MX2007011132A - Medical uses of 39-desmethoxyrapamycin and analogues thereof. - Google Patents

Abstract

The present invention relates to medical uses of 39-desmethoxyrapamycin analogues.

Description

USE OF A COMPOUND BACKGROUND OF THE INVENTION Rapamycin (sirolimus) (formula A) is a lipophilic macrolide produced by Streptomyces hygroscopicus NRRL 5491 (Sehgal et al., 1975, Vecina et al., 1975; US 3,929,992; US 3,993,749), with a 1, 2,3-tricarbonyl bound portion to a pipecolic acid lactone (Paiva et al., 1991). For the purposes of this invention, rapamycin is described by the numbering convention of McAlpine et al., (1991), preferably with the numbering conventions of Findlay et al. (1980) or Chemical Abstracts (11th cumulative index, 1982- 1986, p60719CS).

Formula A Rapamycin has a significant therapeutic value due to its broad spectrum of biological activities (Huang et al., 2003). He compound is a potent inhibitor of the mammalian rapamycin target (mTOR), a serine-threonine kinase at the end of the signaling pathway of phosphatidylinositol 3-kinase (PI3K) / Akt (protein B kinase), which mediates survival and the proliferation of cells. This inhibitory activity is obtained after rapamycin binds to immunophilin binding protein 12 FK506 (FKBP12) (Dumont, F. J. and Q. X. Su, 1995). In T cells, rapamycin inhibits IL-2 receptor signaling and subsequent selfproliferation of T cells, resulting in immunosuppression. Rapamycin is marketed as an immunosuppressant for the treatment of organ transplant patients to prevent graft rejection (Huang et al., 2003). In addition to immunosuppression, rapamycin has a potential therapeutic use in the treatment of various diseases, for example cancer, cardiovascular diseases such as restenosis, autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, fungal infection, and neurodegenerative diseases such as Alzheimer, Parkinson's disease and Huntington's disease. Despite its utility in a variety of diseases, rapamycin has several major disadvantages. First, it is a substrate of the P glycoprotein of the cell membrane effusion pump (P-gp, LaPlante et al., 2002, Crowe et al., 1999) which pumps the compound out of the cell, making the penetration deficient from rapamycin to cell membranes. This causes poor absorption of the compound after its administration. Also, since a main mechanism Of the multidrug resistance of cancer cells is through the effusion pump of the cell membrane, rapamycin is less effective against cancer cells with multidrug resistance (MDR). Second, rapamycin is extensively metabolized by cytochrome P450 enzymes (Lampen et al., 1998). Its loss in the first hepatic step is high, which contributes more to its low oral bioavailability. The role of CYP3A4 and P-gp in the low bioavailability of rapamycin has been confirmed in studies demonstrating that the administration of CYP3A4 and / or P-gp inhibitors reduces the effusion of rapamycin in Caco-2 cells transfected with CYP3A4 ( Cummins et al., 2004), and the administration of CYP3A4 inhibitors decreases the metabolism of rapamycin in the small intestine (Lampen et al., 1998). The low oral bioavailability of rapamycin causes significant variability among individuals, giving an inconsistent therapeutic result and difficulty in clinical management (Kuhn et al., 2001, Crowe et al., 1999). Therefore, there is a need to develop novel compounds similar to rapamycin that are not substrates of P-gp, which may be metabolically more stable and therefore have a better bioavailability. When used as anticancer agents, these compounds must be more effective against MDR cancer cells, in particular against cancer cells expressing P-gp. A range of synthetic analogs of rapamycin have been reported using the chemically available sites of the molecule. The Description of the following compounds was adapted to the numbering system of the rapamycin molecule shown in formula A. Chemically available sites of the molecule for modification or replacement include C40 and C28 hydroxyl groups (eg, US 5,665,772; 5,362,718), the C39 and C16 methoxy groups (for example, WO 96/41807; US 5,728,710), the C32, C26 and C9 keto groups (for example, US 5,378,836; US 5,138,051; US 5,665,772). Hydrogenation at C17, C19 and / or C21, making white in the triene, results in retention of antifungal activity but relative loss of immunosuppression (eg, U.S. 5,391, 730; U.S. 5,023,262). Significant improvements in the stability of the molecule have been achieved by modification (eg, formation of oximes at C32, C40 and / or C28, US 5,563,145, US 5,446,048), resistance to metabolic attack (eg, US 5,912,253), bioavailability (eg, US 5,221,670; US 5,955,457; WO 98/04279); and prodrug production (eg, US 6,015,815; US 5,432,183). Two of the most advanced rapamycin derivatives in clinical development are 40-O- (2-hydroxy) ethyl-rapamycin (RAD001, Certican, everolimus), a semisynthetic analogue of rapamycin that exhibits immunosuppressive pharmacological effects (Sedrani, R. others, 1998; Kirchner and others, 2000; US 5,665,772) and 40-O- [2,2-bis (hydroxymethyl) propionyloxy] rapamycin, CCI-779 (Wyeth-Ayerst), a rapamycin ester that inhibits cell growth in vitro and inhibits tumor growth in vivo (Yu et al., 2001). Currently CCI-779 is in several clinical trials as a drug anticancer potential. A recent publication of the phase II study of CCI-779 in patients with recurrent glioblastoma multiforme (Chang et al, 2005), suggests that the low efficacy of this drug in these patients may be due to its poor penetration of the blood-brain barrier. Studies investigating the pharmacokinetics of RAD001 have shown that, similarly to rapamycin, it is a substrate for P-gp (Crowe et al., 1999, LaPlante et al., 2002). Despite their great structural similarity to rapamycin, the compounds of the invention exhibit a surprisingly different pharmacological profile. In particular, they show a significantly greater penetration of the cell membrane and decreased effusion compared to rapamycin, and are not a substrate for P-gp. In addition, 39-demethoxy-rapamycin exhibits more potent activity against cancer-resistant and multi-drug cancer cell lines expressing P-gp, than rapamycin. Compared to rapamycin, 39-demethoxyrapamycin shows a significantly different inhibitory profile against panels of the NCl 60 cell line. Furthermore, in clinical trials the 39-demethoxy-rapamycin analogues show a significantly different pharmacokinetic profile of rapamycin and the outstanding derivatives . Unexpectedly, the analogues of 39-demethoxyrapamycin show a greater capacity to cross the blood-brain barrier and therefore show a better bioavailability in the brain.

Therefore, the present invention provides the medical use of 39-demethoxyrapamycin analogs, these rapamycin analogs have significantly altered pharmacokinetics, a greater ability to cross the blood-brain barrier, greater metabolic stability, greater penetration of the cell membrane, a faster decreased effusion and a tumor cell inhibitory profile different from that of rapamycin. These compounds are useful in medicine, in particular for the treatment of cancer and / or the malignancy of B cells, for the induction or maintenance of immunosuppression, stimulation of neuronal regeneration, treatment of fungal infections, rejection of transplantation, graft disease against host, autoimmune disorders, diseases of vascular inflammation and fibrotic diseases. The present invention particularly provides for the use of 39-demethoxyrapamycin in the treatment of cancer and / or the malignancy of B cells. Rapamycin has been shown to stimulate autophagy (Raught et al., 2001). Deterioration of autophagy or uncontrolled autophagy have been implicated in several disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease and prion diseases (which include Creutzfeldt-Jacob disease), suggesting that the manipulation of this route can be beneficial in these diseases. A recent in vitro study showed that the administration of rapamycin was able to reduce the appearance of aggregates and cell death associated with the expansions of poly (Q) and poly (A) in transfected COS-7 cells (Ravikumar et al. others, 2002). Therefore, if rapamycin was able to cross the blood-brain barrier, these results indicate that it would be a suitable candidate for the treatment of Huntington's disease and other related disorders. This suggests that there is a need to develop rapamycin analogs that are capable of crossing the blood-brain barrier. The hyperphosphorylation of tau protein associated with microtubules and their subsequent aggregation in insoluble paired helical filaments, which form intracellular "tangles", are one of the salient features of Alzheimer's disease, and the accumulation of this neurofibrillary pathology and associated death of neuronal cells is closely related to cognitive decline. A recent study by An et al. (2003) showed that activated p70 S6 kinase is co-distributed with neurofibrillary pathology in the brains of people with Alzheimer's, and in particular there was obviously an increase in activated p70 S6 kinase in neurons before the tangle development (An et al., 2003). In an in vitro test where the administration of zinc sulfate results in the activation of p70 S6 kinase and the increase in total, normal and hyperphosphorylated tau concentration, the pretreatment of the cells with rapamycin was shown to reduce the activation of p70 S6 kinase, and significantly reduces the concentration of total, normal and hyperphosphorylated tau. Therefore, these studies indicate that the administration of rapamycin or rapamycin analogs may be beneficial in reducing the neurofibrillary pathology of Alzheimer's disease, provided that the compounds are able to reach the site of action. Additionally, rapamycin has been reported to increase neuritic growth and neuronal survival in several in vitro and in vivo models (Avramut and Achim, 2002), indicating that rapamycin and its analogues may be useful in the treatment of disorders where Neuronal regeneration can be of significant therapeutic benefit. However, this utility depends on the ability to reach the site of action, and therefore rapamycin analogs with a better ability to cross the blood-brain barrier would be particularly preferred. The present invention provides the novel and surprising use of 39-demethoxyrapamycin analogues in medicine, in particular the use of 39-demethoxyrapamycin, particularly in the treatment of cancer or malignancy of B cells, in the induction or maintenance of immunosuppression, stimulation of neuronal regeneration, treatment of fungal infections, rejection of transplant, graft disease against host, autoimmune diseases, neurodegenerative conditions, diseases of vascular inflammation and fibrotic diseases. In particular, the present invention provides the use of 39-demethoxyrapamycin analogues in the treatment of cancer and malignancy of B cells. In a preferred embodiment, the present invention provides the use of 39-demethoxyrapamycin analogs in the treatment of neurological disorders. or neurodegenerative. In a preferred additional embodiment, the present invention provides the use of 39-demethoxyrapamicycin analogs in the treatment of brain tumors, in particular glioblastoma multiforme. In a specific aspect of the present invention, the 39-demethoxy-rarapamycin analog is 39-demethoxy-rapamycin.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to the medical use of 39-demethoxy-rarapamycin analogs, in particular 39-demethoxy-rapamycin, particularly in the treatment of cancer and / or malignancy of B cells, the induction or maintenance of immunosuppression, the rejection treatment of transplant, graft disease against host, autoimmune diseases, neurodegenerative conditions, inflammation diseases, vascular disease and fibrotic diseases, the stimulation of neuronal regeneration or the treatment of mycotic infections. In particular, this invention relates to the use of 39-demetoxirrapamycin analogs for the treatment of cancer and malignancy of B cells. In a specific embodiment, the present invention relates to the use of 39-demethoxyrapamcin in the treatment of cancer and the malignancy of B cells. Specifically, the present invention also provides the use of 39-demethoxyrapamicyn analogs in the treatment of brain tumors or neurodegenerative conditions. In a specific embodiment, the present invention provides the use of 39-demethoxyrapamycin in the treatment of brain tumors or neurodegenerative conditions.

Specifically, the present invention also provides for the use of 39-demethoxyrapamycin analogs in the treatment of neurodegenerative conditions. In particular, the present invention provides the use of 39-demethoxyrapamycin in the treatment of neurodegenerative conditions.

