Synthesis and in vitro cysticidal activity of new benzimidazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.05.005

Despite albendazole being the drug of choice in neurocysticercosis treatment, its low solubility limits its bioavailability; therefore, more research is required in order to find new molecules with cestocidal activity and adequate aqueous solubility. A set of 13 benzimidazole derivatives were synthesized and their in vitro activities were evaluated against Taenia crassiceps cysts, using albendazole sulfoxide as reference molecule, showing that two of them exhibited good activity. Molecular modelling revealed that the cysticidal efficacy depends on the presence on the molecule of an H in the 1-position, a planar carbamate group at 2-position, and if the substituent in 5-position is voluminous, it should be orthogonal to the benzimidazole ring.

Full text of DOI:10.1016/j.ejmech.2008.05.005

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 1794e1800
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and in vitro cysticidal activity of new
benzimidazole derivatives
a,c
Francisca Palomares-Alonso , Helgi Jung-Cook , Jaime Perez-Villanueva ,
a,c
c
´
Juan Carlos Piliado , Sergio Rodrıguez-Morales , Guadalupe Palencia-Hernandez ,
a
c
b
´
Nayeli Lopez-Balbiaux , Alicia Hernandez-Campos , Rafael Castillo ,
´
c
c
c
´
´
Francisco Hernandez-Luis
c,
*
´
a Laboratorio de Neuropsicofarmacolog´ıa, Instituto Nacional de Neurolog´ıa y Neurocirug´ıa, Me´xico DF 14269, Mexico
b Laboratorio de Neuroinmunolog´ıa, Instituto Nacional de Neurolog´ıa y Neurocirug´ıa, Me´xico DF 14269, Mexico
c Facultad de Qu´ımica, Departamento de Farmacia, Universidad Nacional Auto´noma de Me´xico, Me´xico DF 04510, Mexico
Received 14 November 2007; received in revised form 18 April 2008; accepted 5 May 2008
Available online 15 May 2008
Abstract
Despite albendazole being the drug of choice in neurocysticercosis treatment, its low solubility limits its bioavailability; therefore, more
research is required in order to find new molecules with cestocidal activity and adequate aqueous solubility. A set of 13 benzimidazole deriv-
atives were synthesized and their in vitro activities were evaluated against Taenia crassiceps cysts, using albendazole sulfoxide as reference
molecule, showing that two of them exhibited good activity. Molecular modelling revealed that the cysticidal efficacy depends on the presence
on the molecule of an H in the 1-position, a planar carbamate group at 2-position, and if the substituent in 5-position is voluminous, it should be
orthogonal to the benzimidazole ring.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Benzimidazole analogues; Cysticidal activity; Molecular modelling
1. Introduction
drug of choice for neurocysticercosis treatment, however, its
bioavailability is limited by its low solubility in the gastroin-
testinal tract; therefore, a high inter-individual variability in
plasma levels and in efficacy has been found [7]. Considering
that the search for new alternatives is still necessary, we
synthesized 13 benzimidazole derivatives and their activities
against Taenia crassiceps cysts were evaluated using albenda-
zole sulfoxide 1, which is the active metabolite of ABZ [8], as
reference molecule. Also to understand the requirements for
activity a molecular modelling study was performed.
Cysticercosis, the infection caused by the larval stage of the
tapeworm Taenia solium, is the most common parasitic
disease of the nervous system in humans and the single most
common cause of acquired epileptic seizures in the developing
world, specially Latin America, India, Africa, and China [1,2].
The treatment of neurocysticercosis has evolved from surgical
therapy in the past to anticysticercal chemotherapy. Praziquan-
tel and albendazole (ABZ) are the drugs commonly used for
the treatment of this disease [3,4], and both undergo extensive
metabolism by cytochrome P450 [5,6]. Up-to-date ABZ is the
2. Chemistry
Compound 1 was obtained by the treatment of ABZ with 3-
chloroperbenzoic acid. Compound 2 was prepared by the meth-
ylation of ABZ with methyl iodide in methanol in the presence
of potassium hydroxide. For the synthesis of compound 3, the
* Corresponding author. Tel.: þ55 56063822.
