Pyridazinone-based derivatives as anticancer agents endowed with anti-microbial activity: molecular design, synthesis, and biological investigation

Abstract

Cancer patients undergoing chemotherapy are highly susceptible to infections owing to their compromised immune system, which also promotes cancer progression through inflammation. Thus, this study aimed to develop novel chemotherapeutic agents with both anticancer and antimicrobial properties. A series of diarylurea derivatives based on pyridazinone scaffolds were designed, synthesized, and characterized as surrogates for sorafenib. The synthesized compounds were tested for their antimicrobial activity and screened against 60 cancer cell lines at the National Cancer Institute (NCI). Compound 10h exhibited potent antibacterial activity against Staphylococcus aureus (MIC = 16 μg mL⁻¹), whereas compound 8g showed significant antifungal activity against Candida albicans (MIC = 16 μg mL⁻¹). Additionally, ten compounds were further evaluated for VEGFR-2 inhibition, with compound 17a showing the best inhibitory activity. Compounds 8f, 10l, and 17a demonstrated significant anticancer activity against melanoma, NSCLC, prostate cancer, and colon cancer, with growth inhibition percentages (GI%) ranging from 62.21% to 100.14%. Compounds 10l and 17a were selected for five-dose screening, displaying GI₅₀ values of 1.66–100 μM. Compound 10l induced G0–G1 phase cell cycle arrest in the A549/ATCC cell line, increasing the cell population from 85.41% to 90.86%. Gene expression analysis showed that compound 10l upregulated pro-apoptotic genes p53 and Bax and downregulated the anti-apoptotic gene Bcl-2. Molecular docking studies provided insights into the binding modes of the compounds to the VEGFR-2 enzyme. In conclusion, the pyridazinone-based diarylurea derivatives developed in this study show promise as dual-function antimicrobial and anticancer agents, warranting further investigation.

Discovery of novel pyridazinone derivatives with dual antimicrobial and anticancer activities.

1. Introduction

The spread of infectious diseases triggered by microbes, particularly bacteria and fungi, is entrenched as a significant health threat. Specifically, the overuse, abuse, and extensive application of antibiotics have resulted in significant issues with the management of numerous microbial diseases.¹ Gram-positive bacteria such as Staphylococcus aureus (S. aureus) continue to rank highly among the leading causes of healthcare-associated infections. Similarly, ailments triggered by Gram-negative pathogens place a high premium on their treatment in the drug development process.²S. aureus is abundantly observed on the human skin, skin glands, and mucous membranes.^3,4 However, it was noted in the 1950s that β-lactam-resistant S. aureus strains, such as methicillin-resistant Staphylococcus aureus (MRSA), have emerged and quickly spread to many hospitals throughout the world. MRSA is a concerning bacteria because of its high pathogenic capacity and its ability to produce a wide range of potentially fatal infections and adapt to various environmental settings.⁵ The resistance of S. aureus to many antibiotics has made treatment challenging, resulting in prolonged hospital stays and increased mortality rates.⁶ Antibiotic resistance to bacterial infections, particularly S. aureus, has been primarily linked to three factors: changes in drug binding sites on molecular targets, enhanced expression of efflux pumps, and the acquisition of genetic determinants through the horizontal gene transfer of mobile genetic elements.⁷ Consequently, there is a pressing need for the discovery of novel antibacterial medicines to combat S. aureus and other bacteria resistant to most antibiotics.⁶ Furthermore, persistent infection exposure causes inflammation, which aids in the development of cancer.⁸

Globally, the uncontrolled and rapid pathological proliferation of abnormal cells in the body that spread to different organs makes cancer one of the most deadly, serious, and potentially fatal diseases worldwide.¹ Tumorigenesis is a multi-stage, complex process in which a collection of cells in one area of the body begins unrestricted replication as a result of genetic and epigenetic changes. There are over a hundred distinct types of cancer. For instance, lung cancers are categorized histologically into non-small-cell lung carcinoma (NSCLC) and small-cell lung carcinoma (SCLC), which are distinguished for therapeutic purposes. Aggressive SCLCs are often treated non-surgically, while NSCCs are treated with a hybrid of adjuvant therapy and surgery. NSCLC is the collective term for several subtypes, which originate from distinct types of lung cells and account for 80–85% of lung cancer cases. Adenocarcinoma, squamous cell carcinoma, and large cell carcinoma are the three primary subtypes of NSCLC.⁹

Almost two million new cases of cancer patients were caused by infectious agents including bacteria, viruses, and fungi. Multi-drug therapy for cancer combined with microbial infections was reported as harmful to human health, especially patients who suffer from liver or kidney problems.⁸ Patients with cancer are more vulnerable to infectious diseases due to the chemotherapy effect, which weakens the immune system and impairs the patient's capacity to fight against infections.¹⁰ Patients diagnosed with lung cancer are frequently at risk for infection, which is exacerbated by tumor-associated immunosuppression and treatment side effects. Historically, there has been ample evidence linking cytotoxic chemotherapy-induced neutropenia and respiratory symptoms to infection risk.¹¹

An attempt was established to discover novel effective antimicrobial drugs by repurposing well-established candidates. This was carried out through the screening of various therapeutic classes for their antimicrobial activities against MRSA. This screening program led to the discovery of sorafenib (I), which is a well-known multitarget kinase inhibitor, where it displayed significant anti-MRSA activity with MIC = 3 μg mL⁻¹.¹² Subsequently, several sorafenib analogues were synthesized based on the antimicrobial activity of the diarylurea derivative triclocarban (II) and tested as antibacterial agents although their specific modes of action are still unclear.¹³ Modifying some structural features of sorafenib led to the synthesis of several structural analogues including compounds SC72 (III), SC78 (IV), SC79 (V), SC80 (VI), SC005 (VII), and PK150 (VIII). All these compounds exhibited potent dual antimicrobial and anticancer activities against VEGFR-2 except for compound PK150 (VIII), which demonstrated amazing sub-micromolar antibacterial activity against multiple pathogenic strains with a tenfold increase in anti-MRSA activity compared sorafenib but with reduced anticancer activity.¹² In the same direction, a group of researchers tested a series of previously reported pyridazinone-based congeners, IX(a–c), for their potential antimicrobial activities. These derivatives exhibited promising antimicrobial activity with MIC values = (0.5–128 μg mL⁻¹) against S. aureus and MRSA besides their remarkable VEGFR-2 inhibitory activity with IC₅₀ values = (60.70–1800 nM) [Fig. 1].^14,15

Fig. 1. N,N⁻-Diarylurea derivatives acting as anticancer agents with antimicrobial activity.

Subsequently, another series of chloropyridine-based derivatives with a benzoylthiourea linker revealed potent antimicrobial activity. Among these derivatives, compound MAC-545496 (X) displayed the highest activity as a glycopeptide-resistance associated protein R (GRAR) inhibitor, which is essential for S. aureus virulence.¹⁶

In this work, we pursue the design and synthesis of pyridazinone-based derivatives as dual antimicrobial and anticancer agents. Pyridazine-based small molecules emerged as aza analogues of pyridine with the aim to discover novel and safer drug candidates with advantageous biological activities.¹⁷ Indeed, several pyridazine derivatives have been elaborated for a wide range of crucial biological activities, such as anti-microbial,¹⁸ anti-inflammatory,^16,19,20 anti-hypertensive,^16,20 anti-diabetic,^20,21 anti-obesity,^16,21 neuroprotective,^16,22 anti-Alzheimer's,^16,23 anti-tubercular,^16,24 antifungal,¹⁷ anti-HIV^16,25 and anticancer activities.^16,26

Due to the favorable physicochemical and biological features of pyridazine, it has become very popular among medicinal chemists.^27,28 Consequently, herein, we aimed to develop novel pyridazine-based derivatives as antimicrobial agents with anticancer activity.

2. Rationale and design

Considering the previous findings, our scope was based on designing novel pyridazinone-based compounds exhibiting both antimicrobial and anticancer activities. The novel targeted compounds were designed to mimic dual anticancer and antimicrobial agents, namely, sorafenib (I), MAC-545496 (X) and pyridazine compounds IX(a–c). This was accomplished via the bio-isosteric replacement of the pyridine ring of (I) and (X) by a pyridazine ring. Similar to pyridazine compounds IX(a–c), the extra nitrogen of the pyridazine ring is intended to retain the essential activity through extra hydrogen bonding interactions. In this regard, this ring, with a pK_α of 2.3, represents the most basic aromatic skeleton with high π deficiency. Its nitrogen atoms aid in protonation, hydrogen bond formation, and chelation.²⁹ The terminal amide attached to the pyridine ring of sorafenib was inserted inside the pyridazine ring to explore the effect of the internal amide on the activity of the pyridazinone ring. It was also reported that the urea/thiourea moieties are essential for the anti-VEGFR-2 activity, which are responsible for the key interactions with the ASP996 and GLU885 residues.^30,31 Therefore, the phenylurea moiety of sorafenib (I) and the relevant analogues were kept in [series I, 8a–j] of the designed compounds. Another strategy for the synthesis of compounds [series II, 9a–d and series III, 10a–l] was adopted through the elongation of the urea/thiourea moieties by one carbon through the insertion of a carbonyl group to form benzoylthiourea instead of phenylthiourea mimicking MAC-545496 (X). The aromaticity of the pyridazine ring of the designed compound was altered in [series II, 9a–d], giving [series III, 10a–l] to assign the necessity of the aromaticity for both activities. Finally, bio-isosteric replacement of the urea/thiourea moieties with a sulfonamide group was implemented, yielding [series IV, 11a–f] to explore its potential to develop antimicrobial and anticancer activities [Fig. 2].

Fig. 2. Rational design of pyridazinone-based compounds to develop novel agents with antimicrobial and antiproliferative activities.

3. Results and discussion

3.1. Chemistry

The synthetic strategies for the key intermediates and targeted compounds are outlined in Schemes 1–5, respectively. Scheme 1 describes two pathways for the preparation of intermediate 2, which is the key molecule for the synthesis of the targeted derivatives [8a–j, 10a–l, and 11a–f]. Also, the main synthetic route for intermediate 6 was described as crucial for the synthesis of dihydropyridazinone derivatives [9a–d]. Initially, the commercially available p-nitroacetophenone was reacted with glyoxylic acid in the presence of K₂CO₃ at room temperature for 24 h. Subsequently, the as-produced intermediate was treated with AcOH (to pH 4–5), conc. NH₄OH to pH 8–9, and then heated with hydrazine hydrate in isopropanol under refluxing conditions to afford nitrophenylpyridazinone intermediate 1.³² Subsequently, the freshly prepared intermediate 1 was subjected to metal/acid reduction in aqueous ethanol under reflux to give the corresponding aminopyridazine intermediate 2.³³ Intermediate 6 was obtained in multiple steps, where initially, successful Friedel–Crafts acylation reaction of commercial acetanilide with succinic anhydride using the Lewis acid anhydrous AlCl₃ and DMF as the solvent gave the anticipated acid derivative with an approximate yield of 58%.³⁴ Subsequently, the freshly prepared intermediate 3 was refluxed with hydrazine hydrate to give the acetamido derivative of dihydropyridazine 4 in 95% yield.³⁵ Different attempts were made to obtain the aromatic analogue of intermediate 4, but the successful trial was performed by refluxing with hydrated CuCl₂ to obtain compound 5 in 89% yield.³⁶ The ¹H NMR spectral data were in agreement with the anticipated structures. In particular, two new aromatic protons appeared at δ = 6.95 and 7.95 ppm, respectively. Compound 2 was prepared via another route through the acid hydrolysis of intermediate 5, which was very obvious by the change in the IR spectra with the appearance of a forked band corresponding to the NH₂ group at 3343 cm⁻¹. It is worth mentioning that the first method gave the pure form of compound 2 with a better yield than the second method. Alternatively, compound 6, which is the dihydropyridazine analogue of 2, was prepared through the direct hydrolysis of 4 in 87% yield,³⁴ as shown in Scheme 1.

Scheme 1. Reagents and conditions: (a) K₂CO₃, H₂O, rt, 24 h; (b) conc. aqueous NH₄OH, AcOH, NH₂NH₂·H₂O, isopropanol, reflux, 4 h, 39%; (c) Fe/2 N HCl, EtOH, reflux, 6 h, 50%; (d) anhydrous AlCl₃/80 °C, DMF, 1 h, 58%; (e) NH₂NH₂·H₂O, EtOH, reflux, 6 h, 95%; (f) CuCl₂·H₂O, DMSO, reflux, 3 h, 89%; and (g) conc. HCl, reflux, 30 min, 87%.

Scheme 2. Reagents and conditions: (a) oxalyl chloride, DCM, reflux, and 4 h; (b) NH₄SCN, acetone, reflux, and 10 min.

Scheme 3. Reagents and conditions: (a) appropriate phenyl isocyanate or isothiocyanate, THF, rt, 48 h; (b) appropriate benzoyl isothiocyanates (7a–l), acetone, rt, 48 h; (c) appropriate sulfonyl chloride, pyridine, rt, 48 h; and (d) appropriate benzoyl isothiocyanates (7a–d), acetone, rt, 48 h.

Scheme 4. Reagents and conditions: (a) ethyl levulinate, AcOH, piperidine, toluene, reflux, 12 h, and 51%; (b) levulinic acid, AcOH, piperidine, toluene, reflux, 18 h, 40%; (c) NH₂NH₂·H₂O, AcOH, reflux, 6 h, 43–56%; (d) CuCl₂·H₂O, DMSO, reflux, 3 h, 74% and (e) Fe/2 N HCl, EtOH, H₂O, reflux, and 3 h.

Scheme 5. Reagents and conditions: (a) appropriate phenyl isocyanate or isothiocyanate, THF, rt, and 48 h. (b) Appropriate benzoyl isothiocyanates (7h and 7k), acetone, rt, and 48 h. (c) Appropriate sulfonyl chloride, pyridine, rt, and 48 h.

The benzoyl isothiocyanate derivatives [7a–l] were obtained through the reflux of different benzoyl chlorides with ammonium thiocyanate under dry nitrogen, as shown in Scheme 2.³²

As shown in Scheme 3, intermediate 2 was reacted with phenyl iso/thiocyanates in dry tetrahydrofuran (THF) at room temperature to give the phenylurea/thiourea of pyridazinone derivatives, [series I, 8a–j]. The structures of the synthesized compounds were confirmed by IR, ¹H NMR, mass spectroscopy, elemental analysis, high-resolution mass spectroscopy (HRMS) and representative ¹³C NMR, which agreed with the designed compound structures. ¹H NMR revealed two singlet signals at δ = 10.40–8.95 and 10.40–8.83 ppm, which are the characteristic signals of the phenylurea/thiourea moieties, respectively, in addition to the amidic NH appearing within range of 13.17–13.06 ppm.

Furthermore, the resulting benzoyl isothiocyanate derivatives [7a–d], as shown in Scheme 2, were reacted with compound 6 in dry acetone to produce the dihydropyridazine derivatives of benzoylthiourea [series II, 9a–d], respectively. The IR spectra showed an absorption band in the range of 3346–3370, 3336–193 and 1675–1667 cm⁻¹ due to the amidic NH, thiourea NH and amidic carbonyl group, respectively. The ¹H NMR spectra exhibited common signals for the two-methylene groups of dihydropyridazine ring, which both appeared as a triplet signal at δ = 2.46–2.47 and 2.96–2.97 ppm, respectively. Furthermore, three singlet signals of the amidic and two thiourea NH groups appeared at δ = 10.94–10.95, 11.98–11.59, and 12.71–12.49 ppm, respectively, confirming the formation of the benzoylthiourea moiety. Moreover, ¹³C NMR revealed a signal at δ = 181.0 ppm, corresponding to the thiourea carbon.

