted analogues were found to be the least σ12 receptor selective ligands (K i : σ21 = 2). We conclude that replacement of the spirofusion in spipethiane with a hydroxymethylene or carbonyl group preserves affinity and selectivity for σ1 receptors.
Table 1

Binding affinity of methanone analogues

Compound

R

σ1 (K i nM)

σ2 (K i nM)

Selectivity ratio (K i σ21)

7a

4″-F

2.96 ± 0.5

221.64 ± 8.0

75

7b

3″,4″-Cl2

>434

>854

2

7c

4″-Cl

5.98 ± 0.41

554.03 ± 34.22

93

7d

2″-NO2

>434

>854

2

7e

4″-Br

1.40 ± 0.5

>854

610

7f

4″-Me

11.58 ± 0.26

151.47 ± 7.79

13

Spipethianea

0.50

416

208

(+)-pentazozcineb

3.58

1932

540

aData available from Ref. [24]

bData available from Ref. [24]

Table 2

Binding affinity of methanol analogues

Compound

R

σ1 (K i nM)

σ2 (K i nM)

Selectivity ratio (K i σ21)

8a

4″-F

1.41 ± 0.22

>854

606

8b

3″,4″-Cl2

>434

>854

2

8c

4″-Cl

2.49 ± 0.24

>854

343

8d

2″-NO2

526.53 ± 69

>854

2

8e

4″-Br

5.22 ± 0.3

>854

164

8f

4″-Me

1.45 ± 0.4

>854

589

Table 3

Binding affinity of bromobenzylamine analogues

Compound

R

σ1 (K i nM)

σ2 (K i nM)

Selectivity ratio (K i σ21)

9d

2″-NO2

>434

>854

2

9e

4″-Br

2.95 ± 0.57

>854

289

SAR and QSAR study

Gund et al. have reported the molecular modeling of several σ1 receptor specific ligands: PD144418, spipethiane, haloperidol and pentazocine in a bid to develop a ph4 for σ1 receptor-ligand binding under the assumption that all the compounds interact at the same receptor site [13]. The primary ph4 for the σ binding sites was defined by mapping the topographic arrangements of the phenyl ring, the N-atom, and N lone pair vector; a point was placed 2.8 Å tetrahedrally from N atoms to represent an interaction between a protonated N atom and its binding site; dummy atoms were built 3.5 Å above and below a phenyl ring to represent hydrophobic binding to a receptor. The distance from the C-center to the N atom was 7.14 Å, while that from O and C-center was 3.68 Å and from O to N atom was 4.17 Å. The choice of ligands used in the study was based on their potency, selectivity and structural diversity with their affinity ranging from 0.08 to 5.8 nM.

Correlation of binding affinity to σ1 receptor and van der Waals surface areas, dipole moments and water accessible surface areas of target compounds

Table 4 shows the computed values for 3D van der Waals surface areas (S vdW ), the 2D van der Waals surface areas (A vdW ), the AM1 dipole moments (μ D(AM1)), the densities (d) and 3D water accessible surface areas (S wat ), as well as the experimentally derived binding affinities (ΔG exp) and the predicted binding affinities (ΔG pred ) obtained from the most significant derived QSAR equation. The three most significant QSAR Eqs. (1) to (3), were derived for 14 molecules (N = 14) and three molecular descriptors (k = 3):
Table 4

Computed molecular descriptors, experimental and theoretically obtained binding affinities for σ1 receptor (obtained with the best model, Eq. 3)

Compd

d

S vdW

S wat

A vdW

μ D(AM1)

ΔG exp

ΔG pred

ΔG res

7a

1.03

328.65

550.09

304.36

1.55

−0.47

−1.02

0.55

7b

1.01

356.64

591.24

335.12

2.16

−2.64

−1.95

−0.69

7c

1.05

343.45

565.21

317.53

1.69

−0.78

−1.16

0.38

7d

1.07

347.51

564.98

328.09

6.67

−2.64

−2.78

0.14

7e

1.14

354.30

588.50

329.31

1.58

−0.15

−1.19

1.04

7f

0.97

345.98

575.78

317.19

3.13

−1.06

−0.33

−0.73

8a

1.03

341.05

554.61

309.59

1.64

−0.15

−0.30

0.15

8b

1.09

365.46

594.35

340.35

2.07

−2.64

−1.75

−0.89

8c

1.03

353.34

575.63

322.77

1.39

−0.39

−0.53

0.14

8d

1.05

356.39

568.95

333.33

4.69

−2.72

−2.54

−0.18

8e

1.12

363.84

588.79

334.55

1.55

−0.72

−1.02

0.3

8f

0.97

356.88

581.58

322.42

0.36

−0.16

0.24

0.4

9d

1.11

491.05

756.77

345.12

4.60

−2.64

−2.79

0.15

9e

1.18

502.38

783.44

459.34

1.64

−0.47

−0.49

0.02

These are the structures of the compounds with the assigned positions. Preferable to be inserted in the scheme

$$\Delta G^{\exp } = 0.11 + 0.19S_{vdW} - 0.21A_{vdW} - 0.001d;R^{2} = 0.71,RMSE = 0.58,F = 7.9$$
(1)
$$\Delta G^{\exp } = 0.22\,+\,0.14S_{vdW}\,-\,0.16A_{vdW}\,-\,0.018\mu_{D(AM1)} ; R^{2} = 0.74, RMSE = 0.54, F = 9.4$$
(2)
$$\Delta G^{\exp } = - 4.19\,+\,0.13S_{vdW}\,-\,0.20A_{vdW}\,+\,0.04S_{wat} ;R^{2} = 0.77, RMSE = 0.51, F = 11.2$$
(3)
where RMSE is the root mean square error and F is the Fischer statistic level of significance. It was observed that there was more than 50 % correlation with the different descriptors combined. This implies that there is a relationship between the σ1 receptor binding affinity of the target compounds and the selected parameters for the study. Multilinear regression analysis showed that the three dimensional hydrophobic (S vdW ) and solvent accessible surface (S wat ) parameters are important factors in the binding affinity of the 1,4-disubstituted piperidine analogues towards the σ1 receptor, because they have positive coefficients compared to densities and dipole moments. The influence of hydrophobic constants confirms the presence of a hydrophobic binding site at the σ1 receptor. The respective R 2 values of 0.71, 0.74 and 0.77 indicate that we can account for about 70–80 % of the variability in binding affinity and the remaining 20–30 % of the variability in affinity cannot be accounted for by the use of the two to four parameters.
The correlation plots for QSAR Eqs. (1), (2) and (3) have been respectively shown in Fig. 4a–c. Interestingly, these plots showed similarity wherein points are grouped into two clusters. The clusters are formed such that the least potent σ1 ligands (characterized by substitution at the ortho and meta positions) are at the bottom left while the most potent ligands (characterized by substitution at the para position) are at the top right. Thus, the QSAR equations are able to discriminate between the active and inactive σ1 binders.
Fig. 4

Correlation plot for three-descriptor QSAR relations a relation 1, b relation 2 and c relation 3

Molecular electrostatic potential maps

Further structure–activity evaluation was performed by studying the electronic distribution of analogues through the use of molecular electrostatic potentials (MEPs). Electrostatic potential is the energy of interaction of a positive charge with the nuclei and electrons of a molecule. The MEP surfaces are color coded, with light brown indicating the hydrophobic regions, red the acceptor regions and blue, the donor regions (availability of lone pairs of electrons). The MEPs will be subsequently discussed for spipethiane (cyan carbons), the most potent ligand (purple carbons) (7e) and the least potent ligand (green carbons) (8d) for the σ1 binding affinity. These are illustrated in Fig. 5.
Fig. 5

MEP maps for a spipethiane and the most potent σ1 ligand 7e, b spipethiane and the least potent σ1 ligand 8d and c the most potent 7e and its corresponding alcohol, 8e

The main difference between the MEPs of spipethiane, the most potent and least potent ligands is observed around the secondary hydrophobic site (Ar1 region) where there is an additional field generated around the substituent of the pendant phenyl ring of the least potent ligand. This could be probably due to the fact that the nitro-group on the pendant phenyl group of the least potent ligand is strongly electron withdrawing and ortho substituted thereby pulling the electrons from the pendant phenyl group onto itself and as a result deactivating the ring.

