EP0000053B1

EP0000053B1 - Somatostatin analogs, process for their preparation and pharmaceutical compositions containing them

Info

Publication number: EP0000053B1
Application number: EP78100095A
Authority: EP
Inventors: Daniel Frank Veber
Original assignee: Merck and Co Inc
Current assignee: Merck and Co Inc
Priority date: 1977-06-08
Filing date: 1978-06-06
Publication date: 1981-05-27
Also published as: US4146612A; IT1105131B; IT7849736A0; EP0000053A1; DE2860734D1; DK252778A; JPS543085A; IE47421B1; IE781151L

Description

BACKGROUND OF THE INVENTION:

Somatostatin is a tetradecapeptide having the structure:
and has the properties of inhibiting the release of growth hormone, inhibiting the release of insulin and glucagon and reducing gastric secretion. This lack of specificity of the biological activity of somatostatin has led to an intensive search for analogs which exhibit a more specific biological activity. Somatostatin itself has a short duration of action because it is inactivated, inter alia, by aminopeptidases and carboxypeptidases present in vivo. This problem of the short duration of action has been partially solved in the prior art by preparing derivatives of somatostatin which have low solubility, thus attaining a slow release on subcutaneous injection. Once dissolved, however, the derivatives are no more stable to inactivation by aminopeptidases and carboxypeptidases than somatostatin itself. The present invention provides somatostatin analogs having no material affect on gastric secretion and a longer duration of action and a novel method for preparing said analogs.
The present invention further provides somatostatin analogs which are easier to prepare because they contain only 26 atoms in the peptide backbone.

SUMMARY OF THE INVENTION:

This invention is concerned with novel somatostatin analogs having a more specific biological activity and duration of activity than naturally occurring somatostatin and which are easier to prepare because of the smaller ring size and having the structural formula:
wherein

