COMPOUNDS, COMPOSITIONS AND METHODS

OF TREATING CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Patent Application No. 62/268,266, filed December 16, 2015, the disclosure of which is incorporated herein in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with partial support under NIH/NCI Grant Numbers CA 155638 and CA 148817. Therefore, the U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Cancer is a leading cause of death in the developed world, with over one million people diagnosed and more than 500,000 deaths per year in the United States alone. Overall it is estimated that at least one in three people will develop some form of cancer during their lifetime. There are more than 200 different histopathological types of cancer, four of which (breast, lung, colorectal, and prostate) account for over half of all new cases in the U.S.

(Jemal et al., Cancer J. Clin., 53, 5-26 (2003)).

[0004] Many of these tumors arise from mutations that activate Ras proteins, which control critically important cellular signaling pathways that regulate growth and other processes associated with tumorigenesis. The name "Ras" is an abbreviation of "Rat sarcoma" reflecting the way the first members of the Ras protein family were discovered. The name "ras" also is used to refer to the family of genes encoding these proteins.

[0005] Ras proteins are key regulators of several aspects of normal cell growth and malignant transformation, including cellular proliferation, survival and invasiveness, tumor angiogenesis and metastasis (Downward, Nature Rev. Cancer, 3, 11-22 (2003)). Ras proteins are abnormally active in most human tumors due to mutations in the ras genes themselves, or in upstream or downstream Ras pathway components, or other alterations in Ras signaling. Targeted therapies that inhibit Ras-mediated pathways therefore are expected to inhibit the growth, proliferation, survival and spread of tumor cells having activated or mutant Ras. Some such new experimental therapeutic agents have shown promising activity in preclinical studies, albeit with only modest activity in human clinical trials.

[0006] Genetic mutations in ras genes were first identified in human cancer over 3 decades ago. Such mutations result in the activation of one or more of three major Ras protein isoforms, including H-Ras, N-Ras, or K-Ras, that turn on signaling pathways leading to uncontrolled cell growth and tumor development. Activating ras gene mutations occur de novo in approximately one third of all human cancers and are especially prevalent in pancreatic, colorectal, and lung tumors. Ras mutations also develop in tumors that become resistant to chemotherapy and/or radiation, as well as to targeted therapies, such as receptor tyrosine kinase inhibitors (Gysin et al., Genes Cancer, 2, 359-372 (2011)). While ras mutations are relatively infrequent in other tumor types, for example, breast cancer, Ras can be pathologically activated by certain growth factor receptors that signal through Ras.

[0007] Although ras gene mutations have been known for many years, there currently are no available cancer therapeutics approved by the U.S. Food and Drug Administration that are known to selectively suppress the growth of tumors driven by activated Ras. In fact, Ras has been described as "undruggable" because of the relative abundance in cells and high affinity for its substrate, GTP (Takashima and Faller, Expert Opin. Ther. Targets, 17, 507-531 (2013)).

[0008] In addition to its role in cancer, activated Ras is important in a variety of other diseases, collectively referred to as "rasopathies." One such disease, neurofibromatosis type 1 (NFl), a very prevalent autosomal dominant heritable disease, is caused by a mutation in neurofibromin, a Ras GAP (inactivating protein), which results in Ras hyperactivation in the relatively common event of loss of the second NFl allele. Such mutations reportedly affect 1 :3000 live births. The most dire symptoms associated with NFl include numerous benign tumors (neurofibromas) arising from precursor nerve cells and Schwann cells of the peripheral nervous system. These tumors can cause severe problems depending on their location within the body, such as hearing or vision loss, as well as disfiguring masses on visible areas. Less common but extremely serious complications may arise when central nervous system gliomas develop or plexiform neurofibromas become transformed, resulting in the development of metastatic peripheral nerve sheath tumors (Tidyman and Rauen, Curr. Opin. Genet. Dev., 19, 230-236 (2009)). Another rare developmental disease which is attributable to hyperactive H-Ras is Costello syndrome. This condition causes a range of developmental abnormalities as well as predisposing patients to a variety of benign and malignant neoplasms (Tidyman and Rauen, supra).

[0009] Several approaches to treat diseases that arise from activating ras mutations have been undertaken. Because full maturation of the Ras protein requires lipid modification, attempts have been made to target this enzymatic process with inhibitors of farnesyl transferase and geranylgeranyltransferase, but with limited success and significant toxicity. Targeting of downstream components of Ras signaling with inhibitors of Raf/Mek/Erk kinase components of the cascading pathway has been an extremely active area of pharmaceutical research, but also fraught with difficulties and paradoxes arising from complex feedback systems within the pathways (Takashima and Faller, supra).

[0010] Inhibitors targeting components within the PI3K/Akt pathway also have not been successful as single agents, but presumably might synergize with Raf/Mek/Erk pathway inhibitors to block Ras-dependent tumor growth and survival. Similarly, several other molecular targets have been identified from RNAi screening, which might provide new opportunities to inhibit the growth of Ras-driven tumors; such other potential targets include CDK4, Cyclin Dl, Tiaml, Myc, STK33, and TBK, as well as several genes involved in mitosis (Takashima and Faller, supra).

[0011] Certain other compounds have been described with selective toxicity toward cells expressing activated Ras. A high-throughput phenotypic screen of over 300,000 compounds was conducted within N H Molecular Libraries Screening Center program to identify compounds which were synthetically lethal to cells expressing oncogenic H-Ras. A lead compound, ML210 (Fig. 1), inhibited growth of cells expressing mutant Ras with an IC50 of 71 nM, and was 4-fold selective versus cells lacking oncogenic Ras. Though the specific molecular target of ML210 is unknown, the compound was chemically optimized to eliminate reactive groups and improve pharmacologic properties (ML210, 12/12/2011 update, Probe Reports from NIH Molecular Libraries Program, Bethesda,

http://www.ncbi.nlm.nih.gov/books/NBK98919/).

[0012] A separate high-throughput screen identified two compounds, RSL3 and RSL5 (Fig. 1) which induce non-apoptotic, Mek-dependent, oxidative cell death (Yang and

Stockwell, Chem. Biol., 15, 234-245 (2008). RSL5, like a previously identified Ras synthetic lethal compound, erastin (Fig. 1), binds the voltage-dependent anion channel (VDAC) (Dolma et al., Cancer Cell, 3, 285-296 (2003)). Yet another small-molecule screen identified oncrasin, a compound selectively active against K-Ras mutant cell lines (Guo et al., Cancer Res., 68, 7403-7408 (2008)). One analog, NSC-743380 (Fig. 1), is highly potent and has shown anti -tumor activity in a preclinical model of K-Ras driven renal cancer (Guo et al., PLoS One, 6, e28487 (2011)). A prodrug approach has recently been described for oncrasin derivatives, to improve stability, pharmacokinetics, and safety (Wu et al., Bioorg. Med.

Chem., 22, 5234-5240 (2014)). A synthetic lethal screen using embryonic fibroblasts derived from mice expressing the oncogenic K-Ras (G12D) identified a compound, lanperisone (Fig. 1), that induced non-apoptotic cell death via a mechanism involving oxidative stress (Shaw et al., Proc. Natl. Acad. Sci. USA, 108, 8773-8778 (2011)). In contrast to the synthetic lethal approach, a fragment-based screening approach paired with crystallographic studies has been used to identify compounds which irreversibly bind to and inhibit K-Ras in lung tumor cells having the relatively rare G12C ras gene mutation (Ostrem et al., Nature, 503, 548-551 (2013)). While compounds of this series potently inhibit Ras through a covalent interaction, the low frequency of this mutation may limit the utility of such compounds. Finally, a new investigational strategy for targeting oncogenic Ras has been described (Zimmerman et al., J. Med. Chem., 57, 5435-5448 (2014)) which involves structure guided design and kinetic analysis of benzimidazole inhibitors targeting the PDE5 prenyl binding site.

[0013] Ras-driven cancers have remained the most intractable diseases to any available treatment. New therapeutic and preventative strategies are urgently needed for such cancers (Stephen et al., Cancer Cell, 25, 2Ί2-2%\ (2014)). Drug discovery programs worldwide have sought Ras-selective drugs for many years, but heretofore no avail (Spiegel, et al., Nature Chem. Biol., 10, 613-622 (2014)). New drugs that selectively target abnormal or mutant Ras and/or Ras-mediated pathological processes in patients' tumors will enable highly efficacious treatments of such patients while minimizing toxicity to cells and tissues with normal Ras functions (Stephen et al., supra; Spiegel et al., supra).

[0014] The foregoing shows that there exists an unmet need for compounds that are suitable for treating or preventing cancers. There further exists an unmet need for compounds that inhibit Ras-dependent diseases or undesirable conditions.

BRIEF SUMMARY OF THE INVENTION The invention provides a compound of formula I or II,

in which R, R ₀ , R ₁ -R4, R7-R ₁₀ , n, X, Y, Y', and E are as described herein.

[0016] The present invention further provides a pharmaceutical composition comprising a compound described above and a pharmaceutically acceptable carrier.

[0017] It is contemplated that the compound of formula I or II can effectively inhibit Ras. Thus, the invention further provides a method of inhibiting a human or nonhuman

mammalian Ras-mediated biological process, which method comprising administering in vivo or in vitro a Ras-inhibitory amount of at least one compound of formula I or II, or the corresponding Z- or E-isomer, pharmaceutically acceptable salt, or prodrug thereof.

[0018] In one aspect, the present invention provides a method of therapeutically or prophylactically treating a human or nonhuman mammalian patient with a disease or condition treatable by the inhibition of one or more neoplastic or cancerous process, which method comprises administering to a patient in need thereof a therapeutically or

prophylactically effective amount of at least one neoplastic or cancerous inhibitory compound of formula I or II, as described above, or the corresponding Z- or E-isomer, pharmaceutically acceptable salt, or prodrug thereof, either alone or in combination with at least one therapeutic agent which is not a compound of formula I or II as described above, or the corresponding Z- or E-isomer, pharmaceutically acceptable salt, or prodrug thereof. The compounds or salts thereof and the pharmaceutical composition containing such compounds or salts herein are for use in the treatment of a patient with cancer.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0019] FIG. 1 shows chemical structures of Ras-inhibitory compounds identified by screening.

[0020] FIG. 2 depicts the results of a Ras Binding Domain (RBD) pulldown assay paired with Western blot showing the relative levels of Ras activation in a panel of colorectal cancer cell lines. [0021] FIGS. 3 A-3E illustrate antitumor cell growth inhibitory activities of representative compounds of formula I or II against HT-29 colon cancer cells (which lack activated Ras) compared to HCT-116 colon cancer cells (which harbor activated Ras). The data are shown for compounds 083 (FIG 3A), 084 (FIG. 3B), 088 (FIG. 3C), 260 (FIG. 3D) and 270 (FIG. 3E).

[0022] FIGS. 4A-4E illustrate antitumor cell growth inhibitory activities of

representative compounds of formula I or II against HT-29 colon cancer cells (which lack activated Ras) compared to A549 lung cancer cells (which harbor activated Ras). The data are shown for compounds 083 (FIG 4A), 084 (FIG. 4B), 088 (FIG. 4C), 260 (FIG. 4D) and 270 (FIG. 4E).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with an embodiment, the invention provides a compound of I or II:

[0024] wherein:

[0025] R and Ro are independently selected from hydrogen, hydroxyl, alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, alkenyl, alkenylalkyl, alkynyl, alkynylalkyl, cyano, cyanoalkyl, halogen, azido, alkoxy, haloalkyl and a substituted or unsubstituted group selected from aryl, arylalkyl, aryloxy, aminocarbonyl, aminocarbonylalkyl, aminosulfonyl, aminosulfonylalkyl, alkylcarbonyl, alkylcarbonylalkyl, amino, alkylamino, dialkylamino, aminocarbonylalkylamino, and carbocyclylamino, carbocyclylalkylamino, heterocyclylamino and heterocyclylalkylamino wherein the ring structures are saturated or unsaturated; or R and Ro together is double-bonded oxygen or double-bonded sulfur, or R and Ro together is a double-bonded nitrogen bonded to one of hydrogen, hydroxyl, alkyl, or haloalkyl, or R and Ro together is a double-bonded carbon bonded to two substituents independently selected from hydrogen, hydroxyl, alkyl and haloalkyl, or R and Ro together is a substituted or unsubstituted, saturated or unsaturated 3-, 4-, 5-, 6- or 7-membered heterocyclic ring, or Ro is nitrogen which is part of a substituted or unsubstituted, saturated or unsaturated, 3-, 4-, 5-, 6- or 7- membered heterocyclic ring;

[0026] n is 0, 1 or 2;

[0027] Ri, R ₂ , R ₃ , and R4 are independently selected from hydrogen, halogen, alkyl, cycloalkyl, haloalkyl, hydroxyl, carboxyl, alkoxy, formyloxy, alkylcarbonyloxy,

hydroxyalkyl, aldehydo, amino, alkylamino, aminoalkyl, alkylaminoalkyl, dialkylamino, mercapto, alkylmercapto, cyano, cyanoalkyl, nitro, azido, and substituted or unsubstituted groups selected from alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido,

alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl, alkylcarbonyloxy, arylcarbonyloxy, arylalkylcarbonyloxy, aryloxycarbonyloxy, heterocyclylcarbonyloxy,

heterocyclylalkylcarbonyloxy, phosphonooxy, phosphonoalkyloxy and sulfonamido, or any two of Ri, R ₂ , R ₃ , and R ₄ form an alkylenedioxy group;

[0028] R ₇ , R _8; R9 and Rio are independently selected from hydrogen, alkyl, haloalkyl, and alkoxy;

[0029] Y is hydrogen, alkyl, or haloalkyl, and Y' is hydrogen, alkyl, haloalkyl, amino, alkylamino, or alkoxy, or Y and Y together is double-bonded oxygen or double-bonded sulfur, or Y and Y together is a double-bonded nitrogen bonded to hydrogen, hydroxyl, alkyl, or haloalkyl;

[0030] X is selected from hydrogen, alkyl, cycloalkyl, haloalkyl, alkoxy, alkylmercapto, and hydroxyl, or X is R'R", where R and R" are independently selected from the group consisting of hydrogen, hydroxyl, alkyl, aryloxy, cyanoalkyl, haloalkyl, alkoxy, alkenyl, alkynyl, hydroxyalkyl, polyhydroxyalkyl, alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, alkylamino, aryl, aryloxy, arylalkyl, arylalkenyl, arylcycloalkyl, arylcycloalkenyl, carbocyclyl, and carbocycloalkyl where the carbocycle of the carbocyclyl and the

carbocycloalkyl is selected from 7-membered carbocyclic rings containing no double bond, or one, two or three double bonds, 6-membered carbocyclic rings containing no double bond, or one, two or three double bonds, 5-membered carbocyclic rings containing no double bond, or one or two double bonds, 4-membered carbocyclic rings containing no double bond or one double bond and 3 -membered carbocyclic rings containing no double bond, heterocyclyl, and heterocyclylalkyl, where the heterocycle of the heterocyclyl and heterocyclylalkyl is selected from 7-membered heterocyclic rings, 6-membered heterocyclic rings, and 5-membered heterocyclic rings, and the aryl of the aryl, arylalkyl, arylalkylenyl, arylcycloalkyl, or arylcycloalkenyl structure or the carbocyclic or heterocyclic structure may optionally be substituted with one or more of halo, alkyl, haloalkyl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano, oxo,

alkylcarbonyloxy, sulfonamido and CORn, wherein Rn is selected from hydrogen, amino, alkyl, haloalkyl, alkoxy, alkylmercapto, and aryl; or R' and R" together form a 5-, 6- or 7- membered, saturated or unsaturated, heterocyclic ring containing at least one nitrogen and optionally oxygen or sulfur, and the heterocyclic ring may optionally be substituted with one or more of halo, alkyl, haloalkyl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano, oxo, alkylcarbonyloxy, and sulfonamido; and

[0031] E is a substituted or unsubstituted, saturated or unsaturated, 7- membered, 6- membered, 5-membered or 4-membered carbocyclic or heterocyclic ring; or

[0032] a pharmaceutically acceptable salt thereof or a prodrug thereof.

In an embodiment, E is a carbocyclic or heterocyclic ring, optionally substituted with one or more substituents selected from hydroxyl, halogen, alkyl, alkenyl, haloalkyl, carboxyl, cyano, cyanoalkyl, nitro, oxo, imino, hydroxyimino, alkylimino, haloalkylimino, alkenyl, alkylalkenyl, haloalkenyl, hydroxyalkenyl, alkoxy, formyloxy, amino, alkylamino, dialkylamino, aminoalkyl, alkylaminoalkyl, arylalkylamino, hydroxyalkyl, aldehydo, mercapto, alkylmercapto, azido, and substituted or unsubstituted groups selected from alkylsulfonyl, alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, alkylcarbonyloxy, alkylcarbonyloxyalkyloxy, aminocarbonyloxyalkyloxy, arylcarbonyloxy,

arylalkylcarbonyloxy, aryloxycarbonyloxy, heterocyclylcarbonyloxy,

heterocyclylalkylcarbonyloxy, phosphonooxy, phosphonoalkyloxy, carbamate, carbamido, alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl, sulfonamido, and alkylenedioxy spanning two substituent positions.

[0033] In an embodiment, E is selected from 7-membered carbocyclic rings containing no double bond, or one, two or three double bonds, 7-membered heterocyclic rings containing one, two or three nitrogen atoms and no double bond, or one, two or three double bonds, 6- membered carbocyclic rings containing no double bond, or one or two double bonds, 6- membered heterocyclic rings containing one, two, or three nitrogen atoms and no double bond, or one or two double bonds, 5-membered carbocyclic rings containing no double bond, or one or two double bonds, and 5-membered heterocyclic rings containing one or two nitrogen atoms and no double bond, or one or two double bonds, and 4-membered carbocyclic rings containing no double bond or one double bond, each of said ring is substituted or unsubstituted.

