Synthesis and evaluation of 4-acyl-2-thiazolylhydrazone derivatives for anti-Toxoplasma efficacy in vitro

Article abstract of DOI:10.1021/jm9005862

A new series of 4-acyl-2-thiazolylhydrazone derivatives was synthesized and screened for its in vitro activity against Toxoplasma gondii. We evaluated parasite growth inhibition and cytotoxicity, inhibition of replication, and inhibition of parasite invasion of host cells. The biological results indicated that some substances had an antiproliferative effect against intracellular T. gondii tachyzoites cultivated in vitro.

Full text of DOI:10.1021/jm9005862

4574 J. Med. Chem. 2009, 52, 4574–4577
DOI: 10.1021/jm9005862
by a sequential release of secretory proteins such as cysteine
protease. Studies performed on Trypanosoma species report
that cysteine protease inhibitors can be potent antiprotozoal
agents.11,12 In particular, thiosemicarbazones (a) and 4-thia-
zolidinone derivatives (b) possess a better in vitro specific
activity against T. gondii than hydroxyurea (Chart 1).13,14
In the search for new anti-infective agents, we took into
account the importance of a thiazole ring, which is one of the
possible molecular complications of thiosemicarbazone moi-
ety. It is present in the structure of many antimicrobial and
antiparasitic substances like the antihelmintic thiabendazole
(c) and the antischistosomial myridazole (d) (Chart 1).15 The
contribution of the presence of a hydrazine moiety is also
important, as reported before.
Synthesis and Evaluation of 4-Acyl-2-
thiazolylhydrazone Derivatives for
Anti-Toxoplasma Efficacy in Vitro
Franco Chimenti,† Bruna Bizzarri,*,† Adriana Bolasco,†
Daniela Secci,† Paola Chimenti,† Simone Carradori,†
Arianna Granese,† Daniela Rivanera,‡ Nathan Frishberg,§
§
§
ꢀ
Claudia Bordon, and Lorraine Jones-Brando
†Dipartimento di Chimica e Tecnologie del Farmaco and
‡Dipartimento di Scienze di Sanitaꢁ Pubblica, Universitaꢁ “La
Sapienza”, P.le A. Moro 5, 00185 Rome, Italy, and Stanley Medical
§
Research Institute, Johns Hopkins University School of Medicine,
600 North Wolfe Street, Blalock 1105, Baltimore, Maryland 21287
As a result of these observations, we decided to synthesize a
series of new thiazolylhydrazone derivatives bearing different
substitutions at 2 and 4 positions of the thiazole ring. To
increase the variability of the imine position and to improve
the drug penetration within the parasite-infected cell, we
designed a series of 2-hydrazothiazoles substituted at the
N-hydrazone moiety with cycloaliphatic rings or heterocyclic
rings (all structures provided with a suitable lipophilic
character) such as thiophene, furan, pyridine, naphthalene,
indole, and coumarin. The introduction of an ester or an acid
group in the 4 position of the thiazole ring gave us insight into
the influence of this substitution on the anti-Toxoplasma
activity. Thus, our objective was to deepen the study by
evaluating these new compounds for activity againstT. gondii.
We found that some analogues could moderately block the
parasite’s invasion and replication in vitro.
The synthesis of new derivatives was performed knowing
that different carbonyl compounds react directly with thiose-
micarbazide in the presence of catalytic amounts of acetic acid
in ethanol. The obtained thiosemicarbazones were subse-
quently condensated with ethyl ester of bromopyruvic acid
to give the 2,4-disubstituted 1,3-thiazoles (Hantzsch reaction)
as shown in Scheme 1 (1-22). Our choice of ethanol as solvent
allowed the final products to precipitate without further
purification. Subsequently some of the derivatives were hy-
drolyzed tothe corresponding carboxylic acidbymild alkaline
Received May 6, 2009
Abstract: A new series of 4-acyl-2-thiazolylhydrazone derivatives
was synthesized and screened for its in vitro activity against
Toxoplasma gondii. We evaluated parasite growth inhibition and
cytotoxicity, inhibition of replication, and inhibition of parasite
invasion of host cells. The biological results indicated that some
substances had an antiproliferative effect against intracellular
T. gondii tachyzoites cultivated in vitro.
The intracellular protozoan Toxoplasma gondii is an op-
portunistic pathogen responsible for asymptomatic parasitic
infection. It is generally acquired by ingesting food or water
contaminated with oocysts shed by cats or by eating under-
cooked or raw meat containing tissue cysts. After host cell
invasion, the rapidly replicating form of T. gondii, the tachy-
zoite, localizes in the cytoplasm within a membrane-bounded
vacuole. It multiplies at intervals of 6-8 h and thus initiates
the acute stage of infection.1 Following the acute stage, the
parasite forms cysts (latent stage) in various organs, especially
the brain, heart, and skeletal muscle. Primary infection in
immunocompetent individuals results in chronic infection. In
chronically infected people, immunosuppression may result in
reactivation of a latent infection that can cause life-threaten-
ing encephalitis.2-4 In pregnant women, T. gondii infection
may cause severe congenital defects in the fetus.5
The standard chemotherapy against human toxoplasmosis
is a combination of pyrimethamine and sulfa drugs like
sulfadiazine.6,7 This combination therapy targets folate me-
tabolism enzymes such as dihydrofolate reductase. However,
current therapy has been limited by high toxicity, resistance,
and poor absorption of efficient drugs. In addition, this
traditional therapeutic regime is not always suitable for pro-
longed treatment because of the appearance of undesirable
side effects like allergic reactions, which are common
among patients and which further limit the treatment in some
cases.8-10 Identifying novel antitoxoplasmic compounds
effective against chronic stages of the parasite would help in
overcoming such disadvantages.
hydrolysis (LiOH H2O in H2O/MeOH, 1/4) to obtain the
3
derivatives 23-30.
All synthesizedcompounds, whichshowcalculated log P <
5, were fully characterized by analytical and spectral data as
listed in Tables 1, 2, and 3 (see also Supporting Information).
The synthesis of 10 and 13 has been described and was
performed with slight changes; their analytical and spectral
data were in full agreement with those reported in the
literature.16,17
For all the synthesized compounds, the relative ability to
inhibit the growth of the tachyzoites of T. gondii was deter-
mined using a published method.18 The tests were performed
on genetically modified parasites constitutively expressing
β-galactosidase (β-gal) (strain 2F derived from strain RH;
ATCC, VA) and human foreskin fibroblast (HFF; ATCC)
host cells plated in 96-well (12 columns ꢀ 8 rows) plates. Prior
to adding the parasite to cells and beginning with column 2 of
the plate, drugs were serially diluted across the plate by
dilutions of 0.5 log10, leaving column 11 drug-free (parasite
control). Following this, T. gondii tachyzoites were added to
As an obligate intracellular parasite, T. gondii depends on
an efficient host cell invasion strategy to support its survival
and transmission. In this process, an important role is played
*To whom correspondence should be addressed. Phone: þ39 064
9693242. Fax: þ39 064 991 3772. E-mail: bruna.bizzarri@uniroma1.it.
r
pubs.acs.org/jmc
Published on Web 07/20/2009
2009 American Chemical Society

