WO2000064917A2 - Synthetic indolocarbazole regioisomers and uses thereof - Google Patents

Abstract

This invention relates to synthetic indolocarbazole regioisomers and uses thereof. More particular, this invention relates to 3,9-dihydroxy and 2,10-dihydroxy indolocarbazoles of formulae (I) and (II) capable inhibiting Topoisomerase (I) activity. This invention also relates to methods of using such indolocarbazoles.

Description

SYNTHETIC INDOLOCARBAZOLE REGIOISOMERS AND USES THEREOF

CROSS-REFERENCE

This application is a continuation-in-part of U.S. Ser. No. 09/299,813, filed April 26, 1999. This application also claims the benefit of U.S. Provisional Application No. 60/083,316, filed April 28, 1998. The work in this application was supported in part by NIH grant number

1R43 CA72328-01 Al. Accordingly, the United States government may have certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to synthetic indolocarbazole regioisomers and uses thereof. More particularly, this invention relates to 3,9-dihydroxy and 2,10-dihydroxy indolocarbazoies capable of inhibiting Topoisomerase I activity. This invention also relates to methods of using such indolocarbazoies.

BACKGROUND OF THE INVENTION

Topoisomerase types I and II are important enzymatic targets in the design of antineoplastic agents.¹'² These enzymes are responsible for topological isomerization of double-stranded DNA, and are critical for cellular viability. Topoisomerase I (Topo I) is the target of camptothecin and its semi-synthetic derivatives, particularly topotecan

(Hycamtin) and CPT-11 (Camptosar), which are approved for clinical use by the Food and Drug Administration. Other than camptothecin and its analogues, few specific Topo I poisons are known, and topotecan and CPT-11 are the only drugs of this class approved for clinical use as antitumor agents.

Certain representatives of the indolocarbazole family have been shown to be potent inhibitors of Topo I. ED-110 and NB-506, both semi-synthetic derivatives of the microbial metabolite BE-13793C, induced Topo I-mediated DNA cleavage, in vitro at concentrations similar to those required for camptothecin. Both drugs were specific for Topo I.

BE-13793C

ED-110 showed in vitro antitumor activity against a panel of twelve human tumor cell lines, nine of which were sensitive. Murine in vivo studies involving i.p. implanted leukemia cells showed increased survival period by more than two-fold following treatment with ED-110. ED-110 also inhibited the growth of solid murine tumors, as well as prevented the spontaneous metastasis of Meth A fibrosarcoma. The drug suppressed the growth of MKN-45 human stomach cancer cells xenotransplanted into B ALB/c nude mice.

NB-506 inhibited the growth of human tumor cells xenotransplanted into nude mice. A dose of 90 mg/m² rapidly reduced tumor size of human PC-13 lung and MKN-45 stomach cancer nodules in nude mice, with low toxicity. A Phase I clinical trial of NB- 506 showed reduction of tumor-specific markers in ovarian and breast cancer patients who were clinically resistant to taxol.

Topoisomerase I

Topoisomerase I (Topo I) is a 100 kD monomeric protein which catalyzes changes in the topological state of double-stranded DNA (dsDNA) in steps of one linking number.³ Topo I can relax both positively and negatively supercoiled DNA and does not require an energy cofactor. The mechanism by which Topo I relaxes DNA is postulated to proceed through a transient single-stranded break in the dsDNA via formation of a covalent protein-DNA complex known as the cleavable complex, so named because these complexes are detected as DNA breaks upon treatment with denaturing agents or alkali. The cleavable complex is formed upon transesterification of a DNA phosphodiester linkage by the active-site tyrosine-723 residue on human Topo I, resulting in an ester linkage between the tyrosine phenolic group and the 3' phosphoryl end of the broken DNA strand. This allows free rotation of the protein-bound 3' end of the broken DNA strand about the intact complementary DNA, resulting in relaxation of the duplex in steps of one linking number. Religation of the broken strand (via a second transesterification reaction) and subsequent dissociation of Topo I completes the catalytic cycle.

Topo I inhibitors typically act by one of two mechanisms: stabilization of the cleavable complex, thus promoting DNA strand breakage (likely via inhibition of religation), or inhibition of the binding of Topo I to DNA.⁴ The latter mechanism is generally non-specific for Topo I and involves DNA intercalating agents, which interfere with the ability of the enzyme to interact with DNA via either direct blockage of access to the duplex DNA or disruption of tertiary DNA structure. The former mechanism is regarded as the most important for specific inhibitors of Topo I. These types of compounds are generally referred to as Topo I poisons, rather than Topo I inhibitors, because they essentially convert Topo I into a DNA damaging agent.

Stabilization of the cleavable complex is mediated by formation of a ternary complex, consisting of drug, Topo I, and DNA. Agents such as camptothecin (the prototype Topo I poison) do not bind to DNA directly, nor to Topo I alone, but only to Topo I complexed with DNA. It has been postulated that the stabilized DNA-protein-drug complex causes lethal DNA strand breaks upon collision with the advancing replication fork. It is by this mechanism that the Topo I poison converts the Topo I molecule into a DNA damaging agent, resulting in disruption of DNA replication and, eventually, cell death. This postulate is supported by the fact that camptothecin is highly phase-specific, only killing cells in S-phase. Camptothecin and a number of semi-synthetic derivatives are currently being evaluated in clinical trials for their antitumor activity.⁵ Topotecan (Hycamtin) and CPT-11 (Camptosar), derivatives of camptothecin, are the only Topo I poisons approved for clinical use as antitumor agents.

Indolocarbazoies as Topoisomerase I Inhibitors

Indolocarbazole alkaloids have been a subject of chemical interest for many years, and a large number of naturally occurring examples are known.⁷ Recently, indolocarbazoies were found to possess a number of interesting biological activities.

