Vol. 1, 103–120, December 2001 Molecular Cancer Therapeutics 103 Design and Synthesis of Fluoroquinophenoxazines That Interact with Human Telomeric G-Quadruplexes and Their Biological Effects1 Wenhu Duan,2,3 Anupama Rangan,2 center of the external tetrad but within the loop region. Hariprasad Vankayalapati,2,3 Mu-Yong Kim,3 Molecular modeling using simulated annealing was 4 Qingping Zeng, Daekyu Sun, Haiyong Han, performed on the FQP-parallel G-quadruplex complex to 5 Oleg Yu. Fedoroff, David Nishioka, determine the optimum FQP orientation and key 6 Sun Young Rha, Elzbieta Izbicka, molecular interactions with the telomeric G-quadruplex Daniel D. Von Hoff, and Laurence H. Hurley3,7,8 structure. On the basis of the results of these studies, College of Pharmacy, The University of Texas, Austin, Texas 78712 two additional FQP analogues were synthesized, which [W. D., A. R., M-Y. K., Q. Z., O. Y. F., L. H. H.]; Institute for Drug Development, San Antonio, Texas 78245 [D. S., S. Y. R., E. I., were designed to test the importance of these key D. D. V. H.]; Department of Biology, Georgetown University, interactions. These analogues were evaluated in the Taq Washington, DC 20057 [D. N.]; and Arizona Cancer Center, Tucson, Arizona 85724 [H. H., D. D. V. H., L. H. H.] polymerase stop assay for G-quadruplex interaction. The data from this study and the biological evaluation of these three FQPs, using cytotoxicity and a sea urchin Abstract embryo system, were in accord with the predicted more In this study we have identified a new structural potent telomeric G-quadruplex interactions of the initial motif for a ligand with G-quadruplex interaction lead compound and one of the analogues. On the basis that results in biological effects associated with G- of these structural and biological studies, the design of quadruplex-interactive compounds. Fluoroquinolones more potent and selective telomeric G-quadruplex- have been reported to possess weak telomerase interactive compounds can be envisaged. inhibitory activity in addition to their better known bacterial gyrase poisoning. Starting with a Introduction fluoroquinobenzoxazine, which has modest potency Fluoroquinolones are best known therapeutically as bacterial in a human topoisomerase II assay, we have gyrase inhibitors (1, 2) but more recently have also been designed a more potent inhibitor of telomerase that demonstrated to be active against eukaryotic topoisomerase has lost its topoisomerase II poisoning activity. II, either as catalytic inhibitors or topoisomerase II poisons This fluoroquinophenoxazine (FQP) interacts with (3), or as telomerase inhibitors (4). One such group of com- G-quadruplex structures to inhibit the progression pounds, the quinobenzoxazines, typified by A-62176 of Taq polymerase in a G-quadruplex polymerase (Fig. 1B), has served as a starting point for the design of more 1 stop assay. In addition, we demonstrate by H NMR potent topoisomerase II inhibitors using a structure-based studies that this compound interacts with telomeric approach (5). The extended aromatic conjugation system of G-quadruplex structures by external stacking to the A-62176 suggested to us that quinobenzoxazines may also G-tetrad with both the unimolecular fold-over and the intercalate with the more expansive system of G-quadruplex parallel G-quadruplex structures. A photocleavage assay DNA and thereby be telomerase inhibitors in parallel with other confirms the FQP interaction site, which is located off G-quadruplex-interactive compounds, such as the anthraqui- nones (6–10), cationic porphyrins (11–13),9 piperazines (14), ethidium compounds, (15), and a pentacyclicacridinium com- pound (16). After we started this project, an independent report Received 8/17/01; revised 9/21/01; accepted 10/2/01. The costs of publication of this article were defrayed in part by the that bacterial quinolones may be weak telomerase inhibitors (4) payment of page charges. This article must therefore be hereby marked also supported the idea that a ring-extended fluoroquinolone advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- would be a more potent telomerase inhibitor. cate this fact. 1 Supported by NIH Grants CA88310 and CA67760. G-quadruplexes (Fig. 1A) were first identified in 1962 (17); 2 These authors contributed equally to this paper. however, their possible biological significance was only sug- 3 Present address: College of Pharmacy, The University of Arizona, Tuc- son, AZ 85721. gested starting in the late 1980s (18–23), and they were first 4 Present address: Monsanto Co., St. Louis, MO 63167. proposed to be potential targets for drug design in 1989 (24). 