New 1,3-diaryl-5-thioxo-imidazolidin-2,4-dione derivatives: synthesis, reactions and evaluation of antibacterial and antifungal activities

2.1 Chemistry

N-(3,5-Dichlorophenyl)-cyanothioformamide (1) was prepared by treating 3,5-dichlorophenyl isothiocyanate with potassium cyanide [12]. It is noteworthy to mention that analysis of the cyanothioformanilide derivatives by NMR indicated the presence of tautomeric mixtures of thiol and thione forms, in which the latter predominates. The tautomeric nature of the cyanothioformanilides reflects their nucleophilic character in which they may react via the sulfur or nitrogen atom leading to different heterocyclic cores in ring closing reactions. A comprehensive study on the tautomeric nature of the cyanothioformanilides was carried out by El-Sharief and Moussa [10]. Treating an ethereal solution of N-cyanothioformanilide 1 with an equimolar amount of phenyl isocyanate, followed by the addition of a few drops of triethylamine as a catalyst, furnished the corresponding 5-imino-4-thioxo-2-imidazolidinone 2 as the sole product. Hydrolysis of 2 with dilute hydrochloric acid in boiling ethanol afforded the corresponding dione derivative 3 in which the imino group has been hydrolyzed (Scheme 1). The structures of compounds 2 and 3 were inferred from their correct elemental analyses and spectral data. Infrared measurements of 2 showed diagnostic absorbance bands at 3234 cm⁻¹ for the imino NH, 1766 cm⁻¹ for the C=O group and 1654 cm⁻¹ for the C=N function. The thione stretch appeared at its expected frequency at 1102 cm⁻¹. A ¹H NMR spectrum of 2 exhibited the usual broad exchangeable singlet for the imine proton at δ = 9.79 ppm. Integration values and multiplicities were in agreement with the assigned structure. The IR spectrum of 3 has no absorption band characteristic to an NH group. The hydrolysis product 3 was characterized by similar means where all spectroscopic and microanalytical data corroborated the proposed structure. Most notable, the disappearance of the NH resonance from the proton NMR spectrum was observed.

Scheme 1:

Synthesis of imidazolidinedione derivative 3.

Upon treatment of the dione 3 with one mole of hydrazine hydrate or phenyl hydrazine, the reaction proceeds via elimination of H₂S to produce the hydrazono imidazolidine derivatives 4a, b. Furthermore, thiosemicarbazone derivatives 5a, b were prepared by treatment of dione derivative 3 with different N-substituted thiosemicarbazide derivatives. Condensation of dione 3 with cyanoacetohydrazide in ethanol containing a catalytic amount of acetic acid afforded 2-cyano-acetohydrazide derivative 6 (Scheme 2). The chemical structures of 4–6 were elucidated on the basis of elemental analyses and spectral data. A ¹H NMR spectrum of 4a as a representative example revealed a broad signal (cancelled with D₂O) at δ = 8.32 ppm due to NH₂ protons. A ¹H NMR spectrum of 5a as a representative example revealed three broad singlet signals at 8.52, 9.33 and 11.87 due to NH₂ and NH protons. An IR spectrum of 6 exhibited absorption bands at 3265 and 2264 cm⁻¹ for NH and nitrile groups, respectively. A ¹H NMR spectrum of 6 exhibited a singlet signal at δ = 4.08 ppm for methylene protons and another broad singlet appeared at δ = 11.28 ppm for an NH proton.

Scheme 2:

Reaction of imidazolidinedione 3 with different types of hydrazine derivatives.

The reaction of 2-cyanoacetohydrazide 6 with arylidene malononitrile upon heating under reflux in the presence of piperidine as a catalyst afforded a product which was identified as the unexpected arylidene derivative 8 rather than the expected pyridine derivative 7 (Scheme 3). An important evidence for the latter product through its synthesis via another synthetic route was provided. Thus, condensation of the 2-cyanoacetohydrazide 6 with p-anisaldehyde in ethanol containing piperidine as a catalyst furnished the corresponding arylidene 8. A ¹H NMR spectrum of 8 displayed singlet signal at δ = 8.33 ppm due to the azomethine moiety and broad signals at δ = 12.35 assignable to the NH proton.

