Investigation of NPB Analogs That Target Phosphorylation of BAD-Ser99 in Human Mammary Carcinoma Cells

The design and development of a small molecule named NPB [3-{(4(2,3-dichlorophenyl)piperazin-1-yl}{2-hydroxyphenyl)methyl}-N-cyclopentylbenzamide], which specifically inhibited the phosphorylation of BAD at Ser99 in human carcinoma cells has been previously reported. Herein, the synthesis, characterization, and effect on cancer cell viability of NPB analogs, and the single-crystal X-ray crystallographic studies of an example compound (4r), which was grown via slow-solvent evaporation technique is reported. Screening for loss of viability in mammary carcinoma cells revealed that compounds such as 2[(4(2,3-dichlorophenyl)piperazin-1-yl][naphthalen-1-yl]methyl)phenol (4e), 5[(4(2,3-dichlorophenyl)piperazin-1-yl][2-hydroxyphenyl)methyl)uran-2-carbaldehyde (4f), 3[(2-hydroxyphenyl][4(p-tolyl)piperazin-1-yl)methyl)benzaldehyde (4i), and NPB inhibited the viability of MCF-7 cells with IC50 values of 5.90, 3.11, 7.68, and 6.5 µM, respectively. The loss of cell viability was enhanced by the NPB analogs synthesized by adding newer rings such as naphthalene and furan-2-carbaldehyde in place of N-cyclopentyl-benzamide of NPB. Furthermore, these compounds decreased Ser99 phosphorylation of hBAD. Additional in silico density functional theory calculations suggested possibilities for other analogs of NPB that may be more suitable for further development.


Results and Discussion
The NPB analogs were synthesized based on the Petasis borono−Mannich reaction using N-substituted-piperazines, salicylaldehyde, and various boronic acids as the nucleophilic reagent [17][18][19][20][21]. In this multicomponent reaction, the iminium ion formation occurs initially, which reacts with boronic acid to form a tetracoordinate boronate in situ intermediate, and eventually the product formation occurs by intramolecular delivery of the organic group to iminium carbon (Scheme 01). The structures of all NPB analogs were characterized by LCMS, 1 H NMR, and 13 C NMR spectroscopic techniques (Table 1, refer supplementary spectra of all the compounds). Scheme 1. Petasis reaction between substituted (R3) phenyl piperazine, various (R2) salicylaldehydes, and (R1) different boronic acids to obtain NPB analogs (4a-r).

Results and Discussion
The NPB analogs were synthesized based on the Petasis borono−Mannich reaction using N-substituted-piperazines, salicylaldehyde, and various boronic acids as the nucleophilic reagent [17][18][19][20][21]. In this multicomponent reaction, the iminium ion formation occurs initially, which reacts with boronic acid to form a tetracoordinate boronate in situ intermediate, and eventually the product formation occurs by intramolecular delivery of the organic group to iminium carbon (Scheme 1). The structures of all NPB analogs were characterized by LCMS, 1 H NMR, and 13  RAS/RAF/MAPK and PI3K/AKT/mTOR pathways, respectively [5]. In addition, all three PIM kinase family members may also phosphorylate hBAD on multiple sites but require prior phosphorylation of Ser-75 or Ser-99 and function as rescue kinases for BAD phosphorylation upon inhibition of RAS/RAF/MAPK and PI3/AKT/mTOR pathways [5][6][7][8].