E-mail addresses: ingpalomares@yahoo.com.mx (F. Palomares-Alonso),
´
franher@servidor.unam.mx (F. Hernandez-Luis).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.05.005

F. Palomares-Alonso et al. / European Journal of Medicinal Chemistry 44 (2009) 1794e1800
1795
used as a model for the evaluation of cestocidal drugs [15],
and albendazole sulfoxide 1 was used as the standard drug.
We found that among the 13 investigated compounds, only 5,
6 and 8 showed activity against T. crassiceps cysts of ORF strain
(Table 1). The cysticidal activity of 5 and 6 was close to that
exhibited by 1; while 8 showed a lower activity. Compound 9
was not evaluated due to its insolubility in the culture media.
Those compounds that exhibited activity against T. crassiceps
cysts of ORF strain were evaluated on WFU strain (which has
scolex). We found that 5, 6 and 8 also exhibited activity against
T. crassiceps of WFU strain. To note, 5 behaved similarly as 1,
while the activity of 6 and 8 was lower. Interestingly the activity
of compounds in this strain was lower. This fact could be due to
the size of the cysts, since cysts of WFU strain are larger (3e
5 mm) than ORF cysts (2e3 mm) or due to the presence of the
scolex, which has been suggested to have a specialized nutri-
tional function and could help the parasite to resist the drugs’
action [16,17].
The in vitro study results showed that the introduction of
a methyl group instead of an H moiety at 1-position
completely abolished the activity (3 and 4). In the case of 2
when the H atom in the N-carbamate was replaced with
a methyl group, the activity decreased drastically, indicating
that the protons in N-carbamate and in 1-position are neces-
sary for the cysticidal activity. Also the derivatives containing
methylthio instead of N-carbamate group (10e13) at 2-
position were all inactive, confirming the importance of this
group for the activity of the molecule. When the effect of
the substituents at the 5-position was evaluated, we found
that when the propylthio group at 5-position was replaced
with larger groups such as 4-nitrobenzoyl carboxylate or
piperidinyl-carboxamide (5 or 6) the activity was conserved
and was close to that exhibited by 1. In the case of 8, the
presence of a chlorine atom in the benzimidazole ring reduced
the activity significantly; while the introduction of the N-
methylcarboxamide abolished the activity of 7.
Scheme 1. Synthesis of compound 3. Reagents and conditions: (a)
(CH3CO)2O, HNO3; (b) (CH3)2SO4/NaOH; (c) H2SO4, D; (d) C3H7SH; (e)
H2/CePd 5%; (f) 2-methyl-2-thiopseudourea hemisulfate salt, methyl chloro-
formate, pH ¼ 8, D.
sequence shown in Scheme 1 was followed. The synthesis began
with the nitration of 3-chloroaniline by using fuming nitric acid
and acetic anhydride, the product was treated with dimethyl
sulfate and KOH to give the N-methylated acetamide 15. The
hydrolysis of this compound with H2SO4 led to N-methyl-2-
nitroaniline 16. Aromatic nucleophilic substitution in 16 with
1-propanethiol afforded 17 which upon catalytic reduction
with hydrogen and CePd 5% gave 1,2-phenylenediamine 18.
Reaction of this compound with 2-methyl-2-thiopseudourea
hemisulfate salt, methyl chloroformate and NaOH yielded 3.
Compounds 4e9 were prepared as described in Scheme 2.
