Antibacterial and antifungal activity of some thiosemicarbazones
and 1,3,4-thiadiazolines

Beatriz N. Brousse,¹ Rosana Massa,² Albertina G. Moglioni,¹ Miriam Martins Alho,³
Norma D´Accorso,³ Gabriel Gutkind ² and Graciela Y. Moltrasio.¹*

Facultad de Farmacia y Bioquímica, Buenos Aires.
Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires-Argentina
E-mail: gmoltra@ffyb.uba.ar
(Received: August 12, 2003- Accepted: October 2, 2003)

ABSTRACT

A series of thiosemicarbazones and thiadiazolines was evaluated for biological activity against various microorganisms such as Bacillus subtilis, Micrococcus luteus, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, Trichophyton mentagrophites and Aspergillus niger. Some of the assayed compounds showed interesting activity against Bacillus subtilis, Candida albicans, Micrococcus luteus, Trichophyton mentagrophites and Aspergillus niger.

Keywords: Thiosemicarbazones; thiadiazolines; antibacterial activity; antifungal activity

INTRODUCTION

For some years our group has been engaged in the synthesis of 1,3,4-thiadiazolines (TDZ) through the appropriate thiosemicarbazones (TSC) [1, 2]. Certain thiadiazolines and thiosemicarbazones have been reported to exhibit antiviral and antibacterial properties [3-5]. In continuation of our work on the synthesis of these heterocycles of pharmaceutical interest we report here the characterization and antimicrobial evaluation of several thiosemicarbazones and thiadiazolines.

EXPERIMENTAL

1. Chemistry

The compounds had been synthesized following Scheme 1 and the experimental details of some of them have been described previously [1, 2].

The TSC were synthesized from the corresponding ketones (Figure 1) by treatment with thiosemicarbazide, then the TSC were treated with acetic anhydride and pyridine to afford the heterocyclic compounds (TDZ). The TSC and TDZ are numbered the same as the ketones.

Fig. 1.

From the reaction of TSC-8 with pyridine/acetic anhydride, three compounds TDZ-8, [2] 12 [2] and 13, could be isolated and characterized; these compounds were also tested for antimicrobial activity (Figure 2).

Fig. 2.

Here we describe two new compounds: verbenone thiosemicarbazone (TSC-11) and 4-N-acetyl camphor thiosemicarbazone 13.

M.p.´s were determined in an Electrothermal 9100 apparatus and are uncorrected. IR spectra were measured as mulls with a Nicolet IRFT spectrophotometer;¹H and ¹³C NMR spectra, using the solvent signal as internal standard, were obtained using a Bruker 400 spectrometer at 400 MHz and 100 MHz, respectively (chemical shifts in d ppm). HRMS were recorded on a VG AutoSpec (Micromass Instruments).

1.1. Cis- and trans-thiosemicarbazones of (-)(S)-verbenone (TSC-11). C₁₁H₁₇N₃S, m.w. 223.3. Title compounds were prepared from equimolecular quantities of (-)(S)-verbenone and thiosemicarbazide dissolved in ethanol. The mixture was heated at reflux monitoring the reaction by tlc. Precipitation with water gave the corresponding thiosemicarbazones as an inseparable mixture. Yield: 95%. IR: (n , cm-1) 3405-3153 (NH), 1609 (C=N), 1501 (C=S), 1242 (S=C-NH₂). 1H NMR (DMSO-d₆) d (ppm) (as a mixture of geometric isomers in a 3:2 ratio) 0.81 (for both isomers) (s, 3H, H-8), 1.39 (major isomer) (s, 3H, H-9) and 1.42 (minor isomer) (s, 3H, H-9), 1.49 (minor isomer) (d, 1H J 8.9 Hz, H-7a) and 1.58 (major isomer) (d, 1H J 8.5 Hz, H-7a), 1.87 (minor isomer) (s, 3H, H-10) and 1.90 (major isomer) (s, 3H, H-10), 2.24 (minor isomer) (dd, 1H, J 5.6 and 5.3 Hz, H-1 or H-5), 2.27 (major isomer) (dd, 1H, J 6.1 and 5.3 Hz, H-1 or H-5), 2.55-2.70 (both isomers) (unresolved multiplets, 2H, H-5 or H-1 and H-7b), 5.77 (minor isomer) (s, 1H, H-3), 6.70 (major isomer) (s, 1H, H-3), 7.50 (major isomer) (broad s, 1H, NH), 7.95 (both isomers) (broad s, 2H, NH), 8.64 (minor isomer) (broad s, 1H, NH), 10.27 (minor isomer) (s, 1H, NH), 10.37 (major isomer) (s, 1H, NH). 13C NMR (DMSO-d₆) d (ppm) (major isomer) 21.8 (C-8), 23.7 (C-9), 26.0 (C-10), 37.4 (C-7), 50.9 (C-1), 48.0 (C-6), 48.9 (C-5), 110.9 (C-3), 154.1 (C-4), 160.4 (C=N), 178.2 (C=S); (minor isomer) 22.1 (C-8), 23.0 (C-9), 25.7 (C-10), 36.7 (C-7), 43.3 (C-1), 47.9 (C-6), 48.6 (C-5), 118.7 (C-3), 156.8 (C-4), 155.8 (C=N), 178.4 (C=S); HRMS (FAB) m/z: MH+ exp. 224.1221, calcd. 224.1225.

