Claims:We claim,
1. Method for synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative comprising condensation of 2-fluoro benzoyl glycine with various substituted aromatic aldehyde catalysed by biowaste as an efficient heterogeneous catalyst under microwave irradiation to form the 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative of Formula I

Formula I
Wherein R is selected from H, 4-F, 4-Cl, 4-OCH3, 2-NO2, 2-OH and 4-CH3.

2. Method for synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative as claimed in claim 1 wherein the biowaste selected is eggshell.
3. Method for synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative as claimed in claim 2 wherein the eggshell is eggshell powder.
4. Method for synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative as claimed in claim 3 wherein the eggshell powder is prepared by collecting the chicken eggshells and washing, drying and grinding eggshells to form the eggshell powder.
5. Method for synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative as claimed in claim 1 wherein the 2-fluoro benzoyl glycine is prepared by dissolving the Glycine in sodium hydroxide and then adding 2-Fluoro benzoyl chloride by continuous stirring at 0 ºC and incubating the mixture in refrigerator for overnight to form the 2-fluoro benzoyl glycine crystals.

6. Method for synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative as claimed in claim 1 wherein the 2-fluoro benzoyl glycine condensation is carried by mixing substituted aromatic aldehyde, 2-fluoro benzoyl glycine, and acetic anhydride in a dry flask with eggshell powder and then stirring under microwave irradiation to form 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives.
7. Method for synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative as claimed in claim 6 wherein the microwave irradiation is carried at 300W power fitted with a reflux condenser.
8. Compounds of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative of Formula I

Formula I
Wherein R is selected from H, 4-F, 4-Cl, 4-OCH3, 2-NO2, 2-OH and 4-CH3.

9. Compounds of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative as claimed in claim 8 wherein the compounds selected from:
4-Benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one;
4-(4-Fluorobenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one;
4-(4-Chlorobenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one;
4-(4-Methoxybenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one; and
4-(4-Methylbenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one.

10. Pharmaceutical composition for treatment of cancer comprising compounds as claimed in claim 8 or 9 and suitable pharmaceutical excipients/carriers.
Dated 30th day of August, 2020 APPLICANT’S AGENT

SHARANABASAVA, PATENT AGENT (IN/PA-1375)
, Description:
FORM-2

THE PATENTS ACT, 1970
&
THE PATENTS RULES, 2003

COMPLETE SPECIFICATION

SYNTHESIS OF 4-BENZYLIDENE-2-(2-FLUOROPHENYL)OXAZOL-5(4H)-ONE DERIVATIVES CATALYSED BY BIOWASTE

K. KANTHARAJU & PRASHANT B. HIREMATH

DEPARTMENT OF CHEMISTRY,
RANI CHANNAMMA UNIVERSITY, P-B, NH-4, BELAGAVI-591156, KARNATAKA INDIA

The following specification describes the invention and the manner in which it is performed

FIELD OF THE INVENTION
The present invention relates to development of solvent free synthetic process by using the bio-waste as catalyst. It particularly relates to the development of synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives by using biowaste as catalyst under microwave irradiation. It specifically relates to the synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives catalysed by biowaste under microwave irradiation. The present invention also relates to the development of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives as anticancer agents. The invention further relates to method for treatment of cancer by using synthesized 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives.
BACKGROUND OF THE INVENTION

Friedrich Gustav Carl Emil Erlenmeyer [Erlenmeyer, E. Ueber die Condensation der Hippursäure mit Phtalsäureanhydrid und mit Benzaldehyd. Annalen. Der. Chem., 1893, 1, 1-8] discovered the synthetic route to azlactones in 1893 though the reaction of benzaldehyde with N-acetyl glycine [Baltazzi, E.Q. The chemistry of 5-oxazolones. Rev. Chem. Soc., 1955, 9, 150-173] in presence of acetic anhydride and sodium acetate which termed as Erlenmeyer reaction.

1
Due to the employment of 2-oxazolin-5-ones (azlactones) as an important precursor, researchers made numerous biologically prominent heterocyclic compounds are gained huge attention to the chemists. Their versatile wide spectrum of pharmacological scope (Figure 1) has witnessed the huge attention of chemists to focus for their synthesis. As azlactones are reactive compounds, which possessing C=N and C=O as multifunctional groups, which can lead to diverse reactivity to involve in the cycloaddition, replacement, dimerization and other reaction towards the formation of various heterocyclic synthons. The basic skeleton of azlactone has a huge attention, due to its drug like characteristics [Takenaka, K.; Tadakazu, J. Synthesis of [1,3,4]thiadiazolo[3,2-a]pyrimidines in the presence of formic acid. Heterocycl. Chem., 1996, 33, 1367-1370 (Figure 1).

