Design and synthesis of new pyrazolylbenzimidazoles as sphingosine 1-kinase inhibitors

Sphingosine-1 Kinase (SphK-1) is one of the important enzymes of phospholipids and it is inhibition is one of the therapeutic strategies for different diseases. SphK1 over expression is observed in different types of cancer that indicating its important role in tumor growth. In search of effective SphK-1 inhibitors, a new series of pyrazolylbenzimidazoles was synthesized and evaluated as sphingosine1-kinase (Sphk-1) inhibitors. In order to evaluate the binding anities of all the synthesized compounds, all compounds were subjected to docking analysis and uorescence quenching. The results indicated that there is a consistency between the docking and the uorescence quenching results which revealed that compounds 47 and 48 exhibited signicant decrease in the uorescence intensity of SphK1 as well as they formed stable protein–ligand complexes. In addition, Enzyme inhibition assay was performed and showed effective inhibitory potential towards SphK-1. Moreover, IC 50 values was calculated and displayed that compounds 47 and 48 were the most promising compounds. In addition, antiproliferation study for all the synthesized compounds was performed against NCI 60-cell line panel. The target compounds 47 and 48 demonstrated effective antitumor activity inhibitory potential to the SphK-1. Most of these compounds t well into the ATP-binding site of SphK1 and form signicant hydrogen-bonding interactions with catalytically relevant residues as predicted by molecular docking. In this article, insight has been given for the importance of pyrazolylbenzimidazoles as Sphk1 inhibitors and the perspectives that they hold for future research. activity on a panel of 60 cell lines, compounds 47 and 48 exhibited an effective cytotoxic activity against several NCI cell panels. Based on molecular-docking study, most these synthesized as ATP-binding and several important and hydrogen interactions with the active site A considerable correlation was noticed between the docking results and the IC 50 values and its that, compounds and act as promising inhibitors of with low IC 50 values in the single-digit.


Introduction
Sphingolipid is a kind of phospholipid which is a major component of all cell membranes and can form lipid bilayers that maintain the uidity of membranes [1]. Ceramide (Cer), sphingosine (Sph) and sphingosine1-phosphate (S1P) are metabolites of sphingomylein which play an important role in different diseases such as cancer [2,3], brosis [4] and Alzheimer's disease [5]. The balance between the sphingolipid metabolites which acting in two opposite ways is crucial in the determination of the cell fate [6,7]. Ceramide and sphingosine [8,9] are a pro-apoptotic molecule to mediate the cell cycle and induce apoptosis, while S1P promotes cell proliferation and acts as a 'pro-survival' molecule [10,11].
The evolution of SphK1 and SphK2 inhibitors has been recently reported [51,52]. Many SphK inhibitors were designed to have a polar head group and a lipophilic tail region. Studying all structural variations in the sphingosine-based SphKIs and their resulting biological effects in the earlier work, several points will be taken into consideration to improve the potency of the new target compounds possessing both pharmacophoric moieties, benzimidazole and pyrazole: (1) In the lipophilic region: increasing lipophilicity by introducing saturated heterocycles to the pyrazole ring as R 2 = morpholinyl, piperidinyl, and pyrrolidinyl in the lipophilic tail as well as the introduction of a phenyl ring will enhance the lipophilicity and the bioavailability by producing a better drug-like pro le. (2) In the polar head: replacing the hydroxyl group to prevent its phosphorylation by other polar groups seems to be vital in the design of new sphingosine-based SKIs. Modi cation of the polar head to possess the following groups: R 1 = NO 2, COOH, was planned so that the nitro group and the carboxylic group will be the polar heads. (Fig. 3) Results And Discussion Chemistry The present study aimed to synthesize new benzimidazole candidates as sphingosine kinase 1 inhibitors ( Figure 3) and it was guided by molecular docking study assessing their binding energies taking into consideration that polar substitution of the benzimidazole ring at 5-position was essential for activity. The target pyrazolylbenzimidazoles derivatives were synthesized through two main schemes. The rst part of this synthesis was demonstrated in scheme 1 which deals with the preparation of pyrazole derivatives via three steps starting by the synthesis of 3-methyl-1-phenylpyrazol-5-one 13 by the reaction of phenyl hydrazine with ethyl acetoacetate in the presence of glacial acetic acid and ethanol according to the procedure described by Khanal et al [53]. The Vilsmeier-Haack reaction of the previous step Based on the predicted binding a nities and interactions, compounds 47 and 48 were selected as top-scoring compounds and modi cation of the polar head to possess the NO 2, group, was planned. Unfortunately, the low yield and poor solubility caused no synthesis of other derivatives of nitro-analogues.

