Comparative In Silico drug likeness and In vitro study of some Schiff’s bases as potent COX-II Inhibitors

 

P. P. Chinchole¹*, S. B. Wankhede²

¹Padmashree Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune. India – 411018.

²JSPM’s Charak College of Pharmacy and Research, Gate No. 720/1&2, Wagholi, Pune-Nagar Road, Pune. India – 412207

 

ABSTRACT:

In the present study a series of 3-substituted isatin derivatives were screened in silico by docking method for anti-inflammatory activities. The screened compound shows optimum binding energy in the range of -7.72 to -10.64kcal/mol. The compound P40 have shown the significant binding energy of -10.64kcal/mol. Most of the compounds shown significant anti-inflammatory activity compared with the Indomethacin as standard drugs. Furthermore bioactive analogue showing maximum in-silico activity were synthesized and confirmed by physical and spectral analysis and subjected to in vitro study of anti-inflammatory activity. Results are expressed by using one way ANOVA with Dunnet’s t test. Compounds P26, P28, P30, P34, P37 and P40 were found to have significant analgesic and anti-inflammatory activity.

 

 

INTRODUCTION:

Inflammation is one of the protective consequences related to tissue homeostasis which involve the sequential biochemical reaction in response to cellular injury. This biochemical reaction results in the synthesis of mediator of inflammation. Mediators formed may be the component of metabolism, immune system or endocrine (hormone) system. Tissue injury decides extend of inflammation. Generally the word “itis” is suffixes after the injured body part to which inflammation occurs, for example “nephritis” indicating that inflammation of kidney.

 

Roman scientist Aulus Cornelius Celsus 20 A.D. were focused the four cardinal sign and symptoms of inflammation. The magnitude of the injury define the way to express the inflammation as

 

1.     Tumor:

Tumor termed for Swelling. Collection of extra vascular fluid along with the inflammatory mediators migrating into damaged part results in the formation of edema and is the mechanism for Swelling.

 

2.     Rubor:

Due to dilation of small blood vessels within the injured area tissue appears to be red termed as hyperemia.

 

3.     Calor:

Blood flow increases due to dilation of small blood vessels within the injured part. This increases the temperature as supply of warm blood to the area.

 

The knowledge of the Pathophysiology of disease and biological target related to concern disease is the key to innovation for the drug design. The Drug Design refers to the designing of the chemical structure term as ligand that interacts with biomolecule present in biological system that evokes pharmacological action. Optimization of structure of ligand with help of software on computer is known as Computer Aided Drug Design (CADD). Therapeutic action of drug candidate is related to pharmacokinetic and pharmacodynamic feature. The pharmacokinetic process includes absorption, distribution, metabolism and excretion (ADME) where as pharmacodynamics means the ligand-protein interaction and toxicity¹. These features depend upon the Drug-likeness. Drug-likeness may be defined as a delicate balance among molecular properties affecting pharmaco-dynamics and pharmacokinetics of molecules. Molecular properties include molecular weight, electronic distribution, hydrophobicity, hydrogen bond donors/acceptors, solubility, viscosity, excess volume and other related properties.

 

Selection of suitable molecular descriptors for correctly predicting the drug likeness of an analogue is of prime importance for drug design. Methods for drug-likeness prediction include from simple counting schemes like Lipinski’s “rule of five” to Molinspiration software. Lipinski’s “rule of five” predicts drug-likeness on the basis of various molecular descriptors Milog P, n violations, molecular mass, n rotb No. hydrogen bond donors and No. hydrogen bond acceptor, Bioactivity score².

 

Due to the vast spectrum of biological activities, Isatin and benzothiazole derivatives have achieved big hopes which are reflected by their use as anti-convulsant, antimicrobial, analgesic and anti-inflammatory activity^3-4. In the present study, synthesized series of Schiff’s base of isatin and 2-carbomoyl 1-3 benzothiazole^5-6 is subjected to docking procedure by AutoDock Tools v1.5.6. The crystal structure of the enzyme Cyclooxygenase-2 (Prostaglandin Synthase-2) was obtained from RCSB Protein data bank (PDB code: 1CX2)⁷. The analogues showing optimum binding energy were screened for drug likeness on the Molinspiration software⁸. Data predicted by the docking method and Molinspiration software is compare with in vitro anti-inflammatory activity.

