NOVEL 3-BENZYLIDINE AND 3-BENZYL SUBSTITUTED

CHROMANONES

FIELD OF THE INVENTION The present invention relates generally to the synthesis of novel derivative compounds. Such compounds can inhibit several different biological processes involved in disease, including proliferation, migration, inflammation and angiogenesis. In particular, the invention provides novel 3-benzylidine and 3-benzyl substituted chromanone derivative compounds. BACKGROUND INFORMATION

The preparation of 4-chromanones has been taught, for example, by U.S. Patent Nos. 4,625,042 and 4,918,203. More specifically, methods for the preparation of 3- benzylidine-chroman-4-ones and 3-benzyl-chroman-4-ones are well known in the literature. For example, methods of acid and base catalyzed preparation of 3- benzylidine-chroman-4-ones from benzaldehydes and chroman-4-ones have been reviewed. Levai, Arkivoc, Part (vii) 15-33, SA-894BR, ISSN 1424-6376 (2004). 3- benzyl-chroman-4-ones have been isolated as natural products or produced by chemical methods. For example, the isolation of 3-(3,4-dihydroxyphenyl)methyl- chroman-4-one from the dried heart wood of Caesalpinia Sappan has been reported. Saitoh, T., Chem Pharm Bull 34:2506-2511(1986).

The preparation of 3-(3-hydroxy-4-methoxybenzyl)-7-methoxy-6-hydroxy- A- chromanone has been reported as a proof of the structure assigned to natural product. Jain, S.C., Indian J Chem. 36B:860-863 (1997). In addition, 3-benzyl-5,7- dimethoxychromanone has been prepared with the reaction of methylene iodide with the dihydrochalcone. Makrandi, J.K., Ind J Chem. 19B:739-743 (1980). The preparation of (±)-3-(3,4-dimethoxybenzyl)-7-methoxy-4-chromanone as an intermediate for the preparation of O-trimethylsappanone was also reported. Davis and Chen, J. Org. Chem.. 58:1751-53 (1993).

An alternate route to (±)-3-(3,4-dimethoxybenzyl)-7-methoxy-4-chromanone by the hydroxymethylation of the dihydrochalcone and treatment with aqueous diethylamine has also been taught. Pinkey, P.K.J. , Indian J Chem, 25B:365-67 (1986). A similar reaction can convert a o-hydroxy dihydrochalcone to a 3- benzylchromanone in one step. Jaspal, S., Indian J Chem, Sect B: Org Chem Incl Med Chem, 43:1782-83 (2004).

The 3-benzyl-chroman-4-one has been prepared as well through a radical cyclization process. Bachi. M.D.. Pure & Appl. Chem.. 65:595-601 (1993). A multi- step procedure to prepare 3-benzyl-4-chromanones from chromanones through the use of an intermediate 3-phenylsufonyl-4-chromanone has also been taught. Santhosh, K.C.. Tetr. Lett.. 32:7727-30 (1991).

While chemical methods have been established for the preparation of diverse 3- benzylidinechroman-4-ones and 3-benzylchroman-4-ones, no specific information on the activity of the compounds as mediators of biological processes or their utility to treat diseases is provided by these papers or patents on chemical methods. Several chromanone compounds have been available for purchase in milligram quantities through MicroSource Discovery Systems, Inc. (Gaylordsville, CT). These include the 3-(4-methoxy-3hydroxybenzyl)chroman-4-one listed as deoxysappanone b 7,3'-dimethyl ether, derived from ex Caesalpinia Sappan, as well as the 3-(4-methoxy- 3-hydroxybenzylidine)chroman-4-one listed as dehydrodeoxysappanone b dimethyl ether and the 3-(3-acetoxy-4-methoxybenzylidine)chroman-4-one listed as deoxysappanone b 7,3'-dimethyl ether acetate. The compound deoxysappanone b 7,3'-dimethyl ether is also listed for screening at the ICCB-Longwood Screening Facility, Harvard Medical school.

In view of the lack of diversified compounds in this area as well as a lack of information about therapeutic activity, there is a need to make additional novel chromanone derivative compounds with therapeutic activity. The present invention satisfies these needs and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides novel chromanone derivatives with the following formula:

with the meaning of the variables described below.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows that a compound of the invention, designated "IMS 1998" and described in Example ID, caused significant G2/M arrest as early as three days post- treatment.

Figure 2 shows the inhibitory effect on tube formation of HUVEC cells (an angiogenesis process) of two different concentrations of IMS 1998.

Figure 3 shows that a compound of the invention, designated "IMS 1999" and described in Example IE, significantly decreased VEGF-induced blood vessel formation, with local delivery (s.c.) of the compound having an even stronger effect than systemic (i.p.) delivery.

Figure 4 shows that, in the first chamber slide experiment with HUVEC cells, IMS 1998 inhibited VEGF induced cell migration.

Figure 5 shows the results of the second chamber slide experiment with THP-I cells. Specifically, two compounds of the invention, "IMS2217" (Figure 5A and described in Example 4) and "IMS2186" (Figure 5B and described in Example 2) inhibit cell migration, with IMS2217 being even more potent that IMS2186.

Figure 6 shows the results of the wound healing experiment. Specifically, IMS2186 inhibited the migration of cells into the wound (scratch) - (Figure 6A and Figure 6B). Control cells that were not exposed to IMS2186 migrated and covered the wound within 20 hours (Figure 6C).

Figures 7A and Figure 7B each show that IMS2217 significantly decreased PGE2 production on both undifferentiated THP-I cells and differentiated THP-I cells in a dose dependent manner, with the levels of inhibition higher on differentiated THP-I cells.

Figure 8 shows that IMS 1998 inhibited iNOS mediated nitric oxide production in a dose dependent manner.

Figures 9, 10 and 11 show that IMS 1999 caused a significant reduction in all inflammatory indicators measured (TNF-α, IL-6 and PGE2, respectively), both in plasma and in the peritoneal fluid.

Figure 12 shows the results of mice inoculated with SKO V3 human ovarian cancer cells (ATCC) subcutaneously, with one group injected intraperitoneally (i.p.) with IMS2217 at 50mg/kg, and the other group injected with 5% dextrose (Sigma).

Tumor size (V= (length X width ² )/2 and body weight were measured twice/week. The results are shown in Figure 12A (tumor size) and Figure 12B (body weight). Figure 13 shows the results of mice inoculated with human fibrosarcoma HT 1080 cells subcutaneously at 3X10 ⁶ cells/site. Once the average tumor size reached 50 ³ mm, the mice were divided into two groups evenly (n=10). For the test group, IMS2186 was administered directly into the tumor (intratumoral injections) twice at weekly intervals. Tumor growth in the injected mice and mouse body weight was measured twice/week post injection. The results are shown in Figure 13A (tumor size) and Figure 13B (body weight).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel chromanone derivatives with the following formula:

wherein, the dotted lines represent an optional second bond;

Ri is Ci to C ₁₂ alkyl, C ₂ to Ci ₂ alkenyl, C ₂ to Ci ₂ alkynyl, Ci to Ci ₂ substituted alkyl, C ₂ to Ci ₂ substituted alkenyl, C ₂ to Ci ₂ substituted alkynyl or the formula -OR, where R is Ci to Ci ₂ alkyl, Ci to Ci ₂ substituted alkyl, heterocycle or substituted heterocycle;

R ₂ , R3, R ₄ and R5 are, independently, hydrogen, halogen, cyano, Ci to Ci ₂ alkyl, C ₂ to Ci ₂ alkenyl, C ₂ to Ci ₂ alkynyl, Ci to Ci ₂ substituted alkyl, C ₂ to Ci ₂ substituted alkenyl, C ₂ to Ci ₂ substituted alkynyl or the formulas -OR, -NHC(O)R, -NHC(O)OR, -C(O)R, -C(O)OR or -SO ₂ NRY, where R and Y are, independently, Ci to Ci ₂ alkyl, Ci to Ci ₂ substituted alkyl, heterocycle or substituted heterocycle; and

R ₆ is hydrogen, phosphate, sulfate, the formulas -C(O)X or -C(O)CH(R ₇ )NH ₂ , where X is Ci to Ci ₂ alkyl, Ci to Ci ₂ substituted alkyl, heterocycle or substituted heterocycle and where R ₇ is hydrogen, Ci to Ci ₂ alkyl, Ci to Ci ₂ substituted alkyl, heterocycle or substituted heterocycle.

In a preferred embodiment, R ₂ is hydrogen, R ₅ is hydrogen or both positions are hydrogen.

In another preferred embodiment, R ₆ is -C(O)CH(Ry)NH ₂ and, even more preferably, R ₇ is hydrogen, methyl, -CH(CH ₃ ) ₂ , -CH ₂ CH(CH ₃ ) ₂ , -CH(CH ₃ )CH ₂ CH ₃ , -CH ₂ OH, -CH(OH)CH ₃ , benzyl, 4-hydroxybenzyl, indol-3-ylmethyl, -CH ₂ SH, - CH ₂ CH ₂ SCH ₃ , -CH ₂ C(O)NH ₂ , -CH ₂ CH ₂ C(O)NH ₂ , -CH ₂ -C(O)OH, -CH ₂ -C(O)O ^" , - CH ₂ CH ₂ -C(O)OH, -CH ₂ CH ₂ -C(O)O ^" , (CH ₂ ) ₄ NH ₃ ⁺ , guaninidino-1 -butyl or imidazol- 4-ylmethyl.

In another preferred embodiment, R ₆ is -C(O)X and, more preferably, X is 2- pyrrolidyl or 2-piperidinyl.

In a further preferred embodiment, Ri is -OR and, more preferably, R is Ci to Ci ₂ alkyl, and even more preferably, R is methyl. In yet another embodiment, R ₃ is -OR and, more preferably, R is Ci to Ci ₂ alkyl and, even more preferably, R is methyl or ethyl. In another embodiment of the subject invention, R ₆ is phosphate or hydrogen.

In still a further embodiment, R ₄ is halogen and, more preferably, is chloro or fluoro.

The compounds of the invention can be used against a variety of biological processes, including cell proliferation (see Example 16), cell cycle progression (see Example 17), angiogenesis (see Examples 18 and 19), cell migration (see Example 20) and inflammation (see Examples 21 and 22). Thus, the compounds of the invention can be used to treat or assist in inhibiting a variety of diseases, including, but not limited to, cancer (see Example 23), keloids or hypertrophic scars and other conditions associated with fibroblast activity (see Example 24), inflammatory diseases and wet AMD (age-related macular degeneration, see Example 25). Accordingly, the present invention provides a method for reduction of the growth or proliferation of mammalian cancer cells, comprising applying to the cancer cells a therapeutically effective amount of the novel compound of the invention.

Furthermore, the present invention provides a method for the treatment of mammalian cancer, comprising administering to the mammalian subject a pharmaceutical composition containing as an active ingredient a therapeutically effective amount of the novel compound of the invention.

In addition, the present invention provides similar methods for treating or preventing the biological processes or diseases described herein.

The term "reduction of growth" in relation to cancer cells, in the context of the

present invention refers to a decrease in at least one of the following: number of cells (due to cell death which may be necrotic, apoptotic or any other type of cell death or combinations thereof) as compared to control; decrease in growth rates of cells, i.e. the total number of cells may increase but at a lower level or at a lower rate than the increase in control; decrease in the invasiveness of cells (as determined for example by soft agar assay) as compared to control even if their total number has not changed; progression from a more differentiated cell type to a less differentiated cell type; a deceleration in the neoplastic progress; or alternatively the slowing of the progression of the cancer cells from one stage to the next. Reduction of growth of cancer cells may be utilized for the treatment of cancer by the administration, to an individual in need of such treatment, of a therapeutically effective amount of the compound of the present invention, as described herein.

The present invention additionally discloses use of a composition of the invention, as described above, for preparing a medicament for the treatment of cancer in mammals.

The term "treatment of cancer" in the context of the present invention includes at least one of the following: a decrease in the rate of growth of the cancer (i.e. the cancer still grows but at a slower rate); cessation of growth of the cancerous growth, i.e., stasis of the tumor growth, and, in preferred cases, the tumor diminishes or is reduced in size. The term also includes reduction in the number of metastasis, reduction in the number of new metastasis formed, slowing of the progression of cancer from one stage to the other and a decrease in the angiogenesis induced by the cancer. In most preferred cases, the tumor is totally eliminated. Additionally included in this term is lengthening of the survival period of the subject undergoing treatment. This term also encompasses prevention for prophylactic situations or for those individuals who are susceptible to contracting a tumor. The administration of the compounds of the present invention will reduce the likelihood of the individual contracting the disease. In preferred situations, the individual to whom the compound is administered does not contract the disease. The term "cancer" in the context of the present invention includes all types of neoplasm whether in the form of solid or non-solid tumors, from all origins, and includes both malignant and benign conditions as well as their metastasis. In particular this term refers to: carcinoma, sarcoma, adenoma, hepatocellular carcinoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid

carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphagiosarcoma, synovioama, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hematoma, bile duct carcinoma, melanoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell and non-small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma, ependynoma, pinealoma, retinoblastoma,, multiple myeloma, rectal carcinoma, cancer of the thyroid, head and neck cancer, brain cancer, cancer of the peripherial nervous system, cancer of the central nervous system, neuroblastoma, cancer of the edometrium, myeloid lymphoma, leukemia, lymphoma, lymphoproliferative diseases, acute myelocytic leukemia, chronic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma as well as metastasis of all the above.

