Synthesis, Purity Verification and Comparison of Antiplasmodial and Antitrypanosomal Activities of Hydrazone Derivatives and Corresponding Thiosemicarbazones

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Journal of Pharmaceutical, Chemical and Biological Sciences ISSN: 2348-7658 Impact Factor (GIF): 0.615 Impact Factor (SJIF): 2.092 June-August 2015; 3(2): 279-294 Available online at http://www.jpcbs.info

Original Research Article Synthesis, Purity Verification and Comparison of Antiplasmodial and Antitrypanosomal Activities of Hydrazone Derivatives and Corresponding Thiosemicarbazones

Bardieu Atchade1,2, Salomé D.S. Kpoviessi1,3*, Raymond H. Fatondji1, Léon A. Ahoussi1, Joachim Gbenou2, Georges C. Accrombessi1, Jacques H. Poupaert3, Mansourou Moudachirou2

1Laboratory of Physic and Synthesis Organic Chemistry (LaCOPS), Department of Chemistry, Faculty of Sciences and Technics, University of Abomey-Calavi, 01 PB : 4166 Cotonou, Benin 2Laboratory of Pharmacognosy and Essential Oils (LaPHE) Faculty of health Sciences (FSS), Faculty of Sciences and Technics (FAST) 01BP: 188 Cotonou, Benin 3 Louvain Drug Research Institute (LDRI), School of Pharmacy, Université catholique de Louvain, B1 7203 Avenue Emmanuel Mounier 72, B-1200 Brussels, Belgium

*Corresponding Author: Salomé D.S. Kpoviessi, Laboratory of Physic and Synthesis Organic Chemistry (LaCOPS), Department of Chemistry, Faculty of Sciences and Technics, University of Abomey-Calavi, 01 PB : 4166 Cotonou, Benin

Received: 22 July 2015 Revised: 31 July 2015 Accepted: 03 August 2015 ABSTRACT

Hydrazones and thiosemicarbazones are molecules that inhibit the development of several microbes and parasites. The present work investigates comparative study of the in vitro antitrypanosomal and antiplasmodial activity of hydrazones and thiosemicarbazones synthesized from the same ketones: S- (+)-carvone, 4'-methylacetophenone, 2-acétonaphthone and 7-methoxy-1-tetralone. A series of salicylhydrazones (1a-4a), of p-tosylhydrazones of (1b-4b) and of thiosemicarbazones (1c-4c) were synthesized with good yields (57-93%). Purity of synthesized compounds was checked by elemental and HPLC analysis and their structures were confirmed using spectrometric methods such as HRMS, 1H NMR and 13C. All of the compounds were evaluated for their antitrypanosomal and antiplasmodial activities on Trypanosoma brucei brucei and Plasmodium falciparum respectively and their selectivity was assessed by analyzing of their toxicity against Artemia salina Leach. Salicylhydrazones and

thiosemicarbazones were the most actives series on the studies parasites but salicylhydrazone compounds could be a most promising approach to the treatment of malaria and trypanosome.

Keyword: Synthesis, hydrazones, thiosemicarbazones, antitrypanosomal and antiplasmodial properties, toxicity

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INTRODUCTION MATERIAL AND METHODS The thiosemicarbazones and hydrazones are Chemistry derivatives of aldehydes or ketones. They are Melting points (m.p.) were determined on a small molecules (compared to peptides) with fusionometer of the type electrothermal 1A interesting biological activities, because they 9000 and were not corrected. For checking are many protease and DNA replication purity of synthesized compounds, we used a inhibitors’ *1-4]. Their inhibitory activity justifies Thermo Electron Corporation Flash EA 1112 the importance of the interest given to them as series analyzer equipped with a micro-balance part of the fight against microbial and parasitic Mettler Toledo MX5 and a logiciel : Eager 300. diseases. Molecules used to treat these We used also a Accela type (Thermo Fisher diseases have costs out of reach of poor and in Scientifique, Bremen, Germany) HPLC system some cases have high risk of toxicity [5]. equipped with a Phenomenex type C18 column. Thiosemicarbazones and hydrazones have All synthesized compounds were characterized biological properties: Antiviral [6,7], by Nuclear Magnetic Resonance spectra using antibacterial [8-10], antitumor [11-16], Bruker Avance 400 UltraSheild with anticonvulsant [17,18], antitrypanosomal [19] dimethylsufoxide (DMSO)-d6 or chloroform and antimalarial [20] etc. According to Eman et CDCl3 and the Mass spectra were acquired in al. [21], they served as models for the enzymes positive mode using an LTQ-Orbitrap XL hybride and were used as effective oxidants and (Thermo Fisher Scientifique, Bremen, Germany) catalysts [22,23]. They were not commercially equipped with an electrospray ionization ESI available and can only be obtained by synthesis. and high resolution mass was given in m/z of In addition, they were important compounds in [MH+]. the synthesis of drugs and the formation of The frequencies for 1H and 13C are 400.130 and complexes with metals [24]. 100.612 MHz respectively. Chemical shifts are Previous studies indicated antitrypanosomal given in parts per million (ppm) relative to and antiplasmodial properties of some tetramethylsilane as internal standard. thiosemicarbazones and hydrazones [19,25-26], Multiplicity was designated as singlet (s), but present work investigates comparative redoubled doublet (dd) triplet (t), quintuplet study of the in vitro antitrypanosomal and (qi) and multiplet (m). antiplasmodial activity of hydrazones and thiosemicarbazones synthesized from the same Reagents ketones in the same conditions. All reagents were obtained from chemical Salicylhydrazones and p-tosylhydrazones with societies: Sigma-Aldrich, Acros Organic, Janssen corresponding thiosemicarbazones, synthesized Chimica, Prolabo and Riedel-de Haen. from the same arylketones and S-(+)-carvone, Substrates, reagents, catalysts and solvents were their structures confirmed by different were used directly for syntheses without any spectrometric analysis methods after the further purification. They were: S-(+)-carvone, verification of their purity by elemental and 4'-methylacetophenone, 7-methoxy-1- HPLC analysis. They were evaluated for their tetralone, 2-acetylnaphthalene; glacial acetic antitrypanosomal and antiplasmodial activities acid, Technical ethanol (EtOH) and and their selectivity was assessed by analyzing salicylhydrazide. The para-toluenesulfonyl of their toxicity against Artemia salina Leach. hydrazide (p-tolylhydrazide) was prepared from the hydrazine monohydrate with the p-