Scheme 3:

Reaction of 2-cyanoacetohydrazide 6 with arylidene malononitrile.

The cyclocondensation reaction of 2-cyanoacetohydrazide derivative 6 with salicylaldehyde derivatives in ethanol under reflux in the presence of ammonium acetate furnished smoothly 2-imino-chromene derivatives 9a, b in good yield (Scheme 4). An IR spectrum of chromene derivative 9a showed absorption bands at 3320 (NH), and 1770, 1732, 1698 cm⁻¹ (3 C=O). A ¹H NMR spectrum of 9a exhibited a singlet signal at δ = 8.84 ppm for the chromene proton 4-H and another broad singlet appeared at δ = 9.30, 14.70 ppm for two 2NH protons.

Scheme 4:

Reaction of 2-cyanoacetohydrazide 6 with salicylaldehyde derivatives.

2.2 Antimicrobial activity

The search for newer antibacterial and antifungal agents is still in continuation due to the rapid development of the resistance among bacteria and fungi. Imidazolidinedione derivatives may comprise a new class of antimicrobial agents with diminished resistance. Therefore, the aim of the present investigation is to synthesize different series of imidazolidinedione derivatives which bear various substituents at C-4. Accordingly, these compounds were synthesized and tested for their expected effects against selected Gram-positive bacteria, Gram-negative bacteria and fungi species.

2.3 Antibacterial and antifungal activities

The synthesized compounds were tested in vitro for antibacterial and antifungal activities against the following strains: three Gram-positive bacteria, Staphylococcus aureus RCMB 010027, Staphylococcus epidermidis RCMB 010024 and Bacillus subtilis RCMB 010063; three Gram-negative bacteria, Neisseria gonorrhoeae RCMB 010079, Escherichia coli RCMB 010052 and Klebsiella pneumoniae RCMB 010093; and three Fungi, Aspergillus fumigatus RCMB 02564, Aspergillus clavatus RCMB 02593 and Geotrichum Candidum RCMB 05096. The synthesized compounds were tested for antimicrobial activity by the agar diffusion method [15] using a microplate with 1 cm well diameter and a 100 μL of each concentration. The antifungal agents were evaluated against clinical isolates of standard strains of fungi by the broth dilution method according to National Committee for Clinical Laboratory Standards (NCCLS) [16, 17]. Antimicrobial tests were carried out using 100 μL of tested compound solution prepared by dissolving 5 mg of the chemical compound in 1 mL of dimethyl sulfoxide (DMSO). Ampicillin, gentamicin and amphotericin B (1 mg mL⁻¹) were used as standard references for Gram-positive bacteria, Gram-negative bacteria and antifungal activity, respectively. The results are summarized in Table 1.

The aim of the present investigation was to synthesize a series of imidazolidin-2-one derivatives which contain a 3,5-dichlorophenyl moiety at N¹ and a phenyl moiety at N³ and various substituents at C-4. The effect of each substituent at C-4 was studied and allows a comparative study between them to deduce a structure activity relationship. Using the general structure provided in Fig. 1, certain aspects of the structure activity relationships for these compounds can be more clearly highlighted.

Fig. 1:

General formula of the synthesized compounds under antimicrobial investigation.

5-Imino-4-thioxo-2-imidazolidinone 2 showed moderate activity with all the tested Gram-positive and -negative bacteria. Changing the substituent on C-4 from imino to oxo (2 → 3) led to higher activity than imino analog 2 and gave activities against all the tested strains which were near to those of the reference drug. When the substituent at C-4 was changed from thioxo to hydrazono (3 → 4a), a lower activity than for imino analog 3 was found. The presence of a hydrazono moiety caused moderate to good activity against most of the tested organisms. In structure 5 the substituent on C-4 has been changed from thioxo to thiosemicarbazono (3 → 5a). Compound 5a showed high activities against all the tested strains near to those of the reference drug.

