Synthesis and First-Time Assessment of o-Eugenol Derivatives against Mycobacterium tuberculosis

Article abstract of DOI:10.1007/s10600-020-03110-2

In this work, we report the first-time assessment of o-eugenol, 6-allyl-2-methoxyphenol, and their selected derivatives, against Mycobacterium tuberculosis H₃₇RV, using the MABA susceptibility test. The bromo, nitro, O-alkylated, and reduced derivatives were obtained by standard methods and were characterized by spectroscopic and mass spectral data. Structure–activity relationships were investigated, with the most active derivatives being 4,5-dibromo-2-methoxy-6-propylphenol (139 μM) and 2-methoxy-3-nitro-6-propylphenol (237 μM). This study provides important information on the rational design of new lead anti-TB drugs based on o-eugenol derivatives.

Full text of DOI:10.1007/s10600-020-03110-2

DOI 10.1007/s10600-020-03110-2
Chemistry of Natural Compounds, Vol. 56, No. 4, July, 2020
SYNTHESIS AND FIRST-TIME ASSESSMENT OF o-EUGENOL
DERIVATIVES AGAINST Mycobacterium tuberculosis
1,2
2
Adriana Marques Moraes, Emerson Teixeira da Silva,
3
2*
James L. Wardell, and Marcus V. N. de Souza
In this work, we report the first-time assessment of o-eugenol, 6-allyl-2-methoxyphenol, and their selected
derivatives, against Mycobacterium tuberculosis H RV, using the MABA susceptibility test. The bromo,
37
nitro, O-alkylated, and reduced derivatives were obtained by standard methods and were characterized
by spectroscopic and mass spectral data. Structure–activity relationships were investigated, with the most
active derivatives being 4,5-dibromo-2-methoxy-6-propylphenol (139 μM) and 2-methoxy-3-nitro-6-propylphenol
(237 μM). This study provides important information on the rational design of new lead anti-TB drugs based
on o-eugenol derivatives.
Keywords: eugenol, o-eugenol, synthesis, tuberculosis, Mycobacterium tuberculosis, nitration, bromination, large scale.
Eugenol (1, 4-allyl-2-methoxyphenol) (Scheme 1), is a natural aromatic product, obtained as an oil from various
plant species such as cloves (Syzygium aromaticum), and cinnamon (Cinnamomum tamala). Its biosynthetic path involves the
amino acid tyrosine [1], and this natural product has several applications in different fields, e.g., as an oxidant, a pheromone,
a stabilizing agent, an anesthetic, an antiseptic, an antibacterial, a flavoring agent, an essential oil, and in perfumery [2].
An isomer of eugenol is o-eugenol, (2, 2-allyl-6-methoxyphenol) (Scheme 1), a natural aromatic product either,
found in species such as Miscanthus x giganteus [3], in cade oil of Juniperus oxycedrus L. [4, 5], and Mosla chinensis [6].
However, this isomer is readily available synthetically as the major product from a reaction involving guaiacol (3, 2-methoxyphenol)
and allyl bromide in a two-step process, which also produces eugenol (1) as a minor product. Astudy done with this regioisomer,
o-eugenol, has already highlighted the antinociceptive and anti-inflammatory activity [7], but little is known about other
biological uses of this compound as well as its derivatives.
Despite the broad spectrum of applications of eugenol (1), few studies have been made on the use of 1 or 2 and
their derivatives in the search for new antitubercular agents. Tuberculosis (TB) continues today as a significant global
public health problem, being responsible for 1.7 million deaths worldwide in 2016 [8] (WHO, 2017). The resurgence of this
disease is due to different factors, with a major one being the emergence of bacteria becoming increasingly resistant to the
standard array of drugs used to treat this disease [9].
OH
O
OH
HO
H CO
MeO
MeO
MeO
b
Br
+
a
3
3a
1
3
2
a. K CO ; acetone, reflux, 8 h, 85%; b. 200°C, 90 min
2
3
Scheme 1
1) Universidade Federal do Rio de Janeiro, Instituto de Quimica, Departamento de Quimica Organica, 68563,
Zip Code 21945-970, Rio de Janeiro, Brazil, e-mail: m_drik@yahoo.com.br; 2) Fiocruz–Fundacao Oswaldo Cruz, Instituto
de Tecnologia em Farmacos – Farmanguinhos, Sizenando Nabuco 100, Manguinhos, Zip Code 21041-250, Rio de Janeiro,
Brazil, e-mail: emerson.silva@far.fiocruz.br; mvndesouza@gmail.com; 3) Department of Chemistry, University ofAberdeen,
Aberdeen AB2 3UE, Scotland, UK, e-mail: j.wardell@abdn.ac.uk. Published in Khimiya Prirodnykh Soedinenii, No. 4,
July–August, 2020, pp. 549–554. Original article submitted July 1, 2019.
