Synthesis and anti-HIV-1 integrase activitiy of cyano pyrimidinones

Article abstract of DOI:10.1002/ardp.200900066

A series of 2-phenethyl/benzylthio-6-oxo-4-phenyl-1,6-dihydropyrimidine-5- carbonitrile were synthesized and tested against recombinant HIV-1 integrase in an enzyme assay. 2-(Phenethylthio)-4-(4-chlorophenyl)-6-oxo-1,6- dihydropyrimidine-5-carbonitrile 4m

Full text of DOI:10.1002/ardp.200900066

710
Arch. Pharm. Chem. Life Sci. 2009, 342, 710–715
Full Paper
Synthesis and Anti-HIV-1 Integrase Activitiy of Cyano
Pyrimidinones
R. Ramajayam1, Nilesh B. Mahera1, Nouri Neamati2, Mange Ram Yadav1, and Rajani Giridhar1
1 Pharmacy Department, Faculty of Technology and Engineering, Kalabhavan, The M.S. University of Baroda,
Vadodara, India
2 Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern
California, Los Angeles, CA, USA
A series of 2-phenethyl/benzylthio-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile were syn-
thesized and tested against recombinant HIV-1 integrase in an enzyme assay. 2-(Phenethylthio)-4-
(4-chlorophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 4m and 2-(phenethylthio)-4-(3-
chlorophenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile 4o showed significant inhibition
against integrase in the assay (strand transfer: IC50 values of 16 and 17 lM, respectively).
Keywords: Cyano pyrimidinone / HIV-1 integrase / Phenyl acrylonitrile /
Received: March 30, 2009; accepted: April 29, 2009
DOI 10.1002/ardp.200900066
costs are some of the reasons to develop novel drugs tar-
Introduction
geting other steps in the viral replication process [5]. In
this context, HIV-1 integrase (IN), the enzyme which
mediates a mandatory step in the viral replication proc-
ess by catalyzing the integration of viral cDNA into the
host genome, represents a validated target for the devel-
opment of new drugs against HIV-1 infection [6–10]. The
integration process is catalyzed by IN through two differ-
ent reactions: 39-processing and strand transfer [11–13].
Following reverse transcription, the viral cDNA is primed
for integration in the cytoplasm by endonucleolytic
cleavage of the 39-viral DNA ends by removing the last
two nucleotides (GT). This step is referred to as the 39-proc-
essing reaction and generates two CA-39-hydroxyl
recessed ends, which are the reactive intermediates
required for the next step, the strand transfer. After 39-
processing, IN remains bound to the viral cDNA as a mul-
timeric complex that bridges both ends of the viral DNA
within intracellular particles called pre-integration com-
plexes. The pre-integration complex then translocates
into the nucleus, where IN catalyzes joining of the proc-
essed 39-OH DNA ends to the 59-DNA phosphate of the host
chromosome (strand-transfer reaction). Viral integration
is completed by the removal of unpaired nucleotides and
gap repair, carried out by cellular enzymes, leading to a
stable provirus formation.
The acquired immunodeficiency syndrome (AIDS), which
is the final and most serious stage of human immunode-
ficiency virus (HIV) infection, renders the body suscepti-
ble to a variety of infections, cancers, and other diseases.
To date FDA-approved anti-HIV therapies are primarily
target at two viral enzymes, HIV reverse transcriptase [1]
and HIV protease [2], to block the viral life cycle. Multi-
drug cocktails consisting of a protease inhibitor (PI) or a
non-nucleoside reverse transcriptase inhibitor (NNRTI) in
combination with two nucleoside reverse transcriptase
inhibitors (HAART, highly active antiretroviral therapy)
are now the standard for treatment. HAART remarkably
decreases the viral load and provides a significant
improvement in the life expectancy of HIV / AIDS patients
[3]. However, several factors [4], including the emergence
of multi-drug-resistant HIV strains, drug toxicity, and
Correspondence: Rajani Giridhar, Pharmacy Department, Faculty of
Technology and Engineering, Kalabhavan, The M.S. University of Baro-
da, Vadodara-390 001, India.
E-mail: rajanimsu@rediffmail.com
Fax: + 91 0265 2423898
Abbreviations: HAART, highly active antiretroviral therapy; IN, HIV-1 in-
tegrase; NNRTI, non-nucleoside reverse transcriptase inhibitor; PI, pro-
tease inhibitor
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2009, 342, 710–715
Synthesis of Cyano Pyrimidinones
711
Figure 2. Drug design of HIV integrase inhibitors containing pyr-
imidinone and phenylacrylonitrile component.
