Design, synthesis, in vitro and in silico studies of novel 4-oxoquinoline ribonucleoside derivatives as HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112255

Human immunodeficiency virus type 1 (HIV-1) is a public health problem that affects over 38 million people worldwide. Although there are highly active antiretroviral therapies, emergence of antiviral resistant strains is a problem which leads to almost a million death annually. Thus, the development of new drugs is necessary. The viral enzyme reverse transcriptase (RT) represents a validated therapeutic target. Because the oxoquinolinic scaffold has substantial biological activities, including antiretroviral, a new series of 4-oxoquinoline ribonucleoside derivatives obtained by molecular hybridization were studied here. All synthesized compounds were tested against human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), and 9a and 9d displayed the highest antiviral activities, with IC₅₀ values of 1.4 and 1.6 μM, respectively. These compounds were less cytotoxic than AZT and showed CC₅₀ values of 1486 and 1394 μM, respectively. Molecular docking studies showed that the most active compounds bound to the allosteric site of the enzyme, suggesting a low susceptibility to the development of antiviral resistance. In silico pharmacokinetic and toxicological evaluations reinforced the potential of the active compounds as anti-HIV candidates for further exploration. Overall, this work showed that compounds 9a and 9d are promising scaffold for future anti-HIV-1 RT drug design.

Full text of DOI:10.1016/j.ejmech.2020.112255

Journal Pre-proof
Design, synthesis, in vitro and in silico studies of novel 4-oxoquinoline ribonucleoside
derivatives as HIV-1 reverse transcriptase inhibitors
Luana da S.M. Forezi, Mariana M.J. Ribeiro, Andressa Marttorelli, Juliana L.
Abrantes, Carlos R. Rodrigues, Helena Carla Castro, Thiago Moreno L. Souza,
Fernanda da C.S. Boechat, Alessandra M.T. de Souza, Maria Cecília B.V. de Souza
PII:
S0223-5234(20)30222-1
DOI:
https://doi.org/10.1016/j.ejmech.2020.112255
EJMECH 112255
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 November 2019
Revised Date: 19 February 2020
Accepted Date: 17 March 2020
Please cite this article as: L.d.S.M. Forezi, M.M.J. Ribeiro, A. Marttorelli, J.L. Abrantes, C.R. Rodrigues,
H.C. Castro, T.M.L. Souza, F.d.C.S. Boechat, A.M.T. de Souza, Maria.Cecí.B.V. de Souza, Design,
synthesis, in vitro and in silico studies of novel 4-oxoquinoline ribonucleoside derivatives as HIV-1
reverse transcriptase inhibitors, European Journal of Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.ejmech.2020.112255.
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DESIGN, SYNTHESIS, IN VITRO AND IN SILICO STUDIES OF NOVEL 4-
OXOQUINOLINE RIBONUCLEOSIDE DERIVATIVES AS HIV-1 REVERSE
TRANSCRIPTASE INHIBITORS
Luana da S. M. Forezi,1,# Mariana M. J. Ribeiro,2,# Andressa Marttorelli,3,4 Juliana L.
Abrantes,3,4 Carlos R. Rodrigues,2 Helena Carla Castro,5 Thiago Moreno L. Souza,3,4
Fernanda da C. S. Boechat,1 Alessandra M. T. de Souza,2,* Maria Cecília B. V. de
Souza1,*
1
Laboratório LNHC, Departamento de Química Orgânica, Instituto de Química,
Universidade Federal Fluminense, Campus Valonguinho, 24020-150, Niterói-RJ,
Brazil.
2Laboratório de Modelagem Molecular e QSAR (ModMolQSAR), Faculdade de
Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
3Laboratório de Imunofarmacologia, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo
Cruz (Fiocruz), Rio de Janeiro, RJ, Brazil.
4National Institute for Science and Technology on Innovation on Neglected Diseases
(INCT/IDN), Center for Technological Development in Health (CDTS), Fiocruz, Rio de
Janeiro, RJ, Brazil.
5Laboratório de Antibióticos, Bioquímica, Ensino e Modelagem Molecular
(LABiEMol), Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ,
Brazil.
#these authors contributed equally
Corresponding Authors
Alessandra Mendonça Teles de Souza, Laboratório de Modelagem Molecular e
QSAR (ModMolQSAR), Faculdade de Farmácia, Universidade Federal do Rio de
Janeiro, Rio de Janeiro-RJ, Brazil; E-mail: amtsouza@pharma.ufrj.br
Maria Cecília B. V. de Souza, Laboratório LNHC, Departamento de Química
Orgânica, Instituto de Química, Universidade Federal Fluminense, Campus
Valonguinho, 24020-150, Niterói-RJ, Brazil; E-mail: mceciliabvs@gmail.com

Abstract
Human immunodeficiency virus type 1 (HIV-1) is a public health problem that affects
over 38 million people worldwide. Although there are highly active antiretroviral
therapies, emergence of antiviral resistant strains is a problem which leads to almost a
million death annually. Thus, the development of new drugs is necessary. The viral
enzyme reverse transcriptase (RT) represents a validated therapeutic target. Because the
oxoquinolinic scaffold has substantial biological activities, including antiretroviral, a
new series of 4-oxoquinoline ribonucleoside derivatives obtained by molecular
hybridization were studied here. All synthesized compounds were tested against human
immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), and 9a and 9d
displayed the highest antiviral activities, with IC50 values of 1.4 and 1.6 μM,
respectively. These compounds were less cytotoxic than AZT and showed CC50 values
of 1486 and 1394 µM, respectively. Molecular docking studies showed that the most
active compounds bound to the allosteric site of the enzyme, suggesting a low
susceptibility to the development of antiviral resistance. In silico pharmacokinetic and
toxicological evaluations reinforced the potential of the active compounds as anti-HIV
candidates for further exploration. Overall, this work showed that compounds 9a and 9d
are promising scaffold for future anti-HIV-1 RT drug design.
Keywords: 4-oxoquinoline; ribonucleoside; antiviral; HIV-1; molecular docking
1. Introduction
Human immunodeficiency virus (HIV) has a global incidence, affecting over
37.9 million people [1]. Highly active antiretroviral therapy (HAART) allows efficient
patient management. HAART reduced viremia and HIV-infected individuals may live a
normal life. Nevertheless, 770,000 AIDS-related deaths were reported in 2018 [2].
These deaths are associated with the emergence of drug-resistant strains of HIV-1,
which jeopardizes the HAART-suppressed viremia. Therefore, the development of new
agents is urgently necessary.
HIV-1 enzyme reverse transcriptase (RT) is a validated therapeutic target aimed
by Nucleoside and Non-Nucleoside Reverse Transcriptase Inhibitors (acronymed as
NRTI and NNRTI, respectively). NRTIs and NNRTIs block RT ability to convert

single-stranded RNA genome to double-stranded DNA during HIV-1replication cycle
[3].
NNRTIs are structurally diverse noncompetitive inhibitors that bind onto the
hydrophobic pocket located 10 Å from the polymerase active site [4]. Nevirapine,
delavirdine, efavirenz, etravirine, rilpivirine and doravirine are exemples of currently
approved NNRTIs. Besides these drugs, more than 55 classes of structurally unrelated
NNRTIs have already been tested, including clinical trials [5, 6]. Altogether, these
information mean the new NNRTIs may pave the way from discovery to clinic.
Previous efforts from our group showed that the chloroxoquinolinic
ribonucleoside
6-chloro-1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-quinoline-3-
carboxylic acid (1) inhibits HIV-1 replication through inhibition of both the wild-type
(WT) and antiviral resistant RT [8]. This substance synergizes with both classes of RT
inhibitors, represented by the NNRTI and NRTI efavirenz and AZT, respectively [9].
Additionally, the bioisosteric substitution of the carboxylic acid by an amide jointly
with molecular simplification of the riboside ring of the 4-oxoquinoline moiety (2 and
3) resulted in a lead compound with low toxicity [10]. Thus, our group have
demonstrated capacity to discover safe compounds endowed with ability to circumvent
antiviral resistance.
The allosteric site of RT was used to rationalize the design of a new series of 4-
oxoquinoline derivatives by molecular hybridization (Figure 1). Since this allosteric site
is large and hydrophobic, our strategy was to evaluate the increase of the contact surface
area within the site by inserting ribofuranosidic units at the N-1 position of 4-
oxoquinoline and involving alterations to the type of substituent group at the C-3
position. Toward this end, a new series of 22 4-oxoquinoline ribonucleoside derivatives
were synthesized. The anti-HIV activity evaluation demonstrated that derivatives
containing the benzoyl substituents showed potent inhibitory activity in the micromolar
range against the WT strains of HIV-1. Structure–activity relationship (SAR), molecular
docking, in silico pharmacokinetics and toxicology studies were also carried out.

