Synthesis, characterization and pharmacological evaluation of novel spiro heterocyclic compounds as anti diabetic agents

 

K. Kishore Kumar, R.S.K. Sharma, P. Chanti Babu, M. Sreenivasa Rao, K. Durga Prasadu,

D. Ravi Kumar^*

Department of Chemistry, CMJ University, Shillong-793101, India

*Corresponding Author E-mail: kishor_kumar2827@rediffmail.com

Natural products possessing a spiro carbon in their molecules have proven their biological activity in agriculture and medicine. The present paper describes the synthesis, characterization of five newly synthesized Spiro-compounds characterized by means of chromatography, IR, ¹H-NMR and Mass spectral analysis. The author carried in vitro anti diabetic methods like alpha amylase and alpha glucosidase methods for anti diabetic activity of tilted compounds. The investigation of anti diabetic activity revealed that the test compounds of Spiro compounds showed favorable anti diabetic activity. Among the titled compounds some were having potent antidiabetic activity.

 

 

 

1. INTRODUCTION:

Spiro hetero cyclic compounds are present in a large number of plants and animals. They also act as nicotinic receptor antagonists and have anticancer, antibiotic and antimicrobial properties. Existing literature survey reveals that very few spiro hetrerocyclics were prepared. The review on some spiro heterocyclics and the details of such compounds were mentioned. Spiro [pyrrolidin-3,3-indole] ring system is a recurring structural motif in a number of natural products which function as cytostatics and are of prime importance in cancer chemotherapy [1].

 

The derivatives of spiro-oxindole find very wide biological application [2]. Bodo et al [3] prepared spiro compound, Horsfiline, an oxindole alkaloid containing a spiro-[indole-pyrrolidone] nucleus and is a novel Ca₂C antagonist, norsesquiterpenoid spirolactones isolated from marine sources and was reported [4]. Maligre et al[5] synthesized a non-peptidal (K)-spirobyclic NK-1 receptor antagonist. Hobert and co-workers [6] have reported the synthesis of spirodiones by using α-hydroxy allyl ester. Pigge et al [7] synthesised novel cyclohexadienyl azaspiro cyclic ruthenium complexes by using the corresponding N- benzyl acetoacetamide derivatives as the synthon component. Pardasani et al [8] described the synthesis of spiro-oxazolidinone and spiro-pyrrolidine derivatives. Chen et al [9] have reported an efficient synthesis of Alantrypinone by a hetero Diels-Alder reaction of a novel pyrazine diene with 3-alkylidene oxindole through a spiro intermediate. Schiff bases followed by cycloaddition reaction with chloroacetyl chloride and or mercaptoacetic acid to give spiro β-lactam derivatives and spiro thiazolidinone[10]. α-arylidene cyclohexanones had been prepared [11] via Claisen-Schmidt condensation, while N-arylidene benzylamines Schiff bases had been synthesized. The 1, 3-anionic cycloaddition of Schiff bases to α-arylidene cyclohexanones gives the pyrrolidines. An N-spiro quaternary ammonium salt was synthesized [12] and was found to catalyse α-alkylation of glycine schiff bases with enantioselectivities. Based on the review of spiro heterocyclics, there is a need to focus on the synthesis and characterization of some novel spiro compounds possessing biological activity. The author in the present work aimed and succeded in synthesis, characterization of some newer spiro compounds possessing anti diabetic activity. 

 

2. EXPERIMENTAL:

2.1 Materials and methods:

All chemical in the present study for synthesis of new spiro heterocyclic compounds from Sigma Aldrich (LR grade) and Merck were used in the present synthesis work.

 

2.2 Instruments used:

The instruments such as Nuclear Magnetic Resonance spectra are recorded on varian Gemini-200 and ¹³C-NMR spectra are recorded on an avance-500 MHZ instrument. The samples are made in chloroform-d (1:1) or/and DMSO-d6 using tetra methyl silane (Me₄Si) as the internal standard. Elemental analysis is carried out on VARFIO EL, se Elementor. Analytical Thin-layer Chromatography (TLC) is performed on pre coated silica-gel-60 F254 (0.5mm) glass plates. Yields of materials judged homogenous by TLC and NMR spectroscopy. Melting points were recorded on Melter Fp-51 instrument and were uncorrected were used for the characterization of synthesized compounds.