Changing the substituent at C-4 from thioxo to cyanoacetohydrazide (3 → 6) had a detrimental effect on antimicrobial activity. The presence of the cyanoacetohydrazide moiety led to a moderate activity against the tested fungi only and no activity against all tested bacterial organisms. Compound 8, where the X substituent contains an arylidene moiety, exhibited moderate to high activity against all the tested organisms. Finally, incorporation of a chromene moiety as in structure 9 had a high effect on antimicrobial activity. Compound 9a led in the highest antimicrobial activity among all the compounds investigated in this study. The presence of a chromene moiety exhibited activities near those of the reference drug against all the tested organisms.

2.4 MIC of the most active compounds

The minimum inhibitory concentration (MIC) of the most active synthesized compounds 2a, 5a and 5b was evaluated in vitro using the broth dilution method according to NCCLS [16, 17]. The results of MIC are depicted in Table 2. Regarding the effect of each substituent at C-4 against bacterial and fungal strains, the antimicrobial activity of the studied compounds is as follows: Compound 3 is about 25% less potent than ampicillin against S. epidermidis, about 30% less potent than ampicillin against B. subtilis, 25% less potent than gentamicin against K. pneumoniae and 25% less potent than amphotericin B against A. clavatus. Compound 3 was equipotent to amphotericin B in inhibiting the growth of G. Candidum (MIC 0.12 µg mL⁻¹). Compound 5a showed an about 50% less potent effect than amphotericin B against A. clavatus. Compound 8 showed an about 50% less potent effect than amphotericin B against A. clavatus and G. candidum. Finally, compound 9a showed an about 30% less potent effect than ampicillin against B. subtilis and 25% less potent effect than amphotericin B against A. clavatus. Compound 3 was equipotent to amphotericin B in inhibiting the growth of G. candidum (MIC 0.12 µg mL⁻¹).

3.1 Synthesis of 1-(3,5-dichlorophenyl)-4-imino-3-phenyl-5-thioxo-imidazolidin-2-one (2)

A solution of the cyanothioformanilide (1) (0.01 mol) and phenyl isocyanate (0.01 mol) in dry diethyl ether (30 mL) was treated with three drops of triethylamine. The reaction mixture was stirred for 15 min. The obtained product was filtered off, washed with ether, air-dried and recrystallized from chloroform-n-hexane to give imidazolidineiminothione derivative 2. Yield: 88%; m.p. 160–162°C. – IR: ν = 3234 (NH), 1766 (C=O), 1654 (C=N), 1102 (C=S) cm⁻¹. – ¹H NMR (500.14 MHz, [D₆]DMSO, 25°C, TMS): δ = 7.43–7.48 (m, 1H, Ar-H), 7.51–7.61 (m, 5H, Ar-H), 7.71 (d, 1H, J = 3.0 Hz, Ar-H), 7.85 (d, 1H, J = 3.5 Hz, Ar-H), 9.79 (br, 1H, NH) – C₁₅H₉Cl₂N₃OS (350.22): calcd. C 51.44, H 2.59, N 12.00; found C 51.53, H 2.44, N 12.08.

3.2 Synthesis of imidazolidin-2,4-dione derivative 3

The imidazolidineiminothione derivative 2 (0.01 mol) was dissolved in boiling ethanol (20 mL) and treated with dilute aqueous HCl (1:1 molar ratio). The obtained product was filtered off, washed with cold water, air-dried and recrystallized from chloroform-n-hexane to give the corresponding dione derivative 3. Yield 83%; m.p. 175–177°C. – IR: ν = 1779, 1738 (C=O) cm⁻¹. – ¹H NMR (500.14 MHz, [D₆]DMSO, 25°C, TMS): δ = 6.85–7.74 (m, 8H, Ar-H) – C₁₅H₈Cl₂N₂O₂S (351.21): calcd. C 51.30, H 2.30, N 7.98; found C 51.46, H 2.14, N 7.76.

3.3 Synthesis of imidazolidine-2,4-diones 4a, b

Dione 3 (0.01 mol) was dissolved in absolute ethanol, stirred to complete solubility and warmed if needed. Then hydrazine or phenyl hydrazine (0.01 mol) was added drop by drop until a precipitate was formed, which was recrystallized from ethanol-dioxane to give hydrazono imidazolidines 4a, b.

