Synthesis and highly potent hypolipidemic activity of alpha-asarone- and fibrate-based 2-acyl and 2-alkyl phenols as HMG-CoA reductase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.09.022

In the search for new potential hypolipidemic agents, the present study focused on the synthesis of 2-acyl phenols (6a-c and 7a-c) and their saturated side-chain alkyl phenols (4a-c and 5a-c), and on the evaluation of their hypolipidemic activity using a

Full text of DOI:10.1016/j.bmc.2014.09.022

Bioorganic & Medicinal Chemistry 22 (2014) 5871–5882
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and highly potent hypolipidemic activity
of alpha-asarone- and fibrate-based 2-acyl and 2-alkyl
phenols as HMG-CoA reductase inhibitors
Aarón Mendieta a, Fabiola Jiménez b, Leticia Garduño-Siciliano c, Angélica Mojica-Villegas c,
Blanca Rosales-Acosta d, Lourdes Villa-Tanaca d, Germán Chamorro-Cevallos c, José L. Medina-Franco e,
,
Nathalie Meurice f, Rsuini U. Gutiérrez a, Luisa E. Montiel a, María del Carmen Cruz b, , Joaquín Tamariz
a,
⇑
⇑
a Depto. de Química Orgánica, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional (IPN), Prol. Carpio y Plan de Ayala S/N, 11340 México, D.F., Mexico
b Centro de Investigación en Biotecnología Aplicada, IPN, Carretera Estatal Santa Inés Tecuexcomac-Tepetitla, Km 1.5, Tepetitla de Lardízabal, 90700 Tlaxcala, Mexico
c Laboratorio de Toxicología Preclínica, Escuela Nacional de Ciencias Biológicas, IPN, Prol. Carpio y Plan de Ayala S/N, 11340 México, D.F., Mexico
d Laboratorio de Genética Microbiana, Escuela Nacional de Ciencias Biológicas, IPN, Prol. Carpio y Plan de Ayala S/N, 11340 México, D.F., Mexico
e Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, 04510 México, D.F., Mexico
f Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, AZ 85259, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 May 2014
Revised 4 September 2014
Accepted 11 September 2014
Available online 19 September 2014
In the search for new potential hypolipidemic agents, the present study focused on the synthesis of 2-acyl
phenols (6a–c and 7a–c) and their saturated side-chain alkyl phenols (4a–c and 5a–c), and on the eval-
uation of their hypolipidemic activity using a murine Tyloxapol-induced hyperlipidemic protocol. The
whole series of compounds 4–7 greatly and significantly reduced elevated serum levels of total choles-
terol, LDL-cholesterol, and triglycerides, with series 6 and 7 showing the greatest potency ever found
in our laboratory. At the minimum dose (25 mg/kg/day), the latter compounds lowered cholesterol by
68–81%, LDL by 72–86%, and triglycerides by 59–80%. This represents a comparable performance than
that shown by simvastatin. Experimental evidence and docking studies suggest that the activity of these
derivatives is associated with the inhibition of HMG-CoA reductase.
Keywords:
Hypolipidemic
Docking
2-Acyl phenols
2-Alkyl phenols
Phenoxyacetic
HMG-CoA reductase
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
glucose intolerance and insulin resistance.5 In 2007, the Frimex
(Risk Factors in Mexico) survey data showed that 71.9% of the
Hyperlipidemia represents a great burden for the health care
systems of many countries. A third of adults meet the criteria for
metabolic syndrome,1 the principal symptom of which is central
obesity. Recent studies suggest extraordinarily high prevalence
rates of hypercholesterolemia (HC) and hypertriglyceridemia
(HTG),2 also associated with obesity. In the year 2000, about 30
million adults (60.5% of the adult population) in Mexico had at
least one cardiovascular risk factor,3 and ischaemic heart disease
was the second global leading cause of general mortality.4 Obesity
is an independent risk factor for cardiovascular disease (CVD) and
mortality, being associated with hypertension, dyslipidemia,
140,017 participants were overweight or obese, 26.5% had hyper-
tension and 40% hypercholesterolemia.6 The prevalence of obesity
in Mexico is currently about 65% in the adult population.7,8
Hyperlipidemia is a risk factor for cardiovascular disease,
including atherosclerosis.9,10 Genetic and environmental factors
associated with obesity and diabetes may contribute to the high
prevalence of dyslipidemia in Mexico and worldwide.11 Lifestyle
interventions—including diet, exercise and weight loss—represent
the primary strategy during the initial stages of dyslipidemia treat-
ment.9,12 However, if this strategy is ineffective and/or patients
begin to exhibit multiple risk factors for chronic disease, healthcare
practitioners turn to lipid-lowering pharmaceuticals.12,13
Many synthetic drugs are available for the treatment of hyper-
lipidemia, but all have serious side effects. Muscle (myopathy,
rhabdomyolysis) and liver toxicity are the most common adverse
effects reported with hypolipidemic therapy.14–21 Many other
adverse effects have been attributed to statins such as
⇑
Corresponding authors. Tel.: +52 55 57296300x87805 (M.C.C.), +52 55
57296300x62411 (J.T.).
E-mail addresses: carmencruz25@yahoo.com.mx (M.C. Cruz), jtamarizm@gmail.
com (J. Tamariz).
Current address: Mayo Clinic, 13400 East Shea Boulevard, Scottsdale, AZ 85259,
USA.
http://dx.doi.org/10.1016/j.bmc.2014.09.022
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

5872
A. Mendieta et al. / Bioorg. Med. Chem. 22 (2014) 5871–5882
rhabdomyolysis, cognitive decline, neuropathy, pancreatic and
hepatic dysfunction, and increased risk of cancer.22 Thus, there is
a need to develop new synthetic hypolipidemic agents with fewer
or no side effects.
a
-Asarone (1) is a naturally occurring compound exhibiting
antihyperlipidemic,23 antifungal24,25 and antithrombotic26 activity
(Fig. 1). The mechanism of its hypolipidemic effect has been
established as the inhibition of the 3-hydroxy-3-methylglutaryl-
coenzyme A (HMG-CoA) reductase,27 which is involved in the
biosynthesis of cholesterol.28,29
Scheme 1. Retrosynthesis of acyl phenols and derivatives.
Previously, we reported the synthesis of a series of
a-asarone
(1) analogues with hypolipidemic activity.30,31 Docking studies
conducted to explore the binding mode of 132 showed the potential
pharmacophore groups, such as the polar methoxy groups at C-1,
C-2 and C-4, and the hydrophobic side-chain, which may be
responsible for the main interactions with the active site of the
enzyme.32 Moreover, it was observed that the analogues increased
their affinity for the enzyme when the C-4 methoxy group was
substituted with a hydroxy group.33
It is noteworthy that the size of the active site cavity allows the
entry of a long polar group at C-2 of the benzene ring (the acetic
group), which can then stabilize the interaction with the enzyme32
(Fig. 1). In this same sense, previous studies on the synthesis of
The synthetic approach was envisioned starting from simple
materials such as phenols (Scheme 1). Actually, compounds 4
and 6 are phenols ortho substituted with an alkyl or alkanoyl
group, respectively, and may be found as raw materials in the per-
fume industry or as precursors in the synthesis of natural products.
It has been observed that the aryl alkanoyl fragment is present in
some biologically active molecules, though pharmacological stud-
ies on such molecules are scarce.38,39
The series of compounds 6a–c and 7a–c were readily prepared
by acylation of the corresponding phenols 8 and 9, respectively,
using the appropriate acyl chloride or acetic anhydride in the pres-
ence of boron trifluoride diethyl etherate (BF3ꢀEt2O),30,40 to give the
corresponding 2-acyl phenols in high yields (81–97%) (Scheme 2).
Reduction of compounds 6a–-c and 7a–c was carried out under
Clemmensen conditions (Zn–Hg, HCl) to give the respective alkyl
phenols 4a–c and 5a–c in modest to fairly good yields (52–77%)
(Scheme 2). Unexpectedly, when the reaction was carried out with
6a and 6c, quinones 10a–b were isolated as by-products in low
yields. The structures of all compounds were established by 1H
NMR, 13C NMR and mass spectrometry. The preparation of deriva-
analogues of
a-asarone (1) showed that the oxyacetic group at
the C-2 position,30 as similar to the structure found in fibrates,
had a significant effect on lipid-lowering activity.34,35 Therefore,
in the current contribution we describe the synthesis of the series
of compounds 4–7 and assess their in vivo hypolipidemic effect. An
exploration of the possible action mechanism and an analysis of
the docking results are provided with the aim of deepening the
understanding of the molecular structure interactions between
these substrates and the active site of the enzyme, which could
improve the drug design and the effectiveness for this class of
potential drugs.
tives 4b and 5b has already been reported via
a different
approach.30 Phenol 9 was not commercially available, and conse-
quently its synthesis was carried out according to the previously
developed procedure in three steps, starting from isovanilline.30
The 1H NMR spectra of the acyl phenols 6a–c show the presence
of a single and fine signal of the hydroxyl proton in the 12.65–
12.79 ppm range. The same protons in compounds 7a–c appear
at 12.57–12.68 ppm. Characteristic signals for the protons of the
tetrasubstituted aromatic ring were observed as singlets. The 13C
NMR spectra of compounds 6 and 7 show a signal at around
204 ppm, which is characteristic of the ketonic carbonyl group. In
the series of compounds 4 and 5, the hydroxyl proton appears as
a broad simple signal in the 4.53–4.97 ppm range. These signals
are at low-frequency because of the lack of hydrogen bonding with
the carbonyl group. Contrarily, such hydrogen bonding is present
with derivatives 6 and 7. The full assignment of proton and carbon
signals was achieved by 2D NMR experiments (HMQC and HMBC).
The unambiguous structural determination of compound 7b
was carried out by X-ray diffraction analysis (Fig. 2). One can see
that the acetyl and hydroxyl groups, as well as the methoxy
group,41 are coplanar with respect to the benzene ring. In this
structure hydrogen bonding is formed and the methyl acetate
side-chain adopts an orthogonal conformation. It is supposed that
these groups are anchored to the polar amino acid residues of the
enzyme active site, as suggested by the docking analysis (vide
infra).
2. Results and discussion
2.1. Chemistry
Upon considering the previous biological and docking studies,
the design of compounds 4–7 was achieved on the basis of two cri-
teria: (1) The series of compounds 4 and 6 retain the basic struc-
ture of
a-asarone (1), but they substitute the C-4 methoxy group
with a more polar hydroxy group (Scheme 1). (2) The propenyl
chain is replaced by an analogous saturated hydrocarbon chain
(series 4)36 or by a chain bearing a carbonyl group (series 6).37
For compounds 5 and 7, the changes were similar to those for 4
and 6, but a methyl acetate fragment was introduced (as a fibrate)
in place of the methyl group at the C-2 position of the aromatic
ring.
