Synthesis and HIV-1 integrase inhibitory activities of caffeic acid dimers derived from Salvia officinalis

Article abstract of DOI:10.1016/j.bmcl.2005.07.091

The synthesis of two caffeoyl-coumarin conjugates, derived from sagecoumarin, has been accomplished, starting from ferulic acid, isoferulic acid and sesamol. Both compounds exhibited potent inhibitory activities at micromolar concentrations against HIV-1 integrase in 3′-end processing reaction but were less effective against HIV-1 replication in a single-round infection assay of HeLa-β-gal-CD4+ cells.

Full text of DOI:10.1016/j.bmcl.2005.07.091

Bioorganic & Medicinal Chemistry Letters 15 (2005) 5053–5056
Synthesis and HIV-1 integrase inhibitory activities of caffeic
acid dimers derived from Salvia officinalis
a
b
Fabrice Bailly,a, Clemence Queffelec, Gladys Mbemba,
*
´
Jean-Franc¸ois Mouscadetb and Philippe Cotellea
a
´ ´
Laboratoire de Chimie Organique et Macromoleculaire, UMR CNRS 8009, Universite des Sciences et Technologies de Lille I,
59655 Villeneuve dÕAscq, France
b
´ ´
´
Laboratoire de Biotechnologies et Pharmacologie Genetique Appliquee, UMR CNRS 8113, ENS Cachan,
´
61 Avenue du President Wilson, 94325 Cachan, France
Received 17 June 2005; revised 28 July 2005; accepted 29 July 2005
Abstract—The synthesis of two caffeoyl-coumarin conjugates, derived from sagecoumarin, has been accomplished, starting from
ferulic acid, isoferulic acid and sesamol. Both compounds exhibited potent inhibitory activities at micromolar concentrations against
HIV-1 integrase in 30-end processing reaction but were less effective against HIV-1 replication in a single-round infection assay of
HeLa-b-gal-CD4+ cells.
Ó 2005 Elsevier Ltd. All rights reserved.
Sage (Salvia officinalis) is a popular herb which is widely
cultivated in various parts of the world and used in fla-
vouring and folk medicines. In various studies, sage has
been shown to be the most potent natural antioxidant of
the common spices.1 Its antioxidant effect has been
attributed to the main phenolic compounds, rosmarinic
acid, a caffeic acid dimer and carnosic acid.2 Sage also
contains an impressive array of biologically active caffeic
acid oligomers, ranging from trimers, tetramers and
higher oligomers, such as salvianolic acids, lithospermic
acids and yunnaneic acids.3 Along with salvianolic acid
K (a caffeic acid trimer), sagerinic acid (a tetramer) and
rosmarinic acid, Lu et al. also characterized three
further caffeic acid trimers, melitric acid A, methyl
melitrate A and sagecoumarin.4 This last compound
was particularly interesting as it was the first caffeic acid
oligomer with a coumarin structure. The full assignment
HO
HO
O
O
O
O
O
COOH
OH
OH
HO
Sagecoumarin
O
HO
HO
O
CO2H
O
8a
HO
O
CO2H
HO
HO
O
O
8b
HO
Figure 1. Structures of sagecoumarin and caffeoyl-coumarin
conjugates.
1
of the H and 13C NMR resonances of sagecoumarin
showed that it is a 6,7-dihydroxycoumarin linked via
an ether bond on its C-3 position to a rosmarinic acid,
as shown in Figure 1. It exhibited strong antioxidant
activities.5
Amongst the several varied biological responses elicited
by the caffeic acid derivatives, the inhibition of human
immunodeficiency virus type 1 (HIV-1), integrase ap-
peared recently as their most promising biological
activity. Apart from reverse transcriptase and protease,
the two enzymes targeted by clinically used anti-HIV
drugs, integrase has recently emerged as an attractive
and alternative target. This led to the elaboration of
a great number of HIV-1 integrase inhibitors.6–9 The
Keywords: Sagecoumarin; Caffeic acid dimers; HIV-1 integrase;
Antiviral.
