Design and synthesis of N-methylpyrimidone derivatives as HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.12.059

A series of novel β-diketo derivatives which combined the virtues of dihydroxypyrimidine carboxamide derived from the evolution of DKA and polyhydroxylated aromatics moieties, were designed and synthesized as potential HIV-1 integrase (IN) inhibitors and

Full text of DOI:10.1016/j.bmc.2014.12.059

Accepted Manuscript
Design and synthesis of N-methylpyrimidone derivatives as HIV-1 integrase
inhibitors
Yujie Wang, Jie Rong, Bin Zhang, Liming Hu, Xiaoli Wang, Chengchu Zeng
PII:
S0968-0896(14)00917-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.12.059
BMC 11991
Reference:
Bioorganic & Medicinal Chemistry
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20 October 2014
25 December 2014
26 December 2014
Please cite this article as: Wang, Y., Rong, J., Zhang, B., Hu, L., Wang, X., Zeng, C., Design and synthesis of N-
methylpyrimidone derivatives as HIV-1 integrase inhibitors, Bioorganic & Medicinal Chemistry (2015), doi: http://
dx.doi.org/10.1016/j.bmc.2014.12.059
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Design and synthesis of N-methylpyrimidone
derivatives as HIV-1 integrase inhibitors
Leave this area blank for abstract info.
Yujie Wanga, Jie Ronga, Bin Zhanga, Liming Hua,*, Xiaoli Wanga, Chengchu Zenga
College of Life Science and Bioengineering, Beijing University of Technology, Beijing, P.R.China.
0

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Design and synthesis of N-methylpyrimidone derivatives as HIV-1 integrase
inhibitors
Yujie Wang, Jie Rong, Bin Zhang, Liming Hu*, Xiaoli Wang, Chengchu Zeng
College of Life Science and Bioengineering, Beijing University of Technology, Beijing, P. R. China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of novel β-diketo derivatives which combined the virtues of dihydroxypyrimidine
carboxamide derived from the evolution of DKA and polyhydroxylated aromatics moieties,
were designed and synthesized as potential HIV-1 integrase (IN) inhibitors and evaluated their
inhibition to the strand transfer process of HIV-1 integrase and anti-HIV-1 activity. The result
indicates that 3,4,5-trihydroxylated aromatic derivatives exhibit good inhibition to HIV-1
integrase, but dihydroxylated aromatic derivatives appear little inhibition to HIV-1 integrase. In
addition, the preliminary structure-activity relationship (SAR) of these new derivatives was
rationalized by docking studies.
Available online
Keywords:
HIV-1 integrase
Inhibitors
N-methylpyrimidone
Synthesis
2014 Elsevier Ltd. All rights reserved.
1. Introduction
The viral enzyme integrase (IN), which mediates integration
of the viral cDNA into the host genome by catalytic reactions-
first catalyzes removal of the terminal dinucleotide from each 3’-
end of the viral DNA (3’-processing) and subsequently medicates
joining of the 3’-end of the viral DNA to the host DNA (strand
transfer) during the viral replication cycle1,2. Because of lack of a
cellular homologue and its essential role in viral replication, it is
an attractive target for the development of new anti-HIV-1
inhibitors with high selectivity and low toxicity3,4. In the past two
decades, IN-targeted antiviral research has led to the
identification of various inhibitor types, of which the most
prominent scaffolds feature is a diketo acid (DKA) structure or
its heterocyclic bioisosteres5-9. The dihydroxypyrimidine carbox-
amide derived from the evolution of DKA is a potent, reversible,
and selective HIV-1 integrase strand transfer inhibitor10.
Extensive research on these chemotypes culminated with
raltegravir11,12 (Fig. 1), it is the first drug approved for clinical
use target to HIV integrase. The pharmacophoric center 5-
hydroxypyrimidone carboxamide could bind to divalent metal
ions (Mg2+ or Mn2+) to block the strand transfer (ST) in the HIV-
1 integrase catalytic site. Unfortunately, continuous mutation of
viral genome leads to multi-drug resistant viral strains that are no
Figure 1. Structure of Raltegravire (MK-0518)
too toxic to be pursued as therapeutic IN inhibitors16,17. In order
to study possible interaction between the two main classes of
inhibitors (polyphenols and DKAs), a kind of catechol or
dicaffeoyltartaric-DKA hybrids have been designed and tested as
IN inhibitors following the goals of refining the pharmacophore,
enhancing antiviral potency and improving cellular membranes
permeability18,19
. The majority of the hybrid compounds
exhibited submicromolar potency against strand transfer and
modest antiviral activities, thus, there is a clear need to optimize
the structure of compounds into the highly potent inhibitory
activity derivatives to HIV integrase. Despite the high interest
generated by these compounds, little number of the compounds
results in a lake of structure-activity relationships information for
the class of inhibitors.
In our previous studies, a series of polyhydroxylated aromatics
were designed and synthesized as potential HIV-1 integrase
inhibitors and evaluated their inhibitory activity to the strand
transfer process of HIV-1 integrase17,20. On the basis of the
consideration as well as on our interest in development of new
anti-IN agents, we planned to explore the effect of
polyhydroxylated aromatics-dihydroxypyrimidine hybrids to gain
new insight in the fundamental structural requirements for anti-
longer susceptible to current therapy including raltegravir13-15
.
The polyhydroxylated aromatics were also the first HIV
integrase inhibitor identified, but the early polyhydroxylated
derivatives were later demonstrated to inhibit viral entry or to be
———
* Corresponding author. Tel.: +86-10-67396211; fax: +86-10-67396211; e-mail: huliming@bjut.edu.cn(L. Hu)
1

bonyl)-piperazin-1-yl)alkyl)benzamide is shown in Scheme 1.
The synthesis started with the commercial available polymethoxy
benzoic acid 1, which reacted with thionyl chloride, to give
benzoyl chloride 2 in excellent yield. It was treated with 3-
chloropropylamine or 2-chloroethyamine to give benzamide 3.
Direct mono-amination of 3 with piperazine in chloroform obtain
intermediate 4 in a good yield.
Figure 2. Structure of designed integrase inhibitors
IN activity. Keeping in mind the pharmacophoric groups of
hydroxyl-pyrimidone carboxamide and polyhydroxylated
aromatics, adjusting length of carbon chain of linker between two
pharmacophoric group, we designed and synthesized compound
3,4,5-polyhydroxy-N-(3-(4-(5-methoxy-1-methyl-2-(4-substitute-
dbenzyl)-6-oxo-1,6-dihydropyrimidine-4-carbonyl)-piperazin-1-
yl)alkyl)benzamide(Fig.2).