 

Experimental:

All synthetic grade chemicals used in the synthesis were procured from Rajesh Chemical; Mumbai. The designed analogues were evaluated by the docking method firstly. In this procedure ligand pdb format created by Marvin sketch software. Protein structure (PDB ID: 1CX2) obtained from RCSB protein data bank (https://www.rcsb.org/) were docked with series of ligand (analogue) designed and evaluated by binding energy⁹.

 

The compounds showing optimum binding energy were screen for the drug likeness and bioactivity score by using Molinspiration software and subjected to in vivo and in vitro analgesic and anti-inflammatory activity study after synthesis and spectral characterization.  

 

 

General method of synthesis:

The general synthetic approach involved condensation of an equimolar mixture of corresponding substituted indole 2, 3- dione (0.01 mol) and substitute 2 carbomoyl 1-3 benzothiazole (0.01 mol) in absolute ethanol in the presence of 2, 3-drops of glacial acetic acid for 3–4 h. On cooling, flakes separated out which were filtered and recrystallized from hot ethanol to give shining brightly colored needles of Schiff’s base. Synthesized compounds will characterized by their spectral studies¹⁰.

 

In vitro evaluation of Anti-inflammatory activity by protein albumin denaturation method:

All synthesized compounds were screened for anti-inflammatory activity by using in vitro method reported earlier by Muzushima and Kabayashi¹¹ with slight modification. Accordingly, inhibition of albumin denaturation technique was studied, a 5.0ml reaction mixture was prepared consisting of 0.2ml of egg albumin (obtained from fresh hen’s egg), 2.8ml phosphate buffered saline (pH: 6.4) and 2.0ml of varying concentration of test compounds so that final concentrations become 25, 50, 100, 200μg/ml. Similar volume of double distilled water served as control. Then the mixtures were incubated at 37±2°C in an incubator for 15 minutes and then heated at 70ºC for 5 minutes. After cooling, their absorbance was measured at 660 nm by using vehicle as blank. Indomethacin with final concentration of 50 and 100μg/ml was used as reference drug and treated similarly for determination of absorbance^12-15. The percent inhibition of protein denaturation was calculated as follows:

 

Percentage inhibition = (Abs _control – Abs _sample) X 100/ Abs _control

 

RESULTS AND DISCUSSION:

In-silico molecular characterization was the most important preliminary step in the rational drug designing of novel drugs. In the present study different proposed analogues are screened for various molecular descriptors by using Auto Dock tool1.5.6 and Molinspiration software. Marvin Sketch was used for 3-D drawing.

 

The data predicted by In-silico molecular characterization for the drug likeness is cited in the following table no. 1 and 2

 

Table No.1: Docking Analysis

Code	Structure	Amino Acid	Bond	Binding Energy	Inhibitory constant	Inter molecular energy	Electrostatic Energy
P25		ASN43	HN	-8.35	755	-8.95	00
P26		ASN43	HN	-9.39	131.24	-9.99	-0.18
P27		ASN43; ASN43	HN; HO	-8.00	1.38	-8.59	-0.09
P28		LYS68; GLU65	HO; HS	-9.66	83.82	-10.55	-1.56
P29		UNKO	HN	-8.19	992.81	-8.79	-0.32
P30		LYS468; GLU465	HO; HS	-8.29	837.27	-9.19	-0.29
P31		UNKO	HN	-8.42	671.53	-9.02	0.11
P32		ASN43; LYS468; ASN43	HN; HO; HO	-8.02	1.32	-8.62	-0.14
P33		ASN43; ASN43	HN; HO	-8.02	1.32	-8.62	-0.11
P34		ASN43; LYS473	HN; HO	-9.41	125.91	-10.13	-1.71
P35		ASN43; LYS468; ASN43	HN; HO; HO	-7.79	1.96	-8.38	-0.11
P36		CYS41	HS	-8.06	1.24	-8.95	-0.06
P37		ARG44	HO	-9.54	101.5	-10.14	-0.14
P38		LYS468; GLU465	HN; HS	-9.06	230.41	-9.65	-0.22
P39		GLN42; LYS468	HO; HO	-7.72	2.2	-8.32	-0.05
P40		LYS468	HN	-10.64	15.18	-11.54	-1.76
P41		LYS468	-	-8.99	257.2	-9.59	-0.07
P42		LYS468; GLU465	HS; HO	-9.05	233.37	-9.94	-0.43
P43		ASN43; CYS41	HN; HN	-9.2	180.09	-9.8	0.01
P44		ASN43	HO	-7.83	1.82	-8.43	-0.06
P45		CYS41	HO	-9.4	128.57	-10.0	-0.09
P46		LYS468; GLU465	HN; HS	-9.41	127.11	-10.3	-1.11
P47		ASN43	HN	-9.43	122.82	-10.02	-0.17
P48		LYS468; GLU465	HN; HN	-8.42	670	-9.32	-0.3
	Indomethacin	CYS57	NH	-6.31	23.78	-7.5	-0.31

