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Journal of the Brazilian Chemical Society

Print version ISSN 0103-5053On-line version ISSN 1678-4790

J. Braz. Chem. Soc. vol.28 no.2 São Paulo Feb. 2017

https://doi.org/10.5935/0103-5053.20160166 

Articles

Ultrasound-Promoted Synthesis of 3-(Thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboximidamides and Anticancer Activity Evaluation in Leukemia Cell Lines

Eric F. S. dos Santosa 

Nathália M. Curyb  c 

Tainara A. do Nascimentoa 

Cristiano Raminellid 

Gleison A. Casagrandea 

Claudio M. P. Pereirae 

Euclésio Simionattof 

José A. Yunesb  c 

Lucas Pizzuti*  a 

aGrupo de Pesquisa em Síntese e Caracterização Molecular do MS, Universidade Federal da Grande Dourados, Rua João Rosa Góes, 1761, 79825-070 Dourados-MS, Brazil

bDepartamento de Genética e Biologia Molecular, Instituto de Biologia, Universidade Estadual de Campinas, Rua Monteiro Lobato, 255, 13083-970 Campinas-SP, Brazil

cLaboratório de Biologia Molecular, Centro Infantil Boldrini, Rua Dr. Gabriel Porto, 1270, 13083-210 Campinas-SP, Brazil

dDepartamento de Ciências Exatas e da Terra, Universidade Federal de São Paulo, Rua Prof. Artur Riedel, 275, 09972-270 Diadema-SP, Brazil

eLaboratório de Lipidômica e Bio-Orgânica, Centro de Ciências Químicas, Farmacêuticas e dos Alimentos, Universidade Federal de Pelotas, Campus Universitário, 96001-900 Pelotas-RS, Brazil

fUniversidade Estadual de Mato Grosso do Sul, Rua Emilio Mascolli, 275, 79950-000 Naviraí-MS, Brazil


ABSTRACT

3-(Thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboximidamides were efficiently prepared through a cyclocondensation of thiophenylchalcones with aminoguanidine hydrochloride under ultrasonic conditions in the presence of KOH and ethanol as a green solvent in short reaction times (15-35 min) and good yields (62-95%). All compounds produced were evaluated against the human Jurkat and RS4;11 acute lymphoblastic leukemia cell lines of T- and B-cell origin, respectively, and the K562 myelogenous leukemia cell line. Six compounds presented half maximal inhibitory concentration (IC50) values around 15 µmol L-1 and five compounds presented IC50 values around 40 µmol L-1 for at least one of the three cell lines analyzed. One compound was not significantly cytotoxic, presenting IC50 value > 100 µmol L-1.

Keywords: amidinopyrazole; pyrazoline; cytotoxic activity; leukemia; ultrasonic irradiation

Introduction

Amidine functional group has proven to be an important fragment in compounds with recognized bioactivities. In this sense, several amidines have been prepared and their antifungal,1 antiprotozoal,2 antibacterial,3 anti-HIV,4 antithrombotic,5 and antidegenerative6 potentials have been evaluated. Pentamidine is clinically used for treatment of pneumonia and first stage human African tripanosomiasis.7 That drug is on the WHO's List of Essential Medicines for the basic health system.8 Moreover, amidine containing molecules have been pointed as proeminent prototypes in the search for new anticancer agents.9 In addition, thiophene-2-carboximidamides have shown potent and selective inhibitory activities of nitric oxide synthases for the treatment of human melanoma.10

In the same context, pyrazoles are recognized as a fundamental class of heterocyclic compounds because of their well-established applicability in several areas as agrochemicals, functional materials and medicines.11 Dihydro-1H-pyrazole derivatives have been reported as antiproliferative agents12 and inhibitors of vascular endothelium growth factors,13 mitotic kinesin spindle protein (KSP),14 and multidrug resistance protein 1 (MDR1).15 Pyrazoline derivatives have shown high selectivity against leukemia cell lines (the concentration for 50% of maximal inhibition of cell proliferation, GI50 = 0.69-3.35 µmol L-1) in comparison with eight other tumor cell lines.16

Taking into account the valuable pharmacological properties of pyrazoline and amidine scaffolds, we envisioned that hybrid molecules could be active against leukemia cells. Analogous 4,5-dihydro-1H-pyrazole-1-carboximidamides have been already prepared by the cyclocondensation of α,β-unsaturated ketones with aminoguanidine free base under conventional17 and sonochemical18 conditions. However, to the best of our knowledge there are not reported studies about the antileukemic potency of these hybrid molecules.

Thus, in our continuous efforts to develop sonochemically promoted reactions in environmentally benign solvents19 and synthetic methodologies for preparation of heterocyclic compounds20 selected on the basis of their biological activity,21 we describe herein a rapid and efficient synthetic method for the preparation of 3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboximidamide hydrochlorides under ultrasonic conditions. In addition, all compounds synthetized were evaluated against the human Jurkat and RS4;11 acute lymphoblastic leukemia cell lines of T- and B-cell origin, respectively, and the K562 myelogenous leukemia cell line.

