HK1166953B - Compositions and methods of use - Google Patents

Description

Compositions and methods of use

Technical Field

The present invention relates to plant extract compositions, isolated active agents, and methods of use for cancer therapy.

Background

The identification of new therapeutically useful extracts and compounds of continuing biomedical importance, natural products continue to be of interest for this purpose. Proliferative disorders continue to provide challenges to human and animal health, and treatment strategies for these diseases remain an unmet need in a wide range. Due to changes in the targets involved in mechanisms, signal processing and proliferative disorders, the identification of more new and effective compositions, compounds and therapeutic approaches is of great interest to the medical community and the general public.

Lycopodium clavatum (also known as Wolf's Foot) and Lycopodium clavatum (Club Moss Lycopodiaceae) belong to the genus Lycopodium of the family Lycopodiaceae. It gets the name of shisong because its branches are shaped like wolf's claws (lukos refers to wolf, podos refers to claw). Such plants are found throughout the world in subtropical and tropical forests. After meiosis, its sporophytes produce spores within the sporangia. The sporangia cluster in the stone pine produces cone-shaped cones, where each sporangia is protected by phylliform sporophylls. In the search for new compositions of interest with new therapeutic activity, extracts derived from spores of plants were fractionated to obtain isolated extracts and compounds.

Disclosure of Invention

The present invention relates to methods of using the plant extracts herein to treat a proliferative disorder in a subject. One aspect of the invention is a method of treating a proliferative disorder in a subject using a compound isolated from a plant extract herein. In one embodiment, the proliferative disorder is cancer. In certain embodiments, the cancer is leukemia, lung cancer, liver cancer, or colon cancer. In one embodiment, the cancer is non-small cell lung cancer. In another embodiment, the cancer is liver cancer.

In one embodiment, the compound is 8-hydroxypalmitic acid ("8-HHA"), or salts, prodrugs, prodrug salts, solvates, hydrates, and polymorphs thereof. In another embodiment, the compound used in the process according to the invention is a racemic mixture of the R-and S-enantiomers of 8-hydroxypalmitic acid. In one embodiment, the compound is the S-enantiomer of 8-hydroxypalmitic acid ("(S) -8-HHA"). In another embodiment, the compound is the R-enantiomer of 8-hydroxypalmitic acid ("(R) -8-HHA").

Another aspect of the invention is a composition comprising a plant extract herein (e.g., an extract of lycopodium clavatum). Another aspect is a composition comprising an isolated compound from a plant extract herein. Another aspect is a composition comprising an isolated compound from a plant extract herein, the isolated compound having one or more substituents.

Another aspect is a compound (or combination of compounds) described herein (or a method of using a compound/combination described herein), wherein the compound or combination of compounds has been demonstrated to have anti-cancer activity in a subject (e.g., an animal model, a mouse, a rat, a rabbit, a primate, a human). To determine such effects, the examples herein are instructive with respect to representative methods.

In one embodiment, the compound (or combination of compounds) described herein is obtained from a process comprising extraction from a plant. In certain embodiments, the procedure used in obtaining the compound (or combination of compounds) further comprises any of the steps of isolation, evaporation and partitioning (fractionation) of the plant extract.

In another embodiment, the compounds (or combinations of compounds) described herein are obtained from synthetic methods.

Another aspect of the invention is a pharmaceutical composition comprising a plant extract herein (e.g., BCP-21 extract, any of the BCP-21 extract fractions in table 1 or 2) or a compound present in a plant extract herein (e.g., BCP-21 extract, any of the BCP-21 extract fractions in table 1 or 2) (e.g., 8-hydroxyhexadecanoic acid, any of the BCP compounds 1-10 herein). In one embodiment, the pharmaceutical composition comprises a racemic mixture of 8-HHA, or salts, prodrugs, prodrug salts, solvates, hydrates, and polymorphs thereof. In one embodiment, the pharmaceutical composition comprises the S-enantiomer of 8-hydroxypalmitic acid in significant enantiomeric purity ("(S) -8-HHA"). In another embodiment, the pharmaceutical composition comprises the R-enantiomer of 8-hydroxypalmitic acid in substantial enantiomeric purity ("(R) -8-HHA").

In another aspect, the composition can be contained in a kit.

The above compositions (and methods of treatment described herein) can be further combined with appropriate chemical or biological therapeutic agents. Examples of such formulations include, but are not limited to, Aldesleukin (Aldesleukin), Alemtuzumab (Alemtuzumab), Alitretinoin (alitretinoid), Altretamine (Altretamine), Aminolevulinic acid (Aminolevulinic acid), Anagrelide (Anagrelide), Anastrozole (Anastrozole), arsenic trioxide (arsenicrrioxide), Asparaginase (aspargine), bacillus calmette-guerin (bacilmetal-guerin, BCG), Betamethasone (Betamethasone), Bexarotene (Bexarotene), Bicalutamide (Bicalutamide), Bleomycin (Bleomycin), Busulfan (Busulfan), Capecitabine (Capecitabine), Carboplatin (Carboplatin), Carmustine (Carmustine), Chlorambucil (cil), chromophorin (clavam), Cytarabine (clavam), copoviductine (clavine), Cisplatin (clavulane), clavulan (clavulane), clavulane (clavulan), clavulane (clavulane), clavulane (c acid), c acid (c acid, ara-, Dacarbazine (Dacarbazine), Dactinomycin (Dactinomycin), actinomycin D (actinomycin D), Daunorubicin (Daunorubicin), Daunorubicin liposome (Daunorubicin clinopome), dinil (Denileukin difitox), Dexamethasone (Dexamethasone), Diclofenac (Diclofenac), Docetaxel (Docetaxel), Doxorubicin (Doxorubicin), Doxorubicin liposome (Doxorubicin lipome), Epirubicin (Epirubicin), Esterified estrogen (esterifed estranges), Estradiol (Estradiol), Estradiol valerate (Estradiol), Estramustine (Estramustine), Estrone (estriol), ethisterone (Etoposide), Etoposide (Etoposide), flunomide (flunomide), flunomide (flunaridine), flunaridine (Etoposide (flunaridine), flunaridine (flunaricide (flunaridine), flunaringine (flunaringine), flunaringine (flunaringine), flunaringin (flunaringi, Goserelin acetate (Goserelin acetate), Granisetron (Granistron), Hydrocortisone (Hydrocortisone), Hydroxyprogesterone (Hydroxyprogesterone), Hydroxyurea (Hydroxyurea), Ibritumomab tiuxetan (Ibritumumab tiuxetan), Idarubicin (Idarubicin), Ifosfamide (Ifosfamide), imatinib mesylate (Imatinibmesylate), Interferon alpha-2 a (Interferon alfa-2a), Interferon alpha-2 b (Interferon alfa-2b), Irinotecan (Irinotecan), Letrozole (Letrozole), Leuprolide acetate (Leuprolide acetate), Levothyroxine (Levoyrosine), Lomustine (Lostine), Mechlorethamine (Melochlorestine), megestrone acetate (Methoxyprogesterone acetate), Megestrol acetate (Methoxyprogesterone acetate), mesterone acetate (Methylosterone), Methylosterone acetate (Methylosterone acetate), Methylosterone (Methylosterone acetate (Methylosterone), Methylosterone acetate (Methylosterone), Methylosterone acetate), Methylosterone (Methylosterone, Methylosterone acetate), Methylosterone (Methylosterone acetate), Methylosterone (Methylosterone, Methylosterone (Methylostero, Mitotane (Mitotane), Mitoxantrone (Mitoxantrone), Nandrolone phenylpropionate (Nandrolone phenopionate), Nilutamide (Nilutamide), Octreotide acetate (Octreotide acetate), Omphalin (Oprelekin), Oxymetholone (Oxymethol), pemetrexed (Pegaspargase), Pentostatin (Pentostatin), Plicamycin (Plicamycin), Polifeprosan 20 and carmustine implants (Polifrosan 20/carmustine), Porfimer Sodium (Porfimer Sodium), Prednisolone (Prednisone), Prednisone (Prednisone), Procarbazine (Procarbazine), Progesterone (Progesterone), Rituximab (Rituximab), Lysoxib [ Saxib ] Sodium (Saxib ] 153-Sodium citrate [32 ], Sodium citrate [32 ] Sodium chloride (Sodium citrate), Sodium citrate [89 ], Sodium citrate [32 ], Sodium chloride [32 ] and Sodium citrate [32 ] (Sodium citrate [32 ]), Strontium chloride [32 ], Sodium citrate [32 ] and Sodium citrate [32 ], Temozolomide (Temozolomide), Teniposide (Teniposide), lactone (Testolactone), Testosterone heptanoate (Testosterone enanthate), Thioguanine (Thioguanine), Thiotepa (Thiotepa), Topotecan (Topotecan), toremifene citrate (toremifene citrate), Trastuzumab (Trastuzumab), Tretinoin (Tretinoin), Triamcinolone (Triamcinolone), Triptorelin pamoate (Triptorelin pamoate), Valrubicin (Valrubicin), Vinblastine (vinlasine), Vincristine (vincrisristine), and Vinorelbine (Vinorelbine).

In a further aspect, the present invention provides the use of a compound of any of the formulae herein, alone or in combination with one or more additional therapeutic agents, in the manufacture of a medicament, as a single composition or in separate dosage forms, for the treatment or prevention of a disease, disorder or condition described herein in a subject. Another aspect of the invention is a compound of the general formula herein (isolated natural or synthetic) for use in the treatment or prevention of a disease, disorder or symptom thereof described herein in a subject.

Drawings

FIGS. 1 to 3 show NMR spectra of compounds isolated from BCP-21 extract.

FIGS. 4 to 5 are MS spectra of compounds isolated from BCP-21 extract.

FIG. 6 shows that BCP-21 dose-dependently induced apoptosis in Hela (HeLa) cells.

FIG. 7 is a result of immunoblotting (western blot), which shows that extract of Lycopodium clavatum induces PARP cleavage (PARP cleavage) time-dependently.

FIG. 8 shows that (S) -8-HHA dose-dependently inhibited the survival of HOP-92 lung cancer cell line.

FIG. 9 shows that 100. mu.M of 8-HHA (racemic mixture, S-or R-enantiomer) resulted in decreased survival of PCL5, Hep3B and SNU liver cancer cell lines using MTT colorimetric analysis.

FIG. 10 demonstrates that (S) -8-HHA dose-dependently inhibited the survival of PLC-5 hepatoma cell line.

FIG. 11 shows that 100. mu.M of (S) -8-HHA causes cleavage of PARP in SNU and PCL5 liver cancer cell lines.

FIG. 12 demonstrates that (S) -8-HHA treatment results in a substantial reduction in FAS receptor levels compared to controls.

FIG. 13 shows that 8-HHA reduces tumor growth in PLC5 liver cancer cells in a mouse model of xenotransplantation of liver.

FIG. 14 shows the antitumor efficacy of 8-HHA in HOP92 lung cancer cells in a xenograft mouse model.

Detailed Description

Definition of

The terms "ameliorating" and "treating" are used interchangeably and refer to reducing, inhibiting, attenuating, reducing, arresting, or stabilizing the development or progression of a disease (e.g., a disease or disorder as described herein).

"disease" refers to any condition or disorder that impairs or interferes with the normal function of a cell, tissue or organ.

"marker" refers to any change associated with a disease or disorder. For example, any protein or polynucleotide whose expression level or activity is altered in association with a disease or disorder.

The term "cancer" generally refers to a class of diseases in which a group of cells exhibits uncontrolled growth (division outside the normal range), invasion (invasion and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body). Examples of cancer include, but are not limited to, leukemia, lung cancer, liver cancer, colon cancer, melanoma, breast cancer, CNS cancer, ovarian cancer, renal cancer, and prostate cancer. In the present disclosure, words "comprising", "containing", "having", and the like may have meanings given to them by us patent law, and may mean "including", and the like; "consisting essentially of … …" or "consisting essentially of has the meaning attributed to them by U.S. patent law, and the term is open-ended, allowing the presence of objects other than those listed, provided that the underlying or novel features are not altered by the presence of objects other than those listed, but excluding prior art embodiments.

The compounds herein may also be used in the form of compounds including salts, prodrugs, and prodrug salts, solvates, hydrates, and polymorphs of the compounds of the general formula herein.