J Pharm Chem Biol Sci, March-May 2015; 3(1):279-294 Koviessi et al 281 toluenesulfonyl chloride following the method Characterization of synthesized compounds described in the literature [27]. S-(+)-carvone salicylhydrazone (1a) O OH General procedure for the preparation of N N hydrazones and thiosemicarbazones. Synthesis of p-tolylhydrazide (p-toluenesol H fonylhydrazide)

Into a 1 L round-bottomed three-necked flask Yield : 78% ; m.p. : 189-190°C ; Rf (Hex/AcOEt, fitted with a thermometer, a mechanical stirrer, 13 v/v, 7/3) : 0.57 ; NMR C (DMSO-d6, δ in ppm) : and a dropping funnel was placed 200 g (1.05 161,78 (N-CO-Ar); 147.53 (C=N); 156.36 (C‒OH moles) of p-toluenesulfonyl chloride and 350 phenolic); 133.87, 130.45, 119.59, 117.95, mL of tetrahydrofuran. The stirred mixture is 116.77 (other C-Ar); 153.99, 133.16, 132.28, cooled in an ice bath to 10–15°C; then a 110.32, 40.10, 29.57, 29.45, 20.44, 17.85 (C- solution of hydrazine in water (135 mL of 85% 1 carvone). NMR H (DMSO-d6, δ in ppm) : 11.75 hydrazine hydrate, 2.22 moles) was added at (s, 1H, OH); 11.20 (s, 1H, NH); 7.97-6.95 (m, 4H, such a rate that the temperature was H-Ar); 6.25 (t, 1H, C=CH‒); 4.84 (d, 2H, C=CH2); maintained between 10° and 20°C. Stirring was 2.75 (qi, 1H, CH2-CH-CH2); 2.45 (t, 2H, C=CH- continued for 15 minutes after the addition was CH2-CH); 2.25 (d, 2H, HC-CH2-C=N); 1.90 (s, 3H, complete. The reaction mixture was transferred + CH3); 1.78 (s, 3H, CH3). MS m/z [MH ]found : to a separator funnel. The lower layer was 285.37 ; [M]theoretical : 284.35 ; Molecular drawn off, and discarded. The upper formula : C17H20N2O2. tetrahydrofuran layer was filtered by suction through a bed of Celite to remove suspended 4’-methylacetophenone salicylhydrazone (2a) particles and foreign matter (if any). The Celite H was washed with a little tetrahydrofurane to N remove any absorbed tosylhydrazide. The clear, N colorless filtrates are stirred vigorously during O OH the slow addition of two volumes of distilled water. p-toluenesulfonylhydrazide separates as Yield : 79% ; m.p. : 237-238°C ; Rf (Hex/AcOEt, 13 fluffy white crystalline needles. The product v/v, 7/3) : 0.63 ; NMR C (DMSO-d6, δ in ppm): was filtered through a Büchner funnel; washed 163.14 (N-CO-Ar); 153.51 (C=N); 157.67 (C‒OH several times with distilled water, and air-dried. phenolic); 140.18, 136.27, 134.47, 131.69, 130.12, 127.52, 120.82, 119.02, 118.01 (other Synthesis of salicylhydrazones C-Ar); 21.99 (H3C-Ar); 14.93 (CH3). 1 A salicylhydrazine solution (1.52 g in 10 mL of NMR H (DMSO-d6, δ in ppm): 11.80 (s, 1H, OH); ethanol) was prepared in a 100 mL flask in 11.30 (s, 1H, NH); 8.00-6.97 (m, 8H, H-Ar); 2.45 which was gradually add, a solution of ketone (s, 3H, H3C-Ar), 2.31 (s, 3H, CH3). MS m/z + (0.01 mole) dissolved in 10-40 ml of ethanol [MH ]found : 269.27 ; [M]theoretical : 268.31 ; and 2 mL of glacial acetic acid. The mixture was Molecular formula : C16H16N2O2. brought to reflux for 2 hours and the reaction was followed by TLC (Hex / AcOEt: 8/2 or 7/3). After cooling, the precipitate was filtered off, washed with distilled water and dried and then was recrystallized from technical ethanol.

