New thiazolyl-pyrazoline derivatives bearing nitrogen mustard as potential antimicrobial and antiprotozoal agents

Article abstract of DOI:10.1002/ardp.201900351

A new series of N-substituted pyrazoline derivatives 6a–g, 7a–g, 8a–g, and 9a–g was synthetized by reaction of hydrazine derivatives and chalcone–thiazole hybrids bearing nitrogen mustard 5a–g. The chalcones 5a–g were obtained by Claisen–Schmidt condensation of thiazole-2-nitrogen mustard 3 and selected acetophenones 4a–g. These new compounds 6/7/8/9a–g were screened for their antifungal activity against Cryptococcus neoformans, with IC₅₀ values of 3.9–7.8 μg/ml for the N-3,5-dichlorophenyl pyrazolines 9e–g. Interestingly, those compounds show low cytotoxic effects toward erythrocytes (RBC). In addition, N-acetyl (6a,b) and N-formyl pyrazolines (7a, 7b, 7c, and 7g) showed inhibitory activity against methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, and vancomycin-intermediate S. aureus, with the most important minimum inhibitory concentration values ranging from 31.25 to 125 μg/ml. Regarding the antiprotozoal activity, thiazolyl-pyrazolines 9g, 8f, and 7c display high activity against Plasmodium falciparum, Leishmania (V) panamensis, and Trypanosoma cruzi, with EC₅₀ values of 11.80, 6.46, and 4.98 μM, respectively, and with 7c being approximately 2.6-fold more potent than benznidazole with a selectivity index of 1.61 on U-937 human cells, showing promising potential as a novel antitrypanosomal agent.

Full text of DOI:10.1002/ardp.201900351

Received: 25 November 2019
DOI: 10.1002/ardp.201900351
Revised: 24 February 2020
Accepted: 25 February 2020
|
|
F U L L P A P E R
New thiazolyl‐pyrazoline derivatives bearing nitrogen
mustard as potential antimicrobial and antiprotozoal agents
1
,2
3
3
Viviana Cuartas
| Sara M. Robledo
| Iván D. Vélez
|
4
5
5
María del Pilar Crespo
| Maximiliano Sortino
| Susana Zacchino
|
6
6
3
3
Manuel Nogueras
| Justo Cobo
1,2
| Yulieth Upegui
| Tatiana Pineda
|
3
Lina Yepes | Braulio Insuasty
1
Grupo de Investigación de Compuestos
Heterocíclicos, Departamento de Química,
Abstract
Universidad del Valle, Cali, Colombia
A new series of N‐substituted pyrazoline derivatives 6a–g, 7a–g, 8a–g, and 9a–g was
synthetized by reaction of hydrazine derivatives and chalcone–thiazole hybrids
bearing nitrogen mustard 5a–g. The chalcones 5a–g were obtained by Claisen–
Schmidt condensation of thiazole‐2‐nitrogen mustard 3 and selected acetophenones
2
Centre for Bioinformatics and
Photonics‐CIBioFI, Universidad del Valle,
Cali, Colombia
3
PECET, Instituto de Investigaciones Médicas,
Facultad de Medicina, Universidad de
Antioquia, Medellín, Colombia
4
a–g. These new compounds 6/7/8/9a–g were screened for their antifungal activity
4
Grupo de Biotecnología e Infecciones
against Cryptococcus neoformans, with IC50 values of 3.9–7.8 µg/ml for the N‐3,
5‐dichlorophenyl pyrazolines 9e–g. Interestingly, those compounds show low cyto-
toxic effects toward erythrocytes (RBC). In addition, N‐acetyl (6a,b) and N‐formyl
pyrazolines (7a, 7b, 7c, and 7g) showed inhibitory activity against methicillin‐
susceptible Staphylococcus aureus, methicillin‐resistant S. aureus, and vancomycin‐
intermediate S. aureus, with the most important minimum inhibitory concentration
values ranging from 31.25 to 125 µg/ml. Regarding the antiprotozoal activity,
thiazolyl‐pyrazolines 9g, 8f, and 7c display high activity against Plasmodium falci-
parum, Leishmania (V) panamensis, and Trypanosoma cruzi, with EC50 values of 11.80,
Bacterianas, Departamento de Microbiología,
Universidad del Valle, Cali, Colombia
5
Área Farmacognosia, Facultad de Ciencias
Bioquímicas y Farmacéuticas, Universidad
Nacional de Rosario, Rosario, Argentina
6
Department of Inorganic and Organic
Chemistry, Universidad de Jaén, Jaén, Spain
Correspondence
Braulio Insuasty, Departamento de Química,
Facultad de Ciencias Naturales y Exactas,
Universidad del Valle, Cali A. A. 25360, Colombia.
Email: braulio.insuasty@correounivalle.edu.co
6
.46, and 4.98 μM, respectively, and with 7c being approximately 2.6‐fold more
potent than benznidazole with a selectivity index of 1.61 on U‐937 human cells,
Funding information
Science, Technology, and Innovation Fund‐
General Royalties System (FCTeI‐SGR),
Grant/Award Number: BPIN 2013000100007;
Consejería de Innovación, Ciencia y Empresa
showing promising potential as a novel antitrypanosomal agent.
K E Y W O R D S
(
Junta de Andalucía, Spain); Universidad de
antibacterial activity, antifungal activity, antiprotozoal activity, chalcone, pyrazoline
Jaén; Universidad del Valle; Agencia Nacional
de Promoción Científica y Tecnológica,
Grant/Award Number: PICT2016‐1833;
Departamento Administrativo de Ciencia,
Tecnología e Innovación,
Grant/Award Number: 110680864255;
National University of Rosario Argentina
1
| INTRODUCTION
mechanisms to avoid antibiotic actions such as reduced permeability,
[
4]
mutations in the target site, and enzymatic modifications. Ne-
Antimicrobial infections are a major concern for global public health
glected tropical diseases are an important cause of mortality in tro-
[5,6]
[
1–3]
due to the emergence of multidrug‐resistant strains.
These pa-
pical and subtropical regions from 149 countries,
affecting more
[
6]
thogens are known as “superbugs” and they exhibit different
than one billion people with malaria, leishmaniasis, and Chagas
Arch Pharm. 2020;e1900351.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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[
34]
[29]
disease, among others, which require toxic and expensive che-
motherapy agents, such as pentavalent antimonials, miltefosine,
derivatives show potent antifungal,
antibacterial,
and anti-
[
35]
oxidant
activities, in addition to inhibitory effects on the epi-
[
5,7]
amphotericin B, benznidazole, and nifurtimox.
Unfortunately,
dermal growth factor receptor tyrosine kinase as a target for
[
36–39]
[18]
these treatments are contraindicated and in some cases are not ef-
anticancer therapy,
[34]
among others.
For example, Altintop
[
7]
fective in the chronic phase due to parasite resistance. However,
these emerging resistances could be overcome by the synthesis and
modification of new biologically active molecules. Several re-
searchers have demonstrated a broad spectrum of biological activ-
et al.
reported that novel thiazolyl‐pyrazolines bearing thiophenes
showed antifungal activity against different Candida spp. and a no-
table antiproliferative effect on A549 human lung adenocarcinoma
cell line with low cytotoxicity against NIH/3T3 cells.
[
8]
ities of the 2‐aminothiazole (2‐AT) derivatives as antibacterial,
Given the global public health problems that generate anti-
microbial and parasitic infections and the wide medical application of
thiazoles, nitrogen mustard, and 2‐pyrazoline derivatives, we de-
scribe here the synthesis of novel thiazole‐2‐nitrogen mustards for
the design of new series of N‐substituted thiazolyl‐pyrazolines as
potential antimicrobial and antiprotozoal agents.
[
9]
[10]
[11–13]
[14]
antifungal, antitumoral,
antimalarial,
[16]
antidiabetic,
anti‐
[
15]
inflammatory,
and antiviral
agents (Figure 1). Many authors
have suggested that the 2‐AT moiety may be used for fragment‐
based drug discovery strategy, because it plays an important role in
[
17,18]
the treatment of different diseases.
Additionally, nitrogen mustards are an important class of alky-
lating agents, because they show interesting biological properties due
to their high chemical reactivity with nucleophiles such as nitrogen
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
[
19]
atoms of nucleic acids and sulfhydryl groups in proteins.
More-
over, they lead to ADN/ARN alkylation, protein cross‐linking, and
[19–21]
depletion of glutathione, thus promoting apoptotic cell death.
However, N,N‐bis(2‐chloroethyl)amine pharmacophore requires
The synthetic routes employed for the synthesis of the target deri-
vatives 5/6/7/8/9a–g are illustrated in Schemes 1–4. In Scheme 1,
the intermediate 2,4‐dichlorothiazole‐5‐carbaldehyde 2 was pre-
pared by Vilsmeier–Haack reaction of 1,3‐thiazolidine‐2,4‐dione 1
[
19,22]
adequate carriers to reduce the toxicity against normal cells
[
20,21]
and hydrolysis rate
by increasing its specificity during drug
delivery. In the last few years, a number of modifications have been
made, including the binging of the nitrogen mustard moiety to a
3
with dimethylformamide and POCl according to the methodology
[
22,23]
[24]
[21]
[40]
conjugated linker (carbonyl
/aryl /heterocycle ) to reduce
reported in the literature.
The preparation of the thiazole‐
[
21,22]
the basicity of nitrogen mustard and enhance drug delivery.
2‐nitrogen mustard 3 was initially conducted in dichloromethane and
triethylamine, the reaction mixture was stirred at room temperature
for 7 days, and the residue was purified by column chromatography
Although nitrogen mustards are widely used in the design of com-
pounds with antitumor activity such as melphalan, chlorambucil, and
[
19,25–27]
cyclophosphamide,
potential of this moiety in the inhibition of microorganisms.
‐Pyrazolines are important anti‐inflammatory and analgesic
agents, and some of them are potent drugs such as metamizol, phe-
there are few reports that explore the
3
using CHCl as eluent to provide compound 3 in 45% yield. In an
[
22,28]
attempt to develop an efficient procedure to obtain compound 3, the
2
reaction was conducted under basic reaction conditions using NaOH
or K
2
CO
3
as reported for the synthesis of 2‐substituted 4‐chloro‐1,
[
29]
[40–43]
nylbutazone, and morazone.
Also, pyrazoline derivatives exhibit
3‐thiazole‐5‐carbaldehydes with amines and thiophenols.
However, when the reaction mixture was refluxed in the presence of
NaOH in acetonitrile for 3 hr, the thin‐layer chromatography (TLC)
analysis showed the formation of by‐products without consumption
[
30]
[31]
pharmacological activities such as antitumor,
antibacterial,
and antidepressant activities
[32]
[33]
[15]
anti‐inflammatory,
antifungal,
(
Figure 2). Recent studies have revealed that thiazolyl‐pyrazoline
FIGURE 1 Structures of drugs
containing the 2‐aminothiazole scaffold

CUARTAS ET AL.
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|
FIGURE 2 Structures of thiazolyl‐pyrazolines derivatives with notable biological activity. EGFR, epidermal growth factor receptor; MIC,
minimal inhibitory concentration; TK, tyrosine kinase
SCHEME 1 Synthesis of aldehyde 3 from 1,3‐thiazolidine‐2,4‐dione 1
of aldehyde 2. Better results were obtained with K
2
CO
3
in acetoni-
Prompted by the above‐mentioned biological activity, thiazole‐
2‐nitrogen mustard 3 was used as a core scaffold to prepare new
chalcone–thiazole hybrids 5a–g. Thus, compound 3 was subjected to
a Claisen–Schmidt condensation with acetophenone 4a using NaOH
in ethanol at room temperature for 0.5 hr (Scheme 2). Upon con-
sumption of compound 3 (TLC analysis), the reaction mixture was
trile under reflux, reducing the reaction time to 7 hr and affording the
thiazole‐2‐nitrogen mustard 3 in 44% yield. The structure of com-
pound 3 was unambiguously determined by single‐crystal X‐ray dif-
fraction. The Oak Ridge Thermal Ellipsoid Plot (ORTEP) drawing of
the structure for compound 3 is shown in Scheme 1.
SCHEME 2 Synthesis of substituted chalcones 5a–g by Claisen–Schmidt condensation of compound 3 and acetophenones 4a–g

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SCHEME 3 Synthesis of N‐acetyl and N‐formyl pyrazolines 6/7a–g
SCHEME 4 Synthesis of compounds 8a–g and 9a–g

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filtered and washed with ethanol and water to afford 5a in a 90%
yield. It should be mentioned that longer reaction times led to the
formation of some by‐products. By using this methodology and ap-
propriate acetophenones 4a–g, we synthesized the set of hybrids
In addition, these derivatives show an improvement in metabolic
stability and pharmacokinetic profile, as recently demonstrated by
[
49]
Eggert et al.,
who identified that the 3,4‐dichloro derivative
(IC50 = 0.08 μM) is more active than the corresponding 4‐chloro
(IC50 = 0.8 μM) and unsubstituted phenyl derivatives (IC50 > 20 μM)
against SMYD2 for the treatment of cancer. To test this hypothesis,
we decided to synthesize the novel N‐phenyl and N‐3,
5‐dichlorophenyl derivatives to evaluate the effect of the aryl and
the chlorine substituents on biological activities.
5a–g in good yields (90–50%).
The structural elucidation of compounds 5a–g was performed by
spectroscopic techniques, Fourier‐transform infrared spectroscopy
1
13
(
FTIR), H NMR (nuclear magnetic resonance), C NMR, and mass
spectrometry. Compounds 5a–g showed similar spectroscopic data;
therefore, compound 5a is discussed here as the representative of
this series. Infrared (IR) spectrum of compound 5a showed mainly
Therefore, we carried out the reaction of chalcone–thiazole hy-
brids 5a–g with phenylhydrazine in the presence of acetic acid in
MeOH under reflux, which afforded N‐phenylpyrazoline 8a–g
(Scheme 4) in good yields (66–50%). A similar reaction with
3,5‐dichlorophenylhydrazine showed the formation of two products
corresponding to the analogous N‐3,5‐dichlorophenyl pyrazoline 9a
and pyrazole 10a (Scheme 4). Then, we attempted the reaction in
glacial acetic acid as a solvent, and after 8 hr, 3‐(4‐chlorophenyl)‐
1‐(3,5‐dichlorophenyl)‐1H‐pyrazole 10a was obtained with the ab-
sence of N‐3,5‐dichlorophenyl pyrazoline 9a. However, when the
reaction was performed in MeOH, compound 9a was obtained in
88% yield (Scheme 4). Therefore, compounds 9a–g were prepared
from chalcones 5a–g and 3,5‐dichlorophenylhydrazine hydrochloride
in MeOH (Scheme 4). Compounds with electron‐withdrawing groups
5a (4‐Cl), 5b (4‐Br), and 5c (4‐F) had to be kept 12 hr under reflux to
allow the cyclization of the hydrazine intermediate to the desired
products 9a, 9b, and 9c, respectively. However, the reactions of
3,5‐dichlorophenylhydrazine hydrochloride and chalcones with
−1
absorption bands at 1,645, 1,586, and 1,529 cm , assigned to the
1
stretching vibration of C═O, C═N, and C═C bonds. In the H NMR
spectrum, a multiplet between 7.96 and 7.87 ppm integrating for
6
three protons corresponding to protons H and HBo, a doublet at
3
7
.45 ppm with J = 8.3 Hz assigned to two protons HBm, and a doublet
3
at 6.82 ppm with coupling constant J = 15.0 Hz for proton H
7
were
observed, indicating the E configuration of H and H
6
7
in the
1
3
α,β‐unsaturated fragment. The C NMR spectrum showed 12 signals
for carbons of compound 5a. In addition, the molecular structure and
conformation were confirmed for X‐ray analysis, and its ORTEP
drawing is shown in Scheme 2.
Once we obtained the chalcone–thiazole hybrids bearing nitro-
gen mustard 5a–g, we performed the synthesis of diverse N‐acetyl
[
44,45]
and N‐formyl pyrazolines 6/7 by using reported procedures.
For
the synthesis of N‐acetyl derivatives 6a–g, chalcones 5a–g were
treated with hydrazine hydrate in EtOH under reflux, resulting in the
formation of the corresponding N‐H pyrazolines, followed by addition
of acetic anhydride via “one‐pot” reaction, affording the pyrazoline
derivatives 6a–g in good yields (70–52%). On the basis of these re-
sults, the reaction was extended to the synthesis of N‐formyl pyr-
azolines (Scheme 3) in the presence of formic acid in place of acetic
anhydride and the desired products 7a–g were successfully obtained
in good yields (76–52%).
electron‐donating groups 5d (3,4‐OCH
2
O), 5e (4‐OCH
3
), and 5f
(3,4,5‐(OCH ), unsubstituted on phenyl ring 5g (4‐H), occurred
3 3
)
more easily under reflux for 4 hr because their corresponding hy-
drazones were more soluble in the reaction medium, allowing their
cyclization in shorter reaction times. Under these conditions, the
N‐3,5‐dichlorophenyl pyrazolines 9a–g were obtained in good to
excellent yields (90–59%).
1
13
Compounds 6/7a–g were fully characterized by FTIR, H NMR,
C
The structural assignment of compound 10a was performed on
1
NMR, and mass spectrometry. The IR spectra of the series 6/7a–g show
the basis of NMR data and mass spectrometry. In the H NMR
–1
a sharp band at 1653–1678 cm , assigned to the stretching vibration of
spectrum, two doublets for the protons of pyrazole ring H5′ and H4′
1
2
the C═O bond. In the H NMR spectra for N‐acetyl pyrazolines 6a–g,
at 8.73 and 7.15 ppm, with coupling constant J = 2.6 Hz, and two
a singlet for methyl protons of acetyl group was observed at
doublets at 8.05 and 7.56 ppm, with coupling constant J = 1.8 Hz,
corresponding to HCo and HCp, respectively, were observed. At 7.99
and 7.52 ppm, two doublets with coupling constant J = 8.5 Hz, which
were assigned to aromatic protons HBo and HBp, were observed.
1
2
.24–2.27 ppm, whereas the H NMR spectra for N‐formyl pyrazolines
7a–g showed a singlet for formyl proton at 8.86–8.90 ppm. In all cases,
A M
the methylene diastereotopic protons H4a′ and H4b′ (H and H ), and
1
3
the stereogenic proton H5′ (H ) appear as three double‐doublets, which
X
Furthermore, the C NMR spectrum of compound 10a showed all
the expected signals.
13
form AMX spin systems. The C NMR spectra showed all the expected
signals for carbons of compounds 6/7a–g. Compound 6g gave suitable
crystals for X‐ray analysis by slow evaporation technique from EtOH,
confirming the proposed structure and its conformation, which is shown
in Scheme 3.
Regarding the structural elucidation of N‐aryl pyrazolines 8a–g
and 9a–g, we will discuss compound 8a as the representative of these
series (8/9a–g). The IR spectrum of 8a showed absorption bands
−
1
between 1,522 and 1,597 cm , associated to the stretching vibra-
1
According to recent papers on N‐aryl pyrazolines, the introduc-
tion of the aryl group at the N‐1 position of the dihydropyrazole ring
tions of the C═N and C═C bonds. In the H NMR spectrum, the
signals of H4a′ (H ) appeared as a double‐doublet at 3.17 ppm with
A
[
33,45–48]
2
3
leads to biological activity improvement.
However, chlor-
coupling constant JAM = 17.1 Hz and JAX = 6.6 Hz; the signals of H4b′
(H ) and CH –N appeared as a multiplet between 3.78 and 3.70 ppm
integrating for five protons, and the corresponding signal of H5′ (H
ophenyl derivatives may enhance potency and binding efficiency
M
2
[
2,49,50]
compared to their unsubstituted phenyl derivatives.
X
)

