LIPOPHILIC AND STRUCTURE ACTIVITY RELATIONSHIPS STUDY OF THIOSEMICARBAZONES AND DERIVATIVES

Glinma Bienvenu 1 , Medegan Sèdami 2 , Yayi Eléonore 1 , Agnimonhan F. Hyacinthe 1 , Kpoviessi D.S. Salomé 1 , Quetin-leclercq Joëlle 3 , Accrombessi C. Georges 1 , Kotchoni O. Simeon 4 , Poupaert H. Jacques 3 and Gbaguidi A. Fernand 1,2 . 1. Laboratoire de Chimie Organique Physique et de Synthèse (LaCOPS), Département de Chimie, Faculté des Sciences et Techniques (FAST), Université d’Abomey-Calavi, 01 BP 4521 Cotonou, Bénin. 2. Laboratoire de Chimie Organique Pharmaceutique, Ecole de Pharmacie, Faculté des Sciences de la Santé, Université d'Abomey-Calavi, Campus du Champ de Foire, 01 BP 188, Cotonou, Bénin. 3. Louvain Drug Research Institute (LDRI), School of Pharmacy, Université Catholique de Louvain, B1 7203 Avenue Emmanuel Mounier 72, B-1200 Brussels, Belgique. 4. Department of Biology and Center for Computational and Integrative Biology, Rutgers University, Camden, NJ 08102, USA. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 05 September 2019 Final Accepted: 07 October 2019 Published: November 2019

Traditionally, small molecules have been a reliable source for discovering novel biologically active compounds because these molecules are easily synthesized and their smooth structural optimization would usually lead to a feasible candidate compound. Here, some thiosemicarbazones, N(4)-methyl and N(4)-phenyl-3thiosemicarbazones were synthesized in good yield (52-84%), characterized and then their anti-parasitic activity were evaluated. The structure and lipophilic-activity relationships of compounds were particularly studied. Among them, some products exhibited trypanocidal activity with their half inhibitory concentration (IC 50 ≤ 10 micromolar "µM") especially compounds L 1-3 , D 2 , B 3 , C 3 , D 1 (from 2 to 8.73 µM). Other showed moderate antitrypanosomal activity with their IC 50 between 12 to 87 µM (L 4 , C 2 , C 1 , B 2 ) while certain showed little activity (IC 50 ≥ 100µM). Some active products turned out quick selective on the parasite with their selectivity index greater than to unit (SI ≥ 1). Several factors including lipophilicity, steric and electronic effects of the substituents have played a vital role in this activity. The elongation of the carbon chain of the carbonyl, the substitution on a phenyl radical, the fixing of a methyl or phenyl on the N(4) nitrogen atom induced significantly the increased trypanocidal activity of compounds. This is the case specifically of N(4)-methyl and especially of N(4)-phenylsubstituted thiosemicarbazones. Such compounds could be able to have applications in the treatment of parasitic diseases. Nowadays, microbial and parasitic diseases are resistant to existing treatments. For these reasons, chemists have been made a great effort to the development of compounds with biological activity that will be used in pharmaceutical chemistry. Currently, most antiparasitic drugs are considered orphan drugs, with the main exception of antimalarials. The pharmaceutical considerations outweigh all others, because the economic return on the development of anti-parasitic drugs is limited. Therefore, it is necessary to find less expensive alternatives for the treatment of parasitic disease (Soates et al., 2011). Recently, due to having wide-spectrum biological activity of thiosemicarbazones derivatives that synthesis studies made, interest on these compounds has been considerably increased in the pharmaceutical sector at the present time (Rogolino et al., 2015 ;Büscher et al., 2017 ;Zani et al., 2017). Thiosemicarbazones and semicarbazones due to be a small molecule widely used in the treatment of antiviral, anticancer and antiparasital disease. More recently, it has been found that they are highly effective antiparasital compounds against Trypanasoma cruzi parasites that cause especially Malaria and Chagas diseases. Generally, thiosemicarbazones show this effect by causing the inhibition of cysteine proteases in this type of parasites and derivatives (Du et al., 2002 ;Beraldo and Gambino, 2004 ;Greenbaum et al., 2004 ;Fujii et al., 2005 ;Jeremy et al., 2008).
In the light of this important data which have been achieved with the literature survey considering that the thiosemicarbazones are biologically active compounds, synthesis of the thiosemicarbazone derivatives expected to show positive activity was carried out.
African trypanosomiases are still a serious health and economic problem that requires not only the application of the knowledge and resources currently available, but also their improvement through multidisciplinary research (Simarro et al., 2012 ;Grant et al., 2015). Animal trypanosomiasis is a major constraint for the livestock industry in developing countries (Amer et al., 2011). In East Africa, animal trypanosomiasis is caused by numerous protozoan parasites transmitted by tsetse flies, including Trypanosoma vivax, T. congolense and subspecies of T. brucei sl (T. brucei and T. b zoonotic rhodesiense infectious for humans) that can co-circulate in domestic and wild animals (Cox et al., 2010).
In this paper, we described the synthesize of thiosemicarbazones, N(4)-methyl and N(4)-phenyl-3thiosemicarbazones of benzaldehyde, 2'-methylacetophenone, propiophenone, benzophenone with its substituted derivatives. The compounds were tested for their antitrypanosomal activity against Trypanosoma brucei brucei and their toxicity on larvae shrimp Artemia salina Leach. And then, some structure-activity and lipophilicity-activity relationships were examined.