Substituted-benzylidene-2-(phenyl)oxazol-5-one derivatives synthesized through the condensation of unsubstituted or substituted hippuric acid with aryl aldehyde in presence of acetic anhydride under numerous catalytic conditions such as anhydrous sodium acetate and evaluated them for in vivo antidiabetic activity, using molecular sieves (4Å) and evaluated them for in vitro antitubercular activity against M. tuberculosis H37RV (ATCC 27294) by Resazurin Micro titre Assay (REMA) method [Mariappan, G.; Saha, B.P.; Datta, S.; Kumar, D.; Haldar, P.K. Design, synthesis and antidiabetic evaluation of oxazolone derivatives, J. Chem. Sci., 2011, 123(3), 335–341], anti-oxidant activity [Suhasini, K.P.; Chintakindi, P.K.; Chaguruswamya, K.; Murthya, Y.L.N. 5(4H)-Oxazolones as Novel Antitubercular Agents: Synthesis, Characterisation and Structure Activity Study. J. Chin. Chem. Soc., 2015, 62, 855-860], anti-inflammatory activity using Human red blood cell (HRBC) membrane stabilization method [Canan, K.; Ezgi U.; Elçin D.; Benay C. Synthesis and Antioxidant Properties of New Oxazole-5(4H)-one Derivatives. Turk. J. Pharm. Sci., 2017, 14(2), 174-178]. Apart from these there are several other catalytic approaches reported for the synthesis of 4-arylidene-2-phenyl-5(4H)-oxazolones because of the potential biological applications that oxazolones pocess. Some of the catalysts employed for the synthesis of 4-arylidene-2-phenyl-5(4H)-oxazolones through the condensation of aromatic aldehydes and hippuric acid are (Scheme 1) Calcium acetate [Muthuboopathi, G.; Shanmugarajan TS. Synthesis, Characterization, and Biological Evaluation Of Oxazolone Analogs. Asian J. Pharm. Clin. Res., 2018, 11(4), 159-162], ionic liquid-[bmim][PF6] [Paul, S.; Nanda, P.; Gupta, R.; Loupy, A. Calcium acetate catalyzed synthesis of 4-arylidene-2-phenyl-5(4H)-oxazolones under solvent-free conditions. Tetrahedron Lett., 2004, 45, 425–427], nano silica supported Tungstophosphoric acid [Heravi, M.R.P. Erlenmeyer synthesis of azlactones by sonochemical reaction in ionic liquids. J. Uni. Chem. Tech. Met., 2009, 44(1), 86-90], zwitter ionic imidazolium salt [Taki B.S.G.; Mirkhani V.; Baltork, I.M.; Moghadam, M.; Tangestaninejad, S.; Rostami, M.; Khosropour, A.R. Synthesis and characterization of nano silica supported tungstophosphoric acid: an efficient reusable heterogeneous catalyst for the synthesis of azlactones. J. Inorg. Organomet. Polym., 2013, 23, 758–765], nano-Fe2O3 [Baocheng, Z.; Wenxing C. The Zwitterionic Imidazolium Salt: First Used for Synthesis of 4-Arylidene-2-phenyl-5(4H)-oxazolones under Solvent-Free Conditions. J. of Chem., 2014, 1-5, Article ID 280585, Hindawi Publishing Corporation], MgO/Al2O3 [Ahmadi, S.J.; Sadjadi, S.; Hosseinpour. M. A green protocol for Erlenmeyer–Plöchl reaction by using iron oxide nanoparticles under ultrasonic irradiation. Ultrason. Sonochem., 2013, 20, 408–412], graphine oxide zinc oxide supported on silica gel [Rostamizadeh, N.; Khajeh-Amiri, A.; Moghanian, H. Microwave-Assisted Erlenmeyer Synthesis of Azlactones Catalyzed by MgO/Al2O3 under Solvent-Free Conditions. Synth. Reac. Inorg. Metal-Org. Nano-Met. Chem., 2015, 46(5), 631-634], [bmim]OH@agar gel ionic liquid [Sadjadi, S. Graphene–ZnO@SiO2 hybrid: An efficient and solid acid catalyst for synthesis of azlactones under ultrasound irradiation. Iran. J. Cat., 2018, 8(2), 189-194] and many more.
Even though the above-mentioned catalytic approaches have their own advantages yet, most of them suffer from various drawbacks such as hazardous solvents/material usage, expensive approach for the preparation of catalysts, longer reaction time, lesser yield, and so on. Solvent-free reaction in organic synthesis has gained more attention. Since from last few decades, this approach enhanced every aspects of reaction like lower the reaction time, better yield, easier workup and sometimes also observed regio and stereo selectivity. Organic transformations under microwave irradiation have gained significant advantages such as reaction go cleaner, faster and atom economical. The goal of the current green chemistry is replacement of hazardous organic solvents, which lead to unavoidable toxic, solvent wastage or emission. The usage of reagents like solid supported inorganic catalyst has gained more attention in organic synthesis, such type of approaches