Experimental

Chemistry

General

3-Aryl-1-(thiophen-2-yl)prop-2-en-1-ones 1a-l were prepared by us following reported procedures.22 All the chemicals were used without purification as purchased from commercial suppliers. The sonicated reactions were carried out with a microtip probe connected to a 500 W Sonics Vibracell ultrasonic processor operating at 20 kHz at 20% of the maximum power output. Reaction progresses were monitored by gas chromatography (GC). Melting point values were determined in open capillary on an Instrutherm DF-3600 II apparatus and are uncorrected. Infrared spectra (IR) were acquired on a JASCO-4100 spectrophotometer as KBr pellets. 1H and 13C nuclear magnetic resonance (NMR) spectra were acquired on a Bruker DPX400 instrument (400 MHz for 1H and 101 MHz for 13C) in 5 mm sample tubes at 298 K in dimethyl sulfoxide (DMSO-d6) using tetramethylsilane (TMS) as internal reference standard. High resolution mass spectra were recorded on a Bruker microTof (Q-TOF) mass spectrometer in electrospray ionization (ESI) mode.

General procedure for the ultrasound-promoted synthesis of 5-aryl-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamides (2a-l)

To a 50 mL vial containing a solution of 3-aryl-1-(thiophen-2-yl)prop-2-en-1-ones 1a-l (1 mmol) in ethanol (15 mL), the aminoguanidine hydrochloride (0.22 g, 2 mmol) and KOH (0.11 g, 2 mmol) were added. The reaction mixture was sonicated for the time indicated in Table 1 and the reaction temperature reached 55-60 ºC after 10 minutes. The resulting solution was cooled to room temperature and acidified using 10% HCl (10-15 mL). The salts were extracted with chloroform (3 × 20 mL) and the combined organic layer was dried over anhydrous magnesium sulfate. Removal of solvent under vacuum afforded a crude material. The pure products 2a-l were obtained as amorphous solids with yields of 62-95% after recrystallization from ethyl acetate and drying in a desiccator.

Table 1 Cyclocondensation reactions between 1a-l and aminoguanidine hydrochloride in the presence of KOH for the preparation of carboximidamides 2a-l 

entry Product R1 R2 R3 R4 timea / min Yieldb / %
1 2a H H H H 20 88
2 2b OMe H H H 25 80
3 2c Br H H H 30 70
4 2d H NO2 H H 15 65
5 2e H H Me H 35 80
6 2f H H CF3 H 15 62
7 2g H H OMe H 25 86
8 2h H H F H 30 73
9 2i H H Cl H 30 71
10 2j H H Br H 30 75
11 2k Cl H Cl H 30 66
12 2l H OMe OMe H 25 83

aSonication time;

byields of the isolated compounds.

5-Phenyl-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2a)

Yellowish solid; mp 266-268 ºC; IR (KBr) ν / cm-1 3337, 3115, 1609, 1430; 1H NMR (400 MHz, DMSO-d6) δ 7.97 (bs, 4H, C(NH2)2), 7.86 (d, 1H, J 5.0 Hz, T-H*), 7.57 (d, 1H, J 3.6 Hz, T-H), 7.39 (d, 2H, J 7.6 Hz, Ph-H), 7.35-7.32 (m, 1H, Ph-H), 7.25 (d, 2H, J 7.4 Hz, Ph-H), 7.20-7.17 (m, 1H, T-H), 6.02 (dd, 1H, J 2.4, 11.0 Hz, Hx†), 4.11 (dd, 1H, J 11.2, 17.8 Hz, Hm†), 3.36 (dd, 1H, J 2.7, 17.8 Hz, Ha†); 13C NMR (101 MHz, DMSO-d6) δ 153.2, 153.0, 139.6, 132.7, 132.0, 131.3, 129.0, 128.2, 125.3, 60.4, 44.4; HRMS m/z, [M + H]+ calcd. for C14H15N4S: 271.1017; found: 271.1015. *T-H: thiophene hydrogens; Ha, Hm and Hx: pyrazolyl ring hydrogens.

5-(2-Methoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2b)

Yellowish solid; mp 287-288 ºC; IR (KBr) ν / cm-1 3255, 3190, 3103, 1618, 1423, 1243; 1H NMR (400 MHz, DMSO-d6) δ 7.85 (bs, 4H, C(NH2)2), 7.82 (dd, 1H, J 0.9, 5.0 Hz, T-H), 7.54 (dd, 1H, J 0.8, 3.6 Hz, T-H), 7.34 (m, 1H, Ph-H), 7.16 (dd, 1H, J 3.8, 5.0 Hz, T-H), 7.11 (d, 1H, J 8.2 Hz, Ph-H), 6.97-6.89 (m, 2H, Ph-H), 5.87 (dd, 1H, J 3.2, 11.1 Hz, Hx), 4.04 (dd, 1H, J 11.1, 17.7 Hz, Hm), 3.83 (s, 3H, Ph-OCH3), 3.24 (dd, 1H, J 3.2, 17.7 Hz, Ha); 13C NMR (101 MHz, DMSO-d6) δ 156.2, 153.8, 152.9, 132.8, 131.8, 131.1, 129.6, 128.2, 126.3, 124.9, 120.3, 111.8, 57.2, 55.7, 43.3; HRMS m/z, [M + H]+ calcd. for C15H17N4OS: 301.1123; found: 301.1148.