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7-methoxy-1-tetralone salicylhydrazone (3a) in 10 mL ethanol was gradually added. The

H mixture was maintained at reflux for 2 hours

N and the reaction was followed by Thin Layer N Chromatography TLC (Hex / AcOEt: 8/2 or 7/3). O O OH The crystals formed were filtered, washed with distilled water and dried before being recrystallized from technic ethanol. Yield : 85% ; m.p. : 223-224°C ; Rf (Hex/AcOEt, 13 v/v, 7/3) : 0.47 ; NMR C (DMSO-d6, δ in ppm): Characterization of synthesized compounds 161.82 (N-CO-Ar); 151.75 (C=N); 157.64 (C‒OH S-(+)-carvone p-tosylhydrazone (1b) phenolic); 156.42 (CAr‒OCH3); 133.31, 133.04, O O 132.56, 130.57, 129.62, 119.69, 117.90, 116.83, N S N 116.35, 108.18 (other C-Ar); 55.10 (O-CH3); 1 H 27.92, 25.55, 21.53 (3s, 6H, 3“–CH2-”). NMR H

(DMSO-d6, δ in ppm): 11.80 (s, 1H, OH); 11.33 (s, 1H, NH); 8.00-6.90 (m, 7H, H-Ar); 3.80 (s, 3H, Yield : 57% ; m.p. : 167-168°C ; Rf (Hex/AcOEt, O-CH3); 3.35, 2.69, 1.89 (s, 6H, 3CH2). MS m/z v/v, 8/2) : 0.46 ; NMR 13C (CDCl , δ in ppm) : [MH+]found : 311.33 ; [M]theoretical : 310.34 ; 3 154.80 (C=N); 144.02, 132.46, 129.38, 128.26 Molecular formula : C18H18N2O3. (C-Ar); 147.11, 135.17, 133.59, 110.40, 40.36,

29.96, 29.09, 21.64, 17.65 (C-carvone); 20.65 2-acetynaphthalene salicylhydrazone (4a) 1 (H3C-Ar). NMR H (CDCl3, δ in ppm) : 7.90 (s, 1H, H NH); 7.65 & 7.30 (2s, 4H, H-Ar); 6.05 (t, 1H,

N C=CH‒); 4.75 (dd, 2H, C=CH2); 2.60 (m, 1H, CH2- N CH-CH2); 2.45 (t, 2H, C=CH-CH2-CH); 2.25 (d, 2H, OH O HC-CH2-C=N); 1.95 (m, 3H, CH3); 1.77 (s, 3H, + CH3); 1.70 (s, 3H, CH3). MS m/z [MH ]found :

Yield : 91% ; m.p. : 241-242°C ; Rf (Hex/AcOEt, 319.33 ; [M]theoretical : 318.43 ; Molecular 13 v/v, 7/3) : 0.33 ; NMR C (DMSO-d6, δ in ppm) : formula : C17H22N2O2S. 162.01 (N-CO-Ar); 151.83 (C=N); 156.47(C‒OH phenolic); 135.26, 133.39, 133.28, 133.05, 4’-methylacetophenone p-tosylhydrazone (2b) 132.74, 130.66, 128.54, 127.73, 127.48, 126.89, H 123.63, 119.72, 117.92, 116.87 (other C-Ar); N 13.60 (CH3). N S 1 NMR H (DMSO-d6, δ in ppm): 11.80 (s, 1H, OH); O O 11.42 (s, 1H, NH); 8.36-7.05 (m, 11H, H-Ar); 2.33 + (s, 3H, CH3). MS m/z [MH ]found : 305.32 ; Yield : 81% ; m.p. : 190-191°C ; Rf (Hex/AcOEt, [M]theoretical : 304.34 ; Molecular formula : 13 v/v, 7/3) : 0.51 ; NMR C (DMSO-d6, δ in ppm) : C19H16N2O2. 153.17 (C=N); 143.26, 138.98, 136.21, 134.62,

129.40, 128.90, 127.57, 125.86 (C-Ar); 20.97, Synthesis of p-tosylhydrazones 20.75 (H C-Ar); 14.17 (CH ). NMR 1H (DMSO-d , In a 100 mL flask, a solution of 0.01 mole of 3 3 6 δ in ppm) : 10.45 (s, 1H, NH); 7.82-7.15 (m, 8H, ketone in 10-40 mL of ethanol and 2 mL of H-Ar); 2.37 (s, 3H, H C-Ar); 2.30 (s, 3H, H C-Ar); glacial acetic acid (GAA) was prepared and then 3 3 2.17 (s, 3H, CH ). MS m/z [MH+]found : 303.37 ; a solution of p-tosylhydrazide (1.76 g) dissolved 3

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[M]theoretical : 302.39 ; Molecular formula : and the reaction was followed by Thin Layer

C16H18N2O2S. Chromatography TLC (CH2Cl2/AcOEt, v/v, 2/1;

CH2Cl2/MeOH, v/v, 9/1). The precipitate was 7-methoxy-1-tetralone p-tosylhydrazone (3b) filtered, dried and then recrystallized in ethanol O (96°C) to give thiosemicarbazone. O O S N N Characterization of synthesized compounds H S-(+)-carvone thiosemicarbazone (1c) S

N N NH Yield : 90% ; m.p. : 200-201°C ; Rf (Hex/AcOEt, 2 13 v/v, 8/2) : 0.17 ; NMR C (CDCl3, δ in ppm) : H 152.57 (C=N); 157.99 (CAr‒OCH3); 144.20, 135.42, 132.42, 132.41, 129.57, 129.42, 128.23,

117.08, 108.21 (other C-Ar); 55.30 (O-CH3); 28.43, 25.31, 21.64 (3CH & 1CH ). NMR 1H Yield : 68% ; m.p. : 109-110°C ; Rf (CH2Cl2/AcOEt, 2 3 13 v/v, 2/1) : 0,91; RMN C δ (CDCl3, ppm): 178,98 (CDCl3, δ in ppm) : 8.10 (s, 1H, NH); 7.91-6.84 (C=S); 146,82 (C=N); 149,97; 135,36; 132,01; (m, 7H, H-Ar); 3.85 (s, 3H, H3C-O); 2.63, 2.45, 1.83 (m, 6H, 3CH ); 2.34 (s, 3H, H C-Ar). MS m/z 110,86; 40,62; 30,10; 29,07; 20,60; 17,73 (C- 2 3 1 [MH+] found : 345.41 ; [M]theoretical : 344.43 ; carvone). RMN H δ (CDCl3, ppm): 8,71 (s, 1H, C=NNH‒); 7,25 (s, 1H, NH2); 6,40 (s, 1H, NH2); Molecular formula : C18H20N2O3S. 6,25 (s, 1H, CH=C); 4,79-4,75 (d, 2H, C=CH2); 2-acetylnaphthalene p-tosylhydrazone (4b) 2,70 (q, 1H, H2C-CH-CH2); 2,15 (m, 4H, 2CH2); 1,90 (s, 3H, CH3); 1,76 (s, 3H, CH3). MS m/z H [MH+] found: 224,31; [M]theoretical : 223,34 ;

