Nano-sized formazan analogues: Synthesis, structure elucidation, antimicrobial activity and docking study for COVID-19.

Three series of nanosized-formazan analogues were synthesized from the reaction of dithiazone with various types of α-haloketones (ester and acetyl substituted hydrazonoyl chlorides and phenacyl bromides) in sodium ethoxide solution. The structure and the crystal size of the new synthesized derivatives were assured based on the spectral analyses, XRD and SEM data. The antibacterial and antifungal activities were evaluated by agar diffusion technique. The results showed mild to moderate antibacterial activities and moderate to potent antifungal activities. Significant antifungal activities were observed for four derivatives 3a, 3d, 5a and 5g on the pathogenic fungal strains; Aspergillus flavus and Candida albicans with inhibition zone ranging from 16 to 20 mm. Molecular docking simulations of the synthesized compounds into leucyl-tRNA synthetase editing domain of Candida albicans suggested that most formazan analogues can fit deeply forming stable complexes in the active site. Furthermore, we utilized the docking approach to examine the potential of these compounds to inhibit SARS-CoV-2 3CLpro. The results were very promising verifying these formazan analogues as a hopeful antiviral agents.

Introduction

According to World Health Organization (WHO) reports, the misuse of the antibiotics have led to development of Multidrug Resistance among various strains of microorganisms [1]. Accordingly, new antimicrobial agents acting on novel targets have to be developed to overwhelm the increased incidence of microbial resistance to antibiotic remedy. From the best validated antimicrobial targets are the aminoacyl-tRNA synthetase enzymes which are key enzymes in the protein translation, producing the charged tRNAs required for proper assembly of peptide chains. From these enzymes, LeuRS have been considered as a drug target in fungi and bacteria, it is reported to be inhibited in the editing site by the potent antifungal 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690) which is in clinical phases [2], [3], [4].

Recently, the outbreak of COVID-19, a new coronavirus pneumonia, caused by a new coronavirus (SARS-CoV-2) has emerged as a pandemic according to WHO in March 2020 [5]. To date, millions of infections and thousands of deaths have been recorded all over the world. In this scenario, there is a crucial need for developing antiviral agents interfering with the life cycle of the virus or with the virus replication or membrane fusion [6]. Many researchers have considered the SARS-CoV-2 major protease (Mpro), in addition called chymotrypsin-like protease (3CLpr-2) as a potential target to anti-COVID-19 drugs [7], [8], [9].

Literature survey demonstrates that a lot of formazans have been described to possess broad spectrum of biological activities and pharmacological applications [10] such as antimicrobial [11], [12], [13], [14], antiviral [15], [16], [17], anticancer [18], and anti-inflammatory [19], [20].

As for instance, the formazan derivative I was reported to display 100% inhibition of the Ranikhet diseases virus [21]. Additionally, the two formazans II and III synthesized by Misra and Dhar [22] showed 87% and 83% protections against the Ranikhet disease virus, respectively. Furthermore, many formazans IV were reported by Lakshmi et al. [23] to have significant antibacterial and antifungal activities. Also, Uraz et al. [24] synthesized some formazan derivatives V, VI with different substituents and evaluated their antibacterial and antifungal activities against some selected microorganism species. The results revealed high activity against C. tropicalis, C. kefir, S. cerevisiae and C. neofarmans (Fig. 1 ). Recently, the synthesized drugs in the nanoscale have demonstrated a superior ability to penetrate the DNA of various diseases, which have increased their biological activity and their ability to inhibit the diseases [25], [26].

Fig. 1

Some reported antiviral and antimicrobial formazans.

Inspired by the promising previously reported antimicrobial and antiviral activities of formazans and the urgent need of new effective antimicrobial agent and antiviral drugs to act mainly against COVID-19 and in continuation of our research work in synthesis of bioactive compounds [27], [28], [29], [30], [31], [32], we synthesized herein a new series of nano-sized formazan analogues to study their potential as antibacterial and antifungal agents in vitro. Additionally, molecular docking simulations were done to propose their mode of binding in the editing domain of LeuRS and to show the possibility of these compounds to act against SARS-CoV-2 main protease enzyme.

Results and discussion

Chemistry

The first series of formazan analogues 3a-f were synthesized in sodium ethoxide solution at room temperature with continuous stirring from dithiazone 1 and ester-hydrazonoyl chlorides 2a-f (Scheme 1 ). The structure of the formed s-substituted-dithiazone derivatives 3a-f (Formazan analogues) were confirmed based on studying their spectral data. Figs. 2 and 3 are examples for IR(3b) and 1H NMR(3a) of the synthesized series 3a-f. For instance, IR spectra of the formazan analogues 3a-f revealed the appearance of two NH-absorption bands at ν = 3525–3410 & 3425–3178 cm−1 as well as the characteristic bands at ν = 1743–1671 cm−1 refer to the carbonyl group of COOEt. Furthermore, the 1H NMR of all formazan analogues 3a-f were decorated with the triplet (CH3) and quartet (CH2) signals for the ester COOEt protons and the two NH groups at δ = 9.09–8.56 and 8.78–7.36 ppm in addition to the aliphatic and aromatic protons remarked for each derivatives. The calculated molecular weight for all s-substituted-dithiazones 3a-f were in agreement with the appeared molecular ion peaks in their mass spectra.

Scheme 1

The reaction of dithiazone 1 with ester hydrazonoyl chlorides 2a-e.

