Original article Effect of conjugates of all-trans-retinoic acid and shorter polyene chain analogues with amino acids on prostate cancer cell growth Eldem Sadikoglou a,1 , George Magoulas b,1 , Christina Theodoropoulou a , Constantinos M. Athanassopoulos b , Efstathia Giannopoulou a, 2 , Olga Theodorakopoulou a , Denis Drainas c , Dionysios Papaioannou b, * , Evangelia Papadimitriou a, ** a Laboratory of Molecular Pharmacology, Department of Pharmacy, School of Health Sciences, University of Patras, University Campus, GR-26504 Patras, Greece b Department of Chemistry, School of Natural Sciences, University of Patras, University Campus, GR-26504 Patras, Greece c Department of Biochemistry, School of Medicine, University of Patras, University Campus, GR-26504 Patras, Greece article info Article history: Received 8 January 2009 Received in revised form 12 March 2009 Accepted 16 March 2009 Available online 28 March 2009 Keywords: Retinoid Amino acid Conjugate Prostate cancer cells Retinoid receptors abstract In the present work, a series of conjugates of amino acids with all-trans-retinoic acid (ATRA) and shorter polyene chain analogues were rationally designed, synthesized by coupling the succinimidyl active esters of the acidic retinoids with appropriately protected amino acids or peptides followed by deprotection, and examined for their possible effect on viability of human prostate cancer LNCaP cells. In contrast to ATRA, all conjugates bearing amino acids with polar side chains showed no inhibitory effect on LNCaP cell proliferation, while conjugates with a-amino acids with lipophilic side chain, such as 7 , or linear amino acids, such as 9, significantly decreased prostate cancer LNCaP cell number. Interestingly, while the effect of ATRA was RARa-dependent, the effect of its active analogues was not inhibited by a selective RARa antagonist. Cell cycle analysis showed no effect on cell cycle, while quantitative analysis by annexin V-propidium iodide staining revealed that neither ATRA nor its analogues affected LNCaP cell apoptosis or necrosis. These results demonstrate that compounds 7 and 9 are potentially useful agents that warrant further preclinical development for treatment of prostate cancer. Ó 2009 Elsevier Masson SAS. All rights reserved. 1. Introduction Retinoids are lipophilic molecules composed of three distinct structural domains: a b-ionone ring, an isoprenoid tail and a polar end group. These small molecules exhibit multiple and diverse biological activities through their nuclear receptors, Retinoic Acid Receptors (RARs) and Retinoid X Receptors (RXRs) [1]. RARs and RXRs are encoded from different genes, have many isoforms and control the expression of a large number of genes [2]. Moreover, retinoids inhibit the expression of several genes through inhibition of transcription factor activating protein-1, which seems to be responsible for the anti-tumor effects of retinoids [3]. The induction of differentiation [1,4,5], apoptosis [5–7] and cell cycle arrest [5,7] by retinoids have made them important pharmaceutical candidates against dermal dysfunctions and certain cancer types, such as chronic leukaemia, where the use of all-trans-retinoic acid (ATRA) leads to 90% complete remission rate [8]. Due to problems, however, such as development of resistance [9] or toxicity [10], analogues of ATRA are widely being tested as alternatives. Shealy et al. have reported the synthesis of a series of N-(all- trans-retinoyl)amino acids, as analogues of ATRA (1) with extended polyene chain and increased water solubility, and tested their capacity to reverse keratinisation in vitamin A deficient hamster trachea in culture. The amino acids used were Gly, Ala, Leu, Phe, Tyr, and Glu. All of the thus examined conjugates were less active than ATRA [11]. In that bioassay, most active amino acid was Leu, incorporating a lipophilic side chain, and least active Glu, with a polar side chain. Even between Phe and Tyr most active was Phe, Abbreviations: Asp, aspartic acid; ATRA, all-trans-retinoic acid; DCC, N,N 0 -dicy- clohexylcarbodiimide; DCU, N,N 0 -dicyclohexylurea; HOBt, N-hydroxybenzotriazole; HOSu, N-hydroxysuccinimide; bAla, b-alanine; 3Aca, 3-aminocaproic acid; FCC, flash column chromatography; DEAD, diethyl azodicarboxylate; Lys, lysine; RT, room temperature; Boc, tert-butoxycarbonyl; DIEA, ethyldiisopropylamine; Tfa, tri- fluoroacetyl; DMAP, 4-dimethylaminopyridine; DCM, dichloromethane; TFA, tri- fluoroacetic acid; Trt, triphenylmethyl (trityl); RAR, Retinoic Acid Receptor; RXR, Retinoid X Receptor. * Corresponding author. Tel./fax: þ30 2610 997156. ** Corresponding author. Tel./fax: þ30 2610 969336. E-mail addresses: dapapaio@chemistry.upatras.gr (D. Papaioannou), epapad@ upatras.gr (E. Papadimitriou). 1 These authors have equally contributed to this work. 2 Present address: Clinical Oncology Laboratory, Division of Oncology, Depart- ment of Medicine, University Hospital of Patras, Patras Medical School, 26504 Rio, Greece. Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech 0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2009.03.029 European Journal of Medicinal Chemistry 44 (2009) 3175–3187