6
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CUARTAS ET AL.
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appeared as a double‐doublet at 5.51 ppm with coupling constant
These results clearly show that all thiazolyl‐pyrazolines, 8a–g,
3
3
13
J
MX = 11.8 Hz and
JAX = 6.6 Hz. The C NMR spectrum showed
containing an unsubstituted phenyl ring as the azole N‐substituent R
display high activities against C. neoformans with IC50 in the range
5.6–125 µg/ml. The substituent R does not appear to have a key
1
16 signals for carbons of compound 8a.
1
role in the anticryptococcal activity of compound 8, as the IC50 values
of 8a (4‐Cl) and 8b (4‐Br) derivatives (IC50 = 15.6 µg/ml) differ in only
2
.2 | Biology
one dilution with the IC50 of 8g (4‐H), 8d (3,4‐OCH
(4‐OCH ) derivatives (IC50 = 31.25 µg/ml), which is considered the
error of the method.
2
O–), or 8e
2
.2.1 | Antifungal activity
3
[55]
So, almost all compounds 8 can be con-
According to the literature, the 2‐pyrazoline moiety confers inter-
sidered with similar anticryptococcal activity.
esting biological properties to compounds, including the antifungal
However, it is important to note that among N‐3,
[18,33,34]
activity.
Compounds 3, 5a–g, 6a–g, 7a–g, 8a–g, and 9a–g
5‐dichlorophenyl pyrazolines, 9a–g, compounds 9e (4‐OCH
3
), 9f
were tested for antifungal properties against two clinically important
fungal species, Candida albicans and Cryptococcus neoformans, which
are involved in invasive fungal infections in immunocompromised
(3,4,5‐(OCH ), and 9g (4‐H) possess the highest activities of all
3 3
)
studied compounds against C. neoformans, with IC50 values of 7.8, 3.9,
and 7.8 µg/ml, respectively. These results suggest that the occur-
[
51–53]
patients, leading to high risk of morbidity and mortality.
rence of 3,5‐dichlorophenyl as the substituent R
1
along with 4‐OCH
3
The antifungal activity was evaluated by the Broth Microdilution
(9e) or 3,4,5‐(OCH (9f) or 4‐H (9g) in the same molecule leads to
3 3
)
Method, M27 (4th ed.) for yeasts of Clinical and Laboratory Stan-
an increase in the anticryptococcal activity of these compounds, thus
opening an avenue for further research. Table 2 shows the percen-
tages of inhibition of these three compounds at different con-
centrations, which are also reflected in the dose–response curves of
Figure 3.
[
54]
dards Institute (CLSI).
The percentages of growth inhibition for
each fungus were determined for all compounds at the concentra-
tion range of 3.9–250 µg/ml, which allowed to establish IC50 values
of each compound. IC50 represents the minimum inhibitory con-
centration of each compound that inhibits 50% of fungal growth.
Compounds with IC50 > 250 µg/ml were considered inactive;
with 250 ≥ IC50 > 125 µg/ml, marginally active; with 125 ≥ IC50
1.2 µg/ml, moderately active, and with IC50 ≤ 31.2 µg/ml, highly
active. Amphotericin B was used as drug control and displayed
>
2.2.2 | Antibacterial activity
3
All synthesized compounds (3, 5a–g, 6a–g, 7a–g, 8a–g, 9a–g) were
tested for antibacterial activity against wild‐type and multidrug‐
resistant Gram‐negative and ‐positive bacteria including methicillin‐
susceptible Staphylococcus aureus (MSSA), methicillin‐resistant S. aureus
(MRSA), vancomycin‐intermediate S. aureus (VISA), Escherichia coli,
Pseudomonas aeruginosa, Klebsiella pneumoniae, K. pneumoniae BAA
1705, and Neisseria gonorrhoeae. Minimum inhibitory concentration
(MIC) was determined by broth microdilution in those compounds
with reproducible antimicrobial effects in the screening, as reported in
Table 3. The agar microdilution test was performed for N. gonorrhoeae.
Compounds N‐acetyl 6 and N‐formyl pyrazoline 7 were more
active than their N‐aryl derivatives 8/9, particularly against MSSA,
MRSA, VISA, and N. gonorrhoeae, where 8a and 8b showed discrete
inhibition against VISA with MIC values of 250 and 500 µg/ml, and
inhibited N. gonorrhoeae with MIC values of 250 and 500 µg/ml, re-
spectively. However, the introduction of chlorine atoms in the N‐aryl
group abolishes the antibacterial effects of compounds 9a–g, which
were inactive against all bacterial strains tested, which is in contrast
to those results obtained for antifungal activity.
1
00% inhibition at all the concentrations tested.
Among the synthesized compounds, aldehyde 3 and chalcone–
thiazole hybrids 5a–g were inactive (IC50 > 250 µg/ml) against
C. albicans and C. neoformans, suggesting that thiazole‐2‐nitrogen
mustard and prop‐2‐en‐1‐one moieties do not confer an antifungal
effect. Table 1 shows the IC50 values of compounds 6/7/8/9a–g
against C. albicans and C. neoformans.
Regarding the behavior of 6/7/8/9a–g against C. albicans, almost all
the compounds were inactive (IC50 > 250 µg/ml), and only compound 8e
(
4‐OCH
were highly sensitive to compounds 8a–g and 9d–g but not to N‐acetyl
a–g and N‐formyl pyrazolines 7a–g. From these results, it is clear that
3
) was marginally active (IC50 = 125 µg/ml). Instead, C. neoformans
6
cyclization of the chalcones–thiazole hybrids 5a–g into their pyrazoline
derivatives increases the antifungal effects of the compounds.
For a better analysis of the results, the percentages of inhibition
of compounds 8a–g and 9a–g against C. neoformans were recorded in
dose–responses curves (Figure 3).
Figure 3 clearly shows that five compounds (8a,b,d,e,g), over the
seven compounds, 8a–g, display high C. neoformans inhibition with
IC50 = 15.6–31.2 µg/ml. The remaining two compounds, 8c (4‐F) and 8f
Compounds 6a (4‐Cl, N‐acetyl), 6b (4‐Br, N‐acetyl), 7a (4‐Cl,
N‐formyl), 7b (4‐Br, N‐formyl), 7c (4‐F, N‐formyl), and 7g (4‐H,
N‐formyl) evidenced good or discrete activity against MSSA, MRSA,
VISA, and N. gonorrhoeae, ranging from 31.5 to 250 µg/ml, indicating
that the presence of halogens in the structure improves the anti-
bacterial effect. The best antibacterial activity was displayed by
compound 7a (4‐Cl), which showed inhibitory activity against MSSA
ATCC 25923 (MIC = 62.5 µg/ml), as well as two multidrug‐resistant
(
3,4,5‐(OCH
respectively. Interestingly enough, three compounds, 9e–g (4‐OCH
,4,5‐(OCH 3, and 4‐H), over the seven compounds, 9a–g, showed the
highest activity (IC50 = 3.9–7.8 µg/ml) of all series. Compound 9d
3,4‐OCH O–) showed marginal activity (IC50 = 250 µg/ml) and com-
pounds 9a–c (4‐Cl, 4‐Br, 4‐F) are inactive (IC50 > 250 µg/ml).
3 3
) ), were moderately active with IC50 = 62.5 and 125 µg/ml,
3
,
3
3
)
(
2

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|
TABLE 1 Antifungal activity of thiazolyl‐pyrazoline derivatives 6/9a–g against Candida albicans ATCC 10231 and Cryptococcus neoformans
ATCC 32264 expressed in IC50 (μg/ml)
Compound
R
R
1
C. albicans ATCC 10231
C. neoformans ATCC 32264
6
6
6
6
6
6
6
a
b
c
d
e
f
4‐Cl
–COCH
–COCH
–COCH
–COCH
–COCH
–COCH
–COCH
3
3
3
3
3
3
3
250
250
4‐Br
>250
>250
>250
>250
>250
>250
250
4‐F
>250
>250
>250
>250
>250
2
3,4‐OCH O–
4‐OCH
3
3,4,5‐(OCH
4‐H
3
)
3
3
3
3
g
7
7
7
7
7
7
7
a
b
c
d
e
f
4‐Cl
4‐Br
4‐F
–CHO
–CHO
–CHO
–CHO
–CHO
–CHO
–CHO
>250
>250
>250
>250
>250
>250
>250
250
250
250
>250
250
250
250
3,4‐OCH
2
O–
4‐OCH
3
3,4,5‐(OCH
4‐H
3
)
g
8
8
8
8
8
8
8
a
b
c
d
e
f
4‐Cl
4‐Br
4‐F
–C
6
–C
6
–C
6
–C
6
–C
6
–C
6
–C
6
H
5
H
5
H
5
H
5
H
5
H
5
H
5
250
250
>250
250
125
250
250
15.6
15.6
62.5
3,4‐OCH
4‐OCH
2
O–
31.25
31.25
125
3
3,4,5‐(OCH
4‐H
3
)
g
31.25
9
9
9
9
9
9
9
a
b
c
d
e
f
4‐Cl
4‐Br
4‐F
3,5‐Cl
3,5‐Cl
3,5‐Cl
3,5‐Cl
3,5‐Cl
3,5‐Cl
3,5‐Cl
2
2
2
2
2
2
2
C
6
6
6
6
6
6
6
H
3
H
3
H
3
H
3
H
3
H
3
H
3
>250
>250
>250
250
>250
>250
>250
250
7.8
C
C
C
C
C
C
3,4‐OCH
2
O–
4‐OCH
3
250
3,4,5‐(OCH
4‐H
3
)
250
3.9
g
250
7.8
Amphotericin B
Note: Active compounds are highlighted.
Abbreviations: ATCC, American Type Culture Collection. IC50, inhibitory concentration of each compound that inhibits 50% of fungal growth.
0.5
0.5
strains, MRSA ATCC 43300 (MIC = 125 µg/ml) and VISA (MIC =
1.5 µg/ml), and showed discrete inhibition against N. gonorrhoeae
MIC = 500 µg/ml). Compound 7b (4‐Br) was also active against the
great interest due to the emergence of multidrug‐resistant strains for
[
56]
3
almost all antibiotics.
None of the tested compounds were active
(
against E. coli, P. aeruginosa, and K. pneumoniae. However, it should be
noted that compounds 6a (4‐Cl), 6b (4‐Br), 7a (4‐Cl), 7b (4‐Br), 7c
(4‐F), and 7g (4‐H) inhibited the multidrug‐resistant strain VISA.
Therefore, those compounds have good potential as a starting point
to develop novel anti‐VISA agents.
multidrug‐resistant bacterium, VISA (MIC = 31.5 µg/ml), and showed
discrete inhibition against N. gonorrhoeae (MIC = 250 µg/ml).
For N. gonorrhoeae, compounds 7g and 8a exhibited MIC values
of 125 µg/ml, being the most active against this bacterium, which is of

8
of 24
CUARTAS ET AL.
|
(
a)
(b)
100
100
80
60
40
20
0
80
60
40
20
0
8
8
8
8
a
b
c
9
a
9b
9
9
9
9
c
d
e
f
d
8e
8
g
f
8
9g
0
50
100
150
200
250
0
50
100
150
200
250
Concentration (µg/ml)
Concentration (µg/ml)
FIGURE 3 Comparative antifungal activities of (a) N‐phenyl 8a–g and (b) N‐3,5‐dichlorophenyl 9a–g pyrazolines against Cryptococcus
neoformans ATCC 10231. The horizontal dotted lines show 50% inhibition. The vertical dotted lines show the concentration 31.25 µg/ml.
Compounds with IC50≤31.2 µg/ml are considered highly active
2
.2.3 | Hemolytic activity
for the development of anticryptococcal agents. However, compounds
a, 6b, 7a, 7b, and 7g that showed antibacterial activity against MSSA,
6
All compounds were tested on human red blood cells (RBCs) to
evaluate the toxicity of each compound to induce irreversible damages
in the membrane of erythrocytes by the release of hemoglobin and
MRSA, VISA, and N. gonorrhoeae exhibited little or no toxicity on red
blood cells and membranes at concentrations used in therapeutics.
These low hemolytic activities offer a promising potential to the de-
velopment of biological agents.
[57,58]
intracellular components.
Results show that compounds 3, 5a–d,
5f–g, 6a–c, 6e–g, 7a–b, 7d–g, 8c–g, and 9a–g lead to 100% hemolysis
at a concentration of ≥ 200 μM, indicating that these compounds
would be nontoxic. Compounds 5e, 6d, 7c, and 8a–b were considered
moderately toxic. However, no hemolytic toxicity was observed below
2.2.4 | Antiprotozoal activity
1
00 μM, as shown in Table 4. Interestingly, compounds 9e–g were
highly active against C. neoformans, with IC50 values between 3.9 and
.8 µM and low cytotoxic effects on erythrocytes, which suggests that
these compounds can be considered as important structural scaffolds
Cytotoxic activity in human macrophages U‐937
All synthesized compounds were screened for their cytotoxicity on
U‐937 human cells at four serial dilution concentrations (200, 50,
12.5, and 3.125 µM) to determine the 50% lethal concentration
7
TABLE 2 Percentages of inhibition of N‐3,5‐dichlorophenyl pyrazolines 9e–g against Cryptococcus neoformans ATCC 32264
Concentrations (μg/ml)
R
250
100
100
100
100
125
100
100
100
100
62.5
100
100
100
100
31.25
100
100
100
100
15.62
100
7.81
3.9
9
9
9
e
4‐OCH
3
97.99 ± 0.81
82.3 ± 1.06
62.7 ± 0.31
100
11.31 ± 0.39
49.55 ± 0.45
41.86 ± 0.19
100
f
3,4,5‐(OCH
3
)
3
100
g
4‐H
67.73 ± 0.26
100
Amphotericin B

CUARTAS ET AL.
9 of 24
|
TABLE 3 Antibacterial activity of novel thiazole derivatives 3,
TABLE 4 Hemolytic activity for compounds 3, 5a–g, 6a–g, 7a–g,
8a–g, and 9a–g expressed in LC50 (μM)
a,b
a
5a–g, 6a–g, 7a–g, 8a–g and 9a–g expressed in MIC (μg/ml)
a
S. aureus
S. aureus
ATCC
Compound
LC50
Compound
7f
LC50
ATCC
Neisseria
3
>200
242.77 ± 50.82
25923
43300
gonorrhoeae
ATCC 31426
Compound (MSSA)
(MRSA)
VISA
5a
>200
7g
8a
8b
8c
8d
8e
8f
>200
5
5
5
5
5
5
b
c
d
e
f
>200
100.00
6
6
6
6
6
a
250
250
–
250
250
–
125
125
–
250
–
>200
134.64 ± 8.32
796.97 ± 230.92
352.23 ± 76.89
338.63 ± 76.13
730.56 ± 253.93
b
>200
c
–
198.74 ± 37.28
867.03 ± 265.41
>200
d
500
–
500
–
250
–
–
e–g
–
g
7
7
7
7
7
a
62.5
125
250
–
125
125
250
–
31.25
31.25
125
–
500
250
250
–
6
6
6
6
6
6
6
a
b
c
d
e
f
293.31 ± 36.77
526.00 ± 26.24
>200
8g
9a
9b
9c
9d
9e
9f
419.68 ± 143.67
>200
b
c
>200
d–f
g
100.00
>200
250
–
62.5
125
269.28 ± 37.76
>200
278.80 ± 4.18
>200
8
8
8
a
500
–
500
–
250
500
–
125
250
–
b
g
>200
867.03 ± 187.03
c–g
–
–
7
7
7
7
7
a
b
c
464.12 ± 94.89
225.43 ± 12.75
161.02 ± 16.48
>200
9g
300.30 ± 76.02
9
a–g
–
–
–
–
Abbreviations: ATCC, American Type Culture Collection; MIC, minimal
inhibitory concentration; MRSA, methicillin‐resistant Staphylococcus
aureus; MSSA, methicillin‐susceptible Staphylococcus aureus; VISA,
vancomycin‐intermediate Staphylococcus aureus.
d
e
>200
a
Minimal inhibitory concentration of each compound that inhibits visible
Note: Data represent mean value ± standard deviation. Bold values are the
bacterial growth. Active compounds are highlighted.
nontoxic compounds.
b
a
Compounds 3 and 5a–g were inactive against wild‐type and multidrug‐
Lethal concentration on human red blood cells.
resistant Gram‐negative and ‐positive bacteria‐inactive compound
(
MIC > 1,000 μg/ml). None of the tested compounds were active against
aldehyde 3, suggesting that thiazole‐2‐nitrogen mustard moiety does
not confer antiplasmodial activity, but the activity can be tuned with
the introduction of the prop‐2‐en‐1‐one and pyrazoline moieties. The
Escherichia coli ATCC 25922, Pseudomonas aeruginosa, Klebsiella
pneumoniae, K. pneumoniae BAA 1705.
(LC50). Compounds with LC50 < 100 µM were considered as poten-
1
substituents R and R do not seem to have a role in the activity.
tially cytotoxic, with 100 ≤ LC50 < 200 µM as mildly cytotoxic, and
with LC50 ≥ 200 µM as noncytotoxic. As can be seen in Table 5, al-
most all the thiazole derivatives, 3, 5a–g, 6a–g, 7a–c, 7e–g, 8a–g, and
Compound 8g (4‐H) was the most active among the thiazole deri-
vatives, with EC50 value of 10.28 μM. However, compounds 7d
(3,4‐OCH O–) and 9g (4‐H) showed a selective index (SI) higher than
2
9
a–e, displayed high cytotoxic activity on human macrophages
10, being more active against P. falciparum than U‐937 human cells,
with EC50 values of 12.24 µM (LC50 = 142.82 µM, SI > 11.66) and
11.80 µM (LC50 = 182.98 µM, SI = 15.50), respectively, indicating that
both compounds (7d and 9g) have potential for the development of
U‐937. However, compounds 7d (3,4‐OCH O–), 9f (3,4,5‐(OCH ),
2
3 3
)
and 9g (4‐H) were mildly cytotoxic (LC50 > 100 µM). Also, it is
relevant to highlight the potentially cytotoxic activity of amphoter-
icin B (LC50 = 49.78 µM) and doxorubicin (LC50 = 0.90 µM), which are
important commercial drugs.
[
59]
antimalarial agents.
Antileishmanial activity
Antiplasmodial activity
Antileishmanial activity of 3, 5a–g, 6a–g, 7a–g, 8a–g, and 9a–g were
evaluated in intracellular amastigotes of Leishmania (V) panamensis
(MHOM/CO/87/UA140–EpiR–GFP strain), and the results are sum-
marized in Table 7. Most compounds exhibited high activity against
Leishmania amastigotes (EC50 ≤ 25 μM), showing an SI ≥ 1. Interest-
ingly, chalcone–thiazole hybrids (5) showed higher antileishmanial
activity than N‐aryl pyrazolines (8/9). As can be seen in Table 7,
On the basis of the large number of biological agents containing
thiazole ring and the increasingly numerous examples of thiazole
[11–13]
derivatives that are active as antimalarial agents,
it was en-
visaged that the antiplasmodial activity of these novel thiazole de-
rivatives would be worthwhile. The antiplasmodial activity was tested
on asynchronic cultures of Plasmodium falciparum (3D7 strain, sen-
sitive to chloroquine [CQ]) at serial dilution for all compounds, which
allowed to determine the EC50 values of each compound (Table 6).
Compounds with EC50 ≤ 25 µM were considered highly active. From
these results, it is found that compounds 5a–g, 6a–g, 7a–g, 8a–g, and
compounds 8b (4‐Br), 8f (3,4,5‐(OCH
)
3 3
2
), and 9d (3,4‐OCH O–) also
showed remarkable activity, with EC50 values of 10.62, 6.46, and
10.56 μM, respectively. The results obtained with aldehyde 3 indicate
that thiazole‐2‐nitrogen mustard moiety does not confer antil-
eishmanial activity, but its derivatives are highly active against
9a–g were highly active against P. falciparum, being more active than

