CN101600429A - The Cholesterylamines of treatment and infection prevention disease - Google Patents

Abstract

The present invention relates to the purposes of Cholesterylamines in pharmaceutical compositions.These pharmaceutical compositions are used for the especially medical intervention of virus or the caused disease of antibacterial of infectious disease.

Description

The Cholesterylamines of treatment and infection prevention disease

The present invention relates to the purposes of Cholesterylamines (cholesterylamines) in pharmaceutical compositions.These pharmaceutical compositions are used for the especially medical intervention (medical intervention) of virus or the caused disease of antibacterial of infectious disease.

The lipid bilayer (lipid bilayer) that forms cell membrane is the bidimensional fluid, and its organizational structure has been the problem of biochemist and biophysicist further investigation many decades.Though most lipid bilayer is considered to homogeneous fluid, but someone once constantly attempted with horizontal heterogeneity (lateralheterogeneities), the little territory of fat (lipid microdomain) introduce we the lipid bilayer structure and kinetic model in (Glaser, Curr.Opin.Struct.Biol.3 (1993), 475-481; Jacobson, Comments Mol.Cell Biophys.8 (1992), 1-144; Jain, Adv.Lipid Res.15 (1977), 1-60; Winchil, Curr.Opin.Struct.Biol.3 (1993), 482-488).

Epithelial cell is polarized to top and substrate outboard structure territory with its cell surface, each of these domains all has different albumen and lipid components, this understanding has caused the new development (Simons that causes " Lipid Rafts (lipidraft) " notion, Biochemistry 27 (1988), 6197-6202; Simons, Nature 387 (1997), 569-572).The assembly of sphingolipid and cholesterol (assemblies) survives this observed result of extraction of detergent, promoted the notion of these equipment performance memebrane protein platform features, and described assembly is called detergent resistance film (detergent resistant membrane, DRM) (Brown, Cell 68 (1992), 533-544).This is exercisable breakthrough, wherein raft coalition (raft-association) is converted into the resistance to 4 ℃ of following Triton-X100 extractions.Second foundation promptly come loss cholesterol (Ilangumaran, Biochem.J.335 (1998), 433-440 with methyl-β-cyclodextrin; Scheiffele, EMBO be (1997) J.16,5501-5508) cause the adding of detergent resistant lose, impel several research groups of this area to probe into the effect of the little territory of fat in the broad-spectrum biological reaction.The lipid assembly comprises the evidence that works in a large amount of cell processes of cell polarity, protein transportation and processing and signal transduction in adjusting at present, increases day by day.

Lipid Rafts is a kind of lipid platform (being rich in sphingomyelins and cholesterol in the skin (outer leaflet) of cell membrane) of special chemical constituent, and its function is the membrane component in the isolated cell film.Raft is considered to less relatively (diameter is 30 to 50nm, and its big or small valuation changes, and depends on used probe and the cell type of being analyzed to a great extent), but can make them coalescent under certain conditions.The specificity that they are relevant with lipid components makes us associating its behavior that is separated in heterogeneous model membrane systems.

For host cells infected, several groups of pathogen, antibacterial, Protein virus, virus and parasites controlled Lipid Rafts (Van der Goot, Semin.Immunol.13 (2001), 89-97).For example by influenza virus, HIV-1, Measles virus (measles virus), respiratory syncytial virus (respiratory syncytialvirus), filamentous virus section (Filoviridae) such as Ebola virus (Ebola virus) and Marburg virus (Marburg virus), Papillomaviridae (Papillomaviridae) and polyoma virus section (polyomaviridae), epstein-Barr virus (Epstein-Barr virus), hepatitis C virus, the viral infection that echovirus 1 (Echovirus 1) causes, represented raft and/raft albumen is the disease of target.In addition, by escherichia coli (Escherichia coli), mycobacterium tuberculosis (Mycobacteriumtuberculosis) and Mycobacterium bovis (Mycobacterium bovis), campylobacter jejuni (Campylobacter jejuni), vibrio cholera (Vibrio cholerae), clostridium difficile (Clostridiumdifficile), clostridium tetani (Clostridium tetani), streptococcus (Streptococci species), Salmonella (Salmonella), the bacterial infection that shigella (Shigella) causes relates to the pathological process (Simons relevant with raft, J.Clin.Invest.110 (2002), 597-603).

First example that is characterized is influenza virus (Scheiffele, J.Biol.Chem.274 (1999), 2038-2044; Scheiffele, EMBO be (1997) J.16,5501-5508).This virus comprises two kinds of complete glycoproteins, i.e. hemagglutinin and neuraminidase are judged according to cholesterol-dependent detergent resistance, the both relevant with raft (Zhang, J.Virol.74 (2000), 4634-4644).The integrity of cholesterol and raft be with neuraminidase be transported to plasma membrane necessary (Keller, J.Cell Biol.140 (1998), 1357-1367).Influenza virus sprouts from epithelial top film, the enrichment in Lipid Rafts of described top.The peplos of influenza virus is to be formed by coalescent raft between budding time, in the process of sprouting, the cytosol microcell (cytosolic leaflet) that M is proteic to be assemblied in nascent peplos located to form the layer that orders about raft and troop (Zhang, J.Virol.74 (2000), 4634-4644).According to nearest model, viral M2 albumen, it is a periphery raft albumen, promoted ripe influenza virus particles pinch off (pinching-off) (Schroeder, Eur.Biophys.J.34 (2005), 52-66).

Equally host's raft lipid and raft albumen are incorporated into the HIV-1 in its peplos, in at least four critical events in its life cycle, used raft (Schroeder, Eur.Biophys.J.34 (2005), 52-66): the passage, the virus that form by new host's mucosa enter immunocyte, send host cell changing function and the viral signal that leaves from cell, and scatter by host's vascular system.

Virus, antibacterial and parasite can enter host cell by the signal condition that changes cell or have an effect with host cell.Situation also is like this between the HIV infection period.Nef is early stage HIV gene outcome, and it promotes infectivity (Zheng, the Curr.Biol.11 (2001) of virus via Lipid Rafts, 875-879), and infect the HIV-1 virion lack Nef can not develop into AIDS (Kirchhoff, N.Engl.J.Med.332 (1995), 228-232).Carry the relevant proteic Lipid Rafts of host cell surface and troop by making, the oligomerization of Nef can help to set up T-cell signal complex and HIV sprout site (Zheng, Curr.Biol.11 (2001), 875-879; Wang, Proc.Natl.Acad.Sci.USA 97 (2000), 394-399).It is another raft dependency step that HIV leaves from cell, depend critically upon viral Gag albumen (Ono, Proc.Natl.Acad.Sci.USA 98 (2001), 13925-13930; Lindwasser, J.Virol.75 (2001), 7913-7924): Gag albumen is tied to the raft that contains HIV spike protein (spike protein) and combines specifically, and this is clustered in together raft, thereby promotes the virus assembling.Interaction between HIV-1 albumen and the Lipid Rafts can cause the Gag that peplos assembling is required conformation change (Campbell, J.Clin.Virol.22 (2001), 217-227).

Another example that raft is trooped is the mechanism of causing a disease of pore-forming toxin (pore-forming toxins), described pore-forming toxin, except that other antibacterials, also secrete (Lesieur by the kind of fusobacterium (Clostridium), Streptococcus (Streptococcus) and Aeromonas (Aeromonas), MoI.Membr.Biol.14 (1997), 45-64).These toxin can bring out from slight liparitosis (mild cellulites) to gas gangrene (gaseous gangrene) and the disease of pseudomembranous enteritis (pseudomembranous colitis).Best object of study is the toxin from marine bacteria Aeromonas hydrophila (Aeromonashydrophila), i.e. aerolysin.Aerolysin is secreted and is combined with the raft albumen (GPI-anchored raft protein) of the GPI grappling of host cell surface.This toxin is be integrated into behind proteolysis in the film, and it carries out seven dimerizations in raft dependency mode then, thereby has formed the passage relevant with raft, and micromolecule and ion flow are crossed this passage, thereby triggers pathogenic variation.The oligomerization of aerolysin can trigger in solution, but in the concentration low 10 than living cells surface ⁵Take place under many times the concentration.The huge raising of this efficient is owing to forming the activation that the raft cluster causes by the raft combination with by concentrating, and described activation is subjected to the driving of toxin oligomerization.Moreover little variation can produce huge effect (Lesieur, MoI.Membr.Biol.14 (1997), 45-64 by the amplification that raft is trooped; Abrami, J.Cell Biol.147 (1999), 175-184).

The film heterogeneity of antibacterial is existing in the prior art to be described, referring to Fishov, and Mol.Microbiol.32 (1999), 1166-1172; Mileykovskaya, J.Bacteriol.182 (2000), 1172-1175.Different albumen in the bacterial membrane have demonstrated the different sensitivity to the fat flowability, the lipid conformation territory of isolated, different flowabilities and composition is the (Linden of coexistence on this explanation physical arrangement, Proc.Natl.Acad.Sci.USA 70 (1973), 2271-2275; Morrisett, J.Biol.Chem.250 (1975), 6969-6976), and also there is the antibacterial plasma membrane to be organized into the evidence (Myers of functional domain, Curr.Microbiol.19 (1989), 45-51), in described domain, find that the plasma membrane component is subjected to the alienation location, and, and membrane phospholipid is separated into all visibly different domain (Vanounou aspect composition, albumen-lipid interaction, the degree of order, MoI.Microbiol.49 (2003), 1067-1079).In this respect, the membrane structure territory of antibacterial is being similar to mammiferous Lipid Rafts on the function and on part-structure.Recently, it is reported, antibacterial peptide (antimicrobial peptides) is discrepant to the toxicity of the antibacterial that has high-load phosphoric acid ethanolamine in the film, this has emphasized the potential importance (Epand of lipid components in the selection toxicity of measuring antibacterial (antimicrobial agents) of cell surface, Biochim Biophys Acta.1758 (2006), 1343-1350).

(Biochemistry 43 (2004), 1010-1018) studied the structure of sterin/steroid and the relation between the participation Lipid Rafts for Wang.Because the biological process that sterin can be used for depending on the cell cholesterol distinguishes from the process that those can obtain the support of any raft environment, these authors have just considered this problem of discussing.Ironically, Wang and colleagues have found to promote the steroid that the ordered structure territory forms in biomembrane.

The purposes of ganglioside in regulating sphingolipid/cholesterol micro structure territory described among the WO 01/22957.

The problem that becomes basis of the present invention is, is infectious disease/disease, and particularly those are associated and/or relevant infectious disease/disease with biology/Biochemical processes that Lipid Rafts is regulated, and the means and the method for clinical and/or pharmaceutical intervention is provided.

The solution of this technical problem is by providing hereinafter and the embodiment that claims characterized realizes.

In environment of the present invention, it is found that particularly in the medical supervision by viral vector and caused disease of antibacterial or disease, Cholesterylamines is better than other cholesterol derivatives unexpectedly, especially is better than cholesterol sulfate in infectious disease.At environment of the present invention, the chemical compound that the meaning of term " Cholesterylamines " also contains the chemical compound that is sometimes referred to as " amino cholesterol derivative " and is sometimes referred to as " amino cholestane and amino cholestene derivant ".In addition, at environment of the present invention, the meaning of term " Cholesterylamines " also contains the derivant of cholesterol, and wherein the A ring is by the hereinafter replacement of the nitrogen heterocyclic ring shown in the formula 2.

Therefore, in one embodiment, the invention provides the chemical compound of following formula 1 or its pharmaceutically acceptable salt, derivant, solvate or prodrug is used for the treatment of, prevents in preparation and/or improve purposes in the pharmaceutical composition of infectious disease/disease.Described disease or disease can be by virus or bacterial.