[0034] For example, E can be

[0035] wherein

[0036] V is hydrogen, alkyl or haloalkyl, and V is hydrogen, alkyl, haloalkyl, amino, alkylamino, or alkoxy, or V and V together is double-bonded oxygen or double-bonded sulfur, or V and V together is a double-bonded nitrogen bonded to one of hyrdrogen, hydroxy, alkyl, or haloalkyl, or carbon bonded to two substituents independently selected from hydrogen, hydroxyl, alkyl, and haloalkyl, or V and V together form an alkylenedioxy group; and

[0037] Ri2, Ri3, Ri4, Ri5, Ri6, Ri7, Ri9, and R ₂ o are independently selected from hydrogen, hydroxyl, halogen, alkyl, haloalkyl, carboxyl, cyano, cyanoalkyl, nitro, alkoxy, formyloxy, amino, dialkylamino, aminoalkyl, alkylaminoalkyl, arylalkylamino, hydroxyalkyl, aldehydo, alkylamino, mercapto, alkylmercapto, azido, and substituted or unsubstituted groups selected from alkylsulfonyl, alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, alkylcarbonyloxy, carbamate, carbamido, alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl, and

sulfonamide

[0038] In any of the foregoing embodiments, X is R'R", in which R is selected from alkyl, trifluoromethyl, alkenyl, alkynyl, hydroxyalkyl, alkylaminoalkyl, dialkylaminoalkyl, arylalkyl (e.g., benzyl, phenylalkyl), indanyl, heterocyclyl, and heterocyclylalkyl, where the heterocycle is selected from pyridinyl, furanyl, pyrrolyl, thiophenyl, and imidazolyl, and the cyclic structure of heterocyclyl and heterocyclylalkyl is optionally substituted with one or more of halo, alkyl, trifluoromethyl, hydroxy, alkoxy, amino, alkylamino, dialkylamino, mercapto, alkylmercapto, and carboxamido; R" is selected from hydrogen, alkyl, trifluoromethyl, cyanoalkyl, and dialkylaminoalkyl, or R and R" together form a 5-, 6-, or 7- member heterocyclic ring, saturated or unsaturated, substituted or unsubstituted, that contains at least one nitrogen and optionally oxygen.

[0039] In a preferred embodiment, X is R'R", in which R' is selected from

alkylaminoalkyl, dialkylaminoalkyl, arylalkyl, heterocyclyl, and heterocyclylalkyl where the heterocycle is selected from furanyl, pyrrolyl, and thiophenyl, and the cyclic structure of heterocyclyl and heterocyclylalkyl is optionally substituted with one or more of halo, alkyl, trifluoromethyl, hydroxy, alkoxy, amino, alkylamino, and dialkylamino; R" is selected from hydrogen, alkyl, trifluoromethyl or dialkylaminoalkyl, or R and R" together form a 5, 6, or 7- member heterocyclic ring, saturated or unsaturated, substituted or unsubstituted, that contains at least one nitrogen and optionally oxygen. More preferably, X is NR'R", in which R is selected from dialkylaminoalkyl, arylalkyl, benzyl, heterocyclyl, and heterocyclylalkyl where the heterocycle of the heterocyclyl and heterocyclylalkyl is selected from pyridinyl, furanyl, and pyrrolyl, and the cyclic structure may optionally be substituted with one or more of halo, alkyl, trifluoromethyl, alkoxy, alkylamino and dialkylamino; and R" is selected from hydrogen, alkyl, trifluoromethyl or dialkylaminoalkyl. Even more preferably, X is NR'R", in which R is benzyl, or a heterocyclyl or heterocyclylalkyl selected from 2-pyridinylmethyl, 3- pyridinylmethyl, 2-furanyl, 2-furanylmethyl, 3 -furanyl, 3-furanylmethyl, 2-pyrrolylmethyl, and (l-methyl-lH-pyrrol-2-yl)methyl; and R" is hydrogen. A compound in which X is NR'R" and R is heterocyclyl or heterocyclylalkyl selected 2-pyridinylmethyl, 3-pyridinylmethyl, 2- furanylmethyl, (lH-pyrrol-2-yl)methyl, and (l-methyl-lH-pyrrol-2-yl)methyl; and R" is hydrogen is especially preferred.

[0040] In any of the foregoing embodiments, R and R ₀ are independently selected from hydrogen and hydroxyl; Ri, R ₂ , R ₃ and R ₄ are independently selected from halogen, alkoxy, alkyl and trifluoromethyl; n is 1; and Ri ₂ , Ri ₄ , R½, Rn, Ris and R19 are independently selected from hydrogen, halogen, alkyl, trifluoromethyl, hydroxyl, alkoxy, formyloxy,

alkylcarbonyloxy, hydroxyalkyl, aldehydo, amino, alkylamino, aminoalkyl, alkylaminoalkyl, dialkylamino, mercapto, alkylmercapto, azido, and substituted or unsubstituted groups selected from alkylsulfonyl, alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido, and sulfonamido, or any two of R12, Ri ₄ , Ri6, Rn, Ris and R19 form an

alkylenedioxy group. [0041] In an embodiment, Ri, R ₂ , R ₃ and R ₄ are independently selected from halogen, alkoxy, alkyl and trifluorom ethyl; three of R12, R14, R½, Rn, Ris and R19 are independently selected from hydrogen, halogen, alkyl, trifluoromethyl, hydroxyl, alkoxy, formyloxy, alkylcarbonyloxy, hydroxyalkyl, aldehydo, amino, alkylamino, aminoalkyl, alkylaminoalkyl, dialkylamino, mercapto, alkylmercapto, azido, and substituted or unsubstituted groups selected from alkylsulfonyl, alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido, and sulfonamido, and one of R _i2 , R ₁₄ , R½, Rn, Ri ₈ and Ri ₉ is independently selected from hydroxyl, hydroxyalkyl, amino, alkylamino, dialkylamino, mercapto, and alkylmercapto. Preferably, three of R12, R14, Ri6, Rn, Ri ₈ and R19 are independently selected from hydrogen, halogen, alkyl, trifluoromethyl, alkoxy, amino, alkylamino, aminoalkyl, alkylaminoalkyl, dialkylamino, mercapto, and alkylmercapto, and one of R _i2 , R ₁₄ , R½, R ₁₇ , Ri ₈ and Rig is independently selected from hydroxyl, hydroxyalkyl, aldehydo, amino and alkylamino, dialkylamino, mercapto, and alkylmercapto, and R ₈ is methyl.

[0042] In any of the foregoing embodiments, R ₂ is selected from halogen, alkoxy and alkylmercapto, Ri and R ₃ are hydrogen; and three of R12, R14, Ri6, Rn, Ri ₈ and R19 are independently selected from hydrogen, halogen, alkyl, trifluoromethyl, alkoxy, alkylamino, alkylaminoalkyl, dialkylamino, and alkylmercapto, and one of R12, R14, Ri6, Rn, Ri ₈ and R19 is independently selected from hydroxyl, hydroxyalkyl, amino, alkylamino, dialkylamino, mercapto, and alkylmercapto. Preferably, R ₂ is selected from halogen (e.g., fluoro) and alkoxy (e.g., methoxy), and Ri and R ₃ are hydrogen.

[0043] Structures and identification numbers of exemplary compounds of formula I or II are set forth as follows. The corresponding Z- or E-isomers thereof, prodrugs, or salts of these compounds are also envisioned.

12

13

14

15

[0044] The IUPAC names for the corresponding structures as as follows:

[0045] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- m ethyl- lH-inden- 1 -ylidene)-N-(( 1 -methyl- lH-pyrrol -2 -yl)methyl)acetamide (080),

[0046] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-ylidene)-N-(pyridin-2-ylmethyl)acetamide (081),

[0047] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-ylidene)-N-(pyridin-2-ylmethyl)acetamide (082),

[0048] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)acetamide (083),

[0049] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)acetamide (084),

[0050] 2-(6-fluoro-3-((4-imino-3,5-dimethoxycyclohexa-2,5-dien-l-yl idene)methyl)-2- methyl-lH-inden-l-ylidene)-N-(pyridin-3-ylmethyl)acetamide (085),

[0051] 2-(6-fluoro-3-((Z)-((Z)-4-(hydroxyimino)-3,5-dimethoxycycloh exa-2,5-dien-l- ylidene)methyl)-2-methyl-lH-inden-l-ylidene)-N-(pyridin-3-yl methyl)acetamide (086) [0052] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)acetamide (087),

[0053] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)propanamide (088),

[0054] 2-(6-fluoro-3-((4-imino-3,5-dimethoxycyclohexa-2,5-dien-l-yl idene)methyl)-2- methyl-lH-inden-l-yl)-N-(pyridin-2-ylmethyl)propanamide (089), [0055] N-benzyl-2-(6-fluoro-3-((Z)-((Z)-4-(hydroxyimino)-3,5-dimeth oxycyclohexa-2,5- dien- 1 -ylidene)methyl)-2-methyl- IH-inden- 1 -yl)propanamide (090),

[0056] 2-(6-fluoro-3-((Z)-((Z)-4-(hydroxyimino)-3,5-dimethoxycycloh exa-2,5-dien-l- ylidene)methyl)-l,2-dimethyl-lH-inden-l-yl)-N-(pyridin-3-ylm ethyl)acetamide (091),

[0057] 2-(3-((3,5-dimethoxy-4-(propan-2-ylidene)cyclohexa-2,5-dien- l-ylidene)methyl)-

6-fluoro- 1 ,2-dimethyl- IH-inden- 1 -yl)-N-(furan-2-ylmethyl)acetamide (092),

[0058] 2-(3-((3,5-dimethoxy-4-(propan-2-ylidene)cyclohexa-2,5-dien- l-ylidene)methyl)-

6-fluoro- 1 ,2-dimethyl- IH-inden- 1 -yl)-N-(pyridin-3 -ylmethyl)acetamide (093),

[0059] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-ylidene)-N-phenoxyacetamide (157),

[0060] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-yl)-N-phenoxypropanamide (158),

[0061] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)-N-(trifluoro methyl)acetamide (159),

[0062] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)-N-(trifluoromethy l)propanamide (160),

[0063] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)-N-(trifluoro methyl)acetamide (161),

[0064] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)-N-(trifluoromethy l)propanamide (162),

[0065] N-benzyl-2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylid ene)methyl)-6- fluoro-2-methyl-lH-inden-l-ylidene)-N-methylacetamide (163),

[0066] N-benzyl-2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylid ene)methyl)-6- fluoro-2-methyl-lH-inden-l-yl)-N-methylpropanamide (164),

[0067] 2-(6-cyano-3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylide ne)methyl)-2- methyl-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)acetamide (165),

[0068] 2-(6-cyano-3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylide ne)methyl)-2- methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)propanamide (166),

[0069] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-2-methyl-6- (trifluoromethyl)-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)ac etamide (167),

[0070] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-2-methyl-6- (trifluoromethyl)-lH-inden-l-yl)-N-(furan-2-ylmethyl)propana mide (168), [0071] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)propanamide (170),

[0072] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)-N-methylpropanami de (171),

[0073] N-(furan-2-ylmethyl)-2-(3-((4-hydroxy-3,5-dimethoxy-4-methyl cyclohexa-2,5- dien- 1 -ylidene)methyl)-6-m ethoxy-2-m ethyl- lH-inden- 1 -ylidene)acetamide (172),

[0074] (Z)-2-(3-((3,5-dimethoxy-6-methyl-4-oxocyclohex-2-en-l-ylide ne)methyl)-6- methoxy-2-methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)propanam ide (176),

[0075] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- methoxy-2-methyl-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)ace tamide (177),

[0076] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- methoxy-2-methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)acetamid e (178),

[0077] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- fluoro-2-methyl-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)acet amide (179),

[0078] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- fluoro-2-methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)acetamide (180),

[0079] 2-(3 -((3 , 5 -dimethoxy-4-oxocy clohexa-2, 5 -dien- 1 -ylidene)methyl)-6-fluoro- 1,2- dimethyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)acetamide (181),

[0080] 2-(6-fluoro-3-((4-hydroxy-3,5-dimethoxy-4-methylcyclohexa-2, 5-dien-l- ylidene)methyl)-2-methyl-lH-inden-l-ylidene)-N-(furan-2-ylme thyl)acetamide (182),

[0081] (Z)-2-(3-((3,5-dimethoxy-6-methyl-4-oxocyclohex-2-en-l-ylide ne)methyl)-6- fluoro-2-methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)propanami de (185),

[0082] N-(( 1 H-pyrrol-2-yl)methyl)-2-(3 -((3,5 -dimethoxy-4-oxocy clohexa-2, 5 -dien- 1 - ylidene)methyl)-6-methoxy-2-methyl-lH-inden-l-ylidene)acetam ide (186),

[0083] N-(( 1 H-pyrrol-2-yl)methyl)-2-(3 -((3,5 -dimethoxy-4-oxocy clohexa-2, 5 -dien- 1 - ylidene)methyl)-6-methoxy-2-methyl-lH-inden-l-yl)propanamide (187),

[0084] N-((lH-pyrrol-2-yl)methyl)-2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9- dien-8-ylidene)methyl)-6-fluoro-2-methyl-lH-inden-l-ylidene) acetamide (188),

[0085] N-((lH-pyrrol-2-yl)methyl)-2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9- dien-8-ylidene)methyl)-6-fluoro-2-methyl-lH-inden-l-yl)aceta mide (189),

[0086] N-(( 1 H-pyrrol-2-yl)methyl)-2-(3 -((3,5 -dimethoxy-4-oxocy clohexa-2, 5 -dien- 1 - ylidene)methyl)-6-fluoro-2-methyl-lH-inden-l-yl)acetamide (190), [0087] N-(( 1 H-pyrrol-2-yl)methyl)-2-(3 -((3,5 -dimethoxy-4-oxocy clohexa-2, 5 -dien- 1 - ylidene)methyl)-6-fluoro-2-methyl-lH-inden-l-yl)propanamide (191),

[0088] N-(( 1 H-pyrrol-2-yl)methyl)-2-(3 -((3,5 -dimethoxy-4-oxocy clohexa-2, 5 -dien- 1 - ylidene)methyl)-6-fluoro-2-methyl-lH-inden-l-ylidene)acetami de (192),

[0089] 2-(6-fluoro-3-((4-hydroxy-3,5-dimethoxy-4-methylcyclohexa-2, 5-dien-l- ylidene)methyl)-2-methyl- IH-inden- 1 -ylidene)-N-(( 1 -methyl- lH-pyrrol-2- yl)methyl)acetamide (193),

[0090] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-yl)-N-((l -methyl- IH-pyrrol -2 -yl)methyl)propanamide(194),

[0091] (Z)-2-(3-((3,5-dimethoxy-6-methyl-4-oxocyclohex-2-en-l-ylide ne)methyl)-6- fluoro-2-methyl-lH-inden-l-yl)-N-((l -methyl- IH-pyrrol -2 -yl)methyl)propanamide (197),

[0092] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- fluoro-2-methyl-lH-inden-l-ylidene)-N-((l -methyl- lH-pyrrol-2-yl)methyl)acetamide (198),

[0093] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- fluoro-2-methyl- IH-inden- 1 -yl)-N-(( 1 -methyl- lH-pyrrol-2-yl)methyl)acetamide (199),

[0094] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-yl)-N-((l-methyl-lH-pyrrol-2-yl)methyl)ace tamide (200),

[0095] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)-N-methylacet amide (220),

[0096] 3 -(3 -((3 , 5 -dimethoxy-4-oxocy clohexa-2, 5 -dien- 1 -ylidene)methyl)-6-fluoro-2- methyl-lH-inden-l-yl)-4-((furan-2-ylmethyl)amino)-4-oxobutan oic acid (222),

[0097] 3 -(3 -((3 , 5 -dimethoxy-4-oxocy clohexa-2, 5 -dien- 1 -ylidene)methyl)-6-fluoro-2- m ethyl- IH-inden- 1 -yl)- 1 -(furan-2-ylmethyl)pyrrolidine-2, 5-dione (223),

[0098] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)acetamide (224),

[0099] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl)acetamide (225),

[0100] (Z)-3-(5-fluoro-l-(4-hydroxy-3,5-dimethoxybenzylidene)-2-met hyl-lH-inden-3- yl)-l-(furan-2-ylmethyl)pyrrolidine-2,5-dione (226),

[0101] 2-(6-fluoro-3-((Z)-((Z)-4-(hydroxyimino)-3,5-dimethoxycycloh exa-2,5-dien-l- ylidene)methyl)-2-methyl-lH-inden-l-yl)-N-(furan-2-ylmethyl) propanamide (227),

[0102] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-ylidene)-N-phenylacetamide (230), [0103] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-yl)-N-phenylpropanamide (231),

[0104] 2-(3-((4-hydroxy-3,5-dimethoxy-4-methylcyclohexa-2,5-dien-l- ylidene)methyl)- 6-methoxy-2-methyl-lH-inden-l-ylidene)-N-phenylacetamide (233),

[0105] (Z)-2-(3-((3,5-dimethoxy-6-methyl-4-oxocyclohex-2-en-l-ylide ne)methyl)-6- methoxy-2-methyl-lH-inden-l-yl)-N-phenylpropanamide (237),

[0106] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-ylidene)-N-(3-methoxyphenyl)acetamide (240),

[0107] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden- 1 -yl)-N-(3 -methoxyphenyl)propanamide(241),

[0108] (Z)-2-(5-fluoro-l-(4-hydroxy-3,5-dimethoxybenzylidene)-2-met hyl-lH-inden-3- yl)-N-(furan-2-ylmethyl)propanamide (242),

