Synthesis of Novel 3-(5-(Alkyl/arylthio)-1,3,4-Oxadiazol-2-yl)-8-Phenylquinolin-4(1H)-One Derivatives as Anti-HIV Agents

Article abstract of DOI:10.1080/10426507.2017.1394302

Novel quinolone derivatives featuring an 1,3,4-oxadiazole ring as a metal-chelating component and a benzyl group base on HIV-1 integrase inhibitors pharmacophore were designed and synthesized. An antiviral assay revealed that most analogues inhibited HIV-

Full text of DOI:10.1080/10426507.2017.1394302

Phosphorus, Sulfur, and Silicon and the Related Elements
ISSN: 1042-6507 (Print) 1563-5325 (Online) Journal homepage: http://www.tandfonline.com/loi/gpss20
Synthesis of novel 3-(5-(alkyl/arylthio)-1,3,4-
oxadiazol-2-yl)-8-phenylquinolin-4(1H)-one
derivatives as anti-HIV agents
Niloofar Parizadeh, Eskandar Alipour, Sepehr Soleymani, Rezvan
Zabihollahi, Mohammad Reza Aghasadeghi, Zahra Hajimahdi & Afshin
Zarghi
To cite this article: Niloofar Parizadeh, Eskandar Alipour, Sepehr Soleymani, Rezvan Zabihollahi,
Mohammad Reza Aghasadeghi, Zahra Hajimahdi & Afshin Zarghi (2017): Synthesis of novel 3-
(5-(alkyl/arylthio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4(1H)-one derivatives as anti-HIV agents,
Phosphorus, Sulfur, and Silicon and the Related Elements, DOI: 10.1080/10426507.2017.1394302
To link to this article: http://dx.doi.org/10.1080/10426507.2017.1394302
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Running title: 8-Phenylquinolin-4(1H)-ones as anti-HIV agents
Synthesis of Novel 3-(5-(Alkyl/arylthio)-1,3,4-Oxadiazol-2-yl)-8-Phenylquinolin-4(1H)-One
Derivatives as Anti-HIV Agents
Niloofar Parizadeh1, Eskandar Alipour1, Sepehr Soleymani2, Rezvan Zabihollahi2, Mohammad
Reza Aghasadeghi2, Zahra Hajimahdi3* and Afshin Zarghi3*
1Department of Organic Chemistry, Azad University, Tehran North Branch, Tehran, Iran
2Hepatitis and AIDS department, Pasteur institute of Iran, Tehran, Iran;
3Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
Corresponding authors: zarghi@sbmu.ac.ir, z.hajimahdi@sbmu.ac.ir
Abstract
Novel quinolone derivatives featuring an 1,3,4-oxadiazole ring as a metal-chelating component
and a benzyl group base on HIV-1 integrase inhibitors pharmacophore were designed and
synthesized. An antiviral assay revealed that most analogues inhibited HIV-1 replication in the
cell culture. Our results showed that compounds bearing small alkyl groups as R group were
inactive in anti-HIV-1 assay, whereas compounds possessing benzyl or substituted benzyl at the
same position showed good anti-HIV activity with the range of 20-57% at 100 M
concentration.
Among
them,
3-(5-((2-fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-8-
phenylquinolin-4-(1H)-one (compound 13) showed reasonable cell-based antiviral activity (EC50
= 50 M) with no considerable cytotoxicity (CC50 > 100 M) in the cell viability assay,
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suggesting that it may be amenable to further development for identifying new anti-HIV-1
agents. Docking studies using the later crystallographic data available for PFV integrase
corroborate favorable binding to the active site of HIV integrase, providing a basis for the design
of more potent analogues.
Graphical Abstract
Keywords
Anti-HIV-1, Evaluation, Design, Synthesis, 8-Phenylquinolin-4(1H)-ones, Docking studies.