Figure 1. Structure of some representative HIV-1 integrase
inhibitors.
recently bicyclic pyrimidones were identified as potent
In the past several years, a plethora of integrase inhibi- inhibitors of HIV-1 integrase [23, 24].
tors (b-diketo acid, hydrazides, catechols, DNA binders,
Herein, we report the preparation and preliminary bio-
and nucleotide based inhibitors) have been reported [14]. logical evaluation of pyrimidine derivatives obtained by:
Several of them inhibit both the viral enzyme and viral (1) keeping the common structural motif, pyrimidine, as
replication in cell-based assays as well as in animal mod- observed in literature; (2) replacement of the hydroxy by
els. However, as of today, no inhibitor of HIV-1 IN has a cyano group, and (3) introduction of benzylthio or phe-
been approved. Figure 1 shows some potential HIV-1 IN nylpropylthio for N-benzyl carboxamide. Overall, 5-
inhibitors like 5CITEP, L-731,988, Shionogi / Glaxo- cyano-6-oxo-4-phenyl pyrimidine derivatives 4 can be
SmithKline's (S/GSK) S-136043, and Merck's L-870,810. viewed as simplified variants of tyrphositin 2, HIV-1 inte-
Until now, only two compounds, the S-136043 [15, 16] grase inhibitors (Fig. 2) [25, 26]. Tyrphositins belong to
and L-870,810 [17, 18] both belonging to b-diketo acid catechol derivatives of IN inhibitors. These compounds
class of compounds, have entered clinical trials [19]. How- contain two aryl units, of which at least one contains a
ever, due to pharmacokinetic problems, S/GSK decided to 1,2-dihydroxy substitution, separated by a linker. Most of
end the development of S-1360 [20]. Although several syn- the 1,2-dihdroxy-containing inhibitors display a toxic
thetic and biological studies for diketo acid compounds effect on the cell culture which may be related to the
have been reported, the mechanism by which they bind cross reactivity with other metal-requiring enzymes or
IN has not been well understood. So there is a great need covalent protein modification by the catechol unit [27–
to discover novel lead compounds with diverse structural 29]. Such alterations should cause negligible variation in
scaffolds and promising pharmacokinetic properties, to both lipophilicity and overall topology of the rings, with
overcome the difficulties observed with first-generation a potential to coordinate a metal cofactor on the IN active
IN inhibitors.
site and be devoid of cytotoxicity.
Generally, most of the anti-HIV agents like NNRTIs
were constructed by using pyrimidine as a common het-
erocyclic core but very few are reported as IN inhibitors.
Recently, Summa et al. proposed the dihydroxypyrimi-
Results and discussion
dine-4-carboxylic acid, as a suitable replacement of the Synthesis
chelating motif for b-diketo acid, as HIV-1 IN inhibitors The synthesis of compounds 4a–p was carried out via a
[21]. Whereas they found the dihydroxypyrimidine car- two-step reaction which involved substituted benzalde-
boxylic acid derivatives were almost inactive in the HIV hyde, ethyl cyanoacetate, and thiourea to afford the cor-
integrase-mediated strand transfer assay which was origi- responding 6-aryl-5-cyano-2-thiouracil 3a–p in accord-
nally reported for HCV polymerase activity. Later, dihy- ance with the literature procedure [30, 31] followed by
droxypyrimidine-4-carboxamides 1 (Fig. 2) were discov- the regioselective alkylation of 2-thiouracil 3a–p
ered as potent reversible inhibitors of HIV integrase, by achieved by slow addition of the respective halides to a
the same research group [22]. In continuation, very solution of 3a–p in DMF using K2CO3 as a base at 0 to 58C
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R. Ramajayam et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 710–715
Scheme 1. Synthesis of cyano pyrimidinones.
Table 1. Inhibition of HIV-1 integrase catalytic activities (lM) of
the compounds 4a–p.
assay, compound 4m showed significant strand transfer-
and 39-processing activity with IC50 values of 16 and
58 lM, respectively. Introduction of a chloro group at the
meta position of the aryl ring as in compound 4o showed
a similar profile (strand transfer: IC50 = 16 lM and 39-proc-
essing 51 lM, respectively). In parallel, all compounds
were tested for their cytotoxicity against prostate cancer
cell lines (data not shown). None of the compounds
showed cytotoxicity (CC50 A 10 lM) at concentration val-
ues about 2.5 to 15-fold higher than their enzymatic
inhibition-activity values. The cytotoxicity of the test
compounds was tested by performing the MTT assay. All
compounds are devoid of cytotoxicity towards the cell
culture which is commonly observed in catechol deriv-
atives.