[0109] 2-(3 -((3 , 5 -dimethoxy-4-oxocy clohexa-2, 5 -dien- 1 -ylidene)methyl)-6-fluoro-2- methyl-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)propanamide (243),

[0110] 2-(3 -((3 , 5 -dimethoxy-4-oxocy clohexa-2, 5 -dien- 1 -ylidene)methyl)-6-fluoro-2- methyl-lH-inden-l-yl)-2-(ethylamino)-N-(furan-2-ylmethyl)pro panamide (244),

[0111] N-(furan-2-ylmethyl)-2-(6-methoxy-2-methyl-3-((3,4,4,5-tetra methoxycyclohexa-

2, 5-dien- 1 -ylidene)m ethyl)- lH-inden- 1 -ylidene)acetamide (245),

[0112] N-(furan-2-ylmethyl)-2-(6-methoxy-2-methyl-3-((3,4,4,5-tetra methoxycyclohexa-

2, 5-dien- 1 -ylidene)methyl)- lH-inden- 1 -yl)acetamide (246),

[0113] 2-(6-fluoro-2-methy 1-3 -((3,4,4,5 -tetramethoxycy clohexa-2, 5 -dien- 1 - ylidene)methyl)-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)acet amide (247),

[0114] 2-(6-fluoro-2-methyl-3-((3,4,4,5-tetramethoxycyclohexa-2,5-d ien-l- ylidene)methyl)-lH-inden-l-yl)-N-(furan-2-ylmethyl)acetamide (248),

[0115] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-ylidene)-N-(l-methylpyrrolidin-3-yl)acetam ide (250),

[0116] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-yl)-N-((R)-l-methylpyrrolidin-3-yl)propana mide (251),

[0117] 2-(3-((4-hydroxy-3,5-dimethoxy-4-methylcyclohexa-2,5-dien-l- ylidene)methyl)-

6-methoxy-2-methyl-lH-inden-l-ylidene)-N-(l-methylpyrroli din-3-yl)acetamide (253)

[0118] (Z)-2-(3-((3,5-dimethoxy-6-methyl-4-oxocyclohex-2-en-l-ylide ne)methyl)-6- methoxy-2-methyl-lH-inden-l-yl)-N-(l-methylpyrrolidin-3-yl)p ropanamide (258), [0119] 2-(3 -((3 , 5 -dimethoxy-4-oxocy clohexa-2, 5 -dien- 1 -ylidene)methyl)-6-fluoro-2- methyl-lH-inden-l-ylidene)-N-(pyridin-3-ylmethyl)acetamide (260),

[0120] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-yl)-N-(pyridin-3-ylmethyl)propanamide (261),

[0121] 2-(6-fluoro-3-((4-hydroxy-3,5-dimethoxy-4-methylcyclohexa-2, 5-dien-l- ylidene)methyl)-2-methyl-lH-inden-l-ylidene)-N-(pyridin-3-yl methyl)acetamide (263),

[0122] (Z)-2-(3-((3,5-dimethoxy-6-methyl-4-oxocyclohex-2-en-l-ylide ne)methyl)-6- fluoro-2-methyl- IH-inden- 1 -yl)-N-(pyridin-3 -ylmethyl)propanamide (267),

[0123] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- fluoro-2-methyl-lH-inden-l-ylidene)-N-(pyridin-3-ylmethyl)ac etamide (268),

[0124] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- fluoro-2-methyl- IH-inden- 1 -yl)-N-(pyridin-3 -ylmethyl)acetamide (269),

[0125] N-benzyl-2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylid ene)methyl)-6- fluoro-2-methyl- IH-inden- 1 -ylidene)acetamide (270),

[0126] N-benzyl-2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylid ene)methyl)-6- fluoro-2-methyl- IH-inden- 1 -yl)propanamide (271),

[0127] N-benzyl-2-(6-fluoro-3-((4-hydroxy-3,5-dimethoxy-4-methylcyc lohexa-2,5-dien-

1 -ylidene)methyl)-2-m ethyl- IH-inden- 1 -ylidene)acetamide (273),

[0128] (Z)-N-benzyl-2-(3-((3,5-dimethoxy-6-methyl-4-oxocyclohex-2-e n-l- ylidene)methyl)-6-fluoro-2-methyl-lH-inden-l-yl)propanamide (277),

[0129] N-benzyl-2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8- ylidene)methyl)-6-fluoro-2-m ethyl- IH-inden- 1 -ylidene)acetamide (278),

[0130] N-benzyl-2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8- ylidene)methyl)-6-fluoro-2-methyl-lH-inden-l-yl)acetamide (279),

[0131] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-yl)-N-(pyridin-2-ylmethyl)propanamide (280),

[0132] 2-(6-fluoro-3-((4-hydroxy-3,5-dimethoxy-4-methylcyclohexa-2, 5-dien-l- ylidene)methyl)-2-methyl-lH-inden-l-ylidene)-N-(pyridin-2-yl methyl)acetamide (282),

[0133] (Z)-2-(3-((3,5-dimethoxy-6-methyl-4-oxocyclohex-2-en-l-ylide ne)methyl)-6- fluoro-2-methyl- IH-inden- 1 -yl)-N-(pyridin-2-ylmethyl)propanamide (286),

[0134] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- fluoro-2-methyl-lH-inden-l-ylidene)-N-(pyridin-2-ylmethyl)ac etamide (287), [0135] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- fluoro-2-methyl- lH-inden- 1 -yl)-N-(pyridin-2-ylmethyl)acetamide (288),

[0136] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-yl)-N-(pyridin-2-ylmethyl)acetamide (289),

[0137] N-benzyl-2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylid ene)methyl)-6- fluoro-2-methyl- lH-inden- 1 -yl)acetamide (290),

[0138] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-yl)-N-(pyridin-3-ylmethyl)acetamide (291),

[0139] 2-(6-fluoro-2-methy 1-3 -((3,4,4,5 -tetramethoxy cy clohexa-2, 5 -dien- 1 - ylidene)methyl)-lH-inden-l-ylidene)-N-(pyridin-2-ylmethyl)ac etamide (292),

[0140] 2-(6-fluoro-2-methyl-3-((3,4,4,5-tetramethoxycyclohexa-2,5-d ien-l- ylidene)methyl)- lH-inden- 1 -yl)-N-(pyridin-2-ylmethyl)acetamide (293),

[0141] 2-(6-fluoro-2-methyl-3-((3,4,4,5-tetramethoxycyclohexa-2,5-d ien-l- ylidene)methyl)-lH-inden-l-ylidene)-N-(pyridin-3-ylmethyl)ac etamide (294),

[0142] 2-(6-fluoro-2-methyl-3-((3,4,4,5-tetramethoxycyclohexa-2,5-d ien-l- ylidene)methyl)- lH-inden- 1 -yl)-N-(pyridin-3 -ylmethyl)acetamide (295),

[0143] N-benzyl-2-(6-fluoro-2-methyl-3-((3,4,4,5-tetramethoxycycloh exa-2,5-dien-l- ylidene)methyl)-lH-inden-l-ylidene)acetamide (296),

[0144] N-benzyl-2-(6-fluoro-2-methyl-3-((3,4,4,5-tetramethoxycycloh exa-2,5-dien-l- ylidene)methyl)-lH-inden-l-yl)acetamide (297),

[0145] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl-lH-inden-l-ylidene)-N-(pyridin-3-yl)acetamide(298),

[0146] 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-methoxy-2- methyl- lH-inden- 1 -yl)-2-(ethylthio)-N-(pyridin-3 -yl)acetamide (299),

[0147] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- methoxy-2-methyl-lH-inden-l-ylidene)-N-(pyridin-3-yl)acetami de (300),

[0148] 2-(3-((6, 10-dimethoxy-l,4-dioxaspiro[4.5]deca-6,9-dien-8-ylidene)meth yl)-6- methoxy-2-methyl-lH-inden-l-yl)-N-(pyridin-3-yl)acetamide (301).

[0149] As used herein, the "alkyl" part of any of the substituents described herein, e.g., but not limited to, alkyl, alkoxy, alkylamino, alkylmercapto, hydroxyalkyl, polyhydroxyalkyl, alkylaminoalkyl, aminoalkyl, arylalkyl, arylcycloalkyl, heterocyclylalkyl, arylalkylenyl, arylcycloalkyl, dialkylamino, alkylcarbonyloxy, dialkylaminoalkyl, cyanoalkyl, haloalkyl, alkylcarbonylalkylcarbonyloxy, dialkylalkylaminoalkyl, alkylsulfonyl, alkylsulfinyl, alkylsulfinyloxy, alkylsulfonyloxy, alkylenedioxy, carbocycloalkyl, and phenylalkyl, means a straight-chain or branched-chain saturated alkyl which can contain from 1-20 carbon atoms, for example from 1 to about 10 carbon atoms, or from 1 to about 8 carbon atoms, or, preferably, lower alkyl, i.e., from 1 to 6 carbon atoms.

[0150] Examples of alkyls include methyl, ethyl, propyl, isopropyl, «-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, octyl, dodecanyl, octadecyl, and the like. Alkyl substituents can be unsubstituted or substituted, for example with at least one substituent selected from the group consisting of a halogen, a nitro, an amino, a hydroxyl, a thio, an acyl, a mercapto, and a cyano.

[0151] The term "alkenyl" means a straight-chain or branched-chain alkenyl having one or more double bonds. Unless otherwise specified, the alkenyl can contain from 2 to about 10 carbon atoms, for example from 2 to about 8 carbon atoms, or preferably from 2 to about 6 carbon atoms. Examples of alkenyls include vinyl, allyl, 1,4-butadienyl, and isopropenyl substituents, and the like.

[0152] The term "alkynyl" means a straight-chain or branched-chain alkynyl having one or more triple bonds. Unless otherwise specified, alkynyls can contain from 2 to about 10 carbon atoms, for example, from 2 to about 8 carbon atoms, or preferably, from 2 to about 6 carbon atoms. Examples of alkynyls include ethynyl, propynyl (propargyl), butynyl, and the like. Alkenyl or alkynyl substituents can be unsubstituted or substituted, for example, with at least one substituent selected from the group consisting of a halogen, a nitro, an amino, a hydroxyl, a thio, an acyl, an alkyl, and a cyano.

[0153] The term "aryl" means an aromatic carbocyclic radical, as commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl and naphthyl rings. Preferably, the aryl comprises one or more six-membered rings including, for example, phenyl, naphthyl, biphenyl, and the like. Typically, the aryl comprises six or more carbon atoms in the ring skeleton thereof (e.g., from 6 to about 10 carbon atoms making up the ring). Unless specified otherwise, "aryl" by itself refers to unsubstituted aryl groups and does not cover substituted aryl groups. Substituted aryl can be an aryl substituted, for example, with at least one substituent selected from the group consisting of a halogen, a nitro, an amino, a hydroxyl, a thio, an acyl, and alkyl, and a cyano. It is to be noted that arylalkyl, benzyl, or heteroaryl groups are not considered "aryl" in accordance with the present invention. [0154] In accordance with the invention, the term "heteroaryl" refers to a cyclic aromatic radical having from five to ten ring atoms of which at least one atom is O, S, or N, and the remaining atoms are carbon. Examples of heteroaryl radicals include pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, and isoquinolinyl.

[0155] In further accordance with the invention, the term "heterocyclyl" refers to a stable, saturated, partially unsaturated or unsaturated monocyclic, bicyclic, or spiro ring system containing 3 to 7 ring members of carbon atoms and other atoms selected from nitrogen, sulfur, and/or oxygen. The term "heterocyclyl" includes "heteroaryl groups. Preferably, a heterocyclyl is a 5, 6, or 7-membered monocyclic ring and contains one, two, or three heteroatoms selected from nitrogen, oxygen, and/or sulfur. The heterocyclyl may be attached alone or via an alkyl linker (thus becoming a "heterocyclylalkyl") to the parent structure through a carbon atom or through any heteroatom of the heterocyclyl that results in a stable structure. Examples of such heterocyclyl rings are isoxazolyl, thiazolinyl, imidazolidinyl, piperazinyl, homopiperazinyl, pyrrolyl, pyrrolinyl, pyrazolyl, pyranyl, piperidyl, oxazolyl, and morpholinyl.

[0156] Whenever a range of the number of atoms in a structure is indicated (e.g., a C _1-12 , Ci-8, Ci-6, or Ci-4 alkyl, alkylamino, etc.), it is specifically contemplated that any sub-range or individual number of carbon atoms falling within the indicated range also can be used. Thus, for instance, the recitation of a range of 1-8 carbon atoms (e.g., Ci-C ₈ ), 1-6 carbon atoms (e.g., Ci-C ₆ ), 1-4 carbon atoms (e.g., C ₁ -C4), 1-3 carbon atoms (e.g., C ₁ -C3), or 2-8 carbon atoms (e.g., C ₂ -C ₈ ) as used with respect to any chemical group (e.g., alkyl, alkylamino, etc.) referenced herein encompasses and specifically describes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12 carbon atoms, as appropriate, as well as any sub-range thereof (e.g., 1-2 carbon atoms, 1-3 carbon atoms, 1-4 carbon atoms, 1-5 carbon atoms, 1-6 carbon atoms, 1-7 carbon atoms, 1-8 carbon atoms, 1-9 carbon atoms, 1-10 carbon atoms, 1-11 carbon atoms, 1-12 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5 carbon atoms, 2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms, 2-9 carbon atoms, 2-10 carbon atoms, 2-11 carbon atoms, 2- 12 carbon atoms, 3-4 carbon atoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8 carbon atoms, 3-9 carbon atoms, 3-10 carbon atoms, 3-11 carbon atoms, 3-12 carbon atoms, 4-5 carbon atoms, 4-6 carbon atoms, 4-7 carbon atoms, 4-8 carbon atoms, 4-9 carbon atoms, 4-10 carbon atoms, 4-11 carbon atoms, and/or 4-12 carbon atoms, etc., as

appropriate). [0157] In any of the embodiments above, the phrase "salt" or "pharmaceutically acceptable salt" is intended to include nontoxic salts synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. For example, an inorganic acid (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, or hydrobromic acid), an organic acid (e.g., oxalic acid, malonic acid, citric acid, fumaric acid, lactic acid, malic acid, succinic acid, tartaric acid, acetic acid,

trifluoroacetic acid, gluconic acid, ascorbic acid, methylsulfonic acid, or benzylsulfonic acid), an inorganic base (e.g., sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or ammonium hydroxide), an organic base(e.g., methylamine, diethylamine, triethylamine, triethanolamine, ethylenediamine,

tris(hydroxymethyl)methylamine, guanidine, choline, or cinchonine), or an amino acid (e.g., lysine, arginine, or alanine) can be used. Generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, PA, 1990, p. 1445, and Journal of Pharmaceutical Science, 66, 2-19 (1977). For example, they can be a salt of an alkali metal (e.g., sodium or potassium), alkaline earth metal (e.g., calcium), or ammonium of salt.

[0158] A compound of the present invention can also be provided as a prodrug, which is a drug derivative or drug precursor compound that typically is inactive or less than fully active until it is converted in the body through a normal metabolic process such as, for example, hydrolysis of an ester or amide form of the drug, to the active drug. A prodrug may be selected and used instead of the parent drug because, for example, in its prodrug form it is less toxic, and/or may have better absorption, distribution, metabolism and excretion

(ADME) characteristics, and the like, than the parent drug. A prodrug might also be used to improve how selectively the drug interacts with cells or processes that are not its intended target. This approach may be employed particularly, for example, to prevent or decrease adverse effects, especially in cancer treatments, which may be especially prone to having severe unintended and undesirable side effects.

[0159] The term "prodrug" denotes a derivative of a compound, which derivative, when administered to warm-blooded animals, e.g. humans, is converted into the compound (drug). For example, the enzymatic and/or chemical hydrolytic cleavage of a derivative compound of the present invention occurs in such a manner that the proven drug form is released, and the moiety or moieties split off remain nontoxic or are metabolized so that nontoxic metabolites are produced. For example, a carboxylic acid group can be esterified, e.g., with a methyl group or ethyl group to yield an ester. When an ester is administered to a subject, the ester is cleaved, enzymatically or non-enzymatically, reductively, oxidatively, or hydrolytically, to reveal the anionic group. An anionic group can be esterified with moieties (e.g.,

acyloxymethyl esters) which are cleaved to reveal an intermediate compound which subsequently decomposes to yield the active compound.

[0160] The prodrug can be prepared in situ during the isolation and purification of the compound of formula I or II, or by separately reacting the purified compound with a suitable derivatizing agent. For example, hydroxy groups can be converted into esters via treatment with a carboxylic acid in the presence of a catalyst. Examples of cleavable alcohol prodrug moieties include substituted or unsubstituted, branched or unbranched alkyl ester moieties, e.g., ethyl esters, alkenyl esters, di-alkylamino alkyl esters, e.g., dimethylaminoethyl ester, acylamino alkyl esters, acyloxy alkyl esters (e.g., pivaloyloxy methyl ester), aryl esters, e.g., phenyl ester, aryl-alkyl esters, e.g., benzyl ester, optionally substituted, e.g., with methyl, halo, or methoxy substituents aryl and aryl-alkyl esters, amides, alkyl amides, di-alkyl amides, and hydroxy amides.

[0161] Knowing the disclosures herein, it will be appreciated also that a compound of the present invention can be in the form of a prodrug, and that such prodrugs can be prepared using reagents and synthetic transformations that are well-known to those having ordinary skill in the art. The effectiveness of a particular prodrug can be determined using one or more analytical methods (e.g. pharmacokinetics, bioassays, in vivo efficacy studies, and the like) that are well-known to those of ordinary skill in the art.

