Synthesis, crystal structure and molecular docking study of novel isoxazole derivatives as CYP450 inhibitors in search of anticancer agents

Abstract Synthesis of some novel isoxazole derivatives and their molecular docking with enzymes from CYP450 family carried out using erlotinib, gemcitabine and ketoconazole as reference drugs are reported in this work. Eight isoxazole derivatives of 3,4-substituted phenyl 3-chloroacrylaldehyde and one isoxazole derivative of cinnamaldehyde were synthesized. A molecular docking study of all nine compounds shows good docking score compared to standard drugs erlotinib, gemcitabine and ketoconazole. 4-OH and 4-F derivatives were found to have strong affinity for all six CYP450 proteins under study in the present work. 4-F and 3-NO2 derivatives could be a suitable lead compound inhibitor to CYP1A2 followed by 4-OH derivatives. 4-OH derivative with significant binding affinity showed encouraging inhibition of CYP1A2, CYP2C9, CYP2C8, CYP2C19 and CYP2D6. The current predictions over these nine isoxazole derivatives of 3,4-substituted phenyl 3-chloroacrylaldehyde will be needed to be further investigated in vivo and in vitro conditions to identify the optimum therapeutic efficacy. Synthesis of the isoxazole derivatives is the first known report of the Knoevenagal condensation of acrylaldehyde derivatives to form isoxazole derivatives as per the literature survey. A detailed crystal structure study of five analogues gives insight into the solid-state structural features of this new framework with isoxazole moieties. Communicated by Ramaswamy H. Sarma


Introduction
Uncontrollable division of cells due to multifactor reasons leads to cancer.This disease has been characterized by the presence of mutations, independent cell proliferation of mitogen, high genetic instability and invasion of other tissues.
Many chemotherapeutic drugs are developed to treat cancer that includes DNA-alkylating agents such as oxaliplatin, antimetabolites such as gemcitabine (Ishiguro & Toi, 2012), antitumor antibiotics such as doxorubicin, antimitotic agents as paclitaxel (Reyes-Habito & Roh, 2014).Oxaliplatin is commonly used as a reference drug due that it is a platinum-based chemotherapeutic agent with in vitro and in vivo efficacy against many tumor cell lines, including some that are resistant to cisplatin and carboplatin (Raymond et al., 1998).However, the effectiveness of chemotherapy has suffered diminution by many factors including systemic toxicity due to lack of specificity, rapid drug metabolism, high treatment cost and both intrinsic and acquired drug resistance (Alfarouk et al., 2015;Tartarone et al., 2013;Zhu et al., 2013).The difficulties encountered in the treatment available demonstrate the importance of design and development of novel therapeutic molecules.
Earlier published work shows that isoxazole-naphthalene derivatives as tubulin polymerization inhibitors were evaluated for their anti-proliferative activities against human breast cancer cell line MCF-7 (Wang et al., 2020).
While studying the physiology and type of cells in expression in breast, prostate, lung, endometrial and ovarian tumors the role of cytochrome 450 is important.Enzymes of the cytochrome P450 (CYP) subfamily 3A and 2C play a major role in the metabolism of taxane anticancer agents.
The expression of these enzymes in solid tumors may play a role in the in-situ metabolism of drugs.Epoxyeicosatrienoic acid (EET) production via cytochrome P450 (CYP) epoxygenases closely correlates with the progression of breast cancer (Phuong et al., 2017).Cytochrome P450s (CYPs) are a complex group of enzymes that are required for the metabolism of diverse xenobiotics which includes therapeutic agents and environmental procarcinogens (Guengerich & Shimada, 1991;Gonzalez & Gelboin, 1994).The expression of P450s correlates with both the response to chemotherapy and the initiation or promotion of tumorigenesis (Guengerich & Shimada, 1991;Gonzalez & Gelboin, 1994).Human cytochrome P450 (or P450s), a hemeprotein superfamily containing 57 isoforms, is responsible for the oxidation of xenobiotic chemicals including clinical drugs and environmental chemicals (Guengerich, 2008).
Six CYP isoforms (CYP1A2, CYP2C8, 2C9, 2C19, 2D6 and 3A4) are responsible for more than 90% of all metabolic reactions and are involved in carcinogen metabolism (Nebert et al., 2013;Zhang et al., 2006;Shimada, 2006).CYP1A2 is located predominantly in the liver and plays an important role in the metabolism of a variety of compounds including the activation of carcinogenic aryl and heterocyclic amines (Shimada et al., 1989).CYP3A4 is the most abundant CYP enzyme present in both the liver and small intestine and is of great interest because the enzyme has been known to catalyze the metabolism of approximately 50% of therapeutic agents (Tang & Stearns, 2001).Also, expression of CYP3A has been detected in various tumors including breast, hepatic, lung and stomach cancers.CYP3A levels are higher in breast tumors than in normal tissues, suggesting that CYP3A contributes to tumor progression (Kapucuoglu et al., 2003;Murray et al., 1998).CYP3A4 gene polymorphisms are also suggested to be related with prostate or breast cancer risk (Murray et al., 1998).P450s contribute to carcinogenesis by activating procarcinogens but, in prostate and breast cancer, the metabolism of steroid hormones by CYP3A is thought to contribute to carcinogenesis (Murray et al., 1998).CYP3A4 is also expressed in several cancer cell lines such as MCF-7 (breast), Caco-2 (colon) and HepG2 (liver) (Chen et al., 2009;Yasuda et al., 2008;Pfrunder et al., 2003).The effects of overexpression of CYP3A4 and CYP2D6 on the growth and hypoxic response of Hep3B cells were examined earlier (Oguro et al., 2011).CYP2C8 plays a crucial role in the biotransformation of several xenobiotics and endogenous compounds and has high hepatic expression with lower expression in the kidney, adrenal gland, mammary gland, brain, ovary, uterus and duodenum (Hakkola et al., 2020).
The objective of the present study was to synthesize the novel framework using isoxazole moiety fused with a benzene ring through a C3 connector.This skeleton structure was targeted as it was observed to give good results in docking.Gemcitabine and erlotinib showed primary binding affinity with the hem group in six CYP450 enzymes considered in this study.The isoxazole derivatives of chlorovinyl aldehydes also showed similar interactions with hem groups of most of the CYP450 enzymes out of the six enzymes considered in this study.In search of structural and dimensional similarity with gemcitabine, we finalized the synthesis of the series of compounds.In this work, a five-membered isoxazole ring is connected with a phenyl or substituted phenyl ring through a propene kind of fraction.This C3 connector gives dimensional similarity to these compounds with gemcitabine.The five-membered ring on one end and a six-membered ring on the other end gives structural similarity.Though the aromatic ring has planarity which differs from the saturated ring in gemcitabine but the compounds resemble closely to known compounds in structure and dimensions.The simplicity of synthesis and less steps involved in synthesis encouraged us to proceed for synthesis.
In the present study, the isoxazole derivative of cinnamaldehyde along with eight other isoxazole derivatives of beta chlorovinyl aldehydes were synthesized, and the crystal structure of five of them is reported.Also, the in silico molecular docking study of all nine analogues with five members of Cytochrome 450 family is reported.