Figure 1. Design strategy of 4-oxoquinoline ribonucleoside 3-carboxamide derivatives
by molecular hybridization
2. Experimental Section
2.1. Chemistry
The reagents were purchased from Sigma-Aldrich Brazil and were used without
further purification. Analytical thin layer chromatography was performed with silica gel
plates (Merck, TLC silica gel 60 F254), and the spots were visualized using UV light.
Melting points were obtained on a Fisher-Johns apparatus and were uncorrected.
Infrared spectra were measured with KBr pellets on a Perkin-Elmer model 1420 FT-IR
spectrophotometer, and the spectra were calibrated relative to the 1601.8 cm-1
absorbance of polystyrene. NMR spectra were recorded on a Varian Unity Plus VXR
(500.00 and 300.00 MHz) instrument in solutions of DMSO-d6 or CDCl3. The chemical
shift data were reported in units of δ (ppm) downfield from the solvent, which was used
as the internal standard; coupling constants (J) are reported in hertz and refer to
apparent peak multiplicities.
2.1.1. General Procedure for the synthesis of 1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-
β-D-ribofuranosyl)-quinoline-3-carboxamides (9a-k).
A stirred solution of carboxamides 8 (1 equiv.), anhydrous acetonitrile (5.0 mL)
and BSTFA (1.0 mL) containing 1 % TMCS, was heated at 60-70 °C under nitrogen for
1 h. The resulting mixture was allowed to cool to room temperature, and a solution of 1-
O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose 10 (1 equiv.) in 5 mL of acetonitrile was
added, followed by the addition of TMSOTf (0.1 mL). After stirring for 4 h at room

temperature, the solution was poured into ice-cold water (20 g) and neutralized with
saturated aqueous sodium bicarbonate solution. The resulting mixture was extracted
with methylene chloride (3 × 20 mL), and the combined organic layers were washed
with water (3 × 20 mL) and dried over anhydrous magnesium sulfate. The solvent was
removed under reduced pressure, and the corresponding products were recrystallized
from ethanol.
1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-N’-(4-chlorobenzyl)-
1
quinoline-3-carboxamides (9a). White solid, 76 % yield. m.p.: 168-171 °C. H NMR
(500.00 MHz, CDCl3) δ (ppm): 10.26 (1H, t, J 5.5 Hz, C=ONH), 9.29 (1H, s, H-2),
9.51 (1H, dd, J 8.1 and 1.6 Hz, H-5), 8.10 (2H, d, J 7.3 Hz, H-2’’’ and H-6’’’), 7.93-
7.89 (4H, m, H-2’’’ and H-6’’’), 7.75 (1H, d, J 8.6 Hz, H-8), 7.60-7.52 (4H, m, H-4’’’
and H-7), 7.46 (1H, t, J 7.5 Hz, H-6), 7.42-7.29 (6H, m, H-3’’’ and H-5’’’), 7.30 (4H,
d, J 3.0 Hz, H-2’’, H-3’’, H-5’’ and H-6’’), 6.51 (1H, d, J 4.7 Hz, H-1’), 6.03 (1H, t, J
5.3 Hz, H-2’), 5.91-5.88 (1H, m, H-3’), 4.92 (3H, sl, H-4’, H-5’a and H-5’b), 4.60 (2H,
13
d, J 6.0 Hz, CH2NH). C/APT NMR (125.00 MHz, CDCl3) δ (ppm): 177.2 (C=O),
166.2 (C=OOBz), 165.1 (C=OOBz), 164.8 (C=OOBz), 164.5 (C=ONH), 143.3 (C-2), 138.6
(C-8a), 137.6 (C-1’’), 134.1 (CH’’’), 134.0 (CH’’’), 133.6 (CH’’’), 133.2 (C-7), 132.9
(C-4’’), 130.0 (CH’’’), 129.9 (CH’’’), 129.8 (CH’’’), 129.3 (C-1’’’), 129.1 (C-3’’ and
C-5’’), 128.8 (CH’’’), 128.7 (CH’’’), 128.6 (C-2’’ and C-6’’), 128.5 (C-1’’’), 128.2 (C-
1’’’), 127.9 (C-4a), 127.8 (C-5), 125.7 (C-6), 115.5 (C-8), 112.7 (C-3), 92.0 (C-1’), 80.9
(C-4’), 74.0 (C-2’), 70.7 (C-3’), 63.3 (C-5’), 42.7 (CH2NH). IV (KBr) ν (cm-1): 3424,
+
1742, 1728, 1671, 1540. HRMS-ESI Calcd. for C43H33ClN2NaO9 : 779,17723. Found
+
for C43H33ClN2NaO9 : 779.174003.
6-chloro-1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-N’-(4-
chlorobenzyl)-quinoline-3-carboxamides (9b). White solid, 73 % yield. m.p.: 171-173
1
°C. H NMR (300.00 MHz, CDCl3) δ (ppm): 10.08 (1H, t, J 5.9 Hz, C=ONH), 9.23
(1H, s, H-2), 8.42 (1H, d, J 2.5 Hz, H-5), 8.07 (2H, d, J 8.4 Hz, H-2’’’ and H-6’’’), 7.89
(4H, d, J 6.9 Hz, H-2’’’ and H-6’’’), 7.70 (1H, d, J 9.2 Hz, H-8), 7.59-7.50 (3H, m, H-
4’’’), 7.41-7.31 (7H, m, H-3’’’, H-5’’’ and H-7), 7.29 (4H, sl, H-2’’, H-3’’, H-5’’and
H-6’’), 6.42 (1H, d, J 4.9 Hz, H-1’), 5.99 (1H, t, J 5.7 Hz, H-2’), 5.85 (1H, t, J 5.7 Hz,
13
H-3’), 4.87 (1H, sl, H-4’, H-5’a and H-5’b), 4.57 (2H, d, J 6.0 Hz, CH2NH). C/APT
NMR (75.0 MHz, CDCl3) δ (ppm): 175.9 (C=O), 166.2 (C=OOBz), 165.2 (C=OOBz),

164.8 (C=OOBz), 164.1 (C=ONH), 143.6 (C-2), 137.5 (C-1’’), 137.0 (C-8a), 134.3
(CH’’’), 134.0 (CH’’’), 133.6 (CH’’’), 133.4 (C-7), 133.0 (C-4’’), 132.1 (C-6 or C-4a),
130.0 (CH’’’), 129.9 (CH’’’), 129.2 (C-1’’’), 129.1 (C-3’’ and C-5’’), 129.0 (C-6 or C-
4a), 128.8 (CH’’’), 128.7 (CH’’’), 128.6 (C-2’’ and C-6’’), 128.4 (C-1’’’), 128.1 (C-
1’’’), 127.2 (C-5), 117.4 (C-8), 113.1 (C-3), 92.2 (C-1’), 81.1 (C-4’), 73.8 (C-2’), 70.7
(C-3’), 63.2 (C-5’), 42.7 (CH2NH). IV (KBr) ν (cm-1): 3424, 1729, 1714, 1668, 1548.
+
+
HRMS-ESI Calcd. for C43H32Cl2N2NaO9 : 813,13771. Found for C43H32Cl2N2NaO9 :
813.134898.
6-fluoro-1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-N’-(4-
chlorobenzyl)-quinoline-3-carboxamides (9c). White solid, 60 % yield, m.p.: 139-140
1
°C. H NMR (500.00 MHz, CDCl3) δ (ppm): 10.08 (1H, t, J 5.5 Hz, C=ONH), 9.24
(1H, s, H-2), 8.39 (1H, dd, J 9.0 and 7.0 Hz, H-5), 8.02 (2H, d, J 6.5 Hz, H-2’’’ and H-
6’’’), 7.97 (2H, d, J 7.0 Hz, H-2’’’ and H-6’’’), 7.91 (2H, d, J 7.1 Hz, H-2’’’ and H-
6’’’), 7.67-7.59 (3H, m, H-4’’’ and H-8), 7.49-7.41 (6H, m, H-3’’’, H-5’’’ and H-7),
7.38 (2H, d, J 8.3 Hz, H-2’’ and H-6’’), 7.33 (2H, d, J 8.3 Hz, H-3’’ and H-5’’), 7.07
(1H, d, J 5.0 Hz, H-1’), 5.99 (1H, t, J 5.5 Hz, H-2’), 5.91 (1H, t, J 5.5 Hz, H-3’), 5.01
(1H, dd, J 8.0 and 4.4 Hz, H-4’), 4.86 (1H, dd, J 12.7 and 4.4 Hz, H-5’a or H-5’b), 4.78
(1H, dd, J 12.7 and 4.4 Hz, H-5’a or H-5’b), 4.49 (2H, dd, J 8.0 and 2.0 Hz, CH2NH).
1
13C/APT NMR (125.00 MHz, CDCl3) δ (ppm): 175.3 (C=O), 165.6 (d, JC-F = 240.1
Hz, C-6), 165.5 (C=OOBz), 164.6 (C=OOBz), 164.3 (C=OOBz), 163.4 (C=ONH), 143.1
3
(C-2), 140.2 (d, JC-F = 12.2 Hz, C-4a), 138.5 (C-8a), 134.0 (CH’’’), 133.9 (CH’’’),
3
133.4 (CH’’’), 131.4 (C-4’’ or C-1’’), 129.6 (d, JC-F = 11.0 Hz, C-8), 129.5 (CH’’’),
129.4 (CH’’’), 129.3 (CH’’’), 129.2 (C-3’’ e C-5’’), 129.0 (C-1’’’), 129.0 (CH’’’),
128.7 (CH’’’), 128.6 (CH’’’), 128.5 (C-1’’’), 128.3 (C-2’’ and C-6’’), 128.1 (C-1’’’),
2
2
123.8 (C-4’’ or C-1’’), 114.0 (d, JC-F = 23.2 Hz, C-7), 112.1 (C-3), 103.3 (d, JC-F
=
28.1 Hz, C-5), 89.6 (C-1’), 80.4 (C-4’), 74.4 (C-2’), 70.3 (C-3’), 63.5 (C-5’), 41.6
(CH2NH). IV (KBr) ν (cm-1): 3424, 1741, 1732, 1721, 1675, 1548. HRMS-ESI Calcd.
+
+
for C43H32ClFN2NaO9 : 797,16726. Found for C43H32ClFN2NaO9 : 797.163311.
6-methyl-1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-N’-(4-
chlorobenzyl)-quinoline-3-carboxamides (9d). White solid, 86 % yield, m.p.: 211-212
1
°C. H NMR (500.00 MHz, CDCl3) δ (ppm): 10.31 (1H, t, J 5.9 Hz, C=ONH), 9.25
(1H, s, H-2), 8.29 (1H, sl, H-5), 8.10 (2H, d, J 7.3 Hz, H-2’’’ and H-6’’’), 7.93 (2H, d, J

