Kuo-Hsiung Lee, Yizhou Dong, Kenneth F. Bastow,

Eva Y.-H. P. Lee, and Kyoko Nakagawa-Goto

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under NIH grant CA 17625. The Government has certain rights to this invention. FIELD OF THE INVENTION

The present invention concerns active compounds, formulations thereof, and methods of use thereof, particularly in methods of treating cancer.

BACKGROUND OF THE INVENTION

In 2004, our group first isolated and synthesized neo-tanshinlactone (1). (Wang, X.; Bastow, K. F.; Sun, C. M.; Lin, Y. L.; Yu, H. J.; Don, M. J.; Wu, T. S.; Nakamura, S.; Lee, K. H. Antitumor Agents. 239. Isolation, structure elucidation, total synthesis, and anti-breast cancer activity of neo-tanshinlactone from Salvia miltiorrhiza. J Med Chem 2004, 47, 5816-9) Compound 1 was 10-fold more potent and 20-fold more selective as compared to tamoxifen citrate against ER+ human breast cancer cell lines: MCF-7 and ZR-75-1. Further structural optimization led to its 4-ethyl analog 2, which displayed significant and selective anti-breast cancer activity both in vitro and in vivo. (Wang, X.; Nakagawa-Goto, K.; Bastow, K. F.; Don, M. J.; Lin, Y. L.; Wu, T. S.; Lee, K. H. Antitumor agents. 254. Synthesis and biological evaluation of novel neo-tanshinlactone analogues as potent anti-breast cancer agents. J Med Chem 2006, 49, 5631-4; Dong, Y.; Shi, Q.; Pai, H. C; Peng, C. Y.; Pan, S. L.; Teng, C. M.; Nakagawa-Goto, K.; Yu, D.; Liu, Y. N.; Wu, P. C; Bastow, K. F.; Morris-Natschke, S. L.; Brossi, A.; Lang, J. Y.; Hsu, J. L.; Hung, M. C; Lee, E. Y.; Lee, K. H. Antitumor Agents. 272. Structure-Activity Relationships and In Vivo Selective Anti-Breast Cancer Activity of Novel Neo-tanshinlactone Analogues. J Med Chem 2010, 53, 2299-8) Moreover, 2 was selective for a subset of breast cancer-derived cell lines and significantly less active against normal breast-derived tissue. In order to explore the effect of the individual rings on the anticancer activity, identify new lead compounds, and discover new chemical entities, we designed and reported five classes of novel anticancer agents, including (1) 2-(furan-2-yl) naphthalen-l-ol (FNO) (Dong, Y.; Shi, Q.; Liu, Y.-N.; Wang, X.; Bastow, K. F.; Lee, K.-H. Antitumor Agents. 266. Design, Synthesis, and Biological Evaluation of Novel 2-(Furan-2- yl)naphthalen-l-ol Derivatives as Potent and Selective Antibreast Cancer Agents. J Med. Chem. 2009, 52, 3586-3590), (2) 6-phenyl-4H-furo[3,2-c]pyran-4-one (AFPO) (Dong, Y.; Shi, Q.; Nakagawa-Goto, K.; Wu, P. C; Morris-Natschke, S. L.; Brossi, A.; Bastow, K. F.; Lang, J. Y.; Hung, M. C; Lee, K. H. Antitumor agents 270. Novel substituted 6-phenyl-4H- furo[3,2-c]pyran-4-one derivatives as potent and highly selective anti-breast cancer agents. Bioorg Med Chem 2010, 18, 803-8), (3) tetrahydronaphthalene-l-ol (TNO) (Dong, Y.; Shi, Q.; Nakagawa-Goto, K.; Wu, P.-C; Bastow, K. F.; Morris-Natschke, S. L.; Lee, K.-H. Antitumor agents 269. Non-aromatic ring-A neotanshinlactone analog, TNO, as a new class of potent antitumor agents. Bioorg. Med. Chem. Lett. 2009, 19, 6289-6292), (4) 4-amino-2H- benzo[ ?]chromen-2-one (ABO, 3, Figure 1), (Dong, Y.; Nakagawa-Goto, K.; Lai, C. Y.; Morris-Natschke, S. L.; Bastow, K. F.; Lee, K. H. Antitumor agents 278. 4-Amino-2H- benzo[h]chromen-2-one (ABO) analogs as potent in vitro anticancer agents. Bioorg Med Chem Lett 2010, 20, 4085-7), and (5) 4-amino-7,8,9,10-tetrahydro-2H-benzo[//]chromen-2- one (ATBO 4, Scheme A) analogs.

