Antimicrobial Evaluation of Some Styryl Ketone Derivatives and Related Thiol Adducts

Antimicrobial Evaluation of Some Styryl Ketone Derivatives and Related Thiol Adducts E. ERCIYAS'~,H. I. ERKALELI', AND G. COSAR' Received March 29, 1993, from the 'Ege Universitesi Eczacilk Fakuitesi, 35 100 Bornova-Izmir, Turkey. publication August 30, 1993@. Abstract 0 Acyclic a,@-unsaturatedketones were synthesized and

treated with either 2-rnercaptoethanol or cystenarnine hydrochloride under the simulated physiological conditions. The thiol group of these model biological nucleophiles underwent Michael type addition to the activated double bond. The incubation of the bis-Mannich base of 3-benzylidene-2,4-pentanedionewith 2-mercaptoethanol, surprisingly, gave rise to the formation of 5-[(2-hydroxyethyl)thio]-l-phenyl-l-penten3-one (8) in low yield. Evaluation of the compounds versus Grarnpositive and Gram-negativebacteria and also a type of fungus indicated that the conjugated ketones and their adducts, except the bis-Mannich base, have antimicrobial activity at 10 pg/mL. The Mannich base, 3, showed antibacterial property against only Escherichia coli at 1000 pg/mL in spite of containing a bioactive styryl ketone structure and having dearnination ability. However, the thiol adducts, which do not contain any a,&unsaturated ketone function, exhibited similar antimicrobial potency to the conjugated ketone derivatives, possibly due to the exchange reaction with enzymes or coenzymes in t h e microorganisms.

Introduction

Accepted for

The objectives of the present investigation can be outlined briefly as follows: firstly, to investigate the correlation between the chemicalreactivity of the a,p-unsaturated ketones and related Mannich bases toward biomimetic nucleophiles with antimicrobial activity; secondly, to observe the stability of a representative bis-Mannich base 3, which is able to produce additional alkylating centers under the simulated physiological conditions; and thirdly, to compare antimicrobial activity of the unsaturated ketones and their adducts. 0

-1

R'=CH,;

z

R'=CH(CH,)~;

R'=H

3

R'=CH2CH2

"3;

R2=COCH3

RZ=COCH,CH2 N

Z .2 HCI . ' 1 2 H2O

3 R'=C(CH3)2CH2N=). HCI ; R2=H a,@-Unsaturatedketones and their Mannich bases are known to possess alkylating potential which has been associated with their biological properties. In an examination of some cyclic and acyclic non-nitrogeneous antibacterial compounds, it was suggested that the styryl ketone structure was responsible for the biological activity. A number of acyclic compounds containing an ethylenic linkage conjugated to an electron-attracting I R'=CH, ;R'=COCH, ;X=OH group, like a carbonyl function, demonstrated significant antibacterial' and antifungal2 activities. Antibacterial and antisi R'=CH, ; R'=COCH, ; X=NH, HCI fungal activities are associated with the tendency to withdraw electrons from the double bond and thus render the olefinic function susceptible to nucleophilicattack by electron-releasing z R'=CH(CH,)~ ; R'=H ; X=OH groups of biologicaily important constituent^.^ The activity of a,@-unsaturatedketones is probably due to their reactions with Experimental Section the essential thiol groups in bacteria and fungi to give @-keto thioeters.' However, this theory is still speculative and needs Chemistry-Melting points (mp) are uncorrected. Microanalyses further chemical testing. A recent study4 showed that electron (C, H, N) were performed on a Perkin-Elmer elemental analyzer 240C densities on @-carbonsare lower than on a-carbons in various and are within f0.4% for 5-8. TLC plates were composed of silica gel with a fluorescent indicator. IR spectra were recorded from KBr pellets unsaturated ketones, and thus the attack of the nucleophiles on a Perkin-Elmer 1430 IR spectrophotometer. lH NMR spectra were should occur at @-carbonatoms. Similarly,a number of Mannich obtained with a Bruker AM 400 F T (400 MHz) and a Varian T-60 bases of a,p-unsaturated ketones have alkylating ability against spectrometer (60 MHz) in CDC13. TMS was used as internal standard. the biologicallyimportant nucleophiles to produce cyt~toxicity~*~ Chemical ionization (CI) mass spectra were undertaken on a Finnigan and antimicrobial activity.7 Because an amino ketone containing 1015 D instrument with NH3 as the reagent, and the electron-impact at least one activated hydrogen atom at the @ position to an (EI) mass spectrum was obtained on VG Micromass MM 16F specamino function can undergo deamination to liberate the cortrometers. UV spectra were recorded in MeOH on Shimadzu doubleresponding a,@-unsaturated ketone.8 Thus, it is possible to beam spectrophotometer UV-150-02. Column chromatography was generate an additional center for nucleophilic attack.9 It was performed using Merck silica gel (70-230 mesh). Synthesis of Unsaturated Ketones 1 and 2-Compounds 1 and 2 shown that increased antimicrobial activity was associated with were prepared by a literature methodlo from benzaldehyde and approan increased breakdown of the Mannich bases.I In addition, priate ketones in 71 % and 87% yields, respectively, as yellow oily liquids. the amino group in Mannich bases would increase the waterBoth of them were pure as determined by TLC with a solvent system solubilizing property of the molecule which may assist transof hexane-ethyl acetate (l:l), and all had 'H NMR spectra (60 MHz) portation of the compound to a site of action. in accord with the proposed structures. ~

