Efficient 1,3,4-Thiadiazole-4,5-dihydropyridazin-3(2H)-ones as Antimicrobial Agents

Abstract

A set of novel series of 1,3,4-thiadiazolyl-sulfanyl-4,5-dihydropyridazin-3(2H)-ones with anticipated antimicrobial activity has been synthesized. The synthetic protocol of the targeted compounds was accomplished by treating β-aroylacrylic acid 1 with 5-amino-1,3,4-thiadiazole-2-thiol (2) to afford the thia-Michael adduct 3. Afterwards, the obtained thia-Michael adduct 3 was cyclized to 4,5-dihydropyridazin-3(2H)-ones 4a–d and the non-cyclized product hydrazone 5 by using different hydrazines. Moreover, adduct 3 was reacted with esters like diethyl malonate and ethyl acetoacetate affording 1,3,4-thiadiazolobutanamides 6a, b. Furthermore, the concurrent reaction of later butamides 6a, b with the hydrazine derivatives furnished thiadiazolopyridazin-3(2H)-ones 7a–d, 8, and butanoic acid 9.

Antibiotic resistance is a major public health problem. Once confirmed primarily to hospitals it is now increasingly common in first care. The prevalence of resisting bacteria is rising, and organisms’ resistance to almost all antibiotics are obviously identified. The main causes are indiscriminate prescribing and the use of antibiotics in animal feeds and other agricultural applications. Policies to restrict use of antibiotics have had limited success.^1,2)

Heterocyclic compounds containing the pyridazinone backbone are cited as an important biologically active pharmacophores possessing the wide range of various bioorganic and medicinal chemistry applications, which provided an extended range of effective, safe and valuable drugs. Many Comprehensive reviews, as well as several research articles, have been accounted in detail for the significant role of pyridazinone derivatives in pharmacological and biological actions.^3–7)

A substantial number of pyridazinone-based frameworks are widely distributed in pharmaceutical active products with efficient cardiovascular action,⁸⁾ anti-convulsant,^9–11) anti-viral,^12–15) anti-cancer,^16–20) anti-tubercular,^21,22) anti-hypertensive,^23,24) anti-Alzheimer’s,^25–27) platelet aggregation,^28–30) and cholesterol acyltransferase inhibitors.³¹⁾ Several pyridazinone derivatives are showed as N-formyl peptide receptors agonists,³²⁾ and others are announced as selective calcium channel blockers.^33–35)

The majority of current studies in pharmacology have been reported pyridazin-3(2H)-ones as promising class of non-steroidal analgesic and anti-inflammatory agents without gastrointestinal side effect.^36–39) Furthermore, they are characterized as non-ulcerogenic and anti-nociceptive agents,^40–42) particularly pyridazinone derivatives with alkyl and hydrazone moieties linked to 2-nitrogen.^43,44) In addition, pyridazin-3(2H)-one derivatives bearing a thiourea or thioamide moiety possess antiulcer activity without anticholinergic effects.^45,46)

A set of 6-substituted pyridazinones have been explored as potent anti-microbial agents^47–49) as well as some of them, are optimized as Staphylococcus aures sortase A inhibitors.⁵⁰⁾

Recently, several exploratory studies of substituted 3(2H)-pyridazinones for their quantum chemical calculations, electronic, conformational, detailed structural, spectroscopic and optical properties are reported in the literature.^51–55)

Among the wide variety of synthetic protocols the application of 4-(4-chloro-3-methylphenyl)-4-oxobutanoic acid as key starting material play an important role in the functionalization and synthesis of a diverse of pyridazinone derivatives. In view of such observations, much interest has been devoted to synthesize some novel 2,6-disubstituted dihydropyridazinones bearing 1,3,4-thiadiazole as a great important moiety affecting their biocidal activities.

Results and Discussion

Chemistry

The titled compound 4-oxobutanoic acid 3 was obtained by treating β-aroylacrylic acid 1 with a solution of 5-amino-1,3,4-thiadiazole-2-thiol (2) in absolute EtOH.^56,57) Where the β-aroylacrylic acid 1 adds the sulfur nucleophile stereoselectively at a β-carbon atom with respect to α,β-unsaturated ketone. Alkylation of the thiadiazole 2 occurs readily at the S-atom to afford the thia-Michael adduct 3. The IR spectrum of the thia-Michael adduct 3 showed strong absorption peaks at 1678, 1725, and 3084 cm⁻¹ attributed to υ_C=O (carbonyl), υ_C=O (carboxylic), and υ_OH, respectively, as well as a basin peak at 3428 cm⁻¹ for NH₂. ¹H-NMR displayed multiplet signals at δ 2.33–2.50 ppm characteristic to both diastereotopic (CH₂) protons at the α-carbon atom to the (COOH) group and singlet signals at δ 6.71 and 9.40 ppm (Exchangeable in D₂O) for primary amine (NH₂) and OH protons, respectively. Electron ionization (EI)-MS spectrum of this compound confirmed the structure and showed molecular ion peak m/z 344 (M−NH₂) (cf. Chart 1 and Experimental).

Chart 1. Synthesis of Thia-Michael Adduct 3

Hydrazinolysis of adduct 3 using hydrazine derivatives namely, hydrazine hydrate, acylhydrazine, formohydrazine, and/or thiosemicarbazide afforded dihydropyridazin-3(2H)-one derivatives 4a–d, respectively. The structure of the pyridazinones 4a–d was deduced from correct elemental and spectral analysis. The IR spectra of compounds 4a–d exhibited strong absorption peaks at 1650–1676 cm⁻¹ characteristic for υ_C=O amide groups, devoid any peaks for carboxyl groups. In the ¹H-NMR spectrum of 4a, the two diastereotopic protons (CH₂) at C-5 of pyridazinone showed as multiplet peaks at δ 2.26–2.51 ppm, singlet peak at δ 6.73 ppm for primary (NH₂) protons, while the pyridazinone (NH) proton appeared as singlet peak at δ 13.20 (Exchangeable in D₂O). EI-MS spectra of compounds 4a–d confirmed the proposed structures and revealed correct molecular ions m/z 354, 396, 382, and 413 (M⁺) respectively (cf. Chart 2 and Experimental).

Considering the structure of the obtained dihydropyridazin-3(2H)-one derivatives 4a–d, the reaction route could possibly proceed via the addition of the more nucleophilic primary amine of hydrazine(s) at the carbonyl group forming the corresponding hydrazone, followed by ring closure.