Furthermore, the commercially available phenylacetonitrile 5
firstly conversed to the corresponding amidoxime 6, and
followed by reaction with dimethyl acetylenedicarboxylate and
cyclisation to desire pyrimidine methyl ester 7. The ester 8 was
prepared from 7 via K2CO3-promoted alkylation. Hydrolysis in
standard conditions afforded the required acid 9, which could
then be progressed to amides 10, 11 and 12 with variable amine 4
via pre-activation with CDI. Compounds 13, 14 and 15 were
obtained from compounds 10, 11 and 12 by an exhaustive
demethylation using boron tribromide in 50% yield.
2. Results and discussion
2.1. Chemistry
Preparation of 3,4,5-polyhydroxy-N-(3-(4-(5-methoxy-1-met-
hyl-2-(4-substitutedbenzyl)-6-oxo-1,6-dihydropyrimidine-4-car-
O
O
O
O
O
O
O
O
O
O
OH
N
H
Cl
n
N
H
N
n
Cl
a
b
c
NH
O
O
R3
R3
3
R3
R3
2
4
1
O
N
R1
R2
OH
R1
R2
OH
R1
f
d
HN
7
e
N
O
CN
NH2
H2N
O
6
5
O
O
N
R1
R2
O
O
R1
R2
O
h
g
N
N
O
OH
N
O
8
9
O
O
O
N
O
O
O
OH
OH
N
nN
H
N
nN
H
R1
R2
O
R1
R2
O
i
N
N
N
N
R3
R4
N
O
O
10a-f n=3, R3=OMe;
11a-d n=2, R3=OMe,
12a-d n=3, R3=H
13a-f n=3, R4=OH;
14a-d n=2, R4=OH,
15a-d n=3, R4=H
a R1=H, R2=H; b R1=Cl, R2=H; c R1=F, R2=H; d R1=OMe, R2=H; e R1=Me, R2=H; f R1=Cl, R2=H
.
Scheme 1. Reagant and conditions: (a) SOCl2, reflux; (b) Cl(CH2)nNH2 HCl, Et3N, DCM, rt; (c) piperazine, K2CO3, KI, CHCl3, reflux; (d) NH2OH.HCl, KOH,
reflux; (e) DMAD, CHCl3, reflux; then xylene, reflux; (f) CH3I, K2CO3, DMF, rt; (g) NaOH, H2O/MeOH, rt; then 1N HCl(aq), rt; (h) 4, CDI, DMF, rt; (i) 1N
BBr3, DCM, then MeOH
2.2. Inhibition of HIV IN activity
Table 1 Inhibition of HIV-1 integrase strand transfer catalytic
activitiesa
Compounds R1
R2
H
H
H
H
Cl
H
H
H
H
H
H
R4
n
3
3
3
3
3
2
2
2
3
3
3
IC50b(µM)
0.9
The inhibition effects of pyrimidone carboxamides 13-15 were
measured by HIV-1 integrase strand transfer (ST) activity assay.
Compounds 13-15 and reference compound raltegravir were
tested in vitro for strand transfer in the enzyme inhibition assay,
which was carried out as described previously and data were
summarized in Table 1. 3,4-dihydroxylated aromatics derivatives
15 show less activity towards strand transfer, but 3,4,5-
trihydroxylated aromatics derivatives 13 and 14 exhibit activities
at micromolar concentrations. In comparing the potencies of 13,
14 and 15 in the strand transfer assay, it was seen that
introduction of a hydroxyl at 5-position of the caffeoyl in 15 gave
13 to improve IN inhibition activities. The results indicate the
significance of polyhydroxyphenyl as a core pharmacophore.
However, difference in the potencies between the compounds 13
and 14 is very low, which shows that the length of alkyl in linker
domain isn’t notable on their activities. In addition, in order to
investigate the substituent effect on the phenyl ring in
hydrophobic domain, different groups were utilized. But these
compounds display significant potent inhibitory activities without
any marked fluctuation. The HIV-IN inhibitory effect of
compounds 13 and 14 are nearly as much as the reference
compound raltegravir.
13a
H
OH
OH
OH
OH
OH
OH
OH
OH
H
13b
Cl
F
2.4
13c
18.9
3.5
13e
Me
Cl
H
13f
22.4
18.6
2.5
14a
14c
F
14d
OH
H
0.7
15a
>100
>100
>100
1.8
15c
F
H
15d
OH
H
MK-0518
aHIV-1 IN inhibitory activities were measured according to the procedure
described in Ref.18.
bInhibition of strand transfer.
2

A
B
Figure 3. A, B show the binding model of 14d and 15c respectively. Mg2+ ions (green sphere), oxygen atom (red stick), nitrogen atom (blue stick), H-bond
(green dash line), metal bond (grey dash line), pi-pi stack (purple dash line).
2.3. Anti-HIV-1 activities
Mg2+ ions and the benzene can form pi-pi stacking interactions
with viral nucleotide DA17. In addition, the fourth hydroxyl in it
can also bind to Asp367, Asn368 and Arg326 of integrase
through H-bond. Howere, in 15c there can only one hydroxyl
which can function to Mg2+ as a metal acceptor and the benzyl
just form pi-pi T-shaped stacking interactions with DA17.
Moreover, the difference of AutoDock energy and AutoDock
interaction energy of 14d and 15c can also well explain the
difference of inhibiting activity.
MAGI test, also called single life cycle, reflects only one
round of infection. The test compound was added at different
hours post the virus inoculation to observe which stage of the
viral life cycle could be inhibited by the compound. The cells
used were derived from HeLa cell line that both expresses high
levels of CD4 and contains a single integrated copy of a β-
galactosidase gene under the control of HIV-1 LTR. This cell
line, called TZM-bl, can be used to determine quantitatively the
titer of HIV wild type strains or HIV pseudoviruses. The
inhibitory rate of the test compound could be calculated and the
stage upon which the test compound acted could be determined.
Compounds 13a, 13b, 13c and 13e were evaluated for their
antiviral activity against HIV-1 replication in TZM-bl cells.
TZM-bl cells were infected with HIV-1 and subsequently treated
with increasing concentrations of drugs. The amount of virus was
assayed by β-galactosidase assay, with HeLa-CD4-β-gal cells as
reporting cells. Antiviral properties are report in Table 2.
3. Conclusion
In summary, a series of dihydropyrimidine carboxamides
derivatives were facile synthesized and have been identified as
HIV-1 integrase inhibitors. The biological results showed that the
polyhydroxylated aromatic moiety plays an important role to
inhibit HIV-1 integrase ST reaction. In particular, 3,4,5-
trihydroxylated aromatics subunit is the most potential
compounds. And further work based on these structures is in
progress.