R = Me: Neotaishinlatone (1) ABO (3): R ¹ , R ² = H, ATBO (4)

R = Et: 4-Ethyl neo-tanshinlatone (2) R3= 4-OMePh Scheme A. Structures of neo-tanshinlactone (1), 4-ethyl neo-tanshinlactone (2), previously reported ABO (3) and ATBO (4) scaffolds.

SUMMARY OF THE INVENTION

Importantly, ABO and ATBO compounds displayed much higher potency than 1-analogs, which encouraged us to further investigate these scaffolds. Structure-activity relationship (SAR) studies on 3 and 4 indicated that (1) secondary amine (R ₂ or R ₃ = H) is preferred over tertiary amine (R ₂ and R ₃ ≠ H), (2) bulky groups are favored at R ₂ /R ₃ position, (3) 3'- bromophenyl group can cause dramatic loss of potency, and (4) non-aromatic ring-A can increase potency and cancer cell line selectivity for certain analogs. Previously, our studies indicated that the lactone ring-C is critical to the anticancer activity. Therefore, we designed the structurally simplified scaffold 5, which does not contain the fused ringA and -B, and installed various substituents at the R and R' positions to further explore the contributions of ring-A and -B, develop new chemical entities and new leads, and establish the SAR. Herein, we report the design, synthesis, and biological activity of 4-amino-2H-pyran-2-one (APO) analogs. Lead compound 27 is the most potent analog among all derivatives.

A first aspect of the present invention is a compound of Formula I:

wherein:

Ri, R ₂ , R ₃ , R4, and R ₅ are each independently selected from the group consisting of H, lower alkyl, hydroxy, lower alkoxy, cycloalkyl, cycloalkenyl, halo, sulfhydryl, thioalkyl, cyano, carbonyl, carboxyl, amino, aminoalkyl, alkylamino, nitro, heteroaryl, aryl, heteroaryl,

OC(=0)Rio, OC(=O)OR ₁₀ , OC(=O)N(R ₁₀ ) ₂ , O(CH ₂ ) _m N(R ₁₀ ) ₂ , C(=O)N(R ₁₀ ) ₂ , 0(CH ₂ ) _m COORio, (CH ₂ ), ₁₁ C(=0)N(Rio)OR ₁₀ , C(=O)N(Ri ₀ )OR ₁₀ , and (CH ₂ ) _m C(=O)OR ₁₀ , where m is 1-5 and Rio is H or lower alkyl; or Ri and R ₂ together form a cycloalkyl, cycloalkenyl, heteroaryl, or aryl ring; or R4 and R ₅ together form a cycloalkyl, cycloalkenyl, heteroaryl, or aryl ring;

Xi is selected from the group consisting of N, O, and S, wherein when Xi is O or S then R ₂ is not present;

X ₂ is independently selected from the group consisting of— C(Rn)(R ₁₂ ), O, S, NH, C=0, C=S, C=NH, SO, and S0 ₂ , wherein Rn and Ri ₂ are each independently selected from the group consisting of H, lower alkyl, hydroxy, lower alkoxy, halo, sulfhydryl, thioalkyl, cyano, carbonyl, carboxyl, amino, aminoalkyl, alkylamino, nitro, heteroaryl, aryl, cycloalkyl, cycloalkenyl, OC(=0)R ₁₃ , OC(=0)ORi ₃ , OC(=0)N(R ₁₃ ) ₂ , 0(CH ₂ ) _n N(R _I3 ) ₂ , C(=0)N(Ri ₃ ) ₂ , 0(CH ₂ ) _n COOR ₁₃ , (CH ₂ )„C(=0)N(Ri ₃ )ORi3, C(=0)N(R ₁₃ )OR ₁₃ , and (CH ₂ ) _n C(=0)OR ₁₃ , where n is 1-5 and Ri ₃ is H or lower alkyl;

X ₃ is selected from the group consisting of O, S, NH, and (CH ₂ ) _P , where p is 1-2; or a pharmaceutically acceptable salt or prodrug thereof;

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described more fully hereinafter. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

"Moiety" and "group" are used interchangeably herein to refer to a portion of a molecule, typically having a particular functional or structural feature, e.g. a linking group (a portion of a molecule connecting two other portions of the molecule).

