CN100382795C

CN100382795C - Glycinamide derivative for inhibiting HIV replication

Info

Publication number: CN100382795C
Application number: CNB2004800107802A
Authority: CN
Inventors: J·M·R·巴尔扎里尼; A·瓦尔内; M·赫格贝里; 童卫民
Original assignee: Tripep AB
Current assignee: Tripep AB
Priority date: 2003-02-21
Filing date: 2004-02-19
Publication date: 2008-04-23
Anticipated expiration: 2024-02-19
Also published as: ZA200506496B; CN1777414A

Abstract

The present invention relates to the discovery of a novel class of compounds that inhibit the replication of human immunodeficiency virus (HIV) and approaches to identify these compounds. More specifically, it has been found that enzymatically prepared alpha-hydroxyglycinamide and synthetically prepared alpha-hydroxyglycinamide inhibit the replication of HIV in human serum. Embodiments include methods to identify modified glycinamide compounds that inhibit HIV, methods to isolate and synthesize modified glycinamide compounds, and therapeutic compositions comprising these compounds.

Description

Be used to suppress the glycinamide derivative that HIV duplicates

Invention field

Had been found that the medicine that a new class HIV inhibiting (HIV) duplicates.The method that several evaluations suppress the metabolite of the Aminoacetamide that HIV duplicate has been described.Embodiment comprises to be identified and the method for the Aminoacetamide chemical compound of synthetic modification and the Aminoacetamide compound compositions that comprises modification.

Background of invention

Human immunodeficiency virus (HIV) is to infected person and causes the name of the slow virus of acquired immune deficiency syndrome (AIDS) (AIDS).HIV is the complicated retrovirus that comprises at least 9 genes.Encode the especially respectively viral glycoprotein of viral core protein, reverse transcriptase and peplos of the viral structural gene of its called after gag, pol and env.Remaining HIV gene relates to the secondary gene of virus replication.Gag and env gene code polyprotein, promptly each the synthetic albumen by these genes is cut into several littler albumen after translation.

Although the global shape of HIV is spheric, its nucleocapsid is asymmetrical, the about 100nm of the length of nucleocapsid, the about 40-60nm of wide free-end on the width, the about 20nm of narrow end.Nucleocapsid in each ripe virion is made up of the two molecules virus single stranded RNA genome that is surrounded by albumen, and described albumen gets through protease hydrolysis processing from Gag precursor polypeptide.Gag gene polyprotein Pr55 ^GagProtease (PR) cutting by encoding viral produces sophisticated capsid protein.

Owing to found HIV-1, obtained in the impressive progress of understanding on the diseases induced mechanism of virus as the AIDS pathogen.Although developed many diagnostic test methods, because this viral allos character and lack suitable animal model, the progress of HIV vaccine therapy is extremely slow.(referring to Martin, Nature, 345:572-573 (1990)).

Attempted various medicaments is used for the treatment of AIDS.Hiv reverse transcriptase (RT) because its in virus replication pivotal role and become a medicine target, still, the medicine of many these enzymes of inhibition is if not whole, all as being restricted aspect the therapeutic agent.These medicines are the nucleoside/nucleotide analog RT inhibitor (NRTI:s) of inducing chain termination, and are called the reagent of non-nucleoside like direct this enzyme of inhibition of thing RT inhibitor (NNRTI:s).Nucleoside derivates, for example azidothymidine AZT (AZT,

) and other RT inhibitor cause serious adverse, make many patients be impatient at administration.

Another medicine target is the hiv protease (PR) that virus maturation is played a crucial role.PR is an aspartic protease, and can be synthesized chemical compound and suppress (referring to for example, Richards, FEBS Lett., 253:214-216 (1989)).Protease inhibitor strong inhibition HIV duplicates, but it is but relevant with the metabolic disease such as lipodystrophy, hyperlipemia and insulin resistance to prolong therapy.

In addition, HIV is very fast has produced Drug resistance to NRTI:s, NNRT:s and protease inhibitor.Resistance virus also may be propagated between the patient.Research shows, for example, the U.S. recently the 1/10-1/5 of the individuality of infected by HIV have virus to one or more antiviral agents deposits yields resistances, this may be because they infect the people that comfortable propagation periods is carried the virus that forms resistance.

In the period of nearest 10, have been found that some peptide amides suppress duplicating of HIV.(referring to for example U.S. Patent number 5,627,035; 6,258,932; 6,455,670; With Application No. 09/827,822; 09/938,806; 10/072,783; 10/217,933; With 10/235,158).As if these peptide amides suppress HIV in the mode that is different from reverse transcriptase inhibitors and protease inhibitor and duplicate, and have, if having, also be few side effect.Although these achievements are arranged, also need to suppress the more selective therapy agent that HIV duplicates.

Summary of the invention

The hydroxyl Aminoacetamide that has been found that enzymatic preparation and the preparation of synthesizing property suppresses HIV duplicating in human serum.Therefore, aspect of the present invention comprise by, form or comprise its therapeutic combination basically by the Aminoacetamide chemical compound of modifying.The Aminoacetamide chemical compound (for example, metabolite X, α hydroxyl Aminoacetamide or α HGA) of the modification that will exist with any or both forms of any or both of enantiomer (L or D) or isomers (R or S) provides as the active component of medicine that duplicates and/or breed that suppresses HIV and medicament.The Aminoacetamide chemical compound of modifying is such as Alpha-hydroxy Aminoacetamide (Alpha-hydroxy-glycine-NH ₂), α-peroxide Aminoacetamide dimer (NH ₂-glycine-O-O-glycine-NH ₂), two glycyl amidogen ether (NH ₂-glycine-O-glycine-NH ₂) and α-methoxyl group Aminoacetamide (a-MeO-glycine-NH ₂), or its pharmaceutical salts is to mix the preferred active component of pharmaceutical formulation, described pharmaceutical formulation can be used to suppress duplicating of HIV.

Therefore, Aminoacetamide chemical compound (for example chemical compound that provides with formula A, B, C, D, E, F, G, H or I) or its pharmaceutical salts of the modification that can exist by the form that provides with any or both of any or both of enantiomer (L or D) or isomers (R or S) prepare antiretroviral drugs and medicament.The preferred chemical compound that is used to prepare antiretroviral drugs or medicament comprises, for example, the Alpha-hydroxy Aminoacetamide (formula C) that exists with any or boths' of any or both of enantiomer (L or D) or isomers (R or S) form, α-peroxide Aminoacetamide dimer (formula E), two glycyl amidogen ethers (formula F) and α-methoxyl group Aminoacetamide or its pharmaceutical salts.Retrovirus medicine described herein and medicament can be with unit dosage forms (for example, tablet, capsule, soft capsule, liquid dosage form, injectable dosage form, dosage form transdermal or intranasal) provide, and except that the Aminoacetamide chemical compound of modifying, can comprise pharmaceutical carrier or excipient.The container that comprises the described medicine of in bulk or independent dosage form and medicament (for example, the bottle of aseptic bottle, diaphragm seal, bottle, wide mouthed bottle, syringe, aerosol apparatus, cotton are wiped away) also be embodiment, and preferably, prepare described preparation according to being proved to be to good production method (GMP) (for example be suitable for or accepted such as federal FAD (FDA)), and described container comprises label or other labelling of the described government regulator of reflection to described preparation permission by government regulator.But, the nutritional drugs that comprises described chemical compound that has or do not have structure-functional label also is an embodiment.

Some embodiments (for example also comprise one or more described retrovirus chemical compounds, the metabolite X that exists with any or boths' of any or both of enantiomer (L or D) or isomers (R or S) form, Alpha-hydroxy Aminoacetamide (formula C), α-peroxide Aminoacetamide dimer (formula E), two glycyl amidogen ethers (formula F) and α-methoxyl group Aminoacetamide) precursor or prodrug.This precursor or prodrug comprise, for example, comprise Aminoacetamide (for example, the GPG-NH of peptide or Aminoacetamide itself ₂Or ALGPG-NH ₂).These precursors or prodrug are in conjunction with (for example, in mixture, use altogether or before or after prodrug is sent) a kind of Aminoacetamide chemical compound that precursor or prodrug can be converted into modification (for example, the through type A that exists with any or boths' of any or both of enantiomer (L or D) or isomers (R or S) form, B, C, D, E, F, G, H, or the chemical compound that provides of I, such as metabolite X) material (comprise material such as isolating, the hyclone of spissated or undressed form, Ox blood serum, blood plasma or milk, horse serum, blood plasma or milk, the cofactor of cat or dog serum) provide.As above, can prepare described prodrug/cofactor according to the method that is proved to be to good production method (GMP) (for example be suitable for or is accepted such as federal FAD (FDA)), and described container comprises and reflects that described preparation obtains label or other labelling that described government regulator is permitted by government regulator.The nutritional drugs that comprises described chemical compound that has or do not have structure-functional label also is an embodiment.

Alpha-hydroxyl Aminoacetamide (Alpha-hydroxy Aminoacetamide) or its pharmaceutical salts (also being referred to as " α HGA ") are the preferred active component that mixes medicine and/or medicament, and described medicine and/or medicament can be used to suppress duplicating of HIV.Form or form basically by L-α HGA (with R or S isomers form) or D-α HGA (with R or S isomers form) or both (with R or any one or two kinds of isomers forms of S), or the medicine and the medicament that comprise it are embodiments.These compositionss (for example, injection, capsule, pill, tablet, intravenous solution, transdermal with solution, intranasal with solution and other pharmaceutical formulation) preferably include, provide or send the α hydroxyl Aminoacetamide that duplicates and/or breed of the inhibition HIV of many enzymatics preparations (metabolite X) or synthetic property preparation (α HGA).

Embodiment comprises, for example, form by Aminoacetamide compound or pharmaceutically acceptable salt thereof, amide, ester or prodrug as shown in the formula the modification of (A), by its medicine of forming basically and medicament, or comprise medicine and medicament as shown in the formula Aminoacetamide compound or pharmaceutically acceptable salt thereof, amide, ester or the prodrug of the modification of (A):

Wherein

A) E is selected from by oxygen, sulfur and NR ₇In the group of forming;

B) T is selected from by oxygen, sulfur and NR ₈In the group of forming; With

C) R ₁-R ₈Each be independently selected from by in the following group of forming: hydrogen; The alkyl of Qu Daiing randomly; The alkenyl of Qu Daiing randomly; The alkynyl of Qu Daiing randomly; The cycloalkyl of Qu Daiing randomly; The heterocyclic radical of Qu Daiing (heterocyclyl) randomly; The cycloalkyl-alkyl of Qu Daiing (cycloalkylalkyl) randomly; The heterocyclic radical alkyl of Qu Daiing randomly; The aryl of Qu Daiing randomly; The heteroaryl of Qu Daiing randomly; The alkyl-carbonyl of Qu Daiing randomly; The randomly alkoxyalkyl of Qu Daiing and the randomly fully halogenated alkyl (perhaloalkyl) of replacement.

The Ideal Match thing comprises medicine and medicament, and described medicine and medicament are made up of the Aminoacetamide compound or its salt as shown in the formula the modification of (B), is made up of basically it, or comprises Aminoacetamide compound or its salt as shown in the formula the modification of (B):

R wherein ¹Be hydrogen atom, low-grade alkyl group, low-grade alkenyl group, low-grade alkynyl group, benzyl group or the silicyl or alkyl group and aryl and the R that replace with alkyl ²Be hydrogen atom or amido protecting group or its salt.

Preferred compositions comprises medicine and medicament, described medicine and medicament are made up of Aminoacetamide compound or pharmaceutically acceptable salt thereof, amide, ester or prodrug as shown in the formula the modification of (C), basically form by it, or comprise Aminoacetamide compound or pharmaceutically acceptable salt thereof, amide, ester or prodrug as shown in the formula the modification of (C):

Particularly preferred compositions comprises medicine and medicament, and described medicine and medicament are made up of the Aminoacetamide chemical compound as shown in the formula the modification of (D), forms in fact, or comprises Aminoacetamide chemical compound as shown in the formula the modification of (D):

By the chemical compound of enzymatic method preparation and use cation exchange HPLC separate type (C), α hydroxyl Aminoacetamide is also referred to as metabolite X or α HGA, and has prepared the chemical compound of formula (D) by synthetic method.In some cases, formula (C) and the chemical compounds (D) that exist with any or both forms in any or both in the enantiomer (L or D) or the isomers (R or S) all alternately can be called: " metabolite X ", " α HGA " or " Aminoacetamide of modification ".

Preferred compositions also comprises medicine and medicament, described medicine and medicament are made up of the Aminoacetamide compound or pharmaceutically acceptable salt thereof as shown in the formula the modification of (E) or formula (F), basically form by it, or comprise Aminoacetamide compound or pharmaceutically acceptable salt thereof as shown in the formula the modification of (E) or formula (F):

Preferred compositions also comprises medicine and medicament, described medicine and medicament by, substantially form by Aminoacetamide compound or pharmaceutically acceptable salt thereof, or described medicine and medicament comprise the Aminoacetamide compound or pharmaceutically acceptable salt thereof as shown in the formula the modification of (G) as shown in the formula the modification of (G):

Also synthetic property ground has prepared α-methoxyl group Aminoacetamide, and has been found that this chemical compound is more more stable than Alpha-hydroxy Aminoacetamide.

Embodiment also comprises some evaluations and the method for separating the Aminoacetamide chemical compound that suppresses the modification that HIV duplicates and the method for synthetic these chemical compounds.Some embodiments relate to and suppress the method that HIV duplicates and/or breeds, and wherein the experimenter to the medicament that needs inhibition HIV to duplicate provides many enzymatics that enough suppress the propagation of virus or duplicate to prepare the Alpha-hydroxy Aminoacetamide (α HGA) of (metabolite X) or the preparation of synthetic property.In the certain methods therein, measured the influence (for example, by observing or the minimizing of monitoring in viral lode or its labelling) that HIV is duplicated.Other embodiments comprise the method that HIV infects that treats and/or prevents, provide wherein for the people of the patient suffer from HIV or risky infected by HIV enough to suppress Aminoacetamide (for example, Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer, two glycyl amidogen ethers or the α-methoxyl group Aminoacetamide of many modifications that HIV duplicates.As above, in some embodiments, described chemical compound is provided or comprises the medicine of described chemical compound to the experimenter, described experimenter need suppress the medicament that HIV duplicates, and in other embodiment, measured the influence (for example, by measuring the minimizing of viral lode or its labelling such as p24 gathering or reverse transcriptase activity) that HIV is duplicated.

The accompanying drawing summary

Fig. 1 shows GPG-NH2 (2S)-1-(Aminoacetyl)-N-(carbamoylmethyl)pyrrolidine-2-carboxamide (GPG-NH ₂), sarcosylpyrolylglycinamide (SAR-PG-NH ₂), ring-type pyrroles's glutamy amido prolyl Aminoacetamide (pyrroglutaminylprolylglycinamide) (PyrQPG-NH ₂), Q-PG-NH2 (QPG-NH ₂) and Aminoacetamide (G-NH ₂) structure.

Fig. 2 shows the function as the time, in people T-lymphocyte (CEM, C8166, Molt4/C8, MT-4) and PBMC suspension (figure A) and CD26 activity in some different serum (people (HS), Mus (MS), cattle (BS) (scheming B)).Substrate is glycyl prolyl-p-nitroanilide (GP-pNA).By absorptiometry enzymatic activity at the 400nm place.

Fig. 3 shows that the CD26 of purification mediates unmarked GPG-NH ₂To GP-OH and G-NH ₂Change.Detect by mass spectrography.

Fig. 4 shows in the phosphate buffered saline(PBS) (PBS) 5% Ox blood serum (BS), 5% human serum (HS) and cem cell suspension (10 in PBS ⁶Cell) to radiolabeled [ ¹⁴C] GPG-NH ₂To [ ¹⁴C] G-NH ₂Transformation.

Fig. 5 shows and uses GP-pNA as substrate, and CD26 specific inhibitor IlePyr is among 5% Ox blood serum (BS) among the PBS and the PBS 10 ⁶The active inhibitory action of the dipeptidyl peptidase of CD26 in the cem cell suspension.

Fig. 6 shows that CD26 inhibitor IlePyr is to the active influence of the anti-HIV-1 of GPG-NH2 in the cem cell culture and G-NH2.

Human serum of some batches of Fig. 7 display analysis and hyclone transform G-NH ₂Be the G-NH that modifies ₂The result of the ability of (metabolite X).

The different animal serum of Fig. 8 display analysis transforms G-NH ₂Be the G-NH that modifies ₂The result of the ability of (metabolite X).

Fig. 9 shows the result of competitive trials, has wherein estimated glycine, the L-serine-NH of variable concentrations ₂, L-alanine-NH ₂Or GPG-NH ₂Suppress G-NH ₂Change the G-NH of modification into ₂The ability of (metabolite X).

Figure 10 shows that the different fractions of the hyclone that obtains by size exclusion chromatography transforms G-NH ₂Be the G-NH that modifies ₂The analysis result of (metabolite X).

Figure 11 for example understands the result of reverse transcriptase (RT) active testing, and wherein the Alpha-hydroxy Aminoacetamide (metabolite X or Met-X) of enzymatic preparation is suppressed at duplicating of HIV in the culture that comprises the hyclone that boils, and G-NH ₂Then do not suppress.

Figure 12 shows the result of reverse transcriptase (RT) test, and 5 times the Alpha-hydroxy Aminoacetamide (metabolite X or Met-X) of enzymatic preparation of wherein having dialysed has suppressed the duplicating of HIV in the culture that comprises the hyclone that boils.

Figure 13 shows the result of reverse transcriptase (RT) test, has wherein analyzed the antiretroviral activity (IC of Alpha-hydroxy Aminoacetamide (metabolite X or Met-X) of the enzymatic preparation of various concentration ₅₀).

Figure 14 shows result's (in hyclone) of the accumulative HIV experimental infection of monitoring p24, and wherein the Alpha-hydroxy Aminoacetamide (metabolite X or Met-X) of enzymatic preparation is to inhibition and the GPG-NH of HIV ₂Equally effective.

Figure 15 shows result's (in hyclone) of the accumulative HIV experimental infection of monitoring p24, wherein observes inhibition and the GPG-NH of the Alpha-hydroxy Aminoacetamide (α HGA) of synthetic preparation to HIV ₂Equally effective.

Figure 16 shows the result of the accumulative HIV experimental infection (in hyclone (figure A) and human serum (figure B)) of monitoring p24, wherein in hyclone, the Alpha-hydroxy Aminoacetamide (metabolite X or Met-X) of enzymatic preparation and the Alpha-hydroxy Aminoacetamide (α HGA) of synthetic property preparation are to inhibition and the G-NH of HIV ₂Equally effective (figure A), but in human serum has only the Alpha-hydroxy Aminoacetamide (α HGA) of the Alpha-hydroxy Aminoacetamide (metabolite X or Met-X) of enzymatic preparation and the preparation of synthetic property can suppress duplicate (the scheming B) of HIV.

Figure 17 shows result's (in hyclone) of reverse transcriptase (RT) test, has wherein compared G-NH ₂, fresh dilution the preparation of synthetic property Alpha-hydroxy Aminoacetamide (α HGA) with in the antiretroviral activity of 3 days Alpha-hydroxy Aminoacetamide of 37 ℃ of incubations (α HGA37).

Detailed Description Of The Invention

Had been found that some are metabolised to the tripeptide amide and the glycine amide that suppress the compound that HIV copies is prodrug. These antiviral agents are inhibitor (for example, the GPG-NH of high selectivity in cell culture₂With glycine amide or " G-NH₂" HIV that suppresses in the cem cell culture copies to suitable degree (50% valid density :～30 μ M)). Because G-NH₂Also suppress copying of HIV, the research in this field concentrates on tripeptide amide to glycine amide (G-NH₂) conversion. (referring to Application No. 10/235,158). Present known lymphocytic cell surface glycoprotein mark CD26 transforms GPG-NH effectively₂Become G-NH₂, discharge simultaneously dipeptides GP-OH, and GPG-NH₂Need this cracking to bring into play the effect of its ARV activity.

Also have been found that G-NH₂Himself be prodrug, described prodrug is metabolised to one or more and suppresses the compound that HIV copies (for example ring-type, the charged or glycine amide of charged form not). With these derived from G-NH₂Metabolin be called " glycine amide of modification ", " glycinamide derivative " or " metabolin X ". The mass spectrometry that the glycine amide peak fraction of the modification that separates by chromatography is carried out and the analysis of nuclear magnetic resonance (NMR) spectrometry disclose it and comprise Alpha-hydroxy glycine amide (" α HGA " or (C₂H ₆N ₂O ₂) or (C₂H ₇ClN ₂O ₂)). Alpha-hydroxy glycine amide and α-methoxyl group glycine amide all is prepared by organic synthesis. Find that the Alpha-hydroxy glycine amide (metabolin X) of enzymatic preparation and the Alpha-hydroxy glycine amide (α HGA) of synthetic property preparation suppress the HIV in the human serum effectively. It is simple comprising the medicine of glycine amide of these modifications and the preparation of medicament, and this paper application of providing the HIV in the subject that these compounds are used for suppressing needing them to copy. It is the discovery of G-NH2 that following part is described CD26 conversion GPG-NH2 in detail.

CD26 mediation GPG-NH2 is to the conversion of G-NH2

Originally, lymphocytic cell surface gC D26 is described as T-cell activation/differentiation marker. (seeing Fox etc., J.Immunol., 132:1250-1256 (1984)). The CD26 great expression is on the target cell (that is, lymphocytic CEM, Molt, C8166 and MT-4 and PMBC) of HIV, and it also is present in the serum of ox, mouse, people's origin. It is the relevant peptase of film that is same as two peptidyls-peptase IV (DPP IV, EC3.4.14.5), and for comprising proline or alanine as the amino acid whose peptide of the penult of N-end, it is selective that it has height (but not monopolizing). (see Yaron and Naider, Biochem.Mol.Biol., 28:31-81 (1993); The Immunol.Today such as De Meester, 20:367-375 (1999) and Mentlein, Regul Pept., 85:9-24 (1999)). It not only is expressed on the various leucocyte subgroups, also expresses epithelial cell, endothelial cell and the fibroblast of some types. (Id.). The soluble form of CD26 also exists. Its lack the afterbody in cross-film zone and the born of the same parents and in blood plasma and cerebrospinal fluid, detect to a small amount of existence. (see Yaron and Naider, Biochem.Mol.Biol., 28:31-81 (1993); De Meester etc., Immunol.Today, 20:367-375 (1999)).

