New Imidazolidineiminothione, Imidazolidin-2-one and Imidazoquinoxaline 
Derivatives: Synthesis and Evaluation of Antibacterial and Antifungal Activities

Abstract

A series of new 5-imino-4-thioxo-2-imidazolidinone derivatives 3 with various halogenated and alkylated aromatic substituents at N¹ and N³ was synthesized. Imidazolidineiminothione derivatives 3 were prepared from the reaction of N-arylcyanothioformamide derivatives with aryl isocyanates. These compounds were used as key synthons for the preparation of wide variety of new substituted imidazole compounds. Imine hydrolysis of 3 with ethanolic HCl produced the corresponding 4-thioxo-2,5-imidazolidindiones 4. Condensation of 3 with benzophenonhydrazone furnished the corresponding 4-azine derivatives 5. Monohydrazono and dihydrazono derivatives 6 and 8 were obtained upon treatment of imidazolidinone derivatives 3 with hydrazine hydrate. Finally, imidazolidinones 3 were reacted with o-phenylenediamines or pyrazol-5(4H)-ones and afforded the corresponding imidazoquinoxaline and imidazolidin-4-ylidenepyrazolone-5(4H)-one derivatives 11 and 12, respectively. Evaluation of the antibacterial and antifungal activities for the synthesized compounds was carried out to probe their activities. Most of the tested compounds showed significant activities. The best antimicrobial activity was observed for 1-(3-ethoxyphenyl)-6-methyl-1-phenyl-1H-imidazo[4,5-b]quinoxalin-2(3H)-ones (11c) followed by 5-imino-3-(3-methoxy- phenyl)-1-phenyl-4-thioxoimidazolidin-2-one (3f).

Introduction

Infectious diseases caused by bacteria and fungi remain a major worldwide health problem due to rapid development of resistance to the existing antimicrobial drugs. The increasing use and misuse of the existing antimicrobial drugs have resulted in the development of resistant pathogens. The medical community faces a serious problem when treating infections caused by pathogenic microbes and needs an effective therapy and search for novel antimicrobial agents [1-6]. In addition, systemic and dermal fungal infections have significantly increased, specifically in individuals with suppressed immune systems such as those receiving cancer chemotherapy and AIDS patients. Although there are different antifungal drugs used in the treatment of fungal infections, some of them have undesirable side effects because of the biochemical similarity between human cell and fungi forms. The search for new and effective antimicrobial agents, resistant to the mechanisms of defense of these bacteria, is of paramount importance [1, 2]. Imidazoles and their fused derivatives are keys in many bioactive compounds of both natural and synthetic origins [7] such as histidine, purines, biotin and hydantoin. Nitroimidazoles are a well-known family of antibacterial and antiprotozoal drugs [8, 9], including antitrypanosomal drugs or compounds with known anti-trypanosomal activity [10, 11]. Metronidazole, the first drug to be introduced for this purpose and probably also the best known drug in this class, has been in use for more than 50 years [12]. 4-Oxoimidazolidine-2-thiones and their 2-

alkylthio-3,5-dihydro-4H-imidazol-4-one derivatives are important compounds in the fields of drugs, pharmaceutical intermediates. As an example isatinylidene derivatives exhibit immunosuppressive activity [13]. 2-Thiohydantoin derivatives have not only been used in medicinal chemistry, but have also been developed as fungicides e.g. fenamidone [14, 15].

In light of these facts, and in continuation of our ongoing study on antimicrobial agents [16-19], the present study was designed to synthesize new imidazolinone derivatives and evaluate their antimicrobial activity. Various functional groups were introduced into the target compounds in order to investigate their preliminary structure activity relationships.

Results and Discussion

Chemistry

The starting compounds, N-(4-fluoro, 3-chloro, 4-methyl)-cyanothioformanilides 2a-c were prepared according to the previously reported procedure [20]. The new compounds, N-(3-methoxy, 3-ethoxy)-cyanothioformanilides 2d,e were prepared from the reaction of N-aryl isothiocyanates with potassium cyanide. The elemental analysis and spectroscopic data of 2d,e are consistent with the assigned structure. IR measurement of 2e for example displayed absorption bands at: 3264 and 2227 cm^-1 for NH and C≡N groups. ¹H NMR spectrum revealed triplet and quartet signals at: δ = 1.23 and 4.14 ppm corresponding to CH₃ and CH₂, respectively and the presence of a D₂O exchangeable broad singlet at: δ = 13.36 ppm attributable to the NH proton. In addition, ¹³C NMR spectrum of 2e showed two signals at 14.1 (CH₃), 63.3 (CH₂), also it showed a farthermost downfield signal for the thione group (C=S) at: 161.1 due to the tautomeric thione and thiole mixture and an upfield signal resulting from the nitrile group (107.9). The behavior of the cyanothioformanilides towards isocyanate derivatives was investigated. Thus, reaction of cyanothioformanilides with aryl isocyanates in ether and in the presence of triethylamine caused cyclization to furnish the corresponding 5-imino-4-thioxo-2-imidazoli-dinones 3a-k. The structure of 3 was assigned on the basis of analytical and spectral data. IR spectrum of 3f, as representative example, displayed absorption bands at: 3261 cm^-1 due to NH and at 1777 cm^-1 due to C=O functional group. ¹H NMR spectrum exhibited two singlets at: δ = 3.80 and 9.66 ppm indicative of methoxy protons and NH proton, respectively. ¹³C NMR spectrum of 3f displayed four signals at: 183.3, 160.1, 154.5 and 65.3 for C=S, C=O, C=N and OCH₃, respectively, whereas the aromatic signals ranged from 114.1 to 154.1 corresponding for 12C. Mass spectrum showed a molecular ion peak at m/z = 311, corresponding to a molecular formula C₁₆H₁₃N₃O₂S. Hydrolysis of 3h,i with dilute HCl in boiling ethanol afforded the corresponding dione derivatives 4a,b (Scheme 1). IR spectrum has no absorption band characteristic to an NH group.

Scheme 1.

Synthesis of N-arylcyanothioformanilide, imidazolidin-2-one and imidazolidineiminothione derivatives.

The imidazolidine derivatives contain adjacent imino and thione functional groups in the 5-and 4-positions appear promising for further chemical transformations. Therefore, it was interesting to study the reaction of imidazolidineiminothiones 3 with some amino compounds as nitrogen nucleophiles. Condensation of 3a,d,i with benzophenonehydrazone in boiling ethanol using triethylamine as a basic catalyst furnished the corresponding 4-azine derivatives 5a-c. The analytical and spectral data are in agreement with the proposed structure. Thus IR spectrum of 5a showed the presence of three signals at 3265, 1758 and 1624 cm^-1 due to the presence of three groups NH, C=O and C=N, respectively. ¹H NMR spectrum showed singlet signal at: 10.11 ppm corresponding NH proton. While, ¹³C NMR showed the complete disappearance of the signal in the range around 180 ppm due to C=S and the presence of four signals at 160.4, 160.7, 162.4 and 167.5 corresponding 3C=N and C=O groups. In addition, the reactivity of iminothione derivatives 3 towards binucliophiles was also investigated. Thus equimolecular amounts of 3h,i and hydrazine hydrate furnished the monohydrazono derivatives for which structure 6 or 7 seemed possible. The positive element test for sulfur and spectral data favored the 
5-hydrazono derivatives 6a,b. Elemental analysis, IR and NMR are in agreement with the proposed structure. The formation of 6 may be rationalized via the nucleophilic addition of nitrogen atom of hydrazine to the thio group followed by nucleophilic addition of the other nitrogen to the imino group followed by elimination of ammonia as shown in Scheme 2 Dihydrazono derivatives 8a,b were achieved as a sole product by treating the iminothione 3h,i with two moles of hydrazine hydrate in ethanol (Scheme 2). The product of this reaction was identified on the basis of both elemental analyses and spectral data. ¹H NMR spectrum of 8b displayed triplet and quartet signals at: δ =1.32, 4.01 due to the ethoxy group and broad singlet signals at: δ = 5.65 assignable the 2NH₂. ¹³C NMR showed complete disappearance of the signal in the range around 180 ppm due to the functional group change from C=S to C=N and the presence of signals at: 152.6, 159.0 corresponding C=N and C=O groups.

