Synthesis, biological evaluation, and molecular modeling studies of new 1,3,4-oxadiazole- and 1,3,4-thiadiazole-substituted 4-oxo-4H-pyrido[1,2-a] pyrimidines as anti-HIV-1 agents

Article abstract of DOI:10.1007/s00044-012-0241-5

A new series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives containing 1,3,4-oxadiazole and 1,3,4-thiadiazole rings as a part of the metal chelation motif were synthesized and evaluated for their in vitro anti-HIV-1 activity. Most of the tested compounds displayed moderate inhibitory properties against HIV-1 virus (NL4-3) in Hela cell cultures. Compounds 11e and 11b exhibited the highest activity among the synthesized compounds with inhibition rate of 51 and 48 % at concentration of 100 μM, respectively. Molecular docking study using the later crystallographic data available for PFV integrase (IN) showed that the designed compounds bind into the active site of IN such that the keto oxygen atom at position of C-4 and nitrogen atom of thiadiazole or oxadiazole ring moiety chelate the Mg²⁺ ion. Our results also showed that all tested compounds presented no significant cytotoxicity at concentration of 100 μM. Therefore, these compounds can provide a very good basis for the development of new hits.

Full text of DOI:10.1007/s00044-012-0241-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-012-0241-5
ORIGINAL RESEARCH
Synthesis, biological evaluation, and molecular modeling studies
of new 1,3,4-oxadiazole- and 1,3,4-thiadiazole-substituted
4-oxo-4H-pyrido[1,2-a]pyrimidines as anti-HIV-1 agents
•
•
•
Z. Hajimahdi A. Zarghi R. Zabihollahi
M. R. Aghasadeghi
Received: 15 February 2012 / Accepted: 14 September 2012
Ó Springer Science+Business Media New York 2012
Abstract A new series of 4-oxo-4H-pyrido[1,2-a]pyrimi-
dine derivatives containing 1,3,4-oxadiazole and 1,3,4-
thiadiazole rings as a part of the metal chelation motif were
synthesized and evaluated for their in vitro anti-HIV-1
activity. Most of the tested compounds displayed moderate
inhibitory properties against HIV-1 virus (NL4-3) in Hela
cell cultures. Compounds 11e and 11b exhibited the
highest activity among the synthesized compounds with
inhibition rate of 51 and 48 % at concentration of 100 lM,
respectively. Molecular docking study using the later
crystallographic data available for PFV integrase (IN)
showed that the designed compounds bind into the active
site of IN such that the keto oxygen atom at position of C-4
and nitrogen atom of thiadiazole or oxadiazole ring moiety
chelate the Mg2? ion. Our results also showed that all
tested compounds presented no significant cytotoxicity at
concentration of 100 lM. Therefore, these compounds can
provide a very good basis for the development of new hits.
Introduction
Human immuno-deficiency virus type 1 (HIV-1) is the
etiological agent that causes acquired immuno-deficiency
syndrome (AIDS). HIV infection is a life-threatening health
problem necessitating discovery of novel targets and new
lead molecules. Studies in HIV biology have provided
important information about the main steps of the virus life
cycle which consists of viral entry, reverse transcription,
integration, gene expression, virion assembly, budding, and
maturation. HIV-1 encodes three enzymes that are essential
for retroviral replication: reverse transcriptase (RT), prote-
ase, and integrase (IN) (Moyle et al., 2008). Although drugs
targeting RT and protease are in wide use and have shown
effectiveness particularly when employed in combination,
these highly active antiretroviral therapies (HAART) still
show important limitations. These are costs, occurrence of
various side effects due to drug toxicity and most impor-
tantly, the loss of drug effectiveness over time caused by
development of resistance, including multidrug resistance
and cross-resistance (Barbaro et al., 2005). HIV-1 IN plays
a central role in the insertion of viral DNA into the genome
of host cells, which first catalyzes removal of the terminal
dinucleotide from each 30 end of the viral DNA (30 pro-
cessing) and subsequently mediates joining of the 30 end of
the viral DNA to the host DNA (strand transfer). This
enzymatic process is dependent on an active site containing
dual Mg2? metal ions held in place with a highly conserved
triad of carboxylate amino acid residues (asp64/asp116/
glu152) commonly referred to as a DD(35)E triad (Colicelli
and Goff, 1988; Miller et al., 1997; Pommier et al., 2005).