On the other hand, carrying out hydrazinolysis using semicarbazide under mild reaction condition, the hydrazone 5 was isolated as the final sole product and the pyridazinone cyclization not occurred. IR spectrum of hydrazone 5 was characterized by the presence of strong absorption peak at 1678 cm⁻¹ attributed to υ_C=O (amide) and a strong peak at 1725 cm⁻¹ as an indication of the presence of υ_C=O (carboxyl group), and formation of the corresponding hydrazone without cyclization. EI-MS spectrum confirmed the proposed structure and revealed the correct molecular ion peak at m/z 399 (cf. Chart 2 and Experimental).

Chart 2. Formation of Pyridazin-3(2H)-ones 4a–d and Hydrazone 5

Attempts at generating other interesting chromophores or even extending conjugation (or extending functionalization) beside thiadiazolo-pyridazinones for oxobutanoic acid 3 have been succeeded. In this respects, the thia-Michael adduct 3 was submitted to react with esters like diethyl malonate and/or ethyl acetoacetate in basic medium⁵⁸⁾ and the butanoic acids 6a, b were afforded, respectively. Structures of butanoic acids 6a, b were inferred from their analytical and spectral data. The IR spectra of both compounds were characterized by the presence of strong peaks at 1654–1683 cm⁻¹ attributed to υ_max of the carbonyl. Furthermore, IR spectrum of 6a showed a strong absorption peak at 1736 cm⁻¹ attributed to υ_C=O ester, while the spectrum of 6b showed absorption peak at 1726 cm⁻¹ as evidence of the presence of terminal aliphatic carbonyl group. ¹H-NMR spectrum of compound 6b displayed a multiplet signal at δ 2.30–2.48 ppm characteristic to both diastereotopic (CH₂) protons at the α-carbon atom to the (COOH) group and a singlet signal at δ 3.17 ppm of the aliphatic methylene protons. The secondary amide (CO-NH) and (OH) protons were showed at δ 12.80 and 13.00 ppm respectively (D₂O exchangeable) (cf. Chart 3 and Experimental).

Chart 3. Synthesis and Hydrazinolysis of Butanamides 6a, b

The butanoic acid derivatives 6a, b were subjected to further chemical investigation with the aim of study their behavior toward different N-nucleophiles. In this circumstance, hydrazinolysis of both compounds 6a, b using hydrazine hydrate under mild reaction conditions afforded the pyrazolylamino-1,3,4-thiadiazolyl-4,5-dihydropyridazin-3(2H)-one derivatives 7a and 8 respectively. The ring closure could possibly have occurred via the N-nucleophilic addition of NH₂ of hydrazine at the more reactive aroyl carbonyl as well as the ethoxy carbonyl and/or the acetyl group in compounds 6a, b, respectively. Afterward, the ring closure at both sites has taken place to afford the pyrazolyl-thiadiazolylpyridazinone derivatives 7a and 8. The structures of pyridazinones 7a and 8 were confirmed from their correct analytical and spectral data. Their IR spectra exhibited strong absorption bands within 1661–1671 and 3226–3415 cm⁻¹ attributable to υ_C=O of amides and υ_NH, respectively. ¹H-NMR spectrum of compound 8 was characterized by two singlet signals at δ 3.57 and 3.64 ppm for methyl and methylene protons at the pyrazole ring, while the four NH and cyclic amide protons appeared as singlet signals at δ 11.17 and 13.20 ppm, respectively. EI-MS spectra of both compounds 7a and 8 have confirmed the assumed structure and revealed correct molecular ion peaks m/z 434 and 435, respectively (cf. Chart 3 and Experimental).

In the same fashion, oxobutanoic acid derivative 6a has been submitted to react with hydrazine derivatives namely acetohydrazide, formohydrazide, and/or thiosemicarbazide in N,N-dimethylformamide (DMF) and furnished the pyrazolyl-1,3,4-thiadiazolyl pyridazinone derivatives 7b–d, respectively. In this situation, the reaction proceeded at the aroyl and acetyl carbonyl as well to give the corresponding pyridazinone derivatives 7b–d. The structures of compounds 7b–d were established by their correct analytical and compatible spectroscopic data. Their IR spectra exhibited strong absorption peaks within 1681–1683, 1732–1733, and a broad peak at 3426–3437 cm⁻¹ attributed to υ_C=O (amides), υ_C=O (aliphatic amides), and υ_NH, respectively. EI-MS spectra of compounds 7b–d confirmed the assumed structures and recorded correct molecular ion peaks at m/z 538, 510, 572, respectively (cf. Chart 4 and Experimental). Such results are in consistency with thiosemicarbazone primary aliphatic amide and thioamide.^59,60)

On the other hand, treating oxobutanoic acid derivative 6a with semicarbazide under the same reaction condition mentioned above afforded quite a different result. The corresponding open adduct 9 was obtained without ring closure at both sites. The structure of compound 9 was inferred from its analytical and spectroscopic data. Its IR spectrum was characterized by the presence of peaks at 1679 and 1734 cm⁻¹ attributed to υ_C=O (amide and carboxy). EI-MS spectrum of compound 9 confirmed the proposed structure and showed correct molecular ion peak at m/z 558 (cf. Chart 4 and Experimental).

Chart 4. Reactions of Butanamide 6a with Hydrazine Derivatives

According to the present results, thiosemicarbazide is more highly nucleophilic thioamide afforded the pyridazinone derivatives 4d and 7d. However, acetohydrazide and formohydrazide gave pyridazinones 4b, c and 7b, c instead of hydrazines like semicarbazones 5 or 9 which undergo virtually no hydration under the mentioned reaction conditions. In this respects, the abnormal course of the reaction in the former case cannot be accounted only for the different in nucleophilicity. This is due to steric factor and solvation playing a prominent role in hetero-ring closure.

Antimicrobial Activity

In this study, some of the synthesized compounds were screened for their in vitro antibacterial and antifungal activities. They were tested at (5 mg/mL) concentration against two Gram (G)-positive bacteria (Streptococcus pneumonia RCMB 010010, Bacillus subtilis RCMB 010067), two G-negative bacteria (Escherichia coli RCMB 010052, Pseudomonas aeruginosa RCMB 010043), and four yeast (Aspergillus fumigatus RCMB 02568, Syncephalastrum racemosum RCMB 05992, Geotrichum candidum RCMB 05097, Candida albicans RCMB 05036). Ampicillin, gentamicin, and amphotericin B were used as control drug standards for G-positive bacteria, G-negative bacteria, and fungi references respectively. The zone of inhibition (mm) and minimum inhibitory concentration (MIC) (in mg/mL) of the compounds are presented in Tables 1–4.