5-(2-Bromophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2c)

Yellowish solid; mp 285-288 ºC; IR (KBr) ν / cm-1 3290, 3117, 1603, 1423; 1H NMR (400 MHz, DMSO-d6) δ 8.03 (bs, 4H, C(NH2)2), 7.86 (d, 1H, J 4.9 Hz, T-H), 7.57 (d, 1H, J 3.4 Hz, T-H), 7.55 (d, 1H, J 8.1 Hz, Ph-H), 7.48 (m, 1H, Ph-H), 7.38 (t, 1H, J 7.9 Hz, Ph-H), 7.22-7.18 (m, 2H, T-H and Ph-H), 6.04 (dd, 1H, J 2.8, 11.2 Hz, Hx), 4.11 (dd, 1H, J 11.2, 17.9 Hz, Hm), 3.42 (dd, 1H, J 2.9, 17.9 Hz, Ha); 13C NMR (101 MHz, DMSO-d6) δ 153.2, 153.1, 135.2, 133.4, 132.6, 132.3, 132.0, 131.3, 129.8, 128.1, 127.6, 126.7, 58.6, 43.2; HRMS m/z, [M + H]+ calcd. for C14H14BrN4S: 349.0122; found: 349.0129.

5-(3-Nitrophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2d)

Yellowish solid; mp 288-291 ºC; IR (KBr) ν / cm-1 3363, 3284, 3204, 3103, 1611, 1524, 1423, 1344, 847; 1H NMR (400 MHz, DMSO-d6) δ 8.22 (ddd, 1H, J 0.9, 2.2, 8.2 Hz, Ph-H), 8.10 (t, 1H, J 1.9 Hz, Ph-H), 7.97 (s, 4H, C(NH2)2), 7.87 (dd, 1H, J 1.1, 5.0 Hz, T-H), 7.74 (t, 1H, J 8.0 Hz, Ph-H), 7.65 (d, 1H, J 7.9 Hz, Ph-H), 7.55 (dd, 1H, J 1.1, 3.7 Hz, T-H), 7.20 (dd, 1H, J 3.7, 5.0 Hz, T-H), 6.07 (dd, 1H, J 3.2, 11.3 Hz, Hx), 4.16 (dd, 1H, J 11.4, 18.0 Hz, Hm), 3.48 (dd, 1H, J 3.2, 18.0, Ha); 13C NMR (101 MHz, DMSO-d6) δ 153.2, 152.8, 148.0, 141.5, 132.5, 132.2, 132.0, 131.5, 130.9, 128.2, 123.2, 120.7, 59.6, 44.3; HRMS m/z, [M + H]+ calcd. for C14H14N5O2S: 316.0868; found: 316.0878.

5-(4-Methylphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2e)

Yellowish solid; mp 278-280 ºC; IR (KBr) ν / cm-1 3255, 3096, 1611, 1430; 1H NMR (400 MHz, DMSO-d6) δ 7.91 (bs, 4H, C(NH2)2), 7.84 (d, 1H, J 5.0 Hz, T-H), 7.56 (d, 1H, J 3.6 Hz, T-H), 7.21-7.17 (m, 3H, T-H and Ph-H), 7.13 (d, 2H, J 8.1 Hz, Ph-H), 5.92 (dd, 1H, J 3.0, 11.1 Hz, Hx), 4.07 (dd, 1H, J 11.2, 17.9 Hz, Hm), 3.33 (dd, 1H, J 3.0, 17.7 Hz, Ha), 2.28 (s, 3H, Ph-CH3); 13C NMR (101 MHz, DMSO-d6) δ 153.1, 153.0, 137.6, 136.6, 132.8, 131.9, 131.2, 129.5, 128.2, 125.3, 60.2, 44.3, 20.6; HRMS m/z, [M + H]+ calcd. for C15H17N4S: 285.1174; found: 285.1179.

5-(4-Trifluoromethylphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2f)

Yellowish solid; mp 295-298 ºC; IR (KBr) ν / cm-1 3370, 3290, 3068, 1603, 1423, 1315-1113, 839; 1H NMR (400 MHz, DMSO-d6) δ 8.14 (bs, 4H, C(NH2)2), 7.88 (d, 1H, J 4.7 Hz, T-H), 7.79 (d, 2H, J 8.2 Hz, Ph-H), 7.60 (d, 1H, J 3.1 Hz, T-H), 7.52 (d, 2H, J 8.1 Hz, Ph-H), 7.21-7.19 (m, 1H, T-H), 6.27 (dd, 1H, J 2.4, 11.0 Hz, Hx), 4.19 (dd, 1H, J 11.3, 17.8 Hz, Hm), 3.45 (dd, 1H, J 2.8, 17.9 Hz, Ha); 13C NMR (101 MHz, DMSO-d6) δ 153.1, 153.0, 143.9 (q, J 1.3 Hz), 132.5, 132.0, 131.3, 128.7 (q, J 32.0 Hz), 128.1, 126.3, 125.8 (q, J 3.8 Hz), 123.9 (q, J 272.1 Hz), 60.0, 44.2; HRMS m/z, [M + H]+ calcd. for C15H14F3N4S: 339.0891; found: 339.0881.

