Design and discovery of 5-hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamide inhibitors of HIV-1 integrase

Article abstract of DOI:10.1016/j.bmc.2014.07.036

Raltegravir (RAL) is a first clinically approved integrase (IN) inhibitor for the treatment of HIV but rapid mutation of the virus has led to chemo-resistant strains. Therefore, there is a medical need to develop new IN inhibitors to overcome drug resista

Full text of DOI:10.1016/j.bmc.2014.07.036

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and discovery of 5-hydroxy-6-oxo-1,
6-dihydropyrimidine-4-carboxamide inhibitors of HIV-1 integrase
Daoguang Zhang a, , Bikash Debnath b, , Shenghui Yu a, Tino Wilson Sanchez c, Frauke Christ d, Yang Liu a,
Zeger Debyser d, Nouri Neamati b, , Guisen Zhao a,
⇑
⇑
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shangdong
250012, China
b Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA
c Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA 90089, USA
d Laboratory for Molecular Virology and Gene Therapy, Division of Molecular Medicine, KU Leuven (KULeuven), Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Raltegravir (RAL) is a first clinically approved integrase (IN) inhibitor for the treatment of HIV but rapid
mutation of the virus has led to chemo-resistant strains. Therefore, there is a medical need to develop
new IN inhibitors to overcome drug resistance. At present, several IN inhibitors are in different phases
of clinical trials and few have been discontinued due to toxicity and lack of efficacy. The development
of potent second-generation IN inhibitors with improved safety profiles is key for selecting new clinical
candidates. In this article, we report the design and synthesis of potent 5-hydroxy-6-oxo-1,6-dihydropy-
rimidine-4-carboxamide analogues as second-generation IN inhibitors. These compounds satisfy two
structural requirements known for potent inhibition of HIV-1 IN catalysis: a metal chelating moiety
and a hydrophobic functionality necessary for selectivity against the strand transfer reaction. Most of
the new compounds described herein are potent and selective for the strand transfer reaction and show
antiviral activity in cell-based assays. Furthermore, this class of compounds are drug-like and suitable for
further optimization and preclinical studies.
Received 17 May 2014
Revised 20 July 2014
Accepted 22 July 2014
Available online xxxx
Keywords:
HIV-1 integrase inhibitors
INSTIs
5-Hydroxy-6-oxo-1,6-dihydropyrimidine
analogues
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
stages.6,7 Unfortunately, continuous mutation of the viral genome
leads to multi-drug resistant viral strains that are no longer sus-
Reverse transcriptase, integrase (IN), and protease are the only
viral enzymes responsible for HIV-replication. IN plays a pivotal
role in the integration of the viral genome into the host genome
enabling HIV to efficiently propagate in human CD4+ cells. Because
of the lack of a cellular homologue and its essential role in viral
replication, IN remains a promising target for antiretroviral-based
therapy.1–3 Many efforts have been made to develop antiretroviral
drugs targeting IN and Raltegravir (RAL) was the first FDA
approved IN inhibitor.4,5 Currently, more than thirty drugs have
been approved for treating HIV infection targeting alternate
ceptible to current therapy including RAL.8–10 Therefore, develop-
ment of second-generation inhibitors have been the norm since
the discovery of the first approved drug, AZT, nearly 30 years
ago.11 Currently, two other IN inhibitors (Elvitegravir and Dolute-
gravir) have been approved for clinical use and several other inhib-
itors targeting IN catalysis are in clinical trials.12 Efforts are
ongoing to develop allosteric inhibitors targeting IN dimerization
and LEDGF/p75-IN interaction.13–17
HIV-1 IN is a 32 kDa polynucleotidyl transferase comprising
three domains: the N-terminal domain, the C-terminal domain,
and the catalytic domain. The catalytic domain contains a DDE
motif (D64, D116, and E152) that forms metal chelating interac-
tions with one or two divalent metal ions, such as Mn2+ and
Mg2+. IN catalyzes the insertion of reverse transcribed viral DNA
into the host cell’s chromosomes in two steps: (a) 30-processing,
the excision of two terminal nucleotides leaving 30-hydroxyl
ends of the viral DNA, and (b) strand transfer, insertion of the
30-hydroxyl ends onto the host DNA by a nucleophilic addition.18–20
Abbreviations: CPE, cytopathic effect; LEDGF/p75, lens epithelium-derived
growth factor; DMAD, dimethylacetylenedicarboxylate; IN, integrase; PK, pharma-
cokinetics; PD, pharmacodynamics; RAL, raltegravir; ELV, elvitegravir.
⇑
Corresponding authors. Tel.: +1 734 647 2732; fax: +1 734 763 8152 (N.N.); tel./
fax: +86 531 8838 2009 (G.Z.).
E-mail addresses: neamati@med.umich.edu (N. Neamati), guisenzhao@sdu.edu.
cn (G. Zhao).
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmc.2014.07.036
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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2
D. Zhang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Over the past decade, extensive efforts were made to develop IN
followed by cyclization with xylene at reflux affording the desired
intermediate 4. Finally, the compounds from the 6 series were syn-
thesized by the ammonolysis reaction of methyl ester 4 with substi-
tuted benzylamine (5) as the nucleophilic reagent in a DMF solution.
inhibitors with diverse chemical features, such as oligonucleotides,
peptides, hydroxylated aromatics, hydrazides, quinoline deriva-
tives, and quinolones.21–29 In many cases the development of these
inhibitors stalled due to undesired pharmacokinetics and/or toxic-
ity. In addition to the success of RAL, several IN strand transfer
inhibitors (INSTIs) are in different stages of clinical development
(Fig. 1). Elvitegravir (ELV) and Dolutegravir were approved by the
US FDA more recently and MK-2048 is in Phase II clinical trials,
while MK-0536 and PICA are in pre-clinical stages.30,31
Cross-resistance of INSTIs has also been observed. For example,
clinical isolates and recombinant molecular clones selected by RAL
showed similar resistance to ELV. For instance, an E92Q/N155H IN
double mutant selected by RAL and ELV reduces inhibition efficacy
between 100–300 fold. Subsequent secondary mutations further
diminish inhibitory activity while second-generation inhibitors
are showing improved efficacy against RAL and ELV mutagenic
pathways.32–34 Taken together, new second-generation INSTIs with
metal chelating groups and substituted phenyl rings such as the
compounds discussed here are needed to further investigate the
structure-activity relationships amongst this promising class of
IN inhibitors.
2.2. Inhibition of HIV-1 integrase
The inhibition of HIV-1 IN catalytic activity was determined as
previously described.37 The 6 series of compounds that had para
halogen substitutions in the phenyl ring of the carboxamide side
showed potent activity and selectivity for strand transfer reaction
(6-a1, IC50 = 0.6 lM; 6-a2, IC50 = 0.8 lM, 6-a3, IC50 = 0.5 lM). The
presence of a phenyl halogen flanking metal chelating groups has
been identified as an important component of several INSTIs in
clinical trials. However, 6–a5 had methyl groups and still retained
potency (IC50 = 0.5 lM) suggesting the scaffold can also tolerate
alternate groups (Table 1).