General (material and method)
All the starting materials and the solvents were purchased from commercial sources and are used without further purification.Melting points were determined in open capillaries using electrothermal melting point apparatus and are uncorrected.The progress of reactions was monitored by TLC.Infrared (IR) spectra (4000-600 cm À 1 ) of the samples were recorded using a Perkin-Elmer Spectrum 100, equipped with a Specac Golden Gate Diamond ATR as a solid sample support.Elemental (C, H, N) analyses were obtained using Perkin-Elmer 2400 Series II CHNS/O Elemental Analyzer. 1 H NMR spectra were recorded with a Bruker Avance III 500 nuclear magnetic resonance spectrometer with TMS as an internal reference.MS spectra were recorded with a VG-Analytical AutoSpec Q instrument.Molecular docking was performed using Pyrx platform and Autodock Vina.The mol2 files of all compounds were prepared using Avogadro.The UCSF Chimera 1.15 and Discovery Studio visualizer software were used to study the molecular interactions with proteins.

Experimental
Molecular modeling and docking approach were implemented to compare the binding efficiency between erlotinib, gemcitabine and ketoconazole as standard inhibitors and newly synthesized nine isoxazole derivatives with six enzymes from the CYP450 family.A comparison of binding energies and pharmacophoric interactions showed the interaction of synthesized isoxazole derivatives to these enzymes compared to the standard drugs.Looking at the acceptable docking values and pharmcophoric interactions, these compounds were synthesized and analyzed spectroscopically.A single crystal X-ray diffraction study of five analogues was also carried out to study the bonding interaction of these compounds in the solid state.

Synthesis of (3/4-substituted phenyl)-3-chloroacrylaldehydes from substituted acetophenones using
Vilsmeier-Haack reagent.6 mmol of substituted acetophe- none was dissolved in 6 mL DMF in a 100 mL glass beaker.The solution was cooled in an ice bath.4 mL POCl 3 was added dropwise with stirring to the ice cooled solution in DMF.The reaction mixture was taken out of the ice bath after the complete addition of POCl 3 and kept at room temperature for 10 min.It was then heated at 70-80 � C for 40 min.The temperature of reaction mixture was not allowed to rise above 80 � C at any point of time.After 40 min, heating was stopped and 50-60 mL distilled water was added in three installments with the release of heat.The resultant solution was allowed to stand for 8 h to obtain the product.The product was filtered and used as such without further purification, in the next step (Scheme 1).
3 mmol cinnamaldehyde was added to a 50 mL conical flask.4 mmol ethyl acetoacetate and 4 mmol hydroxylamine hydrochloride were added along with 15 mL water.15 mg sodium benzoate was added as a phase transfer catalyst.The conical flask was covered with parchment paper, tied at the neck with rubber band and stirred for 2 h on a magnetic stirrer at room temperature.It was allowed to stand overnight to obtain the isoxazole derivative (Scheme 1).dd, vinylic proton), 7.50 (5H, m, Ar-H).CHN (%) Anal.Cal.C 13 H 11 NO 2 ; .17,N-6.62.

