Synthesis and biological activity of fibrate-based acyl- and alkyl-phenoxyacetic methyl esters and 1,2-dihydroquinolines

Article abstract of DOI:10.1007/s00044-019-02496-1

A series of highly potent antihyperlipidemic agents constituted by a fibrate-based structure was recently reported by our group, whose synthesis started from isovanillin derivatives. In the interest of evaluating the bioisosteric effect of the vanillin-based isomers on their antihyperlipidemic activity, the present study focuses on the synthesis of 5-acyl-1-phenoxyacetic methyl esters 5a–c and their saturated side-chain 5-alkyl-1-phenoxyacetates 6a–c. Their strong in vivo effect was associated with the inhibition of HMG-CoA reductase. Since 1,2-dihydroquinolines inhibit this enzyme, a series of such heterocycles (9a–d) was prepared by our efficient regioselective, one-step, solvent-free method. Apart from showing hypolipidemic activity in vivo, some of the compounds displayed antifungal, antioxidant and cytotoxic activity in vitro. The binding mode of four compounds at the active site of HMGRh was examined with docking simulations, observing an interaction with most of the amino acids targeted by simvastatin.

Full text of DOI:10.1007/s00044-019-02496-1

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02496-1
ORIGINAL RESEARCH
Synthesis and biological activity of fibrate-based acyl- and alkyl-
phenoxyacetic methyl esters and 1,2-dihydroquinolines
Abraham Pucheta1,2 Aarón Mendieta1,3 Damián A. Madrigal1,2 Roberto I. Hernández-Benitez1 Liseth Romero2
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Leticia Garduño-Siciliano2 Catalina Rugerio-Escalona3 María C. Cruz-López3 Fabiola Jiménez3
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Alejandra Ramírez-Villalva4 Aydeé Fuentes-Benites4 Carlos González-Romero4 Omar Gómez-García1
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Julio López5 Miguel A. Vázquez5 Blanca Rosales-Acosta6 Lourdes Villa-Tanaca6 Alfonso Sequeda-Juárez7
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Eva Ramón-Gallegos7 Germán Chamorro-Cevallos2 Francisco Delgado1 Joaquín Tamariz
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Received: 10 October 2019 / Accepted: 22 December 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
A series of highly potent antihyperlipidemic agents constituted by a fibrate-based structure was recently reported by our
group, whose synthesis started from isovanillin derivatives. In the interest of evaluating the bioisosteric effect of the vanillin-
based isomers on their antihyperlipidemic activity, the present study focuses on the synthesis of 5-acyl-1-phenoxyacetic
methyl esters 5a–c and their saturated side-chain 5-alkyl-1-phenoxyacetates 6a–c. Their strong in vivo effect was associated
with the inhibition of HMG-CoA reductase. Since 1,2-dihydroquinolines inhibit this enzyme, a series of such heterocycles
(9a–d) was prepared by our efficient regioselective, one-step, solvent-free method. Apart from showing hypolipidemic
activity in vivo, some of the compounds displayed antifungal, antioxidant and cytotoxic activity in vitro. The binding mode
of four compounds at the active site of HMGRh was examined with docking simulations, observing an interaction with most
of the amino acids targeted by simvastatin.
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Keywords Antihyperlipidemic activity 2-Acyl-1-hydroxyphenoxyacetic esters 2-Alkyl-1-hydroxyphenoxyacetic esters
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1,2-Dihydroquinolines HMG-CoA reductase Antioxidant activity
Introduction
Hyperlipidemia, which represent a significant cardiovas-
cular risk factor, is a growing public health problem
worldwide (Thagizadeh et al. 2019). It is mainly associated
with diseases such as atherosclerosis (Yuan et al. 2007;
Steinberg 2005) and ischemic heart disease (Mehta et al.
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02496-1) contains supplementary
material, which is available to authorized users.
* Germán Chamorro-Cevallos
4
Departamento de Química Orgánica, Facultad de Química,
Universidad Autónoma del Estado de México (UAEM), Paseo
Tollocan y Colón S/N, 50000 Toluca, Mexico
gchamcev@yahoo.com.mx
* Joaquín Tamariz
jtamarizm@gmail.com
5
Departamento de Química, Universidad de Guanajuato, Noria Alta
S/N, 36050 Guanajuato, Gto, Mexico
1
Departamento de Química Orgánica, Escuela Nacional de Ciencias
Biológicas, Instituto Politécnico Nacional (IPN). Prol. Carpio y
Plan de Ayala S/N, 11340 Mexico City, Mexico
6
Laboratorio de Genética Microbiana, Escuela Nacional de
Ciencias Biológicas, Instituto Politécnico Nacional, Prol. Carpio y
Plan de Ayala S/N, 11340 Mexico City, Mexico
2
Departamento de Farmacia, Escuela Nacional de Ciencias
Biológicas, Instituto Politécnico Nacional, Av. Wilfrido Massieu
S/N, Unidad Adolfo López Mateos, 07738 Mexico City, Mexico
7
Departamento de Morfología, Escuela Nacional de Ciencias
Biológicas, Instituto Politécnico Nacional, Av. Wilfrido Massieu
S/N, Unidad Adolfo López Mateos, 07738 Mexico City, Mexico
3
Centro de Investigación en Biotecnología Aplicada, IPN. Carretera
Estatal Santa Inés Tecuexcomac-Tepetitla, Km 1.5, Tepetitla de
Lardizábal, 90700 Tlaxcala, Mexico

Medicinal Chemistry Research
2014). Obesity and diabetes may contribute substantially to
the elevated prevalence of dyslipidemia (Van Gaal et al.
2006; Aguilar-Salinas et al. 2001). When lifestyle changes
(e.g., diet, exercise, and weight loss) fail to control dysli-
pidemia, healthcare systems turn to the prescription of lipid-
lowering medication (Gielen et al. 2009; Grundy et al.
2005). Although many synthetic drugs are available to treat
hyperlipidemia, they are related with multiple adverse
effects, such as myopathy, rhabdomyolysis, and liver toxi-
city (Chalasani 2005; Graham et al. 2004; Elisaf et al. 2008;
Hodel 2002; Joy and Hegele 2009; Williams and Feely
2002; Davidson et al. 2007).
Statins are among the most commonly used drugs for the
clinical management of hypercholesterolemia (Maron et al.
2000; Wierzbicki 2001; Miyazaki et al. 2004; Vivancos
et al. 1999). Their adverse effects include rhabdomyolysis,
cognitive decline, neuropathy, pancreatic, and hepatic dys-
function, and an increased risk of cancer (Golomb and Evans
2008; Rotta-Bonfin et al. 2015). The mechanism of their
activity has been established as the inhibition of the
3-hydroxy-3-methylglutaryl-coenzyme
A
(HMG-CoA)
reductase (HMGR), an enzyme involved in the biosynthesis
of cholesterol (Singh et al. 2009; Haines et al. 2013).
Among other agents inhibiting HMGR (Menéndez et al.
2001; Liu and Yeh 2002; Parker et al. 1993; Sung et al.
2004; Bradfute and Simoni 1994; Harada-Shiba et al. 1995;
Panini et al. 1986) are cholestin (Man et al. 2002), dios-
genin (Raju and Bird 2007), lanosterol analogs (Trzaskos
et al. 1993), β-sitosterol (Field et al. 1997), and the natural
product α-asarone (1) (active metabolite of the Yucatan
peninsula (Mexico) tree called Elemuy (Mosannona
depressa (Baill.) Chatrou) (Campos-Ríos and Chiang Cab-
rera 2006)) resulting in an antihyperlipidemic effect (Cha-
morro et al. 1993). α-Asarone (1) has also been reported to
inhibit HMGR (Singh et al. 2009; Rodríguez-Páez et al.
2003) and exhibit antifungal (Momin and Nair 2002; Lee
et al. 2004) and antithrombotic (Poplawsky et al. 2000)
activity.
Fibrates such as clofibrate (2a) (Oliver 2012; Moham-
madzadeh et al. 2013), bezafibrate (2b) (BIP study group
2000), and fenofibrate (2c) (Uchida et al. 2011) are
phenoxyacetic-based compounds with a potent hypo-
triglyceridemic activity (Lalloyer and Staels 2010; Jover-
Fernández and Hernández-Mijares 2012). Unlike statins,
their mechanism of action consists of binding to and acti-
Fig. 1 Structures of α-asarone (1), clofibrate (2a), bezafibrate (2b),
fenofibrate (2c), α-asarone analogs 3a–f, and fibrate analogs 4a–f
clofibrate was withdrawn from the market in some countries.
The biological activity of phenoxy acids and their derivatives
is exceptionally wide-ranging, being antihyperlipidemic,
hypoglycemic, antimicrobial, antiviral, antitubercular, anti-
inflammatory, analgesic, antioxidant, anticancer, and anti-
hypertensive (Shaheen et al. 2016).
In the search of new synthetic hypolipidemic agents with
fewer or no side effects, our group previously synthesized a
series of α-asarone (1) analogs that showed the desired activity
in potent form (Argüelles et al. 2010; Cruz et al. 2003).
Docking studies revealed the binding mode of 1 with the
active site of the human HMGR (HMGRh) enzyme (Medina-
Franco et al. 2005). Moreover, the oxyacetic group at the C-2
position of 1 (Argüelles et al. 2010), mimicking the fibrate
structure, proved to have a significant lipid-lowering activity
(Labarrios et al. 1999; Zúñiga et al. 2005). Particularly, the
series of α-asarone- 3a–f and phenoxyacetic-based derivatives
4a–f (Fig. 1) (all prepared from isovanillin) displayed a sharp
reduction in cholesterol, LDL-cholesterol, and triglycerides,
indicating HMGRh inhibition (Mendieta et al. 2014).
vating peroxisome proliferator activated receptor
α
(PPARα), a transcription factor (Schoonjans et al. 1996;
Willson et al. 2000; Fazio and Linton 2004; Shaikh and Ali
2018). Fibrates enhance HDL cholesterol but can cause
myopathy, cholelithiasis, nausea, syndrome of inappropriate
secretion of antidiuretic hormone ADH (SIADH), and liver
injury (The Field Study 2005; Okopień et al. 2018). These
adverse side effects were among other motives that
On the other hand, there is evidence of diverse types of
biological activity of 1,2-dihydroquinolines, found to act as
antidiabetic, anti-inflammatory (Hegedüs et al. 2007), anti-
bacterial (Johnson et al. 1989), antioxidant and cytotoxic
(Dorey et al. 2000; Błaszczyk and Skolimowski 2007;
Błaszczyk et al. 2013; de Koning 2002) (Błaszczyk et al.

