The total synthesis of fukiic acid, an HIV-1 integrase inhibitor

Article abstract of DOI:10.1016/j.ejmech.2007.12.013

A successful synthesis of fukiic acid is described in 7% overall yield (6 steps from veratraldehyde). rac-Fukiic acid was found to be a potent inhibitor of HIV-1 integrase but did not reveal any antiviral activity in the MT-4 cells assay.

Full text of DOI:10.1016/j.ejmech.2007.12.013

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2268e2271
http://www.elsevier.com/locate/ejmech
Short communication
The total synthesis of fukiic acid, an HIV-1 integrase inhibitor
a
Clemence Queffelec , Fabrice Bailly , Gladys Mbemba , Jean-Franc¸ois Mouscadet ,
a
b
b
´
´
Zeger Debyser c, Myriam Witvrouw c, Philippe Cotelle a,
*
a Laboratoire de Chimie Organique et Macromole´culaire, CNRS UMR 8009, Universite´ de Lille 1, 59655 Villeneuve d’Ascq, France
b Laboratoire de Biotechnologies et Pharmacologie Ge´ne´tique Applique´e, CNRS UMR 8113, Ecole Normale Supe´rieure de Cachan,
61 av du Pre´sident Wilson, 94235 Cachan, France
c Molecular Medicine, K.U.Leuven and IRC KULAK, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
Received 27 July 2007; received in revised form 20 November 2007; accepted 11 December 2007
Available online 27 December 2007
Abstract
A successful synthesis of fukiic acid is described in 7% overall yield (6 steps from veratraldehyde). rac-Fukiic acid was found to be a potent
inhibitor of HIV-1 integrase but did not reveal any antiviral activity in the MT-4 cells assay.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Natural polyphenol; Anti-HIV-1; Integrase inhibitors
1. Introduction
activity and specially actein. The latter has also revealed anti-
HIV activity [8]. Other constituents are the phenolic esters that
have no effect on the proliferation of MCF-7 cells [9].
The structural similarities with the known HIV-1 integrase
inhibitor [10], chicoric acid (Scheme 1) prompted us to syn-
thesize rac-fukiic acid 1 and evaluate its antiviral properties.
Indeed, fukiic acid also possesses a tartaric acid moiety and
a 3,4-dihydroxyphenyl group. Tartaric acid and catechol are
known to efficiently bind magnesium ion [11], suggesting an
expected activity as HIV-1 integrase inhibitor via the interac-
tion with the magnesium ion(s) present in the active site of this
enzyme. However, this mechanism has not been fully estab-
lished yet. The synthesized rac-fukiic acid was effectively
found to be active against the reactions catalyzed by HIV-1 in-
tegrase but inactive against the viral replication in MT-4 cell
assay. We present herein the synthesis and the biological prop-
erties of this new IN inhibitor.
In 1969, a new polyphenol, Fukinolic acid, was isolated
from Petasites Japonicus by Sakamura et al. [1]. Its alkaline
hydrolysis gave caffeic acid and a new polyphenol, named fu-
kiic acid. Matsumoto et al. [2] proposed the structure of fukiic
acid and Yoshihara et al. [3,4] determined that fukiic acid has
the same stereochemical features as piscidic acid as (2S,3R)-
2,3-dihydroxy-4-(3,4-dihydroxyphenyl)-3-carboxybutyric acid.
Several other rare hydroxycinnamic acid esters, such as
cimicifugic acids, fukinolic acids and fukiic acid, were also
isolated from some Cimicifuga species [5].
Black cohosh (Cimicifuga racemosa) is a North American
perennial in the buttercup family (Ranunculaceae) that has
been in use in Europe for the last 50 years as a natural alter-
native to hormone replacement therapy for women with breast
cancer. C. racemosa extract inhibits proliferation of MCF-7
and MDA-MB231 breast cancer cells by induction of apopto-
sis [6,7]. Amongst the different components of the extract are
the triterpene glycosides responsible for the growth inhibitory
2. Results and discussion
2.1. Chemistry
The synthesis of rac-fukiic acid dimethyl ether 2 was pre-
viously briefly described in 1972 [2] and we reinvestigated all
* Corresponding author. Tel.: þ33 320434939; fax: þ33 320336309.
E-mail address: Philippe.Cotelle@univ-lille1.fr (P. Cotelle).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.12.013