0009-3130/20/5604-0633 ©2020 Springer Science+Business Media, LLC
633

3''
OH
1''
1
3''
1''
3''
1''
1'
1
3'
O
O
Cl
3'
O
R
3'
6
MeO
1'
1'
1
1'
3'
3
MeO
3
1
MeO
MeO
MeO
e
Br
4
Br
3
5
5
7
8 - 11
5
6
5
c
d
b
OH
OH
OH
1
OH
1'
1
1'
1'
1
3'
3'
MeO
3'
MeO
MeO
O N
f
f
a
5
5
3
5
3
O N
2
2
4
12
2
13
8: R = -N(CH CH ) NH; 9: R = -NHCH ; 10: R = -NH(CH ) CH ; 11: R = -NH(CH ) CH
2
2 2
3
2 2
3
2 3
3
a. H , Pd/C, EtOH, r.t., 21 h, 72%; b. Br , CH Cl , –70°C, 1 h, 79%; c. allyl bromide, K CO , acetone, reflux, 14 h, 89%;
2
2
2
2
2
3
d. 1-bromo-3-chloropropane, K CO , acetone, reflux, 25 h, 86%; e. piperazine, methylamine, propylamine or butylamine,
2
3
K CO , CH CN or MeOH, reflux or r.t., 18 – 67 h, 15 – 39%; f. HNO , CH Cl , r.t., 12: 90 min, 37%; 13: 1 h, 51%
2
3
3
3
2
2
Scheme 1
Thus new drugs and strategies are required, with aims to combat the resistant strains, reduce treatment time with
fewer side effects, and reduce costs. Therefore, we have initiated a study of the anti-TB potential of o-eugenol (2) and its
derivatives. We now report our findings.
Compounds 1 and 2 were obtained synthetically in 10 and 84% yields, respectively, following a published procedure
[10], via a two-step process involving an initial allylation of guaiacol (3) with allyl chloride or bromide, followed by a
Claisen rearrangement. The o-eugenol (2) was prepared on a large scale (59 g) (Scheme 1).
The o-eugenol derivatives 4–13 were synthesized, as shown in Scheme 2, and their anti-TB activity was tested
in vitro against M. tuberculosis ATTC 27294 using the Microplate Alamar Blue Assay (MABA) [11].
For the nitration of o-eugenol and its reduced derivative 4, the reactions were carried out using nitric acid in
dichloromethane at room temperature, since the classical sulfuric/nitric acid nitrating agent resulted in decomposition. The
products o-eugenol and 4 were regioselectively nitrated in the ortho position to the methoxy group, furnishing 12 and 13 in
37 and 51% yields, respectively (Scheme 2). The preferential formations of the o-substituted products using HNO /CH Cl
3
2
2
+
can be explained by the formation of a “pre-complex” involving the active NO cation with the methoxy group as indicated
2
by Strazzolini et al. [12] and is illustrated in Scheme 3.
1
13
All the new compounds were generally characterized from spectral data, including H NMR, C NMR, and IR spectra,
and by mass spectrometry. Specifically, in the IR spectra, compounds 4, 5, 12, and 13 showed O–H stretching vibrations at
–1
–1
3394–3536 cm , while only compound 11 showed the characteristic N–H stretching vibration above 3600 cm . In general,
1
the H NMR spectra showed aromatic protons at 6.70–7.02 ppm in the expected multiplicities, the C–H vinylic proton of
compounds 7–12 as a multiplet at 5.91–6.13 ppm, while the CH vinylic protons were found at 5.02–5.40 ppm, and the
2
chemical shifts of the hydroxyl group of compounds 4, 5, 12, and 13 appeared as singlets at 5.05–6.31 ppm. The methoxy
group protons of compounds 4–13 were found as singlets in the range 3.83–3.99 ppm.
O
N
O
N
O
N
H
3
H
-H O
2
O
O
O
O
H
O
CH
O
CH
CH
O
OH
CH
O
OH
OH
OH
B:
3
3
3
O
12
O N
O
2
O N
2
O N
2
N
H
H
H
HB
O
2
Scheme 3. Mechanistic proposal for formation of 12 or 13.