The aromatic analogues of series II compounds were synthesized via a similar method using intermediate 2, which afforded the benzoylthiourea of the pyridazinone derivatives [series III, 10a–l]. The proposed structures of this series III compounds were confirmed with NMR spectral and elemental analyses. Obviously, in series II, the two aliphatic signals at δ = 2.46–2.47 and 2.96–2.97 ppm for the non-aromatic derivatives of series II disappeared, in addition to an increase in the number of aromatic protons. Moreover, ¹³C NMR revealed an interesting signal for the thiourea carbon at δ = 181 ppm. Furthermore, the same three singlet signals of amidic and two thiourea NH groups appeared, but more downfield at δ = 12.23–10.49, 12.73–12.36 and 13.24–13.14 ppm, respectively, confirming the formation of the benzoylthiourea moiety.

Series IV (11a–f) was synthesized through the reaction of the appropriate sulfonyl chloride derivatives with intermediate 2 in the presence of dry pyridine at room temperature to afford the corresponding benzene sulfonamide derivatives. ¹H NMR revealed only two singlet signals of sulfonamide NH and amidic NH at δ = 10.63–10.72 and 13.16–13.13 ppm, respectively. The elemental, mass and high-resolution mass spectroscopic data (HRMS) were in agreement with the proposed structures.

The main synthetic pathways for the key intermediates (12–16) and the targeted compounds of styrylpyridazinones (17a–b, 18a–b and 19a–b) are outlined in Schemes 4 and 5, respectively.

As shown in Scheme 4, intermediate 12 was prepared through the Claisen condensation between levulinic acid ester and p-nitrobenzaldehyde in the presence of a catalytic amount of glacial acetic acid to activate the aldehyde carbonyl group. Piperidine acted as a base catalyst for the deprotonation of the acidic proton of the ester. This compound was prepared either through refluxing with azeotropic removal of water using a Dean–Stark tube or by heating using a microwave but on a small scale. Under these conditions, the carbanion formed at the α-methyl group of the levulinic acid ester was added to the aldehyde carbon, followed by dehydration to afford an arylidene keto acid derivative. Both the Dean–Stark and microwave methods gave a good yield, but the latter method gave a purer derivative than the former. Although the purification of intermediate 12 was very difficult, column chromatography followed by recrystallization was efficient. The appearance of the olefinic protons in the ¹H NMR spectrum at 7.10 and 7.74 ppm for intermediate 12 confirmed its formation.

Similarly, intermediate 13 was synthesized through the same methods as intermediate 12 but replacing the levulinic acid ester with its acid. The presence of the good leaving group of levulinic acid ester accelerated the rate of the reaction. The purification process for this intermediate was easier with acid–base precipitation using solid Na₂CO₃. After salt formation, the impurities of the starting material were removed by extraction with ethyl acetate. The free acid product was liberated from its salt using dil. HCl till the precipitation of the acid.

Intermediate 14 was synthesized by refluxing with hydrazine hydrate in the presence of acetic acid to obtain the nitro derivative of styryl dihydropyridazine intermediate 14. The ¹H NMR spectrum of compound 14 exhibited the two triplet signals for the dihydropyridazine aliphatic protons at 2.44 and 2.82 ppm. Intermediate 15 was synthesized via the same method as intermediate 5 but with replacing 4 with 14. The product was well purified using a gravity column to remove the unreacted 14. ¹H NMR revealed two new aromatic protons at δ = 6.96 and 7.98 ppm. Subsequently, intermediate 15 was subjected to metal acid reduction in the same manner as intermediate 2 to afford the corresponding aminopyridazine derivative 16. Using butanol instead of ethanol as a solvent increased the rate and the yield of the product. The residual product of this intermediate was used directly in the following step without further purification. The separation of intermediate 16 was useless due to its low yield; therefore, a one-pot reduction was used to obtain a better yield, as shown in Scheme 4.

As shown in Scheme 5, the phenyl urea, benzoylurea and benzene sulfonamide derivatives of styrylpyridazinones (17a–b), (18a–b), and (19a–b) were synthesized in a manner similar to the series I, III and IV compounds, respectively, replacing intermediate 2 with 16.

The proposed structures of all the newly prepared compounds were confirmed by spectral and HRMS analyses. It was very clear that the difference between series I, III and IV and series V, VI and VII is the presence of the two olefinic protons at δ = 7.16–7.37 and 6.71–7.29 ppm with the trans coupling of J = 15.0–18.50 Hz (Fig. S1a–S39b†).

3.2. X-ray powder diffraction (XRD) study

Since its invention, X-ray crystallography has led the field in accurately characterizing the structural properties of materials. Recent developments have expanded the utilization of X-ray crystallography to the characterization of a variety of materials and the ability to determine structures based on X-ray powder diffraction data. The X-ray diffractograms of the intermediate compounds 2 and 6 and selected compounds from each synthesized series 8j, 10h and 11a were compared. It was clear from the X-ray diffraction patterns of intermediates 2 and 6, which possessed a polycrystalline structure, and the selected compounds 8j, 10h and 11a that the X-ray diffraction lines differed in their number, position, relative intensity and broadening. The crystal system of intermediate 2 showed a monoclinic type, which confirmed its aromaticity, while the non-aromatic analogue 6 exhibited a triclinic phase. Moreover, the emergence of additional peaks of varying intensity indicated that the aromaticity was translated into a monoclinic phase and its composition depended on the type of derivative in each series. Compounds 8j and 10h exhibited a monoclinic phase with a different intensity compared to 2, while compound 11a showed the transformation of its crystalline system from a monoclinic to orthorhombic phase [Fig. 3].

Fig. 3. XRD diffractograms of intermediates 2 and 6 and the final compounds 8j, 10h, and 11a (the diffraction peaks were assigned to the corresponding crystal planes by comparing the calculated experimental d-spacing from the diffraction pattern and the standard d-spacing and θ is the angle between the incident ray and the diffracting planes).

The crystal structure and lattice parameters of the basic chemical compounds 2 and 6 and the final compounds (8j, 10h, and 11a) are provided in the ESI† (Table S1).

3.3. Biological evaluation

3.3.1. Evaluation of antimicrobial activity

All the synthesized target compounds were examined for their in vitro antibacterial activity against Staphylococcus aureus ATCC29213 (S. aureus), representing Gram-positive bacteria, and Escherichia coli ATCC25922 (E. coli) as an example of Gram-negative bacteria. They were also evaluated for their in vitro antifungal efficacy against Candida albicans ATCC10231 (C. albicans). The minimal inhibitory concentration (MIC) was calculated to determine the lowest concentration of the substances that inhibited the formation of visible growth in a standardized suspension of tested bacteria after their injection and incubation for 24–48 h at 37 °C. Gentamicin and ketoconazole were used as the reference antibacterial and antifungal drugs, respectively. The outcomes of the antibacterial and antifungal effects are summarized in Table 1. The findings of the antimicrobial screening displayed variable in vitro antibacterial and antifungal activities. Compound 10h exhibited the highest activity against S. aureus with MIC = 16 μg mL⁻¹, followed by compounds 8a, 8b, 8d, 8g, 8i, 8j, 10c, 10e, 10g, 11e, 11f, 17a and 17b with MIC = 128 μg mL⁻¹. In contrast, all the compounds showed weak activity against E. coli, where only the 4-bromo derivative of benzoylthiourea 10c showed MIC = 128 μg mL⁻¹. Obviously, promising activity was shown by the compounds bearing a thiourea rather than urea moiety. Alternatively, halogen substitution at the 3- or 4-position or even both was well tolerated and showed the optimum activity against S. aureus. The meta bromo substitution in compound 10h provided the highest potency against S. aureus. It should be mentioned that the compounds of [series II, 9a–d] exhibited poor activity, indicating the importance of the aromaticity of the pyridazinone ring in the activity. The introduction of a carbonyl group as in compound 10h may have played a role in increasing the antibacterial activity against S. aureus relative to phenylthiourea. In addition, insertion of an olefinic linker in the compounds of series V, VI and VII did not improve the antimicrobial activity with their MIC in the range of 256–1024 μg mL⁻¹.

Antimicrobial screening of the synthesized compounds with minimal inhibitory concentrations (MICs) against the tested pathogenic bacteria and fungi.

Compounds	S. Aureus (ATCC 29213)	E. coli (ATCC 25922)	C. albicans (ATCC 10231)
	(μg mL−1)	(μg mL−1)	(μg mL−1)
8a	128	512	32
8b	128	512	>1024
8c	512	>1024	64
8d	128	256	64
8e	256	256	>1024
8f	256	>1024	>1024
8g	128	256	16
8h	>1024	>1024	512
8i	128	512	256
8j	128	1024	32
9a	512	256	512
9b	512	256	512
9c	512	256	512
9d	512	256	512
10a	256	256	512
10b	256	512	512
10c	128	128	512
10d	>1024	>1024	512
10e	128	256	512
10f	256	256	512
10g	128	256	512
10h	16	256	512
10i	512	256	512
10j	>1024	>1024	512
10k	>1024	>1024	512
10l	512	>1024	512
11a	512	256	64
11b	512	1024	256
11c	512	1024	256
11d	256	1024	256
11e	128	256	256
11f	128	256	256
17a	128	512	512
17b	128	512	512
18a	1024	256	512
18b	256	1024	512
19a	1024	512	512
19b	512	512	512
Sorafenib	4	1	>128
Gentamicin	0.5	0.5	—
Ketoconazole	—	—	2

Alternatively, compound 8g (3-Cl) exhibited the most promising antifungal activity against C. albicans with MIC value = 16 μg mL⁻¹, followed by 8a (3,4-di-Cl) and 8j (3-Br) with MIC = 32 μg mL⁻¹, and then 8c (3-F), 8d (2,4,6-tri-Cl) and 11a (3-Br) with MIC = 64 μg mL⁻¹. The remaining derivatives exhibited lower antifungal activity with MIC values = 512–1024 μg mL⁻¹. Alternatively, the phenylthiourea compounds depicted better antifungal activity than the benzoylthiourea compounds. The antimicrobial screening of the tested compounds against S. aureus, E. coli and C. albicans strains is provided in the ESI† (Fig. S40).

3.3.2. Evaluation of the antiproliferative activity

3.3.2.1. VEGFR-2 kinase inhibition assay

As previously mentioned, our rationale was to design novel derivatives with antimicrobial and anticancer activity based on our lead compounds, namely, sorafenib (I) and pyridazine compounds IX(a–c), which have potent inhibitory activity on VEGFR-2. Therefore, ten compounds (two from each series) were selected for the initial screening at a single dose of 10 μM concentration to test their activities as VEGFR-2 inhibitors depending on their structural similarity and presence of the key features required for activity. Compounds 8a, 8e, 9b, 9c, 10j, 10l, 11c, 11f, 17a and 17b were tested in vitro for their kinase inhibitory activity against the VEGFR-2 enzyme compared to the reference kinase inhibitor staurosporine at a single concentration of 10 μM. The tested compounds showed moderate to weak inhibitory activity against VEGFR-2 kinase. Compound 17a with an ethenyl linker displayed the best inhibitory activity among the tested compounds with the percent inhibition of 60% against VEGFR-2. Although compounds 17b and 8a exhibited moderate inhibitory activity (% inhibition = 38% and 34%, respectively).

Compound 10j of the benzoylthiourea derivatives exhibited moderate inhibitory activity (28%) but still lower than the phenylurea derivatives, which confirmed the importance of the urea moiety, similar to the structure of sorafenib. The replacement of the urea moiety with sulphonamide negatively affected the VEGFR-2 inhibitory activity, where compounds 11c and 11f exhibited lower inhibitory activity (14%), which confirmed the importance of the urea moiety for anti-VEGFR-2 activity. Meanwhile, the non-aromatic analogues 9b and 9c exhibited the weakest inhibitory activity of 7% and 5%, respectively [Table 2]. The higher inhibitory activity of 17a can be attributed to the presence of the olefinic bond, which lengthened the spacer between the pyridazine ring and the thiourea moiety, resulting in better fitting in the VEGFR-2 active site.

VEGFR-2% inhibition by the tested compounds at a single concentration of 10 μM.

Compound	VEGFR-2% inhibition
8a	34
8e	8
9b	7
9c	5
10j	28
10l	5
11c	14
11f	14
17a	60
17b	38
Sorafenib	102
Strausporine14	90a

^aReported VEGFR-2% inhibition value.

3.3.2.2. In vitro single-dose screening of anticancer activity in full NCI 60-cell line panel

The present study explains the antiproliferative activity of the synthesized compounds in each series of 8a–j, 9a–d, 10a–l, 11a–f, 17a–b, 18a–b and 19a–b. All these compounds were selected by the National Cancer Institute (NCI) according to the protocol of the drug evaluation branch of the NCI, Bethesda, USA, for in vitro anticancer screening. The results for each compound are reported as the growth percentage inhibition of the treated cells compared to that of the untreated control cells. A panel of 60 human tumor cell lines was derived from nine different cancer types including leukemia, melanoma, lung, colon, central nervous system (CNS), ovarian, renal, prostate and breast cancers. Within 48 h, the drug treatment protocol was utilized, and the sulforhodamine B (SRB) protein assay was applied to evaluate the cell viability and growth.

The mean percentage of growth inhibition (GI%) of the tested compounds over the full panel of cell lines is provided in the ESI† [Fig. 4]. The obtained data revealed that three compounds, 8f, 10l and 17a, exhibited significant and potent anticancer activity against most of the cell lines.

Fig. 4. Mean growth inhibition (%) at 10 μM of the tested compounds over the NCI 60 cell line panel.

Compound 17a exhibited the best anticancer activity against most cancer cell lines, especially non-small cell lung cancer (HOP-92, HOP-62, NCI-H226, and A549/ATCC), CNS cell line (U251), melanoma cell line (LOX IMIV), ovarian cell lines (NCI/ADR-RES and SK-OV-3), renal cell lines (786-0 and UO-31), prostate cancer cell line (DU-145) and breast cancer cell lines (BT-549, MDA-MB-468 and MCF7) with GI% of 100.14%, 98.09%, 88.22%, 73.95%, 99.98%, 88.79%, 85.72%, 82.62%, 88.25%, 76.22%, 91.48%, 95.48%, 88.08% and 72.68%, respectively (Fig. S41†).

In addition, compound 10l exhibited the best anticancer activity against most of the cancer cell lines, especially the melanoma cell lines (SK-MEL-2 and UACC-62), CNS cell lines (U251, SF-295 and SNB-19) and non-small cell lung cancer (HOP-62) with GI% of 98.02%, 91.02%, 96.09%, 93.02%, 91.18% and 94.60%, respectively (Fig. S42†).

Furthermore, compound 8f exerted admirable anticancer activity against various cell lines such as leukemia (MOLT-4, RPMI-8226, CCRF-CEM and K-562), non-small cell lung cancer (NCI-H522), CNS cancer (SF-295), breast cancer (T-47D and MCF7), prostate cancer (PC-3), colon cancer (KM12) and ovarian cancer (OVCAR-3) with GI% 78.13%, 73.48%, 72.93%, 68.42%, 73.51%, 72.69%, 74.70%, 63.98%, 68.0%, 66.43% and 62.21%, respectively (Fig. S43†).

In addition, compounds 8a, 8b, 10j and 10k exhibited remarkable to moderate activity against most of the cell lines discussed before with the GI% of 51%. The rest of the series (I, III, and IV) showed mild to weak anticancer activity against most of the cancer cell lines.

According to these findings, it can be concluded that the 3-chloro substitution of the benzoyl or phenylthio/urea derivatives has a clear impact on increasing the antiproliferative activity of most of the tested compounds. The percentage of growth inhibition % of the NCI 60 cancer cell lines displayed by the final compounds 8a–j, 9a–d, 10a–l, 11a–f, 17a–b, 18a–b and 19a–b are provided in the ESI† (Tables S2–S4).

