Anti-human immunodeficiency activity of novel 2-arylpyrrolidine analogs

Article abstract of DOI:10.1007/s00044-016-1731-7

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.

Full text of DOI:10.1007/s00044-016-1731-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1731-7
ORIGINAL RESEARCH
Anti-human immunodeficiency activity of novel 2-arylpyrrolidine
analogs
1
Ashot H. Martirosyan1 Sahak P. Gasparyan
Marina V. Alexanyan1
●
●
●
Gohar K. Harutyunyan1 Henry A. Panosyan2 Raymond F. Schinazi3
●
●
Received: 2 July 2016 / Accepted: 7 October 2016
© Springer Science+Business Media New York 2016
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 con-
centration values <20 µM. Moreover, most of the com-
pounds were cytotoxic to human lymphocytes, CEM, and
Vero cells. Our structure activity relationship study identi-
fied 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. Com-
pounds 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 sig-
nificant. This initial information could be used to design
improved anti-human immunodeficiency virus compounds
in this class.
●
Keywords Pyrrolidinecarbonitriles
●
●
Aminomethylpyrrolidines Anti-HIV-1 Cytotoxicity
activity
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 retro-
virus 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 deri-
vatives (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
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1731-7) contains supplementary material,
which is available to authorized users.
* Sahak P. Gasparyan
g_sahak@yahoo.com
Raymond F. Schinazi
rschina@emory.edu
1
Scientific and Technological Centre of Organic and
Pharmaceutical Chemistry, Institute of Fine Organic Chemistry,
National Academy of Sciences of RA, Azatutyan Avenue 26,
Yerevan 0014, Armenia
2
Scientific and Technological Centre of Organic and
Pharmaceutical Chemistry, Molecule Structure Research Centre,
National Academy of Sciences of RA, Azatutyan Avenue 26,
Yerevan 0014, Armenia
3
Center for AIDS Research, Laboratory of Biochemical
Pharmacology, Emory University School of Medicine, 1760
Haygood Drive, Atlanta, GA 30322, USA

Med Chem Res
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 dis-
covered. With the discovery of α-APAs, the era of flexible
derivatives started. These exhibited 50 % effective con-
centration (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 opti-
mization. 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 pyrroli-
dine framework was required as a core structure (Fig. 2).
Fig. 2 New derivatives of 1,2-diarylpyrrolidinecarbonitriles
The mixture was stirred for 10 min and then acetic acid
(1 equiv, 10 mmol) was added. After 10 min, the corre-
sponding 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.
Materials and methods
General
All the chemicals were purchased from Alfa Aesar or Sigma
Compounds 2a, 2b were synthesized by us and described
in Gasparyan et al. (2012).
1
Aldrich and were used without purification. H 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.
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) νmax, 3355 (NH), 2232 (C≡N), 1612 (arom.)
1
cm−1; H NMR (DMSO-d6, 300 MHz): δ = 7.49–7.44 (2H,
Chemistry
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/CCl4 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),
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.

Med Chem Res
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).
white crystalline m.p. 107–108 °C; IR (nujol mull) νmax
1
2229 (C≡N), 1721 (С=О), 1614 (arom.) cm−1; H 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.
2-[4-(2,4-Dichlorobenzyloxy)phenyl]-2-(4-toluidino)acetoni-
trile (2h) was prepared in 68 % of yield, light yellow
crystalline m.p. 142–143 °C; IR (nujol mull) νmax 3340 (NH),
1
2229 (C≡N), 1612 (arom.), 1590 (arom.) cm−1; H 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 3f was synthesized by us and described in
Gasparyan et al. (2014).
2-(4-Benzyloxyphenyl)-1-(4-methylphenyl)-5-oxo-2-pyrro-
lidinecarbonitrile (3g) was prepared in 61 % of yield,
yellow crystalline m.p. 146–147 °C; IR (nujol mull) νmax
2236 (C≡N), 1712 (С=О), 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.
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 2 h. After addition of 1,2-
dichloroethane (20 mL), the mixture was washed with water
and dried over CaCl2. After distillation of the 1,2-dichlor-
oethane dry potassium carbonate (1 equiv, 10 mmol), trie-
thylbenzylammonium chloride (0.5 equiv, 5 mmol)
(TEBA), and acetonitrile (20 mL) were added to the residue
and the solution was stirred for 4 h 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 com-
pound was recrystallized from EtOH.
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)
νmax 2228 (C≡N), 1712 (С=О), 1611 (arom.), 1587 (arom.)
1
cm−1; H 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 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).
Compounds 3i, 3j were synthesized by us and
described in Gasparyan et al. (2014).
2-(4-Isopropoxyphenyl)-1-(4-methylphenyl)-5-oxo-2-pyrro-
lidinecarbonitrile (3e) was prepared in 72 % of yield,

