SYA 013 analogs as sigma-2 (σ₂) selective ligands: structure-affinity relationship studies

Abstract

Several lines of evidence suggest that selective sigma-2 (σ₂) ligands might be useful for the treatment of solid tumors. However, very few selective σ₂ ligands have been identified. This study was aimed at identifying new selective σ₂ receptor ligands using a previously identified agent, SYA 013 as a lead. Four groups, homopiperazine, piperazine, tropane and selected oxime analogs of the homopiperazines were identified, synthesized and subsequently screened at the σ₁ and σ₂ receptors. The results demonstrate that these scaffolds can be modified to obtain selective σ₂ receptor ligands. 1-(5-Chloropyridin-2-yl)-4-(3-((4-fluorophenyl)thio)propyl)-1,4-diazepane, 7 and 3-(4-chlorophenyl)-8-(3-((2-fluorophenyl)thio)propyl)-8-azabicyclo[3.2.1]octan-3-ol, 21 were identified as the highest binding affinity ligands (σ₂Ki=2.2 nM) and (4-(4-(5-chloropyridin-2-yl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)-butan-1-one oxime, 22 as a high affinity and the most selective ligand for the σ₂ receptor (σ₁Ki/σ₂Ki = 41.8).

Graphical Abstract

1. Introduction

Initially, sigma (σ) receptors were designated as a subtype of the opioid receptor family,¹ but were shown later to be distinctive proteins incorporated in plasma, mitochondrial and endoplasmic reticulum membranes of tissues in the brain, kidneys, endocrine system, liver, immune system, and reproductive organs.^{2, 3} Historically, the opioid family included the mu (µ), kappa (κ), and sigma (σ) receptors with morphine as a prototype for the µ receptor, ketocyclazocine for the κ receptor, and SKF-10,047 ((N-allylnormetazocine), for the σ receptor.¹ However, Martin et al., 1976, noted that while morphine (µ) caused excessive pupil constriction, bradycardia, hypothermia and decreases in responsiveness to nerve pain, the ketocyclazocine (κ) syndrome was typified with sedation but did not significantly affect pulse rate. SKF-10,047(σ) on the other hand, produced tachycardia, dilation of pupils, tachypnea, and was insensitive to naloxone, a typical antagonist to opioid receptors. These responses were all in contrast to morphine and ketocyclazocine. The later discovery of ligands more selective to sigma sites such as 1,3-di-o-tolylguanidine (DTG) and phencyclidine, 1-(1-phenylcyclohexyl)piperidine (PCP), indicated that they were distinctive from the binding sites in the central nervous system, confirming that they were non-opioid receptors.^{4, 5} Based on extensive binding studies, it has now been established that σ receptors are distinct from other known neurotransmitter receptors since they have high affinity for 1,3-di(2-tolyl) guanidine (DTG), (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine, [(+)-3-PPP] and haloperidol and therefore, are defined as non-opioid binding sites.⁶ Even though biochemical and pharmacological studies proposed that there are several subtypes of sigma receptors, only two well-defined sigma receptor subtypes, sigma-1 (σ₁) and sigma-2 (σ₂), have been characterized.^7–10

The sigma-1 receptor (σ₁R) has been characterized as a 25 kDa protein, whereas the sigma-2 receptor (σ₂R) has a molecular weight of 18–21 kDa.^{11, 12} The σ₁R was cloned in 1996¹³ and crystallized in 2016,¹⁴ while, σ₂R was cloned in 2017.¹⁵ The σ₂R was identified as a fundamental membrane protein residing at the endoplasmic reticulum (ER). The cloning also revealed σ₂R as a four transmembrane domain protein with cytosolic N and C termini.^{15, 16} Until recently, attention was directed to the role of sigma receptors in the brain,¹² however, pharmacological studies have revealed that both σ₁ and σ₂ receptors are widely expressed in various cell lines. Non-malignant cells from the same tissue displayed less sigma receptor expression than malignant cells^17–19 with increased levels of σ₂ receptors over the σ₁ receptor subtype in general.¹⁸ In addition, several lines of evidence have suggested that the ratio of σ₂ receptors in proliferating tumors is higher than in dormant cells with fast multiplying cells expressing ten times more σ₂ receptors than resting cells.^{18, 20} For example, it was noted that σ receptors were abundant in human breast tumor biopsy tissue, but practically lacking in normal breast tissue.²⁰ Furthermore, the cloning of σ₂R confirmed its overexpression in epithelial, colorectal, ovarian, lung, and breast cancers.^21–25 The σ₂R has also been suggested as a possible drug target in cancer therapy^{26, 27} due to its association with tumor biology.²⁸ Pharmacological targeting of the σ₂ receptor in human cancer cell lines has been shown to have anti-proliferative properties.¹² In addition, σ₂ radiotracers have been developed for tumor imaging.¹⁵ Taken together, the σ₂R offers a great potential as a target for anticancer drug development for cancers such as triple negative breast cancer (TNBC) which is without clear targets and for several tumor cell types with fast proliferation.^{29, 30} In this article, we report the design and synthesis of several homopiperazine, piperazine and tropane analogs and their evaluation at the sigma receptors.