634

TABLE 1. Evaluation of the Antimycobacterial Activity of Compounds 1–13 and Ethambutol (reference drug)
LogPb
LD50
Compound
LogPb
LD50
c
c
MICa, μM
MICa, μM
Compound
Res
610
Res
Res
Res
139
Res
Res
2.57
2.57
1.33
2.47
2.84
4.23
3.53
3.65
1930
880
520
2200
2000
686
1670
1670
Res
Res
Res
361
478
237
15
3.32
2.56
3.39
3.8
2.65
2.9
500
372
184
372
1930
400
1
2
8
9
3
10
3a
4
11
12
13
5
Ethambutol
0.06
998
6
7
______
a
b
The maximum concentration tested equal to 100 μg/mL, considering any value above that as resistant; calculated using
c
ChemBioDraw Program version 12.02.1076; the predicted toxicity (mg/kg) was done using the virtual lab program, PRO-TOX II.
From Table 1, it can be observed that the hydroxyl group seems important for the anti-TB activity, as shown by the
active phenolic compounds 5 (139 μM), 12 (478 μM), and 13 (237 μM) and the inactivity of compounds 7–10. However, the
inactivity of eugenol (1) and the reduced activity of o-eugenol (2), both phenolic compounds, suggests that other factors are
important, such as the presence of strong electron withdrawing groups, which are found in compounds 5 (two bromo groups),
12, and 13 (both having nitro groups). The influence of functional groups such as bromine and nitro on different compounds
can occur in different ways. They confer varying degrees of lipophilicity and solubility to the molecules [13]. On the other
hand, both can have a positive effect on a given biological target if their electron withdrawing effect beneficially influences the
interaction of the substituted compounds with the receptor in question [13]. For various phenolic derivatives, such as those
described herein, it may be that the effect of such groups, through the electron withdrawing effect, is to increase the hydrogen
binding donor capacity of free hydroxyl, allowing an increase to activity due to higher interaction with the receptor in
question [13].
An ortho allyl group influences the activity as shown by comparing results for o-eugenol (610 μM) and eugenol
(resistant, with a value MIC > 100 μg/mL). Comparison of the activities of 12 and 13 indicates that replacing the allyl group
by propyl does increase the activity. The lipophilicity of the compounds is another factor that should be generally considered.
The derivative that displayed the best activity is compound 5 (139 μM), which has the highest log P value. However, no
correlation between activities and log P can be realistically drawn from the data presented in Table 1. Compounds with values
of LD greater than 200 are considered nontoxic.
50
EXPERIMENTAL
General. Melting points were determined using an MQAPF-302 (MicroQuimica Ltd., Santa Catarina, Brazil) apparatus
and are uncorrected. NMR spectra were analyzed using 400 and 500 MHz Bruker AC spectrometers with tetramethylsilane as
internal standard. Infrared spectra were obtained using a Thermo Nicolet 6700 spectrometer. High-resolution mass spectra
were acquired on a Bruker compact-Tof. The progress of the reactions was monitored by thin-layer chromatography (TLC) on
2.0 cm × 6.0 cm aluminum sheets (silica gel 60, HF-254, Merck) with a thickness of 0.25 mm, using ultraviolet light irradiation.
For column chromatography, Merck silica gel (70–230 or 230–400 mesh) was used. Solvents and reagents were used without
further purification.
1-(Allyloxy)-2-methoxybenzene (3a). A reaction mixture of guaiacol (3) (63 g, 0.5 mol), allyl bromide (66 g, 0.55 mol),
and K CO (69 g, 0.5 mol) in CH COCH (100 mL) was refluxed for 8 h. After rotary evaporation, H O (200 mL) was added,
2
3
3
3
2
and the mixture was extracted using Et O (2 × 100 mL). The organic phase was collected, washed with 10% w/v NaOH
2
solution (2 × 100 mL), dried over anhydrous Na SO , and evaporated under reduced pressure to afford 1 (70.8 g, 85%) as a
2
4
yellow oil. Spetral data were compared with the corresponding published data [14].