Description is used the name of following carbon atoms numbered and steroid support ring in the whole text:

In addition, stipulate the general formula that provides in the literary composition of the present invention contain shown in all possible stereoisomer and the non-corresponding isomer of chemical compound.

In formula 1, R ¹, R ², R ³, R ⁴And R ⁵In one of be to be selected from X (CH ₂) _nNH ₂, X (CH ₂) _nNH (C _1-4Alkyl), X (CH ₂) _nN (C _1-4Alkyl) ₂, X (CH ₂) _nN (C _1-4Alkyl) ₃ ⁺Amino-contained group.Preferably, R ⁵It is amino-contained group.

X is chemical bond (direct bond) or is selected from OP (O) (OC _1-4Alkyl) O, OP (O) (O ^-) CH ₂O or OP (O) (OC _1-4Alkyl) CH ₂The phosphorus-containing groups of O.In a preferred embodiment, X is a chemical bond.In one embodiment, X is OP (O) (OC _1-4Alkyl) O.Still in another embodiment, X is OP (O) (OC _1-4Alkyl) CH ₂O.

When X was chemical bond, n was from 0 to 2 integer.In one embodiment, n is 0.In another embodiment, n is 1.Still in another embodiment, n is 2.At one group of preferred chemical compound, n is 0 or 1.Organize preferred chemical compound at another, n is 1 or 2.

When X was phosphorus-containing groups, n was from 2 to 6 integer, preferred 2.

When X is OP (O) (O ^-) CH ₂During O, the chemical compound of formula 1 can exist with the form of salt.Hereinafter listed suitable equilibrium ion, as " pharmaceutically acceptable salt ".Corresponding free alkali, promptly wherein X is (OH) CH of OP (O) ₂O also falls within the scope of the present invention.

In embodiments, R wherein ⁵Be amino-contained group,

Be singly-bound or two key, preferred singly-bound.When

When being two key, there is not R ⁴R ¹, R ², R ³And R ⁴Be H or OH independently.R ⁶Be H.When X be directly in conjunction with and n when being 1 or 2, R ⁶Also can be OH.Preferably, R ⁶Be H.

In embodiments, R wherein ¹Be amino-contained group, R ²And R ⁶Be H or OH independently.R ³, R ⁴And R ⁵Be H.

It is singly-bound.

In embodiments, R wherein ²Be amino-contained group, R ³And R ⁶Be H or OH independently.R ¹, R ⁴And R ⁵Be H.

It is singly-bound.

In embodiments, R wherein ³Be amino-contained group, R ²And R ⁶Be H or OH independently.R ¹, R ⁴And R ⁵Be H.

It is singly-bound.

When X was chemical bond, in preferred embodiments, the chemical compound of formula 1 comprised 1 to 4 hydroxyl.This hydroxyl has improved the solubility of Cholesterylamines in amphipathic or polarizable medium, and this helps medical application.

In one embodiment, the chemical compound of formula 1 comprises 0 or 1 hydroxyl.In another embodiment, the chemical compound of formula 1 does not comprise any hydroxyl.

Formula 1a is the preferred embodiment of formula 1 chemical compound to the following chemical compound of 1l.

Chemical compound 1e and 1l represent cation, and it can use with any pharmaceutically acceptable anion combination such as halogenide, phosphate, sulfate and acetate.

In 1l, chemical compound 1a, 1g, 1i, 1j and 1k are preferred at chemical compound 1a.

In another embodiment, the invention provides the chemical compound of following formula 2 or its pharmaceutically acceptable salt, derivant, solvate or prodrug is used for the treatment of, prevents in preparation and/or improve purposes in the pharmaceutical composition of infectious disease/disease.Described disease or disease can be by virus or bacterial.

In formula 2, Y is NH, N (C _1-4Alkyl) or N (C _1-4Alkyl) ₂ ⁺, preferred NH.

P is from 0 to 2 integer, and q is 1 or 2 integer, if p is 2, q is 1 so.Preferably, p be 0 and q be 2.

Be singly-bound or two key, preferred singly-bound.

The following chemical compound of formula 2a and 2b is the preferred embodiment of formula 2 chemical compounds.

Used chemical compound can adopt standard method preparation known in the art according to the present invention.

The chemical compound of general formula 1, wherein X is chemical bond and has primary amine functional group on 3, can utilize synthetic order alcohol-methanesulfonic acid-azide-amine, from 5-cholestene-3 β-alcohol or the 5 α-cholestane-3 β-alcohol preparation that is purchased.The spatial chemistry of final amine depends on the spatial chemistry of initial alcohol: 3 β-alcohol can provide 3 α-amine, and vice versa.5-cholestene-3 α-alcohol or 5 α-cholestane-3 α-alcohol can be by experimental program (Boonyarattanakalin, J.Am.Chem.Soc.126 (2004), 16379-16386) acquisitions of document.Alternatively, these amine can prepare by the reduction amination strategy: for example, replace amine by adopting alkylamine or dialkylamine as reactant, perhaps by adopting azanol as reactant and subsequently with drawing Buddhist nun's nickel (Raney-Nickel) to handle primary amine.Amine can obtain with the form of free alkali, or obtains with the form of corresponding hydrochloride, and this hydrochloride is by coming sedimentary with the HCl that is dissolved in the diethyl ether.Adopt epoxidation and subsequently by come open loop (ring-opening) or the bishydroxy strategy by the osmium mediation with hydride, can be from the 4-cholestene-3-ketone or 1 of commercially available acquisition, the 4-cholestadien-3-one prepares the derivant of corresponding hydroxyl modified.As Oldenziel, J.Org.Chem.42 (1977) is described in the 3114-3118, by (tosylmethylisocyanide TosMIC) handles ketone, obtains the aminomethyl derivant with tosyl methyl isonitrile.Description (Drefahl, Chem.Ber.97 (1964), 2011-2013 according to various document experiment scheme records; Kargiozov, Synth.Commun.34 (2004), 871-888; Shen, Bioorg.Med.Chem.Lett.15 (2005) 4564-4569), adopts with Horner-Wadsworth-Emmons (HWE) method of cyanogen methyl phosphonate as reactant, from corresponding ketone, can prepare the cholesterol derivative that aminoethyl is modified.Carry out the hydrogenization of two keys that form subsequently, carry out the reduction reaction of the hydride mediation of nitrile afterwards again, thereby obtain the cholesterol derivative that aminoethyl replaces.

If amino-contained group is connected to the C2 position of cholestane support, then can be with corresponding ketone as synthetic precursor, described corresponding ketone can by the preparation of the known step of various optional documents (Barillier, Tetrahedron 50 (1994), 5413-5424; Penz, Monatshefte fuer Chemie112 (1981), 1045-1054; Lightner, Steroids 35 (1980), 189-207; Nakai, Tetrahedron Lett. (1979), 531-534).Introducing amino-contained group subsequently can realize by the general policies of above-mentioned 3 cholestanones.Same principle can be used for as substrate the 4-cholestanone (can be from document Nakai, Tetrahedron Lett. (1979), 531-534; Sondheimer, J.Org.Chem.26 (1961), 630-631; Shoppee, J.Chem.Soc. (1959), description among the 630-636 obtains), perhaps being applied to the 1-cholestanone (can be from document Shoppee, J.Chem.Soc.C (1968), description among the 245-249 obtains) have the C4 that is connected to the cholesterol support or the corresponding derivative that contains amine functional group of C1 so that provide respectively.

The chemical compound of general formula 1, wherein X is chemical bond and has primary amine functional group on 1,2 or 4, can be from corresponding allyl amine preparation.Alternatively, the epoxidation of two keys and open loop subsequently or bishydroxy interaction energy produce the chemical compound of described hydroxyl modified.Described allyl amine can be from 2 α, 3 α-epoxy-5 α-cholestane by coming epoxy moieties is carried out open loop with benzyl amine, carry out the debenzylation preparation afterwards, perhaps by handling, handle with azide afterwards and carry out the lithium aluminium hydride reduction prepared in reaction subsequently with mesyl chloride.2 α, 3 α-epoxy-5 α-cholestane, for example, can obtain by the epoxidation of 5 α-cholestane-2-alkene being carried out metachloroperbenzoic acid (meta-chloroperbenzoic acid) mediation, described 5 α-cholestane-2-alkene can be according to the described preparation of document (Cruz Silva, Tetrahedron 61 (2005), 3065-3073).

The chemical compound of general formula 1, wherein X is the O-alkyl phosphate, can adopt document known phosphoramidite method (Beaucage, S.L.; J.Org.Chem.2007,72 (3), 805-815; Noyori, R.; J.Am Chem.Soc.2001,723 (34), 8165-8176; Hayakawa, Y.; Bull.Chem.Soc.Jpn.2001,74 (9), 1547-1565; Noyori, R.; Tetrahedron Lett.1986,27 (35), 4191-4194; Reviews:Bannwarth W.; HeIv.Chim.Acta 1987,70,175-186 and Beaucage, S.L.; Tetrahedron 1993,49 (10), 1925-1963), and by corresponding cholesterol or beta-cholestanol (can obtain) preparation according to said method.Compare with the conventional measures of using phosphoryl chloride phosphorus oxychloride, the phosphoramidite method is more effective and more reliable result is provided.

The chemical compound of general formula 1, wherein X is phosphate ester or O-alkyl phosphate, can, adopt document (Rejman, D.; Nucleosides Nucleotides Nucleic Acids 2001,20,1497-1522 and Holy, A.; J.Med.Chem.2001,44, the 4462-4467) strategy described in is from corresponding cholesterol or beta-cholestanol (can obtain according to above-mentioned method) preparation.Can adopt C _1-4Alkyl halide according to standard test scheme well known by persons skilled in the art, makes the phosphate ester O-alkylation of gained.

The chemical compound of general formula 2 can be according to the general introduction preparation of document.For example, Shoppee etc. have described the synthetic method of preparation 3-and 4-azepine-A-cholesterol derivative (3-and 4-aza-A-cholesteryl derivative), this method is ring expansion (Shoppee, the CW. that adopts Beckmann rearrangement (Beckmann rearrangements) to follow nitrogen-atoms to mix; J.Chem.Soc.1962,105).Alternatively, show that cholesterol derivative expansion, nitrogenous A ring can adopt Schmidt's type to reset (Schmidt-type rearrangements) and produce (Doorenbos, N.J.; J.Org.Chem.1961,26,2548-2549).Prepare 4-azepine-cholesterol derivative and do not expand the synthetic method that steroid A encircles, utilize oxidisability A ring open loop oxidation (oxidative A-ring opening, Bo ú cza-Tomaszewski, Z.; Tetrahedron Lett.1986,27,3767-3770) obtain corresponding seco-cholesterol derivative (seco-cholesteryl derivatives) and in the environment that nitrogenous source exists, carry out ring-closure reaction (Doorenbos, N.J. subsequently; J.Org.Chem.1961,26, dual strategy 2546-2548), the lactam derivatives with gained is reduced to corresponding amine (Shoppee, C.W. afterwards; J.Chem.Soc.1962,2275-2285 and Kim, J.C.; Bull.Korean Chem.Soc.1993,14 (2), 176).For example, can be by Favorskii type ring contraction (Favorski-type ring contraction) (Edwards, the O.E. of corresponding 4-azepine-steroid; Can.J.Chem.1997,75 (6), 857-872), perhaps by utilizing seco-cholesterol derivative mentioned above amination-cyclic condensation ring formation order (amination-cyclocondensation sequence) (Chupina, L.N. as substrate; Khimiko-Farmatsevticheskii Zhurnal1982,16 (5), 563-567 and Rulin, V.A.; Zhumal Organicheskoi Khimii 1975,11 (8), 1763-1766), obtain A-just-azasteroid.