Table 1. Antibacterial Activity of Some Synthesized Compounds Using Agar Well Diffusion Method (5 mg/mL)

Comp. No.	Zone of inhibition
	Gram-positive		Gram-negative
	Streptococcus pneumonia	Bacillus subtilis	Escherichia coli	Pseudomonas aeruginosa
	(RCMB 010010)	(RCMB 010067)	(RCMB 010052)	(RCMB 010043)
4a	21.7±2.10	23.2±0.57	20.8±0.63	NA
4b	19.4±0.63	21.2±0.63	18.6±1.20	NA
4c	17.9±0.58	18.2±0.53	17.7±1.50	NA
4d	29.4±2.10	34.6±1.50	27.2±0.72	NA
5	24.6±1.00	28.6±0.72	23.2±0.58	NA
6a	16.0±0.44	18.3±0.67	13.0±0.46	NA
6b	13.6±0.44	15.6±0.63	10.3±0.25	NA
7c	22.5±1.50	24.2±1.00	19.3±1.20	NA
7d	25.1±1.00	27.4±0.72	23.4±0.63	NA
Ampicillina)	23.8±0.44	32.4±0.3
Gentamicina)			19.9±0.3	17.3±0.1

Data are expressed in the form of mean±standard deviation (S.D.). NA, no activity. a) Control drug concentrations (1 mg/mL), 6.00 mm, (100 µL was tested).

Table 2. Antifungal Activity of Some Synthesized Compounds Using Agar Well Diffusion Method (5 mg/mL)

Comp. No.	Zone of inhibition
	Aspergillus fumigatus	Syncephalastrum racemosum	Geotrichum candidum	Candida albicans
	(RCMB 02568)	(RCMB 05992)	(RCMB 05097)	(RCMB 05036)
4a	22.6±1.50	26.7±0.25	23.3±2.10	NA
4b	20.3±0.72	19.2±0.34	20.7±1.50	NA
4c	18.2±0.58	17.6±1.60	18.9±0.38	NA
4d	26.4±0.72	27.3±0.58	31.2±0.63	22.4±1.50
5	22.8±2.10	21.8±0.58	24.2±1.30	17.3±0.63
6a	10.6±0.25	11.7±0.34	16.5±0.58	NA
6b	NA	NA	NA	NA
7c	19.6±2.10	20.4±0.72	23.6±0.72	NA
7d	22.3±1.50	23.4±0.58	27.3±0.72	NA
Amphotericin Ba)	23.7±0.10	19.7±0.20	28.7±0.20	25.4±0.10

Data are expressed in the form of mean±standard deviation (S.D.). NA, no activity. a) Control drug concentrations (1 mg/mL), 6.00 mm, (100 µL was tested).

Table 3. Antibacterial Minimum Inhibitory Concentration MIC (mg/mL)

Comp. No.	Gram-positive		Gram-negative
	Streptococcus pneumonia	Bacillus subtilis	Escherichia coli
	(RCMB 010010)	(RCMB 010067)	(RCMB 010052)
4d	0.60	1.25	0.60
6a	2.50	1.25	1.25
6b	2.50	2.50	2.50
7d	0.60	0.60	0.60
Ampicillin	0.60	0.60
Gentamicin			0.60

Table 4. Antifungal Minimum Inhibitory Concentration MIC (mg/mL)

Comp. No.	Aspergillus fumigatus	Syncephalastrum racemosum	Geotrichum candidum
	(RCMB 02568)	(RCMB 05992)	(RCMB 05097)
4d	1.25	1.25	1.25
6a	5.00	2.50	5.00
7d	2.50	1.25	2.50
Amphotericin B	0.60	0.60	0.60

Most of the tested compounds showed excellent antimicrobial activities with respect to the control drugs. Regarding antibacterial activity (Table 1), compounds 4d, 5 and 7d showed potent activity against B. subtilis. Compounds 4d, 5 and 7d revealed powerful activity and their effect exceeded the control drug against S. pneumonia. Against E. coli, compounds 4a, 4d, 5, and 7d exhibited strong potent activity and their bacteriostatic effect has overtaken the control drug. Moreover, compounds 4b, 4c, and 7c recorded potent activity, while 6a and 6b were the less activity against all strains. All compounds were inactive against P. aeruginosa.

Table 2 showed the antifungal activity of the tested compounds, where compounds 4a, 4b, 4c, 5, 7c, and 7d displayed strong activity against A. fumigatus. Compound 4d was the most potent and its effect exceeded the control drug. Compounds 4b and 4c were potent against S. racemosum, while the biocidal effect of compounds 4a, 4d, 5, 7c, and 7d has overtaken the control drug. Compound 4d showed superior antifungal activity on G. candidum and preceded the control drug. Compounds 4b and 4c revealed moderate activity on G. candidum, while 4a, 5, 7c, and 7d were more potent. Regarding C. albicans strain, only compound 4d was the most potent while 5 is the less effective. The rest compounds were inactive against such strain. Among all compounds tested for antifungal activity, compounds 6a and 6b were inactive against all strains.

According to the obtained results and the structure of the tested compounds, it was inferred that; pyridazinone rings 4a–d and 5 resulted from thia-Michael adduct 3 were generally very potent against all bacterial and fungal strains except for P. aeruginosa and C. albicans. The presence of either carbamoyl or thiocarbamoyl moiety at N-2 significantly increased the activity to precede the control drug.

Conclusion

The authors successfully endeavor to design, synthesize and evaluate new series of antimicrobial 4,5-dihydropyridazin-3(2H)-one derivatives, based on thia-Michael addition and producing the more interesting functionalized heterocycles containing the important moiety (1,3,4-thiadiazole). The structure of the newly synthesized compounds was elucidated using different spectral and analytical analyses. We hereby highlighted the potential of such new heterocycles as antimicrobial agents.

Experimental

Reagents and Instruments

Reagents and solvents were dried and purified before use by the usual procedures. mp: Büchi^® melting point apparatus; uncorrected. TLC: Merck TLC aluminium sheets, silica gel 60F₂₅₄ with detection by UV quenching at 254 nm. IR spectra: FT-IR Nicolet Impact 400D; KBr pellets; ν in cm⁻¹. The ¹H- and ¹³C-NMR spectra were recorded on a Varian at 300 and 75 MHz, respectively; in dimethyl sulfoxide (DMSO)-d₆; δ in ppm relative to Me₄Si as an internal standard, J in Hz. EI-MS were recorded on a gas chromatographic GCMS-HP model MS5988. Elemental analyses carried out at the Micro Analytical Center, Cairo University, Giza, Egypt. Antimicrobial analyses carried out at Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo Egypt.

