FI67368C - PROCEDURE FOR THE PREPARATION OF A THERAPEUTIC POLICEPTIDER - Google Patents

Description

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(32) (33) (31) t> rrit * tY «Tuoik * / * - Baglrt prtorttet 08.12.77 08.09.78, Γ / .11.78 USA (U5) 858996, 990531, 960550 Proven-Styrkt (71) Ortho Pharmaceutical Corporation, Raritan, New Jersey, USA (72) Gideon Goldstein, Short Hills, New Jersey, USA (79) Oy Koister Ab (59) Method for the preparation of new therapeutically useful polypeptides - Förfarande för framstälining av nya, therapeutically active polypeptide

The invention relates to a process for the preparation of new therapeutically useful polypeptides.

Many polypeptides have been isolated from various tissues and organs (e.g., blood) of animals. Many of these polypeptides have an effect on the immune function of the body, e.g., various immunoglobulins, thymic hormone-thymopoietin, etc. I have not actually isolated and synthesized several of these polypeptides, as described in U.S. Patent Nos. 4,002,602 and 9,002,740, as well as several scientific papers.

Until about the last decade, very little was known about the thymus, although it is now understood that the thymus is one of the organs primarily responsible for the immune function of mammals and birds. Despite great interest in the possible functions of the thymus and previous theories and experiments, very little was known about the function of the thymus before 2,636,368 than recently. However, it has now been realized that the thymus is a connecting organ with both epithelial (endocrine) and suction (immunological) components and thus the thymus is involved in the body's immune functions. The thymus comprises epithelial basal tissue from the third gingival arch and lymphocytes from stem cells originating in blood-forming tissues, Goldstein, et al., The Human Thymus, Heinemann, London, 1969. Lymphocytes differentiate in the thymus and leave the thymus mature as native cells called T cells that circulate in the blood, lymph, spleen and lymph nodes. The induction of stem cell differentiation within the thymus appears to be mediated by thymic epithelial cell secretions.

It has been known for some time that the thymus is associated with the immune properties of the body and therefore great interest has been shown in substances isolated from the thymus. In this regard, a relatively large number of articles have been published in recent years based on scientific work on substances present in the thymus of cattle. In fact, the applicant has published a series of articles focusing on research in this field. Writings on this can be found, for example, in The Lancet, July 20, 1968, pp. 119-122; Triangle, Vol. II, No. 1, pp. 7-14, 1972; Annals of the New York Academy of Sciences, Vol. 183, pp. 230-240, 1971; and Clinical and Experimental Immunology, Vol. 4, No. 2, pp. 181-189, 1969; Nature, Vei. 247, pp. 11-14, 1974; Proceeding of the National Academy of Sciences USA, Vol. 71, pp. 1474-1478, 1974; Cell, Vol. 5, pp. 361-365 and 367-370, 1975; Lancet, Vol. 2, pp. 256-259, 1975; Proceedings of the National Academy of Sciences USA, Vol. 72, pp. 11-15, 1975; Biochemistry, Vol. 14, pp. 2214-2218, 1974; Nature, Vol. 255, pp. 423-424, 1975.

The second class of lymphocytes with immune function are B lymphocytes, or B cells. These are differentiated in birds in the "Bag of Fabricius" (Bursa of Fabricius) and in mammals in a hitherto unidentified organ. T cells and B cells work together in many aspects of immunity. See. for example, the applicant's writings in Science, 19 3, 319 (July 23, 1976), and Cold Spring Harbor · Symposium on Quantitative Biology, Vol. XLI, 5 (1 977).

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67368 Recently, J.F.Bach, et al. isolated a nonapeptide substance known as "facteur thy-mique serique" (FTS) from porcine serum. The isolation of this material and its structure are described in C. R. Acad. Sc. Paris, vol. 283 (November 29, 1976), Series D-1605 and Nature 266, 55 (March 3, 1977). The structure of this nonapeptide has been defined as GLX-ALA-LYS-SER-GLN-GLY-SER-ASN, where "GLX" represents either glutamine or pyroglutamic acid. A substance in which GLX is glutamine or pyroglutamic acid has been synthesized. In these papers, Bach suggested that this nonapeptide FTS selectively differentiated T cells (rather than B cells) using the E-Roset ti assay. Thus, Bach concluded that this substance was thyroid hormone. Recently, a more in-depth study of the activity of this nonapeptide has shown that FTS differentiated both T and B cells and was therefore more ubiquitin-like in activity than thymopoietin, Brand, Gilmour and Foldstein, Nature, 269: 597 (1977).

It has now been found that the synthesized 4-amino acid polypeptide segment of this FTS nonapeptide has many of the properties of the nonapeptide disclosed in the aforementioned publications.

