Somatostatin analogs and intermediates thereto

The present invention relates to a process for the preparation of a tetradecapeptide with varied activity, including inhibition of gastric acid secretion and reduction of intestinal motility, of formula I:

H - D- Val - Gli -l- Cis / & \ Cis - OH, where Q represents -L - Lis -l- Asp - L Fen -L-Fen-L Tri - l-Lîs-l-Țre-L -Fenl-Tre L-Ser-L-. (I) wherein the tetradecapeptide chain amino acids represented by the usual abbreviations are Va] valine, glycine glycocol), Cis - cysteine, Lysine, lysine, Asp - asparagine, Phen - phenylalanine, Tri -. tryptophan, Tre - threonine, and Ser - serine, and the connection between the cysteine ​​residues represents a bridge of disuifure.

A tetradecapeptide of the formula J.-Ala-Gli-L-Cis z8 \ Cis-OH is known, wherein Q has the aforementioned significance (wherein Ala represents aniline and the link between cystic residues represents a disuifure bridge) called somatostatin. or somatotropin formation inhibition factor. This tetradecapeptide was isolated from hypothalamic ovine extracts, being observed to be active in inhibiting growth hormone secretion, also known as somatotropin,

Natural somatostatin is known as a general class of other compounds having the dodecapeptide sequence represented by positions 3 ... 14 of the hormone._1Q of the natural cloud.

The compound designated as ρ -Ala is also known¹ -somatostatin. and - Allah¹ - somatostatin, although structured is a stereoisomer of Ala¹ - natural somatostatin, manifests about half of the activity of natural somatostatin in inhibiting gastric acid secretion in vivo,

The process according to the invention allows the preparation of a tetradecapeptide of the formula I above, in that a tetradecapeptide is treated with a chain ₂₅ linear of formula II:

Hl -Val-Gli - ^ - Ciș / ^ \ Cis-OH, in which Qare the meaning shown above. (II)³⁰ with excess air, as an oxidizing agent, at room temperature in acidic medium

PRICE LEI 12.48 acetic acid at pH = 2.5 ... 9, in the absence of light, after which the reaction mixture is known to be processed in order to separate the reaction product, which can be transformed, if desired, into the salt of addition with an acid.

The following is an example of embodiment of the invention:

Dilute a solution of '- Val¹ - reduced somatostatin (374 ml, theoretically 175 mg] (- vals - glycyl - - cysteinyl - i. - lysyl - - asparaginyl - - phenylalanyl - i - phenylalanyl - - tryptophil - z. - lysyl - i - threonyl - - phenylalanil - - threonyl - i - series - - cysteine) with 147 ml of 0.2 M acetic acid and 2967 ml of distilled water to obtain a concentration of 50 i^itg / ml. Concentrated ammonium hydroxide is added to adjust the pH of the mixture to 6.7, The solution is stirred at room temperature in the dark for 64 hours until Ellman titration determines that the oxidation is complete.

The mixture is concentrated in vacuo to a volume of about 10 ml. The concentrate was diluted with 10 ml of glacial acetic acid and then the salts were removed by passing on a column with sefadex G-25 ion exchangers F. The chromatographic conditions were as follows: solvent, acetic acid 50 ° /₀ degassed, column dimensions 5.0X90 cm, temperature 26^toC, flow rate 246 ml / h, volume fraction 16.4 ml.

The absorption at 280 ηΐμ for each fraction represented by the number of fractions indicated two broad peaks. The first peak represented the aggregate forms of the product, and the second peak represented the monomer product. The material represented by the second tip / fractions 49 ... 64 (787 ... 1050 ml) were collected and lyophilized to dryness in the dark. The resulting solid was dissolved in 15 ml degassed 0.2 M acetic acid, and then passed through a column with sefadex G-25 F ion exchangers. The oromatographic conditions were as follows: solvent, 0.2 M degassed acetic acid , column dimensions 5.0X150 cm, temperature 26 ° C, flow rate 475 ml / h, volume fraction 16.6 ml.