7.3 Hz, H-2’’’ and H-6’’’), 7.89 (2H, d, J 7.3 Hz, H-2’’’ and H-6’’’), 7.64-7,53 (3H, m,
H-4’’’ and H-8), 7.42-7.29 (11H, m, H-3’’’, H-5’’’, H-2’’, H-3’’, H-5’’, H-6’’ and H-
7), 6.47 (1H, d, J 4.9 Hz, H-1’), 6.02 (1H, t, J 4.9 Hz, H-2’), 5.87 (1H, t, J 5.9 Hz, H-
3’), 4.91 (3H, sl, H-4’, H-5’a and H-5’b), 4.61 (2H, d, J 5.9 Hz, CH2NH), 2.44 (CH3).
13C/APT NMR (125,00 MHz, CDCl3) δ (ppm): 177.1 (C=O), 166.2 (C=OOBz), 165.1
(C=OOBz), 164.8 (C=OOBz), 164.7 (C=ONH), 143.9 (C-2), 137.7 (C-8a or C-1’’), 136.6
(C-8a or C-1’’), 135.1 (C-4’’), 134.6 (CH’’’), 134.2 (CH’’’), 133.9 (CH’’’), 133.5 (C-
7), 132.9 (C-6 or C-4a), 130.1 (CH’’’), 130.0 (CH’’’), 129.9 (CH’’’), 129.3 (C-6 or C-
4a), 129.0 (C-3’’ and C-5’’), 128.8 (C-1’’’), 128.7 (C-1’’’), 128.6 (C-2’’ and C-6’’),
128.5 (CH’’’), 128.2 (CH’’’), 127.8(CH’’’), 127.3 (C-5), 115.4 (C-8), 112.5 (C-3), 92.1
(C-1’), 80.8 (C-4’), 74.0 (C-2’), 70.6 (C-3’), 63.3 (C-5’), 42.7 (CH2NH), 21.1 (CH3). IV
(KBr) ν (cm-1): 3424, 1740, 1725, 1655, 1549. HRMS-ESI Calcd. for
+
+
C44H35ClN2NaO9 : 793,19288. Found for C44H35ClN2NaO9 : 793.189613.
6-trifluoromethyl-1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-N’-(4-
chlorobenzyl)-quinoline-3-carboxamides (9e). White solid, 74 % yield, m.p.: 208-210
1
°C. H NMR (500.00 MHz, CDCl3) δ (ppm): 10.05 (1H, t, J 5.5 Hz, C=ONH), 9.28
(1H, s, H-2), 8.78 (1H, sl, H-5), 8.10 (2H, d, J 7.0 Hz, H-2’’’ and H-6’’’), 7.93 (2H, d, J
7.3 Hz, H-2’’’ and H-6’’’), 7.90 (2H, d, J 7.3 Hz, H-2’’’ and H-6’’’), 7.64-7.55 (4H, m,
H-4’’’ and H-7), 7.43-7.37 (6H, m, H-3’’’, H-5’’’), 7.29 (4H, sl, H-2’’, H-3’’, H-5’’, H-
6’’), 6.47 (1H, d, J 4.9 Hz, H-1’), 6.03 (1H, t, J 6.1 Hz, H-2’), 5.90 (1H, t, J 5.4 Hz, H-
3’), 4.99-4.89 (1H, m, H-4’, H-5’a and H-5’b), 4.60 (2H, dd, J 6.1 and 2.4 Hz,
CH2NH). 13C/APT NMR (125.00 MHz, CDCl3) δ (ppm): 176.4 (C=O), 166.2
(C=OOBz), 165.2 (C=OOBz), 164.9 (C=OOBz), 163.8 (C=ONH), 144.4 (C-2), 140.5 (C-
1’’), 137.3 (C-8a), 134.3 (CH’’’), 134.1 (CH’’’), 133.7 (CH’’’), 133.1 (C-4’’), 130.0
3
(CH’’’), 129.9 (CH’’’), 129.4 (q, JC-F = 2.9 Hz, C-7), 129.2 (C-4a), 129.1 (C-3’’ and
C-5’’), 128.9 (C-2’’ and C-6’’), 128.8 (CH’’’), 128.7 (CH’’’), 128.6 (CH’’’), 128.4 (C-
1’’’), 128.0 (C-1’’’), 127.8 (q, 2JC-F = 34.0 Hz, C-6), 127.7 (C-1’’’), 125.7 (q, 3JC-F = 3.8
1
Hz, C-5), 123.5 (q, JC-F = 273.0 Hz, CF3), 116.8 (C-8), 113.8 (C-3), 92.5 (C-1’), 81.2
(C-4’), 73.7 (C-2’), 70.7 (C-3’), 63.1 (C-5’), 42.8 (CH2NH). IV (KBr) ν (cm-1): 3424,
+
1744, 1727, 1672, 1548. HRMS-ESI Calcd. for C44H32ClF3N2NaO9 : 847,16406. Found
+
for C44H32ClF3N2NaO9 : 847.165322.

6-chloro-1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-N’-(4-
methylphenyl)-quinoline-3-carboxamides (9f). White solid, 62 % yield, m.p.: 94-96 °C.
1H NMR (500.00 MHz, CDCl3) δ (ppm): 11.77 (1H, s, C=ONH), 9.32 (1H, s, H-2),
8.50 (1H, d, J 2.4 Hz, H-5), 8.11 (2H, d, J 7.1 Hz, H-2’’’ and H-6’’’), 7.92 (4H, d, J 7.2
Hz, H-2’’’ and H-6’’’), 7.74 (1H, d, J 9.2 Hz, H-8), 7.62-7.52 (5H, m, H-2’’, H-6’’ and
H-4’’’), 7.44-7.34 (7H, m, H-3’’’, H-5’’’ and H-7), 7.15 (2H, d, J 8.2 Hz, H-3’’ and H-
5’’), 6.47 (1H, d, J 4.8 Hz, H-1’), 6.04 (1H, t, J 5.4 Hz, H-2’), 5.91 (1H, t, J 5.4 Hz, H-
3’), 4.98-4.88 (3H, m, H-4’, H-5’a and H-5’b), 2.34 (3H, s, CH3). 13C/APT NMR (75.0
MHz, CDCl3) δ (ppm): 176.0 (C=O), 166.3 (C=OOBz), 165.2 (C=OOBz), 164.8
(C=OOBz), 161.7 (C=ONH), 143.7 (C-2), 137.0 (C-8a), 136.1 (C-1’’), 134.3 (CH’’’),
134.0 (CH’’’), 133.7 (CH’’’), 133.6 (C-4’’), 133.5 (C-7), 132.2 (C-6 or C-4a), 130.0
(CH’’’), 129.9 (CH’’’), 129.5 (C-3’’ and C-5’’), 129.2 (C-1’’’), 128.9 (C-1’’’), 128.8
(CH’’’), 128.7 (CH’’’), 128.6 (CH’’’), 128.5 (C-6 or C-4a), 128.1 (C-1’’’), 127.3 (C-5),
120.5 (C-2’’ and C-6’’), 117.5 (C-8), 113.5 (C-3), 92.3 (C-1’), 81.2 (C-4’), 74.0 (C-2’),
70.6 (C-3’), 63.1 (C-5’), 21.1 (CH3). IV (KBr) ν (cm-1): 3424, 1728, 1550. HRMS-ESI
+
+
Calcd. for C43H33ClN2NaO9 : 779,17668. Found for C43H33ClN2NaO9 : 779.175082.
6-chloro-1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-N’-(4-
chlorophenyl)-quinoline-3-carboxamides (9g). White solid, 46 % yield, m.p.: 184-186
1
°C. H NMR (500.00 MHz, CDCl3) δ (ppm): 11.88 (1H, s, C=ONH), 9.30 (1H, s, H-
2), 8.48 (1H, sl, H-5), 8.09 (2H, d, J 7.5 Hz, H-2’’’ and H-6’’’), 7.93-7.91 (4H, m, H-
2’’’ and H-6’’’), 7.72 (1H, d, J 9.0 Hz, H-8), 7.64 (2H, d, J 8.3 Hz, H-2’’ and H-6’’),
7.61-7.51 (3H, m, H-4’’’ and H-7), 7.46-7.35 (7H, m, H-3’’’, H-5’’’ and H-4’’’), 7.28
(2H, d, J 8.4 Hz, H-3’’ and H-5’’), 6.48 (1H, d, J 3.7 Hz, H-1’), 6.05-6.04 (1H, m, H-
13
2’), 5.91 (1H, t, J 5.0 Hz, H-3’), 5.99-4.88 (3H, m, H-4’, H-5’a and H-5’b). C/APT
NMR (125.00 MHz, CDCl3) δ (ppm): 175.9 (C=O), 166.2 (C=OOBz), 165.2 (C=OOBz),
164.8 (C=OOBz), 161.0 (C=ONH), 143.5 (C-2), 137.2 (C-4’’), 137.0 (C-8a), 134.3
(CH’’’), 134.0 (CH’’’), 133.6 (CH’’’), 133.5 (C-7), 132.3 (C-1’’), 130.0 (CH’’’), 129.3
(C-6 or C-4a), 128.9 (CH’’’), 128.8 (C-3’’ and C-5’’), 128.7 (C-1’’’), 128.6 (CH’’’),
128.4 (C-1’’’), 128.0 (C-6 or C-4a), 127.2 (C-5), 121.6 (C-2’’ and C-6’’), 117.4 (C-8),
113.0 (C-3), 92.2 (C-1’), 81.3 (C-4’), 74.1 (C-2’), 70.6 (C-3’), 63.1 (C-5’). IV (KBr) ν
+
(cm-1): 3420, 1728, 1546. HRMS-ESI Calcd. for C42H30Cl2N2NaO9 : 799,12261. Found
+
for C42H30Cl2N2NaO9 : 799.119327.