2.2. Hypolipidemic activity
Pharmacological screening for the hypolipidemic activity of
these series of compounds was performed on male ICR mice with
Tyloxapol-induced hyperlipidemia.42,43 Tyloxapol (Triton WR
1339) is a non-ionic surfactant that is widely used to explore pos-
sible mechanism of lipid lowering drugs.44–46 It causes a drastic
Figure 1. Structures of HMG-like moiety present in the substrate (I) (in bold),
fragment of a-asarone (1) (in bold), and fragment of the fibrate-like analogues (II)
(in bold), which interact with the HMGR (the numbering of the structures was
chosen for simplifying the comparison between the series of analogues, but it does
not correspond to that established by the IUPAC nomenclature).

A. Mendieta et al. / Bioorg. Med. Chem. 22 (2014) 5871–5882
5873
Scheme 2. Synthesis of acyl phenols 6a–c and 7a–c, reduced derivatives 4a–c and 5a–c, and quinones 10a–b.
Figure 2. X-ray diffraction molecular structure of 7b (ellipsoids at 30% probability level).
rise in serum triglyceride and cholesterol levels, mainly due to an
increase in hepatic cholesterol synthesis. This in turn results from
augmented HMG Co-A reductase activity47,48 and the inhibition of
the lipoprotein lipase enzyme.49–53
serum levels (mg/dL). This procedure is similar to that used in pre-
vious studies with identical (4b and 5b) and related compounds.30
With respect to the hyperlipidemic control group (without
treatment), levels of cholesterol, LDL-cholesterol and triglycerides
were sharply reduced (by 49–89%) after treatment with each of
the test compounds, including simvastatin (11) (Table 1). This
reduction was statistically significant for most of the determined
values. However, there is no lineal dose–effect correlation.
Although an increase in HDL-cholesterol was registered for each
series of test compounds, the differences were not significant. It
is noteworthy that these series display the highest activity for
According to Korolenko et al.,54 reducing the concentration of
blood serum lipids requires the inhibition of endogenous choles-
terol synthesis (elevated after treatment with Tyloxapol),
a
decrease in the absorption of lipoprotein lipids in the blood
serum, and/or the stimulation of their excretion from the same
body fluid. Several groups of compounds hold promise in this
respect: fibrates (PPAR-a agonists), inhibitors of cholesterol
absorption (Ezetimibe), fatty acid scavengers, and statins
(HMG-CoA reductase inhibitors).
the a-asarone- and fibrate-based synthetic analogues previously
prepared in our laboratories.30,31,34–37
Compounds 4a–c, 5a–c, 6a–c and 7a–c (at doses of 25, 50 and
100 mg/kg) were intraperitoneally (ip) administered to hyperlipi-
demic mice, and then an evaluation was made of their effect on
total cholesterol, LDL-cholesterol, HDL-cholesterol and triglyceride
With respect to the dose in mg/kg/day, acyl phenols 6 and 7
were the most active compounds from the series. Since the reduc-
tion in serum cholesterol levels was similar for the 6a–c and 7a–c
series, the hypocholesterolemic activity cannot be associated with

5874
A. Mendieta et al. / Bioorg. Med. Chem. 22 (2014) 5871–5882
Table 1
Effect of the test compounds on the change in the percentage of serum lipids in male ICR micea
Compound
Dose mg (mmol)/kg/day
Cholesterol
LDL-Chol
HDL-Chol
Triglycerides
Normal diet
Simvastatin
Tyloxapol
ꢁ85.18 3.90*
ꢁ63.46 3.15*
100 24.49b
ꢁ93.17 11.8*
ꢁ84.31 4.13*
100 27.05c
52.54 4.90
292.20 3.10*
100 20.34d
33.90 6.44
25.42 1.68
71.19 6.77
49.15 8.94
25.42 3.26
15.25 3.81
23.73 7.48
11.86 2.88
23.73 5.85
40.68 6.37
28.81 10.62
ꢁ3.39 0.59
91.53 10.53
57.63 10.53
50.85 6.86
11.86 2.88
ꢁ3.39 0.65
89.83 12.03
ꢁ4.75 1.70
44.07 16.10
55.93 28.81
6.78 1.29
ꢁ82.75 7.48*
ꢁ56.96 1.72*
100 24.40e
17 (0.04)
400
4a + Tyloxapol
25 (0.14)
50 (0.28)
100 (0.56)
25 (0.13)
50 (0.26)
100 (0.52)
25 (0.12)
50 (0.24)
100 (0.48)
25 (0.10)
50 (0.20)
100 (0.40)
25 (0.10)
50 (0.20)
100 (0.40)
25 (0.09)
50 (0.18)
100 (0.36)
25 (0.13)
50 (0.26)
100 (0.52)
25 (0.12)
50 (0.24)
100 (0.48)
25 (0.12)
50 (0.24)
100 (0.48)
25 (0.10)
50 (0.20)
100 (0.40)
25 (0.10)
50 (0.20)
100 (0.40)
25 (0.09)
50 (0.18)
100 (0.36)
ꢁ67.13 2.70*
ꢁ73.41 4.62*
ꢁ75.85 1.74*
ꢁ71.84 2.79*
ꢁ66.70 3.75*
ꢁ69.22 5.23*
ꢁ70.10 2.96*
ꢁ67.04 4.36*
ꢁ70.10 2.18*
ꢁ79.08 3.57*
ꢁ69.22 7.06*
ꢁ78.20 2.62*
ꢁ79.77 2.79*
ꢁ80.21 2.44*
ꢁ74.02 9.33*
ꢁ70.62 7.50*
ꢁ59.46 8.81*
ꢁ79.95 1.83*
ꢁ71.84 6.19*
ꢁ80.09 1.61*
ꢁ80.12 2.7*
ꢁ81.69 2.79*
ꢁ81.95 1.22*
ꢁ81.43 1.74*
ꢁ74.28 5.75*
ꢁ81.40 1.05*
ꢁ83.26 0.96*
ꢁ74.72 2.62*
ꢁ72.71 4.18*
ꢁ75.33 5.67*
ꢁ68.18 5.84*
ꢁ66.87 5.32*
ꢁ74.46 4.80*
ꢁ78.38 4.10*
ꢁ49.26 13.16*
ꢁ73.23 5.23*
ꢁ72.47 3.85*
ꢁ78.34 4.62*
ꢁ83.93 1.54*
ꢁ78.15 3.85*
ꢁ71.61 4.14*
ꢁ73.63 5.97*
ꢁ74.78 3.56*
ꢁ70.93 5.10*
ꢁ74.69 2.50*
ꢁ86.14 4.43*
ꢁ75.26 8.37*
ꢁ82.58 3.27*
ꢁ89.32 2.60*
ꢁ87.97 2.41*
ꢁ81.23 10.39*
ꢁ77.77 9.34*
ꢁ62.75 9.82*
ꢁ89.51 1.92*
ꢁ76.23 7.8*
ꢁ87.01 2.12*
ꢁ87.68 1.92*
ꢁ86.72 3.37*
ꢁ87.68 1.64*
ꢁ88.35 1.44*
ꢁ80.65 7.41*
ꢁ87.97 1.73*
ꢁ88.74 1.35*
ꢁ81.62 4.52*
ꢁ76.90 4.14*
ꢁ85.76 6.26*
ꢁ72.18 6.83*
ꢁ76.03 8.28*
ꢁ81.33 4.43*
ꢁ84.70 4.91*
ꢁ54.28 15.59*
ꢁ79.40 6.83*
ꢁ76.79 4.95*
ꢁ86.97 1.19*
ꢁ81.65 1.47*
ꢁ82.75 1.38*
ꢁ73.85 2.75*
ꢁ77.98 3.58*
ꢁ82.57 3.21*
ꢁ79.17 2.94*
ꢁ83.94 1.47*
ꢁ72.94 11.0*
ꢁ59.63 17.90
ꢁ75.87 3.39*
ꢁ84.13 2.84*
ꢁ81.93 2.01*
ꢁ71.38 11.56*
ꢁ20.46 11.7
ꢁ49.82 16.3
ꢁ82.75 2.2*
ꢁ59.63 11.38*
ꢁ80.92 2.29
ꢁ82.11 2.29*
ꢁ80.09 2.11*
ꢁ81.01 2.11*
ꢁ80.92 3.03*
ꢁ69.54 8.26*
ꢁ77.25 4.59*
ꢁ79.36 3.30*
ꢁ28.62 5.12
ꢁ74.77 9.72*
ꢁ51.38 27.1
ꢁ59.82 21.28*
ꢁ31.93 39.81
ꢁ75.41 3.48*
ꢁ73.12 1.18*
ꢁ27.80 2.47
ꢁ73.58 7.79*
4b + Tyloxapol
4c + Tyloxapol
5a + Tyloxapol
5b + Tyloxapol
5c + Tyloxapol
6a + Tyloxapol
6b + Tyloxapol
6c + Tyloxapol
7a + Tyloxapol
7b + Tyloxapol
7c + Tyloxapol
16.95 3.93
40.68 5.88
33.90 9.87
30.51 6.34
8.47 0.98
8.47 1.46
1.69 0.34
13.56 3.85
ꢁ3.39 0.77
66.10 11.47
45.76 10.64
30.51 7.92
20.34 9.74
35.59 8.90
a
b
c
Expressed as percentage of the group treated with Tyloxapol (mean standard error); n = 6.
114.7 mmol/L.
103.9 mmol/L.
5.9 mmol/L.
d
e
*
10.9 mmol/L.
Significantly different from the Tyloxapol group, p <0.05.
the length of the side-chain.55 Although the hypolipidemic activity
of all compounds in these series was very high, the best activity
pattern was found for compound 6b. The latter (at 25 mg/kg) sig-
nificantly decreased total cholesterol (81%), LDL-cholesterol (86%)
and triglycerides (80%).
7a–c proved to be the most potent in lowering the cholesterol
and LDL levels. Therefore, the molar dose scale shows a different
potency sequence of the four series with respect to the weight dose
scale. Although the members of each series have relative analogous
molar doses, their potency follows a similar pattern to that
observed from the point of view of the weight dose scale.
The effect of the carbonyl group in 6b can be assessed by com-
paring the activity of this compound with that of analogue 4b.
With both these compounds, triglyceride levels were reduced in
almost equal magnitude. However, compound 4b (with a saturated
chain) had a lesser effect than 6b at the same dosage (25 mg/kg),
lowering total cholesterol by 71% and LDL-cholesterol by 78%.
The opposite hypolipidemic effect was previously observed for
structurally close homologues,55 compounds with an alkenyl
side-chain that were more active than the acyl analogues.
A dose–effect relationship cannot be established for the series
as a whole. However, in some cases the highest dose (100 mg/kg)
produced the highest effect (Table 1). Moreover, the significant
decrease in LDL levels with almost all compounds is relevant, con-
sidering that this type of lipid is regarded as an important risk fac-
tor for the development of atheroma formation and coronary
disease.56
2.3. Stability of compounds 4–7 under pH-variable enzymatic
conditions
For the in vivo administration route, the enzymes of the gastro
intestinal tract represent the first physiological barrier to the
potential activity of the test compounds. Therefore, in order to
determine if such compounds undergo structural changes in the
presence of these enzymes, an in vitro digestion simulation was
carried out using pepsin and trypsin/chymotrypsin.57 Compounds
4a, 4c, 5a, 5c, 6a, 6c, 7a and 7c were selected for these tests.
