*Corresponding Author:
Bhawani singh
Organic Research Laboratory, Department of Chemistry, Banasthali University, Banasthali-304 022
E-mail: bsyadav[email protected]
Date of Submission 30 December 2009
Date of Revision 9 September 2010
Date of Acceptance 25 September 2010
Indian J. Pharm. Sci., 2010, 72 (5): 607-612  

Abstract

Thiazolidin-4-one fused pyrimidines, [1,5]-benzodiazepines and their oxygen substituted hydroxylamine derivatives have been screened for antibacterial, antifungal and antimalarial activity. Bacillus subtilis, Escherichia coli, Proteus mirabilis and Salmonella typhi were used for antibacterial screening. Aspergillus fumigatus and Candida albicans were used for antifungal screening and Plasmodium species were used for antimalarial screening. The antibacterial and antifungal activities are expressed in terms of zone of inhibition and antimalarial activity is expressed in IC 50 value. Fifteen compounds 2Xa, 2Xb, 2Xc, 2Xs, 3IV, 3Va, 3Vc, 3VIIIa, 3VIIIh, 3IXa, 3IXb, 3IXc, 3Xa, 4IXa and 4Xa were tested for antibacterial as well as antifungal activity and seven compounds 2IXb, 2Xb, 3VIIIc, 3Xc, 4IXa, 4Xa and 4IXw were tested for antimalarial activity. Streptomycin, griseofulvin and chloroquine were taken as standard drugs in antibacterial, antifungal and antimalarial activity, respectively. The compound 2Xs was found significant antimicrobial against Bacillus subtilis, E. coli, Aspergillus fumigatus and Candida albicans as well as compound 3Xa was significant antimicrobial against Bacillus subtilis, E. coli, Salmonella typhi, Aspergillus fumigatus and Candida albicans. The compound 2Xb showed significant antimalarial activity.

Keywords

[1,5]-benzodiazepines, IC50 value, oxygen substituted hydroxylamine derivatives, pyrimidines, quantitative structure activity relationship (QSAR), Thiazolidin-4-ones, zone of inhibition

Literature survey revealed that thiazolidin-4-ones and their derivatives, which have been synthesized and screened for antimicrobial activity, are found biologically active with anticonvulsant [1], antitubercular [2], antifungal [3], local anesthetic/ antiHIV [4], and antiinfl ammatory activity [5] with dual cyclooxygenase/lipooxygenase inhibition [6]. Literature survey also revealed that thiazolidin-4-ones have been used as new SHP-2 [7], and protein tyrosine phosphatase-1B inhibitors [8]. Like thiazolidin-4- ones, pyrimidines also play a vital role in many biological processes and have potential anticancer, antiinflammatory and analgesic activities [9,10]. Some pyrimidines and their derivatives are found to possess antiHIV activity [11]. It has also been found that 2-phenylaminopyrimidines possess protein tyrosine kinase inhibitor activity [12]. [1,5]-benzodiazepines are widely used as sedatives, sleep inducers, anesthetics, anticonvulsants, muscle relaxants and also as tranquilizers since 1960 when chlordiazepoxide was introduced as a tranquilizer.

Dilazep, a non-nucleoside reverse transcriptase inhibitor (NNRTI) has also been evaluated for the treatment of kidney disease such as chronic nephritis and its tablets (containing dilazep HCl, lactose, starch and cellulose-20:20:50:5) are used for improving hematopoiesis. Some arylpyridodiazepine and thidiazepine derivatives are highly selective HIV-1 inhibitors [13]. The antiarrhythmic activity of derivatives of dibenzazepine have been studied and found that the most active compounds are those in which carbethoxyamine groups in position-3 in combination with dimethylamino or diethylaminoacetyl groups in position-5 of dibenzazepine ring are present [14]. Literature survey revealed that hydroxylamines and their oxygen-substituted derivatives are reversible inhibitors of allinase. These are also potent inhibitors of aminobutyrate and aminotransferase in Pseudomonas.

The aminooxy compounds possess broad spectrum antibacterial activity. Aminooxy compounds have also been known for some time as potent inhibitors of pyridoxal-5-phosphate dependent enzymes such as aminotransferase, serine hydroxymethyltransferase, tyrosine decarboxylase, cystathionase and ornithine decarboxylase [15,16]. It is mostly true that even minor changes in structure of a molecule with appropriate substituent may change its pharmacological profile appreciably. Keeping in mind this concept, thiazolidin- 4-one condensed pyrimidines, thiazolidin-4-one condensed [1,5]-benzodiazepines and their oxygen substituted hydroxylamine derivatives have been synthesized [17-19] and screened for antimicrobial activity to study the quantitative structure activity relationship between substituents present on the basic nucleus and activity alteration.

