Structure-activity relationships of some 3-substituted-4-hydroxycoumarins as HIV-1 protease inhibitors

Article abstract of DOI:10.1016/S0014-827X(02)01264-8

The screening of the HIV-1 protease (PR) inhibitory activity (IC-50) of various substituted 3-phenyl-4-hydroxycoumarins, 3-benzyl-4-hydroxycoumarins, 3-phenoxy-4-hydroxy-coumarins, 3-benzenesulfonyl-4-hydroxycoumarins and 3-(7-coumarinyloxy)-4-hydroxycoum

Full text of DOI:10.1016/S0014-827X(02)01264-8

Il Farmaco 57 (2002) 703ꢀ708
/
www.elsevier.com/locate/farmac
Structureꢀ
/
activity relationships of some
3-substituted-4-hydroxycoumarins as HIV-1 protease inhibitors
Serge Kirkiacharian a,*, Duong Thanh Thuy a, Sames Sicsic b, Robert Bakhchinian a,
Raffi Kurkjian a, Thierry Tonnaire b
a Laboratoire de Chimie The´rapeutique, Faculte´ de Pharmacie de Paris-Sud, 5, rue Jean-Baptiste Cle´ment, 92296 Chatenay Malabry Cedex, France
b BIOCIS, UPRES A CNRS 8076, 5, rue Jean-Baptiste Cle´ment, 92296 Chatenay Malabry Cedex, France
Received 26 October 2001; received in revised form 23 February 2002; accepted 17 March 2002
Abstract
The screening of the HIV-1 protease (PR) inhibitory activity (ICꢀ50) of various substituted 3-phenyl-4-hydroxycoumarins, 3-
/
benzyl-4-hydroxycoumarins, 3-phenoxy-4-hydroxy-coumarins, 3-benzenesulfonyl-4-hydroxycoumarins and 3-(7-coumarinyloxy)-4-
hydroxycoumarins was performed. The data indicate the importance of substituents at positions 5 and 7 of the coumarin ring on the
´
inhibitory potency of the HIV-1-PR. # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: 4-Hydroxycoumarins; Structure-HIV-1; Protease inhibition
1. Introduction
need for new nonpeptidomimetic drugs devoid from
economic and pharmacokinetic drawbacks led to the
identification of the 3-substituted-4-hydroxycoumarins
Phenprocoumon 1a [7], Warfarin 1b, substituted 4-
hydroxy-2-pyrone derivatives of type 2 as possible first
The availability of new chemotherapeutic agents for
the treatment of the human acquired immune deficiency
syndrome (AIDS) constitutes an important target due to
the worldwide spreading of this infection. Since the
isolation of the causative human immunodeficiency
virus (HIV), favourable results were obtained with the
use of inhibitors of the virally encoded enzymes reverse
transcriptase (RT) and protease (PR), which are indis-
pensable, respectively for the viral replication and
maturation. Indeed, the clinical use of associations of
generation HIV-PR inhibitors [8ꢀ14] and more recently,
/
the sulfamide containing derivative 3 as a second gene-
ration inhibitor [15].
HIVꢀ
/
RT or HIVꢀPR inhibitors (combination therapy)
/
showed to present a good efficiency leading to the
decrease of the viral load and to the increase of the
number of CD4 lymphocytes. However, these drugs
became less efficient and led to therapeutic failures [1]
due particularly to the ability of the virus to generate
resistant mutants [2ꢀ4].
/
The available peptidomimetic protease inhibitors [5]
present low oral bioavailibility and their synthesis
involves expensive multiple steps [6]. Therefore, the
Since the first crystallographic determination of the
3D structure of HIV-1 protease [16,17] showing that the
active HIV-1-PR is formed by two assembled monomers
in a C2-symmetric axis, a lot of works concerning the
structure of HIV-1 protease-inhibitor complexes were
published. More particularly, the work of Thaisrivongs
* Corresponding author
E-mail address: serge.kirkiacharian@cep.u-psud.fr (S. Kirkiachar-
ian).
