CN101472579A - Drugs and uses - Google Patents

Abstract

The invention relates to methods to treat specified clinical disorders such as hyperglycemia, type 2 diabetes, arthritis and multiple sclerosis. The invention also provides methods to identify and characterize drugs, which are characterized in part by eliciting a variable biologic or therapeutic effect on a biomolecule at one time and relative normalization of the biomolecule at another time point. Compounds include 17alpha-ethynylandrost-5-ene-3beta,7beta, 17beta-triol or androst-5-ene-3beta,4beta, 16alph, 17beta-tetrol, which can be used as reference standards to facilitate assessing and characterizing such candidate drugs.

Description

Medicine and purposes

Invention field

The present invention relates to method and chemical compound, androstane-5-alkene-3 β for example, 4 β, 16 α, 17 β-tetrol are used to regulate inflammation, metabolic disorder and described other patient's condition herein.Treatment, the improvement of using chemical compound to regulate inflammation to can be used for the following patient's condition, prevent or delay its progress, for example multiple sclerosis, arthritis or lupus erythematosus of type 2 diabetes mellitus, hyperglycemia, lung inflammation state (for example, cystic fibrosis), acute or chronic asthma/adult respiratory distress syndrome (ARDS) or chronic obstructive pulmonary disease (COPD) and autoimmune disorder for example.

Background of invention

There are many factors to help the formation of many chronic autoimmune disorders and inflammation and keep.Usually, the etiology of this disease is not fully understood.Tumor necrosis factor-alpha (TNF α) is at first to reply the panimmunity stimulant by mononuclear phagocyte and the cytokine that discharges.When the animal or human is given, it causes inflammation, fever, cardiovascular effect, hemorrhage, hemopexis with to actute infection and shock state process in those similar acute stages of being seen reply.Therefore, TNF α excessive or that do not regulated generates and involves the numerous disease state.This is comprising endotoxemia and/or toxic shock syndrome, for example, and people such as Tracey, people such as Nature330:662-664 (1987) and Hinshaw, Circ.Shock 30:279-292 (1990); Cachexia, for example, people such as Dezube, Lancet, 335 (8690): 662 (1990); And ARDS, wherein in ARDS patient's lung extract, found high TNF α concentration.For example, people such as Millar, Lancet 2 (8665): 712-714 (1989).

As if TNF α also involves bone resorption disease, comprises arthritis.When being activated, leukocyte can produce bone-absorption again, and TNF α can contribute to some extent to this activity, for example, people such as Bertolini, people such as Nature 319:516-518 (1986) and Johnson, Endocrinology 124 (3): 1424-1427 (1989).TNF α has also shown in vitro and in vivo to be stimulated bone resorption and suppresses bone formation by stimulating osteoclast to form and activating and be suppressed to bone cell function.Proved with monoclonal anti-TNF alpha antibody blocking TNF α rheumatoid arthritis (people such as Elliot, Int.J.Pharmac.17 (2): 141-1451995) and crohn (people such as von Dullemen, Gastroenterology, 109 (1): be favourable 129-1352005).

Nuclear Factor-Kappa B (NF-κ B) molecule is the mediator of inflammation in many clinical patient's condition.For example dexamethasone, prednisone or hydrocortisone are glucocorticoid receptor (GR) (GR) agonist to be used for the treatment of some therapeutic agents of inflammation, they suppress NF-κ B indirectly by the activity that increases GR, for example, people such as H.Harkonarson, Am.J.Respir.Cell Mol.Biol.25:761-771,2001.Yet, the elevated levels of natural GR agonist and pharmacology's level of synthetic GR agonist produce unwanted toxicity usually, comprise loss of significant immunosuppressant and bone mass or osteopenia, for example, people such as T.L.Popper, Antiinflammatory agents:Anti-inflammatory steroids, RA.Scherer﹠amp; M.W.Whitehouse edits, Academic Press, New York, the 9th chapter, the 1st volume, 245-294 page or leaf, 1974.The many unwanted toxicity relevant with glucocorticoid all is that the activation by GR causes.Therefore, evaluation can suppress the NF-kB activity and obstructed overactivation GR cause these toxic compounds represented one class can be used for treating inflammation and related symptoms for example pain, fever or tired medicine.

Inflammation unwanted or damageability occurs in many chronic or acute patient's condition, for example, and in ARDS, COPD and the sepsis.Activatory mononuclear cell works in the relevant pathology of transmitting inflammation with neutrophil cell.Particularly importantly activatory neutrophil cell, it shows the accumulation increase of the NF κ-B transcription regulaton factor in the nucleus and the generation of proinflammatory cytokine increases.Neutrophil cell also is the source of toxicity oxygen species, and the generation of toxicity oxygen species mediates tumor necrosis factor-alpha (TNF-α) secretion by activated macrophage at least in part, and this may be a organ injury seen in sepsis and depleted necessary.

Can take place by different approach with the signal transduction of inflammation-related, and this can improve the NF-kB activity in the affected cell.NF-κ B is incorporated into TNF-α receptor on the cell membrane by tumor necrosis factor-alpha (TNF-α) activation from TNF-α, follows by the activation of (comprising map kinase) of a series of signal transducer.The activation of NF-κ B in Cytoplasm causes that it translocates in the nucleus and causes and comprise the gene activation that NF-κ B replys composition in promoter.Cytoplasmic NF-κ B is incorporated into Toll sample receptor 4 on the cell surface by bacteria lipopolysaccharide (LPS) activation from LPS, and the activation of intracellular signal transduction thing takes place subsequently, comprises the activation of phosphatidylinositol-3-kinase.Known TNF-α and LPS induce intensive struvite replying in vivo and external in cell.The cell of replying this pro-inflammatory signal comprises the immunocyte of macrophage, mononuclear cell and other type.

There is multiple T cell subsets as if in the evolution of some morbid state, to work.Proposed to comprise the important function of T cell mass in the various aspects of mediation immunity of the uniqueness of modulability and/or suppressor T lymphocyte, for example, people such as E.Suri-Payer, J.Immunol., 160 (3): 1212-1218,1998; People such as J.Shimizu, J.Immunol., 163 (10): 5211-5218,1999; People such as M.Itoh, J.Immunol., 162 (9): 5317-5326,1999; People such as A.M.Miller, J.Immunol., 177:7398-7405,2006.CD4 ⁺CD25 ⁺The T cell can work in suppressing some immunne response.

Described in animal model some researchs carried out in these T cell subsets, for example at United States Patent (USP) 6,593, in 511.For example, at scid/scid CD4 ⁺CD45Rb ^HiExamined or check effect in the model for the research human autoimmune patient's condition.This animal model has been used to study the immunne response of imbalance, inflammatory conditions and be used for evaluation experimental medicine and treatment protocol for example, for example, people such as K.Hong, J.Immunol., 162:7480-7491,1999; People such as Powrie, J.Exp.Med., 183 (6): 2669-2674,1996.

Can form CD4 at inducing T cell by the inductive Foxpro3 gene of thymus epithelial ⁺CD25 ⁺Or CD4 ⁺CD25 ^HighWork in (TregT cell or regulatory T cells) Phenotype.The CD25 surface antigen is IL-2 receptor α-chain.In the animal model of some autoimmune diseases, the Foxpro3 gene deletion is relevant with the generation of autoimmune disease, for example, and U.S. Patent application 2006/0111316.It is unusual that the recovery of this gene seemingly reduces autoimmune.Described and be used for CD4 ⁺CD25 ⁺The all ingredients of cell or testing regulations, for example, H.Yagietal., International Immunol., 16 (11): 1643-1656,2004; People such as W.R.Godfrey, Blood, 105 (2) 750-758,2005.

Recognized the insulin resistant that does not tolerate the experimenter of glucose already.People such as Reaven (American Journal of Medicine, 60 (1): 80-88,1976) glucose of use continuous infusion proves in different non-obesities, non-ketosis subject group with oral glucose tolerance test with insulin (insulin/glucose clamp technology) and has insulin resistant.These experimenters tolerate tangible empty stomach hyperglycemia and difference from critical glucose.Diabetic groups in these researchs comprises (IDDM) of insulin-dependent and the experimenter of non-insulin-depending type (NIDDM).

Consistent with the insulin resistant that continues is easier definite hyperinsulinemia, and it can be measured by the circulating plasma insulin concentration in the accurate mensuration experimenter blood plasma.The result that hyperinsulinemia can be used as insulin resistant occurs, for example in obesity and/or diabetes (NIDDM) experimenter and/or glucose do not tolerate among the experimenter, in IDDM experimenter, be to occur perhaps as the result who compares excessive insulin injection with the insulin that endocrine gland pancreas normal physiologic discharges.

By experiment clinical the and epidemiological study of property described hyperinsulinemia and obesity and with trunk ischemic diseases () association (Stout, Metabolism, 34:7,1985 for example, atherosclerosis; People such as Pyorala, Diabetes/Metabolism Reviews, 3:463,1987).After oral glucose load, 1 rise relevant with the coronary heart disease danger of increase with the plasma insulin of the significance,statistical that occurred in 2 hours.

A kind of model of people's diabetes is db/db mices.The db/db mouse model is described to some extent, for example, people such as D.Koya, The FASEB Journal, 14:439-447,2000; People such as K.Kobayashi, Metabolism, 49 (1): 22-31,2000; People such as J.Berger, J.Biol.Chem., 274 (10): 6718-6725,1999.The db/db mice has sudden change on the gene of coding leptin receptor, along with its functional pancreas beta cell agglomerate worsens in time, having given with bulimia nerovsa, obesity, insulin resistant and diabetes is the phenotype of feature, particularly for the animal of C57BL/Ks genetic background.The db/db mice is typically becoming during ages obviously fat and is beginning to rise at 10 to 14 days plasma insulins in about 3 to 4 weeks.Can observe blood sugar increasing ages in 4 to 8 weeks, having blood glucose out of control increases, and the beta cell of the production insulin of islets of langerhans seriously exhausts, and death when about 10 monthly ages.The progress that this model has been used to characterize the formation of drug candidates influence and diabetes and has kept relevant parameter (for example, hyperglycemia and weight increase) begins or the ability of its speed.

Related to cardiac hypertrophy with PPAR-gamma agonist treatment diabetes, perhaps cardiac weight increases.Show that with rosiglitazone maleate (a kind of PPAR-gamma agonist) treatment the patient may experience hydrops and liquid capacity dependent event, for example edema and congestive heart failure.The cardiac hypertrophy relevant with the PPAR-gamma agonist treatment typically handled by ending treatment.

The physiological action of hydrocortisone is its antagonism to insulin.High hydrocortisone concentration can reduce the insulin sensitivity in this organ in the liver, and it tends to increase gluconeogenesis and increases blood sugar level people such as (, Front Neuroendocrine., 14:303-347,1993) M.F.Dallman.This effect has increased the weight of impaired glucose tolerance or diabetes.In the storehouse Cotard that the circulation composition by too much hydrocortisone causes, the antagonism to insulin in susceptible individual can cause diabetes (people such as E.J.Ross, Lancet, 2:646-649,1982).

Hydrocortisone can be in vivo 11 β-dehydrogenase activity by 11 beta-hydroxysteroid dehydrogenases be converted into cortisone.Its back reaction, promptly the cortisone of non-activity is converted into active hydrocortisone, is to be realized by the 11 β-reductase activity of these enzymes in some organ.This activity is called corticosteroid 11 β-reductase activity again.11 beta-hydroxysteroid dehydrogenases have the isozyme of at least two kinds of uniquenesses.The expression of 11 β-HSD 1 type produces two-way enzyme or dominant 11 β-reductase in various kinds of cell system, its can from otherwise be the 11-ketosteroid parent of non-activity 11 beta-hydroxysteroids of regenerating.

The mitochondrion PCK (is called the PEPCK-mitochondrion again, PEPCK-M, PCK2 and mtPEPCK) in various people's tissues, express, mainly in liver, kidney, pancreas, intestinal and fibroblast, express (people such as Modaressi, Biochem.J., 333:359-366,1998).Although not fully record, the PEPCK-mitochondrion lacks and has related to arrest of development, hypoglycemia and liver anomalies.(PEPCK-C) is different with the endochylema form, and mitochondrion form (PEPCK-mitochondrion) is a constitutive expression, and is not subjected to hormonal stimulation to regulate (Hanson and Patel, Adv.Enzymol.Relat.Areas Mol.Biol., 69:203-281,1994).Two kinds of forms all are positioned on the independent chromosome, are positioned at chromosome 14q11, and PEPCK-C is present in chromosome 20q11 and goes up people such as (, Hum.Mol.Genet, 2:1-4,1993) Stoffel.

Multiple sclerosis (MS) is a kind of autoimmune disease (Bar-Or, A., J.Neuroimmunol.100:252-259,1999) as inflammatory disease of central nervous system.Although the natural process of this disease is by for example interferon (IFN)-β, copolymer, cyclophosphamide and mitoxantrone are treated the (Hafler that improves with immune regulative-inhibitive ability of immunity chemical compound recently, D.A. and Weiner, H.L., Immunological Reviews 144:75,1995; Goodkin, D.E., Lancet 352:1486,1998), but neither one can be blocked the progress of disease in these medicines, and have some to have the serious side effects that limits its prolonged application among them.In addition, significantly the patient of quantity performance go on business to the replying of IFN-β, comprise recurrence remission form patient and secondary progress type MS patient.Therefore, need new chemical compound, be used for treating separately or improving its process by the progress that for example delays MS in therapeutic alliance.

Need effective medicine of cost or Therapeutic Method at present and more effectively treat the above-mentioned patient's condition.The invention provides one or more therapeutic agent and the Therapeutic Method that is used for the treatment of in these patient's condition.Therefore, described medicine and method can be used for reducing and one or more relevant symptoms of the described patient's condition herein.In addition, the application of medicine of the present invention and method can be united with one or more routine treatments that are used for these diseases.

Detailed Description Of The Invention

The embodiment summary

The invention provides the progress of the inflammatory conditions of not expecting, autoimmune disease or metabolic disorder in the mammal that is used for the treatment of, prevents, delays to have these needs or its symptom or improve the chemical compound or the compositions of the described inflammatory conditions of not expecting, autoimmune disease or metabolic disorder or its symptom, randomly, wherein said mammal is people or inhuman primate, wherein compositions comprises formula 1 chemical compound (" F1C ")

One of them R ¹Be-H or optional substituted alkyl another R ¹Be-H ,-OH, ester or ether, perhaps two R ¹Be together=O or oxime for example=NOH or=NOCH ₃Ester or ether; A R ²For-H or optional substituted alkyl, another R ²For-H ,-OH, ester or ether, perhaps two R ²Be together=O or oxime for example=NOH or=NOCH ₃A R ³For-H or optional substituted alkyl, another R ³For-H ,-OH, ester or ether or optional substituted alkyl, perhaps two R ³Be together=O or oxime for example=NOH or=NOCH ₃A R ⁴For-H or optional substituted alkyl, another R ⁴Be-OH, ester or ether, perhaps two R ⁴Be together=O or oxime for example=NOH or=NOCH ₃R ⁵For optional substituted alkyl, randomly be selected from-CH ₃,-C ₂H ₅With-CH ₂OH; R ⁶For-H or optional substituted alkyl, randomly be selected from-CH ₃,-C ₂H ₅With-CH ₂OH; A R ⁷For-H or optional substituted alkyl, another R ⁷For-H ,-OH, ester or ether, perhaps when not having two key for 4, two R ⁷Be together=O or oxime for example=NOH or=NOCH ₃R ⁹For-O-or-C (R ¹²) (R ¹²One of them R of)-, ¹²For-H ,-F ,-Br or optional substituted alkyl, another R ¹²For-H ,-OH, ester or ether or optional substituted alkyl, or two R ¹²Be together=O or oxime for example=NOH or=NOCH ₃R ¹⁰For-H or halogen for example-F or-Cl; With a R ¹¹For-H or optional substituted alkyl, another R ¹¹For-H ,-OH, ester or ether or optional substituted alkyl, perhaps two R ¹¹Be together=O or oxime for example=NOH or=NOCH ₃The embodiment of these chemical compounds comprises following chemical compound, wherein (i) R ², R ⁷, R ¹¹And R ¹²In one, two or three are-OH, C independently _2-8Ester, C _1-8Ether or=O, (ii) R ¹, R ⁷, R ¹¹And R ¹²In one or two be-OH, C _2-8Ester or C _1-8Ether and (iii) R ¹, R ², R ⁷, R ¹¹And R ¹²In one or two be=O or=NOH, and remaining one, two or three are-OH, C independently _2-8Ester or C _1-8Ether.5 hydrogen atom can be in α or beta comfiguration when existing.When having two key for 4, a R is arranged ⁷Group is non-existent.

The present invention also provides the bioactive method of identifying or characterizing the chemical compound with the potentiality that are used for the treatment of or improve mammiferous metabolic disorder, comprise and select following chemical compound (i) to compare, do not make PPAR-α, PPAR-γ in people or the mammalian cell and a kind of, two or three activation among the PPAR-δ surpass about 30% external with external suitable negative control people or mammalian cell; (ii) compare, make transcriptional activity or the level inhibition of the NF-κ B in people or the mammalian cell or reduce about 20-80% external with external suitable negative control people or mammalian cell; (iii) compare with suitable negative control or normal control, alleviate hyperglycemia, delay the progress of hyperglycemia or postpone its morbidity, increase insulin sensitivity, reduce the glucose intolerance, delay the disabled progress and the speed of pancreas β-islet cell quantity or their excreting insulins, increase the ability of pancreas β-islet cell quantity or their excreting insulins, delay the db/db mice or suffer from the experimenter's of the relevant obesity of the inductive or meals of meals weight increase speed, reduce the triglyceride levels that raises, low total blood or the serum cholesterol level that raises, reduce the normal or LDL that raises in blood or the serum, VLDL, apoB-100 or apoB-48 level, or increase HDL normal or lower in blood or the serum or apoA1 level or reduce the fibrinogen level that raises in blood or the serum; (iv) randomly, compare with external suitable negative control people or mammalian cell, do not make one or more the following activation in people or the mammalian cell surpass about 30% external: the biologic activity variant of any in glucocorticoid receptor (GR), mineralcorticoid receptor, progesterone receptor, androgen receptor, estrogen receptor-α, estrogen receptor-beta or these biomolecule.Described method allows to identify or characterize has the chemical compound that is used for the treatment of or improves the potentiality of the metabolic disorder in people or the other mammal.

Embodiment also comprises and comprises formula 1 compound compositions, is used to prevent or treat the symptom of the autoimmune patient's condition, the inflammatory conditions of not expecting or metabolic disorder or any of these patient's condition.

The description in other place in other embodiment such as the description is included in embodiment described herein.

Definition.Unless with context explanation or hint are arranged in addition as used in this article, term used herein has the implication of definition herein.Described explanation to embodiment and embodiment is used to illustrate the present invention, and they do not plan to be construed as limiting by any way.Unless in addition restricted or hint, for example, by element or the option that comprises mutual repulsion, in these definition and in this manual, term " " and " one " are meant one or more, term " or " be meant and/or.

" preparation " and similarly term be meant the compositions that can give experimenter (for example, human or animal).Described preparation is suitable for people or veterinary to be used and typically has feature desired for described preparation, for example, is used for people's normally aseptic solution or the suspending agent of parenteral formulation.

" excipient ", " carrier " " pharmaceutically acceptable carrier " or similar terms are meant that be acceptable one or more components or composition on meaning compatible with other composition of compositions of the present invention or preparation and that can too not injure patient, animal, tissue or the cell generation that gives described preparation.

" experimenter " is meant the human or animal.Usually, animal is a mammal, perhaps for example inhuman primate, rodent, lagomorph, domestic animal or hunting animal.Primate comprises chimpanzee, machin, sapajou and macaque, and for example, Rhesus Macacus or chimpanzee belong to.Rodents and lagomorph comprise mice, rat, marmot, ferret, rabbit and hamster.

" alkyl " used herein is meant the normal chain carbon of connection, secondary carbon, tertiary carbon or ring-type carbon atom, that is, and and straight chain, side chain, ring-type or its any combination.Alkyl can be saturated or unsaturated as used in this article, that is, described group can comprise one, two or more two keys or triple bonds of independently selecting.Undersaturated alkyl comprises following for thiazolinyl and the described group of alkynyl.Unless otherwise mentioned, the carbon atom number in the alkyl is 1 to about 50, for example, and about 1-30 or about 1-20, for example, C _1-8Alkyl is meant the alkyl that comprises 1,2,3,4,5,6,7 or 8 carbon atom.When describing alkyl in detail, described group can comprise methyl, ethyl, 1-propyl group (n-pro-pyl), 2-propyl group (isopropyl ,-CH (CH ₃) ₂), 1-butyl (normal-butyl), 2-methyl isophthalic acid-propyl group (isobutyl group ,-CH ₂CH (CH ₃) ₂), the 2-butyl (sec-butyl ,-CH (CH ₃) CH ₂CH ₃), 2-methyl-2-propyl group (tert-butyl group ,-C (CH ₃) ₃), 1-amyl group (n-pentyl), 2-amyl group (CH (CH ₃) CH ₂CH ₂CH ₃), 3-amyl group (CH (CH ₂CH ₃) ₂), 2-methyl-2-butyl (C (CH ₃) ₂CH ₂CH ₃), 3-methyl-2-butyl (CH (CH ₃) CH (CH ₃) ₂), 3-methyl isophthalic acid-butyl (CH ₂CH ₂CH (CH ₃) ₂), 2-methyl-1-butene base (CH ₂CH (CH ₃) CH ₂CH ₃), 1-hexyl, 2-hexyl (CH (CH ₃) CH ₂CH ₂CH ₂CH ₃), 3-hexyl (CH (CH ₂CH ₃) (CH ₂CH ₂CH ₃)), 2-methyl-2-amyl group (C (CH ₃) ₂CH ₂CH ₂CH ₃), 3-methyl-2-amyl group (CH (CH ₃) CH (CH ₃) CH ₂CH ₃), 4-methyl-2-amyl group (CH (CH ₃) CH ₂CH (CH ₃) ₂), 3-methyl-3-amyl group (C (CH ₃) (CH ₂CH ₃) ₂), 2-methyl-3-amyl group (CH (CH ₂CH ₃) CH (CH ₃) ₂), 2,3-dimethyl-2-butyl (C (CH ₃) ₂CH (CH ₃) ₂), 3,3-dimethyl-2-butyl (CH (CH ₃) C (CH ₃) ₃), cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, suberyl, ring octyl group ,-(CH ₂) n-(CHCH ₃) m-(CH ₂) o-CH ₃With-(CH ₂) n-(CHC ₂H ₅) m-(CH ₂) o-CH ₃, wherein n, m and.Be 0,1,2,3,4,5,6,7 or 8 independently.

" thiazolinyl " used herein is meant the group of the normal chain carbon that comprises connection, secondary carbon, tertiary carbon or ring-type carbon atom, promptly, straight chain, side chain, ring-type or its any combination, it (for example comprises one or more pairs of keys,-CH=CH-), for example, 1,2,3,4,5,6 or more a plurality of pairs of keys, typically be 1 or 2 two key.Unless otherwise mentioned, the carbon atom number in the thiazolinyl is 2 to about 50, for example, and about 2-30 or about 2-20, for example, C ₂- ₈Thiazolinyl or C2-8 thiazolinyl are meant the thiazolinyl that comprises 2,3,4,5,6,7 or 8 carbon atoms.When describing thiazolinyl in detail, this material can comprise vinyl, pi-allyl ,-(CH ₂) n-(CH=CH)-(CH ₂) m-CH ₃,-(CH ₂) n-(CCH ₃=CH)-(CH ₂) m-CH ₃,-(CH ₂) n-(CH=CCH ₃)-(CH ₂) m-CH ₃With-(CH ₂) n-(CH=CH) _0-1-(CH ₂) m-CH ₂CH=CH ₂, wherein n and m are 0,1,2,3,4,5,6,7 or 8 independently.

" alkynyl " used herein is meant the group of the normal chain carbon that comprises connection, secondary carbon, tertiary carbon or ring-type carbon atom, promptly, straight chain, side chain, ring-type or its any combination, it comprises one or more triple bonds (C ≡ C-), for example, 1,2,3,4,5,6 or more a plurality of triple bond typically are 1 or 2 triple bonds, randomly comprise 1,2,3,4,5,6 more a plurality of pairs of keys, remaining key is a singly-bound.Unless otherwise mentioned, otherwise the carbon atom number in the alkynyl is 2 to about 50, for example, and about 2-30 or about 2-20, for example, C _2-8Alkynyl or C2-8 alkynyl are meant the alkynyl that comprises 2,3,4,5,6,7 or 8 carbon atoms.When describing alkynyl in detail, described group can comprise-CCH ,-CCCH ₃,-CCCH ₂CH ₃,-CCC ₃H ₇,-CCCH ₂C ₃H ₇,-(CH ₂) n-(C ≡ C)-(CH ₂) m-CH ₃With-(CH ₂) n-(C ≡ C) _0-1-(CH ₂) m-CH ₂C ≡ CH, wherein n and m are 0,1,2,3,4,5,6,7 or 8 independently.

" alkyl of replacement ", " thiazolinyl of replacement ", " alkynyl of replacement " and similar term are meant alkyl, thiazolinyl, alkynyl or other group of definition herein, the substituent group that it has substituent group or comprises the substituent group that replaces hydrogen atom and be incorporated into carbon atom or interrupt carbon atom chain.Substituent group comprise 1,2,3,4,5,6 or more a plurality of independent select-F ,-Cl ,-Br ,-I ,-OH ,-OR ^PR,-SH ,-SCH ₃,-O-,-S-,-NH-,-C (O)-,-C (O) OR ^PR,-CHO ,-CH ₂SH ,-C=N-,-C (O) OR ^PR,-C (O) CH ₃, R wherein ^PRBe hydrogen, protecting group or two R independently ^PRAll be hydrogen or all be protecting group.

" halogen " is meant fluorine, chlorine, bromine or iodine.

" ester " is meant and comprises-group of C (O)-O-structure.Typically, ester used herein comprises and contains about 1-50 carbon atom (for example, about 2-20 carbon atom) and 0 () organic group for example, O, S, N, P, Si, wherein organic group is at for example R to about 10 independent hetero atoms of selecting ¹Or R ²The place is examined by the steroid that-C (O)-O-structure is incorporated into formula 1, for example, and organic group-C (O)-O-steroid or organic group-O-C (O)-steroid.Organic group generally includes one or more in above-mentioned any organic group, for example, and C _1-20Alkyl, C _2-20Thiazolinyl, C _2-20Alkynyl, aryl, C _2-9Heterocycle or any substituted derivant in these for example, comprise 1,2,3,4 or more a plurality of substituent group, and wherein each substituent group is independent the selection.Ester comprises succinic acid, dicarboxylic acids and amino acid whose ester, for example-and O-C (O)-(CH ₂) n-C (O)-OR ^PR,-O-C (O)-(CH ₂) n-NHR ^PR, and-NH-(CH ₂) n-C (O)-OR ^PR, wherein n is 1,2,3,4,5,6,7 or 8 and R ^PRFor-H or protecting group, for example C _1-4Alkyl.Ester comprises that also structure example is as-O-C (O)-O-(CH ₂) n-H and-O-C (O)-NH-(CH ₂) n-H.

The exemplary replacement of the hydrogen of these organic groups or carbon atom such as above-mentioned described for the alkyl that replaces, and comprise 1,2,3,4,5,6 or more a plurality of, common 1,2 or 3-O-,-S-,-NR ^PR-(comprise-NH-) ,-C (O)-,-CHO ,-CHS ,-C=NH ,-C (S) ,=O ,=S ,-N (R ^PR) ₂(comprise-NH ₂) ,-C (O) OR ^PR(comprising-C (O) OH) ,-OC (O) R ^PR(comprise-O-C (O)-H) ,-OR ^PR(comprise-OH) ,-SR ^PR(comprise-SH) ,-NO ₂,-CN ,-SCN ,-C ₆H ₅,-CH ₂C ₆H ₅,-NHC (O)-,-C (O) NH-,-OC (O)-,-C (O) O-,-O-A8 ,-S-A8 ,-C (O)-A8 ,-OC (O)-A8 ,-C (O) O-A8 ,=N-,-N=,=N-OH ,-OPO ₃(R ^PR) ₂,-OSO ₃H ₂Or halogen group or atom, wherein each R ^PRFor-H, independent protecting group or two RPR that select comprise protecting group, and A8 is C _1-8Alkyl, C _2-8Thiazolinyl, C _2-8Alkynyl, C _1-4Alkyl-aryl (for example, benzyl), aryl (for example, phenyl) or C _0-4Alkyl-C _2-9Heterocycle.Replacement is independent the selection.Organic group comprises and passes through R ⁴The chemical compound of variable-definition.Described organic group is got rid of obvious unsettled group, for example,-O-O-unless this unsettled group is the transition material that is used to prepare the chemical compound that is used for one or more application as herein described with enough chemical stabilities, comprises being used for synthesis type 1 chemical compound or other chemical compound.The above-mentioned replacement of enumerating can be used for replacing the substituent group of one or more carbon atoms typically, for example ,-O-or-C (O)-, or be used to replace the substituent group of one or more hydrogen atoms, for example, halogen ,-NH ₂Or-OH.Exemplary ester comprises one or more independent acetas, heptanoate, propionic ester, isopropyl acid ester, cyclopropionate ester, isobutyrate, n-butyric acie ester, valerate, caproate, dissident's acid esters, alkyl caproate, heptanoate, caprylate, pelargonate, decanoin, hendecane acid esters, phenylacetate or benzoate of selecting, and it is hydroxy ester typically.Ester also comprises amino-acid ester, carbonic ester and carbamate, comprises-O-C (O)-CH ₂-NHR ^PR,-O-C (O)-CH ₂CH ₂-NHR ^PR,-O-C (O)-CH (CH ₃)-NHR ^PR,-O-C (O)-CH ₂CH (CH ₃)-NHR ^PR,-O-C (O)-CH (NHR ^PR)-CH (OR ^PR)-CH ₃,-O-C (O)-O-(CH ₂) m-H and-O-C (O)-NH-(CH ₂) m-H, wherein R ^PRBe-H or protective agent C for example _1-4Alkyl (CH ₃,-C ₂H ₅,-C ₃H ₇Deng) or-C (O)-CH ₃Or-CH ₂CH ₂-O-CH ₃And m is 0,1,2,3,4,5 or 6.Ester also comprises-O-C (O)-(CF2) n-CF ₃,-O-C (O)-(CH ₂) n-CH ₃,-O-C (O)-CH (CH ₃)-(CH ₂) n-CH ₃With-O-C (O)-C (CH ₃) ₂-(CH ₂) n-CH ₃, wherein n is 0,1,2,3,4,5 or 6.

" ether " be meant comprise 1,2,3,4 or more a plurality of (common 1 or 2)-O-groups as for the described organic group of ester.In some embodiments ,-the O-group is at different groups (R for example ¹, R ², R ³, R ⁴Or R ¹¹) locate to be connected in steroid nuclear, for example, organic group-O-steroid.Described organic group such as above-mentioned described for ester.Ether comprises-O-(CH ₂) n-CH ₃,-O-CH ₂(CH ₂) n-O-CH ₃,-O-CH ₂(CH ₂) n-S-CH ₃With-O-CH (CH ₃)-(CH ₂) n-CH ₃, wherein n is 0,1,2,3,4,5 or 6.

Formula 1 chemical compound.In some embodiments, formula 1 chemical compound has 3,4 or 5 hydroxyls, randomly, one of them, two or more are by identical or different ester group esterification.In in these embodiments some, hydroxyl or ester are in 3-, 4-, 16-and 17-position, and the hydroxyl or the ester that wherein are in 3-, 4-and 16-position are in β, β respectively, α, β, β, β, α, β, α, α, β, β, β, α, α, β, α, β, α, α, α or α, α, beta comfiguration.The hydroxyl of 17-position or ester typically are in beta comfiguration, but can be the α configuration.R in these chemical compounds ¹⁰Typically be-H or halogen, for example-F, R ⁵Randomly be-CH ₃Or-C ₂H ₅, and R ⁶Randomly be-H or-CH ₃For in these chemical compounds some, can be in the 7-position or the 11-position have the other hydroxyl or the ester of beta comfiguration or α configuration.

For formula 1 chemical compound, the C of replacement _1-8Alkyl comprises-CH ₂F ,-CF ₃,-CH ₂OH and-C ₂F ₅Optional substituted C _2-8Alkynyl comprises-CCH ,-CCCH ₃,-CCCH ₂OH ,-CCCH ₂Cl and-CCCH ₂Br.

Exemplary formula 1 chemical compound comprises androstane-5-alkene-3 β, 4 β, 7 β, 16 α, one of them of 17 β-pentol and this chemical compound or the epimer of two hydroxyl epimerizations, for example, androstane-5-alkene-3 α, 4 β, 7 β, 16 α, 17 β-pentol, androstane-5-alkene-3 β, 4 α, 7 α, 16 α, 17 β-pentol and androstane-5-alkene-3 β, 4 β, 7 α, 16 β, 17 β-pentol.Other formula 1 chemical compound comprise wherein have 5 independent select-those of OH, ester or ether group, for example, androstane-5-alkene-3 β, 4 β, 11 β, 16 α, the epimer of one of them of 17 β-pentol and this chemical compound or two hydroxyl epimerizations, for example, androstane-5-alkene-3 α, 4 β, 11 β, 16 α, 17 β-pentol, androstane-5-alkene-3 β, 4 β, 11 β, 16 α, 17 α-pentol, androstane-5-alkene-3 β, 4 β, 11 α, 16 α, 17 β-pentol and androstane-5-alkene-3 β, 4 α, 11 β, 16 β, 17 β-pentol.For this chemical compound, in 5 hydroxyls one, two or more can be by derivatization, for example, deriving is ester or ether, for example methoxyl group, ethyoxyl, acetoxyl group, propionyloxy ,-O-C (O)-(CH ₂) ₃-H ,-O-C (O)-(CH ₂) ₄-H or-O-C (O)-(CH ₂) ₅-H derivant.Other ester comprises-O-C (O)-CH ₂-C (O) OH ,-O-C (O)-(CH ₂) ₂-C (O) OH ,-O-C (O)-(CH ₂) ₃-C (O) OH ,-O-C (O)-(CH ₂) ₄-C (O) OH ,-O-C (O)-(CH ₂) ₅-C (O) OH and-O-C (O)-(CH ₂) ₆-C (O) OH.