The term "substituted" as used herein to describe chemical structures, groups, or moieties, refers to the structure, group, or moiety comprising one or more substituents. As used herein, in cases in which a first group is "substituted with" a second group, the second group is attached to the first group whereby a moiety of the first group (typically a hydrogen) is replaced by the second group. The substituted group may contain one or more substituents that may be the same or different.

"Substituent" as used herein references a group that replaces another group in a chemical structure. Typical substituents include nonhydrogen atoms (e.g. halogens), functional groups (such as, but not limited to amino, sulfhydryl, carbonyl, hydroxyl, alkoxy, carboxyl, silyl, silyloxy, phosphate and the like), hydrocarbyl groups, and hydrocarbyl groups substituted with one or more heteroatonis. Exemplary substituents include but are not limited to alkyl, lower alkyl, aryl, aralkyl, lower alkoxy, thioalkyl, hydroxyl, thio, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, boronyl, and modified lower alkyl.

The term "alkyl," as used herein, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n- pentyl, iso-pentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3- dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like.

"Loweralkyl," as used herein, is a subset of alkyl and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso- butyl, tert-butyl, and the like.

"Alkenyl," as used herein, refers to a straight or branched chain hydrocarbon containing from 2 to 10 carbons and containing at least one carbon-carbon double bond formed by the removal of two hydrogens. Representative examples of "alkenyl" include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5- hexenyl, 2-heptenyl, 2-methyl-l-heptenyl, 3-decenyl and the like. "Loweralkenyl" as used herein, is a subset of alkenyl and refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms.

"Alkoxy," as used herein, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through an oxy group, as defined herein. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert- butoxy, pentyl oxy, hexyloxy and the like. "Lower alkoxy," as used herein, is a subset of alkoxy and refers to a lower alkyl group, as defined herein, appended to the parent molecular moiety through an oxy group, as defined herein. Representative examples of lower alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, and the like.

"Alkylthio," as used herein refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a thio moiety, as defined herein. Representative examples of alkylthio include, but are not limited, methylthio, ethylthio, tert-butylthio, hexylthio, and the like.

"Alkylogen," as used herein means alkyl or loweralkyl in which one, two, three or more (e.g., all) hydrogens thereon have been replaced with halo. Examples of alkylogen include but are not limited to trifluoromethyl, chloromethyl, 2-chloroethyl, 2-bromoethyl, and 2-iodoethyl. Alkylogens may also be referred to as haloalkyl or perhaloalkyl (e.g. fluoroalkyl; perfluoroalkyl).

"Cycloalkyl," as used herein, refers to a saturated cyclic hydrocarbon group containing from 3 or 4 to 6 or 8 carbons. Representative examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. A cycloalkyl group may be unsubstituted or substituted and when substituted the substituents may be the same or different.

"Cycloalkenyl," as used herein, refers to an unsaturated cyclic hydrocarbon group containing from 3 to 8 carbons and having at least one double bond. Representative examples include cyclobutenyl, cyclopentenyl, cyclohexenyl, and cycloheptenyl. A cycloalkenyl group may be unsubstituted or substituted and when substituted the substituents may be the same or different. Cycloalkenyl groups herein may or may not be aromatic. "Heterocycle," as used herein, refers to a monocyclic- or a bicyclic-ring system.