not only avoid the use of toxic solvents to carry out reaction, but also create convenient procedure for the reactions. The synthesis of the oxazolones under microwave irradiation has been previously reported using acetic anhydride [Jagadalea, M.; Naikwadea, A.; Salunkhea, R.; Rajmaneb, M.; Rashinkar, G. Ionic liquid gel: A heterogeneous catalyst for Erlenmeyer-Plochl and Henry reaction. New J. Chem., 2018, 42, 10993-11005; Bodaghifard, M.A.; Moghanian, H.; Mobinikhaledi, A.; Esmaeilzadeh, F. Microwave-assisted efficient synthesis of azlactones using zeolite NaY reusable heterogeneous catalyst. Inorg. Nano-Met. Chem., 2017, 47(6), 845-849], which plays crucial role as a reagent as well as medium. As we know acetic anhydride is highly hazardous and its usage for carrying out organic transformation would harm the cavity of the oven, when used in open vessel and it is inevitable to handle toxic vapors that generated during the reaction, which is also harmful to environment. Even though the usage of the acetic anhydride used as a reagent in our on-going work, but by using custom-made microwave oven overcome those problems associated with previous reported methods.
The report by Haboub A et al., 2015 [Abdeljabar Haboub, Meryem Hamlich, Souad Harkati, Yassine Riadi, Rachid Slimani, Mina Aadil, Anouzla Abdelkader, Saïd Lazar, Mohamed Safi. New Methodologie Eco-Friendly Using Calcined Eggshell Meal Catalyst for Synthesis of Benzimidazoles, Benzoxazoles and Benzothiazoles. American Journal of Environmental Protection. Special Issue: Cleaner and Sustainable Production.Vol. 4, No. 5-1, 2015, pp. 28-32.] discloses the Calcined Eggshell Meal (CEM) doped is a new solid support has been employed as a catalyst for efficient synthesis of benzimidazoles, benzoxazoles and benzothiazoles. Taking into account environmental and economic considerations, the handling of CEM used here presents many advantages such as simple and convenient procedure, easy purification and shorter reaction time and enhanced recycling possibilities, which are now well established in fine organic synthesis.
The report of Suresh Patil et al., 2013 [Suresh Patil, Swati D Jadhav and M B Deshmukh, J. Chem. Sci. Vol. 125, No. 4, July 2013, pp. 851–857] discloses a convenient, eco-friendly and economic method for Knoevenagel condensation of aromatic aldehydes with active methylene compounds using calcined eggshell (CES) as an efficient natural catalyst in aqueous medium has been reported. CES is a new, ecologically safe and inexpensive green catalyst obtained from renewable resources.
The report of Jat et al., 2019 [L.R. Jat, R. Mishra and D. Pathak, Int J Pharm Pharm Sci, Vol 4, Issue 1, 378-380] discloses a series of oxazolone derivatives (1-14) have been synthesized as potential anticancer agent. The newly synthesized compounds were evaluated for cytotoxicity in the A549 cell line by SRB (Sulphorhodamine B) assay. Compound 1 was found to be most potent with 25 µg/ ml CTC50 value. Titled compounds have been prepared by the condensation of benzoylglycine with aromatic aldehydes in the presence of ethanol, acetic anhydride and sodium acetate. The newly synthesized compounds were characterized by FTIR, 1HNMR, Mass spectroscopy and Elemental analysis.
Though there prior art reports which reported the use of biowastes as catalsts and calcined egg shells as catalysts but none of the prior arts in the literature reported or disclosed the synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives by using inexpensive biowaste of egg shell as efficient catalyst under microwave irradiation. The major problem associated in the preparation of azlactones required very mild reaction condition; strong basic nature condition may influence the self-condensation of the aldehydes. Hence, mild basic catalytic developments are the permanent need for the establishment of alternative catalytic approach that replace the usage of strong base. Therefore, the present inventors have developed inexpensive biowaste of egg shell as efficient catalyst for the synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives by using inexpensive biowaste of egg shell as efficient catalyst under microwave irradiation and achieved excellent yield and purity. The invention also includes the development of synthesized compounds as anticancer agents and compositions.