@Abstractpublished in Advance ACS Abstracts, January 1, 1994.

0 1994, American Chemical Society and American Pharmaceutical Association

Synthesis of Mannich Bases 3 and 4 4 o m p o u n d 3 wassynthesized by a literature procedure" with a slight modification related to the length

0022-3549/94/ 1200-545$04.50/0

Journal of Pharmaceutical Sciences / 545 Vol. 83, No. 4, April 1994

of reflux time of the reaction mixture. In the case of 3, the mixture was heated under reflux for 15 min instead of a few minutes as reported in the literature method. The reaction was completed in 2 h in 59% yield; mp 166"C (lit.11mp 166 "C). Compound 3 also had a 'H NMRspectrum (400 MHz) in good agreement with the structure of 3. Several attempts were made to prepare 4 by using the traditional Mannich reaction and also the method used for 3. The resultant compound did not give satisfactory combustion analyses, although the 1H NMR spectrum supported the proposed structure. Reaction of 1 and 2 w i t h 2-Mercaptoethanol (5,7)-2-Mercaptoethanol (0.7 mL, 0.01 mol) was added to the solution of ketones (0.005 mol) in phosphate buffer (pH 7.4,20 mL), and the mixture was incubated at 37 "C on a shaking constant-temperature bath. The reactions were monitored by TLC using haxane-ethyl acetate (l:l),and disappearance of the starting ketones was noted for 5 (12 h) and 7 (4 h). The reaction mixtures were extracted with chloroform (3 X 5 mL), the chloroform layers were dried over sodium sulfate and filtered, and the solvents were removed in uacuo. In the case of 5, the solid residue obtained from the reaction of 1 was passed through a column of silica gel by the elution with benzene-methanol (9.5:0.5). Removal of the solvent gave a colorless solid which was recrystallized from ethanol in 56% yield, mp 68 "C. U V ,A, (log c) 223 (2.6) nm. I R Y- 3325 (0-H), 1720 and 1692 (C=O), 1150,1040cm-l. NMR (400 MHz): 6 7.41-7.19 (m, 5H, phenyl protons), 4.53 (d, J = 12 Hz, l H , P-H), 4.24 (d, J = 12 Hz, l H , a-H), 3.59 (t, J = 6 Hz, 2H, methylene protons), 2.50 (t,J = 6 Hz, 2H, methylene protons), 2.32 (s,3H, COCH3, trans to phenyl), 1.83 (s,3H, cocH3, cis to phenyl). MS (EI): m/z (relative intensity) 266 (M+,
Microbiology-Antimicrobial screening was carried out using an agar dilution technique. Bacteria strains were obtained from the American Type Culture Collection, and yeast was provided from the Pasteur Institute. In antimicrobial testing for bacteria, a loopful of a 1 / 1 ~ suspension of 24 h broth culture of each bacterium was streaked on double plates of DST agar (Difco) containing 10,100,200,400,600,800, and 1000pg/mL of eachcompound. The inoculatedplates were incubated a t 37 "C for 24-48 h.I2 Plates Containing 10 pg/mL streptomycin sulfate and no compound were inoculated for the control tests. No growth of bacteria at the end of incubation period was considered as inhibitory activity of the compound. Yeast plates of modified Sabouraud dextrose agar (pH 7) containing 10, 100, 200,400,600,800, and 1000 pg/mL of eachcompound were prepared. The 48-h cultures of yeast in Sabouraud dextrose broth, suspended accordingtoMacFarland No. 1,werestreaked on double test plates containing the seven different concentrations of the compounds. The yeast plates were incubated a t 37 "C for 3 days. For control, all yeasts were inoculatedon two plates of Sabouraud dextrose agar containing no compound. No growth of yeast on test plates a t the end of the incubation period was considered as indicative of the compound activity. Streptomycin sulfate inhibits all the test microorganismsexcept for Candida albicans at used concentration.