 

2.3 General procedure for Ugi reaction and ipsocyclization:

Solution of p-anisidine (2.43 mmol) in methanol (8 mL), aldehyde (2.43 mmol) was added in continuous stirring at room temperature and allowed to stir for 4-5 min. To that mixture, propionic acid (2.43 mmol) fallowed by isocyanide (2.43 mmol) was added and stirred for 12-18 hrs. After consumption of all the substrates based on TLC, methanol was evaporated. And the compound was dissolved in small amount of DCM and afterwards precipitated was collected on a filter paper, and dried under vacuum. (Scheme-1)

 

Ugi product was dissolved in acetonitrile (8 mL), to this iodine (4.86 mmol) was added fallowed by solid sodium bicarbonate (7.29 mmol) and the mixture was allowed to stir for 6-8 hrs at room temperature. After completion of the reaction, the solvent was removed under vacuum then added water to the residue and the mixture was extracted with ethyl acetate (50mL×4). The organic layer was dried on anhydrous Na₂SO₄and evaporated the solvent to obtain crude product that was further purified by column chromatography using ethyl acetate and hexane. The pure compounds were eluted at 30-40% of ethyl acetate with good yields. (Scheme-2)

 

2.4 Procedure for Suzuki coupling to synthesize final compounds: Azaspiro (4.5)-dieneone (0.16 mmol) was dissolved in toluene (4 mL) and palladium catalyst (0.016 mmol) was followed by boronic acid (0.32 mmol) and aqueous sodium carbonate solution at room temperature. The mixture was stirred at 60⁰ C for 6 hours and after conformed by TLC, The organic layer was dried on anhydrous Na₂SO₄ and evaporated the solvent to obtain crude product that was further purified by column chromatography using ethyl acetate and hexane. (Scheme-3). The various substituents present in the final compounds (A-F) were incorporated in the Table-1.

 

Scheme-1

 

Scheme-2

 

Scheme-3

 

 

2.5 Spectral characterization: All the synthesized compounds were purified by chromatographic techniques like TLC and column chromatography and were characterized by proton NMR and ¹³C-NMR spectral characteristics.

 

Compound-A:

¹H-NMR (500 MHz, CDCl₃); δ 8.05 (s, 1H), 7.87 (s, 1H), 7.46 (d, J = 8.1 Hz, 1H), 7.40 – 7.28 (m, 4H), 7.16 (m, 2H), 7.11 – 6.90 (m, 5H), 5.44 (s, 2H). ¹³C-NMR (125 MHz, CDCl₃); δ 151.16, 136.14, 135.78, 133.92, 129.25, 128.03, 126.23, 125.87, 123.35, 123.20, 121.38, 120.60, 118.44, 111.79, 111.05, and 48.37.

 

Compound-B:

¹H-NMR (500 MHz, CDCl₃); δ 7.70 (s, 1H), 7.40 – 7.29 (m, 3H), 7.23 – 7.05 (m, 6H), 6.56 (s, 1H), 6.29 (s, 1H), 5.62 (s, 2H). ¹³C-NMR (125 MHz, CDCl₃); δ 142.35, 135.28, 134.41, 129.28, 128.74, 128.06, 125.88, 123.17, 122.70, 119.93, 117.98, 111.44, 110.59, 110.56, 48.54, 48.43.

 

Compound-C:

¹H-NMR (500 MHz, CDCl₃); δ 8.66 (s, 1H), 8.57 (s, 1H), 7.96 – 7.86 (m, 2H), 7.39 (m, 1H), 7.31 – 7.19 (m, 5H), 7.17 – 7.10 (m, 2H), 6.22 (s, 2H). ¹³C-NMR (125 MHz, CDCl₃); δ 148.90, 137.28, 128.78, 127.74, 126.76, 125.36, 124.66, 119.31, 111.87, 49.31.

 

Compound-D:

¹H-NMR (500 MHz, CDCl₃); δ 7.70 (s, 1H), 7.40 – 7.29 (m, 3H), 7.23 – 7.05 (m, 6H), 6.56 (s, 1H), 6.29 (s, 1H), 5.62 (s, 2H).¹³C-NMR (125 MHz, CDCl₃); δ 142.35, 135.28, 134.41, 129.28, 128.74, 128.06, 125.88, 123.17, 122.70, 119.93, 117.98, 111.44, 110.59, 110.56, 48.54, 48.43.

 

Compound-E:

¹H-NMR (500 MHz, CDCl₃); δ 8.00 – 7.82 (d, 2H), 7.76 – 7.61 (d, 2H), 7.60 – 7.50 (d, 1H), 6.98 (m, J = 21.5, 9.8, 6.4 Hz, 2H), 6.19 (d, J = 1.2 Hz, 1H), 5.29 (s, 2H), 4.89 (s, 2H), 2.41 (d, J = 1.2 Hz, 3H).¹³C-NMR (126 MHz, CDCl₃); δ 165.69, 162.76, 160.88, 160.07, 155.06, 152.22, 133.54, 132.39, 129.96, 125.98, 114.90, 112.92, 112.26, 102.29, 59.84, 41.27, 30.94, 29.72, 18.69.