2. Chemistry

Compounds 1, 5, 6, 11–15, 17–21 were synthesized in our lab and previously reported. ^31–35 Compound 9 was synthesized and reported.³⁶ Homopiperazine analogs 2, 3, 4 were synthesized as described in Scheme 1 by refluxing a mixture of 4-chloro-4’-fluoro-butyrophenone 25, homopiperazines 26a-c, KI, and NaHCO₃ in isopropanol. Refluxing each of homopiperazines 2, 3 and 4 with hydroxylamine hydrochloride under basic conditions in EtOH afforded the corresponding oximes 22, 23, and 24.

Scheme 1.

Synthesis of homopiperazine analogs and their corresponding oximes. Reagents and conditions: i) KI, NaHCO₃, ⁱPrOH, reflux; ii) NH₂OHHCl, KOH, EtOH, reflux.

Analog 7 was synthesized using the synthetic route we previously reported.³⁷ 1-(5-chloropyridin-2-yl)piperazine and 1-(5-chloropyridin-2-yl)-1,4-diazepane were prepared by reaction of the corresponding diamine with 2,5-dichloropyridine. Piperazine analogs 8 and 10 were synthesized via coupling reactions of halides and 1-(5-chloropyridin-2-yl)piperazine. Alkylating agent 27, which was prepared from butyrophenone 25,³² was used to prepare analog 10. The coupling reaction proceeded under basic conditions in the presence of KI as depicted in Scheme 2.

Scheme 2.

Synthesis of piperazine analogs in Group 2. Reagents and conditions: i) KI, NaHCO₃, ⁱPrOH, reflux.

3. Results and Discussion

To identify new σ₂R selective ligands we reviewed our CNS database and identified the previously reported 4-(4-(4-chlorophenyl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one (1), a homopiperazine analog of haloperidol (SYA013, 1),³¹ as binding selectively to σ₂ receptors (σ₁Ki=24 nM and σ₂Ki=5.6 nM, K_i(σ₁R)/Ki(σ₂R) = 4.3 nM). The aim of this research was to synthesize a limited number of SYA013 analogs, evaluate the structural elements that impact σ₂ selectivity, and optimize their binding affinity to the σ₂ receptor. We now report several such analogs of SYA013 that selectively bind to σ₂ receptors. The structure of SYA013 and several designed analogs are shown in Fig. 1.

Fig. 1.

Homopiperazine analogs of group 1.

To obtain selective σ₂ receptor ligands using SYA013 as a lead compound, four groups of analogs were identified for evaluation: homopiperazine analogs (group 1), piperazine analogs (group 2), tropane analogs (group 3), and indanone analogs (group 4). In group 1, initially, the 4-chlorophenyl moiety was replaced by 4-chloropyridine 2, pyridine 3 and pyrimidine 4 moieties. Subsequently, the ketone on the buta-1-one linker was deoxygenated to form compound 5, replaced with an oxygen to form ether 6, and the carbonyl in compound 2 was replaced with sulfur to obtain thioether 7.

Replacing the 4-chlorophenyl moiety with the chloro-pyridine to obtain 2 did not significantly diminish affinity while still retaining selectivity for the σ₂ receptor. Removing the chloro from 2, led to the pyridine analog 3 with significantly diminished affinity as well as the selectivity for σ₂ receptor, suggesting the chloro plays an important role in binding affinity. Introduction of an additional nitrogen to form the pyrimidine analog, 4 also decreased binding at the σ₂ receptor. Thus, it would appear that decreasing lipophilicity is associated with reduction in binding to the σ₂R. Deoxygenation of the carbonyl of compound 1 to form 5 or replacement of the carbonyl group with an oxygen to form 6 both improved affinity overall but led to reversal of selectivity for the σ₂ receptor. However, replacement of the carbonyl in compound 2 with a sulfur atom to form thioether 7, improved affinity at both sigma receptors and also retained selectivity (Table 1).

Table 1.

