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

Sphingosine-1 kinase (SphK1) is one of the important enzymes of phospholipids and its inhibition is one of the therapeutic strategies for different diseases. SphK1 over expression is observed in different types of cancer indicating its important role in tumor growth. In search of effective SphK1 inhibitors, a new series of pyrazolylbenzimidazoles was synthesized and evaluated as sphingosine kinase-1 (SphK1) inhibitors. In order to evaluate the binding affinities of all the synthesized compounds, all compounds were subjected to docking analysis and fluorescence quenching. The results indicated that there is a consistency between the docking and the fluorescence quenching results, which revealed that compounds 47 and 48 exhibited significant decrease in the fluorescence intensity of SphK1 as well as they formed stable protein–ligand complexes. In addition, enzyme inhibition assay was performed which showed effective inhibitory potential toward SphK1. Moreover, IC50 values 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 and growth inhibitory potential toward cancer cell lines. Most of these compounds fit well into the ATP-binding site of SphK1 and form significant 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.


Introduction
Sphingolipid is a kind of phospholipid, which is a major component of all cell membranes and can form lipid bilayers that maintain the fluidity of membranes [1]. Ceramide (Cer), sphingosine (Sph), and sphingosine-1 phosphate (S1P) are metabolites of sphingomylein, which play an important role in different diseases such as cancer [2,3], fibrosis [4], and Alzheimer's disease [5]. The balance between the sphingolipid metabolites, which act in two opposite ways, is crucial in the determination of the cell fate [6,7]. Cer and Sph [8,9] are a proapoptotic molecule to mediate the cell cycle and induce apoptosis, while S1P promotes cell proliferation and acts as a "pro-survival" molecule [10,11]. Phosphorylation of the proapoptotic D-erythro-Sph to the promitogenic S1P is catalyzed by Sphingosine kinases (SphK). The two SphK isoforms (SphK1 and SphK2) are known to catalyze this transformation and regulate the sphingolipid metabolism. In particular, SphK1 is more closely linked in many diseases such as cancer, rheumatoid arthritis, diabetes, asthma, and pulmonary fibrosis [12][13][14]. Studies have shown that over expression of SphK1 is observed in many tumor tissues and regulating tumorigenesis, angiogenesis, and chemotherapy resistance, which play an important role in cancer progression [15][16][17][18][19][20]. Hence, inhibition of SphK1 is considered a new therapeutic strategy in the treatment of metastatic cancer and other diseases [21,22].
The evolution of SphK1 and SphK2 inhibitors has been recently reported [50,51]. Many SphK inhibitors were designed to have a polar head group and a lipophilic tail region. Studying all structural variations in the Sph-based SphKIs and their resulting biological effects in the earlier work, several points were 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 profile. (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 Sph-based SKIs. Modification 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).

Chemistry
The present study aimed to synthesize new benzimidazole candidates as Sph kinase-1 inhibitors (Fig. 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 first 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-phenyl-pyrazol-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 Prajuli et al. [52]. The Vilsmeier-Haack reaction of the previous step afforded 5-chloro-3-methyl-1-phenylpyrazole-4-carboxaldehyde 14, which followed by introduction of nucleophiles i.e., secondary amines or phenol derivatives to give compounds 15-21 (Scheme 1) [52][53][54][55]. The second part of this synthesis was the coupling of pyrazole-4carboxaldehydes 15-21 with 1,2-benzendiamine 22, 3,4diaminobenzoic acid 23, 4-methylbenzene-1,2-diamine 24, or 4-nitrobenzene-1,2-diamine 25 to afford different derivatives 26-48 (Scheme 2). Based on the predicted binding affinities and interactions, compounds 47 and 48 were selected as top-scoring compounds and modification 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-analogs.