Definitions Articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical objects of the article. By way of example, "an analogue" means an analog or more than an analogue. As used herein, the term "autoimmune disorders" includes, without limitation: systemic lupus erythematosus (SLE), rheumatoid arthritis, myasthenia gravis, and multiple sclerosis. As used herein, the term "inflammatory diseases" includes, without limitation: psoriasis, dermatitis, eczema, seborrhea, inflammatory bowel disease (including without limitation ulcerative colitis and Crohn's disease), lung inflammation (including asthma, disease chronic obstructive pulmonary disease, emphysema, acute respiratory distress syndrome and bronchitis), rheumatoid arthritis and uveitis. As used herein, the term "cancer" refers to a malignant or benign growth of cells in the skin or in organs of the body, for example, without limitation, the breast, prostate, lung, kidney, pancreas, brain, stomach or intestine. A cancer tends to infiltrate the tissue adjacent and disseminate to distant organs (metastases), for example bone, liver, lung or brain. As used herein, the term "cancer" includes tumor cells of the metastatic type, such as, for example, without limitation, melanoma, lymphoma, leukemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma, and tissue-type carcinoma, such as, for example, without limitation. , colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, kidney cancer, gastric cancer, glioblastoma, primary liver cancer and ovarian cancer. The term also specifically encompasses brain tumors, as described more fully below. As used herein, the term "brain tumors" refers to a malignant or benign growth of cells in the brain; includes primary and secondary (metastatic) tumors. Primary brain tumors include, without limitation, gliomas (eg, glioblastoma multiforme, astrocytoma, brain stem glioma, ependymoma and oligodendrogloma), medulloblastoma, meningoma, schwannoma (or acoustic neuroma), craniopharyngioma, germ cell tumor of the brain ( example, germinoma), or tumor of the pineal region. The term "brain cancer" is also used to describe the previous group of disorders and these terms are used interchangeably herein. As used herein, the term "B-cell malignancy" includes a group of disorders including chronic lymphocytic leukemia (CLL), Multiple myeloma and non-Hodgkin lymphoma (NHL). They are neoplastic diseases of the blood and blood-forming organs. They cause dysfunction of the bone marrow and the immune system, which makes the host highly susceptible to infection and bleeding. As used herein, the term "vascular disease" includes without limitation: hyperproliferative vascular disorders (e.g., restenosis and vascular occlusion), vascular graft atherosclerosis, cardiovascular disease, cerebral vascular disease, and peripheral vascular disease (e.g., artery disease). coronary, arteriosclerosis, atherosclerosis, non-atherosclerotic arteriosclerosis or vascular wall lesion). It is also used to refer to diseases that involve the neogenesis or proliferation of blood vessels in the eye, in particular choroidal neovascularization. As used herein, the term "neuronal regeneration" refers to the stimulation of neuronal cell growth and includes neurite growth and functional recovery of neuronal cells. Diseases and disorders where neuronal regeneration can be of significant therapeutic benefit include, without limitation, Alzheimer's disease, Parkinson's disease, Huntington's chorea (disease), amyotrophic lateral sclerosis, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's palsy, muscular dystrophy, stroke, progressive muscular atrophy, progressive bulbar muscular atrophy, cervical spondylosis, Gullain-Barre syndrome, dementia, peripheral neuropathy and peripheral nerve damage, caused by physical injury (for example injury or trauma to the spinal cord, injury or damage to the sciatic or facial nerve) or by a pathological condition (for example, diabetes). As used herein, the term "medical condition originating from damage or neural disease" includes, without limitation, neurodegenerative conditions, brain tumors, infection or inflammation of the brain, and other conditions that can lead to cell death or dysfunction. or nerve or glial tissues. As used herein, the term "neurodegenerative conditions" includes, without limitation, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), muscular dystrophy (oculopharyngeal) (which includes oculopharyngeal muscular dystrophy), sclerosis multiple, prion diseases (eg, Creutzfeldt-Jacob disease (CJD)), Pick's disease, Lewy body dementia (or Lewy body disease), and / or motor neuron disease. As used herein, the term "medical condition affecting the central nervous system which requires the drug to cross the blood-brain barrier" includes, without limitation, medical conditions arising from neural damage or diseases, and any other condition for which it is intended. it requires the access of the medicine to the neuronal cells for an effective therapy. As used herein, the term "fibrotic diseases" refers to diseases associated with excessive matrix production extracellular, and include (without limitation) sarcoidosis, keloids, glomerulonephritis, end-stage renal disease, hepatic fibrosis (including but not limited to cirrhosis, liver disease due to alcohol and steatohepatitis), chronic graft nephropathy, surgical adhesions, vasculopathy, cardiac fibrosis , pulmonary fibrosis (which includes without limitation idiopathic pulmonary fibrosis and cryptogenic fibrosing alveolitis), macular degeneration, retinal and vitreal retinopathy and fibrosis induced by chemotherapy or radiation. As used herein, the term "graft-versus-host disease" refers to a complication that is observed after transplantation of allogeneic stem cells / bone marrow. It occurs when the cells fighting the donor's infection recognize the patient's body as different or strange. These infection-fighting cells then attack the tissues of the patient's body as if they were attacking an infection. GvHD is classified as acute when it occurs during the first 100 days after transplantation and chronic if it occurs more than 100 days after transplantation. The tissues involved normally include the liver, gastrointestinal tract and skin. Chronic graft-versus-host disease occurs in approximately 10-40 percent of patients after stem cell / bone marrow transplantation. As used herein, the term "bioavailability" refers to the degree or rate at which a drug or other substance is absorbed or makes available at the site of biological activity after its administration. This property depends on several factors including the solubility of the compound, the rate of absorption in the intestine, the gado of protein binding and metabolism, etc. Several bioavailability tests that would be familiar to the person skilled in the art are described herein (see also Trepanier et al., 1998, Gallant-Haidner et al., 2000). As used herein, the term "cancer or malignancy of B cells resistant to one or more of the existing anticancer agents" refers to the types of cancer or malignancy of B cells for which at least one therapy used normally is ineffective. . These types of cancer are characterized because they survive after the administration of at least one neoplastic agent, where the counterpart of normal cells (i.e., cells with regular growth of the same origin) would show signs of cellular toxicity, cell death or cellular quiescence. (that is, they would not be divided). In particular, this includes cancer or malignancy of MDR B cells; particular examples are the types of cancer and malignancy of B cells expressing high amounts of P-gp. The identification of this resistant type of cancer or malignancy of B cells is of the domain and usual activity of a physician or other similarly trained person. As used herein, the term "39-demethoxy-rarapamycin analog" refers to a compound according to the following formula (I), or a pharmaceutically acceptable salt thereof. where R-, represents (H, H) or = O, and R2 and R3 represent, each independently, H, OH or OCH3. These compounds are also referred to as "the compounds of the invention" and these terms are used interchangeably in the present application. In the present application, the term "39-demethoxy-rapamycin analog" includes 39-demethoxy-rapamycin itself. As used herein, the term "39-demethoxyrapamycin" refers to a compound according to formula (I) above, or a pharmaceutically acceptable salt thereof, wherein Ri represents = O, and R2 and R3 represent, each , OCH3. The pharmaceutically acceptable salts of the 39-demethoxyrapamycin analogs include conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases, as well as quaternary ammonium acid addition salts. More examples specific to suitable acid salts include hydrochloric, bromohydric, sulfuric, phosphoric, nitric, perchloric, fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic, tartaric, citric, pamoic, malonic, hydroximic, phenylacetic, glutamic. , benzoic, salicylic, fumaric, toluenesulfonic, methanesulfonic, naphthalene-2-sulphonic, benzenesulfonic, hydroxynaphthoic, hydriodic, malic, steroic, tannic and the like. Other acids such as oxalic acid, although not by themselves are not pharmaceutically acceptable, may be useful in the preparation of the salts useful as intermediates for obtaining the compounds of the invention and their pharmaceutically acceptable salts. More specific examples of the suitable basic salts include the sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N, N'-dibenzylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine salts. Hereinafter, references to a compound according to the invention include both 39-demethoxy-rapamycin and its pharmaceutically acceptable salts.

DESCRIPTION OF THE INVENTION The present invention relates to the use of a 39-demetoxirrapamycin analogue in medicine, in particular in the treatment of cancer, malignancy of B cells, in the induction or maintenance of immunosuppression, the treatment of transplant rejection, the disease of graft against host, autoimmune disorders, neurodegenerative conditions, inflammatory diseases, vascular disease and flbrotic diseases, the stimulation of neuronal regeneration, the treatment of neurological diseases or neurodegenerative conditions, or the treatment of fungal infections. Therefore, the present invention provides the use of a 39-demethoxy-rarapamycin analog, or a pharmaceutically acceptable salt thereof, in the treatment of a medical condition that results from nerve injury or disease. In a specific embodiment, the present invention provides the use of 39-demethoxyrapamcincin, or a pharmaceutically acceptable salt thereof, in the treatment of a medical condition that is caused by injury or nerve disease. In a further embodiment, the present invention provides the use of a 39-demethoxyrapamicycin analog that additionally differs from rapamycin in one or more of positions 9, 16, or 27, in the treatment of a medical condition that results from injury or nervous disease. The present invention also provides the use of a 39-demetoxirrapamycin analog, ie, a rapamycin analog with increased penetration of the blood-brain barrier, or a pharmaceutically acceptable salt thereof, in the treatment of medical conditions affecting the central nerves that they require the medicine to cross the blood-brain barrier; that is, medical conditions where the blood-brain barrier prevents the supply of the compound. In a specific embodiment, the present invention provides the use of 39-demethoxyrapamycin, or a pharmaceutically acceptable salt thereof, in the treatment of medical conditions that affect the central nervous system, where the blood-brain barrier prevents the supply of the compound. In a further embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog which additionally differs from rapamycin in one or more of positions 9, 16 or 27, in the treatment of medical conditions affecting the central nervous system, wherein the blood-brain barrier prevents the supply of the compound. In a particular embodiment, this invention relates to the use of a 39-demethoxyrapamycin analogue for the treatment of cancer and malignancy of B cells. In a further embodiment, this invention relates to the use of 39-demethoxyrapamycin for the treatment of cancer and the malignancy of B cells. In a further embodiment, the present invention relates to the use of a 39-demethoxyrapamycin analog that additionally differs from rapamycin in one or more of positions 9, 16 or 27, for the treatment of cancer and malignancy of B cells. Specifically, the present invention also provides for the use of a 39-demethoxyrapamycin analog in the treatment of brain tumors. The present invention also provides the use of 39-demethoxyrapamycin in the treatment of brain tumors. In a further embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog that additionally differs from rapamycin in one or more of positions 9, 16 or 27, in the treatment of brain tumors. In particular, this invention provides the use of a 39-demethoxyrapamycin analog in the treatment of glioblastoma multiforme. In a specific embodiment, the present invention provides the use of 39-demethoxyrapamycin in the treatment of glioblastoma multiforme. In a further embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog that additionally differs from rapamycin in one or more of positions 9, 16 or 27, in the treatment of glioblastoma multiforme. The present invention also provides the use of a 39-demethoxyrapamycin analog in the treatment of neurodegenerative conditions. In a further embodiment, the present invention provides the use of 39-demethoxyrapamycin in the treatment of neurodegenerative conditions. In a further embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog that additionally differs from rapamycin in one or more of positions 9, 16 or 27, in the treatment of neurodegenerative conditions. In particular, the neurodegenerative condition can be selected from the group consisting of Alzheimer's disease, multiple sclerosis and Huntington's disease. Therefore, in one embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog in the treatment of Alzheimer's disease. In a further embodiment, the present invention provides the use of 39-demethoxyrapamycin in the treatment of Alzheimer's disease. In a further embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog which additionally differs from the rapamycin in one or more of positions 9, 16 or 27, in the treatment of Alzheimer's disease. In a further embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog in the treatment of multiple sclerosis. In a further embodiment, the present invention provides the use of 39-demethoxyrapamycin in the treatment of multiple sclerosis. In a further embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog which additionally differs from rapamycin in one or more of positions 9, 16 or 27, in the treatment of multiple sclerosis. In an alternative embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog in the treatment of Huntington's disease. In a further embodiment, the present invention provides the use of 39-demethoxyrapamycin in the treatment of Huntington's disease. In a further embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog that additionally differs from rapamycin in one or more of positions 9, 16 or 27, in the treatment of Huntington's disease. In an alternative embodiment, the present invention provides a method for the treatment of cancer or malignancy of B cells, a method of induction or maintenance of immunosuppression, stimulation of neuronal regeneration, a method for the treatment of fungal infections, rejection of transplantation, graft-versus-host disease, autoimmune disorders, neurodegenerative conditions, vascular inflammation diseases, or fibrotic diseases, which comprises administering a patient an effective amount of a 39-demethoxyrapamycin analog, in particular 39-demethoxyrapamycin, or a 39-demethoxyrapamycin analog which additionally differs from rapamycin in one or more of positions 9, 16 or 27. Specifically, the present invention provides a method of treating a medical condition that is caused by nerve injury or disease, which comprises administering a 39-demethoxyrapamycin analog, or a pharmaceutically acceptable salt thereof. In a particular embodiment, the present invention provides a method of treating a medical condition that results from nerve injury or disease, which comprises administering 39-demethoxyrapamcincin. In an additional mode, the present invention provides a method of treating a medical condition that results from nerve injury or disease, which comprises administering a 39-demethoxyraprapamicine analogue which additionally differs from rapamycin in one or more of positions 9, 16 or 27. The present invention also provides a method of treating medical conditions affecting the central nervous system wherein the blood-brain barrier prevents delivery of the compound, by administering an effective amount of a 39-demethoxy-rapamycin analog, ie, a rapamycin analogue with greater penetration of the blood-brain barrier, or a pharmaceutically acceptable salt thereof. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the analogue of 39-demethoxyrapamycin is an analogue of 39-demethoxyrapamycin which additionally differs from rapamycin in a or more than positions 9, 16 or 27. Preferably, the present invention provides a method of treating cancer or malignancy of B cells, which comprises administering to a patient an effective amount of a 39-demethoxyrapamycin analog. In a further preferred embodiment, the present invention provides a method of treating brain tumors, comprising administering to a patient an effective amount of a 39-demethoxyrapamycin analog. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the 39-demethoxy-rarapamycin analog is an analogue of 39-demethoxy-rapamycin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. In a particular embodiment, the present invention provides a method of treatment of glioblastoma multiforme, which comprises administering to a patient an effective amount of a 39-demethoxyrapamycin analogue. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the 39-demethoxy-rarapamycin analog is a 39-demetoxirrapamycin analog which additionally differs from rapamycin in one or more of positions 9, 16 or 27. In a further preferred embodiment, the present invention provides a method of treatment of a neurodegenerative condition, comprising administering to a patient an effective amount of a 39-demethoxy-rarapamycin analogue. In a specific aspect, the analog of 39- Demethoxyrapamycin is 39-demethoxyrapamycin. In a further aspect, the analog of 39-demethoxyrapamycin is an analogue of 39-demethoxyrapamycin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. Particularly, the neurodegenerative condition can be selected from the group consisting of Alzheimer's disease, multiple sclerosis and Huntington's disease. Therefore, in one embodiment, the present invention provides a method of treating Alzheimer's disease comprising administering to a patient an effective amount of a 39-demethoxy-rapamycin analog. In a specific aspect, the analogue of 39-demethoxy-rapamycin is 39-demethoxy-rapamycin. In a further aspect, the 39-demethoxy-rapamycin analog is a 39-demethoxy-rarapamycin analog which additionally differs from rapamycin in one or more of positions 9, 16 or 27. In a further embodiment, the present invention provides a method of treatment of multiple sclerosis, which comprises administering to a patient an effective amount of an analogue of 39-demethoxyrapamicycin. In a specific aspect, the 39-demethoxyrrapamycin analogue is 39-demethoxyrapamycin. In a further aspect, the 39-demethoxy-rapamycin analog is an analogue of 39-demethoxyrapamycin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. In an alternative embodiment, the present invention provides a method of treatment of Huntington's disease, which comprises administering to a patient an effective amount of an analogue of 39-demethoxyrapamycin.

In a specific aspect the analogue of 39-demethoxyrapamycin is 39-demethoxyrapamycin. In a further aspect, the 39-demethoxy-rarapamycin analog is an analogue of 39-demethoxyrapamycin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. The present invention also provides for the use of a 39- demethoxyrapamycin in the manufacture of a medicament for the treatment of cancer or malignancy of B cells, for the induction or maintenance of immunosuppression, for the stimulation of neuronal regeneration, for the treatment of fungal infections, rejection of transplantation, graft disease against host, autoimmune disorders, neurodegenerative conditions, diseases of vascular inflammation, or fibrotic diseases. Specifically, the present invention provides the use of a 39-demetoxirrapamycin analog, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a medical condition that is caused by nerve injury or disease. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the 39-demethoxy-rarapamycin analog is an analogue of 39-demethoxyrapamycin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. The present invention also provides for the use of a 39- demetoxirrapamycin, that is, a rapamycin analog with greater penetration of the blood-brain barrier, or a pharmaceutically acceptable salt thereof, into the preparation of a medicament for the treatment of medical conditions affecting the central nervous system where the blood-brain barrier prevents delivery of the compound. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the 39-demethoxy-rarapamycin analogue is an analogue of 39-demethoxyrapamycin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. Specifically, the present invention also provides for the use of an analogue of 39-demethoxyrapamycin in the manufacture of a medicament for the treatment of brain tumors. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the analogue of 39-demethoxy-rapamycin is a 39-demethoxy-rarapamycin analog which additionally differs from rapamycin in one or more of positions 9, 16 or 27. In a particular embodiment, the present invention specifically provides for the use of an analog of 39-demethoxyrapamycin in the manufacture of a medicament for the treatment of glioblastoma multiforme. In a specific aspect, the analogue of 39-demethoxy-rapamycin is 39-demethoxy-rapamycin. In a further aspect, the analogue of 39-demethoxyrapamicycin is an analogue of 39-demethoxyrapamcinin which additionally differs from rapamycin in one or more of positions 9, 16, or 27.

