Printed in the Republic of Korea
http://dx.doi.org/10.5012/jkcs.2013.57.6.755
Synthesis and Pharmacological Evaluation of Some Novel 2-Mercapto Benzimidazole Derivatives
Sidram A. Nevade*, Sachin G. Lokapure, and Navanath V. Kalyane
Department of Pharmaceutical Chemistry, BLDEA’s College of Pharmacy, Bijapur-586101, Karnataka, India.
*Email: [email protected]
(Received June 11, 2013; Accepted November 4, 2013)
ABSTRACT. The present study is synthesis of derivatives of N'-(4-amino-5-sulfanyl-4H-1,2,4-triazole-3-yl)-2-(1H-benzimi- dazole-2-ylsulfanyl) acetohydrazide (IV). Antibacterial activity tested against the E. coli and A. Substilis. Biological activities conducted by disc diffusion method. Compound 2MB1, 2MB3, 2MB5 inhibit the appreciable microbial growth while rest of the compound possess the moderate activities. Anti-inflammatory activity tested by reduces local edema induced in the rat paw by injection of phlogestic agent. Compound 2MB1, 2MB8, 2MB5, 2MB3 and 2MB6 exhibit satisfying anti-inflammatory activity while analgesic activity conducted by acetic acid induced writhing effect in mice while compound 2MB1, 2MB4 and 2MB7 having the good analgesic activity. The chemical structures of all newly synthesized compounds were confirmed by their IR, 1H NMR and mass spectral data.
Key words: 2-Mercaptobenzimidazole, Dimethyl sulfoxide,Anti-inflammatory, Analgesic and antibacterial activity
INTRODUCTION
The development of resistance to current anti-bacterial therapy continues to stimulate the search for more effec- tive agents. Exclusive literature survey of benzimidazole and triazole1,2 brevealed that it has diversified activities such as anticonvulsant,3−5 antihelmenthic,6 antiamoebic,7 antiparasitic,8 as potent H3 antagonist,9 potential anxiolytic agent,10 cyclooxygenase inhibitors,11 anticancer,12 spas- molytic activity,13 antithyroid activity,14 antimicrobial and antifungal,15 androgen receptor antagonist.16 The antiproto- zoal activity of substituted 2-trifluorobenzimidazoles has been reported17 consistent with several earlier studies on the anti-giardial activity of various benzimidazole deriv- atives.18,19 However, the general antimicrobial activity of benzimidazole derivatives has not been extensively inves- tigated. Development of the synthetic method for the syn- thesis of the titled 2-mercapto benzimidazole compounds has revealed in the methodology, the characterization of synthesized compound has done by IR, NMR and Mass spectral data.
Regarding this, we have synthesized some novel 2- mercapto benzimidazole derivatives or some novel benzimidazole analogues combining with different substituted aromatic and hetero cyclic aldehydes ring system with view to get a potential anti-inflammatory, analgesic and antibacterial activity with less toxic and side effects.
EXPERIMENTAL
Melting points were determined with a Melt-Temp apparatus and are uncorrected. TLC was performed on Silica gel plate by using chloroform and ethanol as devel- oping solvents, and the spot were detected by UV light absorption. IR spectra were recorded with by using KBR pellets method on SHIMADZU instrument. 1H NMR spec- tra were recorded on Bruker 300. Chemical shifts δ are given in ppm relative to the signal for TMS as internal standard. Mass spectral data collected of synthesized com- pound from Jassco instrument. The protocol for the syn- thesis of title compounds is presented in Scheme 1.
Synthesis of Ethyl(1H-benzimidazol-2-ylsulfanyl) Ace- tate (I)
A stirred mixture containing 4.5 gm of (0.03 mole) of 2-mercaptobenzimidazole, 60 ml of ethanol and 1.68 gm of (0.03 mole) potassium hydroxide was added and heated at 78−80oC for 10 min. Then ethyl chloro acetate (3.66 ml, 0.03 mole) was added in one portion, an exothermic reac- tion set in causing a temperature rise from 30−40oC. After stirring at 25−30oC for 18 h, the reaction mixture was added to 100 gm of ice-water and stirred for 30 min at 0−10oC.