Materials and Methods

Antibacterial activity

Nutrient agar medium was used for culture of bacteria in which beef extract (3 g), peptone (5 g), sodium chloride (5 g) and agar-agar (15 g) were mixed in 1000 ml distilled water. The nutrient agar medium was sterilized by autoclaving at 15 psi and 121° for 20 min. The medium was poured in Petri dishes and left for some time to solidify. These Petri dishes were inoculated with 0.2 ml suspension of organisms using the cup-plate method [20]. Four wells were made in the medium with the help of a sterile borer and subsequently these wells were filled with four different concentrations (250, 500, 750 and 1000 ppm) of the synthesized compounds using well diffusion method [21]. Streptomycin (250, 500, 750 and 1000 ppm) was used as reference drug. The Petri dishes were incubated at 37° in an incubator and examined for the zone of inhibition [22] after 24-48 h. Bacillus subtilis (Org. 1), Escherichia coli (Org. 2), Proteus mirabilis (Org. 3) and Salmonella typhi (Org. 4) were taken for antibacterial activity.

Antifungal activity

Sabouraud agar medium was used for culture of fungi in which glucose (20 g), peptone (10 g) and agar-agar (15 g) were mixed in 1000 ml distilled water. The procedure explained in the antibacterial activity was followed. Aspergillus fumigatus (Org. 5) and Candida albicans (Org. 6) were used for antifungal activity.

In vitro antimalarial activity

Plasmodium falsiparum species were grown, adapted and maintained in vitro routinely. For conducting the experiment, the P. falciparum culture was synchronized to ring stage and diluted with fresh human erythrocytes to adjust the level of parasitaemia between 1000 to 80 000/μl of blood. The in vitro assay of inhibition of schizont maturation was followed for screening the antimalarial activity of the compounds. The experiment was done in 24 well microtire plates in the presence of various concentrations of drugs followed by standard method[23,24]. The control wells were without drug. The growth was monitored after 24-36 h of culture when the parasites would have developed to schizont stage. Thick smear was prepared from each well. The schizont maturation in experimental well was compared with control well. Percent inhibition was calculated according to the following formula: % inhibition=100–(No. of schizonts or 200 asexual parasites in test well/ No. of schizonts or 200 asexual parasites in control well)×100.

In vivo antimalarial screening (Blood schizontocidal activity)

Healthy Swiss albino mice (4-6 weeks old) and P. berghei, sensitive to chloroquine, were used in the study. Peter’s 4-day test [25] was followed to evaluate the blood schizontocidal action against P. berghei in Swiss albino mice. The animals were divided into groups consisting of 5 mice each. All the groups were injected P. berghei infected red blood cells in a volume of 0.1 ml (diluted in phosphate buffer solution, PBS) on day 0 intraperitoneally. All the experimental groups received the drugs in different concentration on day 0 to day 3. Control group received only PBS and one group was given chloroquine (IC50 value= 0.03 μg/ml) 3 mg/kg as a standard antimalarial drug. On day 4, thin blood smears were prepared from the tail vein to monitor the parasitaemia. Percent inhibition of the parasitaemia is calculated against the control group. The chloroquine recipients group was negative throughout.

Results and Discussion

Results of antibacterial activity are summarized in Table 1. The zone of inhibition was measured in mm for each concentration. All of the screened compounds were found to have moderate to significant antibacterial activity. Compound 2Xa showed very significant activity against Bacillus subtilis and Proteus mirabilis but moderate activity against E. coli and Salmonella typhi. Compounds 2Xb, 2Xc and 2Xs were found to exhibit very significant activity against Bacillus subtilis and moderate activity against E. coli, Proteus mirabilis and Salmonella typhi. It was also observed that antibacterial activity was increased with introducing chloro group at para-position in phenylimino moiety but decreased with nitro group at same position. Antibacterial activity was also increased when number of carbon atoms increased in alkoxyphthalimide moiety. Compounds 3IV, 3Va, 3Vc, 3VIIIa, 3VIIIh, 3IXa, 3IXb and 3IXc showed signifi cant antibacterial activity while compound 3Xa was found to show more significant antibacterial activity than standard drug. It was also observed that when alkoxyphthalimide group was introduced in thiazolidinone unit as well as number of carbon atoms was increased in alkoxyphthalimide group, the antibacterial activity was also increased. Introduction of arylidene unit at position-5 in thiazolidinone moiety also increases the activity but hydroxyl group at p-position in arylidene unit the decreases activity. Thiazolidinopyrimidines were found to show higher activity than 5-arylidene-4-thiazolidinones. Compounds 4IXa and 4Xa were exhibited moderate to signifi cant activity against all of the organisms. Activity was also increased with introduction of alkoxyphthalimide group in the [1,5]-benzodiazepines.