 

Compound-F:

¹H-NMR (500 MHz, CDCl₃) δ 8.51 – 8.29 (m, 2H), 7.84 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 8.7 Hz, 1H), 7.15 (t, J = 5.6, 2.5 Hz, 2H), 6.40 (d, J = 0.9 Hz, 1H), 5.48 (s, 2H), 4.75 (s, 2H), 2.61 (t, J = 9.3 Hz, 3H). ¹³C-NMR (126 MHz, CDCl₃); δ 190.65, 165.72, 162.76, 160.92, 160.07, 155.04, 152.27, 141.00, 133.13, 129.93, 129.40, 125.99, 114.89, 112.90, 112.29, 102.28, 59.86, 41.34, 18.71.

 

 

 

Table-1: List of various substituents present in the final compounds

 

 

2.6 Biological activity:

2.6.1 In vitro methods employed in anti diabetic studies

2.6.1.1 Inhibition of alpha-amylase enzyme: A starch solution (0.1% w/v) was obtained by stirring 0.1g of potato starch in 100 ml of 16 mM of sodium acetate buffer. The enzyme solution was prepared by mixing 27.5 mg of alpha-amylase in100 ml of distilled water. The colorimetric reagent is prepared by mixing sodium potassium tartrate solution and 3, 5 di nitro salicylic acid solution. Both control (Acarbose std. drug) and synthesized compound(s) were added with starch solution and left to react with alpha- amylase solution under alkaline conditions at 25ºC. The reaction was measured over 3 minutes. The generation of maltose was quantified by the reduction of 3, 5 dinitro salicylic acid to 3- amino-5- nitro salicylic acid. This reaction is detectable at 540 nm (Malik CP and Singh MB, 1980). Results of in vitro anti diabetic activity of alpha-amylase method were presented in the Table-2. α-amylase inhibitory activity of test compounds against % inhibition was shown in the Figure-1.

 

 

Figure-1: α-amylase inhibitory activity of test compounds

 

 

Table-2: In vitro anti diabetic activity of alpha-amylase method

 

 

 

 

2.6.1.2. Inhibition of alpha-glucosidase enzyme: The inhibitory activity was determined by incubating a solution of starch substrate (2 % w/v maltose or sucrose) 1 mL with 0.2 M Tris buffer pH 8.0 and various concentration of control (Acarbose std. drug) and the synthesized compound(s) for 5 min at 37°C. The reaction was initiated by adding 1ml of alpha-glucosidase enzyme (1U/mL) to it followed by incubation for 40 min at 35°C. Then the reaction was terminated by the addition of 2 ml of 6N HCl. Then the intensity of the color was measured at 540nm (Krishnaveni S, 1984).The detailed result was incorporated in the Table-3. α-glucosidase inhibitory activity of test compounds were shown in Figure-2.

 

Figure-2: alpha-glucosidase inhibitory activity of test compounds

 

Table-3: In vitro anti diabetic activity of alpha glucosidase method

 

3. RESULTS AND DISCUSSION:

All the synthesized compounds were tested for their in vitro anti diabetic potential at different concentrations from 0.2 to 1.0 mL by using enzymes alpha-amylase and alpha-glucosidase in order to check their percent inhibition. All compounds shows dose dependent increase in percentage inhibition. Results are shown in Table-2 and Table-3.

 

Among the above synthesized compounds data reveals all compounds have significant inhibitory activity, whereas compound E found to be most active against both enzymes.

 

4. CONCLUSION:

Spiro heterocyclic compounds for the present investigation were synthesis, characterization and physiological activity of Spiro derivatives. Synthetic studies could go positive as per the planning and as such in all the reactions carried out. The expected compounds alone could be obtained.

 All the synthesised five spiro compounds were characterized by means of chromatography and ¹H-NMR and ¹³C-NMR analysis. Biological activity data reveals that all the titled compounds have significant inhibitory activity. Further studies are required to validate the data through in-vivo anti diabetic activity.

 

5. REFERENCES:

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10.  Elkanzi NAA. Synthesis of some new isolated/spiro β- lactam and thiazolidinone incorporating fused thieno pyrimidine derivatives. Jour. Appl. Chem. 1(1); 2012:1-12.

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Received on 18.04.2017         Modified on 19.05.2017

Accepted on 15.06.2017         © AJRC All right reserved