5-(4-Methoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2g)

Yellowish solid; mp 296-299°C; IR (KBr) ν / cm-1 3334-3132, 1611, 1438, 1243, 839; 1H NMR (400 MHz, DMSO-d6) δ 7.85 (dd, 1H, J 1.0, 5.0 Hz, T-H), 7.76 (bs, 4H, C(NH2)2), 7.56 (dd, 1H, J 1.1, 3.7 Hz, T-H), 7.19 (dd, 1H, J 3.7, 5.0 Hz, T-H), 7.16-7.09 (m, 2H, Ph-H), 7.01-6.89 (m, 2H, Ph-H), 5.79 (dd, 1H, J 2.4, 11.0 Hz, Hx), 4.05 (dd, 1H, J 11.1, 17.8 Hz, Hm), 3.73 (s, 3H, Ph-OCH3), 3.41-3.26 (m, 1H, Ha); 13C NMR (101 MHz, DMSO-d6) δ 159.2, 153.3, 152.7, 132.8, 132.0, 131.5, 131.3, 128.2, 126.7, 114.4, 60.0, 55.2, 44.4; HRMS m/z, [M + H]+ calcd. for C15H17N4OS: 301.1123; found: 301.1132.

5-(4-Fluorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2h)

Yellowish solid; mp 243-245 ºC; IR (KBr) ν / cm-1 3348-3103, 1596, 1416, 1229, 832; 1H NMR (400 MHz, DMSO-d6) δ 7.93 (s, 4H, C(NH2)2), 7.86 (dd, 1H, J 1.1, 5.0 Hz, T-H), 7.57 (dd, 1H, J 1.1, 3.7 Hz, T-H), 7.33-7.21 (m, 4H, Ph-H), 7.19 (dd, 1H, J 3.7, 5.0 Hz, T-H), 5.94 (dd, 1H, J 2.9, 11.2 Hz, Hx), 4.09 (dd, 1H, J 11.2, 17.9 Hz, Hm), 3.53-3.27 (m, 1H, Ha); 13C NMR (101 MHz, DMSO-d6) δ 163.1, 160.6, 153.0 (d, J 34.2 Hz), 135.8 (d, J 2.7 Hz), 132.7, 132.1, 131.4, 128.2, 127.6 (d, J 8.3 Hz), 115.9 (d, J 21.7 Hz), 59.7, 44.4; HRMS m/z, [M + H]+ calcd. for C14H14FN4S: 289.0923; found: 289.0936.

5-(4-Chlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2i)

Yellowish solid; mp 257-260 ºC; IR (KBr) ν / cm-1 3070, 1606, 1425, 825; 1H NMR (400 MHz, DMSO-d6) δ 7.98 (bs, 4H, C(NH2)2), 7.85 (d, 1H, J 4.8 Hz, T-H), 7.56 (d, 1H, J 3.1 Hz, T-H), 7.47 (d, 2H, J 8.2 Hz, Ph-H), 7.27 (d, 2H, J 8.3 Hz, Ph-H), 7.20-7.18 (m, 1H, T-H), 6.01-5.99 (m, 1H, Hx), 4.10 (dd, 1H, J 11.2, 17.8 Hz, Hm), 3.40-3.35 (m, 1H, Ha); 13C NMR (101 MHz, DMSO-d6) δ 153.2, 152.9, 138.5, 132.9, 132.6, 132.1, 131.4, 129.0, 128.2, 127.4, 59.8, 44.3; HRMS m/z, [M + H]+ calcd. for C14H14ClN4S: 305.0628; found: 305.0629.

5-(4-Bromophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2j)

Yellowish solid; mp 265-266 ºC; IR (KBr) ν / cm-1 3269, 3190, 3103, 1611, 1430; 1H NMR (400 MHz, DMSO-d6) δ 8.01 (bs, 4H, C(NH2)2), 7.85 (d, 1H, J 5.0 Hz, T-H), 7.60 (d, 2H, J 8.3 Hz, Ph-H), 7.56 (d, 1H, J 3.6 Hz, T-H), 7.22 (d, 2H, J 8.4 Hz, Ph-H), 7.20-7.18 (m, 1H, T-H), 6.03 (dd, 1H, J 2.6, 11.1 Hz, Hx), 4.11 (dd, 1H, J 11.3, 17.8 Hz, Hm), 3.38 (dd, 1H, J 2.7, 17.8 Hz, Ha); 13C NMR (101 MHz, DMSO-d6) δ 153.0, 152.9, 138.8, 132.5, 131.9, 131.8, 131.2, 128.1, 127.6, 121.3, 59.7, 44.1; HRMS m/z, [M + H]+ calcd. for C14H15N4SBr: 349.0122; found: 349.0141.