6-chloro-1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-N’-(4-
fluorophenyl)-quinoline-3-carboxamides (9h). White solid, 35 % yield, m.p.: 174-176
1
°C. H NMR (500.00 MHz, CDCl3) δ (ppm): 11.83 (1H, s, C=ONH), 9.32 (1H, s, H-
2), 8.51 (1H, d, J 2.5 Hz, H-5), 8.10 (2H, d, J 8.2 Hz, H-2’’’ and H-6’’’), 7.93-7.91 (4H,
m, H-2’’’ and H-6’’’), 7.74 (1H, d, J 9.2 Hz, H-8), 7.66 (2H, dd, J 9.0 and 5.0 Hz, H-
2’’ and H-6’’), 7.61-7.52 (3H, m, H-4’’’), 7.46 (1H, dd, J 8.5 and 2.5 Hz, H-7), 7.43-
7.36 (6H, m, H-3’’’, H-5’’’), 7.03 (2H, t, J 9.0 Hz, H-3’’ and H-5’’), 6.47 (1H, d, J 4.9
Hz, H-1’), 6.04 (1H, t, J 5.9 Hz, H-2’), 5.91 (1H, t, J 5.4 Hz, H-3’), 4.98-4.90 (3H, m,
H-4’, H-5’a and H-5’b). 13C/APT NMR (125.00 MHz, CDCl3) δ (ppm): 175.9 (C=O),
1
166.2 (C=OOBz), 165.2 (C=OOBz), 164.8 (C=OOBz), 161.8 (C=ONH), 159.27 (d, JC-F
=
4
243.3 Hz, C-4’’), 143.5 (C-2), 137.0 (C-8a), 134.7 (d, JC-F = 2.6 Hz, C-1’’), 134.3
(CH’’’), 134.0 (CH’’’), 133.7 (CH’’’), 133.5 (C-7), 132.3 (C-6 or C-4a), 130.0 (CH’’’),
129.9 (CH’’’), 129.3 (C-1’’’), 128.9 (C-6 or C-4a), 128.8 (CH’’’), 128.7 (CH’’’), 128.4
3
(C-1’’’), 128.1 (C-1’’’), 127.3 (C-5), 122.0 (d, JC-F = 7.8 Hz, C-2’’ and C-6’’), 117.4
(C-8), 115.5 (d, 2JC-F = 22.4 Hz, C-3’’ and C-5’’),113.2 (C-3), 92.2 (C-1’), 81.2 (C-4’),
74.1 (C-2’), 70.6 (C-3’), 63.1 (C-5’). IV (KBr) ν (cm-1): 3420, 1730, 1678, 1558.
HRMS-ESI Calcd. for C42H30ClFN2NaO9: 783,15216. Found for C42H30ClFN2NaO9:
783.149872
6-chloro-1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-N’-(4-
methoxyphenyl)-quinoline-3-carboxamides (9i). Light purple solid, 72 % yield, m.p.:
1
156-159 °C. H NMR (300.00 MHz, CDCl3) δ (ppm): 11.70 (1H, s, C=ONH), 9.31
(1H, s, H-2), 8.47 (1H, d, J 2.7 Hz, H-5), 8.10 (2H, dd, J 8.4 and 1.2 Hz, H-2’’’ and H-
6’’’), 7.93-7.89 (4H, m, H-2’’’ and H-6’’’), 7.72 (1H, d, J 9.0 Hz, H-8). 7.62 (2H, d, J
9.0 Hz, H-2’’ and H-6’’), 7.57-7.51 (3H, m, H-4’’’), 7.43-7.35 (7H, m, H-3’’’, H-5’’’
and H-7), 6.87 (2H, d, J 9.0 Hz, H-3’’ and H-5’’), 6.48 (1H, d, J 4.7 Hz, H-1’), 6.04
(1H, dd, J 5.7 and 4.8 Hz, H-2’), 5.91 (1H, t, J 5.5 Hz, H-3’), 4.93-4.86 (3H, m, H-4’,
13
H-5’a and H-5’b), 3.80 (3H, s, OCH3). C/APT NMR (75.0 MHz, CDCl3) δ (ppm):
175.9 (C=O), 166.2 (C=OOBz), 165.1 (C=OOBz), 164.8 (C=OOBz), 161.5 (C=ONH),
156.2 (C-4’’), 143.6 (C-2), 137.0 (C-8a), 134.2 (CH’’’), 134.0 (CH’’’), 133.6 (CH’’’),
133.4 (C-7), 132.1 (C-1’’), 131.9 (C-6 or C-4a), 130.0 (CH’’’), 129.2 (C-1’’’), 128.8
(CH’’’), 128.7 (CH’’’), 128.4 (C-6 or C-4a), 128.1 (C-1’’’), 127.2 (C-5), 121.9 (C-3’’
and C-5’’), 117.5 (C-8), 114.1 (C-2’’ and C-6’’), 113.3 (C-3), 92.3 (C-1’), 81.1 (C-4’),
74.9 (C-2’), 70.6 (C-3’), 63.1 (C-5’), 55.6 (OCH3). IV (KBr) ν (cm-1): 3420, 1725,

+
1714, 1668, 1558. HRMS-ESI Calcd. for C43H33ClN2NaO10 : 795,17214. Found for
+
C43H33ClN2NaO10 : 795.169872.
6-chloro-1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-N’-phenyl-
1
quinoline-3-carboxamides (9j). White solid, 38 % yield, m.p.: 169-171 °C. H NMR
(500.00 MHz, CDCl3) δ (ppm): 11.85 (1H, s, C=ONH), 9.33 (1H, s, H-2), 8.52 (1H, d,
J 2.5 Hz, H-5), 8.11 (2H, d, J 7.4 Hz, H-2’’’ and H-6’’’), 7.93 (4H, d, J 7.4 Hz, H-2’’’
and H-6’’’), 7.74 (1H, d, J 8.8 Hz, H-8), 7.71 (2H, d, J 7.8 Hz, H-2’’ and H-6’’), 7.60-
7.53 (3H, m, H-4’’’), 7.46-7.33 (9H, m, H-3’’’, H-5’’’, H-3’’, H-5’’ and H-7), 7.11 (1H,
t, J 7.4 Hz, H-4’’), 6.47 (1H, d, J 4.4 Hz, H-1’), 6.04 (1H, t, J 5.9 Hz, H-2’), 5.91 (1H, t,
J 5.9 Hz, H-3’), 4.98-4.88 (3H, m, H-4’, H-5’a and H-5’b). 13C/APT NMR (125.00
MHz, CDCl3) δ (ppm): 176.6 (C=O), 166.2 (C=OOBz), 165.2 (C=OOBz), 164.8
(C=OOBz), 161.9 (C=ONH), 143.7 (C-2), 138.7 (C-8a or C-1’’), 138.7 (C-8a or C-1’’),
134.3 (CH’’’), 134.0 (CH’’’), 133.7 (CH’’’), 133.5 (C-7), 132.3 (C-6 or C-4a), 130.1
(CH’’’), 130.0 (CH’’’), 129.9 (CH’’’), 129.3 (C-1’’’), 129.0 (CH’’’), 128.9 (C-6 or C-
4a), 128.8 (C-3’’ and C-5’’), 128.7 (CH’’’), 128.6 (CH’’’), 128.5 (C-1’’’), 128.1 (C-
1’’’), 127.7 (C-4’’), 124.0 (C-5), 120.6 (C-2’’ and C-6’’), 117.5 (C-8), 113.4 (C-3),
92.3 (C-1’), 81.2 (C-4’), 74.0 (C-2’), 70.6 (C-3’), 63.1 (C-5’). IV (KBr) ν (cm-1): 3420,
+
1731, 1719, 1679, 1557. HRMS-ESI Calcd. for C42H31ClN2NaO9 : 765,16158. Found
+
for C42H31ClN2NaO9 : 765.158561.
6-chloro-1,4-dihydro-4-oxo-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-N’-cyclohexyl-
1
quinoline-3-carboxamides (9k). White solid, 72 % yield, m.p.: 199-200 °C. H NMR
(300.00 MHz, CDCl3) δ (ppm): 9.68 (1H, d, J 8.1 Hz, C=ONH), 9.23 (1H, s, H-2),
8.46 (1H, d, J 2.4 Hz, H-5), 8.09 (2H, d, J 7.2 Hz, H-2’’’ and H-6’’’), 7.92 (4H, d, J 8.1
Hz, H-2’’’ and H-6’’’), 7.72 (1H, d, J 9.3 Hz, H-8), 7.60-7,53 (3H, m, H-4’’’), 7.46-
7.33 (7H, m, H-3’’’, H-5’’’ and H-7), 6.42 (1H, d, J 4.8 Hz, H-1’), 6.02 (1H, t, J 5.4
Hz, H-2’), 5.90-5.86 (1H, m, H-3’), 4.92 (3H, sl, H-4, H-5’a and 5’b), 3.97 (1H, sl, H-
1’’), 1.96 (2H, sl, H-2’’equatorial and H-6’’ equatorial), 1.74 (2H, sl, H-3’’ equatorial and H-5’’
equatorial), 1.61-1.58 (1H, m, H-4’’ equatorial), 1.48-1.25 (5H, m, H-2’’axial, H-3’’axial, H-
4’’axial, H-5’’axial, H-6’’axial). 13C/APT NMR (75.0 MHz, CDCl3) δ (ppm): 176.0
(C=O), 166.2 (C=OOBz), 165.2 (C=OOBz), 164.8 (C=OOBz), 162.9 (C=ONH), 143.5 (C-
2), 134.2 (C-8a), 134.2 (CH’’’), 134.0 (CH’’’), 133.6 (CH’’’), 133.2 (C-7), 132.0 (C-6
or C-4a), 130.0 (CH’’’), 129.9 (CH’’’), 129.3 (C-1’’’), 129.1 (C-6 or C-4a), 128.8

