1.0. INTRODUCTION:

Chemistry of heterocycles is highly essential for the present pathogenic environment to address the real world demands.^1-3 Synthesis of heterocyclic compounds is a great challenge for higher efficiency and eco-friendly point of view.^4-5 As the quest for synthesis of new heterocycles for controlling and curing the emerging diseases continues, Scientists have paid attention to design and development of novel heterocyclic compounds from natural products and synthetic methods^6-7, Particularly nitrogen-containing heterocyclic compounds that occupy the key position in the area of drugs and pharmaceuticals.^8-9 Majority of the pharmaceutical products are heterocycles that meet the expectations of current requirements of people.

Over the last decade, derivatives of benzimidazoles have received very much attention in medicinal chemistry.^10-11 The current study reports the synthesis of most versatile compound benzimidazole containing oxazole, thiazole, pyrazole and isoxazole which exhibit various biological properties like antitubercular^12-13, antifungal¹⁴, anthelmintic¹⁵, analgesic^16-17, antimicrobial^18-19and antibacterial²⁰ and anti-inflammatory activities.²¹

Hence, in this direction efforts have been undertaken to introduce most active and biologically versatile molecules containing nitrogen, oxygen and sulphur heterocyclic compounds like 6-(4-(1H-benzo[d]imidazol-2-yl)oxazol-2-yl)-3,5-diphenyl-3,3a,5,6-tetrahydrothiazolo[4,5-c]isoxazole and 4-(1H-benzo[d] imidazol-2-yl)-2-(3,5-diphenyl-3,3a-dihydro-2H-pyrazolo[3,4-d]thiazol-6(5H)-yl)oxazole derivatives which were synthesized from 4-(1H-benzo[d]imidazol-2-yl)oxazol-2-amine.

2.0. EXPERIMENTAL SECTION:

2.1. Materials and Methods:

The reaction progress was monitored by TLC plates. Infra red Spectrum of compounds were recorded by Perkin Elmer BX series and ¹HNMR spectra were recorded by Bruker 400 MHz instrument using DMSO as solvent and Tetramethylsilane used as an internal standard. Chemical shifts (δ) were expressed in ppm. Mass spectrums of compounds were measured on a GC/MS-QP1000EX (EI, 70 eV) mass spectrometer. Elemental analysis was performed on PerkinElmer 240 CHN analyzer.

Table:1 Physical data of Synthesized compounds 5, 6 and 7 (a-e)

Compounds	5a	5b	5c	5d	5e
R	Ar	4-CH₃- Ar	4-OCH₃- Ar	4-OH- Ar	4-NO₂- Ar
M.P ( ⁰C )	250-52	255-57	256-58-26	251-53	258-60
Yield (%)	78	82	79	80	75
Compounds	6a	6b	6c	6d	6e
R	Ar	4-CH₃- Ar	4-OCH₃- Ar	4-OH- Ar	4-NO₂- Ar
M.P ( ⁰C )	255-57	258-60	260-62	259-61	263-65
Yield (%)	82	78	76	81	74
Compounds	7a	7b	7c	7d	7e
R	Ar	4-CH₃- Ar	4-OCH₃- Ar	4-OH- Ar	4-NO₂- Ar
M.P ( ⁰C )	258-60	261-63	264-66	261-63	265-67
Yield (%)	80	81	76	73	74

2.2.General reaction procedure for Compound 4:

A mixture of Benzaldehyde (0.004 mol) and 4-(1H-benzo[d]imidazol-2-yl) thiazol-2-amine²²(3) (0.004 mol) were refluxed in ethanol for about 4h with few drops of glacial acetic acid. Reaction progress was monitored by TLC method. Reaction mixture was cooled, filtered and dried followed by recrystallisation with methanol to afford the corresponding Schiff’s base (4).

General reaction procedure for Compound 5:

Equimolar mixture of compound 4 (0.01), mercapto acetic acid (0.01), Aromatic aldehyde (0.01) in 1, 4 dioxane (30 ml) containing a pinch of ZnCl₂ was refluxed for 6.5 hours. The resulting solution was filtered and cooled in ice bath. The resulting solid product was filtered, washed with 10% NaHCO₃and recrystallized from Alcohol. The compounds 3(b-e) were prepared by similar procedure with minor changes in reaction conditions.