Fig. 2

The IR spectrum of derivative 3b.

Fig. 3

The 1H NMR spectrum of compound 3a.

Under similar reaction conditions in sodium ethoxide solution another series of formazan analogues 5a-h have been synthesized from the reaction of dithiazone 1 and acetyl-hydrazonyl chlorides 4a-h (Scheme 2 ). The structure of the prepared formazan analogues 5a-h were confirmed depending on studying the information derived from their spectroscopic analyses. For instance, IR spectra of the formazan analogues 5a-h revealed the appearance of broad absorption band refer to the two NH near ν ≈ 3450 cm−1 in addition to the absorption band for the CO near ν ≈ 1660 cm−1. Another spectra as 1H NMR for derivatives 5a-h showed all characteristic signals for the aliphatic and the aromatic protons as indicated in experimental part.

Scheme 2

The reaction of dithiazone 1 with acetyl hydrazonoyl chlorides 4a-e.

Another two derivatives of formazan analogues 7a,b were formed through the substitution reaction of dithiazone 1 with phenacyl bromide derivatives 6a,b on stirring at ordinary temperature in sodium ethoxide solution (Scheme 3 ). The structure of derivatives 7a,b were demonstrated based on the spectral data and elemental CHN-analyses as documented in the experimental part. The IR and 1H NMR of derivative 7a were illustrated in Figs. 4 and 5 .

Scheme 3

The reaction of dithiazone 1 with phenacyl bromide derivative 6a-c.

Fig. 4

The 1H NMR spectrum of compound 7a.

Fig. 5

The IR spectrum of derivative 7a.

XRD and SEM analysis

The XRD diffraction is a good tool to predict the size of the solid sample and the degree crystal regularity. Five sample 3d, 3f, 5g, 5h and 7b of the synthesized formazan analogues were screened over 10° < 2θ < 80° range to determined their crystallographic features (Fig. 6a, Fig. 6b, Fig. 6c, Fig. 6d, Fig. 6e, Fig. 7, Fig. 8, Fig. 9 ). All investigated formazan analogues revealed sharp peaks which indicated the crystalline feature of them. The size of the crystals of the five samples was calculated according to the reported Debye–Scherrer equation [33] and the calculated size was tabulated in Table 1 . The results referred to the formazan analogues moieties synthesized in the nanometer-scale. In addition, SEM is another tool useful to give excellent insight about the crystallinity as well as surface topography for the tested solid samples. Fig. 7 contains two SEM images for two formazan analogues 5h and 7b as examples for the prepared series. The two images indicated that the crystals of the two derivatives are found in the nanometer-scale.

Fig. 6a

XRD Figures of formazan analogue 3d.

Fig. 6b

XRD Figures of formazan analogues 3f.

Fig. 6c

XRD Figures of formazan analogues 5g.

Fig. 6d

XRD Figures of formazan analogues 5h.

Fig. 6e

XRD Figures of formazan analogues 7b.

Fig. 7

Scanning electron micrographs images (SEM) of two derivatives of formazan analogues 5h and 7b.

Fig. 8

The 3D and 2D proposed binding modes of 3a (A), 3d (B), 5a (C) and 5g (D) docked in the active site of leucyl-tRNA synthetase.

Fig. 9

The 3D and 2D proposed binding modes of 3b (A), 5b (B) and 5g (C) docked in the active site of COVID-19 3CLpro.

Table 1

XRD parameters for nano-crystalline formazan analogues.

Compounds	Size (Å)	2θ	Intensity	d-spacing (Å)	FWHM
3d	7.172	6.686	16,068	13.21060	0.2022
3f	11.740	8.002	9628	11.04057	0.1236
5g	7.160	10.022	41,016	8.81878	0.2034
5h	14.088	9.845	4374	8.97683	0.1031
7b	23.356	7.103	8626	12.43540	0.0621

Antimicrobial activity

Antimicrobial activities were carried out at the Regional Center for Mycology and biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. Target compounds 3a-e, 5a-h, 7b were evaluated for their in vitro antibacterial, and antifungal activities, by inhibition zone method against two gram-positive bacteria: Staphylococcus aureus (RCMB 010010) and Bacillus subtilis (RCMB 015), two gram-negative bacteria: Escherichia coli (RCMB 010052) and Proteus vulgaris (RCMB 004) and two fungi: Aspergillus flavus (RCMB 002002) and Candida albicans (RCMB 005003) using gentamycin and ketoconazole as reference antibacterial and antifungal drugs, respectively (Tables 2 and 3 ).

Table 2

Antifungal activity of the target compounds (3a-e, 5a-h and 7a,b) expressed as mean zones of inhibition in mm based on diffusion agar technique.

Sample code	Tested Fungi Microorganisms
	Aspergillus flavus	Candida albicans
3a	17	18
3b	15	16
3c	13	14
3d	20	16
3e	NA	NA
5a	16	19
5b	15	13
5c	15	16
5d	15	14
5e	11	18
5f	NA	NA
5g	20	18
5h	15	13
7b	NA	NA
Ketoconazol	16	20

Table 3

Antibacterial activity of the target compounds (3a-e, 5a-h and 7b) expressed as mean zones of inhibition in mm based on diffusion agar technique.

