Synthesis, antioxidant, anticoagulant, and fibrinolytic activities of new isatin derivative

Weam S El-Serwy; Neama A Mohamed; Walaa S El-Serwy; Emad M.M Kassem; Al Shimaa G Shalaby

doi:10.4103/epj.epj_52_19

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ORIGINAL ARTICLE

Year : 2020 | Volume : 19 | Issue : 2 | Page : 113-123

Synthesis, antioxidant, anticoagulant, and fibrinolytic activities of new isatin derivative

Weam S El-Serwy¹, Neama A Mohamed², Walaa S El-Serwy², Emad M.M Kassem², Al Shimaa G Shalaby¹
¹ Department of Chemistry of Natural and Microbial Products, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo, Egypt
² Department of Therapeutic Chemistry, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo, Egypt

Date of Submission	10-Jul-2019
Date of Acceptance	01-Dec-2019
Date of Web Publication	18-Jun-2020

Correspondence Address:
Assistant Professor (PhD) Weam S El-Serwy
Department of Chemistry of Natural and Microbial Products, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Cairo 12622
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/epj.epj_52_19

Abstract

Background Isatin as a product was first obtained from the oxidation of indigo dye by nitric acid and chromic acids by Otto Linné Erdman and Auguste Laurent in 1841.
Objective This study presents the synthesis of some new isatin derivatives and examines their biological activities.
Materials and methods 2-Cyano-N’-(5-nitro-2-oxoindolin-3-ylidene)acetohydrazide (2) reacted with some reagents, namely, salicylaldehyde, phenyl isothiocyanate, ethyl chloroacetate, ethyl iodide, ethyl cyanoacetate, thioglycolic acid, phenyl isothiocyanate, malononitrile, and hydrazine hydrate to produce compounds 3, 4, 5, 6, 8, 9, 10, 11, and 12, respectively. Moreover, compound 6 reacted with hydrazine hydrate to produce compound 7. Antimicrobial activities of some newly synthesized compounds were studied.
Results Antioxidant, anticoagulant, and fibrinolytic activities of the new synthesized compounds (1–12) were studied. Compound 10 exhibited highest fibrinolytic activity. On the contrary, compound 12 exhibited highest anticoagulant activities. Moreover, it was noticed that compound 9 exhibited highest antioxidant activity.
Conclusion In summary, 14 novel isatin derivatives were synthesized and screened for their antioxidant, anticoagulant, and fibrinolytic activities. Some compounds displayed moderate-to-excellent activities such as antioxidant, anticoagulant, and fibrinolytic agents.

Keywords: anticoagulant, antioxidant, nitro-isatin, pyridine, pyrimidine, thiazole

How to cite this article:
El-Serwy WS, Mohamed NA, El-Serwy WS, Kassem EM, Shalaby AG. Synthesis, antioxidant, anticoagulant, and fibrinolytic activities of new isatin derivative. Egypt Pharmaceut J 2020;19:113-23

How to cite this URL:
El-Serwy WS, Mohamed NA, El-Serwy WS, Kassem EM, Shalaby AG. Synthesis, antioxidant, anticoagulant, and fibrinolytic activities of new isatin derivative. Egypt Pharmaceut J [serial online] 2020 [cited 2023 Mar 29];19:113-23. Available from: http://www.epj.eg.net/text.asp?2020/19/2/113/287192

