Organotin (IV) complexes with sulphonyl hydrazide moiety. Design, synthesis, characterization, docking studies, cytotoxic and anti-leishmanial activity

Abstract Due to a lack of therapeutic options for the pathological condition of leishmaniasis, which is characterized by polymorphic lesions and skin surface infections, Leishmania genus parasites damaged dermis and mucosa. There was a need to synthesize and characterize some new complexes. This study evaluated the biological activities preferably anti-Leishmanial activity of organotin (IV) containing sulphonyl hydrazide derivatives. A series of six new organotin (IV) complexes 1–6 labeled as R2SnL2; R = Methyl (1), Butyl (2), Phenyl (3) and R3SnL; R = Methyl (4), Butyl (5), Phenyl (6) has been synthesized as reflux method derived from N'‐ (2,4‐dinitrophenyl)‐4‐methylphenylsulfonylhydrazide (L). All compounds were characterized through FT-IR, 1HNMR, 13CNMR, and elemental analysis. Structural analysis confirms the formation of six complexes (1–6). All derivatives have been screened for their pharmacological activities. Interestingly, compound 1 showed promising activity against leishmania promastigotes with low cytotoxicity. All results were further elaborated through docking studies performed on leishmania donovoni synthetase PDB: ID 3QW3 that acts as an essential building block for the viability of Leishmania promastigotes. This research effectively synthesized sulphonyl hydrazide ligand and its six new organotin (IV) derivatives, which were tested for biological properties such as antibacterial, anti-fungal, anti-oxidant, and ideally anti-leishmanial activity and cytotoxicity. Studies have confirmed that these compounds have the potency to be a good candidate against leishmaniasis. Computational studies were carried out to recognize the binding affinities for leishmania donovoni synthetase. Communicated by Ramaswamy H. Sarma