HIV-1 IN represents a crucial target for antiretroviral drugs,
because it has no counterpart in mammalian cells. A
plethora of HIV-1 IN inhibitors were discovered in the last
decade but only one compound, Raltegravir, was FDA
Keywords 4-Oxo-4H-pyrido[1,2-a]pyrimidines ꢀ
Oxadiazoles ꢀ Thiadiazoles ꢀ Anti-HIV-1 activity ꢀ
Molecular modeling
Z. Hajimahdi ꢀ A. Zarghi (&)
Department of Medicinal Chemistry, School of Pharmacy,
Shahid Beheshti University of Medical Sciences,
P.O. Box 14155-6153, Tehran, Iran
e-mail: azarghi@yahoo.com
R. Zabihollahi ꢀ M. R. Aghasadeghi
Hepatitis and AIDS Department, Pasteur Institute of
Iran, Tehran, Iran
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Med Chem Res
approved in October 2007. This is the unique currently used
clinical drug targeting IN, which is administered as a new
addition to HAART regimens. Only two other compounds
are in clinical development: GS-9137 (Elvitegravir) is in the
late stages of clinical development (Shimura et al., 2008),
whereas S/GSK-1349572 is in phases I and II clinical trials
(Min et al., 2010). Raltegravir’s clinical use has already
evidenced the development of viral resistances (Charpentier
et al., 2008). This evidently shows that there is a strong need
to discover novel IN targeting hits with different structural
scaffolds. In this research, aimed at the discovery of new
compounds as anti-HIV-1 agents, we selected the HIV-1 IN
inhibitors as a lead to design new analogs. Among numer-
ous attempts to develop IN inhibitors, the keto-enol acid
class (often referred to as the diketo acid class) of com-
pounds has been most aggressively developed because of
the marked antiretroviral activities exhibited (Pommier
et al., 2005). Since the keto-enol acid compounds often
exhibit adverse hepatic effects (Kirschberg and Parrish,
2007), there is an attempt to find new bioiosters for keto-
enol acid motif. The carboxylic acid in the keto-enol acid
motif can be replaced with not only well-known bioisosters
of a carboxylic acid group, such as triazole and tetrazole
(Herr, 2002), but also by a basic aromatic heterocycle
bearing a lone pair donor atom, such as a pyridine ring
(Zhuang et al., 2003) and oxadiazole (1, Fig. 1) (Johns
et al., 2009). On the other hand, our literature studies
showed that bicyclic pyrido[1,2-a]pyrimidin-4-one deriva-
tives are promising anti-HIV agents due to their ability to
inhibit HIV-1 IN (Donghi et al., 2009) (2, Fig. 1). Based
upon this, we decided to design and synthesize a new series
of pyrido[1,2-a]pyrimidin-4-one derivatives possessing
1,3,4-oxadiazole and 1,3,4-thiadiazole rings as a part of the
metal chelation motif. According to HIV-1 IN inhibitors
structure–activity relationship, we incorporated different
substituted phenyl rings to evaluate the effect of these
substituents on anti-HIV-1 activity. We also performed
docking studies to predict the interaction of newly synthe-
sized compounds into the active site of IN and their prob-
able mechanism of action.
O
OH
O
N
H
F
O
N
O
N
N
F
Cl
OH
O
OH
NH
F
O
O
O
N
H
N
O
H
HO
N
N
N
O
O
F
Raltegravir
Elvitegravir
S/GSK-1349572
O
N
OH
F
N
N
H
N
OH
N
N
O
O
N
O
O
Et
N
Et
N
F
R
Et
1
2
N
O
N
R
N
X
N
Designed compounds
Fig. 1 HIV-1 IN inhibitors (Raltegravir, Elvitegravir, S/GSK-1349572), lead compounds (1, 2), and our designed scaffold
123

Med Chem Res
O
N
O
OEt
a
N
OEt
b
c
N
N
H
O
N
NH2
EtO
O
3
4
5
R
N
O
N
N
O
O
O
N
O
H
N
N
O
g
N
NHNH2
N
N
H
R
f
O
N
6
9a-e
10a-e
h
e
d
N
O
N
N
O
N
N
SH
N
O
N
S
R
N
N
O
N
N
8
NH2
11a-e
N
O
9a, 10a, 11a; R = H
9b, 10b, 11b; R = F
9c, 10c, 11c; R = Cl
7
9d, 10d, 11d; R = OMe
9e, 10e, 11e; R = Me
Scheme 1 Reagents and conditions: a EMME, 120 °C, 1 h; b 2-
chlorobenzoic acid, Ph2O, 250 °C, 1 h; c NH2NH2.OH, DMF, rt,
30 min; d BrCN, MeOH, reflux, 2 h; e CS2, KOH, MeOH, reflux, 4 h;
f 4-substituted benzoyl chloride, DMF, rt, 6 h; g P2O5, toluene, reflux,
5 h; h LR, THF, reflux, 24 h
Result and discussion
substituted benzoyl chlorides in DMF to give acylated
compounds (9a–e). Compounds (10a–e) were synthesized
by dehydrocyclization of diacylhydrazines (9a–e) using
P2O5 in toluene. Dehydrosulfurization of diacylhydrazines
(9a–e) by the Lawesson’s reagent (LR) in THF led to
formation of derivatives containing 1,3,4-thiadiazole ring
(11a–e). All compounds were stable and kept in dry place
at 25 °C. The structure of the synthesized compounds was
Chemistry
The target 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives
were synthesized via the route outlined in Scheme 1.