The required substituted 1,2-phenylenediamines (23e26)
were prepared by the reduction of the corresponding 2-
nitroanilines (19e22) with hydrogen in the presence of 5%
palladium on carbon. The products of reduction without isola-
tion were cyclocondensed with 2-methyl-2-thiopseudourea
hemisulfate salt, methyl chloroformate and NaOH to give the
corresponding methyl benzimidazole-2-carbamates 4, 8, 9 and
27. Compound 27 was converted to acylchlorides with SOCl2
and then dehydrohalogenation between this acylchloride and
4-nitrobenzyl alcohol or piperidine or diethylamine gave 5, 6
or 7, respectively. Finally compounds 8e14 were prepared by
known procedures [9,10,14].
3.2. Molecular modelling studies
In an attempt to identify the structural requirements for the
cysticidal activity, molecular studies were performed on
compounds 1e9. Compounds 10e14 were excluded since
these molecules with methylthio group at 2-position were
ineffective. Although compounds 2e4 resulted ineffective,
they were included in the analysis due to the presence of the
methyl carbamate group at 2-position.
3. Results and discussion
The conformational analysis was performed and the main
molecular descriptors were obtained. All the molecular de-
scriptors were correlated with the in vitro activity against T.
crassiceps cysts using the QSAR module in Sybyl V. 7.3.
Fig. 1 represents the lowest energy conformer of compounds
1e4 for comparison of the substituents at 1- and 2-positions.
Fig. 2 shows the lowest energy conformer of compounds 1
and 5e9. These figures provide visual evidence for the struc-
tural similarities and differences between these compounds. It
can be seen that the introduction of the methyl group in both
positions, N-carbamate (2) or at benzimidazole 1-position
All compounds synthesized (1e14) were obtained as solids
with sharp melting points and their structures were established
by IR, 1H NMR and MS. Compounds 1, 8e14 were character-
ized and the melting points and spectroscopic data were com-
pared with those previously reported by our group [11e14].
3.1. In vitro cysticidal activity
The in vitro cysticidal activity evaluations were performed on
T. crassiceps cysts (ORF strain), which have been previously

1796
F. Palomares-Alonso et al. / European Journal of Medicinal Chemistry 44 (2009) 1794e1800
R1
R1
NO2
NH2
a
R2
R2
NH
R3
NH
R3
19: R1 = SC3H7, R2 = H, R3 = CH3
20: R1 = Cl, R2 =R3 = H
23
: R1 = SC3H7, R2 = H, R3 = CH3
24: R1 = Cl, R2 = R3 = H
21: R1 = 1-naphthyloxy, R2 = Cl, R3 = H
22: R1 = COOH, R2 = R3 = H
25: R1 = 1-naphthyloxy, R2 = Cl, R3 = H
26: R1 = COOH, R2 = R3 = H
b
27
c
R1
N
R1
NHCOOCH3
N
R2
N
NHCOOCH3
R3
N
H
4: R1 = SC3H7, R2 = H, R3 = CH3
8: R1 = Cl, R2 = R3 = H
5: R1 = COOCH2C6H4NO2
6: R1 = CONC5H10
9: R1 = 1-naphthyloxy, R2 = Cl, R3 = H
27: R1 = COOH, R2 = R3 = H
7: R1 = CONHCH3
Scheme 2. Synthesis of compounds 4e9. Reagents and conditions: (a) H2/CePd 5%; (b) 2-methyl-2-thiopseudourea hemisulfate salt, methyl chloroformate,
pH ¼ 8, D (c) thionyl chloride.
(compounds 3 and 4) (Fig. 1a), precludes the planarity of the
carbamate group which is observed in 1 (Fig. 1b). This feature
could be important since the theoretical studies have shown
that a planar conformation of 1 could facilitate its accommo-
dation into the cleft of the Haemonchus contortus b-tubulin
model [18].
McCracken and Lipkowitz in H. diminuta [19], which reported
a good correlation between the HOMO and PSA descriptors
with the anthelmintic activity. This fact could be due to the
differences in the biological evaluation parameters used, since
the authors of Ref. (9) used the dose that is required to reduce
the worm burden by 75%, while we used a fixed concentration
of all compounds. Other factors could be due to the differences
between the cestodes. More studies should be done with the
active compounds in order to evaluate the molecular descrip-
tors for cysticidal activity.
