WO2010006153A2 - Topopyrones: dual topoisomerase inhibitors - Google Patents

Abstract

The present invention provides for topopyrones and their derivatives which act as poisons of both human DNA topoisomerase I and human DNA topoisomerase II. The topopyrones thus represent a rare example of molecules capable of interacting effectively with more than one DNA topoisomerase.

Description

TOPOPYRONES: DUAL TOPOISOMERASE INHIBITORS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Set. No. 61/079,172, filed July 9, 2008.

FIELD OF THE INVENTION

The present invention relates to molecular genetics, molecular biology, enzymology, recombinant DNA, and molecular mechanisms of cancer therapies. More specifically, the invention provides for topopyrones that inhibit both human DNA topoisomerase I and topoisomerase II.

BACKGROUND According to the most recent data from the World Health Organization, ten million people around the world were diagnosed with cancer in 2000, and six million people died from it that year. Moreover, statistics indicate that the cancer incidence rate is rising globally. In America, for example, projections suggest that forty percent of those alive today will be diagnosed with some form of cancer at some point in their lives. By 2OW, thai number >viJJ have climbed to fifty percent. This grim scenario shows the great need for new cancer therapies. Topoisomerase poisons, such as etoposide, doxorubicin and camptothecin, have been used as chemotherapeutic agents to treat a wide spectrum of human cancers. The primary cytotoxic target for etoposide is topoisomerase H₅ a ubiquitous enzyme that regulates genomic DNA tertiary structure by generating transient double-stranded breaks in the double helix. Etoposide kills cells by stabilizing this otherwise transient cleavage complex of topoisomerase II, causing an accumulation of permanent DNA strand breaks which eventually overwhelm the cell and initiate cell death. Thus, etoposide converts topoisomerase II from an essential enzyme to a potent cellular poison that fragments the genome. Although the topoisomerase II-DNA cleavage complex is an important target for cancer chemotherapy, there also is evidence that topoisomerase Il-mediated DNA strand breaks induced by etoposide and other agents can trigger chromosomal translocations that lead to specific types of leukemia.

The primary target of camptothecin is topoisomerase I. Typically, agents targeting either topoisomerase I or topoisomerase II are delivered simultaneously or sequentially in combination to cancer patients. The toxicity levels reported frequently for sequential or simultaneous combinations of the topoisomerase I and II poisons include typically severe to life-threatening (grade 3 to grade 4) neutropenia and anemia. Hence, a need exists for novel anti-cancer agents, for anti-cancer agents that exhibit improved anti-topoisomerase activity, and for topoisomerase poisons that exhibit fewer side- effects or improved activity compared to existing agents. More specifically, there is a need for a single anti-cancer agent that targets both topoisomerase I and topoisomerase II.

SUMMARY

An object of the present invention provides for a topoisomerase poison that targets both topoisomerase I and topoisomerase Iϊ, poisoning both in any ratio. One embodiment of the present invention is a composition that inhibits or poisons both topoisomerases I and II, comprising a topopyrone or a structural analogue of a topopyrone. Another embodiment provides for a pharmaceutical composition or medicament comprising a topopyrone or a structural analogue of a topopyrone.

Another embodiment of the present invention provides a method for modulating topoisomerase I and topoisomerase II activity in a mammal, by administering to the mammal an amount of a topopyrone or structural analogue of a topopyrone effective to provide a topoisomerase modulating effect in both topoisomerase I and topoisomerase II, in any ratio. Another embodiment provides a method of inhibiting cancer cell growth, comprising administering to a mammal afflicted with cancer an amount of a topopyrone or a structural analogue of a topopyrone effective to inhibit the growth of the cancer cells. A related embodiment provides inhibiting cancer cell growth by contacting a cancer cell in vitro or in vivo with an amount of a topopyrone or topopyrone structural analogue, effective to inhibit the growth of the cancer cell.

DESCRIPTION OF THE DRAWINGS Figure 1 presents the 2-D chemical structures of topopyrones A-D, two of their chloro-analogues, and five synthetic derivatives.

Figures 2(a)-2(d), and reflects the effect of topopyrones on topoisomerase I-mediated DNA cleavage/re-ligation of a 23 bp dsDNA derived from Tetrαhymenα thermophilus rDNA spacer sequence, known to contain a strong topoisomerase I cleavage site. Figure 2(c) shows the effect of the chloro-derivatives of topopyrones on topoisomerase I-mediated DNA cleavage/re- ligation. Figure 2(d) presents the stability of topopyrone-induced topoisomerase ϊ-DNA cleavage complexes, R: NaCl 0.35 M (5 min); A-D: topopyrone (5.0 μM) ME (chloro- derivatives) (5.0 μM). See also, Khan et a]., 130 J. Am. Chem. Soc'y 12888-89 (2008).

Figure 3 shows the sequence of a 34-bp oligonucleotide derived from the simian virus 40 (SV40) nuclear matrix-associated region (NMAR). The topoisomerase Ilα poison VPl 6 (etoposide phosphate) stabilizes a cleavage site at position 4265, generating a 16-mer when the upper strand is [5'-³²P] end-labeled and a 14-rner when the lower strand is 5'-end labeled.

Figure 4a shows the effect of topopyrones on human topoisomerase II-mediated DNA cleavage/re-ligation reaction (upper strand). Figure 4b reflects the effect of topopyrone chloro-derivatives on human topoisomerase Iϊ-mediated DNA cleavage/re-ligation reaction (upper strand). Figure 4c shows the effect of topopyrones on human topoisomerase II-mediated DNA cieavage/re-ligation reaction (lower strand). Figure 4d shows the effect of topopyrone chloro-derivatives on human topoisomerase U-mediated DNA cleavage/re-ligation reaction (lower strand). Figure 4e shows that the divalent cation, Mg²⁺, promotes the topoisomerase II- mediated forward (cleavage) reaction as indicated by EDTA chelation (10 min). Figure 4f illustrates that NaCl facilitates the topoisomerase-rnediated backward (re-ligation) reaction. N: 0.5 M NaCl; A-D: topopyrone (20 μM).

Figure 5 evidences the effect of topopyrone A on DNA relaxation.

Figure 6 presents the distribution of topoisomerase II cleavage sites stabilized by etoposide (VP 16) or a topopyrone on phagemid BlueScript SK (-) DNA. pBS SK(-) fragments were subjected to topoisomerase II-mediated DNA cleavage-religation reactions in the presence of VPIό or a topopyrone at room temperature for 30 min. Reactions were terminated by adding SDS to 3 Tina) concentration of 0.5%. DNA fragments were separated on a 12% polyacrykinide gel. The sequence shown corresponds to the region encompassing positions 734-780 of pBS SK (-) DNA. G+A is the purine ladder following sequencing of the control DNA with formic acid. Arrows to the left and right of the gel indicate the migration positions of topoisomerase II- mediated DNA fragments. Figure 6a presents the sequencing of topoisomerase II cleavage sites induced by VP 16 and topopyrones; Figure 6b shows sequencing of topoisomerase II cleavage sites induced by VP 16 and topopyrone chloro-derivatives. Figure 7a shows the effect of topopyrones on the cell cycle progression of human leukemia cell CEM, Figure 7b shows the effect of topopyrone chloro-derivatives on the cell cycle progression of human leukemia cell CEM, ME217 = 5-chlorotopopyrone C; ME218 ~ 5- chlorotopopyrone D,