Perhaps the most well known example is staurosporine, a microbial natural product which is the most powerful naturally-occurring inhibitor of protein kinase C known.⁸

In 1993 it was reported that the indolocarbazole analogue ED-110 induced single- stranded DNA breaks via Topo I.⁹ ED-110 is a semi-synthetic derivative of BE-13793C (the aglycone of ED-110),¹⁰ which was isolated from a streptomycete.¹¹ Treatment of pBR322 supercoiled DNA and Topo I (isolated from P388/S cells) with ED-110 resulted in formation of nicked circular DNA following treatment with sodium dodecylsulfate (SDS, a denaturing agent routinely used to "trap" cleavable complexes). This result suggests that ED-110 stabilizes the cleavable complex formed between Topo I and duplex DNA. No nicked DNA was produced upon treatment with ED-110 in the presence of Topo II, and no DNA damage was caused by ED-110 alone. ED-110 competed with ethidium bromide for DNA binding, suggesting that this agent intercalates DNA. ED-110 was cytotoxic against the P388/S murine leukemia cell line in vitro, with an IC₅₀ of 44 nM. Additionally, ED-110 was cytotoxic against the multi-drug resistant cell lines P388/NCR and P388/ADM, (which are resistant to vincristine and adriamycin, respectively), the former of which overexpresses the P-glycoprotein multi-drug transporter (gpl70). In cell cycle inhibition studies, ED-110 slowed the progress of exponentially dividing cells through the S phase early in the incubation period, and eventually caused cell cycle arrest in G₂.

In a subsequent study, ED-110 was tested for cyto toxicity against a panel of human tumor cell lines in vitro. ^X1 Interestingly, it was found that cytotoxicity varied widely depending upon the cell line. ED-110 was potent against stomach (MKΝ-28, MKN-45 and MKN-74), colon (LS 180, HCT 116), epidermal (A 431 ), oral (KB), lung (PC- 13) and

breast (MCF7) derived cell lines, with IC₅n values ranging from 0.135 to 22 μm. Several

cell lines, such as colon (DLD-1, WiDr) and pancreatic (PSN 1) were resistant, with no

observed effects at concentrations up to 193 μM. Animal studies confirmed that ED-110

possesses antitumor activity in vivo, providing an increase in life span (ILS) in mice implanted i.p. with either P388, L1210, L5178Y or EL4 murine leukemia cells. Additionally, ED-110 inhibited growth of the mouse solid tumors colon 26 and IMC carcinoma by 83% and 91%, respectively when the drug was given i.p. at 160mg/kg. Studies with metastatic murine fibrosarcoma cells (Meth A) implanted s.c. indicated that treatment with 40 mg/kg ED-110 prolonged survival time by 47%. A dose of 160 mg/kg resulted in two of the five test mice surviving until the end of the test period (60 days), while mice treated with vehicle alone survived a mean of 18.4 days. Finally, ED-110 inhibited the growth of MKN-45 human stomach cancer cells xenografted into BALB/c nude mice at a dose of 2.5 mg/kg. In all the above studies, it was observed that drug toxicity was low, and in preliminary safety studies, all mice treated with 500 mg/kg remained alive during the ten day observation period.

The indolocarbazole analogue NB-506, also a semi-synthetic derivative of BE-

13793C, induced Topo I-mediated DNA breaks at a drug concentration of 10 nM, but

showed no effects in the presence of Topo II at drug concentrations as high as 300 μm.¹³

NB-506 was cytotoxic against a variety of cell lines in vitro, exhibiting cell line selectivity against a panel of 11 human tumor lines. However, the cytotoxicity did not correlate well with levels of Topo I activity in the different cell types, but rather correlated with the accumulation of drug in the different cell lines. This correlation was not likely related to the gpl 70 multidrug resistant transporter, because NB-506 effectively inhibited the growth of cell lines actively expressing the gpl 70 protein. A related study involved development of NB-506-resistant cell lines, which showed cross-resistance to other Topo I inhibitors

(such as SN-38, an active metabolite of CPT-11) but not to other common anticancer drugs such as cisplatin, etoposide, teniposide, vinblastine and vincristine.¹⁴ The NB-506- resistant cells were found to express only one-tenth the amount of Topo I as the parent cell line, and it was concluded that down-regulation of Topo I expression was the mechanism of drug resistance.

Like ED-110, NB-506 inhibited the growth of solid human tumors xenotransplanted into nude mice, with impressive therapeutic ratios.¹⁵ A dose of 90 mg/m² was sufficient to cause rapid reduction in tumor size of human PC- 13 lung cancer and MKN-45 stomach cancer nodules in nude mice. The drug's toxicity levels were encouraging, with LD₅o values of 990 mg/m for a single iv injection and 810 mg/m for repeated iv injections (once daily for 10 days). Interest-ingly, little cumulative toxicity was observed, and more than eight times as much drug could be administered by daily injections than could be administered by a single injection. A Phase I clinical trial of NB- 506 showed reduction of tumor-specific makers in ovarian and breast cancer patients who were clinically resistant to taxol treatment.¹⁵ Several other indolocarbazoies were reported to induce DNA damage via Topo I.

