Anti-human immunodeficiency activity of novel 2-arylpyrrolidine analogs

Abstract

A series of 26 new compounds were synthesized and screened for their anti-human immunodeficiency virus-1 and cytotoxicity activity. Of these, 14 were found to be inhibitors of human immunodeficiency virus replications in primary human lymphocytes with 50 % effective concentration values <20 μM. Moreover, most of the compounds were cytotoxic to human lymphocytes, CEM, and Vero cells. Our structure activity relationship study identified different patterns. Compounds 2g–j and 4 (whose structure is closer to the loviride structure) were very potent. Comparing the activity of the compounds containing the 2-aryl substituents, we observed that compounds with benzyloxyphenyl groups were more potent. Compounds in which the 1-aryl moiety contained methyl group in 4- or 3,5-positions also showed high activity. In the series of compounds containing the nitrile, amine, and amide groups, we observed a decrease in activity with CN > NH₂ > C(O)NH₂. The difference of activity between the 5-membered and 4-membered rings compounds was not significant. This initial information could be used to design improved anti-human immunodeficiency virus compounds in this class.

Introduction

Acquired immunodeficiency syndrome (AIDS) has become the leading pandemic disease, and the cause of death worldwide. Human immunodeficiency virus (HIV) is the etiological agent of AIDS. Following infection, this retrovirus uses three key enzymes to complete its replication cycle: the reverse transcriptase (RT), the integrase, and the protease (Sarafianos et al. 2004).

The first RT inhibitors approved were nucleoside derivatives (NRTIs), which compete as 5′-triphosphates with normal nucleoside substrates for incorporation into the viral genome, thus behaving as chain terminators. Unlike nucleoside analogs, non-nucleoside RT inhibitors (NNRTIs) bind in a non-competitive manner to a specific pocket of the HIV-1 RT, which is closely associated with, but distinct from the substrate binding site, altering its ability to function. NNRTIs gained the greatest importance because of their specificity and low cytotoxicity. All NNRTIs bind to a hydrophobic pocket near the polymerase active site. NNRTIs were found to be a more potent class of compounds than the NRTIs and nucleotide RT inhibitors because they differ structurally from the nucleoside analogs. NNRTIs do not interfere with the human cell cycle and are specific inhibitors of HIV-1 RT (Prajapati et al. 2009).

The era of NNRTIs began two decades ago, with the discovery of HEPT and TIBO, two specific inhibitors of the HIV RT. Shortly thereafter, loviride, a representative of the α-anilinophenylacetamides (α-APA) family, was discovered. With the discovery of α-APAs, the era of flexible derivatives started. These exhibited 50 % effective concentration (EC₅₀) values in the nanomolar range. The most active compound in this series is loviride with an EC₅₀ of 13 nM, and an inhibitory concentration 10,000-fold less than the cytotoxic concentration (Fig. 1) (De Corte 2005; Pauwels et al. 1993).

Fig. 1.

Prototype α-APA compounds and their inhibition of HIV-1 replication

The simplicity of its structure and the relative ease of its synthesis made the α-APA series attractive for lead optimization. The compounds in this series were found to bind to the allosteric pocket of RT (Ding et al. 1995).

The purpose of our studies was to discover new antiviral compounds, identify fragments responsible for biological/antiviral/anti-HIV activity in the synthesized compounds to efficiently design and synthesize compounds of this class with high activity.

Herein, we report the synthesis and the biological study of new derivatives of 1,2-diarylpyrrolidinecarbonitriles, which contain fragments of the known RT inhibitor—loviride. During the course of our efforts toward the development of our drug candidate, a synthesis of pyrrolidine framework was required as a core structure (Fig. 2).

Fig. 2.

New derivatives of 1,2-diarylpyrrolidinecarbonitriles

Materials and methods

General

All the chemicals were purchased from Alfa Aesar or Sigma Aldrich and were used without purification. ¹H and ¹³C NMR spectra were recorded on a Varian Mercury 300Vx instrument at 300 and 75 MHz, respectively. Chemical shifts are reported in ppm referenced to the residual solvent signal. FT-IR spectra were recorded on a Nicolet Avatar 330 spectrometer. Melting points were recorded with a Boetius PHMK 77/1479 (Veb Analytik Dresden, Germany) apparatus and are uncorrected.