Salts of the compounds of the invention are formed between an acid and a basic group of the compound, such as an amino functional group, or between a base and an acidic group of the compound, such as a carboxyl functional group. According to another preferred embodiment, the compound is a pharmaceutically acceptable acid addition salt.

As used herein, unless otherwise indicated, the term "prodrug" means a derivative of a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide a compound of the invention. Prodrugs may become active only under biological conditions by this reaction, or they may be active in their unreacted form. Examples of prodrugs contemplated by the present invention include, but are not limited to, analogs or derivatives of compounds of any of the formulae disclosed herein, which include biohydrolyzable moieties such as amide, ester, carbamate, carbonate, and phosphate analogs. Prodrugs can generally be prepared using well known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-; see also Goodman and Gilman's, The pharmaceutical basic of Therapeutics, 8th ed., McGraw-Hill, int. Ed.1992, "Biotransformation of Drugs".

As used herein, unless otherwise indicated, the term "biohydrolyzable moiety" means an analog of a functional group (e.g., amide, ester, carbamate, carbonate, or phosphate) that: or 1) does not destroy the biological activity of the compound and can confer advantageous properties in vivo on the compound, such as uptake, time of action or onset (of action); or 2) a compound which is not biologically active per se but which is converted in vivo to a biologically active compound.

A salt of a prodrug is a compound formed between an acid and a base group, such as an amino functional group, or a base and an acid group, such as a carboxyl functional group, of the prodrug. In one embodiment, the salt of the prodrug is a pharmaceutically acceptable salt.

Particularly preferred prodrugs and salts of prodrugs are those which, when the compounds of the present invention are administered to a mammal, enhance the bioavailability of the compounds (e.g., by allowing an orally administered compound to be more readily absorbed into the blood) or enhance the transport of the parent compound to the biological compartment (e.g., the brain or central nervous system) associated with the parent species (parentspecies). Preferred prodrugs include derivatives in which a group that enhances water solubility or active transport across the intestinal membrane is attached to the structure of the formulae described herein. See, e.g., Alexander, J. et al, Journal of Medicinal Chemistry 1988, 31, 318-; bundgaard, h.design of produgs; elsevier: amsterdam, 1985; pp 1-92; bundgaard, h.; nielsen, N.M. journal of Medicinal Chemistry 1987, 30, 451-; bundgaard, H.A Textbook of Drug Design and Development; harwood A typical publish: switzerland, 1991; pp 113-; digenis, G.A et al, Handbook of Experimental Pharmacology 1975, 28, 86-112; friis, g.j.; bundgaard, H.A Textbook of Drug Design and Development, 2 ed.; overturas pub.: amsterdam, 1996; pp 351-; pitman, i.h. medicinalresearch Reviews 1981, 1, 189-.

The term "pharmaceutically acceptable" as used herein refers to the following components: which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. "pharmaceutically acceptable salt" means any non-toxic salt that, when administered to a subject, provides, directly or indirectly, a compound of the invention or a prodrug of a compound.

Acids commonly used to form pharmaceutically acceptable salts include inorganic acids such as hydrogen disulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, and phosphoric acid; and organic acids such as p-toluenesulfonic acid, salicylic acid, tartaric acid, acid tartaric acid (biartaric acid), ascorbic acid, maleic acid, benzenesulfonic acid (besylic), fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid (benzanesulfonic acid), lactic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid; and related inorganic and organic acids. These pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate (decanoate), octanoate, acrylate, formate, isobutyrate, decanoate (caprate), heptanoate, propiolate (propiolate), oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylenesulfonate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, succinate, iodide, fumarate, maleate, butyrate, and salt thereof, Phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, beta-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Preferred pharmaceutically acceptable acid addition salts include salts formed with inorganic acids such as hydrochloric acid and hydrobromic acid, and especially salts formed with organic acids such as maleic acid.

Suitable bases for forming pharmaceutically acceptable salts with the acid functional group of the prodrugs of this invention include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia and organic amines such as unsubstituted or hydroxy-substituted mono-, di-or trialkylamines; dicyclohexylamine; tributylamine; pyridine; n-methylamine, N-ethylamine; diethylamine; triethylamine; mono-, di-or tri- (2-hydroxy-lower alkyl amines) such as mono-, di-or tri- (2-hydroxyethyl) amine, 2-hydroxy-tert-butylamine or tri- (hydroxymethyl) methylamine, N-di-lower alkyl-N- (hydroxy lower alkyl) -amines such as N, N-dimethyl-N- (2-hydroxyethyl) amine, or tri- (2-hydroxyethyl) amine; N-methyl-D-glucosamine; and amino acids such as arginine, lysine and the like.

The term "hydrate" as used herein means a compound that further includes binding of stoichiometric or non-stoichiometric amounts of water by non-covalent intermolecular forces.

The terms "isolated," "purified," or "biologically pure" refer to a substance that is substantially or essentially free of components that normally accompany it as found in its natural state. Purity and homogeneity are typically determined by analytical chemical techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Specifically, in embodiments the compound is at least 85% pure, more preferably at least 90% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

The term "solvate" as used herein is meant to further include compounds that combine stoichiometric or non-stoichiometric amounts of solvents such as water, acetone, ethanol, methanol, dichloromethane, 2-propanol, and the like, by non-covalent intermolecular forces.

The term "polymorph" as used herein denotes a solid crystalline form of a compound or complex thereof, which may be characterized by physical means, such as X-ray powder diffraction patterns or infrared spectroscopy. Different polymorphs of the same compound may exhibit different physical, chemical and/or optical properties. Different physical properties include, but are not limited to, stability (e.g., stability to heat, light, or humidity), compressibility and density (important in formulation and product manufacture), hygroscopicity, solubility, and dissolution rate (which can affect bioavailability). Differences in stability can result from changes in chemical reactivity (e.g., differential oxidation reactions such that a dosage form discolors more rapidly when comprising one polymorph than when comprising another polymorph) or mechanical properties (e.g., upon storage, tablets break when a kinetically favored polymorph converts to a thermodynamically more stable polymorph) or both (e.g., tablets of one polymorph break more easily at high humidity). Different physical properties of polymorphs can affect their processing. For example, a polymorph may be more prone to solvate formation or may be more difficult to filter or wash free of impurities due to, for example, its particle shape or particle size distribution.

The term "substantially free of other stereoisomers" as used herein means less than 25% of other stereoisomers, preferably less than 10% of other stereoisomers, more preferably less than 5% of other stereoisomers, and most preferably less than 2% of other stereoisomers, or less than "X"% of other stereoisomers (where X is a number between 0 and 100, including 0 and 100). Methods of obtaining or synthesizing diastereomers are well known in the art and may be readily applied to the final compounds or starting materials or intermediates. Other embodiments are those wherein the compound is an isolated compound. The term "enriched in at least X% of an enantiomer" as used herein means that at least X% of the compound is in a single enantiomeric form, wherein X is a number between 0 and 100, including 0 and 100.

The term "stable compound" as used herein refers to a compound that has sufficient stability to permit manufacture and that maintains the integrity of the compound for a sufficient period of time for the purposes described in detail herein (e.g., formulation into a therapeutic product, intermediate for the preparation of a therapeutic compound, isolatable or storable intermediate compound, treatment of a disease or condition responsive to a therapeutic agent).

"stereoisomers" refers to both enantiomers and diastereomers.

The term "halo" or "halogen" as used herein refers to any group of fluorine, chlorine, bromine or iodine.

The term "alk" or "alkyl" refers to a straight or branched chain hydrocarbyl group having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms. The expression "lower alkyl" refers to an alkyl group of 1 to 4 carbon atoms (including 1 and 4). The term "arylalkyl" refers to a moiety in which an alkyl hydrogen atom is replaced with an aryl group. The term "alkenyl" refers to a straight or branched chain hydrocarbyl group of 2 to 10, preferably 2 to 4, carbon atoms having at least one double bond. When an alkenyl group is bonded to a nitrogen atom, it is preferable that such a group is not directly bonded through a carbon having a double bond.

The term "alkoxy" refers to an-O-alkyl group. The term "alkylenedioxy" refers to a divalent species of the structure-O-R-O-, wherein R represents an alkylene group.

The term "alkynyl" refers to a straight or branched chain hydrocarbyl group of 2 to 10, preferably 2 to 4, carbon atoms having at least one triple bond. Where the alkynyl group is bonded to a nitrogen atom, it is preferred that such groups are not directly bonded through a carbon bearing triple bond.

The term "alkylene" refers to a divalent straight chain bridge of 1 to 5 carbon atoms connected by single bonds (e.g., - (CH)₂)_X-, wherein X is 1 to 5), which may be substituted with 1 to 3 lower alkyl groups.

The term "alkenylene" refers to a straight chain bridge of 2 to 5 carbon atoms with one or two double bonds connected by single bonds and may be substituted with 1 to 3 lower alkyl groups. Typical alkenylene radicals are-CH-, -CH₂-CH＝CH-、-CH₂-CH＝CH-CH₂-、-C(CH₃)₂CH-and-CH (C)₂H₅)-CH＝CH-。

The term "alkynylene" refers to a straight chain bridge of 2 to 5 carbon atoms having a triple bond therein, connected by a single bond, and may be substituted with 1 to 3 lower alkyl groups. Typical alkynylene groups are-C.ident.C-, -CH₂-C≡C-、-CH(CH₃) -C.ident.C-and-C.ident.C-CH (C ≡ C-CH)₂H₅)CH₂-。

The terms "cycloalkyl" and "cycloalkenyl" as used herein include cyclic hydrocarbyl groups having from 3 to 12 carbons, preferably from 3 to 8 carbons, more preferably from 3 to 6 carbons, which are saturated and partially unsaturated, respectively. The term "Ar" or "aryl" refers to an aromatic cyclic group (e.g., a 6-membered monocyclic, 10-membered bicyclic, or 14-membered tricyclic ring system) containing 6 to 14 carbon atoms. Typical aryl groups include phenyl, naphthyl, biphenyl, and anthracene.

"heteroaryl" refers to a monocyclic or fused ring (i.e., rings that share a pair of adjacent atoms) group of 5 to 12 ring atoms, containing 1, 2, 3, or 4 heteroatoms selected from N, O or S, with the remaining ring atoms being C, and additionally, having a fully conjugated pi-electron system, wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted with a substituent. Examples of heteroaryl groups are, but are not limited to, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, quinazol, isoquinazol, purine, and carbazole.

The term "heterocycle", or "heterocyclic" refers to a fully saturated or partially unsaturated cyclic group, for example, a 3-to 7-membered monocyclic, 7-to 12-membered bicyclic, or 10-to 15-membered tricyclic ring system, which contains at least one heteroatom in at least one ring, wherein 0, 1, 2, or 3 atoms of each ring may be substituted by a substituent. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system.

The term "substituent" refers to a group that is "substituted" on any of the functional groups described herein, for example, an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, or heteroaryl group on any atom of the group. In some aspects, functional groups described herein, such as alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heterocyclyl, or heteroaryl groups, can be substituted with substituents (e.g., the following substituents). Suitable substituents include, but are not limited to, halogen, CN, NO₂、OR¹⁵、SR¹⁵、S(O)₂OR¹⁵、NR¹⁵R¹⁶、C₁-C₂Perfluoroalkyl group, C₁-C₂Perfluoroalkoxy, 1, 2-methylenedioxy, C (O) OR¹⁵、C(O)NR¹⁵R¹⁶、OC(O)NR¹⁵R¹⁶、NR¹⁵C(O)NR¹⁵R¹⁶、C(NR¹⁶)NR¹⁵R¹⁶、NR¹⁵C(NR¹⁶)NR¹⁵R¹⁶、S(O)₂NR¹⁵R¹⁶、R¹⁷、C(O)R¹⁷、NR¹⁵C(O)R¹⁷、S(O)R¹⁷、S(O)₂R¹⁷、R¹⁶Oxo, C (O) R¹⁶、C(O)(CH₂)_nOH、(CH₂)_nOR¹⁵、(CH₂)_nC(O)NR¹⁵R¹⁶、NR¹⁵S(O)₂R¹⁷Wherein n is independently 0 to 6, including 0 and 6. Each R is¹⁵Independently of each other is hydrogen, C₁-C₄Alkyl or C₃-C₆A cycloalkyl group. Each R is¹⁶Independently is hydrogen, alkenyl, alkynyl, C₃-C₆Cycloalkyl, aryl, heterocyclyl, heteroaryl, C₁-C₄Alkyl or substituted by C₃-C₆C of cycloalkyl, aryl, heterocyclyl or heteroaryl₁-C₄An alkyl group. Each R is¹⁷Independently is C₃-C₆Cycloalkyl, aryl, heterocyclyl, heteroaryl, C₁-C₄Alkyl or substituted by C₃-C₆C of cycloalkyl, aryl, heterocyclyl or heteroaryl₁-C₄An alkyl group. At each R¹⁵、R¹⁶And R¹⁷Each C in₃-C₆Cycloalkyl, aryl, heterocyclyl, heteroaryl and C₁-C₄Alkyl may be optionally substituted with halogen, CN, C₁-C₄Alkyl, OH, C₁-C₄Alkoxy, NH₂、C₁-C₄Alkylamino radical, C₁-C₄Dialkylamino radical, C₁-C₂Perfluoroalkyl group, C₁-C₂Perfluoroalkoxy or 1, 2-methylenedioxy.