Table 2 Antiviral activities of compounds 13a, 13b, 13c and
13ea
4. Experimental section
Compounds
EC50(µM)
1.13
4.1. Chemistry
13a
Unless otherwise noted, all materials were obtained from
commercial suppliers and dried and purified by standard
procedures. Melting points were determined on a Beijing Keyi
elec-opti instrument factory melting point apparatus. Proton
nuclear magnetic resonance (1H NMR) spectra were recorded
with a Bruker Avance DRX400 spectrometer with CDCl3,
DMSO-d6 or MeOH-d4 as the solvent and tetramethyl-silane
(TMS) as the internal standard. The chemical shifts were reported
in δ (ppm). Mass spectra (MS) data were obtained using Esquire
6000 Mass Spectrometer. Petroleum ether used for column
chromatography had a boiling range of 60-90oC. Compounds 6
and 7, as well as compounds 2, 3, and 4 were prepared according
13b
4.27
13c
13e
4.39
2.76
a Effictive concentration 50%
2.4. Docking Studies
To investigate the possible bonding mechanism of these
compounds, an active compound and an inactive compound (14d
and 15c) were selected for docking studies. The binding mode of
selected molecule was studied by Autodock 4.0 with the help of
AutodockTools. The crystallographic structure of prototype
foamy virus integrase (PFV-IN) with a double chains DNA and
two magnesium ions (PDB:3OYA) was selected as the receptor
for docking study21, since it has been demonstrated that PFV-IN
is a good model for HIV-1 integrase core domain22.
to the corresponding literature procedures12,17
.
4.1.1. General procedure for the synthesis of dihydropyrimidine-
4-carboxylate derivatives 8.
To a suspension of 5,6-dihydroxypyridine-4-carboxylate 7
(3.0mmol) in DMF (50mL) was added K2CO3 (9.0mmol) and
CH3I (9.0mmol) and the mixture was stirred overnight at room
temperature. The mixture was extracted with EtOAc, and washed
with H2O and brine. The organic layer was separated, dried
(Na2SO4), filtered, and concentrated by rotary evaporation. The
residue was purified by column chromatography on silica gel
eluting with petroleum ether/ether acetate (5:1).
In order to validate the docking protocol, the extracted ligand
raltegravir (RAL, from 3OYA) was also re-docked into active
site of the crystallographic structure of 3OYA. The best docking
pose of RAL can higher superimposed with experimental
crystallographic RAL with a calculated RMSD of 1.69.
The docking results are shown (Fig.3), the three hydroxylate
ions of trihydroxybenzene in 14d function to chelate with two
3

4.1.1.1. Methyl 2-benzyl-5-methoxy-1-methyl-6-oxo-1,6-dihydro
pyrimidine-4-carboxylate (8a).
7.07 (t, J = 8.4 Hz, 2H), 7.19(dd, J = 5.2 Hz and 8.4 Hz, 2H). MS
(ESI): m/z 292.7 [M+H]+.
1
Brown oil, yield: 30.0%; H NMR (400 MHz, CDCl3, δ ppm)
4.1.2.4. 2-(4-methoxybenzyl)-5-methoxy-1-methyl-6-oxo-1,6-dih-
ydropyrimidine-4-carboxylic acid (9d).
δ: 3.36 (s, 3H), 3.92 (s, 3H), 3.95 (s, 3H), 4.13 (s, 2H), 7.15 (d, J
= 7.2 Hz, 2H), 7.21 (m, 1H), 7.28 (t, J=6.8, 2H). MS (ESI): m/z
288.9 [M+H]+.
o
1
Yellow solid; yield: 96.3%; m.p. 133-134 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 3.47 (s, 3H), 3.79 (s, 3H), 4.09 (s, 3H),
4.10 (s, 2H), 6.87 (dd, J = 2.0 Hz and 6.8 Hz, 2H), 7.09 (d, J =
8.8 Hz, 2H).
4.1.1.2. Methyl 2-(4-chlorobenzyl)-5-methoxy-1-methyl-6-oxo-1,
6-dihydropyrimidine-4-carboxylate (8b).
Brown oil; yield: 31.2%; 1H NMR (400 MHz, CDCl3, δ ppm)
δ: 3.42 (s, 3H), 3.98 (s, 3H), 4.01 (s, 3H), 4.15 (s, 2H), 7.15 (d, J
= 8.4 Hz, 2H), 7.32 (d, J = 6.4 Hz, 2H). MS (ESI): m/z 322.9
[M+H]+.
4.1.2.5. 2-(4-methylbenzyl)-5-methoxy-1-methyl-6-oxo-1,6-dihy-
dropyrimidine-4-carboxylic acid (9e).
Yellow solid; yield: 94.0%; m.p. 162-164 oC; 1H NMR
(400MHz, CDCl3, δ ppm) δ: 3.49 (s, 3H), 4.15 (s, 3H), 4.16 (s,
2H), 7.16 (d, J = 8.4 Hz, 2H), 7.36(d, J = 8.0 Hz, 2H).
4.1.1.3. Methyl 2-(4-fluorobenzyl)-5-methoxy-1-methyl-6-oxo-1,6
- dihydropyrimidine-4-carboxylate (8c).
4.1.2.6. 2-(3,4-dichlorobenzyl)-5-methoxy-1-methyl-6-oxo-1,6-
dihydropyrimidine-4-carboxylic acid (9f).
Brown oil; yield: 31.0%; 1H NMR (400 MHz, CDCl3, δ ppm)
δ: 3.42 (s, 3H), 3.97 (s, 3H), 4.00 (s, 3H), 4.14 (s, 2H), 7.05-7.15
(m, 2H), 7.17-7.28 (m, 2H). MS (ESI): m/z 306.7 [M+H]+.
o
1
Yellow solid; yield: 95.2%; m.p. 180-182 C; H NMR (400
MHz, DMSO-d6, δ ppm) δ: 3.41 (s, 3H), 3.77 (s, 3H), 4.18 (s,
2H), 7.21(dd, J = 2.0 Hz and 8.4 Hz, 1H), 7.54 (s, 1H), 7.58 (d, J
= 8.4 Hz, 1H).
4.1.1.4. Methyl 2-(4-methoxybenzyl)-5-methoxy-1-methyl-6-oxo-
1,6-dihydropyrimidine-4-carboxylate (8d).
Brown oil; yield: 28.8%; 1H NMR (400 MHz, CDCl3, δ ppm)
δ: 3.40 (s, 3H), 3.78 (s, 3H), 3.96 (s, 3H), 3.98 (s, 3H), 4.10 (s,
2H), 6.85(d, J = 8.8 Hz, 2H), 7.13(d, J = 8.4 Hz, 2H).