The analogues KT6006 and KT6528, which are semi-synthetic derivatives of K252a, stabilized the cleavable complex formed between calf thymus Topo I and pBR322 supercoiled DNA, resulting in single-stranded DNA breaks following treatment with SDS.¹⁶ KT6006 and KT6528 both produced DNA damage in a dose-dependent manner at drag concentrations

up to 50 μM, comparable to camptothecin. However, DNA cleavage induced by KT6528

was suppressed at higher concentrations (50 μm and above) because of distortion of the

DNA caused by intercalation of the drug. KT6006 was a weak intercalator, while KT6528 was a strong intercalator. Neither compound induced DNA breaks in the presence of Topo

I, nor in the absence of Topo I. Another

KT6006 KT6528

indolocarbazole, KT6124 (a derivative of K252a), also induced DNA breakage, although a mechanism of action involving topoisomerases was not confimed.¹⁷

Other observations related to the potential antitumor effects of indolocarbazoies have been noted. Cell cycle studies using HR-3Y1 cells, a ras-transformed rat 3Y1 cell line, found that indolocarbazoies having differing activities against particular enzymatic targets exerted differential effects upon the normal progression of the cell cycle.¹⁸ Staurosporine and K252a, both non-specific inhibitors of protein kinase C, caused cell cycle blockade at the G2/M interface, while the selective PKC inhibitor UCN-01 (a semi- synthetic derivative of staurosporine) induced Gl blockade. Significantly, KT6124, KT6528 and KT6006, inhibitors of Topo I, all slowed progression of the cell cycle through the S phase, a property also observed with camptothecin.¹⁶ Topoisomerase I has been a critical enzymatic target for the design of potential antitumor agents, since it was established that Topo I is the target of camptothecin. Several semi-synthetic derivatives of camptothecin are in various stages of clinical trials.⁵ Although a variety of Topo II inhibitors are currently used in the clinical setting for the treatment of human cancers,^1,2 very few specific inhibitors of Topo I are known. Only two drugs of this class, topotecan and CPT-11, have been approved by the FDA. Intracellular Topo I levels were reported to vary between differing tumor types,¹⁹ and several groups have determined that tumor cell sensitivity to camptothecin is directly related to Topo I content.^20,21

Indolocarbazoies represent a novel class of Topo I inhibitors. Identification of a novel Topo I poison having desirable antitumor properties and which could be used clinically would have enormous benefit. A limited number of indolocarbazole analogues have been described in the literature, most of which were semi-synthetic derivatives of natural products. Modifications of indolocarbazole structure can result in significant attenuation of biological activity.

SUMMARY OF THE INVENTION

The invention provides synthetic 2,10- and 3,9-dihydroxy indolocarbazole regioisomers. Indolocarbazole regioisomers of the invention have enhanced Topoisomerase I inhibiting activity relative to ED-110. The invention also provides methods of using such indolocarbazoies for inhibiting Topoisomerase I activity.

Thus, in a first aspect the invention provides a synthetic indolocarbazole regioisomer of the formula I:

wherein

Ri and R₂ are independently H, halogen, hydroxyl, amino, Ci-_o alkyl, aryl-Cι-₆ alkyl, mono- or poly-fluorinated Cι-₆ alkyl, hydroxy-Cι-₆ alkyl, dihydroxy-Cι-₆ alkyl, di(hydroxy-Cι-₆ alkyl)-Cι-₆ alkyl, Cι-₆ alkoxy, amino-Cι-₈ alkyl, Cι-₆ alkylamino, di(Cι-6 alkyl)amino, Cι-₈ alkylamino-Cι-₈ alkyl, di(Cι-6 alkyl)amino- Cι- alkyl, cyclohexyl, aryl or heterocycle, wherein aryl or heterocycle may each be unsubstituted or substituted with one or more of the following: alkyl, Cι-₆ alkoxy, hydroxy-Ci--i alkyl, hydroxyl, a ino, Cι-₆ alkylamino, di(Cι-₆ alkyl) amino, amino-Cι-8 alkyl, Ci-g alkylamino-Cι-₈ alkyl, di(Cι-₆ alkyl)amino-Cι-₈ alkyl, nitro, azido or halogen;

or a pharmaceutically acceptable salt thereof.

In another aspect, the invention provides a composition comprising the synthetic indolocarbazole regioisomer of formula I and a pharmaceutically acceptable carrier.

In yet another aspect, the invention provides a synthetic indolocarbazole regioisomer of the formula II:

wherein

Ri and R₂ are independently H, halogen, hydroxyl, amino, Cι-₆ alkyl, aryl-Ci-_ό alkyl, mono- or poly-fluorinated Cι_-6 alkyl, hydroxy-Cι-6 alkyl, dihydroxy-Cι-₆ alkyl, di(hydroxy-Cι_-6 alkyl)-Cι-6 alkyl, Cι-₆ alkoxy, amino-Cι-₈ alkyl, Ci-_o alkylamino, di(Cι-6 alkyl)amino, Cι_-8 alkylamino-Ci-g alkyl, di(Cι-₆ alkyl)amino- Cι_-8 alkyl, cyclohexyl, aryl or heterocycle, wherein aryl or heterocycle may each be unsubstituted or substituted with one or more of the following: Cι-₆ alkyl, Cι-₆ alkoxy, hydroxy-C alkyl, hydroxyl, amino, Cι-₆ alkylamino, di(Cι-₆ alkyl) amino, amino-d-8 alkyl, Cι-₈ alkylamino-Ci-s alkyl, di(Cι-6 alkyl)amino-Cι-g alkyl, nitro, azido or halogen;

or a pharmaceutically acceptable salt thereof.

In a further aspect, the invention provides a composition comprising the synthetic indolocarbazole regioisomer of formula II and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method of inhibiting topoisomerase I activity comprising administering to a mammal an effective amount of at least one synthetic indolocarbazole regioisomer of the formula I:

wherein

Ri and R₂ are independently H, halogen, hydroxyl, amino, d-o alkyl, aryl-Cι-₆ alkyl, mono- or poly-fluorinated Cι-₆ alkyl, hydroxy-Ci-o alkyl, dihydroxy-Cι-6 alkyl, di(hydroxy-Cι-₆ alkyl)-Cι-₆ alkyl, Cι-₆ alkoxy, amino-Cι-₈ alkyl, Cι-6 alkylamino, di(Cι-₆ alkyl)amino, Cι-₈ alkylamino-Cι-₈ alkyl,

alkyl)amino- Cι-8 alkyl, cyclohexyl, aryl or heterocycle, wherein aryl or heterocycle may each be unsubstituted or substituted with one or more of the following: Cι-₆ alkyl, Cι-₆ alkoxy, hydroxy-Ci--, alkyl, hydroxyl, amino, Cι-₆ alkylamino, di(Cι-₆ alkyl) amino, amino-Cι-₈ alkyl, Cι_-8 alkylamino-Cι-₈ alkyl, di(Cι-₆ alkyl)amino-Cι-₈ alkyl, nitro, azido or halogen; or a pharmaceutically acceptable salt thereof.