The term "oxo" refers to an oxygen atom that when attached to carbon forms a carbonyl, when attached to nitrogen forms an N-oxide, and when attached to sulfur forms a sulfoxide or sulfone.

The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted with a substituent.

The recitation herein of a chemical group by any definition of a variable includes the definition of that variable as any individual group or combination of groups listed. Recitation of embodiments of variables herein includes embodiments as any individual embodiment or in combination with any other embodiments or portions thereof.

The compounds of the present invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention. The compounds of the invention may also exist in a variety of tautomeric forms, in which case the invention expressly includes all tautomeric forms of the compounds described herein. All such isomeric forms of these compounds are expressly included in the present invention. All crystalline forms of the compounds described herein are expressly included in the present invention.

Other methods of synthesizing the compounds of the formulae herein can be readily adapted to the references cited herein. Variations of these steps and their optimization are within the skill of the ordinary practitioner.

The particular methods and compounds shown above are not intended to be limiting. Chemical structures in the schemes herein describe various variables that are defined commensurate with the chemical group definitions (moieties, atoms, etc.) at the corresponding positions in the structural formulae of the compounds herein, whether identified by the same variable name (e.g., R)¹、R²R, R', X, etc.). The suitability of a chemical group in a compound structure for use in the synthesis of another compound structure is within the knowledge of one of ordinary skill in the art. Additional methods of synthesizing the compounds herein and their synthetic precursors, including synthetic methods in pathways not explicitly shown in the schemes herein, are within the skill of one of ordinary skill in the art. Methods for optimizing reaction conditions, if necessary to reduce by-product competition, are known in the art. The methods described herein may additionally include steps of adding or removing appropriate protecting groups before or after the steps specifically described herein to ultimately allow for the synthesis of the compounds herein. In addition, the various synthetic steps may be performed in a different order or sequence to obtain the desired compounds. Synthetic chemical Transformations and protecting group methodologies (protection and deprotection) for synthesizing useful compounds are known in the art and include, for example, r.larock, comprehensive organic Transformations, VCH Publishers (1989); T.W.Greene and P.G.M.Wuts, Protective Groups in Organic Synthesis, 3^rdEd, John Wiley and sons (1999); fieser and m.fieser, Fieser and Fieser's Reagents for organic Synthesis, John Wiley and Sons (1994); and L.Patquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent versions thereof.

The synthetic methods described herein may additionally include steps of adding or removing appropriate protecting groups before or after any of the steps described in any scheme to ultimately allow for the synthesis of compounds of the formulae described herein. The methods described herein contemplate the conversion of a compound of one formula to a compound of another formula. The conversion process involves one or more chemical transformations, which may be performed in situ, or accompanied by isolation of intermediate compounds. The conversion may comprise reacting the starting compounds or intermediates with additional reagents using techniques and methods known in the art, including the methods in the references cited herein. The intermediates can be used with or without purification (e.g., filtration, distillation, sublimation, crystallization, trituration, solid phase extraction, and chromatography).

The combinations of substituents and variables contemplated by the present invention are only those combinations that result in the formation of stable compounds.

The present invention also provides compositions comprising an effective amount of a compound herein, or a pharmaceutically acceptable salt, solvate, hydrate, polymorph or prodrug of said compound, if applicable; and an acceptable carrier. Preferably, the compositions of the present invention are formulated for pharmaceutical use ("pharmaceutical composition"), wherein the carrier is a pharmaceutically acceptable carrier. The carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient thereof in the amounts normally employed in a medicament.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of the present invention include, but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

The pharmaceutical compositions of the present invention include compositions suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. In certain embodiments, a compound of the formulae herein is administered transdermally (e.g., using a transdermal patch). Other formulations may be conveniently presented in unit dosage forms such as tablets and sustained release capsules, and in liposomal form, and may be prepared by any of the methods known in the art of pharmacy. See, for example, Remington's Pharmaceutical Sciences, Mack publishing company, Philadelphia, PA (17th ed.1985).

These methods of preparation include the step of bringing into association the ingredients to be administered, such as the carriers that make up one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, liposomes or finely divided solid carriers or both, and then, if necessary, shaping the product.

In certain preferred embodiments, the compounds are administered orally. Compositions of the present invention suitable for oral administration may be presented as discrete units, such as capsules, sachets or tablets, each of which contains a predetermined amount of the active ingredient; in the form of powder or granules; in the form of a solution or suspension in an aqueous liquid or a non-aqueous liquid; or in the form of an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, or packaged in liposomes, or in the form of pellets, etc. Soft gel capsules may be used to contain these suspensions, which may be beneficial to increase the rate of absorption of the compound.

Tablets may optionally be manufactured by compression or moulding with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated for slow or controlled release of the active ingredient therein. Methods of formulating sustained or controlled release compositions of pharmaceutical active ingredients, such as those herein and other compounds known in the art, are known in the art and are described in several issued U.S. patents, some of which include, but are not limited to, U.S. patent nos. 4369172 and 4842866, and references cited therein. Coatings can be used to deliver compounds to the intestine (see, e.g., U.S. patent nos. 6638534, 5217720 and 6569457, 6461631, 6528080, 6800663, and references cited therein). Useful dosage forms of the compounds of the invention are in the form of enteric pellets, wherein the enteric layer comprises hydroxypropylmethylcellulose acetate succinate.

In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also commonly added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

Compositions suitable for topical administration include lozenges comprising the ingredient in a flavored basis, usually sucrose and acacia or tragacanth; and lozenges comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia.

Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which can include suspending agents and thickening agents. The formulations may be presented in unit-dose and multi-dose containers, for example, sealed ampoules and vials, and may 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. Ready-to-use injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

These injection solutions may be in the form of, for example, sterile injectable aqueous or oleaginous suspensions. The suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, ringer's solution and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are conventionally employed as a solvent or suspending medium. Any less irritating, non-volatile oil may be employed for this purpose, including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylene forms. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant.

For rectal administration, the pharmaceutical compositions of the present invention may be administered in the form of suppositories. These compositions can be prepared by mixing the compounds of the invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the active ingredients. These materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of the present invention may be administered by nasal aerosol or inhalation. These compositions may be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as a salt solution using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other dissolving or dispersing agents known in the art.

Topical administration of the pharmaceutical compositions of the present invention is particularly useful when the desired treatment involves areas or organs that are readily accessible by topical application. For topical application to the skin, the pharmaceutical compositions should be formulated in a suitable ointment containing the active ingredient suspended or dissolved in a carrier. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated with a suitable lotion or cream containing the active ingredient suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl (cetearyl) alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of the present invention may also be applied topically to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topical transdermal patches and iontophoretic administration are also included in the present invention.

Particularly preferred derivatives and prodrugs are the following compounds: these compounds increase the bioavailability of the compounds of the invention when administered to a mammal (e.g., by making the orally administered compounds more readily absorbed into the blood), or they enhance the transport of the parent compound to the biological compartment (e.g., the brain or central nervous system) associated with the parent species. Preferred prodrugs include derivatives wherein a group that increases water solubility or active transport across the intestinal membrane is attached to the structure of the formulae described herein. See, e.g., Alexander, J. et al, Journal of medicinal chemistry 1988, 31, 318-; bundgaard, h.design of produgs; elsevier: amsterdam, 1985; pp 1-92; bundgaard, H; nielsen, N.M. journal of medicinal Chemistry 1987, 30, 451-; bundgaard, H.A Textbook of drug Design and Development; harwood Academic publishing: switzerland, 1991; pp 113-; digenis, g.a. et al, Handbook of experimental pharmacology 1975, 28, 86-112; friis, g.j.; bundgaard, H.A Textbook of drug Design and Development; 2 ed.; overturas pub.: amsterdam, 1996; pp 351-; pitman, i.h. medicinal Research Reviews 1981, 1, 189-.

The application of the subject treatment may be topical, so as to be administered at the site of interest. Various techniques may be used to provide the subject compositions at the site of interest, such as injection, use of a catheter, trocar, projection (projile), polyoxypropylene (pluronic) gel, stent, sustained drug release polymer, or other device provided for internal access.

According to another embodiment, the present invention provides a method of implanting an implantable drug delivery device comprising the step of contacting the drug delivery device with a compound or composition of the present invention. Implantable drug delivery devices include, but are not limited to, biodegradable polymer capsules or pellets, non-degradable diffusible polymer capsules and biodegradable polymer sheets.

According to another embodiment, the invention provides an implantable medical device coated with a compound of the invention or a composition comprising a compound of the invention, said compound being therapeutically active.

In another embodiment, the composition of the invention further comprises a second therapeutic agent. The second therapeutic agent includes any compound or therapeutic agent known to have or demonstrated advantageous properties when administered alone or in combination with a compound of any of the formulae herein. Drugs that may be usefully combined with these compounds include other kinase inhibitors and/or other chemotherapeutic agents useful in the treatment of the diseases and disorders discussed above.

These drugs are described in detail in the art. Preferably, the second therapeutic agent is an agent useful in the treatment or prevention of a disease or condition selected from cancer.

More preferably, the second therapeutic agent formulated in synergy with the compounds of the present invention is an agent useful in the treatment of kinase mediated diseases/disorders such as cancer, immune disorders, cardiovascular disease, viral infection, inflammation, metabolism/endocrine disorders and neurological disorders.

In another embodiment, the invention provides separate dosage forms of the compound of the invention and the second therapeutic agent in association with each other. The term "associated with" as used herein means that separate dosage forms are packaged together or otherwise associated with each other such that it is apparent that the separate dosage forms are intended to be sold and administered together (within less than 24 hours of each other, consecutively or simultaneously).

In the pharmaceutical compositions of the present invention, the compounds of the present invention are present in an effective amount. The term "effective amount" as used herein refers to an amount sufficient, when administered in a suitable dosage regimen, to reduce or ameliorate the severity, duration or progression of the disorder being treated, prevent the development of the disorder being treated, cause regression of the disorder being treated, or enhance or ameliorate the prophylactic or therapeutic effect of another treatment.

Dose correlation for animals and humans (on a milligram per square meter body surface basis) in Freireich et al (1966) Cancer chemither Rep 50: 219, is described. The body surface area can be approximately determined from the height and weight of the patient. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardley, n.y., 1970, 537. An effective amount of a compound of the invention may be from about 0.001mg/kg to about 500mg/kg, more preferably 0.01mg/kg to about 50mg/kg, more preferably 0.1mg/kg to about 2.5 mg/kg. The effective dosage may also vary, as recognized by those skilled in the art, depending upon the condition being treated, the severity of the condition, the route of administration, the sex, age and general health of the patient, the use of adjuvants, the possibility of co-administration with other treatments, such as the use of other drugs, and the judgment of the attending physician.

For pharmaceutical compositions comprising a second therapeutic agent, the effective amount of the second therapeutic agent is about 20% to 100% of the dose typically used in a single treatment regimen using only that agent. Preferably, the effective amount is about 70% to 100% of the normal monotherapy dose. Normal monotherapeutic dosages of these second therapeutic agents are well known in the art. See, e.g., Wells et al, eds., Pharmacotherapy Handbook, 2nd Edition, apple and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon PocketPharmacopoeia 2000, Delluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), each of which is incorporated herein by reference in its entirety.

It is expected that certain second therapeutic agents cited above will act synergistically with the compounds of the present invention. If this occurs, it will result in an effective dose of the second therapeutic agent and/or the compound of the invention that is less than that required for monotherapy. This has the advantage of minimizing toxic side effects of the second therapeutic agent or the compound of the invention, synergistically improving efficacy, increasing ease of administration or use, and/or reducing the overall cost of the compound preparation or formulation.

