CH626328A5 - Process for the preparation of somatostatin analogues - Google Patents

Description

The invention thus relates to the preparation of new somatostatin analogs having a longer action than naturally occurring somatostatin and having the following general formulas:

ÇH -X Y-CH-

I I

R-CH-g-Lys-R "'- Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH-R1

and

CH _X Y-ÇH "

I 2 I,

R-CH-Ç-Lys-R "1-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH-R '

in which:

O

X CH2, S, S, S02 or [X] n (where n = O),

O

Y CH2, S, S, S02 or [Y] n (where n = O),

R H, NH2 or R "NH, where R" is Ala-Gly, a lower acyl radical with 2 to 6 carbon atoms or an aroyl radical with 7 to 21 carbon atoms,

20 R 'H or COOH and

R '"are asparagine, alanine or a-aminobutyric acid, where not both X and Y are heteroatoms.

The somatostatin analogs produced according to the invention differ from somatostatin in that the 25 two adjacent heteroatoms of the disulfide bridge (-S-S-) of the cystine amino acid residue of somatostatin have the following structural formula:

S S

t l

Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH

omitted or by the more stable groups containing C and S, such as —CH2 — CH2—, —S—, —CH2 — S—, —S — CH2—, —CH2—,

9 9 9

-S-, -CH2-S-, -S-CH2-, -S02-, -CH2-S02- or -S02-CH2-, which have no adjacent heteroatoms, are replaced. Examples of the somatostatin analogs according to the invention are those compounds in which Ala-Gly and the amino group of Cys3 of somatostatin are eliminated and, by means of a hydrogen atom, an amino group, a lower acylamino radical (the lower acyl radical having 2 to 6 carbon atoms, as in the case of Acetylamino

group) or an aroylamino radical (the aroyl radical having 7 to 35-21 carbon atoms, as in the case of the benzoylamino group), and those compounds in which a hydrogen atom takes the place of the C-terminal carboxyl group of the cystine. Further examples of the somatostatin analogs according to the invention are those in which 40 the L-tryptophan (Trp) located in the 8-position is replaced by D-tryptophan (D-Trp).

The preferred somatostatin analogs obtainable according to the invention have the following general formulas:

45

CÜ2-X R-CH-C-:

-Y-CH,

-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH-R '

in which: X-Y = X-Y = X-Y = X-Y =

and

; H2-X Y "9H2

R-CH-Ç-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH-R '

Somatostatin analogs are X-Y CH2-CH2, R is a hydrogen atom and R 'is a hydrogen atom or a carboxyl-60 group. The structure of these further preferred somatostatin analogs is thus represented by the formulas and designations below:

CH2-CH2, R = H, R '= H, or CH2-CH2, R = NH2 and R' = COOH or CH2-S, R = H and R '= COOH or S-CH2, R = H and R' = COOH.

In further preferred ch2-ch2-

L

2 ~ g-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr

-6H0-CH

I 2

-Ser-NH-CH-COOH

7

626 328

with the designation Cyclo (cy-Asu-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser) or Des- (Ala ', Gly2) -desamino [Cys3] dicarba3a4-somatostatin;

ch2-ch2-

CH2- <jZ-Lys-Asn-Phe-P-D-Trp-Lys-Thr-Phe-Thr-Ser with the designation Des (Ala1, Gly2) -desamino [Cys3] -dicarba3'14- [D-Trp8] -somatostatin and [D-Trp8] -somatostatin and

CH0-CH0 2 i 2

-NH-CH-

COOH

Ì2 2 H „-C-

CH2 "j: H2

2-C-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH2

called Des (Ala \ Gly2) -desamino [Cys3] -descarboxy [Cys14] -dicarba3,14- [D-Trp8] -somatostatin.

The somatostatin analogs produced according to the invention differ from somatostatin in that the component

1 2 ~ £ s ^ 3

Ala -Gly -Cys "

. 14 C ^ S -OH

of somatostatin is eliminated and by component I:

^ H'2

R-CH-C-

X-

-CH,

-NH-CH-R1

(I)

in which X, Y, R and R 'each have the meaning given above is replaced. The new somatostatin analogs containing a-amino acid, e.g. Des- (Ala1, Gly2) -desamino [Cys3] dicarba3,14-somatostatin, Des- (Ala1, Gly2) -desamino [Cys3] dicarba3,14- [D-Trp8] -

somatostatin and des- (AIa1Gly2) -desamino [Cys3] descarboxy [Cys14] -dicarba3,14- [D-Trp8] -somatostatin,

do not have an N-terminal amino group; one of the groups affected by the enzymatic cleavage of the molecule by aminopeptidases is thus eliminated. The somatostatin analogs containing co-aminoheptanoic acid, e.g. Des- (Ala1, Gly2) -desamino [Cys3] descarboxy- [Cys14] dicarba3,14-somatostatin and 25 Des- (Ala1, Gly2) -desamino [Cys3] descarboxy- [Cys14] dicarba3'14- [D-Trp8] -somatostatin,

do not have a C-terminal carboxyl group, which means that one of the groups affected by the enzymatic cleavage by carboxy-30 peptidases is omitted. By replacing the neighboring heteroatoms of the disulfide bridge of somatostatin with the group -CH2-CH2-, -S-CH2 or —CH2-S—, the stability of the analogue in question is also made. Compound increased in vivo by slowing enzymatic degradation through reducing cleavage. Therefore, these analog compounds are more resistant to cleavage in vivo and thus have a longer-term effect.

The following new, protected or blocked peptide intermediates which are suitable for producing the somatostatin analogs according to the invention are also used in the process according to the invention:

rH2 "x

R-CH-Ç- (£ -INOC) Lys-Asn-Phe-Phe-Trp- (^ f-INOC) Lys-Thr-

Y-CH-,

Phe-Thr-Ser-NH-CK-R '

R-CH-g- (£ -INOC) Lys-Asn-Phe-Phe-D-Trp- (€. -INOC) Lys-Thr-

V

x

Phe-Thr-Ser-NH-CH-R '

ch2-x-

CH2 ~ Ç- (é. -INOC) Lys-Asn-Phe-Phe-OMe

CH,

BOC Trp

- (éZ -INOC) Lys-Thr-Phe-Thr-Ser-NH- ^ H-R1

626 328

fH2

CH "-X"

|

CH ç- (£ -INOC) Lys-Asn-Phe-Phe-OMe O

BOC-D-Trp- (£ -INOC) Lys-Thr-Phe-Thr-Ser-NH-CH-R ÇH2-X Y-CH,

l2 *

CH-j-Ç- (6-INOC) Lys-Asn-Phe-Phe-CMe NH ^ CH-R '

In the above formulas, X, Y, R and R 'each have the meaning given above, while R "" is a hydrogen atom or -COO-tert-butyl.

Amino acid components used in the process according to the invention, certain preferred protective groups, amino acid activation groups, condensing agents, reagents and solvents are indicated by the following short names:

Short name

amino acid

Lys

L-lysine

Asn

L-asparagine

Phe

L-phenylalanine

Trp

L-tryptophan

D-Trp

D-tryptophan

Thr

L-threonine

Ser

L-serine

Asu a-amino acid

Short name

Protecting groups

INOC

Isonicotinyloxycarbonyl

BOC

tert-butyloxycarbonyl

OMe

Methyl ester tBu tert-butyl

CBZ

Benzyloxycarbonyl

Short name activating groups

NPE

p-nitrophenyl ester

HSE

N-hydroxysuccinimide ester

HBT

1-hydroxybenzotriazole

15

20th

Short name

Reagents

TFA

Trifluoroacetic acid

TEA

Triethylamine

DIPEA

Diisopropylethylamine

Short name

solvent

EPAW

25 BAW CMW DMF THF

Ethyl acetate / pyridine / acetic acid / water

Butanol / acetic acid / water chloroform / methanol / water dimethylformamide tetrahydrofuran

30th

35

40

The new somatostatin analogs are produced by block synthesis using the process according to the invention. Different peptide segments of the somatostatin analog are produced separately and then linked in the desired order to produce the open-chain form of the desired compound. The open chain connection is then subjected to a ring closure and freed from the protective groups. If desired, the sulfide analog is oxidized to sulfoxide or sulfone, thereby obtaining those compounds in which -X-Y- is a group

Short designation DCCI

Condensing agent dicyclohexylcarbodiimide

O

—Ì- or S02 included. The overall method for producing the somatostatin analogs according to the invention, in which X, Y and R 'each have the meaning given above and R represents a hydrogen atom, is shown schematically in Table 45, Table I. In the first three process steps, this table explains a possible production of starting materials and then the production of somatostatin analogs containing L-tryptophan; it should be noted that analogs containing D-tryptophan can be prepared according to the Me-50 method shown in Table I, with the exception that D-tryptophan is used instead of L-tryptophan.

-Y-CH,

Table I

H "-COOCH-, NH" -CH-R '

tert-butyl acetate, HCIO ^ when R '= COOH

f

H2. "X"

-Y-CH,

CH2-COOCH3 HCl "« 2

I '

NH "-CH-R" "

V

626 328

^ j tert-butyltrichlorophenyl carbonate; 2)! NaOH

: k2-x -: h2-cooh

-Y-CH,

BOC-NH-CH-R "" DCCI / p-nitrophenol

CH2-X-

x>

ch0-c /

2 \

-y-ch,

BOC-NH-CH-R '

° ~ A / ~ N ° 2

(é-INOC) Lys-Asn-Phe-Phe-OMe * 2HC1

çh2-x

1

ch0-ç- (é-ln

-y-ch,

INOC) Lys-Asn-Phe-Phe-OMe BOC-NH-CH-R ""

: 1) TFA;

2) BOC-Trp- (£ -INOC) Lys-Thr-Phe-Thr-Ser-N,

f hj-x-

CH2 ~ Ç- {£ -INOC) Lys-Asn-Phe-Phe-OMe

:H.

BOC-Trp- (£ -INOC) Lys-Thr-Phe-Thr-Ser-NH-CH-R ,:

1) NH2NH2;

2) KCl;

3) isoamyl nitrite

î

: h2-x-

CH, -Ç- (£ -INOC) Lys-Asn-Phe-Phe-Trp- (é-INOC) Lys-Thr- Y-CH-

I.

Phe-Thr-Ser-NH-CH-R *

Zn

?

: h2-x-

-Y-CH,

R-CH-C-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH-R '

626 328

10th

In the above formulas, X, Y and R 'each have the meaning given above, R represents a hydrogen atom and R "" is hydrogen or the group C.