N Molecular formula : C11H17N3S. N S

O O

4’-méthyl acetophenone thiosemicarbazone

Yield : 74% ; m.p. : 181-182°C ; Rf (Hex/AcOEt, (2c) 13 v/v, 7/3) : 0.23 ; NMR C (CDCl3, δ in ppm) : H 152.37 (C=N); 144.26, 135.42, 134.67, 133.85, N S 132.88, 129.67, 128.58, 128.19, 128.04, 127.64, N 126.92, 126.42, 126.29, 123.56 (C-Ar); 21.64 1 NH (H3C-Ar); 13.29 (CH3). NMR H (CDCl3, δ in ppm) 2 : 8.10 (s, 1H, NH); 7.93-7.25 (m, 11H, H-Ar); 2.39 Yield : 82% ; m.p. : 148-149°C ; Rf (CH2Cl2/AcOEt, + 13 (s, 3H, H3C-Ar); 2.25 (s, 3H, CH3). MS m/z [MH ] v/v, 2/1) : 0.78; RMN C δ (DMSO-d6, ppm): found: 339,38; [M] theoretical : 338.42 ; 179,12 (C=S); 148,30 (C=N); 140,29; 134,38;

Molecular formula : C19H18N2O2S. 130,45; 129,32; 126,44; 126,33 (C-Ar); 21,33 (p- 1 CH3-Ar); 13,62 (CH3). RMN H δ (CDCl3, ppm): Synthesis of thiosemicarbazones 8,85 (s, 1H C=NNH−); 7,60-7,15 (m, 4H, H-Ar); A mixture of ketone (20 mmol dissolved in 100 6,62 (s, 2H, CS-NH2); 2,41 (s, 3H, CH3); 2,25 (s, + ml of ethanol) and thiosemicarbazide (20 mmol 3H, CH3). SM m/z [MH ]found: 208,14; dissolved in 20 ml of 1 N hydrochloric acid) was [M]theoretical : 207,30 ; Molecular formula : stirred until the thiosemicarbazone precipitates C10H13N3S.

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7-méthoxy-1-tétralone thiosemicarbazone (3c) [28,29]. HPLC method was also used. It gives H the percentage of each compound in a mixture. With this method, the percentage of the pure N NH2 N synthesized compound must be at least equal to 95% [28,29]. O S

Pharmacological Screening Parasites and media Yield : 89% ; m.p. : 142-143°C; Rf (CH2Cl2/MeOH, Plasmodium falciparum chloroquine-sensitive 13 v/v, 9/1) : 0,81; RMN C δ (DMSO-d6, ppm): strain 3D7 (from Prof. Grellier of Museum 178,64; (C=S); 147,98 (C=N); 157,73; 132,71; d’Histoire Naturelle, Paris-France) asexual 132,55; 129,45; 116,30; 108,85; (C-Ar); 28,11; erythrocytic stages were cultivated 1 25,72; 21,57 (3CH2) 55,27 (O-CH3). RMN H δ continuously in vitro according to the procedure

(DMSO-d6, ppm): 10,09 (s, 1H, C=NNH‒); 8,30 described by Trager and Jensen [30] at 37 °C (s, 1H, H-Ar); 8,05 (s, 1H, H-Ar); 7,75 (s, 1H, H- and under an atmosphere of 5% CO2, 5% O2 and

Ar); 6,90 (s, 1H NH2); 3,80 (s, 3H, O-CH3); 2,65 (t, 90% N2. The host cells were human red blood

2H, CH2); 2,50 (q, 2H CH2); 1,70 (t, CH2). MS m/z cells (A or O Rh+). The culture medium was + [MH ] found: 250,27; [M]theoretical : 249,33 ; RPMI 1640 (Gibco) containing 32 mM NaHCO3,

Molecular formula : C12H15N3OS. 25 mM HEPES and 2.05 mM L-glutamine. The medium was supplemented with 1.76 g/L 2-acétonaphthone thiosemicarbazone (4c) glucose (Sigma–Aldrich), 44 mg/mL hypoxanthin H (Sigma–Aldrich), 100 mg/L gentamycin (Gibco) and 10% human pooled serum (A or O Rh+). N NH2 N Parasites were subcultured every 3–4 days with S initial conditions of 0.5% parasitaemia and 1% haematocrit. Yield : 91% ; m.p. : 181-182°C ; Rf (CH2Cl2/AcOEt, Trypanosoma brucei brucei strain 427 (Molteno 13 v/v, 2/1) : 0,86; RMN C δ (CDCl3, ppm): 179,28 Institute in Cambridge, UK) bloodstream forms (C=S); 147,93 (C=N); 134,42; 133,96; 132,99; were cultured in vitro in HMI9 medium 128,59; 128,33; 127,72; 127,26; 126,84; 126,75; containing 10% heat-inactivated foetal bovine 1 123,18 (C-Ar); 13,45 (CH3). RMN H δ (CDCl3, serum [31]. ppm): 8,90 (C=NNH‒); 8,10-7,45 (m, 7H, H-Ar); The eggs of Artemia salina Leach were obtained 6,55 (s, 1H, NH2); 2,35 (s, 3H, CH3). MS m/z from JBL society (JBL Gmbh&Co.KG, Germany). + [MH ] found: 244,06; [M]theoretical : 243,33 ; Molecular formula : C13H13N3S. In vitro test for Antiplasmodial activity Parasite viability was measured using parasite Purity checking by elementary and HPLC lactate dehydrogenase (pLDH) activity analysis according to the method described by Makler The elementary analysis was used to check et al. [32]. The in vitro test was performed as purity of the synthesized products. This method described by Murebwayire et al. [33]. determines the percentage of the different Chloroquine (Sigma) or artemisinin (Sigma) atoms of the compound except oxygen. The were used as positive controls in all difference between theoretical and experiments with an initial concentration of 100 experimental percentages (δmex-mth) of pure ng/mL. First stock solutions of essential oils and synthesized compound must be less than 0.5 pure compounds were prepared in DMSO at 20

