(1R,2S,5R)-5-Methyl-2-(propan-2-yl)cyclohexyl 4-amino-3-phenylbutanoate hydrochloride: Synthesis and anticonvulsant activity

2.1 General

Phenibut, l-menthol, 4-dimethylaminopyridine (DMAP), N,N′-dicyclohexylcarbodiimide (DCC), and di-tert-butyl dicarbonate were obtained from commercial sources (TCI, Philadelphia, PA, USA; Acros Organics, Geel, Belgium or Darmstadt, Germany). Butyloxycarbonyl (Boc)-protected phenibut is not a commercially obtained substance and was synthesized pursuant to the literature procedure [9]. Progress of reactions was monitored by thin-layer chromatography (TLC) using silica gel 60 F₂₅₄ plates followed by UV light visualization applying methanol‒ethyl acetate (1:1) and n-butanol‒water (3:1) as the eluent systems. Varian MR-400 spectrometer 400 MHz along with Varian MR-400 spectrometer 100 MHz has been exploited in order to record ¹H NMR and ¹³C NMR spectra with the use of tetramethylsilane as an internal standard and dimethyl sulfoxide (DMSO)-d ₆ as a solvent. VG 70-70EQ mass spectrometer equipped with a Xe ion gun (8 kV, VG Analytical Ltd, Manchester, UK) was used to receive fast-atom bombardment (FAB) mass spectra; meanwhile, the samples were combined with the m-nitrobenzyl–alcohol matrix. The purity of synthesized ester was checked by high-performance liquid chromatography (HPLC) on Shimadzu LC-20 Prominence HPLC system (Shimadzu, Kyoto, Japan). Frontier FT-IR spectrometer (Perkin-Elmer, Hopkinton, MA, USA) was utilized for the measurement IR-spectra using KBr pellets.

2.2 Synthesis of (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl 4-amino-3-phenylbutanoate hydrochloride (3)

Boc-protected phenibut (2.334 g, 13.04 mmol) was added to a stirred solution of l-menthol (2.0 g, 12.8 mmol) in CH₂Cl₂ (60 mL) at room temperature along with catalytic amount of DMAP (0.388 g, 3.176 mmol). Reaction mixture was chilled to 0°C, then stirred for 10 min followed by dropwise addition of N,N′-dicyclohexylcarbodiimide (2.908 g, 14.12 mmol). The resulted mixture was further stirred for 30 min, thereafter the flask was steadily warmed to 20°C and the stirring was continued for extra 10 h. TLC with UV light visualization was utilized to monitor the progress of the reaction. The precipitate formed during the reaction was filtered off, the filtrate was diluted to 200 mL and consistently washed with 1 M aqueous HCl, 10% aqueous NaHCO₃, and water. Acid-catalyzed N-Boc group deprotection was performed by HCl/CH₃COOH in compliance with the literature procedure [10]. Purification of crude product was carried out by recrystallization from ethanol 96%:water system (3:1).

White powder (78%). ¹H-NMR (400 MHz, DMSO-d ₆ ) δ: 0.39 (d, J = 6.9 Hz, 3H, CH₃-9), 0.54 (d, J = 6.9 Hz, 3H, CH₃-10), 0.66 (d, J = 6.9 Hz, 3H, CH₃-7), 0.74–0.81 (m, 9H, H-2‒H-6, H8), 2.50 (m, 2H, α-CH₂), 2.87–3.00 (m, 3H, β-CH + γ-CH₂), 4.39–4.41 (m, 1H, H-1), 7.26–7.32 (m, 5H, ArH). ¹³C-NMR (100 MHz, DMSO-d ₆ ) δ: 170.8 (C═O), 140.1 (Ar–C), 128.9 (Ar–C), 128.3 (Ar–C), 127.8 (Ar–C), 73.8 (C-1), 46.7 (C-2), 43.9 (γ-CH₂), 40.8 (α-CH₂), 38.6 (C-6), 38.2 (β-CH), 34.0 (C-4), 31.1 (C-5), 25.8 (C-8), 23.2 (C-3), 21.0 (C-9), 20.9 (C-10), 16.4 (C-7). FTIR (KBr, ν _max, cm⁻¹): 3,423 (NH₃ ⁺); 2,953–2,869 (C–H); 1,725 (C═O); 1,508 (C–C, Ar); 1,297, 1,234 (C‒O‒C); 697, 760, 888 (C–H, Ar). MS (FAB) m/z: 318 [M + H]⁺. MP 210‒211°C. HPLC: t _r = 3.658 min.

2.3 Anticonvulsant screening

Anticonvulsant properties of synthesized phenibut ester have been examined using outbreed male white mice with the mass of 18–22 g as experimental animals. The 12 h light regime was maintained for the animals along with standard animal facility: free access to water and food, pursuant to ARRIVE guidelines, European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Specific Purposes (Strasbourg, 1986), and the principles of the National Ukrainian Bioethics Congress (Kyiv, 2003). Mice were purchased from Odessa National Medical University, Ukraine. The study has been approved by the Animal Ethics Committee (agreement No. 10/2021) of Odessa International Medical University (Ukraine). l-Menthol, phenibut, VPA, and ester 3 have been administered orally (tentatively dissolved in Tween 80/water emulsion): compound 3 at a dose of 200 mg/kg, l-menthol, phenibut, and VPA in equimolar amounts. Anticonvulsant properties of phenibut ester were evaluated both at short (3 h) and long (24 h) time period after its single oral administration on the models of MES and PTZ-triggered seizures in compliance with the earlier reported procedures [11,12].