Syntheses of o-Eugenol (2) and Eugenol (1). Compound 3a (70 g, 0.43 mol) was heated at 200°C with stirring for 1 h
and 30 min, PhCH (100 mL) was added, and the mixture was extracted using 10% w/v NaOH solution. The basic extract was
3
acidified using dilute H SO (200 mL 1:1) and extracted using EtOAc (3 × 100 mL). The organic extracts were evaporated,
2
4
dried over anhydrous Na SO , and distilled under reduced pressure (75–77°C/0.5 mmHg). The oil containing a mixture of the
2
4
635

regioisomers eugenol and o-eugenol was submitted to chromatographic purification on silica gel (SiO , 70–230 mesh,
2
n-hexane–EtOAc, 75.5:2.5) to separate o-eugenol (2), which was obtained as a yellow oil (58.8 g, 84%). Eugenol (1) was
obtained as a yellow oil (7.0 g, 10%). Spetral data were compared with the corresponding published data [14].
2-Methoxy-6-propylphenol (4). A solution of o-eugenol (2, 450 mg, 2.7 mmol) in EtOH (6 mL) was hydrogenated
in the presence of palladium on active carbon (10%, 1 mol%) under stirring at room temperature for 21 h. Then, the reaction
medium was filtered through Celite, and the filtrate was evaporated under reduced pressure to provide 4, a colorless oil
–1
1
(330 mg, 72%). IR spectrum (ν, cm ): 3496 (OH). H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 6.70–6.69 (3H, m, H-3, 4, 5),
3
5.05 (1H, br.s, OH), 3.87 (3H, s, OCH ), 2.61 (2H, t, J = 7.6, H-1′), 1.64 (2H, sex, J = 7.6, H-2′), 0.96 (3H, t, J = 7.3, H-3′).
3
13
C NMR (100 MHz, CDCl , δ, ppm): 146.29 (C-2), 143.55 (C-1), 128.51 (C-6), 122.39 (C-5), 119.04 (C-4), 108.17 (C-3),
55.95 (OCH ), 31.90 (C-1′), 22.91 (C-2′), 14.05 (C-3′). Mass spectrum (EI, 70 eV), m/z (I , %): 166 (M , 84), 138 (21), 137
(100), 122 (17), 77 (13). HR-ESI-MS m/z 165.09 [M – H] (calcd for C H O , 165.09).
3
+
3
rel
–
10 13
2
3,4-Dibromo-6-methoxy-2-propylphenol (5). A solution of compound 4 (150 mg, 0.9 mmol) in CH Cl (5 mL) was
2
2
cooled to –70°C in a dry ice/acetone bath; Br (176 mg, 1.1 mmol) was added to this solution, and the reaction mixture was
2
stirred for 1 h. An aqueous solution of Na S O (5 mL of 10% w/v solution) was added. The reaction mixture was extracted
2 2
3
with CH Cl (3 × 10 mL), and the combined organic phases were dried over anhydrous Na SO and evaporated under reduced
2
2
2
4
pressure. The residue was purified by a chromatographic column on silica gel (70–230 mesh, 10% v/v acetone–n-hexane) to
–1
1
furnish 5 as a yellow oil (231 mg, 79%). IR spectrum (ν, cm ): 3536 (OH). H NMR (500 MHz, CDCl , δ, ppm, J/Hz): 7.01
3
(1H, s, H-5), 5.72 (1H, s, OH), 3.87 (3H, s, OCH ), 2.84 (2H, t, J = 7.85, H-1′), 1.57 (2H, sex, J = 7.37, H-2′), 0.99 (3H, t,
3
13
J = 7.37, H-3′). C NMR (125 MHz, CDCl , δ, ppm): 145.84 (C-6), 143.24 (C-1), 130.27 (C-2), 118.68 (C-3), 114.27 (C-4),
112.88 (C-5), 56.30 (OCH ), 33.25 (C-1′), 21.84 (C-2′), 14.05 (C-3′). Mass spectrum (EI, 70 eV), m/z (I , %): 324 (M , 100),
293 (52), 197 (50), 126 (43). HR-ESI-MS m/z 322.9112 [M – H] (calcd for C H Br O , 322.9105).