A is Phe, Tyr, O-Me-Tyr,
B is Phe, Tyr,
C is Thr, Val,

The preferred somatostatin analogs of the present invention are illustrated by the following structural formula:
and the pharmaceutically acceptable non-toxic acid addition salts thereof.
Illustrative of acid addition salts are hydrochloride, hydrobromide, sulfate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate and the like.
The somatostatin analogs of the present invention differ from somatostatin by virtue of the fact that they lack an N-terminal amino group and a C-terminal carboxyl group thus eliminating the groups involved in enzymic cleavage of the molecule by aminopeptidases and carboxypeptidases. Furthermore, the deletion of the adjacent heteroatoms of the disulfide bridge of somatostatin increases the stability of the analogs in vivo by preventing enzymatic degradation by reductive cleavage. Therefore, the analogs of the present invention are more resistent to cleavage in vivo than somatostatin and thus have a prolonged duration of action.
Somatostatin is a tetradecapeptide having the structure:
The portion of somatostatin extending from amino acid Cys³ to Cys'⁴ forms a dodecapeptide of the following structure:
The peptide backbone and the disulfide bridge consist of a continuous 38 atom ring.
The present invention provides somatostatin analogs wherein the Ala'-Gly², Lys⁴-Asn⁵, Thr^12- Ser¹³ and the amino groups of Cys³ and carboxyl group of Cys¹⁴ are deleted.
Furthermore, the disulfide atoms of the cystine, -S-S-, have been replaced by the dicarba group, ―CH₂―CH₂. Whereas, in somatostatin the amino acids 4 and 13 are bridged by cystine, the present invention provides somatostatin analogs wherein amino acids 6 and 11 are bridged by 7-aminoheptanoic acid. The resulting ring contains 26 atoms. Furthermore, the somatostatin analogs of the present invention include those wherein Phe⁶ is replaced by Tyr or O-Me-Tyr; Phe⁷ is replaced by Tyr; Trp⁸ is replaced by D-Trp and Thr'° is replaced by Val.
The abbreviated designations, which are used herein for the amino acid components, certain preferred protecting groups, amino acid activating groups, condensing agents, reagents and solvents employed in the process of this invention are as follows:
In accordance with the present invention, the novel somatostatin analogs are prepared by cyclizing corresponding linear peptides. The linear peptides are prepared by using the solid phase sequencial synthesis technique. Accordingly, the process for preparing the somatostatin analogs of the present invention comprises a) preparing a corresponding blocked linear peptide attached to a solid phase resin; b) selectively deblocking the N-terminal amine group;.c) removing the linear peptide from the resin; d) treating the linear peptide with a cyclizing agent to obtain the cyclic peptide; and e) removing the remaining blocking groups.
When the linear peptide is prepared on the resin, it is not critical which amino acid is selected to be at the C-terminal position provided only that the sequence of amino acids in the linear peptide corresponds to that in the desired somatostatin analog. Once a linear peptide has been cyclized one can no longer determine which amino acid was at the C-terminus of the linear peptide. As an example to illustrate this, either of the two following linear peptides, when cyclized, will give the identical somatostatin analog:
or
It is evident that since the linear peptide is going to be cyclized, it does not matter which amino acid is used to start the chain. Starting with Phe at the carboxyl end, as illustrated in the first of the two examples above, has an advantage over the second example. In the first example, D-Trp, which reacts with t-butyl carbonium ions formed when BOC groups are removed, is the N-terminal amino acid and thus will be added last and hence will be subjected to the least number of exposures to t-butyl carbonium ion.
The synthesis of the linear peptides by the solid phase technique is conducted in a stepwise manner on chloromethylated resin. The resin is composed of fine beads (20-70 microns in diameter) of a synthetic resin prepared by copolymerization of styrene with 1 to 2 percent divinylbenzene. The benzene rings in the resin are chloromethylated in a Friedel-Crafts reaction with chloromethyl methyl ether and stannic chloride. The Friedel-Crafts reaction is continued until the resin contains 0.5 to 5 mmoles of chlorine per gram of resin.
The amino acid selected to be the C-terminal amino acid of the linear peptide is converted to its amino protected derivative. The carboxyl group of the selected C-terminal amino acid is bound covalently to the insoluble polymeric resin support, as for example, as the carboxylic ester of the resin-bonded benzyl chloride present in chloromethyl-substituted polystyrene-divinylbenzene resin. After the amino protecting group is removed, the amino protected derivative of the next amino acid in the sequence is added along with a coupling agent, such as dicyclohexylcarbodiimide. The amino acid reactant may be employed in the form of a carboxyl-activated amino acid such as the ONp ester, an amino acid azide, and the like. Deprotection and addition of successive amino acids is performed until the desired linear peptide is formed.
The selection of protecting groups is, in part, dictated by particular coupling conditions, in part by the amino acid and peptide components involved in the reaction.
Amino-protecting groups ordinarily employed include those which are well known in the art, for example, urethane protecting substituents such as benzyloxycarbonyl (carbobenzoxy), p-methoxy- carbobenzoxy, p-nitrocarbobenzoxy, t-butyloxycarbonyl, and the like. It is preferred to utilize t-butyloxycarbonyl (BOC) for protecting the a-amino group in the amino acids undergoing reaction at the carboxyl end of said amino acid. The BOC protecting group is readily removed following such coupling reaction and prior to the subsequent step by the relatively mild action of acids (i.e., trifluoro acetic acid, or hydrogen chloride in ethyl acetate).
The -OH group of Thr can be protected by the Bzl group and the _E-amino group of Lys can be protected by the INOC group or the 2-chlorobenzyloxycarbonyl (2-Cl-CBZ) group. In the case of Lys, it is preferred to protect the _E-amino group with 2-Cl-CBZ group as this group is removed simultaneously with the Bzl groups by treatment with HF after the linear peptide has been cyclized.
The INOC group is not removed by HF and requires an additional treatment with Zn. Neither group is affected by TFA, used for removing BOC protecting groups.
After the linear peptide has been formed on the solid phase resin, it may be removed from the resin by a variety of methods which are well known in the art. For example, the peptide may be cleaved from the resin with hydrazine and thus directly form the peptide hydrazide which may be subsequently cyclized via the azide to the desired cyclic peptide. The hydrazide is converted to the corresponding azide by reaction with a reagent which furnishes nitrous acid in situ. Suitable reagents for this purpose include a lower alkyl nitrite (e.g. t-butyl nitrite, isoamyl nitrite) or an alkali metal nitrite salt (e.g., sodium nitrite, potassium nitrite) in the presence of a strong acid such as hydrochloric, phosphoric, sulfonic, etc. This reaction is carried out in the presence of either water and/or a non-aqueous solvent such as dimethylformamide, tetrahydrofuran, dioxane, chloroform, methylene chloride, etc., at a temperature between about -40°C and +20°C. Alternatively, the peptide may be removed from the resin by treatment with a lower alcohol such as methanol in the presence of an organic base such as triethylamine, thus resulting in the formation of the corresponding lower alcohol ester of the linear hexapeptide. In the case wherein the ester is the methyl ester, the resulting compound may be converted to the azide via the hydrazide which may then be cyclized to the desired cyclic peptide. The preferred method in the present invention is the use of hydrazine.
As reference to Table II will show, one preferred overall procedure for preparing the desired cyclic peptides of the present invention involves the stepwise synthesis of the linear peptide on a solid phase resin. More specifically, in the process for preparing cyc/o(Aha-Phe-Phe-D-Trp-Lys-Thr-Phe), the carboxyl end of the N-blocked amino acid phenylalanine is bound covalently to an insoluble polymeric resin support as the carboxylic acid ester of the resin-bonded benzyl chloride. The amino group of Phe is protected by the BOC group. After the attachment of the Phe is completed on the resin, the protecting group BOC is removed by treatment with TFA in CH₂CL₂. The subsequent amino acids are attached, in the form of BOC-amino acid, using DCCI as the condensing agent or an active ester such as ONp. After the desired linear peptide has been prepared, the N-terminal amino group is selectively deblocked and the peptide is removed from the resin by treatment with hydrazine. The resulting linear peptide hydrazide with the N-terminal amino group deblocked having the amino acid sequence: D-Trp-(ε-2-CI-CBZ)Lys-(O-Bzl)Thr-Phe-Aha-Phe-Phe-NH―NH₂ is treated with isoamyl nitrite in acid pH to form the corresponding azide. The azide solution is diluted with solvent and neutralized with an organic base. The linear peptide cyclizes to form cyclo[Aha-Phe-Phe-D-Trp-(ε-2-Cl-CBZ)Lys-(O-Bzl)Thr-Phe]. During the cyclization the "pH" is checked and maintained at neutral by the addition of organic base. The "pH" in organic solvent is determined by the application of an aliquot of the solution to narrow range pH paper.
After the linear peptide is cyclized, the remaining protective groups, 2-Cl-CBZ and Bzl, are removed in one step by treatment with HF in the presence of anisole. The crude cyclic peptides obtained by the processes of Table II are purified by chromatography, preferably on "Sephadex" (a registered trademark in the United Kingdom) eluted with 50% aqueous acetic acid.
The following Examples illustrate methods of carrying out the present invention, but is to be understood that these Examples are given for purposes of illustration and not of limitation. It is to be understood that by changing the amino acid sequence of the polypeptide in accordance with the instructions provided by this disclosure, affords each of the compounds embraced by the description presented herein and embraced by the claims of this application.

