Development of novel antiatherogenic biaryls: Design, synthesis, and reactivity

Article abstract of DOI:10.1021/jm7014793

On the basis of the 5,5′-bisvanillin scaffold, a series of compounds has been synthesized presenting symmetric or dissymmetric frames on each phenolic moiety. These frames are α,β-unsaturated (fluoro)phosphonate and/or α,β-unsaturated hydrazone(s) formed by coupling aldehydic with isoniazid or hydralazine. All compounds were tested for their ability to inhibit cell-mediated low-density lipoprotein oxidation. Oxidized low-density lipoprotein induced cytotoxicity was also evaluated along with the carbonyl scavenger properties of selected compounds. The most efficient agents were found to be those possessing at least one hydralazinone frame, with the most potent being the symmetrical compound: 4,4′-dihydroxy-3,3′-dimethoxy-5, 5′-biphenyl-1,1′-(diphthalazin-1-yl)methylhydrazone hydrochloride.

Full text of DOI:10.1021/jm7014793

J. Med. Chem. 2008, 51, 3171–3181
3171
Development of Novel Antiatherogenic Biaryls: Design, Synthesis, and Reactivity
Me´lanie Delomene`de,† Florence Bedos-Belval,† Hubert Duran,† Ce´cile Vindis,‡ Michel Baltas,*,† and Anne Ne`gre-Salvayre*,‡
UniVersite´ Toulouse 3, Laboratoire de Synthe`se et Physico-Chimie de Mole´cules d’Inte´reˆt Biologique, LSPCMIB, UMR-CNRS 5068, 118 Route
de Narbonne, 31062 Toulouse Cedex 9, France, INSERM I2MR 858, IFR 31 CHU Rangueil, L3 building, AVenue Jean Poulhe`s BP 84225,
31432 Toulouse Cedex 4, France
ReceiVed NoVember 28, 2007
On the basis of the 5,5′-bisvanillin scaffold, a series of compounds has been synthesized presenting symmetric
or dissymmetric frames on each phenolic moiety. These frames are R,ꢀ-unsaturated (fluoro)phosphonate
and/or R,ꢀ-unsaturated hydrazone(s) formed by coupling aldehydic with isoniazid or hydralazine. All
compounds were tested for their ability to inhibit cell-mediated low-density lipoprotein oxidation. Oxidized
low-density lipoprotein induced cytotoxicity was also evaluated along with the carbonyl scavenger properties
of selected compounds. The most efficient agents were found to be those possessing at least one hydralazinone
frame, with the most potent being the symmetrical compound: 4,4′-dihydroxy-3,3′-dimethoxy-5,5′-biphenyl-
1,1′-(diphthalazin-1-yl)methylhydrazone hydrochloride.
Introduction
hemolytic, mutagenic, and carcinogenic properties.9 Other
hydrazine derivatives, such as hydralazine, also behave as
carbonyl scavengers but do not exhibit strong antioxidant
activity.10 Thus, the generation of drugs sharing both antioxidant
and carbonyl scavenger properties represents a new therapeutic
challenge in the prevention and treatment of atherosclerosis.
Atherosclerosis is a multifactorial pathology characterized by
the development of focal lesions in the vascular wall of medium
and large arteries.1 It is a chronic inflammatory disease with
acute complications represented by plaque erosion and rupture,
thrombosis, and cardiovascular and neurovascular events.1 Low-
density lipoproteins (LDL) become atherogenic after undergoing
oxidative modifications in the intima. Oxidatively modified low-
density lipoproteins (oxLDLs) play an important role in the
initiation and progression of atherosclerosis by promoting
cholesterol storage, endothelial dysfunction, lipid deposition,
chronic inflammatory response, and toxic events.1–3 Modification
of LDLs through oxidative damage may increase their athero-
genicity by increasing their receptor-mediated uptake by cells
in the intima of blood vessels, leading to creation of foam cells
in the subendothelium, an early feature of atherosclerotic
plaque.3 The biological effects of oxLDLs depend on their local
concentration and their content of lipid peroxidation products
such as lipid hydro- and lipoperoxides, oxidized phospholipids,
oxysterols, and aldehydes such as 4-hydroxyalkenals and
4-hydroxynonenal (4-HNE), malondialdehyde (MDA) or
acrolein.4,5 Lipid oxidation-derived aldehydes (carbonyl precur-
sors) may react with free amino groups and thiol residues,
thereby forming protein cross-links which induces progressively
protein dysfunction, also called “carbonyl stress”.6 Antioxidants
are antiatherogenic as they inhibit the early steps of the LDL
oxidation process,5 but these agents are inefficient against
carbonyl stress once lipid oxidation products are formed.7
Carbonyl scavengers usually exhibit modulated antioxidant
activities but inhibit the carbonyl stress by blocking the
formation of protein cross-links.7 Dinitrophenylhydrazine (DNPH)
is a powerful carbonyl scavenger agent, largely utilized for the
detection of reactive carbonyl compounds (RCCs).4 DNPH
prevents in vitro the formation of acrolein- and HNE-adducts
on cellular proteins8 but cannot be used in vivo because of its
Many hindered phenol-derived antioxidants possessing both
of these features have been screened since the initial work on
probucol.11,12 In a previous paper,13 we reported synthesis of
phosphonocinnamic analogues. Phosphonodiesters and espe-
cially fluorophosphonoesters have shown really interesting
antioxidant effects. The latter have also presented strong dual
biological antioxidant and cytoprotective properties. To get
insight to families of compounds sharing these two properties,
we have designed biaryl compounds possessing identical or
different substitutions patterns on a judiciously chosen phenolic
scaffold, i.e., bisvanillin. On one hand, the 4-OH and adjacent
3-OMe functionalities are always present in order to confer to
the compound the basis of a good antioxidant activity, as we
have previously noticed by studying the monomeric units. On
the other hand, the presence of the two aldehydic groups allows
us to construct symmetric or dissymmetric entities conjugated
with their phenolic moieties (Scheme 1). These entities may
confer not only antioxidant properties but also cytoprotection.
In that respect, compounds possessing the known phosphono-
diester and/or fluorophosphonoester moiety and also a hydrazone
function could be synthesized.
The hydrazones family may be interesting by various means.
On one hand, hydrazines possessing strong electron withdrawing
groups are known to form hydrazones sufficiently activated to
be hydrolyzed at physiological pH,14 ultimately making these
type of derivatives useful through creation of dynamic libraries.
On the other hand, hydrazines possessing in their frames basic
centers that can be protonated (or complexed by metals) may
be transformed to stable hydrazones under neutral pH conditions
that could release hydrazines under acid assistant conditions.15
Finally, delocalized systems possessing a hydrazone frame may
induce cytoprotective effects by acting through completely
different ways yet to be adressed.
* To whom correspondence should be addressed. Phone: 00 33 (0)5 61
55 62 89 (M.B.); 00 33 (0)5 61 32 20 59 (A.N.S.). Fax: 00 33 (0)5 61 32
29 53 (A.N.S.). E-mail: baltas@chimie.ups-tlse.fr (M.B.); anesalv@
toulouse.inserm.fr (A.N.S.).
In this paper, we report the synthesis of symmetric and
dissymmetric biaryl analogues bearing (fluoro)phoshonate(s)
and/or hydrazone frames (issued from isoniazid and hydralazine)
†
Universite´ Toulouse 3, Laboratoire de Synthe`se et Physico-Chimie de
Mole´cules d’Inte´reˆt Biologique.
‡
INSERM I2MR 858, IFR 31 CHU Rangueil.
10.1021/jm7014793 CCC: $40.75
2008 American Chemical Society
Published on Web 05/09/2008

3172 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Scheme 1. Biaryl Derivatives Generated from 5,5′-Bisvanillin
Delomene`de et al.
Scheme 2a
a Reagents and conditions: (i) imidazole, DMAP, TBDPSCl, DMF, room temperature, 16 h, 85%; (ii) (COCl)2, room temperature, 3 h, quantitative; (iii)
Et3N·HF, THF, room temperature, 1 h, 77%.
Scheme 3a
a Reagents and conditions: (i) Na2S2O8, FeSO4, H2O, 5d, 50 °C, 95%; (ii) imidazole, DMAP, TBDMSCl, DMF, room temperature, 3 h, 70%; (iii) iPr2NH,
n-BuLi, THF, tetraethyl methylenediphosphonate, -78 °C, 3 h, 86%; (iv) (COCl)2, CH2Cl2, room temperature, quantitative; (v) Et3N·HF, THF, room
temperature, 1 h or 30 min respectively for 8 or 9 and 10, 57%, 60%, and 41%, respectively.
and their biological properties, indicating that they can be
considered as lead structures, endowed with antioxidant and
carbonyl scavenger properties.
chlorinated intermediate 3 obtained quantitatively (31P NMR
29.94 ppm) was then directly reacted with the mild fluorinating
reagent Et3N·HF17 in tetrahydrofuran (THF), furnishing fluo-
rophosphonate 4 (31P NMR 19.50 ppm), which was also
deprotected from its silyl group as illustrated in Scheme 2. Other
fluorinating reagents used led to sluggish results and much lower
yields.
So we developed a new shorter synthetic way to obtain the
fluorophosphonate 4 with better global yield (65% instead of
23%). This method was applied to synthesis of the new biaryl
derivatives as depicted in Scheme 3. First, oxidative coupling
of commercial vanillin in the presence of iron sulfate afforded
bisvanillin 5 with excellent yield (95%), improving by 20% on
the literature report.18 Hydroxyl groups were silylated using tert-
butyldimethylsilyl chloride (TBDMSCl) instead of TBDPSCl
Chemistry
To develop an efficient synthetic route to achieve phospho-
nodiester16 and fluorophosphonoester biaryl compounds, we first
focused our efforts on improvement of the monoaryl fluoro-
phosphonate 4 synthesis previously described.13 Diethylphos-
phonate 1 was silylated on the p-hydroxyphenolic position by
using tert-butyldiphenylsilyl chloride (TBDPSCl) in the presence
of imidazole and 4-(N,N-dimethylamino)pyridine (DMAP) in
dimethylformamide (DMF). The protected diethylphosphonate
2 (31P NMR 20.09 ppm) was treated with neat oxalyl chloride,
and the reaction was followed by 31P NMR spectroscopy. The

DeVelopment of NoVel Antiatherogenic Biaryls
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3173
Scheme 4a
a Reagents and conditions: (i) iPr2NH, n-BuLi, THF, tetraethyl methylenediphosphonate, -78 °C, 3 h, 54%; (ii) (COCl)2, room temperature, 3 h, quantitative;
(iii) Et3N·HF, THF, room temperature, 30 min, 75% and 72% for compounds 12, 13, respectively; (iv) hydralazine or isoniazid, EtOH, reflux, 6 h, 81%,
69%, 93%, and 83% for compounds 14, 15, 16, and 17, respectively.
because of the inefficiency of the latter due to steric hindrance.
Silylated 5,5′-bisvanillin 6 was reacted with lithiated tetraethyl
methylenediphosphonate. Thermal treatment of the bis-phos-
phorylated intermediate gave exclusively the double trans-
vinylic phosphonate 7.16 At this stage, compound 7 can be
treated by Et3N·HF complex to afford desilylated bisphospho-
nate 8.
under reflux. Precipitation of double hydrazones 18 and 19
during reaction afforded pure compounds 18 and 19 in good
yield.
Results and Discussion
Symmetric or dissymmetric phosphonocinnamic and fluoro-
phosphonocinnamic derivatives were synthesized. Their anti-
oxidant properties were studied on cell-mediated LDL oxidation.