The present invention relates to development of solvent free synthetic process by using the bio-waste as catalyst. It particularly relates to the development of synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives by using biowaste as catalyst under microwave irradiation. It specifically relates to the synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives catalysed by biowaste under microwave irradiation. The present invention also relates to the development of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives as anticancer agents. The invention further relates to method for treatment of cancer by using synthesized 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives.
OBJECTS OF THE INVENTION
The primary object of the present invention is the development of eco-friendly benign, efficient, greener synthetic process by using the inexpensive bio-waste as catalyst.
The other object of the present invention is the development of synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives by using egg shell powder as catalyst under microwave irradiation.

The other object of the present invention is the development of synthesized compounds of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives as anticancer agents.

The other object of the present invention is the development of compositions of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives for treatment of cancer.
The other object of the present invention is the development of synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives by using egg shell powder as catalyst under microwave irradiation which is cost-effective.

The other object of the present invention is the development of synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives by using egg shell powder as catalyst under microwave irradiation which is easy to use with little technical expertise.

The other object of the present invention is the development of synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives by using egg shell powder as catalyst under microwave irradiation which is safe and practical to use.
Further, the selected compounds 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives are tested in vitro anti-cancer properties and bioassay results showed promising activity compare to standard Paclitaxel and the computational studies also carried out to specific cancer protein with prepared compounds binding energy calculation.

BRIEF DESCRIPTION OF FIGURES
Figure 1: Biologically prominent skeletons of oxazolones
Figure 2 : General reaction for 4-arylidene-2-phenyl-5(4H)-oxazolone synthesis
Figure 3: Preparation of eggshell powder catalyst
Figure 4: XRD spectrum of ESP
Figure 5: FT-IR spectrum of ESP
Figure 6: Synthetic route of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative synthesis (12a-g)
Figure 7: Plausible mechanistic pathway
Figure 8: EGFR Kinase protein (PDB No: 1XKK)
Figure 9: Erlotinib v/s Protein interaction
Figure 10: Gefitinib v/s Protein interaction
Figure 11: Lapatinib v/s Protein interaction
Figure 12 a-g: Ligand interaction with target protein computational generated diagrams (12a-g)
Figure 13: The cytotoxic activity of the synthesized compounds 12a, 12c and 12g.
Figure 14: FT-IR Spectrum of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one (12a)
Figure 15: LC-MS Spectrum of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one (12a)
Figure 16: 1H-NMR Spectrum of 4-benzylidene-2-(2-fluorophenyl)oxazol- 5(4H)-one (12a)
Figure 17: 13C-NMR Spectrum of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one (12a)
Figure 18: FT-IR Spectrum of 4-(4-fluorobenzylidene)-2-(2-fluorophenyl) oxazol 5(4H)-one (12b)
Figure 19: HR-MS Spectrum of 4-(4-fluorobenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one (12b)
Figure 20:1H-NMR Spectrum of 4-(4-fluorobenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one (12b)
Figure 21:13C-NMR Spectrum of 4-(4-fluorobenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one (12b)

SUMMARY OF THE INVENTION

The 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives are synthesized through the condensation of 2-fluoro benzoyl glycine with various substituted aromatic aldehyde catalysed by eggshell powder (ESP) as an efficient heterogeneous catalyst under microwave irradiation (MWI) is described.

The advantage of the present approach is a benign, efficient, greener, solvent-free and less expensive gave 86-89% of product isolation in spectroscopic pure.
All the compounds synthesized were subjected to molecular docking studies for anti-cancer study of breast cancer. Docking study was carried out between EGFR kinase (PDB No: 1XKK) and seven synthesized oxazolone derivatives to know the binding affinity using computational studies.

Among seven synthesized oxazolone derivatives, three derivatives are shown very good binding affinity and those derivatives were screened for anti-cancer activity against breast cancer MCF-7 cell line in vitro.

The biological assay revealed that, two of the compounds 12a and 12g have performed better activity compared to standard drug (Paclitaxel).

STATEMENT OF THE INVENTION

Method for synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative comprising condensation of 2-fluoro benzoyl glycine with various substituted aromatic aldehyde catalysed by bio-waste as an efficient heterogeneous catalyst under microwave irradiation to form the 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative of Formula I

Formula I
Wherein R is selected from H, 4-F, 4-Cl, 4-OCH3, 2-NO2, 2-OH and 4-CH3.