4.1.3. General procedure for the synthesis of N-methylpyri-
midone derivatives 10, 11, 12
To suspension of CDI (2.38mmol) in dry DMF (10mL) was
4.1.1.5. Methyl 2-(4-methylbenzyl)-5-methoxy-1-methyl-6-oxo-1,6
-dihydropyrimidine-4-carboxylate (8e).
slowly added dihydropyrimidine-4-carboxylic acid derivatives 9
(1.83mmol) in dry DMF (25mL) at 0 C and the mixture was
o
stirred at room temperature for 1h, then compound 4 (1.83mL)
was added. The mixture was stirred overnight at room
temperature and the reaction was quenched by adding H2O. The
mixture was extracted with DCM and the organic layer was
washed with H2O, brine, and dried (Na2SO4). The solvent was
evaporated under reduced pressure and crude product was
purified by column chromatography using ethyl acetate/ethanol
(5:1).
1
Brown oil; yield: 29.6% H NMR (400 MHz, CDCl3, δ ppm)
δ: 2.33 (s, 3H), 3.48 (s, 3H), 3.98 (s, 3H), 4.00 (s, 3H), 4.14 (s,
2H), 7.08 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.0 Hz, 2H).
4.1.1.6. Methyl 2-(3,4-dichlorobenzyl)-5-methoxy-1-methyl-6-ox-
o-1,6-dihydropyrimidine-4-carboxylate (8f).
Brown oil; yield: 32.5%; 1H NMR (400 MHz, CDCl3, δ ppm)
δ: 3.42 (s, 3H), 3.98 (s, 3H), 4.01 (s, 3H), 4.15 (s, 2H), 7.04 (d, J
= 7.2 Hz, 1H), 7.30 (s, 1H), 7.40 (d, J = 8.0 Hz, 1H).
4.1.3.1. N-(3-(4-(2-benzyl-5-methoxy-1-methyl-6-oxo-1,6-dihydr-
opyrimidine-4-carbobyl)pinperazin-1-yl)propyl)-3,4,5-trimethox-
ybenzamide (10a).
4.1.2. General procedure for the synthesis of dihydropyrimidine-
4-carboxylic acid derivatives 9
o
1
Yellow solid; yield: 55.4%; m.p. 88-90 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 1.79-1.83 (m, 2H), 2.45 (t, J = 4.8 Hz,
2H), 2.54 (t, J = 6.8 Hz, 4H), 3.35 (t, J = 4.8 Hz, 2H), 3.42 (s,
3H), 3.55 (t, J = 6.0 Hz, 2H), 3.77 (t, J = 4.8 Hz, 2H), 3.87 (s,
3H), 3.89 (s, 6H), 3.94 (s, 3H), 4.12 (s, 2H), 7.00 (s, 2H), 7.10
(br, 1H), 7.17 (d, J = 6.8 Hz, 2H), 7.26-7.28 (m, 1H), 7.30-7.34
(m, 2H). MS (ESI): m/z 594.0 [M+H]+.
To
a suspension of N-methylpyrimidone derivatives 8
(10.4mmol) in MeOH (50mL) was added 0.5N KOH (41.6mmol)
at room temperature, the reaction mixture was acidified with 1N
HCl after stirred one hour and then extracted with EtOAc. The
organic layer was washed with H2O and brine, dried (Na2SO4),
filtered, and concentrated by rotary evaporation.
4.1.2.1. 2-benzyl-5-methoxy-1-methyl-6-oxo-1,6-dihydropyrimidi-
ne -4-carboxylic acid (9a).
4.1.3.2. N-(3-(4-(2-(4-chlorobenzyl)-5-methoxy-1-methyl-6-oxo-
1, 6-dihydropyrimidine-4-carbonyl)piperzin-1-yl)propyl)-3,4,5-
tri-methoxybenzamide (10b).
o
1
Yellow solid; yield: 95%; m.p. 184-186 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 3.47 (s, 3H), 4.14 (s, 3H), 4.16 (s, 2H),
7.18 (d, J=6.8 Hz, 2H), 7.30-7.38 (m, 3H). MS (ESI): m/z 274.7
[M+H]+.
White solid; yield: 53.0%; m.p. 85-88 oC; 1H NMR (400 MHz,
CDCl3, δ ppm) δ: 1.81-1.84 (m, 2H), 2.43 (t, J = 4.8 Hz, 2H),
2.54 (t, J = 6.4 Hz, 4H), 3.34 (t, J = 4.8 Hz, 2H), 3.44 (s, 3H),
3.55 (t, J = 6.0 Hz, 2H), 3.77 (t, J = 4.8 Hz, 2H), 3.87 (s, 3H),
3.89 (s, 6H), 3.94 (s, 3H), 4.08 (s, 2H), 7.03 (s, 2H), 7.13 (d, J =
8.8 Hz, 2H), 7.21 (br, 1H), 7.29-7.32 (m, 2H). MS (ESI): m/z
628.0 [M+H]+.
4.1.2.2. 2-(4-chlorobenzyl)-5-methoxy-1-methyl-6-oxo-1,6-dihyd-
ropyrimidine-4-carboxylic acid (9b).
1
Yellow solid; yield: 96.2%; m.p. 155-157 °C; H NMR (400
MHz, CDCl3, δ ppm) δ: 3.49 (s, 3H), 4.15 (s, 3H), 4.16 (s, 2H),
7.16 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H). MS (ESI): m/z
308.8 [M+H]+.
4.1.3.3. N-(3-(4-(2-(4-fluorobenzyl)-5-methoxy-1-methyl-6-oxo-1,
6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4,5-tri-
methoxybenzamide (10c).
4.1.2.3. 2-(4-fluorobenzyl)-5-methoxy-1-methyl-6-oxo-1,6-dihyd-
ropyrimidine-4-carboxylic acid (9c).
o
1
Yellow solid; yield: 57.4%; m.p. 90-91 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 1.77-1.80 (m, 2H), 2.40 (t, J = 4.8 Hz,
2H), 2.51 (t, J = 6.8 Hz, 4H), 3.34 (t, J = 4.8 Hz, 2H), 3.41 (s,
o
1
Yellow solid; yield: 93.1%; m.p. 137-138 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 3.50 (s, 3H), 4.15 (s, 3H), 4.15 (s, 2H),
4

3H), 3.51 (t, J = 6.4 Hz, 2H), 3.74 (t, J = 4.8 Hz, 2H), 3.84 (s,
3H), 3.86 (s, 6H), 3.91 (s, 3H), 4.06 (s, 2H), 6.99 (s, 2H), 7.01 (d,
J = 8.8 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 7.24 (br, 1H). MS
(ESI): m/z 612.0 [M+H]+.
41.38, 41.55, 46.66, 52.46, 52.92, 55.31, 56.40, 56.53, 60.30,
60.91, 104.52, 114.53, 125.96, 129.41, 130.03, 140.29, 141.10,
144.48, 153.24, 156.09, 158.97, 159.62, 164.18, 167.17. HRMS
(ESI): m/z 610.2877 calcd for C31H40N5O8 [M+H]+, found
610.2877.
4.1.3.4. N-(3-(4-(5-methoxy-1-methyl-2-(4-methylbenzyl)-6-oxo-
1, 6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4,5-
tri-methoxybenzamide (10e).