In in these embodiments some, four positions in 2-, 3-, 4-, 7-, 11-, 16-and the 17-position of formula 1 chemical compound have four independent hydroxyl, ester or ethers of selecting.This substituent group for example can be incorporated into, 2-, 3-, 16-and 17-position; 2-, 3-, 7-and 17-position; 2-, 3-, 11-and 17-position; 3-, 4-, 7-and 17-position; 3-, 4-, 11-and 17-position; 3-, 7-, 16-and 17-position; 3-, 4-, 16-and 17-position or 3-, 11-, 16-and 17-position.When 4-did not have two key, described hydroxyl, ester or ether can be 2-and/or 3-β respectively, β, β, β-17; 2-and/or 3-β, β, β, α-17; 2-and/or 3-β, β, α, β-17; 2-and/or 3-β, α, β, β-17; 2-and/or 3-α, β, β, β-17; 2-and/or 3-β, β, α, α-17; 2-and/or 3-β, α, β, α-17 configuration perhaps is 2-and/or 3-α, β, β, α-17 configuration.Term " 2-and/or 3-β, β, β, β-17 " is meant that the 2-position is optional and is replaced by hydroxyl, ester or ether and 3-position and 17-position are replaced by hydroxyl, ester or ether.When 4-did not have two key, described hydroxyl, ester or ether can be 2-and/or 3-β respectively, α, α, β-17; 2-and/or 3-α, β, α, β-17; 2-and/or 3-α, α, β, β-17; 2-and/or 3-β, α, α, α-17; 2-and/or 3-α, β, α, α-17; 2-and/or 3-α, α, β, α-17; 2-and/or 3-α, α, α, β-17; 2-and/or 3-β, α, α, α-17 configuration.R in these chemical compounds ¹⁰Typically be-H or-F, R ⁵Randomly be-CH ₃Or-C ₂H ₅, and R ⁶Randomly be-H ,-CH ₃,-CH ₂OH ,-CCH or-CCCH ₃For these chemical compounds, in 2-, 3-, 4-, 7-, 11-, 16-and the 17-position one, two or more positions quilt-H or optional substituted alkyl for example-CH ₃,-C ₂H ₅,-CH ₂=CH ₂,-CCH ,-CF ₃Or-C ₂F ₅Replace.

In some embodiments of formula 1 chemical compound, can have five independent hydroxyl, ester and/or ether groups of selecting.For these chemical compounds, hydroxyl, ester and/or ether group are in 3-position and 17-position usually and are in 3 other positions.These substituent groups can be in 2-, 3-, 7-, 11-and 17-position, 2-, 3-, 7-, 16-and 17-position or 2-, 3-, 11-, 16-and 17-position.Independent hydroxyl, ester and/or the ether group of selecting can be in 3-, 4-, 7-11-and 17-position; 3-, 4-, 11-, 16-and 17-position; 3-, 4-, 7-, 16-and 17-position or 3-, 7-, 11-, 16-, 17-position.For these chemical compounds, when not having two key in the 4-position, each group can be in α configuration or beta comfiguration.Therefore, the substituent group in these chemical compounds can be in 2-and/or 3-β, β, β, β, β-17 respectively, 2-and/or 3-β, β, β, β, α-17,2-and/or 3-β, β, β, α, β-17,2-and/or 3-β, β, α, β, β-17,2-and/or 3-β, α, β, β, β-17,2-and/or 3-α, β, β, β, β-17,2-and/or 3-β, β, β, α, α-17,2-and/or 3-β, β, α, β, α-17,2-and/or 3-β, α, β, β, α-17,2-and/or 3-α, β, β, β, α-17,2-5 and/or 3-β, β, α, α, β-17,2-and/or 3-β, α, β, α, β-17,2-and/or 3-α, β, β, α, β-17,2-and/or 3-β, α, α, β, β-17,2-and/or 3-α, β, α, β, β-17,2-and/or 3-α, α, β, β, β-17 configuration or be in 2-respectively and/or 3-β, β, α, α, α-17 configuration.Substituent group in these chemical compounds also can be in 2-and/or 3-β, α, β, α, α-17 respectively, 2-and/or 3-β, α, α, β, α-17,2-and/or 3-β, α, α, α, β-17,2-and/or 3-α, β, β, α, α-17,2-and/or 3-α, β, α, β, α-17,2-and/or 3-α, β, α, α, β-17,2-and/or 3-α, α, β, β, α-17,2-and/or 3-α, α, β, α, β-17,2-and/or 3-α, α, α, β, β-17,2-and/or 3-β, α, α, α, α-17,2-and/or 3-α, β, α, α, α-17,2-and/or 3-α, α, β, α, α-17,2-and/or 3-α, α, α, β, α-17,2-and/or 3-α, α, α, α, β-17 configuration or be in 2-and/or 3-α, α, α, α, α-17 configuration.

For described formula 1 chemical compound herein, the 16-position can be unsubstituted, that is, and and one or two R ³Be-H, and when not having two key in the 17-position, second R ⁴Can be present in the 17-position with α configuration or beta comfiguration.This second R ⁴Group comprises optional substituted C _1-8Alkyl, for example-CH ₃,-C ₂H ₅,-CF ₃,-C ₂F ₅,-C=CH ₂,-CCH ,-CCCH ₃Or-CCCH ₂OH.

For in these chemical compounds any, the 2-position can be unsubstituted, that is, and and R ⁹For-CH ₂-.In some embodiments, R ⁹Be substituted, for example ,-O-,-CH (OH)-,-CH (ester)-or-CH (ether)-, wherein hydroxyl, ester or ether group are in α or beta comfiguration.Exemplary R ⁹Group is-CH (α-OH)-,-CH (β-OH)-,-C (p-CH ₃) (α-OH)-,-C (α-CH ₃) (β-OH)-,-CH (α-OCH ₃)-,-CH (β-OCH ₃)-,-CH (α-OC (O) CH ₃)-and-CH (β-OC (O) CH ₃)-.Other R ⁹Group is-CH (α-OC (O) CH ₂CH ₃)-,-CH (β-OC (O) CH ₂CH ₃)-,-CH (α-OCH ₂CH ₃)-,-CH (β-OCH ₂CH ₃)-,-C (β-CH ₃) (α-OC (O) CH ₃)-and-C (α-CH ₃) (β-OC (O) CH ₃)-.

The biodynamics chemical compound.The method of evaluation or characterising biological kinetics chemical compound can be carried out as mentioned above.Described method comprises in addition randomly carrying out a kind of rules comes the determination test chemical compound whether to make the activity or the level of the mediator of acute biological answer-reply be subjected to about 20% or about 25% to about 70% or about 75% adjusting in vitro tests, randomly, with the suitable reference activator of glucocorticoid receptor (GR) or antagonist for example dexamethasone or hydrocortisone compare, wherein test compound does not make glucocorticoid receptor (GR) be subjected to surpassing about 10%, about 20% or about 30% activation or antagonism.In these embodiments, acute irritation or infringement biology can be to make the experimenter be exposed to ionizing radiation or pro-inflammatory signal, chemical compound or the compositions of abundant amount, randomly, wherein pro-inflammatory signal, chemical compound or compositions be antibacterial LPS or TNF α and/or randomly wherein the mediator of acute biological answer-reply be NF-κ B or I κ B.

The infringement of acute irritation or biology can be that the usefulness vehicle control mice to the mice that heals with medicine of quantity sufficient and quantity sufficient gives the influence to the mediator of acute biological answer-reply of competent antibacterial LPS and experiment with measuring chemical compound, described measurement was carried out in the following time: (i) described acute replying to maximum or approaching maximum, about 1.5 hours after giving antibacterial LPS randomly by peritoneal injection, for example, about 70-110 minute or 75-105 minute and (ii) before giving competent antibacterial LPS and/or afterwards one or two point At All Other Times, time point before giving competent antibacterial LPS and be maximum or randomly near the later time after maximum acute replying, about 2.0 or 2.5 hours after giving antibacterial LPS randomly by peritoneal injection, and randomly wherein the mediator of acute biological answer-reply be NF-κ B or I κ B.

Giving competent antibacterial LPS can be randomly in fact according to described method or its suitable variant carry out herein, and randomly, wherein chemical compound is partly regulated the level of mediator of acute biological answer-reply or active ability in fact according to described method or its suitable variant carry out herein.

Other stimulation that can analyze or infringement biology comprise the perfusion again of ischemia and one or more ischemic tissues; Low relatively, in or high-intensity heat or chemical burn; Or be exposed under other toxin or the poisonous substance.Biology, the influence of infringement can be estimated in multiple tissue or organ, for example, and spleen, blood, bone marrow, lung, brain, liver, intestinal, colon or heart.

The variable that can estimate comprises that typically the experimenter produces maximum time or the time period of replying for infringement biology that is given.Usually, damage the biological answer-reply that produces maximum in preceding 30 minutes to 48 hours that accept to damage the experimenter acute biology.Given biomolecule is replied usually the maximum of acute biology of infringement and is continued about 15 minutes to about 2 hours, although some were replied in the given time period, and accumulates in a couple of days or longer time subsequently.The time period of maximum biological answer-reply is time period easily for the effectiveness of estimating drug candidates or the mechanism of action.

17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols performances of short duration, but the ability of very effective effect (described effect is disappeared and recovered normal function) is called as biodynamics in this article replys (referring to embodiment 9).By ' biodynamics reagent ' 17 α-acetenyl androstane-5-alkene-3 β for example, 7 β, the biodynamics that 17 beta-triols cause reply with chemical compound for example its effector biomolecule of causing of dexamethasone for example ' biostatics is replied ' of glucocorticoid receptor (GR) or NF-κ B is fully different, it is the indirect inhibition of being undertaken by the activation of glucocorticoid receptor (GR) that described biostatics is replied.Biostatics was replied and come down to " all replying " under all time, that is, ' biostatics medicine ' for example the biological efficacy of dexamethasone has relatively little variation under given concentration in target cell or tissue.

Therefore, the drug action of biostatics medicine mainly changes with its concentration or pharmacokinetic property.On the contrary, biodynamics reagent is 17 α-acetenyl androstane-5-alkene-3 β for example, and 7 β, 17 beta-triols are characterised in that drug action is subjected to the concentration of target cell or tissue and as the character that stimulates biology on basis and the influence of combination of strength.Therefore, stimulate biology be by for example be exposed to may lethal dose ionizing radiation for example gamma-radiation or X-ray or contact antibacterial LPS, TNF α, maybe can activate or other reagent of inflammation-inhibiting mediator for example NF-κ B, IKB, IL-6, c reactive protein are caused.In this respect, the biodynamics medicine can show between pharmacokinetics and pharmacodynamic action than the common observed more uncertain statistics dependency of biostatics medicine.

An aspect of biodynamics medicine is that it reduces the potential ability of the system toxicity relevant with passing through adjusting biostatics medicine identical or similar target biomolecule at least in part.Biostatics medicine for example dexamethasone can be used for the treatment of various inflammatory conditions clinically, and still the biological activity of ' all replying under all time ' can cause toxicity.In the situation that suppresses NF-κ B, its activity great majority or institute in a organized way long-time (for example, above 1,2 or 4 hour to about 1,2,3 day or more of a specified duration) constant and relatively complete inhibition (for example, be suppressed about 75%, 80%, 85%, 90%, 95% or in fact 100%) can cause observable unwanted side effect, because the normal biological function that some foundation level of NF-kB activity is great majority to be organized is needed.With use glucocorticoid for example the relevant known toxicity of dexamethasone may occur at least in part when for example NF-κ B is closed relatively completely in affected biomolecule.By contrast, the biodynamics medicine can be brought into play ofer short duration replying, and can improve or reduce observable toxic side effects.

Another aspect of biodynamics medicine is that they may bring into play the ability of therapeutical effect in tissue-specific mode.Therefore, animal is to attacking the NF-kB activation that may show as in various degree of replying for example be exposed to infringement biology (antibacterial LPS that for example may lethal dose or pour into affected tissue after of short duration ischemia again) in different tissues.Biodynamics medicine replying in the relatively large tissue of animal therein works, for example, spleen of mice or heart tissue, and other organizes the NF-κ B function in the brain for example uninfluenced relatively, and for mediating concerning the target biological molecules of replying of infringement biology to small part, relatively low to replying of infringement biology.

The biodynamics medicine can be partly since the ability that they partly suppress target biological molecules work.As described in example 7 above, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols and some other chemical compounds of describing herein suppress the NF-kB activation in the cell on outer body ground, but all do not observe complete inhibition under any concentration.The activity of this and biostatics medicine dexamethasone forms contrast, and biostatics medicine dexamethasone fully suppresses the NF-kB activity in sufficiently high concentration.As if this part of external NF-κ B suppress partly to be reflected by its activity described in this embodiment.Therefore, at least some situations, the biodynamics medicine is characterised in that the ability that partly suppresses or activate target biological molecules in the system of example in vitro tests as described in example 7 above.The inhibition of NF-κ B is seemingly indirect, because do not think 17 α-acetenyl androstane-5-alkene-3 β at present, and 7 β, 17 beta-triols and other chemical compound of describing herein directly are incorporated into the NF-κ B in Cytoplasm or the nucleus.

The variant that is fit to of the rules of Miao Shuing or its in this embodiment can be used for characterizing other chemical compound by regulating for example NF-κ B and as the ability of biodynamics or biostatics agents act of (can detect activatable maybe can detect antagonism or inhibition) molecule in vivo.Chemical compound can also be analyzed in vitro tests, for example in the test described in the embodiment 7, so that further characterize their mechanism of action.The variant that is fit to of rules comprises the test compound and different route of administration that uses various dose in the body of describing among this embodiment, for example about 0.1mg/kg is to the dosage range of about 350mg/kg, with it with per os, give or give or be drawn into nasal passage, vomeronasal organ or alveolar or lead in the air flue (for example, bronchus or bronchioles) of alveolar by intranasal through cheek, Sublingual or non-intestinal (for example Intradermal, subcutaneous, intravenous or intramuscular injection).The test dose that is fit to typically is about 1-150mg/kg.The biomolecule that can measure in vivo is I κ B, kinases such as src kinases, map kinases or other signal mediator of describing herein for example.This characterizing method can be in multiple experimenter one or more in carry out Rodents for example Rhesus Macacus or machin of rat, Canis familiaris L., inhuman primate for example for example.Can comprise about 3-12 animal to every group, for example the animal groups of every group of 4-8 animal is used vehicle or the placebo that is fit to, it may be the test compound of biodynamics medicine, male biodynamics medicine reference examples is as 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols, male biostatics medicine reference examples such as dexamethasone, and will in different cell masses or tissue, compare by the group of replying and one or more matched group of test compound, for example, vehicle control group, contrast of positive or negative biodynamics medicine or the contrast of positive or negative biostatics medicine.

With this analytical applications during in the mankind, the scope of experiment option is compared with other animal naturally and is reduced.People's tissue or sample for example blood, bone marrow or lung-douching fluid than other types of organization that need obtain by invasive technique spleen or liver is easier obtains being used for analyzing for example.Therefore, usually, use the mankind reply evaluation before or carry out zooscopy simultaneously.

Inflammation treatment.An active aspect of formula 1 chemical compound is that for example NF-κ B, IL-6 or TNF α reduce inflammation for mediator that they can be by influencing inflammation.The inflammation mediator that NF-κ B molecule is normally important.The NF-kB activation that increases relates to the multiple diseases associated with inflammation and the autoimmune patient's condition.

The symptom that formula 1 chemical compound can be used for treating or improvement is relevant with the following patient's condition: inflammation, for example pain, fever or fatigue, endometriosis; Fever; Fibromyalgia; Glomerulonephritis; Graft versus host disease; Organ or tissue's transplant rejection, for example, kidney, lung, bone marrow or liver transplantation; Hemorrhagic shock; Fibromyalgia; Hyperpathia; Inflammatory bowel; Gastritis; Irritable bowel syndrome; Ulcerative colitis; Peptic ulcer; Stress ulcer; Hemorrhagic ulcer; Hyperchlorhydria; Dyspepsia; Gastroparesis; Gastroesophageal reflux disease; The struvite patient's condition in joint comprises osteoarthritis, arthritic psoriasis and rheumatoid arthritis; Struvite oculopathy is as may be relevant with the transplanting of for example cornea; Ischemia, comprise cerebral ischemia (for example, wound, epilepsy, hemorrhage or brain injury that apoplexy causes, its each can cause neural degeneration); Mucocutaneous lymphnode syndrome; The study damage; Pneumonopathy (for example, ARDS); Multiple sclerosis; Myopathy (for example, muscle protein metabolism, particularly sepsis); Neurotoxicity (for example, inductive) by HIV; Osteoporosis; Pain comprises the pain that cancer is relevant; Parkinson disease; Alzheimer; Periodontal disease; Premature labor; Psoriasis; Reperfusion injury; Septic shock; Radiocurable side effect; Temporary mandibular joint disease; Alcohol-induced hepatic damage comprises alcoholic cirrhosis; Rheumatic fever; Sarcoidosis; Scleroderma; Chronic fatigue syndrome; The patient's condition coronarius and sign comprise congestive heart failure, coronary restenosis, myocardial infarction, myocardial function unusual (for example, relevant with sepsis) and coronary artery bypass grafting; Sleep disordered; Uveitis; Seronegative polyarthritis; Ankylosing spondylitis; Reiter's syndrome and reactive arthritis; Chauffard-Still disease; Arthritic psoriasis; Enteropathic arthritis; Polymyositis; Dermatomyositis; Scleroderma; Systemic sclerosis; Vasculitis (for example, mucocutaneous lymphnode syndrome); Because for example, tension force, sprain or cartilage damages the inflammation that causes; Wound healing; Skin is thin or skin is fragile; Petechia or ecchymosis; Erythema; And wound.Wound comprise wound, chemical burn, thermal burn, radiation burn with surgical operation for example orthopedics or relevant tissue or the organ injury of abdominal surgery.

Inflammatory conditions can comprise restenosis, myocardial infarction or the cerebral infarction after the inflammation relevant with reperfusion injury, the angioplasty.Inflammatory conditions of not expecting or symptom comprise the inflammatory conditions of lung, for example, cystic fibrosis, acute asthma, chronic asthma, steroid repellence asthma, acute bronchitis, chronic bronchitis, edema due to disorder of QI, psoriasis, eczema, adult respiratory distress syndrome (ARDS) or chronic obstructive pulmonary disease (COPD).

The autoimmune patient's condition.Formula 1 chemical compound (F1C) and described herein compositions can be used for treating, preventing or delay the progress of the autoimmune patient's condition, for example type 1 diabetes, crohn, arthritis, contact dermatitis, lupus and multiple sclerosis (MS) patient's condition.The MS patient's condition comprises recurrence-remission form MS and secondary progress type MS.The lupus patient's condition comprises heart that the relevant arthritis of systemic lupus erythematosus (sle), lupus erythematosus, skin change that lupus erythematosus is relevant, the injury of kidney that the hematology is unusual, lupus erythematosus is relevant, lupus erythematosus that lupus erythematosus is relevant are relevant or lung disease, tissue inflammation, discoid lupus erythematosus, subacute skin lupus erythematosus and drug-induced lupus erythematosus that neuropsychiatry changes, lupus erythematosus is relevant that lupus erythematosus is relevant.Arthritis and related conditions comprise rheumatoid arthritis, osteoarthritis, fibromyalgia, constitutional osteoarthritis, Secondary cases osteoarthritis, arthritic psoriasis, arthritis that lupus erythematosus is relevant, the arthritis relevant with acute or chronic inflammatory bowel disease or colitis, the arthritis relevant with ankylosing spondylitis, tissue inflammation, arthralgia that arthritis is correlated with, are connected sclerosis, joint motions infringement, arthroncus, connection inflammation and synovial membrane inflammation.

F1C or the compositions that comprises F1C and one or more excipient can be used for treating, prevent, postponing the outbreak of the following patient's condition or delay its progress, for example ankylosing spondylitis, psoriasis, eczema, colitis, crohn, acute or chronic inflammatory bowel disease, autoimmunity injury of kidney and hepatic injury.F1C can be used for treating the lung and the air flue patient's condition, comprise the asthma patient's condition, for example steroid non-correlation asthma, severe asthma, specificity asthma, acute asthma or chronic asthma, allergic rhinitis, chronic bronchitis, acute bronchitis, cystic fibrosis, edema due to disorder of QI, lung fibrosis, lung airway high response, chronic obstructive pulmonary disease, pulmonary edema and adult respiratory distress syndrome.

Experimental autoimmune encephalomyelitis (EAE) is the experimental condition that carries out in animal, and it has similar to people MS clinical, histopathology and amynologic characteristic, and the same with MS, shows as in the T cell and mononuclear cell that is penetrated into CNS.Can in the susceptible mice, induce EAE by proteolipid lipoprotein (PLP) immunity that is used in the suitable adjuvants.The EAE animal model is the people MS body inner model that is used to study the pathogeny of MS and is used to characterize the new medicine that is used for the treatment of MS.

The treatment of metabolic disorder.Formula 1 chemical compound can be used for treating, preventing or delay the progress of metabolic disorder, for example type 1 diabetes, type 2 diabetes mellitus, X syndrome, hypercholesterolemia, hyperglycemia, insulin resistant (for example, relevant), glucose intolerance, hypertriglyceridemia, hyperlipoproteinemia, the lipodystrophy patient's condition, X syndrome, arteriosclerosis, atherosclerosis and obesity with obesity.X syndrome (comprising metabolism syndrome) is defined as two or more unusual set, comprises hyperinsulinemia; Obesity; Triglyceride, uric acid, Fibrinogen, low-density LDL particle and plasminogen activator inhibitor 1 (PAI-1) level raise; Reduce with the HDL-c level.Many patients that have insulin resistant but do not form type 2 diabetes mellitus as yet also are in the danger that forms metabolism syndrome, are also referred to as X syndrome, insulin resistant syndrome or many types of metabolism syndrome (plurimetabolic syndrome).X syndrome typically occur in have hyperlipemia, among two or more the patient in hyperinsulinemia, obesity, insulin resistant, the insulin resistant that causes type 2 diabetes mellitus and the diabetic complication (that is, wherein insulin resistant is the disease of the part of pathophysiology) thereof.

The independently risk factor that can relate to the cardiovascular disease relevant with the F1C treatment with metabolic disorder.These risk factors comprise that hypertension, fibrinogen level increase, high triglyceride level, LDL cholesterol raise, T-CHOL raises and low HDL cholesterol levels.Described treatment can cause the stimulating pancreas beta cell to secrete more insulin and/or the speed of the pancreas beta cell loss that delays may be in diabetics or obese patient to take place in time.

Can combine with other Therapeutic Method with formula 1 compounds for treating metabolic disorder.Can treat diabetes with in formula 1 chemical compound and the multiple therapeutic agent one or more, described multiple therapeutic agent comprises insulin sensitizer, for example PPAR-gamma agonist glitazone for example; Biguanides; Protein Tyrosine Phosphatases-1B inhibitor; Inhibitors of dipeptidyl IV; Insulin; Insulin-mimickers; Sulfonylurea; Meglitinides; α-glycoside hydrolase inhibitor; And alpha-amylase inhibitor.Metformin, phenformin, acarbose and rosiglitazone are the medicines that has been used for the treatment of some type diabetes.

As mentioned above, the compositions that comprises F1C can be used for treating, preventing or delay the progress of insulin resistant or its symptom.Insulin resistant is that insulin is brought into play its biological action in the concentration of broad range ability weakens, and produces less than desired biological action.The ability of the suitable metabolizable glucose of people of insulin resistant weakens, and poor to replying of insulin treatment, even there are some to reply.The symptom of insulin resistant comprises in the inadequate insulin activation of glucose absorption, oxidation and storage in the muscle and the fatty tissue in the steatolysis and cell that glucose produces and excretory inadequate insulin suppresses.Insulin resistant can cause or help polycystic ovary hyperstimulation syndrome, impaired glucose tolerance, gravidic diabetes, hypertension, obesity and atherosclerosis.The F1C compositions can be used for reducing insulin resistant patient's triglyceride levels.

As mentioned above, embodiment of the present invention comprise the method for identifying the chemical compound (or " test compound ") with the potentiality that are used for the treatment of, delay its progress, delay its outbreak or alleviation people or other mammiferous metabolic disorder or its symptom.Usually be described to have the disactivation agent of one or more described active external PPAR and the external incomplete inhibitor of NF-κ B by described method compounds identified, described activity typically derives from observed result in the body, for example, the morbidity of hyperglycemia postpones or the progress that has the diabetes patient's condition now delays.Chemical compound with these features is to can be used as the new chemical compound of a class that the medicine that is used for the treatment of these diseases is estimated.

In these embodiments, described method comprises selects following test compound, (i) with at external suitable negative control people or mammalian cell compare, do not make a kind of, two or three activation among PPAR-α, PPAR-κ in people or the mammalian cell and the PPAR-δ surpass about 10%, about 20%, about 30% or about 40% external; (ii) with at external suitable negative control people or mammalian cell compare, make transcriptional activity or the level inhibition of the NF-κ B in people or the mammalian cell or reduce about 20-80% or about 25-75% or about 30-70% or about 35-65% external; (iii) compare with suitable negative control or normal control, alleviate hyperglycemia, delay the progress of hyperglycemia or postpone its morbidity, increase insulin sensitivity, reduce the glucose intolerance, delay the disabled progress or the speed of pancreas β-islet cell quantity or its excreting insulin, increase the ability of pancreas β-islet cell quantity or its excreting insulin, delay the db/db mice or suffer from the weight increase speed of the mice of the inductive obesity of meals, reduce the triglyceride levels that raises, reduce the total blood or the serum cholesterol level that raise, reduce the normal or LDL that raises in blood or the serum, VLDL, the level of apoB-100 or apoB-48, or increase in blood or the serum normal or the HDL or the apoA1 level that raise or reduce the fibrinogen level that raises in blood or the serum; (iv) randomly, with compare at external suitable negative control people or mammalian cell, the activation of the variant of any biologic activity or isoform surpasses about 5%, about 10%, about 20% or about 30% in external glucocorticoid receptor (GR), androgen receptor, estrogen receptor-α, estrogen receptor-beta, mineralcorticoid receptor, progesterone receptor or these biomolecule that does not make in people or the mammalian cell.This makes to allow to identify or characterize at least in part to have the chemical compound that is used for the treatment of or improves the potentiality of mammiferous metabolic disorder.

In some embodiments, can with the activity of test compound and suitable reference compound for example formula 1 chemical compound compare.Formula 1 chemical compound can be used as positive control or the positive reference standard that meets the feature that described method provides in described method.This chemical compound comprises 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols and androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol.Other formula 1 chemical compound can be as 0 kind, a kind or 2 kinds the negative control or the reference standard that may show in three kinds of required features.This chemical compound comprises 16 α-bromine epiandrosterone, 16 α-bromo-3 β, 17 β-dihydroxy androstane-5-alkene and 16 Alpha-hydroxy epiandrosterones.

Embodiment of the present invention comprise determination test chemical compound pair one or more patient's condition relevant with metabolic disorder or disease or the effect of symptom.Typically, this mensuration is compared with suitable negative control or normal control, or compare with suitable positive control, and described being determined in the human or animal body carry out, although described mensuration can carried out external in full cell or cellular lysate sometimes.

Alleviating of hyperglycemia can be observed when blood or serum level of glucose are reduced in the normal empty stomach scope, people at least 2 years old, described scope for about 70mg/dL to 105mg/dL or 115mg/dL, and the fasting glucose level of hyperglycemia is that about 135mg/dL or about 140mg/dL are to 200mg/dL, 300mg/dL or 350mg/dL.The glucose level that surpasses about 400mg/dL is life-threatening.The 2 hour measurements of post-prandial glucose in blood or the serum after taking in carbohydrate, minimum for the people is 75g, extracts the blood measuring glucose subsequently.Blood after meal or the serum level of glucose of people's 140mg/dL to 200mg/dL are represented the hyperglycemia patient's condition, and the glucose level that surpasses 200mg/dL determines that then the people suffers from diabetes.For the people,, can use the oral glucose tolerance test (OGTT) of blood typically for the patient of the normal fasting glucose level of 70-115mg/dL.In people's OGTT, if peak glucose level (typically on the feed after 30 minutes or 1 hour) and the carbohydrate value after 2 hours in two or more situations above 200mg/dL, represent that then this patient suffers from diabetes.

Substitute as people's blood glucose uses the measured value of glycosylated haemachrome or Hb A1c to be used for for example monitoring treating diabetes.Measure Hb A1c and allow 100 to 120 days inner evaluation blood glucose or sugar levels before test, and it is to for example just having had meal or the short term variations of state is insensitive on an empty stomach.The Hb A1c level of 2.2-48% is normal for the adult, and the level of 2.5-5.9% is represented the control that diabetes obtain, the level of 6-8% is represented the control of medium diabetes, surpasses 8% Hb A1c level and represents control to the difference of the diabetes patient's condition.K.D.Pagana and T.J.Pagana, Mosby ' s Diagnostic and Laboratory Test Reference for example have been described in for the method for carrying out with explaining these and relevant rules, the 5th edition, 2001, Mosby Inc. is in 441-448,451-458, the 507-509 page or leaf.Treat with formula 1 chemical compound and to be used to reduce Hb A1c, it is relevant with the diabetes control or the treatment that improve.

Use described method can make glucose, glucose substitute or other for example be worth for example horizontal normalization of T-CHOL of phase reaction albumen (phase reactive proteins) or lipid composition herein; for example; make it get back to normal limit or scope, or near normal limit or scope.Observe in about 1%, about 2%, about 3% or about 5% scope that the normalization of glucose or substitute value typically drops to the normal glucose level as the glucose that raises or substitute level or in about 5% or about 8% scope of normal glucose substitute value.The dextrose equivalent of other thing class is described to some extent, and can be used for the method for the present invention of carrying out for those thing classes with similarly measuring or testing.The normalization of other value typically as unusual high or low level turn back to the described value for experimenter's thing class normal range the upper end or down in about 2% or about 4% to about 6%, about 10% or about 12% scope of end value.

Can be used for delaying the progress of hyperglycemia or postpone its morbidity by the inventive method compounds identified, perhaps be used to increase wherein existing or have reason to expect the patient's that forms insulin resistant insulin sensitivity.Other effect of chemical compound comprises the ability that reduces glucose intolerance, the progress that delays pancreas β-islet cell quantity or their excreting insulin capacity losses or speed or increase pancreas β-islet cell quantity or its excreting insulin.

In some embodiments, can in obese subjects, carry out described method.People's obesity or " overweight " general reference (1) Body Mass Index is about 26kg/m as used in this article ², 27kg/m ², 28kg/m ², 29kg/m ², 30kg/m ², 31kg/m ², 32kg/m ²Or bigger adult male and Body Mass Index are at least about 26kg/m ², 27kg/m ², 28kg/m ², 29kg/m ², 30kg/m ², 31kg/m ², 32kg/m ²Or bigger adult female, or (2) for example doctor or nurse are evaluated as fat or overweight situation by the health care personnel.For for example people, the determining of obesity considered body fat content and distribution possibly because some have the people of high Body Mass Index may be because high muscular tissue amount replaces fat or lipid tissue or owing at the body region that is different from the abdominal part for example body fat of significant quantity or lipid and do not think obesity academicly in buttocks or the pelvis.Obesity and Body Mass Index are described to some extent, for example, and G.A.Colditz, Med.Sci.Sports Exerc, 31 (11), Suppl., S663-S667 page or leaf, 1999; People such as F.J.Nieto-Garcia, Epidemiology, 1 (2): 146-152,1990, R.H.Eckel, Circulation, 96:3248-3250,1999.

In some embodiments, typically measure as in vitro tests, compare with suitable negative control people or mammalian cell, following one or more in people or the mammalian cell are activated significantly surpass about 10%, about 20% or about 30% external by the inventive method compounds identified: the biologic activity variant of mineralcorticoid receptor, progesterone receptor, glucocorticoid receptor (GR), androgen receptor, estrogen receptor-α, estrogen receptor-beta or any of these biomolecule.Measuring these active methods for example has been described in the United States Patent (USP) 5,298,429.In an exemplary method, whether evaluation test is that the functional part of estrogen receptor protein matter or the algoscopy of its functional through engineering approaches or modified forms comprise: (a) cultured cell, described cell comprises: express estrogen receptor protein matter, or the non-interior originality DNA of its functional through engineering approaches or modified forms, the DNA of the action gonadal hormone response element relevant with reporter gene with coding, wherein cultivate in the presence of at least a test compound and carry out, described chemical compound promptly will manage to measure its part or regulator as estrogen receptor protein matter, or the chemical compound of the ability of its functional through engineering approaches or modified forms and (b) measure the evidence that reporter gene is transcribed described in the described cell.This algoscopy typically uses mammalian cell to carry out, for example, and CV-1 or COS cell.Reporter gene can be included in the reporter plasmid, and the non-interior originality DNA of expression estrogen receptor protein matter or its functional modified forms is included in the expression plasmid, and wherein said reporter gene and expression plasmid also comprise the origin of replication of SV-40.In addition, reporter gene can be included in the reporter plasmid, and the non-interior originality DNA that wherein expresses estrogen receptor protein matter or its functional modified forms is included in the expression plasmid, and wherein reporter gene and expression plasmid also comprise selected marker.The related assays method can be used the cell of the stable transfection with detectable reporter gene, for example, for estrogen receptor-beta (based on ER β-UAS-bla GripTite ^TMThe algoscopy of cell, catalog number K1091, Invitrogen Corp.), estrogen receptor-α is (based on ER α-UAS-bla GripTite ^TMThe algoscopy of 293 cells, catalog number K1090, InvitrogenCorp.), androgen receptor is (based on AR-UAS-bla GripTite ^TMThe algoscopy of 293MSR cell, catalog number K1082, Invitrogen Corp.) or progesterone receptor (progesterone receptor-UAS-blaHEK293T test, catalog number K1103, Invitrogen Corp.) described.

