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Synthesis, Spectral Characterization and Biological Evaluation of Organotin(IV) Complexes of Aniline Derivatives of Naturally Occurring Betulinic Acid.

Byline: Sabiha Khanam, Khadija Shahid, Muhammad Sirajuddin, Saqib Ali and Hameed Ullah

Summary: Betulinic acid (triterpene) has shown an immense potential towards the development of anticancer, antiviral, antimalarial, antifungal, antioxidant and antiprotozoal agents. Cis-platin (cytotoxic agent) has diverted attention of chemists towards organotin complexes with marked pharmacological activities. In present work aniline derivatives of Betulinic acid were synthesized followed by the synthesis of diorganotin and triorganotin metal complexes. These complexes were characterized by FT-IR and multinuclear NMR (1 H and 13 C) spectroscopy. The ligands and their organotin(IV) complexes were screened for antibacterial, antifungal and antioxidant activities. Compound L2 2 SnMe 2 was found with maximum antibacterial activity among the screened compounds. Compounds L1 2 SnMe 2 and L1 2 SnPh 2 were found with remarkable antifungal activity. Similarly L1, L1 2 SnBu 2, L2 and L2 2 SnMe 3 were remarked with good antioxidant activity.

Keywords: Betulinic acid; Triterpene; Organotin(IV) Complex; Antimicrobial activity; Antioxidant activity.

Introduction

Betulinic acid (3[beta],hydroxyl-lup-20(29)-en-28-oic acid) (Compound 1 in Scheme-1) is a lupane-structured pentacyclic triterpene that can be obtained from many fruits, plants, and vegetables, and higher amounts can be extracted from birch tree bark (Betula species), sycamore and eucalyptus bark, the bark of planes (Platanus species), and chemical or enzymatic oxidation of betulin (extracted in large amounts from white birch tree bark) [1-4]. A closely related compound, Betulin (Compound 1a in Scheme-1), is an important part of white-barked birch trees (Betula species) with yields up to 22% (dry weight) [5]. Betulinic acid and its derivatives (Synthesized by modification at the C-3, C-20 and C-28 positions) have been attributed various bioactivities, such as antitumor, antidepression, antimalarial, hepatoprotective, anti-inflammatory, anti-HIV, anthelmintic, antimalarial, antimicrobial, antioxidant activities, ECHO-6 enterovirus, influenza FPV/Rostock and influenza A [6-8].

The 3[beta]-hydroxyl found in Betulinic acid represents a readily available position for chemical variation [9]. Betulin, Betulinic acid and Betulonic acid antiviral activity was against herpes simplex type 1 [10].

An acetylated derivative, 3-O-acetylbetulinic acid, shows cytotoxicity related to Betulinic acid [11]. NVX-207 is one of novel, Betulinic acid derivative which potentially showed considerable anti-cancer activity in clinical studies in canine tumor patients who get resistance to treatment [12].

Experimental

Material and methods

Commercially accessible chemicals were utilized without further purification and all reagents were of scientific evaluation. All responses in anhydrous solvents were performed in oven dried containers. Solvents were dried prior to use according to the literature method [13].

Instrumentations

Melting point (Gallenkamp MPA350.BM2.5) apparatus was used to determine the melting points. Shimadzu FT-IR (Prestige-21) Spectrophotometer with ATR 8000A was used to record the infrared absorption spectra by total reflectance method. 1H and 13C NMR spectra were recorded on a Bruker ARX-300 MHz spectrometer at room temperature, with TMS as the internal standard. Shimadzu UV visible spectrophotometer (UV-1800) was used to measure the antioxidant activity of the synthesized compounds.

Procedure for ligand preparation

The ligand was synthesized from the Betulinic acid by the reported method as shown in the scheme-2[14].

Synthesis of Anilines from Ethyl Ester of Betulinic Acid

The reaction of 2-or 3-nitro aniline with Betulinic acid ethyl ester is shown in Scheme-3. 2-or 3-Nitro aniline and Betulinic acid ethyl ester were taken in a round bottom flask (1: 1). A lowest amount of ethyl alcohol was added to the reaction mixture to obtain a clear solution. The mixture was then refluxed for 5h. After the reaction was completed surplus ethanol was evaporated under reduced pressure through rotary evaporator and the desired product was obtained [15].

General Procedure for Preparation of Triorganotin Complexes (R3SnL)

Ligand (l mmol) solution was prepared in dry toluene (50 mL) and was further treated with triethylamine (l mmol) used as a base. The reaction mixture was then refluxed for 3 hours. Triorganotin (l mmol) was added to the reaction mixture and was refluxed for 8-10 hours. Rotary evaporator was used to remove the solvent and the product was recrystallized through chloroform [16-18]. The reactions of triorganotin complexes are shown in Scheme 4.