[0162] More specifically, a prodrug of a compound of formulas I-II may be prepared using routine chemical procedures, such as the exemplary procedures described herein. For instance, any one of R ₁ -R4, " or any substituent on E of formula I or II can be of the

formula Q-U-, for example, wherein U is selected from the group consisting of oxygen, sulfur, nitrogen, OCH ₂ , SCH ₂ and HCH ₂ ; and Q is selected from the group consisting of aminoalkyl, PEG, PEG-CO, -OC(0)CH ₂ -(l-methyl-piperazin-4-yl), HCO, acetyl, amino acid, substituted benzoic acid and phosphoric acid; or, Q-U together is phosphonooxy, phosphonoalkyloxy, formyloxy, alkyloxy, alkylcarbonyloxy, alkylcarbonyloxyalkyloxy, aminocarbonyloxyalkyloxy, alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido, alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl,

arylcarbonyloxy, arylalkylcarbonyloxy, aryloxycarbonyloxy, heterocyclylcarbonyloxy and heterocy cly 1 alky 1 carb ony 1 oxy .

[0163] In a specific example, an exemplary prodrug of compound 083 can be:

[0164] wherein U is nitrogen and Q is selected from the group consisting of aminoalkyl, PEG, PEG-CO, -OC(0)CH ₂ -(l-methyl-piperazin-4-yl), HCO, acetyl, amino acid, substituted benzoic acid and phosphoric acid.

[0165] More specific examples of suitable prodrugs are depicted below for compounds 082, 084 nd 086, respectively:

[0166] In light of the disclosures of the present invention, it will be appreciated that the compounds of the present invention can be made by methods well-known to those of ordinary skill in the art, for example, by structurally modifying a given compound or by direct synthesis from available building blocks using routine synthetic transformations that are well- known in the art. See for example, Sperl et al., U.S. Patent Application Publication No. US 2003/0009033 Al, Jan. 9, 2003; Sperl et al., U.S. Patent No. 6,071,934, June 6, 2000; Sperl et al., International Publication No. WO 97/47303, Dec. 18, 1997; Whitehead et al., U.S. Patent Application Publication No. US 2003/0176316 Al, Sep. 18, 2003; Thompson et al., U.S. Patent No. 6,538,029 Bl, Mar. 25, 2003; Li et al., U.S. Patent Application Publication No. US 2003/0194750 Al, Oct. 16, 2003; Shen et al., U.S. Patent No. 3,888, 902; Alcade, et al., Org. Biomol. Chem., 6, 3795-3810 (2008); Magar and Lee, Org. Lett., 15, 4288-4291 (2013).

[0167] For instance, a key precursor compound for making a compound of formula I or II of the present invention can be synthesized according to the general approach depicted in Scheme I:

Scheme I

Dean-Stark; e. KOH, H ₂ 0; /. NaOCH ₃ , CH ₃ OH, E-CHO; g. (1) CDI, CH ₂ C1 ₂ , (2) R'R"NH, pyridine, warm

[0168] Detailed methods to achieve all of the synthesis steps depicted in Scheme I to make a desired substituted or unsubstituted indene derivative, are extensively documented in the published literature (e.g., see Sperl, et. al., 1997, 2000, 2003; Li, et. el., supra; Thompson, et. al., supra; Whitehead, et. al., supra; Shen, et al., supra; Alcade, et al., supra). In Scheme I the benzaldehyde building block used for step a, and/or the aldehyde building block (E-CHO) used for step and/or the primary or secondary amine (R'R"NH) building block used in step g can independently be unsubstituted, or substituted with any desired substituent(s) required to yield the desired final precursor compound (above) for making a compound of formula I or II of the present invention.

[0169] For example, the benzaldehyde building block as shown in Scheme I having the desired substituents at Ri, R ₂ , R ₃ and R ₄ can be purchased commercially and/or can be prepared routinely by methods well-known to those of ordinary skill in the art. For instance, such optional substituent(s) independently at Ri, R ₂ , R ₃ and R4 in Scheme I include but are not limited to hydrogen, halogen, alkyl, cycloalkyl, haloalkyl, hydroxyl, carboxyl, alkoxy, formyloxy, hydroxyalkyl, aldehyde, amino, alkylamino, aminoalkyl, alkylaminoalkyl, dialkylamino, mercapto, alkylmercapto, cyano, cyanoalkyl, nitro, azido, and substituted or unsubstituted groups selected from aryl, arylalkyl, aryloxy, alkylsulfinyloxy,

alkylsulfonyloxy, carbamate, carbamido, alkoxycarbonyl, alkylaminocarbonyl,

aminocarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyloxy, aminocarbonyloxyalkyloxy, arylcarbonyloxy, arylalkylcarbonyloxy, aryloxycarbonyloxy, heterocyclylcarbonyloxy, heterocyclylalkylcarbonyloxy, phosphonooxy, phosphonoalkyloxy and sulfonamido, or any two of Ri, R-2, R-3 and R4 form an alkylenedioxy group.

[0170] Likewise, the aldehyde building block (E-CHO) as shown in Scheme I having any desired group at E can be purchased and/or can be prepared by methods well-known to those of ordinary skill in the art. For example, such optional groups at E in Scheme I include but are not limited to any desired substituted or unsubstituted, saturated or unsaturated, 7- membered, 6-membered, 5-membered, 4-membered or 3-membered carbocyclic or heterocyclic ring. Substituents on said ring include one or more of hydrogen, halogen, alkyl, cycloalkyl, alkenyl, haloalkyl, hydroxyl, carboxyl, alkoxy, formyloxy, hydroxyalkyl, aldehyde, amino, alkylamino, aminoalkyl, alkylaminoalkyl, arylalkylamino, dialkylamino, mercapto, alkylmercapto, cyano, cyanoalkyl, nitro, azido, and substituted or unsubstituted groups selected from aryl, arylalkyl, aryloxy, alkylsulfinyloxy, alkylsulfonyloxy, carbamate, carbamido, alkoxycarbonyl, alkylaminocarbonyl, aminocarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyloxy, aminocarbonyloxyalkyloxy, arylcarbonyloxy,

arylalkylcarbonyloxy, aryloxycarbonyloxy, heterocyclylcarbonyloxy,

heterocyclylalkylcarbonyloxy, phosphonooxy, phosphonoalkyloxy and sulfonamido, or any two of Ri, R ₂ , R3 and R ₄ form an alkylenedioxy group.

[0171] Furthermore, the primary or secondary amine building block (R'R" H) as shown in Scheme I can be purchased commercially and/or can be prepared routinely by methods well-known to those of ordinary skill in the art. Such optional substituents independently at R and R" in Scheme I for example include but are not limited to hydrogen, hydroxyl, alkyl, cyanoalkyl, haloalkyl, alkoxy, alkenyl, alkynyl, hydroxyalkyl, polyhydroxyalkyl,

alkylaminoalkyl, dialkylaminoalkyl, aminoalkyl, aryl, aryloxy, arylalkyl, arylalkenyl, arylcycloalkyl, arylcycloalkenyl, carbocyclyl, and carbocycloalkyl where the carbocycle of the carbocyclyl and the carbocycloalkyl is selected from 7-membered carbocyclic rings containing no double bond, or one, two or three double bonds, 6-membered carbocyclic rings containing no double bond, or one or two double bonds, 5-membered carbocyclic rings containing no double bond, or one or two double bonds, 4-membered carbocyclic rings containing no double bond or one double bond and 3-membered carbocyclic rings containing no double bond, heterocyclyl, and heterocyclylalkyl, where the heterocycle of the

heterocyclyl and heterocyclylalkyl is selected from 7-membered heterocyclic rings, 6- membered heterocyclic rings, and 5-membered heterocyclic rings, and the aryl of the aryl, arylalkyl, arylalkylenyl, arylcycloalkyl, or arylcycloalkenyl structure or the carbocyclic or heterocyclic structure may optionally be substituted with one or more of halo, alkyl, haloalkyl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano, oxo, alkylcarbonyloxy, and sulfonamide and CORu wherein Rii is selected from hydrogen, amino, alkyl, haloalkyl, alkoxy, alkylmercapto, and aryl; or R and R" together form a 5-, 6- or 7-membered, saturated or unsaturated, heterocyclic ring containing at least one nitrogen, and optionally oxygen and/or sulfur, and the heterocyclic ring may optionally be substituted with one or more of halo, alkyl, haloalkyl, hydroxyl, alkoxy, amino, alkylamino, dialkylamino, mercapto, alkylmercapto, carboxamido, aldehydo, cyano, oxo, alkylcarbonyloxy, and sulfonamide.

[0172] As a more specific example, a particular precursor compound for making a compound of formulas I and II of the present invention can be synthesized according to the general approach depicted in Scheme II, which includes the key intermediate, a substituted indenyl acetic acid:

Scheme II

[0173] For certain substituents at Ri, R ₂ , R ₃ or R ₄ (e.g., in Scheme II above, R ₂ is fluoro), the starting material for preparation of the substituted indenyl acetic acid intermediate, may optionally be different than that shown in Schemes I and II, depending upon the nature of the substituent(s) desired on the intermediate and final product, and the optimum reaction conditions sought. For example, attachment of a cyano group at R ₂ can be accomplished using as starting material a cyano-substituted benzyl halide (e.g., as adapted from Shen, et. al., supra), as illustrated below. Scheme III

a. CH ₃ CH(C0 ₂ Et) ₂ , a, EtOH; b. NaOH, EtOH; c. H ₂ S0 ₄ , aq.; d. PPA, 83-90 °C, 2h; e. BrCH ₂ C0 ₂ CH ₃ , Zn(Hg), I ₂ , benzene; /. NaOH, Ethanol, warm g. NaOCH ₃ , CH ₃ OH, E-CHO; h. (1) CDI, CH ₂ C1 ₂ , (2) R'R"NH, pyridine, warm

[0174] A wide variety of substituents can be introduced at Ri, R ₂ , R ₃ and R ₄ of a

precursor compound for making a compound of formula I or II in the course of synthesis. In addition to the above-described Schemes I-III, the Scheme IV below illustrates yet another approach to making variations in substituents at Ri-R ₄ .

Scheme IV a.KNO _j , H ₂ S0 ₄ , 46%; b. NCCH ₂ C0 ₂ H, AcOH, AcONH ₄ , toluene, Dean-Stark; c. (1) K.OH, EtOH, H ₂ 0, (2) HOAc;

d. NaOCH ₃ , CH _j OH, E-CHO; e. (1) CDI, CH ₂ C1 ₂ , (2) R'R"NH, pyridine, warm;/. Zn, HOAc; g. standard procedure

Z = R'R", HS0 ₂ R, isocyanide, HCOR, azide, urea, carbamate, halogen, OH, Schiff base, etc.

[0175] Furthermore, variations in the length of the side-chain linkers in precursor compounds for making compounds of formula I or II can be introduced by one of ordinary skill in the art by adapting known methods. For example, adaptations of the methods of

Magar and Lee (supra) can be used to produce novel precursors and compounds of formulas I and II wherein n=0.

[0176] Precursor compounds synthesized according to methods described in Schemes I- IV above, or by any other methods known or obvious to those of ordinary skill in the art, can be converted to compounds of formula I and/or II of the present invention as depicted, for example, in Scheme V below: heme V

[0177] As a more specific example, particular compounds (e.g., compounds 083 and 227) of formulas I and II can be synthesized according to the general approach depicted in Scheme VI below, starting with the precursor compound 007 synthesized according to Scheme II above:

Scheme VI

[0178] Variations in substituents at R and Ro can also be made readily by one skilled in the art by using or adapting methods well-known to those skilled in the art. For example, reactions such as employed in the general Scheme VII below, wherein Nu is a nucleophile containing Ro as part of its structure and Nu' is a nucleophile containing R as part of its structure, can be used or adapted to make substitutions at R and/or Ro:

Scheme VII

[0179] Some more specific examples are shown in Scheme VIII below wherein different substituents are introduced at R and R ₀ to produce exemplary novel, medically useful compounds 088, 243 and 244 of formula I or II of the invention:

Scheme VIII

[0180] Yet more specific and detailed illustrations using these general synthetic approaches are provided in the particular Examples that follow herein. Furthermore, one skilled in the art and knowing the disclosures of the present invention will appreciate that any of the precursor compounds for compounds of formula I or II and/or compounds of formula I or II directly can be modified with different substituents as desired to be in the final products, and/or the final product of a synthesis, for example a synthesis according to Schemes I- VIII can be modified with different substituents as desired. Placement, removal and/or inter- conversion of desired substituents on precursors, building blocks, intermediates, penultimate or ultimate product compounds of formulas I and II can be accomplished by routine methods well-known to those of ordinary skill in the art, for example as briefly overviewed in the following.

[0181] One or more hydroxyl groups, for example, can be converted to the oxo derivative by direct oxidation, which can be accomplished using any known method such as, for example, a Swern oxidation, or by reaction with a metal oxidant, such as a chromium oxide (e.g., chromium trioxide), a manganese oxide (e.g., manganese dioxide or permanganate) or the like. Primary alcohols can be oxidized to aldehydes, for example, via Swern oxidation, or they can be oxidized to carboxylic acids (e.g., -CO ₂ H), for example by reaction with a metal oxidant. Similarly, the thiols (e.g., -SR, -SH, and the like) can be converted to oxidized sulfur derivatives (e.g., -S0 ₂ R or the like) by reaction with an appropriate oxidant.

[0182] One or more hydroxyl groups can be converted to an ester (e.g., -C0 ₂ R), for example, by reaction with an appropriate esterifying agent such as for example, an anhydride (e.g., (R(CO)) ₂ 0) or an acid chloride (e.g., R(CO)Cl), or the like. One or more hydroxyl groups can be converted to a sulfonate (e.g., -S0 ₂ R) by reaction with an appropriate sulfonating agent such as, for example, a sulfonyl chloride (e.g., RS0 ₂ C1), or the like, wherein R is any suitable substituent including, for example, organic substituents described herein. Ester derivatives also can be obtained, for example, by reacting one or more carboxylic acid substituents (e.g., -C0 ₂ H) with an alkylating agent such as, for example, a diazoalkane (e.g., diazomethane) an alkyl or aryl iodide, or the like. One or more amides can be obtained by reaction of one or more carboxylic acids with an amine under appropriate amide-forming conditions which include, for example, activation of a carboxylic acid (e.g., by conversion to an acid chloride or by reaction with a carbodiimide reagent) followed by coupling of the activated species with a suitable amine.

[0183] One or more hydroxyl groups can be converted to a halogen using a halogenating agent such as, for example, an N-halosuccinamide such as N-iodosuccinamide, N- bromosuccinamide, N-chlorosuccinamide, or the like, in the presence of a suitable activating agent (e.g., a phosphine, or the like). One or more hydroxyl groups also can be converted to ether by reacting one or more hydroxyls, for example, with an alkylating agent in the presence of a suitable base. Suitable alkylating agents can include, for example, an alkyl or aryl sulfonate, an alkyl or aryl halide, or the like. One or more suitably activated hydroxyls, for example a sulfonate ester, and/or one or more suitably activated halides, can be converted to the corresponding cyano, halo, or amino derivative by displacement with a nucleophile which can include, for example, a thiol, a cyano, a halide ion, or an amine (e.g., H ₂ NR, wherein R is a desired substituent), or the like.

[0184] Amines can be obtained by a variety of methods known in the art, for example, by hydrolysis of one or more amide groups. Amines also can be obtained by reacting one or more suitable oxo groups (e.g., an aldehyde or ketone) with one or more suitable amines under the appropriate conditions, for example, reductive amination conditions, or the like. One or more amines, in turn, can be converted to a number of other useful derivatives such as, for example, amides, sulfonamides, and the like. [0185] Certain chemical modifications of a compound of formula I or II can be introduced as desired to obtain useful new variants with new or modified biological properties such as: new or improved potency and/or selectivity for inhibiting Ras-mediated biological processes, improved efficacy against a disease process such as, but not limited to, tumor cell growth, proliferation, survival, invasion and metastasis, as well as resistance to chemotherapy, other molecularly targeted therapeutics, and radiation, as well as enhanced oral bioavailability, less toxicity in a particular host mammal, more advantageous

pharmacokinetics and/or tissue distribution in a given host mammal, and the like. Therefore, the present invention employs methods for obtaining useful new compounds of formula I-II by applying one or more well-known chemical reactions to a given compound to obtain a derivative wherein, for example, one or more phenolic hydroxyl group(s) may instead be replaced by an ester, sulfonate ester or ether group; one or more methyl ether group(s) may instead be replaced by a phenolic hydroxyl group; one or more phenolic hydroxyl group(s) may instead be replaced by an aromatic hydrocarbon substituent; a secondary amine site may instead be replaced by an amide, sulfonamide, tertiary amine, or alkyl quaternary ammonium salt; a tertiary amine site may instead be replaced by a secondary amine; and one or more aromatic hydrogen substituent(s) may instead be replaced by a halogen, nitro, amino, hydroxyl, thiol or cyano substituent.

[0186] Depending upon the stoichiometric amount of the particular reactant, a compound of formula I or II can be substituted at one, some, or all of the respective available positions. For example, when such a compound is reacted with a certain amount of CH ₃ COCl, an acetate substituent can be introduced a one, some, or all of the available positions, which may include, for example ether or amino positions.