4.1.3.9. N-(3-(4-(2-benzyl-5-methoxy-1-methyl-6-oxo-1,6-dihydr-
opyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4,5-trimethoxy-
benzamide (12a).
o
1
Yellow solid; yield: 55.2%; m.p. 86-88 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 1.81-1.85 (m, 2H), 2.33 (s, 3H), 2.47 (t,
J = 4.8 Hz, 2H), 2.56 (t, J = 6.4 Hz, 4H), 3.37 (t, J = 4.8 Hz, 2H),
3.43 (s, 3H), 3.57 (t, J = 6.0 Hz, 2H), 3.79 (t, J = 4.8 Hz, 2H),
3.88 (s, 3H), 3.91 (s, 6H), 3.94 (s, 3H), 4.09 (s, 2H), 7.02 (s, 2H),
7.07 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 2H), 7.14 (br, 1H).
HRMS (ESI): m/z 608.3084 calcd for C32H42N5O8 [M+H]+, found
608.3097.
o
1
Yellow solid; yield: 54.5%; m.p. 77-80 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 1.77 (m, J = 6.0 Hz, 2H), 2.42 (t, J = 4.8
Hz, 2H), 2.49-2.53 (m, 4H), 3.32 (t, J = 4.8 Hz, 2H), 3.39 (s, 3H),
3.50 (q, J = 6.0 Hz, 2H), 3.74-3.75 (m, J = 4.8 Hz, 2H), 3.86(s,
3H), 3.87 (s, 3H), 3.91 (s, 3H), 4.09 (s, 2H), 6.81 (d, J = 8.4 Hz,
1H), 7.14 (d, J = 6.8 Hz, 2H), 7.22-7.30 (m, 3H), 7.28 (br, 1H),
7.43(s, 2H); HRMS (ESI): m/z 564.2822 calcd for C30H38N5O6
[M+H]+, found 564.2825.
4.1.3.5. N-(3-(4-(2-(3,4-dichlorobenzyl)-5-methoxy-1-methyl-6-o-
xo-1,6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4,
5-trimethoxybenzamide (10f).
4.1.3.10. N-(3-(4-(2-(4-fluorobenzyl)-5-methoxy-1-methyl-6-oxo-
1,6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4-di-
methoxybenzamide (12c).
o
1
Yellow solid; yield: 59.3%; m.p. 105-108 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 1.78-1.84 (m, 2H), 2.39 (t, J = 4.4 Hz,
2H), 2.53 (t, J = 6.0 Hz, 4H), 3.31 (t, J = 4.8 Hz, 2H), 3.46 (s,
3H), 3.55 (t, J = 6.0 Hz, 2H), 3.76 (t, J = 4.8 Hz, 2H), 3.87 (s,
3H), 3.92 (s, 6H), 3.95 (s, 3H), 4.05 (s, 2H), 7.00 (s, 2H), 7.04
(dd, J = 2.0 Hz and 8.4 Hz, 1H), 7.05(br, 1H), 7.27(s, 1H),
7.40(d, J = 8.4 Hz, 1H). 13C NMR (100 MHz, CDCl3, δ ppm) δ:
25.73, 31.22, 39.62, 40.83, 41.49, 46.59, 52.72, 53.21, 56.46,
57.04, 60.24, 60.89, 104.70, 128.00, 130.24, 130.52, 130.95,
131.82, 133.08, 134.40, 140.57, 141.11, 144.07, 153.20, 154.37,
159.35, 163.93, 167.11. HRMS (ESI): m/z 662.2148 calcd for
C31H38ClN5O7 [M+H]+, found 662.2147.
o
1
Yellow solid; yield: 65.3%; m.p. 80-81 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 1.81-1.86 (m, 2H), 2.46 (t, J = 4.4 Hz,
2H), 2.56-2.59 (m, 4H), 3.35 (t, J = 4.8 Hz, 2H), 3.45 (s, 3H),
3.57 (q, J = 6.4 Hz, 2H), 3.80-3.82 (m, 2H), 3.93 (s, 3H), 3.95 (s,
3H), 3.96 (s, 3H), 4.10 (s, 2H), 6.87 (d, J = 8.0 Hz, 1H), 7.13 (t, J
= 8.4 Hz, 2H), 7.16-7.18 (m, 2H), 7.19 (s, 1H), 7.37 (br, 1H),
7.48 (s, 1H). 13C NMR (100 MHz, CDCl3, δ ppm) δ: 25.33,
31.23, 39.86, 41.27, 41.53, 46.59, 52.80, 53.30, 56.01, 56.06,
57.47, 60.26, 110.20, 110.90, 115.93, 116.14, 118.92, 127.48,
129.79, 130.01, 140.37, 144.32, 149.07, 151.69, 155.44, 159.49,
160.86, 163.31, 164.03, 166.97. HRMS (ESI): m/z 582.2728
C30H37FN5O6, [M+H]+, found 582.2726.
4.1.3.6. N-(2-(4-(2-benzyl-5-methoxy-1-methyl-6-oxo-1,6-dihydr-
opyrimidine-4-carbonyl)piperazin-1-yl)ethyl)-3,4,5-trimethoxyb-
enzamide (11a).
4.1.3.11. N-(3-(4-(2-(4-methoxybenzyl)-5-methoxy-1-methyl-6-o-
xo-1,6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4-
dimethoxybenzamide (12d).
o
1
Yellow solid; yield: 62.1%; m.p. 92-94 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 2.51 (t, J = 4.8 Hz, 2H), 2.62 (t, J = 4.8
Hz, 2H), 2.66 (t, J = 6.0 Hz, 2H), 3.39 (t, J = 4.8 Hz, 2H), 3.44
(s, 3H), 3.57 (q, J = 6.4 Hz, 2H), 3.79-3.81 (m, 2H), 3.89 (s, 3H),
3.91 (s, 6H), 3.95 (s, 3H), 4.11 (s, 2H), 6.73 (br, 1H), 7.01 (s,
2H), 7.19 (d, J = 7.2 Hz, 2H), 7.26-7.28 (m, 1H), 7.33(d, J = 7.2
Hz, 2H). MS (ESI): m/z 580.1 [M+H]+.
o
1
Yellow solid; yield: 65.3%; m.p. 75-77 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 1.81 (m, J = 6.0 Hz, 2H), 2.48 (t, J = 4.4
Hz, 2H), 2.54-2.57 (m, 4H), 3.35 (t, J = 4.4 Hz, 2H), 3.42 (s, 3H),
3.55 (q, J = 6.4 Hz, 2H), 3.77 (s, 3H), 3.78-3.79 (m, J = 4.8 Hz,
2H), 3.91 (s, 6H), 3.91 (s, 3H), 4.05 (s, 2H), 6.85 (t, J = 8.4 Hz,
2H), 7.09 (d, J = 8.4 Hz, 2H), 7.24-7.26 (m, 2H), 7.39 (br, 1H),
7.46 (s, 1H). 13C NMR (100 MHz, CDCl3, δ ppm) δ: 25.23,
31.24, 39.99, 41.38, 41.51, 46.59, 52.84, 53.36, 55.30, 56.03,
56.07, 57.61, 60.28, 110.20,110.93, 114.53, 118.84, 125.94,
127.49, 129.40, 140.29, 144.45, 149.11, 151.71, 156.09, 158.97,
159.61, 164.14, 166.97. HRMS (ESI): m/z 594.2928 calcd for
C31H40N5O7, [M+H]+, found 594.2915.
