Design and synthesis of novel β-diketo derivatives as HIV-1 integrase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.11.014

A series of novel β-diketo derivatives which combined the virtues of 1,3-diketo, 1,2,3-triazole and polyhydroxylated aromatics moieties, were designed and synthesized as potential HIV-1 integrase (IN) inhibitors and evaluated their inhibition to the stran

Full text of DOI:10.1016/j.bmc.2011.11.014

Bioorganic & Medicinal Chemistry 20 (2012) 177–182
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of novel b-diketo derivatives as HIV-1 integrase inhibitors
⇑
Liming Hu , Sulei Zhang, Xianzhuo He, Zaigang Luo, Xiaoli Wang, Wei Liu, Xuemei Qin
College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel b-diketo derivatives which combined the virtues of 1,3-diketo, 1,2,3-triazole 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. The result indi-
cates that 3,4,5-trihydroxylated aromatic derivatives exhibit good inhibition to HIV-1 integrase, but dihy-
droxylated aromatic derivatives and corresponding methoxy aromatic derivatives appear little inhibition
to HIV-1 integrase.
Received 15 September 2011
Revised 8 November 2011
Accepted 8 November 2011
Available online 22 November 2011
Keywords:
HIV-1 Integrase
Inhibitor
Ó 2011 Elsevier Ltd. All rights reserved.
1,3-Diketo
Polyhydroxylated aromatics
Strand transfer
1. Introduction
(30-P) and the strand transfer (ST) in the HIV-1 integrase catalytic
site.9
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 30-end
of the viral DNA (30-processing) and subsequently mediates joining
of the 30-end of the viral DNA to the host DNA (strand transfer) dur-
ing the viral replication cycle, is an attractive target for the devel-
opment of new anti-HIV-1 inhibitors with high selectivity and low
toxicity.1 Recently, many structurally diverse compounds have
been reported. Among these, compounds owning the b-diketoacids
(DKA) represent the most convincing anti-integrase activity (Fig. 1,
S-1360, L-731,988, 5-CITEP).2 The diketo acid moiety was believed
to be the most crucial pharmacophore for the inhibition of IN
inhibitors,3 and the structures of diketotriazole,4 diketotetrazole,5
and diketopyridine6 were reported to be bioisosters of the diketo
acid moiety. S-1360, a b-diketo bioisostere, which showed potent
antiviral activity against HIV-1 integrase, is the first IN inhibitor
for human clinical trials.7 Unfortunately, S-1360 was withdrawn
from clinical trials because of failed efficacy tests in infected pa-
tients due to its low concentrations attained in blood samples, ke-
tone reduction and glucuronidation in vivo, and also suffered from
a short half life.8 It takes us great interests to study its structure–
activity relationships (SAR). The structure of S-1360 consists of a
hydrophobic subunit (4-fluoro-benzylfuran) and a pharmacophoric
center (1,3-diketo and triazol moieties) which could binds to
divalent metal ions (Mg2+ or Mn2+) to block the 30-end processing
Amongst the other classes of HIV-1 integrase inhibitors, poly-
hydroxylated compounds represent of the family with the largest
number of members (Fig. 1, CAPE, AG1717, Styrl-quinoline).10,11a,b
Caffeic acid phenethyl ester, one of the typical polyhydroxylated
compounds, displays potent anti-IN activities with weak cytotoxic-
ity. The phenolic hydroxyl group, the pharma-cophoric center of
this sort of compounds, plays an important role,10,12 which can
coordinate with metal ions (Mg2+ or Mn2+) to block the 30-P and
ST reaction.
In previous studies, Catechol-DKA hybrids have been designed
and tested as IN inhibitors following the goals of refining the phar-
macophore, enhancing antiviral potency and improving cellular
membrane permeability.13,14 In order to study the possible interac-
tion between the two main classes of inhibitors (polyphenol and
DKAs), we synthesize new compounds that possess the two func-
tions, that is, 3-hydroxy-1-oxo-triazol propoyl and polyhydroxy-
phenyl. The 1,2,3-triazole ring is not only a hydrogen-bond donor
but also as a linking unit in the structure scaffold, its planar struc-
ture may facilitate the
p stacking interaction with target enzymes
similarly to phenyl ring. 1,2,3-Triazole moiety can be synthesized
by application of synthetic strategy using organoazide and acetyl
acetone.15 Actually, many known 1,2,3-triazoles has been demon-
strated to possess varied biochemical properties, including
anti-HIV.16a,b
Based on the above consideration, we designed (Z)-1-(substi-
tutedbenzyl-5-methyl-1H-1,2,3-triazol-4-yl)-3-hydroxy-3-(polyhy
droxy(or polymethoxy) phenyl) prop-2-en-1-one (Fig. 2) to ex-
plore the influence of chemical structure modification with the
aim of gaining novel potential anti-IN agents. In this paper, we
⇑
Corresponding author. Tel.: +86 10 6739 6211.
