HUT72734A - Proces for producing bone-resorption-inhibiting cyclic compounds and compositions - Google Patents

Description

the patient being treated with a novel cyclic compound or a mixture thereof, optionally in the presence of a pharmaceutically acceptable carrier, in an amount sufficient to prevent bone resorption.

ZOQ, c0> vC / ^c '

S.B.G. & Κ.

International

Patent Office

H-1062 Budapest, Andrássy út 113.

Phone: 34-24-950, Fax: 34-24-323

61 266 / ΡΑ

compounds

preparations for inhibiting bone resorption

CIBA-GEIGY AG, BASEL, SWITZERLAND

Inventors: RINK, Hans, Riehen, ALTMANN, Eve, BASEL, BLOMMERS, Marcel, Bottmingen, MÜLLER, Klaus, Etting,

SWITZERLAND,

HALL, Tony, BARTENHEIM-LA-CHAUSSE,

FRANCE

Date of filing: 1994. 03 28th PRIORITY: 1993. 04 08 (93810254.8 1993. 04 08 (93810255.5 EUROPE

International Application Number: PCT / EP94 / 00977

International Publication Number: WO 94/24153

The present invention relates to cyclic compounds which inhibit excessive bone resorption. The invention also relates to processes for the preparation of the above compounds. Also described are four pharmaceutical compositions containing the above compounds in admixture with pharmaceutically acceptable carriers, and methods for treating diseases associated with excessive bone resorption and / or decreased calcium excretion. These methods of treatment comprise treating a patient in need of such treatment with an effective amount of a novel cyclic compound or a mixture thereof which inhibits bone resorption, preferably in admixture with a pharmaceutically acceptable carrier.

Typical diseases characterized by increased bone resorption are osteoporosis, Paget's disease that damages the bones, malignant humoral hypercalcaemia, and metastatic bone disease. Increased bone resorption makes the bones fragile and weak, which tend to break and deform. Hypercalcemia leads to the elimination of calcium from the bones through the kidneys and ultimately to the kidney.

There are, in principle, two ways to treat diseases associated with increased bone breakdown: either by prevention, when the bone is still sufficiently supplied with estrogens, bisphosphonates, and calcitonin; or medical treatment when there is already significant bone deficiency and fractures have occurred. The essence of the treatment is to promote bone formation with fluoride.

r

The compounds most commonly used to date in the treatment of such diseases are IGF (insulin-like growth factor), PTH (human parathyroid hormone), and PTHrP (human parathyroid hormone-like proteins). PTHrP is a protein produced in several types of cancer cells. For the first time, it was isolated from a squamous cell carcinoma extract and purified to homogeneity [Moseley et al., Proc. Natl. Acad. Sci. (1987) 85: 5048-5052].

More recently, Fenton et al., Endocrinology (1991) 129 3424-3426, found that Thr-Arg-Ser-Ala-Trp (TRSAW), a segment of PTHrP 107-111, is a very potent inhibitor of bone resorption in rats. WO 92/10511 discloses several TRSAW variants. The major disadvantage of TRSAW fragments is their very short half-life in rat serum, which leads to proteolytic degradation.

Surprisingly, it has been found that the cyclic compound of formula (1) wherein

A is a 12-17 membered ring,

B is a linked group attached through ring carbon or nitrogen to ring A, having 1 to 6 carbon atoms, 0 to 2 nitrogen and 0 or 1 oxygen atoms in its chain; wherein the carbon atoms may be substituted with oxo, hydroxy, sulfo, C 1-4 alkyl, morpholino, amino, carboxy, or a radical derived from a natural amino acid or a peptide of 2-5 natural amino acids; the nitrogen atoms may be substituted with C 1 -C 4 alkyl, C 1 -C 4 alkanoyl, i.e. formyl and acetyl groups, and / or acid radicals derived from a natural amino acid or a peptide of 2 to 5 natural amino acids;