Binding affinity of homopiperazine analogs of SYA013 (group 1) at the sigma receptors

Compound	(σ1R, *Ki), nM/pKi ± SEM	(σ2R, Ki), nM/pKi ± SEM	Ki(σ1R)/Ki (σ2R)
1	(24)/7.63 ± 0.07	(5.6)/8.29 ± 0.07	4.3
2	(30)/7.52 ± 0.05	(8.0)/8.1 ± 0.05	3.8
3	(453)/(6.34) ± 0.06	(32)/7.49 ± 0.09	14
4	(63)/7.20 ± 0.09	(44)/7.40 ± 0.10	1.4
5	(3.6)/8.44 ± 0.05	(8.5)/8.07 ± 0.06	0.4
6	(2.9)/8.54 ± 0.07	(5.9)/8.23 ± 0.08	0.5
7	(7.2)/8.14 ± 0.05	(2.2)/8.65 ± 0.05	3.3

^*Ki data are within 20% of the mean value, pKi data are recorded as the Mean ± SEM.

To evaluate the contribution of the homopiperazine ring to binding affinity and selectivity, and to identify other isosteres of the homopiperazine, we evaluated the piperazine analogs of several compounds in group 1 to form group 2 analogs as shown in Fig. 2. First, we evaluated the piperazine analog of compound 2 that is compound 8, and its deschloro equivalent 9. Consistent with the group 1 agents, compound 8 maintained binding similar to compound 2, while 9, like compound 3, resulted in significant reduction in binding affinity at both sigma receptors. Next we evaluated the deoxygenation of compound 8 to obtain 10. Compound 10 showed a 4 to 5-fold reduction of binding to both receptors. Compound 11, the piperazine analog of compound 5, also showed a diminished affinity and a reversed receptor selectivity as in 5. When the carbonyl in compound 8 was replaced with a sulfoxide to form compound 12, there was a greater than 45- and 71-fold loss of affinity at the σ₁ and σ₂ receptors respectively; suggesting the sulfoxide is an undesirable moiety at either of the sigma receptors. Compounds 13 – 15 are ether analogs in which the role of the chloro and fluoro were evaluated. Compound 13, is a desfluoro equivalent of compound 11 with insignificant change in binding affinity, although reversal of selectivity is noted. The deschloro equivalent, compound 14, surprisingly produced an increased binding affinity for both receptors and selectivity for the σ₂ receptor. Removal of both chloro and fluoro substituents to form 15 produced a 2–5 fold decrease in binding affinity at both receptors, as expected (Table 2). Comparing the highest affinity piperazine in group 2, that is compound (σ₂Ki =5.12 nM), with PB28 (σ₂Ki=0.68 nM), one of the highest binding affinity ligands in the literature,³⁸ reveals a 7.5 fold lower binding affinity to the σ₂R.

Fig 2.

Piperazine analogs in Group 2.

Table 2.

Binding affinity constants of piperazine analogs (group 2 agents) at sigma receptors.

Compound	(σ1R, *Ki), nM/pKi ± SEM	(σ2R, Ki), nM/pKi ± SEM	Ki(σ1R)/Ki (σ2R)
8	(25.0)/7.61 ± 0.07	(8.4)/8.07 ± 0.06	3.0
9	(4 0 3)/6.39 ± 0.05	(52.0)/7.3 ± 0.1	7.8
10	(95.0)/7.02 ± 0.07	(43.0)/7.37 ± 0.05	2.2
11	(36.0)	(51.5)	0.7
12	(1148)/5.94 ± 0.04	(600)/6.2 ± 0.01	1.9
13	(51.7)	(33.0)/7.48 ± 0.09	1.6
14	(41.5)	(5.12)	8.1
15	(77.1)	(25.1)	3.1
#PB 28	(0.38)	(0.68)	0.56

^*Ki data are within 20% of the mean value, pKi data are recorded as the Mean ± SEM.

^#From Ref. [38].

In an attempt to identify other isosteres of the homopiperazine ring, we evaluated the tropane analogs (Fig. 3) in group 3. The results of the screening are reported in Table 3.

Fig. 3.

Tropane analogs in group 3.

Table 3.

Binding affinity constants of tropane analogs of group 3 at sigma receptors.

Compound	(σ1R,* Ki), nM/pKi ± SEM	C(σ2R, Ki), nM/p Ki ± SEM	Ki(σ1R)/Ki (σ2R)
17	(22.0)/7.66 ± 0.06	(27.0)/7.60 ± 0.10	0.8
18	(1 6 6)/6.78 ± 0.06	(18.0)/7.70 ± 0.10	3.7
19	(81.0)/7.09 ± 0.07	(29.0)/7.53 ± 0.08	2.8
20	(71.0)/7.15 ± 0.09	(8.1)/8.09 ± 0.06	8.8
21	(57.0)/7.24 ± 0.06	(2.2)78.67 ± 0.09	25.9

^*Ki data are wihin 20% of the mean value. pKi data are recorded as the Mean ± SEM.

