Scopoli and monodisperse water-soluble oligomer, the method of its production, pharmaceutical product, the method of inhibiting the activity of viruses

 

(57) Abstract:

Describes Scopoli and monodisperse water-soluble oligomer, which is the ratio of polydispersity 1.0 to 1.3, which is a polyurea of the formula (I), where R is a hydrogen atom, a C1-C4, an alkyl group, phenyl group or phenyl group substituted 1 - 2 fragments R1and with up to 3 substituents independently selected from chlorine atoms or bromine, or WITH1-C4alkyl group; R1- SO3R2, - CO2R2, - PO3(R2)2or - OPO3R2; R2is a hydrogen atom or a pharmaceutically acceptable cation; m = 0 or 1, provided that when m = 0, R is a hydrogen atom; X is an aromatic Deputy whose values are described in paragraph 1 of the formula; Y IS-CO2-, -C=C-, -N=N - or (a) n = 3 to 15, an integer; R3is-R or-X-NH2where R and X have the above meanings. The compounds exhibit biological activity against the virus of human immunodeficiency. Also describes pharmaceutical drugs based on them, the method of obtaining the oligomer, the method of inhibiting the activity of viruses. 6 C. and 20 C.p. f-crystals, 15 ill., 3 table.

The invention relates to Scopoli and MES is vnesti virus human immunodeficiency. These monodisperse anionic oligomers, thus, applicable in the treatment of acquired immunodeficiency syndrome (AIDS) and in the treatment of diseases caused by the virus Herpes Simplex (HSV) types 1 and 2 and cytomegalovirus.

A large number of studies now under way on the development of therapies and methods of treatment of viral infections in humans and animals, particularly those caused by Herpes Simplex virus (HSV) types 1 and 2 and AIDS and associated with AIDS complex (KCC). The frequency of AIDS and the CMP in humans increases with particularly alarming speed. A five-year term of life of affected AIDS depressing, and patients with AIDS, whose immune system is severely impaired infection, suffer from multiple infections caused by opportunistic pathogens, including Kaposi's sarcoma and pneumonia caused Pheumocystis carinii. A method for the treatment of AIDS is unknown, and current therapy in most cases is applied without sufficient evidence of efficacy and has numerous adverse side effects. Fear of the disease has led to social ostracism and discrimination against patients or suspected disease of the people.

Retroviruses are a class of viruses that contain ribonucleic acid (RNA), kotoroy is produced by double-stranded proviral DNA. This proviral DNA is then randomly included in the chromosomal DNA of the host cell, making possible the replication of the virus by a later broadcast of viral information from the integrated viral genome.

Many of the known retroviruses are carcinogenic or cause swelling. In fact, the first two discovered human retrovirus, designated as viruses I and II human T-cell leukemia or HTLV-I and II were found, a rare cause leukemia in humans after infection of T-lymphocytes. Third, this human virus that was opened, HTLV-III, now called HIV, how it was discovered, causes cell death after infection of T-lymphocytes, and was identified as the etiological agent of AIDS and the CMP.

Envelope protein of HIV is a glycoprotein 160 kDa. This protein is cleaved by a protease to obtain the outer protein of 120 kDa, gp 120, and transmembrane glycoprotein gp 41. Protein gp 120 contains an amino acid sequence that recognizes the antigen CD 4 human T-helper (T4) cells.

One used approach is to prevent binding of HIV to its target, the T4 cells in humans. These T4 cells have spezifikationen host cells can be suppressed.

Interference in the formation of the viral envelope protein could prevent the primary interaction of virus and host cell or subsequent to the merger, or could prevent duplication by preventing build the corresponding glycoprotein required for completion of the membrane of the virus. It was reported (see H. A. Blough et al., Biochem. Biophys, Res. Comm. 141(1), 33-38 (1986), the nonspecific inhibitors of glycosylation, 2-deoxy-D-glucose and-hydroxyvitamin inhibit the expression of the glycoproteins of HIV and block the formation of the syncytium. The replication of the virus in HIV-infected cells treated with these substances, stops, apparently, for lack of a glycoprotein necessary for the formation of the membrane of the virus. In another report (W. Mc Dowell et. al., Biochemistry, 24 (27), 8145-52 (1985), as has been discovered, the glycosylation inhibitor, 2-deoxy-2-fluoro-D-mannose, and inhibits antiviral activity against infected with influenza virus cells through the prevention of glycosylation of viral membrane protein. This message also States that has been studied the antiviral activity of 2-deoxyglucose and 2-deoxy-2-fergusoni and found that each suppressed glycosylase glycosylation does not have antiviral activity. Thus, the antiviral activity against viruses in General and specific antiviral activity of inhibitors of glycosylation completely unpredictable.

Are ongoing worldwide research on the development of therapies and methods of treatment of infections caused by HSV type 1 and 2. Both HSV type 1 and 2 show a predisposition to infection ectodermal tissues, where is the infection by virus causes lesions on the skin, mouth, vagina, conjunctiva and nervous system. In General, infection with HSV type 1 (HSV) is associated with lesions of the mouth, face and eyes. Infection with HSV type 2 (HSV 2) mainly causes genital and anal injuries. HSV infections, left untreated, often lead to blindness, death in infants and encephalitis. Infections caused by HSV type 2, are at epidemic levels in the U.S. as a result of sexual transmission. Currently, more than about twenty million people affected by the disease in this country with over half a million new cases and relapses per year. Annual costs for HSV infections cause significant economic losses on diagnosis and treatment. Epidemiological to the m

Man is the natural host for infections caused by HSV types 1 and 2, and the virus is transmitted during close personal contact. The initial or primary infection with HSV type 1 and 2 occurs through breaks in the mucosa. In a healthy carrier of the virus can be isolated from tears, saliva, vaginal and other secrets, even in the absence of manifest disease. Mucous membrane, they are able to replicate and spread to regional lymph nodes. Sometimes these viruses can infect cells of the system of hemopoiesis and cause viremia.

Part of the difficulty in the treatment of HSV infections occur because of the ability of these viruses to persist in a latent or dormant form. When primary infection subsides or retreats, the virus mainly remains in a latent form in sensory nerve ganglia, which innerviews place of primary infection. When ocular or oral infections caused by HSV type 1, the virus usually remains in the trigeminal ganglia. When infections caused by HSV type 2 virus stays mainly in the sacral ganglia, the technician genitals and lower abdomen. Defines the period of latency of HSV is not known, in addition, this period can IMMUNOSUPRESSIVE means, leading mainly to repeated infections.

Treatment of HSV infections, mainly ineffective. Was developed a number of strategies to stop the virus. These funds mainly inhibit any one of a number of specific functions of the virus, such as (1) adsorption, (2) loss of shell, (3) transcription, (4) protein synthesis, (5) replication of nucleic acids, (6) maturation, and (7) the selection.

Most anti-virus tools used so far for the treatment of HSV infections were substances that interfere with the synthesis of viral DNA. These substances include idoxuridine, cytosine, arabinose, adrenalised and triptorelin. These substances interfere in similar functions in the host cell, which leads to common problems cellular toxicity and systemic use in humans. Now acyclovir is the preferred drug for treatment of infections caused by HSV 1 and HSV 2, thanks to its strong antiviral activity and low toxicity. However, poor solubility of the high-dose and the emergence of drug-resistant viruses restricts the use of this medicine.

A number of RNA - and DNA-containing viruses have a membrane, in koumi viruses. Infection of a host cell enveloped viruses initially based on the interaction of different receptors on the surface of the host cell with shell membrane glycoproteins of the virus. Then the viral and cellular membranes fuse, and the contents of the virion flows into the cytoplasm of the host cell. Containing glycoprotein shell of the virus plays an important role in the initial interaction of the virion and the host cell and subsequent merging of the membranes of the virus and host cell. Apparently the shell of the virus occurs from the cell membrane, but its specificity is associated with the encoded virus glycopeptides. Therefore, the inhibitor can be a hindrance to education virousspecificakih membranes, can prevent the formation of infectious progeny virus.

In European application 04065512, published on 9 January 1991, it was revealed that the purified form of heparin, a sulfated polysaccharide, communicates through interaction with the viral protein, which is responsible for the recognition of cells and creates a limited suppression of infection of the host cell. However, heparin causes some side effects, especially bleeding and increased the formation of a clot, Elijah, purpura, thrombocytopenia, intracranial hemorrhage, bacterial endocarditis, active tuberculosis, increased capillary permeability, ulcerative lesions of the gastrointestinal tract, severe hypertension, threatened abortion or visceral carcinoma. Of particular interest is the contraindication for use in patients with hemophilia, as currently many such individuals are seropositive for HIV.