Fluorescence binding studies
Fluorescence binding studies were performed for evaluating the binding a nity of all the synthesized compounds 26-48 with SphK1. The gradual loss in the uorescence intensity upon addition of the selected compounds, 47 and 48, Figure (4A and 4B) was observed for SphK1, which points towards the formation of a stable protein-ligand complex.
The rest of the compounds did not show any quenching and some of them even perturbed the structure of SphK1 since major red shift and increase in the uorescence intensity was observed when added to protein samples in increasing concentrations (data not shown). The Stern-Volmer plot Figure (4C and 4D) was used to analyze the quenching data to determine the binding a nity (K a ) for each compound. The number of binding sites per SphK1 molecule (n) for these compounds was also determined from the same plot. Compounds 47, while compounds 48 showed binding in the 10 3 micromolar range (Table 1). Thus, hits obtained from the binding studies showed moderate binding with SphK1 and were further tested for inhibitory activity against SphK1.

Enzyme inhibition assay
Enzyme inhibition potential against compounds 26-48 toward SphK1were evaluated by malachite green ATPase inhibition assays. During the initial screening, the maximum concentration of all compounds (100 µM) was used (Table  S2), which revealed that most of the studied compounds inhibited SphK1 activity effectively ( Table 2). The enzyme inhibitory potential of the synthesized compounds that showed good binding a nity towards the SphK1 was determined by malachite-green based ATPase inhibition assay that revealed IC 50 values in the micro-molar range ( Table   3). The kinase activity of SphK1 is measured in terms of picomolar concentration of phosphate released in the reaction mixture which is represented in Figure 5 (A and B). The absorbance value of the malachite-inorganic phosphate green complex so formed at 620 nm is converted with the help of phosphate standard curve as described [57][58][59][60][61][62][63][64]. The loss in the SphK1 activity followed an inverse relationship between percentage inhibition and an increasing concentration of selected compounds as shown in Figure 5 (C and D) which was used for the calculation of IC 50 values (Table 3). The compound 47 e ciently inhibited SphK1 kinase activity with lower IC 50 (2.48 ± 0.05 μM ). The IC 50 value for compound 48 ( Figure 6D) inhibited SphK1 activity with higher IC 50 (4.02 ± 0.16 μM). The enzyme inhibition results overall propose that compounds 47 and 48 act as promising active inhibitors of SphK1.
Anticancer activity against NCI 60-cell line panel: The synthesized new pyrazolylbenzimidazoles were screened for their in vitro antitumor activity by the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI). This involves screening of the compounds at a single dose of 100 µM [65]. From the obtained results in Table 4, the studied compounds showed a broad spectrum of anti-cancer activity against several NCI cell panels. At 100 µM concentration, compounds 47 and 48 showed potent inhibition against the leukemia and breast Cancer cell lines. (Table4).