Chemistry

General procedure for the synthesis of acetonitriles (2a–j)

A solution of sodium cyanide (1 equiv, 10 mmol) in water (10 mL) was added to a solution of the appropriate aldehyde 1 (1 equiv, 10 mmol) in EtOH (20 mL) at room temperature. The mixture was stirred for 10 min and then acetic acid (1 equiv, 10 mmol) was added. After 10 min, the corresponding amine (1 equiv, 10 mmol) in EtOH (10 mL) was added and the resulting mixture was stirred for another 2 h at room temperature. After completion of the reaction, ice water (10 mL) was then added to the mixture. The formed crystals were filtered off, washed with water (10 mL), dried, and recrystallized from EtOH.

Compounds 2a, 2b were synthesized by us and described in Gasparyan et al. (2012).

Compounds 2c, 2d were synthesized by us and described in Gasparyan et al. (2014).

2-(4-Isopropoxyphenyl)-2-(4-toluidino)acetonitrile (2e) was prepared in 80 % of yield, white crystalline m.p. 91–92 °C; IR (nujol mull) ν_max, 3355 (NH), 2232 (C≡N), 1612 (arom.) cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz): δ = 7.49–7.44 (2H, m) and 6.92–6.87 (2H, m, 4-iPrOC₆H₄), 6.96–6.91 (2H, m) and 6.69–6.64 (2H, m, 4-MeC₆H₄), 6.06 (1H, d, J = 9.1 Hz, NH), 5.52 (1H, d, J = 9.1 Hz, CHCN), 4.59 (1H, sp, J = 6.0 Hz, CH-iPr), 2.24 (3H, s, Me), 1.33 (6H, d, J = 6.0 Hz, 2×Me-iPr); ¹³C NMR (DMSO-d₆/CC1₄ 1/3, 75 MHz): δ = 157.5, 143.2, 128.9 (2×CH), 128.1 (2×CH), 126.5, 126.4, 118.6 (CN), 115.2 (2×CH), 113.7 (2×CH), 68.8 (OCH), 48.1 (CH), 21.5 (Me₂), 20.0 (Me); anal. calcd. for C₁₈H₂₀N₂O: C, 77.11; H, 7.19; N, 9.99. Found: C, 77.23; H, 7.31; N, 9.74.

Compound 2f was synthesized by us and described in Gasparyan et al. (2014).

Compound 2g was synthesized by us and described in Gasparyan (2014).

2-[4-(2,4-Dichlorobenzyloxy)phenyl]-2-(4-toluidino)acetonitrile (2h) was prepared in 68 % of yield, light yellow crystalline m.p. 142–143 °C; IR (nujol mull) ν_max 3340 (NH), 2229 (C≡N), 1612 (arom.), 1590 (arom.) cm⁻¹; ¹H NMR (DMSO-d₆/CCl₄ 1/3, 300 MHz): δ = 7.57 (1H, d, J = 8.3 Hz, H-6 C₆H₃), 7.46 (1H, d, J = 2.1 Hz, H-3 C₆H₃), 7.34 (1H, dd, J = 2.1, 8.3 Hz, H-5 C₆H₃), 7.55–7.50 (2H, m) and 7.04–6.99 (2H, m, 4-MeC₆H₄), 6.95–6.90 (2H, m) and 6.68–6.63 (2H, m, C₆H₄O), 6.12 (1H, d, J = 9.2 Hz, NH), 5.56 (1H, d, J = 9.2 Hz, CH), 5.14 (2H, s, OCH₂), 2.23 (3H, s, Me); ¹³C NMR (DMSO-d₆/CCl₄ 1/3, 75 MHz): δ = 157.9, 143.1, 133.4, 132.9, 132.8, 129.9 (CH), 128.9 (2×CH), 128.5 (CH), 128.3 (2×CH), 127.6, 126.8 (CH), 126.5, 118.6 (CN), 114.5 (2×CH), 113.8 (2×CH), 66.0 (OCH₂), 48.1 (CH), 19.9 (Me); anal. calcd. for C₂₂H₁₈Cl₂N₂O: C, 66.51; H, 4.57; N, 7.05. Found: C, 66.38; H, 4.69; N, 7.23.