(CH’’’), 128.7 (CH’’’), 128.6 (CH’’’), 128.5 (C-1’’’), 128.1 (C-1’’’), 127.2 (C-5),
117.4 (C-8), 113.5 (C-3), 92.3 (C-1’), 81.0 (C-4’), 73.7 (C-2’), 70.7 (C-3’), 63.3 (C-5’),
48.0 (C-1’’), 33.0 (C-2’’ and C-6’’), 25.9 (C-4’’), 24.8 (C-3’’ and C-5’’). IV (KBr) ν
+
(cm-1): 3420, 1727, 1661, 1543. HRMS-ESI Calcd. for C42H37ClN2NaO9 : 771,20853.
+
Found for C42H37ClN2NaO9 : 771.205347.
2.1.2. General Procedure for the Synthesis of 1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-
quinoline-3-carboxamides 4a-j.
A mixture of protected nucleosides 9 (0.13 mmol) in 0.5 M ethanolic sodium
carbonate solution (10 mL) was stirred at room temperature for 12-24 h. The resulting
solution was neutralized with Dowex 50H+. After filtration and evaporation, the crude
ribonucleosides were recrystallized from THF/hexane.
1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-N’-(4-chlorobenzyl)-quinoline-3-
carboxamides (4a). White solid, 72 % yield, m.p.: 225-227 °C. 1H NMR (500.00 MHz,
DMSO-d6) δ (ppm): 10.31 (1H, t, J 6.0 Hz, C=ONH), 9.28 (1H, s, H-2), 8.37 (1H, dd,
J 8.1 e 1.4, H-5), 7.97 (1H, d, J 8.7 Hz, H-8), 7.86 (1H, ddd, J 8.6, 7.1 and 1.6 Hz, H-
7), 7.57 (1H, t, J 7.6 Hz, H-6), 7.38 (4H, d, J 5.0 Hz, H-2’’,H-3’’, H-5’’ and H-6’’),
6.16 (1H, d, J 4.0 Hz, H-1’), 5.7 (1H, sl, C2-OH), 5.3 (1H, sl, C3-OH), 5.1 (1H, sl, C5-
OH), 4.55 (2H, d, J 6.0 Hz, CH2NH), 4.22 (1H, t, J 4.5 Hz, H-2’), 4.12 (1H, dd, J 8.6
and 4.1 Hz, H-4’), 4.01 (1H, t, J 5.2 Hz, H-3’), 3.78 (1H, m, H-5’a or 5’b), 3.70 (1H,
13
m, H-5’a or 5’b). C/APT NMR (125.00 MHz, DMSO-d6) δ (ppm): 175.8 (C=O),
164.1 (C=ONH), 142.7 (C-2), 138.8 (C-1’’ or C-8a), 138.6 (C-1’’ or C-8a), 133.0 (C-
7), 131.4 (C-4’’), 129.2 (C-3’’ and C-5’’), 128.3 (C-2’’ and C-6’’), 126.8 (C-4a), 126.1
(C-5), 125.2 (C-6), 116.9 (C-8), 110.8 (C-3), 92.3 (C-1’), 85.4 (C-4’), 74.7 (C-2’), 69.7
(C-3’), 60.8 (C-5’), 41.5 (CH2NH). IV (KBr) ν (cm-1): 3454, 3309, 3100-3200, 1654,
+
1573. HRMS-ESI Calcd. for C22H21ClN2NaO6 : 467,09858. Found for
+
C22H21ClN2NaO6 : 467.097519.
6-chloro-1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-N’-(4-chlorobenzyl)-quinoline-3-
carboxamides (4b). White solid, 74 % yield, m.p.: 128-130 °C. 1H NMR (500.00 MHz,
DMSO-d6) δ (ppm): 10.13 (1H, t. J= 6.0 Hz, C=ONH), 9.27 (1H, s, H-2), 8.28 (1H, d,
J 2.6 Hz, H-5), 8.03 (1H, d, J 9.3 Hz, H-8), 7.88 (1H, dd, J 9.2 and 2.6 Hz, H-7), 7.38
(4H, m, H-2’’,H-3’’, H-5’’ and H-6’’), 6.14 (1H, d, J 4.0 Hz, H-1’), 5.65 (1H, d, J 5.0

Hz, C2-OH), 5.21 (1H, d, J 5.1 Hz, C3-OH), 5.02 (1H, sl, C5-OH), 4.56 (2H, d, J 6.0 Hz,
CH2NH), 4.23 (1H, dd, J 10.1 and 5.2 Hz, H-2’), 4.13 (1H, dd, J 8.3 and 4.0 Hz, H-4’),
4.04 (1H, q, J 5.3 Hz, H-3’), 3.78 (1H, m, H-5’a or 5’b), 3.70 (1H, m, H-5’a or 5’b).
13C/APT NMR (125.00 MHz, DMSO-d6) δ (ppm): 174.5 (C=O), 163.9(C=ONH),
143.0 (C-2), 138.4 (C-1’’), 137.4 (C-8a), 132.6 (C-7), 131.3 (C-4’’), 130.0 (C-6 or C-
4a), 129.0 (C-3’’ and C-5’’), 128.1 (C-2’’ and C-6’’), 128.0 (C-6 or C-4a), 124.8 (C-5),
119.5 (C-8), 111.2 (C-3), 92.4 (C-1’), 85.7 (C-4’), 74.6 (C-2’), 69.7 (C-3’), 60.7 (C-5’),
41.5 (CH2NH). IV (KBr) ν (cm-1): 3305, 1651, 1543. HRMS-ESI Calcd. for
+
+
C22H20Cl2N2NaO6 : 501,05961. Found for C22H20Cl2N2NaO6 : 501.059744.
6-Fluoro-1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-N’-(4-chlorobenzyl)-quinoline-3-
carboxamides (4c). White solid, 81 % yield, m.p.: 222-224 °C. 1H NMR (500.00 MHz,
DMSO-d6) δ (ppm): 10.21 (1H, t, J= 6.0 Hz, C=ONH), 9.27 (1H, s, H-2), 8.40 (1H,
dd, J 9.0 and 6.6 Hz, H-8), 7.80 (1H, dd, J 11.5 and 2.1, H-5), 7.43 (1H, td, J 8.9 and
2.2 Hz, H-7), 7.40-7.35 (4H, m, H-2’’,H-3’’, H-5’’ and H-6’’), 6.09 (1H, d, J 4.7 Hz, H-
1’), 5.76 (1H, sl, C2-OH), 5.28 (1H, sl, C3-OH), 5.10 (1H, sl, C5-OH), 4.54 (2H, d, J 6.0
Hz, CH2NH), 4.21 (1H, d, J 4.3 Hz, H-2’), 4.12 (1H, dd, J 8.4 and 3.9 Hz, H-4’), 4.01
(1H, d, J 4.3 Hz, H-3’), 3.78 (1H, m, H-5’a or 5’b), 3.70 (1H, m, H-5’a or 5’b).
13C/APT NMR (125.00 MHz, DMSO-d6) δ (ppm): 175.3 (C=O), 164.5 (C-6, d, 1JC-F
=
3
247.9 Hz), 164.0 (C=ONH), 143.5 (C-2), 140.3 (d, JC-F = 12.3 Hz, C-4a), 138.6 (C-
8a), 131.5 (C-4’’ or C-1’’), 129.5 (d, 3JC-F = 10.8 Hz, C-8), 129.2 (C-2’ or C-3’ or C-5’
or C-6’), 128.4 (C-2’ or C-3’ or C-5’ or C-6’), 123.8 (C-4’’ or C-1’’), 113.9 (d, 2JC-F
=
2
27.7 Hz, C-7), 111.3 (C-3), 103.6 (d, JC-F = 27.7 Hz, C-5), 92.7 (C-1’), 85.8 (C-4’),
74.8 (C-2’), 69.8 (C-3’), 60.8 (C-5’), 41.6 (CH2NH). IV (KBr) ν (cm-1): 3469, 1657,
+
1551. HRMS-ESI Calcd. for C22H20ClFN2NaO6 : 485,08916. Found for
+
C22H20ClFN2NaO6 : 485.087863.
6-methyl-1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-N’-(4-chlorobenzyl)-quinoline-3-
carboxamides (4d). White solid, 69 % yield, m.p.: 193-195 °C. 1H NMR (500.00 MHz,
DMSO-d6) δ (ppm): 10.42 (1H, t, J= 6.0 Hz, C=ONH), 9.30 (1H, s, H-2), 8.21 (1H,
sl, H-5), 7.93 (1H, d, J 8.9 Hz, H-8), 7.75 (1H, dd, J 8.8 and 2.0 Hz, H-7), 7.45 (4H, d,
J 5.3 Hz, H-2’’,H-3’’, H-5’’ and H-6’’), 6.20 (1H, d, J 4.0 Hz, H-1’), 5.82 (1H, d, J 5.7
Hz, C2-OH), 5.37 (1H, d, J 5.8 Hz, C3-OH), 5.18 (1H, t, J 4.8 Hz, C5-OH), 4.62 (2H, d,
J 6.0 Hz, CH2NH), 4.26 (1H, dd, J 9.7 and 5.2 Hz, H-2’), 4.17 (1H, dd, J 8.6 and 4.1

Hz, H-4’), 4.06 (1H, dd, J 10.8 and 5.4 Hz, H-3’), 3.84 (1H, m, H-5’a or 5’b), 3.75
(1H, m, H-5’a or 5’b), 2.44 (3H, s, CH3). 13C/APT NMR (125.00 MHz, DMSO-d6) δ
(ppm): 175.7 (C=O), 164.3(C=ONH), 142.2 (C-2), 138.7 (C-1’’), 136.9 (C-8a), 134.9
(C-4’’), 134.3 (C-7), 131.4 (C-6 or C-4a), 129.2 (C-3’’ and C-5’’), 128.4 (C-2’’ and C-
6’’), 126.8 (C-6 or C-4a), 125.5 (C-5), 116.9 (C-8), 110.6 (C-3), 92.3 (C-1’), 85.4 (C-
4’), 74.7 (C-2’), 69.8 (C-3’), 60.9 (C-5’), 41.5 (CH2NH), 20.6 (CH3). IV (KBr) ν (cm-
+
1): 3476, 1651, 1574. HRMS-ESI Calcd. for C44H32ClF3N2NaO9 : 847,16461. Found
+
for C44H32ClF3N2NaO9 : 847.165322.
6-trifluoromethyl-1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-N’-(4-chlorobenzyl)-
1
quinoline-3-carboxamides (4e). Light yellow solid, 80 % yield, m.p.: 145-147 °C. H
NMR (500.00 MHz, DMSO-d6) δ (ppm): 10.10 (1H, t, J= 6.0 Hz, C=ONH), 9.36 (1H,
s, H-2), 8.60 (1H, sl, H-5), 8.22-8.14 (2H, m, H-8 and H-7), 7.38 (4H, d, J 5.0 Hz, H-
2’’,H-3’’, H-5’’ and H-6’’), 6.21 (1H, d, J 4.1 Hz, H-1’), 4.56 (2H, d, J 6.0 Hz,
CH2NH), 4.24 (1H, t, J 4.6 Hz, H-2’), 4.14 (1H, dd, J 8.4 and 3.8 Hz, H-4’), 4.04 (1H, t,
J 5.0 Hz, H-3’), 3.79 (1H, dd, J 12.0 and 3.1 Hz, H-5’a or 5’b), 3.70 (1H, dd, J 12.0 and
4.0 Hz, H-5’a or 5’b). 13C/APT NMR (125.00 MHz, DMSO-d6) δ (ppm): 175.2
(C=O), 163.5 (C=ONH), 143.8 (C-2), 141.0 (C-1’’), 138.5 (C-8a), 131.4 (C-4’’), 129.1
(C-3’’ and C-5’’), 128.8 (C-7), 128.3 (C-2’’ and C-6’’), 126.5 (C-4a), 125.4 (q, 2JC-F
=
32.7 Hz, C-6), 123.4 (q, 3JC-F = 3.8 Hz, C-5), 118.9 (C-8), 112.0 (C-3), 92.5 (C-1’), 85.8
(C-4’), 74.9 (C-2’), 69.8 (C-3’), 60.7 (C-5’), 41.5 (CH2NH). IV (KBr) ν (cm-1): 3262,
+
1656, 1553. HRMS-ESI Calcd. for C23H20ClF3N2NaO6 : 535,08597. Found for
+
C23H20ClF3N2NaO6 : 535.085623.
6-chloro-1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-N’-(4-chlorophenyl)-quinoline-3-
carboxamides (4f). White solid, 76 % yield, m.p.: 234-236 °C. 1H NMR (300.00 MHz,
DMSO-d6) δ (ppm): 12.18 (1H, s, C=ONH), 9.42 (1H, s, H-2), 8.33 (1H, d, J 2.5 Hz,
H-5), 8.05 (1H, d, J 9.3 Hz, H-8), 7.94 (1H, dd, J 9.2 and 2.6 Hz, H-7), 7.75 (2H, d, J
9.0 Hz, H-2’’ and H-6’’), 7.40 (2H, d, J 4.5 Hz, H-3’’and H-5’’), 6.19 (1H, d, J 3.7 Hz,
H-1’), 5.81 (1H, sl, C2-OH), 5.32 (1H, sl, C3-OH), 5.14 (1H, sl, C5-OH), 4.23 (1H, t, J
4.2 Hz, H-2’), 4.18-4.13 (1H, m, H-4’), 4.04 (1H, t, J 5.2 Hz, H-3’), 3.83 (1H, d, J 11.9
13
Hz, H-5’a or 5’b), 3.72 (1H, d, J 12.0 Hz, H-5’a or 5’b). C/APT NMR (75.0 MHz,
DMSO-d6) δ (ppm): 174.8 (C=O), 162.1 (C=ONH), 143.4 (C-2), 137.5 (C-1’’ or C-
8a), 137.4 (C-1’’ or C-8a), 133.1 (C-7), 130.5 (C-6 or C-4a or C-4’’), 128.8 (C-3’’ and