Oxadiazole- and thiadiazole-substituted 4-oxo-4H-pyr-
ido[1,2-a]pyrimidine derivatives were prepared starting
from the 2-aminopyridine (3) in five steps. Condensation of
3 with ethoxymethylenemalonate diethyl ester yielded
aminomethylene malonate (4). The compound (4) was
converted to ethyl 4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
carboxylate (5) in Ph2O containing catalytic 2-chloroben-
zoic acid at 250 °C. Building upon the literatures, we found
that the typical reaction time for optimal yields for this
series transformation should be within 1–2 h. The unwan-
ted byproducts will come into being under too higher
temperature beyond 1 h. Also, we found the catalyst 2-
chlorobenzoic acid did limited effects to shorten the reac-
tion time and improve the yields. Compound (5) was
subsequently treated with hydrazine in DMF to form the
corresponding hydrazide intermediate (6). Hydrazide
intermediate (6) was converted to target molecule con-
taining 5-amino-1,3,4-oxadiazole (7) by heating with BrCN
in methanol. The reaction of hydrazide (6) with CS2 in
methanol gave compound (8). To synthesize phenyl oxa-
diazoles and thiadiazoles, compound (6) reacted with 4-
1
confirmed by IR, HNMR, ESI–MS, and CHN analysis.
Anti-HIV-1 activity
The anti-HIV activity of the all compounds was measured
by determining their ability to general inhibition of the
replication of HIV-1 in Hela cell cultures. For comparative
purposes, nucleoside RT inhibitor, AZT, was assayed in the
same cells. The results are listed in Table 1.
All the compounds displayed no significant cytotoxicity
at concentration of 100 lM. Most of the tested compounds
produced inhibitory effects at 100 lM concentration
ranging from 11 to 51 %. However, in all cases, the mea-
sured activities were lower than that of AZT. The results
demonstrate that the anti-HIV-1 activity seems to have a
dependence on the existence of a hydrophobic group at C-5
of oxadiazole and thiadiazole rings. The 1,3,4-oxadiazole
analogs (7 and 8) containing –NH2 and –SH groups
resulted in no considerable activity (0–11 %). These results
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Med Chem Res
Table 1 Anti-HIV activity of synthesized compounds
N
O
N
N
R
N
X
Compounds
X
R
100 lM
(%) inhibition rate
of P24 expression
% cell viability
7
O
O
O
O
O
O
O
S
–NH2
–SH
–
11
–
96
100
98
8
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
AZT
Phenyl
4-Fluorophenyl
4-Chlorophenyl
4-Methoxyphenyl
4-Methylphenyl
Phenyl
18
26
–
100
94
100
94
29
33
48
39
28
51
100
100
92
S
4-Fluorophenyl
4-Chlorophenyl
4-Methoxyphenyl
4-Methylphenyl
S
95
S
97
S
93
100
indicated that chelation of the 2 Mg2? cations by 1,3,4-
oxadiazole and 1,3,4-thiadizole heterocycles as compo-
nents of the chelation motif are not strong enough to allow
for a strong anti-HIV-1 activity by these compounds.
However, the introduction of para-substituted phenyl at C-
5 position of the 1,3,4-oxadiazole ring (10b, 10c, and 10e),
led to increased anti-HIV-1 activity. Our results also
showed that the compounds containing thiadiazole ring
(11a–e) are more potent than the corresponding ones with
oxadiazole ring (10a–e). It was also found that the intro-
duction of methyl or fluoro group at the para position of
the benzene ring appears to considerably benefit anti-HIV-
1 activity as compounds 11e and 11b exhibited signifi-
cantly higher potency (51 and 48 %, respectively). These
findings conform to the common pharmacophore of HIV-1
IN inhibitors in which a fluorobenzyl group with a specific
spatial arrangement to a chelator is a required structural
determinant.
molecular modeling has been used extensively to assist the
understanding of the binding of inhibitors to the IN active
site and to aid in the design of novel inhibitors.
To date, no X-ray structure of the entire HIV-1 IN for
interaction with its cognate DNA with or without substrate
has been solved. Recently, Hare et al. (2010) solved the
crystal structures of full-length prototype foamy virus
(PFV) IN DNA complexes with various HIV-1 IN inhibi-
tors, exhibiting two-metal ions in the strand transfer active
site (Mg2? or Mn2?). This breakthrough now allows for a
better understanding of the binding mode of IN strand
transfer inhibitors. As shown by Hare et al., PFV IN can be
considered as a good model for the development of HIV-1
IN strand transfer inhibitors. The secondary structures of
PFV IN (PDB: 3L2T or 3OYA) and HIV-1 IN catalytic
core domain (PDB:1BL3) have highly conserved archi-
˚
tectures, with a calculated RMSD of 1.04 A. On the basis
of these considerations, we decided to undertake docking
studies of our molecules based on the 3L2T X-ray crys-
tallographic structure of PFV IN.