E-mail address: huliming@bjut.edu.cn (L. Hu).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.014

178
L. Hu et al. / Bioorg. Med. Chem. 20 (2012) 177–182
F
F
N
N
O
O
N
H
O
N
COOH
O
O
N
L-731,988(FBP)
S-1360
O
O
O
Cl
HO
HO
N
N
O
N
N
H
H
5-CITEP
CAPE
O
O
OH
OH
OH
OH
HO
HO
HOOC
N
N
H
N
H
OH
CN
CN
OH
OH
OH
AG1717
Styrlquinoline
Figure 1. Structure of IN inhibitors.
R1
R5
4. Anti-HIV-1 activities
R4
R3
R2
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 b-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 pseudovi-
ruses. The inhibitive rate of the test compound could be calculated
and the stage upon which the test compound acted could be deter-
mined. Compounds 6d and 6h 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 increas-
ing concentrations of drugs. The amount of virus was assayed by
b-galactosidase assay, with HeLa-CD4-b-gal cells as reporting cells.
Toxicity was estimated by MTT transformation assay. Antiviral
properties are reported in Table 2.
N
N
OH
N
O
Figure 2. The structures of designed integrase inhibitors.
present the synthesis, the anti-integrase and antiviral activities
of these new compounds.
2. Synthesis
(Z)-1-(substitutedbenzyl-5-methyl-1H-1,2,3-triazol-4-yl)-3-hy-
droxy-3-(polyhydroxy(or polymethoxy)phenyl)prop-2-en-1-one
outlined in Scheme 1 is typical synthesis of 1,3-diketo derivatives.
Starting from the commercially available benzyl bromide 1 and so-
dium azide, benzyl azide 2 can be facile prepared in excellent
yields.15 The key intermediate, 1-benzyl-4-acetyl-5-methyl-1,2,3-
triazole 3, was obtained by reaction of benzyl azide with acetyl
acetone in the mixed solution of ethanol and acetonitrile under re-
flux in the presence of potassium carbonate with a moderate
yield.15 Compounds 3 underwent Claisen condensation with vari-
able methyl polymethoxy benzoate 4 to afford the 1,3-diketo com-
pounds 5 in the alkaline medium (NaH). The compounds 5 were
isolated, purified, and also tested as IN inhibitors. Compounds 6
were obtained from compounds 5 by an exhaustive demethylation
using boron tribromide in 43–78% yield.
5. Discussion
The selectivity of integrase inhibitors for the strand transfer
reaction is of particular importance since it seem to be a prerequi-
site of an antiviral activity correlated with the integrase inhibi-
tion.18 Catechol-DKA hybrids are the examples of the correlation
between selectivity toward ST and antiviral activity.13 The inhibi-
tion effects of 1,3-diketo derivatives 5 and 6 were measured by
HIV-1 integrase strand transfer (ST) activity assay, which was car-
ried out as described previously with some minor modifications. As
shown in Table 1, compounds 5a–h proved to be inactive in the ST
assay (IC50 >47 lM), has no or variable methoxy substitutes on the
phenyl ring. But replacing methoxy group by a hydroxyl affected
3. Anti-integrase activity
the strand transfer reaction. 4-Hydroxylated aromatics derivatives
6a and 6d have little inhibition activity with IC50 >56 lM. While 3,
4-dihydroxylated aromatics derivatives 6b and 6e show anti-IN
activity in the low micromolar range. The most potent derivatives
are 3,4,5-trihydroxylated aromatics derivatives 6c and 6f, which
The inhibition effects of 1,3-diketo derivatives 5 and 6 were
measured by HIV-1 integrase strand transfer (ST) activity assay,
which was carried out as described previously.17 Compounds 5a–
h, 6a–h and positive control compound raltegravir were tested
against purified IN and the data are summarized in Table 1.

