HIV-1 integrase inhibition of biscoumarin analogues

Article abstract of DOI:10.1248/cpb.54.682

Nineteen biscoumarins bearing free and modified hydroxyl substituents at benzoyloxyphenyl linker have been synthesized by multiple step synthesis. Among these biscoumarins, thirteen were found to be active molecules against HIV-1 integrase (HIV-1 IN). The structure-activity relationship of the nineteen compounds on HIV IN may be useful for the design of potent therapeutic agents.

Full text of DOI:10.1248/cpb.54.682

682
Notes
Chem. Pharm. Bull. 54(5) 682—686 (2006)
Vol. 54, No. 5
HIV-1 Integrase Inhibition of Biscoumarin Analogues
Chang-Xiao SU,a Jean-Francois MOUSCADET,b Chih-Chia CHIANG,c Hou-Jen TSAI,c and
,a
*
Ling-Yih HSU
a School of Pharmacy, National Defense Medical Center; Taipei, 114, Taiwan, ROC; b L.B.P.A, CNRS UMR8532, Ecole
c
Normale Superieure de Cachan; 61, avenue du Président Wilson, F-92335, Cachan France: and Department of Applied
Chemistry, Chung Cheng Institute of Technology; Ta-shi, Tao-yuan, 335, Taiwan, ROC.
Received August 22, 2005; accepted November 21, 2005
Nineteen biscoumarins bearing free and modified hydroxyl substituents at benzoyloxyphenyl linker have
been synthesized by multiple step synthesis. Among these biscoumarins, thirteen were found to be active mole-
cules against HIV-1 integrase (HIV-1 IN). The structure–activity relationship of the nineteen compounds on HIV
IN may be useful for the design of potent therapeutic agents.
Key words biscoumarins; HIV-1 integrase; inhibitor
Current strategies for anti-HIV chemotherapy involve inhi- in the presence of coupling reagents dicyclohexylcarbodi-
bition of virus adsorption, virus-cell fusion, reverse tran- imide (DCC) and catalyst 4-dimethylaminopyridine (4-
scription, integration, translation, proteolytic cleavage, glyco- DMAP) in dichloromethane, followed by 3 N HCl hydrolysis
sylation, assembly, or release.1,2) Because the integration is to remove the protected phenols was unfruitful (Chart 3).
essential for the replication of HIV and the integrase, the en- The product was unexpected 18 other than 45. Alternative
zyme responsive for integration, appears to be absent in the synthesis of target compounds was successful by esterifica-
mammalian host, HIV-1 integrase (HIV-1 IN) represents a tion of 4-hydroxybenzaldehyde with protected polyphenolic
potential target for the development of non-toxic antiviral acids (9—16, 19) and lipoic acid in the presence of coupling
therapeutic agents.3,4) Recently numerous coumarin com-
pounds have been evaluated for inhibitory effects against
HIV replication, and some of them have been found to in-
hibit different stages in the HIV replication cycle.5) A natural
tetrameric coumarin (I) shown potent integrase inhibitory ac-
tivity (IC50ꢀ0.8 mM for integration and 1.5 mM for 3ꢁ-process-
ing) was first reported by Mazumder et al.6) and the parent
compound was then modified into simple components to de-
termine the minimum active pharmacophore essential for po-
tency. They disclosed a biscoumarin unit linked by a phenyl
ring (II) was required for activity.7)
Our previous studies focused mainly on the linker of
phenyl ring. Replacement of the phenyl ring by aniline mus-
tard (III) was achieved.8) We reasoned the mustard moiety
might react with nearby nucleophile of the binding site to
form a covalent bonding and to improve the inhibitory activ-
ity. However, these compounds failed to enhance activity. We
also prepared biscoumarins with a diversified modification
on the linker.9) Structure–activity relationship studies enabled
us to know that the benzoyloxyphenyl linker (IV) seemed im-
portant for the inhibitory activity. These encouraging results
Chart 1. Molecular Structures of I—IV and Target Compounds V
prompted us to modify these lead compounds. Thus, we ex-
amined the effects of substitutions on the benzene ring of
benzoyloxyphenyl linker. In the present paper, we report the
synthesis and biological evaluation of a novel benzoy-
loxyphenyl linker (V) having either a monohydroxy or bishy-
droxy groups on the benzoyl ring.
Chemistry A general synthetic route for the preparation
of target compounds (30—47) is depicted in Charts 2 and 4.
Protection of the phenolic hydroxyl groups of commercially
available polyphenolic acids (1—8) by acetylation with
acetic anhydride in pyridine in the dark at room temperature
gave (9—16). Key intermediate 17 was prepared from 4-
hydroxybenzaldehyde with 4-hydroxycoumarin in ethanol
under reflux. The first attempt to prepare target compounds
by esterification of 17 with protected polyphenolic acids (14)
Chart 2
∗ To whom correspondence should be addressed. e-mail: hly@ndmctsgh.edu.tw
© 2006 Pharmaceutical Society of Japan

May 2006
683
Fig. 1. The Values of Net-Charge at Three Oxygen Atoms of 17 by
Hartree–Fock 3-21G(*) Calculation
Molecular orbital calculation was carried out by using the program SPARTAN ’04
windows, version 1.0.3 of Wavefunction Inc.
were three reactive sites (two hydroxyls of 4-hydroxy-
coumarin and one hydroxyl of phenol) for the reaction of 17
with protected polyphenolic acids, molecular orbital calcula-
tion was carried out by using the program SPARTAN ’04
windows, version 1.0.3 of Wavefunction Inc to verify the
preferred reactive site for esterification. The net-charges by
Hartree-Fock 3-21G(*) calculation for 17 are shown in Fig.