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Introduction
HIV-1 represents the etiological agent of the acquired immune deficiency syndrome (AIDS)
which is a life-threatening health problem throughout the world [1]. Although combinations of
anti-HIV drugs have had considerable success in controlling HIV infection, emergence of drug-
resistant HIV mutant strains, poor bioavailability and cumulative toxicities limited effectiveness
of Highly Active Anti-Retroviral Therapy (HAART) [2]. Therefore, there is a need for
alternative strategies of antiviral drug discovery and new lead molecules with novel action
modes [3]. HIV integrase (IN) which catalyzes the incorporation of viral DNA into the host
chromatin plays critical roles in viral infection and the establishment of proviral latency [4]. At
first, IN removes the two nucleotides from the 3'-ends of viral DNA (3'-processing). Enzyme
then mediates joining of the 3'-end of the viral DNA to the host DNA (strand transfer) [5]. IN has
been the subject of intense research for anti-HIV drug discovery, because it has no host cellular
counterpart and its inhibition should not interfere with cellular functions [6, 7]. The integration
process is dependent on an active site containing dual Mg2+ metal ions held in place with amino
acid residues (asp64/asp116/glu152) [8]. Therefore, the chelation of the Mg2+ cofactors can lead
to the inhibition of IN [9]. Raltegravir [10], elvitegravir [11] and dolutegravir [12] are the first IN
inhibitors to be approved by the FDA (Figure 1). IN inhibitors all feature a chelating motif that
coordinates the two Mg2+ ions in the active site along with a hydrophobic terminal benzyl moiety
that interacts with the cytosine base near the 3'-end of the viral DNA. In the literature different
chelating motifs have been described such as diketo acids, monoketo acids and their heterocyclic
bioisosteres like triazole and oxadiazole (5-substituted-7-(5-(4-fluorobenzyl)-1,3,4-oxadiazol-2-
yl)-1,6-naphthyridin-8-ol, compound 1, Figure 1) [13-16]. We have previously developed some
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compounds featuring the quinolin-4(1H)-one and pyrido[1,2-a]pyrimidine core that effectively
inhibited HIV-1 in cell culture (Figure 1) [17-19]. Among quinolin-4(1H)-one derivatives, 4-
oxo-8-phenyl-1,4-dihydroquinoline-3-carboxylic acid (compound 2) containing a phenyl group
at C-8 position and monoketo acid group as a chelating motif exhibited an inhibition rate of 55%
against single cycle replicable HIV NL4-3 with a considerable safety profile. On the contrary, 3-
(5-mercapto-1,3,4-oxadiazol-2-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (compounds 3) possessing
the 1,3,4-oxadiazole ring as a chelating moiety displayed inhibition rate of 11% (Figure 1). The
SAR studies of reported compounds illustrated that incorporation of an aryl group to the
scaffolds improved compounds potencies which is in agreement with common pharmacophore of
HIV integrase inhibitors [20]. As part of our research program, aimed at discovering new anti
HIV-1 agents, we focused our attention on the synthesis, HIV-1 replication inhibitory and some
molecular modeling studies of 8-phenylquinolin-4(1H)-one derivatives possessing 1,3,4-
oxadiazole ring as metal-chelating moiety. The rational for the design of these compounds was
based on combining 8-phenylquinolin-4(1H)-one core of compound 2 with an 1,3,4-oxadiazole
ring of compound 3 as a metal-chelating component. The new designed scaffold has the
additional benzyl part believed necessary for a superior activity profile (Figure 1). Both quinolin-
4(1H)-one platform and 1,3,4-oxadiazole ring attracted great attention due to their versatile
nature and well-known biochemical properties [21-23]. The aim of this study was to discover
new anti-HIV agents and this was proposed by merging our previous effective scaffolds. The
designed compounds were synthesized and evaluated for activity against HIV NL4-3 infected
cells.
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Results and Discussion
Chemistry
The target 3-(5-(alkyl/arylthio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4(1H)-one analogues
were synthesized according to the pathway described in the Scheme 1. The preparation of target
compounds starts with the synthesis of the ethyl-4-oxo-8-phenyl-1,4-dihydroquinoline-3-
carboxylate 6 using well-known Gould–Jacobs procedure [24] as reported previously [19].
Compound 6 was subsequently refluxed with hydrazine in absolute ethanol to form the
corresponding carbohydrazide intermediate 7. The preparation of 3-(5-mercapto-1,3,4-oxadiazol-
2-yl)-8-phenylquinolin-4(1H)-one 8 as a key intermediate was performed using carbon disulfide
in refluxing ethanol during 24 h to generate the desired derivative in good yield (74%). The final
step of the synthesis involved arylation and alkylation of mercapto group in hydroalcoholic
solution of sodium hydroxide to give target compounds 9-21 [25]. Different alkyl halides and
substituted benzyl halides were used to provide preliminary structure-activity-relationship (SAR)
data. The alkylated and arylated derivatives were isolated in good yields (60-75%) after 1 h at
1
room temperature. The structures of the synthesized compounds were confirmed by IR, H-
NMR, ESI-MS and CHN analysis.