In yet another aspect, the invention provides a method of inhibiting topoisomerase

I activity comprising administering to a mammal an effective amount of at least one synthetic indolocarbazole regioisomer of the formula I in combination with a pharmaceutically acceptable carrier.

In a still further aspect, the invention provides a method of inhibiting topoisomerase I activity comprising administering to a mammal an effective amount of at least one synthetic indolocarbazole regioisomer of the formula II:

wherein

Ri and R₂ are independently H, halogen, hydroxyl, amino, Cι-₆ alkyl, aryl-Cι-₆ alkyl, mono- or poly-fluorinated Cι-₆ alkyl, hydroxy-C_1- alkyl, dihydroxy-d-₆ alkyl, di(hydroxy-Cι-₆ alky-)-Cι-₆ alkyl, Cι-6 alkoxy, amino-Cι-₈ alkyl, d-6 alkylamino, di(C₁-₆ alkyl)amino, Cι-₈ alkylamino-Cι_-8 alkyl, di(Cι-₆ alkyl)amino- Cι-8 alkyl, cyclohexyl, aryl or heterocycle, wherein aryl or heterocycle may each be unsubstituted or substituted with one or more of the following: Cj-₆ alkyl, Cj-₆ alkoxy, hydroxy-Cι--ι alkyl, hydroxyl, amino, Cι-₆ alkylamino, di(Cι-₆ alkyl) amino, amino-Cι-8 alkyl, Cι-₈ alkylamino-Cι-₈ alkyl, di(Cι-₆ alkyl)--mino-d-₈ alkyl, nitro, azido or halogen; or a pharmaceutically acceptable salt thereof, alone or in combination with a carrier.

In yet a further aspect, the invention provides a method of inhibiting topoisomerase

I activity comprising administering to a mammal an effective amount of at least one synthetic indolocarbazole regioisomer of the formula II in combination with a pharmaceutically acceptable carrier.

These and other aspects of the invention will become apparent in light of the detailed description below. All patents, patent applications, and literature references cited herein are incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates a dose response of SC DU-145 Prostate Tumor to treatment with

a compound of the invention and Paclitaxel.

FIG. 2 illustrates a dose response of SC HT29 Colon Tumor to treatment with a compound of the invention and Paclitaxel. DESCRIPTION OF A PREFERRED EMBODIMENT

The present invention relates to indolocarbazole regioisomers demonstrating Topo I inhibiting activity and methods of using such indolocarbazoies. The invention provides indolocarbazole regioisomers obtained via synthesis employing benzyloxyindole precursors.

Synthesis of 4-. 5-. 6- and 7-Benzyloxyindoles as Precursors of Target Indolocaibazoles For Preparation of the 1, 11-, 2,10-, 3,9- and 4,8-dihydroxyindolocarbazole and corresponding bis-indolylmaleimide targets, the 1-, 6-, 5- and 4-benzyloxyindoles were required as precursors, respectively. Syntheses of 7-, 6-, and 4-benzyloxyindoles were achieved via Batcho-Leimgruber synthesis²² from 3-methyl-2-nitrophenol, 4-methyl-3- nitrophenol, and 2-methyl-3-nitrophenol, respectively, illustrated in Scheme 1. 5- Benyloxyindole was prepared by simple benzylation of commercial 5-hydroxyindole (benzyl bromide, acetone, K₂CO₃).

All of the required methylnitrophenols were commercially available except for 4- methyl-3-nitrophenol, which was prepared from commercial 4-methyl-3-nitroaniline via standard diazonium salt decomposition chemistry. Benzylation of the methylnitrophenols 1 with benzylbromide followed by condensation with dimethylformamide dimethylacetal

and pyrrolidine provided the benzyloxynitro-β-pyrrolidinostyrenes 2. Reductive

cyclization of the crude intermediates 2 via catalytic hydrogenation with Raney- nickel/hydrazine afforded the desired benzyloxyindoles 3 in very good yields.

pyrro ne

3a: 4-benzyloxy 3b: 5-benzyloxy 3c: 6-benzyloxy

Scheme 1 3d: 7-benzyloxy

Synthesis of Indolocarbazoies The benzyloxyindoles 3 described above were utilized to prepare the target core indolocarbazole units 8, illustrated in Scheme 2. Treatment of the benzyloxyindoles 3 with methylmagnesium iodide in THF generated the magnesium iodide salt of the indoles. Reaction of these salts with N-benzyloxymethyl-3,4-dibromomaleimide (prepared²³ from benzyl(chloromethyl) ether and dibromomaleimide) provided the intermediate mono- indolylmaleimides 4. Interestingly, use of excess quantities of indole magnesium halide did not provide bis-indolylmaleimides, in contrast to reports by others.^23"28 However, the phenomenon of the reaction stopping at the mono-indolylmaleimide stage 4 has been observed by others,²⁹ and the same authors reported that protection of the indole nitrogen of analogues such as 4 was required before efficient conversion to bis-indolylmaleimides would proceed.

