Methods and compounds for treating proliferative diseases

The compounds disclosed herein are indolocarbazoles of Formula (I), which are potent CDK4 inhibitors, and are useful in the treatment of cell proliferative disorders, including cancer. Formula (I).

The present invention relates to a method of treating proliferative diseases using indolo[6,7-a]pyrrolo[3,4-c]carbazole-6,8-diones as therapeutic agents. Cancer is a heterogeneous group of diseases presenting in various forms in various tissues but having in common the characteristic of uncontrolled cell proliferation. For some time, cancer has been recognized as a disease of uncontrolled cell proliferation. Thus, the rapidly proliferating cell has been the target of cancer chemotherapy. The goal is to find agents that are more effective against cancer cells than against normal cells. As the basic science of the cell progressed, it was shown that certain anticancer agents were more effective against malignant cells at certain stages of the cell cycle than against cells at other stages of the cell cycle.

In addition to the kinases, which can help to move the cell from one phase of division to the next, there are CDK inhibitors (CKIs) that block the actions of specific cyclin-CDK complexes. The CKIs halt cell cycle progression and cause cells to enter the quiescent G0phase. The CKIs of the INK4 group, including p15, p16, p18, and p19, block the cyclin-CDK4 and cyclin-CDK6 complexes.

One researcher (KAMALATI, T., et al.,Clin. Exp. Metastasis16, 415–426 (1998) et al. (1998)) treated normal human epithelial cells so that they overexpressed cyclin D1. These transfected cells had reduced growth factor dependency, a shortened cell cycle time, thus providing the cells with a growth advantage. In 123 colorectal carcinoma specimens, those staining strongly for cyclin D1 corresponded to patients with a 5-year survival rate of 53.3% while those that were negative or weakly staining had 5-year survival rates of 96.2 and 78.8% (MEEDA, K., et al.,Oncology55, 145–151 (1998); PALMQVIST, R., et al.,Europ. J. Cancer34, 1575–1581 (1998)).

Amplification of CCND1 was found in 25% of dysplastic head-and-neck lesions, and 22% of head-and-neck carcinomas. Overexpression of cyclin D1 was found in 53% of head-and-neck carcinomas. This indicates that in this disease, like breast cancer, alterations in cyclin D1 occur at the very earliest stages of tumorigenesis (KYOMOTO, R., et al.,Int. J. Cancer(Pred. Oncol.) 74, 576–581 (1997); PIGNATARO, L., et al.,J. Clin. Oncol. 16, 3069–3077 (1998) et al., 1998). In a study of 218 specimens of esophageal squamous cell carcinoma, patients with cyclin D1-positive tumors had significantly worse survival than patients with cyclin D1-negative tumors (SARBIA, M. et al.,Int. J. Cancer(Pred. Oncol. 84, 86–91 (1999)).

In eight human esophageal carcinoma cell lines, 7 (87.5%) and 6 (75%) cell lines had homozygous deletions of the p16 and p15 genes (KITAHARA, K. et al.,J. Exp. Therap. Oncol. 1, 7–12 (1996)). All of the p16-negative cell lines express high levels of cyclin D1 and CDK4.

The Rustgi laboratory (MUELLER, A, et al.,Cancer Res. 57, 5542–5549 (1997); NAKAGAWA, H, et al.,Oncogene14, 1185–1190 (1997)) developed a transgenic mouse in which the Epstein-Barr virus ED-L2 promoter was linked to human cyclin D1 cDNA. The transgene protein localizes to squamous epithelium in the tongue and esophagus, resulting in a dysplastic phenotype associated with increased cell proliferation and indicating that cyclin D1 overexpression may be a tumor-initiating event. In a series of 84 specimens of soft-tissue sarcomas, there was no amplification of the CCND1 gene but there was overexpression of cyclin D1 in 29% of cases. The overexpression of cyclin D1 was significantly associated with worse overall survival (KIM, S. H., et al.,Clin. Cancer Res. 4, 2377–2382 (1998); YAO, J., et al.,Clin. Cancer Res. 4, 1065–1070 (1998)).

Another researcher (MARCHETTI, A., et al.,Int. J. Cancer75, 187–192 (1998)) found that abnormalities of cyclin D1 and/or Rb at the gene and/or expression level were present in more than 90% of a series of non-small cell lung cancer specimens, indicating that cyclin D1 and/or Rb alterations represent an important step in lung tumorigenesis. In 49 out of 50 pancreatic carcinomas (98%), the Rb/p16 pathway was abrogated exclusively through inactivation of the p16 gene (SCHUTTE, M., et al.,Cancer Res. 57, 3126–3130 (1997)).

Mantle cell lymphoma is defined as a subentity of malignant lymphomas characterized by the chromosomal translocation t(11;14) (q13;q32) resulting in overexpression of cyclin D1 and, in addition, about 50% of these tumors have deletion of the p16 gene (DREYLING, M. H., et al.,Cancer Res. 57, 4608–4614 (1997); TANIGUCHI, T., et al.,Jpn. J. Cancer Res. 89, 159–166 (1998)).

Olomoucine is an inhibitor of Cdc2, CDK2, CDK5 and MAP kinase in micromolar concentrations and has much weaker effects toward CDK4 and CDK6 (GARRETT, M. D.Current Opin. Genetics Develop. 9, 104–111 (1999)). Olomoucine has been reported to arrest several cell lines in G1and G2phases of the cell cycle and block known CDK-dependent cellular activities.

Much has already been published on the antineoplastic properties of certain compounds such as bisindolylmaleimides, indolocarbazoles, and derivations thereof. Staurosporine and UCN-01 are members of this broad molecular class (COLEMAN, K. G., et al.,Ann. Reps. Med. Chem. 32, 171–179 (1997)). For example, U.S. Pat. No. 5,856,517 discloses substituted pyrroles, which are useful as antiproliferative agents in the treatment of cancer. U.S. Pat. No. 5,292,747 discloses substituted pyrroles useful in the prevention or control of oncological disorders. U.S. Pat. No. 5,721,245 discloses indolylpyrrolones useful in controlling oncological disorders. U.S. Pat. No. 5,438,050 (Godecke) discloses indolocarbazole derivatives useful in the prevention and treatment of cancer. U.S. Pat. No. 5,705,511 and U.S. Pat. No. 5,591,855 discloses fused pyrrolocarbazoles for the inhibition of growth associated with hyperproliferative states.

In addition to the kinases, which control the cell division cycle, there are over several hundred other kinases found in the human body. These kinases perform such diverse functions as growth factor and cytokine signal transduction, inflammatory mediators, biochemical routes controlling activity of nuclear transcription factors and apoptotic pathways. Kinase inhibitors tend to be broadly active against all kinases. Treating patients with broadly active kinase inhibitors not only inhibits the kinases, which have a role in the disease state being treated, but also inhibits many other kinases as well. Such treatment leads to unintended side effects and is not generally acceptable. In treating proliferative diseases, it is particularly desirable to use a kinase inhibitor with narrow activity. Anti-cancer agents are generally given at high doses in order to kill as many cancer cells as possible. With such high dosing, side effects due to broad kinase inhibition can become a serious problem. Accordingly, to treat proliferative diseases, it is desirable to use kinase inhibitors, which inhibit the kinases controlling cell division while not inhibiting other unrelated kinases.

The compounds disclosed herein are indolocarbazoles, which are potent CDK4 inhibitors, and are useful in the treatment of cell proliferative disorders, including cancer.

The present invention provides compounds of Formula I

wherein

A and B are independently O or S;

R5is independently at each occurrence hydrogen, C1–C4alkyl, hydroxy(C1–C4)alkyl, (C1–C4alkyl)-NR7R8, C(O)NR7R8, C(O)—(C1–C4alkyl), or optionally substituted C3–C8cycloalkyl; and R6is independently at each occurrence hydrogen or C1–C4alkyl; or R5and R6are taken together with the nitrogen to which they are attached to form an optionally substituted saturated heterocycle;

R7and R8are independently at each occurrence hydrogen or C1–C4alkyl;

or a pharmaceutically acceptable salt thereof.

Furthermore, the present invention provides a method for the inhibition of CDK4 in a mammal comprising administering to a mammal in need of such treatment an effective amount of a compound of Formula I. Furthermore, the present invention provides the use of a compound of Formula I for the preparation of a medicament useful for the inhibition of CDK4.

Additionally, the present invention provides a pharmaceutical formulation comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable carrier, diluent or excipient.

Also, the present invention provides compounds of Formula II

wherein

A and B are independently O or S;

R5is independently at each occurrence hydrogen, C1–C4alkyl, hydroxy(C1–C4)alkyl, (C1–C4alkyl)-NR7R8, C(O)NR7R8, C(O)—(C1–C4alkyl), or optionally substituted C3–C8cycloalkyl; and R6is independently at each occurrence hydrogen or C1–C4alkyl; or R5and R6are taken together with the nitrogen to which they are attached to form an optionally substituted saturated heterocycle;

R7and R8are independently at each occurrence hydrogen or C1–C4alkyl;

or a pharmaceutically acceptable salt thereof, useful as intermediates for making compounds of Formula I.

The following definitions are to set forth the meaning and scope of the various terms used herein. The general terms used herein have their usual meanings.

As used herein, the “hyperproliferative state” refers to those cells whose unregulated and/or abnormal growth can lead to the development of an unwanted condition, for example, a cancerous condition or a psoriatic condition.

As used herein, the term “psoriatic condition” refers to disorders involving keratinocyte hyperproliferation, inflammatory cell infiltration, and cytokine alteration.

As used herein, the term “proliferative diseases” refers to diseases in which some tissue in a patient proliferates at a greater than normal rate. Proliferative diseases may be cancerous or non-cancerous. Non-cancerous proliferative diseases include epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myofibromatosis, osteoplastic tumors, other dysplastic masses and the like.

The types of proliferative diseases which may be treated are epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myofibromatosis, osteoplastic tumors, other dysplastic masses and the like.