Molecular modeling
Docking of the most potent compounds (11b and 11e) in
the newly built model was performed with the AUTO-
DOCK 4.2 program. All these compounds showed a similar
interfacial-binding mode in which the keto oxygen atom at
We performed detailed docking studies to predict interac-
tion of synthesized compounds into the active site of HIV-1
IN. In the absence of key, relevant crystal structures,
123

Med Chem Res
are suitable scaffold to design anti-HIV agents; (ii) all the
target compounds were completely safe and exhibited no
cytotoxicity at concentration of 100 lM; (iii) most of the
compounds displayed moderate HIV-1 inhibition rate at
concentration of 100 lM; (iv) the most active compounds
(11e and 11b) exhibited activity against HIV-1 virus (NL4-
3) with inhibition values of 51 and 48 %, respectively; (v)
docking study revealed that the anti-HIV activity of these
compounds might involve a metal chelating mechanism.
Experimental
Materials
Fig. 2 Binding model of compound 11b in the active site of HIV-1
IN
All reagents purchased from the Aldrich (USA) or Merck
(Germany) Chemical Company and were used without
further purifications.
General
Melting points (mp) were determined using a Thomas
Hoover capillary apparatus (Philadelphia, USA). Infrared
spectra were acquired on a Perkin-Elmer 1420 ratio
recording spectrometer. A Bruker FT-500 MHz instrument
1
(Brucker Biosciences, USA) was used to acquire HNMR
spectra; chloroform-D and DMSO-d6 used as solvents.
Coupling constant (J) values are estimated in hertz (Hz)
and spin multiples are given as s (singlet), d (doublet),
t (triplet), q (quartet), m (multiplet), and br (broad). The
mass spectral measurements were performed on an 6410
Agilent LCMS triple quadrupole mass spectrometer
(LCMS) with an electrospray ionization (ESI) interface.
Fig. 3 Superimposition of compound 11b on the raltegravir molecule
position of C-4 and nitrogen atom of thiadiazole ring
moiety chelate the Mg2? ion (see docking result of 11b,
Fig. 2). The displaced 30 adenosine terminal base (A17)
was involved in a p-stacking interaction with the 4-oxo-
4H-pyrido[1,2-a]pyrimidine ring. The p-fluoro-phenyl
group fitted within a tight pocket formed by cytosine 16
(C16) and guanine 4 (G4). Further docking study revealed
that 11b and raltegravir positioned a similar orientation in
the active site of HIV-1 IN (Fig. 3). These docking results
were in agreement with the common structure–activity
relationships of HIV-1 IN inhibitors.
Diethyl 2-((pyridin-2-ylamino)methylene)malonate (4)
A mixture of 2-aminopyridine 3 (1 eq.) and diethyl eth-
oxymethylenemalonate (EMME) (1 eq.) was heated at
120 °C for 1 h. The mixture was then evaporated to
dryness to give a residue which was triturated with
cyclohexane to give a solid which was filtered and dried to
afford compound 4. Yield, 89 %; white powder; mp:
60–62 °C; IR (KBr disk): t (cm-1) 1400–1600 (aromatic),
1680–1730 (C=O), 3300 (N–H); LC–MS (ESI) m/z: 286.8
(M?23, 100).
Ethyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate (5)
Conclusion
A biphenyl ether solution of 4 containing catalytic 2-chloro-
benzoic acid was heated by microwave irradiation (250 °C)
This study indicates that (i) 1,3,4-oxadiazole- and 1,3,4-
thiadiazole-substituted 4-oxo-4H-pyrido[1,2-a]pyrimidines
123

Med Chem Res
for 1 h. After cooling, the reaction mixture was filtered and
the precipitate 5 was washed with n-hexane and water. Yield,
97 %; cream powder; mp: 99–100 °C; IR (KBr disk): t
(cm-1) 1400–1600 (aromatic), 1730, 1750 (C=O); LC–MS
(ESI) m/z: 219.1 (M?1, 100).
J = 8.1 Hz), 7.77–7.80 (t, 1H, H7, J = 7.3 Hz), 8.34 (s,
1H, H2), 9.13 (d, 1H, H5, J = 12.4 Hz), 11.36 (s, 1H,
oxadiazole N–H); LC–MS (ESI) m/z: 247.1 (M?1, 100);
Anal. Calcd. for C10H6N4O2S: C, 48.78; H, 2.46; N, 22.75.
Found: C, 48.99; H, 2.70; N, 22.68.
