Design and Synthesis of Novel Molecular Scaffolds of Bicaultamide derivatives for the treatment of Prostate Cell Cancer
Subrahmanyam Lanka1*, Vaikuntarao Lakinani1, Sagar Rao Kanaparthi1, Siva Rama Rao Kakani2
1Department of Chemistry, GITAM Institute of Science, GITAM University, Visakhapatnam, India
2North East Frontier Technical University, Arunachal Pradesh
*Corresponding Author E-mail: tvkpnr1985@gmail.com
ABSTRACT:
Prostate cancer is a major cause of male death worldwide and the identification of new efficient treatments is constantly needed. Different non-steroidal androgen receptor antagonists are approved also in the case of castration-resistant cancer forms. Using a rational approach and molecular modelling studies to modify the structure of antiandrogen drug bicalutamide, a new series of phenylsulfonyl-benzamide derivatives was designed and synthesised. Their antiproliferative activities were evaluated in four different human prostate cancer cell lines and several new compounds showed significantly improved IC50 values in the low μM range. The cytotoxicity profile was also evaluated for the novel structures in the HEK293 cell line.
KEYWORDS: Gemcitabine, RP-HPLC, Validation, LOD, LOQ, Robustness.
INTRODUCTION:
Prostate cancer is the development of cancer in the prostate, a gland in the male reproductive system. Most prostate cancers are slow growing; however, some grow relatively quickly2 The cancer cells may spread from the prostate to other area of the body, particularly the bones and lymph nodes.
PC cell growth is strongly dependent on androgens, therefore blocking their effect can be beneficial to the patient's health. Such outcomes can be achieved by antagonism of the androgen receptor (AR) using anti-androgen drugs, which have been extensively explored either alone or in combination with castration3 (1), Flutamide (2) hydroxyflutamide (3), bicalutamide (4), Enzalutamide (5), Curcumin (6), nilutamide (7) and RU56279 (8) are all non-steroidal androgen receptor antagonists (AR) approved for the treatment of PC
Figure 1: Structures of various anti androgens used currently for the treatment of prostate cancer
In many cases, after extended treatment over several years, these anti-androgens become ineffective and the disease may progress to a more aggressive and lethal form, known as castration resistant prostate cancer (CRPC). The major cause of this progressive disease is the emergence of different mutations on the AR, which cause the anti-androgen compounds to function as agonists, making them tumour-stimulating agents4
Among the drugs used for the treatment of PC, bicalutamide and enzalutamide selectively block the action of androgens while presenting fewer side effects in comparison with other AR antagonists5-6. Non-steroidal ligands are more favorable for clinical applications because of the lack of cross reactivity with other steroid receptors and improved oral bioavailability. Among them, Bicalutamide is the most potent and tolerated drug of choice administered either as monotherapy o8ikl\r with adjuvant castration or luteinizing hormone-releasing hormone.
Structurally these are comprised of two differently substituted aromatic rings, named ring A and ring B, connected by a linker, either linear (Bicalutamide-like compounds) or cyclic Enzalutamide (like compounds), recently, a novel 4-(4-benzoylaminophenoxy) phenol anti-androgen scaffold, derived from the natural pigment Curcumin, has been reported, in which a central phenyl group is acting as linker connecting two different aromatic rings.
One of the most common mutations found for bicalutamide is W741L in helix 12 of the receptor,7 which allows the protein to adopt its closed agonist conformation even in the presence of the antagonist: with this mutation, due to some residual structural flexibility in 1, ring B can bend to occupy an inner portion of the ligand-binding domain, thus allowing the closure of the receptor into its agonist conformation. Treatment with enzalutamide induces instead a F876L mutation in the AR, which also confers an antagonist to agonist switch in activity for the drug.8 Second generation antiandrogen ARN-509 (Fig. 1), which is now in Phase III clinical trials.9
However it was reported recently that these anti-androgens tend to become ineffective due to adaptive mutations on the structure of the androgen receptor, which renders them agonistic.
