In vitro and in vivo cytotoxicities and antileishmanial activities of thymol and hemisynthetic derivatives

Article abstract of DOI:10.1128/AAC.49.4.1652-1655.2005

The in vitro and in vivo antileishmanial and cytotoxic activities of thymol and structural derivatives in comparison to those of Glucantime were studied. The results showed here suggest that thymol and hemisynthetic derivatives have promising antileishmanial potential and could be considered as new lead structures in the search for novel antileishmanial drugs. Copyright

Full text of DOI:10.1128/AAC.49.4.1652-1655.2005

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 2005, p. 1652–1655
0066-4804/05/$08.00ϩ0 doi:10.1128/AAC.49.4.1652–1655.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 4
In Vitro and In Vivo Cytotoxicities and Antileishmanial Activities of
Thymol and Hemisynthetic Derivatives
Sara Robledo,1* Edison Osorio,1,2 Diana Mun˜oz,1 Luz Marina Jaramillo,1,2 Adriana Restrepo,1
Gabriel Arango,2 and Iv´an V´elez1
Programa de Estudio y Control de Enfermedades Tropicales PECET1 and Grupo de Investigacio´n en
Sustancias Bioactivas GISB,2 Universidad de Antioquia, Medell´ın, Colombia
Received 25 November 2004/Returned for modification 27 November 2004/Accepted 29 November 2004
The in vitro and in vivo antileishmanial and cytotoxic activities of thymol and structural derivatives in
comparison to those of Glucantime were studied. The results showed here suggest that thymol and hemisyn-
thetic derivatives have promising antileishmanial potential and could be considered as new lead structures in
the search for novel antileishmanial drugs.
Thymol, a p-cymene-derived compound, is widely used in
medicine for its antimicrobial, antiseptic, disinfectant, and
wound-healing properties (1, 3, 6, 10, 12, 16). Because deriv-
atives of p-cymene have leishmanicidal activity (2, 8, 11) and
are considered important basic structures for development of
novel antiparasitic drugs (9), in this study the thymol structure
was chemically modified and its derivated were evaluated for
cytotoxic and antileishmanial activities.
The capacity to kill Leishmania (Viannia) panamensis
(MHOM/CO/87/UA/UA140) promastigotes and U-937 was
determined by the MTT [3-(4,5-dimethylthiazol-2-yl)2,5-di-
phenyltetrazolium bromide] method, following a previously re-
ported methodology (19). After 96 h of incubation in the pres-
ence of the compounds, the viability of promastigotes and cells
was determined according to the formazan produced after
addition of MTT. Parasites and cells cultivated in the absence
of the compounds but maintained under the same conditions
were used as controls. A control of dimethylsulfoxide was also
included.
The effect against intracellular amastigotes was evaluated by
microscope examination of infected U-937 cells incubated in
the presence of the compounds, following a previously re-
ported methodology (15, 19). Cells grown in RPMI medium
were adjusted to 106 cells/ml. In each well of a 24-well cell
culture plate containing a 12-mm-diameter coverslip glass, 105
cells were dispensed and incubated in the presence of phorbol
myristate acetate for 48 h. Adherent cells were exposed to
promastigotes (25:1, parasite/cell) during 2 h (34°C, 5% CO2).
Nonattached cells and free parasites were washed, and fresh
medium was added; 24 h later, the medium was replaced with
medium containing the compound. The range of concentration
for every compound was selected according to the toxicity
previously observed in the U-937 cells. Infected cells were
exposed to the compounds for 96 h, changing the medium after
48 h. Cells were washed, fixed with methanol, and stained with
Giemsa. Infected cells cultivated in the absence of compounds
served as a control of infection. Two hundred cells were eval-
uated in every well. Reduction of the parasite load was deter-
mined by dividing the number of amastigotes obtained in the
presence of each compound by the number of amastigotes
obtained in the absence of them (15, 19). All the compounds
were evaluated in three independent experiments, each in trip-
licate. Their cytotoxic and antileishmanial activities were
compared with those of Glucantime. Results are expressed
as 50% effective concentrations for parasites and 50% le-
thal concentrations for cells, calculated by the probit meth-
od (7).