Figure 8 presents data on the effect of single base substitution at the +1 position on topoisomerase I-mediated DNA cleavage-religation induced by the topopyrones. The 23-bp oligonucleotide and the related DNA sequences altered at the +1 position were 3'-³²P end labeled on the scissile strand (_*)■ The unmodified oligonucleotide is designated as wild type (wt), whereas, +1C, +1 A, and -+TT correspond to oligonucleotides in which the +1 bp (G-C) was altered to C-G, A-T, and T-A base pairs, respectively. Reactions were performed at room temperature for 30 min in a reaction volume of 30 μL. Reactions were stopped by the addition of SDS to a final concentration of 0,5%. The DNA fragments were separated on a 16% denaturing polyacry] amide gel. Lane M contained a 3'-³²P end-labeled 13-nt oligonucleotide standard. Arrows 2 and 5 indicate the new cleavage sites induced by the chlorinated topopyrones (2, 5) and camptothecin (CPT) (5). Figure 9 shows the effect of single base substitution at the -1 position on topoisomerase I-mediated DNA cleavage-religation induced by the topopyrones. The 23-bp oligonucleotide and the related sequences altered at the -1 position were 3'-³²P end labeled on the scissile strand (_*). The unmodified oligonucleotide is designated as wt, whereas, -IG, -1C, and -IA correspond to oligonucleotides in which the -1 bp (T-A) was altered to G-C, C-G, and A-T base pairs, respectively. Reactions were performed at room temperature for 30 min in a reaction volume of 10 μL. Reactions were stopped by the addition of SDS to a final concentration of 0.5%. The DNA fragments were separated on a 16% denaturing polyacrylamide gel. Lane M contained a 3'-³²P end-labeled 13-nt oligonucleotide standard. Arrows 2 and 5 indicate the new cleavage sites induced by the chlorinated topopyrones and CPT. Figure 10 shows the rate of religation of DNA treated with topoisomerase I in the presence of CPT and topopyrones. A 23-bp oligonucleotide was 3'-³²P end labeled on the scissile strand. Reactions were carried out at room temperature for 30 min in presence of CPT or 5-chlorinated topopyrones. The reactions were reversed by the addition of 0.35 M NaCl for the indicated times. Time zero refers to the samples taken immediately prior to the addition of NaCl. Reactions were stopped by the addition of SDS to a final concentration of 0.5%. DNA fragments were separated on a 16% denaturing polyacrylamide gel Lane M is a 3'-³²P end- labeled 13-nt oligonucleotide standard identical with the cleavage product. Percent cleaved product remaining following salt treatment was quantitated using ImageQuant software, The amount of test compound-induced cleaved product at zero time was defined as 100%. Figure 1 1 presents the distribution of topoisomerase I-mediated DNA cleavage sites stabilized by CPT or the topopyrones on phagemid BlueScript SK(-) DNA. The processed pBS SK(-) fragments were subjected to topoisomerase I-mediated DNA cleavage-religation reactions in presence of CPT or chlorinated topopyrines at room temperature for 30 min. Reactions were terminated by adding 0.5% aq SDS. The DNA fragments were separated on a 12% polyacrylamide gel. The DNA shown corresponds to the region encompassing positions 689-2951 of pBS SK (-) DNA.

Figure 12 shows several schematics for the synthesis of synthetic topopyrone derivatives. Scheme I depicts the synthesis of key intermediate "7". Schemes II - Vl show the synthesis of topopyrone derivatives shown in Figure J . Table i presents growth inhibition of recombinant yeast by topopyrones as reported in 53 J. Antibiot. (Tokyo) 863 (2000).

Table 2 gives the results of MTT cytotoxicity assays for topopyrones and their chloro- derivatives in human and murine cell lines. Table 3 shows the percent cleaved products and topopyrones relative to CPT and VP 16,

DETAILED DESCRIPTION

It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims,

As used herein and in the claims, the singular forms include the plural reference and vice versa unless the context clearly indicates otherwise. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about."

All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein. Cellular DNA exists in entangled form. The unwinding of this DNA is essential for supporting cellular metabolism and is achieved by reversible nucleases called topoisomerases Human DNA topoisomerases are essential for the processes of chromosomal segregation and DNA relaxation during replication and translation, and may also be required for recombination. Wang, 3 Nat. Rev. MoL Cell Biol. 430-40 (2002); Pommier, 6 Nat. Rev. Cancer, 789-802 (2006). Human DNA topoisomerases I and II are the targets of numerous anticancer drugs which are used for the treatment of a variety of tumors. Burden & Osheroff, 1400 Biochim. Biophys. Acta 139-54 (1998); Li & Liu, 41 Ann. Rev. Pharmacol. Toxicol. 53-77 (2001); Thomas et al., 12 Bioorg. Med. Chem. 1585-604 (2004); Pommier, 2006.

The topoisomerase-mediated DNA cleavage-religalion mechanism involves a transient reaction intermediate involving a covalent binary complex found between the topoisomerase and DNA. DNA topoisomerase l is a ubiquitous nuclear enzyme that catalyzes the relaxation of superhelical DNA by inducing a transient single-strand nick in the duplex through cycles of cleavage and re-ligation. Champoux, 70 Ann. Rev. Biochem. 369-413 (2001); Wang, 65 Ann. Rev. Biochem. 635-92 (1996); Wang, 2002.

In contrast, DNA topoisomerase II mediates the ATP-dependent induction of two coordinated nicks in both strands of the DNA duplex, followed by passage of another double strand DNA through the transiently broken duplex. Liu et al., 19 Cell, 697-707 (1980). The sites of topoisomerase II-mediated cleavage are staggered by four bases on the opposite DNA strands. The staggered DNA breaks are generated by the formation of a phosphodiester bond between the catalytically essential tyrosine 805 in each homodimer and 5'-phosphate termini of the cleaved strands. Wilsteππann & Osheroff, 3 Curr. Top. Med. Chem. 321-38 (2003).

Human DNA topoisomerase II exists in two isoforms, topoisomerase Ilα and topoisomerase Ilβ, which are encoded by two individual genes. Wang, 2002. Topoisomerase Ilα is largely confined to proliferating cells in eukaryotes (Heck et al., 85 P.N.A.S. USA 1086-90 (2007)), whereas the beta isoform is present in most cell types including post-mitotic cells. Capranico et al., 1 132 Biochim. Biophys. ACTA 43-48 (1992); Watanabe et al., 19 Neurosci Res. 51-57 (1994); Lyu & Wang, 100 P.N.A.S. USA 7123-28 (2003). Topoisomerase II is important for chromosomal segregation, catenation/decatenation, and relaxation of DNA during the processes of replication, transcription, and possibly recombination. Wang, 2002; Wilstermann & Osheroff, 2003. Generally, topoisomerase poisons convert a functional topoisomerase enzyme to a

DNA-damaging agent by trapping the covalent binary complex formed between a topoisomerase and its DNA substrate. Nelson et al., 81 P.N.A.S. USA, 1361-65 (1984); Hsiang et al., 260 J. Biol. Chem. 14873-78 (1985). The topoisomerase II inhibitors trap the enzyme homodimers by inhibiting re-3igation of DNA during the cleavage/re-ligation cycle, leading to DNA double strand breaks and consequent cell death. Several human topoisomerase II-directed agents are used clinically for antitumor therapy, including etoposide, doxorubicin, amsacrine and mitoxantrone. Chen & Liu, 34 Ann. Rev. Pharmacol. Toxicol. 191-218 (1994); Wilstermann & Osheroff, 2003. An apparent drawback of some of the clinically relevant topoisomerase II- targeted drugs such as etoposide (VPl 6), teniposϊde and doxorubicin, is that they exhibit only limited DNA sequence preference, thereby resulting in DNA double strand breaks throughout the genome. Felix et al., 85 Blood 3250-56 (1995); Felix et al., 5 DNA Repair (Amst) 1093-108 (1998). These random breaks can cause chromosomal translocations that may result in treatment-related secondary malignancies, Felix et al., 1998; Felix, 1400 Biochim. Biophys. Acta 233-55 (1998); Pedersen-Bjergaard et al., 7 Leukemia 1975-86 (1993). Hence, there are ongoing efforts to develop novel human topoisomerase II-specific therapeutics which exhibit comparatively milder side effects.

In contrast, only a single family of topoisomerase I-targeting agents, the camptothecins (Hsiang et al., 1985). has attained clinical relevance. Potmesil, 54(6) Cancer Res. 1431-39 (1994). Camptothecin (CPT), a cytotoxic alkaloid, inhibits topoisomerase I activity by blocking re-ligation of transient DNA strand breaks. Hsiang et al., 1985. To date, few CPT derivatives, such as topotecan (Hycamtin®, GlaxoSmithKline) and irinotecan (Camptostar®, Pfizer Inc.), have been approved for clinical use. These CPTs are chemically unstable, however, due to the labile β-hydroxyiactone ring, which is readily hydrolyzed to inactive CPT-carboxylate form at physiological pH. Chourpa et al., 1379 Biochim. Biophys. Acta 353-66 (1998). The carboxylate form binds to human serum albumin resulting in reduction of effective serum concentrations of active drug molecules. Burke & Mi, 37(1) J. Med, Chem. 40-46 (1994).