The bio-waste selected is eggshell powder and it is prepared by collecting the chicken eggshells and washing, drying and grinding eggshells to form the eggshell powder.
The 2-fluoro benzoyl glycine is prepared by dissolving the Glycine in sodium hydroxide and then adding 2-fluoro benzoyl chloride by continuous stirring at 0 ºC and incubating the mixture in refrigerator for overnight to form the 2-fluoro benzoyl glycine crystals.
The 2-fluoro benzoyl glycine condensation is carried by mixing substituted aromatic aldehyde, 2-fluoro benzoyl glycine, and acetic anhydride in a dry flask with eggshell powder and then stirring under microwave irradiation to form 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives. The microwave irradiation is carried at 300W power fitted with a reflux condenser.
Compounds of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative of Formula I

Formula I
Wherein R is selected from H, 4-F, 4-Cl, 4-OCH3, 2-NO2, 2-OH and 4-CH3.

Compounds of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative wherein the compounds selected from:
4-Benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one;
4-(4-Fluorobenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one;
4-(4-Chlorobenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one;
4-(4-Methoxybenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one; and
4-(4-Methylbenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one.

Pharmaceutical composition for treatment of cancer comprising compounds of Formula I and suitable pharmaceutical excipients/carriers.
DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to development of solvent free synthetic process by using the bio-waste as catalyst. It particularly relates to the development of synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives by using biowaste as catalyst under microwave irradiation. It specifically relates to the synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives catalysed by biowaste under microwave irradiation. The present invention also relates to the development of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives as anticancer agents. The invention further relates to method for treatment of cancer by using synthesized 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives.
In the present work described one-pot synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative catalysed by bio-waste chicken eggshell powder as a heterogeneous and recyclable catalyst under microwave irradiation is reported

Preparation and characterization of eggshell powder
The eggshell powder (ESP) has been prepared by biowaste chicken eggshell collected from the household, removed membrane present on it by washing with water, dried at room temperature for about 8 hours, and crushed to powder using pestle mortar until getting fine powder (Figure 2). The resulted powder named as ESP and used directly for the reaction.

The XRF and elemental analysis data revealed high concentrations, 50.4% of Oxygen, 32.7% of Calcium, 13% of Carbon, 0.75% of Phosphorous and small amount 1.6% of Magnessium, 0.5% of Silicon, 0.4% of Strontium, 0.30% of nitrogen, 0.07% of Potassium, 0.06% of Sodium, 0.03% of Iron, 0.02% of Zinc, 0.02% of Cl, 0.05% of hydrogen and 0.01% of Nickel. The composition present in the ESP is examined using X-ray diffraction (XRD) (Figure 3) and FT-IR studies (Figure 4) which is in accordance with the literature data [Jai, P.H.; Wook, J.S.; Kyu, Y.J.; Gil, K.B.; Mok, L.S. Removal of heavy metals using waste eggshell. J. Environ. Sci., 2007, 19, 1436–1441]. The main and prominent characteristic peak for ESP marked by its indices confirms the presence of a calcite structure and which is similar to calcium carbonate mineral. The FT-IR spectrum of ESP indicates some prominent characteristic peaks for H2O, phosphate and carbonate bands in the spectrum. Peak around 3000 cm-1 indicate presence of stretching vibration ?OH of uncoordinated H2O. A broad band around 3400 cm-1 signifies the presence of H2O. Less intense band at 709 cm-1 can be assigned to the stretching vibration of the P-OH functional group. Key role of phosphorus in the form of phosphate present in the ESP was reported in the literature [Mosaddegh, E.; Hassankhani, A. Application and characterization of eggshell as a new biodegradable and heterogeneous catalyst in green synthesis of 7,8-dihydro-4H-chromen-5(6H)-ones. Cat. Commun., 2013, 33, 70-75]. Stretching bands of medium intensity at 1431 cm-1 and 1153 cm-1 indicate for ?C-O group of two different types. The two out of plane of vibration for d (OCO) are observed in the range of 874 cm-1 and 710 cm-1 as medium strong bands. Medium broadband at 2512 cm-1 signifies the presence of HCO3. All the above findings clearly gave the presence of CaCO3 in the eggshell powder and comparable with the prior published data of CaCO3. Thus, the FT-IR spectrum of the ESP clearly indicates the presence of calcite structure in the catalyst and showed it is hygroscopic in nature.
The optimum condition for the efficient synthesis of azlactone (12a) is begin by taking a model reaction of 2-fluoro hippuric acid (9) and benzaldehyde (11a) in presence of acetic anhydride by varying reaction condition in terms of catalyst loading, solvent effect and microwave irradiation over conventional heating is carefully studied and tabulated in Table 1 (Scheme 2).
Table 1: Optimization of catalyst load and reaction condition for the synthesis 12a
Entry Solvent Conditions Time (min) Yielda (%)
1 EtOH rt/eggshell (100mg) 30 38
2 CH2Cl2 rt/eggshell (100mg ) 30 26
3 CH3CN rt/eggshell (100mg ) 30 24
4 H2O 60 ºC/eggshell (100mg ) 30 22
5 EtOH MWIc/eggshell (100mg ) 15 42
6 EtOH MWIc/eggshell (200mg ) 12 66
7 EtOH 80 ºC/eggshell (100mg ) 30 44
8 Neat 80 ºC/eggshell (100mg ) 30 61
9 Neat MWIc/eggshell (50mg) 20 26
10 Neat MWIc/eggshell (100mg) 10 66
11 Neat MWIc/eggshell (200mg) 5 90
12 EtOH 80 ºC 45 Traceb
13 Neat 80 ºC 60 Traceb
14 Neat MWIc 5 18b
aYields refer to the isolated yield, bYields observed in the absence of ESP, cMWI=Microwave irradiation at 300W power, Reaction condition: 2-fluoro benzoyl glycine, benzaldehyde (1mmol scale)