An inflammatory disease, as used herein, includes, but is not limited to, arthritis, including rheumatoid arthritis, inflammation associated with hypersensitivity, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcer, ulcerative colitis and other diseases of the gastrointestinal tract, hepatic fibrosis, liver cirrhosis or other hepatic diseases, thyroiditis or other glandular diseases, glomerulonephritis or other renal and urologic diseases, otitis or other oto- rhino-laryngological diseases, dermatitis or other dermal diseases, periodontal diseases or other dental diseases, orchitis or epididimo-orchitis, infertility, orchidal trauma or other immune-related testicular diseases, placental dysfunction, placental insufficiency, habitual abortion, eclampsia pre-eclampsia and other immune and/or inflammatory-related gynecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitis or cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative

fondus disease, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitro-retinopathies, acute ischaemic optic neuropathy, excessive scarring, e.g. following glaucoma filtration operation, immune and/or inflammation reaction against ocular implants and other immune and inflammatory-related ophthalmic diseases, inflammation associated with autoimmune diseases or conditions or disorders where, both in the central nervous system (CNS) or in any other organ, immune and/or inflammation suppression would be beneficial, Parkinson's disease, complication and/or side effects from treatment of Parkinson's disease, AIDS-related dementia complex HIV -related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases, conditions or disorders of the CNS, inflammatory components of stokes, post-polio syndrome, immune and inflammatory components of psychiatric disorders, myelitis, encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis, pseudo-tumor cerebri; Down's Syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory components of CNS compression or CNS trauma or infections of the CNS, inflammatory components of muscular atrophies and dystrophies, and immune and inflammatory related diseases, conditions or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery; bone marrow transplantation or other transplantation complications and/or side effects, inflammatory and/or immune complications and side effects of gene therapy, e.g. due to infection with a viral carrier, or inflammation associated with AIDS, to suppress or inhibit a humoral and/or cellular immune response, to treat or ameliorate monocyte or leukocyte proliferative diseases, e.g. leukemia, by reducing the amount of monocytes or lymphocytes, for the prevention and/or treatment of graft rejection in cases of transplantation of natural or artificial cells, tissue and organs such as cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue. The term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, substantially ameliorating clinical symptoms of a disease or substantially preventing the appearance of clinical symptoms of a disease.

The term "preventing" refers to a method for barring an organism from acquiring a disorder or disease in the first place.

The term "organism" refers to any living entity comprised of at least one cell. A living organism can be as simple as, for example, a single eukaryotic cell or as complex as a mammal, including a human being.

The term "therapeutically effective amount" refers to that amount of the compound being administered which will relieve to some extent one or more of the symptoms of the disorder being treated.

In other embodiments of the use of preparing a medicament to treat or prevent cancer, the medicament additionally comprises at least one active chemotherapeutic agent other than the compound of the invention. In certain embodiments, the novel compound may be administered alongside with traditional chemotherapeutic drugs that are effective but have considerable side effects. The combination of a compound of the invention and the traditional drug may allow administration of a lesser quantity of the traditional drug, and thus the side effects experienced by the subject may be significantly lower, while a sufficient chemotherapeutic effect is nevertheless achieved. Preferred additional active chemotherapeutics include but are not limited to taxol or doxorubicin.

In preferred embodiments of the methods of the invention, the compound is administered at a dosage selected from lμg-1000 mg/kg body weight.

There is also provided in the present invention a pharmaceutical composition for the indications described herein, comprising as the active ingredient a therapeutically effective amount of a compound of the invention, as described above.

There is also provided in the present invention a pharmaceutical composition for the indications described herein, comprising as the active ingredient a therapeutically effective amount of a compound of the invention, as described herein, and a pharmaceutically acceptable carrier.

The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals and, more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Water

is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, cyclodextrins and the like.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.

The compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions will contain a therapeutically effective amount of a compound of the invention, preferably in a substantially purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

The carrier can be selected at times based on the desired form of the formulation. The carrier may also at times have the effect of the improving the delivery or penetration of the active ingredient to the target tissue, for improving the stability of the drug, for slowing clearance rates, for imparting slow release properties, for reducing undesired side effects etc. The carrier may also be a substance that stabilizes the formulation (e.g. a preservative), for providing the formulation with an edible flavor, etc.

The carriers may be any of those conventionally used and are limited only by chemical-physical considerations, such as solubility and lack of reactivity with the compound of the invention, and by the route of administration. The choice of carrier will be determined by the particular method used to administer the pharmaceutical

composition. Accordingly, the carrier may include additives, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible carriers. In addition, the carrier may be an adjuvant, which, by definition are substances affecting the action of the active ingredient in a predictable way.

Methods of introduction of a pharmaceutical composition comprising a compound of the invention include, but are not limited to, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, and oral routes. The compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other therapeutically active agents. It is preferred that administration is localized, but it may be systemic. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

It may be desirable to administer the pharmaceutical composition of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material. According to some preferred embodiments, administration can be by direct injection e.g., via a syringe, at the site of a tumor or neoplastic or pre-neoplastic tissue.

Pharmaceutical compositions suitable for oral administration may consist of (a) liquid solutions, where an effective amount of the active substance is dissolved in diluents, such as water, saline, natural juices, alcohols, syrups, etc.; (b) solid dosage forms such as capsules (e.g. the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers), tablets, lozenges (wherein the active substance is flavored, such as with sucrose and acacia or tragacanth, or the active substance is in an inert base, such as gelatin and glycerin), and troches, each containing a predetermined amount of the active ingredient as solids or granules; (c)

powders; (d) suspensions in an appropriate liquid; (e) suitable emulsions; (f) liposome formulation; and others.

In yet another embodiment, the composition is prepared for topical administration, e.g. as an ointment, a gel a drop or a cream. For topical administration to body surfaces using, for example, creams, gels, drops, ointments and the like, the compounds of the present invention can be prepared and applied in a physiologically acceptable diluent with or without a pharmaceutical carrier. The present invention may be used topically or transdermally to treat cancer, for example, melanoma. Adjuvants for topical or gel base forms may include, for example, sodium carboxymethylcellulose, polyacrylates, polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol and wood wax alcohols.

For directed internal topical applications, the pharmaceutical composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; or a glidant such as colloidal silicon dioxide. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.

A compound of the present invention can be delivered in a controlled release system. In one embodiment, an infusion pump may be used to administer a compound of the invention, such as one that is used for delivering insulin or chemotherapy to specific organs or tumors (see Buchwald et al, 1980, Surgery 88: 507; Saudek et al, 1989, N. Engl. J. Med. 321 : 574). In a preferred form, a compound of the invention is administered in combination with a biodegradable, biocompatible polymeric implant, which releases the compound over a controlled period of time at a selected site. Examples of preferred polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (See, Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, FIa.). In yet another embodiment, a controlled release

system can be placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose.

At times, the active compound may be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as propane, butane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.

Furthermore, at times, the pharmaceutical compositions may be formulated for parenteral administration (subcutaneous, intravenous, intraarterial, or intramuscular injection) and may include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Oils such as petroleum, animal, vegetable, or synthetic oils and soaps such as fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents may also be used for parenteral administration. The above formulations may also be used for direct intra-tumoral injection. Further, in order to minimize or eliminate irritation at the site of injection, the compositions may contain one or more nonionic surfactants. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol.

The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described and known in the art.

The amount of a compound of the invention that will be effective in the treatment of a particular disorder or condition, including cancer, will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also

depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. A preferred dosage will be within the range of 0.01-1000 mg/kg of body weight, more preferably, 0.1 mg/kg to 100 mg/kg and even more preferably 1 mg/kg to 10mg/kg. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test bioassays or systems.

A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing or eliminating those signs. A "therapeutically effective amount" of a compound of the invention is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.

Patients in need thereof may suffer from a disease such as cancer or may have been determined to have a greater susceptibility to such disease. Thus, the method of treatment according to the present invention includes both therapeutic and prophylactic utility.

A compound of the invention can be tested in vivo for the desired therapeutic or prophylactic activity as well as for determination of a therapeutically effective dosage. For example, such compounds can be tested in suitable animal model systems prior to testing in humans, including, but not limited to, rats, mice, chicken, cows, monkeys, rabbits, and the like. For in vivo testing, prior to administration to humans, any animal model system known in the art may be used.

When the above-described compounds include one or more chiral centers, the stereochemistry of such chiral centers can independently be in the R or S configuration, or a mixture of the two. The chiral centers can be further designated as R or S or R,S or d,D, 1,L or d,l, D,L.

Regarding the compounds and described herein, the suffix "ene" added to any of the described terms means that two parts of the substituent are each connected to two other parts in the compound (unless the substituent contains only one carbon, in which case such carbon is connected to two other parts in the compound, for example, methylene).

The term "Ci to C 12 alkyl" denotes such radicals as methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, amyl, tert-amyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. Preferred "Ci to C ₁₂ alkyl" groups are methyl, ethyl, iso-butyl, sec-butyl and iso-propyl. Similarly, the term "Ci to C ₁₂

alkylene" denotes radicals of 1 to 12 carbons connected to two other parts in the compound.

The term "C ₂ to Ci ₂ alkenyl" denotes such radicals as vinyl, allyl, 2-butenyl, 3- butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5- hexenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, (as well as octenyl, nonenyl, decenyl, undecenyl, dodecenyl radicals attached at any appropriate carbon position and the like) as well as dienes and trienes of straight and branched chains.

The term "C ₂ to Ci ₂ alkynyl" denotes such radicals as ethanol, propynyl, 2- butynyl, 2-pentynyl, 3-pentynyl, 2- hexynyl, 3-hexynyl, 4-hexynyl, 2-heptynyl, 3- heptynyl, 4- heptynyl, 5-heptynyl (as well as octynyl, nonynyl, decynyl, undecynyl, dodecynyl radicals attached at any appropriate carbon position and the like) as well as di- and tri-ynes of straight and branched chains.

The terms "Ci to Ci ₂ substituted alkyl," "C ₂ to Ci ₂ substituted alkenyl," "C ₂ to Ci ₂ substituted alkynyl," "Ci to Ci ₂ substituted alkylene," "C ₂ to Ci ₂ substituted alkenylene" and "C ₂ to Ci ₂ substituted alkynylene" denote groups that are substituted by one or more, and preferably one or two, halogen, hydroxy, protected hydroxy, oxo, protected oxo, C3 to C ₇ cycloalkyl, phenyl, naphthyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, guanidino, protected guanidino, heterocyclic ring, substituted heterocyclic ring, imidazolyl, indolyl, pyrrolidinyl, Ci to Ci ₂ alkoxy, Ci to Ci ₂ acyl, Ci to Ci ₂ acyloxy, nitro, carboxy, protected carboxy, carbamoyl, carboxamide, protected carboxamide, N-(Ci to Ci ₂ alkyl)carboxamide, protected N-(Ci to Ci ₂ alkyl)carboxamide, N ₅ N-(Ii(C ₁ to Ci ₂ alkyl)carboxamide, cyano, methylsulfonylamino, thiol, Ci to C ₁₀ alkylthio or Ci to Cio alkylsulfonyl groups. The substituted alkyl groups may be substituted once or more, and preferably once or twice, with the same or with different substituents. A preferred substitution is halo.

The term "protected oxo" denotes a carbon atom bonded to two additional carbon atoms substituted with two alkoxy groups or twice bonded to a substituted diol moiety, thereby forming an acyclic or cyclic ketal moiety.

The term "Ci to Ci ₂ alkoxy" as used herein denotes groups such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy and like groups. A preferred alkoxy is methoxy. The term "Ci to Ci ₂ substituted alkoxy" means the alkyl portion of the alkoxy can be substituted in the same manner as in relation to Ci to Ci ₂

substituted alkyl. A preferred substitution is halo. Similarly, the term "Ci to C ₁₂ phenylalkoxy" as used herein means "Ci to C ₁₂ alkoxy" bonded to a phenyl radical.

The term "Ci to C 12 acyloxy" denotes herein groups such as formyloxy, acetoxy, propionyloxy, butyryloxy, pivaloyloxy, pentanoyloxy, hexanoyloxy, heptanoyloxy, octanoyloxy, nonanoyloxy, decanoyloxy, undecanoyloxy, dodecanoyloxy and the like. Similarly, the term "Ci to C 12 acyl" encompasses groups such as formyl, acetyl, propionyl, butyryl, pentanoyl, pivaloyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, benzoyl and the like. Preferred acyl groups are acetyl and benzoyl. The term "Ci to C 12 substituted acyl" denotes the acyl group substituted by one or more, and preferably one or two, halogen, hydroxy, protected hydroxy, oxo, protected oxo, cyclohexyl, naphthyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, guanidino, heterocyclic ring, substituted heterocyclic ring, imidazolyl, indolyl, pyrrolidinyl, Ci to C ₁₂ alkoxy, Ci to C ₁₂ acyl, Ci to C ₁₂ acyloxy, nitro, Ci to C ₁₂ alkyl ester, carboxy, protected carboxy, carbamoyl, carboxamide, protected carboxamide, N-(Ci to C ₁₂ alkyl)carboxamide, protected N-(Ci to C ₁₂ alkyl)carboxamide, N ₅ N-CIi(C ₁ to C ₁₂ alkyl)carboxamide, cyano, methylsulfonylamino, thiol, Ci to C ₁₀ alkylthio or Ci to Cio alkylsulfonyl groups. The substituted acyl groups may be substituted once or more, and preferably once or twice, with the same or with different substituents.