Results of antifungal activity are given in Table 2. The zone of inhibition was measured in mm for each concentration. All of the tested compounds were found to exhibit very significant antifungal activity against Aspergillus fumigatus and Candida albicans. Growth of mycelia was inhibited maximum at 1000 ppm while minimum at 250 ppm. Antifungal activity increased with introduction of chloro group at arylimino moiety whereas decreased when nitro group was introduced at the same position. Although all compounds were exhibited strong antifungal activity yet [1,5]-benzodiazepines were found to show lower activity than thiazolidinopyrimidines.

Results for antimalarial activity are mentioned in Table 3. Compounds 2Xb and 3Xc have been found to show signifi cant antimalarial activity than the chloroquine. Other compounds have showed mild to moderate activity. It may further conclude that introduction of alkoxyphthalimide group in thiazolidinopyrimidine moiety increases to antimalarial activity. It was also observed that antimalarial activity was slightly increased on increasing length of alkyl chain in alkoxyphthalimide unit. Presence of chloro group at the para-position also increases to activity. Thiazolidinopyrimidines have been found to possess higher activity than 5-arylidene-4-thiazolidinones.

Alkoxyphathalimide derivative of thiazolidinopyrimidines were found to have signifi cant antifungal, antibacterial and antimalarial activity. Activity was increased with introduction of chloro group at the para-position in arylimino moiety and with increasing of length of alkyl chain in alkoxyphthalimide group whereas it is decreased when hydroxyl group was introduced. Structures of compounds 2Xa, 2Xb, 2Xc and 2Xs are shown in fig. 1. The antibacterial and antifungal activities were increased when the arylidene unit was introduced in thiazolidinone nucleus at position-5 but activity was decreased when hydroxyl group was present at para-position in arylidene unit. Structures of the compounds are shown in figs. 2 and 3. Compounds 2Xs, 3Xa and 4Xa were found to exhibit signifi cant antimicrobial activities. These results suggest that antimicrobial activities are strongly affected by the presence or absense of alkoxyphthalimide moiety and hence compound 3Xa and 4Xa showed signifi cant activity than compounds 3IXa and 4IXa (comparison in figs. 3 and 4). Although [1,5]-benzodiazepines have strong antibacterial and antifungal activity but it was found to show slightly lower activity than alkoxyphthalimide derivatives of thiazolidinopyrimidines.

Code of Bacillus subtilis(Org. 1) Escherichia coli (Org. 2) Proteus mirabilis (Org. 3) Salmonella typhi(Org. 4)
Compounds 250 500 750 1000 250 500 750 1000 250 500 750 1000 250 500 750 1000
  ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm
2Xa 16* 17* 24* 24* 06 07 07 08 15* 17* 19* 20* 04 05 05 06
2Xb 16* 16* 22* 23* 14* 15* 16* 17* 11 12 14 14 06F 07F 08F 08F
2Xc 12* 13* 15* 16* 07 08 11 12 14 18 20 20 05 06 07 08
2Xs 18* 18* 24* 24* 12F 15F 15F 16F 14 18 21 22 07F 06F 07F 07F
3IV 10F 12F 13F 15F 13F 14F 15F 11F 10 11 12 12 04 06 07 08
3Va 12F 16F 18F 18F 09 10 10 11 12F 14F 15F 17F 05 06 08 09
3Vc 17F 21F 20F 24F 10 12 12 12 13F 13F 18F 18F 06F 08F 10F 12F
3VIIIa 20* 22* 25* 26* 11 12 12 14 20* 17* 17* 18 07F 08F 08F 13F
3VIIIh 06 08 11 12 07 08 08 09 13F 13F 16F 17F 07F 06F 07F 11F
3IXa 13F 16F 17F 18F 08 08 09 10 16* 18* 19* 22* 04 06 06 16
3IXb 10 09 11 12 07 08 09 09 10 12 13 14 06F 09F 11F 12F
3IXc 11 10 12 12 08 10 12 14 17* 16* 18* 19* 07 08 09 13
3Xa 25* 27* 27* 27* 11 13 12 15 18* 19* 19* 23* 22* 25* 26* 27*
4IXa 16F 13F 17F 17F 09 08 14 16 11 12 12 13 06F 08F 09F 10F
4Xa 07 08 08 09 12 15 15 16 12 12 14 15 07F 09F 10F 11F
Streptomycin 08 09 10 12 10 10 11 12 10 12 13 14 03 04 06 08

