Design, molecular modelling and synthesis of novel benzothiazole derivatives as BCL-2 inhibitors

Apoptosis plays a crucial role in cancer pathogenesis and drug resistance. BCL-2 family of enzymes is considered as one of the key enzymes which is involved in apoptosis. When there is disruption in the balance between anti-apoptotic and pro-apoptotic members of the BCL-2 family apoptosis is dysregulated in the affected cells. Herein, 33 novel benzothiazole-based molecules 7a-i, 8a-f, 9a-b, 12a-e, 13a-d, 14a,b, and 17a-j were designed, synthesized and tested for their BCL-2 inhibitory activity. Scaffold hopping strategy was applied in designing of the target compounds. Compounds 13c and 13d showed the highest activity with IC50 values equal to 0.471 and 0.363 µM, respectively. Molecular docking studies of the synthesized compounds showed comparable binding interactions with the lead compound. Structure activity relationship study was performed to show the effects of structural modifications on the inhibitory activities on BCL-2.

proteins 18,19 .If there is imbalance between the anti-apoptotic and pro-apoptotic members of the BCL-2 family, it will result in dysregulated apoptosis in the affected cells.This could attributed to the expression of one or more anti-apoptotic proteins or the low expression of one or more pro-apoptotic proteins or a combination of both 20 .
The selective modulation of both apoptotic pathways has been reported to be a challenge in cancer drug development.BCL-2 functions by preventing programmed cell death which differs from other oncogenes that work mainly by enhancing proliferation 21 .Therefore, inhibiting these proteins would lead to apoptosis induction which could provide potential therapeutic target 22 .However, the critical challenge is that many of these targets are protein-protein interactions and it is hard to modulate.Despite this, there are various promising molecules which target the apoptotic pathway components that have now entered the clinic, and are under investigation as single agents or in combination with other anticancer therapies 23 .Anti-sense oligonucleotides (ASOs), peptides and peptidomimetics as well as small molecules (BH3 mimetics) are among the promising BCL-2 Inhibitors.Despite the emergence of various BCl-2 inhibitors, there are many challenges that are facing these inhibitors, including the development of resistance and limitation of their use in certain types of tumors, e.g., solid tumors.These challenges encourage many researchers to search, design and synthesize novel BCl-2 inhibitors with better activity, less resistance and safer profile 24 .

Rationale and design
Our proposed design of novel benzothiazole based inhibitors was according to the strategies shown in (Fig. 2).Scaffold hopping strategy has been used for designing structurally novel compounds by modifying the central core of an active molecule.Thus, the saccharine scaffold in the lead compound 2 has been replaced by benzothiazole scaffold.The introduction of heterocyclic core will lead to increased acidity of the sulfonamide NH which is proposed to influence potency, solubility, and clearance of these inhibitors.Additionally, the benzothiazole scaffold will maintain the proper orientation of the two essential hydrophobic pocket binding moieties of the compound.The sulfonamide moiety has been retained in most of proposed compounds.In other compounds, it has been replaced by its amide bioisostere to maintain crucial hydrogen bond interactions.Furthermore, a number of diverse small and large hydrophobic moieties with different substituents have been utilized, which maintain the reported hydrophobic interaction with P2 pocket, to explore the hydrophobic moiety with optimum BCL-2 inhibitory activity.Similarly, based on the reported hydrophobic groups used as the P4 binding moieties, the naphthyl (as in compound 1) and 3-nitro-4-(phenethylamino)benzene (to mimic the P4 hydrophobic moiety in compound 2) have been used to maintain desirable hydrophobic interaction with P4 pocket and to explore the hydrophobic moiety with optimum BCL-2 inhibitory activity.Finally, the piperazine linker between the core scaffold and the P2 hydrophobic moiety has been retained in some proposed compounds or replaced by other linkers such as urea and amide linkers in other compounds to explore their impact on BCL-2 inhibitory activity.
The aim of the current study is to rationally design, synthesize and biologically evaluate novel small molecule BCL-2 inhibitors (BH3 mimetics) via targeting the BH3 binding groove of anti-apoptotic BCL-2 protein as targeted anti-cancer therapy.