*
Corresponding author. Tel.: +33 320337231; fax: +33 320336309;
e-mail: Fabrice.Bailly@univ-lille1.fr
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.091

5054
F. Bailly et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5053–5056
main classes of inhibitors included mononucleotides,10
dinucleotides,11,12 oligonucleotides,13,14 peptides,15
flavonoids,16 natural polyaromatics,17 geometrically re-
strained bis-catechols,18 salicyl and thiosalicylhydra-
zines,19,20 naphthalenesulfonic acid derivatives,21
diketo derivatives22,23 and diketo acids.24 Caffeic acid
derivatives represented another widely studied class of
natural and synthetic HIV-1 integrase inhibitors.
Dicaffeoyltartaric acids and dicaffeoylquinic acids,25
rosmarinic acid,22 lithospermic acid B and lithospermic
acid,26 L-chicoric acid,25a were found to be potent
inhibitors of 30-processing and strand transfer reactions
of HIV-1 integrase. Furthermore, lithospermic acid was
weakly cytotoxic.
OCH3
OH
OR2
OR1
i
HO2C
H3CO2C
1a (R1 : H, R2 : CH3)
Ferulic acid
ii
2a (R1 : CH2CO2H, R2 : CH3)
3a (R1 : CH2COCl, R2 : CH3)
OR2
iii
OH
i
OCH3
OR1
HO2C
H3CO2C
Isoferulic acid
1b (R1 : CH3, R2 : H)
ii
2b (R1 : CH3, R2 : CH2CO2H)
iii
In our laboratory, we are interested in the design, synthe-
sis and pharmacological evaluation of polyhydroxylated
lead molecules with antioxidant potencies aimed at inhib-
iting HIV-1 integrase.27 In this paper, we report a simple
and efficient synthesis of two caffeoyl-coumarin conju-
gates, derived from sagecoumarin (Fig. 1). The newly
synthesized compounds were tested in vitro for their
HIV-1 integrase inhibitory activities and antiviral effect.
3b (R1 : CH3, R2 : CH2CO2H)
Scheme 1. Reagents and conditions: (i) SOCl2, MeOH, 0 °C then
reflux, 1a 95%, 1b 95%; (ii) BrCH2CO2H, NaH, THF, rt then reflux, 2a
60%, 2b 77%; (iii) SOCl2, AcOEt, 3a 97%, 3b 98%.
O- +NHEt3
CHO
OH
OH
O
O
O
O
i
ii
O
O
CHO
We first attempted to synthesize the coumarin deriva-
tives by simple one-step routes.
5
6
Scheme 2. Reagents and conditions: (i) C6H12N4, TFA, CH2Cl2, 0 °C
then reflux, 65%; (ii) NEt3, CH2Cl2, rt.
The complex of phosphorus oxychloride and N,N-diet-
hylphenoxyacetamides on reaction with substituted sali-
cylaldehydes was shown to yield various 3-substituted
coumarins.28 Unfortunately, only reactants were recov-
ered by this method. The literature also reported another
similar method based on the condensation of salicylalde-
hydes and phenoxyacetic acids using triethylamine, ace-
tic anhydride and DMF or benzene as solvent.29 Here
again, we were not able to isolate the products in satisfac-
tory yields. Then we turned to another phosphorus acti-
vating agent, phenyldichlorophosphate.30 Surprisingly,
heating under reflux of a solution containing salicylalde-
hyde 5, phenoxyacetic acids 2a,b, triethylamine and
phenyldichlorophosphate did not give the desired cou-
marins but the esters 4a,b formed by coupling of the
two main precursors in 17–25% yield. These novel com-
pounds were expected to be excellent precursors of the
coumarins by cyclisation under basic conditions.
using hexamethylenetetramine and trifluoroacetic acid
under reflux afforded the salicylaldehyde derivative 5
in 64% yield.
Scheme 3 shows the final sequence adopted for the syn-
thesis of the coumarin derivatives.
H3CO
OCOCH2O
R1
O
O
i
3a
+
6
R2
(3b)
CHO
4a (R1 : CH=CH-CO2CH3, R2 : H)
4b (R1 : H, R2 : CH=CH-CO2CH3)
O
O
R2
O
O
ii
The final sequence was based on the preliminary isola-
tion of the esters 4a,b as key intermediates. These esters
were obtained from two precursors, namely the phe-
noxyacetic acid chlorides 3a,b (Scheme 1) and the sali-
cylaldehyde derivative 5 (Scheme 2). Synthesis began
from 4-hydroxy-3-methoxycinnamic acid (ferulic acid)
and its isomer, 3-hydroxy-4-methoxycinnamic acid (iso-
ferulic acid). In a first step, they were converted to their
methyl ester, respectively, 1a and b, by heating under re-
flux with a methanolic solution of thionyl chloride. Then
the phenolic groups of 1a,b were reacted with bromoace-
tic acid in the presence of sodium hydride to give the
phenoxyacetic acids 2a,b in 60% and 77% yield.
