In vitro anti-acanthamoeba action by thioureidic derivatives

Article abstract of DOI:10.1016/S0014-827X(03)00138-1

The anti-amoebic power of a series of bis-thioureidic derivatives against amoeba, responsible for a serious form of keratitis of the cornea, has been analysed. The synthesis of these products is also described.

Full text of DOI:10.1016/S0014-827X(03)00138-1

Il Farmaco 58 (2003) 819ꢁ822
/
www.elsevier.com/locate/farmac
Short Communication
In vitro anti-acanthamoeba action by thioureidic derivatives
Carmela Saturnino a,*, Mariafrancesca Buonerba a, Nicola Paesano a, Jean-
Charles Lancelot b, Giovanni De Martino a
a Dipartimento di Scienze Farmaceutiche, Universita` di Salerno, Via Ponte don Melillo, I-84084 Fisciano (SA), Italy
b Centre d’Etudes et de Recherche sur le Me´dicament de Normandie (CERMN), Caen, France
Received 26 November 2002; accepted 25 May 2003
Abstract
The anti-amoebic power of a series of bis-thioureidic derivatives against amoeba, responsible for a serious form of keratitis of the
cornea, has been analysed. The synthesis of these products is also described.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Anti-amoebic; bis-Thioureidic; Keratitis
1. Introduction
tives too, by preparing some derivatives with nꢀ
/
4ꢁ9
/
methylenic groups. The influence of the substituent
(aliphatic or aromatic) present on one of the nitrogen
atoms of the thiourea has been studied too. The
synthesis of the products is reported in Scheme 1. The
amoebicidal power against the three strains of the
Acanthamoeba genus has been extimated for all the
In previous researches [1], some of us had studied the
amoebicidal power of superior homologues of propa-
midine (Brolene†) and hesamidine (Desomedine†),
present in two collyria used for a serious, even if rare,
form of keratitis, due to the invasion of the cornea by
amoeba of the Acanthamoeba genus. These studies were
carried out on the amoeba cysts, which are the resistant
forms originating the infection persistence. These mole-
cules belonged to the to 4-4?-diamidino-difenoxyalkane
series (see Fig. 1). The results [2], showed the superiority
of action of the hepta, octa and nonamidine that make
inactive all the amoeba cysts studied (in 24 h) included
the cysts of the resistant forms. From these interesting
results we have synthesized a series of bis-thioureidic
derivatives (see Fig. 2), in which the phenoxyamidinic
function was replaced by a simple thioureidic function.
We have studied the influence on the biological activity
of the length of the alkyl chain separating the two
thioureidic functions for these bis- thioureidic deriva-
compounds 1aꢁ
/
f; 2aꢁ
/
f; 3aꢁf.
/
2. Material and methods
2.1. Chemistry
Melting points were determined by a Kofler appara-
tus and are reported in Tables 1ꢁ3. IR spectra were
/
1
recorded on a Shimatzu 8000 spectrometer. H NMR
spectra were recorded on a Brucker DRX 600 spectro-
meter in DMSO-d6 solvent. Chemical shifts, expressed
as d (ppm), are shown in Tables 4ꢁ6. The purity of the
/
compounds was checked by TLC (thin layer chromato-
graphy), using ethyl acetate as eluent. The structural
attributions were performed also by the support of the
elemental analysis, in which found and calculated values
* Corresponding author.
E-mail address: saturnino@unisa.it (C. Saturnino).
atom percentages were identical within 90.4%. The
/
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00138-1