We also found that the cysticidal activity was maintained
when groups at 5-position could adopt the orthogonal confor-
mation to the plane of the benzimidazole ring [4-nitrobenzyl
carboxylate (5) and piperidinyl-carboxamide (6) (Fig. 2a)].
Our data are in agreement with those obtained by McCracken
and Lipkowitz, in Hymenolepis diminuta [19], who found that
those anthelmintics which have substituents at 5-position
twisted out-of-plane are more active than those which have
substituents at 5-position in plane. Although, in compound 8
(Fig. 2a), the substituent in 5-position was planar, the modest
cysticidal activity could be explained considering that the
chlorine atom could accommodate into the b-tubulin cleft.
Compound 7 resulted to be inactive mainly due to the presence
of the group N-methylcarboxamide at 5-position of the benz-
imidazole ring that is not orthogonal in the plane. Although
the conformational study showed that 9 complied with the
presence of the protons in N-carbamate and in 1-position of
benzimidazole ring as well as with the ‘‘L’’ shape at 5-position
(Fig. 2b), its activity was limited due to its low solubility. This
characteristic is very common in benzimidazoles and should
also be considered in the design of new derivatives.
4. Conclusion
In summary we found that among the investigated
compounds, 5 and 6 showed good cysticidal activity. These
outcomes are very promising and our current efforts will focus
on further evaluation to explore their in vivo efficacy. Addi-
tionally our results suggest that for cysticidal activity the
molecule must have an H at 1-position, a methyl carbamate
group at 2-position and an orthogonal substituent at 5-position.
5. Experimental protocols
5.1. Chemistry
Melting points were determined in one-end open capillary
tubes on a Buchi B-540 melting point apparatus and are uncor-
rected.
¨
Reactions were monitored by TLC on 0.2 mm
The molecular descriptors of 1 and 5e9 are listed in Table
2. Correlations of descriptors with determined experimental
activity were performed in order to identify which molecular
features have influence on the cysticidal activity, and only
a weak correlation with HOMO was found (r ¼ 0.399). These
results did not agree with a previous study realized by
precoated silica gel 60 F254 plates (E. Merck). The spots
were visualized under ultraviolet lamp. IR spectra were
recorded on a Perkin Elmer FT-IR-1600 spectrophotometer
1
as potassium bromide discs. H NMR and 13C NMR spectra
were measured with a Varian EM-390 (300 and 75.5 MHz)

Table 1
Structure and biological activity of compounds 1e14 against T. crassiceps ORF and WFU strains
R1
N
R4
R2
N
R3
Compound
R1
R2
R3
R4
Cysts’ mortality (%)
ORF strain
WFU strain
0.28 mM
1.70 mM
0.28 mM
1.70 mM
1
2
3
4
S(O)C3H7
SC3H7
H
H
H
NHCO2CH3
N(CH3)CO2CH3
NHCO2CH3
NHCO2CH3
46 ꢀ 5
88 ꢀ 7
25 ꢀ 2.3
ND
ND
35 ꢀ 2.3
ND
ND
H
H
0
0
0
0
0
0
SC3H7
H
CH3
CH3
SC3H7
ND
ND
O
O
5
6
H
H
H
H
NHCO2CH3
NHCO2CH3
41 ꢀ 4.6
37 ꢀ 6.1
68 ꢀ 7
62 ꢀ 8
22.6 ꢀ 2.3
9.3 ꢀ 2.3
26 ꢀ 4
NO
2
O
16.7 ꢀ 3
N
O
CH3
NH
7
8
9
H
H
H
H
NHCO2CH3
NHCO2CH3
NHCO2CH3
0
0
ND
ND
Cl
H
15 ꢀ 2.3
23 ꢀ 2.3
5 ꢀ 2.3
ND
8 ꢀ 0
ND
O
Cl
ND
ND
O
10
11
H
H
H
SCH3
SCH3
0
0
0
0
ND
ND
ND
ND
N
N
O
Cl
O
12
H
H
SCH3
0
0
ND
ND
N
CH3
O
13
14
Cl
H
H
H
SCH3
SCH3
0
0
0
0
ND
ND
ND
ND
N
CH3
CH3
O
N
N
‘‘ND’’ indicates that the in vitro activity was not determined.