Monocyclic ring systems are exemplified by any 5 or 6 membered ring containing 1, 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur. The 5 membered ring has from 0-2 double bonds and the 6 membered ring has from 0-3 double bonds. Representative examples of monocyclic ring systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to an aryl group as defined herein, a cycloalkyl group as defined herein, or another monocyclic ring system as defined herein. Representative examples of bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizne, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline,tetrahydroisoquinoline, tetrahydroquinoline, fhiopyranopyridine, and the like. Heterocycle groups of this invention can be substituted with 1, 2, or 3 substituents, such as substituents independently selected from alkenyl, alkenyloxy, alkoxy, alkoxyalkoxy, alkoxycarbonyl, alkyl, alkylcarbonyl, alkylcarbonyloxy, alkylsulfinyl, alkylsulfonyl, alkylthio, alkynyl, aryl, azido, arylalkoxy, arylalkoxycarbonyl, arylalkyl, aryloxy, carboxy, cyano, formyl, oxo, halo, haloalkyl, haloalkoxy, hydroxy, hydroxyalkyl, mercapto, nitro, sulfamyl,sulfo, sulfonate,— NR R" (wherein, R' and R" are independently selected from hydrogen, alkyl, alkylcarbonyl, aryl, arylalkyl and formyl), and -C(0)NRR' (wherein, R and R' are independently selected from hydrogen, alkyl, aryl, and arylalkyl). "Aryl," as used herein refers to an aromatic species containing 1 to 5 aromatic rings, either fused or linked, and either unsubstituted or substituted with 1 or more groups typically selected from the group consisting of lower alkyl, modified lower alkyl, aryl, aralkyl, lower alkoxy, thioalkyl, hydroxyl, thio, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl; and lower alkyl substituted with one or more groups selected from lower alkyl, alkoxy, thioalkyl, hydroxyl thio, mercapto, amino, imino, halo, cyano, nitro, nitroso, azido, carboxy, sulfide, sulfone, sulfoxy, phosphoryl, silyl, silyloxy, and boronyl. Typical aryl groups contain 1 to 3 fused aromatic rings, and more typical aryl groups contain 1 aromatic ring or 2 fused aromatic rings. Aromatic groups herein may or may not be heterocyclic. "Heteroaryl," as used herein refers to an aryl, as defined herein, that is heterocyclic. "Halo," as used herein refers to any halogen group, such as chloro, fluoro, bromo, or iodo.

"Hydroxyl," as used herein refers to the radical -OH. "Oxo," as used herein refers to a =0 moiety.

"Oxy," as used herein refers to a -O- moiety. "Thio," as used herein refers to a -S- moiety. "Sulfhydryl," as used herein refers to the radical -SH. "Thioalkyl," as used herein refers to a - S-alkyl group. "Cyano," as used herein refers to a -CN group.

"Carbonyl," as used herein refers to a divalent group of the formula -C(=0)-. "Carboxyl," as used herein refers to a -C(=0)OH group. "Nitro," as used herein is intended to mean the radical -N0 ₂ . "Amine," or "amino group," is intended to mean the radical -NH ₂ .

"Substituted amino," or "substituted amine," refers to an amino group, wherein one or two of the hydrogens is replaced by a suitable substituent. Disubstituted amines may have substituents that are bridging, i.e., form a heterocyclic ring structure that includes the amine nitrogen as the linking atom to the parent compound. Examples of substituted amino include but are not limited to alkylamino, dialkylamino, and heterocyclo (where the heterocyclo is linked to the parent compound by a nitrogen atom in the heterocyclic ring or heterocyclic ring system).

"Alkylamino," is intended to mean the radical -NHR', where R' is alkyl.

"Dialkylamino," is intended to mean the radical NR'R", where R' and R" are each independently an alkyl group. "Aminoalkyl," refers to an alkyl substituent which is further substituted with one or more amino groups.

"Treat" or "treating" as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the disease, prevention or delay of the onset of the disease, etc.

"Treatment effective amount" as used herein refers to an amount of the active compound effective to treat the disease, slow or delay the progression of the disease, prevent or delay of the onset of the disease, etc.

"Pharmaceutically acceptable" as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

"Inhibit" as used herein means that a potential effect is partially or completely eliminated.

The present invention is concerned primarily with the treatment of human subjects, but may also be employed for the treatment of other animal subjects (i. e., mammals such as dogs, cats, horses, etc. or avians) for veterinary purposes. Mammals are preferred, with humans being particularly preferred.

A. Active Compounds.

Active compounds of the present invention are described below, and may be formulated and used in the compositions and methods described below.