Accordingly, the present invention provides a method of preparing a therapeutically useful polypeptide of the formula

R-X-LYS-Y-GLN-R 'I

wherein R 'is OH or NH 2, X is sarcosyl, L-alanyl or D-alanyl, Y is seryl, sarcosyl, 2-methylalanyl or D-alanyl and R is H, GLN or SAR.

The process according to the invention is characterized in that the amino-protected L-glutamine is esterified by covalent bonding to an insoluble resin polymer, the O-amino-protecting group is removed from the L-glutamine group, the Y-amino acid whose oC-amino-protecting group is reacted is reacted, with a glutamine resin to attach a Y-amino acid group thereto, a amino-protecting group is removed from the Y-amino acid group, L-lysine having a <* amino group is protected is reacted with a Y-amino acid L-glutamine resin to attach an L-lysine thereto, The O-amino protecting group is removed from the L-lysine group, the NR-substituted-X-676788 amino acid whose oC-amino group is protected is reacted with L-lysine-Y-amino acid-L-glutamine resin to give the NR-substituted To attach an -X-amino acid thereto, the resin is cleaved from the peptide with acid or ammonia and all protecting groups removed to give a compound of formula I wherein R 'is OH or NH2, wherein when ben is used. a hydryrylamine resin polymer, R 'is NH2 regardless of the cleavage agent used.

The groups at the position of the polypeptides of formula I do not substantially affect the biological activity of the active β-amino acid segment, as measured by the ability to induce differentiation of Th-1 + T lymphocytes and Bu-1 + B lymphocytes in the chicken induction assay described below.

Since the active tetrapeptide segment is included in a longer series in a naturally occurring substance isolated by Bach, it is to be understood that the terminal amino and carboxylic acid groups are not essential for the biological activity of the tetrapeptide segment, as is the case with some polypeptides. Therefore, it is contemplated that the scope of the present invention encompasses not only those tetrapeptide segments that are substituted with H and O, respectively, but also those that are substituted at their ends with one or more other functional groups that do not substantially affect the disclosure herein. biological activity.

The activity of a molecule is believed to depend on its stereochemistry, i.e., "folding" of the molecule. The polypeptide bonds are not rigid, but flexible, and the polypeptides may be in the form of sheets, coils, etc., as a result of which the whole molecule is flexible and "folds" in a certain way. It has been found that these novel tetrapeptide segments are likely to "fold" in the same manner as the corresponding tetrapeptide segment in the native nonapeptide, and therefore have the same biological properties as the native 9-amino acid peptide presented by J.F. Bach in FTS. In this nonapeptide, one set of tetrapeptides of this invention can be identified within the molecule, but only in conjunction with the other amino acids described herein. Since the tetrapeptide segments of the present invention confer the same biological activity as the nonapeptide FTS, it is clear that amino acids and peptide chains substituted with terminal amino acid residues of the tetra-peptide segment do not affect its biological properties.

In a preferred embodiment of the invention, X is L-alkanyl, Y is L-seryl, R is hydrogen and R 'is OH. The formula of this preferred embodiment is as follows:

H N-CH-CONH-CH-CONH-CH-CONG-CH-COOH

^ t 1 T T

CH3 (CH2) 4 CH2 (CH2) 2 1 »t nh2 oh conh2

H - ALA - LY S - SEP - GLN - OH

In this patent application, the following abbreviations have been used for the amino acid components of the peptide: D-amino acids are indicated by placing a "D" before the abbreviation, e.g., D-alanine is represented by "D-ALA".

Amino acid Abbreviated entry

L-alanine ALA

L-serine SER

L-glutarine GLN

sarcosine SAR

2-methyl.lanine 2-HE-ALA

The polypeptides of the invention have similar properties to the 9-amino acid polypeptide FTS isolated from porcine blood, as described in the aforementioned Bach et al. writings. I do not particularly characterize these tetrapeptides in that they are able to induce the differentiation of both T precursor cells and B precursor cells. Some of these polypeptides are active at concentrations as low as 1 picograsm (pg) / ml in the following chicken induction assay.

It has been found that the polypeptides of this invention induce the differentiation of immunocyte precursor cells in vitro in the same manner as the nonapeptides presented by Bach. Thus, the polypeptides of this invention have been found to induce both T-precursor cell differentiation as measured by thymic differential antigen uptake TH-1 and also B precursor cell differentiation as measured by differentiation antigen Bu-1 uptake.

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In other words, these polypeptides have the ability to induce differentiation of both Th-1 + T lymphocytes and Bu-1 + B lymphocytes.