10 of 24
CUARTAS ET AL.
|
a
TABLE 5 In vitro cytotoxicity on U‐937
human cells of novel thiazole derivatives 3,
Compound
3
LC50
Compound
7f
LC50
17.68 ± 6.25
21.45 ± 2.52
5
a–g, 6a–g, 7a–g, 8a–g, and 9a–g expressed
a
in LC50 (μM)
5
5
5
5
5
5
5
a
b
c
d
e
f
8.90 ± 0.24
7g
8a
8b
8c
8d
8e
8f
10.64 ± 0.89
24.07 ± 10.72
17.49 ± 0.43
15.81 ± 1.93
17.44 ± 3.02
50.18 ± 9.50
20.64 ± 3.04
13.49 ± 2.27
25.30 ± 7.48
62.61 ± 22.35
10.85 ± 0.55
19.06 ± 3.47
8.51 ± 0.88
g
6
6
6
6
6
6
6
a
b
c
d
e
f
7.80 ± 0.20
13.48 ± 2.19
6.93 ± 0.40
14.21 ± 1.70
11.07 ± 1.50
25.84 ± 3.91
9.73 ± 1.77
8g
9a
9b
9c
9d
9e
9f
22.96 ± 5.18
19.18 ± 2.08
19.17 ± 4.85
23.40 ± 9.71
34.69 ± 6.87
40.25 ± 6.81
101.30 ± 9.67
g
7
7
7
7
7
a
b
c
15.69 ± 3.47
35.15 ± 8.57
8.00 ± 0.86
9g
182.98 ± 1 8.17
0.90 ± 0.07
49.78 ± 6.8
>200
Doxorubicin
b
Amphotericin B
c
d
e
142.82 ± 16.32
21.81 ± 3.02
Benznidazole
d
CQ
178.51 ± 5.98
Note: Data represent mean value ± standard deviation. Bold values are the nontoxic compounds.
a
Lethal concentration on human cell U‐937.
Antileishmanial drug control.
b
c
Antitrypanosomal drug control.
d
Chloroquine diphosphate salt (CQ): Antimalarial drug control.
Leishmania amastigotes. These findings suggest that the prop‐2‐en‐
‐one and pyrazoline fragments significantly improve the antil-
trypanosomicidal activity as compared to the N‐phenyl and N‐3,
5‐dichlorophenyl pyrazolines (8/9a–g). This behavior could be related
to the number of hydrogen‐bond acceptors of these compounds, as
1
eishmanial activity. This could be due to the presence of the
[61]
α,β‐unsaturated moiety of the chalcone that easily reacts with thiol
shown in Figure 4, which was generated using PyMOL.
Moreover,
[
7]
and amino groups. Also, 2‐pyrazolines (nitrogen‐containing het-
the substitution of the halogen atoms (4‐Cl, 4‐Br, and 4‐F) by me-
thylenedioxy, methoxy, and trimethoxy groups slightly diminishes the
activity. An exception to this behavior is 6d versus 6c, where the
methylenedioxy derivative was more active.
[
60]
erocyclic compound) could form hydrogen bonding
in the active
site of the enzymes of Leishmania.
Antitrypanosomal activity
All compounds were screened on intracellular amastigotes of Trypa-
nosoma cruzi, Tulahuen strain transfected with β‐galactosidase gene.
The activity was determined according to the ability of the compound
to reduce the infection of U‐937 cells by T. cruzi. Results are sum-
marized in Table 8. The new 21 compounds evidence good activity
against T. cruzi, with EC50 (EC50 ≤ 25 μM). Compounds 5a (4‐Cl), 5g
3 | CONCLUSIONS
A new series of nitrogen mustard class of thiazolyl‐pyrazoline deri-
vatives 6/9a–g was obtained in good yields by reacting chalcone–
thiazole hybrids 5a–g with hydrazine derivatives under mild condi-
tions. Compounds 3, 5a, and 6g give good‐quality single crystals,
which confirm the molecular structure and conformation of the dif-
ferent derivatives by X‐ray analysis. The antifungal activity studies
revealed that all the N‐aryl pyrazolines 8a–g, possessing an un-
substituted phenyl ring as R1, display high anticryptococcal activities,
with IC50 values between 15.6 and 125 µg/ml. However, the highest
antifungal activity was displayed by the N‐3,5‐dichlorophenyl pyr-
azolines 9e, 9f, and 9g, with IC50 values of 3.9–7.8 µg/ml against C.
(4‐H), 6c (4‐F), and 7c (4‐F) were highly active against T. cruzi, with
EC50 value between 4.98 and 10.37 μM, showing better activity than
the benznidazole (EC50 = 13.34 μM), which was used as drug control.
Compound 7c is a particularly interesting compound, because it ex-
hibits the highest trypanosomicidal activity (EC50 = 4.98 μM), which is
approximately 2.6‐fold more potent than benznidazole with an SI of
1.61 on U‐937 human cells and has potential as a novel anti-
trypanosomal agent.
Finally, if we compare the synthesized series, the presence of
carbonyl group in chalcone–thiazole hybrids (5a–g) and N‐acetyl and
N‐formyl pyrazolines (6/7a–g) increases or maintains the
neoformans and 9f and 9g being important scaffolds with LC50 >
200 μM against human red blood cells. Moreover, the results on
antibacterial activity showed that the N‐formyl pyrazoline 7a was the

CUARTAS ET AL.
11 of 24
|
TABLE 6 In vitro antimalarial screening
of novel thiazole derivatives 3, 5a–g, 6a–g,
a
b
Compound
3
EC50
IS
Compound
7f
EC50
IS
25.35 ± 0.56
0.69
11.01 ± 0.36
1.94
7
a–g, 8a–g, and 9a–g expressed in
a
EC50 (μM)
5
5
5
5
5
5
5
a
b
c
d
e
f
17.46 ± 1.59
14.95 ± 0.92
10.93 ± 0.61
11.95 ± 0.06
11.36 ± 0.75
11.27 ± 0.25
11.26 ± 0.63
0.50
0.90
2.31
5.23
0.95
1.69
0.75
7g
8a
8b
8c
8d
8e
8f
11.00 ± 0.34
10.62 ± 0.15
10.92 ± 0.13
11.23 ± 0.49
10.70 ± 0.12
10.77 ± 0.29
13.93 ± 0.85
0.96
2.27
1.60
1.41
1.63
4.66
1.48
g
6
6
6
6
6
6
6
a
b
c
d
e
f
12.33 ± 0.47
13.54 ± 0.25
10.61 ± 0.11
10.59 ± 0.36
10.60 ± 0.20
10.56 ± 0.35
10.52 ± 0.22
0.63
0.99
0.65
1.34
1.04
2.44
0.92
8g
9a
9b
9c
9d
9e
9f
10.28 ± 0.36
23.78 ± 1.02
19.50 ± 1.18
10.81 ± 0.33
19.66 ± 2.49
19.68 ± 1.90
16.29 ± 1.05
2.23
0.80
0.98
2.16
1.76
2.04
6.21
g
7
7
7
7
7
a
b
c
10.60 ± 0.27
11.25 ± 0.72
10.88 ± 0.41
12.24 ± 0.94
10.89 ± 0.40
1.48
3.12
9g
11.80 ± 0.35
15.50
c
CQ
4.25 ± 0.40
38.2
0.73
d
e
11.66
2.00
Note: Data represent mean value ± standard deviation. Bold values are the most active compounds.
a
Effective concentration (EC50): Concentration of compounds that decrease 50% of intracellular
parasite.
b
Index of selectivity (IS) = LC50/EC50; LC50: Lethal concentration on human cell U‐937.
c
Chloroquine diphosphate salt (CQ): Antimalarial drug control.
most active against MSSA, MRSA, and VISA, with MIC values of 62.5,
25, and 31.25 µg/ml, respectively. Compounds 7d and 9g showed
solvent and tetramethylsilane as an internal standard. Mass
spectra were obtained on a Shimadzu–GCMS–QP2010 spectro-
meter operating at 70 eV. The elemental analyses were per-
formed using a Thermo Finnigan Flash EA1112 CHN (STIUJA)
elemental analyzer. The single‐crystal X‐ray data were collected
1
remarkable activity, with EC50 values of 12.24 and 11.80 µM against
P. falciparum and SI higher than 10. The best antileishmanial activity
against L. panamensis was displayed by 8f, with EC50 = 6.46 μM.
Moreover, compound 7c (4‐F) was highly active against T. cruzi, with
an EC50 value of 4.98 µM, being more active than the drug control
in
a diffractometer Bruker D8 Venture at “Centro de In-
strumentación Científico y Técnico,” (CICT) in “Universidad de
Jaén” (UJA).
(benznidazole). These compounds represent novel hits for the
development of antimicrobial and antiprotozoal agents.
The original NMR spectra of the investigated compounds are
provided as Supporting Information, as are their InChI codes to-
gether with some biological activity data.
4
4
4
| EXPERIMENTAL
.1 | Chemistry
4.1.2 | Preparation of 2‐[bis(2‐chloroethyl)amino]‐
4
‐chlorothiazole‐5‐carbaldehyde (3)
.1.1 | General
A mixture of 2,4‐dichlorothiazole‐5‐carbaldehyde (300 mg, 1.65 mmol),
bis(2‐chloroethyl)amine hydrochloride (588 mg, 3.30 mmol), and K CO
Reagents and solvents used were purchased from Sigma‐Aldrich.
Reactions were monitored by TLC on Merck silica gel 60 F254
aluminum plates. Melting points were measured in open glass
capillaries using a Stuart SMP10 melting point apparatus. Atte-
nuated total reflection (ATR)–FTIR spectra were obtained on a
2
3
(1,140 mg, 8.25 mmol) in acetonitrile (5.0 ml) was heated under reflux
for 7 hr. After cooling, the reaction was quenched by the addition of
water (20 ml) and extracted with dichloromethane (3 × 20 ml). The
4
combined organic layers were dried over anhydrous MgSO , filtered,
1
13
Shimadzu IRAffinity‐1. H and C NMR spectra were run on a
and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography using hexane/dichloromethane (4:10)
to yield the desired product 3 as a pale yellow solid.
Bruker DPX 400 spectrometer operating at 400 and 100 MHz,
respectively, using CDCl
3
or dimethyl sulfoxide (DMSO)‐d
6
as

12 of 24
CUARTAS ET AL.
|
a
b
TABLE 7 Antileishmanial activity of
thiazole derivatives 3, 5a–g, 6a–g, 7a–g,
Compound
3
EC50
IS
Compound
7f
EC50
IS
53.37 ± 45.51
0.33
n.d.
–
a
8a–g, and 9a–g expressed in EC50 (μM)
5
5
5
5
5
5
5
a
b
c
d
e
f
>4
<2.25
<1.92
<1.94
<2.09
<2.17
<1.91
<2.12
7g
8a
8b
8c
8d
8e
8f
n.d.
–
>7
31.49 ± 6.43
10.62 ± 1.06
16.95 ± 4.63
15.76 ± 3.07
20.20 ± 2.13
6.46 ± 0.05
0.76
1.65
0.93
1.11
2.48
3.19
>13
>30
>5
>10
>4
g
6
6
6
6
6
6
6
a
b
c
d
e
f
>4
<1.95
<1.92
<2.31
<2.03
–
8g
9a
9b
9c
9d
9e
9f
10.14 ± 0.81
41.81 ± 23.56
17.78 ± 2.30
20.09 ± 3.54
10.56 ± 0.28
14.42
2.26
0.46
1.08
1.16
3.28
2.77
2.43
>7
>3
>7
n.d.
n.d.
n.d.
–
g
–
41.64 ± 6.63
7
7
7
7
7
a
b
c
n.d.
n.d.
n.d.
n.d.
n.d.
–
–
–
–
–
9g
54.12 ± 10.21
0.7 ± 0.07
3.38
c
Amphotericin B
51.7
d
e
Note: Data represent mean value ± standard deviation. Bold values are the most active compounds.
Maximum concentration evaluated because the host cell dies at higher concentrations.
>
Abbreviation: n.d., not determined.
a
Effective concentration 50 (EC50): Concentration of compounds that decrease 50% of intracellular
parasite.
b
Index of selectivity (IS) = LC50/EC50. LC50: Lethal concentration on human cell U‐937.
c
Antileishmanial drug control.
2
‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazole‐5‐carbaldehyde (3)
filtered and washed with ethanol and water to obtain compounds
Pale yellow crystals; 42% yield; m.p. 115–116°C. FTIR (ATR) ν
5a–g as pure yellow solids.
−
1
1
(
(
cm ): 1,634 (C═O), 1,543 and 1,535 (C═N and C═C). H NMR
400 MHz, CDCl ) δ ppm 9.80 (s, 1H, CHO), 3.95 (t, J = 6.1 Hz, 4H,
×CH –N), and 3.81 (t, J = 6.1 Hz, 4H, 2×CH
100 MHz, CDCl ) δ ppm 180.4 (CHO), 171.4 (C), 147.7 (C), 120.6
C), 54.7 (CH ), and 40.4 (CH ). Mass spectrometry (electron im-
3
(E)‐3‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐1‐(4‐
chlorophenyl)prop‐2‐en‐1‐one (5a)
1
3
2
2
2
–Cl).
C NMR
−
1
(
(
3
Yellow solid; 90% yield; m.p. 148–149°C. FTIR (ATR) ν (cm ): 3,044
2
2
(═C–H), 2,894 (C–H), 1,645 (C═O), 1,586 and 1,529 (C═N and C═C).
+
+
+
+
1
pact) (MS [EI]): m/z 286/288/290/292 [M /M + 2 /M + 4 /M + 6 ]
28/28/9/1), 237/239/241 (100/68/13), 175/177 (93/33), 63 (83),
3 6
H NMR (400 MHz, CDCl ) δ ppm 7.96–7.87 (m, 3H, H , HBo), 7.45
(
(d, J = 8.3 Hz, 2H, HBm), 6.82 (d, J = 15.0 Hz, 1H, H
4H, 2×CH –N), and 3.82 (t, J = 6.0 Hz, 4H, 2×CH
(100 MHz, CDCl ) δ ppm 187.9 (C═O), 167.0 (C), 143.3 (C), 139.2 (C),
136.6 (C), 133.8 (CH), 129.8 (CH), 129.0 (CH), 119.0 (CH), 117.1 (C),
7
), 3.93 (t, J = 6.0 Hz,
13
4
1
1 (18). Anal. calcd. for C
8
H
9
Cl
3
N
2
OS: C, 34.41; H, 3.15; N, 9.74; S,
2
2
–Cl). C NMR
1.15. Found: C, 34.42; H, 3.13; N, 9.73; S, 11.20. Crystals suitable
3
for single‐crystal X‐ray diffractions analysis were obtained from
slow evaporation in dichloromethane/ethanol and deposited in
Cambridge Crystallographic Data Centre (CCDC) with deposition
number 1919158.
54.5 (CH
2
), and 40.6 (CH
2
). MS (EI): m/z 422/424/426/428/430
+
+
+
+
+
[M /M + 2 /M + 4 /M + 6 /M + 8 ] (1.04/2.41/0.10/0.07/0.10), 237/
239/241/243 (16/16/3/1), 97 (50), 83 (66), 57 (97), 43 (100), and
4 2
41 (69). Anal. calcd. for C16H14Cl N OS: C, 45.31; H, 3.33; N, 6.60; S,
7.56. Found: C, 45.29; H, 3.34; N, 6.59; S, 7.58. Crystals suitable for
4
[
.1.3 | General procedure for the synthesis of
bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl‐
chalcones 5a–g
single‐crystal X‐ray diffraction analysis were obtained from slow
evaporation in dichloromethane/ethanol and deposited in CCDC with
deposition number 1919157.
A
mixture of 2‐[bis(2‐chloroethyl)amino]‐4‐chlorothiazole‐
‐carbaldehyde 3 (1.74 mmol), the selected acetophenone 4a–g
1.95 mmol), and a pellet of NaOH in ethanol (20.0 ml) was stirred
at room temperature for 0.5 to 2.0 hr. The precipitate formed was
(E)‐3‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐1‐(4‐
bromophenyl)prop‐2‐en‐1‐one (5b)
5
−
1
(
Yellow solid; 86% yield; m.p. 150–152°C. FTIR (ATR) ν (cm ): 3,045
(═C–H), 2,876 (C–H), 1,647 (C═O), and 1,585 and 1,528 (C═N and