Specifically, the present invention also provides the use of a 39-demethoxyrapamycin analog in the manufacture of a medicament for the treatment of neurodegenerative conditions. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the 39-demethoxy-rarapamycin analog is a 39-demethoxy-rarapamycin analog which additionally differs from rapamycin in one or more of positions 9, 16 or 27. Particularly, the neurodegenerative condition can be selected from the group consisting of Alzheimer's disease, multiple sclerosis and Huntington's disease. Therefore, in one embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog in the manufacture of a medicament for the treatment of Alzheimer's disease. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the 39-demethoxy-rarapamycin analog is an analogue of 39-demethoxyrapamycin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. In a further embodiment, the present invention provides the use of a Analog of 39-demethoxyrapamycin in the manufacture of a medicament for the treatment of multiple sclerosis. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the 39-demethoxy-rarapamycin analog is an analogue of 39-demethoxyrapamycin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. In an alternative embodiment, the present invention provides the use of a 39-demethoxyrapamycin analog in the manufacture of a medicament for the treatment of Huntington's disease. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the analogue of 39-demethoxyrapamycin is an analogue of 39-demethoxyrapamycin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. The analogs of 39-demethoxyrapamycin are close structural analogs of rapamycin. which are prepared using the methods described in WO 04/007709. However, they show a spectrum of activity different from rapamycin, for example, as shown by the COMPARE analysis of the NCl 60 cell line panel for 39-demethoxyrapamycin and related analogues (see Table 1 below). The COMPARE analysis uses a Pearson analysis to compare the activity of two compounds in the NCl 60 cell line panel and produces a correlation coefficient that indicates how similar the activity spectra of the two compounds are, and this may indicate how Its mechanisms of action are related. As a specific example, the Pearson coefficient for rapamycin and 39-demethoxyrapamycin is 0.614; this would be compared with the Pearson coefficient between rapamycin and CCI-779 (a 40-hydroxy ester derivative of rapamycin), which is 0.86 (Dancey, 2002). Therefore, it can be seen that the analogues of 39-demethoxyrapamycin have a different spectrum of activity compared to rapamycin.

TABLE 1 Multidrug resistance (MDR) is a significant problem in the treatment of cancer and the malignancy of B cells. It is the main reason behind the development of drug resistance in many types of cancer (Persidis A, 1999). The drugs that initially worked have become completely ineffective after a certain period. MDR is associated with a greater number of transporters of the adenosine triphosphate binding cassette (ABC transporters), in particular with an increase in the expression of the MDR1 gene encoding the P glycoprotein (P-gp), or the MRP1 gene that encodes MRP1. The degree of expression of the MDR1 gene varies widely between different cell lines derived from cancer; in some cell lines it is undetectable, while in others it can show an expression 10 times or up to 100 times higher than the standard controls. Some of the differences indicated in the activity spectrum between rapamycin and 39-demethoxyrapamycin can be explained because the analogues of 39-demethoxyrapamycin are not a substrate for P-gp. The 39-demethoxyrapamycin analogues have lower effusion of Caco-2 cells compared to rapamycin, and 39-demethoxyrapamcincin showed not to be a substrate for P-gp in a P-gp substrate test in vitro (see examples of the present). Therefore, a further aspect of the invention provides the use of a 39-demethoxyrapamycin analog in the treatment of cancer or malignancy of B cells resistant to one or more of the existing anticancer agents, i.e., cancer or the malignancy of MDR-type B cells. In a specific aspect, the present invention provides the use of 39-demethoxyrapamycin in the treatment of cancer or malignancy of B cells of the types expressing P-gp. In another highly preferred embodiment, the present invention provides the use of 39-demethoxyrapamycin in the treatment of cancer or malignancy of B cells of the types that have a high expression of P-gp. Particularly, the cancer or malignancy of B cells of the types that have a high expression of P-gp can have an expression 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or 100 times higher than controls. In a specific aspect of the above uses, the 39-demethoxyrrapamycin analog is 39-demethoxyrapamycin. In a further aspect of the above uses, the 39-demethoxy-rarapamycin analogue is an analogue of 39-demethoxyrapamcinin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. Suitable controls are cells that do not express P -gp, which have a low degree of expression of P-gp, or which have a low MDR function; he a person skilled in the art knows or can identify such cell lines; by way of example (but not limitation), suitable cell lines include: MDA435 / LCC6, SBC-3 / CDDP, MCF7, NCI-H23, NCI-H522, A549 / ATCC, EKVX, NCI-H226, NCI- H322M, NCI-H460, HOP-18, HOP-92, LXFL 529, DMS 114, DMS 273, HT29, HCC-2998, HCT-116, COLO 205, KM12, KM20L2, MDA-MB-231 / ATCC, MDA- MB-435, MDA-N, BT-549, T-47D, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, IGROV1, SK-OV-3, K-562, MOLT-4, HL- 60 (TB), RPMI-8226, SR, SN12C, RXF-631, 786-0, TK-10, LOX IMVI, MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, M14, UACC-62, UACC-257, PC-3, DU-145, SNB-19, SNB-75, SNB-78, U251, SF-268, SF-539, XF 498. In an alternative aspect, the present invention provides the use of a 39-demethoxy-rarapamycin analog in the preparation of a medicament for use in the treatment of cancer or malignancy of MDR-B cells. In a specific aspect, the present invention provides the use of a 39-demethoxyrapamicycin analog in the preparation of a medicament for use in the treatment of cancer or B-cell malignancy expressing P-gp. In another preferred embodiment, the present invention provides the use of a 39-demethoxy-rarapamycin analog in the preparation of a medicament for use in the treatment of cancer or B-cell malignancy that has high expression of P-gp. Particularly, the cancer or malignancy of B cells that has high expression of P-gp can have an expression 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or 100 times higher than control values. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the 39-demethoxy-rarapamycin analog is an analogue of 39-demethoxyrapamycin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. Appropriate controls were described above. The methods for determining the degree of expression of P-gp in a sample are discussed below. Therefore, in a further aspect, the present invention provides a method for the treatment of cancer or B-cell malignancy expressing P-gp, which comprises administering a therapeutically effective amount of a 39-demethoxy-rapamycin analog. In a specific aspect, the 39-demethoxyrrapamycin analog is 39-demethoxy-rapamycin. In a further aspect, the analogue of 39-demethoxyrapamycin is an analogue of 39-demethoxyrapamicycin which additionally differs from rapamycin in one or more of positions 9, 16 or 27. The degree of expression of the glycoprotein P (P-gp) in a particular type of cancer can be determined by the person skilled in the art using techniques that include without limitation RT-PCR in real time (Szakács et al., 2004; Stein et al., 2002; Langmann et al., 2003), immunohistochemistry (Stein and others, 2002), or microarrays (Lee et al., 2003); these methods are provided only as examples; other suitable methods will be within the domain of the person skilled in the art.

The 39-demethoxyrapamycin shows an improved metabolic stability compared to rapamycin, as shown here in the examples. Several papers have previously identified the 39-methoxy g in rapamycin as a major metabolic attack site for converting rapamycin into 39-O-demethylrapamycin (Trepanier et al., 1998). The major metabolites of rapamycin have significantly decreased activity compared to the original compound (Gallant-Haidner et al., 2000, Trepanier et al., 1998). In contrast, 39-demethoxyrapamycin no longer has the most significant sites of metabolic attack available, resulting in greater stability of the compounds (see examples). This, together with the equivalent or higher potency of 39-demethoxyrapamycin than the original rapamycin compound, provides a longer half-life of the compound of the invention. This is a surprising additional advantage of 39-demethoxyrapamycin over rapamycin. The previously described properties of 39-demethoxyrapamycin (which is not a substrate for P-gp, which has improved metabolic stability and less effusion of cells by means of P-gp), indicate that 39-demethoxyrapamycin has a better bioavailability in comparison with its original compound, rapamycin. Therefore, the present invention provides the use in medicine of 39-demethoxyrapamycin, a rapamycin analog with improved metabolic stability, increased penetration of the cell membrane and a different inhibitory profile of cancer cells, particularly in the treatment of cancer or the malignancy of B cells. The present invention also provides a pharmaceutical composition comprising a 39-demethoxyrapamicyn analog, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier. In a specific aspect, the present invention provides a pharmaceutical composition comprising 39-demethoxyrapamycin. In a further aspect, the present invention provides a pharmaceutical composition comprising a 39-demethoxyrapamycin analog that additionally differs from rapamycin in one or more of positions 9, 16 or 27. In a specific embodiment, the present invention provides a composition pharmaceutical as described above, formulated specifically for intravenous administration. Rapamycin and related compounds that are or have been in clinical trials, such as CCI-779 and RAD001, have poor pharmacokinetic profiles that include poor metabolic stability, poor penetration, large amount of effusion through P-gp, and poor bioavailability. The present invention provides the use of a 39-demethoxyrapamycin analog, or a pharmaceutically acceptable salt thereof, which has improved pharmaceutical properties compared to rapamycin. A further surprising aspect of the present invention is that the 39-demethoxyrapamycin analogues exhibit a pharmacokinetic profile markedly different from existing analogues of Rapamycin In particular, the analogues of 39-demethoxyrapamycin show a greater penetration of the blood-brain barrier, and therefore in the brain a greater exposure to these compounds is observed in comparison with the related analogues, for a given blood concentration. This difference in its pharmacokinetics is completely unexpected and is not suggested anywhere in the prior art. A known disadvantage of currently available therapies for disorders including neurodegenerative conditions and brain tumors is the challenge of putting the drugs in the site of action (see Pardrídge, 2005). It has also been reported that this is a problem with the existing rapamycin analogs used in therapy; In particular, a study investigating the efficacy of CCI-779 in the treatment of glioblastoma multiforme concluded that although systemic concentrations are adequate, the blood-brain barrier prevents drug delivery to the tumor (Chang, 2005). Therefore, the present invention reveals for the first time a rapamycin analog with greater penetration of the blood-brain barrier and therefore is of significant utility for the treatment of brain tumors and neurodegenerative conditions. Preferred 39-demethoxyrapamycin analogs for use in any of the aspects of the invention described above, include those that differ further from rapamycin in any of positions 9, 16 or 27; that is, it is preferable that the analog of the 39- Demethoxyrapamycin is not 39-demethoxy-rapamycin itself. Additional preferred analogs of 39-demethoxyrapamycin include those in which: - The 39-demethoxy-rarapamycin analog has a hydroxyl group at position 27, ie, R3 represents OH; - The analog of 39-demethoxy-rarapamycin has a hydrogen at position 27, that is, R3 represents OH; or - The 39-demethoxy-rarapamycin analog has a hydroxyl group in the 16-position, ie, R2 represents OH; The person skilled in the art will be able to determine the pharmacokinetics and bioavailability of a compound of the invention using known in vivo and in vitro methods including, without limitation, those described below and in Gallant-Haidner et al., 2000, and Trepanier and others, 1998, and references cited therein. The bioavailability of a compound is determined by several factors (for example, the solubility in water, the penetration of the cell membrane, the degree of protein binding and metabolism and stability), each of which can be determined by tests in vitro as described in the examples herein; it will be appreciated by the person skilled in the art that an improvement in one or more of these factors will improve the bioavailability of a compound. Alternatively, the bioavailability of the 39-demethoxyrapamycin or a pharmaceutically acceptable salt thereof can be measured using the methods described below in greater detail, or in the examples of the present.

In vivo tests In vivo tests can also be used to measure the bioavailability of a compound such as 39-demethoxy-rapamycin. In general, said compound is administered to a test animal (for example mouse or rat) intraperitoneally (ip), or intravenously (iv), and orally, and blood samples are taken at regular intervals to examine how it varies with time. plasma concentration of the drug. The variation of the plasma concentration with time can be used to calculate the absolute bioavailability of the compound as a percentage using standard models. An example of a typical protocol is described below. Mice are given 3 mg / kg of 39-demethoxyrapamycin iv, or 10 mg / kg of 39-demethoxyrapamicin orally. Blood samples are taken at intervals of 5 min, 15 min, 1 h, 4 h and 24 h, and the concentration of 39-demethoxyrapamycin in the sample is determined by means of HPLC. Then the variation of the concentration in the plasma or blood can be used to derive key parameters, such as the area under the curve of plasma concentration or blood against time (ABC, which is directly proportional to the total amount of drug without change reaching the general circulation), the maximum concentration (peak) of the drug in the plasma or blood, the time at which the maximum concentration (peak time) of the drug in plasma or blood occurs; in the Accurate determination of bioavailability using additional factors that include: the terminal half-life of the compound, the total clearance in the body, the volume of distribution in stable state and F%. These parameters are then analyzed by means of compartmental or non-compartmental methods to give a calculated percentage of bi- availability; for an example of this type of method see Gallant-Haidner et al., 2000, and Trepanier et al., 1998, and references cited therein. The efficacy of 39-demethoxyrapamycin can be tested in in vivo models for the neurodegenerative diseases described herein and known to those skilled in the art. These models include, without limitation, for Alzheimer's disease: animals that express the p-amyloid precursor (APP) of human familial Alzheimer's disease (ADF), animals that overexpress wild-type human APP, animals that overexpress p-amyloid 1-42 (pA), animals that express presenilin 1 (PS-1) of FAD (for example, Germán and Eisch, 2004). For multiple sclerosis: the experimental model of autoimmune encephalomyelitis (EAE) (see Bradl, 2003, and example 7). For Parkinson's disease: the 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP) model or the 6-hydroxydopamine (6-OHDA) model (see, for example, Emborg, 2004).; Schober A. 2004). For Huntington's disease there are several models that include the model of the R6 lines generated by the introduction of exon 1 of the human Huntington's disease (HD) gene, which carries highly expanded CAG repeats in the mouse germline (Sathasivam). Y others, 1999), and others (see Hersch and Ferrante, 2004). The above-mentioned compound of the invention, or a formulation thereof, can be administered by any conventional method, for example, without limitation, it can be administered parenterally, orally, topically (including buccal, sublingual or transdermal), by a medical device (for example a stent), by inhalation or by injection (subcutaneous or intramuscular). The treatment may consist of a single dose or a plurality of doses over a period. Alth it is possible to administer the 39-demethoxyrrapamycin analog alone, it is preferable to present it as a pharmaceutical formulation together with one or more acceptable carriers. The vehicle must be "acceptable" in the sense of being compatible with the compound of the invention and harmless to the recipient thereof. Examples of suitable vehicles are described below in greater detail. An analogue of 39-demethoxyrapamycin can be administered alone or in combination with other therapeutic agents; the co-administration of two agents (or more) allows to significantly reduce the doses of each compound used, thus reducing the side effects observed. The improved metabolic stability of 39-demethoxyrapamycin has an extra advantage over rapamycin, because it is less likely to cause drug-drug interactions when used in combination with drugs that are substrates of the P450 enzymes, such as rapamycin (Lampen et al. others, 1998).

