WO2013159224A1

WO2013159224A1 - 2-carbamo(thio)yl-1,3- dioxopropyl derivatives in cancer therapy

Info

Publication number: WO2013159224A1
Application number: PCT/CA2013/050310
Authority: WO
Inventors: Gaik-Lean Chee; A. David BREWER; Brian B. HASINOFF
Original assignee: University Of Manitoba; Chemtura
Priority date: 2012-04-25
Filing date: 2013-04-22
Publication date: 2013-10-31

Abstract

Certain 2-carbamo(thio)yl-1,3-dioxopropyl derivatives, or the tautomers and pharmacologically acceptable addition salts thereof are found to be toxic to cancer cells, in part, by inhibiting DNA topoisomerase II enzyme making them candidates for pharmaceutical use as anticancer agents.

Description

2-Carbamo(thio)yl-1,3- dioxopropyl derivatives in cancer therapy

PRIOR APPLICATION INFORMATION

The instant invention claims the benefit of US Provisional Patent Application USSN 61/638,176, filed April 25, 2012.

FIELD OF THE INVENTION

The present invention relates to new 2-carbamo(thio)yl-1 ,3-dioxopropyl derivatives, or the tautomers and pharmacologically acceptable addition salts thereof. More particularly, the invention relates to 2-carbamo(thio)yl-1 ,3-dioxopropyl derivatives which have been found to be toxic to cancer cells, in part, by inhibiting

DNA topoisomerase II enzyme. Therefore, these derivatives may be pharmaceutically useful as anticancer agents.

BACKGROUND OF THE INVENTION DNA topoisomerase 11 is an essential nuclear enzyme required for the regulation of DNA topology. The enzyme can relax the torsional stress of DNA during replication and transcription and unknot entangled daughter strands during mitosis. Topoisomerase II catalyzes these processes by transiently breaking both strands of a DNA double helix through the formation of two covalent bonds between the enzyme and both DNA strands, passing another unbroken DNA double helix through the break, and then resealing the broken DNA strands (Fortune and Osheroff, Prog.

Nucleic Acid Res. Mol. Biol. 64: 2210253, 2000; Li and Liu, Annu. Rev. Pharmacol. Toxicol. 41 :53-77, 2001 ; Wang, Nat. Rev. Mol. Cell Biol. 3:430-440, 2002). Such activity is referred to as decatenation.

Drugs that target topoisomerase II act on different stages of the catalytic cycle.

A number of clinical and non-clinical anticancer agents are known to target this enzyme by inhibiting the ability of topoisomerase II in resealing the cleaved second DNA helix. These clinical agents are amsacrine, daunorubicin, doxorubicin, etoposide and mitoxanthrone. Specifically, these drugs stabilize the intermediate topoisomerase ll-cleaved DNA covalent complexes which, upon the action of DNA metabolic processes, give rise to permanent double strand breaks that are highly toxic to cells (Capranico, Cancer Chemo. Biol. Response Modifiers 18:125-143, 1999; Fortune and Osheroff, Prog. Nucleic Acid Res. Mol. Biol. 64: 2210253, 2000; Li and Liu, Annu. Rev. Pharmacol. Toxicol. 41 :53-77, 2001 ; Zhang et al., Cancer Cells 2:23-27, 1990). This is why these drugs are often referred to as topoisomerase II poisons and may explain why they are highly cytotoxic and remarkably effective clinically. The other class of topoisomerase II inhibitors is referred to as catalytic inhibitors, a class that includes dexrazoxane, aclarubicin, merbarone, and novobiocin. However, these cytotoxic compounds do not increase the levels of DNA breaks in cells at

pharmacologically relevant concentrations. {Andoh and Ishida, Biochim. Biophys. Acta 1400:155-171 , 1998; Hasinoff et al., Biochem. Pharmacol. 50:953-958, 1995; Larsen et al., Pharmacol. Ther. 99:167-181 , 2003; Drake et al., Cancer Res. 49:2578-2583, 1989).

The 2-carbamo(thio)yl dioxopropyl derivatives in the present invention have been found to be cytotoxic and targeting topoisomerase II. Therefore, they have the potential to be therapeutically useful for treating cancers. The 2-carbamo(thio)yl dioxopropyl derivatives of the present invention have not been disclosed as topoisomerase ll-targeting compounds, although a 2-carbamoyl dioxopropyl derivative, merbarone, has been disclosed as a topoisomerase II catalytic inhibitor (Drake et al., Cancer Res. 49:2578-2583 1989).

Merbarone

Certain 2-carbamo(thio)yl-1 ,3-dioxopropyl derivatives in the present invention have been disclosed as antifungal, bactericidal, insecticidal, acaricidal, keratenous material protective, anthelmintic, immunosuppressive, hematopoietic, and voltage- gated potassium channel inactivation inhibiting agents (US Pat. No. 3,961 ,061 ;

German Offen. No. 24 05 732; US Pat. No. 4,283,444; US Pat. Pub. No. 4,628,060; Int. Pat. Pub. No. WO93/14062; Eur. Pat. Pub. No. 0841063; Japanese Pat. No. 51019126; US Pat. Pub. No. 2006/0014826; Bertolini et al., J. Med. Chem. 40:2011- 2016, 1997), or in synthesis studies (Moskvin et ai., Zhurnal Obshchei Khimii

65:2045-2048, 1995; Song et al., Org. Lett. 9:4307-4310, 2007; Elwan et al.,

Heteroatom Chem. 13:585-591 , 2002; Japanese Pat. Pub. No. 2005/225828;

Japanese Pat. Pub. No.1 1217361 ). However, the compounds of the present invention have not been disclosed as anticancer agents. Structurally related compounds that have been disclosed as anticancer or potential anticancer agents but fall outside the scopes of the present invention are 5-(thio)carboxamides of barbituric acid and thiobarbituric acid of general formula:

(I)

(Japanese Pat. Pub. No. 1 ,445/64; US Pat No. 4,634,707; US Pat. No. 4,920,126; Ukita et al., Cancer Chemotherapy Reports 13: 211-220, 1961 ), 2-carboxamides of cyclohexanedione of formula:

(II)

(Ukita et al., Cancer Chemotherapy Reports 13: 21 1-220, 1961 ), 2-carboxamides of 1-3-indanedione of formula:

(Jung et al., Arch. Pharmacal Res. 27:997-1000, 2004; ), and 3-carboxamide-2,4- pyrandione derivative of formula:

(IV)

(Ukita et al., Cancer Chemotherapy Reports 13: 21 1 -220, 1961). SUMMARY OF THE INVENTION

The present invention describes the methods of inhibiting cell growth, such as cancerous cell growth, by 2-carbamo(thio)yl-1 ,3-dioxopropyl derivatives. The cell growth inhibition by these derivatives relates to the novel discovery that 2- carbamo(thio)y[-1 ,3-dioxopropyl derivatives are topoisomerase II targeting

compounds that act as catalytic inhibitors and poisons of the enzyme. Thus, compounds of the present invention may be utilized as anticancer agents in cancer therapies, wherein the cancer is cancer of the head, brain, eye, mouth, gum, tongue, neck, lung, breast, liver, pancreas, kidney, gastrointestinal tract, small intestine, colon, bladder, prostate, testicle, ovary, cervix, bone, skin, soft tissues, connective tissues, lymph system, hematopoietic system or blood.

