meso-Dihydroguaiaretic acid derivatives with antibacterial and antimycobacterial activity

Article abstract of DOI:10.1016/j.bmc.2017.07.047

Thirty-three meso-dihydroguaiaretic acid (meso-DGA) derivatives bearing esters, ethers, and amino-ethers were synthesized. All derivatives were tested against twelve drug-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including sensitive (H37Rv) and multidrug-resistant Mycobacterium tuberculosis strains. Among the tested compounds, four esters (7, 11, 13, and 17), one ether (23), and three amino-ethers (30, 31, and 33) exhibited moderate activity against methicillin-resistant Staphylococcus aureus, whereas 30 and 31 showed better results than levofloxacin against vancomycin-resistant Enterococcus faecium. Additionally, nineteen meso-DGA derivatives displayed moderate to potent activity against M. tuberculosis H37Rv with minimum inhibitory concentration (MIC) values ranging from 3.125 to 50 μg/mL. Seven meso-DGA derivatives bearing amino-ethers (26–31 and 33) exhibited the lowest MICs against M. tuberculosis H37Rv and G122 strains, with 31 being as potent as ethambutol (MICs of 3.125 and 6.25 μg/mL). The presence of positively charged group precursors possessing steric and hydrophobic features (e.g. N-ethylpiperidine moieties in meso-31) resulted essential to significantly increase the antimycobacterial properties of parent meso-DGA as supported by the R-group pharmacophoric and field-based QSAR analyses. To investigate the safety profile of the antimycobacterial compounds, cytotoxicity on Vero cells was determined. The amino-ether 31 exhibited a selectivity index value of 23, which indicate it was more toxic to M. tuberculosis than to mammalian cells. Therefore, 31 can be considered as a promising antitubercular agent for further studies.

Full text of DOI:10.1016/j.bmc.2017.07.047

Accepted Manuscript
meso-Dihydroguaiaretic acid derivatives with antibacterial and antimycobacte-
rial activity
Karen Reyes-Melo, Abraham García, Antonio Romo-Mancillas, Elvira Garza-
González, Verónica M. Rivas-Galindo, Luis D. Miranda, Javier Vargas-
Villarreal, Juan Manuel J. Favela-Hernández, María del Rayo Camacho-Corona
PII:
S0968-0896(17)30334-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.047
BMC 13884
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 February 2017
21 July 2017
26 July 2017
Please cite this article as: Reyes-Melo, K., García, A., Romo-Mancillas, A., Garza-González, E., Rivas-Galindo,
V.M., Miranda, L.D., Vargas-Villarreal, J., Favela-Hernández, J.M.J., del Rayo Camacho-Corona, M., meso-
Dihydroguaiaretic acid derivatives with antibacterial and antimycobacterial activity, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.07.047
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

meso-Dihydroguaiaretic acid derivatives with antibacterial and antimycobacterial
activity
a
Karen Reyes-Melo , Abraham García a,*, Antonio Romo-Mancillas b, Elvira Garza-González
c, Verónica M. Rivas-Galindo d, Luis D. Miranda e, Javier Vargas-Villarreal f, Juan Manuel J.
Favela-Hernández a, María del Rayo Camacho-Coronaa,*
a Universidad Autónoma de Nuevo León, Facultad de Ciencias Químicas. Av. Universidad
s/n, Ciudad Universitaria, C.P. 66455, San Nicolás de los Garza, Nuevo León, México.
b Facultad de Química, Universidad Autónoma de Querétaro. Centro Universitario, Cerro de
las Campanas, C.P. 76010, Querétaro, Querétaro, México.
c Universidad Autónoma de Nuevo León, Servicio de Gastroenterología y Departamento de
Patología Clínica, Hospital Universitario Dr. José Eleuterio González. Madero y Aguirre
Pequeño, Mitras Centro, C.P. 64460, Monterrey, Nuevo León, México.
d Universidad Autónoma de Nuevo León, Facultad de Medicina. Madero y Aguirre Pequeño,
Mitras Centro, C.P. 64460, Monterrey, Nuevo León, México.
e Universidad Nacional Autónoma de México, Instituto de Química. Circuito Exterior s/n,
Ciudad universitaria, Coyoacán, C.P. 04510, Ciudad de México, México.
f Instituto Mexicano del Seguro Social, Centro de Investigación Biomédica del Noreste,
División de Biología Celular y Molecular, Laboratorio de Bioquímica y Fisiología Celular.
Administración de correos No. 4, Apartado Postal 020-E, Col. Independencia, C.P. 64720,
Monterrey, Nuevo León, México.
* Corresponding authors. Tel.: +52 81 83294000x3403, fax: +52 81 835229025.
E-mail
addresses:
edgar.garciazp@uanl.edu.mx
(A.
García);
maria.camachocn@uanl.edu.mx (M.R. Camacho-Corona)
Abstract

Thirty-three meso-dihydroguaiaretic acid (meso-DGA) derivatives bearing esters, ethers, and
amino-ethers were synthesized. All derivatives were tested against twelve drug-resistant
clinical isolates of gram-positive and gram-negative bacteria, including sensitive (H37Rv)
and multidrug-resistant Mycobacterium tuberculosis strains. Among the tested compounds,
four esters (7, 11, 13, and 17), one ether (23), and three amino-ethers (30, 31, and 33)
exhibited moderate activity against methicillin-resistant Staphylococcus aureus, whereas 30
and 31 showed better results than levofloxacin against vancomycin-resistant Enterococcus
faecium. Additionally, nineteen meso-DGA derivatives displayed moderate to potent activity
against M. tuberculosis H37Rv with minimum inhibitory concentration (MIC) values ranging
from 3.125 to 50 µg/mL. Seven meso-DGA derivatives bearing amino-ethers (26-31 and 33)
exhibited the lowest MICs against M. tuberculosis H37Rv and G122 strains, with 31 being as
potent as ethambutol (MICs of 3.125 and 6.25 µg/mL). The presence of positively charged
group precursors possessing steric and hydrophobic features (e.g. N-ethylpiperidine moieties
in meso-31) resulted essential to significantly increase the antimycobacterial properties of
parent meso-DGA as supported by the R-group pharmacophoric and field-based QSAR
analyses. To investigate the safety profile of the antimycobacterial compounds, cytotoxicity
on Vero cells was determined. The amino-ether 31 exhibited a selectivity index value of 23,
which indicate it was more toxic to M. tuberculosis than to mammalian cells. Therefore, 31
can be considered as a promising antitubercular agent for further studies.
Keywords
meso-Dihydroguaiaretic acid; lignans; antimycobacterial; antibacterial; selectivity index.
1. Introduction

Lower respiratory tract infections (LRTI), diarrheal diseases, HIV/AIDS, tuberculosis, and
malaria accounted for more than 7 million deaths worldwide in 2015, with LRTI being the
third-leading cause of death with 3.2 million.1 During the last decades, communicable
diseases have become a worldwide public health problem due to the emergence of drug-
resistant bacterial strains.2-3 In 2017, the World Health Organization (WHO) released a
priority list of antibiotic-resistant bacteria that includes the carbapenem-resistant bacteria
Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, third generation
cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae, vancomycin-resistant
Enterococcus faecium, and methicillin-resistant Staphylococcus aureus.4 In addition to this
priority list, WHO established multidrug-resistant (MDR) Mycobacterium tuberculosis as a
matter of global concern for which new antibiotics are urgently needed.5 WHO’s report
estimated that 580 000 of the 10.4 million people with tuberculosis (TB) were caused by
MDR M. tuberculosis (including rifampicin-resistant M. tuberculosis) and that 250 000 of
the 1.4 million TB deaths were due to MDR-TB.5 The dearth of novel classes of antibacterial
agents worsens the panorama of antibiotic resistance and therefore widens the opportunities
for drug research and development.2, 6-8 The discovery of novel compounds with different
mechanisms of action and the chemical modifications of promising antibacterial natural
products are two strategies that might contribute to overcome the antimicrobial resistance.8-10
In the search for new antibacterial compounds, we recently reported that the chloroform
extract obtained from the aerial parts of Larrea tridentata (Zygophyllaceae) exhibited
activity against M. tuberculosis and against S. aureus.11-12 Further research allowed us to
identify meso-dihydroguaiaretic acid (meso-DGA, Figure 1) to be responsible for the
antibacterial properties with minimum inhibitory concentration (MIC) values of 50 µg/mL
and 12.5–50 µg/mL against methicillin-resistant S. aureus (MRSA) and MDR clinical
isolates of M. tuberculosis, respectively.13 Recently, the synthesis and antibacterial properties
of meso-DGA, (+)-DGA, and (–)-DGA were reported by Yamauchi et al. (Figure 1).14 The
three stereoisomers showed MIC values of 1.6 mM against Bacillus subtilis, whereas meso-
DGA resulted two times more active than (+)-DGA and (–)-DGA against S. aureus with a
MIC of 1.6 mM. Conversely, meso-DGA was four times less active than (+)-DGA and (–)-
DGA against Listeria denitrificans (MICs of 1.6, 0.4, and 0.4 mM, respectively).
Interestingly, the isomer (+)-DGA exhibited high activity against Salmonella choleraesius
with a MIC of 3.1 mM, while its stereoisomers were not active at 50 mM.14 In addition,

findings about the mechanism of action by a M. tuberculosis microarray revealed that meso-
DGA possibly targets coenzyme A transferase, which is different from those mechanism
reported for the antitubercular drugs.15 Taking into account the pharmacological potential of
meso-DGA, the present work was aimed at improving the antibacterial and antimycobacterial
properties of meso-DGA by chemical conversion of its phenols to ( )-mono- and meso-bis-
substituted esters, ethers, and amino-ethers.
H3CO
H3CO
H3CO
HO
HO
HO
OCH3
OCH3
OCH3
OH
OH
OH
meso-Dihydroguaiaretic Acid
meso-DGA
(R,R)-Dihydroguaiaretic Acid
(S,S)-Dihydroguaiaretic Acid
(–)-DGA
(+)-DGA
Figure 1. Stereoisomers of dihydroguaiaretic acid (DGA).
2. Materials and Methods
2.1. General Experimental Procedures
The meso-dihydroguaiaretic acid (meso-DGA) was isolated from L. tridentata as described
by our research group.13 Reactants and reagents were purchased from Sigma Aldrich.
Solvents such as CHCl3, CH2Cl2 and acetone were dried over appropriate drying agents
prior to use. Potassium carbonate was dried in an oven to remove moisture. Reactions were
monitored by precoated thin-layer chromatography (TLC) sheets of silica gel 60 F254
(Fluka®) and observed under UV light at 254 and 366 nm. Staining spots with ceric
ammonium sulfate was performed to follow the course of reactions. Purification of crude
reactions was carried out by column chromatography (CC) on Fluka Analytical silica gel 60
Å (230–400 mesh), using gradient elution mixtures of hexane/EtOAc, hexane/acetone, and
CH2Cl2/MeOH. Melting points were determined on a Fisher-Johns apparatus and are
uncorrected. NMR spectra were recorded on a Bruker NMR spectrometer at 400 MHz for
13
1H and 100 MHz for C using tetramethylsilane as internal standard and CDCl3 as solvent.
Chemical shifts ( ) are reported in parts per million (ppm) downfield from TMS (0.00
ppm). Coupling constants (J) are reported in Hz. Splitting patterns are described as singlet
(s), broad singlet (br s), doublet (d), triplet (t), quartet (q), multiplet (m), doublet of
doublets (dd), triplets of doublets (td). Low- and High-Resolution Mass Spectra (LRMS