METHODOLOGY:
In the present chapter we reported the design and synthesis of novel molecular scaffolds of Bicaultamide, wherein the steric strain in the linker is increased by derivatisation of secondary amine as illustrated in Scheme 1. The retrosynthetic analysis illustrates the synthesis of N-substituted sulphonamides from substituted aromatic amines. Aromatic amines on condensation with methacrolyl chloride, followed by epoxidation and aminolysis with aliphatic amines yielded the amino alcohols. Further sulphonylation with aromatic sulphonyl chlorides gave novel analogues of Bicalutamide. Similar synthesis was repeated with secondary benzyl amines as starting materials. All the compounds were characterized by, IR, mass, 1H and 13 C NMR and then screened for biological activity against anti-cancer cell lines.
Scheme 1: Retrosynthetic analysis of N-Substituted analogues of Bicalutamide from aromatic amines
In the present chapter we reported the design and synthesis of novel molecular scaffolds of Bicaultamide for biological evaluation
Accordingly the substituted phenyl amine 11 was treated with commercially available methyl acrolyl chloride in DMA for 30 min to give substituted amide 12 in 66.8 % yield.
Scheme 2:
In the 1H NMR of 12 signals corresponding to newly introduced olefin group resonated at 5.79-5.74 ppm as a multiplet and the methyl group at 1.98 ppm as a singlet and rest of the protons resonated at expected chemical shifts indicated the methyl acrolyl amide formation. ES1-MS:m/z found 277.32 (M+Na)+ gave further confirmation for the stucture of 12.
The methyl acrolyl amide 12 on treatment with m-chloro perbenzoic acid in CH2Cl2 at 0 oC for 4 h, yielded the corresponding racemic epoxide mixture 10 in 58.8% yield.
Scheme 3:
The epoxide was confirmed by the loss of olefinic protons at 5.79-5.74 ppm and presence of terminal epoxide protons at 2.96-2.66 ppm appear in the 1H NMR and ESI-MS: m/z found 271.02 (M+H)+ further confirmed the product.
The opening of epoxide 10 in base condition epoxide 10 on treatment with isobutyl amine in presence of a potassium tert-butoxide in reflux for 3 h, gave the N-alkyl 1,2-amino alcohol 13 in 71.4% yield.
Scheme 4:
In the 1H NMR of 13 the signals corresponding to the isobutyl group appeared at 1.63-1.59 ppm and 0.92-0.89 ppm respectively and ESI-MS: m/z found 344.12 (M+H)+ further confirmed the product.
The N-alkyl 1,2-amino alcohol 13 which were further converted to the p-fluoro sulphonamide 9 on reaction with sulphonyl chloride in CH2Cl2 at 0oC for 6h in 54% yield.
Scheme 5:
The 1H NMR of 9 the signals corresponding to the newly introduced aromaticgroup appeared at 7.8-7.3 ppm and ESI-MS: m/z found at 524.12 (M+Na)+ confirmed the sulphonamide.
To understand the role of the alkyl chain attached to the amine we prepared various analogues wherein the isobutyl chain was replaced with isopropyl chain. Thus the epoxide mixture 10 on ammonolysis with isopropyl amine in the presence of base i.e potassium tert-butoxide gave the N-isopropyl 1,2-amino alcohols 14 in 70.0 % yield, with loss of epoxide protons at 2.96-2.66 ppm. The amino alcohols on treatment with sulphonyl chloride were converted into sulphonamide 8 in 58.0 % yield.
The opening of epoxide 10 in base condition epoxide on treatment with isopropyl amine in presence of a potassium tert-butoxide in reflux for 3 h, gave the N-alkyl 1,2-amino alcohol 14 in 70.0% yield.
Scheme 6:
In the 1H NMR of 14 the signals corresponding to the isopropyl group appeared at 1.11-0.98 ppm and 2.85-2.83 ppm respectively and ESI-MS: m/z found 330.11 (M+H)+ further confirmed the product.
The N-alkyl 1,2-amino alcohol 14 which were further converted to the p-fluoro sulphonamide 8, on reaction with sulphonyl chloride in CH2Cl2 at 0oC for 6h in 58% yield.