Results and Discussion The unsaturated ketones 1 and 2 were prepared by the Knoevenagel condensation between benzaldehyde and the appropriate ketones. The Mannich base of 1 was synthesized according to the literature methodologyll in a shorter period of time (2 h) than the literature procedure (48 h). Attempts to synthesize the Mannich base of 2 led to a product whose lH NMR spectrum suggested the proposed structure but whose elemental analysis was unsatisfactory. Hence, this compound, 4, was not submitted for antimicrobial evaluation. The thiol adducts 5-8 were prepared from the reaction of requisite unsaturated ketone or the Mannich base with 2-mercaptoethanol or cystenamine hydrochloride. In the formation of 8, P-mercaptoethanol did not attack to olefinic double bond of the styryl moiety of the compound 3 directly. It was proposed that the thiol function was added to the vinylic intermediate formed after deamination in phosphate buffer (pH 7.4) at 37 O C . The possible reaction mechanism of the formation of 8 from 3 was outlined in Scheme 1. Although two possible acyl cleavage mechanisms of a monoMannich base of 6-(dimethylamino)-3-((3,4-dichlorophenyl)methylene)-2,4-hexandionehydrochloride in phosphate buffer (pH 7.4) at 37 "C to give 4-(3,4-dichlorophenyl)-3-buten-2-one have been documented before,l3 the stability of bis-Mannich base, expected to generate a new a,B-unsaturated ketone group, in the presence of the model nucleophiles under the same conditions has not been reported previously. Compound 8 is capable of existing as both E and 2 isomers. The 400-MHz 1H NMR spectrum of 8 containing absorptions at 7.59 and 6.75 ppm resulting from the ethylenic protons on ,8- and a-carbon atoms, respectively, with a coupling constant at 16 Hz proved that 8 has only the E configuration.14J5 Additionally, the UV spectrum of the compound 8 indicated that the absorption maximum at 293 nm was due to 7~ P* transitions of the styryl ketone chromophore.6 A strong IR band at 975 cm-1 was determined to be due to the carbon-hydrogen out of plane vibration characteristic of ethylenic compounds possessing the E ~0nfiguration.l~ It is of interest to note that the double bond in the styryl moiety of 3 was not affected by thiol function, although the mole ratio between 3 and 2-mercaptoethanol was 21. It is conceivable that a second mole of 2-mercaptoethanol might have reacted with the acrylic acid molecule formed after acyl cleavage (Scheme 1). Compound 1 reacted with the thiol function of the model nucleophiles employed. This compound was inactive against Gram-positive and acid fast bacteria and also a fungus with

-

c

9

I t

'b

H

i

HSCHzCHzOH

8 Scheme 1-Possible

reaction mechanism for the formation of 8.