Therefore, in one embodiment, an analogue of 39-demethoxyrapamycin is co-administered with another therapeutic agent for the induction or maintenance of immunosuppression, for the treatment of transplant rejection, graft-versus-host disease, autoimmune disorders, or inflammatory diseases; Preferred agents include, without limitation, immunoregulatory agents such as azathioprine, corticosteroids, cyclophosphamide, cyclosporin A, FK506, Mycophenolate Mofetil, OKT-3 and ATG. In an alternative embodiment, an analogue of 39-demethoxyrapamcinin is co-administered with another therapeutic agent for the treatment of cancer or malignancy of B cells; preferred agents include, without limitation, methotrexate, leucovorin, adriamycin, prednisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-HER2 monoclonal antibody (eg, Herceptin ™), capecitabine, raloxifene hydrochloride, inhibitors of EGFR (eg, Iressa ®, Tarceva ™, Erbitux ™), VEGF inhibitors (eg Avastin ™), proteasome inhibitors (eg Velcade ™), Glivec ® or hsp90 inhibitors (eg 17-AAG ). Additionally, 39-demethoxyrapamycin can be administered in combination with other therapies including, without limitation, radiation therapy or surgery. In one embodiment, an analog of 39-demethoxyrapamycin is co-administered with another therapeutic agent for the treatment of the disease vascular; Preferred agents include, without limitation, ACE inhibitors, angiotensin II receptor antagonists, fibric acid derivatives, HMG-CoA reductase inhibitors, beta-adrenergic blocking agents, calcium channel blockers, antioxidants, anticoagulants and platelet inhibitors. (for example, Plavix ™). In one embodiment, an analogue of 39-demethoxamycin is co-administered with another therapeutic agent for the stimulation of neuronal regeneration; Preferred agents include, without limitation, neurotrophic factors, for example, nerve growth factor, glial cell-derived growth factor, brain-derived growth factor, ciliary neurotrophic factor, and neurotrophin 3. In one embodiment, an analogue of 39- Demethoxamycin is co-administered with another therapeutic agent for the treatment of fungal infections; preferred agents include, without limitation, amphotericin B, flucytosine, echinocandins (eg, caspofungin, anidulafungin or micafungin), griseofulvin, an imidazole or triazole (eg, clotrimazole, miconazole, ketoconazole, econazole, butoconazole, oxiconazole, terconazole, itraconazole , fluconazole or voriconazole). In one embodiment, an analogue of 39-demethoxamycin is co-administered with another therapeutic agent for the treatment of Alzheimer's disease; Preferred agents include, without limitation, cholinesterase inhibitors, for example donepezil, rivastigmine and galantamine; N-methyl-D-aspartate (NMDA) receptor antagonists, for example memantine.

In one embodiment, an analogue of 39-demethoxamycin is co-administered with another therapeutic agent for the treatment of multiple sclerosis; Preferred agents include, without limitation, interferon beta-1 b, interferon beta-1 a, glatiramer, mitoxantrone, cyclophosphamide, corticosteroids (e.g. methylprednisolone, prednisone, dexamethasone). Coadministration includes any means of delivering two or more therapeutic agents to the patient as part of the same treatment regimen, as will be apparent to the person skilled in the art. Although the two or more agents can be administered simultaneously in a single formulation, this is not essential. The agents can be administered in different formulations and at different times. The formulations can be conveniently presented in unit dosage form and can be prepared by any of the methods known in the art of pharmacy. These methods include the step of associating the active ingredient (the compound of the invention) with the vehicle constituting one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately associating the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product. An analog of 39-demethoxamycin will normally be administered intravenously, orally, or any parenterally, in the form of a pharmaceutical formulation comprising the active ingredient, optionally in the form of an organic acid or base addition salt or inorganic, in a pharmaceutically acceptable dosage form. Depending on the disorder or patient to be treated, and the route of administration, the compositions can be administered at varying doses. The pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. In addition, the compositions may be in the form of sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be efficiently fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; in this way, they should preferably be preserved against the contaminating action of microorganisms such as bacteria and fungi. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils and suitable mixtures thereof. For example, an analog of 39-demethoxyrapamycin can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, solutions or suspensions, which may contain flavoring or coloring agents, for immediate, delayed or controlled release applications. Solutions or suspensions of a 39-demethoxyrapamycin analog suitable for oral administration may also be contain excipients such as NN-dimethylacetamide, dispersants such as polysorbate 80, surfactants and solubilizers, for example polyethylene glycol Phosal 50 PG (consisting of phosphatidylcholine, soybean fatty acids, ethanol, mono / diglycerides, propylene glycol and ascorbyl palmitate) . Said tablets may contain excipients such as microcrystalline cellulose, lactose (for example lactose monohydrate or lactose anhydrous), sodium citrate, calcium carbonate, calcium phosphate and glycine, butylated hydroxytoluene (E321), crospovidone, hyperomelose, disintegrants such as starch ( preferably corn starch, potato or tapioca), sodium starch glycolate, croscarmellose sodium, and certain complex silicates, and granulation binders, such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), macrogol 8000, sucrose, gelatin and acacia. Additionally lubricating agents may be included such as magnesium stearate, stearic acid, glyceryl behenate and talc. Solid compositions of a similar type can also be used as fillers in gelatin capsules. In this regard, preferred excipients include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, the compounds of the invention can be combined with various sweetening or flavoring agents, coloring material or dyes, emulsifying and / or suspending agents, and with eluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof. A tablet can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can 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 (for example povidone, gelatin, hydroxypropylmethylcellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate), interlaced povidone, interlaced sodium carboxymethylcellulose), surface active or dispersing agents. The molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid eluent. Optionally, the tablets can be coated or scratched and formulated so as to provide slow or controlled release of the active ingredient, using for example hydroxypropylmethylcellulose in varying proportions to provide the desired release profile. Formulations according to the present invention suitable for oral administration may be presented as separate units such as capsules, wafers or tablets, each containing a predetermined amount of the active ingredient; as powder or granules; as a suspension or solution in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can be presented as a bolus, electuario or paste.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored base, usually sucrose, and acacia or tragacanth; pills comprising the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouth rinses comprising the active ingredient in a suitable liquid vehicle. It should be understood that in addition to the ingredients particularly mentioned, the formulations of this invention may include other conventional agents having the type of formulation in question, for example, those suitable for oral administration may include flavoring agents. The pharmaceutical compositions adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, patches, gels, impregnated bandages, sprays, aerosols or oils, transdermal devices, dusting powders and the like. These compositions can be prepared by conventional methods containing the active agent. In this manner, the compositions may also comprise compatible conventional carriers and additives, such as preservatives, solvents to aid penetration of the drug, emollients in creams or ointments, and ethanol or oleyl alcohol for lotions. Such vehicles may be present in an amount of about 1% to about 98% of the composition. More usually they will form up to about 80% of the composition. Just as an illustration, a cream or ointment is prepared by mixing sufficient amounts of hydrophilic material and water with about 5-10% by weight of the compound, in amounts sufficient to produce a cream or ointment having the desired consistency. Pharmaceutical compositions adapted for transdermal administration may be presented as separate patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period. For example, the active agent can be released from the patch by iontophoresis. For applications to external tissues, for example the mouth and the skin, the compositions are preferably applied as an ointment or topical cream. When formulated as an ointment, the active agent can be used with a paraffinic or water-miscible ointment base. Alternatively, the active agent can be formulated in a cream with an oil-in-water cream base or a water-in-oil base. For parenteral administration, fluid unit dosage forms using the active ingredient and a sterile vehicle are prepared, for example, without limitation, water, alcohols, polyols, glycerin and vegetable oils, with water being preferred. The active ingredient, depending on the vehicle and concentration used, can be suspended or dissolved in the vehicle. To prepare the solutions, the active ingredient can be dissolved in water for injection and can be sterilized by filtration before emptying into a bottle or suitable ampoule and seal. Advantageously, agents such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle. To improve the stability, the composition can be frozen after emptying it in the bottle and the water can be removed under vacuum. The dry lyophilized powder is then sealed in the bottle and an attached bottle of water for injection can be supplied to reconstitute the liquid before use. Parenteral suspensions are prepared substantially the same as the solutions, except that the active ingredient is suspended in the vehicle instead of being dissolved, and sterilization can not be performed by filtration. The active ingredient can be sterilized by exposure to ethylene oxide before suspending it in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active ingredient. An analog of 39-demethoxyrapamycin can also be administered using known medical devices. For example, in one embodiment, a pharmaceutical composition of the invention can be administered with a needleless hypodermic injection device, such as the devices described in U.S. 5,399,163; U.S. 5,383,851; U.S. 6,312,335; U.S. 5,064,413; U.S. 4,941, 880; U.S. 4,790,824 or U.S. 4,596,556. Well-known examples of implants and modules useful in the present invention include: US 4,487,603, which discloses an implantable microinfusion pump for dispensing a medicament at a controlled rate; US 4,486,194, which describes a therapeutic device for administering drugs through the skin; US 4,447,233, which describes a medicament infusion pump for delivering medication at a precise infusion rate; US 4,447,224, which discloses an implantable variable flow infusion apparatus for continuous drug delivery; US 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and US 4,475,196, which describes an osmotic drug delivery system. In a specific embodiment, a 39-demethoxyrapamycin analog can be administered using a drug eluting stent, for example one corresponding to those described in WO 01/87263 and related publications, or those described by Perin (Perin, EC, 2005). . Many other of these implants, delivery systems and modules are known to the person skilled in the art. The dose to be administered of a compound of the invention will vary according to the particular compound, the disease in question, the subject and the nature and severity of the disease, and the physical condition of the subject, and the selected administration route. The appropriate dose can easily be determined by one skilled in the art. The compositions may contain 0.1% by weight of a compound of the invention, preferably 5-60%, preferably 10-30% by weight, depending on the method of administration. It will be recognized by the person skilled in the art that the optimum amount and spacing of the individual doses of a compound of the invention will be determined by the nature and magnitude of the condition treated, the form, route and site of administration, the age and condition of the particular subject treated, and that finally a doctor will determine the appropriate doses to be used. This dose can be repeated as frequently as appropriate. If side effects develop, the amount and / or frequency of the dose can be altered or reduced according to normal clinical practice.

BRIEF DESCRIPTION OF THE FIGURES Figures 1A and 1B show Western blots summarizing the mTOR inhibitory activity of 39-demethoxyrapamycin and rapamycin. Figures 2A-2D show the% T / C values of all test concentrations for paclitaxel (A and C) and 39-demethoxyrapamycin (B and D) in normal cell lines (A and B) or that have high expression of P-gp (C and D). Figure 3A shows the total area under the curve (AUC) of 0-24 h for brain or blood tissue samples after a single iv or oral administration of rapamycin and 39-demethoxyrapamcincin. Figure 3B shows the concentration of 39-demethoxyrapamicycin and rapamycin detected in brain tissue with respect to time after a single iv administration. Figure 4A shows the progress of the disease in the model EAE under the prophylactic regime. The given values are the average of the vehicle or treatment group. Figure 4B shows the progression of the disease in the EAE model under the therapeutic regimen. The given values are the average of the vehicle or treatment group. Figure 5 is a graph indicating the relative survival percentage of mice after glioma induction by stereotaxic injection of U87-MG cells. Bold diamonds represent the untreated group, bold squares represent the group treated with vehicle, and blank circles represent the group treated with 39-demethoxyrapamycin.

EXAMPLES Materials and Methods Materials Unless otherwise indicated, all reagents used in the following examples were obtained from commercial sources. Culture S. hygroscopicus MG2-10 [IJMNOQLhis] was maintained (WO 04/007709; Gregory et al., 2004) on medium 1 agar plates (see below) at 28 ° C. Spore reserve preparations were made in medium 1 after growth; were conserved in 20% w / v of glycerol: 10% w / v of lactose in distilled water, and stored at -80 ° C. In a 250 ml flask vegetative cultures were prepared by inoculating 0.1 ml of Reserve preparation frozen in 50 mL of medium 2 (see below).

The culture was incubated for 36 to 48 hours at 28 ° C, 300 rpm.

Method of production: Vegetative cultures were inoculated at 2.5-5% v / v in the medium 3. Culturing was performed for 6-7 days, 26 ° C, 300 rpm.

Feeding Procedure: The feeding / addition of cyclohexanecarboxylic acid is effected 24-48 hours after inoculation and fed to a final concentration of 1.2 mM unless otherwise indicated.

Medium 1: Component Source # Catalog Per L Corn infusion powder Sigma c-8160 2.5 g Difco yeast extract 0127-17 3 g Sigma C5929 calcium carbonate 3 g Iron sulphate, Sigma F8633 0.3 g BACTO agar 20 g Sigma S2760 wheat starch 10 g Water for 1 L Then, the medium was sterilized in an autoclave at 121 ° C, 20 minutes Medium 2: RapV7 seed medium Component By L Toasted Nutrisol (ADM Ingredients Ltd) 5 g Avedex W80 Dextrin (Deymer Ingredients Ltd) 35 g Corn infusion solids (Sigma) 4 g Glucose 10 g (NH4) 2 SO4 2 g Lactic acid (80%) 1.6 mL CaC03 (Caltec) 7 g Adjust the pH to 7.5 with 1 M NaOH Then, the medium was sterilized in an autoclave at 121 ° C, 20 min.

After sterilization, 0.16 mL of glucose was added to the 40% to every 7 mL of media.

Medium 3: MD6 medium (fermentation medium) Component By L Toasted Nutrisoy (ADM Ingredients Ltd) 30 g Corn Starch (Sigma) 30 g Avedex W80 Dextrin (Deymer I ngredients Ltd). 19 g Yeast (Allinson) 3 g Corn infusion solids (Sig ma) ig KH2PO4 2.5 g K2HPO4 2.5 g (NH4) 2 SO4 10 g NaCl 5 g CaCO3 (Caltec) 10 g MnCl2.4H2O 10 mg FeSO4JH2O 120 mg ZnSO4JH2O 50 mg MES (2-morpholinoethanesulfuric acid 21.2 g monohydrate) The pH is corrected to 6.0 with 1 M NaOH 0.4 mL of α-amylase was added before sterilization Sigma (BAN 250) to 1 L of medium.