5-(2,4-Dichlorophenyl)-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2k)

Yellowish solid; mp 290-293 ºC; IR (KBr) ν / cm-1 3334, 3175, 3103, 1611, 1423, 825; 1H NMR (400 MHz, DMSO-d6) δ 8.00 (bs, 4H, C(NH2)2), 7.85 (dd, 1H, J 1.1, 5.0 Hz, T-H), 7.56 (dd, 1H, J 1.1, 3.7 Hz, T-H), 7.55-7.53 (m, 1H, Ph-H), 7.48-7.47 (m, 1H, Ph-H), 7.38 (t, 1H, J 7.9 Hz, Ph-H), 7.21-7.18 (m, 2H, T-H and Ph-H), 6.02 (dd, 1H, J 3.1, 11.2 Hz, Hx), 4.10 (dd, 1H, J 11.3, 17.9 Hz, Hm), 3.42 (dd, 1H, J 3.1, 17.9 Hz, Ha); 13C NMR (101 MHz, DMSO-d6) δ 153.1, 152.9, 142.0, 132.5, 132.0, 131.2, 131.2, 131.0, 128.4, 128.1, 124.1, 121.9, 59.6, 44.2; HRMS m/z, [M + H]+ calcd. for C14H13Cl2N4S: 339.0238; found: 339.0237.

5-(3,4-Dimethoxyphenyl)-3-(thiophen-2-yl)-4,5-dihydro-1 H-pyrazole-1-carboximidamide hydrochloride (2l)

Yellowish solid; mp 287-291 ºC; IR (KBr) ν / cm-1 3298, 3139, 2830, 1618, 1430; 1H NMR (400 MHz, DMSO-d6) δ 7.84 (dd, 1H, J 1.0, 5.0 Hz, T-H), 7.80 (bs, 4H, C(NH2)2), 7.56 (dd, 1H, J 1.0, 3.6 Hz, T-H), 7.19 (dd, 1H, J 3.7, 5.0 Hz, T-H), 6.96 (d, 1H, J 8.3 Hz, Ph-H), 6.92 (d, 1H, J 1.9 Hz, Ph-H), 6.67 (dd, 1H, J 1.9, 8.3 Hz, Ph-H), 5.78 (dd, 1H, J 2.9, 11.1 Hz, Hx), 4.05 (dd, 1H, J 11.1, 17.8 Hz, Hm), 3.75 (s, 3H, Ph-OCH3), 3.73 (s, 3H, Ph-OCH3), 3.40-3.36 (m, 1H, Ha); 13C NMR (101 MHz, DMSO-d6) δ 153.2, 152.8, 148.9, 148.7, 132.8, 131.8, 131.8, 131.8, 131.1, 128.2, 116.9, 112.3, 110.1, 60.3, 55.6, 44.4; HRMS m/z, [M + H]+ calcd. for C16H19N4O2S: 331.1229; found: 331.1252.

In vitro citotoxicity assay (MTT assay)

In vitro cytotoxicity assay was carried out on three different human leukemia cell lines: K562, erythroleukemia cells bearing the t(9;22)(q34;q11)-derived BCR/ABL1 fusion gene; Jurkat, T-cells acute lymphoblastic leukemia (ALL) cells; and RS4;11, B-cell precursor ALL cells bearing the t(4;11)(q21;q23)-derived KMT2A/AFF1 fusion gene. The cells were maintained in RPMI-1640 (Cultilab) supplemented with 10% fetal bovine serum (FBS; Cultilab) and penicillin/streptomycin, at 37 °C and 5% CO2. For the cytotoxicity assay, compounds 2a-l were dissolved in DMSO in order to obtain a stock solution of 20 mmol L-1. Further dilutions were made in complete culture medium immediately before use. K562 and Jurkat cells were seeded at 3 × 104 cells per well while RS4;11 cells were seeded at 4 × 104 cells per well in a 96-well plate . After that, 20 µL of 10-fold serial dilutions of compounds 2a-l or vehicle (DMSO at a final concentration of 0.5%) were added to each well, in triplicate, at final concentrations of 0.1, 1.0, 10 and 100 µmol L-1. Culture plates were kept at 37 °C and 5% CO2 for 48 h, then, cell viability was measured by adding 20 µL of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent (Sigma-Aldrich) at 5 mg mL-1. After 4 h, the precipitated formazan crystals were dissolved by the addition of 100 µL of an acid sodium dodecyl sulfate solution (10% SDS, 0.01 mol L-1 HCl). Following overnight incubation, absorbance was measured at 570 nm for the MTT reaction and 620 nm as reference on scanning. Viability was calculated as a percentage of viable cells at different test concentrations relative to the control (vehicle-treated) cells. The concentration of compounds 2a-l that resulted in 50% inhibition of cell growth was calculated as the half maximal inhibitory concentration (IC50) by constructing a dose-response curve using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA).

Results and Discussion

Chemistry

The 4,5-dihydro-1H-pyrazole-1-carboximidamides (2a-l) were synthesized in good yields by the ultrasound-assisted cyclocondensation reaction between compounds 1a-l, prepared as described in literature,22 and aminoguanidine hydrochloride in the presence of KOH using ethanol as a green solvent (Table 1). The time required for the completion of the reaction depends on the nature of the groups attached to the benzene ring and it was determined by monitoring de consumption of the carbonyl compound by gas chromatography (GC) in intervals of 5 minutes. After the total consumption of the starting materials 1a-l the reaction solution was acidified with HCl in order to obtain the salts of the products. As showed in Table 1, the reaction tolerate electron-withdrawing and electron-donating groups. In general, substrates that contain electron-withdrawing substituents such as 3-NO2 (Table 1, entry 4) and 4-CF3 (Table 1, entry 6) gave products in shorter reaction times but in lower yields.