CUARTAS ET AL.
13 of 24
|
TABLE 8 Antitrypanosomal activity of
a
b
Compound
3
EC50
>10
IS
Compound
7f
EC50
>10
IS
thiazole derivatives 3, 5a–g, 6a–g, 7a–g,
a
<1.76
<2
8a–g, and 9a–g expressed in EC50 (μM)
5
5
5
5
5
5
5
a
b
c
d
e
f
10.37 ± 1.50
>6
0.86
<2.24
1.15
7g
8a
8b
8c
8d
8e
8f
17.73 ± 3.46
16.86 ± 1.92
19.53 ± 0.73
34.64 ± 3.90
19.02 ± 2.2
75.30 ± 6.22
50.19 ± 8.32
0.6
1.43
0.90
0.46
0.92
0.67
0.41
21.98 ± 4.06
>30
<2.09
0.74
14.67 ± 2.08
27.76 ± 4.31
9.44 ± 0.35
0.69
g
0.90
6
6
6
6
6
6
6
a
b
c
d
e
f
>4
<1.8
0.09
0.88
1.06
0.52
0.23
0.46
8g
9a
9b
9c
9d
9e
9f
37.05 ± 2.67
>10
0.62
<1.92
0.52
145 ± 5
7.87 ± 0.45
13.38 ± 0.84
21.24 ± 3.47
114.01 ± 18.60
21.29 ± 03.10
36.97 ± 0.43
34.85 ± 4.81
>25
0.67
<1.73
<2.01
<2.03
>25
g
>50
7
7
7
7
7
a
b
c
18.02 ± 1.67
23.05 ± 2.54
4.98 ± 0.99
0.87
1.52
1.61
0.95
1.17
9g
118.01 ± 2.61
13.34 ± 0.10
1.55
c
Benznidazole
>14.99
d
e
150.33 ± 20.62
18.71 ± 2.65
Note: Data represent mean value ± standard deviation. Bold values are the most active compounds.
Maximum concentration evaluated because the host cell dies at higher concentrations.
>
a
Effective concentration 50 (EC50): Concentration of compounds that decrease 50% of intracellular
parasite.
b
Index of selectivity (IS) = LC50/EC50. LC50: Lethal concentration on human cell U‐937.
c
Antitrypanosomal drug control.
1
C═C). H NMR (400 MHz, CDCl
3
) δ ppm 7.92 (d, J = 15.0 Hz, 1H, H
6
),
(E)‐3‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐1‐(4‐
fluorophenyl)prop‐2‐en‐1‐one (5c)
7
.83 (d, J = 8.3 Hz, 2H, HBo), 7.62 (d, J = 8.3 Hz, 2H, HBm), 6.81
−1
(
d, J = 15.0 Hz, 1H, H
7
), 3.93 (t, J = 6.0 Hz, 4H, 2×CH
2
–N), and 3.82
Yellow solid; 66% yield; m.p. 153–154°C. FTIR (ATR) ν (cm ): 3,017
1
3
(t, J = 6.0 Hz, 4H, 2×CH
2
–Cl). C NMR (100 MHz, CDCl ) δ ppm
3
(═C–H), 2,940 (C–H), 1,641 (C═O), and 1,595 and 1,525 (C═N and
1
1
1
88.1 (C═O), 167.1 (C), 143.4 (C), 137.1 (C), 133.9 (CH), 132.0 (CH),
C═C).
H
NMR (400 MHz, CDCl
3
)
δ
ppm 7.99 (dd, J = 8.6,
), 7.15 (t, J = 8.6 Hz,
4
30.0 (CH), 127.9 (C), 118.9 (CH), 117.1 (C), 54.6 (CH
2
), and 40.6
J
H–F = 5.5 Hz, 2H, HBo), 7.91 (d, J = 15.0 Hz, 1H, H
6
+
+
+
(CH
2
). MS (EI): m/z 466/468/470/472/474 [M /M + 2 /M + 4 /
7 2
2H, HBm), 6.84 (d, J = 15.0 Hz, 1H, H ), 3.92 (t, J = 6.0 Hz, 4H, 2×CH –
+
+
13
M + 6 /M + 8 ] (0.68/1.62/0.93/0.30/0.04), 431/433/435 (57/100/
9), 183/185 (9/9), 155/157 (9/10), 57 (35), and 43 (39). Anal. calcd.
for C16 14BrCl OS: C, 41.01; H, 3.01; N, 5.98; S, 6.84. Found: C,
1.04; H, 2.99; N, 5.96; S, 6.86.
N), and 3.82 (t, J = 5.9 Hz, 4H, 2×CH
2
3
–Cl). C NMR (100 MHz, CDCl )
1
4
δ ppm 187.6 (C = O), 167.0 (C), 165.6 (d, JC–F = 254.1 Hz, C), 143.1 (C),
4
3
H
3
N
2
134.6 (d,
J
C–F = 2.9 Hz, C), 133.5 (CH), 131.0 (d, JC–F = 9.1 Hz, CH),
2
4
119.1 (CH), 117.1 (C), 115.8 (d,
0.6 (CH
J
C–F = 21.8 Hz, CH), 54.5 (CH
2
), and
+
+
+
+
4
2
). MS (EI): m/z 406/408/410/412 [M /M + 2 /M + 4 /M + 6 ]
(1.19/1.37/0.79/0.03), 371/373/375 (63/46/9), 81 (49), 69 (100), and
3 2
43 (74). Anal. calcd. for C16H14Cl FN OS: C, 47.13; H, 3.46; N, 6.87; S,
7.86. Found: C, 47.11; H, 3.44; N, 6.89; S, 7.89.
(
E)‐1‐(Benzo[d][1,3]dioxol‐5‐yl)‐3‐{2‐[bis(2‐chloroethyl)amino]‐
‐chlorothiazol‐5‐yl}prop‐2‐en‐1‐one (5d)
Yellow solid; 64% yield; m.p. 149–150°C. FTIR (ATR) ν (cm ): 3,011
4
−
1
(═C–H), 2,907 (C–H), 1,610 (C═O), and 1,572 and 1,525 (C═N and
1
C═C). H NMR (400 MHz, CDCl
3
) δ ppm 7.88 (d, J = 15.0 Hz, 1H, H
6
),
7
.57 (dd, J = 8.2, 1.7 Hz, 1H, HBo), 7.47 (d, J = 1.7 Hz, 1H, HBo′), 6.87 (d,
J = 8.2 Hz, 1H, HBm), 6.83 (d, J = 15.0 Hz, 1H, H ), 6.05 (s, 2H, –OCH
O–), 3.92 (t, J = 6.3 Hz, 4H, 2×CH –N), and 3.81 (t, J = 6.3 Hz, 4H,
–Cl). C NMR (100 MHz, CDCl ) δ ppm 187.1 (C═O), 166.7
7
2
FIGURE 4 Three‐dimensional structures of compounds 5c
green), 7c (cyan), and 8c (purple)
2
1
3
(
2×CH
2
3

14 of 24
CUARTAS ET AL.
|
(C), 151.7 (C), 148.4 (C), 142.5 (C), 133.1 (C), 132.8 (CH), 124.5 (CH),
4.1.4 | General procedure for the synthesis of
1
19.4 (CH), 117.3 (C), 108.4 (CH), 108.0 (CH), 102.0 (–OCH
2
O), 54.5
N‐acetyl and N‐formyl pyrazolines 6/7a–g
+
+
(CH
2
), and 40.6 (CH
2
). MS (EI): m/z 432/434/436/438 [M /M + 2 /
+
+
M + 4 /M + 6 ] (0.82/1.15/0.34/0.29), 397/399/401 (40/28/6), 81
51), 69 (100), and 43 (84). Anal. calcd. for C17 15Cl S: C, 47.08;
H, 3.49; N, 6.46; S, 7.39. Found: C, 47.10; H, 3.47; N, 6.44; S, 7.43.
A mixture of the selected chalcone 5a–g (0.24 mmol) and hydrazine
hydrate (4.80 mmol) in ethanol (3.0 ml) was heated under reflux for
30 min. Then, acetic anhydride (2.0 ml) was added dropwise, after
which the mixture was stirred at room temperature for another 1 hr.
(
H
3 2 3
N O
(
E)‐3‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐1‐(4‐
methoxyphenyl)prop‐2‐en‐1‐one (5e)
Yellow solid; 60% yield; m.p. 124–125°C. FTIR (ATR) ν (cm ): 3,010
The reaction mixture was treated with H
chloromethane (3 × 30 ml). The combined organic layers were dried
over anhydrous MgSO , filtered, and concentrated under reduced
2
O and extracted with di-
−1
4
(
═C–H), 2,845 (C–H), 1,641 (C═O), and 1,599 and 1,582 (C═N and
pressure. The residue was purified by silica gel column chromato-
graphy using dichloromethane/ethanol (50:1) to yield compounds
6a–g.
1
C═C). H NMR (400 MHz, CDCl
3
) δ ppm 7.97 (d, J = 8.7 Hz, 2H, HBo),
), 6.96 (d, J = 8.7 Hz, 2H, HBm), 6.89
), 3.92 (t, J = 6.2 Hz, 4H, 2×CH –N), 3.88 (s, 3H,
–Cl). C NMR (100 MHz,
7
.89 (d, J = 15.1 Hz, 1H, H
6
(d, J = 15.1 Hz, 1H, H
7
2
The synthesis of the N‐formyl pyrazolines 7a–g was similar to
that of the N‐acetyl derivatives, except that formic acid (2.0 ml) was
used in place of acetic anhydride and the reaction mixture was stir-
red at room temperature for 5 hr. The residue was purified by silica
gel column chromatography using dichloromethane/ethyl acetate
(20:1) to obtain compounds 7a–g.
1
3
OCH
3
), and 3.81 (t, J = 6.2 Hz, 4H, 2×CH
2
CDCl
3
) δ ppm 187.6 (C═O), 166.7 (C), 163.5 (C), 142.3 (C), 132.5
(
(
[
(
CH), 131.2 (C), 130.7 (CH), 119.7 (CH), 117.4 (C), 114.0 (CH), 55.6
OCH
3
), 54.5 (CH
2 2
), and 40.7 (CH ). MS (EI): m/z 418/420/422/424
+
+
+
+
M /M + 2 /M + 4 /M + 6 ] (0.11/0.41/0.19/0.31), 97 (43), 83 (62), 57
3 2 2
91), and 43 (100). Anal. calcd. for C17H17Cl N O S: C, 48.64; H,
4
.08; N, 6.67; S, 7.64. Found: C, 48.61; H, 4.07; N, 6.64; S, 7.67.
1‐(5‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐(4‐
chlorophenyl)‐4,5‐dihydro‐1H‐pyrazol‐1‐yl)ethan‐1‐one (6a)
−1
(
E)‐3‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐1‐(3,4,5‐
trimethoxyphenyl)prop‐2‐en‐1‐one (5f)
Yellow solid; 54% yield; m.p. 136–137°C. FTIR (ATR) ν (cm ): 3,005
White solid; 65% yield; m.p. 135–137°C. FTIR (ATR) ν (cm ): 3,028
(═C–H), 2,965 (C–H), 1,668 (C═O), and 1,595 and 1,555 (C═N and
−1
1
C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.79 (d, J = 8.6 Hz, 2H,
(═C–H), 2,939 (C–H), 1,676 (C═O), and 1,584 and 1,568 (C═N and
HBo), 7.54 (d, J = 8.6 Hz, 2H, HBm), 5.69 (dd, J = 11.9, 4.8 Hz, 1H, H5′),
1
C═C). H NMR (400 MHz, CDCl
3
) δ ppm 7.89 (d, J = 15.1 Hz, 1H, H
6
3
),
),
2 2
3.88–3.77 (m, 5H, H4b′, 2×CH –Cl), 3.73 (t, J = 6.7 Hz, 4H, 2×CH –N),
13
7
3
2
.20 (s, 2H, HBo), 6.79 (d, J = 15.1 Hz, 1H, H
.93–3.89 (m, 7H, OCH , 2×CH
–Cl). C NMR (100 MHz, CDCl
7
), 3.94 (s, 6H, 2×OCH
3.27 (dd, J = 18.2, 4.8 Hz, 1H, H4a′), and 2.26 (s, 3H, COCH
NMR (100 MHz, DMSO‐d ) δ ppm 167.6 (C═O), 165.6 (C), 153.2 (C),
135.1 (C), 130.8 (C), 129.7 (C), 128.9 (CH), 128.4 (CH), 118.1 (C), 52.9
3
).
C
3
2
–N), and 3.81 (t, J = 6.1 Hz, 4H,
6
13
×CH
2
3
) δ ppm 188.2 (C═O), 166.9
(C), 153.3 (C), 142.9 (C), 142.6 (C), 133.6 (C), 133.3 (CH), 119.5 (CH),
(C5′), 52.3 (CH
2
), 40.7 (CH
2
), 40.4 (C4′), and 21.7 (COCH
3
). MS (EI):
+
+
+
+
+
117.1 (C), 106.2 (CH), 61.1 (OCH
3
), 56.6 (OCH
3
), 54.5 (CH
2
), and
m/z 478/480/482/484/486 [M /M + 2 /M + 4 /M + 6 /M + 8 ] (14/
+
+
+
4
0.6 (CH
2
). MS (EI): m/z 478/480/482/484 [M /M + 2 /M + 4 /
18/9/2/1), 443/445/447/449 (25/25/9/1), 306/308/310 (100/66/
+
M + 6 ] (1.07/1.88/0.58/0.11), 443/445/447 (86/63/15), 83 (62), 57
16), and 43 (13). Anal. calcd. for C18
4 4
H18Cl N OS: C, 45.02; H, 3.78;
3 2 4
(88), and 43 (100). Anal. calcd. for C19H21Cl N O S: C, 47.56; H,
N, 11.67; S, 6.68. Found: C, 45.00; H, 3.77; N, 11.63; S, 6.70.
4.41; N, 5.84; S, 6.68. Found: C, 47.54; H, 4.39; N, 5.83; S, 6.70.
1‐(5‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐(4‐
(
E)‐3‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐
‐phenylprop‐2‐en‐1‐one (5g)
Yellow solid; 50% yield; m.p. 125–127°C. FTIR (ATR) ν (cm ): 3,091
bromophenyl)‐4,5‐dihydro‐1H‐pyrazol‐1‐yl)ethan‐1‐one (6b)
−
1
1
White solid; 64% yield; m.p. 81–83°C. FTIR (ATR) ν (cm ): 3,025
−1
(═C–H), 2,962 (C–H), 1,665 (C═O), and 1,593 and 1,531 (C═N and
1
(
═C–H), 2,951 (C–H), 1,645 (C═O), and 1,585 and 1,520 (C═N and
C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.72 (d, J = 8.8 Hz, 2H,
1
C═C). H NMR (400 MHz, CDCl
3
) δ ppm 7.97 (d, J = 7.1 Hz, 2H, HBo),
), 7.57 (t, J = 7.3 Hz, 1H, HBp), 7.49
t, J = 7.4 Hz, 2H, HBm), 6.89 (d, J = 15.1 Hz, 1H, H ), 3.93 (t, J = 6.2 Hz,
H, 2×CH –N), and 3.82 (t, J = 6.1 Hz, 4H, 2×CH
100 MHz, CDCl
H
Bo), 7.68 (d, J = 8.8 Hz, 2H, HBm), 5.69 (dd, J = 11.9, 4.8 Hz, 1H,
5′), 3.88–3.77 (m, 5H, H4b′, 2×CH –Cl), 3.73 (t, J = 6.8 Hz, 4H,
–N), 3.27 (dd, J = 18.2, 4.8 Hz, 1H, H4a′), and 2.26 (s, 3H,
7
.92 (d, J = 15.1 Hz, 1H, H
6
H
2
(
7
2×CH
2
1
3
13
4
2
2
–Cl). C NMR
) δ ppm 189.3 (C═O), 166.9 (C), 142.9 (C), 138.3 (C),
COCH
3
6
). C NMR (100 MHz, DMSO‐d ) δ ppm 167.6 (C═O), 165.6
(
3
(C), 155.5 (C), 131.8 (CH), 130.7 (C), 130.0 (C), 128.5 (CH), 123.8
(C), 118.1 (C), 52.9 (C5′), 52.3 (CH ), 40.7 (CH ), 40.5 (C4′), and
). MS (EI): m/z 522/524/526/528/530 [M /M + 2 /
1
33.3 (CH), 132.8 (CH), 128.7 (CH), 128.4 (CH), 119.7 (CH), 117.3
2
2
+
+
+
(C), 54.5 (CH
2
), and 40.6 (CH
2
). MS (EI): m/z 388/390/392/394 [M /
22.2 (COCH
3
+
+
+
+
+
+
M + 2 /M + 4 /M + 6 ] (2.30/2.72/1.69/0.94), 353/355/357 (100/66/
M + 4 /M + 6 /M + 8 ] (8.28/18.20/12.08/4.40/0.59), 487/489/
491/493 (26), 306/308/310 (100/69/14), and 43 (2). Anal. calcd.
2
1), 83 (0.66), and 57 (0.87). Anal. calcd. for C16
3 2
H15Cl N OS: C,
49.31; H, 3.88; N, 7.19; S, 8.23. Found: C, 49.33; H, 3.85; N, 7.21;
for C18
3 4
H18BrCl N OS: C, 41.20; H, 3.46; N, 10.68; S, 6.11. Found:
S, 8.28.
C, 41.18; H, 3.47; N, 10.66; S, 6.14.