Some cell factors, hemopoieticgrowth factor, hormone and neuropeptide comprise X-proline or X-alanine motif at their N-end. (seeing De Meester etc., Immunol.Today, 20:367-375 (1999)). Will be near the existence of the proline of N-end as the structural defence for nonspecific proteolytic degradation. (seeing Vanhoof etc., FASEB J., 9:736-744 (1995)). Particularly, can be with relatively little peptide as natural substrate (for example, chemotactic factor (CF) RANTES (68 amino acid) and SDF-1 α (68 amino acid), and hyperglycemic factor/VIP (vasoactive protein) family peptides such as GIP (42 amino acid) and GLP-2 (33 amino acid)). (seeing De Meester etc., Immunol.Today, 20:367-375 (1999)). In some cases, peptide is (for example, neuropeptide endorphine 2 (4 amino acid) and the substrate P (11 amino acid)) that lacks very much. Find that also the Enterostatin that only is comprised of 5 amino acid also is the substrate of CD26.

What is interesting is, in some cases, show that CD26 with after two peptide moieties are under the N-end portion cracking of molecule, has changed the biological function of native peptides. (Oravecz etc., J.Exp.Med., 186:1865-1872 (1997); Proost etc., J.Biol.Chem., 273:7222-7227 (1998)). Discovery is compared with complete RANTES, and the RANTES of brachymemma (3-68) has anti-HIV-1 activity (seeing Schols etc., Antiviral Res., 39:175-187 (1998)) of remarkable increase really; And CD-26 is to the terminal ability that has obviously reduced its anti-HIV-1 of processing of the N-of SDF-1 α. (see Ohtsuki etc., FEBS Lett., 431:236-240 (1998); Proost etc., FEBS Lett., 432:73-76 (1998)). Recently, show that also CD26 regulates the alpha mediated people of SDF-1 and restraints haemocyte CD34⁺The chemotaxis of progenitor cell. (seeing Christopherson etc., J.Immunol., 169:7000-7008 (2002)).

Have been found that tripeptides GPG-NH2 (GPG-NH₂) suppress copying of HIV at non-toxic concentration. (see, for example, Application No. 5,627,035). But until the present invention is open, just know its related with CD26. GPG-NH2 is blocked HIV-1 laboratory strain and the clinical isolates of broad variety in 2-40 μ M scope. Owing in HIVp24, there are two GPG motifs, there is a GPG motif at the V3 of virus envelope protein gp120 in the ring, research originally concentrates on the potential target of these virus proteins as this new tripeptide derivative. (seeing Su, Ph.D.thesis at the Karolinska Institute (ISBN 91-628-4326-5), Stockholm, Sweden (2000) and Su etc., AIDS Res.Human Retrovir., 16:37-48 (2000)).

Although the GPG-NH in high concentration₂Observe the SDS-PAGE mobilance of the increase of gp160/120, find GPG-NH₂Do not affect the early stage event in HIV infection circulation. (seeing Su etc., J.Hum.Virol., 4:8-15 (2001)). In addition, verified GPG-NH₂With the combination of p24 albumen, and at GPG-NH₂Observe the core texture of mistake assembling of the virion of increase in the-HIV-1 infection cell processed. (seeing Hoglund etc., Antimicrob.Agents Chemother., 46:3597-3605 (2002)). In addition, the formation of viral capsid (p24) has been disturbed in the existence of discovery medicine. (seeing Hoglund etc., Antimicrob.Agents Chemother., 46:3597-3605 (2002)). What become apparent is by new machine-processed GPG-NH₂Suppressed copying of HIV.

Consider that proline residue is at GPG-NH₂Existing in the middle of the peptide molecule (equating with aminoterminal penult amino acid), think GPG-NH₂It can be the antiretroviral activity that the enzymatic activity of the substrate of CD26/ DPP IV and CD26 can be regulated described compound. Therefore, test to determine whether the CD26/ DPP IV can be with GPG-NH₂Be converted into G-NH₂, and really find CD26 and optionally and effectively behind proline residue, cut GPG-NH₂To discharge dipeptides GP-OH and G-NH₂ And, confirmed that also this cutting is for GPG-NH₂It is necessary bringing into play its ARV activity. Following embodiment has described these discoveries in more detail.

Embodiment 1

In experiment originally, to some HIV-1 and HIV-2 strain to GPG-NH ₂, G-NH ₂Estimate with the active sensitivity of the inhibition of related compound.(see Table 1 and Fig. 1).GPG-NH2 (2S)-1-(Aminoacetyl)-N-(carbamoylmethyl)pyrrolidine-2-carboxamide (GPG-NH ₂), Q-PG-NH2 (Q-PG-NH ₂), sarcosinylprolylglycinamide (Sar-PG-NH ₂) and Aminoacetamide (G-NH ₂) by TRIPEPAB provide (Huddinge, Sweden); And pyrroles's Q-PG-NH2 (PyrQ-PG-NH ₂) synthetic at Rega Institute.The lymphocytic cem cell of people T-is available from American type culture collection (American Type culture Collection) (Rockvile, MD), and replenishing 10% hyclone (FBS) (BioWittaker Europe, Verviers, Belgium), 2mM L-glutaminate (Gibco) and 0.075 M NaHCO ₃(Gibco) cultivate in the RPMI-1640 culture medium (Gibco, Paisley Scotland).HIV-1 (IIIB) available from Dr.R.C.Gallo and Dr.M.Popovic (at that time at National Cancer Institute, NIH, Bethesda, MD).HIV-1 (NL4.3) from National Institute of Allergy and Infectious Disease AIDSReagent Program (Bethesda, MD).(Pasteur Institute, Paris France) provides by Dr.L.Montagnier for HIV-2 separator ROD and EHO.

With the lymphocytic cem cell (4.5 * 10 of people T- ⁵Cell/ml) is suspended in the fresh cell culture medium and uses 100 CCID ₅₀/ ml cell suspending liquid (1 CCID ₅₀Be the viral dosage that infects 50% cell culture) HIV-1 (III _BAnd NL4.3) or HIV-2 (ROD or EHO) infect.Then, the infected cell suspension of 100 μ l of the suitable dilution (prepared fresh) of mixed test compounds with 100 μ l (promptly 2000,400,80,16,3.2 and 0.62 μ M final concentration) is transferred in the hole of microtest plate, and further be incubated at 37 ℃.After 4-5 days, form with giant cell in the microscope record cem cell culture.50% valid density (EC ₅₀) corresponding to the required compound concentration of syncytium of the formation 50% of the cem cell culture that prevents viral infection.

Table 1

Tripeptide derivative is at the inhibition activity of a few strain virus strains in the cem cell culture

^a50% valid density, or the HIV-that suppresses in the lymphocytic cem cell culture of T-reduces the required compound concentration of plasmodial formation

What is interesting is, regardless of the character of used virus in antivirus test, GPG-NH ₂And G-NH ₂On mole foundation, suppress virus replication equivalently.In the cem cell culture, their EC ₅₀(50% valid density) grade is between 30 and 50 μ M.These two kinds of chemical compounds do not have showed cell toxicity on the concentration up to 1500-2000 μ M.Sar-PG-NH ₂And Q-PG-NH ₂Also suppress duplicating of HIV, although as GPG-NH ₂, the degree of inhibition is lower.Synthesized a kind of new tripeptides (PyrQ-PG-NH ₂) derivant, it comprises G-NH at c-terminus ₂, and comprise cyclic pyrroles's glutamine (pyrroglutamine) at its aminoterminal.With GPG-NH ₂With other tripeptide amide derivant contrast, find PyrQ-PG-NH ₂It is invalid that HIV in the inhibition cell culture is duplicated.

Next, confirmed in the CD26 of purification and cattle, Mus and human serum, to detect the dipeptidyl peptidase enzymatic activity of CD26, and detected with human lymphocyte or peripheral blood lymphocytes suspension.Put down in writing the enzymatic activity of CD26 by will synthesizing property substrate glycyl prolyl p-nitroanilide (GP-pNA) being converted into glycyl proline (GP-OH) and p-nitroaniline (pNA), described p-nitroaniline (pNA) is a kind of weld, and its formation can be monitored by the increase at the absorbance of 400nm.With the CD26 of the purification in phosphate buffered saline(PBS) (PBS) of about 200 microlitres (1 milli unit/ml) or people, Mus or Ox blood serum (PBS 5%), or in PBS solution 10 ⁶The CEM of human lymphocyte, C8166, Molt4/C8, MT-4 or peripheral blood lymphocytes suspension add in the hole of microtitration plate of 200 microlitres, add substrate (glycyl prolyl-para-nitroanilide) (GP-pNA) of the measurement CD26 enzymatic activity of 3mM final concentration thereafter.Glycyl prolyl-p-nitroanilide (GP-pNA) and glycylphenylalaninyl-p-nitroanilide (GF-pNA) available from Sigma Chemicals (St.Louis, MO).Monitor p-nitro-aniline (pNA) release from the amount of (xanchromatic) p-nitroaniline (pNA) of GlyPro-pNA in 37 ℃ of functions by measuring release with the time.By (CA) release of pNA is put down in writing in the increase of last absorbance [in the optical density (OD) of 400nm] for Molecular Devices, Sunnyvale at Spectramax microtest plate spectrometer.Under experiment condition, reaction was carried out 60 minutes linearly at least.As the measurement of enzymatic activity, with the blank reactant mixture OD of (lacking CD26 enzyme, serum or cell) ₄₀₀The OD of value from obtaining ₄₀₀Deduct in the value with expression OD ₄₀₀The real increase of value.

Find owing in fact thoroughly checked the release of p-nitroaniline from synthetic property substrate GP-pNA (hereinafter) after the specific inhibitor of CD26 added cell suspension, so GP-pNA is only changed by CD26 and not by the role transformation of other two peptidyls/peptidase.Be applied all lymphocyte suspensions (CEM, C8166, MT-4, Molt4/C8) of GP-pNA and other PBMC and all effectively GP-pNA changed into the p-nitroaniline in the time-dependent mode.(seeing Fig. 2 A).The activity of CD26 is the highest and minimum in the MT-4 cell suspension in the cem cell suspension.In addition, tire cattle and Mus serum and special human serum discharge p-nitroaniline (seeing Fig. 2 B) effectively from GP-pNA.Therefore, people T-lymphocyte suspension and serum have all shown the enzymatic activity of significant CD26/ dipeptidyl peptidase.Can be monitored effectively in case determined the CD26 activity, experimentized to determine whether CD26 can be with GPG-NH ₂Change G-NH into ₂

In a sample, approx, make 100 μ M GPG-NH ₂Contact with the purification CD26 of 25 units/l and with described mixture in incubated at room temperature as many as 400 minutes.As previously mentioned, purification lymphocytic cell surface gC D26/ DPP IV.(seeing De Meester, J.Immunol.Methods, 189:99-105 (1996)).At different time points, the aliquot of abstraction reaction mixture and at electrojet ion trap mass spectrometer (Bremen analyzes on Gennany) for Esquire, Bruker.Analyze from GPG-NH by electrojet ion trap mass spectrometer in different time points ₂The appearance of dipeptides GP-OH when the amino terminal of molecule discharges, and the complete GPG-NH that comes reaction mixture ₂Disappearance determine and monitor.Under these experiment conditions, CD26 in the time-dependent mode from GPG-NH ₂Discharge GP-OH, and in 4-6 hour of reaction in fact fully with GPG-NH ₂Change GP-OH and G-NH into ₂In contrast, CD26 can not be from pyrroles Q-PG-NH ₂The middle G-NH that discharges ₂

Next, CD26, hyclone (FBS), human serum (HS) and the cem cell suspension of having analyzed purification make radiolabeled [ ¹⁴C] GPG-NH ₂To [ ¹⁴C] G-NH ₂Transformation.By Amersham Pharmacia Biotech (Buckinghamshire, England) the synthesizing radioactive labelling [ ¹⁴C] GPG-NH ₂(radiation specificity: 58mCi/mmol) and [ ¹⁴C] G-NH ₂(radiation specificity: 56 mCi/mmol), described [ ¹⁴C] GPG-NH ₂In, radiolabeled carbon potential on the main chain carbon of the glycine of tripeptides carboxylic acid end, described [ ¹⁴C] G-NH ₂In, carbon 2 is by radioactive label.Make the CD26 of purification, FBS, HS and cem cell suspension and multiple these [ ¹⁴C] GPG-NH ₂The concentration contact, and analyze to G-NH ₂Transformation.

In one group of experiment, for example, make the cem cell culture (5 * 10 of 5ml ⁵Cell/ml) and 20 μ M[ ¹⁴C] GPG-NH ₂Contact 24 hours.Then, in 1, centrifugal 10 minutes of 200rpm washs with cell, and handles cell precipitation 10 minutes with 60% ice-cooled methanol.In 15, centrifugal 10 minutes of 000rpm is expelled to described supernatant cation exchange Partisphere-SCX post (Whattman) thereafter and goes up with from G-NH with the methanol cell extract ₂The middle GPG-NH that separates ₂Used following gradient: 0-15 minute: buffer A such as degree of grade (the 7mM sodium phosphate, pH3.5); 15-40min is (250mM sodium phosphate, linear gradient pH3.5) from the buffer A to the buffer B; The 40-45 minute linear gradient from the buffer B to the buffer A; 45-55 minute: buffer A such as degree of grade.Under these elution requirements [ ¹⁴C] GPG-NH ₂[C ¹⁴] G-NH ₂The time of staying be respectively 26-28 minute and 14-16 minute.

In another group experiment, after one hour contact, as mentioned above, use the sodium phosphate buffer gradient of cation exchange Partisphere SCX post and pH3.5, by HPLC analyze to determine complete [ ¹⁴C] GPG-NH ₂Disappearance.GPG-NH ₂From G-NH ₂In fully separate (time of staying: be respectively 25-27 minute and 15-17 minute).Calculate the catalytic GPG-NH of CD26 ₂To G-NH ₂The K of transformation _mValue is 0.183mM.As from the GPG-NH that describes among Fig. 4 ₂The disappearance curve, for the dipeptidyl peptidase enzymatic activity relevant with HS and FBS, GPG-NH ₂Estimation K _mValue is respectively 0.45 and 1.4mM.By the cem cell suspension to GPG-NH ₂Conversion linear rise to maximum 1.5mM on the ground.Only at higher GPG-NH ₂Concentration (for example, 3 and 5.4mM), the transition curve of cem cell suspension just begins to tend towards stability slightly.

Next, analyzed the inhibitory action of L-isoleucine pyrrolidine (IlePyr) to CD26.Recently, reported that isoleucine pyrrolidine (IlePyr) is the active relatively effectively and optionally inhibitor of the dipeptidyl peptidase related with the CD26 of purification.(seeing De Meester, J.Immunol.Methods, 189:99-105 (1996)).All enzymatic activity tests are carried out in 96 hole microtitration plates.(Falcon，Becton Dickinson，Frankin Lakes，NJ)。In each hole, add the purification CD26 (final concentration be 0.2milliUnits/200 μ l-hole) of 5 μ l in PBS, 10 μ l hyclones (BS) (final concentration: in PBS 5%; 56 ℃ of preheatings 30 minutes) or the IlePyr inhibitor solution in PBS (500 and 200 μ M) of 1,000,000 cem cells in PBS, 5 μ l debita spissitudos and PBS to reach cumulative volume 150 μ l.Come initial action and carry out in the substrate GP-pNA of 4mg/ml concentration (final concentration in 200 μ l reactant mixtures: 1mg/ml or 3mM) by adding 50 μ l at 37 ℃.It is pNA and the required compound concentration of GP-OH with 50% GP-pNA catalyzing hydrolysis that IlePyr is defined as inhibitory enzyme at active 50% inhibition concentration of the dipeptidyl peptidase related with CD26, BS and cem cell suspension.

In initial experiment, analyzed use GP-pNA as substrate, the IlePyr in cem cell suspension (in hyclone) is to the inhibition of CD26.With the CD26 of purification as positive control.(see figure 5).Inhibitor IlePyr dosage relies on ground and stops the release of p-nitroaniline from GP-NA, described GP-NA and 50% inhibition concentration (IC ₅₀) be respectively 110 and contact with cem cell suspension and the hyclone of 99 μ M.At IC ₅₀When value was 22 μ M, the CD26 of purification was suppressed.Therefore, the 50% inhibition concentration (IC of the inhibitor IlePyr that contacts with the cem cell suspension with serum ₅₀) 5 times of required inhibitor concentration of purification CD26 that are higher than inhibition 50% of value.

Then, experimentize to determine whether to use GPG-NH ₂Active and the catalytic G-NH of CD26 of observed antiretroviral ₂Release from tripeptide derivative is relevant.When the IlePyr that has non--toxic concentration (500 μ M and 200 μ M), with the cem cell culture of HIV-1 infection and the GPG-NH of variable concentrations ₂Contact.With similar G-NH ₂Be included in this research with combining of IlePyr.In these experiments, before the cell that adds test compounds and viral infection, CD26 specific inhibitor L-isoleucine pyrrolidine (IlePyr) is added in every hole of microtest plate.

With in the CEM culture, fully preserve the active G-NH of its anti-HIV under the situation about existing at the IlePyr of 200 and 500 μ M ₂(EC ₅₀-35-43 μ M) opposite, in the situation that has specific C D26 inhibitor, GPG-NH ₂Significantly lost its inhibition activity, (see figure 6) to the cytopathogenicity of virus induction.Reversing tripeptides GPG-NH ₂When anti-HIV-1 was active, the efficient of the relatively lower inhibitor concentration (200 μ M) of the highest inhibitor concentration (500 μ M) was higher slightly.Also at Sar-GP-NH ₂Observe similar result, described Sar-GP-NH ₂Be also in cell culture, to be endowed the active another kind of tripeptide amide derivant of antiretroviral.

The result of present embodiment has proved GPG-NH ₂Make it discharge Aminoacetamide before in cell culture, bringing into play HIV (human immunodeficiency virus)-resistant activity thereby need be hydrolyzed.These data produce evidence also to prove that the enzymatic activity of lymphocytic cell surface glycoprotein activation/differentiation marker CD26 induces G-NH ₂From GPG-NH ₂Middle release.Serum with CD26, people T-lymphocyte suspension and the people and the cattle of purification carries out G-NH ₂From GPG-NH ₂In formation.And, the Q-PG-NH that declares ₂Antiviral activity, PyrQ-PG-NH ₂Antiviral activity (it is the resistance that the CD26 enzymatic is attacked) thorough shortage and have GPG-NH under the situation at the CD26 specific inhibitor ₂And Sar-GP-NH ₂The forfeiture of antiviral efficient GPG-NH is provided ₂As G-NH ₂Effective prodrug and CD26 catalysis GPG-NH ₂To G-NH ₂The favourable evidence that changes.

Therefore, find to be responsible for for example GPG-NH as the lymphocytic cell surface gC D26 of the relevant dipeptidyl peptidase of film ₂, QPG-NH ₂, sarcosylprolylglycinamide (SAR-PG-NH ₂) metabolism is G-NH ₂Enzyme.Obtained CD26 and be responsible for the peptide amide metabolism more evidences for the form that suppresses HIV and duplicate from experiment, described experimental applications is CD26 inhibitor L-isoleucine pyrrolidine (IlePyr) optionally, wherein observes GPG-NH ₂And SAR-PG-NH ₂The active obvious minimizing of anti-HIV.Yet described IlePyr inhibitor is for G-NH ₂Suppress the not influence of ability that HIV duplicates.Therefore, the peptide amide that comprises X-proline-Aminoacetamide is retrovirus prodrug or precursor, and it is G-NH by lymphocytic cell surface gC D26 metabolism ₂Ensuing part more detailed description Aminoacetamide suppresses the discovery that HIV duplicates.

Aminoacetamide suppresses duplicating of HIV

Originally, determined G-NH ₂Effectively suppress duplicating of HIV, the chemical compound of structural similarity does not then suppress.With HIV-1 (III _B) the cem cell culture that infects is with the G-NH of various concentration ₂Or various concentration and G-NH ₂Chemical compound with analog structure is cultivated, and the inhibition of using the standard method evaluation that HIV is duplicated.These experiments are described in the following embodiments.

Embodiment 2

With the lymphocytic cem cell of people T-(about 4.5 * 10 ⁵Cell/ml) be suspended in the fresh cell culture medium also with about 100 CCID ₅₀/ ml cell suspending liquid (1 CCID ₅₀Be the viral dosage that infects 50% cell culture) HIV-1 (III _B) infect.Then, the infected cell suspension of 100 μ l is transferred in the hole (100 μ l/ hole) of microtest plate, and mixed test compounds (2000,400,80,16,3.2 or 0.62 μ M) with the fresh dilution of 100 μ l.Subsequently, mixture is incubated at 37 ℃.Cultivate after 4-5 days, form with giant cell in the microscope record cem cell culture.50% valid density (EC ₅₀) corresponding to 50% the required compound concentration of plasmodial formation in the cem cell culture that stops viral infection.

These result of experiment are shown in the table 2.Find that Aminoacetamide is obviously to suppress only chemical compound that HIV duplicates in cell culture.G-NH ₂EC ₅₀Be about 21.3 μ M, and the chemical compound of other test does not have to show the inhibition to HIV.These results have confirmed G-NH ₂Have and suppress the special construction that HIV duplicates.

Table 2

^a50% valid density

Analysis has subsequently disclosed G-NH ₂It is the specific inhibitor of HIV.In with the cell culture of various types of viral infection, estimate the G-NH of various concentration ₂And GPG-NH ₂Cytotoxicity and antiviral activity.Subsequently each dissimilar cell and virus are carried out conventional host cell cultivation, viral infection and analysis of infection.The chemical compound of the virus replication of the specific type that known inhibition is analyzed is with comparing.

Table 3.5 shows these result of experiment.These data show G-NH ₂And GPG-NH ₂Suppressing herpes simplex virus-1 (KOS), herpes simplex virus-2 (G), herpes simplex virus-1TK-KOSACV ^r, the effect on vaccinia virus, vesicular stomatitis virus, Coxsackie B virus 4, respiratory syncytial virus, parainfluenza-3 virus, reovirus-1, sindbis virus and the Punta Toro virus.These

The result has confirmed G-NH ₂And GPG-NH ₂It is the inhibitor of HIV.