1. The values of net-charge at two oxygen atoms from
hydroxyl of 4-hydroxycoumarin in 17 are nearly equal
(ꢂ0.798602 and ꢂ0.798694, respectively), while the oxygen
atom of phenol has a value of ꢂ0.746966. The net-charges at
oxygen atoms from hydroxyls of 4-hydroxycoumarin are
negatively larger than that at oxygen atom of phenol. Thus,
the 4-hydroxycoumarin hydroxyl groups are more reactive
than the phenol hydroxyl group in 17 for esterification, and
these results agree with the experimental fact.
Chart 3
Results and Discussion
HIV-1 IN Inhibition HIV-1 IN inhibition assays were
carried out in the presence of low amounts of purified recom-
binant integrase (50 nM) in the presence of 7.5 mM Mg2ꢃ as
the cationic cofactor, using 21-mer double-strand oligonu-
cleotide substrate. The compounds were screened for their
inhibitory activity in a 3ꢁ-processing assay which was per-
formed as described previously.3,10) Effects against the strand
transfer activity were evaluated during the same assay from
the homologous integration events and found to be no signifi-
cant differences from 3ꢁ-processing inhibition, thus confirm-
ing that coumarins affect equally both steps of the integration
reaction. Table 1 lists the inhibitory activity of a series of
mono- or bis-hydroxylated benzoyl biscoumarins against the
recombinant wild-type HIV-1 IN enzyme. The inhibitory ac-
tivity of NSC 158393 I and II were included for a compari-
son. Among nineteen compounds in this series, all were ac-
tive, thirteen demonstrated activities less than 10 mM, four
had activities in the range of 10 to 27 mM, and two showed
activities at 37 and 53 mM, respectively. Compound 45 was
Chart 4
reagents DCC and DMAP in dichloromethane and then sub- the most potent in this series, with IC50 of 1.5 mM. The sec-
sequently coupling reaction with 4-hydroxycoumarin in ond-most-potent compound 35 was the acetate analog of 45,
ethanol under reflux (Chart 4). All the structures of target with IC50 of 1.9 mM. Compounds were divided into two
compounds were established by NMR, IR, MS spectroscopy classes based on the free (class I) or modified (class II) hy-
and elementary analysis. The proton on the benzylidene tert- droxyl substitute. All the compounds in class I, bearing free
carbon of 45 is found at d 6.3 ppm, in agreement with previ- hydroxyl substitute(s) (40—47), except compound 44 and
ous studies on similar compounds.8,9) However, an unex- 46, had a slight higher IN inhibitory activity in vitro than the
pected benzylidene proton upfield shift from d 6.3 to corresponding compounds with modified hydroxyl substitute
4.9 ppm is observed in 18. The introduction of an ester in- (30—38). The relative potency of the mono-hydroxyl com-
stead of ketone group on the 4-hydroxycoumarin ring in 18 pounds was in the order: 4-OH (42)ꢄ3-OH (41)ꢄ2-OH (40),
decreases electron withdrawing ability, which causes the ben- whereas the bis-hydroxyl compounds was in the order: 3,4-
zylidene proton upfield shifting. Since it was found that there di-OH (45)ꢄ2,4-di-OH (43)ꢄ2,5-di-OH (44)ꢄ3,5-di-OH

684
Vol. 54, No. 5
Table 1. HIV-1 Integrase Inhibitory Activities of Modified Coumarin (10 ml) cold solution. The mixture was extracted with ethyl acetate. The lay-
Dimers
Entry
ers washed with brine and aq saturated sodium bicarbonate. The combined
organic phase was dried under magnesium sulfate, filtered, and the solvent
was removed under vacuo. The residue was crystallized from proper solvent
to afford the corresponding acetoxy polyphenolic acids: 2-acetoxy benzoic
acid (9), 3-acetoxy benzoic acid (10), 4-acetoxy benzoic acid (11), 2,4-di-
acetoxy benzoic acid (12), 2,5-diacetoxy benzoic acid (13), 3,4-diacetoxy
benzoic acid (14), 3,5-diacetoxy benzoic acid (15), 3,4-diacetoxy cinnamic
acid (16). These acetoxy polyphenolic acids were identified by melting point
and 1H-NMR. Data were in agreement with literature values.11,12)
3,3ꢀ-(4-Hydroxybenzylidene)bis-4-hydroxycoumarin (17) A mixture
of 4-hydroxybenzaldeyde (3.66 g, 30 mmol) and 4-hydroxycoumarin (9.73 g,
60 mmol) in absolute ethanol (100 ml) was heated under reflux for 24 h.