J Pharm Chem Biol Sci, June-August 2015; 3(2): 279-294 Koviessi et al 285 mg/mL. The solutions were further diluted in % death = [(nd test - nd control)/ nd medium to give 2 mg/mL stock solutions. The control)]x100 [36] with = number of dead highest concentration of solvent to which the larvae. parasites were exposed was 1%, which was Camptothecin (Sigma) was used as positive shown to have no measurable effect on reference compound. parasite viability. Essential oils were tested in Data (dose-response) were transformed by eight serial threefold dilutions (final logarithm and the half-lethal concentration LC50 concentration rang: 200-0.09 g/mL, two was determined by linear regression [37]. Tests wells/concentration) in 96-well microtiter were carried out in triplicates. All data were plates. The parasitaemia and the haematocrit expressed as mean ± standard deviation of were 2% and 1%, respectively. All tests were triplicate measurements. performed in triplicate. Statistical analysis In vitro test for antitrypanosomal activity Student’s t-test was used to test the The in vitro test was performed as described by significance of differences between results Hoet et al. [34]. Suramine (a commercial obtained for different samples, and between antitrypanosomal drug, MP Biomedicals, results for samples and controls (GraphPad Eschwege, Germany) was used as positive Prism 4.0; GraphPad Software Inc., San Diego, control in all experiments with an initial USA). Statistical significance was set at P < 0.05. concentration of 1 g/ml. First stock solutions of essential oils and compounds were prepared RESULTS in DMSO at 20 mg/ml. The solutions were Chemistry: further diluted in medium to give 0.2 mg/ml Two series of hydrazone derivatives and one stock solutions. Essential oils and compounds series of corresponding thiosemicarbazone were tested in eight serial threefold dilutions derivatives were synthesized with good yields (final concentration range: 100-0.05 g/ml, two (57-91%). There are: S-(+)-carvone wells/concentration) in 96-well microtiter salicylhydrazone (1a), 4’-methylacetophenone plates. All tests were performed in triplicate. salicylhydrazone (2a), 7-methoxy-1-tetralone salicylhydrazone (3a) and 2-acetylnaphthalene Toxicity test salicylhydrazone (4a); S-(+)-carvone p- The toxicity test was performed on larvae of tosylhydrazone (1b), 4’-methylacetophenone p- brine shrimp (Artemia salina Leach) by the tosylhydrazone (2b), 7-methoxy-1-tetralone p- method of [35] A. salina eggs were incubated in tosylhydrazone (3b) and 2-acetylnaphthalene p- seawater until hatching of young larvae (48 tosylhydrazone (4b); S-(+)-carvone hours). Then, series of solutions of test thiosemicarbazone (1c), 4’- compound at varying concentrations were methylacetophenone thiosemicarbazone (2c), prepared in DMSO/seawater. A defined number 7-methoxy-1-tetralone thiosemicarbazone (3c) of larvae were introduced into each solution and 2-acetylnaphthalene thiosemicarbazone and incubated under rocking condition for 24 h. (4c). To evaluate the toxicity of the solution, Their elementary analysis (table1) showed that counting of larvae viability was performed the compounds 2a, 3a, 4b, 3b, 1c, 2c and 3c under microscope by determining the number meet the purity criteria for this analysis with of dead larvae in each solution. In the case the variation of percentage (δ) of each atom where there was death in the control medium, less than 0.5 [28,29]. the data was corrected by Abbott’s formula:

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Table 1: Elementary Analyses of the synthesized products

Compounds Atom Experience Experience Average theoretic δmex-mth N° 1 N°2

1a N 09.64 09.99 09.815 09.85 -0.035 C 71.02 70.76 70.89 71.81 -0.92 H 07.09 07.09 07.09 07.09 0.00

2a N 10.61 09.9 10.255 10.44 -0.44 C 71 71.15 71.075 71.62 -0.445 H 05.96 05.98 05.97 06.01 -0.005

3a N 08.98 08.64 08.81 09.03 -0.22 C 69.26 69.16 69.21 69.62 -0.45 H 05.82 05.83 05.825 05.85 -0.025

4a N 9.21 08.62 08.915 09.20 -0.285 C 74.34 74.34 74.34 74.98 -0.64 H 05.28 05.27 05.275 05.30 -0.025

1b N 8.71 8.69 8.7 8.8 -0.1 C 64.55 63.88 64.215 64.12 0.095 H 6.84 6.88 6.86 6.96 -0.1 S 9.56 8.96 9.26 10.07 -0.81

2b N 9.01 9.09 9.05 9.26 -0.21 C 62.58 62.85 62.715 63.55 -0.835 H 5.94 5.78 5.86 6.00 -0.14 S 10.16 10.96 10.56 10.6 -0.04