Medicinal Chemistry Research
Materials and methods
Chemistry
Melting points were determined with an Electrothermal
capillary melting point apparatus. IR spectra were recorded
on an FT-IR Perkin Elmer 2000 spectrophotometer. 1H (300
or 500 MHz) and 13C (75.4 or 125 MHz) NMR spectra were
recorded on Varian Mercury-300 or Varian VNMR System
instruments, with TMS as internal standard. Mass spectra
(MS) were taken in the electron impact (EI) mode on
Hewlett-Packard 5971A and Thermo-Finnigan Polaris Q
spectrometers. High-resolution mass spectra (HRMS) were
obtained (EI) on a Jeol JSM-GCMateII spectrometer.
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. All
air moisture sensitive reactions were carried out with oven-
dried glassware under nitrogen atmosphere. Prior to use,
acetone and DMF were distilled from 4Å; molecular sieves
and methylene chloride from calcium hydride. K2CO3 was
dried overnight at 120 °C before use. All other reagents
were used without further purification. The preparation of
compounds 12 (Grenier et al. 2000; Kiss et al. 2010; Rashid
et al. 2018) and 9a–d has been previously described
(Gutiérrez et al. 2013).
Fig. 2 Structures of the synthesized fibrate-based analogs 5a–c, 6a–c,
and 7a–c, and the 1,2-dihydroquinolines 9a–d
2006; Błaszczyk and Skolimowski 2005; Błaszczyk and
Skolimowski 2006) agents. Likewise, analogous 1,2,3,4-
tetrahydroquinolines have shown antifungal (Chander et al.
2016a), antimicrobial (Chander et al. 2016b), and anti-
oxidant (Dorey et al. 2000) activity, which has been asso-
ciated with their capacity of lipid peroxidation and the
inhibition of HMGR (Hegedüs et al. 2007). Consequently,
these heterocycles may also produce a general hypolipi-
demic, antioxidant and antifungal effect.
The first aim of the present study was to examine the
possible impact on the antihyperlipidemic activity caused
by the isomeric position of the substituents in the benzene
ring of vanillin-based isomers. Thus, three series of fibrate-
based compounds were synthesized starting from vanillin
(8) and their structure elucidated. The series consisted of 5-
acyl-1-phenoxyacetic methyl esters 5a–c, their saturated
side-chain 5-alkyl-1-phenoxyacetates 6a–c and nitrophe-
noxyacetates 7a–c (Fig. 2). Due to the HMGR-related
activity of hydroquinolines, a series of the latter was syn-
thesized by using our regioselective one-step method
(Gutiérrez et al. 2013). These compounds, 9a–d, were
evaluated in vivo as hypolipidemic agents, and in vitro for
their antifungal, antioxidant, and anticancer effect. The
mechanism of action of the test compounds on the HMGRh
structure was explored with docking simulations.
Methyl 2-(4-formyl-2-methoxyphenoxy)acetate (10)
Under an N2 atmosphere, a mixture of vanillin (8) (10.00 g,
65.72 mmol), methyl bromoacetate (16.463 g, 98.58 mmol),
and dry K2CO3 (18.165 g, 131.44 mmol) in anhydrous
acetone (100 mL) was heated to 60 °C for 4 h. The reaction
mixture was filtered and the solvent removed under
vacuum. The residue was purified by column chromato-
graphy over silica gel (20 g/g of crude, hexane/EtOAc, 8:2)
to give 10 (13.41 g, 91%) as a white solid. Rf 0.48 (hexane/
EtOAc, 6:4); m.p. 92–93 °C. IR (KBr): ν 2994, 1740, 1677,
1591, 1508, 1452, 1404, 1285, 1261, 1224, 1136, 1028,
1
861, 807, 735 cm−1. H NMR (500 MHz, CDCl3): δ 3.81
(s, 3H, CO2CH3), 3.96 (s, 3H, OCH3), 4.80 (s, 2H,
CH2CO2CH3), 6.89 (d, J = 8.5 Hz, 1H, H-6′), 7.42 (dd, J =
8.5, 1.8 Hz, 1H, H-5′), 7.44 (d, J = 1.8 Hz, 1H, H-3′), 9.87
(s, 1H, CHO). 13C NMR (125 MHz, CDCl3): δ 52.4
(CO2CH3), 56.0 (OCH3), 65.8 (CH2CO2CH3), 109.8 (C-3′),
112.3 (C-6′), 126.1 (C-5′), 131.2 (C-4′), 149.9 (C-2′), 152.4
(C-1′), 168.5 (CO2CH3), 190.7 (CHO). MS (70 eV): m/z
224 (M+, 100), 165 (25), 150 (30), 137 (35), 119 (36), 105
(20), 95 (30), 77 (31), 59 (32). HRMS (EI): calcd. for
C11H12O5 [M]+ 224.0685; found: 224.0685.