2.2.1. (Z)-3-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-5-benzylidene-2-phenylthiazolidin-4-one (5a): IR (KBr, cm^-1): 3328 (NH),1548(C=N), 1665 (C=O), 1230(C=S); ¹H NMR (DMSO-d₆, 400MHz, δ in ppm): 5.92(s,1H,N-CH-Ar), 6.55 (S,1H, CH Ar), 7.12-7.22(m,4H, Ar-H), 7.26-7.48(m,5H, Ar-H),7.56-7.74 (m,5H,Ar-H),9.85 (bs,1H,NH). MS, m/z(%),466 (M⁺); Anal. Calcd for C₂₆H₁₇N₃OS: C, 66.93; H, 3.89; N, 12.01%. Found: C, 66.23; H, 3.72; N, 11.68%.

2.2.2. (Z)-3-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-5-(4-methylbenzylidene)-2-phenyl thiazolidin-4-one (5b):

IR (KBr, cm^-1):3337(NH), 2960(C-H), 1561(C=N), 1672 (C=O), 1249 (C=S); 1HNMR (DMSO-d₆, 400MHz, δ in ppm): 2.85(s,3H,CH₃), 5.86(s, 1H, N-CH-Ar) , 6.54(s, 1H,CH Ar); 7.15-7.24(m,4H Ar-H), 7.28-7.38( m, 4H,Ar-H), 7.42-7.64( m,4H,Ar-H), 9.96(bs, 1H,NH): MS,m/z(%), 480(M+); Anal.Calcd for C₂₇H₂₀N₄OS₂: C,67.48; H,4.19;N, 11.66% Found: C, 67.08;H,3.90; N,11.05%.

2.2.3. (Z)-3-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-5-(4-methoxybenzylidene)-2-phenyl thiazolidin-4-one (5c):

IR(KBr,cm^-1):3341(NH),3045(C-H),1561(C=N),1682(C=O),1238(C=S);¹HNMR(DMSO-d₆,400 MHz, δinppm): 3.45(s,3H,OCH₃),5.72(S,1H,N-CH-Ar),6.58(s,1H,CHAr);7.18-7.25(m,4H,Ar-H),7.26-7.51 (m,5H, Ar-H), 7.65-7.84 (m,4H, Ar-H),10.06(bs,1H,NH), MS, m/z (%), 496 (M⁺); Anal. Calcd for C₂₇H₂₀N₄O₂S₂: C, 65.30; H, 4.06; N, 11.28%.Found: C, 65.04; H, 3.86; N, 10.85 %.

2.2.4.(Z)-3-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-5-(4-hydroxybenzylidene)-2-phenylthiazolidin4one (5d):

IR(KBr,cm¹):3514(OH),3346(NH),1568(C=N),1685(C=O),1238(C=S);¹HNMR (DMSO-d₆,400 MHz, δ inppm): 5.88 (s,1H, N-CH-Ar), 6.56 (s ,1H, CH Ar);7.14-7.27(m,4H,Ar-H),7.25-7.49(m,5H,Ar-H),7.58-7.71(m,4H,Ar-H),10.11(bs,1H,NH), 11.28 (s, 1H, OH), ;MS, m/z (%), 482 (M⁺); Anal. Calcd for C₂₆H₁₈N₄O₂S₂: C, 64.71; H, 3.76; N, 11.61%. Found: C, 64.42; H, 3.42; N, 11.38%.

2.2.5.(Z)-3-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-5-(4-nitrobenzylidene)-2-phenyl thiazolidin-4-one (5e):

IR (KBr, cm^-1): 3340 (NH),1572(C=N), 1523 (NO₂), 1685 (C=O), 1238(C=S); ¹HNMR (DMSO-d₆,400MHz, δ in ppm): 5.85 (s,1H, N-CH-Ar), 6.58 (s,1H, CH Ar); 7.13-7.26 (m,4H, Ar-H),7.25-7.48(m,5H, Ar-H), 8.15-8.34 (m,4H, Ar-H),10.18(bs,1H,NH),MS, m/z (%), 511 (M⁺); Anal. Calcd for C₂₆H₁₇N₅O₃S₂C, 61.04; H, 3.35; N, 13.69%. Found: C, 60.84; H, 3.05; N, 13.22%.