4-Oxo-4H-pyrido[1,2-a]pyrimidine-3-carbohydrazide (6)
General procedure for preparation of N0-(4-substituted
benzoyl)-4H-pyrido[1,2-a]pyrimidin-4-one (9a–e)
A mixture of 5 (1 g, 4.5 mmol) and hydrazine hydrate
(3 ml, 45 mmol) in DMF (5 ml) was stirred in room tem-
perature for 30 min. After reaction completion, the solid
product obtained was filtered, washed with diethyl ether to
give 6. Yield, 70 %; cream powder; mp: 150 °C; IR (KBr
disk): t (cm-1) 1400–1600 (aromatic), 1730 (C=O), 2500–
3400 (O–H), 3310 (N–H), 3370 (NH2); LC–MS (ESI) m/z:
205.1 (M?1, 100).
A mixture of 6 (0.3 g, 1.5 mmol) and 4-substituted benzoyl
chlorides (0.2 ml, 1.5 mmol) was dissolved in dry DMF
(10 ml) and stirred in room temperature for 4 h. The
reaction mixture was slowly poured over crushed ice and
kept for 1 h. The solid thus separated out was filtered,
washed with water, and dried to give compounds (9a–e)
(yield, 40–70 %).
3-(5-Amino-1,3,4-oxadiazol-2-yl)-4H-pyrido[1,2-
a]pyrimidin-4-one (7)
N0-Benzoyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
carbohydrazide (9a)
To a solution of 6 (0.6 g, 3 mmol) in methanol, BrCN (3 g,
30 mmol) was added and the solution was refluxed for 2 h.
After completion of the reaction (monitored by TLC), the
solvent was evaporated under reduced pressure and the
residue was extracted with EtOAc. The organic layer was
washed with water (2 9 10 ml) and finally dried over
anhydrous Na2SO4. The solvent was removed under
reduced pressure and the resulting crude compound was
purified by crystallization in ethanol. Yield, 27 %; yellow
powder; mp: 235 °C (decomposed); IR (KBr disk): t
(cm-1) 1400–1600 (aromatic), 1735 (C=O), 2400–3400
(O–H), 3100–3300 (NH2); 1HNMR (DMSO-d6, 500 MHz):
d 7.13 (br s, 1H, oxadiazole N–H), 7.59–7.62 (t, 1H, H6,
J = 8.1 Hz), 7.89 (d, 1H, H8, J = 8.7 Hz), 8.19–8.22 (t,
1H, H7, J = 7.2 Hz), 8.27 (br s, 1H, oxadiazole N–H), 8.78
(s, 1H, H2), 9.17 (d, 1H, H5, J = 6.9 Hz); LC–MS (ESI) m/
z: 230.1 (M?1, 100); Anal. Calcd. for C10H7N5O2: C,
52.40; H, 3.08; N, 30.56. Found: C, 52.26; H, 3.31; N,
30.69.
Yield, 40 %; white powder; mp: 110 °C; IR (KBr disk): t
(cm-1) 1400–1600 (aromatic), 1690 (C=O), 2500–3200
(O–H), 3200, 3440 (N–H); LC–MS (ESI) m/z: 309.0
(M?1, 100).
N0-(4-Fluorobenzoyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-
3-carbohydrazide (9b)
Yield, 70 %; white powder; mp: 170–172 °C; IR (KBr
disk): t (cm-1) 1400–1600 (aromatic), 1690 (C=O), 2500–
3200 (O–H), 3200 (N–H); LC–MS (ESI) m/z: 327.1 (M?1,
100).
N0-(4-Chlorobenzoyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-
3-carbohydrazide (9c)
Yield, 60 %; white powder; mp: 208–210 °C; IR (KBr
disk): t (cm-1) 1400–1600 (aromatic), 1685 (C=O), 2500–
3200 (O–H); LC–MS (ESI) m/z: 343.1 (M?1).
3-(5-Mercapto-1,3,4-oxadiazol-2-yl)-4H-pyrido[1,2-a]
pyrimidin-4-one (8)
N0-(4-Methoxybenzoyl)-4-oxo-4H-pyrido[1,2-a]
pyrimidine-3-carbohydrazide (9d)
A mixture of 6 (0.8 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 4 h. The solution was then concen-
trated, cooled, and acidified with dilute HCl. The solid
mass that separated out was filtered, washed with ethanol,
dried, and recrystallized from ethanol. Yield, 20 %; yellow
powder; mp: 145 °C (decomposed); IR (KBr disk): t
(cm-1) 1400–1600 (aromatic), 1680 (C=O), 2600–3400
(O–H), 3250 (N–H); 1HNMR (DMSO-d6, 500 MHz): d
7.12–7.15 (t, 1H, H6, J = 6.4 Hz), 7.27 (d, 1H, H8,
Yield, 60 %; white powder; mp: 146–149 °C; IR (KBr
disk): t (cm-1) 1400–1600 (aromatic), 1700 (C=O), 2500–
3300 (O–H); LC–MS (ESI) m/z: 339.1 (M?1, 100).