Medicinal Chemistry Research
Methyl 2-(4-hydroxy-2-methoxyphenoxy)acetate
(11)
OCH3), 4.62 (s, 2H, CH2CO2CH3), 6.46 (s, 1H, H-3′), 7.29
(s, 1H, H-6′), 12.61 (s, 1H, OH). 13C NMR (125 MHz,
CDCl3): δ 26.3 (CH3CO), 52.2 (CO2CH3), 56.1 (OCH3),
68.1 (CH2CO2CH3), 100.9 (C-3′), 111.9 (C-5′), 118.3 (C-
6′), 139.6 (C-1′), 157.8 (C-2′), 161.2 (C-4′), 169.5
(CO2CH3), 202.2 (CH3CO). MS (70 eV): m/z 254 (M+, 51),
239 (6), 195 (7), 181 (100), 153 (14), 135 (15), 107 (9).
HRMS (EI): calcd for C12H14O6 [M]+: 254.0790; found:
254.0787.
A mixture of 10 (10,000 g, 44.64 mmol) and mCPBA (77%)
(15,000 g, 66.96 mmol) in CH2Cl2 (100 mL) was stirred at
room temperature (rt) for 12 h. The solvent was removed
under vacuum, and MeOH (100 mL) and HCl (6N) (5 mL)
were added. The reaction mixture was stirred at rt for 1 h
before removing the solvent under vacuum. A saturated
aqueous solution of NaHCO3 was added until neutral, then
extracted with EtOAc (4 × 50 mL). The organic layer was
dried (Na2SO4) and the solvent removed under vacuum. The
residue was purified by column chromatography over silica
gel (20 g/g of crude, hexane/EtOAc, 7:3) to afford 11
(6.814 g, 72%) as a white solid. Rf 0.43 (hexane/EtOAc,
6:4); m.p. 89–90 °C. IR (KBr): ν 3431, 2958, 1732, 1612,
1512, 1479, 1433, 1309, 1208, 1136, 1081, 1032, 953, 837,
793, 766, 712 cm−1. 1H NMR (300 MHz, CDCl3): δ 3.79 (s,
3H, CO2CH3), 3.80 (s, 3H, OCH3), 4.62 (s, 2H,
CH2CO2CH3), 4.96 (br, 1H, OH), 6.30 (dd, J = 8.5, 2.7 Hz,
1H, H-5′), 6.46 (d, J = 2.7 Hz, 1H, H-3′), 6.75 (d, J =
8.5 Hz, 1H, H-6′). 13C NMR (75 MHz, CDCl3): δ 52.2
(CO2CH3), 55.7 (OCH3), 67.6 (CH2CO2CH3), 100.7 (C-3′),
106.0 (C-5′), 116.7 (C-6′), 140.9 (C-1′), 150.7 (C-2′), 151.8
(C-4′), 170.2 (CO2CH3). HRMS (EI): calcd. for C10H12O5
[M]+ 212.0685; found: 212.0681.
Methyl 2-(4-hydroxy-2-methoxy-5-
propionylphenoxy)acetate (5b)
Following the general procedure, 11 (1.000 g, 4.72 mmol)
was mixed with propanoyl chloride (0.654 g, 7.07 mmol)
and BF3·OEt2 (0.670 g, 4.72 mmol) to obtain 5b (0.91 g,
76%) as a white solid. Rf 0.40 (hexane/EtOAc, 7:3); m.p.
105–106 °C. IR (KBr): ν 3482, 2979, 2942, 1748, 1638,
1616, 1565, 1504, 1439, 1375, 1319, 1249, 1193, 1158,
1
1069, 1007, 975, 945, 820, 707 cm−1. H NMR (300 MHz,
CDCl3): δ 1.22 (t, J = 7.2 Hz, 3H, CH3CH2CO), 2.90 (q,
J = 7.2 Hz, 2H, CH3CH2CO), 3.80 (s, 3H, CO2CH3), 3.90
(s, 3H, OCH3), 4.62 (s, 2H, CH2CO2CH3), 6.46 (s, 1H, H-
3′), 7.33 (s, 1H, H-6′), 12.70 (s, 1H, OH). 13C NMR
(75 MHz, CDCl3): δ 8.3 (CH3CH2CO), 31.2 (CH3CH2CO),
52.1 (CO2CH3), 56.0 (OCH3), 68.0 (CH2CO2CH3), 100.9
(C-3′), 111.3 (C-5′), 117.5 (C-6′), 139.5 (C-1′), 157.4 (C-
2′), 161.1 (C-4′), 169.5 (CO2CH3), 204.9 (CH3CH2CO).
MS (70 eV): m/z 268 (M+, 66), 239 (66), 195 (100), 138
(22), 121 (14). HRMS (EI): m/z calcd for C13H16O6 [M]+:
268.0947; found: 268.0946.
General procedure for preparing 5-
acylphenoxyacetic methyl esters 5a–c
Under an N2 atmosphere and at rt, a mixture of 11 (1.0 mol
equiv.) and of the respective acyl chloride (1.5 mol equiv.)
was stirred for 10 min. Subsequently, BF3·OEt2 (1.0 mol
equiv.) was added, stirred at rt for 20 min, and then heated
to 80 °C for 4 h. Ice was poured into the reaction mixture,
followed by adding a saturated aqueous solution of
NaHCO3 dropwise until neutral, then washed with EtOAc
(3 × 50 mL). The organic extract was dried (Na2SO4) and
the solvent removed under vacuum. The residue was pur-
ified by column chromatography over silica gel (20 g/g of
crude, hexane/EtOAc, 85:15).
Methyl 2-(5-butyryl-4-hydroxy-2-methoxyphenoxy)
acetate (5c)
Following the general procedure, 11 (1.000 g, 4.72 mmol)
was mixed with butanoyl chloride (0.753 g, 7.07 mmol) and
BF3·OEt2 (0.670 g, 4.72 mmol) to produce 5c (0.892 g,
67%) as a white solid. Rf 0.46 (hexane/EtOAc, 7:3); m.p.
82–83 °C. IR (KBr): ν 3482, 2954, 1748, 1637, 1511, 1442,
1384, 1336, 1283, 1253, 1197, 1162, 1075, 1019, 986, 900,
848, 793, 703 cm−1
.
1H NMR (300 MHz, CDCl3):
Methyl 2-(5-acetyl-4-hydroxy-2-methoxyphenoxy)
acetate (5a)
δ 1.01 (t, J = 7.5 Hz, 3H, CH3(CH2)2CO), 1.75 (sext, J =
7.5 Hz, 2H, CH3CH2CH2CO), 2.84 (t, J = 7.5 Hz, 2H,
CH3CH2CH2CO), 3.81 (s, 3H, CO2CH3), 3.91 (s, 3H,
OCH3), 4.63 (s, 2H, CH2CO2CH3), 6.46 (s, 1H, H-3′), 7.32
(s, 1H, H-6′), 12.78 (s, 1H, OH). 13C NMR (75 MHz,
CDCl3): δ 13.8 (CH3(CH2)2CO), 18.1 (CH3CH2CH2CO),
39.9 (CH3CH2CH2CO), 52.2 (CO2CH3), 56.1 (OCH3), 68.0
(CH2CO2CH3), 100.87 (C-3′), 111.4 (C-5′), 117.5 (C-6′),
139.5 (C-1′), 157.4 (C-2′), 161.2 (C-4′), 169.5 (CO2CH3),
204.6 (CH3(CH2)2CO). MS (70 eV): m/z 282 (M+, 66), 239
(68), 209 (100), 167 (14), 138 (20), 107 (10), 69 (16).
Following the general procedure, 11 (1.000 g, 4.72 mmol)
was mixed with acetyl chloride (0.555 g, 7.07 mmol) and
BF3·OEt2 (0.670 g, 4.72 mmol) to provide 5a (0.91 g, 76%)
as a white solid. Rf 0.31 (hexane/EtOAc, 7:3); m.p.
125–126C. IR (KBr): ν 3472, 2974, 2372, 1744, 1640,
1614, 1503, 1440, 1365, 1334, 1265, 1202, 1163, 1058,
1
1007, 975, 814, 710 cm−1. H NMR (500 MHz, CDCl3):
δ 2.53 (s, 3H, CH3CO), 3.80 (s, 3H, CO2CH3), 3.90 (s, 3H,

Medicinal Chemistry Research
HRMS (EI): m/z calcd for C14H18O6 [M]+: 282.1103;
found: 282.1100.
67.8 (CH2CO2CH3), 100.9 (C-3′), 118.1 (C-6′), 119.5 (C-
5′), 140.5 (C-1′), 148.5 (C-2′), 149.1 (C-4′), 170.3
(CO2CH3). HRMS (EI): m/z calcd for C13H18O5 [M]+:
254.1154; found: 254.1151.
General procedure for preparing 5-
alkylphenoxyacetic methyl esters 6a–c
Methyl 2-(5-butyl-4-hydroxy-2-methoxyphenoxy)acetate
(6c)
Under an N2 atmosphere and at rt, a mixture of Zn/Hg and
HCl (36%) (1.0 g/1.0 mL, 10.0 mol equiv.) was stirred for
5 min before being poured into a flask. Addition was made
of the corresponding acyl derivative 5a–c (1.0 mol equiv.),
followed by a mixture of MeOH/HCl (1.0/0.2 mL). After
heating to 60 °C for 4 h, ice was poured in and a saturated
aqueous solution of NaHCO3 added dropwise until neutral,
and then washed with EtOAc (3 × 30 mL). The organic
extract was dried (Na2SO4) and the solvent was removed
under vacuum. The residue was purified by column chro-
matography over silica gel (20 g/g of crude, hexane/
EtOAc, 95:5).
Following the general procedure, 5c (1.000 g, 3.55 mmol)
was mixed with Zn/Hg (9.43 g, 35.5 mmol), resulting in 6c
(0.65 g, 68%) as a white solid. Rf 0.31 (hexane/EtOAc, 7:3);
m.p. 49–50 °C. IR (KBr): ν 3448, 3019, 2952, 2860, 1719,
1617, 1529, 1449, 1417, 1367, 1306, 1206, 1123, 1078,
1013, 939, 894, 856, 829, 759, 714 cm−1 1H NMR
.
(500 MHz, CDCl3): δ 0.94 (t, J = 7.5 Hz, 3H, H-4″), 1.35
(sext, J = 7.5 Hz, 2H, H-3″), 1.53 (qu, J = 7.5 Hz, 2H, H-
2″), 2.45 (t, J = 7.5 Hz, 2H, H-1″), 3.79 (s, 3H, CO2CH3),
3.80 (s, OCH3), 4.61 (s, 2H, CH2CO2CH3), 6.42 (s, 1H, H-
3′), 6.70 (s, 1H, H-6′). 13C NMR (125 MHz, CDCl3): δ 13.9
(C-4″), 22.4 (C-3″), 29.0 (C-1″), 32.1 (C-2″), 52.1
(CO2CH3), 56.0 (OCH3), 68.0 (CH2CO2CH3), 101.1 (C-3′),
118.4 (C-6′), 119.4 (C-5′), 141.0 (C-1′), 148.9 (C-2′, C-4′),
170.0 (CO2CH3). HRMS (EI): m/z calcd for C14H20O5
[M]+: 268.1311; found: 268.1303.
Methyl 2-(5-ethyl-4-hydroxy-2-methoxyphenoxy)acetate
(6a)
Following the general procedure, 5a (1.000 g, 3.94 mmol)
was mixed with Zn/Hg (10.47 g, 39.4 mmol) to furnish 6a
(0.68 g, 72%) as a white solid. Rf 0.26 (hexane/EtOAc, 7:3);
m.p. 66–67 °C. IR (KBr): ν 3402, 2961, 2925, 2866, 1750,
1717, 1616, 1525, 1447, 1423, 1367, 1293, 1237, 1204,
1122, 1069, 1007, 857, 747, 706 cm−1. 1H NMR (500 MHz,
CDCl3): δ 1.17 (t, J = 7.5 Hz, 3H, H-2″), 2.52 (q, J =
7.5 Hz, 2H, H-1″), 3.78 (s, 6H, CO2CH3, OCH3), 4.62 (s,
2H, CH2CO2CH3), 4.77–4.84 (m, 1H, OH), 6.42 (s, 1H, H-
3′), 6.73 (s, 1H, H-6′). 13C NMR (125 MHz, CDCl3): δ 14.2
(C-2″), 22.3 (C-1″), 52.1 (CO2CH3), 55.9 (OCH3), 68.0
(CH2CO2CH3), 101.1 (C-3′), 117.7 (C-6′), 120.9 (C-5′),
141.0 (C-1′), 148.8 (C-2′, C-4′), 170.1 (CO2CH3). HRMS
(EI): m/z calcd for C12H16O5 [M]+: 240.0998; found:
240.1000.
Methyl 2-(4-Formyl-2-methoxy-6-nitrophenoxy)acetato (7a)
Under N2 atmosphere,
a mixture of 12 (10.000 g,
50.76 mmol), dry K2CO3 (14,010 g, 101.52 mmol) and
methyl bromoacetate (15.10 g, 98.7 mmol) in anhydrous
DMF (50 mL) was stirred at 60 °C for 4 h (Brown et al.
2010). The reaction mixture was filtered, the solvent
removed under vacuum, and the residue purified by column
chromatography over silica gel (20 g/g of crude, hexane/
EtOAc, 8:2) to give 7a (9150 g, 67%) as a yellow solid. Rf
0.66 (hexane/EtOAc, 1:1); m.p. 96–97 °C. IR (KBr): ν
3385, 3086, 3014, 2956, 2851, 1752, 1701, 1604, 1541,
1466, 1427, 1357, 1289, 1218, 1202, 1141, 1046, 918, 872,
772, 680 cm−1. 1H NMR (300 MHz, CDCl3): δ 3.78 (s, 3H,
CO2CH3), 3.99 (s, 3H, OCH3), 4.90 (s, 2H, CH2CO2CH3),
7.64 (d, J = 1.8 Hz, 1H, H-3′), 7.88 (d, J = 1.8 Hz, 1H,
H-5′), 9.93 (s, 1H, CHO). 13C NMR (75 MHz, CDCl3):
δ 52.3 (CO2CH3), 56.8 (OCH3), 69.4 (CH2CO2CH3), 113.5
(C-3′), 119.7 (C-5′), 131.7 (C-4′), 145.2 (C-1′), 153.6
(C-2′), 168.5 (CO2CH3), 188.9 (CHO). HRMS (EI): m/z
calcd for C11H11NO7 [M]+: 269.0535; found: 269.0541.
Methyl 2-(4-hydroxy-2-methoxy-5-propylphenoxy)acetate
(6b)
Following the general procedure, 5b (1.000 g, 3.73 mmol)
was mixed with Zn/Hg (9.93 g, 37.3 mmol) to form 6b
(0.663 g, 70%) as a white solid. Rf 0.30 (hexane/EtOAc,
7:3); m.p. 54–55 °C. IR (KBr): ν 3390, 3019, 2955, 2867,
1871, 1708, 1619, 1528, 1450, 1419, 1377, 1302, 1198,
1
1120, 1078, 1008, 884, 866, 843, 768, 739, 703 cm−1. H
NMR (300 MHz, CDCl3): δ 0.93 (t, J = 7.4 Hz, 3H, H-3″),
1.57 (sext, J = 7.4 Hz, 2H, H-2″), 2.46 (t, J = 7.4 Hz, 2H,
H-1″), 3.73 (s, 3H, OCH3), 3.78 (s, 3H, CO2CH3), 4.61 (s,
2H, CH2CO2CH3), 5.45 (br s, 1H, OH), 6.42 (s, 1H, H-3′),
6.69 (s, 1H, H-6′). 13C NMR (75 MHz, CDCl3): δ 13.8 (C-
3″), 23.0 (C-2″), 31.3 (C-1″), 52.1 (CO2CH3), 55.7 (OCH3),
Methyl 2-(4-hydroxy-2-methoxy-6-nitrophenoxy)acetate
(7b)
A mixture of 7a (1.000 g, 3.72 mmol) and mCPBA (77%)
(1.240 g, 5.57 mmol) in CH2Cl2 (10 mL) was stirred at rt for
4 h before removing the solvent under vacuum. MeOH