2.3.0. General reaction procedure for compound 6:

A mixture of compound 5a (0.03mol), hydrazine hydrate (0.03mol) and anhydrous CH₃COONa (0.001mol) in glacial acetic acid (30ml) was heated under reflux for about 7 h, it was cooled at room temperature and poured into crushed ice. The solid was filtered, washed with water and recrystallized by using ethanol to afford the pure compound. The compounds 6(b-e) were prepared by similar procedure with minor changes in reaction conditions.

2.3.1. 6-(4-(1H-benzo[d]imidazol-2-yl) thiazol-2-yl)-3,5-diphenyl-3,3a,5,6-tetrahydro-2H-pyrazolo [3,4-d]thiazole (6a):

IR (KBr, cm^-1): 3348 (NH), 3078 (C-H ring), 1546(C=N), 1238 (C=S), 1042(N-N); ¹H NMR (DMSO-d₆, 400MHz, δ in ppm):4.85 (d, 1H, CH-N), 4.62 (d, 1H, CH-S), 5.90 (s,1H,N-CH-Ar), 6.56 (s,1H, CH Ar); 7.10-7.22(m,4H, Ar-H), 7.26-7.45(m,5H,Ar-H),7.55-7.78 (m,5H, Ar-H), 9.75 (bs, 1H,NH). MS, m/z(%),480 (M⁺); Anal. Calcd for C₂₆H₂₀N₆S₂: C, 64.98; H, 4.19; N, 17.49%. Found: C, 64.58; H, 4.02; N, 17.18%.

2.3.2.6-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-5-phenyl-3-(p-tolyl)-3,3a,5,6-tetra hydro -2H-pyrazolo [3,4-d]thiazole (6b):IR (KBr, cm^-1): 3342 (NH),3084(C-H ring), 2963 (C-H), 1565 (C=N), 1249 (C=S), 1052(N-N); ¹H NMR (DMSO-d₆, 400MHz, δ in ppm): 2.82 (s,3H,CH₃),4.82 (d, 1H, CH-N), 4.60 (d, 1H, CH-S), 5.81 (s,1H, N-CH-Ar), 6.51(s,1H, CH Ar); 7.18-7.26 (m,4H, Ar-H), 7.28-7.38(m,4H, Ar-H), 7.45-7.68 (m,4H, Ar-H), 9.98 (bs, 1H,NH).MS, m/z (%), 494 (M⁺); Anal. Calcd for C₂₇H₂₂N₆S₂: C, 65.56; H, 4.48; N, 16.99%. Found: C, 65.34; H, 4.28; N, 16.55%

2.3.3. 6-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-3-(4-methoxyphenyl)-5-phenyl-3,3a,5,6-tetra hydro-2H-pyrazolo[3,4-d]thiazole(6c):IR(KBr,cm^-1):3343(NH),3039(C-H),1559(C=N),1241(C=S)1063(N-N);¹HNMR (DMSO, d₆,400MHz,δinppm):3.48(s,3H,OCH₃),4.82(d,1H,CH-N), 4.60(d,1H,CH-S),5.74(s,1H,N-CH-Ar), 6.58 (s,1H, CH Ar); 7.21-7.28 (m,4H,Ar-H), 7.31-7.58 (m,5H,Ar-H),7.68-7.87 (m,4H,Ar-H),10.04 (bs,1H,NH), MS, m/z (%), 510 (M⁺); Anal. Calcd for C₂₇H₂₂N₆OS₂:C, 63.51; H, 4.34; N, 16.46%. Found: C, 63.31; H, 4.18; N, 16.16 %.