Molecular docking
The molecular docking study of the designed compounds with SphK1 was performed using the AutoDock vina tool [66]. Vina gives the predicted binding poses of the synthesized compounds 26-48 along with the binding a nities in kcal/mol. Based on the predicted binding a nities and interactions, compounds 47 and 48 were selected as top-scoring compounds. The predicted binding a nities of the selected compounds are given in (Table 5). On the basis of nonbonded interactions of compounds with the SphK1, Compounds 47 and 48 showed comparatively better interactions. Figure 6 shows the 2D structure of compound 47 ( Figure 6A) along with its interactions with the PF-543 binding site residues of SphK1 ( Figure 6B). Compound 47 forms hydrogen bonds with the Thr196 of the PF-543 binding pocket. 2D representation of protein-ligand interactions ( Figure 6C) shows compound 47 having π-interactions and van der Waals interaction with the surrounding residues including the Asp178 which is the substrate binding site. These interactions of compound 47 with SphK1 suggest a strong bonding. Surface view of the protein shows compound 47 has strongly occupied the binding cavity of the protein ( Figure 6D). A similar pattern is observed for compound 48 where it forms a hydrogen bond with the Thr196 of the PF-543 inhibitor binding site; in addition, Ile174 and Leu302 are showing π-sigma interaction with the ligand ( Figure 7C). In addition to that, residues Val177, Leu268, Leu259, Ala274, and Leu319 are showing π-alkyl interactions with the ligand. Besides compounds 46 and 47, A detailed interaction pro le of all the synthesized compounds with SphK1 is given in observed the importance of substituting the 5-position of benzimidazoles with nitro group. The correlation between the enzyme inhibition results, in vitro cytotoxic activity and docking results was observed. This proves the importance of polar substituents at position-5 of the benzimidazole ring. Unfortunately, the low yield and poor solubility caused no synthesis of other derivatives of nitro-analogues. The unsubstituted benzimidazoles and 5-methylbenzimidazoles were synthesized as was predicted from the docking studies that they might possess good binding a nity but they were found to be the least active compounds. An increase in the inhibitory activity was observed by substitution at position 5 of the pyrazole moiety with N-methyl piperazinyl in compounds 28, 35 and 42 than pyrrolidinyl derivatives in compounds 29, 36 and 43 (Table 2). An increase in the inhibitory activities were observed by substituting the phenol ring with two methyl as in compounds 32, 39 and 46 (Table 2). These data correlated with our rationale, which depends on replacing the hydroxyl group by other polar groups to prevent its phosphorylation, and this was of great importance in the design of novel SphK-1 inhibitors. The effect of the nitro group in compounds 47 and 48 was the most favorable for activity and led to enhancement of both the binding free energy (Table 1) and the hydrogen bonding. In addition, substitution of the pyrazole ring with piperidine in compound 48 instead of morpholine in compound 47 decreased the inhibitory activity and the IC 50 (Table 3).

Conclusion
Looking at the major challenges involves in the synthesis of novel inhibitors of SphK-1, the current study is a rst step towards the identi cation of different pyrazolylbenzimidazoles that could be useful in the development of potent SphK-1 inhibitors. A new series of benzimidazole derivatives 26-48 was rationally designed and synthesized. Among the studied compounds, compounds 47 and 48 showed an effective binding a nity to the SphK-1 and signi cantly inhibited SphK-1. Also, the synthesized molecules were evaluated by the NCI DPT for testing their antiproliferative activity on a panel of 60 cell lines, compounds 47 and 48 exhibited an effective cytotoxic activity against several NCI cell panels. Based on molecular-docking study, most of these synthesized compounds as 47 and 48 occupy the ATPbinding pocket and existing the deep pocket of SphK1 forming several important non-covalent and hydrogen bonding interactions with the active site residues. A considerable correlation was noticed between the docking results and the IC 50 values and its worth to mention that, compounds 47 and 48 act as promising inhibitors of SphK1 with low IC 50 values in the single-digit.

Material And Methods
Luria broth and Luria agar were purchased from Himedia(Mumbai, India). Plasmid pET28b+, DH5α, and BL21-Gold cells were procured from Invitrogen (USA). Ni-NTA column was purchased from GE Healthcare (GE Healthcare Life Sciences, Uppsala, Sweden). N-Lauroyl sarcosine, Tris buffer, DMSO, and other reagents were purchased from Sigma Aldrich (St. Louis, MO, USA). BIOMOL ® was obtained from Enzo (New York, USA). All the reagents used for buffer and chemical preparation were of analytical grade. Microanalyses and spectral data of the compounds were performed in the Microanalytical center at National Research Centre, and pharmaceutical faculty, Cairo University, Egypt, and Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) − Helmholtz Centre for Infection Research (HZI), Saarbrücken, Germany . The IR spectra (4000-400cm -1 ) were recorded using KBr pellets in a Jasco FT/IR 300E Fourier transform infrared spectrophotometer on a perkin-Elemer FT-IR 1650 spectrophotometer. The 1 HNMR spectra were recorded using 500 MHz and 400 MHz NMR spectrometer. Chemical shifts are reported in parts per million (ppm) from the tetramethylsilane resonance in the indicated solvent. Coupling constants (J) are reported in Hertz (Hz), and integration (where applicable); spectral splitting patterns are designed as follow: singlet (s); doublet (d); triplet (t); quartet (q); multiplet (m) and broad singlet (brs). The samples were referenced to the appropriate internal non-deuterated solvent peak. The data is given as follows: chemical shift (δ) in ppm, multiplicity (where applicable). The mass spectra were recorded using a Finnigan mat SSQ 7000 (Thermo. Inst. Sys. Inc., USA) spectrometer at 70 ev. Chromatography solvents were HPLC grade and were used without further puri cation. Thin-layer chromatography (TLC) analysis was performed using Merck silica gel 60 F-254 thin layer plates. Starting materials, reagents, and solvents for reactions were reagent grade and used as purchased. The petroleum ether had a boiling temperature in the 60-80 °C range.