L. Hu et al. / Bioorg. Med. Chem. 20 (2012) 177–182
179
O
R1
R2
R1
R2
R1
(ii)
(i)
Br
N3
N
N
R2
N
R1
1a R1=F,R2=H
2a R1=F,R2=H
2b R1=H,R2=F
R6
(iii)
3a R1=F,R 2=H
3b R1=H,R2=F
1b R1=H,R2=F
R2
R7
R8
N
O
N
OH
N
O
R6
R7
OMe
5a R1= F, R2=H, R6=H,R7=H, R8=H
5b R1= F, R2=H, R6=H,R7=OMe, R8=H
5c R1= F, R2=H, R6=H,R7=OMe, R8=OMe
R8
6
6
6
6
7
7
8
R1= F, R2=H, R6=OMe,R7=OMe, R8=OMe
4a
4b
4c
4d
R =H,R =H, R =H
5d
8
8
5e R1= H, R2=F, R 6=H,R7=H, R8=H
5f R1= H, R2=F, R6=H,R7=OMe, R8=H
5g R1= H, R2=F, R6=H,R7=OMe, R8=OMe
R =H,R =OMe, R =H
7
R =H,R =OMe, R =OMe
7
8
R =OMe,R =OMe, R =OMe
R1= H, R2=F, R 6=OMe,R7=OMe, R8=OMe
5h
R1
R3
6a R1= F, R2=H, R3=H,R4=OH, R5=H
6b R1= F, R2=H, R3=H,R4=OH, R5=OH
R2
R4
R5
6c R1= F, R2=H, R3=OH,R4=OH, R5=OH
6d R1= H, R2=F, R3=H,R4=OH, R5=H
6e R1= H, R2=F, R 3=H,R4=OH, R5=OH
6f R1= H, R2=F, R3=OH,R4=OH, R5=OH
(iv)
N
N
OH
N
O
Scheme 1. Reagents and conditions: (i) NaN3, CH3COCH3/H2O, rt; (ii) CH3COCH2COCH3, K2CO3, C2H5OH/CH3CN, reflux; (iii) NaH, THF, reflux; (iv) 1 N BBr3, CH2Cl2, rt, then
H2O.
Table 1
introduction of a hydroxyl at 3-position of the phenyl in 6b and 6e
gave 6c and 6f, respectively, which exhibited high IC50 values. The
Inhibition of HIV-1integrase strand transfer catalytic activitiesa
R1
R2
R3
R4
R5
IC50
(
lM)
b
Compounds
result indicated the significance of the pyrogallol, and that the aryl
substitution could assist the inhibitory activities. The fluorine on
the benzyl group exhibit a little influence to inhibitory activity.
The 3-fluoride substitute on benzyl group shows more active than
2-fluoride substitute do. Comparison of CC50 and EC50 of com-
pounds 6c and 6f shows that their antiviral properties are quite
similar. There are almost same CC50 of 6c, 6f and raltegravir, com-
pared to raltegravir, 6c and 6f are less active, and therefore the
therapeutic index of 6c and 6f is low.
In summary, a series of 1,3-diketo derivatives were facile syn-
thesized and have been identified as HIV-1 integrase inhibitors.
The biological results showed that the poly-hydroxylated aromatic
moiety plays an important role to inhibit HIV-1 integrase ST reac-
tion. In particular, derivatives with 3, 4, 5-trihydroxylated aromat-
ics subunit is the most potential compounds. And further work
based on these structures is in progress.
5a
5b
5c
5d
5e
5f
5g
5h
6a
6b
6c
6d
6e
6f
F
F
F
F
H
H
H
H
F
F
F
H
H
H
H
H
H
H
F
F
F
F
H
H
H
F
F
F
H
H
H
OMe
H
H
H
OMe
H
H
H
H
H
OMe
OMe
H
>59
>54
>50
>47
>59
>54
>50
>47
>56
40
OMe
OMe
OMe
H
OMe
OMe
OMe
OH
OH
OH
OH
OH
H
OMe
OMe
H
OH
OH
H
OH
H
H
2.6
>56
32
OH
OH
OH
0.78
0.9
Raltegravir
a
HIV-1 IN inhibitory activities were measured according to the procedure
described in Ref. 12
b
Inhibition of strand transfer.
6. Experimental
Unless otherwise noted, all materials were obtained from com-
mercial suppliers and dried and purified by standard procedures.
