{'fraction_non_alphanumeric': 0.04122440594398411, 'fraction_numerical': 0.028007943573238377, 'mean_word_length': 4.997535934291581, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Diorganotin(IV) Derivatives of N-Methyl p-Fluorobenzo-Hydroxamic Acid: Preparation, Spectral Characterization, X-ray Diffraction Studies and Antitumor Activity

Three diorganotin(IV) complexes of the general formula R2Sn[RcC(O)N(RN)O] (Rc = aryl, RN = Alkyl) have been synthesized by refluxing in toluene the corresponding diorganotin(IV) oxides with the free ligand N-methyl p-fluorobenzohydroxamic acid, using a Dean and Stark water separator. The ligand was derived from the reaction of the corresponding p-fluorobenzoyl chloride and N-methylhydroxylamine hydrochloride in the presence of sodium hydrogen carbonate. The isolated free ligand and its respective diorganotin compounds have been characterized by elemental analysis, IR and 1H-, 13C-, 119Sn-NMR spectroscopies. The crystal structures of the diorganotin complexes have been confirmed by single crystal X-ray diffraction methods. The investigations carried out on the diorganotin(IV) complexes of N-methyl p-fluorobenzohydroxamic acid confirmed a 1:2 stoichiometry. The complex formation took place through the O,O-coordination via the carbonyl oxygen and subsequent deprotonated hydroxyl group to the tin atom. The crystal structures of three diorganotin complexes were determined and were found to adopt six coordination geometries at the tin centre with coordination to two ligand moieties.


Synthesis
The ligand was prepared by the reaction of p-fluorobenzoyl chloride with N-methyl-hydroxylamine hydrochloride in the presence of sodium hydrogen carbonate as catalyst. All the reagents were in the same ratio by weight (i). Diorganotin(IV) complexes were synthesized in 2:1 molar ratio, by refluxing the free ligand with diorganotin(IV) oxides in hot toluene for 5-6 h with stirring and the water formed was removed azeotropically using a Dean-Stark apparatus (ii), as summarized in Scheme 1. The resulting solution was cooled and filtered and the solvent evaporated. The solid was precipitated by adding petroleum ether (60-80 °C) and then recrystallized from ethanol. The purity of the ligand and the diorganotin complexes were assured by TLC analysis using silica gel-G as adsorbent. The newly synthesized ligand and its diorganotin complexes are white or colorless solids, stable in air and soluble in common organic solvents. Tin was determined gravimetrically, by igniting a known quantity of each complex. The calculated values were in a good agreement with the experimental values.

Infra-Red Spectroscopy
Solid state infrared spectra of the N-methyl p-fluorobenzohydroxamic acid and its complexes have been recorded in the range 4,000-400 cm −1 . The principal infrared absorption bands are those due to ν(O-H), ν(C=O), ν(C-N) and ν(N-O) stretching vibrations of the hydroxamate group observed in the spectrum of free hydroxamic acid at 3,175, 1,610, 1,432 and 908 respectively. The ν(O-H) band is observed in the range 3,175 cm −1 appeared as a broad band indicating the presence of extensive hydrogen bonding. The characteristic band ν(C=O) positioned within the range 1,610 cm −1 is notably, below the usual ketonic ν(C=O) range of 1,650 cm −1 [44,45], indicates that in the solid state the ligand exist in the keto form.
The IR spectra of the free ligand and its diorganotin(IV) complexes illustrated clear differences. In all cases, (O-H) stretching modes were absent in the spectra of the complexes, thus suggesting the deprotonation of the hydroxamate group on complexation, Similarly, the (C=O) group are shifted to lower frequencies in the range 1,599-1,602 cm −1 , indicating a further shift of (C=O) to lower energy thus suggesting the predominance of the enolic form to give a five membered chelate rings at the tin centre. Moreover, the (N-O) stretching vibrations occurring at 938-948 cm −1 in the diorganotin(IV) hydroxamates, are shifted to higher frequencies, excluding the coordination via the nitrogen atom [46]. The occurrence of (Sn-O) in the range of 474-453 cm −1 indicates the chelation of the tin centre to the enolate oxygen [45,47].