The substituent term "C ₃ to C ₇ cycloalkyl" includes the cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings. Similarly, a substituent that can be C ₃ to C ₇ cycloalkyl" can also be "C ₅ to C ₇ cycloalkyl," which includes the cyclopentyl, cyclohexyl or cycloheptyl rings. The substituent term "C ₃ to C ₇ substituted cycloalkyl" or "C ₅ to C ₇ substituted cycloalkyl" indicates the above cycloalkyl rings substituted by one or two halogen, hydroxy, protected hydroxy, Ci to C ₁₀ alkylthio, Ci to C ₁₀ alkylsulfoxide, Ci to C ₁₀ alkylsulfonyl, Ci to C ₁₀ substituted alkylthio, Ci to C ₁₀ substituted alkylsulfoxide, Ci to Cio substituted alkylsulfonyl, Ci to Ci ₂ alkyl, Ci to Ci ₂ alkoxy, Ci to Ci ₂ substituted alkyl, Ci to Ci ₂ alkoxy, oxo, protected oxo, (monosubstituted)amino,

(disubstituted)amino, trifluoromethyl, carboxy, protected carboxy, phenyl, substituted phenyl, phenylthio, phenylsulfoxide, phenylsulfonyl, amino, or protected amino groups.

The term "cycloalkylene" means a cycloalkyl, as defined above, where the cycloalkyl radical is bonded at two positions connecting together two separate additional groups. Similarly, the term "substituted cycloalkylene" means a cycloalkylene where the cycloalkyl radical is bonded at two positions connecting together two separate additional groups and further bearing at least one additional substituent.

The term "C ₅ to C ₇ cycloalkenyl" indicates a 1,2, or 3-cyclopentenyl ring, a 1,2,3 or 4-cyclohexenyl ring or a 1,2,3,4 or 5-cycloheptenyl ring, while the term "substituted C5 to C ₇ cycloalkenyl" denotes the above C5 to C ₇ cycloalkenyl rings substituted by a Ci to C ₁₂ alkyl radical, halogen, hydroxy, protected hydroxy, Ci to C ₁₂ alkoxy, trifluoromethyl, carboxy, protected carboxy, oxo, protected oxo, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, phenyl, substituted phenyl, amino, or protected amino.

The term "C5 to C ₇ cycloalkenylene" is a cycloalkenyl ring, as defined above, where the cycloalkenyl radical is bonded at two positions connecting together two separate additional groups. Examples of C ₅ to C ₇ cycloalkenylenes include 1,3-cyclopentylene and 1,2-cyclohexylene.

Similarly, the term "substituted C5 to C ₇ cycloalkenylene" means a cycloalkenylene further substituted by halogen, hydroxy, protected hydroxy, Ci to C ₁₀ alkylthio, Ci to C ₁₀ alkylsulfoxide, Ci to C ₁₀ alkylsulfonyl, Ci to C ₁₀ substituted alkylthio, Ci to Ci ₀ substituted alkylsulfoxide, Ci to Ci ₀ substituted alkylsulfonyl, Ci to C ₁₂ alkyl, Ci to C ₁₂ alkoxy, Ci to C ₁₂ substituted alkyl, Ci to C ₁₂ alkoxy, oxo, protected oxo, (monosubstituted)amino, (disubstituted)amino, trifluoromethyl, carboxy, protected carboxy, phenyl, substituted phenyl, phenylthio, phenylsulfoxide, phenylsulfonyl, amino, or protected amino group.

The term "heterocycle" or "heterocyclic ring" denotes optionally substituted five-membered to eight-membered rings that have 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen, in particular nitrogen, either alone or in conjunction with sulfur or oxygen ring atoms. The term "bicyclic heterocycle" means two such rings fused to each other. These five-membered to eight-membered rings may be saturated, fully unsaturated or partially unsaturated, with fully saturated rings being preferred. Preferred heterocyclic rings include morpholino, piperidinyl, piperazinyl, 2-amino-imidazoyl, tetrahydrofurano, pyrrolo, tetrahydrothiophen-yl, hexylmethyleneimino and heptylmethyleneimino.

The terms "substituted heterocycle" or "substituted heterocyclic ring" and "substituted bicyclic heterocycle" mean the above-described heterocyclic or fused biheterocyclic rings are substituted with, for example, one or more, and preferably one or two, substituents which are the same or different which substituents can be halogen, hydroxy, protected hydroxy, cyano, nitro, Ci to C ₁₂ alkyl, Ci to C ₁₂ alkoxy, Ci to C ₁₂ substituted alkoxy, Ci to C ₁₂ acyl, Ci to C ₁₂ acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino carboxamide, protected carboxamide, N-(Ci to C ₁₂ alkyl)carboxamide, protected N-(Ci to C12 alkyl)carboxamide, N, N- CIi(C ₁ to C ₁₂ alkyl)carboxamide, trifluoromethyl, N-((Ci to Ci ₂ alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino, heterocycle or substituted heterocycle groups.

The term "heteroaryl" means a heterocyclic aromatic derivative which is a fϊve- membered or six-membered ring system having from 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen, in particular nitrogen, either alone or in conjunction with sulfur or oxygen ring atoms. Examples of heteroaryls include pyridinyl, pyrimidinyl, and pyrazinyl, pyridazinyl, pyrrolo, furano, oxazolo, isoxazolo, phthalimido, thiazolo and the like.

The term "substituted heteroaryl" means the above-described heteroaryl is substituted with, for example, one or more, and preferably one or two, substituents which are the same or different which substituents can be halogen, hydroxy, protected hydroxy, cyano, nitro, Ci to Ci ₂ alkyl, Ci to Ci ₂ alkoxy, Ci to Ci ₂ substituted alkoxy, Ci to Ci ₂ acyl, Ci to Ci ₂ substituted acyl, Ci to Ci ₂ acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected

(monosubstituted)amino, (disubstituted)amino, carboxamide, protected carboxamide, N-(Ci to Ci ₂ alkyl)carboxamide, protected N-(Ci to Ci ₂ alkyl)carboxamide, N, N- di(Ci to Ci ₂ alkyl)carboxamide, trifluoromethyl, N-((Ci to Ci ₂ alkyl)sulfonyl)amino or N-(phenylsulfonyl)amino groups. The term "C ₇ to C ₁₈ phenylalkyl" denotes a Ci to Ci ₂ alkyl group substituted at any position within the alkyl chain by a phenyl. The definition includes groups of the formula: -phenyl-alkyl, -alkyl-phenyl and -alkyl-phenyl-alkyl. Examples of such a group include benzyl, 2-phenylethyl, 3-phenyl(n-propyl), 4-phenylhexyl, 3-phenyl(n-

amyl), 3-phenyl(sec-butyl) and the like. Preferred C ₇ to C ₁₈ phenylalkyl groups are any one of the preferred alkyl groups described herein combined with a phenyl group. Similarly, the term "Ci to C ₁₂ heterocycloalkyl" denotes a Ci to C ₁₂ alkyl group substituted at any position within the alkyl chain by a "heterocycle," as defined herein. The definition includes groups of the formula: -heterocyclic-alkyl, -alkyl- heterocyclic and -alkyl-heterocyclic-alkyl. Preferred Ci to C ₁₂ heterocycloalkyl groups are any one of the preferred alkyl groups described herein combined with any one of the preferred heterocycle groups described herein.

The terms "C ₇ to C ₁₈ substituted phenylalkyl" and "Ci to C ₁₂ substituted heterocycloalkyl" denote a C ₇ to C ₁₈ phenylalkyl group or Ci to C12 heterocycloalkyl substituted (on the alkyl or, where applicable, phenyl or heterocyclic portion) with one or more, and preferably one or two, groups chosen from halogen, hydroxy, protected hydroxy, oxo, protected oxo, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, guanidino, protected guanidino, heterocyclic ring, substituted heterocyclic ring, Ci to Ci ₂ alkyl, Ci to Ci ₂ substituted alkyl, Ci to Ci ₂ alkoxy, Ci to Ci ₂ substituted alkoxy, Ci to Ci ₂ acyl, Ci to Ci ₂ substituted acyl, Ci to Ci ₂ acyloxy, nitro, carboxy, protected carboxy, carbamoyl, carboxamide, protected carboxamide, N-(Ci to Ci ₂ alkyl)carboxamide, protected N- (Ci to Ci ₂ alkyl)carboxamide, N, N-(Ci to Ci ₂ dialkyl)carboxamide, cyano, N-(Ci to Ci ₂ alkylsulfonyl)amino, thiol, Ci to C ₁₀ alkylthio, Ci to C ₁₀ alkylsulfonyl groups; and/or the phenyl group may be substituted with one or more, and preferably one or two, substituents chosen from halogen, hydroxy, protected hydroxy, cyano, nitro, Ci to Ci ₂ alkyl, Ci to Ci ₂ substituted alkyl, Ci to Ci ₂ alkoxy, Ci to Ci ₂ substituted alkoxy, Ci to Ci ₂ acyl, Ci to Ci ₂ substituted acyl, Ci to Ci ₂ acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, carboxamide, protected carboxamide, N-(Ci to Ci ₂ alkyl)carboxamide, protected N-(Ci to Ci ₂ alkyl)carboxamide, N, N- CIi(C ₁ to Ci ₂ alkyl)carboxamide, trifluoromethyl, N-((Ci to Ci ₂ alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino, cyclic C ₂ to Ci ₂ alkylene or a phenyl group, substituted or unsubstituted, for a resulting biphenyl group. The substituted alkyl, phenyl or heterocyclic groups may be substituted with one or more, and preferably one or two, substituents which can be the same or different.

The term "C ₇ to C ₁₈ phenylalkylene" specifies a C ₇ to C ₁₈ phenylalkyl, as defined above, where the phenylalkyl radical is bonded at two different positions connecting together two separate additional groups. The definition includes groups of the formula: -phenyl-alkyl-, -alkyl-phenyl- and -alkyl-phenyl-alkyl-. Substitutions on the phenyl ring can be 1,2, 1,3 or 1,4. C ₇ to C ₁₈ phenylalkylenes include, for example, 1,4-tolylene and 1,3-xylylene.

Similarly, the term "Ci to C12 heterocycloalkylene" specifies a Ci to Ci ₂ heterocycloalkyl, as defined above, where the heterocycloalkyl radical is bonded at two different positions connecting together two separate additional groups. The definition includes groups of the formula: -heterocyclic-alkyl-, -alkyl-heterocyclic and -alkyl-heterocyclic-alkyl-.

The terms "C ₇ to Ci ₈ substituted phenylalkylene" and "Ci to Ci ₂ substituted heterocycloalkylene" means a C ₇ to C ₁₈ phenylalkylene or Ci to Ci ₂ heterocycloalkylene as defined above that is further substituted by halogen, hydroxy, protected hydroxy, Ci to C ₁₀ alkylthio, Ci to C ₁₀ alkylsulfoxide, Ci to C ₁₀ alkylsulfonyl, Ci to C ₁₀ substituted alkylthio, Ci to C ₁₀ substituted alkylsulfoxide, Ci to Cio substituted alkylsulfonyl, Ci to Ci ₂ alkyl, Ci to Ci ₂ alkoxy, Ci to Ci ₂ substituted alkyl, Ci to Ci ₂ alkoxy, oxo, protected oxo, (monosubstituted)amino, (disubstituted)amino, trifluoromethyl, carboxy, protected carboxy, phenyl, substituted phenyl, phenylthio, phenylsulfoxide, phenylsulfonyl, amino, or protected amino group on the phenyl ring or on the alkyl group.

The term "substituted phenyl" specifies a phenyl group substituted with one or more, and preferably one or two, moieties chosen from the groups consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, Ci to Ci ₂ alkyl, Ci to Ci ₂ substituted alkyl, Ci to Ci ₂ alkoxy, Ci to Ci ₂ substituted alkoxy, Ci to Ci ₂ acyl, Ci to Ci ₂ substituted acyl, Ci to Ci ₂ acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, carboxamide, protected carboxamide, N-(Ci to Ci ₂ alkyl)carboxamide, protected N-(Ci to Ci ₂ alkyl)carboxamide, N, N-di(Ci to Ci ₂ alkyl)carboxamide, trifluoromethyl, N-((Ci to Ci ₂ alkyl)sulfonyl)amino, N- (phenylsulfonyl)amino or phenyl, wherein the phenyl is substituted or unsubstituted, such that, for example, a biphenyl results.