According to an aspect of the invention, there is provided a method of inhibiting the activity of topoisomerase II in a cell, thereof comprising administering to the cell an effective amount of a compound of formula (V), or the tautomers and salts thereof:

(V)

wherein

X is oxygen or sulfur;

R is C-i-C-6 linear or branched alkyl, C₅-C6 cycloalkyl, benzyl, phenethyl, C1-C5 alkoxy-COCh Ch , phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C1-C-6 linear or branched alkyl, haloalkyl, C1-C4 alkoxy, Ci-C thioalkoxy, phenoxy, C C₅ alkylcarbonyl, and C Cs alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, Ci-C₆ linear or branched alkyl and CrC₅ alkoxycarbonyl;

W is

a)

wherein R¹ and R² are each independently hydrogen, d-C₆ li near or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when X is oxygen, R cannot be phenyl optionally substituted with 0 to 5 substituents; with the proviso that when X is sulfur and R is phenyl, R¹ and R² cannot be hydrogen;

wherein R³ is d-C₄ linear alkyl, C-i-C₄ alkoxy, or C C₄ thioalkoxy;

(c)

wherein Y is absent or present and when present is carbony!; with the proviso that when Y is absent, X must be sulfur; R⁴ and R⁵ are each independently hydrogen, C1-C-6 linear alkyl, Ci-C₆ alkoxy; halogen, or trihalomethyl; d)

wherein R⁶ and R⁷ are each independently hydrogen, Ci-C₆ linear or branched alkyl; with the proviso that when X is oxygen and R⁷ is hydrogen, R cannot be phenyl; e)

wherein R⁸ is hydrogen or Ci-C₆ linear alkyl; or

<f)

R⁹

CH₂-(Q)-CH—

R⁹

wherein n is 0 or 1 ; Q is carbon or sulfur; R⁹ is present when Q is carbon, or absent when Q is sulfur, and when present is hydrogen or Ci-C₆ linear alkyl; with the proviso that when X is oxygen, R cannot be phenyl;

(g) -NH-NR - wherein R¹⁰ is Ci-C₆ linear alkyl or phenyl.

According to another aspect of the invention, there is provided a method of inhibiting the growth of a ceil, thereof comprising administering to the cell an effective amount of a compound of formula (V), or the tautomers and salts thereof:

(V)

wherein

X is oxygen or sulfur;

R is CrC₆ linear or branched alkyl, C₅-C₆ cycloalkyl, benzyl, phenethyl, C C₅ alkoxy-COCH₂CH₂, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally

substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C-i-Ce linear or branched alkyl, haloalkyl, C C alkoxy, Ci-C₄ thioalkoxy, phenoxy, Ci- C₅ alkylcarbonyl, and C1-C5 aikoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C-i-C₆ linear or branched alkyl and C1-C5 aikoxycarbonyl;

W is

wherein R¹ and R² are each independently hydrogen, C C_e linear or branched alkyi, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when X is oxygen, R cannot be phenyl optionally substituted with 0 to 5 substituents; with the proviso that when X is sulfur and R is phenyl, R¹ and R² cannot be hydrogen;

wherein R³ is Ci-C₄ linear alkyl, Ci-C₄ alkoxy, or C1-C4 thioalkoxy;

wherein Y is absent or present and when present is carbonyl; with the proviso that when Y is absent, X must be sulfur; R⁴ and R⁵ are each independently hydrogen, Ci-C₆ linear alkyl, C-i-C-6 alkoxy; halogen, or trihalomethyl; d)

wherein R⁶ and R⁷ are each independently hydrogen, C C₆ linear or branched alkyl; with the proviso that when X is oxygen and R⁷ is hydrogen, R cannot be phenyl; e)

wherein R is hydrogen or CrCg linear alkyl; or

(f)

R

CH₂-(Q)-CH - R⁹

wherein n is 0 or 1 ; Q is carbon or sulfur; R⁹ is present when Q is carbon, or absent when Q is sulfur, and when present is hydrogen or C C₆ linear alkyl; with the proviso that when X is oxygen, R cannot be phenyl;

(g) -NH-NR^1u- wherein R¹⁰ is Ci-C₆ linear alkyl or phenyl.

According to another aspect of the invention, there is provided a method of treating a patient with cancer, thereof comprising administering to the cell an effective amount of a compound of formula (V), or the tautomers and salts thereof:

wherein

X is oxygen or sulfur;

R is Ci-C-6 linear or branched alkyl, C5-C-6 cycloalkyi, benzyl, phenethyl, C1-C5 alkoxy-COCH2CH2, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C C₆ linear or branched alkyl, haloalkyl, C C₄ alkoxy, C C₄ thioalkoxy, phenoxy, C C₅ alkyicarbonyl, and C1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C C₆ linear or branched alkyl and C1-C5 alkoxycarbonyl; W is

a)

wherein R¹ and R² are each independently hydrogen, Ci-C₆ linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when X is oxygen, R cannot be phenyl optionally substituted with 0 to 5 substituents; with the proviso that when X is sulfur and R is phenyl, R¹ and R² cannot be hydrogen;

wherein R³ is C C₄ linear alkyl, C1-C4 alkoxy, or C1-C4 thioalkoxy; c)

wherein Y is absent or present and when present is carbonyl; with the proviso that when Y is absent, X must be sulfur; R⁴ and R⁵ are each independently hydrogen, C1-C-6 linear alkyl, C C₆ alkoxy; halogen, or trihalomethyl; d)

wherein R⁶ and R⁷ are each independently hydrogen, C-i-Ce linear or branched alkyl; with the proviso that when X is oxygen and R⁷ is hydrogen, R cannot be phenyl; e)

wherein R is hydrogen or Ci-C₆ linear alkyl;

wherein n is 0 or 1 ; Q is carbon or sulfur; R⁹ is present when Q is carbon, or absent when Q is sulfur, and when present is hydrogen or C C₆ linear alkyi; with the proviso that when X is oxygen, R cannot be phenyl;

(g) -NH-NR¹⁰- wherein R ⁰ is C C₆ linear alkyl or phenyl.

According to yet another aspect of the invention, there is provided a compound of formula (V), or the tautomers and salts thereof:

(V)

wherein

X is oxygen or sulfur;

R is Ci-Ce linear or branched alkyl, C₅-C₆ cycloalkyl, benzyl, phenethy], C1-C5 alkoxy-COCH₂CH₂, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C-i-C-6 linear or branched alkyl, haioalkyl, C C alkoxy, C C₄ thioalkoxy, phenoxy, Cr C-5 alkylcarbonyl, and C1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C C-6 linear or branched alkyl and C-1-C5 alkoxycarbonyl;

W is

wherein R is hydrogen or d-Ce linear alkyl.

The present invention also relates to a method of inhibiting the growth of a cell, wherein the cell is a cancer cell, thereof comprising administering to the cell an effective amount of a compound of formula (V), or the tautomers and salts thereof, wherein X, R, and W are as defined above.

The present invention also relates to a method of treating a patient with cancer, thereof comprising administering to the cell an effective amount of a compound of formula (V), or the tautomers and salts thereof, wherein X, R, and W are as defined above.

The present invention also relates to a compound of formula (V), or the tautomers and salts thereof, wherein X and R are as defined above; W is

wherein R is as defined above.

As will be readily apparent to one of skill in the art, in other aspects of the invention, there are provided the use of any of the above-described compounds for treating cancer.

According to another aspect of the invention, there is provided the use of the above-described compounds in the manufacture of a medicament for the treatment of cancer.

Addition salts of a compound of formula (V) may be formed with a variety of pharmacologically acceptable organic and inorganic bases. Addition salt may thus be formed by admixture of a compound of formula (V) with one equivalent of an organic base such as triethylamine or an inorganic base such as sodium hydroxide.

The 2-carbamo(thio)yl-1 ,3-dioxopropyl derivatives of the present invention can exist in the form illustrated in formula (V) or in any tautomeric forms. It is understood that the compounds of the invention illustrated herein the form shown in formula (V) embrace the tautomers thereof, or tautomeric mixtures.

The novel compounds of this present invention may be used alone or in combination with other chemotherapeutic agents useful in cancer therapies. The compounds may be formulated in pharmaceutically appropriate form in suitable medium or carrier with appropriate surfactants and dispersing agents, and accordingly may be administered parentarally, intraperitoneally, intravenously, intramuscularly, subcutaneously, orally, or topically.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. Topoisomerase II catalytic decatenation inhibition assay comparing Example 2 with merbarone, a topoisomerase II catalytic inhibitor. Fluorescent image of ethidium bromide-stained agarose gel shows that Example 2 (100 and 50 μΜ, middle two lanes) and merbarone (100 μΜ, second rightmost lane) completely inhibited the enzyme decatenation activity, whereas merbarone at 50 μΜ only inhibited the activity by 65% (rightmost lane). In the absence of the enzyme and compound, kDNA stayed in the origin (leftmost lane). In the absence of compound, the enzyme decatenated kDNA into nicked and open cirlcular DNA (second leftmost lane).