Anti-HIV activity evaluation
The newly synthesized compounds (9-21) were evaluated for anti-HIV-1 activity in single cycle
replication assay which reflects only one round of infection [26, 27]. In this assay, the activity of
target compounds against single cycle replicable HIV NL4-3 was measured by determining their
ability to inhibit p24 expression in Hela cells cultures. For comparative purposes, nucleoside
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reverse transcriptase inhibitor, zidovudine (AZT), and FDA-approved HIV integrase inhibitor,
raltegravir, were assayed in the same cells. The synthesized compounds were also tested for
cytotoxicity in XTT based cell viability assay [28]. The assays results are summarized in Table S
1 (Supplemental Materials). First the screening was conducted under single concentration (100
M) and the percent inhibition of p24 expression and percent cell viability were calculated for
each compound using a virus control and cell control, respectively. For those compounds that
inhibited more than 50%, the EC50 value was determined using five concentrations. The results
display a few prominent structure-activity-relationship (SAR) trends. As expected, most of the
compounds exhibited good anti-HIV-1 activity, with some exceptions (9-11). XTT assay results
indicated that all compounds have no significant cytotoxicity (cell viability > 80%). Therefore, it
can be concluded that the inhibitory activity of the compounds was not a result of their cytotoxic
effects. From the antiviral screening assay, the most striking SAR was that compounds bearing
small alkyl groups as R group were inactive in anti-HIV-1 assay, whereas compounds possessing
benzyl or substituted benzyl at the same position conferred good anti-HIV activity with the range
of 20-57% at 100 M concentration. A comparison of the activity of benzylated compounds and
their alkylated counterparts and also parent compound 3 confirm a relevant role of benzyl
function for the anti-HIV activity. Interestingly, the nature and the position of the substituent on
the benzyl ring exert a significant influence on the antiviral activity of this series of benzylated
compounds. For example, 2- and 4-substituted benzyl groups were better tolerated than 3-
substituted benzyl group. This may be explained by steric inhibition parameter at meta position
of benzyl group in this series of analogues. In addition, among this series, inhibitory activity
decreased in the following order: Me > F > Cl. These results are consistent with previous reports
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that among the diverse substituent patterns which were tested, 2- and 4-fluoro and chloro
substituents show the best potencies in in vitro assays [29, 30]. Unexpectedly, in our research,
chlorobenzyl compounds (16-18) were less potent than methylbenzyl derivatives (19-21) which
may be due to solubility or permeability properties. In according to parent compound 2,
fluorobenzyl and methylbenzyl derivatives exhibited same HIV-1 inhibitory. Hence, it could be
concluded that 1,3,4-oxadiazole would be a promising chelating moiety in the 8-phenylquinolin-
4(1H)-one platform. Fluorobenzyl and methylbenzyl derivatives showed comparable anti-HIV
activities, while fluorobenzyl derivatives (13-15) displayed better cell viability. Compound 13
with EC50 of 50 M and good safety profile (CC50> 100 M) represent an excellent starting point
for development of new anti-HIV agents.
Molecular modeling studies
To illustrate the binding mode of our derivatives at HIV IN active site, we performed molecular
docking on a representative analogue 15 using a PFV intasome model. It has been previously
reported that prototype foamy virus (PFV) IN and HIV IN have very similar structure so that
PFV IN can be regarded as a suitable model for the design of new HIV-1 IN strand transfer
inhibitors [31, 32]. Docking results indicated that compound 15 fit perfectly into the IN binding
site through two major binding moiety: the carbonyl group and oxadiazole ring simultaneously
bind to both Mg2+ ions (at distances of 2.59 and 3.50 Å) while allowing the placement of the
fluorobenzyl group into the protein-DNA interfacial hydrophobic pocket involving π-stacking of
the i) fluorobenzyl side chain with the deoxycytosine C16 (DC16) of viral DNA, and ii) π-
stacking of the central quinoline ring with the terminal 3′-deoxyadenosine A17 (DA17) (Figure S
1a). Compound 15 does not contain equivalent functionality and would not be expected to binds
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exactly same as co-crystalized raltegravir. However, a structure overlay of compound 15 with
raltegravir reveals a similarity between their binding modes (Figure S 1b), suggesting that our
new molecular scaffold may engage with HIV IN active site.