2.3.4.4-(6-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-5-phenyl-3,3a,5,6-tetrahydro-2H-pyrazolo[3,4-d] thiazol -3-yl)phenol (6d): IR (KBr,cm^-1):3516(OH), 3352 (NH),1571 (C=N), 1242 (C=S) 1043 (N-N);¹HNMR(DMSO-d₆,400 MHz, δ in ppm): 4.72(d,1H,CH-N), 4.61 (d,1H, CH-S), 5.92(s,1H,N-CH-Ar),6.53(s,1H,CH Ar);7.18-7.31 (m,4H,Ar-H), 7.28-7.52 (m,5H,Ar-H), 7.61-7.74 (m,4H,Ar-H), 10.15 (bs,1H,NH), 11.35(s, 1H,OH) ;MS, m/z (%), 413 (M⁺); Anal. Calcd for C₂₆H₂₀N₆OS₂: C, 62.88; H, 4.06; N, 16.92%. Found: C, 62.62; H, 3.86; N, 16.52 %.

2.3.5.6-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-3-(4-nitrophenyl)-5-phenyl-3,3a,5,6tetrahydro-2Hpyrazolo [3,4-d]thiazole (6e):IR ( KBr, cm^-1): 3346 (NH), 1575 (C=N), 1528 (NO₂), 1236 (C=S),1052(N-N) ; ¹HNMR (DMSO-d₆,400MHz, δ in ppm):4.91(d,1H,CH-N), 4.75 (d,1H, CH-S), 5.88 (s,1H, N-CH-Ar), 6.62 (s,1H, CH Ar); 7.16-7.29 (m,4H, Ar-H), 7.28-7.52 (m,5H, Ar-H), 8.18-8.41 (m,4H, Ar-H),10.22 (bs,1H,NH),MS, m/z (%), 525 (M⁺); Anal. Calcd for C₂₆H₁₉N₇O₂S₂: C, 59.41; H, 3.64; N, 18.65 %. Found: C, 59.12; H, 3.35; N, 18.34%.

2.4. General reaction procedure for compound 7:

A mixture of compound 5a (0.03 mol), hydroxylamine hydrochloride (0.03mol) and anhydrous CH₃COONa (0.001 mol) in glacial acetic acid (30ml) was heated under reflux for about 7h, cooled at room temperature and poured in to crushed ice. The resulting solid product was filtered, washed with water and recrystallized from ethanol to afford the pure compound. The compounds 7(b-e) were prepared by similar procedure with minor changes as per the reaction conditions.

2.4.1.6-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-3,5-diphenyl-3,3a,5,6-tetrahydrothiazolo[4,5-c] isoxazole (7a): IR (KBr, cm^-1): 3348 (NH), 3078 (C-H ring), 1546(C=N), 1238 (C=S); ¹H NMR (DMSO-d₆, 400MHz, δ in ppm):4.95 (d, 1H, CH-O), 4.72 (d, 1H, CH-S), 5.90 (s,1H,N-CH-Ar), 6.58 (s,1H, CH Ar); 7.10-7.22(m,4H, Ar-H), 7.26-7.45(m,5H,Ar-H),7.55-7.78 (m,5H, Ar-H), 9.75 (bs, 1H,NH). MS, m/z (%), 481 (M⁺); Anal. Calcd for C₂₆H₁₉N₅OS₂: C, 64.84; H, 3.98; N, 14.54%. Found: C, 64.25; H, 3.45; N, 14.24%.

2.4.2. 6-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-5-phenyl-3-(p-tolyl)-3,3a,5,6-tetrahydro thiazolo[4,5-c] isoxazole (7b): IR (KBr, cm^-1): 3342 (NH),3084(C-H ring), 2963 (C-H), 1565 (C=N), 1249 (C=S) ; ¹H NMR (DMSO-d₆, 400MHz, δ in ppm): 2.82 (s,3H,CH₃), 4.98 (d, 1H, CH-O), 4.75 (d, 1H, CH-S), 5.81 (s,1H, N-CH-Ar), 6.61 (s,1H, CH Ar); 7.18-7.26 (m,4H, Ar-H), 7.28-7.38(m,4H, Ar-H), 7.45-7.68 (m,4H, Ar-H), 9.98 (bs, 1H,NH).MS, m/z (%), 495 (M⁺); Anal. Calcd for C₂₇H₂₁N₅S₂: C, 65.43; H, 4.27; N, 14.13%. Found: C, 65.12; H, 4.04; N, 13.89%.