The precipitate was collected by filtration washed with water until free of chloride and air dried at 50oC and recrystallized by water the yield is 6 gm (62.25%). melt- ing point is 105oC.
Synthesis of 2-(1H-benzimidazol-2-ylsulfanyl) Aceto- hydrazide (II)
The mixture of 2-carboxy ethyl thio 1H-benzimidazole 4 gm (0.004 mole) and Hydrazine hydrate 6 ml (0.01 mole) are mixed well in a RBF and heated on water bath for 10 min. then dissolved in 60 ml ethanol, the reaction mixture is heated with reflux condenser for six hours, cooled to room temperature and the reaction mixture was added to 100 gm of ice-water, and kept aside for the crystallization.
The colorless crystals are collected by filtration, and recrys- tallized from water. Melting point is 180−185oC; the yield is 60 to70%.
Synthesis of N'-(4-amino-5-sulfanyl-4H-1,2,4-triazole- 3-yl)-2-(1H-benzimidazole-2-ylsulfanyl) Acetohydrazide (III)
To continuously stirred solution of potassium hydrox- ide (2.565 g, 0.015 mole) and compound II in absolute dry ethanol (100 ml), carbon disulphide (2.7 ml, 0.015 mole) was added drop wise. After the complete addition the mix- ture was diluted the absolute ethanol (75 ml) and agitated for 16 h. It was then diluted with dry ether (100 ml) and precipitates solid collected by filtration washed with dry ether at 65oC. Then obtained crude suspension of potas- sium dithiocarbonate, to above suspension hydrazine hydrate (1.86 ml, 0.02 mole) was added and the mixture was reflux for 2 h the color of reaction mixture changed to green with
the evaluation of hydrogen sulphide gas and homoge- neous mass was obtained. It was then cooled and diluted with cold water (100 ml). The cold mixture was acidified with concentrated hydrochloric acid. The solid separated was filtered, washed with the water, dried and recrystal- lized from water. Melting point is 225oC; yield is 61.11%.
General Procedure for the Preparation of Schiff Bases Compound (2MB1) to (2MB8)
An equimolar solution of Compound III (0.009 mol, 2gm) is dissolved in 10ml of ethanol and to this solution substituted aldehydes in equimolar quantity (0.009mol) is added with 4−6 drops of glacial acetic acid was added, this reaction mixture is kept under reflux for 8 h. After cooling to room temperature was added to ice cold water. Compound gets separated as solid filtered, dried and recrystallized with Chloroform of ethanol. Melting point is noted in oC, the yield is mentioned in. %.
Physical and Spectral Data of Synthesized Compounds 2-(1H-benzimidazol-2-ylsulfanyl)-N'-(4-{[(E)-(4-hydroxy- phenyl) methylidene] amino}-5-sulfanyl-4, 5-dihydro- 3H-1, 2, 4-triazol-3-yl) acetohydrazide
(2MB1)
C18H16O2N8S2, mp 230−235oC, IR (KBr pellets, cm−1) 3116 (Alc OH), 2926 (NH), 2855 (Ar−CH), 1651 (ester >C=O), 1271 (C=N), 569 (C−S), 13C NMR (100 MHz, CDCl3):
Scheme 1. Synthesis of 2-mercapto benzimidazole derivatives, (2MB1) to (2MB8).
δ=31.34, 38.68, 39.70, 54.04, 52.17, 52.31, 56.56, 56.38, 72.28, 109.44, 157.48, 161.20, 165.94, 163.56, 169.38, 177.21. 1H NMR δ (DMSO) 10.8 (S, H, CHO), 9.0 (S, H, NH), 9.7 (S, H, CH2), 6.9−7.3 (M, 8H, Ar), Ms (m/z): 473 (M+).