NMR Spectroscopy
1 H-NMR spectra for the investigated ligand and their organotin(IV) complexes have been recorded in CDCl 3 solution and tetramethylsilane as internal standard at room temperature. In the 1 H-NMR spectra the free ligand show a signal at 10.34 ppm, which is due to the intramolecularly hydrogen bonded hydroxyl proton. The peak disappeared in the 1 H-NMR spectra of the complexes indicating, thereby, the substitution of the hydroxyl proton and chelation of the oxygen to the tin atom. The proton signals appearing in the region 3.40 ppm were attributed to methyl protons attached to the nitrogen atom, which remained unchanged on chelation, supporting further, the non-involvement of this group in complexation. In the dimethyltin(IV) derivative, the proton resonances appeared as a singlet in the region 0.713 ppm, with well-defined satellites. The value of the two bond coupling constant 2 J( 119 Sn-1 H) calculated from tin satellites in the 1 H-NMR spectra of dimethyltin(IV) complex was found in the region of 84.22 Hz, and the estimated C-Sn-C bond angle is 136.4°, based on the equation of Lockhart and Manders [Equation (1)] [48], which fall in the region for six-coordinate tin [49]. In the dibutyltin(IV) complex, the butyl protons were found as a multiplet and a triplet in the regions 1.36-1.84 ppm and 0.88 ppm due to -(CH 2 ) 3 and the terminal CH 3 respectively. A complex multiplet found at 8.17-8.32 ppm for the aromatic protons of the free ligand and all complexes, is due to the overlapping of the signals of the aromatic protons of the ligand and phenyl group protons in diphenyltin(IV) complex [50,51]. (1) 13 C-NMR spectra for the investigated ligand and its organotin(IV) complexes have been recorded in CDCl 3 solution and tetramethylsilane as internal standard at room temperature. 13  The signals appeared at 115-163 ppm, were assigned to the aromatic carbons. By comparing the 13 C-NMR spectra of the free ligand with its diorganotin (IV) complexes, a slight upfield shift has been observed in the position of carbonyl signal, suggesting the bidentate nature of hydroxamate group. One can notice that the oxygen chelated to metal ion reduce the electron density at carbon atom, hence considered the cause for chemical shift [52,53].
The 119 Sn-NMR spectra of diorganotin(IV) complexes studied herein in DMSO, at room temperature. The 119 Sn-NMR chemical shifts of organotin(IV) compounds appear to depend not only on the coordination number, on the other hand also on the alkyl groups bound to the metal ion and the types of donor atoms [54]. The spectra show one sharp signal in dimethyl-, dibutyl-and diphenyltin complexes at = −407 ppm, −367 ppm and −205 ppm respectively, which strongly supports the six coordination around tin in a distorted octahedral geometry [55][56][57]. In the later an associated structure such as the stereoisomers specie is thus present in solution similar to spectra have reported by [56,58].

X-ray Crystallography
The crystal structure of compound (1), (2) and (3) are shown in Figures 1-3, respectively. Selected bond angles and bond lengths are presented in Tables 1 and 2. The molecular structures of these diorganotin complexes showed that the tin atom is bonded to two N-methyl-p-fluorobenzohydroxamic acids via the hydroxyl oxygen and the carbonyl oxygen [30,59]. The two organic groups of the diorganotin fragment complete the six coordination geometry at tin for the three complexes. It is evident that the carbonyl oxygen are weakly coordinated to the tin compared to the covalent Sn-O hydroxyl bonds [compound (1): Sn-O1 2.0921(9), 2.0921(9) and Sn-O2 2.3778(9), 2.3778(9); compound (2) (2)]. The bond distances and angles of the three complexes as given in Tables 1 and 2 revealed that the geometry of the crystals is distorted octahedral around the six coordinated tin(IV) ion, similar to the diphenyltin(IV) bis(N-methyl p-bromobenzohydroxamate) [27] and di-n-butyl-(4-chlorobenzo-hydroxamato)tin(IV) [60]. The distortion in the coordination sphere of the metal ion from the ideal geometry may be due to the structural constraints imposed by the hydroxamic acid ligand framework.      (1), (2) and (3).   (1), (2) and (3).

Antitumor Activity in vitro
The synthesized organotins were evaluated for the biological activity, specifically cytotoxicity on HCT116 colorectal carcinoma cell line. All the tested organotins induced a concentration-dependent anti-proliferative effect towards HCT116 cells upon treatment for 24 h. However, the cytotoxicity of dibutyltin ( Table 3. Our current data are in agreement with previous study, whereby the triphenyltin(IV) complexes exhibit higher antiproliferative effects compare to diphenyltin(IV) complexes [61][62][63].
Similarily, it has also been demonstrated that triphenyltin(IV) complex possess the highest cytotoxic effect whereas the dimethyltin(IV) complex have little or no cytotoxic effect on HCT116 cells upto 250 µM treatment for 24 h [64]. Therefore, triphenyltin(IV) N-methyl p-fluorobenzohydroxamate has the potential to be developed as an anti-tumor agent due to the potent cytotoxic effect at nano molar concentration which warrant further mechanistic studies.