It has long been known that certain synthetic water-soluble polymers exhibit a wide range of biological activity. (R. M. Ottenbrite b "Biological Activities of Polymers". Amer. Chem. Soc. Symp. Ser. N 182, pp. 205-220, eds. C. E. Carsaher and C. G. Gebelein (1982)). As shown, the copolymer deviceloop ether and maleic anhydride active against several viruses, and its use in cancer chemotherapy has been studied for several years (D. S. Breslow Pure and Applied Chem, 46, 103 (1976)). Polyacrylic, polymethacrylates and a number of other aliphatic frame water-soluble polymers have been shown to also possess a wide spectrum of biological activity (W. Regelson et al., Nature 186, 778 (1960)). European application 0043974 reveals from sulphonated polyurethanes which do not have the desired antiviral applications. British patentlicense molecular weight is 10000 or more. Unfortunately, the extreme toxicity of these polymers prevented their clinical use. In addition, these polymers have high molecular weight and are not able to pass through the kidney membrane.

Attempts were made to overcome the problems of toxicity and excretion by the synthesis of aliphatic polymers with a low molecular weight (1000 to 10000) (P. M. Ottenbrite b in "Biological Activities of Polymers".. Ames. Chem. Soc. Symp. Ser. N 182, pp. 205-220, ds. C. B. Cassaher and C. G. Gebelein (1982)). It was found that such polymers are less toxic, but have a much reduced antiviral activity. These aliphatic polymers with low molecular weight can be classified as polymers "statistical tangle". Such polymers are unpredictable configuration due to the flexibility of the frame linking groups. The configuration of polymers, statistical tangle in solution, mainly can be described as spherical. Although the mechanism of action of such water-soluble polymers is unknown, one of the requirements is that the polymer binds to the membrane of the virus, for example, the virus that causes encephalomyocarditis through ionic attraction, thus rendering the virus unable to infect cells of the host.

Synthetic hard water-soluble polymers are much less common, but there are several known examples of high molecular weight (for example, see U.S. patents 4824916 and 4895660). The structure of this class of polymers that do not form a statistical tangle leads to high for a given molecular weight and concentration of viscosity solutions.

It is clear that it would be desirable to find therapy and treatment method AIDS, KCC and HSV infections, which showed minimal side effects or lack of it, and would create a clear superiority over the polymers previously used as pharmaceuticals. In addition, these oligomers should preferably have a narrow range of molecular weights, low toxicity and easily identifiable characteristics.

In the accompanying Fig. 1-4 the vertical axis represents the absorption in the UV region, and the horizontal axis is time in minutes.

Fig. 1 shows the crude polydispersed from sulphonated the polyurea of formula 1 with a broad polydispersity as source material.

Fig. 2 shows the reduction in the dispersion after stage 1, which is defined on page 11, from the rough floor of one of several factions.

Fig. 3 shows the combination of fractions 7 through 17 when gelfiltration chromatography crude polydispersed oligomer with Fig. 1, as the recombination fractions from Fig. 2.

Fig. 4 shows monodisperse oligomer from sulphonated polyurea, fractionated from the crude polydispersed oligomer with Fig. 1, with the use of liquid chromatography with reversed phase.

In Fig. 5-9 on the vertical axis is the relative absorption, and the horizontal axis is time in minutes.

Fig. 5 shows the different profiles HPLC polyurea formula I.

Fig. 6 shows the HPLC profile uzkopolosnoi polyurea formula I, when using an aqueous solution of trimethylacetate (Et3NHOAc) as eluent.

Fig. 7 shows the HPLC profile uzkopolosnoi polyurea formula I, when using an aqueous solution of tetrabutylammonium (n-Bu4N)PO4as eluent.

Fig. 8 shows the HPLC profile of monodisperse fractions polyurea formula I with Fig. 6 using an aqueous solution of tetrabutylammonium (n-Bu4N)PO)4as eluent.

Fig. 9 shows a histogram of the anti-virus kostytsya linking carbonyl parts, moreover, the above-mentioned oligomers have an anionic group and a predominant linear geometry, so between anionic groups in the aquatic environment there are constant intervals. Preferred oligomers of this invention are represented by any one of formulas I-IV, below.

The production method of the present invention allows to obtain limited poly - and monodisperse anionic oligomers of this invention, especially those represented by formulas I-IV, below, using stages:

1) restrictions on raw polydisperse mixture of anionic oligomers to uzkopolosnoi mixture of anionic oligomers, and/or

2) allocation of monodisperse anionic oligomer, and

3) optionally, conversion of salts Scopoli or monodisperse oligomers with stages 1 and 2 in the desired pharmaceutically acceptable salt, specifically, sodium or potassium salt.

In the above-mentioned process has in mind the following combination of stages: stage 1 and 2 separately, stage 1 or 2, followed by stage 3, or are all three stages.

Scopoli and monodisperse oligomers applicable as a means of suppressing the activity of the immunodeficiency virus is by HSV. The present invention includes Scopoli and monodisperse oligomers, obtaining their medicinal forms and use as a means for the treatment of AIDS, KCC and infections caused by HSV and method of their production.

Scopoli and monodisperse anionic oligomers of the present invention are water-soluble oligomers containing repeating unit, from 3 to 50, which are connected to connecting the carbonyl component, and the above-mentioned oligomers have an anionic group and a predominant linear geometry, so in the aquatic environment, there are persistent gaps between anionic groups. Preferably, these oligomers represented by any one of the following formulas:

< / BR>
where R represents a hydrogen atom, a C1-C4alkyl group, phenyl group or phenyl group having from 1 to 2 parts R1and up to 3 substituents independently selected from fluorine atoms, chlorine and bromine or C1-C20alkyl groups

R1is - SO3R2, -CO2R2, -PO3(R2)2or-OPO3R2,

R2represents a hydrogen atom or a pharmaceutically acceptable cation,

m = 0 or 1, provided that when m = 0, R is at the>R3is-R or-X-NH2where R and X is given the definition as before,

B) a polycarbonate of the formula

< / BR>
where X and n gives the definition as in formula I, above,

X1is HO-X group, where X is given the definition as for formula I, above, or C1-C4alkyl group, phenyl group or phenyl group substituted by from 1 to 2 components of R1and up to 3 substituents independently selected from fluorine atoms, chlorine or bromine, or alkyl groups, C1-C20and

X2represents a hydrogen atom or-CO2X1where X1the definition as above,

C) the polyester of the formula

< / BR>
X and n gives the definition as in formula I, above,

R4is-R2that defined in the formula I, or-X1that defined in the formula II, above,

R5is

< / BR>
where R4the definition as in the formula III, above, or R2where

R2the definition as in formula I, above;

X3is (see Fig. 11).

where R1and Y is given the definition as in formula I, above; or

D) the polyamide with the formula:

< / BR>
where X and n gives the definition as in formula I, above,

X
where R, R2and X is given the definition as in formula I,

R7represents a hydrogen atom,

< / BR>
where R and R2the definition as in formula I, above, and

X3the definition as in the formula III, above.

The terminology used in this application, provides the following definitions:

The term "C1-C20alkyl", containing the term "C1-C4alkyl" includes groups with branched and straight chain, such as, for example, methyl, ethyl, isopropyl, t-butyl, n-decyl, n-dodecyl and the like.

The term "pharmaceutically acceptable cation" means a cation that is acceptable for pharmaceutical use. Those cations that are essentially non-toxic at the dosage used to achieve the desired effect and do not independently possess significant pharmacological effects, is included in the term "pharmaceutically acceptable cation". For illustration, these salts include salts of alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, ammonium, light metals of group IIIA including aluminum, and organic cations derived primary, secondary and tertiary amines, ammonium or alkylamine, the min, N,N'-dibenziletilendiaminom, dehydroabietylamine, N-(C1-C4)alkylpiperidines and any other suitable amine. The preferred sodium and potassium salts. The term "pharmaceutically acceptable" means suitable for the introduction of warm-blooded animals, especially humans, and which includes non-toxic, for example, suitable for pharmaceutical use and non-toxic to warm-blooded animals. Pharmaceutically acceptable cations oligomers of the present invention are obtained by conventional ion exchange processes or by processing R1-acid with a suitable base. In particular, triethylammonium salt, obtained as part of the method of production (stage 2) into more preferred pharmaceutically acceptable salts such as the sodium salt.