´
0014-827X/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 0 1 4 - 8 2 7 X ( 0 2 ) 0 1 2 6 4 - 8

704
S. Kirkiacharian et al. / Il Farmaco 57 (2002) 703ꢀ708
/
et al. [18], describing the structure of protease-hydro-
xycoumarin complex by X-ray diffraction method
(2UPJ, Protein Data Bank), showed that the
3-substituted 4-hydroxycoumarins interact with various
sites of the enzyme: the two key catalytic aspartic acid
residues Asp25 and Asp25? through hydrogen bonds
with the 4-hydroxy group, the NH group of the two
isoleucine Ile50 and Ile50? with the lactone oxygens
through additional hydrogen bonds and the hydro-
phobic S1, S1?, S2 pockets with the side chains
[12,19,20].
Table 1
IC50 OF 3-phenyl-4-hydroxycoumarins
Taking into consideration these structural features of
the active HIV-1-PR and in order to contribute to a
better understanding of the structureꢀactivity relation-
/
ships of the inhibitory activity of 4-hydroxycoumarinic
derivatives, we decided the preparation of some sets of
3-substituted 4-hydroxycoumarins with the objective to
investigate the influence of some modifications in the
way of simplification:
R1
R2
R3
IC50 (mM)
4a OH
4b
H
H
5.0
31
H
OH
H
H
ꢀ
/
influence of a phenyl ring directly attached to the 3-
position of 4-hydroxycoumarin or through a me-
thylene group or an oxygen or a sulfonyl group, in
order to examine if flexibility is necessary for this
region;
4c OCH3
4d OCH3
4e OH
H
11
H
OCH3
OH
H
3.5
2.0
7.0
H
4f
OÃCH2ÃC6H5
H
ꢀ
/
presence of various substituents on the aromatic ring
of the 3-substituted 4-hydroxycoumarins (hydroxy,
methoxy, benzyloxy);
Table 2
IC50 of 3-benzyl-, 3-phenoxy- and 3-arylsulfonyl-4-hydroxycoumarins
ꢀ
/
the influence of a sterically hindered (7-coumariny-
loxy) substituent at 3-position of 4-hydroxycoumarin;
finally, as the 4-hydroxy group of the coumarin forms
a hydrogen bond with the key catalytic aspartic acid
residues (Asp 25 and Asp 25?) of the HIV-PR, we
studied the 4,5-dihydroxylated coumarins 4b and 5b
considering that the presence of a second hydroxy
group could lead to increase the strength of hydrogen
bond and hence increase the inhibitory activity.
ꢀ
/
R1
R2
R3
X
IC50 (mM)
5a OH
5b
H
H
CH2
CH2
CH2
CH2
O
9.5
NI
H
OH
H
H
5c OCH3
5d OCH3
5e OCH3
5f OCH3
5g OCH3
5h OCH3
H
2.4
3.0
H
OCH3
H
Cl
H
61
8.0
H
O
H
CH3
Cl
SO2
SO2
38% at 50 mM
28
H
Table 3
IC50 of 3-(7-coumarinyloxy)-4-hydroxycoumarins
Therefore, the preparation of substituted 3-phenyl-4-
hydroxycoumarins 4aꢀ
5aꢀd, 3-phenoxy-4-hydroxycoumarins 5eꢀ
fonyl-4-hydroxycoumarins 5gꢀh, and 3-(7-coumariny-
loxy)-4-hydroxycoumarins 6aꢀc was performed and the
/f, 3-benzyl-4-hydroxycoumarins
/
/
f, 3-arylsul-
/
/
R1
R4
IC50 (mM)
determination of their IC50 HIV-1 antiprotease activity
determined.
6a
6b
6c
OCH3
OH
H
4.0
9.0
7.5
The structures of the studied derivatives 4aꢀ
and 6aꢀc and the data related to their inhibitory
activities (IC50) are indicated in Tables 1ꢀ3.