Sample code	Tested bacteria Microorganisms
	Gram Positive Bacteria:		Gram Negatvie Bacteria:
	Staphylococcus aureus	Bacillus subtilis	Escherichia coli	Proteus vulgaris
3a	13	8	NA	NA
3b	14	NA	NA	NA
3c	13	14	12	14
3d	15	13	11	17
3e	10	NA	NA	11
5a	13	18	15	16
5b	15	18	15	18
5c	15	16	13	12
5d	12	15	12	13
5e	14	17	11	12
5f	8	14	13	12
5g	12	17	14	17
5h	15	18	15	18
7b	8	NA	NA	8
Gentamycin	24	26	30	25

Antibacterial activity

Concerning the antibacterial activity against gram-positive bacteria, most compounds showed mild to moderate activities. The best activities were seen for the acetyl derivatives 5a, 5b, 5c, 5g and 5h with inhibition zones values ranging from 15 to 18 mm, compared to gentamycin with IZ = 24 and 26 mm for S. aureus and B. subtilis, respectively.

Regarding gram-negative bacteria, they were resistant to 3a and 3b, while 3d having 4-Br group revealed moderate activity against P. vulgaris (IZ = 17 mm) compared to gentamycin (IZ = 25 mm). On the other hand, compounds 5a, 5b, 5g and 5h displayed reasonable antibacterial activities against P. vulgaris with IZ values 16, 18, 17 and 18, respectively. Additionally, most compounds revealed mild antibacterial activities against the gram negative E. coli.

Antifungal activity

Most of the tested formazan analogues exhibited promising antifungal activities with the exception of 3e, 5f and 7b that were inactive against used fungal strains.

Focusing on the antifungal activities of compounds 3a-e, compound 3a elicited comparable antifungal activity (IZ = 17 and 18 mm against A. flavus and C. Albicans, respectively) to ketoconazole (IZ = 16 and 20 mm against A. flavus and C. Albicans, respectively). Also, significant activities were observed for compounds 3b and 3c (IZ = 13–16 mm). In addition, compound 3d demonstrated superior antifungal activity against A. flavus (Z = 20 mm) that was more potent than ketoconazole.

Furthermore, by studying the antifungal activities of 5a-h, moderate to potent activities were displayed. Among these compounds, compound 5g with 3-Cl group showed IZ = 20 mm against A. flavus and 18 mm against C. Albicans that were higher than those of ketoconazole. Additionally, compound 5a showed comparble activity (IZ = 16 and 19 mm) to ketoconazole.

These antifungal activities results were highly appreciated since A. fumigatus is the second main cause of invasive aspergillosis and is the first leading cause of cutaneous aspergillosis [34]. In addition to the increased incidence of infections by C. albicans, the most common cause of candidiasis, and the increased resistance of C. albicans to antifungal drugs [35], [36].

Docking study

Docking into Candida albicans leucyl-tRNA synthetase

Leucyl-tRNA synthetase (LeuRS) belongs to the family aminoacyl tRNA synthetases (aaRSs), group of central enzymes that play a crucial role in protein synthesis, which is vital for survival of micro-organism and hence its inhibition presented a novel and attractive target for developing antimicrobials [37]. Many recent reviews have discussed the importance of aaRSs in the discovery and development of antibacterial and antifungal agents [38], [39], [40], [41].

In this study, molecular docking study of the newly synthesized formazan analogues 3a-f, 5a-h and 7a,b have been performed onto the active site of Candida albicans editing domain of cytosolic leucyl-tRNA synthetase to demonstrate their binding affinity and orientation. MOE 2014 program was used for the docking simulation using cytosolic leucyl-tRNA synthetase editing domain cocrystallized with benzoxaborole-AMP adduct (PDB code: 2WFG). Docking of the cocrystallized ligand was done in order to validate the used docking method, it displayed docking score = −6.4725 Kcal/mol and RMSD = 0.654.

Results of the docking simulation were displayed in Table 4 showing the docking scores and the different formed interactions such as hydrogen bond, pi-H and non-polar pi-cation interactions.

Table 4

Docking results of the target compounds against Candida albicans leucyl-tRNA synthetase.

Compound	Docking score (kcal/mol)	Interacting residues (Type of interaction)	Distance (A0)
3a	−7.8168	Ala315 (H bond)Thr316 (H bond)Lys407 (H bond)Lys483 (H bond)Tyr487 (H-Pi)	3.453.123.332.834.29
3b	−7.3313	Ala315 (H bond)Lys407 (Pi-cation)Lys407 (Pi-cation)Tyr487 (H bond)	3.203.914.292.84
3c	−7.1818	Lys407 (H bond)Tyr487 (H bond)	3.362.90
3d	−7.4430	Ala315 (H bond)Leu317 (H bond)Lys407 (H bond)Lys483 (H bond)	3.483.243.262.83
3e	−6.5127	–	–
3f	−7.0200	Lys407 (Pi-cation)Tyr487 (H bond)	4.003.01
5a	−7.5710	Ala315 (H bond)Lys407 (H bond)Lys483 (H bond)Tyr487 (H-Pi)	3.583.162.764.34
5b	−7.1052	Ala315 (H bond)Lys407 (H bond)Lys483 (H bond)	3.463.282.80
5c	−7.0641	Lys407 (H bond)Ser419 (Pi-H)Lys483 (H bond)Tyr487 (H bond)	3.284.403.182.85
5d	−7.2187	Ala315 (H bond)Lys407 (Pi-cation)Lys407 (Pi-cation)Tyr487 (H bond)	3.133.894.342.87
5e	−7.4357	Ala315 (H bond)Arg318 (H bond)Lys407 (Pi-cation)Lys483 (Pi-H)	3.713.444.404.34
5f	−6.6872	Lys483 (H bond)	2.93
5g	−7.7562	Leu317 (H bond)Lys483 (H bond)Tyr487 (H bond)	3.703.412.88
5h	−6.3603	Lys483 (H bond)	3.03
7a	−6.0904	Ser419 (Pi-H)	3.95
7b	−6.1826	Asp421 (H bond)	3.16