CUARTAS ET AL.
15 of 24
|
1
‐(5‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐(4‐
fluorophenyl)‐4,5‐dihydro‐1H‐pyrazol‐1‐yl)ethan‐1‐one (6c)
Pale yellow solid; 58% yield; m.p. 134–136°C. FTIR (ATR) ν (cm ):
1‐(5‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐(3,4,5‐
trimethoxyphenyl)‐4,5‐dihydro‐1H‐pyrazol‐1‐yl)ethan‐1‐one (6f)
−1
−1
Pale yellow solid; 70% yield; m.p. 103–106°C. FTIR (ATR) ν (cm ):
3,016 (═C–H), 2,964 (C–H), 1,668 (C═O), and 1,523 (C═N and C═C).
3,028 (═C–H), 2,838 (C–H), 1,653 (C═O), and 1,570 and 1,531 (C═N
1
4
1
6
H NMR (400 MHz, DMSO‐d ) δ ppm 7.84 (dd, J = 8.8,
JH–F = 5.5 Hz,
and C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.05 (s, 2H, HBo),
2
H, HBo), 7.32 (t, J = 8.9 Hz, 2H, HBm), 5.71 (dd, J = 11.9, 4.7 Hz, 1H,
5.69 (dd, J = 11.8, 4.5 Hz, 1H, H5′), 3.85–3.77 (m, 11H, 2×OCH
2×CH –Cl), 3.76–3.70 (m, 7H, OCH , 2×CH –N), 3.35 (dd, J = 18.1,
4.5 Hz, 1H, H4a), and 2.27 (s, 3H, COCH ). C NMR (100 MHz,
DMSO‐d ) δ ppm 167.4 (C═O), 165.5 (C), 154.3 (C), 153.0 (C), 139.6
(C), 130.5 (C), 126.2 (C), 118.4 (C), 104.2 (CH), 60.1 (OCH ), 56.0
(OCH ), 52.8 (C5′), 52.3 (CH ), 40.7 (CH ), 40.1 (C4′), and 21.6
(COCH ). MS (EI): m/z 534/536/538/540 [M /M + 2 /M + 4 /M + 6 ]
(73/75/29/5), 499/501/503 (100/68/16), 306/308/310 (95/69/15),
148/150 (47/18), and 43 (12). Anal. calcd. for C21 25Cl S: C,
3
, H4b′
,
H
5′), 3.89–3.77 (m, 5H, H4b′, 2×CH
2
–Cl), 3.74 (t, J = 5.8 Hz, 4H, 2×CH
2
–
2
3
2
1
3
N), 3.28 (dd, J = 18.2, 4.7 Hz, 1H, H4a′), and 2.26 (s, J = 9.7 Hz, 3H,
3
13
COCH
3
). C NMR (100 MHz, DMSO‐d
6
) δ ppm 168.0 (C═O), 166.1
6
1
(
(
(
C), 163.8 (d,
JC–F = 248.5 Hz, C), 153.8 (C), 131.2 (C), 129.5
3
3
4
d,
d,
JC–F = 8.7 Hz, CH), 127.9 (d,
JC–F = 3.1Hz, C), 118.7 (C), 116.4
3
2
2
2
+
+
+
+
2 2
JC–F = 21.9 Hz, CH), 53.3 (C5′), 52.8 (CH ), 41.2 (CH ), 41.1 (C4′),
3
+
+
+
and 22.2 (COCH
3
). MS (EI): m/z 462/464/466/468 [M /M + 2 /M + 4 /
+
M + 6 ] (22/22/8/1), 427/430/432 (30/22/5), 306/308/310 (100/67/
H
3 4 4
N O
1
4), 148 (23), and 43 (12). Anal. calcd. for C18
H
18Cl
3
FN
4
OS: C, 46.61;
47.07; H, 4.70; N, 10.46; S, 5.98. Found: C, 47.05; H, 4.68; N, 10.49;
S, 6.01.
H, 3.91; N, 12.08; S, 6.91. Found: C, 46.59; H, 3.94; N, 12.06; S, 6.95.
1
4
‐(3‐(Benzo[d][1,3]dioxol‐5‐yl)‐5‐{2‐[bis(2‐chloroethyl)amino]‐
‐chlorothiazol‐5‐yl}‐4,5‐dihydro‐1H‐pyrazol‐1‐yl)ethan‐1‐one (6d)
1‐(5‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐phenyl‐4,5‐
dihydro‐1H‐pyrazol‐1‐yl)ethan‐1‐one (6g)
−1
−1
Pale yellow solid; 60% yield; m.p. 90–93°C. FTIR (ATR) ν (cm ):
Pale yellow solid; 53% yield; m.p. 125–127°C. FTIR (ATR) ν (cm ):
3,027 (═C–H), 2,963 (C–H), 1,661 (C═O), and 1,558 and 1,531 (C═N
3,019 (═C–H), 2,967 (C–H), 1,668 (C═O), and 1,557 and 1,541 (C═N
1
1
and C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.35 (d, J = 1.6 Hz,
and C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.78 (dd, J = 6.6,
1
H, HBo′), 7.25 (dd, J = 8.1, 1.6 Hz, 1H, HBo), 7.00 (d, J = 8.1 Hz, 1H,
Bm), 6.10 (s, 2H, –OCH O–), 5.66 (dd, J = 11.8, 4.6 Hz, 1H, H5′),
.83–3.77 (m, 5H, H4b′, 2×CH –Cl), 3.74 (t, J = 5.7 Hz, 4H, 2×CH –N),
.22 (dd, J = 18.1, 4.6 Hz, 1H, H4a′), and 2.24 (s, 3H, COCH
) δ ppm 167.3 (C═O), 165.5 (C), 153.8 (C),
49.3 (C), 147.8 (C), 130.6 (C), 124.9 (C), 121.9 (CH), 118.3 (C), 108.5
CH), 105.9 (CH), 101.6 (–OCH O–), 52.6 (C5′), 52.3 (CH ), 40.7
), 40.6 (C4′), and 21.6 (COCH ). MS (EI): m/z 488/490/492/494
3.1 Hz, 2H, HBo), 7.50–7.45 (m, 3H, HBm, HBp), 5.69 (dd, J = 11.9,
4.7 Hz, 1H, H5′), 3.89–3.77 (m, 5H, H4b′, 2×CH –Cl), 3.74 (t, J = 5.8 Hz,
4H, 2×CH –N), 3.27 (dd, J = 18.2, 4.7 Hz, 1H, H4a′), and 2.26 (s, 3H,
H
2
2
3
3
2
2
2
1
3
13
3
).
C
COCH
3
6
). C NMR (100 MHz, DMSO‐d ) δ ppm 167.5 (C═O), 165.6
NMR (100 MHz, DMSO‐d
6
(C), 154.1 (C), 130.8 (C), 130.7 (C), 130.5 (CH), 128.8 (CH), 126.6
1
(CH), 118.2 (C), 52.7 (C5′), 52.3 (CH
(COCH
2
2
), 40.7 (CH ), 40.5 (C4′), and 21.7
+
+
+
+
(
(
[
2
2
3
). MS (EI): m/z 444/446/448/450 [M /M + 2 /M + 4 /M + 6 ]
CH
2
3
(21.24/23.68/8.42/1.89), 409/411/413 (37/25/5), 306/308/310
(100/68/14), 83 (47), and 43 (18). Anal. calcd. for C18 19Cl OS: C,
+
+
+
+
M /M + 2 /M + 4 /M + 6 ] (4.13/4.82/1.63/0.54), 453/455/457 (26),
H
3 4
N
3
06/308/310 (16/11/2), 57 (86), and 43 (100). Anal. calcd. for
19Cl S: C, 46.59; H, 3.91; N, 11.44; S, 6.55. Found: C,
6.62; H, 3.88; N, 11.42, S, 6.51.
48.50; H, 4.30; N, 12.57; S, 7.19. Found: C, 48.48; H, 4.33; N, 12.59; S,
7.23. Crystals suitable for single‐crystal X‐ray diffraction analysis
were obtained from slow evaporation in ethanol and deposited in
CCDC with deposition number 1919156.
C
19
H
3 4 3
N O
4
1
‐(5‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐(4‐
methoxyphenyl)‐4,5‐dihydro‐1H‐pyrazol‐1‐yl)ethan‐1‐one (6e)
Pale yellow solid; 52% yield; m.p. 147–149°C. FTIR (ATR) ν (cm ):
5‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐(4‐
chlorophenyl)‐4,5‐dihydro‐1H‐pyrazole‐1‐carbaldehyde (7a)
−
1
−1
3,028 (═C–H), 2,965 (C–H), 1,662 (C═O), and 1,558 and 1,531 (C═N
Beige solid; 68% yield; m.p. 75–78°C. FTIR (ATR) ν (cm ): 3,035
1
and C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.72 (d, J = 8.8 Hz,
(═C–H), 2,914 and 2,893 (C–H), 1,676 (C═O), and 1,551 and 1,533
1
2
H, HBo), 7.02 (d, J = 8.8 Hz, 2H, HBm), 5.66 (dd, J = 11.7, 4.4 Hz, 1H,
5′), 3.86–3.76 (m, 8H, OCH , H4b′, 2×CH –Cl), 3.73 (t, J = 5.5 Hz, 4H,
×CH –N), 3.23 (dd, J = 18.1, 4.4 Hz, 1H, H4a′), and 2.24 (s, 3H,
COCH
(C═N and C═C). H NMR (400 MHz, CDCl
3
) δ ppm 8.89 (s, 1H, CHO),
H
3
2
7.67 (d, J = 7.7 Hz, 2H, HBo), 7.42 (d, J = 7.7 Hz, 2H, HBm), 5.74 (dd,
2
2
J = 11.7, 4.5 Hz, 1H, H5′), 3.77 (t, J = 5.5 Hz, 4H, 2×CH
(m, 5H, H4b′, 2×CH
NMR (100 MHz, CDCl
137.1 (C), 133.4 (C), 129.3 (CH), 129.2 (C), 128.1 (CH), 116.6 (C), 54.3
2
–N), 3.76–3.69
1
3
13
3
). C NMR (100 MHz, DMSO‐d
6
) δ ppm 167.3 (C═O), 165.5
2
–Cl), and 3.26 (dd, J = 17.9, 4.5 Hz, 1H, H4a′).
C
(
C), 161.0 (C), 153.9 (C), 130.6 (C), 128.3 (CH), 123.3 (C), 118.4 (C),
3
) δ ppm 165.5 (C), 160.1 (CHO), 154.6 (C),
1
14.3 (CH), 55.4 (OCH
3
), 52.5 (C5′), 52.3 (CH
2 2
), 40.7 (CH ), 40.6 (C4′),
+
+
and 21.7 (COCH
3
). MS (EI): m/z 474/476/478/480 [M /M + 2 /
2
(CH ), 52.4 (C5′), 41.1 (C4′), and 40.7 (CH
2
). MS (EI): m/z 464/466/
+
+
+
+
+
+
+
M + 4 /M + 6 ] (40.12/40.44/15.00/2.46), 439/441/443 (72/49/10),
06/308/310 (100/72/15), 148/150 (43/15), and 43 (14). Anal. calcd.
for C19 21Cl S: C, 47.96; H, 4.45; N, 11.78; S, 6.74. Found: C,
7.99; H, 4.42; N, 11.75; S, 6.78.
468/470/472 [M /M + 2 /M + 4 /M + 6 /M + 8 ] (3.11/4.18/2.23/
0.52/0.07), 429/431/433/435 (2.84/2.53/0.81/0.27), 83 (100), and
3
H
3
N
O
4 2
49 (16). Anal. calcd. for C17
4 4
H16Cl N OS: C, 43.80; H, 3.46; N, 12.02,
4
S, 6.88. Found: C, 43.78; H, 3.49; N, 12.04; S, 6.85.

16 of 24
CUARTAS ET AL.
|
1
5
‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐(4‐
(C═N and C═C). H NMR (400 MHz, CDCl
7.67 (d, J = 8.8 Hz, 2H, HBo), 6.95 (d, J = 8.8 Hz, 2H, HBm), 5.72 (dd,
J = 11.6, 4.8 Hz, 1H, H5′), 3.86 (s, 3H, OCH ), 3.78 (t, J = 6.4 Hz, 4H,
2×CH –N), 3.75–3.69 (m, 5H, H4b′, 2×CH –Cl), and 3.25 (dd, J = 17.7,
4.8 Hz, 1H, H4a′). C NMR (100 MHz, CDCl ) δ ppm 165.4 (C), 161.9
(C), 160.0 (CHO), 155.5 (C), 133.1 (C), 128.5 (CH), 123.2 (C), 117.1 (C),
114.5 (CH), 55.6 (OCH ), 54.3 (CH ), 52.1 (C5′), 41.3 (C4′), and 40.7
(CH ). MS (EI): m/z 460/462/464/466 [M /M + 2 /M + 4 /M + 6 ]
(34.73/35.37/12.88/2.56), 425/427/429 (47/31/6), 97 (43), 69 (80), 57
(91), and 43 (100). Anal. calcd. for C18 19Cl S: C, 46.82; H, 4.15; N,
3
) δ ppm 8.87 (s, 1H, CHO),
bromophenyl)‐4,5‐dihydro‐1H‐pyrazole‐1‐carbaldehyde (7b)
−
1
White solid; 62% yield; m.p. 159–160°C. FTIR (ATR) ν (cm ): 3,034
3
(═C–H), 2,914 and 2,893 (C–H), 1,678 (C═O), and 1,551 and 1,535 (C═N
2
2
1
13
and C═C). H NMR (400 MHz, CDCl
.63–7.56 (m, 4H, HBo, HBm), 5.74 (dd, J = 11.7, 5.0 Hz, 1H, H5′), 3.78
t, J = 5.7 Hz, 4H, 2×CH –N), 3.76–3.70 (m, 5H, H4b′, 2×CH –Cl), and 3.26
dd, J = 17.8, 5.0 Hz, 1H, H4a′). C NMR (100 MHz, CDCl ) δ ppm 165.5
C), 160.1 (CHO), 154.6 (C), 133.4 (C), 132.3 (CH), 129.6 (C), 128.3 (CH),
25.4 (C), 116.6 (C), 54.3 (CH ), 52.4 (C5′), 41.0 (C4′), and 40.7 (CH ). MS
EI): m/z 508/510/512/514/516 [M /M + 2 /M + 4 /M + 6 /M + 8 ]
1.73/3.31/2.20/0.67/0.11), 473/475/477 (1.58/2.14/1.07), 167 (40), 149
100), 71 (38), and 57 (34). Anal. calcd. for C17 16BrCl OS: C, 39.98; H,
3
) δ ppm 8.89 (s, 1H, CHO),
3
7
(
2
2
3
2
13
+
+
+
+
(
3
2
(
1
2
2
H
3 4 2
N O
+
+
+
+
+
(
12.13; S, 6.94. Found: C, 46.79; H, 4.12; N, 12.10; S, 6.97.
(
(
H
3
N
4
5‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐(3,4,5‐
trimethoxyphenyl)‐4,5‐dihydro‐1H‐pyrazole‐1‐carbaldehyde (7f)
3.16; N, 10.97; S, 6.28. Found: C, 39.98; H, 3.15; N, 10.98; S, 6.26.
−1
Beige solid; 76% yield; m.p. 162–163°C. FTIR (ATR) ν (cm ): 3,026
5
‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐(4‐
(═C–H), 2,878 and 2,826 (C–H), 1,676 (C═O), and 1,566 and 1,553
1
fluorophenyl)‐4,5‐dihydro‐1H‐pyrazole‐1‐carbaldehyde (7c)
(C═N and C═C). H NMR (400 MHz, CDCl
3
) δ ppm 8.90 (s, 1H, CHO),
−
1
Reddish solid; 52% yield; m.p. 94–97°C. FTIR (ATR) ν (cm ): 3,038
═ C–H), 2,924 and 2,887 (C–H), and 1,668 (C═O), 1,530 (C═N and C═C).
6.96 (s, 2H, HBo), 5.74 (dd, J = 11.7, 4.9 Hz, 1H, H5′), 3.91 (s, 6H,
2×OCH ), 3.89 (s, 3H, OCH ), 3.78 (t, J = 5.6 Hz, 4H, 2×CH –N), 3.76–
–Cl), and 3.27 (dd, J = 17.7, 4.9 Hz, 1H, H4a′).
) δ ppm 165.5 (C), 160.0 (CHO), 155.4 (C),
(
3
3
2
1
H NMR (400 MHz, CDCl
3
) δ ppm 8.89 (s, 1H, CHO), 7.73 (dd, J = 8.9,
H–F = 5.3 Hz, 2H, HBo), 7.14 (t, J = 8.6 Hz, 2H, HBm), 5.74 (dd, J = 11.7,
.0 Hz, 1H, H5′), 3.78 (t, J = 5.6 Hz, 4H, 2×CH –N), 3.76–3.70 (m, 5H, H4b′
×CH –Cl), and 3.26 (dd, J = 17.8, 5.0 Hz, 1H, H4a′). C NMR (100 MHz,
CDCl
54.6 (C), 133.4 (C), 128.9 (d, JC–F = 8.7 Hz, CH), 127.0 (d, JC–F = 3.2 Hz,
3.70 (m, 5H, H4b′, 2×CH
2
4
13
J
C NMR (100 MHz, CDCl
3
5
2
,
153.6 (C), 140.8 (C), 133.2 (C), 126.0 (C), 116.9 (C), 104.2 (CH), 61.1
(OCH ), 56.5 (OCH ), 54.4 (CH ), 52.4 (C5′), 41.3 (C4′), and 40.7
(CH ). MS (EI): m/z 520/522/524/526 [M /M + 2 /M + 4 /M + 6 ]
(64.37/66.67/24.51/3.82), 485/487/489 (31/20/4), 63 (100), 43 (49),
and 36 (90). Anal. calcd. for C20 23Cl S: C, 46.03; H, 4.44; N,
13
2
2
3
3
2
1
+
+
+
+
3
) δ ppm 165.5 (C), 164.4 (d,
JC–F = 252.2 Hz, C), 160.1 (CHO),
2
3
4
1
2
C), 116.7 (C), 116.3 (d,
J
C–F = 22.0 Hz, CH), 54.3 (CH
). MS (EI): m/z 448/450/452/454 [M /M + 2 /
M + 4 /M + 6 ] (13.07/13.61/4.81/0.70), 413/415/417 (11/7/2), 83 (51),
9 (100), and 43 (82). Anal. calcd. for C17 16Cl FN OS: C, 45.40; H, 3.59;
N, 12.46; S, 7.13. Found: C, 45.38; H, 3.57; N, 12.42; S, 7.17.
2
), 52.4 (C5′),
H
3 4 4
N O
+
+
41.2 (C4′), and 40.7 (CH
2
10.74; S, 6.14. Found: C, 46.06; H, 4.41; N, 10.70; S, 6.17.
+
+
6
H
3
4
5‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐phenyl‐4,5‐
dihydro‐1H‐pyrazole‐1‐carbaldehyde (7g)
−1
Beige solid; 70% yield; m.p. 74–77°C. FTIR (ATR) ν (cm ): 3,039
3
4
‐(Benzo[d][1,3]dioxol‐5‐yl)‐5‐{2‐[bis(2‐chloroethyl)amino]‐
(═C–H), 2,926 and 2,876 (C–H), and 1,670 (C═O), 1,527 (C═N and
1
‐chlorothiazol‐5‐yl}‐4,5‐dihydro‐1H‐pyrazole‐1‐carbaldehyde (7d)
C═C). H NMR (400 MHz, CDCl
3
) δ ppm 8.90 (s, 1H, CHO), 7.74
−1
Beige solid; 57% yield; m.p. 163–165°C. FTIR (ATR) ν (cm ): 3,027
═C–H), 2,965 and 2,895 (C–H), 1,657 (C═O), and 1,553 and 1,541 (1‐(5‐
2‐[bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐(4‐bromophenyl)‐4,5‐
(dd, J = 7.6, 2.0 Hz, 2H, HBo), 7.49–7.38 (m, 3H, HBm, HBp), 5.74 (dd,
J = 11.7, 5.0 Hz, 1H, H5′), 3.82–3.76 (m, 5H, H4b′, 2×CH –N), 3.73
–Cl), and 3.29 (dd, J = 17.8, 5.0 Hz, 1H, H4a′).
) δ ppm 165.5 (C), 160.2 (CHO), 155.7 (C),
(
2
{
(s, J = 5.4 Hz, 4H, 2×CH
2
1
13
dihydro‐1H‐pyrazol‐1‐yl)ethan‐1‐one C═C). H NMR (400 MHz, CDCl
ppm 8.86 (s, 1H, CHO), 7.33 (d, J = 1.3 Hz, 1H, HBo′), 7.10 (dd, J = 8.1,
.3 Hz, 1H, HBo), 6.84 (d, J = 8.1 Hz, 1H, HBm), 6.03 (s, 2H, –OCH O–),
.71 (dd, J = 11.6, 4.9 Hz, 1H, H5′), 3.79 (t, J = 5.7 Hz, 4H, 2×CH –N),
–Cl), and 3.22 (dd, J = 17.7, 4.9 Hz, 1H,
4a′). C NMR (100 MHz, CDCl ) δ ppm 165.5 (C), 160.0 (CHO), 155.3
3
) δ
C NMR (100 MHz, CDCl
3
133.2 (C), 131.0 (CH), 130.7 (C), 129.0 (CH), 126.9 (CH), 116.9 (C),
1
2
54.4 (CH ), 52.2 (C5′), 41.2 (C4′), and 40.7 (CH ). MS (EI): m/z 430/
2
2
+
+
+
+
5
2
432/434/436 [M /M + 2 /M + 4 /M + 6 ] (14.80/14.98/5.43/0.79),
395/397/399 (13/8/2), 81 (48), 69 (100), and 43 (42). Anal. calcd. for
3.75–3.67 (m, 5H, H4b′, 2×CH
2
13
H
3
17 3 4
C H17Cl N OS: C, 47.29; H, 3.97; N, 12.98; S, 7.43. Found: C, 47.30;
(
C), 150.2 (C), 148.5 (C), 133.1 (s), 124.9 (C), 122.1 (CH), 116.9 (C), 108.5
CH), 106.4 (CH), 101.8 (–OCH O), 54.3 (CH ), 52.2 (C5′), 41.3 (C4′), and
0.7 (CH ). MS (EI): m/z 474/476/478/480 [M /M + 2 /M + 4 /M + 6 ]
25.75/29.54/10.28/1.97), 439/441/443 (33/21/4), 148/150 (54/20), 69
83), 57 (100), and 43 (95). Anal. calcd. for C18 17Cl S: C, 45.44; H,
H, 3.98; N, 12.99; S, 7.48.
(
2
2
+
+
+
+
4
2
(
4.1.5 | General procedure for the synthesis of
N‐phenyl pyrazolines (8a–g)
(
H
3 4 3
N O
3
.60; N, 11.78; S, 6.74. Found: C, 45.42; H, 3.58; N, 11.80; S, 6.78.
A total of 4 drops of glacial acetic acid were added into a mixture of
the selected chalcone 5a–g (0.24 mmol) and phenylhydrazine
(1.20 mmol) in MeOH (5.0 ml) at room temperature. The reaction
mixture was heated under reflux for 7 hr and then cooled to room
temperature. The resulting solid was filtered and washed with
5
‐{2‐[Bis(2‐chloroethyl)amino]‐4‐chlorothiazol‐5‐yl}‐3‐(4‐
methoxyphenyl)‐4,5‐dihydro‐1H‐pyrazole‐1‐carbaldehyde (7e)
−1
Beige solid; 55% yield; m.p. 134–136°C. FTIR (ATR) ν (cm ): 3,005
═C–H), 2,873 and 2,839 (C–H), 1,670 (C═O), and 1,547 and 1,526
(