Compound 17 is the tropanol analog of compound 5. Compound 17 has a 6- and 3-fold lower affinity for the σ₁ and σ₂ receptors respectively. Compounds 18 and 19, the ether and thioether analogs of compound 17 respectively, display less affinity for the sigma receptors while compounds 20 and 21, the meta and ortho-substituted fluoro analogs show increased affinity and selectivity for the σ₂ receptor. In fact, compound 21 has the strongest affinity (σ₂ Ki=2.2 nM) and selectivity (σ₁Ki/σ₂Ki=25.9) for the σ₂ receptor among all the compounds evaluated including the lead compound SYA013.

Given the literature precedent that oximes may enhance activity of a compound’s antitumor activity³⁹ we synthesized the oximes of compounds 2, 3 and 4 and obtained compounds 22, 23 and 24 as shown in Fig 4. The results of the screening of these compounds at the sigma receptors is reported in Table 4, showing that the oximes maintain their sigma binding affinity and selectivity for the σ₂ receptor.

Fig 4.

Oxime analogs of group 1 agents (Group 4).

Table 4.

Binding affinity constants of oxime analogs in group 4 at σ receptors.

Compds	(σ1R,* Ki), nM/pKi ± SEM	C(σ2R, Ki), nM/pKi ± SEM	Ki(σ1R)/Ki (σ2R)	Compds	(σ1R) * (Ki), nM/pKi ± SEM	(σ2R) Ki, nM/pKi ± SEM
22	(113.0)	(2.7)/8.6 ± 0.1	41.8	2	(30)77.52 ± 0.05	(8.0)/8.1 ± 0.05
23	(70)	(13.1)	5.3	3	(63)/6.34 ± 0.06	(32)77.49 ± 0.09
24	(1 4 8)	(26.0)77.6 ± 0.1	5.7	4	(63)77.20 ± 0.09	(44)77.40 ± 0.10

^*Ki data are wihin 20% of the mean value. pKi data are recorded as the Mean ± SEM.

As indicated in Table 4, and consistent with an overall increase in lipophilicity, all the oximes increased their binding affinities at the σ₂ receptor as compared to their carbonyl counterparts in group 1. The reverse was the case for binding at the σ₁ receptor. In addition, 22, the oxime of compound 2, produced the highest selectivity for the σ₂ receptor (σ₁Ki/σ₂Ki=41.8) among all the compounds evaluated in this study.

3. Conclusions

In this article, we have evaluated four groups of compounds: homopiperazine, piperazine, tropane and oxime analogs of SYA013. Among these groups, the homopiperazine and tropane groups have the highest affinity ligands at the σ₂ receptor (7 and 21; Ki = 2.2 nM), and 21 displays a high selectivity (σ₁Ki_/σ₂Ki =25.9 nM) as well. Overall, oxime 22, displayed the highest selectivity (σ₁Ki_/σ₂Ki=41.8) among all the compounds evaluated in this study. Compared with PB28, one of the highest affinity ligands at the σ₂R (Ki = 0.68 nM),³⁸ compound 22 compares favorably but has a higher selectivity for the σ₂ receptor.

4. Experimental

4.1. General Information

Melting points were determined on a Gallenkamp (UK) apparatus and are uncorrected. All NMR spectra were obtained on either a Varian 300 MHz Mercury Spectrometer or 600 MHz Bruker Spectrometer. Elemental analyses were carried out by Atlantic Microlab, Inc., Norcross, GA, and are within 0.4% of theory unless otherwise noted. Flash chromatography was performed with Davisil grade 634 silica gel. Starting materials were obtained from Sigma–Aldrich and were used without further purification.

4.2. Preparation of 1-(5-chloropyridin-2-yl)-1,4-diazepane (26a) and 1-(5-chloropyridin-2-yl)piperazine (28)

4.2.1. 1-(5-chloropyridin-2-yl)-1,4-diazepane (26a)

A one to one mixture of homopiperazine and 2, 5-dichloropyridine in ethylene glycol was heated at 150 C for 12 hrs. After cooling to room temperature, the mixture was diluted with EtOAc (200 mL) followed by washing with saturated NaHCO₃ (2×200 mL). The organic layer was dried over Na₂SO₄ and filtered. The filtrate was concentrated in vacuo to dryness, followed by column chromatography on silica gel to afford 1-(5-chloropyridin-2-yl)-1,4-diazepane as an oily residue in a yield of 65%. ¹H NMR (300 MHz, CDCl₃): δ 7.94 (1H, d, J = 2.4 Hz), 7.44 (1H, brs), 7.25 (1H, dd, J = 2.4, 9.0 Hz), 6.33 (1H, d, J = 9.0 Hz), 3.89 (2H, t, J = 5.8 Hz), 3.67 (2H, t, J = 5.8 Hz), 3.15 (2H, t, J = 5.8 Hz), 3.01 (2H, t, J = 6.0 Hz), 2.12 (2H, m).