Chemical compound provided herein can be used for the treatment of infectious disease or the disease that (and prevention and/or improvement) resembles viral disease or bacterial infection and so on.In accordingly based on the disease of cell or disease model, assessed chemical compound provided herein.

Viral disease to be treated comprises by viral-induced disease according to the present invention, described virus is selected from influenza virus, HIV, hepatitis virus (first, second, third, fourth), rotavirus, respiratory syncytial virus, herpetoviridae (Herpetoviridae, for example, herpes simplex virus (Herpes simplexvirus), epstein-Barr virus), echovirus 1, Measles virus, Picornaviridae (Picornavirida, for example, enterovirus (Enterovirus), Coxsackie virus (Coxsackievirus)), filamentous virus section (for example, Ebola virus, Marburg virus), Papillomaviridae, polyoma virus section.In preferred embodiments, described virus is influenza virus.

The bacterial infection to be treated according to the present invention comprises, the especially infection of being brought out by Gram-positive bacillus, gram-positive cocci, gram negative bacilli and Gram-negative coccus.Gram-positive bacillus is, for example, the kind (Nocardia spp.) of the kind of fusobacterium (Clostridium spp.), Bacillus anthracis (Bacillusanthracis), bacillus rhusiopathiae suis (Erysipelothrix rhusiopathiae), Listeria monoeytogenes (Listeria monocytogenes), Nocardia, diphtheria corynebacterium (Corynebacterium diphtheriae) and Propionibacterium (Propionibacteriumacnes).Gram-positive cocci is, for example, and the kind (Streptococcus spp.) of staphylococcus aureus (Staphylococcus aureus) and Streptococcus.Gram negative bacilli is, for example, escherichia coli, helicobacter pylori (Heliobacterpylori), the kind of Brucella (Brucella spp.), Aeromonas hydrophila, the kind of shigella (Shigella spp.), the kind of vibrio (Vibriospp.), Yersinia pestis (Yersinia pestis), the kind of Salmonella (Salmonellaspp.), Klebsiella Pneumoniae (Klebsiella pneumoniae), Burkholderia cepacia (Burkhoideria cepacia), the kind of Enterobacter (Enterobacter spp.), bacillus pyocyaneus (Pseudomonas aeruginosa), campylobacter jejuni and bacillus legionnaires,pneumophila (Legionellapneumophila).Gram-negative coccus is, for example, and Diplococcus gonorrhoeae (Neisseriagonorrhoeae) and moraxelle catarrhalis (Moraxella catarrhalis).

The bacterial infection to be treated according to the present invention also comprises the infection of being brought out by inapplicable Gram-stained clinical bacteria.These antibacterials are, for example, the kind of Borrelia (Borrelia spp.), trench fever Bartonella (Bartonella Quintana), Chlamydia pneumoniae (Chlamydiapneumoniae), mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium leprae (Mycobacteriumleprae), mycobacterium buruli (Mycobacterium ulcerans), mycobacterium kansasii (Mycobacterium kanasasii), Mycobacterium avium (Mycobacterium avium), mycobacterium paratuberculosis (Mycobacterium paratuberculosis), lymphoid tuberculosis mycobacteria (Mycobacterium scrofulaceam), the kind (Treponema spp.) of kind of Dermacentroxenus (Rickettsia spp.) and treponema.

In environment of the present invention, " disease that mycobacteria is brought out " can comprise: by infecting condition/disease, particularly mycobacterium tuberculosis that illustrate and/or relevant with infection, Mycobacterium bovis, Mycobacterium avium, mycobacterium africanum (M.africanum), mycobacterium kansasii, Mycobacterium intracellulare (M.intracellular), mycobacterium buruli, mycobacterium paratuberculosis, mycobacterium habana (M.simiae), the lymphoid tuberculosis mycobacteria, Chu Er covers mycobacteria (M.szulgai), mycobacterium xenopi (M.xenopi), mycobacterium fortuitum (M.fortuitum), Mycobacterium chelonei (M.chelonei), the infection that Mycobacterium leprae and Mycobacterium marinum (M.marinum) cause.Additional example provides chemical compound disclosed herein treating and/or preventing the data that pulmonary tuberculosis is an invention purposes in the infection that causes of mycobacterium tuberculosis especially.In " to be treated and/or prevention infection " environment, mentioned, the present invention (for example is not limited to pathogen media itself (pathogen agent), antibacterial) caused treatment of conditions/prevention, but also the medical science as the caused disease of product of for example toxin that comprises that described pathogen produces is improved.

The disease that the mycobacteria of waiting according to the present invention to treat is brought out comprises, pulmonary tuberculosis particularly, leprosy (leprosy), torrid zone skin ulcer (tropical skin ulcer), ulcer (ulceration), abscess (abscess), pneumonopathy (pulmonary disease), granuloma (skin) disease (granulomatous (skin) disease), opportunistic infection that non-tuberculous mycobacteria causes (opportunistic infection) and the disease that causes by anonymous mycobacteria such as Mycobacterium avium, this disease comprises pneumonopathy, lymphadenitis (lymphadenitis), the skin of being suffered from such as the immunocompromised patient and disseminate the type disease.Purposes is not limited to the disease that people's mycobacteria is brought out, but comprises that also the present invention is as the purposes in the Animal diseases of bovine tuberculosis and so on.In preferred embodiments, the disease that mycobacteria is brought out is a pulmonary tuberculosis, also provides documentation for it at additional example.

Under the situation of bound by theory not, this described chemical compound be particularly conducive to treatment, prevention and/or improvement by betide on the Lipid Rafts, among or within infectious disease/disease that biochemistry/biophysics's pathological process is caused.This paper has provided the respective instance of this class disease/disease and this class biochemistry/biophysics's pathological process.Therefore, term betides on the Lipid Rafts, among or within biochemistry/biophysics's pathological process mean, for example, when virus or bacterial infection, the unusual raft that pathogen is brought out troop (pathogen-induced abnormal raft clustering); When pathogenic infection, antibacterial (toxin) forms low poly structure in Lipid Rafts; Perhaps virus or antibacterial cause the increased activity of signaling molecule in Lipid Rafts.And, according to the present invention, think than the normal packing of Lipid Rafts/Lipid Rafts component more closely process be " biochemistry/biophysical pathological process ".

Additional example proves that when using the cholesterol amine material that defines and describe herein, regulating the Lipid Rafts assembling can influence biology and/or the biochemistry relevant with infectious disease and disease.Without being limited by theory, think that concrete cholesterol amine disclosed herein can disturb the distribution of regulating molecule in the Lipid Rafts.Therefore, formation and/or the trooping of Lipid Rafts that can modify the protein complexes of Lipid Rafts may be changed, and disturb or or even the state that wards off disease.This paper provides specific molecular, Cholesterylamines promptly defined above, it is believed to disturb biological process, particularly betide among the Lipid Rafts of the preferred ill cell of cell, on or within pathological process.Can think that these molecules are " raft regulon ".

Except the activity with the inhibition disease of Cholesterylamines is used for the environment of the present invention, also in toxicity detects, described chemical compound is assessed.Toxicity detects to be known in this area, particularly including lactic acid dehydrogenase (dehydrogenase, LDH) or adenylate kinase (adenylate kinase AK) detects or apoptosis detects.Yet as is known to the person skilled in the art, these (cell) toxicity detect and are not limited to these detections, and this point is well known to those skilled in the art.

According to data provided herein and information, the present invention provides chemical compound shown in Chemical formula 1 a-1l and 2a and the 2b being used for the treatment of by virus or disease that bacterial infection caused and the purposes in the medical environment aspect the disease (medical setting) especially.Yet in this paper environment, what especially pay close attention to is influenza infection and pulmonary tuberculosis.

The more details of diseases related and disease have hereinafter been provided.These diseases and disease can be by using Cholesterylamines prevention provided herein, improving or treatment.Particularly, experimental data provided herein proves that chemical compound 1a, 1b, 1f, 1g, 1h, 1i, 1j, 1k, 2a and 2b are particularly preferred chemical compounds in different medical intervention or prevention.

Cholesterylamines of the present invention can be used for 1) formation of adjusting raft and disturb hemagglutinin and neuraminidase to the transhipment of cell surface, 2) prevent trooping of the raft that contains spike glycoprotein that M albumen is brought out, and therefore viral interference assembling, perhaps 3) by increasing the size/volume of Lipid Rafts, perhaps 4) prevent the division (pinch off) of germ pore, it betides the phase boundary place of raft (film of virus) and non--raft (plasma membrane).

In this, preferred chemical compound is chemical compound 1a, 1b, 1g, 1h, 1i, 1j, 1k, 2a and 2b, and chemical compound 1a, 1i, 1k and 2b have represented in the environment of the present invention even preferred embodiment.Corresponding experimental data is provided in the additional example.

In viral infection, trooping of raft is relevant with viral assembling process.Chemical compound 1a-1l and 2a and 2b work in virus replication detects.Under situation without being limited by theory, be considered to comprise the combination representative of cholesterol type side chain existence as the architectural feature on this effect basis by the intra-annular amine replacement of steroid A and cholesterol type B, C, D ring.Adopt 3-aminomethyl or 3-aminoethyl in the A ring to replace the effect increase that causes chemical compound 1i or 1k, thereby explanation 3-aminomethyl or 3-aminoethyl substitute mode are preferred embodiment.The chemical compound that other modifications of tool hydroxy functional group can provide solubility to increase in the A ring, thereby increased bioavailability.As in the test of the virus replication of experimental section, obtain the result proved, these chemical compounds can be used for pharmaceutical intervention.On the contrary, trans-2-aminomethyl-1-Hexalin, cholesterol sulfate and cholesterol do not show significant activity in the model of influenza infection detects.

Because the viral releasing mechanism of HIV-1 and influenza virus is similar, about the participation of raft, above-claimed cpd also can be used for treating HIV-1 to be infected and the particularly medical supervision of AIDS of HIV-1 relevant disease.

Other viral diseases (as limiting examples) that available above-claimed cpd or derivatives thereof is handled are herpes, Ebola virus, enterovirus, Coxsackie virus, hepatitis C, rotavirus and respiratory syncytial virus.Therefore, under specific (virus) detection or checking system environment, particularly preferred chemical compound provided herein and preferred chemical compound also are believed to be used for other infectious diseases especially medical intervention and/or the prevention of viral infection.

Just as noted above, described herein chemical compound also can be applicable to treat the poisoning that bacterial infection or excretory bacteriotoxin are brought out.

Bacteriotoxin as cholera, aerolysin, anthrax and Helicobacterium (Helicobacter) toxin, forms the oligomerization structure in raft, this is very crucial to its function.Combine the targeting raft by ganglioside with raft lipid such as cholera.Under the situation of bound by theory not, in environment of the present invention, prevent from that oligomerization is equal to prevent that raft from trooping, therefore, be used for the same or analogous chemical compound of viral infection should be able to the bacteriotoxic activity of inhibitor.Those skilled in the art, particularly attending doctor at treatment bacterial infection itself and/or in disease and lysis that improvement is caused by corresponding toxin, can be easy to adopt the therapeutic scheme of the Cholesterylamines that uses this paper definition.

In bacterial infection such as pulmonary tuberculosis, shigellosis and the infection that caused by chlamydia and urethra pathogenic bacterium (uropathogenic bacteria), organism is ingested in often relevant with caveolae (caveolae) raft dependency internalization process and enters in the cell.Prevent location or the blocking-up internalization of bacterial receptor in raft, but prevention infection.Under the situation of caveolae, its depend on cholesterol conjugated protein be caveolin (caveolin), with steroid derivatives cholesterol is got rid of from raft, can prevent the picked-up of pathogenic bacterium.