Table 3

Chemical compound is at hel cell cells in culture toxicity and antiviral activity

^aCause the change of the normal cell form that available microscope inspection measures needed

^bVirus-inductive the cytopathogenicity of minimizing 50% is needed

Table 4

Cytotoxicity and the antiviral activity of chemical compound in the HeLa cell culture

^bVirus-inductive the cytopathogenicity of minimizing 50% is needed

Table 5

Chemical compound is cytotoxicity and antiviral activity in the cell culture in the African green monkey kidney cell strain

^bVirus-inductive the cytopathogenicity of minimizing 50% is needed

Also had been found that G-NH ₂Itself be a kind of prodrug or precursor, it is present in enzyme in some animal blood slurrys and the serum or cofactor metabolism and suppresses the chemical compound that HIV duplicates (for example, Aminoacetamide cyclic, charged or uncharged form) for one or more.Hereinafter this discovery of more detailed description.

Be present in cofactor in some animal blood slurrys and the serum with G-NH ₂Change the metabolite that suppresses HIV into

This paper provides evidence, and described evidence is to be present in the serum of some animals and at least one cofactor in the blood plasma, and it is with G-NH ₂Metabolism is activity form (" Aminoacetamide of modification " or metabolite X), and it is transported in the cell and suppresses duplicating of HIV.Therefore, G-NH ₂Be the precursor or the prodrug of antiretroviral chemical compound, and can be to G-NH ₂Prepare to use with described cofactor or the material that comprises described cofactor.Use chromatography to separate this cofactor.Can use methods described herein and molecular biological conventional method to come this cofactor is carried out purification, clone and order-checking.Therefore, some embodiments comprise and comprise G-NH ₂Or metabolism is G-NH ₂Chemical compound (for example, GPG-NH ₂) medicine and nutraceutical formulation, with described G-NH ₂Or metabolism is G-NH ₂Chemical compound (for example, GPG-NH ₂) be formulated in the mixture or with G-NH ₂The material (for example, with purification, concentrate or porcine blood serum, blood plasma or milk that unpack format exists horse serum, blood plasma or milk, Ox blood serum, blood plasma or milk) that changes metabolite X into is in conjunction with (at G-NH ₂Before or after using) use.

By with G-NH ₂In some serum or blood plasma, carry out incubation and easily prepare G-NH ₂Activated form (Aminoacetamide of modification or metabolite X), and easily separate the Aminoacetamide of modification by chromatography hereinafter described.In whole description, Aminoacetamide metabolite (the retrovirus activity form of Aminoacetamide) is referred to as " Aminoacetamide of modification ", " G-NH of modification ₂" or " fast peak Aminoacetamide ".The G-NH that modifies ₂Example include, but are not limited to Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer (NH ₂-glycine-O-O-glycine-NH ₂), two glycyl amidogen ether (NH ₂-glycine-O-glycine-NH ₂), the salt and/or the derivant of α-methoxyl group Aminoacetamide, α-ethyoxyl Aminoacetamide and these chemical compounds.Separating the back with chromatography discloses it to the mass spectrography of the Aminoacetamide peak fraction of isolating modification and the analysis of nuclear magnetic resonance, NMR (NMR) spectrometry and comprises the Alpha-hydroxy Aminoacetamide.Chemical compound α-peroxide Aminoacetamide dimer (NH ₂-glycine-O-O-glycine-NH ₂) can be than Alpha-hydroxy Aminoacetamide stable more and Alpha-hydroxy Aminoacetamide and α-methoxyl group Aminoacetamide can be prepared by organic synthesis.Use method as herein described and other available synthetic method, those skilled in the art can easily prepare the Aminoacetamide chemical compound of other modification.(seeing the JP 5097789A2 that licenses to Hayakawa etc. that for example submitted on October 3rd, 1991, " Alpha-hydroxyglycinamide Derivative andits Preparation ").Under the situation that the α HGA (Alpha-hydroxy Aminoacetamide) of synthetic property or enzymatic preparation exists, the HIV that carries out contaminates Journal of Sex Research and has disclosed chemical compound and suppressed duplicating of HIV in the human serum effectively.

With the G-NH that modifies ₂It is simple being mixed with medicine and medicament, the G-NH that no matter modifies ₂Be by synthesizing the property preparation or passing through with G-NH ₂It is synthetic that incubation carries out enzymatic in serum.Therefore, Aminoacetamide chemical compound (for example chemical compound that provides with formula A, B, C, D, E, F, G, H or I) or its officinal salt of the modification that can exist by the form that provides with any or both of any or both of enantiomer (L or D) or isomers (R or S) prepare retrovirus medicine and medicament.The preferred chemical compound that is used for preparing antiretroviral drugs or medicament comprises, for example, the Alpha-hydroxy Aminoacetamide (formula C) that exists with any or boths' of any or both of enantiomer (L or D) or isomers (R or S) form, α-peroxide Aminoacetamide dimer (formula E), two glycyl amidogen ethers (formula F) and α-methoxyl group Aminoacetamide or its officinal salt.Retrovirus medicine described herein and medicament can be with unit dosage forms (for example, tablet, capsule, soft capsule, liquid dosage form, injectable dosage form, dosage form transdermal or intranasal) provide, and except that the Aminoacetamide chemical compound of modifying, can comprise pharmaceutical carrier or excipient.The container that comprises the described medicine of in bulk or independent dosage form and medicament (for example, the bottle of aseptic bottle, diaphragm seal, bottle, wide mouthed bottle, syringe, aerosol apparatus, cotton are wiped away) also be embodiment, and preferably, prepare described preparation according to being proved to be to good production method (GMP) (for example be suitable for or accepted such as federal FAD (FDA)), and described container comprises label or other labelling of the described government regulator of reflection to described preparation permission by government regulator.But, the nutritional drugs that comprises described chemical compound that has or do not have structure-functional label also is an embodiment.

Some embodiments are to suppress the preparation of HIV, it is made up of the Aminoacetamide chemical compound of modifying or (for example concentrates with the Aminoacetamide chemical compound of modifying, be used to suppress medicine and the medicament of HIV, its by, substantially by the forming of the amount that suppresses virus replication with isolating, purification or the synthetic Aminoacetamide chemical compound that forms the modification that exists, or comprise the amount that suppresses virus replication with isolating, purification or synthesize the Aminoacetamide chemical compound of the modification that formation exists).Embodiment preferred comprises medicine or medicament, described medicine or medicament by, substantially by Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer (NH ₂-glycine-O-O-glycine-NH ₂), two glycyl amidogen ether (NH ₂-glycine-O-glycine-NH ₂), the derivant of α-methoxyl group Aminoacetamide, α-ethyoxyl Aminoacetamide or these chemical compounds forms, or comprises Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer (NH ₂-glycine-O-O-glycine-NH ₂), two glycyl amidogen ether (NH ₂-glycine-O-glycine-NH ₂), the derivant of α-methoxyl group Aminoacetamide, α-ethyoxyl Aminoacetamide or these chemical compounds.

When being used for this paper, " concentrating " refer to described concentration of material than its natural concentration up to 1000 times (for example), 0.01 weight % advantageously is preferably at least about 0.1 weight %.Expect that also spissated preparation is about 0.5 weight %, 1 weight %, 5 weight %, 10 weight % and 20 weight %.Term " isolating " requires material is removed (for example, if naturally occurring, promptly referring to natural surroundings) from its primal environment.Term " purification " does not require absolute purity; More suitably, it refers to a kind of relative definition.Can come the albumen of conventional purifies and separates by chromatography and/or gel electrophoresis.Special expectation is purified at least one order of magnitude with parent material or natural material, preferably two or three orders of magnitude, more preferably four or five orders of magnitude.

Embodiment subsequently describes a kind of method that purification is purchased Aminoacetamide that is used for.As mentioned below, the aspect of this method is the metabolite that is used for the Aminoacetamide that purification produces behind various animal serum incubations.

Embodiment 3

Observe unpurified when separating by cation exchange high performance liquid chromatography (HPLC) [ ¹⁴C] G-NH ₂During preparation, two groups of G-NH ₂Dissolving.(seeing Table 6).Use cation exchange column (as Partisphere SCX-Whattman), separated radiolabeled G-NH by HPLC ₂With radiolabeled GPG-NH ₂Crude preparation by using.Use following gradient: 0-15 minute (buffer A of forming by 5mM ammonium phosphate such as degree such as grade, pH3.5); Linear gradient from the buffer A to the buffer B (was made up of 250mM ammonium phosphate, pH3.5) in 15-40 minute; 40-45 minute buffer B; Linear gradient was to buffer A in 45-55 minute; With the buffer A of 55-60 minute degree such as grade, the post of next time testing with balance.

By this separation method, main thick [ ¹⁴C] GPG-NH ₂, typically at 26-28 minute eluting (fraction 26-28), still, the chemical compound of trace-level activity labelling is at 20-22 minute (fraction 20-22), 15-17 minute (fraction 15-17) and 2-3 minute (fraction 2-3) eluting.About 89% thick [ ¹⁴C] G-NH ₂Typically at 15-17 minute eluting (fraction 15-17) and about 11% thick [ ¹⁴C] G-NH ₂At 2-3 minute eluting (fraction 2-3).Also in fraction 20-22 and fraction 5-6, detect trace thick [ ¹⁴C] G-NH ₂

Slight change in buffer and the gradient has caused the slight change of the elution time of chemical compound, but, in all prepared products, detect two groups of main Aminoacetamides, wherein first group is eluted from the post rapidly and (is called fast peak, fraction 2-3 or fraction 3-4, or radiolabeled G-NH ₂In impurity, or the G-NH that modifies ₂), second group of strong bonded (is called slow peak, fraction 13-14, or fraction 15-17 or G-NH to post ₂).For example, separate the G-NH that modifies ₂Another kind of method also use buffer A (5mM ammonium phosphate pH3.5) and buffer B (250mM ammonium phosphate pH3.5).The gradient of these buffer that use is as follows: 10 minutes buffer A; Linear gradient was to buffer B in 6 minutes; 2 minutes in buffer B; 6 minutes linear gradients are to buffer A then; And in buffer A balance 6 minutes.In addition, by this method, G-NH ₂With at radiolabeled G-NH ₂In impurity eluted 10-11 minute and 2-3 minute respectively.

Table 6

[ ¹⁴C] radiolabeled GPG-NH ₂And G-NH ₂The impurity of stock solution

In this embodiment, the G-NH that provides purification to be purchased ₂Method.As mentioned below, improved this method has been used for the Aminoacetamide that purification is modified.Should be appreciated that for these methods, can utilize many different cation exchange columns and can use many different buffer and gradient.According to this description, those skilled in the art can adopt cation exchange column, FPLC or HPLC, buffer or the gradient of particular type to separate the G-NH that modifies rapidly ₂(metabolite X).That is, the those skilled in the art that are modified in of said method grasp in the scope, and suitable with method as herein described.

Described as part subsequently, discovery can be by the G-NH of incubation unmodified in various serum or blood plasma ₂(fraction 15-17) comes from wherein preparing the G-NH that modifies ₂(fraction 2-3).Then, can use one of said method to separate by this way the G-NH of the modification of (enzymatic preparation) preparation ₂The routine techniques of use in structural analysis determined the G-NH by the isolating modification of above-mentioned chromatography ₂Comprise the Alpha-hydroxy Aminoacetamide.

Originally, heated G-NH if observe the cell culture medium that comprises hyclone 30 minutes at 95 ℃ ₂Suppress the Disability that HIV duplicates.In some experiments, with people T-lymphocyte cem cell (about 4.5 * 10 ⁵Cell/ml) is suspended in the fresh culture and uses 100 CCID ₅₀HIV-1 (the III of/ml cell suspension _B) infect.The G-NH of various concentration is provided to the cell that infects subsequently, ₂, described G-NH ₂Be the G-NH that has been dissolved in the RPMI-1640 culture medium that comprises serum (10% hyclone in PBS) ₂, or be dissolved in the G-NH that comprises the RPMI-1640 culture medium of (at 30 minutes 10% hyclone in PBS of 95 ℃ of heating) in the heat-inactivated serum ₂Then, described cell suspension at 37 ℃ of incubations, after 4-5 days, is estimated duplicating of HIV.Find the G-NH of incubation in comprising the culture medium of heat-inactivated serum ₂Lost the ability that HIV duplicates that suppresses.These results provide favourable evidence, promptly be present in the serum to heat-labile albumen with G-NH ₂Metabolism is the G-NH of the modification of duplicating of inhibition HIV ₂Form.

Can be according to the thermal instability cofactor that is present in the hyclone with G-NH ₂Change this discovery of Aminoacetamide of antiretroviral activity form into, experimentize to determine whether this cofactor is present in human serum and the serum from other animal.The following examples are described these experiments in detail.

Embodiment 4

Logarithm batch human serum and hyclone are analyzed them with G-NH ₂Change the G-NH of modification into ₂Ability.G-NH with radiolabeled cation exchange HPLC purification ₂(seeing embodiment 3) is that 10% various serum carried out incubation 15 minutes and 1,6,24 or 72 hour in 37 ℃ with final concentration in PBS.Subsequently, use above-mentioned cation exchange HPLC method to estimate radiolabeled modification G-NH ₂Amount.The results are shown among Fig. 7 described.Behind the incubation 24 hours, each in 10 different people blood serum samples all shown G-NH ₂To the G-NH that modifies ₂Be less than 10% transformation.The hyclone of all tests has shown G-NH ₂To the G-NH that modifies ₂Remarkable transformation, wherein incubation changed 6-10% after 6 hours, changed 18-32% after 24 hours.These results have proved that hyclone comprises G-NH ₂Significantly metabolism is the G-NH of modification ₂Cofactor, human serum is then different.

Next, analyzed seroconversion G-NH from other animal ₂Be the G-NH that modifies ₂The evaluation carried out of ability.With porcine blood serum (PS), mice serum (MS), dog serum (CS), cat serum (FS), horse serum (ES) and monkey serum (SS) G-NH with the HPLC purification ₂Incubation together, and at 15 minutes, 1 hour, 6 hours and/or took out the mixture of aliquot in 24 hours, by cation exchange HPLC it is analyzed as mentioned above, about 10% serum dilution in PBS used.As shown in Figure 8, available from the serum of pig, Canis familiaris L., cat, horse and monkey rapidly with G-NH ₂Be converted into the G-NH of modification ₂, and mice serum metabolism G-NH ₂Poor ability.Although some animal capables of these data show are with G-NH ₂The G-NH of metabolism for modifying ₂, the cofactor metabolism G-NH in people and the mice ₂Ability on evolving, cannot not be conservative.

Some experiments have also been carried out to characterize the cofactor that is found in the porcine blood serum better.In one group of experiment, porcine blood plasma is dialysed (molecular weight holds back 10,000) and estimated described dialysate with G-NH ₂Change the G-NH of modification into ₂Ability.G-NH with various concentration ₂Mix with the porcine blood plasma of 90% porcine blood plasma or 90% dialysis, and in 37 ℃ of incubations 24 hours.Subsequently, as previously mentioned, separate the described mixture of aliquot, and estimate G-NH by cation exchange HPLC ₂To the G-NH that modifies ₂Transformation.Table 7 has shown these result of experiment.The porcine blood plasma sample of these data show porcine blood plasma and dialysis is with G-NH ₂Change the G-NH of modification into ₂Ability almost be identical.The saturation of the enzymatic activity of the cofactor in the porcine blood plasma (in PBS 90%) is present in 1,000 μ M and 10,000 μ M G-NH ₂Between.These results provide more evidence, are about to metabolism G-NH ₂The G-NH that becomes to modify ₂Described cofactor be the albumen that is found in blood plasma or the serum.

Table 7

The porcine blood serum (24 hours) of dialysis makes G-NH ₂Change the G-NH of modification into ₂

^aBlood plasma: in PBS 90%

In another group experiment, the saturation point of the cofactor in the porcine blood plasma that is found in dialysis is more closely investigated.The porcine blood plasma (in PBS 90%) of dialysis is mixed with the G-NH of concentration between 2,000 μ M and 10,000 μ M ₂Subsequently, as preceding with as described in mixture separate aliquot in 37 ℃ of incubations 6 hours and by cation exchange HPLC.The result who is shown in table 8 has confirmed that the cofactor saturation point in the porcine blood plasma approaches 2,000 μ M G-NH ₂

Table 8

The porcine blood plasma of dialysis ^α(6 hours) make G-NH ₂Change the G-NH of modification into ₂

^aBlood plasma: in PBS 90%

Also used aminoacid competition research and whether be specific to G-NH with the cofactor of determining to be present in the porcine blood serum ₂In these experiments, at 18 μ M G-NH ₂And competitor (10 μ M, 40 μ M, 100 μ M, 400 μ M, 1000 μ M, 4,000 μ M or 10,000 μ M glycine, 10,000 μ M L-serine-NH ₂, 10,000 μ M-alanine-NH ₂, 1000 μ M, 4,000 μ M or 10,000 μ M GPG-NH ₂) under the situation about existing, about 10% porcine blood serum that will be in PBS was in 37 ℃ of incubations 6 hours.Also estimated the contrast that does not contain competitor.Subsequently, as preceding, HPLC has analyzed G-NH by cation exchange ₂To the G-NH that modifies ₂Transformation.The result who is shown among Fig. 9 provides evidence, and promptly the cofactor that exists in the porcine blood serum is specific to G-NH ₂Although, GPG-NH ₂As if also to G-NH ₂Transformation inhibited.

In case verified certain serum comprises can be with G-NH ₂Change the G-NH of modification into ₂Cofactor, experimentize to separate described cofactor.These experiments of the following examples more detailed description.

Embodiment 5

Design in the experiment that separates cofactor at first group, adopt size exclusion chromatography (Superdex200) to separate the component that is present in the hyclone, described cofactor is with G-NH ₂Change the G-NH of modification into ₂Be separated in the milli Q water and continued 60 minutes and collect 30 fraction (0.5ml/min).By with the separation fraction of aliquot and the G-NH of HPLC purification ₂Incubation as measuring by cation exchange HPLC, is analyzed the G-NH that modifies subsequently together ₂Existence or disappearance determine the existence of cofactor in each fraction.As shown in Figure 10, most of cofactor elutes from size-exclusion column in fraction 10-12.As previously mentioned, the G-NH as modifying by the monitoring of HPLC cation-exchange chromatography ₂Gather and measured, fraction 10-12 is found effectively with G-NH ₂Change the G-NH of modification into ₂Find that also fraction 10-12 recovers G-NH in the serum of heating ₂HIV (human immunodeficiency virus)-resistant activity.The activity that detects in fraction afterwards can be such result, i.e. the cofactor of part degraded or with the cofactor of used resin non-specific interaction.This data acknowledgement be separated to G-NH ₂Change the G-NH of modification into ₂Cofactor.Can utilize the conventional art in protein purification and molecular biology that cofactor is carried out purification now, order-checking and clone.

With G-NH ₂Behind the serum incubation, utilize the cation exchange HPLC can be from G-NH by chromatographic technique ₂The middle G-NH that modifies that separates ₂(seeing that for example, embodiment 3), and can be with G-NH purification, that modify ₂The G-NH of (fraction 2-3) and purification ₂The HIV (human immunodeficiency virus)-resistant activity of (fraction 15-17) compares in the infectious test of traditional HIV.Can also analyze Aminoacetamide chemical compound (for example, Alpha-hydroxy Aminoacetamide, the α-peroxide Aminoacetamide dimer (NH of modification in this way ₂-gly-O-O-gly-NH ₂), two glycyl amidogen ether (NH ₂-gly-O-gly-NH ₂), α-methoxyl group Aminoacetamide, α-ethyoxyl Aminoacetamide, and/or its derivant) effect of duplicating for HIV.

For example, utilize the G-NH infectious measurements determination purification of aforesaid HIV, that modify ₂(fraction 2-3), the G-NH of purification ₂(fraction 15-17), Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer (NH ₂-gly-O-O-gly-NH ₂), two glycyl amidogen ether (NH ₂-gly-O-gly-NH ₂), α-methoxyl group Aminoacetamide, and the E of α-ethyoxyl Aminoacetamide or derivatives thereof ₅₀In brief, with human T lymphocyte's cem cell (about 4.5 * 10 ⁵Cell/ml) be suspended in the fresh culture also with HIV-1 (III _B) with about 100CCID ₅₀/ ml cell suspending liquid infects.Subsequently, the infection cell suspension of 100 μ l is transferred to (100 μ l/ hole) in each hole of microtitration plate and with the G-NH of the modification of the new dilution of 100 μ l ₂(fraction 2-3), G-NH ₂(fraction 15-17), Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer (NH ₂-gly-O-O-gly-NH ₂), two glycyl amidogen ether (NH ₂-gly-O-gly-NH ₂), α-methoxyl group Aminoacetamide, α-ethyoxyl Aminoacetamide, or derivatives thereof (for example, 2000,400,80,16,3.2 and 0.62 μ M) mixes.Subsequently, with mixture in 37 ℃ of incubations.After 4 to 5 days, by the formation of giant cell in the microscope record CEM culture.Measure 50% valid density (EC subsequently ₅₀).

To show the G-NH that modifies from this group result of experiment ₂(fraction 2-3), Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer (NH ₂-gly-O-O-gly-NH ₂), two glycyl amidogen ether (NH ₂-gly-O-gly-NH ₂), α-methoxyl group Aminoacetamide, α-ethyoxyl Aminoacetamide, or derivatives thereof has and G-NH ₂(fraction 15-17) is suitable, or compares G-NH ₂(fraction 15-17) lower EC ₅₀For example, the G-NH of modification ₂, Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer (NH ₂-gly-O-O-gly-NH ₂), two glycyl amidogen ether (NH ₂-gly-O-gly-NH ₂), α-methoxyl group Aminoacetamide, α-ethyoxyl Aminoacetamide, and derivant will have about 25 μ M or littler EC ₅₀, and G-NH ₂The EC that will have about 30 μ M ₅₀These experiments will provide more evidences, i.e. G-NH ₂Be metabolised to the G-NH of modification ₂, it is the activity form of antiviral agent.