Introduction

It has been reported that several isatin derivatives have antioxidant [1], anticoagulant [2], fibrinolytic [3], antibacterial, anticonvulsant, antifungal, anti-HIV, and anti-inflammatory activities [4],[5]. Isatin as a product was first obtained from the oxidation of indigo dye by nitric acid and chromic acids by Otto Linné Erdman [6] and Auguste Laurent [7] in 1841. Isatin is a multifaceted heterocyclic compound found in some plants as a natural product, such as genus Isatis and in Couroupita guianensis Aubl [8],[9] and has also been present in humans as a metabolic derivative of adrenaline [10]. With broad clinical and pharmacological applications, a wide variety of isatin derivatives can be synthesized from isatin [8],[11],[12]. Derivatives of isatin have recently drawn considerable attention of researchers worldwide owing to their wide applications as anti-HIV [4],[5], anti-tubercular [13], anti plasmodial [14], anticonvulsant [15], and sedative and hypnotic [16] agents. Triazole as well as another class of azole group is a versatile pharmacophore, possessing diverse pharmacological properties [17],[18] such as herbicidal [19], antitumor [20], antipsychotic [21], anticoagulant [22], antimicrobial [23], and antagonist [24]. Among the available substituted hydrazines, cyanoacetohydrazide is a convenient intermediate for the synthesis of a variety of heterocyclic compounds [25],[26],[27],[28],[29],[30],[31],[32],[33],[34],[35],[36]. Schiff bases are found to be the most potent anticonvulsant agents [37], with a large spectrum in clinical application; they were studied by cyclic voltammetry, square wave, and differential pulse voltammetry over a wide pH range using a general certificate of education. The results of some studies of the electrochemical behavior of some isatin derivatives showed that the semicarbazone, nitro groups attached to the isatin ring and hydrazine give rise to separate and different oxidation and reduction mechanisms [38]. The literature survey revealed that the introduction of electron-withdrawing groups, for example, nitro group at positions 5, 6, and 7, greatly increased activity from that of isatin, with substitution at the fifth position being most favorable. This is not surprising, as C-5 substitution has previously been associated with increased biological activity for a range of indole-based compounds [39],[40]. Therefore, the aim of the present study was to examine the antimicrobial, antioxidant, and fibrinolytic and anticoagulation activities of synthesis of nitro-isatin and their derivatives by modification on structure.

Materials and methods

Chemistry

All melting points are uncorrected and were taken in open capillary tubes using Electrothermal apparatus 9100. Elemental microanalyses were carried out in Microanalytical Unit, Central Services Laboratory, National Research Centre, Dokki, Cairo, Egypt, using VarioElementar and were found within ±0.4% of the theoretical values. Infrared spectra were recorded on a FT/IR-4100 Jasco, Japan, Fourier transform infrared spectrometer at cm⁻¹ scale using KBr disc technique at Central Services Laboratory, National Research Centre, Dokki, Cairo, Egypt. ¹H NMR and ¹³C NMR spectra were determined by using a JEOL AS-500 NMR spectrometer at Central Services Laboratory, National Research Centre, Dokki, Cairo, Egypt. Varian Gemini200-Oxford 300 MHz and Merury Plus-Oxford 400 MHz at Ministry of Defense, Chemical Warfare Department, The Main Chemical Warfare Laboratories, Cairo, Egypt. Chemical shifts were expressed in δ (ppm) downfield from tetramethylsilane as an internal standard. The mass spectra were measured with a GC MSQp1000EX Shimadzu, Cairo University, Cairo, Egypt, and with a Finnigan MAT SSQ-7000 mass spectrometer at Central Services Laboratory, National Research Centre, Dokki, Cairo, Egypt. Follow-up of the reactions and checking the purity of the compounds were made by thin-layer chromatography on silica gel-precoated aluminum sheets (Type 60, F 254; Merck, Darmstadt, Germany) using chloroform/methanol (20 : 2, v/v), and the spots were detected by exposure to UV lamp at λ254 nanometer for few seconds and by iodine vapor. The chemical names given for the prepared compounds are according to the International Union of Pure and Applied Chemistry system.

The following compounds were synthesized:

2-Cyano-N’-(5-nitro-2-oxoindolin-3-ylidene)acetohydrazide (2): it was a mixture of nitro-isatin [41] (1.92 g, 0.01 mol) and cyanoacetohydrazide (0.99 g, 0.01 mol) in 1,4-dioxane (20 ml) warmed for 5 min. After slow evaporation, the solid which separated was collected by filtration and then recrystallized from 1,4-dioxane to give 2. Yield: 85%; M.p. 230–232°C. IR spectrum (KBr, ν, cm⁻¹): 3200, 3189 (2NH), 2218 (C<td:glyph name="tbnd6"/>N), 1714, 1694 (2C=O). ¹H NMR (DMSO-d6) δ: 3.1 (s, 2H, CH₂), 7.8–8.6 (m, 3H, Ar-H), 10.2, 10.5 (2s, 2H, 2NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆): δ 27.1, 117.5, 122.9, 123.0, 124.6, 126.7, 133.9, 142.8, 147.1, 167.5, 171.8. MS: m/z=273 (27.5%) (M ^· ⁺); Anal. Calcd. For C₁₁H₇N₅O₄ (273.2): C, 48.36; H, 2.58; N, 25.63%; found: C, 48.16; H, 2.48; N, 25.59%.