¹³C (75 MHz, CD₃OD): δ 156.0, 145.8, 137.2, 119.3, 106.8, 45.9, 45.7, 45.2, 43.6, 25.3.

4.2.2. 1-(5-chloropyridin-2-yl)piperazine (28)

A one to one mixture of piperazine and 2, 5-dichloropyridine in ethylene glycol was heated at 150 C for 12 hrs. After cooling to room temperature, the mixture was diluted with EtOAc (200 mL) followed by washing with saturated NaHCO₃ (2×200 mL). The organic layer was dried over Na₂SO₄, and filtered. The filtrate was concentrated in vacuo to dryness to obtain a residue that was purified by column chromatography on silica gel to afford 1-(5-chloropyridin-2-yl)piperazine as an oily residue in a yield of 78%.

¹H NMR (300 MHz, CDCl₃): δ 8.10 (1H, d, J = 6.0 Hz), 7.41 (1H, dd, J = 6.0, 9.0 Hz), 6.57 (1H, d, J = 9.0 Hz), 3.46–3.49 (4H, m), 2.96–3.00 (4H, m), 2.67 (1H, brs).

¹³C (75 MHz, CDCl₃): δ 157.6, 146.3, 137.1, 120.4, 107.8, 45.3, 44.8.

4.3. Synthesis of homopiperazine analogs 2, 3, 4 and piperazine analogs (8 and 10)

General Procedure I: A mixture of 4-chloro-1-(4-fluorophenyl)butan-1-one (1.0 g, 5.0 mmol), 1-(5-chloropyridin-2-yl)-1,4-diazepane ( 1.2 g, 5.7 mmol), KI (250 mg), NaHCO₃ (1.0 g, 11.9 mmol) in ⁱPrOH (10 mL) was heated to reflux under N₂ for 12 h. After cooling to rt, the mixture was diluted with EtOAc (500 mL) and washed with water (2×300 mL). The organic layer was dried over Na₂SO₄, filtered and the filtrate was concentrated in vacuo to dryness to obtain a residue which was purified by column chromatography on silica gel to afford 4-(4-(5-chloropyridin-2-yl)-1,4-diazepan-1-yl)-1-(4-fluoro-phenyl)butan-1-one 2 as a free base.

4.3.1. 4-(4-(5-chloropyridin-2-yl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one (2·2HCl)

The free base of 2 was converted into the HCl salt by the addition of ethereal HCl and the salt was purified by recrystallization from MeOH-Et₂O to afford the pure salt in a yield of 32%. Mp 185–187 °C, Calcd for C₂₀H₂₅Cl₃FN₃O. 0.4 H₂O: C 52.68, H 5.70, N 9.22; Found: C 52.64, H 5.81, N 9.14.

¹H NMR (300 MHz, DMSO-d⁶): δ 10.70 (1H, brs), 8.09 (1H, d, J = 2.4 Hz), 8.00–8.07 (2H, m), 7.63 (1H, dd, J = 2.7, 9.0 Hz), 7.31–7.38 (2H, t, J = 9.0 Hz), 6.76 (1H, d, J = 9.0 Hz), 4.26 (1H, dd, J = 4.2, 14.2 Hz), 3.75 (1H, dd, J = 8.4, 15.3 Hz), 3.47–3.61 (4H, m), 3.06–3.18 (6H, m), 2.32–2.43 (1H, m), 2.09–2.17 (1H, m), 1.96–2.06 (2H, m).

¹³C NMR (75 MHz, DMSO-d⁶): δ 197.8, 165.7 (d, J = 250.1 Hz), 155.5, 143.5, 139.0, 133.6 (d, J = 3.0 Hz), 131.3 (d, J = 9.3 Hz), 118.7, 116.2 (d, J = 21.4 Hz), 109.4, 55.9, 54.2, 53.4, 46.5, 41.8, 35.7, 23.4, 18.5.

4.3.2. 1-(4-fluorophenyl)-4-(4-(pyridin-2-yl)-1,4-diazepan-1-yl)butan-1-one (3·2HCl)

The free base was converted into the HCl salt by the addition of ethereal HCl, followed by recrystallization from MeOH-Et₂O to afford the pure salt in a yield of 34.7%. Mp 198–199 °C, Calcd for C₂₀H₂₆Cl₂FN₃O^.0.5 H₂O: C 56.74, H 6.43, N 9.93; Found: C 56.86, H 6.36, N 9.76.