The retention times and recovery percentages of compounds 4,
5, 6 and 7 are summarized in Table 2. The results suggest that the
compounds are highly stable under acidic conditions (pH 2.0) or
pepsin treatment, evidenced by the fact that these conditions did
not cause the formation of by-products. The exceptions were 4a
and 4c, in which quinones 10a and 10b, respectively, were
In Table 1, doses have also been indicated in molar scale. It is
interesting to note that the lowest dose of the series 5a–c and

A. Mendieta et al. / Bioorg. Med. Chem. 22 (2014) 5871–5882
5875
observed in trace amounts, but mostly the test compounds were
recovered (93–98%). When treatment was carried out under basic
by Schizosaccharomyces pombe, which is an analogue of human
HMGR (HMGRh). In the case of the homologue series of 4–7, the
specific enzymatic activity was evaluated for HMGRh, which is
commercially available. Once having established the protocol for
determining the inhibition of HMGRh, the activity of derivatives
4b, 5b, 6b, and 7c was evaluated (Table 3). Simvastatin (11) was
used as the positive control. There was no statistically significant
difference between the test compounds and the positive controls,
1 and 11.
Compared to 11, similar activity was found for 5b and 7c, while
a lower IC50 was observed for compounds 4b and 6b. The higher
activity of compounds 4b and 6b with respect to 5b and 7c may
be associated with the fact that the former two compounds have
a C-2 methoxy group in the benzene ring, while the latter two have
a methyl acetyloxy group. On the other hand, there was no notable
effect caused by the polar acyl moiety or the saturated side-chain
present in some derivatives. There is not a complete correlation
between the inhibitory activity produced by the test compounds
on HMGRh and the in vivo hypolipidemic activity. However, all test
compounds evaluated proved to be inhibitors of HMGRh, which is
probably also true for the other derivatives that were not
evaluated.
conditions (pH 8.3) and with a mixture of trypsin and
a-chymo-
trypsin, additional products were observed only for compounds
4a and 4c. Derivative 4a displayed four peaks, at 6.94 (54.5%,
assigned to 4a), 6.35 (22.7%, assigned to 10a), 2.15 (17.4%, not iso-
lated and characterized) and 1.89 (5.1%, not isolated and character-
ized) min. In the case of 4c, a large quantity (82.8%) was recovered
and an additional low peak (11.4%, assigned to 10b) was detected
at 13.14 min.
Since both
a-chymotrypsin and trypsin are alkaline endopro-
teases that hydrolyze peptide bonds at the C-terminus of aromatic
amino acids (tryptophan, tyrosine and phenylalanine),58 they
should be expected to produce changes in the structure of com-
pounds 5 and 7, such as hydrolysis of the methyl phenoxyacetate
group. However, the most affected compounds were those of series
4. In the case of 4a, we were able to establish only the structure of
the major by-product resulting from digestion, which had a reten-
tion time of 6.35 min that corresponds to quinone 10a (Scheme 2).
The structure of the other two by-products was not established
because of the impossibility of isolating them in a sufficiently pure
quantity. Similarly, the retention time of the by-product of diges-
tion of 4c corresponds to quinone 10b. This suggests that the oxi-
dation process is promoted under both acidic (see reaction in
Scheme 2) and basic (a-chymotrypsin and trypsin) conditions.
Table 3
In spite of the partial decomposition of series 4 by the enzymes,
the hypolipidemic results did not show any anomalous behavior of
this series of compounds compared to the other series. This sug-
gests that there is no significant hydrolysis or decomposition of
series 4–7 during the in vivo protocol. However, we cannot rule
out the formation of quinones 10a–b and the contribution that
they might have on the hypolipidemic effect, since analogous com-
pounds have proved to exhibit potent antitumor59 and botulinum
neurotoxin serotype A inhibition60 activity. Although a detailed
study of these compounds is beyond the scope of the present work,
an assessment of their biological activity is contemplated for the
near future.
The 50% inhibitory concentration (IC50) values of the activity of alpha-asarone- and
fibrate-based 2-acyl and 2-alkyl phenols as human-HMG-CoA reductase inhibitors
Entry
Compound
Enzyme IC50 (lM)
1
2
3
4
5
6
Simvastatin (11)
6.11 1.53
5.86 1.97
4.79 0.68
6.11 1.43
4.94 0.47
6.43 1.21
a-Asarone (1)
4b
5b
6b
7c
Table 4
Glide docking results of a-asarone and fibrate analogues tested with human HMGR
2.4. Evaluation of series 4–7 as human HMG-CoA reductase
inhibitors
Compound
pIC50
Glide XP (kcal/mol)
4b
5b
6b
7c
7.64
7.40
7.61
7.35
7.40
7.44
ꢁ1.49
ꢁ4.52
ꢁ2.24
ꢁ5.68
ꢁ11.44
ꢁ2.84
Similar to the action mechanism of statins and
according to a previous study many series of
fibrate-based analogues act through the inhibition of the HMG-
CoA reductase enzyme.30 The protocol used for determining this
inhibition was based on the enzymatic activity of HMGR shown
a
-asarone (1),
a
-asarone- and
Simvastatin (11)
a-Asarone (1)
Table 2
HPLC analysis of products from enzymatic digestion of compounds 4–7a
Treated compound
Pepsin tr (min)
Recovery treated compound
and side products (%)
Trypsin/
tr (min)
a-chymotrypsin
Recovery treated compound
and side products (%)
4a
6.95
4a (94.6)
6.94
6.35
2.15
4a (54.5)
10a (22.7)
(17.4)b
1.89
(5.1)b
4c
12.45
4c (93.0)
12.44
13.14
7.78
13.80
8.58
13.83
8.68
13.18
4c (82.8)
10b (11.4)
5a (99.0)
5c (95.4)
6a (97.0)
6c (97.5)
7a (97.6)
7c (99.0)
5a
5c
6a
6c
7a
7c
7.78
13.81
8.57
13.84
8.67
5a (96.4)
5c (97.5)
6a (95.3)
6c (95.6)
7a (94.0)
7c (96.5)
13.16
a
Column chromatography: Zorbax Eclipse Plus HT C18 column (4.6 mm ꢂ 100 mm, 3.5 lm); Elution: (a) Flow rate: 1.0 mL/min (40 °C), (b) solvent gradient: solvent B
(acetonitrile) in solvent A (aqueous solution of formic acid, 0.2%) from 20% to 50% for 18 min, and an isocratic stage of 50% of solvent B for 2 min; Detection: UV (254 nm).
b
The structure of the compound was not established.

5876
A. Mendieta et al. / Bioorg. Med. Chem. 22 (2014) 5871–5882
4b
Lys691
Asn755
Asp690
Arg590
Ser684
Glu559
His752
Lys735
Leu853
5b
Lys691
Asn755
Asp690
Arg590
Ser684
Glu559
His752
Lys735
Leu853
6b
Lys691
Asn755
Asp690
Arg590
Ser684
Glu559
His752
Lys735
Leu853
7c
Lys691
Asn755
Asp690
Arg590
Ser684
Glu559
His752
Lys735
Leu853
Figure 3. Predicted binding mode of the newly synthesized and tested compounds 4b, 5b, 6b and 7c, with the catalytic portion of HMGR using the program Glide XP. The 3D
models show selected amino acid residues within 4.5 Å of the ligands. Carbon atoms of residues from one monomer are orange and those from the other monomer are purple.
Non-polar hydrogens are omitted for clarity. The 2D interaction diagrams show all amino acid residues within 4.5 Å of the ligands and are represented as follows:
hydrophobic in green, polar in cyan, negatively charge in red, positively charge in blue and glycine in white.

A. Mendieta et al. / Bioorg. Med. Chem. 22 (2014) 5871–5882
5877
2.5. Docking of the analogs 4–7 with human HMGR
The -asarone analogue compounds tested in the in vitro assay
in this study. In fact, 4b and 6b have a very similar structure as
well as a comparable predicted binding mode and similar docking
scores. The carbonyl group at the C-5 position in 6b does not have
a major effect on the binding orientation.
A second common interaction between all docked compounds
and the catalytic portion of HMGRh is a hydrogen bond between
the C-2 oxygen and the side-chain of Arg590. The same hydrogen
bond interaction with this residue was previously predicted for
a
(4b, 5b, 6b and 7c) were docked with the crystal structure of
HMGRh in complex with simvastatin (PDB: 1HW9)61 using the
Glide Extra Precision (XP) program. Previous to this simulation,
the protocol was validated by docking the co-crystal ligand. The
docking protocol (see Section 4) was able to reproduce the exper-
imental binding orientation of simvastatin (11) with a root mean
square deviation (RMSD) of 0.60 (Fig. S30 in Supporting data).
We previously obtained a similar low RSMD value for 11 with
another docking program.32 The Glide XP docking score for 11
was ꢁ11.44 kcal/mol.
the C-2 methoxy group of
a-asarone (1). The O3-hydroxyl group
of simvastatin (11) (Fig. S31) also forms a hydrogen bond with
the side-chain of Arg590.
Compounds 5b and 7c have very similar structures and also
similar binding modes, making equivalent protein–ligand interac-
tion contacts (with comparable docking energies). A carbonyl
oxygen of 7c forms an additional hydrogen bond contact with
the side-chain of His752. However, this structural difference does
not have a significant impact on the enzymatic activity, as indi-
cated by the comparable IC50 between 5b and 7c. This contrasts
with the in vivo hypolipidemic activity, which was found to be
improved in 7c. This can be ascribed to the lipophilic efficiency
(logP) of the latter compound due to the polar-indice effect of
the carbonyl group in the chain, which improves the absorption
or excretion process of 7c and thus its pharmacological profile.
Table 4 summarizes the docking results of the four analogues
tested with the crystal of HMGRh. The docking results of simvasta-
tin (11) and
a-asarone (1) are shown as a reference. We did not
observe a reasonable correlation between the Glide XP scores
and the IC50 values. This can be attributed to the well-known issues
of some scoring functions due, in part, to the substantial amount of
approximations in automated docking.62
The binding mode of 4b, 5b, 6b and 7c as well as protein–ligand
interaction diagrams are depicted in Figure 3. For the sake of clar-
ity, and in accordance with a common practice in the visualization
of protein–ligand interactions,63 the three-dimensional represen-
tations of the binding models (Fig. 3) show only selected residues
of the binding pocket. In contrast, the interaction diagrams (e.g.,
the two-dimensional representations of the binding models) dis-
play all the binding residues in the pocket. All compounds adopt
a very similar binding orientation, a result that is further illustrated
by the overlay of the four analogues (Fig. 4A).