A recent publication described protection of the indole nitrogen of analogues 4 as their t- butyloxylcarbonyl (Boc) derivatives, followed by successful displacement of the bromide with a second indole unit, using lithium hexamethyldisilazide as base.²⁹ However, using the magnesium iodide salt of the indole in this reaction resulted in removal of the N-Boc group, with no formation of the desired bisondolylmaleimide, and only compounds 4 isolated from the reaction. Following a literature procedure,³⁰ a glucose moiety was attached to the molecule at this stage of the synthesis, using Mitsunobu chemistry to form the required nitrogen-carbon bond. Thus, treatment of intermediates 4 with tetra-O benzyl-D-glucose (Sigma) in the presence of triphenylphosphine and diethylazodicarboxylate (DEAD) in THF (3 equiv each of sugar, PPh₃, and DEAD) provided smooth conversion of intermediates 4 to the sugar-containing derivatives 5. Though a large excess of reagents was required in the Mitsunobu reaction to achieve complete consumption of starting 4, the desired products 5 were readily purified via silica gel column chromatography. By contrast, a published procedure³⁰ indicated that only a 1.5 molar excess of reagents in the Mitsunobu reaction was required: however, in the reaction scheme above any less than 3 equiv resulted in incomplete reaction.

Upon attachment of the sugar moiety to the indole nitrogen, displacement of the bromide with a second indole unit occurs, to provide bis-indolylmaleimide derivatives 6. Oxidative ring closure of bis-indolylmaleimides such as 6 to indolocarbazoies was previously mediated by treatment with palladium(II) acetate in acetic acid,²⁵ palladium(II) trifluoroacetate, and 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ).^27,31 Use of palladium(II) acetate was, in our hands, extremely sluggish. However, cyclization proceeded smoothly upon treatment with stoichiometric amounts of palladium(II) trifluoroacetate in acetic acid, affording the protected indolocarbazoies 7. Finally, catalytic hydrogeno lysis (3 atm H₂) in the presence of 10% palladium on carbon proceeded efficiently to provide the desired ED-110 analogues 8a-8d. The final hydrogenolysis step was incomplete when conducted in other solvents, such as methanol or chloroform, but proceeded readily in acetic acid.

Synthesis of 6-N-Substituted Indolocarbazoies

Comparing the biological activities of differentially substituted indolocarbazoies indicates that modifying the substituent at the maleimide nitrogen of analogues such as ED-110 can profoundly alter anti-Topo I activity. For example, studies of rebeccamycin analogues indicated that appending a methyl group onto the maleimide nitrogen reduced protein kinase C inhibition, but increased anti-Topo I activity³⁴. A comparison of rebeccamycin-derived indolocarbazoies substituted on the maleimide nitrogen with amino and hydroxyl moieties demonstrated a 10-fold increase in anti-Topo I activity, relative to the N-unsubstituted analogue³⁵. Additionally, transformation of ED-110 into ΝB-506 resulted in increased anti-Topo I activity¹³.

Accordingly, 6-N-substituted indolocarbazoies were synthesized by various paths to evaluate the effects of maleimide nitrogen functionalization on anti-Topo I activity. As illustrated in Scheme 3, treatment of 8 with a substituted amine in THF afforded the Ν- substituted analogues 10 via a simple transamination reaction. Similarly, treating an aqueous solution of 8 with a substituted amine also produced the N-substituted analogues 10. Because 8 contains 5-hydroxyindole moieties as part of its core structure, which are known to be fairly sensitive to autoxidation in the presence of base, the reaction solution was carefully deoxygenated before addition of the amine, and the reaction environment kept under an inert atmosphere throughout the reaction. Alternatively, an amine exchange reaction on the protected intermediate 7 afforded compounds 11. Catalytic hydrogenolysis of intermediate 11 then produced the desired maleimide jV-substituted analogues 10. Another synthesis path comprised converting maleimide 7 to the maleic anydride 12. Reaction of maleic anhydrides with amines to form maleimides has been documented as a route to bis-indolylmaleimies , and has been used in the synthesis of NB-506 . Treatment of anhydride 12 with the appropriate amine afforded maleimide 11, which was then deprotected to provide the desired analogues 10. Particular moieties appended onto the maleimide nitrogen included methyl, amino (13), hydroxyl, hydroxyethyl (14), and CH(CH₂OH)₂ (15).

Scheme 3

Scheme 3 cont'd

Cytotoxic agents are often employed as chemotherapeutic agents to control or eradicate tumours. Camptothecin, a natural alkaloid demonstrating cytotoxic activity, functions by inhibiting Topoisomerase I activity i.e. camptothecin 's cytotoxicity activity is directly related to its potency as a Topoisomerase I inhibitor. Indolocarbazoies are a class of novel compounds which also demonstrate Topoisomerase I inhibiting activity. Accordingly, indolocarbazoies of the invention are useful anticancer and antitumour agents. In particular, Topoisomerase I inhibiting compounds of the invention have been shown to inhibit human colon, ovarian, and prostate tumor cells. Indolocarbazole regioisomers of the invention may be formulated as a solution of lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or in buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium choride or sodium citrate.

Alternatively, the compounds of the present invention may be encapsulated, tableted or incorporated into an emulsion (oil-in-water or water-in-oil) syrup for oral administration. Pharmaceutically acceptable solids or liquid carriers, which are generally known in the pharmaceutical formulary arts, may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Solid carriers include starch (com or potato), lactose, calcium sulfate dihydrate, terra alba, croscarmellose sodium, magnesium stearate or stearic acid, talc, pectin, acacia, agar, gelatin, maltodextrins and microcrystalline cellulose, or collodial silicon dioxide. Liquid carriers include syrup, peanut oil, olive oil, com oil, sesame oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 10 mg to about 1 g per dosage unit.

The dosage ranges for administration of indolocarbazole regioisomers of the invention are those to produce the desired affect. The dosage will generally vary with age, body weight, and counterindications, if any. The dosage will also be determined by the existence of any adverse side effects that may accompany the compounds. It is always desirable, whenever possible, to keep adverse side effects to a niinimum. One skilled in the art can easily determine the appropriate dosage, schedule, and method of administration for the exact formulation of the composition being used in order to achieve the desired effective concentration in the individual patient. However, the dosage can vary from between about 1 mg/kg/day to about 500 mg/kg/day, and preferably from between about 1 to about 50 mg kg/day.