¹H NMR (300 MHz, DMSO-d⁶): δ 11.27 (1H, s), 8.00–8.06 (3H, m), 7.94–7.99 (1H, m), 7.34 (2H, t, J 9.0 Hz), 7.25 (1H, brs), 6.91–6.95 (1H, m), 4.20–4.26 (1H, m), 4.00–4.10 (1H, m), 3.62–3.75 (2H, m), 3.53–3.61 (2H, m), 3.09–3.29 (6H, m), 2.50–2.56 (2H, m), 2.14–2.20 (1H, m), 1.96–2.02 (2H, m).

¹³C NMR (75 MHz, DMSO-d⁶): δ 197.8, 165.5 (d, J = 250.7 Hz), 152.5, 143.8, 137.7, 133.5 (d, J = 2.3 Hz), 131.3 (d, J = 9.1 Hz), 116.2 (d, J = 21.8 Hz), 113.0, 112.6, 56.2, 53.3, 52.8, 48.1, 43.3, 35.7, 23.0, 18.5.

4.3.3. 1-(4-fluorophenyl)-4-(4-(pyrimidin-2-yl)-1,4-diazepan-1-yl)butan-1-one (4·2HBr)

The free base was treated with ethereal HBr to form a salt. Recrystallization from MeOH:Et₂O, afforded 1-(4-fluorophenyl)-4-(4-(pyrimidin-2-yl)-1,4-diazepan-1-yl)butan-1-one 4, dihydrobromide, in yield of 33%, Mp 225–230 °C. Calculated for C₁₉H₂₅Br₂FN₄O: C 45.26, H 5, N 11.11; Found: C 45.47, H 5.07, N 11.02.

¹H NMR (600 MHz, DMSO-d⁶): δ 9.80 (1H, s), 8.51 (2H, d, J = 4.9 Hz), 8.21–7.92 (2H, m), 7.52–7.25 (2H, m), 6.81 (1H, t, J = 4.9 Hz), 4.49–4.21 (1H, m), 3.93–3.81 (2H, m), 3.78–3.74 (1H, m), 3.72–3.65 (1H, m), 3.62–3.52 (1H, m), 3.34–3.23 (2H, m), 3.23–3.14 (4H, m), 2.37–2.3 (1H, m), 2.25–2.13 (1H, m), 2.08–1.99 (2H, m).

¹³C NMR (151 MHz, DMSO-d⁶): δ 197.7, 165.1 (d, J = 250.0 Hz), 161.5, 157.6, 133.6, 131.4 (d, J = 9.4 Hz), 116.2 (d, J = 21.7 Hz), 110.7, 56.8, 55.4, 54.78, 4.36, 45.9, 36.0, 27.4, 22.2.

4.3.4. 4-(4-(5-chloropyridin-2-yl)piperazin-1-yl)-1-(4-fluorophenyl)butan-1-one (8·HCl)

The free base was converted into the salt by addition of ethereal HCl and the salt was purified by recrystallization from MeOH-Et₂O to afford the pure salt in a yield of 25%, Mp 183–185 °C. Calcd for C₁₉H₂₂Cl₂FN₃O^. 1.0 H₂O: C 54.81, H 5.81, N 10.09; Found: C 54.89, H 5.47, N 10.09.

¹H NMR (300 MHz, DMSO-d⁶): δ 10.47 (1H, brs), 8.15 (1H, d, J = 2.7 Hz), 8.06 (1H, d, J = 5.7 Hz), 8.03 (1H, d, J = 5.7 Hz), 7.68 (1H, dd, J = 2.7, 9.0 Hz), 7.36 (2H, t, J = 9.0 Hz), 6.98 (1H, d, J = 9.3 Hz), 4.33 (2H, d, J = 14.1 Hz), 3.56 (2H, d, J = 14.1 Hz), 3.21–3.32 (2H, m), 3.00–3.21 (6H, m), 1.91–2.06 (2H, m).

¹³C NMR (75 MHz, DMSO-d⁶): δ 197.7, 165.5 (d, J = 250.1 Hz), 157.0, 145.8, 138.1, 133.6, 131.3 (d, J = 9.3 Hz), 120.5, 116.2 (d, J = 21.4 Hz), 109.8, 55.5, 50.6, 42.3, 35.7, 18.1.

4.3.5. 1-(5-chloropyridin-2-yl)-4-(4-(4-fluorophenyl)butyl)piperazine (10·HCl)

The free base was converted into the salt by addition of ethereal HCl and the residue was purified by recrystallization from MeOH-Et₂O to afford the pure salt in a yield of 31%, Mp 195–196 °C. Calcd for C₁₉H₂₄Cl₂FN₃: C 59.38, H 6.29, N 10.93; Found: C 59.22, H 6.04, N 10.90.