(E)-4-(4-Chloro-3-methylphenyl)-4-oxobut-2-enoic acid (1) and 5-amino-1,3,4-thiadiazole-2-thiol (2) were synthesized according to previously reported studies.^56,57)

2-[(5-Amino-1,3,4-thiadiazol-2-yl)sulfanyl]-4-(4-chloro-3-methylphenyl)-4-oxobutanoic Acid (3)

Aroylacrylic acid 1 (10 mmol) was added to a hot solution of 5-amino-1,3,4-thiadiazole-2-thiol 2 (10 mmol) in absolute EtOH (15 mL) and refluxed for 3h. The excess solvent was evaporated under reduced pressure and cold water was added to the residue. The precipitate that obtained was filtered off, washed with water, and crystallized from EtOH/water to give 3. Orange powder; Yield 68%; mp 150–152°C; ¹H-NMR (DMSO-d₆) δ: 2.19 (3H, s, CH₃), 2.33–2.46 (2H, m, diastereotopic CH₂), 3.98 (1H, t, J=15 Hz, CH), 6.71 (2H, s, D₂O Exch., NH₂), 7.30–8.32 (3H, m, Ar-H), 9.40 (1H, s, D₂O Exch., OH). ¹³C-NMR (DMSO-d₆) δ: 21.6 (CH₃), 31.9 (C-5 pyridazinone), 44.3 (C-4 pyridazinone), 126.8, 131.2, 133.4, 136.9, 137.5, 138.8 (C and CH aromatic), 146.8, 163.7 (C=N thiadiazole), 173.1, 189.3 (C=O). IR (KBr) cm⁻¹: 3428 (br NH), 3084 (OH), 2919 (CH-aliph.), 1725, 1679 (C=O). MS m/z: 344 [M−NH₂], 326, 315, 235, 168, 152. Anal. Calcd for C₁₃H₁₂ClN₃O₃S₂: C, 43.64; H, 3.38; N, 11.74; S, 17.92. Found: C, 43.37; H, 3.27; N, 11.56; S, 17.77.

General Procedure for Synthesis of Pyridazinones 4a–d and Hydrazine 5

A mixture of thia-Michael adduct 3 (10 mmol) in DMF (15 mL) and hydrazine derivatives (hydrazine hydrate, acetohydrazide, formohydrazide, thiosemicarbazide and/or semicarbazide) (10 mmol) was heated under reflux at 85–90°C for 8h. After cooling, the reaction mixture was poured upon cold water and the solid that precipitated was collected, washed with water, and crystallized from the proper solvent MeOH/H₂O to give 4a–d and 5, respectively.

4-[(5-Amino-1,3,4-thiadiazol-2-yl)sulfanyl]-6-(4-chloro-3-methylphenyl)-4,5-dihydropyridazin-3(2H)-one (4a)

Brownish yellow powder; Yield 67%; mp 171–173°C; ¹H-NMR (DMSO-d₆) δ: 1.91 (3H, s, CH₃), 2.26–2.47 (2H, m, diastereotopic CH₂), 3.79 (1H, t, J=15 Hz, CH), 7.18 (2H, s, D₂O Exch., NH₂), 7.30–8.03 (3H, m, Ar-H), 13.20 (1H, s, D₂O Exch., NH). ¹³C-NMR (DMSO-d₆) δ: 20.8 (CH₃), 31.7 (C-5 pyridazinone), 44.4 (C-4 pyridazinone), 125.7, 129.8, 134.6, 136.5, 137.4, 138.9 (C and CH aromatic), 150.1 (C=N pyridazinone), 159.9, 163.4 (C=N thiadiazole), 167.5 (C=O). IR (KBr) cm⁻¹: 3253 (NH₂), 3055 (CH arom.), 2919 (C–H aliph.), 1654 (C=O). MS m/z: 354 [M], 339, 325, 308, 286, 254, 197, 187, 148. Anal. Calcd for C₁₃H₁₂ClN₅OS₂: C, 44.13; H, 3.42; N, 19.79; S, 18.12. Found: C, 43.82; H, 3.38; N, 19.61; S, 17.96.

2-Acetyl-4-[(5-amino-1,3,4-thiadiazol-2-yl)sulfanyl]-6-(4-chloro-3-methylphenyl)-4,5-dihydropyridazin-3(2H)-one (4b)

Brownish yellow powder; Yield 58%; mp 166–168°C; ¹H-NMR (DMSO-d₆) δ: 1.98 (3H, s, Me), 2.25–2.56 (2H, m, diastereotopic CH₂), 3.40 (3H, s, COMe), 4.51 (1H, t, J=15 Hz, CH), 7.14 (2H, s, D₂O Exch., NH₂), 7.50–8.13 (3H, m, Ar-H). IR (KBr) cm⁻¹: 3426 (br. NH), 2921 (CH aliph.), 1679 (CO amide). MS m/z: 396 [M]⁺, 369, 353, 325, 297, 268, 244, 210, 196, 185, 153. Anal. Calcd for C₁₅H₁₄ClN₅O₂S₂: C, 45.51; H, 3.56; N, 17.69; S, 16.20. Found: 45.23; H, 3.51; N, 17.51; S, 16.08.

5-[(5-Amino-1,3,4-thiadiazol-2-yl)sulfanyl]-3-(4-chloro-3-methylphenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-carbaldehyde (4c)

Brownish yellow powder; yield 59%; mp 123–125°C; ¹H-NMR (DMSO-d₆) δ: 1.92 (3H, s, CH₃), 2.23–2.63 (2H, m, diastereotopic CH₂), 3.86 (1H, t, J=15 Hz, CH), 7.19 (2H, s, D₂O Exch., NH₂), 7.34–7.92 (3H, m, Ar-H), 9.08 (1H, s, CHO). IR (KBr) cm⁻¹: 3432 (br. NH), 2921 (CH aliph.), 1680 (CO amide). MS m/z: 381 [M]⁺, 353, 337, 310, 282, 251, 222, 216, 198, 186. Anal. Calcd for C₁₄H₁₂ClN₅O₂S₂: C, 44.04; H, 3.17; N, 18.34; S, 16.79. Found: C, 43.73; H, 3.10; N, 18.26; S, 16.55.

5-[(5-Amino-1,3,4-thiadiazol-2-yl)sulfanyl]-3-(4-chloro-3-methylphenyl)-6-oxo-5,6-dihydropyridazin-1(4H)-carbothioamide (4d)

Brownish yellow powder; yield 65%; mp 208–210°C; ¹H-NMR (DMSO-d₆) δ: 1.89 (3H, s, CH₃), 2.27–2.66 (2H, m, diastereotopic CH₂), 3.92 (1H, t, J=15 Hz, CH), 7.13 (2H, s, D₂O Exch., NH₂), 7.39–7.58 (3H, m, Ar-H), 8.46 (2H, s, D₂O Exch., CS-NH₂). IR (KBr) cm⁻¹: 3431(br NH), 2920 (CH aliph.), 1680 (CO amide). MS m/z: 413 [M]⁺, 387, 355, 314, 296, 278, 266, 245, 209, 184. Anal. Calcd for C₁₄H₁₃ClN₆OS₃: C, 40.72; H, 3.17; N, 20.35; S, 23.29. Found: C, 40.45; H, 3.11; N, 20.21; S, 23.05.