The absorption at 280 ιημ for each fraction represented by the number of fractions indicated a broad peak. Ultraviolet spectroscopy indicated that the main part of the tip was the product. fractions

157., 172 (2590 efflux volume, 2855 ml. Peak = 2667 ml.) Were combined and lyophilized to dryness in the dark. Ultraviolet spectroscopy indicated 95 mg of the desired product. (Reduced form yield = 54.3¼).

A portion of the resulting solid was dissolved in 5 ml of 50¼ acetic acid and was chromatographed on a column with sefadex ion exchangers G-25 F. The chromatographic conditions were as follows: solvent, acetic acid 50 ° /_of degassed, column dimensions 2.5X180 cm, temperature 26 ° C, flow rate 53.2 ml / h, volume fraction. 8.87 ml.

The absorption at 280 sqm for each fraction is represented according to the number of fractions of a wide peak. Ultraviolet spectroscopy indicated that the main portion of the peak was the product. Fractions 56 ... 60 (488 ... 532 ml, tip = 505 ml) were combined and lyophilized in the dark, dry,

Optical rotation («] □ —42.1 ° -e pp^B) cetic lo /_a).

Analysis of amino acids ·: Val = 0.98; Gli = l, 01; 2 Cj ^ = 1.81; 2Lis-l, 99; Asp = 0.95; 3 End = 2.94j Tri = 0.80; 2 Tre-1.91; Ser-0,8,5.

The above results are expressed as ratios at (Gli + Lis) / 3 = 1.0. The following three 21-hour hydrolyses were performed: (1) in the presence of dimethylsulfoxide for cysteine ​​oxidation of cystic acid; (2) for the removal of thiogiicoic acid (3) without removal or oxidation. All the above values ​​represent averages of the hydrolysed irs with the following exceptions: Cis and Ser, only (1) and (3), Tri, only (2), Fen, only (2) and (3).

The compound obtained by the process according to the invention, D - Val¹ - somatostatin, differs from D - Ala¹ - somatostatin mentioned in the prior art by substituting two hydrogen atoms with methyl groups. It can be assumed that the pharmacological activity of Val¹ - somatostatin will be. similar to that of D - Ala¹ - Somatostatin, meaning the compound will be less active than the natural hormone as an inhibitor of gastric acid secretion in vivo. However, a - Val¹ - somatostatin shows a somewhat higher activity than the natural hormone activity. It follows that the activity o - Val¹ - somatostatin compared to the activity of known compounds, similar in structure, is not predictable.

The biologically active tetradecapeptide, of formula I above, can be transformed into non-toxic salts of addition with acids. Its structure differs from that of somatostatin in the presence of a d-valine residue at position 1 instead of 76054's residue of L-alanine. For this reason the tetradecapeptide of formula I is represented in this description by the name of η - Val¹ - somatostatin.

The compound of formula I is prepared from an intermediate of formula: R - d - Val - Gli - L - Cis (R ^ - h - Lis (R_from) -T, - Asp - L - End - l - End - L - Tri (R₅) - τ. - Lis (Ra) - i. - Three (R₃) - L - End -L - Three (R₃) - The Ser (R₄) t . - Cis (R,) - X, wherein R represents hydrogen or a group_α amino protectors, Rt represents hydrogen or a thio-protecting group, R₂ represents hydrogen or an epsilon-amino-protecting group, R₃ and R₄represents each hydrogen or hydroxy-protecting group, R₅ represents hydrogen or formyl and X represents hydroxy "- ^ resin, or a group -O-CH ^ -x wherein the resin is polystyrene, provided that when X represents hydroxy, each of the radicals R, R 1, R 2, R₃, R₄ and R₅ represents hydrogen, and when X represents a group. resin

X / · / X

-O-CH₂Each of the radicals R, Ri, Rs, R3 and Rt is different from hydrogen.

The tetradecapeptide of formula I is prepared by treating the corresponding tetradecapeptide with the linear chain H - D - Val - Gli - τ, - Cis -l - Lis - l - Asp -r, - Fen - ί. - End - r. - Tri -; .- List

- Tre - r. - End - τ. - Three - L - Serum - 1- Cis - OH with an oxidizing agent. Through this reaction, the two sulfhydryl groups are converted to a disulfide bridge.