The types of cancers which may be treated with compounds and compositions of the present invention include:

The term “effective amount” as used in “an effective amount of a compound of Formula I,” for example, refers to an amount of a compound of the present invention that is capable of inhibiting CDK4.

The general chemical terms used herein have their usual. meanings. For example, as used herein, the term “C1–C4alkyl,” alone or in combination, denotes a straight-chain or branched-chain C1–C4alkyl group consisting of carbon and hydrogen atoms, examples of which are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, and the like.

The term “C3–C8cycloalkyl” refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The term “optionally substituted C3–C8cycloalkyl” refers to a C3–C8cycloalkyl as defined herein unsubstituted or substituted once with hydroxy, C1–C4alkyl, hydroxy(C1–C4)alkyl, NR7R8, C(═O)OR7, or C(═O)NR7R8.

The term “C1–C4alkoxy,” alone or in combination, denotes an alkyl group as defined earlier which is attached via an oxygen atom, such as, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, and the like. The term “hydroxy,” alone or in combination, represents an —OH moiety. As used herein, the term “hydroxy(C1–C4)alkyl” represents a straight or branched alkyl group as defined earlier bonded through a carbon containing a —OH group as a substituent on the carbon chain.

As used herein, the term “halo” or “halogen” represents fluorine, chlorine, bromine, or iodine. A halo(C1–C4)alkyl is an alkyl group as defined earlier substituted with one or more halo atoms, preferably one to three halo atoms. However, all the hydrogen atoms in alkyl group may be replaced by halogens. As more halogens are added to an alkyl group, fluorine is preferred over the other halogens. An example of a haloalkyl is trifluoromethyl.

As used herein, the term “saturated heterocycle” is taken to be a 4–9 membered ring containing nitrogen and optionally one other atom selected from oxygen, nitrogen, and sulfur. The term “optionally substituted saturated heterocycle” is taken to be a saturated heterocycle as defined herein unsubstituted or substituted once with hydroxy, C1–C4alkyl, hydroxy(C1–C4)alkyl, NR7R8, C(═O)OR7, or C(═O)NR7R8.

As used herein the term “amino acid residue” means the product of an amino acid derivative coupled to a compound of Formula I through the N-terminus of the amino acid. The term includes both naturally occurring and synthetic amino acids and includes both the D and L form of the acids as well as the racemic form. More specifically, amino acids contain up to ten carbon atoms. They may contain an additional carboxyl group, and heteroatoms such as nitrogen and sulfur. Preferably the amino acids are α- and β-amino acids. The term α-amino acid refers to amino acids in which the amino group is attached to the carbon directly attached to the carboxyl group, which is the α-carbon. The term β-amino acid refers to amino acids in which the amino group is attached to a carbon one removed from the carboxyl group, which is the β-carbon. Some common α-amino acid residues are shown in Table I wherein the residues are given the name of the amino acids from which they are derived.

TABLE I

Suitable β-amino acid residues can be the β-amino derivative of any suitable α-amino acid residue wherein the amino group is attached to the residue through the β-carbon rather than the α-carbon relative to the carboxyl group, for example, 3-aminopropionoic acid, 3-amino-3-phenylpropionoic acid, 3-aminobutyric acid and the like:

As used herein, the term “pharmaceutically acceptable salt” includes acid and basic addition salts. Such pharmaceutically acceptable salts include inorganic acid addition salts such as hydrochloride, sulfate, and phosphate, and organic acid addition salts such as acetate, maleate, fumarate, tartrate, citrate, and methane sulfonate. Examples of pharmaceutically acceptable basic salts include metal salts are alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of pharmaceutically acceptable ammonium salts are ammonium salt and tetramethylammonium salt. Examples of pharmaceutically acceptable amine addition salts are salts with morpholine and piperidine. Examples of pharmaceutically acceptable amino acid addition salts are salts with lysine, glycine, and phenylalanine.

The preceding paragraphs may be combined to define additional preferred classes of compounds.

The compounds of Formula I are useful for the treatment of disorders of mammals, and the preferred mammal is a human.

The skilled artisan will appreciate that the introduction of certain substituents will create asymmetry in the compounds of Formula I. The present invention contemplates all enantiomers and mixtures of enantiomers, including racemates. It is preferred that the compounds of the invention containing chiral centers are single enantiomers. The present invention further contemplates all diastereomers.

The compounds of the present invention can be prepared by a variety of procedures, some of which are illustrated in the Schemes below. It will be recognized by one of skill in the art that the individual steps in the following schemes may be varied to provide the compounds of Formula I. Some of these variations are discussed below. The particular order of steps required to produce the compounds of Formula I is dependent upon the particular compound being synthesized, the starting compound, and the relative lability of the substituted moieties. Some substituents have been eliminated in the following schemes for the sake of clarity, and are not intended to limit the teaching of the schemes in any way.

A maleimide of Formula (5) and iodine and DDQ in a suitable solvent, such as dioxane, may be reacted via irradiation by a medium-pressure mercury lamp. The reaction mixture is irradiated for about 10 minutes to about 24 hours. The resulting carbazole Formula (I) may be isolated and purified by standard techniques.

The requisite maleimides of Formula (5) may be prepared from an appropriately substituted oxoacetic acid ester and an appropriately substituted acetamide as illustrated in Scheme II.

In Scheme II, maleimides of Formula (5) may be prepared from an appropriately substituted oxoacetic acid ester of Formula (2) with an appropriately substituted acetamide of Formula (1) as illustrated in Method A. Alternatively, maleimides of Formula (5) may be prepared from an appropriately substituted oxoacetic acid ester of Formula (3) with an appropriately substituted acetamide of Formula (4) as illustrated in Method B. This reaction involves a base-mediated condensation of the acetamide with the ester as described in FAUL, M. A.; WINNEROSKI, L. L.; KRUMRICH, C. A.J. Org. Chem., 63, 6053–6058 (1998). Potassium t-butoxide is preferred, either as potassium t-butoxide in t-butanol or potassium t-butoxide in THF. Preferred solvents include DMF and THF.

The formation of the indolylacetamides, Formula (1), is depicted in Scheme IIIa and IIIb. In Scheme IIIa, an appropriately substituted indole of Formula (6) is alkylated at the 3-position by N,N-dimethylmethylene ammonium chloride. An exchange of the dimethylamine to cyano is performed in the presence of sodium nitrile at reflux temperatures of dimethylformamide to afford the compounds of Formula (8). Alkylation of the indole of Formula (8) by sodium hydride and an appropriate alkylating reagent is performed to incorporate R2substitution in Formula (8a). Reaction of the acetonitrile group of Formula (8a) with hydroxide results in the formation of a compound of Formula (1).

Alternatively, as in Scheme IIIb, a compound of Formula (9) is acylated with an oxalyl halide reagent, followed by the addition of ammonium hydroxide to give the indole-3-glyoxamides of Formula (10). A selective reduction of the glyoxamide to the acetamide is accomplished by reacting a compound of Formula (10) with palladium on carbon in the presence of sodium dihydrogenphosphate monohydrate, in an aqueous-dioxane solution at elevated temperatures, to afford a compound of Formula (1).

The formation of the indolylacetamides of Formula (4) is depicted in Scheme IIIc. Alkylation of an indole of Formula (11) by sodium hydride and the appropriate alkylating reagent is performed to incorporate R4substitution in Formula (12). With the appropriate substituted 7-carboxaldehyde-indoles, indolylacetamides of Formula (4) are produced by reduction of derived phosphorylated cyanohydrins of Formula (12a) to afford compounds of Formula (13). See, for example, YONEDA, R.; HARUSAWA, S.; KURIHARA, T. Tetrahedron Lett. 1989, 30, 3681–3684; YONEDA, R.; HARUSAWA, S.; KURIHARA, T. J. Org. Chem. 1991, 56, 1827–1832. This is followed by hydrolysis of the acetonitrile group to the acetamides of Formula (4). Similar conversions of Formula (13) to give a compounds of Formula (4) are well known and appreciated in the art (See, for example, LAROCK, R. C.,Comprehensive Organic Transformations, 2ndEd., John Wiley & Sons, pp 1988–1989 (1999)).

The resulting indolylacetamides of Formula (1) and (4) are isolated by standard techniques and may be purified by crystallization or chromatography as described above.

In Scheme IVa, The compounds of Formula (2), are prepared from appropriately substituted 7-bromo-indoles of Formula (15) or appropriately substituted 2-bromo-anilines of Formula (14), which are commercially available. The anilines are subjected to the indole syntheses procedures, as described in ROBINSON,The Fischer Indole Synthesis, Wiley, New York (1983); HAMEL, et al.,Journal of Organic Chemistry, 59, 6372 (1994); and RUSSELL, et al.,Organic Preparations and Procedures International, 17, 391 (1985), to give the 7-bromo-indoles of Formula (15). Alkylation of the indoles of Formula (15), by sodium hydride and the appropriate alkylating agent in dimethylformamide give the appropriate R4substitution groups of Formula (16). A compound of Formula (16) is then subjected to a lithium-halogen exchange, following which the lithium anion is quenched by dimethyl or diethyl oxalate at low temperatures, to give compounds of Formula (2).

In Scheme IVb, the formation of Formula (3) follows the procedure described in FAUL, M. A.; WINNEROSKI, L. L.; KRUMRICH, C. A.J. Org. Chem., 63, 6053–6058 (1998). For example, an indole of Formula (17) is reacted with oxalyl chloride followed by sodium methoxide at low temperatures to give the corresponding indole-glyoxylates of Formula (3).

The resulting indole-glyoxylates of Formula (2) or (3) are isolated by standard techniques and may be purified by crystallization or chromatography as described above.

Schemes Va and Vb depict the formation of the optionally substituted C1–C4alkyl amines or heterocycles. The hydroxy(C1–C4)alkyl-maleimides of Formulae (18) and (21) are converted to the bromoalkyl-indolocarbazoles by a hydroxy to bromide conversion. This type of conversion is well-known and appreciated in the art and is referenced in LAROCK, R. C.,Comprehensive Organic Transformations, 2ndEd., John Wiley & Sons, pp 689–697 (1999). Compounds of Formulae (19) and (22) are then subjected to conditions of Scheme I, followed by a displacement of the bromide with the appropriate amine or heterocyclic-amine to further give compounds of Formula (I).