MEP maps generated for the most potent ligand (7e) and its corresponding alcohol (8e) show no significant difference (Fig. 5c), in agreement with the observation that both ligands are potent σ1 receptor binders. Therefore, the difference between the most potent and least potent ligands lies in the type of substituent and position of substitution on the pendant phenyl group. The superposition of spipethiane (cyan), the most potent (purple) and least potent (green) σ1 receptor ligands is shown in Fig. 6. A difference observed when spipethiane (cyan) and the most potent σ1 ligand (purple) are superimposed is at the Ar2 portion (the primary hydrophobic site). Although the superposition around this site is not perfect, both ligands remain potent binders to the σ1 receptor, with high affinity and selectivity. This is possible because phamacophore studies for σ1 receptor binding have shown that this site is associated with much bulk tolerance [13].
Fig. 6

Superposition of spipethiane (cyan), the most potent (purple) and least potent (green) σ1 receptor ligands

Molecular surfaces of ligands

The molecular surfaces of 1,4-disubstituted piperidine analogues were studied to further evaluate the structure–activity relationships. Molecular surface maps provide an efficient way of comparing molecular shape and property. They are color coded, with blue indicating the mildly polar regions, green indicating the hydrophobic regions and purple indicating the H-bonding regions. Discussion on the MEPs will be for spipethiane, the most potent (7e) and the least potent σ1 ligand (8d), illustrated in Fig. 7.
Fig. 7

Molecular surfaces map for a spipethiane and most potent σ1 ligand, b spipethiane and least potent σ1 ligand and c most potent and least potent σ1 ligands

The molecular shape of the geometry optimized spipethiane structure is different from those of the most potent and least potent ligands in that, the former is linear while the latter are curved. However, the direction of the curvature is not identical for geometry optimized structures of the two compounds. Interestingly, there is some consistency in the hydrophobic regions of spipethiane and the most potent ligand (Fig. 7a), compared to the least potent ligand and spipethiane (Fig. 7b). The molecular surface of the least potent ligand is characterized mostly by the mild polar and H-bond regions instead of the hydrophobic regions as seen for spipethiane and the most potent ligand. Therefore, we can conclude that molecular shape has minimal influence on affinity for this series of compounds since the most potent ligand is different from spipethiane in shape but similar to the least potent ligand.

Pharmacophore study

In this study, a comparison between the ph4 features generated for the target compounds with the existing ph4 model for σ1 receptor ligands by Gund et al. [13] was carried out. Gund et al. had proposed that, the distance from the centroid of the primary hydrophobic site to the N atom was 7.14 Å; from the secondary binding site to centroid of the primary hydrophobic site was 3.68 Å and from the secondary binding site to N atom was 4.17 Å. In our model (Fig. 8), the centroids of the primary and secondary hydrophobic sites were chosen from the phenyl groups Ar2 and Ar1, respectively, and the following dimensions were obtained: the distance from the centroid to N atom is 6.30 Å for the most potent ligand and 6.02 Å for the least potent ligand. The distance from O to centroid of the most potent ligand is 3.71 Å and that of the least potent ligand is 3.68 Å; Distance from O to N atom for most potent ligand is 4.97 Å and that for the least potent ligand is 5.06 Å. Therefore, it could be concluded that the distance from the centroid of the primary hydrophobic site to the N atom may vary between 6.30 and 7.14 Å; between 3.68 and 3.71 Å from the secondary binding site to the centroid of the primary hydrophobic site and between 4.17 and 4.97 Å from O to N atom.
Fig. 8

Pharmacophore generation from most potent and least potent σ1 ligands

Experimental section

Chemistry

The reactions described below were carried out with commercially available chemicals, of reagent grade, that were used without further purification. Reagents were purchased from Sigma-Aldrich Chemical Company, St. Louis, MO, USA. The silica gel (63–200 mesh) which was used as the stationary phase in column chromatography was obtained from Mallinckrodt Baker, Inc. Phillipsburg, New Jersey, USA and Melting points were determined on a Melt—temp II Laboratory device and are uncorrected. All the 1,4-disubstituted piperidine derivatives were converted to their HCl salts by treatment of the corresponding free base with methanolic HCl. Only the HCl salts were submitted for pharmacological evaluation [3946]. 1H and 13C NMR spectra were recorded using VARIAN 400 MHz spectrometer (1H NMR at 399.75 MHz and 13C NMR at 100.53 MHz). Chemical shifts are presented in units of ppm relative to the solvent (1H NMR peak: 7.26 ppm for CDCl3, 3.3 ppm for CD3OD, and 13C NMR peak: 49.1 ppm for CD3OD and 77.2 ppm for CDCl3). Peak multiplicities and characteristics are represented by the following abbreviations: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet). Mass spectra were performed by direct infusion of target compounds. The data was recorded in ESI mode, either ES+ or ES−. TLC analyses were carried out on aluminium plates (Merck) coated with silica gel 60 F254 (0.2 mm thickness). Visualization of spots was performed with UV light and by treatment with iodine. The MS and NMR data are available in the supplementary data (Additional files 1, 2 and 3).

General method for the preparation of compounds

General methods of synthesis for 7a–f

The synthesis followed the procedure described by Wang et al. [36] with some modification. A mixture containing equimolar quantities (8.6 mmol) of 4-(4-fluorobenzoyl)piperidine hydrochloride, the substituted benzyl chloride in EtOH (15 mL) and NaOAc in distilled water (10 mL) was stirred and heated under reflux overnight. The mixture was allowed to cool to room temperature and concentrated under reduced pressure to provide a residue. The residue was neutralized with a saturated solution of NaHCO3 (2 N, 50 mL) and extracted with CH2Cl2 (2 × 50 mL). The organic extracts were subsequently dried over anhydrous CaCl2, concentrated and set aside to give a residue. The product was purified using a short column of silica gel (hexane–ethyl acetate, 3:1). Reaction conditions for compounds: compounds 7a–f were refluxed at 120 °C, while compounds 8a–f were refluxed at 60 °C and compounds 9d–f were refluxed at 120 °C.

