INTRODUCTION:

Bioactive natural products can be considered very promising starting points for the development of new therapeutic agents¹. The biological importance of unsaturated lactones is well known. In particular, the γ-alkylidene butenolides skeleton is a useful entity that is present in natural product such as fibrolides, dihydroxerulin, and protoanemonin and its derivatives possess antiviral, antibiotic, anticancer activity^2,3.

The direct method for the synthesis of γ-butenolides is the addition of 2-trimethylsiloxyfuran to imines in the presence of Lewis acids such as BF₃.OEt₂, TMSOTf, SnCl_4,Bi(OTf)_3, andSiCl₄ under strictly anhydrous and low temperature reaction conditions.^4,5 The presence of even a small amount of water lower the yields of the product probably due to the rapid decomposition or deactivation of the promoters. Therefore, the development of new reagents that are more efficient and provide improved yields and selectivity are well appreciated.⁶

Recently, molecular iodine has received considerable attention as an inexpensive, non-toxic, readily available catalyst for various organic transformations; affording the corresponding products with high selectivity in excellent yields. The mild Lewis acidity associated with iodine enhanced its usage in organic synthesis to perform several organic transformations using stoichiometric levels to catalytic amounts. Owing to advantages associated with this eco-friendly catalyst molecular iodine has been explored as a powerful reagent for various organic transformations.

The unique features of iodine prompted us to explore further applications of iodine as catalyst in various carbon-carbon bond forming reactions. The present work was aimed to develop a novel method for the synthesis of δ-amino γ-butenolides by the condensation of 2-trimethylsiloxyfuran with various imines in presence of iodine under mild conditions and perform insilico evaluation by docking with prostaglandin E synthase1 and phospholipase A2 using Molegro virtual docker.

MATERIALS AND METHODS:

The chemicals used were of synthetic grade purchased from local chemical vendors. Infrared spectra are recorded on Perkin Elmer model 283B and Nicolet 740 FT-IR instruments and values are given in cm^-1Proton magnetic resonance spectra are recorded on Varian Gemini 200, Varian unity-400 and Advance-300 MHz Bruker UX-NMR instrument. The samples are made in CCl₄/chloroform-d (1:1) using tetramethylsilane (Me₄Si) as the internal standard and are given in the scale Mass spectra are recorded on VG micro mass 7070H(ESI and Cl), VG Auto spec (FAB) using Cs⁺ ion gun, m-nitro benzyl alcohol (MNBA) as a matrix and are given in mass units(m/z).Analytical thin-layer chromatography (TLC) is performed on pre coated silica-gel-60 F₂₅₄(0.5mm) glass plates. Visualization of the spots on the TLC plates is achieved either to iodine vapour or UV light. Employing TLC techniques using appropriate solvent system for development monitored all the reasons. Moisture sensitive reactions are carried out by standard syringe-septum techniques. Dry either, dry toluenes are made by distilling them from sodium benzophenone ketyl and dry methanol is prepared by using potassium hydroxide. All extracts are extracted with ethylacetate and water and concentrated at reduced pressure on Buchi-R-3000 rotary evaporated below 50⁰C. Yields reported are isolated. Various databases such as unit prot, PDB, PDBSUM, BLAST and RAPPER were used. Softwares used for docking were Accelrys discovery studio, ACD Chemsketch and MOL GRO virual docker

General procedure for synthesis of Butenolides:

Synthesis of δ-amino γ-butenolides:

To the stirred solution of imine (1mmol) in ether (5mL) added I₂(5 mol%) in ether (0.5mL) and stirred for 10 min at -78⁰C. Then solution of 2-(trimethylsiloxy) furan (1.2mmol) in ether (2mL) was added to the reaction mixture at -78⁰C under nitrogen. After complete conversion as indicated by TLC, the reaction mixture was quenched with saturated Hypo (sodium thiosulphate penta hydrate) solution (5mL) at -78⁰C and the reaction mixture was stirred at room temperature for 10min. The resultant mixture was extracted with ethylacetate (3x15mL), the combined organic layers were dried over anhydrous Na₂SO₄, concentrated under vaccum and purified by column chromatography on silica gel (merck, 60-120 mesh, ethyl acetate-hexane 1:4) to afforded pure product. (scheme-1)