The solvent was removed in vacuo. The residue was recrystallized from
ethanol to give pale yellow powder 17 (11.4 g, 97%). Rf: 0.10 (n-
hexane : EtOAcꢀ1 : 1). mp: 210—212 °C (CH2Cl2). 1H-NMR (DMSO-d6) d:
7.89—6.59 (m, 12H, ArH), 6.22 (s, 1H, CH). FAB-MS m/z: 428.09
(MꢃHꢃ). Anal. Cacld for C25H16O7: C, 70.09; H, 3.76. Found: C, 69.87; H,
4.00.
4-(3,4-Dihydroxybenzoyloxy)coumarin-4ꢀ-hydroxycoumarin-4-hy-
droxyphenylmethane (18) To a solution of 14 (2.3 g, 10 mmol) and DCC
(2.3 g, 11 mmol) in CH2Cl2 (30 ml) were added DMAP (122 mg, 1 mmol)
and 17 (2.14 g, 5 mmol). The reaction mixture was stirred over night at room
temperature. The mixture was filtered, and the solvent was removed in
vacuo. The residue was recrystallized from methanol to give white interme-
diate (1.98 g, 61%). Rf: 0.30 (n-hexane : EtOAcꢀ1 : 1). mp: 230—233 °C
(MeOH). 1H-NMR (CDCl3) d: 8.09—7.12 (m, 15H, ArH), 5.20 (s, 1H, CH),
2.30 (s, 6H, CH3). IR (KBr) cmꢂ1: 3512 (OH), 1725 (CꢀO). UV lmax
Compound
mp (°C)
3ꢁ-Processing (IC50 mM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
18
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
262—263
258—260
270—272
252—254
232—234
242—243
178—180
116—118
118—120
125—126
247—249
238—239
232—234
258—260
ꢄ300
37.0
27.0
7.1
4.6
19.0
9.7
1.9
9.3
2.6
2.6
2.3
10.0
6.1
3.2
4.9
25.0
1.5
53.0
2.6
1.1
43.0
262—264
210—212
177—181
266—268
45
46
47
NSC 158393 (I)
(II)
(46), suggesting the inhibitory activity is associated with the (EtOH) nm (log e): 309 (4.13). FAB-MS m/z: 648.13 (MꢃHꢃ). Anal. Cacld
specific spatial disposition of the hydroxyl groups. Com-
pound 47 (containing free hydroxyl caffeoyl) and 45 (having
catechol) displayed considerable activity, with IC50 of 2.6 and
1.5 mM, respectively. It was of interesting that compound 37
(containing protected methoxy caffeoyl) and 38 (containing
protected acetoxy caffeoyl) were equal potent to 47, implying
modification on bis-hydroxyl substitute tolerates activity.
Class II compounds demonstrated similar trends to class I
compounds on relative potency, except 33 and 36. Addition-
ally, compound 39, replacing the caffeoyl with a potent an-
tioxidative lipoyl moiety remained the potency, with IC50 of
2.3 mM.
In conclusion, we have designed and synthesized nineteen
biscoumarins bearing free and modified hydroxyl substitute
at the benzoyl ring. Among these easily available bis-
coumarins, thirteen were found to be active molecules
against HIV-1 IN. These compounds may prove useful in
structure–activity relationship studies on HIV IN and may ul-
for C36H24O12: C, 66.67; H, 3.73. Found: C, 66.87; H, 4.00. A mixture of the
intermediate (324 mg, 0.5 mmol) and 3 M HCl (10 ml) in THF (30 ml) was
heated under reflux for 3 h and poured onto ice water (50 ml). The mixture
was extracted with EtOAc. The layer was washed with water and brine. The
organic extract was dried over MgSO4, filtered, and the solvent was removed
in vacuo. The residue was recrystallized from CH2Cl2 to give white powder
18 (210 mg, 75%). Rf: 0.15 (n-hexane : EtOAcꢀ1 : 1). mp: 262—263 °C
1
(CH2Cl2). H-NMR (DMSO-d6) d: 9.95, 9.46 (s, 1H each, OH), 8.39—6.82
(m, 15H, ArH), 4.89 (s, 1H, CH). IR (KBr) cmꢂ1: 3624 (OH), 1808 (CꢀO).
UV lmax (EtOH) nm (log e): 322 (4.23). FAB-MS m/z: 565.1 (MꢃHꢃ).
Anal. Cacld for C32H20O10: C, 68.09; H, 3.57. Found: C, 67.87; H, 3.87.
Esterification of Acetoxy (Poly)phenolic Acids or Lipoic Acid with 4-
Hydroxybenzaldehyde, General Procedure to Obtain Compounds 20—
29 To a solution of acid (10 mmol) and DCC (11 mmol) in CH2Cl2 (50 ml)
were added DMAP (1 mmol) and 4-hydroxybenzaldehyde (11 mmol). The
reaction mixture was stirred over night at room temperature. The mixture
was filtered, and the solvent was removed in vacuo. The residue was purified
by flash chromatography on silica gel with CH2Cl2/acetone (9/1) and recrys-
tallized from methanol.
4-(2-Acetoxybenzoyloxy)benzaldehyde (20) as white powder in a yield of
1
79%: mp: 74—75 °C (n-hexane). H-NMR (CDCl3) d: 9.87 (1H, s, CHO),
8.07—7.84 (8H, m, ArH), 2.17 (3H, s, COCH3). FAB-MS m/z: 284.07 (Mꢃ).
timately lead to new classes of potent therapeutic agents. Anal. Cacld for C16H12O5: C, 67.60; H, 4.25. Found: C, 67.29; H, 4.51.