3.3.2.3. In vitro five dose screening of anticancer activity in full NCI 60-cell line panel

Fortunately, compounds 10l and 17a were selected by NCI for further screening at five different concentrations (0.01, 0.1, 1, 10, and 100 μM), where the cytotoxic and/or growth inhibitory effects of the selected compound were tested in vitro against a variety of cell lines. The assessment of five dose assays validated the specific effects on cells observed in the one-dose screen. The panel cell lines used in the NCI screen were the leukemia (L) lines, non-small cell lung cancer (NSCLC) lines, colon cancer (CL) lines, central nervous system cancer (CNSC) lines, melanoma (M) lines, ovarian cancer (OC) lines, renal cancer (RC) lines, prostate cancer (PC) lines, and breast cancer (BC) lines.

Compound 17a exerted broad-spectrum antitumor activity against the nine tumor subpanels tested from cytostatic to cytotoxic at GI₅₀ in the range of 1.66 to 100 μM [Fig. 5]. The best results were recorded for MDA-MB-468, RXF 393, SK-MEL-2, SK-MEL-28, UACC-62, MCF7, HOP-92, M14, CAKI-1, SF-295, SF-268 and SN12C with GI₅₀ values of 1.66, 1.66, 1.86, 2.0, 2.34, 2.44, 2.51, 2.57, 2.57, 2.69, 2.82 and 3.02 μM, respectively. In addition, significant activity was registered for UACC-257, EKVX, NCI-H23, PC-3, OVCAR-8 and SNB-75 with GI₅₀ values of 3.24, 3.54, 3.54, 3.63, 3.47 and 3.98 μM, respectively.

Fig. 5. GI₅₀ of the compound 17a over NCI 60 cell line panel in five-dose screening.

Compound 10l exerted broad-spectrum antitumor activity against the nine tumor subpanels tested at the GI₅₀ range of 3.18 to 100 μM [Fig. 6]. The best results were recorded for HOP-92, SNB-75, UACC-62 and NCI-H522 with GI₅₀ values of 3.18, 3.92, 4.07 and 4.55 μM, respectively. In addition, significant activity was registered for UACC-257, HS 578T, OVCAR-8, RXF 393, MDA-MB-231/ATCC, SF-268, U251, and SF-295 with GI₅₀ values of 5.59, 5.80, 6.24, 6.60, 6.83, 6.91, 6.94 and 7.23 μM, respectively. The in vitro tumor 50% growth inhibition (GI₅₀, μM) of 10l and 17a is provided in the ESI† (Table S5).

Fig. 6. GI₅₀ of compound 10l over NCI 60 cell line panel in five-dose screening.

3.3.2.4. Gene expression of apoptotic markers p53, Bax, and Bcl-2 in NSCLC cell line

One of the cancerous hallmarks that makes it more difficult for damaged cells to undergo apoptosis is the loss of balance between proliferation and apoptosis. Accordingly, one of the most outstanding strategies for cancer treatment is to trigger apoptotic pathways in tumor-affected cells. Numerous genes or signaling cascades, including p53 and Bax, which induce apoptosis, are implicated in the deregulations that lead to the initiation and progression of different malignancies. In addition, numerous carcinomas have been linked to the overexpression of the anti-apoptotic gene Bcl-2.^37,38 In an attempt to further explore the underlying mechanism of action of compound 10l, which exhibited significant antiproliferative activity, its effect on the p53, Bax and Bcl-2 apoptotic genes was investigated in the NSCLC (A549/ATCC) cell line using doxorubicin as the reference drug. The genetic expression analysis of Bcl-2, p53, and Bax in the A549/ATCC cell line treated with 10l and doxorubicin is presented in Fig. 7. The findings revealed that the tested compound decreased the expression level of the anti-apoptotic gene (Bcl-2) to half in the cancer cell line treated with compound 10l and doxorubicin, respectively, compared with the negative control cancer cell line. Alternatively, the expression levels of the pro-apoptotic genes (p53 and Bax) increased by three folds in the cancer cell line treated with compound 10l and the positive cancer cell line (doxorubicin), respectively, compared with the negative control cancer cell line.

Fig. 7. (A) Alterations in Bcl-2 gene expression in the A549/ATCC cell line treated with 10l and doxorubicin (data are presented as mean ± SEM. a–c: Mean values for the tissue with different superscript letters are significantly different (P < 0.05)). (B) Alterations in p53 gene expression in the lung cancer cell line treated with compound 10l and doxorubicin (data are presented as mean ± SEM. a and b: Mean values for the tissue with different superscript letters are significantly different (P < 0.05)). (C) Alterations in Bax gene expression in lung cancer cell lines treated with compound 10l and doxorubicin (data are presented as mean ± SEM. a–c: Mean values in tissue with different superscript letters are significantly different (P < 0.05)).

3.3.2.5. Cell cycle analysis

The design of novel anti-proliferative drugs that can control cell cycle progression and apoptosis is an appealing strategy for lowering the possibility of nonspecific medication side effects during cancer treatment.³⁹ Therefore, the most potent anticancer compound 10l, which was previously selected for the five-dose assay and exhibited significant activity on the A549/ATCC cell line, was also selected for further investigation of its cell cycle arrest potential.

The cell cycle arrest potential of compound 10l was carried out three times for confirmation using the GI₅₀ value of 0.025 μg mL⁻¹, which was represented by the cellular population percentages [Fig. 8 and 9].

Fig. 8. Cell cycle analysis for the A549/ATCC cell line treated with compound 10l.

Fig. 9. Histograms of the cell cycle for (A) the control and (B) compound 10l in the A549/ATCC cell line.

Compound 10l arrested the cell cycle in the G0–G1 phase, where the cell population levels of A549/ATCC increased from 85.41%, 85.46% and 85.84% for the control to 90.86%, 89.51% and 89.87%, respectively, upon treatment with compound 10l. However, compound 10l exhibited additional arrest of the G2-M and S phases of the cell cycle, which decreased the cell population levels to 8.95–10.29% and 0.19–0.27% compared to the control (13.15–13.37%) and (0.31–0.51%), respectively [Fig. 8 and 9].

3.4. Molecular docking studies of the designed compound on VEGFR-2 enzyme

In this work, a molecular docking study was performed to interpret the biological results for the designed compounds (8a–j), (9a–d), (10a–l) and (11a–f), (17a–b), (18a–b) and (19a–b) in comparison to sorafenib. The compounds were modeled using VEGFR-2 in complex with sorafenib (PDB: 3WZE).⁴⁰

The X-ray co-crystal structures of kinase-bound type-II kinase inhibitors and the analysis of the binding modes of sorafenib (I) generally depict two main hydrogen-bond interactions with the backbone-NH of the ASP1046 residue and the carboxylate group of GLU885 residue with the carbonyl and amino groups of the urea moiety, respectively.⁴⁰ The terminal phenyl moiety occupies the hydrophobic pocket obtained by rearrangement of the protein structure.⁴¹ Sorafenib forms two hydrogen bonds with the backbone-NH of CYS919 through the carboxamide NH and the pyridyl ring nitrogen. In the design of our compounds, we used the urea and thiourea moieties and the terminal-substituted phenyl ring with the aim to retain the essential hydrogen bonding and hydrophobic interactions with the key amino acids [Fig. 10].

Fig. 10. Essential pharmacophoric features of sorafenib and the target compounds.⁴².

The most potent compound against VEGFR 2 (17a) exhibited a high docking score of −32.65 kcal mol⁻¹ and almost fulfilled the basic key interactions of our lead compound (sorafenib score: −52.93 kcal mol⁻¹). This may be due to the structural similarity to sorafenib with replacement of the oxygen linker with the olefinic bond, which adopted the volumes and orientation that made this compound fit properly in the active site of the receptor. The decrease in the activity of this series relative to our lead compound may be due to the absence of an important interaction with CYS919 [Fig. 11].

Fig. 11. Docking poses (2D and 3D) and amino acids involved in the binding interactions of the lead compound (sorafenib) [A and B], representative compounds of series V (17a) [C and D] and series II (9c) [E and F] to the active site.

In addition, the series I compounds (8a–j) exhibited the best binding interactions with the highest binding scores ranges among the designed compounds such as compounds 8b (R = 3-Br, X = O), 8f (R = 3-Cl, X = O) and 8e (R = 4-F, X = O), which showed binding scores of 39.83, 38.31 and 36.14 kcal mol⁻¹, respectively. Surprisingly, the biological activities of these compounds (8a and 8e) against VEGFR-2 depicted moderate to poor activity. Although these compounds shared the same phenylurea moiety of sorafenib and showed nearly the same hydrogen bond interactions with the ASP1046, CYS919, and GLU885 residues, their poor biological activity can be attributed to the lack of the oxygen linker between pyridazinone and the urea moiety. The missed linker in the tested compounds may be the reason for these compounds failure to fit well in the active site.

Although the compounds of series II, III and VI compounds such as 9c, 10j, 18a and 18b could fulfill the main key interactions of sorafenib, the inhibitory activity against the VEGFR-2 enzyme was poor. This can be attributed to the extra C O group in the pyridazine benzoylthiourea derivatives. This carbonyl may have increased the volume, size and orientation of these compounds, hindering their good fitting with the receptor.

Finally, the sulfonamide derivatives of series IV and VII compounds (11c and 19a) depicted the lowest binding scores despite the presence of some hydrogen bonds and hydrophobic interactions. The poor docking result and actual anti-VEGFR-2 activity of these sulfonamides among the tested compounds explained and confirmed the importance of the urea/thiourea for VEGFR-2 activity. Conclusively, despite the good docking results of most of the tested compounds, their biological activity against the VEGFR-2 enzyme was unfortunately unexpected except for compound 17a. The docking poses and amino acids involved in the binding interactions of the tested compounds with the VEGFR-2 enzyme are provided in the ESI† (Table S6 and Fig. S44a–f).

3.5. Structure–activity relationship (SAR)

According to the previous findings, the structure–activity relationship for the antimicrobial and anticancer activities of the synthesized pyridazinone derivatives [Fig. 12] can be depicted as follows:

Fig. 12. Structure–activity relationship of the targeted compounds.

• The presence of the heterocyclic ring (pyridazine ring) is essential for both anticancer and antimicrobial activities. The aromaticity of the pyridazinone ring enhances both activities.

• The extra carbon in the benzoyl thiourea compounds potentiates the antibacterial activity than phenyl urea or thiourea compounds. The bromo substitution at the 3-position of the benzoyl urea derivatives enhanced the antibacterial activity against S. aureus (compound 10h).

• The phenyl thiourea compounds exhibited better antifungal activity than the benzoyl thiourea compounds. The chloro substitution at the 3-position of the phenyl urea derivatives boosts the antifungal activity against C. albicans (compound 8g), followed by 3-bromo and 3,4-dichloro substitution (compounds 8j and 8a, respectively).

• The anti-VEGFR-2 activity was potentiated by the presence of phenyl thiourea rather than benzoyl thiourea. Also, the presence of the olefinic bond linker lengthens the spacer between the pyridazine ring and the thiourea moiety and may be a better fit to the VEGFR-2 active site. In addition, the 3-bromo substitution was more preferable for anti-VEGFR-2 activity (compound 17a), followed by the 3,4-dichlrophenyl thiourea derivatives (compounds 17b and 8a).

• The 3-chloro or 3-bromo substitution of the benzoyl or phenyl thiourea derivatives has a clear impact on increasing the antiproliferative activity of most of the tested compounds (compounds 10l and 17a, respectively).

4. Conclusion

Seven series of pyridazinone-based compounds were designed, synthesized, and biologically evaluated for their anti-microbial and anti-proliferative activities. All the compounds were examined for their in vitro antimicrobial activity against two bacteria and one fungal strain. The introduction of a carbonyl group in the benzoylthiourea derivatives, as seen in compound 10h, played a role in increasing the antibacterial activity against S. aureus relative to phenylthiourea as in compound 8g, which interestingly showed the most promising antifungal activity against C. albicans. Having sorafenib as the lead compound and based on keeping the essential features needed for VEGFR-2 inhibitory activity, compounds 8a, 8e, 9b, 9c, 10j, 10l, 11c, 11f, 17a and 17b were tested against VEGFR-2 with compound 17a showing the best% inhibition. Furthermore, the tested compounds were evaluated for their in vitro antiproliferative activities, exhibiting significant anticancer activity against most of the NCI cell lines. Compounds 10l and 17a exhibited broad spectrum and potent anti-proliferative activity against several NCI cell panels with increased GI₅₀ values including the NSCLC, CNS, and breast cancer cell lines.

Additionally, these top-performing anticancer compounds were tested for their potential to induce cell cycle arrest in specific cancer cell lines. Compound 10l induced G0–G1 phase cell cycle arrest in the A549/ATCC cell line, increasing the cell population in this phase from 85.41% in the control cells to 90.86% following treatment with compound 10l.

For the further investigation of the mechanism of its cytotoxic activity, compound 10l was tested for its impact on the apoptotic genes p53, Bax and Bcl-2 in the NSCLC cell line (A549/ATCC). It was found that it induced apoptotic activity through the upregulation and downregulation of the pro-apoptotic genes p53 and Bax and the anti-apoptotic gene Bcl-2, respectively. This strongly suggests that inducing apoptosis is the underlying trigger of the anti-tumor activity of compound 10l. Furthermore, lengthening the spacer between the pyridazinone ring and urea/thiourea moiety as series V, VI and VII compounds potentiated the anti-VEGFR-2 activity more than the compounds without a linker. Our study revealed the potential of pyridazinone-based compounds to act as anti-microbial agents with anti-cancer activity.

Further biological investigations to clarify the intrinsic mechanism of action behind its anti-cancer activity will be attempted together with the synthesis of other derivatives to expand the SAR analysis and explore more potent analogues. In addition, further testing of the compound for its antibiofilm and quorum sensing activity will be done. Furthermore, the effect of the designed compounds when combined with other antibiotics on bacterial resistance will be investigated.

5. Experimental

5.1. Chemistry

5.1.1. Materials and instrumentation

¹H and ¹³C NMR spectra were recorded at room temperature in base-filtered DMSO-d₆ on a spectrometer operating at 400 MHz for proton and 100 MHz for carbon nuclei. The signal due to residual DMSO-d₆ appearing at δ = 2.5 ppm and the central resonance of DMSO-d₆ appearing at δ = 40 ppm were used to reference the ¹H-NMR and ¹³C-NMR spectra, respectively. ¹H-NMR was recorded as follows: chemical shift (δ) [multiplicity, coupling constant(s), J (Hz), relative integral], where multiplicity is defined as s = singlet; d = doublet; t = triplet; m = multiplet or combinations of the above. Infrared spectra (ν_max) were measured using an FT-IR spectrometer and reported in cm⁻¹. High-resolution mass measurements were conducted on a time-of-flight instrument magnetic sector machine. Low-resolution mass spectra were recorded on the direct inlet part to the mass analyzer in the Thermo Scientific GC-MS model ISQ at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Nasr City, Cairo. The low- and high-resolution EI mass spectra were recorded on a magnetic sector machine. Melting points were measured on an automated melting point system and were uncorrected. Elemental microanalysis was performed on an elemental analyzer, Flash 2000 Thermo Fisher at the Regional Center for Mycology and Biotechnology (RCMB), Faculty of Science, Al-Azhar University, Nasr City, Cairo, Egypt. The X-ray diffraction (XRD) patterns in this work were obtained using a JEOL model JSDX-60PA materials research X-ray diffractometer, Minia University, Minia, Egypt. Intermediates were purified using a gravity column with silica gel (60–120 mesh) and hexane/ethyl acetate (1/1 v/v%) as the developing system. The final compounds were purified through flash chromatography columns, which were packed with 230–400 mesh silica gel.

5.1.2. Synthesis

6-(4-Nitrophenyl)pyridazin-3(2H)-one (1)

Compound 1 was prepared according to the reported procedure³² (m.p. 280–282 °C, as reported).