Results and Discussion
The NPB analogs were synthesized based on the Petasis borono−Mannich reaction using N-substituted-piperazines, salicylaldehyde, and various boronic acids as the nucleophilic reagent [17][18][19][20][21]. In this multicomponent reaction, the iminium ion formation occurs initially, which reacts with boronic acid to form a tetracoordinate boronate in situ intermediate, and eventually the product formation occurs by intramolecular delivery of the organic group to iminium carbon (Scheme 01). The structures of all NPB analogs were characterized by LCMS, 1 H NMR, and 13 C NMR spectroscopic techniques (Table 1, refer supplementary spectra of all the compounds). Scheme 1. Petasis reaction between substituted (R3) phenyl piperazine, various (R2) salicylaldehydes, and (R1) different boronic acids to obtain NPB analogs (4a-r). Scheme 1. Petasis reaction between substituted (R3) phenyl piperazine, various (R2) salicylaldehydes, and (R1) different boronic acids to obtain NPB analogs (4a-r).      Furthermore, the crystal structure of an example compound was determined using a slow evaporation technique [22]. Compound 4r emerged as a crystal and therefore its Xray intensity data were recorded at a temperature of 293K on a Bruker AXS kappa Apex2 CCD Diffractometer, with a fine-focus sealed tube radiation source (MoKα) and 0.71073 Å wavelength. The procedure and reduction of the data set was accomplished using SAINT PLUS. SHELXS and SHELXL programs were adopted to solve and refine the structure, respectively [23,24]. The geometrical calculations, molecular figures, and crystal packing were generated and visualized by PLATON and MERCURY software, respectively ( Table 2) [25]. CCDC number 2027110 contains full crystallographic data of 4r and is available online at the Cambridge crystallographic data center. The ORTEP diagram was obtained for compound 4r (Figure 2a). The crystal packing of the structure (Figure  2b) revealed the importance of the oxygen atom in the hydroxy group (i.e., present in all 4a-s) and the chlorine atom in the para position of the phenyl ring (i.e., especially in the more potent compounds 4e and 4f), which confirms their participation in potential hydro- Furthermore, the crystal structure of an example compound was determined using a slow evaporation technique [22]. Compound 4r emerged as a crystal and therefore its Xray intensity data were recorded at a temperature of 293K on a Bruker AXS kappa Apex2 CCD Diffractometer, with a fine-focus sealed tube radiation source (MoKα) and 0.71073 Å wavelength. The procedure and reduction of the data set was accomplished using SAINT PLUS. SHELXS and SHELXL programs were adopted to solve and refine the structure, respectively [23,24]. The geometrical calculations, molecular figures, and crystal packing were generated and visualized by PLATON and MERCURY software, respectively ( Table 2) [25]. CCDC number 2027110 contains full crystallographic data of 4r and is available online at the Cambridge crystallographic data center. The ORTEP diagram was obtained for compound 4r (Figure 2a). The crystal packing of the structure (Figure  2b) revealed the importance of the oxygen atom in the hydroxy group (i.e., present in all 4a-s) and the chlorine atom in the para position of the phenyl ring (i.e., especially in the more potent compounds 4e and 4f), which confirms their participation in potential hydro- Furthermore, the crystal structure of an example compound was determined using a slow evaporation technique [22]. Compound 4r emerged as a crystal and therefore its Xray intensity data were recorded at a temperature of 293K on a Bruker AXS kappa Apex2 CCD Diffractometer, with a fine-focus sealed tube radiation source (MoKα) and 0.71073 Å wavelength. The procedure and reduction of the data set was accomplished using SAINT PLUS. SHELXS and SHELXL programs were adopted to solve and refine the structure, respectively [23,24]. The geometrical calculations, molecular figures, and crystal packing were generated and visualized by PLATON and MERCURY software, respectively ( Table 2) [25]. CCDC number 2027110 contains full crystallographic data of 4r and is available online at the Cambridge crystallographic data center. The ORTEP diagram was obtained for compound 4r (Figure 2a). The crystal packing of the structure (Figure  2b) revealed the importance of the oxygen atom in the hydroxy group (i.e., present in all 4a-s) and the chlorine atom in the para position of the phenyl ring (i.e., especially in the more potent compounds 4e and 4f), which confirms their participation in potential hydro- Furthermore, the crystal structure of an example compound was determined using a slow evaporation technique [22]. Compound 4r emerged as a crystal and therefore its X-ray intensity data were recorded at a temperature of 293K on a Bruker AXS kappa Apex2 CCD Diffractometer, with a fine-focus sealed tube radiation source (MoKα) and 0.71073 Å wavelength. The procedure and reduction of the data set was accomplished using SAINT PLUS. SHELXS and SHELXL programs were adopted to solve and refine the structure, respectively [23,24]. The geometrical calculations, molecular figures, and crystal packing were generated and visualized by PLATON and MERCURY software, respectively (Table 2) [25]. CCDC number 2027110 contains full