FIGURE 2. Topoisomerase ΙΙα-mediated supercoiled pBR322 DNA cleavage assay comparing Example 2 with etoposide, a topoisomerase II poison, and merbarone, a catalytic inhibitor. Fluorescent image of ethidium bromide-stained agarose gel shows the ability of etoposide (middle lane) and Example 2 (rightmost lane) to induce the formation of linear DNA at 100 μΜ in the presence of the enzyme. On the other hand, merbarone (second rightmost lane) did not show such an ability under the same condition. In the absence of drug, the enzyme converted supercoiled pBR322 DNA into catenated DNA (second leftmost lane), whereas in the absence of the enzyme and drug, supercoiled DNA remained unaffected (leftmost lane).

FIGURE 3. The growth inhibitory effects of Example 2 (O) and merbarone (·) on human leukemia K562 cells as measured by an TS assay with three replicates for each compound. Example 2 and merborone inhibited the growth inhibition of K562 cells with IC₅₀ values of 4 ± 1 and 42 + 3 μ , respectively. The solid lines are nonlinear least square fits to a four-parameter logistic equation. Where the error bars are not shown, they are smaller than the symbol.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

The preparation of compounds of formula (V) may be achieved using the following synthetic method:

<^v>

1 ,3-Dione derivative can be reacted with an isocyanate or isothiocyanate derivative in the presence of a suitable base such as 1 ,8-diazabicyclo[5.4.0]undec-7- ene, triethylamine, and pyridine, in the presence or absence of an organic solvent or dispersing medium such as acetonitrile, tetrahydrofuran, dimethyl formamide, and dimethyl sulfoxide, as previously described for related analogs (US Pat No.

4,634,707; US Pat. No. 4,920,126; US Pat. Pub. No. 4,628,060). The reaction may be carried out at 0 to 200 °C, usually at 24 to 100 °C. The formation of the compounds of formula (V) usually completes within 0.5 to 24 hour period. The reaction may then be acidified with a suitable acid such as hydrochloric acid to afford the compounds of formula (V) which may be further purified by recrystallization or chromatography. The detailed description of the general preparation of specific examples of the compounds of formula (V) is provided in the Examples section, including Examples 1 to 20 below, structures of which are summarized in Table .

The compounds of formula (V) are highly cytotoxic and exhibit the ability to inhibit topoisomerase II activity, as illustrated by Example 1 to 20 (Table 1 ). The activities of these compounds are summarized in Table 2. Example 2 is considered to be the most active compound in Table 2, followed by Examples 1 and 10. Examples 2, 1 and 10 have the ability to fully inhibit the catalytic activity of topisomerase II at both 100 and 50 μΜ concentrations tested, as well as act as topoisomerase II poisons by inducing the formation of linear DNA at the tested concentration of 100 μΜ. The enzyme poisoning activity exhibited by these examples is especially desirable because presently used topoisomerase II targeting clinical drugs also exhibit such activity. Examples 2, 1 and 10 also potently inhibit the growth of human leukemia 562 cells at low micromolar concentrations. The detailed results of these activities are shown for Example 2 in Figure 1 to 3 for illustration purpose only. The testing procedures of these compounds are described in Example 21 to 23.

(V)

wherein

X is oxygen or sulfur;

R is C-i-C-6 linear or branched alkyl, C₅-C₆ cycloalkyl, benzyl, phenethyl, C-1-C5 alkoxy-COCH₂CH2, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally

substituted with 0 to 5 substituents independently selected from hydrogen, halogen, Ci-Ce linear or branched alkyl, haloalkyl, C C₄ alkoxy, C C₄ thioalkoxy, phenoxy, C C₅ alkylcarbonyl, and C1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C-i-Ce linear or branched alkyl and C-rC-5 alkoxycarbonyl;

W is

a)

wherein R and R² are each independently hydrogen, Ci-C₆ linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when X is oxygen, R cannot be phenyl optionally substituted with 0 to 5 substituents; with the proviso that when X is sulfur and R is phenyl, R¹ and R² cannot be hydrogen;

wherein R³ is C-|-C₄ linear alkyl, C C₄ alkoxy, or C1-C4 thioalkoxy;

(c)

wherein Y is absent or present and when present is carbonyl; with the proviso that when Y is absent, X must be sulfur; R⁴ and R⁵ are each independently hydrogen, near alkyl, Ci-C₆ alkoxy; halogen, or trihalomethyi; d)

wherein R⁶ and R⁷ are each independently hydrogen, C C₆ linear or branched aikyl; with the proviso that when X is oxygen and R⁷ is hydrogen, R cannot be phenyl; e)

wherein R is hydrogen or C C₆ linear alkyl; or

wherein n is 0 or ; Q is carbon or sulfur; R is present when Q is carbon, or absent when Q is sulfur, and when present is hydrogen or C C-e linear alkyl; with the proviso that when X is oxygen, R cannot be phenyl;

(g) -NH-NR¹⁰- wherein R¹⁰ is C-i-Ce linear alkyl or phenyl.

As will be appreciated by one of skill in the art, an "effective amount" of the compound will be an amount that is sufficient to inhibit topoisomerase II to the degree desired. It is of note that the exact amount may depend on a number of factors, for example, the age, weight and general condition of the patient and/or the type of cell(s) in which inhibition is to be effected. It is however noted that determination of such an effective amount is well within the routine skill of one knowledgeable of the art.

As will be appreciated by one of skill in the art, the celi(s) may be in vitro or may be in vivo. Specifically, as discussed herein, the compounds may be

administered to an animal, for example, a human or patient.

According to another aspect of the invention, there is provided a method of inhibiting the growth of a cell, thereof comprising administering to the cell an effective amount of a compound of formula (V), or the tautomers and salts thereof:

(V)

wherein

X is oxygen or sulfur;

R is Ci-C₆ linear or branched alkyl, C₅-C₆ cycloalkyl, benzyl, phenethyl, CrC₅ a[koxy-COCH₂CH2, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C-i-Ce linear or branched alkyl, haloalkyl, C1-C4 alkoxy, C-1-C4 thioalkoxy, phenoxy, Cr C₅ alkylcarbonyl, and C1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C1-C6 linear or branched alkyl and C1-C5 alkoxycarbonyl;

W is

a)

wherein R¹ and R² are each independently hydrogen, C-i-C₆ linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when X is oxygen, R cannot be phenyl optionally substituted with 0 to 5 substituents; with the proviso that when X is sulfur and R is phenyl, R and R² cannot be hydrogen; b)

wherein R is C C₄ linear alkyl, Ci-C₄ alkoxy, or C C₄ thioalkoxy;

(c)

wherein Y is absent or present and when present is carbonyl; with the proviso that when Y is absent, X must be sulfur; R⁴ and R⁵ are each independently hydrogen, C-i-Ce linear alkyl, C-i-C₆ alkoxy; halogen, or trihalomethyl; d)

wherein R⁶ and R⁷ are each independently hydrogen, C C₆ linear or branched alkyl; with the proviso that when X is oxygen and R⁷ is hydrogen, R cannot be phenyl; (e)

wherein R is hydrogen or C1-C-6 linear alkyl; or

(f)

R⁹

-CH -(Q)-CH - R⁹

wherein n is 0 or 1 ; Q is carbon or sulfur; R⁹ is present when Q is carbon, or absent when Q is sulfur, and when present is hydrogen or C1-C-6 linear alkyl; with the proviso that when X is oxygen, R cannot be phenyl;

(g) -NH-NR¹⁰- wherein R ⁰ is Ci-Ce linear alkyl or phenyl.

wherein

X is oxygen or sulfur;