One skilled in the art will recognize that modifications may be made in the present invention without deviating from the spirit or scope of the invention. The invention is illustrated further by the following examples, which are not to be construed as limiting the invention in spirit or scope to the specific procedures or compositions described in them. EXPERIMENTAL

Evaluation of Topo I inhibition

Prior to use, the activity of each stock of human Topo I (Topogen, Inc., Columbus, OH) was evaluated and normalized by titrating with pBR322 supercoiled plasmid DNA. One unit of enzyme activity was defined as the amount of enzyme which catalyzed the

complete relaxation of 0.4 μg pBR322 DNA in 30 minutes at 37 °C. As another measure

of control, it was verified that one unit of Topo I was completely inhibited by 100 μM camptothecin.

Enzymatic assays were conducted with commercial human Topo I using a modification of a routine literature procedure,³² as follows. The reaction mixture

contained 40 mM Tris-HCI., pH 7.5, 1 mM dithiothreitol, 0.5 mM EDTA, 15 μg/mL BSA,

7.5 mM MgCl₂, 80 mM KCI, 0.4 μg pBR322 plasmid DNA and I unit Topo I, in a total

volume of 600 μL. Test drug was added, dissolved in DMSO, with a final DMSO

concentration of approximately 5%. Reactions were allowed to proceed for 30 minutes at 37 °C, then the reaction was terminated by the addition of proteinase K in SDS (10

mg/mL, 2 μL). After 30 minutes at 37 °C, the samples were cooled to 0 °C and treated

with EtOH (75 μL) and 5 M NaCl (2.2 μL). The reaction vessels were cooled on dry ice for 60 minutes, then the DNA pelleted by centrifugation (16,000x g, 10 minutes). The

supernatant was discarded, and the DNA pellet resuspended in 18 μL reaction buffer,

diluted with 2 μL loading buffer, and then added to appropriate lanes of a 1 % agarose gel

made up with IX TPE buffer containing 2 μg/mL chloroquine. Gels were run for 15 hours

at 15 V. then stained with 0.5 μg/mL ethidium bromide. After destaining in H₂O for 30 minutes, gels were photographed over a UN light box with Polaroid 665 positive/negative film, and negatives were scanned with a UMAX Super Vista S-12 scanner, using Adobe and ΝIH imaging software. The IC ₀ values were assigned as the drug concentration which prevented Topo I-mediated relaxation of pBR322 plasmid DΝA by 50% relative to controls in the absence of enzyme and inhibitor, measured by densitometry of the band area of the unreacted plasmid and comparison with controls.

Evaluation of in vitro cvtotoxicitv

Cytotoxicities of the analogues were evaluated using the MTT cell viability assay.³³ The assay was repeated in triplicate for each analogue, using maximum drag

concentrations of 50 μM, to establish an average IC₅o value based upon the three trials.

The cyto toxicity assays were conducted using the ΝIH:OVCAR-3 ovarian carcinoma, DU-

145 prostatic carcinoma, and HT-29 colon adenocarcinoma cell lines.

EXAMPLE 1

The potencies of the four ED-110 regioisomers 8a - 8d against Topo I-mediated relaxation of supercoiled pBR322 plasmid DNA are shown in Table I. Compound 8d

(ED- 110) exhibited an IC₅o of 13 μM, consistent with concentrations previously reported

by others.⁹ Analogue 8a, which possesses the 4,8-dihydroxy substitution pattern, was less active than EDI 10, by approximately 3-fold. Analogues 8b and 8c, which contain the 3,9- and 2,10-dihydroxy substitution patterns, respectively, were both more active than ED-110

against Topo I. Compound 8b, with an IC₅o of 1.5 μM, was the most active regioisomer, approximately one order of magnitude more potent than ED-110, while 8c exhibited an IC o of 2.3 μM, nearly 6-fold more active than ED-110.

Table I

Inhibition of Human Topoisomerase I (Topo I) by Analogues 8a - 8d

EXAMPLE 2

The four indolocarbazoies 8a - 8d were evaluated for in vitro antitumor activity, against a panel of three human tumor cell lines. HT29 colon, OVCAR-3 ovarian, and DU145 prostate lines were used to determine if the analogues possess differential toxicity to different tumor types. ED-110 was reported to vary greatly in its toxicity to different tumor cell types.⁹

As shown in Table II, indolocarbazole analgoues of the invention also possessed differential toxicity against the three different tumor lines. ED-110 (8d) was fairly

inactive against the colon line (IC₅0 > 10 μM), active against the ovarian line (IC₅₀ 0.92

μM), and moderately active against the prostate line (IC₅o 2.3 μM). ED-110 was

previously reported to be inactive against DLD- 1 and WiDr colon cell lines, consistent with these results.⁹ Compound 8a followed the same trends, showing weak activity against the colon line, potent activity against the ovarian line, and moderate activity against the prostate line. Compounds 8b and 8c, which were the two most active analogues in the Topo I assay, were also the most active analogues in the in vitro antitumor assay. All compounds exhibited the weakest activity against the colon line.

Analogue 8b, which was the most active compound against Topo I, was also the most active compound in the in vitro antitumor study. 8b was active against the colon line

(IC₅o 0.84 μM), the ovarian line (IC₅o 0.19 μM), and very active against the prostate line

(ICso 0.067 μM).