Table 1: Antibacterial Activity

Code of   Aspergillusfumigatus(Org. 5)       Candida albicans(Org. 6)  
Compounds 250 ppm 500 ppm 750 ppm 1000 ppm   250 ppm 500 ppm 750 ppm 1000 ppm
2Xa 18 19 21 21 15 16 18 18
2Xb 19 21 22 23 16 16 19 20
2Xc 17 16 18 18 18 17 19 21
2Xs 20 21 22 22 20 22 30 30
3IV 15 18 19 21 17 18 20 25
3Va 16 17 16 22 17 17 23 23
3Vc 17 19 20 24 18 18 24 24
3VIIIa 15 18 20 24 16 18 24 25
3VIIIh 11 17 19 23 18 22 24 23
3IXa 11 19 20 24 17 18 24 25
3IXb 09 15 18 18 16 18 18 20
3IXc 17 18 19 21 18 22 23 24
3Xa 11 19 20 25 17 25 29 29
4IXa 08 11 21 23 16 16 18 20
4Xa 10 12 14 18 16 18 18 20
Griseofulvin 08 09 10 12 13 13 14 15

Table 2: Antifungal Activity.

The compounds 2Xb, 2Xs, 2Vc and 2Xa were found to possess significant antibacterial activity while compounds 2Xa, 3Va, 3VIIIa, 3IXa, 3IXc, 4IXa and 4Xa showed moderate activity against Bacillus subtilis. The compound 2Xs possessed strong activity but other compounds showed moderate to good activity against E. coli. All of the examined compounds were found to show moderate activity against Proteus mirabilis. The compound 3Xa showed signifi cant activity against Salmonella typhi whereas other compounds possessed moderate to good activity. All compounds were found to possess very strong activity against Aspergillus fumigatus and Candida albicans.

Based on the above study, it can be concluded that the compound 2Xs i.e. 7-N-(4-butoxyphthalimido)- 2-hydroxy-4-phenyl-6-phenyliminothiazolidino[2,3-b] pyrimidine was found to have signifi cant antimicrobial activity against Bacillus subtilis, E. coli, Aspergillus fumigatus and Candida albicans as well as the compound 3Xa i.e. p-Bis-(7-{2-ethoxyphthalimido)- 2-hydroxy-4-phenyl-6-iminothiazolidino[2,3-b] pyrimidine-N2-yl)biphenyl was found to possess significant antimicrobial activity against Bacillus subtilis, E. coli, Salmonella typhi, Aspergillus fumigatus and Candida albicans. The compound 2Xb i.e. 7-N-(4-ethoxyphthalimido)-2-hydroxy-4-phenyl-6-(4- cholrophenyl)iminothiazolidino[2,3-b]pyrimidine was found to possess signifi cant antimalarial activity fig. 5.

Code of Compounds Sample No. IC50 Value (µg/ml)
2IXb 13 19.0
2Xb 16 02.0
3VIIIc 53 15.0
3Xc 55 05.0
4IXa 90 110.0
4Xa 92 80.0
4Xw 98 75.0
Chloroquine SD 0.03
IC50 values of various compounds.

Table 3: Antimalarial Activity.

Figure

Figure 1:General structure of compounds synthesized[17].
2Xa (X=H, Z=H, n=2), 2Xb (X=Cl, Z=H, n=2), 2Xc (X=NO2, Z=H, n=2) and 2Xs (X=H, Z=H, n=4)

Figure

Figure 2:General structure of compounds synthesized[18]
3Va (n=2), 3Vb (n=2) and 3Vc (n=4).

Figure

Figure 3:General structure of compounds synthesized[18] 3VIIIa (Z=H, n=2), 3VIIIh (Z=OH, n=4), 3IXa (Z=H), 3IXb (Z=OH), 3IXc (Z=OCH3) and 3Xa (Z=H, n=2)

Figure

Figure 4:General structure of compounds synthesized[19]
4IXa (X=H, Z=H), 4Xa (X=H, Z=H, n=2) and 4Xw (X=Cl, Z=OH, n=4).

Figure

Figure 5:Compounds with signifi cant antimicrobial activity (a) Antimalarial compound, (b and c) antibacterial and antifungal
compounds.

Acknowledgements

The authors are thankful to the Head, Department of Chemistry, M. L. Sukhadia University, Udaipur (Rajasthan) for providing laboratory facilities to synthesize the compounds, to the Head, Department of Botany, M. L. Sukhadia University, Udaipur (Rajasthan) for providing laboratory facilities to screen the microbiological activity and Dr. C. Usha Devi, Scientist, Malaria Research Centre, Indian Council of Medical Research, Delhi for screening of antimalarial activity of the synthesized compounds.

References