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4575
Table 2. Chemical-Physical Data of Derivatives 9-22
Chart 1. Antimicrobial and Antiparasitic Thiazole-Based
Structures Reported in Literature
compd
Cy
R
mp (°C)
yield (%) ClogP
9
thiophen-20-yl
thiophen-20-yl
fur-20-yl
CH3 205-207
74
89
99
77
80
98
77
98
72
75
99
99
93
99
2.42
2.69
2.12
1.95
1.28
1.66
1.28
1.66
1.28
4.12
3.95
3.19
3.42
1.11
10
11
12
13
14
15
16
17
18
19
20
21
22
H
H
198-199
183-185
fur-20-yl
CH3 208-210
CH3 215-216
pyridin-20-yl
pyridin-30-yl
pyridin-30-yl
pyridin-40-yl
pyridin-40-yl
naphthalen-10-yl
naphthalen-20-yl
benzodioxol-50-yl
indol-30-yl
H
211-212
CH3 210-215
251-254
CH3 215-218
189-190
CH3 228-230
H
Scheme 1a
H
H
217-218
232-235
H
coumarin-30-yl
CH3 193-195
Table 3. Chemical-Physical Data of Derivatives 23-30
a Reagents and conditions: (i) thiosemicarbazide, acetic acid (cat.),
EtOH; (ii) ethyl ester of bromopyruvic acid, EtOH; (iii) (1) LiOH H2O,
H2O/MeOH, (2) HCl, 3 N.
compd
R
n
mp (°C)
yield (%)
ClogP
23
24
25
26
27
28
29
30
H
1
1
1
2
2
2
2
3
242-250
215-217
203-205
242-247
221-224
231-235
236-238
251-253
78
72
77
72
78
75
78
73
2.22
2.73
2.73
2.73
3.29
3.29
3.29
3.29
3
2-CH3
3-CH3
H
Table 1. Chemical-Physical Data of Derivatives 1-8
2-CH3
3-CH3
4-CH3
H
compd
R
n
mp (°C)
yield (%)
ClogP
1
2
3
4
5
6
7
8
H
1
1
1
2
2
2
2
3
153-158
143-148
134-139
146-147
146-148
125-132
109-112
149-140
99
91
79
99
79
74
99
85
1.87
2.39
2.39
2.43
2.95
2.95
2.95
2.99
by viable cells into a soluble, colored formazan product was
captured by reading the plates at 490-650 nm. The median
inhibitory concentration (IC50) and median cytotoxic dose
(TD50) were calculated using CalcuSyn software (Biosoft,
Cambridge, U.K.). For each compound, a therapeutic index
(TI) was calculated with the formula TI=TD50/IC50. This
number reflects the specific activity of a compound against
Toxoplasma. The TIs of the testcompounds were compared to
the TI of trimethoprim, a folate antagonist commonly em-
ployed for toxoplasmosis therapy in humans.
Of the 30 thiazolylhydrazone derivatives tested, the ester
analogues 9 and 15 displayed a moderate efficacy relative to
controltrimethoprim. Theywere similarinpotency, inhibiting
T. gondii growth with IC50 ranging from 34 to 52 μM.
Analogue 22 proved to be relatively more effective in inhibit-
ing T. gondiigrowthwithIC50 = 21μM. In addition, as shown
in Table 4, the four derivatives proved to be noncytotoxic
(TD50 g 320 μM).
Compound 13 appeared to be quite effective against Tox-
oplasma (IC50 =3 μM); however, this was due to its strong
cytotoxic action (TD50=6 μM) (Table 4 and Figure 1).
As shown in Figure 1, when compounds were tested for the
ability to inhibit the intracellular replication of T. gondii
tachyzoites, derivative 22 was found to be moderately inhibi-
tory while the cytotoxic derivative 13 showed the largest
reduction of the number of tachyzoites per vacuole.
2-CH3
3-CH3
H
2-CH3
3-CH3
4-CH3
H
6 out of 8 wells in each column, leaving 2 wells in each column
parasite-free for cytotoxicity testing. The substrate for β-gal,
chlorophenol red-β-D-galactopyranoside (CPRG), was added
to the Toxoplasma wells after 4 days of incubation at 37 °C/
5% CO2. Further incubation for 20 h was followed by
addition of the cell viability reagent, CellTiter 96Aqueous
One Solution Reagent (Promega Corp., WI) to the uninfected
cytotoxicity wells. Color reactions in all wells were read in a
Vmax microplate reader (molecular Devices, CA) after 3 h of
incubation. The amount of absorbance (570-650 nm) in wells
containing drug, Toxoplasma, and CPRG was compared to
that in parasite control wells. The amount of absorbance in
these wells is directly proportional to the amount of β-gal
activity and thus to the amount of viable tachyzoites. Thus, a
decrease in amount of absorbance indicates an inhibition of
enzyme activity and, by extension, parasite growth. In the
cytotoxicity wells, the bioreduction of the cell viability reagent