Scheme 7:
The 1H NMR of 8 the signals corresponding to the newly introduced aromaticgroup appeared at 7.6-7.3 ppm and ESI-MS: m/z found at 510.28 (M+Na)+ confirmed the sulphonamide.
The opening of epoxide 15 in base condition epoxide on treatment with isopropyl amine in presence of a potassium tert-butoxide in reflux for 3 h, gave the N-alkyl 1,2-amino alcohol 16 in 60.3 % yield.
Scheme 8:
In the 1H NMR of 16 the signals corresponding to the isopropyl group appeared at 2.43-2.36 ppm and 0.94-0.91 ppm respectively and ESI-MS: m/z found 223.23 (M+H)+ further confirmed the product.
The N-alkyl 1,2-amino alcohol 16 which were further converted to the methane sulphonamide 17 on reaction with methyl sulphonyl chloride in CH2Cl2 at 0oC for 6h in 53.33% yield.
Scheme 9:
The 1H NMR of 17 the signals corresponding to the newly introduced methyl group appeared at 3.03 ppm and ESI-MS: m/z found at 301.22 (M+H)+ confirmed the methane sulphonamide.
The N-alkyl 1,2-amino alcohol 16 which were further converted to the p-methyl sulphonamide 18 on reaction with sulphonyl chloride in CH2Cl2 at 0 oC for 6h in 82.0% yield.
Scheme 10:
In the 1H NMR of 18 the signals corresponding to the p-methyl group appeared at 3.88 ppm and 0.92-0.89 ppm respectively and ESI-MS: m/z found 399.02 (M+Na)+ further confirmed the product.
The N-alkyl 1,2-amino alcohol 16 which were further converted to the p-fluoro sulphonamide 19 on reaction with sulphonyl chloride in CH2Cl2 at 0 oC for 6h in 65.5% yield.
Scheme 11:
Similarly the racemic epoxide mixture xxx on treatment with isopropyl amine in the presence of potassium tert-butoxide in THF in reflux gave the corresponding 1,2-amino alcohol which was converted to the corresponding sulphonamides xx, xxx, xxx on treatment with methane sulphonyl
In the 1H NMR of 19 the signals corresponding to the aromatic group appeared at 7.87-7.82 ppm and 7.44-7.38 ppm respectively and ESI-MS: m/z found 403.35 (M+Na)+ further confirmed the product.
In the next design we relieved the steric strain in the phenyl ring attached to the amine by removing the methyl group. Thus the racemic epoxide mixture 15 on treatment with isopentyl amine in the presence of potassium tert-butoxidein THF in reflux gave the corresponding 1,2-amino alcohol which was converted to the corresponding sulphonamides 21, 22 and 23 on treatment with methane sulphonyl chloride, p-methyl and p-fluoro sulphonyl chloride respectively in 57.3 %, 62.8 % and 49.7 % yields. These sulphonamides were further confirmed by mass and NMR respectively.
The opening of epoxide 15 in base condition epoxide on treatment with isopentyl amine in presence of a potassium tert-butoxide in reflux for 3 h, gave the N-alkyl 1,2-amino alcohol 20 in 78.0% yield.
Scheme 12:
In the 1H NMR of 20 the epoxy protons are disappeared and newly formation of corresponding to the isopropyl group appeared at 0.89-0.71 ppm respectively and ESI-MS: m/z found 251.25 (M+H)+ further confirmed the product.
The N-alkyl 1,2-amino alcohol 20 which were further converted to the N-methyl sulphonamide 21 on reaction with methane sulphonyl chloride in CH2Cl2 at 0oC for 6h in 57.3 % yield.
Scheme 13:
The 1H NMR of 21 the signals corresponding to the newly introduced sulphonyl methyl group appeared at 3.03 ppm and ESI-MS: m/z found at 329.14 (M+H)+ confirmed the sulphonamide.
The N-alkyl 1,2-amino alcohol 20 which were further converted to the p-methyl sulphonamide 22 on reaction with sulphonyl chloride in CH2Cl2 at 0oC for 6h in 62.8% yield.