As expected from our previous docking studies with a-asarone
(1)32 and phenoxyacetic analogues,30 the acetylated phenoxyacetic
group at the C-2 position of 5b and 7c occupy the same pocket cav-
ity as the carboxylate group of simvastatin (11). Notably, the bind-
ing position of the carboxylate group of 11 is similar to that of the
terminal carboxylate of the HMG moiety of other statins.61 The car-
bonyl oxygen of the ester of 5b and 7c makes a hydrogen bond
with the side-chain of Ser684, similar to the carbonyl oxygen of
11 (Fig. S31). Despite the stability shown by compounds 4–7 under
the enzymatic conditions, the ester group in 5b and 7c is likely to
be hydrolyzed in vivo. As extensively discussed above, the ester
moiety that is exposed in the in vitro inhibition assay with HMGRh
is probably different from the group that is exposed in the in vivo
assay. Therefore, there is not necessarily a direct relationship
between the in vitro and in vivo activity of 5b, 7c and other
compounds.
For all four molecules, hydrogen bond interactions are pre-
dicted between the hydroxyl group (in the equivalent C-4 posi-
tion of
a-asarone (1)) and the side-chains of Glu559, Lys691
and Asn755. The same hydrogen bonds are observed for the O5-
hydroxyl group of simvastatin (11) (Fig. S31 in Supporting data).
Similar interactions are also reported in the crystallographic
structures of compactin, fluvastatin, cerivastatin, atorvastatin
and rosuvastatin.61 These results support our previous hypothesis
that replacing the C-4 methoxy group of 1 by a hydroxyl group
would lead to the formation of a hydrogen bond network. Sur-
Similar to the conclusions reported for the docking of a-asarone
prisingly, only two compounds, 4b (IC50 = 4.79
(IC50 = 4.95 M) showed improved enzymatic inhibitory activity
compared to 1 (IC50 = 5.86 M) under the assay conditions used
l
M) and 6b
(1) and other analogues, the binding mode of 4b, 5b, 6b and 7c
(Fig. 4B) has significant structure-binding contact similarities
when compared to the binding mode of sinvastatin (11) and other
l
l
Figure 4. (A) Overlay of the docking poses of 4b (carbon atoms in dark blue), 5b (light blue), 6b (pink) and 7c (green). (B) Comparison of the binding modes with the
co-crystal position of simvastatin (11) (carbon atoms in yellow).

5878
A. Mendieta et al. / Bioorg. Med. Chem. 22 (2014) 5871–5882
Figure 5. Structure-binding contact similarities of test compounds and simvastatin (11) (in blue bold) and the catalytic portion of HMGRh, and specific structural
requirements for a future plausible design of more active compounds (in blue and red colors).
statins (Fig. 5). Hence, from Figure 4B and the discussion of
Figure 3, it can be appreciated that:
4. Experimental
4.1. Synthesis
(1) The C-4 hydroxyl group of the a-asarone analogues occupies
a similar binding region as the O5-hydroxyl group of statins.
(2) The carbonyl oxygen of the group at the C-2 position of 5b
and 7c has similar interactions as the terminal carboxylate
group of the HMG moiety of the statins.
Melting points were determined on an Electrothermal appara-
tus and are uncorrected. Infrared spectra (IR) were recorded on a
FT-IR 2000 Perkin–Elmer spectrometer. 1H (300 or 500 MHz) and
13C (75 or 125 MHz) NMR spectra were recorded on Varian Mer-
cury-300 or Varian V NMR System instruments, with TMS as inter-
nal standard; chemical shifts (d) are reported in ppm. Mass Spectra
were recorded on a Polaris Q-Trace GC Ultra (Finnigan Co.). High-
resolution mass spectra (HRMS), in electron impact mode, were
obtained on Jeol JSM-GCMateII. Elemental analyses were per-
formed on a CE-440 Exeter Analytical instrument. A Multi-Therm
Benchmark, Model H5000-HC was used for enzymatic stability
assays as a heating and cooling shaker. Commercial reagents were
used as received from Aldrich and anhydrous solvents were
obtained by a distillation process. Thin layer chromatography
was performed on precoated silica gel plates (Merck 60F254). Silica
gel (230–400 mesh) was used for column chromatography. The
(3) The C-2 oxygen of the
a-asarone analogues makes similar
protein–ligand contacts as the O3-hydroxyl group of 11.
The four compounds did not show great differences in potency
(Table 3), which is in agreement with a relatively flat SAR with
respect to the in vitro activity of 4b, 5b, 6b and 7c.
3. Conclusions
The four series of acyl and alkyl phenols herein synthesized
showed high hypolipidemic activity. The lowest dose (25 mg/
kg/day) of the acyl phenols caused a reduction of more than
80% in total cholesterol, LDL-cholesterol and triglycerides in
serum. Actually, series 4–7 proved to be the most active
preparation of compounds
8 and 9 has been previously
described.30
a
-asarone- and fibrate-based analogues ever reported from our
4.1.1. General procedure for the synthesis of 2-acyl phenols 6
and 7
laboratories. Four selected compounds, 4b, 5b, 6b and 7c, dis-
played in vitro inhibition of the human HMG-CoA reductase
(responsible for cholesterol biosynthesis). Docking studies with
this enzyme established multiple molecular modeling interac-
tions between the active site and the different polar and non-
polar functional groups of the derivatives. These results provide
a good SAR image of the structural requirements of more active
compounds to be used in future drug design. Like the highly
active derivatives 5a–c and 7a–c, the new lead compounds
should include polar groups (hydroxyl, amino and thiol groups)
separated at a distance similar to that between the carboxylic
group and both the C-3 and C-5 hydroxyl groups, as established
in the active side-chain of 11 and other statins (Fig. 5). It is also
necessary to include the hydrophobic moiety, which should not
be larger than the decalin skeleton of statins, but instead a sat-
urated side-chain. The benzene ring can be a good and versatile
scaffold to obtain such a setting of substituents, as shown for the
series herein tested.
BF.3Et2O (1.0 mol equiv) was added to a solution of phenol 8 or 9
(1.0 mol equiv) and acetic anhydride or the corresponding acyl
chloride (2.0 mol equiv) under nitrogen atmosphere at 0 °C. The
mixture was stirred at 80 °C for 3 h. The residue was poured into
ice water (20 mL), adjusted to neutral pH with an aqueous satu-
rated solution of NaHCO3, and extracted with EtOAc (3 ꢂ 20 mL).
The organic layer was dried (Na2SO4) and concentrated under
reduced pressure. The residue was purified by column chromatog-
raphy on silica gel (hexane/EtOAc, ca. 9:1).
4.1.1.1. 1-(2-Hydroxy-4,5-dimethoxyphenyl)ethanone (6a). Fol-
lowing the general procedure, a mixture of 8 (1.500 g, 9.74 mmol),
acetic anhydride (1.987 g, 19.48 mmol) and BF.3Et2O (1.383 g,
9.74 mmol) afforded 6a (1.57 g, 82%) as a colorless solid. Rf 0.20
(hexane/EtOAc, 8:2); mp 107–108 °C [Lit.72 112 °C]. IR (KBr):
3449, 2947, 1637, 1510, 1265, 1204, 1062, 838, 808 cmꢁ1 1H
.
NMR (500 MHz, CDCl3): d 2.56 (s, 3H, CH3CO), 3.87 (s, 3H, CH3O-
C5), 3.92 (s, 3H, CH3O-C4), 6.46 (s, 1H, H-3), 7.06 (s, 1H, H-6),
12.65 (s, 1H, OH). 13C NMR (125 MHz, CDCl3): d 26.3 (CH3CO),
56.1 (CH3O-C4), 56.6 (CH3O-C5), 100.5 (C-3), 111.3 (C-6), 111.5
(C-1), 141.9 (C-5), 156.8 (C-4), 160.1 (C-2), 202.0 (CH3CO). MS
(70 eV) m/z 196 (M+, 59), 181 (100), 153 (13), 135 (20), 125 (32),
110 (18), 95 (12), 43 (18).
Evidence from studies carried out with fibrates suggests that
other mechanisms may be responsible for the reduction of tri-
glycerides, such as the peroxisome proliferator-activated receptor
(PPAR) pathway. PPARs are a subfamily of nuclear hormone
receptors, which are ligand-activated transcription factors regu-
lating the metabolism of dietary fats.64 These receptors (particu-
larly PPARa) reduce apoC-III expression and thus result in
increased lipolysis.65–70 Compounds 5 and 7, structurally related
to fibrates, probably reduce lipid levels through a similar mecha-
nism of action.71 Experimental and computational studies on this
mechanism are currently underway and the results will be
reported in due course.
4.1.1.2.
1-(2-Hydroxy-4,5-dimethoxyphenyl)propan-1-one
(6b). Following the general procedure, a mixture of 8 (1.500 g,
9.74 mmol), propanoyl chloride (1.802 g, 19.48 mmol) and BF.3Et2O
(1.383 g, 9.74 mmol) afforded 6b (1.91 g, 93%) as a colorless solid.

A. Mendieta et al. / Bioorg. Med. Chem. 22 (2014) 5871–5882
5879
Rf 0.34 (hexane/EtOAc, 8:2); mp 124–125 °C [Lit.72 125 °C]. IR
3100, 2967, 1772, 1631, 1510, 1384, 1250, 1219, 1193, 1165,
1025, 856, 830 cmꢁ1 1H NMR (500 MHz, CDCl3):
1.02 (t,
(KBr): 3349, 2986, 1615, 1509, 1427, 1205, 1156, 1025,
.
d
816 cmꢁ1
.
1H NMR (500 MHz, CDCl3): d 1.24 (t, J = 7.5 Hz, 3H,
J = 7.5 Hz, 3H, CH3CH2CH2CO), 1.78 (sext, J = 7.5 Hz, 2H, CH3CH2
CH2CO), 2.88 (t, J = 7.5 Hz, 2H, CH3CH2CH2CO), 3.81 (s, 3H, CO2CH3),
3.89 (s, 3H, CH3O-C2), 4.74 (s, 2H, OCH2CO2Me), 6.32 (s, 1H, H-6),
7.16 (s, 1H, H-3), 12.68 (s, 1H, OH). 13C NMR (125 MHz, CDCl3): d
13.8 (CH3CH2CH2CO), 17.9 (CH3CH2CH2CO), 40.0 (CH3CH2CH2CO),
52.4 (CO2CH3), 57.0 (CH3O-C2), 65.4 (OCH2CO2Me), 101.7 (C-6),
112.3 (C-4), 112.4 (C-3), 141.9 (C-2), 154.5 (C-1), 159.6 (C-5),
168.1 (OCH2CO2Me), 204.5 (n-PrCO). MS (70 eV) m/z 282 (M+,
37), 239 (100), 207 (10), 179 (10), 138 (20). HRMS (70 eV) Calcu-
lated for C14H18O6: 282.1103; found: 282.1105. Anal. Calcd for
CH3CH2CO), 2.95 (q, J = 7.5 Hz, 2H, CH3CH2CO), 3.86 (s, 3H, CH3O-
C5), 3.91 (s, 3H, CH3O-C4), 6.46 (s, 1H, H-3), 7.10 (s, 1H, H-6),
12.76 (s, 1H, OH). 13C NMR (125 MHz, CDCl3): d 8.3 (CH3CH2CO),
31.3 (CH3CH2CO), 56.1 (CH3O-C4), 56.7 (CH3O-C5), 100.6 (C-3),
110.9 (C-6), 111.1 (C-1), 141.8 (C-5), 156.5 (C-4), 160.0 (C-2), 204.7
(CH3CO). MS (70 eV) m/z 210 (M+, 23), 182 (12), 181 (100), 149 (4),
138 (9), 125 (72), 121 (9), 110 (28), 93 (7), 91 (8).