3b N 08.02 08.09 08.05 08.13 -0.08 C 62.55 62.55 62.69 62.77 -0.08 H 05.84 05.88 05.86 05.85 -0.01 S 09.16 08.96 09.06 09.31 -0.25

4b N 08.10 08.13 08.115 08.28 -0.165 C 67.71 67.13 67.65 67.13 0.32 H 05.41 05.42 05.415 05.36 0.055 S 09.52 09.48 09.50 09.47 0.03

1c N 18.9 18.79 18.845 18.81 0.035 C 59.32 59.02 59.17 59.16 0.01 H 7.98 7.19 7.585 7.67 -0.085 S 13.8 15.00 14.4 14.36 0.04

2c N 20.03 20.43 20.23 20.27 -0.04 C 58.01 58.00 58.005 57.94 0.065 H 6.42 6.27 6.345 6.32 0.025

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S 15.54 15.3 15.42 15.47 -0.05

3c N 17.01 16.81 16.91 16.85 0.06 C 58.03 57.86 57.945 57.81 0.135 H 5.94 6.04 5.99 6.06 -0.07 S 12.79 12.87 12.83 12.86 -0.03

4c N 18.22 18.08 18.15 17.27 0.88 C 63.18 63.85 63.515 64.17 -0.655 H 5.94 5.78 5.86 5.39 0.47 S 12.66 12.29 12.475 13.17 -0.695

The other compounds (1a, 4a, 1b, 2b and 4c) compounds fulfilled the purity criteria for HPLC were then purified again and subjected to HPLC analysis with percentages of purity higher than analysis for purification checking. These 95 % (figure 1; Table 2) [28, 29].

Fig.1: Chromatogram with percentage of purity of compound 1a

Table 2: Purity checking by HPLC method Compound Parentage of purity 1a 99.028 4a 100 1b 96.432 2b 97.254 4c 98.157

In total, the two purity verification methods spectrometrical analysis HRMS, and NMR 1H showed the purity of all synthesized products. &13C. The scaffold has advantageous properties: These products can therefore be used for low molecular weight, reasonable Clog P, good biological analysis. Their structures were at first hydrogen bond donating and accepting characterized with the TLC frontal rapport (Rf),

J Pharm Chem Biol Sci, June-August 2015; 3(2): 279-294 Koviessi et al 288 capabilities (table 3), easy, and economical their toxicity on larvae of Artemia salina L. The synthetic routes [37]. half-inhibitory concentration (IC50) and half-

lethal concentration (LC50) were respectively Pharmacological Screening determined and expressed in micromolar (µM) All the studied compounds were tested in vitro to be compared with the scales of parasitic for their antitrypanosomal and antiplasmodial activities and toxicity. Then, the selectivity activities respectively on Trypanosoma brucei index (SI) of each compound was calculated as brucei and Plasmodium falciparum 3D7 and summarized in table 4.

Table 3: Properties Compatible with Reasonable Pharmacokinetics and Drug Availability, Rules of Lipinski [38] applied to hydrazones

Compounds Molecular C logP No. of No. of No. of weight H bond H bond criteria (g.mol-1) donors acceptors met Rules < 500 < 5 ≤ 5 < 10 At least 3 1a 284.35 3.09 2 4 all 2a 268.31 2.44 2 4 all 3a 310.34 2.36 2 5 all 4a 304.34 3.11 2 4 all 1b 318.43 4.77 1 4 all 2b 302.39 4.12 1 4 all 3b 344.43 4.20 1 5 all 4b 338.42 4.79 1 4 all 1c 223.33 3.70 3 3 all 2c 217.30 2.90 3 3 all 3c 249.33 3.17 3 4 all 4c 243.08 3.58 3 3 all

Table 4: Antiplasmodial activity, toxicity and selectivity index of synthesized compounds

Compounds Antiplasmodial Antitripanosomal LC50 (µM) Selectivity Index activity activity (SI = LC50 / IC50)

Pf IC50 (µM) Tbb IC50 (µM) Pf Tbb

1a 18.65 ± 2.99b 24.42±3.24 d 219.44 ± 2.17 11.77 9.80

2a 20.30 ±2.82b 101.05±4.61 g 352.57 ± 1.32 17.37 11.62

3a 95.25 ±32.95c 35.23±1.69 e 731.13 ± 3.27 7.68 7.74

4a 13.05 ± 4.84 a 4.65±0.63a 310.50 ± 1.93 23.79 13.97

1b >314.33 >314.00 536.38 ± 2.35 1.70 < 1.70 2b >331.01 >331.01 368.06 ± 1.87 <1.11 < 1.11 3b >290.60 15.03±0.61c 137.32 ± 0.77 <0.47 9.136 4b >198.10 34.81±8.83 e 35.45 ± 1.41 <0.19 < 0.17 1c 17.93 ± 2.22b 77.23±6.21 f 20.58 ±0.07 1.14 0.27

