In Silico Studies, Synthesis and Biological Evaluation of 4,5-Dehydrospisulosine Butyrate Ceramides as Potential Sphingosine Kinase I Inhibitors

In the present work, synthesis of 4,5-dehydrospiulosine and its chain analogues (1-3) as potential Sphingosine Kinase I inhibitors has been achieved via the diasteroselective Grignard reaction, stereoselective cross metathesis reaction followed by N-acylation with p-nitrophenyl butyrate to give the corresponding butyrate ceramides (4-6). All compounds were obtained in high yield and purity followed by molecular docking simulation studies using AutoDock which indicated their varying binding anities with Sphingosine Kinase 1 protein was done. Further, the biological evaluation studies, as potential anti-prostate cancer agents by inhibiting the sphingosine kinase 1 protien of all synthesized compounds (1-6) on PC-3 cell lines by SRB method was done. Compound N-((2S,3S,E)-3 hydroxyheptadec-4-en-2-yl) butyramide (4) exhibited remarkable cytotoxicity with an IC 50 value of 6.06 µM.


Introduction
Sphingolipids are highly bioactive compounds that modulate many cell signalling pathways that are relevant to tumour biology and cancer control. The ceramide analogues of sphingolipids act as the inhibitor of cell growth and hence lead to apoptosis. Sphingosine 1-phosphate (S1P), inhibits apoptosis, induces cell migration and other pro-carcinogenic behaviours. 1,2 However, sphingosine, sphinganine, and other sphingoid bases also have the potential to be useful for cancer control, because they inhibit transformation of normal cells induced by different sources. The effectiveness of sphingoid bases have a limitation as it is phosphorylsed by sphingosine kinase-an enzyme that has been called an oncogene.
Recent literature has shown interest towards targeting sphingosine kinase for cancer therapy. SK1 has been a primary target with a hope to suppress the ceramide/S1P rheostat in an attempt to regulate cell growth. On the basis of this rationale, one would predict that compounds that cannot be phosphorylated would be more effective in cancer suppression than the naturally occurring sphingoid base sphingosine. 2 In the recent past, interest in deoxysphingolipids has evolved due to the lack of the C1-OH group responsible for phosphorylation and also have shown promising biological activity. 2 The interest has further been accelerated with studies of naturally extracted Fumonisin B1 3 (Fig. 1.) that acts as inhibitors of ceramide synthase, a key enzyme in the sphingolipid biosynthetic pathway. 4,5 Delgado et al. 6 reported a sterioselective synthesis of Spisula polynyma Spisulosine (Fig. 1.) spisulosine and its analogues along with its 4,5-dehydrospisulosine( Fig. 1.) analogues for analysing the role of ceramide synthase and their resulting products in cell fate. These group of antiproliferative compounds of marine origin isolated from the clam and its different chain analogues have also been reported 7,8 as an inhibitor of sphingosine kinase, resulting in lower levels of endogenous S1P. Spisolusine was initially developed as anticancer agent due to its ability to inhibit proliferation in the prostate tumor PC-3 and LNCaP cell lines 8 but later discontinued from phase I in 2008. 10,11 The other closely related structure such as obscuraminols, 7,8 claviminols, 9,10 xestoaminols 11 (Fig. 1.) have also shown remarkable cytotoxic propterties. In addition, recent evidence of the presence of 1-deoxysphingolipids in human sensory neuropathy type 1 (HSN1) 12 and for the metabolic syndrome such as type 2 diabetes as a novel class of biomarkers, has also been reported. 12 The chemical composition of this important class of compounds includes long lypophilic carbon chain, vicinal amino alcohol with de ned syn/anti stereochemistry 8,9 , variable number of unsaturation on the lipophilic backbone with de ned cis/trans stereochemistry, presence of amidic linkage with short and long chain fatty acids 10 and other additional functionalities as illustrated in (Fig. 1.). The design and synthesis of new sphingosine kinase inhibitors as anti cancer agents are interesting target for organic chemists. Further the in silico studies support the rationale behind the design the new chemical entities by calculating the binding conformations with respect to amino acid interaction, hydrogen bonding and hydrophobic interaction between the ligand and protein.
Ceramide serves as a central mediator in sphingolipids metabolism and signaling pathways, regulating many fundamental cellular responses. They are referred to as a 'tumor suppressor lipid'. Recently, many studies have focused on the ceramide-induced apoptosis pathway and mitochondria dependent apoptosis. Structurally the ceramide molecule contains a sphingoid long-chain base backbone, linked to a fatty acid molecule through an amide bond. Two main approaches to promote anticancer activity in the ceramide-Sph-S1P axis have been identi ed as use of an exogenous supplement of ceramide to promote apoptosis and inhibition of cermaide-metabolizing enzymes to regulate the ceramide /S1P rheostat. Few short chain ceramide have lead to promising anti-cancer effects due to its more solubility as compared to long chain fatty acids. Thus in most studies mostly short chain ceramides but not naturally occurring ceramides have been used as potential anti-cancer agents. 13 Ceramide molecules contains a long sphingoid chain backbone, linked to a fatty acid molecule through an amide bond with a 4,5-trans double bond. In the present work, keeping in view of the main structural features required and also inspired by the potential biological signi cance of deoxysphingolipids as cancer therapeutics with varying structural features we have undertaken the design of few 4,5dehydrospisulosine and its chain analogues (1-3) followed by a small library of their corresponding butyrate ceramides (4-6) as Sphingosine Kinase I inhibitors via the diasteroselective Grignard reaction, stereoselective cross metathesis reaction followed by N-acylation in good yield and purity. The choice of butyric acid is based on the known anti cancer activity of short chain ceramides 14 itself and can also present an added advantage as chemopreventive agents which may result due to the partial hydrolysis of the ceramide. The in silico studies of the binding conformations with respect to amino acid interaction, hydrogen bonding and hydrophobic interaction between the ligand and protein for the proposed compounds was done. Further the in vitro study on PC-3 cell lines by SRB method has been evaluated.