Moreover, CPTs are substrates for the ABC (ATP binding cassette) transporter BCRP (breast cancer resistance protein), known to be expressed in a variety of tumors, Maliepaard et al., 7 Clin. Cancer Res. 935-41 (2001). Several promising new topoisomerase I poisons have been identified, however, including indolocarbazoles (Bailly et al., 36 Biochem. 3937-29 (1997)); indenoisoquinolines (Marchand et al., 5 MoI. Cancer Ther. 287-95 (2006)); terbenzimidazoles (Khan & Pilch, 365 J. MoL Biol. 561-69 (2007)); aporphine alkaloids (Zhou et al., 63 J. Nat. Prod. 217-21 (2000)); and the topopyrones (Kanai et al., 53 J. Antibiot. (Tokyo) 863-72 (2000); Ishiyama et al., 53 J. Antibiot. (Tokyo) 873-78 (2000)). The topopyrones are planar anthraquinones having a fused 1,4-pyrone ring, bearing structural relationships both to doxorubicin and to some of the pluramycins. Sun et al., 32 Biochem, 8068-74 (1993). Four topopyrones, named A. B, C and D (Figure 1). have been isolated from the culture broths of two fungi, Phαmα sp. BAUA2861 and PenicilHum sp. BAUA4206. Kanai et al,, 2000; Isiyama et al, 2000. The four topopyrones strongly inhibited the growth of yeast harboring a plasmid for human topoisomerase I, and did so only under conditions leading to expression of human topoisomerase I. They were also reported to inhibit DNA relaxation mediated by topoisomerase I, but not by topoisomerase JI, Kanai et al., 2000, Although the study of topopyrones has been limited by their exclusive availability as natural products, recent synthetic efforts have now provided access to all four naturally occurring topopyrones (Tan & Ciufolini, 8 Org. Lett. 4771-74 (2006); Gattinoni et al,, 48 Tetrahedron Lett. 1049-51 (2007); Elban & Hecht, 73 J. Org. Chem. 785-93 (2008)), as well as synthetic analogues. Elban & Hecht, 2008. Two chloro-derivative topopyrone analogs and five additional topopyrone derivatives are depicted in Figures 1 {see also Figure 12).

The present invention provides for topopyrones and their derivative analogs that act as poisons of both topoisoraerase 1 and topoisomerase Oa, both of which are validated targets for antitumor therapy. More specifically, in addition to stabilizing the topoisomerase 1-DNA covalent binary complex, the topopyrones are also strong topoisomerase Il poisons, In characterizing these compounds as topoisomerase I-directed agents, it was discovered, surprisingly, that topopyrones also induce an inhibitory effect on human topoisomerase Ilα- mediated DNA ckavage/re-ligation activity, It has been proposed that formation of drug- topoisomerase-DNA ternary complex is essential for the stabilization of DNA cleavable complexes and eventual cell death. Silber et al,, 4 NCI Monogr. 1 1 1-15 (1987). The present invention provides experimental evidence for the stabilization of such complexes by topopyrones and their derivatives. Despite their potent effect on inhibition of yeast {Saccharomyces cerevisiae), growth under human topoisomerase I-mductive conditions (Kanai et al., 2000; Ishiyama et al., 2000) (Table 1), substantially lower IC₅₀ values (drug concentrations resulting in 50% growth inhibition of cell cultures) were found for topopyrones and their chloro-analogues in human cell lines (Table 2). In cell-free biochemical assays, however, these compounds indeed targeted human topoisomerase 1 by stabilizing topoisomerase I-DNA cleavage complexes.

To study the relative potency of topoisomerase I-mediated DNA cleavage induced by topopyrones and their chloro-analogues, a 22-bp double stranded oligonucleotide substrate derived from the T. thermophilics rDNA spacer sequence (Bonven et al., 41 Cell, 542-51 (1985)), was employed (Figure 2a). This sequence contains a strong topoisomerase I cleavage site (Tanizawa et al., 34 Biochem. 7200-06 (1995)), with guanine (G) at the +1 position and thymidine (T) at the -1 position of the 5'-DNA terminus of the topoisomerase I break. Pommier et al., 92 P.N.A.S. USA, 8861-65 (1995). The results, shown in Figures 2b and 2c, demonstrate that although the stimulation of DNA-cieavable complex formation appears a general phenomenon for topopyrones, including their derivatives, the degree of DNA cleavage induced by each of the four topopyrones or their derivatives varies significantly, thus suggesting that enzyme-drug-DNA interactions are different for each of these chemical molecules. Each of the topopyrones stabilized the enzyme-DNA covalent binary complex at the same site as camptothecin, but did so less efficiently. Interestingly, the concentration dependence of binary complex stabilization by some of the topopyrones was not concentration dependent, perhaps reflecting direct DNA binding at higher concentrations. These results are fully in agreement with the earlier reports for the compounds. Kanai et al., 2000; Ishiyama et al, 2000.

All topopyrones and their derivatives induce recombinant topoisometase I-mediated DNA cleavage by generating a 13-mer cleavage product, however, a dose dependent cleavage pattern was observed only for topopyrone C and its chloro-analogue, 5-chlorotopopyrone C (ME237), and to some extent for topopyrone A. Furthermore, the percent cleaved product relative to CPT varied for each of the four topopyrones and their chloro-analogues (Table 3a). As shown Figure 1, in the structures of topopyrone A, topopyrone C and the topopyrone analogue 5-chlorotopopyrone C, the pyrorve ring is localized between C-I and C- 13 positions. In contrast, in topopyrone B, topopyrone C and the topopyrone chloro-analogue 5- chlorotopopyxone D (ME218), the pyrone ring exists between C- 3 and C- 13 positions on the molecule.

Analysis revealed that topopyrones A, C and the 5-chlorotopopyrone C induced topoisomerase I-mediated DNA cleavage at all concentrations (1 μM, 5 μM, and 10 μM). In contrast, topopyrones B, D (Figure 2b) and the 5-chlorotopopyrone D (Figure 2c) failed to stabilize the topoisomerase I-mediated DNA cleavage complexes at higher concentrations. It is plausible that the variability in their biological activity may arise due to different orientation of the pyrone moiety on the planar anthraquinone system of these compounds. This structural differentiation possibly facilitates strong interaction of topopyrones B, D, and 5 chloro- topopyrone D with DNA due to intercalation at topoisomerase inhibitory sites resulting in the inhibition of enzyme activity. Pommier et al., 97 P.N.A.S. USA, 10739*44 (2000). It is also possible that topopyrones A, C, and 5-chlorotopopyrone C may partially intercalate into the double helix through the planar anthraquinone ring, while the pyrone moiety is- directed into the minor groove, and may contribute to topoisomerase I poisoning. The variability in the relative extent of induced cleavage by each of the four topopyrones and their derivatives may be due to the identity of the base-pair at the +1 or -1 position of the topoisomerase 1 cleavage site. Antony et al., 63 Cancer Res. 7428-35 (2003). For example, a + 1 G-C pair may promote favorable interactions between enzyme-DNA and topopyrones A, C and the analogue 5-chlorotopopyrone C, while inducing negative interactions between topoisomerase I-DNA and topopyrones B, D, and the analogue 5-chlorotopopyrone D. Such differential ligand-enzyme-DNA contacts may be due to the differing structural properties of the two types of ligands (topopyrones A, C versus B, D), Therefore, topopyrones and their derivatives stabilize DNA cleavage complexes by targeting human topoisomerase I, albeit with variable potencies.