Scheme 2.

Reactions of imidazolidineiminothiones with hydrazone derivatives and hydrazine hydrate.

Furthermore, upon reaction of the iminothiones 3a,f,h,i with thiosemicarbazide, the nucleophilic addition occurred at the thio group and the corresponding 4-thiosemicarbazone derivatives 9a-d were obtained in good yield. Compounds 9 were not converted into the triazine derivatives 10 under these conditions. The chemical structure of 9a-d was elucidated on the basis of elemental analyses and spectral data. ¹HNMR spectrum of 9d as a representative example revealed triplet and quartet signals at: δ = 1.30 and 4.03 ppm, respectively, characteristic for ethoxy protons, in addition to three broad singlet signals at: 8.46, 9.47, 12.79 due to NH and NH₂ protons. Now, we have extended our synthetic program to the synthesis of otherwise inaccessible heterocyclic ring system utilizing imidazolidineiminothiones 3 as the key starting material. Thus condensation of the imidazolidineiminothione 3i with o-phenylenediamine derivatives as 1,4-binucleophile in ethanol under reflux afforded yellow products which were identified as imidazo[4,5-b]quinoxalines 11a-c. The spectral data of the isolated product were in complete agreement with structure 11. IR spectrum revealed lack of an absorption band corresponding to a C=NH functional group. Mass spectrum of compound 11c showed molecular ion peak at m/z 396 (base peak) corresponding to molecular formula C₂₄H₂₀N₆O₂. Finally, it was interesting to study the behavior of imidazolidi-

neiminothiones toward carbon nucleophiles. Thus, condensation of the imidazolidineiminothiones 3c,i with 3-methyl-1H-pyrazol-5(4H)-one derivatives in boiling ethanol using triethylamine as a basic catalyst afforded a product which was identified as imidazolidin-4-ylidenpyrazolones 12a-d (Scheme 3 ). IR spectrum of 12d as a representative example revealed absorption bands at: 3280, 1717 and 1603 characteristic to NH, C=O and C=N functional groups, respectively. The ¹H NMR spectrum exhibited triplet, quartet and singlet at: 1.14, 4.19 and 2.41 ppm due to ethoxy and methyl protons, singlet signal at 12.83 specific for NH. Moreover, its ¹³C NMR spectrum revealed signals at: 14.8, 15.1, 63.3 assigned ethoxy and methyl carbons signals 105.0, 144.7 corresponding for C=C and signals at 159.3, 159.4, 162.3 and 166.6 assigned to 2 imino and 2 carbonyl carbons.

Scheme 3.

Reactions of imidazolidineiminothione derivatives with different reagents.

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 Bacillis subtilis RCMB 010063; three Gram-negative bacteria, Neisseria gonorrhoeae RCMB 010079, Esherichia coli RCMB 010052 and Klebsiella pneumoniae RCMB 010093, and three Fungi, Aspergillus fumigatus RCMB 02564, Aspergillus clavatus RCMB 02593 and Geotricum Candidum RCMB 05096, and the results were summarized in Table 1. The synthesized compounds were tested for antimicrobial activity by the agar diffusion method [21] using a 1 cm microplate 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 methods Broth dilution method according to NCCLs [22, 23]. Tested compound solution prepared by dissolving 5 mg of the chemical compound in 1 mL of dimethyl sulfoxide (DMSO). The inoculated plates were then incubated for 24 h at 37 °C. Ampicillin, Gentamycin and Amphotericin B (1 mg/mL) were used as standard references for Gram positive bacteria, Gram negative bacteria and antifungal activity, respectively. After incubation time, antimicrobial activity was evaluated by measuring the inhibition zone diameters against the test organisms and compared with standard zone size ranges that determine susceptibility, intermediate susceptibility, or resistance to the screened compounds. Visual bacterial growth is observed only in areas in which the drug concentrations are below those required for growth inhibition. The experiment was carried out in triplicate and the average zone of inhibition was calculated.

The mean values of the inhibition zone diameter obtained for these compounds suggest that all of the imidazole derivatives evaluated possess significant antibacterial activity against most of the test organisms used in these assays. A moderate difference in antibacterial activity is noted between the tested compounds, this indicate that the main effect related to the presence of the imidazolidinone moiety. 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.

A series of imidazolidineiminothione derivatives 3 which contain 4-fluorophenyl, 3-methoxyphenyl, 3-ethoxyphenyl moieties (Ar) at N¹ and phenyl, 2-fluorophenyl, 4-chlorophenyl, 3,5-di(trifluoromethyl)-phenyl moieties (Ar') at N³, X = O, Y = S and Z = NH was synthesized and their biological activates were measured and studied. Regarding the effect of Ar and Ar' groups. The type of the substitutions on the benzene ring of aryl moiety is important. It was noticed that the presence of 3-methoxyphenyl and phenyl in 3f and 3-ethoxyphenyl and 4-chlorophenyl in 3j showed the maximum activities against most of the test organisms which showed near the reference drug. On the other hand, compounds 3b,g,h,i showed moderate to good activity against most of the tested organisms. Compound 3k with 4-fluorophenyl and 3,5-di(trifluoromethyl)-phenyl moieties has moderate activity the Gram +ve bacteria and no activity toward the Gram -ve bacteria. Regarding the effect of changing the substituents on N¹ and N³, the results of screening demonstrated the presence of good correlation between the antifungal and antibacterial activities. Changing the substituent on C4 from imino to oxo (3h,i → 4a,b) was carried out to show the deference between imino and oxo on the effect of the antimicrobial activity. Compound 4a showed higher activity more than its imino analogues 3h while compound 4b has less activity than its imino analogues compound 3i. Y and Z substituents in imidazolidineiminothione derivatives 3 were changed to monohydrazono, dihydrazono and thiosemicarbazono moieties to do the same study. Compounds 6, 8, 9 with mono hydrazono, dihydrazono and thiosemicarbazono moieties have weak activity against Gram +ve bacteria and no activity against Gram -ve activity. Changing the substituent at C4 from imino to hydrazono (3h,i → 6a,b) showed lower activity than their imino analogues 3h,i. Also, changing the substituents on C4 and C5 from thioxo and imino to hydrazono (3h,i → 8a,b) showed lower activity than its analogues 3h,i. Moreover, changing the substituent on C4 from thioxo to thiosemicarbazono (3a,f,h → 9a,b,c) showed lower activity than their analogues. The authors also studied the effect of changing of the substituent at C4 and C5 to cyclic substituents at imidazolidine ring on the biological activities. Compounds 11a-c with quinoxaline moiety showed strong activity against all of the tested organisms, where compound 11c was equipotent to the reference drug. Finally, the vary substitution on C5 from thioxo to substituted pyrazolone moiety (3i → 12c,d) had slight effect on antibacterial activity. Compounds 12c,d with moiety showed moderate activity with all the tested Gram positive and negative bacteria. The comparison between the antimicrobial activity of our potent synthesized compounds and standard reference drug against the used Gram positive, Gram negative bacteria and fungi is represented graphically in Figure 2.

Fig. (2).

The comparison between the antimicrobial activity of our potent synthesized compounds and standard drug against the used Gram positive, Gram negative bacteria and fungi.