Scheme 14:
The 1H NMR of 22 the signals corresponding to the newly introduced aromatic group appeared two set of proton signal at 7.64-7.61 ppm and 7.39-7.36 ppm. The mass spectrum ESI-MS: m/z found at 405.32 (M+H)+ confirmed the sulphonamide.
The N-alkyl 1,2-amino alcohol 20 which were further converted to the p-fluoro sulphonamide 23, on reaction with sulphonyl chloride in CH2Cl2 at 0 oC for 6h in 49.7% yield.
Scheme 15:
The 1H NMR of 23 the signals corresponding to the newly introduced aromatic group appeared at 7.85-7.80 and 7.44-7.38 ppm and ESI-MS: m/z found at 409.42 (M+H) + confirmed the sulphonamide.
Cytotoxicity assay:
A Cell Titer Blue viability assay was performed in the human embryonic kidney cell line HEK293, in order to evaluate the cytotoxicity profile of the newly prepared compounds. Fig. 5 shows the reduction of cell viability caused by the novel analogues and standard bicalutamide at a fixed concentration of 10 μM. Interestingly, as reported in the graph, only three compounds (11d, 11e and 13e) significantly reduced cell viability if compared to bicalutamide (statistical significance), and only one of them (13e) killed more than 50% of cells. Due to their higher cytotoxicity in comparison with bicalutamide, the antiproliferative effect of these three compounds in the prostate cancer cell lines could also be a consequence of their intrinsic toxicity, besides the canonical AR antagonist activity. Comparing the rest of the compounds with bicalutamide, even if some of them are characterized by an increased cytotoxicity, cell viability reduction is not statistically significant and less than 40% in most cases, therefore the observed cytotoxicity is comparable to the one found for bicalutamide. Moreover, it is worth to note that bicalutamide reaches its antiproliferative IC50 at 50 μM, therefore it would not show reduction of cell viability at 10 μM. On the contrary, most of our new compounds showed antiproliferative activity in the four prostate cancer cell lines at a low μM concentration (<10 μM), and even if some of them show a reduction of cell viability by 35–40%, their anticancer therapeutic window is still wide enough for consideration as a potential treatment for prostate cancer. In particular, compounds 11h, 12h, 13b and 13h are associated with a promising antiproliferative/cytotoxicity profile, with an antiproliferative effect much better than bicalutamide (<16 μM), and with the same cytotoxicity of bicalutamide at 10 μM (none or minimal reduction of cell viability).
RESULTS:
Cytotoxic assay:
HEK293 human embryonic kidney cells were treated at 70% confluence with negative controls (0.1% DMSO, BICAL at 10μM), positive control (10% DMSO) and compounds at 10μM concentration for 24 hours. 20μl of Cell Titer Blue reagent (Promega) was added to each well containing 100 μl media and cell were incubated for 2 h at 37°C 5% CO2. Fluorescence was measured at 560/590 nm using a CLARIO star luminescence plate reader (BMG Labtech). N = 4, n = 6 for controls, n = 3 for drugs. Errors are calculated as standard error of the mean. * = p-value < 0.05, ** = p-value < 0.01, *** = p-value < 0.001 **** = p-value < 0.0001 between bicalutamide and compounds.
DISCUSSION AND CONCLUSION:
In the present chapter we reported the design and synthesis of novel molecular scaffolds of Bicaultamide, wherein the steric strain in the linker is increased by derivatisation of secondary amine as illustrated in Scheme 1. The retrosynthetic analysis illustrates the synthesis of N-substituted sulphonamides from substituted aromatic amines. Aromatic amines on condensation with methacrolyl chloride, followed by epoxidation and aminolysis with aliphatic amines yielded the amino alcohols. Further sulphonylation with aromatic sulphonyl chlorides gave novel analogues of Bicalutamide. Similar synthesis was repeated with secondary benzyl amines as starting materials. All the compounds were characterized by, IR, mass, 1H and 13 C NMR and then screened for biological activity against anti-cancer cell lines.
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Received on 12.04.2019 Modified on 16.05.2019
Accepted on 18.06.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2019; 12(11): 5427-5432.
DOI: 10.5958/0974-360X.2019.00941.7