For majority of compounds, Ala315, Lys407, and Lys483 were recognized as key amino acid residues responsible for hydrogen bonds generation and Thr316, Leu317, Arg318, Asp421 and Tyr487 were identified for minor interaction. Whereas, for pi-H and pi-cation interactions, Lys407, Ser419, Lys483 and Tyr487 were the only observed residues.

Focusing on the binding mode of the most active analogues as antifungals, 3a, 3d, 5a and 5g (Fig. 8), it was observed that they are well stabilized into the active site through strong hydrogen bonds and pi-H interactions with docking scores −7.8168, −7.4430, −7.5710 and −7.7562, respectively. The sulfur atom of 3a, 3d and 5a formed hydrogen bond interactions with Ala315, while the hydrazonoyl nitrogen atoms were involved in hydrogen bonds with Lys407 and Lys483 residues. Additional hydrogen bond was seen between the hydrazonoyl nitrogen atom of 3a and Thr316 and between 3d and Leu317. Furthermore, the methoxy group of 5a formed H-Pi interaction with Tyr487. For 5g, three hydrogen bond interactions were observed between the hydrazonoyl nitrogen atoms and the important Leu317, Lys483 and Tyr487 residues.

Docking into COVID-19 3CLpro-2

Due to both the emergency of COVID-19 outbreak together with previous reports of the antiviral activities of formazan derivatives, we evaluated the antiviral potential of the synthesized formazan analogues against COVID-19 by targeting 3CLpro-2 through molecular docking.

The 3C-like protein (3CLpro-2) is the central protease of SARS-CoV-2. It plays a vital role in viral RNA translation and maturation and, thus, is critical for viral replication rendering it an attractive target for developing anti-coronavirus drug [6].

The protein structure of 3CLpro-2 consists of 9 α-helices and 13 β-strands making together three distinctive domains: Domain I (residues 8–101), Domain II (residues 102–184) and Domain III (residues 201–306) connected to Domain II by an extended loop (residues 185–200). 3CLpro-2 contains a catalytic dyad that is composed of conserved residues His41 and Cys145 and the key substrate-binding site is formed as a split between Domain I and Domain II [42].

To estimate the binding affinity of derivatives 3a-f, 5a-h and 7a,b with COVID-19 3CLpro (PDB code: 6LU7), molecular docking study was done. As shown in Table 5 , our newly synthesized compounds were found to fit deeply in the active site of COVID-19 3CL protease with binding energies ranging from −5.6064 to −8.0555 Kcal/mol. Interestingly, the key amino acid residue Cys145 was involved in hydrogen bond interactions with all of the tested compounds. Moreover, residues His41, Asn142, Gly143, His163, His164, and Gln189 were positively contributed in hydrogen bonding. While, Met49, Glu166 and Gln189 were contributed in the formed Pi-H interactions.

Table 5

Docking results of the target compounds against COVID-19 3CLpro.

Compound	Docking score (kcal/mol)	Interacting residues (Type of interaction)	Distance (A0)
3a	−7.2566	Met49 (Pi-H)Cys145 (H bond)	4.194.41
3b	−7.7700	Cys145 (H bond)His164 (H bond)Glu166 (Pi-H)	3.843.084.60
3c	−7.2756	His41 (H bond)Cys145 (H bond)	3.173.40
3d	−7.2122	Cys145 (H bond)Cys145 (H bond)His164 (H bond)Gln189 (H bond)	3.953.393.372.98
3e	−7.1521	Cys145 (H bond)His163 (H bond)	3.743.40
3f	−6.1250	Cys145 (H bond)Cys145 (H bond)Met165 (H bond)	2.873.244.11
5a	−7.3248	Asn142 (H bond)Asn142 (H bond)Cys145 (H bond)His164 (H bond)Glu166 (Pi-H)	3.182.973.953.034.66
5b	−8.0555	Cys145 (H bond)His163 (H bond)His164 (H bond)	3.353.333.40
5c	−7.1865	Cys145 (H bond)His163 (H bond)	3.743.28
5d	−7.0781	Cys145 (H bond)His164 (H bond)Gln189 (Pi-H)	3.132.923.95
5e	−7.3608	Cys145 (H bond)His163 (H bond)	3.743.31
5f	−5.6064	Gly143 (H bond)Cys145 (H bond)	2.853.95
5g	−7.7868	Cys145 (H bond)His163 (H bond)His164 (H bond)	3.563.263.12
5h	−5.9377	Gly143 (H bond)Cys145 (H bond)	3.203.68
7a	−6.9032	Cys145 (H bond)His164 (H bond)	4.133.9
7b	−6.4258	Cys145 (H bond)	3.91