The term "phenoxy" denotes a phenyl bonded to an oxygen atom, wherein the binding to the rest of the molecule is through the oxygen atom. The term "substituted phenoxy" specifies a phenoxy group substituted with one or more, and preferably one or two, moieties chosen from the groups consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, Ci to C ₁₂ alkyl, Ci to C ₁₂ alkoxy, Ci to C ₁₂ substituted alkoxy, Ci to C ₁₂ acyl, Ci to C ₁₂ acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, carboxamide, protected carboxamide, N-(Ci to C 12 alkyl)carboxamide, protected N-(Ci to C ₁₂ alkyl)carboxamide, N, N-CIi(C ₁ to C ₁₂ alkyl)carboxamide, trifluoromethyl, N-((Ci to C ₁₂ alkyl)sulfonyl)amino and N- (pheny lsulfony l)amino .

The term "aryl" refers to an aromatic group having at least one carbocyclic aromatic group or heterocyclic aromatic group, which may be unsubstituted or substituted by one or more groups selected from halogen, haloalkyl, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxy or thio or thioalkyl. Nonlimiting examples of aryl rings are phenyl, naphthyl, pyranyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyrazolyl, pyridinyl, furanyl, thiophenyl, thiazolyl, imidazolyl, isoxazolyl, and the like. The term "aryloxy" refers to an "aryl" group bonded to an oxygen atom, wherein the binding to the rest of the molecule is through the oxygen atom.

The term "C ₇ to C ₁₈ substituted phenylalkoxy" denotes a C ₇ to C ₁₈ phenylalkoxy group bonded to the rest of the molecule through the oxygen atom, wherein the phenylalkyl portion is substituted with one or more, and preferably one or two, groups selected from halogen, hydroxy, protected hydroxy, oxo, protected oxo, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, guanidino, heterocyclic ring, substituted heterocyclic ring, Ci to C ₁₂ alkoxy, Ci to C ₁₂ acyl, Ci to C ₁₂ acyloxy, nitro, carboxy, protected carboxy, carbamoyl, carboxamide, protected carboxamide, N-(Ci to C ₁₂ alkyl)carboxamide, protected N-(Ci to C ₁₂ alkyl)carboxamide, N, N-(Ci to C ₁₂ dialkyl)carboxamide, cyano, N-(Ci to C ₁₂ alkylsulfonyl)amino, thiol, Ci to C ₁₀ alkylthio, Ci to C ₁₀ alkylsulfonyl groups; and/or the phenyl group can be substituted with one or more, and preferably one or two, substituents chosen from halogen, hydroxy, protected hydroxy, cyano, nitro, Ci to C ₁₂ alkyl, Ci to C ₁₂ alkoxy, Ci to C ₁₂ acyl, Ci to C ₁₂

acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, carboxamide, protected carboxamide, N-(Ci to C ₁₂ alkyl) carboxamide, protected N- (Ci to C ₁₂ alkyl) carboxamide, N, N-di(Ci to C12 alkyl)carboxamide, trifluoromethyl, N-((Ci to C 12 alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or a phenyl group, substituted or unsubstituted, for a resulting biphenyl group. The substituted alkyl or phenyl groups may be substituted with one or more, and preferably one or two, substituents which can be the same or different. The term "phthalimide" means a cyclic imide which is made from phthalic acid, also called 1,2-benzenedicarboxylic acid. The term "substituted phthalimide" specifies a phthalimide group substituted with one or more, and preferably one or two, moieties chosen from the groups consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, Ci to Ci ₂ alkyl, Ci to Ci ₂ alkoxy, Ci to Ci ₂ substituted alkoxy, Ci to Ci ₂ acyl, Ci to Ci ₂ acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, carboxamide, protected carboxamide, N-(Ci to Ci ₂ alkyl)carboxamide, protected N- (Ci to C ₁₂ alkyl)carboxamide, N, N-CIi(C ₁ to Ci ₂ alkyl)carboxamide, trifluoromethyl, N-((Ci to C i2 alkyl)sulfonyl)amino and N-(phenylsulfonyl)amino.

The term "substituted naphthyl" specifies a naphthyl group substituted with one or more, and preferably one or two, moieties either on the same ring or on different rings chosen from the groups consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, Ci to C ₆ alkyl, Ci to C ₇ alkoxy, Ci to C ₇ acyl, Ci to C ₇ acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, carboxamide, protected carboxamide, N-(Ci to C ₁₂ alkyl)carboxamide, protected N-(Ci to Ci ₂ alkyl)carboxamide, N, N- di(Ci to C ₁₂ alkyl)carboxamide, trifluoromethyl, N-((Ci to Ci ₂ alkyl)sulfonyl)amino or N-(phenylsulfonyl)amino. Also, the term "substituted naphthyl" represents disubstituted naphthyl groups wherein the substituents are different.

The term "naphthylene" means a naphthyl radical bonded at two positions connecting together two separate additional groups. Similarly, the term "substituted napthylene" means a naphthylene group that is further substituted by halogen,

hydroxy, protected hydroxy, Ci to C ₁₀ alkylthio, Ci to C ₁₀ alkylsulfoxide, Ci to C ₁₀ alkylsulfonyl, Ci to C ₁₀ substituted alkylthio, Ci to C ₁₀ substituted alkylsulfoxide, Ci to Cio substituted alkylsulfonyl, Ci to C ₁₂ alkyl, Ci to C ₁₂ alkoxy, Ci to C ₁₂ substituted alkyl, Ci to Ci ₂ alkoxy, oxo, protected oxo, (monosubstituted)amino, (disubstituted)amino, trifluoromethyl, carboxy, protected carboxy, phenyl, substituted phenyl, phenylthio, phenylsulfoxide, phenylsulfonyl, amino, or protected amino group.

The terms "halo" and "halogen" refer to the fluoro, chloro, bromo or iodo atoms. There can be one or more halogens, which are the same or different. The term "(monosubstituted)amino" refers to an amino group with one substituent chosen from the group consisting of phenyl, substituted phenyl, Ci to Ci ₂ alkyl, Ci to Ci ₂ substituted alkyl, Ci to Ci ₂ acyl, Ci to Ci ₂ substituted acyl, C ₂ to Ci ₂ alkenyl, C ₂ to Ci ₂ substituted alkenyl, C ₂ to Ci ₂ alkynyl, C ₂ to Ci ₂ substituted alkynyl, C ₇ to Ci8 phenylalkyl, C ₇ to C ₁₈ substituted phenylalkyl, heterocyclic ring, substituted heterocyclic ring, Ci to Ci ₂ heterocycloalkyl and Ci to Ci ₂ substituted heterocycloalkyl. The (monosubstituted)amino can additionally have an amino- protecting group as encompassed by the term "protected (monosubstituted)amino."

The term "(disubstituted)amino" refers to an amino group with two substituents chosen from the group consisting of phenyl, substituted phenyl, Ci to Ci ₂ alkyl, Ci to Ci ₂ substituted alkyl, Ci to Ci ₂ acyl, C ₂ to Ci ₂ alkenyl, C ₂ to Ci ₂ alkynyl, C ₇ to Ci ₈ phenylalkyl, C ₇ to Ci ₈ substituted phenylalkyl, Ci to Ci ₂ heterocycloalkyl and Ci to

Ci ₂ substituted heterocycloalkyl. The two substituents can be the same or different.

The term "amino-protecting group" as used herein refers to substituents of the amino group commonly employed to block or protect the amino functionality while reacting other functional groups of the molecule. The term "protected

(monosubstituted)amino" means there is an amino-protecting group on the monosubstituted amino nitrogen atom. In addition, the term "protected carboxamide" means there is an amino-protecting group on the carboxamide nitrogen. Similarly, the term "protected N-(Ci to Ci ₂ alkyl)carboxamide" means there is an amino-protecting group on the carboxamide nitrogen.

The species of amino-protecting group employed is not critical so long as the derivatized amino group is stable to the conditions of the subsequent reaction(s) and can be removed at the appropriate point without disrupting the remainder of the compounds. Preferred amino-protecting groups are Boc, Cbz and Fmoc. Further

examples of amino-protecting groups embraced by the above term are well known in organic synthesis and the peptide art and are described by, for example, T. W. Greene and P.G.M. Wuts, "Protective Groups in Organic Synthesis," 2nd ed., John Wiley and Sons, New York, NY, 1991, Chapter 7, M. Bodanzsky, "Principles of Peptide Synthesis," 1st and 2nd revised ed., Springer- Verlag, New York, NY, 1984 and 1993, and Stewart and Young, "Solid Phase Peptide Synthesis," 2nd ed., Pierce Chemical Co., Rockford, IL, 1984, each of which is incorporated herein by reference. The related term "protected amino" defines an amino group substituted with an amino- protecting group discussed above. The term "protected guanidino" as used herein refers to an "amino-protecting group" on one or two of the guanidino nitrogen atoms. Examples of "protected guanidino" groups are described by T. W. Greene and PGM. Wuts; M. Bodanzsky; and Stewart and Young, supra.

The term "epimino" means -NH-. The term "substituted epimino" means - N(R)-, where R is a substitution group listed above under the definition of "(monosubstituted)amino."

The term "Ci to Cs alkylene epimino" refers to a one to five carbon alkylene chain with an epimino at any point along the chain. The term "Ci to Cs substituted alkylene epimino" refers to a Ci to Cs alkylene epimino group that is substituted a) at the epimino position (in the same way as "substituted epimino," described above); and/or b) at one or more of the alkylene positions (in the same way as "substituted alkylene," as described above).

The term "thio" refers to -SH or, if between two other groups, -S-. The term "Ci to Cio alkylene thio" refers to a one to ten carbon alkylene chain with a thio at any point along the chain. The term "Ci to C ₁₀ substituted alkylene thio" refers to a Ci to Cio alkylene thio group that is substituted at one or more of the alkylene positions (in the same way as "substituted alkylene," as described above).

The term "sulfonyl" refers to -S(O) ₂ -. The term "Ci to Ci ₀ alkylene sulfonyl" refers to a one to ten carbon alkylene chain with a sulfonyl at any point along the chain. The term "Ci to Cio substituted alkylene sulfonyl" refers to a Ci to Cio alkylene sulfonyl group that is substituted at one or more of the alkylene positions (in the same way as "substituted alkylene," as described above).

The term "sulfmyl" refers to -S(O)-. The term "Ci to Ci ₀ alkylene sulfmyl" refers to a one to ten carbon alkylene chain with a sulfmyl at any point along the

chain. The term "Ci to C ₁₀ substituted alkylene sulfinyl" refers to a Ci to C ₁₀ alkylene sulfmyl group that is substituted at one or more of the alkylene positions (in the same way as "substituted alkylene," as described above).

The term "oxy" refers to -O-. The terms "Ci to Ci ₀ alkylene oxy," "Ci to Ci ₀ alkylene dioxy" and "Ci to C ₁₀ alkylene trioxy" refer to a one to ten carbon alkylene chain with, respectively, one, two or three -O- at any point along the chain, provided that no two oxygen atoms are consecutive, and provided that any two oxygen atoms are separated by at least two carbons. The terms "Ci to C ₁₀ substituted alkylene oxy," "Ci to Cio substituted alkylene dioxy" and "Ci to C ₁₀ substituted alkylene trioxy" refer, respectfully to "Ci to C ₁₀ alkylene oxy," "Ci to C ₁₀ alkylene dioxy" and "Ci to Cio alkylene trioxy" that are substituted at one or more of the alkylene positions (in the same way as "substituted alkylene," as described above).

The term "thiocarbonyl" refers to -C(S)H or, if between two other groups, - C(S)-. The term "thioester" refers to -C(O)SH or, if between two other groups, - C(O)S-.

The term "carboxy-protecting group" as used herein refers to one of the ester derivatives of the carboxylic acid group commonly employed to block or protect the carboxylic acid group while reactions are carried out on other functional groups on the compound. Examples of these groups are found in E. Haslam, "Protective Groups in Organic Chemistry," J. GW. McOmie, Ed., Plenum Press, New York, NY, 1973, Chapter 5, and T. W. Greene and PGM. Wuts, "Protective Groups in Organic Synthesis," 2nd ed., John Wiley and Sons, New York, NY, 1991, Chapter 5, each of which is incorporated herein by reference. A related term is "protected carboxy," which refers to a carboxy group substituted with one of the above carboxy-protecting groups.

The term "hydroxy-protecting group" refers to readily cleavable groups bonded to hydroxyl groups. The species of hydroxy-protecting groups is not critical so long as the derivatized hydroxyl group is stable to the conditions of subsequent reaction(s) and can be removed at the appropriate point without disrupting the remainder of the molecule. Examples of hydroxy-protecting groups are described by CB. Reese and E. Haslam, "Protective Groups in Organic Chemistry," J. GW McOmie, Ed., Plenum Press, New York, NY, 1973, Chapters 3 and 4, respectively, and T. W Greene and PGM. Wuts, "Protective Groups in Organic Synthesis," 2nd ed., John Wiley and Sons, New York, NY, 1991, Chapters 2 and 3. Related terms are "protected hydroxy,"

and "protected hydro xymethyl" which refer to a hydroxy or hydroxymethyl substituted with one of the above hydroxy-protecting groups.