/
f, 5aꢀf
/
CH3
CH3
/
OÃCH2ÃC6H5
/

S. Kirkiacharian et al. / Il Farmaco 57 (2002) 703ꢀ
/708
705
dihydroxycoumarin (5b) [26], 3-(4-methoxybenzyl)-4-
hydroxy-7-methoxycoumarin (5d) [27], 3-phenoxy-4-hy-
droxy-7-methoxycoumarin (5e) [28,29] are known and
their synthesis achieved by the described procedures.
The synthesis of 3-(4-chlorophenoxy-4-hydroxy-7-meth-
oxycoumarin (5f) is obtained by thermal condensation
of diethyl-(4-chlorophenoxy-malonate with O-methylre-
sorcine [29] (Scheme 2).
The synthesis of 3-arylsulfonyl-4-hydroxycoumarins
5gꢀh, will be reported in a coming publication [30].
/
Compound 6a is known and its preparation per-
formed by a described procedure [31]. The derivative 6b
is obtained by the same route than 6a by thermal
condensation of diethyl 7-coumarinyloxy malonate with
2-methylresorcine. The synthesis of 6c was achieved by
benzylation of the corresponding hydroxy-derivative 6b
using the previous alkylation route (Scheme 3).
The chemical shifts of the 13C NMR spectra of these
derivatives are in agreement with those already reported
for 3-substituted-4-hydroxycoumarins [32,33].
Scheme 1.
2. Synthesis
Most of the 3-substituted-4-hydroxycoumarins 4aꢀ
5aꢀf and 6aꢀc were prepared by described procedures:
4,7-dihydroxy-3-phenylcoumarin (4a), 4-hydroxy-7-
methoxy-3-phenylcoumarin (4c) [21,22], 4,5-dihydroxy-
3-phenylcoumarin (4b) [23], 4-hydroxy-7-methoxy-3(4-
methoxyphenyl)coumarin (4d) [24].
/f,
/
/
The 4,7-dihydroxy-3-(4-hydroxyphenyl)coumarin 4c
was obtained from the corresponding dimethoxy deri-
vative 4d by demethylation using pyridine hydrochlo-
ride. The 4-hydroxy-3-phenyl-7-benzyloxycoumarin (4f)
was prepared by benzylation of the corresponding
hydroxylated compound 4a using benzyl chloride as
reagent in acetone as solvent, potassium carbonate as
base and tetrabutylammonium bromide as phase trans-
fer catalyst (Scheme 1).
3. Experimental
3.1. Chemistry
The purity of all the compounds was routinely
checked on the ‘Riedel-de Haen 60 F
special’ silica
gel plates (0.2 mm) and spots were located by UV lamp
¨
254
and or by iodine vapors. M.p.s were taken on a Kofler
The 3-benzyl-4,7-dihydroxycoumarin (5a), 3-benzyl-
4-hydroxy-7-methoxycoumarin (5c) [25], 3-benzyl-4,5-
bench and are uncorrected. Analyses (C,H) are within9
0.5% of the theoretical values.
/
Scheme 2.
Scheme 3.

706
S. Kirkiacharian et al. / Il Farmaco 57 (2002) 703ꢀ708
/
The infrared spectra (n in cmꢁ1) of the 3-substituted
3.1.3. 3-(4-chlorophenoxy)-4-hydroxy-7-methoxy-
coumarin (5g) (C16H11O5Cl)
4-hydroxycoumarins are recorded on a Bruker ‘Vec-
tor22’ spectrophotometer. They present characteristic
bands of the hydroxyl and the conjugated carbonyl
group of the lactone.
The 1H NMR spectra were recorded on a Bruker
AC300 in CDCl3 or DMSO-d6 using tetramethylsilane
(TMS) as internal reference. Chemical shifts d are in
ppm. Splitting patterns are described as follows: (s)
singlet; (d) doublet; (t) triplet; (q) quadruplet; (m)
multiplet.
Prepared by thermal condensation of diethyl (4-
chlorophenoxy)malonate [34] with O-methylresorcine
according to a reported procedure [29]. M.p.: 257 8C
(EtOH). IR: 3025, 1680, 1605, 1486 (CÄ
NMR (CDCl3): 3.9 (s, 3H, OCH3), 6.5ꢀ
arom.), 7.8 (d, 1H, H-5).
