WO2009140467A1

WO2009140467A1 - Oxobenzindolizinoquinolines and uses thereof

Info

Publication number: WO2009140467A1
Application number: PCT/US2009/043905
Authority: WO
Inventors: Mark S. Cushman; Maris A. Cinelli; Andrew E. Morrell; Yves G. Pommier
Original assignee: Purdue Research Foundation; THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY OF THE DEPARTENT OF HEALTH & HUMAN SERVICES
Priority date: 2008-05-15
Filing date: 2009-05-14
Publication date: 2009-11-19

Abstract

The synthesis of aromathecins, substituted 12H-5,l la-diazadibenzo[b,h]fluoren- 11 -ones is described. Use of these cytotoxic compounds and pharmaceutical compositions containing them for the treatment of cancer is described. Two novel processes for the synthesis of this system and a series of 14-substituted aromathecins as novel cytotoxic, topoisomerase I poisons are described.

Description

OXOBENZINDOLIZINOQUINOLINES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U. S. C. § 119(e) to U.S. Patent Application Serial No. 61/053,338, filed May 15, 2008, the disclosure of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This invention relates to oxobenzindolizinoquinoline compounds, their preparation and their uses, such as in treating cancers.

BACKGROUND AND SUMMARY

Topoisomerase I (topi) is an enzyme that is crucial for DNA replication and transcription. Through these normal cellular processes, duplex DNA acquires a considerable degree of both positive and negative supercoiling. Topi solves the topological problems supercoiling causes to allow efficient replication and transcription. Mechanistically, it is believed that topi acts through a nucleophilic tyrosine residue that nicks a single strand of the phosphodiester backbone of DNA and allows a "controlled rotation" of the DNA about the non- scissile strand, thus relaxing the double helix (Pommier, Y., Topoisomerase I inhibitors: camptothecins and beyond. Nature Reviews Cancer. 2006, 6, 789-802; Wang, J. C, Cellular roles of DNA topoisomerases: a molecular perspective. Nature Rev. MoI. Cell Biol. 2002, 3, 430-440.) The disclosure of the foregoing is incorporated herein in its entirety by reference. In addition, the entirety of the disclosures of each of the documents cited herein are also incorporated herein by reference. Topi is preferentially expressed in the S-phase of the cell cycle and has been found in high levels in several solid human tumors, (Luzzio, M. J.; Besterman, J. M.; Emerson, D. L.; Evans, M. G.; Lackey, K.; Leitner, P. L.; Mclntyre, G.; Morton, B.; Myers, P. L.; Peel, M.; Sisco, J. M.; Sternbach, D. D.; Tong, W.; Truesdale, A.; Uehling, D. E.; Vuong, A.; Yates, J. Synthesis and Antitumor Activity of Novel Water Soluble Derivatives of Camptothecin as Specific Inhibitors of Topoisomerase I. J. Med. Chem. 1995, 38, 395-401; Husain, L; Mohler, J. L.; Seigler, H. F.; and Besterman, J. M. Elevation of Topoisomerase I Messenger RNA, Protein, and Catalytic Activity in Human Tumors: Demonstration of Tumor-Type Specficity and Implications for Cancer Chemotherapy. Cancer. Res. 1994, 54 (2), 539-546). It is considered to be an attractive target for the design of cancer chemotherapeutics (Pommier, 2006; Thomas, C. J.; Rahier, N. J.; and Hecht, S. M.; Camptothecin: current perspectives. Bioorg. Med. Chem. 2004, 12, 1585-1604; Meng, L-H, Liao, Y-Z, Pommier, Y. Non- camptothecin DNA topoisomerase I inhibitors in cancer chemotherapy. Curr. Topics. Med. Chem. 2003, 3, 305-20.; Bailly, C. Topoisomerase I poisons and suppressors as anticancer drugs. Curr. Med. Chem. 2000, 7, 39-58). In 1966, Wall and Wani isolated the cytotoxic alkaloid camptothecin (1)

1 from the Chinese tree Camptotheca acuminate (Wall, M. E.; Wani, M. C; Cook, C. E.; Palmer, K. H.; A.T., M.; Sim, G. A. The Isolation and Structure of Camptothecin, a Novel Alkaloidal Leukemia and Tumor Inhibitor from Camptotheca acuminata. J. Am. Chem. Soc. 1966, 88, 3888-3890). Camptothecin and its semi-synthetic analogues such as topotecan (2) and irinotecan (3) inhibit topi by intercalating into the DNA-enzyme complex. It is believed that the steric bulk of the inhibitors prevents re-ligation of the nicked DNA strand by topi and subsequent release of the enzyme, thus "poisoning" the cleavage complex and triggering apoptosis (Pommier, 2006; Staker, B. L.; Hjerrild, K.; Feese, M. D.; Behnke, C. A.; Burgin Jr., A. B.; Stewart, L. The Mechanism of Topoisomerase I Poisoning by a Camptothecin Analog. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15387-15392.; Staker, B. L.; Feese, M. D.; Cushman, M.; Pommier, Y.; Zembower, D.; Stewart, L.; and Burgin, A. Structures of Three Classes of Anticancer Agents Bound to the Human Topoisomerase I-DNA Covalent Complex. J. Med. Chem. 2005, 48, 2336-2345; Li, T. K.; and Liu, L. F. Tumor cell death induced by topoisomerase-targeting drugs. Annu. Rev. Pharmacol. Toxicol., 2001, 41, 53-77).

Efforts to improve the solubility and potency of camptothecin have provided 2 and 3, (Pommier, 2006; Thomas, et al, 2004; Kohn, et al, 2000; Pommier, Y., et al, 1998) the only FDA-approved topi inhibitors for the treatment of cancer (Thomas, et al., 2004; Kohn, K. W.; Pommier, Y. Molecular and biological determinants of the cytotoxic actions of camptothecins. Perspective for the development of new topoisomerase I inhibitors. Ann. N. Y. Acad. Sci. 2000, 922, 11-26; Pommier, Y.; Pourquier, P.; Fan, Y.; Strumberg, D. Mechanism of Action of Eukaryotic DNA Topoisomerase I and Drugs Targeted to the Enzyme. Biochim. Biophys. Acta 1998, 1400, 83-106; Wall, M. E.; Wani, M. C; Nicholas, A. W.; Manikumar, G.; Tele, C; Moore, L.; Truesdale, A.; Leitner, P. and Besterman, J. M.; Plant Antitumor Agents. 30.1a,b Synthesis and Structure Activity of Novel Camptothecin Analogs. J. Med Chem. 1993, 36, 2689-2700. Xie, Z.; Ootsu, K.; and Akimoto, H. Convergent approach to water-soluble camptothecin derivatives. Bioorg. Med. Chem. Lett., 1995, 5(19), 2189-2194; Wani, M. C; Nicholas, A. W.; and Wall, M. E., Plant Antitumor Agents., 231. Synthesis and Antileukemic Activity of Camptothecin Analogues, J. Med Chem., 1986, 29, 2358-2363; Jew, S-s.; Kim, M. G.; Kim, H-J.; Rho, E-Y.; Park, H-g.; Kim, J-K.; Han, H-J.; and Lee, H., Synthesis and In Vitro Cytotoxicity of C(20)(RS)-Camptothecin Analogues Modified at Both B (or A) and E Ring. Bioorg. Med. Chem. Lett. 1998, 8, 1797-1800; Ahn, S. K.; Choi, N. S.; Jeong, B. S.; Kim, K. K.; Journ, D. J.; Kim, J. K.; Lee, S. J.; Kim, J. W.; Hong, C. L; and Jew, S., Practical Synthesis of (S)-7-(2-isopropylamino)ethylcamptothecin Hydrochloride, Potent Topoisomerase I Inhibitor., J. Heterocyclic Chem. 2000, Sep-Oct, 1141; Wani, M. C; Ronman, P. E.; Lindley, J. T.; Wall, M. E., Plant Antitumor Agents; Synthesis and Biological Activity of Camptothecin Analogues., J. Med. Chem., 1980, 23, 554-560.). Despite the clinical success of these compounds, camptothecin derivatives exhibit poor solubility, reversibility of cleavage-complex formation, and dose-limiting toxicity (Pommier, 2006; Luzzio, et al., 1995; Pommier; 1998; Gottlieb, J. A.; Luce, J. K. Treatment of Malignant Melanoma with Camptochecin (NSC- 100880). Cancer Chemother. Rep. 1972, 56, 103-105). Drug efflux pumps and resistance mutations also limit their efficacy (Nakagawa, H.; Saito, H.; Ikegami, Y.; Aida-Hyugaji, S.; Sawada, S.; and Ishikawa, T. Molecular modeling of new camptothecin analogues to circumvent ABCG2 -mediated drug resistance in cancer. Cancer Letters 2006, 234, 81-89; Chrencik, J. E.; Staker, B. L.; Burgin, A. B., Jr.; Pourquier, P.; Pommier, Y.; Stewart, L; and Redinbo, M. R. Mechanism of Camptothecin Resistance by Human Topoisomerase I Mutations. J. MoI. Biol. 2004, 339, 773-784).

Without being bound by theory, it is believed herein that a limitation of the camptothecins, including analogs such as topotecan and irinotecan, is in the E-ring lactone present in that family, which has been reported to exist in equilibrium with its ring-open, hydroxycarboxylate form in vivo (see, e.g., Pommier, 2006; Luzzio, et al., 1995). While that hydroxyacid form retains some of its potency, it possesses a high affinity for human serum albumin (Jaxel, C; Kohn, K. W.; Wani, M. C; Wall, M. E.; Pommier, Y., Structure-Activity Study of the Actions of Camptothecin Derivatives on Mammalian Topoisomerase I: Evidence for a Specific Receptor Site and a Relation to Antitumor Activity, Cancer Res. 1989, 49, 1465- 1469; Haas, N. B.; LaCreta, F. P.; Walczak, J.; Hudes, G. R.; Brennan, J. M.; Ozols, R. F.; O'Dwyer, P. J. Phase-I Pharmacokinetic Study of Topotecan by 24-Hour Continuous-Infusion Weekly. Cancer Res. 1994, 54, (5), 1220-1226). The indenoisoquinolines, a class of cytotoxic non-camptothecin topi poisons based on the lead compound NSC 314622 (6), were developed as an alternative to the camptothecins (Kohlhagen, G.; Paull, K. D.; Cushman, M.; Nagafuji, P.; and Pommier, Y. Protein-Linked DNA Strand Breaks Induced by NSC 314622, a Novel Noncamptothecin Topoisomerase I Poison. MoI. Pharm. 1998, 54, 50-58; Nagarajan, M.; Morrell, A.; Fort, B. C; Meckley, M. R.; Antony, S.; Kohlhagen, G.; Pommier, Y.; and Cushman, M. Synthesis and Anticancer Activity of Simplified Indenoisoquinoline Topoisomerase I Inhibitors Lacking Substituents on the Aromatic Rings. J. Med. Chem. 2004, 47 (23), 5651-5661; Morrell, A.; Placzek, M.; Parmley, S.; Grella, B.; Antony, S.; Pommier, Y.; and Cushman, M. Optimization of the Indenonone Ring of Indenoisoquinoline Topoisomerase I Inhibitors. J. Med. Chem. 2007, 50, 4388-4404; Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; and Cushman, M., Synthesis of nitrated indenoisoquinolines as topoisomerase I inhibitors. Bioorg. Med. Chem. Lett. 2004, 14, 3659-3663). Pre-clinical development of several indenoisoquinolines has recently begun (Antony, S.; Agama, K. K.; Miao, Z-H.; Takagi, K.; Wright, M. H.; Robles, A. L; Varticovski, L.; Nagarajan, M.; Morrell, A.; Cushman, M.; and Pommier, Y., Novel Indenoisoquinolines NSC 725775 and NSC 724998 Produce Persistent Topoisomerase I Cleavage Complexes and Overcome Multidrug Resistance. Cancer Res. 2007, 67 (21), 10397-10405).

The aromathecin class of topi poisons, i.e. those compounds having an oxobenzindolizinoquinoline ring system, have been previously described as stable hybrids of indenoisoquinolines and camptothecins (Fox, B. M.; Xiao, X.; Antony, S., Kohlhagen, G.; Pommier, Y.; Staker, B. L.; Stewart, L.; and Cushman, M. Design, Synthesis, and Biological Evaluation of Cytotoxic 11-Alkenylindenoisoquinoline Topoisomerase I inhibitors and

Indenoisoquinoline-Camptothecin Hybrids. J. Med Chem. 2003, 46, 3275-3282; Xiao, X.; Antony, S.; Pommier, Y.; and Cushman, M., Total Synthesis and Biological Evaluation of 22- Hydroxyacuminatine. J. Med Chem. 2006, 49, 1408-1412; Pin, F.; Comesse, S.; Sanselme, M.; and Daϊch, A. A Domino, N-Amidoacylation/Aldol-Type Condensation Approach to the Synthesis of the Topo-I Inhibitor Rosettacin and Derivatives. J. Org. Chem. 2008, 73 (5), 1975- 1978; Cheng, K.; Rahier, N. J.; Eisenhauer, B. M.; Gao, R.; Thomas, S. J.; and Hecht, S. M. 14- Azacamptothecin: A Potent Water-Soluble Topoisomerase I Poison. J. Am. Chem. Soc. 2005, 127, 3, 838-839). 22-Hydroxyacuminatine (8), a rare natural product isolated from Camptotheca acuminate, contains the 12H-5,l la-diazadibenzo[δ,/z]fluoren-l l-one system, of which the unsubstituted core has been named "rosettacin" (9),

4 3

with substituted compounds being named "aromathecins"(Xiao, et al., 2006; Lin, L. Z.; Cordell, G. A., Quinoline Alkaloids from Camptotheca acuminata. Phytochemistry 1989, 28, 1295-1297; Cheng, et al., 2005; Xiao, et al., 2006; Pin, et al., 2008).

The synthesis of rosettacin 9 and the 8,9-bismethoxy derivative 10 have been described. However, these compounds were weak topi poisons and displayed poor cytotoxicity (Fox, et al., 2003). It has been discovered herein that substitution of the 14-position of aromathecins with amines, amino alcohols, and nitrogenous heterocycles, and polyamines confers both improved cytotoxicity and topi inhibition over currently known aromathecins, and improved topi inhibitory activity over 22-hydroxyacuminatine (8). In one embodiment, a compound of formula I

or a pharmaceutically acceptable salt, crystalline form, non crystalline form, hydrate, or solvate thereof is described, wherein:

R^A is hydrogen; or R^A represents one or more substituents selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted amidine, optionally substituted guanidine, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_n-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof; or R^A represents two or more substituents, where two of said substituents are adjacent and taken together with the attached carbons to form a heterocycle, and the remaining substituents if present are selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted amidine, optionally substituted guanidine, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_D-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof; R^B is hydrogen, alkyl, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or Z, where Z is a carboxylic acid or a derivative thereof;

R^D is alkyl substituted with at least one nitrogen or oxygen containing group; and R^E is hydrogen; or R^E represents one or more substituents selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted amino, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_D-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof; or R^E represents two or more substituents, where two of said substituents are adjacent and taken together with the attached carbons to form a heterocycle, and the remaining substituents if present are selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_n-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof.

In another embodiment, the compound of the preceding embodiment wherein R^A is hydrogen, hydroxyalkyl, optionally substituted alkoxy, Z, or R^A represents two or more substituents, where two of said substituents are adjacent and taken together with the attached carbons to form a heterocycle is described. In another embodiment, the compound of any one of the preceding embodiments wherein R^A is hydrogen is described.

The compound of any one of the preceding embodiments wherein R^E is hydrogen, hydroxy, halo, alkyl, optionally substituted alkoxy, acyloxy, or alkylamino. In another embodiment, the compound of any one of the preceding embodiments wherein R^E is hydrogen is describe.

In another embodiment, the compound of any one of the preceding embodiments wherein R^B is hydrogen, optionally substituted alkoxyalkyl, or Z is described. In still another embodiment, the compound of any one of the preceding embodiments wherein R^B is hydrogen is described. In another embodiment the compound of any of the preceding embodiments wherein R^A, R^E, and R^B are all hydrogen is described.

In another embodiment, the compound of any one of the preceding embodiments wherein R^D is hydroxyalkyl, aminoalkyl, azidoalkyl, optionally substituted alkylaminoalkyl, optionally substituted dialkylaminoalkyl, optionally substituted heterocyclylalkyl, optionally substituted heteroarylalkyl, hydroxyalkylaminoalkyl, optionally substituted alkylaminoalkylaminoalkyl, optionally substituted dialkylaminoalkylaminoalkyl, optionally substituted heterocyclylalkylaminoalkyl, optionally substituted heteroarylalkylaminoalkyl, or hydroxyalkylaminoalkylaminoalkyl is described.

In another embodiment, the compound of any one of the preceding embodiments wherein R^D is alkylene-amino-alkylene-NH₂ or alkylene-amino-heteroalkylene-NH₂ is described. In another embodiment, the compound of any one of the preceding embodiments wherein R^D is CH₂NH(CH₂)_PNH₂, where p is 2 to about 12 is described.

In another embodiment, the compound of any one of the preceding embodiments wherein R^D is (2-hydroxyethyl)aminoalkyl is described. In another embodiment, the compound of any one of the preceding embodiments wherein R^D is heterocycloalkyl is described. In another embodiment, the compound of any one of the preceding embodiments wherein R^D is imidazolylalkyl is described.

In one embodiment, a pharmaceutical composition comprising a therapeutically effective amount of the compound of any one of the preceding embodiments, and one or more pharmaceutically acceptable carriers, diluents, or excipients therefor, or a combination thereof is described.

In another embodiment, a method for treating a cancer, the method comprising the step of administering to a patient in need of relief from the cancer a therapeutically effective amount of the compound or the composition of one of the preceding embodiments is described. In another embodiment, the method of the preceding embodiment wherein the composition is adapted for parenteral or oral administration is described.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. Ligand overlay of aromathecin 27d (b) and indenoisoquinoline 4 (a). The positions of the lactam nitrogen and 14-position are indicated. The carboxylate group of 4 is perpendicular to the plane of the ring system.

FIGURE 2. Topi -mediated DNA cleavage induced by aromathecins 27a, d, g, i, 28d, and 28f. Lane 1 : AG; Lane 2: DNA alone; Lane 3: Topi alone; Lane 4: camptothecin (1), 1 μM; Lane 5: MJ-III-65 (5), 1 μM; Lane 6: rosettacin (9), 100 μM; Lanes 7-30 (for compounds 27a, d, g, i, 28d, and 28f: Topi + indicated compound at 0.1, 1, 10, and 100 μM, respectively.