N’-(5-nitro-2-oxoindolin-3-ylidene)-2-oxochromane-3-carbohydrazide (3): it is a mixture of compound 2 (1.09 g, 0.004 mol) and salicylaldehyde (0.48 ml, 0.004 mol) in 1,4-dioxane (20 ml) and piperidine (0.5 ml) stirred for 3 h at room temperature. The reaction mixture was poured onto ice and acidified with dilute acetic acid. The precipitated was filtered off, and washed with cold water several times and then recrystallized from toluene to give 3. Yield: 75%; M.p. more than 300°C. IR spectrum (KBr, ν, cm⁻¹): 3190, 3176 (2NH), 1710, 1698, 1685 (3C=O). ¹H NMR (DMSO-d6) δ: 3.3 (d, 2H, CH₂), 3.6 (t, 1H, CH), 7.4–8.3 (m, 7H, Ar-H), 10.3, 10.7 (2s, 2H, 2NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆): δ 27.5, 55.9, 116.8, 118.2, 122.6, 122.9, 125.2, 126.3, 126.8, 129.8, 132.5, 133.8, 143.2, 146.5, 152.0, 168.5, 169.8, 174.9. MS: m/z=378 (8.6%) (M⁺-2); Anal. Calcd. For C₁₈H₁₂N₄O₆ (380.3): C, 56.85; H, 3.18; N, 14.73%; found: C, 56.80; H, 3.10; N, 14.69%.

2-Cyano-3-(2-(5-nitro-2-oxoindolin-3-ylidene)hydrazinyl)-3-oxo-N-phenyl propanethioamide (4): compound 2 (2.73 g, 0.01 mol) was added to a stirred solution of potassium hydroxide (0.56 g, 0.01 mol) in N, N-dimethylformamide (20 ml). Phenyl isothiocyanate (1.35 g, 0.01 mol) was added to the resulting mixture after the mixture was stirred for 30 min. Stirring was continued for 12 h at room temperature. The reaction mixture was acidified with cold dilute HCl. The product that separated was filtered, washed with water, and recrystallized. Yield: 75%; M.p. 181–183°C. IR spectrum (KBr, ν, cm⁻¹): 3225, 3200, 3189 (3NH), 2215 (C&8801;N), 1690, 1683 (2C=O). ¹H NMR (DMSO-d6) δ: 3.4 (s, 1H, CH), 7.1–8.4 (m, 8H, Ar-H), 10.5, 10.8, 11.1 (3s, 3H, 3NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆): δ 56.5, 115.3, 117.2, 121.8, 122.8, 126.4, 126.6, 126.9, 127.5, 128.6, 129.1, 134.5, 137.5, 143.2, 146.8, 167.8, 171.2, 193.6. MS: m/z=407 (1.3%) (M⁺-1); Anal. Calcd. For C₁₈H₁₂N₆O₄S (408.3): C, 52.94; H, 2.96; N, 20.58%; found: C, 52.90; H, 2.92; N, 20.50%.