 

From the docking analysis data, it clear that analogue P26, P28, P30, P34, P37, P40 shows maximum binding energy thus this analogue were selected for the further study.

 

Fig. No. 1: Ligand-Protein interaction

Table no.2: Molecular descriptors

Compound	Mi Log P	TPSA	n. atom	Mole. weight	n. ON	N Ohnh	n Violation	n rotb	Bioactive score
P26	3.22	74.32	23	343.80	5	2	0	1	-0.26
P28	2.50	120.15	25	354.35	8	2	0	2	-0.30
P30	2.60	83.56	24	339.38	6	2	0	2	-0.26
P34	2.57	109.29	26	368.37	8	1	0	2	-0.41
P37	3.22	74.32	23	343.80	5	2	0	1	-0.26
P40	3.15	120.15	26	388.79	8	2	0	2	-0.31
Indomethacin	3.99	68.54	25	357.79	5	1	0	4	0.30

 

In silico study confirms the proposed analogues are bioactive and furthermore subjected to synthesis. Melting points were determined in open capillaries and were uncorrected. IR spectra (KBr pellets) were recorded on Shimadzu FT-IR model 8010 spectrophotometer. ¹H NMR spectra (DMSO-d6) were taken a Varian mercury spectrometer (model YH- 300 FT NMR) using Tetramethyl Silane (TMS) as internal standard and chemical shift are expressed in δ ppm.

 

N-(5-Chloro-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P26):

Yield: 57 %. mp 154-156 °C. IR (cm^-1): 3415 (NH stretch), 2890 (C-H stretch), 1712 (carbonyl stretch), 1604(NH bending). ¹H-NMR (400 MHz, DMSO-d6): 7.37-7.44 (m, 1H, Ar-H), 7.53 (m, 1H, Ar-H), 7.62 (m, 1H, Ar. H), 7.42 (m, 1H, benzothiazole -H), 8.1 (d, 1H, J=7.34 Hz, benzothiazole-H), 8.2 (m, 1H, benzothiazole-H), 8.42 (m, 1H, benzothiazole-H), MS: (ESI) m/z [M+H] calcd for (C₁₆H₈ClN₃O₂S): 341.0026; found: 341.7690 Anal. Calcd. for Molecular formula C₁₆H₈ClN₃O₂S C 56.23, H 2.36, N 12.30; Found C 56.25, H 2.37, N 12.28.

 

N-(5-bromo-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P27):

Yield: 57 %. mp 155-157 °C. IR (cm^-1): 3414 (NH stretch), 2891 (C-H stretch), 1714 (carbonyl stretch), 1604 (NH bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 7.21-7.26 (t, 1H, J=5.7Hz, Ar-H), 7.51(m, 1H, benzothiazole-H), 7.53(m, 1H, benzothiazole-H), 7.78(dd, 1H, J=6.8Hz, Ar-H), 7.90(d, 1H, J=7.77Hz, Ar-H), 8.04(m, 1H, benzothiazole-H), 8.15(m, 1H, benzothiazole-H); MS: (ESI) m/z [M+H] calcd for(C₁₆H₈BrN₃O₂S): 384.9519,Found: 386.2169; Anal. Calcd. For Molecular formula C₁₆H₈BrN₃O₂S C 49.76, H 2.09, N 10.88, Found C 49.74, H 2.10, N 10.91.

