Bioorg Med Chem Lett.2017 Mar;27(5):11991204

Design, synthesis and biological evaluation of 2-(4-phenylthiazol-2-yl) isoindoline-1,3-dione derivatives as anti-prostate cancer agents

S. Kabilana, *, K. Saravanana,  R. Elancheranb, S. Divakarc, M. Ramanathanc

 

a Drug Discovery Lab, Department of Chemistry, Annamalai University, Annamalai Nagar – 608002, Tamil Nadu, India

b Drug Discovery Laboratory, Life Sciences Division, Institute of Advanced Study in Science and Technology, Guwahati-781035, Assam, India

c Department of Pharmacology, PSG College of Pharmacy, Coimbatore – 641004, Tamil Nadu, India

*Corresponding author: Tel: 04144-238641

E-mail address: profdrskabilanau@gmail.com

 

Abstract

The structural modification and molecular docking-based screening approaches on thiazole-based isoindolinediones were imposed to find the novel 2-(4-phenylthiazol-2-yl) isoindoline-1,3-dione derivatives. The best-fit compounds (6a-n) were synthesized and evaluated their antiproliferative activities on the prostate cancer cell lines (PC-3 & LNCaP). Among them, the compound, 6m exhibited good activity, particularly on LNCaP (IC50 = 5.96 ± 1.6 µM), moderately active against PC-3 cell lines as compared to bicalutamide. The compound, 6m decreased the androgen-mediated transcription of ARE-mRNA in PSA, TMPRSS2, c-myc and cyclin D1 than R-bicalutamide. The compounds, 6e and 6f were reconfirmed through single crystal XRD analysis. The ADME profiling of the test compounds was evaluated to find the drug-likeness and pharmacokinetic parameters. These findings may provide vital information for the development of anti-prostate cancer agents.

Keywords: Androgen Receptor, Prostate Cancer, AR antagonist, Isoindolinediones, Molecular docking

 

Supplement:

Prostate cancer (PCa) is the most commonly diagnosed cancer among men in the United States. It was figured that there would be 161,360 new cases of PCa and 26,730 people will die of this disease in the year 20171,2. Androgens (dihydrotestosterone (DHT) and testosterone) bind with androgen receptor (AR) and stimulate the AR transcriptional mechanism. Activation of AR leads to the proliferation of PCa cells. The AR antagonists competitively prevent the binding of androgens with AR and hence avoid the activation of the AR. The mutations, T877A, W741L, and F876L, were identified with flutamide, bicalutamide and enzalutamide treated groups respectively3,4. All those mutations convert their respective antagonist into an agonist. However, the mutations nullify the bulkiness by increasing the volume of the ligand binding pocket. It is due to the structurally larger amino acids, Thr877, Trp741, and Phe786 were mutated to smaller amino acids, Ala877, Leu741 and Leu876 respectively. This increase in the volume of the ligand binding pocket accommodates the bulky AR antagonists without affecting the conformation of helix-12. In recent studies, various scaffolds were used to design novel AR antagonists with bulky substitutions effectively towards the helix-12 region. In a previous study, we have reported the design and synthesis of several oxobenzimidazoles and 1, 3-thiazolidine-2, 4-diones that demonstrated the relevant cytotoxicity and pharmacokinetic properties5,6. In this study, we rationally designed novel AR antagonist with phenyl-thiazole analogs. The designed ligands were synthesized and evaluated for their anti-prostate cancer activity in-vitro7. The ligands have been assessed for their binding interaction with AR by docking and molecular dynamics simulation. The in-vitro and dynamics studies were correlated, and the necessary structural properties required for AR antagonist activity was identified. Further, the anti-prostate cancer activity of the best active molecule was confirmed by gene expression and apoptosis assay. The phthalimide had binding affinity for AR, and their docking scores were similar to that of the standard, (R) -bicalutamide. The interaction between the ligand and receptor was dominated by hydrophobic contacts. The Val746, Met742, Met745, Leu707, Leu704, Trp741, Met895, Ile899, Ile898 are some of the amino acids involved in the hydrophobic interactions with the ligand. The H-Bond interaction within the ligand binding pocket was observed with Arg752, HOH, Met745 (bridged through HOH) and Thr877 amino acids (Fig 1a & 1b). The Arg752, HOH, Met745 were present deep inside the binding pocket while the Thr877 lies nearer to the helix 12.

 

Fig 1a: Interaction of R-bicalutamide with AR

Fig 1b: The binding mode of the test compounds

The partial agonist activity of the test and standard compounds represents the interactions between the ligand-receptor interactions; R-Bicalutamide forms H-Bond interaction with Arg752, HOH, Thr877, and Met745 (mediated through HOH). The bicalutamide forms π-π stacking with Trp741.

 

These findings led to the synthesis of thiazole-based isoindolinedione derivatives (6a-n) were obtained by refluxing the compounds (5a-g) with the respective phthalic anhydride in acetic acid in good to excellent yield (60-90 %), 8-12 h. Also, the compounds (5a-g) were synthesized by using commercially available 2-bromo-1-(4-substituted phenyl)ethanone and thiourea in ethanol were refluxed in the presence of triethylamine. The synthesized compounds (6a-n) were well characterized by IR, NMR, and HRMS, which proposed the structure of the compounds.