Com-
pound
R1
R2
n
39 Strand Strand
Processing Transfer
(IC50)
(IC50)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
H
H
H
H
H
4-NO2
H
H
4-NO2
H
H
4-NO2
H
H
1
2
1
1
2
1
1
2
1
1
2
1
2
1
1
1
A100
A100
A100
A100
A100
52
A100
A100
67
A100
84
A100
58
A100
A100
A100
A100
A100
75
A100
35
35
87
58
A100
16
A100
17
A100
4-OCH3
4-OCH3
4-OCH3
4-CH3
4-CH3
4-CH3
3-NO2
3-NO2
3-NO2
4-Cl
Conclusion
In summary, we prepared a focused library of diversely
substituted pyrimidin-4(3H)-ones containing the phenyl-
acrylonitrile as a key pharmacophoric element. The bio-
logical evaluation of these compounds allowed the iden-
tification of new and potent anti-HIV agents. Further
studies are in progress in our laboratories to exploit these
preliminary results for the synthesis of a larger library
incorporating substitution on/and bio-isosteric replace-
ment of the phenyl group in the pyrimidinone system.
4-NO2
H
4-NO2
H
4-Cl
3-Cl
3-Cl
A100
51
A100
4-NO2
to give the corresponding compounds 4a–p (Scheme 1).
All compounds were purified by crystallization and
purity was ascertained by thin-layer chromatography.
Structures of the compounds were established through
IR, 1H-NMR, and mass spectral analyses. The proton NMR
of the compounds –SCH2 shows singlet at around d =
4.5–4.6 ppm. On the other hand, compounds possessing -
SCH2CH2 show two sets of doublet of doublets at d = 3.0–
3.5 ppm, and their coupling constants for the ethylene
protons are in the range of 15.2 to 15.5 Hz.
We thank the Indian Council of Medical Research (ICMR), New
Delhi, India, for the award of a Senior Research Fellowship to
RR, and the All India Council for Technical Education (AICTE),
New Delhi, India, for financial support in form of a research
promotion scheme [File No. 8023/RID/RPS.46/2004-05] to RG.
The authors have declared no conflict of interest.
Biology
A series of substituted 2-benzylthio-6-oxo-4-phenyl-1,6-
dihydro-pyrimidine-5-carbonitrile and 2-phenethylthio-
6-oxo-4-phenyl-1,6-dihydropyrimidine-5-carbonitrile 4a–
p were synthesized with the aim to evaluate their inte-
grase and cytotoxicity activity. The IC50 values of the com-
pound 4a–p are summarized in Table 1. In the integrase
Experimental
Chemistry
Melting points were determined on a Toshniwal melting point
apparatus (Toshniwal, India) and are uncorrected. IR (in cm– 1
)
spectra in KBr pellets were performed on Shimadzu 8300 (Shi-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 710–715
Synthesis of Cyano Pyrimidinones
713
madzu, Austria), and 1H-NMR spectra were recorded on a Bruker
spectrometer (400 MHz) (Bruker, Switzerland) using DMSO-d6 as
solvent and tetramethylsilane as an internal standard. Chemical
shift data are reported in parts per million (d in ppm) where s,
br, d, dd, t, and m designate singlet, broad, doublet, doublet of
doublet, triplet, and multiplet, respectively. Elemental analyses
were recorded on PE 2400 CHNS analyzer (Perkin-Elmer, USA).
Mass spectra were recorded on JEOL SX 102/DA-6000 mass spec-
trometer (Jeol, Japan). Thin-layer chromatography (TLC) was per-
formed on precoated Silica gel Merck plates (Merck, Germany).
Compounds were visualized by illuminating with UV light
(254 nm) or exposure to iodine vapors. Solvents were purified
using standard methods.
2-(Benzylthio)-4-(4-methoxyphenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile 4d
Yield: 52%; m.p.: 248–2508C; IR (KBr): 3018, 2214, 1651, 1598,
1506, 1473, 1369, 1263, 1180, 1024, 929, 840, 781 cm– 1; 1H-NMR
(400 MHz, DMSO-d6) d: 3.89 (s, 3H, OCH3), 4.52 (s, 2H, CH2), 7.03–
7.06 (d, J = 8.92 Hz, 2H, ArH), 7.26–7.33 (m, 5H, ArH), 8.02–8.04
(d, J = 8.88 Hz, 2H, ArH), 12.88 (br, 1H, NH); MS (CI) m/z: 350 [M +
H+]. Anal. calcd. for C19H15N3O2S: C, 65.31; H, 4.33; N, 12.03.
Found: C, 65.61; H, 4.27; N, 11.98.
2-(Phenethylthio)-4-(4-methoxyphenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile 4e
Yield: 64%; m.p.: 210–2128C; IR (KBr): 3000, 2223, 1658, 1602,
1533, 1456, 1373, 1251, 1174, 1022, 997, 840, 788 cm– 1; 1H-NMR
(400 MHz, DMSO-d6) d: 3.07–3.11 (dd, J = 15.52 Hz, 2H, CH2),
3.52–3.56 (dd, J = 15.56 Hz, 2H, CH2), 3.91, (s, 3H, OCH3), 7.01–
7.04 (dd, J = 9.0 Hz, 2H, ArH), 7.22–7.26 (m, 3H, ArH), 7.30–7.33
(m, 2H, ArH), 8.15–8.17 (dd, J = 8.8 Hz, 2H, ArH), 12.7 (br, 1H,
NH); MS (CI) m/z: 364 [M + H+]. Anal. calcd. for C20H17N3O2S: C,
66.10; H, 4.71; N, 11.56. Found: C, 66.04; H, 4.82; N, 11.56.