The in vivo effectivity was evaluated in Golden hamsters
(Mesocricetus auratus) experimentally infected with L. (V.)
panamensis. Male and female outbred adult hamsters (n ϭ 5
per group), maintained according to institutional guidelines,
were inoculated intradermically in the snout (13) with 106
infective promastigotes (14). Treatment schedules began when
lesions were conspicuous (17). Compounds were solubilized in
carboxymethylcellulose and administered orally at a dosage of
40 mg/kg of body weight/day for 10 days. A control group
received carboxymethylcellulose, and a second control group
received Glucantime by intramuscular injections at the same
dose. Animals were evaluated for lesion size prior to treatment
and at days 15 and 30 after treatment was finished by measur-
ing the diameter of the nose. Their parasitological stage was
determined by culture of material obtained from the lesions in
NNN medium (13). Animals were also bled to evaluate serum
level of glucose, uric acid, and bilirubin, and animals were
monitored for changes in behavior or weight. Finally, hamsters
were sacrificed as recommended, and the parasite burden was
estimated by a semiquantitative limiting dilution culture assay
of tissue samples adjusted to 0.1 mg/ml of medium, serially
fourfold diluted, and incubated for 4 weeks at 26°C (13, 18).
Parasite burden was expressed as the maximum dilution in
which parasites were detected by examination under an in-
verted microscope.
The hemisynthesis of the compounds was analyzed by thin-
layer chromatography, silica gel column chromatography, the
combined gas chromatography–mass spectrometry (MS) tech-
nique, and UV spectral analysis. All of the products were
1
identified by the H nuclear magnetic resonance (NMR), 13C
NMR, and MS techniques. The pathways for synthesis, the
structures of the compounds, and their chemical properties are
summarized in Fig. 1.
* Corresponding author. Mailing address: Calle 62 No. 52-59, Lab
632, Medell´ın, Colombia. Phone: (574) 210 6503. Fax: (574) 210 6511.
E-mail: srobledo@guajiros.udea.edu.co.
1652

FIG. 1. The pathway for synthesis, the structure, and the chemical properties of aromatic monoterpenes derived from thymol. T1, 6-isopropyl-
3-methyl-2,4-dinitro-phenol [C10H12N2O5, pf 51 to 52°C; UV (ethyl alcohol [EtOH]) ␭ max (log ε) 211.0 (4.12), 267.0 (3.68), 399.0 (3.87) nm;