N0-(4-Methylbenzoyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-
3-carbohydrazide (9e)
Yield, 63 %; white powder; mp 235 °C; IR (KBr disk): t
(cm-1) 1400-1600 (aromatic), 1685 (C=O), 2500–3200
(O–H), 3250 (N–H); LC–MS (ESI) m/z: 322.9 (M?1, 100).
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Med Chem Res
General procedure for preparation of 3-(5-(4-
substituted benzoyl)-1,3,4-oxadiazol-2-yl)-4H-
pyrido[1,2-a]pyrimidin-4-one (10a–e)
Calcd. for C16H9ClN4O2: C, 59.18; H, 2.79; N, 17.25.
Found: C, 59.31; H, 2.66; N, 17.10.
3-(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)-4H-
pyrido[1,2-a]pyrimidin-4-one (10d)
To a solution of compounds (9a–e) (1 g, 3 mmol) in tol-
uene, P2O5 (3 g, 20 mmol) was added and the solution was
refluxed for 5 h. After completion of the reaction (moni-
tored by TLC), the solvent was evaporated under reduced
pressure and the residue was extracted with EtOAc. The
organic layer was washed with NaHCO3 (10 %,
2 9 10 ml) and water (10 ml), and finally dried over
anhydrous Na2SO4. The solvent was removed under
reduced pressure and the resulting crude compound was
purified by column chromatography (yield, 10–20 %).
Yield, 10 %; cream powder; mp: 245 °C; IR (KBr disk): t
(cm-1) 1400–1600 (aromatic), 1705 (C=O); 1HNMR
(CDCl3, 500 MHz): d 3.89 (s, 3H, OCH3), 7.03 (d, 2H, 4-
methoxyphenyl H3 & H5, J = 8.8 Hz), 7.40–7.43 (t, 1H,
H6, J = 6.9 Hz), 7.93 (d, 1H, H8, J = 8.7 Hz), 7.98-8.01
(t, 1H, H7, J = 7.2 Hz), 8.12 (d, 2H, 4-methoxyphenyl H2
& H6, J = 8.7 Hz), 9.21 (s, 1H, H2), 9.34 (d, 1H, H5,
J = 7.1 Hz); LC–MS (ESI) m/z: 321.1 (M?1, 100); Anal.
Calcd. for C17H12N4O3: C, 63.75; H, 3.78; N, 17.49.
Found: C, 64.01; H, 3.99; N, 17.55.
3-(5-Phenyl-1,3,4-oxadiazol-2-yl)-4H-pyrido[1,2-
a]pyrimidin-4-one (10a)
3-(5-p-Tolyl-1,3,4-oxadiazol-2-yl)-4H-pyrido[1,2-
a]pyrimidin-4-one (10e)
Yield, 10 %; cream powder; mp: 180 °C; IR (KBr disk): t
(cm-1) 1400–1600 (aromatic), 1714 (C=O); 1HNMR
(CDCl3, 500 MHz): d 7.39–7.42 (m, 1H, H6), 7.51–7.55
(m, 3H, phenyl H3, H4 & H5), 7.87 (d, 1H, H8,
J = 8.6 Hz), 7.97–8.00 (m, 1H, H7), 8.18–8.20 (m, 2H,
phenyl H2 & H6), 9.24 (s, 1H, H2), 9.34 (d, 1H, H5,
J = 6.3 Hz); LC–MS (ESI) m/z: 291.1 (M?1, 100); Anal.
Calcd. for C16H10N4O2: C, 66.20; H, 3.47; N, 19.30.
Found: C, 66.36; H, 3.29; N, 19.38.
Yield, 10 %; cream powder; mp: 184 °C; IR (KBr disk): t
(cm-1) 1400–1600 (aromatic), 1700 (C=O); 1HNMR
(CDCl3, 500 MHz): d 2.44 (s, 3H, CH3), 7.33 (d, 2H, 4-
methylphenyl H3 & H5, J = 8.01 Hz), 7.39–7.42 (t, 1H,
H6, J = 5.8 Hz), 7.89 (d, 1H, H8, J = 8.7 Hz), 7.97–8.00
(m, 1H, H7), 8.07 (d, 2H, 4-methylphenyl H2 & H6,
J = 8.1 Hz), 9.22 (s, 1H, H2), 9.33 (d, 1H, H5, J = 7 Hz);
LC–MS (ESI) m/z: 327.1 (M?1, 100); Anal. Calcd. for
C17H12N4O2: C, 67.10; H, 3.97; N, 18.41; O, 10.52. Found:
C, 67.22; H, 3.68; N, 10.40.