Active compounds of the invention include compounds of Formula I:

wherein:

Ri, R , R ₃ , R4, and R ₅ are each independently selected from the group consisting of H, lower alkyl, hydroxy, lower alkoxy, cycloalkyl, cycloalkenyl, halo, sulfhydryl, thioalkyl, cyano, carbonyl, carboxyl, amino, aminoalkyl, alkylamino, nitro, heteroaryl, aryl, heteroaryl, OC(=0)Rio, OC(=O)OR ₁₀ , OC(=0)N(Rio) ₂ , O(CH ₂ ) _m N(R ₁₀ ) ₂ , C(=O)N(R ₁₀ ) ₂ , O(CH ₂ ) _m COOR ₁₀ , (CH ₂ ) _m C(=0)N(Rio)OR ₁₀ , C(=O)N(Ri ₀ )OR ₁₀ , and (CH ₂ ) _m C(=O)ORi ₀ , where m is 1-5 and Rio is H or lower alkyl; or R[ and R ₂ together form a cycloalkyl, cycloalkenyl, heteroaryl, or aryl ring; or R4 and R ₅ together form a cycloalkyl, cycloalkenyl, heteroaryl, or aryl ring; Xi is selected from the group consisting of N, O, and S, wherein when Xi is O or S then R ₂ is not present;

X ₂ is independently selected from the group consisting of— C(Rn)(Ri ₂ ), O, S, NH, C=0, C=S, C=NH, SO, and S0 ₂ , wherein Rn and Ri ₂ are each independently selected from the group consisting of H, lower alkyl, hydroxy, lower alkoxy, halo, sulfhydryl, thioalkyl, cyano, carbonyl, carboxyl, amino, aminoalkyl, alkylamino, nitro, heteroaryl, aryl, cycloalkyl, cycloalkenyl, OC(=0)R ₁₃ , OC(=0)OR ₁₃ , OC(=0)N(Ri ₃ ) ₂ , 0(CH ₂ ) _n N(Ri ₃ ) ₂ , C(=0)N(Ri ₃ ) ₂ , 0(CH ₂ )„COORi3, (CH ₂ )„C(=0)N(Ri ₃ )ORi ₃ , C(=0)N(R ₁₃ )OR ₁₃ , and (CH ₂ ) _n C(=0)OR ₁₃ , where n is 1-5 and Rj ₃ is H or lower alkyl;

X ₃ is selected from the group consisting of O, S, NH, and (CH ₂ ) _P , where p is 1-2; or a pharmaceutically acceptable salt or prodrug thereof;

In some embodiments of Formula I, X ₂ is C¾, S, O, or NH, C=0, C=S, C=NH, SO, or S0 ₂ .

In some embodiments of Formula I, X ₃ is CH ₂ , S, O, or NH,

In some embodiments of Formula I, at least one of Ri, R ₂ , R3, R4, and R ₅ is not H {i.e. , at least one of R _1} R ₂ , R3, R4, and R ₅ is selected from the group consisting of lower alkyl, hydroxy, lower alkoxy, cycloalkyl, cycloalkenyl, halo, sulfhydryl, thioalkyl, cyano, carbonyl, carboxyl, amino, aminoalkyl, alkylamino, nitro, heteroaryl, aryl, OC(=O)Ri ₀ , OC(=0)ORio, OC(=0)N(Rio) ₂ , O(CH ₂ ) _m N(R ₁₀ ) ₂ , C(=O)N(R ₁₀ ) ₂ , O(CH ₂ ) _m COOR ₁₀ ,

(CH ₂ ) _m C(=0)N(Rio)ORio, C(=0)N(R,o)ORio, and (CH ₂ ) _m C(=O)ORi ₀ , where m is 1-5 and Rio is H or lower alkyl).

In some embodiments of Formula I, Ri and R ₂ together form a cycloalkyl, cycloalkenyl, heteroaryl, or aryl ring.

In some embodiments of Formula I, X ₂ is O and X ₃ is O.

B. Formulations and pharmaceutically acceptable salts.

The term "active agent" as used herein, includes the pharmaceutically acceptable salts of the compounds of Formula I and Formula II. Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b) salts formed from elemental anions such as chlorine, bromine, and iodine.

Active agents used to prepare compositions for the present invention may alternatively be in the form of a pharmaceutically acceptable free base of the active agent. Because the free base of the compound is less soluble than the salt, free base compositions are employed to provide more sustained release of active agent to the target area. Active agent present in the target area which has not gone into solution is not available to induce a physiological response, but serves as a depot of bioavailable drug which gradually goes into solution.

