BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 233, 121–125 (1997) ARTICLE NO. RC976414 Action Mechanism of Retinoid-Synergistic Dibenzodiazepines Hiroki Umemiya,* Hiroyuki Kagechika,* Hiroshi Fukasawa,* Emiko Kawachi,* Masayuki Ebisawa,* Yuichi Hashimoto,† Ghislaine Eisenmann,‡ Cathie Erb,‡ Astrid Pornon,‡ Pierre Chambon,‡ Hinrich Gronemeyer,‡ and Koichi Shudo* ,1 *Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan; †Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan; and ‡Institut de Ge ´ne ´tique et de Biologie Mole ´culaire et Cellulaire, 3 Centre National de la Recherche Scientifique, Institut National de la Sante ´ et de la Recherche Me ´dicale, 67404 Illkirch cedex, Strasbourg, France Received February 26, 1997 nuclear receptors, such as the thyroid hormone and 4-[5H-2,3-(2,5-Dimethyl-2,5-hexano)-5-methyldibenzo- vitamin D 3 receptors, as well as certain ‘‘orphan’’ recep- [b,e][1,4]diazepin-11-yl]benzoic acid (HX600), as well as tors. its oxa- (HX620) and thia- (HX630) analogs, enhanced RA and several synthetic retinoids are important the activity of retinoic acid and a receptor a (RARa)- therapeutic agents in the fields of dermatology and on- selective agonist Am80 in HL-60 cell differentiation cology (4,5) and various retinoids, such as receptor iso- assays. HX600 synergizes with Am80 by binding to, and type-selective retinoid agonists and antagonists, have transactivating through, the RXR subunit of the RXR- been developed with the aims of increasing the speci- RAR heterodimer. HX600 exhibited RXR pan-agonist ficity and efficiency of treatment, and reducing RA side activity in transient transfections with a DR1-based effects. (1,6-8) In addition, retinoids that can synergize reporter gene and synergized with RA-bound RARa with other beneficial treatments (9) may lead to novel and RARb in inducing transcription from a DR5-based therapeutic strategies. Recently, we reported that the reporter. In addition, all three compounds at high con- dibenzodiazepine derivative HX600 (Fig. 1) exhibits a centrations acted as RAR pan-antagonists in stably synergistic activity with RA, or the synthetic retinoid transfected RAR ‘‘reporter cells.’’ These efficient syn- Am80, in the differentiation-induction assay with hu- ergists bind only weakly with RXRs in vitro, sug- gesting that they are RXR-RAR heterodimer-selective man HL-60 promyelocytic leukemia cells. (10) In view activators. These HX retinoids exhibited dual func- of the structural similarity between HX600 and other tionality, since they affected signalling through both synthetic retinoids, we anticipated that this synergistic retinoid receptor families (RARs and RXRs).  1997 effect reflects a direct HX600-retinoid receptor interac- Academic Press tion. Several synthetic RXR-selective ligands have re- cently been reported to synergize with RAR-selective ligands. (11-13) Here we describe the structural and biological properties of HX600 and characterize its in- The three retinoic acid (RAR) and three retinoid X teraction with, and transcription regulatory effects via, (RXR) receptors (a, b and g, and their isoforms), belong RARs and RXRs. to the nuclear receptor (NR) superfamily and act as ligand-inducible transcriptional regulators transduc- MATERIALS AND METHODS ing the pleiotropic effects of retinoic acids on morpho- genesis, differentiation and homeostasis during em- Materials. HX600 was prepared similarly to the retinoid antago- bryonal development and post-natal life. (1-3) RARs nist LE135 (an isomer of HX600). (14) Briefly, 6-bromo-1,2,3,4-tet- are activated by both all-trans-retinoic acid (RA) and rahydro-1,1,4,4-tetramethylnaphthalene was coupled with 2-nitro- 9-cis-retinoic acid (9cRA), while RXRs bind to and are aniline in the presence of copper iodide, followed by N-methylation. After reduction of the nitro group, N-methyl-N-(1,2,3,4-tetrahydro- activated only by 9cRA. Although RARs and RXRs can 1,1,4,4-tetramethylnaphthyl)phenylenediamine was condensed with form homodimers, heterodimerization increases the ef- terephthalic acid monomethyl ester chloride. The resultant amide ficiency of binding to the cognate response elements. compound was cyclized in polyphosphoric acid, followed by ester hy- RXR acts as a promiscuous heterodimerization part- drolysis to generate HX600 (mp 282 °C). 4-[2,3-(2,5-Dimethyl-2,5- hexano)dibenzo[b, f ][1, 4]oxazepin-11-yl]benzoic acid (HX620, mp ner, since it also forms complexes with various other 0006-291X/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. 121