Chemistry.
Starting materials and reagents were purchased from Sigma-Aldrich or Alfa-Aesar Organics and used without further purification.Reactions were monitored by analytical TLC, performed on silica gel 60 F 254 packed on Aluminum sheets, purchased from Merck, with visualization under UV light (254 nm).Melting points were recorded on Stuart Scientific apparatus and are uncorrected.Electron-impact ionization mass (EI-MS) spectra were recorded on Thermo Scientific ISQLT mass spectrometer at the Regional Center for Mycology and Biotechnology, Al-Azhar University. 1 H-NMR spectra were recorded using Bruker 400 MHz spectrophotometer (using DMSO as solvent) at Center for Drug Discovery Research and Development, Ain Shams University. 13C -NMR spectra were recorded in δ scale given in ppm on a Bruker 100 MHz spectrophotometer (at 101 MHz, using DMSO as solvent) at Center for Drug Discovery Research and Development, Ain Shams University.Elemental analyses were performed on a Thermo Scientific Flash 2000 elemental analyzer at the Regional Center for Mycology and Biotechnology, Al-Azhar University.
For the detailed synthetic procedures and structural characterization, refer to the supporting information.Assay protocol.The assay was performed by TR-FRET technology using a recombinant BCL-2 and a peptideligand substrate.The TR-FRET signal from the assay is correlated with the amount of Ligand binding to BCL-2.Compounds were diluted in 100% DMSO then tenfold dilution in 10% DMSO in 1X Reaction Buffer. 2 µl of the dilution was added to a 20 µl reaction so that the final concentration of DMSO is 1% in all of reactions.All of the binding reactions were conducted at room temperature.The 20 µl reaction mixture in Assay Buffer contains bcl-2, the indicated amount of the inhibitor, ligand, and the reaction dyes.The reaction mixture incubated for 180 min prior to reading the TR-FRET signal.For the background, ligand was replaced with assay buffer.Fluorescence signals for both the donor and acceptor dyes were measured using a Tecan Infinite M1000 plate reader.TR-FRET was recorded as the ratio of the fluorescence of the acceptor and the donor dyes (acceptor/donor).

Biological evaluation.
Binding experiments were performed in duplicate at each concentration.The TR-FRET data were analyzed using the computer software, Graphpad Prism.In the absence of the compound in wells containing BCL-2 ligand, the TR-FRET signal (Ft) in each data set was defined as 100% activity.In wells without peptide ligand, the TR-FRET signal (Fb) in each data set was defined as 0% activity.The percent activity in the presence of each compound was calculated according to the following equation: % activity = [(F − Fb)/ (Ft − Fb)] × 100, where F = the TR-FRET signal in the presence of the compound.The percent inhibition was calculated according to the following equation: % inhibition = 100-% activity.IC 50 determination for target where, Y = percent activity, B = minimum percent activity, T = maximum percent activity, X = logarithm of compound and Hill Slope = slope factor or Hill coefficient.The IC 50 value was determined by the concentration causing a half-maximal percent activity 30 .
In vitro anti-proliferative activity against NCI leukemia cell line.The national cancer institute (NCI) in vitro anticancer screening is a two-stage process, beginning with the evaluation of the selected compounds 13c and 13d against NCI leukemia cell line at a single dose of 10 µM.The human leukemia cell line was obtained and maintained at the NCI.The output from the single dose screen is reported as a mean graph.The detailed assay protocol is discussed in the supporting material.
Molecular modelling study.Molecular docking was conducted using C-Docker 2.5 software in the interface of Accelry's discovery studio 2.5 (Accelrys Inc., San Diego, CA, USA) at the drug design laboratory of Faculty of pharmacy, Ain Shams university.The X-ray crystal structure of BCL-2 co-crystallized with saccharinebased lead compound 2 was obtained from the Protein Data Bank 31 with PDB code: 4IEH 29 .This PDB code was chosen to compare the docked poses of our designed compounds to the lead compound 2. Protein was prepared in Discovery Studio 2.5 by deleting water molecules and adding hydrogen atoms.A final step of energy minimization for hydrogen atoms was done to relieve the constraints resulting from randomly adding hydrogen atoms with constraining protein heavy atoms.Docking search space was identified by assigning a sphere centred around the co-crystallized ligand including the surrounding amino acids of the binding site.The docking sphere had a centre at coordinates 12.

Results and discussion
Chemistry.The titled compound (5) was prepared in good yield and purity via nucleophilic substitution of the 2-aminobenzothiazole derivatives (4) on 2-naphthylsulfonyl chloride in dry pyridine.
Compound 6 served as a crucial starting point for the synthesis of series 1, 3 and 5 (scheme 2). the titled compounds, series 1, (7a-i) were prepared by direct coupling of equimolar amounts of the carboxylic acid ( 6) and the respective substituted piperazine in presence of EDC.HCl as coupling agent/ DMAP as a base in dry DMF under N 2 atmosphere 34 .Series 3, compounds (8a-f), was prepared though a similar pattern as compounds 7a-i, by the direct coupling of equimolar amounts of the carboxylic acid (10) and the respective amino compounds (Ia-f, Fig. 3) using EDC.HCl as coupling agent/ DMAP as a base in dry DMF under N 2 atmosphere 34 .Using the same coupling procedure 34 , compound (6) and the respective amino compounds (IIa,b, Fig. 3) were reacted yielding 9a,b, series 5, Fig. 4.
According to Fig. 6, compound 11 is considered very important starting building block for the synthesis of series 2, 4 and 6.Compound lists 12a-e, 13a-d and 14a,b were obtained using the same coupling procedure using compound 11 and respective substituted piperazines, amino compounds (Ia-f) and (IIa,b) respectively.
Finally, Fig. 7 illustrates the synthesis of the final compounds 17a-j, series 7. Compound 15 was synthesized according to the reported method where it was produced in good yield and purity 35 .Furthermore, nitro group in compound 15 was reduced following the reported procedure 35 .Compound 16 served as crucial starting material to obtain compounds 17a-j, where it reacted with the respective isocyanate in THF 36 .The urea derivatives 17a-j www.nature.com/scientificreports/were obtained in good yield and purity.The structures of all the newly synthesized compounds were confirmed by their spectral data.