General Procedure for Preparation of Diorganotin Complexes (R2SnL2)

Ligand (L1/L2, 2 mmol) solution was prepared in dry toluene (50 mL) and was further treated with triethylamine (2 mmol). The reaction mixture was then refluxed for 3 hours. Diorganotin (1 mmol) was added to the reaction mixture and was refluxed for 8-10 hours. Rotary evaporator was used to remove the solvent and the product was recrystallized through chloroform [15-18]. The reactions of diorganotin complexes are shown in Scheme 5.

Results and Discussion

Aniline derivative of Betulinic acid (Ligands LI/L2) and its metal complexes were synthesized by the procedures described in the experimental section. Fourier Transform Infrared spectroscopy and NMR spectroscopy (1H, 13C) were used for the spectral characterization of these metal complexes. Solubility behavior of metal complexes is summarized in Table-1. The compounds were soluble in most of the organic solvents like ethanol, toluene, acetone, methanol, DMSO and chloroform.

FT-IR

The most characteristic bands of investigated ligands and metal complexes were recorded on FTIR Spectrophotometer in range of 4000-400 cm-1. Comparison of infrared spectra of free ligands and metal complexes facilitate the determination of coordination mode of ligands with tin.

The disappearance of OH band from C-28 position of Betulinic acid and emergence of new NH band at 3331 cm-1 and 3334 cm-1 verified the bonding of 2-and 3-nitroaniline derivatives of Betulinic acid, respectively. NH and C=O bonds were involved in formation of coordination complexes. Absorption bands corresponding to M-N (metal nitrogen) in range of 456-492 cm-1 and 457-491 cm-1 (nitro aniline derivatives) respectively provide supportive evidence of formation of new metal complexes.

1H-NMR

NMR spectra of synthesized ligands and their complexes were recorded using CDCl3 as solvent. The data of 1H-NMR and 13C-NMR Spectra are depicted in Table-5-8 respectively. The 1H NMR spectra of the metal complexes and ligands showed that most of the peaks are according to proposed structures. Structures of ligands were justified by the presence of proton signals at 7.95ppm and 6.26ppm for NH and NH2 of L1 and 7.96 and 6.25 ppm for NH and NH2 of L2. This slight shift in values revealed the M-N (meta-nitrogen) bond formation. Nitro anilines peaks were also observed in structure of ligands (6.89-8.01 for L1 and 7.02-7.62 for L2). Tri and diphenyltin complexes (2-and 3-nitroaniline derivatives) were confirmed by the proton signals at 7.30-7.33 and 7.29-7.31 ppm for phenyl hydrogens.

The presence of chemical shift values 1.56-1.62, 1.55-1.58, 0.79-0.81, 0.76-0.78 for butyl and methyl substituent in Bu3SnL, Bu2Sn L2, Me3SnL2, Me2SnL complexes of L1 and L2 verified that the proton signals are in agreement with proposed structures of metal complexes.

Table-1: Physical data of Ligands (L1/L2) and their Metal complexes.

Compound###Mol. Formula###Mol. Wt###Physical Appearance###M. P (AdegC)

Betulinic acid###C30H48O3###456###White Crystalline###318

Ligand (LI)###C36H50N204###574###Yellowish Crystalline###275

Ph3SnL1###C54H64N2O4Sn###923.8###Brownish Crystalline###210

Ph2SnL21###C83H108N4O8Sn###1408.4###Pale Yellow Crystalline###190

Bu3SnL1###C48H76N2O4Sn###863.8###Pale Yellow Crystalline###260

Bu2SnL21###C79H116N4O8Sn###1368.4###Yellowish Crystalline###135

Me3SnL1###C39H58N2O4Sn###737.5###Brownish Crystalline###200

Me2SnL21###C73H104N4O8Sn###1284.3###Brownish Crystalline###215

Ligand (L2)###C36H50N204###574###Yellowish Crystalline###275

Ph3SnL2###C54H64N2O4Sn###923.8###Brownish Crystalline###170

Ph2SnL22###C83H108N4O8Sn###1408.4###Pale Yellow Crystalline###185

Bu3SnL2###C48H76N2O4Sn###863.8###Pale Yellow Crystalline###220

Bu2SnL22###C79H116N4O8Sn###1368.4###Yellowish Crystalline###256

Me3SnL2###C39H58N2O4Sn###737.5###Brownish Crystalline###120

Me2SnL22###C73H104N4O8Sn###1284.3###Brownish Crystalline###180

Table-2: FT-IR data of ligands (L1/L2) and their complexes.