C. Queffe´lec et al. / European Journal of Medicinal Chemistry 43 (2008) 2268e2271
HO
2269
O
H
HO
H
HO
COOH
O
OH
OH
OH
COOH
O
HOOC
HO
HO
HOOC
H
O
Fukiic acid
L-Chicoric acid
Scheme 1.
the steps until the final deprotection leading to the target mol-
ecule that had never been addressed (Scheme 2). The synthesis
starts by the conversion of veratraldehyde 3 into the oxazolone
4 by the Erlenmeyer reaction [12]. The acidic hydrolysis of 4
affords the pyruvic acid 5 [12] which is esterified using methyl
iodide and DBU in dry DMF giving 6 [13]. Wittig reaction of
this pyruvic acid methyl ester 6 with methoxycarbonylmethyl-
triphenylphosphorane quantitatively affords a mixture of
dimethyl 3,4-dimethoxyphenylmethylmaleate 7 as the major
isomer (75%) and dimethyl 3,4-dimethoxyphenylmethylfuma-
rate 8 (25%) that can be quantitatively separated by column
chromatography. The Z stereochemistry of 7 was assigned ac-
cording to the characteristic chemical shifts of the methylenic
numerous polyphenols confirms that the IC50 values are 50e
100 folds lower with BSA than without BSA.
The antiviral activity on HIV-1-induced CPE in human
lymphocyte MT-4 cell culture was determined by the MT-4/
MTT-assay [17]. Compound 1 was not found to be active at
concentrations lower than the CC50.
Results reported in Table 1 indicated that fukiic acid and
chicoric acid have very similar activities against IN but their
toxicities are quite different. L- and D-chicoric acids were
found to inhibit the overall integration reaction at submicro-
molar levels via the selective inhibition of the 30-processing
step [10]. This apparent 30-processing inhibition is due to as-
sembly inhibition [18], but the viral entry was demonstrated
to be the primary target for the anti-HIV-1 activity of chicoric
acid [10]. Compared to L- and D-chicoric acids, fukiic acid is
5-fold more toxic and its toxicity did not allow us to evaluate
its potential activity against the viral replication. It is now well
established that selective strand transfer inhibitors also inhibit
HIV-1 replication in cell culture [19] (most of the non-selec-
tive strand transfer inhibitors were found to present antiviral
properties via another target that IN). The lack of cell-based
activity of fukiic acid in MT-4 cells could be therefore due
to the absence of selectivity towards the second step catalyzed
by integrase and/or the absence of another viral target (i.e., en-
try as in the case of chicoric acid).
Whereas chicoric acid presented anti-IN and anti-HIV-1
properties, its acid constituents, i.e. caffeic and tartaric acids
had no antiviral activities [20]. In fukiic acid, the catechol
and tartaric acid moieties are linked by a methylene group.
This link yields submicromolar anti-IN activities comparable
to those of L- and D-chicoric acids. However, the anti-HIV-1
properties of fukiic acid cannot be measured due to cytotoxic-
ity. The link between catechol and tartaric acid moieties will
be modified in order to decrease the cytotoxicity.
(3.58 ppm in CDCl3) and ethylenic (5.63 ppm) proton signals
4
€
and their cisoıd coupling constant ( J ¼ 1.9 Hz), the E stereo-
chemistry of 8 being attested by the downfield shift of the
methylenic proton signal (3.72 ppm), the high field shift of
the ethylenic proton signal (4.12 ppm) and the absence of an
€
observable transoıd coupling constant. The Z and E stereo-
chemistries of 7 and 8 were also confirmed by NOESY exper-
iments. Compound 7 is then oxidized using potassium
permanganate and magnesium sulphate in ethanol at 0 ꢀC in
rac-fukiic acid dimethyl ether 2 in 68% yield. The same reac-
tion on 8 gives 9, the diastereoisomer of 2 in 75% yield. The
critical step in the synthesis of rac-fukiic acid is the final
deprotection and the isolation/purification of 1. Best results
are obtained using boron tribromide yielding 1 in 35% yield.
2.2. Anti-integrase activity and antiviral properties
Since there is no significant difference between the activi-
ties of L- and D-chicoric acids, we have tested fukiic acid as
the racemic form on integrase. rac-1 was tested in IN inhibi-
tion assays which have been recently reviewed [14]. The IN
inhibition assays were done in two independent laboratories
and gave different results mainly due to the presence or the ab-
sence of bovine serum albumin (BSA) in the medium assay.
Other smaller differences due to the IN purification processes
[14,15] and the conditions of the assays did not significantly
modify the IC50’s. The IC50 values were measured for the
overall integration reaction and compared to the inhibitory
properties of L-chicoric acid and D-chicoric acid [10]. Results
are reported in Table 1. With numerous inhibitors, BSA does
not interfere in this assay, but in the present case it seems
that the presence of a catechol function may promote an inter-
action between rac-1 and BSA [16]. Our own experience on
In conclusion, we report the first total synthesis of rac-fu-
kiic acid, a new submicromolar inhibitor of HIV-1 integrase.
3. Experimental protocols
3.1. Chemistry
All reagents and solvents were purchased from Aldrich-
Chimie (Saint-Quentin-Fallavier, France), were of ACS re-
agent grade and were used as provided. TLC analyses were
performed on plastic sheets precoated with silica gel 60F254
(Merck). SiO2, 200e400 mesh (Merck) was used for column