and HRMS) were obtained on a JEOL JMS-AX505HA spectrometer. The 1H and 13C NMR
spectra for target compounds can be found in the Supporting Information.
2.2. General procedure for the isolation of meso-DGA
Larrea tridentata (Zigophyllaceae) leaves were collected in San Rafael, Nuevo León,
México in July 2011. A voucher specimen (No. 027502) was deposited at the Herbario
UNL Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León. Dried
leaves were ground in a blender (1 kg) and macerated six times with 6 L of
dicloromethane/methanol (1:1) for 72 h. Extracts were separated by filtration and
concentrated under vacuum to obtain a brownish residue (286 g). The crude was subjected
to silica gel (2 kg) column chromatography (CC) eluted with hexane–ethyl acetate and
ethyl acetate–methanol gradient yielding 660 fractions of 500 mL. From fractions 106-112
eluted with 80:20 hexane:ethyl acetate an off-white powder (344.2 mg) of meso-
dihydroguaiaretic acid (meso-DGA) precipitated. Fractions 81-133 (12 g), eluted with
90:10 and 80:20 hexane:ethyl acetate, were chromatographed successively on several silica
gel CC eluted with hexane-ethyl acetate gradient, yielding 789 mg of meso-DGA.
2.2.1.
meso-1,4-Bis(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethylbutane
(meso-
dihydroguaiaretic acid, meso-DGA)
Off-white powder; mp 87-88 °C; 1H NMR (CDCl3, 400 MHz) 6.83 (d, J = 8.0 Hz, 2H-Ar),
6.66 (dd, J = 8.0, 1.8 Hz, 2H-Ar), 6.62 (d, J =1.8 Hz, 2H-Ar), 5.48 (s, 2 x OH), 3.87 (s, 6H,
2 x OMe), 2.73 (dd, J = 13.0, 5.0 Hz, 2H), 2.29 (dd, J = 13.0, 9.0 Hz, 2H), 1.75 (m, 2H),
0.84 (d, J = 6.7 Hz, 6H, 2 x Me); 13C NMR (CDCl3, 101 MHz) 146.2, 143.5, 133.7, 121.6,
113.9, 111.4, 55.8, 39.1, 38.8, 16.1. NMR data are consistent with those reported in the
literature.16
2.3. General procedures for the preparation of esters.
Method A (Steglich esterification): A mixture of meso-dihydroguaiaretic acid (meso-
DGA, 0.07 mmol), N,N-dimethyl-4-aminopyridine (DMAP, 0.08 mmol), and
dicyclohexylcarbodiimide (DCC, 0.11 mmol) in anhydrous CH2Cl2 (2.5 mL) was stirred at
0 °C for 2 minutes before addition of acetic acid or butyric acid or pyrazincarboxylic acid
or benzoic acid or nicotinic acid (0.11 mmol). After stirring for 30 minutes at room
temperature, the solvent was removed under reduced pressure. The residue was dissolved in

EtOAc (2 mL) and dicyclohexylurea was removed by filtration. The organic layer was
dried over Na2SO4 and concentrated in vacuum. The residue was purified by column
chromatography (gradient elution of hexane/acetone) to afford the desired compounds (1, 2,
and 9–22). To prepare compounds 17–22, the Steglich esterification was carried out with
higher amounts of N,N-dimethyl-4-aminopyridine (0.12 mmol), dicyclohexylcarbodiimide
(0.15 mmol), and palmitic acid or stearic acid or oleic acid (0.15 mmol).
Method B: To an ice-cooled solution of meso-dihydroguaiaretic acid (meso-DGA, 0.07
mmol) in anhydrous tetrahydrofuran (2 mL), sodium hydride (0.11 mmol) or K2CO3 (0.11
mmol) was added. The reaction mixture was stirred for 5 minutes before addition of 4-
bromobenzoyl chloride or 2-nitrobenzoyl chloride or 2-pyridinecarbonyl chloride (0.11
mmol) at 0 °C. After stirring for 30 minutes at room temperature, the solvent was removed
under reduced pressure. The residue was partitioned between EtOAc (2 mL) and water (1
mL). The organic layer was separated, dried over Na2SO4, and concentrated in vacuum. The
residue was purified by column chromatography with a gradient elution of hexane/EtOAc
(or hexane/acetone), to afford the target compounds (3-8).
2.3.1.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-(4-benzoyloxy-3-
methoxyphenyl)butane (1)
1
Light yellow resin; 28% yield; H NMR (CDCl3, 400 MHz) 8.21 (d, J = 7.8 Hz, 2H-Ar),
7.62 (t, J = 7.6 Hz, 1H-Ar), 7.50 (t, J = 7.6 Hz, 2H-Ar), 7.04 (d, J = 7.9 Hz, 1H-Ar), 6.83
(d, J = 8.0 Hz, 1H-Ar), 6.77 (dd, J = 8.0, 1.4 Hz, 1H-Ar), 6.75 (d, J = 1.4 Hz, 1H-Ar), 6.67
(dd, J = 8.0, 1.6 Hz, 1H-Ar), 6.64 (d, J = 1.6 Hz, 1H-Ar), 5.47 (s, 1H, OH), 3.86 (s, 3H,
OMe), 3.78 (s, 3H, OMe), 2.81 (dd, J = 13.2, 4.6 Hz, 1H), 2.74 (dd, J = 13.4, 5.1 Hz, 1H),
2.35 (dd, J = 13.2, 9.4 Hz, 1H), 2.32 (dd, J = 13.4, 9.1 Hz, 1H), 1.80 (m, 2H), 0.87 (d, J =
6.5 Hz, 3H, Me), 0.86 (d, J = 6.5 Hz, 3H, Me); 13C NMR (CDCl3, 101 MHz) 164.9, 150.9,
146.3, 143.6, 140.9, 137.9, 133.6, 133.3, 130.2, 129.5, 128.5, 128.4, 126.6, 122.3, 121.7,
121.2, 113.9, 113.2, 111.4, 55.8, 39.3, 39.1, 39.0, 38.9, 16.3, 16.1; HRMS (ESI) m/z:
calculated for C27H31O5 [M+H]: 435.21715, found [M+H]: 435.21775.
2.3.2. meso-1,4-Bis(4-Benzoyloxy-3-methoxyphenyl)-(2R,3S)-dimethylbutane (2)
White solid; 17% yield; mp 113-115 °C; 1H NMR (CDCl3, 400 MHz) 8.21 (d, J = 7.3 Hz,
4H-Ar), 7.63 (t, J = 7.4 Hz, 2H-Ar), 7.50 (t, J = 7.6 Hz, 4H-Ar), 7.05 (d, J = 7.7 Hz, 2H-
Ar), 6.80 (dd, J = 8.6, 1.6 Hz, 2H-Ar), 6.78 (d, J = 1.6 Hz, 2H-Ar), 3.79 (s, 6H, 2 x OMe),

2.84 (dd, J = 13.4, 4.8 Hz, 2H), 2.40 (dd, J = 13.4, 9.4 Hz, 2H), 1.86 (m, 2H), 0.91 (d, J =
13
6.6 Hz, 6H, 2 x Me); C NMR (CDCl3, 101 MHz) 164.8, 151.0, 140.8, 137.9, 133.3,
130.2, 129.6, 128.4, 122.4, 121.2, 113.2, 55.9, 39.3, 39.1, 16.2; HRMS (ESI) m/z:
calculated for C34H35O6 [M+H]: 539.24336, found [M+H]: 539.24198.
2.3.3. ( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-[4-(4-bromobenzoyloxy)-3-
methoxyphenyl]butane (3)
Colorless resin; 35% yield; 1H NMR (CDCl3, 400 MHz) 8.06 (d, J = 6.8 Hz, 2H-Ar), 7.64
(d, J = 6.8 Hz, 2H-Ar), 7.03 (d, J = 7.6 Hz, 1H-Ar), 6.83 (d, J = 8.0 Hz, 1H-Ar), 6.77 (dd, J
= 8.0, 1.7 Hz, 1H-Ar), 6.74 (d, J = 1.6 Hz, 1H-Ar), 6.67 (dd, J = 8.0, 1.7 Hz, 1H-Ar), 6.64
(d, J = 1.7 Hz, 1H-Ar), 5.46 (s, 1H, HO), 3.86 (s, 3H, OMe), 3.77 (s, 3H, OMe), 2.82 (dd, J
= 13.6, 4.8 Hz, 1H), 2.74 (dd, J = 13.6, 5.2 Hz, 1H), 2.35 (dd, J = 13.4, 9.4 Hz, 1H), 2.32
(dd, J = 13.3, 9.1 Hz, 1H), 1.80 (m, 2H), 0.87 (d, J = 6.6 Hz, 3H, Me), 0.86 (d, J = 6.6 Hz,
13
3H, Me); C NMR (CDCl3, 101 MHz) 164.1, 150.8, 146.3, 143.6, 141.1, 137.6, 133.5,
131.8, 131.7, 128.5, 128.4, 122.2, 121.6, 121.2, 113.9, 113.1, 111.3, 55.8, 55.8, 39.3, 39.08,
81
39.05, 38.9, 16.3, 16.1; HRMS (ESI) m/z: calculated for C27H30 BrO5 [M+H]: 515.12561,
found [M+H]: 515.12432.
2.3.4. meso-1,4-Bis[4-(4-Bromobenzoyloxy)-3-methoxyphenyl]-(2R,3S)-dimethylbutane (4)
White solid; 11% yield; mp 147-149 °C; 1H NMR (CDCl3, 400 MHz) 8.06 (d, J = 6.8 Hz,
4H-Ar), 7.64 (d, J = 6.8 Hz, 4H-Ar), 7.04 (d, J = 8.0 Hz, 2H-Ar), 6.79 (d, J = 2.0 Hz, 2H-
Ar), 6.78 (dd, J = 8.4, 1.7 Hz, 2H-Ar), 6.77 (d, J = 1.7 Hz, 2H-Ar), 3.79 (s, 6H, 2 x OMe),
2.83 (dd, J = 13.6, 4.8 Hz, 2H), 2.34 (dd, J = 13.4, 9.4 Hz, 2H), 1.86 (m, 2H), 0.90 (d, J =
13
6.8 Hz, 6H, 2 x Me); C NMR (CDCl3, 101 MHz) 164.1, 150.8, 140.9, 137.7, 131.8,
131.7, 128.5, 128.4, 122.2, 121.2, 113.1, 55.8, 39.2, 39.1, 16.2; HRMS (ESI) m/z:
calculated for C34H33Br2O6 [M+H]: 697.06234, found [M+H]: 697.06146.
2.3.5.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-[3-methoxy-4-(2-
nitrobenzoyloxy)phenyl]butane (5)
1
Yellow resin; 26% yield; H NMR (CDCl3, 400 MHz) 8.0 (dd, J = 7.5, 1.1 Hz, 1H-Ar),
7.94 (dd, J = 7.7, 1.4 Hz, 1H-Ar), 7.72 (td, J = 7.7, 1.1 Hz, 1H-Ar), 7.68 (td, J = 7.7, 1.3
Hz, 1H-Ar), 7.14 (d, J = 8.0 Hz, 1H-Ar), 6.83 (d, J = 8.0 Hz, 1H-Ar), 6.79 (dd, J = 8.0, 2.3
Hz, 1H-Ar), 6.75 (d, J = 1.6 Hz, 1H-Ar), 6.66 (dd, J = 8.0, 2.0 Hz, 1H-Ar), 6.62 (d, J = 1.7
Hz, 1H-Ar), 5.48 (s, 1H, OH), 3.86 (s, 3H, OMe), 3.83 (s, 3H, OMe), 2.80 (dd, J =13.3,4.9