6-(4-Aminophenyl)pyridazin-3(2H)-one (2)

Compound 2 was prepared according to the reported procedure^32,33 (m.p. 290–292 °C, as reported). X-ray diffractogram (XRD) characteristic lines at 2θ = [17.2°, 18.0°, 20.1°, 21.6°, 24.2°, 25.2°, 26.5°, 29.2°, 33.6°, 35.0°, 37.0°, 40.2°, 41.5°, 45.7°, 47.6°, 53.7°, 57.6°, 59.5° and 61.6°]; space group = P2₁/a; lattice parameters: [a = 8.875(4) Å, b = 9.498(1) Å, c = 11.655(8) Å, α = 90°, β = 110.97(4)°, γ = 90°]; volume of unit cell (V) = 917.39 (Å)³; crystalline structure: monoclinic.

4-(4-Acetamidophenyl)-4-oxobutanoic acid (3)

Compound 3 was prepared according to the reported procedure^34,43 (m.p. 201–203 °C, as reported).

N-(4-(6-Oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)acetamide (4)

Compound 4 was prepared according to the reported procedure⁴⁴ (m.p. 251–253 °C, as reported).

N-(4-(6-Oxo-1,6-dihydropyridazin-3-yl)phenyl)acetamide (5)

Compound 5 was prepared according to the reported procedure^36,45 (m.p. 298–300 °C, as reported).

6-(4-Aminophenyl)-4,5-dihydropyridazin-3(2H)-one (6)

Compound 6 was prepared according to the reported procedure^34,43 (m.p. 237–239 °C, as reported). X-ray diffractogram (XRD) characteristic lines at 2θ = [15.0°, 15.6°, 16.4°, 21.0°, 22.5°, 24.0°, 26.4°, 27.6°, 32.0°, 32.2°, 33.5°, 39.0°, 40.0°, 44.0°, 46.0°, 52.0°, 58.0° and 60.0°]; space group = P1; lattice parameters: [a = 7.612(4) Å, b = 11.520(2) Å, c = 11.898(4) Å, α = 112.85°, β = 102.06°, γ = 96.57°]; volume of unit cell (V) = 920.34 (Å)³; crystalline structure: triclinic.

Benzoyl isothiocyanates derivatives (7a–l)

Compounds 7a–l were prepared according to the reported procedure⁴⁶ and used directly in the following step.

1-(Substituted)-3-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)urea/thiourea (8a–j)

Under dry nitrogen, compound 2 (0.28 g, 1.5 mmol) was dissolved in dry dioxane or tetrahydrofuran (THF) (10 mL). The appropriate phenyl isocyanate or isothiocyanate (1.5 mmol) was added portion wise and the mixture was stirred at room temperature for 48 h. The formed solid was collected by suction filtration, washed with diethyl ether, ethanol and dilute HCl (10 mL each), and then dried. The afforded compounds were further purified through recrystallization from THF or ethanol for compounds 8e and 8j.

1-(3,4-Dichlorophenyl)-3-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)thiourea (8a)

The title compound was obtained as yellow crystals (0.31 g, 53% yield); m.p. 175–177 °C. M.F. C₁₇H₁₂Cl₂N₄OS; HRMS: calcd.: 390.0109; found: {m/z: [M + H]⁺; 391.0190 (³⁵Cl) and 393.0167 (³⁷Cl)}. Anal. calcd.: C, 52.18; H, 3.09; N, 14.32; S, 8.20; found: C, 52.40; H, 3.23; N, 14.59; S, 8.34. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.17 (s, 1H, NH), 10.24 (s, 1H, NH), 10.14 (s, 1H, NH), 8.03 (d, J = 9.90 Hz, 1H, Ar–H), 7.92 (d, J = 2.05 Hz, 1H, Ar–H), 7.84 (d, J = 8.52 Hz, 2H, Ar–H), 7.77–7.66 (m, 1H, Ar–H), 7.62 (d, J = 8.60 Hz, 1H, Ar–H), 7.59 (d, J = 8.85 Hz, 1H, Ar–H), 7.48 (dd, J = 8.71, 2.22 Hz, 1H), 6.99 (d, J = 9.90 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.89 (C̲ S); 160.63 (C̲ O); 143.88 (C̲ N); 140.52, 140.14; 131.77 (2 × Ar–C̲); 131.12, 130.93, 130.67, 130.60, 126.51, 126.38, 125.22 (2 × Ar–C̲); 123.99, 123.91. FT (IR) ( max, cm⁻¹) 3350 and 3204 (amidic and thiourea NH stretching), 1649 , 1575 ( stretching).

1-(3-Bromophenyl)-3-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)urea (8b)

The title compound was obtained as brown crystals (0.41 g, 71.18% yield); m.p. 188–190 °C. M.F. C₁₇H₁₃BrN₄O₂; HRMS: calcd.: 384.0222; found: {m/z: [M + H]⁺; 385.0303 (⁷⁹Br) and 387.0283 (⁸¹Br)}. Anal. calcd.: C, 53.00; H, 3.40; N, 14.54; found: C, 53.27; H, 3.62; N, 14.75. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.06 (s, 1H, NH), 9.31 (s, 2H, NH), 7.96 (s, 1H, Ar–H), 7.79 (s, 2H, Ar–H), 7.54 (s, 2H, Ar–H), 7.28 (s, 2H, Ar–H), 7.21 (s, 1H, Ar–H), 7.11 (s, 1H, Ar–H), 6.92 (s, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 152.74 (2 × C̲ O), 144.03 (C̲ N); 141.64, 131.80, 131.29, 130.67, 126.79, 125.0, 124.88, 122.19, 120.80, 120.72, 118.6, 118.51, 117.46, 117.38. FT (IR) ( max, cm⁻¹) 3324 and 3290 (amidic NH stretching), 1652 and 1588 .

1-(3-Fluorophenyl)-3-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)thiourea (8c)

The title compound was obtained as reddish-brown crystals (0.21 g, 41.18% yield); m.p. 248–250 °C. M.F. C₁₇H₁₃FN₄OS; HRMS: calcd.: 340.0794; found: {m/z: [M + H]⁺; 341.0879}. Anal. calcd.: C, 59.99; H, 3.85; N, 16.46; S, 9.42; found: C, 60.17; H, 3.98; N, 16.72; S, 9.60. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.16 (s, 1H, NH), 10.36 (d, J = 4.52 Hz, 2H, NH), 8.02 (d, J = 9.66 Hz, 1H, Ar–H), 7.83 (d, J = 8.07 Hz, 2H, Ar–H), 7.74–7.56 (m, 3H, Ar–H), 7.36 (q, J = 7.50 Hz, 1H, Ar–H), 7.29 (d, J = 7.70 Hz, 1H, Ar–H), 6.96 (q, J = 9.70 Hz, 2H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.52 (C̲ S); 163.69, 160.68 (C̲ O); 143.95 (C̲ N); 141.81, 140.46, 131.83, 130.82, 130.59, 130.43, 126.28, 123.52, 119.10, 111.14, 111.30, 110.94, 109.94. FT (IR) ( max, cm⁻¹) 3180 (thiourea NH stretching), 1649 , 1591 ( stretching).

1-(4-(6-Oxo-1,6-dihydropyridazin-3-yl)phenyl)-3-(2,4,6-trichlorophenyl)thiourea (8d)

The title compound was obtained as yellow crystals (0.42 g, 66.04% yield); m.p. 214–216 °C. M.F. C₁₇H₁₁Cl₃N₄OS; HRMS: calcd.: 423.9719; found: {m/z: [M + H]⁺; 424.9802 (³⁵Cl) and 426.9780 (³⁷Cl)}. Anal. calcd.: C, 47.96; H, 2.60; N, 13.16; S, 7.53 found: C, 48.12; H, 2.83; N, 13.40; S, 7.79. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.17 (s, 1H, NH), 10.36 (s, 1H, NH), 9.62 (s, 1H, NH), 8.04 (d, J = 10.0 Hz, 1H, Ar–H), 7.86 (d, J = 8.40 Hz, 2H, Ar–H), 7.76 (s, 2H, Ar–H), 7.66 (d, J = 8.10 Hz, 2H, Ar–H), 6.99 (d, J = 9.90 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 180.74 (C̲ S); 160.63 (C̲ O), 143.84 (C̲ N); 140.42, 136.30, 133.04, 131.76, 131.21, 130.62, 128.70 (2 × Ar–C̲), 127.95, 126.44 (2 × Ar–C̲), 125.25, 123.99, 118.80. FT (IR) ( max, cm⁻¹) 3310 and 3207 (amidic and thiourea NH stretching), 1654 , 1594 ( stretching).

1-(4-Fluorophenyl)-3-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)urea (8e)

The title compound was obtained as yellow crystals (0.22 g, 45.36% yield); m.p. 251–253 °C. M.F. C₁₇H₁₃FN₄O₂; HRMS: calcd.: 324.1023; found: {m/z: [M + H]⁺; 325.1104}. Anal. calcd.: C, 62.96; H, 4.04; N, 17.28; found: C, 62.82; H, 4.15; N, 17.51. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.10 (s, 1H, NH), 8.95 (s, 1H, NH), 8.83 (s, 1H, NH), 7.98 (d, J = 9.90 Hz, 1H, Ar–H), 7.78 (d, J = 8.56 Hz, 2H, Ar–H), 7.57 (d, J = 8.56 Hz, 2H, Ar–H), 7.48 (dd, J = 8.31, 4.77 Hz, 2H, Ar–H), 7.12 (t, J = 8.74 Hz, 2H, Ar–H), 6.96 (d, J = 9.90 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 160.61, 159.07 (amidic and urea C̲ O); 156.70; 152.95; 144.09 (C̲ N); 141.44, 136.35, 136.33, 131.67, 130.52, 128.40, 126.73, 120.56, 120.49, 118.61, 115.87, 115.65. FT (IR) ( max, cm⁻¹) 3294 (amidic NH stretching), 1708 and 1644 .

1-(3-Chlorophenyl)-3-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)urea (8f)

The title compound was separated as brown crystals (0.41 g, 80.39% yield); m.p. 206–208 °C. M.F. C₁₇H₁₃ClN₄O₂; HRMS: calcd.: 340.0727; found: {m/z: [M + H]⁺; 341.0809 (³⁵Cl) and 343.0784 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.10 (s, 1H, NH), 9.00 (s, 2H, NH of urea), 8.00 (d, J = 9.78 Hz, 1H, Ar–H), 7.80 (d, J = 7.70 Hz, 2H, Ar–H), 7.71 (d, J = 7.34 Hz, 1H, Ar–H), 7.58 (br, s, 2H, Ar–H), 7.29 (br, s, 2H, Ar–H), 7.03 (d, J = 6.90 Hz, 1H, Ar–H), 6.97 (d, J = 9.66 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 160.65 (C̲ O); 152.71; 144.06 (C̲ N); 141.47, 140.95, 133.66, 131.68, 130.86, 130.53, 128.65, 126.74, 122.15, 118.78, 118.69, 118.20, 118.10, 117.27. FT (IR) ( max, cm⁻¹) 3300 and 3273 (amidic NH stretching); 1674 and 1655 .

1-(3-Chlororophenyl)-3-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)thiourea (8g)

The title compound was obtained as yellow crystals (0.32 g, 61.54% yield); m.p. 162–164 °C. M.F. C₁₇H₁₃ClN₄OS; HRMS: calcd.: 356.0499; found: {m/z: [M + H]⁺; 357.0581 (³⁵Cl) and 359.0558 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.17 (s, 1H, NH), 10.16 (s, 1H, NH), 10.08 (s, 1H, NH), 8.02 (d, J = 9.90 Hz, 1H, Ar–H), 7.84 (d, J = 8.68 Hz, 2H, Ar–H), 7.72 (t, J = 1.90 Hz, 1H, Ar–H), 7.69–7.64 (m, 1H, Ar–H), 7.63 (d, J = 8.80 Hz, 1H, Ar–H), 7.46–7.40 (m, 1H, Ar–H), 7.36 (t, J = 8.0 Hz, 1H, Ar–H), 7.23–7.15 (m, 1H, Ar–H), 6.99 (d, J = 9.90 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.84 (C̲ S); 160.64 (C̲ O); 143.91 (C̲ N); 141.46, 140.69, 133.00 (2 × Ar–C̲), 131.77, 130.95, 130.59, 130.52, 126.34 (2 × C), 124.53, 123.78, 123.32, 122.30. FT (IR) ( max, cm⁻¹); 3270 and 3100 (amidic NH stretching and thiourea NH stretching), 1650 (NH–C O), 1576 ( stretching).

1-(4-Bromophenyl)-3-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)urea (8h)

The title compound was obtained as yellow crystals (0.38 g, 66% yield); m.p. 276–278 °C. M.F. C₁₇H₁₃BrN₄O₂; HRMS: calcd.: 384.0222; found: {m/z: [M + H]⁺; 385.0307 (⁷⁹Br) and 387.0286 (⁸¹Br)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.10 (s, 1H, NH), 8.96 (s, 1H, NH), 8.92 (s, 1H, NH), 8.00 (d, J = 9.90 Hz, 1H, Ar–H), 7.80 (d, J = 8.80 Hz, 2H, Ar–H), 7.57 (d, J = 8.80 Hz, 2H, Ar–H), 7.45 (s, 4H, Ar–H), 6.97 (d, J = 9.90 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 160.62 (C̲ O); 152.73, 144.08 (C̲ N); 141.05; 139.45, 131.99 (2 × Ar–C̲), 131.68 (2 × Ar–C̲), 130.51 (2 × Ar–C̲), 128.56, 126.74, 120.66, 118.69 (2 × Ar–C̲), 113.84. FT (IR) ( max, cm⁻¹); 3309 and 3275 (amidic NH stretching); 1709 and 1650 .

1-(4-Bromophenyl)-3-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)thiourea (8i)

The title compound was separated as yellow crystals (0.29 g, 48.34% yield); m.p. 158–160 °C. M.F. C₁₇H₁₃BrN₄OS; HRMS: calcd.: 399.9993; found: {m/z: [M + H]⁺; 400.9966 (⁷⁹Br) and 403.0042 (⁸¹Br)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.16 (s, 1H, NH), 10.40 (d, J = 27.60 Hz, 2H, NH), 8.06–7.98 (m, 1H, Ar–H), 7.90 (s, 1H, Ar–H), 7.88–7.78 (m, 2H, Ar–H), 7.77–7.63 (m, 2H, Ar–H), 7.50 (d, J = 2.80 Hz, 1H, Ar–H), 7.29 (t, J = 7.90 Hz, 2H, Ar–H), 7.05–6.94 (m, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.68 (C̲ S), 160.64 (NH–C̲ O), 143.93 (C̲ N); 141.63, 140.73, 131.79, 130.86, 130.79, 130.58, 127.40, 127.33, 126.30, 125.92, 123.57 (2 × Ar–C̲), 122.50, 121.36. FT (IR) ( max, cm⁻¹); 3390, 3230 and 3151 (amidic and thiourea NH stretching), 1647 , 1588 ( stretching).