2270
C. Queffe´lec et al. / European Journal of Medicinal Chemistry 43 (2008) 2268e2271
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
AcNHCH2COOH
HCl 3M
reflux
75%
O
O
CHO
Ac2O, AcONa
65%
O
OH
N
HO
3
5
4
H3CO
H3CO
H3CO
Ph3PCH-COOCH3
DMF
O
5
H3CO
Toluene
99%
DBU/CH3I
81%
COOCH3
COOCH3
H3COOC
OCH3
HO
75%
7
6
H3CO
H3CO
H3COOC
8
25%
H3CO
H3CO
HO
H
H
HO
OH
OH
KMnO4/MgSO4
ethanol 0°C
H3CO
H3CO
H3COOC
HOOC
HO
COOCH3
COOH
HO
BBr3
35%
H3CO
H3CO
HO
HO
COOCH3
H3COOC
7
OH
H
OH
H
HO
H3COOC
HO
COOCH3
COOH
HOOC
Rac-2
68%
Rac-1
H3CO
H3CO
H
COOCH3
OH
KMnO4/MgSO4
ethanol 0°C
COOCH3
H3COOC
H3CO
H3CO
HO
H3CO
H3CO
COOCH3
H
COOCH3
HO
8
OH
H3COOC
Rac-9
75%
Scheme 2.
chromatography. NMR spectra were obtained on an AC 200
Bruker spectrometer in the appropriate solvent with TMS as
internal reference. Melting points were obtained on a Reichert
Thermopan melting point apparatus, equipped with a micro-
scope and are uncorrected. Melting points and spectroscopic
data of compounds 7, 8, rac-9 and rac-2 are in accordance
with the literature [21].
[5]. Mp >250 ꢀC; 1H NMR (acetone-d6) (d): 2.91 (d, 1H,
2J ¼ 13.8 Hz, CH2); 3.12 (d, 1H, 2J ¼ 13.8 Hz, CH2); 4.53
(s, 1H); 6.52e6.78 (m, 3H, ArH); 13C NMR (d): 41.5 (t);
75.7 (d); 80.6 (s); 115.5 (d); 118.35 (d); 122.6 (d); 128.1 (s);
144.6 (s); 145.15 (s); 172.9 (s); 174.2 (s).
Table 1
Inhibition of integration and anti-HIV activity, both expressed in mM
3.1.1. Synthesis of rac-fukiic acid
Compound 2 was treated with boron tribromide 1.0 M in
CH2Cl2 (5 equiv) for 1 h at room temperature. After hydroly-
sis at room temperature, the aqueous layer was separated and
extracted with ethyl acetate. The washed aqueous layer was
concentrated in vacuum and the crude solid was dissolved in
hot acetone. The solution was filtered and the solvent was
evaporated. The residue was then treated in the same manner
with MeOH affording rac-fukiic acid 1 in 35% yield as oily
syrup. Spectroscopic data are in accordance with the literature
Compound
IN inhibitiona
MT-4 cells
EC50
IC50
CC50
L-CAb
D-CAb
rac-Fukiic acid
0.2 ꢁ 0.2
0.3 ꢁ 0.02
0.25 ꢁ 0.02
17.0 ꢁ 0.02c
12.7 ꢁ 8.5
10.9 ꢁ 6.5
>22
115 ꢁ 21
111 ꢁ 25
22 ꢁ 0.02
a
The IN inhibition was assayed on the overall integration reaction.
Biological data of chicoric acids are from Ref. [10].
BSA was added in the medium.
b
c

C. Queffe´lec et al. / European Journal of Medicinal Chemistry 43 (2008) 2268e2271
2271
3.2. Pharmacology
Acknowledgements
3.2.1. HIV-1 integrase inhibition
This work was financially supported by grants from le Cen-
tre National de la Recherche Scientifique (CNRS), le Minis-
`
3.2.1.1. IN inhibition þ BSA. To determine the susceptibility
of the HIV-1 integrase enzyme towards different compounds
we used an enzyme-linked immunosorbent assay. This assay
uses an oligonucleotide substrate of which one oligo (50-
ACTGCTAGAGATTTTCCACACTGACTAAAAGGGTC-30)
is labeled with biotin on the 30 end and the other oligo is
labeled with digoxigenin at the 50 end. For the overall
integration assay the second 50-digoxigenin labeled oligo is 50-
GACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGT-30.
The integrase enzyme [13] was diluted in 750 mM NaCl,
10 mM Tris, pH 7.6, 10% glycerol and 1 mM b-mercaptoetha-
nol. To perform the reaction, 4 mL diluted integrase (1.6 mM)
and 4 mL of oligonucleotides (7 nM) were added in a final vol-
ume of 40 mL containing 10 mM MgCl2, 5 mM DTT, 20 mM
HEPES, pH 7.5, 5% PEG, 15% DMSO and 0.1 mg/mL BSA.
Reaction products were denatured with 30 mM NaOH and
detected by an immunosorbent assay on avidin-coated plates
[22].
tere de l’Enseignement et de la Recherche (MESR),
l’Agence Nationale de la Recherche contre le Sida (ANRS)
and the European Commission (LSHB-CT-2003-503480).
The European TRIoH Consortium is gratefully acknowledged.
We are grateful to Martine Michiels, Nam Joo Vanderveken
and Barbara Van Remoortel for excellent technical assistance.
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mitochondrial dehydrogenase of metabolically active cells to
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of the virus stock using MT-4 cells. For the drug-susceptibility
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HIV strains, which is defined as the EC50 was determined.
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