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F. Palomares-Alonso et al. / European Journal of Medicinal Chemistry 44 (2009) 1794e1800
Fig. 1. Lowest energy conformer of compounds 1e4. (a) Compounds modelled. (b) Superimposition of 1 (yellow), 2 (magenta), 3 (cyan) and 4 (purple), which
were colored for better understanding. Methyl carbamate group of benzimidazole is planar, while the carbamate group of compounds 2e4 is outside the plane due
to the steric effect of methyl group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
spectrometer. Chemical shifts (d) are given in parts per million
(ppm) using tetramethylsilane (Me4Si) as an internal standard;
J values are given in Hz. The following abbreviations are used:
s, singlet; d, doublet; q, quartet; dd, doublet of doublet; t,
triplet; m, multiplet; bs, broad signal. MS were recorded on
a JEOL JMS-SX102A spectrometer by electron impact (EI)
or fast atomic bombarded (FAB). Starting materials 3-
chloroaniline, 20, 22 and ABZ were purchased from Sigma
Aldrich Company.
J ¼ 7.5 Hz), 2.904 (t, 2H, CH3CH2CH2S, J ¼ 7.2 Hz), 3.660
(s, 3H, CH3), 3.796 (s, 3H, OCH3), 7.226 (d, 1H, H-4,
J ¼ 7.8 Hz), 7.249 (dd, 1H, H-5, J5,4 ¼ 7.5, J5,7 ¼ 1.2 Hz),
7.286 (d, 1H, H-7, J5,7 ¼ 1.2 Hz), 9.631 (s, 1H, exchangeable
with D2O) ppm; 13C NMR (CDCl3) d: 13.283, 22.500,
28.581, 37.501, 52.755, 111.126, 111.314, 125.886,
127.062, 131.010, 131.170, 153.673, 163.238 ppm; FAB-MS
m/z: 280 [M þ 1]þ.
5.1.3. Methyl [1-methyl-5-(propylthio)-1H-
5.1.1. Methyl methyl[5-(propylthio)-1H-
benzimidazol-2-yl]carbamate (4)
benzimidazol-2-yl]carbamate (2)
M.p. 120.5e122.1 ꢁC. IR n: 3286.98, 2956.08, 2929.46,
2868.25, 1631.18, 1490.64, 1451.44, 1217.44, 1172.07,
M.p. 80e82 ꢁC. IR n: 3333.18, 2956.25, 2870.21, 1702.88,
1546.67, 1454.16, 1198.50, 1165.06, 1107.82 cmꢂ1; H NMR
1086.47 cmꢂ1 1H NMR (DMSO-d6) d: 0.938 (t, 3H, CH3
;
1
(DMSO-d6) d: 0.935 (t, 3H, CH3CH2CH2S, J ¼ 7.2 Hz), 1.524
(m, 2H, CH3CH2CH2S, J ¼ 6.8 Hz), 2.848 (t, 2H,
CH3CH2CH2S, J ¼ 7.2 Hz), 3.469 (s, 3H, CH3), 3.839 (s,
3H, OCH3), 7.112 (dd, 1H, H-6, J6,7 ¼ 8.4, J6,4 ¼ 1.6 Hz),
7.394 (d, 1H, H-7, J ¼ 8.1 Hz), 7.476 (d, 1H, H-4,
J ¼ 1.6 Hz), 11.992 (s, 1H, exchangeable with D2O) ppm;
13C NMR (DMSO-d6) d: 13.99, 22.078, 33.661, 36.487,
53.768, 111.914, 113.361, 117.544, 119.164, 124.106,
127.078, 132.526, 134.033, 139.626, 141.283, 148.961,
154.203 ppm; EI-MS m/z: 279 [M]þ (100%).