4-(3-Acetoxybenzoyloxy)benzaldehyde (21) as white powder in a yield of
Further detailed studies of the biological activity of these
1
84%: mp: 64—65 °C (n-hexane). H-NMR (CDCl3) d: 10.01 (1H, s, CHO),
compounds will reveal their full potential.
8.08—7.37 (8H, m, ArH), 2.34 (3H, s, COCH3). FAB-MS m/z: 284.07 (Mꢃ).
Anal. Cacld for C16H12O5: C, 67.60; H, 4.25. Found: C, 67.45; H, 4.11.
Experimental
General Melting points (mp) were taken on a BUCHI 530 apparatus 53%: mp: 116—118 °C (EtOAc). H-NMR (CDCl3) d: 9.88 (1H, s, CHO),
and are uncorrected. Merck Art No105554 plates precoated with Silica gel
8.11—7.11 (8H, m, ArH), 2.21 (3H, s, COCH3). FAB-MS m/z: 284.07 (Mꢃ).
60 containing fluorescent indicator were used for thin-layer chromatography, Anal. Cacld for C16H12O5: C, 67.60; H, 4.25. Found: C, 67.61; H, 4.33.
and Silica gel 60 (Merck Art No 109385, 230—400 mesh) was employed for
4-(2,4-Diacetoxybenzoyloxy)benzaldehyde (23) as white powder in a
column chromatography. Evaporations were carried out at ꢅ50 °C using a yield of 53%: mp: 152—154 °C (EtOAc). 1H-NMR (CDCl3) d: 10.00 (1H, s,
4-(4-Acetoxybenzoyloxy)benzaldehyde (22) as white powder in a yield of
1
rotary evaporator at reduced pressure (water aspirator). 1H- and 13C-NMR
CHO), 8.25—7.02 (7H, m, ArH), 2.32, 2.29 (3H each, s, COCH3). FAB-MS
spectra were obtained at Varian 300 NMR spectrometer at 300 and 75 MHz, m/z: 342.07 (Mꢃ). Anal. Cacld for C18H14O7: C, 63.16; H, 4.12. Found: C,
respectively. Where necessary, deuterium exchange experiments were used 63.29; H, 4.31.
to obtained proton shift assignments. Mass spectra were recorded on a JEOL
J.M.S-300 spectrophotometer. Analytical samples were dried under reduced yield of 70%: mp: 124—125 °C (EtOAc). 1H-NMR (CDCl3) d: 10.00 (1H, s,
pressure at 78 °C in the presence of P2O5 for at least 12 h unless otherwise
CHO), 7.97—7.25 (7H, m, ArH), 2.32, 2.29 (3H each, s, COCH3). FAB-MS
specified. Elemental analyses were obtained from Perkin-Elmer 2400 Ele- m/z: 342.07 (Mꢃ). Anal. Cacld for C18H14O7: C, 63.16; H, 4.12. Found: C,
4-(2,5-Diacetoxybenzoyloxy)benzaldehyde (24) as white powder in a
mental Analyzer.
63.12; H, 4.30.
Acetylation of Phenol(s) of Polyphenolic Acid, General Procedure to
Obtain Compounds 9—16 To a solution of polyphenolic acid (20 mmol)
was added acetic anhydride (6 eq) and pyridine (2 ml). The mixture was CHO), 7.94—7.09 (7H, m, ArH), 2.17 (6H, s, COCH3). FAB-MS m/z:
4-(3,4-Diacetoxybenzoyloxy)benzaldehyde (25) as white powder in a
yield of 70%: mp: 100—101 °C (EtOAc). H-NMR (CDCl3) d: 9.86 (1H, s,
1
stirred at room temperature under dark for 4 h. Then poured onto 1 M H3PO4
342.07 (Mꢃ). Anal. Cacld for C18H14O7: C, 63.16; H, 4.12. Found: C, 63.08;

May 2006
H, 4.45.
685
lmax (EtOH) nm (log e): 303 (4.07). FAB-MS m/z: 648.1 (MꢃHꢃ). Anal.
4-(3,5-Diacetoxybenzoyloxy)benzaldehyde (26) as white powder in a Cacld for C36H24O12·H2O: C, 64.88; H, 3.78. Found: C, 64.83; H, 3.88.
1
yield of 59%: mp: 110—111 °C (EtOAc). H-NMR (CDCl3) d: 9.87 (1H, s,
CHO), 7.82—7.08 (7H, m, ArH), 2.17 (6H, s, COCH3). FAB-MS m/z: coumarin (37) as white powder in
342.07 (Mꢃ). Anal. Cacld for C18H14O7: C, 63.16; H, 4.12. Found: C, 63.24; hexane : EtOAcꢀ1 : 4). mp: 125—126 °C (EtOH). 1H-NMR (CDCl3) d:
H, 4.03.