It has also been found that these polypeptides increase the in vivo production capacity of cytotoxic lymphocytes when stimulated with allogeneic antigens. Ts. administration of these polypeptides to e.g. rats promotes the production of cytotoxic lymphocyte precursors as measured in vitro in rat spleen cells. Since the development of cytotoxic lymphocytes and graft rejection in an allogeneic graft-versus-host reaction are similar, the above finding provides additional support for the immunological utility of these polypeptides.

To understand the importance of differentiating the biological properties of the polypeptides of this invention, it should be noted that the function of the thymus in relation to immunity can be broadly considered to produce thymic or lymphocytes, termed T cells. T cells make up a large part of the collection of circulating small lymphocytes. T cells have immunological specificity and are directly involved in cell-mediated immune responses (such as the native transplant response) as enhancer cells. However, T cells do not secrete humoral antibodies.

These antibodies are secreted by cells (called B cells) that come directly from the bone marrow, regardless of the effect of the thymus. However, for many antigens, B cells suitably require the presence of reactive T cells before they can produce antibodies. The mechanism of the cell interaction process is not yet fully understood.

From this description, it can be said in practice that the thymus is necessary for the development of cellular immunity and many humoral antibody reactions and affects these systems by causing the differentiation of blood-forming stem cells within the thymus into T cells. This effect is mediated by the secretions of thymic epithelial cells, i.e., thymic hormones.

In order to understand the cell system of the thymus and lymph cells, as well as the circulation of the lymph cells, it should also be mentioned that stem cells are born in the bone marrow and enter the thymus with the blood flow. Within the thymus, stem cells differentiate immunologically into n 67368-capable T cells that travel into the bloodstream and, together with B cells, circulate between tissues, lymph nodes, and blood flow.

Cells in the body that secrete antibody (B cells) also develop from blood-producing stem cells, but their differentiation is not dictated by the thymus. In birds, they differentiate in a thymus-like organ called the Fabricius bag (Bursa of Fabricius). No similar organ has been found in mammals and it is thought that these cells differentiate in the bone marrow. For this reason, they are called bone marrow-born cells, or B cells. The physiological substances that determine differentiation are completely unknown.

As mentioned above, the polypeptides of the invention are therapeutically useful in the treatment of humans and animals. Because these novel polypeptides are capable of inducing the differentiation of lymphopoietic stem cells produced in blood-forming tissues into both thymic lymphocytes (T cells) and immunoqualified B cells capable of participating in body immune responses, the products of this invention are considered to have a wide variety of therapies. First, because these compounds have the ability to perform certain functions of the thymus, they have utility in different areas of thymus function and immunity. The first field of application is in the treatment of the symptoms of DiGeorge, a condition in which the thymus is congenitally absent. Injecting a polypeptide of the invention, as described below, overcomes this deficiency. Another area of use is in the deficiency of gamma globulin in the blood due to a deficiency of a putative hormone that differentiates B cells in the body. This deficiency is overcome by injecting one of the target polypeptides. Because these polypeptides are extremely active at low concentrations, they are useful for enhancing overall body immunity in that they stimulate the development of cellular immunity and humoral immunity and are thus useful in treating diseases involving chronic infection in vivo, such as fungal or mycoplasma infection. tuberculosis, leprosy, acute or chronic viral infections, etc. In addition, these peptides are considered useful in any region with deficiencies in cellular or humoral immunity, such as the aforementioned DiGeorge o'ireist. In addition, due to their properties, polypeptides have utility in vitro in inducing T cell surface antigens, in inducing functional capacity to develop sensitivity to mitogens and antigens, and in cell interoperability by enhancing the ability of B cells to produce antibodies. They have in vitro use in inducing the development of B cells, as measured by the development of surface complement receptors. These peptides are also useful in inhibiting the unlimited growth of ubiquitin-sensitive lymphocytes (described in Applicant's U.S. Patent No. 4,002,602). An important feature of these polypeptides is their ability to regenerate T cell properties in cells in vivo and also their ability to regenerate 3 cell properties in cells in vivo.

They are therefore useful in the treatment of relative or absolute B cell deficiencies as well as relative and absolute T cell deficiencies, whether these deficiencies are due to deficiencies in B cell differentiating tissue or the thymus, or for some other reason.

Another important property of the polypeptides of this invention is that they are highly active at very low concentrations. Thus, it has been found that polypeptides are generally active at concentrations of about 1 ng / ml, while certain particularly potent polypeptides (H-SAR-LYS-D-ALA-GLN-NH2 and H-SAR-LYS-SAR-GLN- NH 2) are active at concentrations from about 0.1 pg / ml.

To determine the activity and properties of the tetrapeptide of formula H-ALA-LYS-SER-GLN-OH, the following chicken induction assay was performed. This experiment is explained in more detail by Brad et al. in Science 193, 319-321 (July 23, 1976) and references cited therein.