The skilled artisan also will appreciate that the individual steps in Schemes Va and Vb may be varied to provide the compounds of Formula I. For example, hydroxy(C1–C4)alkyl-maleimides of Formulae (18) and (21) may be converted first by a hydroxy to bromide conversion to the bromoalkyl-indolocarbazoles of Formulae (19) and (22), respectively; then bromide displacement by an amine or heterocyclic-amine to form compound of Formulae (19a) and (22a), respectively; and finally cyclization according to Scheme I to form compounds of Formula (I). Alternatively, hydroxy(C1–C4)alkyl-maleimides of Formulae (18) and (21) may be first cyclized according to Scheme I to form compounds of Formulae (18a) and (21a), respectively; then, hydroxy to bromide conversion to the bromoalkyl-indolocarbazoles of Formulae (20) and (23), respectively; and finally bromide displacement by an amine or heterocyclic-amine to form compound of Formula (I). The particular order of steps required to produce the compounds of Formula I is dependent upon the particular compound being synthesized, the starting compound, and the relative lability of the substituted moieties.

The skilled artisan will appreciate that compounds of the invention where variables A and B are independently S may be prepared by treating either the final compound or an appropriate carbonyl starting material with [2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide] (Lawesson's Reagent) or phosphorus pentasulfide.

Many of the compounds of the present invention are not only inhibitors of CDK4, but are also useful intermediates for the preparation of additional compounds of the present invention. For example, secondary amines may be acylated, alkylated or coupled with simple carboxylic acids or amino acids under standard conditions. Furthermore, ester moieties may be reduced to the corresponding alcohols. These alcohols may then be activated and displaced by a number of nucleophiles to provide other compounds of the invention. The skilled artisan will also appreciate that not all of the substituents in the compounds of Formula I will tolerate certain reaction conditions employed to synthesize the compounds. These moieties may be introduced at a convenient point in the synthesis, or may be protected and then deprotected as necessary or desired. Furthermore, the skilled artisan will appreciate that in many circumstances, the order in which moieties are introduced is not critical.

The following preparations and examples further illustrate the preparation of compounds of the present invention and should not be interpreted in any way as to limit the scope. Those skilled in the art will recognize that various modifications may be made while not departing from the spirit and scope of the invention. All publications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains.

The terms and abbreviations used in the instant Preparations and Examples have their normal meanings unless otherwise designated. For example “° C.”, “N”, “mmol”, “g”, “mL”, “M”, “HPLC”, “IR”, “MS(FD)”, “MS(IS)”, “MS(FIA)”, “MS(FAB)”, “MS(EI)”, “MS(ES)”, “UV”, and “1H NMR”, refer to degrees Celsius, normal or normality, millimole or millimoles, gram or grams, milliliter or milliliters, molar or molarity, high performance liquid chromatography, infra red spectrometry, field desorption mass spectrometry, ion spray mass spectrometry, flow injection analysis mass spectrometry, fast atom bombardment mass spectrometry, electron impact mass spectrometry, electron spray mass spectrometry, ultraviolet spectrometry, and proton nuclear magnetic resonance spectrometry respectively. In addition, the absorption maxima listed for the IR spectra are only those of interest and not all of the maxima observed.

Preparation of 2-(1-Methyl-1H-indol-7-yl)-acetamide

To a solution of 1H-indole-7-carboxaldehyde (20.0 g, 137 mmol) and dimethylsulfate (19.1 g, 151 mmol) in DMF (400 mL) at 0° C. was added a 60% dispersion of NaH in mineral oil (6.60 g, 165 mmol). The reaction was stirred and allowed to warm to room temperature over 30 minutes. The reaction was quenched with H2O, diluted with EtOAc (1 L) and H2O. The two layers were separated and the aqueous layer was extracted with EtOAc (250 mL). The combined organic layers were washed with H2O (3×500 mL), brine, saturated aq LiCl, and brine. The solution was dried (MgSO4), filtered and concentrated to afford the title compound as an off white solid of sufficient quality to use directly in the next step. Flash chromatography (80% hexane/EtOAc) can be used to afford the title compound pure, as a white solid.1H NMR (400 MHz, DMSO-d6) 10.39 (s, 1H), 7.91 (d, J=7 Hz, 1H), 7.75 (d, J=7 Hz, 1H), 7.42 (d, J=3 Hz, 1H), 7.21 (dd, J=7, 7 Hz, 1H), 6.60 (d, J=3 Hz, 1H), 4.08 (s, 3H). MS (electrospray, m/z) 160 (M++1).

Preparation of 6-Bromoindole-3-acetamide

To a solution of 6-bromoindole (7.18 g, 36.6 mmol) in CH2Cl2(140 mL) was added N,N-dimethylmethylene ammonium chloride (5.48 g, 58.6 mmol, 1.60 eq.) in one portion and the resultant mixture was stirred at rt for 2 h under N2. Water (150 mL) and 1M NaOH (62 mL, 62 mmol) were added, and the reaction mixture was extracted with EtOAc (300 mL). The organic layer was washed with aqueous saturated NaCl and dried (MgSO4). The solvent was removed in vacuo to give 9.29 g (100%) of the title compound.

6-Bromo-N,N-dimethyl-1H-indole-3-methanamine (7.76 g, 30.6 mmol) and NaCN (1.95 g, 39.8 mmol, 1.30 eq.) were combined with DMF (40 mL) and water (40 mL). The mixture was heated to reflux for 6 hours, then cooled to rt. Aqueous 1N HCl (40 mL) was added, and the product was extracted with EtOAc. The organic layer was washed with water and dried (MgSO4). The solvent was removed in vacuo to give 5.70 g (79%) of the title compound, that was 79% pure by HPLC area, containing 14% 6-bromoindole. The material was used without purification in the subsequent step.

Powdered K2CO3(408 mg, 2.95 mmol, 1.60 eq.) was heated to 50° C. in DMSO (3 mL) for 20 min, then cooled to rt. 6-Bromo-1H-indole-3-acetonitrile (434 mg, 1.85 mmol) was dissolved in DMSO (3 mL) and added to the reaction slurry. Water (0.2 mL) and aqueous 30% H2O2(0.46 mL, 4.0 mmol, 2.2 eq.) were added, and the reaction mixture was stirred for 2–3 hours at rt. The reaction was quenched with 1N HCl and extracted with EtOAc. The organic layer was washed with 1.5 N sodium thiosulfate and dried (MgSO4). The solvent was removed in vacuo, and the residue crystallized from CH2Cl2to give 273 mg (55%) of the title compound.

Preparation of 6-Methoxyindole-3-acetamide

Preparation of N-(3-hydroxypropyl)indole-3-acetamide

To a solution of NaH (3.59 g, 89.6 mmol) in DMF (20 mL) cooled to 0° C. was added a solution of indole-3-acetonitrile (10.0 g, 64.0 mmol) in DMF (80 mL) and the mixture was allowed to come to rt and stir for 30 min, becoming dark brown. It was cooled back to 0° C. and 3-bromopropylacetate (89.6 mmol) was added by syringe, slowly to control foaming. The reaction was stirred vigorously at rt for 4 h and then diluted with EtOAc and quenched with a 0.5 M solution of HCl. The organic layer was washed with a saturated aqueous solution of NaCl, dried (MgSO4) and the solvent removed in vacuo to give a brown oil. This was dissolved in t- butanol (175 mL) and freshly ground KOH (42.2 g, 640 mmol) was added with vigourous stirring. The reaction was heated to reflux for 1.5 h. It was then cooled to 30° C. and poured onto ice (500 mL). 6N HCl was added until the pH of the mixture was 1.0. The reaction was diluted with EtOAc the layers separated. The organic layer was washed with a saturated aqueous solution of NaCl, dried (MgSO4) and the solvent removed in vacuo to give a brown oil which was purified by column chromatography (Start 1:1 hexanes:acetone until product begins eluting, wash off with 95:5 acetone:MeOH) to afford 374076 the title compound (60%) as a creme colored solid.

Preparation of 6-Trifluoromethyl indole-3-acetamide

To a solution of 6-trifluoromethylindole (1.0 g, 5.4 mmol) in CH2Cl2(10 mL) was added N,N-dimethylmethylene ammonium chloride (0.63 g, 6.75 mmol, 1.25 eq.) in one portion and the resultant mixture was stirred at rt for 2 h under N2. 1M NaOH (6.75 mL,) was added, and the reaction mixture was extracted into methylene chloride. The organic layer was washed with aqueous saturated NaCl and dried (MgSO4). The solvent was removed in vacuo to give 1.3 g (100%) of title compound.

6-Trifluoromethyl-N,N-dimethyl-1H-indole-3-methanamine (1.2 g, 4.95 mmol) and NaCN (0.72 g, 14.86 mmol, 3.0 eq.) were combined with DMF (10 mL) and EtOAc (2 mL). The mixture was heated to reflux for 6 hours, then cooled to rt. The product was extracted with EtOAc and the organic layer was washed with water and dried (MgSO4). The solvent was removed in vacuo to give 0.66 g (60%) of the title compound after column chromatography (Hex:EtOAc 1:1).

A 250 mL round bottom flask was charged with 5.0 g of the nitrile and KOH (6.25 g) in 100 mL tert-BuOH. The reaction was heated to reflux until complete. The reaction was extracted with EtOAc and the product purified by column chromatography to afford 1.58 g (28%) the title compound.