MIC of the Most Active Compounds

Minimum inhibitory concentration (MIC) of the most active synthesized compounds 3f, 3k, 4a, 11c, 12a, and 12b was evaluated in vitro using the Broth dilution method according to NCCLs [22, 23]. The results of minimum inhibitory concentration are depicted in Table 2. Regarding the effect of each substituent at C2, C4, C5 and those at N¹ and N³ against bacterial and fungal strains, results of antimicrobial activity in this study revealed that the presence of quinoxaline moiety in compound 11c resulted in highest antimicrobial activity among all the investigated compounds. The latter compound showed better results when compared with standard drugs as revealed from its MIC values (0.12 - 0.98 μg/mL). Compound 11c was equipotent to Ampicillin in inhibiting the growth of B. subtilis (MIC 0.15 μg/mL). This compound was equipotent to Gentamycin in inhibiting the growth of K. pneumonia (MIC 0.12 μg/mL). Also, this compound was equipotent to Amphotericin B in inhibiting the growth of A. clavatus (MIC 0.49 μg/mL) and G. candidum (MIC 0.12 μg/mL). In certain cases, such values are low enough to render such agents as potential candidates for further studies.

Table 2. Minimum inhibitory concentration (µg/mL) of the more potent synthesized compounds against the pathological organisms.

Compd. No.	Gram +ve			Gram -ve			Fungi
	S. aureus	S.epidermidis	B. subtilis	N.gonorrhoeae	E. coli	K.pneumoniae	A. fumigatus	A. clavatus	G. candidum
3f	1.95	0.98	0.49	31.25	7.81	7.81	0.98	7.81	0.49
3k	3.9	1.95	0.98	125	15.63	3.9	7.81	7.81	1.95
4a	1.95	15.63	1.95	125	31.25	15.63	31.25	3.9	1.95
11c	0.49	0.49	0.12	0.98	0.49	0.12	0.98	0.49	0.12
12a	15.63	3.9	1.95	500	125	125	62.5	15.6	1.95
12b	3.9	0.98	0.98	500	31.25	31.25	15.63	7.8	1.95
St. (A)	0.03	0.12	0.15	-----	-----	-----	-----	-----	-----
St. (B)	-----	-----	-----	0.49	0.24	0.12	-----	-----	-----
St. (C)	-----	-----	-----	-----	-----	-----	0.12	0.49	0.12

St. (A): Ampicillin, St. (B): gentamycin and St. (C): Amphotericin b

Conclusion

The aim of the present investigation is to synthesize different series of imidazolidin-2-one bearing various substituents at N¹ and others at N³ beside various substituents at C1, C3 and C4. The authors measured the antibacterial and antifungal activities of these derivatives. They studied the effect of each substituent on these activities and make a comparative study between them to deduce a structure activity relationship. Most compounds displayed antibacterial and antifungal activities. A moderate difference in antimicrobial activity is noted between the tested compounds, this indicates the main effect related to the presence of the imidazolidinone moiety. Our results clearly revealed that the best antimicrobial activity was observed for 1-(3-ethoxyphenyl)-6-methyl-1-phenyl-1H-imidazo[4,5-b]quinoxalin-2(3H)-ones (11c) followed by 5-imino-3-(3-methoxy- phenyl)-1-phenyl-4-thioxoimidazolidin-2-one (3f).

Experimental Section

All melting points are recorded on digital Gallen Kamp MFB-595 instrument and are uncorrected. The IR spectra (KBr) (cm^-1) were measured on a Shimadzu 440 spectrophotometer. NMR spectra (δ, ppm) were obtained in deuterated dimethyl sulfoxide on a Varian Gemini 500 (500 MHz) spectrometer, using TMS as an internal standard; chemical shifts are reported as δ ppm units. Mass spectra (m/z, %) were obtained on GC MS-QP 100 Exmass spectrometer at 70 eV. Elemental analyses were carried out at Microanalytical Unit, Cairo University, Cairo, Egypt.

General Procedure for the Synthesis of Arylcarbamothioyl Cyanide Derivatives 2d,e

To a solution of isothiocyanate derivative (3-methoxyphenyl isothiocyanate or 3-ethoxyphenyl isothiocyanate) (0.01 mol) in ethanol (50 ml), a solution of KCN (0.01 mol) in 5 ml water was added slowly while stirring. After complete addition, stirring of the reaction mixture was continued for additional 1 h. The reaction mixture was poured into acidified crushed ice. The resulting precipitate was filtrated off, dried and crystallized from ethanol to give 2d, e.

(3-Methoxyphenyl)carbamothioyl cyanide (2d): as yellow crystals; yield 65%; mp 75-77 °C; IR: ν/cm^-1 = 3251 (NH), 3041 (CH-Ar), 2996 (CH-aliph.), 2221 (C≡N); ¹H NMR: δ/ppm = 3.83 (s, 3H, CH₃), 6.96 -7.68 (m, 4H, Ar-H), 9.55 (br, 1H, NH; cancelled with D₂O); ¹³C NMR: 55.6 (OCH₃), 113.7, 114.4, 124.2, 129.9, 130.2, 137.4, 159.0, 161.0; Anal. Calcd for C₉H₈N₂OS (192.24): C, 56.23; H, 4.19; N, 14.57; Found: C, 56.44; H, 4.35; N, 14.81%.

(3-Ethoxyphenyl)carbamothioyl cyanide (2e): as yellow crystals; yield 73%; mp 83-85 °C; IR: ν/cm^-1 = 3264 (NH), 3066 (CH-Ar), 2980 (CH-aliph.), 2227 (C≡N); ¹H NMR: δ/ppm = 1.23 (t, 3H, J = 6.9 Hz, CH₃), 4.14 (q, 2H, J = 7.0 Hz, CH₂), 6.83-7.68 (m, 4H, Ar-H), 13.36 (br, 1H, NH; cancelled with D₂O); ¹³CNMR: 14.1 (CH₃), 63.3 (CH₂), 107.9, 113.7, 114.5, 130.3, 138.7, 140.3, 158.8, 161.1; Anal. Calcd. for C₁₀H₁₀N₂OS (206.26): C, 58.23; H, 4.89; N, 13.58; Found: C, 58.42; H, 4.76; N, 13.84%.

General Procedure for the Synthesis of 5-imino-4-thioxoimidazolidin-2-one Derivatives 3

To a solution of 2 (0.0l mo1) in ether (20 ml), isocyanates (0.0l mo1) and triethylamine (0.5 ml) were added; the reaction mixture was stirred at room temperature for 1 hr. The obtained products were crystallized from the chloroform / n-hexane to afford 3.

5-Imino-4-thioxoimidazolidin-2-ones (3a,c-e) were prepared according to the reported method [ 20 ]. 1-(3,5-Bis(trifluoromethyl)phenyl)-3-(4-fluorophenyl)-5-imino-4-thioxoimidazolidin-2-one (3b): as orange crystals; yield 83%; mp 105-107 °C; IR: ν/cm^-1 = 3215 (NH), 1774 (C=O), 1664 (C=N); ¹H NMR: δ/ppm = 7.12-8.35 (m, 7H, Ar-H), 10.44 (br, 1H, NH; cancelled with D₂O); ¹³C NMR: 116.8, 117.0, 120.1, 122.2, 124.4, 126.6, 127.9, 129.7, 130.5, 131.3, 131.5, 134.9, 153.5, 153.8, (12C-Ar + 2CF₃), 161.6 (C=N), 163.6 (C=O), 182.9 (C=S); Anal. Calcd. for C₁₇H₈F₇N₃OS (435.32): C, 46.90; H, 1.85; N, 9.65; Found: C, 46.55;H, 1.87; N, 9.76%.