C. Saturnino et al. / Il Farmaco 58 (2003) 819ꢁ
/822
820
Fig. 1.
synthesis were carried out in the laboratories of
C.E.R.M.N. Caen (France) and in the University of
Pharmacy in Salerno (Italy); the biological tests were
executed by C.E.R.M.N.
2.1.1. Preparation of 1aꢁ
/
f, 2aꢁ
/
f, 3aꢁ
/
f compounds
The compounds 1aꢁf, 2aꢁ
/
/
f and 3aꢁ
/
f (see Fig. 2) have
been synthesized as follows. Two grams of the suitable
Scheme 1.
thiourea (1 equiv.) and dibromoalkane (0.5 equiv.) was
refluxed in anhydrous ethanol for 4ꢁ5 h. The reaction
/
The criteria to assign the protozoas to the Acantha-
moeba genus were: presence of acanthopolia in slowly
moving trophozoites, the use of metamitosis for nuclear
division and the presence of double-walled cysts with
polygonal or stellate endocysts. After silver staining and
microscopic examination, cysts exhibited the features of
group II defined by Pussard and Pons [3], [4] and
Acanthamoeba strains were identified as Acanthamoeba
polyphaga, and Hartmannella varinii and Vahlkampfia
avara.
mixture was evaporated to dryness under vacuum. The
residue was crystallised from suitable solvent to give the
bis-thioureidic salts (Scheme 1).
2.2. Pharmacology
2.2.1. Biological assays
The Amoeba strains used in this study were isolated
from lenses, lenses care systems or corneal scraping of
patients affected by keratitis. Cysts were used as
challenge organisms, being the resistant forms of the
Acanthamoeba.
The strains were grown at 30 8C on a monoexenic
nutrient agar (MNA) medium made of 1.5% agar (Bacto
Agar, Difco) aseptically spread over with a live Esche-
richia Coli suspension.
2.2.2. Trophozoite and cyst harvesting
The Acanthamoeba strains were grown at 30 8C on
MNA medium. Trofozoites were collected from 24 to 48
h cultures and cysts were collected from 3-week cultures.
The agar surfaces were flooded with 5 ml of PBS and
were gently scraped with an inoculating loop. Tropho-
zoites and cysts were harvested from the suspension by
Table 1
Physical and analytical data of compounds 1aꢁf
/
Comp. no.
n
Melting point (8C)
Yield (%)
1a a
1b b
1c b
1d a
1e b
1f b
4
5
6
7
8
9
238
170
80
60
84
37
72
90
84
133
180
130
a
Crystallisation solvent: diethyl ether.
Crystallisation solvent: acetonitrile.
b
Fig. 2.