Single crystal X-ray diffraction study
Single-crystal X-ray diffraction data of for 2a-b, 2e-f and 2i were collected on an Agilent Technologies SuperNova Dual diffractometer using Mo-Ka radiation (k ¼ 0.71073 Å) at 150 K.The data were processed using CrysAlis Pro (2019).The structure was solved by ShelXT (Sheldrick, 2015a) using intrinsic phasing and refined by a full-matrix least-squares procedure based on F 2 with ShelXL (Sheldrick, 2015b) using Olex2 program suite (Dolomanov et al., 2009).All the non-hydrogen atoms were refined anisotropically.Hydrogen atoms were readily located in different Fourier maps and were subsequently treated as riding atoms in geometrically idealized positions with C-H ¼ 0.95 Å (aromatic) or 0.98 Å (methyl), and with U iso (H) ¼ kU eq (C), where k ¼ 1.5 for methyl group and 1.2 for all H atoms.The hydroxy H atom in 2e was refined freely and was treated with U iso (H) ¼ 1.5 U eq (O).The crystallographic data are listed in Table 1.Deposition Numbers 2124298-2124302 (for 2a-b, 2e-f and 2i) contain the supplementary crystallographic data for this article.These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service at www.ccdc.cam.ac.uk/structures.

Molecular docking study
Molecular modeling and docking approach were implemented to compare binding efficacy of standard drug molecules like erlotinib, gemcitabine and ketoconazole and nine synthesized isoxazole derivatives as inhibitors with selected enzymes of cytochrome 450 family.A comparison of binding energies and pharmacophoric interactions showed the strength and stability of isoxazole derivatives to the cytochrome enzymes.3D structures of nine isoxazole derivatives were drawn using software package Avogadro an opensource molecular builder and visualization tool, version 1.2.0.http://avogadro.cc/(Hanwell et al., 2012).All the structure files were used for docking after geometry optimization and saved as mol2 files to be used for docking.Six cytochrome 450 enzymes namely CYP1A2(2HI4), CYP2C9(1OG2), CYP2C19(4GQS), CYP2C8(2NNJ), CYP2D6(4WNW) and CYP3A4(4D78) were used as target protein structures.The .pdb files of these enzymes were sourced from the website rscb.org(Berman et al., 2000).The .pdb files downloaded from the website were prepared for docking by removing the ligand from the complex (if any) and prepared for docking using UCSF Chimera (Pettersen et al., 2004).Solvent molecules and co-crystalized ligand, if any was removed in the process.Docking was performed using Pyrx software (Dallakyan & Olson, 2015).The ID code of the used pdb file is mentioned in parentheses.Interactions between selected ligand and enzymes were calculated and analyzed in UCSF Chimera (Pettersen et al., 2004) and BIOVIA Discovery Studio Visualizer (2020).

In silico based ADMET studies
Assessment of absorption, distribution, metabolism and excretion (ADME) is an important aspect of drug development.Though many organic compounds show encouraging activity in molecular docking studies or in vitro screening, all of the active compounds do not proceed further in the path of drug development.There are many reasons for these compounds to fail as a successful drug, like blood-brain permeation failure, toxicity, poor efficacy, off-target interactions, undesirable interactions and solubility constraints are few of them.
Estimation of ADME in the preliminary stages of drug discovery reduces the failure in clinical phases.At least a lot of energy, time and resources are saved, which would otherwise be wasted on organic compounds which may show activity in terms of molecular docking score and in vitro screening but otherwise fail in the toxicological and solubility front.
In silico models provide an effective substitute to experimental procedures for predictions of ADME.In this work, ADMET properties of synthesized isoxazole derivatives were predicted through SwissADME database which is available freely at http://www.swissadme.ch/(Daina et al., 2017).
In our study, the lipophilicity, iLOGP is seen between 2 and 2.8 whereas XLOGP3 is in the range of 2.8-3.9;MW between 213 and 326 g/mol; TPSA between 38 and 85 Å; solubility log S (calculated with ESOL model) in the class of 'soluble' to 'moderately soluble'.

Synthesis of isoxazole derivative of cinnamaldehyde and (3/4-substituted phenyl)-3chloroacrylaldehydes
All the 3-chloroacrylaldehyde derivatives show IR band in the range of 1650-1680 cm À 1 for carbonyl fraction of aldehyde.
It also shows a band for -C-Cl stretch around 810-820 cm À 1 range.Apart from the common bands compounds 1a, 1b and 1c show bands corresponding to -C-X stretching of fluoro, chloro, bromo and fluoro substituent on phenyl ring at 768.14, 725.63 and 715.826 cm À 1 , respectively.1g and 1h show peaks at 1513.74 and 1517.01 cm À 1 for asymmetrical stretch and at 1350.23 and 1346.96cm À 1 , respectively, for symmetrical stretch of the nitro group.The 1 HNMR spectra of these compounds show peaks for vinylic proton and aldehydic proton confirming the structure of the compounds.
The isoxazole derivatives show IR band in the range of 1740-1760 cm À 1 corresponding to carbonyl -C ¼ O group of the isoxazole ring.All compounds show band for -C ¼ N bond of the isoxazole ring in the 1580-1620 cm À 1 range.Compounds also show band for -C ¼ C bond in ring and vinylic -C ¼ C bond in the IR spectra.All compounds show a band for -C-Cl stretch in the range of 815-840 cm À 1 .In addition to the common band compounds 2a, 2b and 2c show bands corresponding to -C-X stretching for fluoro, chloro and bromo substituents on phenyl ring at 871.73, 864.69 and 892.37 cm À 1 , respectively.2g and 2h show peaks at 1523.55 and 1513.74cm À 1 , respectively, for asymmetrical stretch and at 1350.23 and 1343.69 cm À 1 , respectively, for a symmetrical stretch of the nitro group.
The 1 H NMR spectra of all compounds show a singlet for the methyl group on the isoxazole ring and a singlet for proton of the chlorovinyl group.