5-[(5-Amino-1,3,4-thiadiazol-2-yl)sulfanyl]-4-(2-carbamoylhydrazinylidene)-4-(4-chloro-3-methylphenyl)-butanoic Acid (5)

Dark yellow powder; yield 78%; mp 178–180°C; ¹H-NMR (DMSO-d₆) δ: 1.93 (3H, s, CH₃), 2.31–2.74 (2H, m, diastereotopic CH₂), 3.84 (1H, t, J=15 Hz, CH), 6.78 (2H, s, D₂O Exch.,CO-NH₂), 7.23 (2H, s, D₂O Exch., NH₂), 7.49–8.01 (3H, m, Ar-H), 9.86 (1H, s, D₂O Exch., CO-NH), 13.26 (1H, s, D₂O Exch.,OH). ¹³C-NMR (DMSO-d₆) δ: 22.1 (CH₃), 37.6 (C-5 pyridazinone), 41.9 (C-4 pyridazinone), 127.1, 130.8, 133.6, 136.2, 137.5, 139.1 (C and CH aromatic), 147.2, 163.9 (C=N thiadiazole), 157.3 (C=N), 173.2 (C=O). IR (KBr) cm⁻¹: 3433 (br. NH/NH₂), 2920 (CH aliph.), 1722 (CO carboxyl), 1678 (CO amide). MS m/z: 399 [M⁺−NH₂], 357, 330, 316, 260, 229, 213, 186, 156. Anal. Calcd for C₁₄H₁₅ClN₆O₃S₂: C, 40.53; H, 3.64; N, 20.26; S, 15.45. Found: C, 40.29; H, 3.58; N, 20.11; S, 15.24.

General Procedure for Butamides 6a, b

To a solution of compound 3 (10 mmol) in dry MeOH (80 mL), sodium metal (20 mmol) was added followed by diethylmalonate and/or ethyl acetoacetate (10 mmol) and the mixture was heated under reflux in a water bath at 60°C for 5h. The cooled reaction mixture was concentrated under reduced pressure, and then acidified with dil. HCl. The obtained precipitate was collected, washed with water, and recrystallized from the proper solvent to afford 6a and 6b, respectively.

4-(4-Chloro-3-methylphenyl)-2-({5-[(3-ethoxy-3-oxopropanoyl)amino]-1,3,4-thiadiazol-2-yl}sulfanyl)-4-oxobutanoic Acid (6a)

Orange yellow powder; yield 61% (EtOH/H₂O); mp 130–132°C; ¹H-NMR (DMSO-d₆) δ: 1.38 (3H, t, J=7.2 Hz, CH₃), 1.94 (3H, s, CH₃), 2.31–2.63 (2H, m, diastereotopic CH₂), 3.18 (2H, s, CH₂), 4.08 (1H, t, J=15 Hz, CH), 4.46 (2H, q, J=7.6 Hz, CH₂Me), 7.28–7.79 (3H, m, Ar-H), 12.73 (1H, s, D₂O Exch., CO-NH), 13.18 (1H, s, D₂O Exch.,OH). ¹³C-NMR (DMSO-d₆) δ: 13.8 (CH₃), 20.5 (CH₃), 37.4 (C-5 pyridazinone), 41.7 (C-4 pyridazinone), 52.4 (CH₂), 127.2, 130.8, 133.6, 136.0, 137.6, 138.7 (C and CH aromatic), 148.1, 163.0 (C=N thiadiazole), 162.9, 167.7, 173.4, 189.2 (C=O). IR (KBr) cm⁻¹: 3423 (br. NH), 2922 (CH aliph.), 1736 (CO ester), 1683 (CO carbonyl), 1593 (C=N). MS m/z: 459 [M⁺−CH₃], 358, 259, 249, 224, 183. Anal. Calcd for C₁₈H₁₈ClN₃O₆S₂: C, 45.81; H, 3.84; N, 8.90; S, 13.59. Found: C, 45.59; H, 3.79; N, 8.74; S, 13.44.

4-(4-Chloro-3-methylphenyl)-2-({5-[(3-oxobutanoyl)amino]-1,3,4-thiadiazol-2-yl}sulfanyl)-4-oxobutanoic Acid (6b)

Orange yellow powder; yield 74% (EtOH/H₂O); mp 120–122°C; ¹H-NMR (DMSO-d₆) δ: 1.42 (3H, t, J=7.2 Hz, Me), 1.96 (3H, s, Me), 2.30–2.50 (2H, m, diastereotopic CH₂), 3.17 (2H, s, CH₂), 4.13 (1H, t, J=15 Hz, CH), 7.33–7.67 (3H, m, Ar-H), 12.80 (1H, s, D₂O Exch., CONH), 13.04 (1H, s, D₂O Exch., OH). IR (KBr) cm⁻¹: 3425 (br. NH), 2921 (CH aliph.), 1729 (CO carbonyl), 1654 (CO amide), 1595 (C=N). MS m/z: 441 [M]⁺, 412, 402, 338, 259, 224, 218, 209, 194, 180, 153. Anal. Calcd for C₁₇H₁₆ClN₃O₅S₂: C, 46.21; H, 3.65; N, 9.51; S, 14.51. Found: C, 45.93; H, 3.59; N, 9.43; S, 14.38.

General Procedure for Pyridazin-3(2H)-one Derivatives 7a–d, 8 and 9

A mixture of oxobutanoic acid 6a and/or 6b (10 mmol) and hydrazine derivatives (hydrazine hydrate, acylhydrazine, formylhydrazine, and/or thiosemicarbazide) (20 mmol) in DMF (30 mL) was heated under refluxed (110–120°C) for 8h. The reaction mixture was left to cool to room temperature then poured into ice/water. The solid that formed was filtered off, washed with cold water, dried, and recrystallized from the suitable solvent to give the targeted compounds.