3
+
3
rel
–
10 11
2 2
1-Allyl-2-(allyloxy)-3-methoxybenzene (6). A mixture of o-eugenol (2, 500 mg, 3 mmol), K CO (1.240 mg, 9 mmol),
2
3
and allyl bromide (400 mg, 3.3 mmol) in 20 mL of CH COCH (20 mL) was refluxed for 14 h. After rotary evaporation, H O
3
3
2
(15 mL) was added and the whole extracted with Et O (2 × 15 mL). The organic phase was washed with aqueous NaOH
2
solution (10% w/v, 2 × 15 mL). The organic solution was dried over anhydrous Na SO and evaporated under reduced pressure
2
4
1
to furnish a yellow oil (550 mg, 88%). H NMR (500 MHz, CDCl , δ, ppm, J/Hz): 6.99 (1H, t, J = 7.9, H-5), 6.77–6.80 (2H,
3
m, H-4, 6), 6.08–6.12 (1H, m, H-2′′), 5.94–6.00 (1H, m, H-2′), 5.37 (1H, dd, J = 17.2, 1.6, H-3′′
), 5.22 (1H, dd, J = 10.4,
trans
1.3, H-3′′ ), 5.03–5.07 (2H, m, H-3′), 4.48 (2H, dt, J = 5.7, 1.25, H-1′′), 3.85 (3H, s, OCH ), 3.42 (2H, d, J = 6.6, H-1′).
cis
3
13
C NMR (125 MHz, CDCl , δ, ppm): 152.83 (C-2), 145.84 (C-3), 137.30 (C-2′), 134.48 (C-2′′), 134.16 (C-1), 123.86 (C-6),
3
121.95 (C-5), 117.13 (C-3′′), 115.52 (C-3′), 110.42 (C-4), 73.75 (C-1′′), 55.73 (OCH ), 34.23 (C-1′). Mass spectrum (EI, 70 eV),
3
+
+
m/z (I , %): 204 (M , 72), 163 (100), 135 (40), 103 (72), 91 (43). HR-ESI-MS m/z 227.1062 [M + Na] (calcd for C H NaO ,
rel
13 16
2
227.1048).
1-Allyl-2-(3-chloropropoxy)-3-methoxybenzene (7).Areaction mixture of o-eugenol (2, 300 mg, 1.8 mmol), K CO
2
3
(760 mg, 5.5 mmol), 1-bromo-3-chloropropane (860 mg, 5.5 mmol), and CH COCH (15 mL) was refluxed for 25 h.
3
3
After evaporation of the solvent under reduced pressure, H O (15 mL) was added and extraction was effected with Et O
2
2
(2 × 15 mL). The organic phase was washed with 10% w/v NaOH solution (2 × 15 mL), dried over anhydrous Na SO , and
2
4
1
evaporated under reduced pressure to give a colorless oil (380 mg, 86%), without further purification. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 6.99 (1H, t, J = 7.9, H-5), 6.76–6.80 (2H, m, H-4, 6), 5.90–6.00 (1H, m, H-2′), 5.02–5.07 (2H, m, H-3′),
3
13
4.07 (2H, t, J = 5.8, H-1′′), 3.80–3.83 (5H, m, OCH , H-3′′), 3.41 (2H, d, J = 6.5, H-1′), 2.18–2.30 (1H, m, H-2′′). C NMR
3
(100 MHz, CDCl , δ, ppm): 152.74 (C-2), 145.81 (C-3), 137.23 (C-2′), 133.98 (C-1), 123.98 (C-6), 122.04 (C-5), 115.59 (C-3′),
3
110.41 (C-4), 69.21 (C-1′′), 55.69 (OCH ), 41.92 (C-3′′), 34.03 (C-1′), 33.46 (C-2′′). Mass spectrum (EI, 70 eV), m/z (I , %):
3
rel
+
+
240 (M , 100), 164 (80), 149 (42), 131 (44), 103 (42). HR-ESI-MS m/z 263.0819 [M + Na] (calcd for C H ClNaO ,
13 17
2
263.0815).