Background
Researchers always focus to help the patient to overcome the disease and improve their life styles (Zhu, 2020). The purpose of each research is to ensure that the innovative new molecules are effective, safe and available for patients in a shortest possible time (Ali et al., 2017;Du et al., 2020). The Trypanosomatidae embraces is a very huge number of the parasitic protozoa, in which several are causes a variety of ailments in humans (Aher et al., 2009). Leishmaniasis disease is caused through a group of parasites protozoan called leishmania. It is caused by parasites transmitted by phlebotomy insects (Sabaa et al., 2016). Across the world there are about 1.3 million new cases of leishmaniasis annually causing about 20,000-30,000 deaths (Molina et al., 2020). Over 20 different species of leishmania are there that infects humans and especially it affects poorest countries in world that have low level of hygiene (Desjeux, 2001). It is transmitted to humans by the bite of female sand fly that is already infected. Over 90 different species of sand fly have been recognized to promote leishmaniasis (Santos et al., 2020). Leishmaniasis specifies a poverty-related illness that involves a disease complex having two important epidemiological forms. Visceral leishmaniasis (Leven et al.) and skin leishmaniasis (SL). Cutaneous or skin leishmaniasis is the most common type and is characterized by the sores on skin (Badirzadeh et al., 2020). About a hundred countries, nearly one million cases of leishmaniasis have been reported per year in the last decade (Alvar et al., 2008). Visceral leishmaniasis which is also known as Kala-Azar is more crucial and fatal than cutaneous leishmaniasis and has deteriorated due to better understanding of the disease pathogenesis. It can develop from few days and even up to years after the bite of an infected sand fly (Rogers, 2012). It infects the internal organs usually the spleen, liver and bone marrow. WHO has confirmed that leishmaniasis remains among the most dangerous tropical diseases that need to be cured and signifies major global health issues that gives a mild to wide spectrum of clinical appearances with dangerously deadly outputs (Chapman et al., 2020).
Unfortunately, no vaccine is presently available which exhibited excellent results for the cure and prevention of this ailment (Aloui et al., 2016). Therefore drugs are the merely source of treating this disease. The anti-leishmanial drugs are chiefly based on the antimonial treatment and lately urbanized and the tested medicaments have displayed comparatively less protection (Ouakad et al., 2007). The Pentavalent antimony (SbV) molecules like the meglumine antimoniate and sodium stibogluconate are the primary option of treatment for the leishmaniasis. In spite of their wide clinical utilization for numerous decades, the mechanism of action relics indistinct (Chai et al., 2005). Furthermore, the other drugs which were employed for the treatment of leishmaniasis are, amphotericin B and pentamidine but due to high cost and severe side effects the use of these medicaments has been restricted (Borborema et al., 2005). In recent times, miltefosine which is the oral drug was permitted for the cure and treatment of the human visceral Leishmania disease (Sundar et al., 2002). Along with this for the cutaneous leishmaniasis the anti-fungal drug fluconazole is also efficient result when taken orally (Croft et al., 2006). Because of the worse side effects and expensive cost of these treatment regimen, they are not recommended and substantial interest has been given for the discovery and development of less toxic and new chemo-therapeutic agents (Pitzer et al., 1998).
Free radicals are concerned in the development of a diversity of disorder in the human beings counting arthritis, central nervous system injury, atherosclerosis, gastritis, ischemic heart diseases, reperfusion injury and cancer of various tissues (De Queiroz et al., 2014;Ullah et al., 2017). The free radicals from ecological pollutant, radiations, chemical agents, toxins, deep fried foods and spicy causes the reduction of antioxidants immune scheme change genetic material expression along with the initiation of the irregular proteins (S. Ahmad et al., 2015;Wojtunik-Kulesza et al., 2016). They are produced in the living organism in the process of oxidation. To overcome oxidative pressure, hydro peroxidase and catalase enzymes in the human body change hydro peroxides and hydrogen peroxide to the non-radical form and therefore exertion as antioxidants.
From the last century antibiotics have been lost their potency and efficacy due to resistance of drugs. The appearance of microbial resistance to numerous drugs improved due to the utilization and mistreatment of the various antimicrobials in human medications (A. Ahmad et al., 2020;Ayaz et al., 2019). The appearance of resistance strains and further newly extreme drug-resistant mutants of various bacterial strain, like mycobacterium tuberculosis, pretense main difficulty in management (Ayaz et al., 2016). Though, despite a call for new antibiotic therapy, there has been a continued decline in the number of newly approved drugs. There is no uncertainty that humanity desires new drugs which conquer defiant strains (Jabeen et al., 2018).
The organotin containing compounds is attaining more interest due to their unique structural features making them more prone to be used as anti-microbial, anti-fungal and antimalarial agent (A. M. Khan et al., 2011). Hydrazide groups along with organotin(IV) are a topic of interest for most of the scientist with important reference to their structural characterization and pharmacological evaluation (Khadija Shahid et al., 2006). These types of organotin consisting compounds have also gained considerable concern as anti-cancer and antimitotic agents. The nature and function of organotin complex remarkably depends upon the number of moieties attached to central atom . The tri aryl and tri alkyl organotin derivatives have more energetic hazardous effects towards brain while the di aryl and di alkyl organotin derivatives are more toxic when taken from oral route and toxicity decreases as the length of carbon chain increases Khadija Shahid et al., 2006).
By considering all the information about organotin (IV) complexes, we have reported here a new series of 6 organotin complexes of N'-(2,4-dinitrophenyl)-4-methylphenylsulphonylhydrazide. The characterization of all synthesized compounds was done effectively through FT-IR, 1 HNMR, 13 CNMR and elemental analysis. All compounds were analyzed for anti-microbial, anti-fungal, anti-oxidant, anti-leishmanial and cytotoxic relevant activities.

Chemistry
All the chemicals and reagents utilized were of decent quality and purity without any contamination and impurity. 2,4-dinitrophenyl hydrazine, chlorotrimethylstannane (Me 3 SnCl), chlorotributylstannane (Bu 3 SnCl), chlorotriphenylstannane (Ph 3 SnCl), dichlorodimethylstannane (Me 2 SnCl 2 ), dichlorodibutylstannane (Bu 2 SnCl 2 ), and dichlorodiphenylstannane (Ph 2 SnCl 2 ), acetaldehyde, acetone, chloroform, hexane, methanol and triethylamine were acquired through Sigma Aldrich Ltd USA. Gallenkamp Electro Thermal UK melting point apparatus was utilized to determine the melting points. Bruker FT-IR spectrophotometer was used to examine the IR spectra of synthesized compounds. Elemental analyses was carried out using a CE-440 elemental analyzer and they were determined within 0.4% theoretical values range. The calculated values were found to be in good agreement with the results. NMR spectra were analyzed on Bruker Digital spectrometer 300 MHz (Switzerland) (300 MHz for 1 HNMR and 75 MHz for 13 CNMR). Peaks were described as singlet s, doublet d, triplet t, quartet q and multiplet m along with coupling constant J values in hertz Hz. Chemical shifts values were described in parts per million (ppm). DMSO-d6 (2.50 ppm for 1 HNMR and 39.70 ppm for 13 CNMR) and CDCl 3 (7.26 ppm for 1 HNMR and 76.90 ppm for 13 CNMR).