Method of treatment

According to one aspect, the invention provides a method of treating a subject suffering from or susceptible to a disease or disorder or symptoms thereof (such as those described herein), comprising the step of administering to the subject an effective amount of a compound (e.g., an isolated compound; a compound herein) or composition of the invention. These diseases are well known in the art and are also disclosed herein.

The method may further comprise, wherein the composition is an extract of lycopodium clavatum (e.g., BCP-21 extract, any of the BCP-21 extract fractions in table 1 or 2) or an isolated compound (e.g., 8-hydroxyhexadecanoic acid (8-HHA), any of the BCP compounds 1-10 herein) found in a plant extract herein (e.g., BCP-21 extract, any of the BCP-21 extract fractions in table 1 or 2).

In one embodiment of the invention, the isolated compound is 8-hydroxyhexadecanoic acid ("8-HHA"), or a salt, prodrug salt, solvate, hydrate, and polymorph thereof. In certain embodiments the compound is a racemic mixture of the R-and S-enantiomers of 8-hydroxypalmitic acid. In one embodiment, the compound is the S-enantiomer of 8-hydroxypalmitic acid ("(S) -8-HHA"). In another embodiment, the compound is the R-enantiomer of 8-hydroxypalmitic acid ("(R) -8-HHA").

In another embodiment, the compound (or composition) used in the method of the invention is obtained by synthetic methods.

In one aspect of the invention, the disease or disorder is a caspase (caspase) -mediated disease or disorder.

In one aspect of the invention, the disease or disorder is mediated by caspase-3 mediated cell death.

In one aspect of the invention, the disease or disorder is a caspase-3 mediated disease or disorder.

In one aspect of the invention, the disease or disorder is treated by inducing cell death.

In one aspect of the invention, the disease or disorder is treated by inducing cell death mediated by caspase-3.

In one aspect of the invention, the disease or disorder may be modulated by caspase-3.

In one aspect, the method of treatment involves the treatment of a proliferative disorder or a symptom thereof. These include cancer, tumors, any disease for which an oncology agent is useful.

Examples of cancers that can be treated with the compounds, compositions, and methods of treatment of the present invention include cancers (e.g., leukemia, liver cancer, lung cancer, colon cancer, CNS cancers, melanoma, kidney cancer, and caspase-3 mediated cancers), allergic and inflammatory diseases. A human or animal patient suffering from a proliferative disorder, such as cancer, may thus be treated by a method comprising administering thereto a compound of the invention as defined above. The symptoms of the patient may thus be improved or ameliorated. Diseases and conditions treatable according to the methods of the invention include, but are not limited to, cancer and inflammatory diseases. Cancers that may be treated according to the methods of the present invention include, but are not limited to, liver cancer, hepatocellular carcinoma, leukemia, lung cancer, colon cancer, CNS cancer, melanoma, renal cancer, and the like.

In one embodiment, the cancer treatable according to the methods of the invention is leukemia, lung cancer, liver cancer or colon cancer. In one embodiment, the cancer is liver cancer. In another embodiment, the cancer is lung cancer. In one embodiment, the lung cancer is non-small cell lung cancer. In certain examples, cancers treatable according to the methods of the invention involve HOP-92 mediated diseases (e.g., non-small cell lung cancer).

In yet another embodiment, the cancer treatable according to the methods of the invention is leukemia.

The methods described herein include those in which the subject is identified as in need of a particular prescribed treatment. Identifying a subject in need of such treatment can be within the judgment of the subject or a healthcare professional, and can be subjective (e.g., opinion) or objective (e.g., measurable by a detection or diagnostic method).

In one embodiment, the invention provides a method of modulating the activity of a caspase (e.g., caspase-3) in a cell, comprising contacting the cell with one or more compounds of any of the formulae herein.

In another embodiment, the above method of treatment comprises the further step of co-administering to said patient one or more second therapeutic agents. The second therapeutic agent may be selected from any second therapeutic agent known to be useful for the conditions (indications) herein.

The term "co-administered" as used herein means that the second therapeutic agent can be administered with the compound of the present invention as part of a single dosage form (e.g., a composition of the present invention comprising the compound of the present invention and the second therapeutic agent as described above) or separate multiple dosage forms. Alternatively, the additional agent may be administered prior to, sequentially with, or after administration of the compound of the invention. In such combination therapy treatment, both the compound of the invention and the second therapeutic agent are administered by conventional methods. Administration of a composition of the invention comprising a compound of the invention and a second therapeutic agent to a subject does not preclude separate administration of the same therapeutic agent, any other second therapeutic agent, or any compound of the invention to the subject at another time during the course of treatment.

Effective amounts of these second therapeutic agents are well known to those skilled in the art, and dosage guidance can be found in the patents and published patent applications cited herein, as well as in Wells et al, eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Delluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), and other medical textbooks. However, it is within the ability of one skilled in the art to determine the range of optimal effective amounts of the second therapeutic agent.

In one embodiment of the invention, wherein the second therapeutic agent is administered to the subject, the effective amount of the compound of the invention is less than its effective amount when administered without the second therapeutic agent. In another embodiment, the effective amount of the second therapeutic agent is less than its effective amount when administered without a compound of the present invention. In this way, undesirable side effects associated with high doses of each drug can be minimized. Other potential advantages, including but not limited to improved dosage regimens and/or reduced drug costs, will be apparent to those skilled in the art.

In a further aspect, the present invention provides the use of a compound of any of the formulae herein, alone or in combination with one or more of the second therapeutic agents described above, in the manufacture of a medicament, as a single composition or in separate dosage forms, for the treatment or prophylaxis of a disease, disorder or condition described above in a subject. Another aspect of the invention is a compound of the formulae herein for use in the treatment or prevention of a disease, disorder or symptom thereof described herein in a subject.

In other aspects, the methods herein include methods further comprising monitoring the subject's response to the administration of the treatment. Such monitoring may include periodic sampling of subject tissue, body fluids, samples, cells, proteins, chemical markers, genetic material, etc., as markers or indicators of a treatment regimen. In other methods, subjects in need of such treatment are pre-screened or identified by evaluation of relevant markers or indicators of the suitability of such treatment.

In one embodiment, the invention provides a method of monitoring the progress of a treatment. The method comprises the following steps: determining a level of a diagnostic Marker (Marker) (e.g., any of the targets or cell types described herein modulated by a compound herein) or a diagnostic measurement (e.g., screening, assay) in a subject suffering from or susceptible to a disorder described herein or a symptom thereof, wherein the subject is administered a therapeutic amount of a compound herein sufficient to treat the disease or symptom thereof. The marker levels determined by this method can be compared to known marker levels of healthy normal controls or other diseased patients to establish the disease state of the subject. In a preferred embodiment, a second level of the marker in the subject is determined at a time point later than the time point at which the first level is determined, and the two levels are compared to monitor the course of the disease or the effectiveness of the treatment. In certain preferred embodiments, the pre-treatment level of the marker in the subject is determined prior to the start of treatment according to the invention; this pre-treatment level of the marker can then be compared to the marker level in the subject after treatment has begun to determine the effect of the treatment.

In certain method embodiments, the marker or the level of marker activity in the subject is determined at least once. Comparing the marker level with another marker level measurement, e.g., from the same patient, another patient, or a normal subject before or after, can help determine whether the treatment according to the invention has the expected effect, thereby allowing adjustment to the appropriate dosage level. The determination of the level of marker may be performed using any suitable sampling/expression assay known in the art or described herein. Preferably, the tissue or body fluid sample is first removed from the subject. Examples of suitable samples include blood, urine, tissue, cells of the mouth or cheek, and hair samples containing hair roots. Other suitable samples are known to those skilled in the art. The determination of protein levels and/or mRNA levels (e.g., label levels) can be performed using any suitable technique known in the art, including, but not limited to, enzyme immunoassays, ELISA, radiolabelling/assay techniques, blotting/chemiluminescence methods, real-time PCR, and the like.

Use of the compounds of the invention

The invention also provides kits for treating a disease, disorder, or symptom thereof, comprising the kits described herein. These kits comprise: a) a pharmaceutical composition comprising one or more compounds of any structural formula herein (e.g., a composition herein, any particular compound herein), or a salt thereof; or a prodrug, or a salt of a prodrug thereof; or a hydrate, solvate, or polymorph thereof, wherein the pharmaceutical composition is in a container; and b) instructions for use that describe a method of using the pharmaceutical composition to treat a disease, disorder, or symptom thereof, including those described herein. The compounds/compositions may be administered sequentially or simultaneously with the administration of radiation therapy.

The container may be any container or other sealed or sealable device that can hold the pharmaceutical composition. Examples include bottles, divided or multi-compartment rack bottles, wherein each compartment or chamber contains a single dose of the composition, divided foil packets, wherein each compartment contains a single dose of the composition, or dispensers that dispense a single dose of the composition. The container may be of any conventional shape or form known in the art, made of a pharmaceutically acceptable material, such as a paper or cardboard box, a glass or plastic bottle or can, a resealable bag (e.g., to hold "refill" tablets for placing into different containers), or a blister pack for a single dose pressed out of the pack according to a treatment schedule. The container employed may depend on the exact dosage form involved, e.g. conventional cartons are not normally used to hold liquid suspensions. It is possible that more than one container may be used together in a single package to sell a single dosage form. For example, the tablets may be contained in bottles which in turn are contained in boxes. Preferably, the container is a blister pack.

The kit may additionally include information and/or instructions for use to a physician, pharmacist or subject. Such memory aids include numbers printed on each chamber or compartment containing a dose corresponding to the date of the regimen specifying the tablets or capsules that should be ingested or administered to the patient, or the day of the week printed on each chamber or compartment, or a card containing the same type of information. In one aspect, the instructions also relate to administration of radiation to the subject.

In a further aspect, the invention provides the use of a compound (or combination of compounds) of the invention, alone or with one or more additional therapeutic agents, in the manufacture of a medicament, either as a single composition or as a separate dosage form, for the treatment or prophylaxis of a disease, disorder or condition in a patient as described herein above. Another aspect of the invention is a compound (or combination of compounds) herein for use in the treatment or prevention of a disease, disorder or condition described herein in a patient.

All references cited herein, whether in printed, electronic, computer-readable storage media or other form, are expressly incorporated by reference in their entirety, including without limitation, abstracts, articles, journals, publications, texts, papers, technical data sheets, internet web sites, databases, patents, patent applications, and patent publications.

Examples

General procedure

The solvent used (CDCl) was taken on a JEOL ECLIPSE 400MHz NMR spectrometer₃) Is the NMR spectrum of the reference. ESI-MS (negative ion mode) was obtained with a Hitachi M-8000 mass spectrometer. An Agilent Model 5971A mass spectrometer equipped with a Phenomenex ZB-WAX-column (300 m.times.0.32 mm.times.0.25 m)(range: 70-550amu) and Agilentmodel 5890 gas chromatography.

HPLC detection method 1 (reverse phase method): the gradient method can be used to assay for extract and column fractions. The column was a 4.6X 100mm Phenomenex Luna (2), 3. mu.C 18 column (PIN 00D-4251-EO). The flow rate is 1.0 mL/min; temperature 40 ℃, injection of 10 u L. The detector was ELSD with UV at 200 nm. Prior to injection, the normal phase chromatographic sample was evaporated and reconstituted in acetone.

HPLC assay method 2 (normal phase method): column fractions and final product can be detected using this gradient method. The column was a 4.6X 250mm, 5. mu.l, Alltech, Adsorbosil silica gel column (P/N298017). Mobile phase: hexane/EtOAc, flow rate 1.0 mL/min; temperature 40 ℃, injection of 10 u L. The detector is an ELSD. The normal phase chromatography sample was directly injected and the solid sample was reconstituted in EtOAc prior to injection. Acetone solutions give broad or bimodal peaks.

Bioassay of apoptotic Activity of HPLC fractions: one hundred thousand HeLa cells per well were plated onto 24-well cell culture plates. Cells were cultured in a cell incubator for 24 hours. After 24 hours, the cells were treated with the compound obtained from the purification of BCP 21. Fractions generated by HPLC during purification were dried and resuspended in DMSO. Cells were treated with three different concentrations of the compound in each fraction. Cells were harvested after 24 hours to determine% apoptosis (cell death). Cells were treated with trypan blue to estimate cell death. Less than 10% of cell death is marked as + and 100% is indicated as ++++, with 50 and 75% marked as ++++, respectively.