Hz, 1H), 2.74 (dd, J = 13.4, 5.1 Hz, 1H), 2.36 (dd, J = 13.9, 9.4 Hz, 1H), 2.31 (dd, J = 13.8,
9.1 Hz, 1H), 1.78 (m, 2H), 0.87 (d, J = 6.6 Hz, 3H, Me), 0.86 (d, J = 6.6 Hz, 3H, Me); 13C
NMR (CDCl3, 101 MHz) 163.6, 150.7, 148.2, 146.3, 143.6, 141.5, 137.2, 133.5, 133.0,
131.9, 130.3, 127.3, 124.0, 121.9, 121.7, 121.4, 113.9, 113.1, 111.3, 55.9, 55.8, 39.2, 39.1,
38.9, 16.3, 16.1; HRMS (ESI) m/z: calculated for C27H30NO7 [M+H]: 480.20223, found
[M+H]: 480.20249.
2.3.6. meso-1,4-Bis[3-Methoxy-4-(2-nitrobenzoyloxy)phenyl]-(2R,3S)-dimethylbutane (6)
1
Light yellow solid; 14% yield; mp 165-167 °C; H NMR (CDCl3, 400 MHz) 8.0 (dd, J =
8.0, 1.0 Hz, 2H-Ar), 7.95 (dd, J = 7.5, 1.4 Hz, 2H-Ar), 7.75 (td, J = 8.0, 1.2 Hz, 2H-Ar),
7.68 (td, J = 8.0, 1.5 Hz, 2H-Ar), 7.16 (d, J = 8.0 Hz, 2H-Ar), 6.81 (dd, J = 8.0, 1.7 Hz, 2H-
Ar), 6.76 (d, J = 1.6 Hz, 2H-Ar), 3.84 (s, 6H, 2 x OMe), 2.82 (dd, J = 13.4, 5.0 Hz, 2H),
13
2.40 (dd, J = 13.4, 9.3 Hz, 2H), 1.85 (m, 2H), 0.89 (d, J = 6.6 Hz, 6H, 2 x Me); C NMR
(CDCl3, 101 MHz) 163.6, 150.8, 148.1, 141.3, 137.2, 133.0, 131.9, 130.3, 127.4, 124.0,
122.0, 121.3, 113.1, 55.9, 39.2, 39.0, 16.2; HRMS (ESI) m/z: calculated for C34H33N2O10
[M+H]: 629.21352, found [M+H]: 629.21529.
2.3.7.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-[3-methoxy-4-
(picolinoyloxy)phenyl]butane (7)
White crystals; 46% yield; mp 72-74 °C; 1H NMR (CDCl3, 400 MHz) 8.85 (d, J = 4.0 Hz,
1H-Ar), 8.28 (d, J = 7.8 Hz, 1H-Ar), 7.91 (td, J = 7.7, 1.7 Hz, 1H-Ar), 7.55 (dd, J = 4.7, 1.0
Hz, 1H-Ar), 7.53 (dd, J = 4.7, 1.0 Hz, 1H-Ar), 7.09 (d, J = 7.9 Hz, 1H-Ar), 6.83 (d, J = 7.9
Hz, 1H-Ar), 6.77 (dd, J = 8.0, 1.6 Hz, 1H-Ar), 6.75 (d, J = 1.3 Hz, 1H-Ar), 6.67 (dd, J =
8.0, 1.6 Hz, 1H-Ar), 6.64 (d, J = 1.5 Hz, 1H-Ar), 5.51 (s, 1 H, OH), 3.86 (s, 3H, OMe),
3.78 (s, 3H, OMe), 2.82 (dd, J = 13.2, 4.6 Hz, 1H), 2.74 (dd, J = 13.4, 5.2 Hz, 1H), 2.35
(dd, J = 13.1, 9.4 Hz, 1H), 2.32 (dd, J = 13.7, 9.1 Hz, 1H), 1.81 (m, 2H), 0.87 (d, J = 6.6
13
Hz, 3H, Me), 0.86 (d, J = 6.6 Hz, 3H, Me); C NMR (CDCl3, 101 MHz) 163.3, 150.7,
150.0, 147.5, 146.3, 143.6, 141.1, 137.8, 137.1, 133.6, 127.2, 125.8, 122.2, 121.6, 121.2,
114.0, 113.1, 111.4, 55.9, 55.8, 39.3, 39.0, 38.9, 16.3, 16.1; HRMS (ESI) m/z: calculated
for C26H30NO5 [M+H]: 436.21240, found [M+H]: 436.21121.
2.3.8. meso-1,4-Bis[3-Methoxy-4-(picolinoyloxy)phenyl]-(2R,3S)-dimethylbutane (8)
White solid; 26% yield; mp 132-135 °C; 1H NMR (CDCl3, 400 MHz) 8.85 (d, J = 4.4 Hz,
2H-Ar), 8.28 (d, J = 7.8 Hz, 2H-Ar), 7.90 (td, J = 7.6, 1.7 Hz, 2H-Ar), 7.55 (dd, J = 4.7,

1.0, 1H-Ar), 7.53 (dd, J = 4.7, 1.0 Hz, 1H-Ar), 7.11 (d, J = 8.2 Hz, 2H-Ar), 6.77 (dd, J =
7.9, 1.6 Hz, 2H-Ar), 6.75 (d, J = 1.7 Hz, 2H-Ar), 3.79 (s, 6H, 2 x OMe), 2.84 (dd, J = 13.2,
4.8 Hz, 2H), 2.39 (dd, J = 13.4, 9.4 Hz, 2H), 1.87 (m, 2H), 0.91 (d, J = 6.6 Hz, 6H, 2 x
13
Me); C NMR (CDCl3, 101 MHz) 163.3, 150.7, 150.0, 147.5, 140.9, 137.9, 137.0, 127.2,
125.8, 122.2, 121.1, 113.1, 55.8, 39.3, 39.1, 16.2; HRMS (ESI) m/z: calculated for
C32H33N2O6 [M+H]: 541.23386, found [M+H]: 541.23303.
2.3.9.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-[3-methoxy-4-
(nicotinoyloxy)phenyl]butane (9)
1
White solid; 27% yield; mp 70-71 °C; H NMR (CDCl3, 400 MHz) 9.42 (d, J = 1.5 Hz,
1H-Ar), 8.87 (dd, J = 4.8, 1.6 Hz, 1H-Ar), 8.48 (dt, J = 7.9, 1.9 Hz, 1H-Ar), 7.48 (dd, J =
7.1, 4.9 Hz, 1H-Ar), 7.07 (d, J = 7.9 Hz, 1H-Ar), 6.86 (d, J = 7.9 Hz, 1H-Ar), 6.80 (dd, J =
6.7, 1.6 Hz, 1H-Ar), 6.79 (d, J = 1.5 Hz, 1H-Ar), 6.70 (dd, J = 8.0, 1.7 Hz, 1H-Ar), 6.66 (d,
J = 1.6 Hz, 1H-Ar), 5.54 (s, 1 H, OH), 3.89 (s, 3H, OMe), 3.81 (s, 3H, OMe), 2.84 (dd, J =
13.3, 4.6 Hz, 1H), 2.76 (dd, J = 13.5, 5.3 Hz, 1H), 2.38 (dd, J = 12.7, 9.0 Hz, 1H), 2.35 (dd,
J = 13.1, 9.0 Hz, 1H), 1.82 (m, 2H), 0.90 (d, J = 6.5 Hz, 3H, Me), 0.89 (d, J = 6.6 Hz, 3H,
13
Me); C NMR (CDCl3, 101 MHz) 163.6, 153.8, 151.5, 150.7, 146.3, 143.6, 141.3, 137.7,
137.4, 133.5, 125.6, 123.4, 122.1, 121.7, 121.2, 114.0, 113.2, 111.4, 55.8, 55.8, 39.3, 39.1,
39.0, 38.9, 16.3, 16.1. HRMS (ESI) m/z: calculated for C26H30NO5 [M+H] 436.21240, found
[M+H]: 436.21272.
2.3.10. meso-1,4-Bis[3-Methoxy-4-(nicotinoyloxy)phenyl]-(2R,3S)-dimethylbutane (10)
1
White solid; 12% yield; mp 78-79 °C; H NMR (CDCl3, 400 MHz) 9.40 (d, J = 1.6 Hz,
2H-Ar), 8.85 (dd, J = 4.8, 1.6 Hz, 2H-Ar), 8.46 (dt, J = 8.0, 1.9 Hz, 2H-Ar), 7.46 (dd, J =
7.1, 4.9 Hz, 2H-Ar), 7.07 (d, J = 8.0 Hz, 2H-Ar), 6.81 (d, J = 1.6 Hz, 2H-Ar), 6.79 (s, 2H-
Ar), 3.80 (s, 6H, 2 x OMe), 2.84 (dd, J = 13.4, 4.8 Hz, 2H), 2.41 (dd, J = 13.4, 9.4 Hz, 2H),
1.87 (m, 2H), 0.91 (d, J = 6.6 Hz, 6H, 2 x Me); 13C NMR (CDCl3, 101 MHz) 163.5, 153.8,
151.5, 150.8, 141.1, 137.6, 137.5, 125.6, 123.4, 122.2, 121.2, 113.2, 55.8, 39.3, 39.1, 16.2;
HRMS (ESI) m/z: calculated for C32H33N2O6 [M+H]: 541.23386, found [M+H]: 541.23293.
2.3.11. ( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-[3-methoxy-4-(pyrazine-2-
carbonyloxy)phenyl]butane (11)
1
White solid; 28% yield; mp 95-97 °C; H NMR (CDCl3, 400 MHz) 9.46 (d, J = 1.4 Hz,
1H-Ar), 8.83 (dd, J = 6.8, 2.4, 2H-Ar), 7.10 (d, J = 8.0 Hz, 1H-Ar), 6.84 (d, J = 7.9 Hz,

1H-Ar), 6.79 (dd, J = 8.0, 1.6 Hz, 1H-Ar), 6.76 (d, J = 1.6 Hz, 2H-Ar), 6.67 (dd, J = 8.0,
2.0 Hz, 1H-Ar), 5.50 (s, 1H, OH), 3.87 (s, 1H, OMe), 3.79 (s, 3H, OMe), 2.82 (dd, J =
13.2, 4.6 Hz, 1H), 2.74 (dd, J = 13.5, 5.4 Hz, 1H), 2.36 (dd, J = 13.1, 9.4 Hz, 1H), 2.32 (dd,
13
J = 13.6, 9.1 Hz, 1H), 1.80 (m, 2H), 0.87 (d, J = 6.0 Hz, 6H, 2 x Me); C NMR (CDCl3,
101 MHz) 162.1, 150.5, 147.9, 146.8, 146.3, 144.6, 143.6, 143.1, 141.6, 137.3, 133.5,
122.0, 121.7, 121.2, 114.0, 113.1, 111.4, 55.8, 55.8, 39.3, 39.1, 39.0, 38.9, 16.4, 16.1;
HRMS (ESI) m/z: calculated for C25H29N2O5 [M+H]: 437.20765, found [M+H]: 437.20802.
2.3.12.
meso-1,4-Bis[3-Methoxy-4-(pyrazine-2-carbonyloxy)phenyl]-(2R,3S)-
dimethylbutane (12)
White solid; 20% yield; mp 107-109 °C; 1H NMR (CDCl3, 400 MHz) 9.46 (d, J = 1.2 Hz,
2H-Ar), 8.82 (dd, J = 6.9, 2.4 Hz, 4H-Ar), 7.12 (d, J = 8.4 Hz, 2H-Ar), 6.80 (dd, J = 8, 1.7
Hz, 2H-Ar), 6.79 (d, J = 1.7 Hz, 2H-Ar), 3.81 (s, 6H, 2 x OMe), 2.84 (dd, J = 13.3, 4.8 Hz,
13
2H), 2.41 (dd, J = 13.4, 9.3 Hz, 2H), 1.88 (m, 2H), 0.92 (d, J = 6.6 Hz, 6H, 2 x Me); C
NMR (CDCl3, 101 MHz) 162.0, 150.6, 147.9, 146.8, 144.6, 143.1, 141.3, 137.4, 122.1,
121.2, 113.1, 55.8, 39.3, 39.1, 16.2; HRMS (ESI) m/z: calculated for C30H31N4O6 [M+H]:
543.22436, found [M+H]: 543.22356.
2.3.13.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-(4-acetoxy-3-
methoxyphenyl)butane (13)
1
Light yellow resin; 50% yield; H NMR (CDCl3, 400 MHz) 6.92 (d, J = 7.9 Hz, 1H-Ar),
6.82 (d, J = 7.9 Hz, 1H-Ar), 6.71 (dd, J = 7.9, 1.5 Hz, 1H-Ar), 6.69 (d, J = 1.6 HZ, 1H-Ar),
6.65 (dd, J = 7.9, 1.6 Hz, 1H-Ar), 6.62 (d, J = 1.5 Hz, 1H-Ar), 3.85 (s, 3H, OMe), 3.79 (s,
3H, OMe), 2.77 (dd, J = 13.3, 4.6 Hz, 1H), 2.72 (dd, J = 13.4, 5.2 Hz, 1H), 2.33 (dd, J =
13.5, 9.0 Hz, 1H), 2.30 (s, 3H, MeCO2), 2.28 (m, 1H), 1.76 (m, 2H), 0.85 (d, J = 6.6 Hz,
6H, 2 x Me); 13C NMR (CDCl3, 101 MHz) 169.2, 150.7, 146.3, 143.6, 140.8, 137.7, 133.6,
122.2, 121.6, 121.1, 113.9, 113.1, 111.4, 55.8, 55.8, 39.3, 39.0, 39.0, 38.9, 20.7, 16.3, 16.1;
HRMS (ESI) m/z: calculated for C22H29O5 [M+H]: 373.20150, found [M+H]: 373.20171.
2.3.14. meso-1,4-Bis(4-Acetoxy-3-methoxyphenyl)-(2R,3S)-dimethylbutane (14)
1
Light yellow solid; 21% yield; mp 105-106 °C; H NMR (CDCl3, 400 MHz) 6.92 (d, J =
8.8 Hz, 2H-Ar), 6.72 (dd, J = 8.0, 1.6 Hz, 2H-Ar), 6.71 (d, J = 1.8 Hz, 2H-Ar), 3.79 (s, 6H,
2 x OMe), 2.78 (dd, J = 13.4, 4.8 Hz, 2H), 2.34 (dd, J = 13.6, 9.2 Hz, 2H), 2.30 (s, 6H, 2 x
13
MeCO2), 1.81 (m, 2H), 0.86 (d, J = 6.8 Hz, 6H, 2 x Me); C NMR (CDCl3, 101 MHz)