2-(1H-benzimidazol-2-ylsulfanyl)-N'-(4-{[(E)-(3-bro- mophenyl) methylidene] amino}-5-sulfanyl-4, 5-dihy- dro-3H-1, 2, 4-triazol-3-yl) acetohydrazide
(
2MB2)C18H15ON8S2Br, mp 190−210 oC, IR (KBr pellets, cm−1) 3246 (NH), 2856 (Ar−CH), 1742 (ester >C=O), 1172 (C=N), 626 (C−S), 13C NMR (100 MHz, CDCl3): δ=30.34, 37.68, 37.70, 51.04, 52.17, 52.31, 52.56, 52.38, 72.28, 108.44, 154.48, 160.20, 165.94, 163.56, 169.38, 177.21. 1H NMR δ (DMSO), 8.6 (S, H, CH2), 7.5−7.7 (m, 8H, Ar), 7.2 (S, H, NH), Ms (m/z): 502 (M+).
2-(1H-benzimidazol-2-ylsulfanyl)-N'-(4-{[(E)-(3-chlo- rophenyl) methylidene] amino}-5-sulfanyl-4, 5-dihy- dro-3H-1,2, 4-triazol-3-yl) acetohydrazide
(2MB3)
C18H15ON8S2Cl, mp 170−185oC, IR (KBr pellets, cm−1) 744 (C−Cl), 2922 (Ar−CH), 1710 (ester >C=O), 1177 (C=N), 670 (C−S), 13C NMR (100 MHz, CDCl3): δ=30.34, 37.68, 37.70, 51.04, 54.17, 54.31, 52.56, 52.38, 72.28, 108.44, 154.48, 160.20, 168.94, 163.56, 170.38, 177.21. 1H NMR δ (DMSO), 8.8 (S, H, CH2), 7.3−7.7 (m, 8H, Ar), 7.1 (S, H, NH), Ms (m/z): 458 (M+).
2-(1H-benzimidazol-2-ylsulfanyl)-N'-(4-{[(E)-(3-meth- oxyphenyl) methylidene] amino}-5-sulfanyl-4, 5-dihydro- 3H-1,2,4-triazol-3-yl) acetohydrazide
(2MB4)
C19H19O2N8S2, mp 215−220oC, IR(KBr pellets, cm−1) 3215 (NH), 3001 (Ar−CH), 1699 (ester >C=O), 1320 (C=N), 609(C−S) 13C NMR (100 MHz, CDCl3): δ=30.34, 37.68, 37.70, 57.04, 51.17, 52.31, 52.56, 52.38, 71.28, 106.44, 154.48, 160.20, 166.94, 163.56, 169.38, 177.21, 1H NMR δ (DMSO), 8.1 (S, H, CH2), 7.7 (m, 8H, Ar), 7.1 (S, H, NH), Ms (m/z): 400 (M+).
2-(1H-benzimidazol-2-ylsulfanyl)-N'-(4-{[(E)-(3-nitro- phenyl) methylidene] amino}-5-sulfanyl-4, 5-dihydro- 3H-1,2,4-triazol-3-yl) acetohydrazide
(2MB5)
C19H15O3N9S2, mp 195−200oC, IR (KBr pellets, cm−1) 3315 (NH), 3019 (Ar−CH), 1753 (ester >C=O), 2259 (C=N), 672 (C−S), 13C NMR (100 MHz, CDCl3): δ=37.34, 37.68, 37.70, 51.04, 52.17, 52.31, 52.56, 52.38, 72.28, 108.44,
159.48, 160.20, 167.94, 163.56, 169.38, 177.21, 1H NMR δ (DMSO), 8.2 (S, H, CH2), 7.5 (m, 8H, Ar), 7.0 (S, H, NH), Ms (m/z): 480 (M+).