3-(5-(4-Fluorophenyl)-1,3,4-oxadiazol-2-yl)-4H-
pyrido[1,2-a]pyrimidin-4-one (10b)
Yield, 10 %; cream powder; mp: 24 °C (decomposed); IR
(KBr disk): t (cm-1) 1400–1600 (aromatic), 1705 (C=O);
1HNMR (CDCl3, 500 MHz): d 7.21–7.23 (t, 2H, 4-fluo-
rophenyl H3 & H5, J = 8.8 Hz), 7.39–7.42 (t, 1H, H6,
J = 6.9 Hz), 7.87 (d, 1H, H8, J = 8.5 Hz), 7.97–7.99 (t,
1H, H7, J = 7.3 Hz), 8.18–8.21 (dd, 2H, 4-fluorophenyl H2
& H6), 9.23 (s, 1H, H2), 9.34 (d, 1H, H5, J = 7.1 Hz); LC–
MS (ESI) m/z: 309.1 (M?1, 100); Anal. Calcd. for
C16H9FN4O2: C, 62.34; H, 2.94; N, 18.17. Found: C, 62.48;
H, 3.11; N, 18.29.
General procedure for preparation of 3-(5-4-substituted
benzoyl-1,3,4-thiadiazol-2-yl)-4H-pyrido[1,2-
a]pyrimidin-4-one (11a–e)
To a solution of compounds (9a–e, 1 g, 3 mmol) in THF,
LR (2 g, 5 mmol) was added and the solution was refluxed
for 24 h. After completion of the reaction (monitored by
TLC), the solvent was evaporated under reduced pressure
and the residue was extracted with EtOAc. The organic
layer was washed with HCl solution (20 %, 2 9 10 ml).
The aqueous layer was neutralized with NaOH solution
(10 %) and then it was extracted with EtOAc. The organic
layer was washed with water (10 ml) and finally dried over
anhydrous Na2SO4. The solvent was removed under
reduced pressure and the resulting crude compound was
purified by column chromatography (yield, 5–10 %).
3-(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)-4H-
pyrido[1,2-a]pyrimidin-4-one (10c)
Yield, 20 %; cream powder; mp: 238 °C (decomposed); IR
(KBr disk): t (cm-1) 1400–1600 (aromatic), 1696 (C=O);
1HNMR (CDCl3, 500 MHz): d 7.40–7.43 (t, 1H, H6,
J = 6.9 Hz), 7.50 (d, 2H, 4-chlorophenyl H3 & H5,
J = 7.0 Hz), 7.87 (d, 1H, H8, J = 8.7 Hz), 7.98–8.01 (t,
1H, H7, J = 7.3 Hz), 8.13 (d, 2H, 4-chlorophenyl H2 & H6,
J = 8.7 Hz), 9.23 (s, 1H, H2), 9.33 (d, 1H, H5,
J = 6.9 Hz); LC–MS (ESI) m/z: 347.0 (M?1); Anal.
3-(5-Phenyl-1,3,4-thiadiazol-2-yl)-4H-pyrido[1,2-
a]pyrimidin-4-one (11a)
Yield, 10 %; yellow powder; mp: 190 °C (decomposed);IR
(KBr disk): t (cm-1) 1400-1600 (aromatic), 1670 (C=O);
123

Med Chem Res
1HNMR (CDCl3, 500 MHz): d 7.40–7.43 (m, 1H, H6),
7.49–7.52 (m, 3H, phenyl H3, H4 & H5), 7.90 (d, 1H, H8,
J = 8.8 Hz), 7.94–7.98 (m, 1H, H7), 8.08–8.10 (m, 2H,
phenyl H2 & H6), 9.30 (d, 1H, H5, J = 7.1 Hz), 9.70 (s,
1H, H2); LC–MS (ESI) m/z: 307.1 (M?1, 100); Anal.
Calcd. for C16H10N4OS: C, 62.73; H, 3.29; N, 18.29.
Found: C, 62.51; H, 3.01; N, 18.36.
2H, 4-methylphenyl H3 & H5, J = 9.3 Hz), 7.39–7.42 (t,
1H, H6, J = 6.3 Hz), 7.89 (d, 1H, H8, J = 8.7 Hz), 7.94–
7.98 (m, 3H, H7 & 4-methylphenyl H2 & H6), 9.29 (d, 1H,
H5, J = 6.7 Hz), 9.68 (s, 1H, H2); LC–MS (ESI) m/z: 321.1
(M?1, 100); Anal. Calcd. for C17H12N4OS: C, 63.73; H,
3.78; N, 17.49. Found: C, 64.01; H, 4.05; N, 17.53.
In vitro anti-HIV and cytotoxicity assays
3-(5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-yl)-4H-
pyrido[1,2-a]pyrimidin-4-one (11b)
The inhibitory effect of compounds against HIV-1 was
studied by single-cycle replication assay as previously
described (Zabihollahi et al., 2011). In brief, Hela cells
(6 9 103 per well of 96-well plate) were infected with sin-
gle-cycle replicable HIV NL4-3 virions (200 ng P24) in the
presence of different concentrations of compounds. Addition
of compounds to the cells environment was simultaneous
with viral infection. The supernatants were collected 72 h
postinfection and evaluated for P24 antigen load by capture
ELISA (Biomerieux, France). The inhibition rate (%) of P24
expression was calculated. The cellular toxicity was evalu-
ated by XTT (sodium 3-[1-(phenylaminocarbonyl)-3,4-tet-
razolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid)
proliferation assay (Roche, Germany) according to the kit
instruction (Scudiero et al., 1998; Lin et al., 2002). The HIV
replication assay plates were directly considered for cyto-
toxicity assay after determination of P24 load.