FIGURE 3. Hypothetical model for the binding of aromathecin 27d in the ternary complex of DNA, topi, and the inhibitor. The diagram is programmed for wall-eyed (relaxed) viewing. DETAILED DESCRIPTION

In one embodiment, a compound of formula I

R^A is hydrogen; or R^A represents one or more substituents selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted amidine, optionally substituted guanidine, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_D-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof; or R^A represents two or more substituents, where two of said substituents are adjacent and taken together with the attached carbons to form a heterocycle, and the remaining substituents if present are selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted amidine, optionally substituted guanidine, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_D-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof;

R^B is hydrogen, alkyl, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or Z, where Z is a carboxylic acid or a derivative thereof;

R^D is alkyl substituted with at least one nitrogen or oxygen containing group; and R^E is hydrogen; or R^E represents one or more substituents selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted amino, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_n-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof; or R^E represents two or more substituents, where two of said substituents are adjacent and taken together with the attached carbons to form a heterocycle, and the remaining substituents if present are selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_n-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof.

In this and other embodiments described herein, it is understood that the compounds may be neutral or may be one or more pharmaceutically acceptable salts, crystalline forms, non crystalline forms, hydrates, or solvates, or a combination of the foregoing.

Accordingly, all references to the compounds described herein may refer to the neutral molecule, and/or those additional forms thereof collectively and individually from the context.

In another embodiment, the compound of any one of the preceding embodiments wherein R^E is hydrogen, hydroxy, halo, alkyl, optionally substituted alkoxy, acyloxy, or alkylamino. In another embodiment, the compound of any one of the preceding embodiments wherein R^E is hydrogen is describe.

In another embodiment, the compound of any one of the preceding embodiments wherein R^D is hydroxyalkyl, aminoalkyl, azidoalkyl, optionally substituted alkylaminoalkyl, optionally substituted dialkylaminoalkyl, optionally substituted heterocyclylalkyl, optionally substituted heteroarylalkyl, hydroxyalkylaminoalkyl, optionally substituted alkylaminoalkylaminoalkyl, optionally substituted dialkylaminoalkylaminoalkyl, optionally substituted heterocyclylalkylaminoalkyl, optionally substituted heteroarylalkylaminoalkyl, or hydroxyalkylaminoalkylaminoalkyl is described. In another embodiment, the compound of any one of the preceding embodiments wherein R^D is alkylene-amino-alkylene-NH2 or alkylene-amino-heteroalkylene-NH2 is described. In another embodiment, the compound of any one of the preceding embodiments wherein R^D is CH₂NH(CH₂)pNH2, where p is 2 to about 12 is described.

In another embodiment, the compound of any one of the preceding embodiments wherein R^D is (2-hydroxyethyl)aminoalkyl is described. In another embodiment, the compound of any one of the preceding embodiments wherein R^D is heterocycloalkyl is described. In another embodiment, the compound of any one of the preceding embodiments wherein R^D is imidazolylalkyl is described. In another embodiment, a compound of formula

R = NH₂, NHR', NR'₂, N₃, Cl,

NH(CH₂)_PNH₂, heterocycle; R' = alkyl, heteroalkyl; n = 1 ,3; p = 2-12 is described.

In another illustrative embodiment, a method for treating a cancer, the method comprising the step of administering to a patient in need of relief from the cancer a therapeutically effective amount of the compound of formula I

R^B is hydrogen, alkyl, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or Z, where Z is a carboxylic acid or a derivative thereof; R^D is alkyl substituted with at least one nitrogen or oxygen containing group; and

R^E is hydrogen; or R^E represents one or more substituents selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted amino, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_D-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof; or R^E represents two or more substituents, where two of said substituents are adjacent and taken together with the attached carbons to form a heterocycle, and the remaining substituents if present are selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_D-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof.

In another embodiment, the method of the preceding embodiment wherein R^A is hydrogen, hydroxyalkyl, optionally substituted alkoxy, Z, or R^A represents two or more substituents, where two of said substituents are adjacent and taken together with the attached carbons to form a heterocycle is described. In another embodiment, the method of any one of the preceding embodiments wherein R^A is hydrogen is described.

The method of any one of the preceding embodiments wherein R^E is hydrogen, hydroxy, halo, alkyl, optionally substituted alkoxy, acyloxy, or alkylamino. In another embodiment, the method of any one of the preceding embodiments wherein R^E is hydrogen is describe.

In another embodiment, the method of any one of the preceding embodiments wherein R^B is hydrogen, optionally substituted alkoxyalkyl, or Z is described. In still another embodiment, the method of any one of the preceding embodiments wherein R^B is hydrogen is described. In another embodiment the method of any of the preceding embodiments wherein R^A, R^E, and R^B are all hydrogen is described.

In another embodiment, the method of any one of the preceding embodiments wherein R^D is hydroxyalkyl, aminoalkyl, azidoalkyl, optionally substituted alkylaminoalkyl, optionally substituted dialkylaminoalkyl, optionally substituted heterocyclylalkyl, optionally substituted heteroarylalkyl, hydroxyalkylaminoalkyl, optionally substituted alkylaminoalkylaminoalkyl, optionally substituted dialkylaminoalkylaminoalkyl, optionally substituted heterocyclylalkylaminoalkyl, optionally substituted heteroarylalkylaminoalkyl, or hydroxyalkylaminoalkylaminoalkyl is described. In another embodiment, the method of any one of the preceding embodiments wherein R^D is alkylene-amino-alkylene-NH2 or alkylene-amino-heteroalkylene-NH2 is described. In another embodiment, the method of any one of the preceding embodiments wherein R^D is CH₂NH(CH₂)_pNH₂, where p is 2 to about 12 is described. In another embodiment, the method of any one of the preceding embodiments wherein R^D is (2-hydroxyethyl)aminoalkyl is described. In another embodiment, the method of any one of the preceding embodiments wherein R^D is heterocycloalkyl is described. In another embodiment, the method of any one of the preceding embodiments wherein R^D is imidazolylalkyl is described. In another embodiment, the method of the preceding embodiment wherein the composition is adapted for parenteral or oral administration is described.

Also described herein are pharmaceutical compositions and formulations comprising a therapeutically effective amount of one or more aromathecin compounds for treating a patient having cancer. It is appreciated that mixtures of certain aromathecin compounds may be administered. Such pharmaceutical compositions may also include one or more diluents, carriers, and/or excipients. As used herein, an effective amount of the aromathecin compound is defined as the amount of the compound which, upon administration to a patient, inhibits growth of cancer cells, kills malignant cells, reduces the volume or size of the tumors, and/or eliminates the tumor entirely in the treated patient. It is to be understood that treated patients include humans and other mammals.

As used herein, the term "therapeutically effective amount" refers to the amount to be administered to a patient, and may be based on body surface area, patient weight, and/or patient condition. In addition, it is appreciated that there is an interrelationship of dosages determined for humans and those dosages determined for animals, including test animals (illustratively based on milligrams per meter squared of body surface) as described by Freireich, E. J., et al, Cancer Chemother. Rep. 1966, 50 (4), 219, the disclosure of which is incorporated herein by reference. Body surface area may be approximately determined from patient height and weight (see, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, New York, pages 537-538 (1970)). A therapeutically effective amount of the aromathecin compounds described herein may be defined as any amount useful for inhibiting the growth of (or killing) a population of malignant cells or cancer cells, such as may be found in a patient in need of relief from such cancer or malignancy. Typically, such effective amounts range from about 5 mg/kg to about 500 mg/kg, from about 5 mg/kg to about 250 mg/kg, and/or from about 5 mg/kg to about 150 mg/kg of aromathecin compounds per patient body weight. It is appreciated that effective doses may also vary depending on the route of administration, optional excipient usage, and the possibility of co-usage of the aromathecin compounds with other conventional and non-conventional therapeutic treatments, including other anti-tumor agents, radiation therapy, and the like.

For the treatment of cancer, illustratively the aromathecin compounds described herein may be formulated in a therapeutically effective amount in conventional dosage forms, including one or more carriers, diluents, and/or excipients. Such formulation compositions may be administered by a wide variety of conventional routes in a wide variety of dosage formats, utilizing art-recognized products. See generally, Remington: The Science and Practice of

Pharmacy, (21^st ed., 2005). It is to be understood that the compositions described herein may be prepared from isolated compounds described herein or from salts, solutions, hydrates, solvates, and other forms of the compounds described herein. It is also to be understood that the compositions may be prepared from various amorphous, non-amorphous, partially crystalline, crystalline, and/or other morphological forms of the compounds described herein.

In making the formulations of the aromathecins described herein, a therapeutically effective amount of the inhibitor in any of the various forms described herein may be mixed with an excipient, diluted by an excipient, or enclosed within such a carrier which can be in the form of a capsule, sachet, paper, or other container. Excipients may serve as a diluent, and can be solid, semi-solid, or liquid materials, which act as a vehicle, carrier or medium for the active ingredient. Thus, the formulation compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. The compositions may contain anywhere from about 0.1% to about 99.9% active ingredients, depending upon the selected dose and dosage form. Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates; sweetening agents; and flavoring agents. The compositions can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. It is appreciated that the carriers, diluents, and excipients used to prepare the compositions described herein are advantageously GRAS (Generally Regarded as Safe) compounds. The aromathecin compounds may be administered in a variety of pharmaceutical formulations, including conventional pharmaceutical formulations. The aromathecin compounds, and formulated variants thereof, may also be delivered by a variety of administration routes, including conventional delivery routes. In one embodiment, the aromathecin compounds, and formulated variants thereof, are delivered via a parenteral route, including subcutaneously, intraperitoneally, intramuscularly, and intravenously. Examples of parenteral dosage forms and formulations include aqueous solutions of the aromathecin compounds in isotonic saline, 5% glucose or other conventional pharmaceutically acceptable liquid carrier. In one aspect, the one or more aromathecin compounds are dissolved in a saline solution containing 5% dimethyl sulfoxide and 10% Cremphor EL (Sigma Chemical Company). Additional solubilizing agents such as cyclodextrins, which can form specific, more soluble complexes with the aromathecin compounds described herein, or other conventional solubilizing agents can be included as pharmaceutical excipients for delivery of the compounds.

In another embodiment, the aromathecin compounds, and formulated variants thereof, are delivered via oral administration, such as in a capsule, a gel seal, a tablet, and the like. Capsules may comprise any conventional pharmaceutically acceptable material including gelatin and/or cellulose derivatives. Tablets may be formulated by conventional procedures, including by compressing mixtures of the aromathecin compounds, solid carriers, lubricants, disintegrants, and other conventional ingredients for solid dosage forms, such as starches, sugars, bentonite, and the like. The compounds described herein may also be administered in a form of a hard shell tablet or capsule containing, for example, lactose or mannitol as a binder, and conventional fillers and tableting agents. Solid dosage forms described herein and useful for delivering the aromathecin compounds also include sustained release formulations, such as tablets, caplets, pills, capsules, and the like that include an enteric coating that may delay the release of the aromathecin compounds until the formulation has passed into the intestinal tract.

Any effective regimen for administering the aromathecins can be used. For example, the aromathecin compounds can be administered as single doses, or they can be divided and administered as a multiple-dose daily regimen. Further, a staggered regimen, for example, one to three days per week can be used as an alternative to daily treatment, and for the purpose of defining this invention such an intermittent or staggered daily regimen is considered to be equivalent to every day treatment and within the scope of this invention. In one embodiment of the invention the patient is treated with multiple injections of the aromathecin. In one embodiment, the patient is treated, for example, injected multiple times with the aromathecin at, for example, at 12-72 hour intervals or at 48-72 hour intervals. Additional injections of the aromathecin compound can be administered to the patient at intervals of days or months after the initial injections, and the additional injections may prevent recurrence of disease.

The syntheses of a new series of 14-substituted aromathecins via two novel routes that proceed through tricyclic ketone 23 (Shamma, M.; Novak, L., Synthetic Approaches to Camptothecin. Tetrahedron 1969, 25, 2275-2279) are described. In one embodiment, substitution of the analogous 7-position of camptothecin with hydrogen bond donor-acceptor groups capable of increasing solubility improves biological activity over the parent compound (Luzzio, et al, 1995; Xie, et al, 1986; Jew, et al, 1998; Ahn, et al, 2000). For indenoisoquinolines, groups such as amino, imidazole, morpholine, and Λ/,Λ/-dimethylamine, located on the lactam nitrogen at a distance of 2-3 methylene units from the aromatic core, confer topi inhibition and cytotoxicity (Nagarajan, M.; Morrell, A.; Fort, B. C; Meckley, M. R.; Antony, S.; Kohlhagen, G.; Pommier, Y.; and Cushman, M., Synthesis and Anticancer Activity of Simplified Indenoisoquinoline Topoisomerase I Inhibitors Lacking Substituents on the Aromatic Rings., J. Med. Chem. 2004, 47 (23), 5651-5661; Morrell, A.; Placzek, M. S.; Steffen, J. D.; Antony, S.; Agama, K.; Pommier, Y.; and Cushman, M. Investigation of the Lactam Side Chain Length Necessary for Optimal Indenoisoquinoline Topoisomerase I

Inhibition and Cytotoxicity in Human Cancer Cell Cultures., J. Med. Chem. 2007, 50, 2040- 2048.). Without being bound by theory, it is believed that in addition to solubilizing the aromatic core, molecular modeling studies described herein indicate that substituents at the 14- position of the aromathecin core and camptothecin's 7-position may occupy the same region in space as the lactam substituents of indenoisoquinolines, projecting out into the major groove of the DNA-topl complex. These studies are consistent with the solved crystal structures of indenoisoquinolines in ternary complex with DNA and topi (Staker, et al., 2005). It is believed herein that these substituents hydrogen bond with water and major-groove amino acids and increase the stability of the ternary complex. A ligand overlay of the crystal structure of the indenoisoquinoline (4)-topl-DNA ternary complex (Staker, et al., 2005) and a hypothetical model of the aromathecin 27d-topl-DNA ternary complex, showing this substituent overlap, is displayed in FIGURE 3. In another embodiment mono- and tri-methylene analogues 27a-k and 28a-g were designed and synthesized. As shown in FIGURE 3, the aromatic core of compound 27d is calculated to intercalate between the base pairs without steric hindrance and is likely stabilized by π-stacking interactions. The imidazolyl group projects on the outer range of H-bonding distance with Asn352 (heavy-atom distance of 3.77 A). It is believed that the imidazolyl group may be able to rotate somewhat to make this contact. In another illustrative example, models indicate that the monomethylene analogues may make hydrogen bonding contacts with Asn352, Thr747, and the carbonyl of Ile427 (structures not shown). Without being bound by theory, it is also believed that the pyridine nitrogen of the aromathecin core faces the minor groove, where it appears to interact with Arg364, similar to the interaction seen with the camptothecin class of topi inhibitors. The presence of a lone pair of electrons at this position also appears to be important for many classes of topi inhibitors, such as indenoisoquino lines and indolocarbazoles (Staker, et al, 2005).

It is has been observed by X-ray crystallography that the lactam substituents of indenoisoquinolines project into the major groove of the ternary complex (Staker, et al., 2005). Computer models show that the 14-position substituents of aromathecins can project into the same region, where they are believed to interact favorably with water and amino acids such as ASN 352. Computer modeling of compound 53, the most potent topi inhibitor from the diaminoalkyl series, in ternary complex with topi and DNA shows that the aromathecin core can intercalate between the base pairs, where the quinoline nitrogen faces toward the minor groove and may interact with Arg364. Without being bound by theory, it is believed that, despite being out of hydrogen-bonding distance (4.47 A) in this model (this contact is closer in other aromathecin models) a hydrogen could be possible via induced fit (Cinelli, M. A.; Morrell, A.; Dexheimer, T.; Scher, E.; Pommier, Y.; and Cushman, M. Design, Synthesis, and Biological Evaluation of 14-Substituted Aromathecins as Topoisomerase I Inhibitors. J. Med. Chem. 2008, 51 (15), 4609-4619). In this model, the diamine side chain projects into the major groove, where it is proposed to hydrogen bond with a flanking nucleobase. Without being bound by theory, it is believed that water molecules may participate in these interactions, forming a network of water-mediated H-bonds and polar contacts between the positively charged amines and a nearby nucleobase and Asn352. Without being bound by theory, it is believed that in this well-solvated environment, increasingly hydrophobic side-chains (such as those over eight carbons as in 60-63) would yield unfavorable interactions by possibly disturbing this intricate network of water molecules. It is further appreciated that accomadation of larger side chains in the ternary complex may also be hindered by steric or entropic factors. The discovery that the variation in inhibition of topi by aromathecin diamines is similar to the variation of inhibition of topi by analogous indenoisoquino lines lends additional support to the binding mode proposed herein (where the 14-position faces the major groove). Molecular modeling of a model of 53 in ternary complex with a model of a representative indenoisoquino line, also in a ternary complex was constructed based on overlays of the crystal structures. It is observed that the aromatic regions are approximately in the same position, the side chains occupy nearly identical regions, and both compounds appear to be capable of interacting with Asn352 and nearby water molecules.

Several routes to aromathecin derivatives have been developed. The synthesis of 22-Hydroxyacuminatine (8), rosettacin (9), and compound 10 via the condensation of pyrroloquinoline and appropriately substituted phthalide derivatives has been described (Fox, et al, 2003; Xiao, et al, 2006). Other routes to 22-hydroxyacuminatine and the 12H-5,1 Ia- diazadibenzo[δ,/z]fluoren-l 1-one system include pyridone benzannulation and Ηeck coupling (Babjak, M.; Kanazawa, A.; Anderson, R. J, and Greene, A. E. Concise synthesis of 22- hydroxyacuminatine, cytotoxic camptothecinoid from Camptotheca acuminata, by pyridone benzannulation. Org. Biomol. Chem. 2006, 4, 407-409). Recently, Daϊch, et al., have published a novel route to rosettacin and 14-methyl- and 14-phenylaromathecin employing an N-amidoacylation/aldol condensation with a benzotriazole ester to form the key intermediate (Pin, et al., 2008). In one embodiment, 14-substituted aromathecins 27a-k and 28a-g were prepared from known oxatricyclic ketone 23 ((Shamma, et al., 1969); Scheme 1). This compound was prepared by two new routes, both beginning with commercially available amino acids. The first route is outlined in Scheme 1. Beginning with the ethyl carbamate 12 of methyl glycinate (11), 3-pyrrolidinone ethylene ketal (15) was prepared via a one-pot acrylation-Dieckmann condensation and decarboxylation, followed by ketalization of carbamate 13 to yield 14 (Bhaskar Kanth, J. V.; Periasamy, M., Convenient Method for the Synthesis of N- (Ethyloxycarbonyl) Ester Derivatives from Amino Acids. Synth. Commun. 1995, 25, 1523- 1530; Dyke, S. F.; Sainsbury, M.; Evans, J. R. Lycorine: Studies in Synthesis., Tetrahedron 1973, 29, 213-220; Roglans, A.; Marquet, J.; Moreno-Manas, M., Preparation of 3-Pyrrolidone and 4-Perhydroazepinone. Synth. Commun., 1992, 22, 1249-1258). Removal of the carbamate functionality and reaction with chlorophthalide 17, prepared from 2-carboxybenzaldehyde (16) , yielded ketal 18. Ketal 18 was cyclized directly to 23 using and a combination of polyphosphoric acid and 85% phosphoric acid, following the final step of Shamma and Novak's work (Sloan, K. B.; Koch, S. A. M., Effect of Nucleophilicity and Leaving Group Ability on the SN2 Reactions of Amines with (Acyloxy)alkyl a-Halides: A Product Distribution Study. J. Org. Chem., 1983, 48, 635-640).