Equipment
All synthesized compounds were characterized by Nuclear Magnetic Resonance spectra using Bruker Avance 400. UltraSheild with dimethylsufoxide (DMSO)-d 6 or chloroform CDCl 3 and then Mass Spectrophotometer spectra obtained using the method of Atmospheric-pressure chemical ionization and mass is given in m/z of [MH + ]. The frequencies for 1 H and 13 C are 400.130 and 100.612 MHz respectively. Chemical shifts are given in parts per million (ppm) relative to tetramethylsilane as internal standard. Multiplicity was designated as singlet (s), doublet (d), doublet dedouble (dd), triplet (t), quintuplet (qi) and multiplet (m). Melting points (m.p.) were determined on a fusionometer of the type electrothermal 1A 9000 and were not corrected.

Synthesis of the compounds
An equimolar mixture (0.01 mol) of thiosemicarbazide and analogues dissolved in 10 mL ethanol (EtOH 96°) was added slowly to a solution (0.01 mol) of arylketone dissolved in 20-30 mL of EtOH in presence of acid (HCl, 1 N or 31 GAA). The mixture was heated at reflux for 4 h with stirring. After cooling, the precipitate was filtered, washed with cold distilled water until neutrality, dried and then recrystallized in ethanol.
All compounds after synthesis have been submitted to the in vitro anti-trypanosomal test on the bloodstream form of the strain 427 of Trypanosoma brucei brucei and were evaluated for their in vitro cytotoxicity on Artemia salina Leach following standard biological methods.

Anti-trypanosomal test
The assessment is performed on the bloodstream form of the strain 427 of Trypanosoma brucei brucei by the «LILIT Alamar Blue TM » method (Baltz et al., 1985 ;Räz et al., 1997). The stock solutions of each thiosemicarbozone have been prepared from an initial concentration of 10 mg/mL in dimethylsufoxide (DMSO). The trypanosomes are grown in a medium containing 10% of heat inactivated fetal calf serum and bloodstream form supporting factor. The trypanosome suspensions were adjusted to 5×10 4 tryp/mL. In each well, 50 μL of different dilutions of the stock solution were added to 50 μL of suspension of trypanosomes. The plates were then incubated at 37°C for 72 hours in an atmosphere with 5% CO 2 . 10 μL of dye "Alamar BlueTM" is added to each well and then incubated for 4 hours. The dye "Alamar BlueTM" is a reagent for detecting enzymatic activity. The wells in which the concentration of compound is insufficient to inhibit the proliferation of trypanosomes are stained. The halfinhibitory concentration is the concentration of unstained wells in which there is the lowest amount of thiosemicarbazones. The plate reading is made in comparison with control wells on a fluorescence plate reader using an excitation wavelength of 530 nm and an emission wavelength 590 nm. We carried out the test in triplicate for each compound. All data were expressed as means ± standard deviation of triplicate measurements.