The oligomers of the present invention are Scopoli and monodisperse water-soluble polymers with a low molecular weight. Additionally, the oligomers have an ordered arrangement of anions. The term "orderly arrangement of anions" or "constant spacing between anionic groups" means that the anionic groups (R1) are present on the polymer frame at intervals determined source mA while wanting to be bound by any theory, I believe that the anionic groups of the oligomers are part of, which is associated with HIV, HSV and/or cell membrane and thereby interrupts the ability of the virus to replicate.

The terms "predominantly linear geometry in the aquatic environment refers to the configuration of the oligomer in solution. Method to characterize the configuration of polymer molecules in solution, well known in this field of knowledge is based on the following formula, called the equation of the Mark-Houwink ("Introduction to Phisical Polimer Science ed. L. H. Sperling, pub. John Wiley & Sons; (1985), pp.81-83)

[] = KMd,

where is a proper viscosity, M is srednevekovoi molecular weight, K is a constant related to the dimensions of the chain link, and is a constant determined by the configuration of the polymer. Constant for the polymer, forming a statistical tangle, 0.5 < < 0,8 and for linear polymers is 0.8 < a 1.8. This formula relates the viscosity of a solution with molecular weight "M". For this invention, the definition of linear polymers is given to those that have a value of 0.8. For rigid rod polymer theoretical upper limit is 1.8. For a given molecular weight, the higher the viscosity of the solution will be obtained for polymers with linear confia is what value is a function of the used solvent. for a given water-soluble polymer can be different at different salt concentrations. For this invention, the salt concentration is set at the levels present in serum (example 80 g/l NaCl, 4 g/l KCl).

The term "monodisperse and polydisperse" oligomers (and similar terms) refer to the distribution of oligomers in the sample. The polydispersity in the sample is determined by the ratio srednevekovogo molecular weight, Minto srednecenovom molecular weight, Mh(see G, Odian, Principles of Polymerization Ld, ed, pp.20-25, John Wiley & Jons, 1981). For the purposes of this invention, "roughly polydisperse sample oligomer represented by Min/Mh= 1,3 (see Fig. 1). For the purposes of this invention, the oligomer is a "narrow polydisperse" oligomer, when Min/Mh= 1.0 to 1.3 (see Fig. 2), preferably from 1.0 to 1.2 and more preferably from 1.0 to 1.15. Narrow polydispersed oligomer was obtained from grossly polydisperse mixture of oligomers. For the purposes of this invention, the oligomer is a "monodisperse" oligomer, when Min/Mh= 1,0 to 1,1 (see Fig. 4), which is a narrower range within which ernesti on some size in comparison with the previous sample.

The term "rigid frame" means a repeating unit, also called repeat unit mainly consists of groups that limit the rotation of the distance from the axis of the chain. For example, p-phenylenebis and amide group -(C(O)-OL-) not easy to turn away from the axis of the chain. A limited number of rotated groups have allowed deviation of the axis of the chain, such as m-phenylene and urea. The oligomers of the present invention preferably have a rigid frame.

The purity of each of monodisperse fractions of any desired n fraction is at least 75%, preferably from about 85 to about 100%. Purity is defined as the ratio of the area of the desired oligomer to the area of all peaks observed in the analysis by HPLC.

Used here, the term "oligomer" encompasses all possible values for n, for example, from 3 to 50. The oligomers are preferably linear with n equal to an integer from 3 to 50, preferably from 3 to 15, more preferably from 5 to 10, most preferably, from 6 to 9. Of course, the molecular weight is directly associated with the value n of the resulting oligomer. It is essential that these oligomers are low enough they say the>/P>For the purposes of the present invention, the oligomers described herein, and physiologically acceptable salts are considered equivalent. Physiologically acceptable salts are salts of those bases which will form a salt with at least one acid group of the groups R1 and which will not cause significant adverse physiological effects in the introduction, which is described here. Suitable bases include, for example, hydroxides of alkali and alkaline earth metals, carbonates and bicarbonates, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, potassium carbonate, sodium bicarbonate, magnesium carbonate and the like, ammonia, primary, secondary and tertiary amines and the like. Especially preferred bases are the hydroxides of alkali metals, carbonates and bicarbonates. Physiologically acceptable salts may be obtained using conventional ion exchange processes or by treating the acid P1 corresponding basis. Examples of additional salts are described here.

The preparations of the present invention is represented in solid and liquid form. These drugs can be in the form set so that the components are mixed at an appropriate time before item is Ramsamy carrier or adjuvant.

The oligomers of the present invention are soluble in water and in salt solutions, especially with physiological pH and physiological solutions. Thus these oligomers are easily derived in the form of a convenient water pharmaceutical dosage forms. Moreover, after the introduction of this oligomer he remains dissolved and in vivo.

The preferred values for the previously described formulas I to IV are the following:

R and R3are 4-methylphenylene group

m = 1,

n = 3 to 15,

R4and R5are hydrogen,

R6is phenyl,

R7is benzoyl,

X1is a 4-methylphenylene group

X2is-CO2-(4-were)-group,

X3represents a

< / BR>
< / BR>
X represents (see Fig. 12)

Especially preferred are polyurea formula I, where R and R3are 4-methylphenylene groups, m = 1, n = 3 to 15, X is a (see Fig.13) and R2defined previously. Specific examples of such politician are:

StOS/P/T = poly[imino(3-sulfo-1,4-phenylene)-1,2-ethandiyl-(2-sulfo-1,4-phenylene) aminocarbonyl] , alpha - {[(4-metalfree)amino]-carbonyl}-omega-[(4-were)-amulets

< / BR>
PDS /P/T= poly[imino(2,5-disulfo-1,4-phenylene)aminocarbonyl] , alpha - {[(4-metalfree)amino] carbonyl} -omega-[(4-were)amino] - and is represented by formula I above when R and R3are 4-methylphenylene, R2is hydrogen, X is

< / BR>
BPDS/P/T= poly{ imino[2,2'-disulfo(1,1'-biphenyl)-4,4'-diyl} - aminocarbonyl/, alpha-{[(4-were)amino]-carbonyl}-omega- [(4-were)amino]- and is represented by formula I, when

R is 4-were, R2is hydrogen, X is

< / BR>
and especially when n= 5, 6, 7, 8, 9 or 10.

Particularly preferred polycarbonates with formula II, where X1is a 4-were, X2is-CO2-(4-were), n = 3 to 15, and X is

< / BR>
Specific examples of such polycarbonates are:

HBDS/P/C= poly[oxy(2,5-disulfo-1,4-phenylene)oxycarbonyl] , alpha-[(4-methylphenoxy)-carbonyl] -omega-(4-methylphenoxy)- and is represented by formula II, above, when X1is a 4-were, R2is hydrogen, X is

< / BR>
< / BR>
and n = 3 to 15.

HBPDS/P/C = poly{oxy[2,2'-disulfo(1,1'-biphenyl)-4,4'- diyl]-oxycarbonyl} , alpha-[(4-methylphenoxy)carbonyl]-omega-(4 - methylphenoxy)- and is represented by formula II, above, when X1is a 4-Mei the>Especially preferred polyesters with formula III, where R4and R5are hydrogen, n = 3 to 15, X3is

< / BR>
X represents (see Fig. 14)

Specific examples of such polyesters are:

HBPDS/TPC = poly{oxy[2,2'-disulfo(1,1'-biphenyl)-4,4'- diyl]-oxycarbonyl-1,4-phenylenecarbonyl] - and is represented by formula III, above, when R4and R5are hydrogen, X3is p-phenylene, X is

< / BR>
and n = 3 to 15.

HBDS /TPC = poly[oxy(2,5-disulfo-1,4-phenylene)-oxycarbonyl-1,4-phenylcarbamoyl] - and is represented by formula III, above, when R4and R5are hydrogen, X3is p-phenylene, X is

< / BR>
and n = 3 to 15.

Especially preferred polyamides with formula IV, where R6is phenyl, R7is methylbenzoyl, n = 3 to 15, X3represents a

< / BR>
and X represents a (see Fig. 15).

Specific examples of such polyamides are:

BPDS/TPC/MBC = poly{ imino[2,2'-disulfo(1,1'-biphenyl)- 4,4'-diyl]aminocarbonyl-1,4-phenylenecarbonyl}, alpha-{[(4-were)- amino]carbonyl}-omega-[(4-were)amino] - and is represented by formula IV above, when R6is R-C(O)-NH-X-NH-, R is 4-were, R2is.