2.4.3.6-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-3-(4-methoxyphenyl)-5-phenyl-3,3a,5,6-tetrahydro thiazolo [4,5-c]isoxazole(7c):IR(KBr,cm^-1):3343(NH),3039(C-H),1559(C=N),1241(C=S);¹HNMR(DMSO-d₆,400MHz, δ in ppm):3.48 (s,3H,OCH₃),5.12 (d,1H,CH-O),4.74 (d,1H,CH-S), 5.74(s,1H,N-CH-Ar), 6.64 (s,1H, CH Ar); 7.21-7.28 (m,4H,Ar-H), 7.31-7.58 (m,5H,Ar-H),7.68-7.87 (m,4H,Ar-H),10.04 (bs,1H, NH), MS, m/z (%), 511 (M⁺); Anal. Calcd for C₂₇H₂₁N₅O₂S₂: C, 63.38; H, 4.14; N, 13.69%. Found: C, 63.05; H, 4.05; N, 13.21%.

2.4.4.4-(6-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-5-phenyl-3,3a,5,6-tetrahydro thiazolo [4,5-c]isoxazol-3-yl)phenol(7d):IR (KBr,cm^-1): 3516(OH), 3352 (NH),1571 (C=N), 1242 (C=S); ¹HNMR(DMSO-d₆,400 MHz, δ in ppm): 5.08 (d,1H,CH-O), 4.72 (d,1H, CH-S), 5.92 (s,1H, N-CH-Ar), 6.63 (s,1H, CH Ar); 7.18-7.31 (m,4H,Ar-H), 7.28-7.52 (m,5H,Ar-H), 7.61-7.74 (m,4H,Ar-H), 10.15 (bs,1H,NH), 11.35 (s, 1H,OH) ;MS, m/z (%), 497 (M⁺); Anal. Calcd for C₂₆H₂₀N₆OS₂: C, 62.76; H, 3.85; N, 14.07%. Found: C, 62.76; H, 3.85; N, 14.07%.

2.4.5.6-(4-(1H-benzo[d]imidazol-2-yl)thiazol-2-yl)-3-(4-nitrophenyl)-5-phenyl-3,3a,5,6-tetrahydro thiazolo [4,5 -c] isoxazole(7e): IR ( KBr, cm^-1): 3346 (NH), 1575 (C=N), 1528 (NO₂), 1236 (C=S); ¹HNMR (DMSO-d₆,400MHz, δ in ppm):5.14 (d,1H,CH-O), 4.85 (d,1H, CH-S), 5.88 (s,1H, N-CH-Ar), 6.65 (s,1H, CH Ar); 7.16-7.29 (m, 4H, Ar-H), 7.28-7.52 (m,5H, Ar-H), 8.18-8.40 (m,4H, Ar-H),10.22 (bs,1H,NH),MS, m/z (%), 526 (M⁺); Anal. Calcd for C₂₆H₁₈N₆O₃S₂: C, 59.30; H, 3.45; N, 15.96%. Found: C, 58.90; H, 3.15; N, 15.56%.

3.0. In-Vitro Anti-Inflammatory Activity:

About 5 ml of reaction mixture which includes 0.2 ml of egg albumin, 2.8 ml of phosphate buffered saline (PBS, p^H 6.4) was added with 2 ml of compounds at 50 and 100 µg/ml and double-distilled water with same volume was used as control. The prepared mixtures were incubated at a temperature of (37±2)⁰C in BOD incubator for about 15 min and followed by heating at 70⁰C for about 5 min. After attaining the room temperature, absorbance was measured at 660 nm using vehicle as blank and viscosity was determined by using Ostwald viscometer. Diclofenac sodium was used as standard drug²³and its concentrations were maintained at 50, and100 µg/mL.

The inhibition (%) of protein deanaturation was measured using the following formulae

% Inhibition = 100 X (Vt/Vc-1)

Where, V_t= Test sample Absorbance, V_c= Absorbance of control

Figure 1: Docking pose showing the interaction between the enzyme and the ligand 1