Result And Discussion
Molecular docking is one of the widely implemented approaches for the study of protein-ligand interactions and for drug discovery and development. For molecular docking simulation of the compounds, the position and size of ligand-binding site were determined based on the receptor-bound SKI-II inhibitor in the crystal structure of SPHK1. The information of respective amino acid residues were obtained from the literature associated with the respective PDB structure [PMID: 23602659]. 14  Molecular docking studies were carried out with all the proposed molecules (1)(2)(3)(4)(5)(6) in order to nd their optimal conformations into the ligand binding pocket of SPHK1. Before commencing prospective molecular screening, the docking protocol was benchmarked by comparing the experimental and computationally predicted conformations of inhibitor SKI-II from the crystal complex (PDB ID: 3VZB), which is measured by computing the root mean square deviation (RMSD) between experimentally observed ligand conformation and the one predicted by algorithm. RMSD between experimental and computationally predicted best conformation was 1.581 Å, which is usually in the range 1.5-2 Å [PMID: 15937897], 14 which con rmed the accuracy of docking protocol for predicting the reliable binding conformations of all compounds. Physicochemical properties of the all the synthesized compounds is presented in Table 1.  Table 2. Also a hydrophobic contact with Thr 196 as shown in Fig. 2. was observed and is a critical and important binding site residue due to its prime involvement in establishing SKI-II-SPKH1 complex [PMID: 23602659].

Chemistry
Our synthetic approach, utilizes a reaction sequence starting from commercially available HNBoc amino ester 8, which was successively treated with diisobutylaluminum hydride (DIBAL) and vinylmagnesium bromide in a one-pot reaction to yield vinyl alcohols 9a/9b as syn: anti isomers (dr = 75:25). The spectral analysis was in agreement as reported in literature wherein the syn diastereoselectivity has been established by the chelation-controlled Cram cyclic model. 20 For easy separation the mixture of alcohols was converted to corresponding chromatographically separable tert-butyldimethylsilyl ethers 10a and 10b giving the major isomer 10a followed by treatment with 46% hydro uoric acid in acetonitrile regenerated the pure alcohol 9a. The compound 9a obtained was subjected to ole n metathesis using Grubbs 2nd generation catalyst and respective alkenes to obtain compounds (11-13) as a colorless oil followed by deprotection with 1N HCl in dioxane to obtain intermediates (1-3) which on puri cation were further subjected to N-acylation using p-nitrophenyl butyrate leading to compound (4-6) in good yield after column chromatography as illustrated in Scheme 1. All the synthesized compounds were characterized by 1 H NMR, 13 C NMR, MS, CHN analysis along with the optical rotation.