 

N-(5-Nitro-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P28):

Yield: 59 %. mp 160-162 °C. IR (cm^-1): 3415 (NH stretch), 2893 (C-H stretch), 1711 (carbonyl stretch), 1604(NH bending), 1536 (N-O stretch). ¹H-NMR (400 MHz, DMSO-d6): 7.52 (m, 1H, Ar-H), 7.54 (m, 1H, Ar-H), 7.61 (m, 1H, Ar. H), 7.66 (m, 1H, Ar-H), 8.05 (d, 1H, J=7.34 Hz, Ar-H), 8.3 (m, 1H, Ar-H), 8.47 (m, 1H, Ar-H); MS: (ESI) m/z [M+H] calcd for (C₁₆H₈N₄O₄S): 352.0266; found: 352.3240; Anal. Calcd. for Molecular formula C₁₆H₈N₄O₄S C 54.55, H 2.29, N 15.90, Found C 54.57, H 2.28, N 15.89.

 

N-(5-fluoro-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P29):

Yield: 59 %. mp 157-159 °C. IR (cm^-1): 3416 (NH stretch), 2894 (C-H stretch), 1717 (carbonyl stretch), 1602 (NH bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 7.10-7.18(t, 1H, J=5.7Hz, Ar-H), 7.49(m, 1H, benzothiazole-H), 7.51(m, 1H, benzothiazole-H), 7.78(dd, 1H, J=6.8Hz, Ar-H), 7.91(d, 1H, J=7.77Hz, Ar-H), 8.06(m, 1H, benzothiazole-H), 8.11(m, 1H, benzothiazole-H), 10.03(s, 1H, Ar-NH) ; MS: (ESI) m/z [M+H] calcd for (C₁₆H₈FN₃O2S): 325.0321 , Found: 325.3178; Anal. Calcd. for Molecular formula C₁₆H₈FN₃O2S C 59.07, H 2.48, N 12.92, Found C 59.10, H 2.46, N 12.89.

 

N-(6-Methoxy-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P30):

Yield: 64 %. mp 154-156 °C. IR (cm^-1): 3415 (NH stretch), 2890 (C-H stretch), 1712 (carbonyl stretch), 1604(NH bending). ¹H-NMR (400 MHz, DMSO-d6): 2.49 (s, 3H, CH₃), 7.51 (m, 1H, Ar-H), 7.53 (m, 1H, benzothiazole -H), 7.70 (d, 1H, J=7.34 Hz, benzothiazole -H), 8.2 (m, 2H, Ar-H), 8.42 (m, 1H, Ar-H), 8.6 (m, 1H, benzothiazole -H), MS: (ESI) m/z [M+H] calcd for (C₁₇H₁₁N₃O₃S): 337.0521 , Found: 337.3640. Anal. Calcd. for Molecular formula C₁₇H₁₁N₃O₃S C 60.53, H 3.29, N 12.46, Found: C 60.53, H 3.28, N12.45.

 

N-(1-methyl-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide(P31):

Yield: 62 %. mp 154-156 °C. IR (cm^-1):  3035 (Aryl C-H stretch), 2890 (C-H stretch), 1710 (carbonyl stretch), 815 (Aryl C-H bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 3.46(s, 3H, Ar-CH₃), 7.34(t, 1H, J=5.7Hz, Ar-H), 7.48(m, 1H, benzothiazole-H), 7.53(m, 1H, benzothiazole-H), 7.60(dd, 2H, J=6.8Hz, Ar-H), 7.92(d, 1H, J=7.77Hz, Ar-H), 8.04(m, 1H, benzothiazole-H), 8.17(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for(C₁₇H₁₁N₃O₂S): 321.0569 Found: 321.3536. Anal. Calcd. For Molecular formula C₁₇H₁₁N₃O₂S C 63.54, H 3.45, N 13.08, Found C 63.53, H 3.47, N 13.07.

 

N-(5-chloro-1-methyl-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide(P32):

Yield: 59 %. mp 155-157 °C. IR (cm^-1): 3035 (Aryl C-H stretch), 2890 (C-H stretch), 1710 (carbonyl stretch), 815 (Aryl C-H bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 3.46(s, 3H, Ar-CH₃), 7.37(t, 1H, J=5.7Hz, Ar-H), 7.44(m, 1H, benzothiazole-H), 7.54(m, 1H, benzothiazole-H), 7.90(d, 2H, J=7.77Hz, Ar-H), 8.05(m, 1H, benzothiazole-H), 8.16(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for(C₁₇H₁₀ClN₃O₂S): 355.0202, Found: 355.7989. Anal. Calcd. for Molecular formula C₁₇H₁₀ClN₃O₂S C 57.39, H 2.83, N 11.81, Found C 57.42, H 2.84, N 11.78.