Crystal structure study
In addition to the spectroscopic analysis, we have determined crystal structures of compounds 2a-b, 2e-f and 2i.Structures of all compounds under study are essentially planar with dihedral angles between the isoxazole ring and alkenyl backbone (C2-C3-C5-C6) and between the phenyl ring and alkenyl backbone (C6-C7-C8-C9) deviating from planarity within the range 1-6 � , except in 2f were the C6-C7-C8-C9 angle deviates by 18.2 � (Figure 1).
In 2a infinite chain with a step-like motif is formed through C13-H13���O1 hydrogen-bonding between the phenyl group and the ring oxygen of the isoxazole moiety.Double chain is formed due to head-to-tail p���p interactions between phenyl and isoxazole ring with centroid-to-centroid distance of 3.5266(9) Å (Figure 2).
The presence of a chloro substituent on the phenyl ring significantly alters the packing motif in 2b compared to 2a.This is evident already since compound 2a crystalizes in monoclinic P 2 1 /n space group while 2b crystallizes in the triclinic P-1 space group.The hydrogen-bonded motif observed in 2a is not formed in 2b, instead chain is formed through the Cl1���N1 halogen bond with the distance of 3.171 Å representing 96.1% of the sum of van der Waals radii.Furthermore, two adjacent chains are further connected into a double chain through the p���p interactions between the phenyl rings of the adjacent chains with a centroid-tocentroid distance of 3.8508(12) Å (Figure 3).
The introduction of the hydroxyl group in 2e enables the formation of strong O3-H3���N1 hydrogen bond thus leading to the infinite hydrogen-bonded chain (Figure 4).Chains are connected into layer through the Cl1���O1 halogen bond with the distance of 3.083 Å representing 94.2% of the sum of van der Waals radii.
In 2i the carbonyl oxygen atom of the isoxazole ring is an acceptor of two hydrogen bonds formed by the alkenyl group (C7-H7���O2) and phenyl group (C9-H9���O2) leading to 2D crystal structure.This hydrogen-bonded network is further supported by head-to-tail p���p stacking interactions between phenyl and isoxazole rings with alternating centroid-to-centroid distances of 3.6224(17) and 3.7271( 17) Å (Figure 6).However, this alteration is different as observed in 2f where a molecule interacts with one adjacent molecule with the centroid-to-centroid distances 3.5971(13) Å, while the centroid-to-centroid distances to another adjacent molecule are 3.6534(13) Å (Figure 5).In case of 2i, the molecule interacts with both adjacent molecules with two different centroid-to-centroid distances.

Molecular docking study
A molecular docking study of all 9 isoxazole derivatives with 6 selected enzymes from the CYP450 family is shown in Table 2.The docking score of standard reference drugs, erlotinib, gemcitabine and ketoconazole is also mentioned.

Interaction of ligands with CYP1A2
Ketoconazole shows a docking value of À 7.5 kcal/mol in hydrogen bonding interaction with Asp237 and Asp110.
Gemcitabine shows hydrogen bond interaction with Ser129 and Gly130 through -NH of ligand and with Asp110 through nitrogen of -NH 2 group with a corresponding energy value of À 6.2 kcal/mol.It also shows hydrogen bond interaction with Gly452 through H of -NH 2 with docking value of À 6.0 kcal/ mol.Erlotinib shows interaction with Arg108, Lys59 and Gly58 with docking score in the range of À 6.6 to À 6.4 kcal/mol.
Among 9 isoxazole derivatives 2a, 2c, 2e, 2f, 2g and 2h derivatives show significant values higher than erlotinib and gemcitabine.2h shows hydrogen bonding interaction with Lys404, Glu228 and Ser232 with a docking score of À 7.0, À 6.7 and À 6.6 kcal/mol, respectively.2f shows interaction with Phe451 and Leu450 through the oxygen atom of the -C ¼ O group of the isoxazole ring.The oxygen atom of the -C ¼ O group of the isoxazole ring of 2c shows interaction with Gly452, Leu450 and Phe451.O atom of the isoxazole ring also shows weak interactions with Leu450 and Phe451 in the same position.2a and 2e do not show any significant interaction with CYP1A2.Figure 7 shows p-hydrophobic and alkyl hydrophobic interaction of 2g with CYP1A2.