Embodiment of the present invention comprise the method for determining or characterizing the chemical compound with the potentiality that are used for the treatment of or improve mammiferous metabolic disorder, described method comprises selects following chemical compound: (i) compare with external suitable negative control people or mammalian cell, do not make a kind of, two or three activation among PPAR-α, PPAR-γ and the PPAR-δ surpass about 30% external; (ii) compare, make transcriptional activity or the level inhibition of NF-κ B in people or the mammalian cell or reduce about 20-80% external with external suitable negative control people or mammalian cell; (iii) compare with suitable negative control or normal control, reduce hyperglycemia, delay the progress of hyperglycemia or postpone its morbidity, increase insulin sensitivity, reduce the glucose intolerance, delay the progress or the speed of pancreas β-islet cell quantity or their excreting insulin capacity losses, increase pancreas β-islet cell quantity or their excreting insulin abilities, delay the db/db mice or suffer from the experimenter's of the relevant obesity of the inductive or meals of meals weight increase speed, reduce the triglyceride levels that raises, reduce the total blood or the serum cholesterol level that raise, reduce blood or serum LDL normal or that raise, VLDL, apoB-100 or apoB-48 level, or increase normal or low blood or serum hdl or apoA1 level, or reduce blood or the serum fibrinogen level that raises; (iv) randomly, compare with external suitable negative control people or mammalian cell, one or more activation in the external biologic activity variant that does not make glucocorticoid receptor (GR), mineralcorticoid receptor, progesterone receptor, androgen receptor, estrogen receptor-α, estrogen receptor-beta or any of these biomolecule surpass about 30%; (v) randomly external in hepatocyte or the deutero-cell of liver or in vivo hepatocyte derive from hepatocyte or the hepatocyte of hepatic tissue or tissue in suppress PCK (PEPCK) or 11 beta-hydroxysteroid dehydrogenases (11 β-HSD), optional 11 β-HSD 1 type or the level or the activity of 11 β-HSD 2 types; Described method allows to identify or characterize has the chemical compound that is used for the treatment of or improves the potentiality of the metabolic disorder in people or the other mammal.The PEPCK enzyme can be cytosol or mitochondrion source.

The bone loss patient's condition.The compositions that comprises F1C and one or more excipient can be used for described bone loss or osteopenia treatment of conditions, prevention herein, postpones its morbidity or delay its progress, for example, the osteoporosis patient's condition, for example after primary osteoporosis, the menopause or 1 type osteoporosis, climacteric or 2 type osteoporosis, spontaneous osteoporosis, the secondary osteoporosis bone loss patient's condition that for example glucocorticoid is relevant with for example relevant bone loss of heat, chemistry or radiation burn of first, second or the third level of wound.Can improve bone mass, bone density and/or bone strength with the F1C treatment.

Drug products.In some embodiments, the invention provides the drug products that is used for the treatment of inflammatory conditions.Described drug products comprises that typically (a) is suitable for for example medicine of solid or liquid dosage form of dosage form that for example oral or non-intestinal gives.The packing of medicine and/or package insert or label have the information about the order of severity of the toxicity of pharmaceutical efficacy, the mechanism of action, predetermined patient crowd, dosage, dosage regimen, route of administration, infringement biology or the infringement that described medicine can be used for treating, if these are known.When biology, infringement was roentgenization, package insert or label can comprise the information about radiation dose or the dosage range that can use or go through to use described drug products.Drug products can randomly comprise daily record or use instruction, so that when or how patient's record uses medicine, or in using the medicine process or afterwards user has experienced what symptom or drug effect.This can help the IV phase of pharmaceutical efficacy or side effect or analyze post sales.Described in other embodiment of drug products such as other embodiment of this description of this paper.

Drug products as used in this article is meant to be checked by having the right or ratifies to sell or the administrative organization of the application of medical applications or tissue examination and approval are used for listing or product sold, described administrative organization or for example organize U.S. food and drug administration (U.S.Food andDrug Administration) or European bureau of drug (European Medicines Agency) or European medicine evaluation office (European Medicines Evaluation Agency).The application of medicine product comprises its listing or sale and sale offer or buys and consider (buy it for consideration).These activities are typically observed can influence or manage the qualification of the regulatory approved of listing, sale, purchase or product treatment.Medicine in the medicine product can novel drugs, common medicine, biological product, medical device or is used for the rules of these each application.Drug products has obtained new drug application, the application of biological product licence of U.S. of U.S. food and drug administration or European medicine evaluation office or non-U.S. new drug application, simplification usually or has sold the sale approval of medical device application.The application of drug products comprises sells government or private buyer with it, for example U.S. Department of Defense, USDOE, U.S.'s health and Department of Public Enterprises or private medicine buyer or distributor tissue.Other application comprises uses described Drug therapy to point out or the application of the medical patient's condition ratified and doctor's approval or the outer purposes (off label use) of indication not.Drug products before the approval is others of the present invention, and it may described drug products be identical in fact with herein, but it is used in the medicine that listing approval preparation before is used to sell or be used for administration section's examination.

The predetermined patient crowd that drug products is determined also can specify the crowd who is excluded, and if any, eliminating is applied to for example pediatric patients or gerontal patient.Typically specify dosage every day of medicine about the information of dosage, and dosage regimen is described by frequency and the persistent period that gives or take medicine.Route of administration determines to be suitable for one or more paths of medicinal application, although given preparation typically goes through only to be used for a kind of route of administration.Packing or the confirmable dosage of label, dosage regimen and route of administration are as described in other place herein.

In one embodiment, drug products is used for the treatment of, the prevention or the another kind of patient's condition of improving inflammatory conditions or describing herein, and it comprises or comprises preparation and package insert or label, described preparation comprises and 1,2,3,4 or more kinds ofly (for example be used for oral or non-intestinal, muscle, subcutaneous or corium is injection down) chemical compound of the excipient preparation that gives formula 1 chemical compound for example, described package insert or label be described in disease or comprise begin giving construction 1 chemical compound after making a definite diagnosis or otherwise observing daily dose (for example, 0.5mg, 1mg, 4mg, 5mg, 10mg, 20mg, 25mg, 50mg, 100mg, 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 400mg, 450mg or 500mg), continue 1,2,3,4,5,6,7,8,9,10 or more a plurality of Consecutive Days.The information of package insert or label can comprise about the information to the biological answer-reply of medicine or therapeutic scheme.Described information can comprise the explanation to one or more following situations: (a) for example use relevant one or more side effect or the toxicity of described medicine among the non-human primate with people or mammal, (b) it is to the effect of inflammation or other patient's condition, (c) with the other therapeutic agent rules used with described medicine of dexamethasone or other glucocorticoid or instruct and (d) begin time or time period about described medicine in order to obtain best or known treatment interests for example.

In certain aspects, the invention provides the CD4 that evaluation may be used for regulating mammal with detecting ⁺CD25 ⁺Regulatory T cells, CD4 ⁺CD25 ⁺CD103 ⁺Regulatory T cells, CD4 ⁺CD25 ^HighCD103 ⁺Regulatory T cells or CD4 ⁺CD25 ^HighThe quantity of regulatory T cells or the method for active chemical compound, it comprises selects following chemical compound: (i) compare one or more activation in the external biologic activity variant that does not make glucocorticoid receptor (GR), androgen receptor, estrogen receptor-α, estrogen receptor-beta or any of these biomolecule in people or the mammalian cell or suppress to surpass about 30% with external suitable contrast people or mammalian cell; (ii) molecular weight is about 100-1000 dalton, and randomly, molecular weight is about 250-850 dalton; (iii) compare, in suitable algoscopy, make CD4 with suitable negative control or normal control ⁺CD25 ⁺Regulatory T cells, CD4 ⁺CD25 ⁺CD103 ⁺Regulatory T cells, CD4 ⁺CD25 ^HighCD103 ⁺Regulatory T cells or CD4 ⁺CD25 ^HighThe quantity of regulatory T cells or active increase or minimizing surpass 20%; (iv) compare, randomly make NF-κ B transcriptional activity or the level inhibition in people or the mammalian cell or reduce about 20-80% external with external suitable negative control people or mammalian cell.Formula 1 chemical compound and other chemical compound can be used in fact in these embodiments described in following examples 20 or 21.

In external or body, increase or reduce for example CD4 of some T cell subsets ⁺CD25 ⁺T cell and CD4+CD25 ^HighThe chemical compound of T cell activity or quantity is to be used for the treatment of or to delay cell, organ or tissue in the autoimmune patient's condition, cancer, neurological's wound or disease (for example wound such as ischemia after neurone loss) and metabolic disease such as type i diabetes, atherosclerosis, the autotransplantation to repel and the morbidity of graft versus host disease of position of these situations or the material standed for of progress maybe may take place in existence.Described treatment can be used for improving wound healing, treatment reperfusion injury, narrow, angioplasty restenosis, myocardial infarction or cerebral infarction afterwards.Embodiment comprises that molecular weight is less than about 2,000 dalton, less than about 1,000 dalton or less than about 500 daltonian chemical compounds.The molecular weight of one group of chemical compound is about 285 or 290 to about 500 or 650 dalton.The Treg cell response can be used as to be compared with suitable negative control with the experimenter of compounds for treating or the Treg cell is increased or reduce about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 100%, about 200%, about 400%, about 600%, about 1000%, about 2000%, about 5000%, about 10000% or more and observe, and the Treg cell typically is CD4 ⁺CD25 ⁺T cell or CD4+CD25 ^HighThe T cell.These changes can be used as external or intravital their the active increases at blood circulation or in cell of Treg cell quantity or reduction and observe.

Analyze the cell subset pattern for example the method for T cell pattern can obtain by in the several different methods any, comprise flow cytometry (FACS), for example, people such as Levy, Clin.Immunol.Immunopathol.35:328,1985.In facs analysis, the monoclonal antibody of various cell subsets is incorporated into and identifies the phenotype surface antigen that is present on the cell.Have the commercially available antibody that can detect these indicants existence, making does not need to prepare antibody usually.Expect that the antibody of the antigen indicant that evaluation is identical or be closely related provides similar diagnostic result.Therefore, when quoting with it bonded concrete monoclonal antibody (for example, CD4 or CD25) appointment indicant antigen as proof in description or claims, this appointment comprises this indicant, even used different monoclonal antibodies in authentication.The phenotype indicant of being concerned about comprises the general indicant that is used for multiple cell subset type, comprises the CD3 that is used for total T cell, be used to assist/CD4 of induced T lymphocyte, is used for the CD8 of suppressor T lymphocyte/cytotoxic T cell and is used for the CD16/56 of NK cell; Express the subclass indicant of CD8, for example be used for CD11b, the cd38 that is used for activatory suppressor T lymphocyte/cytotoxic T cell, the HLA-DR that is used for activatory suppressor T lymphocyte/cytotoxic T cell and the CD57 of suppressor T lymphocyte; And the indicant of expressing CD4, for example CD25 and the HLA-DR that is used for activatory assisting/induced T lymphocyte comprise the Treg cell.

Dosed administration rules or method.When treatment any patient's condition disclosed herein or symptom, can be to suffering from the described patient's condition or symptom or to the experimenter of the patient's condition or symptom sensitivity (every day) or giving construction 1 chemical compound periodically continuously.With the formula 1 compounds for treating patient's condition for example when inflammatory conditions or the another kind of patient's condition disclosed herein, some aspects of not expecting relevant with discontinuous dosed administration usually can be avoided or improve to dosed administration intermittently.This aspect of not expecting comprises that patient or experimenter fail to observe the dosage regimen of every day or reduce other therapeutic agent for example dosage of glucocorticoid and/or the side effect do not expected or the toxicity relevant with them, for example bone loss or absorption again.

In some embodiments,, then continue the dosed administration of every day as long as disease or symptom are significantly, typically like this for chronic conditions.In other embodiments, the dosed administration of every day continues 1,2,3,4,5,6,7,8,9 or 10 Consecutive Days, follows no dosed administration by a period of time then, if up to or need dosed administration once more.These embodiments typically relate to treats the acute patient's condition that may recur every now and then or not recur.The treatment of chronic conditions typically relates to long successive every day of dosed administration.

In any (every day) continuously or dosage regimen intermittently, or when treatment described herein any disease, the patient's condition or symptom, formula 1 chemical compound can give by one or more suitable paths, for example, oral, under cheek, Sublingual, part, intramuscular, subcutaneous, corium, intravenous, Intradermal or give by aerosol.

Daily dose is generally about 0.05mg/kg/ days to about 200mg/kg/ days.Typical dosage range is about 0.1 to about 100mg/kg/ days, comprise about 0.2mg/kg/ days, 0.5mg/kg/ days, about 1mg/kg/ days, about 2mg/kg/ days, about 4mg/kg/ days, about 5mg/kg/ days, about 6mg/kg/ days, about 8mg/kg/ days, about 10mg/kg/ days, about 20mg/kg/ days, about 40mg/kg/ days or about 100mg/kg/ days.Also can in the situation that the veterinary uses, adopt higher dosage, for example, about 250mg/kg/ days, about 300mg/kg/ days or about 350mg/kg/ days.Can formula 1 chemical compound is oral or give by non-intestinal with about 2 to about 50mg/kg/ days or about 2-40mg/kg/ days.This dosed administration typically produces about 4ng/mL or the about 8ng/mL serum levels to formula 1 chemical compound of about 125ng/mL or about 250ng/mL, and for example, about 15ng/mL arrives about 120ng/mL or about 20ng/mL arrives about 100ng/mL.This serum levels can be of short duration, for example, continues about 30 minutes or about 60 minutes to about 2 hours or about 8 hours, and I can appear at the same day that gives chemical compound, perhaps occur in the slower time for depot formulations.

Successive every day, dosed administration was generally used for the described chronic conditions of treatment herein.Daily dose gives as independent dosage usually, but daily dose can be divided into again 2 or 3 sub-doses.Dosed administration rules intermittently comprise every 1 day or giving construction 1 chemical compound every three days, continue the reasonable time section.When the treatment hemocyte lacked, dosed administration experienced the clear marrow incident of short-term (short-lived myeloablative event) same day of roentgenization for example from the experimenter usually.For continuing situation more of a specified duration, for example, the chemotherapy of cancer, about 12 hours, about 1 day, about 2 days or beginning in about 3 days that dosed administration formula 1 chemical compound can be after giving chemotherapeutics to the experimenter.The time period that the dosed administration of every day can continue to determine is fixing or the variable time period that does not have dosed administration subsequently.In these embodiments, the disease activity for example active treatment of multiple sclerosis, arthritis, asthma, colitis or crohn can no longer be treated before another disease activity takes place or begins afterwards for continuing dosed administration every day of 3-14 Consecutive Days or 5-10 Consecutive Days.

The clinical patient's condition and symptom.Described herein Compounds and methods for can be used for making the described patient's condition and/or their one or more symptoms to obtain medical treatment, improve, prevent herein or delays its progress.This application comprises the inhibition bone resorption, minimizing and chemotherapy are (for example, the antiinflammatory glucocorticoid) relevant or by its side effect of not expecting that causes, treatment, prevention or delaying osteoporosis disease and fracture, suppress vascular restenosis and treatment diabetic retinopathy, degeneration of macula, inflammation. ^*1 ^*Inflammatory conditions of not expecting or symptom, the inflammatory conditions of lung for example, for example, cystic fibrosis, acute or chronic asthma, bronchial asthma, specificity asthma, ARDS or COPD, or autoimmune disorder osteoarthritis for example, rheumatoid arthritis, for example autoimmune pancreatitis of pancreatitis, systemic lupus erythematosus (sle), the tissue inflammation that lupus erythematosus is relevant, the arthritis that lupus erythematosus is relevant, the skin change that lupus erythematosus is relevant, the hematology that lupus erythematosus is relevant is unusual, the kidney injury that lupus erythematosus is relevant, heart or lung disease that lupus erythematosus is relevant, neuropsychiatry that the lupus erythematosus of not expecting is relevant or neurological change.

The symptom of the treatable patient's condition comprises fever, arthralgia (arthralgia), arthritis and oromeningitis (pleuritis or pericarditis).Also can give other medicines and be used for treatment of the present invention.Therefore, pain can be used NSAID (non-steroidal anti-inflammatory drug) for example aspirin, salicylate, ibuprofen, naproxen, sulindac, oxaprozin and tolmetin are treated.The skin characteristic of systemic lupus erythematosus (sle) can be treated with antimalarial, for example oxychloroquine, chloroquine and quinacrine.Tretinoin for example Accutane (istretinoin) and etretinate also can be used for the treatment of skin symptom with described chemical compound combination herein.Organ damages can be with oral or corticosteroid treatment that intravenous gives usually.Spendable corticosteroid comprises hydrocortisone (Cortisol), corticosterone, aldosterone, ACTH, triamcinolone and derivant be the diacetic acid triamcinolone for example, triamcinolone hexacetonide, and triamcinolone acetonide, betamethasone and derivant be betamethasone dipropionate for example, betamethasone benzoate, betamethasone sodium phosphate, the acetic acid betamethasone, and betamethasone valerate, flunisolide, prednisone and derivant thereof, fluocinonide and derivant be fluocinolone acetonide for example, the for example two acetic acid diflorasones of diflorasone and derivant, halcinonide, dexamethasone and derivant be dexamethasone dipropionate and valeric acid dexamethasone for example, desoximetasone (removing the hydroxyl betamethasone), diflucortolone and derivant be diflucortolone valerate for example), flucloronide, fluocinonide, fluocortolone, fluprednidene, flurandrenolide, clobetasol, clobetasone and derivant be the clobetasone butyrate alclometasone for example, aniprime, and fluocortolone.

When the oral administration of corticosteroid is not competent, intravenous injection medrat therapy pulse (high dose) can be used for treating lupus nephritis and other serious non-kidney performance, for example hemolytic anemia, inflammation of the central nervous system (cerebritis), low platelet counting and serious pleuropericarditis.

Formula 1 chemical compound can be used for making osteoporosis or fracture to obtain medical treatment, prevent or delays its progress.Experimenter's treatment can cause that bone enhancing and/or bone mass or mineral loss reduce, and causes that the resistance to fracture increases.As used in this article, " treatment " described herein those patient's condition are meant that described treatment can cause that the described patient's condition improves, prevents or delays its progress, and/or one or more symptoms of this patient's condition improve, prevent or delay its progress.

Formula I chemical compound can be used for metabolic disease treatment, prevent, delay its progress or postpone its morbidity, for example type i diabetes, type ii diabetes, hyperglycemia, insulin resistant, glucose intolerance, hypercholesterolemia, hypertriglyceridemia, hyperlipoproteinemia, lipodystrophy, X syndrome and atherosclerosis.

Preparation and the compositions that is used to prepare preparation.Preparation that embodiment of the present invention comprise herein and describe in other place of the present disclosure.Although formula 1 chemical compound might give separately, usually it is provided as preparation.Described preparation for veterinary and human the application, comprises at least a formula 1 chemical compound and one or more excipient and one or more optional other therapeutic components.

Preparation comprises and contains 1,2,3,4 or the compositions of acceptable excipient of more kinds of pharmacy or carrier.Compositions is used to prepare the preparation that is suitable for human or animal's application.That the preparation route of administration that is fit to comprises is oral, rectum, per nasal, part (comprising through cheek and Sublingual), vagina, rectum and non-intestinal (comprise in subcutaneous, intramuscular, intravenous, intradermal, the sheath, ophthalmic and epidural).Usually, moisture and on-aqueous liquid or cream preparation are sent by non-intestinal, oral or local approach.In other embodiments, for example in intermittence of the present invention dosed administration method, formula 1 chemical compound can be used as and is adapted to pass through the moisture of any administration disclosed herein or on-aqueous liquid preparation or solid preparation existence, for example, oral, local, through for example subcutaneous warehouse of cheek, Sublingual, non-intestinal, inhalation aerosol or bank or intraperitoneal or intramuscular bank.Should be appreciated that the difference of replying that preferred approach can be treated employed formula 1 chemical compound or other Therapeutic Method with for example experimenter's pathological conditions or body weight or experimenter and different is suitable for described situation perhaps.

Preparation comprises those that are suitable for above-mentioned route of administration.Preparation can exist as unit dosage forms easily, and can be by known any method preparation in the pharmaceutical field.Described technology, excipient and preparation be usually for example, Remington ' s Pharmaceutical Sciences, MackPublishing Co., Easton, PA 1985, the 17 editions; People such as Nema, PDA J.Pharm.Sci.Tech.1997 51:166-171; The Pharmaceutical CoatingTechnology that people such as G.Cole edit, 1995, Taylor ﹠amp; Francis, ISBN0 136628915; The Pharmaceutical Dosage Forms that people such as H.A.Lieberman edit, 1992 revised editions for the second time, the 1st and 2 volumes, Marcel Dekker, ISBN 0824793870; J.T.Carstensen.PharmaceuticalPreformulation, 1998, the 1-306 pages or leaves are found among the Technomic Publishing Co.ISBN1566766907.The Exemplary excipients that is used for preparation comprises emulsifing wax, propyl gallate, citric acid, lactic acid, polyoxyethylene sorbitan monoleate, sodium chloride, isopropyl palmitate, glycerol, white petrolatum and other excipient disclosed herein.

Be adapted to pass through preparation that approach disclosed herein gives or the compositions that is used to produce preparation disclosed herein and randomly comprise about 0.01 to about 500 microns, about 0.1 to about 100 microns or about 0.5 to about 75 microns mean diameter.Mean diameter comprises with 0.05 micron or 0.1 micron or other increment mean diameter of (for example, about 0.05,0.1,0.5,1,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6,7,8,9,10,15,20,25,30,35,40,50,60,75,85,100,120 etc. micron number) in 0.01 to 500 micron scope.At formula 1 chemical compound or when comprising that formula 1 compound compositions is used as intermediate and produces agent next life, they can comprise in these mean diameters or the particle size range a kind of, two kinds, three kinds or more kinds of.Disclosed herein and when comprising any compositions of formula 1 chemical compound (with one or more optional excipient) or preparation in preparation, can randomly chemical compound or compositions be ground, sieve or with other mode pelletize, with the particle diameter that obtains expecting.

In some embodiments, spendable formula 1 chemical compound is characterised in that does not have appreciable androgenicity (androgenicity).In these embodiments, formula 1 chemical compound be characterised in that have androgen for example testosterone, Testosterone Propionate, dihydrotestosterone or propanoic acid dihydrotestosterone about 30% or lower, about 20% or lower, about 10% or lower, about 5% or lower androgenicity, as the algoscopy that is fit to of using the suitable positive and/or negative control to carry out is measured.The algoscopy that is fit to that is used for the androgenicity of all cpds for example has been described in, and J.R.Brooks waits the people, and Prostate 1991,18:215-227; People such as M.Gerrity, Int.J.Androl.19814:494-504; People such as S.S.Rao, Indian J.Exp.Biol.19697:20-22; People such as O.Sunami, J.Toxicol.Sci.2000 25:403-415; People such as G.H.Deckers are among the J.SteroidBiochem.Mol.Biol.2000 74:83-92.The androgenicity of formula 1 chemical compound is randomly as measuring as described in these algoscopys or any other algoscopy one or more as described in these algoscopys or any other algoscopy one or more or in fact.

Therefore, one of this embodiment comprises the treatment method of the described patient's condition herein, comprise formula 1 chemical compound that the experimenter that these needs are arranged is given effective dose, or send formula 1 chemical compound of effective dose for experimenter's tissue, wherein (for example in the algoscopy that is fit to, described in the above-mentioned cited literature 2) in when measuring, formula 1 chemical compound have androgen for example testosterone, Testosterone Propionate, dihydrotestosterone or propanoic acid dihydrotestosterone about 30% or lower, about 20% or lower, about 10% or lower, about 5% or lower androgenicity.When carrying out this method, randomly to experimenter or mammal, for example, rodent, people or primate are for example monitored, and whether disease, the patient's condition or symptom improve, prevent or reduce its order of severity.This monitoring can randomly be included under the reasonable time following one or more of measuring in the circulation: cytokine ((for example, TNF α, IL-13, IL-1 β), WBC, platelet, granulocyte, neutrophil cell, RBC, NK cell, macrophage or other immunocyte type, for example, in this article or in the list of references of quoting, describe, for example, in the baseline before the begin treatment and the different time behind usefulness formula 1 compounds for treating, for example about 2-45 days after finishing with formula 1 compounds for treating.

As mentioned above, in some embodiments, will treat with corticosteroid or glucocorticosteroidsin in combination with formula 1 chemical compound and use.Corticosteroid uses in many clinical settings, for example be used for, for example reduce inflammation or the generation of autoimmune response or the intensity or the frequency of acute attack under ulcerative colitis, the acute or conditions such as chronic asthma, bronchial asthma, psoriasis, systemic lupus erythematosus (sle), hepatitis, pulmonary fibrosis, type i diabetes, type ii diabetes or cachexia in acute or chronic rheumatoid arthritis for example, acute or arthroxerosis, the colitis patient's condition.Yet many corticosteroid have pronounced side effects or toxicity, and this has limited their application and effect.Formula 1 chemical compound can be used for offsetting this side effect or toxicity, and can not resist whole desired treatment ability of corticosteroid.This makes and allows to continue to use corticosteroid or revise its dosage, for example, uses with the dosage that increases, and can not strengthen side effect or toxicity or reduce corticosteroid dosage.Can treat, prevention, side effect that improves or reduce or toxicity comprise following one or more: the bone loss, osteogenesis reduces, bone resorption strengthens, osteoporosis, immunosuppressant, the sensitivity that infects is increased, emotion or personality change, depression, headache, dizzy, hypertension or hypertonicity, the muscle weakness, tired, feel sick, uncomfortable, peptic ulcer, pancreatitis, skin is thin or skin is fragile, child or experimenter's growth inhibited before the manhood, thromboembolism, cataract and edema.Dosage, route of administration and the dosage regimen of formula 1 chemical compound are in fact as described herein.Randomly give about 0.5 formula 1 chemical compound that arrives about 20mg/kg/ days exemplary dose in time period of about 1 week after the dosed administration of corticosteroid finishes to about 6 months or longer time in the process neutralization that gives corticosteroid.Corticosteroid uses known dosage, route of administration and dosage relieve pain in fact, referring to for example, and Physicians Desk Reference, the 54th edition, 2000, the 323-2781 pages or leaves, ISBN 1-56363 2, Medical Economics Co., Inc., Montvale, NJ.Yet, randomly can control the dosage of corticosteroid, for example, surpassing normal dose increases about 10% to about 300%, and whole side effect relevant with corticosteroid or toxicity do not have corresponding increase.This increase is little by little to carry out in the fully long time period and according to experimenter's clinical condition, and for example, in about 2 week to about 1 year times, the daily dose of corticosteroid increases by about 10% to about 20% to about 300% maximum.

Therapeutic Method can be used for treatment, prevents or improves acute wound, for example myocardial infarction, hemorrhage for example cerebral hemorrhage or apoplexy or fracture, osteoporosis or bone resorption excessive or that do not expect or loss.Treatment can be used for promoting the reparation to following damage or damage, described damage or injury are to skin, mucosa, cartilage, liver, heart tissue, bone or CNS or wherein have the damage or the injury of nervous tissue of the position of damage, for example, chemistry or thermal burn, osteoarthritis, rheumatoid arthritis, liver cirrhosis, osteoporosis, fracture, myocardial infarction, apoplexy or head trauma.Described can also be used for reducing because treatment (for example, in the lupus patient's condition suffering from inflammatory bowel, crohn, acute or chronic colitis or kidney disorders is for example acute or the patient of chronic renal failure or autoimmunity injury of kidney in use glucocorticoid treatment) bone that causes loses.

Following embodiment is described one or more aspect of the present invention.

1. identify or the method for characterising biological kinetics chemical compound, comprise: the experimenter be exposed to cause detectable to acute irritation or infringement biology the acute irritation of replying or infringement biology after biological answer-reply in the body of experiment with measuring chemical compound in the experimenter, wherein said test compound following time or time period to stimulating or the mediator of the acute biological answer-reply of infringement biology causes that favourable treatment replys: (i) acute reply into maximum or near maximum, or (ii) acute replying in the long acute biological answer-reply time period increases, and wherein reply and be different from it at one in time (i) or favourable treatment (ii), two, three, or more a plurality of early or the effect of later time or time period to the mediator of acute biological answer-reply, and early or later time or this effect of time period, with identical or identical in fact early or later time or suitable vehicle or the placebo of time period compare, the level of the mediator of acute biological answer-reply or active increase or reduce less than about 50%, thus identify that mediator to acute biological answer-reply produces that favourable treatment is replied and in the time (i) or (ii) produce and be different from it at one, two, three, more a plurality of early or the favourable treatment of later time or the time period chemical compound of replying to the effect of the mediator of acute biological answer-reply be the biodynamics chemical compound.

2. the method for embodiment 1, it comprises in addition carrying out a kind of rules comes the determination test chemical compound whether to make the activity or the level of the mediator of acute biological answer-reply be subjected to about 25% to about 75% adjusting in vitro tests, randomly, wherein compare with suitable the reference activator or the antagonist of glucocorticoid receptor (GR), test compound does not make glucocorticoid receptor (GR) be subjected to surpassing about 20% activation or antagonism.

3. embodiment 1 or 2 method, wherein said acute irritation or infringement biology are to make the experimenter be exposed to ionizing radiation or pro-inflammatory signal, chemical compound or the compositions of abundant amount, randomly wherein pro-inflammatory signal, chemical compound or compositions are antibacterial LPS or TNF α, and/or randomly wherein the mediator of acute biological answer-reply be NF-κ B or I κ B.

4. embodiment 1,2 or 3 method, the infringement of wherein said acute irritation or biology is that the usefulness vehicle control mice to the heal with medicine mice and the quantity sufficient of quantity sufficient gives enough antibacterial LPS, and in of the effect of following measure of time test compound: (i) described acute replying to maximum or when maximum to the mediator of acute biological answer-reply, about 1.5 hours after giving antibacterial LPS randomly by peritoneal injection, (ii) before giving enough antibacterial LPS and/or afterwards one or two point At All Other Times, time point before giving enough antibacterial LPS and reply maximum or near a later time point after maximum randomly acute, randomly about 2.0 or 2.5 hours after giving antibacterial LPS by peritoneal injection and randomly wherein the mediator of acute biological answer-reply be NF-κ B or I κ B.

5. the method for embodiment 4, wherein giving enough antibacterial LPS is to carry out according to the method described in the embodiment 9 or its variant that is fit in fact, and randomly, wherein the chemical compound level of mediator or the active ability of partly regulating acute biological answer-reply is to carry out according to the method described in the embodiment 7 or its variant that is fit in fact.

6. embodiment 1,2,3,4 or 5 method, comprise that using positive biodynamics medicine contrast (randomly is 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols) come the relative potency or the effectiveness of evaluation test chemical compound, and use the biostatics medicine to contrast the relative potency or the effectiveness of evaluation test chemical compound.

Drug products or the approval before drug products, comprise medicine and package insert or label in the dosage form that is in medicine and the packing, described package insert or label comprise the information of effectiveness, the mechanism of action or clinical practice about medicine, and wherein effectiveness, the mechanism of action or clinical practice information derive from the characterizing method of the method that comprises embodiment 1,2,3,4 or 5 at least in part.

Drug products or the approval before drug products, comprise medicine and package insert or label in the dosage form that is in medicine and the packing, described package insert or label comprise the effectiveness about medicine, the mechanism of action, or the information of clinical practice, wherein render a service, the mechanism of action or clinical practice information derive from following characterizing method at least in part, described characterizing method comprises that (a) makes cell contact time enough at the activator of the NF-kB activity of external and q.s, and wherein said cell can be replied the agent of NF-kB activation by NF-κ B level in can detected cell or active increasing.(b) make cell contact time enough external with the medicine of q.s, wherein compare with the contrast that is fit to, described medicine can suppress the activation of NF-kB activity with detecting; (c) ability and the reference compound that randomly medicine is suppressed the NF-kB activation compares, wherein reference compound is described formula 1 chemical compound herein, it has the ability that makes the NF-kB activation be subjected to about 25% to about 75% inhibition in characterizing method, wherein in characterizing method, described medicine make the NF-kB activation suppress about 25% to about 75% and randomly wherein reference compound or medicine can not detect ground or directly be incorporated into glucocorticoid receptor (GR) significantly, perhaps randomly wherein reference compound or medicine can not detect ground or exciting significantly glucocorticoid receptor (GR), randomly, compare with the agonist contrast that is fit to, described medicine does not make glucocorticoid receptor (GR) have to surpass about 20% excitement.

9. embodiment 7 or 8 drug products, wherein dosage form comprises oral, non-intestinal, part or sucks preparation.

10. embodiment 8 or 9 drug products, wherein reference compound or medicine make the NF-kB activation be subjected to about 35% to about 70% or about 40% to about 65% inhibition in characterizing method.

11. the drug products of embodiment 8,9 or 10, wherein the NF-κ B in the cell is by antigen or gene outcome, ultraviolet radiation, heat or temperature rising, lymphokine or oxyradical or the H of TNF-α, TNF-β, TGF-β, IL-1, epidermal growth factor, antibacterial LPS, bacterial peptide polysaccharide, zymosan, bacterial lipoprotein, antibacterial or virus ₂O ₂In a kind of, two kinds, three kinds or more kinds of activation.

12. the drug products of embodiment 8,9,10 or 11, wherein reference compound or medicine be fit in conjunction with test in the k of 10 μ m _dDirectly be incorporated into glucocorticoid receptor (GR), perhaps wherein reference compound or medicine can not detect the exciting glucocorticoid receptor (GR) in ground in the concentration that is equal to or greater than about 10 μ M in the test of the activation of the gene expression that is suitable for detecting the glucocorticoid receptor (GR) mediation or increase.