The structure of the carboximidamides were confirmed by IR, 1H and 13C NMR and HRMS. The IR spectra showed sets of absorption bands in accordance with the proposed structures. In the 1H NMR spectra of the compounds, the characteristic signals of the AMX coupling system between pyrazolyl ring hydrogens, Ha, Hm and Hx, were observed as sets of three doublets of doublets in the d 3.48-3.24, 4.19-4.04, and 6.27-5.78 ppm regions, respectively. Besides, the formation of the salts was confirmed by the presence of broad singlets in the range of d 8.14-7.76 ppm due to the presence of four equivalent hydrogens attached to nitrogens atoms in the carboximidamidyl moieties. The analysis of the 13C NMR and HRMS spectra also confirmed the structure of the products.

Although the mechanism of the cyclocondensation reaction between aminoguanidine and α,β-unsaturated ketones was not yet experimentally established, the literature shows a possible explanation based on the hard and soft acids and bases (HSAB) concept and quantum chemical studies on the aminoguanidine.17,23 Accordingly, a proposed mechanism is shown in Figure 1. Firstly, KOH neutralizes the aminoguanidine salt to give aminoguanidine in its free form, which can exist as two tautomers. Thus Aza-Michael-type addition from the internal imine nitrogen to the β-position of 1 leads to adduct I. In the next step, an intramolecular nucleophilic attack of the primary amino nitrogen to the carbonyl leads to intermediates II, which is dehydrated to give the product III. Finally, the addition of HCl produces the salt 2.

Figure 1 Proposed mechanism for the cyclocondensation reaction between 3-aryl-1-(thiophen-2-yl)prop-2-en-1-ones and aminoguanidine hydrochloride in the presence of KOH. 

In vitro evaluation of antileukemia activity

To gain insight on structure-activity relationship, twelve pyrazoline derivatives (2a-l) had their cytotoxic activity evaluated against three different human leukemia cell lines (Table 2). Compounds 2b, 2c, 2f, 2i, 2j and 2k showed the best cytotoxic activity, with IC50 value around 15 µmol L-1 for at least one of the three leukemia cell lines analyzed. Compounds 2a, 2d, 2e, 2g and 2h showed intermediate cytotoxic activity, with IC50 around 40 µmol L-1. Finally, compound 2l did not present a significant cytotoxic activity, with IC50 value >100 µmol L-1.

Table 2 Cytotoxic activity of compounds 2a-l against different leukemia cell lines 

entry Compound IC50
a / (µmol L-1)
Jurkat RS4;11 K562
1 2a 29.5 70.5 21.26
2 2b 10.2 29.4 5.9
3 2c 21.5 20.5 14.7
4 2d 29.5 46.4 33.4
5 2e 34.2 28.7 24.8
6 2f 24.7 17.3 17.4
7 2g 28.6 47.1 40.0
8 2h 42.4 51.5 36.2
9 2i 37.9 16.7 30.4
10 2j 22.0 14.4 23.7
11 2k 21.3 16.2 5.7
12 2l > 100 > 100 > 100

aThe drug concentration resulting in a 50% of maximal inhibition of cell proliferation (as measured by MTT staining).

The in vitro cytotoxic activity of pyrazolines is in the same range to that of several anti-leukemia drugs used in the clinic: 6-mercaptopurine (20.9 and 233.4 µmol L-1, median IC50 values for a panel of seven T-cell and fifteen B-cell precursor ALL cell lines, respectively), 6-thioguanine (4 and 11.8 µmol L-1), dexamethasone (82.7 and 0.19 µmol L-1), and methylprednisolone (38.6 and 69.8 µmol L-1).24 In conclusion, the cytotoxic activity of most pyrazolines is comparable to that of drugs used in leukemia treatment.

Conclusions

Ultrasound irradiation was efficient to promote the cyclocondensation reaction between thiophenylchalcones and aminoguanidine hydrochloride in the presence of KOH, furnishing a series of twelve 3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboximidamides in short reaction times (15-35 min) and good yields (62-95%). All compounds prepared were evaluated against the human Jurkat and RS4;11 acute lymphoblastic leukemia cell lines of T- and B-cell origin, respectively, and the K562 myelogenous leukemia cell line. Six compounds presented IC50 values around 15 µmol L-1 and five compounds presented IC50 values around 40 µmol L-1 for at least one of the three cell lines analyzed. One compound was not significantly cytotoxic presenting IC50 value > 100 µmol L-1.

Supplementary Information

Supplementary information (1H NMR, 13C NMR and HRMS spectra and dose-response curves) is available free of charge at http://jbcs.sbq.org.br as a PDF file.

0103-5053-jbchs-28-02-0217-suppl01.pdf

FAPESP has sponsored the publication of this article.

FAPERGS/CAPES has sponsored the publication of this article.

Acknowledgments

We acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant 483021/2013-0 for L. Pizzuti), Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT, grant 0180/12 for L. Pizzuti), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 12/12802-1 for J. A. Yunes) for financial support. We also thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and FAPESP for scholarships to E. F. S. dos Santos and N. M. Cury, respectively. J. A. Yunes thanks CNPq for the productivity fellowship.