CUARTAS ET AL.
17 of 24
|
methanol and water to afford compounds 8a–g, which did not require
3 4
(93), and 43 (100). Anal. calcd. For C22H20Cl FN S: C, 53.08; H, 4.05;
further purification.
N, 11.25; S, 6.44. Found: C, 53.04; H, 4.02; N, 11.22; S, 6.49.
4
‐Chloro‐N,N‐bis(2‐chloroethyl)‐5‐[3‐(4‐chlorophenyl)‐1‐phenyl‐
,5‐dihydro‐1H‐pyrazol‐5‐yl]thiazol‐2‐amine (8a)
5‐[3‐(Benzo[d][1,3]dioxol‐5‐yl)‐1‐phenyl‐4,5‐dihydro‐1H‐pyrazol‐
5‐yl]‐4‐chloro‐N,N‐bis(2‐chloroethyl)thiazol‐2‐amine (8d)
4
−1
−1
Pale yellow solid; 66% yield; m.p. 146–148°C. FTIR (ATR) ν (cm ):
Pale yellow solid; 57% yield; m.p. 177–180°C. FTIR (ATR) ν (cm ):
3
,036 (═C–H), 2,958 and 2,914 (C–H), and 1,595 and 1,527 (C═N and
3,018 (═C–H), 2,961 and 2,910 (C–H), and 1,597 and 1,526 (C═N
1
1
C═C). H NMR (400 MHz, CDCl
3
) δ ppm 7.64 (d, J = 8.6 Hz, 2H, HBo),
and C═C). H NMR (400 MHz, CDCl
3
) δ ppm 7.40 (d, J = 1.4 Hz, 1H,
7
.36 (d, J = 8.6 Hz, 2H, HBm), 7.26 (t, J = 7.5 Hz, 2H, HCm), 7.16
d, J = 7.8 Hz, 2H, HCo), 6.88 (t, J = 7.3 Hz, 1H, HCp), 5.51 (dd, J = 11.8,
H
Bo′), 7.26 (t, J = 7.9 Hz, 2H, HCm), 7.16 (d, J = 7.9 Hz, 2H, HCo), 7.05
(dd, J = 8.1, 1.4 Hz, 1H, HBo), 6.90–6.80 (m, 2H, HBm, HCp), 6.00
(s, 2H, –OCH O–), 5.46 (dd, J = 11.6, 6.6 Hz, 1H, H5′), 3.81–3.73 (m,
5H, H4b′, 2×CH –N), 3.72–3.65 (m, 4H, 2×CH –Cl), and 3.15 (dd,
J = 17.0, 6.6 Hz, 1H, H4a′). C NMR (100 MHz, CDCl ) δ ppm 166.2
(
6
.6 Hz, 1H, H5′), 3.78–3.70 (m, 5H, H4b′, 2×CH
×CH –Cl), and 3.17 (dd, J = 17.1, 6.6 Hz, 1H, H4a′).
) δ ppm 166.2 (C), 146.7 (C), 144.5 (C), 134.9 (C),
32.3 (C), 130.9 (C), 129.3 (CH), 129.0 (CH), 127.2 (CH), 120.5 (CH),
19.3 (C), 114.1 (CH), 58.0 (C5′), 54.3 (CH ), 41.7 (C4′), and 40.7 (CH ).
2
–N), 3.70–3.65 (m, 4H,
2
1
3
2
2
C NMR
2
2
1
3
(100 MHz, CDCl
3
3
1
1
(C), 148.8 (C), 148.3 (C), 147.8 (C), 144.9 (C), 136.5 (C), 129.2 (CH),
126.8 (C), 120.6 (CH), 120.2 (CH), 119.7 (C), 114.0 (CH), 108.3 (CH),
2
2
+
+
+
+
MS (EI): m/z 512/514/516/518/520 [M /M + 2 /M + 4 /M + 6 /
106.0 (CH), 101.5 (–OCH
2
O–), 58.00 (C5′), 54.26 (CH
2
–N), 42.02
+
+
M + 8 ] (86/100/50/12/3), 477/479/481/483 (89/82/48/4), 274/276
(C4′), and 40.75 (CH
2
–Cl). MS (EI): m/z 522/524/526/528 [M /
+
+
+
(
49/21), and 173 (4). Anal. calcd. for C22
H
20Cl
4
N
4
S: C, 51.38; H, 3.92;
M + 2 /M + 4 /M + 6 ] (21/24/9/2), 487/489/491 (11/9/2), 91 (52),
57 (94), and 43 (100). Anal. calcd. for C23 21Cl S: C, 52.73; H,
.04; N, 10.69; S, 6.12. Found: C, 52.71; H, 4.07; N, 10.72; S, 6.09.
N, 10.89; S, 6.23. Found: C, 51.41; H, 3.91; N, 10.84; S, 6.28.
H
3 4 2
N O
4
5
4
‐[3‐(4‐Bromophenyl)‐1‐phenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐yl]‐
‐chloro‐N,N‐bis(2‐chloroethyl)thiazol‐2‐amine (8b)
4‐Chloro‐N,N‐bis(2‐chloroethyl)‐5‐[3‐(4‐methoxyphenyl)‐1‐phenyl‐
4,5‐dihydro‐1H‐pyrazol‐5‐yl]thiazol‐2‐amine (8e)
−
1
Pale yellow solid; 60% yield; m.p. 132–133°C. FTIR (ATR) ν (cm ):
−1
3,038 (═C–H), 2,972 and 2,903 (C–H), and 1,593 and 1,535 (C═N
White solid; 50% yield; m.p. 150–152°C. FTIR (ATR) ν (cm ): 3,031
1
and C═C). H NMR (400 MHz, CDCl
3
) δ ppm 7.60 (d, J = 8.6 Hz, 2H,
(═C–H), 2,956 and 2,901 (C–H), and 1,595 and 1,533 (C═N and C═C).
1
H
Bo), 7.53 (d, J = 8.6 Hz, 2H, HBm), 7.29 (t, J = 7.4 Hz, 2H, HCm), 7.17 (d,
J = 8.1 Hz, 2H, HCo), 6.90 (t, J = 7.2 Hz, 1H, HCp), 5.53 (dd, J = 11.7,
H NMR (400 MHz, CDCl
Cm), 7.17 (d, J = 7.9 Hz, 2H,
(d, J = 8.8 Hz, 2H, HBm), 6.87 (t, J = 7.2 Hz, 1H, HCp), 5.46 (dd, J = 11.6,
6.7 Hz, 1H, H5′), 3.85 (s, 3H, OCH ), 3.80–3.72 (m, 5H, H4b′, 2×CH –N),
3.71–3.66 (m, 4H, 2×CH –Cl), and 3.19 (dd, J = 17.0, 6.7 Hz, 1H, H4a′).
C NMR (100 MHz, CDCl ) δ ppm 166.2 (C), 160.6 (C), 148.0 (C),
145.1 (C), 132.0 (C), 129.2 (CH), 127.6 (CH), 125.1 (C), 120.1 (CH),
3
) δ ppm 7.68 (d, J = 8.8 Hz, 2H, HBo), 7.27
(t, J = 7.6 Hz, 2H,
H
HCo), 6.94
6
.6 Hz, 1H, H5′), 3.83–3.72 (m, 5H, H4b′, 2×CH
×CH –Cl), and 3.19 (dd, J = 17.1, 6.6 Hz, 1H, H4a′).
) δ ppm 160.6 (C), 146.7 (C), 144.4 (C), 135.8 (C),
31.9 (CH), 131.3 (C), 129.3 (CH), 127.4 (CH), 124.8 (C), 120.5 (CH),
19.2 (s), 114.0 (CH), 58.0 (C5′), 54.3 (CH ), 41.6 (C4′), and 40.7
). MS (EI): m/z 556/558/560/562/564 [M /M + 2 /M + 4 /
2
–N), 3.72–3.63 (m, 4H,
1
3
2
2
C NMR
3
2
(
100 MHz, CDCl
3
2
13
1
1
3
2
+
+
+
(CH
2
3 2
119.9 (C), 114.2 (CH), 114.0 (CH), 57.9 (OCH ), 55.5 (C5′), 54.3 (CH ),
+
+
+
+
M + 6 /M + 8 ] (9/15/9/3/1), 521/523/525/527 (10/13/6/1), 97 (60),
and 57 (100). Anal. calcd. for C22 20BrCl S: C, 47.29; H, 3.61; N,
0.03; S, 5.74. Found: C, 47.32; H, 3.59; N, 10.01; S, 5.78.
42.0 (C4′), and 40.8 (CH
2
). MS (EI): m/z 508/510/512/514 [M /M + 2 /
+
+
H
N
3 4
M + 4 /M + 6 ] (27/35/12/3), 473/475/477 (14/8/1), 91 (72), 63 (100),
and 43 (81). Anal. calcd. for C23 23Cl OS: C, 54.18; H, 4.55; N,
0.99; S, 6.29. Found: C, 54.14; H, 4.57; N, 10.96; S, 6.31.
1
H
3 4
N
1
4
‐Chloro‐N,N‐bis(2‐chloroethyl)‐5‐[3‐(4‐fluorophenyl)‐1‐phenyl‐4,5‐
dihydro‐1H‐pyrazol‐5‐yl]thiazol‐2‐amine (8c)
Pale yellow solid; 61% yield; m.p. 134–136°C. FTIR (ATR) ν (cm ):
4‐Chloro‐N,N‐bis(2‐chloroethyl)‐5‐[1‐phenyl‐3‐(3,4,5‐
−
1
trimethoxyphenyl)‐4,5‐dihydro‐1H‐pyrazol‐5‐yl]thiazol‐2‐amine (8f)
−1
3,021 (═C–H), 2,961 and 2,935 (C–H), and 1,597 and 1,530 (C═N
White solid; 58% yield; m.p. 144–145°C. FTIR (ATR) ν (cm ): 3,032
1
and C═C). H NMR (400 MHz, CDCl
3
) δ ppm 7.72 (dd, J = 8.9,
(═C–H), 2,955 and 2,920 (C–H), and 1,591 and 1,530 (C═N and
4
1
J
H–F = 5.4 Hz, 2H,
HBo), 7.28 (t, J = 7.3 Hz, 2H,
HCm), 7.17
C═C). H NMR (400 MHz, CDCl
3
) δ ppm 7.29 (t, J = 7.3 Hz, 2H, HCm),
(
d, J = 7.7 Hz, 2H, HCo), 7.10 (t, J = 8.7 Hz, 2H, HBm), 6.89 (t, J = 7.3 Hz,
7.18 (d, J = 7.7 Hz, 2H, HCo), 6.97 (s, 2H, HBo), 6.89 (t, J = 7.3 Hz, 1H,
Cp), 5.51 (dd, J = 11.7, 6.6 Hz, 1H, H5′), 3.93 (s, 6H, 2×OCH ), 3.89
1
2
6
H, HCp), 5.50 (dd, J = 11.7, 6.7 Hz, 1H, H5′), 3.78–3.73 (m, 5H, H4b′
,
H
3
×CH –N), 3.72–3.67 (m, 4H, 2×CH –Cl), and 3.19 (dd, J = 17.1,
2
2
(s, 3H, OCH
CH
CDCl
132.1 (C), 129.3 (CH), 128.0 (C), 120.4 (CH), 119.6 (C), 114.0 (CH),
3
), 3.85–3.71 (m, 5H, H4b′, 2×CH
2
–N), 3.73–3.64 (m, 4H,
1
3
13
.7 Hz, 1H, H4a′). C NMR (100 MHz, CDCl
3
) δ ppm 166.2 (C), 163.3
2
–Cl), and 3.21 (dd, J = 17.0, 6.6 Hz, 1H, H4a′). C NMR (100 MHz,
1
(
d,
J
C–F = 232.5 Hz, C), 147.0 (C), 144.8 (C), 132.2 (C), 129.3 (CH),
3
) δ ppm 166.2 (C), 153.5 (C), 147.7 (C), 144.7 (C), 139.4 (C),
4
3
1
1
28.7 (d,
J
C–F = 3.0 Hz, C), 127.9 (d, JC–F = 8.3 Hz, CH), 120.4 (CH),
2
19.5 (C), 115.9 (d,
JC–F = 21.9 Hz, CH), 114.0 (CH), 58.1 (C5′), 54.3
103.4 (CH), 61.1 (OCH
(C4′), and 40.7 (CH
3
), 58.0 (C5′), 56.4 (OCH
3
), 54.3 (CH
2
), 42.0
+
+
+
(
CH
2
), 41.9 (C4′), and 40.7 (CH
2
). MS (EI): m/z 496/498/500/502 [M /
2
). MS (EI): m/z 568/570/572/574 [M /M + 2 /
+
+
+
+
+
M + 2 /M + 4 /M + 6 ] (6/6/1/1), 461/463/465 (5/3/1), 97 (44), 57
M + 4 /M + 6 ] (57/61/24/4), 533/535/537 (17/10/3), 91 (40), and 63