Complex###E(cm-1)

###C=C###C=O###C-N###NH###C-O###N-Sn

Ligand(L1)###1472###1653###1315###3334###1275###-

Me2SnL1 2###1477###1652###1322###-###1270###456

Me3SnL1###1463###1674###1324###-###1274###465

Bu2SnL1 2###1472###1648###1312###-###1260###492

Bu3SnL1###1473###1656###1310###-###1262###488

Ph2SnL1 2###1477###1654###1312###-###1272###487

Ph3SnL1###1465###1674###1311###-###1244###482

Ligand(L2)###1473###1653###1315###3331###1275###-

Me2SnL12###1469###1662###1320###-###1260###457

Me3SnL1###1475###1675###1321###-###1256###462

Bu2SnL12###1473###1654###1322###-###1234###491

Bu3SnL1###1472###1655###1310###-###1262###487

Ph2SnL12###1486###1658###1325###-###1272###488

Ph3SnL1###1469###1662###1320###-###1260###483

Table-3: 1H NMR Spectroscopic data, I' (ppm), of Ligand L1 and its complexes.

H. No.a###Comp. Noa###L1###L1SnPh3###L12SnPh2###L1SnBu3###L12SnBu2###L1SnMe 3###L12SnMe 2

1###a. CH###1.24###1.25###1.21###1.22###1.24###1.21###1.22

###b. CH###1.18###1.21###1.19###1.18###1.19###1.2###1.19

2###a.CH###1.22###1.22###1.21###1.22###1.21###1.09###1.20

###b.CH###1.19###1.23###1.24###1.21###1.22###1.21###1.45

3###CH###2.8###2.96###2.98###2.96###2.98###2.88###3.01

3'###OH###3.15###3.19###3.18###3.19###3.19###3.17###3.18

4###-###-###-###-###-###-###-###-

5###CH###1.43###1.47###1.44###1.47###1.48###1.49###1.43

6###a.CH###1.20###1.06###1.05###1.07###1.08###1.06###1.08

###b.CH###1.07###1.03###1.02###1.01###1.02###1.02###1.01

7###a.CH###1.22###1.22###1.19###1.21###1.22###1.21###1.0

###b.CH###1.08###1.16###1.17###1.18###1.17###1.19###1.16

8###-###-###-###-###-###-###-###-

9###CH###1.43###1.44###1.42###1.44###1.43###1.41###1.42

10###-###-###-###-###-###-###-###-

11###a.CH###1.22###1.25###1.24###1.25###1.24###1.27###1.23

###b.CH###1.17###1.20###1.18###1.19###1.21###1.20###1.20

12###CH###5.2###5.20###5.17###5.20###5.22###5.24###5.23

13###C###-###-###-###-###-###-###-

14###C###-###-###-###-###-###-###-

15###a. CH###1.22###1.20###1.22###1.23###1.20###1.24###1.21

###b. CH###1.18###1.17###1.18###1.18###1.16###1.18###1.19

16###a. CH###1.24###1.21###1.21###1.23###1.24###1.22###1.23

###b. CH###1.18###1.19###1.18###1.17###1.18###1.18###1.20

17###-###-###-###-###-###-###-###-

18###CH###2.69###2.77###2.76###2.75###2.76###2.75###2.76

19###a.CH###1.50###1.63###1.62###1.59###1.60###1.54###1.53

###b.CH###1.23###1.33###1.32###1.31###1.30###1.28###1.27

20###-

21###a.CH###1.30###1.30###1.29###1.30###1.31###1.29###1.30

###b.CH###1.21###1.21###1.20###1.23###1.22###1.21###1.22

22###a.CH###1.30###1.30###1.31###1.31###1.32###1.30###1.31

###b.CH###1.21###1.22###1.20###1.21###1.21###1.22###1.21

23###CH3###0.71###0.71###0.73###0.71###0.72###0.71###0.74

24###CH3###1.70###0.72###0.73###0.74###0.75###0.73###0.75

25###CH3###0.65###0.66###0.67###0.62###0.63###0.60###0.61

26###CH3###0.85###0.85###0.88###0.86###0.87###0.86###0.86

27###CH3###1.07###1.07###1.06###1.07###1.08###1.07###1.07

28###CO###-###-###-###-###-###-###-

29###CH3###0.65###0.66###0.68###0.64###0.65###0.62###0.63

30###CH3###0.71###0.71###0.72###0.73###0.74###0.70###0.71

31###-###-###-###-###-###-###-###-

32###CONH###7.95###-###-###-###-###-###-

33###C###-###-###-###-###-###-###-

34###CH###6.89###6.62###6.64###6.62###6.61###6.63###6,61

35###CH###7.59###7.21###7.20###7.22###7.21###7.22###7.23

36###CH###7.40###6.82###6.81###6.83###6.82###6.81###6.83

37###CH###8.01###7.21###7.22###7.23###7.21###7.22###7.21

38###CH###-###-###-###-###-###-###-

1'###NH 2 (aniline)###6.26###6.24###6.26###6.26###6.25###6.24###6.25

2'###Sn-Ph###-###7.30-7.33###7.29-7.31###-###-###-###-

3'###Sn-Bu and Sn-Me###-###-###-###1.56-1.6###1.55-1.58###0.79-0.81###0.76-0.78

13C NMR spectroscopy

Structures of Ligands (L1/L2) were confirmed by the formation of amide bond between Betulinic acid and 2 and 3-nitroaniline. It was further confirmed by presence of aniline (2 and 3-nitroaniline) carbon peaks (C-33 to C-38). The CH3 peaks showed almost same values in all compound. At C-3 a peak value 75.2-79.2 ppm indicated that OH is present in all complexes of L1 and L2. Peak at 144 ppm assigned to C-13showed the double bond in all complexes and ligands (L1/L2). The further study of these spectra showed valuable data for the structure confirmation of the synthesized complexes.

In case of LSnPh3, L2SnPh2, LSnBu3, L2SnBu2, LSnMe3, L2Sn Me2, complexes of L1 and L2 the chemical shift value for carbonyl group at C-28 was in the range of 177.5-179.5 ppm. In LSnPh3, L2SnPh2 the chemical shift value 134.5, 129.8, 128.8, 128.8, 129.0 and 134.2, 129.6, 128.4, 128.4, 128.4, 128.6 for phenyl carbon showed structural determination of complexes of L1 and L2. Similarly chemical shift values 28.6, 34.8, 21.5, 16.8 and 28.2, 35.6, 22.4, 15.4 for butyl and methyl confirmed the structures of LSnBu3, L2SnBu2 L2SnMe2, LSnMe3 L2SnMe2 and L2SnBu2 complexes (L1 and L2, aniline derivatives of Betulinic acid).

Table-4: 1H NMR Spectroscopic data, I' (ppm), of Ligand L2 and its complexes.