[0187] Other examples may include, but are not limited to: (1) conversion to ester, sulfonate ester, and ether substituents at one or more phenolic hydroxyl positions in compounds of formulas I and II; for instance, for preparation of esters or sulfonate esters a given compound can be reacted with an acid halide (e.g., RCOX or RS0 ₂ X, where X is CI, Br or I, and R is a Ci-C ₆ aliphatic or aromatic radical) in anhydrous pyridine or triethylamine; alternatively, the given compound may be reacted with an acid (RCO ₂ H or RSO3H) wherein R is an aliphatic or aromatic radical and dicyclohexylcarbodiimide in triethylamine to prepare the ester or sulfonate ester; for preparation of ethers, the given compound is reacted with an organic halide (e.g., RX or RCH ₂ X, where X is CI, Br or I, and R is a Ci-C ₆ aliphatic or aromatic radical) in anhydrous acetone with anhydrous potassium carbonate; (2) removal of an ether methyl group(s) to provide a phenolic hydroxyl functionality and/or conversion of that moiety to an ester, sulfonate, or other ether in a compound or derivative of formula I or II: for instance, for hydrolytic cleavage of a methyl ether substituent and conversion to a phenolic hydroxyl moiety, the given compound is reacted with BBr ₃ or BX ₃ ^» (CH ₃ ) ₂ S in CH ₂ C1 ₂ (where X is F, CI or Br); the resulting phenol can be converted to an ester, sulfonate ester or ether as described above; (3) preparation of amide or sulfonamide derivatives at an amine site in a compound of formula I or II: for instance, for preparation of amides or sulfonamide derivatives, the same general procedures described above in (1) apply; in either case (procedure (1) or (3)), an appropriate functional group protection strategy

(blocking/deblocking of selected group(s)) may need to be applied; (4) conversion of a secondary amine functionality in a compound of formula I or II to a tertiary amine: for instance, for preparation of a tertiary amine, the given compound is reacted with an aldehyde, and the resulting product is then reduced with NaBH ₄ ; alternatively, for preparation of an alkyl ammonium salt, the given compound is reacted with an alkyl halide (RX, where X is CI, Br or I, and R is a Ci-C ₆ aliphatic radical) in an anhydrous aprotic solvent; (5) conversion of a tertiary amine functionality in a compound of formula I or II to a secondary amine; for instance, for preparation of a secondary amine, the given compound is reacted with cyanogen bromide to give a cyanamide derivative which is then treated with LiAlH ₄ ; (6) conversion of one or more phenolic hydroxyl groups in a given compound of formula I or II to an aromatic hydrogen substituent: for instance, the given compound is converted (after suitable protection of any amine substituent(s) if necessary) to the triflate ester to give the corresponding deoxy compound; (7) substitution of one or more hydrogen substituent(s) on the aryl system(s) on a compound of formula I or II by halogen, nitro, amino, hydroxyl, thiol, or cyano groups: for instance, for preparation of a bromine-substituted derivative, the given compound is reacted with Br ₂ in H ₂ 0; for the preparation of other substituted derivatives, the given compound is treated with HN0 ₃ /HOAc to provide a nitro-substituted (-N0 ₂ ) derivative; in turn, the nitro- derivative can be reduced to an amino derivative, and the amino derivative is the point of origin of the chloro, iodo, cyano, thiol and hydroxyl substitution via well-known and practiced diazonium substitution reactions.

[0188] It will be appreciated that certain compounds of formula I or II can have one or more asymmetric carbon(s) and thus such compounds are capable of existing as enantiomers or diastereomers. Unless otherwise specified, the present invention includes such

enantiomers or diastereomers, including any racemates thereof. If desired, the separate enantiomers or diastereomers can be synthesized from appropriate chiral starting materials, or the racemates can be resolved by conventional procedures, which are well-known to those skilled in the art, such as chiral chromatography, fractional crystallization of diastereomers or diastereomeric salts, and the like. Certain compounds can exist as geometrical isomers, such as, for example, compounds with double-bonded substituents with geometrical isomers Z and E, and the present invention includes all such isomers, including certain isomers, for example the Z isomers, which are preferred. Also, certain compounds may contain substituents wherein there is restricted rotation and/or other geometric isomers are possible. For example, certain oxime substituents may exist in syn or anti configurations. The present invention includes all such configurations, including all possible hindered-rotational isomers, and other geometric isomers.

[0189] It will be appreciated by one skilled in the art that the proof or confirmation of the chemical structure of a compound provided by or used in the present invention can be demonstrated using at least one or more well-known and established, convergent methods including, but not limited to, for example: proton and/or carbon MR spectroscopy, mass spectrometry, x-ray crystallography, chemical degradation, and the like.

[0190] The present invention further provides a pharmaceutical composition comprising at least one compound of formula I or II, a pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier. In that respect, the pharmaceutical composition includes an effective amount of at least one compound of formula I or II, which may be in the form of pharmaceutically acceptable salt(s) or prodrug(s) thereof and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, for example, vehicles, adjuvants, excipients, or diluents, are accessible to those of skill in the art and are typically available commercially. One skilled in the art can easily determine the appropriate method of administration for the exact formulation of the composition being used.

[0191] The composition of the present invention can be produced by combining one or more compound(s) of formula I or II with an appropriate pharmaceutically acceptable carrier, and can be formulated into a suitable preparation, which may include, for example, preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powers, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols, and other formulations known in the art for their respective routes of administration. In pharmaceutical dosage forms, a compound of formula I or II can be used alone or in appropriate association, as well as in combination, with other pharmacologically active compounds, including other compounds, e.g., other Ras-inhibitory compounds, as described herein.

[0192] Any suitable pharmacologically or physiologically acceptable carrier can be utilized. The following methods and carriers are merely exemplary and are in no way limiting. In the case of oral preparations, a compound of formula I or II can be administered alone or in combination with a therapeutically or prophylactically effective amount of at least one other compound. The active ingredient(s) can be combined, if desired, with appropriate additives to make tablets, powders, granules, capsules or the like.

[0193] Suitable additives can include, for example, lactose, mannitol, corn starch or potato starch. Suitable additives also can include binders, for example crystalline cellulose, cellulose derivatives, acacia, or gelatins; disintegrants, for example, corn starch, potato starch or sodium carboxymethylcellulose; or lubricants such as talc or magnesium stearate. If desired, other additives such as, for example, diluents, buffering agents, moistening agents, preservatives, and/or flavoring agents, and the like, can be included in the composition.

[0194] The Ras-inhibitory compounds used in accordance with the present invention can be formulated into a preparation for injection or infusion by dissolution, suspension, or emulsification in an aqueous or non-aqueous solvent, such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acid or propylene glycol (if desired, with conventional additives such as solubilizers isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives).

[0195] The compounds of formulas I and II also can be made into an aerosol formulation to be administered by inhalation. Such aerosol formulations can be placed into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0196] The compounds can be formulated into suppositories by admixture with a variety of bases such as emulsifying bases or water-soluble bases. The suppository formulations can be administered rectally, and can include vehicles such as cocoa butter, carbowaxes, and polyethylene glycols, which melt at body temperature but are solid at room temperature.

[0197] Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions can be provided wherein each dosage unit, e.g., teaspoonful, tablet, or suppository contains a predetermined amount of the composition containing the compound of formula I or II. Similarly, unit dosage forms for injection or intravenous administration can comprise a composition as a solution in sterile water, normal saline, or other

pharmaceutically acceptable carrier. [0198] The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a

predetermined quantity of at least one compound(s) of formula I or II (alone, or if desired, with another therapeutic or prophylactic agent). The unit dosage can be determined by methods known to those of skill in the art, for example, by calculating the amount of active ingredient sufficient to produce the desired effect in association with a pharmaceutically acceptable carrier. The specifications for the unit dosage forms that can be used in accordance with the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the compound(s) in the individual host. Any necessary adjustments in dose can readily be made by an ordinarily skilled practitioner to address the nature or severity of the condition being treated. Adjustments in dose also can be made on the basis of other factors such as, for example, the individual patient's overall physical health, sex age, prior medical history, and the like.

[0199] The composition of the present invention preferably includes a therapeutically or prophylactically effective amount of at least one Ras-inhibitory compound of formula I or II. The therapeutically or prophylactically effective amount can include an amount that produces a therapeutic or prophylactic response (e.g., treats or prevents cancer or a Ras-mediated biological process, attenuates, ameliorates, or eliminates one or more symptoms of cancer or a Ras-mediated biological process, and/or prevents or delays the onset of one or more symptoms of cancer or a Ras-mediated biological process) in a patient to whom a compound or composition of the present invention is administered. A therapeutically or prophylactically effective amount can include, for example, a Ras-inhibitory and/or an anticancer effective amount that is effective to inhibit one or more conditions selected from the group consisting of tumor cell growth, proliferation, survival, invasion and metastasis, as well as resistance to chemotherapy, other molecularly targeted therapeutics, and radiation.

[0200] By way of example and not intending to limit the invention, the dose of the pharmaceutically active agent(s) described herein for methods of preventing or treating a disease or disorder can be, in embodiments, about 0.001 to about 1 mg/kg body weight of the subject being treated per day, for example, about 0.001 mg, 0.002 mg, 0.005 mg, 0.010 mg, 0.015 mg, 0.020 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.5 mg, 0.75 mg, or 1 mg/kg body weight per day. In certain embodiments, the dose of the pharmaceutically active agent(s) described herein can be about 1 to about 1000 mg/kg body weight of the subject being treated per day, for example, about 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 0.020 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 500 mg, 750 mg, or 1000 mg/kg body weight per day.

[0201] The terms "treat," "prevent," "ameliorate," and "inhibit," as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment, prevention, amelioration, or inhibition. Rather, there are varying degrees of treatment, prevention, amelioration, and inhibition of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods can provide any amount of any level of treatment, prevention, amelioration, or inhibition of the disorder in a mammal. For example, a disorder, including symptoms or conditions thereof, may be reduced by, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%). Furthermore, the treatment, prevention, amelioration, or inhibition provided by the inventive method can include treatment, prevention, amelioration, or inhibition of one or more conditions or symptoms of the disorder, e.g., cancer. Also, for purposes herein, "treatment," "prevention," "amelioration," or "inhibition" can encompass delaying the onset of the disorder, or a symptom or condition thereof.

[0202] When the effective level is used as the preferred endpoint for dosing, the actual dose and schedule can vary depending, for example, upon inter-individual differences in pharmacokinetics, drug distribution, metabolism, and the like. The effective level also can vary when one or more compound(s) of formula I or II are used in combination with other therapeutic agents, for example, one or more additional anticancer compound(s), or a combination thereof. Moreover, the effective level can vary depending upon the particular disease (e.g., cancer or neurofibromatosis) or biological process (e.g., tumor cell growth, proliferation, survival, invasion and metastasis, as well as resistance to chemotherapy, other molecularly targeted therapeutics, and radiation) for which treatment is desired. Similarly, the effective level can vary depending on whether the treatment is for therapy or prevention of a particular disease such as, for example, cancer.

[0203] An anticancer effective amount can be determined, for example, by determining an amount to be administered effective to produce a Ras-inhibiting-effective blood or tissue level and/or intracellular target-inhibiting "effective level" in the subject patient. The effective level can be chosen, for example, as that blood and/or tissue level (e.g., 10 ^"12 - 10 ^"6 M) effective to inhibit the proliferation of tumor cells in a screening assay. Similarly, the effective level can be determined, for example, on the basis of the blood, tissue or tumor level in a patient that corresponds to a concentration of a therapeutic agent that effectively inhibits the growth of a human cancer in any assay that is clinically predictive of anticancer activity. Further, the effective level can be determined, for example based on a concentration at which certain markers of cancer in a patient's blood or tumor tissue (e.g., mutant or hyperactive ras gene(s) and/or Ras protein(s) and/or aberrant Ras-mediated biological pathway(s)) are inhibited by a particular compound that inhibits cancer. Alternatively, the effective level can be determined, for example, based on a concentration effective to slow or stop the growth of a patient's cancer, or cause a patient's cancer to regress or disappear, or render a patient asymptomatic to a particular cancer, or improve a cancer patient's subjective sense of condition. The anticancer effective level can then be used to approximate (e.g., by extrapolation) or even to determine precisely, the level which is required clinically to achieve a Ras-inhibiting-effective blood, tissue, tumor and/or intracellular level to cause the desired medical treatment. It will be appreciated that the determination of the therapeutically effective amount clinically required to effectively inhibit Ras-mediated processes also requires consideration of other variables that can influence the effective level, as discussed herein. When a fixed effective amount is used as a preferred endpoint for dosing, the actual dose and dosing schedule for drug administration can vary for each patient depending upon factors that include, for example, inter-individual differences in pharmacokinetics, drug absorption, drug disposition and tissue distribution, drug metabolism, drug excretion, whether other drugs are used in combination, or other factors described herein that influence the effective level.

[0204] One skilled in the art and knowing and understanding the disclosures of the present invention can readily determine the appropriate dose, schedule, or method of administering a particular formulation, in order to achieve the desired effective level in an individual patient. Given the disclosures herein, one skilled in the art also can readily determine and use an appropriate indicator of the effective level of the compound(s) of formulas I and II. For example, the effective level can be determined by direct analysis (e.g., analytical chemistry) or by indirect analysis (e.g., with clinical chemistry indicators) of appropriate patient samples (e.g., blood and/or tissues). The effective level also can be determined, for example, if the compound in question has antitumor activity, by direct or indirect observations, such as, for example, observing the shrinkage, slowing or cessation of growth or spreading of a tumor in a cancer patient. There are many references to the art that describe the protocols used in administering and monitoring responses to active compounds in a patient in need thereof. For example, drug-appropriate protocols used in the administration of different types of anticancer agents to patients are described in "Cancer Chemotherapy and Biotherapy: Principles and Practice ^' " eds. Chabner and Longo,

Lippincott, Williams and Wilkins (2011), and citations therein.

[0205] In accordance with an embodiment, the pharmaceutical composition can further include a therapeutically or prophylactically effective amount of at least one additional compound other than a compound of formula I or II, which may or may not be another Ras- inhibitory compound, and may be an anticancer compound. When the additional compound is a Ras-inhibitory compound other than a compound of formula I or II, it is preferably present in the composition in a Ras-inhibiting amount. When the additional compound is an anticancer compound in general, it is preferably present in the composition in an anticancer effective amount.

[0206] In a preferred embodiment, a Ras-inhibitory compound can be identified from one or more compounds of formula I or II by an assay of Ras inhibition. Some representative assays of selective Ras inhibition are illustrated in the examples that follow herein. As used herein, the terminology selective "Ras inhibition" means selective, preferential or specific inhibition of aberrant Ras-mediated cellular processes, such as, for example, accelerated or aberrant cell growth, proliferation, survival, and invasiveness, relative to these processes in cells or tissues with normal or non-aberrant Ras and Ras-mediated processes.

Experimentally, selective Ras inhibition can be shown, for example, by determining the ratio (numerator/denominator) of a given compound's potency (e.g., IC ₅₀ ) to inhibit the growth of cells with "normal" or "wild-type" Ras and/or lacking activated (numerator) relative to that of cells with mutated and/or activated Ras (denominator). The terminology used herein for such an experimentally determined ratio is "selectivity" or "selectivity index," which may be further denoted by showing the respective cell types used to determine the numerical ratio (e.g., HT-29/A549; Caco-2/SW-480; HT-29/SW-480; HT-29/HCT-116). For a given compound, a "selectivity" value or "selectivity index" of greater than 1 (one), preferably greater than 10 (ten), more preferably greater than 100 (one hundred) and even more preferably greater than 1000 (one thousand) indicates said compound selectively inhibits hyperactive Ras and/or Ras-mediated cellular functions, such as those which may drive or accelerate cancer cell growth, proliferation, metastasis, resistance to drugs or radiation, and the like.

[0207] The assay of Ras inhibition preferably employs one or more isogenic cell line pair(s), in which both of the lines share the same genetic background except that one of the lines ("mutant line") contains one or more mutated or hyperactive ras gene(s), Ras protein(s) and/or aberrant Ras-mediated biological process(es), and the other line ("normal line") lacks such mutation(s) or aberrant function(s).

[0208] In a preferred embodiment of the present invention, the aforementioned assay employing isogenic cell line(s) enables the determination and calculation of a Ras-Inhibitory Specificity Index (RISI). One experimental approach to determination of such a RISI may, for example, comprise determining the ratio of the concentration of a compound producing a specified effect on the normal line, such as, for example, 50% growth inhibition in a specified period of time, divided by the concentration of the same compound producing the same specified effect (e.g., 50% growth inhibition in the same specified period of time) on the mutant line.

[0209] Whereas in the aforementioned approach, the 50% growth inhibition values may be obtained by testing the compound against both normal and mutant cell lines at multiple concentrations over a specified concentration range, for example 10 nM-10,000 nM, an alternate, more streamlined approach to determining a RISI value could comprise measuring the ratio of percentage growth inhibition in a given period of time by a specified single concentration of the compound, for example 250 nM, selected from within a range of concentrations, for example from within a range of 10 nM-10,000 nM, against the mutant (numerator) relative to the normal cell line (denominator). This approach may be generally more applicable to larger-scale or preliminary screening of groups of individual compounds or mixtures thereof to obtain a preliminary or screening RISI, whereas a RISI determined using concentration ranges to determine 50% growth inhibition values may be more precise. A RISI value obtained for a given compound by either approach may be less than, equal to or greater than 1 (one), and a RISI value of greater than 1 (one) indicates said compound selectively inhibits Ras or Ras-mediated cellular functions.

[0210] In an aspect of this embodiment, the employed assay of Ras inhibition enables identification of a compound from one or more compounds of formulas I-II having a RISI of greater than 1, preferably greater than 10, more preferably greater than 100, and even more preferably greater than 1000.

[0211] In view of the biological activity, the compounds of formulas I and II can be utilized in a variety of therapeutic and prophylactic (disease preventing) applications, and also in certain non-therapeutic or non-prophylactic applications. It will be appreciated that one or more of these compounds can be used, for example, as a control in diagnostic kits, bioassays, or the like. Preferably the method of the present invention is applied therapeutically or prophylactically, for example, toward treatment or prevention of cancer or toward treatment or prevention of a condition (e.g. an abnormal condition or disease) treatable by the inhibition of Ras-mediated biological process(es). The compounds of formulas I and II can be administered alone, or in combination with a therapeutically or prophylactically effective amount of at least one additional compound other than a compound of formula I or II. For example, the disease or condition treatable by the inhibition of one or more Ras-mediated biological process is a disease wherein hyperactive Ras (e.g., including mutant Ras) is implicated, such as cancer, neurofibromatosis, or Costello syndrome.