169.2, 150.7, 140.7, 137.7, 122.2, 121.1, 113.0, 55.8, 39.2, 39.0, 20.7, 16.2; HRMS (ESI)
m/z: calculated for C24H31O6 [M+H]: 415.21206, found [M+H]: 415.21305. NMR data are
consistent with those reported in the literature.17
2.3.15.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-(4-butyryloxy-3-
methoxyphenyl)butane (15)
Beige solid; 33% yield; 1H NMR (CDCl3, 400 MHz) 6.93 (d, J = 7.9 Hz, 1H-Ar), 6.84 (d,
J = 8.0 Hz, 1H-Ar), 6.73 (dd, J = 8.0, 1.7 Hz, 1H-Ar), 6.72 (d, J =1.5 Hz, 1H-Ar), 6.68
(dd, J = 8.0, 1.6 Hz, 1H-Ar), 6.64 (d, J = 1.7 Hz, 1H-Ar), 5.47 (s, 1H, OH), 3.88 (s, 3H,
OMe), 3.80 (s, 3H, OMe), 2.80 (dd, J = 13.7, 4.9 Hz, 1H), 2.75 (dd, J = 13.6, 5.3 Hz, 1H),
2.57 (t, J = 7.4 Hz, 2H, CH2-CO2R), 2.34 (dd, J = 13.4, 9.4 Hz, 2H), 1.82 (m, 4H), 1.07 (t, J
= 7.4 Hz, 3H, Me), 0.87 (d, J = 6.6 Hz, 6H, 2 x Me).
2.3.16. meso-1,4-Bis(4-Butyryloxy-3-methoxyphenyl)-(2R,3S)-dimethylbutane (16)
Beige solid; 20% yield; 1H NMR (CDCl3, 400 MHz) 6.93 (d, J = 8.1 Hz, 2H-Ar), 6.75 (d,
J = 1.8 Hz, 1H-Ar), 6.73 (br s, 3H-Ar), 3.80 (s, 6H, 2 x OMe), 2.80 (dd, J = 13.4, 4.8 Hz,
2H), 2.57 (t, J = 7.3 Hz, 4H, 2 x CH2-CO2R), 2.37 (dd, J = 13.5, 9.3 Hz, 2H), 1.82 (m, 6H),
13
1.07 (t, J = 7.4 Hz, 6H, 2 x Me), 0.88 (d, J = 6.6 Hz, 6H, 2 x Me); C NMR (CDCl3, 101
MHz) 171.8, 150.8, 140.5, 137.8, 122.3, 121.1, 113.0, 55.8, 39.2, 39.0, 35.9, 18.6, 16.2,
13.5.
2.3.17.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-(3-methoxy-4-
palmitoyloxyphenyl)butane (17)
White solid; 26% yield; mp 103-105 °C; 1H NMR (CDCl3, 400 MHz) 6.90 (d, J = 7.9 Hz,
1H-Ar), 6.82 (d, J = 8.0 Hz, 1H-Ar), 6.71 (dd, J = 8.0, 1.7 Hz, 1H-Ar), 6.69 (d, J = 1.7 Hz,
1H-Ar), 6.66 (dd, J = 8.0, 1.8 Hz, 1H-Ar), 6.62 (d, J = 1.7 Hz, 1H-Ar), 3.85 (s, 3H, OMe),
3.78 (s, 3H, OMe), 2.77 (dd, J = 13.3, 4.7 Hz, 1H), 2.72 (dd, J = 13.4, 5.1 Hz, 1H), 2.56 (t,
J = 7.6 Hz, 2H), 2.31 (dd, J = 13.5, 9.3 Hz, 2H), 1.76 (m, 2H), 1.63 (m, 2H), 1.41 (m, 2H),
1.26 (br s, 22H), 0.88 (t, J = 7.1 Hz, 3H, Me), 0.87 (d, J = 6.6 Hz, 3H, Me), 0.84 (d, J = 6.6
13
Hz, 3H, Me); C NMR (CDCl3, 101 MHz) 172.0, 150.7, 146.3, 143.5, 140.7, 137.7,
133.6, 122.2, 121.6, 121.1, 113.9, 113.0, 111.4, 55.8, 55.7, 39.3, 39.0, 38.9, 34.0, 31.9,
30.9, 29.7, 29.6, 29.5, 29.4, 29.3, 29.3, 29.0, 25.0, 24.7, 22.7, 16.3, 16.1, 14.1; HRMS (ESI)
m/z: calculated for C36H57O5 [M+H]: 569.42060, found [M+H]: 569.42035.
2.3.18. meso-1,4-Bis(3-Methoxy-4-palmitoyloxyphenyl)-(2R,3S)-dimethylbutane (18)

1
White solid; 22% yield; mp 48-49 °C; H NMR (CDCl3, 400 MHz) 6.91 (d, J = 8.0 Hz,
2H-Ar), 6.71 (dd, J = 8.0, 1.7 Hz, 2H-Ar), 6.70 (d, J = 1.7 Hz, 2H-Ar), 3.78 (s, 6H, 2 x
OMe), 2.78 (dd, J = 13.4, 4.8 Hz, 2H), 2.56 (t, J = 7.4 Hz, 4H), 2.34 (dd, J = 13.4, 9.4 Hz,
2H), 1.82 (m, 2H), 1.75 (m, 4H), 1.41 (m, 4H), 1.28 (br s, 44H), 0.88 (t, J = 6.7 Hz, 6H, 2 x
Me), 0.86 (d, J = 6.5 Hz, 6H, 2 x Me); 13C NMR (CDCl3, 101 MHz) 172.0, 150.8, 140.5,
137.8, 122.3, 121.1, 113.0, 55.8, 39.2, 39.1, 34.0, 31.9, 29.7, 29.6, 29.6, 29.5, 29.3, 29.3,
29.0, 25.0, 22.7, 16.2, 14.1; HRMS (ESI) m/z: calculated for C52H87O6 [M+H]: 807.65026,
found [M+H]: 807.64797.
2.3.19.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-(3-methoxy-4-
stearoyloxyphenyl)butane (19)
White resin; 22% yield; 1H NMR (CDCl3, 400 MHz) 6.90 (d, J = 7.9 Hz, 1H-Ar), 6.82 (d,
J = 8.0 Hz, 1H-Ar), 6.72 (dd, J = 8.0, 1.6 Hz, 1H-Ar), 6.69 (d, J = 1.6 Hz, 1H-Ar), 6.66
(dd, J = 8.0, 1.8 Hz, 1H-Ar), 6.62 (d, J = 1.8 Hz, 1H-Ar), 5.46 (s, 1H, OH), 3.85 (s, 3H,
OMe), 3.78 (s, 3H, OMe), 2.77 (dd, J = 13.3, 4.6 Hz, 1H), 2.72 (dd, J = 13.4, 5.1 Hz, 1H),
2.56 (t, J = 7.4 Hz, 2H), 2.31 (dd, J = 13.4, 9.2 Hz, 2H), 1.76 (m, 4H), 1.41 (m, 2H), 1.26
(br s, 26H), 0.88 (d, J = 6.6 Hz, 3H, Me), 0.86 (t, J = 7.2 Hz, 3H, Me), 0.84 (d, J = 6.6 Hz,
13
3H, Me); C NMR (CDCl3, 101 MHz) 172.0, 150.7, 146.3, 143.6, 140.7, 137.8, 133.6,
122.2, 121.6, 121.1, 113.9, 113.0, 111.3, 55.8, 55.7, 39.3, 39.0, 38.9, 34.0, 31.9, 30.9, 29.7,
29.6, 29.6, 29.5, 29.3, 29.3, 29.0, 26.4, 25.0, 22.7, 16.3, 16.1, 14.1; HRMS (ESI) m/z:
calculated for C38H61O5 [M+H]: 597.45190, found [M+H]: 597.45059.
2.3.20. meso-1,4-Bis(3-Methoxy-4-stearoyloxyphenyl)-(2R,3S)-dimethylbutane (20)
1
White solid; 48% yield; mp 53-55 °C; H NMR (CDCl3, 400 MHz) 6.91 (d, J = 7.9 Hz,
2H-Ar), 6.71 (dd, J = 8.0, 1.6 Hz, 2H-Ar), 6.70 (d, J = 1.6 Hz, 2H-Ar), 3.78 (s, 6H, 2 x
OMe), 2.78 (dd, J = 13.4, 4.8 Hz, 2H), 2.56 (t, J = 7.6 Hz, 4H), 2.34 (dd, J = 13.4, 9.4 Hz,
2H), 1.81 (m, 2H), 1.75 (m, 4H), 1.41 (m, 4H), 1.26 (br s, 52H), 0.87 (t, J = 6.7 Hz, 6H, 2 x
Me), 0.86 (d, J = 6.6 Hz, 6H, 2 x Me); 13C NMR (CDCl3, 101 MHz) 172.0, 150.8, 140.5,
137.8, 122.3, 121.1, 113.0, 55.8, 39.2, 39.1, 34.0, 31.9, 29.7, 29.6, 29.6, 29.5, 29.3, 29.3,
29.0, 25.1, 22.7, 16.2, 14.1; HRMS (ESI) m/z: calculated for C56H95O6 [M+H]: 863.71286,
found [M+H]: 863.71237.
2.3.21.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-(3-methoxy-4-
oleoyloxyphenyl)butane (21)