The medium was sterilized for 20 minutes at 121 ° C.

After sterilization, 0.35 mL of fructose was added sterile at 40% and 0.10 mL of L-lysine (140 mg / mL in water, sterilized by filtration), at every 7 mL.

Medium 4: RapV7a seed medium Component By L Toasted Nutrisoy (ADM Ingredients Ltd) 5 g Avedex W80 Dextrin (Deymer Ingredients Ltd) 35 g Corn infusion solids (Sigma) 4 g (NH4) 2 SO4 2 g Lactic acid (80%) 1.6 mL CaCO3 (Caltec) 7 g Adjust to pH 7.5 with 1 M NaOH Then, the medium was sterilized in an autoclave at 121 ° C, 20 minutes Medium 5: MD6 medium / 5-1 (fermentation medium) Component By L Toasted Nutrisoy (ADM Ingredients Ltd) 15 g Avedex W80 Dextrin (Deymer Ingredients Ltd) 50 g Yeast (Allinson) 3 g Corn infusion solids (Sigma) ig KH2PO4 2.5 g K2HPO4 10 g (NH4) 2SO4 13 g NaCl 10 g CaCO3 (Caltec) 3.5 mg MnCl2.4H2O 15 mg MgSO4JH2O 150 mg FeS04JH2O 60 mg SAG 471 0.1 ml The medium was sterilized for 30 minutes at 121 ° C.

After sterilization, 15 g of fructose was added per L.

After 48 hours 0.5 g / L or L-lysine was added.

Analytical Methods Method A Injection volume: 0.005-0.1 mL (as required, depending on the sensitivity). HPLC was performed on Agilent cartridges "Spherisorb" "Rapid Resolution" SB C8, 3 microns, 30 mm x 2.1 mm, running a mobile phase of: Mobile phase A: 0.01% formic acid in pure water Mobile phase B: 0.01% formic acid in acetonitrile Flow rate: 1 mL / minute. A linear gradient of 5% B at 0 min was used, at 95% B at 2.5 min, retaining 95% B up to 4 min, returning to 5% B until the next cycle. The detection was by UV absorbance at 254 nm and / or electroaspersion ionization in mass spectrometry (positive or negative) using a Micromass Quattro-Micro instrument.

Method B Injection volume: 0.02 mL. HPLC was performed on a HDSSIL (ThermoHypersil-Keystone Ltd) BDS C18 column, 150 x 4.6 mm, maintained at 50 ° C, running a mobile phase of: Mobile phase A: acetonitrile (100 mL), trifluoroacetic acid (1 mL), 1 M ammonium acetate (10 mL), brought to 1 L with deionized water. Mobile phase B: deionized water (100 mL), trifluoroacetic acid (1 mL), 1 M ammonium acetate (10 mL), brought to 1 L with acetonitrile. Flow rate: 1 mL / minute. A linear gradient of 55% B - 95% B was used for 10 minutes, followed by 2 minutes at 95% B, 0.5 minutes at 55% B, and 2.5 minutes more at 55% B. compound was by UV absorbance at 280 nm.

Method C The HPLC system comprised an Agilent HP1100 and was performed on a BDS C18 Hypersil (ThermoHypersil-Keystone Ltd) column of 3 microns, 150 x 4.6 mm, maintained at 40 ° C, running a mobile phase of: Mobile phase A: deionized water. Mobile phase B: acetonitrile. Flow rate: 1 mL / minute. This system was coupled with a Bruker Daltonics Esqire3000 electrospray mass spectrometer. Positive-negative switching was used on a scan scale of 500 to 1, 000 Dalton. A linear gradient of 55% B - 95% B was used for 10 minutes, followed by 2 minutes at 95% B, 0.5 minutes at 55% B, and 2.5 minutes more at 55% B.

In vitro bioenzyme of anticancer activity The in vitro anticancer activity of the compounds was evaluated in a panel of 12 human tumor cell lines in a monolayer proliferation test at the Oncotest Testing Facility, Institute for Experimental Oncology, Oncotest GmbH, Freiburg . The characteristics of the 12 selected cell lines are summarized in Table 2.

TABLE 2 Test cell lines # Cell line Features 1 MCF-7 Breast, standard NCL 2 MDA-MB-231 Breast-PTEN positive, resistant to 17-AAG 3 MDA-MB-468 Mama-PTEN negative, resistant to 17-AAG 4 NCI-H460 Lung, standard NCI 5 SF-268 SNC, standard NCI 6 OVCAR-3 mutated ovarian-p85, AKT amplified 7 A498 Renal, high MDR expression 8 GXF 25 1 L Gastric 9 MEXF 394NL Melanoma 10 UXF 1138L Uterus 1111 LLNNCCAAPP Prostate-PTEN negative 12 DU145 Prostate-positive PTEN The Oncotest cell lines were established from xenografts of human tumor as described by Roth et al., 1999. The origin of donor xenografts was described by Fiebig et al., 1992. Other lines of Cells were obtained from NCI (H460, SF-268, OVCAR-3, DU145, MDA-MB- 231, MDA-MB-468), or were purchased from DSMZ, Braunschweig, Germany (LNCAP). All cell lines, unless specified otherwise Thus, they were developed at 37 ° C in a humid atmosphere (95% air, 5% of CO2), in a "ready mix" medium containing RPMI 1640 medium, 10% of fetal calf serum and 0.1 mg / mL of gentamicin (PAA, Coibe, Germany).

Monolayer Test - Protocol 1: A modified propidium iodide test was used to determine the effects of test compounds on the growth of twelve human tumor cell lines (Dengler et al., 1995). Briefly, the cells were harvested from exponential phase cultures by trypsinization, counted and deposited in 96-well flat bottom microtiter plates at cell density dependent on the cell line (5-10,000 viable cells / well). After recovery for 24 hours to allow the cells to resume exponential growth, 0.01 mL of culture medium (6 control wells per plate) or culture medium containing 39-demethoxyrapamycin was added to the wells. Each concentration was deposited in triplicate. The 39-demethoxyrapamycin was applied in two concentrations (0.001 mM and 0.01 mM). After 4 days of continuous incubation, the cell culture medium with or without 39-demethoxyrapamycin was replaced with 0.2 mL of an aqueous solution of propidium iodide (Pl) (7 mg / L). To measure the proportion of living cells, the cells were permeabilized by freezing the plates. After thawing the plates the fluorescence was measured using the Cytofluor 4000 microplate reader (excitation 530 nm, emission 620 nm), giving a direct relationship for the total number of viable cells. Growth inhibition was expressed as treatment / control x 100 (% T / C). For the active compounds, the IC50 and Cl70 values were estimated by plotting the concentration of compound against the viability of the cells Monolayer Test - Protocol 2: The human tumor cell lines of the National Cancer Institute (NCI) cancer screening panel were developed in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine ( Boyd and Paull, 1995). The cells were inoculated into 96-well microtiter plates in 0.1 mL, at plaque densities ranging from 5,000 to 40,000 cells / well, depending on the doubling time of the individual cell lines. After inoculation of the cells, the microtiter plates were incubated for 24 hours at 37 ° C, 5% CO2, 95% air and 100% relative humidity, before adding the experimental drugs. After 24 hours, two plates of each cell line were fixed in situ with trichloroacetic acid (TCA) to represent a measurement of the cell population of each cell line at the time of drug addition (Tz). The experimental drugs were solubilized in dimethyl sulfoxide at 400 times the final maximum concentration of the desired test and stored frozen before use. At the time of drug addition, an aliquot of the frozen concentrate was thawed and diluted to 2 times the final maximum concentration of the desired test with complete medium containing 0.05 mg / mL of gentamicin. Four additional 10-fold serial dilutions or Vz log were made to provide a total of 9 drug concentrations plus control. Aliquots of 0.1 mL were added of these different dilutions of drug to the appropriate microtitre wells, which already contained 0.1 mL of medium, resulting in the final concentrations of drug required. After the addition of the drug the plates were incubated an additional 48 hours at 37 ° C, 5% C02, 95% air and 100% relative humidity. For adherent cells, the test was terminated by the addition of cold TCA. The cells were fixed in situ by the gentle addition of 0.05 mL of cold TCA at 50% (w / v) (final concentration 10% TCA), and incubated 60 minutes at 4 ° C. The supernatant was discarded and the plates were washed 5 times with tap water and air dried. To each well was added solution of sulforhodamine B (SRB) (0.1 mL) at 0.4% (w / v) in 1% acetic acid, and the plates were incubated 10 minutes at room temperature. After staining, the unbound dye was removed by washing three times with 1% acetic acid and the plates were air-dried. Subsequently the bound dye was solubilized with 10 mM trizma base, and the absorbance was read in an automatic plate reader at a wavelength of 515 nm. For cells in suspension, the method was the same except that the test was terminated by fixing the sedimented cells at the bottom of the wells by gently adding 0.05 mL of 80% TCA (final concentration 16% TCA). Using the seven absorbance measurements [zero time, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration values (Ti)], the growth percentage was calculated for each one. of the drug concentration values. The inhibition percentage of growth was calculated as: [(Ti-Tz) / (C-Tz)] x 100, for concentrations where Ti = Tz [(Ti-Tz) / Tz] x 100, for concentrations where Ti < Tz. Three parameters of dose response were calculated for each experimental agent. The inhibition of 50% growth (Gl50) was calculated from [(Ti-Tz) / (C-Tz)] x 100 = 50, which is the concentration of drug that results in a 50% reduction in net protein increase in the control cells during incubation with the drug (measured by SRB staining). The drug concentration that results in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (drug concentration that results in a 50% reduction of the protein, measured at the end of the drug treatment compared to the beginning), which indicates a net loss of cells after treatment, was calculated from [(Ti -Tz) / Tz] x 100 = -50. Lines of cells resistant to multiple drugs in the panel 60 cell lines were identified by NCI as cell lines with high P-gp content, identified by rhodamine B effusion (Lee et al., 1994) and by PCR detection of mdr-1 mRNA (Álvarez et al. nineteen ninety five).

Pharmacokinetic Analysis - Protocol 1 The test compounds were prepared in a vehicle consisting of 4% ethanol, 5% Tween-20, 5% polyethylene glycol 400 in NaCl 0.15 M. A single dose of 10 mg / kg was administered orally, or 3 mg / kg iv to groups of female CD1 mice (3 mice for each compound per time point). At 0 min, 4 min, 15 min, 1 hour, 4 hours and 24 hours the groups of animals were sacrificed and the blood and brain of each mouse were extracted for further analysis. The brain samples were frozen in liquid nitrogen and stored at -20 ° C. A minimum of 0.2 mL of whole blood was collected from each animal in tubes containing ethylenediaminetetraacetic acid (EDTA) as an anticoagulant; mixed well and stored at -20 ° C.

Pharmacokinetic Analysis - Protocol 2 To prepare the dosing solution, 5 mg of the test compound was dissolved in 100 μL of ethanol, resulting in a 50 mg / mL compound solution. Then, the solution was diluted to 2 mg / mL adding approximately 2.4 mL of 0.5 M NaCl (0.95 w / v saline), 5% v / v of Tween 20 and 5% v / v of PEG 400 (final concentration of ethanol of 4% v / v). Groups of 3 female Balb C mice were administered a single dose of 10 mg / kg orally, or 2 mg / kg iv of test compound, at a concentration of 10 mg / kg (oral) or 2 mg / kg (iv). At 5 min, 15 min, 60 min, 4 h, 8 h and 24 h, the groups were sacrificed and samples of whole blood of approximately 0.2 mL were recovered in EDTA, to give a final concentration of 0.5 mM; additionally the brains were extracted.

Both the whole blood and the brains were rapidly frozen in liquid nitrogen and stored at -20 ° C until they were released in solid carbon dioxide for analysis.

Analysis of the pharmacokinetic study samples The analysis was performed by ASI Limited, (St. George's Hospital Medical School, London). The concentration of the test compound in the blood and brain samples was determined by HPLC with MS detection. Control blood samples were obtained free of test compound from Harian Sera-Lab Limited (Loughborough, England). Zero-time brain samples were provided as control brain samples without test compound.

Preparation of brain samples: One hemisphere of each brain was homogenized with 5 mL of water. Extraction of samples The control or test sample from the brain or mouse blood (0.05 L), standard internal solution (0.1 mL), 5% zinc sulfate solution (0.5 mL) and acetone (0.5 mL), are pipetted into a 2 mL polypropylene tube (Sarstedt Limited, Beaumont Leys, Leicester, United Kingdom), and then the contents were mixed for a minimum of 5 minutes (IKA-Vibrax-VXR mixer, Merck (BDH) Limited, Poole Dorset, United Kingdom). Afterwards, the tubes centrifuged in a microcentrifuge for a minimum of 2 minutes. The solvent layer was decanted into a 4.5 mL polypropylene tube containing sodium hydroxide (0.1 M, 0.1 mL) and methyl tert-butyl ether (MTBE, 2 mL). Then the tube was mixed for a minimum of 5 minutes (IKA-Vibrax-VXR mixer) and then centrifuged at 3,500 rpm for 5 minutes. The solvent layer was transferred to a 4.5 mL polypropylene conical tube, placed in a SpeedVac®, and evaporated to dryness. The dried extracts were reconstituted with 0.15 mL of 80% methanol and mixed for a minimum of 5 minutes (IKA-Víbrax-VXR mixer) and centrifuged at 3,500 rpm for 5 minutes. The extract was transferred to tubes of an autosampler (NLG Analytical, Adelphi Mill, Bollington, Cheshire, United Kingdom) and placed in the autosampler tray which was placed at room temperature. The autosampler was programmed to inject an aliquot of 0.03 mL of each extract into the analytical column.