[0212] In an aspect, the present invention provides a method of inhibiting a human or nonhuman mammalian Ras-mediated biological process, which method comprises administering in vivo or in vitro a Ras-inhibitory amount of at least one compound of formula I or II, the corresponding Z- or E-isomer, pharmaceutically acceptable salt, or prodrug thereof. In an embodiment, the Ras-mediated biological process is selected from growth, proliferation, survival, metastasis, drug resistance and radiation resistance of a tumor cell.

[0213] In another embodiment, the present invention provides a method of

therapeutically or prophylactically treating a human or nonhuman mammalian patient with cancer, which method comprises administering to a patient in need thereof a therapeutically or prophylactically effective amount of at least one compound of formula I or II. The cancer can be any suitable cancer harboring hyperactive or mutant Ras, such that the cancer is treatable by a Ras inhibitor. Compounds of formula I or II can be expected to have efficacious actions in patients with cancer, especially in patients whose cancers have underlying hyperactive, over-expressed or mutant Ras-mediated pathological processes that are inhibited by a compound(s) of formula I or II. For example, the cancer can be pancreatic cancer, lung cancer, colorectal cancer, melanoma, ovarian cancer, renal cancer, prostate cancer, head and neck cancer, endocrine cancer, uterine cancer, breast cancer, sarcoma cancer, gastric cancer, hepatic cancer, esophageal cancer, central nervous system cancer, brain cancer, hepatic cancer, germline cancer, lymphoma, or leukemia. Preferably the cancer is pancreatic cancer, colorectal cancer, or lung cancer. In accordance with an embodiment, the cancer is drug-resistant or radiation-resistant.

[0214] In a further embodiment, the invention provides a method of therapeutically or prophylactically treating a human or nonhuman mammalian patient with a disease or condition treatable by the inhibition of one or more neoplastic or cancerous process, which method comprises administering to a patient in need thereof a therapeutically or prophylactically effective amount of at least one neoplastic or cancerous inhibitory compound of formula I or II, or a compound selected from (080)-(086), (091)-(093), (157)- (168), (170)-(178), (181)-(187), (191)-(197), (202), (203), (220), (221), (230)-(237), (240), (241), (250)-(257), (260)-(267), (270)-(277), and (280)-(286), or a pharmaceutically acceptable salt or prodrug thereof, either alone or in combination with one other therapeutic agent other than a compound of formula I or II, or a compound selected from (080)-(086), (091)-(093), (157)-(168), (170)-(178), (181)-(187), (191)-(197), (202), (203), (220), (221), (230)-(237), (240), (241), (250)-(257), (260)-(267), (270)-(277), and (280)-(286), or a pharmaceutically acceptable salt or prodrug thereof. In a preferred embodiment of the above method, the neoplastic or cancerous process is selected from growth, proliferation, survival, metastasis, drug resistance and radiation resistance of a tumor cell.

[0215] It is contemplated that a compound of formula I or II may promote broader sensitivity of cancer to other drugs and/or radiation therapy by inhibiting the ability of cancer cells to develop or express resistance to such drugs and/or radiation therapy making possible the effective chemotherapeutic and/or radiotherapeutic treatment of cancer.

[0216] Any of the methods of the present invention further includes administering a Ras- inhibiting effective amount of at least one additional compound other than a compound of formula I or II. In some instances, the method of the present invention can be made more effective by administering one or more other Ras-inhibitory compound(s), along with a compound of formula I or II. One or more Ras-inhibitory compound(s) of formula I or II also can be co-administered in combination with an anticancer agent other than a compound of formula I or II, for example, to cause anticancer chemotherapy-resistant and/or radiation- resistant tumor cells to become chemotherapy-sensitive and/or radiation-sensitive and/or to inhibit de novo the development of cancer cell resistance to the anticancer agent and/or to cancer cell resistance to radiation treatment. Alternatively, or in addition, one or more compound(s) of formula I or II can be co-administered with radiation therapy, in which case the effective level is the level needed to inhibit or reverse the ability of the cancer to develop resistance to the radiation therapy.

[0217] Examples of anticancer compounds include reversible DNA binders, DNA alkylators, and DNA strand breakers. Examples of suitable reversible DNA binders include topetecan hydrochloride, irinotecan (CPT11 - Camptosar), rubitecan, exatecan, nalidixic acid, TAS-103, etoposide, acridines (e.g., amsacrine, aminocrine), actinomycins (e.g., actinomycin D), anthracyclines (e.g., doxorubicin, daunorubicin), benzophenainse, XR 11576/MLN 576, benzopyridoindoles, Mitoxantrone, AQ4, Etopside, Teniposide, (epipodophyllotoxins), and bisintercalating agents such as triostin A and echinomycin.

[0218] Examples of suitable DNA alkylators include sulfur mustard, the nitrogen mustards (e.g., mechlorethamine), chlorambucil, melphalan, ethyleneimines (e.g.,

triethylenemelamine, carboquone, diaziquone), methyl methanesulfonate, busulfan, CC-1065, duocarmycins (e.g., duocarmycin A, duocarmycin SA), metabolically activated alkylating agents such as nitrosoureas (e.g., carmustine, lomustine, (2-chloroethyl)nitrosoureas), triazine antitumor drugs such as triazenoimidazole (e.g., dacarbazine), mitomycin C, leinamycin, and the like.

[0219] Examples of suitable DNA strand breakers include doxorubicin and daunorubicin (which are also reversible DNA binders), other anthracyclines, belomycins, tirapazamine, enediyne antitumor antibiotics such as neocarzinostatin, esperamicins, calicheamicins, dynemicin A, hedarcidin, C-1027, N1999A2, esperamicins, zinostatin, and the like.

[0220] Examples of anticancer agents include abarelix, aldesleukin, alemtuzumab, altretamine, amifostine, aminoglutethimide, anastrazole, arsenic trioxide, asparaginase, azacitidine, azathioprine, BCG vaccine, bevacizumab, bexarotene, bicalutamide, bleomycin sulfate, bortezomib, bromocriptine, busulfan, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, chloroquine phosphate, cladribine, cyclophosphamide,

cyclosporine, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, daunorubicin citrate liposomal, dexrazoxane, docetaxel, doxorubicin hydrochloride, doxorubicin hydrochloride liposomal, epirubicin hydrochloride, estramustine phosphate sodium, etoposide, estretinate, exemestane, floxuridine, fludarabine phosphate, fluorouracil, fluoxymesterone, flutamide, fulvestrant, gefitinib, gemcitabine hydrochloride, gemtuzumab ozogamicin, goserelin acetate, hydroxyurea, idarubicin hydrochloride, ifosfamide, imtinib mesylate, interferon alfa-2a, interferon alfa-2b, irinotecan hydrochloride trihydrate, letrozole, leucovorin calcium, leuprolide acetate, levamisole hydrochloride, lomustine, lymphocyte immune anti-thymocyte globulin (equine), mechlorethamine hydrochloride,

medoxyprogestone acetate, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone hydrochloride, nilutamide, oxaliplatin, paclitaxel, pegaspargase, pentostatin, plicamycin, porfimer sodium, procarbazine hydrochloride, streptozocin, tamoxifen citrate, temozolomide, teniposide, testolactone, testosterone propionate, thioguaine, thiotepa, topotecan hydrochloride, tretinoin, uracil mustard, valrubicin, vinblastine sulfate, vincristine sulfate, and vinorelbine.

[0221] Suitable forms of radiation therapy include, for example, all forms of radiation therapy approved for commercial use in the United States, and those forms that will become approved in the future, for which radiation resistance thereto can be controlled by a Ras- inhibitory compound of formula I or II.

[0222] The subject to be treated in any of the methods described herein is any human or nonhuman mammalian patient with a condition (e.g. an abnormal condition or disease) treatable by the inhibition of Ras-mediated biological process(es). The term "subject" includes humans, sheep, horses, cattle, pigs, dogs, cats, rats, and mice. In embodiments of the invention, the subject is a human.

[0223] In an embodiment, the patient's tissue, blood or tumor contains an abnormal, mutant or hyperactive ras gene or Ras protein, or aberrant Ras-mediated biological process. Often the patient is pre-selected by utilizing an assay of the patient's tissue, blood or tumor, which is tested for an abnormal, mutant or hyperactive ras gene or Ras protein, or an aberrant Ras-mediated biological process.

[0224] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0225] This example illustrates the synthesis of an essential precursor compound, (Z)-2- (5-fluoro-l-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl-lH -inden-3-yl)-N-(furan-2- ylmethyl)acetamide (007), for use in synthesis of certain exemplary compounds of formulas I and II of the present invention.

[0226] (A) p-fluoro-a-methylcinnamic acid: p-fluorobenzaldehyde (200 g, 1.61 mol), propionic anhydride (315 g, 2.42 mol) and sodium propionate (155 g, 1.61 mol) in a 1L three- necked flask in an atmosphere of argon was stirred in an oil bath to 140°C for 36 hours. The clear solution was cooled to 100°C and poured into 8 L of water. The precipitate was collected and dissolved by adding potassium hydroxide to 2 L of ice water to pH 12. The aqueous solution was extracted with ether, and the extracts washed with potassium hydroxide solution (200 mL><2). The combined aqueous solution was acidified with concentrated HC1. The precipitate was collected by filtration, washed with water, ethanol and hexane, dried under air to give p-fluoro-a-methylcinnamic acid, which was used for next step reaction without further purification.

[0227] (B) p-fluoro-a-methylhydrocinnamic acid: A 2L catalytic hydrogenation flask containing p-fluoro-a-methylcinnamic acid (180 g, 0. 987 mol), 5%-Pd/C (1.2g) and 1.2 L ethanol was flushed with argon and warmed to 65-70 °C. The mixture was treated with hydrogen (40 psi) until the hydrogen uptake ceases (about 30 min). The catalyst was filtered off, and the filtrate was concentrated in vacuum to give p-fluoro-a- methylhydrocinnamic acid as an oil.

[0228] (C) 5-fluoro-2-methylindanone: polyphosphoric acid (PPA 85%, 650 g) was warmed in a 80°C water bath for lh, then transferred to a 1L 3-necked flask equipped with a mechanical stirrer, a dropping funnel, and a thermometer. The flask was warmed in a 70°C oil bath and p-fluoro-a- methylhydrocinnamic acid (93.2 g, 0 5 mol) was added in about 5 minutes with stirring. The temperature was gradually raised to 90°C, and kept there for about 30 min. The reaction mixture was poured into 2 L of ice water, the aqueous layer extracted with ether, and the solution washed twice with saturated sodium chloride solution, 5% Na2C03 solution, water, dried over Na2S04, and then concentrated to give a milky oil. The oil was dissolved in 100 mL of methylene chloride and 200 mL of hexane, and the solution was loaded to a dry -packed silica gel flash column (800g of TLC grade silica gel tightly packed in a 2L fritted funnel, vacuum), eluted with 5% ether- hexane to give 5-fluoro-2- methylindanone as a clear oil.

[0229] (D) 5-fluoro-2-methylindenyl-3-acetic acid: A mixture of 5-fluoro-2- methylindanone (184 g, 1.12 mol), cyanoacetic acid (105 g, 1.23 mol), acetic acid (130 g), and ammonium acetate (34 g) in dry toluene (about 600 ml) was refluxed for 48 to 72 hours, and the liberated water/acetic acid was collected in a Dean Stark trap. To the cooled reaction mixture was added 600 mL of methylene chloride, the solution washed with water (200 mLx3), the organic layer concentrated, and the residue treated with 150g of potassium hydroxide in 300 ml of ethanol and 200 ml of water. The mixture was refluxed overnight under nitrogen, the ethanol removed under vacuum, 500 ml water added, the aqueous solution washed well with ether and then boiled with charcoal. The aqueous filtrate was acidified to pH 2 with 50% hydrochloric acid, and extracted with methylene chloride (300 mLx 3). The solvent was evaporated, and the residue treated with acetone in a sonicator bath until precipitate formed. The mixture was stored in a -20°C freezer overnight, and the precipitate collected by filtration. The procedure gave 5-fluoro-2-methylindenyl-3-acetic acid as a colorless solid (mp 164-166°C).

[0230] (E) (Z)-2-(5-fluoro-l-(4-hydroxy-3,5-dimethoxybenzylidene)-2-met hyl-lH-inden- 3-yl)acetic acid: The 5-fluoro-2-methyl-3-indenylacetic acid (0.54g, 2.62 mmol), 4-acetoxy- 3,5-dimethoxybenzaldehyde (0.60 g, 2.67 mmol) and potassium butoxide (0.69g, 7.7 mmol) in DMSO (6 ml) were stirred in a microwave synthesizer at 75°C under argon for 2h. After cooling, the reaction mixture was poured into 50 ml of ice-water, and was acidified with 2N hydrochloric acid. The mixture was extracted with methylene chloride (25 mL><2), the combined organic layer washed with water (25 mL><2), and concentrated. The residue was purified on a silica gel column three times to produce the titled compound (E, 117 mg) as a yellow/orange solid.

[0231] (F) (Z)-2-(5-fluoro-l-(4-hydroxy-3,5-dimethoxybenzylidene)-2-met hyl-lH-inden- 3-yl)-N-(furan-2-ylmethyl)acetamide (007): The (Z)-2-(5-fluoro-l-(4-hydroxy-3,5- dimethoxybenzylidene)-2-methyl-lH-inden-3-yl)acetic acid (120 mg, 0.324 mmol), 1, 1'- Carbonyldiimidazole (100 mg, 0.61 mmol) in 5 mL of anhydrous methylene chloride was stirred for 30 min at room temperature. Furfuryl amine (100 μΐ., 1.13 mmol) was added, the reaction mixture stirred for 2 h, and quenched with 1 ml of 30% potassium hydroxide solution, which was diluted with 25 mL of methylene chloride, neutralized with 2 mL of acetic acid, washed with water (20 mL><3), dried with sodium sulfate, then concentrated. The residue was purified with silica gel column, eluted with hexane/acetone. The major yellow fraction was collected and after concentration the residue (120 mg) was stored under argon in a freezer, and after 2 weeks crystals formed. The mixture containing the crystals was suspended in 2 mL of ethyl ether and 3 mL of hexane, treated with sonicator for lh, then stored in a -20°C freezer overnight. The precipitate was collected by filtration, and the titled compound (007) was obtained as a yellow solid (41 mg).

EXAMPLE 2

[0232] This example illustrates the synthesis of another essential precursor compound, (Z)-2-(5-methoxy-l-(4-hydroxy-3,5-dimethoxybenzylidene)-2-me thyl-lH-inden-3-yl)-N- (furan-2-ylmethyl)acetamide (006), for use in synthesis of certain exemplary compounds of formulas I and II of the present invention. [0233] (A) p-methoxy-a-methylcinnamic acid: p-methoxybenzaldehyde (219 g, 1.61 mol), propionic anhydride (315 g, 2.42 mol) and sodium propionate (155 g, 1.61 mol) in a 1L three-necked flask in an atmosphere of argon was stirred in an oil bath at 140°C for 36 hours. The clear solution was cooled to 100°C, poured into 8 L of water, and the precipitate collected and dissolved by adding potassium hydroxide to 2L of ice water to pH 12. The aqueous solution was extracted with ether, the extracts washed with potassium hydroxide solution (200 mL><2) and the combined aqueous solution acidified with concentrated HC1. The precipitate was collected by filtration, washed with water, ethanol and hexane, then dried over air to give p-methoxy-a-methylcinnamic acid (235 g) which was used for next step reaction without further purification.

[0234] (B) p-methoxy-a-methylhydrocinnamic acid: A 2L catalytic hydrogenation flask containing p-methoxy-a-methylcinnamic acid (192 g, 1.00 mol), 5%-Pd/C (1.2g) and 1.2 L ethanol was flushed with argon and warmed to 70°C. The mixture was treated with hydrogen (40 psi) until the hydrogen uptake ceased (about 30 min). The catalyst was filtered off, and the filtrate concentrated in vacuum to give p-methoxy-a- methylhydrocinnamic acid as an oil.

[0235] (C) 5-methoxy-2-methylindanone: Polyphosphoric acid (PPA 85%, 650 g) was warmed in a 60°C water bath for lh, and transferred to a 1L 3-necked flask equipped with a mechanical stirrer, a dropping funnel, and a thermometer. The flask was warmed to 50°C in oil bath and p-methoxy-a- methylhydrocinnamic acid (96 g, 0.50 mol) was added in about 5 minutes with stirring. The temperature was gradually raised to 70°C for about 15 min, and the solution was poured into 2 L of ice water. The aqueous layer was extracted with ether, and the solution was washed twice with saturated sodium chloride solution, 5% Na2C03 solution, water, dried over Na2S04, and then concentrated to give a milky oil. The oil was dissolved in 100 mL of methylene chloride and 200 mL of hexane, and applied to a dry- packed silica gel flash column (800g of TLC grade silica gel tightly packed in a 2L fritted funnel, vacuum), eluted with 5% ether-petroleum ether to give 6- methoxy-2-methylindanone as a clear oil.

