Screening of Some Pyrazole Derivatives as Promising Antileishmanial Agent

Pyrazole derivatives (I-VII) were prepared in good yields using aldol condensation followed by cyclization and were characterized by elemental analysis, IR and 1H NMR spectroscopy. In vitro antileishmanial activity test was conducted using Alamar blue reduction method. The test revealed that the synthesized compounds (except compound IIb) exhibit better antileishmanial activity than the standard drug miltefosine and lower antileishmanial activity (except compounds III and IIIb) compared to the standard drug amphotericin B deoxycholate. Compound IIIb, phenyl pyrazoline with propanoyl side chain, 1-(3-phenyl-5-(1-phenyl-3-p-tolyl1H-pyrazol-4-yl)-4,5-dihydropyrazol-1-yl)propan-1-one, was found to be the most active (IC50= 0.0112μgml) than the standards miltefosine(IC50= 0.3±0.04 μgml) and amphotericin B deoxycholate (IC50= 0.2±0.02 μgml) for L. dovani. Compound III is found to be the most active (IC50= 0.28±0.03 μgml) and has comparable antileishmanial activity to the standard miltefosine (IC50= 0.3±0.04 μgml) and amphotericin B deoxycholate (IC50= 0.2±0.02 μgml) on L. aethiopica amastigote.


I. Introduction
eishmaniasis is a group of vector-borne diseases caused by species of the genus Leishmania, a compulsory intra-cellular parasite of the mammalian host cell [1,2].Leishmania parasites exist in two forms: amastigote in the mammalian host and a flagellated promastigote in the insect vector [1].Clinical manifestation of leishmaniasis occur in four major forms in humans: (i) visceral, the most severe and lifethreatening form; (ii) cutaneous, originating as nodules and ulcers that may persist for years; iii) diffuse cutaneous leishmaniasis, which is a long-lasting disease due to a deficient cellular-mediated immune response and (iv) mucocutaneous, causing permanent lesions in the mouth, nose or genital mucosa [1][2][3].This lifethreatening disease that affects about 12 million people worldwide with 1.5 million to 2 million new cases of cutaneous leishmaniasis (CL) and 500,000 new cases of visceral leishmaniasis (VL) each year is endemic in the tropical and sub-tropical regions [4].
Endemic Human leishmaniasis is reported in 88 countries, majority of them are low-income countries [5].East Africa is one of the world's main endemic areas for VL, and over the last 20 years has gained dramatic increase in the number of VL cases, due to a complexity of factors [6].Several studies have convincingly shown that malnutrition, HIV and genetic susceptibility are individually responsible for VL [7].The epidemiological pattern of Leishmania species is changing, with a tendency to urbanization and geographic expansion.Despite the high worldwide prevalence, no vaccine for Leishmaniasis and complex vector control, few advances were made in the treatment of this disease [8].
The difficulty to control this parasitic disease remains a serious problem mainly due to the diversity of mammalian reservoirs (wild and domestic animals), species of vectors and Leishmania species [1].
Chemotherapy for leishmaniasis is generally ineffective mainly due to the emergence of drug-resistant strains and toxicity of the therapeutics agents [1,8].The pentavalent antimonials compounds such as sodium stibogluconate (pentostan) and meglumine antimoniate (glucantime) are widely used as primary therapy but they induce toxic side effects together with drug resistance [1,9].Amphotericin (AmBisome) is now the treatment of choice.Its failure in some cases to treat visceral leishmaniasis (L.donovani) has been reported in Sundar [10], but this may be related to host factors such as co-infection with HIV or tuberculosis rather than parasite resistance.Miltefosine (Impavido) is a new drug for visceral and cutaneous leishmaniasis.Paromomycin drug is though to be an inexpensive and effective treatment for leishmaniasis [11].
Pyrazole derivatives were found to possess various important biological activities such as; antibacterial [12,13], antiinflammatory [14,15], antioxidant [16], ACE inhibitory [17], anti-cancer [18], MAO-B inhibitory [19], antidepressant [20], antiviral [21], antimycobacterial [22,23], antileishmanial [24,25], and antimalarial [26,27] activities.These reports have been useful for biologist, chemists and pharmacists engaged in the development of new drugs and/or synthetic routes from pyrazoline derivatives.Pyrazoline derivatives were reported to possess significant in vitro anti-leishmanial activity [28].This has prompted the synthesis and investigation of safe, effective and cheap antileishmanial agent from pyrazoline derivatives containing phenyl or thiophenyl moiety in this research laboratory.L. donovani, a leishmanial parasite that causes visceral leishmaniasis in Africa and L. aethiopica the leading cause of cutaneous leishmaniasis in Ethiopia were used for the antileishmanial testing.c) Standard Drugs Amphotericin B deoxycholate (Fungizone®, E R Squibb, UK) and miltifosine/ hexadecylphosphocholine (A G Scientific, San Diego, CA, USA) were used as standard drugs in the determination of the antileishmanial activity of the synthesized compounds.