O
R1
H3CO
7a (R1 : CH=CH-CO2CH3, R2 : H)
7b (R1 : H, R2 : CH=CH-CO2CH3)
O
O
iii
HO
HO
R2
O
R1
HO
8a (R1 : CH=CH-CO2H, R2 : H)
8b (R1 : H, R2 : CH=CH-CO2H)
For the elaboration of the other precursor 5, we turned
to a simple one-step sequence starting from the commer-
cially available sesamol (Scheme 2). Duff formylation31
Scheme 3. Reagents and conditions: (i) 0 °C then rt, 4a 82%, 4b 65%;
(ii) DBU, toluene, reflux, 7a 25%, 7b 20%; (iii) a—BBr3, CH2Cl2, rt
then reflux; b—H2O, rt then reflux, 8a 90%, 8b 68%.

F. Bailly et al. / Bioorg. Med. Chem. Lett. 15 (2005) 5053–5056
Table 1. HIV-1 integrase inhibitory potencies (IC50) and antiviral
5055
In summary, a concise and rapid synthesis of 8a,b was
achieved in six steps from ferulic and isoferulic acids
with 10% and 6.3% overall yields, respectively. The
present synthetic route could be used for the elaboration
of novel oligomeric caffeic acid derivatives. Although
the compounds were effective in inhibiting in vitro
HIV-1 integrase and presented low cytotoxicities,
they were modest inhibitors of HIV-1 replication in
HeLa-b-gal-CD4+ cells.
activities (EC50
)
Entry
IC50 (lM)
EC50 (lM)
3,6,7-Trihydroxycoumarin
2.0
2.0
1.0
12.0
37.0
31.0
8a
8b
For the condensation of the phenoxyacetic acid chlo-
rides 3a,b and the salicylaldehyde 5, 5 was converted
to its phenolate 6 and then mixed with 3a,b to give 4a
(82%) and 4b (65%). Cyclisation of 4a,b to the corre-
sponding coumarins 7a,b was ineffective with sodium
methylate or sodium hydride. But refluxing with DBU
(1,8-diazabicyclo-[5.4.0]-7-undecene; ca. 1.6 mol equiv)
in toluene as previously described32 afforded the couma-
rins 7a,b in modest but reproducible yields (25% and
20%). Final deprotection of 7a,b to their corresponding
polyhydroxylated counterparts 8a,b was accomplished,
using boron tribromide. Standard conditions with an ex-
cess of BBr3 at ꢀ78 °C then room temperature did not
give the fully deprotected coumarins 8a,b. More drastic
conditions were necessary with the reflux of the solution
after the addition of boron tribromide and hydrolysis.
The coumarins 8a,b were obtained in 90% and 68%
yield, respectively. Intermediates and final compounds
of both series were fully characterized by IR, 1H
Acknowledgments
This work was supported by funds from the Centre
National de la Recherche Scientifique (CNRS) and the
Agence Nationale de la Recherche sur le SIDA (ANRS).
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13
NMR, C NMR and MS.33
Each new compound was screened for inhibitory activity
against HIV-1 integrase in Mg2+-dependent 30-end pro-
cessing reaction. 3,6,7-Trihydroxycoumarin27b was also
tested for comparative studies. Table 1 shows that
micromolar activities (IC50) were obtained for the three
compounds.
They were also evaluated for their antiviral activities
against the HIV-1 early replicative steps in HeLa-b-
gal-CD4+ (P4) reporter cells. P4 cells were infected with
HIV-1 for 2 h and subsequently treated with increasing
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three times less potent than 3,6,7-trihydroxycoumarin.