A 60% dispersion of sodium hydride in mineral oil (1.40 g, 97 mmol) was washed with hexanes (3×10 mL, 1×5 mL), suspended in THF (50 mL) and CH3I added (1.64 mL, 26.3 mmol) followed by a solution of 7-bromoindole (4.06 g, 20.7 mmol) in THF (15 mL), with vigorous gas evolution. After 1 h at rt, the mixture was treated with additional CH3I (0.28 mL, 4.5 mmol) and stirred overnight. Saturated aq NaHCO3was added and the mixture poured into EtOAc, which was separated, washed with saturated aq NaHCO3, dried (MgSO4) and concentrated to afford 1-methyl-7-bromoindole as an off-white semisolid which was used without further purification.

A solution of 7-[2-(tert-butyl-dimethyl-silanyloxy)-ethyl]-1H-indole (6.17 g, 22.4 mmol) in Et2O (50 mL) was cooled to 0° C. (ice bath) and oxalyl chloride (2.0 mL, 24 mmol) added dropwise. The ice bath was removed and the reaction was allowed to warm to room temperature (1.5 h). The reaction was then cooled to −78° C. and solution of NaOMe in MeOH (10.8 mL, 25% wt, 47.3 mmol) was introduced over 5 minutes. The cold bath was removed and the reaction was allowed to warm to room temperature (1 h). The reaction was quenched with H2O and poured into EtOAc. The water layer was separated and extracted with EtOAc (20 mL) and the combined organic layer was washed with saturated aq NH4Cl and brine, dried over MgSO4, filtered and concentrated. Flash chromatography (50% EtOAc/CH2Cl2) gave the title compound (2.79 g, 50%) as a yellow solid.1H NMR (400 MHz, DMSO-d6) 3.04 (t, J=6.8 Hz, 1H), 3.70 (m, 2H), 3.88 (s, 3H), 4.72 (t, J=4 Hz, 1H), 7.10–7.22 (m, 2H), 8.00 (d, J=7.6 Hz, 1H), 8.36 (d, J=3.2 Hz, 1H), 12.38 (s, 1H). MS (electrospray, m/z) 248 (M++1).

Also a product of chromatography was a mixture of the product and the starting acetamide (10:1, 0.365 g). The yield of the product based on recovered starting material was 72%.

To a 60% dispersion of sodium hydride in mineral oil (0.80 g, 21 mmol), suspended in DMF (50 mL), was added 7-bromoindole (2.0 g, 10 mmol) in DMF (15 mL) followed by a solution of iodoethane (1.64 mL, 26.3 mmol). After 20 minutes, the mixture was quenched with saturated NaHCO3, and poured into EtOAc. The organic layer which was separated, washed with saturated NaHCO3, dried (MgSO4) and concentrated to afford 1-ethyl-7-bromoindole as a brown oil which was used without further purification.

A 1.0 M solution of KOtBu in THF (8.1 mL, 8.1 mmol) was added to a solution of 2-(1H-indol-3-yl)-acetamide (417 mg, 2.4 mmol) and {1-(2-trimethylsilanyl-ethoxymethyl)-1H-indol-7-yl}-oxo-acetic acid methyl ester (774 mg, 2.32 mmol) in DMF (50 mL). The deep red-orange solution was stirred 16 h at room temperature and then 1 h at 65° C. The heating bath was removed and pH 7.0 buffer added. The mixture was poured into EtOAc, the layers separated and the EtOAc solution washed with 0.1 N HCl, water (3×), saturated aq NaHCO3and brine (3×), dried (MgSO4), filtered and concentrated onto SiO2. Flash chromatography afforded 3-(1H-Indol-3-yl)-4-[1-(2-trimethylsilanyl-ethoxymethyl)-1H-indol-7-yl]-pyrrole-2,5-dione (141 mg, 13%) as a bright orange solid which was dried under vacuum overnight at 70° C., and 3-(1H-Indol-7-yl)-4-(1H-indol-3-yl)-pyrrole-2,5-dione (165 mg, 22%) as a bright red solid which was dried under vacuum overnight at 70° C.

Oxalyl chloride (1.8 mL, 20.7 mmol) was added dropwise to a solution of 7-bromoindole (3.65 g, 18.6 mmol) in Et2O (100 mL) cooled in an ice bath. After the addition was complete, the ice bath was removed and the mixture was allowed to warm to room temperature and stirred for 8 h. The mixture was then cooled in a dry ice-acetone bath and a 25% by wt solution of sodium methoxide in methanol (9.5 mL, 41.8 mmol) added dropwise over 5 min. The suspension was allowed to warm to room temperature affording a yellow suspension. After stirring 1 h at rt, the suspension was treated with water and the mixture poured into EtOAc. The EtOAc solution was separated and washed with pH 7.0 buffer and brine, dried (MgSO4) and concentrated to ˜75 mL. The solution was diluted with Et2O and allowed to stand. Filtration afforded the title compound as pale yellow crystals (0.61 g, 12%). Concentration of the mother liquor to a slurry followed by addition of a small amount of Et2O (20 mL) and filtration afforded a second crop (2.33 g, 44%).1H NMR (400 MHz, DMSO-d6) 12.68 (br s, 1H), 8.46 (br s, 1H), 8.17 (d, J=8 Hz, 1H), 7.54 (d, J=8 Hz, 1H), 7.23 (dd, J=8, 8 Hz, 1H), 3.90 (s, 3H).13C NMR (75.5 MHz, DMSO-d6) 178.63, 163.34, 138.72, 135.06, 127.16, 126.45, 124.27, 120.51, 113.22, 105.03, 52.59. IR (CHCl3, cm−1) 3450, 1731, 1654. MS (electrospray, m/z) 284/282 (M++1), 282/280 (M−−1). HRMS 281.9786 (M++1, calcd for C11H9NO3Br 281.9766). Anal. Calcd for C11H8BrNO3: C, 46.84; H, 2.86; N, 4.97. Found: C, 46.81; H, 2.72; N, 4.91.

Using procedures similar to those detailed in Example 12, except for using in (a) 4-(t-butyldimethylsilyl)oxymethyl-indole, in (b) (4-hydroxymethyl-1H-indol-3-yl)-oxo-acetic acid methyl ester and in (c) 3-(4-(t-butyldimethylsilyl)oxymethyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-7-yl)-pyrrole-2,5-dione, affords the title compound; MS (electrospray, m/z) 368.1 (M−−1).

Using procedures similar to those detailed in Example 12, except for using in (a) 4-bromo-indole, in (b) (4-bromo-1H-indol-3-yl)-oxo-acetic acid methyl ester and in (c) 3-(4-brommo-1H-indol-3-yl)-4-(1-methyl-1H-indol-7-yl)-pyrrole-2,5-dione, affords the title compound; MS (electrospray, m/z) 420/418 (M++1), 418/416 (M−−1).

Using procedures similar to those detailed in Example 11, except for using in (a) 4-(t-butyldimethylsilyl)oxymethyl-indole, in (b) 1-[3-(tert-Butyl-dimethyl-silanyloxy)-propyl]-4-(t-butyldimethylsilyl)oxymethyl-1H-indole and in (c) 3-[1-(3-Hydroxy-propyl)-4-hydroxymethyl-1H-indol-3-yl]-4-(1-methyl-1H-indol-7-yl)-pyrrole-2,5-dione, affords the title compound; MS (electrospray, m/z) 410.1 (M++1).

Using procedures similar to those detailed in Example 11, except for using in (a) 4-bromo-indole, in (b) 1-[3-(tert-Butyl-dimethyl-silanyloxy)-propyl]-4-bromo-1H-indole and in (c) 3-[1-(3-Hydroxy-propyl)-4-bromo-1H-indol-3-yl]-4-(1-methyl-1H-indol-7-yl)-pyrrole-2,5-dione, affords the title compound; MS (electrospray, m/z) 478.1/476.1 (M++1), 476.1/474.1 (M−−1).

Using procedures similar to those detailed in Example 11, except for using in (a) 4-methoxy-indole, in (b) 1-[3-(tert-Butyl-dimethyl-silanyloxy)-propyl]-4-methoxy-1H-indole and in (c) 3-[1-(3-Hydroxy-propyl)-4-methoxy-1H-indol-3-yl]-4-(1-methyl-1H-indol-7-yl)-pyrrole-2,5-dione, affords the title compound; MS (electrospray, m/z) 428.2 (M++1), 426.3 (M−−1).

Using procedures similar to those detailed in Example 10, except for using in (a) (1-ethyl-1H-indol-7-yl)-oxo-acetic acid methyl ester, in (b) 3-[1-(3-hydroxypropyl)-1H-indol-2-yl]-4-(1-ethyl-1H-indol-7-yl)-pyrrole-2,5-dione, affords the title compound; MS (electrospray, m/z) 412.2 (M++1), 410.2 (M−−1).

2-Bromo-4-methyl-aniline (10.0 g, 53.7 mmol) was added dropwise to a solution of boron trichloride in methylene chloride (1.0 M, 60 mL, 60 mmol) cooled with ice water. The reaction mixture was warmed to room temperature, stirred for 30 min, and chloroacetonitrile (10 mL, 64.4 mmol) and aluminum chloride (10 g, 59.8 mmol) added, followed by 1,2-dichloroethane (70 mL). The reaction mixture was heated to 70° C. to distill off methylene chloride, and then refluxed for 24 hrs. The mixture was cooled to 0–5° C., treated with 25 M HCl (96 mL) at 0˜5° C. carefully, and then heated to 80° C. for 1 h until all solids dissolved. The aqueous layer was separated and extracted with methylene chloride. The combined organic layers were washed with water and brine, dried (sodium sulfate) and concentrated a yellow solid (11.6 g, 82.3%), that was used without-further purification. The crude product was taken into dioxane (76 mL) and water (8.5 mL), and treated with sodium borohydride (1.75 g, 46.3 mmol) in portions. After 30 min at room temperature, all the starting material was consumed, and the reaction mixture was heated to reflux for 14 h. The mixture was then cooled to room temperature, treated with 0.1 N HCl (74 mL), and extracted with ethyl acetate. The extract was washed with water and brine, dried (Na2SO4) and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) gave 5-methyl-7-bromoindole (8.0 g, 90%). 1H NMR (400 MHz, CDCl3) 2.43 (s, 3H), 6.54 (dd, J1=1.94 Hz, J2=3.13 Hz, 1H), 7.20 (d, J=0.78 Hz, 1H), 7.22 (m, 1H), 7.36 (m, 1H), 8.22 (br, 1H); MS (ES, m/z): C9H8BrN: 210 (M+(79Br)+1), 212.0 (M+(81Br)+1).