The remaining compounds screened in this series were less
potent IN inhibitors. Lengthening the distance between the carbox-
amide and the phenyl moiety with an additional carbon decreased
potency against strand transfer seven-fold (6-a7, IC50 = 4 lM)
while 30-processing was unaffected. When the fluorine was chan-
ged to the 2 or 3 position of the phenyl, the inhibitory activity
decreased depending on the position. Substitution of the phenyl
halogen with a hydroxy or methoxy group also affected strand
2. Results and discussion
transfer inhibition (6-a6, IC50 = 2 lM; 6-a9, IC50 = 6 lM) while
non-substituted phenyl rings (6-b1) led to an eight-fold reduction
in inhibition compared to the para-fluorinated analogue.
Previously, Summa et al. reported on the synthesis, optimiza-
tion, and PK properties of a series of 4,5-dihydroxypyrimidine car-
boxamides and N-alkyl-5-hydroxypyrimidinone carboxamides.35,36
On the basis of these reports, other studies leading to the develop-
ment of RAL5 and a number of other IN inhibitors in different stages
of clinical development, we designed a series of 5-hydroxy-6-
oxo-1,6-dihydropyrimide-4-carboxamide analogues including
metal chelating groups and substituted phenyl rings.
The 6 series of compounds followed a similar pattern of activity
as RAL and other INSTIs in clinical trials. The overall scaffold of
these compounds had the two basic moieties indicative of the
INSTI class, a metal-chelating group flanked by a fluorinated
phenyl. The distance between the fluorine and the nearest metal
chelating member in all of the INSTIs in clinical trials remained
at seven atoms that helps explaining the altered activity observed
in compounds with halogens in the 2 or 3 position of phenyl ring.
The scaffold also included a third ring opposite of the fluorinated
phenyl linking the carboxamide. The addition of a methylene linker
between the phenyl and pyrimidine ring (6-c1), opposite of the
carboxamide, resulted in nearly a 20-fold reduction in strand
2.1. Chemistry
The general synthetic route to target compounds 6 is depicted in
Scheme l. The raw materials 1 were obtained from commercial
sources (TCI, Tokyo kasei kogyo co., ltd). The key intermediates, ami-
doximes 2, were synthesized from the corresponding nitrile 1 with
hydroxylamine hydrochloride and potassium carbonate in an
alcoholic solution. The synthesis of the key core methyl 5-hydroxy-
6-oxo-1,6-dihydropyrimidine-4-carboxylate was achieved by
conversion of the amidoximes 2 with DMAD into intermediate 3,
transfer inhibition (IC50 = 10 lM).
The in vitro data suggest that the pyrimidines in this series dis-
played selectivity for the strand transfer reaction. Like other INSTIs,
the more active compounds had a halogen in the para position,
Figure 1. IN inhibitors in different stages of clinical development.
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D. Zhang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
Scheme 1. Reagents and conditions: (a) K2CO3, C2H5OH, room temp, 30 min, then reflux for 12 h; (b) DMAD, anhyd CH3OH, room temp for 12 h; (c) xylene, room temp for 2 h,
90 °C for 2 h, then reflux for 24 h; (d) anhyd DMF, reflux for 14 h.
Table 1
profile in MT-4 cells (therapeutic index, TI). Although the in vitro
activities of the para-halogenated compounds were equally potent,
the antiviral profile of the compounds with chlorine and bromine in
the para position had a significant decrease in inhibition and thus a
Inhibition of HIV-1 integrase catalytic activities by select 5-hydroxy-6-oxo-1,6-
dihydropyrimidine-4-carboxamides
O
OH
HN
n1
less favorable TI.
H
R1
R2
N
The dimethylated compound, 6-a5, had a slightly improved
antiviral inhibitory profile compared with a diflourinated ana-
logue, a pattern also observed against IN catalysis in vitro.
Although altering the 3-fluorine to chlorine of the difluorinated
analogue reduced inhibition in vitro, 6-a13 had the best antiviral
N
n2
O
Code
n1
n2
R1
R2
Inhibition of HIV-1 IN (IC50 lM)
30-Processing
Strand transfer
activity and the greatest therapeutic index (EC50 = 1.3
lM and
6-a1
6-a2
6-a3
6-a4
6-a5
6-a6
6-a7
6-a8
6-a9
6-a10
6-a11
6-a12
6-a13
6-a14
6-b1
6-b2
6-c1
6-d1
6-e1
6-f1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
4-F
H
4-F
4-Cl
12
9
6
0.6 0.2
0.8 0.2
0.5 0.1
7
CC50 = 45.5 M). Another compound 6-a8 had a chlorine in the 3
l
4-Br
3,4-F
3,4-CH3
4-OCH3
4-F
3-Cl
4-OH
3-F
11
17
16
16
17
20
>20
20
20
6
5
6
6
5
position and showed significant inhibition of HIV-1 replication
with minimal cytotoxicity in MT-4 cells, while the fluorinated ana-
logue did not exhibit the same potency in vivo. Altering the dis-
tance between the carboxamide carbonyl and the fluorinated
phenyl with an additional carbon (6-a7) failed to show anti-HIV
activity at 30 lM, reinforcing the notion that a fixed distance
between the metal chelating moiety and the phenyl fluorine is
essential for improved inhibition (Table 2).
1
0.4
0.5 0.04
2
4
0.3
1
1.4 0.9
6
3
7
3
0.1
4
1
2
8
2-F
2-F-3-Cl
4-F-3-Cl
3,4,5-F
H
4-F
4-F
4-F
4-F
4-F
4-F
13
6
1.4 0.1
4
4
2
14
>20
17
>20
5
16
3
5
8
8
0.1
5
H
1
10
2
2
1
0.1
2
0.4
0.1
0.1
Table 2
4-F
4-Cl
2-F
4-Br
3-F
Antiviral activities of 5-hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamides
6
Compound
Activity in MT-4 cells
Cytotoxicity
TIc
6-g1
1
HIV-1
EC50
(
l
M)a
CC50
(l
M)b
6-b1
6-a1
6-a2
6-a3
6-a4
6-a5
6-a6
6-a7
6-a8
6-a9
6-a10
6-a11
6-a12
6-a13
6-a14
5.65 1.19
1.72 1.22
8.1 2.2
11.03 0.73
5.17 0.45
2.7 0.29
7.61 0.07
>30
1.91 0.25
6.71 0.73
7.34 0.49
7.81 2.34
2.61 0.27
1.3
50.7
7
9
26
5
4
6
44 15
44
47.5 1.5
31
33.5 10.5
44
30
35
49
49.5 1.5
28.5 4.5
48
45.5 1.5
34.5 9.5
although a dimethylated phenyl displayed a comparable inhibitory
profile. This similarity in activity observed with the dimethylated
compound and the difluorinated analogue suggests this compound
should be explored further.