Pulmonary tuberculosis is the example of the bacterial infection disease relevant with raft.At first, 3 type complement receptors (Complement receptor type 3, CR3) be the receptor that can make granule (C3bi-coated particles) internalization of zymosan and C3bi bag quilt, and in people's neutrophil cell, be responsible for the non-opsonin phagocytosis (nonopsonicphagocytosis) of mycobacterium kansasii (Mycobacterium kansasii).In these cells, have been found that CR3 is relevant with the several GPI anchorins that are positioned at the plasma membrane Lipid Rafts, the cholesterol loss suppresses the phagocytosis of mycobacterium kansasii significantly, and does not influence the phagocytosis of the mycobacterium kansasii of zymosan or serum conditioning.When with the GPI protein binding, CR3 reorientates in the domain of the enrichment cholesterol that makes the mycobacterium kansasii internalization.As CR3 during not with the GPI protein binding, it is present in outside these domains, and the particulate phagocytosis that mediates zymosan and handle through opsonin, but do not mediate isopentenylpyrophosphate (the isopentenyl pyrophosphate of mycobacterium kansasii, IPP) phagocytosis, described IPP are the antigen of mycobacterium of differential stimulus V γ 9V δ 2T cell.Therefore, the present invention also provides chemical compound disclosed herein in treatment and/or improve the particularly purposes in the m tuberculosis infection of mycobacteria (Mycobacterium).

Chemical compound as herein described can be used with chemical compound itself as pharmacophore or pharmaceutical composition the time, perhaps is mixed with medicine.One of comprise among the formula 1 or 2 formula 1a-1l particularly defined above and the chemical compound of 2a and 2b as the pharmaceutical composition of active component, be located within the scope of the present invention.Pharmaceutical composition can randomly comprise pharmaceutically acceptable excipient, as carrier, diluent, filler, disintegrating agent, lubricant, binding agent, coloring agent, dyestuff, stabilizing agent, antiseptic or antioxidant.

Pharmaceutical composition can adopt technology well known by persons skilled in the art preparation, as the technology of announcing among Remington ' the sPharmaceutical Sciences (Lei Mingdun materia medica) the 20th edition.The dosage form that pharmaceutical composition can be configured to is Orally administered, gastrointestinal tract is used outward, described gastrointestinal tract are used outward as intramuscular, intravenous, subcutaneous, Intradermal, intra-arterial, rectum, nose, surface, aerosol or vaginal application.Orally administered dosage form comprises, bag by or do not wrap tablet, soft capsule (soft gelatincapsules), hard capsule (hard gelatin capsules), lozenge (lozenges), tablet (troches), solution, Emulsion, suspension, syrup, elixir (elixirs), the powder that is used to reformulate and granule, dispersible powder or granule, medicine chewing gum (medicated gums), chewable tablet (chewingtablets) and the effervescent tablet (effervescent tablets) of quilt.Be used for the dosage form that gastrointestinal tract uses outward and comprise solution, Emulsion, suspension, dispersant and powder that is used to reformulate and granule.Emulsion is the preferred dosage form that gastrointestinal tract is used outward.The dosage form of rectum and vaginal application comprises suppository (suppositories) and bolt (ovula).The dosage form of intranasal administration can for example be passed through dosage inhaler (metered inhaler) through sucking and spray application.The dosage form of surface applied comprises, Emulsion, gel, ointment (ointments), ointment (salves), patch (patches) and stride the skin delivery system.

The pharmaceutically acceptable salt that can be used for chemical compound of the present invention can be formed by various organic and inorganic bronsted lowry acids and bases bronsted lowries.Exemplary base addition salts for example comprises the alkaline metal salt such as sodium salt or potassium salt; Alkali salt is as calcium salt or magnesium salt; Ammonium salt; Fatty amine salt is as trimethylamine, triethylamine, hexanamine, ethanolamine, diethanolamine, triethanolamine, procaine salt, flunixin meglumine salt, diethanolamine salt or ethylenediamine salt; Aralkyl amine salt (aralkyl amine salts), as N, the N-dibenzyl ethylenediamine salt; The heteroaromatic amine salt is as pyridiniujm, methyl pyrrole salt, quinolinium or isoquinolin salt; Four ammonium salts are as tetramethyl ammonium, tetraethyl ammonium salt, phenyltrimethyammonium salt, phenyl triethyl ammonium salt, phenyl tributyl ammonium salt, methyl trioctylphosphine ammonium salt or 4-butyl ammonium; And alkaline amino acid salt, as arginine or lysinate.Exemplary acid-addition salts comprises acetate, adipate, alginate, Ascorbate, benzoate, benzene sulfonate, disulfate, borate, bromide, butyrate, chloride, citrate, carbonate, camphorate (camphorate), camsilate, cyclopentane propionate (cyclopentanepropionate), digluconate (digluconate), lauryl sulfate, ethane sulfonate, fumarate, glucose enanthate (glucoheptanoate), glycerophosphate, Hemisulphate (hemisulfate), enanthate, caproate, hydrochlorate, hydrobromate, hydriodate (hydroiodide), 2-hydroxyl ethanedisulphonate (2-hydroxyethanesulfonate), lactate, maleate, methane sulfonates (methanesulfonate), 2-naphthalene sulfonate salt (2-naphthalenesulfonate), nicotinate, nitrate, oxalates, pectate (pectinate), high-sulfate, the 3-benzene sulfonate, phosphate, hydrophosphate, dihydric phosphate, picrate, pivalate, propionate, Salicylate, sulfate, sulfonate (sulfonate), tartrate, rhodanate, toluene fulfonate such as p-methyl benzenesulfonic acid (tosylate), undecylate etc., and amino acid salts.

Can be used for chemical compound of the present invention pharmaceutically acceptable solvate can with the solvate of water for example the form of hydrate exist, or exist with the form of the solvate of organic solvent such as methanol, ethanol or acetonitrile, promptly exist as methoxide (methanolate), ethylate (ethanolate) or acetonitrile salt (acetonitrilate) respectively.

The pharmaceutically acceptable prodrug that can be used for chemical compound of the present invention is such derivant, and promptly this derivant has chemically or the group that can cut in the metabolism and become the chemical compound of the present invention that has pharmaceutical active in the body by solvolysis or under physiological condition.The prodrug that can be used for chemical compound of the present invention, can with the compound functions group as with amino or hydroxyl as carbaminate or ester, form in the mode of routine.The form of prodrug derivant, in the mammal organism, the advantage that dissolubility, histocompatibility often is provided or delays to discharge (consult Bundgaard, H., Design ofProdrugs, pp.7-9,21-24, Elsevier, Amsterdam 1985).

Pharmaceutical composition described herein can be applied to individuality with appropriate dosage.Dosage is determined by attending doctor and clinical factor.As known in medical domain, the dosage that is used for arbitrary patient, depend on many factors, comprise patient's size, body surface area, age, particular compound, sex, the time of using and approach, general health situation to be administered and the other drug of using simultaneously.Usually, as the therapeutic scheme that the pharmaceutical composition routine is used, should be μ g to 15000mg unit every day 0.1.If therapeutic scheme is a continuous infusion, then it can be respectively per minute, every kg body weight 0.1ng to 10 μ g.Can monitor progress by periodical evaluation.

Purposes as herein described, i.e. the purposes of Cholesterylamines treatment, improvement and/or infection prevention disease has medical science and medicinal benefit.

Therefore, the present invention also relates to treat the method for the individuality that needs this type of therapeutic treatment, described method comprises, promptly is enough to improve or cure the amount that medical conditions state that described individuality suffers from particularly resists infectious disease to be enough to illustrate drug effect, uses the Cholesterylamines that this paper limits.In preferred embodiments, individuality to be treated is the people.

Because the medical importance of described Cholesterylamines herein, the present invention also provides the method for pharmaceutical compositions, and described pharmaceutical composition comprises the chemical compound that this paper limits and the mixture of one or more pharmaceutically acceptable excipient.Corresponding excipient is above mentioned, includes but not limited to, is used for the lipid derivate that liposome forms.As noted above, if pharmaceutical composition of the present invention is used by injection or infusion, this pharmaceutical composition is Emulsion preferably.

For example, Emulsion can be by stirring 60 minutes or until even matter, made by 67w-% lipoid S100 (lipoid (Lipoid) catalog number (Cat.No.) 790), ethanol (25w-%) and glycerol (8.33w-%) down at 37 ℃.With test-compound (test compound), as 14.1mg chemical compound 1i, by at hot mixed instrument (thermomixer, Eppendorf) (the Agilent glass tubes of the Agilent teat glass in, volume: stirred 90 minutes down in 37 ℃ 1.7mL), be dissolved in the preparation of 185.9mg.This liplid emulsions is represented the pro-liposome concentrate (pro-liposomal concentrate) of test-compound., 200mg liplid emulsions in 1.04g 0.536%NaCl solution diluted, produce isotonic solution thereafter, and vortex 20 seconds.With this liposome suspension (test-compound of 11.35mg/mL) further dilution in 0.9%NaCl solution, obtain the concentration of expectation.

Following examples are illustrated the present invention.

All following chemical reactions all are in anhydrous solvent, carry out under noble gas (argon or nitrogen) condition.Utilize solvent purification system (MBraun-SPS) dry THF and diethyl ether.Employed chemical drugs and solvent are buied from commercial source.TLC:Merck TLC aluminium foil silica gel 60F ₂₅₄Flash chromatography: Merck silica gel (0.040-0.063mm).Mass spectrum: Bruker HP-Esquire LC.NMR spectrum: Bruker DRX 500 or DRX 300; The unit of δ is ppm, and the unit of J is Hz.

The preparation of embodiment 1,2 and 3:3 beta-amino-5 α-cholestane 1a, 3 alpha-amidos-5 α-cholestane 1b and 3 beta-amino gallbladder steroids-5-alkene 1f

By the initial synthetic compound 1a of dihydro epicholesterol (dihydroepicholesterol), adopt Boonyarattanakalin, J.Am.Chem.Soc.126 (2004), the L-selectride experimental program that 16379-16386 describes prepares the dihydro epicholesterol from the 3-cholestanone that is purchased.Then the dihydro epicholesterol is changed into corresponding methanesulfonic acid, (Davis, Tetrahedron Lett.38 (1997) 4305-4308), and are reduced into amine by handling with lithium aluminium hydride reduction with azide subsequently with the azide replacement afterwards.

From the initial synthetic compound 1b of the beta-cholestanol that is purchased, described beta-cholestanol is changed into corresponding methanesulfonic acid, replace (Davis with azide afterwards, Tetrahedron Lett.38 (1997), 4305-4308), and subsequently azide is reduced into amine by handling with lithium aluminium hydride reduction.

From the initial synthetic compound 1f of epicholesterol (epicholesterol), (Boonyarattanakalin, J.Am.Chem.Soc.126 (2004) 16379-16386) adopt synthetic epicholesterol of two steps from the cholesterol that is purchased according to the document experiment step.Epicholesterol is changed into corresponding methanesulfonic acid, and (Davis, Tetrahedron Lett.38 (1997) 4305-4308), and are reduced into amine by handling with lithium aluminium hydride reduction with azide subsequently with the azide replacement afterwards.

The exemplary steps that is used for synthetic compound 1a, 1b and 1f is described below.