4.1.1.3.
1-(2-Hydroxy-4,5-dimethoxyphenyl)butan-1-one
(6c). Following the general procedure, a mixture of 8 (1.500 g,
9.74 mmol), butanoyl chloride (2.075 g, 19.48 mmol) and BF.3Et2O
(1.383 g, 9.74 mmol) afforded 6c (1.77 g, 81%) as a colorless solid.
Rf 0.25 (hexane/EtOAc, 8:2); mp 76–77 °C [Lit.72 81 °C; 77–
78 °C73]. IR (KBr): 3300, 2960, 2931, 1630, 1603, 1508, 1432,
1267, 1204, 1147, 1031, 833, 789 cmꢁ1. 1H NMR (500 MHz, CDCl3):
d 1.03 (t, J = 7.5 Hz, 3H, CH3CH2CH2CO), 1.78 (sext, J = 7.5 Hz, 2H,
CH3CH2CH2CO), 2.88 (t, J = 7.5 Hz, 2H, CH3CH2CH2CO), 3.87 (s, 3H,
CH3O-C5), 3.91 (s, 3H, CH3O-C4), 6.45 (s, 1H, H-3), 7.10 (s, 1H,
H-6), 12.79 (s, 1H, OH). 13C NMR (125 MHz, CDCl3): d 13.8 (CH3CH2
CH2CO), 18.0 (CH3CH2CH2CO), 39.9 (CH3CH2CH2CO), 56.1 (CH3O-
C4), 56.7 (CH3O-C5), 100.6 (C-3), 111.1 (C-6), 111.3 (C-1), 141.8
(C-5), 156.5 (C-4), 160.2 (C-2), 204.3 (CH3CO). MS (70 eV) m/z
224 (M+, 34), 193 (10), 181 (100), 125 (35), 110 (17), 95 (10).
C14H18O6: C, 59.57; H, 6.43. Found: C, 59.54; H, 6.39.
4.1.2. General procedure for the synthesis of 2-alkyl phenols 4
and 5
A mixture of Zn–Hg (10.0 mol equiv) and conc. aqueous solu-
tion of HCl (36%) (6.0 mL) was heated to 65 °C, and a solution of
the corresponding acyl phenol 6a–c or 7a–c (1.0 mol equiv) in
MeOH (2 mL) and HCl (36%; 1.0 mL) was added dropwise. The mix-
ture was stirred for 4 h and extracted with EtOAc (5 ꢂ 10 mL). The
organic layer was washed with a 5% aqueous solution of NaHCO3
until neutral, dried (Na2SO4), and the solvent was removed under
vacuum. The residue was purified by column chromatography over
silica gel (20 g, hexane/EtOAc, ca. 9:1) to give the respective alkyl
phenols 4a–c or 5a–c.
74
4.1.1.4. Methyl 2-(4-acetyl-5-hydroxy-2-methoxyphenoxy)ace-
tate (7a). Following the general procedure, a mixture of 9 (1.000 g,
4.72 mmol), Ac2O (0.963 g, 9.44 mmol) and BF.3Et2O (0.670 g,
4.72 mmol) afforded 7a (1.08 g, 90%) as a white solid. Rf 0.30 (hex-
ane/EtOAc, 6:4); mp 132–133 °C. IR (KBr): 2943, 1770, 1633, 1510,
1397, 1374, 1226, 1197, 1168, 1023, 832 cmꢁ1. 1H NMR (500 MHz,
CDCl3): d 2.58 (s, 3H, CH3CO), 3.82 (s, 3H, CO2CH3), 3.89 (s, 3H,
CH3O-C2), 4.75 (s, 2H, OCH2CO2Me), 6.32 (s, 1H, H-6), 7.11 (s, 1H,
H-3), 12.57 (s, 1H, OH). 13C NMR (125 MHz, CDCl3): d 26.4 (CH3CO),
52.5 (CO2CH3), 56.8 (CH3O-C2), 65.3 (OCH2CO2Me), 101.4 (C-6),
112.5 (C-3), 112.6 (C-4), 141.8 (C-2), 154.8 (C-1), 159.4 (C-5),
168.0 (OCH2CO2Me), 202.3 (CH3CO). MS (70 eV) m/z 254 (M+, 89),
239 (100), 179 (49), 165 (13), 151 (24), 135 (19), 43 (16). HRMS
(70 eV) Calculated for C12H14O6: 254.0790; found: 254.0782. Anal.
Calcd for C12H14O6: C, 56.69; H, 5.55. Found: C, 56.70; H, 5.51.
4.1.2.1. 2-Ethyl-4,5-dimethoxyphenol (4a).
2-Ethyl-5-meth-
oxycyclohexa-2,5-diene-1,4-dione (10a). Following the general
procedure, a mixture of 6a (1.200 g, 6.12 mmol), Zn–Hg (16.28 g,
61.2 mmol) and HCl (16 mL) afforded 4a (0.92 g, 83%) as a pale yel-
low solid, and 10a (0.04 g, 4%) as a yellow solid. Data of 4a: Rf 0.63
(hexane/EtOAc, 7:3); mp 43–44 °C. IR (KBr): 3276, 2959, 1618,
1528, 1451, 1410, 1204, 1170, 1106, 990, 828 cmꢁ1 1H NMR
.
(500 MHz, CDCl3): d 1.22 (t, J = 7.5 Hz, 3H, CH3CH2), 2.56 (q,
J = 7.5 Hz, 2H, CH3CH2), 3.81 (s, 3H, CH3O-C5), 3.83 (s, 3H, CH3O-
C4), 4.53 (br s, 1H, OH), 6.43 (s, 1H, H-6), 6.66 (s, 1H, H-3). 13C
NMR (125 MHz, CDCl3): d 14.4 (CH3CH2), 22.6 (CH3CH2), 56.0
(CH3O-C5), 56.6 (CH3O-C4), 100.9 (C-6), 113.1 (C-3), 120.6 (C-2),
143.1 (C-4), 147.0 (C-1), 147.8 (C-5). MS (70 eV) m/z 182 (M+,
79), 167 (100), 139 (33), 111 (58), 93 (15), 77 (25). HRMS (70 eV)
Calculated for C10H14O3: 182.0943; found: 182.0949.
Data of 10a: Rf 0.65 (hexane/EtOAc, 7:3); mp 155–156 °C [Lit. 75
57–58 °C]. 1H NMR (500 MHz, CDCl3): d 1.14 (t, J = 7.5 Hz, 3H, CH3
CH2), 2.48 (qd, J = 7.5, 1.5 Hz, 2H, CH3CH2), 3.82 (s, 3H, CH3O), 5.92
(s, 1H, H-6), 6.50 (t, J = 1.5 Hz, 1H, H-3). 13C NMR (125 MHz, CDCl3):
d 11.7 (CH3CH2), 22.0 (CH3CH2), 56.2 (CH3O), 107.7 (C-6), 129.6
(C-3), 151.9 (C-2), 158.6 (C-5), 182.4 (C-4), 187.4 (C-1). HRMS
(70 eV) Calculated for C9H10O3: 166.0630; found: 166.0632.
4.1.1.5. Methyl 2-(5-hydroxy-2-methoxy-4-propionylphen-
oxy)acetate (7b). Following the general procedure, a mixture of 9
(1.00 g, 4.72 mmol), n-propanoyl chloride (0.873 g, 9.44 mmol)
and BF.3Et2O (0.670 g, 4.72 mmol) afforded 7b (1.22 g, 97%) as a
white solid. Rf 0.50 (hexane/EtOAc, 6:4); mp 119–120 °C. IR
(KBr): 3101, 2978, 1754, 1639, 1511, 1453, 1390, 1257, 1223,
1168, 1081, 864, 803 cmꢁ1 1H NMR (500 MHz, CDCl3): d 1.24 (t,
.
J = 7.5 Hz, 3H, CH3CH2CO), 2.95 (q, J = 7.5 Hz, 2H, CH3CH2CO), 3.81
(s, 3H, CO2CH3), 3.88 (s, 3H, CH3O-C2), 4.74 (s, 2H, OCH2CO2Me),
6.33 (s, 1H, H-6), 7.16 (s, 1H, H-3), 12.62 (s, 1H, OH). 13C NMR
(125 MHz, CDCl3): d 8.3 (CH3CH2CO), 31.4 (CH3CH2CO), 52.4 (CO2
CH3), 57.0 (CH3O-C2), 65.4 (OCH2CO2Me), 101.7 (C-6), 112.1
(C-3), 112.2 (C-4), 141.9 (C-2), 154.5 (C-1), 159.5 (C-5), 168.1
(OCH2CO2Me), 205.0 (CH3CH2CO). MS (70 eV) m/z 268 (M+, 39),
240 (14), 239 (100), 138 (19). HRMS (70 eV) Calculated for
4.1.2.2. 4,5-Dimethoxy-2-propylphenol (4b). Following the gen-
eral procedure, a mixture of 6b (0.620 g, 2.95 mmol), Zn–Hg
(7.85 g, 29.5 mmol) and HCl (3.0 mL) afforded 4b (0.34 g, 60%) as
a white solid. Rf 0.50 (hexane/EtOAc, 7:3); mp 75–76 °C [Lit.30
75–76 °C].
4.1.2.3. 2-Butyl-4,5-dimethoxyphenol (4c). 2-Butyl-5-methoxy-
cyclohexa-2,5-diene-1,4-dione (10b). Following the general pro-
cedure, a mixture of 6c (1.000 g, 4.46 mmol), Zn–Hg (11.875 g,
44.64 mmol) and HCl (5.0 mL) afforded 4c (0.66 g, 70%) as a pale
yellow solid, and 10b (0.05 g, 6%) as a yellow solid. Data of 4c: Rf
0.45 (hexane/EtOAc, 7:3); mp 54–55 °C. IR (KBr): 3325, 2948,
C13H16O6: 268.0947; found: 268.0955. Anal. Calcd for C13H16O6:
C, 58.20; H, 6.01. Found: C, 58.20; H, 6.03.
4.1.1.6.
Methyl
2-(4-butanoyl-5-hydroxy-2-methoxyphen-
oxy)acetate (7c). Following the general procedure, a mixture of 9
(1.00 g, 4.72 mmol), n-butanoyl chloride (1.005 g, 9.44 mmol) and
BF.3Et2O (0.670 g, 4.72 mmol) afforded 7c (1.26 g, 95%) as a white
solid. Rf 0.50 (hexane/EtOAc, 6:4); mp 122–123 °C. IR (KBr):
1619, 1527, 1457, 1417, 1198, 1116, 1006, 854 cmꢁ1 1H NMR
.