As another example, for the G-NH that modifies ₂Hot inactivated serum (in 95 ℃ 30 minutes) or comprise and suppress the ability that HIV duplicates in the culture medium of human serum and compare.With human T lymphocyte (for example, about 4.5 * 10 ⁵The cem cell of cell/ml) is suspended in the fresh culture that comprises hyclone also with HIV-1 (III _B) with about 100CCID ₅₀/ ml cell suspending liquid infects.Subsequently, the cell that infects wash in PBS and is resuspended in comprise 10% in 95 ℃ of culture medium that heat 30 minutes hyclone or human serum.Next, the G-NH that the infection cell suspension of 100 μ l is transferred to (100 μ l/ hole) in each hole of microtitration plate and modified with the purification of the new dilution of 100 μ l ₂(fraction 2-3), Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer (NH ₂-gly-O-O-gly-NH ₂), two glycyl amidogen ether (NH ₂-gly-O-gly-NH ₂), α-methoxyl group Aminoacetamide, α-ethyoxyl Aminoacetamide, or derivatives thereof, or the G-NH of purification ₂(fraction 15-17) (for example, 2000,400,80,16,3.2 and 0.62 μ M) mixes.Subsequently, with mixture at 37 ℃ of incubations.Behind the incubation 4 to 5 days, by the formation of giant cell in the microscope record culture.Measure 50% valid density (EC subsequently ₅₀).The G-NH that will show the modification of purification from this group result of experiment ₂(fraction 2-3), Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer (NH ₂-gly-O-O-gly-NH ₂), two glycyl amidogen ether (NH ₂-gly-O-gly-NH ₂), α-methoxyl group Aminoacetamide, α-ethyoxyl Aminoacetamide, or derivatives thereof effectively are suppressed at duplicating of HIV among the hyclone that boiled or the human serum sample, and the G-NH of purification ₂(fraction 15-17) then can not.The following example is described such experiment, and its Alpha-hydroxy Aminoacetamide (metabolite X) that shows the enzymatic preparation effectively suppresses duplicating of HIV.

Embodiment 6

The Aminoacetamide of modifying is carried out zymetology production, separate, and analyze it and suppress the ability that HIV duplicates.Bag filter (the 3500kD molecular weight is held back) was shaken 30 minutes in room temperature in the distilled water with PEST buffer (RPMI with streptomycin and penicillin), in 2% sodium bicarbonate and 1mM EDTA, shook 30 minutes subsequently in 60 ℃.With bag filter twice of rinsing in having the distilled water of PEST.Bag filter during having the distilled water of PEST boiled 5 minute thereafter.After boiling, bag filter is transferred in the beaker that is full of PBS+PEST, and is stored in+4 ℃ stand-by.

After boiling 20 days, use bag filter.On aseptic platform, with aseptic and deionized water wash bag filter.The porcine blood serum (Promeda corp.) that approximately in bag filter, adds 10ml.Bag filter is put in the glass beaker that has been full of 200ml PBS-A/PEST (1mlPEST+1L PBS-A).Beaker is taken out from aseptic and place on the track shaking table.After 1 hour, replace PBS-A/PEST=" prewashing " with the fresh PBS-A of 200ml.As mentioned above, with five parts of PBS-A with bag filter prewashing five times 1 hour.After the prewashing, the bag filter that will comprise serum is transferred in the aseptic vial of the 1mM Aminoacetamide (Bachem) that is full of 100ml aseptic filtration and magnetic agitation bar.The bottle that will comprise Aminoacetamide and serum carries out incubation in 37 ℃ on the magnetic agitation plate.After about 48 hours, stop dialysis, dialysis solution is divided into three parts (10ml+38ml+50ml) and transfers in the vial that labelling crosses, bottle sealing and freezing in-85 ℃.Get a refrigerated dialysis solution subsequently and carry out lyophilizing.

Prepare freeze-drying system (Vacuum oil (Heto 88900100), Milli-Q water, water purification equipment, freeze dryer and-85 ℃ of refrigerators).Refrigerated dialysis solution (from 38ml part of 1-1) is transferred in the cryodesiccation chamber from-85 ℃ of refrigerators.Place lid on the chamber and open vacuum.Stop freeze-drying process after about 72 hours.Close vacuum and vial is removed from cryodesiccation chamber.

Next, by the freeze dried product of HPLC purification.By taking by weighing 27.22g KH ₂PO ₄And it is dissolved in the 2L water 0.1M KH of the about 2L of preparation ₂PO ₄(Merck no.14873-250/Lot:A397373251) (pH about 4.06).With mobile phase (90%0.1M KH ₂PO ₄/ 10% acetonitrile (Scharlau AC0329/Batch:57048)) with 5ml/min with post (Hypersil SCX ion exchange column 5 μ m/250 * 10mm (ThermoQuest 3-34087/ lot number: 5/100/5580) and comprise the HPLC system of software D-7000 HSM) balance 60 minutes.The ultraviolet detector wavelength is made as 206nm.Exsiccant dialysis " sample " is dissolved in the 2ml water (having the 19mM Aminoacetamide during dialysis beginning) and inject and with 5ml/min with mobile analyze (RUN-1) of degree such as mobile phase (see above) 10 minutes.The volume injected of RUN-1 is about 100 μ l.

After the calibration, inject the sample (RUN-2 → 10) of nine times 200 μ l again and utilize the gathering-device of TIME pattern each run to be collected in the fraction of 2.5-3.1 minute eluting with the 0.1min/ fraction.Between RUN-8 and 9, by taking by weighing 13.61g KH ₂PO ₄And it is dissolved in the 1L water 0.1M KH of preparation 1L ₂PO ₄Be incorporated in the corresponding fraction of collecting in RUN-2 → 10 and be injected in (RUN-11 → 16) on the post.In RUN-11 → 16, per injection all comprises about 100 μ l.Collect the fraction between 2.6 to 2.8 minutes and merge.As by the amount of initial Aminoacetamide with collect that the area at peak measures, obtained the modification Aminoacetamide (metabolite X) of about 1.25mg.With 7.5ml mobile phase (90%0.1M KH ₂PO ₄The 2.6-2.8 of the merging/10% acetonitrile) minute fraction is transferred in the vial that labelling crosses, with described bottle sealing and freezing in-85 ℃.In addition, 7.5ml mobile phase is freezingly contrasted as salt in-85 ℃.HPLC analyzes and shows that all detectable Aminoacetamides (about 5.9min of holdup time) have all changed the Aminoacetamide (about 2.7min) of modification into.Behind the analysis/purification, with 40% acetonitrile/water column scrubber 31 minutes, and utilize said method that the Aminoacetamide (" metabolite X ") of the modification of enzymatic preparation is carried out lyophilizing with 5ml/min.

Aminoacetamide (MetX) with the modification of enzymatic preparation carries out the infectious test of HIV subsequently.Freeze dried MetX (1.25mg) is dissolved in the 7.5ml sterile distilled water (2.24mM MetX).Every kind of RPMI++ (the RPMI-culture medium with 10%FCS and 0.1%PEST) normal and that boil of 2.24mM MetX and 4.8ml of about 3.7ml is mixed.That is the 1mM MetX of two crowdes of 8.5ml of preparation.Subsequently, 1mM MetX and the every kind of RPMI++ (that is the 500 μ M MetX of 2 * 6ml) normal and that boil of 3ml with about 3ml mixes.Every kind of RPMI++ normal and that boil of 500 μ M MetX and 4ml with about 1ml mixes subsequently, produces the 100 μ M MetX of 2 * 5ml.Freeze dried salt contrast is accurately dissolved in the same manner and diluted with top MetX.Also as described for MetX, the unmodified Aminoacetamide stock solution of utilizing 1mM prepares the Aminoacetamide of 100 μ M in normal and the RPMI++ (contrast) that boils.

In three A of Burke chamber are square, the H9 cell is counted (average 1.2 * 10 ⁶Cell/ml, it is 4 * 10 ⁶Cell is in 3.3ml).In two 50ml test tubes, add about 4 * 10 ⁶Cell (3.3ml).Next, the normal RPMI++ of about 14.7ml is joined in first test tube and the RPMI++ that boils of about 14.7ml is joined in second test tube (that is the RPMI++ of 18mlH9 cell+normal/boil).Then the virus of about 2ml is preserved liquid (SF2+H9, the 9th day: 22/3-02 2) and join in the 50ml test tube of each cell that comprises about 20ml/ pipe and culture medium, and solution is mixed.Two parts of viruses/cell mixture branch is gone in two new 50ml test tubes (that is four test tubes with 10ml cell/virus (two test tube with normal RPMI++ and two have the test tube that boils RPMI++))))))))).After 50 minutes with cell/viral test tube mixing incubation in 37 ℃ 90 minutes.Stop to infect (in 1200rpm5 minute) by collecting cell.Subsequently that cell is resuspended and transfer in 12 10ml test tubes (0.5 * 10 ⁶Cell/pipe).That is, the cell suspension of six test tubes in normal RPMI++ and the cell suspension of six test tubes in the RPMI++ that boils.With RPMI (no additive) washed cell and collect (in 1500rpm5 minute).Abandoning supernatant also is resuspended in cell in following every kind of 4.5ml:

Normal RPMI++

The RPMI++ that boils

100 μ M Aminoacetamides are in normal RPMI++

100 μ M Aminoacetamides are in the RPMI++ that boils

500 μ M MetX are in normal RPMI++

500 μ M MetX are in the RPMI++ that boils

100 μ M MetX are in normal RPMI++

100 μ lM MetX are in the RPMI++ that boils

500 μ M salt are in normal RPMI++

500 μ M salt are in the RPMI++ that boils

100 μ M salt are in normal RPMI++

100 μ M salt are in the RPMI++ that boils

Approx, the following every kind of cell suspending liquid (every kind of four times of repetitions) that adds the 0.9ml/ hole to 48 orifice plates:

Plate 1:

4 holes have 100 μ M Aminoacetamides in normal RPMI++

4 holes, 100 μ M Aminoacetamides are in the RPMI++ that boils

4 holes have 500 μ M MetX in normal RPMI++

4 holes have 500 μ M MetX in the RPMI++ that boils

4 holes have 100 μ M MetX in normal RPMI++

4 holes have 100 μ M MetX in the RPMI++ that boils

Plate 2:

The untreated normal RPMI++ in 4 holes

The untreated RPMI++ that boils in 4 holes

4 holes " 100 μ M " salt is in normal RPMI++

4 holes " 100 μ M " salt is in the RPMI++ that boils

4 holes " 500 μ M " salt is in normal RPMI++

4 holes " 500 μ M " salt is in the RPMI++ that boils

Remaining hole is full of sterile distilled water.With Tissue Culture Plate in 37 ℃ and 5%CO ₂Middle incubation.Change culture medium after four days, change culture medium and collecting cell after eight days.After 11 days, stop to infect observation of cell and collect every kind of cell conditioned medium liquid of 650 μ l and be frozen in-80 ℃ and be used for further analysis in 10 * zoom microscope.After five days, melt supernatant and be used for conventional reverse transcription (RT) activity analysis (for example, Roche AMPLICOR MONITOR ^TM) or the p24 quantitative analysis (for example, Abbott Laboratories, Chicago).(see U.S. Patent number 6,258,932 and Application No. 10/235,158).The results are shown among Figure 11 and the TABLE 9.

Table 9

Sample	The syncytium that can see
Sample	The syncytium that can see	100 μ M MetX are in normal RPMI++	Negative
100 μ M MetX are in the RPMI++ that boils	Negative	100 μ M MetX are in normal RPMI++	Negative
100 μ M MetX are in the RPMI++ that boils	Negative	500 μ M MetX are in normal RPMI++	Negative
500 μ M MetX are in the RPMI++ that boils	Negative	500 μ M MetX are in normal RPMI++	Negative
500 μ M MetX are in the RPMI++ that boils	Negative	100 μ M Aminoacetamides are in normal RPMI++ contrast	Negative
100 μ M Aminoacetamides are in the RPMI++ contrast of boiling	Positive	100 μ M Aminoacetamides are in normal RPMI++ contrast	Negative
	Positive	Untreated normal RPMI contrast	Positive
The untreated RPMI that boils contrasts	Positive	Untreated normal RPMI contrast	Positive
The untreated RPMI that boils contrasts	Positive	100 μ M salt contrast in normal RPMI++	Positive
100 μ M salt contrast in the RPMI++ that boils	Positive	100 μ M salt contrast in normal RPMI++	Positive
100 μ M salt contrast in the RPMI++ that boils	Positive	500 μ M salt contrast in normal RPMI++	Negative
500 μ M " salt is in the RPMI++ that boils	Negative	500 μ M salt contrast in normal RPMI++	Negative

Check that by the educational inspector Aminoacetamide of modification (metabolite X) effectively suppresses duplicating and/or breeding of HIV in the hyclone that boiled, but Aminoacetamide then can not (table 9).Reverse transcription (RT) activity data (Figure 11) has been proved conclusively the Aminoacetamide of modifying (Met-X or metabolite X) and effectively suppressed duplicating of HIV in the hyclone sample that boiled, even G-NH ₂Can not suppress duplicating of HIV under these conditions.That is, the antiviral activity of the Aminoacetamide of modification (MetX) does not need to be present in cofactor in the hyclone but the Aminoacetamide needs.These data also show feasible enzyme (cofactor) degeneration that Aminoacetamide is changed into the Aminoacetamide of modification of heating hyclone.

In the relevant experiment of another group, compare with the metabolite X for preparing by above method five times the anti-retroviral of metabolite X of dialysing is active.In brief, use G-NH ₂, as above, in hyclone, carry out the infectious test of HIV with the metabolite X of five dialysis with by the metabolite X that above method prepares.These result of experiment are shown among Figure 12.The standardized products of the Alpha-hydroxy Aminoacetamide (metabolite X) that produces with zymetology is compared, and does not observe the active significant change of Alpha-hydroxy Aminoacetamide (metabolite X) of five dialysis.

By mass spectral analysis and NMR the Aminoacetamide of the modification that obtains according to above-mentioned Enzymology method is analyzed, structural analysis discloses Alpha-hydroxy Aminoacetamide (" α HGA ").Thereby the experiment in the present embodiment has shown that the Aminoacetamide (Alpha-hydroxy Aminoacetamide or metabolite X) of modification effectively suppresses duplicating of HIV under the situation of disappearance cofactor, and described cofactor is present in the hyclone, is G-NH ₂The anti-retroviral activity necessary.Alpha-hydroxy Aminoacetamide (" α HGA ") also also as described below being found in of being synthesized property ground preparation suppresses duplicating of HIV under the situation that lacks cofactor.

In more experiments, in comprising the cell culture of hyclone, analyze the 50% inhibition concentration (IC of metabolite X ₅₀).Following examples are introduced these experiments in further detail.

Embodiment 7

With about 0.1 * 10 ⁶ H9 cell 50 TCID ₅₀HIV (SF2 virus) infects and the metabolite X (see embodiment 6) of infected cell with the enzymatic preparation is handled with various concentration.Hyclone is included in the test.With cell culture 10 days (the 7th day in culture, add fresh culture), collect supernatant thereafter and quantize to test and analyze by classical inverse transcriptase (RT).Data show is in Figure 13.The result shows that effective inhibition that HIV duplicates betides among the metabolite X of low concentration (for example, between 3.9 μ M-15.6 μ M) and reaches 15.6 μ M or when higher, do not observe the inhibition that HIV duplicates when concentration.

In more experiment, H9 cell (SF2 virus) incubation that the Aminoacetamide (metabolite X) of the modification of enzymatic preparation is infected with HIV also will be handled viral morphology and send to by ultramicroscope and analyze.As positive control, use GPG-NH ₂(seeing U.S. Patent number 6,258,932, a kind of method of carrying out the ultramicroscope experiment of these types).The following examples are introduced these experiments in further detail.

Embodiment 8

By a kind of method, by the purification G-NH of anti-porcine blood serum ₂The Aminoacetamide (metabolite X) (seeing embodiment 6) modified of dialysis enzymatic preparation; Utilize the Aminoacetamide of described modification to handle the H9 cell that HIV (SF2 virus) infects subsequently, and the cell that infects sent to carry out electron-microscopic analysis.In brief, bag filter (3500MW holds back-composes) loaded with porcine blood serum (Biomedia) and with porcine blood serum with respect to the pre-dialysis of RPMI 1640 buffer four times, each one hour to remove less than 3500 daltonian molecules.Subsequently with the serum of pre-wash G-NH with respect to the 1mM purification ₂In RPMI 1640, dialysed 48 hours for 37 ℃.Carry out aseptic filtration as dialysis buffer liquid subsequently, be divided into aliquot and freezing at the Aminoacetamide that will comprise modification described in the embodiment 6 (metabolite X).

Next, four (each) contain in 10mlRPMI (containing hyclone) about 0.5 * 10 ⁶100 μ M metabolite X or 100 μ MGPG-NH have been established in the bottle of individual H9 cell ₂Concentration.Cell in the sample is counted and centrifugal subsequently.Subsequently cell is resuspended in RPMI 1640 (containing hyclone) and 100 μ M metabolite X or the 100 μ M GPG-NH of 10ml ₂In.The contrast and the untreated control sample that do not infect are also included within the experiment.Subsequently with sample in 37 ℃ of 5%CO ₂Carry out incubated overnight.

Subsequently, the p24 that utilizes conventional p24 to detect in the test analysis sample measures (seeing U.S. Patent number 6,258,932).As shown in Figure 14, Aminoacetamide (metabolite X) or the 100 μ M GPG-NH that 100 μ M modify when hyclone exists ₂Effectively suppress duplicating of HIV; And untreated control sample shows that observable HIV duplicates.These results are proved conclusively by a kind of reverse transcription of routine (RT) active testing, and described test is presented at the reverse transcriptase activity of observable amount in the untreated control sample, but at the Aminoacetamide or the 100 μ MGPG-NH that modify with 100 μ M ₂There is not reverse transcriptase activity in the sample of handling.Verified Aminoacetamide or the 100 μ M GPG-NH that modify with 100 μ M ₂After the sample of handling comprises repressed virus, sample sent to by ultramicroscope analyze.

By a kind of method, can will be subjected to the H9 cell fixation of SF2 viral infection in 2.5% glutaraldehyde by conventional means.Subsequently with fixed cell at 1%OsO ₄In to carry out the back fixing and dewater, carry out embedding with epoxy resin, make the piece polymerization.It is thin that about 60-80nm is made in the cell epoxylite section of viral infection, so that hold the width of nucleocapsid.Section is placed with analyzing with the accelerating potential of 80kV on the painted grid of 1.0% uranium acetate and at Zeiss CEM 902 microscopes.Described microscope is equipped with spectrometer to improve picture quality and to use a kind of cooled with liquid nitrogen device to reduce the electron beam damage.In several blind tests to having contrast GPG-NH ₂The grid of the section of incubation cell and metabolite X incubation cell is checked.

The particulate electron micrograph of untreated HIV is with the RNA of the conical nucleocapsid of indicating characteristic and the level dyeing of enclosing, and described RNA stretches the length of nucleocapsid; And use GPG-NH ₂Or the particulate cell of HIV-1 that has that metabolite X handles has the HIV-1 granule of complete relatively conical shell body structure with demonstration, but RNA is gathered in the top (wide end) of hull outside or housing with the configuration of ball sample.Can observe some from GPG-NH ₂Or the housing of the sample handled of metabolite X has odd-shaped structure,, its with normal nucleocapsid seldom or do not have morphology similar, and RNA can be outside structure, structure inboard that also can be at one end.

Still in more experiment, to G-NH ₂, GPG-NH ₂, the anti-retroviral activity of the Aminoacetamide (α HGA) of the Aminoacetamide (metabolite X) of the modification of enzymatic preparation and the modification of synthetic property preparation compares.The following examples are introduced these experiments in further detail.

Embodiment 9

(seeing embodiment 6-8) described in pro-embodiment carries out the infectious test of HIV under the situation that hyclone exists, but is to use the G-NH of various concentration ₂, GPG-NH ₂And the Aminoacetamide (α HGA) of the modification of the Aminoacetamide (metabolite X) of the modification of enzymatic preparation and the synthetic property production of 100 μ M.(seeing Table 10).The triple samples (" repetition ") that do not infect sample and untreated samples are also included within the experiment in contrast.Monitor the inhibition that HIV duplicates by the level of utilizing the conventional sense test kit to quantize p24.

Table 10

Peptide	Concentration	Sample
Peptide	Concentration	Sample	GPG-NH ₂	100μM 50μM 25μM 12.5μM 6.25μM 3.1μM 1.6μM 0.8μM	The treble of each concentration is multiple
G-NH ₂	100μM 50μM 25μM 12.5μM 6.25μM 3.1μM 1.6μM 0.8μM	The treble of each concentration is multiple	GPG-NH ₂	100μM 50μM 25μM 12.5μM 6.25μM 3.1μM 1.6μM 0.8μM	The treble of each concentration is multiple

Met-X (preparing) by the dialysis enzymatic	100μM 50μM 25μM 12.5μM 6.25μM 3.1μM 1.6μM 0.8μM	The treble of each concentration is multiple
Met-X (preparing) by the dialysis enzymatic	100μM 50μM 25μM 12.5μM 6.25μM 3.1μM 1.6μM 0.8μM	The treble of each concentration is multiple	α HGA (producing) by chemical method is synthetic	100μM	Treble is multiple

Figure 15 shows some result of these experiments.As directed, at the 11st day of experiment, synthetic Alpha-hydroxy Aminoacetamide (α HGA) and the GPG-NH that produces ₂Suppressing HIV equally effectively in containing the culture medium of hyclone duplicates.Similar result also observed at the 7th day.The synthetic Alpha-hydroxy Aminoacetamide of producing (α HGA) of this data show suppresses HIV effectively and duplicates.

Still in more experiment, under the situation of people or hyclone existence, the enzymatic anti-retroviral activity with the Alpha-hydroxy Aminoacetamide preparation of synthetic property preparation is compared.The following examples are introduced these experiments in further detail.

Embodiment 10

(seeing embodiment 6-8) described in pro-embodiment carries out the infectious test of HIV under the situation of human serum or hyclone existence, but is to use the G-NH of various concentration ₂, the Aminoacetamide (α HGA) of the modification of the Aminoacetamide (metabolite X) of the modification of enzymatic preparation and the synthetic property production of 100 μ M.(seeing Table 11 and 12).The treble that does not infect sample and untreated samples is also included within the experiment in contrast again.