1-(3-Bromophenyl)-3-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)thiourea (8j)

The title compound was separated as yellow crystals (0.42 g, 70% yield); m.p. 165–167 °C. M.F. C₁₇H₁₃BrN₄OS; HRMS: calcd.: 399.9993; found: {m/z: [M + H]⁺; 401.0075 (⁷⁹Br) and 403.0059 (⁸¹Br)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.17 (s, 1H, NH), 10.22 (s, 1H, NH), 10.13 (s, 1H, NH), 8.03 (d, J = 9.90, 1H, Ar–H), 7.84 (d, J = 8.68 Hz, 3H, Ar–H), 7.63 (d, J = 8.68 Hz, 2H, Ar–H), 7.48 (dt, J = 6.63, 2.19 Hz, 1H, Ar–H), 7.36–7.26 (m, 2H, Ar–H), 6.99 (d, J = 9.90 Hz, 1 H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.84 (C̲ S), 160.64 (NH–C̲ O), 143.91 (C̲ N); 141.60, 140.68, 131.78 (2 × Ar–C̲), 130.95, 130.81, 130.59, 127.43, 126.34 (2 × Ar–C̲), 126.19, 123.78, 122.75, 121.38. FT (IR) ( max, cm⁻¹) 3270 and 3230 (amidic and thiourea NH stretching), 1649 , 1577 ( stretching). X-ray diffractogram (XRD) characteristic lines at 2θ = [9.2°, 10.0°, 14.5°, 17.0°, 18.5°, 19.0°, 20.7°, 22.0°, 24.5°, 25.2°, 26.0°, 27.2°, 28.7°, 33.0°, 35.8°, 37.2°, 40.0°, 41.6°, 46.8°]; space group = P2; lattice parameters: [a = 10.960(2) Å, b = 9.697(6) Å, c = 13.5885(8) Å, α = 90°, β = 98.644°, γ = 90°]; volume of unit cell (V) = 1428.3 (Å)³; crystalline structure: monoclinic.

4-Substituted/unsubstituted-N-((4-(6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)carbam-othioyl)benzamide (9a–d)

The appropriate freshly prepared benzoyl isothiocyanate derivative (7a–d) was added using a dropping funnel to compound 6 (0.47 g, 2.5 mmol) in freshly dried acetone with a few drops of DMF and continued stirring for 48 h at room temperature. The product was poured into 30 mL of cold water and stirred for 10 min. The precipitated solid was filtered and washed with diethyl ether, then 80% aqueous ethanol, and finally diluted HCl (10 mL), and then left to dry. The afforded compounds (9a–d) were further purified through ethanol crystallization.

N-((4-(6-Oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (9a)³⁵

The title compound was obtained as dark-yellow crystals (0.31 g, 35.22% yield); m.p. 238–240 °C, as reported.³⁵ M.F. C₁₈H₁₆ N₄O₂S; HRMS: calcd.; 352.0994; found: {m/z: [M + H]⁺; 353.1065}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 12.71 (s, 1H, NH), 11.59 (s, 1H, NH), 10.94 (s, 1H, NH), 7.99 (d, J = 7.46 Hz, 2H, Ar–H), 7.81 (s, 4H, Ar–H), 7.67 (t, J = 7.21 Hz, 1H, Ar–H), 7.55 (t, J = 7.52 Hz, 2H, Ar–H), 2.97 (t, J = 8.19 Hz, 2H, CH₂), 2.47 (t, J = 8.19 Hz, 2H, CH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.27 (C̲ S); 167.45 (NH–C̲ O); 149.26 (C̲ N); 140.62, 139.22, 134.11; 133.64, 132.58, 129.16, 128.93, 128.16, 126.46 (2 × Ar–C̲); 124.28, 120.34, 119.89 (Ar–C̲s); 26.19; 22.16 (Aliph. C̲s). FT (IR) ( max, cm⁻¹) 3346 and 3193 (amidic and thiourea NH stretching), 2921 (sp³-CH stretching), 1667 , 1591 ( stretching).

4-Chloro-N-((4-(6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (9b)

The title compound was separated as white crystals (0.45 g, 46.63% yield); m.p. 213–215 °C. M.F. C₁₈H₁₅ClN₄O₂S; HRMS: calcd.; 386.0604; found: {m/z: [M + H]⁺; 387.0680 (³⁵Cl) and 389.0654 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 12.62 (s, 1H, NH), 11.70 (s, 1H, NH), 10.94 (s, 1H, NH), 8.0 (d, J = 8.30 Hz, 2H, Ar–H), 7.80 (s, 4H, Ar–H), 7.61 (d, J = 8.30 Hz, 2H, Ar–H), 2.96 (t, J = 8.13 Hz, 2H, CH₂), 2.46 (t, J = 8.13 Hz, 2H, CH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.14 (C̲ S); 167.65, 167.44, (NH–C̲ O); 149.24 (C̲ N); 139.20, 138.48, 134.11, 131.47, 131.15 (2 × Ar–C̲); 129.00 (2 × Ar–C̲); 126.46 (2 × Ar–C̲); 124.26 (2 × Ar–C̲); 26.44, 22.27 (Aliph. C̲s). FT (IR) ( max, cm⁻¹) 3397 and 3336 (amidic and thiourea NH stretching), 1674 , 1589 ( stretching).

4-Bromo-N-((4-(6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (9c)

The title compound was separated as white crystals (0.75 g, 69.70% yield); m.p. 218–220 °C. M.F. C₁₈H₁₅BrN₄O₂S; HRMS: calcd.; 430.0099; found: {m/z: [M + H]⁺; 431.0177 (⁷⁹Br) and 433.0163 (⁸¹Br)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 12.61 (s, 1H, NH), 11.70 (s, 1H, NH), 10.95 (s, 1H, NH), 7.91 (d, J = 8.44 Hz, 2H, Ar–H), 7.79 (s, 4H, Ar–H), 7.75 (d, J = 8.31 Hz, 2H, Ar–H), 2.96 (t, J = 8.13 Hz, 2H, CH₂), 2.46 (t, J = 8.13 Hz, 2H, CH₂).¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.11 (C̲ S); 167.81, 167.43 (NH–C̲ O); 149.23 (C̲ N); 139.19, 134.12; 131.94 (2 × Ar–C̲); 131.81; 131.25 (2 × Ar–C̲); 127.60; 126.45 (2 × Ar–C̲) 124.26 (2 × Ar–C̲); 26.44, 22.28 (Aliph. C̲s). FT (IR) ( max, cm⁻¹) 3387 and 3336 (amidic and thiourea NH stretching), 1675 , 1585 ( stretching).

4-Nitro-N-((4-(6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (9d)

The title compound was separated as yellow crystals (0.45 g, 45.34% yield); m.p. 214–216 °C. M.F. C₁₈H₁₅N₅O₄S; HRMS: calcd.; 397.0845; found: {m/z: [M + H]⁺; 398.0921}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 12.49 (s, 1H, NH), 11.98 (s, 1H, NH), 10.95 (s, 1H, NH), 8.35 (d, J = 8.30 Hz, 2H, Ar–H), 8.17 (d, J = 8.20 Hz, 2H, Ar–H), 7.80 (s, 4H, Ar–H), 2.97 (t, J = 8.01 Hz, 2H, CH₂), 2.46 (t, J = 8.10 Hz, 2H, CH₂). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 178.92 (C̲ S); 167.44, 167.14 (NH–C̲ O); 150.28; 149.2 (C̲ N); 139.13, 138.53; 134.21; 130.73 (2 × Ar–C̲); 126.48 (2 × Ar–C̲); 124.28 (2 × Ar–C̲); 123.83 (2 × Ar–C̲); 26.43, 22.27. FT (IR) ( max, cm⁻¹) 3370 (NH stretching), 1674 , 1585 ( stretching).

Substituted/unsubstituted-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothio-yl)benzamide (10a–l)

Compounds 10a–l were prepared from compound 2 (0.28 g, 1.5 mmol) according to the same steps for the preparation of compounds 9a–d. The afforded compounds were further purified through crystallization using an ethyl acetate/hexane mixture except for compounds 10b, 10c and 10d, which were purified using flash chromatography (ethyl acetate : hexane; 6 : 4).

N-((4-(6-Oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10a)

The title compound was separated as yellow crystals (0.25 g, 47.71% yield); m.p. 242–244 °C. M.F. C₁₈H₁₄N₄O₂S; HRMS: calcd.; 350.0837; found: {m/z: [M + H]⁺; 351.0913}. Anal. calcd.: C, 61.70; H, 4.03; N, 15.99; S, 9.15; found: C, 61.94; H, 4.21; N, 16.25; S, 9.23. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.22 (s, 1H, NH); 12.73 (s, 1H, NH), 11.61 (s, 1H, NH), 8.06 (d, J = 9.78 Hz, 1H, Ar–H), 8.00 (d, J = 6.97 Hz, 2H, Ar–H), 7.81–7.95 (m, 4H, Ar–H), 7.66 (d, J = 6.36 Hz, 1H, Ar–H), 7.56 (d, J = 6.85 Hz, 2H, Ar–H), 7.01 (d, J = 9.66 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.27 (C̲ S); 168.69, 160.67 (NH–C̲ O); 143.66 (C̲ N); 139.19, 133.69, 132.83, 132.52, 131.82, 130.62; 129.18 (2 × Ar–C̲); 128.94, 128.74, 126.85, 126.44, 124.88, 120.36. FT (IR) ( max, cm⁻¹) 3434 (amidic NH stretching), 1676 and 1656 , 1597 ( stretching).

4-Chloro-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10b)

The title compound was separated as brown crystals (0.25 g, 43.47% yield); m.p. 207–209 °C. M.F. C₁₈H₁₃ClN₄O₂S; HRMS: calcd.: 384.0448; found: {m/z: [M + H]⁺; 385.0520 (³⁵Cl) 387.0516 (³⁷Cl)}. Anal. calcd.: C, 56.18; H, 3.40; N, 14.56; S, 8.33; found: C, 56.40; H, 3.63; N, 14.80; S, 8.45; ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.22 (s, 1H, NH); 12.62 (s, 1H, NH), 11.70 (s, 1H, NH), 8.05 (d, J = 9.90 Hz, 1H, Ar–H), 8.00 (d, J = 8.56 Hz, 2H, Ar–H), 7.91 (d, J = 8.60 Hz, 2H, Ar–H), 7.85 (d, J = 8.40 Hz, 2H, Ar–H), 7.62 (d, J = 8.44 Hz, 2H, Ar–H), 7.00 (d, J = 9.90 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 181.01 (C̲ S); 167.63, 160.66 (NH–C̲ O); 149.57; 143.10 (C̲ N); 139.70, 135.43, 133.89, 133.20; 131.16 (2 × Ar–C̲); 130.57, 130.16, 129.01, 128.97, 126.51, 126.42, 120.84. FT (IR) ( max, cm⁻¹) 3301 and 3200 (amidic NH and thiourea NH stretching), 1632 , 1590 ( stretching).

4-Bromo-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10c)

The title compound was separated as brownish-yellow crystals (0.31 g, 38.75% yield); m.p. 210–212 °C. M.F. C₁₈H₁₃BrN₄O₂S; HRMS: calcd.: 427.9943; found: {m/z: [M + H]⁺; 429.0018 (⁷⁹Br) and 430.9998 (⁸¹Br)}. Anal. calcd.: C, 50.36; H, 3.05; N, 13.05; S, 7.47; found: C, 50.59; H, 3.23; N, 13.29; S, 7.60. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.22 (s, 1H, NH), 12.61 (s, 1H, NH), 11.73 (s, 1H, NH), 8.05 (t, J = 10.80 Hz, 1H, Ar–H), 7.90 (dd, J = 14.40, 6.30, 3H, Ar–H), 7.83 (dd, J = 14.50, 8.50, 2H, Ar–H), 7.77 (d, J = 8.50 Hz, 2H, Ar–H), 7.67 (d, J = 8.50 Hz, 1H, Ar–H), 7.00 (t, J = 8.60 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.00 (C̲ S); 166.39, 158.38 (NH–C̲ O); 155.92; 143.71 (C̲ N); 139.15, 133.44, 132.75, 131.84; 131.30 (2 × Ar–C̲); 126.43 (2 × Ar–C̲), 124.73 (2 × Ar–C̲); 120.36, 117.65, 117.42. FT (IR) ( max, cm⁻¹) 3301 (amidic NH stretching), 1651 , 1587 ( stretching).

4-Nitro-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10d)

The title compound was obtained as yellow crystals (0.21 g, 35.53% yield); m.p. 200–202 °C. M.F. C₁₈H₁₃N₅O₄S; HRMS: calcd.: 395.0688; found: {m/z: [M + H]⁺; 396.0763}. Anal. calcd. C, 54.68; H, 3.31; N, 17.71; S, 8.11; found: C, 54.90; H, 3.47; N, 18.00; S, 8.23. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.22 (s, 1H, NH), 12.49 (s, 1H, NH), 11.99 (s, 1H, NH), 8.36 (d, J = 8.60 Hz, 2H, Ar–H), 8.18 (d, J = 7.09 Hz, 2H, Ar–H), 8.07 (d, J = 9.54 Hz, 1H, Ar–H), 7.93 (d, J = 7.58 Hz, 2H, Ar–H), 7.86 (d, J = 8.30 Hz, 1H, Ar–H), 7.81 (s, 1H, Ar–H), 7.01 (d, J = 9.17 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.02 (C̲ S); 167.12, 160.67 (NH–C̲ O); 158.28, 152.68, 150.30; 143.65 (C̲ N); 138.55, 134.81, 131.82; 130.74 (2 × Ar–C̲); 126.45 (2 × Ar–C̲); 124.77 (2 × Ar–C̲); 123.85. FT (IR) ( max, cm⁻¹) 3418, 3242, 3108 (amidic and thiourea NH stretching), 1678 , 1595 ( stretching).

2,4,6-Trichloro-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10e)

The title compound was obtained as yellow crystals (0.35 g, 51.62% yield); m.p. 212–214 °C. M.F. C₁₈H₁₁Cl₃N₄O₂S; HRMS: calcd.: 451.9668; found: {m/z: [M + H]⁺; 452.9744 (³⁵Cl) and 454.9720 (³⁷Cl)}. Anal. calcd.: C, 47.65; H, 2.44; Cl, 23.44; N, 12.35; S, 7.07; found: C, 47.92; H, 2.60; N, 12.59; S, 7.21. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.24 (s, 1H, NH), 12.36 (s, 1H, NH), 12.23 (s, 1H, NH), 8.08 (d, J = 9.90 Hz, 1H, Ar–H), 7.93 (d, J = 8.0 Hz, 2H, Ar–H), 7.86 (d, J = 8.30 Hz, 3H, Ar–H), 7.81 (s, 1H, Ar–H), 7.01 (d, J = 10.0 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 178.53 (C̲ S); 167.95, 160.68 (NH–C̲ O); 143.60 (C̲ N); 138.90, 136.20, 133.06; 132.39 (2 × Ar–C̲); 131.83 (2 × c); 130.64; 128.55 (2 × Ar–C̲); 126.43 (2 × Ar–C̲); 125.18 (2 × Ar–C̲). FT (IR) ( max, cm⁻¹) 3430 and 3200 (amidic and thiourea NH stretching), 1654 , 1601 ( stretching).

4-Fluoro-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10f)

The title compound was obtained as yellow crystals (0.5 g, 90.90% yield); m.p. 218–220 °C. M.F. C₁₈H₁₃FN₄O₂S; HRMS: calcd.; 368.0743; found: {m/z: [M + H]⁺; 369.0820}. Anal. calcd.: C, 58.69; H, 3.56; N, 15.21; S, 8.70; found: C, 58.90; H, 3.43; N, 15.49; S, 8.67. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.22 (s, 1H, NH), 12.66 (s, 1H, NH), 11.67 (s, 1H, NH), 8.08 (s, 3H, Ar–H), 7.92 (d, J = 8.10 Hz, 2H, Ar–H), 7.85 (d, J = 7.50 Hz, 2H, Ar–H), 7.39 (t, J = 8.19 Hz, 2H, Ar–H), 7.01 (d, J = 9.90 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.31 (C̲ S); 167.59, 166.68 (NH–C̲ O); 164.18, 160.65 (C̲–F); 143.66 (C̲ N); 139.25, 132.75, 132.26, 132.17, 131.81, 130.62; 129.13, 129.10, 126.42, 124.74, 116.10, 115.88. FT (IR) ( max, cm⁻¹) 3270 and 3200 (amidic and thiourea NH stretching), 1655 , 1580 ( stretching).