We also performed docking protocol on a selected set of compounds (9, 12, 13, 17, 19). The
docking outcome displayed that there was a correlation between affinity binding and anti-HIV-1
activities (Table S 2). For example, compounds 19 and 13 with high affinity binding (-10.7 and -
10.6 kcal/mol, respectively) were more potent in anti-HIV assay (57 and 48%, respectively)
suggesting that our docking protocol provides a useful in silico approach for predicting anti-
HIV-1 activity for new designed compounds. From Table 2, it is obvious that affinity binding of
designed compouds showed lower value than that of reference compound. These observations
suggest that our compounds may confer antiviral activity via inhibiting IN in a manner similar to
that of INST inhibitors.
Conclusions
In conclusion, we designed and synthesized novel anti-HIV-1 agents using the two-metal binding
pharmacophore model and incorporating structural elements from two different scaffolds. Key
features of designed compounds include the use of 1,3,4-oxadiazole ring as a metal-chelating
motif and the inclusion of substituted benzyl functionality with the goal of achieving more
potency. Anti-HIV-1 assay results revealed the requirement of a substituted benzyl group on
mercapto group for HIV-1 replication inhibition. Docking studies showed that designed
compounds binds to IN with a mode similar to raltegravir. Among designed compounds,
compound 13 is favored due to good anti-HIV-1 activity (EC50 = 50 M) and the lack of
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cytotoxicity at concentrations up to 100 M and represents a promising starting point further
development.
Experimental
Chemistry
All chemicals and solvents used in this study were purchased from Merck AG and Aldrich
Chemical. Melting points were determined with a Thomas–Hoover capillary apparatus. Infrared
spectra were acquired using a Perkin Elmer Model 1420 spectrometer. A Bruker FT-500 MHz
1
instrument (Brucker Biosciences, USA) was used to acquire H-NMR spectra with TMS as
internal standard. Chloroform-D and DMSO-d6 were used as solvents. Coupling constant (J)
values are estimated in hertz (Hz) and spin multiples are given as s (singlet), d (double), t
(triplet), q (quartet), m (multiplet), and br (broad). The mass spectral measurements were
performed on an 6410Agilent LCMS triple quadrupole mass spectrometer (LCMS) with an
electrospray ionization (ESI) interface. Microanalyses, determined for C and H, were within ±
0.4% of theoretical values.
The synthesis of diethyl 2-((biphenyl-2-ylamino)methylene)malonate (5)
Compound 5 was prepared according to the reported procedure [19].
The synthesis of ethyl 4-oxo-8-phenyl-1,4-dihydroquinoline-3-carboxylate (6)
Compound 6 was prepared according to the reported procedure [19].
The synthesis of 4-oxo-8-phenyl-1,4-dihydroquinoline-3-carbohydrazide (7)
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A mixture of 4 (1 g, 3.5 mmol) and hydrazine hydrate (2 mL, 35 mmol) in ethanol (10 mL) was
refluxed for 4 h. After reaction completion, solvent was evaporated under reduced pressure and
obtained solid product washed with water and diethyl ether to give compound 7.
Yield: 75%; m. p.: 313-314 oC; IR (KBr): υ (cm-1) 1400-1600 (aromatic), 1650 (C=O), 3170,
3320 (N-H), 3390 (NH2); LC-MS (ESI) m/z: 279.9 (M+1, 100); Anal. Calcd. for C16H13N3O2:
C, 68.81; H, 4.69; N, 15.05. Found: C, 68.61; H, 4.53; N, 15.23.
The synthesis of 3-(5-mercapto-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4(1H)-one (8)
A mixture of 7 (1 g, 4 mmol), KOH (1.1 g, 20 mmol) and carbon disulfide (3 mL) in ethanol (50
mL) was refluxed on a steam bath for 24 h. The solution was then concentrated, cooled and
acidified with dilute HCl. The solid mass that separated out was filtered, washed with ethanol,
dried and recrystallized from ethanol [25].
○
Yield: 74%; m. p.: 370-372 C (decomposed); IR (KBr): υ (cm-1) 1400-1600 (aromatic), 1650
1
(C=O), 3170 (N-H); H-NMR (DMSO-d6, 500 MHz) ppm 7.27-7.30 (t, 1H, H6, J = 7.5 Hz),
7.34-7.44 (m, 5H, 8-phenyl), 7.53 (d, 1H, H7, J = 7.5 Hz), 8.19 (d, 1H, H5, J = 7.3 Hz), 8.43 (s,
1H, H2), 9.70 (br s, 1H, NH); LC-MS (ESI) m/z: 320.0 (M-1, 100); Anal. Calcd. for
C17H11N3O2S: C, 63.54; H, 3.45; N, 13.08. Found: C, 63.29; H, 3.26; N, 13.14.