Table 11

Human serum

Peptide	Concentration	Sample
Peptide	Concentration	Sample	G-NH ₂	100μM 50μM	The treble of each concentration is multiple

Met-X (preparing) by the dialysis enzymatic	100μM 50μM	The treble of each concentration is multiple
Met-X (preparing) by the dialysis enzymatic	100μM 50μM	The treble of each concentration is multiple	α HGA (by chemical method synthetic property preparation)	50μM	Treble is multiple
The not contrast of Gan Raning	0μM	Treble is multiple	α HGA (by chemical method synthetic property preparation)	50μM	Treble is multiple
The not contrast of Gan Raning	0μM	Treble is multiple	The contrast of infecting	0μM	Treble is multiple

Table 12

Hyclone

Peptide	Concentration	Sample
Peptide	Concentration	Sample	G-NH ₂	100μM 50μM	The treble of each concentration is multiple
Met-X (preparing) by the dialysis enzymatic	100μM 50μM	The treble of each concentration is multiple	G-NH ₂	100μM 50μM	The treble of each concentration is multiple
Met-X (preparing) by the dialysis enzymatic	100μM 50μM	The treble of each concentration is multiple	α HGA (by chemical method synthetic property preparation)	50μM	Treble is multiple
The not contrast of Gan Raning	0μM	Treble is multiple	α HGA (by chemical method synthetic property preparation)	50μM	Treble is multiple
The not contrast of Gan Raning	0μM	Treble is multiple	The contrast of infecting	0μM	Treble is multiple

These result of experiment are provided in table 13 and 14 and in Figure 16 A and 16B.Data show is at the 12nd day, the Aminoacetamide (metabolite X) of the modification of enzymatic preparation and the Alpha-hydroxy Aminoacetamide (α HGA) and the G-NH of synthetic property production ₂Suppressing HIV equally effectively in containing the culture medium of hyclone duplicates; Yet, have only the Aminoacetamide (metabolite X) of modification of enzymatic preparation and the Alpha-hydroxy Aminoacetamide (α HGA) of synthetic property production can in human serum, suppress duplicating of HIV.That is G-NH, ₂Can not in human serum, suppress duplicating of HIV, but the Aminoacetamide (metabolite X) of the modification of enzymatic preparation and the Alpha-hydroxy Aminoacetamide (α HGA) of synthetic property production all are effective inhibitor that HIV duplicates in human serum.Similar result observed at the 7th day.These data provide strong evidence, i.e. the Aminoacetamide (metabolite X) of the modification of enzymatic preparation and the Alpha-hydroxy Aminoacetamide (α HGA) of synthetic property production all are the strong inhibitor that HIV duplicates in the people who infects.

Table 13

Hyclone

	OD1	OD2	Average OD	Average OD-blank	P24 concentration (ng/ml)
	OD1	OD2	Average OD	Average OD-blank	P24 concentration (ng/ml)	100μM G-NH2(1)	0.078	0.075	0.077	0.035	0.09
100μM G-NH2(2)	0.071	0.069	0.070	0.028	0.08	100μM G-NH2(1)	0.078	0.075	0.077	0.035	0.09
100μM G-NH2(2)	0.071	0.069	0.070	0.028	0.08	100μM G-NH2(3)	0.077	0.071	0.074	0.032	0.09
50μM G-NH2(1)	0.319	0.335	0.327	0.285	0.49	100μM G-NH2(3)	0.077	0.071	0.074	0.032	0.09
50μM G-NH2(1)	0.319	0.335	0.327	0.285	0.49	50μM G-NH2(2)	0.182	0.183	0.183	0.141	0.26
50μM G-NH2(3)	0.105	0.103	0.104	0.062	0.14	50μM G-NH2(2)	0.182	0.183	0.183	0.141	0.26
50μM G-NH2(3)	0.105	0.103	0.104	0.062	0.14	100μM Met-X(1)	0.193	0.343	0.268	0.226	0.40
100μM Met-X(2)	0.081	0.107	0.094	0.052	0.12	100μM Met-X(1)	0.193	0.343	0.268	0.226	0.40
100μM Met-X(2)	0.081	0.107	0.094	0.052	0.12	100μM Met-X(3)	0.144	0.152	0.148	0.106	0.21
50μM Met-X(1)	1.105	1.089	1.097	1.055	1.71	100μM Met-X(3)	0.144	0.152	0.148	0.106	0.21
50μM Met-X(1)	1.105	1.089	1.097	1.055	1.71	50μM Met-X(2)	1.895	1.887	1.891	1.849	2.98
50μM Met-X(3)	2.351	2.230	2.291	2.249	3.61	50μM Met-X(2)	1.895	1.887	1.891	1.849	2.98
50μM Met-X(3)	2.351	2.230	2.291	2.249	3.61	50μM αHGA (1)	0.183	0.185	0.184	0.142	0.26
50μM αHGA(2)	0.232	0.216	0.224	0.182	0.33	50μM αHGA (1)	0.183	0.185	0.184	0.142	0.26
50μM αHGA(2)	0.232	0.216	0.224	0.182	0.33	50μM αHGA (3)	0.147	0.139	0.143	0.101	0.20
0μM(1/500)(1)	0.691	0.717	0.704	0.662	544.90	50μM αHGA (3)	0.147	0.139	0.143	0.101	0.20
0μM(1/500)(1)	0.691	0.717	0.704	0.662	544.90	0μM(1/500)(2)	0.673	0.637	0.655	0.613	505.98
0μM(1/500)(3)	0.544	0.568	0.556	0.514	427.33	0μM(1/500)(2)	0.673	0.637	0.655	0.613	505.98
0μM(1/500)(3)	0.544	0.568	0.556	0.514	427.33	Contrast 1)	0.042	0.039	0.041	-0.001	0.04
Contrast (2)	0.042	0.037	0.040	-0.002	0.03	Contrast 1)	0.042	0.039	0.041	-0.001	0.04
Contrast (2)	0.042	0.037	0.040	-0.002	0.03	Contrast (3)	0.046	0.045	0.046	0.004	0.04

Table 14

Human serum

	OD1	OD2	Average OD	Average OD-blank	P24 concentration (ng/ml)
	OD1	OD2	Average OD	Average OD-blank	P24 concentration (ng/ml)	100μM G-NH2(1/500) (1)	1.194	1.196	1.195	1.111	780.21
100μM G-NH2(1/500) (2)	1.184	1.221	1.203	1.119	785.24	100μM G-NH2(1/500) (1)	1.194	1.196	1.195	1.111	780.21
100μM G-NH2(1/500) (2)	1.184	1.221	1.203	1.119	785.24	100μM G-NH2(1/500) (3)	1.315	1.362	1.339	1.255	876.34
50μM G-NH2(1/500)(1)	1.079	1.114	1.097	1.013	714.23	100μM G-NH2(1/500) (3)	1.315	1.362	1.339	1.255	876.34
50μM G-NH2(1/500)(1)	1.079	1.114	1.097	1.013	714.23	50μM G-NH2(1/500)(2)	0.996	1.015	1.006	0.922	653.27
50μM G-NH2(1/500)(3)	1.176	1.194	1.185	1.101	773.51	50μM G-NH2(1/500)(2)	0.996	1.015	1.006	0.922	653.27
50μM G-NH2(1/500)(3)	1.176	1.194	1.185	1.101	773.51	100μM Met-X(1/100) (1)	0.117	0.114	0.116	0.032	11.41
100μM Met-X(1/100) (2)	0.269	0.281	0.275	0.191	32.78	100μM Met-X(1/100) (1)	0.117	0.114	0.116	0.032	11.41
100μM Met-X(1/100) (2)	0.269	0.281	0.275	0.191	32.78	100μM Met-X(1/100) (3)	0.377	0.378	0.378	0.294	46.52
50μM Met-X(1/500)(1)	0.698	0.728	0.713	0.629	457.33	100μM Met-X(1/100) (3)	0.377	0.378	0.378	0.294	46.52
50μM Met-X(1/500)(1)	0.698	0.728	0.713	0.629	457.33	50μM Met-X(1/500)(2)	0.676	0.662	0.669	0.585	427.85
50μM Met-X(1/500)(3)	0.418	0.422	0.420	0.336	261.05	50μM Met-X(1/500)(2)	0.676	0.662	0.669	0.585	427.85
50μM Met-X(1/500)(3)	0.418	0.422	0.420	0.336	261.05	50μM αHGA(1)	1.546	1.546	1.546	1.462	2.03
50μM αHGA(2)	1.183	1.219	1.201	1.117	1.57	50μM αHGA(1)	1.546	1.546	1.546	1.462	2.03
50μM αHGA(2)	1.183	1.219	1.201	1.117	1.57	50μM αHGA(3)	0.665	0.679	0.672	0.588	0.86
0μM(1/1000)(1)	0.887	0.857	0.872	0.788	1127.68	50μM αHGA(3)	0.665	0.679	0.672	0.588	0.86
0μM(1/1000)(1)	0.887	0.857	0.872	0.788	1127.68	0μM(1/1000)(2)	0.827	0.791	0.809	0.725	1043.27
0μM(1/1000)(3)	0.472	0.472	0.472	0.388	591.77	0μM(1/1000)(2)	0.827	0.791	0.809	0.725	1043.27
0μM(1/1000)(3)	0.472	0.472	0.472	0.388	591.77	Contrast (1)	0.095	0.089	0.092	0.008	0.08
Contrast (2)	0.091	0.089	0.090	0.006	0.08	Contrast (1)	0.095	0.089	0.092	0.008	0.08
Contrast (2)	0.091	0.089	0.090	0.006	0.08	Contrast (3)	0.081	0.089	0.085	0.001	0.07

In the experiment of another series, the Alpha-hydroxy Aminoacetamide (α HGA) of the involutory preparation that becomes second nature is analyzed for prolonging heating stability at 37 ℃.To synthesize α HGA (C ₂H ₇ClN ₂O ₂) dilute sample compare in the activity of the α HGA of 37 ℃ of incubations period and subsequently that the anti-retroviral of incubation chemical compound is active and new dilution.Introduce these experiments in the following embodiments in further detail.

Embodiment 11

(seeing embodiment 6-8) described in pro-embodiment carries out the infectious test of HIV under the situation that hyclone exists, but is to use the G-NH of various concentration ₂, the Aminoacetamide (α HGA) of the modification of synthetic property production, and at the Aminoacetamide (α HGA 37) of the modification of three days synthetic property production of 37 ℃ of incubations.(seeing Table 15).The treble that does not infect sample and untreated samples is also included within the experiment in contrast again.

Table 15

Peptide	Concentration	Sample
Peptide	Concentration	Sample	αHGA	32μM 16μM 8μM 4μM 2μM 1μM 0.5μM	The treble of each concentration is multiple
α HGA 37 (37 ℃ of incubations three days)	32μM 16μM 8μM 4μM 2μM 1μM 0.5μM	The treble of each concentration is multiple	αHGA	32μM 16μM 8μM 4μM 2μM 1μM 0.5μM	The treble of each concentration is multiple

G-NH ₂

32μM 16μM 8μM 4μM 2μM 1μM 0.5μM

The treble of each concentration is multiple

These result of experiment are shown among Figure 17 and the table 16.Figure 17 is presented at the 7th day active chart of RT that detects.When analyzing the RT activity, obtain similar result at the 11st day.The α HGA of the synthetic property preparation of data show is suitable for 37 ℃ of incubations three days at least.In the anti-retroviral activity of the chemical compound that the α of fresh dilution HGA and incubation are crossed, observe minimum difference.In addition, the observable inhibition that these data show HIV duplicates betides among the synthetic property α HGA that concentration is higher than 8 μ M (carry out Overheating Treatment or do not have), better the anti-retroviral activity is observed on the concentration that is higher than 16 μ M, and the extremely effectively inhibition that HIV duplicates sees on the concentration that is higher than 30 μ M.Interesting is to change G-NH by hyclone in analysis ₂Formed metabolite X (sees for G-NH ₂The data of sample) the α HGA than synthetic property purification has more activity, and this provides evidence, promptly a kind of enantiomer of α HGA and/or ratios of the isomers other have a higher anti-retroviral activity.

Table 16

Following joint is described the application of duplicating of system each and these combination treatments, prevention and/or inhibition HIV of the medicine of the Aminoacetamide that comprises modification.

The chemical compound that suppresses HIV

As discussed above, except G-NH ₂With the G-NH that modifies ₂, G-NH ₂Some derivant and metabolite suppress that HIV duplicates and these chemical compounds can ingredients become medicament or medicine, it can be used in and suppresses HIV and duplicate and treat and/or prevent HIV and infect.Some medicament or medicine are made up of compound or pharmaceutically acceptable salt thereof, amide, ester or the prodrug of chemical formula A, basic composition, or comprise them:

Wherein:

A) E is selected from by oxygen, sulfur and NR ₇In the group of forming;

B) T is selected from by oxygen, sulfur and NR ₈In the group of forming; With

Therefore as described herein, the term " G-NH of modification ₂Or the Aminoacetamide chemical compound of modifying " comprise the derivant and the metabolite of Aminoacetamide, all suc as formula A those, no matter concentrating or separate from cell still is synthetic property preparation (for example, Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer (NH ₂-gly-O-O-gly-NH ₂), α-methoxyl group Aminoacetamide, α-ethyoxyl Aminoacetamide and/or its derivant).

As mentioned above, Aminoacetamide extracts some these chemical compounds and by mass spectrography and nuclear magnetic resonance, NMR (NMR) spectra methods it is identified from the HPLC post in serum behind the incubation.As described below, can be from synthetic these chemical compounds of obtainable parent material and derivant or related compound.

Term " pharmaceutical salts " refers to a kind of compound formulation, and it does not cause being applied biological obvious stimulation and not eliminating biologic activity and compound property.Can obtain pharmaceutical salts by making reactions such as all example hydrochloric acids of The compounds of this invention and mineral acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethyl sulfonic acid, right-toluenesulfonic acid, salicylic acid.Can also obtain pharmaceutical salts by The compounds of this invention and alkali reaction are waited with the salt that forms salt such as ammonium salt, alkali metal salt such as sodium salt or potassium salt, alkali salt such as calcium salt or magnesium salt, organic alkali salt such as hexanamine, N-methyl D-glycosamine, trihydroxymethylaminomethane and have aminoacid such as arginine, lysine.

Term " ester " refers to have formula-(R) _nThe chemical part of-COOR ', wherein R and R ' are independently selected from the group of being made up of alkyl, cycloalkyl, aryl, heteroaryl (combination of process ring carbon) and heterolipid ring (through the combination of ring carbon), and wherein n is 0 or 1.

Term " amide " refers to have formula-(R) _n-C (O) NHR ' or-(R) _nThe chemical part of-NHC (O) R ', wherein R and R ' are independently selected from the group of being made up of alkyl, cycloalkyl, aryl, heteroaryl (combination of process ring carbon) and heterolipid ring (through the combination of ring carbon), and wherein n is 0 or 1.Amide can be to be attached to aminoacid or the peptide molecule that forms prodrug on the molecule of the present invention.

Can carry out esterification or amidatioon to any amine, hydroxyl or carboxylic side-chain on the The compounds of this invention.It is known and easily at list of references such as Greene and Wuts for those skilled in the art to be used to obtain this result's method and specificity group, Protective Groups in Organic Synthesis, third edition John Wiley ﹠amp; Sons, New York, NY finds in 1999.

" prodrug " refers to be changed in vivo the medicament of parent's medicine.In some cases, prodrug often is useful, because they are easier to use than parent medicine.For example, by oral administration, they can then cannot by parent's medicine that biology utilized.With respect to parent's medicine, prodrug also has the dissolubility or the stability of improvement in pharmaceutical composition.The example of prodrug is, and be not limited to chemical compound of the present invention, described chemical compound is used as ester (" prodrug ") and uses to promote it to pass through cell membrane, water solublity is disadvantageous for flowability in described cell membrane, in a single day and enter water solublity is favourable cell interior, its then by metabolic be hydrolyzed to carboxylic acid, active entity.Another example of prodrug can be a kind of and the bonded small peptide of acid groups (polyamino acid), wherein peptide by metabolism to expose active part.The conventional method of preparation of selecting and preparing suitable prodrug derivant is for example, Design ofProdrugs, description to some extent in (H.Bundgaard edit, Elsevier, 1985).

Term " aromatic " refers to have the aromatic group that at least one has the ring of the pi electronic system of puting together and comprises isocyclic aryl (for example, phenyl) and heterocyclic aryl group (for example, pyridine).This term comprises monocycle or condensed multi-ring (that is, sharing the right ring of carbon atom that adjoins) group.Term " carbocyclic ring " refers to a kind of chemical compound, and described chemical compound comprises the ring structure of one or more covalently closed circles, and the atom of formation ring skeleton all is a carbon atom.Therefore this term comes carbocyclic ring and heterocycle difference, and wherein said heterocyclic ring skeleton comprises the atom that at least one is different from carbon.Term " heteroaromatics " refers to comprise the aromatic group of at least one heterocyclic ring.

When being used for this paper, term " alkyl " refers to aliphatic hydrocarbon group.Moieties can be " saturated alkyl " group, and it means and does not comprise any alkene or alkynyl moiety.Moieties can also be " unsaturated alkyl " part, this means that it comprises at least one alkene or alkynyl moiety." alkene " partly refers to the group is made up of at least two carbon atoms and at least one carbon-to-carbon double bond, and " alkynes " partly refers to the group be made up of at least two carbon atoms and at least one carbon-to-carbon triple bond.Described moieties no matter be saturated or undersaturated, can be side chain, straight chain or cyclic.

Alkyl group can have 1-20 carbon atom and (no matter when occur at this paper, refer to each integer in given range such as the digital scope of " 1-20 "; For example, " 1-20 carbon atom " means that alkyl group can be by 1 carbon atom, 2 carbon atoms, 3 carbon atoms, Deng, up to as many as with comprise that 20 carbon atoms form, exist although present definition also comprises the term " alkyl " of not indicating digital scope).Alkyl group can also be the alkyl of the middle-sized 1-10 of a having carbon atom.Alkyl group can also be the low-grade alkyl group with 1-5 carbon atom.The alkyl group of The compounds of this invention can be called " C _1-6Alkyl " or similar title.Only according to example, " C _1-6Alkyl " be illustrated in and have 1-6 carbon atom in the alkyl chain, promptly alkyl chain is selected from by in methyl, ethyl, propyl group, isopropyl, normal-butyl, isobutyl group, sec-butyl, the tert-butyl group, amyl group (straight or branched) and base (straight or branched) has been formed the group.

Alkyl group can be substituted or unsubstituted.When being substituted; described substituent group group is separately and is independently selected from following one or more groups: cycloalkyl; aryl; heteroaryl; the heterolipid ring; hydroxyl; alkoxyl; aryloxy group; sulfydryl; alkylthio group; arylthio; cyano group; halogen atom; carbonyl; thiocarbonyl; the O-carbamyl; the N-carbamyl; the O-thiocarbamoyl; the N-thiocarbamoyl; the C-amide groups; the N-amide groups; the S-sulfonamido; the N-sulfonamido; the C-carboxyl; the O-carboxyl; isocyano; thiocyano; the isocyanide sulfenyl; nitro; silicyl; three halogen methylsulfonyls and the amino that comprises the amino group of list and two-replacement, and protected derivant.Typical alkyl group comprises, but is in no way limited to methyl, ethyl, propyl group, isopropyl, butyl, isobutyl group, the tert-butyl group, amyl group, hexyl, vinyl, acrylic, cyclobutenyl, cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl etc.No matter wherein, substituent group is described as by " optional replacement ", promptly substituent group can replace with one of above-mentioned substituent group.

The substituent group that only has substituent group " R " itself to occur and do not indicate numeral be meant be selected from by alkyl, cycloalkyl, aryl, heteroaryl (through ring carbon in conjunction with) and heterolipid ring (through the combination of ring carbon) form the substituent group in the group.

" O-carboxyl " group refers to RC, and (=O) O-group, wherein R as defined herein.

" C-carboxyl " group refers to-(=O) OR group, wherein R is as defined herein for C.

" acetyl group " group refers to-C (=O) CH ₃, group.

" three halogen methylsulfonyls " group refers to X ₃CS (=O) ₂-group, wherein X is a halogen atom.

" cyano group " group refers to-the CN group.

" isosulfocyanate radical " group refers to-the NCO group.

" thiocyano " group-CNS group.

" isocyanide sulfenyl " group refers to-the NCS group.

" sulfinyl " group refers to-S (=O)-and the R group, wherein R is as defined herein.

" S-sulfonamido " group refers to-S (=O) ₂NR, group, wherein R is as defined herein.

" N-sulfonamido " group refer to RS (=O) ₂The NH-group, wherein R as defined herein.

" three halogen methylsulfinyl amino " group refers to X ₃CS (=O) ₂The NR-group, wherein X and R are as defined herein.

" O-carbamyl " group refers to-OC (=O)-and the NR group, wherein R is as defined herein.

" N-carbamyl " group refers to ROC, and (=O) NH-group, wherein R as defined herein.

" O-thiocarbamoyl " group refers to-OC (=S)-and the NR group, wherein R is as defined herein.

" N-thiocarbamoyl " group refers to ROC, and (=S) NH-group, wherein R as defined herein.

" C-amide groups " group refers to-C (=O)-NR ₂Group, wherein R as defined herein.

" N--amide groups " group refers to RC, and (=O) NH-group, wherein R as defined herein.

Term " perhaloalkyl radical " refers to a kind of alkyl group, and wherein all hydrogen atoms are all replaced by halogen atom.

Term " aryl " is meant aromatic ring carbon ring or member ring systems herein.In addition, term " aryl " comprises the fused rings system, wherein at least two aryl rings or at least one aryl and at least one C _3-8-cycloalkyl is shared at least one chemical bond.Some examples of " aryl " ring comprise optional phenyl, naphthyl, phenanthryl, anthryl, 1,2,3,4-tetralin base, fluorenyl, the indenyl and 2 that replaces, 3-indanyl.Term " aryl " relates to aromatic, the preferred benzenoid group that becomes ring carbon atom to connect by; and choose wantonly and contain one or more substituent groups, described substituent group is selected from heterocyclic radical, heteroaryl, halogeno-group, hydroxyl, amino, cyano group, nitro, alkylamidoalkyl, acyl group, C _1-6-alkoxyl, C _1-6-alkyl, C _1-6-hydroxyalkyl, C _1-6-aminoalkyl, C _1-6-alkyl amino, alkyl sulphinyl, alkyl sulfinyl, alkyl sulphonyl, sulfamoyl or trifluoromethyl.Aryl can replace in a para-position and/or a position.The representative example of aryl includes but not limited to: phenyl, the 3-halogenophenyl, the 4-halogenophenyl, the 3-hydroxy phenyl, the 4-hydroxy phenyl, the 3-aminophenyl, the 4-aminophenyl, the 3-aminomethyl phenyl, the 4-aminomethyl phenyl, the 3-methoxyphenyl, the 4-methoxyphenyl, the 4-Trifluoromethoxyphen-l, the 3-cyano-phenyl, the 4-cyano-phenyl, 3,5-dimethylphenyl, naphthyl, the hydroxyl naphthyl, hydroxymethyl phenyl, trifluoromethyl, alkoxyl phenyl, 4-morpholine-4-base phenyl, 4-pyrrolidine-1-base phenyl, 4-pyrazolyl phenyl, 4-triazolyl phenyl and 4-(2-oxo-pyrrolidine-1-yl) phenyl.

