Design, Synthesis, and Biological Evaluation of Indenoisoquinoline

Mar 8, 2013 - Nuclear receptors, such as the retinoid X receptor (RXR), are proteins that regulate a myriad of cellular processes. Molecules that func...
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Design, Synthesis, and Biological Evaluation of Indenoisoquinoline Rexinoids with Chemopreventive Potential Martin Conda-Sheridan,†,∥ Eun-Jung Park,§,∥ Daniel E. Beck,† P. V. Narasimha Reddy,† Trung X. Nguyen,† Bingjie Hu,† Lian Chen,‡ Jerry J. White,‡ Richard B. van Breemen,‡ John M. Pezzuto,§ and Mark Cushman*,† †

Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, and the Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana, 47907, United States ‡ Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, United States § College of Pharmacy, University of Hawaii at Hilo, Hilo, Hawaii 96720, United States ABSTRACT: Nuclear receptors, such as the retinoid X receptor (RXR), are proteins that regulate a myriad of cellular processes. Molecules that function as RXR agonists are of special interest for the prevention and control of carcinogenesis. The majority of these ligands possess an acidic moiety that is believed to be key for RXR activation. This communication presents the design, synthesis, and biological evaluation of both acidic and nonacidic indenoisoquinolines as new RXR ligands. In addition, a comprehensive structure−activity relationship study is presented that identifies the important features of the indenoisoquinoline rexinoids. The ease of modification of the indenoisoquinoline core and the lack of the necessity of a carboxyl group for activity make them an attractive and unusual family of RXR agonists. This work establishes a structural foundation for the design of new and novel rexinoid cancer chemopreventive agents.



INTRODUCTION Nuclear receptors are cellular proteins that control gene expression1 and regulate cellular functions such as growth, differentiation, apoptosis, and metabolism.2 There are 48 nuclear receptors,3 all of which share a similar structural organization.4−6 The preferred binding partner for one-third of all nuclear receptors is retinoid X receptor (RXR). For this reason, RXR has been called the “master partner.”7,8 The RXR heterodimers can be classified into two distinct groups: permissive and nonpermissive. The former group is activated by agonists of RXR or the other nuclear receptor partner, as in the case of RXR−liver X receptor (LXR) heterodimers. The latter group requires the presence of the ligand of the heterodimerization partner to be activated. This group is further divided into two subgroups: conditional, where the full response to the RXR ligand occurs in the presence of the partner’s ligand, as in the case of the RXR−retinoid acid receptor (RAR) partnership; and nonconditional, where RXR− ligands cannot activate the dimer even if an agonist of the partner receptor is present, as in the case of RXR−vitamin D receptor (VDR).9 RXR also has the ability to form homodimers that contain ligand-binding and DNA-binding domains. There are three isoforms of RXR: α, which is mainly found in the kidney, liver, and intestine and is the major isotype found in the skin; β, which can be detected in nearly every tissue; and γ, which is found in the pituitary gland, brain, and muscles.10−14 Literature reports suggest that there is overlap between the © 2013 American Chemical Society

functions of the three isoforms, but malfunction of RXRα has far worse consequences than those of the other two types. For example, knockout mouse studies have shown that absence of the α isoform is fatal to fetal life, produces cardiac failure, and results in ocular malformations. Inactivation of the α type has an effect similar to the one observed in vitamin A-deficient fetuses, implying that this isoform is key for retinoid signaling.15 Retinoids are natural or synthetic vitamin A derivatives. The effects of retinoids, such as 9-cis-retinoic acid (9cRA, 1, Figure 1), are modulated by two families of nuclear receptors, the retinoic acid receptor (RAR) and the retinoid X receptor (RXR). When 9cRA or other retinoids bind to RAR/RXR heterodimers, the receptor is activated and conformational changes take place. The nuclear receptor then binds to its cisacting response element initiating transcriptional activity. This up-regulates the production of the cyclin-dependent kinase p21, which produces chemopreventive effects such as stopping the cell cycle progression of cancer cells and apoptosis.16−18 The RAR/RXR heterodimer binds to the retinoic acid response element (RARE) and, with lesser affinity, to the retinoid X receptor response element (RXRE). The RAR/RXR heterodimer binds RXRE with higher affinity than the RXR homodimer. Therapies based on 9cRA and other retinoids have two limitations: (1) a high dose is needed to achieve a Received: January 5, 2013 Published: March 8, 2013 2581