General procedure for the synthesis of 3-(5-(alkyl/arylthio)-1,3,4-oxadiazol-2-yl)-8-
phenylquinolin-4(1H)-one (9-21)
To the mixture of compound 8 (0.4 g, 1.24 mmol) and sodium hydroxide (0.4 g, 12 mmol) in
ethanol 96% (20 mL), alkyl/benzyl halide derivative (1.5 mmol) was added slowly. The mixture
was stirred at room temperature for 1 h, progress of the reaction was monitored by TLC. In case
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that product had precipitated, the solid was collected by filtration. In the other situations, some
water was added and allowed to stir at room temperature. After 1 hour, the precipitated product
was isolated. The crude solid was purified with crystallization from ethanol.
3-(5-(Methylthio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4(1H)-one (9)
○
Yield: 61%; m. p.: 547-549 C (decomposed); IR (KBr)  υ (cm-1) 1400-1600 (aromatic), 1664
1
(C=O), 2900-3400 (O-H), 3406 (N-H); H-NMR (DMSO-d6, 500 MHz) ppm 2.68 (s, 3H,
CH3), 7.27-7.30 (t, 1H, H6, J = 7.5 Hz), 7.32-7.35 (t, 1H, 8-phenyl H4', J = 7.2 Hz), 7.40-7.52 (m,
4H, 8-phenyl), 7.52-7.54 (d, 1H, H7, J = 7.5 Hz), 8.19 (d, 1H, H5, J = 7.3 Hz), 8.43 (s, 1H, H2);
LC-MS (ESI) m/z: 336.0 (M+1, 100); Anal. Calcd. C18H13N3O2S; C, 64.46; H, 3.91; N, 12.53.
Found: C, 64.12; H, 3.71; N, 12.64.
3-(5-Ethylthio-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4(1H)-one (10)
○
Yield: 74%; m. p.: 249-251 C; IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1660 (C=O), 2900-
1
3300 (O-H), 3341 (N-H); H-NMR (CDCl3, 500 MHz) ppm 1.29-1.32 (s, 3H, CH3), 3.08-3.10
(2, 2H, CH2), 7.48-7.57 (m, 6H, H6 & 8-phenyl), 7.66 (d, 1H, H7, J = 7.2 Hz), 8.59 (d, 1H, H5, J
= 8.1 Hz), 8.91 (d, 1H, H2, J = 6.1 Hz), 9.74 (s, 1H, NH); LC-MS (ESI) m/z: 350.0 (M+1, 100);
Anal. Calcd. For C19H15N3O2S; C, 65.31; H, 4.33; N, 12.03. Found: C, 65.55; H, 4.41; N, 11.92.
8-Phenyl-3-(5-(propylthio)-1,3,4-oxadiazol-2-yl)quinolin-4(1H)-one (11)
○
Yield: 66%; m. p.: 297-300 C; IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1650 (C=O), 2900-
3209 (O-H), 3334 (N-H); 1H-NMR (CDCl3, 500 MHz)  ppm 1.08-1.10 (t, 3H, CH3, J = 7.3 Hz),
1.64 (s, 2H, CH2), 1.85-1.90 (q, 2H, -CH2-, J = 7.3 Hz), 7.44-7.47 (m, 6H, H6 & 8-phenyl), 7.59
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(d, 1H, H7, J = 7.0 Hz), 8.50 (d, 1H, H5, J = 8.0 Hz), 8.55 (s, 1H, H2, J = 4.33 Hz), 9.80 (s, 1H,
NH); LC-MS (ESI) m/z: 364.0 (M+1, 100); Anal. Calcd. for C20H17N3O2S; C, 66.10; H, 4.71; N,
11.56. Found: C, 66.22; H, 4.91; N, 11.87.
3-(5-(Benzylthio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4-(1H)-one (12)
○
Yield: 68%; m. p.: 283-286 C; IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1657 (C=O), 2900-
1
3200 (O-H), 3340 (N-H); H-NMR (DMSO-d6, 500 MHz) ppm 4.51 (s, 2H, CH2), 7.27-7.66
(m, 12H, H7, H6, phenyl & benzyl), 8.45 (s, 1H, H5), 8.49 (d, 1H, H2, J = 7.2 Hz), 8.77 (s, 1H,
NH); LC-MS (ESI) m/z: 413.0 (M+1, 100); Anal. Calcd. for C24H17N3O2S; C, 70.06; H, 4.16; N,