4-(4-Chloro-3-methylphenyl)-4-({5-[(5-oxo-4,5-dihydro-1H-pyrazol-3-yl)amino]-1,3,4-thiadiazol-2-yl}sulfanyl)-4,5-dihydropyridazin-3(2H)-one (7a)

Yellow powder; yield 73% (EtOH/H₂O); mp 162–164°C; ¹H-NMR (DMSO-d₆) δ: 1.93 (3H, s, CH₃), 2.24–2.58 (2H, m, diastereotopic CH₂), 3.43 (2H, s, CH₂), 3.98 (1H, t, J=15 Hz, CH), 6.08 (1H, s, D₂O Exch., NH), 7.43–7.92 (3H, m, Ar-H), 9.82 (1H, s, D₂O Exch., NH), 12.67 (1H, s, D₂O Exch., NH). ¹³C-NMR (DMSO-d₆) δ: 21.9 (CH₃), 31.6 (C-5 pyridazinone), 44.7 (C-4 pyridazinone), 56.6 (CH₂), 126.7, 131.4, 133.5, 135.9, 137.3, 138.9 (C and CH aromatic), 147.3 (C=N pyrazole), 149.1, 161.6 (C=N thiadiazole), 162.0 (C=N pyridazinone), 167.5, 175.1 (C=O). IR (KBr) cm⁻¹: 3226 (br. NH), 2921 (CH aliph.), 1671 (CO amide), 1592 (C=N). MS m/z: 435 [M]⁺, 395, 351, 314, 284, 252, 220, 180, 153. Anal. Calcd for C₁₆H₁₄ClN₇O₂S₂: C, 44.09; H, 3.24; N, 22.49; S, 14.71. Found: C, 43.85; H, 3.18; N, 22.31; S, 14.53.

N-(5-{[2-Acetyl-6-(4-Chloro-3-methylphenyl)-3-oxo-2,3,4,5-tetrahydropyridazin-4-yl]sulfanyl}-1,3,4-thiadiazol-2-yl}-3-(2-acetylhydrazinyl)-3-oxopropanamide (7b)

Beige powder; yield 64% (EtOH/H₂O); mp 146–148°C; ¹H-NMR (DMSO-d₆) δ: 1.78, 1.92, 2.16 (9H, s, 3CH₃), 2.26–2.61 (2H, m, diastereotopic CH₂), 3.24 (2H, s, CH₂), 4.02 (1H, t, J=15 Hz, CH), 6.09 (3H, s, D₂O Exch., 3NH), 7.49–7.91 (3H, m, Ar-H). IR (KBr) cm⁻¹: 3426 (br. NH), 2924 (CH aliph.), 1733 (CO aliph. amide), 1682 (CO amide), 1595 (C=N). MS m/z: 538 [M]⁺, 509, 493, 479, 459, 423, 395, 383, 353, 292, 278. Anal. Calcd for C₂₀H₂₀ClN₇O₅S₂: C, 44.65; H, 3.75; N, 18.22; S, 11.92. Found: C, 44.43; H, 3.69; N, 18.02; S, 11.83.

N-(5-{[2-Formyl-6-(4-chloro-3-methylphenyl)-3-oxo-2,3,4,5-tetrahydropyridazin-4-yl]sulfanyl}-1,3,4-thiadiazol-2-yl}-3-(2-formylhydrazinyl)-3-oxopropanamide (7c)

Beige powder; yield 67% (EtOH/H₂O); mp 152–154°C; ¹H-NMR (DMSO-d₆) δ: 1.97 (3H, s, CH₃), 2.31–2.58 (2H, m, diastereotopic CH₂), 3.19 (2H, s, CH₂), 3.96 (1H, t, J=15 Hz, CH), 6.17, 6.24 (3H, s, D₂O Exch., 3NH), 7.46–7.94 (3H, m, Ar-H), 9.38, 9.65 (2H, s, CHO). IR (KBr) cm⁻¹: 3424 (br. NH), 2921 (CH aliph.), 1733 (CO aldehyde), 1683 (CO amide), 1595 (C=N). MS m/z: 510 [M]⁺, 480, 470, 466, 449, 463, 407, 385, 360, 337, 282, 279. Anal. Calcd for C₁₈H₁₆ClN₇O₅S₂: C, 42.40; H, 3.16; N, 19.23; S, 12.57. Found: C, 22.78; H, 3.09; N, 19.01; S, 12.38.

N-(5-{[2-Carbamothioyl-6-(4-chloro-3-methylphenyl)-3-oxo-2,3,4,5-tetrahydropyridazin-4-yl]sulfanyl}-1,3,4-thiadiazol-2-yl}3-(2-carbamothioylhydrazinyl)-3-oxopropanamide (7d)

Yellow powder; yield 58% (EtOH/H₂O); mp 161–163°C; ¹H-NMR (DMSO-d₆) δ: 2.01 (3H, s, CH₃), 2.33–2.64 (2H, m, diastereotopic CH₂), 3.21 (2H, s, CH₂), 4.05 (1H, t, J=15 Hz, CH), 6.08 (2H, s, D₂O Exch., NH), 7.46–8.01 (3H, m, Ar-H), 9.84 (1H, s, D₂O Exch., NH), 12.73 (4H, s, D₂O Exch., 2NH₂). IR (KBr) cm⁻¹: 3431 (br. NH/NH₂), 2925 (CH aliph.), 1682 (CO amide), 1594 (C=N). MS m/z: 572 [M]⁺, 554, 541, 511, 496, 480, 469, 436, 423, 412, 372, 331, 292, 278. Anal. Calcd for C₁₈H₁₈ClN₉O₃S₄: C, 37.79, H, 3.17; N, 22.04; S, 22.42. Found: C, 37.53; H, 3.10; N, 21.89; S, 22.31.

6-(4-Chloro-3-methylphenyl)-4-({5-[(5-methyl-4H-pyrazol-3-yl)amino]-1,3,4-thiadiazol-2-yl}sulfanyl)-4,5-dihydropyridazin-3(2H)-one (8)

Yellow powder; yield 73% (EtOH/H₂O); mp 131–133°C; ¹H-NMR (DMSO-d₆) δ: 1.92, 2.13 (6H, s, 2CH₃), 2.25–2.60 (2H, m, diastereotopic CH₂), 3.26 (2H, s, CH₂), 4.19 (1H, t, J=15 Hz, CH), 6.19 (1H, s, D₂O Exch., NH), 7.36–7.82 (3H, m, Ar-H), 12.41 (1H, s, D₂O Exch., NH). ¹³C-NMR (DMSO-d₆) δ: 21.8 (CH₃), 36.9 (C-5 pyridazinone), 40.8 (C-4 pyridazinone), 51.4 (CH₂), 126.7, 131.1, 133.5, 136.3, 137.5, 139.2 (C and CH aromatic), 146.7, 158.9 (C=N thiadiazole), 158.4 (C=N), 159.2, 159.8, 163.9, 165.1, 173.4 (C=O). IR (KBr) cm⁻¹: 3226 (br. NH), 2921 (CH aliph.), 1671 (CO amide), 1592 (C=N). MS m/z: 434 [M]⁺, 395, 351, 314, 284, 252, 220, 180, 153. Anal. Calcd for C₁₇H₁₆ClN₇OS₂: C, 47.05; H, 3.72; N, 22.60; S, 14.78. Found: C, 46.85; H, 3.66; N, 22.51; S, 14.53.

