Abstract
A series of germanium and silicon incorporated diorganotin derivatives of general formula 
where R1 = H3C, C6H5, p-CH3C6H4, p-FC6H4; R2 = H2CSi(CH3)2C6H5, H2CC6H5, p-CH3C7H7 were synthesized by the reaction of appropriate diorganotin dichlorides and germatranyl (substituted) propionic acid in 1:2 mole ratio, respectively. The evidence regarding their structure is mainly based on spectroscopic data obtained by multinuclear (1H, 13C, 29Si, 119Sn) NMR and 119mSn Mössbauer, IR and mass spectral studies in combination with melting points and elemental analyses. The compounds have been screened for in vitro anti-leishmanial activity against promastigotes of Leishmania major and the results offer potent activities which are better than the standard drug, pentamidine, for one compound.
Introduction
There is a continuing interest in the syntheses and biological studies of organotin compounds because of inherent potential chemotherapeutic applications and structural diversity present in their molecules [Citation1,Citation2]. Organotin carboxylates are the most ubiquitous owing to their important biocidal activity [Citation3,Citation4]. Toxicological and pharmaco-kinetic studies of germanium revealed its low mammalian toxicity, after these informations, developments were oriented towards its biological applications as chemotherapeutic agents Citation5, Citation6, Citation7. Germatranes have been examined for their neurotropic, antitumour, and radioprotective properties [Citation8]. The existence of co-relation between the bioactive properties of organogermanium and organotin compounds and the close analogy between the organic chemistry of germanium and silicon paved the way for the syntheses of useful mixed metal complexes Citation9, Citation10, Citation11, Citation12, Citation13, Citation14.
The toxicity of organotin deivatives containing silicon in the alkyl group bonded to tin has been comprehensively lowered by the introduction of germatranyl moiety as a part of carboxylate ligand in these compounds Citation12, Citation13, Citation14. The toxicity of the germatranyl propionic acids were determined as their LD50 vales and found in range 60–80 ug/mL. The LD50 values of the synthesized compounds are over 200 ug/mL and their activities show the potential to be used as drugs. Unfortunately, epidemic diseases such as leishmaniasis, caused by the parasite protozoa in the genus Leishmania, are spreading in different parts of the world as a result of non-availability of the high priced drugs or more seriously due to the development of parasitic resistance against the available drugs. In continuation of our previous works Citation14, Citation15, now we report herein the syntheses, spectroscopic characterization, and antileishmanicial activities of some germanium and silicon substituted diorganotin dicarboxylates (Scheme ).
Scheme 1. Synthesis of Diorganotin derivatives.
Experimental
Materials and instruments
Phenyldimethyl chlorosilane and methyl substituted cinnamic acid were purchased from Aldrich, UK and used as received. Germanium dioxide, all diorganotin dichlorides and triethylamine were purchased from Aldrich, USA and used without further purification. All the chemicals used were of reagent grade and all organic solvents were dried prior to use according to the reported methods [Citation16]. For the details of apparatus and instruments used see elsewhere [Citation14,Citation15,Citation17].
General synthetic procedures
Synthesis of precursors
Germatranyl-3-propionic acids and bis[phenyl(dimethysilylmethylene)]tin dichloride were synthesized and characterized in accordance to our earlier report [Citation15].
Synthesis of compounds
The target compounds (1–8) were synthesized by following the general procedure. The appropriate amount of the respective germatranyl substituted propionic acid (2 mmol) was suspended in a flask containing ethanol and fitted with a reflux condenser. The stoichiometric amount of triethylamine was added followed by dropwise addition of the respective diorganotindichloride (1 mmol) at 0°C and the mixture was refluxed for 6–8 h. After cooling, solid triethylamine hydrochloride was filtered off and the solvent was removed under vacuum to yield crude solid product. The solid was dissolved in dichloromethane and recrystallized using n-hexane:chloroform mixture (1:3) as white solid but unfortunately could not obtain fine crystals for single crystal X-ray analysis.
In Vitro Antileishmanial assay
Leishmania major (MHOM/PK/88/DESTO) promastigotes, cultivated in bulk were aseptically be sedimented down at 300 rpm, counted with the help of improved Neubaver chamber under the microscope and diluted with the fresh medium to a final concentration of 2 × 106 parasites/mL. The compounds to be checked were dissolved to a final concentration of 1.0 mg in 0.1 mL of PBS (Phosphate Buffered Saline, pH 7.4 containing 0.5% MeOH, 0.5% DMSO).