The compounds of the present invention are useful as pharmaceutically active agents and may be utilized in bulk form. More preferably, however, these compounds are formulated into pharmaceutical formulations for administration. Any of a number of suitable pharmaceutical formulations may be utilized as a vehicle for the administration of the compounds of the present invention. The compounds of the present invention may be formulated for administration for the treatment of a variety of conditions in the manufacture of a pharmaceutical formulation according to the invention, the compounds of the present invention and the physiologically acceptable salts thereof, or the acid derivatives of either (hereinafter referred to as the "active compound") are typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation, for example, a tablet, which may contain from 0.5% to 95% by weight of the active compound. One or more of each of the active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well-known techniques of pharmacy consisting essentially of admixing the components, optionally including one or more accessory ingredients.

The formulations of the invention include those suitable for oral, rectal, topical, buccal (e.g., sub-lingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.

Formulations suitable for oral administration may be presented in discrete units, such as capsules, cachets, lozenges, or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above).

In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet may be prepared by compressing or molding a powder or granules containing the active compound, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the compound in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Molded tablets may be made by molding, in a suitable machine, the powdered compound moistened with an inert liquid binder.

Formulations suitable for buccal (sub-lingual) administration include lozenges comprising the active compound in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatin and glycerin or sucrose and acacia.

Formulations of the present invention suitable for parenteral administration conveniently comprise sterile aqueous preparations of the active compound, which preparations are preferably isotonic with the blood of the intended recipient. These preparations may be administered by means of subcutaneous, intravenous, intramuscular, or intradermal injection. Such preparations may conveniently be prepared by admixing the compound with water or a glycine buffer and rendering the resulting solution sterile and isotonic with the blood.

Formulations suitable for rectal administration are preferably presented as unit dose suppositories. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include vaseline, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. Formulations suitable for transdermal administration may also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 :318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.

Suitable formulations comprise citrate or bisXtris buffer (pH 6) or ethanol/water and contain from 0.01 to 0.2M active ingredient.

C. Methods of Use.

In addition to the compounds of the formulas described herein, the present invention also provides useful therapeutic methods. For example, the present invention provides a method of inducing cytotoxicity against tumor cells, or treating a cancer or tumor in a subject in need thereof.

Cancer cells which may be inhibited include cells from skin cancer, small cell lung cancer, non-small cell lung cancer, testicular cancer, lymphoma, leukemia, Kaposi's sarcoma, esophageal cancer, stomach cancer, colon cancer, breast cancer, endometrial cancer, ovarian cancer, central nervous system cancer, liver cancer and prostate cancer.

Subjects which may be treated using the methods of the present invention are typically human subjects although the methods of the present invention may be useful for veterinary purposes with other subjects, particularly mammalian subjects including, but not limited to, horses, cows, dogs, rabbits, fowl, sheep, and the like. As noted above, the present invention provides pharmaceutical formulations comprising the compounds of formulae described herein, or pharmaceutically acceptable salts thereof, in pharmaceutically acceptable carriers for any suitable route of administration, including but not limited to oral, rectal, topical, buccal, parenteral, intramuscular, intradermal, intravenous, and transdermal administration.

The therapeutically effective dosage of any specific compound will vaiy somewhat from compound to compound, patient to patient, and will depend upon the condition of the patient and the route of delivery. As a general proposition, a dosage from about 0.1 to about 50 mg/kg will have therapeutic efficacy, with still higher dosages potentially being employed for oral and/or aerosol administration. Toxicity concerns at the higher level may restrict intravenous dosages to a lower level such as up to about 10 mg/kg, all weights being calculated based upon the weight of the active base, including the cases where a salt is employed. Typically a dosage from about 0.5 mg/kg to about 5 mg/kg will be employed for intravenous or intramuscular administration. A dosage from about 10 mg/kg to about 50 mg/kg may be employed for oral administration.

The present invention is explained in greater detail in the following non-limiting examples. Example 1 4-Amino-2H-pyran-2-one Analogs (APO)

This example describes our design and synthesis of novel 4-amino-2H-pyran-2-one (APO) analgos, cytotoxicity evaluation against several human cancer cell lines, and structure- activity relationships (SAR) of this new compound class. The goal is to generate and optimize 4-amino-2H-pyran-2-one (APO) analogs as promising clinical trial candidates for treating cancer.

