Docking Studies , Synthesis , and Evaluation of Antioxidant Activities of N-Alkylated , 1 , 2 , 4-Triazole , 1 , 3 , 4-Oxa-, and Thiadiazole Containing the Aminopyrazolopyridine Derivatives

Synthesis of some 1,3,4thia-, oxadiazol and 1,2,4 triazole incorporated the biologically active and the pyrazolopyridine derivative. Molecular modeling and docking of the active compounds into AKR1C3 complexed with its bound inhibitor indomethacin using Molsoft ICM 3.4-8C program were performed in order to predict the affinity and orientation of the synthesized compounds at the active site.

Design of drug targets containing two carboxylic groups appropriately attached to the opposite sides of the aromatic fragment (e.g., naphthalene ring) as shown by our synthesized compounds to interact with the active site hydrophobic pocket while the first carboxylate may occupy the oxyanion hole and the second forms H-bonds with oxygen of coenzyme's diphosphate moiety will be a good rational for potent inhibitors.

Experimental
Melting points were determined on a Buchi melting point.IR spectra were recorded with a Perkin-Elmer model 1720 FTIR (KBr), 1H NMR spectra were recorded with Bruker AC 250 FT NMR spectrometer at 250 MHz with TMS as an internal standard.EIMS and FABMS spectra were recorded with a Finnigen MAT 312 = AMD.The microanalyses were performed at the microanalytical unit, Cairo University.

Generation of Ligand and Enzyme Structures
The crystal structure of AKR1C3 complexed with its bound inhibitor Indomethacin was downloaded through the Protein Data Bank PDB/ RCSB site and saved as * .pdbfile [18].A set of novel N-Alkylated, 1,2,4-triazole, 1,3,4-oxa-, thiadiazole and complexes containing the aminopyrazolopyridine derivatives were designed to inhibit AKR1C3.All compounds were built in ChemDraw Ultra version 8.0.3 and their energy minimized through Chem3D Ultra version 8.0.3/MM2, Jop Type: minimum RMS Gradient of 0.100, and saved as MDL MolFile ( * .mol).

Docking Using Molsoft ICM 3.4-8C Program
The novel energy-minimized Indomethacin analogues were docked into the active site of AKR1C3 crystal structure using ICM-Pro software version 3.4 -8 C. ICM-Pro scores the binding of a ligand to a receptor based upon the comparison of a series of small molecule/ protein interactions that have been reported in the PDB database.A rigid receptor/flexible ligand approach was adopted that uses five potential energy maps combining hydrophobicity, electrostatics, hydrogen bond formation, and two van-der-Waals parameters.In all cases, the program's default parameters were used.
The acetohydrazide 3 was treated with phenylisothio-its molecular formula (Scheme 3).cyanate to give the corresponding phenylhydrazinecarbothioamide derivative 6. which was elucidated, besides the 1 H NMR and IR, by the mass spectrum which showed the molecular ion peak at m/z 270 corresponding to [M + + 1], m/z When the thiosemicarbazide 6 was reacted with concentrated sulfuric acid.1,3,4-thiadiazole derivative 7 was obtained which showed in its 1 H NMR spectrum of the broadband of the NHPh at δ 11.55 ppm.The formation of the thiadiazole ring, under such acidic conditions, is due to the loss of nucleophilicity of N-4 as a result of its protonation leading to an increase in the nucleophilicity of the sulfur atom toward the attack of the carbonyl carbon as shown in Scheme 2. On the other hand, when the cyclization of 6 was carried out under basic conditions, the nucleophilicity of N-4 was enhanced and affording cyclization with carbonyl carbon atom to afford 1,2,4-triazole derivative in 67% yield.In the treatment of thiosemicarbazide 6 with mercuric oxide, the cyclization was performed, affording the 1,3,4oxadiazole derivative 9.The method of cyclization includes desulfurization by HgO.The 1 H NMR of triazole 8 showed a broad singlet at δ 12.05 ppm corresponding to the SH group.The oxadiazole derivatve 9 was elucidated besides, NMR, elemental analysis, by the mass spectrum which showed a [M + ] peak, in agreement with

Molecular Modeling Studies
To pre-assess the anti-tumorigenic behavior of our N-Alkylated, 1,2,4-triazole, 1,3,4-oxa-, thiadiazole and complexes containing the aminopyrazolopyridine derivatives 5(a-c) and (7-9) on a structural basis, automated docking studies were carried out using MOLSOFT ICM 3.4 -8C program [21].The scoring functions and hydrogen bonds formed with the surrounding amino acids are used to predict their binding modes, their binding affinities and orientation of these compounds at the active site of AKR1C3 enzyme.The protein-ligand complex was constructed based on the X-ray structure (PDB entry 1S2A) AKR1C3 with its bound inhibitor indomethacin [22].
The scoring functions of the compounds were calculated from minimized ligand protein complexes.The X-ray crystal structure of AKR1C3 reveals a substratebinding site that consists mainly of: hydrophobic aromatic amino acid side chains (Tyr24, Tyr55, Leu54, Trp227, and Phe306).An oxyanion hole, which is located at the bottom of the hydrophobic pocket, is formed by active site tyrosine (Tyr55), histidine (His117), and the coenzymes nicotinamide ring [21].Thiosemicarbazide derivative Scheme 2. Schematic investigation according to the condition applied.
In our investigation, the 3D-coordinates in X-ray crystal structure of AKR1C3 in complex with the ligand, indomethacin (PDB entry 1S2A) [21] was used as the receptor model in AKR1C3 docking simulation.The docked model of indomethacin with AKR1C3 (Figure 1) was consistent with the previously reported X-ray analysis [22] and revealed the following binding mode: The carbonyl oxygen is far away from Tyr 55 or His 117 to H-bond directly.Instead, the carboxylate group points toward and interacts with the oxygen atoms O 1 n, O 2 n from the nicotinamide half of the NADP + diphosphate moiety, forming two hydrogen bonds, and additional H-bond if formed between indomethacin O 2 of COOH and He 2 of Glu 222.

Figure 1 .
Figure 1.The proposed binding mode of original ligand indomethacin into the active binding site of AKR1C3 active site.It has ICM score of −80.45, it forms three hydrogen bonds, two of them between H of COOH and O1n, O2n of Phosphate moiety of NADP.And another hydrogen bond between O2 of COOH and He2 of amino acid Glu222….indomethacin.It is interesting to point out that comounds 5(a-c) and (7-9) are found to be very promising p

Figure 2 .
Figure 2. The proposed binding mode of 5a into the active binding site of AKR1C3 active site.It has ICM score of −67.08, it forms three hydrogen bonds, two of them between O2 of OH and He2 and He3 of Tyr24 and Glu58…One hydrogen bond between O3 of SO2 and Hh1 of amino acid Arg 226.And hydrogen bond between O4 of SO2 and H1n1 of Phosphate moiety of NADP….

Figure 3 .
Figure 3.The proposed binding mode of 5b into the active binding site of AKR1C3 active site.It has ICM score of −88.58, it forms 13 hydrogen bonds, one between O1 of OCH3 and Ha1 of Phosphate moiety of NADP.Two hydrogen bonds between O4 of SO2 and Hd2 of Arg31 and H7n7 of Amide of Nicotinamide ring … three hydrogen bonds, two of them between O2 of OH and He2 and He3 of Tyr24 and Another two hydrogen bonds is between H14, H15 of benzene ring of sulfonamide and He2 of amino acid Tyr55.