SYNTHESIS OF NOVEL SPIROTHIAZOLIDINONE DERIVATIVES AND EVALUATION OF ANTI-INFLAMMATORY ACTION

SYNTHESIS OF NOVEL SPIROTHIAZOLIDINONE DERIVATIVES AND EVALUATION OF ANTI-INFLAMMATORY ACTION

Ashish Kumar Yadav, Muraree Lal, Munesh Singh Bhadauria, Narottam Singh and Avinash K. Kondalkar *

Sun Institute of Pharmaceutical Education and Research, Lahar, Madhya Pradesh, India.

ABSTRACT: The objective of this investigation was to synthesize spirothiazolidinone-chalcone derivatives and evaluate them for anti-inflammatory action. Five 4-substituted benzaldehydes were utilized for the aldol condensation leading to chalcones of spirothiazolidinone. The compounds were obtained in yield of 64-72% and displayed varying solubility with all compounds soluble in chloroform and DMSO. The NMR spectra revealed protons of amine, imine, hydroxy and aromatic groups. The presence of the molecular ion peak of the isotopic peak was found in the mass spectra of the compounds confirming the formation of the compounds. The anti-inflammatory activity was determined using the albumin denaturation method and antiprotease method. All the compounds exhibited dose dependent inhibition of albumin denaturation with 7d having the highest capacity to cause the inhibition (61.56 ± 1.033 %) at the concentration of 500µg/mL. The antiprotease action was also dose dependent and 7d at 500µg/mL was able to inhibit (46.32±3.011 %) of protease activity. The type of substitution on the chalcone phenyl ring played a vital role in the activity of the compounds. The results led to the conclusion that newer chalcone based molecules with anti-inflammatory activity were obtained from the current work.

Keywords: Anti-inflammatory, Chalcone, Spirothiazolidinone, Albumin Denaturation, Protease

INTRODUCTION: Spirocyclic compounds isolated from plant and animal origins have important applications in medicinal chemistry ¹. Spiro compounds having cyclic structures fused at a central carbon are of recent interest because of their interesting conformational features and their structural implications for biological systems. The asymmetric characteristic of the molecule due to the chiral spiro carbon is one of the important criteria of biological activities.

Spiro compounds represent an important class of naturally occurring substances characterized by their highly pronounced biological properties ^2-6. Although, a number of molecules have been developed as antimicrobial agents. But the demand for the effective and potent antimicrobial agent is always on high priority due to the development of resistant for the current drugs.

Spiro moiety is part of various natural products and medicinal agents. In literature, it has been reported that sharing of the indole-3 carbon atom during formation of spirothiazolidinone derivatives greatly enhances its biological activity. Thus, considering this fact, we planned to synthesize some spirothiazolidinone derivatives to improve antimicrobial efficacy.

 MATERIAL AND METHODS:

Materials: All the reagents and chemicals were used as obtained. All the synthesized compounds were characterized for melting point, solubility, yield and elucidation of the structure ⁷. The structure elucidation was performed by spectroscopic analysis (NMR, Mass and IR). Melting point were determined on Biotechnics melting point apparatus using open capillary. The purity and homogeneity of the compounds was determined by thin layer chromatography, using silica gel G as the stationary phase on glass plates using hexane: ethylacetate in the ratio 8:2.

The synthesis of the desired compounds was achieved using scheme 1.

SCHEME 1: SYNTHETIC SCHEME

Preparation of 3,4-thiazolidinedione: To 0.6 mole of chloroacetic acid, 1 in 60 ml of water, followed by the addition of 0.6 mole of thiourea, 2. The reaction mixture was stirred until the completion of the reaction (white precipitate was formed, approximately 15 min.) and refluxed under microwave irradiation at 240 Watt power until the completion of the reaction (approximately 12 min). On cooling, a solid separated, which was recrystallized from ethyl alcohol to give the pure thiazolidine-2,4-dione, 3 ⁸.

Preparation of Hydrazine Carbothioamide Derivative, 5: Thiazolidine-3,4-dione, 3 (0.94 mmol) was taken in a mixture of thiosemicarbazide, 4 (0.95 mmol) and glacial acetic acid (3.5 mL) and the reaction mixture was irradiated under microwave for 50 sec pulses.

The progress of the reaction was monitored by TLC (EtOAc: Hexane, 1:4). On completion of reaction (3-4 min), the reaction mixture was cooled to room temperature. A yellow solid was obtained, which was filtered off and washed with hexane and recrystallized from methanol to give the hydrazinecarbothioamide compound as a pale yellow crystalline solid.