LDL oxidation by vascular cells and the uptake of oxLDL by
macrophages are involved in the formation of fatty streaks,
which constitute the early atherosclerotic lesions.1–3 Thus we
aimed to determine the antioxidant ability of the newly
synthesized compounds in inhibiting LDL oxidation mediated
by human microvascular endothelial cells (HMEC-1) and the
formation of foam cells.
LDL oxidation was determined by using the thiobarbituric
acid reactive substance (TBARS) assay and expressed as a
percentage of TBARS formed in LDL in contact with HMEC-1
in the absence of antioxidant. HMEC-1 were incubated for 6 h
in Roswell Park Memorial Institute (RPMI) 1640 culture
medium containing native LDL (100 µg/mL) and the different
agents solubilized in dimethylsulfoxide (DMSO) (less than 0.1%
final concentration) tested at a final concentration of 10 µM.
LDL oxidation was monitored by the TBARS content.20 As
shown in Table 1, only compounds 16, 17, and 19 exhibited
strong antioxidant properties by comparison to probucol and
R-tocopherol tested at the same concentration (10 µM).
The other agents were not (or very slightly) able to inhibit
LDL oxidation by HMEC-1 in this system.
Because LDL oxidation by cultured cells renders them
cytotoxic21 and because antioxidants are able to block the LDL
oxidation process by cells,22 we evaluated the residual cyto-
toxicity of LDL previously oxidized by HMEC-1 in presence
or absence of the synthesized biaryls agents by the MTT assay.
The data were expressed as a percentage of the unstimulated
control (cell incubated without LDL). The data presented in
Table 1 are in agreement with the TBARS content of LDL,
indicating a strong cytotoxicity of LDL incubated without any
agent (less than 15% of residual cell viability), which was
efficiently protected by the compounds 16, 17, 19. Interestingly,
compounds 8 and12, found noneffective as antioxidants, were
able to protect in part against oxLDL cytotoxicity, thus
suggesting that they may neutralize the toxic effect of newly
formed oxidized lipids either through direct interactions or by
blocking the apoptotic signaling of oxLDL.
Compound 7 was also used in order to introduce one or two
fluorine atom(s) on the phosphorus, following the chlorination-
fluorination procedure. Extensive experimental conditions were
checked and reactions were quantified after each final workup
and purification as 31P NMR spectroscopy gave in this case only
qualitative but not quantitative information on the constitution
of the reaction mixtures.
For the difluorinated addent 9, best results were obtained
when compound 7 reacted with 6 equiv of oxalyl chloride in
methylene chloride for 40 h followed by Et3N·HF treatment.
Compound 9 was thus obtained in 60% yield after purification
by silica-gel chromatography. Using 4.5 equiv of oxalyl chloride
in CH2Cl2 and modifying the reaction time gave the monoflu-
orinated adduct 10 in 41% yield along with desilylated starting
material 8 (15%) and the difluorinated compound 9 (32%).
To introduce dissymmetrization on the phosphorus atoms (as
for compound 10) and on the aldehydic frame of the 5,5′-
bisvanillin, we synthesized key intermediate 11. Scheme 4
details the formation of 11 and its use in the synthesis of
compounds 12-17.
Protected bisvanillin 6 reacted with strictly 1 equiv of lithiated
tetraethyl methylenediphosphonate. The reaction, carefully
controlled by TLC, afforded mainly phosphonate-aldehyde 11
in 54% yield along with starting material (21%) and the
symmetrical functionalized compound 7 (12%). Compound 11
could be either deprotected through desilylation, affording 12
in 75% yield or used in the chlorination-fluorination process,
yielding compound 13 in 72% total yield after silica-gel
purification.
Compounds 11, 12, and 13 were used in order to introduce
hydrazone functionality on the remaining aldehydic frame. Two
examples are presented here where the reagents are the known
drugs, i.e., isoniazid19 and hydralazine8 (antimalarial and
hypotensor, respectively). Filtration and purification on silica
gel chromatography afforded in good yields compounds 14 and
16 (starting from 12) and 15 and 17 (starting from 13).
Finally, synthesis of symmetrical hydrazones 18 and 19 was
undertaken as depicted in Scheme 5. 5,5′-Bisvanillin 5 reacted
with 2 equiv of hydrazine (hydralazine or isoniazid) in ethanol
Considering the compounds synthesized, results are parti-
tioned in four groups. In the first group the 5,5′-biaryl
compounds issued from the oxidative coupling of vanillin

3174 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Delomene`de et al.
Scheme 5a
a Reagents and conditions: (i) hydralazine or isoniazid, EtOH, reflux, 6 h, 77% and 87% for compounds 18 and 19, respectively.
Table 1. Antioxidant Effect (TBARS) and Residual Cytotoxicity (MTT)
of Biaryls Derivatives
(identical activities for 12 and 14). Finally, the fourth group of
compounds (16, 17, and 19) present a hydrazone bond provided
by the condensation between the aldehydic moiety (moieties)
of the 5,5′-biaryl compounds and hydralazine. Gratifyingly, all
compounds present an extremely potent antioxidant activity
between 2.6% and 4.1% in the TBARS assay. These are the
most potent antioxidants we have synthesized. These results
contrast with those where the hydrazone link is obtained through
introduction of isoniazid. It should be noted also that compounds
16, 17, and 19 present much better antioxidant and cytoprotec-
tive effects values than the corresponding parent compounds
12 and hydralazine alone. In fact, pure hydralazine tested alone
did not exhibit any antioxidant effect at this concentration (data
not shown). We do not know yet the reason for these excellent
activities. A possible explanation could be as a result of their
hydrazone link. Dolenc and al.23 recently reported that hydra-
zones deriving from primary hydrazines are prone to autoxi-
dation reactions, this depending strongly on the nature of the
substituents on the two nitrogen atoms.
TBARS %a
MTT %b
control
native LDL
5
13
12
8
9
10
14
15
18
16
17
19
0 ( 5
100 ( 5
100 ( 5
15 ( 5
86.2 ( 7.4
135.1 ( 8.9
67.5 ( 8.4
67.4 ( 8.6
76.5 ( 7.9
74.6 ( 9.4
67.5 ( 6.9
95.8 ( 9.1
83.0 ( 2.1
4.1 ( 0.6
2.6 ( 0.6
2.8 ( 0.1
55 ( 2.1
40.5 ( 8.6
26.8 ( 4.6
63.5 ( 9.5
66.0 ( 5.5
30.2 ( 2.5
40.9 ( 8.7
31.2 ( 5.5
43.3 ( 6.2
13.5 ( 0.3
61.2 ( 1.5
68.9 ( 0.9
82.0 ( 8.6
29 ( 2.8
R-tocopherol
probucol
10 ( 0.7
92 ( 11.3
a The different compounds were tested at 10 µM on LDL oxidation
mediated by HMEC-1 in RPMI culture medium in presence of traces of
CuSO4(µM). LDL oxidation was monitored after 6 h of incubation by the
determination of TBARS in the culture medium. The results are expressed
as % of the TBARS content in LDL-containing culture medium incubated
with HMEC-1 in the absence of any agent (15 ( 2.4 nmol TBARS/mg
apoB). b The toxicity of LDL oxidized by HMEC-1 in presence or absence
of the different compounds was evaluated by the MTT test after 24 h of
incubation. The results are expressed as % of the unstimulated cell control
(MTT done on control cells incubated in fresh RPMI culture medium in
the absence of LDL and antioxidant).
Concerning the first three groups of compounds, we can
conclude that these 5,5′-biaryl derivatives present lower anti-
oxidant activities than the related phosphonate or fluorophos-
phonate monomers 1 and 4 reported earlier. This is in agreement
with the few studies reported on polyphenolic systems where
the hydroxy groups do not belong in the same aromatic ring.
In a recent paper, Pedulli and al.24 reported that 2,2′-dihydroxy-
biphenyl systems presented low reactivity toward peroxy radicals
and were weak antioxidants, attributed to the formation of twin
intramolecular hydrogen bond in the starting biphenyl frame
and to strong H-bond interaction between the quinone adduct
and the remaining OH phenol group. The second OH group is
thus less reactive, resulting in a reduction of its antioxidant
activity.
present all an aldehydic moiety. While vanillin itself is a known
antioxidant, the diethyl phosphonate monomer 1 and the
corresponding fluorinated adduct 4 previously synthesized and
tested13 present good antioxidant activities, compounds 5, 12,
and 13 are much less efficient.
In the second group, the 5,5′-biaryl compounds from the
oxidative coupling of vanillin present in both parts the R,ꢀ-
unsaturated phosphonate moiety that might be symmetrically
substituted as for compounds 8 and 9 or dissymmetrically
substituted on the phosphorus atom as for compound 10. Bis-
phosphonodiester 8 presents the best result and is similar to
that of compound 12. As previously, compound 8 is less efficient
than its corresponding monomer 1.
The third group of compounds present a hydrazone bond
provided by the condensation between the aldehydic moiety of
the 5,5′-biaryl compounds and isoniazid. Compounds are
symmetrically substituted as for 18 or dissymmetrically as for
14 and 15. The latter two possess also a R,ꢀ-unsaturated
(fluoro)phosphonate moiety. Results are also very modest; the
best activity is observed for the dissymmetrically substituted
compound 14 possessing the diethylphosphonate functionality.
Note that pure isoniazid tested alone did not exhibit any
antioxidant effect at this concentration (data not shown). Thus
we can say that introduction of isoniazid through a hydrazone
link on compound 12 did not modify its antioxidant activity
To make an exploration of molecular geometry in relation to
the dihedral angle of interest, a conformation sampling was
carried using a short molecular dynamics on each compound
synthesized. The temperature was increased from 298 to 450
K during 50 ps in order to provide a range of 5000 conformers.
Software used was Discover from Accelrys and extensible and
systematic force field (ESFF) in vacuum. Energy versus time
graphs were plotted. Lower energy conformers (6-8) with
energies varying up to 30% comparing to the lowest value, were
selected and each one of them was subjected to a new dynamic
(Discover/ESFF). This process is repeated until reaching the
minimum energy conformer. Selected dihedral angles
(H1-O2-H2-OCH3) and hydrogen bond lengths of potent (16,
17, 19) and less potent (12, 14) derivatives are reported in Ta-
ble 2.
Results indicated, as in the case of Pedulli’s et al. compounds,
the adoption of cisoid geometry with the two aromatic rings
making a dihedral angle of ca. 50° and where each of the two

DeVelopment of NoVel Antiatherogenic Biaryls
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3175
Table 2. Computational Modelling Studies of Biaryl Dihedral Angle
and Hydrogen Bond Distances
Altogether, these data indicate that the newly synthesized
agents (16, 17, and 19) exhibited potent antioxidant properties
against cell-mediated LDL oxidation and subsequent cytotoxicity
as well as the formation of foam cells, properties that are well
beyond the individual considered frames (phosphonate, fluo-
rophosphonate, or hydrazine). Our results show that at least in
vitro, these compounds are efficient for preventing the early
steps of atherosclerosis.
dihedral
angle, deg
hydrogen bond
hydrogen bond
(Å) H1-O2
(Å) H2-OCH3
Although compound 19 exhibited antioxidant and carbonyl
scavenger properties as well, it is likely that its antioxidant
activity was not involved in its protective effect against carbonyl
stress because 19 prevented the toxicity and the carbonyl stress
mediated by 4-HNE, which is a potent carbonyl stress inducer,
independently of any reactive oxygen species increase. It is
noteworthy that hydralazine was also able to prevent the
carbonyl stress but at higher concentrations (>100 µM).
In conclusion, on the basis of the bisvanillin scaffold for
which the synthesis has been greatly improved, we have
synthesized different biaryls compounds, symmetrically or
dissymmetrically substituted on aromatic frames by a vi-
nyl(fluoro)phosphonate and/or hydrazino functionalities.