Chemistry
General Procedure for the Synthesis of compounds 26-48. A suspension of benzene-1,2-diamine derivatives 22-25 (9.2 mmol) and sodium metabisul te (7 g, 36.8 mmol), dissolved in absolute ethanol (40 mL) was added to a solution of compounds 15-21 [53][54][55] 9.2 mmol) dissolved in absolute ethanol (30 mL). The mixture was stirred for 6-10 h and monitored by TLC. The reaction mixture was poured onto crushed ice; the resulting precipitate was collected by ltration, dried, and recrystallized from ethanol to give compounds 26-48. (26). Molecular-docking studies of the newly synthesized compounds with SphK1 were performed to gain insight into the predicted binding a nity and interaction patterns. The 2D and 3D structures of the synthesized compounds were generated using the Chem Draw Ultra v12.0. AutoDock Tools [66] was used for the preparation of docking les supported by AutoDock Vina [66] . In this study; we have performed site speci c molecular docking. The docking was done within 15 Å diameters from the reference PF-543 ligand. The binding site of the crystal structure of SphK1 is composed of the following amino acids: Leu167, Ser168, Ala170, Phe173, Ile174, Val177, Asp178, Phe192, Thr196, Leu259, Leu261, Leu268, Ala274, Phe288, Val290, Leu302, Phe303, Met306, His311, and Ala339. The binding site was de ned by including all residues constituting the binding pocket of reference PF-543 ligand.
AutoDock Vina was used for running molecular docking. The docked poses of the newly synthesized compounds with SphK1 were ranked based on the predicted binding a nity and interaction patterns. Intermolecular interactions were studied using PyMol molecular [67] The 2D plots for protein ligand interaction were created using the Discovery Studio Visualizer. The top-ranked compounds selected from the analysis are listed in Table 2.

Expression and puri cation of SphK1
The secondary cultures of SphK1 were induced by 1 mM IPTG for 4 h followed by centrifugation at 7000 rpm for 15 minutes to get the cell pellet, which was later resuspended in the lysis buffer and inclusion bodies were prepared as described [57] . Finally, inclusion bodies were solubilized in the solubilization buffer (pH 8.0) comprising 0.5% sarcosine, 50 mMTris and 150 mMNaCl. SphK1 was puri ed using Ni-NTA a nity chromatography, followed by dialysis for 24 h to get the refolded native protein. The puri ed protein was loaded on SDS-PAGE and the concentration was calculated using a molar absorption coe cient of 48275 M −1 cm −1 at 280 nm on the Jasco V-660 UV-visible spectrophotometer.

Fluorescence binding studies
The Jasco Spectro uorometer at 25 °C was used for the binding studies of all the synthesized compounds. The compounds were rst dissolved in DMSO to get the 20 mM stock solution and then diluted to a working concentration of 1mM in 20 mMTris and 100 mMNaCl buffer (pH 8.0). The quenching studies were performed with a xed concentration of SphK1 (5μM) and the compounds were added gradually in increasing concentration from the 1 mM stocks into the protein solution until the achievement of saturation point. The emission spectra were recorded from 300-400 nm with excitation of SphK1 at 280 nm. The blank titrations (buffer with selected compounds) were subtracted to obtain the nal spectra and the quenching data was corrected for the inner lter effect according to the formula, F = F obs antilog [(A ex + A em )/2], where A ex and A em is the absorbance of the selected compound at the excitation and emission wavelength respectively [68]. The quenching spectra obtained for selected compounds were plotted and the inverse correlation between the gradual decrease in the uorescence intensity with increasing concentration of compounds was used for determining the kinetic parameters (K a and n) from a modi ed Stern-Volmer equation (Equation (1) as described [69] where F o denotes uorescence intensity of SphK1 without the compound and F denotes the uorescence intensity of SphK1 at a speci c concentration of compound at λ max .

Enzyme inhibition assay
A standard Malachite Green (BIOMOL ® GREEN reagent) microtitre-plate assay was performed to evaluate the inhibitory potential of all the synthesized compounds against SphK1. Brie y, compounds were incubated with SphK1 (4 μM) for 1 h at 25 °C and then later freshly prepared ATP (200 μM) and 10 mM MgCl 2 were added to the protein-ligand mixture.