4-(2-Carbamoylhydrazinylidene)-2-[(5-{[3-(2-carbamoylhydrazinyl)-3-oxopropanoyl]amino}-1,3,4-thiadiazol-2-yl)sulfanyl]-4-(4-chloro-3-methylphenyl)butanoic Acid (9)

Yellow powder; yield 55% (EtOH/H₂O); mp 163–165°C; ¹H-NMR (DMSO-d₆) δ: 1.94 (3H, s, CH₃), 2.36–2.61 (2H, m, diastereotopic CH₂), 3.16 (2H, s, CH₂), 4.07 (1H, t, J=15 Hz, CH), 6.37 (3H, s, D₂O Exch., 3NH), 7.49–8.03 (3H, m, Ar-H), 9.47 (1H, s, D₂O Exch., NH), 12.66 (4H, s, D₂O Exch., 2NH₂), 13.12 (1H, s, D₂O Exch., OH). IR (KBr) cm⁻¹: 3439 (br. NH/NH₂), 2953 (CH₂), 1733 (CO aliph. amide), 1640, 1679 (CO amide). MS m/z: 558 [M]⁺, 539, 514, 499, 460, 448, 414, 372, 343, 315, 275. Anal. Calcd for C₁₈H₂₀ClN₉O₆S₂: C, 38.75; H, 3.61; N, 22.59, S, 11.49. Found: C, 38.58; H, 3.57; N, 22.32; S, 11.33.

Antimicrobial Activity

The antimicrobial activity of all compounds was determined by agar well diffusion method⁶¹⁾; the microbial inocula were uniformly spread using sterile L-shaped rod on sterile Petri dishes loaded with nutrient agar and potato dextrose agar for antibacterial and antifungal tests respectively. The screened compounds were dissolved in DMSO in order to prepare a solution of (5 mg/mL) concentration. Reference substances were amphotericin B for fungi, ampicillin, and gentamicin for Gram-positive and Gram-negative bacteria respectively, all with (1 mg/mL) concentration.

A Hundred micrograms of solution were added to 5 wells (6 mm diameter holes scooped out with sterile cork borer); the system was then incubated under aerobic conditions (24h at 37°C for bacteria and 48 h at 28°C for fungi). Inhibition zones were determined and the diameter was then expressed in mille meter. Furthermore, MIC⁶²⁾ for substances with potent antimicrobial activity were determined. Thus, the compounds were dissolved in DMSO in order to prepare a series of different concentration solutions (0.60, 1.25, 2.50, and 5 mg/mL).

Conflict of Interest

The authors declare no conflict of interest.