H₃CO₂C^NH₂ ^ _o ^_NXO₂B ^ °γ^

HCI ^{3 2} H ^N-CO₂Et

1

^aReagents and Conditions: (a) EtOCOCI, CHCI₃, reflux; (b) ;. NaH, benzene, reflux, ;;. methyl acrylate, reflux, ///^'. 6 M HCI, reflux; (c) ethylene glycol, benzene, reflux; (d) KOH, H₂O, reflux; (e) cat. FeCI₃, SOCI₂, reflux; (f) THF, Et₃N, r.t.; (g) polyphosphoric acid, 85% phosphoric acid, CH₂CI₂, 100 ⁰C.

SCHEME 1

Prior routes give variable yields and loss of material associated with protection- deprotection steps, the route depicted in Scheme 2, a protecting group-free pathway to the key intermediate is described. Preparation of 23 began with catalytic decarboxylation of commercially available £rα/?s-4-hydroxy-L-proline (19) to yield the amino alcohol 20 as its hydrogen maleate salt (Houghton, P. G.; Humphrey, G. R.; Kennedy, D. J.; Roberts, D. C; Wright, S. H. B. Enantiospecifϊc Synthesis of the (4R)-1-Azabicyclo[2.2.1]heptane Ring System. J. Chem. Soc. Perkin Trans. 1. 1993, 1421-1424). Condensation of 20 with 17 provided hydroxyamide 21. Swern oxidation of 21 followed by heating under reflux of the intermediate aldehyde, 22, yielded 23.

Reagents and Conditions: (a) i. cat. 2-cyclohexen-l-one, cyclohexanol, reflux, ii. maleic acid, EtOAc, r.t.; (b) FeCl₃, SOCl₂, reflux; (c) MeHO, Et₃N, r.t.; (d) i. DMSO, (COCl)₂, CH₂Cl₂, -78°C, ii. Et₃N, -78°C to r.t.; reflux

SCHEME 2

In additional embodiments, 14-chloromethylaromathecin 27a and 14- chloropropylaromathecin 28a were prepared by Friedlander condensation of 23 with aminoacetophenone 25 or aminobutyrophenone 26, respectively (see SCHEME 3). These acetophenones were prepared from aniline (24) and chloroacetonitrile or 4-chlorobutyronitrile via the aminohaloborane modification of the Friedel-Crafts acylation (Sugasawa, T.; Toyoda, T.; Adachi, M.; Sasakura, K. Aminohaloborane in Organic Synthesis. 1. Specific Ortho Substitution Reaction of Anilines. J. Am. Chem. Soc, 1978, 100, 4842-4852; Cevasco, A. A. Process for the Manufacture of Cycloalkyl and Haloalkyl o-Aminophenyl Ketones., 5,405,998, 1995). It is anticipated that the synthesis of future aromathecin derivatives can become, in essence, modular, enabling access to numerous substituted aromathecins through 23 and various substituted acetophenones.

aReagents and Conditions: (a) /. BCI₃-Me₂S, 1 ,2,-dichloroethane, 0 ⁰C, //. chloroacetonitrile (25), 4-chlorobutyronitrile (26), AICI₃, reflux. ///. 2 M HCI, reflux; (b) p-TsOH, benzene, reflux.

SCHEME 3

In another embodiment, the benzylic chloride of intermediate 27a is substituted by a variety of nucleophiles in DMSO. Displacement of the chloride by sodium azide yielded 27b, which was converted to amine 27c by Staudinger reduction. Although substitution of 27a with imidazole to provide 27d required higher temperatures, displacement by the remaining amines at room temperature readily afforded analogues 27e-27k (Scheme 4).

27c

aReagents and Conditions: (a) amine or amine salt + Et₃N, DMSO, r.t; (b) NaN₃, DMSO; (c) /. (EtO₃)P, benzene, reflux, /7. 3 M HCI, MeOH, reflux

SCHEME 4

Amine 28c was prepared from azide 28b using the Staudinger methodology as described above (Scheme 5). Without being bound by theory, it is believed that due to the decreased electrophilicity of the terminal chloride of 28a, increased reaction temperatures were required for substitution. In another embodiment, substitution with the selected amines was assisted by adding sodium iodide along with excess amine. Without being bound by theory, it is believed that in situ Finkelstein reaction, followed by displacement of the resulting iodides with the illustrative amines yielded analogues 28d-g, which were isolated as their trifluoroacetate salts.

28d, R = -N b.

28e, R = -N O CF,COOH

^aReagents and Conditions: (a) amine, NaI, DMSO, 100 ⁰C; (b) NaN₃, DMSO, 100 ⁰C (c) /. (EtO₃)P, benzene, reflux, /7. 3 M HCI, MeOH, reflux

SCHEME 5

30a: P = 2 31 P = 2 42 P = 2

30b: P = 3 32 P = 3 43 P = 3

30c: P = 4 33 P = 4 44 P = 4

3Od: P = 5 34 P = 5 45 P = 5

3Oe: P = 6 35 P = 6 46 P = 6

3Of: P = 7 36 P = 7 47 P = 7

3Og: P = 8 37 P = 8 48 P = 8

30h: P = 9 38 P = 9 49 P = 9

30i: P = 10 39 P = 10 50 P = 10

30j: P = 11 40 P = 11 51 P = 11

53 P = 2

54 P = 3

55 P = 4

56 P = 5

57 P = 6

58 P = 7

59 P = 8

60 P = 9

61 P = 10

62 P = 11

63 P = 12

Reagents and Conditions: (a) BoC₂O, CHCl₃, r.t.; (b) DMSO, r.t.; (c) 3 M HCl, MeOH, CHCl₃, or 3 M HCl, MeOH, benzene, reflux

SCHEME 6

Illustrative diaminoalkyl examples 53-63 were prepared by the process shown in SCHEME 6. In another embodiment, the compounds described herein can be prepared via intermediate A shown in SCHEME 7 using the process shown in SCHEME 7.

SCHEME 7

In one embodiment, the aromathecin analogues were assayed for cytotoxic activity in the National Cancer Institute's Developmental Therapeutics screen. Each compound was evaluated against approximately 60 cell lines originating from various human tumors (Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J. New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening. J. Natl. Cancer Inst. 1990, 82, (13), 1107- 1112; Boyd, M. R.; Paull, K. D. Some Practical Considerations and Applications of the

National Cancer Institute In- Vitro Anticancer Drug Discovery Screen. Drug Development Res. 1995, 34, 91-109). After an initial one-dose assay, selected compounds were tested at five concentrations encompassing the range of 10^"8 to 10^"4 molar. Cytotoxicity results are reported as GI₅₀ values for selected cell lines from each sub-panel, and overall cytotoxicity is quantified as a Mid-Graph Midpoint (MGM) in Table 1. The MGM is a measure of the average GI50 against all cell lines tested. The activities of camptothecin (1), indenoisoquinoline MJ-III-65 (5) (Pommier, 2006; Nagarajan, et al., 2004; Antony, S.; Jayaraman, M.; Laco, G.; Kohlhagen, G.; Kohn, K. W.; Cushman, M.; Pommier, Y. Differential Induction of Topoisomerase I-DNA Cleavage Complexes by the Indenoisoquinoline MJ-III-65 (NSC 706744) and Camptothecin: Base Sequence Analysis and Activity against Camptothecin-Resistant Topoisomerase I. Cancer Res. 2003, 63, 7428-7435), NCS314622 (6) (Kohlhagen, et al, 1998), rosettacin (9) and dimethoxyaromathecin (10) (Fox, et al., 2003), in addition to the inhibitory activity of 22- hydroxyacuminatine (8), are described.

In another embodiment, topi inhibition was assayed by measurement of topi - mediated DNA cleavage and inhibition data are expressed semi-quantitatively as follows: 0, no inhibitory activity; +, between 20 and 50% the activity of 1 μM camptothecin (1); ++, between 50 and 75% the activity of 1 μM camptothecin; +++, between 75-100% the activity of 1 μM camptothecin; and ++++, equipotent to or more potent than 1 μM camptothecin. Topi inhibitory data for aromathecins and comparative compounds are also included in Table 1.

Compounds 27b, 27k, and 28a-b, at the rates tested, do not show inhibition of a topi . Nonetheless, compounds 27b, 27k, and 28a were tested in the National Cancer Institute's 60-cell line screen at an initial high dose (10 μM) and were not selected for further testing because of their low cytotoxicities. Compounds 27f, 27k, and 28g, although not selected for 5- dose testing, induced 14%, 25.5%, and 21.4% average growth inhibition, respectively, in the presence of the inhibitor at a concentration of 10 μM across all cell lines tested.

Without being bound by theory, it is believed that among the mono-methylene aromathecin series, 27a-j, substitution at the 14-position of the aromathecin "core" with solubilizing groups capable of forming hydrogen bond donor/acceptor interactions improves topi inhibitory activity relative to 9 and the 14-unsubstituted dimethoxyaromathecin 10. In addition, most aromathecins are better topi inhibitors than 8 (a one "+" inhibitor). FIGURE 4 indicates the presence of topi -mediated DNA breaks induced by aromathecins 27a, d, g, i and 28d and 28f. It has also been observed that the cleavage patterns appear to resemble both those induced by camptothecins and by indenoisoquino lines. Active aromathecin (28f) displays a predominant topi cleavage site at position 62 (FIGURE 4, lanes 27-30), also observed for the identically-substituted indenoisoquinoline 5 (Nagarajan, et al., 2004; Antony, et al., 2003). MGM values were improved over 9 and 10 for the majority of 14-substituents tested. Without being bound by theory, it is believed that these groups vary considerably in size and conformational flexibility, indicating a tolerance by the ternary complex at this position. In one embodiment, the imidazolyl moiety of 27d is effective at improving anti-top 1 activity in the aminomethylene series. The chiral hydroxypyrrolidinyl group of 27i, a group not previously investigated in the development of camptothecins or indenoisoquinolines, also conferred increased anti-top 1 activity relative to rosettacin.

22-Hydroxyacuminatine (8) was also not tested in the National Cancer Institute assay, although previous studies report activity against murine leukemia KB and P388 cell lines (Lin, et al, 1989; Zhou, et al, 2007). However, it was determined in 2006 that 22- hydroxyacuminatine's cytotoxicity did not appear to be topi dependent (Xiao, et al., 2006).

Without being bound by theory, it is believed that the improved topi inhibitory activity and cytotoxicity of 14-substituted aromathecins over the parent compound may be due, in part, to improved solubility as the substituents at the 7-position of camptothecin and the substituents of irinotecan and topotecan greatly enhance activity through solubilizing the aromatic core (Pommier, 2006; Luzzio, et al., 1995; Xie, et al., 1995; Jew, et al., 1998; Ahn, et al., 2000). Without being bound by theory, it is believed that for the aromathecins, this observation may be corroborated, in part, by the inactivity of compounds 27b and 28a-b. No correlation between cytotoxicity, topi inhibition, or cLogP has been observed for the aromathecin class, although it has been observed for certain indenoisoquinolines (Morrell, et al., 2007). For indenoisoquinolines and other compounds, it was observed that an increasing cLogP correlated well with decreasing cytotoxicity, it is believed that this may result from poor solubility, inefficient DNA-targeting, or other hydrophobic effects. Compound 27e, despite having a higher cLogP (3.67) than 9 (3.37), is nearly ten times as cytotoxic and is also a more potent topi inhibitor. Conversely, compound 27k has a lower cLogP (3.32), but is inactive against topi compared to rosettacin 9. In addition, it is noted that compounds 53 and 63 are equipotent, and compound 57, believed to be more hydrophobic than 53, is more active than 53. In other embodiments, analogues 28a-g were prepared. In another embodiment, compound 28f is described. Without being bound by theory, it is believed that certain amino alcohol substitutions, as represented by compound 28f, confer more potent activity (Nagarajan, M., Xiao, X., Antony, S., Kohlhagen, G., Pommier, Y., and Cushman, M. Design, Synthesis, and Biological Evaluation of Indenoisoquinoline Topoisomerase I Inhibitors Featuring Polyamine Side Chains on the Lactam Nitrogen. J. Med. Chem. 2003, 46, 5712-5724). Without being bound by theory, it is also believed that it is possible that 27f acts in a manner similar to indenoisoquinoline 5, which bears an identical side chain (Nagarajan, et al., 2004; Antony, et al., 2003).

Other observations between the cytotoxicity and topi inhibition with some aromathecin analogues have been made. Compound 27a, despite its lower anti-top 1 activity, is more cytotoxic than rosettacin (9). In addition, preliminary assays indicate intense cytotoxicity for compound 28c (-47.5% cell growth in the presence of 10 μM inhibitor, indicating a net cell kill). It appears that the antiproliferative activity of these two compounds is not due to inhibition of topi . It is unknown how these compounds exert their cytotoxic effect. Without being bound by theory, it is believed that the mechanism may be similar to that described for 8. Compounds 56, 57, 59, and 63 were assayed for the ability to inhibit top2-mediated DNA cleavage, however, little inhibitory activity was observed. In addition, at high concentrations, 14-(alkanediaminomethyl)aromathecins do not suppress the formation of topi -DNA cleavage complexes, suggesting that a mechanism of action proceeding through either suppression of topoisomerase binding or though non-specific DNA intercalation is unlikely. (Kohlhagen, et al, 1998), (Boyd, et al, 1995; Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.; Scudiero, D. A.; Rubinstein, L.; Plowman, J.; and Boyd, M. R. Display and Analysis of Patterns of Differential Activity of Drugs Against Human Tumor Cell Lines: Development of Mean Graph and COMPARE Algorithm. J. Natl. Cancer. Inst. 1989, 81, 1088-1092; Paull, K. D.; Hamel, E.; and Malspeis, L.; Prediction of Biochemical Mechanism of Action from the In Vitro

Antitumor Screen of the National Cancer Institute. In Cancer Chemotherapeutic Agents, Foye, W. O., Ed. American Chemical Society: Washington, DC, 1995: pp 9-45).

For topi inhibition, the shorter diaminoalkanes confer good anti-top 1 activity upon the aromathecin core, inducing topi -mediated DNA breakage. Activity generally decreases with increasing side-chain length, however. With the exception of 56, up to six atoms between proximal and distal amine are tolerated readily for aromathecins. Activity decreases beyond this length, and compounds with ten or more carbons are inactive. A similar trend is noted for indenoisoquino lines.

Two novel synthetic routes to aromathecin analogues from inexpensive, commercially available precursors are described. Multiple series of 14-substituted aromathecins,prepared via these routes, and bearing nitrogenous substituents separated from the aromatic core by short "linker" regions are described. A topi inhibitor equipotent to camptothecin is described. These novel "composite" structures of camptothecin and indenoisoquino lines have shown inhibition of human topi and control of numerous human tumor cell lines. These results show that 14-substitution can improve topi inhibitory potency and cytotoxicity. Without being bound by theory, it is believed that this effect may result from a combination of increased solubility (as seen with 7-substituted camptothecins), charge complementarity with DNA, and hydrogen bonding (as proposed for indenoisoquinolines). The results of side-chain elongation were largely variable with respect to both topi inhibition and antiproliferative activity.

It is also appreciated that in the foregoing embodiments, certain aspects of the compounds are presented in the alternative, such as selections for any one or more of Z, R^A, R^B, R^D, R^E, b, y, n, and p. It is therefore to be understood that various alternate embodiments of the invention include individual members of those lists, as well as the various subsets of those lists.

Each of those combinations are to be understood to be described herein by way of the lists.

The compounds described herein may contain one or more chiral centers, or may otherwise be capable of existing as multiple stereoisomers. Accordingly, it is to be understood that the present invention includes pure stereoisomers as well as mixtures of stereoisomers, such as enantiomers, diastereomers, and enantiomerically or diastereomerically enriched mixtures. The compounds described herein may be capable of existing as geometric isomers.

Accordingly, it is to be understood that the present invention includes pure geometric isomers or mixtures of geometric isomers. It is appreciated that the compounds described herein may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention.

The compounds of the present invention may exist in multiple crystalline or amorphous forms.

In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

In another embodiment, the compounds described herein include the following examples. The examples further illustrate additional features of the various embodiments of the invention described herein. However, it is to be understood that the examples are illustrative and are not to be construed as limiting other embodiments of the invention described herein. In addition, it is appreciated that other variations of the examples are included in the various embodiments of the invention described herein.

METHODS AND MATERIALS

General Procedures. Melting points were determined in capillary tubes using a Mel-Temp apparatus and are not corrected. Infrared spectra were obtained as films on salt plates using CHCI3 as the solvent except where otherwise specified, using a Perkin-Elmer Spectrum One FT-IR spectrometer, and are baseline-corrected. ¹H NMR spectra were obtained at 300 or 500 MHz, using a Bruker ARX300 and Bruker Avance 500 (TXI 5 mm probe), respectively. Mass spectral analyses were performed at the Purdue University Campus-Wide Mass Spectrometry Center. Combustion microanalyses were performed at the Purdue University Microanalysis Laboratory and reported values are within 0.4% of calculated values. Analytical thin-layer chromatography was performed on Baker-flex silica gel IB2-F plastic-backed TLC plates. Preparative thin-layer chromatography was performed on Analtech silica gel G 1200 uM glass plates. Compounds were visualized with both short and long-wavelength UV light. Silica gel flash chromatography was performed using 40-63 μm, flash grade silica gel.