The stability of topopyrone-induced topoisomerase-cleavage complexes was also explored by examining the effect of salt concentration. NaCl initiates the reversal of drug- stabilized topoisomerase I cleavage complexes by dissociating topoisomerase 1 from the drug- DNA binary complex. Figure 2d demonstrates the effect of 0.35 M NaCl on the reversibility of topopyrone-induced topoisomerase I-cleavage complexes. As shown in Figure 2d, the reversal of topoisomerase I DNA cleavage for topopyrones or their 5-chloroanalogues was as rapid as it was observed for CPT. A purified Hind III and Pvu II 161 -bp restriction fragment from pBJueScript SK(-) phagemid may be used to determine the distribution of cleavage sites induced by topopyrones or their 5-chloroanaloguεs. CPT is used as a positive control in these experiments. Additionally, topoisomerase I-mediated DNA cleavage assay on phagemid Bluescript SK(-) DNA was carried out in the presence of CPT and the two chlorinated topopyrones. As shown in Figure 11, both CPT and the two 5-chlorinated topopyrones exhibited similar DNA cleavage patterns, albeit with differences in the relative intensities of bands at individual cleavage sites.

To further evaluate the effect of high ionic strength on the CPT or the chlorotopopyrone- stabilized topoisomerase I-covalent binary complexes, 0,35 M NaCl was added to the reaction mixtures for varying time periods prior to termination of the reaction. Figure 10 shows that the addition of NaCl shifted the reaction equilibrium from cleavage to ligation as reflected by a decrease in the cleavage band intensities. It was observed that at equimolar concentrations, the rate of reversal of thε- fopojsomerøse J-DNA covalent binary complex stabilized by 5-chlorotopopyrone C was greater than that stabilized by 5-chlorotopopyrone D, indicating that the topoisomerase I cleavage complexes formed by 5-chlorotopopyrone D are comparatively more stable. The latter complex was also more stable than the complex containing CPT.

Topopyrones and their unnatural derivatives also stabilize human topoisomerase llα DNA cleavage complexes. Topoisomerase Ilα is associated with mitototic chromosomes and is expressed preferentially in proliferating cells (Heck et al., 85 P.N.A.S. USA, 1086-90 (1988), although topoisomerase Hβ levels do not change significantly during the cell cycle. Capranico et al., 1 132 Biochim. Biophys, Acta, 43-48 (1992). More specifically, topoisomerase Ilα is associated with mitotic chromosomes and is preferentially expressed in proliferating cells with levels increasing in S-phase, peaking in G₂/M and diminishing in Gi phase. Heck et al., 1988. In contrast, the levels of topoisomerase Ilβ do not change significantly during cell cycle. Capranico et al., J 992. The relative potency of topoisomerase Ilα-mediated DNA cleavage by topopyrones and their derivatives was studied using a 34-base pair oligonucleotide substrate derived from the SV40 nuclear matrix associated region. Pommier et al., 222 J. MoI Biol 909-24 (1991); Capranico et al., 18 Nucleic Acids Res. 6611-19 (1992). The topoisomerase II cleavage site, stabilized by VP- 16 at position 4265 (Figure 3), generates a 16-mer oligonucleotide when the upper strand is 5'~end labeled and a 14-mer when the lower strand is 5'-end labeled. Figure 4a shows topoisomerase ll-mediated DNA cleavage induced by VP- 16 (etoposide phosphate) and topopyrones on the upper strand, generating a 16-mer cleavage product. As shown in the Figure, the extent of topopyrone-induced cleavage was comparable to that induced by etoposide (a clinically relevant topoisomerase II poison) at the tested concentrations. More specifically, the topopyrone-induced cleavage was quite prominent at both 5 μM and 20 μM concentrations of the drugs, but the effect of the drugs was not dose-dependent for topopyrone D (Table 3d). Similar results were obtained with the chioro-analogues, except for 5-chloro-topopyrone D (ME218, Figure 4b), where the percentage of cleaved product was substantially reduced at 20 μM concentration of the drug. Type II topoisomerases, such as human topoisomerase IIα_; catalyze DNA transactions by an ATP-dependent induction of two coordinated nicks in the complementary strands of the duplex DNA substrate. Therefore, it was plausible to examine the topoisomerase II-mediated effect of these compounds on the lower strand of the 34-mer oligonucleotide substrate. The lower strand of the 34-bp oligonucleotide was labeled at the 5'-end with [γ-^j2P]~ATP. This single stranded ³ P-iabeled oligonucleotide was subjected to topoisomerase II-mediated DNA cleavage reaction after being annealed to its complementary strand. Results shown in Figure 4c illustrate that this reaction generated a 14-mer cleavage product in response to both VP-16 and the topopyrones A-D. As shown ϊn Figure 4d, in contrast to VP 16, the stabilization topoisomerase II-mediated DNA cleavage by the chlorotopopyrones was not dose-dependent. When employed at 20 μM concentrations, both topopyrones derivatives failed to stabilize topoisomerase II-mediated DNA covalent binary complex formation.

Earlier studies had proposed that a single drug molecule can efficiently trap both subunits of a topoisomerase Il homodimer leading to a concerted DNA double-strand break. Capranico et al., 1990. The four-base-stagger is characteristic of concerted cleavage by a topoisomerase II homodimer. The results demonstrate that topopyrone molecule enhanced both upper strand cleavage to 16-mer (Figure 4a) as well as lower strand cleavage to 14-mer (Figure 4c), which is a characteristic of concerted cleavage by a topoisomerase II homodimer. Khan et al., 100 P.N.A.S. USA, 12498-503 (2003); Bromberg et al., 43 Biochem. 13416-23 (2004). Figure 4c illustrates the effect of topopyrones A-D on the topoisomerase Ilα-mediated cleavage of a 34-bp oligonucleotide derived from the Simian Virus 40 (SV40) nuclear matrix associated protein. Pommier et al., 1991. The topoisomerase Ilα poison, VP-16, stabilizes a cleavage site at position 4265 (upper strand of DNA duplex shown in Figure 3), generating a free 16-mer product when the upper strand is [5'-³²P]-end labeled. At the concentrations tested, the four topopyrones were as potent as VP 16, a clinically used antitumor agent, in stabilizing the topoisomerase II-DNA covalent binary complex. Assay of topoisomerase Ilα-mediated π cleavage of the lower strand of the same DNA substrates (Figure 4a) indicated precisely the same effect. In contrast to VPl 6, the topoisomerase ll-mediated DNA cleavage was not dose- dependent in response to treatment with topopyrones B and D (Table 3h). The two 5-chloro- analogues completely inhibited topoisomerase II-mediated DNA cleavage complex formation at higher concentrations (Figure 4d and Table 3h). These results reveal that, in general, topopyrones B, D, and the analogue 5-chlorotopopyrone D suppress the topoisomerase-mediated DNA cleavage at higher concentrations. This inhibition may be attributed to strong DNA intercalating property of these compounds as revealed by an increase in the relaxed form of pBlueScript phagemid supercoiled DNA upon incubation with Topopyrones (Figure 5), As noted previously herein, the topopyrones and their chloro-analogues are planar molecules with an anthraquinone ring system that is possibly involved in intercalation.

Numerous studies have shown that DNA intercalation is required but not sufficient for topoisomerase II targeting activity. Bodley et al,, 49(21) Cancer Res. 5969-78 (1989). An increasing number of intercalated drug molecules may eventually lead to saturation thereby, however, preventing the stabilization of cleavable complexes by interfering with the catalytic activity of the enzyme. Intercalation located immediately outside of the topoisomerase II stagger, i.e., two bases away from both the upper- and lower-strand cleavage sites, reduces topoisomerase II-mediated DNA cleavage in either the absence or presence of VPl 6. Khan et al., 2003. The observation that strong intercalators such as doxorubicin and its analogues are known to inhibit topoisomerase fl-mediated DNA cleavage activity at concentrations as Jow as 1 μM (Bodley et al., 1989), suggests that topopyrones may function through a mechanism that is similar to known DNA intercalators such as doxorubicin and mitoxantrone, which also possess an anthraquinone ring system.