EXAMPLE 1 Fermentation and isolation of test compounds 1. 1 Fermentation and isolation of 39-demethoxyrapamycin 39-demethoxyrapamycin was produced by growing cultures of S. hygroscopicus MG2-10 [IJMNOQLhis] and feeding with cyclohexanecarboxylic acid (CHCA) as described below. S. hygroscopicus MG2-10 [IJMNOQLhis] was produced introducing into the MG2-10 strain described in WO 2004/007709 a plasmid containing the rapl, rapJ, rapM, rapN, rapO, rapQ and rapL genes. The gene cassette was constructed with the rapL gene containing a histidine tag in a 5 'frame. As described in WO 2004/007709, the plasmid also contained a transfer origin and an apramycin resistance marker for the transformation of MG2-10 by conjugation and selection of the exconjugates, and a phiBTI binding site for specific integration of site on the chromosome. The isolation of each of these genes and the method used to construct the gene cassettes containing post-PKS gene combinations were performed as described in WO 2004/007709. Liquid culture A vegetative culture of S. hygroscopicus MG2-10 [IJMNOQLhis] was cultivated as described in the Materials and Methods. Production cultures were inoculated with a vegetative culture at 0.5 mL in 7 mL of medium 3 in 50 mL tubes. Culturing was carried out for 7 days, 26 ° C, 300 rpm. Samples of 1 mL were extracted with acetonitrile 1: 1 with stirring for 30 min; they were centrifuged 10 min, 13,000 rpm, and analyzed and quantified according to method B of analysis (see Materials and Methods). The product was confirmed by mass spectrometry using the C method of analysis (see Materials and Methods). It was proposed that the rapamycin analog observed was the desired 39-demethoxyrapamycin based on the analytical data described below in the Characterization. Fermentation A primary vegetative culture of S. hygroscopicus MG2-10 [IJMNOQLhis] was grown in medium 4, essentially as described in the Materials and Methods. A secondary vegetative culture was inoculated in 4 to 10% v / v medium, 28 ° C, 250 rpm, for 24 h. The vegetative cultures were inoculated at 5% v / v in medium 5 (see Materials and Methods) in a 20 L fermentor. Cultivation was carried out for 6 days at 26 ° C; it remained 0.5 vvm. > 30% of dissolved oxygen altering the peripheral speed of the impeller, minimum peripheral speed of 1.18 ms "1, maximum peripheral speed of 2.75 ms'1.The supply of cyclohexanecarboxylic acid was made at 24 hours and 48 hours after the inoculation to give a final concentration of 2 mM Extraction and purification The fermentation broth (30 L) was stirred with an equal volume of methanol for 2 hours and then centrifuged to pellet the cells (10 min, 3500 rpm) .The supernatant was stirred with DiaIon® HP20 resin (43 g / L) for 1 hour and then filtered.The resin was washed in batches with acetone to entrain the rapamycin analog and the solvent was removed in vacuo, then the aqueous concentrate was diluted to 2 ml. L with water and extracted with EtOAc (3 x 2L) The solvent was removed in vacuo to give a brown oil (20.5 g) The extract was dissolved in acetone, dried on silica, applied to a silica column (6 x 6.5 cm in diameter) and eluted with a step gradient of acetone / hexane (20% -40%). The fractions containing the rapamycin analog were combined and the solvent was removed in vacuo. The residue (2.6 g) was further subjected to chromatography (in three batches) on Sephadex LH20, eluting with 10:10 chloroform / heptane / ethanol: 1. The semipurified rapamycin analogue (1.7 g) was purified by reverse phase preparative HPLC. (C18) using a Gilson HPLC kit, eluting a BDS C18 Phenomenex 21.2 x 250 mm Luna, 5 μm column, with 21 mL / min of 65% acetonitrile / water. The purest fractions (identified by analytical HPLC, method B) were combined, and the solvent was removed in vacuo to give 39-demethoxyrapamycin (563 mg). Characterization The 1H NMR spectrum of 39-demethoxyrapamycin was equivalent to that of a standard (P. Lowden, Ph.D Dissertation, University of Cambridge, 1997). Analysis of LCMS and LCMSn of the culture extracts showed that the ratio m / z of the rapamycin analog is 30 units lower than rapamycin, consistent with the absence of a methoxy group. Observed ions: [M-H] 882.3, [M + NH4] + 901.4, [M + Na] + 906.2, [M + K] + 922.2. Fragmentation of the sodium adduct gave the predicted ions for 39-demethoxyrapamycin following a previously identified fragmentation path (J. A. Reather, Ph.D Dissertation, University of Cambridge, 2000). This mass spectrometry fragmentation data narrows the rapamycin analog region where the loss of a methoxy occurs to fragment C28-C42, which contains the cyclohexyl moiety. Fragmentation pathway of 39-demethoxyrrapamycin C32H1Í [INNGOS? - Moso exncto: 614.33 Or seivntlo: 614.0 These mass spectrometry fragmentation data are completely consistent with the structure of 39-demethoxyrapamycin. 1. 2 Fermentation and isolation of 27-O-desmethyl-39-demethoxyrapamycin 27-0-desmethyl-39-demethoxyrapamycin was produced by growing cultures of S. hygroscopicus MG2-10 [JMNOLhis] and feeding with cyclohexanecarboxylic acid (CHCA) as described below. S. hygroscopicus MG2-10 [JMNOLhis] was produced by introducing into the strain MG2-10 described in WO 2004/007709 a plasmid containing the rapJ, rapM, rapN, rapO, and rapL genes. The gene cassette was constructed with the rapL gene containing a histidine tag in a 5 'frame. As described in WO 2004/007709, the plasmid also contains a transfer origin and apramycin resistance marker for the transformation of MG2-10 by conjugation and selection of exconjugates, and a binding site of phiBT1 for an integration site-specific chromosome. The isolation of each of these genes and the method used for the construction of the gene cassettes containing the post-PKS gene combinations were made as described in WO 2004/007709. Liquid culture A vegetative culture of S. hygroscopicus MG2-10 [JMNOLhis] was cultivated as described in the Materials and Methods. They were inoculated production cultures with vegetative culture at 0.5 mL in 7 mL of medium 3 in 50 mL tubes. Culturing was carried out for 7 days, 26 ° C, 300 rpm. Samples of 1 mL were extracted with acetonitrile 1: 1 with stirring for 30 min; they were centrifuged 10 min, 13,000 rpm, and analyzed and quantified according to method B of analysis (see Materials and Methods). The product was confirmed by mass spectrometry using the C method of analysis (see Materials and Methods). It was proposed that the rapamycin analog observed was the desired 27-O-desmethyl-39-demethoxyrapamycin based on the analytical data described below in the Characterization. Fermentation A primary vegetative culture of S. hygroscopicus MG2-10 [JMNOLhis] was grown in medium 2, essentially as described in the Materials and Methods. A secondary vegetative culture was inoculated in medium 2 to 10% v / v, 28 ° C, 250 rpm, for 24 h. The vegetative cultures were inoculated at 10% v / v in medium 5 (see Materials and Methods) in a 20 L fermentor. Cultivation was carried out for 6 days at 26 ° C; it remained 0.75 vvm. > 30% of dissolved oxygen altering the peripheral speed of the impeller, minimum peripheral speed of 1.18 ms "1, maximum peripheral speed of 2.75 ms" 1. The cyclohexanecarboxylic acid feed was made at 24 hours and 48 hours after inoculation to give a final concentration of 2 mM.

Extraction and purification The fermentation broth (15 L) was stirred with an equal volume of methanol for 2 hours and then centrifuged to pellet the cells (10 min, 3500 rpm). The supernatant was stirred with Diaion® HP20 resin (43 g / L) for 1 hour and then filtered. The resin was washed in batches with acetone to entrain the rapamycin analog and the solvent was removed in vacuo. Then, the aqueous concentrate was diluted to 2 L with water and extracted with EtOAc (3 x 2L). The solvent was removed in vacuo to give a brown oil (12 g). The extract was dissolved in acetone, dried on silica, applied to a silica column (4 x 6.5 cm in diameter) and eluted with a step gradient of acetone / hexane (20% -40%). The fractions containing the rapamycin analog were combined and the solvent was removed in vacuo. The residue (0.203 g) was enriched by preparative reverse phase HPLC (C18) using a Gilson HPLC kit, eluting a BDS C18 Phenomenex 21.2 x 250 mm Luna, 5 μm column, with 21 mL / min of acetonitrile 65 % /Water. The purest fractions (identified by analytical HPLC, method B) were combined, and the solvent was removed in vacuo to give a residue (25.8 g). The residue was purified by reverse phase preparative HPLC (C18) using a Gilson HPLC kit, eluting a BDS C18 Hypersil 4.6 x 150 mm 3 μm column, with 1 mL / min of 60% acetonitrile / water. The purest fractions (identified by analytical HPLC, method B) were combined, and the solvent was removed in vacuo to give 27-O-demethyl-39- Demethoxyrapamycin (19.9 mg). Characterization The 1H and 13C NMR spectra were consistent with the structure of 27-0-desmethyl-39-demethoxyrapamycin, and the assignments are shown in Table 3 below.

TABLE 3 NMR data of 27-O-demethyl-39-demethoxyrapamycin in 500 MHz CDCI3 for 1 H-NMR and 125 for 13 C-NMR Position? -RMN C-RMN Correlations d ppm Multiplicity, Hz COZY d ppm HMBC 1H to 13C - - 169.3 - 5.21 br. d, 5 H-3 51.3 C-1, C-3, C-4, C-6 and C-8 2.30 m, complex H-2, H-4 27.0 C-1.C-2.C-4 and C- 5 1J8 m, complex H-3, H-5 20.7 C-2.C-3.C-5, and C-6 1.43 m, complex 1.67 m, complex H-4, H-6 25.1 C-3.C -4, and C-6 1.36 m, complex 3.50 ddd, 16, 10.5, 5 H-5 46.3 C-2.C-4.C-5, and C-8 3.30 ddd, 16,9.5.6 -. N. - - 166.5 - TABLE 3 (Continued) Position 'H-NMR "C-NMR Correlations d ppm Multiplicity, COZY d ppm HMBC 1H at 13C Hz 9 - - 194.2 - 10 - - 98.5 - 11 2.02 m, complex H-11CH3, 32.0 C-9.C-10. C-12.C- H-12 13y H-CH3 11-CH3 0.91 d, 6.5 H-11 16.0 C-10, C-11, and C-12 12 1.61 m, complex H-11.H- 26.8 C-10, C-11, C-13 13, C-14y11-CH3 13 1.66 m, complex H-12, H-30.5 C-1, C-3, C-4, C-6y 1. 43 m, complex 14 C-8 14 3.95 m, complex H-13, H-70.8 C-11, C-12, C-14 and 15 C-15 15 1.83 m, complex H-14, H-35.1 C-13 .C-14.C-16, and 1. 44 m, complex 16 C-17 16 4.11 dd, 5.5, 5.5 H-15 83.6 C-1, C-3, C-4, C-6 C-8 I6-OCH3 3.11 br. s - 55.9 C-16, C-15 and C-17 17 -. 17 - - - 135.6 - I7-CH3 1.77 s - 13.3 C-16, C-17 and C-18 18 6.09 d, 11 H-19 130.1 C-16, C-17, C-19, C-20y17-CH3 19 6.35 dd, 14.5, 11 H-18, H- 126.8 C-17, C-18, C-20y 20 C-21 20 6.24 dd, 14.5, 10.5 H-19, H- 132.8 C-18.C-19 .C-21 and 21 C-22 21 5.99 dd, 15, 10.5 H-20, H-128.2 C-19, C-20, C-22 and 22 C-23 22 5.48 dd, 15.8 H-21.H - 137.0 C-20.C-21.C- 23 23.C-24 and 23-CH3 23 2.29 m, complex H-22, 23-35.2 C-21.C-22.C- CH3, H- 24.C-25 and 23-CH3 24 TABLE 3 (Continued) Position? -RMN "C-RMN Correlations d ppm Multiplicity, COZY d ppm HMBC 1H at 13C Hz 31 3.62 dq, 11, 6.5 H-30, 31-44.2 C-29, C-30, C-32, C-CH3 33 and 31-CH3 3I-CH3 1.00 d, 6.5 H-31 15.8 C-30, C-31 and C-32 32 -. 32 - - - 208.4 - 33 2.70 dd, 17.5, 5.5 H-34 40.5 C-31, C-32, C-34 and 2. 52 dd, 17.5, 4 C-35 34 5.10 ddd, 7, 5.5, 4 H-33, H- 67.3 C-1, C-32, C-33, C-35 35, C-36 and 35-CH3 35 1.90 m, complex H-34, 35-34.1 C-33.C-34.C-36.C- CH3, H- 37 and 35-CH3 36 35-CH3 0.84 d, 6.5 H-35 15.2 C-34 .C-35 and C-36 36 1.44 m, complex H-35, H-39.6 C-34, C-35, C-37, C-1.20 m, complex 37 38, C-42 and 35-CH3 37 1.35 m, complex complex 39.0 C-35.C-36.C-38.C- 39, C-41 and C-42 38 1.46-m, complex complex 33.6 * - 0.69 39 1.46-m, complex complex 40.7 - 0.69 40 3.99 m, complex complex 75.5 C-38, C-39, C-41 and C-42 41 1.46-m, complex complex 40.8 - 0.69 42 1.46-m, complex complex 33.6 * - 0.69 * Value shown as double integration compared to other 13C values in the 13C-NMR spectrum. * The stereochemistry has not been determined, since more NMR experiments are required (such as NOESY 1 D and 2D) because the axial and equatorial methylene 1H has not been assigned. The LCMS and LCMSn analysis of the culture extracts showed that the m / z ratio for the rapamycin analog is 44 units of mass lower than rapamycin, which is consistent with the absence of a methyl and methoxy group. Observed ions: [M-H] 868J, [M + NH 4] + 887.8, [M + Na] + 892.8. Fragmentation of the sodium adduct gave the predicted ions for 27-O-demethyl-39-demethoxyrapamycin following a previously identified fragmentation path (shown above) (J. A. Reather, Ph.D Dissertation, University of Cambridge, 2000). These mass spectrometry fragmentation data narrow the rapamycin analog region where the loss of a methoxy occurs to the C28-C42 fragment, which contains the cyclohexyl moiety, and narrows the rapamycin analog region where the loss of a O-methyl to the C15-C27 fragment. These mass spectrometry fragmentation data are completely consistent with the structure of 27-O-demethyl-39-demethoxyrapamycin. 1. 3 Fermentation and isolation of 16-0-desmethyl-27-0-desmethyl-39-demethoxyrapamycin 16-0-desmethyl-27-0-desmethyl-39-demethoxyrapamycin was produced by growing cultures of S. hygroscopicus MG2-10 [IJNOLhis] and feeding with cyclohexanecarboxylic acid (CHCA) as described below. S. hygroscopicus MG2-10 [IJNOLhis] was produced by introducing into the strain MG2-10 described in WO 2004/007709 a plasmid containing the rapl, rapJ, rapN, rapO and rapL genes. The gene cassette was constructed with the rapL gene containing a histidine tag in a 5 'frame. As described in WO 2004/007709, the plasmid also contains a transfer origin and an apramycin resistance marker for the transformation of MG2-10 by conjugation and selection of exconjugates, and a binding site of phiBT1 for a site-specific integration in the chromosome. The isolation of each of these genes and the method used for the construction of the gene cassettes containing the post-PKS gene combinations were made as described in WO 2004/007709. Liquid culture A vegetative culture of S. hygroscopicus MG2-10 was cultivated [IJNOLhis] as it is deciphered in the Materials and Methods. Production cultures were inoculated with a vegetative culture at 0.5 mL in 7 mL of medium 3 in 50 mL tubes. Culturing was carried out for 7 days, 26 ° C, 300 rpm. HE extracted 1 mL samples with acetonitrile 1: 1 with shaking for 30 min; they were centrifuged 10 min, 13,000 rpm, and analyzed and quantified according to method B of analysis (see Materials and Methods). The product was confirmed by mass spectrometry using the C method of analysis (see Materials and Methods). It was proposed that the rapamycin analog observed was the desired 16-O-desmethyl-27-O-demethyl-39-demethoxyrapamycin based on the analytical data described below in the Characterization. Fermentation A primary vegetative culture of S. hygroscopicus was grown MG2-10 [IJNOLhis] in medium 2, essentially as described in Materials and methods. A secondary vegetative culture was inoculated in medium 2 to 10% v / v, 28 ° C, 250 rpm, for 48 h; and a tertiary culture was inoculated 10% v / v, 28 ° C, 250 rpm, for 24 h. The vegetative cultures were inoculated at 10% v / v in medium 5 (see Materials and Methods) in 3 fermentors of 7 L. Cultivation was carried out for 6 days at 26 ° C; it remained 0.75 vvm. > 30% dissolved oxygen by altering the peripheral speed of the impeller, minimum peripheral speed of 0.94 ms "1, maximum peripheral speed of 1.88 ms" 1.