2-(1H-benzimidazol-2-ylsulfanyl)-N'-(4-{[(E)-(4-meth- oxyphenyl) methylidene] amino}-5-sulfanyl-4,5-dihydro- 3H-1,2,4-triazol-3-yl) acetohydrazide
(2MB6)
C19H19O2N8S2, mp 210−235oC, IR (KBr pellets, cm−1) 3001 (NH), 1660 (ester >C=O), 1157 (CN), 1041 (−O−), 609 (C−S) 13C NMR (100 MHz, CDCl3): δ=37.34, 37.68, 37.70, 50.04, 51.17, 52.31, 52.56, 52.38, 71.28, 106.44, 154.48, 160.20, 165.94, 163.56, 169.38, 177.21. 1H NMR δ (DMSO), 8.0 (S, H), 7.1 (m, 8H, Ar), 7.7 (NH), Ms (m/
z): 455 (M+).
2-(1H-benzimidazol-2-ylsulfanyl)-N'-(4-{[(E)-phenyl- methylidene] amino}-5-sulfanyl-4,5-dihydro-3H-1,2, 4-triazol-3-yl) acetohydrazide
(2MB7)
C18H15ON8S2, mp 210−215oC, IR (KBr pellets, cm−1) 3500 (NH), 3128 (Ar−CH), 1672 (ester >C=O), 1274 (CN), 2384 (CH=O), 688 (C−S), 13C NMR (100 MHz, CDCl3):
δ=30.34, 38.68, 39.70, 51.04, 55.17, 52.31, 55.56, 52.38, 72.28, 108.44, 154.48, 162.20, 165.94, 163.56, 169.38, 177.21, 1H NMR δ (DMSO), 8.1 (S, CH2), 7.5−7.7 (m, 8H, Ar), 7.6 (S, H, NH), Ms (m/z): 423 (M+).
2-(1H-benzimidazol-2-ylsulfanyl)-N'-(4-{[(E)-(4-meth- ylphenyl) methylidene] amino}-5-sulfanyl-4,5-dihydro- 3H-1,2,4-triazol-3-yl) acetohydrazide
(2MB8)
C19H19ON8S2, mp 185−190oC, 3127 (Ar−CH), 1604 (ester >C=O), 1341 (CN), 2704 (HC=O), 749 (C−S), 13C NMR (100 MHz, CDCl3): δ=32.34, 32.68, 36.70, 52.04, 52.17, 52.31, 52.56, 52.38, 72.28, 108.44, 157.48, 160.20, 165.94, 163.56, 169.38, 177.21, 1H NMR δ (DMSO), 8.2 (S, CH2), 7.2 (m, 8H, Ar), 7.1 (S, H, NH), Ms (m/z): 440 (M+).
RESULTS AND DISCUSSION
In this article, we wish to report the synthesis of novel’- (4-amino-5-sulfanyl-4H-1,2,4-triazole-3-yl)-2-(1H-benz- imidazole-2-ylsulfanyl) acetohydrazide (III) i.e., 2-mer- capto benzimidazole derivatives is in good yields which synthesized from 2-(1H-benzimidazol-2-ylsulfanyl) ace- tohydrazide. The un-substituted 2-mercapto benzimida- zole was prepared from o-phenylene diamine and carbon disulfide in presence of KOH in single step. The synthesis
of N4-amino-1,2,4-triazoles with unsymmetrical substit- uents at 3,5-positions proceeds through the formation of the intermediate unsymmetrical N,N-diacyl hydrazines.
The N4-amino-1,2,4-triazoles can be condensed with carbo- nyl compounds to give a type of sterically crowded hydra- zones where the terminal nitrogen of the hydrazone is locked in a ring system which represent a group of inter- esting compounds for stereochemical studies. The benz- imidazole derivatives further converted in to respective ethyl (1-H-benzimidazol-2-ylsulfanyl)-acetate by react- ing with Ethyl Chloro-acetate in presence of KOH. The 2- mercapto benzimidazole acetate further converted to hydrazides by reacting with Hydrazine Hydrate. Then the resultant compound stirred for 16 h in presence of carbon disulphide and potassium hydroxide, this sus- pension shifted for reflation with the hydrazine hydrate for 2 h. Then the expected final product is further reacted with different substituted aromatic and hetero cyclic alde- hydes in the presence of acetic acid as catalyst in ethanol by refluxing for 8 h to yield the different derivatives of benzimidazoles. The synthesized compounds were char- acterized by TLC, Melting point and Spectral data.