Cytotoxicity screen
The cytotoxicity test was performed on larvae of brine shrimp (Artemia salina Leach) by the method of Sleet and Brendel (1983). Artemia salina eggs were incubated in seawater until hatching of young larvae (48 hours). Then, series of solutions of test compound at varying concentrations were prepared in DMSO/seawater. A defined number of larvae were introduced into each solution and incubated under rocking condition for 24 h. To evaluate the toxicity of the solution, counting of larvae viability was performed under microscope by determining the number of dead larvae in each solution. In the case where there was death in the control medium, the data was corrected by Abbott's formula: % death = [(nd test -nd control)/ nd control)] x 100 (Abott, 1925) with nd = number of dead larvae. Data (dose-response) were transformed by logarithm and the halflethal concentration LC 50 was determined by linear regression (Hafner et al., 1977). Tests were carried out in triplicates. All data were expressed as mean ± standard deviation of triplicate measurements.

Chemistry
Before synthesizing the compounds, we carried out a theoretical study based on the pharmacokinetic properties rules (Lipinski et al., 1997 ;2001) and results are summarized in the table 1. Fifteen compounds were synthesized and their physico-chemical properties are described in the table 2.
Compounds were synthesized following the condensation reaction (figure 1).
GAA or HCl EtOH, 

Pharmacology
The antiparasitic activity of products was evaluated on the strain 427 of Trypanosoma brucei brucei using lapachol as witness. The toxicity activity of antitrypanosomal compounds was screened on Artemia salina Leach. The results of biological activities of products were obtained and expressed in IC 50 and LC 50 respectively. Selectivity of actives products are determined (table 3). Discussion:-Synthetic molecules have physical properties compatible with reasonable pharmacokinetics and drug availability. The scaffold (Figure 1) has advantageous properties: low molecular weight, reasonable C.logP, good hydrogen bond donating and accepting capabilities (Table 1), easy and economical synthetic routes (Lipinski et al., 1997(Lipinski et al., , 2001. The analysis of spectrometrical data gave especially in 13 C NMR spectra, peaks of C=S from 179.13 to 175.47 ppm and of C=N between 150.47 and 143.74 ppm in all molecules. All aromatics carbons of the compounds ranged from 159.04 to 115.49 ppm. 1 H NMR spectra gave the characteristic protons in each structure: signals of protons (HC=N) were observed between 8.06 and 7.97 ppm (products K 1 & K 2 ), we generally noted the disappearance of the peaks for the proton NH2 of thiosemicarbazide, except the protons in the amino group H 2 N in molecules L 1 and L 4 respectively were identified at 6.87 and 6.70 and 6.78 and 6.35 ppm. In the products L 2 and L 3 , the signal of the typical proton HO was observed respectively at 8.70 and 8.80 ppm. Aromatics protons in the compounds were obtained between 7.80 and 6.70 ppm. It is worth mentioning that substituents used here, OH and NH 2 , are both electron donating in the ortho and para positions. The analysis of these spectral data further confirms the structure of each molecule synthesized. In mass spectrometry, mass of each molecular ion peak (parent peak) obtained is very consistent and comparable to the theoretically estimated mass.
Pharmacological tests (table 3) showed that compounds presented interesting activity. On the parasites, some molecules  (table 4).
Lipophilicity is a main physico-chemical determinant influencing the bioavailability, permeability and frequently the toxicity of drugs (Lipinski et al., 2001). A substance is all the more lipophilic as log P is positive. The calculation of the log P (C log P) involves the additivity rules of the hydrophobic constants of Rekker (Rekker, 1977). The higher the logP (C logP) the lower IC 50 and the more active substance (Du et al., 2002 ;Fujii et al., 2005).