Yield, 8 %; yellow powder; mp: 167 °C (decomposed); IR
(KBr disk): t (cm-1) 1400–1600 (aromatic), 1670 (C=O);
1HNMR (CDCl3, 500 MHz): d 7.18–7.21 (t, 2H, 4-fluo-
rophenyl H3 & H5, J = 8.6 Hz), 7.40–7.43 (m, 1H, H6),
7.90 (d, 1H, H8, J = 8.8 Hz), 7.95–7.98 (m, 1H, H7), 8.06–
8.09 (dd, 2H, 4-fluorophenyl H2 & H6), 9.30 (d, 1H, H5,
J = 7.1 Hz), 9.68 (s, 1H, H2); LC–MS (ESI) m/z: 325.1
(M?1, 100); Anal. Calcd. for C16H9FN4OS: C, 59.25; H,
2.80; N, 17.27. Found: C, 59.54; H, 2.68; N, 17.41.
3-(5-(4-Chlorophenyl)-1,3,4-thiadiazol-2-yl)-4H-
pyrido[1,2-a]pyrimidin-4-one (11c)
Yield, 9 %; yellow powder; mp: 250 °C (decomposed); IR
(KBr disk): t (cm-1) 1400–1600 (aromatic), 1680 (C=O);
1HNMR (CDCl3, 500 MHz): d 7.42–7.43 (m, 1H, H6),
7.48 (d, 2H, 4-chlorophenyl H3 & H5, J = 8.5 Hz), 7.91 (d,
1H, H8, J = 8.7 Hz), 7.97 (m, 1H, H7), 8.01–8.03 (d, 2H,
4-chlorophenyl H2 & H6, J = 8.5 Hz), 9.30 (d, 1H, H5,
J = 7.0 Hz), 9.68 (s, 1H, H2); LC–MS (ESI) m/z: 341.0
(M?1); Anal. Calcd. for C16H9ClN4OS: C, 56.39; H, 2.66;
N, 16.44. Found: C, 56.25; H, 2.77; N, 16.31.
Molecular modeling (docking) studies
The active compoundwas selected for docking studies against
HIV-1 IN. 3OYA is used for binding mode analysis of HIV-1
IN inhibitory activity. All the compounds were built using
Chem Draw and subsequently minimized. The protein struc-
ture was prepared for docking using AUTODOCK Tool.
Docking was performed by AutoDock 4.0 program using the
implemented empirical free energy function and the
Lamarckian genetic algorithm (LGA) (Morris et al., 1998).
Co-crystallized ligand and all water molecules were removed
3-(5-(4-Methoxyphenyl)-1,3,4-thiadiazol-2-yl)-4H-
pyrido[1,2-a]pyrimidin-4-one (11d)
Yield, 5 %; yellow powder; mp: 254 °C (decomposed); IR
(KBr disk): t (cm-1) 1400–1600 (aromatic), 1705 (C=O);
1HNMR (CDCl3, 500 MHz): d 3.93 (s, 3H, OCH3), 7.01 (d,
2H, 4-methoxyphenyl H3 & H5, J = 8.8 Hz), 7.39–7.41 (m,
1H, H6), 7.89 (d, 1H, H8, J = 8.7 Hz), 7.93-7.96(m, 1H, H7),
8.02 (d, 2H, 4-methoxyphenyl H2 & H6, J = 8.8 Hz), 9.29
(d, 1H, H5, J = 7.0 Hz), 9.68 (s, 1H, H2); LC–MS (ESI) m/z:
337.1 (M?1, 100); Anal. Calcd. for C17H12N4O2S: C, 60.70;
H, 3.60; N, 16.66. Found: C, 60.88; H, 3.97; N, 16.40.
from crystal protein (3OYA), while a magnesium ion (Mg2?
)
attheactivesiteofHIV-1INwasmaintained. Polarhydrogens
wereaddedand non-polar hydrogensweremerged, and finally
Kallman united atom charge and atom type parameter was
added to 3OYA. Grid map dimensions (20 9 20 9 20) were
set surrounding active site. Lamarckian genetic search algo-
rithm was employed and docking run was set to 50.
Acknowledgments This study was financially supported by
Research Deputy of Shahid Beheshti University of Medical sciences
as part of Ph.D. thesis of Z. Hajimahdi.
3-(5-p-Tolyl-1,3,4-thiadiazol-2-yl)-4H-pyrido[1,2-
a]pyrimidin-4-one (11e)
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