3-Fluoro-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10g)

The title compound was separated as yellow crystals (0.25 g, 45.45% yield); m.p. 235–237 °C. M.F. C₁₈H₁₃FN₄O₂S; HRMS: calcd.: 368.0743; found: {m/z: [M + H]⁺; 369.0818}. Anal. calcd.: C, 58.69; H, 3.56; F, 5.16; N, 15.21; S, 8.70; found: C, 58.87; H, 3.67; N, 15.43; S, 8.76. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.22 (s, 1H, NH), 12.60 (s, 1H, NH), 11.70 (s, 1H, NH), 8.06 (d, J = 9.90 Hz, 1H, Ar–H), 7.92 (d, J = 8.20 Hz, 2H, Ar–H), 7.85 (d, J = 7.10 Hz, Ar–H, 2H), 7.82 (d, J = 9.0 Hz, Ar–H, 2H) 7.60 (dd, J = 13.90, 7.40 Hz, 1H, Ar–H), 7.52 (t, J = 8.10 Hz, 1H, Ar–H), 7.0 (d, J = 9.8 Hz, Ar–H, 1H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.25 (C̲ S); 167.26, 163.28 (NH–C̲ O); 160.70 (C̲–F); 143.66 (C̲ N); 139.11, 134.91, 132.78, 131.80, 131.22, 130.70; 126.55, 125.39, 124.74, 120.57, 120.57, 116.10, 115.87. FT (IR) ( max, cm⁻¹) 3410 and 3214 (amidic and thiourea NH stretching); 1662 , 1597 ( stretching).

3-Bromo-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10h)

The title compound was separated as yellow crystals (0.23 g, 35.93% yield); m.p. 216–218 °C. M.F. C₁₈H₁₃BrN₄O₂S; HRMS: calcd.: 427.9943; found: {m/z: [M + H]⁺; 429.0023 (⁷⁹Br) and 430.9994 (⁸¹Br)}. Anal. calcd.: C, 50.36; H, 3.05; N, 13.05; S, 7.47; found: C, 50.57; H, 3.22; N, 13.29; S, 7.55. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.22 (s, 1H, NH), 12.57 (s, 1H, NH), 11.78 (s, 1H, NH), 8.17 (s, 1H, Ar–H), 8.06 (d, J = 9.6 Hz, 1H, Ar–H), 7.99–7.89 (m, 3H, Ar–H), 7.86 (s, 3H, Ar–H), 7.51 (s, 1H, Ar–H), 7.0 (d, J = 9.80 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.16 (C̲ S); 167.28, 160.65 (NH–C̲ O); 143.64 (C̲ N); 139.22, 136.15, 134.86, 132.78, 131.82, 131.77, 131.09, 130.64; 128.28 (2 × Ar–C̲); 126.44 (2 × Ar–C̲); 124.74, 121.96. FT (IR) ( max, cm⁻¹) 3370 and 3233 (amidic and thiourea NH stretching), 1656 , 1588 ( stretching). X-ray diffractogram (XRD); characteristic lines at 2θ = [7.2°, 12.0°, 14.2°, 18.2°, 19.5°, 22.5°, 24.0°, 25.0°, 26.5°, 29.2°, 30.1°, 32.0°, 38.0°, 53.7°]; space group = P2₁; lattice parameters: [a = 10.826(2) Å, b = 9.769(6) Å, c = 18.349(4) Å, α = 90°, β = 98.644°, γ = 90°]; volume of unit cell (V) = 1918.53 (Å)³; crystalline structure: monoclinic.

3-Chloro-4-fluoro-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10i)

The title compound was separated as yellow crystals (0.42 g, 70.0% yield); m.p. >300 °C. M.F. C₁₈H₁₂ClFN₄O₂S; HRMS: calcd.: 402.0354; found: {m/z: [M + H]⁺; 403.0427 (³⁵Cl) and 405.0406 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.20 (s, 1H, NH), 12.53 (s, 1H, NH), 11.76 (s, 1H, NH), 8.24 (d, J = 6.9 Hz, 1H, Ar–H), 8.05 (d, J = 9.29 Hz, 1H, Ar–H), 8.00 (s, 1H, Ar–H), 7.91 (d, J = 8.10 Hz, 1H, Ar–H), 7.84 (d, J = 8.20 Hz, 2H, Ar–H), 7.65–7.46 (m, 2H, Ar–H), 7.0 (d, J = 9.50 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.10 (C̲ S); 166.36, 160.70 (NH–C̲ O); 143.64 (C̲ N); 139.17, 136.15, 134.86, 132.78, 131.89, 131.82, 130.76, 130.66; 126.44 (2 × Ar–C̲); 124.73, 120.25, 117.67, 117.46. FT (IR) ( max, cm⁻¹) 3410 and 3282 (amidic and thiourea NH stretching), 1698 and 1679 , 1597 ( stretching).

4-Chloro-3-fluoro-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10j)

The title compound was obtained as dark-yellow crystals (0.39 g, 65% yield); m.p. 203–205 °C. M.F. C₁₈H₁₂ClFN₄O₂S; HRMS: calcd.: 402.0354; found: {m/z: [M + H]⁺; 403.0436 (³⁵Cl) and 405.0389 (³⁷Cl)}. Anal. calcd.: C, 53.67; H, 3.00; N, 13.91; S, 7.96; found: C, 53.51; H, 3.24; N, 14.15; S, 8.03. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.14 (s, 1H, NH), 10.49 (s, 1H, NH), 7.99 (t, J = 8.62 Hz, 2H, Ar–H), 7.90–7.85 (m, 3H, Ar–H), 7.83 (s, 1H, Ar–H), 7.75 (t, J = 7.64 Hz, 2H, Ar–H), 6.96 (d, J = 9.54 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 178.99 (C̲ S); 166.91, 161.27 (NH–C̲=O); 155.92 (F–C̲H); 143.74 (C̲ N); 139.15, 133.43, 133.39, 132.74, 131.85, 131.29, 130.64, 126.83, 126.42, 124.74, 120.36, 117.67, 117.40. FT (IR) ( max, cm⁻¹) 3351, 3270 and 3120 (amidic and thiourea NH stretching), 1686 and 1662 , 1596 ( stretching).

3,4-Dichloro-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10k)

The title compound was separated as yellow crystals (0.24 g, 38.27% yield); m.p. 202–204 °C. M.F. C₁₈H₁₂Cl₂N₄O₂S; HRMS: calcd.: 418.0058; found: {m/z: [M + H]⁺; 419.0135 (³⁵Cl) and 421.0106 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.22 (s, 1H, NH), 12.52 (s, 1H, NH), 11.83 (s, 1H, NH), 8.29–8.21 (m, 1H, Ar–H), 8.19 (s, 1H, Ar–H), 8.06 (d, J = 9.80 Hz, 1H, Ar–H), 7.92 (d, J = 6.0 Hz, 1H, Ar–H), 7.85 (dd, J = 15.70, 8.60 Hz, 3H, Ar–H), 7.78 (d, J = 8.40 Hz, 1H, Ar–H), 7.01 (d, J = 9.50 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 182.30 (C̲ S); 166.11, 160.66 (NH–C̲ O); 143.64 (C̲ N); 139.19, 136.13, 133.21, 131.81, 131.72, 131.66, 131.21, 131.17, 131.10, 130.63, 129.43, 129.34, 126.44, 124.75. FT (IR) ( max, cm⁻¹) 3400, 3225 and 3154 (amidic and thiourea NH stretching), 1682 and 1661 , 1587 ( stretching).

3-Chloro-N-((4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)carbamothioyl)benzamide (10l)

The title compound was obtained as yellow crystals (0.45 g, 78.29% yield); m.p. 232–234 °C. M.F. C₁₈H₁₃ClN₄O₂S; HRMS: calcd., 384.0448; found: {m/z: [M + H]⁺; 385.0522 (³⁵Cl) and 387.0500 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.23 (s, 1H, NH), 12.59 (s, 1H, NH), 11.77 (s, 1H, NH), 8.07 (s, 1H, Ar–H), 8.04 (d, J = 3.06 Hz, 1H, Ar–H), 7.92 (d, J = 8.80 Hz, 3H, Ar–H), 7.85 (d, J = 8.44 Hz, 2H, Ar–H), 7.73 (d, J = 7.21 Hz, 1H, Ar–H), 7.58 (t, J = 7.89 Hz, 1H, Ar–H), 7.01 (d, J = 9.9 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 179.15 (C̲ S); 167.29, 160.66 (NH–C̲ O); 143.64 (C̲ N); 139.21, 134.67, 133.58, 133.27, 132.78, 131.80 (2 × Ar–C̲); 130.85, 130.62, 128.95, 127.91, 126.43, 124.74 (2 × Ar–C̲). FT (IR) ( max, cm⁻¹) 3409, 3210 and 3100 (amidic and thiourea NH stretching), 1660 , 1596 ( stretching).

Substituted/unsubstituted-N-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)benzenesulfonamide (11a–f)

Under dry nitrogen, compound 2 (1.5 mmol, 0.28) was dissolved in dry pyridine (5 mL). The sulfonyl chloride derivative was added portion-wise (1.5 mmol), and the mixture was stirred at room temperature for 48 h. Diluted HCl (20 mL) was added to the mixture and continued stirring for about 15 min. The formed solid was collected by filtration, washed with ethanol (10 mL), then dried, and finally crystalized using THF.

3-Bromo-N-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)benzenesulfonamide (11a)

The title compound was obtained as yellow crystals (0.23 g, 37.70% yield); m.p. 265–267 °C. M.F. C₁₆H₁₂BrN₃O₃S; HRMS: calcd.: 404.9783, found: {m/z: [M + H]⁺; 405.9865 (⁷⁹Br) and 407.9849 (⁸¹Br)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.14 (s, 1H, NH), 10.65 (s, 1H, NH), 7.94 (d, J = 8.40 Hz, 2H, Ar–H), 7.84 (d, J = 7.95 Hz, 1H, Ar–H), 7.77 (t, J = 8.00 Hz, 3H, Ar–H), 7.53 (t, J = 8.0 Hz, 1H, Ar–H), 7.21 (d, J = 8.56 Hz, 2H, Ar–H), 6.95 (d, J = 9.90 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 160.60 (NH–C̲ O); 152.33; 143.56 (C̲ N); 141.77, 138.60, 136.43, 132.12, 131.64, 131.02, 130.56, 129.45, 127.19, 126.20, 122.65, 120.54 (2 × Ar–C̲). FT (IR) ( max, cm⁻¹) 3330, 3210 and 3094 (amidic and sulfonamide NH stretching), 1656 . X-ray diffractogram (XRD); characteristic lines at 2θ = [8.6°, 9.5°, 11.0°, 14.5°, 16.0°, 18.0°, 19.8°, 21.0°, 22.4°, 24.0°, 25.0°, 26.0°, 29.0°, 32.0°, 33.8°, 35.0°, 37.6°, 40.2°, 42.8°, 46.8°]; space group = Phca; lattice parameters: [a = 7.360(2) Å, b = 7.469(3) Å, c = 33.061(7) Å, α = 90°, β = 90°, γ = 90°]; volume of unit cell (V) = 1817.5 (Å)³; crystalline structure: orthorhombic.

3,4-Dichloro-N-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)benzenesulfonamide (11b)

The title compound was separated as yellow crystals (0.33 g, 55% yield); m.p. 226–228 °C. M.F. C₁₆H₁₁Cl₂N₃O₃S; HRMS: calcd.: 394.9898; found: {m/z: [M + H]⁺; 395.9984 (³⁵Cl) and 397.9956 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.15 (s, 1H, NH), 10.72 (s, 1H, NH), 8.02–7.91 (m, 2H, Ar–H), 7.84 (d, J = 8.10 Hz, 1H, Ar–H), 7.74 (d, J = 8.20, 1H, Ar–H), 7.72 (d, J = 7.20 Hz, 1H, Ar–H), 7.68–7.53 (m, 1H, Ar–H), 7.28–7.14 (m, 2H, Ar–H), 6.95 (d, J = 9.80 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 160.59 (NH–C̲ O); 143.51 (C̲ N); 140.04, 138.34, 136.71, 132.72, 132.31, 131.64, 131.22, 130.56; 128.85 (2 × Ar–C̲); 127.23 (2 × Ar–C̲); 120.78 (2 × Ar–C̲). FT (IR) ( max, cm⁻¹) 3220 and 3086 (amidic and sulfonamide NH stretching), 1658 .

3-Fluoro-N-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)benzenesulfonamide (11c)

The title compound was separated as yellow crystals (0.38 g, 73.64% yield); m.p. 230–232 °C. M.F. C₁₆H₁₂FN₃O₃S; HRMS: calcd., 345.0583; found: {m/z: [M + H]⁺; 346.0666}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.13 (s, 1H, NH), 7.93 (d, J = 10.03 Hz, 1H, Ar–H), 7.75 (d, J = 8.80 Hz, 2H, Ar–H), 7.67–7.61 (m, 2H, Ar–H), 7.61–7.55 (m, 1H, Ar–H), 7.54–7.44 (m, 1H, Ar–H), 7.21 (d, J = 8.80 Hz, 2H, Ar–H), 6.95 (d, J = 9.90 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 160.60 (NH–C̲ O); 143.56 (C̲ N); 141.81, 138.66, 132.36, 132.28, 131.64, 130.95, 130.55, 127.16, 123.46, 120.90, 120.69, 120.48, 114.28, 114.03. FT (IR) ( max, cm⁻¹) 3210 and 3094 (amidic and sulfonamide NH stretching), 1656 .

3-Chloro-N-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)benzenesulfonamide (11d)

The title compound was separated as yellow crystals (0.39 g, 72.23% yield); m.p. 235–237 °C. M.F. C₁₆H₁₂ClN₃O₃S; HRMS: calcd.: 361.0288; found: {m/z: [M + H]⁺; 362.0368 (³⁵Cl) and 364.0346 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.13 (s, 1H, NH), 10.67 (s, 1H, NH), 7.93 (s, 1H, Ar–H), 7.80 (s, 2H, Ar–H), 7.74 (s, 3H, Ar–H), 7.60 (s, 1H, Ar–H), 7.21 (br, s, 2H, Ar–H), 6.95 (s, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 160.60 (NH–C̲ O); 143.54 (C̲ N); 141.65, 138.58 (2 × Ar–C̲); 134.38, 133.59, 131.97, 131.66, 131.01, 130.57, 127.19 (2 × Ar–C̲); 126.66, 125.88, 120.53. FT (IR) ( max, cm⁻¹) 3270 and 3076 (amidic and sulfonamide NH stretching), 1655 .

3-Chloro-4-fluoro-N-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)benzenesulfona-mide (11e)

The title compound was separated as yellow crystals (0.42 g, 74% yield); m.p. 280–282 °C. M.F. C₁₆H₁₁ClFN₃O₃S; HRMS: calcd.: 379.0194; found: {m/z: [M + H]⁺; 380.0272 (³⁵Cl) and 382.0227 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.16 (s, 1H, NH), 10.68 (s, 1H, NH), 7.97 (dd, J = 6.70, 2.20 Hz, 1H, Ar–H), 7.94 (d, J = 9.90 Hz, 1H, Ar–H), 7.82–7.78 (m, 1H, Ar–H), 7.76 (d, J = 8.60 Hz, 2H, Ar–H), 7.64–7.59 (m, 1H, Ar–H), 7.22 (t, J = 7.80 Hz, 2H, Ar–H), 6.95 (d, J = 9.90 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 160.67 (NH–C̲ O); 143.63 (C̲ N); 138.44, 137.22, 131.70, 131.12, 130.55, 129.67, 128.69, 128.60, 127.22, 121.45, 121.26, 120.71, 118.80, 118.58. FT (IR) ( max, cm⁻¹) 3230 and 3095 (amidic and sulfonamide NH stretching), 1658 .