The present invention relates to a method for Scopoli and monodisperse anionic oligomers, which have a narrow range of molecular weights, which makes possible the use of these oligomers as pharmaceuticals, especially humans, for the treatment of AIDS or KCC. The advantages resulting from the presence of a narrow range of molecular weight of these oligomers represent a higher activity than the corresponding mixtures, and the best characteristics of the oligomers.

The present invention is directed to a method of separating the above-mentioned oligomers on their individual components, for example, with a narrow range of molecular weights, and thus obtained product selected oligomer.

When the process of obtaining Scopoli and monodisperse anionic oligomers of the present invention can be performed in different stages, as follows.

Stage I restrictions rough mixture of polydispersed anionic oligomer to uzkopolosnoi mixture of anionic oligomer - may be carried out by gelfiltration, selective precipitation, membrane separation or by using chromatography with reversed phase, and/or

Stage 2 - videografia with reversed phase. Monodisperse purity of each of the desired n fraction is at least 75%, preferably from about 85 to about 100%, and

Stage 3 - optionally, conversion of salt Scopoli or monodisperse anionic oligomer with stage 1 or 2 in the desired pharmaceutically acceptable salt, especially sodium or potassium salt can be performed with or ion exchange, especially when formed tetrabutylammonium salt, or by adding a salt of the weak volatile acid.

When the process of the present invention uses the conventional and known in the art methods. All these techniques are known, some examples of the descriptions of these methods can be found in the following:

Hellfire - "Protein Purification", ed.Charles R. Cantor, Springer - Verlag, 1987, Chapter by Robert K. Scopes, "Separation in Solution", pp. 186-198.

Selective deposition - "Polymer Fractionation", ed. Manfred J. R., Cantow, Academic Press, 1967, Chapter Akira Kotera, Fractional Precipitation, pp. 43-65.

Membrane separation - "Polymer Fractionation", ed. Manfred J. K. Cantow, Academic Press, 1967, pp. 462.

Chromatography with reversed phase - J. Chrom, Library, 41 A, "High-Performance Liguid Chromatography of Biopolymers and Bioligomers", ed. O. Mikes Elsevier, 1988, pp. A 127 - A 208, A 303 - A 399.

The electrophoresis gel "Hotein Purification", ed. Charles. R. Cantor, Springer-Verlag, 1987, in th.

The salt of the weak volatile acid oligomers in solution in the form of ammonium salts of volatile amines can turn into a preferred pharmaceutically acceptable salts such as the sodium or potassium salt by treating a solution of a salt of an alkali metal and a weak volatile acid. When the concentration of the solution by evaporation or lyophilization amine and a weak acid are removed, and the oligomers are in the form of their alkali metal salts. Relevant examples of ammonium salts that may become at this stage, are ammonium salts, monoethylamine, triethylamine or dimethylamine (here called "ammonium salts"). Examples of salts of alkali metals are sodium and potassium hydroxide, bicarbonate, acetate, or propionate.

Obtaining raw materials gross polydispersed anionic oligomer

Rough polydispersed anionic oligomers used as starting materials in the process of the present invention to obtain Scopoli and monodisperse anionic oligomers of the present invention, obtained using the production method described in European application 0467185, published on 22 January 1992, When the process of the floor is 895660 and described further below) by replacing part of one of bifunctional monomers, monofunctional substance, a locking end, and a reaction time in the absence of surfactants. Srednetsenovoj molecular weight (Mh) is determined by the stoichiometry of the reacting substances.

Polydispersed anionic oligomers obtained using the various reactions described below.

Polydisperse polyureas and polyamides (formulas I and III, above)

The preferred method for the production of polydisperse politician and polyamides of formula I and III are described in this technology (Kershner, U.S. patent 4824916) and, in addition, is explained in the following. Also describes the various reagents and conditions.

The diamines: this includes a wide variety of aromatic diamines. The range of possible substituents is also broad and includes hydroxyl, alkenyl, lower alkyl compounds, carboxylate, sulfonate, and halogen. Deputies are not necessarily anionic at neutral pH in water.

Defunctionalize the electrophiles: phosgene (carbonyl dichloride), carbonyl dibromide, Cl3COCOCl, carbonyldiimidazole, Cl3COCO2CCl3, dicyclomine halides of aromatic dibasic acids, such as cartaleva, terephthalic, 2,6-Natalegawa acid.

The acceptors K2">

Various additives can be added to the various surfactants. Suitable surfactants can be nonionic, such as sorbitan monolaurate, sorbitan the monostearate, ethylene glycol distearate, polietilene/polipropilene-polymer. Such surfactants can be Tsunozaki of the product, and therefore, the use of surfactants is not preferred.

Solvents: when processes with a single solvent used polar aprotic solvents such as N,N-dimethylacetamide and N,N-dimethylformamide. Also apply a combination of water and a second solvent, such as toluene, carbon tetrachloride, benzene, acetone, ethylene dichloride and the like. Typical ratios of organic and aqueous solvents are about 0.5 to about 2.

When the processes described in this area, galogenangidridy added to stir the solution or suspension of other

the source materials. In some examples, while adding halogenanhydrites is added to the base. The temperature is maintained between 0 and 50oC, preferably from 20 to 30oC. Can use the reading, essentially equimolar amounts.

The reaction mixture was stirred at a rate sufficient to achieve mixing of the reagents. The reaction rate depends in part on the area of the interfacial surface, and therefore, preferably vigorous movement. For this purpose, can be used industrial mixers.

The process used to produce polydisperse politician, is a modification of the process described above.

The diamines include Diamines of the present invention are primary aromatic, with formulas described in the previous sections. Such diamines substituted by at least one group that has a charge at neutral reaction, preferably a sulfonate. Acceptable monovalent aliphatic substituents. You can use a small set of aliphatic linking groups that link together aromatic radicals, such as the TRANS-substituted ethylene and acetylene. The preferred diamines are diamines, in which the carbon-nitrogen strengthened, as they are parallel, such as 2,5-diamines-1,4-benzolsulfonat acid (PDS), 4,4'-diamino-(1,1'-biphenyl)-2,2'-disulfonate acid (BPDS), TRANS-2,2'-(1,2-ethandiyl)bis(5-AMI is Ofili: To obtain politician can be used phosgene (carbonyl dichloride) and carbonyl dibromide and other precursors of urea, such as carbonyldiimidazole, hexachloroacetone, Cl3COCO2CCl3, CCl3COCl and Cl3COCOCl.

The acid acceptors: you Can use a number of inorganic bases, such as hydroxides, carbonates, bicarbonates, phosphates of alkali metals or divalent metals. The acid acceptors with buffer properties preferred when the base is added before adding a bifunctional electrophile. Organic bases, such as trialkylamine can be used, but they are not preferred.

Monofunctional blocking the end of the matter: you Can use a number of tools such limiting molecular weight. These compounds may be aliphatic or aromatic compounds that react with diamines or bifunctional electrophiles. Examples of suitable monofunctional substances are amines, such as aniline, methylaniline, methylamine, ethylamine, butylamine, diethylamine, ammonia, N-methyl aniline, phenol and cresol. Examples of monofunctional reactive with amines substances are benzoyl chloride, acetylchloride and phenylcarbamate. These locking end of the connection may also contain charged substituents, for example, potassium-2-sulfone is required or preferred and can complicate the process of selection.

Solvents: only Preferred solvent, water, when bifunctional electrophile is liquid at the reaction temperature. An example of such difunctional the electrophile is phosgene. When using a solid water-insoluble reagents, preferably a small amount is not miscible with water co-solvent. Examples of such is not miscible with water co-solvents are chloroform, carbon tetrachloride, toluene, and methylene chloride. Typical relations of organic solvent to water is 0 to 1, with the preferred 0 to 0.1. The process is conducted at temperatures which give the opportunity to complete the reaction, typically from about 0 to 100oC. the Preferred temperatures are from 0 to 25oC. When using raw materials with low boiling point, for example, phosgene (since 6oC), it is advantageous to work at temperatures equal to or below the boiling point. Pressure is not of great importance, and in the typical case, is used atmospheric pressure. For optimal process must be carefully maintained the pH of the reaction. At low pH (< 6) the reaction is very slow, while at high pH (> 10) disfunctionally Aleksa degradation of polyurea. Preferably, the pH is maintained between 7 and 9.