References

• 1) Viksveen P., Homeopathy, 92, 99–107 (2003).
• 2) Sönmez M., Berber I., Akbaş E., Eur. J. Med. Chem., 41, 101–105 (2005).
• 3) Akhtar W., Shaquiquzzaman M., Akhter M., Verma G., Khan M. F., Alam M. M., Eur. J. Med. Chem., 123, 256–281 (2016).
• 4) Asif M., Sop. Trans. Org. Chem., 1, 1–10 (2014).
• 5) Bansal R., Thota S., Med. Chem. Res., 22, 2539–2552 (2013).
• 6) Costas T., Besada P., Piras A., Acevedo L., Yaneez M., Orallo F., Laguna R., Teraen C., Bioorg. Med. Chem. Lett., 20, 6624–6627 (2010).
• 7) Asif M., Singh A., Int. J. ChemTech Res., 2, 1112–1128 (2010).
• 8) Betti L., Floridi M., Giannaccini G., Manetti F., Strappaghetti G., Tafi A., Botta M., Bioorg. Med. Chem. Lett., 13, 171–173 (2003).
• 9) Partap S., ShaharYar M., Hassan M. Z., Akhtar M. J., Siddiqui A. A., Arch. Pharm. Chem. Life Sci., 350, e1700135 (2017).
• 10) Sharma B., Verma A., Sharma U. K., Prajapati S., Med. Chem. Res., 23, 146–157 (2014).
• 11) Banerjee P., Sharma P., Nema R., Int. J. ChemTech Res., 1, 522–525 (2009).
• 12) Felfel E. M., Tantawy W. A., El-Sofany W. I., El-Shahat M., El-Sayed A. A., Abd-Elshafy D. N., Molecules, 22, 148–162 (2017).
• 13) Li D., Zhan P., Liu H., Pannecouque C., Balzarini J., de Clercq E., Liu X., Bioorg. Med. Chem., 21, 2128–2134 (2013).
• 14) Wang Z., Wang M., Yao X., Li Y., Tan J., Wang L., Qiao W., Geng Y., Liu Y., Wang Q., Eur. J. Med. Chem., 54, 33–41 (2012).
• 15) Ferro S., Agnello S., Barreca M. L., de Luca L., Christ F., Gitto R., J. Heterocycl. Chem., 46, 1420–1424 (2009).
• 16) Yue Q., Zhao Y., Hai L., Zhang T., Guo L., Wu Y., Tetrahedron, 71, 7670–7675 (2015).
• 17) Zhou S., Liao H., He C., Dou Y., Jiang M., Ren L., Zhao Y., Gong P., Eur. J. Med. Chem., 83, 581–593 (2014).
• 18) Rathish I. G., Javed K., Ahmed S., Bano S., Alam M. S., Akhter M., Pillai K. K., Ovais S., Samim M., Eur. J. Med. Chem., 49, 304–309 (2012).
• 19) Murty M., Rao B. R., Ram K. R., Yadav J., Antony J., Anto R. J., Med. Chem. Res., 21, 3161–3169 (2012).
• 20) Csoekaes D., Zupkoe I., Kaerolyi B. I., Drahos L., Holczbauer T., Palloe A., Czugler M., Csaempai A., J. Organomet. Chem., 743, 130–138 (2013).
• 21) Husain A., Ahmad A., Bhandari A., Ram V., J. Chil. Chem. Soc., 56, 778–780 (2011).
• 22) Siddiqui A. A., Islam M., Kumar S., Der Pharma Lett., 2, 319–327 (2010).
• 23) Costas T., Besada P., Piras A., Acevedo L., Yaneez M., Orallo F., Laguna R., Teraen C., Bioorg. Med. Chem. Lett., 20, 6624–6627 (2010).
• 24) Demirayak S., Karaburun A. C., Beis R., Eur. J. Med. Chem., 39, 1089–1095 (2004).
• 25) Oezdemir Z., Yilmaz H., Sari S., Karakurt A., Senol F. S., Uysal M., Med. Chem. Res., 26, 2293–2308 (2017).
• 26) Lin H., Fang H., Meng Q., Dai X., Wu S., Luo J., Pu D., Chen L., Minick D., Bioorg. Med. Chem. Lett., 25, 3748–3753 (2015).
• 27) Contreras J.-M., Parrot I., Sippl W., Rival Y. M., Wermuth C. G., J. Med. Chem., 44, 2707–2718 (2001).
• 28) Kumar D., Carron R., LaCalle C., Jindal D., Bansal R., Acta Pharm., 58, 383–405 (2008).
• 29) Sotelo E., Fraiz N., Yáñez M., Terrades V., Laguna R., Cano E., Ravinea E., Bioorg. Med. Chem., 10, 2873–2882 (2002).
• 30) Laguna R., Rodrigues-Lineares B., Cano E., Estevez I., Ravinea E., Sotelo E., Chem. Pharm. Bull., 45, 1151–1155 (1997).
• 31) Gelain A., Barlocco D., Kwon B.-M., Jeong T.-S., Im K. R., Legnani L., Toma L., J. Med. Chem., 51, 1474–1477 (2008).
• 32) Vergelli G., Schepetkin I. A., Ciciani G., Cilibrizzi A., Crocetti L., Giovannoni M. P., Guerrini G., Iacovone A., Kirpotina L. N., Khlebnikov A. I., Ye R. D., Quinn M. T., Bioorg. Med. Chem., 24, 2530–2543 (2016).
• 33) El-Moselhy T. F., Sidhom P. A., Esmat E. A., El-Mahdy N. A., Chem. Pharm. Bull., 65, 893–903 (2017).
• 34) Zamponi G. W., Nat. Rev. Drug Discov., 15, 19–34 (2016).
• 35) Tarr T. B., Malick W., Liang M., Valdomir G., Frasso M., Lacomis D., Reddel S. W., Garcia-Ocano A., Wipf P., Meriney S. D., J. Neurosci., 33, 10559–10567 (2013).
• 36) Abouzid K., Khalil N. A., Ahmed E. M., Zaitone S. A., Chem. Pharm. Bull., 61, 222–228 (2013).
• 37) Öezadali K., Öezkanli F., Jain S., Roa P. P. N., Velaezquez-Martienez C. A., Bioorg. Med. Chem., 20, 2912–2922 (2012).
• 38) Abouzid K., Bekhit S. A., Bioorg. Med. Chem., 16, 5547–5556 (2008).
• 39) Dogruer D. S., Sahin M., Turk. J. Chem., 27, 727–738 (2003).
• 40) Ibrahim T. H., Loksha Y. M., Elshihawy H. A., Khodeer D. M., Said M. M., Arch. Pharm. Chem. Life Sci., 350, e1700093 (2017).
• 41) Saeed M. M., Khalil N. A., Ahmed E. M., Eissa K. I., Arch. Pharm. Res., 35, 2077–2092 (2012).
• 42) Malinka W., Redzicka A., Jastrzebska M., Filipek B., Dybala M., Karczmarzyk Z., Urbaneczyk-Lipkowska Z., Kalicki P., Eur. J. Med. Chem., 46, 4992–4999 (2011).
• 43) Harris R., Black L., Surapaneni S., Kolasa T., Majest S., Namovic M. T., Grayson G., Komater V., Wilcox D., King L., Marsh K., Jarvis M. F., Nuss M., Nellans H., Pruesser L., Reinhart G. A., Cox B., Jacobson P., Stewart A., Coghlan M., Carter G., Bell R. L., J. Pharmacol. Exp. Ther., 311, 904–912 (2004).
• 44) Rubat C., Coudert P., Tronche P., Bastide J., Bastide P., Privat A. M., Chem. Pharm. Bull., 37, 2832–2835 (1989).
• 45) Yamada T., Nobuhara Y., Yamaguchi A., Ohki M., J. Med. Chem., 25, 975–982 (1982).
• 46) Yamada T., Nobuhara Y., Shimamura H., Tsukamoto Y., Yoshihara K., Yamaguchi A., Ohki M., J. Med. Chem., 26, 373–381 (1983).
• 47) Faidallah H. M., Rostom S. A., Basaif S. A., Makki M. S., Khan K. A., Arch. Pharm. Res., 36, 1354–1368 (2013).
• 48) Tan O. U., Ozadali K., Yogeeswari P., Sriram D., Balkan A., Med. Chem. Res., 21, 2388–2394 (2012).
• 49) El-Mariah F., Hosny M., Deeb A., Phosphorus Sulfur Silicon Relat. Elem., 181, 809–818 (2006).
• 50) Chan A. H., Yi S. W., Weiner E. M., Amer B. R., Sue C. K., Wereszczynski J., Dillen C. A., Senese S., Torres J., McCammon J. A., Miller L. I. S., Jung M. E., Clubb R. T., Chem. Biol. Drug Des., 90, 327–344 (2017).
• 51) Desai V. R., Hunagund S. M., Basanagouda M., Kadadevarmath J. S., Sidarai A. H., J. Fluoresc., 27, 1839–1846 (2017).
• 52) Jones R. A., Whitmore A., ARKIVOC, 11, 114–119 (2007).
• 53) Bachceli S., Gokce H., Indian Pure Appl. Phys., 52, 224–235 (2014).
• 54) Gokce H., Bahceli S., Spectrochim. Acta [A], 133, 741–751 (2014).
• 55) Avci D., Bahceli S., Tamer O., Atalay Y., Can. J. Chem., 93, 1147–1156 (2014).
• 56) Papa D., Schwenk E., Villani F., Klingsberg E., J. Am. Chem. Soc., 70, 3356–3360 (1948).
• 57) Janniah S. L., Guha P. C., J. Am. Chem. Soc., 52, 4860–4866 (1930).
• 58) Kornis G., Marks P. J., Chidester C. G., J. Org. Chem., 45, 4860–4863 (1980).
• 59) Davison W., Christie P., J. Chem. Soc., 3389 (1955).
• 60) Chandra S., Gupta L. K., Spectrochim. Acta A Mol. Biomol. Spectrosc., 62, 1089–1094 (2005).
• 61) Valgas C., De Souza S., Smaenia E., Smaenia A., Braz. J. Microbiol., 38, 369–380 (2007).
• 62) Andrews J. M., J. Antimicrob. Chemother., 48 (Suppl. S1), 5–16 (2001).