This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Bioorganic & Medicinal Chemistry 18 (2010) 5535–5552 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc The structure–activity relationships of A-ring-substituted aromathecin topoisomerase I inhibitors strongly support a camptothecin-like binding mode Maris A. Cinelli a, Andrew E. Morrell a, Thomas S. Dexheimer b, Keli Agama b, Surbhi Agrawal b, Yves Pommier b, Mark Cushman a,* a Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and the Purdue Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA b Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892-4255, USA article info a b s t r a c t Article history: Aromathecins are inhibitors of human topoisomerase I (Top1). These compounds are composites of sev- Received 3 May 2010 eral heteroaromatic systems, namely the camptothecins and indenoisoquinolines, and they possess nota- Revised 11 June 2010 ble Top1 inhibition and cytotoxicity when substituted at position 14. The SAR of these compounds Accepted 14 June 2010 overlaps with indenoisoquinolines, suggesting that they may intercalate into the Top1-DNA complex Available online 20 June 2010 similarly. Nonetheless, the proposed binding mode for aromathecins is purely hypothetical, as an X-ray structure is unavailable. In the present communication, we have synthesized eight novel series Keywords: of A-ring-substituted (positions 1–3) aromathecins, through a simple, modular route, as part of a compre- Topoisomerase 1 hensive SAR study. Certain groups (such as 2,3-ethylenedioxy) moderately improve Top1 inhibition, and, Aromathecins Anticancer often, antiproliferative activity, whereas other groups (2,3-dimethoxy and 3-substituents) attenuate bio- Camptothecin-like activity. Strikingly, these trends are very similar to those previously observed for the A-ring of camptot- hecins, and this considerable SAR overlap lends further support (in the absence of crystallographic data) to the hypothesis that aromathecins bind in the Top1 cleavage complex as interfacial inhibitors in a ‘cam- ptothecin-like’ pose. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction the interface of Top1-DNA cleavage complexes in an intercalative mode,10–12 where it stacks between the base pairs of the Top1- Topoisomerase I (Top1) is an enzyme that is critical for efficient DNA cleavage complex and, stabilized chiefly by pi–pi stacking,13 DNA replication and cell division. As DNA is highly supercoiled, it sterically prevents the re-ligation reaction. It also forms key hydro- must also be relaxed prior to cellular processes such as replication gen bonds with Top1 amino acid residues.1,10–12 The resulting and transcription. The enzyme acts by binding to and nicking dou- covalent Top1-DNA adduct then produces collisions with advanc- ble-stranded DNA through the action of a nucleophilic tyrosine res- ing replication forks and transcription complexes, which triggers idue (Tyr723). Within the covalent Top1-DNA cleavage complex, irreversible DNA damage and apoptosis.14,15 the scissile DNA strand undergoes ‘controlled rotation’1–3 around Although the camptothecins are potent and possess high cyto- the nonscissile strand, relieving the supercoils. The hydroxyl group toxicity, they also suffer from well-identified drawbacks, including 16 of the scissile strand’s 50 end then re-ligates the broken strand and short duration of action, poor solubility, resistance mutants, and the enzyme is released.1 As it plays a pivotal role in cellular prolif- high toxicity.17,18 Additionally, the E-ring lactone of camptothecin eration, Top1 is often overexpressed in human tumors. High levels is readily opened to its hydroxycarboxylate form in vivo.19 This of this enzyme have been found in lung, colorectal, and ovarian form is less active and binds strongly to human blood proteins.20 cancers4–6 and elevated Top1 levels in breast cancers are also asso- One promising class of noncamptothecin Top1 poisons is the ciated with poor patient prognosis.7 indenoisoquinolines, such as MJ-III-65 (4).21,12 These compounds The only FDA-approved Top1 inhibitors are topotecan (2) and possess high anti-Top1 activity, are cytotoxic, and are more stable irinotecan (3),8 drugs based on the pentacyclic antitumor alkaloid because they lack the hydroxylactone. Through comprehensive camptothecin (1), (Fig. 1) which was originally isolated from the SAR studies,21–24 two clinical candidates, indotecan (5) and indim- Chinese tree Camptotheca acuminata.8,9 Camptothecin binds at