General
The chemicals were purchased from Aldrich and were used as received. All the chemicals were of analytical grade. The triphenyltin(IV) N-methyl p-fluorobenzohydroxamate was success-fully prepared according to a standard method reported in the literature [65]. The melting points were determined in open capillary tubes using an Electrothermal 9300 digital melting point apparatus. The percentage compositions of the elements (CHN) for the compounds were determined using an elemental analyzer CHNS-O Model Fison EA 1108. Solid state infrared spectra of the compounds are recorded in the range 4000-400 cm −1 . The infrared spectra were recorded as potassium bromide discs using a Perkin-Elmer spectrophotometer GX. The 1 H-, 13 C-and 119 Sn-nuclear magnetic resonance spectra were recorded using the Bruker FT-NMR 600 MHz Cryo-Prob spectrometer and the JEOL JNM-ECP 400 spectrometer using DMSO/CDCl 3 as a solvent and tetramethylsilane as an internal standard. Crystals structures determination were carried out on a Bruker Smart APEX CCD area detector diffractometer equipped with graphite mono-chromatised Mo-Kα (λ = 0.71073Å) radiation in each case. All data collection was carried out at 100K. The program APEX2 (Bruker [66]) was used for collecting frames of data, indexing of reflections and determination of lattice parameters, SAINT (Bruker 2008) for absorption correction, and SHELX97 (Sheldrick [67]). HCT116 human colorectal carcinoma cells were obtained from the American Type Culture Collection (Manassas, Virginia, USA). The cells were grown in McCoy's 5A medium (Invitrogen Cooperation, Paisley, UK) supplemented with 10% FBS (PAA Laboratories, Morningside, QLD, Australia) and maintained at 37 °C with 5% CO 2 in humidified incubator.

Synthesis of Ligand
p-Fluorobenzoyl chloride (0.01 mol) was poured down drop by drop to a stirred cold solution of N-methylhydroxylamine hydrochloride (0.01 mol) containing sodium hydrogen carbonate (0.01 mol) and was further stirred for 30 min below 4 °C. The solution was filtered and reduced to evaporate at low pressure. The precipitate was then dissolved in boiling ethyl acetate to remove any undissolved substance and then the filtrate is placed in the fridge overnight to obtain the crystals.

Synthesis of Complexes
Diorganotin(IV) complexes were synthesized by 2:1 molar ratio, dissolving the free ligand (0.005 mol) in hot toluene and then added the diorganotin(IV) oxides (0.0025 mol) to the solution. The solution was refluxed for 5-6 h with magnetic stirrer and the water formed during the course of reaction was removed azeotropically using a Dean-Stark apparatus. The solution was then cooled and filtered. The filtrate was placed under vacuum to evaporate the solvent and the solid was precipitated by adding petroleum ether (60-80 °C) and then recrystallized in ethanol.

X-ray Crystallography
The single crystals of dimethyltin, dibutyltin and diphenyltin complexes of N-methyl p-fluorobenzohydroxamic acid of suitable quality were each mounted on a fine glass capillary and aligned on the Bruker SMART APEX2 diffractometer, equipped with graphite monochromated Mo-Kα radiation source (λ = 0.71073 Å). The range of theta for data collections together with other crystallographic information are given in Table 4. All calculations were performed using the SHELXTL-97 package [68]. Crystallographic data for the compounds (1), (2) and (3)

MTT Cytotoxicity Assay
The antitumor activity against carcinoma cells was assayed by the MTT method [69]. Cells were seeded in 96-well plate at a density of 5 × 10 4 cells per well in a volume of 200 mL and were treated with various concentrations of the compounds for 24 h. After treatment, 20 µL of 5 mg/mL MTT (Sigma-Alrich, St. Louis, MO, US) was added to each treated cells and further incubated for 4 h at 37 °C. Subsequently the medium was discarded from each well before adding 200 µL DMSO (Fisher Scientific, Loughborough, UK). For complete dissolution, the plate was incubated for 15 min followed with gentle shaking for 5 min. The cytotoxic effect of the organotins on HCT116 cells was assessed by measuring the absorbance of each well at 570 nm. Mean absorbance for each concentration was expressed as a percentage of vehicle control absorbance and plotted versus compound concentration.

Conclusions
In this work, we have successfully synthesized a novel ligand and its three diorganotin(IV) hydroxamates, which gave fairly sharp melting points indicating that the compounds were pure and were characterized by elemental analyses, IR, NMR and X-ray single-crystal diffraction. The structural analyses of complexes 1-3 reveal that the coordination mode observed in metal-hydroxamic acid complexes is the O,O-bidentate chelation and a five membered chelated ring was assembled. The NMR and X-ray studies were in full concurrent with the IR spectral evidences. The crystal structures of the three diorganotin complexes adopted a six coordination geometry at tin which is coordinated to the carbonyl oxygen and hydroxyl oxygen of two benzohydroxamic acid ligands and the two organic substituent of the diorganotin(IV) fragment. The diphenyltin(IV) and triphenyltin(IV) complexes demonstrated promising antiproliferative activities whereas dimethyltin(IV) shows very little cytotoxic effect at μM concentration on human HCT116 cells.