Compound 2i was synthesized by us and described in Gasparyan et al. (2014).

Compound 2j was synthesized by us and described in Gasparyan (2014).

Preparation for 2-aryl-5-oxo-2-pyrrolidinecarbonitriles (3a–j)

3-Chloropropanoyl chloride (1 equiv, 10 mmol) was added to a mixture of dry potassium carbonate (1 equiv, 10 mmol) and the appropriate acetonitrile 2 (1 equiv, 10 mmol) in 1,2-dichloroethane (20 mL) at 10–15 °C. The mixture was stirred at room temperature for 30 min and stirring was continued at 40–45 °C for 2h. After addition of 1,2-dichloroethane (20 mL), the mixture was washed with water and dried over CaCl₂. After distillation of the 1,2-dichloroethane dry potassium carbonate (1 equiv, 10 mmol), triethylbenzylammonium chloride (0.5 equiv, 5 mmol) (TEBA), and acetonitrile (20 mL) were added to the residue and the solution was stirred for 4h at 45–50 °C. Product was filtered, the filtrate was evaporated, and the residue was dissolved in chloroform, washed with water, and dried over sodium sulfate. Chloroform was distilled off and the compound was recrystallized from EtOH.

Compounds 3a, 3b were synthesized by us and described in Gasparyan et al. (2012).

Compounds 3c, 3d were synthesized by us and described in Gasparyan et al. (2014).

2-(4-Isopropoxyphenyl)-1-(4-methylphenyl)-5-oxo-2-pyrrolidinecarbonitrile (3e) was prepared in 72 % of yield, white crystalline m.p. 107–108 °C; IR (nujol mull) ν_max 2229 (C≡N), 1721 (C═O), 1614 (arom.) cm⁻¹; ¹H NMR (DMSO-d₆/CCl₄ 1/3, 300 MHz): δ = 7.40–7.35 (2H, m, H-2, H-2′ 4-iPrOC₆H₄), 7.08–7.00 (4H, m, 4-MeC₆H₄), 6.85–6.80 (2H, m, H-3, H-3′ 4-iPrOC₆H₄), 4.56 (1H, sp, J = 6.0 Hz, OCH), 2.92–2.67 (3H, m) and 2.62–2.53 (1H, m, CH₂CH₂), 2.29 (3H, s, Me), 1.31 (6H, d, J = 6.0 Hz, 2×CH₃-iPr); ¹³C NMR (DMSO-d₆/CCl₄ 1/3, 75 MHz): δ = 172.2, 157.8, 135.7, 133.0, 128.7 (2×CH), 127.3, 126.9 (2×CH), 125.4 (2×CH), 118.8 (CN), 115.2 (2×CH), 68.9 (OCH), 65.5 (CCN), 36.6 (CH₂), 29.2 (CH₂), 21.4 (Me₂), 20.4 (Me); anal. calcd. for C₂₁H₂₂N₂O₂: C, 75.42; H, 6.63; N, 8.38. Found: C, 75.49; H, 6.51; N, 8.28.

Compound 3f was synthesized by us and described in Gasparyan et al. (2014).

2-(4-Benzyloxyphenyl)-1-(4-methylphenyl)-5-oxo-2-pyrrolidinecarbonitrile (3g) was prepared in 61 % of yield, yellow crystalline m.p. 146–147 °C; IR (nujol mull) ν_max 2236 (C≡N), 1712 (C═O), 1605 (arom.), 1584 (arom.) cm⁻¹; ¹H NMR (DMSO-d₆/CCl₄ 1/3, 300 MHz): δ = 7.44–7.27 (7H, m) and 6.98–6.93 (6H, m, Ar–H), 5.05 (2H, s, OCH₂), 2.93–2.67 (3H, m) and 2.62–2.54 (1H, m, CH₂CH₂), 2.29 (3H, s, Me); ¹³C NMR (DMSO-d₆/CCl₄ 1/3, 75 MHz): δ = 172.1 (CO), 158.6, 136.1, 135.7, 132.9, 128.7 (2×CH), 128.0, 127.8 (2×CH), 127.3 (CH), 127.0 (2×CH), 126.9 (2×CH), 125.4 (2×CH), 118.8 (CN), 114.7 (2×CH), 69.2 (OCH₂), 65.5 (CCN), 36.6 (CH₂), 29.2 (CH₂), 20.4 (Me); anal. calcd. for C₂₅H₂₂N₂O₂: C, 78.51; H, 5.80; N, 7.32. Found: C, 78.33; H, 5.72; N, 7.45.