 

N-(5-bromo-1-methyl-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P33):

Yield: 57 %. mp 161-162 °C. IR (cm^-1): 3032 (Aryl C-H stretch), 2896 (C-H stretch), 1715 (carbonyl stretch), 821 (Aryl C-H bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 3.42(s, 3H, Ar-CH₃), 7.35(t, 1H, J=5.7Hz, Ar-H), 7.45(m, 1H, benzothiazole-H), 7.52(m, 1H, benzothiazole-H), 7.92(d, 2H, J=7.77Hz, Ar-H), 8.07(m, 1H, benzothiazole-H), 8.14(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for(C₁₇H₁₀BrN₃O₂S): 398.9667, Found: 400.2501, Anal. Calcd. For Molecular formula C₁₇H₁₀BrN₃O₂S C 51.01, H 2.52, N 10.50, Found C 51.03, H 2.51, N 10.49.

 

N-(1-Methyl-5-nitro-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P34):

Yield: 55 %. mp 144-146 °C. IR (cm^-1): 2890 (C-H stretch), 1712 (carbonyl stretch), 1525 (N-O stretch). ¹H-NMR (400 MHz, DMSO-d6): 3.46 (s, 3H, CH₃), 7.51 (m, 1H, Ar-H), 7.56 (m, 1H, Ar-H), 7.63 (m, 1H, Ar. H), 7.66 (m, 1H, Ar-H), 8.32 (d, 1H, J=7.34 Hz, Ar-H), 8.42 (m, 1H, Ar-H), 8.63 (m, 1H, Ar-H). MS: (ESI) m/z [M+H] calcd for (C₁₇H₁₀N₄O₄S): 366.0423, found: 366.3510. Anal. Calcd. For Molecular formula C₁₇H₁₀N₄O₄S C 55.74, H 2.75, N 15.29, Found C 55.76, H 2.74, N 15.30.

 

N-(5-fluoro-1-methyl-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P35):

Yield: 59 %. mp 145-147 °C. IR (cm^-1): 3030 (Aryl C-H stretch), 2892 (C-H stretch), 1711 (carbonyl stretch), 825 (Aryl C-H bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 3.40(s, 3H, Ar-CH₃), 7.32(t, 1H, J=5.7Hz, Ar-H), 7.44(m, 1H, benzothiazole-H), 7.51(m, 1H, benzothiazole-H), 7.94(d, 2H, J=7.77Hz, Ar-H), 8.08(m, 1H, benzothiazole-H), 8.17(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for(C₁₇H₁₀FN₃O₂S): 339.0471, Found: 339.3445. Anal. Calcd. For Molecular formula C₁₇H₁₀FN₃O₂S C 60.17, H 2.97, N 12.38, Found C 60.19, H 2.98, N 12.39.

 

N-(5-methoxy-1-methyl-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P36):

Yield: 61 %. mp 162-164 °C. IR (cm^-1): 3032 (Aryl C-H stretch), 2895 (C-H stretch), 1715 (carbonyl stretch), 830 (Aryl C-H bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 3.43(s, 3H, Ar-CH₃), 3.77(s, 3H, Ar-OCH₃), 7.32(t, 1H, J=5.6Hz, Ar-H), 7.41(m, 1H, benzothiazole-H), 7.50(m, 1H, benzothiazole-H), 7.91(d, 2H, J=7.72Hz, Ar-H), 8.08(m, 1H, benzothiazole-H), 8.17(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for (C₁₈H₁₃N₃O₃S): 351.0672. Found: 351.3798. Anal. Calcd. For Molecular formula C₁₈H₁₃N₃O₃S C 61.53, H 3.73, N 11.96, Found C 61.52, H 3.74, N 11.94.