itecan (6) were developed and have begun Phase 1 clinical trials at the National Cancer Institute.25,26 27,28 * Corresponding author. Tel.: +1 765 494 1465; fax: +1 765 494 6790. We described in two previous communications the design, E-mail address: [email protected] (M. Cushman). synthesis, and biological evaluation of substituted 12H-5,11a- 0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2010.06.040 Author's personal copy 5536 M. A. Cinelli et al. / Bioorg. Med. Chem. 18 (2010) 5535–5552 12 11 rationale provided by existing camptothecin and indenoisoquino- 1 HO OH line SARs and the ‘overlapping’ SAR hypothesis, we prepared 8 no- OH N 10 N O 21 14 O vel series of aromathecins (series 27 through 34) substituted on 20 9 N both the A-ring and position 14. The A-ring substituents encom- O 7 O N 19 N pass a variety of steric, electronic, and H-bonding properties, 17 5 O O allowing for maximum exploration of chemical space and elucida- Camptothecin Topotecan tion of those elements required for binding and optimal bioactivity. 1 2 2. Chemistry O N N Although there are several routes to rosettacin and aromathecin O HO N derivatives,32,33 our route proceeds through tricyclic synthon 8.34 O The previously developed route to 828 (see Scheme 3 for structure) O N proceeds in good yield from commercially available starting materials and can be scaled up readily. This ketone can then be con- O densed with substituted o-aminochloroaceto- or -chlorobutyr- Irinotecan O O ophenones to provide versatile aromathecin cores that can be 3 O readily functionalized, making the assembly of substituted aromat- O H3CO hecins rapid and modular. Following existing camptothecin SARs, a O O variety of groups (halogens, ethers, the methylenedioxy group, and O N N H3CO a methyl group) were chosen for the study. To vary the nature of the H3CO O A-ring substituent, substituted precursor amino aryl ketones were H Indotecan N first prepared. H CO N 5 3 OH The preparation of some of these ketone coupling partners is O MJ-III-65 described in Scheme 1. To install the 2,3-ethylenedioxy group, 4 1,4-benzodiozan-6-amine (9) was chloroacetylated19 using Sugas- awa’s Friedel–Crafts conditions to yield 10, and chlorobutyrated O 4 3 likewise to afford 11.35–37 As the methylenedioxy group (for 2,3- 5 O methylenedioxyaromathecins) is not compatible with the strong O 7 6 N 2 Lewis acids utilized in the chloroacetylation, the modified zinc-cat- H CO 8 1 3 alyzed procedure of Luzzio et al.19 was employed, and beginning 9 N 14 N N N with 3,4-methylenedioxyaniline (12) the ketone 15 was eventually H CO 10 11 12 3 O obtained in low yield but high purity. The chloroacetylation of O Indimitecan Rosettacin 4-aminoveratrole (16) proceeded in the absence of additional cat- 6 7 alyst (due to the electron-donating effects of the methoxy groups) to afford 17. 3-Fluoro-4-methylaniline (18) and 4-chloroaniline Figure 1. Representative Top1 poisons. (19) were also chloroacetylated to afford their respective ketones 20 and 21, albeit in low yield. The chloroacetylation of 3-chloroan- iline (22), however, afforded an inseparable mixture of 23 and 24, 27–30 diazadibenzo[b,h]fluoren-11-ones, called ‘aromathecins’. which were converted into their respective acetanilides 25 and 26 These compounds can be thought of as composites of the camptot- to aid in purification. These compounds were separated and hecins and indenoisoquinolines, in which the E-ring of the former hydrolyzed to yield 23 and 24, in a ratio consistent with that re- has been ‘aromatized’ (replaced by a benzene ring). The majority of these compounds, when substituted at position 14, possess greater Top1 inhibitory and antiproliferative activity than the unsubstitut- 31 H2N R1 ed core compound, rosettacin (7). Molecular models indicate that H2N R1 a these 14-substituents overlap spatially with the lactam substitu- O ents of indenoisoquinolines, which are both proposed to project R2 R2 n into the major groove of the DNA-Top1 complex and hydrogen 9, R , R = -O(CH ) O- Cl bond to Top1 amino acids and water in the DNA major groove.27,28 1 2 2 2 16, R1, R2 = -OCH3 10, R1, R2 = -O(CH2)2O-, n = 1 Because of the high degree of SAR overlap at these positions (down 18, R1 = F, R2 = CH3 11, R1, R2 = -O(CH2)2O-, n = 3 to specific substituents), it was proposed that ‘common’ SAR 19, R1 = H, R2 = Cl 17, R1, R2 = -OCH3, n = 1 elements are shared between the indenoisoquinolines and aroma- 20, R1 = F, R2 = CH3, n = 1 12, R1, R2 = -OCH2O- 21, R = H, R = Cl, n = 1 thecins.