¹H NMR (300 MHz, DMSO-d⁶): δ 10.30 (1H, brs), 8.15 (1H, d, J = 1.8 Hz), 7.68 (1H, dd, J = 2.7, 9.0 Hz), 7.21–7.26 (2H, m), 7.09 (2H, t, J = 9.0 Hz), 6.97 (1H, d, J = 8.4 Hz), 4.32 (2H, d, J = 14.2 Hz), 3.47–3.51 (2H, m), 3.17–3.26 (2H, m), 2.93–3.12 (4H, m), 2.56–2.61 (2H, m), 1.62–1.71 (2H, m), 1.52–1.60 (2H, m).

¹³C NMR (75 MHz, DMSO-d⁶): δ 161.1 (d, J = 239.5 Hz), 157.0, 146.1, 138.2, 138.0, 130.5 (d, J = 7.7 Hz), 120.5, 115.4 (d, J = 20.3 Hz), 109.6, 55.6, 50.6, 42.2, 34.1, 28.6, 23.0.

4.4. Synthesis of 1-(5-chloropyridin-2-yl)-4-(3-((4-fluorophenyl)thio)propyl)-1,4-diazepane hydrochloride (7· HCl)

The method previously reported in Sampson et al.³⁴ was used. A mixture of 3-((4-fluorophenyl)-thio)propyl methanesulfonate (0.86 g, 3.5 mmol), 1-(5-chloropyridin-2-yl)-1,4-diazepane (0.85 g, 4.0 mmol) 26a, KI (150 mg), NaHCO₃ (1.0 g, 11.9 mmol) in ⁱPrOH (10 mL) was heated to reflux under N₂ for 12 h. After cooling to rt, the mixture was diluted with EtOAc (500 mL), followed by washing with water (2×300 mL), the organic layer was separated, dried over Na₂SO₄, and filtered. The filtrate was concentrated in vacuo to dryness, followed by column chromatography on silica gel to afford 1-(5-chloropyridin-2-yl)-4-(3-((4-fluorophenyl)thio)propyl)-1,4-diazepane 7. The product was converted into the salt by the addition of ethereal HCl and the salt was purified by recrystallization from MeOH-Et₂O to afford the HCl salt, in a yield of 42%, Mp 191–193 °C. Calcd for C₁₉H₂₅Cl₃FN₃S^. 0.5 H₂O: C 49.41, H 5.67, N 9.10; Found: C 49.41, H 5.65, N 9.08.

¹H NMR (300 MHz, DMSO-d⁶): δ 10.91 (1H, brs), 8.08 (1H, d, J = 2.4 Hz), 7.63 (1H, dd, J = 3.0, 9.0 Hz), 7.38–7.45 (2H, m), 7.14–7.21 (2H, m), 6.75 (1H, d, J = 9.0 Hz), 4.20–4.27 (1H, m), 3.67–3.75 (1H, m), 3.41–3.53 (4H, m), 3.11–3.17 (2H, m), 3.03–3.07 (2H, m), 2.95–3.00 (2H, m), 2.31–2.41 (1H, m), 2.04–2.16 (1H, m), 1.90–2.00 (2H, m).

¹³C NMR (75 MHz, DMSO-d⁶): δ 161.4 (d, J = 241.7 Hz), 154.5, 141.4, 140.1, 131.9 (d, J = 7.6 Hz), 131.1 (d, J = 2.8 Hz), 118.7, 116.7 (d, J = 21.4 Hz), 110.6, 52.3, 54.0, 53.5, 47.0, 42.2, 30.9, 23.6, 23.3.

4.5. Synthesis of oximes 22–24

General Procedure II: A mixture of 4-(4-(5-chloropyridin-2-yl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one 2 (0.65g, 1.73 mmol), hydroxylamine hydrochloride (0.25g, 3.46 mmol), KOH (0.19g, 3.46 mmol) in EtOH (10 mL) was heated to reflux at 90˚C for 5 h. After cooling to room temperature, H₂O was added to the reaction mixture to precipitate the compound. The residue was washed with water and EtOAc and the aqueous layer was extracted twice with EtOAc. The combined organic layer was dried over anhydrous Na₂SO₄ and filtered. The filtrate was evaporated and the residue purified by flash chromatography using (90:10% EtOAc: Hexane).