R is C1-C6 linear or branched alkyl, C₅-Ce cycloalkyl, benzyl, phenethyl, C C₅ alkoxy-COCH₂CH₂₎ phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C-i-C-6 linear or branched alkyl, haloalkyl, C C₄ alkoxy, C C₄ thioalkoxy, phenoxy, C-r C₅ alkylcarbonyl, and C1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, CrCe linear or branched alkyl and C1-C5 alkoxycarbonyl;

W is

(a)

I I

G

wherein R¹ and R² are each independently hydrogen, C C₆ linear or branched alkyl, ally], or phenyl; G is oxygen or sulfur; with the proviso that when X is oxygen, R cannot be phenyl optionally substituted with 0 to 5 substituents; with the proviso that when X is sulfur and R is phenyl, R¹ and R² cannot be hydrogen;

wherein R is C-1-C4 linear alkyl, C1-C4 alkoxy, or C C₄ thioalkoxy; c)

wherein Y is absent or present and when present is carbonyl; with the proviso that when Y is absent, X must be sulfur; R⁴ and R⁵ are each independently hydrogen, CrCe linear alkyl, C1-C-6 alkoxy; halogen, or trihalomethyl;

wherein R is hydrogen or Ci-C₆ linear aikyl; or

(f)

R⁹

— CH₂-(Q)-CH—

R

wherein n is 0 or ; Q is carbon or sulfur; R⁹ is present when Q is carbon, or absent when Q is sulfur, and when present is hydrogen or C-t-Ce linear alkyl; with the proviso that when X is oxygen, R cannot be phenyl; (g) -NH-NR¹⁰- wherein R¹⁰ is C C₆ linear alkyl or phenyl.

The individual to be treated may be an individual in need of such treatment, such as an individual who has, has been diagnosed with or is suspected of having cancer. As will be appreciated by one of skill in the art, a wide variety of cancers may be treated in this manner, for example but by no means limited to cancer of the head, brain, eye, mouth, gum, tongue, neck, lung, breast, liver, pancreas, kidney, gastrointestinal tract, small intestine, colon, bladder, prostate, testicle, ovary, cervix, bone, skin, soft tissues, connective tissues, lymph system, hematopoietic system and blood.

According to one embodiment of the invention, there is provided a method of: inhibiting the activity of topoisomerase II in a cell; inhibiting the growth of a cell; or treating a patient with cancer comprising administering to the cell or patient an effective amount of a compound of formula (V) or the tautomers and salts thereof,

(V)

wherein

X is sulfur;

R is Ci-Ce linear or branched alkyl, C₅-Ce cycloalkyl, benzyl, phenethyl, C-1 -C5 alkoxy-COCH₂CH2, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C1-C-6 linear or branched alkyl, haloalkyi, Ci-C₄ alkoxy, C C thioalkoxy, phenoxy, C C5 alkylcarbonyl, and C1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C-i-Ce linear or branched alkyl and C1-C5 alkoxycarbonyl;

W is

wherein R¹ and R² are each independently hydrogen, CrC linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when R is phenyl, R and R² cannot both be hydrogen.

According to one embodiment of the invention, there is provided a method of: inhibiting the activity of topoisomerase II in a cell; inhibiting the growth of a cell; or treating a patient with cancer comprising administering to the ceil or patient an effective amount of a compound as set forth in Examples 2, 1 or 10

(V)

wherein

X is oxygen or sulfur;

R is C-i-Ce linear or branched alkyl, C₅-Ce cycloalkyl, benzyl, phenethyl, C1-C5 alkoxy-COCH2CH2, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally

substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C1-C-6 linear or branched alkyl, haloalkyi, C-i-C₄ alkoxy, C1-C4 thioalkoxy, phenoxy, C-i- C₅ alkylcarbonyl, and C1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C-1-C6 linear or branched alkyl and C1-C5 alkoxycarbonyl;

W is

wherein R is hydrogen or C Ce linear alkyl. The present invention also relates to a method of inhibiting the growth of a cell, wherein the cell is a cancer cell, thereof comprising administering to the cell an effective amount of a compound of formula (V), or the tautomers and salts thereof, wherein X, R, and W are as defined above.

wherein R is as defined above.

The invention will now be further illustrated by way of examples; however, the invention is not necessarily limited to the examples.

EXAMPLES

Examples of compound synthesis General procedure used for the preparation of Examples 1-20

A mixture of 1 ,3-dione derivative (0.2 - 0.7 mmol) and 1 ,8- diazabicyclo[5.4.0]undec-7-ene (1 .1 eqv) in acetonitrile (2 ml) was stirred at 50 °C for 10 min and cooled to room temperature before adding an isocyanate or

isothiocyanate derivative (1 .0 eqv). The resulting mixture was allowed to react at room temperature or 80 °C overnight. With stirring at room temperature, 1 M HCI (aq) was added dropwise to the reaction mixture until pH was lower than 1. The mixture was then worked up using one of the following procedures:

Procedure A: Precipitate formed was filtered, washed with water, dried under vacuum, and may be further purified by recrystallization or column chromatography.

Procedure B: If titration to acidic pH did not give rise to precipitation, the mixture was extracted with ethyl acetate and the organic phase was then washed with water, dried (Na₂S0₄), and concentrated to a residue which was further

chromatographed on silica gel.

Example 1

Preparation of 1 .S-dimethyl^^^-trioxo-N-phenyl-l ,3-diazinane-5-carbothioamide The reaction of 1 ,3-dimethyl-2-thiobarbituric acid (0.1 1 g, 0.72 mmol) with phenyl isothiocyanate (0.086 ml, 0.72 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a yellow solid (0.18 g, 81 % yield) after

recrystallization (ethyl acetate/hexane). ¹H-N R (DMSO-d₆) δ 13.95 (1 H, s), 7.46 (4H, m), 7.36 (1 H, m), 3.29 (6H, s). Example 2

Preparation of 1 ,3-dimethyl-N-phenyl-4,6-dioxo-2-sulfanylidene-1 ,3-diazinane-5- carbothioamide

The reaction of ,3-dimethyl-2-thiobarbituric acid (0.1 1 g, 0.72 mmol) with phenyl isothiocyanate (0.086 g, 0.72 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a yellow solid (0.16 g, 72% yield) after

recrystallization (dichloromethane/hexane). H-NMR (DMSO-d₆) δ 3.74 (1 H, s), 7.51 (4H, m), 7.35 (1 H, m), 3.72 (6H, s).

Example 3

Preparation of 1 ,3-dimethyl-4,6-dioxo-N-(2-phenylethyl)-2-sulfany!idene-1 ,3- diazinane-5-carbothioamide

The reaction of 1 ,3-dimethyl-2-thiobarbituric acid (0.12 g, 0.72 mmol) with beta- phenylethyl isothiocyanate (0.11 ml, 0.73 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a beige solid (0.17 g, 70% yield). ¹H-N R (D SO-d₆) 5 12.25 (1 H, s), 7.31 (4H, m), 7.24 (1 H, m), 3.86 (2H, q), 2.99 (2H, t). Example 4

Preparation of ethyl 3-[(1 ,3-dimethyl-4,6-dioxo-2-sulfanylidene-1 ,3-diazinan-5- yl)formamido]propanoate

The reaction of 1 ,3-dimethyl-2-thiobarbituric acid (0.11 g, 0.64 mmol) with ethyl

3-isocyanatoproprionate (0.09 ml, 0.69 mmol) was carried out at room temperature and worked up according to Procedure A to afford a beige solid (0.18 g, 90% yield). H-N R (DMSO-de) δ 10.14 (1 H, s), 4.09 (2H, q), 3.64 (2H, q), 3.61 (6H, s), 2.67 (2H, t), 1.20 (3H, t).