C-5’’), 127.9 (C-6 or C-4a or C-4’’), 127.1 (C-6 or C-4a or C-4’’), 125.0 (C-5), 121.2
(C-2’’ and C-6’’),119.8 (C-8), 110.9 (C-3), 92.8 (C-1’), 85.6 (C-4’), 75.0 (C-2’), 69.6
(C-3’), 60.6 (C-5’). IV (KBr) ν (cm-1): 3409, 1674, 1546. HRMS-ESI Calcd. for
+
+
C21H18Cl2N2NaO6 : 487,04396. Found for C21H18Cl2N2NaO6 : 487.042773.
6-chloro-1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-N’-(4-fluorophenyl)-quinoline-3-
carboxamides (4g). White solid, 82 % yield, m.p.: 232-234 °C. 1H NMR (500.00 MHz,
DMSO-d6) δ (ppm): 12.11 (1H, s, C=ONH), 9.42 (1H, s, H-2), 8.35 (1H, d, J 2.6 Hz,
H-5), 8.06 (1H, d, J 9.3 Hz, H-8), 7.95 (1H, dd, J 9.2 and 2,6 Hz, H-7), 7.75 (2H, dd, J
9.0 and 5.0 Hz, H-2’’ and H-6’’), 7.10 (2H, t, J 9.0 Hz, H-3’’and H-5’’), 6.20 (1H, d, J
3.8 Hz, H-1’), 5.80 (1H, d, J 5.6 Hz, C2-OH), 5.30 (1H, d, J 5.8 Hz, C3-OH), 5.14 (1H,
t, J 4.6 Hz, C5-OH), 4.24 (1H, dd, J 9.3 and 5.2 Hz, H-2’), 4.17-4.13 (1H, m, H-4’),
4.04 (1H, dd, J 10.8 and 5.5 Hz, H-3’), 3.82 (1H, m, H-5’a or 5’b), 3.73 (1H, m, H-5’a
or 5’b). 13C/APT NMR (125.00 MHz, DMSO-d6) δ (ppm): 174.8 (C=O), 161.9
(C=ONH), 158.15 (d, 1JC-F = 239.3 Hz, C-4’’), 143.3 (C-2), 137.4 (C-8a), 134.9 (C-1’’),
133.0 (C-7), 130.4 (C-6 or C-4a), 127.9 (C-6 or C-4a), 125.0 (C-5), 121.4 (d, 3JC-F = 7.8
Hz, C-2’’ and C-6’’), 119.8 (C-8), 115.5 (d, 2JC-F = 22.2 Hz, C-3’’ and C-5’’), 111.0 (C-
3), 92.8 (C-1’), 85.6 (C-4’), 75.0 (C-2’), 69.6 (C-3’), 60.6 (C-5’). IV (KBr) ν (cm-1):
+
3401, 1669, 1570. HRMS-ESI Calcd. for C21H18ClFN2NaO6 : 471,07351. Found for
+
C21H18ClFN2NaO6 : 471.072074.
6-chloro-1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-N’-(4-methoxyphenyl)-quinoline-3-
carboxamides (4h). Light yellow solid, 83 % yield, m.p.: 193-195 °C. 1H NMR (300.00
MHz, DMSO-d6) δ (ppm): 11.95 (1H, s, C=ONH), 9.39 (1H, s, H-2), 8.34 (1H, d, J
2.4 Hz, H-5), 8.06 (1H, d, J 9.3 Hz, H-8), 7.84 (1H, dd, J 9.3 and 2.4 Hz, H-7), 7.64
(2H, d, J 9.0 Hz, H-3’’ and H-5’’), 6.93 (2H, d, J 9.0 Hz, H-2’’and H-6’’), 6.19 (1H, d,
J 3.9 Hz, H-1’), 5.95 (1H, sl, C2-OH), 5.51 (1H, sl, C3-OH), 5.20 (1H, sl, C5-OH), 4.24
(1H, t, J 5.1 Hz, H-2’), 4.15 (1H, m, H-4’), 4.04 (1H, t, J 5.4 Hz, H-3’), 3.82 (1H, dd, J
11.9 and 3.0 Hz, H-5’a or 5’b), 3.75 (3H, s, OCH3), 3.72 (1H, dd, J 10.4 and 2.4 Hz,
13
H-5’a or 5’b). C/APT NMR (75.0 MHz, DMSO-d6) δ (ppm): 175.7 (C=O), 161.4
(C=ONH), 155.4 (C-4’’), 143.1 (C-2), 137.4 (C-8a), 132.9 (C-7), 131.7 (C-6 or C-4a or
C-1’’), 130.3 (C-6 or C-4a or C-1’’), 127.9 (C-6 or C-4a or C-1’’), 124.9 (C-5), 121.0
(C-3’’ and C-5’’), 119.7 (C-8), 114.1 (C-2’’ and C-6’’), 111.2 (C-3), 92.7 (C-1’), 85.7
(C-4’), 74.9 (C-2’), 69.7 (C-3’), 60.6 (C-5’), 55.1 (OCH3). IV (KBr) ν (cm-1): 3429,

+
1670, 1559. HRMS-ESI Calcd. for C22H21ClN2NaO7 : 483,09350. Found for
+
C22H21ClN2NaO7 : 483.092458.
6-chloro-1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-N’-(phenyl)-quinoline-3-
carboxamides (4i). White solid, 79 % yield, m.p.: 188-190 °C. 1H NMR (500.00 MHz,
DMSO-d6) δ (ppm): 12.12 (1H, s, C=ONH), 9.42 (1H, s, H-2), 8.35 (1H, d, J 2.6 Hz,
H-5), 8.05 (1H, d, J 9.3 Hz, H-8), 7.94 (1H, dd, J 9.2 and 2.6 Hz, H-7), 7.72 (2H, d, J
7.5 Hz, H-2’’ and H-6’’), 7.37 (2H, t, J 7.5 Hz, H-3’’and H-5’’), 7.10 (1H, t, J 7.4 Hz,
H-4’’), 6.19 (1H, d, J 3.8 Hz, H-1’), 5.78 (1H, d, J 5.5 Hz, C2-OH), 5.29 (1H, d, J 5.7
Hz, C3-OH), 5.14 (1H, t, J 4.6 Hz, C5-OH), 4.24 (1H, dd, J 9.2 and 5.0 Hz, H-2’), 4.17-
4.14 (1H, m, H-4’), 4.04 (1H, dd, J 11.0 and 5.4 Hz, H-3’), 3.84-3.80 (1H, m, H-5’a or
5’b), 3.75-3.71 (1H, m, H-5’a or 5’b). 13C/APT NMR (125.00 MHz, DMSO-d6) δ
(ppm): 174.9 (C=O), 161.9 (C=ONH), 143.3 (C-2), 138.5 (C-1’’), 137.4 (C-8a), 133.0
(C-7), 130.5 (C-6 or C-4a), 128.9 (C-3’’ and C-5’’), 127.9 (C-6 or C-4a), 125.0 (C-5),
123.5 (C-4’’), 119.7 (C-8), 119.6 (C-2’’ and C-6’’), 111.2 (C-3), 92.8 (C-1’), 85.7 (C-
4’), 75.0 (C-2’), 69.7 (C-3’), 60.6 (C-5’). IV (KBr) ν (cm-1): 3400, 1672, 1559. HRMS-
+
+
ESI Calcd. for C21H19ClN2NaO6 : 453,08293. Found for C21H19ClN2NaO6 :
453.082354.
6-chloro-1,4-dihydro-4-oxo-1-(β-D-ribofuranosyl)-N’-(cyclohexyl)-quinoline-3-
carboxamides (4j). Light yellow solid, 86 % yield, m.p.: 198-199 °C. 1H NMR (500.00
MHz, DMSO-d6) δ (ppm): 9.79 (1H, d, J 7.8 Hz, C=ONH), 9.25 (1H, s, H-2), 8.27
(1H, d, J 2.7 Hz, H-5), 8.02 (1H, d, J 9.3 Hz, H-8), 7.89 (1H, dd, J 9.2 and 2.6 Hz, H-
7), 6.13 (1H, d, J 4.2 Hz, H-1’), 5.94 (1H, sl, C2-OH), 5.50 (1H, sl, C3-OH), 5.21 (1H,
sl, C5-OH), 4.21 (1H, t, J 4.6 Hz, H-2’), 4.12 (1H, dd, J 8.4 and 4.0 Hz, H-4’), 4.01 (1H,
t, J 5.1 Hz, H-3’), 3.84 (1H, m, H-1’’), 3.77 (1H, d, J 11.8 Hz, H-5’a or 5’b), 3.69 (1H,
d, J 10.5 Hz, H-5’a or 5’b), 1.86 (2H, m, H-2’’equatorial and H-6’’ equatorial), 1.69 (2H, m,
H-3’’ equatorial and H-5’’ equatorial), 1.56 (1H, m, H-4’’ equatorial), 1.34 (5H, m, H-2’’axial, H-
3’’axial, H-4’’axial, H-5’’axial, H-6’’axial). 13C/APT NMR (125.00 MHz, DMSO-d6) δ
(ppm): 174.6 (C=O), 162.4 (C=ONH), 142.9 (C-2), 137.4 (C-8a), 132.7 (C-7), 131.7
(C-6 or C-4a), 130.0 (C-6 or C-4a), 128.1 (C-6 or C-4a), 124.9 (C-5), 119.5 (C-8),
111.5 (C-3), 92.5 (C-1’), 85.6 (C-4’), 74.7 (C-2’), 69.8 (C-3’), 60.8 (C-5’), 46.8 (C-1’’),
32.3 (C-2’’ and C-6’’), 25.1 (C-4’’), 24.0 (C-3’’ and C-5’’). IV (KBr) ν (cm-1): 3444,