Addition salts with non-toxic, pharmaceutically acceptable acids are addition salts with organic or inorganic acids, for example, salts with hydrochloric, sulfuric, sulfonic, tartaric, fumaric, hydrobromic, glycolic, citric, maleic, phosphoric, succinic, acetic , azotic, benzoic, ascorbic, p-toluenesulfonic, benzenesulfonic, naphthalene sulfonic and propionic. Preferably, the addition salts are those prepared with acetic acid, All the above mentioned salts are prepared by conventional methods.

The starting material of formula II is a straight-chain peptide containing 14 amino acid residues. In order to obtain the final product of formula I it is necessary to treat the tetradecapeptide with a linear chain under conditions, which will carry out its oxidation by converting the two sulfhydryl groups present in the molecule, one to each cysteineyl residue, in a disulfide bridge. This conversion can be achieved by treating a dilute solution of the linear tetradecapeptide with an oxidizing agent, such as, for example, iodine or potassium ferricide. The air can also be used as an oxidizing agent, the pH of the mixture being with: pi:) s generally between about 2.5 and lime and, preferably, between about 7.0 and Xșgpa 7.6. When using air as an oxidizing agent, the concentration of the peptide solution generally does not exceed about 0.4 mg of peptide per ml of solution, usually being 50 FgZmI.

The compound of formula I may be administered to warm-blooded mammals, including humans, by multiple methods, including orally, sublingually, subcutaneously, intramuscularly, intravenously, or any other appropriate route of administration. The compound is active in inhibiting growth hormone secretion. This inhibitory effect is useful in cases where the treated subject requires therapeutic treatment of excessive secretion of somatotr'opin, a secretion associated with various conditions, such as juvenile diabetes and acromegaly. The compound also has other physiological effects, including inhibition of gastric acid secretion, effect useful in treating ulcerative conditions, inhibition of exocrine pancreas secretion, potentially useful effect in treating pancreatitis, inhibition of insulin and glucagon secretion, and reduction of intestinal motility, useful effect in gastrointestinal radiology, preferably, The dose of sublingual or oral route is about 1 mg to about 100 mg / kg body weight per day. In general, the dose of intravenous, suheutane or intramuscular administration is from about 10 jig to about 1 mg / kg body weight per day, and preferably from about 50 rg to about 100 S'-g / kg. of body weight per day. It is obvious that the size of the dose varies widely, depending on the particular conditions to be treated and the severity of these conditions.

It is also possible to administer the compound of formula I associated with a pharmaceutical carrier, for example in the form of tablets or capsules. Inert diluents or carriers, for example, magnesium carbonate or Iactose, may be used together with conventional disintegrating agents764, for example, corn starch and alginic acid, and lubricating agents, for example, magnesium stearate, in Typically, the amount of carrier material or diluent varies⁵from about 5 to about 95% of the final composition, and preferably from about 50 to about 85¾ from the final composition.

Suitable ar'omatlzQre agents may also be used in the preparation of ¹⁰nai, to make the composition more pleasing to the administration.

Suitable carriers, such as, for example, isotonic saline and phosphoric buffer solutions may be used in the intravenous administration of the compound of formula I.

and - Val¹ - Somatostatin was test ed in dogs to determine the effect of inhibiting gastric acid secretion in vivo. In six dogs with chronic fistula and Heidenhain bag, gastric secretion of HCl was induced by injection of C-terminal tetrapeptide of gastrin in 25 doses of 0.5 rg / kg / h. Each dog served as its own martpr, receiving on one separate day only the tetrapeptide, on another day, the six dogs received the tetrapeptide and, after one hour of stationary secretion of HCl, injected j - Val¹ - somatostatin at a dose of 0.75 ng / kg / h for one hour. Gastric acid sample collection continued for an additional H /₂ h at intervals of 15 min. Samples 35 were titrated to pH = 7 with an automatic titration device. The maximum inhibitory effect of 3 - Val was extrapolated¹ - somatostatin versus the dose-response curve of .somatostatin and the relative effect of! 40 analogous to that of somatostatin was expressed as a percentage activity. o - Val¹ - somatostatin inhibited stationary acid secretion caused by the C-terminal tetrapeptide a. gas- 45 slips to 85.1 + 6.0¾ standard mean error. This effect is equivalent to that given by 0.935 µg / kg / h of somatostafin. The activity relative to somatostatin is therefore 125%. 50 d - Go¹ - somatostatin was also tested for its action on intestinal motility in unstressed dogs. Three dogs were used with intralu-⁵⁵menials placed in the antrum, duodenum and furs. Pressure changes in the intestinal hymen were recorded on a foloSid viscorder recording device.⁶⁰beams with light beam, After establishing a steady state, i) - Val¹ - intravenous somatostatin for 10 min. The compound caused an initial increase in intraluminal pressure-⁶⁵ be 1 in the piles and. then a decrease of a-J this, while-pressure in douden!