As a first step in the current work, we designed two model compounds 12 and 13 by removing the fused ring-A and -B of the ABO and ATBO analogs, and instead installing phenyl and styryl groups, respectively. 6-Substituted 4-hydroxy-2H-pyran-2-one 9 was synthesized according to the method reported by Bach et al. (Bach, T.; Kirsch, S. Synthesis of 6-substituted 4-hydroxy-2-pyrones from aldehydes by addition of an acetoacetate equivalent, Dess-Martin oxidation and subsequent cyclization. Synlett 2001, 1974-1976) An aldehyde (6) and an acetoacetate equivalent (7) underwent a vinylogous Mukaiyama aldol addition to give 8, which was oxidized to ketone 9 using the Dess-Martin method. A thermal cyclization of 9 yielded 10 (R group based on identity of aldehyde 6). Chlorination and amination with cyclohexylamine provided model compounds 12 and 13. (Dong, Y.; Nakagawa-Goto, K.; Lai, C. Y.; Morris-Natschke, S. L.; Bastow, K. F.; Lee, K. H. Antitumor agents 278. 4- Amino-2H-benzo[h]chromen-2-one (ABO) analogs as potent in vitro anticancer agents. Bioorg Med Chem Lett 2010, 20, 4085-7) Analogs 12 andl3 were tested for in vitro cytotoxic activity against a panel of human tumor cell lines according to previously published methods (Table 1). (Dong, Y.; Shi, Q.; Liu, Y.-N.; Wang, X.; Bastow, K. F.; Lee, K.-H. Antitumor Agents. 266. Design, Synthesis, and Biological Evaluation of Novel 2-(Furan-2- yl)naphthalen-l -ol Derivatives as Potent and Selective Antibreast Cancer Agents. J Med. Chem. 2009, 52, 3586-3590). Cell lines included A549 (non small cell lung cancer), DU145 (prostate cancer cell line), KB (nasopharyngeal carcinoma), and KB-vin (vincristine-resistant MDR KB subline), SK-BR-3 (estrogen receptor negative, HER2 over-expressing breast cancer). Importantly, 13 showed significant inhibition of all human cancer cell lines tested with ED ₅₀ values from 1.23-2.02 μΜ, and 12 displayed moderate activity. Encouraged by these promising results, analogs 14-30 were designed for further structure optimization in order to establish SAR correlations and to identify more active derivatives with the desired biological properties.

Scheme 1

12 13

R eagents and conditions: (a) TiCl ₄ , CH ₂ C1 ₂ , -78 °C; (b) Dess-Martin reagent, rt; (c) toluene, reflux; (d) POCl ₃ , Et ₃ N, reflux, lh; (e) aliphatic amines, EtOH, reflux, 2h; (f) aromatic amines, ethylene glycol, 160 °C, lh. Firstly, we installed various groups at the R position to explore the effect of group size, ring size, and aromaticity. Secondly, different substituents at the R' position were investigated, while maintaining the best group found at the R position. New analogs 14-30 were obtained similarly to 12-13 through the five step procedure shown in Scheme 1, and evaluated against five human tumor cell lines from different tissues.

As shown in Table 1, with the R' substituent fixed as j7-methoxyanilino and varying R substituents, compounds 14-15 with methyl and isopropyl groups were not active, and 16-18 with pentyl, cyclopentyl, and cyclohexyl groups showed moderate activity. These results suggested that short alkyl groups were not favored at the R position. However, 21-23 with benzyl, furan, and naphthalene groups displayed significant activity, and 19-20 with phenyl and styryl groups were the most potent analogs with ED ₅₀ values of 0.079-0.163 μΜ, equipotent or slightly more potent compared with ABO analog 3. Overall, the results indicated that an aromatic ring at the R position may be critical to the antitumor activity.

With a phenyl group always present at the R position, analogs 24-30 with various substituents at the R' position were designed based on structural simplicity and chemical availability. Compounds 24 and 25 with cyclopropylamine and piperidine groups, respectively, were not active. The rank order of potency for all aromatic analogs against KB was 27 (4' -Me) > 19 (4'-OMe) > 26 (Ph) > 28 (4'-Br) ~ 29 (2'-OMe) ~ 30 (3'-OMe). Compound 27 was the most potent analog with ED ₅₀ values of 0.059-0.090 μΜ, and was about twofold more potent than ABO analog 3.

Table 1. Cytotoxicity of 12-30 against Human Tumor Cell Line Panel"

a mean ED50 (μΜ), from 3 or more independent tests.

The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.