Example 1

Preparation of Cyc/o(Aha-Phe-Phe-D-Trp-Lys-Thr-Phe)

Step a) - Preparation of D-Trp-(ε-2-Cl-CBZ)Lys-(O-Bzl)Thr-Phe Aha-Phe-Phe-O-CH₂-Ø-Resin

Chloromethyl resin (2% cross-linked Merrifield resin), 862.0 g. (2.37 moles), having 2.75 meq. chlorine/g., and 607.0 g. (2.37 moles, 1 equivalent) of BOC-Phe were added to 4320 ml. of peroxide- free tetrahydrofuran. The mixture was stirred in an oil bath at 80°C. bath temperature for 45 minutes. Triethylamine, 310.0 ml., was added and the reaction mixture stirred at 80°C. bath temperature for 70 hours, cooled to 25°C. and transferred to a stirred solid phase reaction column with 2000 ml. of tetrahydrofuran. After removal of the solvent, the resin was washed using the stirred column with:
The BOC-Phe-O-CH₂-Ø-resin was dried in vacuo at 25°C. for 16 hours, giving 1203 g. of BOC-Phe-O-CH₂-Ø-resin containing 0.937 mmole of phenylalanine/g. of resin.
BOC-Phe-O-CH₂-Ø-resin (2.84 g.; 2.0 mmole) was carried through the procedures in Tables III and IV using 2 deblockings (2 minutes and 25 minutes) with 25% TFA in methylene chloride, and 2.5 equivalents of BOC-amino acid in the required sequence until the desired BOC-octapeptide-O-CH2-Ø- resin was obtained.
DCCI was used as the coupling agent in every step except the coupling of BOC-Phe-to Aha-Phe-Phe-O-CH₂-Ø-resin in which case the coupling was carried out in the presence of DCCI and 1-hydroxybenzotriazole monohydrate (HBT. H₂0).
The coupling- of each amino acid proceeded smoothly. Best yields were obtained when the coupling was repeated in each step. When the coupling was repeated, the initial two chloroform washes, the deblocking step and the succeeding three chloroform washes were all omitted and replaced by a single chloroform wash.
The coupling reactions were carried out in methylene chloride, freshly degassed DMF or a mixture of these two solvents. The N-terminal amino group was blocked with a BOC group in each case; the hydroxy group of Thr was blocked with Bzl and the e-amino group of Lys with 2-Cl-CBZ.
When the desired BOC-heptapeptide-O-CH₂-Ø-resin was obtained, the N-terminal BOC group was removed by the terminal deblocking procedure set forth in Table V.
After the sequence of Tables III, IV and V were completed, the blocked heptapeptide-O-CH₂-Ø- resin was filtered, washed with MeOH 3 x 40 ml. (3 minutes per wash) and dried overnight in vacuo. It weighed 3.41 g.