4-Imino-1-(3-methoxyphenyl)-3-phenyl-5-thioxoimida-zolidin-2-one (3f): as orange crystals; yield 78%; mp 118-120 °C; IR: ν/cm^-1 = 3261 (NH), 2988, 2933 (CH-aliph), 1777 (C=O), 1600 (C=N); ¹H NMR: δ/ppm = 3.80 (s, 3H, OCH₃), 7.10-7.50 (m, 9H, Ar-H), 9.66 (br, 1H, NH; cancelled with D₂O); ¹³C NMR: 65.3 (OCH₃), 115.3, 118.5, 120.3, 122.3, 128.5, 129.3, 130.3, 133.1, 134.9, 140.1, 153.0, 154.1 (12C-Ar), 154.5 (C=N), 160.1 (C=O), 183.3 (C=S); MS, m/z (%): 311 (M⁺, 1.30), 261 (13.67), 205 (2.4), 180 (13.25), 166 (14.11), 165 (81.01), 119 (20.49), 104 (13.67), 91 (46.48), 77 (100); Anal. Calcd. for C₁₆H₁₃N₃O₂S (311.36): C, 61.72; H, 4.21; N, 13.50; Found: C, 61.53; H, 4.36; N, 13.62%.

1-(2-Fluorophenyl)-5-imino-3-(3-methoxyphenyl)-4-thioxoimidazolidin-2-one (3g): as orange crystals, yield 62%; mp 143-145 °C; IR: ν/cm^-1 = 3264 (NH), 3064 (CH-Ar), 2986, 2936 (CH-aliph), 1778 (C=O), 1602 (C=N); ¹H NMR: δ/ppm = 3.80 (s, 3H, OCH₃), 7.10-7.69 (m, 8H, Ar-H), 9.82 (br, 1H, NH; cancelled with D₂O), ¹³CNMR 65.3 (OCH₃), 115.7, 116.9, 117.1, 120.2, 125.1, 125.5, 130.3, 131.8, 134.3, 153.5, 153.5, 153.5 (12C-Ar), 154.0 (C=N), 160.1 (C=O), 183.1 (C=S); Anal. Calcd. for C₁₆H₁₂FN₃O₂S (329.35): C, 58.35; H, 3.67; N, 12.76; Found: C, 58.53; H, 3.84; N, 12.54%.

1-(4-Chlorophenyl)-5-imino-3-(3-methoxyphenyl)-4-thioxoimidazolidin-2-one (3h): as yellow crystals; yield 68%; mp 127-129 °C, IR: ν/cm^-1 = 3231 (NH), 2934 (CH-aliph.), 1772 (C=O), 1665 (C=N); ¹H NMR: δ/ppm = 3.79 (s, 3H, OCH₃), 7.11-7.66 (m, 8H, Ar-H), 9.74 (s, 1H, NH; cancelled with D₂O), ¹³C NMR: 55.9 (OCH₃), 114.0, 115.5, 120.2, 122.3, 129.1, 129.3, 129.4, 130.5, 131.9, 133.0, 134.8, 153.9 (12C-Ar), 154.3 (C=N), 160.1 (C=O), 183.2 (C=S); MS, m/z (%): 345 (M⁺, 100), 347 (M+2; 39.0), 314 (2.0), 192 (22.1), 191 (20.4), 165 (44.1), 161 (10.8), 135 (10.2), 125 (4.3), (3.8), 77 (15.9); Anal. Calcd. for C₁₆H₁₂ClN₃O₂S (345.80): C, 55.57; H, 3.50; N, 12.15; Found: C, 55.76; H, 3.37; N, 12.23%.

1-(3-Ethoxyphenyl)-4-imino-3-phenyl-5-thioxoimidazolidin-2-one (3i): as orange crystals; yield 82%; mp 100-102 °C; IR: ν/cm^-1 = 3217 (NH), 2974, 2879 (CH-aliph), 1769 (C=O), 1599 (C=N); ¹H NMR: δ/ppm = 1.34 (t, 3H, J = 6.9 Hz, CH₃), 4.05 (q, 2H, J = 6.9 Hz, CH₂), 7.08-7.59 (m, 9H, Ar-H), 9.67 (br, 1H, NH; cancelled with D₂O); ¹³C NMR: 15.0 (CH₃), 63.9 (CH₂), 114.4, 116.0, 120.1, 127.6, 128.6, 129.3, 130.4, 130.5, 131.9, 133.0, 134.9, 154.1 (12C-Ar), 154.6 (C=N), 159.3 (C=O), 183.3 (C=S); Anal. Calcd. for C₁₇H₁₅N₃O₂S (325.38): C, 62.75; H, 4.65; N, 12.91; Found: C, 62.84; H, 4.42; N, 12.75%.

1-(3-Ethoxyphenyl)-3-(2-fluorophenyl)-4-imino-5-thioxoimidazolidin-2-one (3j): as orange crystals; yield 71%, mp 105-107 °C; IR: ν/cm^-1 = 3262 (NH), 2969, 2886 (CH-aliph.), 1775 (C=O), 1597 (C=N); ¹H NMR: δ/ppm = 1.34 (t, 3H, J = 6.8 Hz, CH₃), 4.04 (q, 2H, J = 6.9 Hz, CH₂), 7.08-7.68 (m, 8H, Ar-H), 9.81 (br, 1H, NH; cancelled with D₂O); ¹³C NMR: 14.3 (CH₃), 63.9 (CH₂), 114.3, 116.1, 116.9, 117.1, 120.3, 125.5, 130.5, 133.5, 130.9, 131.9, 134.7, 153.5 (12C-Ar), 157.0 (C=N), 159.4 (C=O), 183.1 (C=S); MS, m/z (%): 343 (M⁺, 100), 344 (M+1; 20.6), 324 (12.9), 205 (7.6), 179 (16.0), 161 (8.2), 151 (28.8), 93 (6.4), 65 (9.3); Anal. Calcd. for C₁₇H₁₄FN₃O₂S (343.38): C, 59.46; H, 4.11; N, 12.24; Found: C, 59.65; H, 4.32; N, 12.42%.

1-(4-Chlorophenyl)-3-(3-ethoxyphenyl)-5-imino-4-thioxoi-midazolidin-2-one (3k): as golden crystals; yield 78%; mp 142-144 °C; IR: ν/cm^-1 = 3242 (NH), 2980, 2921 (CH-aliph), 1770 (C=O), 1604 (C=N); ¹H NMR: δ/ppm = 1.36 (t, 3H, J = 7.0 Hz, CH₃), 4.09 (q, 2H, J = 6.9 Hz, CH₂), 7.07-7.63 (m, 8H, Ar-H), 9.72 (s, 1H, NH; cancelled with D₂O); ¹³C NMR: 14.9 (CH₃), 63.9 (CH₂), 114.4, 116.0, 120.1, 120.3, 125.9, 129.0, 129.4, 130.5, 133.0, 134.1, 139.1, 152.4 (12C-Ar), 153.3 (C=N), 154.3 (C=O), 183.3 (C=S); Anal. Calcd. for C₁₇H₁₄ClN₃O₂S (359.83): C, 56.74; H, 3.92; N, 11.68; Found: C, 56.91; H, 3.75; N, 11.76%.

Synthesis of Imidazolidin-2,5-diones 4a,b

The imidazolidineiminothione derivative 3h or 3i (5 mmol) was dissolved in boiling ethanol (20 mL) and treated with dil. HCl (1:1 molar ratio). The obtained products were filtered off, washed with cold water, air-dried, and recrystallized from chloroform/n-hexane to give the corresponding diones 4a,b, respectively.

3-(4-Chlorophenyl)-1-(3-methoxyphenyl)-5-thioxoimida-zolidine-2,4-dione (4a): as orange crystals; yield 62%; mp 100-102 °C; IR: ν/cm^-1 = 2918, 2826 (CH-aliph), 1749, 1797 (C=O); ¹H NMR: δ/ppm = 3.82 (s, 3H, OCH₃), 7.06-7.68 (m, 8H, Ar-H); Anal. Calcd. for C₁₆H₁₁ClN₂O₃S (346.79): C, 55.41; H, 3.20; N, 8.08; Found: C, 55.64; H, 3.42; N, 8.26%.