[0236] (D) 5-methoxy-2-methylindenyl-3-acetic acid: A mixture of 6-methoxy-2- methylindanone (197 g, 1.12 mol), cyanoacetic acid (105 g, 1.23 mol), acetic acid (130 g), and ammonium acetate (34 g) in dry toluene (about 600 ml) was refluxed for 48 to 72 hours, until the liberated water collected in a Dean Stark trap ceased. To the cooled toluene reaction mixture was added 600 mL of methylene chloride. The solution was washed with water (200 mLx3), the organic layer concentrated, and the residue treated with 150g of potassium hydroxide in 300 ml of ethanol and 200 ml of water. The mixture was refluxed overnight under nitrogen, the ethanol removed under vacuum, 500 ml water added, the aqueous solution washed well with ether and then boiled with charcoal. The aqueous filtrate was acidified to pH 2 with 50% hydrochloric acid, extracted with methylene chloride (300 mLx 3), solvent evaporated, and the residue treated with acetone in a sonicator bath until a precipitate formed. The mixture was stored at -20°C overnight, and the precipitate collected by filtration. The procedure gave 5-methoxy-2-methylindenyl-3-acetic acid as a colorless solid (mp 164- 166°C).

[0237] (E) (Z)-2-(5-methoxy-l-(4-hydroxy-3,5-dimethoxybenzylidene)-2-me thyl-lH- inden-3-yl)acetic acid: 5-methoxy-2-methyl-3-indenylacetic acid (0.50g, 2.29 mmol), 4- acetoxy-3,5-dimethoxybenzaldehyde (0.60 g, 2.67 mmol) and potassium butoxide (l .Og, 8.9 mmol) in anhydrous DMSO (5 ml) and pyridine (10 mL) were stirred at 95°C under argon for lh. The reaction mixture was cooled to 65°C. 1.0 mL of methanol was added and stirred for 30 min. After cooling, the mixture was poured into 50 ml of ice-water, acidified with 2N hydrochloric acid, and extracted with methylene chloride (25 mL><2), and the combined organic layer washed with water (25 mL><2), and concentrated. The residue was purified on a silica gel column twice, the first eluted with methylene chloride/methanol, followed by washing with hexane, acetone/acetic acid, to produce the title compound (E, 152 mg) as a yellow/ orange solid.

[0238] (F) (Z)-2-(5-methoxy-l-(4-hydroxy-3,5-dimethoxybenzylidene)-2-me thyl-lH- inden-3-yl)-N-(furan-2-ylmethyl)acetamide (006): (Z)-2-(5-methoxy-l-(4-hydroxy-3,5- dimethoxybenzylidene)-2-methyl-lH-inden-3-yl)acetic acid (123 mg, 0.324 mmol), 1, 1'- carbonyldiimidazole (100 mg, 0.61 mmol) in 5 mL of anhydrous methylene chloride was stirred for 30 min at room temperature, furfuryl amine (100 μΕ, 1.13 mmol) was added, and the reaction mixture stirred for 2 h, then quenched with 1 ml of 30% potassium hydroxide solution. The solution was diluted with 25 mL of methylene chloride, neutralized with 2 mL of acetic acid, washed with water (20 mLx 3), dried with sodium sulfate, and concentrated. The residue was purified over silica gel, eluted with hexane/acetone, and the major yellow fraction was collected. After concentrating, the residue was stored in a freezer under argon until a crystal seed formed (~2 weeks). The mixture was suspended in 2 mL of ethyl ether and 3 mL of hexane, treated with sonicator for lh, then stored in a -20°C freezer overnight. The precipitate was collected by filtration. The title compound (006) was obtained as a yellow solid (36 mg). EXAMPLE 3

[0239] This example illustrates the synthesis of another essential precursor compound, (Z)-2-(5 -fluoro- 1 -(4-hydroxy-3 , 5 -dimethoxybenzylidene)-2-methyl- 1 H-inden-3 -yl)-N-(( 1 - methyl-lH-pyrrol-2-yl)methyl)acetamide (019) for use in synthesis of certain exemplary compounds of formulas I and II of the present invention.

[0240] (Z)-2-(5-fluoro-l-(4-hydroxy-3,5-dimethoxybenzylidene)-2-met hyl-lH-inden-3- yl)acetic acid (120 mg, 0.324 mmol) (see (E) under Example 1 above), and 1, 1'- carbonyldiimidazole (100 mg, 0.61 mmol) in 5 mL of anhydrous methylene chloride was stirred for 30 min at room temperature. To the reaction mixture (l-methyl-lH-pyrrol-2- yl)methanamine (110 μΕ, 1.0 mmol) and 0.5 mL of pyridine were added. The mixture was stirred for 2 h, quenched with 1 ml of 30% potassium hydroxide solution, diluted with 25 mL of methylene chloride, neutralized with 2 mL of acetic acid, washed with water (20 mL><3) and dried with sodium sulfate. The organic layer was concentrated and the residue was purified with silica gel column eluted with hexane/acetone. The major yellow fraction was collected, and after concentration the residue was treated with acetone/hexane, sonicated for lh, and stored in a -20 °C freezer for 2h. The precipitate was collected by filtration, and the titled compound (019) was obtained as a yellow solid (75mg).

EXAMPLE 4

[0241] This example illustrates the synthesis of various other exemplary essential precursor compounds that can be used in synthesis of diverse compounds of formulas I and II of the present invention. For instance, when the same synthetic approach (e.g., Scheme I) is used as illustrated in Examples 1-3, except in the acetamide-forming step (e.g., step F, in Example 1) using the appropriate precursor acetic acid derivative reacted with any of a wide variety of primary or secondary amines to produce the corresponding acetamide compounds having a wide variety of substituents at R' and R". Then, these variously substituted acetamide compounds can be used as precursors for synthesis of compounds of formulas I and II having a wide variety of substituents at R. EXAMPLE 5

[0242] This example illustrates the synthesis of another specific exemplary precursor compound, 049, that can be used in synthesis of certain specific exemplary compounds of formulas I and II of the present invention.

[0243] To a stirred solution of (Z)-2-(5-methoxy-l-(4-hydroxy-3,5- dimethoxybenzylidene)-2-methyl-lH-inden-3-yl)acetic acid (synthesized as in EXAMPLE 1) (100 mg, 0.27 mmol) in dichloromethane (5 ml), Ι, -carbonyl diimidazol (48 mg, 0.30 mmol) was added in one portion resulting in the evolution of C0 ₂ gas. The yellow solution was allowed to stir for 45 minutes, then N-benzyl methyl amine (40 mg, 0.32 m mol.) was added and the reaction was heated to 45 °C for 2 hrs. Pyridine (3 ml) was added and the mixture was further heated for three more hrs at 45 °C. Pyridine was removed under vacuum and the residue was extracted with dichloromethane, followed by washing the organic layer with water and brine, and then dried (anhydrous MgS04). The filtered organic layer was evaporated under vacuum and the residue was purified by silica column chromatography eluting with hexane- acetone (1 : 1). The compound 149 was recrystallized from EtOAc- hexane as a yellow solid (45 %). Scheme IXa below summarizes and illustrates the synthesis of 149:

Scheme IXa

EXAMPLE 6

[0244] This example illustrates synthesis of some additional specific exemplary precursor compounds used for synthesis of certain other exemplary compounds of formulas I and II of the present invention. In particular, shown here are syntheses of precursor compounds 151, 152, 153 and 155 (a synthesis of 154 was illustrated previously herein, under Scheme III). These compounds were chosen to further illustrate and reinforce that extensive variations in substituents on the aryl and heteroaryl rings, and variations of E, R' and R" can be achieved readily using methods well known to those of ordinary skill in the art. The synthesis Schemes IXb-e below all start with the key intermediate, the desired substituted or unsubstituted indenyl acetic acid, synthesis of which is illustrated in detail in previous examples herein.

Scheme IXb

Scheme IXc

o

Scheme IXe

[0245] Additional experimental details for the above-described syntheses of 151-155 are as follows, using synthesis of 151 to illustrate. To a stirred solution of (Z)-2-(5-fluoro-l-(4- hydroxy-3,5- dimethoxybenzylidene)-2-methyl-lH-inden-3-yl)acetic acid (prepared as in EXAMPLE 1) (100 mg, 0.27 mmol) in dichloromethane (5 ml) , Ι, Ι '-carbonyl diimidazol (48 mg, 0.30 mmol) was added in one portion. The solution was stired for 45 minutes before O- phenyl hydroxylamine hydrochloride (47 mg, 0.32 mmol) was added, and the reaction was heated to 45 °C for 2 hrs. Pyridine (3 ml) was added and the mixture was further heated for three more hrs at 45 °C. Pyridine was removed under vacuum and the residue was extracted with dichloromethane. The organic layer was washed with water, brine and dried (anhydrous MgS04) .The filtered organic layer was evaporated under vacuum and the residue was purified by silica column chromatography eluting with hexane- acetone (1 : 1). The compound 151 was recrystallized from EtOAc- hexane as a yellow solid in 40% yield. Similar experimental details are employed in the syntheses of compounds 152-155 except using the appropriate precursor compounds and routine minor procedural modifications as needed, which are familiar to those skilled in the art.

EXAMPLE 7

[0246] This example illustrates the synthesis of other specific exemplary precursor compounds, 214 and 210, which can be used in synthesis of other specific exemplary compounds of formulas I and II of the present invention.

[0247] To a stirred mixture of (Z)-2-(5-fluoro-l-(4-hydroxy-3,5-dimethoxybenzylidene)- 2-methyl-lH-indene-3-yl)acetic acid (prepared according to EXAMPLE 1) ( 370 mg, lmmol) , sodium hydroxide ( 160 mg, 4 mmol), and H ₂ 0 ( 10 mL), benzoyl chloride (1 mmol) was added dropwise at 0°C. The reaction mixture was stirred for 2 hrs, acidified, filtered, the precipitate extracted with 20 ml hot water and the insoluble part was recrystallized from ethyl acetate-hexane to give (Z)-2-(l-(4-(benzoyloxy)-3,5-dimethoxybenzylidene)-5-fluoro- 2- methyl-lH-inden-3-yl)acetic acid in 70 % yield. To a stirred solution of the latter compound (100 mg, 0.21 mmol) in dichloromethane ( 5 ml), Ι, -carbonyl diimidazol (38 mg,

0.23mmol) was added in one portion resulting in the evolution of C0 ₂ gas. The yellow solution was allowed to stir for 45 minutes, then N-methyl furfurylamine (28 mg, 0.25 mmol) was added, and the reaction was heated to 45 °C for 2 hrs. Pyridine (3 ml) was added and the mixture was heated for three more hrs at 45 °C. Pyridine was removed under vacuum and the residue was extracted with dichloromethane. The organic layer was washed with water and brine, and dried (anhydrous MgS04). The filtered organic layer was evaporated under vacuum and the residue was purified by silica column chromatography eluting with hexane- acetone (1 : 1). The product 214 was recrystallized from EtOAc- hexane in 65 % yield as a yellow solid.

[0248] Compound 214 (35 mg) was dissolved in methanolic ammonia (12 mL, 7N) and the solution stirred overnight. Solvent was evaporated under vacuum and the residue was purified by silica column chromatography by eluting with hexane-acetone (1 : 1). The product 210 was recrystallized from ethyl acetate-hexane as a yellow solid (55% yield). The synthesis is further illustrated and summarized in Scheme IXf below:

Scheme IXf

EXAMPLE 8

[0249] Table 1 provides the 1H- MR data confirming structures of representative precursor compounds (007 and 006; see Examples 1 and 2) used for synthesis of compounds of formulas I and II of the invention. All spectra were recorded, using DMSO-d ⁶ as solvent, at 400 MHz. Table 1. ^-NMR data of exemplary precursor compounds that can be used for synthesis of certain exemplary compounds of formulas I and II of the present invention.

EXAMPLE 9

[0250] This example illustrates synthesis of a compound of formula II of the present invention, 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6-fluoro-2- methyl-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)acetamide (083), starting from compound 007 which is prepared according to EXAMPLE 1. This example furthermore illustrates that a compound of formula II, here specifically compound 083, can be converted to a compound of formula I of the present invention, here specifically compound 088.

[0251] To a solution of 007 (120 mg, 0.27 mmol) in dichloromethane ( 20 ml) at -15 °C, a dichloromethane solution of DDQ (30 mg, 0.14 mmol, 12 ml) was slowly added under argon. The reaction mixture was stirred at 0-4 °C for 10 min, then diluted with hexane and immediately applied to a silica column and eluted with hexane-acetone (1 : 1). The product 083, which is a compound of formula II, was recrystallized as an orange solid from acetone- hexane (50 % yield). For preparation of compound 088, which is a compound of formula I, the following procedure was typically used.

[0252] To a -40 °C suspension of copper (1) iodide (52 mg) in 3 mL of anhydrous ether was added methyl lithium (400 uL, 3 % solution in 2-Me THF ) dropwise over the course of 15 min, until the initially formed yellow precipitate completely dissolved. The reaction mixture was then allowed to warm to 0°, then a solution of 2-(3-((3,5-dimethoxy-4- oxocyclohexa-2,5-dien-l-ylidene)methyl)-6-fluoro-2-methyl-lH -inden-l-ylidene)-N-(furan- 2-ylmethyl)acetamide (083), 25 mg, 0.056 mmol) in 2 mL of anhydrous THF/ether (3 :2) was added dropwise. The reaction mixture was allowed to warm to ambient temperature for 2h, then cooled to 0°C. Acetic acid (15 %, 0.4 mL) was added slowly and the mixture was then washed with saturated H ₄ C1 solution, dilute H ₄ OH solution and water. The organic phase was dried over Na ₂ S0 ₄ , filtered, and concentrated under vacuum. The residue was crystalized from acetone-hexane to obtain 088 as a light orange solid in 60 % yield.

[0253] These syntheses of 083 and 088 are summarized and illustrated in Scheme Xa below:

Scheme Xa

[0254] This example illustrates synthesis of another compound of formula II of the present invention: 2-(3-((3,5-dimethoxy-4-oxocyclohexa-2,5-dien-l-ylidene)methy l)-6- methoxy-2-methyl-lH-inden-l-ylidene)-N-(furan-2-ylmethyl)ace tamide (084), starting from compound 006 prepared according to EXAMPLE 2. This example furthermore illustrates that a compound of formula II, here specifically compound 084, can be converted to a compound of formula I of the present invention, here specifically compound 170.

[0255] To a solution of 006 (100 mg, 0.22 mmol) in dichloromethane (15 ml) at 0 °C, a dichloromethane solution of DDQ (25 mg, 0.11 mmol, 12 ml) was slowly added under nitrogen. The reaction mixture was stirred at 0-4 °C for 10 min, then diluted with hexane and immediately applied to a silica column and eluted with hexane-acetone (1 : 1). The product 084 was recrystallized as a red solid from acetone-hexane (35 % yield). For preparation of compound 170, which is a compound of formula I, the following is a typical procedure used.

[0256] To a -40 °C suspension of copper (1) iodide (52 mg) in 3 mL of anhydrous ether was added methyl lithium (400 μΕ, 3 % solution in 2-Me THF) dropwise over the course of 15 min, until the initially formed yellow precipitate completely dissolved. The reaction mixture was then allowed to warm to 0°, then a solution of 2-(3-((3,5-dimethoxy-4- oxocyclohexa-2,5-dien-l-ylidene)methyl)-6-methoxy-2-methyl-l H-inden-l-ylidene)-N- (furan-2-ylmethyl)acetamide (084) (25 mg, 0.054 mmol) in 2 mL of anhydrous THF/ether (3 :2) was added dropwise. The reaction mixture was allowed to warm to ambient temperature for 2h, then cooled to 0°C. Acetic acid (15 %, 0.4 mL) was added slowly, and the mixture was then washed with saturated H ₄ C1 solution, dilute H ₄ OH solution and water. The organic phase was dried over Na ₂ S0 ₄ , filtered, and concentrated under vacuum. The residue was crystalized from acetone-hexane to obtain 170 as a light orange solid in 60 % yield.

[0257] These syntheses of 084 and 170 are summarized and illustrated in Scheme Xb below:

Scheme Xb

EXAMPLE 11

[0258] This example illustrates synthesis of another compound, 270, which is of formula II of the present invention, starting from compound 015 which was prepared as described below. This example furthermore illustrates that a compound of formula II, here specifically compound 270, can be converted to a compound of formula I of the present invention, here specifically compound 271.

[0259] To a stirred solution of (Z)-2-(5-fluoro-l-(4-hydroxy-3,5-dimethoxybenzylidene)- 2-methyl-lH-inden-3-yl)acetic acid (prepared as in EXAMPLE 1) (100 mg, 0.27 mmol ) in dichloromethane (5 ml), Ι, -carbonyl dimidazol (48 mg, 0.30 mmol) was added in one portion resulting in the evolution of C02 gas. The yellow solution was allowed to stir for 45 minutes, then benzylamine (0.32 mmol) was added, and the reaction was heated to 45 °C for 2 hrs. Pyridine (3 ml) was added and the mixture was further heated for three more hrs at 45 °C. Pyridine was removed under vacuum and the residue was extracted with

dichloromethane. The organic layer was washed with water and brine, and dried (anhydrous MgS04). The filtered organic layer was evaporated under vacuum and the residue was purified by silica column chromatography eluting with hexane-acetone ( 1 : 1 ). (Z)-N-Benzyl- 2-(5-fluoro-l-(4-hydroxy-3,5-dimethoxybenzylidene)-2-methyl- lH-inden-3-yl)acetamide (015) was recrystallized from EtOAc- hexane as a yellow solid (45 % yield).

[0260] To a solution of 015 (30 mg, 0.065 mmol) in dichloromethane (5 ml) at -15 °C, a solution of DDQ in dichloromethane (8.0 mg, 0.035mmol) was slowly added under nitrogen. The reaction was stirred at 0-5 °C for 10 min, then the reaction mixture was diluted with hexane and immediately applied to a silica column, eluted with hexane-acetone ( 1 : 1). The product compound 270 was recrystallized from acetone-hexane as a red solid (35%).