(500 MHz, CDCl3): d 0.94 (t, J = 7.5 Hz, 3H, CH3(CH2)3), 1.38 (sext,
J = 7.5 Hz, 2H, CH3CH2(CH2)2), 1.57 (qu, J = 7.5 Hz, 2H, CH3CH2CH2
CH2), 2.53 (t, J = 7.5 Hz, 2H, CH3(CH2)2CH2), 3.80 (s, 3H, CH3O-C5),

5880
A. Mendieta et al. / Bioorg. Med. Chem. 22 (2014) 5871–5882
3.82 (s, 3H, CH3O-C4), 4.61 (s, 1H, OH), 6.42 (s, 1H, H-6), 6.64 (s, 1H,
H-3). 13C NMR (125 MHz, CDCl3):
14.0 (CH3(CH2)3), 22.5
(CH3CH2(CH2)2), 29.3 (CH3(CH2)2CH2), 32.3 (CH3CH2CH2CH2), 55.9
(CH3O-C5), 56.6 (CH3O-C4), 100.9 (C-6), 113.8 (C-3), 119.3 (C-2),
142.9 (C-4), 147.2 (C-1), 147.8 (C-5). MS (70 eV) m/z 210 (M+,
32), 167 (100), 139 (21), 79 (12). HRMS (70 eV) Calculated for
(graphite crystal monochromator, k = 71073 Å) at 19 °C. Two stan-
dard reflections, which were monitored periodically, showed no
change during data collection. Unit cell parameters were obtained
from least-squares refinement of 3005 reflections in the range of
d
2 < 2
H < 20°. Intensities were corrected for Lorentz and polariza-
tion effects. Multi-scan absorption correction was applied. Aniso-
tropic temperature factors were introduced for all non-hydrogen
atoms. Hydrogen atoms were placed in idealized positions and
their atomic coordinates refined. Unit weights were used in the
refinement. Details of data collection and refinement for this crys-
tal are listed in Tables S1–S5 (Supplementary data), which include
bond distances and angles, atomic coordinates, and anisotropic
thermal parameters. The structure was solved using the SHEL-
XS9777 program as implemented in the WinGX suite,78 and refined
using SHELXL9779 within WinGX, on a personal computer. In all
cases ORTEP and packing diagrams were made with ORTEP-3.80
The structure was submitted to Cambridge Crystallographic Data
Centre: 7b, CCDC No. 977212.
C12H18O3: 210.1256; found: 210.1256. Anal. Calcd for C12H18O3:
C, 68.54; H, 8.63. Found: C, 68.51; H, 8.58.
Data of 10b: Rf 0.47 (hexane/EtOAc, 7:3); mp 128–129 °C [Lit. 76
129–130 °C]. 1H NMR (500 MHz, CDCl3): d 0.93 (t, J = 7.5 Hz, 3H,
CH3(CH2)3), 1.38 (sext, J = 7.5 Hz, 2H, CH3CH2(CH2)2), 1.45–1.52
(m, 2H, CH3CH2CH2CH2), 2.43 (td, J = 7.5, 1.5 Hz, 2H, CH3(CH2)2
CH2), 3.82 (s, 3H, CH3O), 5.92 (s, 1H, H-6), 6.50 (t, J = 1.5 Hz, 1H,
H-3). 13C NMR (125 MHz, CDCl3):
d 13.8 (CH3(CH2)3), 22.4
(CH3CH2(CH2)2), 28.6 (CH3(CH2)2CH2), 30.0 (CH3CH2CH2CH2), 56.2
(CH3O), 107.8 (C-6), 130.4 (C-3), 150.7 (C-2), 158.5 (C-5), 182.4
(C-4), 187.5 (C-1). HRMS (70 eV) Calculated for C11H14O3:
194.0943; found: 194.0939.
4.1.2.4. Methyl 2-(4-ethyl-5-hydroxy-2-methoxyphenoxy)ace-
4.2. Hypolipidemic activity
tate (5a). Following the general procedure,
a mixture of 7a
(1.200 g, 4.72 mmol), Zn–Hg (12.56 g, 47.2 mmol) and HCl
(12 mL) afforded 5a (0.60 g, 52%) as a white solid. Rf 0.25 (hex-
ane/EtOAc, 7:3); mp 55–56 °C. IR (KBr): 3585, 3252, 2962, 1748,
Hypolipidemic activity was studied in (ICR) male mice weighing
25–30 g (Birmex, S.A. de C.V., Mexico City). All animals were
housed in hanging metal cages and maintained at 24 2 °C and
50 10% relative humidity, with 12 h light/dark cycle (lights on
at 8:00 a.m.). They were fed on standard pellet diets (Rodent Diet
5001, PMI Nutrition International, Inc., Brenwood, MO) and drink-
ing water was freely available. All animals appeared healthy
throughout the dosing period, maintaining normal food intake
and weight gain. At the time of sacrifice, no gross abnormalities
were observed in any treated mice. All animals were treated in
accordance with ethical principles and regulations specified by
the Bioethics Committee of our Institution and the Standards of
the National Institutes of Health of Mexico.
1616, 1522, 1420, 1203, 1120, 1019, 845 cmꢁ1
.
1H NMR
(500 MHz, CDCl3): d 1.19 (t, J = 7.5 Hz, 3H, CH3CH2), 2.55 (q,
J = 7.5 Hz, 2H, CH3CH2), 3.77 (s, 3H, CO2CH3), 3.82 (s, 3H, CH3O-
C2), 4.62 (s, 2H, OCH2CO2Me), 4.97 (br s, 1H, OH), 6.40 (s, 1H, H-
6), 6.68 (s, 1H, H-3). 13C NMR (125 MHz, CDCl3): d 14.2 (CH3CH2),
22.6 (CH3CH2), 52.2 (CO2CH3), 56.8 (CH3O), 66.8 (OCH2CO2Me),
104.0 (C-6), 114.1 (C-3), 123.3 (C-4), 143.6 (C-2), 145.7 (C-1),
147.1 (C-5), 169.8 (CO2Me). MS (70 eV) m/z 240 (M+, 80), 225
(41), 167 (72), 165 (40), 151 (13), 139 (44), 121 (14), 111 (100),
107 (22), 93 (22), 79 (23), 77 (24). HRMS (70 eV). Calculated for
C
12H16O5: 240.0998; found: 240.0998. Anal. Calcd for C12H16O5:
An aqueous solution of Tyloxapol was given ip to mice (400 mg/
kg) and one hour later the test compounds (25, 50 and 100 mg/kg),
dissolved in saline or saline-tween were ip administered. Simva-
statin (11) (17 mg/kg) was used as a positive control group. After
24 h, blood was taken from the retro-orbital puncture and centri-
fuged at 13,000 rpm for the determination of serum levels of total
cholesterol (TC), LDL-cholesterol (LDL-C) and triglyceride (TG),
using a Wiener Lab Selectra 2 instrument. Serum LDL-cholesterol
levels were calculated using the Friedewald equation.81 Levels of
serum lipids were determined in duplicate and values represent
the mean from 6 mice (per compound). All data were statistically
analyzed by Student–Newman–Keuls (SNK) test. P values less than
0.05 were considered as indicative of significance.
C, 59.99; H, 6.71. Found: C, 59.96; H, 6.68.
4.1.2.5. Methyl 2-(5-hydroxy-2-methoxy-4-propylphenoxy)ace-
tate (5b). Following the general procedure, a mixture of 7b
(1.000 g, 3.73 mmol), Zn–Hg (9.93 g, 37.3 mmol) and HCl (5.0 mL)
afforded 5b (0.63 g, 67%) as a white solid. Rf 0.38 (hexane/EtOAc,
7:3); mp 57–58 °C [Lit.30 57–58 °C].
4.1.2.6. Methyl 2-(4-butyl-5-hydroxy-2-methoxyphenoxy)ace-
tate (5c). Following the general procedure,
a mixture of 7c
(1.000 g, 3.55 mmol), Zn–Hg (9.44 g, 35.5 mmol) and HCl (5.0 mL)
afforded 5c (0.73 g, 77%) as a white solid. Rf 0.41 (hexane/EtOAc,
7:3); mp 74–75 °C. IR (KBr): 3581, 3457, 3123, 2952, 1771, 1625,
1528, 1422, 1217, 1128, 1021, 856 cmꢁ1
.
1H NMR (500 MHz,
4.3. Enzymatic evaluation
CDCl3): d 0.93 (t, J = 7.5 Hz, 3H, CH3(CH2)3), 1.37 (sext, J = 7.5 Hz,
2H, CH3CH2(CH2)2), 1.55 (qu, J = 7.8 Hz, 2H, CH3CH2CH2CH2), 2.52
(t, J = 7.5 Hz, 2H, CH3(CH2)2CH2), 3.78 (s, 3H, CO2CH3), 3.82 (s, 3H,
CH3O-C2), 4.63 (s, 2H, OCH2CO2Me), 4.79 (br s, 1H, OH), 6.40 (s,
1H, H-6), 6.66 (s, 1H, H-3). 13C NMR (125 MHz, CDCl3): d 13.9
(CH3(CH2)3), 22.5 (CH3CH2(CH2)2), 29.4 (CH3(CH2)2CH2), 32.2
(CH3CH2CH2CH2), 52.2 (CO2CH3), 56.8 (CH3O-C2), 66.8 (OCH2CO2
Me), 103.9 (C-6), 114.7 (C-3), 121.9 (C-4), 143.6 (C-2), 145.8 (C-
1), 147.1 (C-5), 169.8 (CO2Me). MS (70 eV) m/z 268 (M+, 34), 225
(100), 195 (19), 167 (17), 137 (21), 111 (16). HRMS (70 eV) calcu-
lated for C14H20O5: 268.1311; found: 268.1311. Anal. Calcd for
4.3.1. In vitro digestion of compounds 4a, 4c, 5a, 5c, 6a, 6c, 7a
and 7c by pepsin and trypsin/a-chymotrypsin
In an Eppendorf tube, compounds were dissolved at 10 mM
(1 mL) in 10 mM HCl (pH 2.0) and pepsin was added at an
enzyme/substrate (E/S) ratio of 1:200 (w/w). The solution was cen-
trifuged at 37 °C. After 12 h of hydrolysis by pepsin, an aliquot of
500
stopped in the aliquot by adding 500
100 mM buffer (pH 8.3), and the mixture was extracted with
L). The solvent was removed under vacuum and
lL was taken to assess the process. The peptic reaction was
l
L of sodium phosphate
CH2Cl2 (3 ꢂ 200
l
C14H20O5: C, 62.67; H, 7.51. Found: C, 62.67; H, 7.46.
the residue kept at 5 °C until further analysis.