Interaction of ligands with CYP2C9
Erlotinib and gemcitabine show interaction with CYPC9 with the highest docking values of À 7.0 and À 7.1, respectively,   while ketoconazole shows energy values of À 8.3 and À 8.3 kcal/mol.All the 9 isoxazole derivatives show docking score higher than erlotinib and gemcitabine in interaction with CYP2C9.
2h shows interaction with Lys423 through the oxygen atom of nitro group and with Ser343 through the oxygen atom of -C ¼ O group of the isoxazole ring with an energy value À 8.6.It shows hydrogen bond interaction with Lys423 through the nitrogen of the nitro group and oxygen of the nitro group with an energy value of À 8.4.-NH hydrogen shows bonding interaction with Phe419 with an energy value of À 8.2 kcal/mol and through the oxygen atom of the isoxazole ring with Lys423 with an energy value of À 8.1 kcal/mol.Figure 8 shows the two-dimensional graphic of this interaction.
2g shows interaction with CYP2C9 with highest docking values of À 8.4 and À 8.0 kcal/mol.2a, 2b and 2c show strong hydrogen bond interaction with Lys322 with -NH hydrogen of the isoxazole ring.2e and 2f also show strong interaction with Lys423 with highest docking energy values of À 8.2 and À 8.3, respectively.All the isoxazole derivatives show interaction with Lys423 of CYP2C9 in strong to moderate strength.

Interaction of ligands with CYP2C19
The reference standard compounds, erlotinib, gemcitabine and ketoconazole show interaction with CYP2C19 with highest docking energy values À 7.4, À 6.8 and À 8.2 kcal/mol, respectively.Erlotinib interacts with the protein through  Gln193 through the -NH of ligand with an energy value of À 7.4 and À 7.2.
Gemcitabine interacts with the hem group of the protein through hydrogen of the -OH group.It also shows interaction with Ala292 through the -NH group.Ketoconazole shows interaction with Pro471 of protein through the ring nitrogen atom.
2a, 2b and 2c show highest docking values À 7.4, À 8.2 and À 7.4, respectively, 2f shows a highest docking energy of À 8.1 kcal/mol whereas 2e shows a highest docking energy of À 8.8 kcal/mol.Both 2d and 2i show À 7.9 as highest docking energy value.2d shows interaction with all four nitrogen atoms of the hem group of protein through the oxygen atom of the -OH group.2g and 2h show highest docking energy values of À 8.0 and À 8.7, respectively.2g shows interaction with Ser460 of protein through H of -NH from the Isoxazole ring whereas 2h shows interaction with Ser460 of protein through oxygen of the -C ¼ O group of the isoxazole ring.2g also shows interaction with Ser460 at lower energy values through the nitrogen atom of the -NH group and through the oxygen atom of -C ¼ O group.It also binds to Phe134 and Leu458 with energy values of À 7.9 and À 7.2, respectively.Figure 9 shows the docking interactions of 2g with CYP2C19.

Interaction of ligands with CYP2C8
The reference standard compounds, erlotinib, gemcitabine and ketoconazole show interaction with CYP2C8 with docking energy in the range of À 8.1 to À 7 kcal/mol.Erlotinib binds through Val296 and Leu208.Gemcitabine binds through His396, Asp398 and Val296.Ketoconazole binds through Asp398.The isoxazole derivatives 2b and 2c do not show any interaction with CYP2C8 but the rest of the ligands shows a common interaction through Gly98.H of -NH of the isoxazole ring shows a strong hydrogen bonding tendency with Gly98 in these ligands.The energy values associated with this interaction are among the highest of the docking values for these compounds.Apart from the hydrogen bonding interaction with Gly98 2d, 2e and 2g derivatives show other interactions as well.2e and 2h derivatives also show interaction with Val296 which can be correlated to interaction of standard drugs.Figure 10 shows the docking interactions of 2d with CYP2C8.

Interaction of ligands with CYP2D6:
The reference standard compounds, erlotinib, gemcitabine and ketoconazole show interaction with CYP2D6 with highest docking energy values À 7.5, À 7.0 and À 8.0 kcal/mol, respectively.Erlotinib interacts with the protein through Gln193 through the -NH of ligand with an energy value of À 7.4 and À 7.2.Erlotinib shows binding with Pro55 through H of -NH whereas gemcitabine shows bonding through   Ala209, Leu121, Arg101, Pro102 and Val104 with a docking score of À 7.0 kcal/mol.Gemcitabine shows these interactions through ring nitrogen, terminal -NH 2 group and the oxygen atom of the ring.Ketoconazole shows binding with Gly479 through the oxygen atom of the ring and with His477, Gly367 and Phe366 through nitrogen of one of the rings.2a, 2b and 2c show highest docking values of À 9.4, À 8.1 and À 8.5, respectively.All these ligands show binding with Ala209 through hydrogen of -NH of the isoxazole ring.Additionally, 2a shows binding with Gly212 and 2b shows binding with Leu208 through the hydrogen of -NH of the isoxazole ring.2h interacts with Gly373 and Gly479 through oxygen of the nitro group and through hydrogen of -NH of the isoxazole ring with highest docking energy values of À 9.1, À 7.6 and À 7.4, respectively.2f shows binding with Ala209 of the protein with energy value of À 8.7 kcal/mol.2e also shows binding affinity with Ala209 with docking energy value of À 8.8 and À 8.5 kcal/mol, respectively, through the oxygen atom of the -OH group and the hydrogen atom of the -NH of the isoxazole ring.It also shows interaction with Gly479 through an oxygen atom of the -C ¼ O group of the isoxazole ring, oxygen of the isoxazole ring and hydrogen of the -NH of isoxazole ring.2i also shows interaction with Ala209 through hydrogen of the -NH of isoxazole ring with docking energy score of À 8.0 kcal/mol.In all it is observed that the prominent interactions of most of the ligands with protein are through Ala209 and Gly479.Figure 11 shows the docking interactions of 2a with CYP2D6.