H. No.a###Comp. Noa###L2###L2SnPh3###L22SnPh2###L2SnBu3###L22SnBu2###L2SnMe 3###L22SnMe 2

1###a. CH###1.25###1.24###1.22###1.21###1.23###1.22###1.21

###b. CH###1.18###1.21###1.18###1.18###1.19###1.23###1.19

2###a.CH###1.23###1.23###1.21###1.22###1.21###1.09###1.20

###b.CH###1.19###1.22###1.24###1.21###1.22###1.21###1.46

3###CH###2.8###2.96###2.98###2.96###2.98###2.88###3.02

3'###OH###3.15###3.18###3.17###3.18###3.17###3.18###3.17

4###-###-###-###-###-###-###-###-

5###CH###1.43###1.47###1.45###1.47###1.47###1.48###1.43

6###a.CH###1.20###1.07###1.05###1.08###1.08###1.07###1.07

###b.CH###1.08###1.03###1.02###1.01###1.02###1.02###1.01

7###a.CH###1.22###1.23###1.18###1.21###1.23###1.21###1.02

###b.CH###1.09###1.16###1.17###1.18###1.17###1.19###1.16

8###-###-###-###-###-###-###-###-

9###CH###1.42###1.43###1.42###1.42###1.41###1.43###1.42

10###-###-###-###-###-###-###-###-

11###a.CH###1.22###1.25###1.23###1.25###1.22###1.27###1.24

###b.CH###1.18###1.20###1.18###1.19###1.21###1.20###1.20

12###CH###5.2###5.21###5.17###5.21###5.21###5.24###5.24

13###C###-###-###-###-###-###-###-

14###C###-###-###-###-###-###-###-

15###a. CH###1.23###1.20###1.22###1.23###1.20###1.24###1.21

###b. CH###1.18###1.17###1.18###1.18###1.16###1.18###1.19

16###a. CH###1.23###1.21###1.21###1.23###1.24###1.22###1.23

###b. CH###1.18###1.19###1.18###1.17###1.18###1.18###1.20

17###-###-###-###-###-###-###-###-

18###CH###2.68###2.77###2.76###2.75###2.76###2.75###2.76

19###a.CH###1.50###1.63###1.62###1.59###1.60###1.54###1.53

###b.CH###1.22###1.34###1.32###1.32###1.30###1.28###1.26

20###-

21###a.CH###1.31###1.31###1.29###1.31###1.32###1.26###1.30

###b.CH###1.21###1.21###1.21###1.20###1.22###1.23###1.22

22###a.CH###1.31###1.30###1.31###1.31###1.32###1.30###1.31

###b.CH###1.21###1.21###1.20###1.20###1.21###1.22###1.21

23###CH3###0.72###0.71###0.73###0.71###0.72###0.71###0.74

24###CH3###1.70###0.72###0.73###0.74###0.75###0.73###0.75

25###CH3###0.65###0.66###0.68###0.62###0.63###0.61###0.60

26###CH3###0.86###0.85###0.88###0.86###0.87###0.86###0.86

27###CH3###1.08###1.07###1.07###1.06###1.08###1.06###1.08

28###CO###-###-###-###-###-###-###-

29###CH3###0.66###0.66###0.68###0.64###0.65###0.62###0.63

30###CH3###0.72###0.72###0.71###0.73###0.75###0.71###0.70

31###-###-###-###-###-###-###-###-

32###CONH###7.96###-###-###-###-###-###-

33###C###-###-###-###-###-###-###-

34###CH###7.02###7.03###7.03###7.01###7.03###7.04###7.05

35###CH###7.46###7.21###7.20###7.22###7.21###7.22###7.23

36###CH###7.62###7.82###7.81###7.83###7.82###7.81###7.83

37###CH###-###-###-###-###-###-###-

38###CH###7.56###6.61###6.62###6.63###6.62###6.61###6.22

1'###NH2 (aniline)###6.25###6.24###6.26###6.26###6.25###6.24###6.25

2'###Sn-Ph###-###7.30-7.33###7.29-7.31###-###-###-###-

3'###Sn-Bu and Sn-Me###-###-###-###1.56-1.62###1.55-1.58###0.79-0.81###0.76-0.78

Biological activities

Antibacterial activity

Agar well diffusion method was employed for determination of antibacterial activity. The agar well diffusion assay consisted of making 10 mL aliquot nutrient broth. This broth was inoculated with bacteria and incubated at 37 +- 1 AdegC for 1 day. A sterilized pipette was utilized to put 0.6mL of broth culture of tested bacteria to molten agar cooled at 45AdegC contained in 9 cm petri dish. Agar was solidified then a sterile cork awl was used to prepare holes of 10 mm. Appropriate allocation of holes in margins and one in midpoint were made by taking out agar plugs. Each organism duplicate plates were prepared. Solution of ligand and metal complexes and tetracycline (reference drug) were prepared having concentration of l mg/mL. The test sample (100uL) was dissolved in proper solvent and added into appropriately labeled well. Tetracycline (l mg/mL) solution was utilized as a standard.

Samples were permitted to diffuse by placing the plates at room temperature for 2 hours and were placed in incubator at 37 +- 1 AdegC for one day faced upwards. Zone of inhibition in millimeters was measured and antibacterial activity was determined [19]. Aniline derivative of Betulinic acid (ligand) and their metal complexes were assessed for their antibacterial activity in vitro and zone of inhibition in mm was measured and antibacterial activity was determined [20]. It can be seen from Table-7 that all the synthesized compounds are against the studied bacterial strains. Against Streptococcus only L1 SnPh 3 is inactive while L2 2 SnMe 2 has the maximum activity among the screened compounds even than that of the standard drug, Tetracycline. The activity of L2 2 SnMe 2 and L2 2 SnPh 2 against B. Subtilis is higher even than that of the standard drug, Tetracycline. Against S. Aureus L2 SnPh 3 is the most active compound.

Table-5: 13C NMR data, I' (ppm), of Ligand L1 and its complexes.

###H. No.a###Comp. Noa###BA###L1###L1SnPh3###L12SnPh2###L1SnBu3###L12SnBu2###L1SnMe 3###L12SnMe 2