Table 2: Synthesis of various 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivative and their physical constant
Entry Aldehyde Product Time (min) Yield (%) m.p. (ºC)
1

12a 6 90 156-158
2

12b 7
90 206-208
3

12c 8 89 196-198
4

12d 6 89 126-128
5

12e 7 88 160-163
6

12f 10 88 178-180
7

12g 10 89 106-108

aProducts were characterized by FT-IR, 1H- and 13C-NMR, LC/HR-MS.
bYields refer to the isolated yields.
The homogeneity and purity of the synthesized compound is determined by various spectroscopic techniques. The FT-IR spectrum of 12a has shown characteristic peak at 1795.13 cm-1 for carbonyl functional group (C=O), C=N stretching frequency observed at 1651.96 cm-1 and C=C stretching frequency observed at 1489.18 and 1453.06 cm-1. 1H-NMR spectrum of product 12a has shown characteristic peaks as singlet at d 7.23 for HC=C, multiplet at d 7.23-7.33 for 3 aromatic protons, multiplet at d 7.46-7.50 for 2 aromatic protons, multiplet at d 8.10-8.22 for 4 aromatic protons. 13C-NMR spectrum also shown characteristic peaks for the respective carbon atom at various chemical shift such as 76.91, 77.22, 77.43, 77.54, 117.43, 117.64, 124.72, 124.76, 129.20, 131.02, 131.67, 132.86, 133.11, 133.63, 135.04 and 135.13. LC-MS spectrum of product 12a showed molecular peak at 268.0664 for (M+H)+, which is in accordance with calculated [M]+ as 267.2545.

Plausible mechanism
The catalytic activity of eggshell powder to Erlenmeyer–Plöchl reaction for the synthesis of azlactone derivative is being surprising, but still from the observed findings, we have described plausible reaction mechanism in Figure 3.
Initially 2-fluoro hippuric acid (10) react with acetic anhydride in presence of eggshell powder to produce intermediate 12a, which undergoes intramolecular cyclization to produce another intermediate 10b. In the final step, eggshell powder act as a Lewis acid type catalyst, which activate the carbonyl functional group of aldehyde carbon followed by nucleophilic attack on the intermediate 36b via aldol condensation to form product 12.

Molecular Docking Studies: As an extension of the present work, docking study was carried out between EGFR Kinase (PDB No: 1XKK) and seven synthesized oxazolone derivatives to know the binding affinity parameter. In brief, molecular docking was carried out by Glide (Glide, Version 6.0, Schrodinger, LLC, New York, NY, 2013) with extra precision (XP) mode. We have utilized X-ray crystallographic structure of the EGFR kinase protein. The structure of protein was retrieved from PDB (Protein Data Bank), PDB ID: 1XKK for molecular docking (Table 3). The ligands were sketched in Maestro 9.3 (Maestro Version 9.3, Schrodinger, LLC, New York) and Lig Prep modulae used to optimize potential ligand simulation OPLS3 force field to rectify the molecular geometries to retain the specific chirality of the molecule and to get least energy conformation. Protein structure used protein preparation wizard by removing water, addition of explicit hydrogen atoms and optimizing it with the hydrogen bonds. Receptor grid was generated using 1.0 Vander Waal’s (VdW) radius scaling factor and with 0.25 partial charge cut off. Ligand was docked with XP mode and post-docking minimization was carried out. The binding energy calculation was carried out by Prime Molecular Mechanics/Generalized born surface area (MM-GBSA) (Prime, Version 3.3, Schrodinger, LLC, New York, NY, 2013) that calculate ligand binding and ligand strain energies for the ligand-protein complex.