10.21. Found: C, 70.19; H, 4.41; N, 10.36.
3-(5-((2-Fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4-(1H)-one (13)
○
Yield: 66%; m. p.: 294-296 C; IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1651 (C=O), 2800-
1
3170 (O-H), 3329 (N-H); H-NMR (CDCl3, 500 MHz) ppm 4.42 (s, 2H, CH2, JF,H = 39.4),
7.02-7.52 (m, 10H, H6, 8-phenyl & 2-F-benzyl), 7.59 (d, 1H, H7, J = 7.3 Hz), 8.28 (d, 1H, H5, J
= 7.93), 8.55 (s, 1H, H2), 10.55 (s, 1H, NH); 13C-NMR (DMSO-d6, 100 MHz)  110.7,
125.1, 125.5, 127.0, 129.0, 129.7, 130.0, 132.6, 134.2, 136.5, 136.6, 144.1, 164.2, 176.1; LC-MS
(ESI) m/z: 430.0 (M+1, 100); Anal. Calcd. for C24H16FN3O2S; C, 67.12; H, 3.76; N, 9.78.
Found: C, 67.45; H, 3.61; N, 10.02.
3-(5-((3-Fluorophenyl)thio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4(1H)-one (14)
○
Yield: 67%; m. p.: 282-284 C (decomposed); IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1685
1
(C=O), 2900-3331 (O-H), 3429 (N-H); H-NMR (DMSO-d6, 500 MHz) ppm 4.47 (s, 2H,
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CH2), 6.80-7.40 (m, 5H, H6 & 3-F-benzyl), 7.50-7.90 (m, 6H, H7 & 8-phenyl), 8.20 (m, 1H, H5),
8.44 (s, 1H, H2), 8.60 (s, 1H, NH); 13C-NMR (DMSO-d6, 100 MHz) 115.6, 115.7,
125.1, 125.8, 126.2, 129.1, 129.7, 130.2, 131.0, 131.1, 132.5, 134.1, 134.6, 134.9, 135.9, 161.6,
163.8, 176.1 LC-MS (ESI) m/z: 430.0 (M+1, 100); Anal. Calcd. for C24H16FN3O2S; C, 67.12; H,
3.76; N, 9.78. Found: C, 67.33; H, 3.51; N, 10.01.
3-(5-((4-Fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4-(1H)-one (15)
○
Yield: 75%; m. p.: 227-230 C; IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1662 (C=O), 2950-
1
3243 (O-H), 3207 (N-H); H-NMR (CDCl3, 500 MHz) δ ppm 4.11 (s, 2H, CH2), 6.94-7.53 (m,
10H, H7, 8-phenyl & 4-F-benzyl), 7.58 (d, 1H, H7, J = 6.8 Hz), 8.37 (d, 1H, H5, J = 7.3 Hz), 8.63
(s, 1H, H2), 9.08 (s, 1H, NH); 13C-NMR (DMSO-d6, 100 MHz) 115.2, 115.4,
125.2, 125.5, 126.8, 128.9, 129.7, 130.0, 131.0, 131.1, 132.6, 134.2, 134.3, 134.4, 136.4, 160.6,
163.8, 176.1; LC-MS (ESI) m/z: 430.0 (M+1), 452.0 (M+23); Anal. Calcd. for C24H16FN3O2S;
C, 67.12; H, 3.76; N, 9.78. Found: C, 67.23; H, 3.89; N, 9.56.
3-(5-((2-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4-(1H)-one (16)
○
Yield: 70.3%; m. p.: 273-275 C; IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1660 (C=O), 2860-
3200 (O-H), 3368 (N-H); 1H-NMR (CDCl3) ppm 4.41 (s, 2H, CH2), 7.16-7.41 (m, 10H, H6, 8-
phenyl & 2-Cl-benzyl), 7.58 (d, 1H, H7, J = 7.2 Hz), 8.26 (d, 1H, H5, J = 7.9 Hz), 8.56 (s, 1H,
13
H2), 10.66 (br s, 1H, NH); C-NMR (DMSO-d6, 100 MHz) 59.7, 111.5, 124.5, 125.9, 126.8,
128.0, 128.5, 128.8, 129.8, 130.9, 131.5, 133.7, 134.6, 140.5, 163.6, 176.2; LC-MS (ESI) m/z:
446.0 (M+1, 100); Anal. Calcd. for C24H16ClN3O2S; C, 64.64; H, 3.62; N, 9.42. Found: C, 64.44;
H, 3.51; N, 9.22.