13. the drug products of embodiment 8,9,10,11 or 12, wherein cell in vitro is mammal, rodent or people's a cell, randomly is selected from people THP-1 cell, rat RAW cell, macrophage, mononuclear cell, T-lymphocyte, B-lymphocyte, arborescent cell, glial cell, Kupffer Cell, hepatocyte, neutrophil cell, leukocyte and derives from the cell of whole blood.

14. the drug products of embodiment 7 or 8, comprising about the information of effectiveness, the mechanism of action or the clinical practice of medicine to meet administrative organization or to check tissue examination or ratify the commercial use of described drug products or the standard of listing.

15. the inflammatory conditions in the treatment mammal or the method for autoimmune disease, comprise the biodynamics chemical compound that the experimenter is given or sends for experimenter's tissue the method evaluation of passing through embodiment 1,2,3,4,5 or 6 of effective dose, wherein use positive biodynamics chemical compound as the reference standard, perhaps wherein the biodynamics chemical compound partly suppresses the mediator of acute biological answer-reply in the in vitro tests that is fit to, and wherein the in vitro tests of Shi Heing randomly comes down to method or its suitable variant of embodiment 7.

16. have the pure chemical compound of following structure

Wherein, R ¹For-H or optional substituted C _1-8Alkyl, another R ¹For-OH, C _2-8Ester or C _1-8Ether; A R ²For-H or optional substituted C _1-8Alkyl, another R ²For-H ,-OH, C _2-8Ester or C _1-8Ether; A R ³For-H or optional substituted C _1-8Alkyl, another R ³For-OH, C _2-8Ester, C _1-8Ether or optional substituted C _1-8Alkyl; A R ⁴For-H or optional substituted C _1-8Alkyl, another R ⁴For-OH, C _2-8Ester or C _1-8Ether; R ⁵For-CH ₃,-C ₂H ₅Or-CH ₂OH; R ⁶For-H ,-CH ₃,-C ₂H ₅Or-CH ₂OH; A R ⁷For-H or optional substituted C _1-8Alkyl, another R ⁷For-H ,-OH, C _2-8Ester or C _1-8Ether; R ¹⁰For-H or halogen; With a R ¹¹For-H or optional substituted C _1-8Alkyl, another R ¹¹For-H ,-OH, C _2-8Ester, C _1-8Ether or optional substituted C _1-8Alkyl.

17. the chemical compound of the purification of embodiment 16, wherein R ², R ⁷Or R ¹¹In one, two or three are-OH, C _2-8Ester, C _1-8Ether ,=O or=NOH.

18. the chemical compound of the purification of embodiment 17, it is selected from 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols, androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 7 α, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 11 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 11 β, 16 α, 17 β-tetrol, 3 β, 11 β, 16 β, 17 β-tetrol, androstane-5-alkene-3 α, 11 β, 16 β, the C of 17 β-tetrol or any of these chemical compound _2-4Monoesters or C _2-4Two ester analogs, randomly, (1) C wherein _2-4Monoesters is acetas or propionic ester or (2) C on 3-position or the 17-position _2-4Diester is acetas or the propionic ester on 3-position and the 17-position.

19. the chemical compound of the purification of embodiment 17 or 18, wherein chemical compound for (a) is minimum be 80% pure, minimum be 95% pure or be to the maximum 98% pure powder or granule or (b) minimum be 80% pure, minimum be 95% pure or be 98% pure solution or suspension to the maximum.These chemical compounds comprise 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols, androstane-5-alkene-3 β, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 11 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 7 β, 16 α, the configuration of 17 β-tetrol and one of them or two hydroxyls becomes to β or becomes the epimer of these chemical compounds of α from β from α, for example, 17 β-acetenyl androstane-5-alkene-3 β, 7 β, 17 α-triol, androstane-5-alkene-3 β, 4 β, 16 α, 17 α-tetrol, 17 α-acetenyl androstane-5-alkene-3 α, 7 β, 17 beta-triols or androstane-5-alkene-3 α, 4 β, 16 α, 17 β-tetrol.

20. the chemical compound of the purification of embodiment 17,18 or 19, wherein said chemical compound is about 80%, about 85%, about 90%, about 95%, about 97% or about 98% to about 99.5% or about 99.9% pure, randomly, wherein said chemical compound is powder or particulate form, randomly, wherein by suitable algoscopy for example the mean diameter of the powder measured of light scattering method arrive about 5 μ m, about 10 μ m or about 25 μ m for about 50nm or about 100nm.

21. the method for authenticating compound, described chemical compound have the CD4 that can regulate in the mammal with detecting ⁺CD25 ⁺Regulatory T cells, CD4 ⁺CD25 ⁺CD103 ⁺Regulatory T cells, CD4+CD25 ^HighCD103 ⁺Regulatory T cells or CD4+CD25 ^HighThe quantity of regulatory T cells or active potentiality, described method comprises selects following chemical compound: (i) with at external the contrast people or the mammalian cell that are fit to compare, one or more in the biologic activity variant of the glucocorticoid receptor (GR) in external people of making or mammalian cell, androgen receptor, estrogen receptor-α, estrogen receptor-beta or any of these biomolecule are subjected to surpassing about 30% activation or inhibition; (ii) molecular weight is about 100-1000 dalton, and randomly molecular weight is about 250-850 dalton; (iii) compare, in the test that is fit to, make CD4 with the negative control or the normal control that are fit to ⁺CD25 ⁺Regulatory T cells, CD4 ⁺CD25 ⁺CD103 ⁺Regulatory T cells, CD4+CD25 ^HighCD103 ⁺Regulatory T cells or CD4+CD25 ^HighThe quantity of regulatory T cells or active increase or the reduction that has above 20%; (iv) compare, randomly make transcriptional activity or the level inhibition of NF-κ B in people or the mammalian cell or reduce about 20-80% external with external suitable negative control people or mammalian cell; Thereby authenticating compound.

22. the method for embodiment 21, wherein mammal is rodent or people.

23. the method for embodiment 21 or 22 is wherein measured CD4 by a kind of rules ⁺CD25 ⁺Regulatory T cells, CD4 ⁺CD25 ⁺CD103 ⁺Regulatory T cells, CD4+CD25 ^HighCD103 ⁺Regulatory T cells or CD4+CD25 ^HighThe quantity of regulatory T cells or activity, described rules comprise following a kind of, two or three: (a) method of embodiment 20 or its variant that is fit to; (b) method of embodiment 21 or its variant that is fit to; Or the method in the list of references of (c) quoting in this article or its variant that is fit to, wherein said method or its variant that is fit to allow to estimate CD4 ⁺CD25 ⁺Regulatory T cells, CD4 ⁺CD25 ⁺CD103 ⁺Regulatory T cells, CD4+CD25 ^HighCD103 ⁺Regulatory T cells or CD4+CD25 ^HighThe quantity of regulatory T cells or activity.

24. embodiment 21,22 or 23 method, wherein said chemical compound is used for following treatment of diseases or prevention: autoimmune disease or the inflammatory conditions of not expecting, and it randomly is a for example osteoarthritis (constitutional or Secondary cases osteoarthritis) of the arthritis patient's condition, rheumatoid arthritis, for example ankylosing spondylitis of the arthritis relevant with spondylitis, multiple sclerosis, Alzheimer, tenosynovitis, the lupus patient's condition is systemic lupus erythematosus (sle) or discoid lupus erythematosus for example, tendinitis, bursitis, the lung inflammation situation is asthma for example, edema due to disorder of QI, chronic obstructive pulmonary disease, pulmonary fibrosis, cystic fibrosis, acute or adult respiratory distress syndrome, chronic bronchitis, acute bronchitis, bronchiolitis, bronchiolitis fibrosa obliterans, bronchiolitis obliterans with organized pneumonia.Described chemical compound can be described herein formula 1 chemical compound.

The variant of these embodiments and other parts of the present disclosure and improvement are conspicuous for having read those skilled in the art of the present disclosure.This variant and improvement are all within the scope of the present invention.All cited literature 2s quoted herein or list of references are all incorporated into this paper as a reference in full in this position or the other paragraph in this section back.

Embodiment.Following examples further illustrate the present invention, and they are not intended to by any way and limit it.

Embodiment 1.The treatment of lung inflammation.In fact as (people such as D.Auci, Ann.New YorkAcad.Sci.1051:730-742 2005, newly quote) described in, use three chemical compounds, 3 β, 16 alpha-dihydroxy-s-17-oxo androstane, 3 α, 16 β, 17 β-trihydroxy androstane and 3 α, 16 α, the inflammation of 17 α-trihydroxy androstane treatment mice.Use under study for action 5 to 8 the week ages the CD1 male mice (Charles River, Calco, Italy).Animal is housed in the controlled environment and for it provides the rodent food and the water of standard.Animal care is followed the regulations about watching for animals applicatory.During mice was assigned to one of following group: (1) was used in the mice (carrageenin-λ treats matched group) that the 2% carrageenin-λ in the saline handles, (2) 24 hours before giving carrageenin-λ and 1 hour 3 β by subcutaneous injection (s.c.) 0.1mg, 0.01mg or 0.001mg, the mice that 16 alpha-dihydroxy-s-17-oxo androstane is handled, (3) 24 and 1 hours 3 α before carrageenin by s.c. injection 0.1mg, 0.01mg or 0.001mg, 16 α, the mice that 17 α-trihydroxy androstane is handled; (4) 24 hours before giving carrageenin-λ and 1 hour 3 α by s.c. injection 0.1mg, 0.01mg or 0.001mg, 16 β, the mice that 17 β-trihydroxy androstane is handled; (5) 24 hours before giving carrageenin-λ and 1 hour mice by s.c. injection vehicle (0.1% carboxymethyl cellulose, 0.9% saline, 2% Tween 80,0.05% phenol) processing; (6) 24 hours before giving carrageenin-λ and 1 hour by with the anti-mice polyclone of rabbit anti-TNF-Alpha antibodies (200 μ g) as the concentrate mice (positive controls) of injection treatment of intraperitoneal; (7) the pseudo-operation mice of carrageenin of no use-λ processing.Each group is made up of 10 mices.All processing all give with the final volume of 100 μ L.Following lung (pleural space) inflammation of inducing.With mice with isoflurane anesthesia and in the left side horizontal plane skin incision of the 6th intercostal space make skin incision.Underlying muscle cut open and with 0.1mL saline (contrast) or the 0.1mL saline injection that contains 2% λ-carrageenin in pleural space.Carrageenin-λ is effective inducer of inflammation, and its hydrops and neutrophil cell that shows as in these rules in the pleural space gathers.Close otch and make the animal recovery by stitching.

In injection 4 hours after carrageenin-λ, by being exposed to CO ₂With the painless execution of animal.Carefully chest is opened and pleural space usefulness is comprised the 1mL saline solution rinsing of heparin (5U/mL) and indomethacin (10 μ g/mL).By extracting out cumulative volume is removed and measured to exudate and cleaning mixture.To be abandoned by any exudate of blood contamination.Deduct the amount of the 1mL volume calculation exudate of injection by the total pleural space volume that reclaims.Be suspended in the neutrophil cell in the exudate in the phosphate buffered saline (PBS) and after trypan blue staining, in the Burker chamber, count by optical microscope.The result analyzes by single factor ANOVA, is used for the Bonferroni post-hoc check of multiple comparisons subsequently.Think that less than 0.05 p-value be significant.For statistical analysis, with each group with compare with carrageenin-λ and the matched group of not accepting the mice of other treatment.

All develop into acute pleurisy with what carrageenin-λ attacked with undressed all mices, the neutrophil cell quantity that produces muddy exudate and pleura increases.In the pleura of the mice of handling with vehicle, the increase of observing exudate volume and quantity of leucocyte is to similar with the control mice of not receiving treatment with carrageenin-λ attack.With respect to these two control mice groups, use 3 β, the animal that 16 alpha-dihydroxy-s-17-oxo androstane is handled shows the number of neutrophil cell in the pleura, the obvious minimizing of pleural exudate volume under 0.1mg and 0.01mg dosage, and does not have activity under the lower 0.001mg dosage.With 3 β of 0.1mg dosage, the pleural exudate volume that 16 alpha-dihydroxy-s-17-oxo androstane is handled significantly reduces, but this situation do not occur under lower 0.01mg and 0.001mg dosage.Use 3 α, 16 α, the animal that 17 α-trihydroxy androstane is handled shows the neutrophil cell quantity in the pleura under 0.1mg and 0.01mg dosage obvious minimizing.Use 3 α, 16 β, 17 β-trihydroxy androstane handle also the obvious minimizing of revealing neutrophil cell quantity in the pleura at 0.1mg and 0.01mg dose form.3 α, 16 α, 17 α-trihydroxy androstane and 3 α, 16 β, the effectiveness of 17 α-trihydroxy androstane is similar to the observed result of using polyclonal anti-TNF-Alpha antibodies contrast, and 3 β, 16 alpha-dihydroxy-s-17-oxo androstane is renderd a service relatively poor relatively.

Following table has been described the animal groups of acceptance processing with respect to being exposed to the neutrophil cell quantity that carrageenin-λ does not still use the undressed control animal (negative control group) of any other mass treatment.The neutrophil cell quantity of negative control group is set to 100%, and with other group by comparison.With 29% of the neutrophil cell quantity of the negative matched group of neutrophil cell quantity of the animal groups (positive controls) of anti-TNF-Alpha antibodies treatment, show that this antibody has the antiinflammatory action that exposes at carrageenin-λ.Vehicle control group does not show the remarkable minimizing (91%) with respect to the neutrophil cell quantity of negative control group, shows that independent vehicle does not have significant antiinflammatory action.

Other chemical compound that has the anti-inflammatory activity of statistically significant in this model is 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols (by oral gavage gives 1mg and 0.1mg) and 17 beta-aminos androstane-5-alkene-3 β-alcohol (by oral gavage gives 40mg/kg, about 0.5mg/ mice).Compare with the mice group as the vehicle contrast, these chemical compounds are activated.

Other formula 1 chemical compound of describing herein can be used by this way, treat or improve the relative ability of inflammation to characterize their.These chemical compounds comprise 3 β, 16 β, 17 β-trihydroxy androstane, 3 β, 16 α, 17 α-trihydroxy androstane, 3 β, 16 β, 17 α-trihydroxy androstane, 3 β, 16 beta-dihydroxies androstane-5-alkene-17-oxime, 3 β, 16 alpha-dihydroxy-s androstane-5-alkene-17-oxime, 3 α, 16 alpha-dihydroxy-s androstane-5-alkene-17-oxime, 3 β, the following analog (1) of 16 alpha-dihydroxy-s androstane-17-oxime and these chemical compounds comprise in the 7-position hydroxyl of α configuration or beta comfiguration and/or (2) in the 5-position or the 4-position comprise two keys, and/or (3) any ester in these, ether, aminoacid, carbamate or oxime (=NOH) derivant, conjugate, or analog.

Embodiment 2.The analysis of immunne response.Find chemical compound 3 α, 16 α, 17 α-trihydroxy androstane have makes described chemical compound be used for the treatment of for example biological characteristics of the medicine of asthma of inflammatory conditions as excellence.Particularly, described application of compound is not attended by the bounce-back of IL-13, and the bounce-back of IL-13 is for example known side effect of dexamethasone of antiinflammatory glucocorticoid compound.IL-13 after using glucocorticoid rebounds and makes asthmatic patient be more prone to take place acute onset subsequently, and therefore, the anti-inflammatory agent that does not have this side effect is favourable.It is unexpected that the IL-13 bounce-back does not take place.

In the asthma mouse model of ovalbumin (OVA) sensitization, 3 α have been observed, 16 α, the ability of 17 α-trihydroxy androstane restriction eosinocyte load and the crucial inflammation mediator of minimizing (IL-5, IL-13, cysteinyl-leukotriene).By the 1st and the 12nd day peritoneal injection OVA (in the Alumen adjuvant) make the BALB/c mouse sensitization.Attack air flue by sending OVA to lung with OVA, perhaps send saline to lung at the 28th and the 30th day.At the 31st day, will put to death and analyze lung tissue with 6 mices of saline treatment with 6 mices that OVA attacks.Remaining animal is divided into 6 groups (every group of 6 mices).Following group to mice is treated by subcutaneous injection once a day.The 1st group of vehicle contrast (0.1% carboxymethyl cellulose, 0.9% saline, 2% Tween 80,0.05% phenol).The 2nd group of dexamethasone (5mg/kg).The 3rd group of 3 α, 16 α, 17 α-trihydroxy androstane (1mg/ mice).Three sacrifice of animal in the 1-3 group were put to death three animals of residue in the 1-3 group at the 38th day in 1 hour after the 35th day treats the last time.

As shown in the table, 3 α, 16 α, 17 α-trihydroxy androstane are not created in observed IL-13 increase in the animal for the treatment of with dexamethasone.

The treatment group	IL-13(pg/mL)
		Saline control	220
Ovalbumin	230
		Vehicle (the 35th day)	220
Dexamethasone (the 35th day)	340
		3 α, 16 α, 17 α-trihydroxy androstane (the 35th day)	195
Vehicle (the 38th day)	190
		Dexamethasone (the 38th day)	390
3 α, 16 α, 17 α-trihydroxy androstane (the 38th day)	210

After attacking the 38th day, except the IL-13 bounce-back reduces, compare with dexamethasone treatment group (145pg/mL), use 3 α, 16 α, the IL-5 level reduction (90pg/mL) in the animal lung tissue that 17 α-trihydroxy androstane is treated.At the 38th day, the IL-5 level in the vehicle control group was 75pg/mL.Other formula 1 chemical compound as herein described is used by this way,, comprise 3 β to identify their treatments or to improve inflammation and do not have the ability of IL-13 and/or IL-5 rebound effect, 16 β, 17 β-trihydroxy androstane, 3 β, 16 α, 17 α-trihydroxy androstane, 3 β, 16 β, 17 α-trihydroxy androstane, androstane-5-alkene-2 α, 3 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol and 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols.These results show that F1C is used in interior therapeutic lung inflammation or asthma.

In another kind of rules, following from murine bone marrow cultivating fertilizer maxicell group.In brief, use PBS and 27g syringe needle to derive from the bone marrow of Balb/C mice from femur flushing (flush).Cell is cultivated in the 2/3RPMI-1640 of the cell that contains 19%FBS and secretion IL-3.Before being used for testing, make medullary cell the mixture differentiation that comprises IL-3 18-25 days.So the medullary cell of cultivating has with the similar Phenotype of mucosal mast cell and is called as the mastocyte (BMMC) of derived from bone marrow.

Check by the stream cell counting technology of routine external breeding mastocyte homogeneity and dye with the cell type specificity marker.At the 14th and 21 day of breeding, gather in the crops sophisticated mastocyte and preparation and be used for the test cultivation.Purpose be want assessing compound for example dehydroepiandrosterone mastocyte is stimulated the influence of bonded threshing.With the preparation mastocyte with 1 * 10 ⁷The density distribution of cell/mL is in the test culture hole.In contrast culture, after IgE receptor and IgE antigen-antibody complex are crosslinked, induce the mastocyte threshing.In parallel cultivation group,, use anti--IgE antibody activation subsequently with the dehydroepiandrosterone preincubate of mastocyte and various dose.Measured as discharging beta-glucuronidase by the cytosol storage granule from cell, in the presence of not having to stimulate, mastocyte does not have can detected threshing.Introduce anti--IgE receptor antibody to culture and cause the remarkable release of beta-glucuronidase.When mastocyte is exposed to independent dehydroepiandrosterone, there is not measurable threshing.Yet before with anti--IgE antigen-antibody complex activation, 5 to 10 minutes the mastocyte of dehydroepiandrosterone that is exposed to 100 μ M dosage in advance shows about 70% threshing inhibition.The dehydroepiandrosterone of reduced levels shows the ability that suppresses threshing and diminishes pro rata.In similar rules, F1C is 17 α-acetenyl androstane-5-alkene-3 β for example, 7 β, 17 beta-triols, androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol or androstane-5-alkene-3 α, 7 β, 16 α, 17 β-tetrol is than the big 10-1000 of effectiveness times of dehydroepiandrosterone.

Embodiment 3.The treatment of mortality inflammation/shock.In mortality shock rules, use two kinds of chemical compounds, 16 α-bromine epiandrosterone (3 beta-hydroxies-16 α-bromine androstane-17-ketone) and 3 β, 16 alpha-dihydroxy-s-17-oxo androstane.In rules, give a treated animal with 16 α-bromine epiandrosterone by oral gavage of 3mg, and another group is accepted 16 α-bromine epiandrosterone of 3mg by subcutaneous injection.One group of control animals receive placebo contrast.In these rules, give 16 α-bromine epiandrosterone to mice giving the bacteria lipopolysaccharide of lethal dose (LPS) before 24 hours and afterwards 1 hour.When the observation period (after giving LPS 72 hours) finishes, do not have a survival in the placebo animal of vehicle treatment, and accept 65% survival is arranged by the animal of orally give 16 α-bromine epiandrosterone.The animal of accepting 16 α bromine epiandrosterones by subcutaneous injection has 50% survival.The animal that survived 72 hours all gets well from the LPS contact.

In second test, before giving the LPS of lethal dose 24 hours and 1 hour by oral gavage afterwards give 16 α-bromine epiandrosterone or 3 β to mice, 16 alpha-dihydroxy-s-17-oxo androstane.The animal of vehicle treatment group is used as placebo.At 72 hours, the placebo mice had 25% survival, used 3 β, and the mice of 16 alpha-dihydroxy-s-17-oxo androstane treatment has 50% survival, and the mice for the treatment of with 16 α-bromine epiandrosterone has 80% survival.

In another experiment, shown 16 α-bromine epiandrosterone and 3 β, 16 alpha-dihydroxy-s-17-oxo androstane prevents by making mice be exposed to the LPS of sublethal dose and the ability of inductive injury of lung.In this test, before the LPS that gives 100 μ g to trachea and the lung of animal under the shallow degree anesthesia situation 24 hours and 1 hour by oral gavage afterwards give chemical compound, Sterile Saline (negative control) or vehicle (vehicle contrast) to the group of 5 mices.At 48 hours, obtain sample from the lung of animal with sacrifice of animal and by alveolar wass (BAL).With the cell counting in the BAL liquid, the cell number multilist shows the inflammation and the damage of lung.In this test, the cell response of transmitting inflammation and injury of lung penetrates in the lung in the existence of LPS.In negative control and vehicle control group, BAL liquid comprises about 6 * 10 ⁷Cell/mL.With 16 α-bromine epiandrosterone (p=0.02) or 3 β, the cell number of the animal groups of 16 alpha-dihydroxy-s-17-oxo androstane (p=0.04) treatment has the cell counting (about 4.4 * 10 in the BAL fluid of remarkable minimizing ⁷Cell/mL).This result shows, in for example asthma of chemical compound injury of lung or infringement therein and uncontrolled or excessive inflammation-related or the clinical patient's condition of COPD activity arranged.Can characterize other chemical compound in a similar manner, for example, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols or 17 beta-aminos androstane-5-alkene-3 β-alcohol.

Embodiment 4.Remove antibacterial from lung tissue.Use disclosed in advance rules to prove that 16 α-bromine epiandrosterone removes the ability that Pseudomonas aeruginosa (Pseudomonas aeruginosa) infects from lung tissue, people such as A.van Heeckeren, J.Clin.Invest., 100 (11): 2810-2815 1977; People such as A.van Heeckeren, Am.J.Respir.Crit.Care Med., 161:271-2792000.Described rules are carried out in the CFTR mice, and it is used as the animal model of people's cystic fibrosis, people such as S.D.Freedman, and Proc.Natl.Acad.Sci.USA, 96 (24): 13995-14000 1999; People such as W.Zeng, Am.J.Physiol.Cell.Physiol.273:C442-C455 1997.Use comprises the agarose beads of antibacterial, and (50 μ L comprise about 6.1 * 10 ⁴The CFU/ animal) set up chronic charrin disease and formerly announce to some extent, people such as J.R.Starke, Pediatr.Res., 22:698-702 1987.With 16 αs-bromine epiandrosterone or vehicle (contrast) processing and the antibacterial load 10 day measured in animal lungs agarose beads be incorporated in lung after of two groups of mices (every group of n=9) with 40mg/kg.At the 10th day, the antibacterial load in the lung of vehicle control animal was about 6 * 10 ⁶The CFU/ animal, and the animal of handling with 16 α-bromine epiandrosterone has the antibacterial load (p=＜0.04) of reduction.This result shows that 16 α-bromine epiandrosterone is antiinflammatory not only, and it also can be used for treatment or reduce pulmonary infection, and this is desirable character for the medicine that is used for the treatment of inflammation wherein and infects the patient's condition that can compatible for example cystic fibrosis.

Embodiment 5.Extracorporeal anti-inflammatory activity in people's cell.People's whole blood that use is exposed to LPS proves 16 α-bromine epiandrosterone and 3 β, the ability of the inflammation of 16 alpha-dihydroxy-s-17-oxo androstane in external minimizing people cell.Compare with the cell that is exposed to independent LPS (positive control) or does not contain the vehicle (dimethyl sulfoxide) (vehicle contrast) of chemical compound, at 16 α-bromine epiandrosterone (100ng/mL) and 3 β, observe the gamma interferon minimizing that cell produces under the existence of 16 alpha-dihydroxy-s-17-oxo androstane.When cell being cultivated 24 hours in the presence of LPS, measure the amount of the gamma interferon in the growth medium.

Embodiment 6.The neurodegenerative treatment of autoimmune.To three kinds of chemical compounds, the amino androstane of 17 beta-aminos androstane-5-alkene-3 β-alcohol, the 17 beta-dimethyl-s-amino androstane of 5-alkene-3 β-pure and mild 17 Beta-methyls-5-alkene-3 β-alcohol characterizes the ability of its experimental allergic encephalomyelitis of improving mice (EAE).This demyelination patient's condition is widely used as people's multiple sclerosis model and is used for the model of the Therapeutic Method of test of cure multiple sclerosis, for example, people such as B.F.Bebo Jr., J.Neurosci.Res.52:420-426 1998; People such as R.R.Voskuhl, Neuroscientist, 7:258-270 2001; People such as H.Offner, J.Neuroimmunol., 130:128-139 2002.Activity in these models is represented the neuronal death that the test compound prevention is relevant with the EAE progression of disease or is delayed the ability of neuronal death speed.

In this rules, by oral gavage gives chemical compound to female SJL/J mice when disease symptoms shows effect.Use antigen causes the EAE patient's condition in the mice.The antigen that is used for active immunization is murine protein lipid protein (PLP) 139-151 (HCLGKWLGHPDKF).Use this peptide antigen to carry out the demyelination that immunity causes central nervous system's autoimmune Th1 mediation.Described antigen is by the solid phase synthesis preparation and by the high performance liquid chromatography purification.Carry out immunity by PLP 139-151 peptide and in female SJL/J mice, cause the EAE patient's condition with 150 μ g in the complete Freund's adjuvant that comprises 200 μ g mycobacterium tuberculosis (Mycobacterium tuberculosis).The immunity rules are subcutaneous injections on the 0th day four position at aft rib.After immunity, use the EAE clinical indication of following standard evaluation mice every day: 0=does not have clinical indication or symptom; The 1=afterbody is unable; The hind leg that 2=is slight is weak and afterbody is unable; Weak and the unable or slight ataxia of afterbody of the hind leg of 3=moderate; Weak and the slight forelimb weakness of the hind leg that 4=is serious is followed the ataxia of moderate; 5=paraplegia is with the forelimb weakness that is no more than moderate; 6=paraplegia is weak or serious ataxia or moribund condition with the forelimb that is no more than severe.

The mice of vehicle control group is showing observable EAE symptom with about 10-11 days after the PLP antigen immune, and this is typical for the EAE disease model.Since the 1st day, by oral gavage gives 17 beta-aminos androstane-5-alkene-3 β-alcohol, the amino androstane of 17 beta-dimethyl-s-5-alkene-3 β-alcohol or the amino androstane of 17 Beta-methyls-5-alkene-3 β-alcohol every day to animal, is meant afterwards first day of immunity in described first day.When the observation period finished, all three kinds of chemical compounds all had activity under the dosage of 5mg/kg, and they are reduced in the clinical order of severity of observed symptom before the 26th day.In mice, observe the therapeutic activity of chemical compound in the blood levels of about 10ng/mL.These results show that chemical compound has biologic activity for this chronic autoimmune neurodegenerative disease of treatment.

Embodiment 7.The vitro inhibition of NF-κ B.There is chemical compound lot to be used in the vitro inhibition people cell NF-κ B by TNF-α or LPS activation.The activation of NF-κ B increases many expression of gene of transmitting inflammation.These rules end user THP-1 cell, it is to have the phenotypic people's monokaryon of mononuclear cell hemocyte.The cell line that is called as NF-κ B-bla THP-1 is included in beta-lactamase reporter gene (Invitrogen, the CellSensor under the control of NF-κ B response element ^TM, production code member K1176).In this cell line, the beta-lactamase reporter gene is incorporated in the THP-1 cell with being stabilized.This cell line is used to detect the agonist or the antagonist of NF-κ B signal transduction passage.These NF-κ B-bla THP-1 cell responses are in the existence of tumor necrosis factor (TNF α) or bacteria lipopolysaccharide (LPS) and increase the expression of beta-lactamase reporter gene.The level of beta-lactamase enzymatic activity detects by fluorescence resonance energy transmission ratio and measures.TNF α and LPS are the effective inflammation-induced reagent of the NF-κ B in the activation THP1 cell.In this test, thereby the chemical compound that reduces NF-kB activity and minimizing beta-lactamase in the presence of TNF α or LPS has anti-inflammatory activity.

By going down to posterity or feeding as required and keep NF-κ B-bla THP-1 cell.The cell that to grow in suspension remains on 2 * 10 ⁵Individual cell/mL to 2 * 10 ⁶The density of individual cell/mL.Cell with 20,000 cells/well bed board in the bread board (Costar# 3712-TC hangs down the fluorescence background plate) of 384 hole black walls, clear bottom, was added the TNF α of 10ng/mL or the LPS of 0.2ng/mL in about afterwards 24 hours, with activation NF-κ B.In the positive control test of activation NF-κ B, after the beta-lactam zymolyte is cultivated 1 hour, the EC of TNF α ₅₀Concentration is 0.20ng/mL.The EC of LPS ₅₀Dosage is 0.15ng/mL.TNF-α in this test or the EC of LPS ₅₀Concentration is meant 50% of the concentration that causes maximum activated T NF-α of NF-κ B or LPS.Synthetic glucocorticoid dexamethasone (a kind of anti-inflammatory agent of brute force) in this test with the EC of 0.47nM (5 times test meansigma methodss) ₅₀Reduce the influence of TNF α.In other cell in vitro test, reported similar dexamethasone biologic activity, at the IC of about 1nM ₅₀Observe inhibition fully (people such as M.K.A.Bauer, Eur.J.Biochem.243:726-731,1977) down to the NF-kB activation.

Use this test, after LPS stimulates, in NF-κ B-bla THP-1 cell, suppress the IC of the chemical compound of NF-kB activation ₅₀Show below.The IC that is used for the chemical compound of this test ₅₀Concentration is meant and causes that chemical compound can the inductive 50% maximum compound concentrations that suppresses to the NF-kB activation.Described test is carried out 2-4 time for every kind of chemical compound usually, and value as follows is the meansigma methods of every kind of chemical compound.Data in the following table 1 show that many NF-κ B that can suppress in extremely low level in these human macrophage cells are arranged in these chemical compounds.

Table 1

^*μM＝10 ^-6M；nM＝10 ^-9M；pM＝10 ^-12M；fM＝10 ^-15M

Other chemical compound that shows anti-inflammatory activity in these rules is 3 α-pentafluoroethyl group androstane-4-alkene-3 β, 17-isoallopregnane-3 (IC ₅₀3.1nM), 3 α-pentafluoroethyl group androstane-5-alkene-3 β, 17-isoallopregnane-3 (IC ₅₀17nM; It is 50% that maximum NF-κ B suppresses), 3 α/β, 17 α-acetenyl androstane-3 α/β, 17-isoallopregnane-3 (IC ₅₀200pM), 17 α-trifluoromethyl androstane-3 α, 17-isoallopregnane-3 (IC ₅₀190nM), 17 β-glycyl androstane-3 β-alcohol (IC ₅₀0.42pM), 3 β-glycyl androstane-17 β-alcohol (IC ₅₀1nM), androstane-3 β, 16 beta-diols-17-oxime (IC ₅₀1.9fM) 17 α-acetenyl androstane-4-alkene-3-ketone-17 β-alcohol (IC ₅₀2.9fM; It is 80% that maximum NF-κ B suppresses), 16 α-fluorine androstane-5-alkene-17-ketone (IC ₅₀30pM), 16 β-fluorine androstane-5-alkene-7 β-alcohol-17-ketone (IC ₅₀1.5nM), androstane-3 α, 16 α, 17 beta-triol (IC ₅₀6.9fM), androstane-3 α, 16 β, 17 beta-triol (IC ₅₀19fM), androstane-5-alkene-3 β-alcohol-17 β-succinyl group ester (IC ₅₀0.2nM), 3 β-acetoxyl group-7 β, 17 beta-dihydroxies-11-oxo androstane-5-alkene (IC ₅₀1fM; It is 65% that maximum NF-κ B suppresses).The NF-κ B of these chemical compounds is maximum to be suppressed for about 25% to 80%, 100% of NF-kB activation is suppressed to have any different in these rules with synthetic glucocorticoid dexamethasone.

In these rules, there are two chemical compounds to increase the NF-kB activity, are androstane-5-alkene-3 β, 7 α, 16 α-triol-17-ketone (IC ₅₀1.3nM; Be 140% NF-kB activity with respect to control cells) and 3 β, 17 alpha-alpha-dimethyl androstane-3 α, 17-isoallopregnane-3 (IC ₅₀40nM).