 

6-chloro-N-(2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P37):

Yield: 62 %. mp 168-170 °C. IR (cm^-1): 3415 (NH stretch), 2891 (C-H stretch), 1712 (carbonyl stretch), 1604(NH bending). ¹H-NMR (400 MHz, DMSO-d6): 7.53 (m, 1H, Ar-H), 7.57 (m, 1H, Ar-H), 7.64 (m, 1H, Ar. H), 7.69 (m, 1H, Ar-H), 8.1 (d, 1H, J=7.38 Hz, Ar-H), 8.2 (m, 1H, Ar-H), 8.42 (m, 1H, Ar-H). MS: (ESI) m/z [M+H] calcd for (C₁₆H₈ClN₃O₂S): 341.0026; Found: 341.3460; Anal. Calcd. For Molecular formula C₁₆H₈ClN₃O₂S C 56.23, H 2.36, N 12.30, Found C 56.24, H 2.35, N 12.31.

 

6-chloro-N-(5-chloro-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P38):

Yield: 62 %. mp160-162 °C. IR (cm^-1): 3417 (NH stretch), 2894 (C-H stretch), 1719 (carbonyl stretch), 1610 (NH bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 7.13(t, 1H, J=5.1Hz, Ar-H), 7.49(m, 1H, benzothiazole-H), 7.51(m, 1H, benzothiazole-H), 7.91(d, 2H, J=7.73Hz, Ar-H), 8.11(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for (C₁₆H₇Cl₂N₃O₂S): 374.9633, Found: 376.2159, Anal. Calcd. For Molecular formula C₁₆H₇Cl₂N₃O₂S C: 51.08, H 1.88, N 11.17, Found C 51.07, H 1.86, N 11.19.

 

6-chloro N-(5-bromo-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P39):

Yield: 57 %. mp 155-157 °C. IR (cm^-1): 3415 (NH stretch), 2895 (C-H stretch), 1720 (carbonyl stretch), 1609 (NH bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 7.11(t, 1H, J=5.1Hz, Ar-H), 7.48(m, 1H, benzothiazole-H), 7.58(m, 1H, benzothiazole-H), 7.90(d, 2H, J=7.70Hz, Ar-H), 8.12(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for (C₁₆H₇BrClN₃O₂S): 418.9131; Found: 420.6659. Anal. Calcd. For Molecular formula C₁₆H₇BrClN₃O₂S C 45.68, H 1.68, N 9.99, Found C 45.69, H 1.69, N 9.98.

 

6-Chloro-N-(5-nitro-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P40):

Yield: 64 %. mp 177-179 °C. IR (cm^-1): 3415 (NH stretch), 2893 (C-H stretch), 1712 (carbonyl stretch), 1604(NH bending), 1536 (N-O stretch). ¹H-NMR (400 MHz, DMSO-d6): 7.61 (m, 1H, Ar-H), 7.66 (d, 1H, J=7.43, Ar-H), 7.99 (m, 1H, Ar-H), 8.12 (d, 1H, J=7.34 Hz, Ar-H), 8.62 (m, 1H, Ar-H), 8.47 (m, 1H, Ar-H). MS: (ESI) m/z [M+H] calcd for (C₁₆H₇ClN₄O₄S): 385.9877; Found: 386.0120. Anal. Calcd. For Molecular formula C₁₆H₇ClN₄O₄S C 49.69, H 1.82, N 14.49 Found C 49.69, H 1.81, N 14.48

 

6-chloro-N-(5-fluoro-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide(P41):

Yield: 61 %. mp167-169 °C. IR (cm^-1): 3417 (NH stretch), 2894 (C-H stretch), 1719 (carbonyl stretch), 1610 (NH bending).¹H NMR (DMSO-d6, 400 MHz) δ: 7.11(t, 1H, J=5.6Hz, Ar-H), 7.44(m, 1H, benzothiazole-H), 7.50(m, 2H, benzothiazole-H), 7.92(d, 2H, J=7.71Hz, Ar-H). MS: (ESI) m/z [M+H] calcd for (C₁₆H₇ClFN₃O₂S): 358.9929, Found: 359.6624. Anal. Calcd. For Molecular formula C₁₆H₇ClFN₃O₂S C 53.42, H 1.96, N 11.68, Found C 53.44, H 1.96, N11.67.

 

6-chloro-N-(5-methoxy-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P42):

Yield: 55 %. mp157-159 °C. IR (cm^-1): 3032 (Aryl C-H stretch), 2895 (C-H stretch), 1715 (carbonyl stretch), 1610 (NH bending), 830 (Aryl C-H bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 3.77(s, 3H, Ar-OCH₃), 7.32(t, 1H, J=5.6Hz, Ar-H), 7.41(m, 1H, benzothiazole-H), 7.50(m, 1H, benzothiazole-H), 7.91(d, 2H, J=7.72Hz, Ar-H), 8.08(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for (C₁₇H₁₀ClN₃O₃S) 371.0131, Found: 371.6981. Anal. Calcd. For Molecular formula C₁₇H₁₀ClN₃O₃S C 54.92, H 2.71, N 11.30, Found C 54.93, H 2.72, N 11.30.