With respect to the topoisomerase II-mediated DNA cleavage results, the intensity of the cleaved product in the control (topoisomerase II alone) was similar to that induced by topopyrones in presence of 10% DMSO. It appears that DMSO somehow promotes topoisomerase II-mediated DNA cleavage of the 34-mer oligonucleotide substrate. Therefore, to suppress the effect of DMSO, the concentration of DMSO was reduced to 5% in the final reaction mixtures. An increase in the intercalator-induced DNA breakage in response to treatment with DMSO has been reported earlier. Pommier et al., 43 Cancer Res. 5718-24 (1983). In this connection, the extent of cleavage of DNA by topoisomerase II alone also depends on the activity of topoisomerase II enzyme preparation.

Topoisomerase II needs a divalent cation for its catalytic function. Mg^{2^} appears to be the co-factor that enzyme employs in vivo to promote forward-reaction. Osheroff, 26 Biochem. 6402-06 (1987). Thus, to investigate if topopyrone-induced DNA cleavage (as reflected in Figures 4a and 4c) was topoisomerase II -mediated, the effect of chelating agent, EDTA, on topopyrone-induced topoisomerase II-mediated DNA cleavage/re-ligation reaction was examined. EDTA shifts the equilibrium of topoisomerase II-DNA complexes from cleavage to ligation by chelating Mg²⁺, Robinson & Osheroff, 29 Biochem. 2511-15 (1990). The results in Figure 4e show that a significant fraction of the binary complex stabilized b> the topopyrones remained (Figure 4e: compare the percent cleaved product remaining) following EDTA exposure, although the major product in each case was ligated DNA, as reflected by a decrease in the intensity of the cleaved bands (Table 3e). Similarly, the chloro-derivatives also showed that a significant fraction of the covalent binary complex stabilized by either of the two chlorinated topopyrones remained following incubation of the reaction mixtures with EDTA for 10 min. These observations suggest that topopyrone-induced accumulation of cleavage complexes was indeed topoisomerase II -mediated, and that the topopyrones act by stabilizing the transient topoisomerase II-DNA covalent binary complexes, thereby stimulating enhanced enzyme-mediated DNA cleavage. It is known that topoisomerase II-mediated DNA strand breaks are reversible. Liu et al., 258 J. Biol. Chem. 15365-70 (1983). Therefore, to determine the stability of topopyrone- induced DNA cleavage complexes, 0.5 M NaCl was introduced for 10 min prior to termination of the topoisomerase II-mediated DNA cleavage reaction. The results, shown in Figure 4f, demonstrate that the addition of NaCl did not completely reverse the topopyrone-induced cleavage sites but led to a decrease of the cleaved DNA products (Table 3f), suggesting that topopyrone-induced topoisomerase II cleavage complexes are more stable than topoisomerase I cleavage complexes alone. Regarding the 5-chloro derivatives, the addition of NaCl led to reversal of topoisomerase II-DNA covalent binary complexes stabilized by VP 16 as well as the two chlorinated topopyrones. The stabilities of the topoisomerase II-DNA covalent binary complexes formed by the two 5-chlorinated topopyrones and VP 16 were in the same range. This result highlights the fact that topopyrones trap topoisomerase II more efficiently than topoisomerase I.

The distribution of topopyrone- or their chloro-analogue-induced topoisomerase II cleavage sites were also studied on a purified Pst I-Hind III 2936 bp restriction fragment isolated from pBϊueScript SK (-) phagemid. VP 16 was used as a positive control in these experiments. The results shown in Figure 6a demonstrate that topopyrones and their 5-chloroanalogues (Figure 6b) exhibit low topoisomerase Ilα poisoning (-10%- 15% cleaved product relative to VP16). Nevertheless, the cleavage profile of these compounds resembles that of VP16 as these compounds exhibited at least four cleavage sites which are common to VP 16 (common cleavage sites are indicated by arrows). Topopyrones and their unnatural chloro-analogues also affect the progress of the human cell cycle. To understand the effect of topopyrones and their 5-chloroanalogues on the progress of ceils through the cell cycle, exponentially growing human leukemia cells (CEM. cells), were treated with varying concentrations of the drugs for 18 hr, after which the cells were processed for flow cytometry analysis. Figure 7a shows the flow cytometry profiles of CPT- and topopyrone-treated CEM cells. It is apparent from these results that CPT induced an S-phase accumulation and G2/M arrest at 10 μM concentrations, at which only 7% of cells were apoptotic. In contrast, at similar concentrations the majority of topopyrone-treated cells were arrested in Gl (-45%) or underwent apoptosis (~24%-26%). The only exception was topopyrone A, which induced a minor effect on the progress of cells through the cell cycle. At higher concentrations of topopyrones (100 μM), however, the degree of apoptosis was lower and the number of ceils accumulated in the S- and G2/M phases was only modestly increased (Figure 7a). These profiles were clearly different from those observed for CPT, which induces dose-dependent accumulation of cells in the S- and G2/M phases of the cell cycle. Shao et al., 57 Cancer Res. 4029-35 (1997); Shao et al.. 18 Embo J. 1397-406 (1999). The delay in S-phase and the replication block due to the formation of topoisomerase-drug-DNA ternary complexes are the major cytotoxic mechanisms associated with both CPT and VP- 16. Kalwinsky et al., 43 Cancer Res. 1592-97 (1983); Chen & Liu. 1994). Although, at higher concentrations, CPT may also kill tumor cells through apoptosis. Id. These results therefore suggest that topopyrones may possibly target other cellular entities in addition to topoisomerase I and topoisomerase II enzymes.

The two 5-chloroanalogues also exhibited unique cell cycle profiles (Figure 7b). At 10 μM concentration, a majority of cells were arrested in Gl in response to treatment with 5-chlorotopopyrone C, and about 18% of the cells underwent apoptosis. At a 10-fold higher concentration of this analog no obvious change in the cell cycle profile was observed, however. In response to treatment with a 10 μM concentration of 5-chlorotopopyrone D, about 41% of the cells were arrested in Gi phase, ~ 8% lower as compared to 5-chlorotopopyrone C, but the number of cells accumulated in the S-phase was increased by - 6% as compared to 5-chlorotopopyrone C (Figure 7b). No substantial change was observed in the ceil cycle profile of 5-chlorotopopyrone D at a 10- fold higher concentration (100 μM). These results suggest that in addition to inducing an S-phase block, both the 5-chlorinated topopyrones cause cell cycle arrest in the Gi phase.