2-Cyano-N’-(5-nitro-2-oxoindolin-3-ylidene)-2-(4-oxo-3-phenylthiazolidin-2-ylidene) acetohydrazide (5): to a stirred solution of potassium hydroxide (0.56 g, 0.01 mol) in N, N-dimethylformamide (20 ml), compound 2 (2.73 g, 0.01 mol) was added. After the mixture was stirred for 30 min phenyl isothiocyanate (1.35 g, 0.01 mol) was added to the resulting mixture. Stirring was continued at room temperature for 12 h and then ethyl chloroacetate (1.07 ml, 0.01 mol) was added and stirring was continued for additional 6 h. The reaction mixture was acidified with cold dilute acetic acid. The separated solid was filtered off, washed several times with cold water and recrystallized. Yield: 80%; M.p. 191–193°C. IR spectrum (KBr, ν, cm⁻¹): 3224, 3190 (2NH), 2217 (C&8801;N), 1715, 1695, 1689 (3C=O). ¹H NMR (DMSO-d6) δ: 4.1 (s, 2H, CH₂), 6.8–7.9 (m, 8H, Ar-H), 9.7, 10.5 (2s, 2H, 2NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆): δ 32.5, 70.1, 114.5, 117.8, 122.6, 123.5, 125.6, 128.3, 128.5, 128.6, 128.8, 128.9, 133.6, 138.9, 143.7, 146.5, 167.3, 168.1, 168.6, 177.5. MS: m/z=447 (1.5%) (M⁺-1); Anal. Calcd. For C₂₀H₁₂N₆O₅S (448.4): C, 53.57; H, 2.70; N, 18.74%; found: C, 53.52; H, 2.68; N, 18.71%.

2-Cyano-3-(ethylthio)-N’-(5-nitro-2-oxoindolin-3-ylidene)-3-(phenylamino) acrylohydrazide (6): compound 2 (2.73 g, 0.01 mol) was added to a stirred solution of potassium hydroxide (0.56 g, 0.01 mol) in N, N-dimethylformamide (20 ml). Phenyl isothiocyanate (1.35 g, 0.01 mol) was added after the mixture was stirred for 30 min. Stirring was continued for 12 h at room temperature and then ethyl iodide (0.62 ml, 0.01 mol) was added and stirring was continued for additional 6 h. The separated solid was filtered off, washed with cold water several times, and recrystallized. Yield: 86%; M.p. 170–172°C. IR spectrum (KBr, ν, cm⁻¹): 3228, 3200, 3189 (3NH), 2215 (C&8801;N), 1710, 1690 (2C=O). ¹H NMR (DMSO-d6) δ: 1.2 (t, 3H, CH₃), 3.2 (q, 2H, CH₂), 6.9–8.1 (m, 8H, Ar-H), 9.6, 10.1, 10.8 (3 s, 3H, 3NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆): δ 15.6, 25.8, 70.3, 114.5, 117.3, 121.5, 121.9, 123.3, 125.1, 125.2, 126.8, 128.7, 129.3, 133.6, 135.3, 142.6, 146.9, 167.3, 167.6, 177.9. MS: m/z=436 (1.1%) (M⁺); Anal. Calcd. For C₂₀H₁₆N₆O₄S (436.4): C, 55.04; H, 3.70; N, 19.26%; found: C, 54.99; H, 3.68; N, 19.24%.

5-Amino-N’-(5-nitro-2-oxoindolin-3-ylidene)-3-(phenylamino)-1H-pyrazole-4-carbohydrazide (7): it is a mixture of hydrazine hydrate 80% (0.005 mol) and 6 (0.87 g, 0.002 mol) in ethanol (20 ml), which was heated under reflux for 4 h. The solvent was evaporated in vacuo, and the solid product obtained was collected and recrystallized. Yield: 75%; M.p. 187–189°C. IR spectrum (KBr, ν, cm⁻¹): 3350, 3330 (NH₂), 3270, 3250, 3200, 3189 (4NH), 1699, 1685 (2C=O). ¹H NMR (DMSO-d6) δ: 6.8–8.3 (m, 8H, Ar-H), 9.6 (s, 2H, NH₂, D₂O exchangeable), 10.4, 10.7, 10.9, 11.1 (4 s, 4H, 4NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆): δ 85.6, 116.3, 116.5, 117.5, 122.3, 122.6, 123.5, 125.8, 128.4, 128.6, 133.6, 140.9, 142.3, 146.5, 151.2, 151.7, 162.8, 167.9. MS: m/z=406 (1.2%) (M⁺); Anal. Calcd. For C₁₈H₁₄N₈O₄ (406.3): C, 53.20; H, 3.47; N, 27.58%; found: C, 53.18; H, 3.44; N, 27.53%.