References

1 Manetti, F.; Castagnolo, D.; Raffi, F.; Zizzari, A. T.; Rajamaki, S.; D'Arezzo, S.; Visca, P.; Cona, A.; Fracasso, M. E.; Doria, D.; Posteraro, B.; Sanguinetti, M.; Fadda, G.; Botta, M.; J. Med. Chem. 2009, 52, 7376. [ Links ]

2 dos Santos, M. S.; Gomes, A. O.; Bernardino, A. M. R.; de Souza, M. C.; Khan, M. A.; de Brito, M. A.; Castro, H. C.; Abreu, P. A.; Rodrigues, C. R.; de Léo, R. M. M.; Leon, L. L.; Canto-Cavalheiro, M. M.; J. Braz. Chem. Soc. 2011, 22, 352; Soeiro, M. N. C.; Werbovetz, K.; Boykin, D. W.; Wilson, W. D.; Wang, M. Z.; Hemphill, A.; Parasitology 2013, 140, 929. [ Links ]

3 Gobis, K.; Foks, H.; Wiśniewska, K.; Dąbrowska-Szponar, M.; Augustynowicz-Kopeć, E.; Napiórkowska, A.; Arch. Pharm. Chem. Life Sci. 2012, 345, 911; Nguyen, S. T.; Williams, J. D.; Butler, M. M.; Ding, X.; Mills, D. M.; Tashjian, T. F.; Panchal, R. G.; Weir, S. K.; Moon, C.; Kim, H. -O.; Marsden, J. A.; Peet, N. P.; Bowlin, T. L.; Bioorg. Med. Chem. Lett. 2014, 24, 3366; Stolić, I.; Paljetak, H. Č.; Perić, M.; Matijašić, M.; Stepanić, V.; Verbanac, D.; Bajić, M.; Eur. J. Med. Chem. 2015, 90, 68. [ Links ]

4 Echevarria, A.; Santos, L. H.; Miller, J.; Mahmood, N.; Bioorg. Med. Chem. Lett. 1996, 6, 1901. [ Links ]

5 Liebeschuetz, J. W.; Jones, S. D.; Morgan, P. J.; Murray, C. W.; Rimmer, A. D.; Roscoe, J. M. E.; Waszkowycz, B.; Welsh, P. M.; Wylie, W. A.; Young, S. C.; Martin, H.; Mahler, J.; Brady, L.; Wilkinson, K.; J. Med. Chem. 2002, 45, 1221. [ Links ]

6 Panico, A.; Vicini, P.; Incert, M.; Cardile, V.; Gentile, B.; Ronsisvalle, G.; Farmaco 2002, 57, 671; Vicini, P.; Incerti, M.; Cardile, V.; Garufi, F.; Ronsisvalle, S.; Panico, A. M.; ChemMedChem 2007, 2, 113; Woltering, T. J.; Wostl, W.; Hilpert, H.; Rogers-Evans, M.; Pinard, E.; Mayweg, A.; Göbel, M.; Banner, D. W.; Benz, J.; Travagli, M.; Pollastrini, M.; Marconi, G.; Gabellieri, E.; Guba, W.; Mauser, H.; Andreini, M.; Jacobsen, H.; Power, E.; Narquizian, R.; Bioorg. Med. Chem. Lett. 2013, 23, 4239; [ Links ]

7 Huang, T. L.; Eynde, J. J. V.; Mayence, A.; Collins, M. S.; Cushion, M. T.; Rattendi, D.; Londono, I.; Mazumder, L.; Bacchi, C. J.; Yarlett, N.; Bioorg. Med. Chem. Lett. 2009, 19, 5884. [ Links ]

8 http://www.who.int/medicines/publications/essentialmedicines/en/, accessed in May 2016. [ Links ]

9 Bhongade, B. A.; Gadad, A. K.; Bioorg. Med. Chem. 2004, 12, 2797; Özdemir, A.; Altıntop, M. D.; Kaplancıklı, Z. A.; Turan-Zitouni, G.; Çiftçi, G. A.; Yıldırım, Ş. U.; J. Enzyme Inhib. Med. Chem. 2013, 28, 1221; Houck, J. D.; Dawson, T. K.; Kennedy, A. J.; Kharel, Y.; Naimon, N. D.; Field, S. D.; Lynch, K. R.; Macdonald, T. L.; ACS Med. Chem. Lett., in press, DOI: 10.1021/acsmedchemlett.6b00002. [ Links ]

10 Huang, H.; Li, H.; Yang, S.; Chreifi, G.; Martásek, P.; Roman, L. J.; Meyskens, F. L.; Poulos, T. L.; Silverman, R. B.; J. Med. Chem. 2014, 57, 686. [ Links ]

11 Küçükgüzel, S. G.; Şenkardeş, S.; Eur. J. Med. Chem. 2015, 97, 786; Dias, D.; Pacheco, B. S.; Cunico, W.; Pizzuti, L.; Pereira, C. M. P.; Mini-Rev. Med. Chem. 2014, 14, 1078; Kumar, H.; Saini, D.; Jain, S.; Jain, N.; Eur. J. Med. Chem. 2013, 70, 248; Alex, J. M.; Kumar, R.; J. Enzyme Inhib. Med. Chem. 2014, 29, 427. [ Links ]