crystallographic data of 4r and is available online at the Cambridge crystallographic data center. The ORTEP diagram was obtained for compound 4r (Figure 2a). The crystal packing of the structure (Figure 2b) revealed the importance of the oxygen atom in the hydroxy group (i.e., present in all 4a-s) and the chlorine atom in the para position of the phenyl ring (i.e., especially in the more potent compounds 4e and 4f), which confirms their participation in potential hydrogen bond formation via O and Cl atoms to form a three-dimensional supramolecular hydrogen-bonded network. This reflects the importance for compound stability and its role in interaction with other molecules. Such information is helpful in structure and activity relationships that provide a solid basis for structure-based optimization in the future design of further compounds. Structural analysis revealed that both phenyl and thiophene rings exhibited planar conformation, while the six-membered piperazine is in a chair conformation exhibiting puckering parameters: amplitude (Q) = 0.5535 Å, Theta = 4.61 • , and Phi = 154.0425 • . The planarity conformation of the phenyl and thiophene rings allows for the partial overlapping of aromatic rings, which play an important role in biological activity [26]. In addition, the hybridization of the C-C bond which is considered as one of the most important and common chemical elements, especially for organic connections, is usually formed by s and p orbitals of the second shell in carbon and lead to the formation of different bonds. Among several types of this hybridization, the 4r molecule exhibited sp 2 hybridization that formed with two single bonds and one double bond between three atoms showing a 120 • angle value between bonds. This type of hybridization was observed in phenyl and thiophene rings. On the other side, the piperazine ring exhibited sp 3 hybridization in which the carbon atom is bonded to four other atoms forming only a single bond. Here, 1s orbital and 3p orbitals in the same shell of an atom combine to form four new equivalent orbitals. The presence of different types of hybridization enhances the bond strength, stability, and reactivity of the molecule. sp 2 hybridization that formed with two single bonds and one double bond between three atoms showing a 120° angle value between bonds. This type of hybridization was observed in phenyl and thiophene rings. On the other side, the piperazine ring exhibited sp 3 hybridization in which the carbon atom is bonded to four other atoms forming only a single bond. Here, 1s orbital and 3p orbitals in the same shell of an atom combine to form four new equivalent orbitals. The presence of different types of hybridization enhances the bond strength, stability, and reactivity of the molecule.  Since NPB was previously reported to inhibit the viability of various carcinoma cells, its analogs were tested for their ability to inhibit mammary carcinoma cell viability using the reported protocol [27,28]. The results of the study revealed that the compounds such as 2[(4(2,3-dichlorophenyl)piperazin-1-yl][naphthalen-1-yl]methylphenol (4e), 5[(4(2,3-dichlorophenyl)piperazin-1-yl][2-hydroxyphenyl]methylfuran-2-carbaldehyde (4f), 3[(2-hydroxyphenyl)4(p-tolyl)piperazin-1-yl]methylbenzaldehyde (4i) as well as NPB inhibited the viability of MCF-7 cells with an IC 50 values of 5.9, 3.11, 7.68, and 6.53 µM, respectively (Table 1). Interestingly, the compounds 4e, 4f, 4i, and NPB inhibited the viability of normal breast cell MCF10A, with higher IC 50 values of 33.8, 61.4, 28.5, and 110.6 µM, respectively. The NPB analogs synthesized by adding newer substituents such as naphthalene and furan-2-carbaldehyde in place of N-cyclopentylbenzamide of NPB slightly enhanced the loss of cell viability in MCF-7 cells. It was important to note that the dichlorophenyl group in NPB and its analogs seems quite important; however, some of the tested NPB analogs, which were synthesized by replacing the dichlorophenyl, and N-cyclopentylbenzamide group of NPB with 4-p-tolyl-group and benzaldehyde substitution, also exhibited enhanced loss of cell viability. Among 4-chloro-phenyl group containing piperazine compounds, 4b and 4c showed better inhibitory effects on viability of MCF-7 cells with IC 50 values of 20.91 and 23.83 µM, whereas for the tolyl group added piperazine compounds such as 4l and 4m, IC 50 values were observed to be 63.66, and 45.73 µM, respectively.
As the tested NPB analogs produced loss of cell viability to variable extents in mammary carcinoma cells, in silico density functional theory calculations were performed in order to understand the structure activity relationship of NPB analogs against the loss of cell viability (Supplementary Figure S1). The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy values showed the electron donating and accepting ability of NPB analogs, respectively. Computed HOMO and LUMO eigen functions indicated that the donor-acceptor nature of NPB analogs localized with MOs at different regions (Figures 3 and 4). Therefore, the substitutions at both ends were observed to significantly alter the electronic density levels. The computed data indicated that IC 50 decreases with decreasing E HOMO for NPB analogs, which bear electronically similar functional groups. Calculated HOMO and LUMO