4.1.3.7. N-(2-(4-(2-(4-fluorobenzyl)-5-methoxy-1-methyl-6-oxo-1,
6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)ethyl)-3,4,5-trim-
ethoxybenzamide (11c).
o
1
Yellow solid; yield: 65.7%; m.p. 92-94 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 2.51 (t, J = 4.8 Hz, 2H), 2.62 (t, J = 4.8
Hz, 2H), 2.66 (t, J = 6.0 Hz, 2H), 3.39 (t, J = 4.8 Hz, 2H), 3.43
(s, 3H), 3.57 (q, J = 6.4 Hz, 2H), 3.79-3.81 (m, 2H), 3.89 (s, 3H),
3.91(s, 6H), 3.95(s, 3H), 4.11(s, 2H), 6.73(br, 1H), 7.01(s, 2H),
7.19(d, J = 7.2 Hz, 2H), 7.26-7.28 (m, 1H), 7.33 (d, J = 7.2 Hz,
2H). HRMS (ESI): m/z 598.2677 calcd for C30H37FN5O7 [M+H]+,
found 598.2678.
4.1.4. General procedure for demethylation of N-methylpyrimi-
done derivative 13, 14, 15
To a suspension of N-methylpyrimidone derivatives 10, 11, 12
(0.76mmol) in dry DCM (20mL) was added dropwise to a
solution of BBr3 (10mL, 1N) in dry DCM at 0 oC and the mixture
was stirred overnight at room temperature, and then MeOH
4.1.3.8. N-(2-(4-(5-methoxy-2-(4-methoxybenzyl)-1-methyl-6-ox-
o-1,6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)ethyl)
benzamide (11d).
o
(30mL) was slowly added to above solution at 0 C and the
mixture was stirred continually for 1h. The solvent was
evaporated under reduced pressure and crude product was
purified by column chromatography using chloroform/methanol
(3:1).
o
1
Yellow solid; yield: 61.0%; m.p. 89-92 C; H NMR (400
MHz, CDCl3, δ ppm) δ: 2.52 (t, J = 4.4 Hz, 2H), 2.63 (t, J = 4.4
Hz, 2H), 2.67 (t, J = 6.0 Hz, 2H), 3.39 (t, J = 4.4 Hz, 2H), 3.45
(s, 3H), 3.57 (q, J = 6.4 Hz, 2H), 3.80 (s, 3H), 3.80-3.82 (m, 2H),
3.90 (s, 3H), 3.92 (s, 6H), 3.96 (s, 3H), 4.07 (s, 2H), 6.64 (br,
1H), 6.87 (d, J = 8.4 Hz, 2H), 7.01 (s, 2H), 7.11 (d, J = 8.4 Hz,
2H). 13C NMR (100 MHz, CDCl3, δ ppm) δ: 30.90, 31.24, 36.48,
4.1.4.1. N-(3-(4-(2-benzyl-5-methoxy-1-methyl-6-oxo-1,6-dihydro
pyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4,5-trihydroxy
benzamide (13a).
5

White solid; yield: 50.1%; m.p. 200.2-203.2 oC; 1H NMR (400
MHz, MeOH-d4, δ ppm) δ: 2.04-2.07 (m, 2H), 2.70-2.80 (m, 2H),
3.00-3.10 (m, 2H), 3.15-3.30 (m, 2H), 3.30-3.40 (m, 2H), 3.40-
3.60 (m, 4H), 3.52 (s, 3H), 3.90 (s, 3H), 4.20 (s, 2H), 6.90 (s,
2H), 7.17 (t, J = 7.2 Hz, 2H), 7.24-7.28 (m, 3H). MS (ESI): m/z
552.1 [M+H]+.
4.1.4.8. N-(2-(4-(2-(4-hydroxybenzyl)-5-methoxy-1-methyl-6-oxo-
1,6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)ethyl)-3,4,5-tri-
hydroxybenzamide (14d).
o
1
White solid; yield: 55.0%; m.p. 145-147 C; H NMR (400
MHz, DMSO-d6, δ ppm) δ: 2.80-3.00 (m, 2H), 3.00-3.20 (m,
2H), 3.30-3.50 (m, 8H), 3.29 (s, 3H), 3.80 (s, 3H), 4.01 (s, 2H),
6.71 (d, J = 8.4 Hz, 2H), 6.84 (s, 2H), 7.01 (d, J = 8.4 Hz, 2H).
MS (ESI): m/z 551.9 [M-H]-.
4.1.4.2. N-(3-(4-(2-(4-chlorobenzyl)-5-methoxy-1-methyl-6-oxo-
1, 6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4,5–
tr-ihydroxybenzamide (13b).
4.1.4.9. N-(3-(4-(2-benzyl-5-methoxy-1-methyl-6-oxo-1,6-dihydro
pyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4-dihydroxyben-
zamide (15a).
o
1
White solid; yield: 55.0%; m.p. 180-182 C; H NMR (400
MHz, DMSO-d6, δ ppm) δ: 1.55-1.75 (m, 2H), 2.10-2.45 (m,
4H), 3.10-3.30 (m, 4H), 3.30-3.40 (m, 2H), 3.50-3.70 (m, 2H),
3.43 (s, 3H), 3.77 (s, 3H), 4.16 (s, 2H), 6.81 (s, 2H), 7.26 (d, J =
8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 8.08 (br, 1H), 8.61 (s, 1H),
8.97 (s, 2H). MS (ESI): m/z 584.9 [M-H]-.
White solid; yield: 57.5%; m.p. 162-164 oC; 1H NMR
(400MHz, MeOH-d4, δ ppm) δ: 2.00-2.10 (m, 2H), 2.80-2.90 (m,
2H), 3.00-3.15 (m, 2H), 3.20-3.28 (m, 2H), 3.30-3.45 (m, 2H),
3.45-3.60 (m, 4H), 3.53 (s, 3H), 3.92 (s, 3H), 4.23 (s, 2H), 6.81
(d, J = 8.0 Hz, 1H), 7.25-7.30 (m, 4H), 7.30-7.37 (m, 3H); 13C
NMR (100 MHz, MeOH-d4, δ ppm) δ: 22.83, 25.27, 29.33,
30.62, 37.35, 40.10, 40.93, 45.24, 51.87, 52.37, 55.34, 59.44,
114.40, 114.51, 119.30, 125.40, 126.98, 128.50, 128.63, 134.75,
140.41, 143.38, 144.96, 148.87, 156.92, 159.90, 164.65, 169.04;
MS (ESI): m/z 536.2 [M+H]+.