4-(4-fluorobenzoyl)-1-[(4-fluorophenyl)methyl]piperidine (7a)

Yield [from 4-(4-fluorobenzoyl)piperidine hydrochloride (2.0 g, 8.6 mmol), 4-fluorobenzyl chloride (1.2 g, 8.6 mmol) and NaOAc (1.8 g, 8.6 mmol): sweet smelling shiny cream solid (0.7 g, 51 %). m.p. 103–105 °C. 1H NMR (CDCl3) δ 1.86 (br. s., 4H, H-3/H-5), 2.17 (br. s., 2H, Hax-2/Hax-6), 2.97 (d, 2H, J = 11.2 Hz, Heq-2/Heq-6), 3.22 (br. s., 1H, H-4), 3.54 (br. s., 2H, H-7″), 6.99 (t, 2H, J = 8.5 Hz, H-3′/H-5′), 7.13 (t, 2H, J = 8.5 Hz, H-3″/H-5″), 7.31 (br. s., 2H, H-2″/H-6″), 7.95 (dd, 2H, J = 8.2, 5.7 Hz, H-2′/H-6′). 13C NMR (CDCl3) δ 28.4 (C-3/C-5), 43.9 (C-4), 52.7 (C-2/C-6), 62.2 (C-7″), 115.2 (C-3′/C-5′), 115.9 (C-3″/C-5″), 130.7 (C-2″/C-6″), 130.8 (C-2′/C-6′), 130.9 (C-1′), 132.4 (C-1″), 164.4 (C-4″), 166.9 (C-4′), 201.0 (C-7′). [TOF MS ES+] calcd for C19H19F2NO m/z 315.14, found 338.16 (M + Na)+.

1-[(3,4-dichlorophenyl)methyl]-4-(4-fluorobenzoyl)piperidine (7b)

Yield [from 4-(4-fluorobenzoyl)piperidine hydrochloride (2.0 g, 8.6 mmol), 3,4-dichlorobenzyl chloride (1.7 g, 8.6 mmol) and NaOAc (1.8 g, 8.6 mmol): sweet smelling shiny white solid (1.4 g, 44 %). m.p. 104–108 °C. 1H NMR (CDCl3) δ 1.77 (br. s., 4H, H-3/H-5), 2.07 (br. s., 2H, Hax-2/Hax-6), 2.85 (d, 2H, J = 11.3 Hz, Heq-2/Heq-6), 3.14 (m, 1H, H-4), 3.41 (s, 2H, H-7″), 7.03–7.15 (m, 3H, H-3′/H-5′, H-6″), 7.31 (d, 1H, J = 8.2 Hz, H-5″), 7.37 (s, 1H, H-2″), 7.89 (dd, 2H, J = 8.4, 5.7 Hz, H-2′/H-6′). 13C NMR (CDCl3) δ 28.6 (C-3/C-5), 43.5 (C-4), 53.0 (C-2/C-6), 61.8 (C-7″), 115.9 (C-3′/C-5′), 128.1 (C-3″), 130.2 (C-6″), 130.6 (C-5″), 130.8 (C-2′/C-2″), 130.9 (C-1′), 132.3 (C-4″), 132.4 (C-1″), 166.9 (C-4′), 201.0 (C-7′).

1-[(4-chlorophenyl)methyl]-4-(4-fluorobenzoyl)piperidine (7c)

Yield [from 4-(4-fluorobenzoyl)piperidine hydrochloride (2.0 g, 8.6 mmol), 4-chlorobenzyl chloride (1.4 g, 8.6 mmol) and NaOAc (1.8 g, 8.6 mmol): sweet smelling shiny white solid (1.4 g, 44 %). m.p. 115–117 °C. 1H NMR (CDCl3) δ 1.77 (m, 4H, H-3/H-5), 2.05 (br. s., 2H, Hax-2/Hax-6), 2.87 (d, 2H, J = 11.7 Hz, Heq-2/Heq-6), 3.13 (m, 1H, H-4), 3.43 (s, 2H, H-7″), 7.06 (t, 2H, J = 8.4 Hz, H-3′/H-5′), 7.21 (m, 4H, H-2″/H-6″, H-3″/H-5″), 7.89 (dd, 2H, J = 8.6, 5.5 Hz, H-2′/H-6′). 13C NMR (CDCl3) δ 28.7 (C-3/C-5), 43.6 (C-4), 53.0 (C-2/C-6), 62.4 (C-7″), 115.9 (C-3′/C-5′), 128.4 (C-3″/C-5″), 130.3 (C-2′/C-6′), 130.8 (C-1′), 130.9 (C-2″/C-6″), 132.5 (C-1″), 132.7 (C-4″), 166.9 (C-4′), 201.0 (C-7′). [TOF MS ES+] calcd for C19H19ClFNO m/z 331.11, found 354.14 (M + Na)+.

4-(4-fluorobenzoyl)-1-[(2-nitrophenyl)methyl]piperidine (7d)

Yield [from 4-(4-fluorobenzoyl)piperidine hydrochloride (2.0 g, 8.6 mmol), 2-nitrobenzyl bromide (1.9 g, 8.6 mmol) and NaOAc (1.8 g, 8.6 mmol): sweet smelling shiny brownish-yellow solid (1.3 g, 46 %). m.p. 95–97 °C. 1H NMR (CDCl3) δ 1.80 (br. s., 4H, H-3/H-5), 2.19 (br. s., 2H, Hax-2/Hax-6), 2.87 (d, 2H, J = 11.0 Hz, Heq-2/Heq-6), 3.18 (m, 1H, H-4), 3.80 (s, 2H, H-7″), 7.11 (t, 2H, J = 8.6 Hz, H-3′/H-5′), 7.37 (t, 1H, J = 7.4 Hz, H-4″), 7.53 (t, 1H, J = 7.4 Hz, H-5″), 7.68 (d, 1H, J = 7.4 Hz, H-6″), 7.82 (d, 1H, J = 7.8 Hz, H-3″), 7.94 (dd, 2H, J = 8.6, 5.5 Hz, H-2′/H-6′). 13C NMR (CDCl3) δ 28.7 (C-3/C-5), 43.4 (C-4), 53.3 (C-2/C-6), 59.0 (C-7″), 115.9 (C-3′/C-5′), 124.3 (C-3″), 127.7 (C-5″), 130.7 (C-6″), 130.8 (C-1′), 130.9 (C-2′/C-6′), 132.4 (C-1″), 132.5 (C-4″), 149.6 (C-2″), 166.9 (C-4′), 201.0 (C-7′). [TOF MS ES +] calcd for C19H19FN2O3 m/z 342.14, found 365.14 (M + Na)+.

1-[(4-bromophenyl)methyl]-4-(4-fluorobenzoyl)piperidine (7e)

Yield [from 4-(4-fluorobenzoyl)piperidine hydrochloride (2.0 g, 8.6 mmol), 4-bromobenzyl chloride (1.2 g, 8.6 mmol) in EtOH (15 mL) and NaOAc (1.8 g, 8.6 mmol): shiny white solid (0.8 g, 48 %). m.p. 125–126 °C. 1H NMR (CDCl3) δ 1.77 (m, 4H, H-3/H-5), 2.05 (br. s., 2H, Hax-2/Hax-6), 2.86 (d, 2H, J = 11.3 Hz, Heq-2/Heq-6), 3.13 (m, 1H, H-4), 3.41 (s, 2H, H-7″), 7.05 (t, 2H, J = 8.6 Hz, H-3′/H-5′), 7.14 (d, 2H, J = 8.2 Hz, H-3″/H-5″), 7.36 (d, 2H, J = 8.2 Hz, H-2″/H-6″), 7.88 (dd, 2H, J = 8.4, 5.7 Hz, H-2′/H-6′). 13C NMR (CDCl3) δ 28.7 (C-3/C-5), 43.5 (C-4), 53.0 (C-2/C-6), 62.4 (C-7″), 115.8 (C-3′/C-5′), 120.8 (C-4″), 130.6 (C-2′/C-6′), 130.9 (C-2″/C-6″), 131.3 (C-3″/C-5″), 132.4 (C-1′), 137.4 (C-1″), 166.9 (C-4′), 201.0 (C-7′). [TOF MS ES+] calcd for C19H19BrFNO m/z 375.06, found 400.08 (M + 2 + Na)+.