Table 1-Mlcroblological

Results of the Compounds8 Antimicrobial Activity

Compd

Yield % (Reaction Period)

1 2 3

71 87 59

5 6

56 (12 h) 69 (9 h) 74 (4 h) 18 (>28 h)

7 8

b

c

d

e

f

+ + + +

+ + + + + + +

+ + + +

+ + + +

+ + + + +

a The minimum inhibitory concentrations are 10 pg/mL for 1-2 and 5-8 and 1000 pg/mL for 3;the reference compound is streptomycin sulfate. Staphylococcusaureus. Escherichia coli. Pseudomonas aeruginosa. * Mycobacterium smegmatis. Candida albicans.

*

tested concentrations. The most active compound, 2, reacted rapidly and in high yield with 2-mercaptoethanol. For compound 2, it could be assumed that on increasing the electron release from the isopropyl group found in the molecule and rendering the oxygen atom more electronegative the rate of proton attachment resulting from the dissociation of the thiol group of 2-mercaptoethanol will be increased.16 Compound 3 underwent a deamination reaction liberating the conjugated ketone centers, and then one of the unsaturated ketone intermediates was reacted with the thiol function over a very long period and in very poor yield under the conditions used. Compound 3 was found to be inactive against test microorganisms, possibly due to the lowest fragility of the compound in basic aqueous media. However, this compound was active against Escherichia coli at 1000 pg/ mL. The yields, durations of the reactions, and microbiological results are given in Table 1. In view of these findings, along with contributions from other sources,1,2J6 it can be hypothesized that the antimicrobial activity of the compounds may be due to their ability to react with the thiol groups in the biological constituents. It seems likely that rapid reaction in high yield with a thiol function indicates more potential antimicrobial activity. In addition, the formation ofthioether function in all adducts, 5-8, confirmed that the thiol group of the biomimetic

nucleophiles can react with a conjugated ketone much more quickly than amine17 and hydroxyl type nucleophiles under simulated physiological conditions. Especially noteworthy is the observation that both the a,@-unsaturatedketone, 2, and its thiol adduct, 7, showed similar antimicrobial activity with high potency. In some cases, it might be expected that the adducts could be responsible for the biological action due to dethiolation mechanism. However, a solution of the thiol adducts in phosphate buffer (pH 7.4) showed that no reversion to the corresponding a,@-unsaturatedketone occurred after 24 h at 37 "C. Clarke and co-workers2 found that the thiol adducts had the greatest fungitoxicity, which was probably due to their reaction with the thiol groups of enzymes through an exchange reaction. Recent works have demonstrated that conjugates of electrophilic xenobiotics with certain naturally occurring thiols have marked bioactivities.18 It was also reported that thiol adducts of the a,@-unsaturatedketones could contribute to the alkylating potency.lg Meanwhile, it was observed that there is no difference between the hydroxyl and amino groups of the adducts in their contribution to the dethiolation procedure on comparison the adducts of 2-mercaptoethanol and cystenamine hydrochloride in this study. However, it is of interest to note that the adduct of 2-mercaptoethanol, 5, displayed antimicrobial activity against Gram-positive, Gram-negative, and acid fast bacteria, whereas the cystenamine hydrochloride adduct, 6, affected only Escherichia coli and Candida albicans. The another observation is that the activity of all of the compounds against the Gram-negative Escherichia coli was generally more easily and completely abolished than that against other microorganisms tested in this study. Some anomolies in the biological results may be explained by noting that the inhibition of Gramnegative bacteria may be due to reaction with the thiol group of a different enzyme than found in Gram-positive bacteria. Alternatively, the same enzyme present in both Gram-negative and Gram-positive bacteria may be less accessible in the case of Gram-negative or Gram-positive bacteria to the compounds tested in this study. The microbiological results are summarized in Table 1. Compounds 5-8 are being reported for the first time. Journal of Pharmaceutical Sciences / 547 Vol. 83, No. 4, April 1994