/
C aromatic). 1H
7.5 (m, 6H,
/
3.1.4. 3-(7-coumarinyloxy)-4,7-dihydroxy-8-methyl-
coumarin (6b) (C19H12O7)
This compound is prepared by thermal condensation
of diethyl (7-coumarinyloxy)malonate (3.04 g, 10 mmol)
and 2-methylresorcine (1.24 g, 10 mmol) at 250 8C,
during 4 h. After cooling, the product was washed twice
with a small portion (5 ml) of Et2O to remove impurities
The following derivatives 3-(4-methoxyphenyl)-4-hy-
droxy-7-methoxycoumarin (4d) [24], 3-(phenoxy)-4-hy-
droxy-7-methoxycountarin (5f) [29], 3-(7-coumariny-
loxy)-4-hydroxy-7-methoxycoumarin (6a) [31], diethyl
p-chlorophenoxymalonate [34] and diethyl (7-coumar-
inyloxy)malonate [35] are prepared according to the
described procedures.
and the derivative 6a recrystallized in an EtOHꢀTHF
/
water mixture (8/1/1). Yield: 80%; m.p.: 231 8C; IR:
3256, 1726, 1672, 1593. H NMR (DMSO-d6): 2.15 (s,
1
3H, CH3), 6.3ꢀ8.1 (m, 7H, arom.), 10.4 (s, 1H, OH,
/
exchangeable with D2O; the other OH group is mixed in
the aromatic protons and is also exchangeable with
D2O).
3.1.1. Synthesis of the new 3-substituted 4-hydroxy-
coumarins 3-(4-hydroxyphenyl)-4,7-dihydroxycoumarin
(4e) (C15H10O5)
This compound was obtained from 3-(4-methoxy-
phenyl)-4-hydroxy-7-methoxycoumarin (4d) by deme-
thylation.
In a round bottom flask, fitted with a stirring ma-
gnetic bar, a mixture of 2 g of 3-(4-methoxyphenyl)-7-
methoxy-4-hydroxycoumarin (4d) and l6 g pyridine
hydrochloride were heated at 180 8C in an oil bath
during 6 h. After cooling, water (50 ml) was added and
3.1.5. 3-(7-coumarinyloxy)-4-hydroxy-7-benzyloxy-8-
methyl-coumarin (6c) (C26H18O7)
The preparation of compound 6c was performed with
the same procedure than compound 4f, by refluxing
under anhydrous conditions compound 6b (1.0 mmol),
benzylchloride (2.2 mmol), K2CO3 as base (10 mmol)
and a pinch of tetrabutylammonium bromide as phase
transfer catalyst in dry C3H6O as solvent (100 ml)
during 4 h (end of the reaction monitored by TLC).
the reaction mixture extracted with EtOAc (3ꢂ
The resulting organic solution was washed with water
(2ꢂ50 ml), dried over sodium sulfate and evaporated.
The remaining solid was recrystallized.
Yield: 92%; m.p.: 327 8C in EtOHꢀ
/50 ml).
Yield: 95%; m.p.: 228ꢀ
mixture (8/1/1). IR: 3297, 3089, 1712, 1607. H NMR
(DMSO-d6): 2.15 (s, 3H, CH3), 5.2 (s, 2H CH2). 6.3ꢀ8.1
/
229 8C in EtOHꢀ
/
THFꢀ
/
water
1
/
/
(m, 7H, arom.).
/
water (9/1). IR:
1
3311, 3069, 1670. H NMR (CDCl3): 6.6ꢀ7.6 (m, 6H,
/
3.2. Biological assay
arom) 7.9 (d, 1H, H-5); 9.4 (s, 1H, OH), 10.4 (s, 1H,
OH).