In a 96 well microtiter plate, 90 μL of the parasite culture (2.0 × 106 parasites/mL) was added in different wells. 10 μL of the experimental compound was added in culture and serially diluted so that minimum concentration of the compound is 0.1 μg/mL. 10 μL of PBS (Phosphate buffered saline, pH 7.4 (containing 0.5% MeOH, 0.5% DMSO) was added as negative control while pentamidine to a final concentration of 1.0 mg/mL was added separately as positive control. The plate was incubated between 21–22°C in dark for 5 days during which control organisms multiply 6 times. The culture was examined microscopically on an improved Neubaver chamber and IC50 values of compounds possessing antileishmanial activity were calculated [Citation18]. All assays were performed in triplicate.
Results and discussion
Some germanium and silicon substituted organotincarboxylates of general formula 
were synthesized by the reaction of diorganotin dichlorids and germatranyl substituted propionic acid (1:2 mole ratio) in the presence of triethylamine as a base. The compounds (1–8) are quite stable in moist-air and their physico-analytical data are presented in . The molecular structures of the synthesized compounds were established on the basis of the data obtained by elemental analyses and spectroscopic studies like multinuclear (1H,13C,29Sn,119Sn) NMR, 119mSn Mössbauer, IR and mass spectrometry.
Table I. Physico-analytical data for compounds*of general formula. 
Spectroscopic investigations
NMR spectrscopy
The 1H NMR data of compounds 1–8 are given in . The CH2 group attached to silicon and tin resonated up-field in the region of 0.06–0.8 ppm which is due to the electropositive nature of both the two atoms. The cyclic skeleton of germatranes, comprises an AABB spin system, give two triplets around 3.6–3.9 ppm for OCH2 and 2.7–2.9 ppm for NCH2 moiety with vicinal coupling constant, 3J (AB) value of 5–6 Hz in solution (CDCl3) [Citation15]. Methyl group attached to silicon absorbs at 0.1–0.3 ppm. The unit CH2CHGe, having three non-equivalent protons in germatranyl propionate framework, comprises an ABX system by presenting CHGe a chiral centre and CH2 as a prochiral centre. The methylene protons (A,B) resonate at 2.9–3.2 ppm showing two pseudoquartets along with geminal coupling constant (Jgem 14.8 ± 1 Hz) and two different vicinal coupling constants (Jvic 4.5, 12.5 ± 1 Hz). The HX of the chiral centre resonate further downfield at about 3.8–4.1 ppm. For figure and more details see elsewhere [Citation19]. The CH2 group of benzyl moiety resonated at 2.5 ppm as a singlet for compounds (5–8). Phenyl group of the benzyl moiety and phenyl attached to germanium overlap in aromatic region and pose some difficulty to be differentiated. Thus, all the protons in the compounds have been identified and the total numbers of protons estimated from the peak height of the integration curves agree well with their expected molecular formulae.
Table II. 1H NMR data*ab of organotin (IV) derivatives of general formula.
The 13C NMR spectral data are presented in . The methylenic carbon directly attached to tin and/or silicon atoms resolved at up-field due to the electropositive nature of these two atoms. The carbon atoms linked to germanium atom through oxygen and nitrogen in germatranyl moiety resonate at 56 and 51 ppm, respectively. The aromatic carbon resonances were assigned by comparison of experimental chemical shift values with those calculated from incremental method [Citation20] and then compared with the literature values [Citation21]. The chemical shifts values of carboxylate carbon for these compounds appear in range 172–182 ppm. The 119Sn chemical shift data for some selected compounds are given in . Now it is well established that compounds with different geometries about tin atom produce chemical shift in moderately well defined ranges, and for these compounds δ (119Sn) values lie in between 99 to 123 ppm [Citation15,Citation22]. In conclusion NMR parameters suggested that there are dynamic processes connected with the coordination of the carboxylate oxygens to the tin atom in these compounds. It appears that a weakly hexacoordinated tin generates a deformed octahedron or skew trapezoidal bipyramid geometry with four strong and two somewhat weaker bonds. The data, thus, reflect consistency with the literature values Citation22, Citation23, Citation24.
Table III. 13C, 29Si NMR dataab of organotin (IV) derivatives of general formula.
Table IV. 119mSn Mössbauer and 119Sn NMR data (in CDCl3) of the selected compounds.