Melting points were determined on a Beijing keyi elec-opti instru-
ment factory melting point apparatus. Proton nuclear magnetic
resonance (1H NMR) spectra were recorded with a Bruker Avance
DRX400 spectrometer with CDCl3 or DMSO-d6 as the solvent and
tetramethyl-silane (TMS) as the internal standard. The chemical
shifts were reported in d (ppm). Mass spectra (MS) data were ob-
tained using Esquire 6000 Mass Spectrometer. HRMS data were
measured using Bruker APEX IV Fourier Transform Ion Cyclotron
Resonance Mass Spectrometer. Petroleum ether used for column-
chromatography had a boiling range of 60–90 °C. Purities of all tar-
get compounds 5 and 6 were determined by an Agilent 1200 HPLC
system with a Agilent Zorbax Extend C-18 column, UV detector at
340 nm, mobile CH3CN/H2O (50–100%) and flow rate of 1 mL/min.
Compounds 2 and 3 were prepared according to the corresponding
literature procedures.15,19
Table 2
Antiviral activities of compounds 6d and 6h compared to that of raltergravir
TIc
a
b
Compound
EC50
(
l
M)
CC50 (lM)
6c
6f
8.92
7.32
33.4
36.6
>30
3.7
5.0
>1250
Raltegravir
0.025
a
b
c
Cytotoxic concentration 50%.
Effective concentration 50%.
Therapeutic index = CC50/EC50
.
IC50 values for strand transfer are 2.6 and 0.78
l
M, respectively.
They are nearly 15-fold and 41-fold higher than 6b and 6e. The re-
sults indicate that the 1,3-diketo and triazol moieties are not the
key active site for the compound. And the inhibitory activity corre-
lates closely to the number of hydroxyl group on phenyl ring. The

180
L. Hu et al. / Bioorg. Med. Chem. 20 (2012) 177–182
6.1. General procedure for the synthesis of 1,3-diketo
derivatives 5
6.87–7.05 (m, 3H, Ar-H), 7.30–7.32 (m, 1H, Ar-H), 7.33 (s, 1H, –
COCH–), 7.45–7.55 (m, 3H, Ar-H), 7.99–8.01 (m, 2H, Ar-H); 13C
NMR (CDCl3, 100 MHz, d ppm) 9.4, 51.2, 94.4, 114.2, 114.5, 115.6,
115.9, 122.8, 127.1, 128.7, 130.9, 132.3, 134.5, 136.8, 142.2,
181.1, 184.2; HRMS: m/z calcd for C19H17FN3O2: 338.1299; found:
338.1304; calcd for C19H16FN3NaO2: 360.1118; found: 360.1123;
HPLC purity 98.83%.
To a suspension of sodium hydride (60% dispersion in oil)
(20 mmol) in dry tetrahydrofuran (10 mL) was slowly added 1,2,3-
triazoles 3 (10 mmol) in dry THF (10 mL) at 0 °Cand the mixture
was stirred for 5 min. After that, variable methoxy substituted ethyl
benzoate 4 (15 mmol) in dry THF (15 mL) was added to the above
solution at 0 °Cand then the reaction mixture was slowly heated to
refluxing for about 90 min with stirring till TLC confirmed that the
reaction had finished. And then the cooled mixture was poured into
a mixture of ice-water (20 mL) and concentrated HCl (5 mL), ex-
tracted with EtOAc and purified by flash chromatography on silica
gel eluting with petroleum ether/ethyl acetate (4:1).
6.1.6. (Z)-1-(1-(3-Fluorobenzyl)-5-methyl-1H-1,2,3-triazol-4-
yl)-3-hydroxy-3-(4-methoxyphenyl)prop-2-en-1-one (5f)
White powder; yield 63%; mp 127–128 °C; 1H NMR (CDCl3,
400 MHz, d ppm) 2.57 (s, 3H, triazole-CH3), 3.89 (s, 3H, Ar-OCH3),
5.55 (s, 2H, Ar-CH2–), 6.91–6.96 (m, 2H, Ar-H), 6.98 (d, 2H,
J = 8.8 Hz, Ar-H), 7.02–7.07 (m, 1H, Ar-H), 7.28 (s, 1H, –COCH–),
7.32–7.38 (m, 2H, Ar-H), 8.02 (d, 2H, J = 9.2 Hz, Ar-H); 13C NMR
(CDCl3, 100 MHz, d ppm) 9.3, 51.1, 55.5, 93.3, 113.9, 114.2, 115.8,
122.7, 127.0, 129.2, 130.9, 136.5, 142.0, 163.2, 182.0, 182.5; HRMS:
m/z calcd for C20H19FN3O3: 368.1410; found: 368.1405; HPLC pur-
ity 100%.