/ 0

'OtBu and the amino acid Trp can be replaced by D-Trp.

If the group —X — Y— includes a group -S— or -S02-, an additional oxidation step using a suitable oxidizing agent such as performic acid or hydrogen peroxide is required.

The somatostatin analogs in which X, Y and R 'each have the meaning given above and R is -NH2 5 and R "NH can be prepared by the process shown in Table II. Table II illustrates the synthesis of somatostatin analogs containing L-tryptophan It should be noted that analogs containing D-tryptophan can be prepared according to the procedure shown in Table II, with the exception that 10 D-tryptophan is used instead of L-tryptophan.

Table II

fH2-

X-

ÇH-

cbz-nh-ck-cooh.

nh2-ch-r *

tert-butyl acetate, HCIO ^ when R '= COOH

:H;

• X

T:

cbz-nh-ch-cooch3 hcl * nh2-ch-r ""

i 1.) tert. -Butyltrichlorophenyl carbonate; 2) NaOH

:H;

-X-

:H.

CBZ-NH-CH-COOH BOC-NH-CH-R ""

DCCI / p-N itrophenol

CR ~ X

boc-nh-ch-r ""

(é-INOC) Lys-Asn-Phe-Phe-OMe ^ 2HC1

f

H2-x-

-y-çh f

CBZ-NH-CH-Ç- (é-INOC) Lys-Asn-Phe-Phe-OMe BOC-NH-CH-R ""

1) TFA;

2) BOC-Trp- (é-INOC) Lys-Thr-Phe-Thr-Ser-N,

11

626 328

CBZ-NH-CH-Ç- (é. -INOC) Lys-Asn-Phe-Phe-OMe

<fH2

BOC-Trp- (é: -INOC) Lys-Thr-Phe-Thr-Ser-NH-CH-R1

1) NH2NH2;

2) HCl;

v 3) isoamylriitrite f

H - X-

cbz-nh-ch - ^ - (£ -inoc) Lys-Asn-Phe-Phe-Trp - inoc) Lys-Thr- ¥ -ch "

Phe-Thr-Ser-NH-CH-R *

f

H2-x-

hf y

NH2 ~ CH-g- (£ -INOC) Lys-Asn-Phe-Phe-Trp- (-INOC) Lys-Thr- Y-CH-

Phe-Thr-Ser-NH-CH-R1

VK

Acetyl-A, Benzoyl-A or CBZ-Ala-Gly-A and HF Zn

î

H2-x-

• y-ch,

r "-nh-ch-c-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-nh-ch-r '

In the above formulas, X, Y, R ', R "and R" "each have the meaning given above, A represents any suitable known group for forming an amide bond (such as a halogen atom or an anhydride or active ester group) and the amino acid Trp can be replaced by D-Trp. qu

If the group -X-Y- contains the group -S- or -S02-, an additional oxidation step with a suitable oxidizing agent such as performic acid or hydrogen peroxide is required.

The starting compounds of the following general formula used for the process shown in Table I:

X-

CH,

ch2-cooch3

nh2-ch-r '

Jost and F. Sorm, Collect. Czech. Chem. Comm. 36 (1971), page 238.

The starting materials of the general formula below used for the process shown in Table I:

ch;

-X

-ch,

ss ch2-cooch3

nh2- ch-r '

60

in which R 'is a hydrogen atom and -X-Y CH2-CH2- are known from the following references: C.F.

Horn et al., Angew. Chem. 74 (1962), page 531 and Chemical Abstract, volume 57, 11411 (c).

The starting compounds of the following general formula used for the process shown in Table II:

in which R 'denotes COOH and -X-Y CH2-S- or -S-CH2- are described in the following publications: K. Jost, Collect. Czech. Chem. Comm. 36 (1971), page 223 and K.

T'd

X-

:H.

cbz-nh-ch-c00ch,

nh2-ch-r '

626 328

12th

in the R 'COOH and —X-Y CH2-S- or —S-CH2- mean- all other amino acid required according to the invention

ten, are described in the following publications: K. Jost derivatives (see Tables I and II), with the exception of and F. Sorm, Collect. Czech. Chem. Comm. 36 (1971), page from Table II intermediate products of the next-238 and K. Jost and J. Rudinger, Collect, Czech. Chem. General formula:

Comm. 32 (1967), page 2488. s ch;

-X-

cbz-nh-ch-cooh

-CH,

boc-nh-ch-c

^ DtBu in which -X-Y CH2-CH2- is also known. The intermediates mentioned can be obtained by electrolytic oxidative decarboxylation (according to Kolbe) of two molecules

CH2-CH2-COOH

of suitably protected glutamic acid are produced according to the following reaction scheme hooc-ch2-ch2

CBZ-NH-CH-COOMe (IV)

BOC-NH-CH-C

'OtBu

(V)

V

ÇH ^ CHj-

Kolbe ch - çh

CBZ-NH-CH-COOMe boc

2 f'V

-nh-ch-cç

UtBu

(IV + V) +

Î

H2-CH2 "

CB Z-NH-CH-COOMe

(IV + IV) +

- CH2- ^ 2

CBZ-NH-CH-COOME

CH2-CH2

BOC-NH-

UtBu

-ch2-ch2

BOC-NH-CH-C

OtBu

(V + V)

diluted base

Acid ch2-ch2-

■ ch2-ch2

cbz-nh-ch-cooh boc

L /

-nh-ch-c ^

T3tBu

(IV + V) 1 +

CH ^ CH ^ -

-CH2-ÇH2

cbz-nh-ch-cooh cbz-nh-ch-cooh

(IV + IV) '

13

626 328

ch2-c%

boc-nh-Ch boc-nh ch2-çh2

-LS

OtBu

The desired monocarboxylic acid (IV + V) 'can be obtained from The compound (IV + V)' is shown in Table II

the dicarboxylic acid (IV + IV) 'and the neutral compound (V + V)' easily by conventional methods.

presented method for the production of ch2-ch2

nh.

r

, -ch-c-

-ch0-ch0 2 | 2nd

-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH-COOH

and

Î

K2 ~ CE2

NH0-CH-C-Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser ch ~ -ch0 2 î 2

-nh-ch- '

used cooh.

The peptide segments shown in Tables I and II (£ -INOC) -Lys-Asn-Phe-Phe-OMe • 2HC1, BOC-Trp- (e-. INOC) -Lys-Thr-Phe-Thr-Ser-N3 and BOC-D-Trp- (e-INOC) -Lys-Thr-Phe-Thr-Ser-N3 are themselves expediently prepared by stepwise synthesis in solution using the HSE, NPE or HBT active ester method or the azide method.

When performing these step-by-step or block syntheses, which are based on the reaction of a carboxyl group (or activated carboxyl group) of an amino acid with an amino group of another amino acid, the amino groups of the amino acid or peptide that react on the terminal carboxyl group of the molecule become as well as other functional groups of both reaction components which are reactive under the conditions of such syntheses are preferably protected. The protective groups must retain their protective action under the conditions of the peptide linkage and must be able to be split off selectively without influencing the peptide bonds. The protective groups to be split off after a certain level must also be able to be selectively separated without other protective groups to be retained for later linkage levels being recorded.

Examples of commonly used amino protecting groups are acyl-type substituents such as the formyl, phthalyl, trifluoroacetyl, toluenesulfonyl, dibenzylphosphoryl, nitrophenylsulfenyl, tritylsulfenyl or o-nitrophenoxyacetyl group, and urine substituents, such as the benzyloxycarbonyl, (carbobenzoxy), p-methoxycarbobenz-oxy, p-nitrocarbobenzoxy, tert-butyloxycarbonyl, 2- (p-bi-phenylyl) -2-isopropyloxycarbonyl or isonicotinyloxycarbo-nyl group, and substituents from Alkyl type, such as the triphenylmethyl group, trialkylsilyl radicals or the trimethylsilyl group. The preferred protecting group for the e-amino group of lysine is the isonicotinyloxycarbonyl group. To protect the a-amino group of the amino acids (or peptides) participating in the reaction at the carboxyl end of the molecules, the tert-butyloxycarbonyl group (BOC) is preferably used, since this occurs after the reaction mentioned and before the subsequent step (where the a-amino group itself reacts) easily due to relatively mild exposure to acid (e.g. with

With the help of trifluoroacetic acid or HCl in ethyl acetate), whereby the groups serving to protect other amino groups (such as the basic amino group of lysine), such as the carbobenzoxy group (CBZ) and the isonicotinyloxycarbonyl group, are not significantly attacked. The carbobenzoxy group can be split off with the help of hydrogen bromide in glacial acetic acid or hydrogen fluoride, whereby the Isoni-35 cotinyloxycarbonyl group is not attacked significantly. Elimination with hydrogen fluoride is facilitated by the presence of anisole. The isonicotinyloxycarbonyl group can be split off with the help of zinc.

The protection of the carboxyl groups is usually u. a. 40 by forming amides, salts or esters, such as the methyl and ethyl esters (which is preferred if a subsequent conversion via the hydrazide to the azide is desired), the benzyl, p-nitrobenzyl or tert-butyl esters . The hydroxyl groups are generally not protected in the synthesis of the somatostatin analogs according to the invention, in which the linking reactions take place in solution. If necessary, however, to protect the hydroxyl groups e.g. use a tetrahydropyranyl, benzyl, trifluoroacetyl or tert-butyl group.

The choice of protective groups is determined in part by the special linking or coupling conditions and in part by the amino acid and peptide components participating in the implementation.

Detailed guidelines for the selection of certain protective groups to be used according to the invention can be found in FR-PS 1 496 536; reference is expressly made here to the protective groups described in this patent.

An overall method for making the preferred 60 new somatostatin analogs

Des- (Ala1, Gly2) -desamino [Cys3] dicarba3'14-

somatostatin and Des- (Ala1, Gly2) desamino [Cys3] dicarba3'14- [D-Trp8] somatostatin 65 is shown schematically in Table III. This table illustrates the preparation of a somatostatin analog containing L-tryptophan; it should be noted that the compound containing D-tryptophan is as shown in Table III

626 328

14

Method can be produced with the exception that in step 5a BOC-D-tryptophan-HSE is used instead of BOC-L-tryptophan-HSE.