Term " heteroaryl " is meant heterocyclic aromatic group herein, and wherein one or more hetero atoms of the selected self-contained nitrogen of the one or more carbon atoms in the aromatic ring, sulfur, phosphorus and oxygen replace.

Term " heteroaryl " comprises the fused rings system herein in addition, wherein at least one aryl rings and at least one hetero-aromatic ring, at least two hetero-aromatic rings, at least one hetero-aromatic rings and at least one heterocycles or at least one hetero-aromatic ring and at least one C _3-8-cycloalkyl ring is shared at least one chemical bond.

Term " heteroaryl " is interpreted as relating to and also comprises an oxygen or sulphur atom or 4 nitrogen-atoms at the most, perhaps oxygen or sulphur atom and the aromatic C of the combination of 2 nitrogen-atoms at the most _3-8-cyclic group, and their replace and benzo-, pyrido-condensed derivant, this group preferably becomes ring carbon atom to connect by one.Described heteroaryl can contain one or more substituent groups, and substituent group is selected from halogeno-group, hydroxyl, amino, cyano group, nitro, alkylamidoalkyl, acyl group, C _1-6-alkoxyl, C _1-6-alkyl, C _1-6-hydroxyalkyl, C _1-6-aminoalkyl, C _1-6-alkyl amino, alkyl sulphinyl,, alkyl sulfinyl, alkyl sulphonyl, sulfamoyl or trifluoromethyl.In certain embodiments, heteroaryl can be to contain 0,1 or 2 substituent five yuan and six-membered aromatic heterocyclic system, and substituent group can be same to each other or different to each other, and is selected from above-mentioned substituent group.The representative example of heteroaryl includes but not limited to: one of unsubstituted furan and furan replaces or disubstituted derivatives, benzofuran, thiophene, benzothiophene, the pyrroles, pyridine, indole oxazole benzoxazole isoxazole, benzoisoxazole, thiazole, benzothiazole, isothiazole, imidazoles, benzimidazole, pyrazoles, indazole, tetrazolium, quinoline, isoquinolin, pyridazine, pyrimidine, purine and pyrazine, these all are preferred, and furazan (furazan), 1,2, the 3-oxadiazole, 1,2, the 3-thiadiazoles, 1,2, the 4-thiadiazoles, triazole, benzotriazole, pteridine Fen oxazole oxadiazole, benzopyrazoles, quinolizine, cinnolines, 2, quinazoline and quinoxaline.In certain embodiments, substituent group is halogeno-group, hydroxyl, cyano group, O-C _1-6-alkyl, C _1-6-alkyl, hydroxyl-C _1-6-alkyl, amino-C _1-6-alkyl.

Term " alkyl " and " C herein _1-6-alkyl " be meant linearity or branching saturated hydrocarbon chain, wherein long-chain contains 1 to 6 carbon atom, for example methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, sec-butyl, the tert-butyl group, amyl group, isopentyl, neopentyl and hexyl.Alkyl chain can be chosen replacement wantonly.

Term " heterocyclic radical " is meant three-, four-, five-, six-, seven-and eight-unit ring, wherein carbon atom and 1 to 3 hetero atom constitute described ring together.Heterocyclic radical can be chosen wantonly and comprise one or more unsaturated bonds, but described unsaturated bond is positioned on the position that does not produce aromatic series π-electron system.Described hetero atom is independently selected from oxygen, sulfur and nitrogen.

Heterocyclic radical can also comprise one or more carbonyls or thiocarbonyl functionality, makes its definition comprise oxo-system and sulfo--system, for example lactams, lactone, cyclic imides, ring-type thioimides, cyclic carbamate etc.

Heterocycle can also be chosen wantonly with aryl rings and condense, thereby its definition comprises twin nuclei.Preferred condensed heterocycle base like this and the optional shared key of phenyl ring that replaces.The example of benzo-fused heterocycle base includes but not limited to: benzimidazole alkane ketone, tetrahydroquinoline and methylenedioxy benzene ring structure.

Some examples of " heterocyclic radical " include but not limited to: tetrahydric thiapyran, the 4H-pyrans, Pentamethylene oxide., piperidines, 1, the 3-dioxin, 1, the 3-diox, 1, the 4-dioxin, 1, the 4-diox, piperazine, 1, the 3-thioxane, 1, the 4-oxathiin, 1, the 4-thioxane, tetrahydrochysene-1, the 4-thiazine, 2H-1, the 2-oxazine, maleimide, butanimide, barbituric acid, thiobarbituric acid, the dioxo piperazine, hydantoin, dihydrouracil, morpholine trioxane, six hydrogen-1,3, the 5-triazine, Tetramethylene sulfide, oxolane, pyrrolin, pyrrolidine, ketopyrrolidine, pyrrolinone, pyrazoline, pyrazolidine, imidazoline, imidazolidine, 1, the 3-dioxole, 1, the 3-dioxolanes, 1, the 3-dithiole, 1,3-dithiolane isoxazoline isoxazole alkyl oxazoline oxazolidine oxazolidone, thiazoline, Thiazolidine and 1,3-oxathiolane.Can or pass through heterocyclic carbon atom on heteroatomic position with heterocyclic combination, perhaps for benzo-fused derivant, by the carbon atom of benzenoid ring.

Term " (heterocyclic radical) C _1-6-alkyl " be interpreted as the heterocyclic radical of substituent group each all so place definition of these groups by the alkyl connection.(heterocyclic radical) C _1-6The heterocyclic radical of-alkyl can be replacement or unsubstituted.Term " (heterocyclic radical) C _1-6-alkyl " be meant at least by heterocyclic radical and replace once alkyl chain that its typical the position of substitution is at the end of alkyl chain.

Herein, term " C _2-8-thiazolinyl " be meant the linearity or the branched hydrocarbyl radical that contain 2 to 8 carbon atoms and contain one or more pairs of keys.C _2-8Some examples of-thiazolinyl comprise pi-allyl, height-pi-allyl, vinyl, crotyl, cyclobutenyl, pentenyl, hexenyl, heptenyl and octenyl.Contain the C that surpasses two keys _2-8Some examples of-thiazolinyl comprise butadiene, pentadiene, hexadiene, heptadiene, heptantriene and sarohornene, and the branching form of these chemical compounds.The position of unsaturated (two key) can be in any position of carbochain.

Term " C herein _2-8-alkynyl " be meant and contain 2 to 8 carbon atoms and contain one or more triple-linked linearities or branched hydrocarbyl radical.C _2-8Some examples of-alkynyl comprise acetenyl, propinyl, butynyl, pentynyl, hexin base, heptyne base and octyne base, and the branching form of these chemical compounds.The position of unsaturated (triple bond) can be in any position of carbochain.It can be undersaturated surpassing a key, " C _2-8-alkynyl " be two-alkynes or alkene two-alkynes as is known to persons skilled in the art.

Herein, term " C _3-8-cycloalkyl " comprise only comprise three of carbon atom-, four-, five-, six-, seven-and eight-unit ring.C _3-8-cycloalkyl can be chosen wantonly and comprise one or more unsaturated bonds, but contained unsaturated bond does not produce aromatic series π-electron system.

Preferred " C _3-8-cycloalkyl " some examples be carbocyclic ring cyclopropane, Tetramethylene., Pentamethylene., cyclopentenes, cyclopentadiene, cyclohexane extraction, cyclohexene, 1,1, cycloheptane, cycloheptene.

Term " (aryl) C _1-6-alkyl " be meant as substituent group and pass through C _1-6The aryl that-alkyl connects, each all so place definition of these groups.(aryl) C _1-6The aryl of-alkyl can be replacement or unsubstituted.The example comprises benzyl, substituted benzyl, 2-phenylethyl, 3-phenyl propyl and naphthyl alkyl.

Term " (cycloalkyl) C _1-6-alkyl " be meant the cycloalkyl that connects by alkyl as substituent group, each place all like this of these groups defines.

When using herein, term " O-C _1-6-alkyl " be meant C _1-6-alkyl oxy or alkoxyl, for example, methoxyl group, ethyoxyl, positive propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, amoxy, isoamoxy, neopentyl oxygen and hexyloxy.

Term " halogen " comprises fluorine, chlorine, bromine and iodine.

Herein, promptly and term " C _1-6-alkyl ", " aryl ", " heteroaryl ", " heterocyclic radical ", " C _3-8-cycloalkyl ", " heterocyclic radical (C _1-6-alkyl) ", " (cycloalkyl) alkyl ", " O-C _1-6-alkyl ", " C _2-8-thiazolinyl " and " C _2-8-alkynyl " during logotype, term " the optional replacement " is meant that the group in the formula can be replaced 1 time by one or more groups or repeatedly, such as 1 to 5 time, or 1 to 3 time, or 1 to 2 time, described group is selected from: C _1-6-alkyl, C _1-6-alkoxyl, oxo base (this group can be represented with tautomeric enol form), carboxyl, amino, hydroxyl (when this group is present in the enol system, can represent), nitro, alkyl sulphonyl, alkyl sulphinyl, alkyl sulfinyl, C with tautomeric ketone form _1-6-alkoxy carbonyl group, C _1-6-alkyl-carbonyl, formoxyl, amino, one-and two (C _1-6-alkyl) amino, carbamyl ,-and two (C _1-6-alkyl) amino carbonyl, amino-C _1-6-alkyl-amino carbonyl, one-and two (C _1-6-alkyl) amino-C _1-6-alkyl-amino carbonyl, C _1-6-alkyl-carbonyl-amino, C _1-6-alkyl hydroxy imino group, cyano group, guanidine radicals, urea groups, C _1-6-alkanoyloxy, C _1-6-alkylsulfonyloxy, dihalo-C _1-6-alkyl, three halos-C _1-6-alkyl, heterocyclic radical, heteroaryl and halogeno-group.Usually, above-mentioned substituent group can be easy to carry out further optional the replacement.

Unless otherwise indicated; when substituent group is considered to by " the optional replacement "; then mean substituent group be can by one or more separately and be independently selected from the group that the group of following group replaces: cycloalkyl; aryl; heteroaryl; the heterolipid ring; hydroxyl; alkoxyl; aryloxy group; sulfydryl; alkylthio group; arylthio; cyano group; halo; carbonyl; thiocarbonyl; the O-carbamyl; the N-carbamyl; the O-thiocarbamoyl; the N-thiocarbamoyl; the C-amide groups; the N-amide groups; the S-sulfonamido; the N-sulfonamido; the C-carboxyl; the O-carboxyl; isocyano; thiocyano; the isocyanide sulfenyl; nitro; silicyl; three halo mesyl and amino; comprise one-and dibasic amino, and their protected derivant.The protecting group that can form above-mentioned substituent protection derivant is well known by persons skilled in the art, and can find in above-mentioned list of references such as Greene and Wuts.

In certain embodiments, the E in the formula A chemical compound is an oxygen.In certain embodiments, T also is an oxygen.

In certain embodiments, term " heterocyclic radical " refers to the substituent group that is selected from following group: tetrahydric thiapyran, the 4H-pyrans, Pentamethylene oxide., piperidines, 1, the 3-dioxin, 1, the 3-diox, 1, the 4-dioxin, 1, the 4-diox, piperazine, 1, the 3-thioxane, 1, the 4-oxathiin, 1, the 4-thioxane, tetrahydrochysene-1, the 4-thiazine, 2H-1, the 2-oxazine, maleimide, butanimide, barbituric acid, thiobarbituric acid, the dioxo piperazine, hydantoin, dihydrouracil, morpholine trioxane, six hydrogen-1,3, the 5-triazine, Tetramethylene sulfide, oxolane, pyrrolin, pyrrolidine, ketopyrrolidine, pyrrolinone, pyrazoline, pyrazolidine, imidazoline, imidazolidine, 1, the 3-dioxole, 1, the 3-dioxolanes, 1, the 3-dithiole, 1,3-dithiolane isoxazoline isoxazole alkyl oxazoline oxazolidine oxazolidone, thiazoline, Thiazolidine and 1,3-oxathiolane.

In certain embodiments, term " heteroaryl " refers to the substituent group that is selected from following group: furan, benzofuran, thiophene, benzothiophene, the pyrroles, pyridine, indole oxazole benzoxazole isoxazole, benzoisoxazole, thiazole, benzothiazole, isothiazole, imidazoles, benzimidazole, pyrazoles, indazole, tetrazolium, quinoline, isoquinolin, pyridazine, pyrimidine, purine, pyrazine, furazan, 1,2, the 3-oxadiazole, 1,2, the 3-thiadiazoles, 1,2, the 4-thiadiazoles, triazole, benzotriazole, pteridine Fen oxazole oxadiazole, benzopyrazoles, quinolizine, cinnolines, 2, quinazoline and quinoxaline.

In certain embodiments, term " aryl " refers to the substituent group that is selected from following group: phenyl, naphthyl, phenanthryl, anthryl, 1,2,3,4-tetralin base, fluorenyl, indenyl and 2,3-indanyl.

In other embodiments, term " cycloalkyl " refers to the substituent group that is selected from following group: cyclopropane, Tetramethylene., Pentamethylene., cyclopentenes, cyclopentadiene, cyclohexane extraction, cyclohexene, 1,3-cyclohexadiene, 1, cycloheptane, cycloheptene.

Some embodiment of formula A chemical compound comprises such chemical compound, wherein R ₁Be selected from hydrogen; C _1-6Alkyl; C _2-6Thiazolinyl; C _2-6Alkynyl; C _3-8Cycloalkyl; C _3-8Heterocyclic radical; Cycloalkyl (C _1-6) alkyl; Heterocyclic radical (C _1-6) alkyl; Aryl; Heteroaryl; (C _1-6) alkyl-carbonyl; (C _1-6) alkoxyl (C _1-6) alkyl and perhalogeno (C _1-6) alkyl.In some of these embodiments, the alkyl in above-mentioned each substituent group is selected from methyl, ethyl, propyl group, normal-butyl, sec-butyl and the tert-butyl group.

But R in certain embodiments, ₁Be hydrogen.

In certain embodiments, R ₂Be selected from hydrogen; C _1-6Alkyl; C _2-6Thiazolinyl; C _2-6Alkynyl; C _3-8Cycloalkyl; C _3-8Heterocyclic radical; Cycloalkyl (C _1-6) alkyl; Heterocyclic radical (C _1-6) alkyl; Aryl; Heteroaryl; (C _1-6) alkyl-carbonyl; (C _1-6) alkoxyl (C _1-6) alkyl and perhalogeno (C _1-6) alkyl.In some of these embodiments, the alkyl in above-mentioned each substituent group is selected from methyl, ethyl, propyl group, normal-butyl, sec-butyl and the tert-butyl group.

But R in certain embodiments, ₂Be hydrogen.

In certain embodiments, R ₃-R ₆Be selected from hydrogen independently of one another; C _1-6Alkyl; C _2-6Thiazolinyl; C _2-6Alkynyl; C _3-8Cycloalkyl; C _3-8Heterocyclic radical; Cycloalkyl (C _1-6) alkyl; Heterocyclic radical (C _1-6) alkyl; Aryl; Heteroaryl; (C _1-6) alkyl-carbonyl; (C _1-6) alkoxyl (C _1-6) alkyl and perhalogeno (C _1-6) alkyl.In some of these embodiments, the alkyl in above-mentioned each substituent group is selected from methyl, ethyl, propyl group, normal-butyl, sec-butyl and the tert-butyl group.

But R in certain embodiments, ₃-R ₆Be hydrogen.

In further embodiment, R ₇And R ₈Be selected from independently of one another: hydrogen and C _1-6Alkyl.In some of these embodiments, R ₇And R ₈All be hydrogen.

Preferred medicine or medicament are made up of formula C compound or pharmaceutically acceptable salt thereof, amide, ester or its prodrug, or are made up of it substantially, or comprise it:

。(referring to embodiment 6) as described here uses cation exchange HPLC, cultivates the G-NH of unmodified in containing the serum of cofactor ₂After, isolate this chemical compound.After above-mentioned chromatographic isolation, utilize the NMR spectrogram with the property G-NH of formula C compound identification for modifying ₂(metabolite X).

Analysis based on double labelling is ¹³C/ ¹⁵The sample of N labelling. ¹H NMR spectrogram is by being positioned at 7.65 and two wide NH-amide signals of 7.15ppm and be that the CH-proton bimodal (J=163Hz) at center is formed with 5.21ppm.The strength ratio of all three signals was near 1: 1: 1.In the spectrogram that under not having aqueous solvent signal presaturation situation, obtains, can observe～the extra NH at 7.4ppm place ₃ ⁺Signal.A proton in this expression glycine methylene is replaced by electronegative substituent, and cause with original glycine amide and compare, ¹H NMR spectrogram is obviously to low field displacement.

¹³C NMR spectrum demonstrates two groups of equicohesive signals: 177.6 ppm places ¹³Bimodal three the different coupling constant (J=7.1 that have with 89.0ppm place aliphatic carbon signal of C=O (J=62Hz); 62 and 163Hz) eight lines.J=163Hz is a key ¹³C- ¹The H coupling, J=62Hz is a key ¹³C- ¹³The C coupling, and the third a coupling 7.1Hz and a key ¹⁵N- ¹³C coupling phase unanimity.As expection theoretically, all possible pair of key coupling is all near zero. ¹H- ¹³C and ¹³C- ¹³The C coupling is all relatively large, and is consistent with the substituent introducing of strong electronegativity on the glycine aliphatic carbon.Use the existing scheme (additive schemes) that adds of chemical shift prediction, from aliphatic carbon ¹³The C chemical shift is analyzed and is also obtained same conclusions.

¹⁵N- ¹The H hsqc spectrum by be positioned at～20ppm from ¹⁵The strong signal of the amine of N labelling and～the weak signal composition from the nitrogen of unmarked amide at 105ppm place.These are NH ₃ ⁺And CONH ₂The prediction exemplary value of the resonance of nitrogen.The overall measurement time of this double labelling sample is～10 hours.

So, the structure for formula C chemical compound obtains best ¹H and ¹³Concordance between the C.Therefore, preferred embodiment comprises to be made up of formula C chemical compound and its derivant, or (enrichment or the isolating preparation that for example contain the formula C chemical compound of enantiomer (D or L) and/or isomer (R or S) form) formed by formula C chemical compound and its derivant substantially, or comprising formula C chemical compound and its derivant, described derivant is the wherein medicine and the medicament of the derivant that replaced by methoxyl group, ethyoxyl or alkoxyl of hydroxyl particularly.

Other preferred embodiment comprises medicine and medicament that be made up of it or that comprise it that be made up of following chemical compound or basic: the α-peroxide Aminoacetamide dimer (NH with structure shown in the formula E ₂-gly-O-O-gly-NH ₂), the two glycyl amidogen ether (NH that perhaps have structure shown in the formula F ₂-gly-O-gly-NH ₂):

Preferred composition also comprises by the α with structure shown in the formula (G)-methoxyl group Aminoacetamide (α-MeO-gly-NH ₂) that form or basic medicine and medicament that form by it or that comprise it:

The whole bag of tricks of the Aminoacetamide of synthetic modification all is known in the art.(referring to, the JP 5097789A2 of Hayakawa etc. for example, on October 3rd, 1991 submitted to, title is " Alpha-hydroxy glycinamide derivative and preparation thereof ").Adopt a kind of method, be prepared as follows the Alpha-hydroxy glycinamide derivative and the salt thereof of formula (B) expression:

(R wherein ¹Be hydrogen atom, low alkyl group, low-grade alkenyl, low-grade alkynyl, benzyl, perhaps by the silicyl of alkyl or alkyl and aromatic group replacement; R ²Be hydrogen atom or amino protecting group).

Adopt another kind of method, will as shown in the formula (H) expression Alpha-hydroxy glycinamide derivative or its salt:

(R wherein ¹And R ²Suc as formula defining in (B); R ³Be hydrogen atom or carboxyl-protecting group), handle in solvent with ammonia, remove protecting group if desired, and if desired, the chemical compound that obtains is further transformed salify.

According to some preferred embodiments described herein, reference marks R ₁The low alkyl group of expression is to contain to be no more than 6, preferably to be no more than the alkyl of 4 carbon atoms.Such examples of groups comprises methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, the tert-butyl group, can be the amyl group of branching and can be the hexyl of branching.

Reference marks R ₁The low-grade alkenyl of expression is to contain to be no more than 6, preferably to be no more than the thiazolinyl of 4 carbon atoms.Such examples of groups comprises vinyl, pi-allyl and two key cyclobutenyl at an arbitrary position.Reference marks R ₁The low-grade alkynyl of expression is to contain to be no more than 6, preferably to be no more than the alkynyl of 4 carbon atoms.Such examples of groups comprises acetenyl etc.

Reference marks R ₁The silicyl that the silicyl that is replaced by low alkyl group of expression is replaced by 1 to 3 low alkyl group.The low-grade alkyl substituent of Shi Yonging is about R in this case ₁Above-mentioned low alkyl group in any one or its combination.The silicyl that is replaced by low alkyl group is t-butyldimethylsilyl preferably.The silicyl that the silicyl that is replaced by alkyl and aromatic group is replaced by abovementioned alkyl and phenyl, for example t-butyldiphenylsilyl.

Can will be used for the protecting group in aminoacid or chemistry of peptides field as R ₂The amino protecting group of expression.Such examples of groups comprises: oxygen carbonyl-type protecting group, for example two propyloxy phenyl oxygen carbonyls (Bpoc-) of benzyloxycarbonyl group (Cbz-), right-methoxyl group benzyloxy carbonyl [Z (OMe)-], tertbutyloxycarbonyl (Boc-) or 2-etc.; Acyl group protecting group, for example HCO-, phthalic acid ester group (Pht-) or neighbour-nitrophenylsulfenyl (Nps-) etc.; With the alkyl protecting group, trityl group (Trt-) etc. for example.

According to some embodiments described herein, the salt of Alpha-hydroxy glycinamide derivative is acid-addition salts, and inorganic salt for example is such as hydrogen halide salt, for example hydrofluoride, hydrochlorate, hydrobromate, nitrate, sulfate or phosphate, perhaps acylate, such as fumarate, acetate etc.