The chemical compound that does not show anti-inflammatory activity at this rules invading the exterior is 3 α, 17 Alpha-Methyl androstane-3 β, 17-isoallopregnane-3,3 β-acetoxyl group androstane-5-alkene-3 β, 17-isoallopregnane-3,17 Alpha-Methyls androstane-5-alkene-3 β, 17-isoallopregnane-3-7-ketone, 16 α-fluorine androstane-5-alkene-7 β-alcohol-17-ketone, 16 α-fluorine androstane-5-alkene-7 α-alcohol-17-ketone, 17 Alpha-Methyl androstane-3 β, 7 α, 17 beta-triols, androstane-5-alkene-3 β, 11 β, 17 beta-triols, 16 α-fluorine androstane-17-ketone, androstane-5-alkene-3 α, 17-isoallopregnane-3, androstane-2 β, 3 α, 16 α, 17 β-tetrol and androstane-3 α, 16 α, 17 α-triol, all tool is greater than the IC of 10 μ M ₅₀

Chemical compound reduces the NF-kB activity under low level ability shows that they can be used for treating inflammation, particularly the excessive levels of NF-κ B or played in the situation of main effect in the pathology of the described disease or the patient's condition by the nucleus transcriptional activity of NF-κ B mediation therein.

In the said determination method, it is 100% that the maximum of the NF-κ B that is caused by dexamethasone, 16 α-bromine epiandrosterone and 16 β-bromine epiandrosterone suppresses, and surpasses the IC of these chemical compounds in these compound concentrations ₅₀The time do not have can detected NF-kB activation.By contrast, other chemical compound for example, 3 β, 7 β, 16 α, 17 β-tetrahydroxy androstane-5-alkene, 3 α, 7 β, 16 α, 17 β-tetrahydroxy androstane-5-alkene or 3 β, 7 β, it is about 80% that the maximum of the NF-κ B that 17 β-trihydroxy-17 Alpha-Methyl androstane-5-alkene causes suppresses to be lower than, and the amount that increases chemical compound is to surpassing its IC ₅₀Level can not provide significant other inhibition activity to the NK-kB activation.For the great majority in these chemical compounds, the maximum inhibition degree of the NF-κ B in this algoscopy is about 25-80%, is mainly about 30-65% or about 30-70%.These results show that chemical compound is 3 β for example, 7 β, and 16 α, 17 β-tetrahydroxy androstane-5-alkene suppresses the NF-kB activation by can not fully stoping its bioactive mechanism.

Several chemical compounds in the table 1 do not have can the detected ability of bringing into play anti-inflammatory activity in the cell in vitro algoscopy.Carry out test and in described algoscopy, do not had activity (IC ₅₀10 μ M) other chemical compound comprise 3 β, 17 alpha-dihydroxy-s androstane-5-alkene, dehydroepiandrosterone (3 beta-hydroxies androstane-5-alkene-17-ketone), 3 beta-hydroxy androstanes-7,17-diketone, 16 α-bromo-3 β, 17 beta-dihydroxies androstane-5-alkene and 16 β-bromo-3 beta-hydroxies androstane-5-alkene-17-ketone.However, have in those chemical compounds of non-activity in this cell in vitro test, for example, 16 Alpha-hydroxy epiandrosterones find that still it has interior anti-inflammatory activity in animal.This result shows that described chemical compound may work by different mechanism or their activity need be from the cell of more than cell line.The interior anti-inflammatory activity of this chemical compound can come from, and for example, causes synthetic and other activity in liver of prostaglandin, replys thereby produce systemic antiinflammatory.Perhaps, the anti-inflammatory activity of this chemical compound can come from chemical compound and suppresses owing to be different from the ability that NF-κ B that the source of LPS causes stimulates.There are many different materials can activate the NF-kB activity, comprise that the activation of existence, B cell or T cell of LPS, TNF-α, IL-1, some virus or bacterial gene product or cellular exposure are in ultraviolet radiation.Not every cellular type can be replied all these to stimulate, because not every cellular expression is replied each the required signaling mechanism in these stimulations.The most cells type can respond to one or more in these signals, and is rarely found but certain cellular type responds to all signals.In algoscopy used herein, the activation of NF-κ B comes from the inductive stimulation of antibacterial LPS-, and therefore, the chemical compound that has limited inhibition LPS signal transduction channel capacity with it has the ability of limited reduction NF-kB activity in this algoscopy.

The method of having described to some extent and can having incorporated the present invention into or be used for putting into practice adjusting NF-κ B of the present invention is included in those that following discloses describe.United States Patent (USP) 5,989,835,6,410,516,6,545,027,6,831,065 and 6,998,383.The others of NF-kB activity are described to some extent, and also can be incorporated in the method for the present invention, for example, and A.S.Baldwin, Annual Rev.Immunol.14:649-6831996; People such as M.Muller., Mol.Cell.Biol.22 ((4) 1060-1072 2002; P.A.Baeuerle, Cell 95:729-731 1998.

Embodiment 8.By examining or check the ability of the inductive shock/inflammation of LPS in the selected compounds for treating mice to above-mentioned similar rules.With five groups of the ICR mice of every group of three about 30g of body weight separately by peritoneal injection 120 μ L vehicles (water that contains 30% sulfo group butyl ether-cyclodextrin), contain androstane-5-alkene-3 α, 7 β, 16 α, the vehicle of 17 β-tetrol, contain androstane-5-alkene-3 β, 4 β, 16 α, the vehicle of 17 β-tetrol or contain 4 β-acetoxyl group androstane-5-alkene-3 β, 16 α, the vehicle of 17 beta-triols is handled.All medicines and vehicle preparation all are solution rather than suspension.Sulfo group butyl ether-cyclodextrin obtains (Captisol by purchase ^TM, www.cydexinc.com).Two vehicle control group are arranged, and a winding is subjected to independent vehicle, and another winding is subjected to vehicle and LPS.Giving LPS (about LD by peritoneal injection to mice _50/24Dosage, that is, after giving LPS 24 hours have 50% cause death) before 24 hours and afterwards 1 hour gives vehicle and medicine.Medicine with about 40mg/kg (for medicine to give be 1.2mg medicine/animal at every turn) give.Gather in the crops spleen in injection 1.5 hours after the LPS from animal, with the molten born of the same parents of splenocyte and by measuring activatory NF-κ B from the splenocyte separating nucleus and from molten born of the same parents' nucleus measuring N F-κ B.The result shows, compares with the LPS+ vehicle control group, and three kinds of all chemical compounds all make the level of NF-kB activation reduce about 50%.Must use by oneself vehicle and not have the activatory NF-κ B level in the spleen cell of activatory NF-κ B level and the animal that derives from drug treating in the spleen cell of the animal that LPS handles identical in fact.These results have shown the effective antiinflammatory action in animal, and this is by comparing with control animal, and activatory NF-κ B reduces that institute revealed in the animal that heals with medicine.

Analyzing other chemical compound that uses in the similar rules of NF-κ B or TNF α in 1.5 hours after LPS attacks is: androstane-3 α, 16 α, 17 beta-triols, androstane-3 α, 17-isoallopregnane-3-16-ketone, 17 α-trifluoromethyl androstane-5-alkene-3 β, 17-isoallopregnane-3, androstane-5-alkene-3 α, 17-isoallopregnane-3, androstane-5-alkene-3 α, 16 α, 17 beta-triols, 3 α-trifluoromethyl androstane-5-alkene-3 β, 17-isoallopregnane-3, androstane-3 α, 17-isoallopregnane-3-16-oxime and androstane-5-alkene-3 β, 17-isoallopregnane-3-7-ketone.All these chemical compounds all are to give as the solution (but not suspension) of chemical compound in the water that contains sulfo group butyl ether-cyclodextrin.These chemical compounds before LPS attacks 24 hours and when LPS attacks (replaces aforesaid after LPS attacks 1 hour) give NF-κ B in 1.5 hours analysis spleens after the PLS attack or the TNF α in the blood subsequently.Chemical compound gives with the dosage of 40mg/kg, and for 3 α-trifluoromethyl androstane-5-alkene-3 β, 17-isoallopregnane-3 also gives its dosage with 4mg/kg, 0.1mg/kg and 0.05mg/kg.With respect to the vehicle contrast, all observe NF-κ B and TNF α inhibition for all these chemical compounds.For 3 α-trifluoromethyl androstane-5-alkene-3 β, 17-isoallopregnane-3 suppresses in the maximum of observing NF-κ B and TNF α under the dosage level of 0.1mg/kg in the animal of treatment.

Embodiment 9.The dynamic analysis that NF-κ B suppresses in the body.Examined or check the kinetics that the NF-κ B after bacterial injection LPS suppresses in the mice, so that further explore for example 17 α-acetenyl androstane-5-alkene-3 β of chemical compound, 7 β, the mechanism of action of 17 beta-triols, as described in example 7 above, described chemical compound just partly suppresses in the immunocyte (macrophage or mononuclear cell) by LPS or the inductive NF-kB activation of TNF α external.In this research, with mice by the solution (be not suspension) of peritoneal injection chemical compound as described in example 8 above in vehicle with 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols (about 40mg/kg, about 1.2mg/ animal) are handled.At peritoneal injection antibacterial LPS (about LD _50/24) before 24 hours injectable drugs.Two groups of every group of 12 animals are used in described research, give vehicle and contrast or medicine in 24 hours before LPS attacks.Gathered in the crops spleens from 3 animals of two groups respectively in 1.5,2.0 and 2.5 hours before being about to carry out the LPS attack and after giving LPS.Results spleen cell and by measuring the level that NF-κ B in the nucleus measures activatory NF-κ B in fact as described in example 8 above.

The maximum of the NF-kB activation in the vehicle contrast after 1.5 hours occur giving LPS, it has 4 times rising with respect to the level that gives LPS activatory NF-κ B before.The result shows below.The numerical value of the animal of vehicle contrast and drug treating all is that the NF-κ B in the nucleus that derives from spleen cell is carried out the relative optical density unit that ELISA measures.

In 1.5 hours time points the far-reaching inhibition of NF-κ B is shown with relative normal level at At All Other Times NF-kB activity, but the material time of chemical compound after the LPS contact brought into play of short duration effective inhibition to the inductive wound of LPS.Similar algoscopy in other research shows, in injection 30 minutes and 60 minutes after the LPS, the preceding time point of LPS processing is similar in the level of the activatory NF-κ B in the vehicle control mice and this research.This result shows, in this model, and LPS maximum during to after LPS attacks about 1.5 hours of the influence of NF-kB activation in the spleen cell.This time point has disclosed and has been used for active appropriate time of interior evaluating anti-inflammatory drug material standed for or window, that is, and and after LPS attacks about 75 minutes to about 105 minutes.Described window can depend on the route of administration of biology infringement and change, for example, LPS that gives by peritoneal injection or TNF α with compare by LPS or TNF α subcutaneous or that intramuscular injection gives.Similarly, biology, infringement can be other situation, and for example, the experimenter is exposed to about 50-60cGy/ minute ionizing radiation is exposed to about 500-1000cGy/ minute or about 5-10cGy/ minute with respect to the experimenter ionizing radiation.

To the analysis showed that of the inductive TNF alpha expression of the LPS in the mice, the TNF alpha levels is attacked (giving the LPS of 500 μ g by peritoneal injection) afterwards 1.5 hours at LPS and is reached peak, the top level of observing TNF α in 1-2 hour after LPS attacks.After LPS 30 minutes and lower at 2.5 hours TNF alpha levels.

Other chemical compound that can analyze the ability that works as the biodynamics medicine comprises androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 7 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 7 α, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 4 α, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 4 β, 16 α, 17 β-tetrol, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols, 17 α-acetenyl androstane-5-alkene-3 β, 7 α, 17 beta-triols, 17 α-acetenyl androstane-5-alkene-3 β, the acceptable analog of pharmacy of 17 beta-triols-7-ketone and any of these chemical compound, for example, at 1,2 or the hydroxy ester at more a plurality of hydroxyls place or the analog of ether derivant.Ester that is fit to and ether comprise acetas, n Propanoic acid, isopropyl acid ester, succinate ,-O-C (O)-(CH ₂) n-CH ₂R ,-O-C (O)-O-(CH ₂) n-CH ₂R ,-O-C (O)-NH-(CH ₂) n-CH ₂R, aminoacid is glycine and alanine (O-C (O)-CHCH for example ₃-COOH), hydroxy ester and methyl, ethyl, n-pro-pyl, isopropyl-O-(CH ₂) n-CH ₂R ,-O-(CH ₂) n-O-CH ₂R (for example ,-O-CH ₂CH ₂-O-CH ₃) ether, wherein n is 1,2,3,4,5 or 6, and R be-H ,-F ,-Cl ,-Br ,-I ,-OH ,-C (O) OH (or acceptable salt, for example, sodium salt or potassium salt) ,-C (O) OCH ₃,-C (O) OC ₂H ₅

Embodiment 10.Examining or check formula 1 chemical compound in fact as previously mentioned influences ability (people such as E.Simelyte, the Arthritis ﹠amp of arthritis process in passive collagen-induced arthritis model; Rheumatism, 52 (6): 1876-1884,2005; People such as Z.Han, Arthritis ﹠amp; Rheumatism 46 (3): 818-823,2002; People Clin.Immunol.ImmunopathoL such as H.Miyahara, 89 (1): 89-76,1993).In these rules, in the DBA/1 mice, induce passive collagen-induced arthritis by giving anti-II Collagen Type VI antibody, described anti-II Collagen Type VI antibody is induced the immunne response at joint tissue in animal.Effectiveness in this arthritis model represents to be primarily aimed at the effectiveness of inflammation, estimates in the separator of the cytosis that works in getting comfortable arthritis.Use the arthritic order of severity of semiquantitative clinical score system evaluation.With 17 α-acetenyl androstane-5-alkene-3 β of 40mg/kg/ days of the group by oral gavage of every group of 8 animals, 7 β, 17 beta-triols handled 14 days or handled 14 days with vehicle.Vehicle is the water that contains 30% cyclodextrin-sulfo group butyl ether, and drug solution is the vehicle that contains the 20mg/mL medicine.

Animal is checked the arthritis that higher arthritis score (4 minutes/pawl) expression is more serious by measuring ankle thickness.Experiment finished after about 14 days, and carried out histology and gene expression measurement.For the histology, gather in the crops left back pawl, in 10% formalin, fix 24 hours, decalcification, and be embedded in the paraffin.Tissue slice is used for green hematoxylin of safranin O-Nai and eosin dyeing, to measure proteoglycan content.Use the sxemiquantitative marking system to estimate inflammation outside synovial fluid inflammation, the joint, corrosion and proteoglycan loss.

After giving antibody, begin to use compound treatment.Described rules allow to observe the influence of treatment to the arthritis progress.The result shows, compares with the 4th treated animal, and at 7-14 days, the collagen-induced arthritis in the 1st group reduced.Compare the 7th day 5.1 maximum clinical scoring with the 1st treated animal, the maximum clinical scoring at the 8th day in the animal of handling with vehicle is 10.2.When the 14th day rules finish, to compare with 4.1 scoring of matched group, the clinical score of vehicle processed group is 7.8.Difference at 7-14 days clinical score in the animal of accept handling is significantly, and this shows, compares with vehicle control animal group, has occurred the reduction of level of inflammation in the animal of receiving treatment.With 17 α-acetenyl androstane-5-alkene-3 β, 7 β, the effect that 17 beta-triols are handled to handle similarly with dexamethasone, it is inflammation-inhibiting and reduce the arthritic order of severity in this animal model also.17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols reduce the ability and for example methotrexate or anti-TNF alpha medicine formation contrast of the cellular immunization inhibitor that does not almost have to render a service of the arthritis order of severity in this arthritis model.

Embodiment 11.Studied the ability that LPS in the formula 1 compounds affect mice induces injury of lung.The inductive injury of lung model of LPS had been used for estimating treatment for acute lung injury (ALI), acute adult respiratory distress syndrome (ARDS) and endotoxin shock or sepsis in the past, and (Chest 100 (4): 1110-9,1991 for people such as Metz, C; Windsor, people such as A.C., Ann.J.Med.Sci.306 (2): 111-6,1993; People such as Brigham K.L., Am.Rev.Respir.Dis.133 (5): 913-27,1986).

Described rules are in fact as Su, people such as X., and Intenstive Care Med.30:133-140 carries out described in 2004.(Bar Harbor, 6-8 week female C57/BL6 mice in age (average weight 25g) ME) is divided into seven animal groups at random and the stable breeding and the food supply of standard is provided will to derive from Jackson Laboratory.The mice that described each group is handled with saline and LPS for (1), (2) mice of handling with vehicle and LPS, (3) mice of handling with 125 μ g dexamethasone, (4) with 40mg/Kg androstane-5-alkene-3 β, 7 β, 16 α, the mice that 17 β-tetrol and LPS handle, (5) are used 40mg/Kg5 α-androstane-3 β, the mice that 16 salmefamols-17-ketone and LPS handle, (6) with 40mg/Kg5 α-androstane-3 β, the mice that 17 beta-dihydroxies-16-oxime is handled, (7) use 40mg/Kg androstane-5-alkene-3 α, 7 β, 16 α, the mice that 17 β-tetrol is handled.

At the 1st day, mice was with chemical compound or vehicle pretreatment.At the 0th day, mice was handled with the chemical compound or the vehicle of second dosage.At the 0th day+60 minutes, the bacillus coli LPS (Sigma) with 100 μ g under the direct developing situation at trachea under the situation of shallow degree anesthesia attacked with mice.At the 2nd day (that is, the 48th hour time point after LPS attacks), mice is put to death, and obtain BAL (with cell counting and measurement TNF α/IL6 level).Take out lung, pulverize and be used for myeloperoxidase (MPO) research.By (bacillus coli 0111:B4,100mLPBS Sigma-Aldrich) splash in the trachea of shallow degree anesthesia (isoflurane) and be pre-formed the inductive acute pneumonia of LPS containing 50mg LPS under the situation of direct developing.Time point at the 48th hour is put to death mice.After this, the trachea of cervical region carries out tracheotomy after exposing making down.No. 20 blunt nosed syringe needles are inserted in the trachea of exposure, then with its ligation and be used to carry out bronchoalveolar lavage (BAL).Air flue is hemorrhage to minimize with wound in order to make, and uses aseptic PBS * 3 of 0.5mL to carry out BAL.Typically reclaim 1300mL altogether from this process.Use the cell divide numeration of leukocyte in the hematimeter mensuration BAL fluid (BALF).80-100 cell classified.After obtaining BAL, the thoracic cavity is opened and is used by the right ventricle puncture Sterile Saline perfusion heart/lung of 3mL.Gather in the crops all lung tissues and preparation then and be used for MPO mensuration.Algoscopy hereto is with lung homogenize in kaliumphosphate buffer (pH6.0 comprises 0.5% cetyl trimethyl ammonium bromide) respectively.F joins the supernatant of 50 μ L in the 950 μ L kaliumphosphate buffers that comprise 0.2mg/mL dianisidine dihydrochloride (Sigma-Aldrich) and 0.00002% hydrogen peroxide in centrifugal (14,000Xg, 10 minutes, 4 ℃) afterwards.Measure the variation of absorbance at 460 η m.Use ELISA (the R ﹠amp of commercially available ELISA by being used for TNF α, IL-6; D Systems) cytokine levels in the mensuration acellular supernatant of BALF (200Xg, 10 minutes, 4 ℃).That surprising especially is androstane-5-alkene-3 β, 7 β, and 16 α, the result of 17 β-tetrol, discovery is compared reduction by MPO, TNF α among the BAL of the animal of oral this compound treatment with the animal of IL-6 level and vehicle processing.Influence to MPO (it is the tolerance of neutrophil cell load in the lung) and pro-inflammatory cytokine TNF α is far-reaching especially.The short inflammatory cell of this prompting compounds block is moved in the inflammation tissue and the ability that reduces the pro-inflammatory cytokine signal transduction.In this model, the chances are by the LPS of innate immunity composition stimulates and drive for acute inflammation.These identical medium diseases have some to increase to some extent, and are considered to relevant with the pneumonia that relates to several diseases, comprise cystic fibrosis, chronic obstructive pulmonary disease, acute and chronic bronchitis or even some infectious disease such as pulmonary tuberculosis.With androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol handle the observed result that significantly reduced MPO and pro-inflammatory cytokine level among the BALF at 48 hours with herein about androstane-5-alkene-3 β, 7 β, the anti-inflammatory activity unanimity that 16 α, 17 β-tetrol report in the disease specific model (comprising EAE) of chronic inflammatory disease.

Embodiment 12.People's mixed lymphocyte reaction (MLR).3 β, 16 alpha-dihydroxy-s-17-oxo androstane, 3 β, 17 beta-dihydroxies-16-oxo androstane, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols and 17 beta-aminos androstane-5-alkene-3 β-alcohol influence the ability that human T lymphocyte wherein responds to the antigen-specific sexual stimulus of specificity heterologous antigen (major histocompatibility complex).The influence that external model that MLR replys as delayed hypersensitivity and expression chemical compound can be replied human antigen's specific T-cells in vivo.Chemical compound suppresses MLR and represents that chemical compound is to lymphocytic immunosuppressive action.The chemical compound that does not suppress MLR is lymphocytic for responsiveness not to be inhibitive ability of immunity antigen-specific, activated.

Obtain blood samples from the healthy human volunteers of 3 (2 male, 1 women) fasting in 23-31 year.The experimenter did not use immunomodulator, antiallergic agent or antibiotic three middle of the month before research.The experimenter gets blood between 9 and 10 of the mornings, may may fluctuate to hormone or cytokine levels in the influential circulation of lymphocyte vitro responses with restriction.By in Ficoll-Hypaque (density 1.077, Biochrom AG, Berlin, Germany) gradient is carried out centrifugalize peripheral blood lymphocytes (PBMC), and it is resuspended in culture medium ((RPMI1640, be supplemented with 2mM L-glutaminate, penicillin (100U/mL) and streptomycin (100mg/mL) (Invitrogen s.rl., Milan, Italy) in.Use 10% blood plasma from body (effector) inactivation.With 500,000 effector PBMC (PBMCr) and 500, the stimulus object PBMC (PBMCs) of 000 allos radiation (30Gy) is that the concentration of 300nM or 30nM is at flat 96 orifice plate (Nunc with the mixed of 1:1 in 200 μ L culture medium and with each of 4 kinds of chemical compounds, Roskilde, Denmark) the middle cultivation 6 days.Compound dissolution in the ethanol kind, is diluted to desired concn with culture medium then, and generation comprises 0.01% alcoholic acid final solution.With this vehicle with comparing.Contrast also comprises PBMCr and the PBMCs that cultivates respectively.In last 8 hours processes of culture period, with PBMC with 1 μ Ci/ hole [ ³H] thymidine (Amersham, Milan, Italy) burst process.Harvesting and the combination of use beta cell counter measures radioactivity then.Calculate the average cpm in quadruplicate hole.The propagation of T cell is expressed as stimulation index: and S1=cpm (PMBCs * PBMCr)/cpm (PBMCr)+cpm (PBMCs).Use student t check carrying out statistical analysis.Reply and compare deriving from the propagation that obtains among the quadruplicate cpm number of every kind of test compound and contrast PBMCr that in the presence of vehicle, cultivates and the PBMCs.O'clock think that difference is significant in p＜0.05.

The result shows except 17 beta-aminos androstane-5-alkene-3 β-alcohol of 300nM, do not have a kind of MLR of showing to suppress in 4 kinds of chemical compounds.This shows, 3 β, 16 alpha-dihydroxy-s-17-oxo androstane, 3 β, 17 beta-dihydroxies-16-oxo androstane and 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols do not have measurable inhibitive ability of immunity (p〉0.05) under 300nM or 30nM in this is measured, and the 17 beta-aminos androstane of 300nM-5-alkene-3 β-alcohol is compared with control reaction and had suitable inhibitive ability of immunity (p＜0.05).These results show, 3 β, and 16 alpha-dihydroxy-s-17-oxo androstane, 3 β, 17 beta-dihydroxies-16-oxo androstane and 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols do not have inhibitive ability of immunity to human lymphocyte in vivo.These results and chemical compound are that the ability that do not have the anti-inflammatory agent of inhibitive ability of immunity (referring to for example, embodiment 7) is consistent.

Embodiment 13.Immunosuppressant is analyzed.Glucocorticoid steroids for example dexamethasone or hydrocortisone typically has inhibitive ability of immunity and has the significant toxicity relevant with its use.Examine or check immunosuppressant (people such as C.Goebel, Inflamm.Res., 45 (Suppl.2): S85-S90,1996 in fact as previously mentioned in the reporter gene Kang Yuan lymphonodi poplitei algoscopy in mice; People such as R.Pieters, Environmental Health Perspectives 107 (Suppl.5): 673-677,1999).These rules are used to analyze 17 α-acetenyl androstane-5-alkene-3 β, 7 β, and the activity in 17 beta-triol Zai lymphonodi poplitei (PLN) algoscopys does not have tangible immunosuppressive activity in vivo to show this chemical compound.In these rules, vehicle is 0.1% carboxymethyl cellulose, 0.9% saline, 2% Tween 80 and 0.05% phenol, uses to comprise 17 α-acetenyl androstane-5-alkene-3 β, 7 β, the suspension of 17 beta-triols in the animal that heals with medicine.Active evaluation comprises (measures the inhibition to total lymphocyte count, antigen-specific IgM, IgG1 and IgG2a antibody secreting cell (ASC) (ELISPOT algoscopy) in the 1) Zai lymphonodi poplitei cell; (2) express by the living cells in the suspension being flowed cell counting analysis of cells surface marking thing (CD4, CD8, CD19, F480, CD80, CD86); (3) the lymphocytic IL-4 of in-vitro measurements (ELISA), TNF α and IFN γ produce.

Use the group (every group of n=5 only) of specific pathogen free BALB/C mice.Positive controls is handled with vehicle (oral gavage) and the dexamethasone by subcutaneous injection 5ng/ days suppresses with induction of immunity.Vehicle control animal (negative control) is handled with independent vehicle (oral gavage).17 α-acetenyl androstane-5-alkene-3 β of 0.1mg/ days of an animal groups by oral gavage, 7 β, 17 beta-triols are handled.Another animal groups gives 17 α-acetenyl androstane-5-alkene-3 β of 1mg/ days with by oral gavage to animal, 7 β, and 17 beta-triols are handled.Two tail student t check analysis results of variance such as pass through.Inject the freshly prepd TNP-OVA of the sensitizing dose of 50 μ L for the right back sole of animal.Every day with after the TNP-OVA sensitization immediately by subcutaneous injection with dexamethasone (phosphoric acid Decadron injection; Dexamethasone sodium phosphate) is administered in the cervical region.Give 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols by gavage immediately afterwards.In injection five days after the TNP-OVA, extract blood by the eye socket puncture, and by the cervical region dislocation with the painless execution of mice, take out lymph node and except that the fatty tissue of attachment removal.Preparation single-cell suspension liquid is resuspended among the PBS-BSA (1%) of 1mL and calculates.Measure cell number IL-4, IL-5 and IFN γ.

The lymphocyte average that derives among the PLN of vehicle control group is each lymph node 7.8 * 10 ⁶Individual, become 2.9 * 10 with each lymph in the animal groups for the treatment of with dexamethasone ⁶Individual comparing.This minimizing that lymphocyte calculates shows the significant immunosuppressant of typically seeing when using dexamethasone or other glucocorticoid compound significantly.On the contrary, with 17 α-acetenyl androstane-5-alkene-3 β of 1mg/ days, 7 β, each lymph node of the group that 17 beta-triols are handled has 8.2 * 10 ⁶Individual lymphocyte, with 17 α-acetenyl androstane-5-alkene-3 β of 0.1mg/ days, 7 β, each lymph node of the group that 17 beta-triols are handled has 11.1 * 10 ⁶Individual lymphocyte.The result shows, 17 α-acetenyl androstane-5-alkene-3 β, and 7 β, 17 beta-triols do not have inhibitive ability of immunity, but have the immunostimulant performance.Compare with vehicle control group, with 17 α-acetenyl androstane-5-alkene-3 β of 1.0mg/ days and 0.1mg/ days, 7 β, 17 beta-triols are handled IFN γ, IL-4 and IL-5 level are increased, and have also shown the immunostimulant performance.0.1mg/ it 17 α-acetenyl androstane-5-alkene-3 β, 7 β, the group that 17 beta-triols were handled greater than usefulness the influence of IFN γ, IL-4 and IL-5 level in 1.0mg/ days.On the contrary, compare with vehicle control group or with the drug treating group, IFN γ, IL-4 and the IL-5 level of the group of handling with dexamethasone reduce.

Embodiment 14.Immunosuppressant is analyzed.Characterized the ability of several compounds affect immunne response.This rules examination chemical compound immunization in the standard immunoassay algoscopy.Ovalbumin (OVA) specific immune response test is the stable system that is used for measuring previously (cell-mediated with antibody-mediated) immunne response.The sedimentary 100 μ g OVA in Alumen (25mg/mL) that comprised in saline by peritoneal injection (cumulative volume 200 μ L) at the 0th and 7 day make the BALB/c mouse immunity.Mice (every group of n=5 only) was carried out 20 days with compound treatment (oral gavage, 40mg/kg, about 1mg/ animal) every day.At the 20th day, extract to be permitted also and in ELISA, measured antibody titer to OVA.The chemical compound of test is 3 β, 16 alpha-dihydroxy-s-17-oxo androstane, 16 α-bromine epiandrosterone, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols, 3 β, 16 alpha-dihydroxy-s androstane-17-oxime, 17 beta-aminos androstane-5-alkene-3 β-pure and mild 3 α, 16 α, 17 β-trihydroxy androstane.Neither one is found and has far-reaching inhibitive ability of immunity in these chemical compounds, and the OVA antibody titer is similar to the result in the vehicle control group.

Embodiment 15.Reduce glucose and improve insulin resistant.In the diabetic db/db of people's diabetes and insulin resistant mouse model, estimate and reduce the glucose effect and improve insulin resistant.In these researchs, the db/db C57BL/Ks mice in about 8 to 10 ages in week is divided into 10 every group group, by oral gavage is with vehicle contrast (not having medicine) or 17 α-acetenyl androstane-5-alkene-3 β then, 7 β, 17 beta-triols are handled.Give twice with chemical compound every day with 20mg/kg/ days (give with 10mg/kg every day twice), 40mg/kg/ days (give with 20mg/kg every day twice), 80mg/kg/ days (give with 40mg/kg every day twice), up to 28 days.Use small amounts of blood (tail cant is got blood), twice monitor blood glucose level weekly in the dosed administration process is so that by saccharometer reagent paper measure glucose concentration.Special time in the dosed administration process (the 14th day and the 28th day), also the standard oral dose by giving 1g/kg glucose (being about 40mg) for the mice of 40mg and then behind 15,30,60 and 120 minutes after glucose dosage gives the fluctuation of monitor blood glucose level promptly carry out oral glucose tolerance test (OGTT).In the drug treating group, 40% the reduction of having an appointment of the blood sugar level of in the db/db mice, observing hyperglycemia.In vehicle control group, blood glucose is near 380mg/dL, and in the drug treating group after at least 10 days of dosed administration blood glucose be＜230mg/dL.Depart from the peak value hyperglycemia of the about 400mg/dL of oral glucose dosed administration after 30 minutes seen in the vehicle processed group with drug treating 28 angels of 80mg/kg b.i.d. and to be reduced in the drug treating group＜200mg/dL.

Embodiment 16.The hyperglycemia treatment of the inductive obesity of meals (DIO) mice.Can be therein strengthen the effect to the sensitivity of insulin of periphery by research medicine in the mouse model of the insulin resistant states that obtained at least 6 weeks for feeding animal high fat diet (total amount of heat take in 60%).This model for example has been described in, people such as J.N.Thupari, Proc.Natl.Acad.Sci.USA, 99 (14): 9498-9502,2002, people such as H.Xu, J.Clin.Invest, 112:1821-1830,2003, people such as H.Takahashi, J.Biol.Chem., 278 (47): 46654-46660, in 2003.Under these meals conditions, mice show weight increase (+35g) and the state of glucose intolerance, the checkout time that shows as the glucose of orally give in standard OGTT (oral glucose tolerance test) process has significant delay.

For these research, the animal in about 4 ages in week is divided into the groups of every group of 10 animals, by oral gavage is with vehicle contrast (not having medicine) or 17 α-acetenyl androstane-5-alkene-3 β then, 7 β, 17 beta-triols are handled.17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols give with 20mg/kg, 40mg/kg or 80mg/kg, and every day twice was up to 28 days.OGTT is carried out in the dosed administration process the 14th day and the 28th day.In the DIO of this insulin resistant model, shown in the remarkable improvement of OGTT blood glucose deviation, compare with the vehicle control animal, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols significantly reduce the glucose intolerance.These discoveries show that with 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols are handled insulin sensitivity or the absorption that has strengthened periphery, and it improves the glucose intolerance in these animals.

Embodiment 17.The db/db mice younger than animal described in the embodiment 15 carries out and similar treatment protocol described in the embodiment 15 in use.With animal (every group n=8 to 10) by oral gavage twice usefulness 40mg/kg/ every day days (20mg/kg dosage, give every day twice) and 80mg/kg/ days (40mg/kg dosage, give every day twice) 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols or vehicle are handled.When dosed administration began, before glucose level rising or hyperglycemia morbidity, animal was 6 ages in week.Vehicle or drug dose administration were kept 32 days, to measure treatment to the morbidity of the hyperglycemia of animal and the influence of progression rates.In matched group, observe the hyperglycemia morbidity after 25 days at dosed administration, and it continues to worsen, that is, when 32 days dosed administration time finished, blood sugar level rose to tangible hyperglycemia from normal level.On the contrary, the glucose level of two medication therapy groups does not all have to be elevated to above normal level when 32 days dosed administration time finished, and shows that in the process of described rules, Drug therapy has postponed the morbidity of hyperglycemia.