When not in use, blocking the end of the matter, the regulation of the molecular weight can be achieved by careful adjustment of the stoichiometry of the reagents. The diamine or disfunctionally the electrophile can be used in excess, for example, from 1 to 100% molar excess. Stoichiometry must be calculated for any of difunctional electrophiles, which are destroyed by hydrolysis, before the reaction with the diamine. For example, when used at high pH required large excess to compensate for rapid reaction with hydroxide, which destroys it. Since the extent of this side reaction is difficult to control, preferably used monofunctional blocking the end of the substance to adjust the molecular weight. Although to adjust srednecenovogo molecular weights may be used the above-mentioned methods, the products are mixtures of polymers with multiple molecular weights, which are characterized by their distribution.

The order of addition of reagents is not critical. However, the preferred procedure is to add in the first place defunctioning of the electrophile. When use is to be part of the beginning, to achieve the desired pH and then add the remainder simultaneously with difunctional the electrophile.

Finally, it is desirable to conduct these polymerization at high concentrations. This reduces the amount of solvent that must be removed to separate the product. Moreover, in some cases, the product precipitates from the reaction solution closer to the end of the reaction, and can be separated by simple decantation of the solvent. A large part of the inorganic salt, which is obtained by reaction of the acid acceptor is removed during this process. Concentration is not a critical factor and may be from 0.5 to 50 weight %, expressing the weight of a diamine with respect to the weight of the solvent. The preferred range is from 1 to 10 weight %.

Rough polydisperse product can be distinguished by precipitation from the reaction solution with a solvent that is miscible with water but is a poor solvent for the product. Examples of such solvents are acetone, methanol, ethanol, isopropanol.

Polycarbonates and polyesters (formulas II and IV, above)

We used the process described previously for politician and polyamides, with the following exceptions: Vestitel, which are anionic at pH 7. These definaly have a structure identical structures diamines, except that amines substituted with a hydroxyl group. It is possible to pre-process diols with one or two moles of a base, to form a mono - or diperoxide. Some specific examples are dicale-4,4'-dihydroxy(1,1'-biphenyl)-2,2'-disulfonate (HBPDS) and dicale-2,5-dihydroxy-1,4-bansilalpet (HBDS).

The process conditions are significantly more crucial, due to the instability of the products in aqueous solutions. Of particular importance is the control of pH. At pH < 7 the rate of polymerization is very low, whereas at high pH (> 9) carbonate and ether groups in the polymer are subjected to hydrolysis. The preferred interval pH is from 7 to 8, and it is desirable to use automatic pH Adjuster to maintain. The applicable temperature range, which can be used for polymerization, is narrower, from 8 to 40oC, and preferably from 0 to 25oC.

After adding decolletage chloride is completed, it is advisable to wait for some time, usually from 15 to 120 minutes, to ensure that the transformation of the raw materials completed. In order to yatsa over the previously described limits. Rough polydisperse product stands out as described above.

Applications

These Scopoli and monodisperse anionic oligomers can be used to prevent syncytium formation in cells infected with HIV-1 virus, or related viruses. Anti-HIV anionic oligomers can be used to treat AIDS and the CMP and other diseases caused by the retrovirus HIV-1 or other related viruses. Scopoli and monodisperse anionic oligomers of this invention can be used in a mixture where the ratio of polydispersity 1, 3, or in the form of monodisperse oligomer, such as oligomers with selected values and in particular with formulas I through IV, or a mixture of substances with more than one formula, for example, represented by formula I and formula II compounds, or as mixtures with other known means for this antiviral applications, such as 3'-azido-3'-deoxythymidine (AZT).

The number of anti-HIV Scopoli and monodisperse anionic oligomers, which is necessary to prevent syncytium formation in HIV-infected cells can be any effective amount. It has been experimentally determined that the suppression of the formation of a syncytium, and also reduced the presence of antigen p 24.

The number of anti-HIV anionic oligomers, which must be entered for the treatment of AIDS, KCC or other diseases caused by HIV infection can vary within wide limits in accordance with the specific used unit dosing, duration of treatment, age and gender of the patient, which are treated, the nature and degree of the disease that is being treated, and other factors well known for working in the field of medicine. In addition, the anti-HIV anionic oligomers can be used in combination with other agents that are known to be useful in the treatment of retroviral diseases, and drugs, which are known to be useful for the treatment of symptoms and complications associated with diseases and conditions caused by retroviruses.

Effective anti-HIV amount of anti-HIV Scopoli and monodispersity anionic oligomers, which will be introduced in accordance with this invention, will largely vary from about 0.1 mg/kg to 500 mg/kg body weight of the patient and can be administered one or more times a day. Anti-HIV anionic oligomers can be administered with a pharmaceutical carrier using conventional forms to which this invention to act as antiviral agents can be demonstrated by their ability to inhibit the growth and replication of the virus HSV. Used here, the term "method of treating herpes viral infection" refers to a patient who is infected with herpes virus type 1 or type 2, and the introduction of a specified patient effective against the virus in the number Scopoli and monodisperse compounds of formulas I-IV. In addition, it is also understood that the term "viral infection" refers to any condition characterized by the presence of the virus in cells or body specified patient.

Antiviral activity of the oligomers of this invention can be assessed using research to reduce belascoaran that previously described Tyms et. al., J. Antimicrobial chemotherapy, 8, 65-72 (1981). Briefly, human embryonic fibroblast cells (MPC5) were cultured in 24-cell boards in the presence of minimum essential eagle medium (ISI), supplemented with 10% fetal calf serum. When cell monolayers were poluchivshiisya, they were infected 30-50 plaque-forming units of HSV 2 strain HG52 or HSVI, strain 17 i (Dvison & Wilkie, J. General Virology, 55, 315-331 (1981)). At the end of adsorption for one hour at room temperature, the infected monolayers were covered with MCI containing 2% fetal calf serum, 0.5% agarose, the curable at a low temperature, and protivovirusnoe were stained with 0.3% methylene blue. Line according to response dose-response was drawn according to the average number present plaques depending on the log concentration of the substance. 50% effective dose (ED50) was calculated by linear regression analysis.

Application Scopoli or monodisperse anionic oligomers of this invention for the treatment of infections caused by humans, is of great importance. The term "herpes viral infection" means infection or herpes virus type 1 or herpes type 2. The term "patient" used here is taken to refer to mammals such as primates, including humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice.

The number Scopoli or monodisperse anionic oligomer formulas 1 to 4, which you need to enter may vary within wide limits in accordance with the specific used unit dosing, duration of treatment, age and gender are treated the patient, the nature and degree of the disease being treated and the particular selected anionic oligomer. Additionally, anionic oligomer can be used in combination with other known means, which are useful in the treatment of HSV and CMV infections, and known means, what irusa. Effective against herpes virus and cytomegalovirus number of anionic oligomer of formula I, want to enter, will generally range from about 15 kg/kg to 500 mg/kg Unit dosage may contain from 25 to 500 mg of anionic oligomer and may be taken one or more times a day. Anionic oligomer may be administered with a pharmaceutical carrier using conventional dosage form or orally, or parenterally, or topically.

For oral administration the anti-HIV or anti-HSV anionic oligomers can be prepared in solid or liquid preparations such as capsules, pills, tablets, lozenges, cakes, syrups, powders, solutions, suspensions or emulsions. Solid dosage form can be a capsule which can be of conventional hard or soft gelatin capsules, containing, for example, surfactants, lubricants and inert fillers such as lactose, saccharose, sorbitol, calcium phosphate and corn starch. In another implementation, anionic oligomers of this invention can tabletroute with generally accepted bases for tablets, such as lactose, sucrose and corn starch, mixed with svyazyami, designed to help the disintegration and dissolution of tablets after administration such as potato starch, alginic acid, corn starch and the guar gum (guar), sliding substances intended to improve the flow tableting granules and to prevent adhesion of tableting material to the surface of the tablet strains and punches, for example, talc, stearic acid, stearates of magnesium, calcium or zinc, coloring agents, flavoring means, correcting means, intended to enhance the aesthetic qualities of the tablets and make them more acceptable to the patient. Suitable fillers for use in oral liquid dosage forms include solvents such as water and alcohols, for example, ethanol, benzyl alcohol and polyethylene glycol with addition or without a pharmaceutically acceptable surfactant, suspending or emulsifying means.