Biological evaluation. Initial in vitro BCL-2 inhibitory screening at single dose of 10 µM concentration.
Our novel synthesized compounds were screened for their potential BCL-2 anti-apoptotic effects.The in vitro BCL-2 inhibition assay was executed at the BPS Bioscience Corporation, San Diego, CA, USA.Table 1 and Fig. 8 illustrate the inhibitory effects of the compounds against BCL-2 protein, which happened to be either very poor or good inhibitory activity.

Measurement of potential enzyme inhibitory activity (IC 50 ).
Promising candidates, which exhibited BCL-2 inhibition percent above 60% at 10 µM concentration (8d, 13b-d & 17f) were further investigated for their doserelated BCL-2 inhibitory activity at 5 different concentrations (1 nM-10 nM-100 nM-1 µM-10 µM) to subsequently calculate their IC 50 values, Table 2.  www.nature.com/scientificreports/ In vitro anti-proliferative activity against leukemia NCI cell lines.This assay was performed for compounds 13c and 13d by the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI), division of cancer treatment and diagnosis, NIH, Bethesda, Maryland, USA (www.dtp.nci.nih.gov).The operation of this screen utilizes leukemia human tumor cell line 37 .The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine.
Results for the two compounds were reported as a mean graph of the percent growth of the treated cells when compared to the untreated control cells.The results are expressed as cell growth percent for the tested compounds on each of the used NCI cell line panels.Table 3 shows the results for the compounds 13c and 13d.
Compound 13d, which demonstrated the highest inhibitory activity against BCL-2 with IC 50 value of 0.363 µM, exhibited moderate inhibition against leukemic SR cell lines with growth inhibition 62.87.While compound 13c exhibited poor inhibition against the used leukemia cell line.

Molecular modelling study.
The validity of the proposed design was evaluated by molecular docking study through predicting the binding modes and binding affinities of the designed compounds.These predicted properties should meet the minimum desirable requirements to validate the hypothesis that the designed  BCL-2 inhibiƟon %   compounds have proposed biological activities against BCL-2.The docking study was performed in order to test for a comparable binding mode to the saccharine lead compound 2 and investigate the binding affinities of the designed compounds to BCL-2 binding groove.The major goal of a good docking protocol is to discriminate between the groups of true solutions (proposed poses), usually defined as poses within 2.0 Å root mean square deviations (RMSD) from the X-ray geometry, and false solutions or misdocked structures 38 .In order to validate the C-DOCKER protocol's predictability of the correct poses, we re-docked the co-crystallized ligand using C-DOCKER, and aligned the pose retrieved from docking to the X-ray geometry (The co-crystallized bioactive conformation) to calculate the RMSD.The docking of this compound in BCL-2 binding groove generated RMSD of 0.6656 Å (Fig. 10), therefore, the C-DOCKER protocol is reliable for predicting the poses of the tested compounds in the X-ray crystal structure of BCL-2 protein.
The docking study of the synthesized compounds into BCL-2 binding groove revealed comparable binding modes of the docked molecules to the lead compound.Docking of the target compounds showed that the core scaffold adopted volumes and orientation as the lead compound.The docking results showed the designed compounds to have C-Docker interaction energies ranging from − 67.57 to − 40.31 kcal/mole which came comparable to the docking score of the co-crystallized ligand with docking score of − 71.89 kcal/mol.The docking score of the 5 most active compounds come in a good agreement with their IC 50 , where they are ranked in the same order according to the IC 50 order.They are arranged from the most active to the least active as follows: 13d > 13c > 13b > 8d > 17f, as shown in Table 4.
P-substituted-benzyloxyphenyl derivatives (series 3 & 4), exhibited the best BCL-2 inhibitory activity as they fulfilled most of the key interactions with BCL-2, where the key hydrogen bonds with Gly 104 and Arg 66 residues were established (as seen in compounds 13c & 13d, which are the most active compounds against BCL-2), as well as a pi-pi interaction with Tyr67 and Tyr161.Also, pi-alky interactions were observed with various amino acid residues (Arg 98, Ala 59 & Pro163) resulting in better docking scores as well, which could explain the good BCL-2 inhibitory activity of those compounds (Fig. 11A and B).
Regarding the moderately active compounds (8a,c,d, 13b, 14b & 17f), they showed the same binding mode and interactions as the most active compounds (13c & 13d) but they missed a key HB with Gly 104 which could explain its moderate activity (Fig. 12).However, they formed another H-bond with Arg 105 residue compensating the loss of the main HB with Gly 104 (Table S1).
On the contrary, piperazine derivatives (series 1 & 2) failed to exhibit BCL-2 inhibitory activity as they missed the key interaction with Gly104 residue which seemed to be an essential feature for BCL-2 inhibitory activity, although they maintained the same binding mode and the same key hydrogen bond with Arg 66 and hydrophobic interactions (Pi-Pi) with Tyr 67, Tyr 161.Generally, most of inactive compounds missed this key hydrogen bond interaction with Gly 104 residue.Moreover, they showed lower docking scores relative to that of the docked lead    S1 in supporting info.
Physicochemical and drug likeness properties.The insilico physicochemical properties of the reported compounds were calculated using SwissADME web server.Six parameters that detect the oral bioavailability were calculated namely number of rotatable bonds, Solubility Log S, fraction of sp3 carbons, Topological polar surface area (TPSA), molecular weight (MW), and XLOGP3 for lipophilicity, as shown in Fig. 13.Most of the compounds showed acceptable number of rotatable bonds (< 9), TPSA (< 130 Å 2 ), and sp3 carbon fraction (< 1).Most of the compounds showed moderate water solubility (ESOL Log S < -6), slightly high molecular weight (> 500), and high lipophilicity (XLOGP3 > 5),.These results gave a basis for further drug development for the reported series to adjust the lipophilic/hydrophilic balance, solubility, and molecular weight.The drug likeness for the reported compounds was also predicted using SwissADME web server by calculating five filters namely, Lipinski, Ghose, Veber, Egan, and Muegge.The results were reported by the number of violations each compound commits per filter in Table 5.The results are depicted as a heat map.Most compounds fulfil all the filters except for the Ghose filter which come from higher molecular weight and molar refractivity.This also can be exploited in prospective drug development projects by adjusting the molecular weight of the designed compounds.