The treatment of imines with 2-trimethylsiloxyfuran in the presence of 5 mol% Iodineafforded the corresponding α,β-unsaturated- γ-lactones in 84-91% yield. Similarly a variety of imines were converted to their corresponding lactones in high yields by using this procedure. The reactions proceeded smoothly at ambient temperature under mild conditions. Generally the products were obtained as a mixture of threo and erythro isomers favouring threo diastereomers, which cannot be separated by column chromatography. The threo and erythro stereochemistry (fig-1) of the products was assigned on the basis of coupling constants of the protons in the ¹HNMR spectra of products

Fig. 1:

Databases used:

Uni Prot Consortium hosts a large resource of bioinformatics databases and services. SIB, located in Geneva, Switzerland, maintains the ExPASy (Expert Protein Analysis System) servers that are a central resource for proteomics tools and databases. PDB (Protein Data Bank) is a database in which the details about all the known proteins will be stored and it is available for all the users across the world at free of cost. PDBSUM provides an at-a-glance overview schematic diagram of the molecules in each structure and of the interactions between them. BLAST Basic Local Alignment Search Tool is an algorithm for comparing primary biological sequence information and identifies the library sequences that resemble the query sequence above a certain threshold. RAPPER, an abinitio conformational search algorithm for restraint-based protein modeling. It is used for all-atom loop modeling. It also mainly used for structure validation of modeled protein through ramachandran plot analysis.

Softwares used:⁷

Accelrys Discovery Studio⁸ used to examine the properties of large and small molecules, study systems, identify leads and optimize candidates. Drive scientific exploration from target identification to lead optimization with a wealth of trusted life science modeling and simulation tools. ACD/Chemsketch freeware was used to draw chemical structures, reactions and calculation of basic molecular properties. MOL GRO VIRUAL DOCKER^9,10 was used for predicting protein - ligand interactions. it includes all the steps from preparation of the molecules to determination of the potential binding sites of the target protein, and prediction of the binding modes of the ligands. The Molegro Virtual Docker (MVD) has been shown to yield higher docking accuracy than other state-of-the-art docking products (MVD: 87%, Glide: 82%, Surflex: 75%, FlexX: 58%

RESULTS AND DISCUSSION:

The methodology offers very attractive features such as reduced time, higher yields, and relatively best and thus a wide scope in synthesis. The possible mechanism for the synthesis of the compounds described in Scheme 2. The compounds synthesized from various intermediates were tabulated in the table-1 and properties of the five compounds were discussed.

Compound-1: IUPAC name: 5-[phenyl (phenyl amino) methyl] furan-2(5H)-one Molecular Formula: C₁₇H₁₅NO₂, Molecular weight: 265, ¹H-NMR (300MHz, CDCl₃) : δ4.45(d, 1H, J = 6.4 Hz), 4.76 (d, 1H, J = 3.39 Hz), 5.14 (d, 1H, J = 6.2 Hz), 5.28-5.39 (m, 1H), 6.04 (dd, 1H, J = 3.9, 5.6 Hz), 6.13 (dd, 1H, J = 3.7, 5.6 Hz), 6.39-6.52 (m, 4H), 6.67 (t, 2H, J = 7.3 Hz), 7.05 (q, 4H, J = 6.0, 13.2 Hz), 7.15-7.41 (m, 11H), IR (neat): 3377, 2924, 2855, 1757, 1601, 1498, 1096, 753 cm^-1, MS (ESI): m/z 266 (M+H)⁺

Compound-2: IUPAC name: 5-[(3-bromophenyl) (phenylamino) methyl] furan-2(5H)-one

Molecular Formula: C₁₇H₁₄BrNO_2,Molecular Weight: 343¹H-NMR (500MHz, CDCl₃) : δ 4.21-4.62 (brs, 2H (NH)), 4.44 (d, 1H, J = 6.9 Hz), 4.77 (d, 1H, J =3.9 Hz), 5.14 (d, 1H, J = 5.9 Hz), 5.33 (t, 1H, J = 1.9 Hz), 5.99 (dd, 1H, J = 3.9, 5.9 Hz), 6.06 (dd,1H, J = 3.9, 5.9 Hz), 6.48 (t, 4H, J = 6.9 Hz), 6.64 (t, 2H, J = 7.9 Hz), 7.02 (q, 4H, J = 8.9, 17.8 Hz), 7.19-7.42 (m, 10H) , IR (neat) :3385, 3026, 2923, 1756, 1601, 1502, 1160, 752 cm^-1, MS (ESI) :m/z 366 (M+Na)⁺