Example 1.Extracting and separating from stone pine

Separation of BCP-21: extracting spore of Lycopodium clavatum with 95% ethanol. The resulting extracts were combined and stored as a solution in 95% ethanol, or they may be concentrated.

Evaporation and isolation of the extract: the ethanol solution of BCP21 (1L) was evaporated in vacuo at 45 deg.C to give a suspension containing a small amount of precipitate. To the suspension was added 50mL of water and partitioned with 200mL of dichloromethane (MC). The MC layer was separated in a separatory funnel and evaporated to dryness to give 18.9g of an oily residue. The aqueous layer was dried to give 2.84g of a solid. HPLC profiles of the ethanol extract of BCP21 and the MC soluble residue were prepared.

Example 2.40M silica gel Biotage fractionation

Since bioassay showed that the above MC-soluble residue contained the active compound, 5g of this material were dissolved in 15mL of MC and loaded into a 40M silica gel tube which had been equilibrated with 800mL of hexane. The column was eluted with 10% acetone/hexane (A/H) (1L), 20% A/H (1L), 25% A/H (1.2L) and acetone (0.5L). According to the bioassay results, F5 to F8, and F9 to F11 of 25% A/H eluate were combined and dried with a nitrogen stream to obtain 2615-182-8(43.4mg) and 2615-182-9(36.3mg), respectively. Table 1 details the results of the fraction samples indicated by the apoptosis assay.

Table 1 weight of fractions from 40M column and bioassay results.

Example 3.12M silica gel Biotage fractionation:

the combined active product 2615-182-8(43.4mg) was dissolved in 2mL 30% EtOAc/hexane at 50 ℃ and the solution was injected onto a 12M Biotage silica gel tube equilibrated with 100mL 30% EtOAc/hexane. The column was eluted with 60mL of 35% EtOAc/hexane, 100mL of 40% EtOAc/hexane, 60mL of 45% EtOAc/hexane, 60mL of 80% EtOAc/hexane to yield 26 fractions. After drying, 1mL fractions were sent to bioassay. The remaining solution of each fraction was dried to give weight. Table 2 details the results of the fraction samples indicated by the apoptosis assay.

Table 2 weight of fractions from 12M column and bioassay results.

Sample #	Fraction(s) of	Weight (mg)	Biological detection results
				1	35% EtOAc/Hexane F1	2.5	-
2	F2	0.14	-
				3	F3	0	-
4	F4	0	-
				5	40% EtOAc/Hexane F1	0.27	-
6	F2	0.44	++
				7	F3	0.67	++
8	F4	0.71	+++
				9	F5	0.72	+++
10	F6	0.69	+++
				11	F7	0.59	+++
12	F8	0.48	+++
				13	F9	0.85	++
14	F10	0.51	++

15	45% EtOAc/Hexane F1	0.67	+
				16	F2	0.44	-
17	F3	0.63	-
				18	F4	0.82	+
19	F5	0.64	-
				20	F6	0.45	-
21	80% EtOAc/Hexane F1	0.59	-
				22	F2	1.00	+
23	F3	2.72	+++
				24	F4	3.25	+++
25	F5	1.60	+++
				26	F6	1.23	+++

Table 3: BCP-compounds

BCP-compounds	TABLE 2 samples
		1	1
2	3
		3	7
4	13
		5	14
6	18
		7	22
8	-
		9	-
10	-

Example 4.Preparation of test Compound samples for detection

The preparation of the above compound fractions was carried out according to the following table 4:

table 4: compound preparation & dilution

Example 5.Synthesis of racemic 8-Hydroxypalmitic acid ("8-HHA")

Synthesis scheme for Synthesis of racemic 8-Hydroxypalmitic acid

Gas inlet fittings, spacers and stir bars were mounted to two three-necked flasks, and the flasks were then purged with argon. Monomethyl suberate (10.0g, 53.1mmol, 1 eq.) was dissolved in THF (380mL, 0.17M) in a flask. Gradually add threeEthylamine (0.95mL, 64.1mmol, 1.2 eq) (4 min in duration using a gas-tight syringe) and the mixture was stirred at room temperature for 1.25 h. From PPh in anhydrous DCM₃Cl₂(24.4g, 73.2mmol, 1.4 equiv.) A separate solution was prepared which was then cooled to < 30 ℃. The suberic acid solution was added dropwise to the yellow PPh₃Cl₂To the solution and stirred at-35 to-20 ℃ for 2 h. The grignard reagent was then added dropwise over 45 minutes while maintaining the temperature at-41 to-7 ℃. The reaction mixture was stirred for 1.5h, then quenched with 2N HCl (75mL) while still cold. The yellow color faded after stirring for 20 minutes. Thin layer chromatography (silica gel 60, 25% acetone/hexane) showed the reaction mixture to be two major product spots and PPh₃And OPPh₃。

The reaction mixture was added to a separatory funnel along with ethyl acetate (200ml) and 2N HCl (200 ml). The organic layer was collected and the aqueous layer was back-extracted with 2X 80ml of ethyl acetate. By using NaHCO₃(saturated solution, 2X 90mL) wash to neutralize the combined organic phases, then wash with brine (90mL), MgSO₄(20g, 30 min) dried, filtered, rotary evaporated at 25 ℃ and dried under high vacuum for 3 h. To remove ethyl acetate, the material was mixed with 20mL DCM and evaporated again to give 33g of crude material.

The crude material was taken up in 10% acetone/DCM, loaded into a silica gel plug (240ml) and rinsed with 4CV of 10% acetone/DCM. First plug (plug) removal of 10.4g OPPh₃Second removal of 6.8g OPPh₃. Purification was then carried out by flash column chromatography, wherein a 5X 29cm column was packed with silica gel 60 in 10% acetone/hexane. The crude material was charged at a minimum volume of 55% DCM/hexane. The title compound was eluted with 10% DCM/5% acetone/85% hexane. Evaporation and drying of one fraction gave 2.7g of Compound 1 (methyl 8-ketopalmitate), and re-column of several impure fractions gave 2.0g of Compound 1. The total yield was 31% based on suberic acid monomethyl ester. By using¹H NMR(CDCl₃Middle) characterization of compound 1 (8-ketopalmitate methyl ester): 3.65(s, 3H, OCH)₃)，2.37(m，2H，O＝CCH₂)，2.29(t，2H，O＝CCH₂)，157 and 1.28, (br., 22H, CH)₂)，0.87(s，3H，CH₃-terminal).

Compound 1(2.86g, 10.1mmol) was dissolved in 50mL ethanol and cooled in an ice bath. Sodium borohydride (1.9g, 59.5mmol) was added in 1-17C portions. The reaction was monitored by TLC (plates developed in 25% acetone/hexane) where the ketone had an Rf of 0.56 and the methyl ester of 0.28. Solid NaBH was added over 2h₄Until no starting material was detected, another portion (60 eq total) of (c) was used. The reaction was quenched by slowly adding 2N HCl (60ml) to the cold solution, transferred to a separatory funnel, and washed with DCM (3 × 35 ml). With NaHCO₃(saturated, 35ml) and brine (35ml) the combined organic layers were washed and then over MgSO₄(7g, 30 min) dried, filtered and rotary evaporated to give a clear oil which turned into a white waxy solid when stored at 4 ℃ (yield 2.86 g).

The material was purified by flash chromatography (silica gel 60, 20% acetone in heptane). After rotary evaporation to dryness, the compound was an oil and the product was a white waxy solid after high vacuum drying.¹HNMR Spectroscopy (CDCl)₃) The major product was shown to be compound 2, with a small amount of unknown impurities. By using¹H NMR(CDCl₃Middle) characterization of compound 2 (methyl 8-hydroxypalmitate): 3.66(s, 3H, OCH)₃) Br, 1H, methine), 2.29(t, 2H, O ═ CCH₂) 1.62 and 1.31, (br., 28H, CH)₂)，0.87(s，3H，CH₃-terminal).

Compound 2(1.97g, 6.9mmol) was dissolved in THF (28 mL). Lithium hydroxide was dissolved in water (Nanopure, 15mL) and stirred for 5 minutes to dissolve it. The LiOH solution was added slowly to the ester solution over 5 minutes and stirred at room temperature. The reaction was monitored by TLC (20% acetone/hexane) until the product disappeared and a new spot appeared at baseline (4 h). Work up (work up) involves rotary evaporation of the reaction mixture to remove volatiles to give a large amount of white solid. The solid was rinsed with DCM and then mixed with 2N HCl (solid almost insoluble) and chloroform. Aqueous layer with CHCl₃(3×75mL)³And (4) extracting. The combined organic phases were washed with brine and MgSO₄Drying (30 minutes)Then filtered, rotary evaporated and dried under high vacuum overnight to give 1.67g of white solid as final product (89% yield).

Analysis of the final product confirmed that it was 8-hydroxy-palmitic acid as the major product.¹H NMR(CDCl₃The following are added: 5.5 (very broad, 1H, OH) (3.58br, 1H, methine), 2.34(t, 2H, O ═ CCH₂) 1.64 and 1.33, (br., 28H, CH)₂)，0.87(s，3H，CH₃-terminal).¹³C NMR(CDCl₃The following are added: 178.40, 72.08, 37.59, 37.43, 33.79, 31.97, 29.79, 29.68, 29.37, 29.09, 25.73, 25.51, 24.69, 22.76, 14.20. LC-MS (ESI-mode, ACN-NH)₄In OH). Parent ion 271.13m/z, theory: 272 (100%) 273 (17%). Melting point: 70.5 ℃.

Example 6.Synthesis of (S) -and (R) -8-Hydroxypalmitic acid

Formation of R, S or racemic 1, 2-epoxydecane from 1, 2-decanediol

It is known that the reaction of racemic 1, 2-decanediol with p-toluenesulfonyl chloride in the presence of a base yields a mixture of the desired primary tosylate, bismesylate and a small amount of secondary tosylate (Bull. Korean chem. Soc.2009 Vol.30, No.7, 1671-4). The production of any secondary tosylate can adversely affect the stereochemical purity of the resulting epoxide due to the inversion of the stereocenter during the 2-position ring closure.

In this reaction, 23.9ml (0.172mol, 3.0 equivalents) of triethylamine in 10ml of anhydrous dichloromethane was slowly added to a solution of 10.00g (0.0574mol) of 1, 2(S) -decanediol (99.5% "S"), 13.7g (0.0717mol, 1.25 equivalents) of p-toluenesulfonyl chloride, 0.70g (0.00574mol, 0.10 equivalents), and 30ml of anhydrous dichloromethane at < 10 ℃ over a period of 40 minutesAn alkane. After stirring for 20 min at < 10 ℃ according to TLC (100% CH)₂Cl₂) The reaction was complete. After warming to room temperature, the reaction mixture was cooled by adding 200ml MTBE and 120ml 1MHCl partitioned the product mixture into the organic phase. With 1X 80ml of saturated NaHCO₃The organic layer was washed with the solution, 1X 80ml of water and 1X 80ml of saturated NaCl solution. Drying over sodium sulphate and concentration gave 21.7g of residue which was directly subjected to conversion to epoxide. Without cooling at room temperature, the residue was dissolved in 217ml of anhydrous THF and 9.64g (0.086mol, 1.30 eq.) of potassium tert-butoxide were added. After 30 min, the reaction was complete according to TLC (10: 1 heptane/ethyl acetate). The reaction was quenched by the addition of 100ml of water and the THF was removed in vacuo. The product mixture was partitioned into 300ml of MTBE and the aqueous layer was back-extracted with 1X 50ml of MTBE. The combined organics were then washed with 1X 80ml of saturated NaCl solution. Dried over sodium sulfate and concentrated to give 10.4g of residue. The residue was distilled in a short path, and 6.11g of 1, 2(S) -epoxydecane was collected at about 60 ℃ and 1mmg Hg., giving a recovery of 68% and a GC-MS purity of > 99%.

Reaction of alkenylmagnesium bromide with R, S or racemic 1, 2-epoxydecane

Grignard reagents were prepared according to literature procedures (j. org. chem.2009, 74, 5063-za 5066) and added to racemic or chiral 1, -epoxydecane. Preparation at < 10 ℃ of about 0.5MThereby minimizing self-coupling products. Without any titration of the grignard reagent, it was considered to be 0.5M. Without the need to add I₂To activate the magnesium to start the grignard reaction.