Preparation of Triazaspiro Derivative, 6: Compound 5 (0.53 mmol) and freshly distilled acetic anhydride (2.5 mL) were irradiated under microwave for 5 minutes. The progress of the reaction was monitored by TLC (EtOAc: CHCl₃, 1:3). The reaction mixture was cooled to room temperature. A yellow solid was obtained, which was filtered off and crude solid was recrystallized from MeOH to give compound 6.

General Method for Synthesis of Compounds 7a-e: In a fume hood was placed a two-necked flask, equipped with a magnetic stirring bar, and reflux condenser was added 0.2g of the compound 6 and 10 mL of dried THF. The round bottom flask is placed in an ice bath kept on a stirring plate and 2 equivalent of desired benzaldehyde was slowly added to it. The two-necked flask is then placed in the microwave and irradiate at constant power of 400 W for 15 minutes. After this period the reaction mixture was poured into a vessel containing ice (30 g) and hydrochloric acid was added to it to adjust pH to 2.5. The formed solid is filtered using a vacuum filtration apparatus.

Inhibition of Albumin Denaturation: The technique of inhibition of albumin denaturation reported previously ^{9, 10} was used with slight modifications. The synthesized molecules were individually dissolved in DMSO and appropriately diluted to prepare solutions of 100, 200, 300, 400 and 500 µg/mL concentration.

A solution of 1% BSA in deionized water was prepared for the test. The reaction vessel was filled with 200 µL of BSA, 1400 µL of PBS and 1000 µL of the test solutions. Ibuprofen solution (1 µg/mL) was used in the positive control and distilled water was used in the negative control vessels instead of test solution. The reaction mixtures were incubated at 37°C for 15 min and then heated at 70°C for 5 min. The mixtures were then allowed to cool to room temperature and the absorbance of constituent of each vessel were analyzed in UV-Visible spectrophotometer at 660 nm. The inhibition of percent denaturation of albumin was determined using the following formula:

% Denaturation inhibition = (1−D/C) × 100%

Where D is the absorbance reading of the test sample, and C is the absorbance reading without test sample (negative control).

Antiprotease Action Method: The technique of antiprotease action reported by Oyedepo et al ¹¹ and Sakat et al ¹² was used with slight modifications. The reaction mixture was prepared with 0.06 mg trypsin, 1 mL 20 mMTris-HCl buffer (pH 7.0) and 1 mL test sample of different concentrations (100 - 500 µg/mL).

The mixture was incubated at 37°C for 5 min followed by the addition of 1 mL of 0.8% w/v solution of casein in water. The mixture was incubated additionally for 20 min. In order to stop the reaction, 2 mL of 70% perchloric acid was added to the mixture. The turbid suspension obtained after the reaction was centrifuged and the absorbance of the supernatant was recorded at 210 nm against buffer as blank. The percentage inhibition of protease inhibitory activity was calculated by the following formula:

Percentage inhibition = (Abs control –Abs sample) x 100/ Abs control

RESULTS: Five 4-substituted benzaldehydes were utilized for the aldol condensation leading to chalcones of spirothiazolidinone. The compounds were characterized for yield, melting point, solubility and retention factor (by TLC). The physicochemical properties of the compounds 7a-e using the various aromatic aldehydes is presented in Table 1.

TABLE 1: PHYSICOCHEMICAL FEATURES OF SPIROTHIAZOLIDINONE-CHALCONE COMPOUNDS, 7A-E

The solubility of the synthesized compounds 7a-e was determined in water, methanol, chloroform and DMSO. The compounds displayed varying solubility with all compounds soluble in chloroform and DMSO.

The structure of the compounds was determined using proton NMR in CDCl₃ solvent, IR and Mass study.

(E)-N-(2-cinnamoyl-7-oxo-1, 8-dithia-2, 3, 6-triazaspiro [4.4] non-3-en-4-yl) acetamide, 7a: Molecular Formula: C₁₅H₁₄N₄O₃S₂; FT-IR (cm^-1): 3342 (N-H), 3907 (C-H aliphatic), 1690.78 (C=O); 1H-NMR (DMSO-d6) (d ppm): 2.087 (N-H), 2.541 (CH methylene), 3.881 (CH methoxy), 6.962-7.980 (CH Aromatic); Mass (m/e): 365.1 (M⁺+2).