The remaining new somatostatin analogs can be prepared analogously to the method shown in Table III, by using a compound of the general formulas VI and VII instead of the a-amino acid used in step 10:

V '

x-

-ch.

ch2-cooch3

nh2-ch-r *

ch;

-x-

-ch.

cbz-nh-cooch3 nh2-ch-r '

(vi)

(vii)

in which X, Y and R 'each have the meaning given above, and carry out the reactions described in steps 10 to 15 and 17 to 19. If RR "is, however, further synthesis steps for the condensation of the N-terminal amine with an acetyl or benzoyl group or with CBZ-Ala-Gly are required. In the latter case, the protective group of the a-amino group of Ala must be split off in an additional step Furthermore, in that case,

O

10 If the group —X-Y- contains a sulfoxide group, -S- or —S02—, oxidation is carried out as a further step using a suitable oxidizing agent (such as performic acid or hydrogen peroxide). The corresponding D-tryptophan-analogs can be prepared by using BOC-D-tryptophan-HSE instead of BOC-L-tryp-tophan-HSE in stage 5a.

Table III

Phe-OMe / m> | a) BOC-PheHSE; b) HCl Phe-Phe-OMe • HCl 7 1 a) BOC-AsnNPE; b) HCl Asn-Phe-Phe-OMe • HCl

8th

a) a-BOC- (e-INOC) LysHBT;

b) HCl a-amino acid 9 | CH3OH, HCl 10 X tert-butyl acetate, HCIO4 a-tert-butyl-cu-methyl-aaminosuberate 11 | tert-butyltrichlorophenyl carbonate

12 l NaOH

a-tert-butyl-BOC-a-aminosuberate

13 l DCCI / p-nitrophenol a-tert-butyl- <o-p-nitrophenyl BOC-a-aminosuberate

14 I (f-INOC) Lys-Asn-Phe-Phe-OMe • 2HCK a-tert-butyl-BOC-cü-Asu- (£ -INOC) Lys-Asn-Phe-Phe-OMe

15 1TFA

ai -Asu- (f-IN OC) Lys-Asn-Phe-Phe-OMe • 2TFA

17 i BOC-Trp- (£ -INOC) Lys-Thr-Phe-Thr-Ser-N3 <=

BOC-Trp- (£ -INOC) Lys-Thr-Phe-Thr-Ser-cu-Asu (£ -INOC) Lys-Asn-Phe-Phe-OMe

18 4 a) NH2NH2; b) HCl; c) isoamyl nitrite

Cyclo [co-Asu- (£ -INOC) Lys-Asn-Phe-Phe-Trp- (£ -INOC) Lys-Thr-Phe-Thr-Ser]

19 iZn

Des (Ala1, Gly2) desamino [Cys3 "dicarba3'14-somatostatin

Ser-OMeHCl

1 4 a) BOC-ThrHSE; b) TFA Thr-Ser-OMe • TFA

2 4 a) BOC-PheHSE; b) TFA Phe-Thr-Ser-OMe • TFA

3 4 a) BOC-ThrHSE; b) HCl Thr-Phe-Thr-Ser-OMe • HCl

4 4 a) a-BOC- (£ -INOC) LysHBT; b) TFA (£ -INOC) Lys-Thr-Phe-Thr-Ser-OMe • 2TFA

5 4 a) BOC-TrpHSE; b) NH2NH2 BOC-Trp- (£ -INOC) Lys-Thr-Phe-Thr-Ser

16

Isoamyl nitrite

H2NHN

In Table III, the numbers to the left of the arrows each relate to the exemplary embodiment from which the conditions specified in the conversion mentioned are apparent.

The overall method mentioned is based on combinations of a step and a block synthesis, with certain peptide segments of

Des (AIa \ Gly2) -desamino [Cys3] -dicarba3'14-somatostatin and

Des- (Ala1, Gly2) -desamino [Cys3] -dicarba3'14-

[D-Trp8] somatostatin was initially prepared by the step method by successive synthesis in solution, these segments were then linked in the correct order and the two ends of the peptide obtained were combined to form the desired cyclic peptide. In this method, the BOC substituent serves to protect the a-amino groups, the isoni-cotinyloxycarbonyl group (INOC) to protect the £ -amino group of lysine and the methyl ester group to protect the carboxyl group of phenylalanine, phenylalanylphenylalanine, asparaginylphenylalanylphenylalanine, (E- ) -Lysylasparaginylphenylalanylphenylalanin and a-tert-butyl ester-BOC-to-Asu- (£ -ÌNOC) -lysyl-asparaginylphenylalanyïphenylalanin.

45 In the case of "-tert.-butyl ester-BOC-o" -Asu- (£ -INOC) -lysyI-asparaginylphenylalanylphenylalanin-OMe, the methyl ester is also used to provide the intermediate for the preparation (via the hydrazide) of Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-a-Asu (g-INOC) -50 lysylasparaginylphenylalanylphenylalaninazid or D-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-ai-Asu - (£ -INOC) -

lysylasparaginylphenylalanylphenylalaninazid.

In addition to this preferred method, the invention also encompasses various modifications or alternative methods and the use of other protective groups which meet the aforementioned criteria. The alternative methods are also based on step-by-step syntheses in solution as well as combinations of step-by-step and block syntheses.

As can be seen from Table III, an overall method involves the production of

Des- (Ala1, Gly2) -desamino [Cys3] dicarba314-

somatostatin or Des- (Ala1, Gly2) -desamino [Cys3] dicarba3-14- [D-Trp8] -somatostatin 65 specifically (a) the step-by-step synthesis of the N-terminal He-xapeptide segments BOC-Trp- (£ -INOC) - Lys-Thr-Phe-Thr-Ser-azide and BOC-D-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-azide and (b) the C-terminal pentapeptide segment m-

15

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Aus (e-INOC) -Lys-Asn-Phe-Phe-OMe, (c) the condensation of the peptides produced in steps (a) and (b) to form the peptide BOC-Trp- (£ -INOC) -Lys- Thr-Phe-Thr-Ser-oj-Asu-

£ -INOC) -Lys-Asn-Phe-Phe-OMe or BOC-D-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-a »-Asu-

f-INOC) -Lys-Asn-Phe-Phe-OMe,

(d) the conversion of -OMe to the hydrazide, the elimination of the BOC protective group and (f) the cyclization and deprotection of the undecapeptide obtained, the new somatostatin analogue Des- (Ala1, Gly2) -desamino [Cys3] dicarba3 , 14-

somatostatin or Des- (Ala1, Gly2) -desamino [Cys3] dicarba3,14-

[D-Trp8] somatostatin is obtained.

The N-terminal hexapeptide segment is produced by reacting Ser-OH • HCl with BOC-ThrHSE. The Ser-OMe hydrochloride is converted into the free amine using a weak base, which is reacted with BOC-ThrHSE in a suitable organic solvent. To carry out the reaction, the solutions are combined and the mixture is stirred at room temperature and approximately neutral pH, but preferably at a pH of 6.8 to 8, under which conditions the reaction is usually complete after about 4 hours. The product is then extracted with water and the solution is evaporated to dryness. The residue is dissolved in a suitable organic solvent and the insoluble salt is filtered off. The crude product is purified by chromatography, whereby essentially pure BOC-Thr-Ser-OMe is obtained. This dipeptide is reacted with TFA, which cleaves the BOC substituent to form Thr-Ser-OMe TFA.

The dipeptide Thr-Ser-OMe ■ TFA is converted with the help of a weak base into the free amine, which is reacted with a slurry of BOC-PheHSE in a suitable organic solvent. To carry out the reaction, the reactants are vigorously agitated for 2 hours to overnight at room temperature. The reaction solution is then washed with water and the organic layer is evaporated to dryness. The crude product obtained is purified by chromatography; essentially pure BOC-Phe-Thr-Ser-OMe is obtained. This tripeptide is converted to the essentially pure tripeptide Phe-Thr-Ser-OMe • TFA by reaction with TFA.

The tripeptide Phe-Thr-Ser-OMe - TFA is converted into the free base using a weak base, which is reacted with BOC-ThrHSE in a suitable organic solvent. To carry out the reaction, the reactants are vigorously agitated for about 1 hour to overnight at room temperature. The precipitated crude product is isolated and reprecipitated from methanol / ether; the essentially pure tetrapeptide BOC-Thr-Phe-Thr-Ser-OMe is obtained. The tetrapeptide is reacted with anhydrous hydrogen chloride in ethyl acetate, whereby the BOC substituent is split off; Thr-Phe-Thr-Ser-OMe hydrochloride is obtained.

a-BOC- (£ -INOC) -Lys-OH is reacted with N-hydroxybenzotriazole monohydrate in the presence of DCCI. The ct-BOC- (e-INOC) -LysHBT obtained is reacted with the tetrapeptide Thr-Phe-Thr-Ser-OMe hydrochloride. To this end, the reactants are allowed to react in a suitable organic solvent, such as freshly degassed DMF / methylene chloride, for a period of about 1 hour to overnight. The insoluble constituents are then filtered off and the filtrate is concentrated considerably. The product is precipitated by adding petroleum ether. The crude product is purified by chromatography; the essentially pure protected pentapeptide a-BOC- (E-INOC) -Lys-Thr-Phe-Thr-Ser-OMe is obtained. The protected pentapeptide is reacted with TFA, which cleaves the BOC substituent. The pentapeptide (£ -INOC) -Lys-Thr-Phe-Thr-Ser-OMe • 2TFA is obtained.

The pentapeptide (e-INOC) -Lys-Thr-Phe-Thr-Ser-OMe -2TFA is converted into the free base with the help of a weak base, which is dissolved in a suitable organic solvent (such as DMF) at approximately neutral pH ( preferably at about 7.5 to about 7.1) with BOC-D-TrpHSE or BOC-TrpHSE. Under the conditions mentioned, the reaction is complete after stirring the mixture overnight. The product is then precipitated by adding ether. The crude product is chromatographed; the essentially pure protected hexapeptide BOC-Trp- (e-INOC) -Lys-Thr-Phe-Thr-Ser-OMe or BOC-D-Trp- (e-ESIOC) -Lys-Thr-Phe-Thr is obtained -Ser-OMe. The protected hexapeptide is reacted with anhydrous hydrazine in methanol, whereby essentially pure BOC-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-hydrazide or BOC-D-Trp- (e-INOC) - Lys-Tfir-Phe-Thr-Ser hydrazide obtained.

The C-terminal tetrapeptide (£ -INOC) -Lys-Asn-Phe-Phe-OMe • 2HC1 is produced by reacting Phe-OMe-hydrochloride with BOC-PheHSE. The reaction is carried out overnight in the alkaline pH range in the presence of a suitable organic solvent, such as chloroform or methylene chloride. The insoluble parts are then separated off. The organic solution is evaporated to dryness and the residue is recrystallized. The protected dipeptide is reacted with anhydrous hydrogen chloride in ethyl acetate, whereby the BOC substituent is split off to form Phe-Phe-OMe-hydrochloride.