Male diabetes db/db mice to 8 ages in week gives 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols suppress the hyperglycemia level of basic blood glucose significantly, this acts on dosed administration and becomes apparent after 10 days, and continues other 18 days in the 40mg/kg dosage group of successive, twice processing every day.In younger 6 week ages male db/db mices, 17 α-acetenyl androstane-5-alkene-3 β with 40mg/kg, 7 β, 17 beta-triols handle at dosed administration and have blocked the progress that in the vehicle processed group observed animal is developed into hyperglycemia state after 25 days fully.Animal through treatment keeps and the suitable blood sugar level of the brood mice of thin type db/+.In addition, the result of the OGTT that carries out in the animal model through treatment shows that comparing the glucose intolerance with the vehicle control animal has significant improvement.

Embodiment 18.Glucose reduces in 8 week age db/db diabetic mices.Carry out hyperinsulinism-positive sugared clamp rules, so that measure intravital insulin sensitivity.In this operation, give insulin so that insulin concentration is risen, the infusion glucose is to keep the normal or fixed euglycemia level of blood glucose (about 180mg/dL) simultaneously.Keep the normal required glucose infusion speed (GIR) of blood glucose and shown insulin action in these animals.The purpose of these rules is to investigate or to characterize 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols and androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol improve systemic insulin resistant and improve the ability that overall glucose is eliminated in hyperinsulinism-positive sugared clamp model.Also estimated the degree of the insulin sensitivity and the tissue-specific glucose uptake of skeletal muscle and liver.The 14th day by oral gavage to dosed administration every day.At the 10th day that handles, implantation catheter in carotid artery and jugular vein.On the same day (the 14th day) of clamp, 7:30 gives chemical compound in the morning.

Estimated body weight and concentration of glucose at the 0th, 7 and 14 day that handles.Carried out the positive sugared clamp of hyperinsulinism handled at the 14th day.Take food away and continue infusion [3-at 7:30 at 10:30 ³H]-glucose (0.05 μ C/ minute) strengthens.Obtain baseline blood samples at 12:50 (10 minutes), and cause the positive sugared clamp of hyperinsulinism at 1:00 (0 minute) by the insulin that gives 10mU/kg/ minute and handle.With different speed infusion glucoses, so that concentration of glucose is fixed on～180mg/dl.When research finishes, give [ ¹⁴C] the 2-deoxyglucose concentrate the injection with the specific glucose uptake of evaluation of tissue.Estimated blood plasma at 122,125,130,135,145 minutes ¹⁴The C2-deoxyglucose.Then with the pentobarbital sodium of intravenous infusion with Animal Anesthesia and obtain selected tissue, freezing and before analyzing, be stored in-70 ℃ in liquid nitrogen immediately.

Analysis is carried out as follows.With plasma sample Ba (OH) ₂(0.3N) and ZnSO ₄(0.3N) slough albumen, drying, and in scintillation counter (Packard TRICARB 2900 TR, Meriden, CT) the last radioactivity of estimating.With the homogenize in 0.5% perchloric acid of refrigerated tissue sample, centrifugal and neutralization.Directly a supernatant is counted, with measure from [ ¹⁴C] DG and [ ¹⁴C] the two radioactivity of DGP.With second aliquot Ba (OH) ₂And ZnSO ₄Processing is to remove ¹⁴C DGP and be attached to any tracer in the glycogen, measure then from free [ ¹⁴C] radioactivity of DG (2).[[ ¹⁴C] DGP is calculated as two differences between the aliquot.Will [ ¹⁴C] accumulation of DGP is with respect to the injection normalization of concentrating of tissue weight and tracer.Computation organization's specificity glucose uptake index Rg (people such as E.W.Kraegen, Am.J.Physiol., 248:E353-E362,1985) as previously mentioned.The overall glucose turnover is calculated as ³Ratio between H glucose infusion speed (dpm/kg/ minute) and the tremulous pulse plasma glucose specific activity (dpm/mg).Interior originality glucose is produced the difference that is calculated as between overall glucose turnover and allos glucose infusion speed people such as (, Endocr.Rev., 6:45-86,1985) R.N.Bergman.Processed group is summarized in the table shown below.

Group	Handle	Dosed administration volume and dosed administration solution concentration	N
				The contrast of A-vehicle *	Vehicle 8mL/kg, po, bid kept 13 days, at the 14th day qd	8mL/kg	10
B-chemical compound 1 *	40mg/kg, po, bid kept 13 days, at the 14th day qd	4mL/kg, the 10mg/mL stock solution in vehicle	10
				C-chemical compound 1 **	80mg/kg, po, bid kept 13 days, at the 14th day qd	8ml/kg, the 10mg/ml stock solution in vehicle	10
D-chemical compound 2 **	40mg/kg, po, bid kept 13 days, at the 14th day qd	4mL/kg, the 10mg/ml stock solution in vehicle	10
				The E-positive control ***	25mg/kg, po, bid kept 13 days, at the 14th day qd	5mL/kg, the 5mg/mL+1%CMC in water	10

^*Vehicle: 30% sulfo group butyl ether in water (, in solution, containing the medicine of 20mg/mL) for the B-D group

^*Chemical compound 1:17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols

Chemical compound 2: androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol

^{* *}Rosiglitazone maleate (31493r, AApin Chemicals Limited (UK),

The CMC-carboxymethyl cellulose (middle rank, C4888, Sigma)

Insulin dose is 10mU/kg/ minute.In the intact animal, the glucose that this insulin dose needs infusion～90mg/kg/ minute is fixed on～150mg/dl to keep glucose level.Average glucose requirement in all processed group is normal value～50%.The result shows, compares 17 α-acetenyl androstane-5-alkene-3 β with the vehicle contrast, 7 β, 17 beta-triols and androstane-5-alkene-3 β, 7 β, 16 α, the two all increases glucose infusion speed 17 β-tetrol, this means that the insulin action in B, C, D and E group improves.

Use 3- ³H glucose tracer calculates the speed of hepatic glucose generation and the ability of insulin inhibition hepatic glucose generation in the clamp process in the process in period of basis.In serious insulin resistant animal, at employed insulin dose, endogenous glucose produces and reduces about 50%.In C, D and E group, insulin fully suppresses endogenous glucose and produces (p＜0.05), the improvement of this expression liver insulin action.

In order to estimate the insulin action of periphery, use ¹⁴The C-2-deoxyglucose is estimated the tissue specificity glucose uptake in the positive sugared clamp process of hyperinsulinism.Gave at 120 minutes ¹⁴The injection of concentrating of C-2-deoxyglucose.Collection organization after 25 minutes.The analysis tissue ¹⁴The total accumulation of C-2-deoxyglucose phosphate ester.In these rules, the brain glucose uptake is not subjected to the influence of most of therapeutic schemes, so used as internal contrast.The result shows that the glucose uptake of the brain between all groups all has comparability.In processed group, the glucose uptake in heart and barrier film is higher than vehicle control group.Androstane-5-alkene-3 β, 7 β, 16 α, the two all more effectively increases muscle glucose uptake (p＜0.05) in the gastrocnemius muscle 17 β-tetrol and rosiglitazone.In white vastus muscle as non-oxidizable muscle group, remove androstane-5-alkene-3 β, 7 β, 16 α beyond the difference between 17 β-tetrol and the rosiglitazone, do not detect difference.

Embodiment 19.Rat is freely contacted comprise androstane-5-alkene-3 β of 0.45% w/w, 7 β, the standard laboratory food of 17 beta-triols 6 days, at the 6th day liver organization is analyzed subsequently, analyzed the PCK (" PEPCK ") of liver and the level of 11beta-Hydroxysteroid dehydrogenase (" 11 β-HSD ").The control animal normal diet of feeding, and measured messenger RNA (mRNA) at the 6th day by RT-PCR and examine or check PEPCK and 11 β-HSD level.Contrast and the animal of handling are all freely obtained water.Discovery gives the level that chemical compound 6 days reduces by 11 β-HSD 1 type (" 11 β HSD1 ") and PEPCK in the liver organization in food, as follows.PPAR α mRNA level in these animals androstane-5-alkene-3 β that is not subjected to feeding, 7 β, the influence of 17 beta-triols.

In another research, discovery gives chemical compound 17 α-acetenyl androstane-5-alkene-3 β to mice, 7 β, it is about 50% that 17 beta-triols reduce the 11 β-HSD1 in the osteoblast, this with mice that chemical compound induces the glucocorticoid dexamethasone of bone loss to handle in body in to have the observed result of the effect (bone-sparing effects) that bone keeps consistent.

In another research, from 17 α-acetenyl androstane-5-alkene-3 β with 20mg/kg, 7 β, fatty tissue around thin type db/+ that 17 beta-triols are handled or the gonad of diabetes db/db mice is separated total RNA and processing and is used for quantitative RT/PCR, described quantitative RT/PCR uses iCycler iQ polychrome real-time detecting system (Bio-Rad), uses for monocyte chemoattractant protein-1 (MCP-1) and carries out for specific primer.Make the rna expression level contrast normalization with respect to vehicle.Find that chemical compound makes the level of monocyte chemoattractant protein-1 (MCP-1) reduce about 50%.For this research, also to giving vehicle with the heterozygote db/+ mice of thin type matched group (n=7) of the same age.

Examined or check for example 17 α-acetenyl androstane-5-alkene-3 β of other chemical compound in a similar fashion, 7 β, 17 beta-triols, androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 7 α, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 7 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 4 β, 16 α, the monoesters of 17 β-tetrol or these chemical compounds or diester, for example, at 3 or 17 chemical compounds that comprise one or two acetas or propionic ester, examine or check them and reduce in the cell in hepatocyte or liver source or other tissue or cell (kidney for example, muscle, osseous tissue or cell, fatty tissue or cell, or CNS tissue or cell for example neuron or neuroglia) in PEPCK or level or the active ability of 11 β-HSD (for example 11 β-HSD 1 type or 11 β-HSD 2 types).

Embodiment 20.Suppress CD4 in vivo ⁺CD25 ⁺The generation of T regulatory cell or their activity.To derive from the CD4 of the purification of every group of congenic strain B6.SJL mice (CD45.1) ⁺CD25 ^-T cell (5 * 10 ⁶Individual cell) each of adoptive transfer to five a B6 mice (CD45.2) is merely hit.Obtain the CD4 of purification at least by the cell sorting (FACS) twice that donorcells is carried out fluorescent activation ⁺CD25 ^-The T cell.In subcutaneous injection vehicle (0.1% carboxymethyl cellulose, 0.9% saline, 2% Tween 80,0.05% phenol) before CD45.1 donor animal transitional cell or contain vehicle (the 1mg/ animal/sky of 16 α-bromine epiandrosterone, in 100 μ L vehicles), and every day, injection continued 14 days.Collected thymus, lymph node and spleen at the 15th day from animal.Obtain the sample of thymus, lymph node and spleen from individuality, and cell usefulness is incorporated into the fluorescent antibody labelling of CD4, CD25, CD103 or Foxp3.Then by stream cell counting analysis of cells, so that count the number of different cellular types.Compile preconcentration CD4 with deriving from the lymph node of each processed group and all the other cells of spleen ⁺CD25 ⁺Cell is analyzed then by host's (endogenous CD45.2 cell) and donorcells and (is stopped 15 days in vivo afterwards from CD4 ⁺CD25 ^-The donor Phenotype is converted into CD4 ⁺CD25 ⁺Phenotypic CD45.1 cell) CD4 of Chan Shenging ⁺CD25 ⁺By the cell sorter analysis of cells.For the test of regulatory function, with the conversion CD45.1CD4 of the purification of varying number ⁺CD25 ⁺Cell or endogenous CD45.2 CD4 ⁺CD25 ⁺Cell and 2000 CD4 ⁺CD25 ^-Effector cell, as 1 * 10 of antigen presenting cell ⁵The anti-cd 3 antibodies of individual radiating spleen cell and 0.5mg/ml is cultivated altogether.Use fresh CD4 ⁺CD25 ⁺Cell in contrast.4 days absorb after cultivating beginning by measuring ³The H-thymidine is measured propagation.

The result shows, with the CD45.1 CD4 of the spleen that derives from the vehicle control animal ⁺CD25 ⁺The Treg cell number is compared, and derives from the donor CD45.1CD4 of the spleen of Drug therapy treated animal ⁺CD25 ⁺The Treg cell number is lower.Vehicle contrast CD45.1CD4 ⁺CD25 ⁺The average cell number be 1.97 * 10 ⁵Individual cell, and derive from the CD45.1CD4 of Drug therapy treated animal ⁺CD25 ⁺Cell average is 0.62 * 10 ⁵Individual cell.

CD45.1 CD4 with the spleen that derives from the vehicle control animal ⁺CD25 ⁺The Treg cell number is compared, and derives from the endogenous CD45.2 CD4 of the spleen of Drug therapy treated animal ⁺CD25 ⁺The number of Treg cell is also lower.Vehicle contrast CD45.2 CD4 ⁺CD25 ⁺The average cell number be 9.54 * 10 ⁶Individual cell, and the average of Drug therapy treated animal is 5.49 * 10 ⁶Individual CD45.2CD4 ⁺CD25 ⁺Cell.

Endogenous CD45.2 CD4 in the thymus of vehicle control animal ⁺CD25 ⁺The average of cell is 3.10 * 10 ⁵, and the Drug therapy treated animal is 1.59 * 10 ⁵

With the CD45.1 CD4 in the spleen of vehicle control animal ⁺CD25 ⁺CD103 ⁺The Treg cell number accounts for total donor CD4 ⁺CD25 ⁺The percentage ratio of cell is compared, the donor CD45.1 CD4 in the spleen of Drug therapy treated animal ⁺CD25 ⁺CD103 ⁺Cell accounts for total donor CD4 ⁺CD25 ⁺The percentage ratio of cell is lower.Derive from the endogenous CD45.2CD4 in the spleen of vehicle control animal ⁺CD25 ⁺CD103 ⁺The ratio of Treg cell (13.85%) is compared approximately identical with Drug therapy treated animal (13.40%).The donor CD45.1 CD4 of vehicle contrast ⁺CD25 ⁺CD103 ⁺Cell average is 29.06%, and the average of Drug therapy treated animal is 9.63%.The CD103 surface antigen is by activated T reg cellular expression.This shows, the activatory CD45.1 CD4 in the Drug therapy treated animal ⁺CD25 ⁺The relative scale of cell is than lower in the vehicle contrast, and this is consistent with the Treg cytoactive of inhibition donorcells in the body.

The variant that is fit to of these rules comprises (1) donor CD4 to every bigger quantity of animal use ⁺CD25 ^-The T cell, for example, 1 * 10 ⁶Individual/animal, 1.5 * 10 ⁶Individual/animal or 2 * 10 ⁶Individual/animal, (2) the different daily doses of medicine, (3) different way of administration of medicine, (4) different chemical compounds are as medicine, (5) comprise other animal groups, for example, accept for example anti-inflammatory agent or the inhibitive ability of immunity glucocorticoid group of dexamethasone or hydrocortisone for example of another kind of therapeutic agent.There are some can be applied to the rules of embodiment 3 or the list of references that some are quoted in these variants.

Embodiment 21.Increase CD4 ⁺CD25 ⁺T regulatory cell or their activity in vivo.Give chemical compound 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols to mice described in the collagen-induced in fact as previously described arthritis animal model.People such as H.Offner., Clin.Immunol., 110:181-190,2006.

The DBA/1Lac/J mice is used in this research.Mice derives from Jackson Laboratories, and (BarHarbor, Harbor is MA) and according to the system principle stable breeding that is suitable for.By using ratio with 1:1 with comprising 200 μ g mycobacterium tuberculosis (100 μ L; Difco, Detroit, the bCII of the emulsive 200 μ g of CFA MI) make 8 age in week mouse immune, use cattle II Collagen Type VI (bCII) to induce collagen-induced arthritis (CIA).The antigen intradermal is expelled to the tail root.Monitor animal 4-7 week is so that observe the morbidity and the progress of immunity back disease.With hierarchy system each pawl being estimated the arthritic order of severity: 0=according to following standard does not have rubescent or swelling; Swollen ankles or human hair combing waste that 1=is slight are red; The swelling of 2=development and inflammation and from the ankle to the foot middle part rubescent; Swelling and inflammation appear in the whole foot of 3=; Swelling and inflammation appear in the whole foot of 4=(comprising toe).

After immunity, when occurring, observable clinical disease begins (after immunity about 26-27 days) by oral gavage with 40mg/kg/ days the Drug therapy mice that is included in vehicle week.The vehicle that is used for these rules is the water that contains 30% cyclodextrin-sulfo group butyl ether.Cyclodextrin-sulfo group butyl ether is the (Captisol that is commercially available ^TM, derive from cydexinc.com).Pharmaceutical preparation is 20mg medicine/mL vehicle.

The result who derives from the Drug therapy treated animal shows, gives 17 α-acetenyl androstane-5-alkene-3 β, and 7 β, 17 beta-triols increase in the full splenocyte and express Foxp3 ⁺And CD4+Foxp3 ⁺The frequency of cell, as follows.

Vehicle (n=3) medicine (n=3) p value

Total Foxp3 ⁺1.6% ± 0.16 2.39% ± 0.16 0.00001

CD4+Foxp3 ⁺ 1.1％±0.09 1.34％±0.05 <0.00001

Cell divide the analysis showed that, the Foxp3 of Drug therapy treated animal value ⁺CD4+ cell (1.4%) increases to some extent with respect to control animal (1.0%).Foxp3 albumen relates to CD4 ⁺CD25 ^-Differentiation or be converted into CD4 ⁺CD25 ⁺The Treg cell, and the cell number increase of expressing Foxp3 shows to grow from the precursor of Treg cell and increases to some extent for the Treg cell.After immunity, beginning (after immunity about 26-27 days) by oral gavage when observable clinical disease occurs shows with the result that 40mg/kg/ days the Drug therapy mice that is included in vehicle week derives from the Drug therapy treated animal, give 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols increase in the full splenocyte and express Foxp3 ⁺And CD4 ⁺Foxp3 ⁺The frequency of cell, as follows.Consistent with this result is, compares with the vehicle contrast, and the 44-49 after immunity days, the clinical score of Drug therapy treated animal had statistical improvement.At the 34th day to the 49th day, the average clinical score of vehicle control animal was about 6.8-8, and the Drug therapy treated animal is about 5 at the 34th day maximum average clinical score, and the average score that slowly was reduced to the 49th day is about 3.These results show, compare the arthritic progress of compound retards and reduce its maximum order of severity with the vehicle control animal.

Embodiment 22.Chemical compound synthetic as described below.

Androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol (7)

5-androstene-3 β, 16 salmefamols-17-ketone diacetate esters (3).Be prepared as follows 16 α-bromine dehydroepiandrosterone 2: DHEA (1) is refluxed with copper bromide (II) in methanol.In the pyridine (129mL) of 2 (40.8mmol) that contain 15.0g and water (309mL), add the 1N sodium hydrate aqueous solution of 120mL and mixture was stirred in air 15 minutes.Be poured over sodium chloride reactant mixture in saturated and ice/water that comprise excessive hydrochloric acid.With filtration of crude product, wash with water up to neutrality, and at 55-60 ℃ of vacuum drying under anhydrous calcium chloride.Obtain 16 Alpha-hydroxies-DHEA (Mp 194.4-195.1 ℃) of 8.21g from recrystallizing methanol.By handling product is converted into and acetas then, and passes through purified by flash chromatography with the excessive acetic acid in the pyridine.

5-androstene-3 β, 16 salmefamols-7,17-diketone (5).3 (20.1g, 70% tert-butyl hydroperoxide of solution adding 22mL 51.7mmol) in the benzene that is comprising kieselguhr (60g) and pyridinium dichromate (75g).After at room temperature stirring 2 days, add ether (600mL), and wash (2 * 100mL) with sedimentation and filtration with ether.Residue obtains 5 of 16.0g (39.8mmol, 77%) by purified by flash chromatography (hexane that contains 60% ethyl acetate) and recrystallization, is prism.Mp 205.6-206.2℃。

5-androstene-3 β, 7 β, 16 α, 17 β-tetrol (7).(10.0g, 24.8mmol) solution in dichloromethane (75mL) and methanol (255mL) adds the sodium borohydride of 1.5g and mixture was stirred 1 hour at 0 ℃ to 5 at 0 ℃.After with acetic acid (3.5mL) quencher, reactant mixture is distributed between dichloromethane and water.Mixture with organic matter layer simmer down to 7 α and 7 β diacetate esters tetrols.This by flash chromatography and HPLC purification, is obtained 7 β-epimer (9.5mmol, 38%) of 2.90g.Mp 216.8-220.8℃。At room temperature in methanol (100mL), use 1N sodium hydroxide (60mL) saponification 2 days and pass through the HPLC purification, obtain 7 (1.41g, 4.4mmol, 46%) and obtain, be fine needle crystal from acetonitrile solution.Mp 202.1-206.4 ℃; [a] D+1.35 (methanol, c=1).Select ¹H NMR peak (CD ₃OD): d 0.77 (s, 3H), 1.01 (s, 3H), 3.39 (d, 1H), 3.46 (m, 1H), 3.74 (t, 1H), 4.04 (m, 1H), 5.55 (dd, 1H).

3 α, 7 α, 17 β-triacetyl oxygen base androstane-5-alkene-16 α-alcohol (8), androstane-5-alkene-3 α, 7 α, 16 α, 17 β-tetrol (9)

16 α-bromo-5-androstene-3 α-alcohol-17-ketone (2).(17.8g, (36.4g 163mmol) refluxed 19 hours for (1.35L) solution of methanol 61.7mmol) and copper bromide (II) under agitation to make 5-dehydroandrosterone (1).Add entry (1.35L) and dichloromethane (1.5L) to refrigerative reactant mixture.Organic layer is filtered by anhydrous sodium sulfate and product as fine needle crystal from methanol crystallization (16.7g, 45.5mmol, 74%).Mp 195-207℃。

3 α, 16 α-diacetoxy-5-androstane-17-ketone (4).Under air to 2 (12.0g, 32.7mmol) solution in pyridine (1.032L) and water (0.247L) add 1N sodium hydrate aqueous solution (90mL) and with mixture stirring at room 15 minutes.Reactant mixture is joined in the ice/aqueous mixtures of the 1N hydrochloric acid that comprises 1.2L.After solution usefulness sodium chloride is saturated, it is used ethyl acetate extraction (2 * 1L).The organic layer that merges passes through anhydrous sodium sulfate and concentrates with saline (250mL) washing, filtration.Thick 5-androstene-3 α, 16 salmefamols-17-ketone (3) at room temperature spend the night with the pyridine processing that contains the excessive acetic acid acid anhydride and cross column purification, obtain 4 (7.46g, 19.2mmol, 59%) for deriving from the prism of methanol.Mp 172.7-173.7℃。

5-androstene-3 α, 16 α, 17 beta-triols 3,16-diacetate esters (5).(7.46g, 19.2mmol) solution in dichloromethane (45mL) and methanol (120mL) adds sodium borohydride (950mg) to alkene diketone (enediolone) diacetate esters 4 at 0 ℃.Solution was stirred 1 hour at 0 ℃.After adding excessive acetic acid, reactant mixture is distributed between dichloromethane and water.Organic layer is filtered by anhydrous sodium sulfate and concentrated, obtain the mixture of 17 α (less important) and 17 β (mainly) epimer.This mixture by flash chromatography (hexane that contains 25% ethyl acetate) purification, is obtained the 17 β epimers 5 of 6.1g (15.6mmol, 81%).Mp126.9-128.6℃。By at room temperature spending the night and passing through column purification,, obtain 6.0g (13.9mmol, 89%) from 5 preparation triacetates 6 with the pyridine processing that contains the excessive acetic acid acid anhydride.

5-androstene-3 α, 16 α, 17 beta-triols-7-ketone triacetate (7).(6.0g, benzene 13.9mmol) (255mL) solution is handled and was at room temperature stirred 19 hours with kieselguhr (25.5g), pyridinium dichromate (31.5g) and 70% tert-butyl hydroperoxide (9.0mL) with triacetate 6.Add absolute ether (255mL) and reactant mixture was cooled off in ice bath 1 hour.The solid that produces filtered and with ether washing (2 * 50mL).The organic moiety that merges is concentrated and, obtain 7 (7.7mmol, 55%) of 3.45g by purified by flash chromatography (hexane that contains 29% ethyl acetate).

5-androstene-3 α, 7 α, 16 α, 17 β-tetrol (9).(3.45g, 7.7mmol) solution in dichloromethane (15mL) and methanol (30mL) adds sodium borohydride (1.0g) and solution was stirred 2 hours at 0 ℃ to 7 at 0 ℃.Adding excessive acetic acid (1.5mL) afterwards, reactant mixture is distributed between dichloromethane and water.Organic layer is filtered by anhydrous sodium sulfate and concentrated, obtain the mixture of 7 α (less important) and 7 β (mainly) epimer.In methanol (100mL), at room temperature use 1N sodium hydroxide (60mL) saponification to spend the night in this mixture.By being distributed, saponification mixture reclaims thick tetrol between ethyl acetate and saline.Separate epimer by HPLC, obtain 9 (0.68mmol, 9%) of 220mg.Mp 243-248.3℃。。Select ¹H NMR peak (CD ₃OD): δ 0.77 (s, 3H), 1.02 (s, 3H), 2.11 (m, 1H), 2.57 (m, 1H), 3.34 (s, 1H), 3.44 (d, 1H), 3.70 (brt, 1H), 4.04 (m, 2H), 5.55 (dd, 1H).Separate 8 epimer by HPLC, obtain 8 and the 7 β-acetas epimer of purification.

Androstane-5-alkene-3 β, 7 β, 11 β, 17 β-tetrol-3 β-acetas (8), androstane-5-alkene-3 β, 7 β, 11 β, 17 β-tetrol (9), androstane-5-alkene-3 β, 7 β, 11 β, 17 β-tetrol-3 β-acetas-11-oxime (10).

I: to 1 (4g) at 150ml Ac ₂Solution among the O adds p-TsOH 2.8g, at room temperature, spends the night, and post processing, leaches solid up to forming solid for adding the 700mL frozen water, stirring 1 hour, obtains solid product 2,4.55g.

II: at 0 ℃ to 1.5g NaBH ₄Solution in 35ml EtOH and 5ml MeOH slowly adds the solution of 2 (1.2g) in 30ml EtOH and 10ml chloroform.Solution is continued to stir 2 hours at 0 ℃, at room temperature stirred then 2 hours.After this time, add 4ml acetic acid with quencher NaBH ₄, add 50ml water then.By extract 50ml * 3 separated products with EtoAc, solvent is removed in a vacuum, obtains crude product.By the column chromatography purification, obtain 3,250mg.

III: add 0.36g H to the solution of 3 (200mg) in 8ml MeOH ₅IO ₆Solution in 2ml water at room temperature stirred 1 hour, and solvent removed in vacuo adds entry and uses dichloromethane extraction.The column chromatography purification obtains product 4,60mg.

IV: slowly add the 0.5ml chloroacetic chloride at 0 ℃ in the 5ml pyridine solution of 4 (0.4g), continue to stir 15 minutes at 0 ℃, stirring at room is 30 minutes then.To react quencher by adding 20ml water, extract 15ml * 3,, use Na then with 1N HCl, saturated NaHCO3, salt water washing with EtoAc ₂SO ₄Dry.Vacuum concentration obtains 5,520mg.

V: the solution in the 15ml acetonitrile slowly adds 70% t-BuOOH of 3ml to 5 (0.5g) and 0.13g CuI, stirs 1 hour, stirs 2 hours at 50 ℃ then.Add 12ml 10%Na ₂S ₂O ₅Solution extracts with EtOAc, uses Na ₂SO ₄Drying is removed and is desolvated, and crosses post, obtains 6,80mg.

VI: add 260mgCeCl to the solution of 6 (50mg) in 1.5ml THF and 3ml MeOH ₃7H ₂O slowly adds 75mg NaBH at 0 ℃ then ₄, stirred 30 minutes, add 0.5mL 1N HCl and 5ml water, extract 5ml * 3 with EtoAc, use Na ₂SO ₄Drying is removed and is desolvated, and obtains 7,49mg.

VII: to 300mg NaBH ₄Solution in 4ml EtOH and 1ml MeOH adds the solution of 7 (40mg) in 0.5ml EtOH and 0.5ml chloroform, stirs at 0 ℃ and spends the night in cryoprobe then in 8 hours.Add acetic acid and react, add entry and, obtain 8,30mg with the EtoAc extraction with quencher.mp>250℃； ¹H NMR(CD ₃OD)δ 0.86(s，3H)，1.35(s，3H)，1.95(s，3H)，3.55(t，1H，J＝7.5Hz)，3.71(dd，1H，J＝7Hz，J＝2.5Hz)，4.32(d，1H，J＝2.7Hz)，4.55(m，1H)，5.21(s，1H)。

VIII: add the 0.25mL aqueous solution that contains 50mg NaOH to the solution of 8 (30mg) in 1mL MeOH.Stirred 15 minutes at 50 ℃, add 1N HCl1mL, water 5mL then, extract 5mL * 3, remove and desolvate, obtain 9,20mg with EtoAc.mp 170-172℃； ¹HNMR(CD ₃OD)δ 0.95(s，3H)，1.32(s，3H)，3.41(m，1H)，3.51(t，1H，J＝8.0Hz)，3.78(dd，1H，J＝7.1Hz，J＝2.5Hz)，4.31(d，1H，J＝2.5Hz)，5.15(s，1H)。

IX: to 29mg NH ₂OHHCl and the 17mg NaOH solution in the 1mL of heat EtOH adds the solution of 9 (50mg) in the 1mL of heat EtOH, refluxes 2 hours at 100 ℃, salt is leached, at EtOH/H ₂Recrystallization among the O obtains 10,40mg.mp>250℃； ¹HNMR(CD ₃OD)δ 0.72(s，3H)，1.02(s，3H)，2.03(s，3H)，3.86(t，1H，J＝8.5Hz)，4.08(dd，1H，J＝8.0Hz，J＝2.6Hz)，4.60(m，1H，)，5.19(s，1H)。

17 Alpha-Methyls androstane-5-alkene-3 β, 17-isoallopregnane-3-3 β-acetas-7,11-diketone (7), 17 Alpha-Methyls androstane-5-alkene-3 β, 7 β, 17 beta-triols-3 β-acetas-11-ketone (8), methyl androstane-5-alkene-3 β, 7 β, 17 beta-triols-11-ketone (9).

I: to 1 (4g) at 150ml Ac ₂Solution among the O adds p-TsOH2.8g, ambient temperature overnight, and post processing leaches solid for adding the 700mL frozen water, stirring 1 hour, obtains white product 2,4.55g.

II: at 0 ℃ slowly to 1.5g NaBH ₄Solution in 35mL EtOH and 5mL MeOH adds the solution of 2 (1.2g) in 30mL EtOH and 10mL chloroform, continues to stir 2 hours at 0 ℃, and stirring at room 2 hours, add 4mL acetic acid with quencher NaBH ₄, add 50mL water, extract 50Ml * 3 with EtoAc, remove then and desolvate, obtain thick product.Cross post and obtain 3,250mg.

III: add 0.36g H to the solution of 3 (200mg) in 8ml MeOH ₅IO ₆Solution in 2ml water stirring at room 1 hour, removes and desolvates, and adds entry and extracts with DCM, crosses post then, obtains product 4,60mg.

IV: at-78 ℃ at N ₂Slowly add the THF that 1mL contains 22% MeMgCl to the solution of 4 (250mg) in 1.5mL THF and 3.5mL ether down, stirred 1.5 hours at-78 ℃, stirring at room is 1 hour then, refluxes 1 hour at 75 ℃ then.Add 4mL 1N HCl and 10mL water at 0 ℃.Extract with EtoAc, remove and desolvate, obtain crude product 249mg.Cross post, obtain 5,46mg.

V: slowly add the 1.1mL chloroacetic chloride at 0 ℃ to 5 (1.0g) solution in the 15mL pyridine, stirred 15 minutes at 0 ℃, stirring at room is 30 minutes then.Add 50mL water, extract 50mL * 3, with 1N HCl, saturated NaHCO with EtoAc ₃, salt water washing and use Na ₂SO ₄Dry.Remove and desolvate, obtain 6,1.02g.

VI: the solution in the 40mL acetonitrile slowly adds 6mL70% t-BuOOH to 6 (1.0g) and 0.3g CuI, stirring at room 1 hour, stirs 2 hours at 50 ℃ then.Add 24mL 10% Na ₂S ₂O ₅Solution extracts with EtoAc, uses Na ₂SO ₄Drying is removed and is desolvated, and crosses post, obtains 7,285mg.mp>250℃； ¹H NMR(CD3Cl)δ 0.82(s，3H)，1.29(s，3H)，2.05(s，3H)，4.70(m，1H，)，5.75(s，1H)。

VII: add 150mg CeCl to the solution of 7 (45mg) in 1.5mL THF and 3mL MeOH ₃7H ₂O slowly adds 30mg NaBH at 0 ℃ then ₄, stirred 10 minutes, add 0.5mL1N HCl and 5mL water, extract 5mL * 3 with EtoAc, use Na ₂SO ₄Drying is removed and is desolvated, and obtains 8,41mg.mp108-110℃； ¹H NMR(CD ₃OD)δ0.725(s，3H)，1.25(s，3H)，2.01(s，3H)，4.02(dd，1H，J＝8.2Hz，J＝2.4Hz)，4.53(m，1H，)，5.29(s，1H)。

VIII: add the solution of 23mg NaOH in 0.1mL water to the solution of 8 (22mg) in 1mL MeOH.Stirred 10 minutes at 50 ℃, add 1N HCl 1mL, water 5mL then, extract 5mL * 3 with EtoAc.Remove and desolvate, obtain 9,10mg.mp>250℃； ¹H NMR(CD ₃OD)δ 0.75(s，3H)，1.24(s，3H)，3.41(m，1H)，3.99(dd，1H，J＝8.2Hz，J＝2.5Hz)，5.23(s，1H)。

17 α-acetenyl androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol (8), 17 β-acetenyl androstane-5-alkene-3 β, 7 β, 16 α, 17 α-tetrol (9)

I: the solution in 15ml DMF adds TBDMS-Cl1.24g to 1 (1.0g) and 0.56g imidazoles, ambient temperature overnight, and post processing comprises adding 50ml water, has solid to form, and filters and obtains white solid product 2,1.75g.