3
9
12
6
5
2.3. Lead compounds show antiviral activity in cell-based assays
9
5
18
7
7
To assess whether the inhibition of the in vitro enzymatic activ-
ities can be translated into phenotypic activity, compounds were
screened during HIV IIIB infection in MT-4 lymphocytes. The inhib-
itory effect of these compounds on the HIV-1-induced cytopathic
effect (CPE) was determined by the MTT assay.38 Table 2 lists antivi-
ral and cytotoxic activities of select compounds. Compound 6-a1
4
0
18
35
7
4.64 1.76
a
Effective concentration required to reduce HIV-1-induced cytopathic effect by
50% in MT-4 cells.
b
Cytotoxic concentration to reduce MT-4 cell viability by 50%.
Therapeutic index: ratio CC50/EC50
inhibited HIV-1 replication with an EC50 value of 1.72
lM and
c
.
showed a 26 fold therapeutic advantage compared to its cytotoxicity
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4
D. Zhang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 2. Binding poses for IN inhibitors (yellow colored). Inhibitors form metal chelation with Mg2+ (pink) along with key IN residues (cyan). (a) RAL; (b) 6-a1; (c) 6-a2; (d)
6-a3; (e) 6-a5.
2.4. Molecular docking study
4. Experimental section
4.1. Chemistry
Compounds 6-a1, 6-a2, 6-a3, and 6-a5 were docked on the
active site of IN to better understand their binding properties
(Fig. 2). The docking pose of RAL reveals a similar binding mecha-
nism with the exception of an aromatic ring absent in the 6 series
that comes in close contact with IN Y143, an important residue in
another mutagenic pathway resistant to RAL.39 The two carbonyl
oxygens and the hydroxylate ion of the dihydropyrimidine-4-
carboxamide moiety of these active compounds function to chelate
with two Mg2+ ions along with IN residues D64, D116, and E152.
The docking pose shows the dihydropyrimidine ring can form
pi–pi interactions with the viral nucleotide DA17. These docking
results also suggest the phenyl ring attached to the carboxamide
can form pi–pi stacking interactions with the viral nucleotide
DC16. Thus, the binding mechanism of these compounds likely
displaces the reactive DNA 30OH from the metal chelation site
and blocks the intasome activity.
Unless otherwise noted, all materials were obtained from com-
mercial suppliers and used without further purification. All melt-
ing points were determined in a Büchi capillary melting point
apparatus and are uncorrected. The proton nuclear magnetic reso-
nance (1H NMR) spectra were recorded with a Bruker Avance
DRX600 instrument with tetramethyl-silane (TMS) as the internal
standard. The chemical shifts (d) were reported in parts per million
(ppm) and were relative to the central peak of DMSO-d6. Coupling
constants (J) are given in Hz. and reported as follow: chemical
shift, multiplicity (s = singlet, d = doublet, t = triplet, br = broad,
m = multiplet), coupling constants, and number of protons. The
13C NMR spectra were recorded with a Bruker Avance DRX400
instrument. The chemical shifts (d) are reported in ppm relative
to the centerline of the septet at 40.0 ppm for DMSO-d6. The
high-resolution mass spectra data were obtained using an Accela
UPLC-LTQ Orbitrap Mass Spectrometer. Column chromatography
was carried out with silica gel in the solvents indicated. Thin-layer
chromatography (TLC) was performed on silica gel GF254 plates
(layer thickness, 0.2 mm) and compounds were visualized using
UV light. Petroleum ether used for TLC and column chromatogra-
phy had a boiling range of 60–90 °C. All tested compounds showed
more than 95% purity on the basis of HPLC analysis. The specific
method for HPLC analysis and a table of the data for all tested
compounds are presented in the Supporting information.
3. Conclusion
The present study focuses on the design and synthesis of cata-
lytic IN inhibitors based on RAL as well as other agents in clinical
trials. Basic structural frameworks of these designed compounds
include metal chelating groups and substituted phenyl rings likely
forming interactions with the IN active site residues and viral DNA
nucleotides. As expected, these 5-hydroxy-6-oxo-1,6-dihydropy-
rimidine-4-carboxamide analogs showed significant inhibition
and strand transfer selectivity by chelation of metal ions perhaps
by binding to key IN residues (D64, D116, and E152) at the
catalytic binding site. Molecular docking suggests the binding
mechanism could evade the Y143R/C mutagenic pathway that is
observed after long exposure to RAL. Analogues from this series
of INSTIs show antiviral activity without significant cytotoxicity
in HIV-1 infected cells. Further preclinical PK/PD studies as well
as lead optimization are warranted to select clinically viable
compound for development.
4.1.1. Synthesis of the compound 2 series
Hydroxylamine hydrochloride (200 mmol) and K2CO3
(200 mmol) were dissolved in 200 mL of alcohol and stirred for
30 min at room temperature. The corresponding nitriles (75 mmol)
1 were added and the reaction mixture was heated to reflux for
12 h. After filtration of inorganic salts, the solvent was evaporated
under reduced pressure. The products 2 were purified by column
chromatography (petroleum ether/acetone, 2:1, v/v).
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D. Zhang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
4.1.1.1. 4-Fluoro-N-hydroxybenzimidamide (2a).
White
(br, 1H), 10.28 (br, 1H), 7.32 (dd, J = 7.8 Hz, J = 6.0 Hz, 2H), 7.15
(t, J = 9.0 Hz, 2H), 3.82 (s, 3H), 3.80 (s, 2H).
powder, yield 66.5%, mp: 79.4–80.1 °C. 1H NMR (DMSO-d6) d
(ppm): 9.66 (s, 1H), 7.71 (m, 2H), 7.20 (m, 2H), 5.84 (s, 2H).
4.1.3.4. Methyl 2-(4-chlorophenyl)-5-hydroxy-6-oxo-1,6-dihy-
4.1.1.2. N-Hydroxybenzimidamide (2b).
White powder, yield
dropyrimidine-4-carboxylate (4d).
Pink powder, yield
65.8%, mp: 72.1–73.2 °C. 1H NMR (DMSO-d6) d (ppm): 9.62 (s, 1H),
43.2%, mp: 271.7–273.6 °C. 1H NMR (DMSO-d6) d (ppm): 13.17
(br, 1H), 10.58 (br, 1H), 8.03 (d, J = 8.4 Hz, 2H), 7.58 (dt,
J = 9.0 Hz, J = 2.4 Hz, 2H), 3.86 (s, 3H).
7.67(m, 2H), 7.36 (m, 3H), 5.80 (s, 2H).