The analysis of the binding poses of compounds 3b, 5b and 5g having the best binding scores −7.7700, −8.0555 and −7.7868 Kcal/mol, respectively revealed closed binding pattern (Fig. 9). Concerning the ethyl ester formazan derivative 3b, the sulfur atom formed hydrogen bond with Cys145, while the hydrazonoyl NH group is involved in hydrogen bonding with His164. Moreover, Pi-H interaction was observed between the tolyl phenyl moiety of 3b and Glu166. On the other hand, the acetyl groups of 5b and 5g were bonded to His163 through hydrogen bonds. Additional hydrogen bonds connected 5b and 5g with His163 and His164 residues. Moreover, hydrophobic interactions were noticed between the tolyl moieties of 3b and 5b/the 3-chlorophenyl group of 5g and Glu166, Pro168, Gln192 and Gln189 indicating the importance of these hydrophobic moieties.

Conclusion

Finally, we succeeded to synthesize a new series of nano-sized formazan analogues in excellent yield via the reaction of dithiazone with different types of hydrazonoyl chlorides and phenacyl bromide derivatives. Characterization of the crystal size and the structure of the formed formazan analogues were achieved with the aid of XRD, SEM and the spectral information. Evaluation of the antibacterial and antifungal activities demonstrated that most derivatives have mild or moderate antibacterial activity, while significant antifungal activity was observed for most compounds. Some derivatives like 3a, 3d, 5a and 5g displayed potent antifungal activity against tested fungal strains (IZ = 16–20 mm) compared to the reference drug ketoconazole (IZ = 16 and 20 mm against A. flavus and C. Albicans, respectively). Molecular modeling study revealed that these compounds could bind properly to C. albicans leucyl-tRNA synthetase editing domain. Additionally, docking simulation into the active site of COVID-19 3CL protease showed superior fitting into the active site with binding scores from −5.6064 to −8.0555 Kcal/mol.

Experimental

General methods

The melting points of all new formazan analogues were recorded using a SMP3 melting point apparatus (the diameter of the glass capillaries is 0.5 mm). The 1430-Perkin-Elmer infrared-spectrophotometer was utilized to record the IR-spectra for all new formazan analogues in the range of wavenumber from 4000 cm−1 to 200 cm−1 as through the formation of sample-KBr discs. The well-known Bruker Avance-300 instrument was utilized to record the 1H NMR-spectra for all new formazan analogues at 300 MHz DMSO‑d solutions. Ppm and Hz characterize the chemical shifts (δ) and coupling constants, respectively. To record the molecular weight of all new formazan analogues we used a Finnigan-MAT8222 spectrometer at 70 eV in Micro-analytical center at Cairo University. Also, measuring of CHN elemental analyzes were investigated on Elementar vario-LIII C—H—N—S analyzer.

Synthesis of formazan analogues 3a-e, 5a-f, and 7a,b

To a stirred solution of 0.56 g of dithizone 1 (1 mmol) in sodium ethoxide solution (1 mmol of Na in 20 mL absolute EtOH) was added portion wise the suitable hydrazonyl chlorides 2a-f or 4a-h or the phenacyl derivatives 6a,b, then the mixture was left to stirred for 2 h. The formed colored formazan analogues solid was filtered, washed with MeOH and recrystallized from the suitable solvent to give the corresponding formazan analogues 3a-f, 5a-h and 7a,b, respectively.

2-Ethoxy-N′-(4-methoxyphenyl)-2-oxoacetohydrazonic-N′,2-diphenyldiazenecarbo-hydrazonic thioanhydride (3a)

Orange solid, yield (0.42 g, 90%), mp 158–160 °C (ethanol /dioxane), IR ύ: 3425, 3178 (2NH), 1681 (CO), 1579 (CN), 1535, 1465, 1338, 1288, 1226, 1134, 1064 cm−1. 1H NMR (DMSO‑d 6) 1.32 (t, J = 8.4 Hz, 3H, CH2 CH), 3.81 (s, 3H, OCH3), 4.36 (q, J = 8.4 Hz, 2H, CHCH3), 6.86–7.83 (m, 14H, Ar H), 7.36 (s, 1H, NH), 8.56 (s, 1H, NH). M/z (%) 477 (M++1, 12), 476 (M+, 6), 446 (54), 402 (3), 370 (74), 342 (4), 312 (3), 279 (4), 242 (2), 224 (5), 210 (10), 166 (55), 147 (12), 133 (9), 121 (45), 105 (27), 97 (10), 77 (100). Anal. Calcd. for C24H24N6O3S (476.55) Calcd: C, 60.49; H, 5.08; N, 17.64. Found: C, 60.62; H, 5.28; N, 17.45%.

2-Ethoxy-2-oxo-N′-(p-tolyl)acetohydrazonic-N′,2-diphenyldiazenecarbohydrazonic thioanhydride (3b)

Orange solid, yield (0.39 g, 85%), mp 152–154 °C (ethanol /dioxane), IR ύ: 3448, 3178 (2NH), 1681 (CO), 1597 (CN), 1535, 1473, 1388, 1303, 1273, 1234. 1188, 1157, 1134. cm−1. 1H NMR (DMSO‑d 6) 1.32 (t, 3H, CH2 CH), 2.26(s, 3H, CH3), 4.36 (q, 2H, CHCH3), 6.76–7.83 (m, 14H, Ar H), 7.36 (s, 1H, NH), 8.59 (s, 1H, NH). Anal. Calcd. for C24H24N6O2S (460.55) Calcd: C, 62.59; H, 5.25; N, 18.25. Found: C, 62.39; H, 5.29; N, 18.16%.