4.5.1. (4-(4-(5-chloropyridin-2-yl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one oxime (22)

The product was recrystallized using EtOAc/Hexane to afford (4-(4-(5-chloropyridin-2-yl)-1,4-diazepan-1-yl)-1-(4-fluorophenyl)butan-1-one oxime 22 in a yield of 37%, Mp 148–150 °C. Calculated for C₂₀H₂₄ClFN₄O: C 61.46, H 6.19, N 14.33; Found: C 61.68, H 6.28, N 14.33. ¹H NMR (600 MHz, DMSO-d⁶); δ 11.16 (1H, s), 8.03 (1H, d, J = 2.7 Hz), 7.83–7.58 (2H, m), 7.51 (1H, dd, J = 2.7, 9.1 Hz), 7.42–6.94 (2H, m), 6.63 (1H, d, J = 9.2 Hz), 3.71–3.59 (2H, m), 3.56 (2H, t, J 6.1 Hz), 2.72–2.65 (2H, m), 2.65–2.60 (2H, m), 2.49–2.44 (2H, m), 2.43 (2H, dd, J = 8.7, 15.6 Hz), 1.92–1.69 (2H, m), 1.69–1.49 (2H, m).

¹³C NMR (151 MHz, DMSO-d⁶): δ 162.8 (d, J = 244.3 Hz), 156.9, 156.4, 146.0, 137.3, 133.2, 128.3 (d, J = 8.3 Hz), 117.4, 115.7 (d, J = 21.6 Hz), 107.2, 57.0, 55.0, 54.5, 47.3, 46.6, 27.3, 24.4, 23.4.

4.5.2. 1-(4-fluorophenyl)-4-(4-(pyridin-2-yl)-1,4-diazepan-1-yl)butan-1-one oxime (23)

Using the general method II, 1-(4-fluorophenyl)-4-(4-(pyridin-2-yl)-1,4-diazepan-1-yl)butan-1-one, 3 was treated with hydroxylamine hydrochloride to form the product which was recrystallized from EtOAc/Hexane to afford 1-(4-fluorophenyl)-4-(4-(pyridin-2-yl)-1,4-diazepan-1-yl)butan-1-one oxime 23 in a yield of 48%, Mp 108–112 °C. Calculated for C₂₀H₂₅FN₄O: C 67.39, H 7.07, N 15.72; Found: C 67.43, H 6.90, N 15.66.

¹H NMR (600 MHz, DMSO-d⁶): δ 11.16 (1H, s), 8.43–7.87 (1H, m), 7.89–7.54 (2H, m), 7.46–7.43 (1H, m), 7.36–7.08 (2H, m), 6.57 (1H, d, J = 8.7 Hz), 6.54–6.46 (1H, m), 3.78–3.63 (2H, m), 3.57 (2H, t, J = 6.2 Hz), 2.76–2.66 (2H, m), 2.68–2.59 (2H, m), 2.48–2.44 (2H, m), 2.43 (2H, t, J = 7.0 Hz), 1.80 (2H, dt, J = 6.0, 11.7 Hz), 1.69–1.49 (2H, m).

¹³C NMR (151 MHz, DMSO-d⁶): δ 162.8 (d, J = 244.3 Hz), 158.3, 156.4, 148.1, 137.7, 133.2 (d, J = 2.4 Hz), 128.3 (d, J = 8.3 Hz), 115.7 (d, J = 21.4 Hz), 111.5, 105.8, 57.0, 55.3, 54.6, 46.9, 46.2, 27.5, 24.4, 23.4.

4.5.3. 1-(4-fluorophenyl)-4-(4-(pyrimidin-2-yl)-1,4-diazepan-1-yl)butan-1-one oxime (24)

Using the general method II, 1-(4-fluorophenyl)-4-(4-(pyrimidin-2-yl)-1,4-diazepan-1-yl)butan-1-one 4 was treated with hydroxylamine hydrochloride to form the product which was recrystallized from EtOAc/Hexane to afford 1-(4-fluorophenyl)-4-(4-(pyrimidin-2-yl)-1,4-diazepan-1-yl)butan-1-one oxime 24 in a yield of 20%, Mp 126–128 °C. Calculated for C₁₉H₂₄FN₅O: C 63.85, H 6.77, N 19.59; Found: C 63.49, H 6.79, N 19.57.

¹H NMR (600 MHz, CDCl₃): δ 8.32 (2H, d, J = 4.7 Hz), 7.79–7.57 (2H, m), 7.17–7.00 (2H, m), 6.48 (1H, t, J = 4.7 Hz), 4.12–3.91 (2H, m), 3.84 (2H, t, J = 6.4 Hz), 2.80 (3H, dd, J = 4.5, 8.8 Hz), 2.75–2.62 (m, 2H), 2.62–2.50 (2H, m), 2.16–1.98 (2H, m), 1.98–1.74 (3H, m).

¹³C NMR (151 MHz, CDCl₃): δ 163.3 (d, J = 247.3 Hz), 161.5, 158.3, 157.7, 132.1 (d, J = 3.2 Hz), 127.9 (d, J = 8.3 Hz), 115.3 (d, J = 21.7 Hz), 109.2, 56.8, 55.6, 54.6, 45.8, 29.7, 27.0, 23.9, 23.5.4.6.