Colorless resin; 20% yield; 1H NMR (CDCl3, 400 MHz) 6.90 (d, J = 7.9 Hz, 1H-Ar), 6.82
(d, J = 8.0 Hz, 1H-Ar), 6.71 (dd, J = 7.8, 1.6 Hz, 1H-Ar), 6.69 (d, J = 1.4 Hz, 1H-Ar), 6.66
(dd, J = 8.0, 1.7 Hz, 1H-Ar), 6.62 (d, J = 1.7 Hz, 1H-Ar), 5.46 (br s, 1H, OH), 5.35 (td, J =
5.52, 1.8 Hz, 2H), 3.85 (s, 3H, OMe), 3.78 (s, 3H, OMe), 2.77 (dd, J = 13.2, 4.8 Hz, 1H),
2.72 (dd, J = 13.5, 4.8 Hz, 1H), 2.56 (t, J = 7.4 Hz, 2H), 2.31 (dd, J = 13.4, 9.3 Hz, 2H),
2.02 (m, 4H), 1.76 (m, 4H), 1.41 (m, 2H), 1.31 (br s, 8H), 1.26 (br s, 10H), 0.88 (d, J = 6.6
Hz, 3H, Me), 0.86 (t, J = 7.3 Hz, 3H, Me), 0.84 (d, J = 6.6 Hz, 3H, Me); 13C NMR (CDCl3,
101 MHz) 172.0, 150.7, 146.3, 143.6, 140.7, 137.7, 133.6, 130.0, 129.7, 122.2, 121.6,
121.1, 113.9, 113.0, 111.3, 55.8, 55.7, 39.3, 39.0, 38.9, 34.0, 31.9, 29.7, 29.7, 29.5, 29.3,
29.2, 29.1, 29.0, 27.2, 27.1, 25.0, 22.6, 16.3, 16.1, 14.1; HRMS (ESI) m/z: calculated for
C38H59O5 [M+H]: 595.43625, found [M+H]: 595.43631.
2.3.22. meso-1,4-Bis(3-Methoxy-4-oleoyloxyphenyl)-(2R,3S)-dimethylbutane (22)
Colorless resin; 17% yield; 1H NMR (CDCl3, 400 MHz) 6.91 (d, J = 8.2 Hz, 2H-Ar), 6.71
(dd, J = 8.0, 1.2 Hz, 2H-Ar), 6.70 (d, J = 1.24 Hz, 2H-Ar), 5.35 (m, 4H), 3.78 (s, 6H, 2 x
OMe), 2.78 (dd, J = 13.4, 4.8, 2H), 2.56 (t, J = 7.6 Hz, 4H), 2.34 (dd, J = 13.4, 9.0 Hz,
2H), 2.01 (m, 8H), 1.76 (m, 6H), 1.42 (m, 4H), 1.33 (br s, 16H), 1.26 (br s, 22H), 0.88 (d, J
13
= 6.6 Hz, 6H, 2 x Me), 0.87 (t, J = 6.8 Hz, 6H, 2 x Me); C NMR (CDCl3, 101 MHz)
172.0, 150.7, 140.5, 137.8, 130.0, 129.7, 122.3, 121.1, 113.0, 55.8, 39.2, 39.1, 34.0, 31.9,
29.7, 29.7, 29.5, 29.3, 29.3, 29.2, 29.1, 29.0, 27.2, 27.2, 25.0, 22.6, 16.2, 14.1; HRMS (ESI)
m/z: calculated for C56H91O6 [M+H]: 859.68156, found [M+H]: 859.68129.
2.4. General procedure for the preparation of ethers
To a mixture of meso-dihydroguaiaretic acid (meso-DGA, 0.07 mmol) and K2CO3 (0.11
mmol) in anhydrous N,N-dimethylformamide (2 mL), iodomethane (0.11 mmol) was
added. After stirring for 3 hours at room temperature, the reaction mixture was
concentrated under reduced pressure. The residue was purified by column chromatography
with a gradient elution of hexane/acetone, to afford the desired compounds (23-24).
2.4.1.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-(3,4-
dimethoxyphenyl)butane (23)
Colorless resin; 50% yield; 1H NMR (CDCl3, 400 MHz) 6.82 (d, J = 8.0 Hz, 1H-Ar), 6.78
(d, J = 8.1 Hz, 1H-Ar), 6.69 (dd, J = 8.2, 1.8 Hz, 1H-Ar), 6.66 (dd, J = 8.0, 1.8 Hz, 1H-Ar),

6.63 (d, J = 1.8 Hz, 1H-Ar), 6.62 (d, J = 1.8 Hz, 1H-Ar), 5.47 (s, 1H, OH), 3.86 (s, 3H,
OMe), 3.85 (s, 3H, OMe), 3.84 (s, 3H, OMe), 2.75 (dd, J = 13.1, 5.5 Hz, 1H), 2.72 (dd, J =
13.1, 5.7 Hz, 1H), 2.30 (dd, J = 13.1, 9.1 Hz, 1H), 2.27 (dd, J = 13.1, 9.3 Hz, 1H), 1.76 (m,
13
2H), 0.85 (d, J = 6.6 Hz, 3H, Me), 0.84 (d, J = 6.6 Hz, 3H, Me); C NMR (CDCl3, 101
MHz) 148.7, 147.0, 146.3, 143.5, 134.5, 133.7, 121.7, 120.9, 113.9, 112.2, 111.4, 111.0,
55.9, 55.8, 55.8, 39.2, 39.1, 38.9, 38.7, 16.2, 16.1; HRMS (ESI) m/z: calculated for C21H29O4
[M+H]: 345.20658, found [M+H]: 345.20656. NMR data are consistent with those reported
in the literature.16
2.4.2. meso-1,4-Bis(3,4-Dimethoxyphenyl)-(2R,3S)-dimethylbutane (24)
White solid; 21% yield; mp 104-106 °C; 1H NMR (CDCl3, 400 MHz) 6.79 (d, J = 8.0 Hz,
2H-Ar), 6.70 (dd, J = 8.0, 2.0 Hz, 2H-Ar), 6.65 (d, J = 2 Hz, 2H-Ar), 3.86 (s, 6H, 2 x
OMe), 3.85 (s, 6H, 2 x OMe), 2.76 (dd, J = 13.4, 5.0 Hz, 2H), 2.31 (dd, J = 13.5, 9.2 Hz,
13
2H), 1.77 (m, 2H), 0.85 (d, J = 6.6 Hz, 6H, 2 x Me); C NMR (CDCl3, 101 MHz) 148.7,
147.0, 134.4, 120.9, 112.2, 112.2, 110.9, 55.9, 55.8, 39.1, 38.8, 16.2; HRMS (ESI) m/z:
calculated for C22H31O4 [M+H]: 359.22223, found [M+H]: 359.22263. NMR data are
consistent with those reported in the literature.16
2.5. General procedure for the preparation of amino-ethers
To a mixture of meso-dihydroguaiaretic acid (meso-DGA, 0.07 mmol) and K2CO3 (0.11
mmol) in anhydrous acetone (2 mL), 2-chloro-N-N-dimethylethylamine hydrochloride or 2-
chloro-N-N-diethylethylamine hydrochloride or 4-(2-chloroethyl)morpholine hydrochloride
or 1-(2-chloroethyl)pyrrolidine hydrochloride or 1-(2-chloroethyl)piperidine hydrochloride
(0.11 mmol) was added. The reaction mixture was refluxed under vigorous stirring for 24 h.
The solvent was removed under reduced pressure. The residue was dissolved in EtOAc (2
mL) and washed with water (1 mL) three times. The organic layer was dried over Na2SO4
and concentrated in vacuum. The residue was purified by column chromatography (gradient
elution of CH2Cl2/MeOH) to afford the desired compounds (25-33).
2.5.1.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-[4-(2-
(dimetilamino)ethoxy)-3-methoxyphenyl]butane (25)
1
Light yellow resin; 26% yield; H NMR (CDCl3, 400 MHz) 6.82 (d, J = 7.8 Hz, 1H-Ar),
6.80 (d, J = 6.9 Hz, 1H-Ar), 6.74 (d, J = 1.8 Hz, 1H-Ar), 6.66 (d, J = 2.0 Hz, 1H-Ar), 6.64
(dd, J = 7.9, 1.8 Hz, 2H-Ar), 4.16 (t, J = 5.8 Hz, 2H, CH2O), 3.86 (s, 3H, OMe), 3.82 (s,

3H, OMe), 2.93 (t, J = 5.6 Hz, 2H, CH2N), 2.75 (dd, J = 13.0, 4.8 Hz, 1H), 2.72 (dd, J =
13.1, 5.3 Hz, 1H), 2.48 (s, 6H, 2 x NCH3), 2.31 (dd, J = 13.2, 9.2 Hz, 1H), 2.26 (dd, J =
13.1, 9.3 Hz, 1H), 1.76 (m, 2H), 0.85 (d, J = 6.3 Hz, 3H, Me), 0.83 (d, J = 6.3 Hz, 3H,
13
Me); C NMR (CDCl3, 101 MHz) 149.3, 146.3, 145.8, 143.5, 135.5, 133.7, 121.6, 121.0,
113.9, 113.7, 112.7, 111.4, 66.5, 57.7, 55.8, 55.7, 45.2, 39.3, 39.0, 38.7, 16.2, 16.1; HRMS
(ESI) m/z: calculated for C24H36NO4 [M+H]: 402.26443, found [M+H]: 402.26413.
2.5.2. meso-1,4-Bis[4-(2-(Dimetilamino)ethoxy)-3-methoxyphenyl]-(2R,3S)-dimethylbutane
(26)
White solid; 22% yield; mp 156-157 °C; 1H NMR (CDCl3, 400 MHz) 6.80 (d, J = 8.0 Hz,
2H-Ar), 6.66 (dd, J = 8.0, 1.8 Hz, 2H-Ar), 6.64 (d, J = 1.6 Hz, 2H-Ar), 4.08 (t, J = 6.2 Hz,
4H, 2 x CH2O), 3.83 (s, 6H, 2 x OMe), 2.77 (t, J = 6.0 Hz, 4H), 2.74 (m, 2H), 2.34 (s, 12H,
4 x NCH3), 2.30 (m, 2H), 1.76 (m, 2H), 0.84 (d, J = 6.6 Hz, 6H, 2 x Me); 13C NMR (CDCl3,
101 MHz) 149.3, 146.3, 135.0, 121.0, 113.3, 112.7, 67.1, 58.2, 55.8, 45.9, 39.2, 38.8,
16.2; HRMS (ESI) m/z: calculated for C28H45N2O4 [M+H]: 473.33793, found [M+H]:
473.33768.
2.5.3. meso-1,4-Bis[4-(2-(Dietilamino)ethoxy)-3-methoxyphenyl]-(2R,3S)-dimethylbutane
(27)
Colorless resin; 16% yield; 1H NMR (CDCl3, 400 MHz) 6.83 (d, J = 8.1 Hz, 2H-Ar), 6.70
(dd, J = 8.1, 1.9 Hz, 2H-Ar), 6.67 (d, J = 1.8 Hz, 2H-Ar), 4.14 (t, J = 6.6 Hz, 4H, 2 x
CH2O) 3.85 (s, 6H, 2 x OMe), 3.02 (t, J = 6.6 Hz, 4H, 2 x CH2N), 2.76 (m, 10H), 2.31 (dd,
J = 13.4, 9.3 Hz, 2H), 1.78 (m, 2H), 1.13 (t, J = 7.2 Hz, 12H, 4 x Me), 0.86 (d, J = 6.6 Hz,
6 H, 2 x Me); 13C NMR (CDCl3, 101 MHz) 149.2, 146.2, 135.0, 121.0, 113.2, 112.8, 67.0,
55.8, 51.5, 47.7, 39.2, 38.8, 16.2, 11.4; HRMS (ESI) m/z: calculated for C32H53N2O4 [M+H]:
529.40053, found [M+H]: 529.40194.
2.5.4.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-[3-methoxy-4-(2-
(pyrrolidin-1-yl)ethoxy)phenyl]butane (28)
1
Light yellow resin; 28% yield; H NMR (CDCl3, 400 MHz) 6.82 (d, J = 8.0 Hz, 1H-Ar),
6.81 (d, J = 8.0 Hz, 1H-Ar), 6.67 (d, J = 1.7 Hz, 1H-Ar), 6.66 (d, J = 1.8 Hz, 1H-Ar), 6.63
(dd, J = 8.0, 1.8 Hz, 2H-Ar), 4.15 (t, J = 6.3 Hz, 2H, CH2O), 3.86 (s, 3H, OMe), 3.82 (s,
3H, OMe), 3.0 (t, J = 6.2 Hz, 2H, CH2N), 2.74 (dd, J = 13.7, 5.1 Hz, 2H), 2.71 (t, J = 5.2
Hz, 4H), 2.30 (dd, J = 13.6, 9.1 Hz, 1H), 2.26 (dd, J = 13.5, 9.2 Hz, 1H), 1.84 (br s, 4H),