4576 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15
Table 4. Biological Data of 1-30 Derivatives and Drug Reference
Chimenti et al.
compd
IC50 (μM)
IC90 (μM)
TD50 (μM)
TIa
1
92
239
248
190
638
508
46
g320c
g320
g320
g320
g320
84
6
2
130
59
4
3
10
4
4
160
185
15
5
3
6
6
7
60
77
151
226
136
NDb
ND
161
7
g320
g320
g320
g320
g320
241
9
8
7
9
37
15
ND
ND
5
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Td
g320
g320
49
3
6
2
162
34
398
86
g320
g320
g320
g320
g320
127
4
17
ND
9
g320
64
52
ND
188
182
88
11
3
43
479
280
21
2590
2235
55
g320
g320
g320
g320
g320
82
1
2
27
ND
ND
2
g320
g320
38
ND
ND
70
Figure 2. Invasion assay. (A) The red/green invasion assay was
performed with published methods.20 Tachyzoites were mixed with
DMSO or compound (final 25 μM), left at room temperature for
20 min, and then added to HFF monolayers. After 1 h at 37 °C, 5%
CO2, the cells were rinsed and fixed. Attached/extracellular para-
sites were detected using Rb anti-p30 followed by Alexa Fluoro
594 (red). After permeabilization, penetrated/intracellular parasites
were stained with mAb 9e11 anti-SAG1 (Argene Inc., NY) followed
by Gt antimouse Alexa Fluoro 488 (green). DAPI was added to
secondary antibody. Slides were visualized as in Figure 1. Numbers
of green and red tachyzoites per host cell nucleus were enumerated.
(B) Representative fluorescence images of fibroblast monolayers
infected with strain 2F tachyzoites that have been treated with
experimental compounds or DMSO. Attached/extracellular para-
sites are stained red, penetrated/intracellular parasites are stained
green, and host nuclei are stained blue.
g320
128
g320
g320
179
14
ND
310
ND
ND
3872
ND
g320
g320
g320
g320
g320
g320
ND
4
ND
ND
3
41
a TI, therapeutic index determined by TD50/IC50
.
b ND, not deter-
mined. c Toxicity endpoint not reached; a value of 1/4 log10 greater
than the concentration tested was used to compute the TI. d T, trimetho-
prim.
The sequential staining of the red/green invasion assay20
permitted us to evaluate the compounds for inhibitory effects
on tachyzoite host cell invasion and to distinguish T. gondii
tachyzoites that had actively penetrated the cells from those
parasites that were simply attached to host cells. A moderate
decrease in the number of penetrated parasites relative to the
control penetrated (indicating inhibition of invasion) was
observed with 3 (48%), 4 (45%), 6 (48%), 16 (49%), 18
(35%), 19 (45%), 25 (47%), and 26 (45%), while derivative
13 appeared to enhance invasion (147%) (Figure 2A). As
demonstrated by previous studies,21 an overall decrease in
both attached and invaded parasites strongly indicates an
inhibitory effect on parasite attachment, an essential prerequi-
site for penetration. A moderate decrease in the number of
total parasites (penetrated þ attached relative to same of
control) was observed with 18 (47%) and 23 (47%) (Figure 2A
and Figure 2B). An increased number of attached vs penetrated
parasites (within one compound) indicates inhibition of penetra-
tion. No compounds showed such activity.
Figure 1. Replication assay. Compounds were tested using a con-
ventional method.19 HFF monolayers were inoculated with tachy-
zoites and then incubated for 2 h. Compounds (final 25 μM) or
DMSO (control) was then added to the medium. Parasite replica-
tion proceeded for 26 h, after which time the monolayers were fixed,
permeabilized, and immunolabeled with Rb anti-p30 (SAG1) (AbD
Serotec, Oxfordshire, U.K.) followed by goat antirabbit Alexa
Fluoro 594 (red) (Invitrogen, CA). DAPI (Invitrogen) was used
for visualizing host cell nuclei. Slides were examined by reflected
fluorescence. The numbers of vacuoles containing 1, 2, 4, or 8þ
parasites/vacuole were enumerated. Shown are representative fluor-
escence images of the most common sized vacuole for the active
compounds 13 and 22, an example of an inactive compound 10, and
control. Tachyzoites within vacuoles are immunostained red and
host nuclei stained blue.
In conclusion we report herein the synthesis and the
characterization of 4-acyl-2-thiazolylhydrazone derivatives
based on their physical, analytical, and spectral data. The
prepared compounds were evaluated for their in vitro
antiprotozoal properties against T. gondii. The results con-
firm that the 2,4-disubstituted 1,3-thiazole moiety could be
an important pharmacophoric requirement in displaying