To a suspension of sodium hydride (60%, 0.91 g, 22.9 mmol) in DMF (70 mL) was added a solution of 5-methyl-7-bromoindole (4.0 g, 19.05 mmol) in DMF (10 mL). The mixture was stirred at room temperature for 1 h, and then methyl iodide (1.78 mL, 28.6 mmol) was added with cooling in an ice-water-bath. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched with water and extracted with ethyl acetate. The extract was washed with water and brine, dried (sodium sulfate) and concentrated to give a crude product (4.1 g, 96%), which was used without further purification. 1H NMR (400 MHz, CDCl3) δ 2.38 (s, 3H), 4.12 (s, 3H), 6.36 (d, J=3.12 Hz, 1H), 6.95 (d, J=3.13 Hz, 1H), 7.18 (d, J=1.56 Hz, 1H), 7.31 (dd, J1=0.79 Hz, J2=1.56 Hz, 1H).

2-Bromo-4-methoxy-aniline (5.0 g, 24.7 mmol) was added dropwise to a solution of boron trichloride in methylene chloride (1.0 M, 27 mL, 27 mmol) cooled with ice water. The reaction mixture was warmed to room temperature, stirred for 30 min, and chloroacetonitrile (4.55 mL, 29.7 mmol) and aluminum chloride (4.54 g, 27.2 mmol) added, followed by 1,2-dichloroethane (32 mL). The reaction mixture was heated to 70° C. to distill off methylene chloride, and then refluxed for 24 h. The mixture was cooled to 0–5° C., treated with 2.5 M HCl (44 mL) at 0˜5° C. carefully, and then heated to 80° C. for 1 h until all solids dissolved. The aqueous layer was separated and extracted with methylene chloride. The combined organic layers were washed with water and brine, dried (sodium sulfate) and concentrated a yellow solid, that was used without further purification. The crude product was taken into dioxane (19 mL) and water (2.1 mL), and treated with sodium borohydride (0.46 g, 12.2 mmol) in portions. After 30 min at room temperature, all the starting material had been consumed, and the reaction mixture was heated to reflux overnight. The mixture was then cooled to room temperature, treated with 0.1 N hydrochloric acid (195 mL), diluted with ethyl acetate, treated with concentrated hydrochloric acid and trifluoroacetic acid and stirred until all the hydroxy-intermediate was converted to the final product. The reaction mixture was then extracted with ethyl acetate, and the extract was washed with sodium bicarbonate, water and brine, dried (Na2SO4) and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) gave 4-methoxy-7-bromoindole (1.5 g, 59.8%).1H NMR (400 MHz, CDCl3) 3.77 (s, 3H), 6.47˜6.49 (m, 1H), 6.99 (s, 2H), 7.17(m, 1H), 8.11 (br, 1H); MS (ES, m/z): C9H8BrNO: 228 (M+(79Br)+1), 226.01 (M+(81Br)+1), 224.03 (M+(79Br)−1), 226.04 (M+(81Br)−1).

To a suspension of sodium hydride (60%, 0.32 g, 7.96 mmol) in DMF (18 mL) was added a solution of 5-methoxy-7-bromoindole (1.5 g, 6.63 mmol) in DMF (25 mL). The mixture was stirred at room temperature for 1 h, and then methyl iodide (0.62 mL, 9.95 mmol) was added with cooling in an ice-water-bath. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched with water and extracted with ethyl acetate. The extract was washed with water and brine, dried (sodium sulfate) and concentrated to give a crude product (1.6 g, 100%), which was used without further purification.1HNMR (400 MHz, CDCl3) 3.82 (s, 3H), 4.10 (s, 3H), 6.36 (d, J=3.13 Hz, 1H), 6.96 (d, J=2.74 Hz, 1H), 7.0 (d, J=2.35 Hz, 1H), 7.03 (d, J=2.35 Hz, 1H); MS (ES, m/z): C10H10BrNO: 240.08 (M+(79Br)+1), 242.1 (M+(81Br)+1).

2-Bromo-4-fluoro-aniline (13.8 g, 72.6 mmol) was added dropwise to a solution of boron trichloride in methylene chloride (1.0 M, 81.1 mL, 81.1 mmol) cooled with ice water. The reaction mixture was warmed to room temperature, stirred for 30 min, and chloroacetonitrile (13.5 mL, 87.1 mmol) and aluminum chloride (13.5 g, 98.4 mmol) added, followed by 1,2-dichloroethane (95 mL). The reaction mixture was heated to 70° C. to distill off methylene chloride, and then refluxed for 24 h. After cooling to 0–5° C., the mixture was treated with 2.5 M HCl (130 mL) carefully, and then heated to 80° C. for 1 h until all solids dissolved. The aqueous layer was separated and extracted with methylene chloride. The combined organic solutions were washed with water and brine, dried (sodium sulfate) and concentrated to a yellow solid (11, 56.9%), which was used without further purification. The crude product was taken into dioxane (68 mL) and water (7.6 mL), and treated with sodium borohydride (1.64 g) in portions. After 30 min at room temperature, all the starting material was consumed, and the reaction mixture was heated to reflux for 18 h. The mixture was cooled to room temperature, treated with 0.1 N HCl (70 mL), 2N HCl (20 mL) and concentrated HCl (15 mL), and extracted with ethyl acetate. The organic layer was separated and washed with water and brine, dried (sodium sulfate), and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) gave 5-fluoro-7-bromoindole (4.4 g, 52.2%).1H NMR (400 MHz, CDCl3) 6.49, (dd, J1=2.35 Hz, J2=3.13 Hz, 1H), 7.06 (d, J1=2.35 Hz, J2=8.60 Hz, 1H), 7.14˜7.17 (m, 2H), 8.18 (br, 1H); MS (ES, m/z): C8H5BrFN: 211.9 (M+(79Br)−1), 214.0 (M+(81Br)−1).

To a suspension of sodium hydride (60%, 1.75 g, 43.8 mmol) in DMF (134 mL) was added a solution of 5-fluoro-7-bromoindole (7.8 g, 36.4 mmol) in DMF (10 mL). After 1 h at room temperature, the mixture was treated with methyl iodide (3.4 mL, 54.6 mmol) with cooling in an ice-water bath. The reaction mixture was allowed to warm up room temperature and stirred overnight. The reaction was quenched with water, extracted with ethyl acetate, and the organic layer was washed with water and brine, dried (sodium sulfate) and concentrated to give a crude product, which was used without further purification.1H NMR (400 MHz, CDCl3) 4.06 (s, 3H), 6.34 (d, J=3.13 Hz, 1H), 6.96 (d, J=3.13 Hz, 1H), 7.07 (dd, J1=2.34 Hz, J2=8.60 Hz, 1H), 7.12 (dd, J1=2.34 Hz, J2=9.0 Hz, 1H).

2-Bromo-5-methoxy-aniline (4.4 g, 21.8 mmol) was added dropwise to a solution of boron trichloride in methylene chloride (1.0 M, 24 mL, 24 mmol) cooled with ice water. The reaction mixture was warmed to room temperature, stirred for 30 min, and chloroacetonitrile (4.01 mL, 26.2 mmol) and aluminum chloride (4.01 g, 24.0 mmol) were added, followed by 1,2-dichloroethane (28.5 mL). The reaction mixture was heated to 70° C. to distill off methylene chloride, and then heated to reflux for 24 hrs. After cooling to 0–5° C., the mixture was treated with 2.5 M HCl (38.4 mL) carefully, and then heated to 80° C. for 1 h until all solids dissolved. The aqueous layer was separated, extracted with methylene chloride and the combined extracts washed with water and brine, dried (sodium sulfate) and evaporated to a yellow solid, which was used without further purification. The crude product was taken into dioxane (37 mL) and water (4.2 mL), and treated with sodium borohydride (0.91 g, 24.0 mmol) in portions. After stirring at room temperature for 30 min, all the starting material was consumed and the reaction mixture was then heated to reflux for 14 hrs. After cooling to room temperature, the mixture was treated with concentrated HCl, and extracted with ethyl acetate. The organic extract was washed with water and brine; dried (sodium sulfate), and concentrated. Column chromatography on silica gel (hexanes/ethyl acetate) gave 4-methoxyl-7-bromoindole (1.2 g, 24%).1H NMR (400 MHz, CDCl3) 3.94 (s, 3H), 6.44 (d, J=8.21 Hz, 1H), 6.73 (m, 1H), 7.17 (t, J=2.74 Hz, 1H), 7.22˜7.26 (m, 1H), 8.32 (br, 1H); MS (ES, m/z): C9H8BrNO: 227.99 (M+(79Br)+1), 230.0(M+(81Br)+1).

To a suspension of sodium hydride (60%, 0.23 g, 5.75 mmol) in DMF (18 mL) was added a solution of 4-methoxyl-7-bromoindole (1.1 g, 4.87 mmol) in DMF (2 mL). After 1 h at room temperature, the mixture was treated with methyl iodide (0.45 mL, 7.23 mmol) with cooling in an ice-water bath. The reaction mixture was allowed to warm up room temperature and stirred overnight. The reaction was quenched with water, extracted with ethyl acetate and the extract washed with water and brine, and dried (sodium sulfate). Evaporation of solvent gave a crude product, which was used without further purification.1H NMR (400 MHz, CDCl3) 3.92(σ,3H), 4.13 (s, 3H), 6.35 (d, J=8.21 Hz, 1H), 6.55 (d, J=3.13 Hz, 1H), 6.89 (d, J=3.52 Hz, 1H), 7.22 (d, J=8.21 Hz, 1H).