The cyclohexanecarboxylic acid feed was made 24 hours after the inoculation to give a final concentration of 1 mM. L-lysine was fed at t = 0. Extraction and purification The fermentation broth (12 L) was stirred with an equal volume of methanol for 2 hours and then centrifuged to pellet the cells (10 min, 3500 rpm). The supernatant was stirred with Diaion® HP20 resin (43 g / L) for 1 hour and then filtered. The resin was washed in batches with acetone to entrain the rapamycin analog and the solvent was removed in vacuo. Then, the aqueous concentrate was diluted to 2 L with water and extracted with EtOAc (3 x 2L). The solvent was removed in vacuo to give a brown oil (8.75 g). The extract was dissolved in acetone, dried on silica, applied to a silica column (4 x 6.5 cm in diameter) and eluted with a step gradient of acetone / hexane (20% -40%). The fractions containing the rapamycin analog were combined and the solvent was removed in vacuo. The residue (0.488 g) was subjected to chromatography (in three batches) on Sephadex LH20, eluting with chloroform / heptane / ethanol, 10: 10: 1. The fractions containing the rapamycin analog were combined and the solvent was removed in vacuo. The semipurified rapamycin analog (162 mg) was purified by reverse phase preparative HPLC (C18) using a Gilson HPLC kit, eluting a BDS C18 Phenomenex 21.2 x 250 mm Luna, 5 μm column, with 21 mL / min of acetonitrile. % /Water. The purest fractions (identified by analytical HPLC, method B) were combined, and the solvent was removed in vacuo to give 16-O-demethyl-27-O-demethyl-39-demethoxyrapamycin (44J mg). Characterization The LCMS and LCMSn analysis of the culture extracts showed the presence of a new rapamycin analog that elutes much earlier than the other 39-demethoxy analogs. The m / z ratio for the various ions of the rapamycin analog is 58 units of mass lower than rapamycin, which is consistent with the absence of two O-methyl groups and one methoxy group. Observed ions: [MH] "854.7, [M + NH4] + 877.8, [M + Na] + 892.7, [M + K] + 908.8 Fragmentation of the sodium adduct gave the predicted ions for 16-0-demethyl- 27-O-desmethyl-39-demethoxyrapamycin following a previously identified fragmentation path (shown above) (JA Reather, Ph.D Dissertation, University of Cambridge, 2000) These mass spectrometry fragmentation data narrows the analogue region of rapamycin where the loss of a methoxy occurs to the C28-C42 fragment, which contains the cyclohexyl moiety, and narrows the region of the rapamycin analogue where the loss of the O-methyl groups occurs to the C15-C27 fragment. Fragmentation of mass spectrometry are completely consistent with the structure of 16-O-desmethyl-27-O-desmethyl-39-demethoxyrapamycin.

EXAMPLE 2 In vitro bioassays of the anticancer activity In vitro evaluation of the anticancer activity of 39-demethoxyrapamicin An in vitro evaluation of the anticancer activity of 39-demethoxyrapamycin in a panel of 12 human tumor cell lines in a monolayer proliferation test, using a modified propidium iodide test as described above in Protocol 1 of the General Methods. The results are presented in table 4 below; each result represents the average of duplicate experiments. Table 5 shows the IC50 and IC or the compounds and rapamycin in all the cell lines tested.

TABLE 4 TABLE 5 In vitro evaluation of the selective anticancer activity of multidrug resistance (MDR) of 39-demethoxyrapamycin The selective MDR anticancer activity of 29-demethoxyrapamycin was evaluated in the panel of human NCL 60 tumor cells in a proliferation test of monolayer, using a SRB-based test as described in Protocol 2 of the Materials and Methods. The results are presented in table 6 below.

TABLE 6 In vitro activity against cell lines expressing MDR It can be seen that, with the exception of one cell line, 39-demethoxyrapamycin has equivalent or greater efficacy against cell lines expressing MDR compared to rapamycin.

EXAMPLE 3 ADME tests in vitro Penetration test of Caco-2 confluent Caco-2 cells (Li, AP, 1992; Grass, GM et al., 1992, Volpe, DA and others, 2001), in a 24-well Corning Costar Transwell format, were provided by In Vitro Technologies Inc. (IVT Inc., Baltimore, Maryland, USA). The apical chamber contained 0.15 mL of Hank's balanced buffer solution (HBBS) pH 7.4, 1% DMSO, 0.1 mM Lucifer yellow. The basal chamber contained 0.6 mL of HBBS pH 7.4, 1% DMSO. Controls and tests were incubated at 37 ° C in a humid incubator, which was shaken at 130 rpm for 1 h. The yellow Lucifer penetrates only through the paracellular way (between the narrow junctions); a high apparent penetration (Pap) for yellow Lucifer indicates cell damage during the test and all these wells were rejected. Propranolol (good passive penetration without known carrier effects) and acebutalol (low passive penetration attenuated by active effusion by P-glycoprotein) were used as reference compounds. The compounds were tested in a uni- and bidirectional format by applying the compound to the apical chamber or basal (at 0.01 mM). The compounds of the apical or basal chambers are analyzed by means of HPLC-MS (Method A, see Materials and Methods).

The results were expressed as apparent penetration, Pap (nm / s), and as the Flow Ratio (A to B versus B to A).

Volume of receptor? [Receptor] Pap (nm / s) = x Area x [donor]? Time Recipient volume: 0.6 ml (A> B) and 0.15 ml (B> A) Monolayer area: 0.33 cm2 ? Time: 60 min A positive value of the Flow Ratio indicates active effusion from the apical surface of the cells.

Stability test of human liver microsomes (HLM) The liver homogenates provide a measure of the inherent vulnerability of the compounds to Phase I (oxidative) enzymes including CYP450's (eg, CYP2C8, CYP2D6, CYP1A, CYP3A4, CYP2E1), esterases, amidases and flavin monooxygenases (FMOs).

The half-life (T? / 2) of the test compounds was determined by exposure to human liver microsomes, monitoring their disappearance over time through LC-MS. The compounds at 0.001 M were incubated 40 min at 37 ° C, 0.1 M Tris-HCl, pH 7.4, with the fraction microsomal subcellular human liver at 0.25 mg / mL protein and saturation concentrations of NADPH as a cofactor. At intervals they are added to the acetonitrile test samples to precipitate the protein and stop the metabolism. The samples were centrifuged and analyzed for the original compound using analytical method A (see Materials and Methods). TABLE 7 Results of the ADME test in vitro EXAMPLE 4 In vitro binding tests FKBP12 The FKBP12 is reversibly split into the chemical denaturing chemical guanidinium hydrochloride (GdnHCI), and the unfolding can be monitored by changing the protein's fluorescence of the protein.

(Main and others, 1998). The ligands that bind specifically to the FKBP12 and stabilize it in its native state, deviating the denaturation curve in such a way that the protein unfolds at higher concentrations of the chemical denaturant (Main et al., 1999). From the difference in stability, the binding constant of the ligand can be determined using equation 1. where AGpp is the apparent difference of free energy of splitting between the free form and the bound form,? G ° is the free energy of unfolding in water of the free protein, [L] is the concentration of the ligand and Kd is the dissociation constant of the protein-ligand complex (Meiering et al., 1992). The free energy of the split can be related to the midpoint of the splitting transition using the following equation:? G? J? = D??, [] S0% (2) where mo-N is a constant for a given protein and given denaturant, and is proportional to the change in the degree of exposure of the residues to the cleavage (Tanford, 1968, and Tanford, 1970), and [D] s or% is the concentration of denaturant that corresponds to the midpoint of the split. It is defined here ?? G ^, the stability difference of FKBP12 with rapamycin and an unknown ligand (at the same concentration of ligand), as: "G¿_" = < mD_N > ? [] 50O / 0 (3) where < mD.N > is the average m value of the split-off transition and? [D] 50% is the difference in midpoints for the split-rapamycin-FKBP12 transition and the unfolding transition of the unknown ligand-FKBP12 complex. Under the conditions where [L] > K, then ?? GD_N can be related to the relative Kd of the two compounds by means of equation 4: where Kdrap is the dissociation constant for rapamycin and K * is the dissociation constant for unknown ligand X. Therefore, For the determination of the Kd of 39-demethoxyrapamycin, the denaturation curve was adjusted to generate values for mD.N and [D] 5o% > which were used to calculate an average m value, < mo-N > V? [£ > ], and By '° so much K. The value of the literature of? ^ Of 0.2 nM is used.

TABLE 8 Results of the binding test of FKBP12 in vitro mTOR Inhibition of mTOR can be established indirectly by measuring the degree of phosphorylation of the surrogate markers of the mTOR pathway and p70S6 kinase and S6 (Brunn et al., 1997, Mothe-Satney et al., 2000; Tee and Proud , 2002; Huang and Houghton, 2002). HEK293 cells were cotransfected with mTOR labeled with FLAG and Raptor labeled with myc, were cultured for 24 h, and then deprived of serum overnight. The cells were stimulated with 100 nM insulin and then harvested and lysed by 3 freeze / thaw cycles. The lysates were pooled and equal amounts were immunoprecipitated with FLAG antibody to the mTOR / Raptor complex. The immunoprecipitates were then processed: the samples treated with compound (0.00001 to 0.003 mM) were preincubated 30 min at 30 ° C with FKBP12 / rapamycin, FKBP12 / 39-demethoxyrapamycin, or vehicle (DMSO); the untreated samples were incubated in kinase buffer: The immunoprecipitates were then subjected to an in vitro kinase test in the presence of 3 mM ATP, 10 mM Mn2 + and GST-4E-BP1 as substrate. The reactions were stopped with 4x sample buffer and then subjected to 15% PAGE / SDS; they were transferred wet to a PVDF membrane and then examined for phospho-4E-BP1 (T37 / 46). Alternatively, HEK293 cells were seeded in 6-well plates and pre-incubated for 24 h and then deprived of serum overnight. Then, the cells were pretreated with vehicle or compound for 30 min at 30 ° C; then they were stimulated with 100 nM insulin for 30 min at 30 ° C and lysed by 3 freeze / thaw cycles; then they were analyzed to determine the protein concentration. Equal amounts of protein were loaded and separated on PAGE / SDS gels. Then, the protein was transferred wet to PVDF membranes and examined for phospho-S6 (S235 / 36) or phospho-p70 S6K (T389). The results of these experiments are summarized in Figures 1A and 1B. EXAMPLE 5 In vitro P-gp substrate test Cell lines The cell lines used in the present study (MACL MCF7 and MACL MCF7 ADR) were provided by the National Cancer Institute, USA. UU The cells were routinely passed once or twice a week. They were kept in culture no more than 20 passes. All cells were grown at 37 ° C in a humid atmosphere (95% air, 5% CO2) in RPMI 1640 medium (PAA, Cólbe, Germany) supplemented with 5% fetal calf serum (PAA, Cólbe, Germany ) and 0.1% gentamicin (PAA, Cólbe, Germany). Test Protocol To determine the effects of 39-demethoxyrapamycin, a modified propidium iodide test based on protocol 1 described above was used (Dengler et al., 1995). Briefly, exponentially cultured cells were harvested by means of trypsinationwere counted and deposited in 96-well flat bottom microtiter plates at a cell density of 5,000 cells / well. After 24 h recovery to allow the cells to resume exponential growth, 0.01 mL of verapamil was added to the cells at a concentration of 0.18 mg / mL or 0.01 mL of culture medium, to produce a final concentration of verapamil in the wells of 0.01 mg / mL. In previous experiments it was found that this concentration is safe for the cells. 0.01 mL per well of culture medium containing 39-demethoxyrapamycin, taxol or culture medium alone (for the control wells) was added. The compounds were applied in triplicate in 8 concentrations in semilogarithmic steps from 0.03 mM to 10 nM. After 3 days of continuous exposure to the drug, the medium or more compound medium was replaced with 0.2 mL of an aqueous solution of propidium iodide (Pl) (7 mg / L). Since the Pl only goes through the lysed or leaking membranes, the DNA of the dead cells is stained and measured, while the living cells do not stain. To measure the proportion of living cells, the cells permeabilized freezing the plaques, killing all the cells. After thawing the plates, the fluorescence was measured using a Cytofluor 4000 plate reader (excitation 530 nm, emission 620 nm), giving a direct relationship with the total number of cells. Growth inhibition was expressed as Test / Control x 100 (% T / C). The tests were considered valuable only when the positive control (taxol) induced a change in the inhibition of tumor growth in the presence and absence of verapamil, and only when the control cells treated with vehicle had a fluorescence intensity > 500. Preparation of 39-demethoxyrapamycin test solutions A stock solution of 3.3 mM 39-demethoxiropamycin in DMSO was prepared and stored at -20 ° C. Then, the stock solution was thawed on the day of use and stored at room temperature before and during dosing. The dilution steps were made using RPMI 1640 medium and solutions of 18 times the final concentration were obtained. Results Figures 2A-2D show four graphs showing the values of% T / C at all test concentrations for paclitaxel (A and C) and 39-demethoxyrapamycin (B and D), in normal cell lines (A and B) or cell lines with high expression of P-gp (C and D). The diamonds in black represent the values after the administration of paclitaxel or 39-demethoxyrapamycin alone; the blank squares represent the values after the administration of paclitaxel or 39-demetoxirrapamycin in the presence of 0.01 mg / mL of verapamil (a P-gp inhibitor). Paclitaxel, a known P-gp substrate, showed reduced potency to inhibit the MCF7 ADR cancer cell line expressing P-gp, and this power reduction was restored by co-administering verapamil, a P-gp inhibitor (Figs. and 2C). The 39-demethoxyrapamycin did not show a significant change in growth-proliferation curves in the MCF7 ADR cell line, expressing P-gp, with or without verapamil (Figures 2B and 2D), demonstrating that 39-demethoxyrapamycin does not it is a substrate for P-gp.