Anti-HIV or anti-HSV Scopoli and monodisperse anionic oligomers of this invention can also be administered parenterally, that is, subcutaneously, intravenously, intramuscularly or intraperitoneally, in the form of injectable doses shall be a sterile liquid or mixture of liquids, such as water, saline, aqueous dextrose and similar solutions of sugars, alcohol, such as ethanol, isopropanol or hexadecylamine alcohol, glycols, such as propylene glycol or polyethylene glycol, glycerokinase, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such as poly(ethylene glycol)400, oils, fatty acids, ester of fatty acids or glycerides, or acetylated fatty acid glycerides with addition or without a pharmaceutically acceptable surfactant such as a soap or a detergent, suspending means, such as pectin, carbomer, methylcellulose, hypromellose, or carboxymethylcellulose, or emulsifying funds and other pharmaceutical adjuvants.

Illustrations for oils that can be used in parenteral preparations of this invention are oils from petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, cottonseed oil, corn oil, olive oil, vaseline and mineral oil. Suitable fatty acids include oleic acid, stearic acid and isostearoyl acid. Suitable fatty acid esters are, napr and triethanolamine, and suitable detergents include cationic detergents, for example, dimethyldiallylammonium, alkylpyridinium and acetates alkylamines followed, anionic detergents, for example, alkyl-, aryl - and reincorporate, sulfates Akilov, olefins, ethers and monoglycerides, and sulfosuccinate, non-ionic detergents, for example, oxides, fatty amines, alkanolamide fatty acids and polyoxyethylene-polypropylene copolymer, and amphoteric detergents, for example, alkyl-beta-aminopropionic and 2-alkylimidazole salts of Quaternary ammonium compounds, and mixtures. Compositions for parenteral administration of this invention will usually contain from about 0.5 to about 25% by weight of anti-HIV anionic oligomer in solution. Also can be used preservatives and buffers. In order to minimize or eliminate irritation at the site of injection, such compositions may contain nonionic surfactants having a hydrophilic-lipophilic balance (products HLB) of from about 12 to about 17. The amount of surfactant in such preparations ranges from about 5 to about 15% by weight. Surfactant can be a single component having wisenut the but-active substances, used in parenteral preparations are belong to the class of esters polyethylenimine and fatty acids, for example, servicemanual.

Scopoli and monodisperse anionic oligomers of this invention can also be used prophylactically, that is, to prevent transmission of the virus from an infected individual to an uninfected target. The virus spreads partially by blood transfusion, but may also be transmitted through other body fluids. Thus, the oligomers of this invention may be incorporated in the preparations together with standard products detergent for use in cleaning, especially in research and clinical laboratories and in hospitals, where they are processed blood products infected individuals. Drugs, containing oligomers of the present invention can be used for cleaning medical/surgical equipment and instruments, as well as hands and other skin surfaces health care workers. The oligomers of this invention can also be applied in the form of liquid or powder composition, to the surface of the prophylactic drugs during sex, such as condoms, or by a user or proizvoditelnosti to sprinceana for use by women prior to sexual contact with an infected individual. The oligomers of this invention can also be included in the lubricants and spermatocytic jellies and lotions. And finally, the oligomers of this invention can also be formulated in compositions that will be added in hot tubs, whirlpool tubs and swimming pools, to inactivate possible activity of the virus.

The invention will be further clarified by consideration of the following examples, which are intended to be merely illustrative for the present invention.

Definition

The terminology used in these examples, the definition of the following, if not worded differently, for example, represent a sample of the appropriate equipment or resins, but can be used similar equipment or resins with different parameters:

Diode multiple detector = 280 nm or 320 nm or 340 nm or 550 nm UV with a bandwidth of 4 nm or 100,

Column for gelfiltration = Sephadex G-25 column to gelfiltration (3.0 cm x 94 cm)

HPLC = high performance liquid chromatography,

IC50= inhibitory dose that blocks 50% the growth of virus,

Liquid chromatograph = Hewlett PackardTM1090,

Membrane filter with cutoff of 10,000 daltons = Method the line of human T cells,

MTT = tetrazolyl the reducing reagent, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide,

The peristaltic pump = Gilson Minipuls peristaltic pump,

PFU = plaque-forming unit,

Column I for chromatography with reversed phase = Alltech HyperprepTM1200,

Column II for chromatography with reversed phase = Alltech AbsorbosphereTMHS with high filling coal C18,

RPMI 1640 = standard environment for cell cultures obtained from GIBCO,

Column syringe cartridge = Alltech Max-CleanTMCX;

TCID50 = infectious dose for tissue culture, i.e., the amount of the culture fluid, effective for the infection of 50% of the cells (50% causing disease cells effect on day 7 after infection), and

UV detector = ISCO model UA-5 UV detector.

Obtaining raw materials

Example a: Blocking end bromine

4-necked flask equipped syringe input cell for a thermometer, mechanical stirrer, pH electrode, refrigerator with dry ice, the inlet tube for gas phosgene. Into the flask was loaded 8.00 g (23, 23 mmol) 4,4'-diaminobiphenyl-2,2'-disulfonic acid, 0,961 g (5,162 mmol) of 2-bromo-4-methylaniline and 250 ml of water. The flask was cooled to 12oC, paramasivan reached the 11.5. To the mixture over a 10 minute period was added 4.5 g (45 mmol) of phosgene with additional 5M sodium hydroxide to maintain the pH between 8 and 9. After 3 hours stirring was further added of 1, 848 g Ltd (9.93 mmol) of 2-bromo-4-methylaniline with 100 ml of water. Then was added 3.7 g (37 mmol) additional quantity of phosgene with a sufficient quantity of 5M sodium hydroxide to maintain the pH between 8 and 9. Stirring was continued overnight (about 16 h), and then the contents were besieged in 1000 ml of acetone. The product was filtered and washed 3 times with 100 ml of acetone. The precipitate from the filter is then transferred to the mold and dried under vacuum overnight at 50oC, receiving 11,249 g crude not quite white powder. The resulting product has the formula

< / BR>
and rough polydispersity when Min/Mhequal to about 1.4.

Example B:

The method of obtaining the crude polydispersed oligomers of formulas I-IV have been described in European application 0467185, published September 9, 1992

The limitation of the crude mixture of oligomers up to a more narrow polydispersed mixture of oligomers; stage I

Example I: Gelfiltration

A:

A. a Solution of 8.34 per ml of water 0,542 g neochimiki polymerization degree, equal to 9 (shown in Fig. 1, Mh= 2348, Min= 3408, Min/Mh= 1,45) was passed through the column to gelfiltration. A constant flow of 5.0 ml/min of deionized water maintained using a peristaltic pump. Eluent was monitored using UV detector at 310 nm in the presence of a mixture of anionic oligomers. After 40 min began to receive the sample, and collecting 4 ml fractions. Analysis by analytical HPLC showed that the dispersion of each fraction was significantly reduced compared with the dispersion of the source material. For example, fraction 14 (shown in Fig. 2) consisted of 25 mg of oligomer with a narrow dispersion (Mh= 2549, Min= 2880, Min/Mh= 1,13). Fractions 7 through 17 were combined to obtain 271 mg of the sample (shown in Fig. 3, Mh= 2860, Min= 3830, Min/Mh= 1,34) with a reduced amount of impurities with a low retention time.

In Fig. 1 - 4 peaks in the time interval from 0 to 3 min represents the oligomers without block on the end, with 3 to 5 min is monobromobimane at the end of the oligomers, with 5 to 14 min is desired diplomirovannyi at the ends of the oligomers with n = 1 at 14 min, n = 2 at 13 min, n = 3 at 12 min, and similarly, considering n from right to left.

B:

Column for gelfiltration prepared, giving 115 g resin Pharmacia SephadexTMG-10 (40-120 μm bead size) to swell in example 500 ml of distilled water for one hour. Pulverized part decentralise with a mixture of resin, as the suspension was allowed to settle in one litre cylinder. A glass column was filled with a slurry of resin and let in 500 ml of distilled water through the column, resulting in the layer 2.5 x 50 cm Constant flow rate through the column was maintained using a peristaltic pump (flow rate of approximately 3 to 4 ml/min). Eluent was monitored using UV detector at 310 nm in the presence of product mix more narrow polydispersed anionic oligomers.

A solution of 50 mg of polydispersed anionic polyurea of formula I from example B of source materials with Mh2730, as defined using the1NMR, was dissolved in 2.5 ml of distilled water and made the top of the column. The material was suirable distilled water, and collected three fractions of 25 ml each. Analysis of the fractions by HPLC showed that fraction 1 was enriched in oligomers with high mo is part of the source material.