General procedure for the synthesis of 2-phenethylthio/
benzylthio-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-
carbonitrile 4a–p
To a mixture of 6-aryl-5-cyano-2-thiouracil (1 mmol) and K2CO3
(1.5 mmol) in DMF (10 mL), alkyl iodide (1.2 mmol) was added
dropwise with stirring while maintaining the temperature of
the reaction mixture at 0–58C. Stirring was continued for 3 h at
this temperature and continued for additional 2 h at room tem-
perature. Water was added to the mixture and filtered. The aque-
ous filtrate was neutralized with acetic acid and the precipitate
was filtered and purified.
2-(4-Nitrobenzylthio)-4-(4-methoxyphenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile 4f
Yield: 70%; m.p.: 241–2438C; IR (KBr): 2920, 2214, 1656, 1598,
1467, 1379, 1259, 1178, 997, 844, 781 cm– 1; 1H-NMR (400 MHz,
DMSO-d6) d: 3.89 (s, 3H, OCH3), 4.61 (s, 2H, CH2), 7.01–7.03 (d, J =
8.92 Hz, 2H, ArH), 7.61–7.63 (d, J = 8.72 Hz, 2H, ArH), 7.98–8.00
(d, J = 8.88 Hz, 2H, ArH), 8.12–8.15 (d, J = 8.72 Hz, 2H, ArH,), 12.56
(br, 1H, NH); MS (CI) m/z: 395 [M + H+]. Anal. calcd. for
C19H14N4O4S: C, 57.86; H, 3.58; N, 14.21. Found: C, 58.01; H, 3.56;
N, 14.12.
2-(Benzylthio)-6-oxo-4-phenyl-1,6-dihydropyrimidine-5-
carbonitrile 4a
Yield: 54%; m.p.: 202–2048C; IR (KBr): 3000, 2219, 1660, 1531,
1475, 1378, 1261, 1115, 1002, 912, 848, 765 cm– 1 1H-NMR
;
(400 MHz, DMSO-d6) d: 4.51 (s, 2H, CH2), 7.25–7.33 (m, 3H, ArH),
7.37–7.39 (d, J = 8.04 Hz, 2H ArH), 7.51–7.60 (m, 3H, ArH), 7.96–
7.98 (d, J = 8.48 Hz, 2H, ArH), 12.56 (br, 1H, NH); MS (CI) m/z: 320
[M + H+]. Anal. calcd. for C18H13N3OS: C, 67.69; H, 4.10; N, 13.16.
Found: C, 67.74; H, 3.98; N, 13.24.
2-(Benzylthio)-6-oxo-4-(4-methylphenyl)-1,6-
dihydropyrimidine-5-carbonitrile 4g
Yield: 51%; m.p.: 229–2328C; IR (KBr): 3000, 2221, 1652, 1539,
1456, 1377, 1244, 1186, 1001, 900, 829, 779 cm– 1 1H-NMR
;
(400 MHz, DMSO-d6) d: 2.44 (s, 3H, CH3), 4.51 (s, 2H, CH2), 7.24–
7.38 (m, 7H, ArH), 7.86–7.90 (m, 2H, ArH), 12.42 (br, 1H, NH); MS
(CI) m/z: 334 [M + H+]. Anal. calcd. for C19H15N3OS: C, 68.45; H,
4.53; N, 12.60. Found: C, 68.48; H, 4.58; N, 12.74.
2-(Phenethylthio)-6-oxo-4-phenyl-1,6-dihydropyrimidine-
5-carbonitrile 4b
2-(Phenethylthio)-6-oxo-4-(4-methylphenyl)-1,6-
Yield: 62%; m.p.: 236–2378C; IR (KBr): 3008, 2223, 1650, 1523,
1473, 1375, 1257, 1120, 999, 852, 763 cm– 1; H-NMR (400 MHz,
1
dihydropyrimidine-5-carbonitrile 4h
DMSO-d6) d: 3.07–3.10 (dd, J = 15.52 Hz, 2H, CH2), 3.52–3.56 (dd,
J = 15.56 Hz, 2H, CH2), 7.20–7.31 (m, 5H, ArH), 7.52–7.63 (m, 3H,
ArH), 8.07–8.10 (m, 2H, ArH), 12.98 (br, 1H, NH,); MS (CI) m/z: 334
[M + H+]. Anal. calcd. for C19H15N3OS: C, 68.45; H, 4.53; N, 12.60.
Found: C, 68.48; H, 4.48; N, 12.72.