2c 21.12 ±0.82 b 8.73±0.63b 317.52 ± 0.13 21 36.37

3c >325.79 >401.00 231.13 ± 3.27 <0.71 <0.58

4c 11.22 ±4.23a 4.58±1.69a 480.85 ± 1.51 42.86 104.98

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DISCUSSION 11.75 ppm whereas the NH proton singlet The hydrazine, hydrazide or thiosemicarbazide occurred at 11.42 to 11.20 ppm. The aromatic having the same radical H2N―NH―R reacts protons appeared between 8.36 and 6.90 ppm. with a carbonyl compound according to the In the series of p-tosylhydrazones compounds same mechanism. Steric and electronic effects (1b - 4b), 13C NMR spectral analysis presented of the various substituents of the carbonyl are the group C=N characteristic of the formation of responsible for the difference in reactivity and products between 154 and 152 ppm. Aromatic yields when the same hydrazine derivative was carbons were in the region 144-108 ppm, reacted with various substrates and vice versa. depending on the structure of the test In the literature, it was described the synthesis compound. We remarked in 1H NMR, the single of acetophenone p-tosylhydrazone without proton of the internal nitrogen NH in the form catalyst for 5.5 hours with 68% yield [39]; the of a singlet at 10.45 ppm for the 4'- synthesis using benzaldehyde salicylic acid methylacetophenone p-tosylhydrazone (2b) in hydrazide and 4-dimethylaminobenzaldehyde DMSO-d6 and 8.10 ppm (3b & 4b) and 7.90 ppm or 4-nitrobenzaldehyde for 4 hours [40]. To (1b) ppm in CDCl3. This difference was due to enhance the reaction, we used during our work the effect of DMSO solvent which generated a in the synthesis of the salicylhydrazones (a) and strong hydrogen bond between oxygen and the p-tosylhydrazones (b) the glacial acetic acidic NH proton [43] causing a chemical shift of and technical ethanol. The mixture is heated to hydrogen downfield. Aromatic protons were reflux for 2 hours and the reaction followed by observed as bedding from 7.93 to 6.84 ppm. TLC with yields ranging from 57 to 90%. The low In the series of thiosemicarbazone, the analysis yield 57% obtained of S-(+)-carvone p- of the 13C NMR spectra results confirmed the tosylhydrazone (1a) was due to the nature of presence of fundamentally functions. The C=S this α-β unsaturated ketone. We note well the peaks appeared in the range from 179.12 to presence of adduct 1-4 minority (Michael 175.47 ppm, peaks of C=N between 150.21 and addition) which was removed after purification. 146.82 ppm, aromatic carbons were shown Molecules of each series (a, b, c) showed similar from 123.05 to 140.29 ppm, the C=C carbons in spectrometric data. The analysis of the 13C NMR the structures of products 1c and 3c were spectra of molecules from salicylhydrazones shown in 110, 128, 133, 135, 148 and 149 ppm series (1a - 4a) showed the peak of the amide and methyl carbons of compounds in 12, 13, 21, carbon function (N-CO-Ar) between 163 and 24, 29, 40 ppm. 1H NMR spectra analysis gives 161 ppm. The carbon of the imine’s function, the characteristic protons existing in each characteristic group obtaining during the structure; δ: 6.3-6.90 ppm for protons signals in reaction (C=N) appeared around 153-147 ppm. H2NCS. In =NNH− and the protons signals were Other peaks between 158 and 108 ppm shown from 10.3-8.6 ppm. correspond to the aromatic ring; phenolic The molar mass of each synthesized molecule carbon (aromatic C-OH) had a high value given by mass spectrometry is consistent with between 157 and 156 ppm. In 1H NMR, there theoretical mass found. Various spectrometrical are two types of protons: the phenolic OH analyses done on each compound have really proton and that of NH. The phenolic OH proton confirmed the presence of functional groups having acidic properties and may establish a and different types of protons and carbons in hydrogen bond with the carbonyl in ortho was each structure. The spectrometric data were in to a lower field than the NH proton [41,42]. So conformity with the structures suggested for it will be assigned to OH, the higher δ value. the products. Therefore, it was a singlet between 11.80 and

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The antiplasmodial test reveals that molecules conserved their first place in the order of from p-tosylhydrazide series b were less active actives compounds on the two studied on this parasite than those from parasites and were more antrytripanosomal salicylhydrazide ones (b) that showed (IC50 < 5 M) than antiplasmodial (IC50 > 10 M). antiplasmodial activites closed to that of the All synthesized compounds were none toxic compounds from thiosemicarbazone series (c) against larvae of Artemia salina L. except 4b except 3c which presented an IC50 value higher and 1c which exhibited IC50 values lower than than 300. This could be explained by the camptothecin one (IC50 = 38.09±0.06 M) that S was the positive reference used for this test presence of the sulfone group ( O O ) in p- tosylhydrazide compounds. The replacement of (table 4). From the analyses of their selectivity C index, the most actives compounds (4a, 4c) also this group by the carbonyl group ( O ) in the showed good selectivity on the parasites (SI1) passage of series b compounds to series a ones (table 4). All synthesized compounds of the seemed to enhance antiplasmodial activity of series a exhibited a good selectivity on the these compounds. S-(+)-carvone studied parasites contrary to compounds of the salicylhydrazone (1a) (IC = 18.65±2.99 M) 50 others series (b and c). This could be due to the and 2-acetylnaphthalene salicylhydrazone (4a) presence of sulphur atom in structure of (IC = 13.05±4.84 M) were the most actives 50 compounds b and c. Antiparasitic activity of compounds followed by 4’- these compounds was previously reported in methylacetophenone salicylhydrazone (2a), and the literature [19,25-26] but this work showed 7-methoxy-1-tetralone salicylhydrazone (3a). that salicylhydrazone compounds are more Among the synthesized arylketone compounds, promising than thiosemicarbazone ones in the the increasing of conjugation with C=N groups study of these parasites. These results were in seemed to enhance antiplasmodial activity from perfect agreement with the work of Tiuman et 3a to 4a. 1a, 1c, 4a and 4c showed moderate al. [45] in which if the SI value is higher than activity with IC values between 2 and 20 M 50 unity, the test compound is considered to be and the others compounds, lower activities (IC 50 selective on the parasite and if SI value is lower > 20 µM) (table 4) according to the scale of than unity, the test compound is more cytotoxic Bero and al. [44]. Among all synthesized than antiparasitic. compounds, 4a and 4c were the most actives compounds with IC value of 13.05±4.84 M 50 CONCLUSION and 11.22±4.23 M respectively. Their In this study, three series of compounds are antiplasmodial activites were not statistically study, salicylhydrazones, p-tosylhydrazones and different by Student’s t-test (P < 0.05). thiosemicarbazones, from the same ketones Salicylhydrazone and thiosemicarbazone were synthesized, purified and characterized. derivatives seemed to be promising for the Their biological activities were evaluated and struggle against Plasmodium falciparum. products showed interesting antiplasmodial and On Trypanosoma brucei brucei, the evolution of antitrypanosomal activity on the studded activities seemed to be the same as on parasite with good selectivity. Salicylhydrazones Plasmodium falciparum for all synthesized series and thiosemicarbazones series were the compounds. But the compounds 3 and 4 of the most actives on the studies parasites but series b which were not actives on Plasmodium salicylhydrazone compounds are more falciparum exhibited antitrypanosomal activity promising. To our knowledge, this is the first with IC values of 15.03±0.61 M and 50 time that comparative antiparasitic activities of 34.81±8.83 M respectively. 4a and 4c these molecules are evaluated on Plasmodium

