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BY 4.0 license Open Access Published by De Gruyter Open Access July 25, 2022

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

  • Mariia Nesterkina EMAIL logo , Vladimir Musatov , Olena Honcharova and Iryna Kravchenko
From the journal Open Chemistry


Ester based on l-menthol and phenibut ‒ (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl 4-amino-3-phenylbutanoate hydrochloride was obtained in 78% yield using N,N′-dicyclohexylcarbodiimide as a coupling reagent along with catalytic amount of 4-dimethylaminopyridine. The obtained product was characterized by FT-IR, fast-atom bombardment-mass spectrometry along with 1H-NMR and 13C-NMR spectral analysis; the purity was assessed using high-performance liquid chromatography. Phenibut ester has been examined on the models of chemically- and electrically-induced seizures for potential anticonvulsant profile.

1 Introduction

To date, the concept of polypharmacology (or multidrugs) offers the novel prospects for rational drug design aimed at synthesis of compounds capable to interact with multiple targets [1]. The polypharmacological approaches also include drug repurposing and pleiotropy as the ability of compounds to implement more than one mechanism of action [24]. More impactful, however, is a multidrug idea directed towards transporting compounds through membranes by their conjugation with penetration enhancer. Within this framework, specific concern is concentrated on terpenoids that were confirmed to significantly enhance the permeability of hydrophilic drugs [5,6]. In the present study, the aforementioned concept has been applied to anxiolytic and nootropic drug phenibut (β-phenyl-γ-aminobutyric acid) by its conjugation with monocyclic terpenoid l-menthol with a view to increase the anticonvulsant potency. Remarkably, conjugation of biologically active molecules with terpenoid scaffold is feasible by the formation of –NH‒N═C–, –CO‒OR–, and –CO‒NH– bonds which are capable of undergoing enzymatic cleavage in the mammals’ body.

Taking the above-mentioned aspects into consideration, monocyclic terpenoid l-menthol possessing its own anticonvulsant effect [7,8] was applied for the synthesis of ester containing phenibut moiety. Thereby, the present investigation focused on the synthesis and structure confirmation of (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl 4-amino-3-phenylbutanoate hydrochloride comprising both l-menthol and phenibut residues. The synthesized derivative has been subsequently tested in vivo for its anticonvulsant properties on the models of pentylenetetrazole (PTZ)-triggered seizures and maximal electroshock stimulation (MES).

2 Materials and methods

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 1822 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 Results and discussion

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.
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.
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).

Table 1

Anticonvulsant effect of ester 3, l-menthol, phenibut, and VPA against MES-induced seizures in mice (n = 10 animals in each group)

Compound Control l-Menthol Phenibut Compound 3 VPA
3 h after single oral administration
% Mortality protection 0 60 60 100 80
24 h after single oral administration
% Mortality protection 0 40 40 100 60

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).

4 Conclusion

The title compound, (1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl 4-amino-3-phenylbutanoate hydrochloride was successfully synthesized by Steglich esterification, with subsequent structure identification by 1H-NMR, and 13C-NMR spectral analysis along with FT-IR spectroscopy and FAB-mass spectrometry. The title compound has been revealed to possess prolonged antiseizure effect both on chemical (PTZ)- and electrical (MES)-triggered seizures.

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  1. Funding information: No funding to declare.

  2. Author contributions: All authors contributed to the study conception and design. M.N., V.M., and O.H. performed material preparation, data collection, and writing of original draft. I.K.: supervision and editing of original draft. All authors read and approved the final manuscript.

  3. Conflict of interest: No potential conflict of interest was reported by the authors.

  4. Ethical approval: The conducted research is related to animal use. The study has been approved by the Animal Ethics Committee (agreement No. 10/2021) of Odessa International Medical University (Ukraine).

  5. Data availability statement: All data generated are included in this article.


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Received: 2022-03-25
Revised: 2022-06-08
Accepted: 2022-06-29
Published Online: 2022-07-25

© 2022 Mariia Nesterkina et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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