and in the antrum it remained reduced all the time of the test, the minimum effective dose required to increase the pyloric pressure and decrease the pressure in the duodenum and antrum was less than 0.125 ng / kg for 10 min. This value is comparable to the activity of somatostatin itself, which is 0.125 to 0.25 rg / kg for i min. · R> - Val¹ - somaiosi.atiuui also showed an activity of inhibiting pancreatic secretion ©, in three dogs having both. pancreatic fistula as well as total gastric fistula induced pancreatic secretion;

of injectin secretin 2 units / kg / h and cholecystokinin 0.45 units / kg / h, and gastric secretion of HCl by injection of tetragastrin 0.5 rg / kg / h. After installing stationary reactions, each holding received a> - Val¹ - somatostatin:

hourly dose at 0.75 cg / kg / h. \

The maximum inhibitory effect expressed as percentage of control relative to control for total protein was 51%. n - Val¹ - somatostatin was also tested for its activity on growth hormone secretion. Rats were used

Normal male Sprague-Dawley with weight:

100,., 120 g. utensils were used 5 groups per 8 rats. Pentobarbital sodium salt was administered intraperitoneally to all rats to stimulate growth hormone secretion.

One group served as a witness and received only physiological serum. In two groups, somatostatin was administered :, one, group 2 ing / rat, subcutaneous, the other;

also rat / subcutaneous.

In two other groups it was administered .0 - Val¹ - somatostatin, O: group 2 rg / rat, subcutaneous: the other 50 µg / rat, subcutaneous. 20 minutes later. Simultaneous administration of pentobarbital-sodium and test compound measured serum growth hormone concentration. The degree of inhibition of serum growth hormone concentration in relation to the control group was then determined and the relative activities of π - Val were compared¹ - somatostatin and somatostatin itself.

At the dose of 2 -pg / rat, p - Val¹ - somatostatin inhibited the growth of growth hormone secretion by 2% compared to the control, while somatostatin produced a 44% inhibition. At the dose of 50 Rg / rat, P - Val¹ - Somatostatin inhibited growth hormone secretion growth by 73¾ compared to control, while somatostatin itself produced 79% inhibition.

D - Val¹ - somatostatin was tested for in vivo activity In inhibiting glucagon and insulin secretion upon stimulation with L-alanine. There were 5 normal sting dogs of both sexes overnight. Blood samples were taken and then intravenous injection of physiological serum, somatostatin or d - Val¹ - somatostaitin. After 30 min, a new amount of l - alanine was administered intravenously over a period of 15 min. The injection of physiological serum, somatostatin or D - Val was continued¹ - somatostatin for 15 minutes after the end of the injection - ¹⁵of the country of I - alanine. The injection of τ - alanine produced a sharp increase in the concentration of glucagon and insulin in the serum, which returned to the concentration of the control sample at the end of the injection of i.²⁰dyestuffs. From the above, it was determined that the minimum dose of D - Val¹ - somatostatin for glucagon inhibition is 0.06 to 0.11 hg / kg / mm and for insulin inhibition is 0.006 25 to 0.03 lig / kg / min, while the minimum dose of somatostatin for inhibition of glucagon is from 0.10 to 0.12 hg / kg / min and for insulin inhibition it is 0.03 to 0.10 ng / kg / ³⁰min.

The process according to the invention has the advantage that it allows the preparation of a tetradecapeptide with physiological activities ³⁵ especially useful for inhibiting gastric acid secretion and reducing intestinal motility.