1-(3-(2-Allyl-6-methoxyphenoxy)propyl)piperazine (8). A reaction mixture of compound 7 (300 mg, 1.2 mmol),
K CO (520 mg, 3.8 mmol), and 210 mg of piperazine (2.4 mmol) in CH CN (10 mL) was refluxed, with magnetic stirring for
2
3
3
19 h. The solvent was removed under reduced pressure, H O (20 mL) was added, and the mixture was extracted with EtOAc
2
(3 × 15 mL). The organic phase was dried over anhydrous Na SO and evaporated under reduced pressure. The residue was
2
4
purified by a chromatographic column on silica gel (230–400 mesh; 10% v/v MeOH–CHCl ) to furnish the product as a
3
1
yellow oil (127 mg, 35%). H NMR (500 MHz, CDCl , δ, ppm, J/Hz): 6.98 (1H, t, J = 7.9, H-4), 6.76–6.78 (2H, m, H-3, 5),
3
5.93–5.98 (1H, m, H-2′), 5.02–5.07 (2H, m, H-3′), 3.97 (2H, t, J = 6.4, H-1′′), 3.83 (3H, s, OCH ), 3.41 (2H, d, J = 6.4, H-1′),
3
13
2.94 (4H, t, J = 4.7, H-5′′), 2.57 (2H, t, J = 7.4, H-3′′), 1.95 (3H, qt, J = 6.4, H-2′′). C NMR (125 MHz, CDCl , δ, ppm):
3
636

152.81 (C-1), 146.07 (C-6), 137.36 (C-2′), 134.03 (C-2), 123.77 (C-3), 121.99 (C-4), 115.51 (C-3′), 110.43 (C-5), 71.20 (C-1′′),
55.73 (C-3′′), 55.71 (C-4′′), 54.04 (OCH ), 45.80 (C-5′′), 34.07 (C-1′), 27.41 (C-2′′). Mass spectrum (EI, 70 eV), m/z (I , %):
3
rel.
+
+
290 (37), 127 (12), 99 (M , 100), 84 (12), 70 (14). HR-ESI-MS m/z 291.2105 [M + H] (calcd for C H N O , 291.2073).
17 27
2 2
3-(2-Allyl-6-methoxyphenoxy)-N-methylpropan-1-amine (9). To a solution of compound 7 (300 mg, 1.2 mmol) in
MeOH (5 mL) was added a 40% aqueous solution of CH NH (7.5 mL). The reaction mixture was stirred at room temperature
3
2
for 48 h, H O (10 mL) was added, and the mixture was extracted with EtOAc (3 × 15 mL). The organic phase was dried over
2
anhydrous Na SO and evaporated under reduced pressure, affording a residue which was purified by a chromatography
2
4
1
column on silica gel (230–400 mesh; 10% v/v MeOH–CHCl ) to furnish 9 as a yellow oil (56 mg, 19%). H NMR (500 MHz,
3
CDCl , δ, ppm, J/Hz): 7.02 (1H, t, J = 7.9, H-4), 6.78–6.80 (2H, m, H-3, 5), 5.90–5.98 (1H, m, H-2′), 5.02–5.06 (2H, m, H-3′),
3
4.02 (2H, t, J = 5.6, H-1′′), 3.90 (3H, s, OCH ), 3.40 (2H, d, J = 6.1, H-1′), 3.12 (2H, t, J = 6.2, H-3′′), 2.65 (3H, s, H-4′′), 2.13
3
13
(2H, qt, J = 5.97, H-2′′). C NMR (125 MHz, CDCl , δ, ppm, J/Hz): 147.61 (C-1), 140.72 (C-6), 132.35 (C-2′), 129.20 (C-2),
3
119.54 (C-3), 117.52 (C-4), 111.03 (C-3′), 105.52 (C-5), 66.74 (C-1′′), 51.09 (OCH ), 44.05 (C-3′′), 29.84 (C-4′′), 29.15 (C-1′),
3
23.16 (C-2′′). Mass spectrum (EI, 70 eV), m/z (I , %): 164 (44), 103 (20), 91 (24), 72 (100), 70 (30). HR-ESI-MS m/z 236.1776
rel
+
[M + H] (calcd for C H NO , 236.1651).
14 22
2
3-(2-Allyl-6-methoxyphenoxy)-N-propylpropan-1-amine (10). A reaction mixture of compound 7 (200 mg,
0.8 mmol), propylamine (229 mg, 1.7 mmol), K CO (740 mg, 12.5 mmol), and CH CN (3.5 mL) was refluxed with stirring
2
3
3
for 67 h. After evaporation of the solvent under reduced pressure, H O (10 mL) was added, and the mixture was extracted with
2
EtOAc (3 × 15 mL). The organic phase was dried over anhydrous Na SO and evaporated under reduced pressure. The residue
2
4
was purified by a chromatographic column on silica gel (230–400 mesh; 10% v/v MeOH–CHCl ) to furnish 10 as a yellow oil.