6.1.1. (Z)-1-(1-(4-Fluorobenzyl)-5-methyl-1H-1,2,3-triazol-4-
yl)-3-hydroxy-3-phenylprop-2-en-1-one (5a)
White powder; yield 67%; mp 128–130 °C; 1H NMR (CDCl3,
400 MHz, d ppm) 2.56 (s, 3H, triazole-CH3), 5.51 (s, 2H, Ar-CH2–),
7.07 (t, 2H, J = 8.4 Hz, Ar-H), 7.18–7.21 (m, 2H, Ar-H), 7.33 (s, 1H,
–COCH–), 7.45–7.55 (m, 3H, Ar-H), 8.01 (t, 2H, J = 6.4 Hz, Ar-H);
13C NMR (CDCl3, 100 MHz, d ppm) 9.4, 51.0, 94.5, 116.3, 116.6,
127.0, 129.1, 129.2, 129.9, 132.3, 134.5, 136.6, 142.1, 181.0,
6.1.7. (Z)-3-(3,4-Dimethoxyphenyl)-1-(1-(3-fluorobenzyl)-5-
methyl-1H-1,2,3-triazol-4-yl)-3-hydroxyprop-2-en-1-one (5g)
White powder; yield 61%; mp 122–124 °C; 1H NMR (CDCl3,
400 MHz, d ppm) 2.57 (s, 3H, triazole-CH3), 3.97 (s, 3H, Ar-OCH3),
3.99 (s, 3H, Ar-OCH3), 5.55 (s, 2H, Ar-CH2–), 6.89–6.92 (m, 1H, Ar-
H), 6.98 (t, 2H, J = 8.4 Hz, Ar-H), 7.02–7.07 (m, 1H, Ar-H), 7.28 (s,
1H, –COCH–), 7.32–7.38 (m, 1H, Ar-H), 7.55 (d, 1H, J = 2.0 Hz, Ar-
H), 7.70 (dd, 1H, J = 2.0 and 8.4 Hz, Ar-H); 13C NMR (CDCl3,
100 MHz, d ppm) 9.3, 51.1, 56.1, 77.3, 93.5, 109.7, 110.1, 110.6,
114.2, 115.8, 121.4, 122.9, 127.4, 130.8, 136.5, 141.9, 149.1,
152.7, 181.9, 182.5; HRMS: m/z calcd for C21H21FN3O4: 398.1516;
found: 398.1514; HPLC purity 99.54%.
184.3; HRMS: m/z calcd for
C19H17FN3O2: 338.1299; found:
338.1304; calcd for C19H16FN3NaO2: 360.1118; found: 360.1122;
HPLC purity 99.26%.
6.1.2. (Z)-1-(1-(4-Fluorobenzyl)-5-methyl-1H-1,2,3-triazol-4-
yl)-3-hydroxy-3-(4-methoxyphenyl)prop-2-en-1-one (5b)
White powder; yield 56%; mp 145–146 °C; 1H NMR (CDCl3,
400 MHz, d ppm) 2.55 (s, 3H, triazole-CH3), 3.88 (s, 3H, Ar-OCH3),
5.51 (s, 2H, Ar-CH2–), 6.96 (d, 2H, J = 9.6 Hz, Ar-H), 7.06 (t, 2H,
J = 6.4 Hz, Ar-H), 7.17–7.21 (m, 2H, Ar-H), 7.25 (s, 1H, –COCH–),
7.99 (d, 2H, J = 6.4 Hz, Ar-H); 13C NMR (CDCl3, 100 MHz, d ppm)
9.3, 51.0, 55.5, 93.3, 113.9, 116.3, 129.3, 129.9, 131.0, 136.3,
142.0, 163.2, 181.9, 182.6; HRMS: m/z calcd for C20H19FN3O3:
368.1410; found: 368.1408; HPLC purity 98.99%.