2-[4-(2,4-Dichlorobenzyloxy)phenyl]-1-(4-methylphenyl)-5-oxo-2-pyrrolidinecarbonitrile (3h) was prepared in 81 % of yield, yellow crystalline m.p. 161–162 °C; IR (nujol mull) ν_max 2228 (C≡N), 1712 (C═O), 1611 (arom.), 1587 (arom.) cm⁻¹; ¹H NMR (DMSO-d₆/CCl₄ 1/3, 300 MHz): δ = 7.53 (1H, d, J = 8.3 Hz, H-6 C₆H₃), 7.45 (1H, d, J = 2.1 Hz, H-3 C₆H₃), 7.32 (1H, dd, J = 2.1, 8.3 Hz, H-5 C₆H₃), 7.07–6.99 (4H, m, 4-MeC₆H₄), 7.47–7.42 (2H, m) and 6.99–6.94 (2H, m, C₆H₄O), 5.10 (2H, s, OCH₂), 2.94–2.86 (1H, m), 2.84–2.71 (2H, m) and 2.69–2.55 (1H, m, CH₂CH₂), 2.29 (3H, s, Me); ¹³C NMR (DMSO-d₆/CCl₄ 1/3, 75 MHz): δ = 172.1 (CO), 158.1, 135.7, 133.5, 132.8, 132.6, 129.9 (CH), 128.7 (2×CH), 128.5, 128.4 (CH), 127.1 (2×CH), 126.8 (CH), 125.4 (2×CH), 118.7 (CN), 114.7 (2×CH), 65.9 (OCH₂), 65.4 (CCN), 36.6 (CH₂), 29.2 (CH₂), 20.4 (Me); anal. calcd. for C₂₅H₂₀Cl₂N₂O₂: C, 66.53; H, 4.47; N, 6.21. Found: C, 66.38; H, 4.32; N, 6.14.

Compounds 3i, 3j were synthesized by us and described in Gasparyan et al. (2014).

2-(2,6-Dichlorophenyl)-2-(3,5-dimethylanilino)acetamide (4)

2-(2,6-Dichlorophenyl)-2-(3,5-dimethylanilino)acetonitrile (2c) (1 equiv, 10 mmol) was dissolved in conc. H₂SO₄ (10 mL) at 0–5 °C, left at room temperature for 3h, and slowly poured into a beaker with ice. The resulting crystals were filtered off, washed with dilute NaHCO₃ solution and H₂O, and recrystallized from EtOH. Compound (4) was obtained in 94 % yield, white crystalline m.p. 147–148 °C; IR (nujol mull) ν_max 3453 (NH₂), 3364 (NH), 1704 (C═O), 1602 (arom.) cm⁻¹; ¹H NMR (DMSO-d₆, 300 MHz): δ = 7.33 (2H, d, J = 7.8 Hz, H-3,5 C₆H₃Cl₂), 7.23 (1H, t, J = 7.8 Hz, H-4 C₆H₃Cl₂), 7.35 (1H, br) and 6.88 (1H, br., NH₂), 6.32 (2H, s, H-2,6 C₆H₃Me₂), 6.23 (1H, s, H-4 C₆H₃Me₂), 5.65 (1H, d, J = 8.7 Hz, CH), 5.41 (1H, d, J = 8.7 Hz, NH), 2.16 (6H, s, 2×Me); ¹³C NMR (DMSO-d₆/CCl₄ 1/3, 75 MHz): δ = 170.3 (CO), 146.0, 137.4 (2C), 135.3, 135.0 (br.), 128.8 (CH), 128.4 (CH), 119.4 (CH), 111.1 (CH-2,2′ C₆H₃Me₂), 57.7 (CH), 20.9 (2×Me); anal. calcd. for C₁₆H₁₆Cl₂N₂O: C, 59.46; H, 4.99; N, 8.67. Found: C, 59.38; H, 4.77; N, 8.54.