11.57, 11.30 (s, 1H each, OH), 8.18—6.86 (m, 15H, ArH), 7.78, 6.47 (d,
3,3ꢁ-[4-(3,4-Dimethoxycinnamoyloxy)-benzylidene]bis-4-hydroxy-
a
yield of 50%: Rf: 0.15 (n-
4-(3,4-Dimethoxycinnamoyloxy)benzaldehyde (27) as white powder in a Jꢀ7.8 Hz, 1H each, CH), 6.07 (s, 1H, CH), 3.91 (s, 6H, OCH3). IR (KBr)
yield of 59%: mp: 88—90 °C (MeOH). 1H-NMR (CDCl3) d: 9.99 (s, 1H, cmꢂ1: 3471, 1821. UV lmax (EtOH) nm (log e): 313 (4.21). FAB-MS m/z:
CHO), 7.94—6.88 (m, 7H, ArH), 7.83, 6.48 (d, Jꢀ7.8 Hz, 1H each, CH),
3.92 (s, 6H, OCH3). FAB-MS m/z: 284.07 (Mꢃ). Anal. Cacld for C18H16O5: 69.91; H, 4.35.
619.16 (MꢃHꢃ). Anal. Cacld for C36H26O10: C, 69.90; H, 4.24. Found: C,
C, 69.22; H, 5.16. Found: C, 69.35; H, 5.31.
3,3ꢁ-[4-(3,4-Diacetoxycinnamoyloxy)]bis-4-hydroxycoumarin (38) as
4-(3,4-Diacetoxycinnamoyloxy)benzaldehyde (28) as white powder in a white powder in a yield of 43%: Rf: 0.20 (n-hexane : EtOAcꢀ1 : 4). mp:
yield of 59%: mp: 91—92 °C (MeOH). H-NMR (CDCl3) d: 10.00 (s, 1H, 118—120 °C (EtOHc). 1H-NMR (CDCl3) d: 8.05—7.11 (m, 15H, ArH),
1
CHO), 7.95—7.25 (m, 7H, ArH), 7.81, 6.56 (d, Jꢀ7.8 Hz, 1H each, CH),
7.77, 6.54 (d, Jꢀ8.1 Hz, 1H each, ꢀCH), 6.07 (s, 1H, CH), 2.30 (s, 6H,
CH3). IR (KBr) cmꢂ1: 3607, 1804. UV lmax (EtOH) nm (log e): 313 (4.28).
FAB-MS m/z: 675.15 (MꢃHꢃ). Anal. Cacld for C38H26O12: C, 67.66; H,
2.31, 2.30 (s, 3H each, CH3). FAB-MS m/z: 368.09 (Mꢃ). Anal. Cacld for
C20H16O7: C, 65.22; H, 4.38. Found: C, 65.29; H, 4.31.
1
4-(Liponyloxy)benzaldehyde (29) as an yellow oil in a yield of 83%: H- 3.88. Found: C, 67.49; H, 4.06.
NMR (CDCl3) d: 10.03 (s, 1H, CHO), 7.96, 7.31 (d, Jꢀ4.2 Hz, 2H each,
3,3ꢁ-[4-(Liponyloxy)benzylidene]bis-4-hydroxycoumarin (39) as yellow
ArH), 3.61—3.55 (m, 1H, H-6), 3.23—3.08 (m, 2H, H-8), 2.44—2.38 (m, powder in a yield of 50%: Rf: 0.15 (n-hexane : EtOAcꢀ1 : 4). mp: 247—
1
1H, H-7), 2.35 (t , Jꢀ7.2 Hz, 2H, H-2), 1.92—1.88 (m, 1H, H-7), 1.70— 249 °C (EtOAc). H-NMR (DMSO-d6) d: 7.81—6.86 (m, 12H, ArH), 6.24
1.60 (m, 4H, H-3 and H-5), 1.53—1.40 (m, 2H, H-4). 13C-NMR (CDCl3) d: (s, 1H, CH), 3.61—3.55 (m, 1H, H-6), 3.23—3.08 (m, 2H, H-8), 2.44—2.38
190.66, 171.07, 155.37, 133.99, 131.09, 122.26, 56.23, 40.20, 38.47, 34.53, (m, 1H, H-7), 2.35 (t, Jꢀ7.2 Hz, 2H, H-2), 1.88—1.92 (m, 1H, H-7), 1.70—
34.11, 28.61, 24.50. IR (KBr) cmꢂ1: 3425, 3120, 1676, 1076. FAB-MS m/z:
1.60 (m, 4H, H-3 and H-5), 1.53—1.40 (m, 2H, H-4). IR (KBr) cmꢂ1: 3611,
310.07 (Mꢃ). Anal. Cacld for C15H18O3S2: C, 58.04; H, 5.84. Found: C, 1752. UV lmax (EtOH) nm (log e): 307 (4.15). FAB-MS m/z: 617.13
58.35; H, 5.51.