Table 3: Docking scores of synthesized molecules and standard drug inhibitors of EGFR Kinase
S. No Ligands G-Score
1 Erlotinib -2.9
2 Gefitinib -3.8
3 Lapatinib -3.5
4 12a -3.5
5 12b -2.5
6 12c -3.4
7 12d -3.2
8 12e -3.0
9 12f -3.2
10 12g -5.9

The EGFR kinase co-crystallized with compounds 12a-g with standard drugs are computed. Compound 12g was found to be interacting with amino acids with high binding affinity than the standard drug and followed by 12a, which showed similar binding affinity as the standard drug Lapatinib.
In view of the above computational results obtained from the molecular docking studies of synthesized compounds for the EGFR kinase of breast cancer cells. We concluded that, out of seven synthesized compounds (12a-g), only 12a and 12g got excellent binding energy, and 38c moderate binding energy compared to standard drugs. Thus, we further extended our studies to investigate in vitro anti-cancer activity for 12a, 12g and 12c compounds.

MTT assay: This is a colorimetric assay, that measure the reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria, where it is reduced to an insoluble colored (dark purple) formazan product. The cells are then solubilized with an organic solvent (DMSO or isopropanol) and released, the solubilized formazan reagent is measured spectrophotometrically. Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells. The newly synthesized three-oxazolone derivatives (12a, 12g & 12c) were screened for in vitro cytotoxic against human breast carcinoma (MCF-7) cell lines. Paclitaxel, which is one of the most effective anticancer agents, was used as a reference drug (Table 3). The obtained in vitro results revealed that, all three synthesized oxazolone product exhibited a moderate to strong growth inhibition on the tested cell lines between 12.5 and 651µg/mL concentration in comparison with the reference anticancer drug. A graph of survival curve is plotted to state the relationship between surviving fraction and concentration of drug molecule of the MCF-7 cell line. The IC50 values were calculated and tabulated in Table 3, which describes the amount of drug in µg/mL concentration required for the 50% inhibition of cell viability. The synthesized oxazolones 12a & 12g showed strong cytotoxic activity, and 12c is having moderate activity compared to standard Paclitaxel drug against MCF-7 cancer cells. The experimental data is summarized for the cytotoxic activity of the synthesized compounds 12a, 12c and 12g against human breast carcinoma (MCF-7) cell lines.

Table 4: IC50 value of compounds in µg/mL
Compound MCF-7 (µg/ml)
12a 19.01
12c 651.4
12g 27.47
Paclitaxel 298.8671

Example of Procedure and description:
Preparation of ESP catalyst: Chicken eggshells are collected from the household, washed with tap water, and separated membrane on it. Then, it was again washed with distilled water and dried at room temperature for about 8 hr. After drying, ground in mortar and pestle into fine powder, the resulted white powder is used directly for the reaction.
Preparation of 2-fluoro benzoyl glycine: Glycine (20 mmol) dissolved in 8 mL of 2N sodium hydroxide in a 100 mL round bottomed flask and stirred vigorously in mechanical stirrer until it is totally dissolved. 2-Fluoro benzoyl chloride (20 mmol) was added in five portion and continued stirring vigorously further 1hr in ice bath by maintaining temperature 0 ºC. The mixture left in the refrigerator overnight, and formed crystals are filtered, washed with ice-cold water and dried at 60 ºC. The melting point of the compound found 145–149 ºC with isolated yield of 80%.

Microwave-assisted synthesis of 4-benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one derivatives: Aldehyde (1 mmol), 2-fluoro benzoyl glycine (1mmol), and acetic anhydride (3.3 mmol) taken in a dry 50 mL round bottomed flask followed by eggshell powder (200 mg). The mixture was stirred under microwave irradiation at 300W power fitted with a reflux condenser for the appropriate time. The progress of the reaction was monitored by TLC. After completion of the reaction, hot ethanol was added, the catalyst is separated by filtration, and final product obtained in pure form by recrystallization using absolute ethanol gave chromatographically pure product.