3
1
H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 7.01 (1H, t, J = 7.9, H-4), 6.77–6.80 (2H, m, H-3, 5), 5.92–5.98 (1H, m, H-2′), 5.06
3
(1H, t, J = 1.44, H-3′), 5.02–5.05 (1H, m, H-3′), 4.23 (1H, br.s, NH), 4.01 (2H, t, J = 5.8, H-1′′), 3.88 (3H, s, OCH ), 3.40 (2H,
3
d, J = 6.5, H-1′), 3.06 (2H, t, J = 6.7, H-3′′), 2.78 (2H, t, J = 7.5, H-4′′), 2.09 (2H, qt, J = 6.1, H-2′′), 1.68 (2H, sex, J = 7.4,
13
H-5′′), 0.95 (3H, t, J = 7.4, H-6′′). C NMR (100 MHz, CDCl , δ, ppm): 152.42 (C-1), 145.57 (C-6), 137.13 (C-2′), 134.00
3
(C-2), 124.22 (C-3), 122.23 (C-4), 115.73 (C-3′), 110.28 (C-5), 71.74 (C-1′′), 55.80 (OCH ), 50.70 (C-4′′), 47.04 (C-3′′),
3
33.91 (C-1′), 28.42 (C-2′′), 21.82 (C-5′′), 11.54 (C-6′′). Mass spectrum (EI, 70 eV), m/z (I , %): 263 (29), 234 (23), 100 (48),
rel
+
+
72 (M , 100), 70 (22). HR-ESI-MS m/z 264.1986 [M + H] (calcd for C H NO , 264.1964).
16 26
2
N-(3-(2-Allyl-6-methoxyphenoxy)propyl)butan-1-amine (11). A solution of compound 7 (200 mg, 0.8 mmol) and
butylamine (3 mL, 30 mmol) was stirred at 70°C for 18 h. After addition of H O (15 mL), the reaction mixture was extracted
2
with EtOAc (3 × 15 mL). The organic phases were collected, dried over anhydrous Na SO , and evaporated under reduced
2
4
pressure. The residue was purified by a chromatographic column on silica gel (230–400 mesh; 10% v/v MeOH–CHCl ) to
3
1
furnish 11 as a yellow oil (90 mg, 39%). H NMR (500 MHz, CDCl , δ, ppm, J/Hz): 7.00 (1H, t, J = 7.9, H-4), 6.77–6.79 (2H,
3
m, H-3, 5), 5.93–5.99 (1H, m, H-2′), 5.02–5.06 (2H, m, H-3′), 4.00 (2H, t, J = 5.9, H-1′′), 3.87 (3H, s, OCH ), 3.41 (2H, d,
3
J = 6.5, H-1′), 2.95 (2H, t, J = 6.7, H-3′′), 2.72 (2H, t, J = 7.4, H-4′′), 2.02 (2H, qt, J = 6.3, H-2′′), 1.56 (2H, qt, J = 7.4, H-5′′),
13
1.36 (2H, sex, J = 7.6, H-6′′), 0.92 (3H, t, J = 7.35, H-7′′). C NMR (100 MHz, CDCl , δ, ppm): 152.60 (C-1), 145.82
3
(C-6), 137.23 (C-2′), 134.01 (C-2), 124.00 (C-3), 122.12 (C-4), 115.63 (C-3′), 110.34 (C-5), 71.62 (C-1′′), 55.76 (OCH ),
3
49.25 (C-4′′), 47.15 (C-3′′), 33.97 (C-1′), 31.52 (C-5′′), 29.51 (C-2′′), 20.41 (C-6′′), 13.93 (C-7′′). Mass spectrum (EI, 70 eV),
+
+
m/z (I , %): 277 (63), 234 (51), 114 (68), 86 (M , 100), 70 (31). HR-ESI-MS m/z 278.2148 [M + H] (calcd for C H NO ,
rel
16 26
2
278.2120).
6-Allyl-2-methoxy-3-nitrophenol (12). Fuming HNO (230 mg, 3.6 mmol) was added carefully to a solution of
3
o-eugenol (2, 200 mg, 1.2 mmol) in CH Cl (5 mL). The reaction mixture was stirred at room temperature for 1 h and 30 min.
2
2
After addition of an aqueous solution of K CO (20 mL, 10% w/v), the reaction mixture was extracted with CH Cl
2
3
2
2
(3 × 20 mL). The reunited organic phase was dried over anhydrous Na SO and evaporated under reduced pressure. The
2
4
residue obtained was subjected to column chromatographic purification on silica gel (230–400 mesh, EtOAc–n-Hex 2%) to
–1
1
furnish 12 as a yellow solid (94 mg, 37%), mp 67–68°C (EtOAc). IR spectrum (ν, cm ): 3394 (OH). H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 7.79 (1H, d, J = 2.5, H-5), 7.66 (1H, d, J = 2.5, H-4), 6.31 (1H, s, OH), 5.90–5.98 (1H, m, H-2′), 5.08–5.16
3
13
(2H, m, H-3′), 3.99 (3H, s, OCH ), 3.45 (2H, d, J = 6.6, H-1′). C NMR (100 MHz, CDCl , δ, ppm): 149.36 (C-1), 145.86
3
3
(C-2), 140.56 (C-3), 134.82 (C-6), 126.16 (C-2′), 119.07 (C-5), 116.99 (C-3′), 104.41 (C-4), 56.58 (OCH ), 33.50 (C-1′).