 

6-chloro-N-(1-methyl-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P43):

Yield: 65 %. mp 161-163 °C. IR (cm^-1): 3030 (Aryl C-H stretch), 2892 (C-H stretch), 1711 (carbonyl stretch), 825 (Aryl C-H bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 3.40(s, 3H, Ar-CH₃), 7.32(t, 2H, J=5.7Hz, Ar-H), 7.44(m, 1H, benzothiazole-H), 7.94(d, 2H, J=7.77Hz, Ar-H), 8.08(m, 1H, benzothiazole-H), 8.17(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for (C₁₇H₁₀ClN₃O₂S): 355.0162, Found: 355.5986. Anal. Calcd. For Molecular formula C₁₇H₁₀ClN₃O₂S C 57.39, H 2.83, N 11.81, Found C 57.40, H 2.82, N 11.83.

 

6-chloro-N-(5-chloro-1-methyl-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P44):

Yield: 65 %. mp 167-169 °C. IR (cm^-1): 3030 (Aryl C-H stretch), 2892 (C-H stretch), 2251(C-N stretch), 1711 (carbonyl stretch), 825 (Aryl C-H bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 3.40(s, 3H, Ar-CH₃), 7.32(t, 1H, J=5.7Hz, Ar-H), 7.44(m, 1H, benzothiazole-H), 7.94(d, 2H, J=7.77Hz, Ar-H), 8.08(m, 1H, benzothiazole-H), 8.17(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for(C₁₇H₉Cl₂N₃O₂S):388.9791, Found: 390.3435. Anal. Calcd. For Molecular formula C₁₇H₉Cl₂N₃O₂S C 52.32, H 2.32, N 10.77, Found C 52.31, H 2.33, N 10.77.

 

N-(5-bromo-1-methyl-2-oxoindolin-3-ylidene)-6-chlorobenzothiazole-2 carboxamide (P45):

Yield: 55 %. mp 163-165 °C. IR (cm^-1): 3031 (Aryl C-H stretch), 2890 (C-H stretch), 2255(C-N stretch), 1710 (carbonyl stretch), 825 (Aryl C-H bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 3.40(s, 3H, Ar-CH₃), 7.31(t, 1H, J=5.7Hz, Ar-H), 7.43(m, 1H, benzothiazole-H), 7.93(d, 2H, J=7.77Hz, Ar-H), 8.07(m, 1H, benzothiazole-H), 8.17(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for(C₁₇H₉BrClN₃O₂S): 432.9286, Found: 434.6940. Anal. Calcd. For Molecular formula C₁₇H₉BrClN₃O₂S C 46.97, H 2.09, N 9.67, Found C 46.98, H 2.08, N 9.68.

 

6-chloro-N-(1-methyl-5-nitro-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P46):

Yield: 61%. mp 158-160 °C. IR (cm^-1): 3034 (Aryl C-H stretch), 2895 (C-H stretch), 2257(C-N stretch), 1714 (carbonyl stretch), 829 (Aryl C-H bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 3.40(s, 3H, Ar-CH₃), 7.31(t, 1H, J=5.7Hz, Ar-H), 7.43(m, 1H, benzothiazole-H), 7.93(d, 2H, J=7.77Hz, Ar-H), 8.07(m, 1H, benzothiazole-H), 8.17(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for(C₁₇H₉ClN₄O₄S): 400.0123, Found: 400.7963. Anal. Calcd. For Molecular formula C₁₇H₉ClN₄O₄S C 50.95, H 2.26, N 13.98, Found C:50.94, H 2.28, N 13.96.