Preparation for 5-aminomethyl-5-aryl-2-pyrrolidinones (5a–g)

To a solution of appropriate 2-aryl-5-oxo-2-pyrrolidinecarbonitrile 3 (1 equiv, 3 mmol) in methylene chloride (10 mL), a mixture of CoCl₂·6H₂O (0.2 equiv, 0.6 mmol) and PEG-400 (1 equiv, 3 mmol) was added. The mixture was stirred and sodium borohydride (5 equiv, 15 mmol) was slowly added at −5 to 0 °C. The stirring was continued at −5 to 0 °C for 2 h and then the reaction mixture was slowly warmed up to room temperature. After completion of the reaction (Thin-layer chromatography), ice water (10 mL) was added. After filtration, and extraction with methylene chloride (20 mL), the organic layer was washed with water, dried over CaCl₂, and distilled off. Product was purified by crystallization from the appropriate solvent or was prepared by forming the corresponding salt.

5-Aminomethyl-1-(4-methylphenyl)-5-phenyl-2-pyrrolidinone (5a) was prepared in 55 % of yield, white crystal m. p. 130 °C; IR (nujol mull) ν_max 3400 broad (NH₂), 1650 (C═O) cm⁻¹; ^JH NMR (DMSO-d₆/CCl₄ 1/3, 300 MHz): δ = 7.38–7.23 (5H, m, C₆H₅), 7.02–6.97 (2H, m) and 6.90–6.85 (2H, m, C₆H₄), 3.27 (1H, d, J = 13.5 Hz) and 3.20 (1H, d, J = 13.5 Hz, NCH₂), 2.92 (2H, br., NH₂+H₂O), 2.79–2.68 (1H, m, CH₂), 2.53–2.47 (2H, m, CH₂), 2.28 (3H, s, Me), 2.01–1.92 (1H, m, CH₂); ¹³C NMR (DMSO-d₆/CCl₄ 1/3, 75 MHz): δ = 174.0 (CO), 143.9, 134.6, 134.0, 128.4 (2×CH), 128.2 (2×CH), 126.8 (CH), 125.3 (2×CH), 125.2 (2×CH), 70.0 (N-CPh), 44.1 (NCH₂), 31.0 (CH₂), 29.4 (CH₂), 20.4 (Me); anal. calcd. for C₁₈H₂₀N₂O: C, 77.11; H, 7.19; N, 9.99. Found: C, 77.32; H, 7.28; N, 10.15.

Compounds 5b–e were synthesized by us and described in Gasparyan et al. (2014).

5-Aminomethyl-5-(4-benzyloxyphenyl)-1-(4-methylphenyl)-2-pyrrolidinone (5f) was prepared in 52 % yield, which gives the corresponding hydrochloric salt, yellow crystal m.p. 169–170 °C; IR (nujol mull) δ_max 3214 broad (NH₂), 1680 (C═O), 1608 (arom.) cm⁻¹; ¹H NMR (DMSO-d₆/CCl₄ 1/3, 300 MHz): δ = 8.65 (3H, br., NH₂, and HCl), 7.43–7.25 (5H, m, C₆H₅), 7.25–7.20 (2H, m), 7.05–7.00 (2H, m), 6.98–6.93 (2H, m) and 6.84–6.79 (2H, m, 2×C₆H₄), 5.08 (2H, s, OCH₂), 3.61 (1H, d, J = 13.6 Hz) and 3.28 (1H, d, J = 13.6 Hz, NCH₂), 2.80–2.53 (3H, m) and 2.33–2.19 (1H, m, CH₂CH₂), 2.29 (3H, s, Me); ¹³C NMR (DMSO-d₆/CCl₄ 1/3, 75 MHz): δ = 173.6 (CO), 157.7, 136.4, 135.5, 133.8, 132.8, 128.7 (2×CH), 127.8 (2×CH), 127.2 (CH), 127.0 (2×CH), 126.8 (2×CH), 126.6 (2×CH), 111.6 (2×CH), 69.1 (OCH₂), 68.9 (NC), 41.5 (NCH₂), 30.3 (CH₂), 29.1 (CH₂), 20.4 (Me); anal. calcd. For C₂₅H₂₆N₂O₂·HCl: C, 70.99; H, 6.43; N, 6.62. Found: C, 71.26; H, 6.59; N, 6.45.