D is aryl-C 1-4 -alkyl, where the aryl may be mono- or bicyclic and may be substituted by hydroxy, mercapto, amino, or halogen, i.e. fluorine, chlorine, bromine or iodine;

E may be amino; or amino substituted with C 1-4 alkyl or amino substituted with C 1-4 alkyl; C 1-4 alkyl substituted by one or more, in particular one or two amino groups; A 5- or 6-membered heterocyclic group containing one or two nitrogen atoms; an amino group substituted by an acidic radical derived from a natural amino acid or from 2 to 5 natural amino acid peptides;

F is NH or CH ₂ ;

x is 1-6;

y is 0-4;

with the proviso that the - (CH ₂ ) _X -FC (= NH) NH ₂ , - (CH ₂ ) y -OH and the BD groups are located above the plane of ring A and the group E is below the plane of ring A ; or a pharmaceutically acceptable salt thereof;

they have excellent stability and inhibitory effect on excessive bone resorption.

Rings A within the scope of the invention include, for example, crown ethers, steroids, porphyrins, and cyclic peptides. Preferred rings are those having a distance of 0.50-0.65 nm, preferably 0.55-0.60 nm, between the BD group and the point of attachment of the group E, between the point of attachment of the (CH ₂ ) y and the group E a distance of 0.50-0.62 nm, preferably 0.55-0.60 nm, and a distance of 0.55-0.75 nm, preferably 0, between the point of attachment of the (CH ₂ ) _X and BD group, 60-0.70 nm.

Particularly preferred are cyclic peptides (head-to-foot linkages) or peptides in which two amino acid side chains, or one amino acid side chain and another amino acid are linked directly or via a linker group. In order to avoid too rapid degradation in the body, i.e. the action of proteolytic enzymes, one or more, i.e., 1-5 genetically encoded amino acids may be replaced by non-genetically encoded amino acids; or one or more, i.e. 2 to 3 amino acids may be substituted with suitable -aminocarboxylic acids which, when incorporated, fulfill the steric requirements of formula (1). The invention is advantageous

realization of the ring amino acids D and L forms build up. Examples of linking groups are the They are as follows: CH ₂ ; C ₂ H ₄ , C ₂ H ₂ ; CH (CH ₃ ) -CH ₂ , o, ch ₂ -o, c ₂ h ₄ - -She, ch ₂ -o-ch ₂ , . and, ch ₂ -s, ch ₂ -ss, ss-ch ₂ , S-S, S = O, O = S = Ό, NH, NH-CO, and Neither .

Examples of amino acids are naturally occurring amino acids, i.e., genetically encoded amino acids such as alanine, arginine, aspartic acid, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine and valine; D-forms of these amino acids or amino acids such as 2-amino-adipic acid, 3-amino-adipic acid, beta-alanine, 2-amino-butyric acid, 4-amino-butyric acid,

6-aminocaproic acid, 2-amino-heptanoic acid, 2-amino-isobutyric acid, 3-amino-isobutyric acid, 2-amino-pimelic acid, 2,4-diaminobutyric acid, desmosin, 2,2 -diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethyl aspartic acid, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodmesosin , allo-isoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, ornithine, citrulline, gamma -carboxyglutamic acid, o-phosphoserine, homoserine, homocysteine, carboxyethylpiperidine, aminoisobutyric acid, diaminopropionic acid or N- (3-glycinyl) propylguanidine. The conformation of the amino acids is chosen so that the steric requirements of the compound of formula (I) are met.

In the case where the variable group is attached to ring A via a carbon atom, the inserted group B may be a C 1-6 alkyl group, i.e., methylene, ethylene, propylene, butylene, pentylene, or hexylene. The variable group may contain 1 or 2 nitrogen in the chain and / or an oxygen, i.e. it may be -CH 2 -NH-, -CH ₂ -CH ₂ -NH-, -CH ₂ -NH-CH ₂ -.