12 Johnson, M.; Younglove, B.; Lee, L.; LeBlanc, R.; Holt Jr., H.; Hills, P.; Mackay, H.; Brown, T.; Mooberry, S. L.; Lee, M.; Bioorg. Med. Chem. Lett. 2007, 17, 5897; Havrylyuk, D.; Zimenkovsky, B.; Vasylenko, O.; Zaprutko, L.; Gzella, A.; Lesyk, R.; Eur. J. Med. Chem. 2009, 44, 1396; Shaharyar, M.; Abdullah, M. M.; Bakht, M. A.; Majeed, J.; Eur. J. Med. Chem. 2010, 45, 114; Insuasty, B.; Tigreros, A.; Orozco, F.; Quiroga, J.; Abonıa, R.; Nogueras, M.; Sanchez, A.; Cobo, J.; Bioorg. Med. Chem. 2010, 18, 4965. [ Links ]

13 Kumar, S.; Bawa, S.; Drabu, S.; Kumar, R.; Gupta, H.; Recent Pat. Anti-Infect. Drug Discovery 2009, 4, 154. [ Links ]

14 Roecker, A. J.; Coleman, P. J.; Mercer, S. P.; Schreier, J. D.; Buser, C. A.; Walsh, E. S.; Hamilton, K.; Lobell, R. B.; Tao, W.; Diehl, R. E.; South, V. J.; Davide, J. P.; Kohl, N. E.; Yan, Y.; Kuo, L. C.; Li, C.; Fernandez-Metzler, C.; Mahan, E. A.; Prueksaritanont, T.; Hartman, G. D.; Bioorg. Med. Chem. Lett. 2007, 17, 5677. [ Links ]

15 Kobayashi, H.; Dorai, T.; Holand, J. F.; Ohnuma, T.; Cancer Res. 1994, 54, 1271; Manna, F.; Chimenti, F.; Bolasco, A.; Secci, D.; Bizzarri, B.; Befani, O.; Turini, P.; Mondovi, B.; Alcaro, S.; Tafi, A.; Bioorg. Med. Chem. Lett. 2002, 12, 3629. [ Links ]

16 Havrylyuk, D.; Kovach, N.; Zimenkovsky, B.; Vasylenko, O.; Lesyk, R.; Arch. Pharm. (Weinheim, Ger. ) 2011, 344, 514. [ Links ]

17 Světlík, J.; Šallai, L.; J. Heterocycl. Chem. 2002, 39, 363. [ Links ]

18 Pizzuti, L.; Martins, P. L. G.; Ribeiro, B. A.; Quina, F. H.; Pinto, E.; Flores, A. F. C.; Venzke, D.; Pereira, C. M. P.; Ultrason. Sonochem. 2010, 17, 33. [ Links ]

19 Ferreira, I. M.; Casagrande, G. A.; Pizzuti, L.; Raminelli, C.; Synth. Commun. 2014, 44, 2094; Franco, M. S. F.; Casagrande, G. A.; Raminelli, C.; Moura, S.; Rossatto, M.; Quina, F. H.; Pereira, C. M. P.; Flores, A. F. C.; Pizzuti, L.; Synth. Commun. 2015, 45, 692; Kuhn, B. P.; Malavolta, J. L.; Casagrande, G. A.; Raminelli, C.; Quina, F. H.; Pereira, C. M. P.; Flores, A. F. C.; Pizzuti, L.; J. Braz. Chem. Soc. 2015, 26, 1306. [ Links ]

20 Flores, A. F. C.; Pizzuti, L.; Piovesan, L. A.; Flores, D. C.; Malavolta, J. L.; Pereira, C. M. P.; Tetrahedron Lett. 2010, 51, 4908; Flores, A. F. C.; Piovesan, L. A.; Pizzuti, L.; Flores, D. C.; Malavolta, J. L.; Martins, M. A. P.; J. Heterocycl. Chem. 2014, 51, 733. [ Links ]

21 Silva, F. A. N.; Pizzuti, L.; Quina, F. H.; Souza, S. P.; Rosales, P. F.; Siqueira, G. M.; Pereira, C. P. M.; Barros, S. B. M.; Rivelli, D. P.; Lett. Drug Des. Discovery 2010, 7, 657; Oliveira, S.; Pizzuti, L.; Quina, F.; Flores, A.; Lund, R.; Lencina, C.; Pacheco, B. S.; Pereira, C. M. P.; Piva, E.; Molecules 2014, 19, 5806. [ Links ]

22 Li, R.; Kenyon, G. L.; Cohen, F. E.; Chen, X.; Gong, B.; Dominguez, J. N.; Davidson, E.; Kurzban, G.; Miller, R. E.; Nuzum, E. O.; Rosenthal, P. J.; McKerrow, J. H.; J. Med. Chem. 1995, 38, 5031. [ Links ]

23 Koskinena, J. T.; Koskinen, M.; Mutikainen, I.; Tilus, P.; Mannfors, B.; Elo, H.; Z. Naturforsch. 1997, 52B, 1259. [ Links ]

24 Beesley, A. H.; Palmer, M. L.; Ford, J.; Weller, R. E.; Cummings, A. J.; Freitas, J. R.; Firth, M. J.; Perera, K. U.; de Klerk, N. H.; Kees, U. R.; Br. J. Cancer 2006, 95, 1537. [ Links ]

Received: March 03, 2016; Accepted: March 31, 2016

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