Interaction of ligands with CYP3A4
Erlotinib interacts -N A -H of the hem group of the protein through oxygen atom with a docking value of À 7.7 kcal/mol.It also interacts with Arg105 and Arg106 through nitrogen and oxygen atoms.Gemcitabine interacts with CYP3A4 with a docking energy value of À 7.2 kcal/mol.It binds with protein through Leu196 with oxygen of the -OCH 3 group.It shows binding to Asn198 through oxygen and nitrogen atoms and with Ser195 through nitrogen atoms.Ketoconazole shows the highest docking energy of À 10.7 kcal/mol in binding to CYP3A4.Ketoconazole binds with the hem group through the oxygen atom of the -C ¼ O  group in various positions with a docking energy value of À 10.7, À 10.5 and À 9.9 kcal/mol.Apart from interactions with the hem group, it also shows binding through Gly481, Thr224, Arg372 and Thr309.
All the synthesized isoxazole derivatives show strong binding with CYP3A4 through the hem group at more than one position.The hem interaction is mainly through oxygen of -C ¼ O group of isoxazole ring and through oxygen of isoxazole ring in some cases.The 2a shows a binding energy score of À 7.5 in hem interactions.It also shows binding to Arg105, Arg375, Glu374, Arg372 and Met371.2b shows interaction with Arg372, Glu374 and Met371 along with hem interactions with a docking energy value of À 7.2 kcal/mol.2a shows the highest docking score of À 8.3 kcal/mol while interacting with hem group.It also shows interaction with Glu374, Arg375 and Arg105 with a docking energy value of À 8.0 kcal/mol.2g shows the highest docking energy score of À 8.0 while binding with Met371, Arg372 and Thr274 whereas hem group interactions of 2g show a docking energy value of À 7.9 kcal/mol.2g shows hem interaction through oxygen of the -NO 2 group with a docking energy value of À 8.6 kcal/ mol.It also shows interaction with Arg105, Glu374 and Arg375 through oxygen of -C ¼ O group of the isoxazole ring.2d, 2e and 2f also show similar hem interactions with a docking scores of, À 8.0, À 8.2 and À 7.6 kcal/mol.Figure 12 shows the docking interactions of 2f with CYP3A4.

In silico drug likeness and toxicity assessment
Assessment of toxicity of newly synthesized compounds involves over usage of chemicals and man-power.It also costs time.To reduce all these efforts at the preliminary drug screening level, 9 synthesized isoxazole derivatives were subjected to assessment of toxicity prior to their synthesis by adopting a computational approach using Swiss ADME online service.
The prediction data for all 9 isoxazole derivatives shows that lipophilicity lies between the ranges of 2.47-3.65;molecular weight ranging 213.23-326.57;total polar surface area (TPSA) from 38.66 to 84.48 Å; and log S (ESOL) À 4.49 to À 3.12; considering the flexibility, the list of molecule bears minimum 2 and maximum 3 rotatable bonds.All 9 compounds show high GI absorption; all molecules except 2g and 2h show ability to cross the blood-brain barrier.Further, there is no violation for Lipinski rule of 5.Both 2g and 2h showing negative results in crossing blood-brain barrier are nitro derivatives and these two compounds have highest TPSA (Supplementary data 01).
Further results show that none of the compounds is substrate for P-gp.Comparing the activity of compounds on CYP450 class enzymes, it is observed that all compounds from 2a to 2i show inhibition of CYP1A2.Expression of CYP1A2 is induced by some polycyclic aromatic hydrocarbons (PAHs), some of which are found in cigarette smoke.All compounds except 2d show inhibition of CYP2C19.Inhibition to CYP2C19 may affect the serotonergic synapse, chemical carcinogenesis, drug metabolism, linoleic acid metabolism and arachidonic acid metabolism.2a, 2b, 2c, 2f and 2g shows inhibition for CYP2C9.Overexpression of CYP2C9 may increase COX-2 promoter activity which may lead to a significant increase in COX-2 protein expression and elevated prostacyclin production (Michaelis et al., 2005).None of the compounds shows inhibition to CYP2D6 and CYP3A4 enzymes.Erlotinib showed more than one docking score with acceptable RMSD value in interaction with CYP1A2, CYP2C9, CYP2C8, CYP2D6 and CYP3A4.Docking of gemcitabine showed only 1 docking score with acceptable RMSD value in interaction with CYP2C9 and CYP3A4.Docking of ketoconazole showed only 1 docking score with acceptable RMSD value in interaction with CYP2C8 and more than one docking score with acceptable RMSD value in interaction with CYP3A4 (Supplementary data 2).