4-(4-fluorobenzoyl)-1-[(4-methylphenyl)methyl]piperidine (7f)

Yield [from4-(4-fluorobenzoyl)piperidine hydrochloride (2.0 g, 8.6 mmol), 4- methylbenzyl chloride(1.7 g, 8.6 mmol) and NaOAc (1.8 g, 8.6 mmol): sweet smelling colorless shiny solid (0.7 g, 46 %). m.p. 108–110 °C. 1H NMR (CDCl3) δ 1.82 (m, 4H, H-3/H-5), 2.09 (td., 2H, J = 10.8, 3.5 Hz, Hax-2/Hax-6), 2.32 (s, 3H, 4″-CH 3 ), 2.95 (d, 2H, J = 11.7 Hz, Heq-2/Heq-6), 3.17 (m, 1H, H-4), 3.50 (s, 2H, H-7′), 7.11 (m, 4H, H-3′/H-5′, H-3″/H-5″), 7.20 (d, 2H, J = 7.3 Hz, H-2″/H-6″), 7.94 (dd, 2H, J = 8.4, 5.7 Hz, H-2′/H-6′). 13C NMR (CDCl3) δ 21.1 (4″-CH3), 28.7 (C-3/C-5), 43.7 (C-4), 53.0 (C-2/C-6), 62.9 (C-7″), 115.8 (C-3′/C-5′), 128.9 (C-3″/C-5″), 129.1 (C-2″/C-6″), 130.9 (C-2′/C-6′), 132.5 (C-1′), 135.1 (C-1″), 136.6 (C-4″), 166.8 (C-4′), 201.1 (C-7′). [TOF MS ES+] calcd for C20H22FNO m/z 311.17, found 334.16 (M + Na)+.

General method for the preparation of compounds 8a–f

The synthesis followed the procedure described by Mach et al. [33] with some modification.

Added to a suspension each of 7a–f in THF (10 mL) was four equivalents of hydrogen from LiBH4 all in equimolar quantities in THF (10 mL). The mixture was stirred for 30 min, heated at reflux overnight and allowed to cool to room temperature. The mixture was then concentrated to remove the THF and then treated with distilled water. The organic phase extracted with CH2Cl2 (2 × 20 mL), washed with brine, dried over CaCl2 and evaporated to dryness. The product crystallized spontaneously, was washed with hexane, filtered and air dried.

(4-fluorophenyl)({1-[(4-fluorophenyl)methyl]piperidin-4-yl})methanol (8a)

Yield [from 7a (0.4 g, 1.2 mmol), LiBH4 (0.03 g, 1.2 mmol). Solid (0.4 g, 97 %). m.p. 133–134 °C. 1H NMR (CD3OD) δ 1.14 (m, 2H, Hax-3/Hax-5), 1.29 (m, 2H, Heq-3/Heq-5), 1.46 (m, 1H, H-4), 1.81 (m, 2H, Hax-2/Hax-6), 2.71 (d, 1H, J = 11.3 Hz, Heq-2), 2.82 (d, 1H, J = 11.3 Hz Heq-6), 3.35 (s, 2H, H-7″), 4.20 (d, 1H, J = 7.8 Hz, H-7′), 6.93 (m, 4H, H-3′/H-5′, H-3″/H-5″), 7.20 (m, 4H, H-2′/H-6′, H-2″/H-6″). 13C NMR (CDCl3) δ 27.7 (C-3), 27.9 (C-5), 43.0 (C-4), 52.9 (C-2), 53.0 (C-6), 61.9 (C-7″), 77.2 (C-7′), 114.3 (C-3″/C-5″), 114.5 (C-3′/C-5′), 128.1 (C-2″/C-6″), 131.1 (C-2′/C-6′), 133.0 (C-1″), 139.5 (C-1′), 160.9 (C-4″), 163.4 (C-4′). [TOF MS ES+] calcd for C19H21F2NO m/z 317.16, found 318.18 (M + H)+.

{1-[(3,4-dichlorophenyl)methyl]piperidin-4-yl}(4-fluorophenyl)methanol (8b)

Yield [from 7b (0.4 g, 1.0 mmol), LiBH4 (0.02 g, 1.0 mmol). Solid (0.4 g, 99 %). m.p. 84–86 °C.1H NMR (CD3OD) δ 1.14 (m, 2H, Hax-3/Hax-5), 1.27 (m, 2H, Heq-3/Heq-5), 1.46 (m, 1H, H-4), 1.83 (m, 2H, Hax-2/Hax-6), 2.69 (d, 1H, J = 11.3 Hz, Heq-2), 2.80 (d, 1H, J = 11.3 Hz Heq-6), 3.34 (s, 2H, H-7″), 4.21 (d, 1H, J = 7.4 Hz, H-7′), 6.93 (t, 2H, J = 8.6 Hz, H-3′/H-5′), 7.12 (d, 1H, J = 8.2 Hz, H-6″), 7.20 (dd, 2H, H-2′/H-6′), 7.34 (d, 1H, J = 8.2 Hz, H-5″), 7.38 (s, 1H, H-2″). 13C NMR (CDCl3) δ 29.5 (C-3), 29.6 (C-5), 44.6 (C-4), 54.6 (C-2), 54.8 (C-6), 62.9 (C-7″), 78.8 (C-7′), 115.9 (C-3′/C-5′), 129.7 (C-2′/C-6′), 130.5 (C-3″), 131.5 (C-6″), 132.2 (C-5″), 132.6 (C-2″), 133.3 (C-4″), 140.1 (C-1″), 141.1 (C-1′), 164.8 (C-4′). [TOF MS ES+] calcd for C19H20Cl2FNO m/z 367.09, found 388.12 (M + Na)+.

{1-[(4-chlorophenyl)methyl]piperidin-4-yl}(4-fluorophenyl)methanol (8c)

Yield [from 7c (0.40 g, 1.1 mmol), LiBH4 (0.02 g, 1.1 mmol): Solid (0.4 g, 99 %). m.p. 113–116 °C. 1H NMR (CD3OD) δ 1.15 (m, 2H, Hax-3/Hax-5), 1.28 (m, 2H, Heq-3/Heq-5), 1.47 (m, 1H, H-4), 1.84 (m, 2H, Hax-2/Hax-6), 2.72 (d, 1H, J = 11.3 Hz, Heq-2), 2.83 (d, 1H, J = 11.3 Hz, Heq-6), 3.37 (s, 2H, H-7″), 4.21 (d, 1H, J = 7.4 Hz, H-7′), 6.93 (t, 2H, J = 8.6 Hz H-3′/H-5′), 7.20 (m, 6H, H-2′/H-6′, H-2″/H-6″, H-3″/H-5″). 13C NMR (CDCl3) δ 29.3 (C-3), 29.5 (C-5), 44.5 (C-4), 54.6 (C-2), 54.7 (C-6), 63.4 (C-7″), 78.7 (C-7′), 115.8 (C-3′/C-5′), 129.5 (C-3″/C-5″), 129.8 (C-2′/C-6′), 132.5 (C-2″/C-6″), 134.4 (C-4″), 137.3 (C-1″), 141.1(C-1′), 164.8 (C-4′).

