l'olia Microbiol.

40 (6), 611-614 (1995)

Antibacterial Effect of Substituted 4-Q.uinazolylhydrazines and "l-heirArylhydrazones Determined by a Modified Microdilution Method
S. JANTOV,A,a,D. HUDECOV,A,a,~. STANKOVSK~, K. ~PIRKOVA and g. RU~EKOV/t,a b
aDepanmem of Microbiology, Biochemistry and Biology bDepartment of Organic Chemistry, Faculty of Chemical Technology, Slovak Technical University, 812 37 Bratislava, SIovakia
Dedicated to Professor Vladimfr lklina, DSc., on the occasion of his 65th birthday

Received .Tune 14, 1995

ABKrRACT. Eight 4-quinazolylhydrazines and eleven their arylhydrazones have been tested for antibacterial effects and for structure-activity relationships by a modified microdilution method. The derivative 6-chloro-2-morpholino-4-quinazolyt5'-nitro-2'-furylhydrazone had the highest antibacterial effect, the MIC values being 100 mg/L for E. fcecalis, 250 mg/L for S. aureus, 200 mg/L for P. aeruginosa and 350 mg/L forE. coil The most effective derivatives were those with the benzene ring substituted with chlorine or methyl group in position 6 or 8 and with pyrimidine ring substituted with a secondary amine in position 2. The modified microdilution method did not give rise to any statistically significant deviations in the MIC values for ampicillin in comparison with reported reference collection values.

Many derivatives of quinazoline, which have a substituent with -N-N- grouping on their pyrimidine ring, were tested for a biological effect. Quinazolines substituted with diazo-, hydrazido-, and hydrazino- groups were reported to exhibit antibacterial effects, action on CNS and inhibition of monoamine oxidase (Saksena et al. 1988). Also 2-alkylhydrazinoquinazolines act as inhibitors of monoamine oxidase and have a spasmolytic effect (Kottke and Kiihmstedt 1978). Certain antibacterial activity of 2- or 4-hydrazinoquinazoline thiosemicarbazides was also described (Omar et aL 1981; Jantov~i et al. 1994). To study this broad-range biological activity of quinazoline derivatives, Stankovsky and Sokyrov~i (1984), and ~pirkov~i et aL (1993, 1994) prepared a series of hydrazinoquinazolines (1-8) and quinazolylarylhydrazones (9-19), substituted by known pharmacophores (halogen, nitro group, on secondary amines such as morpholine and piperidine).

X
1-8

NH --NH 2

NH-N=CH-Z
9-19

X
1 2 3 6-CI 6-CI 6-CI H H 8-Me 8-Me

Y
Mo Pi Ph Pi Ph Mo Ph 9 10 11 12 13 14 15 16 17 18 19

X
6-CI 6-CI 6-CI 6-CI 6-CI 6-CI 6-CI 8-Me 8-Me 8-Me 8-Me

Y
Mo Mo Mo Mo Mo Mo Mo Ph Ph Ph Ph

4
5 6 7 8

6-Br Mo

Mo - morpholinyl Pi - piperidinyl

Z 2'-furyl 4'-nitrophenyl 2'-nitrophenyl 2'-chlorophenyl 4'-acetamidophenyl 5'-bromo-2'-furyl 5'-nitro-2'-furyl 2'-furyl phenyl 4'-N,N-dimethylaminophenyl 4'-nitrophenyl

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The antibacterial effect of these eight hydr~ones and eleven arylhydr~ones was studied by a modified microdilution method and the relationship between this antibacterial effect and the structure of synthetically prepared quinazolines was evaluated.