Anti-bacterial assay
Ligand containing hydrazide and its organotin (IV) complexes were analyzed for having anti-bacterial activity against the gram þ ve bacteria like Streptococcus, Bacillus subtilis Staphylococcus aureus and Gram À ve bacteria like Salmonella Typhi, Shigella and Escherichia Coli using method of well diffusion and results were obtained by measuring the percentage growth inhibition (N'Guessan et al., 2015). This method consist of making 10 ml nutrient broth aliquot inoculated with bacterial species i.e. species which had to be tested, and incubated for 24 h at 37 C ± 1. Afterwards, 0.6 ml of broth culture of test organism with the help of previously incubated sterile pipette was poured into molten agar that was contained in a 9 cm petridish cooled at 45 C, mixed well. After this, nutrient agar was allowed to solidify. After solidification, required number of holes of 10 mm each were cut using the sterile cork borer and agar plugs from these holes were removed. Make sure that all holes were properly distributed and one hole was present in the center. Each organism duplicate plate was also prepared . Ligand and organotin (IV) complexes and tetracycline (which was taken as a standard), solutions and dilutions were prepared having concentration of 1 mg/ml. 100 ml of test organism was poured into each hole that was previously prepared and labelled. Diffusion of samples was allowed to take place by placing the plates near room temperature up to two hours with placement of plates in upside position in incubator for 24 h. Zone of inhibition in millimeters of tetracycline, ligand and complexes was determined.

Anti-fungal assay
Anti-fungal in vitro assay of ligand and its organotin (IV) complexes was examined against different strains of fungi counting Candida alibican, Fusarium solani, Tricophyton longifusus, Microsporum canis, Bipol and Aspergillus flavus by agar tube diffusion way and results were measured by examining the percent growth inhibition (Aher et al., 2009;E. Khan et al., 2017). Sabouraud dextrose agar was prepared by heating and stirring the Sabouraud 4% glucose agar with water. Afterwards, this media was poured into test tubes and autoclaved at 121 C for 15 min for sterilization. After that, the autoclaved test tubes were settle down to 50 C. Accordingly, test samples of ligand and organotin (IV) complexes were prepared in DMSO having the concentration of 2, 4, 8, 16, 32, 64, 256, 512 mg/ml. Miconazole having the concentration of 20 mg/ml was taken as standard. 100 ml of standard and test samples were transferred into different Sabouraud tubes using the micropipette. Tubes were permitted to solidify in angled position. From the 7 days old culture of fungal strains, 4 mm diameter piece of inoculums was removed and instilled into each tube. These culture containing tubes were then allowed to incubate at 28-30 C for 7-10 days. These cultures were than examined after every 3 days during their incubation period. After 7-10 days of incubation, test tubes were analyzed for their percentage of growth inhibition.

Anti-oxidant assay
Proton atom giving ability of synthesized compound and all the organotin (IV) derivatives was measured through 2, 2diphenyl-1-picryl-hydrazyl (DPPH) as by procedure described in literature (A. Ahmad et al., 2019;Hussain et al., 2019). DPPH 3.2 mg was poured in 100 ml of methanol (82%). Stock solution was synthesized in a vial by taking 0.1 mM DPPH solution 2800 ll along with 200 ll of methanolic test sample. The stock solution was serially diluted in methanol to obtain 1000 lg/ml, 500 lg/ml, 250 lg/ml, 125 lg/ml, 62.5 lg/ml and 31.25 lg/ml dilutions. Mixture was mixed well and positioned away from light temperature (25-28 C) up to60 minutes. The DPPH free radical color change was measured by placing this mixtures in UV/Visible spectrophotometer at the absorbance of 517 nm. 2800 ml of DPPH in 82% methanol and 200 ml of methanol solution was taken as standard. Inhibition percentage of DPPH radical was measured and recorded according to formula and graphical method through Microsoft excel was used to calculate IC 50 value.
where Ac ¼ Absorbance of standard at 517 nm and As ¼ Absorbance of test sample at 517 nm.