Earlier studies have revealed that CPT exhibits site- selectivity of covalent binary complex stabilization with a preference for T at the -1 position and G at the ⁴^l position of topoisomerase I cleavage sites. Thomsen et al., 6 EMBO J. 1817-23 (1987); Jaxel et al., 266 J. Biol. Chem. 20418-23 (1991). To determine the sequence preferences of the topopyrone derivatives, the effect of single-base substitutions at positions flanking the topoisomerase 1 cleavage site on the scissile strand was studied. A series of 23 base-pair double stranded oligonucleotides were synthesized in which the G-C base pair at the +1 position of the topoisomerase 1 cleavage site on the scissile strand was systematically substituted with C-G, A-T or T-A base pairs. These oligonucleotides were then subjected to topoisomerase I-mediated DNA cleavage-rεligation reaction in presence of CPT or the chlorinated topopyrones. Figure 8 illustrates the pattern of topoisomerase I-mediated DNA cleavage induced by the chlorinated typopyrones at a concentration of 5.0 μM on each of the three mutated oligonucleotides. The results were compared with the wild-type sequence (Figure 8a). Although the degree of cleavage induced by the four topopyrones was similar in the wild-type sequence, each of the four compounds induced a differential pattern of topoisomerase I-mediated DNA cleavage in the mutant oligonucleotides (compare Figure 8a with 8b, 8c, and 8d). It is apparent from these results that topopyrone B induced the greatest degree of topoisomerase I-mediated DNA cleavage irrespective of the identity of the base at the +1 position in all four oligonucleotides, whereas topopyrone C was the weakest inducer of DNA cleavage by topoisomerase I at the same concentration. Further analysis of these results suggested that a new site two base pairs upstream from the existing topoisomerase I cleavage site was stimulated by the topopyrones but did not appear in the presence of CPT (Figure 8a). A second cleavage site was observed five base pairs upstream from the existing topoisomerase I cleavage site and appeared to be at the same position as that induced by CPT (Figure 8a). Although substitution of G to C at the +1 position enhanced the new cleavage sites, these sites were suppressed when G was substituted by A or T at this position. These results suggest that in this 23-bp double stranded oligonucleotide, topopyrones exhibit a cleavage pattern partially resembling that of CPT. Regarding the chlorotopopyrone derivatives, although the differences were not great, 5- chlorotopopyrone C induced somewhat greater topoisomerase I-mediated DNA cleavage regardless of the identity of the base at the +1 position in all the four oligonucleotides. Further, a new site two base pairs upstream from the existing topoisomerase I cleavage site was stimulated by both of the chlorinated topopyrone but did not appear in the presence of CPT (Figure 8, arrow 2). A second cleavage site was observed five base pairs upstream from the existing topoisomerase I cleavage site and appeared to be at the same position as that induced by CPT (Figure 8, arrow 5). The extent of cleavage induced by the two chlorinated topopyrones in relation to the base pairs at the +1 position was in the order G-C > T-A > A-T >C-G. Thus, while 5-chIorotopopyrones C and D can accommodate a cytidine, adenosine, or a thymidine at the +1 position, guanosine remains the preferred base at this position, as it is for CPT (Figure 8). Next the effect of single -base substitution at the -1 position of the cleavage site on the interaction of topopyrones with the enzyme-DNA covaSent binary complex was examined. The results shown in Figure 9b indicate that substitution of T (wild-type sequence) at the -1 position with G suppressed cleavage at the normal topoisomerase I cleavage site but substantially enhanced cleavages at the new sites two and five base pairs upstream from the normal topoisomerase 1 cleavage site. This pattern was observed with both CPT and the topopyrones in the -1 G mutant oligonucleotide, indicating that topopyrones can enhance or abolish topoisomerase I-mediated DNA cleavage at specific sequences. In contrast, substitution of T with C or A at the -1 position altered cleavage at both the existing topoisomerase I cleavage site as well as the new sites induced by the topopyrones (Figures 9c and 9d). It is also clear from these results that topopyrones B and D were generally the strongest in stimulating topoisomerase I-mediated DNA cleavage at the tested concentration. The rank order of cleavage potency by the topopyrones in relation to the identity of base pairs flanking the topoisomerase I cleavage site is as follows: base pair at the +1 position - G-C > T-A > A-T > C-G; base pair at the -1 position - T-A > C-G > A-T > G-C. These results suggest that topopyrones have a general preference for a purine base relative to a pyrimidine at the +1 position and a pyrimidine base relative to a purine at the -1 position of the topoisomerase I cleavage site. Interestingly, the relative extents of cleavage of the three sites differed somewhat for the individual topopyrones.

The effect of single-base substitution at the -1 position of the topoisomerase I cleavage site on the interaction of the two chlorinated topopyrones with the enzyme-DNA binary complex was also examined. The results shown in Figure 9d indicate that substitution of T (wild-type sequence) at the - 1 position with guanosine (G) suppressed the existing topoisomerase I cleavage site but significantly enhanced new cleavage sites two and five base pairs upstream from the existing topoisomerase I cleavage site. This pattern was observed for both CPT and the two chlorinated topopyrones, indicating that both analogues exhibited a cleavage pattern similar to that of CPT in this regard. In contrast, substitution of T with adenosine (A) or cytidine (C) had no significant effect on the existing topoisomerase I cleavage site induced by the two chlorinated topopyrones but did diminish CPT-stabilized cleavage. The new cleavage site induced five base pairs upstream of the existing topoisomerase 1 cleavage site was significantly enhanced in the presence of CPT and 5-chlorotopopyrone C (Figures 9b and 9c). The extent of cleavage induced by the two chlorinated topopyrones in relation to the identity of base pairs at the -1 position was in the order T-A > A-T > C-G > G-C

The experiments discussed herein establish unequivocally that topopyrones A - D and their derivatives act as poisons both of topoisomerase I and topoisomerase Ha, both of which are validated targets for antitumor therapy. The results obtained in comparison with the clinically used agents camptothecin (a specific topoisomerase I poison) (Hsiang et al., 1985) and VPl 6 (a specific topoisomerase llα poison) suggest that the actions of topopyrones may be more potent at the locus of topoisomerase Ilα-DNA interaction.

The topopyrones thus represent a rare example of molecules capable of interacting effectively with more than one DNA topoisomerase. The exploration of modified topopyrones optimized for interaction at both loci offers an interesting opportunity to enhance the antitumor activity of these interesting dual DNA topoisomerase poisons.

This study provides first evidence that topopyrones and their 5-chloroanaiogues possess the rare properties of dual topoisomerase inhibitors. To date only a limited number of dual topoisomerase inhibitors have been identified, some of which are being developed as anti-cancer drugs. Denny, 9 Expert Opin. Emerg. Drugs, 105-33 (2004); Leteurtre et al., 269. J. Biol. Chem. 28702-07 (1994); Rao et al., 67 Cancer Res. 9971 -79 (2007). Structurally, topopyrones and their choloro-analogues contain a planar anthraquinone ring system that is similar to other clinically active Topo2 inhibitors such as doxorubicin and mitoxantrone, both of which exhibit strong DNA intercalating properties. It is plausible that inhibition of topoisomerase-mediated DNA cleavage at higher concentrations of topopyrones and their chloro-analogues may be due to excessive intercalation of the drug molecules on additional sites upstream or down-stream of the cleavage sites, which are essential for enzyme DNA interaction, Pommier et al., 2000.

Deregulation of cell cycle control is the basis for abnormal cell proliferation resulting in tumor growth. Hakem, 27 EMBO J. 589-605 (2008); 41 1 Nature 366-74. (2001). The progression of cells through the cell cycle is monitored by checkpoints which sense cellular damage during the growth (Gi), DNA synthesis (S), or chromosomal segregation (G₂/M) phases and consequently induce cell cycle arrest. Hakem, 2008; Zhou & Elledge, 408 Nature 433-39 (2000); Fei & El-Deiry, 22 Oncogene 5774-83 (2003). Cells that experience genotoxic stress during DNA replication, transiently delay their progression through S-phase, however, if the damage is not repaired during S-phase, cells exit this phase and arrest in the G₂/M phase of the cell cycle. Hakem, 2008; Zhou & Elledge, 2000. The cell cycle profiles obtained in response to treatment with the chlorinated analogues of topopyrones C and D were clearly different from those observed for CPT (Figure 7), which is known to induce a dose-dependent accumulation of cells in the S and G₂/M phases of the cell cycle. Shao et al., 18 EMBO J. 1397-406 (1999); Shao et al., 57 Cancer Res, 4029-35 (1997). No significant G₂/M arrest in CEM cells treated with either of the two chlorinated topopyrones was observed; however, the degree of sub-Gj cell population representing dead cells was substantially higher in CEM cells treated with the chlorinated topopyrones as compared to those exposed to CPT at the same concentrations. A delay in S-phase and the replication block due to the formation of topoisomerase-drug-DNA ternary complexes are the major cytotoxic mechanisms associated with both CPT and VP 16. Li & Liu, 2001; Horwitz &, Horwitz, 33 Cancer Res. 2834-36 (1973); Kalwinsky et al., 43 Cancer Res. 1592-97 (1983). At higher concentrations, however, CPT may also kill tumor cells through apoptosis. Li & Liu, 2001. These results suggest that topopyrones possibly initiate alternative signaling pathways leading to DNA damage in addition to targeting topoisomerases I and II, Further studies are needed to understand the effects of these potential anticancer agents on cell cycle perturbation and cell death, stabilization of tumor suppressor protein p53 and the induction of its downstream genes such as p2/^wα-^/7'α/)/, box, bid and puma.

The instant invention thus provides for methods of treating cancer by treating subjects (mammals or humans) in need thereof with at least one topopyrone. As discussed herein, several topoisomerase inhibitors are already used in humans for treating cancer, and several have been used for over a decade. These medicaments may be prepared in various formulations, which are well known in the art.