EXAMPLE 1 Methyl TV-Ethoxycarbonylglycinate (12) (Zhou, et al, 2007). The hydrochloride salt of methyl glycinate (11) (12.6 g, 0.1 mol) was diluted with CHCl₃ (200 mL). Triethylamine (25.26 g, 0.250 mol) was added, and the reaction mixture was cooled to 0 ⁰C. Ethyl chloroformate (22.21 g, 0.205 mol) was then added, and the mixture was heated at reflux for 24 h. The solution was washed sequentially with H₂O (2 x 150 mL), 10% aq HCl (100 mL) and sat NaCl (100 mL), dried over anhydrous sodium sulfate, and concentrated to provide an orange oil (14.8 g, 92%). ¹H NMR (300 MHz, CDCl₃) δ 5.15 (bs, 1 H), 4.17 (q, J= 7.1 Hz, 2 H), 3.98 (d, J= 5.6 Hz, 2 H), 3.76 (s, 3 H), 1.30 (t, J= 7.1 Hz, 3 H).

EXAMPLE 2 7V-Ethoxycarbonyl-3-pyrrolidinone (13) (Bhaskar Kanth, et al., 1995; Dyke, et al., 1973) Compound 12 (8.350 g, 29.59 mmol) was diluted with benzene (50 mL). Sodium hydride (1.420 g, 59.18 mmol) was added and the mixture was heated at reflux for 30 min. Methyl acrylate (3.057 g, 35.51 mmol) was added and the reaction mixture was heated at reflux for 8 h and then allowed to stir at room temperature for 16 h. 6 M HCl (30 mL) was added and the reaction mixture was heated at reflux for 16 h. The reaction mixture was allowed to cool to room temperature and the aqueous and organic phases were separated. The aqueous phase was extracted with EtOAc (3 x 30 mL) and the combined organic layer was washed with sat NaHCO₃ (3 x 30 mL) and sat NaCl (30 mL). The organic layer was dried over sodium sulfate and concentrated to provide a yellow oil (3.089 g, 73%). ¹H NMR (300 MHz, CDCl₃) δ 4.39- 4.01 (m, 2 H), 3.82-3.78 (m, 4 H), 2.60 (t, J= 7.8 Hz, 2 H), 1.27 (q, J= 7.2 Hz, 3 H); CIMS m/z (rel intensity) 158 (MH⁺, 100).

EXAMPLE 3

7V-Ethoxycarbonyl-3-pyrrolidinone Ethylene Ketal (14). (Dyke, et al., 1973) Compound 15 (2.858 g, 18.18 mmol) was diluted with benzene (50 mL). Ethylene glycol (2.257 g, 36.37 mmol) was added, followed by/?-TsOH (0.346 g, 1.82 mmol). A Dean-Stark trap was affixed and the reaction mixture was heated at reflux for 20 h, allowed to cool to room temperature, and washed with water (3 x 25 mL) and sat NaCl (25 mL). The organic layer was dried over sodium sulfate and concentrated to provide a yellow oil (3.108 g, 81%). IH NMR (CDC13) δ 4.14 (q, J = 7.1 Hz, 2 H), 3.99-3.94 (m, 4 H), 3.56 (q, J = 7.5 Hz, 2 H), 3.43 (d, J = 7.6 Hz, 2 H), 2.05-2.00 (m, 2 H), 1.28 (t, J= 7.1 Hz, 3 H).

EXAMPLE 4

3-Pyrrolidinone Ethylene Ketal (15). (Shamma, et al, 1969) Compound 14 (3.108 g, 14.71 mmol) was diluted with water (25 mL). Potassium hydroxide (2.800 g, 49.91 mmol) was added and the solution was heated at reflux for 16 h. The reaction mixture was allowed to cool to room temperature, extracted with CH₂Cl₂ (4 x 30 mL), and the combined organic layer was washed with sat NaCl (30 mL). The organic layer was dried over sodium sulfate and concentrated to provide a light yellow oil (0.887 g, 44%). ¹H NMR (300 MHz, CDCl₃) δ 3.97-3.86 (m, 4 H), 3.07 (t, J= 7.2 Hz, 2 H), 2.89 (s, 2 H), 2.15 (s, 1 H), 1.98 (t, J = 7.4 Hz, 2 H).

EXAMPLE 5

3-Chloro-l(3H)-isobenzofuranone (17). (Sloan, et al, 1983) 2-

Carboxybenzaldehyde (16, 10.00 g, 66.61 mmol) and ferric chloride (0.030 g, 0.185 mmol) were diluted with thionyl chloride (25 mL) and the mixture was heated at reflux for 1 h. The reaction mixture was allowed to cool to room temperature and concentrated to provide a brown oil. The oil was diluted with hexanes (20 mL) and concentrated to provide a brown solid. The solid was extracted with boiling hexanes (5 x 50 mL), and concentration of the extract provided a white solid (10.96 g, 98%): mp 52-56 ⁰C (Sloan, et al., 1983; mp 57-59 ⁰C). ¹H NMR (300 MHz, CDCl₃) δ 7.95-7.93 (m, 1 H), 7.82 (t, J= 7.4 Hz, 1 H), 7.68-7.60 (m, 2 H), 7.10 (s, 1 H). EXAMPLE 6

2,3-Dihydropyrrolo[l,2-6]isoquinoline-l,5-dione (23, Method 1). (36. Shamma, et al., 1969) Compound 15 (2.167 g, 15.69 mmol) was diluted with THF (30 mL) and triethylamine (10 mL). Compound 17 was then added and the solution was allowed to stir at room temperature for 30 min. The reaction mixture was concentrated, diluted with water (50 mL) and extracted with CHCl₃ (4 x 40 mL). The combined organic layers were washed with sat NaCl (40 mL) and dried over sodium sulfate. The solution was filtered and concentrated to provide compound 18, which was used without further purification in the next step. Compound 18 was diluted with polyphosphoric acid (10.00 g), dichloromethane (5 mL) and phosphoric acid (85%, 5 mL). The reaction mixture was heated at 100 ⁰C for 3 h and then allowed to cool to room temperature. The reaction mixture was diluted with ice water (100 mL) and extracted with CHCI3 (7 x 100 mL). The combined organic layer was washed with sat NaCl (100 mL), dried over sodium sulfate, and concentrated to provide the product 23 as an orange-brown solid (2.117 g, 88%). ¹H NMR (300 MHz, CDCl₃) δ 8.53 (d, J= 7.9 Hz, 1 H), 7.79-7.64 (m, 3 H), 7.27 (s, 1 H), 4.42 (t, J= 6.9 Hz, 2 H), 7.98 (t, J= 7.3 Hz, 2 H).

EXAMPLE 7

(S)-Pyrrolidin-3-ol Hydrogen Maleate (20). (Houghton, et al, 1993) trans-4- Hydroxy-L-proline (19) (10.00 g, 76.26 mmol) was diluted with cyclohexanol (60 mL). 2- Cyclohexene-1-one (1.0 mL) was added, and the mixture heated at reflux for 3 h. After cooling the dark red solution to room temperature, maleic acid (8.95 g, 77.09 mmol) was added portionwise over a 30-min period, keeping the internal temperature below 35 ⁰C. Ethyl acetate (140 mL) was then added dropwise over 1 h to precipitate a pale orange amorphous solid (12.4615 g, 80%) after filtration: mp 83-86 ⁰C (Houghton, et al., 1993; mp 90-91 ⁰C). ¹H NMR (300 MHz, CD₃OD) δ 6.25 (s, 2 H), 4.60-4.50 (m, 1 H), 3.30-3.29 (bm, 2 H), 3.22-3.10 (bm, 2 H), 2.10-2.00 (m, 2H).

EXAMPLE 8

7V-(ø-Formylbenzoyl)-(S)-pyrrolidin-3-ol (21). Compound 20 (3.000 g, 14.62 mmol) was diluted with MeOH (30 mL) and Et₃N (13 mL). Compound 17 (2.096 g, 12.43 mmol) was added, and the mixture was stirred overnight at room temperature for 22 h. The solution was concentrated, and the brown residue dissolved in H₂O (20 mL) and extracted with CHCl₃ (4 x 50 mL). The organic layers were dried over anhydrous sodium sulfate, concentrated, and the resulting brown oil flash chromatographed (SiO₂), eluting with 20:1 CH₂Cl₂-MeOH, to afford an orange-yellow amorphous solid (2.16 g, 79%): mp 71-73 ⁰C. IR (film) 3379, 2947, 2886, 1698, 1613, 1597, 1439 cm^"1; ¹H NMR (300 MHz, CD₃OD) δ 10.0 (s,

1 H), 8.01 (d, J= 7.7 Hz, 1 H), 7.75 (dt, J= 26.1 Hz and 6.7 Hz, 2 H), 7.49 (dt, J= 6.4 Hz and 1.3 Hz), 4.50 (m, 0.5 H), 4.35 (m, 0.5 H), 3.79-3.65 (m, 2 H), 3.31-3.00 (m, 2 H), 2.20-1.80 (m,

2 H); CIMS m/z (rel intensity) 220 (MH⁺, 100).

EXAMPLE 9 2,3-Dihydropyrrolo[l,2-6]isoquinoline-l,5-dione (23, Method 2). (36.

Shamma, et al., 1969) Pyridinium dichromate (5.147 g, 13.68 mmol) was diluted with anhydrous CH₂Cl₂ (30 mL) under an argon atmosphere. A solution of 20 (2.00 g, 9.12 mmol) in anhydrous CH₂Cl₂ (15 mL) was added, and the mixture was heated at reflux for 19.5 h. The mixture was cooled, filtered, and the dark brown filter cake was washed with CHCI3 (4 x 30 mL). The filtrate was filtered through a pad of Celite, and the pad was washed with CHCI3 (3 x 30 mL). The filtrate was concentrated to yield a dark brown oil that was then diluted with CHCI₃ (40 mL). Polyphosphoric acid (6.15 g) was added, and the mixture was heated at reflux for 2 h 10 min. The mixture was cooled and poured into ice water (100 mL). The residue in the flask was stirred with ice water (3 x 40 mL). The aqueous mixture was extracted with CHCI3 (5 x 100, 1 x 50 mL). The organic layers were washed with sat NaCl (250 mL), dried over anhydrous sodium sulfate, adsorbed onto SiO₂ (7.2735 g), and purified by flash column chromatography (SiO₂), eluting with a 1% MeOH in CHCI3. The solvent was evaporated and the resulting solid was recrystallized from boiling EtOH (20 mL) to yield an iridescent orange solid (0.590 g, 33%): mp 180-184 ⁰C (Shamma, et al, 1969; lit. mp 191-192 ⁰C). The ¹H NMR spectrum was identical to compound 23 prepared by method 1 above.

EXAMPLE 10 2-Amino-α-chloroacetophenone (25). (Sugasawa, et al., 1978) Boron trichloride -methyl sulfide complex (1.059 g, 5.906 mmol) was diluted with dichloroethane (15 mL) and cooled to 0 ⁰C. Aniline (24, 0.500 g, 5.369 mmol) was added dropwise and the solution was allowed to stir at 0 ⁰C for 10 min. Chloroacetonitrile (0.507 g, 6.711 mmol) was added, followed by aluminum chloride (0.787 g, 5.906 mmol), and the solution was allowed to gradually warm to room temperature. After 10 min, the reaction mixture was heated at reflux for 3 h. The solution was allowed to cool to room temperature, 2 M HCl (15 mL) was added, and the reaction mixture was heated at reflux for 30 min. The reaction mixture was diluted with water (20 mL) and extracted with CH₂Cl₂ (3 x 20 mL). The combined organic layers were washed with sat NaCl (25 mL) and dried over sodium sulfate. Concentration provided a yellow solid (0.194 g, 21%) that was isolated by washing with hexanes: mp 106-109 ⁰C (Sugasawa, et al., 1978; lit. mp 112-113 ⁰C). ¹H NMR (300 MHz, CDCl₃) δ 7.65 (dd, J= 8.2 Hz and 1.4 Hz, 1 H), 7.34-7.28 (m, 1 H), 6.72-6.64 (m, 2 H), 4.70 (s, 2 H).

EXAMPLE 11 l-(ø-Aminophenyl)-4-chloro-l-butanone (26). (Cevasco, et al., 1995) Boron trichloride -methyl sulfide complex (6.353 g, 35.43 mmol) was diluted with dichloroethane (70 mL) and cooled to 0 ⁰C. Aniline (24, 3.000 g, 32.21 mmol) was added dropwise and the solution was allowed to stir at 0 ⁰C for 10 min. 4-Chlorobutyronitrile (4.170 g, 40.27 mmol) was added, followed by aluminum chloride (4.724 g, 35.43 mmol), and the solution was allowed to gradually warm to room temperature. After 10 min, the reaction mixture was heated at reflux for 2.5 h. The solution was allowed to cool to room temperature, 10% aq HCl (70 mL) was added, and the reaction mixture was heated at reflux for 30 min. The reaction mixture was allowed to stir at room temperature for 24 h and the organic layer was separated. The aqueous layer was extracted with CH₂Cl₂ (3 x 50 mL). The combined organic layers were washed with sat NaCl (50 mL) and dried over sodium sulfate. Concentration provided a crude yellow-brown oil that was purified by flash column chromatography (SiO₂), eluting with a gradient of hexanes to 50% EtOAc-hexanes. The solvent was evaporated and the resulting product was diluted with Et₂O (50 mL) and treated with 3 M HCl in MeOH (10 mL) and allowed to stir at room temperature for 20 min. The salt was filtered and washed with Et₂O (50 mL) to provide a white solid. The solid was dissolved in sat NaHCO₃ (150 mL) and extracted with CHCl₃ (3 x 50 mL). The combined organic layers were washed with sat NaCl (50 mL), dried over sodium sulfate, filtered, and concentrated to provide a yellow oil (2.022 g, 32%) that solidified upon standing: mp 51-55 ⁰C. ¹H NMR (300 MHz, CDCl₃) δ 7.79 (dd, J= 8.5 Hz and 1.6 Hz, 1 H), 7.31-7.25 (m, 1 H), 6.70-6.65 (m, 2 H), 3.70 (t, J= 6.3 Hz, 2 H), 3.18 (t, J= 7.1 Hz, 2 H), 2.25 (pent, J = 6.9 Hz, 2 H).

EXAMPLE 12

14-Chloromethyl-12H-5,lla-diazadibenzo[6,/j]fluoren-ll-one (27a). Compound 23 (0.176 g, 0.884 mmol) and compound 25 (0.150 g, 0.884 mmol) were diluted with benzene (100 mL). /?-Toluenesulfonic acid monohydrate (0.168 g, 0.884 mmol) was added and the solution was heated at reflux for 24 h using a Dean-Stark trap to collect the azeotroped water. The solution was concentrated, diluted with NaHCO₃ (150 mL) and extracted with CHCl₃ (6 x 100 mL) and sat NaCl (100 mL). The organic layer was dried over sodium sulfate, concentrated, and purified by flash column chromatography (SiO₂), eluting with a gradient of CHCl₃ to 5% MeOH in CHCl₃, to provide a yellow solid (0.174 g, 59%) after washing with MeOH: mp 270 ⁰C (dec). IR (KBr) 1661, 1638, 761, 754, and 688 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.56 (d, J= 8.1 Hz, 1 H), 8.27 (d, J= 7.6 Hz, 1 H), 8.19 (d, J= 8.4 Hz, 1 H), 7.86-7.69 (m, 4 H), 7.65 (s, 1 H), 7.62-7.56 (m, 1 H), 5.44 (s, 2 H), 5.05 (s, 2 H); ESIMS m/z (rel intensity) 333/335 (MH⁺, 100/35). Anal. (C₂₀Hi₃ClN₂O) C, H, N.

EXAMPLE 13 14-Azidomethyl-12H-5,lla-diazadibenzo[ό,Λ]fluoren-ll-one (27b).

Compound 27a (0.070 g, 0.210 mmol) and sodium azide (0.0225 g, 0.3456 mmol) were diluted with DMSO (20 mL) and the mixture was stirred at room temperature for 19 h. The solution was diluted with CHCl₃ (30 mL) and more CHCl₃ was then added until the organic phase was clear. The organic layer was washed with H₂O (4 x 25 rnL) and sat NaCl (25 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated, and the residue washed and filtered with MeOH, re-dissolved in CHCI3, and purified by flash column chromatography (SiO₂), eluting with CHCl₃ to provide a pale yellow amorphous solid (0.0509 g, 71%) after washing with MeOH: mp 210-212 ⁰C (dec). IR (film) 2918, 2102, 1665, 1639, 1605, 745, 685 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.58 (d, J= 7.6 Hz, 1 H), 8.28 (d, J= 8.2 Hz, 1 H), 8.11 (d, J= 8.3 Hz, 1 H), 7.84-7.60 (m, 5 H), 7.67 (s, 1 H), 5.50 (s, 2 H), 5.01 (s, 2 H); ESIMS m/z (rel intensity) 340 (MH⁺, 100). Anal. (C₂₀Hi₃N₅O-O-O H₂O) C, H, N.