Medicinal Chemistry Research
(10 mL) and HCl (37%) (1 mL) were added, the mixture
was stirred at rt for 2 h, and the solvent was removed under
vacuum. An aqueous saturated solution of NaHCO3 was
added until neutral, then extracted with EtOAc (4 × 50 mL).
The organic layer was dried (Na2SO4), the solvent removed
under vacuum, and the residue purified by column chro-
matography over silica gel (20 g/g of crude, hexane/EtOAc,
1:1) to afford 7b (0.580 g, 61%) as a pale yellow solid. Rf
0.33 (EtOAc); m.p. 110–111 °C. IR (KBr): ν 3070, 2952,
2643, 1732, 1609, 1542, 1490, 1466, 1421, 1354, 1289,
1222, 1202, 1114, 1066, 1017, 918, 897, 865, 854, 814,
showed no gross abnormalities in any treated mice. All
animals were handled and maintained in accordance with
ethical principles and regulations specified by the Animal
Care and Use Committee of our institution and the stan-
dards of the National Institutes of Health of Mexico.
An aqueous solution of Triton WR 1339 was adminis-
tered intraperitoneally (ip) to mice (400 mg/kg) and after 1 h
the test compounds (25, 50, or 100 mg/kg, dissolved in
saline, or saline-Tween) were injected ip. Blood was taken
from a retro-orbital puncture 24 h later, and the plasma
levels of total cholesterol (TC) and triglycerides (TG) were
determined in duplicate with commercially available kits.
Values are expressed as the mean from 6 animals per
compound.
1
775, 755, 736, 700 cm−1. H NMR (500 MHz, CDCl3): δ
3.79 (s, 3H, CO2CH3), 3.99 (s, 3H, OCH3), 4.89 (s, 2H,
CH2CO2CH3), 7.80 (d, J = 2.0 Hz, 1H, H-3′), 8.12 (d, J =
2.0 Hz, 1H, H-5′). 13C NMR (125 MHz, CDCl3): δ 52.3
(CO2CH3), 56.8 (OCH3), 69.5 (CH2CO2CH3), 116.8 (C-3′),
118.7 (C-5′), 124.6 (C-6′), 144.6 (C-1′), 153.0 (C-2′), 168.6
(CO2CH3), 169.1 (C-4′). HRMS (EI): m/z calcd for
C10H11NO7 [M]+: 257.0536; found: 257.0531.
Antifungal activity
The in vitro antimicrobial effect of compounds 5a–c, 6a–b,
7a, 7c, and 9a–d was evaluated with susceptibility assays
based on the microdilution techniques of the Antimicrobial
Susceptibility Testing Protocols standardized by the Clin-
ical and Laboratory Standards Institute (CLSI). The M38-
A2 method was used for the four strains of filamentous
fungi (CLSI 2002; Espinel-Ingroff and Canton 2007a):
Aspergillus fumigatus ATCC-16907, Trichosporon cuta-
neum ATCC-28592, Rhizopus oryzae ATCC-10329 and
Mucor hiemalis ATCC-8690. The M27-A3 method was
applied for the three strains of ATCC Candida yeasts (CLSI
2008; Espinel-Ingroff and Cantón 2007b; Pfaller and Die-
kema 2012): C. albicans ATCC-10231, C. utilis ATCC-
9226 and C. tropicalis ATCC-13803. The test compounds
were soluble in dimethyl sulfoxide (DMSO), in accordance
with the Antimicrobial Susceptibility Testing Protocols.
Hence, the antifungal assays were performed with this
solvent.
Methyl 2-(4-(hydroxymethyl)-2-methoxy-6-nitrophenoxy)
acetate (7c)
Under N2 atmosphere and at 0 °C, a mixture of 7a (1.000 g,
3.71 mmol) and NaBH4 (0.070 g, 1.85 mmol) in MeOH
(10 mL) was stirred for 2 h before removing the solvent
under vacuum. The residue was purified by column chro-
matography over silica gel (20 g/g of crude, hexane/EtOAc,
1:1) to provide 7c (0.86 g, 86%) as a white solid. Rf 0.16
(hexane/EtOAc, 1:1); m.p. 97–98 °C. IR (KBr): ν 3481,
2921, 1747, 1535, 1452, 1390, 1355, 1276, 1241, 1208,
1135, 1062, 972, 919, 850, 810, 780 cm−1 1H NMR
.
(500 MHz, CDCl3): δ 2.47 (br, 1H, OH), 3.79 (s, 3H,
CO2CH3), 3.90 (s, 3H, OCH3), 4.68 (s, 2H, CH2OH), 4.73
(s, 2H, CH2CO2CH3), 7.14 (d, J = 1.5 Hz, 1H, H-3′), 7.29
(d, J = 1.5 Hz, 1H, H-5′). 13C NMR (125 MHz, CDCl3): δ
52.2 (CO2CH3), 56.4 (OCH3), 63.7 (CH2OH), 69.6
(CH2CO2CH3), 113.7 (C-5′), 114.2 (C-3′), 138.1 (C-4′),
139.2 (C-1′), 144.5 (C-6′), 153.3 (C-2′), 169.1 (CO2CH3).
HRMS (EI): m/z calcd for C11H13NO7: 271.0692; found:
271.0687.
The minimum inhibitory concentration (MIC) of all
compounds and the reference drug (itraconazole), expressed
in µg/mL, was determined in 96-well plates, utilizing
RPMI-1640 culture medium buffered with 3-(N-morpho-
lino)propanesulfonic acid (MOPS, Sigma) and dilutions of
16 to 0.03 µg/mL (according to the procedure outlined in
the instructions for each type of fungus).
Hypolipidemic activity
The inoculum for the yeasts was prepared with culture
colonies of 24-h growth strain in SDA medium and resus-
pending them in a tube of saline solution (0.85% NaCl) to
obtain a concentration of ~1–5 × 106 CFU/mL. The neces-
sary amount of saline solution was added to adjust the
optical density to 0.5 McFarland. Subsequently, a 1:1000
dilution was made with RPMI medium. For filamentous
fungi, the inoculum was elaborated from a culture having
undergone 7 days of growth at 35 °C on PDA agar, a
medium that induces the formation of conidia or spor-
angiospores. To remove the conidia, the culture was
The hypolipidemic effect of the compounds was studied on
male ICR mice weighing 25–30 g (Birmex, Mexico City),
which were housed in hanging metal cages and maintained
at 24 2 °C and 50 10% relative humidity on a 12 h light/
dark cycle (lights on at 8:00 a.m.). Food (a standard pellet
diet, Rodent Diet 5001, PMI Nutrition International, Bren-
wood, MO) and water were freely available. All animals
appeared healthy throughout the dosing period, maintaining
normal food intake and weight gain. Post-sacrifice analysis