Example 5

N-cydohexyl-1 ,3-dimethyl-4,6-dioxo-2-sulfanylidene-1 ,3-diazinane-5-carbothioamide

The reaction of 1 ,3-dimethyl-2-thiobarbituric acid (0.10 g, 0.59 mmol) with cyclohexyl isothiocyanate (0.09 ml, 0.69 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a beige solid (0.13 g, 70% yield). ¹H-NMR (DMSO-de) δ 12.35 (1 H, d), 4.20 (1 H, m), 3.70 (6H, s), 1.94 (2H, m), 1.33-1.65 (8H, m).

Example 6

Preparation of 1 ,3-dimethyl-4,6-dioxo-N-pentyl-2-sulfanylidene-1 ,3-diazinane-5- carbothioamide

The reaction of ,3-dimethyl-2-thiobarbituric acid (0.08 g, 0.47 mmol) with pentyl isothiocyanate (0.06 ml, 0.47 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a colorless solid (0.10 g, 70% yield). ¹H-NMR (DMSO-de) δ 12.24 (1 H, br s), 3.70 (6H, s), 3.60 (2H, q), 1.67 (2H, p), 1.34 (4H, m), 0.89 (3H, t).

Example 7

N-benzyl-1 ,3-dimethyl-4,6-dioxo-2-sulfanylidene-1 ,3-diazinane-5-carbothioamide

The reaction of 1 ,3-dimethyl-2-thiobarbituric acid (0.12 g, 0.70 mmol) with benzyl isothiocyanate (0.06 ml, 0.47 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a beige solid (0.07 g, 30% yield) after

recrystaliization (dichloromethane/hexane). ¹H-N R (D SO-d₆) δ 12.51 (1 H, s), 7.38 (4H, m), 7.33 (1 H, m), 4.86 (2H, d), 3.61 (6H, s).

Example 8

Preparation of 2-(methylsulfanyl)-4,6-dioxo-N-phenyl-1 ,4,5,6-tetrahydropyrimidine-5- carbothioamide

The reaction of 4,6-Dihydroxy-2-methythiopyrimidine (0.139 g, 0.88 mmol) with phenyl isothiocyanate (0.11 ml, 0.88 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a yellow solid (0.20 g, 78% yield). ¹H-NMR

(DMSO-d₆) δ 14.10 (1 H, s), 7.49 (4H, m), 7.34 (1 H, m), 2.56 (3H, s).

Example 9

Preparation of 4,6-dioxo-N,1 ,3-triphenyl-2-sulfanylidene-1 ,3-diazinane-5- carbothioamide

The reaction of 1 ,3-diphenyl-2-thiobarbituric acid (0.088 g, 0.30 mmol) with phenyl isothiocyanate (0.04 ml, 0.30 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a yellow solid (0.10 g, 78% yield). ¹H-N R

(DMSO-de) δ 13.55 (1 H, s), 7.42 (15 H, m).

Example 10

Preparation of 4,6-dioxo-N-phenyl-1 -{prop-2-en-1 -yl)-2-sulfanylidene-1 ,3-diazinane-5- carbothioamide

The reaction of 1 -allyl-2-thiobarbituric acid (0.10 g, 0.54 mmol) with phenyl isothiocyanate (0.07 ml, 0.55 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a yellow solid (0.09 g, 55% yield) after

recrystaliization (dichloromethane/hexane). ¹H-N R (DMSO-d₆) δ 13.59 (1 H, s), 7.48 (4H, m), 7.34 (1 H, m), 5.87 (1 H, ddt), 5.20 (1 H, dd), 5.16 (1 H, dd), 4.96 (2H, d). Example 11

Preparation of 1-methyl-4,6-dioxo-N-phenyl-2-sulfanylidene-1 ,3-diazinane-5- carbothioamide

The reaction of 1-methyl-2-thiobarbituric acid (0.10 g, 0.63 mmol) with phenyl isothiocyanate (0.08 ml, 0.63 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a yellow solid (0.12 g, 65% yield). ¹H-NMR

(DMSO-de) δ 13.67 (1 H, s), 7.46 (4H, m), (7.36 (1 H, m), 3.60 (3H, s).

Example 12

Preparation of 2,6-dioxo-N-phenylcyclohexane-1-carbothioamide

The reaction of cycIohexane-1 ,3-dione (0.25 g, 2.2 mmol) with phenyl isothiocyanate (0.297 g, 2.2 mmol) was carried out at room temperature and worked up according to Procedure B to afford a brown solid (0.35 g, 65% yield) after chromatography on silica gel (33% ethyl acetate/hexane). ¹H-N R (DMSO-d₆) δ 13.80 ( H, s), 7.48 (4H, m), 7.34 ( H, m), 2.68 (4H, m), 1 .93 (2H, p).

Example 13

Preparation of 2,5-dioxo-N-phenylcyclopentane-1 -carbothioamide

The reaction of cyclopentane-1 ,3-dione (0.20 g, 2.0 mmol) with phenyl isothiocyanate (0.27 g, 2.0 mmol) was carried out at room temperature and worked up according to Procedure A to afford a brown solid (0.23 g, 50% yield). ¹H-NMR

(DMSO-de) δ 12.49 (1 H, br s), 7.67 (2H, d), 7.48 (2H, t), 7.34 (1 H, t), 2.70 (4H, br s).

Example 14

Preparation of 1 ,3-dioxo-N-phenyl-2,3-dihydro-1 H-indene-2-carbothioamide

The reaction of 1 ,3-indanedione (0.14 g, 0.94 mmol) with phenyl isothiocyanate (0.12 g, 0.99 mmol) was carried out at room temperature and worked up according to Procedure A to afford a beige solid (0.21 g, 82% yield). H-NMR (DMSO-d₆) δ 1 1.99 (1 H, br s), 7.46 (9H, m). Example 15

Preparation of 3,5-dioxo-N,1 -diphenylpyrazolidine-4-carbothioamide

The reaction of 1-phenylpyrazolidine-3,5-dione (0.06 g, 0.34 mmol) with phenyl isothiocyanate (0.042 ml, 0.035 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a beige solid (0.053 g, 50% yield) after

recrystallization (dichloromethane/hexane). ¹H-NMR (D SO-d₆) δ 11.90 (1 H, s), 7.71 (4H, d), 7.47 (2H, t), 7.44 (2H, t), 7.27 (1 H, t), 7.24 (1 H, t).

Example 16

Preparation of 6,7-dimethoxy-1 ,3,4-trioxo-N-phenyl-1 ,2,3,4-tetrahydronaphthalene-2- carbothioamide

The reaction of 2-hydroxy-6,7-dimethoxy-p-naphthoquinone (0.07 g, 0.30 mmol) with phenyl isothiocyanate (0.036 ml, 0.30 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a beige solid (0.026 g, 22% yield) after recrystallization (dichloromethane/hexane). H-NMR (DMSO-d₆) δ 13.28 (1 H, s), 7.63 (1 H, s), 7.45 (1 H, s), 7.03 (5H, m), 3.96 (3H, s), 3.94 (3H, s).

Example 17

Preparation of 3,5-dioxo-N-phenylthiane-4-carboxamide

The reaction of thiane-3,5-dione (0.22 g, 1.7 mmol) with phenyl isocyanate

(0.20 ml, 1.8 mmol) was carried out at room temperature and worked up according to Procedure A to afford a yellow solid (0.32 g, 76% yield). ¹H-NMR (DMSO-d₆) δ 11.76 (1 H, s), 7.55 (2H, d), 7.41 (2H, t), 7.23 (1 H, t), 3.68 (4H, s). Example 18

Preparation of 3,5-dioxo-N-phenylthiane-4-carbothioamide

The reaction of thiane-3,5-dione (0.26 g, 2.0 mmol) with phenyl isothiocyanate (0.23 ml, 1.8 mmol was carried out at room temperature and worked up according to Procedure A to afford a beige solid (0.42 g, 79% yield). H-NMR (D SO-d₆) δ 14.20 (1 H, br s), 12.39 (1 H, s), 7.70 (2H, d), 7.42 (2H, t), 7.27 (1 H, t), 3.60 (4H, s). Example 19

6-methyl-2,4-dioxo-N-phenyl-3,4-dihydro-2H-pyran-3-carbothioamide

The reaction of 6-methyl-3,4-dihydro-2H-pyran-2,4-dione (0.065 g, 0.52 mmol) with phenyl isothiocyanate (0.068 ml, 0.53 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a beige solid (0.047 g, 35% yield) after chromatography on silica gel (50% ethyl acetate/hexane). ¹H-NMR (DMSO-d₆) δ 15.69 (1 H, br s), 12.89 (1 H, s), 7.57 (2H, d), 7.47 (2H, t), 7.34 (1 H, t), 6.41 (1 H, s), 2.30 (3H, s).