2-Ethoxy-2-oxo-N′-phenylacetohydrazonic-N′,2-diphenyldiazenecarbohydrazonic thioanhydride (3c)

Orange solid, yield (0.42 g, 95%), mp 138–140 °C (ethanol /dioxane), IR ύ: 3446 (br2NH), 1671 (CO), 1601 (CN), 1536, 1474, 1383, 1277, 1237, 1128, 1059 cm−1. 1H NMR (DMSO‑d 6) 1.32 (t, 3H, CH2 CH), 4.39 (q, 2H, CHCH3), 6.74–7.98 (m, 15H, Ar H), 7.39 (s, 1H, NH), 8.63 (s, 1H, NH). M/z (%) 448 (M++1, 0.63), 429 (19), 416 (14), 243 (4), 208 (2), 195 (10), 183 (20), 167 (100), 152 (7), 118 (5), 105 (14), 91 (28), 77 (78). Anal. Calcd. for C23H22N6O2S (446.52) Calcd: C, 61.87; H, 4.97; N, 18.82. Found: C, 61.77; H, 4.85; N, 18.64%.

N′-(4-Bromophenyl)-2-ethoxy-2-oxoacetohydrazonic-N′,2-diphenyldiazenecarbo-hydrazonic thioanhydride (3d)

Orange solid, yield (0.49 g, 90%), mp 162–164 °C (ethanol /dioxane), IR ύ: 3510, 3464 (2NH), 1674 (CO), 1604 (CN), 1527, 1473, 1388, 1234, 1126, 1064, 1010 cm−1. 1H NMR (DMSO‑d 6) 1.32 (t, 3H, CH2 CH), 4.37 (q, 2H, CHCH3), 6.75–7.99 (m, 14H, Ar H), 7.36 (s, 1H, NH), 8.68 (s, 1H, NH). M/z (%) 525 (M+, 0.02),480 (1), 420 (19), 392 (2), 340 (13). 214 (4), 170 (7), 150 (4), 135 (4), 105 (29), 92 (35), 77 (100). Anal. Calcd. for C23H21BrN6O2S (525.42) Calcd: C, 52.58; H, 4.03; N, 15.99. Found: C, 52.33; H, 4.22; N, 16.08%.

2-Ethoxy-N′-(3-nitrophenyl)-2-oxoacetohydrazonic-N′,2-diphenyldiazenecarbo-hydrazonic thioanhydride (3e)

Orange solid, yield (0.44 g, 90%), mp 252–254 °C (ethanol /dioxane), IR ύ: 3525, 3425 (2NH), 1743 (CO), 1589 (CN), 1535, 1489, 1350, 1265, 1157, 1103, 1010 cm−1. 1H NMR (DMSO‑d 6) 1.34 (t, 3H, CH2 CH), 4.39 (q, 2H, CHCH3), 6.77–8.42 (m, 14H, Ar H), 8.78 (s, 1H, NH), 9.09 (s, 1H, NH). M/z (%) 492 (M++1, 0.41), 491 (M+, 0.73), 474 (2), 393 (3), 368 (5), 354 (2), 339 (8), 313 (19), 292 (4), 264 (20), 239 (16), 183 (12), 167 (36), 152 (7), 135 (12), 129 (14), 123 (13), 109 (23), 95 (37), 83 (54), 77 (69), 69 (67), 57 (100). Anal. Calcd. for C23H21N7O4S (491.52) Calcd: C, 56.20; H, 4.31; N, 19.95. Found: C, 56.33; H, 4.54; N, 19.76%.

2-Ethoxy-N′-(4-nitrophenyl)-2-oxoacetohydrazonic-N′,2-diphenyldiazenecarbo-hydrazonic thioanhydride (3f)

Dark red solid, yield (0.41 g, 85%), mp ˃ 300 °C (ethanol /dioxane), IR ύ: 3410, 3263 (2NH), 1728(CO), 1604 (CN), 1496, 1458, 1357, 1288, 1234, 1149, 1080, 1056 cm−1. 1H NMR (DMSO‑d 6). M/z (%) 492 (M++1, 0.11), 491 (M+, 0.09), 310 (39), 282 (6), 221 (5), 177 (32), 150 (14), 135 (8), 119 (11), 108 (21), 105 (21), 93 (83), 77 (100). Anal. Calcd. for C23H21N7O4S (491.52) Calcd: C, 56.20; H, 4.31; N, 19.95. Found: C, 56.23; H, 4.54; N, 19.86%.

N′-(4-Methoxyphenyl)-2-oxopropanehydrazonic-N′,2-diphenyldiazenecarbo-hydrazonic thioanhydride (5a)

Orange solid, yield (0.41 g, 93%), mp 140–142 °C (ethanol /dioxane), IR ύ: 3451 (br. 2NH), 1658 (CO), 1600 (CN), 1533, 1481, 1460, 1386, 1269, 1228, 1190, 1156, 1129, 1020 cm−1. 1H NMR (DMSO‑d 6) 2.36 (s, 3H, CH3), 3.71 (s, 3H, OCH3), 6.96–7.66 (m, 14H, Ar-H), 10.93 (s, 1H, NH), 11.30 (s, 1H, NH). M/z (%) 446 (M+, 1), 255 (0.36), 339 (6), 136 (1.2), 122 (45), 107 (16), 106 (10), 105 (31), 92 (43), 91 (8), 77 (100), 65 (28).Anal. Calcd. for C23H22N6O2S (446.52) Calcd: C, 61.87; H, 4.97; N, 18.82. Found: C, 61.99; H, 4.88; N, 18.64%.