To a solution of 1-[3-(tert-butyldimethylsilyloxy) propyl]-1H-indole (4.99 g, 17.2 mmol) in diethyl ether (20 mL) at 4° C. was added a 2.0 M solution of oxalyl chloride in methylene chloride (9.5 mL, 19.0 mmol). The resulting solution was allowed to warm to room temperature. After 2 h, the solution was cooled to −78° C. To this solution was added a 25% (w/w) solution of sodium methoxide in methanol (8.7 mL, 38.0 mmol). The resulting mixture was allowed to warm to room temperature and stirred for 3 h. The reaction was quenched with water (10 mL) and poured into EtOAc (100 mL). The aqueous layer was extracted with EtOAc (20 mL). The combined organic layers were washed with saturated aq NH4Cl (100 mL) and brine (100 mL), dried (MgSO4), filtered, and concentrated. Flash chromatography (SiO2, 20% to 80% EtOAc/hexanes) gave the title compound (3.24 g, 71%) as a white solid.1H NMR (300 MHz, CDCl3) 8.42 (m, 2H), 7.47 (m, 1H), 7.36 (m, 2H), 4.38 (m, 2H), 3.94 (s, 3H), 3.67 (m, 2H), 2.25 (m, 2H), 1.50 (m, 1H). MS (atmospheric pressure chemical ionization, m/z) 262 (M++1).

This compound is also prepared via condensation of methyl[1-(3-hydroxypropyl)-1H-indol-3-yl]oxoacetate with 2-(1-methyl-1H-indol-7-yl)acetamide.

DDQ is also used to produce this compound.

A solution of 1-[2-(tert-butyldimethylsilyloxy)ethyl]-6-methoxy-1H-indole (14.6 g, 46.5 mmol) in Et2O (300 mL) was cooled to 4° C. (ice bath) and 2.0 M solution of oxalyl chloride in THF (25.6 mL, 51.2 mmol) was added dropwise. The ice bath was removed and the reaction was allowed to warm to room temperature (1.5 h). The reaction was then cooled to −78° C. and a solution of 25% (w/w) NaOMe in MeOH (23.2 mL, 107.0 mmol) was introduced over 5 min. The reaction was allowed to warm to room temperature. The reaction was quenched with H2O (300 mL) and poured into EtOAc (300 mL). The water layer was separated and extracted with EtOAc (2×200 mL). The combined organic solution was washed with saturated aqueous NH4Cl (300 mL) and brine (400 mL), dried (MgSO4), filtered and concentrated. Flash chromatography (SiO2, 50% hexanes/EtOAc) gave the title compound (16.2 g, 89%) as a yellow solid.1H NMR (300 MHz, CDCl3) 8.44 (m, 2H), 7.12 (d, J=7.2 Hz, 1H), 6.98 (s, 1H), 4.38 (m, 2H), 4.10–4.00 (m, 5H), 3.96 (s, 3H), 0.94 (s, 9H), 0.02 (s, 6H).

11-Methoxy-5-methyl-13-(N,N-dimethyl-N′-propyl-ethane-1,2-diamine)-7H-indolo[6,7a] pyrrolo[3,4-c]carbazole-6,8-dione (50 mg, 0.1 mmol) was dissolved in CH3CN (15 mL) and 0.1 N HCl (1 mL) added. The mixture was purified by reverse phase HPLC as described above. The title compound (15 mg, 26%) was obtained as an orange solid.

Method B: To a solution of 3-[7-(2-hydroxyethyl)-1H-indol-3-yl]-4-(1-methyl-1H-indol-7-yl)-pyrrole-2,5-dione (0.5 g, 1.3 mmol) in solution of CH2Cl2/THF (40/30 ml) was added PPh3(0.3 g, 1.3 mmol) and CBr4(0.4 g, 1.3 mmol). The reaction mixture was stirred at room temperature and under nitrogen for 1 h. Another equivalent of PPh3(0.3 g, 1.3 mmol) was added followed by an equivalent of CBr4(0.4 g, 1.3 mmol). This operation was repeated one more time after 1 h. The reaction was monitored by TLC and was completed after 1 h. The reaction mixture was diluted with EtOAc (100 ml) and washed with saturated aq NaHCO3(100 mL), brine (100 ml). The organic solution was dried (MgSO4), filtered and concentrated. Flash chromatography (SiO2, 50% EtOAc/hexanes) gave the title compound (0.5 g, 87%) as a red solid.

A mixture of 5-Methyl-12-(2-bromoethyl)-7H,13H-indolo[6,7-a]pyrrolo[3,4-c]carbazole-6,8-dione (150 mg, 0.33 mmol), 4-hydroxypiperidine (33 mg, 3.3 mmol), and DMF (10 mL) in a manner analogous to that described for the preparation of Example 61. The mixture was concentrated and the crude product was purified by reverse phase preparatory HPLC to give title compound (94 mg, 56%) as an orange solid. MS (electrospray, m/z) 466.2 (M++1).

Using procedures similar to those detailed in Example 84, except for using thiomorpholine, affords the title compound; MS (electrospray, m/z) 469.2 (M++1), 467.2 (M−−1).

Using procedures similar to those detailed in Example 84, except for using diethylamine, affords the title compound; MS (electrospray, m/z) 439.2 (M++1), 437.2 (M−−1).

Using procedures similar to those detailed in Example 84, except for using thiomorpholine, affords the title compound; MS (electrospray, m/z) 483.1 (M++1), 481.1 (M−−1).

Using procedures similar to those detailed in Example 75, except for using morpholine, affords the title compound; MS (electrospray, m/z) 467.1 (M++1), 465.2 (M−−1).

Using procedures similar to those detailed in Example 81, except for using diethylamine, affords the title compound; MS (electrospray, m/z) 453.2 (M++1), 451.2 (M−−1).

Using procedures similar to those detailed in Example 75, except for using piperidine, affords the title compound; MS (electrospray, m/z) 465.2 (M++1), 463.2 (M−−1).

To a solution of 5-methyl-1H-indole (1.00 g, 7.6 mmol) in diethyl ether (10 mL) at 4° C. was added a 2.0 M solution of oxalyl chloride in methylene chloride (4.0 mL, 8.0 mmol). The resulting solution was allowed to warm to room temperature and stirred for 1.5 h. The solution was cooled to −78° C. To this solution was added 25% (w/w) solution of sodium methoxide in methanol (3.65 mL, 16.0 mmol). The resulting solution was allowed to warm to room temperature. After one hour, the solution was filtered to give the title compound (1.16 g, 70%) as a light brown solid.1H NMR (DMSO-d6) δ 8.31 (m, 1H), 7.95 (s, 1H), 7.42 (m, 1H), 7.11 (m, 1H), 3.88 (s, 3H), 2.41 (s, 3H).

To a solution of 6-methylindole (1.00 g, 7.62 mmol) in diethyl ether (10 mL) at 4° C. was added a 2.0 M solution of oxalyl chloride in methylene chloride (4.0 mL, 8.0 mmol). The resulting solution was allowed to warm to room temperature. After 90 min, the solution was cooled to −78° C. To this solution was added 25% (w/w) solution of sodium methoxide in methanol (3.65 mL, 16.0 mmol). The resulting solution was allowed to warm to room temperature. After 1 h, the solution was filtered to give the title compound (894 mg, 54%) as a beige solid.

To a solution of 1H-indole-5-carbonitrile (1.14 g, 8.04 mmol) in diethyl ether (12 mL) at 4° C. was added a 2.0 M solution of oxalyl chloride in methylene chloride (4.2 mL, 8.4 mmol). The resulting solution was allowed to warm to room temperature. After 90 min, the solution was cooled to −78° C. To this solution was added 25% (w/w) solution of sodium methoxide in methanol (3.83 mL, 16.8 mmol). The resulting solution was allowed to warm to room temperature. After one hour, the solution was filtered to give the title compound (768 mg, 42%) as a light brown solid.1H NMR (DMSO-d6) δ 9.39 (s, 1H), 8.67 (m, 1H), 8.50 (s, 1H), 7.29 (m, 1H), 3.87 (s, 3H).

To a solution of 5-trifluoromethylindole (2.0 g, 10.8 mmol) in diethyl ether (30 mL) at 4° C. was added a 2.0 M solution of oxalyl chloride in methylene chloride (5.5 mL, 11.0 mmol). The resulting solution was allowed to warm to room temperature. After 90 min, the solution was cooled to −78° C. To this solution was added 25% (w/w) solution of sodium methoxide in methanol (5.0 mL, 21.9 mmol). The resulting solution was allowed to warm to room temperature. After one hour, the solution was filtered to give the title compound (1.24 g, 43%) as a yellow solid.1H NMR (DMSO-d6) δ 8.66 (m, 1H), 8.45 (s, 1H), 7.78 (m, 1H), 7.60 (m, 1H), 3.89 (s, 3H).

A 2.0 M solution of oxalyl chloride in methylene chloride (4.50 mL, 9.0 mmol) was added to a solution of 5-fluoroindole (1.14 g, 8.4 mmol) in ether (19 mL) dropwise over 10 min at 4° C. under N2. The ice bath was removed and the resulting mixture was stirred for 2 h. A 25% sodium methoxide in methanol solution (4.3 mL, 18.8 mmol) was added at −78° C. over 5 min. The mixture was allowed to warm up to room temperature and stirred for 1 h. The reaction was quenched with water (50 mL), filtered and rinsed with cold ether to afford the title compound (1.34, 72%) as a yellow solid.1H NMR (DMSO-d6) δ 12.51 (bs, 1H), 8.56 (s, 1H), 7.86 (m, 1H), 7.60 (m, 1H), 7.15 (m, 1H), 3.90 (s, 3H).