Altogether, biological data indicate that the newly synthesized
biaryl agents exhibit both antioxidant and carbonyl scavenger
properties, thus suggesting a potential strong antiatherogenic
effect. In that respect, compounds 16, 17, and 19 are shown to
be very interesting. The reason for their strong antioxidant and
cytoprotective efficiency will be addressed in terms of physi-
cochemical properties and of biological future of these com-
pounds (reactivity against radicals, prodrug nature of the
compounds, interaction with metals, activity against others
biological targets, . . .).
Concerning the biological aspect of our research program,
further investigations in apoE-/- mice as an animal model for
atherosclerosis, should support the antiatherogenic efficiency
of these agents.
Moreover, to increase the antioxidant activities of the three
first groups of compounds synthesized, we envisage to introduce
a new dissymmetry on the phenolic parts of the bisvanillin in
order to eliminate the strong double OH interaction leading to
another interesting scaffold for further elaboration of hydrazone-
like antiatherogenic derivatives.
12
14
16
17
19
52.18
56.26
51.83
50.79
53.66
1.90
1.89
1.87
1.92
1.89
2.10
2.14
2.08
2.05
2.12
OH protons is hydrogen bonded to the oxygen atom of the other
hydroxy group. So, the strong biological activities found for
compounds 16, 17, and 19 may be due to another structural
and/or physicochemical properties of these derivatives yet to
be adressed.
Concerning some further biological experiments, we tested
the ability of the selected compounds 8, 15, and 19, as
representatives of the newly synthesized derivatives, to inhibit
the formation of foam cells. For this purpose, human macroph-
age-like U937 cells were incubated for 18 h with native LDL
(100 µg/mL) in the presence of each selected agent (10 µM).
The formation of foam cells was evaluated fluorimetrically after
Nile Red staining, in the following reported conditions.25
Alternatively, foam cells were microscopically characterized in
terms of red lipid droplet formation after staining with the neutral
lipid probe, oil red O. As shown in Figure 1, LDL incubated
with U937 were oxidized (TBARS content 26.9 ( 3.4 nmol/
mg protein) and taken up by the cells, resulting in a huge
increase in Nile Red fluorescence (Figure 1a) and characteristic
lipid droplet accumulation in the cytosol (Figure 1b). As
expected, compound 19 efficiently inhibited foam cells forma-
tion, this was also partially prevented by compound 8 (in
agreement with its protective effect against oxLDL toxicity).
In contrast, compound 15 did not exhibit any protective effect.
We then aimed to evaluate the direct cytoprotective effect of
all synthesized compounds, toward LDL oxidized by UV-
irradiation, in the absence of any agent. As shown in Figure 2,
the first three groups of compounds are not very efficient, with
better results obtained for the bis(fluoro)phosphonate derivatives.
Interestingly, the fourth group (compounds 16, 17, and 19) was
shown to be very potent, with compound 19 possessing the
highest efficiency (more than 80% of cell survival).
Finally, we evaluated the ability of some newly synthesized
agents in preventing cell death induced by 4-HNE and in
neutralizing the formation of carbonyl proteins (carbonyl stress).
Previous experiments have shown that high hydralazine and
isoniazid concentrations (100 µM) may protect against 4-HNE-
induced cell death. We tested the protective effect of the most
representative compounds on the toxicity induced by 4-HNE
(50 µM).
As shown in Figure 3a, the compound 19 was highly efficient
in protecting cells against 4-HNE-induced cell death. Compound
8 was mildly protective, while compound 15 was ineffective.
Inhibitory effect of these agents on carbonyl stress evaluated
as DNPH-labeled proteins in HMEC-1 incubated with oxLDL
(200 µg/mL) or 4-HNE (50 µM) for 14 h (Figure 3b), showed
also that compound 19 is very efficient in preventing the increase
in carbonyl proteins, and thus the formation of adducts resulting
in carbonyl stress. The other agents were almost ineffective.
Experimental Section
Materials. All chemicals are of the highest commercially
available purity. 2-Thiobarbituric acid and diagnostic kits for total
cholesterol, LDL, and triglyceride determination are purchased from
Sigma Chemical Co. (St. Louis, MO). All other reagents were
purchased from Aldrich-Chemie (Steinheim, Germany). Com-
mercial 2-piperidinemethanol corresponds to the racemate.
Synthesis. Organic solvents were purified when necessary by
methods described by Perrin, Armarengo, and Perrin (Purification
of Laboratory Chemicals; Pergamon: Oxford, 1986) or were
purchased from Aldrich Chemie.
Melting points (mp) were obtained on a Buchi apparatus and
are uncorrected. Infrared (IR) spectra were recorded on a Perkin-
Elmer 1725 infrared spectrophotometer, and the data are reported
in inverse centimeters. UV spectra were recorded on a Cary 100/
300 spectrophotometer. Proton nuclear magnetic resonance (1H
NMR) spectra were obtained with Bruker AC-200, AC-250, AC-
300, and AC 400 MHz spectrometers. Chemical shifts were reported
in parts per million (ppm) relative to tetramethylsilane (TMS), and
signals are given as follows: s, singlet; d, doublet; t, triplet; m,
multiplet. Mass spectra were recorded on an R 10-10 C Nermag
(70 eV) quadripolar spectrometer using desorption chemical ioniza-
tion (DCI) or electrospray (ES) techniques. Elemental analyses were
performed with a Perkin-Elmer 2400 CHN analyzer.

3176 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Delomene`de et al.
Figure 1. Inhibition of foam cell formation. (1A) Human U937 monocyte/macrophage cells were incubated with oxLDL (100 µg/mL) for 14 h,
in RPMI 1640 medium containing 1 µM CuSO4, in the absence (0) or in the presence of the newly synthesized biaryl compounds 8, 15, and 19 (10
µM). The control (C) was performed in the absence of oxLDL and of any agent. Foam cells formation was monitored fluorometrically by the
uptake of Nile Red, as described in the Experimental Section. These data are a mean of three separate experiments. (1B) Pictures showing the
accumulation of oil Red O-stained lipid droplets, in U937 incubated with oxLDL as in (1A), and in the presence of agents 15 and 19 (10 µM).
C2 + C6); 111.13 (d, 1C, 1JC-P ) 192.0 Hz, CdC-P); 126.36 (d,
1C, 3JC-P ) 20.3 Hz, C1); 137.02 (s, 1C, C4); 147.20 (s, 2C, C3 +
C5); 148.95 (d, 1C, 2JC-P ) 6.8 Hz, CdC-P). 31P NMR (CDCl3,
81 MHz) δ ppm: 20.36 (s, P-OEt). MS (DCI, NH3, pos) m/z: 317.0
(MH+, 100%), 334.0 (MNH4+, 36%). Anal. (C14H21O6P) C, H calcd
53.16, 6.69; found 52.91, 6.76. IR (KBr) ν cm-1: 3188 (O-H);
3082 (C-H ethyl); 1620 (CdC ethyl); 1594 (CdC arom); 1242
(PdO); 1162 (P-O). UV (EtOH, 22 µM): λ ) 232 nm, ꢀ ) 27600
mol-1 ·L·cm-1
.
Diethyl(E)-2-(4-tert-butyldiphenylsilyloxy-3,5-dimethoxyphe-
nyl)vinylphosphonate (2). To vinylphosphonate (1) (2 g, 6.33 mmol)
in dried DMF (29 mL) were added imidazole (0.86 g, 12.66 mmol),
DMAP (0.77 g, 6.33 mmol), and TBDPSCl (2.6 g, 9.5 mmol) under
N2. The reaction mixture was stirred for 16 h at room temperature.
Aqueous NaHCO3 5% (30 mL) was added, and the aqueous layer
was extracted with Et2O (3 × 30 mL). The combined extracts were
dried (Na2SO4) and concentrated under vacuo. Silica gel chroma-
tography (EtOAc/petroleum ether 6/4) gave 2 as white crystals (5.33
g, 78%); mp )155-157 °C. 1H NMR (CDCl3, 300 MHz) δ ppm:
Figure 2. Direct cytoprotective effect towards oxLDL. Cytotoxic effect
of UV-oxidized LDL and cytoprotective effect of the synthesized agents
(10 µM). HMEC-1 was incubated for 24 h with UV-oxidized LDL
(200 µg/mL), alone (0) or in the presence of the newly synthesized
agents (10 µM). The cytotoxicity was evaluated by the MTT assay.
Results are expressed as % of the unstimulated control (cell incubated
in LDL-free medium). Each bar represents the mean for four separate
determinations.
3
1,11 (s, 9H, tBu); 1,34 (t, 6H, JH-H ) 7.0 Hz, CH3CH2O); 3.47
3
3
(s, 6H, CH3O); 4.13 (qd, 4H, JH-H ) 7.0 Hz, JH-P ) 7.0 Hz,
3
2
CH3CH2O); 6.05 (dd, 1H, JH-H ) 13.8 Hz, JH-P ) 17.4 Hz,
CHdCH-P); 6.60 (s, 2H, H2 + H6); 7.36 (m, 7H, CHdCH-P +
Diethyl(E)-2-(4-hydroxy-3,5-dimethoxyphenyl)vinylphospho-
nate (1). To iPr2NH (5 mL, 36.26 mmol) dissolved in dried THF
(8 mL/mmol) stirred at -78 °C under nitrogen, n-BuLi (1.6 M in
hexane: 22.7 mL, 36.26 mmol) was added. The reaction mixture
was stirred for 30 min at -78 °C under N2. Tetraethyl methylene-
diphosphonate (9 mL, 36.26 mmol) in THF was added to formed
lithium diisopropylamide (LDA) solution, and the reaction mixture
was stirred 1 h at -78 °C. Syringaldehyde (17.6 mmol, 3.21 g) in
THF was added, and mixture was stirred for 45 min at -78 °C,
then allowed to warm up to room temperature and refluxed. After
2 h, saturated aqueous NH4Cl (150 mL) was added, and the aqueous
layer was extracted with Et2O (3 × 200 mL). The combined extracts
were dried (Na2SO4) and concentrated. The residue was purified
by recrystallization from CH2Cl2 to give 1 (3.75 g, 67%) as a white
3
Hp and Hm phenyl); 7.72 (d, 4H, JHo-Hm ) 7.5 Hz, Ho phenyl).
13C NMR (CDCl3, 75 MHz) δ ppm: 16.43 (d, 2C, 3JC-P ) 6.8 Hz,
CH3CH2O); 20.15 (s, 1C, Cq tBu); 26.17 (s, 3C, CH3 tBu); 55.21
2
(s, 2C, CH3O); 61.73 (d, 2C, JC-P ) 5.3 Hz, CH3CH2O); 104.77
1
(s, 2C, C2 + C6); 111.18 (d, 1C, JC-P ) 192.0 Hz, CdC-P);
3
127.09 (s, 4C, Cm phenyl); 127.51 (d, 1C, JC-P ) 24.0 Hz, C1);
129.20 (s, 2C, Cp phenyl); 134.23 (s, 1C, C4); 135.06 (s, 4C, Co
phenyl); 136.63 (s, 2C, C phenyl); 149.13 (d, 1C, 2JC-P ) 6.8 Hz,
CdC-P); 151.06 (s, 2C, C3 + C5). 31P NMR (CDCl3, 81 MHz) δ
ppm: 20.09 (s, P-OEt). MS (DCI, NH3, pos) m/z: 555.0 (MH+).