The bone marrow of newly hatched chicks was chosen as the source of inducible cells because it contains hardly any Bu-1 ^ · -j ·. f,,, or Th-1 cells. The pooled cells from the femoral and tibial ankles of five newly hatched SC (Hy-Line) strains were fractionated in an ultracentrifuge with a five-layer discontinuous bovine serum albumin (BSA) gradient. Cells from the two lighter layers were pooled, washed, and suspended for incubation in g at a concentration of 5 x 10 cells / ml at the appropriate concentration of polypeptide.

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67368 in RPMI 163O medium supplemented with 15 mM HEPES, 5% Y'-globulin-free fetal calf serum, deoxyribonuclease (14-18 units / ml), heparin (5 units / ml), penicillin ( 100 units / ml) and streptomycin (100 μg / ml). Control subjects were incubated with BSA (1 μg / ml) or medium alone. After incubation, cells were subjected to cytotoxicity analysis using chicken C1 and guinea pig C2-C9 complement fractions as described in the reference. The proportion of Bu-1 + or Th-1 + cells in each layer was calculated as the cytotoxicity factor, 100 (a-b) / a, where a and b are the percentages of viable cells in the complement and test preparations, respectively. The percentage of induced cells was obtained by subtracting the mean values of the control incubations (without inducers, usually 1-3%) from the means of the experimental inductions.

The specificity of the effect of the test polypeptides and its similarity to ubiquitin was demonstrated by inhibiting the induction of Bu-lf B cells and Th-1 + T cells by the test polypeptides after the addition of ubiquitin at a concentration of 100 μg / ml · This high dose of ubiquitin inactivates ubiquitin receptors and inhibits thus inducing cells by any agent that acts through these receptors.

As a result of this experiment, it was found that the tetrapeptide of Example I had similar biological activity to that caused by ubiquitin and the differentiation of Th-1 + T and 3u-1 + B lymphocytes at ng / ml concentrations.

To further illustrate the utility of the polypeptides, a cytotoxic allogeneic antigen assay is described below. lymphocytes prepared by excitation »to assess sea buckthorn densities. The frequencies of cytotoxic precursors between control animals and animals injected at different concentrations, i.e., injected at different concentrations, were compared in a limiting dilution experiment.

Substances and methods

mice

Inbreeding C57 BL / 6J (females, 8 weeks) was obtained from Jackson Laboratory, Bar Harbor, Maine.

Inbreeding DBA / 2J (male and female) was also obtained from Jackson Laboratory, Bar Harbor, Maine.

10 67368 Media

Phosphate buffered saline (PBS), RPMI 1640, fetal calf serum (FCS) - (lot number R776116), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer were obtained from Gibo, Grand; 2-Mercaptoethanol was obtained from Eastman Kodak, Rochester, N.Y. Cells were washed with PBS and cultured in RPMI containing 10% FCS, 10 mM HEPES buffer and 5 x 10 2-mercaptoethanol.

Pharmaceutical processing

Experimental animals (C57 BL / 6J) were injected (intravenously or intraperitoneally) with 0.2 ml of various concentrations of H-SAR-LYS-SAR-GLN-NH2 (with drug; identification number G040) 24 hours before killing for experiments.

Cell preparations Cell suspensions from the spleens of C57 / 6J mice or DBA / 2J mice were prepared by comminuting the organs and pressing them through a metal wire mesh (number SO) with a 5 ml syringe plunger into a Falcon-Petri dish (Falcon 3002, 15 x 60 mm). The cell suspensions were allowed to stand at room temperature for 10 minutes and large lumps of tissue were allowed to settle. The cell suspensions were then transferred to 15 ml Corning centrifuge tubes (Corning 25310) and centrifuged for 10 minutes at 1,500 rpm in a Beckman TJ-6 centrifuge. All cell suspensions were washed at least three more times with PBS. After the third wash, response cells (C57 BL / 6J) were resuspended in culture medium and counted on a Coulter counter. DBA / 2J (stimulating cells) was resuspended in RPMI at a concentration of 10 cells / ml. 30 μg mitomycin C was added to each ml of DBA / 2J spleen cells and incubated at 37 ° C for 30 minutes. After mitomycin C treatment, spleen cells were washed three times with PBS to remove excess miromycin C. DBA cells were then resuspended in culture medium and counted on a Coulter counter.