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3-(5-((3-Chlorophenyl)thio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4(1H)-one (17)
○
Yield: 70.8%; m. p.: 105-108 C; IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1672 (C=O), 2800-
1
3078 (O-H), 3198 (N-H); H-NMR (CDCl3, 500 MHz) ppm 4.23 (s, 1H, CH2), 7.26-7.51 (m,
9H, 8-phenyl & 3-Cl-benzyl), 7.54-7.57 (t, 1H, H6, J = 7.4 Hz), 8.45 (d, 1H, H7, J = 7.3 Hz),
8.56 (d, 1H, H5, J = 7.9 Hz), 8.82 (d, 1H, H2, J = 6.5 Hz), 12.47 (s, 1H, NH); 13C-NMR (DMSO-
d6, 100 MHz) 59.8, 110.5, 125.1, 125.4, 126.9, 127.5, 127.7, 128.8, 129.6, 130.0, 130.4, 133.3,
134.1, 140.8, 164.0, 176.0; LC-MS (ESI) m/z: 445.0 (M+1, 100); Anal. Calcd. for
C24H16ClN3O2S; C, 64.64; H, 3.62; N, 9.42. Found: C, 64.39; H, 3.50; N, 9.31.
3-(5-((4-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4-(1H)-one (18)
○
Yield: 72%; m. p.: 144-146 C; IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1660 (C=O), 2936-
1
3150 (O-H), 3240 (N-H); H-NMR (CDCl3, 500 MHz) ppm 4.32 (s, 2H, CH2), 7.24-7.46 (m,
10H, H6, 8-phenyl & 4-Cl-benzyl), 7.58 (d, 1H, H7, J = 7.2 Hz), 8.42 (d, 1H, H5 J = 7.9 Hz), 8.62
13
(s, 1H, H2); C-NMR (DMSO-d6, 100 MHz) 59.4, 125.2, 128.5, 128.6, 128.8, 128.9, 130.5,
131.3, 131.4, 131.6, 131.8, 132.7, 136.6, 164.0, 172.4; LC-MS (ESI) m/z: 446.0 (M+1, 100);
Anal. Calcd. for C24H16ClN3O2S; C, 64.64; H, 3.62; N, 9.42. Found: C, 64.43; H, 3.48; N, 9.29.
3-(5-((2-Methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4(1H)-one (19)
○
Yield: 70%; m. p.: 197-199 C; IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1626 (C=O), 2850-
1
3047 (O-H), 3341(N-H); H-NMR (CDCl3, 500 MHz) ppm 2.47 (t, 3H, CH3), 4.54 (s, 2H,
CH2), 7.19-7.51 (m, 10H, H6, 8-phenyl & 2-Me-benzyl), 7.59 (d, 1H, H7, J = 7.0 Hz), 8.47 (d,
1H, H5, J = 7.6 Hz), 8.54 (s, 1H, H2), 9.45 (s, 1H, NH); 13C-NMR (DMSO-d6, 100 MHz) 
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19.1, 59.3, 125.5, 125.8, 129.0, 129.7, 130.0, 130.3, 130.9, 134.2, 137.7, 164.2, 176.1; LC-MS
(ESI) m/z: 426.0 (M+1, 100); Anal. Calcd. for C25H19N3O2S; C, 70.57; H, 4.50; N, 9.88. Found:
C, 70.25; H, 4.26; N, 9.67.
3-(5-((3-Methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4(1H)-one (20)
○
Yield: 62%; m. p.: 270-273 C (decomposed); IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1625
1
(C=O) 2900-3068 (O-H), 3263 (N-H); H-NMR (CDCl3, 500 MHz) ppm 4.39 (s, 1H, CH2),
7.09-7.46 (m, 2H, H6, H7, 8-phenyl & 3-Me-benzyl), 8.28-8.39 (m, 2H, H2 & H5); 13C-NMR
(DMSO-d6,
100
MHz)

; LC-MS (ESI) m/z: 425.0 (M+1), 448.0 (M+23), 464 (M+39); Anal. Calcd. for
C25H19N3O2S; C, 70.57; H, 4.50; N, 9.88. Found: C, 70.40; H, 4.34; N, 10.05.