Step b) ―Preparation of D-Trp-(ε-2-Cl~CBZ)-Lys-(O-Bz)Thr-Phe Aha-Phe-Phe-NH-NH₂

To a mixture of 3.20 g. D-Trp-(ε-2-Cl-CBZ)Lys-(O-Bzl)Thr-Phe-Aha-Phe-Phe-O-CH₂-Ø-resin in 33 ml. freshly degassed DMF was added 3.3 ml. NH₂―NH₂. The mixture was magnetically stirred at room temperature for 1 hour. The mixture was filtered to remove the resin. The resin was washed with 4 x 10 ml. DMF. The filtrate and washings were concentrated in vacuo to near dryness. The semi-solid residue was triturated with ether to obtain a solid. The solid was collected by filtration and dried in vacuo for 45 min. to yield 3.14 g, crude product. The solid was slurried with 4 x 20 mi. water to remove all traces of formyl hydrazide and dried in vacuo overnight to give 1.81 g. of product.

Step cJ - Preparation of D-Trp-(ε-2-Cl-CBZ-)Lys-(O-Bzl)Thr-Phe-Aha-Phe-Phe-N₃

D-Trp-(ε-2-Cl-CBZ)Lys-(O-Bzl)Thr-Phe-Aha-Phe-Phe-NH-NH₂ (1.75 g., 1.39 mmole), prepared by the process set forth in Step b), was suspended in 20 mi. freshly degassed DMF. The turbid solution was stirred magnetically at -40°C. under a nitrogen atmosphere. To the suspension was added 1.94 ml., 5.07N HCI in THF (8.34 mmole), 6.0 equivalents). The resulting clear acidic solution, "pH" 1.0 to 1.5, was warmed to -25°C. and 0.215 ml. isoamyl nitrite (1.39 mmole, 1.0 equivalents) was added and stirring continued for 30 minutes. This solution of D-Trp-(ε-2-Cl-CBZ)Lys-(O-Bzl)-Thr-Phe-Aha-Phe-Phe-N₃ was used immediately in step d).

Step d) - Preparation of Cyclo[Aha-Phe-Phe-D-Trp-(ε-2-Cl-CBZ)Lys-(O-Bzl)Thr-Phe)

The solution of D-Trp-(_E-2-CI-CBZ)Lys-(0-Bzl)Thr-Phe-Aha-Phe-Phe-N₃ in DMF, obtained by the process set forth in Step c), was diluted in 1200 ml. freshly degassed DMF, precooled to -40°C. The solution was maintained under a nitrogen atmosphere and allowed to warm to -20°C. during which time the "pH" was maintained at 7.2 to 7.6 by the addition of 1.62 ml. N,N-diisopropylethylamine. The solution was maintained at -16°C. for 24 hours and then kept at 5°C. for an additional 24 hours. During this period 1.95 ml. of N,N-diisopropylethylamine was added to maintain a "pH" of 7.2 to 7.6.
The solution was concentrated in vacuo to a thick oil, washed twice with ether and once with ethyl acetate and triturated with water to give a solid. The solid was collected by filtration and dried in vacuo overnight to give 1.17 g. of product.

Step e) - Preparation of Cyclo(Aha-Phe-Phe-D-Trp-Lys-Thr-Phe)

Cyclo[Aha-Phe-Phe-D-Trp-(ε-2-Cl-CBZ)Lys-(O-Bzl)-Thr-Phe], 1.15 g., obtained by the process set forth in Step d), was dissolved in 2 mi. anisole and 20 ml. hydrogen fluoride at dry ice-acetone bath temperature. The solution was stirred magnetically at ice-bath temperature for 1 1/2 hours. The excess hydrogen fluoride was removed in vacuo at ice-bath temperature. The resulting oily residue was maintained in vacuo for an additional 3/4 hour at ice-bath temperature and triturated with ethyl acetate to give a solid. The solid was collected by filtration and dried in vacuo to give 674 mg. of product.