Figure 1 .Figure 2 .
Figure 1.The common structural features and the possible key interactions of prominent BCL-2 inhibitors with BCL-2 protein.Figure 1 was generated using Chemdraw Professional 16.0.

Figure 3 . 2 - 4 Figure 4 .Figure 5 .
Figure 3. Structures of reported intermediates Ia-f and IIa,b used in the synthesis of the final compounds.

Figure 8 .
Figure 8. Percent inhibition of BCL-2 inhibitory activity achieved by the newly synthesized compounds.

Figure 9 .
Figure 9. Structure activity relationship diagram showing the most potent compound 13.

Figure 10 .
Figure10.The alignment between the co-crystallized bioactive conformer of compound (blue) and the pose of the same compound retrieved from docking using CDOCKER (orange).Figure10was generated using Discovery studio 2.5.

Figure 10
Figure10.The alignment between the co-crystallized bioactive conformer of compound (blue) and the pose of the same compound retrieved from docking using CDOCKER (orange).Figure10was generated using Discovery studio 2.5.

Table 4 .Conclusion
Docking scores, 2D interaction diagrams, and interacting amino acids with compounds 8d, 13b-d and 17fOur main approach was to design new small molecules as BCL-2 inhibitors with potential anti-cancer activity based on literature review and SAR studies, novel benzothiazole-based compounds were designed, synthesized and evaluated for their in vitro BCL-2 inhibitory activity.The 4-CH 3 and 4-CF 3 benzyloxy derivatives (13c & 13d) having 3-nitro-4-phenethyl amino moiety as the P4 hydrophobic binding group, exhibited the highest inhibitory activity against BCL-2 protein with IC 50 values of 0.471 and 0.363 µM, respectively.The designed and synthesized compounds could act as a potential cornerstone for future design of more potent and selective BCL-2 inhibitors and hence promising anticancer agents.
2, 25.8, 11.8, and a radius of 60 Å.Ligands were sketched in ChemDraw and minimized in Discovery Studio 2.5 using CHARMm force field adjusting the ionization pH at 7.4, with no isomers or tautomers generated for the ligands.Figures were generated using UCSF Chimera 32 and Discovery Studio 2.5.Physicochemical and drug likeness properties are predicted using SwissADME webserver (https:// www.nature.com/ artic les/ srep4 2717).

Table 2 .
The IC 50 values for compounds 8d, 13b-d and 17f.Significant values are in bold.

Table 3 .
Cell growth percentage of NCI leukemia cancer cell line exhibited by the final compounds 13c and 13d.Significant values are in bold.