Yield: 73%; m.p.: 213–2148C; IR (KBr): 3018, 2223, 1657, 1533,
1473, 1377, 1257, 1182, 1001, 918, 858, 781 cm– 1 1H-NMR
;
(400 MHz, DMSO-d6) d: 2.46 (s, 3H, CH3), 3.04–3.07 (dd, J =
15.48 Hz, 2H, CH2), 3.46–3.49 (dd, J = 15.44 Hz, 2H, CH2), 7.20–
7.24 (m, 3H, ArH), 7.27–7.29 (dd, J = 7.12 Hz, 2H, ArH), 7.31–7.33
(dd, J = 7.84 Hz, 2H, ArH), 7.96–7.98 (dd, J = 8.12, 2H, ArH), 12.84
(br, 1H, NH); MS (CI) m/z: 348 [M + H+]. Anal. calcd. for C20H17N3OS:
C, 69.14; H, 4.93; N, 12.09. Found: C, 69.10; H, 4.93; N, 12.00.
2-(4-Nitrobenzylthio)-6-oxo-4-phenyl-1,6-
dihydropyrimidine-5-carbonitrile 4c
2-(4-Nitrobenzylthio)-6-oxo-4-(4-methylphenyl)-1,6-
Yield: 63%; m.p.: 247–2488C; IR (KBr): 2980, 2219, 1654, 1515,
1413, 1346, 1257, 1107, 1001, 929, 852, 763 cm– 1 1H-NMR
;
dihydropyrimidine-5-carbonitrile 4i
(400 MHz, DMSO-d6) d: 4.60 (s, 2H, CH2), 7.51–7.63 (m, 5H, ArH),
7.90–7.92 (d, J = 8.64 Hz, 2H, ArH), 8.12–8.14 (d, J = 8.76 Hz, 2H,
ArH), 12.85 (br, 1H, NH); MS (CI) m/z: 365 [M + H+]. Anal. calcd. for
C18H12N4O3S: C, 59.33; H, 3.32; N, 15.38. Found: C, 59.30; H, 3.44;
N, 15.16.
Yield: 55%; m.p.: 253–2548C; IR (KBr): 3000, 2219, 1660, 1539,
1479, 1348, 1267, 1120, 1004, 914, 833, 783 cm– 1 1H-NMR
;
(400 MHz, DMSO-d6) d: 2.44 (s, 3H, CH3), 4.62 (s, 2H, CH2), 7.33–
7.35 (d, J = 8.08 Hz, 2H, ArH), 7.64–7.66 (d, J = 8.76 Hz, 2H, ArH),
7.81–7.84 (d, J = 8.24 Hz, 2H, ArH), 8.12–8.15 (d, J = 8.76 Hz, 2H,
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714
R. Ramajayam et al.
Arch. Pharm. Chem. Life Sci. 2009, 342, 710–715
ArH), 13.12 (br, 1H, NH); MS (CI) m/z: 379 [M + H+]. Anal. calcd. for
C19H14N4O3S: C, 60.31; H, 3.73; N, 14.81. Found: C, 60.32; H, 3.68;
N, 14.94.
2-(Phenethylthio)-4-(3-chlorophenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile 4o
Yield: 69%; m.p.: 241–2428C; IR (KBr): 2997, 2221, 1643, 1523,
1473, 1375, 1255, 1120, 1008, 887, 779 cm– 1; 1H-NMR (400 MHz,
DMSO-d6) d: 3.02–3.06 (dd, J = 15.52 Hz, 2H, CH2), 3.44–3.48 (dd, J
= 15.52 Hz, 2H, CH2), 7.18–7.30 (m, 5H, ArH), 7.52–7.62 (m, 2H,
ArH), 7.93–8.00 (m, 2H, ArH), 13.1 (br, 1H, NH); MS (CI) m/z: 368
[M + H+]. Anal. calcd. for C19H14ClN3OS: C, 62.04; H, 3.84; N, 11.42.
Found: C, 61.98; H, 3.78; N, 11.58.
2-(Benzylthio)-4-(3-nitrophenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile 4j
Yield: 27%; m.p.: 256–2578C; IR (KBr): 3008, 2218, 1651, 1544,
1514, 1473, 1344, 1257, 1190, 1001, 906, 856, 777 cm– 1; 1H-NMR
(400 MHz, DMSO-d6) d: 4.50 (s, 2H, CH2), 7.26–7.40 (m, 5H, ArH),
7.77 (t, J = 8.04 Hz, 1H, ArH), 8.33–8.35 (d, J = 7.88 Hz, 1H, ArH),
8.43–8.41 (dd, J = 8.16 Hz, 1H, ArH), 8.82 (m, 1H, ArH), 12.64 (br,
1H, NH); MS (CI) m/z: 365 [M + H+]. Anal. calcd. for C18H12N4O3S: C,
59.33; H, 3.32; N, 15.38. Found: C, 59.08; H, 3.48; N, 15.28.