1.2. 4-N-Acetyl camphor thiosemicarbazone (13). C₁₃H₂₁N₃OS, m.w. 267.4. This compound was obtained together with TDZ-8 and compound 12 [2]. It was purified by column chromatography (Silicagel; chloroform and ethyl acetate 2:1), compound 13 eluting first. Yield: 30%, m. p. 167 ºC- 170 ºC (CHCl₃:EtOAc). Elemental analysis (%Calc./Found): 58.39/58.35 (C), 7.92/7.94 (H), 15.71/15.75 (N), 11.99/11.97 (S). IR: (n , cm-1) 3253 -3180 (NH), 1694 (C=O), 1538 (C=S), 1457 (C=N). 1H NMR (CDCl₃) d (ppm) 0.76 (s, 3H, H-8), 0.93 (s, 3H, H-9), 1.12 (s, 3H, H-10), 2.17 (s, 3H, CH₃CO), 1.10-1.25 (m, 1H), 1.40-1.55 (m, 1H), 1.60-2.05 (unresolved multiplets, 4H), 2.26-2.50 (m, 1H), 9.35 (broad s, 2H, NH). 13C NMR (CDCl₃) d (ppm) 11.1 (C-8), 18.6 (C-9), 19.6 (C-10), 24.2 (CH₃CO), 27.0 (C-5), 32.4 and 35.1 (C-6, C-3 exchangeable), 44.0 (C-4), 48.5 (C-7), 53.5 (C-1), 171.3 (C=N), 175.7 (C=O) and 176.5 (C=S).

2. Microbiology

Initial screening for antimicrobial activity was performed by impregnating 6 mm sterile paper disks with 100 Ïg of each substance, dissolved in ethanol, and drying at 45 °C. Microbial inocula were prepared as previously described with minor modifications [6]. Essentially, 2.5 ml of 1x108 microorganisms/ml suspensions were inoculated in 25 ml Antibiotic Medium (AM 1) pH 6.6 (DIFCO) or (AM 11) pH 8.0, and poured into 90 mm dishes. Once solidified, impregnated disks were distributed on the surface of the agar plates, which were incubated at 37 ºC for 24 h. Lacking previous information on the diffusion pattern for each substance, inhibition (+) was recorded when the absolute inhibition zone, around the disk, was seen with the naked eye (Tables 1, 2, 3 and 4). Some of the substances that exhibited considerable activity against Aspergillus niger were assayed on a quantitative basis determining the Minimal Inhibitory Concentration (MIC) for that microorganism (Table 5). For these tests, each substance was dissolved in ethanol. Dilutions incorporated in molten Sabouraud agar were homogenized and poured into 90 mm Petri dishes. Then each plate was allowed to solidify and inoculated in duplicate with 10 Ïl 5 x 105 cfu/ml by means of a Steers multi-inoculator. A negative control of ethanol was tested against all the microorganisms used.