1-(3-Ethoxyphenyl)-3-phenyl-5-thioxoimidazolidine-2,4-dione (4b): as orange crystals; mp 157-159 °C; yield 76%; IR: ν/cm^-1 = 2982, 2895 (CH-aliph), 1745, 1802 (C=O); ¹H NMR: δ/ppm = 1.34 (t, 3H, J = 6.8 Hz, CH₃), 4.11 (q, 2H, J = 6.9 Hz, CH₂), 7.03-7.65 (m, 8H, Ar-H); Anal. Calcd. for C₁₇H₁₃ClN₂O₃S (360.81): C, 56.59; H, 3.63; N, 7.76; Found: C, 56.75; H, 3.84; N, 7.45%.

Synthesis of Azine Derivatives 5a-c

To a solution of 3a, 3d, 3i (0.0l mo1) in ethanol (20 ml), benzophenonehydrazone (0.01mol) was added and the reaction mixture was heated under reflux for 3hr. The obtained product was filtered, washed with ethanol and crystallized from ethanol/dioxane to afford 5a-c.

4-((Diphenylmethylene)hydrazono)-3-(4-fluorophenyl)-5-imino-1-phenylimidazolidin-2-one (5a): as yellow crystals; yield 62%, mp 170-172 °C, IR: ν/cm^-1 = 3265 (NH), 1758 (C=O), 1624 (C=N); ¹H NMR: δ/ppm = 7.34-7.62 (m, 19H, Ar-H), 10.11 (br, 1H, NH; cancelled with D₂O); ¹³C NMR: 115.6, 126.5, 127.3, 128.1, 128.3, 128.4, 128.5, 128.8, 129.2 (3C), 129.4 (2C), 130.0, 130.1, 130.2, 133.7, 134.4, 134.8, 137.4, 142.4, 150.7, 152.1, 152.8 (24 C-Ar), 160.4, 160.7, 162.4 (3C=N), 167.5 (C=O); Anal. Calcd. For C₂₈H₂₀FN₅O (461.49): C, 72.87; H, 4.37; N, 15.18; Found: C, 72.77; H, 4.27; N, 15.29%.

4-((Diphenylmethylene)hydrazono)-5-imino-1-phenyl-3-p-tolylimidazolidin-2-one 5b: as yellow crystals; mp 162-164 °C; yield 72%; IR: ν/cm^-1 = 3249 (NH), 1757 (C=O), 1612 (C=N); ¹H NMR: δ/ppm = 2.27 (s, 3H, CH₃), 7.13-7.61 (m, 19H, Ar-H), 10.04 (br, 1H, NH; cancelled with D₂O); ¹³C NMR: 21.1 (CH₃), 115.4, 126.5, 127.3, 128.1, 128.3, 128.4, 128.5, 128.8, 129.1 (2C), 129.2, 129.3, 129.9, 130.0, 130.1, 131.3, 132.8, 134.3, 134.4, 137.7, 137.5, 142.2, 145.3, 148.1 (24 C-Ar), 150.8, 153.7, 153.0 (3C=N), 167.3 C=O); Anal. Calcd. for C₂₉H₂₃N₅O (457.53): C, 76.13; H, 5.07; N, 15.31; Found: C, 76.28; H, 5.19; N, 15.43%.

4-((Diphenylmethylene)hydrazono)-3-(3-ethoxyphenyl)-5-imino-1-phenylimidazolidin-2-one (5c): as yellow crystals; yield 67%; mp 165-167 °C; IR: ν/cm^-1 = 3240 (NH), 1751 (C=O), 1609 (C=N); ¹H NMR: δ/ppm = 1.23 (t, 3H, J = 6.8 Hz, CH₃), 3.86 (q, 2H, J = 6.8 Hz, CH₂), 6.86-7.61 (m, 19H, Ar-H), 10.07 (br, 1H, NH; cancelled with D₂O); ¹³C NMR: 14.9 (CH₃), 63.6 (CH₂), 114.5, 120.0, 125.9, 127.6, 127.7, 128.1, 128.2, 128.3, 128.5, 128.7, 128.9, 129.0, 129.1, 129.2, 129.4, 129.8, 130.0, 130.7, 131.3, 132.8, 133.5, 134.2, 137.6, 142.3 (24 C-Ar), 150.7, 152.9, 158.8 (3C=N), 167.4 (C=O); MS, m/z (%): 487 (M⁺, 3.0), 488 (M+1, 1.6), 459 (41.2), 458 (100), 430 (14.5), 410 (30.9), 368 (15.9), 323 (51.6), 291 (18.6), 135 (11.0), 119 (14.8), 77 (44.3); Anal. Calcd. for C₃₀H₂₅N₅O₂ (487.55): C, 73.90; H, 5.17; N, 14.36; Found: C, 73.76; H, 5.29; N, 14.44%.

Synthesis of Hydrazone Derivatives 6a,b

A solution of 3h, 3i (0.01 mol) in ethanol (30 ml) was treated with hydrazine hydrate (0.012 mol), and the reaction mixture was stirred at room temperature for 15 min. The product was collected and recrystallized from ethanol/dioxane to give 6a,b.

1-(4-Chlorophenyl)-5-hydrazono-3-(3-methoxyphenyl)-4-thioxoimidazolidin-2-one (6a): as yellow crystals; yield 61%; mp 213-215 °C; IR: ν/cm^-1 = 3372, 3119 (NH₂), 2965, 2886 (CH-aliph), 1725 (C=O), 1598 (C=N); ¹H NMR: δ/ppm = 1.22 (s, 3H, CH₃), 5.78 (br, 2H, NH₂), 7.02-7.87 (m, 8H, Ar-H); MS, m/z (%): 360 (M⁺, 100), 361 (M+1, 22.7), 362 (M+2, 36.4), 334 (17.1), 332 (40.8), 166 (11.0), 164 (9.4), 148 (9.1), 134 (60.2), 111 (11.7), 107 (18.4), 77 (14.3); Anal. Calcd. for C₁₆H₁₃ClN₄O₂S (360.82): C, 53.26; H, 3.63; N, 15.53; Found: C, 53.42; H, 3.76; N, 15.48%.

1-(3-Ethoxyphenyl)-4-hydrazono-3-phenyl-5-thioxoimidazolidin-2-one (6b): as yellow crystals; yield 65%; mp 158-160 °C; IR: ν/cm^-1 = 3327, 3169 (NH₂), 3109 (CH-Ar), 2984, 2884 (CH-aliph.), 1722 (C=O), 1598 (C=N); ¹H NMR: δ/ppm = 1.42 (t, 3H, J = 7.1 Hz, CH₃), 4.05 (q, 2H, J = 7.0 Hz, CH₂), 5.56 (br, 2H, NH₂), 7.01-7.62 (m, 9H, Ar-H); ¹³C NMR: 14.7 (CH₃), 63.6 (CH₂), 113.7, 114.0, 116.1, 120.7, 126.1, 127.8, 128.9, 130.1, 132.1, 133.1, 135.2, 150.1, 152.6, 159.0, 183.6 (C=S); MS, m/z (%): 340 (M⁺, 100), 341 (M+1, 25.1), 313 (17.2), 312 (82.1), 295 (19.7), 164 (11.4), 149 (14.2), 148 (36.7), 136 (20.4), 135 (17.3), 121 (20.1), 104 (16.8), 93 (11.6), 77 (38.8); Anal. Calcd. for C₁₇H₁₆N₄O₂S (340.40): C, 59.98; H, 4.74; N, 16.46; Found: C, 59.75; H, 4.63; N, 16.73%.

Synthesis of Dihydrazone Derivatives 8a,b

A mixture of 3h, 3i (0.01 mol) and hydrazine hydrate (0.03 mol) in ethanol (30 ml) was stirred for 30 min. at room temperature. The product was recrystallized from ethanol/dioxane to give 8a, b.