The dipeptide Phe-Phe-OMe hydrochloride is reacted with BOC-AsnNPE overnight in the presence of a suitable organic solvent (such as DMF) at neutral pH (preferably at a pH of about 7.2). The reaction solution is then evaporated and the residue is dissolved in chloroform. The organic solution is washed and evaporated to dryness. By recrystallization of the residue, essentially pure BOC-Asn-Phe-Phe-OMe is obtained. This protected tripeptide is reacted with anhydrous hydrogen chloride in ethyl acetate, whereby the BOC substituent is split off; this gives Asn-Phe-Phe-OMe hydrochloride.

a-BOC- (£ -INOC) -Lys is reacted with 1-hydroxybenzotriazole (HBT) in the presence of DCCI in a suitable organic solvent (such as DMF) to a-BOC- (£ -INOC) -LysHBT. The solution obtained is mixed with Asn-Phe-Phe-OMe hydrochloride. Then the mixture is adjusted to an approximately neutral pH and stirred overnight. The dicyclohexylurea is then filtered off from the reaction mixture and the filtrate is evaporated to dryness. The residue is suspended in a suitable organic solvent and then filtered off. The protected tetrapeptide is reacted with anhydrous hydrogen chloride in ethyl acetate, as a result of which the BOC substituent is split off; one obtains (£ -INOC) -Lys-Asn-OMe-dihydrochloride.

a-tert.-Butyl- (wp-nitrophenyl ester-BOC-a-amino-suberate is prepared by first a-amino-coric acid for 30 min at 25 ° C with a methanol solution of anhydrous HCl to œ-methyl- a -aminosuberat • HCl The hydrochloride is reacted in tert-butyl acetate with perchloric acid by extraction of the Reak5

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16

tion solution at neutral pH with an organic solvent gives a-tert-butyl - &> -methyl-a-amino-SUBATE.

The a-amino group of the a-tert-butyl-a> -methyl-a-amino-suberate is protected by conversion into the BOC derivative, for which purpose the compound is reacted with tert-butyltrichlorophenyl carbonate. The reaction is carried out overnight at 25 ° C. in the presence of a suitable organic solvent (such as DMF) in the weakly alkaline pH range from about 8 to 9 (preferably at a pH of 8.5). The reaction solution is then evaporated to dryness. The residue is dissolved in the smallest possible amount of a suitable organic solvent, the solution is washed with dilute acid and base and the organic solvent is evaporated off. Essentially pure a-tert-butyl-cy-methyl-N-BOC-a-amino-suberate is obtained.

The w-methyl ester group of the a-tert.-butyl-cy-methyl-N-BOC-a-aminosuberate is selectively saponified by reaction for 1 4 hours at 25 ° C. with lN NaOH in dioxane. The organic solvent is then evaporated off, the remaining solution is acidified and extracted with a suitable organic solvent. The crude product obtained in the solvent evaporation is purified by chromatography; this gives pure a-tert-butyl-BOC-a-aminosuberate.

The üj - carboxyl group of the a-tert-butyl-BOC-a-aminosu-berate is converted into an active ester group by reaction with p-nitrophenol in the presence of DCCI. The solid fractions are then filtered off and the filtrate is strongly concentrated. The desired a-tert-butyl-co-p-nitrophenyl-BOC-a-aminosuberate is obtained in the form of an oil.

A solution of (e-INOC) -Lys-Asn-Phe-Phe-OMe-dihydrochloride is in the alkaline pH range (preferably at a pH of about 8) overnight with a-tert-butyl-wp -nitro-phenyl-a-aminosuberat implemented. The reaction solution is then evaporated and the residue is purified by chromatography; the essentially pure pentapeptide a-tert-butyl-BOC-w-Asu (£ -INOC) -Lys-Asn-Phe-Phe-OMe is obtained.

The protected pentapeptide is freed from both the a-N-BOC and the a-tert-butyl group by reaction with TFA; this gives w-Asu (f-INOC) -Lys-Asn-Phe-Phe-OMe • 2TFA.

A solution of the pentapeptide a> -Asu- (t-INOC) -Lys-Asn-Phe-Phe-OMe • 2TFA is mixed with a cold solution of BOC-Trp- (£ -INOC) -Lys-Thr-Phe-Thr- Ser-N3 or BOC-D-10 Trp- (e-INOC) -Lys-Thr-Phe-Thr-Ser-N3, which by reaction of BOC-Trp- (£ -INOC) -Lys-Thr-Phe-Thr -Ser-NHNH2 or BOC-D-Trp- (e-INOC) -Lys-Thr-Phe-Thr-Ser-NHNH2 with isoamyl nitrite freshly prepared at -25 ° C, to the new protected undecapeptide 15 BOC-Trp- (e -INOC) -Lys-Thr-Phe-Thr-Ser- {o-Asu- (£ -INOC) -Lys-Asn-Phe-OMe or BOC-D-Trp- (£ -INOC) -Lys-Thr-Phe -Thr-Ser-cü-Asu- (£ -INOC) -Lys-Asn-Phe-Phe-OMe Lys-Asn-Phe-Phe-OMe 20 implemented.

The undecapeptide BOC-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser - «> - Asu- (£ -INOC) -

Lys-Asn-Phe-Phe-OMe or BOC-D-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-a> -Asu-25 (£ -INOC) -Lys-Asn-Phe- Phe-OMe is cyclized by creating a peptide bond between the carboxyl group of phenylalanine and the a-amino group of L-tryptophan or D-tryptophan. The ring closure is carried out by (a) converting the 30 —OMe group into a hydrazide group by reaction with a methanol solution of hydrazine, (b) converting the BOC protective group of L-tryptophan or D-tryptophan by reacting the undecapeptide hydrazide with anhydrous HCl in the presence of mercaptoethanol, where-35 at man

Trp- (e-INOC) -Lys-Thr-Phe-Thr-Ser-a> -Asu- (£ -INOC) -

Lys-Asn-Phe-Phe-hydrazide-4HCl or D-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-w-Asu- (£ -INOC) -Lys-Asn-Phe-Phe- hydrazide • 4HC1 40 with the structure:

f

H2 "" CH2

ch2-,

L

4HC1

§- (& -INOC) Lys-Asn-Piie-Pne-NHNH2 £ H • 4hc

I 2 iJ

Trp - (& - INOC) Lys-Thr-Phe-Thr-Ser-NH-CH-C ^

NîH

or

<jH2-CHs CH2]

CH2-C - INOC) Lys-Asn-Phe-Phe-NHNH2 * 4HC1

oil 2 X)

D-Tro {Ç. -INOC) Lys-Thr-Phe-Thr-Ser-NH-CH-C '

uh is obtained and (c) the desired cyclic peptide is produced by converting the hydrazide group into an azide group by reaction with isoamyl nitrite at −25 ° C. in the strongly acidic pH range, the solution with a suitable organic solvent to a concentration of about 1 mg / ml diluted to favor cyclization over polymerization, and adjusted to a weakly alkaline pH to release the a-amino group of L-tryptophan or D-tryptophan and to initiate the ring closure reaction. The ring closure is complete after the batch has been left at −20 ° C. for about 4 days.

The solvent is then evaporated off and the rear edge is overturned from methanol / ethyl acetate; this gives 60 the essentially pure, protected cyclic undecapeptide

Cyclo [tu-Asu- (f-INOC) -Lys-Asn-Phe-Phe-Trp- (£ -INOC) -

Lys-Thr-Phe-Thr-Ser] or Cyclo [(u-Asu- (f-INOC) -Lys-Asn-Phe-Phe-D-Trp-65 (£ -INOC) -Lys-Thr-Phe-Thr -Ser].

The protected undecapeptide is treated in 50% aqueous acetic acid with freshly activated zinc dust, whereby the -INOC substituents of the lysine building blocks are split off

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be. The unreacted zinc is filtered off from the mixture and the crude product contained in the filtrate is purified by chromatography; the new somatostatin analogs are obtained

Des- (Ala ', Gly2) -desamino [Cys3] dicarba3-14-

somatostatin or Des- (Ala1, Gly2) -desamino [Cys3] dicarba3'14-

[D-Trp8] somatostatin in essentially pure form.

Further new somatostatin analogs according to the invention can be prepared analogously to the method described above, the equivalent amount of a compound of the following general formula being used instead of a-aminocorkic acid:

R-CH-COOH NH2-CH-R '

in which X, Y, R and R 'each have the meaning given above.

If L-tryptophane is mentioned in the descriptions and examples, this can always be replaced by D-tryptophan.

Production of Thr-Ser-OMe • TFA stage a

A mixture of 3.50 g Ser-OMe • HCl and 7.47 g BOC-ThrHSE in 70 ml methylene chloride is stirred in a 250 ml round bottom flask. The solution is then adjusted to a pH of 7 with triethylamine and the batch is stirred for 90 minutes. The reaction mixture is then extracted three times with 75 ml of water each time. The aqueous extracts containing the product are combined and re-extracted once with 45 ml of methylene chloride. Freeze drying gives 14.2 g of the crude product. It is dissolved in 200 ml of 10% methanol (in ether) and the salt is filtered off from the solution. The crude product obtained is applied to a column containing 725 g of silica gel 60 and eluted with a chloroform / methanol / water mixture (80: 20: 2). Those fractions which contain the product on the basis of thin layer chromatography are combined and evaporated to dryness. The residue is dissolved in water. Freeze drying gives 6.2 g of BOC-Thr-Ser-OMe.

Stage b

5.9 g of BOC-Thr-Ser-OMe are suspended in 55 ml of trifluoroacetic acid at 0 ° C. The solid goes into solution by stirring for 10 minutes at room temperature. The solution is stirred for a further 3 minutes, then cooled in an ice bath and the product is precipitated by adding 230 ml of ether and cold petroleum ether. The solid is filtered off, washed out with a mixture of equal parts of ether and petroleum ether and dried in vacuo overnight. 6.2 g of Thr-Ser-OMe - TFA are obtained.

Production of Phe-Thr-Ser-OMe • TFA stage a

6.2 g of Thr-Ser-OMe TFA and 6.7 g of BOC-Phe-HSE are suspended in 100 ml of methylene chloride. After adjusting the pH to 7 with triethylamine, the reaction mixture is stirred for 2 hours at 25 ° C. and then extracted three times with 100 ml of water each time. The precipitated crude product is filtered off. The combined aqueous solutions are re-extracted with 100 ml of methylene dichloride and the extracts are combined, dried over magnesium sulfate and filtered.

