BACKGROUND OF THE INVENTION:
[0001] Somatostatin is a tetradecapeptide having the structure: Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH
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.
[0002] 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:
[0003] 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,
wherein the ring formed by the peptide backbone contains 26 atoms and pharmaceutically
acceptable non-toxic acid addition salts thereof.
[0004] 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.
[0005] Illustrative of acid addition salts are hydrochloride,hydrobromide, sulfate, phosphate,
maleate, acetate, citrate, benzoate, succinate, malate, ascorbate and the like.
[0006] 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.
[0007] Somatostatin is a tetradecapeptide having

tostatin extending from amino acid Cys
3 to Cys
14 forms a dodecapeptide of the following structure:

bridge consist of a continuous 38 atom ring.
[0008] The present invention provides somatostatin analogs wherein the
Ala
l-Gly
2,
Ly
s4-Asn5, Thr
12-Ser
13 and the amino group of Cys3and carboxyl group of Cys
14 are deleted.
[0009] Furthermore, the disulfide atoms of the cystine, -S-S-, have been replaced by the
dicarba group, -CH
2-CH
2. 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 Phe is replaced by Tyr or O-Me-Tyr;
Phe is replaced by Tyr; Trp
8 is replaced by D-Trp and Thr
10 is replaced by Val.
[0010] 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:

[0011] 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.
[0012] 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:
D-Trp-(∈-2-C1-CBZ)Lys-(0-Bzl)Thr-Phe-Aha-Phe-Phe-N3 or
Aha-Phe-Phe-D-Trp-(E -2-Cl-CBZ)Lys-(O-Bzl)Thr-Phe-N3
cyclization cyclo[Aha-Phe-Phe-D-Trp-(∈ -2-Cl-CBZ)Lys-(0-Bzl)Thr-Phe].
[0013] 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.
[0014] 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
ofchlorine per gram of resin.
[0015] 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.
[0016] 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.
[0017] 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).
[0018] The -OH group of Thr can be protected by the Bzl group and the ∈ -amino group of
Lys can be protected by the INOC group or the 2-chlorobenzyloxycarbonyl (2-C1-CBZ)
group. In the case of Lys, it is preferred to protect the ∈ -amin group with 2-C1-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.
[0019] 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.

[0020] 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 cyclo(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 CH
2C1
2. 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-termi
- nal amino group deblocked having the amino acid sequence: D-Trp-(f.-2-Cl-CBZ)Lys-(O-Bzl)Thr-Phe-Aha-Phe-Phe-NH-NH
2 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-(∈-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.
[0021] 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 eluted with 50% aqueous acetic acid.
[0022] 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 Cyclo(Aha-Phe-Phe-D-Trp-Lys-Thr-Phe) Step a) - Preparation of D-Trp-(∈-2-Cl-CBZ)Lys-(O-Bzl)Thr-Phe-Aha-Phe-Phe-O-CH2-0-Resin
[0023] 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-CH
2-0-resin was dried in vacuo at 25°C. for 16 hours, giving 1203 g. of BOC-Phe-O-CH
2-φ-resin containing 0.937 mmole of phenyl- alanine/g. of resin.

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-CH
2-ø-resin was obtained.
[0024] DCCI was used as the coupling agent in every step except the coupling of BOC-Phe
to Aha-Phe-Phe-O-CH
2-Ø-resin in which case the coupling was carried out in the presence of DCCI and 1-hydroxybenzotriazole
monohydrate (HBT.H
20).
[0025] 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.
[0026] 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∈-amino
group of Lys with 2-Cl-CBZ.
[0027] When the desired BOC-heptapeptide-O-CH
2- 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-CH
2-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.,
Steb b) - Preparation of D-Trp-(∈-2-C1-CBZ)-Lys-(O-Bzl)Thr-Phe-Aha-Phe-Phe-NH-NH2
[0028] To a mixture of 3.20 g. D-Trp-(∈-2-Cl-CBZ)Lys-(O-Bzl)Thr-Phe-Aha-Phe-Phe-O-CH
2-Ø- resin in 33 ml. freshly degassed DMF was added 3.3 ml. NH
2-NH
2. 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 ml. water to remove all traces of formyl hydrazide and dried in vacuo
overnight to give 1.81 g. of product.
Step c) - Preparation of D-Trp-(E - 2-Cl-CBZ-)Lys-(O-Bzl)Thr-Phe-Aha-Phe-Phe-N3
[0029] D-Trp- (∈ -2-Cl-CBZ)Lys-(O-Bzl)Thr-Phe-Aha-Phe-Phe-NH-NH
2 (1.75 g., 1.39 mmole), prepared by the process set forth in Step b), was suspended
in 20 ml. 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 HC1 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-(∈-2-C1-CBZ)Lys-(O-Bzl)-Thr-Phe-Aha-Phe-Phe-N
3 was used immediately in step d).
Step d) - Preparation of Cyclo[Aha-Phe-Phe-D-Trp-(∈-2-C1-CBZ)Lys-(O-Bzl)Thr-Phe]
[0030] The solution of D-Trp-(E -2-Cl-CBZ) Lys-(O-Bzl)Thr-Phe-Aha-Phe-Phe-N
3 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.
[0031] 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)
[0032] Cyclo[Aha-Phe-Phe-D-Trp-(∈ -2-Cl-CBZ) Lys-(O-Bzl)-Thr-Phe], 1.15 g., obtained by
the process set forth in Step d), was dissolved in 2 ml. 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)
[0033] The cyclo(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.
[0034] 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

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.
[0035] 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.
[0036] 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.
[0037] The following example is included to illustrate the preparation of a representative
dose of cyclo(Aha-Phe-Phe-D-Trp-Lys-Thr-Phe) suitable for subcutaneous injection.
EXAMPLE 2
[0038]
1 ml. sterile saline;
1 mg. cyclo(Aha-Phe-Phe-D-Trp-Lys-Thr-Phe).