The term "Ci to C ₁₀ alkylthio" refers to sulfide groups such as methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, t-butylthio and like groups. The term "Ci to C ₁₀ alkylsulfoxide" indicates sulfoxide groups such as methylsulfoxide, ethylsulfoxide, n-propylsulfoxide, isopropylsulfoxide, n- butylsulfoxide, sec-butylsulfoxide and the like. The term "Ci to C ₁₀ alkylsulfonyl" encompasses groups such as methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, isopropylsulfonyl, n-butylsulfonyl, t-butylsulfonyl and the like, it should also be understood that the above thio, sulfoxide or sulfonyl groups can be at any point on the alkyl chain (e.g., 2-methylmercaptoethyl).

The terms "Ci to Ci ₀ substituted alkylthio," "Ci to Ci ₀ substituted alkylsulfoxide," and "Ci to C ₁₀ substituted alkylsulfonyl," denote the Ci to C ₁₀ alkyl portion of these groups may be substituted as described above in relation to "substituted alkyl."

The terms "phenylthio," "phenylsulfoxide," and "phenylsulfonyl" specify a thiol, a sulfoxide, or sulfone, respectively, containing a phenyl group. The terms "substituted phenylthio," "substituted phenylsulfoxide," and "substituted phenylsulfonyl" means that the phenyl of these groups can be substituted as described above in relation to "substituted phenyl."

The term "Ci to Ci ₂ alkylaminocarbonyl" means a Ci to Ci ₂ alkyl attached to a nitrogen of the aminocarbonyl group. Examples of Ci to Ci ₂ alkylaminocarbonyl include methylaminocarbonyl, ethylaminocarbonyl, propylaminocarbonyl and butylaminocarbonyl. The term "Ci to Ci ₂ substituted alkylaminocarbonyl" denotes a substituted alkyl bonded to a nitrogen of the aminocarbonyl group, which alkyl may be substituted as described above in relation to Ci to Ci ₂ substituted alkyl.

The term "Ci to Ci ₂ alkoxycarbonyl" means a "Ci to Ci ₂ alkoxy" group attached to a carbonyl group. The term "Ci to Ci ₂ substituted alkoxycarbonyl" denotes a substituted alkoxy bonded to the carbonyl group, which alkoxy may be substituted as described above in relation to "Ci to Ci ₂ substituted alkyl."

The term "phenylaminocarbonyl" means a phenyl attached to a nitrogen of the aminocarbonyl group. The term "substituted phenylaminocarbonyl" denotes a substituted phenyl bonded to a nitrogen of the aminocarbonyl group, which phenyl may be substituted as described above in relation to substituted phenyl.

The term "Ci to C ₁₂ alkylaminothiocarbonyl" means a Ci to C ₁₂ alkyl attached to an aminothiocarbonyl group, wherein the alkyl has the same meaning as defined above.

The term "Ci to C ₁₂ substituted alkylaminothiocarbonyl" denotes a substituted alkyl bonded to an aminothiocarbonyl group, wherein the alkyl may be substituted as described above in relation to Ci to C12 substituted alkyl.

The term "phenylaminothiocarbonyl" means a phenyl attached to an aminothiocarbonyl group, wherein the phenyl has the same meaning as defined above.

The term "substituted phenylaminothiocarbonyl" denotes a substituted phenyl bonded to an aminothiocarbonyl group, wherein phenyl may be substituted as described above in relation to substituted phenyl.

The term "phenylene" means a phenyl group where the phenyl radical is bonded at two positions connecting together two separate additional groups.

The term "substituted phenylene" means a phenyl group where the phenyl radical is bonded at two positions connecting together two separate additional groups, wherein the phenyl is substituted as described above in relation to "substituted phenyl."

The term "substituted Ci to Ci ₂ alkylene" means a Ci to Ci ₂ alkyl group where the alkyl radical is bonded at two positions connecting together two separate additional groups and further bearing an additional substituent.

The terms "cyclic C ₂ to C ₇ alkylene," "substituted cyclic C ₂ to C ₇ alkylene," "cyclic C ₂ to C ₇ heteroalkylene," and "substituted cyclic C ₂ to C ₇ heteroalkylene," defines such a cyclic group bonded ("fused") to the phenyl radical resulting in a bicyclic ring system. The cyclic group may be saturated or contain one or two double bonds. Furthermore, the cyclic group may have one or two methylene or methine groups replaced by one or two oxygen, nitrogen or sulfur atoms that are the cyclic C ₂ to C ₇ heteroalkylene.

The cyclic alkylene or heteroalkylene group may be substituted once or twice by the same or different substituents which, if appropriate, can be connected to another part of the compound (e.g., alkylene) selected from the group consisting of the following moieties: hydroxy, protected hydroxy, carboxy, protected carboxy, oxo, protected oxo, Ci to C ₄ acyloxy, formyl, Ci to Ci ₂ acyl, Ci to Ci ₂ alkyl, Ci to C ₇ alkoxy, Ci to C ₁₀ alkylthio, Ci to C ₁₀ alkylsulfoxide, Ci to C ₁₀ alkylsulfonyl, halo,

amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, hydroxymethyl or a protected hydroxymethyl.

The cyclic alkylene or heteroalkylene group fused onto the benzene radical can contain two to ten ring members, but it preferably contains three to six members. Examples of such saturated cyclic groups are when the resultant bicyclic ring system is 2,3-dihydro-indanyl and a tetralin ring. When the cyclic groups are unsaturated, examples occur when the resultant bicyclic ring system is a naphthyl ring or indolyl. Examples of fused cyclic groups which each contain one nitrogen atom and one or more double bond, preferably one or two double bonds, are when the benzene radical is fused to a pyridino, pyrano, pyrrolo, pyridinyl, dihydropyrrolo, or dihydropyridinyl ring. Examples of fused cyclic groups which each contain one oxygen atom and one or two double bonds are when the benzene radical ring is fused to a furo, pyrano, dihydrofurano, or dihydropyrano ring. Examples of fused cyclic groups which each have one sulfur atom and contain one or two double bonds are when the benzene radical is fused to a thieno, thiopyrano, dihydrothieno or dihydrothiopyrano ring. Examples of cyclic groups that contain two heteroatoms selected from sulfur and nitrogen and one or two double bonds are when the benzene radical ring is fused to a thiazolo, isothiazolo, dihydrothiazolo or dihydroisothiazolo ring. Examples of cyclic groups which contain two heteroatoms selected from oxygen and nitrogen and one or two double bonds are when the benzene ring is fused to an oxazolo, isoxazolo, dihydrooxazolo or dihydroisoxazolo ring. Examples of cyclic groups which contain two nitrogen heteroatoms and one or two double bonds occur when the benzene ring is fused to a pyrazolo, imidazolo, dihydropyrazolo or dihydroimidazolo ring or pyrazinyl. The term "carbamoyl" means an -NC(O)- group where the radical is bonded at two positions connecting two separate additional groups.

The term "organic or inorganic cation" refers to counter-ions for the carboxylate anion of a carboxylate salt. The counter-ions are chosen from the alkali and alkaline earth metals, (such as lithium, sodium, potassium, barium, aluminum and calcium); ammonium and mono-, di- and tri-alkyl amines such as trimethylamine, cyclohexylamine; and the organic cations, such as dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium, phenylethylbenzylammonium, dibenzylethylenediammonium, and like cations. See, for example, "Pharmaceutical Salts," Berge et al, J. Pharm. ScL, 66:1-19 (1977),

which is incorporated herein by reference. Other cations encompassed by the above term include the protonated form of procaine, quinine and N-methylglucosamine, and the protonated forms of basic amino acids such as glycine, ornithine, histidine, phenylglycine, lysine and arginine. Furthermore, any zwitterionic form of the instant compounds formed by a carboxylic acid and an amino group is referred to by this term. For example, a cation for a carboxylate anion will exist when a position is substituted with a (quaternary ammonium)methyl group. A preferred cation for the carboxylate anion is the sodium cation.

The compounds of the invention can also exist as solvates and hydrates. Thus, these compounds may crystallize with, for example, waters of hydration, or one, a number of, or any fraction thereof of molecules of the mother liquor solvent. The solvates and hydrates of such compounds are included within the scope of this invention.

One or more compounds of the invention can be in the biologically active ester form, such as the non-toxic, metabolically-labile ester- form. Such ester forms induce increased blood levels and prolong the efficacy of the corresponding non-esterified forms of the compounds. Ester groups which can be used include the lower alkoxymethyl groups, for example, methoxymethyl, ethoxymethyl, isopropoxymethyl and the like; the -(Ci to C12) alkoxyethyl groups, for example methoxyethyl, ethoxyethyl, propoxyethyl, isopropoxyethyl and the like; the 2-oxo-l,3-diooxlen-4- ylmethyl groups, such as 5-methyl-2-oxo-l,3-dioxolen-4-ylmethyl, 5-phenyl-2-oxo- l,3-dioxolen-4-ylmethyl and the like; the Ci to C ₁₀ alkylthiomethyl groups, for example methylthiomethyl, ethylthiomethyl, iso-propylthiomethyl and the like; the acyloxymethyl groups, for example pivaloyloxymethyl, pivaloyloxyethyl, - acetoxymethyl and the like; the ethoxycarbonyl-1 -methyl group; the -acetoxyethyl; the 1-(Ci to C ₁₂ alkyloxycarbonyloxy)ethyl groups such as the 1- (ethoxycarbonyloxy)ethyl group; and the 1-(Ci to Ci ₂ alkylaminocarbonyloxy)ethyl groups such as the l-(methylaminocarbonyloxy)ethyl group.

The term "amino acid" includes any one of the twenty naturally-occurring amino acids or the D-form of any one of the naturally-occurring amino acids. In addition, the term "amino acid" also includes other non-naturally occurring amino acids besides the D-amino acids, which are functional equivalents of the naturally-occurring amino acids. Such non-naturally-occurring amino acids include, for example, norleucine ("NIe"), norvaline ("Nva"), L- or D- naphthalanine, ornithine ("Orn"), homoarginine

(homoArg) and others well known in the peptide art, such as those described in M. Bodanzsky, "Principles of Peptide Synthesis," 1st and 2nd revised ed., Springer- Verlag, New York, NY, 1984 and 1993, and Stewart and Young, "Solid Phase Peptide Synthesis," 2nd ed., Pierce Chemical Co., Rockford, IL, 1984, both of which are incorporated herein by reference. Amino acids and amino acid analogs can be purchased commercially (Sigma Chemical Co.; Advanced Chemtech) or synthesized using methods known in the art.

It should be understood that any position of the claimed invention has up to three serial "substitutions." For example, a "substituted alkyl" that is substituted with a "substituted phenyl" that is, in turn, substituted with a "substituted alkyl" can, in turn, be substituted by one more group and no longer further substituted. However, it should also be understood that the invention contemplates, if appropriate, more than three parallel substitutions. For example, if appropriate, more than three hydrogens on an alkyl moiety may be substituted with any one or more of a variety of groups, including halo and hydroxy.

Example 1: Synthesis of Phosphoric acid mono-{2-methoxy-5-[7-methoxy-4-oxo- chroman-(3E)-ylidenemethyl] -phenyl} ester disodium salt.

A. 3-Chloro-l-(2,4-dihydroxy-phenyl)-propan-l-one

Resorcinol (10.0 g, 90.9 mmol, Spectrum chemicals,) and 3-chloropropionic acid (10.0 g, 92.0 mmol, Aldrich,) were charged into a 250 ml round-bottomed flask with magnetic stirring bar. Trifluoromethanesulfonic acid (50.0 g, Aldrich,) was added to the flask in one portion. The solution was heated at 85°C for 30 min, cooled to room temperature, and poured into chloroform (200 ml). The solution was slowly poured into H ₂ O (200 ml) and the layers were separated. The aqueous layer was further extracted with chloroform (2 x 100 ml). The combined organic layers were dried over Magnesium sulfate and filtered. Concentration in vacuum yielded 3-

Chloro-l-(2,4-dihydroxy-phenyl)-propan-l-one as orange oil, which was used crude in the next step. ¹ H NMR (CDCl ₃ , 500 MHz): δ 12.48 (s, IH), 7.61 (d, J= 8.7 Hz, IH), 6.40 (m, 3H), 3.90 (t, J= 6.8 Hz, 2H), 3.39 (t, J= 6.8 Hz, 2H).

B. 7-hydroxychroman-4-one

3-Chloro-l-(2,4-dihydroxy-phenyl)-propan-l-one (crude, see Example 1, part A) and a magnetic stirring bar were placed in a 1 L round-bottomed flask. A solution 2 N Sodium hydroxide (500 ml) was cooled at 0 ⁰ C and poured to the flask in one portion. The solution was stirred at room temperature for 2 h and cooled to 5°C. The pH of the solution was adjusted to ca. 2.0 by slowly adding 6 M H ₂ SO ₄ (ca. 50 ml). The mixture was extracted with ethyl acetate (4 x 100 ml). The combined organics were washed with brine (100 ml) and dried over Magnesium sulfate. Concentration in vacuum yielded a yellowish solid. This solid was triturated with hexanes (200 ml) and recrystallized from water (ca. 200 ml) to give an off-white solid 7- hydroxychroman-4-one (7.0 g, 48%). m.p. 145.2°C (lit. m.p. 145°C). ¹ H NMR (CDCl ₃ , 500 MHz): δ 7.83 (d, J= 8.8 Hz, IH), 6.52 (dd, Ji = 8.8 Hz, J ₂ = 2.3 Hz, IH), 6.39 (d, J= 2.3 Hz, IH), 5.98 (s, IH), 4.51 (t, J= 6.0 Hz, 2H), 2.76 (t, J= 6.4 Hz, 2H).