Lymphocyte mixed cultures (MLC) were performed in microtiter cells (Linbro Chemicals, New Haven, Conn., IS-MCV-96). Each cell contained a 96-V bottom well. The outer side wells surrounding the edge of the plate were not used for cell culture but were filled with PBS to avoid evaporation from the culture wells. 60 samples were placed in each V-bottom cell. Typically, each cell contained three counter cell concentrations (20 replicates each) and one stimulation cell concentration. Response cells were usually suspended at concentrations of 7.5 x 10 4, 5 x 10 4, and 2.5 x 10 4 / ml, and 0.1 ml of suspension was added to each well. The stimulation cell concentrations used were 10 °, 2.5 x 10 ° and 5 x 10 / ml, and 0.1 ml was also added to each well. The same stimulation cell concentration was used throughout the plate. The control plate contained only response cells without stimulation cells to assess basal stimulation from the medium. The cells were cultured for six days at 37 ° C in a humidified soaker containing 5% CO 2.

Brand cells

The marker cell used in the cytotoxic assay was the DBA mastosy-Toma cell line P815. This cell line was maintained at normal

O

passing through DBA / 2J mice. 5 x 10 P815 cells were used for each passage, and tumor cells from the peritoneal cavity of the carriers were used 4-5 days after passage. Tumor cells from the peritoneal cavity were washed three times with PBS and then labeled 51 7

Cr at a concentration of 100 μl / 10 cells. Labeling was performed for one hour at 37 ° C in a humidified incubator. The labeled label cells were then washed three times with BPS to remove excess label.

Cytotoxic test

Six days after culture, 0.1 ml of medium was removed from each well by mixing cell pellets. Using an automated micropipette (MLA pipette), • 4 51 100 μΐ of marker cells containing 2.5 x 10 Cr-labeled marker cells were pipetted into each well, resuspending the cell pellet during the process (a new pipette tip had to be used for each well). The microtiter cells were then centrifuged at 1,000 rpm for seven minutes at room temperature.

Lathe in GLC-2B. The cells were incubated for four hours at 37 ° C.

100 microliters of supernatant was then removed to Gamma downcomers (Amershen 196271). The tubes were then counted on a Beckman Gamma Counter (Beckman 310). The tubes were usually lowered in one minute.

Determination of frequencies of cytotoxic lymphocyte (CLP) precursors

The limiting dilution analysis is the total or 0-response test, which is explained by the Poisson probability distribution. The probability of non-response is given by the zero-order term Po = e where <£ - CLP frequency and N = number of lymphocytes per well. Thus, plotting the logarithm of the unreacted cultures / cell dose ratio in the coordinate system would give a line with a slope of -, the frequency of CLP occurrence.

In the example, background chromium release (spontaneous release) was calculated from 20 wells containing only response cells (C57 BL / 6J) and no stimulation cells at all. Test wells were considered positive if their calculated numbers were greater than the mean spontaneous value with a standard deviation of more than 2.07 (P 0.05). Spontaneous lysis was usually 9-15% of the total counts associated with the marker cells. Poisson's equation po = ^^ ^, when PO = e ^ = 0.37 (corresponding to 37% of unresponsive cultures), & - 1 / N, thus the number of response cells corresponding to 37 i of non-reactive cultures, the inverse value is the CLP occurrence density. In general, the number of cells per well and their corresponding value for the percentage of unresponsive cells was fitted to the computer that calculates the most appropriate regression line through these points and the number of cells per well corresponding to 37% unresponsive cultures, the inverse of this value being CLP: n frequency.

Score

The frequencies of cytotoxic lymphocyte precursors for each stimulation cell concentration were plotted as a function of drug dose. The mean and standard deviation of 20 replicates were also calculated for comparison. The three stimulation cell concentrations used were 10 (suboptimal stimulation), 2.5 κ 5 5 10 (optimal stimulation), and 5 x 10 (top-optimal stimulation). It was found that the test drug promoted the production of cytotoxic lymphocyte precursors at concentrations of 1 pg / mouse to 100 ng / mouse (corresponding to about 50 pg / kg to about 5 μg / kg body weight) in the presence of suboptimal stimulation cell concentrations. The test drug therefore acts as an immunoregulator to increase the cellular immune responses of mice treated at these concentrations.

According to the invention, polypeptides were prepared by the method of Merrifield, Journal of the American Chemical Society, 85, pp. 2149-2154, 1963. The synthesis involved the gradual addition of protected amino acids to a growing peptide chain covalently bonded to a solid resin moiety. In this method, reactants and by-products were removed by filtration and recrystallization of intermediates was omitted. In this method, the C-terminal amino acid of the fox is attached to the solid polymer by a covalent bond and the following amino acids are added one at a time, stepwise until the desired series is assembled, the peptide is removed from the solid polymer and deprotected. This method results in a growing pepti chain attached to an insoluble solid support, making it easy to filter and wash free of reagents and by-products.