Ethyl(E)-2-(4-tertiobutyldiphenylsilyloxy-3,5-dimethoxyphenyl)vi-
nylphosphonochloridate (3). To phosphonodiester (2) (1.25 g, 2.26
mmol) in CH2Cl2 (15 mL) under N2 was added freshly distilled
oxalyl chloride (0.89 mL, 10.2 mmol). The reaction mixture was
stirred at room temperature. Reaction was monitored by 31P NMR
until completion. CH2Cl2 and oxalyl chloride excess were removed
under vacuo. Phosphonochloridate was obtained as yellow solid
(1.23 g, 100%) and used without purification in fluorination step.
31P NMR (D2O, 81 MHz) δ ppm: 29.94 (s, P-Cl).
1
solid; mp ) 181-183 °C. H NMR (CDCl3, 250 MHz) δ ppm:
1.35 (t, 6H, 3JH-H ) 7.0 Hz, CH3CH2O); 3.91 (s, 6H, CH3O); 4.13
3
3
(qd, 4H, JH-H ) 7,2 Hz, JH-P ) 7.2 Hz, CH3CH2O); 5.78 (s,
3
2
1H, OH); 6.08 (dd, 1H, JH-H ) 17.2 Hz, JH-P ) 17.2 Hz,
3
CHdCH-P); 6.74 (s, 2H, H2 + H6); 7.39 (dd, 1H, JH-H ) 17.5
Hz, 3JH-P ) 24,5 Hz, CHdCH-P). 13C NMR (CDCl3, 75 MHz) δ
3
ppm: 16.42 (d, 2C, JC-P ) 6.5 Hz, CH3CH2O); 56.34 (s, 2C,
2
CH3O); 61.78 (d, 2C, JC-P ) 5.4 Hz, CH3CH2O); 104.72 (s, 2C,

DeVelopment of NoVel Antiatherogenic Biaryls
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3177
Figure 3. Direct cytoprotective effect towards 4-HNE-induced cell death proteins carbonylation at 10 µM. (3A) HMEC-1 were incubated overnight
in pure fetal calf serum (FCS)-free RPMI-1640 culture medium containing 4-HNE (50 µM). The cells were carefully washed with warm PBS,
scrapped, and the carbonyl protein content was determined in presence of DNPH as described in the Experimental Section. The results are expressed
as % of the unstimulated control ( (structural equation modelling) SEM. These data are a mean of four separate experiments. (3B) Cytotoxicity
of UV-oxidized LDL (200 µg/mL) and of 4-HNE (5 µM) was evaluated by the MTT test on HMEC-1 after 24 h of incubation. The results are
expressed as % of the unstimulated control (cell incubated in LDL-free medium). Each bar represents the mean for four separate determinations.
Ethyl(E)-2-(4-hydroxy-3,5-dimethoxyphenyl)vinyl phosphono-
fluoridate (4). To phosphonochloridate (3) (0.3 g, 0.42 mmol) in
freshly distilled THF (4.2 mL) under N2 were added Et3N (0.2 mL,
1.4 mmol) and Et3N·3HF (0.11 mL, 0.7 mmol). The reaction
mixture was stirred at room temperature and monitored by 31P NMR
and then quenched by addition of EtOAc. Precipitate formed was
filtered off. Water was added to the filtrate. Aqueous layer was
extracted with EtOAc (3 × 10 mL). The combined extracts were
dried (Na2SO4) and concentrated under vacuo. Silica gel chroma-
tography (EtOAc/petroleum ether 4/6) gave 4 as a white solid (93
mg, 77%); mp ) 132-134 °C. 1H NMR (CDCl3, 250 MHz) δ
ppm: 1.38 (t, 3H, 3JH-H ) 7.0 Hz, CH3CH2O); 3.88 (s, 6H, CH3O);
1672 (CdO); 1587 (CdC arom); 1454 (CdC arom). UV (EtOH
+ 0.2% DMSO, 25 µM): λ ) 303 nm, ꢀ ) 14300 mol-1 ·L·cm-1
.
5,5′-bis(4-tert-butyldimethylsilyloxy-3-methoxybenzaldehyde) (6).
Tert-butyldimethylsilyl chloride (1.25 g, 8.28 mmol) and imidazole
(1.13 g, 16.56 mmol) were added to a solution of 5,5′-bisvanillin
5 (1.0 g, 3.31 mmol) in DMF (10 mL) under N2. The reaction
mixture was stirred at room temperature for 3 h, then poured into
5% aqueous NaHCO3. The aqueous layer was extracted with EtOAc
(3 × 50 mL) and the combined organic extracts were washed with
water, dried (Na2SO4), and concentrated as brown oil. The residue
was dissolved in EtOAc (10 mL), filtered through a short pad of
silica, and concentrated. Silica gel chromatography (7/3 petroleum
ether/EtOAc) gave 6 (1.24 g, 70%) as a crystals; mp ) 183-185
3
3
4.27 (qd, 2H, JH-H ) 7.0 Hz, JH-P ) 7.0 Hz, CH3CH2O); 5.81
3
2
(s, 1H, OH); 6.07 (dd, 1H, JH-H ) 17.5 Hz, JH-P ) 19.2 Hz,
1
°C. H NMR (CDCl3, 300 MHz) δ ppm: 0.02 (s, 12H, Si(CH3)2);
3
CHdCH-P); 6.72 (s, 2H, H2 + H6); 7.49 (dd, 1H, JH-H ) 17.5
0.61 (s, 18H, tBu); 3.90 (s, 6H, CH3O); 7.41 (d, 2H, 4JH2-H6 ) 2.0
Hz, 3JH-P ) 24.5 Hz, CHdCH-P). 13C NMR (CDCl3, 75 MHz) δ
4
Hz, H2); 7.47 (d, 2H, JH6-H2 ) 1.8 Hz, H6); 9.84 (s, 2H, CHO).
3
ppm: 16.30 (d, 1C, JC-P ) 6.0 Hz, CH3CH2O); 56.35 (s, 2C,
13C NMR (CDCl3, 75 MHz) δ ppm: -4.28 (s, 4C, Si(CH3)2); 18.34
(s, 2C, Cq tBu); 25.16 (s, 6C, CH3 tBu); 55.20 (s, 2C, CH3O); 108.68
(s, 2C, C2); 129.40 (s, 2C, C6); 129.46 (s, 2C, C5); 129.67 (s, 2C,
C1); 149.10 (s, 2C, C4); 151.06 (s, 2C, C3); 190.96 (s, 2C, CHO).
MS (ES, pos) m/z: 531.5 (MH+).
2
CH3O); 63.41 (d, 1C, JC-P ) 6.0 Hz, CH3CH2O); 105.11 (s, 2C,
1
2
C2 + C6); 106.75 (dd, 1C, JC-P ) 207.0 Hz, JC-F ) 33.0 Hz,
3
CdC-P); 125.50 (d, 1C, JC-P ) 25.5 Hz, C1); 137.86 (s, 1C,
2
C4); 147.32 (s, 2C, C3 + C5); 151.62 (dd, 1C, JC-P ) 7.5 Hz,
3JC-F ) 3.8 Hz, CdC-P). 31P NMR (CDCl3, 81 MHz) δ ppm:
19.50 (d, 1JP-F ) 1023.0 Hz, P-F). 19F NMR (CDCl3, 188.5 MHz)
Diethyl(E)-2-[4,4′-ditertiobutyldimethylsilyloxy-1′-(E)-(diethoxy-
phosphoryl)vinyldimethoxy-5,5′-biphenyl]vinylphosphonate (7). Com-
pound 7 was synthesized from 6 (0.2 g, 3.77 ×10-4 mol) as
compound 1. The residue was purified by silica gel chromatography
(EtOAc) to give pure 7 (0.26 g, 86%) as a white solid; mp )
1
δ ppm: 11.88 (d, JF-P ) 1021.0 Hz, F-P). MS (DCI, NH3, pos)
+
m/z: 308.0 (MNH4
,
100%), 291.0 (MH+, 38%). Anal.
(C12H16FO5P) C, H calcd 49.66, 5.56; found 49.66, 5.70. IR (KBr)
ν cm-1: 3270 (O-H); 3014 (C-H ethyl); 1616 (CdC ethyl); 1597
(CdC arom); 1253 (PdO); 1159 (P-O). UV (EtOH, 26 µM): λ )
181-183 °C. 1H NMR (CDCl3) δ ppm: -0.11 (s, 6H, SiCH3);
3
318 nm, ꢀ ) 27000 mol-1 ·L·cm-1
.
-0.02 (s, 6H, SiCH3); 0.57 (s, 18H, tBu); 1.29 (t, 12H, JH-H
)
7.3 Hz, CH3CH2O); 3.79 (s, 6H, CH3O); 4.05 (q, 8H, 3JH-H ) 7.3
Hz, CH3CH2O); 6,02 (dd, 2H, 3JH-H ) 2JH-P ) 17.7 Hz, CHdCH-
P); 6.91 (s, 2H, H2); 7.05 (s, 2H, H6); 7.36 (dd, 2H, 3JH-H ) 17.4
Hz, 3JH-P ) 22.5 Hz, CHdCH-P). 13C NMR (CDCl3, 75 MHz) δ
ppm: -4.54 (s, 2C, SiCH3); -4.22 (s, 2C, SiCH3); 16.40 (d, 4C,
3JC-P ) 6.0 Hz, CH3CH2O); 18.35 (s, 2C, Cq tBu); 25.29 (s, 6C,
5,5′-Bisvanillin (5). To vanillin (10.64 g, 70 mmol) in 700 mL
of water was added FeSO4 (0.4 g, 1.4 mmol) under stirring. After
heating during 10 min at 50 °C, Na2S2O8 (8.93 g, 37.5 mmol) was
added. The reaction mixture was stirring at 50 °C for 5 days. The
brown precipitate formed was filtered off. The solid was dissolved
in NaOH (2 M) solution. HCl (2 M) solution was added and
precipitate formed was isolated by filtration (10 g, 95%); mp >270
2
CH3 tBu); 55.10 (s, 2C, CH3O); 61.70 (d, 4C, JC-P ) 5.3 Hz,
1
1
CH3CH2O); 109.56 (s, 2C, C2); 111.00 (d, 2C, JC-P ) 191.0
°C. H NMR (DMSO-d6, 250 MHz) δ ppm: 3.94 (s, 6H, CH3O);
Hz, CdC-P); 124.36 (s, 2C, C6); 127.15 (d, 2C, 3JC-P ) 23.3 Hz,
7.42 (s, 4H, H2 + H6); 9.80 (s, 2H, CHO); 9.89 (s, 2H, OH). 13C
NMR (DMSO-d6, 75 MHz) δ ppm: 56.50 (s, 2C, CH3O); 109.70
(s, 2C, C2); 125.00 (s, 2C, C5); 128.23 (s, 2C, C1); 128.62 (s, 2C,
C6); 148.60 (s, 2C, C4); 150.90 (s, 2C, C3); 191.60 (s, 2C, CHO).
MS (FAB, MNBA) m/z: 303.1 (MH+). Anal. (C16H14O6) C, H calcd
63.52, 4.63; found 63.46, 4.61. IR (KBr) ν cm-1: 3186 (O-H);
C1); 130.19 (s, 2C, C5); 145.01 (s, 2C, C4); 148.83 (d, 2C, 2JC-P
)
6.8 Hz, CdC-P); 150.59 (s, 2C, C3). 31P NMR (CDCl3, 81 MHz)
δ ppm: 20.53 (s, P-OEt). MS (DCI, NH3, pos) m/z: 799.9 (MH+).
IR (KBr) ν cm-1: 2928 (C-H ethyl); 1616 (CdC ethyl); 1573
(CdC arom); 1495 (CdC arom); 1233 (PdO); 1155 (P-O).

3178 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Delomene`de et al.