4-Chloro-N-(4-(6-oxo-1,6-dihydropyridazin-3-yl)phenyl)benzenesulfonamide (11f)

The title compound was separated as yellow crystals (0.40 g, 74% yield); m.p. 217–220 °C. M.F. C₁₆H₁₂ClN₃O₃S; HRMS: calcd.: 361.0288; found: {m/z: [M + H]⁺; 362.0376 (³⁵Cl) and 364.0352 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.13 (s, 1H, NH), 10.63 (s, 1H, NH), 7.93 (d, J = 9.90 Hz, 1H, Ar–H), 7.79 (d, J = 8.40 Hz, 2H, Ar–H), 7.74 (d, J = 8.50 Hz, 1H, Ar–H), 7.69–7.57 (m, 3H, Ar–H), 7.27–7.11 (m, 2H, Ar–H), 6.95 (d, J = 9.90 Hz, 1H, Ar–H). ¹³C NMR (100 MHz, DMSO-d₆, δ = ppm) 160.60 (NH–C̲ O) 143.57 (C̲ N); 138.74, 138.66, 138.34, 131.64, 130.88; 130.55 (2 × Ar–C̲); 130.13, 130.01; 129.07 (2 × Ar–C̲); 127.14; 120.45 (2 × Ar–C̲). FT (IR) ( max, cm⁻¹) 3270 and 3088 (amidic and sulfonamide NH stretching), 1652 .

(E)-Ethyl 6-(4-nitrophenyl)-4-oxohex-5-enoate (12)

Intermediate 12 was prepared according to the reported procedure^47,48 (m.p. 104–106 °C, as reported).

Other method assisted by microwave radiation for the preparation of intermediate (12)

In a dry rounded flask, p-nitrobenzaldehyde (0.01 mol, 1.51 g) was dissolved in (10 mL) toluene containing glacial acetic acid (0.7 mL). Levulinic acid ethyl ester (0.02 mol, 2.92 mL) was added to the solution followed by piperidine (5 drops). The mixture was subjected to continuous automatic stirring for 10 min, while being exposed to microwave radiation of 200–400 W at 120 °C. The reaction was followed step by step using TLC until complete disappearance of the aldehyde. The solvent was evaporated in vacuo, cooled and washed twice with 2 M HCl. The oily product was carefully extracted with ethyl acetate, dried over anhydrous magnesium sulfate and recrystallized using aqueous ethanol to obtain yellow crystals of intermediate 12 (4.21 g, 50.66% yield); m.p. 104–106 °C. This method did not require any purification of the product in comparison to the former method. M.F.: C₁₄H₁₅NO₅; HRMS: calcd.: 277.0950; found: {m/z: [M + H]⁺; 278.1022}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 8.26 (d, J = 8.19 Hz, 2H, Ar–H), 8.00 (d, J = 8.19 Hz, 2H, Ar–H), 7.74 (d, J = 16.38 Hz, 1H, C–H̲), 7.10 (d, J = 16.38 Hz, 1H, C–H̲), 4.06 (q, J = 6.77 Hz, 2H, CH₂), 3.02 (t, J = 5.90 Hz, 2H, CH₂), 2.59 (t, J = 5.87 Hz, 2H, CH₂), 1.18 (t, J = 6.91 Hz, 3H, CH₃). FT (IR) ( max, cm⁻¹); 3100 (sp² stretching), 1716 , 1689 , 1509 and 1339 .

(E)-6-(4-Nitrophenyl)-4-oxohex-5-enoic acid (13)

Intermediate 13 was prepared according to the reported procedure^47,48 (m.p. 169–171 °C, as reported); M.F.: C₁₂H₁₁NO₅; HRMS: calcd.: 249.0637; found: {m/z: [M + H]⁺; 250.0707}.

(E)-6-(4-Nitrostyryl)-4,5-dihydropyridazin-3(2H)-one (14)

Hydrazine hydrate 80% (0.15 mol, 7.40 mL) was slowly added to a solution of intermediate 12 or its ester 13 (0.015 mol, 4.16 g or 3.74 g, respectively) in acetic acid (20 mL). The reaction mixture was refluxed for 6 h. The solvent was removed under reduced pressure. The residue was triturated with cold distilled water (20 mL) and filtered after 10 min to obtain pure yellow crystals of intermediate 14 (2.70 g, 42.58% and 3.21 g, 56.22% yield, respectively); m.p. 240–242 °C; M.F.: C₁₂H₁₁N₃O₃; HRMS: calcd.: 245.0800; found: {m/z: [M + H]⁺; 246.0874}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 10.98 (s, 1H, NH), 8.18 (d, J = 8.60 Hz, 2H, Ar–H), 7.83 (d, J = 8.30 Hz, 2H, Ar–H), 7.16 (d, J = 17.80 Hz, 1H, C–H̲), 7.08 (d, J = 17.50 Hz, 1H, C–H̲), 2.79 (t, J = 8.20 Hz, 2H, CH₂), 2.40 (t, J = 8.0 Hz, 2H, CH₂). FT (IR) ( max, cm⁻¹); 3206 (NH stretching), 1671 , 1513 and 1342 .

(E)-6-(4-Nitrostyryl)pyridazin-3(2H)-one (15)

A mixture of intermediate 14 (0.01 mol, 2.45 g) and CuCl₂·2H₂O (0.02 mol, 3.41 g) was stirred in DMSO (50 mL) at 110 °C for 3 h. The completion of the reaction was monitored by TLC. The resultant mixture was poured onto crushed ice containing distilled water (20 mL). The precipitated compound was filtered and washed with aqueous ethanol and purified using flash chromatography (ethyl acetate : hexane; 6 : 4) to obtain pure yellow crystals of intermediate XV (1.80 g, 74.07% yield); m.p. 165–167 °C; M.F.: C₁₂H₉N₃O₃; HRMS: calcd.: 243.0644; found: {m/z: [M + H]⁺; 244.0720}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.22 (s, 1H, NH), 8.23 (d, J = 8.30 Hz, 2H, Ar–H), 7.98 (d, J = 9.78 Hz, 1H, Ar–H), 7.89 (d, J = 8.30 Hz, 2H, Ar–H), 7.52 (d, J = 16.63 Hz, 1H, C–H), 7.30 (d, J = 16.63 Hz, 1H, C–H), 6.96 (d, J = 9.78 Hz, 1H, Ar–H). FT (IR) ( max, cm⁻¹); 3442 (NH stretching), 3101 and 3046 (sp² CH stretching), 1682 , 1502 and 1347 .

(E)-6-(4-Aminostyryl)pyridazin-3(2H)-one (16)

Intermediate 15 (0.015 mol, 3.65 g) was heated in a mixture of butanol (100 mL) and water (30 mL). Iron metal (0.18 mol, 10.05 g) and 2 N HCl (2.5 mL) were added sequentially to the hot mixture. The reaction was monitored by TLC until a new spot of the corresponding amine intermediate (16) was formed. After 3 h of reflux, the suspension was cooled to room temperature and the resultant product was filtered and washed with hot butanol to remove the exhausted iron. The filtrate was concentrated under vacuum, the residual solid was diluted with ethyl acetate and washed with 1 N HCl, and then with aqueous NaHCO₃ until basic pH = 7–8. The organic layer was dried over anhydrous magnesium sulphate and concentrated under reduced pressure. The residual product was used directly in the following step without further purification.

(E)-1-(Substitutedphenyl)-3-(4-(2-(6-oxo-1,6-dihydropyridazin-3-yl)vinyl)phenyl)thiourea (17a and 17b)

General procedure

Compounds 17a and 17b were prepared according to the same procedure for the preparation of compounds 8a–j, which were further purified using ethanol.

(E)-1-(3-Bromophenyl)-3-(4-(2-(6-oxo-1,6-dihydropyridazin-3-yl)vinyl)phenyl)thiourea (17a)

The title compound was obtained as brownish yellow crystals (0.14 g, 30.37% yield); m.p. 175–177 °C. M.F.: C₁₉H₁₅BrN₄OS; HRMS: calcd.: 426.0150; found: {m/z: [M + H]⁺; 427.0221 (⁷⁹Br) and 429.0215 (⁸¹Br)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 12.93 (s, 1H, NH), 10.44 (s, 1H, NH), 7.85 (s, 2H, Ar–H), 7.53 (s, 4H, Ar–H), 7.23 (s, 4H, Ar–H and C–H̲), 6.96 (s, 1H, Ar–H), 6.85 (s, 1H, C–H). FT (IR) ( max, cm⁻¹) 3423 and 3231 (amidic and thiourea NH stretching), 1652 , 1585 ( stretching).

(E)-1-(3,4-Dichlorophenyl)-3-(4-(2-(6-oxo-1,6-dihydropyridazin-3-yl)vinyl)phenyl)thiourea (17b)

The title compound was obtained as brown crystals (0.22 g, 53.53% yield); m.p. 200–202 °C. M.F.: C₁₉H₁₄Cl₂N₄OS; HRMS: calcd.: 416.0265; found: {m/z: [M + H]⁺; 417.0335 (³⁵Cl) and 418.9624 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.01 (s, 1H, NH), 10.92–10.29 (m, 1H, NH), 8.05–7.80 (m, 2H, Ar–H), 7.64 (d, J = 6.10 Hz, 1H, Ar–H), 7.57 (d, J = 7.40 Hz, 1H, Ar–H), 7.54–7.41 (m, 2H, Ar–H), 7.37 (d, J = 15.0 Hz, 1H, C–H̲), 7.29 (d, J = 18.50 Hz, 1H, C–H̲), 7.19 (d, J = 7.70 Hz, 1H, Ar–H), 7.09–6.96 (m, 1H, Ar–H), 6.91 (d, J = 7.90 Hz, 1H, Ar–H). FT (IR) ( max, cm⁻¹) 3366, 3233 and 3093 (amidic and thiourea NH stretching), 1651 , 1585 ( stretching).

(E)-Substituted-N-((4-(2-(6-oxo-1,6-dihydropyridazin-3-yl)vinyl)phenyl)carbamothioyl)benzamide (18a and 18b)

General procedure

Compounds 18a and 18b were prepared according to the same procedures for the preparation of compounds 9a–d or 10a–l, which were further purified using an ethyl acetate/hexane mixture.

(E)-3-Bromo-N-((4-(2-(6-oxo-1,6-dihydropyridazin-3-yl)vinyl)phenyl)carbamothioyl)benzamide (18a)

The title compound was obtained as buff crystals (0.19 g, 47% yield); m.p. 220–222 °C. M.F.: C₂₀H₁₅BrN₄O₂S; HRMS: calcd.: 454.0099; found: {m/z: [M + H]⁺; 455.0174 (⁷⁹Br) and 456.9701 (⁸¹Br)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.05 (s, 1H, NH), 12.54 (s, 1H, NH), 11.73 (s, 1H, NH), 8.16 (s, 1H, Ar–H), 8.04–7.60 (m, 7H, Ar–H), 7.49 (s, 1H, Ar–H), 7.37 (d, J = 13.7 Hz, 1H, C–H̲), 7.07 (d, J = 16.50 Hz, 1H, C–H̲), 6.93 (d, J = 9.0 Hz, 1H, Ar–H). FT (IR) ( max, cm⁻¹) 3417, 3266 and 3151 (amidic and thiourea N̲H̲ stretching), 1674 and 1655 , 1585 ( stretching).

(E)-3,4-Dichloro-N-((4-(2-(6-oxo-1,6-dihydropyridazin-3-yl)vinyl)phenyl)carbamothioyl)benzamide (18b)

The title compound was obtained as brown crystals (0.20 g, 45.66% yield); m.p. 207–209 °C. M.F.: C₂₀H₁₄Cl₂N₄O₂S; HRMS: calcd.: 444.0215; found: {m/z: [M + H]⁺; 445.0287 (³⁵Cl) and 447.0263 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 12.85 (s, 1H, NH), 8.24 (d, J = 8.50 Hz, 1H, Ar–H), 8.01–7.81 (m, 2H, Ar–H), 7.64–7.47 (m, 1H, Ar–H), 7.36–7.24 (m, 2H, Ar–H), 7.16 (d, J = 16.50 Hz, 1H, C–H̲), 6.86 (d, J = 9.70 Hz, 1H, Ar–H). 6.71 (d, J = 16.50 Hz, 1H, C–H̲), 6.57 (d, J = 8.20 Hz, 2H, Ar–H). FT (IR) ( max, cm⁻¹) 3341 and 3230 (amidic and thiourea NH stretching), 1671 and 1651 , 1587 ( stretching).

(E)-(Substituted)-N-(4-(2-(6-oxo-1,6-dihydropyridazin-3-yl)vinyl)phenyl)benzenesulfonamide (19a and 19b)

General procedure

Compounds 19a and 19b were prepared according to the same procedure for the preparation of compounds 11a–f, which were further purified using methanol.

(E)-3-Bromo-N-(4-(2-(6-oxo-1,6-dihydropyridazin-3-yl)vinyl)phenyl)benzenesulfonamide (19a)

The title compound was obtained as yellow crystals (0.11 g, 28.57% yield); m.p. 264–266 °C. M.F.: C₁₈H₁₄BrN₃O₃S; HRMS: calcd.: 430.9939; found: {m/z: [M + H]⁺; 432.0013 (⁷⁹Br) and 433.9980 (⁸¹Br)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.01 (s, 1H, NH), 10.59 (s, 1H, NH), 7.91 (s, 1H, Ar–H), 7.88 (d, J = 9.90 Hz, 1H, Ar–H), 7.84 (d, J = 7.82 Hz, 1H, Ar–H), 7.76 (d, J = 7.58 Hz, 1H, Ar–H), 7.52 (d, J = 7.70 Hz, 3H, Ar–H), 7.25 (d, J = 16.51 Hz, 1H, C–H̲), 7.13 (d, J = 8.19 Hz, 2H, Ar–H), 6.95 (d, J = 16.75 Hz, 1H, C–H̲), 6.90 (d, J = 9.90 Hz, 1H, Ar–H). FT (IR) ( max, cm⁻¹) 3441 and 3265 (amidic and sulfonamide NH stretching), 1678 .

(E)-3,4-Dichloro-N-(4-(2-(6-oxo-1,6-dihydropyridazin-3-yl)vinyl)phenyl)benzenesulfonamide (19b)

The title compound was obtained as yellow crystals (0.13 g, 31.25% yield); m.p. 245–247 °C. M.F.: C₁₈H₁₃Cl₂N₃O₃S; HRMS: calcd.: 421.0055; found: {m/z: [M + H]⁺; 422.0128 (³⁵Cl) and 423.9671 (³⁷Cl)}. ¹H NMR (400 MHz, DMSO-d₆, δ = ppm) 13.02 (s, 1H, NH), 10.63 (s, 1H, NH), 7.95 (s, 1H, Ar–H), 7.86 (dd, J = 14.50, 9.20 Hz, 2H, Ar–H), 7.70 (d, J = 8.50 Hz, 1H, Ar–H), 7.52 (d, J = 8.30 Hz, 2H, Ar–H), 7.24 (d, J = 16.60 Hz, 1H, C–H̲), 7.13 (d, J = 8.20 Hz, 2H, Ar–H), 7.03–6.85 (m, 2H, Ar–H and C–H̲). FT (IR) ( max, cm⁻¹); 3300 (amidic NH stretching), 1652 .