2-Oxo-N′-(p-tolyl)propanehydrazonic-N′,2-diphenyldiazenecarbohydrazonic thioanhydride (5b)

Orange solid, yield (0.38 g, 90%), mp 132–134 °C (ethanol /dioxane), IR ύ: 3452 (br. 2NH), 1649 (CO), 1601 (CN), 1540, 1489, 1459, 1388, 1273, 1233, 1191, 1154, 1130, 1070, 1021 cm−1. 1H NMR (DMSO‑d 6) 2.26 (s, 3H, CH3), 2.38 (s, 3H, CH3), 6.47–7.90 (m, 14H, Ar-H), 10.89 (s, 1H, NH), 11.40 (s, 1H, NH). M/z (%)432 (M++2, 0.26), 310 (0.32), 223 (3), 175 (1.34), 120 (1.12), 118 (4), 106 (14), 105 (49), 92 (40), 91 (46), 77 (73), 65 (45). Anal. Calcd. for C23H22N6OS (430.53) Calcd: C, 64.16; H, 5.15; N, 19.52. Found: C, 64.23; H, 5.30; N, 19.45%.

2-Oxo-N′-phenylpropanehydrazonic-N′,2-diphenyldiazenecarbohydrazonic thioanhydride (5c)

Orange solid, yield (0.39 g, 95%), mp 156–158 °C (ethanol /dioxane), IR ύ: 3449 (br. 2NH), 1662 (CO), 1598 (CN), 1534, 1474, 1439, 1382, 1249, 1269, 1228, 1190, 1150, 1127, 1070, 1020 cm−1. 1H NMR (DMSO‑d 6) 2.37 (s, 3H, CH3), 7.07–7.63 (m, 15H, Ar-H), 10.93 (s, 1H, NH), 11.26 (s, 1H, NH). M/z (%)416 (M+, 0.36), 339 (4), 223 (2), 161 (0.5), 150 (14), 105 (52), 92 (31), 77 (100), 65 (27). Anal. Calcd. for C22H20N6OS (416.50) Calcd: C, 63.44; H, 4.84; N, 20.18. Found: C, 63.23; H, 4.65; N, 20.22%.

N′-(4-Chlorophenyl)-2-oxopropanehydrazonic-N′,2-diphenyldiazenecarbohydrazonic thioanhydride (5d)

Orange solid, yield (0.4 g, 90%), mp 146–148 °C (ethanol /dioxane), IR ύ: 3451 (br. 2NH), 1657 (CO), 1597 (CN), 1535, 1486, 1464, 1381, 1337, 1269, 1228, 1189, 1156, 1131, 1021 cm−1. 1H NMR (DMSO‑d 6) 1.60 (s, 3H, CH3), 7.11–7.93 (m, 14H, Ar-H), 10.92 (s, 1H, NH), 11.58 (s, 1H, NH). M/z (%)452 (M++2, 0.15), 451 (M++1, 0.09), 450 (M+, 0.27), 346 (12), 344 (31), 228 (2), 139 (1.8), 126 (16), 111 (12), 106 (5), 105(30), 92 (52), 91 (9), 77 (100), 65 (28). Anal. Calcd. for C22H19ClN6OS (450.94) Calcd: C, 58.60; H, 4.25; N, 18.64. Found: C, 58.88; H, 4.45; N, 18.45%.

N′-(4-Nitrophenyl)-2-oxopropanehydrazonic-N′,2-diphenyldiazenecarbohydrazonic thioanhydride (5e)

Orange solid, yield (0.39 g, 85%), mp ˃ 300 °C (ethanol /dioxane), IR ύ: 3479 (br. 2NH), 1662 (CO), 1596 (CN), 1536, 1503, 1479, 1422, 1384, 1336, 1266, 1228, 1186, 1170, 1127, 1072, 1021 cm−1. 1H NMR (DMSO‑d 6) 1.94 (s, 3H, CH3), 7.0–8.28 (m, 14H, Ar-H), 11.20 (s, 1H, NH), 11.47 (s, 1H, NH). M/z (%)461 (M+, 0.18), 310 (1.9), 255 (1.1), 206 (0.74), 137 (3.5), 122 (1.7), 106 (9), 105 (27), 92 (64), 91 (11), 77 (100), 65 (40). Anal. Calcd. for C22H19N7O3S (461.50) Calcd: C, 57.26; H, 4.15; N, 21.25. Found: C, 57.34; H, 4.26; N, 21.19%.