Pharmacophore study of the ligands
It is observed in general for all the compounds (2a to 2i) that the elements and substituents on the isoxazole ring are prominently involved in interaction with receptor enzymes as compared to the phenyl group and its substituents.Methyl and carbonyl groups on the isoxazole ring, ring oxygen and ring nitrogen are actively involved in hydrogen bond interaction irrespective of the substituents on the phenyl ring in the moiety.Thus, the isoxazole ring can be considered the pharmacophore center of this framework.In addition to the isoxazole ring and its substituent methyl group, the double bond (-C ¼ C-) adjacent to phenyl ring is also significantly involved in bonding interaction with receptor proteins.
All these facts are observed in the receptor-ligand interaction study.In interaction of various ligands with CYP1A2, the -N-H of isoxazole ring is seen forming conventional hydrogen bond with one of the amino acids from asparagine, glutamic acid or aspartic acid whereas the methyl group are involved in alkyl interaction with valine in some cases.Also, the isoxazole ring electrons show a pi-donor interaction with glutamine, arginine and leucine in some cases.
In CYP2C8 as receptor, the isoxazole ring electrons show pi-donor interaction with alanine, valine and isoleucine.The alkyl group is seen to form alkyl interaction with isoleucine, alanine and leucine.In receptor-ligand interaction of CYP2C9, the isoxazole ring oxygen shows the formation of conventional hydrogen bond with asparagine and arginine in most of the cases whereas carbonyl oxygen forms a conventional hydrogen bond with at least one of the amino acids from glycine, asparagine, arginine and lysine.The alkyl group of ligands shows interaction with CYP2C9 through phenylalanine in most of the cases.
The receptor-ligand interaction of CYP2C19 with ligands shows that isoxazole ring nitrogen forms a pi-donor hydrogen bond with the Hem group in 2g.It forms a conventional hydrogen bond with asparagine, serine and threonine.The isoxazole ring is involved in pi-pi interaction with hem, alanine and leucine in 2g and shows a pi-pi interaction with phenylalanine in case of 2d.It also shows pi-pi interaction with two leucine fragments and one valine in case of 2b and with valine alone in case of 2a.CYP2C19 also shows pi-pi interaction through cysteine and glutamine in with 2f and through leucine and proline with 2i.Thus, in all almost all the ligands show pi-pi interaction with CYP2C19.The isoxazole ring shows interaction through its alkyl group with Isoleucine, leucine, alanine, valine, cysteine and phenylalanine.
In receptor-ligands interactions of CYP2D6, it is observed that the phenyl ring of ligand is involved in pi-pi interaction in many examples.The pi-pi interaction is through the hem group, phenylalanine, alanine, valine, leucine, etc. in almost three to four ligands.The isoxazole ring atoms and groups also show interactions in case of CYP2D6 as seen in earlier cases.
In receptor-ligand interaction of CYP3A4 with ligands, the carbonyl oxygen of the isoxazole ring show conventional hydrogen bond through arginine, glutamine and hem group.The alkyl group shows interaction with alanine and arginine.
In most of the receptor-ligand interactions, the beta unsaturation in the aliphatic part of the ligand is involved in pi-alkyl interactions through amino acids like valine, leucine and isoleucine.This is observed in interaction with all the six receptor enzymes.Interestingly, the beta unsaturation in 2i is not involved in such pi-alkyl interaction with any of the receptor.Thus, the chlorine substitution on beta unsaturation seems to have a role in interaction of pi electrons of the unsaturation with amino acids in receptor.
The pharmacophore study of all the ligands shows that derivation of these ligands on the phenyl ring side can enhance the receptor-ligand interaction.Isoxazole part shows significant involvement in interaction with receptor through the two hetero atoms (-N and -O) and the two groups (carbonyl and alkyl).Replacement of phenyl ring with five-or six-membered heterocyclic ring with functional groups like -C ¼ O, -OH can significantly increase the activity of these scaffolds.The future scope for study lies in the direction to substitute the phenyl ring with such heterocyclic ring like pyrrole, pyridine or thiophene, etc. Figure 13 shows detail of interaction of each fragment of the ligands with receptor enzymes.