(E)-N-(2-(3-(4-hydroxyphenyl) acryloyl)-7-oxo-1, 8-dithia-2, 3, 6-triazaspiro[4.4] non-3-en-4-yl) acetamide. 7b: Molecular Formula: C₁₅H₁₄N₄O₄S₂; FT-IR (cm^-1): 2925 (C-H aliphatic), 1741 &1683.50 (C=O); 1H-NMR (DMSO-d6) (d ppm): 2.087 (N-H), 2.750-2.541 (CH methylene), 3.881 (CH methoxy), 6.962-7.980 (CH Aromatic); Mass (m/e): 379.0 (M⁺).

(E)-N-(2-(3-(4-chlorophenyl)acryloyl)-7-oxo-1,8-dithia-2, 3, 6-triazaspiro[4.4] non-3-en-4-yl) acetamide, 7c: Molecular Formula: C₁₅H₁₃ClN₄O₃S₂;FT-IR (cm^-1): 3340.46 (N-H), 2756.18 (C-H aliphatic), 1699.89 (C=O); 1H-NMR (DMSO-d6) (d ppm): 2.082 (N-H), 2.539 (CH methylene), 3.884 (CH methoxy), 6.952-7.983 (CH Aromatic); Mass (m/e): 517.0 (M⁺).

(E)-N-(2-(3-(4-nitrophenyl) acryloyl)-7-oxo-1, 8-dithia-2, 3, 6-triazaspiro[4.4] non-3-en-4-yl) acetamide, 7d: Molecular Formula: C₁₅H₁₃N₅O₅S₂; FT-IR (cm^-1): 2759.22 (C-H aliphatic), 1741.65 &1682.94 (C=O); 1H-NMR (DMSO-d6) (d ppm): 2.085 (N-H), 2.576 (CH methylene), 3.980 (CH methoxy), 6.807-8.018 (CH Aromatic); Mass (m/e): 407.5 (M⁺).

(E)-N-(7-oxo-2-(3-(p-tolyl)acryloyl)-1, 8-dithia-2, 3, 6-triazaspiro[4.4]non-3-en-4-yl)acetamide, 7e: Molecular Formula: C₁₆H₁₆N₄O₃S₂; FT-IR (cm^-1): 2756.14 (C-H aliphatic), 1748.01 &1690.93 (C=O); 1H-NMR (DMSO-d6) (d ppm): 2.085 (N-H), 2.576 (CH methylene), 3.980 (CH methoxy), 6.807-8.018 (CH Aromatic); Mass (m/e): 377.0 (M⁺).

Anti-inflammatory Action: The anti-inflammatory action of the synthesized compounds was evaluated using two of the well established in-vitro methods viz., antiprotease activity and inhibition of albumin denaturation. The results are presented in table 2 and 3 respectively.

TABLE 2: INHIBITION OF ALBUMIN DENATURATION BY TEST COMPOUNDS

ND-Not Determined; n=5; Values are Mean ± SD

TABLE 3: PERCENT INHIBITION OF PROTEASE ACTION BY TEST COMPOUNDS

ND-Not Determined; n=5; Values are Mean±SD

DISCUSSION: The synthesis of the spirothiazolidinone compounds was done via the reaction of thiazolidine-3,4-dione with thiosemicarbazide under MW-irradiation to yield Schiff bases which were subjected to intramolecular cyclization in presence of acetic anhydride resulting in the formation of the spiro compound. The spiro compound was reacted with various aromatic aldehydes using aldolcondenstation resulting in the formation of the desired chalcone derivatives. Previous study has shown that microwave assisted synthesis of spirothiazolidines using water as solvent resulted in 90-95% yield of the compound1 ¹³.

In the IR spectrum, the sharp absorption bands at 3340 corresponding to NH stretching and 1740 to 1680 corresponding to carbonyl groups were prominent along the fingerprint bands of the aromatic system. The NMR spectra revealed protons of amine, imine, hydroxy and aromatic groups. The presence of the molecular ion peak of the isotopic peak was found in the mass spectra of the compounds confirming the formation of the compounds.

The anti-inflammatory activity was determined using the albumin denaturation method and antiprotease method. Protein denaturation has been significantly correlated with the occurrence of the inflammatory response and may lead to various inflammatory diseases including arthritis. It has been said that tissue injury might be due to denaturation of the protein constituents of cells or of intercellular substance. Hence, the ability of the test compounds to inhibit the denaturation of protein signifies obvious potential for anti-inflammatory activity. It has also been reported that leukocytes protease has an important role in the development of tissue damage during inflammatory reactions and significant level of protection could be provided by protease inhibitors. Hence the inhibition of protease action by test compounds signifies its role as anti-inflammatory molecules.