Anti-leishmanial activity
For the antileishmanial activity, stock solution of ligand and its organotin (IV) complexes were prepared by dissolving 1 mg or 0.001 g of each per ml of DMSO. After that dilutions of each stock solution was prepared in a serial (100 ll, 10 ll, 1 ll, 0.1 ll, 0.01 ll, and 0.001 ll) by diluting each in DMSO to get the desired values (Yasinzai et al., 2013). Filtration was done on each dilution by using the syringe filter having 0.45 lm diameter. Leishmania tropica KWH23 isolation through patient which was appeared to be suffering from leishmaniasis and characteristics of this species was done. Leishmania promastigotes forms were grown up in M199 medium having the HEPES buffer, penicillin, fetal calf serum (FCS) 10% and streptomycin (N. A. Shah et al., 2014). 1 Â 10 6 cells/ml of promastigote form were prepared from the log phase culture after counting from the hemocytometer. After that 180 ll of leishmania tropica KWH23 obtained from the log phase culture, along with the M199 media and 20 ll of the each dilution was distributed to microtitter plate wells. Dimethylsulfoxide and Glucantime were utilized as a negative and positive control respectively. Make sure that all the procedure had been done in Laminar flow Hood. After that the microtitter plate was incubated for 24-72 h at 24 C. Afterwards, 15 ll of dilution from each chamber was taken on the neubauer counting chamber and counting was done under the microscope (S. Majid Shah et al., 2019).

Cytotoxicity
A compound having the property of degradation of blood or lysis of blood cells is said to be having the hemolytic activity. Hemolytic activity was done to determine the blood lysing potential of synthesized hydrazide derivative and its organotin (IV) complexes, if carries the stuff to burst the red blood cells. The results were compared with that of 0.5% Triton-X 100 which gives the 100% hemolysis. 5-10 ml of fresh human blood was taken in an EDTA tube (Ethylene Diamine Tetra Acetic acid)(K. M. Khan et al., 2000). Blood was mixed with 10-15 ml of 4% PBS (Phosphate Buffer Saline). Stock solution of ligand and its organotin (IV) complexes was synthesized by dissolving 0.001 gm of each compound in unit ml of DMSO and then making the dilutions i.e. 100 ll, 10 ll, 1 ll, 0.1, 0.01 and 0.001 ll by diluting them in the same solvent. 900 ll of red blood cell suspension in PBS was poured in the 96-well plates each well and 100 ll of each dilution was added in it (Yasinzai et al., 2013). The plates were incubated at 37 C for 60 min. After the incubation period, cell suspensions were taken to the centrifuge machine and centrifugation was done for 5 min at 1500 rpm. Each well supernatant was acquired and placed in cuvette and hemoglobin release was detected at 576 nm in UV spectrophotometer. As a negative control, red blood cells suspended in PBS were used. The Triton X-100 was utilized as standard. Hemolysis percentage was calculated by using the following formula.

AbsorbanceofTritonXÞÂ100
Calculations were done and results were drawn in a graph showing the hemolytic activity of hydrazide derivative and its metal complexes (Nederberg et al., 2011).

Statistical analysis
Results of all the experiments were taken as triplicate and mean ± standard deviation (Mean ± SD). Probability tests and ANOVA, two-way variance analysis was done in order to analyze significant values. Calculations were carried out at GraphPad Prism 8 software to draw the charts.

Docking studies
Molecular docking studies (MD) are the fundamental tools that are utilized to get the better understanding of synthesized compounds bindings with receptors more precisely and accurately. The structure of leishmania was downloaded as PDB ID: 3QW3 having leishmania donovani synthase that is essential for the viability of leishmania promastigotes. The docking procedure was performed through Auto Dock Vina 1.1.2 software (Zafar et al., 2021). All the synthesized compounds were prepared as ligand using the Dollegro Data Modeler software and open babel. Further sketching of structures was carried out through Marvin Sketch version 20.21. After docking, visualization of interaction was carried out through BIOVIA 2020 Discovery studio visualizer (DSV) showing the best images as two dimensional (2D) and three dimensional (3D). Pymol 1.8 and Ligplot þ software was utilized to understand the binding pocket and binding site of synthesized compounds with receptors.