1.75 (m, 2H), 0.84 (d, J = 6.5 Hz, 3H, Me), 0.83 (d, J = 6.5 Hz, 3H, Me); 13C NMR (CDCl3,
101 MHz) 149.2, 146.3, 146.2, 143.5, 135.1, 133.7, 121.6, 121.0, 114.0, 113.4, 112.7,
111.4, 67.7, 55.8, 55.8, 54.8, 54.5, 50.8, 39.3, 39.0, 39.0, 38.7, 29.6, 23.4, 16.2, 16.1;
HRMS (ESI) m/z: calculated for C26H38NO4 [M+H]: 428.28008, found [M+H]: 428.27994.
2.5.5.
meso-1,4-Bis[3-methoxy-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl]-(2R,3S)-
dimethylbutane (29)
Colorless resin; 17% yield; 1H NMR (CDCl3, 400 MHz) 6.81 (d, J = 8.0 Hz, 2H-Ar), 6.67
(dd, J = 8.0, 1.8 Hz, 2H-Ar), 6.65 (d, J = 1.8 Hz, 2H-Ar), 4.14 (t, J = 6.4 Hz, 4H, 2 x
CH2O), 3.83 (s, 6H, 2 x OMe), 2.96 (t, J = 6.4 Hz, 4H, 2 x CH2N), 2.74 (dd, J = 13.4, 5.0
Hz, 2H), 2.66 (t, J = 5.4 Hz, 8H, 4 x CH2N), 2.29 (dd, J = 13.4, 9.3 Hz, 2H), 1.82 (br s,
13
8H), 1.77 (m, 2H), 0.84 (d, J = 6.6 Hz, 6H, 2 x Me); C NMR (CDCl3, 101 MHz) 149.2,
146.3, 135.0, 121.0, 113.3, 112.8, 67.9, 55.9, 54.9, 54.5, 39.2, 38.8, 23.5, 16.2; HRMS
(ESI) m/z: calculated for C32H49N2O4 [M+H]: 525.36923, found [M+H]: 525.36929.
2.5.6. ( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-[3-methoxy-4-(2-(piperidin-
1-yl)ethoxy)phenyl]butane (30)
1
Light yellow resin; 28% yield; H NMR (CDCl3, 400 MHz) 6.82 (d, J = 7.9 Hz, 1H-Ar),
6.79 (d, J = 8.1 Hz, 1H-Ar), 6.67 (d, J = 1.8 Hz, 1H-Ar), 6.66 (d, J = 1.9 Hz, 1H-Ar), 6.63
(dd, J = 8.0, 1.7 Hz, 2H-Ar), 4.13 (t, J = 6.3 Hz, 2H, CH2O), 3.85 (s, 3H, OMe), 3.82 (s,
3H, OMe), 2.84 (t, J = 6.3 Hz, 2H, CH2N), 2.75 (dd, J = 13.3, 4.6 Hz, 1H), 2.71 (dd, J =
13.3, 9.6 Hz, 1H), 2.55 (br s, 4H, 2 x CH2N), 2.30 (dd, J = 13.6, 9.1 Hz, 1H), 2.26 (dd, J =
13.0, 9.0 Hz, 1H), 1.75 (m, 2H), 1.63 (quint, J = 5.6 Hz, 4H), 1.46 (m, 2H), 0.84 (d, J = 6.5
13
Hz, 3H, Me), 0.83 (d, J = 6.6 Hz, 3H, Me); C NMR (CDCl3, 101 MHz) 149.1, 146.3,
146.2, 143.5, 134.9, 133.7, 121.6, 121.0, 114.0, 113.0, 112.7, 111.4, 66.3, 57.7, 55.8, 54.9,
39.3, 39.1, 39.0, 38.7, 25.6, 24.0, 16.2, 16.1; HRMS (ESI) m/z: calculated for C27H40NO4
[M+H]: 442.29573, found [M+H]: 442.29490.
2.5.7.
meso-1,4-Bis[3-methoxy-4-(2-(piperidin-1-yl)ethoxy)phenyl]-(2R,3S)-
dimethylbutane (31)
White solid; 20% yield; mp 147-148 °C; 1H NMR (CDCl3, 400 MHz) 6.80 (d, J = 8.0 Hz,
2H-Ar), 6.65 (dd, J = 8.1, 1.8 Hz, 2H-Ar), 6.64 (d, J = 1.7 Hz, 2H-Ar), 4.13 (t, J = 6.3 Hz,
4H, 2 x CH2O), 3.85 (s, 6H, 2 x OMe), 2.84 (t, J = 6.3 Hz, 4H, 2 x CH2N), 2.73 (dd, J =
13.4, 4.8 Hz, 2H), 2.55 (br s, 8H, 4 x CH2N), 2.28 (dd, J = 13.4, 9.2 Hz, 2H), 1.75 (m, 2H),

13
1.63 (quint, J = 5.6 Hz, 8H), 1.46 (m, 4H), 0.84 (d, J = 6.6 Hz, 6H, 2 x Me); C NMR
(CDCl3, 101 MHz) 149.1, 146.3, 134.8, 121.0, 113.1, 112.8, 66.6, 57.8, 55.9, 55.0, 39.2,
38.8, 25.8, 24.2, 16.2; HRMS (ESI) m/z: calculated for C34H53N2O4 [M+H]: 553.40053,
found [M+H]: 553.40106.
2.5.8.
( )-1-(4-Hydroxy-3-methoxyphenyl)-(2R,3S)-dimethyl-4-[3-methoxy-4-(2-
morpholinoethoxy)phenyl]butane (32)
White solid; 22% yield; mp 124-125 °C; 1H NMR (CDCl3, 400 MHz) 6.81 (d, J = 8.0 Hz,
2H-Ar), 6.66 (dd, J = 8.0, 1.8 Hz, 2H-Ar), 6.64 (d, J = 1.8 Hz, 2H-Ar), 5.51 (s, 1H, OH),
4.13 (t, J = 6.0 Hz, 2H, CH2O), 3.86 (s, 3H, OMe), 3.82 (s, 3H, OMe), 3.74 (t, J = 4.6 Hz,
4H, 2 x CH2O), 2.84 (t, J = 6.0 Hz, 2H, CH2N), 2.75 (dd, J = 12.7, 4.9 Hz, 1H), 2.71 (dd, J
= 12.8, 5.3 Hz, 1H), 2.60 (t, J = 4.3 Hz, 4H, 2 x CH2N), 2.30 (dd, J = 13.4, 9.8 Hz, 1H),
2.26 (dd, J = 13.3, 9.9 Hz, 1H), 1.78 (m, 2H), 0.84 (d, J = 6.2 Hz, 3H, Me), 0.83 (d, J = 5.9
13
Hz, 3H, Me); C NMR (CDCl3, 101 MHz) 149.3, 146.3, 146.2, 143.5, 135.2, 133.7,
121.6, 121.0, 114.0, 113.6, 112.8, 111.4, 66.9, 66.8, 57.6, 55.8, 55.8, 54.0, 39.3, 39.0, 39.0,
38.7, 16.2, 16.1; HRMS (ESI) m/z: calculated for C26H38NO5 [M+H]: 444.27500, found
[M+H]: 444.27524.
2.5.9.
meso-1,4-Bis[3-methoxy-4-(2-morpholinoethoxy)phenyl]-(2R,3S)-dimethylbutane
(33)
White solid; 48% yield; mp 165-168 °C; 1H NMR (CDCl3, 400 MHz) 6.80 (d, J = 7.9 Hz,
2H-Ar), 6.67 (dd, J = 7.9, 1.8 Hz, 2H-Ar), 6.65 (d, J = 1.8 Hz, 2H-Ar), 4.13 (t, J = 6.0 Hz,
4H, 2 x CH2O), 3.83 (s, 6H, 2 x OMe), 3.73 (t, J = 4.7 Hz, 8H, 4 x CH2O), 2.83 (t, J = 4.7
Hz, 4H, 2 x CH2N), 2.74 (dd, J = 13.4, 4.8 Hz, 2H), 2.59 (t, J = 4.5 Hz, 8H, 4 x CH2N),
13
2.29 (dd, J = 13.4, 9.0 Hz, 2H), 1.77 (m, 2H), 0.84 (d, J = 6.6 Hz, 6H, 2 x Me); C NMR
(CDCl3, 101 MHz) 149.3, 146.4, 135.2, 121.0, 113.7, 112.9, 66.9, 57.6, 55.9, 54.0, 39.2,
38.8, 16.2; HRMS (ESI) m/z: calculated for C32H49N2O6 [M+H]: 557.35906, found [M+H]:
557.35927.
2.6. In Vitro Antibacterial Assay
Twelve drug-resistant clinical bacterial isolates were provided and used at the Hospital
Universitario “Dr. José Eleuterio González”, Monterrey, Nuevo León, México. Gram-
negative bacteria: carbapenem-resistant (CR) Acinetobacter baumannii (12-666), extended-

spectrum β-lactamase-positive (ESBL) Escherichia coli (14-2081), oxacillin-resistant E.
coli (OXA-48), CR Pseudomonas aeruginosa (13-1391), Klebsiella pneumoniae NDM-1
positive (14-3335), ESBL K. pneumoniae (14-2081), Providencia rettgeri NDM-1 positive,
trimethoprim-sulfamethoxazole-resistant Stenotrophomonas maltophilia. Gram-positive
bacteria: methicillin-resistant Staphylococcus aureus (MRSA, 14-2095), linezolid-resistant
(LR) S. epidermis (14-583), LR S. haemolyticus (2975), vancomycin-resistant
Enterococcus faecium (10-984). Bacterial strains were inoculated on plates, prepared with
5% blood agar, and grown at 37 °C for 24 h (except CR P. aeuroginosa that needed 48 h).
Three to five colonies of each culture were transferred into tubes with sterile saline solution
and turbidity was adjusted by nephelometer to 0.5 McFarland standard (1.5 x 108 colony
forming units [CFU]/mL). Then, 10 µL of bacterial suspension was transferred to 11 mL of
Mueller–Hinton broth to achieve 5 x 105 CFU/mL. The microdilution assay was performed
as previously described in literature.12, 18 The range of concentrations tested of each analog
was 50 to 0.78 µg/mL. Each concentration was tested in triplicate and levofloxacin was
used as the reference drug. Each experiment was performed twice.
2.7. In Vitro Antimycobacterial Assay
Mycobacterium tuberculosis H37Rv ATCC 27294 (sensitive to first line antitubercular
drugs) and two MDR clinical isolates of M. tuberculosis (isoniazid/rifampicin/ethambutol-
resistant G122 strain and isoniazid/rifampicin/streptomycin-resistant G133 strain) were
used. The strains of M. tuberculosis were cultured in Middlebrook 7H9 broth supplemented
with 0.2% glycerol and 10% OADC at 37 °C for 2 weeks in order to reach logarithmic
phase growth. The inoculum for assay was prepared by diluting the bacterial suspension
with supplemented Middlebrook 7H9 broth adjusting turbidity to standard No. 1 of
McFarland’s nephelometer. Then it was further diluted 1:20 with the same culture medium.
Microplate Alamar Blue Assay (MABA) was carried out as previously described in
literature.11, 19 The dilution range for each analog was from 50 to 1.56 µg/mL. Levofloxacin
and ethambutol were used as positive controls. The assays were performed in duplicate.
2.8. In Vitro Cytotoxicity Assay
Cytotoxicity assay was performed on the Vero cell line (ATCC CCL-81), as previously
described in literature.20-21 Briefly, Vero cells were cultivated in RPMI medium