ANTIMICROBIAL ACTIVITY
The antibacterial activity of synthesized compounds was evaluated by the agar well diffusion method. All the bacterial cultures were adjusted to 0.5 McFarland stan- dards, which is visually comparable to a bacterial suspension of approximately 1.5×108 cfu/ml. 10 mL of nutrient agar medium was poured into each Petri plate and plates were swabbed with 100 µL inocula of the test microorganisms and kept for 10 to 15 min for adsorption. Using sterile cork borer of 8 to 10 mm diameter, wells were bored into the seeded agar plates and these were loaded with a 100 µL volume with concentration of 2.0 mg mL−1 of each com- pounds reconstituted in the dimethylsulphoxide (DMSO).
All the plates were incubated at 37 ºC for 24 h. Antibacterial activity of each complex was evaluated by measuring the zone of growth inhibition against the test organisms with zone reader (HiAntibiotic zone scale). DMSO was used as a negative control whereas Ciprofloxacin was used as pos- itive control.20 This procedure was performed in three rep- licate plates for each organism.
The antimicrobial screening results presented in Table 1 reveal that compounds 2MB1, 2MB3, 2MB5 exhibited sat- isfactory effect against S.aureus, and E.coli, while the compounds 2MB2, 2MB6, 2MB7 have shown the moder- ate activity against the same organism. The zone inhibition of
synthesized compound compared with the standard. The same Compounds also screened for the anti-fungal activity against Candida albicans the compounds 2MB3, 2MB2, 2MB4 Showed highest degree of inhibition and the com- pound 2MB5, 2MB1 against C.albicans when compared with the Standard drug ketoconazole.
ANTI-INFLAMMATORY ACTIVITY
The suspensions of test compounds were prepared in sterile 0.9% NaCl solution. In all cases control received the same quantity of sterile 0.9% NaCl solution as vehicle.
Anti-inflammatory activity was evaluated by carrageenan induced rat paw edema method of Winter et al. albino rats of either sex weighing between 150−200 g were ran- domly distributed in control and experimental group of six animals. At 0 hr the test compounds (20 mg/kg) and standards (5 mg/kg) doses were administered orally. 1 h after compounds and standards were administered orally, carrageenan in distilled water 0.1 ml of 1% (w/v) suspension was injected into the planter tissue of right paw of rat.
The paw was measured by plethysmometer within 30 sec.
of injection. The relative increase in paw volume was found by re-measuring the paw volume after 3 of carrageenan injection. The % inhibition of edema was calculated by following formula and the results are shown in Table 2.
ANALGESIC ACTIVITY
Analgesic activity was determined in vivo by calculating total number of writhing, following intraperitonial (I.P.) Table 1. Anti-bacterial activity of synthesized 2-Mercaptobenzim- idazole derivatives
Sr. No. Compounds Zone of Inhibition (in mm) E.Coli S. Aureus C. Albicans
1 2MB1 15 13 18
2 2MB2 13 11 12
3 2MB3 17 16 14
4 2MB4 12 13 16
5 2MB5 13 17 09
6 2MB6 10 08 11
7 2MB7 08 11 12
8 2MB8 12 07 10
9 S 24 25 --
10 K − − 20
11 B − − −
S−Standard–Ampicilline, B–Blank–DMSO, K−Ketokonazole, Zone of inhibition of synthesized compounds: 6−8 mm poor activity, 9−11 mm moderate activity, 12−15 above good
administration of 0.6% (0.1 ml/10 g) acetic acid in mice.