Ethyl-5′-amino-5-nitro-2,7′-dioxo-4′,7′-dihydro-1′H-spiro[indoline-3,2′-pyrazolo- [1,5-a]pyrimidine]-3′-carboxylate (8): ethyl cyanoacetate (0.45 g, 0.004 mol) was added to a solution of compound 2 (1.09 g, 0.004 mol) in ethanol (20 ml) containing piperidine (0.5 ml). The reaction mixture was refluxed for 3 h, and then poured on ice and acidified with dilute acetic acid. The precipitated solid was filtered off, washed with cold water several times and recrystallized. Yield: 70%; M.p. more than 300°C. IR spectrum (KBr, ν, cm⁻¹): 3322, 3315 (NH₂), 3250, 3200, 3195 (3 NH), 1705, 1689 (3C=O). ¹H NMR (DMSO-d6) δ: 1.8 (t, 3H, CH₃), 3.3 (q, 2H, CH₂), 3.9 (s, 1H, CH), 6.4 (s, 2H, NH₂, D₂O exchangeable), 6.9–7.9 (m, 3H, Ar-H), 10.5, 10.9, 11.2 (3 s, 3H, 3NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆): δ 16.3, 60.5, 72.5, 78.9, 83.8, 110.2, 122.9, 126.7, 127.8, 140.5, 143.6, 146.8, 152.3, 159.6, 167.2, 168.6. MS: m/z=386 (8.3%) (M⁺); Anal. Calcd. For C₁₆H₁₄N₆O₆ (386.3): C, 49.74; H, 3.65; N, 21.75%; found: C, 49.71; H, 3.62; N, 21.72%.

N’-(5-nitro-2-oxoindolin-3-ylidene)-2-(4-oxo-4,5-dihydrothiazol-2-yl)aceto hydrazide (9): it is a mixture of compound 2 (1.09 g, 0.004 mol) and thioglycolic acid (0.3 ml, 0.004 mol) in dry pyridine (20 ml) that was refluxed for 3 h. The reaction mixture was poured on ice cold acetic acid after cooling. The solid separated was filtered off, washed with cold water several times, and recrystallized from toluene to give 9. The remaining solid which was insoluble in toluene was recrystallized from 1,4-dioxane to give 9. Yield: 80%; M.p. more than 300°C. IR spectrum (KBr, ν, cm⁻¹): 3208, 3189 (2 NH), 1700, 1670, 1655 (3C=O). ¹H NMR (DMSO-d6) δ: 3.1 (s, 2H, CH₂), 4.1 (s, 2H, CH₂), 7.1–8.4 (m, 3H, Ar-H), 10.4, 10.6 (2 s, 2H, 2NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆): δ 38.5, 43.2, 119.1, 122.5, 123.6, 125.3, 134.5, 142.7, 146.8, 162.3, 168.2, 171.3, 175.8. MS: m/z=347 (1.2%) (M⁺); Anal. Calcd. For C₁₃H₉N₅O₅S (347.3): C, 44.96; H, 2.61; N, 20.17%; found: C, 44.92; H, 2.59; N, 20.15%.

4-Imino-N’-(5-nitro-2-oxoindolin-3-ylidene)-3-phenyl-2-thioxothiazolidine-5-carbohydrazide (10): to a solution of compound 2 (2.73 g, 0.01 mol) in ethanol (15 ml) containing triethylamine (0.5 ml), elemental sulfur (0.32 g, 0.01 mol) and phenyl isothiocyanate (1.35 g, 0.01 mol) were added. The reaction mixture heated with continuous stirring for 2 h at 60°C. The reaction mixture was acidified with cold dilute acetic acid after cooling. The precipitated product was filtered off, washed with cold water several times and recrystallized. Yield: 75%; M.p. 170–172°C IR spectrum (KBr, ν, cm⁻¹): 3223, 3210, 3189 (3 NH), 1715, 1695 (2C=O). ¹H NMR (DMSO-d6) δ: 3.3 (s, 1H, CH), 7.9-8.1 (m, 8H, Ar-H), 10.2, 11.0, 11.3 (3s, 3H, 3NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆): δ 58.6, 117.2, 122.3, 123.6, 125.1, 127.6, 129.1, 129.3, 133.0, 132.3, 132.5, 133.6, 143.6, 146.3, 165.9, 167.3, 171.2, 192.4. MS: m/z=440 (0.95%) (M ^· ⁺); Anal. Calcd. For C₁₈H₁₂N₆O₄S₂ (440.4): C, 49.09; H, 2.75; N, 19.08%; found: C, 49.01; H, 2.71; N, 19.01%.