supplemented with 10% fetal bovine serum (FBS). The most active analogs were tested,
each of these analogs was dissolved and sterilized in DMSO and brought to a final
concentration of 487, 243.9, 121.95 and 66.97 µg/mLL with RPPMI meddiium suppplemented
with 10% FBS. The cell suspension was adjusted to 12.62 x 104/mL with RPMI medium
supplemented with 10% FBS. Cell suspension (200 µL) was placed into the wells of a 96
well microplate. The cells were incubated at 37 °C in a 5% CO2 atmosphere for 24 h. Each
analog solution was added to each well containing the Vero cells and incubated for 24 h.
The assay was carried out in triplicate. The number of cells was determined in a Newbauer
chamber, while the percentage of viable cells was measured with the Trypan blue staining
method. Medium inhibitory concentrations (IC50) were calculated by Probit method.
2.9. Selectivity Index (SI)
SI was calculated dividing the IC50 value by the MIC value for each compound.22
2.10. Structure-Activity Relationships Analysis
The tridimensional structure of all compounds was built in Maestro 10.423 with their
geometry optimized using DFT-B3LYP 6-31G theory with implicit water solvent
simulation, available in Jaguar.24-25 Later, the compounds were aligned using an algorithm
based in the maximum common structure based on SMILES code of meso-DGA.
2.10.1. Activity-Cliff Search
The search of activity cliffs, along with R-group pharmacophoric analysis, was performed
based on molecular fingerprints on Canvas.26-28 The Mahalanobis distance of the difference
of the antimycobacterial activity on the three strains and pharmacophoric components of
pairs were used as cut-off criteria to find activity cliffs.
2.10.2. 3D-QSAR
Field-based quantitative structure-activity relationships were performed using QSAR model
available on Maestro.29-31 For each model, 50% of the compounds were used for the model
building and 50% for the model internal validation; these groups were assigned randomly.
The models were built using PLS, and were validated using the leave-one-out algorithm.
The molecular fields calculated were electrostatic, steric, hydrophobic, hydrogen bond
donors and acceptors.

3. Results and discussion
3.1. Synthesis of ( )-mono- and meso-bis-O-substituted meso-DGA derivatives
The starting material for reactions, meso-dihydroguaiaretic acid (meso-DGA), was isolated
from the aerial parts of Larrea tridentata according to our previously described protocol
with slight modifications.13 The meso-DGA was structurally confirmed by its experimental
spectroscopic data and by comparison with those reported in the literature.16 The target
compounds 1-33 were designed to improve the antibacterial and antimycobacterial
properties of parent meso-DGA by increasing lipophilicity and donor/acceptor capacity to
form hydrogen bonds. To this end, we postulated the chemical conversion of one or both
phenols in meso-DGA to esters, ethers, and amino-ethers. The esters 3–8 were synthesized
by the nucleophilic acyl substitution between meso-DGA and different acyl chlorides such
as 4-bromobenzoyl, 2-nitrobenzoyl, and 2-pyridinecarbonyl chlorides (Scheme 1). It is
worth mentioning that the reaction conditions were set to obtain the mixture of mono- and
bis-O-acylated products. The mono-substituted derivatives were obtained in racemic
mixtures [( )-3, ( )-5, and ( )-7)], while bis-O-acylated compounds retained the C2-
symmetry (meso-4, meso-6, and meso-8, Scheme 1). In addition, esters 1, 2, and 9–22 were
prepared by the Steglich reaction of meso-DGA with a variety of carboxylic acids such as
acetic, butyric, palmitic, stearic, oleic, benzoic, nicotinic, and pyrazincarboxylic acids. As
described above, the esterification protocols were carried out to obtain the mixture of ( )-
mono-O-acylated (1, 9, 11, 13, 15, 17, 19, and 21) and meso-bis-O-acylated (2, 10, 12, 14,
16, 18, 20, and 22) products (Scheme 1).

H3CO
H3CO
HO
H3CO
HO
O
O
R
R
X
O
OCH3
OCH3
K2CO3 or DCC/DMAP
OCH3
O
O
OH
O
O
R
R
meso-DGA
X = Cl or OH
R =
( )-1
( )-3
meso
meso
-
-
2
4
R =
R =
Br
meso
meso
-
-
6
8
( )-5
NO2
( )-7
( )-9
R =
R =
R =
N
N
N
meso
-10
( )-11
meso
meso
-
12
14
N
-
( )-13
( )-15
R =
R =
H3C
meso
meso
-
16
18
R =
( )-17
-
12
14
R =
R =
( )-19
( )-21
meso
meso
-
20
22
-
6
6
Scheme 1. Synthesis of ( )-mono- and meso-bis-O-acylated compounds from meso-DGA.
On the other hand, the ( )-mono- and meso-bis-O-alkylated compounds 23 and 24 were
synthesized by the Williamson reaction of meso-DGA and iodomethane using K2CO3 as base
(Scheme 2). In a similar scenario, the ( )-mono- and meso-bis-O-alkylaminated products 25-
33 were also prepared by the SN2 reaction of meso-DGA and different aminoalkyl halides
such as 2-chloro-N,N-dimethylethylamine, 2-chloro-N,N-diethylethylamine, 4-(2-
chloroethyl)morpholine, 1-(2-chloroethyl)pyrrolidine, and 1-(2-chloroethyl)piperidine. The
reaction conditions of these nucleophilic substitutions were set to obtain the mixture of ( )-
mono- and meso-bis-O-alkyl(amin)ated products (Scheme 2).

H3CO
H3CO
HO
H3CO
HO
O
R
R
X
DMF or Acetone
K2CO3
OCH3
OCH3
OCH3
O
O
OH
R
R
X = I or Cl
R = H
meso-DGA
( )-23
( )-25
meso
-
24
26
N
R =
R =
meso
meso
-
-27
N
N
N
( )-28
( )-30
meso
meso
-
29
31
R =
R =
R =
-
O
meso
-33
( )-32
N
Scheme 2. Synthesis of mono- and bis-O-alkyl(amin)ated compounds from meso-DGA.
To summarize, a series of twenty-two esters, two ethers, and nine amino-ethers were
synthesized and structurally characterized by 1H and 13C NMR and HRMS (see Experimental
Section and Supplementary Information). The chemical structures of ( )-13, meso-14, ( )-23
and meso-24 have been reported in the literature and the spectroscopic data of meso-14, ( )-
23 and meso-24 are consistent with those obtained in the present work.16-17
3.2. Antibacterial activity
The ( )-mono- and meso-bis-substituted analogs of meso-DGA were tested against twelve
drug-resistant clinical isolates of gram-negative (carbapenem-resistant A. baumannii,
extended-spectrum β-lactamase-positive E. coli, oxacillin-resistant E. coli, CR P.
aeruginosa, K. pneumoniae NDM-1 positive, ESBL K. pneumoniae, P. rettgeri NDM-1
positive, trimethoprim-sulfamethoxazole-resistant S. maltophilia) and gram-positive bacteria
(methicillin-resistant S. aureus, linezolid-resistant S. epidermis, LR S. haemolyticus,

vancomycin-resistant E. faecium) to determine the lowest compound concentration at which
bacterial growth is inhibited (MIC). Among the tested compounds (1-33), eight derivatives
(7, 11, 13, 17, 23, 30, 31, 33) showed activity against gram-positive bacteria (Table 1), while
no activity was observed against gram-negative bacteria up to 50 µg/mL. Four ( )-mono-O-
acylated compounds (7, 11, 13, 17), two ( )-mono-O-alkyl(amin)ated products (23 and 30),
and two meso-bis-O-alkylaminated derivatives (31 and 33) exhibited moderate activity with
MIC values in the range of 25 to 50 µg/mL against MR S. aureus.
Table 1. Antibacterial and cytotoxic activity of some meso-DGA derivatives.
Selectivity
Index
Cytotoxicity
IC50 (µg/mL)
Antibacterial activity (MIC, µg/mL)
IC50/MICa
VR E.
faecium
MR S.
aureus
LR S. LR S.
epidermidis haemolyticus
Vero Cells
Analog
> 50
> 50
> 50
> 50
> 50
50
> 50
> 50
> 50
> 50
> 50
50
> 50
> 50
> 50
> 50
> 50
50
65.04
43.34
111.58
ND
2.60
0.87
4.46
ND
25
50
( )-7
( )-11
( )-13
( )-17
( )-23
( )-30
meso-31
meso-33
Lev
25
50
47.75
62.57
73.23
71.65
293.16
1.91
1.25
1.46
1.43
93.81
25
50
> 50
> 50
12.5
50
50
50
> 50
>50
> 50
12.5
50
3.125
VR: Vancomycin-resistant. MR: Methicillin-resistant. LR: Linezolid-resistant. Lev: Levofloxacin. ND: Not Determined. a MR S. aureus.
Additionally, amino-ethers 30 and 31 showed weak activity (MIC = 50 µg/mL) against VR
E. faecium, LR S. epidermis, and LR S. haemolyticus. It is worth mentioning that analogs 30
and 31 resulted more active than the positive control levofloxacin (MIC > 50 µg/mL) against
VR E. faecium, but much less active than levofloxacin against MR S. aureus, LR S.
epidermidis, and LR S. haemolyticus. The meso-DGA derivatives 7, 11, 13, 17, 23, 30, 31,
and 33 were evaluated for cytotoxicity on Vero cells (derived from the kidney of an African
green monkey) and the medium inhibitory concentrations (IC50) are reported in Table 1. The
calculated selectivity index (SI) indicated that these eight meso-DGA analogs are less toxic
to bacteria (SI < 10) than to mammalian cells. Regarding antistaphylococcal activity listed in
Table 1, the pyrazincarbonyl-containing ester 11 showed less antibacterial activity than the
pyridine-containing compound 7, which may be correlated to its higher lipophilicity (Table

2) as suggested for some antibacterial studies.32-34 However, a larger increase in lipophilicity
can diminish the antibacterial properties of a molecule. Particularly, the palmitoyl ester 17
with a clogP value of 11.0 resulted less active against MR S. aureus than the acetyl ester 13
possessing less lipophilic character (clogP = 4.9). It seems that a clogP value around 5.0 may
confer activity against MR S. aureus. As observed for esters 7 and 13, the ether 23 with a
clogP of 5.2 exhibited the same MIC value (25 µg/mL) against MR S. aureus. Conversely,
the ethylpiperidine-containing analogs 30 and 31 with clogP values of 6.0 and 7.0,
respectively, showed less antistaphylococcal activity (MIC = 50 µg/mL).
3.3. Antimycobacterial activity
The results in Table 2 showed that 19 meso-DGA analogs were active against sensitive and
resistant M. tuberculosis strains, while 12 derivatives (2, 4, 6, 8, 15-22, 24, and 32) did not
exhibit antimycobacterial activity up to 50 µg/mL. The ( )-mono- and meso-bis-O-acylated
products 1, 3, 5, 7, and 9–14 exhibited activity against the sensitive H37Rv strain with MIC
values in the range of 12.5 to 50 µg/mL. In particular, compounds bearing one benzoyl (1, 3,
and 5) and two nicotinoyl (10) groups resulted four times more active than esters 7, 9, and
11–14, but fourfold less active than ethambutol (MIC = 3.125 µg/mL). Previous studies have
reported that fatty acids such as palmitic or linoleic acids inhibited M. tuberculosis H37Rv.35-
36 Herein, we supposed the meso-DGA derivatives bearing long-chain fatty acids (compounds
17–22) to be more potent than parent meso-DGA, but no antimycobacterial activity was
observed up to 50 µg/mL. The incorporation of short chain carboxylic acids such as butyric
and acetic acid on meso-DGA decreased lipophilicity of derivatives 13–16 (clogP = 4.9–7.1)
in comparison with 17–22 (clogP > 11), but no influence on the antimycobacterial properties
of parent meso-DGA was observed. If we compare MIC values of esters bearing
hydrocarbon chains (13 and 14) with those bearing benzene rings (1, 3, and 5), it appears that
the incorporation of a benzoyl group is required for good activity against M. tuberculosis
H37Rv. It is noteworthy that benzoyl-containing esters 1, 3, and 5 are more lipophilic than
the acetylated esters 13 and 14, which may be related to easier diffusion through the
mycobacteria cell wall.21