For example, in cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate,

Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α- glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal, for example, sodium, potassium or Jithium. or alkaline earth metai, for example calcium, salts of carboxylic acids can also be made. The topopyrones and topopyrone derivatives (collectively "topopyrones") can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, that is, orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

The pharmaceutical compositions of this invention may be in the dosage form of solid, semi-solid, or liquid such as, e.g., suspensions, aerosols or the like. Preferably the compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts. The compositions may also include, depending on the formulation desired, pharmaceutically-acceptable, nontoxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. Effective amounts of such diluent or carrier will be those amounts which are effective to obtain a pharmaceutically acceptable formulation in terms of solubility of components, or biological activity, and the like.

Useful dosages of topopyrones can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art. See, e.g., U.S. Patent No. 4,938,949; Cannistra et al., 57 Cancer Res., 1228-32 (1997). Additionally, given the comparisons between clinically used topoisomerase inhibitors and the topopyrones presented herein, the skilled practitioner can readily extrapolate between used and present agents. See, e.g., Saijo, 922 Annals NY Acad. Sci. 92-99 (2000).

The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about 0,01 mg/kg to about 500 mg/kg per day. The compound may conveniently be administered in unit dosage form; for example, containing 5 mg to 1 ,000 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 0.5 μM to about 75 μM. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the active ingredient, optionally in saline, or orally administered as a bolus containing about 1 -100 mg of the active ingredient. Desirable blood levels may be maintained by continuous infusion to provide about 0.01-5.0 mg/kg/hr or by intermittent infusions containing about 0.4-15 mg/kg of the active ingredient(s).

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two. three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.

In therapeutic applications, the dosages of the agents used in accordance with the invention vary depending on the agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. Generally, the dose should be sufficient to result in slowing, and preferably regressing, the growth of the tumors and also preferably causing complete regression of the cancer. An effective amount of a pharmaceutical agent is that which provides an objectively identifiable improvement as noted by the clinician or other qualified observer. Regression of a tumor in a patient is typically measured with reference to the diameter of a tumor. Decrease in the diameter of a tumor indicates regression. Regression is also indicated by failure of tumors to reoccur after treatment has stopped.

Additionally, the appropriate route of administration, dose, and dosing schedule may be determined by the skilled practitioner in light of the particular patient needs, as are also well known to practitioners. See, e.g., Becerra et al., 31 J. Clin. Oncol. 219-25 (2008), Thus, for example, the practitioner may determine whether the topopyrone should be administered before, with, or following administration of another chemotherapeutic agent. See, e.g., U.S. Patent No. 6.875,745. Any necessary modifications to these known approaches for use in cancer therapies are well within the grasp of those of ordinary skill in light of the present specification.

EXAMPLES Example 1. In vitro characterization of topopyrone activities. Chemicals and enzymes

CPT was synthesized essentially as described earlier. Wani et al., 94(10) J. Am. Chem. Soc. 3631-32 (1972), Total synthesis of Topopyrones was achieved by employing Diels-Alder reactions and Friedel-Crafts acylation. Elban & Hecht, 2008. Etoposide (VP-16) was purchased from Sigma, St Louis, MO. Terminal deoxynucleotidyl transferase (Tdt) and T4 polynucleotide kinase were purchased from Roche Applied Science (Indianapolis, IN). Human Topoisomerase 1 enzyme was purchased from Topogen Inc. (Port Orange, FL). Human Topoisomerase II enzyme was purchased from USB (Cleveland, OH). Phosphate Buffered Saiine (PBS) (Invjtrogen, Carlsabad, CA). All synthetic oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Radiolabeled nucleotides were purchased from Perkin Elmer, Life Sciences (Waltham, MA),

Isolation and purification ofphagemidBlueScript (pBS)DNA

Preparation and transformation of competent cells was performed as described in Sambrook & Russell, MOLECULAR CLONING: A LAB. MANUAL (Cold Spring Harbor Lab., Cold Spring Harbor, NY, 2001). Briefly, 100 ng of pBS DNA was incubated with 0.2 mL of the host XLl -Blue MRF' E. coli host strain for 30 min at 4⁰C followed by a 2 min heat shock at 42°C. Then, 0.8 mL of Luria Broth (LB) was added to the transformed cells and the bacterial culture was incubated at 37°C for 1 hr for the expression of antibiotic resistance. Transformed cells were then plated on LB plates supplemented with 100 μg/mL of ampicillin. Plates were incubated over night at 37°C. A single bacterial colony was cultured to log phase (-3-4 hr) with vigorous shaking to attain an OD600= -0.4. DNA was isolated and purified by GenElute Plasmid Miniprep system (Sigma- Aldrich, St. Louis, MO).

3 '- and 5 '-¹²P end labeling and purification of oligonucleotides

The 3'- and 5^"-end labeling of oligonucleotides was performed according to the protocols provided by the manufacturer (Roche Applied Science, Indianapolis, IN). The labeled oligonucleotides were purified using Sephadex G-25 mini Quick Spin Oligo columns (Roche). Briefly, 5'-end labeling was done by adding 10 pmol of template DNA, 50 μci [γ-³²P] ATP (specific activity 30G0Ci/mmol), 10 units of T4 PNK in 50 niM Tris-HCl, 10 mM MgC12, 5 mM dithioihreitol, pH 7.5 reaction buffer. dHiO was added to a final volume of 40 μL, The reaction mixture was incubated at 37°C for 1 hr. Reactions were terminated by heating at 70°C for 10 min. The 3'-end labeling was done by adding 10 pmol of template DNA, 50 μCi [α-³²P] cordycepin (specific activity 5000 Ci/mmol), 400 units of recombinant terminal transferase in 200 mM potassium cacodylate, 25 mM Tris-HCl, and 0.25 mg/ml BSA (pH 6,6) reaction buffer, df^O was added to a final volume of 40 μLs. The reaction mixture was incubated at 37°C for 1 hr. Reactions were terminated by SDS to a final concentration of 0.5%.

The 5 ^!-³'P end labeling and purification ofphagemid BlueScript (pBS) DNA Two (2) μg of pBS (SK-) was digested for Ih at 37°C with PsM (20 U) in a final volume of 20 μLs, Then, 3 μL of 1OX shrimp alkaline phosphatase buffer (100 mM glycine, pH 10.4, ImM MgCl₂, 1 mM ZnCl^, 10 mM nitrophenyl phosphate), 1 U of alkaline phosphatase, and dHiO was added to 30 μL. The mixture was incubated at 37°C for 1 h. Alkaline phosphatase was heat inactivated for 20 min at 65°C. An aliquot of the aforementioned mixture (~500 ng pBS) was 5'-end labeled as described earlier. The 5^;-end labeled product was digested with Hind III (20 U). This step generates a 5'-end labeled fragment of 2936 bp. Unincorporated [γ-³²P] ATP was removed by Sephadex G-25 Quick Spin DNA columns (Roche).

Topoisomerase I-medi cited DNA cleavage assay Single stranded DNA oligos were labeled at their 3' ends with [α-³²P] cordycepin as described previously. Khan & Pilch, 2007. The labeled DNA strands were annealed with their complementary strands in buffer containing 10 mM Tris-HCl (pH 7.8). 100 mM NaCl, and 1 mM EDTA. The annealing process entailed the heating of the reaction mixture at 95°C for 5 mm, followed by slow cooling to room temperature. Duplex DNA substrates (at approximately 100 fmol per reaction), 5 Units of topoicomerase I, and either CPT or a topopyrone at the indicated concentrations were incubated at room temperature for 30 min. The reaction volume was 10 μL and the reaction buffer contained 10 mM Tris-HCl (pH 7.9), 50 mM KCl, 1.5 M NaCl, 0.1 mM EDTA, 15 μg/mL bovine serum albumin (BSA)₃ and 1 mM spermidine. The reactions were stopped by addition of SDS to a final concentration of 0.5%. For the NaCl-induced religation reactions, the SDS stop was preceded by adding NaCl for the indicated times. The reaction mixtures were then diluted in 3.3 volumes of buffer containing 98% (v/v) formamide, 10 mM EDTA, 10 mM NaOH, 1 mg/mL of xylene cyanol, and 1 mg/mL of bromophenol blue, Five micro liters (5 μL) of each sample was then loaded onto a denaturing (7 M urea) 16% (w/v) polyacrylamide gel and electrophoresed at 40 V/cm and 55°C for 3 hr. Imaging was performed with a phosphorlmager (Molecular Dynamics).

