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l'olia Microbiol. 40 (6), 611-614 (1995)

Antibacterial Effect of Substituted 4-Q.uinazolyl- hydrazines and "l-heirArylhydrazones Determined by a Modified Microdilution Method

S. JANTOV,A,a,D. HUDECOV,A,a,~. STANKOVSK~, K. ~PIRKOVAb and g. RU~EKOV/t,a

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 .Tune14, 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-quinazolyt- 5'-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 pyri- midine 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; Jan- tov~iet al. 1994). To study this broad-range biological activity of quinazoline derivatives, Stankovsky and Soky- rov~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 sec- ondary amines such as morpholine and piperidine).

X

NH --NH 2

X

NH-N=CH-Z

 
 

1-8

9-19

X

Y

X

Y

Z

1

6-CI

Mo

9

6-CI

Mo

2'-furyl

2

6-CI

Pi

10

6-CI

Mo

4'-nitrophenyl

3

6-CI

Ph

11

6-CI

Mo

2'-nitrophenyl

4

6-Br

Mo

12

6-CI

Mo

2'-chlorophenyl

5 H

Pi

13

6-CI

Mo

4'-acetamidophenyl

6 H

Ph

14

6-CI

Mo

5'-bromo-2'-furyl

7 8-Me Mo

15

6-CI

Mo

5'-nitro-2'-furyl

8 8-Me

Ph

16

8-Me Ph

2'-furyl

 

17

8-Me

Ph

phenyl

18

8-Me

Ph

4'-N,N-dimethylaminophenyl

Mo -

morpholinyl

19

8-Me Ph

4'-nitrophenyl

Pi - piperidinyl

612 S. JANTOV.~` etal.

Vol. 40

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 quinazoline- 4-thiones. Treatment of hydrazinoderivatives with corresponding arylcarbaldehydes yielded arylalde- hyde 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 bacte- ria 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 8- channel 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

with the tested derivatives, the bacteria were inoculated to a solid culturing medium and cultured stati- cally for 24 h with effective derivatives at 37 ~ The microbistatic (MBS) or microbicide (MBC) con- centration was determined. The antibacterial effect was characterised by IC50values, 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 IC50and MIC values were determined from toxicity curves.

used as standard. After 8-1] culturing

RESULTS

AND

DISCUSSION

IC50 and MIC values obtained in the tests are summarized in Table I. The broadest antibacte- rial 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 § bacte- ria 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

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

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, deriva- tives 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-

hydrazones against G § bacteria was several times lower than that of the

18.5-175 mg/L. A bacteriocide effect was

1995

ANTIBACTERIAL

EFFECI"

OF

SUBSqTI'WI'ED

4-QUINAZOLYLIIYDRAZINES

613

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.

Table !.

Antibacterial activity of individual compounds a

E. coli

P. aeruginosa

S. aureus

E. fcecalis

Compound

 

IC50

MIC

IC5o

MIC

IC50

MIC

IC50

MIC

1

>1000

>1000

>1000

>1000

330

>1000

>1000

>1000

2

540

> 1000

275

500*

18.5

> 1000

175

500

3

160

500"

280

> 1000

60

500*

43

250*

4

170

500

>1000

>1000

90

250"

>1000

>1000

5

> 1000

> 1000

> 1000

> 10013

27

250*

820

> 1000

6

370

> 1000

490

1000

140

500"

430

> 1000

7

160

1000

155

> 1000

140

1000

125

500

8

130

1000

400

250

1000

94

1000

1000

10

> 1000

> 1000

> 1000

> 1000

> 1000

> 1000

670

> 1000

11

400

>1000

500

>1000

890

>1000

>1000

>1000

12

>1000

>1000

>1000

>1000

>1000

>1000

>1000

>1000

13

> 1000

> 1000

830

> 1000

> 1000

> 1000

88

> 1000

14

400

1000

400

> 1000

700

> 1000

350

> 1000

15

350

500

200

500

15

250"

10

100"

16

450

>1000

940

>1000

630

>1000

65

1000

17

98

1000

300

1000

620

> 1000

> 1000

> 1000

18

300

5O0

30O

500

96

500

91

500

19

500

> 1000

> 1000

> 1000

> 1000

> 1000

1000

> 1000

AMPI b

0.28

1"*

500

500

0.015

0.04**

1

1"*

AMP2 c

-

1

-

-

-

0.03125

-

0.5-2.0

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.

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 com-

pared with reference MIC values for ampicillin. Students's test showed no statistically significant differ- ences (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

most effective against G § bacteria were those with chlo-

rine 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.

The hydrazones most effective against

G + bacteria (S. aureus and E. faecalis) were

both groups which were

Mutual comparison of antibacterial activity of substituted hydrazones showed that their effec-

tiveness is probably due to the nitro-group.

pyrimidine ring positively influenced the antibacterial activity of both the hydrazines and the hydra-

zones.

Based on these results, 6-chloro-2-morpholino-4-quianazolyl-5'-nitro-2'_furyl_hydrazone can be classed among new potential antibacterial substances.

a phenyl group substituted in position 2 of the

Likewise,

(15)

We wish to thank K. Bt~nyovfi, MSc., for technical assistance in measurements on the Elisa iluman reader.

614 S. JANTOV/~ eta/.

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Vol. 40

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~PIRKOVAIC, STANKOVSlr ~;., HOR~A~"EKJ.: Preparation of s-triazolo[4,3-c]quinazolines. ColI.Czech.Chem.Commun. 59,

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