###1###CH2###39.1###39.07###38.2###38.1###38###38.2###37.8###39.1

###2###CH2###28.2###28.1###26.5###26.2###26.4###26.5###26.2###28.7

###3###CH###78.1###77.2###75.4###75.3###75.2###75.4###75.3###79.2

###4###C###39.4###39.3###37.8###37.5###37.4###37.8###37.5###39.4

###5###CH###55.8###55.2###55.6###54.7###55.1###55.6###54.7###55.2

###6###CH2###18.8###18.8###15.9###15.2###16.4###15.9###15.2###19.3

###7###CH2###33.3###33.3###32.7###31.9###32.2###32.7###31.9###33.2

###8###C###39.8###39.6###38.7###38.4###38.5###38.7###38.4###39.6

###9###CH###48.2###47.2###47.1###46.8###47.8###47.1###46.8###47.5

###10###C###37.4###38.2###35.6###35.2###35.8###35.6###35.2###37.1

###11###CH2###23.7###23.6###21.4###20.8###21.5###21.4###20.8###23.5

###12###CH###122.6###122.1###119.5###119.2###120.5###119.5###119.2###122.1

###13###C###144.7###144###144.2###144.3.###144.1###144.1###144.3###144.0

###14###C###42.2###42.09###40.7###40.2###41###40.7###40.2###41.7

###15###CH2###28.4###28.3###27.2###27.5###27.2###27.2###27.5###27.2

###16###CH2###23.8###23.7###22.4###22.6###21.9###22.4###22.6###23.8

###17###C###46.7###46.6###45.8###45.9###46.3###45.8###45.9###47.1

###18###CH###42###42.09###40.7###40.3###40.1###40.7###40.3###41.8

###19###CH###46.5###46.4###43.8###43###44.2###43.8###43###45.6

###20###CH###31###31.1###29.8###29.4###29.8###29.8###29.4###30.7

###21###CH2###34.3###34.2###32.9###32.5###33.2###32.9###32.5###34.4

###22###CH2###33.2###33.1###31.5###31.2###32.1###31.5###31.2###33.2

###23###CH3###28.8###28.7###25.4###25###25.1###25.4###25###28.5

###24###CH3###16.6###16.4###13.4###13.8###13.9###13.4###13.8###16.3

###25###CH3###15.6###15.5###12.8###12.9###13.1###12.8###12.9###15.5

###26###CH3###17.5###17.5###14.9###15.1###14.5###14.9###15.1###17.3

###27###CH3###26.2###26.1###25.7###25.4###25.2###25.7###25.4###26.1

###28###C###180.2###179.1###179.5###178.9###178.7###177.5###179.9###179.1

###29###CH3###33.3###33.2###30.7###30.6###30.8###30.7###30.6###33.2

###30###CH3###23.8###23.7###22.6###22.4###22.5###22.6###22.4###23.3

###31###CH3###33.3###33.2###30.7###30.6###30.8###30.7###30.6###33.2

###33###C###-###144.9###144.5###145.3###144.8###144.5###144.9###144.7

###34###CH###-###117.2###117.1###117.2###117.3###117.0###117.1###117.2

###35###CH###-###135.6###135.5###135.4###135.3###135.2###135.4###135.3

###36###CH###-###119.6###119.3###119.4###119.2###119.4###119.2###119.1

###37###CH###-###125.9###125.5###125.4###125.3###125.2###125.4###125.3

###38###CH###-###133.2###133.1###133.2###133.3###133.0###133.1###133.2

###1'###C###-###-###134.5###134.2###28.6###28.2###-###-

###2'###CH###-###-###129.8###129.6###34.8###35.6###-###-

###3'###CH###-###-###128.8###128.4###21.5###22.4###-###-

###4'###CH###-###-###128.8###128.4###16.8###15.4###-###-

###5'###CH###-###-###128.8###128.4###-###-###-###-

###6'###CH###-###-###129###128.6###-###-###-###-

Table-6: 13C NMR data, I'(ppm), of Ligand L2 and its complexes.

###H. No.a###Comp. Noa###BA###L2###L2SnPh3###L22SnPh2###L2SnBu3###L22SnBu2###L2SnMe 3###L22SnMe 2