When the filtrate is evaporated to dryness, further crude product is obtained. The combined crude product (7.9 g) is dissolved in the smallest possible amount of a chloroform / isopropanol mixture (95: 5) and the solution is added to a column with 1200 g of silica gel 60. It is eluted with chloroform / isopropanol (95: 5) until no further product runs off. The mixture is then eluted with a chloroform / methanol / water mixture (90: 10: 1), the product (identified by thin layer chromatography) running out. The fractions containing the product are combined and evaporated to dryness. 6.73 g of BOC-Phe-Thr-Ser-OMe are obtained.

Stage b

6.63 g of BOC-Phe-Thr-Ser-OMe are suspended in 67 ml of trifluoroacetic acid. The suspension is stirred at 25 ° C. until completely dissolved. The solution is then cooled to 0 ° C. and the product is precipitated by adding 300 ml of ether and 600 ml of petroleum ether. The product is filtered off, washed with ether / petroleum ether and dried in vacuo. 6.4 g of essentially pure Phe-Thr-Ser-OMe TFA are obtained.

Production of Thr-Phe-Thr-Ser-OMe • TFA

Stage a

6.4 g of Phe-Thr-Ser-OMe • TFA and 4.6 g of BOC-Thr-HSE are suspended in 100 ml of methylene dichloride. After adjusting the pH to 7 with triethylamine, the reaction mixture is stirred at 25 ° C. for 90 minutes. The crude product is then filtered off, partially dissolved in 50 ml of methanol and precipitated by adding 450 ml of ether. The partially purified product is filtered off and the reprecipitation process is repeated four times. 6.04 g of essentially pure BOC-Thr-Phe-Thr-Ser-OMe are then obtained.

Stage b

5.93 g of BOC-Thr-Phe-Thr-Ser-OMe are suspended in 45 ml of ethyl acetate. The suspension is cooled to 0 ° C. in an ice bath and a strong stream of anhydrous hydrogen chloride gas is passed in for 9 minutes and then a stream of nitrogen for 4 minutes. 170 ml of ether and 285 ml of petroleum ether are then added to completely precipitate the product. The product is filtered off, washed out with an ether / petroleum ether mixture and dried in vacuo. 5.66 g of Thr-Phe-Thr-Ser-OMe HCl are obtained.

Production of (e-INOC) -Lys-Thr-Phe-Thr-Ser-OMe • 2TFA

Stage a

A mixture of 5.63 g Thr-Phe-Thr-Ser-OMe • HCl, 4.27 g BOC- (e-INOC) -Lys, 2.57 g N-hydroxybenzotriazole monohydrate, 20 ml DMF and 30 ml methylene dichloride is carried out. Then add one while stirring. Solution of 2.42 g of dicyclohexylcarbodiimide in 4.8 ml of methylene dichloride. Then 3.7 ml of triethylamine in the form of three portions are added within 90 minutes with vigorous stirring. The precipitated dicyclohexylurea is filtered off from the reaction mixture and washed with chloroform. The combined filtrates and washings are concentrated to 20 ml. The product is separated by adding petroleum ether. The separated oil is washed five times by decanting with petroleum ether and then suspended in about 100 ml of chloroform. The organic mixture is washed by decanting with three 100 ml portions of water. The organic phase is filtered and the solid product washed with ether and dried in vacuo.

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This gives 8.62 g of crude product. This is dissolved in 150 ml of a chloroform / isopropanol / water mixture (85: 19: 1.2) and the solution is applied to a column filled with 1.3 kg of silica gel 60. The product is eluted with the same solvent mixture. The fractions containing the product (identified on the basis of thin layer chromatography) are combined and evaporated in vacuo. This gives 6.72 g of essentially pure BOC- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-OMe.

Stage b

6.65 g of BOC- (e-INOC) -Lys-Thr-Phe-Thr-Ser-OMe are dissolved in 65 ml of trifluoroacetic acid. The solution is stirred for 3 minutes at 25 ° C., then cooled in an ice bath and the product is precipitated by adding 250 ml of cold ether and 400 ml of cold petroleum ether. The mixture is then filtered and the filter residue is washed out with an ether / petroleum ether mixture. After drying the solid in vacuo, 8.1 g of essentially pure (e-INOC) -Lys-Thr-Phe-Thr-Ser-OMe • 2TFA are obtained.

Preparation of BOC-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser hydrazide

Stage a

8.1 g (£ -INOC) -Lys-Thr-Phe-Thr-Ser-OMe • 2TFA are dissolved in the smallest possible amount of DMF. 2.98 g of BOC-Trp-HSE are added to the solution while stirring, it is adjusted to a pH of 7 with triethylamine and the reaction mixture is stirred at 25 ° C. for 3 hours. The reaction mixture is then adjusted to a pH of 7 by adding further triethylamine and the mixture is stirred at 25 ° C. for 18 hours. After renewed pH adjustment to 7 with triethylamine, the mixture is stirred for a further 3 hours at 25 ° C., after which the reaction is essentially complete. The product is then precipitated by adding ether and the solvent is decanted off. The solidification of the oil-like product is stimulated by rubbing with ether. Then the product is filtered off and dried in vacuo; 8.31 g of crude product are isolated. The crude product is dissolved in the smallest possible amount of a chloroform / methanol / water mixture (85: 15: 1.5), the solution is added to a column loaded with 1 kg of silica gel 60 and eluted with the same solvent mixture. The fractions containing the product (based on thin-layer chromatographic identification) are combined and evaporated to dryness in vacuo. This gives 5 g of essentially pure BOC-Trp (e-INOC) -Lys-Thr-Phe-Thr-Ser-OMe.

Stage b

2 g of BOC-Trp- (f-INOC) -Lys-Thr-Phe-Thr-Ser-OMe are suspended in 3 ml of a methanol / hydrazine mixture (2: 1) at 0 ° C. The suspension is stirred until it is homogeneous. Then anhydrous hydrazine is added dropwise until the solution is complete. The reaction solution is then stirred for a further 15 minutes at 25 ° C. and then evaporated to dryness in vacuo. The solid residue is rinsed twice with 5 ml of methanol in vacuo. The product is suspended in methanol, the suspension is stirred and the product is centrifuged off. The product is then washed with 30 ml of methanol, isolated by centrifugation five times and dried in vacuo. This gives 1.6 g of essentially pure BOC-Trp (£ -INOC) -Lys-Thr-Phe-Thr-Ser-hydrazide.

Production of

BOC-D-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser hydrazide

The compound is prepared as described above, stages a and b, using BOC-D-Trp-HSE instead of BOC-Trp-HSE.

Manufacture of Phe-Phe-OMe hydrochloride

Stage a

A suspension of 10.78 g of Phe-OMe hydrochloride in 5,500 ml of methylene dichloride is mixed with 18 g of BOC-PheHSE. The pH is adjusted to 9 with triethylamine, a clear solution being formed. The solution is left to stand overnight, after which the precipitated solids are filtered off and washed with methylene dichloride. The filtrate and the washing water containing the product are combined and washed with saturated sodium chloride solution (2 x 500 ml), 10% sodium bicarbonate solution (2 x 500 ml) and 0, in sulfuric acid (2 x 250 ml). The washings are washed back with methylene dichloride (200 ml). The combined methylene dichloride solutions are dried over anhydrous sodium sulfate and filtered. The residue obtained by evaporating the filtrate to dryness is recrystallized from methylene dichloride / hexane. This gives 9 g of the protected dipeptide BOC-Phe-Phe-OMe, mp. 122.0 20 to 122.5 ° C.

BOC-Phe-Phe-OMe:

calculated: C 67.57 H 7.09 N 6.57 found: C 67.67 H 7.37 N 6.31

A second crystal yield (11 g) 25 is obtained from the filtrates.

Stage b

3 g of the protected dipeptide are suspended in 100 ml of ethyl acetate. The suspension is cooled in an ice bath and anhydrous hydrogen chloride gas is passed in for 15 minutes. The hydrochloric acid is then flushed out with nitrogen.

The solid is then filtered off, washed with ethyl acetate and dried in vacuo at room temperature. 2.1 g of Phe-Phe-OMe hydrochloride are obtained. Phe-Phe-OMe • HCl:

35 calculated: C 63.06 H 6.41 N 7.74 found: C 63.00 H 6.52 N 7.66

Production of Asn-Phe-Phe-OMe hydrochloride

40 level a

12.3 g of Phe-Phe-OMe hydrochloride are dissolved in 102 ml of freshly degassed DMF. The solution is mixed with 12.86 g (7% excess) BOC-AsnNPE. The reaction solution is then adjusted to a pH of 7.2 by adding 4.73 ml of TEA and stirred overnight. The reaction solution is then evaporated to dryness in vacuo and the residue is taken up in chloroform. The chloroform solution is washed with water, saturated sodium bicarbonate solution, 0.1 µm sulfuric acid and again water. Then the solution is dried over anhydrous magnesium sulfate and evaporated to dryness. The remaining solid crude product is dissolved in the smallest possible amount of hot ethanol. Upon cooling, the product crystallizes out of the solution. The crystals are filtered off, washed with cold ethanol and dried in vacuo. This gives 9.18 g of BOC-Asn-Phe-Phe-OMe.

Stage b

8.5 g of BOC-Asn-Phe-Phe-OMe are slurried in 110 ml of water-60 free ethyl acetate. The slurry is cooled to 0 ° C in an ice bath and a strong stream of anhydrous hydrogen chloride gas is introduced for 15 minutes. This creates a clear solution, which is flushed using a nitrogen stream. After adding 100 ml of ether, the 65 solid product separates from the solution. The solid is filtered off, washed with ether (3 x 100 ml) and dried in vacuo. 7.27 g of Asn-Phe-Phe-OMe hydrochloride are obtained.

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Production of (£ -INOC) -Lys-Asn-Phe-Phe-OMe-dihydrochloride

Stage a

1.9 g of a-BOC- (£ -INOC) -Lys are dissolved in 50 ml of freshly degassed DMF. The solution is mixed with 0.675 g of 1-hydroxybenzotriazole, stirred and cooled in an ice bath. The cooled solution is mixed with 1.13 g DCCI. The ice bath is removed and the reaction mixture is stirred for a further 30 min. Then 2.38 g of Asn-Phe-Phe-OMe hydrochloride are added. The reaction mixture is then adjusted to a pH of 7.6 by adding 1.92 ml of TEA and stirred overnight at room temperature. Any solids are then filtered off from the reaction mixture. The filtrate is evaporated to dryness in vacuo. The residue is stirred with 800 ml of chloroform, the mass is filtered off, the gelatinous solid is washed out with chloroform and dried in vacuo. This gives 1.8 g of a-BOC- (fi-INOC) -Lys-Asn-Phe-Phe-OMe. The filtrate and the wash water are evaporated to dryness in vacuo. The residue is suspended again in chloroform, the suspension is filtered off, the solid is washed out with chloroform and dried in vacuo. This gives a further 1.82 g of the product.