(MꢃHꢃ). Anal. Cacld for C33H28O8S2·H2O: C, 62.45; H, 4.76. Found: C,
Condensation of Substituted Aromatic Aldehydes with 4-Hydroxy- 6.27; H, 4.81.
coumarin (30—39),6—8) General Procedure The reaction mixture of 4-
Hydrolysis of the Acetate of 30—38, General Procedure to Obtain
hydroxycoumarin (2.2 eqivalent), aromatic aldehyde (1 equivalent) in Compounds 40—47 The polyphenolic acids esters (5 mmol) was dis-
ethanol was heated under reflux for 48 h. The mixture was concentrated
under reduced pressure to furnished product. Pure products were obtained
under recrystalliztion with certain solvents.
solved in a mixture of THF (30 ml) and aqeous 3 N HCl (30 ml). The reac-
tion mixture was stirred for 2 d at room temperature and then extracted with
ethyl acetate three times. The combined organic layers were washed with
3,3ꢁ-[4-(2-Acetoxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin (30) brine and water. The organic phase was dried under magnesium sulfate, fil-
as white powder in a yield of 63%: Rf: 0.1 (n-hexane : EtOAcꢀ1 : 2). mp: tered, and the solvent was removed in vacuo to give the residue. Pure prod-
258—260 °C (hexane). 1H-NMR (DMSO-d6) d: 8.18—6.98 (m, 16H, ArH),
6.28 (s, 1H, CH), 2.23 (s, 3H, CH3). IR (KBr) cmꢂ1: 3545, 1745. UV lmax
(EtOH) nm (log e): 305 (4.18). FAB-MS m/z: 586.98 (MꢃHꢃ). Anal. Cacld
for C34H22O10·2H2O: C, 65.60; H, 4.18. Found: C, 65.62; H, 3.99.
ucts were obtained under recrystalliztion with certain solvents.
3,3ꢁ-[4-(2-Hydroxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin (40)
as white powder in a yield of 96%: Rf: 0.2 (n-hexane : EtOAcꢀ1 : 2). mp:
238—239 °C (MeOH). 1H-NMR (CDCl3) d: 11.57, 11.30, 10.96 (s, 1H
3,3ꢁ-[4-(3-Acetoxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin (31) each, OH), 8.15—6.95 (m, 16H, ArH), 6.10 (s, 1H, CH). IR (KBr) cmꢂ1
:
as white powder in a yield of 60%: Rf: 0.15 (n-hexane : EtOAcꢀ1 : 2). mp: 3479, 1808. UV lmax (EtOH) nm (log e): 316 (4.31). FAB-MS m/z: 549.0
270—272 °C (hexane). 1H-NMR (DMSO-d6) d: 8.00—7.06 (m, 16H, ArH),
6.28 (s, 1H, CH), 2.28 (s, 3H, CH3). IR (KBr) cmꢂ1: 3437, 1752. UV lmax
(EtOH) nm (log e): 304 (4.19). FAB-MS m/z: 586.99 (MꢃHꢃ). Anal. Cacld
for C34H22O10·2H2O: C, 65.60; H, 4.18. Found: C, 65.36; H, 4.19.
(MꢃHꢃ). Anal. Cacld for C32H20O9: C, 70.07; H, 3.68. Found: C, 70.45; H,
3.55.
3,3ꢁ-[4-(3-Hydroxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin (41)
as white powder in a yield of 71%: Rf: 0.05 (CH2Cl2 : MeOHꢀ19 : 1). mp:
1
3,3ꢁ-[4-(4-Acetoxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin (32) 232—234 °C (EtOAc). H-NMR (DMSO-d6) d: 7.89—7.07 (m, 16H, ArH),
as white powder in a yield of 58%: Rf: 0.20 (n-hexane : EtOAcꢀ1 : 4). mp: 6.33 (s, 1H, CH). IR (KBr) cmꢂ1: 3611, 1782. UV lmax (EtOH) nm (log e):
252—254 °C (AcOEt). 1H-NMR (DMSO-d6) d: 8.14—7.01 (m, 16H, ArH),
6.28 (s, 1H, CH), 2.30 (s, 3H, CH3). IR (KBr) cmꢂ1: 3432, 1732. UV lmax
(EtOH) nm (log e): 305 (4.18). FAB-MS m/z: 587.05 (MꢃHꢃ). Anal. Cacld
for C34H22O10·2.5H2O: C, 64.26; H, 4.12. Found: C, 64.08; H, 4.08.
322 (4.31). FAB-MS m/z: 549.0 (MꢃHꢃ). Anal. Cacld for C32H20O9: C,
70.07; H, 3.68. Found: C, 70.05; H, 4.00.
3,3ꢁ-[4-(4-Hydroxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin (42)
as white powder in a yield of 69%: Rf: 0.1 (CH2Cl2 : MeOHꢀ19 : 1).
1
3,3ꢁ-[4-(2,4-Diacetoxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin mp: 258—260 °C (CH2Cl2). H-NMR (DMSO-d6) d: 7.95—6.88 (m, 16H,
(33) as white powder in a yield of 76%: Rf: 0.20 (n-hexane : EtOAcꢀ1 : 4). ArH), 6.29 (s, 1H, CH). IR (KBr) cmꢂ1: 3479, 1823. UV lmax (EtOH) nm
mp: 232—234 °C (AcOEt). 1H-NMR (DMSO-d6) d: 8.18—6.98 (m, 15H, (log e): 321 (4.36). FAB-MS m/z: 549.0 (MꢃHꢃ). Anal. Cacld for
ArH), 6.27 (s, 1H, CH), 2.23, 2.29 (s, 3H each, CH3). IR (KBr) cmꢂ1: 3436, C32H20O9·2.5H2O: C, 64.76; H, 3.82. Found: C, 64.49; H, 4.01.