 

6-Chloro-N-(5-fluoro-1-methyl-2-oxoindolin-3-ylidene) benzothiazole-2-carboxamide (P47):

Yield: 55 %. mp 144-146 °C. IR (cm^-1): 2891 (C-H stretch), 1712 (carbonyl stretch). ¹H-NMR (400 MHz, DMSO-d6): 3.39 (S, 3H, CH₃), 7.47 (m, 1H, Ar-H), 7.54 (m, 1H, Ar-H), 7.69 (m, 1H, benzothiazole -H), 7.76 (m, 1H, Ar-H), 7.91 (m, 1H, Ar-H), 8.11 (dd, 1H, J=7.34, J=3.5 Hz, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for (C₁₇H₉ClFN₃O₂S): 373.0087; found: 373.801; Anal. Calcd. For Molecular formula C₁₇H₉ClFN₃O₂S C 54.63, H 2.43, N 11.24 Found C 54.65, H 2.43, N 11.21. 

 

6-chloro-N-(5-methoxy-1-methyl-2-oxoindolin-3-ylidene) benzothiazole-2carboxamide (P48):

Yield: 57 %. mp 152-154 °C. IR (cm^-1): 3032 (Aryl C-H stretch), 2895 (C-H stretch), 1715 (carbonyl stretch), 830 (Aryl C-H bending). ¹H NMR (DMSO-d6, 400 MHz) δ: 3.43(s, 3H, Ar-CH₃), 3.77(s, 3H, Ar-OCH₃), 7.32(t, 1H, J=5.6Hz, Ar-H), 7.41(m, 1H, benzothiazole-H), 7.50(m, 1H, benzothiazole-H), 7.91(d, 2H, J=7.72Hz, Ar-H), 8.08(m, 1H, benzothiazole-H). MS: (ESI) m/z [M+H] calcd for(C₁₈H₁₂ClN₃O₃S):385.0289 Found 385.8244. Anal. Calcd. For Molecular formula C₁₈H₁₂ClN₃O₃S C 56.04, H 3.14, N 10.89, Found C 56.06, H 3.15, N 10.87.

Table No. 3: In- vitro Anti-inflammatory activity

S. N.	Compound	Absorbancea	% Denaturation (Mean ± S. E. M.)
01	P26	0.0293	72.09±0.339
02	P27	0.0425	59.52±0.331
03	P28	0.0384	63.42±0.228
04	P29	0.0442	57.90±0.230
05	P30	0.0309	70.57±0.132
06	P31	0.0342	67.42±0.164
07	P32	0.0378	64.00±0.117
08	P33	0.0345	67.13±0.182
09	P34	0.0225	78.57±0.173
10	P35	0.0394	62.47±0.124
11	P36	0.0426	59.42±0.223
12	P37	0.0204	80.57±0.169
13	P38	0.0458	56.38±0.236
14	P39	0.0398	62.09±0.264
15	P40	0.0246	76.57±0.191
16	P41	0.0338	67.80±0.212
17	P42	0.0299	71.52±0.123
18	P43	0.0405	61.42±0.150
19	P44	0.0430	59.04±0.268
20	P45	0.0389	62.95±0.196
21	P46	0.0326	68.95±0.108
22	P47	0.0242	76.95±0.095
23	P48	0.0398	62.09±0.184
24	(Indomethacin)	0.0150	85.71±0.851

 

CONCLUSION:

Finally, we concluded from the results obtained by in silico, in vitro and compared with the standard drug Indomethacin. We utilized molecular docking and the Molinspiration software for the calculation of molecular descriptor of the proposed analogues. The study reveals for better activity and indicating that analogues P26, P28, P30, P34, P37, and P40 influences the optimum drug likeness property when compared with the Indomethacin. Furthermore this analogue also shows the better in vitro pharmacological activity.

 

Values are expressed as mean ± S.E.M. (N=5)

^*Groups P26, P28, P30, P34, P37 and P40 compared to Indomethacin

(One-way ANOVA followed by Dunnett’s test.).^* p < 0.05, ^**p < 0.01 and ^***p < 0.001.                            

 

ACKNOWLEDGEMENT:

We, the authors are thankful to Dr. S. S. Chitlange, Principal, Padmashree Dr. D. Y. Patil Pratishthan’s Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri Pune – 411018 for providing research facilities and encouragement and to our friends those helped us for completion of this research.

 

CERTIFICATE OF CONFLICT OF INTEREST/ PLAGIARISM REPORT:

Authors has declared that no conflict of interest in submission of manuscript for publication.

 

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Received on 19.03.2019           Modified on 23.04.2019

Accepted on 21.05.2019         © RJPT All right reserved