Step f) - Purification of Cyclo(Aha-Phe-Phe-D-Trp-Lys-Thr-Phe)

The cyc/o(Aha-Phe-Phe-D-Trp-Lys-Thr-Phe), 654 mg., obtained by the process set forth in step e), was dissolved in 12 ml. 50% aqueous acetic acid and charged to a column of Sephadex G-50, (5 cm. x 115 cm., 2260 ml.) packed in 50% aqueous acetic acid. The column was eluted with 50% aqueous acetic acid at the rate of 21 ml./10 min./fraction. The effluent was monitored at 280 nm.
Fractions 83 to 91 were combined, concentrated to dryness in vacuo and the residue lyophilized from 20 ml. 10% aqueous acetic acid to give 369 mg. of substantially pure product. A 20 hour acid hydrolysate showed the following amino acid analysis:
The somatostatin analogs of the present invention and the non-toxic pharmaceutically acceptable salts thereof, are useful in humans and animals for inhibiting growth hormone release as in the treatment of acromegaly, inhibiting the release of glucagon and alone or in conjunction with insulin, for lowering blood glucose as in the treatment of diabetes with reduced gastrointestinal side effects. In the treatment of diabetes, the number and size of daily doses and the time of administration are determined by an individual study of each subject. The method of determining these factors is known to those skilled in the art.
The somatostatin analogs described herein may be administered to warm blooded animals, including humans, either intravenously, subcutaneously, intramuscularly or orally. The contemplated dose range for oral administration in tablet or capsule form to large mammals is about 0.001 mg. to about 7 mg./kg. of body weight per day. These somatostatin analogs are preferably administered by injection. A therapeutically effective amount of an analog is ordinarily supplied at a dosage level of from about 0.001 mg. to about 2 mg./kg. of body weight. Preferably the range is from about 0.00142 mg. to about 0.428 mg./kg. of body weight administered by intravenous infusion or by subcutaneous injection. The required dosage will vary with the particular condition being treated, the severity of the condition and the duration of treatment.
If the active ingredient is administered in tablet form, the tablet may contain: a binder such as gum tragacanth, corn starch, gelatin, an excipient such as dicalcium phosphate; a disintegrating agent such as corn starch, and alginic acid; a lubricant such as magnesium stearate; and a sweetening and/or flavoring agent such as sucrose, lactose and wintergreen. Suitable liquid carriers for intravenous administration include sterile water, isotonic saline and phosphate buffer solutions or other pharmaceutically acceptable injectable carriers.
The following example is included to illustrate the preparation of a representative dose of cyc/o(Aha-Phe-Phe-D-Trp-Lys-Thr-Phe) suitable for subcutaneous injection.

Example 2

1 ml. sterile saline;
1 mg. cyclo(Aha-Phe-Phe-D-Trp-Lys-Thr-P"he).

Claims

1. Compounds of the formula:

wherein

A is Phe, Tyr, O-Me-Tyr,

B is Phe, Tyr,

C is Thr, Val,

wherein the ring formed by the peptide backbone contains 26 atoms and pharmaceutically acceptable non-toxic acid addition salts thereof.

2. The compound according to Claim 1 having the formula:

3. The process for preparing the peptides having the formula:

wherein

A is Phe, Tyr, O-Me-Tyr,

B is Phe, Tyr,

C is Thr, Val,

which comprises:

a) preparing a corresponding blocked linear peptide attached to a solid phase resin wherein when C is Thr the -OH group of Thr is blocked by Bzl; the _E-amino group of Lys is blocked with 2-CI-CBZ and the N-terminal amino group is blocked with BOC;

b) selectively removing the BOC group from the N-terminal amino group;

c) treating the resin bound linear peptide with hydrazine to remove the peptide from the resin to obtain the linear peptide hydrazide with the N-terminal group deblocked;

d) treating the linear peptide with isoamyl nitrite at acid pH to obtain the corresponding azide; diluting and neutralizing the azide solution to obtain the corresponding cyclic peptide, and;

e) treating the cyclic peptide with HF to simultaneously remove the Bzl and 2-CI-CBZ blocking groups.

4. A composition comprising a therapeutically effective amount of the peptides having the structure:

wherein

A is Phe, Tyr, O-Me-Tyr,

B is Phe, Tyr,

C is Thr, Val,

wherein the ring formed by the peptide backbone contains 26 atoms and pharmaceutically acceptable non-toxic acid addition salts thereof in a pharmaceutically acceptable excipient.