Medicinal Chemistry Research
introduced and handled in Tween 20, then resuspended in
saline solution. The particles were allowed to settle for
5 min, the supernatant transferred to another tube and sha-
ken vigorously for 15 s. Because the size of the conidia is
different for each species, the optical density required to
each well was measured on an ELISA microplate reader at
595 nm. The result was expressed as the percentage of the
viability of control cells and the LC50 was calculated (van
Meerloo et al. 2011).
reach
a
concentration of 1–5 × 106 CFU/mL varied.
Data analysis
Adjustment to the desired value was accomplished with the
McFarland Turbidity Standard No. 0.15, based on the
methods of the CLSI.
Data analysis was carried out by nonparametric one-way
ANOVA and a post hoc with Dunn’s test, run on Sigma Plot
software version 12 (Motulsky 1999). All data are expressed
as the mean SD, with significance set at p < 0.05. Each
experiment was performed in triplicate.
Compounds 5–7 and 9 were prepared (at 10 mM) and
tested for their inhibition of Fusarium oxysporum with the
PDA dilution method (Lalitha 2004). Briefly, PDA medium
was sterilized in an autoclave at 121 °C for 20 min, fol-
lowed by the addition of one of the compounds diluted in
DMSO (2%). The plates were inoculated with F. oxysporum
and then incubated at 26–28 °C for 72 h. All experiments
were carried out in triplicates (n = 12), and the data are
expressed as the mean. The growth inhibition efficiency of
each compound was compared with captan (10 mM), the
reference drug, and validated by ANOVA and the Tukey
test (p < 0.05) on the SAS V9.0 program (SAS 2014).
Antioxidant activity
DPPH radical scavenging assay
The scavenging of free radicals by the chromones was
assessed by using the 2,2-diphenyl-1-picrylhydrazyl radical
(DPPH•) assay, as previously described (Cevallos-Casals
and Cisneros-Zevallos 2003) with slight modifications. A
concentration of 10 mM of each compound was prepared, to
which a solution of DPPH• (133.33 µM) was added at a ratio
of 1:3 (v/v). The mixture was incubated at 37 °C for 30 min
and read at 517 nm. Scavenging capacity (SC) was
expressed as the percentage decrease in DPPH at 10 mM:
Cytotoxic activity
Cell culture
Cell lines of human cervical cancer (HeLa), prostate cancer
(DU-145), breast cancer (MDA-231), and no tumoral cell line
(HaCaT) were cultivated in Dulbecco’s Minimum Essential
Medium (DMEM) containing 10% fetal bovine serum (FBS)
and 1% penicillin/strectomycin (100/100 U/mL). Cells were
cultured as adherent monolayers and maintained at 37 °C
and 5% humidity (Freshney 2010).
SC% ¼ ½ðAcontrol À AtestÞ=AcontrolŠ  100
where Acontrol is the absorbance of the DPPH solution
(control) and Atest is the absorbance of the DPPH solution
plus a compound.
ABTS radical activity
Cell viability assay
The free radical scavenging capacity was quantified based
on a slightly modified version of the previously reported
Cell viability was determined by an MTT (3-[4,5-dime-
thylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.
Cells were harvested, counted, and transferred to 96-well
plates at 3000 cells per well to be incubated for 24 h. Upon
completion of this time, cells were treated with compounds
5a–b, 6a–b, and 9a–c at concentrations of 10, 20, 40, 60,
80, and 100 μg/mL with 1% DMSO in cell culture medium,
then again incubated for 24 h. Subsequently, microscope
images were obtained for each cell line exposed to each
compound. Cells without treatment were used as the via-
bility control, 1% DMSO as the vehicle control, and
methotrexate as the control for comparison of the respective
median lethal concentrations (LC50).
2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid)
radical cation (ABTS•+) assay (Gallardo et al. 2016; Zhang
et al. 2013). The radical cation was prepared by dissolving
the stock solution of ABTS (7 mM in distilled water) with
K2S2O8 solution (2.45 mM) at ratio of 1:1 (v/v) and then
leaving the mixture to stand in the dark at rt for 16 h. For the
evaluation of antioxidant activity, the ABTS•+ solution was
diluted with absolute ethanol until reaching the absorbency
of 0.700 0.02 at 734 nm. Taking 10 µL of the resulting
solution at different concentrations (10, 1.0 and 0.1 mM),
1 mL of ABTS solution was added and after 6 min absor-
bance was read at 734 nm. All tests were performed in tri-
plicate and the mean was centered. Finally, the percentage
inhibition of ABTS absorbance was calculated by the
aforementioned formula for the DPPH• assay. The data are
expressed as Trolox equivalent antioxidant capacity (TEAC),
developing a standard curve in a range of 0.5 to 3.5 mM.
In each well, 50 μL of a solution of MTT in culture
medium (100 μg/μL) were added and cells were incubated at
37 °C for 4 h. The culture medium was removed and 100 μL
of isopropanol was added to each well. Optical density in

Medicinal Chemistry Research
Molecular docking study
Results and discussion
Protein and ligand structures
Chemistry
The crystallographic structures of human HMGRh in
complex with a selective inhibitor (simvastatin) were
retrieved from the protein data bank (PDB) (http://www.
rcsb.org/) with the code 1HW9 (Istvan and Deisenhofer
2001). Before docking simulations were run, the coordi-
nates of the protein were set, water molecules removed,
hydrogen atoms added to the polar atoms (considering pH at
7.4), and Kollman charges assigned. The 3D structures of
simvastatin were downloaded from the Zinc database (Irwin
and Shoichet 2005). The structures of the ligands were
sketched in 2D with ChemSketch (https://www.acdlabs.
com/resources/freeware/chemsketch/) and optimized with
AM1 on Gaussian 98 software (Frisch et al. 2004) to obtain
the minimum energy conformation for docking studies.
The synthesis of the series of methyl phenoxyacetates 5a–c
and 6a–c was based on the functionalization of vanillin (8)
as the starting material (Scheme 1). Firstly, the hydroxy
group was protected by the acetate moiety through a Wil-
liamson alkylation with methyl bromoacetate under basic
conditions (Argüelles et al. 2010) to give compound 10 in
high yield. The conversion of the formyl group to the
hydroxy group was carried out by a Baeyer–Villiger rear-
rangement with mCPBA, followed by hydrolysis of the
formyl ester (Argüelles et al. 2010), to afford the phenol
derivative 11 in 72% yield. Acylation of the latter in the
ortho position of the hydroxy group was achieved by a
Lewis acid (BF3·OEt2)-catalyzed reaction (probably through
a Fries rearrangement) in the presence of the acid chloride
to furnish the corresponding acylphenoxyacetic esters 5a–c
in fairly good yields (Scheme 1). Conversion of this series
into the alkylphenoxyacetic esters 6a–c took place through
a Clemmensen reduction to provide the expected products
in good yields.
Molecular docking
The protein-ligand interaction was observed on Autodock
version 4.0 and AutoDockTools (Morris et al. 2009). All the
possible rotatable bonds, torsion angles, atomic partial
charges and non-polar hydrogens were determined for each
ligand. For HMGRh, the grid dimensions in Auto-
DockTools were 62 × 82 × 106 Å3 with points separated by
0.375 Å, centered at X = 5.564, Y = −6.681, and Z = 4.637.
The hybrid Lamarckian Genetic Algorithm was applied for
minimization, utilizing default parameters. A total of one
hundred docking runs were conducted, adopting the con-
formation with the lowest binding energy (kcal/mol) for all
further simulations. The script and files were prepared and
the docking results visualized on AutoDockTools, then
edited in Discovery 4.0 Client (Dassault Systèmes 2016).
1
The structures of all compounds were established by H
NMR, 13C NMR, and HRMS. The full assignment of proton
and carbon signals was achieved by 2D NMR experiments
(HMQC and HMBC). The selective formation of the tet-
rasubstituted benzene ring was ascertained by the two
characteristic singlets for the para aromatic protons. The
presence of the carbonyl group of the series of acyl deri-
vatives 5a–c was verified by the signal appearing at around
204 ppm in the 13C NMR spectra.
The series of nitro compounds 7a–c was readily prepared
by direct nitration of vanillin (8) to provide the known 5-
nitrovanillin (12) (Grenier et al. 2000; Kiss et al. 2010;
Scheme 1 Synthesis of phenol 10, acyl phenols 5a-c, and alkyl phenols 6a-c