To the acidic pepsin solution containing the corresponding
4.1.3. Single-crystal X-ray crystallography
A single crystal of compound 7b, obtained from recrystallization
of EtOAc/CH2Cl2, 9:1, was mounted on a glass fiber. Crystallo-
compound, one volume (500
100 mM) was added and then a mixture of trypsin and
trypsin at an E/S ratio of 1:50 (w/w) was added. The mixture was
l
L) of the buffer (sodium phosphate,
-chymo-
a
graphic measurements were performed using MoK
a
radiation
maintained at 37 °C for 24 h, then heated to 90 °C for 15 min to

A. Mendieta et al. / Bioorg. Med. Chem. 22 (2014) 5871–5882
5881
inactivate the enzymes. The pH was adjusted to 7.0 with 1.0 M HCl
L). The sol-
vent was removed under vacuum and the residue kept at 5 °C for
further analysis.
4.4. Docking
and the mixture was extracted with CH2Cl2 (3 ꢂ 200
l
The crystallographic structure of human HMGR in complex with
simvastatin (11) was retrieved from the Protein Data Bank (PDB),
with the code 1HW9.61 The protein was prepared using the Protein
Preparation Wizard implemented in Maestro 9.3,83 which opti-
mizes H-bond networks and flip orientations/tautomeric states of
Gln, Asn and His residues. Geometry optimization was performed
to a maximum root mean square deviation (RMSD) of 0.3 Å with
the OPLS2005 force field. We recently employed a similar proce-
dure to prepare the structure of other proteins.84 During the pro-
tein preparation all water and adenosine-50-diphosphate
molecules were removed from the original PBD file. The binding
site was established by grids with a default rectangular box cen-
tered on the co-crystal ligand simvastatin. XP descriptors were
generated to obtain atom-level energy terms for the docking run,
such as hydrogen bond interaction, electrostatic interaction,
In an Eppendorf tube, a mixture of 4a (18.2 mg) (or of 4c
(21.0 mg)) with 1 mL of HCl 10 mM (pH 2.0) was agitated at
300 rpm for 12 h at 37 °C. Afterwards, an aliquot (0.5 mL) was
taken and a 100 mM aqueous solution of NaOH was added until
neutral, and then extracted with CH2Cl2 (3 ꢂ 200
lL). The solvent
was removed under vacuum and the residue kept at 5 °C until fur-
ther analysis.
To the remaining centrifuged mixture, a 1.0 M aqueous solution
of NaOH was added to adjust pH to 8.3, and the solution was main-
tained in agitation at 300 rpm for 24 h at 37 °C. The pH was
adjusted to 7.0 with 1.0 M HCl and the mixture was extracted with
CH2Cl2 (3 ꢂ 200
lL). The solvent was removed under vacuum and
the residue kept at 5 °C until further analysis.
Reaction products were analyzed by HPLC under gradient con-
hydrophobic enclosure and p–p stacking interaction. The structure
ditions using
(4.6 mm ꢂ 100 mm, 3.5
and filtered through a 0.22
a
Zorbax Eclipse Plus HT C18 column
m). Samples were dissolved in methanol
m syringe filter, and then 10 L was
of the ligands was generated with Molecular Operating Environ-
ment (MOE), version 2011.1,85 by starting from the coordinates
of the crystallographic structure of compound 7b. Docking was
performed with the program Glide Extra Precision (XP).86,87 Up
to ten binding poses were generated per molecule and the top
ranked binding pose was selected for analysis.
l
l
l
injected into the column. Elution was carried out at 40 °C. The flow
rate was 1.0 mL/min with a linear gradient of solvent B (acetoni-
trile) in solvent A (aqueous solution of formic acid, 0.2%) from
20% to 50% for 18 min, and an isocratic stage with 50% of solvent
B for 2 min. Absorbance peaks were monitored at 254 nm by a
DAD detector. Calibration curves of pure compounds 4a, 4c, 5a,
5c, 6a, 6c, 7a and 7c were obtained under the same chromato-
graphic conditions at concentrations of 1.0, 2.5, 5.0, 7.5, and
10.0 mM.
Acknowledgments
We thank Bruce Allan Larsen for reviewing the use of English in
this manuscript. M.C.C. and J.T. gratefully acknowledge Secretaría
de Investigación
y
Posgrado (SIP)-IPN (Grants 20090326,
20100236, 20110172, 20120830, 20130686, and 20140858) and
CONACYT (Grants 80508, 83446, and 178319) for financial support.
A.M., A.M.-V., B.R.-A., R.U.G. and L.E.M. thank CONACYT for gradu-
ate scholarships awarded and also thank SIP-IPN (PIFI) for a schol-
arship complement. A.M. also thanks CONACYT (Grant 178319) for
a partial scholarship. F.J., L.G.-S., L.V.-T., G.C.-C., M.C.C. and J.T. are
fellows of the Estímulos al Desempeño de los Investigadores
(EDI)-IPN and Comisión de Operación y Fomento de Actividades
Académicas (COFAA)-IPN programs.
4.3.2. Assay of human HMG-CoA reductase activity
NADPH, human HMG-CoA reductase (HMGRh), simvastatin
(11),
a-asarone (1), and dimethyl sulfoxide (DMSO) were pur-
chased from Sigma (Sigma, Saint Louis, MO, USA). The human
HMG-CoA reductase (HMGRh) activity and inhibition assays were
performed using the HMGRh Assay Kit from Sigma–Aldrich (Sigma
CS-1090, Saint Louis, MO, USA), according to the manufacturer’s
instructions, using HMGRh, NADPH and Tris–HCl, pH 7.5. The oxi-
dation of NADPH was spectrophotometrically monitored at 340 nm
in a BioSpectometer-Kinetic from Eppendorff. HMG-CoA reductase
activity was assayed at least six times. The reaction mixture con-
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.09.022.
tained: 0.13 mM HMG-CoA, 1
itor, and 50 mM Tris–HCl, pH 7.5, to a final volume of 100
l
L of HMGRh with or without inhib-
l
L. After
15 min incubation at 37 °C, the reaction was started with the addi-
tion of 0.13 mM NADPH, and monitored for 10 min. For all six reac-
tions, 1 unit of enzyme activity is defined as the amount of enzyme
required to catalyze the oxidation of 1 mmol of NADPH per min
References and notes
1. Goonasekara, C. L.; Balse, E.; Hatem, S.; Steele, D. F.; Fedida, D. Expert Rev.
Cardiovasc. Ther. 2010, 8, 965.
2. Ashraf, H.; Rashidi, A.; Noshad, S.; Khalilzadeh, O.; Esteghamati, A. Expert Rev.
Cardiovasc. Ther. 2011, 9, 309.
3. Oláiz, G.; Rojas, R.; Barquera, S.; Shamah, T.; Aguilar, C.; Cravioto, P.; López, P.;
Hernández, M.; Tapia, R.; Sepúlveda, J. Encuesta nacional de salud 2000. Tomo 2.
La salud de los adultos. Cuernavaca, Morelos, México, Instituto Nacional de
Salud Pública, 2003.
4. Estadísticas de mortalidad en México: muertes registradas en el año 2000. Salud
Pública Méx. 2002, 44, 266.
5. Van Gaal, L. F.; Mertens, I. L.; De Block, C. E. Nature 2006, 444, 875.
6. Meaney, E.; Lara-Esqueda, A.; Ceballos-Reyes, G. M.; Asbun, J.; Vela, A.;
Martínez-Marroquín, Y.; López, V.; Meaney, A.; de la Cabada-Tamez, E.;
Velásquez-Monroy, Ó.; Tapia-Conyer, R. Public Health 2007, 121, 378.
7. World health statistics 2013 (www.who.int). World Health Organization, 2013,
p. 115.
8. Gutiérrez, J. P.; Rivera-Dommarco, J.; Shamah-Levy, T.; Villalpando-Hernández,
S.; Franco, A.; Cuevas-Nasu, L.; Romero-Martínez, M.; Hernández-Ávila, M.
Encuesta nacional de salud y nutrición 2012. Resultados nacionales. Cuernavaca,
México, Instituto Nacional de Salud Pública, 2012, p. 185.
(1 lU catalyzes 1 lM of NADPH, in 1 min), modified from Bischoff
and Rodwell82 and Argüelles.30
4.3.3. Effect of synthetic compounds on HMGRh activity
The HMGRh (HMG-CoA Reductase Assay Kit from Sigma–
Aldrich) enzyme was pre-incubated with the respective com-
pounds for 30 min at 37 °C, followed by the standard enzyme
assay. The synthetic compounds, 4b, 5b, 6b and 7c, and positive
controls of inhibition,
prepared in DMSO at the final concentrations of 2.5, 5.0, 10.0 and
15.0 M. DMSO alone was tested with the enzyme to exclude the
a-asarone (1) and simvastatin (11), were
l
possibility that this solvent may have an inhibitory effect. After
measuring HMGR inhibition, the mean IC50 values were compared
to each other and evaluated by the Tukey post hoc test with the
two-way ANOVA. Significant differences were determined with a
value of p <0.05. Statistical analysis was carried out using the
SigmaStat software.
9. Yuan, G.; Al-Shali, K. Z.; Hegele, R. A. Can. Med. Assoc. J. 2007, 176, 1113.
10. Steinberg, D. Mol. Nutr. Food Res. 2005, 49, 995.
11. Aguilar-Salinas, C. A.; Olaiz, G.; Valles, V.; Ríos-Torres, J. M.; Gómez-Pérez, F. J.;
Rull, J. A.; Franco, A.; Sepulveda, J. J. Lipid Res. 2001, 42, 1298.

5882
A. Mendieta et al. / Bioorg. Med. Chem. 22 (2014) 5871–5882
12. Gielen, S.; Sandri, M.; Schuler, G.; Teupser, D. Eur. J. Cardiovasc. Prev. Rehabil.
2009, 16, S29.
13. Grundy, S. M.; Cleeman, J. I.; Daniels, S. R.; Donato, K. A.; Eckel, R. H.; Franklin,
B. A.; Gordon, D. J.; Krauss, R. M.; Savage, P. J.; Smith, S. C., Jr.; Spertus, J. A.;
Costa, F. Circulation 2005, 112, 2735.
44. Levine, S.; Saltzman, A. J. Pharmacol. Toxicol. Methods 2007, 55, 224.
45. Zeniya, M.; Reuben, A. Am. J. Physiol. 1988, 254, G346.
46. Edelstein, C.; Byrne, R. E.; Yamamoto, K.; Zarins, C.; Scanu, A. M. J. Lipid Res.
1985, 26, 351.
47. Kuroda, M.; Tanzawa, K.; Tsujita, Y.; Endo, A. Biochim. Biophys. Acta 1977, 489,
119.
14. Chalasani, N. Hepatology 2005, 41, 690.
15. Graham, D. J.; Staffa, J. A.; Shatin, D.; Andrade, S. E.; Schech, S. D.; La Grenade,
L.; Gurwitz, J. H.; Chan, K. A.; Goodman, M. J.; Platt, R. P. JAMA 2004, 292, 2585.
16. Elisaf, M. S.; Florentin, M.; Liberopoulos, E. N.; Mikhailidis, D. P. Curr. Pharm.
Des. 2008, 14, 574.