(EtOH ϩ NaOH, 0.1 M) ␭ max (log ε) 209.0 (4.11), 262.0 (3.72), 383.0 (4.12) nm; IR (KBr) vmax 3,401 (OH), 3,037 (Ar-H), 2,969 (CH), 2,875
(CH3), 1,547 (NO2), 1,333 (NO2), 1,235 (CO, phenol); MS m/z 241 [M ϩ 1]ϩ (52), 240 [M]ϩ (15), 223 [M-OH]ϩ (100), 177 [M-OH-NO2]ϩ (19),
1
149 (12), 119 (13), 104 (7), 91[C7H7]ϩ (35), 39 (13); H NMR (CDCl3) ␦ 7.90 (1H, s, H-5), 3.40 (1H, sep, J ϭ 7.0 Hz, H-7), 1.30 (6H, d, J ϭ 7.0
Hz, H-8,9), 2.60 (3H, s, H-10), 9.80 (1H, br s, OH); 13C NMR JMOD (CDCl3) ␦ 154.12 (C-1), 138.18 (C-2), 126.40 (C-3), 138.18 (C-4), 127.44
(C-5), 129.00 (C-6), 27.73 (C-7), 22.24 (C-8), 22.24 (C-9), 16.86 (C-10)]; T2: 1-Isopropyl-2-methoxy-4-methyl-3,5-dinitro-benzene [C11H14N2O5. pf
41 to 42°C; UV (EtOH) ␭ max (log ε) 213.0 (4.11), 258.0 (3.65) nm; IR (KBr) vmax 3037 (Ar-H), 2970 (CH), 2873 (CH3), 1540 (NO2), 1340 (NO2),
1
1277 (CO, ether), 1,091 (COC, ether) cmϪ1; MS m/z 254 [M]ϩ (21), 237 (100), 220 (21), 205 (22), 162 (8), 121 (10), 91 (24); H NMR (CDCl3)
␦ 8.00 (1H, s, H-6), 3.35 (1H, sep, J ϭ 7.0 Hz, H-7), 1.30 (6H, d, J ϭ 7.0 Hz, H-8,9), 2.45 (3H, s, H-10), 3.90 (3H, s, OCH3); 13C NMR JMOD (CDCl3)
␦ 131.00 (C-1), 152.50 (C-2), 143.52 (C-3), 124.96 (C-4), 143.52 (C-5), 125.24 (C-6), 27.31 (C-7), 23.59 (C-8), 23.59 (C-9), 14.58 (C-10), 64.25 (OCH3)];
T3A: 5-Isopropyl-4-methoxy-2-methyl-3-nitro-phenylamine [C11H16N2O3. pf 80 to 82°C; UV (EtOH) ␭ max (log ε) 214.0 (4.11), 238.0 (4.04), 291.0 (3.41)
nm; IR (KBr) vmax 3,473 (NH2), 3,386 (NH2), 3,035 (Ar-H), 2,964 (CH), 2,870 (CH3), 1,630 (NH2), 1,526 (NO2), 1,377 (NO2), 1,240 (CO, ether), 1,109
(COC, ether) cmϪ1. MS m/z 224 [M]ϩ (100), 209 (15), 207 (17), 175 (3), 164 (10), 147 (11), 91 (3), 39 (3); 1H NMR (CDCl3) ␦ 7.24 (1H, s, H-6), 7.88
(2H, br s, NH2), 3.83 (1H, sep, J ϭ 6.9 Hz, H-7), 1.82 (6H, d, J ϭ 6.9 Hz, H-8,9), 2.60 (3H, s, H-10), 4.35 (3H, s, OCH3); 13C NMR JMOD (CDCl3) ␦
143.00(C-1), 140.80 (C-2), 146.60 (C-3), 154.10 (C-4), 141.20 (C-5), 114.53 (C-6), 26.73 (C-7), 24.04 (C-8), 24.04 (C-9), 12.18 (C-10), 64.16 (OCH3)]; T3B,
N,NЈ-bis-(5-isopropyl-4-methoxy-2-methyl-3-nitro-phenyl)-N-hydroxy-hydrazine [C22H30N4O7, pf 140 to 142°C; UV (EtOH) ␭ max (log ε) 211.0 (4.41),
238.0 sh (4.10), 320.0 (4.08) nm; IR (KBr) vmax 3,036 (Ar-H), 2,968 (CH), 2,872 (CH3), 1,532 (NO2), 1,455 (NOH), 1,340 (NO2), 1,266 (CO, ether), 1,102
(COC, ether), 1,054 (NOH) cmϪ1. MS m/z 462 [M]ϩ (12), 429 (86), 355 (88), 73 (100); 1H NMR (CDCl3) ␦ 8.10 (1H, s, H-6), 7.20 (1H, s, H-6Ј), 2.90