C. 7-methoxychroman-4-one

7-hydroxychroman-4-one (1.0 g, 6.1 mmol) was dissolved in 20 ml acetone in a 250 ml round-bottomed flask. Potassium carbonate (1.68 g, 12.2 mmol, Aldrich) and iodomethane (2.4 g, 17.1 mmol, Aldrich) were added successively. The solution was refluxed for 4 h, cooled to room temperature, and diluted with dichloromethane. The solid was removed by filtration. The filtrate was concentrated in vacuum to yield

yellowish clear oil. The oil was dissolved in dichloromethane (100 ml) and extracted with H ₂ O (2 x 50 ml). The organic was dried over magnesium sulfate and concentrated in vacuum to give 7-methoxychroman-4-one as a white waxy solid (1.05 g, 97%), which was of great purity indicated by NMR. ¹ U NMR (CDCl ₃ , 500 MHz): δ 7.84 (d, J= 8.8 Hz, IH), 6.58 (dd, Ji = 8.8 Hz, J ₂ = 2.4 Hz, IH), 6.40 (d, J = 2.4 Hz, IH), 4.52 (t, J= 6.5 Hz, 2H), 3.84 (s, 3H), 2.76 (t, J= 6.5 Hz, 2H).

D . 3 - [ 1 -(3 -Hydroxy-4-methoxy-phenyl)-meth-(E)-ylidene] -7-methoxy-chroman-4- one

7-methoxychroman-4-one (534 mg, 3 mmol) and isovanillin (456 mg, 3 mmol, Aldrich) were dissolved in methanol (20 ml) in a 100 ml round-bottomed flask with a magnetic stirring bar. To this solution, 10 drops of concentrated hydrochloric acid (37%) was added. The solution was refluxed for 19 h and cooled to room temperature. The solution was concentrated to dryness in vacuum. The residue was dissolved in small amount of ethyl acetate and passed through a silica gel (ca. 50 ml) plug with ethyl acetate. Concentration in vacuum yielded a yellow solid, which was recrystallized from methanol to give 3-[l-(3-Hydroxy-4-methoxy-phenyl)-meth-(E)- ylidene]-7-methoxy-chroman-4-one. (700 mg, 75%). ¹ H NMR (CDCl ₃ , 500 MHz): δ 7.95 (d, J= 8.8 Hz, IH), 7.76 (t, J= 1.6 Hz, IH), 6.90 (m, 2H), 6.86 (dd, Ji = 8.4 Hz, J ₂ = 2.0 Hz, IH), 6.62 (dd, Ji = 8.8 Hz, J ₂ = 2.4 Hz, IH), 6.40 (d, J= 2.0 Hz, IH), 5.69 (s, IH), 5.36 (d, J= 1.8 Hz, 2H), 3.95 (s, 3H), 3.85 (s, 3H).

E. Phosphoric acid mono-{2-methoxy-5-[7-methoxy-4-oxo-chroman-(3E)- ylidenemethyl] -phenyl} ester disodium salt

3-[l-(3-Hydroxy-4-methoxy-phenyl)-meth-(E)-ylidene]-7-methox y-chroman- 4-one (3.12 g, 10 mmol) was dissolved in dry pyridine (20 ml) in a 100 ml round- bottomed flask. The solution was cooled to 0 ⁰ C in an ice-water bath. Phosphorous oxy chloride (1.84 g, 12 mmol, Alfa Aesar) was slowly added to the flask via a syringe. After stirred at 0 ⁰ C for 1 hr, the reaction was quenched by adding 5 ml ice- water to the flask. The resulting solution was stirred for another 1 hr at 0 ⁰ C. The solvent was removed in vacuum. The resulting solid was suspended in 3M HCl (40 ml). An orange solid was collected by filtration and air-dried. Assuming the yield of this step was quantitative, 3.1 g above crude product was suspended in H ₂ O (100 ml). Sodium bicarbonate (1.34 g, 16 mmol) was added to the solution with vigorous stirring. The solvent was removed in vacuum. The resulting yellow solid 983-107-14 was suspended in methanol (20 ml) and collected by filtration. Yield 2.1 g (72%). ¹ H NMR (D ₂ O, 500 MHz): δ 7.81 (d, J= 8.9 Hz, IH), 7.71 (s, IH), 7.48 (s, IH), 7.09 (m, 2H), 6.68 (dd, Ji = 9.0 Hz, J ₂ = 2.2 Hz, IH), 6.52 (d, J= 2.5 Hz, IH), 5.44 (s, 2H), 3.88 (s, 3H), 3.84 (s, 3H). ESI Mass for Ci ₈ Hi ₅ Na ₂ O ₈ P: calculated 436.0, found 437.2 [M+H] ⁺ .

Example 2: 3-[l-(3-Hydroxy-4-methoxy-phenyl)-meth-(E)-ylidene]-6-methyl - chroman-4-one

A 20 X 200 mm test tube was loaded with 6-methylchroman-4-one, 4.7 g, then 2-methoxyethanol, 6 ml, then 3-hydroxy-4-methoxybenzaldehyde. The mixture was treated with concentrated hydrochloric acid, 0.1 ml, and warmed to 80° C. After 4 days the solid orange mass was washed into a 500 ml Erlenmeyer flask and suspended in 95% ethanol, 200 ml. The mix was warmed to reflux for 2 hours, dissolving all the solids. The hot solution was treated slowly with water, and at 60 ml a precipitate

began to form. The suspension was vigorously stirred and allowed to cool to room temperature for two hours. The yellow solids were collected by filtration, washed with 2 X 25 ml of 1 : 1 95% ethano I/water. The solids were air dried to provide 3-[l- (3-Hydroxy-4-methoxy-phenyl)-meth-(E)-ylidene]-6-methyl-chro man-4-one, 6.73 g, mp 160.3-161.3 ⁰ C.

¹ H NMR (CDCl ₃ , 500 MHz): 7.796(1H, d, J=2.1), 7.77(1H t, J=I.8), 7.28,(lH,dd, Jl=I.9, J2=8.4) , 6.92-6.90(2H ,m), 6.87-6.85(2H, m), 5.34(2H,d, J=I.9) 3.94(3H,s) , 2.33(3H, s)

Example 3: 3-[l-(3-Hydroxy-4-methoxy-phenyl)-meth-(E)-ylidene]-6-chloro - chroman-4-one

Method A:

A 20 X 200 mm test tube was loaded with 6-chlorochroman-4-one, 1.00 g, and 3-hydroxy-4-methoxybenzaldehyde, 0.9 g, and a magnetic stirring bar. Then 4 ml of butanol was added, followed by 4 drops of concentrated hydrochloric acid. The mixture was warmed to 100 ⁰ C for 24 hours, the tube full of solids was cooled to 22 ⁰ C. After standing three days at RT, 12 ml of ethano 1 was added, and the tube was warmed to 85 ⁰ C to dissolve the solids. The mix was then treated slowly with 4 ml of water, and cooled to 3O ⁰ C with stirring. The resulting solids were collected by filtration, washed with 4 ml of 1 :1 ethanol/water, and air dried to give 3-[l-(3- Hydroxy-4-methoxy-phenyl)-meth-(E)-ylidene]-6-chloro-chroman -4-one, 1.126 g, mp 155-156 C, elemental analysis C17H13C11O4 calc C 64.46%, H 4.14%, Found C 64.33%, H 4.32%.

Method B:

A 20 X 200 mm test tube was loaded with 6-chlorochroman-4-one, 3.64 g, and 3-hydroxy-4-methoxybenzaldehyde, 3.04 g, and a magnetic stirring bar. Then 4 ml of methoxyethanol, followed by 0.1 ml of concentrated hydrochloric acid. The tube was then placed in a 100 ⁰ C heat block, and heated with stirring for 18 hours. The mix was then cooled, and the resulting solids transferred to a 500 ml Erlenmeyer flask. The

solids were suspended wit 150 ml of ethanol, and warmed to reflux. Once all the solids had dissolved, 50 ml of water was slowly added, at which point solids began to separate from the hot solution. The mixture was stirred and allowed to cool. After 2 hours stirring at room temperature, the solids were collected by filtration, washed with 1 :1 ethanano I/water, 20 ml, and air dried to give of 3-[l-(3-Hydroxy-4-methoxy- phenyl)-meth-(E)-ylidene]-6-chloro-chroman-4-one, 4.6 grams, a red/orange solid, mp 156-157 ⁰ C.

Example 4: 3-[l-(3-Hydroxy-4-methoxy-phenyl)-methyl]-6-chloro-chroman-4 - one

Method A:

3-[l-(3-Hydroxy-4-methoxy-phenyl)-meth-(E)-ylidene]-6-chloro -chroman-4- one, 0.312 g, and [l,r-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex, 0.022 g were placed in a 20 ml screw cap vial with a stirring bar. The vial was then closed with a silicon/Teflon septum screw cap, and flushed with nitrogen gas for 10 minutes. The solids were then treated with 3 ml of dry tetrahydrofuran, and stirred until the solids had dissolved. The solution was then cooled by placing the flask in an ice water bath, and then 9-Borabicyclo[3.3.1 Jnonane, 0.5 M in tetrahydrofuran, 5 ml, was added over 5 minutes. The mixture was stirred for one hour at O ⁰ C, and then 30 minutes at 2O ⁰ C. The mixture was then treated with 0.15 ml of ethanolamine and stirred over night. The cloudy solution was filtered through celite, abd the filtrate was then evaporated in vacuum. The residue was dissolved in 5 ml of dichloromethane, and flash chromatographed on silica, 1 X 4 inch column, with 100 ml each of dicloromethane, 2% acetone in dichloromethane, and 4 % acetone in dichloromethane. The major peak elutes with the dichloromethane and early 2% acetone in DCM. The factions containing pure major spot were combined and evaporated in vacuum. The residue was sisolved hot with 10 ml of 95% ethanol, and the solution cooled to -20 C for one hour. The resulting solids were collected by filtration, and washed with 2 ml of 1 ethanol and air dried to give 3-[l-(3-Hydroxy-4-

methoxy-phenyl)-methyl]-6-chloro-chroman-4-one as white solids, 0.202 g, mp 126.4-126.8 C

¹ H NMR (CDCl ₃ , 500 MHz): 7.87( IH, d, J=2.6), 7.74(1H, dd, Jl=2.7, J2=8.8), 6.93(1H, d, J=8.8), 6.80-6.78(2H, m), 6.69(1H, dd, Jl=2.0, J2=8.2), 4.37(1H, dd, Jl=4.4, J2=11.6), 4.17(1H, dd, J=8.3, J2=11.7), 3.88(3H, s), 3.17(1H, dd, Jl=4.6, J2=14.1), 2.90-2.85 (IH, m), 2.61(1H, dd, Jl=10.4, J2=14.1)

Method B:

3-[l-(3-Hydroxy-4-methoxy-phenyl)-meth-(E)-ylidene]-6-chl oro-chroman-4- one, 0.624 g, was placed in a 40 ml screw cap vial with a stirring bar. Then [1,1 '-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex, 0.043 g was added. The vial was closed with a silicon/Teflon septum screw cap, and flushed with nitrogen gas for 10 minutes. The solids were then treated with 5 ml of dry tetrahydrofuran, and stirred until the solids had dissolved. The solution was then cooled by placing the flask in an ice water bath, and then 9-

Borabicyclo[3.3.1]nonane, 0.5 M in tetrahydrofuran, 10 ml, was added over 5 minutes. The mixture was stirred for one hour at O ⁰ C, and then warmed to 2O ⁰ C over one hour as the water bath warmed to RT. The mixture was then treated with 0.3 ml of ethanolamine and stirred 5 minutes. The solvents were then evaporated in vacuum, and the residue diluted with 20 ml of 95% ethanol, and the solution cooled to O ⁰ C. The resulting solids were collected by filtration, and washed with 10 ml of 1 : 1 ethano I/water and air dried to give 3-[l-(3-Hydroxy-4-methoxy-phenyl)-methyl]-6- chloro-chroman-4-one as pink solids, 0.402 g, mp 126.4-126.8 C

Example 5: 3-[l-(3-Hydroxy-4-methoxy-phenyl)-methyl]-7-methoxy-chroman- 4- one

A sample of 3-[l-(3-Hydroxy-4-methoxy-phenyl)-meth-(E)-ylidene]-7- methoxy-chroman-4-one (see Example Id), 0.092 g, was treated with 1 ml of ethanol, and then 1 ml of dimethylacetamide to provide a clear solution. The solution was

treated with 9.7 mg of 5 % palladium on carbon, followed by 2.5 ml of 98% formic acid. The mix was stirred in a screw cap 20 ml vial on a 50° C heating stir plate. After 15 more minutes the yellow solution was treated with an additional 46 mg of 5% palladium on carbon. After stirring an additional 40 minutes, TLC showed SM consumed, and two new spots with 5% acetone in dichloromethane on silica. The catalyst was filtered off, and the filtrate was diluted with 40 ml of water. The resulting cloudy mix was allowed to stand overnight. The next day the aqueous layer was decanted from the gummy residue. The residue was dissolved in 6 ml of dichloromethane, and dried with granular anhydrous magnesium sulfate. The dry solution was loaded on to a IX 2 inch silica flash column, and eluted with 50 ml each of 0%, 2 % and 4% acetone in dichloromethane. A faint yellow band eluted with 2% acetone in dichloromethane. Pure fractions were combined and evaporated to give 3- [l-(3-Hydroxy-4-methoxy-phenyl)-methyl]-7-methoxy-chroman-4- one, 47 mg, as a faintly yellow solid.