Results and discussions
Spectroscopic analysis is done by using FT-IR Fourier transform Infrared spectrophotometer. Data shows the most important bands for ligand and its organotin (IV) complexes. When finding for synthesis of ligand through starting materials, the presence of N-S stretching vibrations appearing at 820 cm À1 confirms the formation. The presence of the S ¼ O bands at 1115 cm À1 and the N-H bands at 1028 cm À1 in the same compound confirms the presence of sulfonyl hydrazide formation. On comparing the spectrum of free ligand with organotin (IV) complexes, the guest appearance of Sn-N stretching in case derivatives confirms the formation of organotin containing compounds (Sirajuddin et al., 2018). The oxygen atom attached to sulphonyl group forms a linkage with Sn giving it a complex moiety shape. It has been noticed from the literature preview that when the structure of compound moves to higher complexity, symmetric and asymmetric frequencies variates which indicates the chelation of compound. Furthermore appearance of some unique peaks of Sn-C ranging from 590 cm À1 to 525 cm À1 for Snalkyl groups and 278 cm À1 to 270 cm À1 for Sn-phenyl groups admires the formation of organotin complexes. The proton NMR data of ligand and derivatives give further evidence of formation of complexes. The peak of hydrogen attached to Nitrogen 2 appeared in the spectra of ligand but disappears in all other spectra of complexes reliable of deprotonation of nitrogen atom of hydrazide. The numbering pattern for proton NMR and Carbon NMR is given in Scheme 1. The peaks of proton 1 appear as singlet in all the compounds with the value in the range of 2.20-2.30 ppm (Sirajuddin et al., 2014). The characteristic peak of N 2 hydrogen (proton for N2) appears in case of ligand but disappears in organotin (IV) confirming the formation of complexes. There is no as such change in the position of N 1 hydrogen (proton for N1) indicating no involvement of this nitrogen in formation of complexes. The methyl protons, butyl protons and phenyl protons positions are elaborated in the synthesis, indicating the presence of complexes. The carbon NMR spectra strongly agreed with the results of FT-IR and 1 HNMR. The carbon 5 and carbon 6 appears up field due to shifting of electron density towards organotin. Furthermore, positions of all other carbon atoms along with appearance of carbons of methyl, butyl and phenyl in case of complexes justifies organotin (IV) complexes formation.

Anti-bacterial activity
Anti-bacterial screening of ligand and its organotin (IV) complexes was done In-vitro against Gram positive strain (Streptococcus, Bacillus subtilis, Staphylococcus aureus) and Gram negative strains (Escherichia coli, Salmonella typhi, Shigella). Results are tabulated in Table 1. Anti-bacterial activity data of ligand and its organotin (IV) complexes for different Gram positive and Gram negative bacteria strains showed that all the complexes have the capacity as bactericidal especially compound 1. Ligand showed promising results against all the stains when comparing with the positive control tetracycline while the derivatives (1-6) displayed moderate activities.

Anti-fungal activity
Ligand and its organotin (IV) complexes anti-fungal activity was evaluated In-vitro against different fungal strains which includes the Bipol, Aspergillus flavus, Microsporum canis, Fusarium solani, Tricophyton Longifor, Candida Albicans by the reported method as described in the experimental section. The results are tabulated in Table 2. Antifungal activities of the ligand and its metal complexes is shown in the table in terms mean of percent growth inhibition.
The stricture activity relationship data showed that all the complexes had activity against the fungal strains. Compound   Observed values were taken as triplicate and mean as mean ± SEM. Compounds were taken in concentration of 1000, 500, 250, 125, 62.5, 31.25 mg/ml.  Triton-X was taken as standard which has 100 percent toxicity.    1 and 5 showed great activity against the Tricophyton Longifor and Candida Albicans. Excellent activity against the Microsporum canis and Fusarium solani was shown by compound 1 and 6 respectively. Compound 5 also displayed promising results against Bipol and Aspergillus Flavus. Due to the presence of dimethyl organotin complex, compound 1 justified to be satisfactory in case of anti-bacterial activity. In comparison with compound 5 and 6, the binding interaction, geometrical structure and complexity of compound 1 were more acceptable.