###1###CH2###39.1###39.1###38.1###38.0###38.01###38.1###37.7###39.2

###2###CH2###28.2###28.2###26.4###26.2###26.4###26.4###26.2###28.6

###3###CH###78.1###77.2###75.4###75.2###75.2###75.3###75.4###79.2

###4###C###39.4###39.2###37.8###37.5###37.4###37.8###37.6###39.3

###5###CH###55.8###55.1###55.5###54.5###55.2###55.6###54.7###55.1

###6###CH2###18.8###18.7###15.9###15.2###16.4###15.9###15.2###19.2

###7###CH2###33.3###33.3###32.7###31.7###32.1###32.5###31.9###33.1

###8###C###39.8###39.7###38.6###38.4###38.5###38.7###38.3###39.6

###9###CH###48.2###47.3###47.1###46.6###47.8###47.2###46.8###47.5

###10###C###37.4###38.2###35.5###35.2###35.7###35.6###35.2###37.2

###11###CH2###23.7###23.6###21.4###20.7###21.5###21.3###20.6###23.5

###12###CH###122.6###122.2###119.4###119.2###120.5###119.5###119.2###122.2

###13###C###144.7###144.1###144.3###144.2.###144.0###144.2###144.1###144.0

###14###C###42.2###42.08###40.7###40.2###41###40.7###40.2###41.6

###15###CH2###28.4###28.3###27.1###27.6###27.1###27.1###27.6###27.2

###16###CH2###23.8###23.6###22.4###22.6###21.9###22.4###22.6###23.7

###17###C###46.7###46.6###45.8###45.9###46.3###45.7###45.8###47.1

###18###CH###42###42.08###40.7###40.2###40.1###40.7###40.3###41.7

###19###CH###46.5###46.3###43.8###43###44.1###43.7###43###45.6

###20###CH###31###31.1###29.6###29.3###29.8###29.6###29.3###30.6

###21###CH2###34.3###34.3###32.8###32.5###33.1###32.8###32.5###34.4

###22###CH2###33.2###33.1###31.4###31.1###32.1###31.5###31.2###33.1

###23###CH3###28.8###28.6###25.4###25###25.1###25.3###25.2###28.5

###24###CH3###16.6###16.4###13.4###13.7###13.8###13.3###13.8###16.4

###25###CH3###15.6###15.6###12.8###12.9###13.2###12.8###12.9###15.5

###26###CH3###17.5###17.5###14.8###15.2###14.5###14.8###15.2###17.2

###27###CH3###26.2###26.2###25.7###25.4###25.1###25.7###25.4###26.1

###28###C###180.2###179.1###179.6###178.5###178.7###177.4###179.9###179.2

###29###CH3###33.3###33.2###30.6###30.6###30.8###30.6###30.5###33.2

###30###CH3###23.8###23.6###22.6###22.3###22.4###22.6###22.4###23.4

###31###CH3###33.3###33.2###30.6###30.6###30.8###30.7###30.6###33.2

###33###C###-###149.3###148.5###148.3###147.8###147.5###146.9###147.7

###34###CH###-###121.1###121.1###121.2###121.3###121.0###121.1###121.2

###35###CH###-###130.3###129.5###129.4###129.3###129.2###129.4###129.3

###36###CH###-###113.9###113.3###113.4###113.2###113.4###113.2###113.1

###37###CH###-###148.7###148.5###148.4###148.3###148.2###148.4###148.3

###38###CH###-###108.1###108.1###108.2###108.3###108.0###108.1###108.2

###1'###C###-###-###134.5###134.2###28.6###28.2###-###-

###2'###CH###-###-###129.8###129.6###34.8###35.6###-###-

###3'###CH###-###-###128.8###128.4###21.5###22.4###-###-

###4'###CH###-###-###128.8###128.4###16.8###15.4###-###-

###5'###CH###-###-###128.8###128.4###-###-###-###-

###6'###CH###-###-###129###128.6###-###-###-###-

Table-7: Antibacterial activity of Ligands (L1/L2) and their complexes.

###Zone of inhibition

###Compound