Amino acids can be incorporated into any suitable polymer, which need only be readily separable from unreacted reagents. The polymer is insoluble in the solvent used or may be soluble in certain solvents and insoluble in others. The polymer should have a permanent physical form to make it easy to filter. It must contain a functional group to which the first protected amino acid can be firmly attached by a covalent bond. Various insoluble polymers suitable for this purpose include, for example, cellulose, polyvinyl alcohol, polymethacrylate and sulfonated polystyrene. In the synthesis according to the invention, a chloromethylated copolymer of styrene and divinylbenzene was generally used. Polymers that are soluble in organic solvents but insoluble in aqueous solvents can also be used. One such polymer is polyethylene glycol, which has a molecular weight of about 20,000 and is soluble in methylene chloride but insoluble in water. The use of this polymer in peptide synthesis is described by F. Bayer and M. Mutter in Nature 237, 512 (1972) and references therein.

The various functional groups in the amino acid were protected with conventional protecting groups used in polypeptide chemistry. For example, the lysine functional group was protected with a protecting group that could be removed upon replenishment of the kit without adversely affecting the final polypeptide product. Fluorescamine was used in the synthesis to determine the success of the coupling as indicated by positive fluorescence (see Felix, et al., Analyt, Biochem., 52, 377, 1973). If the coupling was not complete, the coupling was repeated with the same protected amino acid before deprotection.

The C-terminal amino acid can be incorporated into the polymer in a variety of well-known ways. Summaries of methods using halomethyl resins have been published by Horiki, et al. in Chem. Letters, pp. 165-168 (1978) and Gis in Helv. Chim. Acta, 56, 1476 (1973).

The general procedure is as follows: L-glutamine, the amino groups of which are protected, is esterified in an absolute alcohol containing an amine. The coupled amino acid resin is filtered, washed with alcohol and water and dried. The protecting group on the ε-amino group of the glutamic amino acid (e.g., t-BOC, i.e., t-butyloxycarbonyl) is then removed. The resulting coupled amino acid resin having a free amino group is then reacted with protected L-serine, preferably alpha-t-BOC-O-benzyl-L-serine, to attach L-serine. The reactions are then repeated with protected L-lysine and L-alanine until the complete molecule is prepared. The reaction sequence was performed as follows:

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15

Har.tsi 673 6 8

at' R-Gln-OH

cC-R 2 -Gln resin

Remove the o-amino-I protecting group H-Gln resin σ,? 2

oC -R-, -L-Ser-OH

* 2 i c * (- R 2 -Ser-Gln resin

Remove the ε-amino-D protecting group, 2 + H-Ser-Gln resin? 3

R ^ o-Lys-OH

R0 R0 t 3 t 2 oC -R1 ~ Lys-Ser-Gln resin

Οό-amino protecting group removed * 3 h 1H-Lys-Ser-Gln resin

06-R, -Ala-OH

R R

I I * cC -R 2 -Ala-Lys-Ser-Gln resin

Remove all protecting groups and resin

V

H-Ala-Lys-Ser-Gln-OH

In this series of reactions, R 1 is a protecting group of the α-amino group, and R 2 and R 2 are protecting groups of the reactive side chains of L-serine and L-lysine, respectively, which are not removed when R 1 is removed to carry out the next reaction. In the above pentapeptide-resin intermediate, the term is preferably tert-butyloxycarbonyl, R 2 is benzyl or substituted benzyl, (e.g.

4-chlorobenzyl), and R 1 is substituted benzyloxycarbonyl (e.g. 2,6-dichlorobenzyloxycarbonyl). The resin is one of the above-mentioned resins.

16 67368

Once the final intermediate is prepared, the peptide resin is cleaved with the R 1 and R 2 protecting groups and the resin. The protecting groups are removed by conventional methods, e.g. by treatment with anhydrous hydrogen fluoride, and the resulting free peptide is then recovered.

As mentioned above, the amino groups must be protected when carrying out the process. Strongly basic amino groups can be protected by salt formation or using p-methoxybenzyloxycarbonyl and / or tert-butyloxycarbonyl protecting groups. It is preferred to use tert-butyloxycarbonyl (BOC) or tert-amyloxycarbonyl (AOC) to protect the o6 amino group in amino acids where the reaction occurs at the carboxyl terminus of the molecule, as the BOC and AOC protecting groups can be easily removed after such reaction and before the next step ( wherein such an o 2 '- amino group itself is reacted) by allowing an acid (e.g., trifluoroacetic acid) to act under mild conditions, which treatment does not affect other groups protecting other reactive side chains. Thus, it is to be understood that the o (, -) amino groups may be protected by reaction with any substance which protects the amino groups with respect to the subsequent reaction (s) but which may subsequently be removed under conditions which would not otherwise affect the molecule. acid derivatives which acylate an amino group.