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180 (12%), 179 (100%), 175 (8%), 166 (11%), 165 (13%), 145
(7%). Compound 4b: beige solid; mp 180–184 °C; IR mmax
(KBr/cmꢀ1) 1785, 1721, 1707, 1676, 1609, 1505, 1491, 1436,
1274, 1175, 1143, 1034; 1H NMR (DMSO-d6): 3.72 (s, 3H),
3.83 (s, 3H), 5.23 (s, 2H), 6.22 (s, 2H), 6.62 (d, 1H,
3
3J = 16.0 Hz), 6.91 (d, 1H, J = 8.3 Hz), 6.93 (s, 1H), 7.34
(m, 2H), 7.53 (s, 1H), 7.60 (d, 1H, 3J = 16.0 Hz), 9.95 (s, 1H);
13C NMR (DMSO-d6): 45.1, 49.5, 58.8, 97.0, 98.3, 100.4,
105.9, 106.0, 109.4, 115.7, 117.7, 120.6, 138.2, 139.9, 140.8,
141.9, 144.8, 146.8, 160.7, 161.4, 181.7; IEMS (70 eV): 414
(M+Å, 12%), 221 (10%), 208 (10%), 207 (8%), 206 (40%), 189
(12%), 180 (11%), 179 (100%), 175 (9%), 166 (13%), 165
(14%), 161 (11%), 145 (7%). Compound 7a: white solid; mp
234 °C; IR mmax (KBr/cmꢀ1) 1722, 1640, 1600, 1505, 1487,
1455, 1430, 1352, 1306, 1261, 1196, 1175, 1156, 1133, 1070;
1H NMR (CDCl3): 3.79 (s, 3H), 3.87 (s, 3H), 6.02 (s, 2H),
6.38 (d, 1H, 3J = 16.0 Hz), 6.68 (s, 1H), 6.84 (s, 1H), 6.86 (s,
3
1H), 7.03 (d, 1H, J = 8.0 Hz), 7.10–7.13 (m, 2H), 7.65 (d,
1H, 3J = 16.0 Hz); 13C NMR (CDCl3): 51.8, 56.1, 98.2,
102.2, 104.4, 111.9, 112.5, 117.7, 120.4, 121.7, 122.2, 132.2,
140.2, 144.0, 145.2, 145.4, 147.3, 149.6, 150.8, 157.2, 167.2;
IEMS (70 eV): 397 (M+Å+H, 11%), 396 (M+Å, 45%), 365
(7%), 309 (10%), 204 (27%), 203 (100%), 177 (25%), 175
(14%). Compound 7b: beige solid; mp 199–203 °C; IR mmax
(KBr/cmꢀ1) 1716, 1637, 1599, 1519, 1488, 1451, 1329, 1263,
1
1223, 1176, 1164, 1132, 1023; H NMR (CDCl3): 3.75 (s,
3H), 3.82 (s, 3H), 6.02 (s, 2H), 6.26 (d, 1H,3J = 16.0 Hz),
6.66 (s, 1H), 6.76 (s, 1H), 6.82 (s, 1H), 6.99 (d, 1H,
3J = 8.6 Hz), 7.22 (s, 1H), 7.35 (d, 1H, 3J = 8.6 Hz), 7.60 (d,
1H, 3J = 16.0 Hz); 13C NMR (CDCl3): 45.4, 50.0, 92.0, 96.0,
98.2, 106.4, 106.8, 110.4, 113.4, 115.1, 120.5, 121.7, 134.4,
137.3, 137.5, 139.0, 141.0, 143.3, 146.3, 151.0, 161.1; IEMS
(70 eV): 397 (M+Å+H, 11%), 396 (M+Å, 49%), 365 (10%), 204
(21%), 203 (100%), 177 (24%), 175 (16%). Compound 8a:
brown solid; mp 248 °C (dec); IR mmax (KBr/cmꢀ1) 1670,
1605, 1523, 1444, 1406, 1346, 1278, 1262, 1228, 1217, 1136,
1100; 1H NMR (acetone-d6): 6.42 (d, 1H, 3J = 15.9 Hz),
6.84 (s, 1H), 7.01 (s, 1H), 7.09 (d, 1H, 3J = 8.4 Hz), 7.16 (dd,
1H, 3J = 8.4 Hz, 4J = 1.8 Hz), 7.28 (s, 1H), 7.30 (d, 1H,
4J = 1.8 Hz), 7.09 (d, 1H, 3J = 15.9 Hz); 13C NMR (ace-
tone-d6): 103.3, 112.1, 112.5, 117.1, 118.5, 120.6, 121.5,