A 2.0 M solution of oxalyl chloride in methylene chloride (4.75 mL, 9.6 mmol) was added to a solution of 6-ethylindole (1.39 g, 9.6 mmol) in ether (20 mL) dropwise over 10 min at 4° C. under N2. The ice bath was removed and the resulting mixture was stirred for 2 h. A 25% sodium methoxide in methanol solution (4.3 mL, 18.8 mmol) was added at −78° C. over 5 min. The mixture was allowed to warm up to room temperature and stirred for 1 h. The reaction was quenched with water (10 mL), filtered and rinsed with cold ether to afford the title compound (1.34, 72%) as a yellow solid.1H NMR (CDCl3) δ 9.07 (bs, 1H), 8.43 (m, 1H), 8.32 (m, 1H), 7.29–7.21 (m, 2H), 3.95 (m, 3H), 2.78 (m, 2H), 1.29 (m, 3H).

To a solution of 5-bromolindole (2.05 g, 10.5 mmol) in diethyl ether (20 mL) at 4° C. was added a 2.0 M solution of oxalyl chloride in methylene chloride (5.5 mL, 11.0 mmol). The resulting solution was allowed to warm to room temperature. After 90 min, the solution was cooled to −78° C. To this solution was added 25% (w/w) solution of sodium methoxide in methanol (5.0 mL, 21.9 mmol). The resulting solution was allowed to warm to room temperature. After 1 h, the solution was filtered to give the title compound (2.235 g, 76%) as a brown solid.1H NMR (DMSO-d6) δ 8.53 (s, 1H), 8.28 (s, 1H), 7.54 (m, 1H), 7.43 (m, 1H), 3.90 (s, 3H).

To a solution of 2-(1-methyl-1H-indol-7-yl)acetamide (200 mg, 1.06 mmol) and methyl 2-(5-bromo-1H-indol-3-yl)oxoacetate (300 mg, 1.06 mmol) in DMF (5.4 mL) was added a 1.0 M solution of potassium tert-butoxide in THF (1.06 mL, 1.06 mmol) at 4° C. under N2. The reaction was stirred for 20 min at this temperature, and additional KOtBu (2.12 mL, 2.12 mmol) was added. The resulting dark red solution was heated up to 50° C. overnight. The mixture was cooled to room temperature, quenched with 1N HCl (9 mL) and poured into EtOAc (300 mL). The organic layer was separated, washed with saturated aqueous NaHCO3(150 mL), and brine (3×150 mL). The solution was dried (MgSO4), filtered, and concentrated to an orange solid. Flash chromatography (SiO2, 30% EtOAc/hexanes) gave the title compound (201 mg, 45%) as an orange solid, mp 270–290° C. dec.

A 2.0 M solution of oxalyl chloride in methylene chloride (6.65 mL, 13.3 mmol) was added to a solution of 6-ethoxyindole (2.0 g, 12.4 mmol) in ether (25 mL) dropwise over 10 min at 4° C. under N2. The resulting mixture was stirred for 1 h at 4° C. A 25% (w/w) sodium methoxide in methanol solution (6.2 mL, 28.5 mmol) was added at −78° C. The mixture was allowed to warm up to room temperature and stirred for 4 h. The reaction was quenched with water (50 mL) and extracted with methylene chloride. The organic solution was dried (MgSO4) and concentrated at reduced pressure. The residue was stirred in a solution of EtOAc (50 mL) and ether (50 mL) for 10 min. Solid was collected by filtration to give the title compound (2.2 g, 73%) after drying.1H NR (CDCl3) δ 8.20 (s, 1H), 7.90 (d, J=9 Hz, 1H), 6.92 (s, 1H), 6.82 (d, J=9 Hz, 1H), 4.00 (q, J=6.5 Hz, 2H), 3.78 (s, 3H), 1.30 (t, J=6.5 Hz, 3H)

2-{11-Methoxy-5-methyl-7-[3-(t-butyldimethylsilanoxy)-3-phenylpropyl]indolo[6,7-a]pyrrolo[3,4-c]carbazole-6,8-dione}-acetamide (1.08 g, 1.60 mmol), anhydrous tetrahydrofuran (25 mL), and 3 N HCl (8.3 mL) are combined and stirred for 8 hours. Dilution with ethyl acetate (200 mL) and water (30 mL) separated the two layers and the organic layer was washed with saturated sodium bicarbonate and brine. The resulting extract was dried with magnesium sulfate, filtered, and concentrated to 50 mL. Crystallization by addition of hexane, filtration, and drying gave 0.688 g (72% yield) of the title compound. MS (electrospray, m/e): 559.2 (M−−1).

2-{11-Methoxy-5-methyl-7-(3-hydroxy-3-phenylpropyl)indolo[6,7-a]pyrrolo[3,4-c]carbazole-6,8-dione}-acetamide (0.543 g, 0.968 mmol), pyridinium chlorochromate (0.313 g, 1.45 mmol), silica gel (60 Å, 0.800 g), and methylene chloride (100 mL)are combined and stirred for 18 hours. Additional pyridinium chlorochormate (0.052 g, 0.242 mmol) is added and the mixture is stirred for 24 more hours. Purification by flash chromatography and eluting with ethyl acetate:hexane and ethyl acetate:methyl alcohol gave 0.305 g of the ketone product. This ketone (0.305 g, 0.546 mmol) was combined with cesium carbonate (0.534 g, 1.63 mmol) and anhydrous dimethylformamide (20 ml) and heated at 80° C. for 3 hours. Purification by flash chromatography and eluting with ethyl acetate:hexane plus reverse phase preparatory high pressure chromatography and eluting with acetonitrile and water gave 0.041 g (10% yield) of the title compound. NS (electrospray, m/e)425.1 (M−−1).

A 1.0 M solution of KOtBu in THF (4.77 ml, 4.77 mmol) was added to a solution of 2-(1-methyl-1H-indol-7-yl-acetamide (0.3 g, 1.59 mmol) and [7-(tert-Butoxycarbonylamino-methyl)-1H-indol-3-yl]-oxo-acetic acid methyl ester (0.53 g, 1.59 mmol) in DMF (15 ml) at room temperature. The reaction was heated at 140° C. for 4 h, allowed to cool to room temperature quenched with pH 7 buffer. The mixture was poured into EtOAc, extracted, washed with saturated aq NaHCO3, brine, dried (MgSO4), filtered, and concentrated onto SiO2. Flash chromatography (50:50 ethyl acetate: hexanes) afforded the title compound (0.1 g, 14%) as an orange solid. MS (electrospray, m/z) 442.2 (M++1), 440.3 (M−−1).

A solution of 1,1-Dimethyl-3-{3-[4-(1-methyl-1H-indol-7-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-1H-indol-7-ylmethyl}-urea (50 mg, 0.11 Mmol), I2(28 mg, 0.11 mmol) and dioxane (200 ml) was photolyzed with a 450 W Hanovia lamp fitted with a pyrex filter for 2 h. During the irradiation, the temperature of the reaction rose to 90° C. The solution was concentrated to ˜5 ml and poured into ethyl acetate (300 ml) and washed with 10% NaHSO3(3×50 ml), saturated aqueous NaCl, dried (MgSO4), and concentrated to yield the title compound (30 mg, 0.06 mmol) as a dark yellow solid. MS (electrospray, m/z) 440.3 (M++1).

KOtBu in THF (1.32 ml, 1.32 mmol, 1M solution in THF) was added to a solution of 2-(1-methyl-1H-indol-7-yl-acetamide (0.25 g, 1.32 mmol) and (7-{[acetyl-(2-hydroxy-ethyl)-amino]-methyl}-1H-indol-3-yl)-oxo-acetic acid methyl ester (0.42 g, 1.32 mmol) in DMF (10 ml) at room temperature. Additional KOtBu in THF (2.64 ml, 2.64 mmol, 1M solution in THF) was added and the reaction was heated at 50° C. overnight. The reaction was quenched with 1N HCl, poured into EtOAc, extracted, washed with saturated aqueous NaHCO3, brine, dried (MgSO4), filtered, and concentrated onto SiO2. Flash chromatography and successive washings repeatedly gave impure material (0.15 g, 25%) that was taken onto the next step without further purification. MS (electrospray, m/z) 457.0 (M++1), 455.1 (M−−1).

A solution of N-2-Hydroxy-ethyl)-N-{3-[4-(1-methyl-1H-indol-7-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl]-1H-indol-7-ylmethyl}-acetamide (100 mg, 0.22 Mmol), I2(56 mg, 0.22 mmol) and (200 ml) was photolyzed with a 450 W Hanovia lamp fitted with a pyrex filter for 1 h. During the irradiation, the temperature of the reaction rose to 47° C. The solution was concentrated to ˜5 ml and loaded onto a bed of SiO2. Chromatography, elution with 40% Acetone/CH2Cl2gave 90% pure material. The solid was re-dissolved in CH2Cl2 and to it was added tert-Butyl-chloro-dimethyl-silane (50 mg, 0.33 mmol) and imidazole (23 mg, 0.33 mmol). Aqueous work-up followed by SiO2chromatography gave the pure compound (60 mg, 59%). The compound was then dissolved in THF (10 ml) and 1N HCl was added. The reaction was poured into ethyl acetate, washed with saturated aqueous NaHCO3, brine, dried (MgSO4), filtered, and concentrated. SiO2flash chromatography gave the title compound (37 mg, 91%) as an orange solid. MS (electrospray, m/z) 371.2 (M++1), 370.2 (M−−1).

The compounds of Formula I were tested using in vitro. An important in vitro test is the ability of the compound to inhibit the activity of the CDK4 enzyme. This test is done using RbINGand/or Rb21substrates as described in the literature (KONSTANTINIDIS, A. K., et al,J. Biol. Chem. 273, 26506–26515 (1998)). The test yields the micromolar concentration of the test compound, which produces a 50% inhibition of the CDK4 enzyme activity. The lower the value in this test, the more active the test compound is. Representative examples are shown in Table 1.