Compound 5g was synthesized by us and described in Gasparyan et al. (2014).

Compounds 6a, b were synthesized by us and described in Gasparyan (2014).

Compound 7 was synthesized by us and described in Martirosyan et al. (2000).

Compounds 8a–c, 9a–c were synthesized by us and described in Gasparyan et al. (2012).

2-Aminomethyl-2-phenyltetrahydro-1H-1-pyrrolyl-4-bromophenylmethanone (10) was prepared in 44 % yield according to the method described for the preparation of compounds (5a–g), which gives the corresponding hydrochloric salt, white crystal m.p. 216–218 °C; IR (nujol mull) ν_max 3302 (NH₂), 1645 (C═O) cm⁻¹; ^:H NMR (DMSO-d₆, 300 MHz): δ = 11.28 (1H, br., HCl), 9.36 (1H, br.) and 9.05 (1H, dd, J = 5.6, 7.2 Hz, NH₂), 7.88 (2H, m), 7.56–7.49 (4H, m) and 7.40–7.27 (3H, m, Ar–H), 3.96 (1H, dd, J = 7.2, 14.6 Hz) and 3.82 (1H, dd, J = 5.6, 14.6 Hz, NH₂CH₂), 3.58 (1H, m) and 3.35 (1H, m, NCH₂CH₂, 2.56 (1H, m), 2.32 (1H, m), 2.21 (1H, m) and 2.02 (1H, m, N CH₂CH₂CH₂); ¹³C NMR (DMSO-d₆/CCl₄ 1/3, 75 MHz): δ = 165.9 (CO), 138.2, 133.3, 130.5, 127.9 (2×CH), 127.5 (CH), 127.5 (2×CH), 127.2 (2×CH), 126.0 (2×CH), 72.6 (C-Ph), 45.0 (NCH₂), 43.3 (NCH₂),33.4 (CH₂), 21.7 (CH₂); anal. calcd. for C₁₈H₁₉BrN₂O·HCl: C, 54.63; H, 5.09; N, 7.08. Found: C, 54.29; H, 5.34; N, 7.13.

Anti-HIV-1 assay

Primary human peripheral blood mononuclear (PBM) cells were stimulated with phytohemagglutinin A for 2–3 days prior to use. HIV-1/LAI obtained from the Centers for Disease Control and Prevention (Atlanta, GA) was used as the standard reference virus for the antiviral assays (Schinazi et al. 1990). The antiviral EC₅₀ and 90 % effective concentration (EC₉₀) were determined from the concentration-response curve using the median effect method (Table 1).

Table 1.