(4-fluorophenyl)({1-[(2-nitrophenyl)methyl]piperidin-4-yl})methanol (8d)

Yield [from 7d (0.5 g, 2.0 mmol), LiBH4 (0.03 g, 2.0 mmol): yellow oil (0.5 g, 98 %). was obtained, washed with hexane and air dried. 1H NMR (CD3OD) δ 1.08 (m, 2H, Hax-3/Hax-5), 1.21 (m, 2H, Heq-3/Heq-5), 1.40 (m, 1H, H-4), 1.83 (m, 2H, Hax-2/Hax-6), 2.59 (d, 1H, J = 11.0 Hz, Heq-2), 2.70 (d, 1H, J = 11.0 Hz, Heq-6), 3.60 (s, 2H, H-7″), 4.16 (d, 1H, J = 7.8 Hz, H-7′), 6.92 (t, 2H, J = 8.8 Hz H-3′/H-5′), 7.18 (dd, 2H, J = 8.0, 5.7 Hz, H-2′/H-6′), 7.33 (dt, 1H, J = 8.3, 4.3 Hz, H-4″), 7.46 (d, 2H, J = 4.3 Hz, H-5″/H-6″), 7.68 (d, 1H, J = 7.8 Hz, H-3″). 13C NMR (CDCl3) δ 29.8 (C-3), 29.9 (C-5), 44.7 (C-4), 54.9 (C-2), 55.0 (C-6), 60.3 (C-7″), 78.9 (C-7′), 116.0 (C-3′/C-5′), 125.4 (C-3″), 129.4 (C-5″), 129.7 (C-2′/C-6′), 132.7 (C-6″), 133.5 (C-1″), 134.8 (C-4″), 141.2 (C-1′), 151.7 (C-2″), 164.8 (C-4′).

{1-[(4-bromophenyl)methyl]piperidin-4-yl}(4-fluorophenyl)methanol (8e)

Yield [from 7e (0.5 g, 1.3 mmol), LiBH4 (0.03 g, 1.3 mmol): solid (0.4 g, 95 %). m.p. 75–78 °C. 1H NMR (CD3OD) δ 1.14 (m, 2H, Hax-3/Hax-5), 1.26 (m, 2H, Heq-3/Heq-5), 1.46 (m, 1H, H-4), 1.82 (m, 2H, Hax-2/Hax-6), 2.71 (d, 1H, J = 11.3 Hz, Heq-2), 2.82 (d, 1H, J = 11.3 Hz, Heq-6), 3.34 (s, 2H, H-7″), 4.20 (d, 1H, J = 7.8 Hz, H-7′), 6.93 (t, 2H, J = 8.8 Hz, H-3′/H-5′), 7.12 (d, 2H, J = 8.2 Hz, H-2″/H-6″), 7.19 (dd, 2H, J = 8.2, 5.5 Hz, H-2′/H-6′), 7.35 (d, J = 8.2 Hz, H-3″/H-5″). 13C NMR (CDCl3) δ 29.3 (C-3), 29.5 (C-5), 44.5 (C-4), 54.6 (C-2), 54.7 (C-6), 63.5 (C-7″), 78.8 (C-7′), 116.0 (C-3′/C-5′), 122.3 (C-4″), 129.8 (C-2′/C-6′), 132.5 (C-2″/C-6″), 132.8 (C-3″/C-5″), 138.0 (C-1″), 141.1(C-1′), 164.8 (C-4′). [TOF MS ES+] calcd for C19H21BrFNO m/z 377.08, found 378.11 (M + H)+.

(4-fluorophenyl)({1-[(4-methylphenyl)methyl]piperidin-4-yl})methanol (8f)

Yield [from 7f (0.4 g, 1.2 mmol), LiBH4 (0.02 g, 1.2 mmol). Solid (0.4 g, 98 %). m.p. 94–95 °C. 1H NMR (CD3OD) δ 1.13 (m, 2H, Hax-3/Hax-5), 1.27 (m, 2H, Heq-3/Heq-5), 1.45 (m, 1H, H-4), 1.80 (m, 2H, Hax-2/Hax-6), 2.20 (s, 3H, 4″-CH 3 ), 2.72 (d, 1H, J = 11.3 Hz, Heq-2), 2.83 (d, 1H, J = 11.3 Hz, Heq-6), 3.33 (s, 2H, H-7″), 4.19 (d, 1H, J = 7.4 Hz, H-7′), 6.93 (t, 2H, J = 8.6 Hz, H-3′/H-5′), 7.01 (d, 2H, J = 7.8 Hz, H-3″/H-5″), 7.06 (d, 2H, J = 7.8 Hz, H-2″/H-6″), 7.19 (dd, J = 8.2, 5.5 Hz, H-2′/H-6′). 13C NMR (CDCl3) δ 21.3 (4″-CH3), 29.2 (C-3), 29.4 (C-5), 44.6 (C-4), 54.5 (C-2), 54.6 (C-6), 64.1 (C-7″), 78.8 (C-7′), 116.0 (C-3′/C-5′), 129.8 (C-2′/C-6′), 130.0 (C-2″/C-6″), 131.0 (C-3″/C-5″), 135.1 (C-1″), 138.3 (C-4″), 141.1(C-1′), 164.8 (C-4′). [TOF MS ES+] calcd for C20H24FNO m/z 313.18, found 314.19 (M + H)+.

General method for the preparation of compounds 9d–e

The synthesis followed the procedure described by Abdel-Magid et al. [34] with some modification. Equimolar quantities of each 7d–e and 3-bromobenzylamine hydrochloride were weighed in a round bottom flask. Added into the flask was THF (15 mL), equimolar quantity of LiBH4 and acetic acid (2 mL). The mixture was stirred and heated under reflux for 3 days and allowed to cool to room temperature. The mixture was then concentrated to remove the THF and then washed with NaHCO3 (2 N, 30 mL). The organic phase extracted with CH2Cl2 (2 × 30 mL) and dried over CaCl2 and evaporated to dryness. The product crystallized spontaneously, was washed with hexane, filtered and air dried.

[(3-bromophenyl)methyl][(4-fluorophenyl)({1-[(2-nitrophenyl)methyl]piperidin-4-yl})methyl]amine (9d)

Yield [from 7d (0.4 g, 1.0 mmol), 3-bromobenzylamine hydrochloride (0.2 g, 1.0 mmol), LiBH4 (0.02 g, 1.0 mmol), AcOH (2 mL), THF (15 mL). Yellow oil (0.4 g, 48 %) was obtained, washed with hexane and air dried. 1H NMR (CD3OD) δ 1.15 (m, 2H, Hax-3/Hax-5), 1.31 (m, 2H, Heq-3/Heq-5), 1.49 (m, 1H, H-4), 1.92 (m, 2H, Hax-2/Hax-6), 2.68 (d, 1H, J = 11.0 Hz, Heq-2), 2.79 (d, 1H, J = 11.0 Hz, Heq-6), 3.69 (s, 2H, H-7″), 4.24 (d, 1H, J = 7.4 Hz, H-7′), 4.31 (s, 1H, Ha-7‴), 4.76 (s, 1H, Hb-7‴), 7.01 (t, 2H, J = 8.8 Hz, H-3′/H-5′), 7.19-7.44 (m, 7H, H-2′/H-6′, H-4″, H-5″, H-6″, H-5‴, H-6‴), 7.54 (m, 2H, H-3″, H-2‴), 7.77 (d, 1H, J = 8.2 Hz, H-4‴). 13C NMR (CDCl3) δ 29.9 (C-3/C-5), 44.7 (C-4), 54.9 (C-2), 55.0 (C-6), 60.3 (C-7″), 65.0 (C-7‴), 78.9 (C-7′), 116.0 (C-3′/C-5′), 125.4 (C-3″), 127.5 (C-3‴), 128.0 (C-6‴), 128.5 (C-5‴), 129.4 (C-5″), 129.7 (C-2′/C-6′), 131.4 (C-4‴), 132.2 (C-2‴), 132.7 (C-6″), 133.5 (C-1″), 134.8 (C-4″), 135.2 (C-1‴) 141.2 (C-1′), 151.7 (C-2″), 163.8 (C-4′).