Conclusion
Among synthesized ligands 2a, 2e and 2g show highest affinity and docking score with CYP1A2 whereas 2b, 2c, 2d, 2f, 2h and 2i show moderate affinity to CYP1A2.All the docking values are higher than those of erlotinib and gemcitabine but lower than that of ketoconazole.
Interaction of the synthesized ligands with CYP2C9 shows that the 2d, 2e, 2f, 2g and 2h show highest affinity whereas 2a, 2b, 2c and 2i show moderate activity.But all the ligands show higher docking score than erlotinib and gemcitabine but lower affinity than ketoconazole.
2b, 2e, 2f and 2h show highest affinity for CYP2C19 which is higher in terms of docking score than all three standard drug molecules.2a, 2c, 2d, 2g and 2i show lower affinity than that of ketoconazole but higher than that of erlotinib and gemcitabine.
With CYP2C8, all 9 ligands show affinity comparable to Erlotinib but lower than that of ketoconazole and higher affinity than Gemcitabine.The docking score of gemcitabine is À 6.2 kcal/mol whereas all ligands show a docking score above À 8.1 kcal/mol.2a, 2 b, 2c and 2g in interaction with CYP2D6 show docking scores higher than all three standard drug molecules.Only ketoconazole shows comparable docking scores whereas erlotinib and gemcitabine show much lower docking scores than the ligands under study.
2a, 2e and 2g show highest activity in interaction with CYP3A4 whereas 2b, 2c, 2d, 2f, 2h and 2i show moderate affinity.Affinity of all the ligands is comparable to erlotinib and higher than gemcitabine.Ketoconazole shows much higher docking score than all the 9 ligands in this case.But all the ligands show significant bonding interactions with the hem group in more than one docking positions.All the 9 ligands show binding with CYP3A4 through a similar set of amino acids and all ligands show strong interaction with the hem group.
In all, it is observed that the isoxazole derivatives of substituted phenyl 3-Chloroacrylaldehyde synthesized and studied for molecular docking in this study shows higher binding affinity than erlotinib and gemcitabine and comparable affinity to ketoconazole in most of the cases.As ketoconazole is not an approved drug for later stage ovarian cancer, the isoxazole derivatives of this kind of framework may give the lead for some potential molecules with enhanced anticancer activity.Of course, further investigation and in vitro study on cancer cell lines is required for further conclusions.

Figure 1 .
Figure 1.Molecular structures and atom numbering schemes for 2a-b, 2e-f and 2i.Probability ellipsoids are drawn at the 50% level.

Figure 2 .
Figure 2. (a) Top view on chain formation in 2a generated by C13-H13���O1 hydrogen bonding.Hydrogen-bonding is indicated by blue dashed lines.Hydrogen atoms not involved in the motif shown have been omitted for clarity.(b) Side view on a chain with a step-like motif.(c) Double chain formation due to the p���p interactions indicated by green dashed lines.

Figure 3 .
Figure 3. Double chain formation in 2b through Cl1���N1 halogen bonding and p���p interactions indicated by green dashed lines.Hydrogen atoms have been omitted for clarity.

Figure 5 .
Figure 5. (a) Chain formation in 2f generated by C9-H9���Cl1 hydrogen bonding.Hydrogen-bonding is indicated by blue dashed lines.Hydrogen atoms not involved in the motif shown have been omitted for clarity.(b) 2D structure formation through p���p stacking interactions indicated by green dashed lines.Only p���p stacking interactions part of a central pillar are presented for clarity.

Figure 6 .
Figure 6.2D structure formation in 2i through C7-H7���O2 and C9-H9���O2 hydrogen bonding and p���p stacking interactions.Hydrogen-bonding and p���p stacking interactions are indicated by blue and green dashed lines, respectively.Hydrogen atoms not involved in the motif shown have been omitted for clarity.Only p���p stacking interactions part of a central pillar are presented for clarity.

Figure 8 .
Figure 8.Molecular docking interactions of 2h with CYP2C9 (a) 2-dimensional representation of interaction of ligand molecule with amino acids (b) 3-dimensional view of the interaction.

Figure 9 .
Figure 9. Molecular docking interactions of 2g with CYP2C19 (a) 2-dimensional representation of interaction of ligand molecule with amino acids (b) 3-dimensional view of the interaction.

Figure 7 .
Figure 7. Molecular docking interactions of 2g with CYP1A2 (a) 2-dimensional representation of p-hydrophobic and alkyl hydrophobic interactions of ligand molecule with amino acids (b) 3-dimensional view of the interaction.

Figure 10 .
Figure 10.Molecular docking interactions of 2d with CYP2C8 (a) 2-dimensional representation of interaction of ligand molecule with amino acids (b) 3-dimensional view of the interaction.

Figure 11 .
Figure 11.Molecular docking interactions of 2a with CYP2D6 (a) 2-dimensional representation of interaction of ligand molecule with amino acids (b) 3-dimensional view of the interaction.
Compounds 2b, 2c and 2i show lead likeliness.RMSD values of docking calculations show that in case of CYP1A2 interaction of 2d, 2e, 2i show a next value in consistency to the highest docking score.For CYP2C9, interaction with 2b, 2d, 2f, 2h shows consistency of highest docking score with one next value.Interaction of CYP2C9 with 2c shows a second and third lower values (À 7.1 and À 7 kcal/mol) in close consistency with the higher score.2c, 2d, 2f, 2g show at least one value consistent with the higher docking score.In the docking study of CYP2C9 2a, 2d, 2f and 2i show at least one value in close consistency with the highest docking score. 2 g shows four other docking scores consistent with the highest score in interaction with CYP2D6.

Figure 12 .
Figure 12.Molecular docking interactions of 2f with CYP3A4 (a) 2-dimensional representation of interaction of ligand molecule with amino acids (b) 3-dimensional view of the interaction.

Figure 13 .
Figure 13.Pharmacophore study of ligand showing role of various elements, groups and fragments in interaction with receptor enzymes.