Diethyl(E)-2-[4,4′-dihydroxy-1′-(E)-(diethoxyphosphoryl)vinyl-
3,3′-dimethoxy-5,5′-biphenyl]vinylphosphonate (8). To bisphos-
phonate 7 (53 mg, 6.63 × 10-2 mmol) in freshly distilled THF (1
mL) under N2 were added Et3N (15 µL, 11.07 × 10-2 mmol) and
Et3N·3HF (9 µL, 5.5 × 10-2 mmol). The reaction mixture was
stirred at room temperature for 1 h and then quenched by addition
of EtOAc. Aqueous HCl (1 M) was added until pH ) 4. Aqueous
layer was extracted with EtOAc (3 × 2 mL). The combined extracts
were dried (Na2SO4) and concentrated under vacuum. Silica gel
chromatography (EtOAc/MeOH 95/5) gave 8 as a white solid (21
mg, 57%); mp ) 187-189 °C. 1H NMR (CDCl3, 250 MHz) δ
ppm: 1.34 (t, 12H, 3JH-H ) 7.0 Hz, CH3CH2O); 3.95 (s, 6H, CH3O);
δ ppm: 1.34 (t, 6H, 3JH-H ) 7.0 Hz, CH3CH2O); 1.40 (t, 3H, 3JH′-H′
) 7.0 Hz, CH′3CH′2O); 3,95 (s, 3H, CH3O); 3.95 (s, 3H, CH′3O);
3
3
4.12 (q, 4H, JH-H ) JH-P ) 7.0 Hz, CH3CH2O); 4.29 (q, 2H,
3
3
3JH′-H′ ) JH′-P′ ) 7.0 Hz, CH′3CH′2O); 6.09 (dd, 1H, JH-H
)
)
2JH-P ) 17.6 Hz, CHdCH-P); 6.10 (dd, 1H, JH′-H′ ) JH′-P′
17.6 Hz, CH′dCH′-P′); 7.02 (s, 1H, H2); 7.03 (s, 1H, H2′); 7.09
(d, 1H, JH6-H2 ) 1.5 Hz, H6); 7.13 (d, 1H, JH6′-H2′ ) 1.8 Hz,
3
2
4
4
3
3
H6′); 7.43 (dd, 1H, JH-H ) 17.3 Hz, JH-P ) 22.2 Hz, CHdCH-
P); 7.56 (dd, 1H, 3JH′-H′ ) 17.4 Hz, 3JH′-P′ ) 24.4 Hz, CH′dCH′-
P′). 13C NMR (CDCl3, 75 MHz) δ ppm: 16.33 (d, 2C, 3JC-P ) 5.3
Hz, CH3CH2O); 16.41 (d, 1C, 3JC′-P′ ) 6.0 Hz, C′H3C′H2O); 56.20
2
(s, 1C, CH3O); 56.23 (s, 1C, C′H3O); 61.81 (d, 2C, JC-P ) 5.3
3
3
4.11 (q, 8H, JH-H ) JH-P ) 7.0 Hz, CH3CH2O); 6.10 (dd, 2H,
Hz, CH3CH2O); 63.42 (d, 1C, 2JC′-P′ ) 6.0 Hz, C′H3C′H2O); 106.96
3JH-H ) 2JH-P ) 17.4 Hz, CHdCH-P); 7.02 (d, 2H, 4JH2-H6 ) 1.8
(dd, 1C, JC′-P′ ) 206.0 Hz, JC′-F ) 33.0 Hz, C′dC′-P); 108.82
(s, 1C, C2); 108.87 (s, 1C, C2′); 111.39 (d, 1C, 1JC-P ) 191.0 Hz,
CdC-P); 123.47 (s, 1C, C5); 123.91 (s, 1C, C5′); 124.12 (s, 1C,
C6); 124.79 (s, 1C, C6′); 126.25 (d, 1C, 3JC-P ) 25.5 Hz, C1); 127.11
1
2
4
3
Hz, H2); 7.10 (d, 2H, JH6-H2 ) 1.8 Hz, H6); 7.43 (dd, 2H, JH-H
) 17.4 Hz, JH-P ) 22.6 Hz, CHdCH-P). 13C NMR (CDCl3, 75
3
MHz) δ ppm: 16.40 (d, 4C, 3JC-P ) 6.0 Hz, CH3CH2O); 55.19 (s,
2
3
2C, CH3O); 61.81 (d, 4C, JC-P ) 5.3 Hz, CH3CH2O); 108.83 (s,
(d, 1C, JC′-P′ ) 24.0 Hz, C1′); 145.20 (s, 1C, C4); 145.98 (s, 1C,
C4′); 147.34 (s, 1C, C3); 147.46 (s, 1C, C3′); 148.58 (d, 1C, JC-P
1
2
2C, C2); 111.32 (d, 2C, JC-P ) 191.0 Hz, CdC-P); 123.84 (s,
3
2
3
2C, C5) 124.14 (s, 2C, C6); 127.13 (d, 2C, JC-P ) 24.0 Hz, C1);
) 6.8 Hz, CdC-P); 151.33 (dd, 1C, JC′-P′ ) 7.5 Hz, JC′-F )
145.18 (s, 2C, C4); 147.41 (s, 2C, C3); 148.64 (d, 2C, 2JC-P ) 7.5
Hz, CdC-P). 31P NMR (CDCl3, 81 MHz) δ ppm: 20.31 (s, P-OEt).
MS (APCI, pos) m/z: 571.1 (MH+). Anal. (C26H36O10P2) C, H calcd
54.69, 6.31; found 54.51, 6.19. IR (KBr) ν cm-1: 3223 (O-H);
2980 (C-H ethyl); 1612 (CdC ethyl); 1590 (CdC arom); 1464
(CdC arom); 1235 (PdO); 1165 (P-O). UV (EtOH, 25 µM): λ )
3.8 Hz, C′dC′-P). 31P NMR (CDCl3, 81 MHz) δ ppm: 19.42 (d,
1P, JP′-F ) 1023.0 Hz, P′-F); 20.32 (s, 1P, P-OEt). 19F NMR
1
(CDCl3, 188.5 MHz) δ ppm: 11.85 (d, 1JF-P′ ) 1022.0 Hz, F-P′).
MS (APCI, pos) m/z: 545.3 (MH+). Anal. (C24H31FO9P2) C, H calcd
52.95, 5.74; found 51.92, 5.83. IR (KBr) ν cm-1: 3400 (O-H);
2987 (C-H ethyl); 1615 (CdC ethyl); 1592 (CdC arom); 1494
(CdC arom); 1270 (PdO); 1147 (P-O). UV (EtOH, 20 µM): λ )
296 nm, ꢀ ) 29000 mol-1 ·L·cm-1
.
318 nm, ꢀ ) 30700 mol-1 ·L·cm-1
.
Ethyl(E)-2-[4,4′-dihydroxy-1′-(E)-(fluoroethoxyphosphoryl)vinyl-
3,3′-dimethoxy-5,5′-biphenyl]vinylphosphonofluoridate (9). To bis-
phosphonodiester 7 (0.1 g, 0.125 mmol) in CH2Cl2 (1 mL) under
N2 was added freshly distilled oxalyl chloride (65 µL, 0.75 mmol).
The reaction mixture was stirred at room temperature for 40 h.
CH2Cl2 and oxalyl chloride excess were removed under vacuum.
Phosphonochloridate was obtained as a yellow solid (0.097 g,
100%) and used without purification in fluorination step. 31P NMR
(D2O, 81 MHz) δ ppm: 29.95 (s, 2P, P-Cl).
Diethyl(E)-2-[4,4′-ditertiobutyldimethylsilyloxy-1′-formyl-3,3′-
dimethoxy-5,5′-biphenyl]vinylphosphonate (11). To iPr2NH (0.25
mL, 1.88 mmol) dissolved in dried THF (15 mL) stirred at -78
°C under nitrogen n-BuLi (1.6 M in hexane: 1.2 mL, 1.88 mmol)
was added. The reaction mixture was stirred for 30 min at -78 °C
under N2. Tetraethyl methylenediphosphonate (0.47 mL, 1.88 mmol)
in THF (15 mL) was added to formed LDA solution, and the
reaction mixture was stirred 1 h at -78 °C. Bisaldehyde 6 (1 g,
1.88 mmol) in THF was added and mixture was stirred for 4 h at
-78 °C then allowed to warm up to room temperature, and refluxed.
After 2 h, saturated aqueous NH4Cl (8 mL) was added, and the
aqueous layer was extracted with Et2O (3 × 15 mL). The combined
extracts were dried (Na2SO4) and concentrated. The residue was
purified by silica gel chromatography (EtOAc/petroleum ether 6/4)
Bisfluorophosphonate 9 was synthesized from bischlorophos-
phonate just obtained as for compound 4. The residue was purified
by silica gel chromatography (EtOAc/MeOH 95/5) to give pure
(9) (39 mg, 60%) as a white solid. 1H NMR (CDCl3, 250 MHz) δ
ppm: 1.41 (t, 6H, 3JH-H ) 7.0 Hz, CH3CH2O); 3.99 (s, 6H, CH3O);
3
3
4.30 (q, 4H, JH-H ) JH-P ) 7.0 Hz, CH3CH2O); 6.12 (dd, 2H,
1
3JH-H ) 2JH-P ) 17.4 Hz, CHdCH-P); 7.05 (d, 2H, 4JH2-H6 ) 1.8
to give pure 11 (0.45 g, 54%) as colorless oil. H NMR (CDCl3,
4
3
300 MHz) δ ppm: -0.02 (s, 12H, Si(CH3)2); 0.61 (s, 9H, tBu);
Hz, H2); 7.15 (d, 2H, JH6-H2 ) 1.8 Hz, H6); 7.58 (dd, 2H, JH-H
3
) 17.4 Hz, JH-P ) 24.4 Hz, CHdCH-P). 13C NMR (CDCl3, 75
3
0.63 (s, 9H, tBu′); 1.33 (t, 6H, JH-H ) 7.1 Hz, CH3CH2O); 3.85
(s, 3H, CH3O); 3.88 (s, 3H, CH′3O); 4.10 (q, 4H, 3JH-H ) 3JH-P
)
MHz) δ ppm: 16.34 (d, 2C, 3JC-P ) 6.0 Hz, CH3CH2O); 56.27 (s,
3
7.3 Hz, CH3CH2O); 6.11 (dd, 1H, JH-H
)
2JH-P ) 17.5 Hz,
2C, CH3O); 63.42 (d, 2C, 2JC-P ) 6.0 Hz, CH3CH2O); 107.20 (dd,
4
1
2
CHdCH-P); 7.01 (d, 1H, JH2-H6 ) 1.9 Hz, H2); 7.11 (d, 1H,
2C, JC-P ) 206.0 Hz, JC-F ) 33.0 Hz, CdC-P); 108.89 (s, 2C,
4
4JH6-H2 ) 1.9 Hz, H6); 7.38 (d, 1H, JH2′-H6′ ) 1.9 Hz, H2′); 7.43
C2); 123.40 (s, 2C, C5); 124.71 (s, 2C, C6); 126.33 (d, 2C, 3JC-P
)
(dd, 1H, 3JH-H ) 17.3 Hz, 3JH-P ) 22.4 Hz, CHdCH-P); 7.45 (d,
1H, 4JH6′-H2′ ) 1.9 Hz, H6′); 9.82 (s, 1H, CHO). 13C NMR (CDCl3,
75 MHz) δ ppm: -4.54 (s, 2C, Si(CH3)2); -4.32 (s, 2C, Si(C′H3)2);
25.5 Hz, C1); 145.81 (s, 2C, C4); 147.29 (s, 2C, C3); 151.22 (dd,
2C, 2JC-P ) 7.5 Hz, 3JC-F ) 3.8 Hz, CdC-P). 31P NMR (CDCl3,
81 MHz) δ ppm: 19.30 (d, JP-F ) 1024.0 Hz, P-F). 19F NMR
1
3
1
16.35 (d, 2C, JC-P ) 6.4 Hz, CH3CH2O); 18.28 (s, 1C, Cq tBu);
(CDCl3, 188.5 MHz) δ ppm: 11.93 (d, JF-P ) 1022.0 Hz, F-P).