CH2CH2S, J ¼ 7.5 Hz), 1.532 (m, 2H, CH3CH2CH2S, J ¼
7.5 Hz), 2.858 (t, 2H, CH3CH2CH2S, J ¼ 6.9 Hz), 3.468 (s,
3H, CH3), 3.607 (s, 3H, OCH3), 7.190 (dd, 1H, H-6, J6,7
¼
8.4, J6,4 ¼ 1.5 Hz), 7.326 (d, 1H, H-7, J ¼ 8.4 Hz), 7.427 (d,
1H, H-4, J ¼ 1.4 Hz), 11.981 (s, 1H, exchangeable with D2O)
ppm; 13C NMR (DMSO-d6) d: 13.019, 21.994, 28.035,
36.162, 51.722, 109.645, 112.782, 124.351, 128.713, 129.214,
129.562, 153.511, 162.517 ppm; FAB-MS m/z: 280 [M þ 1]þ.
5.1.4. 4-Nitrobenzyl-2-[(methoxycarbonyl)amino]-1H-
benzimidazole-5-carboxylate (5)
5.1.2. Methyl [1-methyl-6-(propylthio)-1H-
benzimidazol-2-yl]carbamate (3)
M.p. 319.6e320.3 ꢁC. IR n: 3382.02, 1708.07, 1652.23,
1272.12, 1186.56, 852.11 cmꢂ1 1H NMR (DMSO-d6) d:
;
M.p. 142.1e143.4 ꢁC. IR n: 3255.97, 2964.50, 2869.96,
3.776 (s, 3H, OCH3), 5.496 (s, 2H, CH2O), 7.491 (dd, 1H,
H-6, J6,7 ¼ 8.5, J6,4 ¼ 1.0 Hz), 7.752 (d, 2H, H-20,
J ¼ 8.6 Hz), 7.801 (d, 1H, H-7, J ¼ 8.5 Hz), 8.119 (d, 1H,
H-4, J ¼ 1.0 Hz), 8.274 (d, 2H, H-30, J ¼ 8.6 Hz), 11.981
1628.59,
1088.48 cmꢂ1
CH3CH2CH2S, J ¼ 7.2 Hz), 1.650 (m, 2H, CH3CH2CH2S,
1590.14,
;
1450.93,
1219.37,
1173.80,
1H NMR (CDCl3) d: 1.022 (t, 3H,

F. Palomares-Alonso et al. / European Journal of Medicinal Chemistry 44 (2009) 1794e1800
1799
Fig. 2. Lowest energy conformer of compounds 1 and 5e9. (a) Compounds modelled. (b) Superimposition of 1 (blue), 5 (cyan), 6 (orange), 7 (yellow), 8 (violet)
and 9 (red), which were colored for better understanding. In all the molecules, except 7 and 8, substituent at 5-position was orthogonal to benzimidazole ring. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(bs, 1H, exchangeable with D2O) ppm; FAB-MS m/z: 371
[M þ 1]þ.
NHCO2CH3), 7.440 (d, 1H, H-7, J ¼ 8.4 Hz), 7.810 (dd, 1H,
H-6, J1 ¼ 8.4, J2 ¼ 1.5 Hz), 7.980 (d, 1H, H-4, J ¼ 1.2 Hz),
8.390 (d, J ¼ 4.8 Hz, 1H, exchangeable with D2O, NHCH3),
12.21 (bs, 1H, exchangeable with D2O, NHCO2CH3) ppm;
EI-MS m/z: 232 ([M]þ 5%), 200 (100%).