+
1651, 1563. HRMS-ESI Calcd. for C21H25ClN2NaO6 : 459,12988. Found for
+
C21H25ClN2NaO6 : 459.128318.
2.2. Biological Assays
2.2.1. Reverse Transcriptase (RT) inhibition assay.
The inhibitory effect of the compounds on the RNA-dependent DNA
polymerase (RDDP) activity of the RT clone HXB2 was evaluated using purified
recombinant HIV-1 enzyme as reported [10], with modifications. RDDP activity was
assayed with a colorimetric assay from Roche Life Sciences. In brief, 3 U of enzyme
(one unit is the enzyme concentration that incorporates 1 pmol of dTTP per minute per
mg of enzyme at 37 °C under the standard assay conditions) was incubated with
different compounds and polirA-oligodU. Incorporation of the incoming free dU labeled
with biotin and digoxin (equimolar mixture) was blocked by the drugs. To reveal the
incorporated dU, plates were coated with streptavidin and an antibody against digoxin
tagged horseradish used. The reactions were initiated at 37 °C, incubated for 30 min,
and arrested with 0.5 M EDTA. An automatic ELISA plate reader with a 570 nm test
wavelength and 690 nm reference wavelength was used. The 50 % inhibitory
concentration (IC50) was calculated by linear regression analysis of the dose-response
curves generated from the data.
2.2.2. Cytotoxicity assay
Human primary peripheral blood mononuclear cells (PBMCs), at density of 2 ×
104 cell/well in 96-well culture plates, were incubated with the compounds at different
concentrations for 72 h. Then, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxanilide (XTT; 5 mg/mL) was added to DMEM in the presence of
0.01 % N-methyl-dibenzopyrazine methyl sulfate (PMS). After incubation for 4 h at
37°C, the absorbance of the plates were measured in a spectrophotometer at 492 nm and
620 nm. The 50 % cytotoxic concentration (CC50) was calculated by linear regression
analysis of the dose-response curves generated from the data.
2.3. In silico studies
2.3.1. Molecular Modeling and SAR analysis

Three-dimensional structures of all compounds (9a-k and 4a-j) were built and
optimized using Spartan 10 software. The structures were minimized, and a
conformational analysis was performed using molecular mechanics force field (MMFF).
Then, the lowest energy conformation obtained was subjected to geometric optimization
in a vacuum using the semiempirical RM1 method and single point ab initio Hartree-
Fock with the 6-31G* basis set.
The stereoelectronic properties (HOMO energy,
HOMO orbital coefficients distribution, LUMO density, dipole moment, dipole moment
vector, lipophilicity (clogP), volume, area, and polarizability) were calculated for all
compounds. The theoretical logP (clogP) was calculated at the AM1 semiempirical
level using the Villar method included in Spartan.
2.3.2. Molecular docking
To investigate the binding mode of the 4a, 9a, 9d and 9j compounds, we
performed molecular docking studies on the allosteric site of HIV-1 RT. The AutoDock
4.2 [11] program was used on a Windows-based PC.
The three-dimensional structures of these compounds were built and optimized
using Spartan 10 software as described above. The coordinates from HIV-1 RT were
obtained from the Protein Data Bank (PDB code 1KLM) [12]. The cocrystallized ligand
and solvent molecules were removed with PyMOL 1.3 (The PyMOL Molecular
Graphics System, Version 1.3, Schrödinger, LLC, San Francisco, CA, USA). The
ligands were flexibly docked to HIV-1 RT and the docking files were prepared using
AutoDock Tools. The protein was treated as rigid, polar hydrogen atoms were added,
nonpolar hydrogen atoms were merged and Gasteiger charges were assigned by default.
The grid center was established on the allosteric site with 0.375 Å spacing and
52×32×40 points. Docking studies were carried out using the empirical free energy
function and the Lamarckian Genetic Algorithm. A total of 50 independent docking
runs were carried out using the program’s default parameters. The results of the most
favorable free energies of binding in the most populated clusters were selected as
possible structures of the resultant complex.
2.3.3. Pharmacokinetics and toxicity analyses
ADMET parameters were evaluated using the ADMET Predictor™ version 9.5
(Simulations Plus, Inc., Lancaster, CA, USA) qualitative and quantitative models.

Compounds 4a, 9a, 9d, 9j, AZT and nevirapine were submitted for in silico analysis of
their pharmacokinetic and toxicity parameters [13].
3. Results and Discussion
3.1. Chemistry
Initially, we promoted the synthesis of the key intermediates of 4-oxoquinolines
8 using a procedure involving the condensation of anilines 5 with diethyl
ethoxymethylenemalonate (EMME) in refluxing ethanol followed by the thermal
cyclization of the aniline acrylate intermediates 6 [14]. A nucleophilic substitution
reaction between oxoquinolines 7 and the appropriate amines in diphenyl ether afforded
the respective carboxamides 8, as previously reported. Ribonucleosides were prepared
according to the Vorbruggen methodology adapted by us for the synthesis of
oxoquinoline ribonucleosides [15-17]. Previous silylation of 4-oxoquinoline-3-
carbomides 8 with bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1 %
trimethylchlorosilane and the subsequent condensation reaction with 1-O-acetyl-2,3,5-
tri-O-benzoyl-β-D-ribofuranose
10
in
acetonitrile
under
trimethylsilyl-
trifluoromethanesulfonate catalysis led to the corresponding protected ribonucleosides
9. The treatment of compounds 9 with ethanolic sodium carbonate solution produced
the free ribonucleosides 4 in good yields (Figure 2). The structures of the new
ribonucleosides 4 and 9 were confirmed by IR, NMR and high-resolution mass
spectrometry.
Figure 2. Synthesis of ribonucleosides 9 and 4. Reagents and conditions: (a) EMME,
ethanol, reflux, 6 h; (b) diphenyl ether, reflux, 20 min; (c) diphenyl ether, amine, 210°C,

1 h; (d) 1- BSTFA/1 % TMCS, acetonitrile, 60 °C, 2 h; 2- 10, TMSOTf, rt, 4 h; (e)
Na2CO3, ethanol, rt, 24 h
3.2. Biology and Structure-Activity Relationships
The 22 newly synthesized compounds were evaluated for their biological
activity against HIV-1 RT. The FDA-approved drug azidothymidine (AZT) was
included as the reference (Table 1). All compounds exhibited good anti-HIV activity,
ranging from 1.4ꢀto 29ꢀµM, among which compounds 9a and 9d displayed the highest
antiviral activity profiles, with IC50 values of 1.4 and 1.6 μM, respectively.
Previous work from our group showed that chlorine substituents on the 4-
oxoquinolic derivatives enhanced the antiviral activity against HIV-1 [8, 9, 10].
Nevertheless, the absence of halogenation at the R1 position rendered 9a and 9d with
improved antiviral activity when compared to the other compounds (Table 1). Although
all tested compounds were less potent than AZT, IC50 values in the low micromolar
range indicates good candidates for further pharmacological development since
resistance is a current problem for HIV treatment that requires new antiviral options.
The compounds were also assayed for cytotoxicity using human primary cells,
peripheral blood mononuclear cells (PBMCs). Our compounds were more than 10-fold
less cytotoxic than AZT (Table 1), with CC50 values above 1000 µM. Most active
candidates, compounds 9a and 9d, showed CC50 values of 1486 and 1394 µM,
respectively. Compounds 9a and 9d presented selectivity indexes (SI = CC50/EC50) of
998 and 830, respectively, indicating they are as safe as AZT.
Table 1. HIV-1 RT inhibitory effects (IC50 µM), cytotoxicity (CC50 µM) and volume
(ų) of 4-oxoquinoline ribonucleosides (4a-j and 9a-k)
COMPOUND R1
R2
R3
Cl
Cl
R4
H
IC50
(µM)
29.2 ±
4.6
18.4 ±
6.7
CC50
(µM)
>1000
Volume
(ų)
409.63
n
4a
4b
H
phenyl
phenyl
1
Cl
H
1
>1000
422.84
4c
4d
F
CH3
phenyl
phenyl
Cl
Cl
H
H
1
1
3.9 ± 0.3 >1000
4.3 ± 0.7 >1000
414.19
427.59