(2H, sep, H-7,7Ј), 0.80 (12H, d, H-8,9,8Ј,9Ј), 1.95 (3H, s, H-10), 1.87 (3H, s, H-10Ј), 3.53 (3H, s, OCH3), 3.48 (3H, s, OCH3), 3.02 (1H, br s, NH); 13
C
NMR JMOD (CDCl3) ␦ 143.28 (C-1), 123.62(C-2), 155.03 (C-3), 148.62 (C-4), 126.90 (C-5), 124.23 (C-6), 27.27 (C-7), 23.88 (C-8), 23.88 (C-9), 13.52
(C-10), 64.24 (OCH3), 139.00 (C-1Ј), 123.50 (C-2Ј), 157.16 (C-3Ј), 141.78 (C-4Ј), 126.00 (C-5Ј), 122.62 (C-6Ј), 27.27 (C-7Ј), 23.72 (C-8Ј), 23.72 (C-9Ј), 13.14
(C-10Ј), 64.10 (OCH3)]; T3C, 5-isopropyl-4-methoxy-2-methyl-benzene-1,3-diamine [C11H18N2O. pf 94 to 95°C; UV (EtOH) ␭ max (log ε) 212.0 (4.08),
290.0 (3.52) nm; IR (KBr) vmax 3467 (NH2), 3376 (NH2), 3246 (NH2), 3035 (Ar-H), 2961 (CH), 2870 (CH3), 1610 (NH2), 1225 (CO, ether), 1151 (COC,
ether) cmϪ1; MS m/z 195 [M ϩ 1]ϩ (60), 194 [M]ϩ (100), 179 (95), 162 (21), 136 (10), 91 (7), 65 (7), 39 (5); 1H NMR (CDCl3) ␦ 5.80 (1H, s, H-6), 3.40
(1H, sep, J ϭ 6.9 Hz, H-7), 1.20 (6H, d, J ϭ 6.9 Hz, H-8,9), 2.20 (3H, s, H-10), 3.90 (3H, s, OCH3), 3.09 (4H, br s, NH2); 13C NMR JMOD (CDCl3) ␦
142.08 (C-1), 112.35 (C-2), 140.79 (C-3), 142.21 (C-4), 131.20 (C-5), 114.55 (C-6), 26.73 (C-7), 24.00 (C-8), 24.00 (C-9), 12.12 (C-10), 64.13 (OCH3)]; T4,
5-isopropyl-4-methoxy-2-methyl-3-nitro-phenol [C11H15NO4, pf 149 to 151°C; UV (EtOH) ␭ max (log ε) 214.0 (3.98), 278.0 (3.40) nm; (EtOH ϩ NaOH
0.1 M) ␭ max (log ε) 213.0 (4.10), 241.0 (3.93), 297.0 (3.50) nm; IR (KBr) vmax 3403 (OH), 3036 (Ar-H), 2,967 (CH), 2,872 (CH3), 1530 (NO2), 1374
(NO2), 1,197 (CO, phenol) cmϪ1; MS m/z 226 [M ϩ 1]ϩ (58), 225 [M]ϩ (100), 208 (58), 193 (2), 176 (4), 164 (8), 150 (10), 121 (18), 105 (8), 91 (18);
1H NMR (CDCl3) ␦ 6.75 (1H, s, H-6), 3.25 (1H, sep, J ϭ 6.8 Hz, H-7), 1.21 (6H, d, J ϭ 6.8 Hz, H-8,9), 2.12 (3H, s, H-10), 3.79 (3H, s, OCH3), 5.40 (1H,
br s, OH); 13C NMR JMOD (CDCl3) ␦ 151.02 (C-1), 115.21 (C-2), 142.70 (C-3), 142.70 (C-4), 129.15 (C-5), 115.50 (C-6), 27.11 (C-7), 24.26 (C-8), 24.26
(C-9), 14.60 (C-10), 64.49 (OCH3)]; T5: 1-isopropyl-2,5-dimethoxy-4-methyl-3-nitro-benzene [C12H17NO4, pf 54 to 56°C; UV (EtOH) ␭ max (log ε) 209.0
(4.01), 276.0 (3.33) nm; IR (KBr) vmax 3036 (Ar-H), 2966 (CH), 2871 (CH3), 1534 (NO2), 1373 (NO2), 1264 (CO, ether), 1091 (COC, ether) cmϪ1; MS
m/z 240 [M ϩ 1]ϩ (58), 239 [M]ϩ (100), 224 (12), 190 (3), 178 (12), 162 (17), 147 (4), 105 (7), 91 (6), 79 (2), 39 (3); 1H NMR (CDCl3) ␦ 6.80 (1H, s, H-6),