Table 4: Free energy of binding between the enzyme and the ligand 1

Est. Free Energy of Binding		vdW + Hbond + desolv Energy			Electrostatic Energy		Frequency		Interact. Surface
-6.06 kcal/mol		-7.15 kcal/mol			-0.04 kcal/mol		50%		689.87
Various types of interactions between the amino acids of the enzyme and the ligand 1
polar			hydrophobic			pi-pi		other
N5 (20)	GLU67 (OE1, OE2)		C23 (31)	CYS57 (CB)		C18 (26)		C19 (27)		GLN54 (CB)
[2.88]			[3.89]			[3.24]		[3.79]
N6 (21)	GLU67 (OE1, OE2)					C19 (27)		N4 (15)		GLU67 (CB, CG)
[3.22]						[3.88]		[3.63]
H9 (43)	GLU67 (OE1, OE2)							C11 (16)		GLU67 (CB, CD, CG, OE1, OE2)
								C9 (12)		GLU67 (CB)
								[3.69]
								N3 (11)		GLU67 (CB)
								[2.93]
								N2 (9)		GLU67 (CB)
								[3.69]
								C12 (17)		GLU67 (CD, CG, OE2)
								N5 (20)		GLU67 (CD, CG)
								[3.11]
								C22 (30)		GLU67 (CG)
								[3.77]
								N6 (21)		GLU67 (CD)
								[3.41]
								H9 (43)		GLU67 (CD)
								[3.08]
								C14 (22)		GLU67 (OE2)
								[3.48]
								C15 (23)		GLU67 (OE2)
								[3.32]
								C17 (25)		GLU67 (OE2)
								[3.28]
								C3 (3)		THR71 (CG2)
								[3.42]
								C2 (2)		THR71 (CG2)
								[3.19]
								C1 (1)		THR71 (CG2)
								[3.69]

Docking studies of 6-(4-(1H-benzo[d]imidazol-2-yl) thiazol-2-yl)-3, 5-diphenyl-3, 3a, 5, 6-tetrahydrothiazolo [4, 5-c] Isoxazole (7a):

Figure 2: Docking pose showing the interaction between the enzyme and the ligand 2

Table 5: Free energy of binding between the enzyme and the ligand 2:

Est. Free Energy of Binding

vdW + Hbond + desolv Energy

Electrostatic Energy

Frequency

Interact. Surface

-6.40 kcal/mol

-6.93 kcal/mol

-0.51 kcal/mol

50%

568.269

Interactions between the amino acids of the enzyme and the ligand 2:

hydrogen bonds

polar

hydrophobic

pi-pi

Other

N5 (20)

TYR55 (CD2, CE1, CE2, CZ, O, OH)

H5 (39)

GLU67 (OE2)

C18 (25)

CYS57 (CB)

C11 (14)

TYR55 (CD1, CE1)

C11 (14)

GLN54 (CB)

[3.12]

[3.86]

[3.25]

[3.61]

[3.88]

N4 (15)

GLU67 (CD, OE2)

C19 (26)

CYS57 (CB)

C10 (13)

TYR55 (CD1, CE1)

C10 (13)

GLN54 (CB)

[3.24]

[3.63]

[3.35]

[3.80]

N5 (20)

GLU67 (CD, CG, OE2)

C19 (26)

CYS59 (SG)

O1 (21)

TYR55 (CE2)

[3.35]

[3.56]

[3.45]

N3 (11)

GLU67 (OE2)

C17 (24)

LYS56 (NZ)

[3.08]

[3.81]

C22 (29)

LYS56 (NZ)

[3.48]

O1 (21)

CYS57 (SG)

[3.88]

C12 (16)

GLU67 (CD, CG, OE2)

[3.28]

C13 (17)

GLU67 (CG)

[3.45]

O1 (21)

GLU67 (CG)

[3.29]

C23 (30)

GLU67 (CD, OE1, OE2)

[3.39]

C24 (31)

GLU67 (OE1)

[3.68]

C9 (12)

GLU67 (OE2)

[3.20]

C8 (10)

GLU67 (OE2)

[3.89]