Medicinal Chemistry Research
Scheme 3 Synthesis of 1,2-dihydroquinolines 9a-d
Scheme 2 Synthesis of methyl nitrophenoxyacetates 7a-c from
vanillin (8)
(tyloxapol), a non-ionic surfactant (Levine and Saltzman
2007; Zeniya and Reuben 1988; Edelstein et al. 1985). It
causes a drastic rise in serum triglycerides and cholesterol
levels by increasing HMGR activity (Kuroda et al. 1977;
Goldfard 1978). To diminish the concentration of blood
serum lipids, it is necessary to inhibit the synthesis of endo-
genous cholesterol (elevated after treatment with tyloxapol),
limit the absorption of lipoprotein lipids, and stimulate the
excretion of the latter (Korolenko et al. 2010). Many clinically
used drugs accomplish some of these functions, such as
ezetimibe (inhibitor of cholesterol absorption), fibrates
(PPAR-α agonists) and statins (HMGR inhibitors).
The series of analogs 5a–c, 6a–c, 7a, and 9a–d were
administered ip at doses of 25, 50, and 100 mg/kg to
hyperlipidemic mice, and their effect on the plasma levels
(mg/dL) of total cholesterol, and triglycerides was examined
with a procedure similar to that employed in previous studies
with related compounds (Argüelles et al. 2010). Since pre-
liminary assays showed a very modest decrease in cholesterol
by 7a–c, and considering that the nitro group can be cytotoxic
(Chung et al. 2011; Olender et al. 2018), only 7a was sub-
jected to further assessment of hypolipidemic activity.
All compounds evaluated, including the reference (sim-
vastatin), sharply depleted plasma levels of cholesterol and
triglycerides, in the range of 44–75% and 51–93%,
respectively, vs. the hyperlipidemic control animals. A
significant difference existed between the values of the
control and most treatments (Table 1). Moreover, it is worth
noticing that almost all compounds significantly decrease
triglyceride levels, since it is critical to diminish the con-
centration of the latter because in excess they induce the
formation of atheroma, leading to an imminent cardiovas-
cular disease (Veseli et al. 2017).
Rashid et al. 2018) (Scheme 2). The latter was treated with
methyl bromoacetate under basic conditions to obtain the
nitrophenoxyacetate 7a in a 67% yield for the two steps.
Baeyer–Villiger rearrangement produced the respective
formate, which was hydrolyzed under acid conditions to
deliver 7b in 61% yield. Moreover, reduction of the formyl
group of 7a with sodium borohydride resulted in the
hydroxymethyl derivative 7c in 86% yield.
The synthesis of 1,2-dihydroquinolines 9a–d was
achieved through a single-step reaction, according to the
published procedure (Gutiérrez et al. 2013) (Scheme 3).
Thus, analogs 9a–b were prepared in high yields by react-
ing m-anisidine (13a) with ketones 14a–b, under solvent-
free conditions and in the presence of magnesium bromide
as the catalyst and lithium carbonate as the base. Mean-
while, 1,2-dihydroquinoline 9c was formed by using p-
anisidine (13b) and methyl pyruvate (14c) under similar
reaction conditions. Finally, the spiro-polycyclic 1,2-dihy-
droquinoline 9d was furnished in high yield via the reaction
of 13a with cyclopentanone (14d). The physical data and
NMR spectra of the resulting 1,2-dihydroquinolines were in
agreement with the previous report (Gutiérrez et al. 2013).
Hypolipidemic activity
The in vivo hypolipidemic screening of the compounds 5a–c,
6a–c, 7a, and 9a–d was performed on male ICR mice sub-
jected to tyloxapol-induced hyperlipidemia (Silva et al. 2001;
Kourounakis et al. 2002). The possible mechanism of lipid-
lowering agents is commonly evaluated with Triton WR 1339

Medicinal Chemistry Research
Table 1 Effect of the test compounds on the serum lipid profile of
derived from isovanillin 4a–f (Mendieta et al. 2014), as well
as for the analogs to α-asarone 3a–f (Mendieta et al. 2014).
However, an inverse hypolipidemic effect was found for
other homologs structurally closer to α-asarone (Cruz et al.
2001), among which the most potent activity was displayed
by those containing an alkyl vs. acyl side chain.
Actually, the alkyl side chain would be expected to act as
an important hypocholesterolemic pharmacophore, because
of its strong lypophilic binding interaction with the HMGR
active site (Argüelles et al. 2010). Nevertheless, there was
no relation between the side chain length and the hypo-
cholesterolemic effect, evidenced by the comparable
reduction in serum cholesterol values of the homologs in the
5a–c and 6a–c series. Regardless of the possible synergetic
effect of the acyl and alkyl side chains on the test com-
pounds, the phenoxyacetic ester frame appears to play the
main role as the pharmacophore moiety (Table 1), as has
been reported (Argüelles et al. 2010; Cruz et al. 2003;
Labarrios et al. 1999; Zúñiga et al. 2005; Mendieta et al.
2014).
Interestingly, among the four tested 1,2-dihy-
droquinolines, 9a, 9b, and 9d displayed high hypocholes-
terolemic and hypotriglyceridemic activity, which is
relevant and almost unique for this kind of heterocycles
(Lagu et al. 2007; Matsuda et al. 2007; Guo et al. 2017).
The change of substituents in the A ring of the heterocyclic
frame does not seem to have a significant influence on the
hypolipidemic effect of the compounds, suggesting that the
1,2-dihydroquinoline scaffold could be the active pharma-
cophore of these potential hypolipidemic agents. Despite
the non-significant hypocholesterolemic activity of 9c, it
was able to diminish the level of triglycerides significantly.
Although a dose-response relationship cannot be estab-
lished for each member of the series, in many cases the
25 mg/kg dose (the closest to that of the control group)
produced the greatest effect (Table 1).
male ICR micea
Compound
Dose mg/ Cholesterol
Kg/day
Triglycerides
Normal diet
Simvastatin
Tyloxapol
−78.15 0.07* −80.02 0.08*
−76.38 0.19* −79.10 0.17*
17
400
100 1.03b
100 0.33c
5a + Tyloxapol 25
−67.29 6.78* −83.74 2.44*
−75.44 6.27* −93.40 1.25*
−58.5 7.35* −86.46 2.31*
−75.20 3.52* −81.42 2.51*
−57.49 7.74* −87.49 1.25*
−52.53 11.61* −88.95 1.31*
−73.43 4.70* −92.04 2.51*
−64.93 11.06* −91.4 1.79*
−74.02 5.14* −85.70 1.84*
−72.84 0.25* −20.77 0.79
−74.73 0.09* −80.59 0.11*
−65.40 0.69* −73.95 0.38*
50
100
5b + Tyloxapol 25
50
100
5c + Tyloxapol 25
50
100
6a + Tyloxapol 25
50
100
6b + Tyloxapol 25
−69.89 0.49*
−61.03 0.60*
−70.89 0.58*
4.11 1.14
4.11 2.18
50
100
−9.55 0.83
6c + Tyloxapol 25
−71.07 0.70* −76.25 0.34
−69.30 0.30* −75.72 0.19*
−72.72 0.41* −78.22 0.29*
50
100
7a + tyloxapol 25
−58.43 0.53
−59.03 0.78
−17.46 2.68
−67.13 0.40*
−66.98 0.60*
−43.01 0.37
50
100
9a + tyloxapol 25
−72.27 0.11* −76.22 0.24*
−63.16 0.62* −63.26 0.62*
−72.72 0.24* −70.47 0.44*
−68.82 0.29* −67.37 0.55*
−44.98 1.45* −34.11 0.54
−73.78 0.33* −75.25 0.22*
50
100
9b + tyloxapol 25
50
100
9c + tyloxapol 25
−51.70 1.01
28.22 7.82
−65.99 0.4
−60.13 0.46*
−66.94 0.57*
−82.79 0.20*
50
Antifungal activity
100
9d + tyloxapol 25
−72.72 0.42* −73.92 0.21*
−63.99 0.72* −61.10 0.75*
−62.01 0.39* −51.36 0.39*
Statins have been reported to produce in vitro activity
against several human pathogenic fungi, including Candida
spp. and Aspergillus spp. (Qiao et al. 2007; Cabral et al.
2013). Hence, the test compounds (except 6c and 7b) and
itraconazole (the reference) were tested in vitro against four
filamentous fungi (Aspergillus fumigatus ATCC-16907,
Trichosporon cutaneum ATCC-28592, Rhizopus oryzae
ATCC-10329, and Mucor hiemalis ATCC-8690) and three
yeast specimens (Candida albicans ATCC-10231, C. utilis
ATCC-9226, and C. tropicalis ATCC-13803) (Table 2). The
MIC values (expressed in μg/mL) of all compounds were
determined in 96-well plates with 3-(N-morpholino)propa-
nesulfonic acid (MOPS) as the buffer. Standardized micro-
biological methods developed by the CLSI (Fothergill 2012;
50
100
*Significantly different from the tyloxapol group (p < 0.05)
aExpressed as a percentage of the group treated with tyloxapol only
(mean standar error, n = 6)
b141.16 mmol/L
c1.26 mmol/L
The best hypolipidemic agents were the acylphenox-
yacetic esters 5a–c, depleting cholesterol and triglycerides
to almost the same extent as the positive control (simvas-
tatin). A similar result was observed for the analogous series