3.35 (1H, sep, J ϭ 6.9 Hz, H-7), 1.20 (6H, d, J ϭ 6.9 Hz, H-8,9), 2.10 (3H, s, H-10), 3.80 (3H, s, OCH3), 3.86 (3H, s, OCH3); 13C JMOD (CDCl3)
␦ 130.01 (C-1), 142.00 (C-2), 142.00 (C-3), 118.15 (C-4), 154.52 (C-5), 110.15 (C-6), 27.28 (C-7), 24.30 (C-8), 24.30 (C-9), 15.01 (C-10), 64.41
(OCH3), 56.81 (OCH3)]; and T6, 3-isopropyl-2,5-dimethoxy-6-methyl-phenylamine [C12H19NO2, pf 67 to 69°C; UV (EtOH) ␭ max (log ε) 208.0
(4.02), 275.0 (3.30) nm. IR (KBr) 3,469 (NH2), 3,379 (NH2), 3,036 (Ar-H), 2,966 (CH), 2,870 (CH3), 1,534 (NH2), 1,132 (CO, ether), 1,091 (COC,
1
ether) cmϪ1. MS m/z 209 [M]ϩ (100), 194 (49), 177 (7), 162 (4), 134 (8), 119 (5), 91 (1), 65 (1); H NMR (CDCl3) ␦ 6.20 (1H, s, H-6), 3.30 (1H,
m, H-7), 1.20 (6H, d, H-8,9), 2.00 (3H, s, H-10), 3.80 (3H, s, OCH3), 3.76 (3H, s, OCH3), 3.34 (2H, br s, NH2); 13C NMR JMOD (CDCl3) ␦ 140.01
(C-1), 156.10 (C-2), 139.86 (C-3), 98.38 (C-4), 156.00 (C-5), 109.70 (C-6), 27.15 (C-7), 24.58 (C-8), 24.58 (C-9), 15.01 (C-10), 61.13 (OCH3), 56.43
(OCH3)]. Reaction conditions: (a) HNO3/CH3COOH; (b) (CH3)2SO4/Na2CO3-acetone; (c) Fe/HCl-CH3OH; (d) NaNO2/H2SO4.
1653

1654
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. In vitro cytotoxicities and anti-leishmanial activities of aromatic monoterpene isoprenoidsa
EC50
ISd
c
b
Compound
R1
R2
R3
LC50
Prom
Am
Pro
Am
Thymol
T1
OH
H
H
Ͼ400.0 Ϯ 0
101.7 Ϯ 0.7
176.3 Ϯ 18.7
410.4 Ϯ 2.4
220.2 Ϯ 25.7
87.1 Ϯ 6.3
194.3 Ϯ 3.9
0.4 Ϯ 0
0.31 Ϯ 0.03
17.1 Ϯ 1.0
10.0 Ϯ 1.0
8.2 Ϯ 1.2
Ͼ100.0 Ϯ 0.0
Ͼ100.0 Ϯ 0
Ͼ100.0 Ϯ 0
400.0 Ϯ 1.2
Ͼ400.0 Ϯ 0
58.8 ؎ 7.7
176.3 Ϯ 0
318.0 Ϯ 34.3
210.0 Ϯ 0
26.7 ؎ 1.0
43.4 ؎ 1.5
131.5 Ϯ 0.9
13.6 ؎ 4.0
6.7 Ϯ 0.1
Ͼ2.0
148.1
568.7
24.0
22.0
10.6
NCe
NC
Ͼ1.0
1.7
OH
NO2
NO2
NO2
NO2
NH2
NO2
NO2
NH2
NO2
NO2
NH2
T2
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
1.0
T3A
T3B
T3C
T4
T5
T6
1.3
C
11N3H16O4
1.0
NH2
3.3
OH
376,9 Ϯ 74,4
368.3 Ϯ 85.0
23.8 Ϯ 0.4
8.7
OCH3
OCH3
2.8
1.8
62.1
NC
Glucantime
416.4 Ϯ 0
1.0
a All the data reported are the average of three independent experiments conducted in triplicate and are expressed as the mean plus or minus the standard deviation.
R, chemical substitution.
b Cytotoxic activity (50% lethal concentration) in U-937 human promonocytic cells (␮g/ml) Ϯ SD.
c Leishmanicidal activity (␮g/ml) Ϯ SD; Prom, promastigotes; Am, amastigotes. Boldface indicates the most active compounds.
d IS, index of selectivity (LC50 in U-937/50% effective concentration [EC50] in Prom or Am).
e NC, not calculated.