5.1.5. Methyl [5-(piperidin-1-ylcarbonyl)-1H-
benzimidazol-2-yl]carbamate (6)
M.p. 296e297 ꢁC. IR n: 3339.01, 2932.17, 2849.02,
1734.11, 1627.01, 1596.02, 1423.11, 1259.10, 1237.02 cmꢂ1
;
1H NMR (DMSO-d6) d: 1.406 (bs, 2H, H-30), 1.652 (bs, 4H,
H-20, H-40), 3.551 (bs, 4H, H-10, H-60), 3.802 (s, 3H, OCH3),
7.011 (dd, 1H, H-6, J6,7 ¼ 8.5, J6,4 ¼ 1.0 Hz), 7.452 (d, 2H,
H-4), 11.670 (bs, 1H, exchangeable with D2O) ppm; EI-MS
m/z: 301 ([M]þ 50%), 269 (100%).
5.2. In vitro activity
The in vitro cysticidal activity evaluations were performed
on T. crassiceps cysts due to its similarity with T. solium cysts
[16,20]. Cysts were obtained from experimentally infected
male BALB/c mice (two months old). After three months
the mice were killed by cervical dislocation and the cysts
were removed from the peritoneal cavity. The parasites were
washed with sterile 0.9% saline solution and only those cysts
exhibiting intact bladder surface were used for the
5.1.6. Methyl {5-[(methylamino)carbonyl]-1H-
benzimidazol-2-yl}carbamate (7)
M.p. 368 ꢁC. IR n: 3298, 2947, 1638, 1587 cmꢂ1; 1H NMR
(DMSO-d6) d: 3.520 (s, 3H, CONHCH3), 3.620 (s, 3H,

1800
F. Palomares-Alonso et al. / European Journal of Medicinal Chemistry 44 (2009) 1794e1800
Table 2
Molecular descriptors retrieved from molecular modelling calculations
3
MV (A )
3
PSA (A )
˚
˚
Compound
HF (kcal/mol)
HOMO (eV)
LUMO (eV)
IP (eV)
DM (Debye)
log P
1
5
6
7
8
9
ꢂ53.20
ꢂ60.45
ꢂ50.17
ꢂ43.18
ꢂ15.71
12.65
832.18
1030.07
899.12
735.57
633.97
965.00
ꢂ8.88
ꢂ9.14
ꢂ8.88
ꢂ8.93
ꢂ8.84
ꢂ8.35
ꢂ0.26
ꢂ1.15
ꢂ0.32
ꢂ0.33
ꢂ0.31
ꢂ0.31
8.88
9.14
8.88
8.93
8.84
8.35
5.35
8.55
5.00
5.28
3.93
3.44
1.54
3.12
1.65
0.68
2.09
3.56
150.77
216.07
144.19
164.02
112.84
115.98
HF: heat of formation, MV: molecular volumes, log P: water/octanol partition coefficient, IP: ionization potential, DM: dipolar moment, PSA: polar surface area.
experiments. Stock solution of each compound was prepared
in dimethyl sulfoxide (DMSO), and working solutions were
prepared with culture medium to obtain concentrations of
0.28 and 1.7 mM (which represent the effective concentration
50 and 99 of 1, obtained previously by us in T. crassiceps cysts
of strain ORF) [14]. Culture medium containing 0.25% DMSO
was prepared as control. Each cell culture flask contained 25
cysts in 5 mL of culture medium and was incubated for 11
days at 37 ꢁC, with 5% CO2. The parasites were observed
every day for 11 days with an inverted light microscope
(Reichert 569) and the mortality of the cysts was registered.
The criteria to assess cyst mortality in vitro were loss of vesic-
ular fluid, paralysis of membrane and cyst collapse. The
mortality was confirmed on day 11 using the Trypan Blue
exclusion test. Each experiment was performed in triplicate.
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Acknowledgment
This work was supported by grants from PAPIIT-UNAM
IN210805. We are grateful to Rosa Isela del Villar, Georgina
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