2-(4-Nitrobenzylthio)-4-(3-chlorophenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile 4p
Yield: 33%; m.p.: 245–2468C; IR (KBr): 2980, 2227, 1658, 1520,
1471, 1343, 1217, 1109, 1008, 887, 785 cm– 1; 1H-NMR (400 MHz,
DMSO-d6) d: 4.57 (s, 2H, CH2), 7.47–7.51 (m, 1H, ArH), 7.54–7.57
(m, 1H, ArH), 7.60–7.62 (d, J = 8.68 Hz, 2H, ArH), 7.74 (t, 1H, ArH),
7.83–7.85 (m, 1H, ArH), 8.16–8.18 (d, J = 8.72 Hz, 2H, ArH), 12.96
(br, 1H, NH); MS (CI) m/z: 399 [M + H+]. Anal. calcd. for
C18H11ClN4O3S: C, 54.21; H, 2.78; N, 14.05. Found: C, 54.36; H,
2.64; N, 14.24.
2-(Phenethylthio)-4-(3-nitrophenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile 4k
Yield: 46%; m.p.: 267–2688C; IR (KBr): 3018, 2221, 1645, 1537,
1475, 1352, 1255, 1153, 1012, 914, 833, 786 cm– 1 1H-NMR
;
(400 MHz, DMSO-d6) d: 3.04–3.08 (dd, J = 15.28 Hz, 2H, CH2),
3.48–3.52 (dd, J = 15.32 Hz, 2H, CH2), 7.18–7.27 (m, 5H, ArH),
7.82 (t, 1H, ArH), 8.39–8.46 (m, 2H, ArH), 8.85–8.86 (m, 1H, ArH),
12.78 (br, 1H, NH); MS (CI) m/z: 401 [M + Na+]. Anal. calcd. for
C19H14N4O3S: C, 60.31; H, 3.73; N, 14.81. Found: C, 60.42; H, 3.70;
N, 14.94.
Biology
All compounds were dissolved in DMSO and the stock solutions
were stored at 208C. The c[32P]-ATP was purchased from either
Amersham Biosciences or ICN (Germany). The expression system
for wild-type IN was a generous gift of Dr. Robert Craigie, Labora-
tory of Molecular Biology, NIDDK, NIH, Bethesda, MD, USA.
2-(4-Nitrobenzylthio)-4-(3-nitrophenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile 4l
Preparation of oligonucleotide substrates
Yield: 51%; m.p.: 236–2378C; IR (KBr): 3078, 2221, 1666, 1521,
The oligonucleotides 21top, 59-GTGTGGAAAATCTCTAGCAGT-39
and 21bot, 59-ACTGCTAGAGATTTTCCACAC-39 were purchased
from Norris Cancer Center Core Facility (University of Southern
California) and purified by UV shadowing on polyacrylamide
gel. To analyze the extent of 39-processing and strand transfer
using 59-end labeled substrates, 21top was 5'-end labeled using
T4 polynucleotide kinase (Epicentre, Madison, WI, USA) and
c[32P]-ATP (Amersham Biosciences or ICN). The kinase was heat-
inactivated and 21bot was added in 1.5-molar excess. The mix-
ture was heated at 958C, allowed to cool slowly to room temper-
ature, and run through a spin 25 mini-column (USA Scientific)
to separate annealed double-stranded oligonucleotide from
unincorporated material.
1473, 1346, 1249, 1112, 1010, 916, 891, 785 cm– 1 1H-NMR
;
(400 MHz, DMSO-d6) d: 4.59 (s, 2H, CH2), 7.63–7.65 (d, J = 8.72 Hz,
2H, ArH), 7.79 (t, J = 8.04 Hz, 1H, ArH), 8.17–8.20 (d, J = 8.72 Hz,
2H, ArH), 8.31–8.33 (d, J = 8.0 Hz, 1H, ArH), 8.41–8.44 (m, 1H,
ArH), 8.69–8.70 (m, 1H, ArH), 12.9 (br, 1H, NH); MS (CI) m/z: 410
[M + H+]. Anal. calcd. for C18H11N5O5S: C, 52.81; H, 2.71; N, 17.11.
Found: C, 52.68; H, 2.85; N, 17.04.
2-(Phenethylthio)-4-(4-chlorophenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile 4m
Yield: 61%; m.p.: 199–2008C; IR (KBr): 3025, 2218, 1678, 1533,
1471, 1375, 1257, 1093, 1001, 914, 840, 783 cm– 1 1H-NMR
;
(400 MHz, DMSO-d6) d: 3.02–3.06 (dd, J = 15.28 Hz, 2H, CH2),
3.44–3.48 (dd, J = 15.32 Hz, 2H, CH2), 7.19–7.23 (m, 3H, ArH),
7.26–7.30 (m, 2H, ArH), 7.48–7.50 (dd, J= 8.64 Hz, 2H, ArH),
7.98–8.00 (dd, J = 8.64 Hz, 2H, ArH), 12.92 (br, 1H, NH); MS (CI)
m/z: 368 [M + H+]. Anal. calcd. for C19H14ClN3OS: C, 62.04; H, 3.84;
N, 11.42. Found: C, 62.08; H, 3.98; N, 11.40.