Infrared spectroscopy
Important IR data for the compounds are presented in . A characteristic strong band at about 700 cm− 1 demonstrates N → Ge coordination mode for germatrane derivatives. Two absorption bands at about 900 and 820 cm− 1 are of typical for the Ge-O skeleton. Absorptions around 610 and 520 cm− 1 are assigned to Sn-C asymmetric and symmetric modes respectively whereas a weak band around 440 cm− 1 indicates Sn-O stretching vibration [Citation13,Citation21]. Another important parameter, Δν ( = ν(COO)asym − ν(COO)sym] described effectively the nature of bonding of the carboxylate group to tin(IV) [Citation19]. The Δν values lie in range 202–222 cm− 1 which suggests bidentate nature of the carboxylate ligand in these diorganotin derivatives.
Table V. Characteristic IR data (cm(−1) of organotin(IV) derivatives (1-8).
119mSn Mössbauer spectroscopy
The Mössbauer parameters, quadrupoles splitting (QS) and isomer shift (IS), provided useful indirect evidence for proposing the solid-state structure of organotin compounds and the 119mSn Mössbauer data are given in Table IV. The observed QS values range 3.21–3.29 mms− 1 which correspond to monomeric hexacoordinated trans-R2SnO4 geometry around tin in these compounds. The IS values for these lie in range 1.22–1.26 mms.− 1
The ρ-values (QS/IS) for the compounds fall in range 2.46–2.68 which demonstrated higher than four coordination at tin [Citation19,Citation25]. There is a distortion from perfect octahedral geometry in these diorganotin derivatives due to high electronegativity of carboxylate oxygens which give QS values similar to those for trigonal bipyramidal geometry.
Mass spectrometry
Main fragment ions observed in the mass spectra of a selected compound are listed in . Here primary decomposition occurs due to the elimination of one ligand, followed by loss of R-group attached to tin or CO2 molecules. The base peak is at m/z 220 which corresponds to the germatranyl ion in the compound [Citation25]. This species might result from the cleavage of Ge-C bond in parent ion. It has been reported [Citation21] that the fragmentation of germatrane takes place via two routes; the first of which is the loss of three formaldehyde molecules (m/z = 30) in succession from the molecular ion while the second route is associated with the elimination of OCH2CH2 moiety (m/z = 44) successively to give metal ion. Thus mass spectral data are supportive to the proposed molecular structures of the compounds and the absence of molecular ion peaks demonstrated that there is no intermolecular coordination.
Table VI. Mass fragment of a selected compound 2.
Biological studies
Antileishmanicidal data is presented in . The compounds (1–8) were screened for their antileishmanial activity against promastigotes of Leishmania major in vitro and demonstrated good activity. Leishmaniasis is group of human parasitic diseases caused by a group of parasitic protozoa in the genus Leishmania. Leishmania are digenetic in their life cycle i.e., they exist in two different morphological forms; the promastigotes and the amastigotes present in the insect and the mammalian host, respectively. The female sandfly injects these promastigotes into the skin of the mammalian host during blood meal whereas humans are accidental hosts.
Table VII. In vitro antileishmanial activity data for compounds (1–8).
Compounds 1–8 were randomly screened for their antileishmanial activity and almost all of these germatranyl substituted organotin carboxylates exhibited potential leishmanicidal activity and their IC50 values fall in the range of 1.3–12.25 μg/mL. The antileishmanial activities of newly synthesized complexes 1–8 was compared with the standard drug pentamidine (IC50 = 2.56 μg/mL). The activity of each compound was determined in triplicate. Compound 2 was found to be the most active compound of the series having IC50 value 1.30 μg/mL, which is even better than standard pentamidine. A limited structure activity relationship (SAR) study suggests that the activity of compounds largely depends upon the R1 and R2 groups. In compound 1, where R1 and R2 are phenyl and (CH2Si(CH3)2C6H5) showed less activity (IC50 = 12.15 μg/mL) as compared to compound 2, where R1 is p-CH3C6H4 and R2 is (CH2Si(CH3)2C6H5), which clearly indicates that suitable R1 substitution is responsible for antileishmanial activity. However, comparison of activities of compounds 4 and 5 suggests that activity is also depends upon R2 groups as reflected by less activity of compound 5 (R2 = CH2C6H5) than compound 4 (R2 = CH2Si(CH3)2C6H5). The study demonstrated that the nature and position of the substituent on the phenyl ring is crucial for displaying the antileishmanial activity as in case of compound 3 (R1 p-FC6H4) which is found to be inactive. However, there is notable variation in the activity with changes in the nature of R1 and R2 groups present in the complex. In conclusion, compound 2 was found to be a potential lead compound for further study in the search for a better antileishmanial chemotherapeutic agent.
Acknowledgements
U.S. is thankful to MoST. Govt. of Pakistan for the award of S & T Scholarship and Dr. Imtiaz-ud-Din is grateful to the Punjab Education Department for the grant of study leave for doing post-doctoral research.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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