In vitro biological evaluation
The effect of chain length and butyrate ceramide formation on Human Prostate cancer cell toxicity was tested in vitro using sulforhodamine-B (SRB) assay. Androgen independent prostate cancer cells (PC-3) were treated for 48 hr with compound (1-3) and their ceramides (4-6) as 1:1 molar complex with fatty acid free bovine serum albumin. The concentration dependent cytotoxic effect of these compounds was screened in vitro at various concentrations ie. 100µM, 50µM, 25µM, 12.5µM and 6.25µM, respectively. From Fig. 3. it is evident that among the compound (1-3) compound (2) shows higher cytotoxicity while among the corresponding ceramide (4-6), compound (4) displayed the best cytotoxicity result.
Compound 4(N-((2S,3S,E)-3 hydroxyheptadec-4-en-2-yl) butyramide), the shorter chain C 17 ceramide analogue exhibited good cytotoxic activity against PC-3 cell line with the IC 50 value of 6.06 µM as shown in Table 3. Hence, it is observed that by shortening of sphingoid backbone and keeping the 4, 5-trans double bond intact with an amide linkage, plays an important role in promoting cytotoxic acitivity ( Fig. 3.). In vitro results corroborate the observations of molecular docking studies and suggest the anticancer potential of compound (4). The balance between the levels of interconvertible sphingosine-1-phosphate and ceramide is a crucial determinant of cell fate. Sphingosine-1-phosphate promotes cell proliferation while ceramide is pro-apoptogenic and arrest cell growth. This sphingolipid rheostat is regulated by the action of SPHK1. Inhibition of SPHK1 may lead to ceramide accumulation and cause cell death in the cancer cell lines. In the current study the hypothesis suggest that the C 17 ceramide analogue (4) incorporates itself in the complex sphingolipids pathway and thereby, modulate the signaling cascades involved in carcinogenesis. Earlier reports have shown that the 4,5dehydrospisolusine ceramides have been synthesized and used as probes for pro ling ceramide synthase activities in intact cells. 15 Studies have also utilized C 17 ceramide analogues to probe the sphingolipid metabolism and have shown to be e ciently taken up by the cells and converted to complex sphingolipids. 16 However, further work needs to be carried out to prove our hypothesis and understand the mechanism of action of these compounds.

Conclusion
We

Docking methodology
The intermolecular interactions between receptor and ligands was done by a three-step computational approach that included (i) receptor-and ligand-preparation, (ii) de ning the grid box, (iii) molecular docking, and (iv) assessing the intermolecular interactions. AutoDockTools and AutoDock v 4.2.0 (ADT) [PMID: 19399780] program was used to perform all molecular docking simulation studies. The docking studies were carried out with all the proposed ligand molecules in order to nd their optimal conformations into the ligand binding pocket of SPHK1.

Chemicals and intsruments
The 13 C and 1 H NMR spectra were obtained on a Bruker Avance-400 spectrometer operating at 100 and 400 MHz, respectively. Chemical shifts are down eld relative to TMS. The couplings are given in Hz.
Mass spectra were performed on a Thermo Q-Exative GC Orbitrap mass spectrometer (Thermo Fisher Scienti c, Bremen, Germany).
In vitro cytotoxicity of the compound was determined by sulforhodamine B (SRB) assay. 100 μl cell suspension containing 7.5 × 10 3 cells were added in each well of 96-well plate. The plate was incubated for 48 h to allow the cells to attach and grow. The cells were then treated with different concentrations of the compound conjugated with equimolar amount of Bovine Serum Albumin, preincubated at 37 °C. After incubation for 48 h, the cells were xed with 10% (w/v) ice-cold trichloroacetic acid (TCA) at 4 °C for 2 h. 18 The wells were washed with water, air dried and 100 μl SRB solution (0.057% w/v SRB in 1% acetic acid) was added in each well. The plate was incubated at room temperature for 1 h and the unbound SRB solution was removed by washing with 1% acetic acid solution followed by the air drying. Thereafter, 100 μl of 10 mM Tris solution (pH 10.5) was added to each well and shaken on a gyratory shaker for 5 min. The absorbance was measured at 510 nm using a microplate reader. 19 Inhibition of cell growth was calculated as: % cell growth = (absorbance of treated cells / absorbance of control) × 100; % cell inhibition = 100 − % cell growth.

Declarations Competing interest
The authors declare that they have no known competing nancial interest or personal relationships that could have appeared to in uence the work reported in this paper. Cytotoxicity (% cell death) data of compound (1-6) at concentration 100 µM (blue) and 50 µM (red).

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