MS (APCI pos) m/z: 519.0 (MH+). Anal. (C22H26F2O8P2) C, H calcd
50.93, 5.02; found 50.85, 5.52. IR (KBr) ν cm-1: 3291 (O-H);
3033 (C-H ethyl); 1612 (CdC ethyl); 1590 (CdC arom); 1494
(CdC arom); 1223 (PdO); 1174 (P-O). UV (EtOH, 25 µM): λ )
18.37 (s, 1C, C′q tBu); 25.14 (s, 3C, CH3 tBu); 25.15 (s, 3C, C′H3
tBu); 55.03 (s, 1C, CH3O); 55.07 (s, 1C, C′H3O); 61.63 (d, 2C,
2JC-P ) 5.2 Hz, CH3CH2O); 108.33 (s, 1C, C2); 109.69 (s, 1C,
1
C2′); 111.16 (d, 1C, JC-P ) 192.5 Hz, CdC-P); 124.07 (s, 1C,
C6); 127.26 (d, 1C, 3JC-P ) 23.7 Hz, C1); 129.31 (s, 1C, C5); 129.57
(s, 1C, C1′); 129.63 (s, 1C, C6′); 130.15 (s, 1C, C5′); 144.97 (s, 1C,
C4); 148.62 (d, 1C, 2JC-P ) 6.7 Hz, CdC-P); 148.96 (s, 1C, C4′);
150.54 (s, 1C, C3); 151.01 (s, 1C, C3′); 190.90 (s, 1C, CHO). 31P
NMR (CDCl3, 81 MHz) δ ppm: 20.59 (s, P-OEt). MS (DCI, NH3,
pos) m/z: 665.0 (MH+). IR (NaCl) ν cm-1: 2930 (C-H aliph);
2857 (C-H ald); 1690 (CdO); 1575 (CdC arom); 1487 (CdC
arom); 1251 (PdO); 1158 (P-O).
318 nm, ꢀ ) 22600 mol-1 ·L·cm-1
.
Diethyl(E)-2-[4,4′-dihydroxy-1′-(E)-(fluoroethoxyphosphoryl)vi-
nyl-3,3′-dimethoxy-5,5′-biphenyl]vinylphosphonate (10). To bis-
phosphonodiester 7 (0.1 g, 0.125 mmol) in CH2Cl2 (1 mL) under
N2, was added freshly distilled oxalyl chloride (43 µL, 0.56 mmol).
The reaction mixture was stirred at room temperature for 15 h.
CH2Cl2 and oxalyl chloride excess were removed under vacuum.
Monophosphonochloridate was obtained as yellow solid (0.068 g,
100%) and used without purification in fluoration. 31P NMR (D2O,
81 MHz) δ ppm: 20.56 (s, 1P, P-OEt); 29.95 (s, 1P, P′-Cl).
Monophosphonofluoridate 10 was synthesized from monophospho-
nochloridate just obtained as for compound 4. The residue was
purified by silica gel chromatography (EtOAc/MeOH 95/5) to give
pure 10 (28 mg, 41%) as white solid. 1H NMR (CDCl3, 250 MHz)
Diethyl(E)-2-[4,4′-dihydroxy-1′-formyl-3,3′-dimethoxy-5,5′-bi-
phenyl]vinylphosphonate (12). Compound 12 was synthesized from
11 (0.7 g, 1.06 mmol) as compound 8. The residue was purified by
silica gel chromatography (EtOAc) to give pure 12 (0.35 g, 75%)
1
as a brown solid; mp ) 72-74 °C. H NMR (CDCl3, 300 MHz)
3
δ ppm: 1.34 (t, 6H, JH-H ) 6.0 Hz, CH3CH2O); 3.93 (s, 3H,

DeVelopment of NoVel Antiatherogenic Biaryls
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3179
3
3
CH3O); 3.95 (s, 3H, CH′3O); 4.12 (q, 4H, JH-H ) JH-P ) 6.0
Hz, CH3CH2O); 6.09 (dd, 1H, 3JH-H ) 2JH-P ) 18.0 Hz, CHdCH-
P); 7.02 (s, 1H, H2); 7.15 (s, 1H, H6); 7.40 (s, 1H, H2′); 7.45 (dd,
1H, 3JH-H ) 18.0 Hz, 3JH-P ) 21.0 Hz, CHdCH-P); 7.50 (s, 1H,
H6′); 9.82 (s, 1H, CHO). 13C NMR (CDCl3, 75 MHz) δ ppm: 16.40
CH3CH2O); 107.54 (s, 1C, C2); 109.40 (s, 1C, C2′); 110.41 (d, 1C,
1JC-P ) 188.3 Hz, CdC-P); 121.99 (s, 2C, C2′′ + C6′′); 123.96 (s,
2C, C5 + C5′); 124.85 (s, 1C, C1′); 126.29 (d, 1C, 3JC-P ) 8.3 Hz,
C1); 125.16 (s, 1C, C6′); 125.26 (s, 1C, C6); 141.21 (s, 1C, C1′′);
2
148.81 (d, 1C, JC-P ) 6.4 Hz, CdC-P); 149,02 (s, 2C, C4 +
3
(d, 2C, JC-P ) 6.8 Hz, CH3CH2O); 56.12 (s, 1C, CH3O); 56.13
C4′); 149.18 (s, 2C, C3 + C3′); 150.38 (s, 2C, C3′′ + C5′′); 150.72
(s, 1C, HCdN); 161.73 (s, 1C, CdO). 31P NMR (DMSO-d6, 121,5
MHz) δ ppm: 20.86 (s, P-OEt). MS (DCI, NH3, pos) m/z: 556.2
(MH+). IR (NaCl) ν cm-1: 3426 (O-H); 3018 (C-H ethyl); 1661
(CdN); 1652 (CdO); 1615 (CdC ethyl); 1552 (CdC arom); 1489
(CdC arom); 1272 (PdO); 1151 (P-O). UV (EtOH, 25 µM): λ )
(s, 1C, C′H3O); 61.82 (d, 2C, 2JC-P ) 5.3 Hz, CH3CH2O); 107.65
(s, 1C, C2′); 108.60 (s, 1C, C2); 110.61 (d, 1C, 1JC-P ) 191.3 Hz,
CdC-P); 124.31 (s, 1C, C6); 124.52 (s, 1C, C4 or C4′); 124.82 (s,
1C, C4′ or C4); 126.49 (d, 1C, 3JC-P ) 23.3 Hz, C1); 128.23 (s, 1C,
C1′); 129.91 (s, 1C, C6′); 146.83 (s, 1C, C3); 148.17 (s, 1C, C3′ or
2
327 nm, ꢀ ) 22000 mol-1 ·L·cm-1
.
C5); 148.70 (s, 1C, C5 or C3′); 148.90 (d, 1C, JC-P ) 6.8 Hz,
CdC-P); 151,71 (s, 1C, C5′); 190.92 (s, 1C, CHO). 31P NMR
(CDCl3, 121 MHz) δ ppm: 20.51 (s, P-OEt). MS (DCI, NH3, pos)
m/z: 454.3 (MNH4+). IR (KBr) ν cm-1: 3353 (O-H); 2989 (C-H
ethyl); 1678 (CdO); 1613 (CdC ethyl); 1589 (CdC arom); 1280
(PdO); 1142 (P-O). UV (EtOH, 25 µM): λ ) 311 nm, ꢀ ) 20600
Ethyl(E)-2-[4,4′-dihydroxy-1′-isonicotinoylhydrazonomethyl-3,3′-
dimethoxy-5,5′-biphenyl]vinylphosphonofluoridate (15). Compound
15 was synthesized from 13 (0.045 g, 0.11 mmol) as compound
14. The residue was purified by silica gel chromatography (EtOAc)
to give pure 12 (0.04 g, 69%) as a yellow solid. 1H NMR (CD3OD,
mol-1 ·L·cm-1
.
3
300 MHz) δ ppm: 1.12 (t, 3H, JH-H ) 7.0 Hz, CH3CH2O); 3.66
Ethyl(E)-2-[4,4′-dihydroxy-1′-formyl-3,3′-dimethoxy-5,5′-biphe-
nyl]vinylphosphonofluoridate (13). To phosphonodiester 12 (0.1
g, 0.151 mmol) under N2 was added freshly distilled oxalyl chloride
(2.4 mL, 30.2 mmol). The reaction mixture was stirred at room
temperature for 3 h. Oxalyl chloride excess was removed under
vacuo. Phosphonochloridate was obtained as yellow solid and used
without purification in fluorination step. 31P NMR (D2O, 81 MHz)
δ ppm: 29.27 (s, P-Cl). Monophosphonofluoridate 13 was syn-
thesized from phosphonochloridate just obtained as for compound
4. The residue was purified by silica gel chromatography (EtOAc/
MeOH 95/5) to give pure 13 (45 mg, 72%) as a white solid; mp )
84-86 °C. 1H NMR (CDCl3, 300 MHz) δ ppm: 1.33 (t, 3H, 3JH-H
) 7.0 Hz, CH3CH2O); 3.89 (s, 3H, CH3O); 3.91 (s, 3H, CH′3O);
3
(s, 3H, CH3O); 3.68 (s, 3H, CH′3O); 3.98 (qd, 2H, JH-H ) 7.1
3
3
Hz, JH-P ) 8.6 Hz, CH3CH2O); 6.05 (dd, 1H, JH-H ) 17.5 Hz,
2JH-P ) 20.3 Hz, CHdCH-P); 6.80 (d, 1H, 4JH2-H6 ) 1.8 Hz, H2);
6.82 (d, 1H, 4JH6-H2 ) 1.7 Hz, H6); 6.97 (d, 1H, 4JH2′-H6′ ) 1.8 Hz,
4
3
H2′); 7.39 (d, 1H, JH6′-H2′ ) 1.8 Hz, H6′); 7.58 (dd, 1H, JH-H
17.5 Hz, JH-P ) 25.0 Hz, CHdCH-P); 7.86 (d, 2H, JH2′′-H3′′
5.3 Hz, H2′′); 7.98 (s, 1H, HCdN); 8.56 (d, 2H, JH3′′-H2′′ ) 3.4
Hz, H3′′). 13C NMR (CD3OD, 75 MHz) δ ppm: 15.91 (d, 1C, 3JC-P
) 6.0 Hz, CH3CH2O); 55.87 (s, 1C, CH3O); 56.00 (s, 1C, C′H3O);
)
)
3
3
3
2
1
63.76 (d, 1C, JC-P ) 6.0 Hz, CH3CH2O); 105.97 (dd, 1C, JC-P
) 206.0 Hz, 2JC-F ) 32.6 Hz, CdC-P); 107.82 (s, 1C, C2′); 109.45
(s, 1C, C2); 122.45 (s, 2C, C2′′ + C6′′); 124.96 (s, 1C, C5); 125.08
3
(s, 1C, C5′); 125.35 (s, 1C, C1′); 125.39 (d, 1C, JC-P ) 25.8 Hz,
3
3
4.22 (q, 2H, JH-H ) JH-P ) 7.0 Hz, CH3CH2O); 6.05 (dd, 1H,
C1); 125.62 (s, 1C, C6); 125.75 (s, 1C, C6′); 142.13 (s, 1C, C1′′);
147.17 (s, 1C, C4); 147.58 (s, 1C, C4′); 148.38 (s, 1C, C3); 148.51
(s, 1C, C3′); 149.41 (s, 1C, HCdN); 150.65 (s, 2C, C3′′ + C5′′);
152.02 (dd, 1C, 2JC-P ) 7.7 Hz, 3JC-F ) 4.1 Hz, CdC-P); 162.16
(s, 1C, CdO). 31P NMR (CD3OD, 81 MHz) δ ppm: 22.65 (d, 1JP-F
) 1017.0 Hz, P-F). 19F NMR (CD3OD, 188.5 MHz) δ ppm: 12.75
3JH-H ) 17.5 Hz, JH-P ) 19.3 Hz, CHdCH-P); 6.98 (d, 1H,
2
4JH2-H6 ) 1.7 Hz, H2); 7.09 (d, 1H, JH6-H2 ) 1.6 Hz, H6); 7.37
4
4
4
(d, 1H, JH2′-H6′ ) 1.7 Hz, H2′); 7.41 (d, 1H, JH6′-H2′ ) 1.8 Hz,
3
3
H6′); 7.49 (dd, 1H, JH-H ) 17.5 Hz, JH-P ) 24.6 Hz, CHdCH-
P); 9.76 (s, 1H, CH′O). 13C NMR (CDCl3, 75 MHz) δ ppm: 16.31
3
(d, 1C, JC-P ) 6.0 Hz, CH3CH2O); 56.26 (s, 1C, CH3O); 56.35
1
(d, JF-P ) 1015.0 Hz, F-P). MS (DCI, NH3, pos) m/z: 530.5
(s, 1C, C′H3O); 63.48 (d, 1C, 2JC-P ) 6.0 Hz, CH3CH2O); 107.02
(dd, 1C, 1JC-P ) 206.0 Hz, 2JC-F ) 33.0 Hz, CdC-P); 108.05 (s,
1C, C2); 108.95 (s, 1C, C2′); 123.10 (s, 1C, C5); 123.16 (s, 1C,
C5′); 124.80 (s, 1C, C6); 126.23 (d, 1C, 3JC-P ) 26.0 Hz, C1); 129.19
(s, 1C, C1′); 129.44 (s, 1C, C6′); 146.01 (s, 1C, C4); 147.36 (s, 1C,
C4′); 147.69 (s, 1C, C3); 149.08 (s, 1C, C3′); 151.31 (dd, 1C, 2JC-P
(MH+). IR (NaCl) ν cm-1: 3410 (O-H); 2927 (C-H ethyl); 1667
(CdN, CdO); 1615 (CdC ethyl); 1587 (CdC arom); 1494 (CdC
arom); 1284 (PdO); 1152 (P-O). UV (EtOH, 25 µM): λ ) 325
nm, ꢀ ) 18000 mol-1 · L· cm-1
.