48. Goldfard, S. J. Lipid Res. 1978, 19, 489.
49. Hermier, D.; Hales, P.; Brindley, D. N. FEBS Lett. 1991, 286, 186.
50. Borensztajn, J.; Rone, M. S.; Kotlar, T. J. Biochem. J. 1976, 156, 539.
51. Schurr, P. E.; Schultz, J. R.; Parkinson, T. M. Lipids 1972, 7, 68.
52. Schotz, M. C.; Scanu, A.; Page, I. H. Am. J. Physiol. 1957, 188, 399.
53. Abe, C.; Ikeda, S.; Uchida, T.; Yamashita, K.; Ichikawa, T. J. Nutr. 2007, 137, 345.
54. Korolenko, T. A.; Tuzikov, F. V.; Vasil’eva, E. D.; Cherkanova, M. S.; Tuzikova, N.
A. Bull. Exp. Biol. Med. 2010, 149, 567.
55. Cruz, A.; Garduño, L.; Salazar, M.; Martinez, E.; Jiménez-Vázquez, H. A.; Diaz, F.;
Chamorro, G.; Tamariz, J. Arzneim.-Forsch. Drug Res. 2001, 51, 535.
56. Chen, W.; Guo, J. X.; Chang, P. Mol. Nutr. Food 2012, 56, 681.
57. Hedstrom, L. Chem. Rev. 2002, 102, 4501.
58. Kumar, A.; Venkatesu, P. Chem. Rev. 2012, 112, 4283.
59. Yusufi, M.; Banerjee, S.; Mohammad, M.; Khatal, S.; Swamy, K. V.; Khan, E. M.;
Aboukameel, A.; Sarkar, F. H.; Padhye, S. Bioorg. Med. Chem. Lett. 2013, 23, 3101.
60. Bremer, P. T.; Hixon, M. S.; Janda, K. D. Bioorg. Med. Chem. 2014, 22, 3971.
61. Istvan, E. S.; Deisenhofer, J. Science 2001, 292, 1160.
17. Hodel, C. Toxicol. Lett. 2002, 128, 159.
18. Joy, T. R.; Hegele, R. A. Ann. Intern. Med. 2009, 150, 858.
19. Williams, D.; Feely, J. Clin. Pharmacokinet. 2002, 41, 343.
20. Davidson, M. H.; Armani, A.; McKenney, J. M.; Jacobson, T. A. Am. J. Cardiol.
2007, 99, S3.
21. Keech, A.; Simes, R. J.; Barter, P.; Best, J.; Scott, R.; Taskinen, M. R.; Forder, P.;
Pillai, A.; Davis, T.; Glasziou, P.; Drury, P.; Kesäniemi, Y. A.; Sullivan, D.; Hunt,
D.; Colman, P.; d’Emden, M.; Whiting, M.; Ehnholm, C.; Laakso, M. Lancet 2005,
366, 1849.
22. Golomb, B. A.; Evans, M. A. Am. J. Cardiovasc. Drugs 2008, 8, 373.
23. Chamorro, G.; Salazar, M.; Salazar, S.; Mendoza, T. Rev. Invest. Clin. 1993, 45,
597.
24. Momin, R. A.; Nair, M. G. J. Agric. Food Chem. 2002, 50, 4475.
25. Lee, J. Y.; Lee, J. Y.; Yun, B.-S.; Hwang, B. K. J. Agric. Food Chem. 2004, 52, 776.
26. Poplawsky, J.; Lozowicka, B.; Dubis, A. T.; Lachowska, B.; Witkowski, S.; Siluk,
D.; Petrusewicz, J.; Kaliszan, R.; Cybulski, J.; Strzalkowska, M.; Chilmonczyk, Z.
J. Med. Chem. 2000, 43, 3671.
62. Plewczynski, D.; Lazniewski, M.; Augustyniak, R.; Ginalski, K. J. Comp. Chem.
2011, 32, 742.
63. Medina-Franco, J. L.; Méndez-Lucio, O.; Martinez-Mayorga, K. In Advances in
Protein Chemistry and Structural Biology: Biomolecular Modelling and
Simulations; Karabencheva-Christova, T., Ed.; Elsevier: New York, 2014; Vol.
96,. in press.
27. Rodríguez-Páez, L.; Juárez-Sánchez, M.; Antúnez-Solís, J.; Baeza, I.; Wong, C.
Phytomedicine 2003, 10, 397.
28. Singh, N.; Tamariz, J.; Chamorro, G.; Medina-Franco, J. L. Mini-Rev. Med. Chem.
2009, 9, 1272.
64. Willson, T. M.; Brown, P. J.; Sternbach, D. D.; Henke, B. R. J. Med. Chem. 2000, 43,
527.
29. Haines, B. E.; Wiest, O.; Stauffacher, C. V. Acc. Chem. Res. 2013, 46, 2416.
30. Argüelles, N.; Sánchez-Sandoval, E.; Mendieta, A.; Villa-Tanaca, L.; Garduño-
Siciliano, L.; Jiménez, F.; Cruz, M. C.; Medina-Franco, J. L.; Chamorro-Cevallos,
G.; Tamariz, J. Bioorg. Med. Chem. 2010, 18, 4238.
31. Cruz, M. C.; Salazar, M.; Garcíafigueroa, Y.; Hernández, D.; Díaz, F.; Chamorro,
G.; Tamariz, J. Drug Dev. Res. 2003, 60, 186.
32. Medina-Franco, J. L.; López-Vallejo, F.; Rodríguez-Morales, S.; Castillo, R.;
Chamorro, G.; Tamariz, J. Bioorg. Med. Chem. Lett. 2005, 15, 989.
33. Medina-Franco, J. L. Diseño de fármacos asistido por computadora. Inhibidores de
65. Fruchart, J.-C. Am. J. Cardiol. 2001, 88, 24.
66. Gouni-Berthold, I.; Krone, W. Curr. Drug Targets Cardiovasc. Hematol. Disord.
2005, 5, 513.
67. Israelian-Konaraki, Z.; Reaven, P. D. Cardiol. Rev. 2005, 13, 240.
68. Duval, C.; Müller, M.; Kersten, S. Biochim. Biophys. Acta 2007, 1771, 961.
69. Zandbergen, F.; Plutzky, J. Biochim. Biophys. Acta 2007, 1771, 972.
70. Cheng, A. Y. Y.; Leiter, L. A. Diabetes Obes. Metab. 2008, 10, 691.
71. Medina-Franco, J. L.; Giulianotti, M. A.; Welmaker, G. S.; Houghten, R. A. Drug
Discov. Today 2013, 18, 495.
la transcriptasa reversa del virus del VIH, hipocolesterolemiantes
antiparasitarios. PhD Thesis, UNAM, México, 2005.
y
72. Jones, H. G.; Mackenzie, D. B.; Robertson, A. J. Chem. Soc. 1949, 562.
73. Moffet, B. R.; Hanze, R. A.; Seay, H. P. J. Chem. Soc. 1964, 178.
74. Späth, E.; Pailer, M.; Gergely, G. Ber. Deut. Chem. Ges. 1940, 73, 935.
75. Davidge, H.; Davies, A. G.; Kenyon, J.; Mason, R. F. J. Chem. Soc. 1958, 4569.
76. Bieber, L. W.; Rolin Neto, P. J.; Generino, R. M. Tetrahedron Lett. 1999, 40, 4473.
77. SHELXS97, Programs for Crystal Structure Analysis. Release 97–2. Institüt für
Anorganische Chemie der Universität, Tammanstrasse 4, D-3400 Göttingen,
Germany.
34. Labarrios, F.; Garduño, L.; Vidal, M. R.; García, R.; Salazar, M.; Martínez, E.; Díaz,
F.; Chamorro, G.; Tamariz, J. J. Pharm. Pharmacol. 1999, 51, 1.
35. Zúñiga, C.; Garduño, L.; Cruz, M. C.; Salazar, M.; Pérez-Pastén, R.; Chamorro, G.;
Labarrios, F.; Tamariz, J. Drug Dev. Res. 2005, 64, 28.
36. Cruz, A.; Garduño, L.; Salazar, M.; Martínez, E.; Diaz, F.; Chamorro, G.; Tamariz,
J. Med. Chem. Res. 2001, 10, 587.
37. Diaz, F.; Muñoz, H.; Labarrios, F.; Chamorro, G.; Salazar, M.; Morelos, M. E.;
Tamariz, J. Med. Chem. Res. 1993, 3, 101.
38. Brown, F. J.; Bernstein, P. R.; Cronk, L. A.; Dosset, D. L.; Hebbel, K. C.; Maduskuie,
T. P., Jr.; Shapiro, H. S.; Vacek, E. P.; Yee, Y. K.; Willard, A. K.; Krell, R. D.; Snyder,
D. W. J. Med. Chem. 1989, 32, 807.
39. Ferreira, E. A.; Fortes Gris, E.; Matos Rebello, J.; Gomes Correia, J. F.; Souza de
Oliveira, L. F.; Wilhelm Filo, D.; Curi Pedrosa, R. Planta Med. 2011, 77, 1569.
40. Ploypradith, P.; Jinaglueng, W.; Pavaro, C.; Ruchirawat, S. Tetrahedron Lett.
2003, 44, 1363.
41. Anderson, G. M., III; Kollman, P. A.; Domelsmith, L. N.; Houk, K. N. J. Am. Chem.
Soc. 1979, 101, 2344.
78. WinGX Farrugia, L. J. J. Appl. Cryst. 1999, 32, 837.
79. SHELXL97, Sheldrick, G. M. Acta Cryst. 2008, A64, 112.
80. ORTEP-3, Farrugia, L. J. J. Appl. Cryst. 1997, 30, 565.
81. Friedewald, W. T.; Levy, R. I.; Fredrickson, D. S. Clin. Chem. 1972, 18, 499.
82. Bischoff, K. M.; Rodwell, V. W. J. Bacteriol. 1996, 178, 19.
83. Suite 2012: Maestro, version 9.3, Schrödinger, LLC, New York, NY, 2012.
84. Yoo, J.; Choi, S.; Medina-Franco, J. L. PLoS ONE 2013, 8, e62152.
85. Molecular Operating Environment (MOE), version 2011.10; Chemical
Computing Group, Inc.: Montreal, Quebec, Canada. Available at http://
www.chemcomp.com (accessed Aug. 2013).
86. Suite 2012: Glide, version 5.8, Schrödinger, LLC, New York, NY, 2012.
87. Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.;
Halgren, T. A.; Sanschagrin, P. C.; Mainz, D. T. J. Med. Chem. 2006, 49, 6177.
42. Silva, R. M.; Santos, F. A.; Maciel, M. A. M.; Pinto, A. C.; Rao, V. S. N. Planta Med.
2001, 67, 763.
43. Kourounakis, A. P.; Victoratos, P.; Peroulis, N.; Stefanou, N.; Yiangou, M.;
Hadjipetrou, L.; Kourounakis, P. N. Exp. Mol. Pathol. 2002, 73, 135.