Albino mice of either sex (25−30 g) were used. Synthe- sized compounds 2MB1-2MB8 were administered intra- peritonial (20 mg/kg) as a suspension in sterile 0.9% NaCl solution as vehicle. Pentazocine (5 mg/kg p.o.) was used as the standard drug under same condition. Acetic acid solu- tion was administered intraperitonial 30 min after admin- istration of the compounds. 10 min after intraperitonial injection of the acetic acid solution, the number of writings’
per animal was recorded for 20 min. Control animals received an equal volume of vehicle. Analgesic activity was expressed as percentage of inhibition of number of writhing when compared with the vehicle control group. The Analgesic activity of synthesized compounds in Mice shown in Table 3.
CONCLUSION
In conclusion, N'-(4-amino-5-sulfanyl-4H-1,2,4-triazole- 3-yl)-2-(1H−benzimidazole-2-ylsulfanyl) acetohydrazide (2MB1 to2MB8) has been designed and synthesized. From the data of the Table 1, 2 and 3, the pharmacological and bio- logical screening it is clearly concluded that the synthesized compounds are promisingly significant good antimicro- bial, antifungal, anti-inflammatory and analgesic agent. As per the results of screening it is clearly indicated that the compounds of the scheme have shown good antibacterial and antifungal activity equipotent with the standard drugs.
This is because of the presence of groups like −OCH3,
−NO2, −Br, −N−CH3, at the different positions of phenyl nucleus and heterocyclic system attached to benzimida- zole nucleus which is attached to benzimidazole mole-
cule. The substituted benzimidazole moieties are already known for different biological activities.
Acknowledgments. Author is thankful to the Principal Prof Dr. N.V. Kalyane and Management of B.L.D.E.A college of Pharmacy, Bijapur for providing the necessary facilities to carry out this work. And the publication cost of this paper was supported by the Korean Chemical Society.
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Table 2. Anti-inflammatory activity of synthesized 2-mercaptoben- zimidazole derivatives
Compound code Change in Paw edema
at the end inhibition % Inhibition
Controle 0.42 ± 0.07 −
Standard 0.16 ± 0.01 61.9**
2MB1 0.23 ± 0.04 45.2**
2MB2 0.48 ± 0.02 21.31*
2MB3 0.28 ± 0.03 33.3*
2MB4 0.31 ± 0.06 26.2*
2MB5 0.39 ± 0.01 36.06**
2MB6 0.41 ± 0.01 32.78*
2MB7 0.47 ± 0.01 22.95*
2MB8 0.24 ± 0.02 42.9*
Standard drug = Diclofenac sodium, Dose = 5 mg/kg. Synthesized compound = 20 mg/kg. One way ANOVA followed by multiple tukeys comparison test *P≤0.05, **P≤0.01, ***P≤0.001 when com- pared with control
Table 3. Analgesic activity of synthesized compound in Mice Sr.
No. Derivatives Dosage
Number of Writhing in 10 minutes (mean ± SEM)
% Inhibition
1 Control Vehicle 79.60 ± 1.98 --
2 2MB1 20 mg/kg 32.33 ± 2.30*** 59.68
3 2MB2 20 mg/kg 49.16 ± 1.018* 59.68
4 2MB3 20 mg/kg 57.50 ± 2.57 27.76
5 2MB4 20 mg/kg 35.5 ± 1.88*** 55.40
6 2MB5 20 mg/kg 58.50 ± 2.58 26.50
7 2MB6 20 mg/kg 52.66 ± 3.77 33.84
8 2MB7 20 mg/kg 33.66 ± 3.47*** 57.78
9 2MB8 20 mg/kg 45.5 ± 1.88 ** 42.83
10 Pentazocine 5 mg/kg 21.66 ± 1.06*** 72.78 One way ANOVA followed by multiple tukeys comparison test.
*P≤0.05, **P≤0.01, ***P≤0.001 when compared with control
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