4.1.1.3. 2-(4-Fluorophenyl)-N-hydroxyacetimidamide (2c). White
powder, yield 69.7%, mp: 79.3–81.2 °C. 1H NMR (DMSO-d6) d (ppm):
8.89 (s, 1H), 7.30(m, 2H), 7.10 (m, 2H), 5.41 (s, 2H), 3.24
(s, 2H).
4.1.3.5. Methyl 2-(2-fluorophenyl)-5-hydroxy-6-oxo-1,6-dihy-
dropyrimidine-4-carboxylate (4e).
Yellowish powder, yield
39.7%, mp: 235.1–236.7 °C. 13.14 (br, 1H), 10.63 (br, 1H), 7.65 (t,
J = 7.8 Hz, 1H), 7.59 (m, 1H), 7.35 (m, 2H), 3.83 (s, 3H).
4.1.1.4. 4-Chloro-N-hydroxybenzimidamide (2d). White powder,
yield 71.6%, mp: 124.3–126.4 °C. 1H NMR (DMSO-d6) d (ppm): 9.73
(s, 1H), 7.69 (dd, J = 6.6 Hz, J = 2.4 Hz, 2H), 7.44 (dd, J = 6.6 Hz,
J = 1.8 Hz, 2H), 5.87 (s, 2H).
4.1.3.6. Methyl 2-(4-bromophenyl)-5-hydroxy-6-oxo-1,6-dihy-
dropyrimidine-4-carboxylate (4f).
Yellowish powder, yield
44.5%, mp: 260.6–261.9 °C. 1H NMR (DMSO-d6) d (ppm): 13.18
(br, 1H), 10.61 (br, 1H), 7.95 (d, J = 9.0 Hz, 2H), 7.72 (d, J = 8.4 Hz,
2H), 3.85 (s, 3H).
4.1.1.5. 2-Fluoro-N-hydroxybenzimidamide (2e). Yellowish oil,
yield 65.4%. 1H NMR (DMSO-d6) d (ppm): 9.64 (s, 1H), 7.50 (t,
J = 7.2 Hz, 1H), 7.43 (m, 1H), 7.24 (m, 2H), 5.82 (s, 2H).
4.1.3.7. Methyl 2-(3-fluorophenyl)-5-hydroxy-6-oxo-1,6-dihy-
dropyrimidine-4-carboxylate (4g).
Yellowish powder, yield
4.1.1.6. 4-Bromo-N-hydroxybenzimidamide (2f). White powder,
yield 67.8%, mp: 142.9–144.5 °C. 1H NMR (DMSO-d6) d (ppm): 9.74
(s, 1H), 7.62 (dt, J = 8.4 Hz, J = 2.4 Hz, 2H), 7.57 (dt, J = 9.0 Hz,
J = 2.4 Hz, 2H), 5.87 (s, 2H).
39.7%, mp: 262.3–264.1 °C. 1H NMR (DMSO-d6) d (ppm): 13.19
(br, 1H), 10.64 (br, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.83
(d, J = 10.8 Hz, 1H), 7.56 (m, 1H), 7.38 (td, J = 8.4 Hz, J = 2.4 Hz,
1H), 3.88 (s, 3H).
4.1.1.7. 3-Fluoro-N-hydroxybenzimidamide (2g).
Yellowish
4.1.4. Synthesis of the compounds in the 6 series
powder, yield 67.3%, mp: 70.1–71.8 °C. 1H NMR (DMSO-d6) d
(ppm): 9.80 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.47 (dt, J = 9.6 Hz,
J = 1.2 Hz, 1H), 7.42 (m, 1H), 7.21 (td, J = 8.4 Hz, J = 2.4 Hz, 1H), 5.91
(s, 2H).
To a stirred solution of compounds 4 (5 mmol) in N,N-dimethyl-
formamide (20 mL), substituted benzylamine (3 equiv) was added
and the resulting mixture was stirred at 90 °C for 12 h under a
nitrogen atmosphere. After the mixture was cooled at room
temperature, distilled water (150 mL) and 1 N HCl (15 mL) was
added. The resulting solid was collected by filtration and then
was re-crystallized from N,N-dimethylformamide/water to afford
compounds 6.
4.1.2. Synthesis of the compound 3 series
Compounds in series 2 (20 mmol) were dissolved in 50 mL of
distilled methanol under anhydrous conditions. 1.2 equiv of dim-
ethylacetylenedicarboxylate (24 mmol) was added drop-wise with
external cooling. The reaction mixture was stirred for 14 h at room
temperature and then concentrated under vacuum. Brown oil (3)
was obtained, which was used for the next step without further
purification.
4.1.4.1.
N-(4-Fluorobenzyl)-2-(4-fluorophenyl)-5-hydroxy-
(6-a1). White
6-oxo-1,6-dihydropyrimidine-4-carboxamide
powder, yield 94.2%, mp: 262.8–264.6 °C. 1H NMR (DMSO-d6) d
(ppm): 12.96 (br, 1H), 12.55 (br, 1H), 9.65 (s, 1H), 8.33 (dd,
J = 9.0 Hz, J = 6.0 Hz, 2H), 7.40 (dd, J = 9.0 Hz, J = 6.0 Hz, 2H), 7.35
(t, J = 9.0 Hz, 2H), 7.17 (t, J = 9.0 Hz, 2H), 4.52 (d, J = 6.6 Hz, 2H).
13C NMR (100 MHz, DMSO-d6) d (ppm): 42.00, 115.47, 115.68,
115.77, 115.99, 127.38, 128.75, 129.84, 129.92, 130.51, 130.60,
135.30, 135.33, 145.66, 148.41, 158.92, 160.55, 162.96, 163.01,
165.48, 169.16. HRMS (ESI) m/z calcd for C18H14F2N3O3 [M+H]+:
358.0998, found 358.1000.
4.1.3. Synthesis of the compound 4 series
An amount of 20 mmol of compounds 3 were dissolved in
100 mL of xylene, obtaining a brown solution. The brown solution
was stirred at room temperature for 2 h, 90 °C for 2 h, and then
refluxed for 24 h under a nitrogen atmosphere. The reaction mix-
ture was stirred at room temperature for 2 h to allow the precipi-
tation of the product as a light-brown solid. The resulting solid was
collected by filtration and then was washed with ether to afford
compounds 4.
4.1.4.2.
N-(4-Chlorobenzyl)-2-(4-fluorophenyl)-5-hydroxy-
Yellow-
6-oxo-1,6-dihydropyrimidine-4-carboxamide (6-a2).
ish powder, yield 92.6%, mp: 273.8–274.9 °C. 1H NMR (DMSO-d6) d
(ppm): 12.96 (br, 1H), 12.52 (br, 1H), 9.67 (t, J = 6.6 Hz, 1H), 8.34
(dd, J = 9.0 Hz, J = 6.0 Hz, 2H), 7.38 (m, 6H), 4.53 (d, J = 6.6 Hz, 2H).