dx.doi.org/10.1021/jm400026k | J. Med. Chem. 2013, 56, 2581−2605

Journal of Medicinal Chemistry

Article

binding site using GOLD, and the top binding poses calculated for each ligand were energy-minimized with SYBYL. The obtained binding poses suggest that the aminopropyl side chain of compound 3 does not interact with any key residues of the protein, and thus, it might possibly be removed without loss of activity. The computational studies also suggested that the addition of an acidic substituent in the 3 position of the indenoisoquinoline core may interact with the Arg316 and Ala 327 residues as seen with other rexinoids. Thus, a working hypothesis was proposed that addition of carboxylic acid side chains at C-3 would improve activity. Several indenoisoquinolines were designed and docked inside the binding pocket of RXR (PDB code 1FBY). Figure 2 shows one of the docked compounds, indenoisoquinoline 4, inside the RXR cavity where 9cRA (1) binds. In order to prepare indenoisoquinolines with carboxylic acid side chains of various lengths and flexibilities that could mimic the structure of 1, possible precursors such as halides were synthesized. Compounds 6−8 (Table 1) were prepared from 3amino-6-methyl-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (5) by modifying a previously reported method33 using Sandmeyer chemistry (Scheme 1). Precursor 8 was reacted with acrylonitrile (9) or methyl acrylate (10), using Heck coupling conditions, to provide compounds 11 and 12, which feature an ethylene linker between the functional group and the indenoisoquinoline (Scheme 2). The ester group of compound 12 was hydrolyzed to provide the acid analogue 13. Precursor 8 was also used to synthesize compounds with acetylene and propyl linkers between the carboxylic acid and the indenoisoquinoline. Iodide 8 was reacted with methyl propiolate (14) using Sonogashira cross-coupling conditions to provide indenoisoquinoline 15, which was hydrolyzed to furnish 16 (Scheme 3). Indenoisoquinoline 8 was also reacted with methyl but-3-enoate (17) using Heck coupling conditions. The obtained intermediate (not shown) was reduced by catalytic hydrogenation to provide 18, which was hydrolyzed to yield acid 19. Next, an indenoisoquinoline with a single methylene linker between the functional group and the 3-position was prepared. Indenoisoquinoline 7 was coupled with ethyl cyanoacetate (20), using tetrakis(triphenylphosphine)palladium(0) (Scheme 4). The position α to the carbonyl of 20 was deprotonated with sodium hydride to generate the anion coupling partner in situ.34,35 The intermediate 21 was hydrolyzed and decarboxylated in the presence of sodium hydroxide in aqueous ethanol at reflux to provide the indenoisoquinoline 4.36 Esterification of this indenoisoquinoline afforded the methyl ester 22. The next challenge was to attach a carboxylic acid substituent directly to the A ring of the indenoisoquinoline core. Ethyl 3-

Figure 1. RXR ligands.

biological response,19,20 and (2) a variety of side effects are observed including hypothyroidism, liver toxicity, and teratogenicity.21−25 Rexinoids are RXR-specific ligands having the potential to function as cancer-preventive agents without the serious side effects associated with retinoids. One rexinoid, bexarotene (2, Figure 1), is used to treat cutaneous T-cell lymphoma and holds promise as a chemopreventive agent against various cancers.26−29 The RXR binding pocket is composed of hydrophobic amino acids except for an Arg316 residue that is believed to be important for transcriptional activation. A common characteristic of RXR agonists is the presence of an acidic moiety, the conjugate base of which can bind to the positively charged Arg316.22,26,30−32 The indenoisoquinoline 3 (Figure 1) was synthesized in our laboratory and found to bind to RXRα and induce apoptosis in MCF-7 breast cancer cells in a dose-dependent manner.33 The rexinoid activity of the lead compound 3 was discovered through its inclusion in a 5000compound library that was screened for RXR agonist activity in an RXRE-luciferase reporter gene assay, and it proved to be the only active compound in the whole array.33 It also inhibited HL-60 cell proliferation with an IC50 of 92 nM after a 96 h incubation and caused the accumulation of these cells in the subG1 phase, from 4.5% at 13 nM to 28.7% at 250 nM.33 These results were unexpected given the lack of an acidic substituent in this indenoisoquinoline. In order to validate the indenoisoquinolines as RXRα ligands and to understand the key features for RXR agonist activity, an extensive SAR study was undertaken.



CHEMISTRY Exploratory docking studies were performed on RXRα in order to facilitate the rational design of indenoisoquinoline rexinoids. Various indenoisoquinolines were docked into the 9cRA

Figure 2. Model of indenoisoquinoline 4 inside RXR binding pocket. The image is programmed for walleyed (relaxed) viewing. 2582

dx.doi.org/10.1021/jm400026k | J. Med. Chem. 2013, 56, 2581−2605

Journal of Medicinal Chemistry

Article

Table 1. Screening N-Methylated Indenoisoquinolines vs RXR

a

compd

R1

IRa

compd

R1

IRa

4 6 7 8 11 12 13 15 18 19 22 29 30 31

CH2CO2H Cl Br I CHCHCN CHCHCO2CH3 CHCHCO2H CCCO2CH3 (CH2)3CO2CH3 (CH2)3CO2H CH2CO2CH3 CO2CH3 CO2H CO2NH2