[(3-bromophenyl)methyl]({1-[(4-bromophenyl)methyl]piperidin-4-yl}(4-fluorophenyl)methyl)amine (9e)

Yield [from 7e (0.4 g, 1.1 mmol), 3-bromobenzylamine hydrochloride (0.3 g, 1.1 mmol), LiBH4 (0.02 g, 1.1 mmol), AcOH (2 mL), THF (15 mL). Yellow oil (0.3 g, 42 %) was obtained, washed with hexane and air dried. 1H NMR (CD3OD) δ 1.13 (m, 2H, Hax-3/Hax-5), 1.28 (m, 2H, Heq-3/Heq-5), 1.47 (m, 1H, H-4), 1.86 (m, 2H, Hax-2/Hax-6), 3.10 (d, 1H, J = 12.1 Hz, Heq-2), 3.18 (d, 1H, J = 12.1 Hz, Heq-6), 3.87 (s, 2H, H-7″), 4.09 (s, 1H, Ha -7‴), 4.24 (m, 2H, H-7′, Hb -7‴), 6.96 (t, 2H, J = 8.8 Hz, H-3′/H-5′), 7.12 (d, 1H, J = 6.7 Hz, H-6‴), 7.21-7.32 (m, 6H, H-2′/H-6′, H-2″/H-6″, H-3″/H-5″), 7.34 (m, 1H, H-5‴), 7.49 (m, 2H, H-2‴, H-4‴). 13C NMR (CDCl3) δ 27.5 (C-3), 27.8 (C-5), 43.7 (C-4), 52.1 (C-7‴), 53.6 (C-2), 53.8 (C-6), 61.5 (C-7″), 77.7 (C-7′), 116.1 (C-3′/C-5′), 124.4 (C-4″), 127.5 (C-3‴), 128.0 (C-6‴), 128.5 (C-5‴), 129.8 (C-2′/C-6′), 131.7 (C-4‴), 132,1 (C-2‴), 133.1 (C-2″/C-6″), 133.7 (C-1‴), 133.8 (C-3″/C-5″), 134.0 (C-1″), 143.8 (C-1′), 164.9 (C-4′).

Sigma receptor binding

These experiments were performed as described by Jinbin et al. [48] with some modification. Different concentrations of test samples were achieved by diluting stock solutions with a solution containing 50 mM Tris–HCl, 150 mM NaCl and 100 mM EDTA at pH 7.4. Rat liver membrane homogenates (~300 μg protein) were diluted with 50 mM Tris–HCl buffer, pH 8.0 and incubated in a total volume of 150 μL with the radioligand at 25 °C in 96 well plates. The incubation time was 60 min for test compounds and 120 min for [3H] DTG and [3H] (+)-pentazocine.

For determination of sigma 1 binding affinities, the σ2 sites were masked in the presence of 1 μM [3H]DTG to determine the σ1 receptor binding characteristics of [3H] (+)-pentazocine while the σ1 sites were masked in the presence of 1 μM (+)-pentazocine to determine the σ2 receptor binding characteristics of [3H]DTG. It is worth mentioning that, this was done one at a time. The final concentration of the radioligand in each assay was ~1 nM for [3H] test compounds and ~5 nM for [3H] (+)-pentazocine and [3H]DTG. Nonspecific binding was determined from samples that contained 10 μM of cold haloperidol.

The reaction was started by adding 0.2 mL of the membrane preparation to the 50 mM Tris–HCl (pH 8.0) buffer containing 3H-labeled ligand with a final concentration of 5 nM and cold ligand ranging from 0.01 to 0.1 mM in a final volume of 1.0 mL. Incubations were carried out at 37 °C for 150 min in the binding study with [3H] (+)-pentazocine and at 25 °C for 90 min in the study with [3H] DTG. Inhibitor concentrations ranging from 0.1 nM to 10 μM were added to acquire the inhibition curves. After the reaction was completed, the samples were harvested, washed three times, and the bound radioactivity counted and analyzed. Data from the competitive inhibition experiments were modeled using nonlinear regression analysis to determine the concentration of inhibitor that inhibits 50 % of the specific binding of the radioligand (IC50 value) and the competitive inhibition constants (Ki values) were calculated from the IC50.

Computational methodology

All molecular modeling was carried out using the software, MOE [49]. Initially, each compound was sketched using the Builder module of MOE package. Energy minimization was carried out using the MOPAC module of MOE at the AM1 level of theory using a minimization gradient of 0.001 kcal/mol. For compounds with chiral centres, only the R-isomers were considered. In the generation of MEPs, the cut-offs were set at 1.62 Å. Pharmacophore models were generated using the polarity-charge-hydrophobicity (PCH) scheme implemented in MOE. The binding sites were defined by mapping the topographical arrangement of the phenyl rings, N-atoms and the electronegative atoms as described by Gund et al. [13].

The binding affinities to the σ1 receptor were computed using Eq. 4:
$$\Delta G^{\exp } = - RT \ln K_{i}$$
(4)
where R is the ideal gas constant and T is the absolute temperature. The residual binding affinities were computed as:
$$\Delta G^{res} = \Delta G^{\exp } - \Delta G^{pred}$$
(5)

These values give a measure of the error estimates for individual values calculated by the regression equation for the dataset. Similarly, the residual values for experimental and predicted activities were obtained from the difference between \(pIC_{50}^{\exp }\) and \(pIC_{50}^{{\text{pred}}}\). This value gives a measure of the error in estimates for individual values calculated by the regression equation for the data set.

Conclusions

The replacement of spirofusion in the lead compound 1 by either a hydroxymethylene or carbonyl bridge led to 4-aroylpiperidines and 4-(α-hydroxyphenyl)piperidines. Most of the compounds have high affinity for σ1 receptors and fit well into the Gund’s pharmacophore model for σ1 receptor ligands; they also display poor affinity for σ2 receptors, and finally, some of them have a higher selectivity for the σ1 receptor compared to the lead compound 1. Thus, spirofusion confers no particular advantage in 1 over its ring open analogues. These analogues with secondary binding sites like H-bond acceptors as well as H-bond donors both emerged as potent σ1 receptor ligands. Therefore, the secondary binding site proposed by Lu et al. [7], may either be a H-bond donor or acceptor. Following the ph4 models generated in this study, potential σ1 binders could be virtually screened from our recently developed natural product libraries from African medicinal plants [5053].