7′-Ethoxy-5-nitro-2,5′-dioxo-1′,5′-dihydro-3′H-spiro[indoline-3,2′-[1],[2],[4]triazolo- [1,5-a]pyridine]-6′,8′-dicarbonitrile (11): a mixture of 2-(3,4-dimethoxybenzylidene) malononitrile (0.94 g, 0.004 mol) and 2 (1.09 g, 0.004 mol) in 1,4-dioxane (25 ml) in the presence of triethylamine (0.5 ml) was refluxed for 3 h. Then, it was concentrated and acidified with cold dilute acetic acid. The solid separated out was filtered off, washed with cold water several times and recrystallized. Yield: 80%; M.p. 241–243°C. IR spectrum (KBr, ν, cm⁻¹): 3222, 3215, 3187 (3 NH), 2215, 2225 (2C&8801;N), 1701, 1690 (2C=O). ¹H NMR (DMSO-d6) δ: 1.2 (t, 3H, CH₃), 4.2 (q, 2H, CH₂), 7.1-7.9 (m, 3H, Ar-H), 10.4, 10.6, 10.9 (3 s, 3H, 3NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆): δ 15.8, 65.4, 65.8, 68.3, 104.6, 108.2, 115.2, 115.6, 122.7, 125.6, 130.7, 143.9, 147.8, 158.2, 160.4, 167.8, 184.3. MS: m/z=392 (1.2%) (M⁺-1); Anal. Calcd. For C₁₇H₁₁N₇O₅ (393.3): C, 51.91; H, 2.82; N, 24.93%; found: C, 51.89; H, 2.78; N, 24.90%.

N’-(6-nitro-3-oxo-2,3-dihydrocinnolin-4(1H)-ylidene)hydrazinecarbohydrazide (12): a mixture of hydrazine hydrate 80% (0.01 mol) and 2 (1.09 g, 0.004 mol) and in 1,4-dioxane (20 ml) was refluxed for 2 h. The solid precipitated after cooling was collected by filtration and recrystallized. Yield: 85%; M.p. more than 300°C. IR spectrum (KBr, ν, cm⁻¹): 3370, 3333 (NH₂), 3255, 3222, 3200, 3189 (4 NH), 1701, 1695 (2C=O). ¹H NMR (DMSO-d6) δ: 4.3 (s, 2H, NH₂, D₂O exchangeable), 7.0–8.0 (m, 3H, Ar-H), 10.1, 10.7, 10.8, 11.2 (4s, 4H, 4NH, D₂O exchangeable). ¹³C NMR (DMSO-d₆): δ 111.7, 114.3, 121.5, 125.3, 135.6, 144.3, 146.7, 153.2, 157.4. MS: m/z=278 (4.4%) (M⁺-1); Anal. Calcd. For C₉H₉N₇O₄ (279.2): C, 38.72; H, 3.25; N, 35.12%; found: C, 38.70; H, 3.21; N, 35.10%.

Pharmacology

Antioxidant activity (DPPH assay)

From modified synthesized isatin compounds, stock solutions will be prepared by dissolving 10 mg of samples in 1 ml DMSO is an organo sulfur compound with the formula (CH₃)₂SO, and then diluted to several dilutions. From each concentration, a triplicate of 10 μl will be prepared and then 90 μl of DPPH was added on Eliza plate, and after that plate will be stored in dark cover with aluminum foil for 30 min and then measured at 520 nm on ELISA reader.

Anticoagulation activity

The anticoagulation activities of the different synthesized compounds adopting the method of USA, Pharmacopoeia [42], for the assay of sodium heparin were evaluated as follows:

Reagents

The following reagents were used: standard heparin sodium preparation, human plasma and calcium chloride solution 1% (w/v), and saline solution 0.9% (w/v).