4.1.4.3. N-(3-(4-(2-(4-fluorobenzyl)-5-methoxy-1-methyl-6-oxo-1,
6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4,5-tr-
ihydroxybenzamide (13c).
White solid; yield: 52.4%; m.p. 187-190 oC; 1H NMR
(400MHz, MeOH-d4, δ ppm) δ: 2.05-2.08 (m, 2H), 2.80-2.90 (m,
2H), 3.00-3.15 (m, 2H), 3.15-3.20 (m, 2H), 3.30-3.35 (m, 2H),
3.40-3.55 (m, 4H), 3.57 (s, 3H), 3.92 (s, 3H), 4.18 (s, 2H), 6.89
(s, 2H), 7.08 (t, J = 8.4 Hz, 2H), 7.27 (q, J = 8.0 Hz, 2H). MS
(ESI): m/z 567.9 [M-H]-.
4.1.4.10. N-(3-(4-(2-(4-fluorobenzyl)-5-methoxy-1-methyl-6-oxo-
1,6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4-di-
hydroxybenzamide (15c).
o
1
4.1.4.4. N-(3-(4-(2-(4-methylbenzyl)-5-methoxy-1-methyl-6-oxo-
1,6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4,5-
trihydroxybenzamide (13e).
White solid; yield: 53.0%; m.p. 156-158 C; H NMR (400
MHz, MeOH-d4, δ ppm) δ: 2.07-2.12 (m, 2H), 2.75-2.95 (m, 2H),
3.05-3.15 (m, 2H), 3.20-3.30 (m, 2H), 3.30-3.40 (m, 2H), 3.50-
3.65 (m, 4H), 3.56 (s, 3H), 3.91 (s, 3H), 4.19 (s, 2H), 6.80 (d, J =
8.0 Hz, 1H), 7.09 (t, J =8.8 Hz, 2H), 7.25-7.33 (m, 4H). 13C
NMR (100MHz, MeOH-d4, δ ppm) δ: 22.84, 25.15, 29.35, 30.55,
37.24, 39.92, 40.13, 45.05, 51.83, 52.26, 55.26, 59.49, 78.11,
114.44, 114.55, 115.38, 119.37, 125.23, 125.33, 129.72, 140.42,
143.24, 144.95, 148.88, 156.35, 157.36, 159.95, 164.67, 169.09.
MS (ESI): m/z 554.1 [M+H]+.
o
1
White solid; yield: 51.0%; m.p. 253-255 C; H NMR (400
MHz, MeOH-d4, δ ppm) δ: 2.05-2.08 (m, 2H), 2.29 (s, 3H), 2.90-
3.00 (m, 2H), 3.00-3.20 (m, 2H), 3.20-3.25 (m, 2H), 3.30-3.40
(m, 2H), 3.50-3.60 (m, 4H), 3.50 (s, 3H), 3.90 (s, 3H), 4.15 (s,
2H), 6.90 (s, 2H), 7.11 (d, J = 8.0 Hz, 2H), 7.15(d, J = 8.0 Hz,
2H). MS (ESI): m/z 563.9 [M-H]-.
4.1.4.5. N-(3-(4-(2-(3,4-dichlorobenzyl)-5-methoxy-1-methyl-6-o-
xo-1,6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)-propyl)-
3,4, 5-trihydroxybenzamide (13f).
4.1.4.11. N-(3-(4-(2-(4-hydroxybenzyl)-5-methoxy-1-methyl-6-ox-
o-1,6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)-propyl)-3,4-
dihydroxybenzamide (15d).
o
1
White solid; yield: 56.6%; m.p. 207-209 C; H NMR (400
MHz, DMSO-d6, δ ppm) δ: 1.60-1.75 (m, 2H), 2.10-2.50 (m,
4H), 3.10-3.25 (m, 4H), 3.25-3.40 (m, 2H), 3.40-3.60 (m, 2H),
3.46 (s, 3H), 3.77 (s, 3H), 4.17 (s, 2H), 6.81 (s, 2H), 7.23 (d, J =
8.0 Hz, 1H), 7.58 (d, J = 8.0 Hz, 2H), 8.06 (br, 1H), 8.60 (s, 1H),
8.97 (s, 2H). MS (ESI): m/z 617.9 [M-H]-.
o
1
White solid; yield: 53.6%; m.p. 162-165 C; H NMR (400
MHz, MeOH-d4, δ ppm) δ: 2.05-2.10 (m, 2H), 2.70-2.90 (m, 2H),
3.00-3.10 (m, 2H), 3.15-3.25 (m, 2H), 3.30-3.40 (m, 2H), 3.45-
3.55 (m, 4H), 3.49 (s, 3H), 2.89 (s, 3H), 4.09 (s, 2H), 6.08 (q, J =
8.4 Hz, 3H), 7.07 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.4 Hz, 1H),
7.33 (s, 1H). 13C NMR (100MHz, MeOH-d4, δ ppm) δ: 22.84,
25.06, 29.36, 30.59, 37.16, 39.76, 40.03, 44.90, 51.76, 52.25,
55.17, 59.49, 60.16, 78.12, 114.54, 115.13, 119.39, 125.31,
140.51, 143.19, 144.94, 148.89, 156.75, 159.87, 160.79, 163.22,
164.64, 169.09. MS (ESI): m/z 549.9 [M-H]-.
4.1.4.6. N-(2-(4-(2-benzyl-5-methoxy-1-methyl-6-oxo-1,6-dihydr-
opyrimidine-4-carbonyl)piperazin-1-yl)ethyl)-3,4,5-trihydroxybe-
nzamide (14a).
o
1
White solid; yield: 55.7%; m.p. 193-194 C; H NMR (400
MHz, MeOH-d4, δ ppm) δ: 3.10-3.20 (m, 2H), 3.20-3.40 (m, 2H),
3.45-3.60 (m, 8H), 3.50 (s, 3H), 3.91 (s, 3H), 4.22 (s, 2H), 6.93
(s, 2H), 7.27 (q, J = 7.2 Hz, 3H), 7.35(t, J = 7.2 Hz, 2H). MS
(ESI): m/z 535.9 [M-H]-.