1-(4-Chlorophenyl)-4,5-dihydrazono-3-(3-methoxyphenyl)imidazolidin-2-one (8a): as yellow crystals; yield 63%; mp 193-195 °C; IR: ν/cm^-1 = 3373, 3193 (NH₂), 2978 (CH-aliph), 1724 (C=O), 1603 (C=N); ¹H NMR: δ/ppm = 1.21 (s, 3H, CH₃), 5.65 (br, 4H, 2NH₂), 6.91-7.58 (m, 8H, Ar-H); MS, m/z (%): 358 (M⁺, 1.76), 343 (32.7), 177 (12.8), 173 (15.6), 161 (16.7), 151 (100), 136 (18.7), 135 (37.4), 122 (33.6), 107 (41.8), 80 (13.3); Anal. Calcd. for C₁₆H₁₅ClN₆O₂ (358.78): C, 53.56; H, 4.21; N, 23.42; Found: C, 53.78; H, 4.45; N, 23.56%.

1-(3-Ethoxyphenyl)-4,5-dihydrazono-3-phenylimidazolidin-2-one (8b): as yellow crystals; yield 77%; mp 150-152 °C, IR: ν/cm^-1 = 3372, 3210 (NH₂), 3067 (CH-Ar), 2925, 2833 (CH-aliph), 1724 (C=O), 1600 (C=N); ¹H NMR: δ/ppm = 1.32 (t, 3H, J = 7.0 Hz, CH₃), 4.01 (q, 2H, J = 6.9 Hz, CH₂), 5.65 (br, 4H, 2NH₂), 6.91-7.58 (m, 9H, Ar-H); ¹³C NMR: 15.0 (CH₃), 63.6 (CH₂), 113.8, 114.5, 116.0, 120.2, 127.4, 128.4, 129.0, 129.7, 131.2, 133.0, 134.7, 150.1 (12C-Ar), 152.6 (2C=N), 159.0 (C=O); MS, m/z (%): 338 (M⁺, 100), 339 (M+1; 22.5), 307 (34.0), 175 (11.9), 148 (53.3), 135 (13.4), 119 (14.2), 77 (32.4); Anal. Calcd. for C₁₇H₁₈N₆O₂ (338.36): C, 60.34; H, 5.36; N, 24.84; Found: C, 59.95; H, 5.53; N, 24.73%.

Synthesis of Thiosemicarbazone Derivatives 9a-d

To a solution of 3a, 3f, 3h, 3i (0.0l mo1) in ethanol (20 ml), thiosemicarbazide (0.01mol) was added and the reaction mixture was heated under reflux for 3hr. The obtained product was filtered, washed with ethanol and crystallized from ethanol/dioxane to afford 9a-d.

2-(3-(4-Fluorophenyl)-5-imino-2-oxo-1-phenylimidazolidin-4- ylidene)hydrazinecarbothioamide (9a): as orange crystals; yield 62%; mp 215-217 °C; IR: ν/cm^-1 = 3426, 3260, 3148 (NH₂, NH), 1758 (C=O), 1611 (C=N); ¹H NMR: δ/ppm = 6.87-7.74 (m, 9H, Ar-H), 8.43, 9.72, 11.63 (3br, 4H, NH₂+2NH); Anal. Calcd. For C₁₆H₁₃FN₆OS (356.38): C, 53.92; H, 3.68; N, 23.58; Found: C, 53.69; H, 3.88; N, 23.76%.

2-(5-Imino-3-(3-methoxyphenyl)-2-oxo-1-phenylimidazolidin-4-ylidene)hydrazinecarbothioamide (9b): as yellow crystals; yield 61%; mp 218-220 °C; IR: ν/cm^-1 = 3442, 3251, 3203 (NH₂, NH), 2964, 2930 (CH-aliph), 1765 (C=O), 1645 (C=N); ¹H NMR: δ/ppm = 1.23 (s, 3H, CH₃), 6.91-7.63 (m, 9H, Ar-H), 8.52, 9.33, 11.87 (3br, 4H, NH₂+2NH); Anal. Calcd. for C₁₇H₁₆N₆O₂S (368.41): C, 55.42; H, 4.38; N, 22.81; Found: C, 55.67; H, 4.69; N, 22.75%.

2-(1-(4-Chlorophenyl)-5-imino-3-(3-methoxyphenyl)-2-oxoimidazolidin-4-ylidene)hydrazinecarbothioamide (9c): as yellow crystals; mp 222-224 °C; yield 59%; IR: ν/cm^-1 = 3421, 3238, 3139 (NH₂, NH), 2934 (CH-aliph), 1757 (C=O), 1640 (C=N); ¹H NMR: δ/ppm = 1.21 (s, 3H, CH₃), 6.95-7.87 (m, 8H, Ar-H), 8.53, 9.34, 11.92 (3br, 4H, NH₂+2NH); MS, m/z (%): 402 (M⁺, 9.2), 346 (21.3), 255 (37.0), 253 (100), 252 (50.6), 249 (32.0), 180 (11.9), 166 (13.7), 155 (31.6), 153 (99.0), 119 (13.5), 77 (49.4); Anal. Calcd. For C₁₇H₁₅ClN₆O₂S (402.86): C, 50.68; H, 3.75; N, 20.86; Found: C, 50.39; H, 3.86; N, 20.79%.

2-(3-(3-Ethoxyphenyl)-5-imino-2-oxo-1-phenylimidazolidin-4-ylidene)hydrazinecarbothioamide (9d): as yellow crystals yield 75%, mp 188-190 °C, IR: ν/cm^-1 = 3382, 3254, 3162 (NH₂, NH), 2978 (CH-aliph), 1775 (C=O), 1607 (C=N); ¹H NMR: δ/ppm = 1.30 (t, 3H, J = 7.0 Hz, CH₃), 4.03 (q, 2H, J = 7.0 Hz, CH₂), 6.91-7.63 (m, 9H, Ar-H), 8.46, 9.47, 12.79 (3s, 4H, NH₂+2NH); MS, m/z (%): 382 (M⁺, 16.5), 383 (M+1, 8.1), 365 (38.9), 348 (15.8), 323 (1.5), 263 (17.3), 219 (66.4), 218 (50.2), 180 (28.7), 163 (27.9), 135 (47.5), 119 (62.0), 93 (100), 77 (45.0); Anal. Calcd. for C₁₈H₁₈N₆O₂S (382.44): C, 56.53; H, 4.74; N, 21.97; Found: C, 56.34; H, 4.84; N, 21.76%.

Synthesis of imidazo[4,5-b]quinoxalin-2(3H)-one derivatives 11a-c

A solution of (3i, 0.0l mo1) in ethanol (20 ml) was treated with o-phenylenediamine derivatives (0.0l mo1) and triethylamine (0.5 ml). The reaction mixture was heated under reflux for 3hr and the solid that obtained after filtration was crystallized from ethanol/dioxane to give 11a-c.

1-(3-Ethoxyphenyl)-3-phenyl-1H-imidazo[4,5-b]quinoxalin-2(3H)-one (11a): as white crystals; yield 62%; mp 160-162 °C; IR: ν/cm^-1 = 3055 (CH-Ar), 2975 (CH-aliph.), 1744 (C=O), 1597 (C=N); ¹H NMR: δ/ppm = 1.37 (t, 3H, J = 7.0 Hz, CH₃), 4.10 (q, 2H, J = 7.0 Hz, CH₂), 7.06-7.98 (m, 13H, Ar-H); ¹³C NMR: 15.0 (CH₃), 63.8 (CH₂), 113.6, 114.6, 119.1, 119.2, 127.2, 127.5, 128.6, 129.3, 129.6, 130.3 (2C), 132.9, 133.9, 136.9, 137.0, 137.4, 138.8, 140.6 (18C-Ar), 152.6 (2C=N), 159.2 (C=O); Anal. Calcd. for C₂₃H₁₈N₄O₂ (382.41): C, 72.24; H, 4.74; N, 14.65; Found: C, 72.36; H, 4.44; N, 14.76%.

