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

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

Viviana Cuartas

| Sara M. Robledo

| Iván D. Vélez

|

4

5

María del Pilar Crespo

| Maximiliano Sortino

| Susana Zacchino

|

6

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 IC₅₀values 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.

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https://doi.org/10.1002/ardp.201900351

2

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CUARTAS ET AL.

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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.

3 of 24

|

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

4

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CUARTAS ET AL.

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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

CUARTAS ET AL.

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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.

|

appeared as a double‐doublet at 5.51 ppm with coupling constant

These results clearly show that all thiazolyl‐pyrazolines, 8a–g,

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

)

(

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

1

C. albicans ATCC 10231

C. neoformans ATCC 32264

6

a

b

c

d

e

f

4‐Cl

–COCH

3

250

4‐Br

>250

250

4‐F

>250

2

3,4‐OCH O–

4‐OCH

3

3,4,5‐(OCH

4‐H

3

)

3

g

7

a

b

c

d

e

f

4‐Cl

4‐Br

4‐F

–CHO

>250

250

>250

250

3,4‐OCH

2

O–

4‐OCH

3

3,4,5‐(OCH

4‐H

3

)

g

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

125

250

15.6

62.5

3,4‐OCH

4‐OCH

2

O–

31.25

125

3

3,4,5‐(OCH

4‐H

3

)

g

31.25

9

a

b

c

d

e

f

4‐Cl

4‐Br

4‐F

3,5‐Cl

2

C

6

H

3

H

3

H

3

H

3

H

3

H

3

H

3

>250

250

>250

250

7.8

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

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

80

60

40

20

0

80

60

40

20

0

8

a

b

c

9

a

9b

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)

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

125

100

62.5

100

31.25

100

15.62

100

7.81

3.9

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

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

b

c

d

e

f

>200

100.00

6

a

250

–

250

–

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

a

62.5

125

250

–

125

250

–

31.25

125

–

500

250

–

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

a

500

–

500

–

250

500

–

125

250

–

b

g

>200

867.03 ± 187.03

c–g

–

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

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

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

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

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

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

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

| 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

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

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.

–

g

–

41.64 ± 6.63

7

a

b

c

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

(

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

–Cl).

C NMR

−

1

(

3

Yellow solid; 90% yield; m.p. 148–149°C. FTIR (ATR) ν (cm ): 3,044

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

–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

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

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

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

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

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

–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

+

(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

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

4

d,

JC–F = 8.7 Hz, CH), 127.9 (d,

JC–F = 3.1Hz, C), 118.7 (C), 116.4

3

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

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

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

3

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

), 40.7 (CH ), 40.5 (C4′), and 21.7

+

(

[

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

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

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

3

2

13

+

(

3

2

(

1

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

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

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

+

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

+

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

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

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

C NMR

2

1

3

(100 MHz, CDCl

3

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

+

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

C NMR

3

2

(

100 MHz, CDCl

3

2

13

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

(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

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

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

H

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

), 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

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

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

.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

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.

23 of 24

|

Susana Zacchino

Manuel Nogueras

http://orcid.org/0000-0002-7112-5500

http://orcid.org/0000-0001-5976-0387

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24] B. Marvania, R. Kakadiya, W. Christian, T. L. Chen, M. H. Wu, S.

Justo Cobo

http://orcid.org/0000-0001-9184-2390

http://orcid.org/0000-0002-9348-6227

http://orcid.org/0000-0001-8371-3310

Suman, K. Tala, T. C. Lee, A. Shah, T. L. Su, Eur. J. Med. Chem. 2014, 83,

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Braulio Insuasty

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How to cite this article: Cuartas V, Robledo SM, Vélez ID,

et al. New thiazolyl‐pyrazoline derivatives bearing nitrogen

mustard as potential antimicrobial and antiprotozoal agents.

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