Med Chem Res
2-(2,6-Dichlorophenyl)-2-(3,5-dimethylanilino)acetamide
(4)
(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.
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 3 h, 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) νmax 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.
Compounds 5b–e were synthesized by us and
described in Gasparyan et al. (2014).
5-Aminomethyl-5-(4-benzyloxyphenyl)-1-(4-methylphe-
nyl)-2-pyrrolidinone (5f) was prepared in 52 % yield,
which gives the corresponding hydrochloric salt, yellow
crystal m.p. 169–170 °C; IR (nujol mull) νmax 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.
Preparation for 5-aminomethyl-5-aryl-2-pyrrolidinones
(5a–g)
Compound 5g was synthesized by us and described in
Gasparyan et al. (2014).
To a solution of appropriate 2-aryl-5-oxo-2-pyrrolidine-
carbonitrile 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.
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-bro-
mophenylmethanone (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) νmax 3302 (NH2), 1645 (C=O) cm−1; 1H
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),
5-Aminomethyl-1-(4-methylphenyl)-5-phenyl-2-pyrrolidi-
none (5a) was prepared in 55 % of yield, white crystal m.
p. 130 °C; IR (nujol mull) νmax 3400 broad (NH2), 1650
1
(С=О) cm−1; H 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
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.

Med Chem Res
Anti-HIV-1 assay
Culture Collection, Rockville, MD), and Vero (African
green monkey kidney) cells. The 50 % inhibition con-
centration (IC50) was determined from the concentration-
response curve using the median effect method (Table 1)
(Stuyver et al. 2002).
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).
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).
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
Table 1 Anti-HIV-1 and
Compounds R, R1
cytotoxic activities of
synthesized compounds
Anti-HIV-1
activity in human
PBM cells (µM)
Cytotoxicity (IC50, µM)
EC50
EC90
PBM
CEM
Vero
2g
2h
2i
4-PhCH2O, 4-MeC6H4
4.7
>100
22.4
23.5
15.0
14.0
15.6
59.0
95.8
>100
5.9
20.1
11.7
16.8
3.8
69.6
39.7
80.9
23.7
>100
47.6
46.5
3.0
4-(2,4-Cl2C6H3CH2O), 4-MeC6H4 3.1
4-(2,6-Cl2C6H3CH2O), 4-MeC6H4 9.6
30.0
2j
2-PhCH2O, 4-MeC6H4
3,5-Me2C6H3, 2,6-Cl2
4-iPrO, Ph
14.4
50.1
3c
3d
3e
3f
72.2
5.2
>100
29.8
20.3
>100
17.0
7.0
4-iPrO, 4-MeC6H4
4-PhCH2O, PhCH2
4-PhCH2O, 4-MeC6H4
37.3
9.8
>100
30.7
3g
3h
3i
16.7
>100
24.4
>100
9.7
30.3
6.6
53.9
21.8
52.9
77.9
68.7
>100
>100
>100
33.0
13.1
12.3
>100
6.2
4-(2,4-Cl2C6H3CH2O), 4-MeC6H4 5.3
4-(2,6-Cl2C6H3CH2O), 4-MeC6H4 2.0
51.9
14.1
22.8
6.1
16.6
16.1
5.8
3j
2-PhCH2O, 4-MeC6H4
2,6-Cl2, 3,5-Me2C6H3
H, 4-MeC6H4
17.3
>100
3.1
4
0.43
5a
5b
5c
5d
5e
5f
32.6
>100
>100
>100
>100
>100
41.5
54.1
>100
>100
48.3
15.7
15.9
45.2
>100
>100
>100
>100
>100
>100
>100
53.6
>100
≥100
32.9
23.6
33.7
47.9
29.4
70.1
>100
>100
>100
96.1
14.3
H, PhCH2
>100
>100
>100
33.4
2,6-Cl2, 3,5-Me2C6H3
4-iPrO, Ph
4-PhCH2O, PhCH2
4-PhCH2O, 4-MeC6H4
2-PhCH2O, 4-MeC6H4
4-PhCH2O, PhCH2
4-PhCH2O, 4-MeC6H4
2-Br
10.8
5g
6a
6b
9a
9b
9c
10
AZT
11.5
>100
>100
>100
>100
>100
>100
>100
0.015
>100
8.9
38.3
>100
>100
>100
>100
50.6
50.3
4-Br
>100
>100
>100
0.0018
4-MeO-3-NO2
4-Br