6.1.8. (Z)-1-(1-(3-Fluorobenzyl)-5-methyl-1H-1,2,3-triazol-4-
yl)-3-hydroxy-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (5h)
White powder; yield 72%; mp 112–113 °C; 1H NMR (CDCl3,
400 MHz, d ppm) 2.57 (s, 3H, triazole-CH3), 3.94 (s, 3H, Ar-OCH3),
3.96 (s, 6H, Ar-OCH3), 5.55 (s, 2H, Ar-CH2–), 6.89–7.07 (m, 3H, Ar-
H), 7.25 (s, 2H, Ar-H), 7.28(s, 1H, –COCH–), 7.31–7.38 (m, 1H, Ar-
H); 13C NMR (CDCl3, 100 MHz, d ppm) 9.3, 51.1, 56.3, 61.0, 93.9,
104.4, 114.2, 114.4, 115.6, 115.8, 122.7, 129.8, 130.9, 136.7,
141.9, 153.2, 164.3, 181.8, 182.9; HRMS: m/z calcd for
6.1.3. (Z)-3-(3,4-Dimethoxyphenyl)-1-(1-(4-fluorobenzyl)-5-
methyl-1H-1,2,3-triazol-4-yl)-3-hydroxyprop-2-en-1-one (5c)
White powder; yield 60%; mp 141–142 °C; 1H NMR (CDCl3,
400 MHz, d ppm) 2.56 (s, 3H, triazole-CH3), 3.96 (s, 3H, Ar-OCH3),
3.98 (s, 3H, Ar-OCH3), 5.51 (s, 2H, Ar-CH2–), 6.94 (d, 1H,
J = 8.8 Hz, Ar-H), 7.07 (t, 2H, J = 6.4 Hz, Ar-H), 7.17–7.21 (m, 2H,
Ar-H), 7.27 (s, 1H, –COCH–), 7.53 (d, 1H, J = 2.0 Hz, Ar-H), 7.68
(dd, 1H, J = 2.0 and 8.4 Hz, Ar-H); 13C NMR (CDCl3, 100 MHz, d
ppm) 9.4, 51.0, 56.0, 93.4, 109.5, 110.1, 110.6, 116.3, 121.4,
129.1, 129.3, 129.9, 136.3, 141.9, 149.0, 152.8, 161.5, 181.9,
C22H23FN3O5: 428.1622; found: 428.1619; HPLC purity 99.76%.
6.2. General procedure for the synthesis of 1,3-diketo
derivatives 6
To a suspension of 1,3-diketo derivatives 6 with methyloxy
groups (5 mmol) in dry CH2Cl2 (20 mL) was added 1 N tribromobo-
rane solution (15 mL) at 0 °C and the mixture was stirred overnight
at room tempreture. After that, water (15 mL) was slowly added to
the above solution at 0 °C with stirring, followed by evaporation of
the sovent under reduced pressure, extracted with EtOAc and puri-
fied by flash chromategraphy on silica gel eluting with petroleum
ether/ethyl acetate (1:1).
182.5; HRMS: m/z calcd for
C21H21FN3O4: 398.1516; found:
398.1513; HPLC purity 98.45%.
6.1.4. (Z)-1-(1-(4-Fluorobenzyl)-5-methyl-1H-1,2,3-triazol-4-
yl)-3-hydroxy-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (5d)
White powder; yield 52%; mp 156–158 °C; 1H NMR (CDCl3,
400 MHz, d ppm) 2.57 (s, 3H, triazole-CH3), 3.94 (s, 3H, Ar-OCH3),
3.96 (s, 6H, Ar-OCH3), 5.52 (s, 2H, Ar-CH2-), 7.08 (t, 2H, J = 8.4 Hz,
Ar-H), 7.19–7.22 (m, 2H, Ar-H), 7.24 (s, 2H, Ar-H), 7.25 (s, 1H, –
COCH–); 13C NMR (CDCl3, 100 MHz, d ppm) 9.3, 51.1, 56.4, 60.9,
93.9, 104.6, 116.0, 116.3, 129.0, 129.2, 129.9, 136.4, 141.8, 153.1,
161.5, 181.7, 182.8; HRMS: m/z calcd for C22H23FN3O5: 428.1622;
found: 428.1619; HPLC purity 98.89%.