The chemical compound of preparation formula (C) expression with the following method: will be as shown in the formula the Alpha-hydroxy glycine derivative of (H) expression:

(R wherein ¹And R ²Suc as formula defining in (B); R ³Be hydrogen atom or carboxyl-protecting group) handle in solvent with ammonia, and the optional amino protecting group of removing.

Carbonyl-protection base R ³Be can be by the common carboxyl-protecting group that is replaced by amino with ammonia treatment.Such examples of groups comprises: lower alkoxy, for example methoxyl group (OMe), ethyoxyl (OEt), benzyloxy (OBzl) or tert-butoxy (OtBu), perhaps aryloxy group, such as right-nitro-phenoxy (ONp) etc.

Can be with ordinary organic solvents such as lower alcohol, methanol for example, ethanol, propanol, ether such as methyl ethyl ether, Anaesthetie Ether, isopropyl ether etc. are as reaction dissolvent.Can by will in above-mentioned solvent and at pressure, reduce by the compound dissolution of formula (H) expression, under the normal or increase condition and for example from-78 ℃-40 ℃, under preferred 0 ℃-25 ℃ temperature, for example be blown into ammonia under the room temperature condition and carry out described reaction.

This reaction makes acquisition chemical compound (B) become possibility, wherein R ²It is the amido protecting group.In order from this chemical compound, to remove amido protecting group R ²And acquisition chemical compound (B), wherein R ²Be H, can be according to amido protecting group R ²Type carry out common going protection to handle.For example, as blocking group R ²When being benzyloxycarbonyl, right-methoxyl group benzyloxy base carbonyl etc., can for example, under the situation that palladium/carbon or analog exist, go protection by at hydrogenation catalyst with hydrogen treat.In addition, as blocking group R ²When being uncle-butoxy carbonyl, can go protection with hydrochloric acid-dioxs.Can pass through, for example, under the situation that all example hydrochloric acids of acid exist, carry out the above-mentioned protection of going and handle the salt for preparing chemical compound (B).

Can pass through, two kinds of for example following methods are prepared the chemical compound according to formula (H), in described chemical compound, and R ¹It or not hydrogen atom.With regard to first method, it can pass through R ¹Rather than hydrogen introduces in the chemical compound and is prepared, described chemical compound in chemical compound by formula (H) representative, R wherein ¹Be hydrogen.Can utilize the correlation function derivant of group, for example, halogen derivatives carries out R ¹And non-hydrogen introducing.For example, the introducing of the silyl-group that replaces for low alkyl group can be used the halogenide of silicyl, for example, for the introducing of uncle-butoxy dimetylsilyl group, can use tert-butyl dimetylsilyl chloride.Described reaction can be in carrying out in such as dimethyl formamide at solvent on 0 ℃-30 ℃ the temperature.

In addition, for low-grade alkenyl or low-grade alkynyl group are introduced, can use the halogen derivatives of alkene or alkyl respectively.For example, under situation about existing, use allyl halide, allyl group can be introduced such as pi-allyl iodate thing at catalyst such as silver oxide.Can be on-10 ℃-50 ℃ temperature, on preferred 0 ℃-25 ℃ temperature, carry out this reaction in such as dimethyl formamide at solvent.

Just prepare R ¹Be not hydrogen and during with other method of the chemical compound of formula (H) expression, by with lower alcohol, for example methanol or ethanol as solvent with thionyl chloride to by formula (H) expression and R wherein ¹And R ²The chemical compound that all is hydrogen atom is handled.In this case, can obtain chemical compound, wherein R by formula (H) expression ¹And R ²It is identical low-grade alkyl group corresponding to lower alcohol solvent.Described reaction can preferably be carried out on 0 ℃-25 ℃ the temperature-10 ℃-40 ℃ temperature.

For example, can be by following two kinds of methods preparation by formula (H) expression and R wherein ¹It is the chemical compound of hydrogen.With regard to first method, can by make glyceraldehyde CHO-COOH with by amido protecting group R ²The amine R of protection ²NH ₂Reaction obtains described chemical compound.For example, this reaction can be by being described in the U.S. Patent number 3,668,12 that licenses to Philip X.Masciantonio etc. and Stanlen D.Young etc., J.Am.Chem.Soc. 111, the method in 1933 (1989) is carried out on 20 ℃-75 ℃ temperature in the solvent such as acetone, ether etc.In this case, can obtain by formula (H) expression and R wherein ¹And R ³It all is the chemical compound of hydrogen atom.

Just be used for preparation by formula (H) expression and R wherein ¹Be other method of the chemical compound of hydrogen, make by as shown in the formula (I) (R wherein ³Such as about formula (H) definition, and R ⁴Be low-grade alkyl group) expression chemical compound with by amido protecting group R ²The amine R of protection ²NH ₂Reaction:

This reaction can be in 20 ℃-80 ℃ temperature, for example in carrying out in such as the solvent of oxolane on the reflux temperature of used solvent.As low-grade alkyl group R ¹Equally to low-grade alkyl group R ⁴Define.In these synthetic methods of embodiment more detailed description subsequently some.

Embodiment 12

12-1

With Alpha-hydroxy-uncle N--butoxy carbonyl methyl aminoacetate (4.11g, 20mmol) and imidazoles be dissolved among the DMF and be cooled to 0 ℃ in room temperature.In this temperature, chlorating tert-butyl dimetylsilyl is added this solution then, and composition was stirred 10 minutes.The temperature of solution is back to room temperature and continues stirring 1 hour.Then, add saturated saline and carry out extracting with ethyl acetate.Remove with the anhydrous magnesium sulfate drying organic layer and with the solvent distillation.

Then the oily matter that obtains is dissolved in the ethanol (50mL), and under 0 ℃ of temperature, excess ammonia is blown in the described solution.Next, under reduced pressure, remove excess ammonia and ethanol distillation removed.Thus obtained crude product is carried out purification by silica gel column chromatography and obtained α-tert-butyl dimethyl methyl siloxy-uncle N--butoxy carbonyl Aminoacetamide (6.10g, quantitative).The pattern of expection comprises: ¹HNMR δ (CDCl ₃) 0.16 (s, 3H), 0.21 (s, 3H), 0.92 (s, 9H), 5.46 (d, 1H, J=9Hz), 5.63 (d, 1H, J=9Hz), 6.22-6.82 (br, 2H).

12-2

Prepare as follows as above-mentioned 12-1The Alpha-hydroxy-uncle N--butoxy carbonyl methyl aminoacetate of initial substance: with the tert-butyl carbamate (2.83g, 23.6mmol) and the Acetic acid,oxo-,monohydrate monohydrate (2.02g 21.5mmol) is dissolved in the acetone (50mL) and refluxes and spend the night.Then described solution being cooled to 0 ℃ of temperature also handles in room temperature with excessive Azimethylene .-ethereal solution.Then, the solvent distillation is removed.

Add saturated brine then, carry out extracting, remove with the anhydrous magnesium sulfate drying organic layer and with the solvent distillation with chloroform.Thus obtained crude product is carried out purification by silica gel column chromatography, and obtained Alpha-hydroxy-uncle N--butoxy carbonyl methyl aminoacetate (2.56g, 58%).The collection of illustrative plates of expection comprises: ¹HNMR δ (CDCl ₃) 1.46 (s, 9H), 1.65 (br s, 1H), 3.84 (s, 3H), 5.27-5.52 (br, 1H), 5.59-5.90 (br, 1H) .IR (NaCl) 1755 (s), 1690 (s), 1528 (s) cm ^-1

12-3

Can be by being different from 12-2Method preparation as above-mentioned 12-1The Alpha-hydroxy-uncle N--butoxy carbonyl methyl aminoacetate of initial substance.Therefore (11.35g, 95.0mmol) (14.35g 119.5mmol) is dissolved among the anhydrous THF (50mL) and backflow is spent the night with 1-hydroxyl-1-methoxy menthyl acetate with the tert-butyl carbamate.Cool the temperature to room temperature then, (1.15g 9.6mmol) and with composition further refluxed 8 hours then to add 1-hydroxyl-1-methoxy menthyl acetate.Reaction liquid is remained unchanged drop to room temperature, then the solvent distillation is removed up to temperature.Thus obtained crude product recrystallize from chloroform-hexane solution is come out and obtain pure Alpha-hydroxy-uncle N--butoxy carbonyl methyl aminoacetate (16.42g, 84%).

Embodiment 13

Will 12-2Or 12-3In obtain Alpha-hydroxy-uncle N--butoxy carbonyl methyl aminoacetate (1.21g 5.9mmol) is dissolved among the DMF (10mL), then in room temperature add silver oxide (1.04g, 4.5mmol) and iodobenzene (benzene iodide) (1.99g, 9.1mmol).In room temperature the composition stirring is spent the night, filtering precipitate is added to water in the mother solution, carries out extracting with ethyl acetate.With the extractive solution of anhydrous magnesium sulfate drying, then the solvent distillation is removed, and carry out thick purification with silica gel column chromatography.

Thus obtained oily matter is dissolved in the ethanol (50mL) and under 0 ℃ of temperature excess ammonia is blown into solution.Under reduced pressure remove excess of ammonia then, and the solvent distillation is removed.By the crude product of silica gel column chromatography purification acquisition, and obtained α-benzyloxy-uncle N--butoxy carbonyl Aminoacetamide (0.397g, 22%).The collection of illustrative plates of expection comprises: m.p.115-120 ℃, ¹HNMR δ (CDCl ₃) 1.44 (s, 9H), 4.61 (d, H, J=11.3Hz), 4.79 (d, 1H, J=11.3Hz), 5.4 (d, 1H, J=9.0Hz), 5.75 (brd, 1H, J=9.0Hz), 6.00 (br, 1H), 6.52 (br, 1H), 7.35 (s, 5H) .IR (NaCl) 1698 (s), 1664 (s), 1502 (s), 732 (m), 695 (m) cm ^-1Expection element (C ₁₄H ₂₀O ₄N ₂) assay value; Value of calculation C:59.99, H:7.19, N:9.99 measured value C:59.94, H:7.33, N:10.28.

Embodiment 14

Will be according to as above 12-2Or 12-3Alpha-hydroxy-uncle N--butoxy carbonyl the methyl aminoacetate of preparation (2.07g 10.1mmol) is dissolved among the DMF (20mL), and in room temperature add silver oxide (1.39g, 6.0mmol) and pi-allyl iodate thing (1.2mL, 12.9mmol).After stirred overnight at room temperature, precipitate is leached, in mother solution, add entry, and carry out extracting with ethyl acetate.Use the anhydrous magnesium sulfate drying extracted solution, then the solvent distillation is removed, and add sodium thiosulfate solution, carry out extracting with ethyl acetate then and remove iodine as byproduct of reaction.

Thus obtained oily matter is dissolved in the ethanol, and under 0 ℃ of temperature, in solution, is blown into excess ammonia, under reduced pressure remove excess of ammonia thereafter, and the solvent distillation is removed.The crude product that obtains with the silica gel column chromatography purification is to obtain α-allyloxy-uncle N--butoxy carbonyl Aminoacetamide (0.625g, 27%).The collection of illustrative plates of expection comprises: ¹HNMR δ (CDCl ₃) 1.45 (s, 9H), 4.14 (dd, 2H, J=7.2,1.8Hz), 5.11-5.56 (m, 3H), 5.70-6.20 (m, 2H), 6.33-7.01 (m, 2H).IR(CDCl ₃)2975(w)，1705(s，br)，1498(m)，990(sh.w)cm ^-1。

Embodiment 15

15-1

(4.44g 19.7mmol) is dissolved in the methanol (20mL) with Alpha-hydroxy-N-benzyloxycarbonyl glycine.(2.9mL 40.0mmol) dropwise adds in the solution under 0 ℃ of temperature, stirs under this temperature 30 minutes, then in room temperature restir 2 hours with thionyl chloride.Then the solvent distillation is removed, and the crude product that obtains is dissolved in the methanol (50mL).This solution is cooled to 0 ℃, is blown into excess ammonia then therein.

After reaction finishes, under reduced pressure remove excess of ammonia, the solvent distillation is removed and with silica gel column chromatography purification white crystal with acquisition α-methoxyl group-N-benzyloxycarbonyl Aminoacetamide (3.42g, 73%).The collection of illustrative plates of expection comprises: m.p.110-112 ℃, ¹HNMR δ (CDCl ₃) 3.44 (s, 3H), 5.16 (s, 2H), 5.31 (d, 1H, J=8.8Hz), 5.45-5.98 (br, 2H), 6.28-6.68 (br, 1H), 7.36 (s, 5H).IR(NaCl)1680(s.br)，1540(s)，1520(s)，860(m)，700(m)cm ^-1。Expection element (C ₁₁H ₁₄O ₄N ₂) assay value be: value of calculation C:55.46, H:5.92, N:11.76 measured value C:55.70, H:5.94, N:11.58.

15-2

Parent material Alpha-hydroxy-N-benzyloxycarbonyl glycine above preparing in such a way among the 12-4.With the benzylamino formic acid esters (30.24g, 0.2mol) and the Acetic acid,oxo-,monohydrate monohydrate (20.26 g 0.22mol) are dissolved in the ether (200mL) and with solution and stir in ambient temperature overnight.The crystal that filter to produce and washing to obtain pure Alpha-hydroxy-N-benzyloxycarbonyl glycine (33.78g, 75%) with ether subsequently.The collection of illustrative plates of expection comprises: m.p.200-205 ℃, ¹HNMR δ (CD ₃OD) 5.12 (s, 2H), 5.40 (s, 1H), 7.34 (s, 5H).

Embodiment 16

(2.26g 10.0mmol) is dissolved in (20mL) in the ethanol to the Alpha-hydroxy-N-benzyloxycarbonyl glycine that will produce according to top 15-2.(2mL 27.4mmol) dropwise joins in the solution in-10 ℃ temperature, stirs in ambient temperature overnight with thionyl chloride.Subsequently solvent is distilled and with silica gel column chromatography to thus obtained raw product carry out purification with obtain α-ethyoxyl-N-benzyloxycarbonyl ethyl aminoacetate (2.81g, quantitatively).The collection of illustrative plates of expection comprises: m.p.66-68 ℃, ¹HNMR δ (CDCL ₃) 1.22 (t, 3H, J=7.2Hz), 1.30 (t, 3H, J=7.2Hz),, 3.70 (q, 2H, J=7.2Hz), 4.24 (q, 2H, J=7.2Hz), 5.15 (s, 2H), 5.33 (d, 1H, J=9.7Hz), 5.93 (brd, 1H, J=9.7Hz), 7.35 (s, 5H).IR(NaCl)1740(s)，1700(s)，，1540(s)，760(m)，700(m)cm ^-1。Expection element (C ₁₄H ₁₉O ₅N) assay value; Value of calculation C:59.78, H:6.81, N:4.98, observed value C:60.03, H:6.88, N:4.89.

Embodiment 17

(2.26g 10.0mmol) is dissolved in (20mL) in the isopropyl alcohol to the Alpha-hydroxy-N-benzyloxycarbonyl glycine that will produce according to top 15-2.(2mL 27.4mmol) dropwise joins in the solution in-10 ℃ temperature, stirs in ambient temperature overnight with thionyl chloride.Subsequently solvent is distilled and with silica gel column chromatography to thus obtained raw product carry out purification with obtain α-isopropoxy-N-benzyloxycarbonyl glycine isopropyl ester (2.81g, quantitatively).The collection of illustrative plates of expection comprises: ¹HNMR δ (CDCL ₃) 1.16-1.37 (m, 12H), 3.87-4.22 (m, 1H), 4.57-5.20 (m, 1H), 5.14 (s, 2H), 5.33 (d, 1H, J=9.7Hz), 5.93 (brd, 1H, J=9.7Hz), 7.35 (s, 5H).IR (net value) 1728 (s, br), 1508 (m), 740 (m) cm ^-1

Embodiment 18

(2.29g 8.1mmol) is dissolved in the ethanol (80mL) and be cooled to 0 ℃ to the α-ethyoxyl-N-benzyloxycarbonyl ethyl aminoacetate that will produce according to embodiment 16.In this temperature excess of ammonia is blown in the solution subsequently.After finishing reaction, under the pressure that reduces, remove excess of ammonia, solvent is distilled, wash thus obtained white crystal to obtain pure α-ethyoxyl-N-benzyloxycarbonyl Aminoacetamide (1.51g, 77%) with hexane-ethyl acetate mixed solution.The collection of illustrative plates of expection comprises: m.p119-121 ℃, ¹HNMR δ (CDCL ₃) 1.23 (t, 3H, J=7.1Hz), 3.50-3.90 (m, 2H), 5.14 (s, 2H), 5.37 (d, 1H, J=9.0Hz), 5.65-5.96 (br, 2H), 6.41-6.71 (br, 1H), 7.35 (s, 5H).IR(NaCl)1680(s)，1664(s)，1542(m)，1524(m)，760(w)，740(w)，700(m)cm ^-1。Expection element (C ₁₂H ₁₆O ₄N ₂) assay value; Value of calculation C:57.13, H:6.39, N:11.10, observed value C:57.09, H:6.34, N:11.37.

Embodiment 19

(2.48g 8.0mmol) is dissolved in the ethanol (40mL) and be cooled to 0 ℃ to the α-isopropoxy-N-benzyloxycarbonyl glycine isopropyl ester that will produce according to embodiment 16.In this temperature excess of ammonia is blown in the solution subsequently and continues 5 hours, in the ammonia saturation, continue to stir 2 days.After finishing reaction, under the pressure that reduces, remove excess of ammonia, solvent is distilled, wash thus obtained white crystal to obtain pure α-isopropoxy-N-benzyloxycarbonyl Aminoacetamide (1.64g, 77%) with hexane-ethyl acetate mixed solution.The collection of illustrative plates of expection comprises: m.p111-113 ℃, ¹HNMR δ (CDCL ₃) 1.18 (d, 3H, J=4.4Hz), 1.25 (d, 3H, J=4.4Hz), 3.81-4.20 (m, 1H), 5.15 (s, 2H), 5.44 (d, 1H, J=9.0Hz), 5.53-5.86 (br, 2H), 6.37-6.73 (br, 1H), 7.35 (s, 5H).IR(NaCl)1668(s)，1660(s)，1538(m)，1530(m)，760(w)，740(w)，700(m)cm ^-1。Expection element (C ₁₃H ₁₈O ₄N ₂) assay value; Value of calculation C:58.63, H:6.81, N:10.52. observed value C:58.60, H:6.82, N:10.54.

Embodiment 20

(5.08g 16.7mmol) is dissolved in the diox (10mL) and be cooled to 0 ℃ to α-tert-butyl dimetylsilyl oxygen base-uncle N--butoxy (buthoxy) carbonyl Aminoacetamide that will produce according to embodiment 12 (12-1).Add 4N hydrochloric acid-dioxane solutions (17mL) subsequently and stirred 1 hour in this temperature.

In order to finish reaction, further add 4N hydrochloric acid-dioxane solution, temperature is risen to room temperature and stirred 1 hour.In solution, add ether subsequently, with ether sedimentation, filtration and the big product amount of washing possibility.Drying precipitated to obtain pure Alpha-hydroxy glycyl amide hydrochloride (1.86g, 88%) under the pressure that reduces subsequently.The collection of illustrative plates of expection comprises: ¹HNMR δ (DMSO-d ₆) 4.99 (brsd, 1H), 7.62-8.03 (br, 2H), 8.32-8.85 (br, 3H) .IR (KBr) 1686 (s), 1581 (m), 1546 (m), 1477 (s), 843 (m) cm ^-1

Embodiment 21

Will be according to the α-methoxyl group-N-benzyloxycarbonyl Aminoacetamide (0.24g of embodiment 15 (15-1) preparation, 1.0mmol) be dissolved in the methanol, 12N hydrochloric acid (0.1mL) and palladium-carbon (50mg) are added in the solution in room temperature, and under nitrogen atmosphere, stir 30 minutes.Subsequently palladium-carbon is leached and with the solvent of mother solution distill with obtain α-methoxyl group glycyl amide hydrochloride (0.14g, quantitatively).The collection of illustrative plates of expection comprises: ¹HNMR δ (CD ₃OD) 3.35 (s, 3H), 5.01 (s, 1H), ¹³CNMR δ (CD ₃OD) 42.1,84.3 (d, J=159.8Hz), 170.3.Ensuing embodiment introduces and a kind ofly is used for synthetic Alpha-hydroxy-glycyl amide hydrochloride to make the method for medicine or medicament.

Embodiment 22

The preparation of Alpha-hydroxy-glycyl amide hydrochloride

22.1 Acetic acid,oxo-,monohydrate methyl ester hemiacetal

Will (7.0g, 76mmol) solution refluxes and to spend the night at the Acetic acid,oxo-,monohydrate monohydrate in the methanol (35mL).Use saturated NaHCO subsequently ₃Solution is neutralized and evaporates.Residue is dissolved in CH ₂Cl ₂In and at Na ₂SO ₄On carry out drying.Evaporation provides the raw oil of 3.23g (40.0%), and it is used to need not in the following reaction further purification.

22.2 uncle N--butoxy carbonyl-Alpha-hydroxy methyl aminoacetate

Will be in toluene (45mL) The Acetic acid,oxo-,monohydrate methyl ester(2.0g, 18.9mmol) (2.0g, 17.18mmol) solution refluxes and spends the night hemiacetal with the tert-butyl carbamate.Evaporation provides oil.As eluent this raw oil is carried out purification by silica gel column chromatography EtOAc/ heptane 1/9 to 2/8.Pure fraction provides the oily product of 0.6g, with ether/heptane it is carried out crystallization subsequently.Output 0.39g (10.1%).Observed ¹The HNMR spectrum is: NMR (300MHz, CDCl ₃) δ 5.74 (br s, 1H), 5.44 (br s, 1H), 3.84 (s, 3H), 1.46 (s, 9H).