Anti-HIV-1 and cytotoxic activities of synthesized compounds

Compounds	R, R1	Anti-HIV-1 activity in human PBM cells (μM)		Cytotoxicity (IC50, μM)
		EC50	EC90	PBM	CEM	Vero
2g	4-PhCH2O, 4-MeC6H4	4.7	>100	23.5	20.1	69.6
2h	4-(2,4-Cl2C6H3CH2O), 4-MeC6H4	3.1	22.4	15.0	11.7	39.7
2i	4-(2,6-Cl2C6H3CH2O), 4-MeC6H4	9.6	30.0	14.0	16.8	80.9
2j	2-PhCH2O, 4-MeC6H4	14.4	50.1	15.6	3.8	23.7
3c	3,5-Me2C6H3, 2,6-Cl2	72.2	>100	59.0	20.3	>100
3d	4-iPrO, Ph	5.2	29.8	95.8	>100	47.6
3e	4-iPrO, 4-MeC6H4	37.3	>100	>100	17.0	46.5
3f	4-PhCH2O, PhCH2	9.8	30.7	5.9	7.0	3.0
3g	4-PhCH2O, 4-MeC6H4	16.7	>100	>100	30.3	53.9
3h	4-(2,4-Cl2C6H3CH2O), 4-MeC6H4	5.3	24.4	9.7	6.6	21.8
3i	4-(2,6-Cl2C6H3CH2O), 4-MeC6H4	2.0	51.9	14.1	16.6	52.9
3j	2-PhCH2O, 4-MeC6H4	17.3	>100	22.8	16.1	77.9
4	2,6-Cl2, 3,5-Me2C6H3	0.43	3.1	6.1	5.8	68.7
5a	H, 4-MeC6H4	32.6	>100	54.1	53.6	>100
5b	H, PhCH2	>100	>100	>100	>100	>100
5c	2,6-Cl2, 3,5-Me2C6H3	>100	>100	>100	≥100	>100
5d	4-iPrO, Ph	>100	>100	48.3	32.9	33.0
5e	4-PhCH2O, PhCH2	33.4	>100	15.7	23.6	13.1
5f	4-PhCH2O, 4-MeC6H4	10.8	41.5	15.9	33.7	12.3
5g	2-PhCH2O, 4-MeC6H4	11.5	>100	45.2	47.9	>100
6a	4-PhCH2O, PhCH2	>100	>100	>100	29.4	6.2
6b	4-PhCH2O, 4-MeC6H4	8.9	>100	>100	70.1	38.3
9a	2-Br	50.3	>100	>100	>100	>100
9b	4-Br	>100	>100	>100	>100	>100
9c	4-MeO-3-NO2	>100	>100	>100	>100	>100
10	4-Br	>100	>100	>100	96.1	>100
AZT		0.0018	0.015	>100	14.3	50.6

Cytotoxicity assay

Compounds were evaluated for their potential toxic effects on uninfected PHA-stimulated human PBM cells, in CEM (T-lymphoblastoid cell line obtained from American Type Culture Collection, Rockville, MD), and Vero (African green monkey kidney) cells. The 50 % inhibition concentration (IC₅₀) was determined from the concentration-response curve using the median effect method (Table 1) (Stuyver et al. 2002).

Results and discussion

Chemistry

We have previously developed a method for the synthesis of various cyclic α-amino acids and their analogs, particularly 2-phenylproline derivatives, by intramolecular cyclization of corresponding N-(3-chloro- or 1-oxo-3-chloropropyl)-α-phenylglycines under phase-transfer catalytic conditions in acetonitrile in the presence of potassium carbonate and TEBA as catalyst (Martirosyan et al. 2000).

α-Aminonitriles 2a–j were synthesized in high yields by treatment of appropriate aldehydes 1a–g, amines and NaCN in presence of acetic acid in solution in EtOH/H₂O. By acylation of 2-substituted acetonitriles 2a–j with 2-chloropropanoyl chloride (in 1,2-dichloroethane and in presence of K₂CO₃) and further intramolecular cyclization under phase-transfer catalytic conditions, in presence of K₂CO₃, TEBA, and acetonitrile afforded the corresponding 1,2 diaryl substituted pyrrolidinecarbonitriles 3a–j in high yields (Scheme 1) (Gasparyan et al. 2012).

Scheme 1.

Synthesis of 1,2-diaryl substituted 5-oxopyrrolidinecarbonitrile analogs. Reagents and conditions: a NaCN/RNH₂/H⁺/EtOH/H₂O, room temp., 3 h; b ClCH₂CH₂COCl/ClCH₂CH₂Cl/K₂CO₃, 10–15 °C to 40–45 °C, 3 h; c CH₃CN/TEBA/K₂CO₃, 45–50 °C, 4 h; d conc. H₂SO₄, 0–5 °C, 3 h; e NaBH₄/PEG-400/CoCl₂/CH₂Cl₂, −5–0 °C, 8 h; f ClCH₂COCl/ClCH₂CH₂Cl/K₂CO₃, 10–15 °C to 40–45 °C, 3 h

2-(2,6-Dichlorophenyl)-2-(3,5-dimethylanilino)acetamide (4) was synthesized for biological evaluation by reacting 2-(2,6-dichlorophenyl)-2-(3,5-dimethylanilino)acetonitrile (2c) in conc. H₂SO₄ (Gasparyan et al. 2012).