Declarations

Authors’ contributions

This work was carried out in collaboration between all authors. Authors MNN, ZT, RHM and SMNE designed the study. Authors HIN, FNK, MNN and ZT carried the experiments, respective the synthesis, computational studies and bioassays. All authors contributed to the analysis of results, while authors HIN, FNK and MNN wrote the first draft manuscript. All authors read and approved the final manuscript.

Acknowledgements

Computational resources were made available by the Molecular Simulations Laboratory, University of Buea, Cameroon. The author FNK is currently a Georg Forster fellow of the Alexander von Humboldt Foundation, Germany. The authors acknowledge the technical assistance of Mr. Smith B. Babiaka, Ph.D. student, Chemistry Department, University of Buea, Cameroon.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Chemistry, Faculty of Science, University of Buea
(2)
Department of Pharmaceutical Chemistry, Martin-Luther University of Halle-Wittenberg
(3)
Biotechnology Unit, Department of Biochemistry and Molecular Biology, Faculty of Science, University of Buea
(4)
Department of Radiology, University of Washington School of Medicine

References

  1. Walker JM, Bowen WD, Walker FO, Matsumoto RR, de Costa B et al (1990) Sigma receptors: biology and function. Pharmacol Rev 42(2):355–402Google Scholar
  2. Martin WR, Eades CG, Thompson JA, Huppler RE, Gilbert PE (1976) The effect of morphinand morphin-dependent chronic spinal dog. J Pharmacol Exp Ther 197:517–532Google Scholar
  3. Aydar E, Palmer CP, Djamgoz MBA (2004) Sigma receptors and cancer: possible involvement of ion channels. Cancer Res 64:5029–5035View ArticleGoogle Scholar
  4. Su TP (1982) Evidence for sigma opoid receptor: binding of [3H] SKF-10047 to etorphin-inaccessible sites in guinea pig brain. J Pharmacol Exp Ther 223(2):284–290Google Scholar
  5. Tam SW, Cook L (1984) Sigma opiates and certain antipsychotic drugs mutually inhibit (+)-[3H]SKF-10047 and [3H] haloperidol binding in guinea pigbrain membranes. Proc Natl Acad Sci USA 81(17):5618–5621View ArticleGoogle Scholar
  6. Hellewell SB, Bowen WD (1990) A sigma-like binding site in rat pheochromocytoma (PC12) cells: decreased affinity for (+)-benzomorphans and lower molecular weight suggest a different sigma receptor form from that of guinea pig brain. Brain Res 527(2):244–253View ArticleGoogle Scholar
  7. Yu Y, Zhang L, Yin X, Sun H, Uhl GR et al (1997) μ opioid receptor phosphorylation, desensitization, and ligand efficacy. J Biol Chem 272:28869–28874View ArticleGoogle Scholar
  8. Hanner M, Moebius FF, Flandorfer A, Knaus HG, Striessnig J et al (1996) Purification, molecular cloning, and expression of the mammalian sigma1-binding site. Proc Natl Acad Sci USA 93(15):8072–8077View ArticleGoogle Scholar
  9. Pan YX, Mei J, Xu J, Wan BL, Zuckerman A et al (1998) Cloning and characterization of a mouse σ1 receptor. J Neurochem 70(6):2279–2285View ArticleGoogle Scholar
  10. Seth P, Fei YJ, Li HW, Huang W, Leibach FH et al (1998) Cloning and functional characterization of a σ receptor from rat brain. J Neurochem 70(3):922–931View ArticleGoogle Scholar
  11. Prasad PD, Li HW, Fei YJ, Ganapathy ME, Fujita T et al (1998) Exon-intron structure, analysis of promoter region, and chromosomal localization of the human type 1 σ receptor gene. J Neurochem 70(2):443–451View ArticleGoogle Scholar
  12. Hornick JR, Xu J, Vangveravong S, Tu Z, Mitchem JB et al (2010) The novel sigma-2 receptor ligand SW43 stabilizes pancreas cancer progression in combination with gemcitabine. Mol Cancer 9:298View ArticleGoogle Scholar
  13. Gund TM, Floyd J, Jung D (2004) Molecular modeling of sigma 1 receptor ligands: a model of binding conformational and electrostatic considerations. J Mol Graph Model 22(4):221–230View ArticleGoogle Scholar
  14. Booth RG, Wyrick SD (1994) Development of Phenylaminotetralin ligands for novel sigma (σ3) receptor in brain. Med Chem Res 4:225–237Google Scholar
  15. De Haven-Hudkins DL, Fleissner LC (1992) Competitive interactions at [3H]1,3-di(2-tolyl)guanidine (DTG)-defined sigma recognition sites in guinea pig brain. Life Sci 50(9):PL65–PL70Google Scholar
  16. Leonard BE (2004) Sigma receptors and sigma ligands: background to a pharmacological enigma. Pharmacopsychiatry 37(Suppl 3):S166–S170View ArticleGoogle Scholar
  17. Matsuno K, Kobayashi T, Tanaka MK, Mita S (1996) Sigma 1 receptor subtype is involved in the relief of behavioral despair in the mouse forced swimming test. Eur J Pharmacol 312(3):267–271View ArticleGoogle Scholar
  18. Bermack JE, Haddjeri N, Debonnel G (2004) Effects of the potential antidepressant OPC-14523[1-[3-[4-(3-chlorophenyl)-1-piperazinyl]propyl]-5-methoxy-3,4-dinydro-2-quinolinone monomethanesulfonate] a combined σ and 5-HT1A ligand: modulation of neuronal activity in the dorsal raphe nucleus. J Pharmacol Exp Ther 310:578–583View ArticleGoogle Scholar
  19. Müller WE, Siebert B, Holoubek G, Gentsch C (2004) Neoropharmacology of the anxiolytic drug apipramol, a sigma site ligand. Pharmacopsychiatry 37(Suppl 3):S189–S197View ArticleGoogle Scholar
  20. Maurice T (2001) Beneficial effect of the sigma-1 receptor agonist PRE-084 against the spatial learning deficits in aged rats. Eur J Pharmacol 431(2):223–227View ArticleGoogle Scholar
  21. Mach RH, Wu L, West T, Whirrett BR, Childers SR (1999) The analgesic tropane analogue (±)-SM 21 has a high affinity for σ2 receptors. Life Sci 64:131–137View ArticleGoogle Scholar
  22. Shin EJ, Nah SY, Kim WK, Ho KK, Jhoo WK et al (2005) The dextromethorphan analog dimemorfan attenuates kainite-induced seizures via σ1 receptor activation: comparison with effects of dextromethorphan. Br J Pharmacol 144:908–918View ArticleGoogle Scholar
  23. Bowen WD (2000) Sigma receptors: recent advances and new clinical potentials. Pharm Acta Helv 74(2–3):211–218View ArticleGoogle Scholar
  24. Fontanilla D, Johannessen M, Hajipour AR, Cozzi NV, Jackson MB, Ruoho AE (2009) The hallucinogen N, N-dimethyltryptamine (DMT) is an endogenous sigma-1 receptor regulator. Science 323(5916):934–937View ArticleGoogle Scholar
  25. Bonifazi A, Del Bello F, Mammoli V, Piergentili A, Petrelli R et al (2015) Novel potent N-methyl-D-aspartate (NMDA) receptor antagonists or σ1 receptor ligands Based on properly substituted 1,4-dioxane ring. J Med Chem 58(21):8601–8615View ArticleGoogle Scholar