Table II

IC₅₀ Values (μM) for Analogues 8a - 8d Against Human Tumor Cell Lines in vitro

(average of three trials)

In vivo Evaluation

Compound 8b was evaluated against the human tumor lines DU-145 (prostate) and

HT-29 (colon) for in vivo antitumor activity. The compound exhibited excellent activity against the prostate tumor, and moderate to good activity against the colon tumor. Example 3

The potency of 8b (also termed ALS-007) against DU-145 prostate tumor was evaluated. Human prostate tumor DU-145 was implanted subcutaneously in male athymic NCR-NU mice, and the experiments began on day 21, when the implanted tumors had achieved a median mass of 158-179 mg. The control tumors grew well, with a doubling time of approximately 6 days (200 to 400 mg), which was consistent with historical values. Twelve control mice were used, all of which received injections of 50% DMSO/50% water Q4H X 2 for 10 consecutive days.. The positive control was paclitaxel, formulated in 12.5% cremophor/12.5% ethanol/75% saline. 8b was formulated in 50% DMSO/50% water. Both agents were administered on the basis of exact body weight (0.1 mL/20 g body weight for 8b and 0.1 mL/10 g body weight for paclitaxel). The dosages for 8b were 50, 33.5, and 22.5 mg/kg/dose, whereas a single dosage of paclitaxel (15 mg/kg/dose) was used. Paclitaxel was administered intraveneously once daily for five consecutive days (QID x 5). 8b was administered ip twice daily, separated by four hours between dosings (Q4H X 2), for 10 consecutive days. The original intention was to administer 8b once daily for 10 days, at dosages of 100, 67, and 45 mg/kg/dose. However, the mice did not tolerate the first treatment well, so the daily dosage was fractionated into two treatments separated by four hours. All treatment groups consisted of six mice.

The highest dosage of 8b (50 mg/kg/dose twice daily) was toxic, eliciting 4 of 6 deaths. As summarized in Table III, both of the other dosage levels of 8b exhibited excellent activity against the tumor, evidenced by delays in tumor growth (T-C of >26.7 and >27.9 days, respectively, for dosages of 33.5 and 22.5 mg/kg/dose twice daily). As expected, paclitaxel was active in this model, effecting a tumor growth delay of >26.7 days. An examination of Figure 1 shows that the lowest dosage of 8b appeared to have comparable activity to that observed for paclitaxel, whereas the highest tolerated dosage appeared to be more efficacious than paclitaxel.

Table III

Summary of response of SC DU-145 Prostate tumor to treatment with 8b and Paclitaxel

I

GO Cύ

I

Example 4

The potency of 8b (also termed ALS-007) against HT29 colon tumor was evaluated. The control, vehicles, dosages, and dosing schedules were identical to that described for the DU-145 prostate tumor study. HT29 colon tumor was implanted subcutaneously (sc) in female athymic NCR-NU mice. Treatment began on day 12 post- implant, when the median tumor mass ranged from 167 to 180 mg. The control tumors grew well, with a doubling time of approximately 4.5 days, which is consistent with historical values.

As summarized in Table IV, the highest dosage of 8b (50 mg/kg/dose, twice daily) was toxic, eliciting 4 of 6 deaths. At a dosage of 33.5 mg/kg/dose twice daily, 8b exhibited good antitumor response, with a delay in tumor growth (T-C) of 16.3 days. However, 1 of 6 mice in this group died. The lowest dosage of 8b (22.5 mg/kg/dose, twice daily) elicited a minimal antitumor response (T-C value of 6.5 days). As expected, paclitaxel was active in this model, effecting a tumor growth delay of 13.6 days. An examination of Figure 2 shows that 8b at a dosage of 33.5 mg/kg/dose twice daily appeared to have activity comparable to paclitaxel, whereas the highest tolerated dosage of 8b was less efficacious than paclitaxel.

Table IV

Summary of response of SC HT29 colon tumor to treatment with 8b and Paclitaxel

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Claims

WHAT IS CLAIMED IS:

A synthetic indolocarbazole regioisomer of the formula I:

wherein

Ri and R₂ are independently H, halogen, hydroxyl, amino, C]-₆ alkyl, aryl-Cι-₆ alkyl, mono- or poly-fluorinated Cι-₆ alkyl, hydroxy-Cι-₆ alkyl, dihydroxy-Ci-₆ alkyl, di(hydroxy-Cι-₆ alkyl)-Cι-₆ alkyl, Cι-₆ alkoxy, amino-Cι-₈ alkyl, d-₆ alkylamino, di(Cι-₆ alkyl)amino, C]-₈ alkylamino-Cι-₈ alkyl, di(C₁-₆ alkyl)amino- Cι-8 alkyl, cyclohexyl, aryl or heterocycle, wherein aryl or heterocycle may each be unsubstituted or substituted with one or more of the following: Cι-₆ alkyl, d- alkoxy, hydroxy-C alkyl, hydroxyl, amino, Cι-₆ alkylamino, di(d-₆ alkyl) amino, amino-Cι-₈ alkyl, Cι-₈ alkylamino-Cι-₈ alkyl, di(Cι-₆ alkyl)amino-Cι-₈ alkyl, nitro, azido or halogen; or a pharmaceutically acceptable salt thereof.

The compound of claim 1 , wherein Ri is H.

3. The compound of claim 1, wherein R₂ is H.

4. The compound of claim 1, wherein Ri and R₂ are both H.

5. The compound of claim 1, wherein Ri is NHCHO and R₂ is H.

6. The compound of claim 1 , wherein Ri is NH₂ and R₂ is H.

The compound of claim 1, wherein Ri is CH₂CH₂OH and R₂ is H.