For each of these series, it is noted that in general the introduction of the alkyl or aryl group on the nitrogen N(4) induces an increase in lipophilicity and also activity : B 3 and C 3 with ClogP = 4.600 and 5.400 respectively are all trypanocidal. In the Di series, the two trypanocidal compounds have almost the same degree of lipophilicity.
In the L 1-4 series of 4-phenyl-3-thiosemicarbazones of the benzophenone derivatives, a significant increase in activity is also seen. Among these molecules, 4-hydroxybenzophenone 4-phenyl-3-thiosemicarbazone (L 3 ) is the most active displaying an IC 50 of 2.76 μM. A comparative study between 4-phenyl-3-thiosemicarbazones of benzophenone C 3 and its L 1-3 derivatives shows that the substitution of an ortho or para proton of a phenyl radical of benzophenone by an amino or a hydroxy group significantly enhanced trypanocidal activity ( Table 3). The IC 50 values found for the substituted L 1-3 derivatives are still low compared to that of the unsubstituted C 3 compound (IC 50 = 8.48 μM). It appears that it is the substitution of the hydroxyl group in para position L 2 which gives the best activity (IC 50 = 2.76 μM). This group enriches its nucleus by its mesomeric donor effect. The ortho amino and 38 hydroxy benzophenone derivatives are also more active than C 3 . The difference in activity between these L 1 and L 2 molecules could be explained by the effect of the amino group or the substituted hydroxy group on the phenyl radical. Between ortho and para hydroxy-substituted products, it is para the most active L 2 . This could be explained by the steric OH effect in the ortho position. There is a slight decrease in activity (IC 50 p-OH = 2.76 and o-OH = 3.86 μM) (Du et al., 2002).
In our study, we have remarked that the substitution of the imine's hydrogen CH=N of benzaldehyde 4-phenyl-3thiosemicarbazone (K 2 ) by the radical ethyl (propiophenone B 3 ) and phenyl (benzophenone C 3 ) induced a trypanocidal activity. This observation confirms the work of Du et al. (2002). The IC 50 value goes from more than 300 μM (for K 2 ) to 8.48 μM (for C 3 ) and 7.63 μM (for B 3 ). The last two compounds are also the most lipophilic (table 1). We then studied the larval toxicity of the active compounds on the trypanosome using the cytotoxicity of the lapachol (LC 50 = 281 µM) as referred (Santos et al., 2003 ;Graminha et al., 2008). Shrimp larvae were selected in this study as biological model. Our different LC 50 values obtained using the synthesized products presented toxic or no activity than lapachol. Among compounds, it is B 2 , C 1 , D 2 and the series L 1-4 which showed toxic activity, particularly the 4-phenyl-3-thiosemicarbazones of benzophenone derivatives L 1-4 (Table 3). Indeed, there is a correlation between the toxicity of the compounds on shrimp larvae and their cytotoxicity on 9KB and 9PS cells (human carcinoma nasopharygien) (Pelka et al., 2000), and on A-549 cells of lung carcinoma and HT-29 cells of colon carcinoma (Carballo et al., 2002). In addition, we noticed that the most strongly trypanocidal compounds were also the most cytotoxic. With their LC 50 and IC 50 values, we have determined the selectivity indexe of each trypanocidal compound. Except C 1 all products displayed greater selectivity, especially B 3 with SI = 119 and compounds C 2 , C 3 , D 2 , D 1 and L 1 (SI > 10). These results are in perfect agreement with the work of Tiuman et al., (2005) in which if the SI value obtained is greater than unity, the test compound is considered to be selective on the parasite and if SI value is less than unity, the test compound is more cytotoxic than anti-parasitic.

Conclusion:-
In this work, fifteen thiosemicarbazones and derivatives were synthesized and studied. Their biological activities were evaluated and products showed interesting trypanocidal activity on the parasite study and were selective. Some factors including lipophilicity, steric and electronic effects of the substituents have played a vital role in this activity.
This study could open an interesting opportunity to the treatment of the trypanomiasis sickness.