13C NMR (100 MHz, DMSO-d6) d (ppm): 42.10, 115.75, 115.97,
127.50, 128.79, 129.71, 129.81, 130.51, 130.60, 132.07, 138.14,
145.84, 148.31, 158.96, 163.01, 165.48, 169.22. HRMS (ESI) m/z calcd
for C18H14ClFN3O3 [M+H]+: 374.0702, found 374.0709.
4.1.3.1. Methyl 2-(4-fluorophenyl)-5-hydroxy-6-oxo-1,6-dihydro-
pyrimidine-4-carboxylate (4a). Yellowish powder, yield 38.6%,
mp: 262.3–264.1 °C. 1H NMR (DMSO-d6) d (ppm): 13.11 (br, 1H),
10.54 (br, 1H), 8.07 (dd, J = 7.8 Hz, J = 6.0 Hz, 2H), 7.35 (t,
J = 8.4 Hz, 2H), 3.85 (s, 3H).
4.1.3.2. Methyl 5-hydroxy-6-oxo-2-phenyl-1,6-dihydropyrimi-
dine-4-carboxylate (4b).
Pink powder, yield 46.4%, mp:
4.1.4.3.
N-(4-Bromobenzyl)-2-(4-fluorophenyl)-5-hydroxy-6-
Yellow-
243.4–245.1 °C. 1H NMR (DMSO-d6) d (ppm): 13.11 (br, 1H),
10.52 (br, 1H), 8.06 (d, J = 6.0 Hz, 2H), 7.53 (m, 3H), 3.88 (s, 3H).
oxo-1,6-dihydropyrimidine-4-carboxamide (6-a3).
ish powder, yield 90.3%, mp: 269.4–271.5 °C. 1H NMR (DMSO-d6)
d (ppm): 12.98 (br, 1H), 12.51 (br, 1H), 9.67 (s, 1H), 8.32 (dd,
J = 7.8 Hz, J = 5.4 Hz, 2H), 7.54 (d, J = 9.0 Hz, 2H), 7.34 (m, 4H),
4.51 (d, J = 6.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) d (ppm):
42.15, 115.77, 115.98, 120.52, 127.38, 128.74, 130.07, 130.52,
4.1.3.3. Methyl 2-(4-fluorobenzyl)-5-hydroxy-6-oxo-1,6-dihy-
dropyrimidine-4-carboxylate (4c).
Gray powder, yield
41.3%, mp: 261.3–263.4 °C. 1H NMR (DMSO-d6) d (ppm): 12.95
Please cite this article in press as: Zhang, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.036

6
D. Zhang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
130.60, 131.71, 138.57, 145.71, 148.39, 158.92, 163.01, 165.49,
4.1.4.9. N-(4-Hydroxybenzyl)-2-(4-fluorophenyl)-5-hydroxy-6-
169.22. HRMS (ESI) m/z calcd for
C
18H14BrFN3O3 [M+H]+:
oxo-1,6-dihydropyrimidine-4-carboxamide (6-a9).
Brown
418.0197, found 418.0201.
powder, yield 92.4%, mp: 281.3–282.7 °C. 1H NMR (DMSO-d6) d
(ppm): 12.95 (br, 1H), 12.70 (br, 1H), 9.54 (s, 1H), 9.33 (s, 1H),
8.32 (dd, J = 8.4 Hz, J = 5.4 Hz, 2H), 7.34 (t, J = 9.0 Hz, 2H), 7.17 (d,
J = 8.4 Hz, 2H), 6.72 (d, J = 9.0 Hz, 2H), 4.42 (d, J = 6.0 Hz, 2H). 13C
4.1.4.4. N-(3,4-Difluorobenzyl)-2-(4-fluorophenyl)-5-hydroxy-6-
oxo-1,6-dihydropyrimidine-4-carboxamide (6-a4).
Yellow-
ish powder, yield 93.3%, mp: 278.2–279.6 °C. 1H NMR (DMSO-d6)
d (ppm): 12.98 (br, 1H), 12.43 (br, 1H), 9.60 (s, 1H), 8.33 (dd,
J = 8.4 Hz, J = 6.0 Hz, 2H), 7.45 (dd, J = 15.0 Hz, J = 8.4 Hz, 1H), 7.36
(t, J = 9.0 Hz, 2H), 7.25 (td, J = 8.4 Hz, J = 2.4 Hz, 1H), 7.09 (td,
J = 8.4 Hz, J = 2.4 Hz, 1H), 4.56 (d, J = 6.0 Hz, 2H). 13C NMR
NMR (100 MHz, DMSO-d6) d (ppm): 42.23, 115.57, 115.75,
115.96, 127.43, 128.72, 129.26, 130.52, 130.61, 145.62, 148.44,
156.93, 158.90, 162.76, 163.00, 165.47, 168.93. HRMS (ESI) m/z
calcd for C18H15FN3O4 [M+H]+: 356.1041, found 356.1044.
(100 MHz, DMSO-d6)
d
(ppm): 36.06, 36.10, 103.90, 104.16,
4.1.4.10. N-(3-Fluorobenzyl)-2-(4-fluorophenyl)-5-hydroxy-6-
oxo-1,6-dihydropyrimidine-4-carboxamide (6-a10). Yellowish
powder, yield 93.3%, mp: 286.1–288.1 °C. 1H NMR (DMSO-d6) d
(ppm): 12.92 (br, 1H), 12.51 (br, 1H), 9.68 (s, 1H), 8.34 (dd,
J = 9.0 Hz, J = 6.0 Hz, 2H), 7.26 (dd, J = 13.8 Hz, J = 7.2 Hz, 1H), 7.35
(t, J = 8.4 Hz, 2H), 7.19 (m, 2H), 7.10 (m, 1H), 4.55 (d, J = 6.0 Hz, 2H).
13C NMR (100 MHz, DMSO-d6) d (ppm): 42.23, 114.15, 114.36,
114.41,114.62, 115.78,115.99, 123.77, 123.80, 127.49, 128.80,
130.51, 130.60, 130.78, 130.86, 142.04, 142.11, 145.74, 148.36,
158.99, 161.46, 163.01, 163.88, 165.48, 169.27. HRMS (ESI) m/z
calcd for C18H14F2N3O3 [M+H]+: 358.0998, found 358.1001.
104.41, 111.78, 111.82, 111.99, 112.03, 115.77, 115.99, 122.00,
122.04, 122.15, 122.19, 127.31, 128.69, 130.52, 130.60, 131.14,
131.20, 131.24, 131.30, 145.72, 148.38, 158.91, 159.05, 159.18,
160.64, 160.76, 161.51, 161.64, 163.01, 163.08, 163.20, 165.49,
169.29. HRMS (ESI) m/z calcd for
376.0904, found 376.0906.