Integrase assay [32]
To determine the extent of 39-processing and strand transfer,
wild-type IN was preincubated at a final concentration of
200 nM with the inhibitor in reaction buffer (50 mM NaCl,
1 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid
(HEPES), pH 7.5, 50 lM EDTA, 50 lM dithiothreitol, 10% glycerol
(w/v), 7.5 mM MnCl2, 0.1 mg/mL bovine serum albumin, 10 mM
2-mercaptoethanol, 10% DMSO, and 25 mM 3-(N-morpholino)-
propanesulfonic acid (MOPS), pH 7.2) at 308C for 30 min. Then,
20 nM of the 59-end 32P-labeled linear oligonucleotide substrate
was added, and incubation was continued for an additional 1 h.
Reactions were quenched by the addition of an equal volume
(16 lL) of loading dye (98% deionized formamide, 10 mM EDTA,
0.025% xylene cyanol, and 0.025% bromophenol blue). An ali-
quot (5 lL) was electrophoresed on a denaturing 20% polyacryl-
amide gel (0.09 M Tris-borate pH 8.3, 2 mM EDTA, 20% acryl-
amide, 8 M urea). Gels were dried, exposed in a PhosphorImager
2-(4-Nitrobenzylthio)-4-(4-chlorophenyl)-6-oxo-1,6-
dihydropyrimidine-5-carbonitrile 4n
Yield: 66%; m.p.: 254–2578C; IR (KBr): 3000, 2218, 1651, 1517,
1467, 1346, 1244, 1009, 1004, 997, 856, 779 cm– 1 1H-NMR
;
(400 MHz, DMSO-d6) d: 4.60 (s, 2H, CH2), 7.51–7.53 (d, J = 8.64 Hz,
2H, ArH), 7.60–7.62 (d, J = 8.76 Hz, 2H, ArH), 7.89–7.91 (d, J =
8.64 Hz, 2H, ArH), 8.13–8.15 (d, J = 8.72 Hz, 2H, ArH), 12.88 (br,
1H, NH); MS (CI) m/z: 399 [M + H+]. Anal. calcd. for C18H11ClN4O3S:
C, 54.21; H, 2.78; N, 14.05. Found: C, 54.18; H, 2.85; N, 14.01.
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2009, 342, 710–715
Synthesis of Cyano Pyrimidinones
715
cassette, analyzed using a Typhoon 8610 Variable Mode Imager
(Amersham Biosciences), and quantitated using Image Quant
5.2. Percent inhibition (% I) was calculated using the following
equation:
[7] J. d'Angelo, J. F. Mouscadet, D. Desmaele, F. Zouhiri, H.
Leh, Pathol. Biol. 2001, 49, 237–246.
[8] N. Neamati, Expert Opin. Inv. Drug 2001, 10, 281–296.
[9] N. J. Anthony, Curr. Top. Med. Chem. 2004, 4, 979–990.
% I = 1006[1 – (D – C)/(N – C)]
(1)
[10] Y. Pommier, A. A. Johnson, C. Marchand, Nat. Rev. Drug Dis-
cov. 2005, 4, 236–248.
where C, N, and D are the fractions of 21-mer substrate con-
verted to 19-mer (39-processing product) or strand transfer prod-
ucts for DNA alone, DNA plus IN, and IN plus drug, respectively.
The IC50 values were determined by plotting the logarithm of
drug concentration versus percent inhibition to obtain concen-
tration that produced 50% inhibition.
[11] A. Engelman, K. Mizuuchi, R. Craigie, Cell 1991, 67, 1211–
1221.
[12] P. O. Brown, Integration, Cold Spring Harbor Press, Cold
Spring Harbor, NY, USA, 1999.
[13] E. Asante-Appiah, A. M. Skalka, Adv. Virus Res. 1999, 52,
351–369.
Cell culture
[14] S. Dubey, Y. D. Satyanaraya, H. Lavania, Eur. J. Med. Chem.
2007, 42, 1159–1168.
Human prostate cancer cells were obtained from the American
Type Cell Culture (ATCC). Cells were maintained as monolayer
cultures in RPMI 1640 media supplemented with 10% fetal
bovine serum (Gemini-Bioproducts, Woodland, CA, USA) and
2 mM L-Glutamine at 378C in a humidified atmosphere of 5%
CO2. To remove the adherent cells from the flask for passaging
and counting, cells were washed with phosphate buffered saline
(PBS) without calcium or magnesium, incubated with a small
volume of 0.25% trypsin-EDTA solution (Sigma) for 5–10 min,
and washed with culture medium and centrifuged. All experi-
ments were performed using cells in exponential cell growth.