To N₂A250 ml dry flask under atmosphere was charged with about 0.5M hept-6-enemagnesium bromide (prepared as above) in 98ml (0.049mol, 1.5 equivalents) THF. The contents of the flask were allowed to cool to-40 ℃ with stirring. The flask was charged with 1.24g (0.00653mol, 0.20 eq.) of cuprous iodide. At-40 deg.CAfter the next 0.5 h, 51ml of an anhydrous THF solution containing 5.10g (0.0326mol) of 1, 2(S) -epoxydecane was added slowly over a period of at least 1h at-40. + -. 5 ℃. After the addition was complete, stirring was continued at-40 ℃ for 2 hours, after which the reaction was generally complete according to TLC (40% EtOAc in heptane). After completion, 500ml of MTBE, 125ml of saturated ammonium chloride and 50ml of water were added and mixed well for 15 minutes. The layers are separated and the organic phase is washed with 100ml of a mixture of 1 part saturated ammonium chloride and 1 part water. The organic phase is then washed with 3X 100ml of water, 1X 50ml of saturated NaCl solution, dried over sodium sulfate, filtered and concentrated to an oil which solidifies on standing (8.6g of product). The product was chromatographed on 220g silica gel eluting first with 500ml heptane to remove any non-polar impurities and then with 10: 1 (V: V) heptane/ethyl acetate. The product containing fractions were combined and concentrated to give 8.11g (97.6% yield) of 9(S) -hydroxyheptadec-1-ene as a waxy solid with a purity of 96.7% according to GC-MS.

Protection of the alcohol is achieved by adding 1.50 equivalents of tert-butyldimethylchlorosilane to a THF solution of the enol, with 2.0 equivalents of imidazole as the base. Typically, the reaction is 92-96% complete after 16-20 hours at room temperature. Additional TBS chloride and imidazole did not drive the reaction to completion. Acidic water treatment and partitioning into heptane enabled removal of imidazole without decomposition of acid sensitive TBS protected alcohol. Residual unreacted alcohol was easily removed by flash chromatography on silica gel with heptane and the intermediates were isolated in high purity (> 99% according to GC-MS) (NB 1362-42, 45, 86; 1341-89; 1380-15).

ozonolysis/Pinnick oxidation route to TBS protected acids

A solution of 9.83g (0.0267mol)9(S) - (tert-butyldimethylsilyloxy) -heptadeca-1-ene, 0.10g (0.00027mol, 0.01 equiv.) Sudan Red 7B in 138ml dichloromethane and 138ml methanol was treated with ozone at-25. + -. 5 ℃. Ozone is added for 6-7 minutes after the indicator color fades. Then the solution is treated with N₂Purge for at least 30 minutes. A solution of 13.99g (0.0533mol, 2.00 eq.) of triphenylphosphine was then added over 4-5 minutes at < -15 deg.C and then allowed to warm to room temperature. After stirring at room temperature for 30 minutes, the reaction was checked by GC-MS and checked again at 1.25 hours and 3 hours. All three tests typically showed the same levels of triphenylphosphine, triphenylphosphine oxide and product, indicating that the ozonide had been consumed. The reaction mixture was then concentrated to a pasty solid and concentrated once more from 100ml dichloromethane (to remove methanol). The residue is then chromatographed on 300g of silica gel, eluting with heptane followed by 20: 1 heptane/acetoacetate. The product containing fractions were combined and concentrated to give 13g of an oil which still contained 31% triphenyl phosphine by GC-MS. The oil was chromatographed again on 500g of silica gel, at which point the triphenylphosphine level was 13%. This oil was dissolved in 100ml dichloromethane. 0.46ml (0.0074ml) of iodomethane were added and the mixture was stirred overnight, after which all the triphenylphosphine had been consumed (monitored by GC-MS). Finally, the product was flash chromatographed on 200g silica gel, eluting with 10: 1 heptane/ethyl acetate. After concentration, 8.00g (89% yield) of 8(S) - (tert-butyldimethylsilyloxy) hexadecal with a GC-MS purity of 98.7% was obtained.

To 315ml of a solution of 3.79g (0.0102mol) of 8(S) - (tert-butyldimethylsilyloxy) hexadecanal in 315ml of tert-butanol at room temperature were added 125ml of an aqueous solution of 8.49g (0.0939mol, 9.17 equivalents) of sodium chlorite and 8.50g (0.0708mol) of sodium dihydrogen phosphate. The mild exotherm was controlled by a water bath. After stirring overnight, the reaction was complete according to TLC (1: 0.01 heptane: EtOAc: HOAc). The tert-butanol was removed in vacuo. The residue was dissolved in 200ml of water and 300ml of heptane, and 80ml of 1.0 was addedMHCl. The heptane layer obtained was washed with 3X 100ml of water, 1X 75ml of saturated sodium chloride solution, dried over sodium sulfate, filtered and concentrated to give 3.79g (95.7% yield) of 8(S) - (tert-butyldimethylsilyloxy) hexadecanoic acid,according to GC-MS (as from TMSCHN₂Methyl ester of (d) purity was 99.4%.

Removal of TBS protecting group and purification of 8(S) -hydroxypalmitic acid

3.79g (0.00980mol) of 8(S) - (tert-butyldimethylsilyloxy) hexadecanoic acid, 48ml of ACN and 2.13ml (0.059mol, 6 equivalents) of 48% hydrofluoric acid were mixed and stirred for 1 hour. After 20-30 minutes, the oily mixture becomes a suspension. After 1 hour, the starting material was completely consumed (monitored by TLC (1: 0.01 heptane: EtOAc: HOAc)). After the reaction was complete, 300ml MTBE and 300ml water were added and mixed well. The aqueous layer was discarded and the organic layer was washed with 3X 100ml of water, 1X 100ml of saturated sodium chloride, dried over sodium sulfate, filtered and concentrated to give 2.50g of a solid. The solid was then 95.6% ee (NB 1380-51). The solid was dissolved in 40ml hot ACN and stirred until cooled. The resulting suspension was cooled for 30 min to < 10 ℃, collected by filtration, washed with 20ml cold ACN and dried under vacuum at 40 ℃ to give 2.18g (82% yield) of 8(S) -hydroxypalmitic acid which was > 99% according to GC-MS (as from TMSCHN)₂Methyl ester of (4), 98.4% ee according to HPLC-MS.

Transformation of Mitsunobu to give the 8(R) enantiomer

To convert the stereocenter of an 8-hydroxy acid under Mitsunobu conditions, the carboxylic acid is protected as its methyl ester. Initially at small scale, diazomethane was used as a fast and efficient conversion process. TMS diazomethane is used on a large scale because of its hazardous, safer alternative. Complete esterification of this acid in MTBE/methanol requires 2.6 equivalents of TMS diazomethane. Attempts to convert stereocenters with acetic acid under Mitsunobu conditions are not ideal. Substitution of 4-nitrobenzoic acid (Organic Synthesis, Collective Volume 9, page 607) under the same conditions provided cleanly 8(R) -4-nitrobenzoate. Saponification with LiOH in THF/water followed by recrystallization from ACN gave the desired 8(R) -hydroxypalmitic acid in 100% ee according to HPLC-MS.

Example 7:general assay for determining HeLa cell viability by Alamar blue assay. Assessment of cell viability by alamar blue reduction method is routinely used to elucidate the cytotoxic potential of drug candidates. Alamar blue was used to measure cell viability by using the blue non-fluorescent dye Resazurin, which is converted by cellular metabolic intermediates to the pink fluorescent dye, resorufin (resorufin). The fluorescent signal generated in the assay is proportional to the number of viable cells in the sample. Confluent HeLa cells (70-80%) grown on T-75 flasks were detached with trypsin-EDTA. After centrifugation, the cell pellet was resuspended in DMEM medium and counted with a hemocytometer. Cells were seeded at a density of 10,000 cells/100. mu.l/well in 96-well plates and at 37 ℃/5% CO₂And (5) culturing for 8 hours. The medium was discarded and a volume of 100. mu.l of test compound was added. Plates were incubated for 22, 46 and 70 hours, after which 10. mu.l of alamar blue was added per well. After 24, 48 and 72 hours treatment with test compounds, fluorescence was measured with a microplate reader (micro plate reader) at an excitation wavelength of 530nm and an emission wavelength of 590 nm. Survival as% control was plotted against drug concentration. Terfenadine (Terfenadine) was used as a reference compound in the experiment and showed dose-dependent inhibition and reproducible IC at all time points (24, 48 and 72 hours)₅₀The value is obtained. Different compounds showed different survival rates.

Example 8:example 7 analysis of the data detected. The average of duplicate wells was calculated. Viability of control cells was 100% for all concentrations tested. The ratio of cell viability for the test samples was calculated as:

IC was determined by non-linear regression analysis (curve fitting) of percent survival data versus drug concentration using the sigmoidal dose-response (variate) equation (Graph Pad Prism 4 software)₅₀The value is obtained. BCP-6 and BCP-7 show specific effects on cells.

Quality control: the following quality tests for this test were evaluated:

i: IC of reference Compound₅₀The value: IC of terfenadine₅₀Value of

ii: coefficient of variance between replicates%: the% CV between replicates was within acceptable limits (10%).

Example 9:preparation of test compound samples. The preparation of the above compound fractions was carried out according to the contents described in Table 5.

Table 5: compound preparation & dilution

Example 10:general assay for detecting caspase-3 Activity on HepG2 cells

Cells suspected of or having been induced to undergo apoptosis are first lysed to collect their intracellular material. In our test system, staurosporine (staurosporin) -treated cell lysates were tested for protease activity by adding a caspase-specific peptide that binds to the fluorescent reporter molecule 7-ammoniaOn the radical-4-methylcoumarin (AMC). Caspase decomposes the polypeptide releasing fluorescent material which emits 460nm fluorescence when excited by 380nm wavelength. The level of caspase activity in the cell lysate is directly proportional to the fluorescent signal detected with a fluorescent microplate reader. Fused HepG2 cells (70-80%) grown in T-75 flasks were detached with trypsin-EDTA. After centrifugation, the cell pellet was resuspended in DMEM medium and counted with a hemocytometer. Cells were seeded at a density of 40,000 cells/100. mu.l/well in 96-well plates and at 37 ℃/5% CO₂And then the mixture is incubated for 8 hours. The medium was discarded and a volume of 100. mu.l of test compound was added. After 30 minutes of pretreatment as above, 50. mu.l of 3 Xconcentration staurosporine (20. mu.M final concentration) or 50. mu.l of medium (untreated control) were added to each well. After 16h induction with staurosporine, cells were lysed by adding 50. mu.l lysis buffer followed by incubation for 30 min. After complete lysis was observed under the microscope, 100. mu.l of ice-cold buffer (detection buffer) was added to each well. DEVD-AMC (substrate, final concentration 15uM) was then added and the reaction was allowed to continue for 2 hours. Fluorescence was detected by excitation at 380nm with a BMG Polartar luciferase reader and capture of luminescence at 460 nm.

Example 11:example 10 analysis of the data detected. Duplicate relative fluorescence values (RFU) were averaged for each standard, blank and sample. The enzyme activity of the control (staurosporine, not treated with inhibitor) was taken as 100% activity (0% inhibition). The fluorescence value (RFU) of the test/reference compounds was compared to calculate% inhibition. The percent inhibition of the test/reference compounds was calculated according to the following:

% inhibition of 100 [% activity

I was determined by non-linear regression analysis (curve fitting) of percent survival data versus drug concentration using the sigmoidal dose-response (variate) equation (Graph Pad Prism 4 software)C₅₀The value is obtained. BCP-6 and BCP-7 show a particular effect on caspase-3 inhibition.

Quality control: the following quality tests for this test were evaluated:

i: IC of reference Compound₅₀The value: IC of Ac-DEVD-CHO₅₀Value of

ii: coefficient of variance% between replicate experiments: the% CV between replicates was within acceptable limits (10%).