All the compounds exhibited dose dependent inhibition of albumin denaturation with 7d having the highest capacity to cause the inhibition (61.56 ± 1.033 %) at the concentration of 500µg/mL. The antiprotease action was also dose dependent and 7d at 500µg/mL was able to inhibit (46.32±3.011 %) of protease activity. The type of substitution on the chalcone phenyl ring played vital role in the activity of the compounds. It was evident from the results that the higher the electron withdrawing ability of the substitution attached on this ring, the better was the anti-inflammatory activity of the compound. The order of activity was 7d>7c>7e>7a>7b.

CONCLUSION: In the present study, spriothiazolidinone-chalcone compounds were synthesized using the reaction of thiazolidinedione and thiosemicarbazide to prepare the reactive intermediate which underwent intramolecular cyclization and finally aldol condensation. The compounds were found to be of good purity and yield. The compounds exhibited good antibacterial potential against gram negative bacterium tested. Two compounds 7d & 7c were found to be significantly potent. These compounds would be used for designing newer similar compounds with the aid of computer aided drug design techniques like pharmacophore modeling or docking which might lead to generation of a new lead molecule for antiinflammatory activity.

ACKNOWLEDGMENT: The authors would like to thank RB Science, Bhopal for their kind help in interpretation of the results and performing the experiments.

CONFLICT OF INTEREST: The authors have no conflicts of interest regarding this investigation.

REFERENCES:

1. Khurshid IM, Navedul H, Bahlul ZSA and Mohammad Z: World J Pharm Pharmaceut Sci 2014; 3(12): 536–563.
2. Berghot MA and Moawad EB: Eur J Pharm Sci 2003; 20(2): 173–179.
3. Vidya S, Priya K, Jayasree DV, Deepthi A and Biju PG: Synthesis of heterocycle appended spiro(oxindole-3,2’-pyrrolidine) derivatives from heterocyclic ylidenes and azomethineylide through 1,3-dipolar cycloaddition reactions. Synthetic Communications 2019; 49(12): 1592-1602.
4. Mali PR, Shirsat PK, Khomane N, Nayak L, Nanubolu JB and Meshram HM: 1,3-Dipolar cycloaddition reactions for the synthesis of novel oxindole derivatives and their cytotoxic properties. ACS Combinatorial Science 2017; 19: 633-639.
5. Romo PE, Insuasty B, Abonia R, Crespo MDP and Quiroga J: Synthesis of new oxindoles and determination of their antibacterial properties. Heteroatom Chemistry 2020; 8021920.
6. Oktavia SH, Cahyana AH, Hapsari M, Tri Yunarti R and Rimus Liandi A: Synthesis and antimicrobial activity of spirooxindole-chromene derivative compounds based curcuminoid and chalcone. RJC 2021; 14(3): 1990-1997
7. Mishra R and Jain S: Investigation of antimicrobial potential of some thiazolylchalcone derivatives. Pharmacology Online 2013; 1: 190-193.
8. Meng G and Zheng ML: An efficient one-step method for the large- scale synthesis of 2,4-thiazolidinedione. CSIR J Consortium 2008; 40 (60): 572-573.
9. Singh AP and Mishra B: Evaluation of anti-inflammatory potential of Rutin using in-vitro J Pharmacol Biomed 2020; 4(1): 211-217.
10. Kumari S, Yasmin N, Hussain MR and Babuselvam M: In-vitro antiinflammatory and anti-arthritic property of Rhizoporamucronata leaves. International J Pharm Sci Res 2015; 6: 482-485.
11. Oyedepo OO and Femurewa AJ: Anti‐protease and membrane stabilizing activities of extracts of Fagraz-anthoxiloides, Olaxsubscorpioides and Tetra-pleuratetraptera. Inter J Pharmacogn 1995; 33: 65‐69.
12. Sakat S, Juvekar AR and Gambhire MN: In-vitro antioxidant and anti-inflammatory activity of methanol extract of Oxalis corniculata International J Pharma Pharmacological Sci 2010; 2: 146-155.
13. Mamum HM, Foysal MA and Mahabub M: Al-Amin. microwave-assisted efficient synthesis of isatins and spiro-thiadiazolines under green chemistry protocol. Journal of Scientific Research 2010; 2(2): 322-329.
    

    ---

    How to cite this article:

    Yadav AK, Lal M, Bhadauria MS, Singh N and Kondalkar AK: Synthesis of novel spirothiazolidinone derivatives and evaluation of anti-inflammatory action. Int J Pharm Sci & Res 2025; 16(3): 727-32. doi: 10.13040/IJPSR.0975-8232.16(3).727-32.

    ---

    All © 2025 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.