C:

A solution of the crude mixture, 1,078 g in 10 ml of water, previously obtained, is locked at the end of bromine product from example A source material was passed through gelfiltration column. A constant flow of 5 ml/min of deionized water was maintained with the help of peristaltic pump. Eluent was monitored using UV detector at 310 nm in the presence of a mixture of oligomers. After 40 minutes began to receive the sample, and collected fractions of 3 ml Analysis using analytical HPLC showed that the dispersion of each of the fractions was significantly reduced compared with the dispersion of the source material. Fractions 8 to 11 were combined to obtain approximately 300 mg of the sample, blocked at the ends of bromine, with a reduced amount of impurities with a low retention time. So, was dedicated blocked at the ends of bromine polydisperse oligomer with improved purity.

Example 2: Selective deposition

Preparing a solution of 40.0 ml of water 3,93 g of a mixture of the crude polydispersed from sulphonated of politician formula I from example B of source material with a number average polymerization degree equal to 6. To this solution was added to 40.0 ml of acetone, causing the formation of a precipitate, which was separated price is th average degree of polymerization of about 4.

Example 3: Membrane separation

167 μl of solution with a concentration of 10 mg/ml of the crude mixture of polydisperse from sulphonated ureas with formula I from example B of source material with a number average degree of polymerization equal to 6 was passed through a membrane filter with a cutoff of substances with 10,000 daltons, using a centrifuge. The portion that passed through this filter was similar fractionally using a membrane with a cutoff of 3000 daltons. This sequence of divisions gives the output of a mixture of oligomers in which the dispersion of each fraction was significantly reduced compared with the dispersion of the source material, and part of the low - and high-molecular, compared with the desired degree of polymerization, was absent. The upper line of Fig. 5 shows the crude polydispersed the polyurea of formula I, and the second line is the material that passed through the filter with a cutoff of 10,000 daltons, but remaining on the filter, cut-off of 3000 daltons.

Example 4: Chromatography with reversed phase

Liquid chromatograph equipped with diode multiple detector (280 nm) modified with 1250 ál loop for the sample. Used column chromatography with reversed phase (9.4 cm x 50 mm), samociani formula I from example B of source materials, with the number average degree of polymerization equal to 6, was introduced in 200 μl. When using a constant ratio of the two eluents, 5 mm of water (n-Bu3N)PO4and acetonitrile 45:55 (volume/volume) was observed broad peak, which was fractionally manually. Each of the fractions was analyzed by HPLC, and as has been shown, was a mixture of oligomers with a polydispersity narrower than the polydispersity rough source material.

The allocation of monodisperse oligomer in the form of its ammonium salt. Stage 2

Example 5: Chromatography with reversed phase

A:

Liquid chromatograph equipped with diode multiple detector (320 nm), modified by using 250 ál loop for the sample. Used column chromatography with reversed phase (10 mm x 250 mm) filled with 3 μm particles. A solution of 18.0 ml of water 905,2 mg of the crude mixture of polydisperse from sulphonated polyurea formula I from example B of source material with a number average degree of polymerization equal to 6, was introduced in 200 μl. The eluent pumped with a speed of 1.5 ml/min, was an aqueous solution of 5 mm Et3NHOAc, acetonitrile and tetrahydrofuran. The starting ratio of the three components was 80:20:0 (on/about/27 min does not become equal to 30:40:30 (about/about/about), which was kept constant up to 36 min, when the flow was stopped. After 5 minutes have introduced an additional sample. Eluent was collected using a fraction collector. After 24 hours of continuous elution fractions were analyzed. There were several fractions containing oligomers (in the region of 85 to 100 percent purity), from 2 to 9 replications. These fractions are evaporated at about 20 mm Hg and 50-55oC and then dried overnight in a vacuum Cabinet at 50oC. Received triethylammonium salt polyurea in the form of light glassy particles amber color.

B:

Liquid chromatograph equipped with diode multiple detector (320 nm), modified by using 250 ál loop for the sample. Used column chromatography with reversed phase 11 (10 mm x 250 mm) filled with 3 μm particles. The rough solution of the mixture from example 1C was diluted with 5 ml of water and filtered. Used 75 ál volume for injection. Eluent was supplied by a pump with a speed of 1.5 ml/min and consisted of a solution of aqueous 50 mm Et3NHOAc and acetonitrile. The ratio of the two components at the beginning were equal to 70:30 (V/V), remained constant for 10 min and linearly changed, has not yet reached 60 the elk. After 5 minutes have introduced an additional sample. Eluent was collected using the collector fractions. After 24 h of continuous operation of the reaction were analyzed. Received nine fractions containing oligomers (with a purity of 95 - 100%) 1 to 9 replications. These fractions are evaporated at 20 mm RT.article at 50 - 55oC and then dried overnight in a vacuum Cabinet at room temperature. Received triethylammonium salt monodisperse polyurea, Min/Mh= 1,1 less, in the form of light amber glass particles exit 5 to 10 mg.

C:

Liquid chromatograph equipped with diode multiple detector (340 nm) modified with 250 ál loop for the sample. Used column II for chromatography with reversed phase (10 mm x 250 mm) filled with 7 μm particles. A solution of 200 mg of the crude mixture of polyamide with formula IV (Mh= about 1300), is obtained analogously to example IC, diluted with 3 ml of water and filtered. Used 75 ál volume for injection. Eluent supplied by a pump at a speed of 1.0 ml/min, consisted of a solution of aqueous 50 mm Et3NHOAc and acetonitrile. The ratio of the two components was 72:28 (on/about). Eluent was collected using the collector fractions. After 24 h continuous is evaporated at about 20 mm RT.article at 60oC and then dried overnight in a vacuum Cabinet at 50oC Received triethylammonium monodisperse salt of the polyamide in the form of a white solid. The resulting product has the formula

< / BR>
If the division proceeded in the presence of Et3NHOAc, chain length of the oligomer decreased with increasing retention time, then how could reverse the order of elution, if the separation was carried out in the presence of tetrabutylammonium phosphate, i.e., the length of the chain increases with increasing time. Fig. 6 shows the HPLC profile with Et3NHOAc. Fig. 7 shows the HPLC profile with tetrabutylammonium. Fig. 8 shows the HPLC profile of purified monodisperse components with Fig. 6.

Received other chromatogram HPLC with tetrabutylammonium phosphate. The different fractions of the polyurea oligomers of formula I are shown in Fig. 8. In Fig. 8, the upper line represents the original raw material polydisperse oligomer. Chain length components increases with increasing retention time (consistent with Fig. 7). The second line represents the oligomer after the limitations of the crude mixture of anionic oligomers to a more narrow polydispersed mixture of anionic oligomers way Izzy 10,000 daltons. Separate fractions of oligomers were collected and then subjected to HPLC with tetrabutylammonium phosphate results, which are shown in other separate profiles Fig. 8, which shows each individual monodisperse substance with n = 1 to 9. The last profile in Fig. 8 shows a mixture of oligomers with n = 10 - 13.

The transformation of the ammonium salt of the oligomer to the corresponding sodium salt. Stage 3

Example 6: Ion exchange

A:

The samples of example 5 were individually dissolved in about 2 ml of deionized water and was passed through the column with a syringe cartridges, which were filled with 0.5 mEq from sulphonated cross-linked polystyrene in the acid form. The column washed twice with 1 ml of water. After evaporation, all samples were analyzed using proton NMR, which showed that ion triethylamine completely removed and replaced by hydrogen.

Samples containing oligomers with more than one repeating unit, were a mixture of diastereomers. Although these fractions, apparently, consist of a single substance, they actually are isomeric mixtures of diastereomers, which are the chiral nature befunolol group in the repeating unit. Typical has only one peak. In this small sample peaks retention time in the beginning of the series are monoblocking oligomer, which is obtained by hydrolysis of the main component at the time of allocation.

Mholigomers was calculated by comparing the integrated area of a methyl group at the ends of such aromatic region with1H NMR. Selected samples were also analyzed by time-of-flight mass spectroscopy, which confirmed the determination of NMR.

B:

Column syringe cartridges that are filled with 0.5 mEq from sulphonated cross-linked polystyrene in acid form, was treated with 2 ml of 5 M NaCl and washed with 5 ml of water. The samples from example 5B were individually dissolved in about 2 ml of deionized water and was passed through these speakers for sharing Et3NH+cation on the Na+. The column washed twice with 1 ml of water. After evaporation, all samples were analyzed using proton NMR, which showed that ion triethylamine was removed. Selected samples were also analyzed using time-of-flight mass spectroscopy, which confirmed the determination of NMR.