RESULTS AND DISCUSSION

The synthesized compounds were evaluated for in vitro antimicrobial activity. Table 1 summarizes the findings against Gram positive and Gram negative bacteria. In those cases where antimicrobial activity was found, the screen showed that the TSC have similar or stronger activity than the TDZ, so for some terpenone derivates we decided to work only with the TSC.

Table 1 Antibacterial activity against Gram negative and Gram positive bacteria.

	Bacillus subtilis		Escherichia coli		Micrococcus luteus genes			Listeria monocyto-		Pseudomonas aeruginosa		Staphylococcus aureus
Compound	ATCC 6633 CCM-A-10		ATCC 11105 CCM-A-424		ATCC 9341 CCM- A-45			ATCC 15313 CCM-A-39		ATCC 9027 CCM- A-39		ATCC 6538P CCM-A-305
	AM 1	AM 11	AM 1	AM 11	AM 1		AM 111	AM 1	A M11	AM 1	AM 11	AM 1	AM 1 1
TSC-1	-	+	-	-	-	-		-	-	-	-	-	-
TDZ-1	-	+	-	-	-	-		-	-	-	+	-	-
TSC-2	-	-	-	-	-	-		-	-	-	-	-	-
TDZ-2	-	-	-	-	-	-		-	-	-	-	-	-
ONE-3	-	-	-	-	-	-		-	-	-	-	-	-
TSC-3	-	-	-	-	-	-		-	-	-	-	-	-
TDZ-3	-	-	-	-	-	-		-	-	-	-	-	-
TSC-4	-	+	-	-	-	-		-	-	-	-	-	-
TDZ-4	-	-	-	-	-	-		-	-	-	-	-	-
TSC-5	-	+	-	-	-	-		-	-	-	-	-	-
TDZ-5	-	-	-	-	-	-		-	-	-	-	-	-
ONE-6	-	-	-	-	-	-		-	-	-	-	+	-
TSC-6	-	+	-	-	-	-		-	-	-	-	-	-
TDZ-6	-	++	-	-	-	-		+	+	-	+	-	-
TSC-7	-	+	-	-	-	-		-	-	-	-	-	-
TDZ-7	-	+	-	-	-	-		-	-	-	-	-	-
TSC-8	+/-	+	-	-	-	-		+	-	-	+/-	-	+/-
TSC-9	+	-	+	-	+	-		-	-	-	-	-	-
TSC-10	+	++	-	-	+	+/-		+	-	-	+/-	-	+/-
TSC-11	++	++	-	-	+/-	++		-	-	-	+/-	-	+/-
12	-	-	-	-	-	-		-	-	-	-	-	-
13	+	+	-	-	+/-	+		-	-	-	+/-	-	+/-
Zone of inhibition measured in mm; +: 6-8; ++: 10-20; +/-: < 6

Þ TDZ-6, -10 and TSC-11 showed activity against Bacillus subtilis (AM 11). TSC-1, -4, -5, -6, -7, TDZ-1, -7, and 13 exhibited weaker antimicrobial activity against Bacillus subtilis (AM 11) than the former. TSC-11 showed activity against Bacillus subtilis when tested in AM 1 medium and against Micrococcus luteus when tested in AM 11 medium.

Þ We tested the activity of all the compounds against Aspergillus niger and Candida albicans (Tables 2 and 3). When the activity was important the compounds were also tested against Trichophyton mentagrophites and Mucor sp. (Table 4).

Table 2
Antifungal activity against Candida albicans and Aspergillus niger

Compound	Candida albicans CCM-A-Y1		Aspergillus niger ATCC 16404
	AM1	AM 11	AM1	AM11
TSC-1	-	-	-	-
TDZ-1	-	-	-	-
TSC-2	-	-	-	-
TDZ-2	-	-	-	-
ONE-3	-	-	+	++
TSC-3	-	-	-	++
TDZ-3	-	-	-	-
TSC-4	-	-	-	-
TDZ-4	-	-	-	-
TSC-5	-	++	++	++
TDZ-5	-	-	-	-
TSC-6	-	+	+	++
TDZ-6	-	-	-	-
TDZ-7	-	-	-	-
Zone of inhibition measured in mm +: 6-8; ++: 10-20; +/-: < 6