EXAMPLE 14 14-Aminomethyl-12H-5,1 la-diazadibenzo [b,h] fluoren-11-one

Dihydrochloride (27c). Compound 27b (0.0562 g, 0.1654 mmol) was diluted with benzene (20 mL), triethyl phosphite (0.0687 g, 0.4135 mmol) was added, and the solution heated at reflux for 19 h. The solution was cooled to room temperature, 3 M methanolic HCl (10 mL) was added, and the dark orange solution was heated at reflux for 3 h. The precipitate was vacuum filtered to yield a bright red flaky solid (0.0464 g, 73%) after washing with MeOH: mp 278-284 ⁰C (dec, salt), 265-269 ⁰C (dec, free base). IR (film) 2920, 1656, 1633, 1597, 751, 690 cm^"1; ¹H NMR (300 MHz, CDCl₃, CD₃OD, Et₃N) δ 8.43 (d, J= 7.4 Hz, 1 H), 8.17 (t, J= 8.0 Hz, 2 H), 7.76-7.54 (m, 5 H), 7.70 (s, 1 H), 5.42 (s, 2 H), 4.37 (2 H); ESIMS m/z (rel intensity) 314 (MH⁺, 100). Anal. ( C₂₀Hi₇Cl₂N₃O), C, H, N. EXAMPLE 15

14-(l-Imidazolylmethyl)-2H-5,lla-diazadibenzo[b,h] fluoren-11-one (27d). Compound 27a (0.085 g, 0.225 mmol) and imidazole (0.052 g, 0.766 mmol) were diluted with DMSO (25 mL), heated to 62 oC for 3 h, and the resultant clear pale orange solution was subsequently stirred at room temperature for 15 h. TLC showed incomplete reaction, so additional imidazole (0.255 mmol) was added, and the solution heated at 100 o C for 1 h. The solution was diluted with CHC13 (50 mL) and washed with H2O (4 x 40 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated, and the residue purified by flash column chromatography (SiO2), eluting with a gradient of CHC13 to CHC13-4% MeOH, to yield a yellow amorphous solid (0.0406 g, 44%) after washing with diethyl ether: mp 294-297 oC (dec). IR (film) 1660, 1631, 1600, 762, 687 cm-1; IH NMR (300 MHz, CDC13) δ 8.52 (d, J = 8.0 Hz, 1 H), 8.28 (d, J = 8.4 Hz, 1 H), 8.02 (d, J = 8.5 Hz, 1 H), 7.82-7.57 (m, 5 H), 7.65 (s, 1 H), 7.60-7.47 (m, 1 H), 7.09 (bs, 1 H), 6.90 (bs, 1 H), 5.67 (s, 2 H), 5.26 (s, 2 H); ESIMS m/z (rel intensity) 365 (MH+, 100). Anal. (C23H16N4O-0.75 H2O) C, H, N. EXAMPLE 16 14-[l-(7V-Methylpiperazinylmethyl)]-2H-5,lla-diazadibenzo[ό,/j]fluoren-ll- one (27e). Compound 27a (0.075 g 0.225 mmol) was diluted with DMSO (25 niL) and N- methylpiperazine (0.0676 g, 0.675 mmol) was added. The solution was stirred at room temperature for 19 h. The solution was diluted with CΗCI3 (50 mL) and washed with H₂O (4 x 40 mL) and sat NaCl (50 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated, and the residue purified by flash column chromatography (SiO₂), eluting with 7% MeOH in CHCI₃ to yield a pale yellow amorphous solid (0.582 g, 65.2%) after washing with hexanes: mp 204-207 ⁰C. IR (film) 2932, 2789, 1661, 1633, 1604, 753, 687 cm-¹; ¹H NMR (300 MHz, CDCl₃) δ 8.57 (d, J= 8.4 Hz, 1 H), 8.36 (d, J= 8.6 Hz, 1 H), 8.23 (d, J= 7.9 Hz, 1 H), 7.78-7.57 (m, 5 H), 7.62 (s, 1 H), 5.47 (s, 2 H), 4.06 (s, 2 H), 2.61 (bs, 4 H), 2.47 (bs, 4 H), 2.29 (s, 3 H); ESIMS m/z (rel intensity) 397 (MH⁺, 80), 297 (MH⁺- C₅HnN₂, 100). Anal. (C₂₅H₂₄N₄O O-O H₂O) C, H, N.

EXAMPLE 17 14-(l-Morpholinomethyl)-2H-5,lla-diazadibenzo[ό,Λ]fluoren-ll-one (27f).

Compound 27a (0.064 g, 0.192 mmol) was diluted with DMSO (25 mL) and morpholine (0.0502 g, 0.576 mmol) was added. The solution was stirred at room temperature for 20 h. The solution was diluted with CHCl₃ (4OmL) and then washed with H₂O (4 x 40 mL) and sat NaCl (40 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated, and the resulting residue purified by flash column chromatography (SiO₂), eluting with EtOAc, which afforded a pale yellow amorphous solid (0.0562 g, 77%) after washing with hexanes: mp 242- 244 ⁰C (dec). IR (film) 2929, 1661, 1602, 756, 689 cm^"1; ¹H NMR (300 MHZ, CDCl₃) δ 8.58 (d, J= 8.0 Hz, 1 H), 8.39 (d, J= 8.3 Hz, 1 H), 8.24 (d, J= 8.5 Hz, 1 H), 7.82-7.58 (m, 5 H), 7.66 (s, 1 H), 5.27 (s, 2 H), 4.06 (s, 2 H), 3.73 (t, J= 4.2 Hz, 4 H), 2.59 (t, J= 4.3 Hz, 4 H); ESIMS m/z (rel intensity) 384 (MH⁺, 100). Anal. (C₂₄H₂iN₃O₂) C, H, N.

EXAMPLE 19

14-(7V-Ethanolaminomethyl)-2H-5,lla-diazadibenzo[ό,/j]fluoren-llone (27g). Compound 27a (0.065 g, 0.195 mmol) was diluted with DMSO (25 mL) and ethanolamine (0.0477 g, 0.781 mmol) was added. The mixture was stirred at room temperature for 20 h, poured into CHCl₃ (40 mL), and washed with H₂O (4 x 40 mL). A small amount of methanol was added to aid solubility. The organic layers were dried over anhydrous sodium sulfate, concentrated, and the residue purified by flash column chromagraphy (SiO₂), eluting with a gradient of 6% MeOH in CHCl₃ to 9% MeOH in CHCl₃, to provide a fine yellow powder (0.0292g, 42%) after washing with hexanes: mp 198.5-204 ⁰C (dec). IR (film) 2925, 1656, 1618, 1599, 753, 689 cm^"1; ¹H NMR (500 MHz, DMSO-J₆) δ 8.38-8.31 (m, 2 H), 8.14 (d, J= 8.1 Hz, 1 H), 7.97 (d, J= 7.9 Hz, 1 H), 7.81 (q, J= 7.2 Hz, 2 H), 7.69-7.56 (m, 3 H), 5.44 (s, 2 H), 4.57 (t, J= 5.3 Hz, 1 H), 4.32 (s, 2 H), 3.54 (q, J= 5.6 Hz, 2 H), 2.73 (t, J= 5.7 Hz, 2 H); ESIMS m/z (rel intensity) 358 (MH⁺, 100). Anal. (C₂₂Hi₉N₃O₂- 1.25 H₂O) C, H, N.

EXAMPLE 20

14-(7V^V-Dimethylaminomethyl)-2H-5,lla-diazadibenzo[ό,Λ]fluoren-ll-one (27h). Compound 27a (0.050 g, 0.150 mmol) was diluted with DMSO (25 mL) and N,N- diethylamine hydrochloride (0.035 g, 0.429 mmol) and Et₃N (0.045 g, 0.445 mmol) were added. The solution was stirred at room temperature for 20 h and then diluted with CHCl₃ (40 mL), and then washed with H₂O (4 x 40 mL) and sat NaCl (40 mL). The organic layer was dried over anhydrous sodium sulfate and concentrated. Flash column chromatography of the residue (SiO₂), eluting with EtOAc, yielded a very pale yellow amorphous solid (0.0329 g, 64%) after washing with hexanes: mp 201-202.5 ⁰C. IR (film) 2942, 2819, 2768, 1661, 1634, 1604, 753, 688 cm^"1; ¹H NMR (300 MHz, CDCl₃,) δ 8.58 (d, J= 8.1, 1 H), 8.37 (d, J= 8.3 Hz, 1 H), 8.23 (d, J= 8.9 Hz, 1 H), 7.80-7.58 (m, 5 H), 7.63 (s, 1 H), 5.45 (s, 2 H), 3.97 (s, 2 H), 2.35 (s, 6 H); ESIMS m/z (rel intensity) 345 (MH⁺, 100). Anal. (C₂₂H₁₉N₃O-(U H₂O) C, H, N.

EXAMPLE 21 14-[7V-(5)-3-Hydroxypyrrolidinomethyl]-12H-5,lla diazadibenzo[6,/ι]fluoren-ll-one (27i). Compound 27a (0.060 g, 0.1803 mmol) and compound 20 (0.111 g, 0.541 mmol) were diluted with DMSO (25 mL) and Et₃N (0.182 g, 1.803 mmol) was added. The solution was stirred at room temperature for 19 h, diluted with CHCl₃ (40 mL), and washed with H₂O (4 x 30 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated, and the residue purified by flash column chromatography (SiO₂), eluting with a gradient of CHCl₃ to 4% MeOH in CHCl₃, to yield a flocculent yellow solid (0.0479 g, 69%) after washing with hexanes: mp 220 ⁰C (dec). IR (film) 2918, 2849, 1658, 1601, 1619, 1479, 1347, 1125, 755, 688 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.52 (d, J= 8.0 Hz, 1 H), 8.36 (d, J= 8.5 Hz, 1 H), 8.20 (d, J= 8.0 Hz, 1 H), 7.78-7.51 (m, 5 H), 7.63 (s, 1 H), 5.43 (s, 2 H), 4.35 (bs, 1 H), 4.18 (s, 2 H), 2.92-2.90 (m, 1 H), 2.73-2.71 (m, 2 H), 2.50-2.48 (m, 2 H), 2.23-2.13 (m, 1 H), 1.78-1.74 (m, 1 H); ESIMS m/z (rel intensity) 384 (MH⁺, 100). Anal. (C₂₄H₂₁N₃O₂-0.25 H₂O) C, H, N. EXAMPLE 22

14-[(l-Imidazolyl)propylaminomethyl]-12H-5,lla- diazadibenzo[6,/ι]fluoren-ll-one (27j). Compound 27a (0.060 g, 0.1803 mmol) was diluted with DMSO (25 mL) and l-(3-aminopropyl)imidazole (0.0677 g, 0.5049 mmol) was added. The solution was stirred at room temperature for 17 h, diluted with CΗCI3 (40 mL), and washed with H₂O (4 x 30 mL). The organic layers were dried over anhydrous sodium sulfate, concentrated, and the resultant dark yellow solid purified by flash column chromatography (SiO₂), eluting with a gradient of CHCl₃ to 6% MeOH- 1% Et₃N in CHCl₃, to yield a pale yellow solid (0.0452 g, 60%) after washing with hexanes: mp 138-140 ⁰C (dec). IR (film) 3413, 3292, 1656, 1620, 1600, 1507, 1451, 1343, 755, 688 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.56 (d, J= 8.0 Hz, 1 H), 8.26 (d, J= 8.7 Hz, 2 H), 7.83-7.56 (m, 5 H), 7.67 (s, 1 H), 7.43 (s, 1 H), 7.03 (s, 1 H), 6.84 (s, 1 H), 5.45 (s, 2 H), 4.36 (s, 2 H), 4.07 (t, J= 6.9 Hz, 2 H), 2.78 (t, J = 6.6 Hz, 2 H), 2.04-1.95 (m, 2 H); ESIMS m/z (rel intensity) 422 (MH⁺, 100). Anal. (C₂₆H₂₃N₅O 0.75 H₂O) C, H, N.

EXAMPLE 23

14-(3-Morpholinopropylaminomethyl)-12H-5,lla-diazadibenzo[6,/j]fluoren- 11-one (27k). Compound 27a (0.055 g, 0.165 mmol) was diluted with DMSO (25 mL) and 3- morpholinopropylamine (0.119 g, 0.8260 mmol) was added. The solution was stirred at room temperature for 19 h, diluted with CHCl₃ (40 mL), and washed with H₂O (4 x 30 mL). The organic layers were dried over anhydrous sodium sulfate, concentrated, and the resultant dark yellow solid purified by flash column chromatography (SiO₂), eluting with a gradient of 1% Et₃N in CHCl₃ to 1% MeOH- 1% Et₃N in CHCl₃ to yield a flaky yellow solid (0.0402 g, 55%) after washing with diethyl ether: mp 172-175 ⁰C. IR (film) 3445, 3302, 2929, 1656, 1619, 1600, 1451, 1344, 1117, 753, 687 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.58 (d, J = 8.3 Hz, 1 H), 8.28 (t, J= 8.5 Hz, 2 H), 7.82-7.58 (m, 5 H), 7.67 (s, 1 H), 5.48 (s, 2 H), 4.37 (s, 2 H), 3.65 (t, J= 4.4 Hz, 4 H), 2.81 (bm, 2 H), 2.42-2.37 (m, 6 H), 1.78-1.68 (m, 2 H); ESIMS m/z (rel intensity) 441 (MH⁺, 100). Anal (C₂₇H₂₈N₄O₂-O^ H₂O) C, H, N.

EXAMPLE 24 14-(3-Chloropropyl)-12H-5,lla-diazadibenzo[ό,/ι]fluoren-ll-one (28a).

Compound 23 (1.000 g, 5.020 mmol) and compound 26 (1.091 g, 5.522 mmol) were diluted with benzene (125 mL). /?-Toluenesulfonic acid monohydrate (0.955 g, 5.020 mmol) was added and the solution was heated at reflux for 5 h using a Dean-Stark trap to collect the azeotroped water. The solution was concentrated and the precipitate was washed with Et₂O (50 mL). The precipitate was dissolved in CHCI3 (500 rnL) and washed with sat NaHCO₃ (3 x 200 rnL). The combined aqueous layer was then extracted with CHCI3 (3 x 200 mL). The organic layers were combined, dried over sodium sulfate, concentrated, and purified by flash column chromatography (SiO₂), eluting with a gradient Of CHCl₃ to 4% MeOH in CHCl₃, to provide an off-white solid (1.642 g, 91%) after washing with Et₂O (50 mL): mp 235-237 ⁰C. IR (KBr) 3465, 1654, 1619, 1601, 756, and 689 cm^"1; ¹H NMR (300 MHz, DMSO-J₆) δ 8.37 (d, J= 8.0 Hz, 1 H), 8.29 (d, J= 7.6 Hz, 1 H), 8.18 (dd, J= 8.5 Hz and 1.0 Hz, 1 H), 8.00 (d, J= 7.8 Hz, 1 H), 7.88-7.67 (m, 3 H), 7.64 (s, 1 H), 7.63-7.58 (m, 1 H), 5.40 (s, 2 H), 3.88 (t, J= 6.3 Hz, 2 H), 3.37-3.31 (m, 2 H), 2.19-2.14 (m, 2 H); ESIMS m/z (rel intensity) 361/363 (MH⁺, 100/33). Anal. (C₂₂Hi₇ClN₂O 0.5 H₂O) C, H, N.

EXAMPLE 25

14-(3-Azidopropyl)-2H-5,lla-diazadibenzo[6,/ι]fluoren-ll-one (28b). Compound 28a (0.150 g, 0.416 mmol) and sodium azide (0.0813 g, 1.25 mmol) were diluted with DMSO (35 mL), and the mixture heated at 100 ⁰C 16 h. The mixture was diluted into H₂O (100 mL) and extracted with CHCl₃ (1 x 100 mL, 1 x 80 mL, 1 x 50 mL). The combined organic layers were washed with H₂O (3 x 200 mL), dried over anhydrous sodium sulfate, and concentrated to afford an off-white amorphous solid, which was isolated by washing with ether and MeOH and purified by preparative TLC (SiO₂, CHCl₃) to yield a pale yellow amorphous solid (0.0618 g, 40%) after washing with ether: mp 185-188 ⁰C (dec). IR (film) 2918, 2108, 1659, 1626, 1604, 1506 1441, 1341, 1287, 1244, 1067, 687 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.56 (d, J= 7.9 Hz, 1 H), 8.24 (d, J= 8.2 Hz, 1 H), 8.11 (d, J= 8.3 Hz, 1 H), 7.79-7.58 (m, 6 H), 5.35 (s, 2 H), 3.50 (t, J= 6.3 Hz, 2 H), 3.29 (t, J= 7.7 Hz, 2 H), 2.20-2.00 (m, 2 H); ESIMS m/z (rel intensity), 368, (MH⁺, 100). Anal. (C₂₂Hi₇N₅O 0.5 H₂O) C, H, N. EXAMPLE 26

14-(3-Aminopropyl)-2H-5,lla-diazadibenzo[6,/ι]fluoren-ll-one

Dihydrochloride (28c). Compound 28b (0.040 g, 0.109 mmol) was diluted with benzene (25 mL) and triethyl phosphite (0.0452 g, 0.272 mmol) was added. The solution was heated at reflux for 19 h, cooled, and methanolic HCl (3 M, 15 mL) was added, followed by heating at reflux for another 3 h. The mixture was cooled and concentrated to afford a bright yellow amorphous solid (0.0398 g, 88%) after washing with CHCl₃ and ether and drying in vacuo: mp 290-295 ⁰C (dec). IR (KBr pellet) 3434, 2923, 2874, 1658, 1620, 1601, 1478, 1343, 755, 689 cm^"1; ¹H NMR (300 MHz, D₂O) δ 7.14-7.11 (m, 3 H), 7.00-6.80 (m, 4 H), 6.60-6.40 (m, 1 H), 6.25 (s, 1 H), 4.26 (s, 2 H), 3.10 (t, J= 7.3 Hz, 2 H), 2.60-2.40 (m, 2 H), 1.80-1.60 (bm, 2 H); ESIMS m/z (rel intensity), 342 (MH⁺, 100). Anal. (C₂₂H₂ICl₂N₃O -H₂O) C, H, N.