In general, amino groups can be protected by reaction with a compound containing a moiety of the formula: 0-10 _C__, which has a moiety that prevents the amino group from reacting in the subsequent coupling reaction and can be removed without cleaving the molecule. may be straight or branched chain alkoxyl which may be unsaturated and contains 1 to 1 carbon atoms and is preferably halogen or cyano substituted; aryl, preferably having 6 to 15 carbon atoms; cycloalkyl, preferably containing 5 to 8 carbon atoms; preferably aralkyl having 7 to 13 carbon atoms; preferably alkaryl having 7 to 13 carbon atoms; or a heterocyclic compound, e.g. isonicotinyl. The aryl, aralkyl and time 17,6368 moieties may also be substituted, such as by one or more alkyl groups having 1 to about H carbon atoms. Preferred moieties are tert-butyl, tert-amyl, tolyl, xylyl and benzyl. Highly preferred specific amino protecting groups are benzyloxycarbonyl; substituted benzyloxycarbonyl, wherein the phenyl ring is substituted by one or more halogens, e.g. Cl or Br; heart medicine; lower alkoxy, e.g. methoxy; lower alkyl; tert-butyloxycarbonyl, tert-amyloxycarbonyl; cyclohexyloxycarbonyl; vinyloxycarbonyl; adamantyloxycarbonyl; biphenyl-isopropoxycarbonyl; etc. Other useful protecting groups include isonicotinyloxycarbonyl, phthaloyl, p-tolylsulfonyl, formyl, etc.

In carrying out the general method of the invention, the peptide is constructed by the reaction of a free amino group with a compound having protected amino groups. For reaction, i.e. the coupling, the carboxyl group of the compound to be coupled is activated so that it can react with the free? The carboxyl group can be activated by converting it into a reactive group such as an ester, anhydride, azide, acid chloride, etc. Alternatively, a suitable coupling reagent can be used in the reaction. Suitable coupling reagents have been proposed, e.g., by Bodanszky et al. in Peptide Synthesis, Interscience, Second Edition, 1975, Chapter 5, and these include carbodiimides (e.g., dicyclocarbodiimide), carbonyldiimidazole, and the like.

It is also to be understood that during these reactions, the amino acid moieties contain both an amino group and a carboxyl group, and usually one moiety is involved in the reaction, while the other is protected. Prior to the coupling step, the alpha or terminal amino group of the peptide is deprotected under conditions that do not substantially affect other protecting groups, e.g., the epsi-Icn amino protecting group of the lysine molecule. Preferably, this step is carried out by treatment with an acid such as trifluoroacetic acid at room temperature.

The peptides prepared were identified and their purity was determined by known methods such as thin layer chromatography, electrophoresis, amino acid analysis, etc.

The following examples are presented to illustrate the invention.

18 67368

In the examples and throughout the specification, parts are by weight unless otherwise indicated.

Example I

In the preparation of the polypeptide, the following substances were commercial products: γ-BOC-L-glutamine-o-nitrophenyl ester oi-BOC-1-2-chloro-benzyloxycarbonyl-L-lysine -> C-BOC-O-benzyl-L-serine U6-B0C-L-alanine.

In these reagents, BOC is t-butyloxycarbonyl. Seque-nal reagents for amino acid series assays, dicyclohexylcarbodiimide, fluorescamine, and resin were also obtained commercially. The resin used was a polystyrene-divinylbenzene resin, grain size 200-400 mesh, and contained 1% divinylbenzene and 0.75 mM chloride per gram of resin.

To prepare the polypeptide, 2 mmol / l of BOC-L-glutamine was coupled by esterification to 2 mmol of chloromethylated resin in absolute alcohol containing 1 mM thiethylamine with heating for 24 hours at 80 ° C. The resulting amino acid-resin ester was filtered, washed with absolute alcohol and dried. The other β-BOC amino acids were then coupled in the same manner using equivalent amounts of dicyclohexylcarbodiimide to the o6-amino group of the peptide-resin, which had been deprotected in the correct order to give the desired polypeptide. After each coupling reaction, a resin sample was examined with fluorescamine, and if positive fluorescence was detected, coupling was considered incomplete and repeated with the same protected amino acid. As a result, an intermediate tetrapeptide-resin was prepared from several coupling reactions.

This peptide-resin was cleaved and deprotected in a Kel-F cleavage apparatus (Peninsula Laboratories, Inc.) using anhydrous hydrogen fluoride at 0 ° C for 60 minutes with 1.2 ml of anisole per gram of peptide-resin. The peptide mixture was washed with anhydrous ether and extracted with aqueous acid. The extract was lyophilized and the peptide was chromatographed on P-6 Bio-Gel in acetic acid (1 mol / l). The resulting polypeptide was 94% pure and comprised the following series:

II

67368 19 H-ALA-LYS-SER-GLN-OH.

For identification, thin layer chromatography and electrophoresis were performed as follows.