3.4 Synthesis of hydrazine-carbothioamides 5a, b

A mixture of imidazolidin-2,4-dione 3 (0.01 mol) and thiosemicarbazide or phenylthiosemicarbazide (0.01 mol) in the presence of triethylamine (3 drops) as a catalyst in absolute ethanol was heated under reflux for 6 h and then allowed to cool. The solid product was collected and recrystallized from the ethanol to give 4-thiosemicarbazone derivatives 5a, b.

3.5 Synthesis of 2-cyano-N′-(3- (3,5-dichlorophenyl)-2,5-dioxo-1- phenylimidazolidin-4-ylidene)- acetohydrazide (6)

A solution of dione 3 (0.01 mol) in absolute ethanol (30 mL) and an equimolar quantity of cyanoacetohydrazide (0.01 mol) with two drops of acetic acid as a catalyst was heated under reflux until all H₂S had evolved (about 12 h). The product precipitated on cooling and was recrystallized from ethanol to give 2-cyano-acetohydrazide derivative 6 as orange crystals; yield 81.2%; m.p. 185–187°C. – IR: ν = 3265 (NH), 2970 (CH-aliph), 2264 (C≡N), 1773, 1693 (C=O) cm⁻¹. – ¹H NMR (500.14 MHz, [D₆]DMSO, 25°C, TMS): δ = 4.08 (s, 2H, CH₂), 7.40–7.78 (m, 8H, Ar-H), 11.28 (br, 1H, NH, cancelled with D₂O). – ¹³C NMR: 24.7 (CH₂), 115.1, 125.2, 125.4, 125.8, 126.0, 126.5, 129.0, 130.1, 127.9, 133.5, 133.8 (2C), 134.1, 150.9, 156.0, 159.8, 164.8 (C=O). – C₁₈H₁₁Cl₂N₅O₃ (415.02): calcd. C 51.94, H 2.66, N 16.83; found C 52.12, H 2.52, N 16.71.

3.6 Synthesis of 2-cyano-N′-(3- (3,5-dichlorophenyl)-2,5-dioxo- 1-phenylimidazolidin-4-ylidene)-3- (4-methoxyphenyl)acrylohydrazide (8)

To equimolar amounts of cyanoacetohydrazide derivative 6 and anisaldehyde or 2-(4-methoxybenzylidene)malononitrile (0.01 mol) in ethanol (30 mL), a few drops of piperidine were added. The reaction mixture was heated under reflux for 3 h, and then allowed to cool. The obtained product was collected by filtration and crystallized from ethanol-dioxane to give arylidene derivative 8 as yellow crystals; yield 78% (arylidene 59%); m.p. 290–292°C. – IR: ν = 3257 (NH), 2210 (C≡N), 1776, 1725, 1706 (C=O) cm⁻¹. – ¹H NMR (500.14 MHz, [D₆]DMSO, 25°C, TMS): δ = 3.87 (s, 3H, OCH₃), 7.15 (d, 2H, J = 13.0 Hz, Ar-H), 7.49–7.82 (m, 8H, Ar-H), 8.08 (d, 2H, J = 9.5 Hz, Ar-H), 8.33 (s, 1H, CH=N), 12.35 (br, 1H, NH, cancelled with D₂O). – C₂₆H₁₇Cl₂N₅O₄ (534.35): calcd. C 58.44, H 3.21, N 13.11; found C 58.67, H 3.08, N 13.24.

3.7 Synthesis of 2-imino-2H-chromene-3-carbohydrazides 9a, b

A solution of cyanoacetohydrazide derivative 6 (0.01 mol) with a 5-substituted salicylaldehyde (0.01 mol) and ammonium acetate (0.01 mol) in absolute ethanol (30 mL) was heated under reflex for 15 min. The product precipitated from the hot solution. It was isolated and recrystallized from ethanol-dioxane to give 2-imino-chromene derivatives 9a, b.