[15] T. S. Yoshinaga, T. Fujishita, T. Fujiwara, Presented at the
9th Conference on “Retroviruses and Opportunistic Infec-
tions”, Seattle, WA, 2002.
[16] A. Billich, Curr. Opin. Invest. Drugs 2003, 4, 206–209.
[17] S. Young, Presented at the “XIV International AIDS Confer-
ence”, West Point, PA, 2002.
[18] D. J. Hazuda, S. D. Young, J. P. Guare, N. J. Anthony, et al.,
Science 2004, 305, 528–532.
[19] A. A. Johnson, C. Marchand, Y. Pommier, Curr. Top Med.
Chem. 2004, 4, 671–686.
Stock solution
[20] M. Witvrouw, B. van Maele, J. Vercammen, A. Hantson, et
al., Curr. Drug. Metab. 2004, 5, 291–304.
A 10 mM stock solution of all compounds were prepared in
dimethylsulfoxide (DMSO) and stored at 208C. Further dilutions
were freshly made in PBS.
[21] V. Summa, A. Petrocchi, V. G. Matassa, M. Taliani, et al., J.
Med. Chem. 2004, 47, 5336–5339; U. Koch, B. Attenni, S.
Malancona, S. Colarusso, et al., J. Med. Chem. 2006, 49,
1693–1707.
Cytotoxicity assay
Cytotoxicity was assessed by MTT assay as previously described
[33]. Briefly, cells were seeded in 96-well microtiter plates and
allowed to attach. Cells were subsequently treated with a contin-
uous exposure to the corresponding drug for 72 h. An MTT solu-
tion (at a final concentration of 0.5 mg/mL) was added to each
well and cells were incubated for 4 h at 378C. After removal of
the media, DMSO was added and the absorbance was read at
570 nm. All assays were performed in triplicate. The 50% inhibi-
tory concentration (IC50) was then determined for each drug
from a plot of log (drug concentration) versus percentage of cell
kill.
[22] P. Pace, M. E. Di Francesco, C. Gardelli, S. Harper, et al., J.
Med. Chem. 2007, 50, 2225–2239.
[23] E. Muraglia, O. D. Kinzel, C. Gardelli, B. Crescenzi, et al., J.
Med. Chem. 2008, 51, 861–874.
[24] M. R. Rico Ferreira, G. Cecere, P. Pace, V. Summa, Tetrahe-
dron Lett. 2009, 50, 148–151.
[25] A. Mazumder, A. Gazit, A. Levitzki, M. Nicklaus, et al., Bio-
chemistry 1995, 34, 15111–15122.
[26] H. Hongbin Yuan, A. L. Parrill, Bioorg. Med. Chem. 2002, 10,
4169–4183.
[27] T. R. Burke Jr., M. R. Fesen, A. Mazumder, J. Wang, et al., J.
Med. Chem. 1995, 38, 4171–4178.
References
[28] C. M. Farnet, R. Lipford, B. Wang, F. D. Bushman, Proc. Natl.
Acad. Sci. U. S. A. 1996, 93, 9742–9747.
[1] M. Hogberg, I. Morrison, Expert Opin. Ther. Pat. 2000, 10,
1189–1199; H. Jonckheere, J. Anne, E. De Clercq, Med. Res.
Rev. 2000, 20, 129–154.
[29] C. M. Farnet, B. Wang, M. Hansen, J. R. Lipford, et al., Anti-
microb. Agents Chemother. 1998, 42, 2245–2253.
[2] F. Lebon, M. Ledecq, Curr. Med. Chem. 2000, 7, 455–477; J.
P. Vacca, J. H. Condra, Drug Discov. Today 1997, 2, 261–272.
[30] V. J. Ram, D. A. Vanden Berghe, A. J. Vlietinck, J. Heterocyclic
Chem. 1984, 21, 1307–1312.
[3] F. J. Palella, K. M. Delaney, A. C. Moorman, M. O. Loveless,
[31] N. Agarwal, P. Srivastava, S. K. Raghuwanshi, D. N. Upad-
et al., N. Engl. J. Med. 1998, 338, 853–860.
hyay, et al., Bioorg. Med. Chem. 2002, 10, 869–874.
[4] J. Cohen, Science 2002, 296, 2320–2324.
[32] C. Marchand, N. Neamati, Y. Pommier, Methods Enzymol.
2001, 340, 624–633.
[5] E. De Clercq, Nat. Rev. Drug Discov. 2002, 1, 13–25.
[6] N. Neamati, C. Marchand, Y. Pommier, Adv. Pharmacol.
2000, 49, 147–165.
[33] J. Carmichael, W. G. DeGraff, A. F. Gazdar, J. D. Minna, J. B.
Mitchell, Cancer Res. 1987, 47, 936–942.
i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com