EXAMPLE 6 Pharmacokinetic analysis 6. 1 PK analyzes of rapamycin and 39-demethoxyrapamycin A pharmacokinetic analysis of rapamycin and 39-demethoxyrapamycin was performed using standard methods previously described (the protocol used for each compound is indicated in table 9). The AUC of each compound in blood or brain tissue was calculated using Kinetica 4.4 (InnaPhase Corporation) and a non-compartmental model and the trapezoidal method for the calculation of AUC. The partition coefficient (R,) of each compound after oral and iv administration was calculated as shown below: A UC 'rCEREBRO R: = ^ A I ^ IC ^ BLOOD The results of this analysis are summarized in Table 9 and Figures 3A and 3B.

TABLE 9 Summary of pharmacokinetic data 6. 2 PK analyzes of rapamycin, 39-demethoxyrapamycin and 27-0-desmethyl-39-demethoxyrapamycin A pharmacokinetic analysis of rapamycin, 39-demethoxyrapamycin and 27-O-demethyl-39-demethoxyrapamycin was performed using the standard methods described above (using the protocol 1 described above).

The AUC of each compound in blood or brain tissue was calculated using Kinetica 4.4 (InnaPhase Corporation) and a non-compartmental model and the trapezoidal method for the calculation of AUC. The partition coefficient (R,) of each compound after iv administration was calculated as shown below: TABLE 10 Pharmacokinetic data EXAMPLE 7 Activity in the experimental allergic encephalomyelitis (EAE) model of multiple sclerosis Experimental allergic encephalomyelitis (EAE) is an inflammatory and demyelinating autoimmune disease of the central nervous system (CNS), and is considered the best available animal counterpart of the Multiple sclerosis (MS). The disease can be induced in genetically susceptible animals by injection of the whole spinal cord, or myelin basic protein (MBP) in complete Freund's adjuvant (CFA). The antigen-specific effector cells involved in the damage to the CNS are the CD4 + T lymphocytes restricted to the major histocompatibility complex (MHO) class II. Recently, the role of cytokines such as interleukin 1 (IL-1), tumor necrosis factors (TNF) or interferons (IFN) in inflammatory responses has received increasing attention. After activation with the antigen, the T cells produce several lymphokines which, in the case of EAE, can be directly or indirectly responsible for damage to the CNS. The lymphokines that are probably involved in the pathogenesis of EAE are IL-2, IFN-α. and TNF-β. IL-2 has an important function in the activation and proliferation of T cells, whereas IFN-α it is a potent mediator of macrophage activation. In addition, the IFN-? induces the production of inflammatory cytokines such as IL-1, TNF, and also the expression of MHC class II molecules, among others, in the endothelial cells of blood vessels in the CNS, and in astrocytes, which are thought to have an important function in the presentation of the antigen to encephalitogenic T cells. 7. 1 Animals and immunization procedure Lewis male rats aged 8 to 10 weeks were kept under standard laboratory conditions (free of nonspecific pathogens) with free access to water and food. EAE was induced by a single injection at the base of the 50 mL tail of incomplete Freund's adjuvant (Difco, Detroit, Michigan) plus 50 mL of saline with 25 mg of guinea pig spinal cord and 1 mg of Mycobacterium tuberculosis strain H37 RA (Difco). 7. 2 Clinical and histological score Rats were examined every day by measuring their body weights and clinical signs of EAE, up to 30 days after immunization. These clinical ratings were performed by an observer without knowledge of the treatment: 0 = no disease; 1 = flaccid tail; 2 = moderate paraparesis; 3 = severe paraparesis; 4 = moribund state; 5 = death. The end of the disease was defined as the complete absence of clinical symptoms and the return to motility of the preimmunization period, the rat being rated as 0 for 5 consecutive days. 7. 3 Experimental treatment The test compounds were given at different doses (5 or 15 mg / kg of body weight) under a prophylactic and therapeutic regimen. For the prophylactic part of the study, the treatment was started one day before the immunization, and for the therapeutic part of the study, the treatment was started 7 days after the immunization (d., I.). Rats treated with vehicle under the same experimental conditions were used as control, either prophylactic or therapeutically. The treatment was given orally every day six times a week up to 30 days d. i. Cyclophosphamide was used as a positive control. The results of the experiment are shown below in Figures 4A and 4B and Table 11. Figure 4A shows the effect of the prophylactic regimen of 39-demethoxyrapamycin at 5 and 15 mg / kg; Figure 4B shows the therapeutic effect of the therapeutic regimen of 39-demethoxyrapamycin at 5 and 15 mg / kg. The effects of 40 mg / kg of cyclophosphamide are shown as positive control for each regimen. In both graphs the average score of each group is shown. It can be seen that in this model 39-demethoxyrapamycin has an efficacy equivalent to cyclophosphamide, and that it not only reduces the severity of the symptoms, but also reduces the duration of the episode. It should be noted that due to death during the study, in 5 of 7 rats treated with vehicle the mean value of this group remained at 5; however, the two surviving rats finally returned to baseline values at 28 days.

TABLE 11 * statistically different from the control treated with vehicle, p < 0.05, Mann Whitney rank sum test EXAMPLE 8 Study of the anti-tumor activity of 39-demethoxyrapamycin in a xenografted glioma model orthotopically in hairless mice 8. 1 Preparation of the study 8. 1.1 Preparation of the samples: The test compound was dissolved in ethanol (0.027 mL / mg of compound) and mixed with vortex for 20 min until the solution was made transparent. Aliquots of the ethanolic solutions were taken as appropriate and stored at -20 ° C. Then, the ethanolic solution carried to the correct concentration with vehicle (4% ethanol, 5% Tween-20, 5% polyethylene glycol 400 in NaCl 0.15 M, prepared whenever possible with sterile components free of endotoxins). 8.1.2 Means of administration The test substance and the control vehicle were administered intravenously (v, bolus) by injection into the caudal vein of the test mice. An injection volume of 10 mL / kg was used based on the most recent body weight of the mouse. 8.1.3 Cancer cell line The cell line used for the study was U87-MG, a glioblastoma cell line initiated by J. Ponten of a grade I glioblastoma of a 44-year-old Caucasian woman (Poten et al., 1968). ). 8.1.4 Cell culture conditions to establish the cell line Tumor cells developed as a monolayer at 37 ° C in a humid atmosphere (5% CO2, 95% air). The culture medium was RPMI 1640 (ref BE12-702F, Cambrex), which contained 2 mM L-glutamine supplemented with 10% fetal bovine serum (ref DE14-801 E, Cambrex). The cells were adherent to plastic flasks. For experimental use, the tumor cells were detached from the culture flask for 5 minutes of treatment with trypsin-versose (ref BE-17-161 E, Cambrex), in Hank's medium without calcium or magnesium (ref. BE10-543F, Cambrex). The cells were counted in a hemocytometer and their viability was determined by means of blue of tripane at 0.25%. 8. 2 Induction of glioma by stereotaxic injection into the brain of hairless mice

[0151] Mice were stereotaxicly injected U87-MG cells in the OD, 24 to 48 hours after full body radiation with a source and (2.5 Gy, Co60, INRA BRETENIERE, Dijon). For the stereotaxic injection of tumor cells, the mice were anesthetized by means of an intraperitoneal injection of ketamine, 100 mg / kg (KetaminedOO®, Ref. 043KET204, Centravet, France) and xylazine, 5 mg / kg (Rompun®, Ref 002ROM001, Centravet, France) in 0.9% NaCl solution at 10 mL / kg / iny. The cells were injected stereotaxically using 3 independent stereotaxic apparatuses (Kopf Instrument, Germany and Stoelting Company, USA) in the right frontal lobe, at 1x10 5 U87-MG tumor cells resuspended in 0.002 mL of RPMI-1640 medium. 0.002 mL of the cell suspension was injected at 500 nL / min. 8. 3 Treatment program In D7 the mice were weighed and randomized into 3 groups of mice according to their individual body weight. Four additional mice were added to each treatment group for MRI imaging. The groups were selected in such a way that the mean body weight of each group was not statistically different from the others (variance analysis). The test substances were administered as defined below: 8.3.1 The mice of group 1 received 5 cycles of daily iv injections of test substances or vehicle for 3 consecutive days (days D7 to D9, D14 to D16, D21 to D23, D28 to D30 and D35 to D37: (Q1 Dx3) x5W). Each cycle was separated by a period of 4 days of purification. 8.3.2 The mice of group 2 received 5 cycles of daily iv injections of 39-demethoxyrapamycin at 3 mg / kg / yn for 3 consecutive days on days D7 to D9, D14 to D16, D21 to D23, D28 to D30 and D35 to D37: (Q1 Dx3) x5W). Each cycle was separated by a period of 4 days of purification. 8.3.3 The mice of group 3 were not treated. The treatment program is summarized in the following table 12: TABLE 12 8. 4 MRI analysis A brain analysis was done on days D23 and D37. All MRI analyzes were done in 4JT on the Pharmascan magnet (Briker, Wissembourg). The mice were placed under continuous isoflurane anesthesia within the dedicated mouse cradle and the 38 mm diameter cylindrical coil. After a tripilot acquisition a weighted turboRare T2 sequence was made. Acquisitions covered the entire brain including the tumor. The volume of the tumor was determined manually by drawing a region of interest (ROI) around the tumor in each slice, and adding up all the surfaces. 8. 5 Results Figure 5 shows the survival graph of each treatment group until day 43. Additionally, the results were expressed as a percentage (T / C%) where T represents the average survival times of the animals treated with 39-demethoxyrapamycin and C represents the average survival times of control animals treated with vehicle. The T / C% was calculated as follows: T / C% = [T / C] x 100 Additionally, the MRI analysis was used to calculate the tumor volume per treatment group; The results are summarized in Table 13 below. As all animals treated with vehicle had died at 37 days, it was not possible to compare the tumor sizes at this stage.

TABLE 13 Each data point represents the average of 4 values.

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Endocrine-Related Cancer 8: 249-258.

Claims (23)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of a 39-demethoxyrrapamycin analog of the formula (I), wherein Ri represents (H, H) or = O, and R2 and R3 represent, each independently, H, OH, or OCH3, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a condition caused by damage or neural disease.

2. The use of a 39-demethoxy-rapamycin analog of the formula (I), wherein Ri represents (H, H) or = O, and R2 and R3 represent, each independently, H, OH, or OCH3, in the preparation of a medicament for the treatment of a medical condition affecting the central nervous system, where the medical condition requires that the drug crosses the blood-brain barrier.

3. The use claimed in claim 1 or 2, wherein the medical condition is selected from the group consisting of brain tumors and neurodegenerative conditions.

4. The use claimed in claim 3, wherein the medicament is for the treatment of brain tumors.

5. The use claimed in claim 4, wherein the brain tumor is glioblastoma multiforme.

6. The use claimed in claim 3, wherein the medicament is for the treatment of neurodegenerative conditions.

7. - The use claimed in claim 6, wherein the neurodegenerative condition is Alzheimer's disease.

8. The use claimed in claim 6, wherein the neurodegenerative condition is multiple sclerosis.

9. The use of a 39-demethoxy-rapamycin analog of the formula (I), wherein "represents" (H, H) or = O, and R2 and R3 represent, each independently, H, OH, or OCH3, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of cancer or the malignancy of B cells, where cancer or malignancy of B cells is resistant to one or more of the existing anticancer agents.

10. The use claimed in claim 9, wherein the cancer, or the malignancy of B cells, expresses the P glycoprotein.

11. - The use claimed in claim 10, wherein the cancer, or the malignancy of the B cells, has a high degree of expression of the glycoprotein P.

12. The use claimed in any of claims 1 to 11, wherein the medicament is for intravenous administration of a 39-demetoxirrapamycin analog or a pharmaceutically acceptable salt thereof.

13. The use claimed in any of claims 1 to 11, wherein the medicament also comprises one or more therapeutically effective agents.

14. The use claimed in any of claims 3-5 or 9-13, wherein the medicament is for the treatment of cancer or malignancy of B cells, and wherein the medicament also comprises one or more agents selected from the group consisting of methotrexate, leukovorin, adriamycin, prenisone, bleomycin, cyclophosphamide, 5-fluorouracil, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine, doxorubicin, tamoxifen, toremifene, megestrol acetate, anastrozole, goserelin, anti-monoclonal antibody HER2 (for example Herceptin ™), capecitabine, raloxifene hydrochloride, EGFR inhibitors, VEGF inhibitors, proteasome inhibitors, and hsp90 inhibitors.

15. The use claimed in any of claims 1 to 14, wherein the analogue of 39-demethoxy-rapamycin is 39-demethoxy-rapamycin.

16. - The use claimed in any of claims 1 to 14, wherein the analog of 39-demethoxyrapamycin differs additionally from rapamycin in one or more of positions 9, 16 or 27. 17.- The use claimed in Claim 16, wherein the analogue of 39-demethoxyrapamycin differs from rapamycin in one or more of positions 16 or 27. 18. The use claimed in claim 16, wherein the analogue of 39-demethoxyrapamycin differs from rapamycin in positions 16 and 27. 19. The use claimed in any of the claims 16 to 18, wherein the 39-demethoxy-rarapamycin analog has a hydroxyl group at position 27, ie, R3 represents OH. 20. The use claimed in any of claims 16 to 18, wherein the analog of 39-demethoxy-rarapamycin has a hydrogen at position 27, ie, R3 represents OH. 21. The use claimed in any of claims 16 to 20, wherein the analogue of 39-demethoxyrapamycin has a hydroxyl group in the 16-position, ie, R2 represents OH. 22. A pharmaceutical composition comprising a 39-demethoxy-rapamycin analog of the formula (I), wherein Ri represents (H, H) or = O, and R2 and R3 represent, each independently, H, OH, or OCH3, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier. 23. The pharmaceutical composition according to claim 22, further characterized in that it is specifically formulated for intravenous administration.