6.2.1. (Z)-1-(1-(4-Fluorobenzyl)-5-methyl-1H-1,2,3-triazol-4-
yl)-3-hydroxy-3-(4-hydroxyphenyl)prop-2-en-1-one (6a)
Yellow powder; yield 78%; mp 208–210 °C; 1H NMR (DMSO-d6,
400 MHz, d ppm) 2.58 (s, 3H, triazole-CH3), 5.67 (s, 2H, Ar-CH2–),
6.93 (d, 2H, J = 9.6 Hz, Ar-H), 7.13 (s, 1H, –COCH–), 7.24 (t, 2H,
J = 6.4 Hz, Ar-H), 7.29–7.33 (m, 2H, Ar-H), 7.91 (d, 2H, J = 6.4 Hz,
Ar-H), 10.41 (s, 1H, Ar-OH); 13C NMR (DMSO-d6, 100 MHz, d
ppm) 9.4, 50.3, 92.6, 115.8, 116.1, 116.3, 124.8, 129.7, 130.2,
131.5, 137.4, 141.0, 161.0, 182.1, 182.7; HRMS: m/z calcd for
6.1.5. (Z)-1-(1-(3-Fluorobenzyl)-5-methyl-1H-1,2,3-triazol-4-
yl)-3-hydroxy-3-phenyl-prop-2-en-1-one (5e)
White powder; yield 75%; mp 105–107 °C; 1H NMR (CDCl3,
400 MHz, d ppm) 2.56 (s, 3H, triazole-CH3), 5.53 (s, 2H, Ar-CH2–),

L. Hu et al. / Bioorg. Med. Chem. 20 (2012) 177–182
181
C19H17FN3O3:
354.1248;
found:
354.1249;
calcd
for
(2)Target DNA: T01: 50TGACCAAGGGCTAATTCACT-digoxin 30;
T02: 30digoxin-ACTGGTTCCCGATTAAGTGA-50.
C19H16FN3NaO3: 376.1067; found: 376.1068; HPLC purity 98.95%.
D01 + D02 and T01 + T02 were mixed in a ratio of 1:1 at a con-
centration of 100 lm/L in purified water, heated to 94 °C for 5 min,
6.2.2. (Z)-3-(3,4-Dihydroxyphenyl)-1-(1-(4-fluorobenzyl)-5-
methyl-1H-1,2,3-triazol-4-yl)-3-hydroxyprop-2-en-1-one (6b)
Yellow powder; yield 43%; mp 192–194 °C; 1H NMR (DMSO-d6,
400 MHz, d ppm) 2.56 (s, 3H, triazole-CH3), 5.66 (s, 2H, Ar-CH2–),
6.96 (d, 1H, J = 8.8 Hz, Ar-H), 7.17 (s, 1H, –COCH–), 7.32 (t, 2H,
J = 8.8 Hz, Ar-H), 7.39–7.49 (m, 4H, Ar-H), 9.75 (br, 2H, Ar-OH);
13C NMR (DMSO-d6, 100 MHz, d ppm) 9.4, 50.3, 92.6, 114.2,
116.1, 116.3, 120.3, 125.2, 130.1, 130.3, 131.8, 137.3, 141.0,
146.0, 151.1, 182.2, 182.4; HRMS: m/z calcd for C19H17FN3O4:
370.1197; found: 370.1203; calcd for C19H16FN3NaO4: 392.1017;
found: 392.1021; HPLC purity 100%.
slowly cooled down to room temperature, and stored at ꢀ20 °C un-
til use.
6.4. HIV-1 integrase inhibitory assay
Compounds diluted in DMSO were pre-incubated with 800 ng
integrase at 37.8 °C in the reaction buffer in the absence of Mn2+
for 10 min. 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 volume. The reactions
were carried out at 37.8 °C for 1 h and subsequent detection proce-
dure was applied to detect the assay signals. Integrase inhibitor,
raltergravir, was used as the control compound (positive control),
whereas no compound but only DMSO in the reaction mixture
was set as the drug-free control (negative control). The inhibition
effects of 1,3-diketo derivatives 5 and 6 were calculated based on
the positive and negative controls.
6.2.3. (Z)-1-(1-(4-Fluorobenzyl)-5-methyl-1H-1,2,3-triazol-4-
yl)-3-hydroxy-3-(3,4,5-trihydroxyphenyl)prop-2-en-1-one (6c)
Yellow powder; yield 57%; mp 187–189 °C; 1H NMR (DMSO-d6,
400 MHz, d ppm) 2.56 (s, 3H, triazole-CH3), 5.65 (s, 2H, Ar-CH2–),
6.99 (s, 2H, Ar-H), 7.00 (s, 1H, –COCH–), 7.23 (t, 2H, J = 8.8 Hz, Ar-
H), 7.29–7.33 (m, 2H, Ar-H), 9.27 (br, 3H, Ar-OH); 13C NMR
(DMSO-d6, 100 MHz, d ppm) 9.4, 50.4, 92.6, 106.7, 108.5, 116.0,
116.3, 123.9, 130.2, 137.3, 139.2, 141.1, 146.5, 182.2, 182.3; HRMS:
m/z calcd for C19H17FN3O5: 386.1146; found: 386.1151; calcd for
6.5. Antiviral assays
C19H16FN3NaO5: 408.0966; found: 408.0972; HPLC purity 100%.