5 Present address: Department of Biomolecular Sciences, University of The existence of chaperone proteins that facilitate their for- Manchester Institute of Science and Technology, Manchester M60 1QD, England. mation (25–27), G-quadruplex binding proteins that recog- 6 Present address: Cancer Metastasis Research Center, Yonsei University College of Medicine, Seoul, Korea. 7 Present address: College of Pharmacy and Department of Chemistry, The University of Arizona, Tucson, AZ 85721 and Arizona Cancer Center, 1515 North Campbell Avenue, Tucson, AZ 85724. 9 D-F. Shi, R. T. Wheelhouse, D. Sun, and L. H. Hurley. Quadruplex- 8 To whom requests for reprints should be addressed, at Arizona Cancer interactive agents as telomerase inhibitors: synthesis of porphyrins and Center, 1515 N. Campbell Ave., Tucson, AZ 85724. Phone: 520-626-5622; structure-activity relationship for the inhibition of telomerase. J. Med. Fax: 520-626-5623; E-mail: [email protected]. Chem., in press, 2001. Downloaded from mct.aacrjournals.org on September 25, 2021. © 2001 American Association for Cancer Research. 104 FQP and G-Quadruplex Interactions Fig. 1. A, G-tetrad and G-quadru- plexes. Panel a, four guanine residues forming a planar structure G-tetrad through Hoogsteen hydrogen bond- ing. Panel b, a parallel G-quadruplex model. Panel c, an intermolecular anti- parallel G-quadruplex model. Panel d, an intramolecular basket G-quadru- plex model. Each parallelogram in panels b– d represents a G-tetrad. B, structures of the topoisomerase II-interactive compound (A-62176) and G-quadruplex-interactive com- pounds QQ58, QQ27, QQ28, and TMPyP4. nize these structures (25, 28–31), and G-quadruplex-selec- G-quadruplex-interactive compounds have aroused tive helicases (32–34) that unwind these structures make more interest recently because it has been demonstrated their existence in vivo in human cells a real possibility. Re- that these compounds not only inhibit telomerase in cently their in vivo existence in ciliates has been supported cell-free (6, 9) and in in vitro systems (36) but also by antibody studies (35). cause telomere shortening and cell crisis in cancer cells Downloaded from mct.aacrjournals.org on September 25, 2021. © 2001 American Association for Cancer Research. Molecular Cancer Therapeutics 105 (14).10 A number of reviews on G-quadruplex and their targeting matography (ethyl acetate:hexane ϭ 2:1), yielding a bright 1 ␦ by small molecules have appeared recently (37–45). yellow powder (3.81 g; 83%). H NMR (CDCl3) (mixture of In this contribution we demonstrate that QQ58 (Fig. 1B), a isomers; multiplet, m; singlet, s) 1.17–1.26 (m, 3H), 2.95 (s, FQP11 that lacks bacterial gyrase or topoisomerase II poi- br, 1H), 4.10–4.25 (m, 2H), 7.05–7.20 (m, 1H), 7.33 (m, 2H), soning activity, is able to bind to G-quadruplex structures 7.43 (m, 1H), 7.52 (m, 3H), 7.85 (m, 1H), 8.69 (s, 0.7H), 8.74 and, by doing so, inhibit telomerase. Structural insight into (s, 0.3H); 19F NMR ␦ Ϫ156.2 (m, 1F), Ϫ154.5 (m, 0.75F), the interactions of this compound with both intermolecular Ϫ135.4 (m, 0.25F), Ϫ140.3 (m, 0.75F), Ϫ139.4 (m, 1F), 139.3 and intramolecular G-quadruplex structures has been obtained (m, 0.25F); and MS (CI): m/z 474 (M ϩ H). by one-dimensional 1H NMR molecular modeling and a photo- Ethyl 1,2-difluoro-4-oxo-4H-pyrido[3,2,1-k,l]-(3-amino- cleavage assay. We also demonstrate that this compound 2-hydroxydibenzofuranyl)[1,2-g]phenoxazine-5-carboxy- induces chromosomal effects and slowing of the division late (3). Ethyl 3-(2-hydroxydibenzofuran-3-amino)-2-[(2,3,4,5,- of sea urchin embryos typical of G-quadruplex-interactive tetrafluorophenyl)carbonyl] prop-2-enoate (2.32 g; 4.9 mmol) compounds. On the basis of molecular modeling studies, two and NaH (60% in mineral oil; 0.45 g; 11.3 mmol) were mixed additional analogues of this compound were designed and with freshly distilled tetrahydrofuran and stirred at Ϫ78°C for 15 synthesized (QQ27 and QQ28; Fig. 1B), and their interactions min. The mixture was allowed to warm to room temperature with G-quadruplex structures and biological effects were ex- gradually and then heated at 65°C for 30 min. The excess NaH amined. The maintenance or loss of biological potency paral- was quenched by addition of 30 ml of methanol. The solution leled their interactions with the G-quadruplex structure, and this was evaporated to dryness, and the product was purified was predicted on the basis of molecular modeling. through flash column chromatography, yielding a yellow pow- der (1.84 g; 89%): mp Ͼ229°C (decomposed); 1H NMR ␦ ϭ ϭ Materials and Methods (CDCl3) 9.01 (s, 1H), 7.90 (d, 1H, J 7.8 Hz), 7.80 (dd, 1H, J ϭ ϭ Synthesis of Extended Fluoroquinolones (QQ58, 7.8, 2.1 Hz), 7.39 (t, 1H, J 6.9 Hz), 4.48 (q, 2H, J 7.2 Hz), 19 QQ27, and QQ28 in Figure 2) 1.49 (t, 3H, J ϭ 7.2 Hz); F NMR Ϫ133.6 (dd, 1F, J ϭ 23, 10 Hz), Ϫ ϭ ϩ General. All of the melting points were recorded on a Tho- 1561.5 (dd, 1F, J 21, 9 Hz); and MS (CI): m/z 434 (M H).