Stage b

1 g of a-BOC- (£ -INOC) -Lys-Asn-Phe-Phe-OMe is slurried in 13 ml of anhydrous ethyl acetate. A vigorous stream of anhydrous hydrogen chloride gas is introduced into the suspension with stirring and ice bath cooling for 15 minutes. The suspension is then flushed with nitrogen for 15 minutes. The solid product is filtered off with suction, washed out with ethyl acetate and dried in vacuo. The product yield is 1.04 g.

Preparation of w-methyl-a-amino suberate • HCl 72 ml of saturated methanolic hydrochloric acid are added to a suspension of 22.8 g of a-amino acid in 450 ml of methanol. The mixture is stirred for 30 minutes at 25 ° C., a complete solution taking place. The reaction solution is then evaporated to dryness and the residue is rinsed by evaporating methanol three times. The product is made to crystallize from methanol by adding ether, filtered off and dried at 25 ° C. in vacuo. The crude product (18 g) is recrystallized from methanol / ether; 13 g of essentially pure methyl a-amino suberate • HCl are obtained.

Preparation of a-tert-butyl-fo-methyl-a-amino suberate 13 ge-methyl-a-aminosuberate-HCl are dissolved in a mixture of 811 ml of tert-butyl acetate and 8.65 ml of perchloric acid. The solution is left to stand at 25 ° C. for 4 days and then extracted twice with 600 ml of 0.5N HCl in each case. The combined aqueous solutions are adjusted to a pH of 7 with sodium bicarbonate and extracted three times with 700 ml of ether each time. The combined ether extracts are dried over anhydrous sodium sulfate, filtered and the filtrate is evaporated. 5.5 g of tert-butyl-w-methyl-a-aminosuberate are obtained in the form of an oil.

Preparation of a-tert-butyl-w-methyl-N-BOC-a-amino suberate 5.5 g of a-tert-butyl-co-methyl-a-amino suberate are dissolved in 80 ml of DMF. 6 g of tert-butyl-trichlorophenyl carbonate are added to the solution, and the pH is adjusted to 8.5 with triethylamine. The pH of 8.5 is maintained by adding further triethylamine for 1 hour. The reaction mixture is then stirred at 25 ° C. overnight and then evaporated in vacuo to a heavy oil. The oil is dissolved in 600 ml of ether and the solution is extracted three times with 600 ml of saturated sodium bicarbonate solution, three times with 600 ml of 0, in sulfuric acid and finally once with 600 ml of saturated sodium chloride solution. Then you evaporate the ether solution to dryness. 8.6 ga-tert-butyl-w-methyl-N-BOC-a-aminosuberate is obtained in the form of a heavy oil.

Preparation of a-tert-butyl-BOC-a-amino suberate 5 g of a-tert-butyl-oj-methyl-N-BOC-a-aminosuberate are mixed in a mixture of 208 ml dioxane and 133 ml ln-sodium hydroxide solved. The mixture is stirred for 75 minutes at 25 ° C. and the organic solvent is then evaporated off in vacuo. After adding 150 ml of water, the solution is adjusted to a pH of 4.6 with dilute sulfuric acid and extracted three times with 150 ml of ether each time. The combined ether extracts are dried over anhydrous sodium sulfate and evaporated in vacuo to a heavy syrup. The crude product (4.5 g) is dissolved in the smallest possible amount of methylene dichloride. The solution is placed on a column charged with 400 g of silica gel 60 and the by-products are eluted with methylene dichloride. If no further by-products are found in the eluate due to thin-layer chromatography, a methylene dichloride / isopropanol mixture (98: 2) is used to elute the product. The fractions containing the product (based on thin-layer chromatographic analysis) are combined and evaporated to dryness in vacuo. By recrystallization from hexane, 1.5 g of a-tert-butyl ester-BOC-a-aminosuberate of mp 72 ° to 73 ° C.

Preparation of a-tert-butyl-wp-nitrophenyl-BOC-a-amino suberate A mixture of 1.38 g of a-tert-butyl-BOC-a-amino suberate, 0.556 g of p-nitrophenol and 0.865 g of DCCI in 30 ml of methylene dichloride is stirred at 25 ° C. for 4 hours. The precipitated dicyclohexylurea is then filtered off from the reaction mixture and washed out with methylene dichloride. The combined organic solutions containing the product are evaporated in vacuo to a heavy oil. The oil is dissolved in 20 ml of ether, 0.1 ml of acetic acid is added to the solution, the mixture is stirred at 25 ° C. for 2 hours, evaporated to a heavy oil and the residue is dried in vacuo. This gives 1.7 g of a-tert-butyl-w-p-nitrophenyl-BOC-a-aminosube-rat.

Example 1 Preparation of a-tert-butyl ester-BOC-a> -Asu- (£ -INOC) -Lys-Asn-Phe-Phe-OMe A solution of 1 g (£ -INOC) -Lys-Asn-Phe- Phe-OMe • 2HC1 in 20 ml DMF is introduced with a solution of 0.755 g of a-tert-butyl-cu-p-nitrophenyl-BOC-a-aminosuberate in 3.5 ml of THF at 25 ° C. with stirring. The solution obtained is adjusted to a pH of 8 with diisopropylethylamine and stirred at 25 ° C. for 18 hours. The reaction mixture is then evaporated in vacuo to give a heavy oil and this is dissolved in 50 ml of methylene dichloride. The solution is extracted with water and then with saturated sodium bicarbonate solution until all of the p-nitrophenol has been eliminated. The solution is then washed with further water and dried over anhydrous sodium sulfate. Then the mixture is filtered, the filtrate is evaporated to dryness in vacuo, the crude product obtained is dissolved in the smallest possible amount of a chloroform / methanol / water mixture (80: 20: 2) and the solution is poured onto a 150 g silica-60 mixture Pillar on. The product is

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15

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35

40

45

50

55

60

65

626 328

20th

same solvent mixture eluted. The fractions containing the product (based on thin-layer chromatography analysis) are combined and evaporated to dryness in vacuo. This gives 0.885 g of cc-tert-butyl ester-BOC-a> -Asu- (£ -INOC) -Lys-Asn-Phe-Phe-OMe.

Example 2 Preparation of w-Asu (e-INOC) -Lys-Asn-Phe-Phe-OMe • 2TFA 0.855 g of the product from Example 1 are dissolved in 5 ml of trifluoroacetic acid. The solution is stirred for 90 minutes at 25 ° C. and the product is then precipitated by adding petroleum ether. The centrifuged solid is washed out with petroleum ether (3 x 10 ml), centrifuged again and dried in vacuo. 0.725 gcy-Asu (e-INOC) -Lys-Asn-Phe-Phe-OMe • 2TFA is obtained.

Example 3 Preparation of BOC-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-azide 0.667 g BOC-Trp- (e-INOC) -Lys-Thr-Phe-Thr-Ser-hydrazide (prepared according to Example 5) are dissolved in 6.3 ml DMF at -25 ° C. with stirring. The solution is mixed with 0.585 ml of 5.5N HCl in THF and then 75 mg of isoamyl nitrite are added in the form of 7 portions with stirring at -25 ° C. in the course of 2 hours. This gives a solution of the He-xapeptidazids.

Example 3 a Preparation of BOC-D-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-azide The desired compound is prepared analogously to Example 3, but instead of BOC-Trp- (e-INOC ) -Lys-Thr-Phe-Thr-Ser-hydrazide uses BOC-D-Trp- (e-INOC) -Lys-Thr-Phe-Thr-Ser-hydrazide prepared according to Example 5a.

Example 4

Preparation of BOC-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-a> -Asu- (£ -INOC) -Lys-Asn-Phe-Phe-OMe The solution of the hexapeptide prepared according to Example 3 Azide is added at —30 ° C. while stirring with a solution of 0.554 g of cy-Asu- (£ -INOC) -Lys-Asn-Phe-Phe-OMe • 2TFA (prepared according to Example 15) in 6 ml. The solution is adjusted to a pH of 7.8 with diisopropylethylamine and left to stand at -20 ° C. for 18 hours. The reaction mixture is then evaporated in vacuo to a heavy oil and rubbed with 30 ml of methanol. The solid product is centrifuged off and washed out six times with 30 ml of warm methanol each time. After drying in vacuo, 0.855 g of the essentially pure, protected undecapeptide is obtained

BOC-Trp- (f-INOC) -Lys-Thr-Phe-Thr-Ser-cy-Asu- (£ -INOC) -Lys-Asn-Phe-Phe-OMe.

Example 4a

Production of BOC-D-Trp- (f-INOC) -Lys-Thr-Phe-

Thr-Ser-cy-Asu- (£ -INOC) -Lys-Asn-Phe-Phe-OMe The desired compound is prepared analogously to Example 17, but instead of the hexapeptide azide BOC-Trp- (f-INOC) -Lys-Thr -Phe-Thr-Ser-azide uses BOC-D-Trp- (e-INOC) -Lys-Thr-Phe-TTir-Ser-azide produced according to Example 16a.

Example 5

Production of Cyclo [«- Asu- (t -INOC) -Lys-Asn-Phe-Phe-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser]

Stage a

The protected undekapeptide methyl ester (0.85 g) prepared according to Example 4 is suspended at 0 ° C. with stirring in 1 ml of a methanol / hydrazine mixture (2: 1). Anhydrous hydrazine is added dropwise to the suspension until the solution is complete, and the solution is then stirred at 25 ° C. for 15 minutes. The reaction solution is then evaporated to dryness in vacuo and the solid product is rubbed with methanol. The solid is then centrifuged off, washed out several times with methanol and dried in vacuo. 0.8 g of essentially pure, protected undecapeptide hydrazide is obtained.

10th

Stage b

The protected undecapeptide hydrazide (0.8 g) prepared according to Example 5, stage a is suspended at 0 ° C. with stirring in a mixture of 8 ml of ethyl acetate and 0.8 ml of mercaptoetha-15 noi. A vigorous stream of anhydrous hydrogen chloride and then a vigorous stream of nitrogen are introduced into the suspension for 25 minutes at 0 ° C. The precipitated product is filtered off, washed with 75 ml of ethyl acetate and dried in vacuo. This gives 0.85 g of 20 essentially pure Trp- (f -INOC) -Lys-Thr-Phe-Thr-Ser-cu - Asu (f-IN OC) -Lys-Asn-Phe-Phe-hydrazide. 4HC1.