Conclusions Acyclic a,@-unsaturatedketones containing styryl moiety, 1 and 2, and their thiol adducts, 5-7, exhibited high-potency antimicrobial activity at the concentration of 10 pg/mL. Compound 7 has potential antimicrobial activity against all of the microorganisms used in this study at the same concentration, althought it does not contain any conjugated ketone structure. While bis-Mannich base 3 showed no significant antimicrobial action, its elimination-addition product, 8, has significant antimicrobial activity toward all of the test microorganisms. The evolution of 2 and 7 from this study permits the isopropyl ketone derivatives to serve as lead compounds in the design of further antimicrobial molecules.

References and Notes Geiger, W. B.; Conn, J. E. J. Am. Chem. SOC.1945, 67,112-116. Clark, N. G.; Hams, A. F.; Le getter, B. E. Nature 1963,200,171. Stack, V. T., Jr. Znd. Eng. Ctem. 1957,49,913-917. Dimmock, J. R.; Erciyas, E.; Sidhu, K. K.; Luo, X.; Mezey, P. G.; Allen, T. M.; Muray, L. Drug Des. Delivery 1990, 7, 45-49. 5. Nyathi, C. B.; Gupta, V. S.; Dimmock, J. R. J. Pharm. Sci. 1979,

1. 2. 3. 4.

68,1383-1386. 6. Dimmock, J. R.; Taylor, W. G. J. Pharm. Sci. 1975, 64, 241-249. 7. Schcnenberger, H.; Bastug, T.; Bindl, L.; Adam, A.; Adam, D.; Petter, A.; Zwez, W. Pharm. Acta Helu. 1969,44,691-714.

548 /Journal of Pharmaceutical Sciences Vol. 83,No. 4, April 1994

8. Carsky, P.; Zuman, P.; Horak, V. Collect. Czech. Chem. Commun. 1964,29,3044-3056. 9. Menper. F. W.: Smith. J. H. J.Am. Chem. soc. 1969.91.4211-4216. 10. Horging, E. C.; Koo,'J.; Fish, M. S.; Walker, G. N. Org. Synth. 1951, 31, 56-58. 11. Dimmock,J.R.;Erciyas,E.;Bigam,G.E.;Kirkpatrick,D.L.;Duke, M. M. Eur. J. Med. Chem. 1989,24, 379-383. 12. Mitscher, L. A.; Leu, R.; Bathala, M. S.; Wu, W.-N.; Beal, J. L. Lloydia 1972,35, 157-166. 13. Dimmock, J. R.; Raghavan, S. K.; Logan, B. M.; Bigam, G. E. Eur. J. Med. Chem. 1983.18. 248-254. Jpn. 1976,49, 1369-1374. 14. Iwata, M.; Emoto, S: Bull. Chem. SOC. 15. Dimmock, J. R.; Taylor, W. G. J. Pharm. Sci. 1974, 63, 69-74. 16. Baluja, G.; Municio, A. M.; Vega, S. Chem. Znd. 1964,2053-2054. 1965, 17. Friedman, M.; Cavins, J. F.; Wall, J. S. J. Am. Chem. SOC. 87. - . 3672-3682. ---18. Anders, M. W.; Lash, L. H.; Dehant, W.; Elfarra, A. A.; Dohn, D. R. CRC Crit. Rev. Toxicol. 1988, 311-341. 19. Dimmock. J. R.: Ercivas.E.:Bieam. G. E.: KirkDatrick.D. L.:Duke. M. M. Drug Des. Ddiuery 1980, 7,51-58. I

Acknowledgments We would like to thank the Research Fund Administration of the University of Ege (Turkey) for financial support of the project (91/ECZ/Oll). In particular, one of the authors (E.E.) appreciates Dr. J.R. Dimmock from the University of Saskatchewan (Canada) for his extraordinary helpfulness in each step. Dr. U. Holzgrabe from the University of Bonn is also thanked for determining the 400-MHz NMR spectra.