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 15 4577
anti-Toxoplasma efficacy in vitro. By comparison of the
biological data of derivatives 1-8 with those of 9-22, it
emerges that the substitution of a cycloaliphatic ring with
heteroaromatic ones could be useful for the activity. The 9, 15,
18, and 22 derivatives, which bear a heterocyclic moiety, were
moderately effective in inhibiting T. gondii growth with IC50
ranging from 21 to 52 μM, and they were also noncytotoxic
(TD50 g 320 μM). Furthermore, derivatives 9, 12, 13, 15, 17,
19, and 22, bearing a methyl substituent at the R position of
the hydrazone moiety, display a better activity than an
unsubstituted one. Derivatives 23-30, bearing a carboxylic
acid function at the C4 position of thiazole ring, show a
decrease of Toxoplasma inhibition or an increase in their
cytotoxicity. Finally compound 22, which had an effective
action on intracellular parasite replication causing a 50%
decrease in the number of tachyzoites per vacuole, could be
used as a tool for determining the mode of action of this new
thiazole-based scaffold. In conclusion, it is possible to state
that some derivatives of this series could act in two different
ways (inhibition of replication and decrease of host cell
invasion).
In the present work, we have focused our attention on the
synthesis and the biological evaluation of new 4-acyl-2-thia-
zolylhydrazone derivatives. We plan to extend our studies on
the substitution of the nucleus linked to the hydrazone, which
seems to have a major impact on the anti-Toxoplasma activity
or cytotoxicity. Furthermore, our efforts will be oriented
toward the comprehension of how these compounds could
inhibit not only the replication of parasite but also host cell
attachment and invasion.
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Supporting Information Available: Synthesis procedures, ana-
lytical and spectral data for new compounds, and pharmacologi-
cal studies. This material is available free of charge via the Internet
at http://pubs.acs.org.
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