1
The spectra of H NMR showed the protons of the isopro-
pylic chain in the form of an A6X system. The protons of the
aromatic methyl in position para resonate as a singlet of about
2.2 to 2.4 ppm. MS studies showed peaks for the oxygenated
derivatives of p-cymene at [M-15]ϩ, due to loss of the methyl
group and the formation of a substituted hydroxytropylium
ion. The presence of the tropylium ion [C7H7]ϩ at m/z ϭ 91 is
also characteristic. Peaks were also observed at [M-43]ϩ, these
being due to the elimination of the isopropylic chain.
promastigotes was substantially different from that detected
against amastigotes (Fig. 2), but unlike Glucantime, most of
the compounds presented stronger activity against promastig-
otes than amastigotes. Given that the amastigote is the para-
sitic form responsible for the disease, it should constitute the
chemotherapeutic target in studies of new antileishmanial
agents. Most of the compounds were shown to be toxic only at
concentrations of Ͼ100 ␮g/␮l (Table 1). Indeed, the toxicity of
the T3A compound was comparable to that of Glucantime.
Although none of the compounds proved to be better than
Glucantime, a similar reduction in lesion size was obtained
after treatment with T3A; however, as can be seen in Table 2,
while Glucantime essentially eradicated the parasite from the
lesion, only thymol and T1 decreased the parasite burden, by
46 and 23%, respectively.
The cytotoxic and antileishmanial activities for each of the
compounds are summarized in Table 1. Only the T6 compound
showed leishmanicidal activity against amastigotes at concen-
trations lower than 20 ␮g/ml, and the T1, T3C, and T4 com-
pounds were active at concentrations less than 60 ␮g/ml. Sim-
ilar to previous observations (4, 5, 14, 15), the activity against
None of the compounds seem to be toxic to the animals
TABLE 2. In vivo cytotoxicities and antileishmanial activities of
aromatic monoterpene isoprenoidsa
%
% Reduction of
parasite burden 30
days posttreatment
Corporal weight (mg)
on dayb:
Reduction
of lesion
size on dayb:
Compound
Giemsa
stain
smears
Limiting
dilution
assay
0
15
30
15
30
Thymol
T1
T2
T3A
T3B
112.3
88.7
119.1
112.4
104.7
102.0
96.8
118.5
90.2
127.5
91.5
0
5.7
0
15.1
0
0
16.7
0
0
5.7
0
26.7
0
0
18.8
0
67.8
33.5
0
0
0
0
100
0
46.0
23.5
0
0
0
0
100
0
124.6
123.8
112.5
116.9
100.1
109.0
133.1
120.2
113.5
121.2
101.8
111.7
FIG. 2. Activities of monoterpenic isoprenoids: correlation be-
tween intracellular amastigotes and promastigotes of L. (V.) panamen-
sis. The mean of the results obtained in triplicate with both promas-
tigotes and intracellular amastigotes is expressed as 50% of the
effective concentration (CE50), calculated by the probit method. Spear-
man r ϭ 0.4068.
T4
Glucantime
Vehicle
110.4
a Mean of five animals per group.
b Days posttreatment.

VOL. 49, 2005
NOTES
1655
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In conclusion, these results suggest that thymol and its deriv-
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and could be considered new lead structures in the search for
novel antileishmanial drugs.
9. Kayser, O., and A. Kiderlen. 2003. Delivery strategies for antiparasitics. Exp.
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Our thanks to Ferney Ospina for synthesis of the compounds and to
Wiston Quin˜ones for 1H NMR and 13C NMR spectra. We gratefully
acknowledge J. Santiago Mej´ıa for valuable suggestions and com-
ments.
This study was funded by Colciencias (contract 1115-05-353-96), the
ECOS-Nord/ICFES/COLCIENCIAS/ICETEX programme (code no.
99SO3), and Universidad de Antioquia (code SCPP33).
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