III. Methods
Synthesis of target compounds: The intermediate α, β unsaturated ketones, (II and III) were synthesized by aldol condensation of 1-phenyl-3-p-tolyl-1H-pyrazole-4carbaldehyde I with 2-acetylthiophene and acetophenone in alcoholic KOH.The target thienyl and phenyl pyrazolines, (Figure 1 and 2) were synthesized by cyclization of the intermediate α, β unsaturated ketones (II and III) with hydrazine hydrate in ethanol or the appropriate aliphatic acid [28].

b) Stock solution and working concentration preparation
All the compounds tested (II, IIa, IIIa, IIb, III, IIIb, IIc) were dissolved in DMSO to a final concentration of 1mgml -1 .Both test and standard solutions were serially diluted to appropriate concentrations using complete media.The test compounds were prepared by three fold serial dilutions from 10 µgml -1 to 0.04 µgml -1 .Amphotericin B deoxycholate and miltefosine which were used as a positive control for comparison of the antileishmanial activities of the test compounds, were also made in three fold serial dilutions [33].c) Biological activity test i.In vitro antipromastigote assay Promastigote forms of L. donovani and standard drugs Amphotericin B deoxycholate and miltefosine were used for the assay.3x 10 6 promastigotes of L. donovani in 100 µl were seeded to each well in a 96 well flat bottom plate.Various dilutions (10, 3.33, 1.11, 0.37, 0.12, 0.04 µgml -1 ) of test compounds were added to the parasites.The tests were done in duplicates.Some of the wells contained only the standard drugs and served as a positive control.The media and DMSO alone were used as a negative control.The plates were kept at room temperature.After 24 hr, 20 µl of Alamar blue (12.5 mg resazurin dissolved in 100 ml of distilled water) [34] was added to each of the wells.Absorbance of the resulting mixture was measured after 48 hr at a wavelength of 540 and 630 nm using Enzyme Linked Immuno Sorbent Assay (ELISA) plate reader [35].A quantitative colorimetric assay using the oxidation-reduction indicator Alamar Blue was developed to measure cytotoxicity of the synthesized compounds against the protozoan parasite Leishmania donovani.The Alamar Blue assay permits a simple, reproducible and reliable method for screening antileishmanial drugs [36,37,38].
ii.In vitro antileishmanial activity on L. aethiopica amastigotes In a 96-well microtitre plate, test substances were serially diluted to final test concentrations of 0.04 to 10 µgml -1 in 50 μl culture medium and 50 μl suspensions of axenic amastigotes containing 2 × 10 7 cells/ml were added to each well.Contents of the plates were then incubated in humidified atmosphere containing 5% CO 2 at 31°C for 72 hour.After 68 hour of incubation, 10 μl of fluorochrome resazurin solution (12.5 μg dissolved in 100 ml of PBS (pH=7.2))was added into each well and the fluorescence intensity was measured after a total incubation time of 72 hour using 37 Victor3 Multilabel Counter at excitation wavelength of 530 nm and emission wavelength of 590 nm.The IC 50 (µgml -1 ) values for each extract were evaluated from sigmoidal doseresponse curves using computer software Graph pad prism 3.0 and values expressed as mean + SD of triplicate experiments with each test concentration in duplicate.Assays with standard anti-leishmanial drugs and negative controls (medium alone and 1% DMSO) were also performed to have reference values.Also the background fluorescence intensity of each extract, essential oil and reference drug were measured [31].