4e
4f
4g
4h
4i
CF3
Cl
Cl
Cl
Cl
phenyl
phenyl
phenyl
phenyl
phenyl
Cl
Cl
F
OCH3
H
H
H
H
H
H
H
1
0
0
0
0
0
3.9 ± 1.0 >1000
8.0 ± 0.8 >1000
5.8 ± 1.3 >1000
3.4 ± 0.3 >1000
2.6 ± 0.3 >1000
441.62
404.86
396.98
417.86
390.79
404.33
4j
Cl cyclohexyl
-
22.4 ±
4.5
>1000
9a
H
phenyl
Cl
OBz
1
1.4 ± 0.3 1486 ±
203
730.81
9b
9c
9d
Cl
F
CH3
phenyl
phenyl
phenyl
Cl
Cl
Cl
OBz
OBz
OBz
1
1
1
8.8 ± 2.1 >1000
2.8 ± 0.2 >1000
1.6 ± 0.3 1394 ±
127
743.56
735.63
747.67
9e
9f
CF3
Cl
phenyl
phenyl
Cl
OBz
1
0
2.9 ± 0.6 >1000
761.24
729.36
CH3 OBz
16.6 ±
3.4
>1000
9g
9h
9i
Cl
Cl
Cl
Cl
phenyl
phenyl
phenyl
phenyl
Cl
F
OBz
OBz
0
0
0
0
9.9 ± 3.1 >1000
7.3 ± 1.2 >1000
6.8 ± 0.9 >1000
724.27
713.66
738.68
711.43
OCH3 OBz
9j
H
OBz
19.5 ±
5.5
>1000
9k
AZT
Cl cyclohexyl
-
-
OBz
-
0
-
8.6 ± 2.2 >1000
0.05 126
724.03
-
-
-
The conformational analysis showed that all derivatives without the methylene
spacer presented a phenyl substituent that was planar to the 4-oxoquinoline moiety,
whereas insertion of the spacer led to the phenyl group adopting a perpendicular
orientation (Figure 3). Afterwards, we have two structural patterns of HIV-1 RT
inhibition where the 4-oxoquinoline ribonucleosides with a 2,3,5-tribenzoyl are more
active than the 2,3,5-trihydroxyl when R2 contains a methylene spacer (9a-e/4a-e), and
in the absence of the methylene spacer, the 4-oxoquinolinic β-D-ribonucleosides with
the 2,3,5-trihydroxyl presented better activity (9f-k/4f-j). This may allow us to infer that
in the absence of volumous and flexible substituents, the planarity is important for
activity. It is worth mentioning that greater flexibility allows compounds to adopt
multiple conformations, support a better fit at the binding site and consequently
increasing inhibition potency (Table 1). This structural feature is important for mutant
strains that have a resilient profile, where more flexible molecules can be better
accommodated at the RT binding site [18]. Also, a larger contact surface area may
allow more Van der Waals contacts, contributing to the activity. This is evidenced in the
most active compounds (9a and 9d) (Figure 3).

Figure 3. Comparison of the conformational and steric parameters by structural
alignment of the 4-oxoquinoline ribonucleosides 9a (green) and 4i (magenta). Van der
Waals volume of 9a can be seen in gray
Electronic parameters calculated by quantum chemical methods showed no
direct correlation with activity, while the volume and flexibility were key features for
the most active compounds (9a and 9d). The allosteric site is largely hydrophobic,
includes only a few nonhydrophobic residues, and the size of ligands varies greatly. The
calculated volume of the allosteric site HIV-1 RT using the DoGSiteScorer server [19]
was approximately 822.01 Å3. Accordingly, this pocket may accommodate bulky
molecules as 9a and 9d (Table 1).
3.3. Mechanistic studies by a molecular docking approach
To gain a better insight about the mechanism of action, we performed molecular
docking studies with the most and the less active compounds 4a, 9a, 9d and 9j into the
allosteric site of HIV-1 RT. Initially, the validation of the docking protocol was carried
out through redocking into the HIV-1 RT crystal structure (PDB code 1KLM).
Comparison of the redocking results with the cocrystallyzed conformation showed a
good success rate with a root-mean-square deviation (RMSD) of 1.08 Å. These data
support the hypothesis that the experimental binding mode could be reproduced
accurately using this protocol.

Then, the most active compounds (9a and 9d) were investigated. Analysis of the
docking complex of 9a within the allosteric site showed that the R2 group of this
compound occupies the subpocket formed by hydrophobic residues Leu100, Tyr181,
Tyr188, Phe227, and Trp229 of HIV-1 RT, whereas the 4-oxoquinoline moiety
effectively occupies the NNIBP entrance channel. Compound 9a interacted mainly by
Van der Waals interactions and hydrophobic contacts with Leu100, Lys101, Lys102,
Lys104, Ser105, Tyr181, Tyr188, Ile195, Glu224, Pro225, Pro226, Phe227, Leu234,
His235, Pro236 and Asp237 (Figure 4). The amide moiety of compound 9a participated
in two hydrogen bonds: one with the carbonyl oxygen of Lys103 (distance NH-O of 3.2
Å) and another with the side chain of Tyr318 (O-HO distance of 3.4 Å). Furthermore, a
hydrogen bond was observed between the oxygen of the ribofuranose ring and the N
from the backbone of Val106 (NH-O distance of 3.2 Å) (Figure 4). The allosteric site of
HIV-1 RT is a highly hydrophobic cavity, which may infer that the presence of these
hydrogen bonds may contribute to the stabilization of the complex. It is interesting to
highlight that no mutations have been described yet for Tyr318, so 9a could be an
inhibitor that is less susceptible to viral resistance [12, 20].
Compound 9d interacted by Van der Waals interactions and hydrophobic
contacts with Leu100, Lys101, Lys102, Lys104, Ser105, Val106, Thr107, Val179,
Gly190, Ile195, Glu224, Pro225, Pro226, Phe227, Leu234, His235, Pro236, Asp237
and Tyr318 residues (Figure 4). The amide moiety of compound 9d participated in an
hydrogen bond with N from the backbone of Lys103 (distance NH-O of 3.1 Å) and the
oxygen of the ribofuranose ring with N from the backbone of Val106 (NH-O distance of
3.4 Å) (Figure 4). The hydrogen bond with Lys103 or Lys103 together with Val106
seems to be important for the activity.
The less active compounds in each series (4a and 9j) were also investigated.
Compound 4a showed Van der Waals interactions and hydrophobic contacts with
Leu100, Lys101, Lys102, Lys103, Lys104, Ser105, Glu224, Pro225, Pro226, Phe227,
His235, Pro236, Asp237 and Tyr318 (Figure 4). Hydrogen bond was formed with
Val106 (NH-O distance of 2.8Å) and Leu234 (OH-O distance of 2.6Å). Interestingly,
due to its conformation in the binding pocket, compound 4a presented intramolecular
Hydrogen bonds were also formed (Figure 4).
Compound 9j interacted by Van der Waals interactions and hydrophobic
contacts with Lys101, Lys102, Lys103, Ser105, Val106, Thr107, Ile195, Glu224,

Pro225, Pro226, Phe227, Leu234, His235, Pro236 and Tyr318 residues. One hydrogen
bond with Lys104 (NH-O distance of 2.7Å) was formed (Figure 4).
Figure 4. Binding mode analysis of compound 4a (pink), 9a (green), 9d (magenta) and
9j (yellow) at the HIV-1 RT allosteric site. Residues involved in the interactions are
shown in blue and hydrogen bonds are colored in yellow dashed lines.
3.4. In Silico Pharmacokinetic and Toxicity Analyses

In this work, we also assessed the pharmacokinetic (PK) and toxicological
properties of compounds 4a, 9a, 9d, 9j, AZT and nevirapine using an in silico
approach. Considering the PK analysis, all compounds of series 9 presented high
flexibility, high numbers of hydrogen bond acceptor groups, high lipophilicity and low
solubility, which may lead to absorption issues. AZT is charged and presented low
permeability; however, good absorption has been reported in the literature [21].
Problems related to absorption were not observed for nevirapine, which is in accordance
with Coffey & Volberding [22]. The evaluation of metabolism showed that series 9
could be metabolized by CYP3A4, whereas AZT and nevirapine are metabolized by
glucuronidation [21, 23].
Toxicological evaluations indicated possible carcinogenicity for all compounds
and hepatic effects only for 9a and 9d. Similar to AZT, mutations were also detected.
These results are corroborated by Chiu & Duesberg [24] and by the National
Toxicology Program [25]. Finally, nevirapine showed only hepatic problems, which is
also corroborated by the literature [22]. These findings highlight the reliability of the in
silico results herein and, due to the resistance profile of HIV, reinforces the potential of
the active compounds as anti-HIV candidates for further exploration.
4. Conclusions
In summary, we synthesized twenty-two 4-oxoquinoline ribonucleoside
derivatives with anti-HIV-1 RT activity and low cytotoxicity. Derivatives 9a and 9d
showed the highest antiviral activities and SAR studies revealed that volume and
flexibility are the key features for for modulating HIV-1 RT. Molecular docking studies
showed that the most active compounds bind to the allosteric site of the enzyme,
interacting with important residues for the enzymatic activity. Interactions with Tyr318,
a conserved residue with no mutations described so far, indicated that 9a as an inhibitor
would be less susceptible to viral resistance. Finally, the in silico ADMET results
showed that 9a and 9d are similar to AZT. Overall, 9a and 9d present a promising
scaffold for future anti-HIV-1 drug design since their mode of HIV-1 inhibition might
differ from the classical NNRTIs and be effective against HIV mutant strains.
Supporting Information

Supporting information includes physical and spectroscopic information for
compounds.
Acknowledgments
The authors would like to acknowledge the agencies that funded our research:
CNPq, CAPES and FAPERJ. The fellowships granted by CNPq, CAPES and FAPERJ
are gratefully acknowledged, as well as and FIOCRUZ for the HRMS analyses.
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HIGHLIGHTS
4-oxoquinoline ribonucleoside with anti-HIV-1 RT activity and low cytotoxicity was prepared
Ribonucleoside derivatives were more than 10-fold less cytotoxic than AZT
SAR studies revealed that volume and flexibility are the key features for modulating HIV-1 RT
Docking studies showed that the most active compounds bind to the allosteric site of the enzyme