Molecular Docking, Pharmacokinetic Properties and Toxicity Studies of Novel Folate Conjugated To α -Tocopherol via Peptide Bond

: A novel folate conjugated to α -tocopherol (VAF) via peptide bond was Successfully synthesized by using three components vitamin e ( α -tocopherol), chloro acetamide and folic acid under mild and simple conditions. The new synthesized compound was characterized by proton nuclear magnetic resonance 1 H NMR, 13 H NMR and Fourier transform infrared (FT-IR) analysis. A molecular docking study for targeted compound (VAF ) was performed and showed growth-inhibitory activity towards the human folate receptor alpha (FRα) that may affectively the folate uptake by cancer cells. Further, The predicted pharmacokinetic properties and toxicity of the compound (VAF) and folic acid (F) were determined using a statistical analysis (ADMET). It was suggested that pharmacokinetic properties of compound (VAF) represents a promising drug and used as anticancer agents.


Introduction:
Vitamin E (tocopherol and tocotrienols) [1] has antioxidant properties that subside unstable free radicals by supplying one of their electrons to the deficient free radical electron via a phenolic hydroxyl group (Fig. 1), allowing it more stable. [2]. Vitamin e (-tocopherol), which was chosen for research, controls vitamin A functions in the body and, in addition, protects the skin from harmful effects due to its anti-inflammatory properties [3]. On the other hand, Folate is one of the B vitamins referred to as vitamin (B 9 ) or folic acid produced by the body that has been converted into folate [2,3].
Furthermore, folate is used by the body to produce DNA and RNA for cell division [4,5] and is used as a supplement by pregnant women to avoid neural tube defects in their children [6][7][8]. Folate (Fig. 2) is based on three chemical moieties. A pterin is bound to p-aminobenzoyl group by a methylene bridge, which in turn is bonded to either glutamic acid or poly-glutamate by an amide linkage [9]. According to a literature survey [10], using conjugated folic acid/cholesterol (CPF) to incorporate folic acid as a ligand in liposome systems allowed highly selective tumor-targeted liposome encapsulated doxorubicin with improved cytotoxicity within tumors. Several attempts to use folic acid as a targeting ligand conjugated to lipid or polymer with polyethylene glycol as a linker (Folate-PEG) have been made in the past [11][12][13].
In a previous paper [14], when vitamin e (-tocopherol) carrying selenated heterocyclic derivatives showed excellent cytotoxicity activity against human breast cancer cell line (MCF-7). Furthermore, Abdel-Hafez et al [15] developed a new class of vitamin e-containing sulfa drug derivatives, with the results showing a major effect on the antibacterial function of sulfa drugs linked to vitamin E. Based on previously reported results [10][11][12][13][14] for vitamin e as well as folic acid's biological activity effects, and in continuation of our attempts to synthesize new system based on vitamins linked together or linked to selenium or sulfur compounds [14,15], the goal of this study was to synthesize and describe a biologically active folate linked with vitamin e (αtocopherol) complex with human folate receptor (FRα) in addition pharmacokinetic calculation hoping to access new classes of compounds, for cancer targeting.

i) Chemistry
Our question that how can linked together vitamin e with folic acid so, the starting compound folate conjugated vitamin e 4 (VAF) was designed and synthesized in two steps: initially, synthesis of α-tocopherol acetamide derivative 2 (VA) by the reaction of α-tocopherol 1(V) with chloroacetamide (A) as similar procedure with some modification [15] and as precursor material to synthesize the final target product 4 (VAF). The all resulted data was in agreements with reported literature [15]. The final step coupling reaction between derivative 2 (VA) and folic acid (

ii) Molecular Modeling
The crystal structure of human folate receptor (α-FR) in complex with folic acid (PDB: 4LRH) and (MOE) software [16] were used for modeling and docking the new folate conjugated α-tocopherol compound. FRα is a folate receptor isoform overexpressed selectively in numerous tumors to mediate cellular uptake of folate under low folate conditions [17,18]. Results revealed that the new compound is docked deep inside the folate-binding pocket with a docking score -9.4939 kcal/mol, forming comparable interactions to those of folic acid with the surrounding amino acid residues ( Fig 4B and 4D). The pteroate moiety is injected into the receptor, while the glutamate-conjugated -6 tocopherol moiety stays out of the pocket entry without disrupting the FR binding affinity. (Fig 4A and 4B). The pterin ring is capped by Tyr175 and stacked between the aromatic chains of Tyr85 and Trp171 forming π-π and π-H interactions with Tyr85 (3.66 and 3.79 Å) ( Fig. 4A and 4C). Also, the hydrophilic pterin ring forms hydrogen bonds with the guanidinium group and indolyl moiety of Arg103 and Trp171, respectively (3.52 and 2.99 Å) ( Fig. 4A and 4C). The aminobenzoate moiety forms π-π with Tyr 60 (3.66 Å) and is fixed by hydrophobic interactions with Tyr60, Trp102 and Ser101, which contour the center of the long binding tunnel ( Fig. 4A

iii) Pharmacokinetic
Studies on absorption, distribution, metabolism, elimination, and toxicity [19]: A reliable methodology for predicting the toxicity and pharmacokinetic properties of the target compound and folic acid have been utilized and assessed by computer program and results were presented in Table 1 However, predicted in-silico toxicity was assessed because AMES toxicity testing revealed that none of the studied compounds had a mutagenic ability.
In general, the target compound 4 (VAF) was found to be an effective hERG II inhibitor but not a hERG I; therefore, it could be linked to subsequent therapeutic cardiac effects [21]. Compound 4 (VAF) demonstrated adequate skin sensitization protection with the risk of inducing hepatotoxicity, as seen in Table 1  Caco2 stands for human colon adenocarcinoma-2, VDss stands for steady-state amount of distribution, and BBB stands for blood-brain barrier. AMES stands for Salmonella typhimurium reverse mutation assay, and hERG stands for human ether-a-go-go-related gene [19].

Conclusion:
A novel system of folate conjugated vitamin e was reported. A molecular docking study predicts highly activity towards the human folate receptor alpha  Centre in Cairo, Egypt, mass spectra were measured using a JEOL-JMS-AX 500.