1648. UV lmax (EtOH) nm (log e): 307 (4.21). FAB-MS m/z: 644.98
(MꢃHꢃ). Anal. Cacld for C36H24O12·2H2O: C, 63.17; H, 4.12. Found: C, (43) as white powder in a yield of 60%: Rf: 0.2 (CH2Cl2 : MeOHꢀ9 : 1). mp:
63.15; H, 3.96.
ꢄ300 °C (CH2Cl2). 1H-NMR (DMSO-d6) d: 10.59, 10.42 (s, 1H each, OH),
3,3ꢁ-[4-(2,4-Dihydroxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin
3,3ꢁ-[4-(2,5-Diacetoxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin 7.85—7.05 (m, 13H, ArH), 6.42 (d, Jꢀ4.2 Hz, 1H, ArH), 6.34 (d, Jꢀ1.2 Hz,
(34) as white powder in a yield of 60%: Rf: 0.20 (n-hexane : EtOAcꢀ1 : 4). 1H, ArH), 6.27 (s, 1H, CH). IR (KBr) cmꢂ1: 3612, 1682. UV lmax (EtOH)
mp: 241—243 °C (AcOEt). 1H-NMR (DMSO-d6) d: 7.86—6.99 (m, 15H, nm (log e): 313 (4.09). FAB-MS m/z: 565.2 (MꢃHꢃ). Anal. Cacld for
ArH), 6.27 (s, 1H, CH), 2.24, 2.28 (s, 3H each, CH3). IR (KBr) cmꢂ1: 3468, C32H20O10·3H2O: C, 62.14; H, 3.74. Found: C, 62.45; H, 3.79.
1702. UV lmax (EtOH) nm (log e): 305 (4.18). FAB-MS m/z: 644.98
3,3ꢁ-[4-(2,5-Dihydroxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin
(MꢃHꢃ). Anal. Cacld for C36H24O12·1.5H2O: C, 64.01; H, 3.80. Found: C, (44) as white powder in a yield of 44%: Rf: 0.12 (CH2Cl2 : MeOHꢀ9 : 1).
1
63.93; H, 3.98.
mp: 262—264 °C (CH2Cl2). H-NMR (DMSO-d6) d: 7.89—6.88 (m, 15H,
3,3ꢁ-[4-(3,4-Diacetoxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin ArH), 6.33 (s, 1H, CH). IR (KBr) cmꢂ1: 3619, 1829. UV lmax (EtOH) nm
(35) as white powder in a yield of 36%: Rf: 0.15 (CH2Cl2 : MeOHꢀ19 : 1).
(log e): 307 (4.13). FAB-MS m/z: 565.0 (MꢃHꢃ). Anal. Cacld for
mp: 178—180 °C (AcOEt). 1H-NMR (DMSO-d6) d: 7.99—7.07 (m, 15H, C32H20O10·0.5H2O: C, 67.01; H, 3.60. Found: C, 66.87; H, 3.65.
ArH), 6.30 (s, 1H, CH), 2.26, 2.29 (s, 3H each, CH3). IR (KBr) cmꢂ1: 3511,
3,3ꢁ-[4-(3,4-Dihydroxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin
1706. UV lmax (EtOH) nm (log e): 301 (4.09). FAB-MS m/z: 645.0 (45) as white powder in a yield of 43%: Rf: 0.10 (CH2Cl2 : MeOHꢀ9 : 1).
1
(MꢃHꢃ). Anal. Cacld for C36H24O12·H2O: C, 64.88; H, 3.78. Found: C, mp: 210—212 °C (CH2Cl2). H-NMR (DMSO-d6) d: 7.87—6.84 (m, 15H,
65.11; H, 4.04.
ArH), 6.31 (s, 1H, CH). IR (KBr) cmꢂ1: 3571, 1722. UV lmax (EtOH) nm
3,3ꢁ-[4-(3,5-Diacetoxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin (log e): 322 (4.33). FAB-MS m/z: 565.2 (MꢃHꢃ). Anal. Cacld for
(36) as white powder in a yield of 33%: Rf: 0.15 (n-hexane : EtOAcꢀ1 : 4). C32H20O10·4.5H2O: C, 59.54; H, 3.95. Found: C, 59.78; H, 4.19.
mp: 116—118 °C (AcOEt). 1H-NMR (CDCl3) d: 8.07—7.12 (m, 15H,
3,3ꢁ-[4-(3,5-Dihydroxybenzoyloxy)benzylidene]bis-4-hydroxycoumarin
ArH), 6.08 (s, 1H, CH), 2.31 (s, 6H, CH3). IR (KBr) cmꢂ1: 3462, 1762. UV (46) as white powder in a yield of 64%: Rf: 0.10 (CH2Cl2 : MeOHꢀ19 : 1).

686
Vol. 54, No. 5
1
fractional product in percent of the control without drug.
mp: 177—181 °C (CH2Cl2). H-NMR (DMSO-d6) d: 7.89—6.95 (m, 14H,
ArH), 6.49 (s, 1H, ArH), 6.33 (s, 1H, CH). IR (KBr) cmꢂ1: 3546, 1722. UV
lmax (EtOH) nm (log e): 313 (4.18). FAB-MS m/z: 565.1 (MꢃHꢃ). Anal.