Procedure

Hard-glass test tubes (31×100 mm) were cleaned overnight by immersion in chromic acid. Either 0.8 ml of sample solution (0.01%), 0.8 ml of standard heparin sodium solution (1.4 U.S.P unit/0.8 ml), or 0.8 ml saline solution as a control to each tube was added. Moreover, 1 ml plasma and 0.2 ml calcium chloride solution were added to each of the prepared tubes. The tubes were incubated in a water bath at 37°C. Each tube was stoppered, and the time was immediately recorded. The average of three readings is the time required for clotting was then determined.

Fibrinolytic activity

By exposing a plasma clot to the effect of an aqueous solution (at suitable concentration) of the investigated, the fibrinolytic activity was determined. Under the same conditions mentioned previously for determination of anticoagulation activity, preparation of the plasma clot was achieved [42].

Procedure

By immersion overnight in chromic acid, sets of three-hard-glass test tubes (31×100 mm) were cleaned. 1 ml plasma and 0.2 ml calcium chloride solution (1% w/v) were added to each tube 0.8 ml saline solution (0.89% w/v). After mixing, the tubes were incubated in a water bath at 37°C, and 1 ml of either the saline solution, Pentosan polysulfate (Hemoclar) preparation (2 mg/tube), or the tested sample (1 mg/tube), was added individually when clotting was complete. The lyses percentages of the plasma clots at 37°C were recorded with each sample and compared with the standard Pentosan polysulfate (Hemoclar).

Results and discussion

Chemistry

Compound 2-cyano-N’-(5-nitro-2-oxoindolin-3-ylidene)acetohydrazide (2) was synthesized by the reaction of nitro-isatin with cyanoacetohydrazide in 1,4-dioxane which then reacted with salicylaldehyde to give N’-(5-nitro-2-oxoindolin-3-ylidene)-2-oxochromane-3-carbohydrazide 3 (Scheme 1). IR spectra of 2 showed absorption bands at 1694, 1714 cm⁻¹ due to the respective (2C=O) and absorption band at 2218 cm⁻¹ due to the presence of (C&8801;N). ¹³C NMR spectrum of compound 2 exhibited confirmatory signal of CH₂ at δ 27.1. The IR spectra of 3 showed absorption bands at 1685, 1698, and 1710 cm⁻¹ due to the respective (3C=O) and disappearance of C&8801;N group band. Moreover, compound 2 reacted with phenyl isothiocyanate to synthesize 2-cyano-3-(2-(5-nitro-2-oxoindolin-3-ylidene)hydrazinyl)-3-oxo-N-phenyl-propane thioamide (4). Furthermore, compound 2 was allowed to react with ethyl choloroacetate to give 2-cyano-N’-(-5-nitro-2-oxoindolin-3-ylidene)-2-(4-oxo-3-phenylthiazolidin-2-ylidene)aceto hydrazide (5). Compound 2-cyano-3-(ethylthio)-N’-)-5-nitro-2-oxoindolin-3-ylidene)-3-(phenyl amino)acrylohydrazide (6) was yielded by the reaction of compound 2 with ethyl iodide, which then reacted with hydrazine hydrate to give 5-amino-N’-(5-nitro-2-oxoindolin-3-ylidene)-3-(phenylamino)-1H-pyrazole-4-carbohydrazide (7) (Scheme 2). ¹³C NMR spectrum of compound 6 exhibited confirmatory signal of CH₃ at δ 15.6. ¹H NMR (DMSO-d₆) spectra of compound 7 revealed signal at δ 6.8–8.3 (m, 8H, Ar-H), 9.6 (s, 2H, NH₂, D₂O exchangeable), 10.4, 10.7, 10.9, 11.1 (4 s, 4H, 4NH, D₂O exchangeable). Moreover, we synthesized ethyl-5′-amino-5-nitro-2,7′-dioxo-4′,7′-dihydro-1′H-spiro[indoline-3,2′-pyrazolo [1,5-a]pyrimidine]-3′-carboxylate (8) from the reaction of compound 2 with ethyl cyanoacetate. Compound N’-(5-nitro-2-oxoindolin-3-ylidene)-2-(4-oxo-4,5-dihydrothiazol-2-yl)acetohydrazide (9) was synthesized by the reaction of thioglycolic acid with compound 2. Moreover, compound 2 reacted with elemental sulfur to yield 4-imino-N’-(5-nitro-2-oxoindolin-3-ylidene)-3-phenyl-2-thioxothiazolidine-5-carbohydrazide (10). Malononitrile reacted with compound 2 to give 7′-Ethoxy-5-nitro-2,5′-dioxo-1′,5′-dihydro-3′H-spiro[indoline-3,2′-[1],[2],[4]triazolo [1,5-a]pyridine]-6′,8′-dicarbonitrile (11). Furthermore, N’-(6-nitro-3-oxo-2,3-dihydrocinnolin-4(1H)-ylidene)hydrazinecarbohydrazide (12) was synthesized from the reaction of compound 2 with hydrazine hydrate (Scheme 3). IR spectra of 9 showed absorption bands at 1655, 1670, and 1700 cm⁻¹ due to the presence of (3C=O) and disappearance of C&8801;N group band. Moreover, ¹H NMR (DMSO-d₆) spectra of compound 12 revealed signal at δ 4.3 ppm representing NH₂ and D₂O exchangeable, and signals at δ 10.1, 10.7, 10.8, 11.2 ppm representing 4NH groups.