EXAMPLE 27

14-[3-(l-Imidazolylpropyl)]-12H-5,lla-diazadibenzo[6,/ι]fluoren-ll-one Trifluoroacetate (28d). Compound 28a (0.100 g, 0.277 mmol), sodium iodide (0.249 g, 1.662 mmol), and imidazole (0.113 g, 1.662 mmol) were diluted with DMSO (30 mL) and the reaction mixture was heated at 100 ⁰C for 24 h and then allowed to cool to room temperature. The reaction mixture was diluted with CHCl₃ (150 mL) and washed with water (3 x 50 mL) and sat NaCl (50 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography (SiO₂), eluting with a gradient of CHC1₃-1% Et₃N to 3% MeOH in CHC1₃-1% Et₃N, to provide a yellow solid. The solid was diluted with CHCl₃ (2 mL) and trifluoroacetic acid (5 mL) was added. The reaction mixture was allowed to stir at room temperature for 2 h, concentrated, and the residue was triturated with diethyl ether. The precipitate was filtered and washed with diethyl ether (50 mL) to provide a yellow solid (0.081 g, 58%): mp 215-220 ⁰C. IR (KBr) 1661, 1631, 1201, and 1128 cm^"1; ¹H NMR (300 MHz, DMSO-J₆) δ 9.10 (s, 1 H), 8.38 (d, J= 8.1 Hz, 1 H), 8.27 (d, J= 8.4 Hz, 1 H), 8.18 (d, J= 7.6 Hz, 1 H), 8.01 (d, J= 7.9 Hz, 1 H), 7.86-7.79 (m, 3 H), 7.75-7.62 (m, 4 H), 5.40 (s, 2 H), 4.45 (t, J= 7.3 Hz, 2 H), 3.27 (t, J= 7.4 Hz, 2 H), 2.28 (bs, 2 H); ESIMS m/z (rel intensity) 393 (MH⁺, 100). Anal (C₂₇H₂1F₃N₄O₃ 0.25 H₂O) C, H, N. EXAMPLE 28

14-[3(l-Morpholinopropyl)]-12H-5,lla-diazadibenzo[6,/ι]fluoren-ll-one trifluoroacetate (28e). Compound 28a (0.100 g, 0.277 mmol), sodium iodide (0.250 g, 1.662 mmol), and morpholine (0.144 g, 1.662 mmol) were diluted with DMSO (30 mL) and the reaction mixture was heated at 100 ⁰C for 48 h and then allowed to stir at room temperature for 16 h. The reaction mixture was diluted with CHCl₃ (150 mL) and washed with water (3 x 50 mL) and sat NaCl (50 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography (SiO₂), eluting with a gradient of CHCl₃- 1% Et₃N to 5% MeOH in CHCl₃- 1% Et₃N, to provide a yellow solid. The solid was diluted with CHCl₃ (2 mL) and trifluoroacetic acid (5 mL) was added. The reaction mixture was allowed to stir at room temperature for 2 h, concentrated, and the residue was triturated with diethyl ether. The precipitate was filtered and washed with diethyl ether (50 mL) to provide a yellow solid (0.140 g, 79%): mp 190-195 ⁰C. IR (KBr) 1662, 1632, 1197, 1134 cm^"1; ¹H NMR (300 MHz, DMSO-J₆) δ 8.39 (d, J= 8.1 Hz, 1 H), 8.33 (d, J= 8.6 Hz, 1 H), 8.20 (d, J= 7.5 Hz, 1 H), 8.02 (d, J= 8.0 Hz, 1 H), 7.90-7.72 (m, 3 H), 7.71 (s, 1 H), 7.65- 7.60 (m, 1 H), 5.43 (s, 2 H), 3.98 (d, J= 12.5 Hz, 2 H), 3.63 (t, J= 11.9 Hz, 2 H), 3.44-3.25 (m, 6 H), 3.16-3.03 (m, 2 H), 2.11 (m, 2 H); ESIMS m/z (rel intensity) 412 (MH⁺, 100). Anal (C₂₈H₂₆F₃N₃O₄) C₅ H₅ N. EXAMPLE 29

14-(3-7V-Ethanolaminopropyl)-12H-5,lla-diazadibenzo[ό,Λ]fluoren-ll-one Trifluoroacetate (28f). Compound 28a (0.100 g, 0.277 mmol), sodium iodide (0.249 g, 1.662 mmol), and ethanolamine (0.100 mL, 1.662 mmol) were diluted with DMSO (30 mL) and the reaction mixture was heated at 100 ⁰C for 16 h and then allowed to cool to room temperature. The reaction mixture was diluted with CHCl₃ (150 mL) and washed with water (3 x 50 mL) and sat NaCl (50 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography (SiO₂), eluting with a gradient of CHCl₃- 1% Et₃N to 9% MeOH in CHCl₃- 1% Et₃N, to provide a white solid. The solid was diluted with CHCl₃ (3 mL) and trifluoroacetic acid (5 mL) was added. The reaction mixture was allowed to stir at room temperature for 2 h, concentrated, and the residue was triturated with diethyl ether. The precipitate was filtered and washed with diethyl ether (50 mL) to provide a yellow solid (0.101 g, 73%): mp 225-227 ⁰C (dec). IR (KBr) 3383, 1661, 1621, 1602, 1201, 1132 cm^"1; ¹H NMR (300 MHz, DMSO-J₆) δ 8.48 (bs, 2 H), 8.39 (d, J= 8.1 Hz, 1 H), 8.34 (d, J= 8.1 Hz, 1 H), 8.19 (d, J= 8.4 Hz, 1 H), 8.02 (d, J= 8.0 Hz, 1 H), 7.89-7.80 (m, 2 H), 7.76-7.71 (m, 1 H), 7.70 (s, 1 H), 7.65-7.60 (m, 1 H), 5.40 (s, 2 H), 3.66 (t, J= 5.0 Hz, 2 H), 3.32 (t, J= 7.1 Hz, 2 H), 3.16 (bs, 2 H), 3.02 (bs, 2 H), 2.06 (bs, 2 H); ESIMS m/z (rel intensity) 386 (MH⁺, 100). Anal. (C₂₆H₂₄F₃N₃O₄ O^ H₂O) C, H, N.

EXAMPLE 30 14-[3-(7V,7V-Dimethylaminopropyl)]-12H-5,lla-diazadibenzo[ό,Λ]fluoren-ll- one Trifluoroacetate (28g). Compound 28a (0.100 g, 0.277 mmol), sodium iodide (0.249 g, 1.662 mmol), and dimethylamine (2 M in TΗF, 1.67 mL, 3.324 mmol) were diluted with DMSO (30 mL) and the reaction mixture was heated at 100 ⁰C for 16 h and then allowed to cool to room temperature. The reaction mixture was diluted with CHCl₃ (150 mL) and washed with water (3 x 50 mL) and sat NaCl (50 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography (SiO₂), eluting with a gradient of CHCl₃- 1% Et₃N to 4% MeOH in CHCl₃- 1% Et₃N, to provide a yellow oil. The oil was diluted with CHCl₃ (3 mL) and trifluoroacetic acid (5 mL) was added. The reaction mixture was allowed to stir at room temperature for 2 h, concentrated, and the residue was triturated with diethyl ether. The precipitate was filtered and washed with diethyl ether (50 rnL) to provide a light yellow solid (0.095 g, 71%): mp 210 ⁰C (dec). IR (KBr) 3434, 1666, 1633, 1199, 1128 cm^"1; ¹H NMR (300 MHz, DMSO-J₆) δ 8.38 (d, J= 7.9 Hz, 1 H), 8.33 (d, J= 8.2 Hz, 1 H), 8.20 (d, J= 7.6 Hz, 1 H), 8.02 (d, J= 8.0 Hz, 1 H), 7.90-7.80 (m, 2 H), 7.76-7.71 (m, 1 H), 7.70 (s, 1 H), 7.65-7.60 (m, 1 H), 5.43 (s, 2 H), 3.28- 3.23 (m, 4 H), 2.78 (s, 6 H), 2.09-2.07 (m, 3 H); ESIMS m/z (rel intensity) 370 (MH⁺, 100). Anal. (C₂₆H₂₄F₃N₃O₃ H₂O) C, H, N.

EXAMPLE 30

General Procedure for Synthesis of Mono-Boc Diaminoalkanes 31-41 (Morrell, et al, 2007). BoC₂O (0.5 g, 2.29 mmol) was dissolved in CHCl₃ (10 rnL) and added dropwise to the diamine (11.4 mmol), dissolved in CHCl₃ (50 mL). The reaction mixture was allowed to stir overnight at room temperature. The solution was concentrated, adsorbed onto SiO₂, and purified by flash column chromatography (SiO₂), eluting with 10% MeOH- 1% Et₃N in CHCl₃ to afford the mono-Boc-protected diamines in acceptable purity after drying in vacuo. EXAMPLE 31

Mono-Boc- 1,2-diaminoethane (31). From 30a, the general procedure afforded the desired product as a clear, pale-yellow viscous oil, (0.382 g, 100% with minor impurities): ¹H NMR (300 MHz, CDCl₃) δ 4.9 (bs, 1 H), 3.19 (q, J= 5.8 Hz, 2 H), 2.80 (t, J = 6.0 Hz, 2 H), 1.43 (s, H H). EXAMPLE 32

Mono-Boc- 1,3-diaminopropane (32). From 30b, the general procedure afforded the desired product as a clear, pale-yellow viscous oil (0.330 g, 83%): ¹H NMR (300 MHz, CDCl₃) δ 4.97 (bs, 1 H), 3.21 (q, J= 6.24 Hz, 2 H), 2.75 (t, J= 6.6 Hz, 2 H), 1.62-1.53 (m, 2 H), 1.41 (s, 1 I H). EXAMPLE 33

Mono-Boc- 1,4-diaminobutane (33). From 30c, the general procedure afforded the desired product as a clear yellow viscous oil (0.443, 100% with minor impurities): ¹H NMR (300 MHz, CDCl₃) δ 4.74 (bs, 1 H), 3.10 (q, J= 6.1 Hz, 2 H), 2.70 (t, J= 6.7 Hz, 2 H), 1.5-1.43 (bm, 4 H), 1.41 (s, H H). EXAMPLE 34

Mono-Boc- 1,5-diaminopentane (34). From 30d, the general procedure afforded the desired product as a viscous yellow oil (0.418 g, 90%): ¹U NMR (300 MHz, CDCl₃) δ 4.56 (bs, 1 H), 3.12 (q, J= 6.3 Hz, 2 H), 2.71 (t, J= 6.7 Hz, 2 H), 1.67-1.28 (m, 17 H).

EXAMPLE 35

Mono-Boc-l,5-diaminohexane (35). From 3Oe, the general procedure afforded the desired product as a yellow semisolid (0.428 g, 86%): ¹H NMR (300 MHz, CDCl₃) δ 4.54 (bs, 1 H), 3.13 (q, J= 6.5 Hz, 2 H), 2.7 (t, J= 6.6 Hz, 2 H), 1.43 (s, 11 H), 1.34-1.30 (m, 8 H).

EXAMPLE 36

Mono-Boc-l,7-diaminoheptane (36). From 30f, the general procedure afforded the desired product as a clear, pale yellow viscous oil, (0.387, 73%): ¹H NMR (500 MHz, CDCl₃) δ 4.54 (bs, 1 H), 3.0-3.2 (bm, 2 H), 2.68 (t, J= 7.0 Hz, 2 H), 1.62-1.55 (bm, 4 H), 1.42 (s, 9 H), 1.1-1.3 (bm, 8 H).

EXAMPLE 37

Mono-Boc-l,8-diaminooctane (37). From 30g, the general procedure afforded the product as a colorless semisolid (0.493 g, 91%): ¹H NMR (300 MHz, CDCl₃) δ 4.49 (bs, 1 H), 3.11 (q, J= 6.8 Hz, 2 H), 2.70 (t, J= 6.8 Hz, 2 H), 1.60-1.50 (bm, 4 H), 1.44 (s, 11 H), 1.40-1.30 (bm, 8 H).

EXAMPLE 38

Mono-Boc-l,9-diaminononane (38). From 30h, the general procedure afforded the product as a viscous, colorless oil which solidified upon standing (0.437g, 74%): ¹H NMR (300 MHz, CDCl₃) δ 4.50 (bs, 1 H), 3.13 (q, J= 6.5 Hz, 2 H). 2.69 (t, J= 6.8 Hz, 2 H), 1.40- 1.30 (bm, 13 H), 1.30-1.20 (bm, 12 H).

EXAMPLE 39

Mono-Boc 1,10-diaminodecane (39). From 30i, the general procedure afforded the product as a colorless semisolid (0.598 g, 96%): ¹H NMR (300 MHz, CDCl₃) δ 4.51 (bs, 1 H), 3.13 (q, J= 6.5 Hz, 2 H), 2.69 (t, J= 6.8 Hz, 2 H), 1.50-1.30 (bm, 15 H), 1.30-1.20 (bm, 12 H).

EXAMPLE 40

Mono-Boc-l,ll-diaminoundecane (40). From 3Oj, the general procedure afforded the product as a colorless semisolid (0.558 g, 90%): ¹U NMR (300 MHz, CDCl₃) δ 4.49 (bs, 1 H), 3.13 (q, J= 6.7 Hz, 2 H), 2.70 (t, J= 6.8 Hz), 1.60-1.20 (m, 29 H). EXAMPLE 41

Mono-Boc-l,12-diaminododecane (41). From 30k, the general procedure afforded the product as a colorless semisolid (0.547 g, 79%): ¹H NMR (300 MHz, CDCl₃) δ 4.50 (bs, 1 H), 3.11 (q, 5.7 Hz, 2 H), 2.70 (t, J= 6.9 Hz), 1.60-1.20 (m, 33 H). EXAMPLE 42

14-(2'-tert-Boc-Aminoethyl-l'-aminomethyl)-12H-5,lla- diazadibenzo[6,/ι]fluoren-ll-one (42). Compound 27a (0.070 g, 0.210 mmol) was diluted in DMSO (20 mL) and 31 (0.101 g, 0.631 mmol), was dissolved in DMSO (1 mL) and added to the suspension. The mixture was sonicated briefly to break up the starting material and stirred at room temperature for 19 h, poured into H₂O (100 mL), and extracted with CHCl₃ (1 x 100 mL, 1 x 60 mL). The organic layers were washed with H₂O (2 x 140 mL, 2 x 280 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was adsorbed onto SiO₂, and purified by flash column chromatography (SiO₂), eluting with 0.4% Et₃N in CHCl₃. The obtained solid was further purified by preparative TLC (SiO₂, 1.5% MeOH, few drops Et₃N in CHCl₃) to yield a yellow amorphous solid (0.059 g, 62%) after washing with ether: mp 145-148 ⁰C. IR (film) 3436, 2975, 1712, 1655, 1598, 1502, 1456, 1364, 1171, 753, 687 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.57 (dd J= 8.0 Hz, 3.9 Hz, 2 H), 8.24 (d, J= 8.5 Hz, 1 H), 7.78-7.56 (m, 6 H), 5.47 (s, 2 H), 4.92 (bs, 1 H), 4.40 (s, 2 H), 3.31 (q, J= 5.5 Hz, 2 H), 2.91 (t, J= 5.5 Hz, 2 H), 1.67 (bs, 1 H), 1.42 (s, 9 H); ESIMS m/z (rel intensity) 457 (MH⁺, 65), 357, (MH⁺- Boc, 100). Anal (C₂₇H₂₈N₄O₃ OJS H₂O) C, H, N.

EXAMPLE 43

14-(3'-tert-Boc-Aminopropyl-l'-aminomethyl)-12H-5,lla- diazadibenzo[ό,Λ]fluoren-ll-one (43). Compound 27a (0.070 g, 0.210 mmol) was diluted in DMSO (25 mL) and compound 32 (0.110 g, 0.631 mmol), was dissolved in DMSO (1 mL) and added, and the mixture was sonicated briefly to break up the starting material. The mixture was stirred at room temperature for 16 h, poured into H₂O (100 mL), and extracted with CHCl₃ (2 x 70 mL, 2 x 40 mL). The organic layers were washed with H₂O (4 x 140 mL), dried over anhydrous sodium sulfate, and concentrated. The residue was adsorbed onto SiO₂, and purified by flash column chromatography (SiO₂), eluting with 0.6% Et₃N in CHCl₃. The obtained solid was further purified by preparative TLC (SiO₂, 1.5% MeOH, few drops Et₃N in CHCl₃) to yield a yellow amorphous solid (0.061 g, 62%) after washing with ether: mp 170-173 ⁰C. IR (film), 3307, 2975, 1696, 1659, 1622, 1602, 1512, 1365, 1345, 1170, 754, 687 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.56 (d, J= 7.9 Hz, 1 H), 8.30 (d, J= 8.5 Hz, 1 H), 8.24 (d, J= 8.2 Hz, 1 H), 7.79-7.57 (m, 6 H), 5.47 (s, 2 H), 4.87 (bs, 1 H), 4.36 (s, 2 H), 3.22 (q, J= 6.4 Hz, 2 H), 2.85 (t, J= 6.4 Hz, 2 H), 1.75-1.70 (m, 3 H), 1.39 (s, 9 H); ESIMS m/z (rel intensity) 471 (MH⁺, 35), 371, (MH⁺- Boc, 100). Anal (C₂₈H₃₀N₄O₃) C, H, N.

EXAMPLE 44 14-(4'-tert-Boc-Aminobutyl-l'-aminomethyl)-12H-5,lla- diazadibenzo[6,/ι]fluoren-ll-one (44). Compound 27a (0.070 g, 0.210 mmol) was diluted in DMSO (20 niL) and compound 33 (0.119 g, 0.630 mmol), was dissolved in DMSO (2 mL) and added. The mixture was stirred at room temperature for 17 h, poured into H₂O (100 mL), and extracted with CHCl₃ (60 mL); additionally, MeOH (70 mL) was added. The organic layer was washed with H₂O (4 x 150 mL), dried over anhydrous sodium sulfate, and concentrated. The obtained residue was adsorbed onto SiO₂ and purified by flash column chromatography (SiO₂), eluting with 0.6% Et₃N in CHCl₃ to yield a yellow amorphous solid (0.069 g, 68%) after washing with ether: mp 175-180 ⁰C (dec). IR (film) 3351, 2930, 1693, 1693, 1657, 1621, 1601, 1609, 1365, 1250, 1173, 753, 687 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.56 (d, J= 8.0 Hz, 1 H), 8.26 (t, J= 8.4 Hz, 1 H), 7.79-7.57 (m, 6 H), 5.47 (s, 2 H), 4.67 (bs, 1 H), 4.37 (s, 2 H), 3.14 (q, J= 6.0 Hz, 2 H), 2.80 (t, J= 6.2 Hz, 2 H), 1.59-1.55 (m, 5 H), 1.42 (s, 9 H); ESIMS m/z (rel intensity) 475 (MH⁺, 27), 385, (MH⁺- BoC, 100).

EXAMPLE 45 14-(5'-tert-Boc-Aminopentyl-l'-aminomethyl)-12H-5,lla- diazadibenzo[6,/ι]fluoren-ll-one (45). Compound 27a (0.081 g, 0.243 mmol) and compound 34 (0.147 g, 0.729 mmol) were diluted with DMSO (25 mL), and the mixture was stirred at room temperature for 19 h. The reaction mixture was diluted with H₂O (100 mL), and extracted with CHCl₃, (2 x 100 mL), with additional H₂O (100 mL) being added to break up the resultant emulsion. The organic layers were washed with H₂O (3 x 100 mL), dried over anhydrous sodium sulfate, concentrated, adsorbed onto SiO₂, and purified by flash column chromatography (SiO₂), eluting with 0.4% Et₃N in CHCl₃ to yield a pale yellow amorphous solid (0.079 g, 66%) after washing with ether: mp 186-189. IR (film) 3368, 2928, 2856, 1693, 1656, 1619, 1600, 1512, 1453, 1365, 1250, 1172, 835, 752, 686 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.56 (d, J= 8.0 Hz, 2 H), 8.26 (t, J= 8.8 Hz, 2 H), 7.78-7.57 (m, 6 H), 5.47 (s, 2 H), 4.66 (bs, 1 H), 4.67 (s, 2 H), 3.12 (q, J= 6.2, 2 H), 2.78 (t, J= 7.0 Hz, 2 H), 1.60-1.35 (m, 16 H); ESIMS m/z (rel intensity) 499 (MH⁺, 56), 997, (2MH⁺ 100). EXAMPLE 46

14-(6'-tert-Boc-Aminohexyl-l'-aminomethyl)-12H-5,lla- diazadibenzo[6,/ι]fluoren-ll-one (46). Compound 27a (0.070 g, 0.210 mmol) was diluted with DMSO (20 mL) and compound 35 (0.136 g, 0.631 mmol) was dissolved in DMSO (3 mL) and pipetted into the suspension. The mixture was stirred at room temperature for 16 h, poured into H₂O (120 mL) and extracted with CHCI3 (2 x 50 mL, 1 x 30 mL). The organic layers were washed with H₂O (1 x 120 mL, 4 x 100 mL), dried over anhydrous sodium sulfate, concentrated, adsorbed onto SiO₂, and purified by flash column chromatography (SiO₂), eluting with 0.4% Et₃N in CHCI3. The obtained residue was further purified by preparative TLC (SiO₂, 1 % MeOH, few drops Et₃N in CHCl₃), to yield a pale yellow amorphous solid (0.055 g, 51 %) after washing with ether: mp 99-104 ⁰C. IR (film) 3437, 3360, 3285, 2928, 1705, 1655, 1619, 1601, 1452, 1365, 1172, 835, 752, 685 cm^"1; ¹H NMR (500 MHz, CDCl₃) δ 8.55 (d, J= 8.1 Hz, 1 H), 8.25 (t, J= 8.1 Hz, 2 H), 7.80-7.54 (m, 6 H), 5.46 (s, 2 H), 4.63 (bs, 1 H), 4.35 (s, 2 H), 3.10 (q, J= 6.3 Hz, 2 H), 2.77 (t, J= 7.0 Hz, 2 H), 1.60-1.30 (m, 18 H); ESIMS m/z (rel intensity), 513, (MH⁺, 80), 413 (MH⁺- Boc, 100).