C
18H13F3N3O3 [M+H]+:
4.1.4.5. N-(3,4-Dimethylbenzyl)-2-(4-fluorophenyl)-5-hydroxy-
6-oxo-1,6-dihydropyrimidine-4-carboxamide (6-a5). Yel-
lowish powder, yield 94.1%, mp: 252.1–254.3 °C. 1H NMR
(DMSO-d6) d (ppm): 12.95 (br, 1H), 12.65 (br, 1H), 9.58 (s, 1H),
8.33 (dd, J = 8.4 Hz, J = 5.4 Hz, 2H), 7.34 (t, J = 9.0 Hz, 2H), 7.08
(m, 3H), 4.45 (d, J = 6.6 Hz, 2H), 2.20 (s, 3H), 2.18 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) d (ppm): 19.45, 19.84, 42.47, 115.75,
115.97, 125.33, 127.61, 128.81, 129.07, 129.89, 130.52, 130.61,
135.26, 136.38, 136.46, 145.71, 148.36, 158.97, 163.00, 165.47,
169.03. HRMS (ESI) m/z calcd for C20H19FN3O3 [M+H]+: 368.1405,
found 368.1408.
4.1.4.11. N-(2-Fluorobenzyl)-2-(4-fluorophenyl)-5-hydroxy-6-
oxo-1,6-dihydropyrimidine-4-carboxamide (6-a11). Yellowish
powder, yield 92.6%, mp: 264.3–265.7 °C. 1H NMR (DMSO-d6) d
(ppm): 12.98 (br, 1H), 12.47 (br, 1H), 9.61 (s, 1H), 8.34 (dd,
J = 8.4 Hz, J = 6.0 Hz, 2H), 7.35 (m, 4H), 7.20 (m, 2H), 7.19 (m,
2H), 4.59 (d, J = 6.6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) d
(ppm): 36.44, 36.49, 115.50, 115.71, 115.77, 115.98, 124.87,
124.91, 125.62, 125.76, 127.38, 128.72, 129.48, 129.56, 129.79,
129.84, 130.53, 130.62, 145.72, 148.38, 158.90, 159.16, 161.59,
163.02, 165.49, 169.30. HRMS (ESI) m/z calcd for C18H14F2N3O3
[M+H]+: 358.0998, found 358.1001.
4.1.4.6.
N-(4-Methoxybenzyl)-2-(4-fluorophenyl)-5-hydroxy-
6-oxo-1,6-dihydropyrimidine-4-carboxamide (6-a6).
Brown
powder, yield 93.2%, mp: 244.6–246.3 °C. 1H NMR (DMSO-d6) d
(ppm): 12.95 (br, 1H), 12.65 (br, 1H), 9.58 (s, 1H), 8.33 (dd,
J = 7.8 Hz, J = 5.4 Hz, 2H), 7.35 (t, J = 9.0 Hz, 2H), 7.29 (d,
J = 8.4 Hz, 2H), 6.90 (d, J = 8.4 Hz, 2H), 4.46 (d, J = 6.6 Hz, 2H),
4.1.4.12.
hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamide
a12).
Brown powder, yield 87.9%, mp: 289.3–291.8 °C. 1H
N-(3-Chloro-2-fluorobenzyl)-2-(4-fluorophenyl)-5-
(6-
3.73 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
d (ppm): 42.15,
55.53, 114.25, 115.75, 115.97, 127.44, 128.73, 129.27, 130.51,
130.60, 131.06, 145.65, 148.45, 158.87, 163.00, 165.48, 169.01.
HRMS (ESI) m/z calcd for C19H17FN3O4 [M+H]+: 370.1198, found
370.1199.
NMR (DMSO-d6) d (ppm): 12.99 (br, 1H), 12.38 (br, 1H), 9.65 (s,
1H), 8.33 (m, 2H), 7.52 (d, J = 7.2 Hz, 1H), 7.36 (m, 3H), 7.22 (t,
J = 8.4 Hz, 1H), 4.62 (d, J = 6.6 Hz, 2H). 13C NMR (100 MHz, DMSO-
d6) d (ppm): 36.58, 36.63, 115.76, 115.98, 119.91, 120.09, 125.75,
125.80, 127.31, 127.79, 127.93, 128.77, 128.81, 129.79, 130.53,
130.61, 145.79, 148.37, 154.30, 156.76, 158.90, 163.03, 165.50,
4.1.4.7. N-(4-Fluorophenethyl)-2-(4-fluorophenyl)-5-hydroxy-6-
oxo-1,6-dihydropyrimidine-4-carboxamide (6-a7). Yellowish
powder, yield 89.7%, mp: 239.5–241.3 °C. 1H NMR (DMSO-d6) d
(ppm): 12.94 (br, 1H), 12.65 (br, 1H), 9.11 (s, 1H), 8.29 (dd,
J = 9.0 Hz, J = 5.4 Hz, 2H), 7.36 (t, J = 9.0 Hz, 2H), 7.31 (dd, J = 9.0 Hz,
J = 5.4 Hz, 2H), 7.14 (t, J = 9.0 Hz, 2H), 3.55 (dd, J = 15.0 Hz,
J = 6.0 Hz, 2H), 2.90 (t, J = 7.2 Hz, 2H). 13C NMR (100 MHz,
DMSO-d6) d (ppm): 34.45, 40.73, 115.48, 115.69, 115.76, 115.98,
127.40, 128.79, 130.44, 130.53, 130.83, 130.91, 135.56, 135.59,
145.67, 148.21, 158.94, 160.19, 162.60, 163.01, 165.48, 168.91.
HRMS (ESI) m/z calcd for C19H16F2N3O3 [M+H]+: 372.1154, found
372.1156.
169.35. HRMS (ESI) m/z calcd for
392.0608, found 392.0612.
C
18H13ClF2N3O3 [M+H]+:
4.1.4.13.
hydroxy-6-oxo-1,6-dihydropyrimidine-4-carboxamide
a13).
Yellowish powder, yield 90.8%, mp: 270.9–272.7 °C. 1H
N-(3-Chloro-4-fluorobenzyl)-2-(4-fluorophenyl)-5-
(6-
NMR (DMSO-d6) d (ppm): 12.98 (br, 1H), 12.44 (br, 1H), 9.66 (s,
1H), 8.33 (m, 2H), 7.57 (d, J = 6.6 Hz, 1H), 7.38 (m, 4H), 4.52 (d,
J = 6.6 Hz, 2H). HRMS (ESI) m/z calcd for C18H13ClF2N3O3 [M+H]+:
392.0608, found 392.0612.