Table 3
Antifungal activity against Candida albicans and Aspergillus niger

Compound	Candida albicans ATCC 10231 Sabouraud Agar	Aspergillus niger ATCC 16404 Sabouraud Agar
ONE-3	-	+/-
ONE-6	-	-
TDZ-6	-	-
TSC-7	-	+/-
TSC-8	-	++/-
TSC-9	-	+
TSC-10	-	++/-
TSC-11	-	+/-
12	-	+/-
13	-	+/-
Zone of inhibition measured in mm +: 6-8; ++: 10-20; +/-: < 6

Table 4
Antifungal activity against Trichophyton mentagrophites and Mucor sp

Compound	Trichophyton mentagrophites	Mucor sp.
	Sabouraud Agar	AM1	AM11
ONE-3	+	+	-
TSC-5	++	+	+
TSC-6	++	+	-
TDZ-6	+	-	-
TSC-7	-	-	-
Zone of inhibition measured in mm +: 6-8; ++: 10-20; +/-: < 6

Þ Only TSC-5 and -6 were active against Candida albicans in AM 11 medium. Ketone 3, TSC-5 and -6 were active against Aspergillus niger in both media (AM 1, AM 11). TSC-3 was only active against Aspergillus niger in AM 11.

Þ Ketone 3, TSC-5, -6, -7 and TDZ-6 were chosen to be tested against Trichophyton mentagrophites and Mucor sp. TSC-5 showed activity in all conditions.

For those compounds with a marked qualitative activity (TSC-3, -5, and -6) the actual MIC value was determined against Aspergillus niger ATCC 16404. For TSC-3- and -5 the observed MIC was 128 mg/ml for both, and the MIC determined for TSC-6 was 64 mg/ml (Table 5).

Table 5
Minimum inhibitory concentration (MIC) mg/mL

Microorganism	3	Compound (TSC) 5	6
Aspergillus niger ATCC 16404	128	128	64

CONCLUSIONS

From the results obtained we conclude that:

Þ TSC of the aromatic ketones (1-4) and the indanones (5-7) possess an interesting antifungal activity.

Þ If we consider the derivates of terpenones (8-11), the TSC tested showed activity towards a larger number of bacteria than the aromatic derivatives (1-7).

Þ The MIC values for compounds TSC-3, TSC-5 and TSC-6 are good enough to consider them as leads for the development of antifungals.

ACKNOWLEDGMENTS

The authors thank CONICET, Universidad de Buenos Aires and Agencia de Promoción Científica y Tecnológica for the financial support which enabled this work to be carried out.

REFERENCES

[1].- M. A. Martins Alho, A. G. Moglioni, B. N. Brousse, G. Y. Moltrasio, and N. D´Accorso, Synthesis and characterization of 2,2-disubstituted thiadiazolines, Arkivoc, 1 (iv) (2000) 627-640.        [ Links ]

[2].- B. N. Brousse, A. G. Moglioni, M. Martins Alho, A. Alvarez-Larena, G. Y. Moltrasio, and N. D´Accorso, Behavior of thiosemicarbazones derived from some terpenones under acetylation conditions, Arkivoc 3 (x), (2002) 14-23.        [ Links ]

[3].- S. R. Turk, Ch. Shipman, Jr. and J. C. Drach, Selective inhibition of Herpes simplex ribonucleoside diphosphate reductase by derivatives of 2-acetylpyridine thiosemicarbazone, Biochem. Pharmac., 35 (1986) 1539-1545.        [ Links ]

[4].- D. J. Bauer, P. W. Sadler; Structure-activity relations of the antiviral chemotherapeutic activity of isatin b-thiosemicarbazone, J. Pharmacol. 15 (1960) 101.        [ Links ]

[5].- S. Ali and M. Alam; Potential antimicrobial agents-I: Structural modifications and antimicrobial activity of some isatin derivatives, Arch. of Pharmaceut. Res. 17 (1994) 131-133.        [ Links ]

[6].- G. O. Gutkind, C. Norbedo, M. Mollerach, G. Ferraro, J. Coussio, R. De Torres, Antimicrobial activity of Achyrocline flaccida, J. Ethnopharmacol., 10 (1984) 319-321.        [ Links ]