EXAMPLE 47

14-(7'-tert-Boc-Aminoheptyl-l'-aminomethyl)-12H-5,lla- diazadibenzo[6,/ι]fluoren-ll-one (47). Compound 27a (0.055 g, 0.165 mmol) was diluted with DMSO (25 mL). Compound 36 (0.114 g, 0.495 mmol) was dissolved in a small amount of DMSO and added to the suspension. The mixture was stirred at room temperature for 22 h, diluted with CHCl₃ (40 mL), and washed with H₂O (4 x 50 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated, and the residue was washed with ether and filtered. The filtrate was diluted with CHCl₃ (30 mL) and washed again with H₂O (3 x 20 mL) to remove residual DMSO, before it was dried over anhydrous sodium sulfate, combined with the filtered residue, adsorbed onto SiO₂, and purified by flash column chromatography (SiO₂), eluting with a gradient of CHCl₃ to 0.5% MeOH-0.5% Et₃N to yield a yellow amorphous solid which was further purified by preparative TLC (SiO₂, 1.2% MeOH in CHCl₃), to yield a pale yellow amorphous solid (0.039 g, 45%) after washing with ether: mp 97-105 ⁰C. IR (film) 3436, 2926, 2855, 1691, 1655, 1619, 1601, 1512, 1453, 1365, 1252, 1174, 836, 753, 685 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.58 (d, J= 7.8 Hz, 1 H), 8.27 (t, J= 9.4 Hz, 2 H), 7.80-7.58 (m, 6 H), 5.48 (s, 2 H), 4.69 (bs, 1 H), 4.37 (s, 2 H), 3.10 (q, J= 6.3 Hz, 2 H), 2.77 (t, J= 7.0 Hz, 2 H), 1.60-1.20 (m, 20 H); ESIMS m/z (rel intensity), 527, (MH⁺, 78), 427 (MH⁺- BoC, 100). EXAMPLE 48

14-(8'-tert-Boc-Aminooctyl-l'-aminomethyl)-12H-5,lla- diazadibenzo[6,/ι]fluoren-ll-one (48). Compound 27a (0.071 g, 0.213 mmol) was diluted with DMSO (25 mL), and compound 37 was dissolved in DMSO (2 mL) and added to the suspension. The mixture was stirred at room temperature for 16 h, diluted with CΗCI3 (40 mL) and washed with H₂O (3 x 50 mL, 1 x 80 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated, adsorbed onto SiO₂, and purified by flash column chromatography (SiO₂), eluting with a gradient of CHC1₃-O.5% MeOH in CHCl₃. The resultant residue was purified further by preparative TLC (1% MeOH in CHCl₃) to yield a yellow amorphous solid (0.048 g, 42%) after washing with ether and hexanes: mp 96-101 ⁰C. IR (film) 3436, 3361, 3287, 1684, 1655, 1618, 1600, 1452, 1364, 1250, 1173, 834, 752, 685 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.56 (d, J= 8.0 Hz, 1 H), 8.26 (t, J= 9.1 Hz, 2 H), 7.78-7.56 (m, 6 H), 5.46 (s, 2 H), 4.61 (bs, 1 H), 4.36 (s, 2 H), 3.12 (q, J= 6.5 Hz, 2 H), 2.76 (t, J= 7.1 Hz, 2 H), 1.70-1.20 (m, 22 H); ESIMS m/z (rel intensity) 541 (MH⁺, 32), 441 (MH⁺- Boc, 100), 563 (MNa⁺).

EXAMPLE 49

14-(9'-tert-Boc-Aminononyl-l'-aminomethyl)-12H-5,lla- diazadibenzo[6,/ι]fluoren-ll-one (49). Compound 27a (0.070 g, 0.210 mmol) was diluted with DMSO (20 mL) and compound 38 (0.163 g, 0.631 mmol) was added as a suspension in DMSO (5 mL). The mixture was stirred overnight at 20 h, diluted with H₂O (100 mL) and extracted with CHCl₃ (2 x 50 mL, 1 x 30 mL). The organic layers were washed with H₂O (1 x 120 mL, 4 x 100 mL), dried over anhydrous sodium sulfate, concentrated, adsorbed onto SiO₂, and purified by flash column chromatography (SiO₂), eluting with 0.2% Et₃N in CHCl₃. The obtained residue was further purified by preparative TLC (SiO₂, 1% MeOH, few drops Et₃N in CHCl₃), to afford the desired product as a yellow amorphous solid (0.054 g, 47%) after washing with ether: mp 90-98 ⁰C. IR (film) 3438, 3360, 2925, 2854, 1693, 1655, 1618, 1600, 1511, 1452, 1365, 1250, 1173, 836, 752, 685 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.58 (J= 7.8 Hz, 1 H), 8.27 (dd, J= 8.2, 5.2 Hz), 7.79-7.56 (m, 6 H), 5.47 (s, 2 H), 4.56 (bs, 1 H), 4.38 (s, 2 H), 3.10 (q, J= 6.7 Hz, 2 H), 2.78 (t, J= 7.1 Hz, 2 H), 1.74-1.26 (m, 24 H); ESIMS m/z (rel intensity), 555 (MH⁺, 100).

EXAMPLE 50

14-(10'-tert-Boc-Aminodecyl-l'-aminomethyl)-12H-5,lla- diazadibenzo[6,/ι]fluoren-ll-one (50). Compound 27a (0.070 g, 0.210 mmol) was diluted with DMSO (20 mL) and compound 39 (0.172 g, 0.630 mmol) was added as a suspension in DMSO (5 mL). The reaction mixture was stirred at room temperature for 17 h, diluted with H₂O (120 mL), and extracted with CHCl₃ (2 x 50 mL, 2 x 80 mL, 1 x 30 mL). The organic layers were washed with H₂O (5 x 200 mL), dried over anhydrous sodium sulfate, concentrated, and the resultant residue was adsorbed onto SiO₂ and purified by flash column chromatography (SiO₂) eluting with 0.2% Et₃N in CHCl₃. The obtained material was further purified by preparative TLC (0.7% MeOH, few drops Et₃N in CHCl₃), to yield a bright pale-yellow amorphous solid (0.062 g, 52%) after washing with ether: mp 88-95 ⁰C. IR (film) 3437, 3285, 2924, 2853, 1703, 1655, 1618, 1600, 1453, 1364, 1172, 834, 752, 685 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.56 (d, J= 8.0 Hz, 1 H), 8.27 (dd, J= 8.4, 5.2 Hz, 2 H), 7.80-7.55 (m, 6 H), 5.47 (s, 2 H), 4.53 (bs, 1 H), 4.37 (s, 2 H), 3.10 (q, J= 5.8 Hz, 2 H), 2.77 (t, J= 7.0 Hz, 2 H), 1.64-1.25 (m, 26 H); ESIMS m/z (rel intensity), 569 (MH⁺, 46), 1137 (2MH⁺, 100).

EXAMPLE 51 14-(ll'-tert-Boc-Aminoundecyl-l^l-aminomethyl)-12H-5,lla- diazadibenzo[6,/ι]fluoren-ll-one (51). Compound 27a (0.070 g, 0.210 mmol) and compound

40 (0.180 g, 0.630 mmol) were diluted with DMSO (25 mL) and the mixture stirred at room temperature for 16 h. The mixture was diluted with H₂O (100 mL), extracted with CHCl₃ (2 x 50 mL, 2 x 90 mL, Ix 50 mL, Ix 30 mL), and the organic layers were washed with H₂O (5 x 200 mL), before they were dried over anhydrous sodium sulfate, concentrated, adsorbed onto SiO₂, and purified by flash column chromatography (SiO₂), eluting with 0.2% Et₃N in CHCl₃, to yield a yellow amorphous solid (0.067 g, 55%) after washing with ether and hexanes: mp 88- 100 ⁰C. IR (film) 3436, 3368, 2923, 2853, 1704, 1655, 1618, 1600, 1453, 1365, 1169, 835, 752, 685 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.58 (d, J= 8.2 Hz, 1 H), 8.28 (dd, J= 8.4, 4.7 Hz, 2 H), 7.82-7.58 (m, 6 H), 5.49 (s, 2 H), 4.52 (bs, 1 H), 4.38 (s, 2 H), 3.10 (q, J= 6.6 Hz, 2 H), 2.77 (t, J= 7.1 Hz, 2 H), 1.59-1.24 (m, 28 H); ESIMS m/z (rel intensity), 583 (MH⁺, 100).

EXAMPLE 52

14-(12 '-før^Boc-Aminododecyl- r-aminomethyl)- 12H-5, 11 a diazadibenzo[6,/ι]fluoren-ll-one (52). Compound 27a (0.070 g, 0.210 mmol) and compound

41 (0.190 g, 0.631 mmol) were diluted with DMSO (25 mL) and the mixture was stirred at room temperature for 17 h. The mixture was diluted with H₂O (100 mL), extracted with CHCl₃ (1 x 50 mL, 3 x 80 mL, 1 x 50 mL) and the organic layers were washed with H₂O (5 x 200 mL), before they were dried over anhydrous sodium sulfate, concentrated, adsorbed onto SiO₂, and purified by flash column chromatography (SiO₂), eluting with 0.2% Et₃N in CHCl₃, to yield a pale-yellow amorphous solid (0.088g, 70%) after partially dissolving in ether and precipitating out with hexanes: mp 81-90 ⁰C. IR (film) 3435, 3369, 2922, 2852, 1689, 1655, 1618, 1600, 1453, 1365, 1168, 634, 752, 685 cm^"1; ¹H NMR (300 MHz, CDCl₃) δ 8.58 (d, J= 7.7 Hz, 1 H), 8.29 (dd, J= 7.9, 4.7 Hz, 2 H), 7.81-7.59 (m, 6 H), 5.49 (s, 2 H), 4.50 (bs, 1 H), 4.39 (s, 2 H), 3.10 (q, J= 6.7 Hz, 2 H), 2.76 (t, J= 7.1 Hz, 2H), 1.57-1.23 (m, 30 H); ESIMS m/z (rel intensity), 587 (MH⁺, 100). Anal (C₃₇H₄₈N₄O₃- 0.5 H₂O) C, H, N.

EXAMPLE 53

14-(l,2-Ethanediaminomethyl)-12H-5,lla-diazadibenzo[6,/ι]fluoren-ll-one Trihydrochloride (53). Compound 42 (0.050 g, 0.110 mmol) was dissolved in CHCl₃ (30 mL). Methanolic HCl (3 M, 10 mL) was added dropwise. The addition was slightly exothermic. The mixture was stirred at room temperature for 2 h and concentrated to yield a red-orange solid after washing with ether and drying in vacuo. The powder was then washed with a solution Of CHCl₃ (containing several drops of MeOH), to remove residual Et₃NHCl, yielding the desired product as a red powder (0.035 g, 69%) after washing with ether: mp 228-235 ⁰C (dec). IR (KBr) 3400, 2991, 2725, 1651, 1595, 1337, 1156, 770, 684 cm^"1; ¹H NMR (300 MHz, D₂O) δ 7.78 (d, J= 8.6 Hz, 1 H), 7.65 (s, 2 H), 7.52-7.39 (m, 4 H), 7.08-7.03 (t, J= 6.6 Hz, 1 H), 6.99 (s, 1 H), 5.12 (s, 2 H), 4.64 (s, 2 H), 3.65 (t, J= 6.8 Hz, 2 H), 3.41 (t, J= 7.0 Hz, 2 H); ESIMS m/z (rel intensity) 356 (MH⁺, 100). Anal (C₂₂H₂₃Cl₃N₄O) C, H, N.

EXAMPLE 54 14-(l,3-Propanediaminomethyl)-12H-5,lla-diazadibenzo[6,Λ]fluoren-ll- one Trihydrochloride (54). Compound 43 (0.052 g, 0.111 mmol) was dissolved in CHCl₃ (30 mL). Methanolic HCl (3 M, 10 mL) was added dropwise. The mixture was stirred at room temperature for 2 h and concentrated to yield a red-orange solid (0.043 g, 82%) after washing with ether and drying in vacuo: mp 232-240 ⁰C (dec). IR (KBr) 3435, 2922, 1659, 1623, 1601, 1479, 1334, 762, 686 cm^"1; ¹H NMR (300 MHz, D₂O): δ 7.75 (d, J= 8.3 Hz, 1 H), 7.61 (s, 2 H), 7.52-7.41 (m, 4 H), 7.04-6.96 (m, 2 H), 5.10 (s, 2 H), 4.60 (s, 2 H), 3.38 (t, J= 7.9 Hz, 2 H), 3.10 (t, J= 7.4 Hz, 2 H), 2.20-2.00 (m, 2 H); ESIMS m/z (rel intensity) 371(MH⁺, 100). Anal (C₂₃H₂₅Cl₃N₄O) C, H, N.

EXAMPLE 55 14-(l,4-Butanediaminomethyl)-12H-5,lla-diazadibenzo[6,/j]fluoren-ll-one

Trihydrochloride (55). Compound 44 (0.055 g, 0.113 mmol) was dissolved in CHCl₃ (30 mL). Methanolic HCl (3 M, 10 mL) was added dropwise. The mixture was stirred at room temperature for 2 h and concentrated to yield a red-orange solid (0.050 g, 90%) after washing with ether and drying in vacuo: mp 222-228 ⁰C (dec). IR (KBr) 3401, 2928, 1657, 1596, 1476, 1334, 762, 681 cm^"1; ¹H NMR (300 MHz, D₂O) δ 7.72 (d, J= 8.2 Hz, 1 H), 7.60-7.45 (m, 3 H), 7.36-7.32 (m, 3 H), 7.10-7.00 (m, 1 H), 6.86 (s, 1 H). 5.05 (s, 2 H), 4.55 (s, 2 H), 3.33 (t, J= 6.8 Hz, 2 H), 3.05 (t, J= 7.6 Hz, 2 H), 1.90-1.70 (m, 4 H); ESIMS m/z (rel intensity) 385 (MH⁺, 100). Anal (C₂₄H₂₇Cl₃N₄O H₂O) C, H, N.

EXAMPLE 56

14-(l,5-Pentanediaminomethyl)-12H-5,lla-diazadibenzo[6,/ι]fluoren-ll-one Trihydrochloride (56). Compound 45 (0.065 g, 0.130 mmol) was diluted in CHCl₃ (40 mL) and filtered to remove particulate matter. Methanolic HCl (3 M, 10 mL) was added dropwise, and the dark red mixture was stirred for 2 h at room temperature and concentrated to provide a bright red amorphous solid (0.056 g, 86%) after washing with ether and drying in vacuo: mp 245-251 ⁰C (dec.) IR (KBr) 3399, 2929, 1654, 1474, 1336, 1217, 766, 683 cm^"1; ¹H NMR (300 MHz, D₂O) δ 7.75-7.45 (m, 7 H), 7.20-7.02 (m, 2 H), 5.12 (s, 2 H), 4.57 (s, 2 H), 3.27 (t, J = 6.9 Hz, 2 H), 2.98 (t, J = 6.4 Hz, 2 H), 1.80-1.60 (m, 4 H), 1.50-1.40 (m, 2 H); ESIMS m/z (rel intensity). 399 (MH⁺, 100). Anal (C₂₅H₂₉Cl₃N₄O^ H₂O) C, H, N.

EXAMPLE 57

14-(l,6-Hexanediaminomethyl)-12H-5,lla-diazadibenzo[6,/ι]fluoren-ll-one Trihydrochloride (57). Compound 46 (0.050 g, 0.097 mmol) was dissolved in CHCl₃ (30 mL) and methanolic HCl (3 M, 10 mL) was added dropwise. The orange cloudy mixture was stirred at room temperature for 2 h and concentrated to afford a dark red amorphous solid (0.043 g, 92%) after washing with ether and drying in vacuo: mp 230-235 ⁰C. IR (KBr) 3412, 2928, 1657, 1622, 1596, 1478, 1337, 765, 682 cm^"1; ¹H NMR (300 MHz, D₂O) δ 7.72 (d, J= 8.4 Hz, 1 H), 7.60 (s, 2 H), 6.51-7.42 (m, 4 H), 7.06-6.95 (m, 2 H), 5.08 (s, 2 H), 4.54 (s, 2 H), 3.25 (t, J = 7.7 Hz, 2 H), 2.97 (t, J= 7.4 Hz, 2 H), 1.80-1.60 (m, 4 H), 1.30-1.40 (bm, 4 H); ESIMS m/z (rel intensity) 413 (MH⁺, 100). Anal (C₂₆H₃iCl₃N₄O H₂O), C, H, N.

EXAMPLE 58

14-(l,7-Heptanediaminomethyl)-12H-5,lla-diazadibenzo[6,/ι]fluoren-ll- one Trihydrochloride (58). Compound 47 (0.035 g, 0.066 mmol) was diluted with benzene (25 mL) and methanolic HCl (3 M, 10 mL) was added dropwise, upon which the solution became a dark red. The mixture was heated at reflux for 3 h, cooled, and concentrated to yield a bright orange solid (0.030 g, 91%) after washing with ether: mp 208-215 ⁰C (dec). IR (KBr) 3400, 2929, 1657, 1620, 1600, 1457, 1340, 1132, 764, 687 cm^"1; ¹H NMR (300 MHz, D₂O) 7.75-7.69 (m, 3 H), 7.58-7.48 (m, 4 H), 7.20-7.08 (m, 2 H), 5.13 (s, 2 H), 4.56 (s, 2 H), 3.23 (t, J= 8.1 Hz, 2 H), 2.95 (t, J= 7.7 Hz, 2 H), 1.80-1.50 (m, 4 H), 1.30-1.20 (m, 6 H); ESIMS m/z (rel intensity) 427 (MH⁺, 100). Anal (C₂₇H₃₃Cl₃N₄O-LS H₂O) C, H, N.