Pharmacology

Fibrinolytic and anticoagulant activities of isatin derivatives

Fibrinolytic activity in isatin derivatives was analyzed and the novel isatin derivatives exhibited the promising results in pharmaceutics, and also anticoagulant activity in isatin derivatives. From the results shown in [Table 1], compound 10 exhibited highest fibrinolytic activity (the percentage of lysis of plasma clot was 80%), whereas compounds 2 and 4 exhibited higher fibrinolytic activity as compared with those of ‘rest compounds’ (50% lysis of plasma clot) and standard ‘Pentosan polysulfate (Hemoclar)’ preparation (75% lysis) ([Figure 1]). However, compound 12 exhibited the highest anticoagulant activity for 29 min ([Figure 2]). Then compounds 1 and 2 considered also exhibited highest anticoagulant activity compared with the rest compounds, despite being still lower than that of standard ‘Heparin’ preparation ([Figure 2]). The significant variation in pharmacological activities of the isatin compounds happened may be owing to the position of branching and chemical structure features.

Table 1 Anticoagulant and fibrinolytic activities of novel compounds

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Figure 1 Results of fibrinolytic activities of novel compounds.

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Figure 2 Results of anticoagulant activities of novel compounds.

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Antioxidant activity of isatin derivatives

The DPPH free-radical scavenging activity of isatin derivatives was evaluated. From data described in [Table 2], it was noticed that compound 9 exhibited highest antioxidant activity at 85% ([Figure 3]). Then, compounds 3, 4, 11, and 12 were considered also good antioxidant compounds as compared with other rest compounds. Despite these results, ascorbic acid standard has slightly higher activity than compound 9.

Table 2 Antioxidant activities of novel compounds

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Figure 3 Results of antioxidant activities of novel compounds.

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Structural-activity relationship

Based on the structural-activity relationship of the prepared compounds, it was revealed that the presence of S groups in compound 10 exhibited highest fibrinolytic activity. However, the presence of NHNH₂ group in compound 12 increased the anticoagulant activity. Moreover, the presence of active methylene group in compound 9 resulted in the highest antioxidant activity.

Conclusion

Some new series of isatin derivatives were synthesized and screened for their antioxidant, anticoagulant, and fibrinolytic activities. Results showed that ([Table 1]) compound 10 exhibited the highest fibrinolytic activity (the lysis of plasma clot percentage was 80%), whereas compounds 2 and 4 exhibited higher fibrinolytic activity compared with those of ‘rest compounds’ (50% lysis of plasma clot) and standard ‘Pentosan polysulfate (Hemoclar)’ preparation (75% lysis). On the contrary, compound 12 exhibited highest anticoagulant activities at 29 min. Moreover, from [Table 2], it was noticed that compound 9 exhibited highest antioxidant activity at 85%.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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Figures

[Figure 1], [Figure 2], [Figure 3]

Tables

[Table 1], [Table 2]

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