Medicinal Chemistry Research
Table 2 In vitro antifungal
activities of most synthesized
compounds (MIC, μg/mL)
Compound
Yeast fungi
Filamentous fungi
C. alb.
C. uti.
C. trop.
M. hie.
A. fum.
R. ory.
T. cut.
5a
8
8
4
16
16
16
16
16
16
16
8
16
16
16
16
16
16
16
16
16
16
16
1
16
8
8
5b
8
8
8
16
16
16
16
2
5c
2
16
8
16
16
16
4
8
6a
0.5
0.5
8
16
1
6b
16
16
8
7a
16
16
8
7c
4
16
16
8
16
16
16
16
16
8
9a
4
16
16
16
8
9b
16
4
16
16
16
4
1
9c
8
16
16
1
9d
Standarda
1
16
0.06
0.03
0.25
C. alb. Candida albicans, C. uti. Candida utilis, C. trop. Candida tropicalis, M. hie Mucor hiemalis, A. fum
Aspergillus fumigatus, R. ory Rhizopus oryzae, T. cut Trichosporon cutaneum
aItraconazole
La Regina et al. 2008; Ramírez-Villalva et al. 2017; García-
Vanegas et al. 2019) were employed. The sensitivity of the
filamentous microorganisms was determined by the micro-
dilution M38-A method (CLSI 2002; Espinel-Ingroff and
Canton 2007a), and that of the yeast fungi with the M27-A3
method (CLSI 2008; Espinel-Ingroff and Cantón 2007b;
Pfaller and Diekema 2012).
A good effect was produced against C. albicans by 6a
and 6b, though not as good as the inhibition elicited by
itraconazole. Contrarily, a lower antifungal activity was
found for 5c and 9d. None of the present derivatives were
active against the yeast fungi C. utilis and C. tropicalis.
Compared with the effect of itraconazole, only 6b and 9b
demonstrated good growth inhibition of the filamentous
fungus R. oryzae, and 5a and 7a of T. cutaneum.
No structure-activity correlation was detected among the
active compounds in regard to the growth inhibition of C.
albicans. However, homologs 6 (containing C-5 alkyl
groups) gave a greater antifungal effect than homologs 5
(with the C-5 acyl moiety). Overall, these results indicate
that the presence of the methyl phenoxyacetate and 1,2-
dihydroquinoline scaffolds seems to impact antifungal
capacity.
Fusariosis is one of the most common infections in
humans (Garnica and Nucci 2013; Guarro 2013). Fusarium
oxysporum is an invasive pathogen known to cause infec-
tions like keratitis and onychomycosis in immunocompetent
individuals, but in immunocompromised patients the
infections are frequently fatal (Garnica and Nucci 2013;
Guarro and Gené 1995; Cordoba-Guijarro et al. 2002;
Olivares et al. 2005). Although the optimal treatment
against fusariosis is unclear, the modestly effective drugs
consist of antifungal azoles and amphotericin B. In vitro
data demonstrates general resistance of Fusarium to the
available antifungal drugs with poor MIC values (Guarro
2013; Cordoba-Guijarro et al. 2002).
Consequently, the series 5–7 and 9 were tested as inhi-
bitors of the mycelial growth of the pathogen F. oxysporum,
using the agar dilution method and captan as the positive
control. Captan, a fungicide utilized in agriculture, cos-
metics and pharmaceuticals (U.S. Environmental Protection
Agency 1984) inhibits the mycelial growth of Fusarium
spp. (Türkkan and Erper 2015). According to previous
studies, it also increases cell vulnerability to oxidative stress
(Moreno-Aliaga et al. 1999; Inoue et al. 2018).
The compounds evaluated induced significant cell
growth inhibition, from 72 to 90% for 6a–c, and 70% for 9a
and 9c (Fig. 3). Interestingly, the presence of the alkyl side
chain of derivatives 6 significantly improved growth inhi-
bition, with 6b displaying the best result (90%). 1,2-Dihy-
droquinolines 9a and 9c contain either an alkyl side chain or
alkyl carboxylate.
Cytotoxic activity
The cytotoxic effect of 5a–b, 6a–b, and 9a–c was deter-
mined through the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) cell viability assay with cell
lines of human cervical cancer (HeLa), prostate cancer (DU-
145), breast cancer (MDA-231) and normal skin keratino-
cytes (HaCaT) (van Meerloo et al. 2011). Death was
observed in up to 90% of the cells exposed to these com-
pounds in concentrations higher than 40 μg/mL. In the
MDA-231 and DU-145 cell lines, there was up to 50% cell
death at 80–100 μg/mL. HeLa and HaCaT cells showed
100% viability following exposure to the non-active

Medicinal Chemistry Research
100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0
Fig. 3 Growth inhibition of F.
oxysporum produced by 5a–c,
6a–c, 7a, 7c, and 9a–d (at
10 mM) after 72 h of incubation.
Data are expressed as the mean
standard deviation (n = 12)
and are significantly different
from the control (p < 0.05)
90.0
86.9
72.1
69.9
70.2
61.6
46.4
43.3
30.3
25.5
23.5
22.8
11.1
captan
5a
5b
5c
6a
6b
6c
7a
7c
9a
9b
9c
9d
Table 3 Median lethal
Compound
Cell line/values
MDA-231
DU-145
HeLa
HaCaTa
concentration (LC50) (μg/mL)
calculated for derivatives 6a–b
in the four cell lines evaluated
6a
LC50 (IC 95%)
LC50 (IC 95%)
LC50 (IC 95%)
24 (8–40)
27 (19–35)
44 (25–62)
73 (60–85)
>100
19 (11–27)
69 (53–85)
>100
6b
–
–
Methotrexateb
>100
>100
A 95% confidential interval (CI 95%) was calculated for each LC50
aNo tumoral cell line
bThe reference drug
compounds. In cells incubated exclusively with the vehicle
(DMSO), no death occurred, and the decrease in cell growth
relative to the control was non-significant.
of α-asarone (1), which has antioxidant properties (Pages
et al. 2010). Secondly, the 1,2-dihydroquinoline ethoxyquin
is a known antioxidant feed additive (Dorey et al. 2000;
Błaszczyk and Skolimowski 2007; Błaszczyk et al. 2013; de
Koning 2002; Błaszczyk et al. 2006; Błaszczyk and Sko-
limowski 2005; Błaszczyk and Skolimowski 2006).
The antioxidant activity of the series of analogs 5a–c,
6a–c, 7a, 7c, and 9a–d was assessed by using the radical
scavenging method with the 2,2-diphenyl-1-picrylhydrazyl
radical (DPPH•) and 2,2′-azino-bis(3-ethylbenzothiazoline-
6-sulphonic acid) radical cation (ABTS•+). Antioxidant
activity was expressed as the percentage of decrease in
DPPH and ABTS (Table 4). Butylated hydroxytoluene
(BHT) was the positive control for the DPPH assay, and the
TEAC (mM) was the unit for expressing the data of the
ABTS assay (Huang et al. 2005).
Only compounds 6a, b exhibited cytotoxicity (Table 3).
Derivative 6a produced an exponential decline in cell via-
bility in all cell lines, including non-tumor cells (HaCaT).
Derivative 6b (at 100 μg/mL) caused greater cytotoxicity in
the cervical carcinoma cell line (HeLa) and HaCaT (control)
than the other cell lines. Hence the activity of these com-
pounds was non-selective, a characteristic that may be
improve by future modifications in their structure. Their
selectivity should be thoroughly studied in various cell lines
(Xiao et al. 2018) to seek specificity for tumor cells.
Antioxidant activity
Antioxidants are agents capable of protecting a biological
target against oxidative stress, DNA mutations and cell
damage, helping to prevent many human diseases. For
example, oxidative stress plays a pivotal role in the devel-
opment of diabetes, cancer, and cardiovascular disease
(Pisoschi and Pop 2015). The antioxidant potential was
herein evaluated for the synthesized compounds for two
main reasons that suggest a free radical scavenging capacity
of these derivatives. Firstly, alkylphenols 6a–c are analogs
The 6a–c and 9a–d series showed good radical scaven-
ging capacity in the DPPH assay. The IC50 values for 6a–c
were in a narrow range (0.24–0.25 mM) and for 9a–d in a
much wider range (0.12–1.52 mM). Moreover, a stronger
antioxidant effect was detected for 6a–c, 9a, and 9c than
BHT (based on the IC50 determination). For 6a–c, the
presence of an alkyl side chain apparently promoted anti-
oxidant activity (Gallardo et al. 2016), while the existence
of an acyl group on the side chain notably diminished it.