Fluorescence binding studies
Fluorescence binding studies were performed for evaluating the binding affinity of all the synthesized compounds 26-48 with SphK1. The gradual loss in the fluorescence intensity upon addition of the selected compounds, 47 and 48 (Figs. 4A, B), was observed for SphK1, which points toward 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 fluorescence intensity was observed when added to protein samples in increasing concentrations (Fig. S24-27). The Stern-Volmer plot (Fig. 4C, D) was used to analyze the quenching data to determine the binding affinity (K a ) for each compound. The number of binding sites per SphK1 molecule (n) for these compounds was also determined  (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 of compounds 26-48 toward SphK1 was 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). Further, the IC 50 values of the synthesized compounds that showed good binding affinity toward the SphK1 were evaluated and found to be in the micromolar 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 Fig. 5A, 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 [56][57][58][59][60][61][62][63]. The loss in the SphK1 activity followed an inverse relationship between percentage inhibition and an increasing concentration of selected compounds as shown in Fig. 5C, D, which was used for the calculation of IC 50 values ( Anticancer activity against NCI-60 cell line panel Most of the synthesized compounds were screened for their in vitro antitumor activity by the Developmental Therapeutics Program of the National Cancer Institute (NCI) in the division of cancer treatment and diagnosis, NIH, Bethesda, Maryland, USA. This involves screening of the compounds at a single dose of 10 µM against a full NCI-60 cell panel including leukemia, lung, colon, brain, melanoma, ovary, kidney, prostate, and breast cancers [66]. From the obtained results in Table 4, it is obvious that each of the screened compounds has a different degree of selectively against 60 cell lines. K-562, MOLT-4, PRMI-8226, and SR from leukemia; SNB-75 from CNS Cancer; UACC-62 from melanoma; A498, ACHN, CAKI-1, and UO-31 from renal cancer; PC-3 from prostate cancer; and T-47D and MDA-MB-468 from breast cancer are the most sensitive cell lines to the tested compounds. The studied compounds showed a broad spectrum of anticancer activity against several NCI cell panels. At 10-µM concentration, compounds 47 and 48 showed potent inhibition against the leukemia and breast cancer cell lines (Table 4).

Molecular docking
The molecular-docking study of the designed compounds with SphK1 was performed using the AutoDock vina tool [67]. Vina gives the predicted binding poses of the synthesized compounds 26-48 along with the binding affinities in kcal/mol. Based on the predicted binding affinities and interactions, compounds 47 and 48 were selected as topscoring compounds. The predicted binding affinities 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 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 that compound 47 has strongly occupied the binding cavity of the protein (Fig. 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 (Fig. 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 profile of all the synthesized compounds with SphK1 is given in

Structure-activity relationship
Structure-activity relationships were illustrated from the previous results. Concerning the percent of inhibition of 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-analogs. The unsubstituted benzimidazoles and 5-methylbenzimidazoles were synthesized as was predicted from the docking studies that they might possess good binding affinity 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 was 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 SphK1 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 SphK1, the current study is a first step toward the identification of different pyrazolylbenzimidazoles that could be useful in the development of potent  (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 are 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 purification. 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.

Expression and purification of SphK1
The secondary cultures of SphK1 were induced by 1-mM IPTG for 4 h followed by centrifugation at 7000 rpm for 15 min to get the cell pellet, which was later resuspended in the lysis buffer and inclusion bodies were prepared as described [56]. Finally, inclusion bodies were solubilized in the solubilization buffer (pH 8.0) comprising 0.5% sarcosine, 50-mM Tris, and 150-mM NaCl. SphK1 was purified using Ni-NTA affinity chromatography, followed by dialysis for 24 h to get the refolded native protein. The purified protein was loaded on SDS-PAGE and the concentration was calculated using a molar absorption coefficient of 48275 M −1 cm −1 at 280 nm on the Jasco V-660 UV-visible spectrophotometer.

Fluorescence binding studies
The Jasco Spectrofluorometer at 25°C was used for the binding studies of all the synthesized compounds. The compounds were first dissolved in DMSO to get the 20-mM stock solution and then diluted to a working concentration of 1 mM in 20-mM Tris and 100-mM NaCl buffer (pH 8.0). The quenching studies were performed with a fixed concentration of SphK1 (5 μM) 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 final spectra and the quenching data were corrected for the inner filter 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 [69]. The quenching spectra obtained for selected compounds were plotted and the inverse correlation between the gradual decrease in the fluorescence intensity with increasing concentration of compounds was used for determining the kinetic parameters (K a and n) from a modified Stern-Volmer equation (Eq. (1) as described [70] where F o denotes fluorescence intensity of SphK1 without the compound and F denotes the fluorescence intensity of SphK1 at a specific concentration of compound at λ max . log 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. Briefly, compounds were incubated with SphK1 (2 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. The reaction was allowed to proceed for 30 min at 25°C. After the required incubation period, the reaction was ended by adding the double amount of BIOMOL ® reagent. Finally, a green-colored complex was formed in 10 min and the absorbance readings were recorded on ELISA reader at 620 nm. The reaction with ligands and no protein was also performed to subtract the background reading of inorganic phosphate. A standard phosphate curve was used to determine the loss in activity of SphK1 in terms of amount of phosphate released on treatment with increasing concentrations of selected compounds. The inhibition in SphK1 activity was plotted for selected compounds in terms of percentage as described [56].