3-(5-((4-Methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)-8-phenylquinolin-4(1H)-one (21)
○
Yield: 72%; m. p.: 144-146 C; IR (KBr) υ (cm-1) 1400-1600 (aromatic), 1660 (C=O), 2936-
1
3154 (O-H), 3240 (N-H); H-NMR (DMSO-d6, 500 MHz)  ppm 2.36-2.38 (s, 3H, CH3), 4.19
(s, 2H, CH2), 7.29-7.63 (m, 10H, H6, 8-phenyl & 4-Me-benzyl), 8.25-8.28 (m, 2H, H5 & H7),
8.52 (s, 1H, H2); 13C-NMR (DMSO-d6, 100 MHz)  20.2, 58.6, 125.1, 125.7, 128.6, 129.3,
130.2, 130.3, 131.2, 134.3, 136.7, 164.3, 176.2; LC-MS (ESI) m/z: 425.0 (M+1, 100); Anal.
Calcd. for C25H19N3O2S; C, 70.57; H, 4.50; N, 9.88; S, 7.53. Found: C, 70.34; H, 4.38; N, 10.15.
In-vitro anti-HIV and cytotoxicity assays
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Anti-HIV activity of synthesized compounds was determined by single cycle replication assay.
Concisely, In the presence of different concentrations of compounds, Hela cells (6 × 103 per well
of 96-wells plate) were infected with single cycle replicable HIV NL4-3 virions (200 ng p24).
Compounds were added to the cells environment simultaneously with viral infection. After 72 h
of infection, the supernatants of cell culture were collected and p24 antigen load was measured
by capture ELISA (Biomerieux, France). Percentage inhibition of p24 expression in treated
culture was calculated as inhibition rate of p24 (%). XTT proliferation method was performed to
evaluate cellular toxicity of compounds. XTT (sodium 3-[1 (phenyl aminocarbonyl)-3,4-
tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid) reagent was used according to the kit
instruction (Roche, Germany). The cytotoxic concentration that reduced number of viable cells
by 50% (CC50) was calculated after determination of p24 load in the HIV replication assay
plates.
Molecular modeling (docking) studies
Molecular docking studies were performed by AUTODOCK 4.2 program [33], using the X-ray
crystal structure of PFV IN with PDB code of 3OYA [34]. The structure of all compounds were
drawn in ChemDraw and subsequently minimized using the Hyperchem 8.0 software [35]. The
protein structure was subjected to preparation procedure in which all water molecules and co-
crystallized ligand were deleted while magnesium ions (Mg2+) were remained. Polar hydrogen
atoms were added and non-polar hydrogens were removed for protein and ligands. Finally,
Kollman and Gasteiger charge were calculated for protein and ligands, respectively. A grid box
with 20 × 20 × 20 Å dimensions was defined around the active site. Lamarckian genetic
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algorithm with 50 generations was applied as a search tool to find possible binding
conformations of ligands.
Conflict of interest
The authors confirm that this article content has no conflict of interest
Supplemental Materials
a)
b)
Figure S 1. (a) Mode of binding of 15 in the active site (b) Compound 15 superimposed on the
raltegravir molecule
Table S 1. Anti-HIV activity of synthesized compounds 9-21
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Compounds
R
100 M
(%) Inhibition Rate of
EC50 (M)
% Cell Viability
p24 Expression
Methyl
Ethyl
NA*
NA
NA
20
89
82
-
9
-
10
Propyl
94
-
11
Benzyl
100
100
93
-
50
-
12
2-F-benzyl
3-F-benzyl
4-F-benzyl
2-Cl-benzyl
3-Cl-benzyl
4-Cl-benzyl
2-Me-benzyl
3-Me-benzyl
4-Me-benzyl
-
48
13
31
14
52
85
53
-
15
25
100
82
16
13
-
17
33
100
80
-
18
57
60
-
19
21
83
20
21
49
82
>100
10
100
100
Raltegravir
-
100
100
0.018
AZT
*NA: Not Active
Table S 2. Affinity binding of a selected set of compounds
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Compound
Affinity Binding (kcal/mol)
-8.5
-10.4
-10.6
-10.0
-10.7
-12.6
9
12
13
17
19
Raltegravir
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Figure 1. HIV-1 integrase inhibitors (Elvitegravir, Raltegravir, 1), Anti-HIV lead compounds (2,
3) and our designed compound.
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Scheme 1. Reagents and conditions: (a) EMME, 120○C, 1h (b) 2-chlorobenzoic acid, Ph2O,
250○C, 1h (c) NH2NH2.OH, EtOH, reflux, 4 h (d) CS2, KOH, EtOH, reflux, 24 h (e)
Alkyl/Benzyl halide, EtOH, NaOH 10%, 1h
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