The exemplary steps for preparing methanesulfonic acid by alcohol: gallbladder steroid-5-alkene-3 β-pure methyl-sulfonate (Cholest-5-en-3 beta-ol methane-sulfonate)

(1.32g, 3.42mmol) (42mg 0.34mmol) is dissolved under argon in the anhydrous methylene chloride (25mL) with 4-dimethylamino naphthyridine (4-dimethylamino pyridine) with epicholesterol.To wherein add while stirring diisopropylethylamine (Diisopropylethylamine) (486mg, 3.76mmol) and methane sulfonyl chloride (methanesulfonyl chloride) (431mg, 3.76mmol).After 100 minutes, the TLC that carries out in petroleum ether/dichloromethane (1: 1)+1% ethanol analyzes and shows, transforms fully.Mixture is distributed between dichloromethane and water (each 100mL).Organic layer is used in succession the NaHSO of dilution ₄, water, saturated NaHCO ₃, water and salt water washing, through Na ₂SO ₄Drying is filtered, evaporation drying, and dried overnight in a vacuum.

The gallbladder steroid of output: 1.54g (96%)-5-alkene-3 β-pure methyl-sulfonate, a kind of linen solid.This material can be used for next step, does not need to be further purified.By carrying out flash chromatography, finish being further purified with petrol ether/ethyl acetate (4: 1).

MS(ESI)：482.3(M+NH4) ⁺。

¹H-NMR(300MHz，CDCl ₃)：δ＝0.67(s，3H)，0.81-2.07(m，38H)，2.36(d，J＝15.6，1H)，2.56(d，J＝15.6，1H)，2.98(s，3H)，4.55(m，1H)，5.35(d，J＝5.1，1H)。

The exemplary steps for preparing azide by methanesulfonic acid: 3 β-azido-5 α-cholestane

(17.2g, 37.0mmol) (12.03g 185mmol) places under the argon, and is suspended in anhydrous N.N '-dimethyl allene urea (240mL) with pulverous anhydrous Hydrazoic acid,sodium salt with gallbladder steroid-5-alkene-3 β-pure methyl-sulfonate.With mixture heated to 46 ℃, effectively stirred 24 hours simultaneously.Crude product is distributed between water (1L) and 1: 1 mixture of diethyl ether/petroleum ether (500mL).The water-bearing layer is washed once more with same mixture.With the organic layer water that merges (4 * 1L) washings, through Na ₂SO ₄Drying, filtration, evaporation drying and vacuum drying.Carry out flash chromatography (carrying out stepwise elution with the petroleum ether that contains 0,5,10,15,20% dichloromethane) and produce 14.9g (97%) 3 β-azido-5 α-cholestane on Silicon stone, it is colourless solid.

MS(ESI)：414.5(M+H) ⁺。

¹H-NMR(500MHz，CDCl ₃)：δ＝0.64(s+m，4H)，0.79(s，3H)，0.81-1.61(m，34H)，1.62-1.70(m，1H)，1.72-1.86(m，3H)，1.96(d?m，J＝12.6，1H)，3.24(m，1H)。

The exemplary steps for preparing amine by azide: 3 beta-aminos-5 α-cholestane 1a

Under the noble gas argon, with lithium aluminium hydride reduction (5.0g, anhydrous diethyl ether 131.6mmol) (250mL) suspension is heated to backflow, stirs simultaneously.So that reaction keeps the speed of gentle reflux, dropwise add 3 β-azido-5 α-cholestane (14.9g, anhydrous diethyl ether 36.04mmol) (110mL) solution.After finishing interpolation, mixture was refluxed 1 hour, the TLC that carries out in petroleum ether/dichloromethane (85: 15) afterwards analyzes and shows initial substance full consumption.With the cooling of mixture ice bath, and dropwise add 40mL methanol, dropwise add the NaOH of 200mL 2M afterwards again.The mixture water that obtains (about 500mL) dilution separates organic layer and repeatedly extracts turbid water mutually with dichloromethane with diethyl ether.With organic layer water and the salt water washing that merges, through Na ₂SO ₄Drying, filtration, evaporation drying and vacuum drying spend the night.3 beta-aminos-5 α of output: 12.1g (87%)-cholestane 1a, it is a white solid.

MS(ESI)：388.3(M+H) ⁺。

¹H-NMR(300MHz，CDCl3)：δ＝0.63(s+m，4H)，0.76(s，3H)，0.70-1.87(m，40H)，1.94(d?m，J＝12.2，1H)，2.62(m，1H)。

The preparation of 3 alpha-amidos-5 α-cholestane 1b

According to the general introduction in mentioned above and the exemplary steps, preparation chemical compound 1b.

MS(ESI)：388.3(M+H) ⁺。

¹H-NMR(300MHz，CDCl ₃)：δ＝0.64(s，3H)，0.66-1.90(m，44H)，1.95(d?m，J＝12.1，1H)，3.16(m，1H)。

The preparation of 3 beta-amino gallbladder steroids-5-alkene 1f

Prepare chemical compound 1f according to exemplary steps above-mentioned and that list.

MS(ESI)：386.4(M+H) ⁺。

¹H-NMR(300MHz，CDCl ₃)：δ＝0.66(s，3H)，0.77-2.21(m，42H)，2.59(m，1H)，5.31(d，J＝5.2，1H)。

The preparation of embodiment 4 and 5:3 β-methylamino-5 α-cholestane 1c and 3 β-dimethylamino-5 α-cholestane 1d

According to following normal experiment scheme, the reduction amination strategy of the THF solution by adopting methylamine or dimethylamine (2M) prepares chemical compound 1c and 1d by 5 α that are purchased-cholestane-3-ketone one-step method.For convenient, obtain the pure amine of corresponding hydrochloride form.

With sodium triacetoxy borohydride (sodium triacetoxyborohydride) (1.4eq) and glacial acetic acid (1.0eq) handle 5 α-cholestane-3-ketone (1.0eq) and amine (1.0eq, as the THF solution of commercially available 2M) anhydrous 1, the 2-dichloroethane solution.With the mixture that obtains at room temperature, stirred 18 hours in the argon, up to as the TLC analysis measure reactant depletion.By in reactant mixture, adding the water cessation reaction, and use the diethyl ether extraction product.With organic layer salt water washing, and through MgSO ₄Dry.With solvent evaporation, thereby obtain crude product (being unhindered amina).By in the diethyl ether solution of amine, adding the diethyl ether solution of 1M HCl, prepare hydrochlorate, and wash with diethyl ether subsequently.This experimental program has obtained the hydrochlorate white crystal of chemical compound 1c and 1d, and productive rate is respectively 60% to 70%.

3 β-methylamino-5 α-cholestane 1c:

MS(ESI)：m/z＝402.5(M ⁺)。

¹H-NMR(300MHz，CDCl ₃)：δ＝0.62(s，3H) ₁0.79(s，3H)，0.84-0.88(m，12H)，0.96-1.32(m，18H)，1.45-1.80(m，12H)，1.90(br?d，J＝10.4Hz，1H)，1.97(br?d，J＝15.5Hz，1H)，2.64(br?s，3H)，3.20(br?s，1H)，9.30(br?s，1H)。

3 β-dimethylamino-5 α-cholestane 1d

MS(ESI)：m/z＝416.4(M ⁺)。

¹H-NMR(300MHz，CDCI ₃)：δ＝0.61(d，J＝5.5Hz，3H)，0.81(m，10H)，0.97-1.31(m，18H)，1.42-2.02(m，15H)，2.73(br?s，3H)，2.82(br?s，3H)，3.03(br?m，1H)，11.33(br?s，1H)。

The preparation of embodiment 6:3 β-trimethyl ammonium-5 α-cholestane base chloride 1e

Anhydrous methylene chloride suspension heating with 3 β-methylamino-5 α-cholestane 1c (1.0eq) and sodium hydride (8.0eq, its content are 60% mineral oil suspension) refluxed 30 minutes.To wherein adding neat methyl iodide (20eq), and reactant mixture is heated to 45 ℃ kept 2 days.Dilute with the reactant mixture cooling and with dichloromethane (50mL) then, reuse saline is washed several times repeatedly, and extracts (3 * 100mL) with dichloromethane.With the organic layer process dried over sodium sulfate that merges, and concentrate in a vacuum.Wash 3 β-trimethyl ammonium-5 α-cholestane base chlorine 1e that crude product has obtained corresponding chloride form with diethyl ether, it is a white crystal.

MS(ESI)：m/z＝430.5(M ⁺)。

¹H-NMR(300MHz，CDCl ₃)：δ＝0.67(m，3H)，0.88(m，12H)，1.13-2.01(m，31H)，3.17(br?s，6H) ₁3.36(br?s，1H)，3.72(br?s，1H)，4.48(br?s，2H)。

Embodiment 7:2 beta-amino-3 Alpha-hydroxy-5 α-cholestane 1g

From 2 α, 3 α-epoxy cholestane prepares chemical compound 1g, described 2 α, 3 α-epoxy cholestane can obtain by known steps, this known steps comprises that making beta-cholestanol carry out dehydration generates the 2-cholestene, (Cruz Silva, Tetrahedron 61 (2005), 3065-3073) to carry out epoxidation subsequently again.

As hereinafter summarizing, carry out the epoxide ring-opening reaction with benzylamine, and carry out debenzylation acquisition amino alcohol 1g with hydrogen and palladium carbon.

The preparation of 2 β-(N-benzylamine)-3 Alpha-hydroxy-5 α-cholestane

Under argon, with 2 α, (217mg 0.56mmol) is suspended in the benzylamine (3ml) 3 α-epoxy cholestane.Mixture was stirred 3 days down at 120 ℃.Then, the TLC that carries out in petrol ether/ethyl acetate (10: 1) the analysis showed that initial substance has consumed about 90%.Under vacuum, remove most of benzylamine.Residue is dissolved in the THF/ isopropyl alcohol (2: 1), with trifluoroacetic acid (TFA) acidify, and with preparing HPLC (preparative HPLC) (chromatographic column: Vydac 208TP 1050 (RP-C8), detector: UV (215nm), flow rate: 50mL/ minute, eluent A: water/acetonitrile (85: 15)+0.1%TFA, eluent B:THF+0.1%TFA, with the eluent B gradient elution of concentration from 38% to 59% 21 minutes, RT=19.3 minute) purification.Collection contains the part of product.Remove most of THF under the reduced pressure atmosphere.With saturated moisture Na ₂CO ₃The alkalization rest solution, and use dichloromethane extraction.With organic layer Na ₂SO ₄Drying, filtration, evaporation drying and dry in a vacuum.Carry out chromatography by going up with petrol ether/ethyl acetate (4: 1)+2% ethanol, be further purified product at Alox-N (activity 4, activity 4).2 β of output: 27mg (10%)-(N-benzylamine)-3 Alpha-hydroxy-5 α-cholestane, it is a colorless oil.