Example 20

Preparation of 7,7,8,8-tetramethyl-2,4-dioxo-N-phenyl-2H,3H,4H,6H,7H,8H- pyrrolo[1 ,2-a]pyrimidine-3-carbothioamide

The reaction of 7,7,8,8-tetramethyl-3H,6H-pyrrolo[1 ,2-a]pyrimidine-2,4-dione (0.045 g, 0.22 mmol) with phenyl isothiocyanate (0.028 ml, 0.22 mmol) was carried out at 80 °C and worked up according to Procedure A to afford a yellow solid (0.048 g, 64% yield). ¹H-NMR (DMSO-d₆) δ 15.69 (1 H, br s), 13.99 (1 H, br s), 7.55 (2H, d), 7.47 (2H, t), 7.34 (1 H, t), 3.85 (2H, s), 1.15 (6H, s), 1.04 (6H, s). The starting material, 7,7,8,8-tetramethyl-3H,6H-pyrrolo[1 ,2-a]pyrimidine-2_I4-dione, was in turn prepared via the following steps: 4-Amino-2,2,3,3-tetramethylbutanenitrile was heated to undergo intramolecular cyclization to afford 3,3,4,4-tetramethyl-5H-pyrrol-2-amine as

previously described for a similar analog (Moriconi and Cevasco, J. Org. Chem.

33:2109-211 1 , 1968). This cyclized product (1.7 g, 12 mmol) was then reacted neat with diethyl ma!onate (2.3 g, 14 mmol) at 170 °C for 1 h and worked up as described by Le Barre and Renault (Bull. Soc. Chim. Fr. 9:3133-3138, 1969) to afford 7,7,8,8- tetramethyl-3H,6H-pyrrolo[1 ,2-a]pyrimidine-2,4-dione as a beige solid (1 .2 g, 50% yield). ¹H-NMR (DMSO-d₆) δ 11.30 (1 H, br s), 5.18 (1 H, s), 3.66 (2H, s), 1.09 (3H, s), 0.98 (3H, s). Examples of biological testing Examples 21

Topoisomerase II Catalytic kDNA Decatenation Inhibition Assay

The catalytic inhibition of human topoisomerase II by a drug was measured by the ATP-dependent decatenation of kDNA {Topogen, Orange Port, FL) into

minicircles of DNA as we previously described (Hasinoff et al., Biochem. Pharmacol. 50: 953-958, 1995). The 20 μΙ reaction mixture contained 0.5 mM ATP, 50 mM Tris- HCI (pH 8.0), 120 mM KCI, 10 mM MgCI₂, 30 μg/ml bovine serum albumin, 40 ng kDNA, drug or DMSO (0.5 μ[), and 200 ng K562 cells topoisomerase II nuclear extract, the amount that gave 80% decatenation. The enzymatic reaction was carried out at 37 °C and was terminated by the addition of 6 μΙ of buffer containing 5 mM Tris pH 8.0, 30% w/v sucrose, 0.5% bromophenol blue, and 125 mM EDTA. The resulting mixture was separated by electrophoresis (2 h at 8 V/cm) on an agarose gel prepared from 1 .2% w/v agarose and 0.5 μg ml ethidium bromide in TAE buffer pH 8.0 (40 mM Tris base, 0.1 14% (v/v) glacial acetic acid, 2 mM EDTA). The DNA in the gel was imaged by its fluorescence on an Alpha Annotech Fluorchem 8900 imaging system equipped with a 365 nm illuminator and a CCD camera. Densitometry scanning of gel photographs was used to obtain the fluorescence intensity of the band corresponding to the DNA minicircles. The percentage inhibition of K562 topoisomerase II catalytic activity at concentrations of 50 and 1 00 μΜ was determined using the following equation: % Inhibition = [(1 - (B_drug - BbackgroundVBdmso] x 1 00, where B_drug is the band intensity value for the enzymatic reaction of sample treated with drug, B _ackground is the band intensity value for sample in the absence of the enzyme, and B_dmso is the band intensity value for the enzymatic reaction of sample treated with DMSO solvent only.

Example 22

Topoisomerase ΙΙα-Mediated DNA Cleavage Assays

Topoisomerase ll-cleaved DNA covalent complexes induced by topoisomerase II poisons such as etoposide may be trapped by rapidly denaturing the complexed enzyme with sodium dodecyl sulfate (SDS), thus releasing the cleaved DNA as linear DNA (Osheroff, Biochemistry 28: 6157-6160, 989). The formation of linear DNA was detected by separating the SDS-treated reaction products using ethidium bromide gel electrophoresis as described previously (Burden et al., Methods Mol. Biol. 95: 283- 289, 2001 ). The band corresponding to linear DNA was identified by comparison with that from linear pBR322 DNA produced by action of the restriction enzyme Hind 111 acting on a single site on pBR322 DNA. The 20 μΙ cleavage assay reaction mixture contained 2 units of topoisomerase I la protein (Topogen), 80 ng pBR322 plasmid DNA ( BI Fermentas, Burlington, ON, Canada), 0.5 mM ATP in assay buffer {10 mM Tris-HCI, 50 mM KCI, 50 mM NaCI, 0.1 mM EDTA, 5 mM MgCI₂₎ 2.5% glycerol, at pH 8.0), and 100 μΜ of the drug (0.5 μΙ in DMSO). The order of addition was assay buffer, DNA, topoisomerase lla, and then drug. The mixture was assembled on ice before the addition of drug solution. The reaction mixture was incubated at 37 °C for 10 min, and then quenched with 0.05% (v/v) SDS/22 mM EDTA. The mixture was subsequently treated with 0.25 mg/ml proteinase K (Sigma) at 55 °C for 30 min to digest the topoisomerase lla protein, and was separated by electrophoresis (2 h at 8 V/cm) on an agarose gel prepared from 1 .2% w/v agarose and 0.5 μg/ml ethidium bromide in TAE buffer pH 8.0 (40 mM Tris base, 0.1 14% (v/v) glacial acetic acid, 2 mM EDTA). The DNA in the gel was imaged by its fluorescence on an Alpha

Annotech Fluorchem 8900 imaging system equipped with a 365 nm illuminator and a CCD camera.

Example 23

Cell Culture and Growth Inhibition Assay

K562 cells were obtained from American Type Culture Collection (Rockville, MD). These cells were maintained as suspension cultures in alpha minimum essential medium ( MEM) (Gibco BRL, Burlington, Canada) containing 2 mM L-glutamine and supplemented with 10% fetal calf serum (Invitrogen, Burlington, ON, Canada), 20 mM NaHCO₃, 20 mM HEPES (Sigma), 100 units/ml penicillin G, and 100 μς/ητιΙ

streptomycin at pH 7.4 in an atmosphere of 5% CO₂ and 95% air at 37 °C. For the measurement of growth inhibition, cells in exponential growth were harvested and seeded at 6000 cells/well in 96-well plates (100 μΐ/well). Drugs were dissolved in DMSO and added to the wells such that the final concentration of DMSO was 0.5% (v/v). After 72 h incubation, 7 μΙ of 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyi)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Cell Titer 96® AQueous One Solution (Promega, MD, Wl) was added to each well and incubated for a further 3 h. The absorbance was measured in a Molecular Devices (Menlo Park, CA) plate reader. The spectrophotometry 96-weli plate cell growth inhibition assay measures the ability of the cells to enzymaticaliy reduce MTS. Three replicates were measured at each drug concentration, and the [C₅₀ values and their SEs for growth inhibition were obtained by fitting the absorbance-drug concentration data to a four- parameter logistic equation.