821
C. Saturnino et al. / Il Farmaco 58 (2003) 819ꢁ822
/
Table 2
Table 4
1H NMR data (d ppm) of compounds 1aꢁ
attribution
Physical and analytical data of compounds 2aꢁf
/
/f (DMSO-d6) and signal
1a 3.35ꢁ
/
3.25 (4H, m, 2Sꢁ
/
CH2); 1.75ꢁ
CH2); 1.70ꢁ
CH2); 1.76ꢁ
CH2); 1.90ꢁ
CH2); 1.78ꢁ
CH2); 1.70ꢁ
/
1.68 (4H, m, 2-CH2); 1.08 (6H,
1.32 (6H, m, 3-CH2); 1.03 (4H,
1.52 (8H, m, 4-CH2); 1.06 (6H,
s, 2CH3)
1b 3.33ꢁ
/
3.23 (4H, m, 2Sꢁ
/
/
s, 2CH3)
1c 3.33ꢁ
/
3.23 (4H, m, 2Sꢁ
/
/
s, 2CH3)
1d 3.40ꢁ
/
3.35 (4H, m, 2Sꢁ
/
/
1.35 (10H, m, 5-CH2); 1.09
1.30 (12H, m, 6-CH2); 1.02
1.27 (14H, m, 7-CH2); 1.00
(6H, s, CH3)
Comp. no.
n
Melting point (8C)
Yield (%)
1e 3.35ꢁ
/
3.23 (4H, m, 2Sꢁ
/
/
2a a
2b a
2c a
2d
2e a
2f
4
5
6
7
8
9
226
192
198
Oil
190
Oil
48
43
53
43
20
88
(6H, s, CH3)
1f 3.32ꢁ
/
3.20 (4H, m, 2Sꢁ
/
/
(6H, s, 2CH3)
ding to a range of 1.52ꢂ
testing on the cysts) and to 100 mg/ml, corresponding to
1.52ꢂ 1.92ꢂ
102ꢁ 102 mol/l (when testing on the tro-
/
103ꢁ
/
1.92ꢂ
103 mol/l (when
/
a
Crystallisation solvent: acetonitrile.
/
/
/
phozoites), by dilution in a suitable volume of sterile
distilled water. Experiments and their controls were
carried out in well plates. Sixteen sets of experimental
plates containing each 100 ml of thioureidic derivatives
were used. Control plates containing 100 mg of phos-
phate-buffered saline (PBS, pH 7.2) were also used. A
100 ml amount of standardised trophozoites or cysts
suspension was inoculated into each plate. All plates
were sealed with Parafilm (American Can Co.) to
prevent evaporation, incubated at 30 8C, and examined
daily with an inverted microscope to determine the
presence of active trophozoites. Each plate was evalua-
ted and scored as either positive or negative. From the
number of positive cultures observed in each set, the
most probable number of viable cysts or trophozoites
was read on statistical tables corresponding to the plated
volumes (American Public Health Association. Stan-
dard method for the Examination of Water and Waste-
centrifugation at 350 mg for 10 min in a C 400-S4
centrifuge (Jouan, France). The supernatant was aspi-
rated and the sediment was washed twice in PBS in
order to eliminate most of the bacteria. The trophozoites
or cysts in the resultant suspension were optically
counted with a Malassez hemacytometer with a phase-
contrast microscope, and the suspension was standar-
dised to 105 trophozoites or cysts for 100 ml.
2.2.3. Drug sensitivity tests
Drug solutions were prepared at a concentration ten
times the desired one by dissolution in sterile distilled
water and were stored at 4 8C until they were tested. The
concentrations were adjusted to 1000 mg/ml, correspon-
Table 3
Physical and analytical data of compounds 3aꢁf
/
water.
Washington DC, 1971). Experiments and their control
American
Public
Health
Association,
were repeated five times. The minimum concentration of
Table 5
1H NMR data (d ppm) of compounds 2aꢁ
f (DMSO-d6) and signal
attribution
/
2a 7.52ꢁ
/
7.40 (10H, m, HAr), 3.42ꢁ
/
3.38 (4H, m, 2Sꢁ
3.20 (4H, m, 2Sꢁ
3.25 (4H, m, 2Sꢁ
3.23 (4H, m, 2Sꢁ
3.20 (4H, 2S-CH2), 1.66ꢁ
3.20 (4H, m, 2S-CH2), 3.12ꢁ
/
CH2), 1.80ꢁ
CH2), 1.71ꢁ
CH2), 1.79ꢁ
CH2), 2.20ꢁ
1.26
1.25
/
1.75
1.34
1.56
1.35
(4H, m, 2CH2)
2b 7.50ꢁ
/
7.39 (10H, m, HAr), 3.30ꢁ
/
/
/
Comp. no.
n
Melting point (8C)
Yield (%)
(6H, m, 3CH2)
3a a
3b
4
5
6
7
8
9
210
Oil
182
Oil
Oil
Oil
47
55
36
68
81
69
2c 7.51ꢁ
/
7.47 (10H, m, HAr), 3.33ꢁ
/
/
/
(8H, m, 4CH2)
3c b
3d
2d 7.56ꢁ
/
7.35 (10H, m, HAr), 3.33ꢁ
/
/
/
(10H, m, 5CH2)
2e 7.40ꢁ
/
7.35 (10H, m, HAr), 3.34ꢁ
/
/
3e
3f
(12H, m, 6CH2)
2f 7.44ꢁ
/
7.36 (10H, m, HAr), 3.35ꢁ
/
/
a
(14H, m, 7CH2)
Crystallisation solvent: chloroform.
b
Crystallisation solvent: carbonꢁtetrachloride.
/