MS(ESI)：494.4(M+H) ⁺。

¹H-NMR(500MHz，CDCl ₃)：δ＝0.63(s，1H)，0.65-0.75(m，1H)，0.80-1.40(m，32H)1.45-1.70(m，8H)，1.71-1.91(m，3H)，1.95(d?m，J＝12.6，1H)，2.84(d?m，J＝4.6，1H)，3.75(d，J＝13.2，1H)，3.83(d?m，J＝4.2，1H)，3.87(d，J＝13.4，1H)，7.25(m，1H)，7.32(m，4H)。

The preparation of the 2 beta-aminos-3 Alpha-hydroxy-5 α-cholestane 1g of hydrochloride form

(25mg, 50.6 μ mol) are dissolved in the dichloromethane (3mL) with 2 β-(N-benzylamine)-3 Alpha-hydroxy-5 α-cholestane, and under the situation that the palladium carbon (10%) of 20mg exists, carry out hydrogenation under the normal pressure (atmosphericpressure), spend the night.The mass spectral analysis demonstration of crude mixture does not transform.Add the 50mg catalyst and the 3mL methylene chloride (4: 1) of another part, carry out 24 hours hydrogenation again.Then, utilize petrol ether/ethyl acetate (4: 1)+10% TLC that dichloromethane+3% methanol carries out to analyze and show, transform fully.Filter by the short liner of Celite (celite 577) (short pad), remove catalyst.Use methylene chloride (70: 30) (300mL) to wash, restore fully to guarantee material.Go down to desolventize in reduced pressure atmosphere, and carry out in a vacuum obtaining 18.4mg (90%) colorless solid after the drying, this colorless solid is dissolved in diethyl ether (4.5mL) and several isopropyl alcohols.Add the diethyl ether solution (60 μ L) of the HCl of 1M, with diethyl ether dilution (4mL), and cooling causes the hydrochlorate precipitation, collects this hydrochlorate precipitate with membrane filtration, washes and dry in a vacuum with diethyl ether.The hydrochlorate of 2 beta-aminos-3 Alpha-hydroxy-5 α-cholestane of output: 17mg (77%), it is a white solid.

MS(ESI)：404.4(M+H) ⁺。

¹H-NMR(500MHz，DMSO-d ₆)：δ＝0.62(s，3H)，0.62-0.69(m，1H)，0.76-1.80(m，39H)，1.92(d?m，J＝12.3，1H)，3.14(m，1H，H-2)，3.72(m，1H，H-3)，5.17(d，J＝3.7，1H，OH)，8.04(br?s，3H ₁NH ₃ ⁺)。

¹H-NMR(500MHz，CDCl ₃/CD ₃OD?8∶2)：δ＝0.53(s，3H)，0.64(m，1H)，0.69-1.60(m，40H)，1.65-1.78(m，3H)，1.85(d?m，J＝12.6，1H)，3.15(d?m，J＝6.1，1H)，3.84(br?s，OH，NH ₂，H-3)。

Embodiment 8 and 9:3 β-aminomethyl-5 α-cholestane 1h and 3 α-aminomethyl-5 α-cholestane 1i

According to document (literature precedence) (Oldenziel formerly, J.Org.Chem.42 (1977), 3114-3118), by handling with the tosyl methyl isonitrile (TosMIC) that is purchased, 5 α-cholestane-3-the ketone that is purchased is changed into i.e. 3 α-and the 3 beta-cyano cholestane (3 α-and 3 β-cyano cholestane) of stereomeric mixture of nitriles, preparation chemical compound 1h and 1i.Cross subsequently stereoisomer is carried out chromatography, and isolating nitrile is carried out the lithium aluminium hydride reduction reaction, obtain chemical compound 1h and 1i.

By utilizing petrol ether/ethyl acetate (10: 1) or petroleum ether/dichloromethane (2: 1) on Silicon stone, to carry out flash chromatography as eluent, perhaps by utilizing water as eluent A, on anti-phase C8 post, surpass 40 minutes preparation HPLC as the gradient of eluent B and 40%-70%B with THF, realize 3 α-with the separating of 3 beta-cyano cholestane.

The preparation of 3 α-aminomethyl-5 α-cholestane 1i

Under noble gas, (1.68g 44.3mmol) is suspended among the anhydrous THF (200mL), and mixture is heated to backflow while stirring with lithium aluminium hydride reduction.In 15 minutes, dropwise by adding 3 alpha-cyanos-5 α-cholestane (7.28g, anhydrous THF (200mL) solution 18.3mmol).With mixture restir 90 minutes under reflux state, the TLC that carries out in petroleum ether/dichloromethane (1: 1) afterwards analyzes demonstration, and initial substance is full consumption.Dropwise add the entry cessation reaction.Mixture is distributed between water and diethyl ether, and diethyl ether and ethyl acetate extraction are used repeatedly in the water-bearing layer.With the organic facies process Na that merges ₂SO ₄Drying is filtered and evaporation drying.Residue is dissolved in the methylene chloride (9: 1), filters, remove inorganic matter by Celite 577 short plugs (short plug).Solvent removed and dry under vacuum, obtain 6.60g (90%) chemical compound 1i, it is a colorless solid.

MS(ESI)：402.5(M+H) ⁺。

¹H-NMR(500MHz ₁CDCl ₃/CD ₃OD?8∶2)：δ＝0.67(s，3H)，0.70-0.73(m，1H)，0.80-1.72(m，41H)，1.75-1.87(m，2H)，1.99(d?m，J＝12.5，1H)，2.08(m，1H)，3.00(m，2H)。

The preparation of 3 β of hydrochloride form-aminomethyl-5 α-cholestane 1h

Adopt the method identical, preparation chemical compound 1h with preparing above-claimed cpd 1i.Crude product is dissolved in the diethyl ether (10mL), and the diethyl ether solution (35mL) of the HCl of the logical 1M of adding precipitates the corresponding hydrochlorate of 1h.Leach formed solid, with the diethyl ether washing, and dry under fine vacuum, to obtain the 1h (0.36g, 68%) of hydrochloride form, it is a colorless solid.

MS(ESI)：402.4(M+H) ⁺。

¹H-NMR(500MHz，CDCl ₃/CD ₃OD?8∶2)：δ＝0.67(s，3H)，0.70(m，1H)，0.79(s，3H)，0.86-1.38(m，31H)，1.49-1.58(m，3H)，1.67(m，3H)，1.77-1.85(m，2H)，1.99(d?m，J＝12.6，1H)，2.77(d，J＝5.2，1H)。Obviously do not observe NH ₃ ⁺

Embodiment 10: the preparation of 3 α of hydrochloride form-aminomethyl-3 beta-hydroxy-5 α-cholestane 1j

Cyaniding 01 derivatives (Evans by corresponding O-three silicyl-protections; J.Org.Chem.39 (1974) 914-917), and handles with lithium aluminium hydride reduction subsequently; reuse HCl precipitation prepares the hydrochlorate of chemical compound 1j by 5 α that are purchased-cholestane-3-ketone afterwards.Purification cyaniding alcohol intermediate does not react and it is carried out hydro-reduction as raw material.

To chemical compound description that 1i does, the derivant by with over hydrogenation aluminum lithium reduction cyaniding alcohol prepares chemical compound 1j, and as to chemical compound description that 1h does, forms corresponding hydrochlorate as in the past.

MS(ESI)：418.4(M+H) ⁺。

¹H-NMR (300MHz, CDCl ₃): δ=0.48 (s, 3H), 0.59-1.52 (m, 42H), 1.58-1.71 (m, 1H), 1.76-1.84 (m, 1H), 2.84 (br s, 2H). obviously do not observe NH ₃ ⁺

Embodiment 11: the preparation of 3 β of hydrochloride form-aminoethyl-5 α-cholestane 1k

Utilize document (Karagiozov, S.K.; Synth.Commun.2004,34 (5), the Horner-Wadsworth-Emmons experimental program of describing 871-888), from 3-cholestanone and the diethyl cyanogen methyl phosphorodithioate (diethylcyanomethyl phosphonate) that is purchased, preparation chemical compound 1k.By using the standard hydrogenization of palladium carbon as catalyst, with the α that obtains, alpha, beta-unsaturated nitriles changes into corresponding nitrile.Subsequently, the lithium aluminium hydride reduction with in the diethyl ether is reduced into corresponding amine with nitrile.At last, utilize the diethyl ether solution of the hydrochloric acid that is purchased,, obtain the chemical compound 1k of very pure hydrochloride form by precipitation.

MS(ESI)：416.4(M+H) ⁺。

¹H-NMR (300MHz, CDCl ₃): δ=0.67 (s, 3H), 0.71-1.69 (m, 44H), 1.78-1.85 (m, 1H), 2.61 (m, 2H). obviously do not observe NH ₃ ⁺

Embodiment 12: the preparation of (5 α-cholestane base-3 β-alcohol) methyl [2-(Trimethylamine)-ethyl] phosphate ester 1l of trifluoracetic acid salt form

From the beta-cholestanol that is purchased, utilize the phosphoramidite method described in above conventional synthetic the discussion, the reagent methyl N that use is purchased, N, N ', N '-tetraisopropylphosph-ro phosphoryl diamine (methylN, N, N ', N '-tetraisopropylphosphorodiamidite), tetrazolium, choline tosilate (choline tosylate), N-phenylimidazole fluoroform sulphonate (N-phenylimidazoliumtrifluoromethanesulfonate) and tert-butyl peroxide (terf-butylperoxide), one kettle way (one-pot procedure) preparation chemical compound 1l.Then, by the preparation HPLC with the crude product purification, thereby separate corresponding trifluoroacetate.

MS(ESI)：568.4M ⁺。

¹H-NMR(300MHz，CDCl ₃)：δ＝0.63(s，3H)，0.63(m，1H)，0.70-2.00(m，42H)，3.22(s，9H)，3.71(d，J＝11.3，3H)，3.76(m，2H)，4.23(m，1H)，4.39(m，2H)。

Embodiment 13: the 3-azepine-A-class-5 α-cholestane of the hydrochloride form (3-Aza-A-homo-5 α-cholestane) preparation of 2a

People (Doorenbos, N.J. such as Doorenbos take place by corresponding oxime; J.Org.Chem.1961,26,2548-2549) described Beckman type rearrangement reaction (Beckmann-typerearrangement reaction), by commercial cholestane base-3-ketone of buying, preparation chemical compound 2a.

The fusing point of gained material and ¹The H-NMR spectrum ( ¹H-NMR spectrum) with the data (Takahashi, the T.T. that have announced; J.Chem.Soc.Perkin I 1980,1916-1919) unanimity.

Embodiment 14: the preparation of 4-azepine-5 α of hydrochloride form-cholestane 2b

By following order: 4, the 5-bishydroxy is reduced into the 3-hydroxyl with 3-ketone, utilizes the lead tetraacetate or the lead perchlorate that are dissolved in the methanol to come 3,4, the 5-trihydroxylic alcohol carries out oxidation scission, and the methyl ester that obtains is carried out saponification, by concentrating 4-azepine gallbladder steroid-4 with ammonia treatment under the pressure, 5-alkene-3-ketone, utilize lithium aluminium hydride reduction to 5, the two keys of 6-carry out hydrogenation and turn usefulness into and the lactams that obtains is reduced into 2b (Bo ú cza-Tomaszewski, Z.; Tetrahedron Lett.1986,27,3767-3770; Doorenbos, N.J.; J.Org.Chem.1961,26,2546-2548; Shoppee, C.W.; J.Chem.Soc.1962,2275-2285), by gallbladder steroid-4,5-alkene-3-ketone prepares chemical compound 2b.The another kind of method for preparing chemical compound 2b is by McKenna and Tulley (McKenna, J.; J.Chem.Soc.1960,945) describe.The fusing point of gains and ¹H-NMR spectrum and the data (Pradhan, the S.K. that announce; Heterocycles 1989,28 (2), 813-839) unanimity.