Stage c

25 The undecapeptide hydrazide (0.425 g) prepared according to Example 5, stage b is dissolved in 3 ml of DMF with stirring at -25 ° C. The solution is mixed with 0.25 ml of 5N HCl in THF. Then 28 mg of isoamyl nitrite in the form of 6 portions are added within 90 minutes at -25 ° C., a solution 30 of the undecapeptide azide being obtained. This solution is added to 420 ml DMF at -50 ° C to give a total concentration of 1 mg / ml. The solution is adjusted to a pH of 7.6 with diisopropylethylamine and left to stand at -20 ° C. for four days. The solution is then evaporated to dryness in vacuo. The residue is dried in vacuo for an additional 18 hours. The oil obtained is dissolved in the smallest possible amount of methanol,

causes the solid product to precipitate by adding ethyl acetate and centrifuges it off. The 40 solid is then washed out three times with 10 ml of ethyl acetate each time and dried in vacuo. 0.272 g of the essentially pure, protected cyclic undecapeptide cyclo [co-Asu- (£ -INOC) -Lys-Asn-Phe-Phe-Trp- (£ -INOC) - ^ Lys-Thr-Phe-Thr-Ser is obtained ].

Example 5a

Preparation of Cyclo [co-Asu- (e-INOC) -Lys-Asn-Phe-Phe-D-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser] so The desired compound is obtained analogously to Example 5 , Stages a, b and c, prepared using the protected undeka peptide methyl ester

BOC-Trp- (£ -INOC) -Lys-Thr-Phe-Thr-Ser-w-Asu- (£ -INOC) -Lys-Asn-Phe-Phe-OMe 55 from stage a by BOC-D-Trp- (£ -INOC) -Lys-Thr-Phr-Thr-Ser-ft> -Asu- (£ -

INOC) -Lys-Asn-Phe-Phe-OMe (prepared according to Example 17a). f

;

60 Example 6

Preparation of Des- (Ala1, Gly2) -desamino [Cys3] dicarba3'14-somatostatin 0.3 g of the product prepared according to Example 5, stage c, is dissolved in 3 ml of 50% aqueous acetic acid. After adding 65 mg of activated zinc, the mixture is stirred vigorously for 2 hours. The excess zinc is then filtered off, the filtrate is applied to a column of Sephadex-Gel-25 Superfine and eluted with 50% aqueous acetic acid. It

21st

626 328

10 ml fractions are collected. The fractions containing the product (based on thin-layer chromatography analysis) are combined and evaporated to dryness. The residue is dissolved in water. Freeze drying gives 0.157 g of essentially pure des (Ala1, Gly2) desamino [Cys3] dicarba3'14 somatostatin. The UV spectrum shows a maximum at 280 nm. After acid hydrolysis, the amino acid analysis gives the following result: Lys2, o4; Asplj03; Thr2! I6; Ser0j998; Phe3i03; Trpo, 74.

Example 6a Preparation of Des- (Ala1, Gly2) -desamino [Cys3] dicarba3'14- [D-Trp8] -somatostatin The desired compound is prepared analogously to Example 6, but using the

Cyclo [<y-Asn- (£ -INOC) -Lys-Asn-Phe-Phe-Trp- (e-INOC) -

Lys-Thr-Phe-Thr-Ser] by Cyclo [w-Asn- (£ -INOC) -Lys-Asn-Phe-Phe-D-Trp- (£ -INOC) -

Lys-Thr-Phe-Thr-Ser]

(prepared according to Example 18a) replaced.

Example 7 Preparation of a'-tert-butyl-N (a) -CBZ-N (a ') -BOC-a, a' -diaminosuberat 8 g Cbz-Glu-a-methyl ester and 8.6 g BOC-Glu a-tert-butyl ester are dissolved in 240 ml of methanol and 80 ml of pyridine. The solution is mixed with 250 mg (0.2 equivalents) of sodium and electrolyzed for 55 min at 23 to 28 ° C (4.0 A; 100 V; 2 mm apart platinum electrodes with a size of 2.5 x 4 cm) . The reaction takes place with stirring in a metal beaker; a dry ice / iso-propanol bath of -40 ° C is used for cooling. The pH of the reaction mixture, determined by moist pH paper, changes from 6.8 to 7.2 to 7.6. The solution is evaporated in vacuo, the oily residue is suspended in 100 ml of chloroform / ethyl acetate (95: 5) and the insoluble sodium salts of the starting compounds are filtered off. Evaporation of the filtrate gives 16.77 g of a dark oil.

The oil, which contains a three-component diaminocork acid derivative mixture, is added in chloroform / ethyl acetate (95: 5) to a column loaded with 1700 g of silica gel 60 and isolated with the same solvent. After a flow of 1200 ml has been collected, 70 ml fractions are removed every 45 seconds. The three-component product is eluted with fractions 125 to 290. After evaporation, 7.82 g of the product are obtained.

The crude N (a) -CBZ-N (a ') - BOC-a, a'-diaminocorkic acid-a-methyl ester-a'-tert-butyl ester (7.82 g) is dissolved in 157 ml of di-oxane . 78.5 ml of In NaOH are added to the solution over 15 minutes at room temperature. Then add 4 ml of acetic acid and evaporate the solution in vacuo. The residue is dissolved in water. Freeze drying gives 14.28 g of crude

N (a) -CBZ-N (a ') - BOC-a, a'-diaminocorkic acid-

a'-tert.-butyl ester.

The crude product is suspended in a solution of 20 ml of methanol, 20 4 ml of concentrated ammonium hydroxide and 70 ml of chloroform. The suspension is filtered, the filtrate is evaporated and the residue is redissolved in a solution of 15 ml of methanol, 2 ml of concentrated NH4OH and 45 ml of CHC13. The solution obtained is applied to a column loaded with 1500 g of 25 silica gel-60 in CHCl3 / CH3OH / NH4OH (80: 20: 2) and eluted with the same solvent. After collecting a flow of 2100 ml, 70 ml fractions are taken every 30 seconds. The desired product is eluted with fractions 35 to 82. 30 These fractions are combined and evaporated to dryness. The residue is suspended in water, acidified with citric acid and the product extracted with ethyl acetate. The extract is dried. After evaporation to dryness, 2.17 g of the desired 35 a'-tert-butyl-N- (a) -CBZ-N (a ') - BOC-a, a'-diaminosuberate are obtained.

The a'-tert.-butyl-N (a) -CBZ-N (a ') - BOC-a, a'-diamino-suberate is used to produce

: h2-ch7

"CH2-? H2

NH2-CH-Ç-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH-COOH

I '

-CH- (

by the process described in Examples 1 to 2 and 4 to 6 and for the preparation of

ÇH2-CH2-

• ch2-ch2

NH2-CH - ^ - Lys-Asn-Phe-Phe-D-Trp-Lys-Thr

-Phe-Thr-Ser-nh-ch-cooh used according to the method described in Examples 1 to 2 and 4a to 6a.

Example 8

Production of Des- (Ala1, Gly2) -desamino [Cys3] des-

carboxy [Cys14] dicarba3'14- [D-Trp8] somatostatin The desired compound is prepared according to the process shown in Table III, but instead of the a-amino acid in step 11, methyl 7-aminohep-tanoate, prepared according to the by CF Horn et al., Angew. Chem. 74 (1962), page 531; Chemical Abstracts, Volume 57, 1141 l (c), uses the method described and carries out the conversions described in Examples 1 to 2 and 4a, 5a and 6a. With the silica gel thin layer chromato-

55 graph using ethyl acetate / pyridine / acetic acid / water (10: 5: 1: 3) as solvent, the compound gives an Rr value of 0.32.

Example 9

60 Production of the sulfone from Des- (Ala ', Gly2) -

desamino [Cys3] -carba3-somatostatin A solution of 14 mg Des- (Ala1, Gly2) -desamino [Cys3] -carba3-somatostatin and 0.1 mg ammonium molybdate in 0.2 ml 60% perchloric acid is mixed with 50, «1 30% H202 ver-65 uses. The product is purified by passing the solution through a column of Sephadex-G-25 using 50% acetic acid as the eluent. The elution of the product is carried out by thin layer chromatography using

626 328

22

Use of ethyl acetate / pyridine / acetic acid / water (10: 5: 1: 3) was found as the solvent system. The fractions containing the product are evaporated to dryness. The sulfone is obtained from Des- (Ala1, Gly2) -desami-no [Cys3] carba3-somatostatin.

Example 9a

Preparation of the sulfone of Des- (Ala1, Gly2) -desamino [Cys3] carba3- [D-Trp8] -somatostatin The desired compound is prepared analogously to Example 9, with the Des (Ala1, Gly2) desamino [Cys3] carba3-somatostatin replaced by Des- (Ala1, Gly2) -desamino [Cys3] carba3- [D-Trp8] somatostatin.

Example 10

Preparation of the sulfoxide of Des- (Ala \ Gly2) -desamino [Cys3] carba3-somatostatin A solution of 14 mg of Des- (Ala \ Gly2) -desamino [Cys3] carba3-somatostatin in 0.5 ml of an O, 2m-phosphate buffer with a pH of 7.0, 0.2 ml of a 0.05m ethanolic iodine solution and 0.1 ml of 0.1m sodium hydroxide solution are added. The mixture is left to stand at 25 ° C. for 5 days. The product is then isolated by passing the solution through a column of Sepha-dex-G-25, eluting with 50% acetic acid. The product elution is determined by thin layer chromatography using ethyl acetate / pyridine / acetic acid / water (10: 5: 1: 3) as the solvent system. The fractions containing the product are evaporated to dryness. The sulfoxide of Des- (Ala1, Gly2) -desamino [Cys3] carba3-somatostatin is obtained.

Example 10a Preparation of the Sulfoxide of Des- (Ala1, Gly2) -desamino [Cys3] carba3- [D-Trp8] -somatostatin The desired compound is prepared analogously to Example 10, the Des- (Ala1, Gly2) -desamino [Cys3 ] carba3-somatostatin replaced by Des- (Ala \ Gly2) -desamino [Cys3] carba3- [D-Trp8] -somatostatin.