4.1.4.14. N-(3,4,5-Trifluorobenzyl)-2-(4-fluorophenyl)-5-hydroxy-
6-oxo-1,6-dihydropyrimidine-4-carboxamide (6-a14). Yellowish
powder, yield 90.3%, mp: 268.7–270.9 °C. 1H NMR (DMSO-d6) d
(ppm): 12.99 (br, 1H), 12.35 (br, 1H), 9.65 (s, 1H), 8.33 (dd,
J = 7.8 Hz, J = 5.4 Hz, 2H), 7.36 (t, J = 9.0 Hz, 2H), 7.31 (t, J = 7.8 Hz,
2H), 4.52 (d, J = 6.6 Hz, 2H). HRMS (ESI) m/z calcd for C18H12F4N3O3
[M+H]+: 394.0809, found 394.0814.
4.1.4.8.
N-(3-Chlorobenzyl)-2-(4-fluorophenyl)-5-hydroxy-6-
oxo-1,6-dihydropyrimidine-4-carboxamide (6-a8). Yellowish
powder, yield 90.9%, mp: 296.2–297.3 °C. 1H NMR (DMSO-d6) d
(ppm): 12.97 (br, 1H), 12.46 (br, 1H), 9.67 (s, 1H), 8.33
(dd, J = 8.4 Hz, J = 5.4 Hz, 2H), 7.37 (m, 6H), 4.54 (d, J = 6.6 Hz,
2H). 13C NMR (100 MHz, DMSO-d6) d (ppm): 42.20, 115.78, 116.00,
126.55, 127.46, 127.66, 128.72, 130.51, 130.60, 130.77, 133.47,
141.70, 145.69, 148.42, 158.95, 163.01, 165.49, 169.26. HRMS
4.1.4.15.
rimidine-4-carboxamide (6-b1).
94.5%, mp: 251.4–252.5 °C. 1H NMR (DMSO-d6) d (ppm): 12.95
N-Benzyl-5-hydroxy-6-oxo-2-phenyl-1,6-dihydropy-
(ESI) m/z calcd for
374.0705.
C
18H14ClFN3O3 [M+H]+: 374.0702, found
White powder, yield
Please cite this article in press as: Zhang, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.036

D. Zhang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
(br, 1H), 12.61 (br, 1H), 9.63 (s, 1H), 8.25 (d, J = 7.2 Hz, 2H), 7.51
4.3. Preparation of oligonucleotide substrate
(m, 3H), 7.35 (m, 4H), 7.27 (m, 1H), 4.54 (d, J = 6.0 Hz, 2H).
HRMS (ESI) m/z calcd for C18H16N3O3 [M+H]+: 322.1186, found
322.1191.
The HIV-1 IN catalytic activity assay uses a 210mer top strand:
(50-GTGTGGAAAATCTCTAGCAGT-30), and a 210mer bottom strand:
(50-ACTGCTAGAGATTTTCCACAC-30). The top strand was labeled at
4.1.4.16.
6-dihydropyrimidine-4-carboxamide (6-b2).
der, yield 92.6%, mp: 248.7–250.3 °C. 1H NMR (DMSO-d6)
N-(4-Fluorobenzyl)-5-hydroxy-6-oxo-2-phenyl-1,
the 50end with 32P]-ATP by T4 polynucleotide kinase (Epicenter,
c [
White pow-
Madison, WI). The mixture was then incubated at 95 °C for 15 min
to inactivate the kinase and the bottom strand was added in 1.5
molar excess. The strands were allowed to anneal by cooling the
mixture slowly to room temperature. Any unincorporated material
was subsequently removed by centrifuging the mixture through a
Spin-25 mini-column (USA Scientific, Ocala, FL).
d
(ppm): 12.94 (br, 1H), 12.56 (br, 1H), 9.62 (t, J = 6.0 Hz, 1H), 8.25
(d, J = 7.8 Hz, 2H), 7.52 (m, 3H), 7.40 (dd, J = 8.4 Hz, J = 6.0 Hz,
2H), 7.17 (m, 2H), 4.52 (d, J = 6.0 Hz, 2H). HRMS (ESI) m/z calcd
for C18H15FN3O3 [M+H]+: 340.1092, found 340.1098.
4.1.4.17. N,2-Bis(4-fluorobenzyl)-5-hydroxy-6-oxo-1,6-dihydro-
4.4. Integrase catalytic activity assay
pyrimidine-4-carboxamide (6-c1).
White powder, yield
87.2%, mp: 220.5–222.2 °C. 1H NMR (DMSO-d6) d (ppm): 12.84
(br, 1H), 12.35 (br, 1H), 9.37 (t, J = 6.0 Hz, 1H), 7.39 (m, 4H), 7.16
(m, 4H), 4.46 (d, J = 6.0 Hz, 2H), 3.81 (s, 2H). HRMS (ESI) m/z calcd
for C19H16F2N3O3 [M+H]+: 372.1154, found 372.1160.
The extent of 30-processing and strand transfer was analyzed by
pre-incubating recombinant wild-type HIV-1 IN, at a final concen-
tration of 200 nM, with the inhibitor in reaction buffer (50 mM
NaCl, 1 mM HEPES, pH 7.5, 50 lM EDTA, 50 lM dithiothreitol,
10% glycerol (w/v), 7.5 mM MnCl2, 0.1 mg/mL bovine serum albu-
min, 10 mM 2-mercaptoethanol, 10% DMSO, and 25 mM MOPS, pH
7.2) at 30 °C for 30 min. Then 20 nM of the 32P 50end-labeled linear
210mer substrate was added, and incubation was continued for an
additional 1 h. Reactions were then quenched by the addition of an
4.1.4.18. N-(4-Fluorobenzyl)-2-(4-chlorophenyl)-5-hydroxy-6-
oxo-1,6-dihydropyrimidine-4-carboxamide (6-d1).
White
powder, yield 89.9%, mp: 290.7–292.2 °C. 1H NMR (DMSO-d6) d
(ppm): 13.00 (br, 1H), 12.61 (br, 1H), 9.67 (s, 1H), 8.29
(d, J = 8.4 Hz, 2H), 7.58 (d, J = 8.4 Hz, 2H), 7.40 (dd, J = 9.0 Hz,
J = 6.0 Hz, 2H), 7.17 (t, J = 9.0 Hz, 2H), 4.52 (d, J = 6.6 Hz, 2H). HRMS
equal volume (16
10 mM EDTA, 0.025% xylene cyanol, and 0.025% bromophenol
blue). An aliquot (7 L) was electrophoresed on a denaturing 20%
lL) of loading dye (98% deionized formamide,
(ESI) m/z calcd for
C
18H14ClFN3O3 [M+H]+: 374.0702, found
l
374.0709.
polyacrylamide gel (0.09 M tris–borate pH 8.3, 2 mM EDTA, 20%
acrylamide, 8 M urea). Gels were dried, exposed in a PhosphorIm-
ager cassette, analyzed using a Typhoon 8610 Variable Mode Ima-
ger (Amersham Biosciences), and quantified using ImageQuant 5.2.