II: add 2.3mlLDA to the solution of 2 (1.64g) in 50ml THF that is cooled to-78 ℃, after 30 minutes, slowly add 0.62ml TMSCl, stirred 30 minutes at-78 ℃, the stirring at room of rising again then 1 hour, TLC shows that RXN finishes, with ether extraction 150mlx 2, Na is used in water and salt water washing ₂SO ₄Drying obtains yellow product 3,1.87g.

III and IV: add m-CPBA 4.2g to the solution of 3 (10g) in 250ml THF that is cooled to-20 ℃, stir 3 hours, slowly add 250ml MeOH then, stirred 30 minutes, slowly add 200ml Na at-20 ℃ then at-20 ℃ to form 4 ₂SO ₃Solution stirred 1 hour.The room temperature of rising again with ether extraction 150mL * 3, is used saturated NaHCO ₃, the salt water washing, use Na ₂SO ₄Drying obtains thick 11g, in order to remove some the extra m-CPBA in the product, crosses short column, with 100% Hex 50ml x, 5 eluting, uses 50%Hex/EtoAc100ml * 5 eluting then, collects crude product 5,8.5g.

V: add the solution of 5 (5g) in the anhydrous THF of 50mL by syringe pump to the solution of 10g 90% acetylene lithium ethylenediamine complex in the anhydrous THF of 250ml, this process spends about 8 hours.Make its stirred overnight at room temperature.Add 500mL water at 0 ℃, extract 150mL * 3, with 200mL 0.1N HCl, the saturated NaHCO of 150mL with EtOAc ₃, the water washing of 100mL salt, use Na ₂SO ₄Drying is removed and is desolvated, and obtains crude product 5.5g, crosses post, obtains two isomers, 17-β-OH, 6 (1.5g) and 17-α-OH, 7 (1.2g).

VI: add 4mL1N HCl to the solution of 6 (265mg) in 4mL MeOH and 3mL THF, room temperature 1.5 hours.Add the saturated NaHCO of 10mL ₃, room temperature is removed organic solvent, adds 10mL water, and being stored in spends the night in the cryoprobe anhydrates to remove, and adds THF to solid, filters, and removes THF, obtains white solid product 8,130mg.mp 214-216℃； ¹H NMR(CD ₃OD)δ 0.92(s，3H)，1.06(s，3H)，2.99(s，1H)，3.42(m，1H)，3.72(dt，1H，J＝7.2Hz，J＝2.5Hz)，4.17(dd，1H，J＝8.2Hz，J＝2.7Hz)，5.24(d，1H，J＝1.0Hz)。

VII: add 8mL1N HCl to the solution of 7 (500mg) in 8mL MeOH and 6mL THF, room temperature 1.5 hours.Add saturated NaHCO ₃Arrive pH=8 with neutralization solution.Add 50ml water, the white solid that obtains filters, and washes with water, and vacuum drying obtains white solid product 9,225mg.mp>250℃； ¹H NMR(CD ₃OD)δ 0.90(s，3H)，1.06(s，3H)，2.75(s，1H)，3.42(m，1H)，3.72(dt，1H，J＝7.0Hz，J＝2.0Hz)，.4.37(dd，1H，J＝8.1Hz，J＝2.6Hz)，5.24(t，1H，J＝2.0，J＝1.0Hz)。

4 β-acetyl group androstane-5-alkene-3 β, 16 α, 17 beta-triols (7), androstane-5-alkene-3 β, 4 β, 16 α, 17 β-tetrol (chemical compound 8).

Step 1: with chemical compound 1 (24.0g, 0.0832mol) and copper bromide (56.0g, 0.20mol) mixture in absolute methanol (800mL) refluxed 16 hours.Remove most of solvent under the vacuum and add entry (500mL).Collect the precipitation that produces by filtering, and wash with water.Make solid recrystallization twice in methanol, obtain chemical compound 2, be light yellow solid (19.7g)

Step 2: to the chemical compound 2 that stirs (22.0g, 0.060mol) at 200mL N, the solution in the dinethylformamide add the 1N sodium hydrate aqueous solution (66mL, 0,066mol).With reactant mixture stirring at room 1 hour.Add 1N hydrochloric acid (8mL) and 400mL water.By filtering the precipitation of collecting generation and washing with water.Crude product obtains chemical compound 3 by carrying out purification from recrystallizing methanol, is white solid (11.8g).

Step 3: 0 ℃ to chemical compound 3 (11.8g, 0.0387mol) solution in pyridine (50mL) add chloroacetic chloride (11.8g, 0.128mol).Reactant mixture was stirred 1 hour at 0 ℃.Make rise again room temperature and stirring other 1 hour of the mixture that obtains.Add entry.By the filtration collecting precipitation, and wash with water.With the solid vacuum drying, obtain chemical compound 4 (12.6g), its not purified being used for afterreaction.

Step 4: under nitrogen to the chemical compound 2 of cooling (0 ℃) (3.10g, 0.00916mol) solution in the 80mL absolute ether add lithium aluminium hydride reduction (1.13g, 0.030mol).Remove ice bath and with the mixture that obtains stirring at room 0.5 hour, refluxed then 1 hour.To react quencher by adding 6N hydrochloric acid.Remove ether under the decompression.With the solid filtering that produces and wash with water.Crude product 5 recrystallization in methanol obtains chemical compound 5 (1.1g), is white solid.

Step 5: to 5 (914mg, 2.98mmol) solution in the 20mL chloroform add bromine (303mg, 3.16mmol).With reactant mixture stirring at room 20 minutes.Removal of solvent under reduced pressure obtains chemical compound 6, and it is used for afterreaction without being further purified.

Step 6:4 β-acetyl group androstane-5-alkene-3 β, 16 α, the preparation of 17 beta-triols (7).

Chemical compound 6 is dissolved in the absolute ether and 10mL anhydrous pyridine of 30mL.Add silver acetate (1.03g, 1 (914mg, 2.98mmol) solution in the 5mL anhydrous pyridine.Reactant mixture was in the dark stirred 0.5 hour.There is a large amount of light green color precipitations to produce.Add ether (50mL) and precipitation is filtered.Filtrate is dry under vacuum.Residue is used the 50:50 ethyl acetate by purified by flash chromatography on the silica gel: the hexane eluting, obtain title compound 7 (124mg), and be white solid.Select ¹H NMR data: (CD ₃OD, 300MHz): δ 5.78 (d, 1H, J=2.2Hz), 5.34 (br, 1H), 4.02 (t, 1H, J=4.5Hz), 3.52 (dt, 1H, J=7.7Hz, 3.0Hz), 3.37 (d, 1H, J=4.9Hz), 2.03 (s, 3H), (1.12 (s, 3H), 0.75 (s, 3H).Fusing point: 152-153 ℃.

Step 7: androstane-5-alkene-3 β, 4 β, 16 α, the preparation of 17 β-tetrol (8).

(50mg 0.137mmol) is dissolved in the methanol (5mL) of 1N sodium hydrate aqueous solution (1mL) and with the solution that obtains and refluxed 1 hour with chemical compound 7.Under vacuum, remove methanol and with residue with ethyl acetate extraction (3 * 30mL).With the extract dried over mgso that merges, filter and vacuum concentration, obtain solid.Crude product obtains title compound 8 (23mg) by carrying out purification from recrystallizing methanol, is white solid.Select ¹H NMR data: (CD ₃OD, 300MHz): δ 5.62 (d, 1H, J=3.2Hz), 4.05 (d, 1H, J=2.4Hz), 4.02 (m, 1H), 3.43 (dt, br, 1H, J=11.7Hz, 3.6Hz), 3.36 (d, 1H, J=4.2Hz), 1.197 (s, 3H), 0.76 (s, 3H).Fusing point: 238-241 ℃.

Androstane-5-alkene-3 β, 4 β, 7 β, 17 β-tetrol (12) (method 2), androstane-5-alkene-3 β, 7 β, 17 β-triacetyl oxygen base-4 β-alcohol (11).

Step 1: 0 ℃ to chemical compound 9 (5.0g, add in pyridine 0.0138mol) (20mL) solution chloroacetic chloride (11.8g, 0.128mol).Reactant mixture was stirred 5 hours at 0 ℃, under vacuum, remove most of solvent then.The slurry of remnants is distributed between ethyl acetate (80ml) and water (20ml).Organic layer is used dried over mgso then with 1N hydrochloric acid, saturated sodium bicarbonate aqueous solution washing, filters and be evaporated to solid.Crude product obtains chemical compound 10 (4.8g) from ethyl acetate and hexane recrystallization, is white solid.

Step 2: to chemical compound 10 (720mg, 1.66mmol) solution in dioxane (15mL) and acetic acid (10mL) be added in selenium dioxide in water (1.5mL) and the dioxane (5mL) (185mg, 1.66mmol).Reactant mixture was heated 36 hours at 95 ℃.The mixture cool to room temperature, with the ethyl acetate dilution, and water, saturated sodium bicarbonate and salt water washing sequentially, use dried over mgso then, filter and be concentrated in vacuo to drying.Crude product is used the hexane of 3:2 by the purified by flash chromatography on the silica gel: eluent ethyl acetate, obtain chemical compound 11 (174mg), and be white solid.

Step 3: (148mg 0.33mmol) is dissolved in 1N sodium hydrate aqueous solution (3ml) and the methanol (10ml) and the solution that obtains was refluxed 1 hour with chemical compound 11.Under vacuum, remove most of methanol.Add entry and with mixture sonication and filtration.Solid vacuum drying with collecting obtains 12 (82mg), is white solid.Select ¹H NMR data: (CD ₃OD, 500MHz) δ 5.48 (d, 1H, J=2.8Hz), 4.04 (d, 1H, J=2.7Hz), 3.74 (dd, J=8.3Hz, J=2.5Hz), 3.56 (t, 1H, J=8.5Hz), 3.43 (dt, 1H, J=7.6Hz, J=4.2Hz), 1.25 (s, 3H), 0.75 (s, 3H).Fusing point: 144-147 ℃.

(VI), 16 α-bromine androstane-5-alkene-3 β-alcohol-11 β-acetoxyl group-17-ketone (VII), (VIII), androstane-5-alkene-3 β, 11 β, 16 α-triacetyl Oxy-1 7-ketone (IX), androstane-5-alkene-3 β, 11 β, 16 α-triacetyl Oxy-1 7 β-alcohol (X), androstane-5-alkene-3 β, 11 β, 16 α, 17 β-tetrol (XI).

II. (4.0g, it is anhydrous 1 11.4mmol) to be dissolved in 100ml, the 4-dioxane with Compound I.(3.0g 55.2mmol) and with mixture w refluxed 3 hours under anhydrous condition, monitored by HPLC to add Feldalat NM.Remove and to desolvate 1/3 volume and mixture is acidified to pH=5～6 with 2N HCl.Mixture extracts with 3 * 50ml DCM.Organic layer merged and with 50ml saturated sodium bicarbonate and the water washing of 50ml salt.With after dried over sodium sulfate and the evaporating solvent, separate the 95% pure product that obtains 3.56g.

III. Compound I I (13.5g) and p-methyl benzenesulfonic acid (2.45g) were stirred 18 hours in 175ml anhydrous acetic acid acid anhydride.Then mixture is poured in the 800g ice and stirred 1 hour.Filter by short silica gel short column, obtain the compound III of 3.14g.

IV. compound III (100mg) is dissolved in the 1.55ml ethylene glycol, adds triethyl orthoformate (3.77ml) and p-methyl benzenesulfonic acid (50mg) subsequently.Then mixture was refluxed 1.5 hours, and be poured in the mixture of heat of 6ml methanol and 0.08ml pyridine.With mixture cooling and adding 15ml water.Mixture washs with 3 * 30ml ethyl acetate extraction and with saturated sodium bicarbonate (20ml) and saline (20ml).With dried over sodium sulfate and evaporating solvent, obtain the 97% pure V of 50mg.

V. in the 2ml of 50mg IV DMF solution, add the 27mg sodium borohydride that is dissolved in the 0.5ml water in room temperature.Under vigorous stirring, solution is heated to 100 ℃, kept 15 minutes, subsequently cool to room temperature.Reaction is poured in the 18ml water, adds 0.2ml acetic acid subsequently.(2 * 10ml) extract and wash with saturated sodium bicarbonate (10ml) and saline (10ml) mixture with ethyl acetate.With dried over sodium sulfate and evaporating solvent, obtain the chemical compound V of 39mg.

VI. chemical compound V (50mg) and p-methyl benzenesulfonic acid (2mg) are suspended in the mixture of 2ml acetone and 0.21ml water, refluxed 1.5 hours.After evaporation acetone, add 10ml water, product is separated out from solution precipitation.Filter, obtain the 95% pure required product of 42mg.

VII. with compound VI (315mg) and CuBr ₂(611mg) join 7ml absolute methanol and refluxing 24 hours.Then the reactant mixture drop is cooled off and is poured in the 15ml hot water and with crude product and leach.Then crude product is dissolved among 25ml methanol/THF (1:1), adds the 200mg active carbon.Solution was boiled 10 minutes and active carbon is filtered.Crude product obtains the product of 75% purity of 426mg from recrystallizing methanol.

VIII. compound VI I (420mg) is dissolved in the mixture of 18ml DMF and 7ml water.Under agitation add sodium hydrate aqueous solution (1N, 1.31ml).After 10 minutes, solution is poured in the ice/aqueous mixtures that comprises 1.5ml 1M HCl.It is saturated and with 2 * 5ml ethyl acetate extraction with NaCl to love and respect that solution.With after dried over sodium sulfate and the evaporating solvent, crude product is by the column chromatography purification, obtains the 95% pure VIII of 300mg.

IX. compound VIII (300mg) is dissolved in the 6ml pyridine, adds the 0.34ml chloroacetic chloride subsequently.To react and stir 18 hours, be poured over then in the 30ml frozen water.Crude product is leached, cross column purification then, obtain the pure products of 180mg.

X. Compound I X (120mg) is dissolved in 5ml methanol and in ice bath, cools off.In 5 minutes, add sodium borohydride (11.5mg) and remove ice bath.After 1 hour, reaction is with 0.2ml acetic acid quencher and add 15ml water.Mixture washs with 3 * 20ml ethyl acetate extraction and with saturated sodium bicarbonate (20ml) and saline (20ml).Use dried over sodium sulfate, carry out the column chromatography purification subsequently, obtain the required product of 86mg.

XI. be dissolved in compounds X (180mg) in the 5ml absolute ether and be cooled to-78 ℃.Drip methyl-magnesium-bromide (1.2ml, 1M is in ether).Make and react the room temperature of rising again, refluxed 3 hours.To react cooling then and neutralize with 1M HCl.Sedimentary product leached and, obtain the pure XI of 36mg from methanol recrystallization 3 times.Fusing point=220.3-221.6 ℃.The NMR displacement of selecting: ¹H NMR (CD ₃OD): 5.20ppm (bs, 1H), 4.26ppm (dd, J=3Hz, 5Hz, 1H), 3.88ppm (m, 1H), 3.32ppm (m, 1H), 3.19ppm (d, J=8Hz, 1H), 1.24ppm (s, 3H), 0.92ppm (s, 3H).

Androstane-5-alkene-3 β, 16 α-diacetoxy-7,17-diketone (4), androstane-5-alkene-3 β, 16 α-diacetoxy-7 β, 17-isoallopregnane-3 (HE3467).

2 synthesize.To be cooled to-78 ℃ 1 (3.44g, 10mmol) and TMS-Cl (2.15ml, drip in THF 16.5mmol) (100ml) solution 2.0M LDA (7.5ml, 15mmol).With the solution stirring 30 minutes and the room temperature of rising again.Reactant mixture is distributed between 100ml 1:1 hexane/ether and 100ml water.(3 * 30mL) wash and use Na to organic layer with saline ₂SO ₄Dry.Obtaining yellow oil except that after desolvating.Crude product is by the silica gel chromatography purification, with 5-20% EtOAc/ hexane eluting, reclaim 1.9g 1 and 2 (600mg 1.54mmol), is white solid, yield 31%.

3 synthesize.To be cooled to 0 ℃ 2 (100mg, add in THF 0.26mmol) (3ml) solution mCPBA (77%, 62.2mg, 0.27mmol) and the room temperature of rising again.Add 0.5NHCl (3ml) and stirred 20 minutes, use ether extraction.Extracting solution is used Na with saturated sodium bicarbonate, salt water washing ₂SO ₄Dry.Remove obtain after desolvating product 3 (90mg, 0.26mmol), 100% yield.

4 synthesize.To 3 (721mg, the dripping acetyl chloride and stirred 2 hours in pyridine 2.0mmol) (10mL) solution that are cooled to 0 ℃ at 0 ℃.Reaction water (300mL) quencher and stirring 15 minutes.There is solid to form, by filtering with its collection.Solid water, 1N HCl and water washing, and vacuum drying obtain pale solid 4 (737mg), yield 90%.

HE3467's is synthetic.In 15 minutes to be cooled to-15 ℃ 4 (300mg, 0.75mmol) solution in 1:1 MeOH/THF (15ml) adds NaBH ₄(42.5mg, 1.12mmol).Add the cerium chloride be cooled to-15 ℃ (300mg, methanol solution 0.81mmol) and stirring 2 minutes.Reaction is poured in the 90mL water then with 1N HCl quencher.There is solid to form, by filtering with its collection.Solid is with 1N HCl and water washing, vacuum drying, obtain HE3467 (183mg, 0.45mmol), yield 60%.1HNMR(CD ₃OD)：δ 5.29(s，1H)，4.89(m，1H)，4.55(m，1H)，3.74(d，1H，J＝6.52)，3.60(d，1H，J＝4.76)，2.36(d，2H，J＝1.27)，2.15-2.18(m，2H)，2.05(s，3H)，2.01(s，3H)，1.9-1.1(m，11H)，1.11(s，3H)，0.82(s，3H)。

5 α-androstane-2 β, 3 α, 16 α, 17 β-tetrol (22).

Step 1: 0 ℃ to 13 (50.0g, add in pyridine 0.172mol) (150mL) solution paratoluensulfonyl chloride (47.0g, 0.24mol).Reactant mixture was stirred 2 hours at 0 ℃, then stirred overnight at room temperature.Add entry.By filter to collect the precipitation that obtains and wash with water.Crude product obtains 14 (75.2g) by carrying out purification from recrystallizing methanol, is white solid.

Step 2: (75g, 0.169mol) mixture in 2 (200mL) refluxed 5 hours with chemical compound 14.After cooling, add entry (500mL), the precipitation that obtains is collected and washed with water by filtering.Solid is recrystallization in methanol, obtains crude product (42.5g).Crude product (20.0g) is dissolved in chloroform (113mL) and the acetic anhydride (37mL).At 0 ℃ of chloroform (37mL) solution and 13mL acetic anhydride that in this solution, adds concentrated sulphuric acid (3mL).Reactant mixture was stirred 0.5 hour at 0 ℃, added 700mL water and stirring at room then 6 hours.The precipitation that obtains is collected and washed with water by filtering, and vacuum drying obtains 15 (17.2g), is white solid.

Step 3: with 15 (8.17g, 0.030mol) and copper bromide (510.8g, 0.046mol) mixture in absolute methanol (220mL) refluxed 18 hours.After cooling, under vacuum, remove most of solvent and add entry (150mL).Collect the precipitation that produces by filtering, and wash with water.Make solid recrystallization in methanol, obtain 16, be white solid (6.76g).

Step 4: to stir 16 (6.69g, N 0.019mol), dinethylformamide (180mL) solution add the 1N sodium hydrate aqueous solution (22mL, 0.022mol).With reactant mixture stirring at room 0.5 hour.Add 1N hydrochloric acid (3ml) and 100ml water.The solution that obtains ethyl acetate extraction (3 * 250mL).With the extract dried over mgso that merges, filter and vacuum concentration, obtain 17 (4.37g), be waxy solid.

Step 5: 0 ℃ to 17 (3.74g, add in pyridine 0.013mol) (20mL) solution chloroacetic chloride (2.18,0.028mol).Reactant mixture was stirred 1 hour at 0 ℃.Make rise again room temperature and stirring other 1 hour of the mixture that obtains.Add entry.By the filtration collecting precipitation, and wash with water.With the solid vacuum drying, obtain 18 (4.25g), be white solid.

Step 6: 0 ℃ to 18 (2.4g, add in methanol 0.0072mol) (80mL) solution sodium borohydride (1.2g, 0.031mol).Reactant mixture was stirred 1 hour at 0 ℃.To react quencher by adding acetic acid (6mL) and water (15mL).Under reduced pressure remove most of methanol.Remaining slurry distributes between ethyl acetate (80mL) and water (20mL).Organic layer with the saturated sodium bicarbonate aqueous solution neutralization, is used dried over mgso with 1N salt acid elution then, filters and evaporation, obtains crude product 19 (1.92g), is white solid.

Step 7: 0 ℃ to 19 (1.9g, add in pyridine 0.0057mol) (20mL) solution chloroacetic chloride (1mL, 0.014mol).Reactant mixture was stirred 1 hour at 0 ℃.With rise again room temperature and stirring other 1 hour of the mixture that obtains, under vacuum, remove most of solvent then.The slurry of remnants is distributed between ethyl acetate (80mL) and water (20mL).Organic layer is used dried over mgso then with 1N hydrochloric acid, saturated sodium bicarbonate aqueous solution washing, filters and evaporation, obtains crude product.Crude product is by the purified by flash chromatography on the silica gel, and uses the 1:10 ethyl acetate: the hexane eluting, obtain 20 (1.4g), and be white solid.

Step 8: (980mg adds metachloroperbenzoic acid (3.6mmol) in chloroform 2.61mmol) (25mL) solution to 20.With reactant mixture stirring at room 2 hours.Organic layer washs with saturated sodium bicarbonate aqueous solution, washes with water, uses dried over mgso then, filters and evaporation, obtains crude product.Crude product is used the 1:10 ethyl acetate by the purified by flash chromatography on the silica gel: the hexane eluting, obtain 21 (780mg), and be white waxy solid.

Step 9: with 21 (625mg, acetic acid 1.61mmol) (8mL) solution backflows 5 hours.After cooling, under vacuum, remove and desolvate, obtain grease, it is further spent the night at vacuum drying.The waxy solid that obtains is dissolved in 2N sodium hydrate aqueous solution (8mL) and the methanol (15mL) and backflow 1 hour will be react.Under vacuum, remove methanol and add entry.Collect the precipitation that produces by filtering, and the washing with acetone of water and heat.Solid vacuum drying with collecting obtains androstane-2 β, 3 α, and 16 α, 17 β-tetrol or 22 (295mg) is white solid.Select ¹H NMR data: (CD ₃OD, 500MHz) δ 3.98 (d, 1H, J=4.8Hz), 3.79 (br, 1H), 3.74 (br, 1H), 3.35 (d, 1H, 3.6Hz), 0.99 (s, 3H), 0.77 (s, 3H).mp：260-263℃。

Obviously, above-claimed cpd can be used for preparing other formula 1 chemical compound, for example, and other ester or the ether of these chemical compounds.The intermediate that is used for preparing title compound also can be used for method as herein described.

Embodiment 23.In fact as described in the above-mentioned embodiment 6 in experimental autoimmune encephalomyelitis (EAE) ability of bounds evaluation 1 compounds for treating multiple sclerosis (MS).The rules of implementing the EAE animal model are described in people such as (, Ann.NY.Acad.Sci.USA, 1051:730-42,2005) D.Auci.The female SJL mice (6-8 week age, average weight 25g) that derives from Charles-River remained under the standard laboratory conditions (do not contain non-specific pathogen antibacterial), free contacting foodstuff and water, and before beginning one's study, allow the week that conforms.Six groups that animal are divided at random every group of seven animals, and following processing: (1) mice is handled with vehicle, (2) mice is used SU5416 (Z-3-[(2,4-dimethyl pyrrole-5-yl) methylene]-the 2-indolone) handle, (3) mice is with 17 β-acetenyl androstane-5-alkene-3 β, 7 β, 17 α-triol is handled, and (4) mice is used androstane-5-alkene-3 α, 7 β, 16 α, 17 β-tetrol is handled, and (5) mice is used androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol handle, and (6) mice is with 3 α-trifluoromethyl androstane-5-alkene-3 β, 17-isoallopregnane-3 is handled, (7) mice is with 17 α-trifluoromethyl-androstane-5-alkene-3 β, 17-isoallopregnane-3 handle and (8) mice with 5 α-androstane-3 β, 17-isoallopregnane-3-16-oxime processing.1:1 Emulsion (75 μ g proteolipid protein(PLP) (PLP) and the 6mg/mL mycobacterium tuberculosis H37RA in complete Freund's adjuvant (CFA)) with 200 μ L is induced EAA.To distribute between four positions of 200 μ L injections in draining into secondary lymph node and inguinal lymph nodes.Pertussis toxin, PT is given with 200ng/ mice i.p. as co-adjuvant and after the 0th day and immunity second day.When the clinical onset of disease, begin each group is handled with the 100 μ L vehicles that contain the 0.1mg chemical compound, perhaps handle, q.d.po (oral gavage), and continuation 30 days after immunity with independent vehicle.The clinical symptoms that clinical onset is defined as disease in 25% mice reaches the time of 2-3 level.Clinical scale is undertaken by the observer who does not recognize treatment: 0=does not have disease, and the 1=tail is soft to hang down the paraparesis of 2=moderate, the paraparesis of 3=severe, the dying state of 4=, 5=death.By the ANOVA of paired data and non-parametric Mann-Whitney check being carried out statistical analysis to the significant difference of clinical score.＜0.05 P value is considered to statistically significant.For statistical analysis, the mice that dies from EAE is 5 in death assignment on the same day only, deletes from experimental group then.

As expected, in the 19th day after immunity, 8/8 (100%) all develops the EAE sign that standard in the mice of vehicle processed group.The average natural law of morbidity is 15.5 ± 3.9 (SD).In this animal groups, the persistent period of disease is 12.3 ± 4.3 days.From the 1st day to the 30th day average accumulated scoring was 24.8 ± 7.8, was 22.7 ± 15.8 from (handling the back) average accumulated scoring of the 31st day to the 54th day.Using SU5416, androstane-5-alkene-3 α, 7 β, 16 α, 17 β-tetrol and 5 α-androstane-3 β, observe in the animal that 17-isoallopregnane-3-16-oxime is handled and observed closely similar EAE process in the mice of vehicle processed group, so the mice of handling shows and can compared with the control cumulative incidence rate, disease persistent period and average accumulated fall ill.By contrast, with androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, 3 α-trifluoromethyl androstane-5-alkene-3 β, 17-isoallopregnane-3 or 17 α-trifluoromethyl androstane-5-alkene-3 β, the mice that 17-isoallopregnane-3 is handled shows with vehicle processed group mice compares the EAE process that significantly improves, and causes average accumulated scoring and one or more all significantly reductions in the persistent period.And in further contrasting, there are not accumulation incidence rate or the fatality rate of a kind of appreciable impact EAE in these chemical compounds.Although last 17 β-acetenyl androstane-5-alkene-3 β, 7 β, 17 α-triol only shows the tendency that reduces with respect to the accumulation EAE of vehicle processed group mice outbreak and persistent period, still this effect seemingly biology important (14.9 ± 17.6 and 7 ± 7.9 pairs 24.8 ± 7.8 and 12.3 ± 4.3).This chemical compound do not have statistical significance to be since the whole observation period a large amount of mices be endowed 0 scoring, therefore cause high standard deviation.

When processing in the 30th day finished, the monitoring mice was up to other 24 days.Might observe that disease becomes chronic disease in the vehicle processed group mice, its accumulation scoring can be compared with the accumulation scoring in the processing procedure.Compare with the processing time, in the SU5416 group, observe the phenomenal growth of accumulation scoring in the follow-up time course, be increased to 35.5 ± 13.2 from 25.5 ± 8.9 average accumulated scoring, and 17 β-acetenyl androstane-5-alkene-3 β, 7 β, the growth of 17 α-triol is then littler, for rising to 18.4 ± 20.6 from 14.9 ± 17.6.With 17 β-acetenyl androstane-5-alkene-3 β, 7 β, in the mice that 17 α-triol is handled, 85.7% when 57.1% in the time of also might observing the EAE sickness rate and finishes from the processing time is increased to follow-up time end.On the other hand, other chemical compound seemingly in the follow-up time, kept to the processing time in similar accumulation scoring.This is for 3 α-trifluoromethyl androstane-5-alkene-3 β, and 17-isoallopregnane-3 is significant especially, and its 11.2 ± 4.8 average accumulated scoring in the processing time process is increased to follow-up time 10.8 ± 10.3 when finishing.

These results show, 17 β-acetenyl androstane-5-alkene-3 β, 7 β, 17 α-triol, androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, 3 α-trifluoromethyl androstane-5-alkene-3 β, 17-isoallopregnane-3 and 17 α-trifluoromethyl-androstane-5-alkene-3 β has been brought into play powerful antiinflammatory performance in the inductive EAE model of the PLP of 17-isoallopregnane-3 in the SJL mice.For these discoveries are converted into clinical setting relevant especially be following observed result, promptly, give when the EAE clinical symptom even developed 24% mice in the rules of beginning in after immunity the 12nd day, chemical compound is still activated in this EAE model.What particularly point out is to find that SU5416 is invalid in this case.Reported before that SU5416 was effectively people such as (, J.Med.Chem.48:5412-5414,2005) L.Bouerat in EAE.Yet in order to obtain such result, the SU5416 chemical compound is to give in the animals received immunity.By contrast, in these rules, after the rank symptom is obvious, just give for example 17 β-acetenyl androstane-5-alkene-3 β of chemical compound, 7 β to animal, 17 α-triol, this shows that chemical compound can be used for treating effectively existing disease and is used for prevention or the delay disease incidence.

Embodiment 25.The treatment that ionizing radiation exposes.The F1C that selects is compared with the control animal of handling with independent vehicle to the effect through the radiating female B6D2F1 mice of lethal.Use ¹³⁷The Cs source makes animal be exposed to the total irradiation of 10Gy with 2.5Gy/ minute dosage.Use every group of 12 animals 5 groups altogether.For the 1st, 2,3 and 5 group, test article gives by subcutaneous injection as 100 μ L volumes, carries out three Consecutive Days, and first dosage 2 to 4 hours after being exposed to radiation give.For the 4th group, test article gives three Consecutive Days as 50 μ L volumes by intramuscular injection.Preparation is the suspension that comprises 0.1%w/v carboxymethyl-cellulose, 0.9%w/v sodium chloride and 0.05%v/v phenol.Before injection, stir preparation so that F1C resuspending equably, and be expelled in the animal in a few minutes after the suction syringe, to prevent sedimentation in syringe.

Animal groups is carried out following processing.Only accept vehicle for the 1st group, every day, subcutaneous injection continued 3 Consecutive Days.The 2nd group by every day subcutaneous injection be received in 3 β of the 0.6mg in the 100 μ L suspensions, 17 beta-dihydroxies androstane-5-alkene continues 3 Consecutive Days.The 3rd group by every day subcutaneous injection be received in 3 β of the 3.0mg in the 100 μ L suspensions, 17 beta-dihydroxies androstane-5-alkene continues 3 Consecutive Days.The 4th group by every day subcutaneous injection be received in 3 β of the 0.6mg in the 50 μ L suspensions, 17 beta-dihydroxies androstane-5-alkene continues 3 Consecutive Days.The 5th group by every day subcutaneous injection be received in the 3 beta-hydroxyl-17 beta-amino androstane-5-alkene of the 0.6mg in the 100 μ L suspensions, continue 3 Consecutive Days.After irradiation, animal is carried out monitoring survive, carried out 21 days, obtain following result.Shown the 6th, 7,12 and 21 day surviving animals number.

Embodiment 25.Gastrointestinal inflammatory treatment.Use the animal model proof formula 1 chemical compound restriction of inflammatory bowel or the ability of inflammation-inhibiting or inflammatory symptom.Every group is used 3 male Wistar rats (180 ± 20 gram), after fasting 24 hours with 2,4-dinitrobenzene sulfonic acid (DNBS) or saline attack.DNBS alcoholic solution by colonic instillation 0.5mL (at the 30mg in the 30% alcoholic acid saline solution of containing of 0.5mL) is induced the colitis of far-end, injects the 2mL air by sleeve pipe afterwards and is retained in colon to guarantee solution.The volume that uses is the chemical compound of 2 and 20mg/mL of the 0.1mL of per injection liquid preparation form, for example, androstane-5-alkene-3 β, 7 β, 17 beta-triols, with its every day subcutaneous injection give, continue in 6 days (0.2mg/ animal/sky or 2.0mg/ animal/sky).Preparation is the chemical compound that comprises 100mg/mL in no water slurry (for example, 2% benzylalcohol w/v, 0.1% Brij 96w/v and isopyknic PEG 300 and propylene glycol).By obtain the concentration of 2mg/mL and 20mg/mL with the vehicle dilution 20mg/mL preparation that does not contain chemical compound.

First dosage 30 minutes after DNBS attacks give.Oral once a day (PO) gives sulfasalazine (30mg/mL is in the distilled water that contains 2% soil temperature 80) (10mL/kg/ days), continues 7 days, 24 hour and the 2 hour beginning of preceding two dosage before DNBS attacks.By checking that anal regions writes down diarrheal and occur every day.With animal fasting 24 hours, put to death then., and take out their colon and weigh sacrifice of animal the 7th day or the 8th day.Before taking out colon, the adhesion sign between record colon and other organ.In addition, the existence of after each colon is weighed, writing down ulcer.Colon is changed the baseline group normalization of attacking with respect to saline to " only " of body weight (BW) ratio.The colon of " only " reduces to be considered to significant to the 25-30% of body weight ratio.The result shows, androstane-5-alkene-3 β, 7 β, 17 beta-triols are for the limited influence of the process of disease (clean colon to body weight ratio have an appointment the reduction of 15-20%), and with 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols or androstane-5-alkene-3 β, it is effectively (clean colon to body weight ratio have an appointment the reduction of 25-35%) that 7 β, 16 α, 17 β-tetrol handle.