values are in the range of −5 to −6.5 eV and −1.5 to 2.5 eV (Table 3). Molecular electrostatic potential shows the charge separation between the two ends within the molecule. Improved loss of cell viability is observed with NPB analogs which possess similar functional groups. Hence, the NPB analogs, which possess electron donor function as three separate classes, were analyzed. In the first set, amongst the tested compounds, i. e., 4a, 4b, 4c and 4r, the compound 4b has comparatively lower E HOMO of -6.16 eV and E LUMO of −2.70 eV values. Due to the relatively smaller HOMO-LUMO gaps, electronegativity and electrophilicity values, and chemical hardness of 4b, the lower the observed IC 50 in mammary carcinoma cells. In the second class of NPB analogs, namely 4d, 4e, 4f, 4g, and 4h, the activities are arranged in ascending order of E HOMO and E LUMO values, i.e., 4g < 4d < 4h < 4e < 4f, which is in accordance with their respective IC 50 values ( Figure 5). In the last class of tolyl group containing molecules such as 4i, 4j, 4k, 4l, 4m, 4n, 4o, 4p, and 4q, the molecule 4i found to be highly effective against MCF-7 cells, which possess lower electronic factors compared to the other similar functional group tagged molecules. DFT studies predicted the activity of the molecule in line with experimental predictions. Among the molecules studied here, 4f exhibits higher activity than other molecules.   Table 3. Computed E HOMO , E LUMO , HOMO-LUMO gap (∆), chemical hardness (η), electronegativity (χ), and electrophilicity index (ω) in eV for all the synthesized molecules.    As phosphorylation of hBAD at Ser-99 promotes cancer cell survival and NPB was reported to specifically inhibit BAD-Ser99 phosphorylation, western blot analysis was performed to evaluate the efficacy of the most active NPB analogs (4f, 4e, and 4i) on BAD-Ser99 phosphorylation in MCF-7 cells. All the tested compounds decreased the pBAD at Ser-99 without change in total BAD expression ( Figure 6). As phosphorylation of hBAD at Ser-99 promotes cancer cell survival and NPB was reported to specifically inhibit BAD-Ser99 phosphorylation, western blot analysis was performed to evaluate the efficacy of the most active NPB analogs (4f, 4e, and 4i) on BAD-Ser99 phosphorylation in MCF-7 cells. All the tested compounds decreased the pBAD at Ser-99 without change in total BAD expression ( Figure 6).
As phosphorylation of hBAD at Ser-99 promotes cancer cell survival and NPB was reported to specifically inhibit BAD-Ser99 phosphorylation, western blot analysis was performed to evaluate the efficacy of the most active NPB analogs (4f, 4e, and 4i) on BAD-Ser99 phosphorylation in MCF-7 cells. All the tested compounds decreased the pBAD at Ser-99 without change in total BAD expression ( Figure 6). Since the active compounds (4e, 4f, 4i, and NPB as a comparison) displayed efficacy against MCF-7 cells, the in silico ADMET properties (8 parameters) of these compounds were determined by using vNN-ADMET online platform [29]. The results are tabulated in Table 4. The in silico analyses of active compounds predicted that the compounds (4e, 4f, 4i) would not exhibit hepatotoxicity.  Since the active compounds (4e, 4f, 4i, and NPB as a comparison) displayed efficacy against MCF-7 cells, the in silico ADMET properties (8 parameters) of these compounds were determined by using vNN-ADMET online platform [29]. The results are tabulated in Table 4. The in silico analyses of active compounds predicted that the compounds (4e, 4f, 4i) would not exhibit hepatotoxicity.
Note: Y b , Yes; N a , No; DILI, drug-induced liver injury; CT, cytotoxicity; CYP, cytochrome P450; HLM, human liver microsomes. Predictions and interpretations using online server and a restricted/unrestricted applicability domain are represented.

Materials and Methods
Materials and reagents were purchased from commercial suppliers and used as instructed. Melting points were determined through an open capillary method using Sigma melting point apparatus (Sigma, Bangalore, India) and are uncorrected. IR spectra were recorded on Shimadzu IR spectrophotometer (Shimadzu USA manufacturing Inc., Canby, OR, USA). 1 H NMR and 13 C NMR spectra were recorded on Bruker/Agilent NMR spectrometer operating at 400 and 100 MHz, respectively, using TMS as internal standard; chemical shifts are in d. Mass spectroscopic analysis was performed on Shimadzu LC-MS. Analytical TLCs were implemented on pre-coated Merck 0.25 mm silica gel 60F254 plates using 40% ethylacetate in n-hexane as eluent and the spots were detected under UV light. All other chemicals were of analytical grade and were purchased from Sisco Research Laboratories (SRL, Mumbai, India).
General procedure for synthesis of NPB analogs. The piperazines (1eq) and salicylaldehydes (1eq) were taken in a round bottom flask and stirred with dioxane as a solvent for 10 min. After 10 min the aryl boronic acid (1eq) was added to the mixture and refluxed Conclusively, the HOMO-LUMO (∆) gap establishes the correlation between chemical structure and biological activity.
Western blot analysis: Western blot analysis was carried out by using the earlier reported protocol. pBad (Ser136: equivalent to human BADSer99) and hBAD antibodies were procured from Cell Signaling and similarly mouse anti-β-ACTIN from Santa Cruz Biotechnology [9].