Embodiment 15: virus breeding and infectious detection the (transforming focus reduces detection (FocusReduction Assay))

Hereinafter, estimated the effect that the chemical compound of above confirming suppresses virus breeding and/or reduces viral infection.Use influenza as corresponding viral vector.By titration of virus assessment antivirus action, described titration of virus is equal to and traditional plaque minimizing detection (plaque reductionassay).This detection is carried out in titer plate (microtiter plate), and develop the same with cell ELISA.(MDCK) with the serial dilution preincubate of test-compound 5 minutes, the virus with serial dilution infected then for the dirty epithelial cell of Testis et Pentis Canis, Madin-Darby canine kidney cells with cell.The effect that virus breeding and infection detect (is characterized as IC ₅₀And IC ₉₀Value, the concentration when promptly 50% or 90% virus breeding is suppressed), and compare with the toxicity of the cell model that also is used for measuring effect by described evaluation the hereinafter.Not observe toxic concentration range, detection compound is to virus breeding and infective inhibition.Concentration when being suppressed by calculating 50% or 90% virus replication is carried out quantitatively.Corresponding value is used " IC respectively ₅₀" or " IC ₉₀" expression.Under this environment, " unrestraint " (for example, zero) is meant the inhibition that is not only promptly added the solvent generation of test-compound by solvent carrier." inhibition fully " or 100% inhibition are meant no transforming focus.

The transforming focus minimizing detects uses following material: the low reservation, managed (low retention tube) and glass dilution plate (glass dilution plate) (after soaking, the covering drying) in 70% ethanol; Eppendorf pipe and the 96 hole depth orifice plates (96-wellblock) of two constant temperature vortex mixers (thermomixer), 1.5mL; The orifice plate (glass-coated plate) (Zinsser or Lab Hut) of preparation test-compound diluent 96 hole glass plates (96-well glass plate) or glass-overlay film; Costar 96 orifice plates (black) of mdck cell in 1-2 days ages or the Lab Hut orifice plate of glass film are housed; The known viral aliquot of tiring; Infection culture medium (infectionmedium, IM) (buy from Celliance, catalog number (Cat.No.) is 82-046-4) of bovine serum albumin (BSA) have been added; Be stored at the insulin mother solution of-80 ℃ 2mg/mL with aliquot; PBS (phosphate buffer, dilution ratio 1: the 500) solution of 0.05% glutaraldehyde (content is 25% in water, and the Sigma catalog number (Cat.No.) is G 5882, is stored in-20 ℃), this solution is freshly prepared with the amount of every plate 250mL; Be used for the antibody that cell Rlisa develops; Pierce SuperSignal (West Dura) substrate.(MD USA) obtains mdck cell and influenza virus A hominis (strain A/PR8/34 (H1N1)) for American Type Culture Collection, Rockville from American type culture collection.

Carry out transforming focus with following operating path and reduce detection:

Step 1: the preparation of test-compound solution

The test-compound that will be stored in-20 ℃ as the mother solution of 10mM, 5mM or 3mM among the DMSO thaws under 37 ℃, and if needs are arranged, carry out ultrasonic Treatment, to obtain clear solutions.In the constant temperature vortex mixer, with IM 37 ℃ of following preheatings in low reservation pipe, and the mother solution (is that example is calculated with 10mM test-compound mother solution) that adds in the following manner, test-compound: for the test-compound mother solution of 100 μ M: the test-compound mother solution of the IM+22 μ L of 1078 μ L; Test-compound mother solution for 50 μ M: the test-compound mother solution of the IM+11 μ L of 1089 μ L; Test-compound mother solution for 25 μ M: the test-compound mother solution of the IM+5.5 μ L of 1094 μ L; Test-compound mother solution for 10 μ M: the test-compound mother solution of the IM+2.2 μ L of 1098 μ L.The test-compound solution that obtains was vibrated 30-60 minute, and forward in the 96 hole glass plates, this 96 hole glass plate is in the 37 ℃ of following preheatings in constant temperature vortex mixer microwell plate district (thermomixermicroplate block).A glass plate is used for two titer plates, and what the left-half of glass plate was accepted titer plate 1 is tried culture medium (test media), and right half part is then accepted the culture medium of being tried of titer plate 2.Test-compound solution or the control medium (referring to following template) of 250 μ L accepted in every hole.At last, with multi-channel loading rifle (multichannel pipette) diluent (every kind 100 μ L) of test-compound is transferred to the mdck cell culture plate from the glass dilution plate.

Step 2: infect

Have the 96 orifice plate edge columns test-compound solution-treated of mdck cell monolayer, and carry out puppet and infect (mock-infected); (" testing result quantitatively " a) (consulted hereinafter) in the hole as the background contrast of densitometry assessment (densitometricevaluation) for they.Fill viral dilution liquid to other 3 kinds of hole b, c and d, as 2 * 10 ^-6Focus forming unit (foci formingunits), 1 * 10 ^-6Focus forming unit or 5 * 10 ^-7Focus forming unit so that 2 * 10 ^-6The focus forming unit diluent produce 50-100 transforming focus.Suitable diluent is measured by titration of virus.The all viral dilution liquid of preparation in IM.Virus was diluted in advance (i.e. 630 μ L IM+10 μ L virus solution) with 1: 64 in IM.1: 64 viral dilution liquid is diluted to cold IM 1: 2000 (=1), carries out 22 times of dilutions subsequently again.For 1 96 orifice plate, this class solution of preparation 3mL, 1.5mL and 1.5mL for 2 96 orifice plates, prepares this class solution of 6mL, 3mL and 3mL, and these solution is stored in 4 ℃.Prepare the trypsin solution of 20 μ g/mL, and make the Millex Syringe Filters (syringe filter) of its 0.2 μ m that flows through, dilution is 4 μ g/mL in IM subsequently.In infection preceding 10 minutes, the trypsin diluent (4 μ g/mL) of equal-volume such as 3mL or 6mL is added viral decorating liquid or IM (being used for pseudo-the infection), and be stored in 4 ℃, up to infection.With 2 * 200 μ L IM, the washing cell monolayer.With 100 μ L test-compound or control solvent among the multi-channel loading rifle adding IM, so that every row (2-11) contain single test-compound diluent.If the edge effect minimum, then the 1st row and the 12nd are listed as and accept IM and as solvent-free contrast.It is capable and H is capable (rows A and H) (the pseudo-infection) with the multi-channel loading rifle 100 μ L IM and 2 μ g trypsin/mL to be added A.Add viral dilution liquid to other row, thus, all change the suction nozzle (pipette tip) of pipet at every turn.After each the adding, draw the content in hole back and forth with pipet.Flat board was hatched 16 hours at 37 ℃.By the toxicity/cellular morphology/precipitation in the pseudo-hole of infecting of microscopic evaluation.By the glutaraldehyde PBS solution chamber relaxing the bowels with purgatives of warm nature with 250mL 0.05% fix, soak/fill whole flat board, stop infecting.

Step 3: detect

Remove glutaraldehyde solution, use PBS rinsing flat board, and in PBS, permeated 30 minutes, and use the PBS rinsing once more with the Triton X-100 of 50 μ L 0.1%.Mixture (confining liquid) with the PBS+10% heat inactivation hyclone of every hole 200 μ L, the hole placed on the vibrator in sealing under the room temperature spend the night under 1 hour or 4 ℃, use antibody (MAb storehouse (pool) 5 of the virus nucleoprotein of every hole 50 μ L subsequently, US Biological I7650-04A) handled 1 hour, described antibody diluted by 1: 1000 in confining liquid.By carry out washing in three times 5 minutes with TBS (Tris buffer salt)+0.1%Tween, remove antibody.Hatched 1 hour with the anti-mice two of 50 μ L and horseradish peroxidase is anti-in every subsequently hole, and described two resist in confining liquid and diluted by 1: 2000.At room temperature, flat board is placed vibrator, with TBS/0.1%Tween washing three times, and with the TBS washing once.

Step 4: imaging

After removing last cleaning mixture, fill 50 μ L substrate solutions (SuperSignal West Dura, Pierce 34076) to the microtitre hole, described solution is only before use by mixing isopyknic two kinds of components preparation.Subsequently flat board is placed the Fresenl mirror headstock (Fresnel lense rack) of CCD camera LAS 3000 (Fuji/Raytest), and with high-resolution exposure 10 minutes.

Step 5: testing result quantitatively

Mode evaluate image with densitometry.At first, subtracting background (hole a consults above).Following bulk density metering intensity:

I=[0.25 * i (hole b)+0.5 * i (hole c)+i (hole d)]/1.75

Wherein i is defined as 10000 times of intensity of each area of associated orifices b, the c of measurement or d.This calculating detects corresponding to the plaque of classics.Factor is represented the weighting of single value.

The % that the result is expressed as definition suppresses.

% inhibition=100-% contrast

Wherein the % contrast given I by test-compound multiply by 100 and calculates divided by the suitable I of control solvent.If I is contrast or control solvent, then its value is set to 100%.

The test-compound of a series of variable concentrations is carried out this detection by quantitative result's assessment,, guarantee that thus maximum concentration used in this series is nontoxic, as at IC as 100 μ M, 50 μ M, 25 μ M, 10 μ M, 2.5 μ M, 0.25 μ M, 0.1 μ M ₅₀/ IC ₉₀Before the assessment, utilize MDCK II cell in toxicity detects, to be assessed.The value of each concentration is the meansigma methods of three repeated experiments.Based on IC is provided ₅₀And IC ₉₀4 kinds of parameter logical functions of value utilize Sigmaplot 9.0 (Systat Software Inc.) software, handle the dose response result who obtains.

The result

Various Cholesterylamines detect in (as the cell model of influenza infection) at PR8 (H1N1) virus replication and show had strong inhibitory effects.Particularly, chemical compound 1a-1l, 2a and 2b have produced good result's (table 1).Be not subject under the theoretical situation, seeming the cholesterol support and be attached to steroid A ring (Cholesterylamines and its derivant) or the combination of the amido functional group of a steroid A ring part (azepine cholestane (azacholestanes), azepine class cholestane (azahomocholestanes) or derivatives thereof) is the structural motif that causes virus replication to suppress.On the contrary, when detecting with the concentration of 10,20 and 50 μ M, cholesterol sulfate does not suppress duplicating of virus, and when 50 μ M or higher concentration, observes toxicity.3 β-the glycolic of performance negative charge function under testing conditions, and the 3-oximido cholestane that shows the nitrogen-atoms of the A ring that is attached to the cholesterol core texture in testing environment as herein described, all do not have inhibitory action.In addition, 3-ketone-4 α, 5 α-cholestane-diol (IC ₅₀16.0 μ M) and 3 β, 4 α, 5 α-cholestantriol (IC ₅₀16.1 μ M) in the virus replication as the influenza infection disease model detects, provide faint effect.Seem that the strong polarity that is positioned at steroid A ring place is not enough to provide antiviral activity.And it is necessary that the existence of amido functional group looks like the specific high activity that obtains chemical compound.Yet trans-2-aminomethyl-1-Hexalin does not show any inhibitory action.This proof, the amino part or the amino alcohol part that are attached to ring type carbohydrate primitive are not the sole causes of anti-influenza activity.

Table 1: the virus replication result (IC of example provided herein ₅₀And IC ₉₀Value)

As this paper present data proved, chemical compound 1a-1l is the preferred compound that is used for pharmaceutical intervention in influenza infection.8 kinds of chemical compounds in these chemical compounds, promptly 1a, 1b, 1g, 1h, 1i, 1j, 1k and 2a provide good especially result in the influenza virus copy detection.These chemical compounds all show good result in dissolubility test and therapeutic index in addition.The increase that the dissolubility of Cholesterylamines in polarizable medium replaced by hydroxyl functional group along with the hydrogen atom that is attached to steroid A ring and increasing, thus the increase of dissolubility remedied the slight reduction of effect, and this can cause the increase of bioavailability.In this connection, chemical compound 1g and 1i are used for the treatment of the particularly preferred molecular entity of influenza infection of viral infection.