While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

Compound w X R

Example 1 -N(CH₃)-C(0)-N(CH₃)- s Phenyl

Example 2 -N(CH₃)-C(S)-N(CH₃)- s Phenyl

Example 3 -N(CH₃)-C(S)-N(CH₃)- s CH₂CH₂Phenyl

Example 4 ~~N(CH₃)-C(S)~N(CH₃)- 0 CH₂CH₂C0₂Ethyl

Example 5 -N(CH₃)-C(S)-N(CH₃)~ s Cylcohexyl

Example 6 -N(CH₃)-C(S)-N(CH₃)- s w-Pentyl

Example 7 -N(CH₃)-C(S)-N(CH₃)- s Benzyl

Example 8 - H-C(SC¾)=N - s Phenyl

Example 9 -N(PhenyI)-C(S)-N(Phenyl)- s Phenyl

Example 10 -NH-C(S)-N(Allyl)- s Phenyl

Example 11 -NH~C(S)-N(CH₃)- s Phenyl

Example 12 -(CH₂)₃- s Phenyl

Example 13 -(CH₂)₂- s Phenyl

Example 14 s Phenyl

Example 15 -NH-N(Phenyl)- s Phenyl

Example 16 s Phenyl ocH₃

Example 17 — CH₂— S— CH₂— 0 Phenyl

Example 18 -CH₂-S-CH₂- s Phenyl

Example 19 -0-C(CH₃) =CH- s Phenyl

Example 20 s Phenyl

TABLE 2: The effects of drugs on human topoisomerase II decatenation catalytic activity and DNA cleavage activities, and human leukemia K562 cell growth.

Inhibition of Formation of linear Median growth topoisomerase II DNA mediated by inhibitory

Compound catalytic activity (%) topoisomerase Hot at concentration (IC₅₀) of

100 μΜ drug leukemia 562 cells

100 μΜ 50 μΜ concentration (μΜ)

Example 1 100 100 Yes 15

Example 2 100 100 Yes 4

Example 3 100 50 No 42

Example 4 100 20 No 31

Example 5 70 0 No 52

Example 6 100 32 No 11

Example 7 100 31 No 15

Example 8 80 20 No 54

Example 9 100 62 No 65

Example 10 100 100 Yes 10

Example 11 100 100 No 8

Example 12 65 0 No 73

Example 13 65 0 No 22

Example 14 100 50 Yes 11

Example 15 70 0 No 15

Example 16 100 50 No 15

Example 17 60 0 No 71

Example 18 80 30 No 85

Example 19 70 0 No 77

Example 20 100 70 No 70

Merbarone 95 65 No 42

Control (no drug) 0 0 No No inhibition

Claims

1. A method of inhibiting the activity of topoisomerase II in a cell, thereof comprising administering to the cell an effective amount of a compound of formula (V), or the tautomers and salts thereof:

(V)

wherein

X is oxygen or sulfur;

R is C-i-C-6 linear or branched alkyl, C₅-Ce cycioalkyl, benzyl, phenethyl, C1-C5 alkoxy-COCH₂CH₂, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, CrC-6 linear or branched alkyl, haloalkyl, C C₄ alkoxy, C1-C4 thioalkoxy, phenoxy, Ci- C₅ alkylcarbonyl, and C C₅ alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C-i-Ce linear or branched alkyl and C C₅ alkoxycarbonyl;

W is

wherein R and R² are each independently hydrogen, Ci-C₆ linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when X is oxygen, R cannot be phenyl optionally substituted with 0 to 5 substituents; with the proviso that when X is sulfur and R is phenyl, R and R² cannot be hydrogen;

(b)

wherein R³ is C C linear alkyl, C C alkoxy, or C1-C4 thioalkoxy; c)

wherein Y is absent or present and when present is carbonyl; with the proviso that when Y is absent, X must be sulfur; R⁴ and R⁵ are each independently hydrogen, C C₆ linear alkyl, CrC₆ alkoxy; halogen, or trihalomethyl; d)

wherein R is hydrogen or C C₆ linear alkyl; or

(f) — CH₂-(Q)-CH₂—

R⁹

wherein n is 0 or 1 ; Q is carbon or sulfur; R⁹ is present when Q is carbon, or absent when Q is sulfur, and when present is hydrogen or CrC₆ linear alkyl; with the proviso that when X is oxygen, R cannot be phenyl;

(g) -NH-NR ⁰- wherein R¹⁰ is C-i-C-e linear alkyl or phenyl.

2. The method according to claim 1 wherein:

X is sulfur;

R is C-i-C-e linear or branched alkyl, C₅-C₆ cycloalkyl, benzyl, phenethyl, C1-C5 alkoxy-COCH2CH₂, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C C-6 linear or branched alkyl, haloalkyl, C C₄ alkoxy, Ci-C₄ thioalkoxy, phenoxy, C C₅ alkylcarbonyl, and Ci-C₅ alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C1-C-6 linear or branched alkyl and C1-C5 alkoxycarbonyl; and

W is

wherein R and R² are each independently hydrogen, CrC₆ linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when R is phenyl, R¹ and R² cannot both be hydrogen.

3. The method of claim 1 , wherein the compound is comprised in a pharmaceutically acceptable composition.

4. A method of inhibiting the growth of a cell, thereof comprising administering to the cell an effective amount of a compound of formula (V), or the tautomers and salts thereof:

(V)

wherein

X is oxygen or sulfur;

R is Ci-C₆ linear or branched alkyl, C₅-C₆ cycloalkyl, benzyl, phenethyl, C1-C5 alkoxy-COCH₂CH2, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C-i-C-6 linear or branched alkyl, haloalkyl, C C₄ alkoxy, C C₄ thioalkoxy, phenoxy, C C₅ alkylcarbonyl, and C-1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, d-C-6 linear or branched alkyl and C1-C5 alkoxycarbonyl;

W is

wherein R¹ and R² are each independently hydrogen, C C₆ linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when X is oxygen, R cannot be phenyl optionally substituted with 0 to 5 substituents; with the proviso that when X is sulfur and R is phenyl, R¹ and R² cannot be hydrogen;

(b)

wherein R³ is C C linear alkyl, C-1-C4 alkoxy, or C C₄ thioalkoxy; c)

wherein Y is absent or present and when present is carbonyl; with the proviso that when Y is absent, X must be sulfur; R⁴ and R⁵ are each independently hydrogen, Ci-C₆ linear aikyl, C1-C-6 alkoxy; halogen, or trihalomethyl;

wherein R is hydrogen or Ci-C₆ linear alkyl; or

wherein n is 0 or 1 ; Q is carbon or sulfur; R is present when Q is carbon, or absent when Q is sulfur, and when present is hydrogen or Ci-C₆ linear alkyl; with the proviso that when X is oxygen, R cannot be phenyl;

(g) -NH-NR - wherein R^1U is C-i-C-e linear alkyl or phenyl.

5. The method according to claim 4 wherein:

X is sulfur;

R is C C₆ linear or branched alkyl, C₅-C₆ cycloalkyl, benzyl, phenethyl, C1-C5 alkoxy-COCH₂CH_2j phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C-i-C-6 linear or branched alkyl, haloalkyl, C-1-C-4 alkoxy, C1-C4 thioalkoxy, phenoxy, C C₅ alkylcarbonyl, and C1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C Ce linear or branched alkyl and C1-C5 alkoxycarbonyl; and

W is

wherein R and R² are each independently hydrogen, C1-C6 linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when R is phenyl, R¹ and R² cannot both be hydrogen.