Med Chem Res
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).
benzenecarbonyl chlorides, also under phase-transfer cata-
lysis 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).
Pharmacology
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 NaBH4/
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 subse-
quently cyclized under phase-transfer catalytic conditions
(Gasparyan 2014).
Acylation of 2-((3-chloropropyl)amino)-2-phenylaceto-
nitrile hydrochloride (7) (Martirosyan et al. 2000)
with 2-bromo-, 4-bromo- and 4-methoxy-3-nitro-
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. Com-
pounds 2h, 3i, and 4 proved to be the most active com-
pounds 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.
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

Med Chem Res
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
Dr. Ashot H. Martirosyan who died after a long illness on June 25,
2016.
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.
Compliance with ethical standards
Based on these findings, we believe that further structural
modifications could be made to obtain new potent anti-HIV-
1 derivatives (Table 1).
Conflict of interest The authors declare that they have no competing
interests.
Cytotoxicity studies
References
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).
De Corte BL (2005) From 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-
jk](1,4)benzodiazepin-2(1H)-one (TIBO) to Etravirine (TMC125):
Fifteen years of research on non-nucleoside inhibitors of HIV-1
reverse transcriptase. J Med Chem 48:1689–1696
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).
Ding J, Das K, Tantillo C, Zhang W, Clark Jr AD, Jessen S, Lu X,
Ysiou Y, Jacobo-Molina A, Andries K, Pauwels R, Moereels H,
Koymans L, Janssen PAJ, Smith Jr RH, Kroeger Smith MB,
Koepke M, Michejda CJ, Hughes SH, Arnold E (1995) Structure
of HIV-1 reverse transcriptase in a complex with the non-
nucleoside inhibitor R-APA R 95845 at 2.8 Å resolution. Struc-
ture 3:365–379
Gasparyan SP, Alexanyan MV, Harutyunyan GK, Hovhannesyan VE,
Martirosyan VV, Paronikyan RV, Stepanyan HM, Martirosyan
AH (2012) Synthesis and biological properties of new analogs of
2-arylpyrrolidinecarbonitriles and pyrrolidinecarboxamides.
Pharm Chem J 46(6):331–333
Gasparyan SP (2014) Synthesis of new analogs of 2-aryl-2-
azetancarbonitriles. Chem J Armenia 67(1):111–118
Gasparyan SP, Alexanyan MV, Harutyunyan GK, Hovhannesyan VE,
Martirosyan AH, Panosyan HA (2014) Metal-complex reduction
of nitrile groupe in substituted pyrrolidinecarbonitriles. Chem J
Armenia 67(2-3):239–246
Martirosyan AH, Gasparyan SP, Hovhannesyan VE, Mnjoyan ShL,
Alexanyan MV, Nikishchenko MN, Babayan GSh (2000) New
method for the synthesis of 2-phenylproline and its derivatives.
Chem Het Comp 36:416–420
Pauwels R, Andries K, Debyser Z, Van Daele P, Schols D, Stoffels P,
De Vreese K, Woestenborghs R, Vandamme AM, Janssen
CGM, Anne J, Cauwenbergh G, Desmyter J, Heykants J, Janssen
MAC, De Clercq E, Janssen PAJ (1993) Potent and highly
selective human immunodeficiency virus type 1 (HIV-1) inhibi-
tion by a series of a-anilinophenylacetamide derivatives targeted
Conclusions
Herein, we report the anti-HIV-1 activity of new 1,2-dia-
rylpyrrolidine 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.
Acknowledgments The authors are grateful to the Civilian Research
and Development Foundation (CRDF) (Grant No. ARB2-2701-YE-
05). This work was also facilitated in part by the NIH grant P30-AI-
050409 (to RFS). This paper is dedicated to our friend and colleague

Med Chem Res
at HIV-1 reverse transcriptase. Proc Natl Acad Sci USA
90:1711–1715
Prajapati DG, Ramajayam R, Yadav MR, Giridhar R (2009) The
search for potent, small molecule NNRTIs. Bioorg Med Chem
17:5744–5762
Sarafianos SG, Das K, Hughes SH, Arnold E (2004) Taking aim at a
moving target: designing drugs to inhibit drug-resistant HIV-1
reverse transcriptases. Curr Opin Struct Biol 14:716–730
Schinazi RF, Sommadossi JP, Saalmann V, Cannon DL, Xie M-W,
Hart GC, Smith GA, Hahn EF (1990) Activities of 3′-azido-3′-
deoxythymidine nucleotide dimers in primary lymphocytes
infected with human immunodeficiency virus type 1. Antimicrob
Agents Chemother 34(6):1061–1067
Stuyver LJ, Lostia S, Adams M, Mathew J, Pai BS, Grier J, Tharnish
P, Choi Y, Chong Y, Choo H, Chu CK, Otto MJ, Schinazi RF
(2002) Antiviral activities and cellular toxicities of modified 2′,3′-
dideoxy-2′,3′-didehydrocytidine analogues. Antimicrob Agents
Chemother 46(12):3854–3860