¹ H NMR (CDCl ₃ , 500 MHz): 7.86(1H, d, J=8.85), 6.81-6.78(2H, m), 6.71(1H, dd, Jl=2.1, J2=8.1), 6.59(1H, dd, Jl=2.3, J2=8.8), 6.4O(1H, d, 2.4), 5.59(1H, s), 4.34(1H, dd, Jl=4.3, J2=11.4), 4.16(1H, dd, Jl=7.9, J2=11.4), 3.88(3H, s), 3.83(3H, s), 3.18(1H, dd, Jl=4.5, J2=14.1), 2.83-2.78(1H, m), 2.59(1H, dd, Jl=10.6, J2=14.1)

Example 6: 3-[l-(3-Hydroxy-4-methoxy-phenyl)-meth-(E)-ylidene]-6-fluoro - chroman-4-one

In a 20 ml vial with stirring bar were combined 6-fluoro-4-chromanone, 1.37g, and 3-hydroxy-4-methoxybenzaldehyde, 1.38 g, and 2-methoxyethanol, 4 ml. The mix was stirred and treated with 60 μl concentrated hydrochloric acid, and placed on a hot plate at 50° C. After 4 days, TLC showed unreacted starting materials. The hot plate was warmed to 70° C. After 24 hours at 70° C, TLC showed unreacted starting materials. The hot plate was warmed to 85° C. After 2 days at 85° C, the vial was cooled to room temperature with stirring. The resulting solids were collected by filtration, and washed 3 X 2 ml 95% ethanol, and air dried to give 3-[l-(3-Hydroxy-4-

methoxy-phenyl)-meth-(E)-ylidene]-6-fluoro-chroman-4-one, yellow solids, 2.115 g, mp 183.2-184.4 ⁰ C. HPLC analysis on an Agilent Zorbax SB-C18, 4.6 X 150 mm 5 micron column with a 15 minute 0 to 100% gradient of acetonitrile in water, 0.05% trifluoroacetic acid as buffer, showed a peak at 10.86 minutes, 99.5 % at 254 nm, and showing UV maxima at 265 and 380 nm.

Example 7

983-57-09 983-70-08

983-57-09 (178 mg, 1 mmol) and p-anisaldehyde (136 mg, 1 mmol, Aldrich,

Lot# 13022 JC) were dissolved in Methanol (5 ml) in a 50 ml round-bottomed flask with a magnetic stirring bar. To this solution, 4 drops of concentrated HCl (37%) was added. The solution was refluxed for 20 hours and cooled to room temperature. A pink crystal was formed after cooling to room temperature and was collected by filtration to give 983-70-08 (210 mg, 71%). ¹ H NMR (CDCl ₃ , 500 MHz): δ 7.96 (d, J = 8.8 Hz, IH), 7.81 (s, IH), 7.27 (d, J = 9.0 Hz, IH), 6.96 (d, J = 8.8 Hz, IH), 6.63 (dd, Ji = 8.8 Hz, J ₂ = 2.4 Hz, IH), 6.40 (d, J= 2.4 Hz, IH), 5.36 (d, J= 1.8 Hz, 2H), 3.86 (s, 3H), 3.85 (s, 3H).

Example 8

983-57-09 983-71 -08

983-57-09 (178 mg, 1 mmol) and 4-[N, N-bis(2- hydroxyethyl)amino]benzaldehyde (209 mg, 1 mmol, Aldrich, Lot# 0633 ITB) were dissolved in methanol (5 ml) in a 50 ml round-bottomed flask with a magnetic stirring bar. To this solution, 200 μl of concentrated HCl (37%) was added. The solution was refluxed for 22 h and cooled to room temperature. The solution was concentrated to dryness in vacuum. The residue was purified by flash column chromatography (10%

Methanol/CH ₂ C1 ₂ ) on silica gel (ca. 50 ml) to yield an orange solid 983-71-08 (220 mg, 60%). ¹ H NMR (CDCl ₃ , 500 MHz): δ 7.95 (d, J= 8.8 Hz, IH), 7.77 (s, IH), 7.22 (d, J= 8.8 Hz, IH), 6.77 (br, IH), 6.75 (br, IH), 6.62 (dd, Ji = 8.8 Hz, J ₂ = 2.3 Hz, IH), 6.40 (d, J= 2.3 Hz, IH), 5.40 (d, J= 2.0 Hz, 2H), 3.93 (t, J= 4.6 Hz, 4H), 3.84 (s, 3H), 3.68 (t, J= 4.7 Hz, 4H).

Example 9

983-57-09 (178 mg, 1 mmol) and 3,4-dihydroxybenzaldehyde (138 mg, 1 mmol, Aldrich, Lot# 08619BC) were dissolved in methanol (5 ml) in a 100 ml round- bottomed flask with a magnetic stirring bar. To this solution, 4 drops of concentrated HCl (37%) was added. The solution was refluxed for 12 hours and cooled to room temperature. The solution was concentrated to dryness in vacuum. The residue was re-crystallized from Methanol to yield an orange solid 983-63-10 (192 mg, 64%). ¹ H NMR (DMSO-d ₆ , 500 MHz): δ 9.63 (s, IH), 9.25 (s, IH), 7.79 (d, J= 8.8 Hz, IH), 7.55 (s, IH), 6.85 (m, 2H), 6.78 (m, IH), 6.68 (dd, J ₁ = 8.5 Hz ₅ J ₂ = 1.6 Hz, IH), 6.56 (m, IH), 5.40 (s, 2H), 3.84 (s, 3H).

Example 10

983-52-16 983-62-08

983-52-16 (164 mg, 1 mmol) and isovanillin (152 mg, 1 mmol, Aldrich, Lot# 11530 JA) were dissolved in methanol (5 ml) in a 100 ml round-bottomed flask with a magnetic stirring bar. To this solution, 4 drops of concentrated HCl (37%) was added. The solution was refluxed for 16 hours and cooled to room temperature. The solution was concentrated to dryness in vacuum. The residue was dissolved in small amount of Ethyl acetate/Methanol and passed through a silica gel (ca. 40 ml) plug with Ethyl acetate as eluant. Concentration in vacuum yielded yellow solid 983-62- 08 (240 mg, 80%). ¹ H NMR (DMSO-d ₆ , 500 MHz): δ 10.63 (s, IH), 9.29 (s, IH),

7.73 (d, J= 8.8 Hz, IH), 7.55 (s, IH), 7.02 (d, J= 8.0 Hz, IH), 6.88 (m, 2H), 6.54 (dd, Ji = 8.8 Hz, J ₂ = 2.4 Hz, IH), 6.32 (d, J= 2.4 Hz, IH), 5.46 (d, J= 2.4 Hz, 2H), 3.83 (s, 3H).

Example 11

983-57-09 983-59-10

983-57-09 (178 mg, 1 mmol) and vanillin (152 mg, 1 mmol, Aldrich, Lot# 05702LB) were dissolved in methanol (5 ml) in a 100 ml round-bottomed flask with a magnetic stirring bar. To this solution, 3 drops of concentrated HCl (37%) was added. The solution was refluxed for 17 hours and cooled to room temperature. The solution was concentrated to dryness in vacuum. The residue was dissolved in small amount of ethyl acetate and passed through a silica gel (ca. 50 ml) plug with ethyl acetate as eluant. Concentration in vacuum yielded yellow oil 983-59-10 (280 mg, 90%). ¹ H NMR (CDCl ₃ , 500 MHz): δ 7.96 (d, J= 8.8 Hz, IH), 7.78 (s, IH), 6.97 (d, J= 8.0 Hz, IH), 6.83 (m, 2H), 6.62 (dd, Ji = 8.0 Hz, J ₂ = 2.4 Hz, IH), 6.40 (d, J= 2.4 Hz, IH), 5.91 (s, IH), 5.36 (d, J= 2.0 Hz, 2H), 3.93 (s, 3H), 3.84 (s, 3H).

Example 12

Samples of 4-chromanone, 50 mg, and 3-hydroxy-4-methoxybenzaldehyde, 51 mg, were combine in 2 ml of methanol, and 1 drop concentrated hydrochloric acid was added. The mixture was refluxed 20 hours. The mix was then cooled to room temperature, and 1.5 ml of water was added, and the mix stirred overnight. The resulting solids were collected by filtration, giving 3-[l-(3-Hydroxy-4-methoxy- phenyl)-meth-(E)-ylidene]-chroman-4-one. TLC of the product gave a visible yellow spot, RF 0.3 with dichloromethane on silica plates.

Example 13

3 - [ 1 -(4-Ethoxy-3 -hydroxy-phenyl)-meth-(E)-ylidene] -7-methoxy-chroman-4- one was produced following the method of Example 3, method A: to provide yellow solids, MP 165-166.2° C.

Example 14

7-Ethoxy-3 - [ 1 -(3 -hydroxy-4-methoxy-phenyl)-meth-(E)-ylidene] -chroman-4- one was produced following the method of Example 3, method A: to provide yellow solids, MP 131-132 ⁰ C.

Example 15

This example shows that compounds of the invention can be micronized to give an injectable suspension.

IMS2186 was micronized using a MC One fluid jet mill reducing the particle size from 850 microns to less than 10 microns. The powder particles are fed into the flat cylindrical milling chamber tangentially through a venturi system by pressurized air or nitrogen. The particles are accelerated in a spiral movement inside the milling chamber by a number of nozzles placed around the periphery of the chamber. The micronizing effect takes place by the collision between the incoming particles and those already accelerated into the spiral path. Whilst centrifugal force retains the larger particles at the periphery of the milling chamber, the smaller particles exit with the exhaust air from the centre of the

chamber. The particle size distribution is controlled by adjusting a number of parameters, two of the main ones being pressure and feed rate.

The micronized IMS2186 with a particle size of less than 10 microns was suspended at 50 mg/ml in PBS containing 0.5% carboxymethylcellulose. The suspension could then pass through a 30 gage needle. A similar suspension of IMS2186 that had not been micronized plugged a 25 gage needle.

Example 16

This example shows the anti-pro liferative activity of compounds of the subject invention.

A. Materials and Methods lOOμl of cell suspension containing 10 ³ cells of each cell line were plated in 96-well flat bottom micro-titer plates and incubated overnight. Different concentrations of the compounds of the subject invention were then added and the cells were allowed to grow for 2-3 days. Cell growth was measured by alamarBlue™ staining (1:10 reagentxell suspension volume). IC50 was defined as that concentration which gave 50% reduction in cell growth when compared to solvent only control treatment (0.1% DMSO in cell culture media).

B. Results As can be seen from Table 1, the compounds of the invention have a marked anti-pro liferative effect with little cell type selectivity. The compounds have an IC50 of less than 10 mM (most between 1-3 mM) in cell lines as diverse as HCTl 16 (colon cancer), MCF-7 (breast cancer), NIH-H460, HOP62 (NSCL cancer), ARPE- 19 (retinal pigmented epithelial cells), and HUVEC (human umbilical vein endothelial cells). In addition, the effect does not appear to be species restricted, since this activity also extends to murine cells (NIH3T3).

Table 1. Anti-Proliferative Activity of Compounds of the Invention in Various Cell Lines (NT= not tested)

Example 17

This example shows that a compound of the subject invention can block cell cycle progression at the G2/M phase.

A. Materials and Methods Cells from the human cancer cell line H460 (non-small lung cancer) were incubated with lOμM and 5μM of the compound described in Example ID (designated "IMS 1998") for either three or five days. After fixation in 75% ethanol, the cells were stained with propidium iodide and the DNA content analyzed by flow cytometry. B. Results

As can be seen from Figure 1, IMS 1998 caused significant G2/M arrest as early as three days post-treatment. This effect was shown to be reversible when the drug was withdrawn.

Example 18

This example shows that compounds of the subject invention have anti- angiogenesis activity in vitro.

A. Materials and Methods

Human umbilical vein endothelial cells (HUVEC) were seeded on Matrigel- coated 96-well plates and incubated with different concentrations of compounds of the subject invention. 10ng/ml VEGF was added to the culture medium to stimulate angiogenesis. 20 hours later, tube formation was observed and quantified manually by counting branch points under the microscope. Paclitaxol, a commercially marketed anti-tumor compound with known anti- angiogenesis activity, served as a positive control.

To assess the cytotoxicity and anti-pro liferative activity of the compounds of the subject invention under these conditions, the cells were allowed to grow for three days in culture and then stained with alamarBlue™ (as described above).

B. Results The results in Table 2 below show that the compounds of the subject invention inhibit tube formation with IC50 values of approximately 0. ImM-0.5mM. Moreover, no significant cytotoxicity was observed, while the anti-pro liferative effect of the

compounds was evident. Figure 2 shows the inhibitory effect on tube formation of HUVEC cells (an angiogenesis process) of two different concentrations of IMST 1998 (compound described in Example ID). It should be further noted that the compound designated "IMS 1999" is described in Example IE; the compound designated "IMS2186" is described in Example 2; and the compound designated "IMS2187" is described in Example 6.