###E. coli###B. subtilis###Streptococcus###S. aureus###S. typhi###Shigella

###Tetracycline###35###28###30###30###32###35

###Ligand (L1)###20###12###14###10###15###20

###L12SnMe 2###11###14###22###13###10###14

###L1SnMe 3###20###15###16###14###12###13

###L12SnBu2###21###10###22###0###10###16

###L1SnBu3###18###12###23###20###16###12

###L12SnPh2###17###18###12###16###21###20

###L1SnPh3###10###14###0###22###20###14

###Ligand (L2)###22###15###20###20###13###22

###L22SnMe 2###21###37###32###19###20###16

###L2SnMe 3###22###15###20###23###18###13

###L22SnBu2###25###15###21###16###16###16

###L2SnBu3###25###17###29###23###26###20

###L22SnPh2###26###33###25###20###25###27

###L2SnPh3###19###27###25###32###23###24

Antifungal Activity

Ligand and its metal complexes were also assessed for antifungal activity in vitro using agar tube diffusion method [21]. The minimum inhibitory concentrations (MIC) of the test sample were obtained. Preparation of Sabouraud dextrose agar was done by mixing Sabouraud 4% glucose agar with distilled water by stirring and providing heat. When media get prepared it was transferred into test tubes and was autoclaved at 121AdegC for 15 minutes. Up to 50AdegC autoclaved tubes were permitted to cool. In sterile DMSO individually having concentration of 20 ug/mL test samples were prepared. Standard solution used was Ketoconazole (20 ug/mL). Using a micropipette l00 uL, test samples solution and ketoconazole solution (standard) was shifted into various non-solidified tubes of Sabouraud agar media. Test tubes were placed in a slanting direction and at room temperature allowed to solidify.

By using 4 mm diameter portion of inoculums removed from seven day old culture of fungal strain each tube was inoculated. All tubes having culture were incubated for 7-10 days at optimal temperature of 28-30AdegC. During the incubation cultures were examined twice weekly. After 7-10 days of time period the incubated test tubes were observed and tubes which have not shown visible fungal growth were used to display the minimum inhibitory concentration (MIC) of the samples to be tested. MIC was measured in ug/mL [21]. Among the studied compounds L1 2 SnMe 2 and L1 2 SnPh 2 were the most potent compounds that shows small MIC compared to standard Nystatin drug.

Table-8: Antifungal activity data of Ligands (L1/L2) and their complexes.

Compound

###MIC (mg/mL)

###A. niger###A. gumigates###F. solani###F. oxysporum

Ligand (L1)###9.8###5.6###6.4###4.2

L12SnMe 2###2.1###1###2###1.2

L1SnMe 3###2.4###9.8###5.2###5

L12SnBu2###5.2###9.8###1.75###2

L1SnBu3###10.5###5###10.3###5

L12SnPh2###1.1###1.85###1.2###1.5

L1SnPh3###2.2###2###2.6###5.8

Ligand(L2)###4.5###7.6###5.5###10.2

L22SnMe 2###5.2###9.5###10.4###5.8

L2SnMe 3###9.8###10.8###5.5###5.4

L22SnBu2###5.1###5###4.85###2

L2SnBu3###4.75###5###1###1

L22SnPh2###10###1.2###1###5

L2SnPh3###1.8###9.5###2###5

Nystatin###0.5###1###0.25###0.25

Table-9: Antioxidant Activity data of Ligands (L1/L2) and their complexes.