Table 2. Antimycobacterial and cytotoxic activity of meso-DGA derivatives.
Activity against
Mycobacterium tuberculosis
(MIC, µg/mL)
Cytotoxicity
IC50 (µg/mL)
Selectivity
Index (SI)
Partition
Coefficient
IC50/MICH37Rv
Analog
Strain
H37Rv
Strain
G122
Strain
G133
Vero cells
cLogPa
6.88
7.71
6.39
5.54
5.54
6.08
4.63
4.25
4.98
4.96
5.27
5.27
5.53
6.88
5.58
6.16
6.00
7.00
4.73
5.01
0.06
1.35
( )-1
( )-3
12.5
12.5
12.5
50
>50
>50
50
>50
> 50
>50
>50
50
124.62
111.58
97.07
65.04
71.42
36.30
43.34
ND
9.97
8.93
( )-5
7.76
25
1.30
( )-7
( )-9
50
50
1.43
12.5
50
>50
>50
> 50
>50
>50
50
>50
>50
>50
> 50
> 50
50
2.90
( )-10
0.86
( )-11
meso-12
( )-13
50
ND
50
111.58
95.13
47.75
326.59
>487
2.23
meso-14
( )-23
50
1.90
50
0.96
( )-25
12.5
6.25
25
>50
12.5
25
>50
12.5
25
26.13
>77.92
1.45
meso-26
meso-27
( )-28
36.20
>487
12.5
25
25
25
>38.96
>19.48
5.01
meso-29
( )-30
12.5
25
12.5
25
>487
12.5
3.125
25
62.57
73.23
71.65
ND
meso-31
meso-33
meso-DGA
EMB
6.25
6.25
>50
6.25
6.25
6.25
25
23.43
2.87
50
>50
3.125
1.56
ND
3.125
0.078
125.10
ND
390.93
293.16
LEV
H37Rv: Sensitive to first line anti-TB drugs. G122: Resistant to isoniazid, rifampicin, and ethambutol. G133: Resistant to
isoniazid, rifampicin, and streptomycin. EMB: Ethambutol. LEV: Levofloxacin. ND: Not Determined. a Predicted values using
ChemBioDraw Ultra 12.0.
Among the M. tuberculosis H37Rv growth inhibitors 1, 3, 5, 7, and 9–14, the picolinoyl-
containing ester 7 resulted two times more active against MDR M. tuberculosis G122 strain
than the nitrobenzoyl- and nicotinoyl-containing esters 5 and 9, respectively. Interestingly,
the substitution of meso-DGA with one nicotinoyl group allowed ester 9 to exhibit higher
activity against the G122 and G133 strains than its analog bearing two nicotinoyl groups

(10). Moreover, the position of the nitrogen atom in the heterocycle of esters 7 and 9
influenced their antimycobacterial properties, with 7 being two times more active against
the G122 strain. In addition, the incorporation of two nitrogen atom-containing
heterocycles on meso-DGA (11 and 12) had no influence on the activity against the
resistant M. tuberculosis strains.
In addition, the results in Table 2 reveal that the O-alkylaminated analogs exhibited better
antimycobacterial activities than the O-alkylated and O-acylated products. Among the
amino-ethers, the meso-31 exhibited the lowest MIC values (3.125 and 6.25 µg/mL) against
sensitive and MDR M. tuberculosis strains, being as potent as the positive control
ethambutol. If we compare the MIC values of the amino-ethers series with the substitution
pattern, it is noteworthy that the meso-bis-substituted derivatives (26, 29, 31) resulted more
potent than their ( )-mono-substituted analogs (25, 28, and 30). Particularly, the ether 26
with two N,N-dimethylethylamine groups resulted twofold more potent than its mono-
substituted analog 25 against H37Rv strain. Moreover, compound 25 showed no activity
against the MDR M. tuberculosis G122 and G133 strains, whereas its analog 26 displayed
MIC values of 12.5 µg/mL, thus being two and four times less active than ethambutol.
Similar results were observed for the meso-bis-N-ethylpiperidine-containing compound 31,
which was four times more potent than the mono-N-ethylpiperidine-containing ether 30
against the three M. tuberculosis strains. Based on these results, a correlation between the
lipophilic and antimycobacterial properties of the amino-ethers 25, 26, 30, and 31 can be
inferred. Compound 31 with a clogP value of 7.0 exhibited lower MIC values than the less
lipophilic compound 30 (clogP = 6.0). Similarly, the amino-ether 26 with a clogP value of
5.5 resulted more potent than its less lipophilic analog 25 (clogP = 5.2). Among these
antimycobacterial compounds, 31 exhibited identical MIC values to ethambutol against
sensitive and G122 M. tuberculosis strains (3.125 and 6.25 µg/mL), despite their
differences in clogP values. Ethambutol is a highly hydrophilic antitubercular drug (clogP =
0.06) that diffuses across the mycobacterial outer-membrane by channels called porins,37
whereas the aminoether 31 presumably crosses by passive diffusion due to its high
lipophilic character (clogP = 7.0). Further research is needed to elucidate not only the
transport mechanism but also the mechanism of action of 31. Although compound 31
resulted two and four times less active than ethambutol and levofloxacin against the G133

strain, respectively, it was equally active as levofloxacin against the G122 strain. Among
the bis-substituted amino-ethers, the replacement of N-ethylpiperidine (meso-31) by N-
ethylmorpholine (meso-33) in meso-DGA diminished the antimycobacterial activity against
H37Rv and G133 strains more than four times, but retained activity against the G122 strain.
Furthermore, the meso-27 whose N-alkyl chains contain two carbon atoms exhibited lower
MIC values than its analog meso-26 whose N-alkyl chains contain one carbon atom.
Conversely, the meso-31 bearing six-membered nonaromatic nitrogen heterocycles resulted
more potent than its analog having five-membered nonaromatic nitrogen heterocycles
(meso-29). It appears that the presence of two N-ethylpiperidine moieties is essential to
significantly increase the antimycobacterial properties of parent meso-DGA.
The safety profile of the most active antimycobacterial compounds was investigated taking
into account the SI.22 The esters 1, 3, 5, 7, 11, 13, and 14 displayed SI values below 10 (SI <
10), being more toxic to Vero cells than to M. tuberculosis. Conversely, amino-ethers 25,
26, 28, 29, and 31 exhibited SI values higher than 10 (SI > 10), being more toxic to M.
tuberculosis than to mammalian cells. Despite analog meso-26 showing the higher SI value
(SI > 77), it resulted twofold less potent than ethambutol, while compound meso-31 with
the second best SI value (SI = 23) resulted as potent as ethambutol against M. tuberculosis
H37Rv and G122.
3.4. Quantitative Structure-Activity Relationships
An activity-cliff search followed by a field-based quantitative structure-activity relationship
analysis was performed for each M. tuberculosis strain, considering the stereochemistry
depicted in Schemes 1-2, in order to find relationships among the structure of the evaluated
compounds and their antimycobacterial activity. The statistics and model fractions of the
best models found for the three strains are depicted in Table 3. Acceptable models were
obtained according to the statistics of the best models. The three models showed poor to
acceptable linearity and predictability (r2>0.7, q2>0.4), and low deviation and error (SD and
RMSE below 15% activity range). The rather narrow activity range (5–140 µM, see page
71 in the Supplementary Information) and the codification assigned to the low-activity
compounds (MIC > 50 µg/mL) could have influenced the quality of the models.
Nevertheless, the statistical quality of the models is adequate to find tendencies, confirm or
refute original hypotheses regarding structure-antimycobacterial activity, and show possible

structural modifications of the most active compounds for the optimization of
antimycobacterial activity.
Table 3. Statistics of the best models found by Field-based QSAR.
M.
tuberculosis
H37Rv
M.
tuberculosis
G122
M.
tuberculosis
G133
Standard Deviation
R2
0.1866
0.1634
0.156
0.6775
25.2
0.794
50.1
0.741
40.0
Variability (F)
Significance level (p)
Test group RMSE
Predictability (Q2)
Pearson’s R of calculated activity
Steric
2.98e-4
0.19
8.28e-6
0.17
1.87e-5
0.15
0.3293
0.5907
0.444
0.114
0.280
0.149
0.013
0.422
0.729
0.560
0.067
0.258
0.101
0.016
0.438
0.746
0.585
0.065
0.253
0.079
0.018
Electrostatic
Hydrophobic
Hydrogen-bond Acceptor
Hydrogen-bond Donor
As stated previously, at least two moieties cause drastic changes in the antimycobacterial
activity against M. tuberculosis strain H37Rv: aromatic groups (compounds 1 and 2) and
nitrogen-containing 6-membered rings (compounds 30–33). According to the R-group
pharmacophoric analysis, the incorporation of hydrogen-bond acceptors like ethers or
amides in meso-DGA could decrease the antimycobacterial activity against the sensitive
strain, whereas the incorporation of positively charged group precursors like amines could
increase it. Furthermore, separate pharmacophoric features like hydrophobic, negative or
aromatic groups have little or no influence over the antimycobacterial activity, but in
combination a synergism could be produced (page 72 in the Supplementary Information).
In the other hand, field-QSAR indicated that steric and hydrophobic factors are the main
contributors (more than 72%) to antimycobacterial activity (Table 4 and page 32 in the
Supplementary Information), with the latter being the most sensitive to the pattern
substitution of the compounds with a great tendency towards mono-substitution. Despite
their relatively weak contribution to activity against the strain H37Rv, the results of the
hydrogen bond acceptor field reinforced the importance of the asymmetrical substitution,
which could be achieved adding steric and hydrophobic groups like alicyclic fragments on

one side and hydrogen bond acceptors like amines on the opposite one in order to increase
antimycobacterial activity.
The substitution pattern of meso-DGA derivatives, together with the incorporation of six-
membered heterocyclic fragments (compounds 7, 8, 30–33) caused noticeable changes in
antimycobacterial activity against the resistant M. tuberculosis strain G122. According to
the R-group pharmacophoric analysis, the meso-DGA could be substituted with positively
charged group precursors like amines and hydrophobic groups like aliphatic groups in order
to increase antimycobacterial activity, while the incorporation of hydrogen bond donors
could decrease it as observed for compounds 30 and 32. Furthermore, aromatic and
hydrogen bond acceptors have weak influence on the antimycobacterial activity, but in
combination for asymmetrically substituted meso-DGA derivatives the activity could be
increased (page 72 in the Supplementary Information). Field-QSAR indicated that steric
and hydrophobic factors are the main contributors (more than 80%) to antimycobacterial
activity against strain G122, similar to those fields in the H37Rv QSAR model (Table 4 and
page 73 in the Supplementary Information), with the former being the most sensitive to the
substitution pattern of the compounds with a preference for hydrophilic substituents like
amines and mono-substitution. According to these fields, adding hydrophilic and
voluminous groups like bulky amines in asymmetrically substituted meso-DGA derivatives,
with a careful balance of hydrogen bond acceptors, could increase the antimycobacterial
activity against strain G122.
Compounds bearing 6-membered heterocyclic moieties (derivatives 30–33) along with the
substitution pattern caused drastic changes in antimycobacterial activity against the
resistant M. tuberculosis strain G133. Using the R-group pharmacophoric analysis, we
found that the meso-DGA could be substituted with positively charged and hydrophobic
groups to increase activity against strain G133. However, inadequate addition of hydrogen
bond acceptors and donors and aromatic groups could decrease activity as found for strain
G122 (page 72 in the Supplementary Information). Field-QSAR analysis indicated that
steric and hydrophobic factors are the main contributors (more than 83%) to
antimycobacterial activity (Table 4 and page 73 in the Supplementary Information), with
the latter being the most sensitive to meso-bis-substituted compounds. Hydrophilic groups
as well as a careful balance of hydrogen bond acceptors and asymmetry could be
considered to design new meso-DGA derivatives with higher activity against strain G133.