2-Oxo-N′-(m-tolyl)propanehydrazonic-N′,2-diphenyldiazenecarbohydrazonic thioanhydride (5f)

Orange solid, yield (0.36 g, 85%), mp 144–146 °C (ethanol /dioxane), IR ύ: 3436 (br 2NH), 1653 (CO), 1594 (CN), 1536, 1472, 1378, 1269, 1194, 1129, 1027 cm−1. 1H NMR (DMSO‑d 6) 1.88 (s, 3H, CH3), 2.19 (s, 3H, CH3), 6.50–7.51 (m, 14H, Ar-H), 9.62 (s, 1H, NH), 10.65 (s, 1H, NH). M/z (%) 431 (M+ +1, 0.14), 430 (M+, 0.44), 324 (40), 310 (9), 222 (3), 194 (1), 166 (3), 150 (20), 132 (8), 106 (43), 92 (44), 77 (100). Anal. Calcd. for C23H22N6OS (430.53) Calcd: C, 64.16; H, 5.15; N, 19.52. Found: C, 64.28; H, 5.36; N, 19.47%.

N′-(3-Chlorophenyl)-2-oxopropanehydrazonic-N′,2-diphenyldiazenecarbohydrazonic thioanhydride (5g)

Orange solid, yield (0.38 g, 85%), mp 160–162 °C (ethanol /dioxane), IR ύ: 3437 (br 2NH), 1659 (CO), 1593 (CN), 1534, 1467, 1387, 1270, 1223, 1187, 1129, 1024 cm−1. 1H NMR (DMSO‑d 6) 2.49 (s, 3H, CH3), 6.78–8.22 (m, 14H, Ar-H), 7.36 (s, 1H, NH), 8.70 (s, 1H, NH). M/z (%) 452 (M++2, 0.16), 451 (M++1, 0.11), 450 (M+, 0.38), 420 (3), 344 (28), 310 (7), 167 (4), 150 (8), 126 (16), 111 (9), 105 (35), 92 (60), 77 (100). Anal. Calcd. for C22H19ClN6OS (450.94) Calcd: C, 58.60; H, 4.25; N, 18.64. Found: C, 58.58; H, 4.37; N, 18.52%.

N′-(3-Nitrophenyl)-2-oxopropanehydrazonic-N′,2-diphenyldiazenecarbohydrazonic thioanhydride (5h)

Red solid, yield (0.39 g, 85%), mp 164–166 °C (ethanol /dioxane), IR ύ: 3430 (br 2NH), 1661 (CO), 1595 (CN), 1531, 1473, 1361, 1272, 1219, 1183, 1130, 1020 cm−1 1H NMR (DMSO‑d 6) 2.49 (s, 3H, CH3), 6.79–8.49 (m, 14H, Ar-H), 8.77 (s, 1H, NH), 9.19 (s, 1H, NH). M/z (%) 461 (M+, 0.18), 431 (3), 355 (65), 208 (2), 181 (18), 167 (4), 150 (7), 118 (4), 105 (27), 92 (64), 77 (100). Anal. Calcd. for C22H19N7O3S (461.50) Calcd: C, 57.26; H, 4.15; N, 21.25. Found: C, 57.19; H, 4.31; N, 21.36%.

2-Oxo-2-phenylethyl N′,2-diphenyldiazenecarbohydrazonothioate (7a)

Red solid, yield (0.31 g, 85%), mp 180–182 °C (ethanol /dioxane), IR ύ: 3410 (NH), 1681 (CO), 1597 (CN), 1496, 1450, 1365, 1303, 1126, 1041 cm−1. 1H NMR (DMSO‑d 6) 3.57 (s, 2H, CH2), 7.08–8.14 (m, 15H, Ar-H), 8.15 (s, 1H, NH). M/z (%) 375 (M++1, 0.03), 374 (M+, 0.14), 267 (21), 207 (0.46), 163 (0.32), 152 (0.59), 109 (0.98), 105 (29), 77 (100). Anal. Calcd. for C21H18N4OS (374.46) Calcd: C, 67.36; H, 4.85; N, 14.96. Found: C, 67.45; H, 4.66; N, 14.72%.

2-(4-Chlorophenyl)-2-oxoethyl N′,2-diphenyldiazenecarbohydrazonothioate (7b)

Red solid, yield (0.34 g, 85%), mp 190–192 °C (ethanol /dioxane), IR ύ: 3424 (NH), 1682 (CO), 1590 (CN), 1524, 1490, 1360, 1303, 1229, 1173, 1130 cm−1. 1H NMR (DMSO‑d 6) 3.80 (s, 2H, CH2), 7.10–8.15 (m, 15H, Ar-H, NH). M/z (%) 409 (M++1, 0.08), 408 (M+, 0.27), 267 (23), 139 (4), 113 (2), 111 (6), 105 (26), 77 (100). Anal. Calcd. for C21H17ClN4OS (408.90) Calcd: C, 61.68; H, 4.19; N, 13.70. Found: C, 61.89; H, 4.34; N, 13.88%.

Antimicrobial activity assay

All the microbial strains that used in the current study have been supplied from Al-Azhar University in Cairo, Egypt from the culture collection of the Regional Center for Mycology and Biotechnology (RCMB). The method used for recording the antimicrobial activity according to the literature method [43].

Docking study

Molecular docking was analyzed through using MOE-Dock 2014 software [44]. Chemical structures of 3a-f, 5a-h and 7a,b were drawn by the MOE builder followed by minimization using the force field MMFF94x in this program. Hydrogen atoms were then added and unwanted water molecules were cancelled. Docking was then performed using London dG for rescoring 1 and GBVI/WSA dG for rescoring 2. At the same time, refinement was done through forcefield. “Ligand Interactions” was utilized for the 2D visualization o the protein–ligand interactions. The best pose was then selected depending on the binding energy and the interactions found in the active site.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.