A major in vitro test is the cell growth inhibition test. In this test, the concentration of the test compound that causes a 50% inhibition of cell growth for a selected cell line. A low value is desirable in this test. This test is done according to a standard protocol (SCHULTZ, R. M., et al,Oncology Res. 5, 223–228 (1993)). Another major in vitro test is the G1 arrest test. This test measures the proportion of the cells that are arrested in the G1 phase relative to control. A result above 1.5 is desirable in this test. This test is done again according to standard protocols (ROBINSON, J. P. and DARZYNKIEWICZ, Z.,Current Protocols in Cytometry(1997). Another major in vitro test is the inhibition of Rb phosphorylation. It is desirable that the test compound be able to inhibit the phosphorylation of the Rb protein. Cyclin D-CDK4 is known to specifically phosphorylate Ser-780 of human pRb with a 25- to 60-fold higher efficiency than either cyclin E-CDK2 or cyclin A-CDK2 (KITAGAWA, M. et al.,EMBO J. 15, 7060–7069 (1996) and BRUGAROLAS, J. et al.,Proc. Natl. Acad. Sci. USA96, 1002–1007 (1999)). A commercially available alpha-Phospho-Ser-780 pRb antibody (New England Biolab, Beverly, Mass.) was used in a pRb phosphorylation assay as a means of detecting CDK4 activity in vivo using a HCT116 cell line. The results are expressed as a percentage inhibition of ser-780 pRb phosphorylation in drug treated cells versus control cells treated with DMSO alone. A greater than 50% inhibition of Rb phosphorylation is desirable in this assay. A representative example is shown in Table 2.

Assay of Cyclin D1-cdk4 Kinase Activity with the ING Peptide as Substrate.

The cyclin D1-cdk4 kinase activity of a compound was assayed by preparing a 100 ul reaction at the following concentrations: 35 mM Hepes pH 7.0, 10 mM MgCl2, 300 uM ATP, 200 uM ING peptide, 1.0 uCi of γ-33P-ATP, 4.34 ug of cyclin D-cdk4 enzyme, 4% DMSO, and various concentrations of inhibitor. The reaction was incubated at room temperature (about 74° F.) for 60 minutes, and then terminated by the addition of 100 ul of 10% phosphoric acid. Next, the reaction was filtered through a Millipore Multiscreen-PH Plate—Catalog number MAPH NOB 10, and the plate was washed 2 times with 320 ul each of 0.5% phosphoric acid, followed by the addition of 100 ul of scintillation fluid and quantitation on a Packard Instruments, Top Count, scintillation counter.

Assay of Cyclin D1-cdk4 Kinase Activity with the Rb21 Protein as Substrate.

The cyclin D1-cdk4 kinase activity of a compound was assayed by preparing a 100 ul reaction at the following concentrations: 20 mM Hepes pH 7.0, 10 mM MgCl2, 30 uM ATP, 5 ug of Rb21 protein (Santa Cruz Biotech, Catalog # sc-4112), 1.0 uCi of γ-33P-ATP, 1.09 ug of cyclin D-cdk4 enzyme, 4% DMSO, and various concentrations of inhibitor. The reaction was incubated at room temperature (about 74° F.) for 60 minutes, and then terminated by the addition of 100 ul of 25% trichloroacetic acid. Next, the reaction was filtered through a Millipore Multiscreen-FC Plate—Catalog number MAFC NOB 10, and the plate was washed 2 times with 320 ul each of 10% trichloroacetic acid, followed by the addition of 100 ul of scintillation fluid and quantitation on a Packard Instruments, Top Count, scintillation counter.

Cell Growth Inhibition Assay.

The MTT assay was used to measure growth inhibitory activity (Schultz, R. M., et. al. Oncology Res. 5, 223–228, 1993). The IC50 was determined as the concentration of drug required to inhibit cell growth by 50% over 72 h of drug exposure. Basically, 1000 HCT-116 or NCI H460 cells were added per well to 96-well flat-bottom plates in 100-ul RPMI 1640 medium containing 10% dialyzed fetal bovine serum. The plates were incubated for 24 h prior to addition of test compounds. A stock solution (10 mM) was prepared in DMSO and serially diluted in medium. Compound dilutions were added to triplicate wells, and the plates were incubated for 72 hours.

Cell Cycle Analysis Using Flow Cytometry.

The HCT-116 and NCI H460 cell lines were seeded in 75 cm2flasks at 5×105cells/25 ml RPMI 1640 medium containing 10% dialyzed fetal bovine serum. They were incubated for 24 hours. Compound is then added at 1× IC50 and 3× IC50 (determined from above section “growth inhibition studies”) and incubated for an additional 24 hours. The cells are subsequently harvested and the protocol (Robinson, J. P. and Darzynkiewicz, Z. Current Protocols in Cytometry. 1997) for staining was followed. DNA histogram analysis was performed using ModFit LT(Verity House).

Inhibition of Rb Phosphorylation Assay.

Human HCT116 colon carcinoma cell line was purchased from American Tissue Culture Collection (Rockville, Md.) and maintained as monolayer in RPMI-1640 with L-Glutamine and 25 mM HEPES supplemented with 10% fetal bovine serum in a 37° C. incubator with a 10% CO2atmosphere. The detection of mycoplasm in cultured cells was performed using Mycoplasma Rapid Detection System (TaKaRa Shuzo Co. Ltd., Shiga, Japan) every 2–3 months and the cells were found consistently negative throughout these experiments. The Rb phosphorylation assay was done be plating 4×105cells/well in 6-well plates. After 24 h, exponentially growing HCT116 cells were treated with compounds at 1×, 2× and 3× IC50(as determined by MMT assay) or DMSO in complete medium for 24 h. At the end of the incubation period the medium was removed and the cells were washed twice with cold PBS containing 1 mM sodium orthovanadate (Na3VO4). Cellular protein lysates were prepared by adding freshly prepared 50 uL/well lysis buffer (50 mM HEPES pH 7.5, 1% Triton X-100, 5 mM EDTA, 50 mM NaCl, 10 mM sodium pyrophospate, 50 mM sodium fluoride, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 ug/mL Aprotinin, 10 ug/mL Leupeptin, and 10 ug/mL Pepstatin). The cell lysates were collected and incubated on ice for 30 min with frequent brief vortexing. Cellular debris was removed by centrifugation at 14000×g for 10 min at 4° C. Protein concentration was determined by the Bio-Rad DC protein assay (Bio-Rad, Hercules, Calif.). To analyze extracts, equal amounts of protein (30 ug) were dissolved in 1× Laemmli sample buffer, bioled for 5 min and resolved by electrophoresis on 10% polyacrylamide gels containing SDS. The proteins were transferred to Immobilon-P membrane (Millipore, Bedford, Mass.). Membranes were incubated with 5% non-fat dried milk in TBS-T (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, and 0.1% Tween-20) for 1 h at room temperature to block non-specific sites. Immunoblotting was done by incubating membranes with alpha-Phospho-Ser-780 pRb (1 ug/mL, New England Biolab, Beverly, Mass.) and alpha-actin (0.2 ug/mL) antibodies in TBS-T containing 5% non-fat dried milk for overnight at 4° C. Membranes were washed three times (15 min each) in TBS-T, and subsequently incubated for 2 h at room temperature with horseradish peroxidase-conjugated anti-rabbit (1:2000) and anti-mouse (1:1000) antibody (Amersham) in TBS-T. Membranes were eashed three times (15 min each) with TBS-T, and incubated for 5 min in SuperSignal West Pico Chemiluminescent reagents (Pierce, Rockford Ill.). Proteins were detected by capturing image of the membrane using Quantity One Software on a Fluor-S multi-Imager (Bio-Rad, Hercules, Calif.) in a linear range. Specific bands were quantified using Quantity One Software. After correcting for variable loading using actin as a control, Ser-780 phosphorylated pRb protein levels in the drug treated samples were compared with that of cells treated with vehicle (DMSO). The results (Table II) were expressed as a percentage inhibition of Ser-780 pRb phosphorylation in drug treated cells versus control DMSO treated cells.

Compounds of Formula I may be administered by the oral, transdermal, percutaneous, intravenous, intramuscular, intranasal or intrarectal route, in particular circumstances. The route of administration may be varied in any way, limited by the physical properties of the drugs, the convenience of the patient and the caregiver, and other relevant circumstances (Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (1990)).

The pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art. The carrier or excipient may be a solid, semi-solid, or liquid material which can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art. The pharmaceutical composition may be adapted for oral, inhalation, parenteral, or topical use and may be administered to the patient in the form of tablets, capsules, aerosols, inhalants, suppositories, solutions, suspensions, or the like.

The compounds of the present invention may be administered orally, for example, with an inert diluent or capsules or compressed into tablets. For the purpose of oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like. These preparations should contain at least 4% of the compound of the present invention, the active ingredient, but may be varied depending upon the particular form and may conveniently be between 4% to about 70% of the weight of the unit. The amount of the compound present in compositions is such that a suitable dosage will be obtained. Preferred compositions and preparations according to the present invention may be determined by a person skilled in the art.

The tablets, pills, capsules, troches, and the like may also contain one or more of the following adjuvants: binders such as microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; and sweetening agents such as sucrose or saccharin may be added or a flavoring agent such as peppermint, methyl salicylate or orange flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or a fatty oil. Other dosage unit forms may contain other various materials that modify the physical form of the dosage unit, for example, as coatings. Thus, tablets or pills may be coated with sugar, shellac, or other coating agents. A syrup may contain, in addition to the present compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.

For the purpose of parenteral therapeutic administration, the compounds of the present invention may be incorporated into a solution or suspension. These preparations typically contain at least 0.1% of a compound of the invention, but may be varied to be between 0.1 and about 90% of the weight thereof. The amount of the compound of Formula I present in such compositions is such that a suitable dosage will be obtained. The solutions or suspensions may also include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Preferred compositions and preparations are able to be determined by one skilled in the art.

The compounds of the present invention may also be administered topically, and when done so the carrier may suitably comprise a solution, ointment, or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bees wax, mineral oil, diluents such as water and alcohol, and emulsifiers, and stabilizers. Topical formulations may contain a concentration of the Formula I, or its pharmaceutical salt, from about 0.1 to about 10% w/v (weight per unit volume).