18 of 24
CUARTAS ET AL.
|
1
(
100). Anal. calcd. for C25
H
3
27Cl N
4 3
O S: C, 52.69; H, 4.78; N, 9.83; S,
and C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.77 (d, J = 7.3 Hz,
5
.63. Found: C, 52.71; H, 4.75; N, 9.87; S, 5.67.
2H, HBo), 7.67 (d, J = 7.4 Hz, 2H, HBm), 7.05 (d, J = 1.5 Hz, 2H, HCo),
.97 (t, J = 1.5 Hz, 1H, HCp), 5.80 (dd, J = 11.6, 4.5 Hz, 1H, H5′), 3.89
(dd, J = 17.8, 11.6 Hz, 1H, H4b′), 3.82–3.76 (m, J = 8.5 Hz, 4H,
6
4
‐Chloro‐N,N‐bis(2‐chloroethyl)‐5‐(1,3‐diphenyl‐4,5‐dihydro‐1H‐
pyrazol‐5‐yl)thiazol‐2‐amine (8g)
White solid; 56% yield; m.p. 144–146°C. FTIR (ATR) ν (cm ): 3,038
2×CH
2
–Cl), 3.77–3.64 (m, 4H, 2×CH
13
2
–N), and 3.41 (dd, J = 17.8,
) δ ppm 166.8 (C),
−1
4.5 Hz, 1H, H4a′). C NMR (100 MHz, DMSO‐d
6
(
═C–H), 2,938 and 2,924 (C–H), and 1,595 and 1,528 (C═N and
149.8 (C), 145.3 (C), 134.6 (C), 132.2 (C), 131.8 (CH), 130.5 (C),
1
C═C). H NMR (400 MHz, CDCl
3
) δ ppm 7.74 (d, J = 8.5 Hz, 2H, HBo),
128.3 (CH), 123.0 (C), 118.3 (CH), 116.3 (C), 111.6 (CH), 56.6 (C5′),
7.44–7.34 (m, 3H, HBm, HBp), 7.29 (t, J = 7.4 Hz, 1H, HCm), 7.19 (d,
2 2
52.2 (CH ), 41.0 (CH ), and 40.7 (C4′). MS (EI): m/z 624/626/628/630/
+
+
+
+
+
+
+
J = 7.7, 1.2 Hz, 2H, HCo), 6.89 (t, J = 7.2 Hz, 1H, HCp), 5.51 (dd, J = 11.7,
632/634/636
[M /M + 2 /M + 4 /M + 6 /M + 8 /M + 10 /M + 12 ]
6
.6 Hz, 1H, H5′), 3.83–3.72 (m, 5H, H4b′, 2×CH
×CH –Cl), and 3.22 (dd, J = 17.1, 6.6 Hz, 1H, H4a′).
) δ ppm 166.2 (C), 147.8 (C), 144.8 (C), 132.4 (C),
32.2 (C), 129.3 (CH), 129.2 (CH), 128.8 (CH), 126.1 (CH), 120.3
2
–N), 3.72–3.66 (m, 4H,
(40/73/66/34/11/2/0.5), 589/591/593/595/597/599 (53/100/81/35/
8/1), 367/369/371/373 (60/86/32/6), and 63 (89). Anal. calcd. for
13
2
2
C NMR
(100 MHz, CDCl
3
22 5 4
C H18BrCl N S: C, 42.10; H, 2.89; N, 8.93; S, 5.11. Found: C, 42.13; H,
1
2.90; N, 8.90; S, 5.16.
(
(
(
2
CH), 119.6 (C), 114.0 (CH), 57.9 (C5′), 54.2 (CH ), 41.8 (C4′), and 40.7
+
+
+
+
CH
2
). MS (EI): m/z 478/480/482/484 [M /M + 2 /M + 4 /M + 6 ]
4‐Chloro‐N,N‐bis(2‐chloroethyl)‐5‐[1‐(3,5‐dichlorophenyl)‐3‐(4‐
fluorophenyl)‐4,5‐dihydro‐1H‐pyrazol‐5‐yl]thiazol‐2‐amine (9c)
10/13/6/1), 443/445/447 (9/14/1), 91 (46), 69 (100), and 43 (80).
21Cl S: C, 55.07; H, 4.41; N, 11.68; S, 6.68.
Found: C, 55.02; H, 4.45; N, 11.62; S, 6.71.
−1
Anal. calcd. for C22
H
N
3 4
Pale yellow solid; 76% yield; m.p. 143–145°C. FTIR (ATR) ν (cm ):
3,074 (═C–H), 2,966 and 2,924 (C–H), and 1,583 and 1,533 (C═N and
1
C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.86 (dd, J = 8.5,
4
J
H–F = 5.6 Hz, 2H, HBo), 7.30 (t, J = 8.8 Hz, 2H, HBm), 7.02 (d, J = 1.3 Hz,
4
.1.6 | General procedure for the synthesis of N‐3,5‐
2H, HCo), 6.94 (t, J = 1.3 Hz, 1H, HCp), 5.76 (dd, J = 11.7, 4.6 Hz, 1H, H5′),
3.87 (dd, J = 17.8, 11.7 Hz, 1H, H4b′), 3.80–3.74 (m, 4H, 2×CH –Cl),
.75–3.64 (m, 4H, 2×CH –N), and 3.40 (dd, J = 17.8, 4.6 Hz, 1H, H4a′).
NMR (100 MHz, DMSO‐d ppm 166.8 (C), 162.9
(d, JC–F = 247.8 Hz, C), 149.9 (C), 145.4 (C), 134.5 (C), 132.1 (C), 128.6
dichlorophenyl pyrazolines (9a–g)
2
3
2
13
A mixture of the selected chalcone 5a–g (0.42 mmol) and 3,
C
1
6
)
δ
5
‐dichlorophenylhydrazine hydrochloride (2.1 mmol) in MeOH
3
4
(
5.0 ml) was heated under reflux for 4–12 hr. The precipitate
(d,
JC–F = 8.6 Hz, CH), 127.8 (d,
JC–F = 3.1 Hz, C), 118.0 (CH), 116.4
2
formed was filtered, dried, and recrystallized from ethanol to
(C), 115.8 (d,
J
C–F = 21.9 Hz, CH), 111.4 (CH), 56.5 (C5′), 52.2 (CH
2
),
afford derivatives 9a–g.
2
41.3 (CH ), and 40.7 (C4′). MS (EI): m/z 564/566/568/570/572/574
+
+
+
+
+
+
[
M /M + 2 /M + 4 /M + 6 /M + 8 /M + 10 ] (1.8/3.8/2.5/0.8/0.5/0.3),
4
‐Chloro‐N,N‐bis(2‐chloroethyl)‐5‐[3‐(4‐chlorophenyl)‐1‐(3,5‐
529/531/533/535/537 (2.3/3.1/1.8/0.7/0.5), 369 (6), 97 (43), 83 (50),
and 69 (100). Anal. calcd. for C22 18Cl FN S: C, 46.62; H, 3.20; N,
dichlorophenyl)‐4,5‐dihydro‐1H‐pyrazol‐5‐yl]thiazol‐2‐amine (9a)
H
5
4
−
1
Pale yellow solid; 88% yield; m.p. 187–189°C. FTIR (ATR) ν (cm ):
9.89; S, 5.66. Found: C, 46.60; H, 3.17; N, 9.86; S, 5.69.
3
,074 (═C–H), 2,964 and 2,922 (C–H), and 1,583 and 1,524 (C═N
1
and C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.82 (d, J = 8.3 Hz,
5‐[3‐(Benzo[d][1,3]dioxol‐5‐yl)‐1‐(3,5‐dichlorophenyl)‐4,5‐dihydro‐1
H‐pyrazol‐5‐yl]‐4‐chloro‐N,N‐bis(2‐chloroethyl)thiazol‐2‐amine (9d)
2
H, HBo), 7.52 (d, J = 8.3 Hz, 2H, HBm), 7.03 (d, J = 1.3 Hz, 2H, HCo),
.95 (t, J = 1.3 Hz, 1H, HCp), 5.78 (dd, J = 11.7, 4.0 Hz, 1H, H5′), 3.87
−
1
6
Pale yellow solid; 77% yield; m.p. 171–173°C. FTIR (ATR) ν (cm ):
(dd, J = 17.8, 11.7 Hz, 1H, H4b′), 3.80–3.74 (m, 4H, 2×CH
2
–Cl), 3.73–
3,068 (═C–H), 2,967 and 2,895 (C–H), and 1,582 and 1,530 (C═N
1
3
1
3.63 (m, 4H, 2×CH
2
–N), and 3.39 (dd, J = 17.8, 4.0 Hz, 1H, H4a′).
C
and C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.44 (d, J = 1.7 Hz,
NMR (100 MHz, DMSO‐d
6
) δ ppm 166.8 (C), 149.7 (C), 145.3 (C),
34.6 (C), 134.2 (C), 132.2 (C), 130.1 (C), 128.8 (CH), 128.0 (CH),
18.2 (CH), 116.3 (C), 111.5 (CH), 56.5 (C5′), 52.2 (CH ), 41.0 (CH ),
1H, HBo′), 7.24 (dd, J = 8.1, 1.7 Hz, 1H, HBo), 7.01 (d, J = 1.8 Hz, 2H,
Co), 6.99 (d, J = 8.1 Hz, 1H, HBm), 6.90 (t, J = 1.8 Hz, 1H, HCp), 6.10
(s, 2H, –OCH O–), 5.71 (dd, J = 11.6, 4.6Hz, 1H, H5′), 3.81 (dd,
J = 16.8, 11.6 Hz, 1H, H4b′), 3.78–3.74 (m, 4H, 2×CH
3.66 (m, 4H, 2×CH
NMR (100 MHz, DMSO‐d
1
1
H
2
2
2
and 40.7 (C4′). MS (EI): m/z 580/582/584/586/588/590/592
2
–Cl), 3.74–
+
+
+
+
+
+
+
13
[
M /M + 2 /M + 4 /M + 6 /M + 8 /M + 10 /M + 12 ] (57/95/76/32/9/
2
–N), and 3.34 (d, J = 16.8, 4.6 Hz, 1H, H4a′).
C
1
3
/0.3), 545/547/549/551/553/555 (66/100/66/24/5/0.6), 323/325/
27/329 (84/66/17/2), 274/276/278 (77/32/3), and 63 (16). Anal.
6
) δ ppm 166.7 (C), 150.5 (C), 148.7 (C),
147.8 (C), 145.5 (C), 134.5 (C), 132.0 (C), 125.4 (C), 121.4 (CH),
117.8 (CH), 116.5 (C), 111.3 (CH), 108.4 (CH), 105.8 (CH), 101.5
6 4
calcd. for C22H18Cl N S: C, 45.31; H, 3.11; N, 9.61; S, 5.50. Found: C,
45.30; H, 3.12; N, 9.62; S, 5.53.
(–OCH
2
O–), 56.2 (C5′), 52.2 (CH
2
2
), 41.4 (CH ), and 40.7 (C4′). MS
+
+
+
+
(
EI): m/z 590/592/594/596/598/600 [M /M + 2 /M + 4 /M + 6 /
+
+
5
‐[3‐(4‐Bromophenyl)‐1‐(3,5‐dichlorophenyl)‐4,5‐dihydro‐1H‐
M + 8 /M + 10 ] (13/19/12/3/1/0.2), 555/557/559 (7/7/3), 368
(12), 97 (36), 83 (40), 57 (85), and 43 (100). Anal. calcd. for
pyrazol‐5‐yl]‐4‐chloro‐N,N‐bis(2‐chloroethyl)thiazol‐2‐amine (9b)
−
1
Pale yellow solid; 90% yield; m.p. 188–190°C. FTIR (ATR) ν (cm ):
,076 (═C–H), 2,963 and 2,922 (C–H), and 1,584 and 1,522 (C═N
C
23
H
19Cl
5 4 2
N O S: C, 46.60; H, 3.23; N, 9.45; S, 5.41. Found: C,
3
46.58; H, 3.22; N, 9.44; S, 5.39.

CUARTAS ET AL.
19 of 24
|
4
‐Chloro‐N,N‐bis(2‐chloroethyl)‐5‐[1‐(3,5‐dichlorophenyl)‐3‐(4‐
4.1.7 | Preparation of 3‐(4‐chlorophenyl)‐1‐(3,5‐
dichlorophenyl)‐1H‐pyrazole (10a)
methoxyphenyl)‐4,5‐dihydro‐1H‐pyrazol‐5‐yl]thiazol‐2‐amine (9e)
−1
White solid; 59% yield; m.p. 146–148°C. FTIR (ATR) ν (cm ): 3,005
(
1
═C–H), 2,957 and 2,899 (C–H), and 1,581 and 1,533 (C═N and C═C).
A mixture of chalcone 5a (0.42 mmol) and 3,5‐dichlorophenylhydrazine
hydrochloride (2.1 mmol) in AcOH (5.0 ml) was heated under reflux for
8 hr. After cooling, the precipitate formed was filtered and washed with
ethanol.
6
H NMR (400 MHz, DMSO‐d ) δ ppm 7.73 (d, J = 8.9 Hz, 2H, HBo), 7.00
(
d, J = 8.9 Hz, 2H, HBm), 6.98 (d, J = 1.8 Hz, 2H, HCo), 6.88 (t, J = 1.8 Hz,
1
H, HCp), 5.68 (dd, J = 11.4, 4.5 Hz, 1H, H5′), 3.82 (dd, J = 17.7, 11.4 Hz,
H, H4b′), 3.80 (s, 3H, OCH ), 3.77–3.73 (m, 4H, 2×CH –Cl), 3.71–3.66
–N), and 3.33 (dd, J = 17.7, 4.5 Hz, 1H, H4a′). C NMR
100 MHz, DMSO‐d ) δ ppm 166.9 (C), 160.7 (C), 150.8 (C), 145.8 (C),
34.6 (C), 132.2 (C), 128.1 (CH), 123.8 (C), 117.8 (CH), 116.6 (C), 114.4
1
3
2
13
(
m, 4H, 2×CH
2
3‐(4‐Chlorophenyl)‐1‐(3,5‐dichlorophenyl)‐1H‐pyrazole (10a)
−
1
(
6
Yellow solid; 72% yield; m.p. 137–138°C. FTIR (ATR) ν (cm ): 3,146,
1
1
3,093 and 3,061 (═C–H), and1,589 and 1,531 (C═N and C═C).
NMR (400 MHz, DMSO‐d
H
(
3 2 2
CH), 111.4 (CH), 56.3 (C5′), 55.4 (OCH ), 52.3 (CH ), 41.5 (CH ), and
6
) δ ppm 8.73 (d, J = 2.6 Hz, 1H, H5′), 8.05
+
+
4
0.8 (C4′). MS (EI): m/z 576/578/580/582/584/586 [M /M + 2 /
(d, J = 1.8 Hz, 2H, HCo), 7.99 (d, J = 8.5 Hz, 2H, HBo), 7.56 (d, J = 1.8 Hz,
+
+
+
+
M + 4 /M + 6 /M + 8 /M + 10 ] (49/79/52/19/3.5/0.6), 541/543/545/
47/549 (39/44/21/6/2), 381 (60), 319 (49), 124 (69), and 57 (100).
Anal. calcd. for C23 21Cl OS: C, 47.73; H, 3.66; N, 9.68; S, 5.54.
Found: C, 47.70; H, 3.68; N, 9.65; S, 5.56.
1H, HCp), 7.52 (d, J = 8.5Hz, 2H, HBm), and 7.15 (d, J = 2.6Hz, 1H, H4′).
1
3
5
6
C NMR (100 MHz, DMSO‐d ) δ ppm 151.7 (C), 141.2 (C), 135.1 (C),
H
5
N
4
133.1 (C), 131.0 (C), 130.4 (CH), 128.9 (CH), 127.5 (CH), 125.4 (CH),
116.7 (CH), and 106.6 (CH). MS (EI): m/z 322/324/326/328
+
+
+
+
[
M /M + 2 /M + 4 /M + 6 ] (100/94/21/4), 252/254 (9/3), 145 (10),
4
‐Chloro‐N,N‐bis(2‐chloroethyl)‐5‐[1‐(3,5‐dichlorophenyl)‐3‐(3,4,5‐
111 (15), and 75 (13). Anal. calcd. for C22 18Cl S: C, 55.68; H,
H
6 4
N
trimethoxyphenyl)‐4,5‐dihydro‐1H‐pyrazol‐5‐yl]thiazol‐2‐amine (9f)
2.80; N, 8.66. Found: C, 55.72; H, 2.77; N, 8.64.
−1
White solid; 81% yield; m.p. 166–167°C. FTIR (ATR) ν (cm ): 3,005
(═C–H), 2,959 and 2,937 (C–H), and 1,584 and 1,539 (C═N and
1
C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.04 (s, 2H, HBo), 7.00
4.2 | Biological assays
4.2.1 | Antifungal activity
Microorganisms and media
(
d, J = 1.4 Hz, 2H, HCo), 6.88 (t, J = 1.4 Hz, 1H, HCp), 5.71 (dd, J = 11.3,
4
.2 Hz, 1H, H5′), 3.89–3.77 (m, 7H, H4b′, 2×OCH
3
), 3.78–3.72 (m, 4H,
), and 3.42 (dd,
) δ ppm
67.1 (C), 153.3 (C), 151.1 (C), 145.6 (C), 139.3 (C), 134.8 (C), 132.4
C), 127.0 (C), 118.2 (CH), 116.7 (C), 111.7 (CH), 104.0 (CH), 60.4
2
×CH –Cl), 3.72–3.66 (m, 7H, 2×CH –N, OCH
2
2
3
1
3
J = 17.8, 4.2 Hz, 1H, H4a′). C NMR (100 MHz, DMSO‐d
6
1
For the antifungal evaluation, standardized strains from the Amer-
ican Type Culture Collection (ATCC, Rockville, MD), C. albicans ATCC
10231 and C. neoformans ATCC 32264, were used. Strains were
grown on Sabouraud chloramphenicol agar slants for 48 hr at 30°C,
and they were maintained on slopes of Sabouraud dextrose agar
(Oxoid) and subcultured every 15 days to prevent pleomorphic
transformations. Inocula were obtained according to reported pro-
(
(
(
OCH
3
), 56.6 (C5′), 56.3 (OCH
3
), 52.5 (CH
2 2
), 41.6 (CH ), and 41.0
+
+
+
C
4′). MS (EI): m/z 636/638/640/642/644/646 [M /M + 2 /M + 4 /
+
+
+
M + 6 /M + 8 /M + 10 ] (19/36/25/9/2/1), 601/603/605/607/609
8/10/5/1/0.2), 441 (14), 193 (15), and 63 (100). Anal. calcd. for
25Cl S: C, 47.00; H, 3.94; N, 8.77; S, 5.02. Found: C, 47.05;
H, 3.97; N, 8.73; S, 4.98.
(
C
25
H
5 4 3
N O
[
54]
3
cedures
CFU)/ml.
and adjusted to 1–5 × 10 cells with colony‐forming units
(
4
‐Chloro‐N,N‐bis(2‐chloroethyl)‐5‐[1‐(3,5‐dichlorophenyl)‐3‐phenyl‐
,5‐dihydro‐1H‐pyrazol‐5‐yl]thiazol‐2‐amine (9g)
4
Fungal growth inhibition percentage determination
−1
White solid; 79% yield; m.p. 152–154°C. FTIR (ATR) ν (cm ): 3,089
Broth microdilution techniques were performed in 96‐well micro-
plates according to the Clinical and Laboratory Standards Institute—
Reference Method for Broth Dilution Antifungal Susceptibility Testing of
(
═C–H), 2,968 and 2,900 (C–H), and 1,585 and 1,537 (C═N and
1
C═C). H NMR (400 MHz, DMSO‐d
6
) δ ppm 7.78 (dd, J = 7.8, 1.7 Hz,
[
54]
2
H, HBo), 7.49–7.39 (m, 3H, HBm, HBp), 7.00 (d, J = 1.6 Hz, 2H, HCo)),
.90 (t, J = 1.6 Hz, 1H, HCp), 5.73 (dd, J = 11.6, 4.3 Hz, 1H, H5′), 3.85
Yeasts, Approved Standard, M27 (4th ed.).
For the assay, compound
6
test wells (CTWs) were prepared with stock solutions of each com-
pound in DMSO (maximum concentration ≤ 1%), diluted with Roswell
Park Memorial Institute (RPMI)‐1640, to final concentrations of
250–3.9 μg/ml. An inoculum suspension (100 μl) was added to each
well (final volume in the well = 200 μl). A growth control well (GCW;
containing medium, inoculum, and the same amount of DMSO used in
a CTW, but compound‐free) and a sterility control well (SCW; sample,
medium, and sterile water instead of inoculum) were included for
each fungus tested. Microtiter trays were incubated in a moist, dark
chamber at 30°C for 48 hr for both yeasts. Microplates were read in
a VERSA Max microplate reader (Molecular Devices, Sunnyvale, CA).
(dd, J = 17.8, 11.6 Hz, 1H, H4b′), 3.77–3.70 (m, 4H, 2×CH
2
–Cl), 3.70–
1
3
3
.64 (m, 4H, 2×CH
2
–N), and 3.35 (dd, J = 17.8, 4.3 Hz, 1H, H4a′).
C
NMR (100 MHz, DMSO‐d
6
) δ ppm 167.1 (C), 151.0 (C), 145.7 (C),
1
1
34.8 (C), 132.5 (C), 131.4 (C), 130.1 (CH), 129.1 (CH), 126.5 (CH),
18.3 (CH), 116.6 (C), 111.7 (CH), 56.6 (C5′), 52.5 (CH
2 2
), 41.4 (CH ),
+
+
and 41.0 (C4′). MS (EI): m/z 546/548/550/552/554/556 [M /M + 2 /
+
+
+
+
M + 4 /M + 6 /M + 8 /M + 10 ] (41/70/48/17/3/0.5), 511/513/515/
17/519 (70/80/40/11/1.2), 351 (30), 289 (86), 240 (66), 124 (85),
and 63 (100). Anal. calcd. for C22 19Cl S: C, 48.15; H, 3.49; N,
0.21; S, 5.84. Found: C, 48.19; H, 3.52; N, 10.20; S, 5.87.
5
H
5 4
N
1