Example 12:lycopodium clavatum extract induces morphological characteristics consistent with apoptosis

HeLa cells (cervical cancer) obtained from American type tissue Culture Collection (ATCC) were treated with an extract of Lycopodium clavatum for 48 hours and stained with an anti-tubulin antibody to examine the cytoskeleton, and the nucleus was stained with 4', 6-diamidino-2-phenylindole, which is a fluorescent dye closely bound to DNA (DAPI). Cells treated with vehicle show intact cytoplasmic structures and nuclei. The loss of DNA appeared to be an empty nucleus, thus demonstrating that the cells treated with the extract lost their cytoplasmic structure (round cells) and lost nuclear DNA in the cells completely. These morphological changes imply that BCP-21 induces apoptosis in cervical cancer cell lines.

Example 13:lycopodium clavatum extract induces the sub-G1 family (sub-G1 popularization)

Since the extract of lycopodium clavatum induces apoptosis-related changes, apoptosis was assessed by flow cytometry to measure DNA content. Apoptotic cells lose DNA by rupture, so the DNA content is less than 2 n. In flow cytometry detection, the cell appeared to be to the left of the G1 peak, and was therefore referred to as the "sub-G1 peak". HeLa cells treated with control and different dilutions (1: 500) and 1: 250 of the extract of Lycopodium clavatum were fixed in absolute ethanol and stained with propidium iodide and RNAse for 30 min. The cells were analyzed in the core laboratory of the Dana Farber cancer institute, Harvard medical institute (Boston, MA). The DNA content histograms of the cells were deconvoluted with the MultiCycle software from Phoenix Flow Systems (San Diego, Calif.) to obtain a quantitative result of the percentage of cells in sub-G1 phase.

The crude extract of lycopodium clavatum increased the sub-G1 peak (23%) in a dose-dependent manner compared to the control (6.7%) (fig. 6). This data strongly suggests that extracts induce HeLa apoptosis in a dose-dependent manner.

Example 14:extract of Lycopodium clavatum induces cleavage of PARP

Cells were lysed with a holoprotease inhibitor (Roche) in 50mM Tris-HCl, pH7.6, 150mM NaCl, 30mM EDTA, 0.5% Triton X-100. Protein samples were separated on gels of appropriate proportions by SDS-PAGE. In general, 20-60. mu.g of protein was analyzed for endogenous protein and 10-15. mu.g for transfected protein. The proteins were transferred to 0.2 μm nitrocellulose membranes (Bio-Rad) for 1 hour at 4 ℃. Membranes were blocked for 45 minutes at Room Temperature (RT) in PBS solution containing 5% milk and 0.1% Tween-20(American Bioanalytical). The nitrocellulose membrane was then incubated in primary antibody diluted at appropriate concentration in PBS containing 1% milk and 0.1% Tween-20 for 2h at room temperature or overnight at 4 ℃. After removal of the primary antibody, the membranes were washed 3 times for 10 minutes in wash buffer (1 XPBS with 1% milk and 0.1% Tween-20) at room temperature. The membrane was then incubated for 1h at room temperature in the appropriate secondary antibody diluted in wash buffer. The membrane was then washed 3 times for 10 minutes. Immunoblotting was performed with ECL (Pierce laboratories) according to the manufacturer's instructions.

The results show that full length PARP (mol.wt.116kDa) is completely cleaved after 24 hours. HeLa cells treated with the extract showed an increase in PARP lysis in a time-dependent manner (fig. 7). This data strongly suggests that the extract of lycopodium clavatum induces apoptosis.

Example 15:screening of racemic mixtures of 8-HHA against human tumor cell lines

The National Cancer Institute (NIH) Cancer Diagnosis and Treatment department (the Division of Cancer Diagnosis and Treatment) received a racemic mixture of synthetic 8-HHA dissolved in DMSO for screening against a panel of 60 human tumor cell lines (panel). This screen is a microplate cytotoxicity assay based on a simple colorimetric (MTT) assay, which relies on the metabolic reduction of tetrazolium dye to a colored formazan (formazan) product in living cells. The improved protocol was used to screen compounds and determine inhibition of growth of various tumor cell lines. The concentration of 8-HHA tested in this assay was 10. mu.M.

The results show that 8-HHA significantly inhibited the growth of leukemia cell lines (e.g., CCRF-CEM, K-562, SR, MOLT-4, RPMI-8226, and HL-60(TB)) in the range of 40-96%. It also showed significant growth inhibition on non-small cell lung cancer cell lines (e.g., HOP-92 and NCI-H460). Likewise, 3 of the 7 colorectal cancer cell lines screened (i.e., HCT-116, HCT-15, and KM12) showed modest evidence of growth inhibition. This data indicates that 8-HHA has growth inhibitory activity against a variety of cancer cell lines, such as leukemia, lung cancer and colorectal cancer cell lines, albeit to varying degrees.

Example 16:effect of 8-HHA on cancer cell line survival

1) For lung cancer cell lines: the effect of racemic mixtures and single enantiomers of 8-HHA, i.e., S and R enantiomers, on the survival of HOP-92 lung cancer cell lines was examined. Cell viability was measured during exponential growth phase using a colorimetric MTT assay with 100. mu.M 8-HHA treatment for 72 hours. In a separate assay, HOP-92 cells were treated with various concentrations of the S-enantiomer of 8-HHA ranging from 20-100. mu.M for 72 hours.

The results show that the racemic mixture of 8-HHA reduced survival of HOP92 cell line by 25%. The R-enantiomer reduced the survival of HOP92 cell line by 13%. In contrast, the S-enantiomer had the greatest effect (72% reduction) on the survival of lung cancer cell lines. This data indicates that the S enantiomer of 8-HHA has a potential inhibitory effect on the survival of HOP92 lung cancer cells. Furthermore, FIG. 8 shows that the enrichment of (S) -8-HHA is increasedThe degree inhibited survival of lung cancer cell lines in a dose-dependent manner. A50% reduction in cell survival (IC) was observed at a dose of about 90. mu.M of the S-enantiomer of 8-HHA₅₀). This data strongly suggests that the S-enantiomer of 8-HHA has a dose-dependent inhibitory effect on lung cancer cell lines.

2) For liver cancer cell lines: the effect of racemic mixtures and single enantiomers of 8-HHA, i.e., the S and R enantiomers, on the survival of PLC/PRF/5 (also known as "PLC-5" or "PLC 5"), Hep3B, and SNU449 (also known as "SNU") liver cancer cell lines was examined. Cell viability was measured during exponential growth phase using a colorimetric MTT assay with 100. mu.M 8-HHA treatment for 72 hours. In a separate assay, PLC-5 cells were treated with different concentrations of the S-enantiomer of 8-HHA ranging from 20-100. mu.M for 72 hours.

The results (FIG. 9) show that the racemic mixture of 8-HHA reduced the survival of PLC-5 and Hep3B cell lines by 52-25%, respectively. It has no major effect on SNU cell lines. On the other hand, the S-enantiomer of 8-HHA had the greatest effect on survival of all liver cancer cell lines (almost 75% decrease in cell survival) relative to the racemic mixture or the R-enantiomer. This data indicates that the S-enantiomer of 8-HHA has potential inhibitory effects on all liver cancer cell lines.

Furthermore, FIG. 10 demonstrates that increasing the concentration of (S) -8-HHA inhibits survival of the PLC-5 liver cancer cell line in a dose-dependent manner. A50% reduction in cell survival (IC) was observed at a dose of about 50. mu.M of the S-enantiomer of 8-HHA₅₀). This data strongly suggests that the S-enantiomer of 8-HHA has a dose-dependent inhibitory effect on lung cancer cell lines.

Example 17:8-HHA Induction of apoptosis in liver and Lung cancer cell lines

PARP cleavage is considered a specific and generally accepted marker of apoptosis. During apoptosis, FAS receptors, which are considered to be recognized indicators of apoptosis, undergo down-regulation. Pathways stimulated by binding of the FAS receptor to the FAS ligand induce apoptosis, a mechanism referred to as exogenous. Apoptosis initiated by this pathway results in a decrease in FAS receptors. Thus, a decrease in protein levels of the FAS receptor indicates that apoptosis is induced by exogenous mechanisms.

1) For liver cancer cell lines: the effect of 8-HHA on apoptosis in liver cancer cell lines was examined. FIG. 11 shows that S-enantiomeric treatment of 100. mu.M 8-HHA results in cleavage of PARP in SNU and PLC-5 liver cancer cell lines. This data indicates that 8-HHA induces apoptosis in liver cancer cell lines.

PLC-5 cell lines were treated with 100. mu.M 8-HHA for 72 hours. Treatment with (S) -8-HHA resulted in a substantial reduction in FAS receptor levels compared to the control (FIG. 12). This data indicates that (S) -8-HHA induces apoptosis in liver cancer cell lines through an exogenous pathway.

2) For lung cancer cell lines: HOP-92 lung cancer cell line was treated with 100. mu.M (S) -8-HHA for 72 hours. (S) -8-HHA treatment resulted in a reduction in FAS protein levels in the HOP92 cell line. This data indicates that 8-HHA induces apoptosis in the HOP92 cell line by an exogenous mechanism.

Example 18:effect of 8-HHA treatment on Pre-apoptotic (pro-apoptotic) Bim1 in Lung cancer cell lines

Bim1 is a pro-apoptotic protein, whose upregulation leads to apoptosis. HOP92 cell line was treated with 100. mu.M of the S-enantiomer of 8-HHA for 72 hours. The results show a 2-fold increase in Bim1 in the 8-HHA treated samples compared to the control samples. This data indicates that 8-HHA induces apoptosis in lung cancer cell lines.

Example 19:determination of the Maximum Tolerated Dose (MTD) of 8-HHA in an in vivo animal model

The objective of this study was to determine the maximum tolerated dose of 8-HHA following Intravenous (IV) administration in ICR mice. 20 female ICR mice, 25-30 g in weight, were administered the racemic mixture of 8-HHA, placebo and vehicle, respectively. 8-HHA was formulated as nanolipoids at concentrations of 0, 10, 20 and 40 mg/ml. 4 animals per group were injected with 8-HHA at doses of 0, 10, 20 and 40 mg/kg. Mice were given a single dose (single bolus) by tail vein according to group. Mice were sacrificed one week after dosing. Daily clinical observations were made 15 minutes and 1 hour after dosing. Mice were observed daily for any adverse reactions until necropsy on study day 8. Body weights of all mice were recorded every other day before and during the test substance. The entire study was performed in Toxikon Corporation, Bedford, Mass. The results are described in table 6.

TABLE 6

Results and analysis: the body weights on day 6 and day 8 were not different from the weights before administration. The body weights on day 6 and day 8 were not different from the weights before administration. Two mice dosed with the high dose (40mg/kg) died soon after dosing. Animals taking placebo, 10mg/kg and 20mg/kg of 8-HHA were well tolerated for 8HHA and did not show any observed clinical abnormalities. The 20mg/kg dose is then considered to be the maximum tolerated dose of 8-HHA.

Example 20:xenogeneic liver transplantation mouse model

The objective of this study was to determine the antitumor efficacy of 8-HHA in liver cancer using the hepatocellular carcinoma cell line PLC 5. Tumor growth and animal survival were considered as the primary endpoints.

Animals and care:30 non-pregnant and non-fertile BABL/c female nude mice 5-6 weeks old and weighing 16-20 grams were used in this study. BALB/c nu/nu mice were used because they were used in xenograft studies to test the antitumor efficacy of drug candidates. Mice were allowed to acclimate for a minimum of 5 days under the same conditions as the actual experiment. Mice were housed at room temperature 68 + -5 ℉ with 30-70% room relative humidity, an hourly gas exchange rate of a minimum of 10 exchanges per hour, 12 hours light/dark cycle illumination, full spectrum fluorescence. Mice were group housed in ventilated mini-isolation cages made of polycarbonate. Mice were padded with autoclaved laboratory grade padding and were supplied with irradiated pellets and autoclaved water ad libitum. A feed,There were no known contaminants in the water or bedding that would be expected to interfere with the experimental data. The laboratory and animal rooms are maintained as restricted access zones.

Route of injection and dosage: 8-HHA was administered by Intraperitoneal (IP) injection. 8-HHA is poorly soluble in water and is therefore suspended in a Nanolipid dispersion (ePhase, Basel, Switerzland) at a concentration of 27.2mg/ml, corresponding to 100 mM. The MTD assay (preliminary data) showed that the mice tolerated the maximum dose of 20 mg/kg. Two doses were used in this study: 10mg/kg and 100 mg/kg.