6-Chloro-1-(3-ethoxyphenyl)-3-phenyl-1H-imidazo[4,5-b]quinoxalin-2(3H)-one (11b): as gray crystals; yield 63%; mp 130-132 °C; IR: ν/cm^-1 = 3064 (CH-Ar), 2960 (CH-aliph.), 1748 (C=O), 1600 (C=N); ¹H NMR: δ/ppm = 1.37 (t, 3H, J = 6.8 Hz, CH₃), 4.09 (q, 2H, J = 7.0 Hz, CH₂), 7.09-7.98 (m, 12H, Ar-H); ¹³C NMR: 16.0 (CH₃), 53.9 (CH₂), 113.6, 115.4, 118.5, 119.3, 126.2, 126.8, 127.3 (3C), 127.8, 128.9, 129.5, 130.9, 131.5, 132.3, 132.5, 138.5, 152.6, 159.3 (C=N), 159.4 (C=N), 160.0 (C=O); MS, m/z (%): 416 (M⁺, 100), 417 (M+1, 54.2), 418 (M+2, 30.3), 402 (11.2), 387 (71.4), 320 (46.1), 253 (20.3), 163 (10.0), 176 (14.5), 163 (17.1), 135 (12.5), 91 (19.5), 77 (58.5); Anal. Calcd. for C₂₃H₁₇ClN₄O₂ (416.86): C, 66.27; H, 4.11; N, 13.44; Found: C, 66.13; H, 4.24; N, 13.21%.

1-(3-Ethoxyphenyl)-6-methyl-3-phenyl-1H-imidazo[4,5-b]quinoxalin-2(3H)-one (11c): as brown crystals; yield 67%; mp 118-120 °C; IR: ν/cm^-1 = 3054 (CH-Ar.), 2975, 2922 (CH-aliph.), 1744 (C=O), 1597 (C=N); ¹H NMR: δ/ppm = 1.37 (t, 3H, CH₃), 2.48 (s, 3H, CH₃), 4.09 (q, 2H, CH₂), 7.09-7.92 (m, 12H, Ar-H); ¹³C NMR: 15.0 (CH₃), 21.4 (CH₃), 63.8 (CH₂), 113.6, 114.7, 119.3, 119.4, 127.3, 127.6, 127.8, 127.9, 128.7, 129.6, 130.4, 132.8, 133.8, 138.9, 139.0, 140.6, 140.7, 150.5 (18C-Ar), 152.6 (2C=N), 159.3 (C=O); MS, m/z (%): 396 (M⁺, 100), 397 (M+1, 31.4), 381 (27.6), 368 (50.7), 320 (46.1), 77 (8.5); Anal. Calcd. for C₂₄H₂₀N₄O₂ (396.44): C, 72.71; H, 5.08; N, 14.13; Found: C, 72.86; H, 5.21; N, 14.24.

Synthesis of Imidazolidin-4-Ylidene Derivatives 12a-d

To a solution of iminothione 3c,i (0.0l mo1) in ethanol (20 ml) pyrazolone derivatives (0.01 mol) and triethylamine (0.5 ml) was added. The reaction mixture was heated under reflux for 10 hr. left to cool, the solid that obtained after filtration was crystallized from ethanol/dioxane to give 12a-d.

4-(3-(3-Chlorophenyl)-1-(4-chlorophenyl)-5-imino-2-oxoimidazolidin-4-ylidene)-3-methyl-1H-pyrazol-5(4H)-one (12a): as red crystals; yield 72%; mp 118-120 °C; IR: ν/cm^-1 = 3335 (NH), 3047 (CH-Ar), 2980, 2927 (CH-aliph.), 1726 (C=O), 1594 (C=N); ¹H NMR: δ/ppm: 2.31 (s, 3H, CH₃), 7.08-8.41 (m, 8H, Ar-H), 8.08 (br, 1H, NH; cancelled with D₂O); 12.72 (br, 1H, NH; cancelled with D₂O); Anal. Calcd. for C₁₉H₁₃Cl₂N₅O₂ (414.24): C, 55.09; H, 3.16; N, 16.91; Found: C, 55.29; H, 3.34; N, 16.71%.

4-(3-(3-Chlorophenyl)-1-(4-chlorophenyl)-5-imino-2-oxoimidazolidin-4-ylidene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (12b): as violet crystals; yield 60%; mp 93-95 °C, IR: ν/cm^-1 = 3345 (NH), 3063 (CH-Ar), 2922 (CH-aliph.), 1718 (C=O), 1621 (C=N); ¹H NMR: δ/ppm: 2.41 (s, 3H, CH₃), 7.11-8.79 (m, 13H, Ar-H), 12.64 (br, 1H, NH; cancelled with D₂O); MS, m/z (%): 490 (M⁺, 1.0), 478 (2.4), 417 (5.1), 405 (3.6), 379 (4.4), 358 (3.1), 355 (5.3), 333 (30.9), 315 (4.3), 304 (6.3), 278 (41.6), 255 (21.2), 208 (38.9), 179 (49.0), 159 (16.73), 122 (60.4), 106 (100), 68 (8.47); Anal. Calcd. for C₂₅H₁₇Cl₂N₅O₂ (490.34): C, 61.24; H, 3.49; N, 14.28; Found: C, 61.39; H, 3.54; N, 14.59%.

4-(3-(3-Ethoxyphenyl)-5-imino-2-oxo-1-phenylimidazolidin-4-ylidene)-3-methyl-1Hpyrazol-5(4H)-one (12c): as red crystals; yield 65%; mp 175-177 °C; IR: ν/cm^-1 = 3291 (NH), 3050 (CH-Ar), 2978 (CH-aliph.), 1713 (C=O), 1610 (C=N); ¹H NMR: δ/ppm: 1.12 (t, 3H, J = 6.9 Hz, CH₃), 2.32 (s, 3H, CH₃), 4.18 (q, 2H, J = 6.8 Hz, CH₂), 7.11-8.79 (m, 9H, Ar-H), 8.11 (br, 1H, NH; cancelled with D₂O); 12.71 (br, 1H, NH; cancelled with D₂O); MS, m/z (%): 389 (M⁺, 3.1), 390 (M+1, 3.1), 309 (12.2), 304 (100), 295 (12.6), 287 (13.6), 275 (13.9), 244 (24.9), 200 (23.2), 163 (13.6), 135 (30.3), 124 (44.1), 119 (18.0), 90 (2.3), 77 (26.3); Anal. Calcd. for C₂₁H₁₉N₅O3 (389.41): C, 64.77; H, 4.92; N, 17.98; Found: C, 64.86; H, 4.72; N, 17.76%.

4-(3-(3-Ethoxyphenyl)-5-imino-2-oxo-1-phenylimidazolidin-4-ylidene)-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (12d): as brown crystals; yield 68%; mp 70-72 °C; IR: ν/cm^-1 = 3280 (NH), 3055 (CH-Ar), 2978, 2914 (CH-aliph.), 1717 (C=O), 1603 (C=N); ¹H NMR: δ/ppm: 1.14 (t, 3H, J = 6.8 Hz, CH₃), 2.41 (s, 3H, CH₃), 4.19 (q, 2H, J = 6.9 Hz, CH₂), 7.13- 8.01 (m, 14H, Ar-H), 12.83 (br, 1H, NH; cancelled with D₂O); ¹³CNMR 14.8 (CH₃), 15.1 (CH₃), 63.3 (CH₂), 105.0, 110.7, 112.8, 115.4, 119.2, 120.7, 122.7, 123.7, 124.2, 127.3, 127.9, 128.8, 129.4, 130.3, 137.3, 139.8, 140.5, 144.7, 147.8, 153.9, 159.34 (C=N), 159.4 (C=N), 162.3 (C=O), 166.6 (C=O); Anal. Calcd. for C₂₇H₂₃N₅O₃ (465.50): C, 69.66; H, 4.98; N, 15.04; Found: C, 69.49; H, 4.76; N, 15.21%.