Determination of cytotoxic studies: The cells were seeded on a 96-well flat-bottom micro plate and maintained at 37 ºC in 95% humidity and 5% CO2 for overnight. Different concentration (400, 200, 100, 50, 25, 12.5 µg/mL) of solution were prepared and treated. The cells were incubated for another 48 hrs. The wells were washed twice with PBS and 20 µL of the MTT staining solution was added to each well and plate was incubated at 37 ºC. After 4h, 100 µL of DMSO was added to each well to dissolve the formazan crystals, and absorbance was recorded with a 570 nm using micro plate reader. The IC50 calculated using following formula: Surviving cells (%) = Mean OD of test compound /Mean OD of Negative control ×100 using graph Pad Prism Version5.1calculated the IC50 values.

Spectral data of representative compounds:
4-Benzylidene-2-(2-fluorophenyl)oxazol-5(4H)-one (12a): Pale yellow solid; Yield 90%; m.p. 156-158 ºC; FT-IR cm–1: 1795.13 (C=O), 1651.96 (C=N), 1489.18, 1453.06 (C=C); 1H-NMR d: 7.23-7.33 (m, 3H, ArH), 7.46–7.50 (m, 2H, ArH), 7.60 (m, 1H, CH), 8.10–8.22 (m, 4H, ArH); 13C-NMR : d = 76.91,77.22, 77.43, 77.54, 117.43, 117.64, 124.72, 124.76, 129.20, 131.02, 131.67, 132.86, 133.11, 133.63, 135.04, 135.13; LC-MS m/z (Cal.) = 267.2545 [M]+; m/z (Obs.) = 268.0664 (M+H)+.

4-(4-Fluorobenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one (12b): Pale yellow solid; Yield 90%; m.p. 206-208 ºC; FT-IR cm–1: 1796.02 (C=O), 1657.33 (C=N), 1597.90, 1548.54, 1499.88 (C=C); 1H-NMR d: 7.15-7.34 (m, 2H, ArH), 7.52 (s, 1H, CH), 7.57-7.62 (m, 2H, ArH), 8.09-8.13 (d, 1H, ArH), 8.22–8.26 (m, 3H, ArH); 13C-NMR: d 76.91, 77.22, 77.54, 116.41, 116.63, 117.46, 117.66, 124.75, 124.8, 131.60, 135.03, 135.1, 135.19; HR-MS m/z (Cal.) = 285.2449 [M]+; m/z (Obs.) = 286.0680 (M+H)+.

4-(4-Chlorobenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one (12c): Yellow solid; Yield 89%; m.p. 196-198 ºC; FT-IR (cm–1): 1799.55 (C=O), 1654.13 (C=N), 1590.73, 1410.58 (C=C); 1H-NMR (d ppm): 7.26-7.37 (m, 3H, ArH), 7.46-7.49 (m, 2H, ArH), 7.61-7.64 (m, 1H, CH), 8.10-8.19 (m, 3H, ArH); 13C-NMR (d ppm): 117.2, 117.50, 124.55, 129.37, 130.81, 131.10, 131.88, 133.01, 133.68, 135.00, 135.12, 163.58; HR-MS m/z (Cal.) = 301.6995 [M]+; m/z (Obs.) = 302.0382 (M+H)+.

4-(4-Methoxybenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one (12d): Yellow solid; Yield 89%; m.p. 126-128 ºC; FT-IR cm–1: 1800.51 (C=O), 1655.17 (C=N), 1592.43, 1411.59, (C=C); 1H-NMR (d ppm): 7.27-7.38 (m, 3H, ArH), 7.44-7.47 (m, 2H, ArH), 7.64-7.67 (m, 1H, CH), 8.11-8.20 (m, 3H, ArH); 13C-NMR (d ppm): 116.29, 117.50, 123.99, 129.39, 130.83, 131.14, 131.88, 133.08, 133.88, 134.99, 136.22, 164.57; LC-MS m/z (Cal.) = 297.2804 [M]+; m/z (Obs.)= 298.0873 (M+H)+.

4-(4-Methylbenzylidene)-2-(2-fluorophenyl)oxazol-5(4H)-one (12g): Pale yellow solid; Yield 89%; m.p. 106-108 ºC; FT-IR cm–1: 1799.46 (C=O), 1646.11 (C=N), 1571.56, 1415.62, (C=C); 1H-NMR (d ppm):7.27-7.34 (m, 3H, ArH), 7.42-7.45 (m, 2H, ArH), 7.59-7.62 (m, 1H, CH), 8.12-8.29 (m, 3H, ArH); 13C-NMR (d ppm): 115.44, 117.48, 124.98, 130.04, 130.99, 131.47, 131.91, 133.22, 133.78, 134.98, 136.24, 164.45; LC-MS: m/z (Cal.) = 281.2810 [M]+; m/z (Obs.) = 282.0946 (M+H)+.