Diethyl(E)-2-[4,4′-dihydroxy-3,3′-dimethoxy-1′-(phthalazin-1-yl)-
hydrazonomethyl-5,5′-biphenyl]vinylphosphonate hydrochloride
(16). Aldehyde 12 (0.092 g, 0.21 mmol) was dissolved in absolute
ethanol (8 mL). Hydralazine hydrochloride (0.041 g, 0.21 mmol)
was added. Mixture was refluxed for 6 h then warmed to room
temperature and concentrated under vacuo. The residue was purified
by silica gel chromatography (EtOAc) to give pure 16 (0.12 g, 93%)
as an orange solid; mp ) 195-197 °C. 1H NMR (DMSO-d6, 300
) 7.9 Hz, JC-F ) 4.0 Hz, CdC-P); 190.93 (s, 1C, C′HO). 31P
3
NMR (CDCl3. 81 MHz) δ ppm: 19.35 (d, 1P, 1JP-F ) 1024.0 Hz,
P-F). 19F NMR (CDCl3, 188.5 MHz) δ ppm: 11.80 (d, JF-P
)
1
1024.0 Hz, F-P). MS (DCI, NH3, pos) m/z: 411.0 (MH+, 100%);
428.0 (MNH4+, 72%). Anal. (C19H20FO7P) C, H calcd 55.61, 4.91;
found 54.33. 5.24. IR (KBr) ν cm-1: 3375 (O-H); 2976 (C-H
ethyl); 1679 (CdO); 1610 (CdC ethyl); 1590 (CdC arom); 1495
(CdC arom); 1269 (PdO); 1175 (P-O). UV (EtOH, 25 µM): λ )
3
MHz) δ ppm: 1.26 (t, 6H, JH-H ) 7.1 Hz, CH3CH2O); 3.90 (s,
3
313 nm, ꢀ ) 20000 mol-1 ·L·cm-1
.
3H, CH3O); 3.96 (s, 3H, CH′3O); 4.00 (qd, 4H, JH-H ) 7.1 Hz,
3
2
3JH-P ) 8.1 Hz, CH3CH2O); 6.41 (dd, 1H, JH-H ) JH-P ) 18.0
Diethyl(E)-2-[4,4′-dihydroxy-1′-isonicotinoylhydrazonomethyl-
3,3′-dimethoxy-5,5′-biphenyl]vinylphosphonate (14). Aldehyde 12
(0.092 g, 0.21 mmol) was dissolved in absolute ethanol (8 mL).
Isoniazid (37 mg, 0.21 mmol) was added. Mixture was refluxed
for 6 h then warmed to room temperature and concentrated under
vacuum. The residue was purified by silica gel chromatography
(EtOAc) to give pure 14 (0.095 g, 81%) as an orange solid; mp )
101-103 °C. 1H NMR (DMSO-d6, 300 MHz) δ ppm: 1.18 (t, 6H,
3JH-H ) 7.0 Hz, CH3CH2O); 3.80 (s, 3H, CH3O); 3.82 (s, 3H,
CH′3O); 3.93 (qd, 4H, 3JH-H ) 7.1 Hz, 3JH-P ) 8.1 Hz, CH3CH2O);
6.28 (dd, 1H, 3JH-H ) 2JH-P ) 18.1 Hz, CHdCH-P); 7.02 (d, 1H,
Hz, CHdCH-P); 7.09 (d, 1H, 4JH2-H6 ) 1.9 Hz, H2); 7.24 (d, 1H,
4JH6-H2 ) 1.8 Hz, H6); 7.34 (dd, 1H, JH-H ) 17.5 Hz, JH-P
)
3
3
4
22.8 Hz, CHdCH-P); 7.35 (d, 1H, JH2′-H6′ ) 1.9 Hz, H2′); 7.81
(d, 1H, 4JH6′-H2′ ) 1.9 Hz, H6′); 7.72 (m, 4H, H4′′-7′′); 8.06 (s, 1H,
HCdN); 8.37 (s, 1H, H2′′). 13C NMR (DMSO-d6, 75 MHz) δ ppm:
3
16.77 (d, 2C, JC-P ) 6.2 Hz, CH3CH2O); 56.54 (s, 1C, CH3O);
2
56.63 (s, 1C, C′H3O); 61.54 (d, 2C, JC-P ) 5.4 Hz, CH3CH2O);
1
109.22 (s, 1C, C2); 109.63 (s, 1C, C2′); 111.02 (d, 1C, JC-P
)
188.5 Hz, CdC-P); 116.72 (s, 1C, C8′′); 123.17 (s, 1C, C3′′); 124.03
(s, 1C, C6′); 125.21 (s, 1C, C6); 125.61 (s, 1C, C7′′); 125.63 (s, 1C,
C1′); 125.65 (d, 1C, 3JC-P ) 23.9 Hz, C1); 126.20 (s, 1C, C5); 126.44
(s, 1C, C5′); 126.86 (s, 1C, C4′′); 132.13 (s, 1C, C6′′); 132.56 (s,
1C, C5′′); 137.80 (s, 1C, C2′′); 147.03 (s, 1C, C4); 147.44 (s, 1C,
4JH2-H6 ) 1.8 Hz, H2); 7.04 (d, 1H, JH6-H2 ) 1.8 Hz, H6); 7.21
4
(dd, 1H, 3JH-H ) 17.4 Hz, 3JH-P ) 22.7 Hz, CHdCH-P); 7.23 (d,
4
4
1H, JH2′-H6′ ) 2.0 Hz, H2′); 7.26 (d, 1H, JH6′-H2′ ) 1.9 Hz, H6′);
2
3
C4′); 148.06 (s, 1C, C3); 148.54 (s, 1C, C3′); 148.69 (d, 1C, JC-P
7.74 (d, 2H, JH2′′-H3′′ ) 6.0 Hz, H2′′); 8.30 (s, 1H, HCdN); 8.70
(d, 2H, JH3′′-H2′′ ) 3.0 Hz, H3′′). 13C NMR (DMSO-d6, 75 MHz)
3
) 5.6 Hz, CdC-P); 153.99 (s, 1C, C1′′); 154.22 (s, 1C, HCdN).
31P NMR (DMSO-d6, 121.5 MHz) δ ppm: 20.67 (s, P-OEt). MS
(ES) m/z: 577.3 M - H+. IR (NaCl) ν cm-1: 3399 (O-H); 1650
3
δ ppm: 16.77 (d, 2C, JC-P ) 6.2 Hz, CH3CH2O); 56.27 (s, 1C,
2
CH3O); 56.43 (s, 1C, C′H3O); 61.49 (d, 2C, JC-P ) 5.4 Hz,

3180 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11
Delomene`de et al.
(CdN); 1206 (PdO); 1144 (P-O). UV (EtOH, 25 µM): λ ) 288
Cells were grown in MCDB-131 supplemented with 10% heat
inactivated fetal calf serum, 100 U/mL penicillin and 100 µg/mL
streptomycin. Then 24 h before LDL incorporation, cells were
starved in serum-free RPMI medium.
nm, ꢀ ) 25400 mol-1 ·L·cm-1
.
Ethyl(E)-2-[4,4′-dihydroxy-3,3′-dimethoxy-1′-(phthalazin-1-yl-
)hydrazonomethyl-5,5′-biphenyl]vinylphosphonofluoridate hydro-
chloride (17). Compound 17 was synthesized from 13 (80 mg, 0.19
mmol) as compound 16. The residue was purified by silica gel
chromatography (EtOAc) to give pure 17 (93 mg, 83%) as a yellow
LDL Isolation and Oxidation. Effect of the Newly
Synthesized Biaryl Agents on Cell Viability. LDL were isolated
by ultracentrifugation from the pooled plasma of healthy normo-
lipidemic human subjects and dialyzed against phosphate buffer
saline (PBS) containing 100 µmol/L ethylene diamine tetraacetic
acid (EDTA), as previously indicated.27 To evaluate cell-mediated
LDL oxidation, HMEC-1 were seeded in 24 multiwell plates. The
standard culture medium (on sparse proliferative cells), was
removed and replaced by serum-free RPMI-1640 containing native
LDL (100 µg apoB/mL) and CuSO4 (1 µM) and incubated at 37
°C for 6 h with cells or in cell-free medium, as previously used.28
The synthesized compounds dissolved in DMSO were added to
the culture medium at variable concentrations, just before beginning
the oxidation and in duplicate. At the end of the incubation, LDL-
containing medium was immediately used for determining thiobar-
bituric acid reactive substances (TBARS) formation using the
fluorimetric procedures of Yagi.20 The subsequent toxicity of cell-
oxidized LDL (in the absence or presence of the different agents)
was evaluated by the MTT test.13
Alternatively, to test the direct cytoprotective effect of the newly
synthesized molecules (independently of their antioxidant proper-
ties), we used either free 4-HNE (50 µM) directly added to the
culture medium, or LDL oxidized by (UV + Cu/EDTA) as
previously described:28 briefly, LDL solution [2 mg apoprotein B
(apoB)/mL, containing 2 µmol/L CuSO4] was irradiated for 2 h, as
a thin film (5 mm) in an open beaker placed 10 cm under the UV-C
source (HNS 30W OFR Osram UV-C tube, λ max 254 nm, 0.5
mW/cm2 determined using a Scientech thermopile model 360001).