The HIV-1-protease was kindly supplied by Rhoˆne-
Poulenc Rorer. The fluorogenic substrate DABCYL-S-
Q-N-Y-P-l-V-Q-EDANS was purchased from Bachem
(France). The fluorescence measurements were per-
3.1.2. 3-phenyl-4-hydroxy-7-benzyloxycoumarin (4f)
(C22H16O4)
formed using a PerkinꢀElmer fluorometer. Enzymatic
/
The preparation of this compound was achieved by
refluxing under anhydrous conditions 4,7-dihydroxy-3-
phenylycoumarin (4a) (254 mg, 1.0 mmol), benzylchlor-
ide (1.52 g, 1.2 mmol) in dry C3H6O as solvent (100 ml),
K CO as base (1.4 g, 10.0 mmol) and a pinch of
assays were performed in 150 mM AcONa, 1 M NaCl,
pH 5.5, 3% DMSO. Inhibitors and the substrate were
dissolved in DMSO before addition to the buffer.
For the determination of the IC50 values, 0.52 ml of a
3 mM solution of substrate (final concentration 5.2 mM)
2
3
tetrabutylammonium bromide as phase transfer cata-
lyst, during 4 h (end of the reaction monitored by TLC.
was added to 8.5 ml of 4ꢀ
/10 different concentrations of
inhibitors (final volume 300 ml). The enzymatic reaction
was initiated by the addition of enzyme (final concen-
tration 7.5 nM). The increase in fluorescence at 490 nm
Yield: 95%, m.p: 227 8C in EtOHꢀ
/
water (9/1). IR:
3063, 1671, 1610. H NMR (CDCl3): 5.2 (s, 2H, CH2),
7.1 (d, 2H, H-6, H-8), 7.3ꢀ7.5 (m, 10H, arom.) 7.9 (d,
1H, H-5).
1
/
(l excꢃ
/
340) was monitored over a period of 8 min at
30 8C.

S. Kirkiacharian et al. / Il Farmaco 57 (2002) 703ꢀ
/708
707
4. Results and discussion
the introduction of an hydroxyl group. Position 3 is also
a sensitive one, since direct fixation of an aromatic
group or through a methylene is preferable to fixation
through an oxygen or a sulfonyl group. Finally, a
substitution by a Cl of this aromatic group in para
position seems favourable to activity.
The results of IC50 of protease inhibition for the
studied substituted 4-hydroxycoumarins are presented
in Table 1 for the 3-phenyl-4-hydroxycoumarins deriva-
tives 4aꢀ
ins, 3-phenoxy-4-hydroxycoumarins and 3-arylsulfonyl-
4-hydroxycoumarins 5aꢀh and Table 3 for the 3-(7-
coumarinyloxy)4-hydroxycoumarins 6aꢀc. Examination
of the data obtained with the 3-phenyl-4-hydroxycou-
marins 4aꢀf (Table 1) indicates that the most active
derivatives are the disubstituted compounds 4e and 4d
(R1, R3ꢃ
OH, OCH3).
The activities increase according to 4bB
4aB4dB4e and the least active compound 4b presents
/
f, Table 2 for the 3-benzyl-4-hydroxycoumar-
More work is now in progress in order to improve our
/
knowledge of the structureꢀanti-HIV-PR activity rela-
/
/
tionships of various 3-substituted-4-hydroxycoumarins.
/
/
References
/
4cB
/
4fB
/
/
/
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indicate that this position is particularly sensitive and
a new hydroxyl group near to the hydroxyl group in
position 4 decreases the strength of the hydrogen bond
between this group and Asp25/Asp25? of HIV-1 pro-
tease. The other positions R1 at 7 and R3 are not very
sensitive to modifications, although hydroxyl group as
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in Table 2.
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d, 3-phenoxy-4-hydroxycoumarins 5eꢀ
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derivatives of this group of compounds are 5e and 5d
where R1 at position 7 is a methoxy group and Xꢃ
CH2). Comparison of compounds 5c, 5e and 5g shows
clearly that the replacement of XꢃCH2 by O or SO2 is
not favourable to activ-ity. However, introduction of Cl
in para position of the 3-phenoxy group allows to
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/
The comparison of compound 4a (3-phenyl) and 5a
(3-benzyl) shows that the flexibility of this latter
substituent at 3-position does not lead to a significative
change in activity.
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observed decrease in activity with its surrogate 4,5-
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