EXAMPLE 59

14-(l,8-Octanediaminomethyl)-12H-5,lla-diazadibenzo[ό,Λ]fluoren-ll-one Trihydrochloride (59). Compound 48 (0.042 g, 0.078 mmol) was dissolved in CHCl₃ (30 niL) and methanolic HCl (3 M, 10 niL) was added dropwise, upon which the solution became a dark orange-red. The mixture was stirred at room temperature for 2 h and concentrated to yield a dark red flaky solid (0.036, 98%) after washing with ether and drying in vacuo: mp 200-203 ⁰C (dec). IR (KBr) 3401, 2929, 2856, 1758, 1656, 1597, 1619, 1479, 1338, 1135, 764, 688 cm^"1; ¹H NMR (300 MHz, D₂O) 7.70-7.38 (m, 7 H), 7.04-7.02 (m, 1 H), 6.89 (s, 1 H), 5.04 (s, 2 H), 4.50 (s, 2 H), 3.22 (t, J= 7.6 Hz, 2 H), 3.94 (t, J= 7.3 Hz, 2 H), 1.80-1.50 (m, 4 H), 1.30-1.20 (m, 8 H); ESIMS m/z (rel intensity) 441 (MH⁺, 100). Anal (C₂₈H₃₅Cl₃N₄O-H₂O) C, H, N.

EXAMPLE 60

14-(l,9-Nonanediaminomethyl)-12H-5,lla-diazadibenzo[6,/ι]fluoren-ll-one Trihydrochloride (60). Compound 49 (0.046 g, 0.083 mmol) was dissolved in CHCl₃ (20 mL) and methanolic HCl (3 M, 10 mL) was added dropwise. The mixture was stirred for 2 h 20 min at room temperature, concentrated, and azeotroped with benzene to afford a bright red amorphous solid (0.038 g, 88%) after washing with ether and drying in vacuo: mp 192-196 ⁰C (dec). IR (KBr) 3414, 2928, 2854, 1657, 1622, 1599, 1478, 1336, 761, 688 cm^"1; ¹H NMR (300 MHz, D₂O) δ 7.73-7.65 (m, 2 H), 7.52-7.45 (m, 3 H), 7.20-7.00 (m, 2 H), 5.10 (s, 2 H), 4.53 (s, 2 H), 3.21 (t, J= 6.9 Hz, 2 H), 2.92 (J= 7.2 Hz, 2 H), 1.70-1.50 (m, 4 H), 1.40-1.20 (bm, 10 H); ESIMS m/z (rel intensity) 455 (MH⁺, 100). Anal (C₂₉H₃₇Cl₃N₄O), C, H, N.

EXAMPLE 61

14-(l,10-Decanediaminomethyl)-12H-5,lla-diazadibenzo[6,/ι]fluoren-ll- one Trihydrochloride (61). Compound 50 (0.056 g, 0.096 mmol) was dissolved in CHCl₃ (30 mL) and methanolic HCl (3 M, 10 mL) was added dropwise. The addition was slightly exothermic. The mixture was stirred for 2 h at room temperature and concentrated to yield a red amorphous solid (0.049 g, 92%) after washing with ether and drying in vacuo: mp 185-190 (dec). IR (KBr) 3433, 2925, 2854, 1657, 1622, 1599, 1469, 1335, 764, 688 cm^"1; ¹H NMR (300 MHz, D₂O) δ 7.75-7.69 (m, 3 H), 7.56-7.49 (m, 4 H), 7.15-7.08 (m, 2 H), 5.13 (s, 2 H), 4.56 (s, 2 H), 3.21 (t, J= 7.4 Hz, 2 H), 2.91 (t, J= 7.5 Hz, 2 H), 1.70-1.50 (m, 4 H), 1.40-1.20 (bm, 12 H); ESIMS m/z (rel intensity) 469 (MH⁺, 100). Anal (C₃₀H₃₉Cl₃N₄O-0.6 H₂O) C, H, N. EXAMPLE 62

14-(l,ll-Undecanediaminomethyl)-12H-5,lla-diazadibenzo[6,Λ]fluoren-ll- one Trihydrochloride (62). Compound 51 (0.056 g, 0.097 mmol) was dissolved in CHCl₃ (30 niL) and methanolic HCl (3 M, 10 niL) was added dropwise. The reaction was slightly exothermic. The mixture was stirred for 2 h at room temperature and concentrated to yield a red-orange amorphous solid (0.052 g, 96%) after washing with ether and drying in vacuo: mp 183-188 ⁰C (dec). IR (KBr) 3431, 2925, 2853, 1657, 1622, 1601, 1469, 1336, 762, 688 cm^"1; ¹U NMR (300 MHz, D₂O) δ 7.75-7.52 (m, 7 H), 7.19-7.13 (m, 2 H), 5.16 (s, 2 H), 4.58 (s, 2 H), 3.19 (t, J= 6.2 Hz, 2 H), 2.91-2.85 (m, 2 H), 1.70-1.50 (m, 4 H), 1.40-1.10 (m, 14 H); ESIMS m/z (rel intensity) 483 (MH⁺, 100). Anal (C₃₁H₄₁CI₃N₄OO.5 H₂O) C, H, N.

EXAMPLE 63

14-(l,12-Dodecanediaminomethyl)-12H-5,lla-diazadibenzo[6,Λ]fluoren-ll- one Trihydrochloride (63). Compound 52 (0.069 g, 0.115 mmol) was dissolved in CHCl₃ (30 mL) and methanolic HCl (3 M, 10 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 h and concentrated to yield a bright red amorphous solid (0.064 g,

97%) after washing with ether and drying in vacuo: mp 200-204 ⁰C (dec). IR (KBr) 3409, 2924, 2851, 1657, 1619, 1598, 1467, 1337, 764, 688 cm^"1; ¹H NMR (300 MHz, D₂O, 60 ⁰C) δ 7.98- 7.81 (m, 4 H), 7.72 (t, J= 7.8 Hz, 3 H), 7.43-7.37 (m, 2 H), 5.29 (s, 2 H), 4.71 (s, 2 H), 3.23 (t, J= 7.6 Hz, 2 H), 3.02-2.92 (m, 2 H), 1.41-1.56 (m, 6 H), 1.30-1.15 (m, 16 H); ESIMS m/z (rel intensity), 447, (MH⁺, 100). Anal (C₃₂H₄₃Cl₃N₄O-H₂O) C, H, N.

EXAMPLE 64

The aromathecin analogues were assayed for cytotoxic activity in the National Cancer Institute's Developmental Therapeutics screen. Each compound was evaluated against approximately 60 cell lines originating from various human tumors (Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J. New Colorimetric Cytotoxicity Assay for Anticancer- Drug Screening. J. Natl. Cancer Inst. 1990, 82, (13), 1107-1112; Boyd, M. R.; Paull, K. D. Some Practical Considerations and Applications of the National Cancer Institute In- Vitro Anticancer Drug Discovery Screen. Drug Development Res. 1995, 34, 91-109). After an initial one-dose assay, selected compounds were tested at five concentrations encompassing the range of 10^"8 to 10^"4 molar. Cytotoxicity results are reported as GI50 values for selected cell lines from each sub-panel, and overall cytotoxicity is quantified as a Mid-Graph Midpoint (MGM) in Table 1. The MGM is a measure of the average GI₅₀ against all cell lines tested. The activities of camptothecin (1), indenoisoquinoline 5 (MJ-III-65) (Pommier, 2006; Nagarajan, et al, 2004; Antony, S.; Jayaraman, M.; Laco, G.; Kohlhagen, G.; Kohn, K.W.; Cushman, M.; Pommier, Y. Differential Induction of Topoisomerase I-DNA Cleavage Complexes by the Indenoisoquinoline MJ-III-65 (NSC 706744) and Camptothecin: Base Sequence Analysis and Activity against Camptothecin-Resistant Topoisomerase I. Cancer Res. 2003, 63, 7428-7435), NCS314622 (6) (Kohlhagen, et al, 1998), rosettacin (9) and dimethoxyaromathecin (10) (Fox, et al., 2003), in addition to the inhibitory activity of 22-hydroxyacuminatine (8), are described.

TABLE 1 Cytotoxicities and Topoisomerase I Inhibitory Activities of 14-Substituted Aromathecin Analogues

cytotoxicity (GI50 in M)^a lung colon CNS melanoma ovarian renal prostate breast Top 1

OVCAR- MDA- Cleavage⁰

Cpd HOP-62 HCT-116 SF-539 UACC-62 3 SN12C DU- 145 MB-435 MGM^b

1 0.01 0.03 0.01 0.01 0.22 0.02 0.01 0.04 0.0405 ++++

5 0.02 0.10 0.04 0.03 0.5 >0.01 >0.01 0.79 0.11 ++++

6 1.3 35 41 4.2 73 68 37 96 20.0 ++

8^d - - - - - - - - - +

9 68.2 32.7 66.7 97.2 39.8 >100 >100 41.8 58.9 ++

10 >100 57.3 >100 >100 >100 >100 >100 >100 91.2 +

27a 26.5 43.4 7.4 >100 >100 >100 >100 >100 40.7 +

27c 9.8 1.5 4.9 11.7 2.9 2.9 2.8 9.4 3.9 ++

27d 10.0 13.2 4.7 3.4 17.0 21.5 11.5 >100 12.6 +++

27e >100 1.9 1.8 >100 - >100 4.9 2.1 6.2 ++(+)

27f - - - - - - - - - ++

27g 3.5 3.6 3.9 2.6 6.3 2.7 2.8 >100 5.2 ++(+)

27h >100 - >100 >100 >100 >100 >100 >100 46.8 ++(+)

27i 28.6 9.0 5.4 2.7 5.5 16.9 7.0 10.3 7.6 ++(+)

27j 4.7 3.2 3.8 6.4 4.4 3.5 3.2 7.0 3.5 ++

28c^g - - - - - - - - - +

28d 2.0 1.6 0.74 1.5 5.0 4.5 3.3 4.2 1.6 +++

28e 5.0 3.9 3.5 1.4 10.6 5.9 20.3 >100 5.9 ++

28f 1.2 1.6 12.9 1.8 6.3 4.1 13.2 1.7 2.8 ++++

28g^f - - - - - - - - - +++

53 1.4 1.5 8.3 12.6 1.7 1.5 1.4 11.0 2.8 ± 0.11 +++

54 9.8 1.5 4.9 4.8 1.4 2.1 1.1 6.8 2.1 ± 0.38 ++(+)

55^e - - - - - - - - - ++(+)

56 2.6 3.2 7.2 1.9 8.1 3.2 1.1 12.6 2.6 ± 1.65 +

57 1.2 0.63 0.74 2.0 1.4 0.51 0.33 1.5 1.0 ^■(+)

58^e - - - - - - - - - ++

59 2.8 1.8 1.9 1.8 2.5 1.8 1.1 3.1 1.9 +

60^e - +

61^d - - - - - - - - - 0

62^e - - - - - - - - - 0

63 6.5 2.0 1.8 1.9 1.9 2.5 2.0 1.7 2.6 0 aThe cytotoxicity GI₅₀ values are the concentrations corresponding to 50% growth inhibition. ^bMean graph midpoint for growth inhibition of all human cancer cell lines successfully tested, ranging from 10^"8 to 10^"4 molar. ^cCompound-induced DNA cleavage due to Topi inhibition is graded by the following rubric relative to 1 uM camptothecin: 0, no inhibitory activity; +, between 20 and 50% activity; ++, between 50 and 75% activity; +++, between 75 and 95% of activity; ++++, equipotent. ^d22-hydroxyacuminatine was not tested in the National Cancer Institute assay. ^{e f}These compounds were not selected for further testing; refer to text for details. ^gCurrently undergoing 5-dose testing.

Ol

-t->

EXAMPLE 65

Topoisomerase I-Mediated DNA Cleavage Reactions. Human recombinant Topi was purified from Baculovirus as described previously (Pourquier, P.; Ueng, L. -M.; Fertala, J.; Wang, D.; Park, H. -K.; Essigmann, J.M.; Bjornsti, M. -A.; Pommier, Y. Induction of Reversible Complexes Between Eukaryotic DNA Topoisomerase I and DNA-containing

Oxidative Base Damages. 7,8-Dihydro-8-Oxoguanine and 5-Hydroxycytosine. J. Biol. Chem. 1999, 274, 8516-8523). The 161 bp fragment from pBluescript SK(-) phagemid DNA (Stratagene, La Jolla, CA) was cleaved with the restriction endonuclease Pvu II and Hind III (New England Biolabs, Beverly, MA) in supplied NE buffer 2 (50 μL reactions) for 1 h at 37 ⁰C, and separated by electrophoresis in a 1% agarose gel made in Ix TBE buffer. The 161 bp fragment was eluted from the gel slice using the QIAEX II kit (QIAGEN Inc., Valencia, CA). Approximately 200 ng of the fragment was 3 '-end labeled at the Hind III site by fill-in reaction with [alpha-³²P]-dGTP and 0.5 mM dATP, dCTP, and dTTP, in React 2 buffer (50 mM Tris- HCl, pH 8.0, 100 mM MgCl₂, 50 mM NaCl) with 0.5 unit of DNA polymerase I (Klenow fragment). Unincorporated ³²P-dGTP was removed using mini Quick Spin DNA columns (Roche, Indianapolis, IN), and the eluate containing the 3 '-end-labeled 161 bp fragment was collected. Aliquots (approximately 50,000 dpm/reaction) were incubated with topoisomerase I at 22 ⁰C for 30 min in the presence of the tested drug. Reactions were terminated by adding SDS (0.5% final concentration). The samples (10 μL) were mixed with 30 μL of loading buffer (80% formamide, 10 mM sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue, pH 8.0). Aliquots were separated in denaturing gels (16% polyacrylamide, 7 M urea). Gels were dried and visualized by using a Phosphoimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Inhibition data are expressed semiquantitatively as follows: 0, no inhibitory activity; +, between 20 and 50% the activity of 1 μM camptothecin (1); ++, between 50 and 75% the activity of 1 μM camptothecin; +++, between 75-100% the activity of 1 μM camptothecin; and ++++, equipotent to or more potent than 1 μM camptothecin. Topi inhibitory data for aromathecins and comparative compounds are also included in Table 1.

EXAMPLE 66 Modeling Studies. The structure of the ternary complex, containing topoisomerase I, DNA, and camptothecin, was downloaded from the Protein Data Bank (PDB code 1T8I; Staker, et al., 2005; Fox, et al., 2003). Several of the atoms were then fixed according to the Sybyl atom types. Hydrogens were added and minimized using the MMFF94s force field and MMFF94 charges. Modeled analogues were constructed in Sybyl 7.3, energy minimized with the MMFF94s force field and MMFF94 charges, overlapped with the crystal structure ligand in the ternary complex, and the crystal structure ligand was then deleted. The new complex was subsequently subjected to energy minimization using MMFF94s force field with MMFF94 charges. During the energy minimization, the structure of the aromathecin and a surrounding 5 A sphere were allowed to move, while the structures of the remaining protein and nucleic acids were frozen. The energy minimization was performed using the Powell method with a 0.05 kcal/mol A energy gradient convergence criterion and a distance-dependent dielectric function. Ligand overlays were constructed using the indenoisoquinoline crystal structure 1SC7, (Staker, et al, 2005).

While certain embodiments of the present invention have been described and/or exemplified above, it is contemplated that considerable variation and modification thereof are possible. Accordingly, the present invention is not limited to the particular embodiments described and/or exemplified herein

Claims

WHAT IS CLAIMED IS:

1. A compound of formula

or a pharmaceutically acceptable salt thereof, wherein:

R^D is alkyl substituted with at least one nitrogen or oxygen containing group; and

R^E is hydrogen; or R^E represents one or more substituents selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted amino, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_n-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof; or R^E represents two or more substituents, where two of said substituents are adjacent and taken together with the attached carbons to form a heterocycle, and the remaining substituents if present are selected from the group consisting of halo, alkyl, optionally substituted hydroxy, optionally substituted hydroxyalkyl, optionally substituted aminoalkyl, or (CH₂)_n-Z, where n is an integer from 0 to about 4, and Z is a carboxylic acid or a derivative thereof.

2. The compound of the preceding claim wherein R^A is hydrogen, hydroxyalkyl, optionally substituted alkoxy, Z, or R^A represents two or more substituents, where two of said substituents are adjacent and taken together with the attached carbons to form a heterocycle.

3. The compound of any one of the preceding claims wherein R^E is hydrogen, hydroxy, halo, alkyl, optionally substituted alkoxy, acyloxy, or alkylamino.

4. The compound of any one of the preceding claims wherein R^B is hydrogen, optionally substituted alkoxyalkyl, or Z.

5. The compound of any one of the preceding claims wherein R^B is hydrogen.

6. The compound of any one of the preceding claims wherein R^E is hydrogen.

7. The compound of any one of the preceding claims wherein R^A is hydrogen.

8. The compound of any one of the preceding claims wherein R^D is hydroxyalkyl, aminoalkyl, azidoalkyl, optionally substituted alkylaminoalkyl, optionally substituted dialkylaminoalkyl, optionally substituted heterocyclylalkyl, optionally substituted heteroarylalkyl, hydroxyalkylaminoalkyl, optionally substituted alkylaminoalkylaminoalkyl, optionally substituted dialkylaminoalkylaminoalkyl, optionally substituted heterocyclylalkylaminoalkyl, optionally substituted heteroarylalkylaminoalkyl, or hydroxyalkylaminoalkylaminoalkyl.

9. The compound of any one of the preceding claims wherein R^D is alkylene-amino-alkylene-NH₂ or alkylene-amino-heteroalkylene-NH₂.

10. The compound of any one of the preceding claims wherein R^D is CH₂NH(CH₂)pNH2, where p is 2 to about 12.

11. The compound of any one of the preceding claims wherein R^D is (2- hydroxyethyl)aminoalkyl.

12. The compound of any one of the proceeding claims wherein R^D is heterocycloalkyl.

13. The compound of any one of the proceeding claims wherein R^D is imidazolylalkyl.

14. A pharmaceutical composition comprising a therapeutically effective amount of the compound of any one of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, or excipients therefor, or a combination thereof.

15. A method for treating a cancer, the method comprising the step of administering to a patient in need of relief from the cancer a therapeutically effective amount of the compound or the composition of one of the preceding claims.

16 The method of claim 8 wherein the composition is adapted for parenteral or oral administration.