The percent inhibition (%I) was calculated using the following
equation:%I = 100*[1ꢀ(D ꢀ C)/(N ꢀ C)] where C, N, and D are the
fractions of 21-mer substrate converted to 19-mer (30-processing
product) or strand transfer products for DNA alone, DNA plus IN,
and IN plus drug, respectively. The IC50 values were determined
by plotting the logarithm of drug concentration against percent
inhibition of enzymatic activity to obtain the concentration that
produced 50% inhibition.
4.1.4.19. N-(4-Fluorobenzyl)-2-(2-fluorophenyl)-5-hydroxy-6-
oxo-1,6-dihydropyrimidine-4-carboxamide (6-e1).
White
powder, yield 93.4%, mp: 214.8–216.1 °C. 1H NMR (DMSO-d6) d
(ppm): 12.95 (br, 1H), 12.63 (br, 1H), 9.46 (t, J = 6.0 Hz, 1H), 7.81
(t, J = 7.2 Hz, 1H), 7.59 (dd, J = 13.8 Hz, J = 6.0 Hz, 1H), 7.36
(m, 4H), 7.16 (t, J = 9.0 Hz, 2H), 4.46 (d, J = 7.8 Hz, 2H). HRMS
(ESI) m/z calcd for
358.1003.
C
18H14F2N3O3 [M+H]+: 358.0998, found
4.1.4.20. N-(4-Fluorobenzyl)-2-(4-bromophenyl)-5-hydroxy-6-
oxo-1,6-dihydropyrimidine-4-carboxamide (6-f1).
White
powder, yield 90.6%, mp: 295.6–297.2 °C. 1H NMR (DMSO-d6) d
(ppm): 13.00 (br, 1H), 12.61 (br, 1H), 9.66 (s, 1H), 8.21 (d,
J = 8.4 Hz, 2H), 7.71 (d, J = 8.4 Hz, 2H), 7.40 (dd, J = 8.4 Hz,
J = 6.0 Hz, 2H), 7.17 (t, J = 9.0 Hz, 2H), 4.52 (d, J = 6.0 Hz, 2H). HRMS
4.5. In vitro anti-HIV and drug susceptibility assays
The inhibitory effect of antiviral drugs on the HIV-1-induced CPE
in human lymphocyte MT-4 cell culture was determined by the
MTT assay.38 This assay is based on the reduction of the yellow col-
ored 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) by mitochondrial dehydrogenase of metabolically
active cells to a blue formazan derivative, which can be measured
(ESI) m/z calcd for
C
18H14BrFN3O3 [M+H]+: 418.0197, found
418.0203.
4.1.4.21.
N-(4-Fluorobenzyl)-2-(3-fluorophenyl)-5-hydroxy-
White
6-oxo-1,6-dihydropyrimidine-4-carboxamide (6-g1).
spectrophotometrically. The 50% cell culture infective dose (CCID50
)
powder, yield 90.6%, mp: 277.3–279.4 °C. 1H NMR (DMSO-d6) d
(ppm): 13.01 (br, 1H), 12.65 (br, 1H), 9.70 (s, 1H), 8.22 (d,
J = 10.8 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 7.55 (dd, J = 15.0 Hz,
J = 7.2 Hz, 1H), 7.39 (m, 3H), 7.18 (t, J = 9.0 Hz, 2H), 4.53 (d,
J = 6.6 Hz, 2H). HRMS (ESI) m/z calcd for C18H14F2N3O3 [M+H]+:
358.0998, found 358.1004.
of the HIV-1 (IIIB) strain was determined by titration of the virus
stock using MT-4 cells. For the drug susceptibility assays, MT-4 cells
were infected with 100–300 CCID50 of the virus stock in the pres-
ence of 5-fold serial dilutions of the antiviral drugs. The concentra-
tion of various compounds achieving 50% protection against the
CPE of the HIV strain, which is defined as the EC50, was determined.
In parallel, the 50% cytotoxic concentration (CC50) was determined.
4.2. Biological materials, chemicals and enzymes
4.6. Molecular docking
All compounds were dissolved in DMSO and the 10 mM stock
solutions were stored at ꢀ20 °C. Further dilutions were also per-
formed in DMSO. The expression system used in purifying IN
was a kind gift from Dr. Robert Craigie, Laboratory of Molecular
Biology, NIDDK, NIH, Bethesda, MD. The oligonucleotides used in
the HIV-1 IN catalytic activity assay were synthesized at the USC
Molecular docking studies were performed using GOLD (genetic
optimization for ligand docking) software package, version 4.0
(Cambridge Crystallographic Data Centre, Cambridge, U.K.) in the
catalytic binding site of a homology model of HIV-1 integrase
reported earlier.37 GOLD uses a genetic algorithm to explore the
conformational space of a compound inside the binding site of a
protein.40,41 The active site was defined as the collection of protein
Norris Cancer Center microsequencing core facility. The
ATP was purchased from Perkin-Elmer (Waltham, MA).
c [
32P]-
Please cite this article in press as: Zhang, D.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.07.036

8
D. Zhang et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
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residues with a sphere of 15 Å radius. Before docking different pos-
sible stereomers, ionized forms and conformations of ligands are
prepared by LigPrep (Schrodinger, LLC). Ionization option, epik
was used to ionize ligands at pH 7.0 0.2.42,43 Docking studies
were performed using the standard default settings with 100
genetic algorithm (GA) runs on each molecule. For each of the
100 independent GA runs, a maximum of 100,000 operations were
performed on a set of 5 groups with a population of 100 individu-
als. With respect to ligand flexibility special care has been taken by
including options such as flipping of ring corners, amides, pyrami-
dal nitrogens, secondary and tertiary amines, and rotation of
carboxylate groups, as well as torsion angle distribution and post-
process rotatable bonds as default. The annealing parameters were
used as default cutoff values of 3.0 Å for H-bonds and 4.0 Å for van
der Waals interactions. Hydrophobic fitting points were calculated
to facilitate the correct starting orientation of the compound for
docking by placing the hydrophobic atoms appropriately in the
corresponding areas of the active site. When the top three solu-
tions attained root-mean-square deviation (rmsd) values within
1.5 Å, docking was terminated. GOLD-Score, a scoring function
within the software, is a dimensionless fitness value that takes into
account the intra- and intermolecular H-bonding interaction
energy, van der Waals energy, and ligand torsion energy.40,41
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Acknowledgments
26. Sato, M.; Motomura, T.; Aramaki, H.; Matsuda, T.; Yamashita, M.; Ito, Y.;
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The work in GZ laboratory was supported by a grant from the
National Natural Science Foundation of China (Grant Numbers:
20872082 and 21272140). The work in NN and ZD laboratories
were supported by funds from an NIH/NIAID (R21 AI081610) grant.
TWS was funded by the NIH F31 (AI082942-01) and CHRP (D08-
USC-321) awards.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.07.036.
33. Shimura, K.; Kodama, E.; Sakagami, Y.; Matsuzaki, Y.; Watanabe, W.; Yamataka,
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