5.0. RESULTS AND DISCUSSION:

The present study reports the synthesis of oxazolo-pyrazole and oxazolo-isoxazole derivatives linked with benzimidazole in good yield. The compound 4 (Schiff base) is synthesized using 2-amino benzimidazole with Benzaldehyde under simple conditions. The compound 5 (Chalcone derivatives of thiazolidinone) is synthesized by cyclization of one-pot three-component method using compound 4, mercaptoacetic acid and substituted aromatic aldehyde using anhydrous Zinc chloride²⁶. Later, the derivative of thiazolidinone (5) undergoes cyclized with hydrazine hydrate and hydroxyl amine hydrochloride in the presence of anhydrous acetic acid to afford the compound 6 and 7respectively. The structures of title compounds were confirmed based on spectral and analytical data. The compounds showed Infra red absorption bands at 3348 cm^-1 (NH), 1564 cm^-1 (C=N), 1238 cm^-1 (C=S), 1042 cm^-1(N=N), 1680 cm^-1 (C=O) respectively. Proton NMR spectra of synthesized compounds showed singlet signals at 5.58 for N-CH-S, 6.56 Singlet thiazole proton signal at 6.56, doublet signals at 4.62 CH-S, 4.82 CH-N, and 9.82ppm for NH protons and also phenylic protons as multiplet in the range of 7.10-8.41ppm. Mass spectra of synthesized compounds showed a molecular ion peak at m/z concerning their molecular weights.

In vitro anti-inflammatory activity:

The synthesized derivatives were tested for their efficacy to inhibit the protein deanaturation. The process is carried out by using egg albumin. All the compounds showed concentration dependent inhibition property. Compounds (7e and 6e) are found to exhibit significant inhibition percentage compared to other synthesized compounds. Nevertheless, Compounds (7c, 6c, 7d and 6d) are competed in exhibiting a notable activity with good inhibition percentage (table 2). On the other hand, the compounds 6b and 7a were showed moderate activity. The variations in the activity might be due to different functional groups present in derivatives. By these studies, it is concluded that benzimidazole derivatives showed preliminary anti-inflammatory activity.

The IC₅₀ values represented in table 3 are shown as mean ±SD. According to the data, the IC₅₀ 24±0.1, 21±0.5 of compounds 7c and 6d are significant and are comparable with standard Diclofenac sodium standard.

In silico anti-inflammatory activity:

Molecular docking studies have shown that the ligand 1 has a binding energy of -6.06 kcal/mol (table 4). Figure 1 shows the docking pose between the enzyme and the ligand 1where, different amino acids are bind to the active site of the ligand. Various types of interactions between the ligand1 and amino acids of enzymes are shown in table 4 where the major amino acids involved in binding are gluatamic acid, cysteine, tyrosine, glutamine and threonine at the active site. Docking studies have shown the ligand 2 has a binding energy of -6.40 kcal/mol (table 5). Figure 2 shows the docking pose between the enzyme and the ligand 2 where various amino acids bound to the active site of the ligand. Various types of interactions between the ligand 2 and amino acids of enzymes are are shown in table 5 where the major amino acids involved in binding are tyrosine, gluatamic acid, glutamine, cysteine and lysine. Compared with ligand 1, ligand 2 has higher free energy of binding and could act as a better anti inflammatory compound.

6.0. CONCLUSION:

It has been concluded that a series of pyrazolo-thiazoles and isoxazole-thiazoles linked with benzimidazole was synthesized and it has been reported in the present work that the title compounds are exhibiting excellent in vitro and in silico anti-inflammatory activity.

7.0. ACKNOWLEDGEMENT:

The authors are grateful to the Principal, Management of MGIT–Hyderabad and IICT- Hyderabad for their constant support and also thankful to the Department of Bio-Chemistry, Mahatma Gandhi University-Nalgonda, India.

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Received on 13.04.2020 Modified on 24.09.2020

Research J. Pharm. and Tech. 2021; 14(8):4253-4260.

DOI: 10.52711/0974-360X.2021.00738

Compounds		6a	6b	6c	6d	6e	7a	7b	7c	7d	7e	Standard
Conc. (µg/mL)	50	40	41	44	43	45	42	43	44	43	46	48
Conc. (µg/mL)	100	74	75	78	76	85	75	77	84	83	88	90

Compounds	6a	6b	6c	6d	6e	Standard
IC ₅₀	5.1±2.4	6.5±2.3	7.9±0.5	21±0.5	17±1.0	28 ± 0.5
Compounds	7a	7b	7c	7d	7e	Standard
IC ₅₀	10.±1.5	09±0.7	24±0.1	15.±2.3	14.5±2.1	32±0.5