d) Data analysis
The IC 50 values for synthesized compounds tested for their in vitro antileishmanial activity were evaluated from sigmoidal dose-response curves using non linear regression software (GraphPad Prism®; GraphPad Software, Inc., San Diego, CA)

a) Biological assays i. In vitro antipromastigote activity
The antipromastigote assay of the synthesized compounds was carried out according to the method described in the experimental part.The results obtained were analyzed and IC 50 (µgml -1 ) for each test compound was calculated using Graph pad prism software (Table 1).The result revealed that the synthesized compounds Screening of Some Pyrazole Derivatives as Promising Antileishmanial Agent except compound IIb possess better antileishmanial activity than the standard drug miltefosine which has IC 50 value 3.1911 µgml -1 .However, synthesized compounds except for compounds III and IIIb exhibited lower antileishmanial activity compared to the standard amphotericin B deoxycholate (IC 50 = 0.0460 µgml -1 ).Compound IIIb, the phenyl pyrazoline with propanoyl side chain, was found to be the most active (IC 50 = 0.0112 µgml -1 ) compound as compared to the standard miltefosine (IC 50 = 3.1911 µgml -1 ) and amphotericin B deoxycholate (IC 50 = 0.0460 µgml -1 ).Compared to study done by Vikramdeep et .al, [39] and Manuel et al [3], this research reveal that the phenyl pyrazoline derivative compound III and IIIb have better antileishmanial activity with IC 50 value of 0.0422 and 0.0112 µgml -1 , respectively.This might be due to the formation of hydrogen bonding between its carbonyl group and backbone of certain receptor active site in the former compound, III, and the presence of propanoyl group in the latter compound, IIIb, may play a role in the interaction with vital important biochemical process.The thienyl pyrazoline derivative, 1-phenyl-4-(3-(thiophen-2yl)-4,5-dihydro-1H-pyrazol-5-yl)-3-p-tolyl-1H-pyrazole (II c) the non-substituted compound IIc (IC 50 = 0.1673 µgml -1 ) seems to affect positively the biological activity leading to the better antileishmanial activity compared with ethanyol (CH 3 CO-) (IIa) and propanoyl (CH 3 CH2CO-) (IIb) substituted compound and study done by Luiz CS Pinheiro et al [2].
The phenyl derivative displayed better activity than the corresponding thienyl derivatives for leishmania activity.Regarding the thienyl derivatives of the pyrazolines, activity decreased with the increase in the carbon number of aliphatic substitution at pyrazoline N 1 from H to propanoyl group.However, the activity increased with increasing the length of the side chain in the phenyl pyrazolines.This could be attributed to the associated increase in hydrophobicity of the compounds that increases hydrophobic interaction with the molecular target site.1).The result showed that the synthesized compounds except for compounds III (IC 50 =0.28±0.03µgml - ), possess lower antileishmanial activity than the standard drug miltefosine and amphotericin B deoxycholate that have IC 50 value 0.3±0.04µgml - and 0.2±0.02µgml -1 respectively.Compound IIIb, which exhibited the highest antipromastigote activity, has shown the least antiamastigote activity, while compound III has almost comparable activity with the standard drug miltefosine and amphotericin B deoxycholate.

V. Conclusion
Seven pyrazole derivatives were synthesized using aldol condensation and subsequent cyclization reactions in a good yield (71.39%-95.20%).The compounds were purified with recrystalization method and were characterized by elemental microanalysis, IR, and 1 H NMR spectroscopy.In vitro antileishmanial activity was conducted using alamar blue reduction method and the results revealed that synthesized compounds showed better antileishmanial activity than the standard drug miltefosine.But all the synthesized compounds except for compounds III and IIIb exhibited lower antileishmanial activity compared with the standard amphotericin B deoxycholate.Moreover, the phenyl pyrazolines showed better antileishmanial activity compared with the thienyl pyrazolines and their activity increased with increase in number of carbons in the side chain.Compound IIIb, 1-(3-phenyl-5-(1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)-4,5-dihydropyrazol-1-yl)propan-1-one phenyl pyrazoline, is found to be the most active (IC 50 = 0.0112 µgml -1 ) and this compound could represent a fruitful matrix for the development of antileishmanial agents that would deserve further derivatization and investigation.Among seven synthesized compounds, compounds III is found to be the most active (IC 50 = 0.28±0.03µgml -1 ) and has comparable antileishmanial activity to the standard miltefosine and amphotericin B deoxycholate on L. aethiopica amastigotes.
b) Parasite strain

Table 1 :
Antipromastigote and Antiamastigote activity (IC 50 µgml -1 ) of the test compounds and reference standards