6. The method of claim 4, wherein the compound is comprised in a pharmaceutically acceptable composition.

7. A method of treating a patient with cancer, thereof comprising administering to the cell an effective amount of a compound of formula (V), or the tautomers and salts thereof:

wherein

X is oxygen or sulfur;

R is Ci-C₆ linear or branched alkyl, C₅-C₆ cycloalkyl, benzyl, phenethyl, C1-C5 alkoxy-COCH₂CH2, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C-1-C-6 linear or branched alkyl, haloalkyl, C1-C4 alkoxy, Ci-C₄ thioalkoxy, phenoxy, C-r C₅ alkylcarbonyl, and C1-C5 aikoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen,

linear or branched alkyl and C1-C5 aikoxycarbonyl;

W is

a)

wherein R¹ and R² are each independently hydrogen, C C₆ linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when X is oxygen, R cannot be phenyl optionally substituted with 0 to 5 substituents; with the proviso that when X is sulfur and R is phenyl, R and R² cannot be hydrogen; b)

wherein R³ is C1-C4 linear alkyl, CrC₄ alkoxy, or C1-C4 thioalkoxy; C)

wherein Y is absent or present and when present is carbonyl; with the proviso that when Y is absent, X must be sulfur; R⁴ and R⁵ are each independently hydrogen, Ci-C₆ linear alkyl, Ci-C₆ alkoxy; halogen, or tri halo methyl; d)

wherein R is hydrogen or Ci-C₆ linear alkyl; or

(f)

R⁹

CH₂-(Q)-CH₂

¹ 9

R

wherein n is 0 or 1 ; Q is carbon or sulfur; R⁹ is present when Q is carbon, or absent when Q is sulfur, and when present is hydrogen or CrC₆ linear alkyl; with the proviso that when X is oxygen, R cannot be phenyl; (g) -NH-NR¹⁰- wherein R ⁰ is C-i-Ce linear alkyl or phenyl.

8. The method according to claim 7 wherein:

X is sulfur;

R is C C-6 linear or branched alkyl, C5-C6 cycloalkyl, benzyl, phenethyl, C1-C5 alkoxy-COCH₂CH₂, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C C₆ linear or branched alkyl, haloalkyl, C C₄ alkoxy, C C thioalkoxy, phenoxy, Ci- C₅ alkylcarbonyl, and C1-C5 aikoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C1-C-6 linear or branched alkyl and C1-C5 aikoxycarbonyl; and

W is

wherein R¹ and R² are each independently hydrogen, d-Ce linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when R is phenyl, R and R² cannot both be hydrogen.

9. The method of claim 7, wherein the cancer is cancer of the head, brain, eye, mouth, gum, tongue, neck, lung, breast, liver, pancreas, kidney, gastrointestinal tract, small intestine, colon, bladder, prostate, testicle, ovary, cervix, bone, skin, soft tissues, connective tissues, lymph system, hematopoietic system or blood.

10. The method of claim 7, wherein the compound is comprised in a pharmaceutically acceptable composition.

11. Use of a compound in the manufacture of a medicament for treating cancer, said compound comprising a compound of formula (V), or the tautomers and salts thereof:

(V)

wherein

X is oxygen or sulfur;

R is C C-6 linear or branched alkyl, C₅-C₆ cycloalkyl, benzyl, phenethyl, C1-C5 alkoxy-COCH₂CH2, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, Ci-C₆ linear or branched alkyl, haloalkyl, C1-C4 alkoxy, Ci-C₄ thioalkoxy, phenoxy, C C₅ alkylcarbonyl, and C1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C1-C6 linear or branched alkyl and C1-C5 alkoxycarbonyl;

W is

a)

wherein R³ is C C₄ linear alkyl, C1-C4 alkoxy, or C C₄ thioalkoxy; C)

wherein Y is absent or present and when present is carbonyl; with the proviso that when Y is absent, X must be sulfur; R⁴ and R⁵ are each independently hydrogen, C1-C-6 linear alkyl, Ci-C₆ alkoxy; halogen, or trihalomethyl; d)

wherein R is hydrogen or Ci-C₆ linear alkyl;

( )

R⁹

-CH₂-(Q)-CH₂-

R⁹

(g) -NH-NR - wherein R ⁰ is C C₆ linear alkyl or phenyl.

12. The use according to claim 11 wherein:

X is sulfur;

R is C-t-Ce linear or branched alkyl, C₅-C6 cycloaikyl, benzyl, phenethyl, C1-C5 alkoxy-COCH2CH2, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C-i-C-6 linear or branched alkyl, haloalkyl, C1-C4 alkoxy, C C₄ thioalkoxy, phenoxy, C C₅ alkylcarbonyl, and C1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, CrC₆ linear or branched alkyl and C-1-C5 alkoxycarbonyl; and

W is

13. Use of a compound for treating cancer, said compound comprising a compound of formula (V), or the tautomers and salts thereof:

(V)

wherein X is oxygen or sulfur;

R is C-t-Ce linear or branched alkyl, C₅-C₆ cycloalkyl, benzyl, phenethyl, C-₁-C5 alkoxy-COCH₂CH₂, phenyl, naphthyl, pyridy!, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C₁-C-6 linear or branched alkyl, haloalkyl, C1-C4 alkoxy, C C₄ thioalkoxy, phenoxy, C C5 alkylcarbonyl, and C₁-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C C₆ linear or branched alkyl and C1-C5 alkoxycarbonyl;

W is

wherein R and R² are each independently hydrogen, C C₆ linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when X is oxygen, R cannot be phenyl optionally substituted with 0 to 5 substituents; with the proviso that when X is sulfur and R is phenyl, R and R² cannot be hydrogen;

wherein R³ is C C₄ linear alkyl, Ci-C₄ alkoxy, or C C₄ thioalkoxy;

wherein Y is absent or present and when present is carbonyl; with the proviso that when Y is absent, X must be sulfur; R⁴ and R⁵ are each independently hydrogen, C C-6 linear aikyl, C1-C-6 alkoxy; halogen, or trihalomethyl; d)

wherein R⁶ and R⁷ are each independently hydrogen, C-i-C₆ linear or branched alkyl; with the proviso that when X is oxygen and R⁷ is hydrogen, R cannot be phenyl; e)

wherein R is hydrogen or C C₆ linear alkyl; or

(f)

R⁹

-CH -{Q)-CH - R⁹

wherein n is 0 or 1 ; Q is carbon or sulfur; R⁹ is present when Q is carbon, or absent when Q is sulfur, and when present is hydrogen or Ci-C₆ linear aikyl; with the proviso that when X is oxygen, R cannot be phenyl;

(g) -NH-NR ⁰- wherein R¹⁰ is C-i-Ce linear alkyl or phenyl.

14. The use according to claim 13 wherei

X is sulfur; R is Ci-C₆ linear or branched alkyl, C₅-C₆ cycloalkyi, benzyl, phenethyl, C1-C5 alkoxy-COCH₂CH₂, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C-i-Ce linear or branched alkyl, haloalkyl, Ci-C alkoxy, C C thioalkoxy, phenoxy, C-i- C₅ alkylcarbonyl, and C C₅ alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C C₆ linear or branched alkyl and C1-C5 alkoxycarbonyl; and

W is

wherein R¹ and R² are each independently hydrogen, C C₆ linear or branched alkyl, allyl, or phenyl; G is oxygen or sulfur; with the proviso that when R is phenyl, R¹ and R² cannot both be hydrogen.

15. A compound of formula (V), or the tautomers and salts thereof:

(V)

wherein

X is oxygen or sulfur;

R is Ci-C₆ linear or branched alkyl, C₅-C₆ cycloalkyi, benzyl, phenethyl, C1-C5 alkoxy-COCH2CH₂, phenyl, naphthyl, pyridyl, or thienyl; phenyl is optionally substituted with 0 to 5 substituents independently selected from hydrogen, halogen, C1-C6 linear or branched alkyl, haloalkyl, C1-C4 alkoxy, C C₄ thioalkoxy, phenoxy, Cr C-5 alkylcarbonyl, and C-1-C5 alkoxycarbonyl; thienyl is optionally substituted with 0 to 3 substituents independently selected from hydrogen, C1-C-6 linear or branched alkyl and C1-C5 alkoxycarbonyl;

W

wherein R is hydrogen or C C₆ linear alkyl.