J Pharm Chem Biol Sci, June-August 2015; 3(2): 279-294 Koviessi et al 291 falciparum and Trypanosoma brucei brucei then thiosemicarbazones. Bioorg Med Chem they could open an interesting opportunity to 2004; 4885-4893. the treatment of these diseases. 6. Garcia CC, Brousse BN, Carlucci MJ, Moglioni AG, Martins Alho M, Moltrasio ACKNOWLEDGMENTS GY, D’Accorso NB, Damonte EB. Inhibitory We thank Professors J. Quetin-Leclercq and D. effect of thiosemicarbazone derivatives Lambert and all the staff of LDRI at the on Junin virus replication in vitro. Antiviral Université Catholique de Louvain, Brussels, Chem Chemother 2003; 14:99–105. Belgium. 7. Zoidis G, Kolocouris N, Foscolos GB, Kolocouris A, Fytas G, Karayannis P, CONFLICT OF INTEREST STATEMENT Padalko E, Neyts J, De Clercq E. Are the 2- None Declared isomers of the drug rimantadine active anti-influenza A agents? Antiviral Chem REFERENCES Chemother 2003; 14:153–164. 8. San DK, Butcher RJ, Chandhuri S. 1. Xu J, Cole DC, Chang CP, Ayyad R, Asselin Spectroscopic, structural and M, Hao W, Gibbons J, Jelinsky SA, Saraf KA antibactrerial properties of copper (II) and Park K. Inhibition of the signal complexes with bio-relevant 5-methyl-3- transducer and activator of transcription-3 formylpyrazole N (4)-benzyl- N (4) (STAT3) signaling pathway by 4-oxo-1- methylthiosemicarbazone. Mol Cell phenyl-1,4-dihydroquinoline-3-carboxylic Biochem 2003; 2: 253 - 261. acid esters. J Med Chem 2008; 51: 4115- 9. Rebolledo AP, de Lima GM, Gambi LN and 4121. al. Tin (IV) Complexes of 2-bnzoylpyridine 2. Hassanien MM, Gabr IM, Abdel-Rhman N(4)-phenylthiosemicarbazone : Spectral MH, El-Asmy AA. Synthesis and structural Characterization, structural studies and investigation of mono- and polynuclear antifungal activity. Appl Organomet Chem copper complexes of 4-ethyl-1-(pyridin-2- 2003; 17: 945-951. yl) thiosemicarbazide. Spectrochimica 10. Kasuga NC, Sekino K, Ishibawa Synthesis, Acta A 2008; 71: 73-79. crystal structure and antibacterial activity 3. Singh PK, Kumar DN. Spectral studies on of monomeric 7-cordinate bismuth (III) cobalt (II), nickel (II) and copper (II) complexes with tridentate and complexes of naphthaldehyde substituted pentadentate thiosemicarbazones ligands. aroylhydrazones. Spectrochimica Acta J Inorg Biochem 2003; 96(2): 298-310. Part A 2006; 64(4): 853-858. 11. Afrasiabi Z, Sinn E, Chen JN, Ma YF. 4. Sreeja PB, Kurup MRP, Kishore A, Jasmin Appended 1, 2-naphthoquinones as C. Spectral characterizatıon, X-ray anticancer agents 1 ; Sythesis, Structural , structure and biological investigations of Spectral and antitumur activities of ortho- copper(II) ternary complexes of 2- naphthaquinone thiosemicarbazone and hydroxyacetophenone 4- hydroxybenzoic its transition metal complexes. Inorg acid hydrazone and heterocyclic bases. Chem Acta 2004; 357 : 271-277. Polyhedron 2004; 23: 575-581. 12. Afrasiabi Z, Sinn E, Newton C, Anson CE. 5. Aguiarre G, Boiani L, Cerecetto and al. In Phenanthraquinone thiosemicarbazone vitro activity and mechanic of action and its transition metal complexes with against the protozoan parasite potential anticancer activity: Synthesis, trypaosoma cruzi of 5-nitrofurylcontaining Structure, Spectral properties and in vitro

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Cite this article as: Bardieu Atchade, Salomé D.S. Kpoviessi, Raymond H. Fatondji, Léon A. Ahoussi, Joachim Gbenou, Georges C. Accrombessi, Jacques H. Poupaert, Mansourou Moudachirou. Synthesis, Purity Verification and Comparison of Antiplasmodial and Antitrypanosomal Activities of Hydrazone Derivatives and Corresponding Thiosemicarbazones. J Pharm Chem Biol Sci 2015; 3(2):279-294

J Pharm Chem Biol Sci, June-August 2015; 3(2): 279-294