Substituted pyrrolidinecarbonitriles 3a–d, f, g, j were reduced to the corresponding aminomethylpyrrolidines 5a–g in methylene chloride at −5–0 °C, using a NaB₄/PEG-400/CoCl₂ system (ratio 1/0.2/1/5) (Gasparyan et al. 2014).

With the aim to ascertain the role of the pyrrolidine ring in the observed inhibitory activity, we have synthesized 1,2 diarylazetidines 6a, b. In the same way, 2-substituted acetonitriles 2f, g were first acylated with 2-chloroethanoyl chloride (instead 2-chloropropanoyl chloride) and subsequently cyclized under phase-transfer catalytic conditions (Gasparyan 2014).

Acylation of 2-((3-chloropropyl)amino)-2-phenylacetonitrile hydrochloride (7) (Martirosyan et al. 2000) with 2-bromo-, 4-bromo- and 4-methoxy-3-nitrobenzenecarbonyl chlorides, also under phase-transfer catalysis conditions, followed by intramolecular cyclization produced proline derivatives 8a–c (Gasparyan et al. 2012), from which were synthesized the corresponding amides 9a–c and amine 10 following the above mentioned methods (Gasparyan et al. 2012, 2014) (Scheme 2).

Scheme 2.

Synthesis of 1,2-diaryl substituted pyrrolidinecarbonitrile analogs. Reagents and conditions: a RCOCl/ClCH₂CH₂Cl/K₂CO₃, 10–15 °C to 40–45 °C, 3 h; b CH₃CN/TEBA/K₂CO₃, 45–50 °C, 4 h; c conc. H₂SO₄, 0–5 °C, 3 h; d NaBH₄/PEG-400/CoCl₂/CH₂Cl₂, −5–0 °C, 8 h

Pharmacology

Anti-HIV activity

Certain synthesized compounds appeared to be active against HIV, and among the 26 compounds in this series, 14 exhibited good activity with EC₅₀ values <20 μM. Compounds 2h, 3i, and 4 proved to be the most active compounds with EC₅₀ value of 3.1, 2.0, and 0.43 μM, respectively, but they were highly toxic in all the cell lines used. Compound 3d in particular showed good activity with an EC₅₀ value of 5.2 μM without marked cytotoxicity against all the cell lines.

In our structure activity relationship study, we identified some interesting patterns. Compounds 2g–j and 4 (whose structure is closer to the loviride structure) were very active.

By comparing the activity of the compounds containing the 2-aryl substituents, we noted that compounds with benzyloxyphenyl groups were more active. Compounds in which the 1-aryl moiety contained methyl group in 4- or 3,5-positions also showed high activity.

We also observed a decrease in activity with CN > NH₂ > C(O)NH₂, however, the difference of activity between 5-membered and 4-membered rings containing compound was not significant.

Based on these findings, we believe that further structural modifications could be made to obtain new potent anti-HIV-1 derivatives (Table 1).

Cytotoxicity studies

In order to determine their spectrum of toxicity, cytotoxicity of the compounds was determined in primary human PBM, human CEM, and Vero cells (Table 1).

Compounds 5b, c and 9a–c were nontoxic in all the cell systems tested; however, they were inactive against HIV-1. Similarly, compound 10 showed no toxicity in any of the cell system and exhibited no inhibition of HIV replication. However, 6a and 6b were not toxic in PBM cells (Table 1).

Conclusions

Herein, we report the anti-HIV-1 activity of new 1,2-diarylpyrrolidine derivatives. Fourteen compounds were active against HIV-1 with EC₅₀ values less than 20 μM, which indicates the potential of these compounds as anti-HIV-1 agents. Among these 26 compounds, 2g, 2h, 3i, and 4 were potent anti-HIV-1 agents with EC₅₀ values <5.0 μM. They were less potent than the 3′-Azido-2′,3′-dideoxythymidine or azidothymidine used as a positive control, however, the activity may be due to the toxicity observed in PBM cells. On these grounds, further structural modifications has to be made to improve potency against HIV and reduce cytotoxicity.