C. Saturnino et al. / Il Farmaco 58 (2003) 819ꢁ
/822
822
Table 9
Vahlkampfia avara
Table 6
1H NMR data (d ppm) of compounds 3aꢁ
f (DMSO-d6) and signal
attribution
/
Comp. no.
CMA (mg/ml)
CMC (mg/ml)
3a 7.57 and 7.38 (8H, Ja,b
CH2), 1.78ꢁ1.75 (4H, m, 2CH2)
3b 7.56 and 7.38 (8H, Ja,b 8 Hz, 8Hꢁ
CH2),1.68ꢁ1.03 (6H, m, 3CH2)
3c 7.65 and 7.48 (8H, Ja,b 8 Hz, 8Hꢁ
CH2), 1.91ꢁ1.68 (8H, m, 4CH2)
3d 7.55 and 7.38 (8H, Ja,b 8 Hz, 8Hꢁ
CH2), 2.06ꢁ1.36 (10H, m, 5CH2)
3e 7.44 and 7.38 (8H, Ja,b 8 Hz, 8Hꢁ
CH2), 1.62ꢁ1.27 (12H, m, 6CH2)
3f 7.47 and 7.37 (8H, Ja,b 8 Hz, 8Hꢁ
CH2), 3.27ꢁ1.27 (14H, m, 7CH2)
ꢀ
/
8 Hz, 8Hꢁ
/
Ar), 3.40ꢁ
Ar), 3.32ꢁ
Ar), 3.43ꢁ
Ar), 3.32ꢁ
Ar), 3.34ꢁ
Ar), 3.35ꢁ
/
3.35 (4H, m, 2Sꢁ
3.25 (4H, m, 2Sꢁ
3.32 (4H, m, 2Sꢁ
3.20 (4H, m, 2Sꢁ
3.23 (4H, m, 2Sꢁ
3.25 (4H, m, 2Sꢁ
/
24 h
48 h
78 h
24 h
48 h
/
ꢀ/
/
/
/
2c
2e
3c
50
20
5
50
10
5
20
10
5
ꢀ/1000
ꢀ/1000
/
ꢀ/1000
ꢀ/1000
ꢀ/
/
/
/
/
ꢀ/
/
/
/
/
ꢀ/
/
/
/
/
rine atom in our case), is on the benzene ring in para
position, a big increase in the activity is observed.
ꢀ/
/
/
/
/
4. Conclusions
Table 7
Acanthamoeba polyphaga
The following conclusions can be drawn out from
these first results:
Comp. no.
CMA (mg/ml)
CMC (mg/ml)
24 h
48 h
78 h
24 h
48 h
. when Rꢀ
tion (MAC) is always ꢀ
length of the alkyl chain separating the two thioureas;
. when RꢀC6H5, the minimal amoebicidal concentra-
tion (MAC) is of 20 mg/ml if nꢀ6 and is halved (10
mg/ml) when nꢀ8. This means that the amoebicidal
/
CH3, the minimal amoebicidal concentra-
/
di 100 mg/ml whatever the
2c
2e
3c
50
25
10
50
20
10
20
20
10
ꢀ/1000
ꢀ/1000
ꢀ/1000
ꢀ/1000
/
/
/
the drugs required to kill 100% of trophozoites is
defined as minimal amoebicidal concentration (MAC).
The minimum concentration of the drug required to kill
100% of cysts is defined as minimal cysticidal concen-
tration (MCC).
power is linked both to the general lipophily of the
molecule and to the alkyl chain separating the two
thioureas;
. the best products 2c, 2e and 3c, have resulted to be
very good antiamoebics as able to destroy the
amoebae completely at the concentration between
50 and 5 mg/ml.
3. Results and discussion
All the products 1aꢁ
/
f, 2aꢁ
/
f, 3aꢁf were tested on three
/
Acknowledgements
different type of amoebae, but here we report only the
most interesting data regarding compounds 2c, 2e and
This work was supported in part by MIUR of Italy.
We also wish to express our appreciation to Celine
Lendormy for the studies in vitro.
3c. The results of these products are shown in Tables 7ꢁ
/
9. All compounds, especially 2e and 3c, were effective on
the A. polyphaga, H. varinii and V. avara. These
compounds might be assumed as leads for developing
new drugs actives against Acanthamoeba. However the
length of alkyl chain may be playing an important role.
Finally when an electron-withdrawing group (the chlo-
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1991, no. 48777, 06.
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48 h
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/