[0261] For conversion of compound 270, which is of formula II, to compound 271, which is of formula I, essentially the same procedures as described in EXAMPLES 9 and 10, above, were followed. The syntheses of compounds 270 and 271 are summarized and illustrated further in Scheme Xc below:

Scheme Xc

EXAMPLE 12

[0262] In a manner analogous to the above, suitable precursor compounds, such as compounds synthesized according to EXAMPLES 1-7, can be converted to compounds of formulas I and II of the present invention by the general approaches analogous to those described in Schemes Xa,b,c above, and as further summarized and illustrated below in

Schemes Xd-q. heme Xd

Scheme Xf

Scheme Xg

Scheme Xh

Scheme Xj

Scheme Xk

Scheme XI

Scheme Xm

Scheme Xn

Scheme Xo

cheme Xq

EXAMPLE 13

[0263] This example illustrates synthesis of various derivative compounds having formula II and I which can be made from precursor compounds having a formula of II or I. These examples, which start with compound 083, which is prepared from compound 007 (see EXAMPLE 9), as well as those shown in EXAMPLES 10, 11 and 12, are provided for illustration only, and, as can be appreciated by one of ordinary skill in the art, these same or similar reaction Schemes XIa, b below can be applied to myriad different starting compounds of formula II or I, to yield the desired derivative compounds of formula II or I, such as for example to produce other exemplary compounds including but not limited to 172, 173, 193, 197, 233, 253, 263, 273, 277, and 282.

Scheme XIa

Scheme Xlb

EXAMPLE 14

[0264] This example illustrates synthesis of various derivative compounds having formula II and I which can be made from precursor compounds having a formula of II or I. These examples, which start with precursor compounds 260, 261 and 181, synthesized according to previous examples, as well as those compounds shown in EXAMPLES 10, 11, 12 and 13, are provided for illustration only, and, as can be appreciated by those of ordinary skill in the art, these same or similar reaction Schemes Xlla-c below can be applied to myriad different starting compounds of formula II or I, to yield the desired derivative compounds of formula II or I, such as for example to produce other exemplary compounds including but not limited to 086 and 093.

Scheme Xlla

Scheme Xllb

Scheme

EXAMPLE 15

[0265] This example illustrates synthesis of various additional derivative compounds having formula II and I which can be made from precursor compounds having a formula of II or I. In particular, this example starts with precursor compound 084, synthesized according to Scheme Illb, and the products in this example are compounds are 177 and 178. These exemplary compounds are provided for illustration only, and, as can be appreciated by those of ordinary skill in the art, this same or similar reaction Scheme XIII below, containing adaptations from the methods of Snyder, H.R.; Putnam, R.E. J. Am. Chem. Soc. 1954, 76, 1893 (amide 1,4 reduction), can be applied to myriad different starting compounds of formula II or I, to yield the desired derivative compounds of formula II or I which contain the alkylenedioxy ring in place of the carbonyl as illustrated in this scheme. Scheme XIII

EXAMPLE 16

[0266] Table 2 provides the 1H-NMR data confirming structures of representative compounds of formulas I and II of the invention. All spectra were recorded, using DMSO-d6 or CDC1 ₃ as solvent, at 400 MHz.

[0267] Table 2. 1H-NMR data of representative compounds of formulas I and II of the invention.

EXAMPLE 17

[0268] This example illustrates typical tumor cell growth assays that are employed in the present invention. Cells used in such assays typically include A-549, HT-29, MDA-MB-231, Colo-205, Caco2, HCT-116, SW-480, and DLD-1 human cancer cells that are obtained from the American Type Culture Collection (ATCC). Human tumor cells are cultured using standard methods in RPMI-1640 growth medium supplemented with 5% fetal bovine serum (FBS). Normal rat kidney (NRK) and Ki-Ras transformed NRK cells (K-NRK) are obtained from ATCC, and are cultured according to supplier recommendations. CellTiter-Glo ATP cell growth assay reagents are obtained from Promega and are used according to the manufacturer's protocol. Inhibitors of EGFR, Raf, and MEK are obtained from Selleck Chemicals. Cells are typically plated at a density of 5,000 cells per well in 96-well microplates or 1,250 cells per well in 384-well plates, and are allowed to attach for at least 4h. Test compounds are dissolved in dimethyl sulfoxide (DMSO), and this working stock is further diluted in growth medium for addition to cell cultures. Serial dilutions of the test compound are prepared in growth medium containing an equal amount of DMSO not exceeding 0.2% final concentration. Each compound concentration is tested in at least 3 separate samples per cell line. At the end of a 3-day treatment period, growth inhibition is analyzed using a bioluminescent assay of ATP concentration (Promega CellTiter-Glo) according to the manufacturer's protocol. Resulting luminescence is measured using the luminescence cartridge of the Molecular Devices Spectramax Paradigm microplate reader. Relative growth inhibition for each sample is determined by comparison with the values that are obtained for vehicle treated control samples. Growth inhibition values are plotted with the GraphPad Prism5 software using the 4-parameter logistic fit to obtain IC50 values, which corresponds to the growth inhibitory potency of the compound.

EXAMPLE 18

[0269] This example illustrates a Ras binding domain assay which can be used in an embodiment of the present invention to measure Ras activation status. The activation state of Ras in cell lines is assayed using the Active Ras Pull-Down and Detection Kit (Thermo Scientific). Cell lines are cultured as described above. Cells are disrupted with non-ionic detergent, and the active (GTP-bound) Ras is isolated by its high affinity for Raf via precipitation with sepharose-bound GST-Raf fusion protein. The precipitated active Ras is then subjected to polyacrylamide gel electrophoresis (PAGE) and is transferred to

nitrocellulose membrane (western blot). Detection is achieved using the anti-Ras mouse primary antibody and anti-mouse-horseradish peroxidase conjugated secondary antibody. Paired samples of whole cell lysate are analyzed by western blot for expression level of total Ras protein as well as a gel loading control, glyceraldehyde 3 -phosphate dehydrogenase (GAPDH). Digital enhanced chemiluminescence imaging of the resulting western blots is performed using a Syngene G:Box. The intensity of Ras bands from each cell line and the corresponding GAPDH bands is quantitated using NIH ImageJ, and expressed as "relative Ras activation." EXAMPLE 19

[0270] This example illustrates the determination of levels of Ras activation in different cancer cell lines. In particular, a panel of human colorectal cancer cell lines was selected to further describe the selectivity of the compounds for cells with activated Ras. Three of the cell lines in the panel have been reported to harbor ras mutations: HCT-116, DLD-1, and SW-480 (Stoneman and Morris, Clin Mol Pathol., 48, M326-332Q995)). Three of the cell lines were reported to express wild type Ras: HT-29, Caco-2, and Colo-205(Stoneman and Morris, supra; Shirasawa et al., Science, 260, 85-88 (1993)). The activation state of Ras in the cell lines was assayed using the Active Ras Pull-Down and Detection Kit. The ratio of the intensity of the Ras to GAPDH bands was expressed as relative Ras activation, which is presented above each lane in FIG. 2. This experiment demonstrated that the level of Ras activation in HCT-116 > DLD-1 ~ SW-480 > Caco2 > HT-29 > Colo205.

EXAMPLE 20

[0271] This example illustrates antitumor activities of compounds of formulas I and II of the invention. Here specifically, well-established human colon tumor cells with widely divergent Ras activation status (e.g., see previous EXAMPLE 19) were used to demonstrate antitumor activity and Ras selectivity of exemplary Ras-inhibitory compounds of formula I or II of the present invention. Cells thus employed in this example were HCT-116, which are highly Ras-driven cancer cells expressing mutant Ras, and HT-29, which are cancer cells lacking activated Ras. Cells were plated at 5000 cells/well in 96-well plates, and viable cell numbers were measured using the Cell Titer Glo ATP luminescence assay (Promega). FIGS. 3 A-3E show the results of these studies for exemplary compounds 083, 084, 088, 260 and 270, respectively. From this experiment, the calculated HT-29/HCT-116 selectivity values for the aforementioned compounds were 104, 83, 43, 192 and 67, respectively. These selectivity values are the ratios of each compound's potency (IC50) to inhibit the growth of cells lacking activated Ras ((HCT-29) relative to that of cells with activated Ras (HCT-116), demonstrating selectivity for the Ras-mutant-containing cells. Thus although human cancer cell growth is markedly inhibited for both of these representative types of cancer cells, which are widely employed in cancer research to measure anticancer activity of compounds, the compounds of formulas I and II of the invention are consistently more potent toward the cells containing activated Ras. EXAMPLE 21

[0272] This example further illustrates antitumor activities of compounds of formulas I and II of the invention. Here specifically, well-established human cancer cells with widely divergent Ras activation status (e.g., for HT-29 cells see previous EXAMPLE 19, and for A549 cells see Okedula et. al., Am. J. Pathol., 164, 91-100 (2001)) were used to demonstrate antitumor activity and Ras selectivity of exemplary Ras-inhibitory compounds of formula I or II of the present invention. Cells thus employed in this example were A549, which are highly Ras-driven human cancer cells harboring activated Ras (Okedula et al., supra), and HT-29, which are cancer cells lacking activated Ras. Cells were plated at 5000 cells/well in 96-well plates, and viable cell numbers were measured using the Cell Titer Glo ATP luminescence assay (Promega). FIGS. 4A-4E show the results of these studies for exemplary compounds 083, 084, 088, 260 and 270, respectively. From this experiment, the calculated HT-29/A549 selectivity values for the aforementioned compounds were, 60, 62, 38, 93 and 31,

respectively. These selectivity values are the ratios of each compound's potency (IC50) to inhibit the growth of cells lacking activated Ras ((HCT-29) relative to that of cells with activated Ras (A549), demonstrating selectivity for the ras-mutant-containing cells. Thus although human cancer cell growth is markedly inhibited for both of these representative types of cancer cells, which are widely employed in cancer research to measure anticancer activity of compounds, the compounds of formulas I and II of the invention are consistently more potent toward the cells containing activated Ras.

EXAMPLE 22

[0273] This example illustrates non-selective growth inhibition with known Ras pathway inhibitors which are not compounds of the present invention. In particular, the growth inhibitory activity of commercially available compounds which are active in the Ras signal transduction pathway were tested in the same panel of cell lines using the CellTiter-Glo assay. Cells were seeded in 384-well plates and allowed to attach. Ten-fold serial dilutions of compounds were tested. Each compound concentration was tested in at least 3 separate samples per cell line. As indicated in Table 3 below, the potency of the EGF receptor inhibitor compounds ranged from 4 μΜ to >20 μΜ, with no pattern of selectivity with regard to Ras activation. Likewise, the C-Raf inhibitor, GW5074, did not show selectivity for cell lines expressing activated Ras. The B-Raf inhibitors tested were generally active in the low micromolar range, but were significantly more potent in Colo-205 cells, which have the lowest level of active Ras. The MEK inhibitor, Selumetinib was also most potent against COLO-205 and HT-29 cell lines showing, if anything, a "reverse" selectivity toward inactive Ras compared with the compounds of this invention.

[0274] Table 3. Non Ras-selective growth inhibition with known Ras pathway inhibitors.

[0275] Together, these data demonstrate the activated Ras selective growth inhibitory activity of the compounds of this invention. This is in contrast to the current clinically used inhibitors of proteins within the Ras signaling cascade that exhibit no selectivity or selectivity for cells lacking activated Ras.

EXAMPLE 23

[0276] This example illustrates the treatment of a mammalian patient with a compound of the present invention. For example, the antitumor activity of a compound of formula I or II of the present invention can be evaluated in an orthotopic mouse model of lung cancer utilizing human A549 lung adenocarcinoma cells. Toxicity and efficacy are assessed by determining treatment effects on weight gain and by necropsy observations. Treatment effects on tumor growth in live mice are also measured with human A549 lung

adenocarcinoma cells that are engineered to contain a luciferase expression vector using an In Vivo Imaging System (IVIS). In brief, female athymic nude-Foxnlnu mice, 6-7 weeks old are randomly divided into two groups that are treated with either vehicle alone or with a compound of formula I or II (e.g., up to 100 mg/kg). Each group typically contains about 15 mice in which about 10 are implanted with non-luciferase A549 cells and 5 are implanted with luciferase A549 cells. Treatment is initiated 5 days before implanting tumor cells and is administrated to both groups by gastric gavage twice a day. For tumor cell implantation, cultured luciferase or non-luciferase human A549 lung tumor cells are collected and are mixed with equal volume of Matrigel at a final concentration of 0.250 mg/mL. The mice are weighed and anesthetized and a mark is placed on the skin at the lateral dorsal line, approximately 1.5 cm above the lower rib line just below the inferior border of the scapula. A pre-cooled 0.5 mL insulin syringe with a permanently attached 28G needle is loaded with 75 microliters of a cell suspension containing about one million cells and is inserted at the mark to a depth of approximately 5 mm. Body weight is measured twice a week and IVIS imaging is done once a week.

EXAMPLE 24

[0277] This example, employing genetically engineered isogenic tumor cell lines, shows how Ras selectivity of compounds of formulas I and II of the present invention can be confirmed. In one approach, human colon HT29 tumor cells are transfected with retroviral mutant H-Ras-G12V (H-Rasm) or retroviral control (Retro) and stable clones are selected by puromycin. The cell lines, including parental lines (Par) are treated with a compound of formula I or II for 72 hours and viable cell numbers are measured using the Cell Titer Glo assay. Ras pull-downs are performed with GST-RBD-C-Raf kits (ThermoSci) following cell extraction and are detected by Ras antibody on Western-blots for the amount of active GTP- Ras. Total Ras levels among these cell extractions are detected by the same Ras antibody. In another approach, human lung H322 tumor cells are stably transfected with retroviral mutant H-Ras-G12V (H-Rasm) or retroviral control (Retro). Cell lines, including parental lines (Par) are treated with a compound of formula I or II for 72 hours and viable cell numbers are measured using the Cell Titer Glo assay. Ras pull-downs are performed with GST-RBD-C- Raf kits (ThermoSci) following cell extraction and are detected by Ras antibody on Western- blots for the amount of active GTP-Ras. Total Ras levels among these cell extractions are detected by the same Ras antibody. EXAMPLE 25

[0278] This example illustrates how representative Ras-inhibitory compounds of formulas I and II can be shown to interact with high affinity directly with activated Ras to disrupt Ras interactions with a normal binding partner. Inhibition studies of Ras binding to Raf by treatment with Ras inhibitors using a Raf pull-down assay is conducted in vitro in cell lysates or in intact cells. For in vitro experiments, whole cell lysates from human H322 lung tumor cells transfected with activated (mutant) H-Ras are incubated for 30 minutes at room temperature with a compound of formula I or II at the desired concentrations. GTP -bound (active) Ras is precipitated with GST-Rafl-RBD/GSH Sepharose (Thermo Scientific) and is detected by western blotting. Ras protein is quantified in the blots by densitometry. As a positive control, lysates are incubated with 5 mM GDP to deactivate Ras. For experiments involving intact cells, the same method as described above is used except intact H322 lung tumor cells are incubated with a compound of formula I or II for 1 hour at 37oC prior to the Raf pull-down assay.

EXAMPLE 26

[0279] This example illustrates how the potential toxicity of administration of therapeutic or preventive doses of representative compounds of formulas I and II of the present invention can be assessed in vivo by measuring animal weight gains over time. Female athymic Nude- Foxnlnu mice, 6 - 7 weeks old are purchased from Harlan and acclimated for 1 week. Mice have access to water and food ad libitum for the duration of the experiment. HCT-116 cells are cultured under optimal conditions (RPMI media, 5% fetal bovine serum, antibiotics, glutamate at 5% C02 and 36°C). On the day of inoculation, cells are collected from flasks 70-80% confluent. Mice are inoculated with about 5x106 cells/100 μΐ _^ in the right flank and about 10x106 cells/100 μΐ. in the left flank. The test compound of formula I or II is administered twice daily by ip injection for 14 days at a dosage of 1-100 mg/kg. The vehicle for all test compounds typically contains ethanol, polyethylene glycol-300, and water, typically at a ratio of about 5:75:20. Percent body weight change of mice treated with vehicle alone or with vehicle with test compounds relative to the body weight of the mice prior to treatment is measured. EXAMPLE 27

[0280] This example further illustrates how the efficacious therapeutic antitumor treatments of a representative Ras-driven tumor in vivo with exemplary Ras-inhibitory compounds of formulas I and II can be demonstrated. Female athymic Nude-Foxnlnu mice, 6-7 weeks old are purchased from Harlan and acclimated for 1 week. Mice have access to water and food ad libitum for the duration of the experiment. HCT-116 cells are cultured in optimal conditions (RPMI media, 5% fetal bovine serum, antibiotics, glutamate at 5% C02 and 36°C). On the day of inoculation, cells are collected from flasks 70-80% confluent. Mice are inoculated with 5x106 cells/100 μΙ_, on the right flank. Treatment is initiated once tumors reach an average size of approximately 50 mm3. The selected test compound of formula I or II is administered twice daily by ip injection for 14 days at a dosage of 1-100 mg/kg. The vehicle for all test compounds typically contains ethanol, polyethylene glycol-300, and water, typically at a ratio of 5:75:20. In another approach, a "prevention" protocol rather than a "therapeutic" protocol is employed. Female athymic Nude-Foxnlnu mice, 6-7 weeks old are purchased from Harlan and acclimated for 1 week. Mice have access to water and food ad libitum for the duration of the experiment. HCT-116 cells are cultured in optimal conditions (RPMI media, 5% fetal bovine serum, antibiotics, glutamate at 5% C0 ₂ and 36°C). On the day of inoculation, cells are collected from flasks 70-80% confluent. Mice are inoculated with 10x106 cells/100 μΙ_, on the left flank, and treatment is initiated one day following tumor implantation. The test compound is administered twice daily by ip injection for 14 days at a dosage of 1-100 mg/kg. The vehicle for all test compounds typically contains ethanol, polyethylene glycol-300, and water, typically at a ratio of 5:75:20. Treatment is initiated one day following tumor implantation.

[0281] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0282] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly

contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0283] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.