Topoisomerase ll-mediated DNA cleavage assay

Oligonucleotides were 5 ^"-end labeled with [γ-³²P]ATP and T4 kinase as described previously. Pommier et al,, 1991. The 5^"-end labeling and purification of BlueScript phagemid (pBS) DNA was performed as described. Rao et al,, 67 Cancer Res. 9971-79 (2007). Annealing to the complementary strand was performed by heating the reaction mixture to 95⁰C and overnight cooling to room temperature in 10 mM Tris.HCl (pH 1.5)1 100 mM NaCl/ ImM EDTA. DNA substrates (-10 pmol per reaction) were incubated with 10 Units of topoisomerase Ha in the presence or absence of VP16 for 30 min at 25°C in 10 uL reaction buffer (100 mM Tris, pH 7.5, 500 mM NaCl, 500 mM KCl, 50 mM MgCl₂, 1 mM EDTA₅ 0.15 mg BSA, 10 mM ATP). Reactions were stopped by adding SDS (final concentration 0.5%). For the EDTA or NaCl-induced religation reactions, the SDS stop was preceded by adding EDTA (10 mM final concentration) or NaCJ (500 mM final concentration) for the indicated times. Before loading the samples for electrophoresis, 3.3 volumes of Maxam-Gilbert loading buffer (98% formamide, 0.01 M EDTA, 10 mM NaOH, 1 mg/mL xylene cyanol, 1 mg/mL bromophenol blue) were added to reaction mixtures. Denaturing 16% polyacrylamide gels (7M urea) were run at 40 V/cm at 50°C for 3hr. Imaging was performed with a phosphorlmager (Molecular Dynamics).

Preparation ofG+A sequencing ladder

G+A sequencing reactions were conducted by combining 3'- or 5'- end-labeled DNA strands (-200 fmol per reaction) with Salmon testis DNA (1 μg/μL) and formic acid (-21%). The reaction mixture was then incubated at 37°C for 30 min. The reaction buffer contained 10 mM Tris.HCl (pH 7.6) and 1 mM EDTA. The reactions were stopped by placement on ice, Then, 135 μL of dH₂θ and 15 μL of piperidine was added to each reaction mixture, followed by incubation at 90⁰C for 30 min. Then 200 μL of buffer containing 3 M sodium hydroxide (pH 5.2), 0.5 M EDTA and 1 μg/μL of yeast tRNA and 950 μL of ethanol was added to each reaction mixture followed by incubation at -20⁰C for 15 min. The reaction mix was then centrifuged at 4°C for 15 min. The supernatant was removed and the pellets were dried in DNA concentrator under vacuum.

Example 2, In vivo characterization of topopyrones

Cell lines

The CEM cell line was obtained from Dr. Yves Pommier (Laboratory of Molecular Pharmacology, NCl, Bethesda, MD). All human cell lines were cultured in RPMI 1640 (Life

Technologies/Invitrogen, Gaithersburg, MD) containing 10% heat-inactivated FBS, and glutamine (2 mM) in a humidified incubator maintained at a temperature of 37°C and 5% CO₂.

XLI -Blue MRF' E. coll host strain (Stratagene, La Jolla, CA) was cultured at 37⁰C in a humidified incubator maintained at 5% CO? .

Cytotoxicity assay

Cytotoxicity was measured by using MTT assay after continuous treatment with the drug for 3 days. 3000 cells/100 uL culture medium/well were seeded in a 96-well microliter plate.

One hundred (100) μL culture medium containing varying drug concentrations was added to the experimental wells. Controls were added with cells only and blank with medium only. The cells were incubated for 72 hr in humified atmosphere in an incubator maintained at 37⁰C and supplemented with 5% CO₂. At the end of drug treatment, 20 μL of MTT (5 mg/mL) was added to each well including the blank. Treated microtiter piates were incubated at 37°C for an additional 4 hr. MTT was vacuumed off and 200 μL of DMSO was added to each well to achieve cell lysis. Microtiter plates were kept on a shaker for 30 min. Absorbance was recorded at 595 nM on a spectrophotometer (Spectramax 190, Molecular Devices),

Flow cytometry analysis

CEM cells (10⁶/sampJe) were treated with increasing concentrations of the drug for 3 hr and then cells were allowed to culture for an additional 15 hr at 37°C in presence or absence of 0.4 μg/mL of nocodazole. Cells were harvested and the cell pellets were washed twice with PBS (pH 7.4) and fixed with 70% ethanol. Fixed cells were washed with 1 X PBS and treated with RNase A solution (3UZmL) at 37°C for 15 min. Cells were stained with 50 μg/mL of propidium iodide for 20 min. DNA content of 10,000 cells/analysis was monitored on a Beckman-Coulter flow cytometer. Example 3, Inhibition of prostate tumor growth in vivo

Male SClD (ICR) mice are inoculated with androgen-independent human prostate cancer cells (DU145; 8 x 10⁶ s.c). Administration of drugs is initiated when tumor nodules reach -0.5 cm in diameter. Four mice per group are used in this experiment. The control group is treated with vehicle alone. The topopyrone groups are treated with 50 mg/kg i.p., followed 24 hr later by i.p. injection of vehicle. There is a 1-day break between each cycle. Mice are treated for a total of six cycles. Pictures are taken three weeks after six cycles of treatment.

In another experiment, four mice per group, androgen-independent DU 145 prostate cancer cells are xenografted into immunocompromised mice. To increase stringency, treatment is delayed until the tumors reached ~0.5 cm in diameter. Effective topopyrones inhibit tumor growth.

Claims

1. A method of inhibiting cancer cell growth, comprising administering to a mammal afflicted 5 with cancer, an amount of topopyrone, effective to inhibit the growth of said cancer cells by poisoning topoisomerase 1 and topoisomerase II.

2. A method for modulating topoisomerase activity in a mammal in need of such treatment comprising administering to the mammal an amount of topopyrone effective to provide a ϊ 0 mammalian topoisomerase I and topoisomerase II modulating effect,

3. A method comprising inhibiting mammalian cancer ceil growth by contacting said cancer cell in vitro or in vivo with an amount of topopyrone, effective to inhibit the growth of said cancer cell by poisoning both topoisomerase I and topoisomerase II.

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4. A composition for the poisoning of both mammalian topoisomerase I and mammalian topoisomerase II comprising a topopyrone or a structural analogue of topopyrone.

5. A composition comprising a topopyrone selected from the group consisting of topopyrones 0 A - D, S-chlorotoropyrone C, 5-chlorotopopyrone D, or the topopyrone derivatives of Figure 12, wherein said topopyrone is the product of chemical synthesis, and wherein said topopyrone poisons both topoisomerase I and topoisomerase IL

6. A composition comprising a topopyrone selected from the group consisting of topopyrones 5 A - D 5-chtorotoropyroπe C, 5-chlorotopopyrone D, or the topopyrone derivatives of Figure 12, wherein said topopyrone is the product of chemical synthesis, and wherein said topopyrone inhibits cancer cell growth by poisoning topoisomerase I and topoisomerase IL

7. The composition of claim 5 or 6 further comprising a carrier. 0

8. A process for treating cancer comprising administering to a patient in need thereof a chemically synthesized topopyrone or derivative thereof in an amount sufficient to poison both topoisomerase I and topoisomerase II.

9. A process for treating cancer cells comprising contacting said cancer cells with a chemically synthesized topopyrone or derivative thereof in an amount sufficient to poison both topoisomerase I and topoisomerase II in said cancer cells.

10. A composition comprising a 5-chlorotopopyrone, wherein said 5-chIorotopopyrone poisons both topoisomerase I and topoisomerase IL

1 1. A composition comprising at least one of the topopyrone derivatives as shown in Figure 12, wherein said topopyrone poisons both topoisomerase I and topoisomerase II.

12. The composition of claim 10, or 11 further comprising a carrier.

13. A pharmaceutical formulation comprising the topopyrone of claims 4, 5, 6, 10, or 1 1.