The variant of these rules comprises and uses above-mentioned dosage level and/or the one or more dosage level in 0.05mg/ animal/sky, 0.1mg/ animal/sky, 0.5mg/ animal/sky and the 1.0mg/ animal/sky to give chemical compound in the water that contains 30% sulfo group butyl ether-cyclodextrin.

Embodiment 26.The treatment of neurone loss relevant and osteoporosis or bone loss situation with wound.Immunocompetence is a kind of function of complexity, and it can be subjected to violent weakening after the inductive endogenous glucocorticoid of wound (GC) level raises.Use chemical compound 5-androstene-3 β, 7 β, 17 beta-triols keep these immunologic functions by performance nutrition or assimilation activity.In the animal model of the acute cerebral ischemia apoplexy that after the carotid artery occlusion of the both sides of gerbil jird, takes place, compare with 5-androstene-3 β with independent apoplexy, 7 β, the treatment of 17 beta-triols improves cognitive ability (p=0.03) significantly.Therefore, for the blank group, be 6.9 ± 0.9 seconds (sec) incubation period of looking for food in every group of measurement, and independent stroke groups is 46.9 ± 13.6sec, and with 5-androstene-3 β, 7 β, the stroke groups of 17 beta-triols treatment is 14.8 ± 4.8sec.Incidentally, the inductive CA1 hippocampal neuron of apoplexy counting loss is significantly by 5-androstene-3 β, and 7 β, 17 beta-triols end (abrogated) (blank=362,247 ± 6,839; Apoplexy=152,354 ± 11,575; And apoplexy+5-androstene-3 β, 7 β, 17 beta-triols=207,854 ± 47,334).

In bone loss situation, 5-androstene-3 β, 7 β, the bone structure that the influence of 17 beta-triols is main, that is, and compacted zone and spongy layer and aufwuchsplate.Using 5-androstene-3 β, 7 β, in the mice (20% total body surface area) of the heat injury that 17 beta-triols are handled, the loss of compact bone (femur) and spongy bone/cancellus (tibia) bone mass, and 5-androstene-3 β is all passed through in the inhibition of the chondrocyte proliferation in the tibia pinus aufwuchsplate of nearside, 7 β, 17 beta-triols handle and are prevented (p＜0.01) significantly.The histomorphometry of femur cortical bone shows that bone formation speed increases to some extent.We have observed the partial prophylaxis effect to the bone mineral content loss, as measuring by dual X ray absorption spectrophotometry.The mice of burning that femur ash weight is handled greater than (p＜0.01) vehicle significantly shows 5-androstene-3 β, 7 β, and 17 beta-triols have kept the mineral content of bone.The consequence that the GC level that the short struvite effect prompting of the high GC level of brain midium or long term raises is damaged the neurological worsens.Proved as 5-androstene-3 β, 7 β, and the involution of thymus of the mice of the adrenal steroid DHEA of the upstream metabolic precursor thereof of 17 beta-triols (5-androstene-3 beta-hydroxyl-17s-ketone) prevention induced by dexamethasone (people such as K.L.Blauer., Endocrinology, 129:3174,1991).Take together, these discoveries show, 5-androstene-3 β, 7 β, 17 beta-triols have suppressed GC inductive functional nervous tissue loss and preserve bone structure after the hot injury.

Proof comprises 5-androstene-3 β in people MG-63 osteosarcoma cell line, 7 β, 17 beta-triols, 17 α-acetenyl-5-androstene-3 β, 7 β, 17 beta-triols and 4-hexestrol-3 α, the ability of the side effect of chemical compound counter-rotating glucocorticoid in osteogenesis of 17-isoallopregnane-3.The MG-63 cell is an osteoblast, is the cell of mediation osteogenesis.These cell lines have been widely used for studying bone biology and have been used for biological activity (for example, people such as B.D.Boyan, J.Biol.Chem., 264 (20): 11879-11886,1989 of characterizing compounds treatment bone loss situation; People such as L.C.Hofbauer, Endocrinology, 140 (10): 4382-4389,1999).Comprise with the glucocorticoid level relevant disadvantageous toxicity that raises: comprise the IL-6 that the osteoblast of MG-63 cell line produces and IL-8 reduces and the expression increase of 11beta-Hydroxysteroid dehydrogenase 1 type enzyme (11P-HSD).11beta-Hydroxysteroid dehydrogenase 1 type enzyme increases by making endogenous cortisone be converted into the level increase that the active hydrocortisone that suppresses osteogenesis causes endogenous glucocorticoid activity.11 β-HSD enzyme is expressed in liver, fatty tissue, brain and osseous tissue.The hydrocortisone that 11 β-HSD-1 produces helps osteoporosis, insulin resistant, type 2 diabetes mellitus, unusual lipidemia, obesity, central nervous system unusually for example apoplexy, neuronal death, depression and parkinson disease.The minimizing of IL-6, IL-8 and osteoprotegerin relates to the osteogenesis that is caused by osteoblast and reduces.Preliminary study shows that dexamethasone suppresses the IC that the MG-63 cell produces IL-6 ₅₀Be 10nM, dexamethasone suppresses the IC of MG-63 cell growth ₅₀Be 15.3nM.

In these rules, make the MG-63 cell have or do not have 30nM concentration the synthetic glucocorticoid dexamethasone in the presence of and be with or without 10nM formula 1 chemical compound in the presence of grow.Chemical compound 1 in the following table is 5-androstene-3 β, and 7 β, 17 beta-triols, chemical compound 2 are 17 α-acetenyl-5-androstene-3 β, and 7 β, 17 beta-triols and chemical compound 3 are 4-hexestrol-3 α, 17-isoallopregnane-3.The result of these chemical compounds shows below.

These results show, partly the reverse side effect of dexamethasone of 30nM of the chemical compound of 10nM, this shows, chemical compound can reverse with osteoblast in the glucocorticoid level relevant multiple toxicity that raises, described osteoblast is the cell of mediation osteogenesis.In relevant rules, chemical compound 17 α of 1nM-acetenyl-5-androstene-3 β, 7 β, 17 beta-triols also make synthetic minimizing of the osteoprotegerin of MG-63 cell be reversed fully after cell was grown in the presence of the 30nM dexamethasone 7 hours.Osteoprotegerin is the factor relevant with osteogenesis, and synthetic minimizing of osteoprotegerin loses relevant with bone.Making synthetic other chemical compound that is reversed wholly or in part of the osteoprotegerin that reduces the MG-63 cell in the presence of the 30nM dexamethasone is 17 α-trifluoromethyl-5-androstene-3 β; 7 β; 17 beta-triols (the normal or baseline osteoprotegerin level in the presence of 1 μ M chemical compound; compare with the vehicle contrast that does not contain chemical compound or dexamethasone); 5-androstene-3 β; 7 β; 16 α; 17 beta-triols (is normal osteoprotegerin level at 1 μ M); 3 β; 7 α; 16 α; 17 β-tetrahydroxy androstane-5-alkene (is near normal osteoprotegerin level at 10nM); 3 α; 7 β; 16 α; 17 β-tetrahydroxy androstane-5-alkene (is normal osteoprotegerin level at 10nM); 17 Alpha-Methyls androstane-5-alkene-3 β; 17-isoallopregnane-3-7-ketone (increasing the osteoprotegerin level) at 100nM; 17 Alpha-Methyls androstane-5-alkene-3 β, 7 β, 17-isoallopregnane-3 (is normal osteoprotegerin level at 10nM).Make osteoprotegerin in the presence of the 30nM dexamethasone reduce other chemical compound obtain partial inversion and comprise androstane-5-alkene-3 β, 17-isoallopregnane-3-7-oxime.

In similar rules, chemical compound 3 α, 17 beta-dihydroxies androstane-4-alkene show dexamethasone-inductive produced the statistically evident counter-rotating that the inhibition of IL-8 and IL-6 and 11 β of induced by dexamethasone-HSD mRNA reduce by the MG-63 cell.

Can obtain corresponding effects in vivo in order to prove, with chemical compound 17 α-acetenyl-5-androstene-3 β, 7 β, 17 beta-triols continue 23 day every day to be handled to reduce the mice of osteoprotegerin level in the animal with dexamethasone.With the osteoprotegerin level in the mice (positive controls) of vehicle and the processing of 10 μ g dexamethasone is the 3.3pmol/L osteoprotegerin; and with 17 α-acetenyl-5-androstene-3 β of vehicle, dexamethasone and 4mg/kg/ days; 7 β, the animal that 17 beta-triols are handled has 6.4pmol/L osteoprotegerin (p＜0.05).

Examined or check the apoptosis degree of osteoblast and osteocyte in the murine vertebrae relevant with estrogen deficiency.With Swiss Webster mice (four months big) spay.After 28 days, with sacrifice of animal, separate vertebra, fixing and embedding is carried out non-decalcification then and is handled (undecalcified) in methacrylate.By adopting CuSO ₄Enhanced TUNEL method is measured the sickness rate of osteoblast and osteocyte apoptosis, and finding does not increase after having estrogen to some extent.Discovery is with reference compound 17 beta estradiols and with F1C 4-hexestrol-3 α for example for example, 17-isoallopregnane-3 and or 17 α-acetenyl-5-androstene-3 β, 7 β, 17 beta-triols handle and reduce apoptosis, this is consistent with bone loss minimizing.

In general, the result of Miao Shuing proves in this embodiment, and chemical compound is 17 α-acetenyl-5-androstene-3 β for example, 7 β, and 17 beta-triols lose the two by increase osteogenesis and inhibition bone influences osseous tissue.Chemical compound is 17 α-acetenyl-5-androstene-3 β for example, 7 β, 17 beta-triols and 5-androstene-3 β, 7 β, 16 α, 17 β-tetrol does not interact with androgen receptor, estrogen receptor-α or estrogen receptor-beta, and this does not bring into play the active ability of the gonadal hormone of not expecting with their treatment bone loss situations is consistent.

Embodiment 27.The ability of the inflammation that hot injury's model characterizing compounds treatment of use mouse ear tissue is relevant with hot wound.Described situation is the minimum damage of burning, and it is tissue necrosis in the 24-72 hour progress in back of burning in the undressed mouse ear that exposes.Balb/c mice to many groups of about nine ages in week is given identification marking, is divided into contrast group and treatment group then.Record will be dipped into the thickness of the ear in the hot water, and the whole ear of anesthetized mice was dipped 24 seconds in 52 ℃ of water then.In injection propylene glycol vehicle (contrast) or after containing the propylene glycol of 100mg chemical compound, every mice is sent back in the cage.A plurality of times before burning and after the hot injury change every mice monitoring ear swelling.1,3,6,9,12,18,24 and 48 hour before damage and after the hot injury to the change of every mice monitoring ear swelling.Animal is handled with the 100mg dehydroepiandrosterone (DHEA) that is dissolved in the propylene glycol.The analysis that in the mice that contrast and DHEA handle edema is formed and disappear shows, handle at DHEA with the undressed mice of burning in generations in six hours after damage as the metric maximum ear swelling of edema.

In undressed matched group, the degree of swelling began to descend in 12 hours, and continued to descend rapidly in the 12 hour time subsequently.After burning 24 and 48 hours, ear tissue shows the microtubule obturation in former zone of stasis.Chemical compound androstane-5-alkene-3 β, the animal that 17-isoallopregnane-3 and 16 α-bromine dehydroepiandrosterone protection is received treatment avoid taking place the many ischemic consequences due to the hot injury of ear.The protectiveness of chemical compound 16 alpha-dehydroepiandrosterones is promptly relatively poor, and it has reduced the degree of carrying out ischemia, but ischemia is carried out in prevention fully, and 16 α-chlorine dehydroepiandrosterone just has slightly protectiveness to carrying out ischemia.

In another rules, examined or check the influence of chemical compound to hemorrhagic shock and ischemia.The use methoxyflurane is with the CF-1 mouse anesthesia at 6-8 monthly age, and the preparation abdominal surgery.Every mice is measured that level, the nictation of breathing reply and to the replying of leather wallet pain, is suitable for surgical operation so that guarantee the level of anaesthetizing.The persistent period of abdominal surgery is about two hours, during this, removes the animal blood volume of 35-40% in 30 minute time period.Remove blood in a controlled manner and simulated the effect of hemorrhagic shock.With the recovery fluid (lactated Ringer's solution) of the blood that removes and 2X volume slowly intravenous infusion in central vein.The recovery fluid is supplemented with dehydroepiandrosterone-3 beta-sulfuric ester of 2mg or as the excipient of placebo.Respectively with the skin closure on peritoneum and upper strata.Animal is remained on 38 ℃-39 ℃, up to recovery fully.Under these conditions, the great majority in the animal of placebo treatment group are dead in 24-48 hour.After surgical operation four hours are carried out colony-forming units (CFU) test of antibacterial and are used conventional counting to measure malonaldehyde in the liver.Mesenteric lymph node (MLN) is taken out and on the blood agar plate, cultivates, after cultivating with the CFU counting number.Liver is taken out and measures the amount of malonaldehyde.Cause the survival of 15/15 mice with the processing of dehydroepiandrosterone-3 beta-sulfuric ester, and the vehicle control animal there is 1/15 survival.

In the rat model of hemorrhagic wound, examined or check therapeutic effect.Make 24 rats that the loss of 40% total blood volume take place, comprise that conduit inserts and wound and hemorrhage is simulated in laparotomy ventrotomy (soft tissue injury).After hemorrhage beginning one hour, make the animal recovery with crystalloid fluid and the erythrocyte (PRBC) that concentrates.After beginning is hemorrhage one hour of 12 animals, but before the fluid recovery with concentration of 100 μ L/kg body weight acceptance be the 40mg/kg body weight contain androstane-5-alkene-3 β, 7 β, the subcutaneous injection of the methylcellulose suspension of 17 beta-triols.The subcutaneous methylcellulose contrast injection of 12 animals received 100 μ L/kg body weight.After inducing hemorrhage three days, accept androstane-5-alkene-3 β, 7 β, the survival rate of 12 animals of 17 beta-triols is 100%; And mortality rate is 25% (the non-conditional test of optimal efficiency Barnard of the difference of two binomial ratios is used in P＜0.04) in undressed group.

Also implemented the model of the hemorrhagic wound rules of blood pressure reduction as second hemorrhagic wound.In these rules, make 15 hemorrhage mean arterial pressures of rat as mentioned above, and recover with crystalloid and PRBC in the time of one hour in hemorrhage beginning to about 35-40mmHg.After beginning is hemorrhage one hour of seven animals, but before the fluid recovery with concentration of 100 μ L/kg body weight acceptance be the 40mg/kg body weight contain androstane-5-alkene-3 β, 7 β, the subcutaneous injection of the methylcellulose suspension of 17 beta-triols.The subcutaneous methylcellulose contrast injection of eight animals received 100 μ L/kg body weight.At two days that induce after hemorrhage, the mortality rate in undressed group (n=8) was 75%.Androstane-5-alkene-3 β, 7 β, the mortality rate of the animal of 17 beta-triol-processing is 43%, proves that this chemical compound has protectiveness in the situation of the hemorrhagic wound that blood pressure reduces.

Embodiment 28.Metabolic stability.Use the metabolic stability of the selected chemical compound of the external examination of microsome that derives from liver organization according to following rules.Microsome in these rules can carry out hydroxylating and make hydroxyl on the steroid molecule and the redox reaction of ketone change.Microsome does not mediate association reaction, for example, and the Sulfation of 3 beta-hydroxies or 3 alpha-hydroxy glucuronic acidizations.

Described rules are following carries out.(1) preparation contains the acetonitrile/water 35:65 of 0.5mM chemical compound.For androstane-5-alkene-3 β, 17-isoallopregnane-3, the 1mg/mL stock solution of preparation 0.145mg/mL or 29.0 μ L adds 171 μ L solvents.For standard curve, the dilution of use 0.5mM stock solution obtains androstane-5-alkene-3 β of 10 μ M, 5 μ M, 1 μ M, the ultimate density of 17 β-diphenol.(2) the following sample of setting up.Each mensuration comprises androstane-5-alkene-3 β, 17-isoallopregnane-3 contrast and 1-8 unknown compound.Pipe for each chemical compound is as follows: 1-0 ' 2-0 ' 3-0 ' 4-0 ' * 5-0 ' * 6-5 μ M7-1 μ M8-30 ' 9-30 ' 10-30 ', wherein * represents the microsome negative control reaction tube of degeneration.For additional compounds, numbering is since 11,21,31 etc.(3) add 315 μ L PBS (pH7.3-7.5) to each pipe.The tester solution that is fit to that adds 10 μ L to each pipe.(4) internal standard/acetonitrile solution.(5) NADPH regenerative system (NRS) is managed 125 μ L for each.Add the 6-glucose 1-phosphate1-of 1.7mg/mlNADP, 7.8mg/ml, the glucose-6-phosphate dehydrogenase of 6 units/mL to PBS.The fresh NRS that is used for each experiment remained on ice before using.(6) each is reflected at the NRS that uses 125 μ L in each pipe.(7) from-80 ℃ of cryoprobes, take out the liver microsome prepared product, and in room-temperature water bath, thaw.The concentration of microsome prepared product is 20mg/ml.0.25mg/ pipe is used in each reaction, and is diluted to the concentration (that is 4 times of dilutions) of 5mg/ml and remains on ice in PBS.(8) for the microsome control valve of zero-time and degeneration, add 500 μ L acetonitriles at-20 ℃.The zero-time pipe is transferred on ice and the microsome of degeneration was hatched 5 minutes in advance to impinging upon 37 ℃.(9) developmental tube that comprises the microsome prepared product was also hatched 5 minutes in advance at 37 ℃.(10) for each incubation tube, microsome prepared product by adding 50 μ L and eddy current mix and start reaction.(11) by finishing each reaction at-20 ℃ of adding 500 μ L acetonitriles and eddy current.(12) after reaction finishes, transfer to the 100 μ L that derive from each reaction tube in the fresh pipe and add methyl-tertbutyl ether of 200 μ L water and 1400 μ L to each pipe.Make the pipe eddy current and on microfuge 13, centrifugal 10 minutes of 000rpm.Then pipe is placed on dry ice-methanol bath, become refrigerated solid up to water layer.(13) transfer to the methyl-tertbutyl ether in each pipe in the fresh pipe and under nitrogen evaporating solvent, then precipitate is resuspended among the 100 μ L acetonitrile/water 35:65 and passes through lcms analysis.The result is illustrated in the following table, and incubation time is as follows.

^*The rat microsome replaces the mice prepared product

The result shows that the tetrol chemical compound has toleration to redox reaction, this and with androstane-5-alkene-3 β, it is consistent that the 17-isoallopregnane-3 reference compound compares that extent of metabolism reduces greatly.This observed result is quite unexpected, because each in four hydroxyls all might be converted into ketone, but in fact neither one is affected.Other chemical compound of examination comprises androstane-5-alkene-3 β, 16 α, 17 beta-triols, androstane-3 β, 16 salmefamols-17-ketone and androstane-3 α, 16 α, 17 α-triol, its all with androstane-5-alkene-3 β, the similar speed of 17-isoallopregnane-3 reference compound is by the microsome metabolism.

Embodiment 29.Measure the drug absorption of CaCo-2 cell.These rules are used for the inflow of metrization compound by the CaCo-2 cell monolayer.The CaCo-2 cell is to show that the enteral absorptive cell is phenotypic, polar, the human cell of well differentiated cell line (people such as J.Hunter, J.Biol.Chem., 268 (20): 14991-14997,1993).This cell line is used to study the speed of all cpds by cell monolayer.Typically, use the monolayer that converges of Caco-2 cell to imitate enteral epithelium and the infiltration coefficient that is used to obtain the test compound steady state flux.This can provide the information about the available probability of the oral biology of chemical compound.

In these rules, cell remained in 37 ℃ the culture medium, use the warm culture medium of the every hole 100 μ L in the sterile tube of 50ml.The division culture medium of cell and every hole 600 μ L is grown on 24 hole sterile plate.Each hole comprises strides hole insert (transwell insert) and to allow the hole two compartments is arranged.The division culture medium of 100 μ L is joined in each hole carefully, make pipette tips contact hole sidewall.With cell at 37 ℃, 5%CO ₂, saturated humidity cultivated 48 hours, to form monolayer.For each plate, pipe is numbered pipe 1-24 number, the bottom side buffer is as bottom side zero-time point (T0).26 to No. 49 pipes are for comprising the upside buffer of trier, as upside T0.The 51-74 pipe is T ₂₀Time point (20 minutes), the 76-99 pipe is T ₄₀Time point, the 76-99 pipe is T ₄₀Time point, the 101-124 pipe is T ₈₀Time point, the 126-149 pipe is T ₁₂₀Time point and 151-174 pipe are to be used for the T that material balance is measured ₁₂₀The upside sample.The 175-179 pipe is 5 standard curves of chemical compound 1, and the 180-184 pipe is 5 standard curves of chemical compound 2, and the rest may be inferred, and the 230-234 pipe is 5 standard curves of chemical compound 12.Place 4 of rack 1 to go the 1-49 pipe, number pipe places rack 2, and the 101-149 pipe places rack 3, and the 151-174 pipe places rack 4 and 175-234 pipe to place rack 5 and 6.

Be prepared as follows buffer: the transfering buffering liquid (being adjusted to pH 7.4) that from fresh 1000mL bottle, takes out 150mL with 1N HCl.This buffer is exactly ' a bottom side buffer '.Remaining 850mL is adjusted to 6.5 with 1N HCl, as ' upside buffer '.The upside buffer of 150mL is placed independent container, and remaining 700mL is used for rinsing.Buffer is stored in 4 ℃, is at room temperature to use for described rules still.

After division culture medium reaches room temperature, about 20mL is poured in the little beaker.Make probe balance 15 minutes in this culture medium.24 orifice plates are taken out and make it get back to room temperature from couveuse.Followingly each hole is measured: probe is inserted in the hole and exposing cell monolayer not with probe; Probe press the TEST button during near media surface and during at the probe contact surface reading become a numeral from 0000; The reading of 1000 Ω is acceptable.Then with the upside buffer from striding hole insert decant and fall and with the rinsing during comprising rinsing buffer 1000mL beaker of whole plate, to remove whole differentiation buffer.To stride the hole insert then and place the T20 plate.Add test compound and contrast (the carbamazepine MW236 of 0.1 μ mol (for example, the 1mg/ml androstane of 29 μ l-5-alkene-3 β, 17-isoallopregnane-3 reference solution) by upside buffer with 10 μ M to 10mL; Hydrochlorothiazide MW351) joins in the upside buffer.The bottom side buffer that adds 0.6mL then to all holes.

Join the 50 μ g/ml3 α that prepare in the 10mL acetonitrile/water (25:75) as internal standard, 7 β, 16 α, 17 β-tetrahydroxy androstane-5-alkene solution by chemical compound (1mg/mL is in ethanol) with 150 μ L.In the buffer of bottom side, form the standard curve of every kind of chemical compound.Because therefore the upside buffer of 10 μ M when adding the bottom side compartment diluted six times forms standard curve under lower six times concentration.

Concentration upside TA (10 μ M) bottom side buffer

2μM 120 480

1μM 60 540

0.5μM 30 570

0.2μM 12 588

0.05 3 597

For T0 contrast, the bottom side buffer of 600 μ l is placed the 1-24 pipe.The upside buffer of 100 μ l is added that trier adds that the upside buffer (making that concentration is in standard curve range) of 500 μ l joins in the 26-49 pipe as upside T ₀Upside buffer with 100 adds that chemical compound places upside.Place the time of plate as time zero (T with striding the hole insert ₀).At T=20, will stride that the hole insert is transferred in the T40 plate and 600 μ L samples of T20 plate be joined in the suitable pipe.At T=40, the hole insert is transferred in the T80 plate and obtain the sample of 600 μ l to the pipe that is fit to from the T40 plate with striding.At T=80, will stride the hole insert and transfer in the T120 plate.From 600 μ l samples are pipetted into the suitable pipe from the T80 plate, the rest may be inferred for remaining time point.The upside buffer of 100 μ l joined in the suitable pipe be used for material balance.Immediately sample is extracted, and place cryoprobe immediately.

Except 151-174 number pipe (it contains 100 μ L), every kind of sample of 300 μ L is transferred to from developmental tube in the 2ml pipe of labelling; 50 μ L of these samples are shifted and join in the bottom side buffer (producing 6 times dilution) of 250 μ L.With 3 α of 20 μ L, 7 β, 16 α, 17 β-tetrahydroxy androstane-5-alkene internal standard adds in each pipe and will add the methyl tertiary butyl ether(MTBE) of 1500 μ L in each pipe.Make the pipe eddy current, in microfuge centrifugal 10 minutes, and place methanol/the dry ice bath, up to freezing.Fall the fresh pipe from each cryovial decant with fresh pipe labelling and with methyl tertiary butyl ether(MTBE).Then under nitrogen with methyl tertiary butyl ether(MTBE) evaporation and reconstruct and pass through lcms analysis in 120 μ L acetonitrile/water (35:65).In following table, chemical compound 1 is 3 β, 7 β, 16 α, 17 β-tetrahydroxy androstane-5-alkene, chemical compound 2 is 17 α-acetenyl androstane-5-3 β, 7 β, 17 beta-triols, chemical compound 3 is 3 α, 17 beta-dihydroxies-17 α-acetenyl androstane, chemical compound 4 is 3 α, 7 β, 16 α, 17 β-tetrahydroxy androstane-5-alkene, chemical compound 5 is 2 β, 3 α, 16 α, 17 β-tetrahydroxy androstane, chemical compound 6 is 3 β, 16 α-diacetoxy-7 β, 17 beta-dihydroxies androstane-5-alkene, chemical compound 7 is 3 β-acetoxyl group-17 α-acetenyl androstane-5-7 β, 17-isoallopregnane-3, chemical compound 8 is 3 β-acetoxyl group androstane-5-7 β, and 16 α, 17 beta-triols and chemical compound 9 are 17 α-acetenyl androstane-5-3 α, 7 β, 17 beta-triols.

Compound concentration (μ M) is at T ₀The time go up the total of in the time of the 80 minutes cumulative transfer of shifting at 80 minutes

Side bottom side concentration (μ M) upside % %

1 2.195 0.017 0.008 0.8％

2 1.911 0.470 0.246 24.6％

3 2.727 0.411 0.151 15.1％

4 1.664 0.019 0.012 1.2％

5 1.817 0.162 0.089 8.9％

6 1.776 0.185 0.104 17.8％

7 1.710 0.195 0.114 31.4％

8 1.724 0.123 0.071 15.9％

9 1.773 0.531 0.299 29.9％

That uses CaCo-2 cell line studies show that the tetrol chemical compound is androstane-5-alkene-3 β for example, 7 β, 16 α, 17 β-tetrol does not have high permeability, therefore estimate be oral can not be by biological utilisation.However, as mentioned above in the treating diabetes model with chemical compound androstane-5-alkene-3 β, 7 β still have activity when 16 α, 17 β-tetrol orally give mice.Other rules show that the tetrol chemical compound is 3 β for example, 7 β, and 16 α, 17 β-tetrahydroxy androstane-5-alkene and 3 α, 7 β, 16 α, the Sulfation degree and the glucuronic acid degree of 17 β-tetrahydroxy androstane-5-alkene are lower than glycols chemical compound.This activity comes from the low metabolism in vivo of tetrol chemical compound at least in part.

Claims (14)

1. be used to prevent or treat the compositions of type 2 diabetes mellitus, hyperglycemia, rheumatoid arthritis, osteoarthritis, multiple sclerosis or bone loss situation, wherein said compositions comprises one or more excipient and the chemical compound with following structure:

One of them R ¹For-H or optional substituted C _1-8Alkyl, another R ¹For-OH, C _2-8Ester or C _1-8Ether;

A R ²For-H or optional substituted C _1-8Alkyl, another R ²For-H ,-OH, C _2-8Ester or C _1-8Ether, perhaps two R ²Be altogether=NOH;

A R ³For-H or optional substituted C _1-8Alkyl, another R ³For-H ,-OH, C _2-8Ester, C _1-8Ether or optional substituted C _1-8Alkyl, perhaps two R ³Be altogether=NOH;

A R ⁴For-H or optional substituted C _1-8Alkyl, another R ⁴For-OH, C _2-8Ester or C _1-8Ether;

R ⁵For-CH ₃,-C ₂H ₅Or-CH ₂OH;

R ⁶For-H ,-CH ₃,-C ₂H ₅Or-CH ₂OH;

A R ⁷For-H or optional substituted C _1-8Alkyl, another R ⁷For-H ,-OH, C _2-8Ester or C _1-8Ether;

R ⁹For-O-or-C (R ¹²) (R ¹²One of them R of)-, ¹²For-H ,-F ,-Br or optional substituted C _1-8Alkyl, another R ¹²For-H ,-OH, C _2-20Ester or C _1-20Ether or optional substituted C _1-8Alkyl, perhaps two R ¹²Be altogether=O ,=NOH or=NOCH ₃

R ¹⁰For-H or-F; With

A R ¹¹For-H or optional substituted C _1-8Alkyl, another R ¹¹For-H ,-OH, C _2-8Ester, C _1-8Ether or optional substituted C _1-8Alkyl, wherein (1) at least one R ²For-OH, C _2-8Ester or C _1-8Ether and R ⁷, R ¹¹And R ¹²In at least one be independently-OH, C _2-8Ester or C _1-8Ether, (2) R ⁴Be substituted C _1-8Alkyl or optional substituted C _2-8Alkynyl and R ², R ³, R ⁷, R ¹¹And R ¹²In at least one be independently-OH, C _2-8Ester or C _1-8Ether, or (3) R ², R ³, R ⁷, R ¹¹And R ¹²In at least two be independently-OH, C _2-8Ester or C _1-8Ether.

2. the compositions of claim 1, wherein said chemical compound has following structure:

Or

One of them R ¹For-H or optional substituted C _1-4Alkyl, another R ¹For-OH ,-OC (O) CH ₃,-OC (O) CH ₂CH ₃Or-OCH ₃

A R ²For-H or optional substituted C _1-4Alkyl, another R ²For-OH ,-OC (O) CH ₃,-OC (O) CH ₂CH ₃Or-OCH ₃

A R ³For-H or optional substituted C _1-4Alkyl, another R ³For-OH ,-OC (O) CH ₃,-OC (O) CH ₂CH ₃Or-OCH ₃With

A R ⁴For-H or optional substituted C _1-4Alkyl, another R ⁴For-OH ,-OC (O) CH ₃Or-OC (O) CH ₂CH ₃

3. the compositions of claim 1 is used for prevention or treatment type 2 diabetes mellitus.

4. the compositions of claim 3, wherein said chemical compound is 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols, androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 7 α, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 11 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 11 β, 16 α, 17 β-tetrol, 3 β, 11 β, 16 β, 17 β-tetrol, androstane-5-alkene-3 α, 11 β, 16 β, 17 β-tetrol, androstane-5-alkene-2 β, 3 α, 16 α, the C of 17 β-tetrol or any of these chemical compound _2-4Monoesters or C _2-4Two ester analogs, randomly wherein monoesters is in the 3-position or the acetas of 17-position, or diester is the acetas in 3-position and 17-position.

5. the compositions of claim 1 is used for prevention or treatment multiple sclerosis.

6. the compositions of claim 5, wherein said chemical compound is 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols, androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 7 α, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 11 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 11 β, 16 α, 17 β-tetrol, 3 β, 11 β, 16 β, 17 β-tetrol, androstane-5-alkene-3 α, 11 β, 16 β, 17 β-tetrol, androstane-5-alkene-2 β, 3 α, 16 α, the C of 17 β-tetrol or any of these chemical compound _2-4Monoesters or C _2-4Two ester analogs, randomly wherein monoesters is in the 3-position or the acetas of 17-position, or diester is the acetas in 3-position and 17-position.

7. the compositions of claim 1 is used for prevention or treatment hyperglycemia.

8. the compositions of claim 7, wherein said chemical compound is 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols, androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 7 α, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 11 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 11 β, 16 α, 17 β-tetrol, 3 β, 11 β, 16 β, 17 β-tetrol, androstane-5-alkene-3 α, 11 β, 16 β, 17 β-tetrol, androstane-5-alkene-2 β, 3 α, 16 α, the C of 17 β-tetrol or any of these chemical compound _2-4Monoesters or C _2-4Two ester analogs, randomly wherein monoesters is in the 3-position or the acetas of 17-position, or diester is the acetas in 3-position and 17-position.

9. the compositions of claim 1 is used for prevention or treatment osteoarthritis.

10. the compositions of claim 1 is used for prevention or treatment rheumatoid arthritis.

11. the compositions of claim 10, wherein said chemical compound are 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols, androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 7 α, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-2 β, 3 α, 16 α, the C of 17 β-tetrol or any of these chemical compound _2-4Monoesters or C _2-4Two ester analogs, randomly wherein (a) monoesters is in the 3-position or the acetas of 17-position, or (b) diester is a acetas in 3-position and 17-position.

12. the compositions of claim 1 is used for prevention or treatment bone loss situation.

13. the compositions of claim 12, wherein said bone loss situation is an osteoporosis, fracture, and thermal burn, radiation burn or chemical burn or have the natural sugar corticoid level and raise or have synthetic glucocorticoid randomly are dexamethasone.

14. the compositions of claim 13, wherein bone loss situation is an osteoporosis, and described chemical compound is 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 17 beta-triols, androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, 17 α-acetenyl androstane-5-alkene-3 β, 7 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 7 α, 16 α, 17 β-tetrol, androstane-5-alkene-3 β, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-3 α, 4 β, 16 α, 17 β-tetrol, androstane-5-alkene-2 β, 3 α, 16 α, the C of 17 β-tetrol or any of these chemical compound _2-4Monoesters or C _2-4Two ester analogs, randomly wherein (a) C _2-4Monoesters is in the 3-position or the acetas of 17-position, or (b) C _2-4Diester is the acetas in 3-position and 17-position.