Antimicrobial Activity

Chemical compounds were individually tested against a panel of gram positive and gram negative bacterial pathogens and fungi. Antimicrobial tests were carried out by the agar well diffusion method using 100 μL of suspension containing 1 x10⁸ CFU/mL of pathological tested bacteria and 1 x10⁶ CFU/ml of fungi spread on nutrient agar and Sabouraud dextrose agar media, respectively. After the media had cooled and solidified, wells (10 mm in diameter) were made in the solidified agar and loaded with 100 μL of tested compound solution prepared by dissolving 5 mg of the chemical compound in one mL of dimethyl sulfoxide (DMSO). The inoculated plates were then incubated for 24 h at 37 °C. Negative controls were prepared using DMSO employed for dissolving the tested compound. Ampicillin, Gentamycin and Amphotericin B (1 mg/mL) were used as standard for antibacterial and antifungal activity, respectively. After incubation time, antimicrobial activity was evaluated by measuring the zone of inhibition against the test organisms and compared with that of the standard. The observed zone of inhibition is presented in Table 1. Antimicrobial activities were expressed as inhibition diameter zones in millimeters (mm). The experiment was carried out in triplicate and the average zone of inhibition was calculated.

Table 1. Antimicrobial activity of the synthesized compounds against the pathological organisms expressed as inhibition diameter zones in millimeters (mm) based on well diffusion assay.

Compd. No.	Gram Positive Bacteria			Gram Negative Bacteria			Fungi
	S. aureus	S. epidermidis	B. subtilits	N. gonorrhoeae	E. coli	K. pneumoniae	A. fumigatus	A. clavatus	G. candidum
3b	0	14.6 ± 0.43	16.2 ± 0.53	10.2 ± 0.72	13.7 ± 0.63	15.6 ± 0.25	12.6 ± 0.58	13.6 ± 0.25	15.2 ± 0.38
3f	20.3 ± 0.63	20.7 ± 0.22	22.4 ± .036	16.2 ± .058	17.6 ± 0.58	18.3 ± 1.2	20.5 ± 0.22	18.3 ± 0.26	22.1 ± 0.15
3g	14.3 ± 0.58	15.9 ± 0.44	17.2 ± 0.58	16.3 ± 0.63	18.9 ± 0.63	20.3 ± 0.63	16.3 ± 0.44	18.4 ± 0.58	19.1 ± 0.37
3h	13.6 ± 0.58	15.2 ± 0.44	16.1 ± 0.67	10.2 ± 0.46	13.0 ± 0.46	14.5 ± 0.46	10.6 ± 0.25	11.7 ± 0.34	16.5 ± 0.58
3i	18.9 ± 0.14	16.2 ± 0.15	19.8 ± 0.42	12.3 ± 0.53	15.3 ± 0.53	16.2 ± 0.53	15.7 ± 0.33	15.9 ± 0.25	16.8 ± 0.34
3j	18.9 ± 0.58	20.3 ± 0.43	21.4 ± 0.53	14.3 ± 0.25	16.9 ± 0.25	19.2 ± 0.25	17.6 ± 0.58	18.2 ± 0.25	20.3 ± 0.38
3k	15.8 ± 0.44	17.8 ± 0.17	19.8 ± 0.22	0	0	0	18.7 ± 0.11	19.3 ± 0.23	20.3 ± 0.27
4a	20.3 ± 0.58	17.3 ± 0.63	20.2 ± 0.44	13.6 ± 0.37	15.9 ± 0.37	16.8 ± 0.37	16.3 ± 0.44	18.6 ± 0.58	19.8 ± 0.25
4b	13.6 ± 0.25	20.6 ± 0.63	22.4 ± 0.44	14.3 ± 0.63	16.9 ± 0.58	17.8 ± 1.2	17.8 ± 0.63	18.9 ± 0.44	20.3 ± 0.25
6a	12.8 ± 0.58	14.3 ± 0.63	15.2 ± 0.72	0	0	0	13.3 ± 0.44	16.2 ± 0.58	17.2 ± 0.37
6b	17.3 ± 0.58	18.4 ± 0.17	20.3 ± 0.22	0	0	0	16.3 ± 0.39	18.2 ± 0.16	20.4 ± 0.58
8a	10.3 ± 0.58	11.6 ± 0.58	13.3 ± 0.72	0	0	0	12.3 ± 0.25	13.2 ± 0.25	15.2 ± 0.58
8b	9.6 ± 1.2	10.6 ± 0.58	12.7 ± 0.72	0	0	0	8.6 ± 0.36	9.3 ± 0.44	11.4 ± 0.58
9a	11.6 ± 0.58	12.4 ± 0.63	14.3 ± 0.63	0	0	0	16.3 ± 0.25	15.2 ± 0.58	17.3 ± 0.17
9b	16.3 ± 0.37	17.2 ± 0.20	19.4 ± 0.29	0	11.2 ± 0.33	15.9 ± 0.77	15.6 ± 0.44	16.2 ± 0.58	17.9 ± 0.37
9c	12.3 ± 0.58	14.2 ± 0.17	16.4 ± 0.22	0	0	0	10.3 ± 0.25	11.6 ± 0.34	13.4 ± 0.58
11a	16.6 ± 0.20	19.8 ± 0.12	21.4 ± 0.17	11.4 ± 0.42	18.3 ± 0.58	16.3 ± 0.81	20.6 ± 0.63	21.1 ± 0.27	21.9 ± 0.35
11b	15.6 ± 0.44	18.6 ± 0.44	21.1 ± 0.58	14.3 ± 0.20	16.3 ± 0.33	17.2 ± 0.34	18.3 ± 0.34	21.1 ± 0.25	21.3 ± 0.38
11c	21.6 ± 0.25	22.3 ± 0.25	25.8 ± 0.25	20.6 ± 0.25	21.6 ± 0.19	23.2 ± 0.42	20.9 ± 0.25	22.3 ± 0.25	24.8 ± 0.58
12a	17.2 ± 0.24	19.2 ± 0.15	19.8 ± 0.42	10.4 ± 0.25	13.4 ± 0.63	14.5 ± 0.51	15.7 ± 0.33	17.2 ± 0.25	19.8 ± 0.34
12b	19.2 ± 0.25	20.6 ± 0.25	20.9 ± 0.63	11.6 ± 0.43	15.7 ± 0.56	15.9 ± 0.77	16.8 ± 025	18.3 ± 0.44	20.4 ± 0.63
St. (A)	28.9 ± 0.14	25.4 ± 0.18	29.8 ± 0.35	-------	-------	-------	-------	-------	-------
St. (B)	-------	-------	-------	22.3 ± 0.58	23.4 ± 0.3	26.3 ± 0.15	-------	-------	-------
St. (C)	-------	-------	-------	-------	-------	-------	23.7 ± 0.10	21.9 ± 0.12	25.4 ± 0.16

St. (A): Ampicillin, St. (B): gentamycin and St. (C): Amphotericin b

Minimal Inhibitory Concentration (MIC) Measurement

Screening was evaluated in vitro using the Broth dilution method according to NCCLs [22, 23]. All the bacteria were incubated and activated at 30 °C for 24 h inoculation into nutrient broth and the fungi were incubated in malt extract broth for 48 h. The compounds were dissolved in DMSO and then diluted using cautiously adjusted Mueller-Hinton broth. Two-fold serial concentrations of the compounds were employed to determine the (MIC). In each case triplicate tests were performed and the average was taken as the final reading. The tubes were then inoculated with the test organisms, grown in their suitable broth at 37 °C for 24 hours for tested microorganisms (1×10⁸ CFU/mL for bacteria and 1 x10⁶ CFU/mL of fungi), each 5 ml received 0.1 mL of the above inoculum and incubated at 37 °C for 24 hours. The lowest concentration showing no growth was taken as the minimum inhibitory concentration (MIC).

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.