At the end of the irradiation, aliquots were taken up for analyses
and oxidized LDL (200 µg apoB/mL under standard conditions)
were immediately incorporated in the culture medium. The cyto-
toxicity of 4-HNE or UV-oxidized LDL was determined after 24 h
by the MTT test as described.13
Determination of Foam Cells. Human monocyte/macrophage
U937 cells (American Type Culture Collection (ATCC), Camden
NJ) were grown in RPMI culture medium containing 10% FCS
and antibiotics. For the experiments, U937 were seeded in Petri
dishes (100 mm diameter). At subconfluence, the standard culture
medium was removed and replaced by fresh RPMI containing native
LDL (100 µg/mL), CuSO4 (1 µM), and the different agents used
at 10 µM). After 14 h incubation, the LDL-containing culture
medium was removed, the cells were fixed in 10% paraformalde-
hyde for 10 min in PBS, then carefully washed in PBS and stained
with 0.5 µg/mL Nile Red as reported.25 After washing in PBS, the
cells were scrapped and the fluorescence was measured spectrof-
luorometrically (excitation and emission wavelength 460 and 550
nm, respectively). Alternatively, U937 were cultured on glass slides,
and after incubation with oxLDL and the different agents were
stained with 0.2% oil red O in 60% 2-propanol for 10 min, then
treated with hematoxylin for 5 min to stain nuclei and observed
microscopically.
Determination of the Protein Carbonyl Content. After incuba-
tion overnight with 4-HNE (50 µM) or UV-oxidized LDL (100
µg/mL), HMEC-1 were washed, scraped in PBS, and centrifuged
for 10 min, 1500 rpm. The pellets were suspended in 0.7 mL PBS.
The protein carbonyl content was analyzed using the conditions
reported by Ichihashi and al.29 Briefly, an aliquot of the protein
samples was incubated for 1 h with an equal volume of 0.1% (w/
v) 2,4-dinitrophenylhydrazine (DNPH) in 2 M HCl at room
temperature. The mixture was treated with 0.5 mL of 20%
trichloroacetic acid (w/v, final concentration), centrifuged, and the
pellet was extracted with ethanol/ethyl acetate (1:1, v/v). The protein
sample was dissolved with 2 mL of 8 M guanidine hydrochloride/
13 mM EDTA/133 mM Tris solution (pH 7.4), and the UV
absorbance was measured at 365 nm. The results were expressed
as % of the unstimulated control expressed as arbitrary unit/mg
protein.
1
solid; mp ) 223-225 °C. H NMR (CD3OD, 300 MHz) δ ppm:
1.23 (t, 3H, 3JH-H ) 7.1 Hz, CH3CH2O); 3.80 (s, 3H, CH3O); 3.87
3
3
(s, 3H, CH′3O); 4.12 (qd, 2H, JH-H ) 7.2 Hz, JH-P ) 8.5 Hz,
3
2
CH3CH2O); 6.21 (dd, 1H, JH-H ) 17.5 Hz, JH-P ) 20.3 Hz,
4
CHdCH-P); 6.99 (d, 1H, JH2-H6 ) 1.6 Hz, H2); 7.13 (d, 1H,
4JH6-H2 ) 1.7 Hz, H6); 7.18 (d, 1H, JH2′-H6′ ) 1.8 Hz, H2′); 7.40
4
(dd, 1H, 3JH-H ) 17.5 Hz, 3JH-P ) 25.0 Hz, CHdCH-P); 7.67 (d,
4
1H, JH6′-H2′ ) 1.8 Hz, H6′); 8.00 (m, 4H, H4′′-7′′); 8.39 (s, 1H,
HCdN); 8.74 (s, 1H, H2′′). 13C NMR (CD3OD, 75 MHz) δ ppm:
15.92 (s, 1C, CH3CH2O); 55.90 (s, 1C, CH3O); 55.93 (s, 1C,
2
C′H3O); 63.73 (d, 1C, JC-P ) 6.8 Hz, CH3CH2O); 105.08 (dd,
1C, 1JC-P ) 206.3 Hz, 2JC-F ) 31.5 Hz, CdC-P); 108.36 (s, 1C,
C2); 108.95 (s, 1C, C2′); 123.89 (s, 1C, C6); 124.31 (s, 1C, C5);
3
125.17 (s, 1C, C7′′); 125.39 (s, 1C, C4′′); 125.64 (d, 1C, JC-P
)
23.5 Hz, C1); 125.77 (s, 1C, C8′′); 126.55 (s, 1C, C6′); 126.57 (s,
1C, C5′); 126.60 (s, 1C, C3′′); 127.44 (s, 1C, C1′); 132.04 (s, 1C,
C6′′); 132.56 (s, 1C, C5′′); 138.87 (s, 1C, HCdN); 146.08 (s, 1C,
C4); 147.00 (s, 1C, C4′); 148.20 (s, 1C, C1′′); 148.27 (s, 1C, C3);
148.98 (s, 1C, C3′); 152.26 (m, 1C, CdC-P); 154.45 (s, 1C, C2′′).
31P NMR (CD3OD, 121.5 MHz) δ ppm: 20.65 (d, 1JP-F ) 1021.6
Hz, P-F). 19F NMR (CD3OD, 188.5 MHz) δ ppm: 14.30 (d, 1JF-P
) 1023.5 Hz, F-P). MS (DCI, NH3, pos) m/z: 553.2 (MH+). IR
(NaCl) ν cm-1: 3399 (O-H); 2937 (C-H ethyl); 1717 (CdN);
1614 (CdC ethyl); 1592 (CdC arom); 1492 (CdC arom); 1272
(PdO); 1147 (P-O). UV (EtOH, 25 µM): λ ) 295 nm, ꢀ ) 28500
mol-1 ·L·cm-1, λ ) 373 nm, ꢀ ) 15000 mol-1 ·L·cm-1
.
4,4′-Dihydroxy-3,3′-dimethoxy-5,5′-biphenyl-1,1′-dimethyliso-
nicotinoylhydrazone (18). 5,5′-Bisvanillin 5 (0.2 g, 0.66 mmol) was
suspended in absolute ethanol (26 mL). Isoniazid (0.23 g, 1.32
mmol) was added. The reaction mixture was refluxed for 6 h and
filtered to give pure 18 (0.275 g, 77%) as a brown solid; mp >270
1
°C. H NMR (DMSO-d6, 300 MHz) δ ppm: 3.85 (s, 6H, CH3O);
7.06 (d, 2H, 4JH2-H6 ) 1.8 Hz, H2); 7.34 (d, 2H, 4JH6-H2 ) 1.7 Hz,
3
H6); 7.65 (d, 4H, JH2′-H3′ ) 6.1 Hz, H2′); 8.31 (s, 2H, HCdN);
8.61 (d, 4H, 3JH3′-H2′ ) 6.1 Hz, H3′). 13C NMR (DMSO-d6, 75 MHz)
δ ppm: 55.97 (s, 2C, CH3O); 107.81 (s, 2C, C2); 121.82 (s, 4C, C2′
+ C6′); 125.00 (s, 2C, C1); 125.11 (s, 2C, C5); 125.21 (s, 2C, C6);
140.96 (s, 2C, C1′); 146.91 (s, 2C, C4); 148.42 (s, 2C, C3); 150.11
(s, 2C, HCdN); 150.45 (s, 4C, C3′ + C5′); 162.11 (s, 2C, CdO).
MS (DCI, NH3, pos) m/z: 541.3 (MH+). IR (KBr) ν cm-1: 3215
(O-H); 2967 (C-H ethyl); 1652 (CdN, CdO); 1594 (CdC); 1550
(CdC arom); 1489 (CdC arom). UV (EtOH + 0.2% DMSO, 25
µM): λ ) 337 nm, ꢀ ) 22000 mol-1 ·L·cm-1
.
4,4′-Dihydroxy-3,3′-dimethoxy-5,5′-biphenyl-1,1′-(diphthalazin-
1-yl)methylhydrazone hydrochloride (19). 5,5′-Bisvanillin 5 (0.2
g, 0.66 mmol) was suspended in absolute ethanol (26 mL).
Hydralazine hydrochloride (0.26 g, 1.32 mmol) was added. The
reaction mixture was refluxed for 6 h and filtrated to give pure 19
1
(0.38 g, 87%) as a yellow solid; mp >270 °C. H NMR (DMSO-
d6, 300 MHz) δ ppm: 3.94 (s, 6H, CH3O); 7.41 (d, 2H, 4JH2-H6
)
4
1.8 Hz, H2); 7.78 (d, 2H, JH6-H2 ) 1.8 Hz, H6); 8.10 (m, 8H,
H4′-7′); 8.68 (s, 2H, HCdN); 8.92 (s, 2H, H2′). 13C NMR (DMSO-
d6, 75 MHz) δ ppm: 56.55 (s, 2C, CH3O); 109.70 (s, 2C, C2); 119.35
(s, 2C, C8′); 123.91 (s, 2C, C3′); 124.26 (s, 2C, C7′); 125.12 (s, 2C,
C6); 125.38 (s, 2C, C1); 126.61 (s, 2C, C4′); 128.25 (s, 2C, C5);
128.62 (s, 2C, C6′); 134.12 (s, 2C, C5′); 136.27 (s, 2C, C2′); 147.70
(s, 2C, C4); 148.29 (s, 2C, C3); 148.46 (s, 2C, C1′); 153.75 (s, 2C,
HCdN). MS (DCI, NH3, pos) m/z: 587.2 (MH+). IR (KBr) ν cm-1
:
3423 (O-H); 1675 (CdN); 1617 (CdC); 1594 (CdC arom); 1473
(CdC arom). UV (EtOH + 0.2% DMSO, 25 µM): λ ) 377 nm, ꢀ
) 34750 mol-1 ·L·cm-1, λ ) 296 nm, ꢀ ) 21500 mol-1 ·L·cm-1
.
Pharmacological Methods. Cell Culture. HMEC-1 line was
obtained from Centers for Disease Control (CDC Atlanta, GA)26
and was a generous gift from Dr. F. Trottein (Institut Pasteur, Lille).

DeVelopment of NoVel Antiatherogenic Biaryls
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 11 3181
(13) Lapeyre, C.; Delomene`de, M.; Bedos-Belval, F.; Duran, H.; Negre-
Salvayre, A.; Baltas, M. Design, Synthesis and Evaluation of
Pharmacological Properties of Cinnamic Derivatives as Antiathero-
genic Agents. J. Med. Chem. 2005, 48, 8115–8124.
(14) Nguyen, R.; Huc, I. Optimizing the Reversibility of Hydrazone
Formation for Dynamic Combinatorial Chemistry. J. Chem. Soc.,
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Computational Chemistry. All molecular modeling was per-
formed using the software programs from Accelrys Software Inc.30
Dynamics calculations performed with the Discover_3 using ESFF
force field and graphical displays generated with Insight Molecular
Modeling System.
Acknowledgment. We thank the European Community
(grant to M.D.) Thanks are also due to the CNRS, INSERM,
ANR (Lisa project no. ANR-05-PCOD-019-01), and the “Uni-
versite´ Paul Sabatier” for financial support.
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Supporting Information Available: Elemental analysis data for
target compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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