Compound-3: IUPAC name: 5-[(3-chlorophenyl) (phenylamino) methyl] furan-2(5H)-one Molecular Formula: C17H14ClNO2, Molecular Weight : 299, ¹H-NMR (300MHz, CDCl₃): δ4.25-4.63 (brs, H (NH)), 4.44 (d, 1H, J = 6.4 Hz), 6.74 (d, 1H, J = 3.6 Hz), 5.13 (d, 1H, J = 6.2 Hz), 5.34 (t, 1H, J = 1.5 Hz), 6.05 (dd, 1H, J = 3.9, 5.6 Hz), 6.13 (dd, 1H, J = 3.9, 5.6 Hz), 6.46 (dd, 4H, J = 5.1, 8.1 Hz), 6.6 (t, 2H, J = 7.3 Hz), 7.05 (q, 4H, J = 5.4, 13.0 Hz), 7.17 (d, 2H, J = 8.3 Hz), 7.22-7.35 (m, 4H), 7.36-7.55 (m, 4H), IR (neat): 3381, 3053, 2924, 1756, 1600, 1497, 1160, 752 cm¹ , IR (neat): 3381, 3053, 2924, 1756, 1600, 1497, 1160, 752 cm¹.MS (EI) : m/z 300 (M+H)⁺

Compound-4: IUPAC name:5-{[(3-chlorophenyl) amino] (phenyl)methyl} furan-2(5H)-one, Molecular Formula : C₁₇H₁₄ClNO_2,Molecular Weight : 299_,¹H-NMR (300MHz, CDCl₃) : δ 4.36(d, 1H, J = 6.9 Hz), 4.76 (d, 1H, J =3.9 Hz), 5.09-5.18(m,1H), 5.36 (t, 1H, J =1.8 Hz), 6.02 (dd, 1H, J = 3.7, 5.6 Hz), 6.12 (dd, 1H, J = 3.7, 5.6 Hz), 6.34-6.48 (m, 4H), 6.89-7.04 (m, 4H), 7.17-7.41 (m, 13H), 8.10 (d, 1H, J = 8.5 Hz), IR : 3382, 2926, 1755, 1598, 1498, 1163, 1095, 815 cm^-1, Yield :91%, MS (ESI) m/z 322 (M+Na)⁺

Compound-5: IUPACname:5-{[(3-methylphenyl) amino] (3, 4, 5trimethoxyphenyl)methyl} furan-2(5H)-one, Molecular Formula :C₂₁H₂₃NO₅,Molecular Weight : 369, ¹H-NMR (300MHz, CDCl₃): δ2.20 (s, 6H), 3.79 (s, 6H), 3.81 (s, 6H), 3.84 (s, 6H), 4.24 (d, 1H, J = 6.8 Hz), 4.65 (d, 1H, J = 3.8 Hz), 5.1 (d, 1H, J = 6.8 Hz), 5.37 (t, 1H, J = 2.3 Hz), 6.05 (dd, 1H, J = 3.7, 5.3 Hz), 6.12 (dd, 1H, J = 3.7, 5.3 Hz), 6.38-6.56 (m, 6H), 6.06 (s, 2H), 6.88 (t, 4H, J = 7.5 Hz), 7.02-7.16 (m, 1H), 7.18-7.36 (m, 3H), IR (neat) 3453, 2924, 2854, 1749, 1637, 1123, 1016, 763 cm^-1, MS (ESI): m/z 392 (M+Na)⁺

Before docking the search for the best template for modeling of prostaglandin E synthase 1 was carried out using BLAST against PDB. Electron crystallographic structure of human microsomal prostaglandin E synthase 1 (PDB code 3DWW was obtained from PDB database) PDB database from BLAST results, 3DWW showed 95% sequence identity with target protein. As the percentage of gaps is 2%. The initial model of prostaglandin E synthase1 was generated using Modeler in Discovery studio 2.1. The initial model was also energy refined during generation itself by selecting the option ”true” in ‘refine loops’ parameter. (fig 2) The generated model was again subjected to loop refinement which will refine all the main chain conformations of those residues lying in the structurally variable regions or loops.