Animal preparation

Tumor induction: cell line PLC5 was cultured according to the recommended protocol in Vanas Oncology, where cells were cultured and stored in RPMI medium containing 10% fetal bovine serum, 2mM L-glutamine, 100 units/ml streptomycin and 100 units/ml penicillin. Cells were trypsinized and counted using a hemocytometer using the trypan blue viability assay. The cell counts expressed in quadrants of the hemacytometer were converted to cell/mL values, which would enable the isolation of a reasonable number of cells per mouse. Each mouse was inoculated subcutaneously with 0.2mL of a tumor-containing cell (5X 10)⁶Cell/mouse) suspension of 50% RPMI/50% Matrigel^TMAnd (3) mixing. Tumors were observed twice weekly until tumor formation. Tumor volume was calculated using the following formula: tumor weight (mg) ═ a × b²And/2) wherein 'b' is the smallest diameter and 'a' is the largest diameter, expressed in millimeters.

Animal distribution: once the tumors formed reached an average calculated volume of about 50-100mg, the mice were randomized into treatment groups using appropriate software to reduce the degree of difference in tumor size among the groups.

The steps before administration are as follows: animals acclimatized to the environment were weighed prior to the first day of administration and observed for clinical signs of toxicity.

Administration: two doses were administered according to MTD testing guidelines: 1 and 10 mg/kg. Day 1 was the first Day of administration. On the first day, 8-HHA and vehicle injections were administered according to the study design of Table 7 below.

Table 7: design of research

The post-administration step: after treatment, measurements of tumor weight and mouse body weight were recorded twice weekly, at least once daily visually. Mice with untouched tumors were considered to be fully recovered. The proportion of mice that died and the time to death were recorded for each group in the study. Animals may be defined as dying or dead, if they meet one or more of the following criteria.

● 1 weight loss was 20% or more over the week.

● mice with impaired normal physiological functions such as diet, drinking, mobility, and ability to urinate and/or defecate.

● calipers measure tumors in excess of 2000mg in their largest size.

● ulcerated tumors, or bleeding or secretion producing tumors.

● Long-term, excessive diarrhea leads to excessive weight loss (. gtoreq.20%).

● persistent wheezing and respiratory distress.

● chronic or excessive pain or distress as defined by clinical observations, such as: collapse, humpback posture, paralysis/paresis, swollen abdomen, ulcers, abscesses, epilepsy, and/or bleeding.

After the dosing was completed, the mice were observed for two more weeks to check for tumor regeneration.

And (3) killing: all animals will be sacrificed after the study is completed and the tumors will be collected. One part of the tumor will be frozen in OCT solution and the other part will be fixed in formalin. The frozen and fixed tissue will be sent to the originator. All animal tests were performed in Toxikon Corporation (Bedford, MA).

As a result: on the first day of 8-HHA treatment, mice were randomized into three groups of 10 mice each: control group with vehicle only (C1), 8-HHA 10mg/Kg body weight (T1) and 8-HHA 100mg/Kg body weight (T2). Both the T1 and T2 groups significantly inhibited mouse tumor growth from day 4 of 8-HHA treatment. In contrast, in the 8-HHA treated group (T1), tumor volume was reduced by 44.5%, 49.8%, 72.3%, 77.7% and 78.4% compared to the control group (on days 4, 9, 11, 16 and 18, respectively). In the T2 group, tumor volume was reduced by 56.3%, 61.1%, 81.1%, 87.9% and 88.3% (on days 4, 9, 11, 16 and 18, respectively) (see fig. 13 and table 8).

These results show that the two concentrations of 8-HHA used in this study reduced tumor growth in a mouse model of liver cancer by anti-proliferative and pro-apoptotic effects, as demonstrated by our in vitro experiments with PLC-5 cells. 8-HHA can thus be proven to be a very effective, safe and inexpensive adjunct (adjunct) to standard chemotherapy.

(^*S: significant compared with the control group; n as 10/group)

Mean + -SEM tumor volume (PLC-5)

TABLE 8

Example 21:mouse model for small cell lung cancer xenograft

The objective of this study was to determine the antitumor efficacy of 8-HHA in lung cancer using HOP92 cells.

Animals and care:30 non-pregnant and non-fertile BABL/c female nude mice 5-6 weeks old and weighing 16-20 grams were used in this study. BALB/c nu/nu mice were used because they have been used in xenograft studies to test candidate drugs againstTumor efficacy. Mice were allowed to acclimate for a minimum of 5 days under the same conditions as the actual experiment. Mice were housed at room temperature 68 + -5 ℉ with 30-70% room relative humidity, an hourly gas exchange rate of a minimum of 10 exchanges per hour, 12 hours light/dark cycle illumination, full spectrum fluorescence. Mice were group housed in ventilated mini-isolation cages made of polycarbonate. Mice were padded with autoclaved laboratory grade padding and were supplied with irradiated pellets and autoclaved water ad libitum. There were no known contaminants in the feed, water or bedding that would be expected to interfere with the experimental data. The laboratory and animal rooms are maintained as restricted access zones.

Route of injection and dosage: 8-HHA was administered by Intraperitoneal (IP) injection. 8-HHA is poorly soluble in water and is therefore suspended in a Nanolipid dispersion (ePhase, Basel, Switerzland) at a concentration of 27.2mg/ml, corresponding to 100 mM. The MTD assay (preliminary data) showed that the mice tolerated the maximum dose of 20 mg/kg.

Design of experiments

Animal preparation

Tumor induction: as discussed in example 20 above, cell line HOP92 was cultured according to the recommended specifications in Vanas aerology. Cells were trypsinized and counted using a hemocytometer using the trypan blue viability assay. The cell counts expressed in quadrants of the hemacytometer were converted to cell/mL values, which would enable the isolation of a reasonable number of cells per mouse. Each mouse was inoculated subcutaneously with 0.2mL of a tumor-containing cell (5X 10)⁶Cell/mouse) suspension of 50% RPMI/50% Matrigel^TMAnd (3) mixing. Tumors were observed twice weekly until tumor formation. Tumor volume was calculated using the following formula: tumor weight (mg) ═ a × b²And/2) wherein 'b' is the smallest diameter and 'a' is the largest diameter, expressed in millimeters.

Animal distribution: once the tumors formed reached an average calculated volume of about 50-100mg, the mice were randomized into treatment groups using appropriate software to reduce the degree of difference in tumor size among the groups.

The steps before administration are as follows: animals acclimatized to the environment were weighed prior to the first day of administration and observed for clinical signs of toxicity.

Administration: two doses were administered: 1 and 10 mg/kg. Day 1 was the first Day of administration. On the first day, 8-HHA and vehicle injections were administered according to the study design of Table 9 below.

Table 9: design of research

The post-administration step: after treatment, measurements of tumor weight and mouse body weight were recorded twice weekly, at least once daily visually. Mice with untouched tumors were considered to be fully recovered. The proportion of mice that died and the time to death were recorded for each group in the study. Animals may be defined as dying or dead, if they meet one or more of the following criteria.

● 1 weight loss was 20% or more over the week.

● mice with impaired normal physiological functions such as diet, drinking, mobility, and ability to urinate and/or defecate.

● calipers measure tumors in excess of 2000mg in their largest size.

● ulcerated tumors, or bleeding or secretion producing tumors.

● Long-term, excessive diarrhea leads to excessive weight loss (. gtoreq.20%).

● persistent wheezing and respiratory distress.

● chronic or excessive pain or distress as defined by clinical observations, such as: collapse, humpback posture, paralysis/paresis, swollen abdomen, ulcers, abscesses, epilepsy, and/or bleeding.

After the dosing was completed, the mice were observed for two more weeks to check for tumor regeneration.

And (3) killing: all animals will be sacrificed after the study is completed and the tumors will be collected. One part of the tumor will be frozen in OCT solution and the other part will be fixed in formalin. The frozen and fixed tissue will be sent to the originator.

As a result: on the first day of 8-HHA treatment, mice were randomized into three groups of 10 mice each: control group with vehicle only (C1), 8-HHA 10mg/Kg body weight (T1) and 8-HHA 100mg/Kg body weight (T2). Both the T1 and T2 groups significantly inhibited mouse tumor growth from day 16 of 8-HHA treatment. In the control group, the tumor volumes at days 16 and 18 were 214.6. + -. 38.65 and 258.4. + -. 64.96mm, respectively³. In contrast, in the 8-HHA-treated group (T1), the tumor volumes on days 16 and 18 were 88.78. + -. 28.61 and 82.02. + -. 25.93mm, respectively³Corresponding to a tumor reduction of 58.6% and 68.2%, 32.63mm on days 16 and 18, respectively³Corresponding to a 56.5% and 73.1% reduction in tumor volume (see table 10 and figure 14).

Watch 10

^*S: is significant compared with a control group

Mean ± SEM tumor volume (HOP-92) (N ═ 10/group)

In summary, the two concentrations of 8-HHA used in this study reduced tumor growth in a mouse model of lung cancer by anti-proliferative and pro-apoptotic effects as demonstrated by our in vitro experiments with HOP92 cells. 8-HHA can thus be proven to be a very effective, safe and inexpensive adjunct to standard chemotherapy.

Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and modifications of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the present invention.

Claims (34)

1. Use of a composition consisting of a therapeutically effective amount of 8-hydroxyhexadecanoic acid, or an enantiomer, a pharmaceutically acceptable salt, or a polymorph thereof, and a pharmaceutically acceptable carrier in the manufacture of a medicament for the treatment of cancer.

2. The use as claimed in claim 1, wherein the 8-hydroxyhexadecanoic acid is isolated from an extract of the plant lycopodium clavatum.

3. The use as claimed in claim 1, wherein the 8-hydroxyhexadecanoic acid is synthetically prepared.

4. The use as claimed in claim 1, wherein the 8-hydroxyhexadecanoic acid is the S-enantiomer of 8-hydroxyhexadecanoic acid.

5. The use of claim 1, wherein the cancer is a caspase-3 mediated cancer.

6. The use of claim 1, wherein the cancer is hepatocellular carcinoma.

7. The use of claim 1, wherein the cancer is liver cancer.

8. The use of claim 1, wherein the cancer is lung cancer.

9. The use of claim 8, wherein the lung cancer is non-small cell lung cancer.

10. The use of claim 8, wherein the lung cancer is associated with HOP-92 cancer cells.

11. The use of claim 1, wherein the cancer is leukemia.

12. A kit comprising 8-hydroxyhexadecanoic acid present in an extract of lycopodium clavatum.

13. The kit of claim 12, further comprising instructions for administering 8-hydroxyhexadecanoic acid to a subject identified as in need of cancer treatment.

14. The kit of claim 12, further comprising instructions for administering an additional anti-cancer agent.

15. A composition for treating cancer in a subject, consisting of 8-hydroxyhexadecanoic acid and a pharmaceutically acceptable carrier.

Use of 8-hydroxyhexadecanoic acid in the manufacture of a medicament useful in the treatment of cancer.

17. The composition of claim 15, wherein the 8-hydroxyhexadecanoic acid is synthetic 8-hydroxyhexadecanoic acid.

18. The composition of claim 15, wherein the 8-hydroxyhexadecanoic acid is the S-enantiomer of 8-hydroxypalmitic acid.

19. The composition of claim 15, wherein the cancer is a caspase-3 mediated cancer.

20. The composition of claim 15, wherein the cancer is hepatocellular carcinoma.

21. The composition of claim 15, wherein the cancer is liver cancer.

22. The composition of claim 15, wherein the cancer is lung cancer.

23. The composition of claim 22, wherein the lung cancer is non-small cell lung cancer.

24. The composition of claim 22, wherein the lung cancer is associated with HOP-92 cancer cells.

25. The composition of claim 15, wherein the cancer is leukemia.

Use of the S-enantiomer of 8-hydroxypalmitic acid in the manufacture of a medicament useful in the treatment of cancer.

27. The use of claim 16, wherein the cancer is a caspase-3 mediated cancer.

28. The use of claim 16, wherein the cancer is hepatocellular carcinoma.

29. The use of claim 16, wherein the cancer is liver cancer.

30. The use of claim 16, wherein the cancer is lung cancer.

31. The use of claim 30, wherein the lung cancer is non-small cell lung cancer.

32. The use of claim 30, wherein the lung cancer is associated with HOP-92 cancer cells.

33. The use of claim 16, wherein the cancer is leukemia.

Use of 8-hydroxyhexadecanoic acid, or an enantiomer, a pharmaceutically acceptable salt, or a polymorph thereof, in the manufacture of a medicament for the treatment of cancer.