4.2. HIV-1 integrase inhibitory assay
Compounds diluted in DMSO were pre-incubated with 800ng
integrase at 37.8 oC in the reaction buffer in the absence of Mn2+
for 10min. Subsequently, 1.5 pmol donor DNA and 9 pmol target
DNA were added and the reaction was initiated by the addition of
10 mmol/L Mn2+ into the final reaction volumn. The reactions
were carried out at 37.8 oC for 1h and subsequent detection
procedure was applied to detect the assay signals. Raltegravir,
current being clinical drug, was used as the positive control,
whereas no compound but only DMSO in reaction mixture was
set as the negative control. The inhibition effects of N-
methylpyrimidone derivatives 13, 14, 15 were calculated based
on the positive and negative controls.
4.1.4.7. N-(3-(4-(2-(4-fluorobenzyl)-5-methoxy-1-methyl-6-oxo-1,
6-dihydropyrimidine-4-carbonyl)piperazin-1-yl)propyl)-3,4,5-tri-
hydroxybenzamide (14c).
o
1
White solid; yield: 52.8%; m.p. 210-213 C; H NMR (400
MHz, MeOH-d4, δ ppm) δ: 3.15-3.25 (m, 2H), 3.25-3.40 (m, 2H),
3.45-3.65 (m, 8H), 3.48 (s, 3H), 3.90 (s, 3H), 4.19 (s, 2H), 6.91
(s, 2H), 7.27 (d, J = 8.0 Hz, 2H), 7.35 (d, J =8.0 Hz, 2H); MS
(ESI): m/z 553.9 [M-H]-.
6

2. Engelman, A.; Englund, G.; Orenstein, J. M.; Martin, M. A. and
Craigie, R. J. Virol., 1995, 69, 2729.
4.3. Antiviral assay
3. Quashie, P. K.; Sloan, R. D. and Wainberg, M. A. BMC Med.,
2012, 10, 34.
4. Neamati, N. HIV-1 Integrase: Mechanism and Inhibition Design,
Wiley, 2011.
5. Johns, B. A.; Weatherhead, J. G.; Allen, S. H.; Thompson, J. B.;
Garvey, E. P.; Foster, S. A.; Jeffrey, J. L. and Miller, W. H.
Bioorg. Med. Chem. Lett., 2009, 19, 1807.
6. Garvey, E. P.; Johns, B. A.; Gartland, M. J.; Foster, S. A.; Miller,
W. H.; Ferris, R. G.; Hazen, R. J.; Underwood, M. R.; Boros, E. E.;
Thompson, J. B.; Weatherhead, J. G.; Koble, C. S.; Allen, S. H.;
Schaller, L. T.; Sherrill, R. G.; Yoshinaga, T.; Kobayashi, M.;
Wakasa-Morimoto, C.; Miki, S.; Nakahara, K.; Noshi, T.; Sato, A.
and Fujiwara, T. Antimicrob. Agents Chemother., 2008, 52, 901.
7. Johns, B. A.; Weatherhead, J. G.; Allen, S. H.; Thompson, J. B.;
Garvey, E. P.; Foster, S. A.; Jeffrey, J. L. and Miller, W. H.
Bioorg. Med. Chem. Lett., 2009, 19, 1802.
The HeLa-CD4-LTR-b-gal indicator cells were plated in 96-
well plates at 6000 cells per well. The highest concentration of
the test compounds was 200 µg/mL, then diluted by four-fold
serially. There were 5 dilutions and 4 duplicates for each dilution.
The wells added only with virus-infected cells and without
compounds were used as viral-control and the wells added only
with mock-infected cells and without virus or compounds were
used as cell-control. The supernatant in the test wells and the
viral-control wells was discarded, and infected with 100 µL 2000
TCID50 HIV-1 pseudoviruses. Then 100 µL diluted test
compounds described above was added to the wells, making the
final concentration of the test compounds decreased by half. The
cell cultures were incubated at 37 oC in 5% CO2 humidified
atmosphere for 40–48 h, then fixed and stained. The blue cells
were counted under an inverted microscope.
8. Metobo, S. E.; Jin, H.; Tsiang, M.and Kim, C. U. Bioorg. Med.
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4.4. Molecular Docking Methods
4.4.1. HIV-1 integrase core initial model building
The HIV-1 integrase core model was generated by Discovery
Studio 4.0 (DS4.0) based on the A, C and D chains of 3OYA
crystallographic structure. Unwanted water and ligands were
removed by the DS4.0, and the lost atoms such as hydrogen were
added to build the initial receptor structure for docking 23
.
4.4.2. Small molecular preparation
The structure of 14d and 15c was drawn by Gaussian03
software, then optimized the molecules to the minimum energy
conformation used the semi-empirical AM1 method .
4.4.3. docking protocol
Docking procedure was performed by AutoDock 4 software
with the help of Autodock Tools. The no-polar hydrogen of
receptor and ligands were removed firstly, then, add the
Computer Gasteiger charge for receptor and ligands. The grid
maps were calculated using AutoGrid4 for three dockings, a grid
map with 60×70×60 points and a grid-point spacing of 0.375 Å
was applied. In order to fully explore the possible binding
conformations, 100 conformations were generated using the
Lamarckian Genetic Algorithm (LGA)24. For other docking
parameters, standard values were used as software default.
Cluster analysis was performed on the results using a root mean
square (RMS) tolerance of 2.0 Å. The conformations and binding
energy for further analysis were obtained from the average of the
biggest cluster.
18. Maurin, C.; Bailly, F.; Mbemba, G.; Mouscadet, J. F. and Cotelle,
P. Bioorg. Med. Chem., 2006, 14, 2978.
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W. E. and Reineck, M. G. J. Med. Chem., 2010, 53, 8161.
20. Hu, L. M.; Zhang S. L.; He X. Z.; Luo Z. G.; Wang X. L.; Liu W.
and Qin X. M. Bioorg. Med. Chem., 2012, 20, 177.
21. Tang, J.; Maddali, K.; Pommier, Y.; Sham, Y. Y. and Wang, Z. Q.
Bioorg. Med. Chem. Lett., 2010, 20, 3275.
Acknowledgments
22. Billamboz, M.; Bailly, F.; Lion, C.; Calmels, C.; Andreola, M. L.;
Witvrouw, M.; Christ, F.; Debyser, Z.; De Luca, L.; Chimirri,
A.and Cotellel, P. Eur. J. Med. Chem., 2011, 46, 535.
23. Accelrys Software Inc., Discovery Studio Modeling Environment,
Release 4.0, San Diego: Accelrys Software Inc., 2013.
24. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart,
W. E.; Belew, R. K.and Olson, A. J. J. Comput. Chem., 1998, 19,
1639.
The authors would like to acknowledge financial support
from the National Science Foundation of China (21272020), the
Project of Construction of Innovative Teams and Teacher Career
Development for Universities and Colleges Under Beijing
Municipality (IDHT20140504).
References and notes
1. Zhu, K.; Doboard, C. and Chow, S. J. Virol., 2004, 78, 5045.
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