###Compound###IC50 (ug/mL)###Compound###IC50 (ug/mL)###Compound###IC50 (ug/mL)

###Ligand (L1)###21###L12SnPh2###33###L22SnBu2###32

###L12SnMe 2###28###L1SnPh3###26###L2SnBu3###43

###L1SnMe 3###31###Ligand (L2)###18###L22SnPh2###36

###L12SnBu2###25###L22SnMe 2###40###L2SnPh3###31

###L1SnBu3###42###L2SnMe 3###27###Ascorbic acid###5.4

Antioxidant Activity

The hydrogen donating property of ligand and all the metal complexes were calculated by using 2,2-diphenyl-1-picryl-hydrazyl (DPPH) method, carried out according to the reported protocol [21-22]. Inhibitory action in percentage of DPPH radical was calculated according to formula and IC 50 value was measured by using graphical method [22-23]. To examine the Scavenging activity of ligands and metal complexes antioxidant assay was performed by using 1-diphenyl, 2-picrylhydrazyl (DPPH) free radical according to the procedure mentioned below.

In 100 mL of methanol 3.2 mg of DPPH was dissolved to get 0.1 mM stock solution. In a glass vial, a stock solution was prepared by dissolving 2800 uL of 0.1 mM DPPH solution (in methanol) with 200 uL of sample to be tested (in methanol). This stock solution was in sequence diluted with methanol to obtain final concentrations of 100 ppm, 50 ppm, 25 ppm, 10 ppm, 5 ppm, 2 ppm and 1 ppm. Mixture was shaken and placed in dark at controlled room temperature 25-28AdegC for 1 hour. The color change of DPPH free radical noticed was from deep violet to light-yellow. It was then calculated by taking the absorbance of the reaction mixtures at 517 nm on a UV/Visible spectrophotometer (Shimadzu 1800). 200 uL of methanol and 2800 uL of DPPH solution in methanol were used as control. Each concentration was assayed in triplicate. Inhibitory action in percentage of DPPH radical was calculated according to formula given below and IC50 value was measured by graphical method [22, 23].

% inhibition = A(C)o - A(A)t / A(C)o x 100

where A(C)o is the absorbance of the control at t = 0 min and AA(t) is the absorbance of the antioxidant at t = 1 h.

Antioxidant activity was measured in terms of IC50 value for ligands and synthesized metal complexes and is given in Table-9. L1 2 SnBu 2 and ligand (L1) exhibited significant antioxidant activity with IC50 value of 21 and 25 ug/mL and when antioxidant activity was observed with L2 metal complexes then L2SnMe3 and ligand (L2) showed significant activity with IC50 value of 18 and 27ug/mL, respectively.

Conclusions

After spectroscopic analysis it was observed that the structure of ligands (L1/L2) and their metal complexes were concurring with proposed structures. In FT-IR spectral analysis appearance of new NH bands at 3331 cm-1 and 3334 cm-1 verified the bonding of 2 and 3-nitroaniline to Betulinic acid and formation of ligands (L1/L2). Also N-M (nitrogen-metal) absorption bands displayed in the range of 456-492 I(cm-1) showed the evidence of metals in the structures of metal complexes of aniline derivatives.

1H NMR additionally fortifies the basic explanation of ligands due the presence of NH peaks at 7.95 ppm and 7.96 ppm (L/L). Aniline NH2 peaks were observed at 6.26 ppm and 6.25ppm (L1 L2). 13C NMR interpretation also provided useful information for structure elucidation of ligands and their metal complexes in agreement with proposed structures. In ligands the change in chemical shift value for carbonyl group at C-28 than the parent compound Betulinic acid and aniline peaks confirmed the structural exposition of ligands (L1/L2). The peaks appeared for phenyl, butyl and methyl groups further supported the structure of Organotin complexes.

The ligands (L1/L2) and their metal complexes were evaluated for biologicval activities like antibacterial, antifungal and antioxidant. The antibacterial activity data shows that L2 SnMe has the maximum activity among the screened compounds even with higher activity higher than that of the standard drug, Tetracycline. Antifungal activity data shows that L1 2 SnMe 2 and L1 2 SnPh 2 are the most active compounds compared to standard Nystatin drug. The antioxidant activity data demonstrate that L1, L1 2 SnBu 2, L2 and L2 2 SnMe 3 have remarkable antioxidant activity.

Acknowledgment

Financial support by the Higher Education Commission (HEC) of Pakistan is gratefully acknowledged.

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