There are three cell lines for the screening process which includes two PCa cell lines (LNCaP, PC-3) and one non-cancerous mouse embryo (3T3) cell line. Of these, LNCaP cell line expresses T877A mutated AR, which was frequently identified in advanced and relapsed PCa patients. The PC-3 and 3T3 cell lines were negative for AR expression, but we included this cell line to find out the molecules which are selective for the AR expressing cell line. The test compounds had IC50 values between 5-36µM in LNCaP cell line, 37-44µM in PC3 cell line and 36-50µM in the 3T3 cell line. The range of IC50 values of the test compounds indicates that the compounds were more selective towards T877A AR expressing LNCaP cell line. The basic 4-phenylthiazole phthalimide scaffold (6a) was marginally selective towards LNCaP cell line. Substitution of the electronegative atom (6b & 6c) at R1 had increased the selectivity towards LNCaP cell line. Substitution of a methyl group at R1 (6d) had again marginally increased the selectivity for LNCaP cell line. The compounds 6a-d had partial agonist activity. Substituting polar groups at R1 (6e-g) had significantly increased the selectivity for LNCaP cell line than the standard R-bicalutamide. Polar group substitution at R1 also decreased the partial agonistic activity. The compounds 6e-g have methoxy, nitrile and nitro group respectively. The compound 6e had partial agonist activity, but with increasing polarity at R1 in compounds 6f & g, the partial agonist activity was eliminated. Substitution of fluorine atom at R2 position in compounds 6a-g yielded compounds 6h-n. Notably, the fluorine substitution at R2 had decreased or eliminated the partial agonistic activity. The compounds 6h-n had better IC50 value in LNCaP cell line than 6a-g and the compounds 6h-n were devoid of partial agonistic activity. From this, we can theorize that the molecules which have polar groups at R1 had shown decreased partial agonist activity. Substitution of fluorine atom at R2 had decreased the partial agonist activity while increased the antagonist activity.

The active molecule 6n was further analyzed for AR specific activity by evaluating the mRNA expression of androgen regulated response elements (AREs), PSA and TMPRSS2 in LNCaP cell line. We also measured the androgen responsive oncogenes, c-myc and cyclin D1. The c-myc and cyclin D1 were highly expressed in androgen-independent prostate cancer. The LNCaP cell has 50 times more expression of c-myc than normal cell lines, indicating the significant contribution by these oncogenes in deregulating the androgen mediated homeostatic control in prostate cells. The 1nM DHT increased the mRNA expression of PSA (4 fold), TMPRSS2 (3 fold), c-myc (2 fold) and cyclin D1 (2 fold) than the solvent control. The standard R-bicalutamide and test compound 6n had decreased the androgen mediated transcription of ARE mRNA (PSA, TMPRSS2) and oncogene c-myc. The notable difference is that the test compound 6n was more potent in antagonizing the androgen-mediated mRNA expression of ARE and oncogenes. Also, the test compound 6n could significantly decrease the mRNA expression of cyclin D1 while the standard R-bicalutamide did not. The cyclin D1 is responsible for G1/S transition during cell division. It indicates that the test compound could limit the cell division better than the standard R-bicalutamide. None of the tested compounds had changed the expression of ER response element, PS2. It indicates that the anti-prostate cancer activity of the tested compound was mediated through AR.

 

 

Fig: 2. The figure (20X) represents the EB/AO fluorescent images of LNCaP cells treated with 5.96 µM of compound 6n. The live cell (LC) stains normal green. The early apoptotic (EA) cells have a condensed or fragmented nucleus and stain bright green. The nucleus of the late apoptotic (LA) cells stains yellow orange. The nucleus of the necrotic cells (N) uniformly stains red.

 

References

1. Howlader N, Noone AM, Krapcho M, Miller D, Bishop K, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2014, National Cancer Institute. Bethesda, MD, https://seer.cancer.gov/csr/1975_2014/, based on November 2016 SEER data submission, posted to the SEER web site, April 2017.
2. Elancheran R, Maruthanila VL, Ramanathan M, et al. Recent discoveries and developments of androgen receptor based therapy for prostate cancer. Med. Chem. Commun. 6, 746–768 (2015).
3. Lu NZ, Wardell SE, Burnstein KL, et al. International Union of Pharmacology. LXV. The Pharmacology and Classification of the Nuclear Receptor Superfamily: Glucocorticoid, Mineralocorticoid, Progesterone, and Androgen Receptors. Pharmacol. Rev. 58, 782–797 (2006).
4. Bohl CE, Gao W, Miller DD, Bell CE, Dalton JT. Structural basis for antagonism and resistance of bicalutamide in prostate cancer. Proc. Natl. Acad. Sci. USA.102, 6201–6206 (2005).
5. Elancheran R, Saravanan K, Choudhury B, et al. Design and development of oxobenzimidazoles as novel androgen receptor antagonists. Med. Chem. Res. 25, 539–552 (2016).
6. Elancheran R, Saravanan K, Divakar S, et al. Design, synthesis and biological evaluation of novel 1, 3-thiazolidine-2, 4-diones as anti-prostate cancer agents. Anticancer Agents Med. Chem. doi: 10.2174/1871521409666170412121820 (2017).
7. Saravanan K, Elancheran R, Divakar S, et al. Design, synthesis and biological evaluation of 2-(4-phenylthiazol-2-yl) isoindoline-1,3-dione derivatives as anti-prostate cancer agents Bioorg. Med. Chem. Lett. 27 (5), 1199-1204 (2017).