Thin layer chromatography was performed on a 30 μg sample on silica gel (Brinkman Silica Gel + fluorescent indicator, 20 x 30 cm, 0.1 mm thick) using the following eluents.

n-butanol: pyridine: acetic acid: water; 30: 15: 3: 12

Rf: ethyl acetate: pyridun: acetic acid: water; 5: 5: 1: 3 r 3 R 3: ethyl acetate: n-butanol: acetic acid: water; 1: 1: 1: 1.

Electrophoresis was performed on a 100 μg sample of Whatman 3 mm paper (11.5 x 56.5 cm) using pyridine-acetate buffer pH 5.6 and 1,000 V for one hour.

Spray reagents for both thin layer chromatography and electrophoresis were Pauly reagent and ninhydrin.

1 2

The following results were obtained: R- = stationary, Rf = luk- 3. . - non-uniform and = 0.336. Electrophoresis gave a migration of 9.4 cm towards its cathode.

Examples II-IV

Using the reaction methods described above to prepare substituted polypeptides, polypeptides of the following formula are prepared: R-X-LYS-Y-GLN-R 'These peptide amides were prepared on a benzhydrylamine resin by a solid phase synthesis method known in the art.

Example number R X Y R 'II H SAR D-ALA NH2 I IA H SAR SAR NH2 III H D-ALA D-ALA NH2 IIIA H D-ALA SAR NH2 IV H SAR 2-Me-ALA NH2

IVA H SAR SAR OH

For identification, thin layer chromatography and electrophoresis were performed as follows.

20 6 7 3 6 8

Thin layer chromatography was performed on 20 μg samples on silica gel (Kieselgel, 5 x 20 cm) using n-butanol: acetic acid: ethyl acetate: water 1: 1: 1: 1 (R 2) and cellulose 6064 (Eastman, 20 x 20 cm) using n-butanol as eluent:. 2 pyridimine: acetic acid: water in a ratio of 15: 10: 3: 12 (Rf).

Electrophoresis was performed on 50 μg samples of Whatman paper No. 3 (5.7 x 55 cm) using pH 5.6 pyridine acetate buffer solution and 1000 V for one hour. The compounds move towards the cathode.

Spray reagents for both thin layer chromatography and electrophoresis were Pauly reagent and ninhydrin.

The following results were obtained (both Rf values and electrophoresis are relative to H-ARG-LYS-ASP-VAL-TYR-OH): Example 12 R ^ Electrophoresis movement purity II 0.44 0.68 2.07 98% IIA 0.88 0.60 1.78 98% III 0.56 0.71 2.10 98%

Following the thin layer chromatography and electrophoresis method described in Example I, the following results were obtained for the compound of Example IV: = (0.155), Rf2 = immobile, Rf3 = 0.265 and the electrophoresis movement per cathode is 13.1 cm.

Examples V-VI

Using the reaction methods described above to extend the polypeptide chain, the following pcpeptides of the formula were prepared: K-ALA-LYS-SER-GLN-Rf

Example R R 'number

V GLN OH

VI SAR OH

The polypeptide derivatives prepared in Examples V and VI retained their biological activity well.

Following the thin layer chromatography and electrophoresis method of Example I, the following result was obtained for the compounds of Examples V and VI: 21 67368 1 2 3

Example R 2. Electrophoretic movement keel cathode V immobile immobile 0.303 7.4 cm VI immobile immobile 0.18 6 8, 3 cm

Claims (1)

A process for the preparation of a therapeutically useful polypeptide of the formula RX-LYS-Y-GLN-R 'I wherein R' is OH or NH 4, X is sarcosyl, L-alanyl or D-alkanyl, Y is seryl, sarcosyl, 2-methylalanyl or D-alanyl, and R is H, GLN or SAR, characterized in that L-glutamine, the amino group of which is protected, is covalently esterified with an insoluble resin polymer, the L-glutamine group is removed The 6-amino protecting group, a γ-amino acid whose β-amino group is protected, is reacted with an L-glutamine resin to attach a Y-amino acid group to it, the γ-amino acid group is removed from the γ-amino acid group, L-lysine with a v> 0-amino the group is protected, is reacted with a Y-amino acid-L-glutamine resin to attach L-lysine thereto, the -amino-amino group is removed from the L-lysine group, the NR-substituted-X-amino acid whose <*. -amino group is protected is reacted to react with L-lysine-Y-amino acid-L-glutamine-resin k to attach an NR-substituted-X-amino acid thereto, the resin is cleaved from the peptide with acid or ammonia and all protecting groups removed to give a compound of formula I wherein R 'is OH or NH 2, wherein when a benzhydrylamine resin polymer is used, R' is NH 4. regardless of which cleavage agents are used. Il