2.4 Statistical analysis

Experimental results are denominated as mean ± standard error mean (SEM). p < 0.05 was considered as significant. In order to identify the statistical significance, one-way analysis of variance was applied followed by Tukey’s post hoc comparison.

3.1 Chemistry

The desired target product (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl 4-amino-3-phenylbutanoate hydrochloride has been obtained using N,N′-dicyclohexylcarbodiimide as a coupling reagent along with catalytic amount of DMAP via Steglich esterification, as illustrated in Scheme 1.

Scheme 1

Synthesis of (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl 4-amino-3-phenylbutanoate hydrochloride.

For this purpose, amino group of phenibut has been subjected to tert-Boc protection under aqueous conditions by reaction with a base and di-tert-butyl dicarbonate (Boc₂O) resulting in the formation of Boc-derivative 1. In the second stage, l-menthol was acylated by compound 1 under mild experimental conditions (DCC/DMAP-mediated coupling) that allows the conversion of acid labile substrates. N-Boc deprotection of ester 2 was performed by acid-induced cleavage using 1 M HCl in glacial acetic acid in compliance with the literature procedure [10].

Target compound 3 was isolated in hydrochloride form at 78% yield as white solid, well soluble in dimethyl sulfoxide and methanol. The structure of synthesized ester 3 was verified by ¹H-NMR, ¹³C-NMR, and FAB-mass spectrometry along with FT-IR spectroscopy. The purity of synthesized ester was estimated by HPLC analysis employing the reversed-phase method with isocratic elution via a system composed of methanol‒1% ammonium formate aqueous solution (90:10). According to the HPLC assay, the observed retention time for target product 3 was 3.658 min; the purity was determined by internal normalization through detection at ultraviolet wavelength (260 nm) as 100%. In the present work, FAB was imposed as an ionization method in mass spectrometry analysis; protonated molecular ion peak [M + H]⁺ at m/z 318 of phenibut ester 3 is observed in the FAB-MS spectrum. The FT-IR spectrum shows absorption bands of N–H bonds (3,423 cm⁻¹), C═O group (1,725 cm⁻¹), C‒O ester group (1,297, 1,234 cm⁻¹), alkyl C–H (2,953‒2,869 cm⁻¹), and Aryl‒H deformation vibrations (697, 760, 888 cm⁻¹). The ¹H NMR spectrum of obtained phenibut derivative contains the proton H-1 of cyclohexane ring resonated at δ 4.39‒4.41 ppm as a multiplet. The methyl groups of l-menthol at C-5 and C-8 are observed as doublets at δ 0.39, 0.54, and 0.66 ppm. In addition, a multiplet observed at 7.26–7.32 ppm was assigned to the protons of aromatic core. ¹H-NMR spectrum also contains the signals from axial and equatorial ring protons, their multiplicity along with the position corresponds to similar signals in l-menthol proton spectrum. Similarly, the ¹³C-NMR spectrum revealed the presence of all carbon atoms present in the synthesized molecule.

3.2 Anticonvulsant activity

Antiseizure action of the synthesized phenibut ester 3 was estimated by two pharmacological seizure models including MES and intravenous PTZ-triggered (i.v. PTZ) tests. Minimum effective doses of PTZ induced clonic–tonic convulsions (DCTC) and tonic extension (DTE) has been identified in chemically-triggered test. As illustrated in Figure 1, ester 3 demonstrated high protection against PTZ-triggered convulsion at 3 h after administration, as proven by the increase of DCTC and DTE values to 312 and 321%, respectively, compared with the control (100%) and reference drug valproic acid (VPA) that was slightly active at this time period (157% of DCTC and 161% of DTE). Moreover, synthesized derivative 3 was revealed to exhibit significant anticonvulsant properties over a long time period (24 h after administration) with the average values of 251% for DCTC and 262% for DTE. As seen, ester 3 exhibited higher potency versus initial phenibut, l-menthol, and VPA (p < 0.05), which indicated its prolonged effect.

Figure 1

Anticonvulsant activity of ester 3, l-menthol, phenibut, and VPA against PTZ-induced seizures in mice at 3 and 24 h after oral administration. Values are given as mean ± SEM, n = 5 mice; for all groups p < 0.01 compared with the control; *p < 0.05 and **p < 0.01 compared with VPA.

In the MES test, phenibut ester 3 considerably averted the animals’ mortality at 3 h after administration, showing 100% protection that exceeds the VPA effect (80%), whereas moderate anticonvulsant effect was observed for the initial phenibut and l-menthol (60%) (Table 1).

In the meantime, the activity of phenibut ester 3 remained for a long time period (24 h) with 100% of mortality protection that reaffirms the idea toward enzymatic cleavage of labile ester bond in synthesized compound, followed by gradual release of pure phenibut and terpenoid. Thus, phenibut ester 3 protects against convulsions triggered both by chemical and electrical stimuli during a long time period (3–24 h after single oral administration).