5.1.3. X-ray powder diffraction (XRD) study

The X-ray diffraction patterns in this work were recorded using a JEOL model JSDX-60PA materials research X-ray diffractometer. This diffractometer can analyze powder samples at room temperature using Cu, K_α-radiation (λ = 0.154184 nm), Ni-filtered; 35 kV, 30 mA. Continuous scanning was applied at a slow scanning speed (1° min⁻¹) and a small time constant (one second) for the 2θ range of 5° to 80°. The samples were mounted on a slide and exposed to an X-ray beam. All the samples were run at room temperature. The idea of XRD relies on Bragg's law and this is applied to all planes. When an X-ray beam with wavelength λ is shined onto a crystalline material, diffraction only occurs when the difference in the distance traveled by the rays, diffracted from tandem planes, n times the wavelength, λ, of the incident beam according to Bragg's law, as shown by the following formula:where λ is the wavelength of the X-ray beam, d is the spacing between the planes in the atomic lattice and θ is the angle between the incident ray and the diffracting planes. The diffraction peaks were assigned to the corresponding crystal planes by comparing the calculated experimental d-spacing with the diffraction pattern and the standard d-spacing PDF card, which the crystal structure system, Miller indices (hkl) and lattice parameters of the compound phase.⁴⁹

5.2. Biological evaluation

5.2.1. Evaluation of the antimicrobial activity

a. Antimicrobial susceptibility testing (AST)

Antimicrobial susceptibility testing was primarily done for the tested compounds by a modified Kirby–Bauer method using sterile discs loaded with 3 μL containing 380 μg of the tested compound. Also, AST was determined by the broth microdilution method to determine the MIC values.

b. Modified Kirby–Bauer method (disc diffusion)

General procedure

A sterile cotton swab was dipped into the bacterial suspension (adjusted to 0.5 McFarland) of the tested isolate and the excess suspension was removed by pressing the cotton swab against the wall of the test tube containing the bacterial suspension. The swab was streaked all over the surface of the Mueller–Hinton agar (MHA) plate three times by rotating the plate at an angle of 60° after each application. Finally, the swab was passed around the edge of the agar surface. The inoculated MHA plates were left to dry for a few minutes at room temperature with the lid closed. The loaded discs with the tested compounds were placed on the inoculated plates using sterile forceps. Each disc was gently pressed down to ensure even contact with the medium. Subsequently, the plates were incubated overnight at 37 °C. The next day, the diameter of the inhibition zones (including the diameter of the discs) was measured with a ruler under the surface of the plate and the results were recorded in mm.⁵⁰

c. Broth micro dilution method

General procedure

The broth micro dilution method was performed in 96-well microtitre plates using different concentrations of the investigated compounds. All columns of the sterile 96-well plates were filled with 100 μL of sterile distilled water (SDW) except column no. 1, which was filled with 159 μL of sterile distilled water, and then 41 μL of the tested compound stock solution was added to obtain a final concentration of 1024 μg/200 μL after addition of the broth containing the tested organism. The last two rows of the 96-well plates were used only as the positive and negative controls, respectively. Subsequently, 100 μL from the tested compound stock solutions or suspensions in column No.1 was withdrawn and added to the next well (containing 100 μL of SDW) and mixed by pipetting 3–6 times, and then 100 μL from the third well was withdrawn and added to the 4th well and mixed well by pipetting 3–6 times. This process was repeated until reaching well no. 12, in which the last 100 μL withdrawn was discarded. After serial dilution of the tested compounds, all the wells of the plate were filled with 100 μL of DSNB (containing the tested isolate) and mixed well by pipetting 3–6 times. The plates were incubated at 37 °C for 18 h.⁵¹

Preparation of compound stock solution

The stock solution was prepared by dissolving 10 mg of the tested compound in 1 mL of DMSO to obtain an initial stock concentration of 10 mg mL⁻¹.⁵¹

Preparation of first concentration for further two-fold dilution

The first desired concentration was achieved using the following equation: C₁V₁ = C₂V₂, where C₁ represents the original tested compound stock concentration, V₁ represents the volume required from the original stock concentration to obtain the desired concentration, C₂ represents the desired concentration and V₂ represents the volume containing the desired concentration. For example, when the first desired concentration is 1024 μg/200 μL, the equation will be as follows: 10 000 μg × V₁ = 1024 × 200 μL, and thus the desired volume was 20.48 μL, this volume was doubled to overcome the dilution in the first well by DSNB, and 40.96 μL from the antibiotic stock solution was completed to 200 μL by adding approximately 159 μL of SDW.

Inoculum preparation (direct colony suspension method)

The direct colony suspension method was performed by suspending a few colonies of tested isolate in sterile normal saline. The prepared suspension was adjusted to achieve turbidity equivalent to 0.5 McFarland standard, which is approximately equivalent to 1 to 2 × 10⁸ CFU mL⁻¹, and further diluted to obtain a final inoculum concentration of 5 × 10⁵ CFU mL⁻¹ in each well of a microtitre plate. The dilution was done by adding 35 μL from the 0.5 McFarland adjusted inoculum to 35 mL of double-strength nutrient broth (DSNB). The inoculated broth was used directly within 15 min of preparation.⁵²

5.2.2. Evaluation of the antiproliferative activity

a. Measurement of inhibitory activity against VEGFR-2

The kinase activity of VEGFR-2 was measured using a phosphotyrosine antibody with the AlphaScreen system (PerkinElmer, United States) according to the manufacturer's instructions. Enzyme reactions were performed in 50 mM Tris–HCl pH 7.5, 5 mM MnCl₂, 5 mM MgCl₂, 0.01% Tween 20, and 2 mM DTT, containing 10 μM ATP, 0.1 μg mL⁻¹ biotinylated poly-GluTyr (4 : 1), and 0.1 nM of VEGFR-2 (Millipore, United Kingdom). Before catalytic initiation with ATP, the tested compounds at final concentrations ranging from 0 to 100 μg mL⁻¹ and the enzyme were incubated for 5 min at room temperature. The reactions were quenched by the addition of 25 μL of 100 mM EDTA, 10 μg mL⁻¹ AlphaScreen streptavidin donor beads, and 10 μg mL⁻¹ acceptor beads in 62.5 mM HEPES pH 7.4, 250 mM NaCl, and 0.1% BSA. The plate was incubated in the dark overnight, and then read using an ELISA Reader (PerkinElmer, United States). The wells containing the substrate and the enzyme without compounds were used as the reaction control. The wells containing biotinylated poly-GluTyr (4 : 1) and enzymes without ATP were used as the basal control. Percent inhibition was calculated by comparing the treatment compounds to the control incubations.

b. In vitro single- and five-dose screening of anticancer activity in full NCI 60-cell line panel

The cytotoxicity assay of the synthesized compounds was studied using high-efficiency biological screening according to the Developmental Therapeutic Program (DTP) of the USA National Institutes of Health, National Cancer Institute (NCI) (Bethesda, Maryland, USA).^53–55 The tested compounds were tested at a single dose (10 μM) in the full NCI 60-cell panel. The operation of this screening utilizes 60 different human tumor cell lines, representing leukemia, melanoma, and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. The results for each compound are reported as a mean graph of the percent growth of the treated cells compared to that of the untreated control cells. The percentage of growth was evaluated spectrophotometrically versus the control, which was not treated with the test agents. The compounds that exhibit significant growth inhibition in the one-dose screen were evaluated against the 60-cell panel at five concentration levels. Compounds 10l and 17a, which gave the maximum activity in the one-dose assay were selected for the five-dose assay. The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% foetal bovine serum and 2 mM l-glutamine. In a typical screening experiment, the cells were inoculated in 96-well microtiter plates in 100 μL at plating densities in the range of 5000 to 40 000 cells per well depending on the doubling time of the individual cell lines. After cell inoculation, the microtiter plates were incubated at 37 °C, 5% CO₂, 95% air, and 100% relative humidity for 24 h prior to the addition of the experimental drugs. After 24 h, two plates of each cell line were fixed in situ with trichloroacetic acid (TCA) to represent the measurement of the cell population for each cell line at the time of added the drug (Tz). The experimental drugs were solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen before use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg mL⁻¹ gentamicin. Additional four-, 10-fold or 1/2 log serial dilutions were made to provide a total of five drug concentrations plus the control. Aliquots of 100 μL of these different drug dilutions were added to the appropriate microtiter wells containing 100 μL of medium, resulting in the required final drug concentrations. Triplicate wells were prepared for each dose. Following the addition of the drug, the plates were incubated for an additional 48 h at 37 °C, 5% CO₂, 95% air, and 100% relative humidity. In the case of adherent cells, the assay was terminated by the addition of cold TCA. The cells were fixed in situ by the gentle addition of 50 μL of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4 °C. The supernatant was discarded, and the plates were washed five times with tap water and air dried. SRB solution (100 μL) at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated for 10 min at room temperature. After staining, the unbound dye was removed by washing five times with 1% acetic acid and the plates were air dried. Subsequently, the bound stain was solubilized with 10 mM Trizma base, and the absorbance was read on an automated plate reader at a wavelength of 515 nm. In the case of suspension cells, the methodology was the same except that the assay was terminated by fixing the settled cells at the bottom of the wells by gently adding 50 μL of 80% TCA (final concentration, 16% TCA). Using the seven absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of the drug at the five concentration levels (Ti)], the percentage growth was calculated at each of the drug concentration levels, as follows:

- [(Ti − Tz)/(C − Tz)] × 100 for concentrations for which Ti ≥ Tz.

- [(Ti − Tz)/Tz] × 100 for concentrations for which Ti < Tz.

Three dose response parameters were calculated for each experimental compound, as follows: GI of 50% (GI₅₀) was calculated when [(Ti − Tz)/(C − Tz)] × 100 = 50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in the control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) was calculated using Ti = Tz. The LC₅₀ (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment compared to that at the beginning) indicating a net loss of cells following treatment was calculated as follows: [(Ti − Tz)/Tz] × 100 = −50. The values were calculated for each of these three parameters if the level of activity was reached; however, if the effect was not reached or exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested.

5.2.3. Gene expression analysis

RNA isolation and reverse transcription (RT) reaction

The gene expression analysis was performed at the National Institute for Research, Cairo, Egypt. The RNeasy Mini Kit (Qiagen, Hilden, Germany) supplemented with the DNase I (Qiagen) digestion step was used to isolate the total RNA from the lung cancer cell lines according to the manufacturer's protocol. The isolated total RNA was treated with one unit of RQ1 RNAse-free DNAse (Invitrogen, Germany) to digest the DNA residues, re-suspended in DEPC-treated water and quantified photospectrometrically at 260 nm. The purity of total RNA was assessed by the 260/280 nm ratio, which was between 1.8 and 2.1.⁵⁶ Additionally, integrity was assured with ethidium bromide-stain analysis of the 28S and 18S bands by formaldehyde-containing agarose gel electrophoresis. Aliquots were used immediately for reverse transcription (RT), otherwise they were stored at −80 °C. Complete Poly(A)⁺ RNA isolated from the lung cancer cell lines was reverse transcribed into cDNA in a total volume of 20 μL using RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Germany). A certain amount of total RNA (5 μg) was used with a master mix. The master mix consisted of 50 mM MgCl₂, 10× RT buffer (50 mM KCl; 10 mM Tris-HCl; pH 8.3), 10 mM of each dNTP, 50 μM oligo-dT primer, 20 IU ribonuclease inhibitor (50 kDa recombinant enzyme to inhibit RNase activity) and 50 IU MuLV reverse transcriptase. The mixture of each sample was centrifuged for 30 s at 1000 g and transferred to a thermocycler. The RT reaction was carried out at 25 °C for 10 min, followed by 1 h at 42 °C, and finished with a denaturation step at 99 °C for 5 min. Afterwards, the reaction tubes containing RT preparations were flash-cooled in an ice chamber until use for cDNA amplification through quantitative real-time-polymerase chain reaction (qRT-PCR).

Quantitative real-time-PCR (qRT-PCR)

Determination of the lung cancer cell line cDNA copy number was carried out using the StepOne™ Real-Time PCR System from Applied Biosystems (Thermo Fisher Scientific, Waltham, MA USA). PCR reactions were set up in 25 mL reaction mixtures containing 12.5 mL 1× SYBR® Premix Ex TaqTM (TaKaRa, Biotech. Co. Ltd.), 0.5 mL 0.2 mM sense primer, 0.5 mL 0.2 mM antisense primer, 6.5 mL distilled water, and 5 mL of cDNA template. The reaction program was allocated to 3 steps. The first step was at 95.0 °C for 3 min. The second step consisted of 40 cycles in which each cycle was divided into 3 steps, as follows: (a) at 95.0 °C for 15 s; (b) at 55.0 °C for 30 s; and (c) at 72.0 °C for 30 s. The third step consisted of 71 cycles, which started at 60.0 °C, and then increased about 0.5 °C every 10 s up to 95.0 °C. Each experiment included a distilled water control. The sequences of specific primers of the lung cancer cell lines were determined (Bcl-2, Bax, and p53 genes).^57,58 At the end of each qPCR, a melting curve analysis was performed at 95.0 °C to check the quality of the used primers. The relative quantification of the target to the reference was determined by using the 2^−ΔΔCT method.^59,60 Cancer-related genes were designed and provided in the ESI† (Table S7).

5.2.4. Cell cycle analysis

The cell cycle arrest of compound 10l against A549/ATCC at its GI₅₀ concentration was carried out according to the Abcam method (code ab139418), (https://www.abcam.co.jp/). Thus, the A549/ATCC cell line was collected using 75% ice-cold ethanol and kept at −20 °C for 1 h following treatment with the GI₅₀ dose of compound 10l. Subsequently, the cells were centrifuged, washed twice with ice-cold PBS, and incubated at 4 °C for 20 min. The cell cycle was assessed using a flow cytometry kit (propidium iodide [ab13941]). Finally, the Cell Quest software was used to undertake statistical analysis of the cell fractions in the sub-G0/G1, S, and G2/M phases,^61,62 as represented in Fig. S45a–f.†

5.2.5. Molecular docking studies

Molecular docking was performed at the Pharmaceutical Chemistry department, Faculty of Pharmacy, Ain Shams University using Discovery Studio 2.5 software provided by Accelrys®.

5.2.5.1. Protein preparation for docking

The X-ray crystal structure of VEGFR-2 co-crystallized with sorafenib was obtained from the Protein Data Bank at the Research Collaboration for Structural Bioinformatics (RCSB) website [https://www.rcsb.org/] (PDB code: 3WZE) and loaded in Accelrys Discovery Studio 2.5. The protein structure was prepared using the default protein preparation protocol integrated in the software. The whole protein structure was minimized using many steps employing the SMART minimizer algorithm. The whole enzyme was defined as the receptor. Also, the binding pocket together with the surrounding amino acid residues was identified. The ligand structures were removed from the binding sites.

5.2.5.2. Ligand preparation for docking

The ligand structures were constructed using the default sketching tools of Accelrys Discovery Studio 2.5, and then the ligands were minimized and prepared using the ligand preparation protocol. The ionization pH was adjusted to 7.4, hydrogen atoms were added, and no isomers or tautomers were generated from the ligands.

5.2.5.3. Docking process

The docking process was carried out using the C-Docker software in the interface of Accelrys Discovery Studio 2.5. The default values of C-Docker were used, but the number of docking iterations was increased to 500 000 to obtain a high number of predictions. After running the protocol, a report is given about the docked ligands with their corresponding docking scores (-C-Docker Energy). Ten docking poses were generated for each ligand docked and thoroughly inspected to obtain the best binding mode.

Data availability

All authors agree that the data included in the ESI† are made available.

Author contributions

Mohamed K. S. El-Nagar: data curation, formal analysis, investigation, methodology, writing – original draft, writing – review and editing. Mai Adel: supervision, investigation, writing – review and editing. Mai I. Shahin: supervision, investigation, writing – review and editing. Mohammed F. El-Behairy: supervision and investigation. Ehab S. Taher: formal high resolution mass analysis. Mohamed F. El-Badawy: antimicrobial analysis. Marwa Sharaky: cell cycle analysis. Dalal A. Abou El Ella: supervision, project administration, writing – review and editing. Khaled A. M. Abouzid: supervision, conceptualization, investigation, methodology, project administration, writing – review and editing.

Conflicts of 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 article.