MATERIALS AND METHODS

Materials. Bacterial strains Escherichia coli CCM 3988, Pseudomonas aeruginosa CCM 3955, Staphylococcus aureus CCM 3953 and Enterococcus faecalis CCM 4224 were used. The preparation of compounds (1-19) has been described by Stankovsl~ and Sokyrov;t (1984) and ~pirkov;t et al. (1993,
1994). Starting 4-hydrazinoquinazolines ( 1 - 8 ) were prepared by hydrazinolysis of quinazoline4-thiones. Treatment of hydrazinoderivatives with corresponding arylcarbaldehydes yielded arylaldehyde 4-quinazolylhydrazones (9-19). The compounds were used at concentrations of 1000, 500, 250, 100 and 10 mg/L. Chromatographically pure derivatives were dissolved in dimethyl sulfoxide whose final concentration never exceeded 1 % (V/V) in either control or treated samples. Antibacterial assay. The antibacterial effect of the quinazoline derivatives was assayed by a microdilution method in 96-well microtitration plates (Kneiflov~i 1988) which we modified. The bacteria were kept on Miiller-Hinton agar or Nutrient Broth agar (E. faecalis) at 4 ~ and were subcultured every 7 d. Two days before the test the bacterial strains were transferred to fresh Miiller-Hinton or Nutrient Broth agars. An overnight inoculum was prepared 12 to 16 h before the test and was cultured overnight on a reciprocal shaker in a thermostat at 30 or 37 ~ (E. ftecalis). The growing inoculum was then filtered through gauze and a 1.5 % suspension of bacteria was prepared for the experiments. The bacterial suspension (180 laL) prepared in this way was then added to 20 laL of the tested derivative which was present in every well of the microtitration plate. This procedure was performed by an 8channel pipette. The first well of each plate was taken as blank and contained 200 o.L liquid culturing medium with 1 % solvent. The time course of absorbance (/1630) was then determined in three or seven (with ampicillin) parallels (reference ~ = 0 nm). To compare the antimicrobial activity, ampicillin (AMP) in concentrations 5.0, 2.5, 1.0, 0.1, 0.05 and 0.01 mg/L was used as standard. After 8-1] culturing with the tested derivatives, the bacteria were inoculated to a solid culturing medium and cultured statically for 24 h with effective derivatives at 37 ~ The microbistatic (MBS) or microbicide (MBC) concentration was determined. The antibacterial effect was characterised by IC50 values, i.e. the minimal inhibition concentration of a substance which inhibits bacterial growth by 50 % relative to the control, and MIC, i.e. the minimal inhibitory concentration of a substance which inhibits the bacterial growth by 100 %. The IC50 and MIC values were determined from toxicity curves.

R E S U L T S A N D DISCUSSION IC50 and MIC values obtained in the tests are summarized in Table I. The broadest antibacterial effect was found with derivatives 3, 7, 8, 15 and 18 which were effective with all the tested bacteria (IC50 < 350 rag/L). No antibacterial activity was found with derivative 12. The sensitivity of G bacteria to the derivatives was higher than that of G - bacteria. The hydrazines which were the most effective in G- bacteria were derivatives 8, 7 and 3. Their IC50 values were 130-180 mg/L. A concentration of 500 mg/L of derivative 3 produced a bacteriocide effect while derivatives 7 and 8 at 1000 mg/L had a bacteriostatic effect. Derivatives 8 and 2 were more effective in the G - bacterium P. aeruginosa than ampicillin. At 500 rag/L, derivative 2 had in fact a bacteriocide effect. Derivatives 1 and 5 were completely inactive in G - strains (IC50 and MIC are higher than 1000 rag/L; Table I). Substances 3, 2, 8 and 7 were the most effective in G bacteria, with IC50 values of 18.5-175 mg/L. A bacteriocide effect was found also in compound 3 at 250 mg/L (E. fcecalis) or 500 mg/L (S. aureus), and in derivative 8 at 500 mg/L (E.faecalis) or 1000 mg/L (S. aureus). The antibacterial activity of the hydrazones against G bacteria was several times lower than that of the standard. Among the eleven arylhydrazones the highest antibacterial effect on G - bacteria was found with derivatives 17, 15 and 18, with IC50 values in the range of 98-350 mg/L. Derivatives 9, 14, 15, 17 and 18 were more effective against P. aeruginosa than the standard (IC5o < 500 mg/L). Also, derivatives 15 and 18 had MIC values <500 mg/L. In contrast, none of the tested hydrazones (compounds 1 - 8 ) had a bacteriocide effect on E. coli and P. aeruginosa. The lowest antibacterial effect on G -