Anti-oxidant activity
Antioxidant activity of ligand ant its metal complexes was evaluated by examining the free radical hydrogen donating ability, which was measured by using the radical 2, 2diphenyl-1-picryl-hydrazyl (DPPH). Antioxidant activity of ligand and its metal complexes with the values of IC 50 is shown in Table 3. Ligand and compound 1 showed excellent activity against the DPPH as an antioxidant agent. The pattern of scavenging activity can be elaborated as Ascorbic acid > Ligand > 1 > 2 > 4 > 5 > 6 > 3.

Anti-leishmanial activity
Anti-leishmanial promastigotes activity of ligand and its complexes was analyzed against Leishmania tropica. Results were taken as triplicate. All the procedure was performed by taking Glucantime having IC 50 of 4.7. Results are tabulated as Table 4. All the synthesized compounds have the potential for leishmania promastigotes inhibition but compound 1 gave excellent results inhibiting 80 percent of the promastigotes with IC 50 of 0.045 and 0.070 in mg/ml and mM respectively. The pattern of inhibition can be drawn as 1 > 6 > 5 > 2 > 4 > 3 > ligand. The results were comparable with standard drug glucantime and inhibition was calculated as IC 50 in mg/ ml and mM.

Cytotoxicity activity
Different concentration of synthesized hydrazide derivative and its organotin (IV) complex were prepared and analyzed for cytotoxic potential. If the compound seems to be toxic than it could be a good lead compound in future. The results were tabulated in Table 5.
In the characterization, Triton-X was taken as standard which has 100 percent toxicity and cause hemolysis of blood within no time. Ligand, 3 and 4 were proved to be more toxic and dangerous for blood as they caused hemolysis in huge amount. Compound 1, 2, 5 and 6 were proved to be less toxic as compared to others.

Docking studies
In order to have better understanding of inhibitory potential of innovative synthesized compounds against leishmania promastigotes, docking studies were performed.
Computational work was carried out using PDB: ID 3QW3. All the synthesized compounds including ligand were docked to get appreciative considerations about the insteractions as shown in Figure 1. Among all the synthesized moities, compound 1 gave good results having the binding energy of À8.2 kcal/mol in best binding mode. Remaining compounds gave results as À7.7 kcal/mol, À7.5 kcal/mol, 7.1 kcal/mol, À6.9 kcal/mol, À6.5 kcal/mol and À6.2 kcal/mol for derivative 6, 5, 2, 4, 3 and ligand respectively. All derivatives give best interaction inside the binding pocket of protein acting as a active site where suitable interactions appear as shown in Figure 2.
Furthermore 3D close-up gave a detailed explanation of binding with amino acid residues. In Figure 3, ligand interaction with amino acids elaborates the best binding relations with GLU 56, ARG 85, LEU 66 and LEU 103. Figure 4 further designed to explain the two dimentional view and bindings in the form of hydrogen bonds, ionic bonding and unconventional bonds.

Conclusions
Herein, we conclude that we have synthesized sulphonyl hydrazide derivative and a new series of six organotin (IV) complexes by using p-toluene sulphonyl chloride and 2, 4dinitrophenyl hydrazine as a starting material. The characterization through FT-IR, 1 H_NMR and 13 C_NMR confirms the formation of complexes. From the pharmacological point of view, it is concluded that all synthesized compounds have good potential to be used as anti-microbial, anti-fungal and anti-oxidant agent but Compound 1 shows more promising results in case of Leishmania and cytotoxicity. Further results were confirmed by computational studies/docking studies inside protein binding pockets that proved the activity against Leishmania promastigotes. We further conclude that if more metals like Fe, Ni and Cu are complexed with ligand in place of organotin (IV), we can get some more interesting derivatives having less toxicity and enhanced activity. Biological activities of compounds always increases when chelation takes place with metals. Metals like copper, zinc, iron and bismuth as part of research have the enhanced biological activities in comparison with ligand. Therefore, authors have suggested that if the organotin is replaced with other metals like Fe, Ni and Cu, the activity could be enhanced but can also be decreased.