124.8, 132.7, 140.0, 143.7, 144.9, 145.8, 146.8, 148.8, 149.2,
158.0, 167.8; IEMS (70 eV): 357 (M+Å+H, 16%), 356 (M+Å,
80%), 339 (7%), 338 (20%), 253 (8%), 192 (8%), 191 (100%),
178 (46%), 177 (94%), 166 (13%), 165 (26%), 163 (7%), 162
(27%), 150 (46%), 137 (10%), 121 (11%), 67 (11%). Com-
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pound 8b: black solid; mp 280 °C (dec); IR mmax (KBr/cmꢀ1
)
1666, 1620, 1572, 1513, 1456, 1435, 1411, 1284, 1215, 1184;
3
1H NMR (acetone-d6): 6.35 (d, 1H, J = 16.0 Hz), 6.81 (s,
1H), 6.88 (s, 1H), 7.03 (d, 1H, 3J = 8.4 Hz), 7.28 (s, 1H), 7.41
(dd, 1H, 3J = 8.4 Hz, 4J = 2.0 Hz), 7.49 (d, 1H, 4J = 2.0 Hz),
7.57 (d, 1H, 3J = 16.0 Hz), 8.38 (br s, 1H), 8.91 (br s, 1H),
9.00 (br s, 1H); 13C NMR (acetone-d6): 97.2, 106.0, 106.3,
110.7, 112.2, 114.2, 118.1, 120.9, 121.8, 134.3, 137.5, 138.0,
138.6, 140.5, 142.6, 145.1, 152.0, 161.8; IEMS (70 eV): 357
(M+Å+H, 11%), 356 (M+Å, 54%), 339 (8%), 338 (33%), 328
(7%), 312 (8%), 191 (45%), 178 (63%), 177 (90%), 165 (23%),
162 (61%), 150 (71%), 137 (9%).
30. Gallastegui, J.; Lago, J. M.; Palomo, C. J. Chem. Res. (S)
1984, 170.
31. (a) Smith, W. E. J. Org. Chem. 1972, 37, 3972; (b) Saito,
N.; Tashiro, K.; Maru, Y.; Yamaguchi, K.; Kubo, A.
J. Chem. Soc., Perkin Trans. 1 1997, 53.
32. Yamaguchi, S.; Saitoh, M.; Kawase, Y. J. Heterocycl.
Chem. 1991, 28, 125.
33. Compound 4a: beige solid; mp 163–167 °C; IR mmax (KBr/
cmꢀ1) 1794, 1701, 1670, 1638, 1623, 1518, 1478, 1438, 1309,
1268, 1202, 1186, 1167, 1137, 1078; 1H NMR (CDCl3): 3.80
(s, 3H), 3.93 (s, 3H), 5.05 (s, 2H), 6.09 (s, 2H), 6.34 (d, 1H,
34. Mahmood, N.; Moore, P. S.; De Tommasi, N.; De
Simone, F.; Colman, S.; Hay, A. J.; Pizza, C. Antivir.
Chem. Chemother. 1993, 4, 235.
35. Pluymers, W.; Neamati, N.; Pannecouque, C.; Fikkert, V.;
Marchand, C.; Burke, T. R., Jr.; Pommier, Y.; Schols, D.;
De Clercq, E.; Debyser, Z.; Witvrouw, M. Mol. Pharma-
col. 2000, 58, 641.
36. Reinke, A. R.; Lee, D. J.; McDougall, B. R.; King, P. J.;
Victoria, J.; Mao, Y.; Lei, X.; Reinecke, M. G.; Robinson,
W. E., Jr. Virology 2004, 326, 203.
3
3J = 15.9 Hz), 6.67 (s, 1H), 7.02 (d, 1H, J = 8.3 Hz), 7.05
(m, 2H), 7.07 (m, 2H), 7.25 (d, 1H, 3J = 1.9 Hz), 7.63 (d, 1H,
3J = 15.9 Hz), 9.87 (s, 1H); 13C NMR (CDCl3): 51.7, 56.0,
66.2, 102.9, 104.3, 108.3, 110.8, 114.4, 116.5, 122.0, 122.1,
129.4, 144.3, 146.4, 147.9, 148.9, 149.8, 153.2, 167.2, 167.5,
186.7; IEMS (70 eV): 414 (M+Å, 12%), 207 (11%), 206 (26%),