20 of 24
CUARTAS ET AL.
|
Amphotericin B (Sigma‐Aldrich, St. Louis, MO) was used as a positive
control. Tests were performed in triplicate. Reduction of growth for
each compound concentration was calculated as follows: % of in-
hibition = 100 − (OD405 CTW − OD405 SCW)/(OD405 GCW − OD405
SCW). The means ± SEM was used to construct the dose–response
curves representing % inhibition versus concentration of each
compound.
For N. gonorrhoeae, those compounds with visible growth in-
hibition in the screening test were further tested for MIC on agar
plates, as described by the Centers for Disease Control and Pre-
[
56]
vention (CDC)
and the Clinical and Laboratory Standards Institute
[
63]
(CLSI)
with modifications. Briefly, GC agar supplemented with 1%
isovitalex was prepared, containing increasing concentrations of the
novel compounds, and it was inoculated with 5 µl of the bacterial
4
[56]
suspension (i.e., 1 × 10 CFU).
The lowest concentration of the
compound that inhibited bacterial growth was determined as the
MIC. Bacterial growth was examined and verified using the oxidase
test. Experiments were performed in duplicate and replicated at least
three times.
4
.2.2 | Antibacterial activity
All the synthesized compounds were tested to determine the anti-
bacterial activity against Gram‐negative and ‐positive bacteria. Wild‐
type and multidrug‐resistant strains were included as follows: MSSA
ATCC 25923, MRSA ATCC 43300, VISA, E. coli ATCC 25922,
carbapenemase‐positive K. pneumoniae BAA 1705, K. pneumoniae
ATCC 700603 (extended‐spectrum β‐lactamase, ESBL positive), P.
aeruginosa ATCC 27853, and N. gonorrhoeae ATCC 31426
4.2.3 | Hemolytic activity
The ability to induce hemolysis was evaluated following the method
of cytotoxicity by spectrophotometry on a 96‐well plate. Human red
blood cells (RBC) were adjusted to 5% hematocrit in RPMI‐1640
medium; 500 μl of RBCs were placed into each well of a 24‐well plate
and subsequently exposed against 200 μg/ml. Detection of free he-
moglobin, after 48 hr of incubation at 37°C, is the evidence that the
compound induces hemolysis. The concentration of free hemoglobin
was performed spectrophotometrically at 542 nm (Varioskan; Ther-
mo Fisher Scientific), and intensity of color (absorbance) was regis-
tered as optical densities (OD). Nonspecific absorbance was
corrected by subtracting the absorbance of the blank. Determina-
tions were done by triplicate in at least two independent
(
β‐lactamase positive). Stock solutions (100 mg/ml) of the compounds
were prepared in DMSO and diluted to a final screening concentra-
tion of 1 mg/ml. An initial screening of bacterial inhibition was per-
formed by the agar diffusion method. Briefly, sterile Mueller Hinton
agar (MHA, BBL) was prepared in Petri dishes and inoculated with a
bacterial suspension prepared in trypticase soy broth and adjusted to
8
[62]
1
.5 × 10 CFU/ml (i.e., 0.08–0.1 OD at 600 nm).
diameter) were punched in the agar and 10 µl of each compound
stock solution) was filled into each well. Dimethyl sulfoxide and
Wells (6 mm in
(
trypticase soy broth were included as negative controls (i.e., no in-
hibition of bacterial growth). Gentamicin and polymyxin B (Sigma‐
Aldrich) were included as positive controls of growth inhibition.
Compounds showing growth inhibition were tested at least twice
before being selected for microdilution testing. For N. gonorrhoeae,
the agar diffusion method was also used in the screening process
with some modifications. For this method, 200 µl of a bacterial sus-
[
65]
experiments.
4.2.4 | Antiprotozoal activity
Compounds storage
8
pension (1.5 × 10 CFU/ml) was inoculated in gonococcal (GC) agar
All compounds were stored at room temperature until use. Before
the biological evaluation, each compound was solubilized in 0.5%
DMSO and then diluted to the appropriate concentration in culture
media.
(
BBL) supplemented with 1% isovitalex (BBL), and then the com-
pounds were added to the wells, as mentioned above, and incubated
at 35–36.5°C in 5% CO atmosphere for 48 hr. Penicillin, ceftriaxone,
2
[63]
and ciprofloxacin (BBL) were used as controls.
Cytotoxic activity
Microdilution test
Cytotoxicity of the compounds was evaluated over human mono-
MIC was determined in those compounds with visible and re-
producible bacterial inhibition by the screening test. Bacterial sus-
pensions were adjusted with Mueller Hinton broth (MHB) to a
cytes (U‐937 ATCC CRL‐1593.2) in an exponential growth phase and
5
adjusted at 1 × 10 cells/ml in RPMI‐1640 medium enriched with
10% fetal bovine serum (FBS). Also, 100 μl of cell suspension was
dispensed in each well of a 96‐well microplate, and then, 100 μl of
200, 50, 12.5, and 3.125 μg/ml concentration of each compound or
standard drugs, chloroquine (malaria), benznidazole (Chagas disease),
and amphotericin B (leishmaniasis), was added. Cells exposed to
compounds or standard drugs were incubated for 72 hr at 37°C and
5
5 [62]
concentration of 5 × 10 –8 × 10 .
Stock solutions of the newly
synthesized compounds were diluted in MHB, containing 5% DMSO
[61]
and 0.1% Tween 80,
and added to 90 µl of the bacterial inoculum.
The microplates were incubated for 24 hr at 35–37°C. MICs were
defined as the lowest concentration with visible inhibition of bacterial
[64]
growth
and/or detection using resazurin (1 mg/ml). Gentamicin
2
with 5% of CO . Cytotoxic activity of each compound was de-
(
Sigma‐Aldrich) was included as control of inhibition growth; MHB and
termined according to the effect on the cell viability by the MTT
microenzymatic method in which 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐
diphenyltetrazolium bromide is reduced to a purple product, named
DMSO were used as bacterial growth controls. Experiments were
performed in duplicate and replicated at least three times.

CUARTAS ET AL.
21 of 24
|
formazan by mitochondrial enzyme succinate dehydrogenase. Thus,
parasites. For this, the antileishmanial activity was tested on in-
10 μl/well was added to each well of exposed and unexposed cells,
tracellular amastigotes of L. panamensis transfected with the green
and plates were incubated at 37°C and with 5% CO for 3 hr. The
2
fluorescent
protein
gene
(MHOM/CO/87/UA140‐EpiR–GFP
[
5]
5
reaction was stopped by adding 100 μl/well of isopropanol with 50%
and 10% of sodium dodecyl sulfate. The concentration of formazan
was determined spectrophotometrically at 570 nm (Varioskan;
Thermo Fisher Scientific), and intensity of color (absorbance) was
registered as OD. Cells exposed to control drugs (chloroquine, am-
photericin B, and benznidazole) were used as control for toxicity
strain). Briefly, U‐937 human cells at a density of 3 × 10 cells/ml in
RPMI‐1640 and 0.1 μg/ml of phorbol‐12‐myristate‐13‐acetate (PMA)
were dispensed on a 24‐well microplate and then infected with
stationary‐phase L. panamensis promastigotes in a 15:1 parasites per
2
cell ratio. Plates were incubated at 34°C and 5% CO for 3 hr and
then cells were washed twice with phosphate‐buffered solution (PBS)
to eliminate not internalized parasites. Fresh RPMI‐1640 was added
into each well (1 ml) and plates were incubated again to complete
infection. After 24 hr of infection, the RPMI‐1640 medium was re-
placed by fresh culture medium containing each compound at four
serial dilutions (50, 12.5, 3.125 and 0.78 μg/ml), and plates were then
(positive control), whereas cell incubated in the absence of any
compound or drug was used as control for viability (negative control).
Nonspecific absorbance was corrected by subtracting absorbance
(OD) of the blank. Determinations were done by triplicate in at least
[66]
two independent experiments.
2
incubated at 37°C and 5% CO during 72 hr. Then, cells were re-
Antiplasmodial activity
moved from the bottom plate with a trypsin/ethylenediaminete-
traacetic acid (250 mg) solution. The cells were centrifuged at
1,100 rpm during 10 min at 4°C, the supernatant was discarded, and
cells were washed with 1 ml of cold PBS and centrifuged at
1,100 rpm for 10 min at 4°C. Cells were washed two times employing
PBS, as previously, and after the last wash, the supernatant was
discarded and cells were suspended in 500 μl of PBS.
The antiplasmodial activity was evaluated in vitro on asynchronic
cultures of P. falciparum (3D7 strain), maintained in standard culture
conditions. The effect of each compound over the growth of the
parasites was determined by quantification of parasite death, based on
the measurement of lactate dehydrogenase (LDH) activity released
from the cytosol of damaged cells into the supernatant. Briefly, un-
synchronized P. falciparum cultures were adjusted to 0.5% parasitemia
and 1% hematocrit in RPMI medium enriched with 3% lipid‐rich bovine
serum albumin—Albumax II. Then, in each well of a 96‐well plate,
Cells were analyzed by flow cytometry employing a flow cyt-
ometer (cytomics FC 500MPL) reading at 488 nm (exciting) and
525 nm (emitting) over an argon laser and counting 10,000 events.
Infected cells were determined according to the events for green
fluorescence (parasites). All determinations for each compound and
standard drugs were carried out by triplicate, in two experiments.
Infected cells exposed to control drugs (amphotericin B) were used as
control for antileishmanial activity (positive control), whereas in-
fected cells incubated in the absence of any compound or drug were
used as a control for infection (negative control). Nonspecific fluor-
escence was corrected by subtracting the fluorescence of unstained
cells. Determinations were done by triplicate in at least two in-
100 μl of parasite suspension was dispensed and subsequently ex-
posed against 100 μl of four serial dilutions of compounds (100, 25,
6
.25, and 1.56 μg/ml). Dilutions were prepared from a stock solution of
,000 μg/ml. CQ was used as positive antiplasmodial drug control.
1
Parasites unexposed to any compound were used as control of both
growth and viability (negative control). Plates were incubated for 48 hr
at 37°C in N
cubation, plates were harvested and parasites were subjected to three
0‐min freeze–thaw cycles. Meanwhile, 100 μl of Malstat reagent
400 μl Triton X‐100 in 80 ml deionized water, 4 g L‐lactate, 1.32 g Tris
2 2 2
(90%), CO (5%), and O (5%) atmosphere. After in-
2
[
5,66]
(
dependent experiments.
buffer, and 0.022 g acetylpyridine adenine dinucleotide in 200 ml
deionized water; pH 9.0), and 25 μl of NBT/PES solution (0.16 g ni-
troblue tetrazolium salt and 0.08 g phenazine ethosulphate in 100 ml
deionizated water) were added to each well of a second flat‐bottom
Antitrypanosomal activity
The effectiveness of synthesized compounds was determined ac-
cording to the ability of the compound to reduce the infection by T.
cruzi parasites. For this, the antitrypanosomal activity was tested on
intracellular amastigotes of T. cruzi (Tulahuen strain transfected with
β‐galactosidase gene, donated by Dr. F. S. Buckner, University of
96‐well microtiter plate. After freeze–thaw cycles, culture in each of
the wells of the first plate was resuspended by pipetting and 15 μl of
each well was taken and added to the corresponding well of the sec-
ond plate (containing Malstat and NBT/PES reagents). After an hour of
incubation in the dark, color development of the LDH reaction was
monitored colorimetrically in a spectrofluorometer (Varioskan; Ther-
mo Fisher Scientific) reading at 650 nm. The intensity of color in each
experimental condition was registered as OD. Nonspecific absorbance
was corrected by subtracting the OD of the blank. Determinations
[
68]
Washington).
For this purpose, 100 μl of U‐937 human cells at a
5
concentration of 2.5 × 10 cells/ml in RPMI‐1640, 10% fetal bovine
serum, and 0.1 μg/ml of PMA were placed in each well of a 96‐well
microplate. Cells were then infected with epimastigotes (24 hr of
growing) in 5:1 parasites per cell relation. Infected cells were in-
cubated with each compound at four concentrations (50, 12.5, 3.125,
and 0.78 μg/ml) using benznidazole as trypanocidal control. After
[67]
were done by triplicate in at least two independent experiments.
72 hr of incubation, the effect of all compounds on intracellular
Antileishmanial activity
amastigotes viability was determined by measuring the
β‐galactosidase activity by colorimetric method. For this assay, CPRG
at 100 μM and Nonidet P‐40 at 0.1% were added, and after 3 hr of
Antileishmanial activity of compounds was determined according to
the ability of the compound to reduce the infection by L. panamensis

22 of 24
CUARTAS ET AL.
|
incubation, measurement was performed at 570 nm in a spectro-
photometer (Varioskan; Thermo Fisher Scientific) and intensity of
color (absorbance) was registered as OD. Infected cells exposed to
control drugs (benznidazole) were used as a control for anti-
trypanosomal activity (positive control), whereas infected cells in-
cubated in the absence of any compound or drug were used as
control for infection (negative control). Nonspecific absorbance was
corrected by subtracting the absorbance (OD) of the blank. De-
terminations were done by triplicate in at least two independent
concentration of compound tested were calculated by the following
equation, where the % of infected cells in the control well corre-
sponds to 100% of parasitemia.
%
Infected cells in compound exposed well
%
Infection =
× 100.
%
Infected cells in control well
(
(
5)
6)
Then, the inhibition percentage was calculated with formula:
[
69]
Inhibition (%) = 100 − Parasitemia(%).
experiments.
Statistical analysis
For trypanosomal activity, the parasitemia percentage
Cytotoxicity was determined according to viability and mortality
percentages obtained for each experimental condition (synthesized
compounds, amphotericin B, benznidazole, chloroquine and culture
medium). Results were expressed as mean lethal concentrations
and parasitemia inhibition were calculated by Equation (5) and
Formula (6).
Results of antileishmanial and antitrypanosomal activities were
expressed as being half of the maximal effective concentrations
(EC50) measured by the Probit method. The activity of each com-
pound was established according to EC50 values, using the following
scale: Active: EC50 < 50 μg/ml; moderately active: EC50 > 50 μg/ml
and <100 μg/ml, and potentially not active: EC50 > 100 μg/ml.
SI was calculated by dividing the cytotoxic activity and the
plasmodium, leishmanicidal, or trypanocidal activities using the fol-
lowing equation:
(LC50), concentration necessary to kill 50% of cells, calculated by
Probit analysis (parametric method of linear regression that permits
[
70]
dose–response analysis).
Initially, viability percentages were calculated by the following
equation, where the OD of control well corresponds to 100% of
viability. In turn, the mortality percentage corresponds to 100% – %
viability.
OD Exposed cells
OD Control cells
LC50
EC50
(1)
(7)
%
Viability =
× 100.
SI =
.
Then, the percentage of cell growth inhibition was calculated
using the following equation:
ACKNOWLEDGMENTS
The authors thank the Universidad del Valle, COLCIENCIAS‐
10680864255 and the Science, Technology and Innovation Fund‐
(2)
%
Cell growth inhibition = 100% Viability.
1
General Royalties System (FCTeI‐SGR) under contract BPIN
2013000100007, Colombia; Universidad de Jaén, Spain, and the
Consejería de Innovación, Ciencia y Empresa (Junta de Andalucía,
Spain) for financial support. SZ and MS acknowledge the Agencia
Nacional de Promoción Científica y Tecnológica (ANPCyT) PICT2016‐
1833 and National University of Rosario Argentina (UNR) for funds.
MS is a member of the CONICET Researcher career. Technical and
The toxicity was defined according to LC50 values, using the
following scale: Toxic: LC50 < 100 μM, moderately toxic: LC50 > 100
μg/ml and <200 μM, and potentially nontoxic: LC50 > 200 μM.
The antiplasmodium activity of each evaluated compound was
evidenced by the reduction of the absorbance (OD). Indeed, the
viability percentage was calculated by the following equation:
OD of parasites exposed to compounds − OD of culture medium
OD of parasites unexposed to compounds − OD of culture medium
(3)
Viability (%) =
× 100.
Then, the inhibition growing percentage was calculated accord-
ing to the following formula:
human support provided by CICT of Universidad de Jaén (UJA,
MINECO, Junta de Andalucía, FEDER) is gratefully acknowledged.
(4)
Inhibition(%) = 100 − Viability(%).
CONFLICT OF INTERESTS
The authors declare that there are no conflicts of interests.
The inhibitory concentration (IC50), that is, the concentration of
the compound that produces a 50% reduction of growth of the
parasite, was determined by the statistical program Probit analysis.
Antileishmanial activity was determined according to the re-
duction of infected cells percentage obtained for each experimental
condition by the cytometer. The parasitemia values for each
ORCID
Viviana Cuartas
Sara M. Robledo
http://orcid.org/0000-0003-3776-9641
http://orcid.org/0000-0003-2752-4931
María del Pilar Crespo
Maximiliano Sortino
http://orcid.org/0000-0003-2160-5167
http://orcid.org/0000-0002-4271-1690

CUARTAS ET AL.
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Manuel Nogueras
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http://orcid.org/0000-0001-5976-0387
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