Medicinal Chemistry Research
Table 4 DPPH• and ABTS•+
cation radical scavenging
activity of 5a–c, 6a–c, 7a, 7c,
and 9a–da
Compound
DPPH Scavenging activity
ABTS Scavenging activity
% (10 mM)
(IC50 mM)
% (10 mM)
(mM Trolox/g)
5a
5b
5c
40.6 0.60
15.9 0.61
19.1 0.60
95.3 1.82
94.2 1.50
94.2 1.10
0.2 0.32
13.40 0.20
11.68 1.38
13.23 0.71
98.80 0.01
98.61 0.01
98.62 2.39
0.91 0.37
0.25 0.48
97.81 3.94
97.53 0.01
98.78 0.01
68.07 0.044
–
0.244 0.002
0.220 0.014
0.216 0.007
1.509 0.001
1.424 0.001
1.349 0.001
0.050 0.004
0.058 0.005
1.552 0.039
1.095 0.001
1.244 0.001
1.00 0.021
–
6a
6b
6c
0.241 0.009
0.255 0.016
0.244 0.012
7a
7c
0.9 0.24
9a
9b
9c
93.5 1.94
93.6 2.49
94.3 1.16
91.0 1.93
85.02 3.33
0.127 0.030
1.519 0.023
0.238 0.022
1.52 0.015
0.84 0.08
9d
BHT
aThe DPPH scavenging activity is calculated as the IC50, and the ABTS scavenging activity as the mM
Trolox equivalent (TEAC, mM). Data are expressed as the mean standard deviation (n = 6), with
significant differences considered at p < 0.05
This characteristic was not affected by the side chain length.
Regarding the 1,2-dihydroquinolines 9a–d, the presence of
a hindered group, such as the phenyl (9b) or the cycloalkyl
(9d) groups, played a negative role, slightly reducing the
DPPH• scavenging activity (IC50 for both 9b and 9d was
1.52 mM). Contrarily, the alkyl (9a) and methoxycarbonyl
(9c) groups improved the antioxidant effect, being almost
sevenfold greater with 9a (IC50 = 0.12 mM) than BHT
(IC50 = 0.84 mM). The significant activity of 9a can be
attributed to the lipophilicity of the 2-ethyl and 2-methyl
groups (Dorey et al. 2000). Interestingly, the methoxy
group attached at either the C-6 or C-7 position of the
quinoline ring did not substantially affect the DPPH• radical
scavenging capacity, although its presence seems to have
contributed considerably to the antioxidant effect (Dorey
et al. 2000; Błaszczyk and Skolimowski 2007; Błaszczyk
et al. 2013; de Koning 2002).
2014). Hence, a docking study was carried out for the most
active hypolipidemic derivatives (5b, 6a, 9a, and 9d) of the
four series herein evaluated.
Docking studies were conducted on Autodock version
4.0 and AutoDockTools (Morris et al. 2009) to explore the
binding mode of the test compounds and the reference drug
(simvastatin) at the active site of HMGRh (retrieved from
the PDB; code: 1HW9) (Istvan and Deisenhofer 2001). The
binding energy was calculated and the interaction residues
identified in each case (Table 5).
The binding modes of 5b, 6a, 9a, 9d, and simvastatin
(Fig. 4) involve the amino acid side chains of the active site
of the enzyme, including Glu559, Arg590, Asp690,
Lys691, and Asn755. In a previous study by our group
(Mendieta et al. 2014), these residues were identified and
Glu559 and Lys691 were found to be the key residues
(Andrade-Pavón et al. 2017; Andrade-Pavón et al. 2019).
The acylphenoxyacetic ester 5b had a better binding energy
(−7.47 kcal/mol) than the alkylphenoxyacetic ester 6a
(−5.93 kcal/mol). The docking data correlate with the
results of the in vivo assessment of their hypolipidemic
activity, since 5a–c decreased the levels of cholesterol and
triglycerides to a greater extent than 6a–c.
Compounds 5b, 6a, and simvastatin share key hydro-
philic interactions, such as conventional hydrogen and
carbon-hydrogen bonding to the active site of the HMGRh
enzyme, mainly with amino acid side chains Glu559,
Ser684, Asp690, and Lys735. There are some key simila-
rities in the interactions. For instance, the hydroxyl group at
C-4 of the aromatic ring of both 5b and 6a interact with the
same residue (Ser684) as the carboxylate group of sim-
vastatin. Moreover, there is an interaction with Lys735 by
Series 6a–c and 9a–d also displayed good ABTS•+
radical inhibition (Table 4) with 6a and 9a giving the best
ABTS•+ scavenging capacity (1.51 and 1.55 mM of TEAC).
There was a similar pattern for the DPPH• radical scaven-
ging activity, finding the best results for 9a and no effect for
compounds 5a–c, 7a and 7c. In summary, the phenoxyacetic
ester and 1,2-dihydroquinoline scaffolds demonstrated a
significant antioxidant activity, which was improved by the
presence of an alkyl side chain or a polar carboxylate group.
Docking of analogs 5, 6, and 9 on human HMGR
The in vitro inhibition of HMGRh has been demonstrated
for 3 and 4, showing a significant structure-binding contact
similarities with respect to simvastatin (Mendieta et al.

Medicinal Chemistry Research
Fig. 4 Predicted binding mode of simvastatin (a), 5b (b), 6a (c), 9a
(d), and 9d (e) at the active site of HMGRh (1HW9) using Autodock
version 4.0 and AutoDockTools. The 3D model portrays select amino
acid residues bound by the ligands at the active site of HMGRh. Only
the hydrophilic bonds are shown for better clarity. In the 2D model, the
following interactions are denoted with dotted lines: conventional
hydrogen bonds (green), carbon-hydrogen (yellow), alkyl (light pink),
π-anion (brown), π-cation (orange), π-sigma (purple), and π-alkyl
(dark pink). The amino acid residues are illustrated as: hydrophobic
(green), polar (cyan), positively charged (blue), and negatively
charged (red)

Medicinal Chemistry Research
Fig. 5 A1 Overlay of the
docking poses of 5b (red) and 6a
(yellow). B1 Overlay of 9a
(purple) and 9d (green). A2–B2
The binding mode is compared
between the two pairs of
compounds and
simvastatin (blue)
the hydroxyl group at the C-4 position of 6a, the carbonyl
groups of 5b and simvastatin. The three compounds also
have comparable hydrophobic interactions, such as π-sigma
with the amino acid Leu853. Due to their strongly activated
aromatic ring, compounds 5b and 6a share electrostatic
interactions of the π-cation type with the Arg590 residue.
The carbonyl oxygen atom of 5b forms an additional
hydrogen bond contact with the side chain of Lys735. This
residue may affect the enzymatic activity of 6a by inter-
acting with the alkyl side chain. The latter observations are
in agreement with the in vivo assessment of hypolipidemic
activity, which was improved by the polar-induced effect of
the carbonyl group in the chain.
The results support our hypothesis that by replacing the
C-4 methoxy group of 1 by a hydroxyl group, and main-
taining a hydrogen bond network comparable to the one
exiting in 3 and 4, the hypolipidemic effect of 5b and 6a
should be conserved.
For 1,2-dihydroquinolines 9a and 9d, on the other hand,
a very similar binding energy was found (−8.22 and
8.21 kcal/mol, respectively) (Table 5). Furthemore, 9a had
the binding energy closest to the value for simvastatin. It is
likely that the hydrocarbon portion present in both 9a, 9d
and the reference drug enhances the number of the inter-
actions within the active site as well as the overall stability
(Figs 4 and 5). In addition, 9a and 9d share hydrophilic
interactions between their polar NH and the Asp690 residue
of the enzyme, involving conventional hydrogen bonds and
carbon-hydrogen bonds. These interactions are comparable
to those observed for the acylphenoxyacetic esters 5, the
alkylphenoxyacetic esters 6, and the hydroxy group of
simvastatin. The presence of a high electron density aro-
matic ring in 9a and 9d led to π-cation interactions with
Arg590, and to π-alkyl and alkyl interactions with the side
chains of Met657 and Leu857, respectively.
Compounds 5b and 6a adopt similar orientation at the
active site of the enzyme, especially for the activated
aromatic ring (Fig. 5). The orientation of 1,2-dihy-
droquinolines 9a and 9d is also alike, mainly due to the
benzoheterocyclic frame. They adopt a conformation in
which the polar and the hydrophobic functional groups
occupy a position similar to some of the groups in sim-
vatatin with comparable polarity, although the structure
of the latter is different. Despite this difference, com-
pounds 5b, 6a, 9a, and 9d interact with most amino acids
in the active site of the enzyme that are targeted by the
reference drug. Based on the docking data, 5a–c, 6a–c,
and 9a–d are likely to have the same mechanism of action
as simvastatin.
To the best of our knowledge, there are no reports of
molecular docking of 1,2-dihydroquinolines like 9a and 9d
in the active site of the HMGRh enzyme. The interaction
energy calculated from docking was correlated with the
in vivo hypolipidemic effect.

Medicinal Chemistry Research
Conclusions
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Most members of the three series of acyl phenols (5a–c) and
alkyl phenols (6a–c) and 1,2-dihydroquinolines (9a–d)
showed a potent hypolipidemic effect, resulting in an over
67% decrease in serum cholesterol and triglycerides at the
lowest doses (25 g/kg). Using the most effective derivatives
(5b, 6a, 9a, and 9d), docking studies were carried out on
human HMGR finding that the different polar and non-polar
functional groups of the ligands exhibited strong and mul-
tiple interactions with the active site of this enzyme. Hence,
the mechanism of action of these compounds probably
involves the inhibition of HMGR. However, due to the
structural similarity of analogs 5 and 6 with fibrates, the
activation of PPARα cannot be ruled out as a competitive
mechanism participating in a reduction of the level of
triglycerides.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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