8. The compound of claim 1, wherein Ri is CH(CH₂OH) and R₂ is H.

9. A synthetic indolocarbazole regioisomer of the formula II:

wherein

Ri and R₂ are independently H, halogen, hydroxyl, amino, Cι-₆ alkyl, aryl-Ci-₆ alkyl, mono- or poly-fluorinated Cι-6 alkyl, hydroxy-Cι-₆ alkyl, dihydroxy-Cι-6 alkyl, di(hydroxy-d-₆ alkyl)-Cι-6 alkyl, Cι-₆ alkoxy, amino-d-₈ alkyl, Cι-6 alkylamino, di(Cι_-6 alkyl)amino, Cι-₈ alkylamino-Cι-₈ alkyl, di(d_-6 alkyl)amino- Cι-₈ alkyl, cyclohexyl, aryl or heterocycle, wherein aryl or heterocycle may each be unsubstituted or substituted with one or more of the following: C_1-6 alkyl, -_ό alkoxy, hydroxy-Cι-₄ alkyl, hydroxyl, amino, Cι-₆ alkylamino, di(Cι-₆ alkyl) amino, amino-Cι-₈ alkyl, Cι-₈ alkylamino-Cι-₈ alkyl, di(Cι-₆ alkyl)amino-Cι-8 alkyl, nitro, azido or halogen; or a pharmaceutically acceptable salt thereof.

10. The compound of claim 9, wherein R] is H.

11. The compound of claim 9, wherein R₂ is H.

12. The compound of claim 9, wherein Ri and R₂ are both H.

13. The compound of claim 9, wherein Ri is NHCHO and R₂ is H.

14. The compound of claim 9, wherein Ri is NH₂ and R₂ is H.

15. The compound of claim 9, wherein Ri is CH₂CH₂OH and R₂ is H.

16. The compound of claim 9, wherein Ri is CH(CH₂OH)₂ and R₂ is H.

17. A method of inhibiting topoisomerase I activity comprising administering to a mammal in need of inhibition of topoisomerase I activity an effective amount of at least one synthetic indolocarbazole regioisomer of the formula I:

wherein

R] and R₂ are independently H, halogen, hydroxyl, amino, Cι-₆ alkyl, aryl-Cι-₆ alkyl, mono- or poly-fluorinated Cι_-6 alkyl, hydroxy-Cι-6 alkyl, dihydroxy-Cι-₆ alkyl, di(hydroxy-d-6 alkyl)-Cι-6 alkyl, Ci-6 alkoxy, amino-Cι-₈ alkyl, Ci-6 alkylamino, di(Cι-₆ alkyl)amino, Cι-₈ alkylamino-Ci-s alkyl, di(Cι-₆ alkyl)amino- Cι-₈ alkyl, cyclohexyl, aryl or heterocycle, wherein aryl or heterocycle may each be unsubstituted or substituted with one or more of the following: Ci- alkyl, Cι-₆ alkoxy, hydroxy-Cι- alkyl, hydroxyl, amino, Ci-6 alkylamino, di(Cι-₆ alkyl) amino, amino-Cι-₈ alkyl, Cι-₈ alkylamino-Cι-₈ alkyl, di(Cι_-6 alky amino-d-s alkyl, nitro, azido or halogen; or a pharmaceutically acceptable salt thereof.

18. The method of claim 17, wherein R] is H.

19. The method of claim 17, wherein R₂ is H.

20. The method of claim 17, wherein Ri and R₂ are both H.

21. The method of claim 17, wherein R, is NHCHO and R₂ is H.

22. The method of claim 17, wherein Ri is NH₂ and R₂ is H.

23. The method of claim 17, wherein R, is CH₂CH₂OH and R₂ is H.

24. The method of claim 17, wherein R, is CH(CH₂OH)₂ and R₂ is H.

25. The method of claims 17, 18, 19, 20, 21, 22, 23, or 24 wherein said regioisomer is administered in combination with a pharmaceutically acceptable carrier.

26. A method of inhibiting topoisomerase I activity comprising administering to a mammal in need of inhibition of topoisomerase activity an effective amount of at least one synthetic indolocarbazole regioisomer of the formula II:

wherein

Ri and R₂ are independently H, halogen, hydroxyl, amino, Cι-₆ alkyl, aryl-d-₆ alkyl, mono- or poly-fluorinated Ci-6 alkyl, hydroxy-C]-₆ alkyl, d-hydroxy-d-₆ alkyl, di(hydroxy-Cι-₆ alkyl)-Cι-6 alkyl, Cι-₆ alkoxy, amino-d-₈ alkyl, Ci-₆ alkylamino, di(Cι-6 alkyl)amino, Cι-₈ alkylamino-Cι-₈ alkyl, di(C]-6 alkyl)amino- d-₈ alkyl, cyclohexyl, aryl or heterocycle, wherein aryl or heterocycle may each be unsubstituted or substituted with one or more of the following: Cι-₆ alkyl, Ci-₆ alkoxy, hydroxy-C alkyl, hydroxyl, amino, Cι.₆ alkylamino, di(d-₆ alkyl) amino, amino-Ci-8 alkyl, Cι-₈ alkylamino-Cι-₈ alkyl, di(Cι-₆ alkyl)amino-d-₈ alkyl, nitro, azido or halogen; or a pharmaceutically acceptable salt thereof.

27. The method of claim 26, wherein Ri is H.

28. The method of claim 26, wherein R₂ is H.

29. The method of claim 26, wherein R| and R are both H.

30. The method of claim 26, wherein R_\ is NHCHO and R₂ is H.

31. The method of claim 26, wherein R_\ is NH₂ and R₂ is H.

32. The method of claim 26, wherein Ri is CH₂CH₂OH and R₂ is H.

33. The method of claim 26, wherein R, is CH(CH₂OH)₂ and R₂ is H.

34. The method of claims 26, 27, 28, 29, 30, 31, 32, or 33 wherein said regioisomer is administered in combination with a pharmaceutically acceptable carrier.

35. A composition comprising the synthetic indolocarbazole regioisomer of claims 1, 2, 3, 4, 5, 6, 7, or 8 and a pharmaceutically acceptable carrier.

6. A composition comprising the synthetic indolocarbazole regioisomer of claims 9, 0, 11, 12, 13, 14, 15, or 16 and a pharmaceutically acceptable carrier.