3
+
Mass spectrum (EI, 70 eV), m/z (I , %): 209 (M , 100), 147 (15), 131 (15), 103 (29), 91 (22). HR-ESI-MS m/z 208.0579
rel
–
[M – H] (calcd for C H NO , 208.0610).
10 10
4
2-Methoxy-3-nitro-6-propylphenol (13). Fuming HNO (230 mg, 3.6 mmol) was added carefully to a solution of
3
compound 4 (200 mg, 1.2 mmol) in CH Cl (5 mL). The reaction mixture was stirred at room temperature for 1 h. After
2
2
addition of an aqueous solution of K CO (30 mL, 10% w/v), the reaction mixture was extracted with CH Cl (3 × 30 mL).
2
3
2
2
637

The organic phases were dried over anhydrous Na SO and evaporated under reduced pressure. The residue was subjected to
2
4
purification by column chromatographic on silica gel (230–400 mesh, EtOAc–n-Hex 2%) to furnish the product as a yellow
–1
1
solid (130 mg, 51%), mp 59–61°C (EtOAc). IR spectrum (ν, cm ): 3408 (OH). H NMR (400 MHz, CDCl , δ, ppm, J/Hz):
3
7.78 (1H, d, J = 2.6, H-5), 7.64 (1H, d, J = 2.6, H-4), 6.27 (1H, s, OH), 3.98 (3H, s, OCH ), 2.67 (2H, t, J = 7.65, H-1′), 1.67
3
13
(2H, sex, J = 7.6, H-2′), 0.97 (3H, t, J = 7.3, H-3′). C NMR (100 MHz, CDCl , δ, ppm): 149.55 (C-1), 145.76 (C-2), 140.40
3
(C-3), 128.56 (C-6), 119.15 (C-5), 104.02 (C-4), 56.52 (OCH ), 31.49 (C-1′), 22.42 (C-2′), 13.83 (C-3′). Mass spectrum
3
+
–
(EI, 70 eV), m/z (I , %): 211 (M , 83), 183 (17), 182 (100), 136 (19), 93 (12). HR-ESI-MS m/z 210.0724 [M – H]
rel.
(calcd for C H NO , 210.0766).
10 12
4
The antimycobacterial activities of the compounds guaiacol (3), intermediate 3a, o-eugenol (2), eugenol (1), and
derivatives 4–13 have been assessed against M. tuberculosis ATTC 27294 using the Micro plate Alamar BlueAssay (MABA).
This methodology is nontoxic, uses a thermally-stable reagent, and shows good correlation with proportional BACTEC
radiometric methods [15, 16]. The method is described as follows: 200 μL of sterile deionized water was added to all
outer-perimeter wells of 96 sterile well plates (falcon, 3072: Becton Dickinson, Lincoln Park, NJ) to minimize evaporation of
the medium in the test wells during incubation. The 96 plates received 100 μL of Middlebrook 7H9 broth (Difco laboratories,
Detroit, MI, USA), and successive dilution of the compounds 1–13 was made directly on the plate. The final drug concentrations
tested were 3.12 to 100 μg/mL. Plates were covered and sealed with parafilm and incubated at 37°C for 5 days. Then 25 μL of
a freshly prepared 1:1 mixture of Alamar Blue (Accumed International, Westlake, Ohio) reagent and 10% Tween 80 was then
added to the plate and the whole incubated for 24 h. A blue color in the well was interpreted as no bacterial growth, and a pink
color was scored as growth. The minimal inhibitory concentration (MIC) was defined as the lowest drug concentration that
prevented a color change from blue to pink. MIC values represent means of three separate experiments, and the variation
coefficient of the method is 9.8 %. The toxicity was predicted using the virtual lab program PRO-TOX II.
ACKNOWLEDGMENT
The authors A. M. Moraes and M. V. N. de Souza are thankful to CNPq for financial support.
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