Cacld for C32H20O10·0.5H2O: C, 67.01; H, 3.60. Found: C, 66.87; H, 3.88.
3,3ꢁ-[4-(3,4-Dihydrox-cinnamoyloxy]bis-4-hydroxycoumarin (47) as yel-
low powder in a yield of 68%: Rf: 0.10 (CH2Cl2 : MeOHꢀ9 : 1). mp: 266—
268 °C (CH2Cl2). 1H-NMR (DMSO-d6) d: 9.65, 9.18 (s, 1H each, OH),
7.81—6.77 (m, 15H, ArH), 7.62, 6.45 (d, Jꢀ8.1 Hz, 1H each, ꢀCH), 6.25
(s, 1H, CH). IR (KBr) cmꢂ1: 3421, 1802. UV lmax (EtOH) nm (log e): 305
(4.18). FAB-MS m/z: 591.1 (MꢃHꢃ). Anal. Cacld for C34H22O10·4H2O: C,
61.64; H, 4.56. Found: C, 61.35; H, 4.32.
HIV-1 Integrase Inhibitory Assay. Oligonucleotides Oligonu-
cleotides were purchased from Eurogentec and further purified on 18% acry-
lamide/urea denaturing gel. U5B: GTGTGGAAAATCTCTAGCA; U5B-2:
GTGTGGAAAATCTCTAG; U5A: 5ꢁ-ACTGCTAGAGATTTTCCACAC;
ST1: AGTGAATTAGCCCTTGGTCA-biotine; ST2: 5ꢁ-TGACCAAGGGC-
TAATTCACT-biotine; U5B and U5B-2 were radiolabeled using T4 polynu-
cleotide kinase for respectively 3ꢁ-processing and strand transfer reactions.
HIV-1 Integrase Assays Wild-type HIV-1 integrase was purified as de-
scribsed previously.8) 3ꢁ-Processing assay was performed in a reaction vol-
ume of 20 ml containing 0.025 pmol of labeled U5A/U5B double-stranded
DNA substrate and 1 pmol of integrase in buffer A [20 mM Hepes (pH 7.2),
10 mM MgCl2, 25 mM NaCl, 1 mM DTT]. Products were separated on a 18%
acrylamide/urea denaturing gel and quantified on a phosphoimager using
Image Quant software (Amersham Pharmacia Biotech). Strand transfer reac-
tions were performed in triplicate in 96-well plates using 0.25 pmol of la-
beled U5A/U5B-2 double-stranded DNA substrate, 12 pmol of ST1/ST2 3ꢁ-
biotinylated target DNA and 2 pmol of integrase in buffer A in a final vol-
ume of 40 ml. Radiolabeled reaction products were bound to Streptavidin-
coated magnetic beads (DynaL), washed twice in buffer B (PBS buffer sup-
plemented with 0.025% tween 20 and 10 mg/ml BSA) and quantified on a
beta radiation counter. Inhibition in the presence of drugs is expressed as the
Acknowledgments Financial support from National Science Council
(NSC 92-2320-B016-048) of the Republic of China is gratefully acknowl-
edged. We thank Ms. Shu-Yun Sun and Ching-Wei Lu, Instrumentation Cen-
ter, NSC for their help in obtaining the mass spectra and elemental analysis.
References
1) Moore J. P., Stevenson M., Nat. Rev. Mol. Cell Biol., 1, 40—49 (2000).
2) Miller M. D., Hazuda D. J., Curr. Opin. Microbiol., 4, 535—539
(2001).
3) Taddeo B., Haseltine W. A., Farnet C. M., J. Virol., 68, 8401—8405
(1994).
4) Wiskerchen M., Muesing M. A., J. Virol., 69, 376—386 (1995).
5) Yu D., Suzuki M., Xie L., Morris-Natschke S. L., Lee K.-H., Med.
Res. Rev., 23, 322—345 (2003).
6) Mazumder A., Wang S., Neamati N., Nicklaus M., Sunder S., Chen J.,
Milne G. W. A., Rice W. G., Pommier Y., J. Med. Chem., 40, 2472—
2481 (1996).
7) Zhao H., Neamati N., Hong H., Mazumder A., Wang S., Sunder S.,
Milne G. W. A., Pommier Y., Burke T. R., Jr., J. Med. Chem., 40,
242—249 (1997).
8) Mao P. C.-M., Mouscadet J.-F., Leh H., Auclair C., Hsu L. Y., Hetero-
cycles, 55, 1263—1270 (2001).
9) Mao P. C.-M., Mouscadet J.-F., Leh H., Auclair C., Hsu L. Y., Chem.
Pharm. Bull., 50, 1634—1637 (2002).
10) Mekouar K., Mouscadet J. F., Desmaele D., Subra F., Leh H., Savoure
D., Auclair C., d’Angelo J., J. Med. Chem., 41, 2846—2857 (1998).
11) Inayama S., Harimaya K., Hori H., Ohkura T., Kawamata T., Hikichi
M., Yokokura T., Chem. Pharm. Bull., 32, 1135—1141 (1984).
12) Andrus M. B., Liu J., Meredith E. L., Nartey E., Tetrahedron Lett., 44,
4819—4822 (2003).