Table 2: Anti- Angiogenesis Activity of Compounds of the Invention

Example 19

This example shows that compounds of the subject invention have anti- angiogenesis activity in vivo.

A. Materials and Methods

0.5ml Matrigel (Matrigel plugs) with or without lOOng vascular endothelial growth factor (VEGF) were subcutaneously (s.c.) injected into nude mice. The compounds of the subject invention were either systemically delivered by intraperitoneal (i.p.) injection or locally administrated by subcutaneous injection (together with the Matrigel plug). One week post-dosing, the Matrigel plugs in the mice were harvested and the hemoglobin measured using the Drabkin method and Drabkin reagent kit 525 (Sigma) for the quantification of blood vessel formation. Paclitaxol and capsaicin were used as positive controls for the i.p. and s.c. delivery routes, respectively. See Min et al., Cancer Research, 64:644-651 (2004).

B. Results

As shown in Figure 3, IMS 1999 significantly decreased VEGF-induced blood vessel formation, with local delivery (s.c.) of the compound having an even stronger effect than systemic (i.p.) delivery.

Example 20

This example shows that compounds of the subject invention can inhibit cell migration. A. Materials and Methods

The ability of compounds of the subject invention to inhibit cell migration was shown by using a chamber slide based assay system (see Yeh et al., Molecular Pharmacology. 59:1333-1342 (2001)). First, HUVEC cells were seeded on the upper chamber together with different concentrations of IMS 1998. Cell migration was induced by the addition of 10ng/ml VEGF in the medium located in the lower chamber. Twenty hours later, the cells that migrated into the lower chamber were quantified by alamarBlue™ staining.

Second, THP-I (monocyte) cells were seeded on the upper chamber together with different concentrations of "IMS2217" (described in Example 4) and "IMS2186" (described in Example 2). MCP-I supernatant from a producer cell line ARPE cells, or SDF-I (5mM) was used as a chemo attractant. Anti-MCP-1 antibody (Fab) was used as a positive control and isotype control IgG was used as a negative control.

The ability of compounds of the invention to inhibit cell migration was also tested in vitro in a wound healing assay. A2058 melanoma cells were grown to confluence in 10cm tissue culture dishes. A "wound" in the confluent cell layer was created by scratching the cell layer using a pipette tip. IMS2186 was added at various concentrations and the cells were incubated overnight at 37 ⁰ C for 20 hours. Photo micrographs were taken at 0 hour (when no compound was added) and 20 hours post compound addition. Control cells which were not exposed to IMS2186 migrated and covered the wound within 20 hours (top two panels)

B. Results

The results of the first chamber slide experiment with HUVEC cells show that IMS1998 inhibits VEGF induced cell migration (see Figure 4). The results of the second chamber slide experiment with THP-I cells show that both IMS2217 (Figure 5A) and IMS2186 (Figure 5B) inhibit cell migration, with IMS2217 being more potent. The results of the wound healing experiment show that IMS2186 inhibited the migration of cells into the wound (scratch) - (Figure 6 A and Figure 6B). Control

cells which were not exposed to IMS2186 migrated and covered the wound within 20 hours (Figure 6C).

Example 21 This example shows that compounds of the subject invention have antiinflammatory activity in vitro.

A. Materials and Methods

Anti-inflammatory effects were measured in vitro by a PGE2 ELISA assay (Assay Design, Ann Arbor, Michigan) and a nitric oxide assay (Promega). Undifferentiated THP- 1 cells or PMA-differentiated THP- 1 cells (1 e4 cells/well) were seeded in a 24-well plate. 10ng/ml PMA (phorbol 12-myristate 13-acetate) was added to the culture medium to induce differentiation. Two days later, fresh medium with lipopolysacharide (LPS) (an inflammatory enhancer) was added to the cells together with different concentrations of IMS2217, and incubated overnight. The medium was collected and subjected to PGE2 ELISA assay according to the manufacturer's instructions.

Murine macrophage RAW cells were seeded and differentiated as described above. After treatment with LPS and different concentrations of IMS 1998, the medium was collected and transferred to a 96-well plate for the nitric oxide assay according to the manufacturer's instructions.

B. Results

As shown in Figure 7A and Figure 7B, IMS2217 significantly decreased PGE2 production on both undifferentiated THP-I cells and differentiated THP-I cells, in a dose dependent manner, with the levels of inhibition higher on differentiated THP-I cells. Little cytotoxicity was observed. Furthermore, as can be seen from Figure 8, IMS 1998 inhibited iNOS mediated nitric oxide production in a dose dependent manner.

Example 22 This example shows that compounds of the subject invention have antiinflammatory activity in vivo.

Al

A. Materials and Methods

The anti-inflammatory effects of the compounds of the present invention were shown in vivo using a peritonitis model of inflammation in athymic nude mice induced by the injection of LPS, with TNF-α and IL-6 (both pro-inflammatory cytokines) and PGE2 as the measured indicators.

Six-week-old female athymic nude mice (Simonsen Laboratories, Inc., CA) were divided into different groups of six animals each (n=6). The mice were injected intraperitoneally (i.p.) with IMS 1999 or IMS 2217 at 50mg/kg, with dexamethasone (Sigma) at 15mg/kg and phosphate buffer solution (PBS) used as controls. After one hour, all the mice except the basal controls received lipopolysacharide (LPS) (Sigma) i.p at 20mg/mouse. Two hours after the LPS injection, the mice were anaesthetized with isoflurane. Blood was collected by cardiac puncture and peritoneal fluid collected and lavaged with PBS. The IL-6, TNF-α and PGE2 levels in both plasma and peritoneal fluids were then measured by enzyme linked immuno-sorbent assays (ELISA) according to the manufacturer's recommendations.

For the IL-6 and TNF-α assay (eBioscience, Inc., San Diego), 96 well ELISA plates were coated with 100 μl/well of captured antibody overnight at 4°C and then washed 5 times with wash buffer. The plates were blocked with 200 μl/well of IX assay diluent at room temperature for one hour and washed five times with wash buffer. The samples were added to the appropriate wells and the plates were incubated at room temperature for two hours. After washing five times, avidin-HRP was added and the plates incubated at room temperature for 30 minutes. The plates were washed a total of seven times and the substrate solution was added to each well and then incubated at room temperature for another 15 minutes. The reaction was stopped by adding 50 μl/well of stop solution and the plates were read with a spectrum densitometer at 450 nm. The concentrations of IL-6 and TNF-α were calculated by comparing with the standard controls.

For the PGE2 assay (Assay Design), the plasma and peritoneal fluid samples were diluted 50 times with PBS. The diluted samples were applied together with a PGE2 monoclonal antibody to a 96-well ELISA plate. After 2 hours of incubation, the plates were washed and substrates were added. The reaction was stopped after 45 minutes, and the yellow color generated were read on a microplate reader at 405nm. The intensity of the bound yellow color is inversely proportional to the concentration

of PGE2 in the samples. The concentration of PGE2 was calculated by comparing with the Standard controls

B. Results

As shown in Figures 9. 10 and 11, IMS 1999 caused a significant reduction in all inflammatory indicators measured (TNF-α , IL-6 and PGE2, respectively), both in plasma and in the peritoneal fluid. IMS2217 also showed reduction.

Example 23

This example shows that a compound of the subject invention inhibited the growth of ovarian cancer using a xenograft mouse model.

A. Materials and Methods

Six-week-old female athymic nude mice (Simonsen Laboratories, Inc., CA) were inoculated with SKO V3 human ovarian cancer cells (ATCC) subcutaneously at 10 ⁷ cells/mouse and then divided into two groups of fifteen animals each (n=15). The mice in the test group were injected intraperitoneally (i.p.) with IMS2217 at 50mg/kg, and the mice in the control group with 5% dextrose (Sigma). Tumor size (V= (length X width ² )/2 and body weight were measured twice/week.

B. Results

The results are shown in Figure 12A (tumor size) and Figure 12B (body weight). As can be seen, the size of the tumors in the mice treated with IMS2217 was significantly smaller than the tumors of the mice in the control group, and the treatment with IMS2217 had no discernible toxic effect on the mice (no measurable change in body weight).

Example 24

This example shows that a compound of the subject invention inhibited the uncontrolled growth of fibroblast cells using a xenograft mouse model, indicating that these compounds can be effective in the treatment of indications such as keloids and hypertrophic scars which are typified by the uncontrolled growth of such cells. A. Materials and Methods

Six-week-old female athymic nude mice (Simonsen Laboratories, Inc., CA) were inoculated with human fibrosarcoma HT 1080 cells subcutaneously at 3X10 ⁶

cells/site. Once the average tumor size reached 50 ³ mm, the mice were divided into two groups evenly (n=10). For the test group, IMS2186 was administered directly into the tumor (intratumoral injections) twice at weekly intervals. Tumor growth in the injected mice and mouse body weight was measured twice/week post injection. B. Results

The results are shown in Figure 13A (tumor size) and Figure 13B (body weight). As can be seen, the size of the tumors in the mice treated with IMS2186 was significantly smaller than the tumors of the mice in the control group, and the treatment with IMS2186 had no discernible toxic effect on the mice (no measurable change in body weight).

All references cited herein are incorporated in their entirety. It is appreciated that the detailed description above is intended only to illustrate certain preferred embodiments of the present invention. It is in no way intended to limit the scope of the invention, as set out in the claims.

Example 25

This example shows that compounds of the invention are useful for the treatment of wet AMD, by reducing proliferation and mobilization of cells, including endothelial cells, in the choroid of the eye, as demonstrated by an in vivo CNV (choroidal neovascularization) model.

A. Materials and Methods

35 Brown Norway rats were randomly divided into three groups of nine rats each and one group of eight rats. An 810 Diode laser was used to place five to seven burns around the disc of one eye of each of the rats, between 2-3 disc diameters away from the optic nerve. Laser parameters were: power - 33OmW, spot size - 75 μm, exposure - 10OmS. Only burns with cavitation air bubble formation (indicating broken Bruch's membrane) were accounted as valid burns. Immediately after the laser burns, the eyes of each group were injected intravitreally with 2 μl of the following compounds: Group 1 - IMS 2217; Group 2 - IMS 2186; Group 3 - Kenalog (positive control); and Group 4 - PBS (negative control). The dosages were: lOOμg/eye for IMS 2217 and 2186 (2μl of the solution of 50mg/ml), 80 μg/eye for Kenalog, and 50 μl of PBS containing 0.5% carboxymethylcellulose. All compounds were delivered through a 30 gauge needle connected to a quantitative Hamilton syringe dispenser.

Two weeks after the laser burns and intravitreal drug injection, all rats had fluorescein angiogram (FA) exams. Two days after FA, the rats were sacrificed and the globes prepared for immunohistochemical processing for FITC-isolectin and Dapi staining. After fixation in 4% paraformaldehyde for 2 to 3 hours, the globes were rinsed with PBS 2 times xlO' and permeabilized in 1% Triton X-IOO for 2 hours. Following 2 times PBS rinse xlO', the anterior segment and neural retina were removed and the eyecups were incubated in 1 :150 fluorescein-labeled isolectin B4 (Vector Laboratories) at 4°C (wrapped with foil paper) overnight. After rinsing again twice with PBS, the eye cups were submerged completely in the ImM DAPI staining solution and covered with foil and left to incubate in 4°C for 1 hour. After further rinsing with PBS, the cups were cut radially (4 to 5 radial cuts), mounted and cover slipped.

For the FA evaluation, the laser burns were graded by two ophthalmologists (who were masked to the study design) into four levels (0, 1, 2, and 3) according to the intensity of the fluorescein (a measure of leakage). Average grades were used for the statistical analysis. For the FITC-isolectin and Dapi staining, the images were acquired from the flat mount of RPE-choroidal complex using a confocal microscope for two dimensional area measurement and three dimensional volume measurement. The area was measured using NIH image software along with a 3-scale intensity grading (area measurement plus intensity measurement =gross volume) and the volume was measured from the serial stacks of images using Auto Visualize & AutoDeblur 9.3 software. For the statistical analysis, differences of the lesion grades and difference of the lesion area from the FITC=isolectin and Dapi measurements among groups were analyzed by repeated- measures for multiple comparisons.

B. Results

The FA analysis (anti-angiogenesis effect) indicated that Kenalog significantly inhibited leakage (p<0.05), with IMS 2186 showing some effect and IMS 2217 having little or no effect. See Table 3.

Table 3. Fluorescein Angiogram

With respect to lesion area and intensity (as indicators of anti-pro liferative effect), as measured by FITC-isolectin staining, IMS2186 was significantly superior to PBS (p=0.05, one tail), with no significant difference between IMS 2186 and Kenalog (p=0.744 two tail or 0.37 one tail). See Table 4.

Table 4. FITC-isolectin Staining

Furthermore, analysis of gross volume (cellularity) of the lesions by Dapi staining (an additional indicator of anti-pro liferative effect) showed that both IMS 2186 and Kenalog were superior to PBS and IMS2217 (p<0.05). See Table 5.

Table 5. Dapi Staining