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bacteria was found with hydrazones 13 and 19, which exhibited a marginal effect on a single bacterial strain, and with 10 and 12, which were completely inactive.
T ab le !. Antibacterial activity of individual compounds a

E. coli
Compound IC50 1 2 3 4 5 6 7 8 10 11 12 13 14 15 16 17 18 19 AMPI b AMP2 c >1000 540 160 170 > 1000 370 160 130 > 1000 400 >1000 > 1000 400 350 450 98 300 500 0.28 MIC >1000 > 1000 500" 500 > 1000 > 1000 1000 1000 > 1000 >1000 >1000 > 1000 1000 500 >1000 1000 5O0 > 1000 1"* 1

P. aeruginosa
IC5o >1000 275 280 >1000 > 1000 490 155 400 > 1000 500 >1000 830 400 200 940 300 30O > 1000 500 MIC >1000 500* > 1000 >1000 > 10013 1000 > 1000 250 > 1000 >1000 >1000 > 1000 > 1000 500 >1000 1000 500 > 1000 500 -

S. aureus
IC50 330 18.5 60 90 27 140 140 1000 > 1000 890 >1000 > 1000 700 15 630 620 96 > 1000 0.015 MIC >1000 > 1000 500* 250" 250* 500" 1000 94 > 1000

E. fcecalis
IC50 >1000 175 43 >1000 820 430 125 1000 670 MIC >1000 500 250* >1000 > 1000 > 1000 500 1000 > 1000 >1000 >1000 > 1000 > 1000 100" 1000 > 1000 500 > 1000 1"* 0.5-2.0

>1000 >1000 >1000 >1000 > 1000 88 > 1000 350 250" 10 >1000 65 > 1000 > 1000 500 91 > 1000 1000 0.04** 1 0.03125 -

aValues are in mg/L. bValues measured for ampicillin. cValues for strains listed in the Collection of Microorganisms in Brno (Czech Republic) for ampicillin. *Concentration inducing a bacteriocide effect (MBC). **Statistically insignificant change in the value relative to the values cited in Collection of Microorganisms in Brno.

The hydrazones most effective against G + bacteria (S. aureus and E. faecalis) were derivatives 15, 18 and 9 with MIC values ranging between 10-250 mg/U The lowest MIC values were found with derivative 15, which at 100 mg/L exhibited a bacteriocide effect on E. fcecalis and at 250 mg/L on S. aureus. Derivatives 12 and 19 exhibited no activity against G + bacteria. Our MIC values were compared with reference MIC values for ampicillin. Students's test showed no statistically significant differences (p = 0.05). Our examination of the structure/antibacterial activity relationship of variously substituted quinazolines showed that certain substituents are specifically effective in producing the desired effect. The derivatives from both groups which were most effective against G bacteria were those with chlorine in position 6 on the be~ene ring and those with a secondary amine in position 2 on the pyrimidine ring (derivatives 2, 3, 15). On the other hand, the derivatives most effective against G - bacteria were those in which the benzene ring is substituted with a methyl group in position 8. The molecule of the most effective quinazoline 15 contained all the pharmacophoric groups. Mutual comparison of antibacterial activity of substituted hydrazones showed that their effectiveness is probably due to the nitro-group. Likewise, a phenyl group substituted in position 2 of the pyrimidine ring positively influenced the antibacterial activity of both the hydrazines and the hydrazones.

Based on these results, 6-chloro-2-morpholino-4-quianazolyl-5'-nitro-2'_furyl_hydrazone can be classed among new potential antibacterial substances.
We wish to thank K. Bt~nyovfi, MSc., for technical assistance in measurements
on

(15)

the Elisa iluman reader.

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Vol. 40

REFERENCES

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