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 lg/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
6.2.4. (Z)-1-(1-(3-Fluorobenzyl)-5-methyl-1H-1,2,3-triazol-4-
yl)-3-hydroxy-3-(4-hydroxyphenyl)prop-2-en-1-one (6d)
Yellow powder; yield 55%; mp 184–186 °C; 1H NMR (DMSO-d6,
400 MHz, d ppm) 2.61 (s, 3H, triazole-CH3), 5.78 (s, 2H, Ar-CH2–),
6.98 (d, 2H, J = 6.4 Hz, Ar-H), 7.08–7.16 (m, 2H, Ar-H), 7.19 (s, 1H,
–COCH–), 7.21–7.26 (m, 1H, Ar-H), 7.46–7.51 (m, 1H, Ar-H), 7.91
(d, 2H, J = 6.4 Hz, Ar-H), 10.49 (s, 1H, Ar-OH); 13C NMR (DMSO-d6,
100 MHz, d ppm) 9.4, 50.4, 92.6, 114.8, 115.4, 115.8, 116.2, 123.9,
124.8, 129.7, 131.5, 137.6, 138.3, 141.0, 162.9, 181.1, 182.4; HRMS:
m/z calcd for C19H17FN3O3: 354.1248; found: 354.1251; calcd for
discarded, and infected with 100
lL 2000 TCID50 HIV-1 pseudovi-
ruses. Then 100 L diluted test compounds described above was
l
added to the wells, making the final concentration of the test com-
pounds decreased by half. The cell cultures were incubated at 37 °C
in 5% CO2 humidified atmosphere for 40–48 h, then fixed and
stained. The blue cells were counted under an inverted microscope.
C19H16FN3NaO3: 376.1067; found: 392.1021; HPLC purity 92.70%.
6.2.5. (Z)-3-(3,4-Dihydroxyphenyl)-1-(1-(3-fluorobenzyl)-5-
methyl-1H-1,2,3-triazol-4-yl)-3-hydroxyprop-2-en-1-one (6e)
Yellow powder; yield 66%; mp 200–201 °C; 1H NMR (DMSO-d6,
400 MHz, d ppm) 2.57 (s, 3H, triazole-CH3), 5.70 (s, 2H, Ar-CH2–),
6.90 (d, 1H, J = 8.4 Hz, Ar-H), 7.05 (d, 1H, J = 7.6 Hz, Ar-H), 7.10 (s,
1H, –COCH–), 7.20 (t, 2H, J = 8.4 Hz, Ar-H), 7.37–7.46 (m, 3H, Ar-
H), 9.95 (br, 2H, Ar-OH); 13C NMR (DMSO-d6, 100 MHz, d ppm)
9.4, 50.5, 92.6, 114.3, 114.9, 115.3, 115.6, 116.3, 120.3, 123.8,
125.2, 131.5, 137.5, 138.1, 141.0, 146.0, 151.1, 182.2, 182.4; HRMS:
m/z calcd for C19H17FN3O4: 370.1197; found: 370.1201; calcd for
Acknowledgments
The authors would like to acknowledge financial support from
the National Key Basic Research Program of China (No.
2009CB930200) and Beijing Natural Science Foundation (No.
7102009).
References and notes
C19H16FN3NaO4: 392.1017; found: 376.1020; HPLC purity 99.67%.
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6.2.6. (Z)-1-(1-(3-Fluorobenzyl)-5-methyl-1H-1,2,3-triazol-4-
yl)-3-hydroxy-3-(3,4,5-trihydroxyphenyl)prop-2-en-1-one (6f)
Yellow powder; yield 59%; mp 213–215 °C; 1H NMR (DMSO-d6,
400 MHz, d ppm) 2.57 (s, 3H, triazole-CH3), 5.71 (s, 2H, Ar-CH2–),
7.00 (s, 2H, Ar-H), 7.02 (s, 1H, –COCH–), 7.04–7.21 (m, 3H, Ar-H),
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386.1146; found: 386.1150; calcd for C19H16FN3NaO5: 408.0966;
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6.3. HIV-1 integrase inhibitory assay: oligonucleotides
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(1)
Donor
DNA:
D01:50biotin-CCCTTTTAGTCAGTGTG-
GAAAATCTCT AGCA30; D02: 30GAAAATCAGTCACACCTTTTAGAGA
TCGTCA50.

182
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