Fig. 2: Representing the loops before and after refinement of prostaglandin E synthase 1

Incorrect main chain and side chain conformations were revealed by protein health check report. The model that resulted after loop refinement was validated as having low energy conformation of all other models generated. The modeled proteins before and after loop refinement were validated primarily by the lowest probability density function energy or lowest DOPE (discrete optimized protein energy) score. The new model name B99990002BL00020001 had lowest PDF total energy (-3262) and lowest DOPE score (-14744.45). The other two models had PDF total energies as -3194.72 and -3148.27 respectively as well as DOPE scores as -14704.36 and -14622.58 respectively. The next validation process was carried out using PROCHECK⁸. The percentage of residues lying in the most favorable regions of Ramachandran plot (Fig-3) was 91.1% and of those lying in disallowed region was 0.8%. The RMSD (root mean square deviation) of the equivalent main chain atoms between the 3DWW chain and the final homology modeled structure of human prostaglandin E synthase1 was 0.21A.

Fig. 3: Showing Ramachandran Plot

Docking results obtained by docking of synthesized compounds with prostaglandin E synthase 1 and phospholipase A2 represented in tables 2 and 3 and the interaction of the compound 3 with the target was represented in figures 4 and 5. Compound 3 was found to be the active as it formed 4 hydrogen bonds with two aminoacid residues lysine 62, aspartate 48. whereas in case of phospholipase A2 formed two hydrogen bonds with three aminoacid residues lysine 62, aspartate 48 and valine30. The results obtained from ADMT studies¹¹ and based on the limits mentioned in the methodology all ligand molecules were having good drug likeliness properties were within the boundary limits. All the molecules posses the good BBB, PPB and Optimal solubility and toxicity properties that is required for biological activity. (fig-6)

Table 2: Docking results of prostaglandin E synthase 1

Compound name	Dock score	Interacting aminoacids	Hydrogen bond
3	-105.128	Lys62,Asp48	4
2	-104.588	Tyr51,phe5,Asp48	4
4	-99.5202	Cys28,lys62,val30	1
1	-97.9594	Leu2,lys62,val30	1
5	-93.407	Asn4,asp48,leu2	2

Fig. 4: Interactions of ligand with modeled protein

Fig. 5: Interactions of ligand with modeled protein

Table 3: Docking results of phospholipase A2

Compound name	Dock score	Interacting aminoacids	Hydrogen bond
3	-99.4808	Lys62,Asp48,Val30	2
2	-96.6399	Tyr51,Gly31,Cys28	3
4	-89.9061	Cys28,His47,Val30	4
1	-91.6475	Asp48,lys62,Gly58	4
5	-90.3287	Asn4,Tyr66,Ser65	3

Fig. 6: representing ellipses in the ADMET_PSA_2D, ADMET_AlogP98 plane:

CONCLUSION:

The synthesized compounds are characterized by spectral means i.e., Mass, IR, NMR spectra and the modeled protein of prostaglandin E synthase1 evaluated for validation by using Ramchandran plot showing 91.1% in allowed regions. The synthesized compounds were evaluated for docking studies by using prostaglandin E synthase1 and phospholipaseA2 by Molegro virtual docker. The compounds 3 and 2 are showing highest interaction with target enzyme, and highest docking score. Concluded that the compounds 3 and 2 showing significant interaction with target enzymes.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS:

The authors are thankful to the Bhaskar Pharmacy College for providing necessary facilities to carry out this research work.

REFERENCES:

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Received on 26.06.2020 Modified on 21.08.2020

Research J. Pharm. and Tech. 2021; 14(7):3921-3926.

DOI: 10.52711/0974-360X.2021.00681

S. No	Imine	Product	Time (h)	YIELD Threo: Erythro
1			2.1	60:40
2			2.4	57:43
3			2.2	65:35
4			2.1	60:40
5			2.0	62:38