¹³C NMR(300 MHz，DMSO-d ₆)δ170.3，154.7，78.6，72.8，51.9，28.1。

22.3 uncle N--butoxy carbonyl-Alpha-hydroxy Aminoacetamide

(0.34g 1.66mmol) is dissolved in 7N NH with uncle N--butoxy carbonyl-Alpha-hydroxy methyl aminoacetate ₃Methanol solution (4mL).Solution is stirred, evaporates and use subsequently twice of acetonitrile coevaporation in ambient temperature overnight.As eluent product is carried out purification by silica gel column chromatography EtOAc/ heptane 3/7 to 5/5.Output 0.1g (31.7%).Observed NMR spectrum is: ¹HNMR (300MHz, DMSO-d ₆) δ 7.28 (br d, 2H), 6.20 (d, 1H), 5.09 (t, 1H), 1.39 (s, 9H). ¹³C NMR(300MHz，DMSO-d ₆)δ171.7，155.0，78.3，73.4，28.2。

22.4 Alpha-hydroxy-Gan ammonia ether amine hydrochlorate

(40mg 0.2mmol) is dissolved in (1.5 mL) in the diox with uncle N--butoxy carbonyl-Alpha-hydroxy Aminoacetamide.In 0 ℃ 4N HCl De dioxane solution (0.5mL) is added in the solution.Remove cooling bath and with solution in stirring at room 40 minutes.Add ether and agitating solution.Ether and residue evaporated inclines.Output is about 40mg.Observed NMR spectrum is:

¹HNMR(500 MHz，DMSO-d ₆)δ8.5-7.1(m，5H)，4.85(s，1H)。

¹³C NMR(500MHz，DMSO-d ₆)δ173.1，87.4。

The following example is introduced the method for a kind of α of preparation-methoxyl group-Aminoacetamide

Embodiment 23

The preparation of α-methoxyl group-Aminoacetamide

23-1 N-(9H-fluorenes (Fluoren)-9-ylmethoxy carbonyl)-α-methoxyl group methyl aminoacetate

(276mg, 3mmol) (320mg 1.33mmol) is dissolved in (10mL) in the anhydrous ether with 9H-fluorenes-9-ylmethyl carbamate with the Acetic acid,oxo-,monohydrate monohydrate.With mixture in stirred overnight at room temperature.Be dissolved in (20mL) in the methanol with solvent evaporation and with residue, add 1 sulphuric acid.With reactant mixture in stirring at room 3 days.In mixture, add saturated NaHCO ₃(100mL) and with ethyl acetate it is extracted, at Na ₂SO ₄On carry out drying and evaporate.Residue is carried out purification to provide the title compound of 250mg (55%) on silicagel column.Observed NMR spectrum is:

¹H NMR(300MHz，CDCl ₃)δ7.76(d，2H)，7.59(d，2H)，7.40(t，2H)，7.31(t，2H)，5.90(br d，1H)，5.35(d，1H)，4.46(m，2H)，4.24(t，1H)，3.82(s，3H)，3.43(s，3H).

¹³C NMR(300MHz，CDCl ₃)δ143.6，143.5，141.2，127.7，127.1，124.9，120.0，80.5，67.2，56.2，52.9。

23-2 α-methoxyl group Aminoacetamide

In room temperature 3N NH ₃Methanol solution (20mL) spend the night handle N-(9H-fluorenes-9-ylmethoxy carbonyl)-α-methoxyl group methyl aminoacetate (240mg, 0.7mmol).By evaporative removal methanol.Solid is dissolved among the THF (30mL) and add morpholine (305mg, 3.5mmol).In stirring at room mixture 5 hours.Solvent evaporation concentrated and on silicagel column purified product to provide the title compound of 5mg (6%).Observed NMR spectrum is: ¹H NMR (300MHz, CDCl ₃) δ 4.40 (brs, 1H), 3.35 (s, 3H).

The Aminoacetamide chemical compound of modification described herein is suitable for use as a kind of biotechnology instrument to study the interaction between described chemical compound and the HIV, also infected the experimenter's of HIV medicine or medicament, or infected to avoid HIV as a kind of preventative goods as treatment.By the obtainable cofactor of methods described herein (separately or associating or in conjunction with G-NH ₂Or comprise G-NH ₂Peptide, as GPG-NH ₂) also be suitable for use as the biotechnology instrument and be used as the medicament for the treatment of and preventing HIV to duplicate.By a kind of method, for example, require a kind of prodrug therapy, wherein G-NH ₂Or comprise G-NH ₂Peptide, as GPG-NH ₂Be provided for the experimenter who needs, and provide cofactor by being total to administration.Perhaps, G-NH ₂Or comprise G-NH ₂Peptide, as GPG-NH ₂And cofactor can be combined in (for example, a kind of G-NH of comprising in the medicine ₂Or comprise G-NH ₂Peptide, as GPG-NH ₂And the pharmaceutical composition of cofactor).When, for example, when needing release on time or long-term treatment, can be at this intravenously administrable cofactor and/or G-NH ₂And/or GPG-NH ₂And/or other peptide that comprises Aminoacetamide is as prodrug.

Although can handle anyone as preventive by enough these anti-HIV compositions, only experimenter is the people who is in the risk from viral infection.These experimenters include, but not limited to the old man, chronic, homosexual person, prostitute, intravenous drug user, hemophiliac, child, and the medical professional of once contacted patient or biological sample.

Preparation and use comprise the G-NH of modification ₂The method of the medicament of (for example, metabolite X or α HGA) also is embodiment of the present invention.Can be according to the traditional method of lid human relations pharmaceuticss (galenic pharmacy) G-NH to the modification that can obtain by methods described herein ₂Handle, to produce medicament to the patient, for example mammal comprises that the people carries out administration.Can be with the G-NH that modifies ₂Through improving or being incorporated in the drug products without improvement.In addition, will transmit the G-NH that modifies by some paths ₂Medicine or the preparation of therapeutic agent be included in the scope of the present invention.

The G-NH of described modification ₂Can mix with conventional excipients, be suitable for promptly that intestinal is outer, intestinal (for example, mouthful) or the medicinal organic or inorganic carrier mass of local application use, described conventional excipients can not carried out deleterious reaction with peptide reagent.Suitable pharmaceutical carrier includes, but are not limited to water, saline solution, alcohol, Radix Acaciae senegalis, vegetable oil, benzylalcohol, Polyethylene Glycol, gelatin, sugar is such as lactose, amylose or starch, magnesium stearate, Talcum, sialic acid, the alkane of viscosity (viscousparaffin), aromatic oil, glycerine monofatty ester and two glyceride, pentaerythritol fatty ester, hydroxy methocel, polyvinylpyrrolidone etc.Can sterilize to pharmaceutical preparation, and if desired, with its mix with not can with the G-NH that modifies ₂Auxiliary reagent with adverse reaction, for example lubricant, antiseptic, stabilizing agent, wetting agent, emulsifying agent, the salt that influences osmotic pressure, buffer, coloring agent, flavoring agent and/or aromatic substance etc.

The G-NH that will comprise in some embodiments, modification ₂Medicine combine preparation with other reagent or in conjunction with using, described other reagent suppresses viral infection, infect to obtain better virus reaction such as HIV.At present, medicine that four classes are different is used for the clinical practice of the antiviral therapy that people HIV-1 infects.These are (i) nucleoside analog reverse transcriptase inhibitors (NRTIs), such as zidovudine, Lamivudine, stavudine, didanosine, Abacavir and zalcitabine; (ii) the nucleotide analog reverse transcriptase inhibitors is such as tenofovir; (iii) non-nucleoside reverse transcriptase inhibitor (NNRTIs) is such as efavirenz, nevirapine and delavirdine; And (iv) protease inhibitor, such as indinavir, viracept see nelfinaivr, ritonavir, Saquinavir and ammonia Pune Wei.By using different medicine of two classes, three classes or four classes and combination simultaneously to use the G-NH of modification ₂, the probability of HIV development resistance reduces because in identical virion at the G-NH of different types of medicine and modification ₂The probability that occurs of a plurality of sudden changes less.

Therefore, the G-NH that comprises modification that preferably prepares jointly or use with dosage well known by persons skilled in the art and method and nucleoside analog reverse transcriptase inhibitors, nucleotide analog reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitor ₂Medicament.Can prepare the G-NH that comprises modification ₂With nucleoside analog reverse transcriptase inhibitors, nucleotide analog reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, thereby and protease inhibitor comprise other one-tenth and assign to assist the G-NH that modifies ₂Transmission, stop or the medicine of stability.

The G-NH of specific modification ₂The effective dose of preparation and application process can be based on individual patient and disease stages, and other factor well known by persons skilled in the art and changing.Operate treatment effective percentage and the toxicity that to determine this chemical compound, for example ED by the standard drug in cell culture or laboratory animal ₅₀And LD ₅₀(colony that causes death 50% dosage).Therapeutic index is toxicity and the dosage rate for the treatment of effect, and it can be expressed as LD ₅₀/ ED ₅₀Ratio.The preferred pharmaceutical composition that shows big therapeutic index.To be used to prepare the used dosage range of people available from the data of cell culture assays and zooscopy.The dosage of this chemical compound preferably is present in and comprises having seldom or do not have toxic ED ₅₀Circulation composition (circulating concentration) scope in.Dosage depends on used dosage form, patient's sensitivity and the approach used and changing.

In view of patient to be treated, select the accurate dose scheme by individual doctor.Dosage and method of application are adjusted with active part that sufficient dosage is provided or kept required effect.Admissible other factor comprises seriousness, age, body weight and the sex of patient disease situation; Diet, time of using and frequency, drug regimen, reaction sensibility and toleration/reaction to treating.The short-acting drug compositions is used and per 2,3 to 4 days of depot drug product compositions every day, and jede Woche is used or every fortnight is once used.The half-life and the clearance rate that depend on particular formulations, pharmaceutical composition of the present invention is used 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more times number every day.

Depend on the path of using, normal dose can change between 000 microgram at about 1-100, and accumulated dose can about 20 grams of as many as.Ideal dosage comprises 250 μ g, 500 μ g, 1mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 1g, 1.1g, 1.2g, 1.3g, 1.4g, 1.5g, 1.6g, 1.7g, 1.8g, 1.9g, 2g, 3g, 4g, 5,6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g and 20g.In addition, the G-NH of modification ₂Concentration in the embodiment of using reagent with localized forms, can be quite high.The molar concentration of peptide reagent can be used with some embodiments.Local application and/or the coating armarium the ideal concentration scope at 100:M between the 800mM.The preferred concentration range for of these embodiments is between 500:M-500mM.For example, be used for topical application and/or the coating armarium preferred concentration comprise 500 μ M, 550 μ M, 600 μ M, 650 μ M, 700 μ M, 750 μ M, 800 μ M, 850 μ M, 900 μ M, 1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, 200mM, 300mM, 325mM, 350mM, 375mM, 400mM, 425mM, 450mM, 475mM and 500mM.At document and hereinafter provide about the method transmitted and the guidance of particular dosage regimen.(for example see U.S. Patent number 4,657,760; 5,206,344; Or 5,225,212).

More specifically, the G-NH of modification ₂Dosage provide the G-NH of enough modifications ₂To obtain to comprise that the virus that suppresses suitable discharges and/or suppress the ideal effect that duplicates of HIV.So G-NH that modifies ₂Dosage preferably produce between about 0.1nM to the tissue between the 500mM or haemoconcentration or both.Ideal dosage produces between about 0.1nM to the tissue between the 800 μ M or haemoconcentration or both.Preferred dosage produces tissue or the haemoconcentration that surpasses the about 300:M of about 10nM-.Preferred dosage is for example, to obtain 10nM, 15nM, 20nM, 25nM, 30nM, 35nM, 40nM, 45nM, 50nM, 55nM, 60nM, 65nM, 70nM, 75nM, 80nM, 85nM, 90nM, 95nM, 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1 μ M, 10 μ M, 15 μ M, 20 μ M, 25 μ M, 30 μ M, 50 μ M, 100 μ M, the G-NH of the tissue of 200 μ M and 300 μ M or haemoconcentration or the modification that both are required ₂Amount.Though generation is not preferred greater than the dosage of the tissue concentration of 800 μ M, they can be used with some embodiments.The G-NH of modification can also be provided ₂Lasting infusion to keep steady concentration the same in the tissue with blood levels.

The G-NH that modifies ₂Use the path of using that the path includes, but not limited to alveolar partial, transdermal, parenteral, gastrointestinal, overdraw trachea and saturating.Local application is by comprising the G-NH of modification ₂The ointment of topical application, gel, rinse etc. carry out.By using the G-NH that can make modification ₂The ointment, rinse, the gel that pass skin and enter in the blood flow are finished using of transdermal.The parenteral path of using includes, but are not limited to such as the Electricinjection or direct injection, intravenous injection, intramuscular injection, peritoneal injection or the subcutaneous injection that directly enter central vein system.The gastrointestinal path of using includes, but are not limited to ingest and rectum.The path of overdraw trachea of using and saturating alveolar includes, but not limited to air-breathing by mouth or intranasal.

Be applicable to the G-NH that comprises modification of topical application ₂Compound compositions include, but not limited to implant, ointment, ointment, rinses and the gel that physiology is accepted.Chemical compound wherein is that the soluble any liquid of bottom line, gel or solid medicinal substrate are applicable to local use the of the present invention at least.The local compositions of using is useful especially for the propagation that prevents HIV in the sexual intercourse process.The suitable compositions of this application includes, but are not limited to vagina or supp anal, ointment, gel, lubricant, oil and irrigating.

Be applicable to the G-NH of the modification of transdermal administration ₂Compositions include, but not limited to directly apply to skin or be incorporated into medicinal suspension, oil, ointment and the ointment of protectiveness carrier such as transdermal device (" percutaneous plaster ").For example, can find in doctor's Desk Reference that the example of suitable ointment, ointment etc. and its are well-known in the art.The case description of suitable transdermal device in, for example licensed on April 4th, 1989 in the U.S. Patent numbers 4,818,540 such as Chinen.

Be applicable to the G-NH of the modification of parenteral administration ₂Compositions include, but are not limited to medicinal sterilization isosmotic solution.The G-NH that these solution include, but are not limited to modify ₂The salt and the phosphate buffered saline(PBS) that are expelled to central vein system, intravenous, intramuscular, intraperitoneal or subcutaneous injection agent.

Be applicable to the G-NH of the modification that overdraw trachea and saturating alveolar are used ₂Compositions includes, but not limited to various types of aerosoies that are used to suck.For example, the pneumonia that causes by pneumocystis carinii with prevention to AIDS patient's intranasal administration pentamidine by aerosol.Be applicable to overdraw trachea and the saturating alveolar that include but not limited to nebulizer and vaporizer are used the G-NH of modification ₂Device be also included within the scope of the invention.The nebulizer of many forms that can obtain and vaporizer can easily be used to transmit the G-NH of modification at present ₂

Be applicable to the G-NH of the modification that gastrointestinal is used ₂Compositions include, but are not limited to pharmaceutical powder, pill, sachet or liquid that is used to ingest and the suppository that is used for rectal administration.According to the easiness that HIV infects modal path and use, preferably pass through gastrointestinal, especially mouth uses.For example, the medicine that gastrointestinal uses be will be used for and capsule, pill or tablet form will be mixed with, active component wherein, the Aminoacetamide of modifying (for example, Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer, two glycyl amidogen ethers or α-methoxyl group Aminoacetamide) exists with the effective dose that inhibition HIV duplicates.

The G-NH that modifies ₂Also be applicable to and wherein prevent HIV to infect to be in the important situation.For example, the healthcare givers contacts with the patient always, and described patient may be that male and its secretions of HIV and body fluid comprise HIV virus.In addition, can be with the G-NH that modifies ₂Be mixed with the propagation of the antiviral composition that in the sexual intercourse process, uses with prevention HIV.These compositionss are known in the art, and be described in May nineteen ninety disclosed Modak on the 3rd etc. the PCT publication number be in the international application of WO90/04390.

Embodiment of the present invention also comprises the coating of protecting armarium such as glove, coverlet and working surface to avoid virus disseminating.Perhaps, can be with the G-NH that modifies ₂Inject polymeric medical treatment device.Particularly preferably be the coating of medical gloves and condom.Can be suspended in wherein the polymer coated coating that is applicable to medical treatment device that provides by the powder that comprises peptide or by peptide reagent.The suitable polymeric material that is suitable for coating or equipment is the G-NH that can be the modification of that physiology is accepted and treatment effective dose ₂Can be by those of its diffusion.The polymer that is fit to includes, but are not limited to polyurethanes, polymethacrylates, polyamide, polyester, polyethylene, polypropylene, polystyrene, politef, polrvinyl chloride, cellulose acetate, silicone elastomer, collagen protein, fibroin etc.These coatings, for example being described in, JIUYUE in 1986 licensed in the U.S. Patent number 4,612,337 of Fox etc. on the 16th.Therefore, as mentioned above, the medicament that preparation inhibition HIV duplicates comprises the G-NH that modification is provided ₂And prepare described medicament to be delivered to the experimenter who comprises the people.

The method of the chemical compound of identifying that inhibition HIV duplicates also is provided.By a kind of method, for example, by making G-NH ₂With serum, blood plasma or cell extract incubation up to being enough to the G-NH that metabolism is modified ₂And the G-NH that separates modification by cation exchange HPLC ₂Identify the chemical compound in the medicine that is suitable for being incorporated into anti-HIV.Preferred end user's serum, porcine blood serum, Ox blood serum, cat serum, dog serum, horse serum, monkey serum or porcine blood plasma.By this method, the G-NH of modification ₂Elute from post rapidly, and unreacted G-NH ₂Be retained in the relative longer time on the post.As mentioned above, in the situation that isolated compound exists, can further confirm the G-NH that modifies by carrying out the infectious research of HIV ₂Separation.Similarly, use the infectious research of HIV as herein described can screen the relevant synthetic compound of derivant with Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer, two glycyl amidogen ethers, methoxyl group Aminoacetamide, α-ethyoxyl Aminoacetamide and these chemical compounds.Depend on isolating G-NH ₂Purity or the structure of the Aminoacetamide of synthetic sex modification, the ED of described chemical compound ₅₀Between being less than 1 μ M and being less than between the 30 μ M.That is the G-NH of pure modification, ₂ED ₅₀Be less than 100nM, 200nM, 300nM, 400nM, 500nM, 600nM, 700nM, 800nM, 900nM, 1 μ M, 2 μ M, 3 μ M, 4 μ M, 5 μ M, 6 μ M, 7 μ M, 8 μ M, 9 μ M, 10 μ M, 11 μ M, 12 μ M, 13 μ M, 14 μ M, 15 μ M, 16 μ M, 17 μ M, 18 μ M, 19 μ M, 20 μ M, 21 μ M, 22 μ M, 23 μ M, 24 μ M, 25 μ M, 26 μ M, 27 μ M, 28 μ M, 29 μ M, or 30 μ M.Therefore, in some embodiments, the G-NH of the modification that will identify by said method ₂Be incorporated in the medicine.In addition, this method can be by providing antiviral compound to replenish to medicine, and described antiviral compound is selected from the group of being made up of nucleoside analog reverse transcriptase inhibitors, nucleotide analog reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitor and protease inhibitor.In addition, said method can replenish by carrier is mixed in the medicine.

Although can be with the G-NH that modifies ₂Research tool with the HIV that performs an analysis suppresses it is desirable to the G-NH that will modify ₂The HIV that is used to suppress the experimenter duplicates and infects.For example, by a kind of method, identify risky infected by HIV or infected by HIV the experimenter and the G-NH of modification is provided for described experimenter ₂By another kind of method, provide the G-NH of modification to the experimenter ₂And determined its influence of HIV being duplicated or infecting (for example, by p24 in the analysis of biological samples amount or reverse transcriptase activity).

The Aminoacetamide that expection is modified suppresses duplicating of HIV by the mechanism that is different from conventional nucleoside analog and protease inhibitor.(see U.S. Patent number US6258932; US6455670; US6537967).Therefore, the experimenter of the medicine of the preferred Aminoacetamide of accepting to comprise modification is the HIV infected individuals that has formed the resistance of nucleoside analog and protease inhibitor.

By a kind of method, the patient who infects for 9 HIV provides the Aminoacetamide (for example, Alpha-hydroxy Aminoacetamide, α-peroxide Aminoacetamide dimer, two glycyl amidogen ethers or α-methoxyl group Aminoacetamide) of not commensurability modification and has analyzed the inhibition that HIV is duplicated.The Aminoacetamide of the modification of 1.0g is provided for the group I that comprises 3 individualities by one day 3 times capsule form; And the Aminoacetamide of the modification of 1.5g is provided for the group II that comprises 3 individualities by one day 3 times capsule form; The Aminoacetamide of the modification of 2.0g is provided for the group III that comprises 3 individualities by the capsule form of whole day.The routine techniques (for example Roche AMPLICOR MONITOR) of the amount by detecting HIV RNA is monitored the minimizing of viral lode every day.Minimizing as the amount by the HIV RNA that detects is indicated, will observe the minimizing of viral lode.

Above-mentioned method can be replenished with giving antiviral therapy, and described antiviral therapy is selected from the group of being made up of nucleoside analog retroviral inhibitors, nucleotide analog reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and protease inhibitor.In addition, use the G-NH of the modification in these methods ₂Can combine with holder or in the medicine that comprises pharmaceutical carrier, use.Although, described the present invention in detail, it will be appreciated by those skilled in the art that in the prerequisite that does not deviate from actual range of the present invention and can make various variations on form and the details for illustrating and understanding purpose of the present invention.

Claims

1. medicine that is used for the treatment of or prevents the compound or pharmaceutically acceptable salt thereof that comprises formula C, amide or the ester of the therapy that HIV infects, its Chinese style C is provided by following formula:

2. the medicine of claim 1, wherein said medicine is the Alpha-hydroxy Aminoacetamide, or its pharmaceutical salts.

3. the medicine of claim 2, wherein said medicine is a val-Alpha-hydroxy Aminoacetamide, or its pharmaceutical salts.

4. the compound or pharmaceutically acceptable salt thereof, amide or the ester that comprise formula C are used to prepare the application for the treatment of or preventing the medicine of HIV infection, and its Chinese style C is provided by following formula:

5. the application of claim 4, wherein said chemical compound are the Alpha-hydroxy Aminoacetamides, or its pharmaceutical salts.

6. the application of claim 4, wherein said chemical compound are val-Alpha-hydroxy Aminoacetamides, or its pharmaceutical salts.

7. method for compositions of duplicating for preparing HIV inhibiting (HIV), described method comprises:

Provide to comprise the Alpha-hydroxy Aminoacetamide, or the chemical compound of its pharmaceutical salts; With

Prepare the described compositions that is used for the people's administration that is subjected to the HIV infection.

8. the method for claim 7, wherein said chemical compound is the Alpha-hydroxy Aminoacetamide, or its pharmaceutical salts.

9. the method for claim 7, wherein said chemical compound is a val-Alpha-hydroxy Aminoacetamide, or its pharmaceutical salts.