Embodiment 16: antibacterial activity detects

The purpose of this detection is to identify to have the active chemical compound of tuberculosis, as utilizes mycobacterium tuberculosis H ₃₇The Rv strain is assessed as phthisical disease model.As mentioned below, assess the effect (MIC in the antibiotic detection ₉₀), and the toxicity of itself and mammal Vrero cell compared.

Microwell plate Alamar Blue as the aerobic copy detection detects (Microplate Alamar Blue Assay, MABA)

As document (Collins, Antimicrob.Agents Chemother.41 (1997) 1004-1009; Franzblau, J.Clin.Microbiol.36 (1998), 362-366; Pauli, Life Sci.78 (2005), 485) described, carry out test-compound to mycobacterium tuberculosis H ₃₇The growth inhibiting mensuration of Rv.Suppressing percentage ratio is defined as: 1-(be subjected to prospect hole flat fluorescent/only contain the mean fluorecence unit of three repeating holes of antibacterial) * 100.Think that the lowest concentration of drug that realization 〉=90% suppresses is MIC ₉₀The value that this paper shows is the meansigma methods of three repeated experiments.

Recover as the non-hypoxia that duplicates persistency detection (non-replicating persistence assay) Detect (Low Oxygen Recovery Assay, LORA)

Non-ly duplicate persistency (non-replicating persistence, physiological status NRP) has caused antibiotic patience in many bacterial infections.In pulmonary tuberculosis, for the therapeutic scheme that shortens 6 months, must this NRP subgroup of targeting.As document (Cho, S.H.; Antimicrob.Agents Chemother.2007,51 (4), 1380-1385) described, with high flux, recover to detect (lowoxygen-recovery assay, LORA) test-compound of the mycobacterium tuberculosis of anti-NRP of screening or stable phase based on the hypoxia of fluorescence.Mycobacterium tuberculosis H ₃₇Rv, it contains the plasmid that carries the acetamidase promoter that drives the bacterial luciferase gene, cultivates by prolonging in fermentation tank, adapts to hypoxia condition, and is determined at the MIC of the microwell plate culture of keeping 10 days under the oxygen free condition ₉₀As NMBA is measured, measure and suppress percentage ratio.

Determination of cytotoxic activity

Utilize the cytotoxic activity of Vero cell assessment test-compound, (Cantrell as mentioned previously, J.Nat.Prod.59 (1996), 1131-1136), utilize CellTiter 96 aqueous non-radioactive cell proliferations to detect (CellTiter 96aqueous non-radioactive cell proliferation assay, Promega Corp., Madison WI) carries out.In the active concentration range of compound exhibits, do not observe toxicity.

The result

When utilizing MABA discussed above as the inhibitory action of various cholestane of m tuberculosis infection model evaluation and cholestene, find, be used for the growth that Cholesterylamines derivant of the present invention also suppresses antibacterial.For example, chemical compound 1a, 1f, 1h, 1i, 2a and 2b provide gratifying result (table 2).The cholestane support is to suppress the particularly preferred structure primitive of mycobacterium tuberculosis growth of mycobacterium growth with the combination of the amido functional group that is attached to steroid A ring.On the contrary, when the concentration determination at paramount 100 μ M, cholesterol sulfate or trans-2-aminomethyl-Hexalin do not provide corresponding effect to mycobacteria.Also find, partly incorporate amino into steroid A ring and produced the azepine cholesterol derivative, as (table 2) of MABA detection illustration, its effective bacteria growing inhibiting.Therefore, the cholestane support with incorporate gallbladder steroid A ring into or incorporate the combination of the nitrogen of like derivatives into A ring expansion or that shrink, be to be used to suppress the particularly preferred construction primitive of mycobacterium tuberculosis growth of mycobacterium growth, this point has carried out illustration by chemical compound 2a and 2b.

Table 2 example provided herein is to mycobacterium tuberculosis (bacterial strain H ₃₇The inhibition of Rv) duplicating

Chemical compound 1a, 1f, 1h, 1i, 2a and 2b are the particularly preferred compounds of pulmonary tuberculosis pharmaceutical intervention of mycobacteria disease.2 kinds of chemical compounds in these chemical compounds, promptly chemical compound 1f and 1h provide gratifying especially result in mycobacteria detects, and this is proved by low-down MIC90 value.Therefore, chemical compound 1f and 1h have represented and have been used for the treatment of as in the pharmaceutical composition of phthisical mycobacteria disease even preferred chemical compound.

When assessing the effect of the persistent bacterial subpopulations of test-compound targeting, find that chemical compound 1a and 2a provide gratifying especially result in the LORA model.Therefore, chemical compound 1a and 2a have represented the particularly preferred chemical compound in the pharmaceutical composition that is used for the treatment of persistency mycobacterium tuberculosis phenotype.

Claims (20)

1. the chemical compound of following formula 1 or its pharmaceutically acceptable salt, derivant, solvate or prodrug are used for the treatment of, prevent in preparation and/or improve purposes in the pharmaceutical composition of infectious disease/disease,

Wherein:

R ¹, R ², R ³, R ⁴And R ⁵In one of be to be selected from X (CH ₂) _nNH ₂, X (CH ₂) _nNH (C _1-4Alkyl), X (CH ₂) _nN (C _1-4Alkyl) ₂, X (CH ₂) _nN (C _1-4Alkyl) ₃ ⁺Amino-contained group;

X is chemical bond or is selected from OP (O) (OC _1-4Alkyl) O, OP (O) (O ^-) CH ₂O or OP (O) (OC _1-4Alkyl) CH ₂The phosphorus-containing groups of O;

When X was chemical bond, n was from 0 to 2 integer; When X was phosphorus-containing groups, n was from 2 to 6 integer;

Work as R ⁵When being amino-contained group, then:

R ¹, R ², R ³And R ⁴Be H or OH independently; And

R ⁶Be H, perhaps, when X is chemical bond and n when being 1 or 2, R ⁶Also can be OH;

Be singly-bound or two key, wherein work as When being two key, there is not R ⁴

Work as R ¹When being amino-contained group, then:

R ²And R ⁶Be H or OH independently;

R ³, R ⁴And R ⁵Be H; And

It is singly-bound;

Work as R ²When being amino-contained group, then:

R ³And R ⁶Be H or OH independently;

R ¹, R ⁴And R ⁵Be H, and

It is singly-bound;

Work as R ³When being amino-contained group, then:

R ²And R ⁶Be H or OH independently;

R ¹, R ⁴And R ⁵Be H, and

It is singly-bound.

2. purposes as claimed in claim 1, wherein,

It is singly-bound.

3. purposes as claimed in claim 1 or 2, wherein, R ⁵It is amino-contained group; X is a chemical bond; R ¹, R ², R ³And R ⁴Be H or OH independently; And R ⁶Be H.

4. purposes as claimed in claim 1 or 2, wherein, R ⁵It is described amino-contained group; X is described phosphorus-containing groups; R ¹, R ², R ³And R ⁴Be H or OH independently; And R ⁶Be H.

5. purposes as claimed in claim 1, wherein, the chemical compound that one of the chemical compound of described formula 1 is following formula 1a in the 1l.

6. the chemical compound of following formula 2 or its pharmaceutically acceptable salt, derivant, solvate or prodrug are used for the treatment of, prevent in preparation and/or improve purposes in the pharmaceutical composition of infectious disease/disease,

Wherein:

Y is NH, N (C _1-4Alkyl) or N (C _1-4Alkyl) ₂ ⁺

P is from 0 to 2 integer;

Q is 1 or 2 integer; And

Be singly-bound or two key.

7. purposes as claimed in claim 6, wherein, Y is NH.

8. as claim 6 or 7 described purposes, wherein, p be 0 and q be 2.

9. purposes as claimed in claim 6, wherein, the chemical compound of described formula 2 is the chemical compound one of among following formula 2a and the 2b.

10. as each described purposes among the claim 1-9, wherein, described infectious disease/disease is by virus or bacterial.

11. purposes as claimed in claim 10, wherein, described virus is selected from influenza virus, HIV, hepatitis virus (first, second, third, fourth), rotavirus, respiratory syncytial virus, herpetoviridae (Herpetoviridae, for example, herpes simplex virus, epstein-Barr virus), echovirus 1, Measles virus, Picornaviridae (Picornaviridae, for example, enterovirus, Coxsackie virus), (the Filoviridae of filamentous virus section, for example, Ebola virus, Marburg virus), Papillomaviridae (Papillomaviridae) and polyoma virus section (Polyomaviridae).

12. purposes as claimed in claim 10, wherein, described antibacterial is selected from Gram-positive bacillus, gram-positive cocci, gram negative bacilli and Gram-negative coccus.

13. purposes as claimed in claim 12, wherein, described Gram-positive bacillus is selected from kind (Clostridium spp.), Bacillus anthracis (Bacillus anthracis), bacillus rhusiopathiae suis (Erysipelothrix rhusiopathiae), the Listeria monoeytogenes (Listeriamonocytogenes) of fusobacterium, kind (Nocardia spp.), diphtheria corynebacterium (Corynebacterium diphtheriae) and the Propionibacterium (Propionibacterium acnes) of Nocardia.

14. purposes as claimed in claim 12, wherein, described gram-positive cocci is selected from the kind (Streptococcusspp.) of staphylococcus aureus (Staphylococcus aureus) and Streptococcus.

15. purposes as claimed in claim 12, wherein, described gram negative bacilli is selected from escherichia coli (Escherichia coli), helicobacter pylori (Heliobacter pylori), the kind of Brucella (Brucella spp.), Aeromonas hydrophila (Aeromonas hydrophila), the kind of shigella (Shigella spp.), the kind of vibrio (Vibrio spp.), Yersinia pestis (Yersinia pestis), the kind of Salmonella (Salmonella spp.), Klebsiella Pneumoniae (Klebsiella pneumoniae), Burkholderia cepacia (Burkhoideria cepacia), the kind of Enterobacter (Enterobacter spp.), bacillus pyocyaneus (Enterobacter spp.), campylobacter jejuni (Campylobacter jejuni) and bacillus legionnaires,pneumophila (Legionella pneumophila).

16. purposes as claimed in claim 12, wherein, described Gram-negative coccus is selected from Diplococcus gonorrhoeae (Neisseria gonorrhoeae) and moraxelle catarrhalis (Moraxellacatarrhalis).

17. purposes as claimed in claim 10, wherein, described antibacterial is selected from the kind (Borrelia spp.) of Borrelia, trench fever Bartonella (Bartonella quintana), Chlamydia pneumoniae (Chlamydia pneumoniae), mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium bovis (Mycobacterium bovis), Mycobacterium leprae (Mycobacterium leprae), mycobacterium buruli (Mycobacterium ulcerans), mycobacterium kansasii (Mycobacteriumkanasasii), Mycobacterium avium (Mycobacterium avium), mycobacterium paratuberculosis (Mycobacterium paratuberculosis), lymphoid tuberculosis mycobacteria (Mycobacteriumscrofulaceam), the kind (Treponema spp.) of kind of Dermacentroxenus (Rickettsia spp.) and treponema Pseudomonas.

18. purposes as claimed in claim 11, wherein, described chemical compound has formula 1a, 1b, 1g, 1h, 1i, 1k, 1j or 2a, and described pharmaceutical composition is used for the treatment of, prevents and/or improves influenza infection.

19. purposes as claimed in claim 17, wherein, described chemical compound has formula 1a, 1f, 1h, 1i, 2a or 2b, and described pharmaceutical composition is used for the treatment of, prevents and/or improves the disease that mycobacteria is brought out.

20. purposes as claimed in claim 19, wherein, the disease that described mycobacteria is brought out is selected from pulmonary tuberculosis, leprosy, tropical skin ulcer, abscess, pneumonopathy or skin and disseminates the type disease.