The somatostatin analogs, which contain alanine or a-amino-butyric acid instead of Asn, can be prepared by the method shown in Tables I, II and III by using the peptide (e-INOC) -Lys-Asn-Phe-Phe- OMe • 2HC1 by the peptides (e-INOQ-Lys-Ala-Phe-Phe-OMe • 2HC1 or (e-INOC) -Lys- [a-aminobutyric acid] -Phe-Phe-OMe • 2HC1 replaced. The peptides ( £ -INOC) -Lys-Ala-Phe-Phe-OMe-2HCl and (e-INOC) -Lys- [a-aminobutyric acid] -Phe-Phe-OMe • 2HC1 can be carried out according to steps 6, 7 and 8 with the exception that in stage 7a BOC-AsnNPE is replaced by BOC-AlaNPE or BOC- [a-aminobutyric acid] NPE.

A specific example of the somatostatin analogs, namely des- (Ala \ Gly2) -desamino [Cys3] -descarboxy [Cys14] -dicarba314- [AIa5] -somatostatin, can be prepared according to the method shown in Table III by using in

Stage 12 a-tert-butyl-BOC-a-aminosuberat replaced by BOC-w-aminoheptanoic acid and in stage 7 BOC-AsnNPE by BOC-AlaNPE.

Another specific example of the So-5 matostatin analogs according to the invention, namely des- (Ala1, Gly2) -desamino [Cys3] descarboxy-

[Cys14] dicarba3,14- [a-aminobutyric acid5] -

[D-Trp8] somatostatin,

can be prepared according to the method illustrated in Table III, by a-tert.-butyl-BOC-a-aminosuberate by BOC-w-aminoheptanoic acid in step 12 and BOC-AsnNPE by BOC- [a- aminobutyric acid] NPE and in stage 5a BOC-TrpHSE replaced by BOC-D-TrpHSE.

The somatostatin analogs according to the invention can be used in human and veterinary medicine to inhibit gastric secretion in the treatment of gastric ulcers, to inhibit growth hormone release (as in the treatment of acromegaly), to inhibit glucagon release and, alone or together with insulin, to lower the blood -20 sugar levels (e.g. in diabetes therapy). In the treatment of diabetes, the number and size of the daily doses and the administration time are determined on the basis of an individual examination of the respective patient. The method for determining these factors is familiar to the person skilled in the art.

The somatostatin analogs described here can be administered to warm-blooded animals (including humans) intravenously, subcutaneously, intramuscularly or orally. In the case of large warm-blooded animals, the dose range for oral administration in the form of tablets or capsules is about 0.001 to about 7 mg / kg of body weight / day. The somatostatin analogs according to the invention are preferably administered by injection. A therapeutically effective amount of such a compound is usually in the dose range from about 0.001 to about 2 mg / kg body weight. The range from about 0.00142 to about 0.428 mg / kg of body weight is preferred, the administration being carried out by intravenous infusion or subcutaneous injection. The dose required depends on the specific disease to be treated, its severity and the duration of the therapy.

When the active ingredient is administered in tablet form, the tablets can contain a binder such as tragacanth, maize starch or gelatin, a filler (excipient) such as dicalcium phosphate, a disintegrant such as corn starch or alginic acid, a lubricant such as magnesium stearate , and contain a sweetener and / or a taste or smell correcting agent (or a coloring agent) such as cane sugar, milk sugar or wintergreen. Suitable liquid carriers (diluents) for intravenous administration are e.g.

sterile water, isotonic saline and phosphate buffer solutions or other pharmacologically acceptable injectable carriers.

s

Claims (3)

626 328 2 PATENT CLAIMS 1. Process for the preparation of the peptides of the formulas: CH0-X- ï r Ì-CH-C- ■ Lys-R "'- Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser - «- CH., -NH-CH-R ' and Î : H2-X- -Y-ÇH ? R-CH-C-Lys-R "'- Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH-R' in which: O X CH2, S, S02 or [X] n, where n = O; O Y CH2, S, â, S02 or [Y] n, where n = O; R H, NH2 or R "NH, where R" Ala-Gly-, a lower is an acyl group having 2 to 6 carbon atoms or an aroyl group having 7 to 21 carbon atoms; R 'HoderCOOH and 20 R' "are asparagine, alanine or a-aminobutyric acid, where not both X and Y are heteroatoms, characterized in that a peptide of the formulas: CH2-X- R-CH-Ç- (£ -INOC) Lys-R "1 -Phe-Phe-Trp- (£ -INOC) Lys-Thr- Y-CH" f Phe-Thr-Ser-NH-CH-R ' or Î H2-X- R-CH-Ç - (^ -INOC) Lys-R "'- Phe-Phe-D-Trp - (^ - INOC) Lys-Thr- Y-ÇH- f Phe-Thr-Ser-NH-CK-R1 in which X, Y, R, R ', R "and R'" each have the meaning given above, reacted with zinc dust in an aqueous solution of a lower alkanoic acid and, if one started from a compound in which O X S or S02 or O Y S or S02 so, the reaction product is oxidized with performic acid or hydrogen peroxide. 2. The method according to claim 1, characterized in that the peptides of the formulas: ÇH2 ~ CH2 ~ -ch2-ÇH2 CH 2 g Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH-COOH and SH2 ~ CH2 ~ ch2-ch2 CH2- £ -Lys-Asn-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH-COOH 626 328 from a peptide of the formulas: CH ^ -CH- CH _c- (£ -INOC) Lys-Asn-Phe-Phe-Trp- (£ -INOC) Lys-Thr-Phe- 2 u 0 CH_-CH_ I. Thr-Ser-NH-CH-COOH respectively. ch0-ch0 - | 2nd cl ^ -jjj- (£ -inoc) Lys-Asn-Phe-Phe-D-Trp- (^ -inoc) Lys-Thr-Phe- ch2- (j: h2 Thr-Ser-NH-CH-COOH manufactures. 3. Application of the method according to claim 1 for the preparation of the peptides of the formulas: CH2-X— Ï-ch2 E-CH-Ç-Lys-Asn -Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-NH-Œ-R • and Ï h2-x- -Y— <jH. H-CH-Ç-Lys- As n-phe-Phe-D-Trp-Lys-Thr-Phe-Thr-Ser-NH-CH-R ' characterized in that a peptide of the formula y-ch. boc-nh-ch-r "" in which R "" represents a hydrogen atom or a group cCbu, reacted overnight with a solution containing (e-INOC) -Lys-Asn-Phe-Phe-Ome • 2HC1 in the alkaline pH range and the protected pentapeptide 60 obtained to split off the BOC group with trifluoroacetic acid, whereupon the peptide of the formula obtained ? : H2-X -y-ch. CH "-C- (£ -INOC) Lys-Asn-Phe-Phe-OMe NH - CH-R ' 626 328 4th in an inert solvent with a solution of BOC agent at about -30 ° C and then Trp- (f-INOC) -Lys-Thr-Phe-Thr-Ser-N3 or BOC-D-Trp- the peptide of the formula (f-INOC) -Lys-Thr-Phe-Thr-Ser-N3 obtained in an inert solution Î H2-x- CH2 "^ ~ î Lys-Asn-Phe-Phe-OMe ^ BOC-Trp- (-INOC) Ly s-Thr-Phc-Thr-Ser-NH -CH-R ' or Ï H2-x- CH - C- (-INOC) Lys -Asn-Phe-Phe-OMe 2 ö p2 BOC-D-Trp- (é -INOC) Lys-Thr-Phe-Thr-Ser-NH-CH-R * 25th a) with a methanolic hydrazine solution to convert to a concentration of about 1 mg / ml of the -OMe group in a hydrazide group, thins, and b) the BOC protective group of tryptophan by reaction d) den Adjust the pH to the weakly alkaline range of the undecapeptide hydrazide with anhydrous chlorine water in order to split off the a-amine group of the tryptophan-amino acid in the presence of mercaptoethanol, so converting it to the free a-amino group and the ring closure c) the desired cyclic Peptide is generated by initiating the ring closure through a hydrazide group of about 4 days by reaction with isoamyl nitrite with the reaction mixture left at about -20 ° C. and a strongly acidic pH value converted into an azide group. will close leads and the solution with a suitable organic Lö- whereby the peptides of the formula 35 CH -X - | 2nd CH2-g- {é -INOC) Lys-Asn-Phe-Phe-Trp- (£ -inoc) Lys-Thr- Phe-Thr-Ser-NK - CH-R1 or ch2-x. ch2-g- (£ -inoc) Lys-Asn-Phe-Phe-D-Trp - (^ - INOC) Lys-Thr- -y-ch2 T Phe-Thr-Ser-NH-CH-R1 are obtained, which are then implemented according to the method of patent application 4. according to claim 3, thereby claim 1 to the desired product. 60 shows that the peptide of the formula cz2-cz2 î V -NH-CH-qf XJtBu 626 328 the peptide of formula f 2-ch2 -: - ch2-ch2 I. CH2-C- (£ -INOC) Lys-Asn-Phe-Phe-OMe NH2-CH-COOH which then adds to the peptides of the formulas I2 ~ CH2 Œ2 n CH2-g- (é-INOC) Lys-Asn-Phe-Phe-OMe ' BOC-Trp- (£ -INOC) Lys-Thr-Phe-Thr-Ser-NH-CH-COOH and f H2-CH2 CH2- CH2-g- (é. -INOC) Lys-Asn-Phe-Phe-OMe CH BOC-D-Trp- ((f-INOC) Lys-Thr-Phe-Thr-Ser-NH-CH-COOH \ is implemented and what to do with these to form the peptides of the formulas CH0-CH,::: - I. CH2-g- (£ -INOC) Lys-Asn-Phe-Phe-Trp- (^ -INOC) Lys-Thr-Phe-CH2- (j: H2 Thr-Ser-NH-CH-COOH and rH2 ~ CH 2 2 2 -Ç- (é -INOC) Lys-Asn-Phe-Phe-D-Trp- (^ -INOC) Lys-Thr-Phe- CH2-CH2 Thr-Ser-NH-CH-COOH implements and what they are implemented according to the method of claim 2. Somatostatin is a tetradecapeptide with the structure: Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH and is known to inhibit growth hormone release. Production of somatostatin derivatives with low solubility Somatostatin itself has a short-term effect because it achieves inability to ensure slow release with subcutaneous inter alia through the in vivo aminopeptidase injection. However, once the derivatives and carboxypeptidases are inactivated. A partial solution that has gone into solution, resist the inactivation of this short-term effect problem has so far not been achieved by aminopeptidases and carboxypeptidases 626 328 6 Somatostatin itself. The present invention provides somatostatin analogs with the biological activity of somatostatin and a longer duration of action, as well as a new process for the preparation of these compounds.