C:

Column syringe cartridges used, as described in example 6A, for preimer 7. The addition of salts of weak volatile acids

Lyophilized polyurea oligomers of formula I (obtained from BPDS and toluidine and selected using preparative HPLC using Et3NHOAc buffer) was dissolved in deionized water and treated with acetone sodium. Equivalent of sodium acetate was added to the equivalent of sulfonate in the oligomer. The solution liofilizirovanny for 20 h to remove the water, triethylamine and acetic acid.

The fraction of oligomer with n = 9,63 mg was dissolved in 10 ml of water and 195 μl of 1.1 M solution of sodium acetate (NaOAc). The solution liofilizirovanny, and received 57 mg of oligomer.

Similarly, the fraction of oligomer with n = 7,82 mg was dissolved in 10 ml of water and 250 μl of 1.1 M solution of NaOAc. The solution liofilizirovanny and received 79 mg of oligomer.

The fraction of oligomer with n = 5,162 mg was dissolved in 10 ml of water and 277 μl, 1.1 M solution of NaOAc. The solution liofilizirovanny and received 144 mg of oligomer.

Biological data

Example I. the Ability of various anti-HIV-oligomers to prevent caused by a virus cell death using MT4 cells and strain of HIV-I RF.

Relapsing and antiviral activity of different monodisperse oligomers polyurea of formula I was determined standard is Hartnoll concentration (approximately 100 μg/ml) and then investigated for anti-HIV activity, making breeding solutions twice in 96-microtiter cell Board. Then in each cell was added 5 104cells MT4 and 100 TClD50 virus (HIV-1 strain RF), and Board were incubated at 37oC for 7 days. In each cell was added MTT and fees were incubated for an additional 2 hours Blue crystals formazan was dissolved with the use of acidic isopropanol and measured the absorbance at 540 nm. The results are presented in table I.

Example II. The ability to handle cells of different oligomers and block HIV-1 infection when using JM cells and strains GB8 HIV-1

Antiviral activity of various monodisperse oligomers polyurea of formula I was determined by a standard breakdown of the formation of syncytium (Cardin et al., Trans. Assoc. Amer. Phys. C. 101-109 (1989) and Cardin et al., J. Biol. Chem. , 266, 13355 - 13363 (1981)). The JM cells were treated at 37oC various substances at various concentrations (μg/ml) or left untreated. Cells in the environment PPMI infected with HIV-1 (GB8) for 2 h at room temperature. Cells then twice washed with environment PPMI and resuspendable in fresh medium containing fractionated oligomers before distribution in double cells, and incubated at 37oC. Che is ogramme antiviral IC50 concentrations of different fractions of the oligomer. Antiviral activity increases with increasing chain length of the oligomer up to n = 9. Anti-HIV-1 activity of polydisperse mixture fraction and fractions with n = 10 - 13 was about the same, but both of these factions were less active than each of the fractions with n = 6 to n = 9 out of monodisperse fractions of oligomers.

Example III: Action uzkopolosnykh and monodisperse politician with the formula I on the replication of HSV-2

Vero cells were grown to confluence in 24-cell Board for culturing tissue. Cells infected HSV-2 at a multiplicity of infection of 50 PFU/cell. Infection was carried out in the presence or absence of various concentrations of the substance, which is BPDS /P/T and is called poly{imino[2,2'-disulfo-(1,1'-biphenyl)-4,4'-diyl]aminocarbonyl}, alpha-{ [(4-were)amino] -carbonyl} -omega-[(4-were)amino]- and is represented by formula I, paragraph 1 of the claims, when R is 4-were, R2is hydrogen, X is

< / BR>
and n are defined as set forth in table II. After 2 h, the absorbance at room temperature, infecting the liquid was removed, and cells were incubated with a covering layer of agarose containing the appropriate concentration of the substance. After 2 Denmark activity was calculated for each concentration of the substance. IC50 for each compound was calculated using analysis by linear regression, and the results are presented in table III.

1. Scopoli and monodisperse water-soluble oligomer, which is the ratio of polydispersity 1.0 to 1.3, which is a polyurea of the formula I

< / BR>
where R is a hydrogen atom, a C1-C4alkyl group, phenyl group or phenyl group substituted 1 - 2 fragments R1and with up to 3 substituents independently selected from chlorine atoms or bromine, or WITH1-C4alkyl groups:

R1- -SO3R2, -CO2R2, RHO3(R2)2or ORO3R2;

R2is a hydrogen atom or a pharmaceutically acceptable cation;

m = 0 or 1, provided that if

m = 0, R is a hydrogen atom,

X is

< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
where Y IS-CO2-, -C=C-, -N=N-,

< / BR>
n = 3 - 15, integer,

R3- R or-X-NH2where R and X have the meanings given above.

2. The oligomer under item 1, where n = 5 to 10.

3. The oligomer under item 2, where n = 6 to 9.

4. The oligomer under item 1, which is a polyurea formula I, where n = 3 to 15, and X is

< / BR>
< / BR>
< / BR>
< / BR>
R2the ima hydrogen, X is

< / BR>
6. The oligomer under item 4, where X is

< / BR>
R2- same as in paragraph 1.

7. The oligomer under item 4, where R is a phenyl group substituted 1 - 2 fragments R1and up to 3 basementalism.

8. Escopyexperry oligomer on PP.1, 4 and 7, which is the ratio of polydispersity 1,0 - 1,2.

9. Monodisperse oligomer on PP.1, 4 and 7, which is the ratio of polydispersity to 1.0 - 1.1.

10. Monodisperse oligomer under item 7, which is the ratio of polydispersity of 1.05.

11. Pharmaceutical drug, which inhibits the activity of HIV-1 and HSV-2, characterized in that it contains a pharmaceutically effective amount Scopoli or monodisperse oligomer according to any one of paragraphs.1 - 10 with a pharmaceutically acceptable carrier.

12. A method of inhibiting the activity of HIV-1 and HSV-2 in vitro using an inhibitor of viral activity, characterized in that as an inhibitor of viral activity using the pharmaceutical preparation according to p. 11 in an effective amount.

13. The method of obtaining Scopoli and monodisperse anionic oligomers under item 1, which consists in the fact that they limit the corresponding crude polydispersed mixture of deposition, membrane separation or chromatography with reversed phase, which result in the desired relation to polydispersity 1,0 - 1,3.

14. The method according to p. 13, characterized in that it comprises the additional step of turning salt uzkopolosnogo anionic oligomer in the desired pharmaceutically acceptable salt.

15. The method according to p. 13, characterized in that the ratio of polydispersed oligomer is 1.0 to 1.2.

16. The method according to p. 13, characterized in that the ratio of polydispersed oligomer is equal to 1.0 - 1.1.

17. The method of obtaining Scopoli and monodisperse anionic oligomers under item 1, namely, that produce monodisperse anionic oligomer by electrophoresis in a gel or chromatography with reversed phase.

18. The method according to p. 17, characterized in that it comprises the additional step of turning salt uzkopolosnogo anionic oligomer in the desired pharmaceutically acceptable salt.

19. The method according to p. 17, characterized in that the purity of monodisperse material is at least 75%.

20. The method according to p. 19, characterized in that the purity of monodisperse fraction is from about 85 to about 100%.

21. The method of obtaining wwwusa crude polydispersed mixture of anionic oligomers to uzkopolosnoi mixture of anionic oligomers by gel filtration, selective precipitation, membrane separation or chromatography with reversed phase, which result in the desired relation to polydispersity 1,0 - 1,3; produce monodisperse anionic oligomer by electrophoresis in a gel or chromatography with reversed phase.

22. The method according to p. 21, characterized in that it comprises the additional step of turning salt Scopoli or monodisperse anionic oligomer in the desired pharmaceutically acceptable salt.

23. The method according to PP.13, 14, 17, 18, 21 and 22, characterized in that the oligomer is a polyurea formula I and n means monodisperse fraction of 5 to 10.

24. The method according to p. 23, characterized in that the oligomer is a polyurea formula I and n means monodisperse fraction 6 - 9.

25. The method according to PP.14, 18 and 22, characterized in that the salt is ammonium, sodium or potassium salt.

26. The method according to PP. 14, 18 and 22, characterized in that an additional step is performed by ion exchange or by adding a salt of the weak volatile acid.

 

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< / BR>
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