  26. Ghandi M, Sherafat F, Sadeghzadeh M, Alirezapour B (2016) One-pot synthesis and sigma receptor binding studies of novel spirocyclic-2,6-diketopiperazine derivatives. Bioorg Med Chem Lett 26:2676–2679View ArticleGoogle Scholar
  27. Ablordeppey SY, Fischer JB, Glennon RA (2000) Is a nitrogen atom an important pharmacophoric element in sigma ligand binding? Bioorg Med Chem 8(8):2105–2111View ArticleGoogle Scholar
  28. Glennon RA (2005) Pharmacophore identification for sigma-1 (sigma1) receptor binding: application of the “deconstruction-reconstruction-elaboration” approach. Mini Rev Med Chem 5(10):927–940View ArticleGoogle Scholar
  29. Toussaint M, Mousset D, Foulon C, Jacquemard U, Vaccher C, Melnyk P (2010) Sigma-1 ligands: Tic-hydantoin as a key pharmacophore. Eur J Med Chem 45(1):256–263View ArticleGoogle Scholar
  30. Cratteri P, Romanelli MN, Cruciani G, Bonaccini C, Melani F (2004) GRIND-derived pharmacophore model for a series of α-tropanyl derivative ligands of the sigma-2 receptor. J Comput-Aided Mol Des 18(5):361–374View ArticleGoogle Scholar
  31. Maeda DY, Williams W, Kim WE, Thatcher LN, Bowen WD et al (2002) N-arylalkylpiperidines as high-affinity sigma-1 and sigma-2 receptor ligands: phenylpropylamines as potential leads for selective sigma-2 agents. Bioorg Med Chem Lett 12(3):497–500View ArticleGoogle Scholar
  32. Wang W, Cui J, Lu X, Padakanti PK, Xu J et al (2011) Synthesis and in vitro biological evaluation of carbonyl group-containing analogues for σ1 receptors. J Med Chem 54(15):5362–5372View ArticleGoogle Scholar
  33. Mach UR, Hackling AE, Perachon S, Ferry S, Wermuth CG et al (2004) Development of novel 1,2,3,4-tetrahydroisoquinoline derivatives and closely related compounds as potent and selective dopamine D3 receptor ligands. ChemBioChem 5(4):508–518View ArticleGoogle Scholar
  34. Abdel-Magid AF, Carson KG, Harris BD, Maryanoff CA, Shah RD (1996) Reductive amination of aldehydes and ketones with sodium triacetoxyborohydride. Studies on direct and indirect reductive amination procedures. J Org Chem 61(11):3849–3862View ArticleGoogle Scholar
  35. Huang YS, Lu HL, Zhang LJ, Wu Z (2014) Sigma-2 receptor ligands and their perspectives in cancer diagnosis and therapy. Med Res Rev 34(3):532–566View ArticleGoogle Scholar
  36. Quaglia W, Giannella M, Piergentili A, Pigini M, Brasili L et al (1998) 1′-Benzyl-3,4-dihydrospiro[2H-1-benzothiopyran-2,4′-piperidine] (spipethiane), a potent and highly selective sigma1 ligand. J Med Chem 41(10):1557–1560View ArticleGoogle Scholar
  37. Wang B, Rouzier R, Albarracin CT, Sahin A, Wagner P et al (2004) Expression of sigma-1 receptor in human breast cancer. Breast Cancer Res Treat 87(3):205–214View ArticleGoogle Scholar
  38. Maier CA, Wünsch B (2002) Novel spiropiperidines as highly potent and subtype selective sigma-receptor ligands. Part 1. J Med Chem 45(2):438–448View ArticleGoogle Scholar
  39. Bowen WD, de Costa BR, Hellewell SB, Walker JM, Rice KC (1993) [3H]-(+)-Pentazocine: a potent and highly selective benzomorphan-based probe for sigma-1 receptors. Mol Neuropharmacol 3(2):117–126Google Scholar
  40. Bedurftig S, Wunsch B (2004) Chiral, nonracemic (piperazin-2-yl)methanol derivatives with s-receptor affinity. Bioorg Med Chem 12(12):3299–3311Google Scholar
  41. Weiner I, Traub A, Rawlins JN, Smith AD, Feldon J (1995) The sigma ligand BMY-14802 as a potential antipsychotic: evidence from the latent inhibition model in rats. Behav Pharmacol 6(1):46–54View ArticleGoogle Scholar
  42. De Costa BR, He XS, Dominguez C, Cutts J, Williams W et al (1994) A new approach to the design of sigma-2-selective ligands: synthesis and evaluation of N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)ethylamine-related polyamines at s-1 and s-2 receptor subtypes. J Med Chem 37(2):314–321View ArticleGoogle Scholar
  43. Maurice T, Su TP (2009) The pharmacology of sigma-1 receptors. Pharmacol Ther 124(2):195–206View ArticleGoogle Scholar
  44. Locher CP, Ruben PC, Gut J, Rosenthal PJ (2003) 5HT1A serotonin receptor agonists inhibit Plasmodium falciparum by blocking a membrane channel. Antimicrob Agents Chemother 47(12):3806–3809View ArticleGoogle Scholar
  45. Costantino L, Gandolfi F, Sorbi C, Franchini S, Prezzavento O et al (2005) Synthesis and structure activity relationships of 1-aralkyl-4-benzylpiperidine and 1-aralkyl-4-benzylpiperazine derivatives as potent sigma ligands. J Med Chem 48(1):266–273View ArticleGoogle Scholar
  46. Piergentili A, Amantini C, Del Bello F, Giannella M, Mattioli L et al (2010) Novel highly potent and selective σ1 receptor antagonists related to spipethiane. J Med Chem 53(3):1261–1269View ArticleGoogle Scholar
  47. Anjaneyulu K, Ram B, Wagh PB, Pudukulathan Z (2012) Synthesis of 1′-(carbo-t-butoxy) spiro[isochroman–1,4′-piperidinyl]-3-carboxylic acid. Chem J 02(03):111–117Google Scholar
  48. Jinbin X, Zhude Tu, Jones Lynne A, Vangveravong Suwanna, Wheeler Kenneth T, Mach Robert H (2005) [3H]N-[4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl) butyl]-2-methoxy-5-methylbenzamide: a novel sigma-2 receptor probe. Eur J Pharmacol 525:8–17View ArticleGoogle Scholar
  49. Chemical Computing Group (2010) Molecular operating environment software. Chemical Computing Group Inc., MontrealGoogle Scholar
  50. Ntie-Kang F, Mbah JA, Mbaze LM, Lifongo LL, Scharfe M et al (2013) CamMedNP: building the Cameroonian 3D structural natural products database for virtual screening. BMC Complement Altern Med 13(1):88View ArticleGoogle Scholar
  51. Ntie-Kang F, Zofou D, Babiaka SB, Meudom R, Scharfe M et al (2013) AfroDb: a select highly potent and diverse natural product library from African medicinal plants. PLoS One 8(10):e78085View ArticleGoogle Scholar
  52. Ntie-Kang F, Amoa Onguéné P, Scharfe M, Owono LCO, Megnassan E et al (2014) ConMedNP: a natural product library from Central African medicinal plants for drug discovery. RSC Adv 4:409–419View ArticleGoogle Scholar
  53. Ntie-Kang F, Amoa Onguéné P, Fotso GW, Andrae-Marobela K, Bezabih M et al (2014) Virtualizing the p-ANAPL library: a step towards drug discovery from African medicinal plants. PLoS One 9(3):e90655View ArticleGoogle Scholar

Copyright

© The Author(s) 2016