When the variable group is attached via nitrogen

to ring A, the variable group B with the following formulas can be represented by: -N (H or C 1 -C 4 alkyl) - (C 1 -C 4) mos) alkyl, i.e., -NH-CH ₂ -, -N (CH ₃ ) -CH ₂ -, -NH-CH ₂ -CH ₂ -,

-N (CH ₃ ) -CH ₂ -CH ₂ - and NH-CH (CH ₃ ) -CH ₂ -.

In a preferred embodiment of the invention, the variable group is

It has a backbone of 2 to 6 atoms and, in a more preferred embodiment, the backbone is an amino acid chain. One or more carbon or nitrogen atoms of this variable group may be substituted with C 1-4 alkyl such as methyl or ethyl; a carboxy group and / or an acyl group derived from a natural amino acid or a peptide of 2-5 natural amino acids. The acyl groups of the amino acid or peptide are preferably attached to the ring as an amide. In addition, the carbon atoms of the inserted group may be substituted with oxo, hydroxy, sulfo and amino. Part of the first amino acid may be part of the heterocyclic ring A.

In group D, the aryl group may be a 5 or 6 membered monocyclic or bicyclic ring, the latter consisting of a combination of 2 5 or 2 6 or a combination of 5 and 1 6 ring. Examples of aryl include phenyl, biphenyl, isobenzofuranyl, chromenylpyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, indazol , purinyl, isoquinolyl, quinolyl, phthalazinyl and naphthyl. These aryl groups may be substituted with C 1-4 alkyl groups such as methyl or ethyl, hydroxy, sulfo, amino and / or carboxy, so that the appropriate radicals of the following compounds are formed: aniline, naphthoquinone, phthalimide, phthalic acid, salicylic acid, sulfanylic acid, picolinic acid or nicotinic acid.

Preferred D groups are aryl-C 2 -C 3 alkyl groups such as phenyl-C 1 -C 3 alkyl, hydroxyphenyl-C 1 -C 3 alkyl, imidazolyl-C 1 -C 3 ··· · * R

m is alkyl or naphthyl-C 1-3 -alkyl. A more preferred group D is the side chain of an aromatic amino acid. Such amino acids include Trp, Phe, Tyr and His. Of these, the side chain of Trp is particularly preferred.

E may be, for example, an amino group optionally substituted by one or two methyl, ethyl, propyl, butyl, aminomethyl or aminoethyl groups, or a five- or six-membered heterocyclic ring containing one or two nitrogen atoms. a group attached via ring nitrogen to ring A, i.e., piperidyl, 2-oxopyrrolidyl or piperazyl. The group E may be an amino group substituted by an acyl group derived from a natural amino acid or from a peptide consisting of 2 to 5 natural amino acids.

(2¾) x (1-3) y groups are straight chain groups such as methylene, ethylene, propylene or butylene.

Ring A atoms are unsubstituted or substituted with small substituents such as C 1-4 alkyl. Preferably, these ring-forming atoms are unsubstituted or substituted with methyl or ethyl.

The parent compounds of formula (1) may form acid addition salts. Pharmaceutically acceptable acid addition salts include, for example, salts of the compound of formula (1) with appropriate mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid, e.g., hydrochlorides, hydrobromides, sulfates, hydrogen sulfates, phosphates. Salts may also be formed from suitable aliphatic or aromatic sulfonic acids or N-substituted sulf 9 minic acids to form methanesulfonates, benzenesulfonates, p-toluenesulfonates or N-cyclohexyl sulfamates (cyclamates).

The salts may be salts with organic acids such as lower alkane carboxylic acids, optionally unsaturated or hydroxylated aliphatic dicarboxylic acids, i.e., acetate, oxalate, maleate, malonate, fumarate, malate or citrate. Particularly preferred are internal salts, where possible, between an amino group and a carboxyl group of the compound of formula (I).

The cyclic compound of formula (Ia) is preferred.

Up to 6, preferably 3, carbon atoms of the ring may be replaced by oxygen, sulfur or nitrogen, which are preferred variants; and the ring-forming atoms may be optionally substituted with up to 6, preferably 3 to 4 C 1-4 -alkyl, oxo, hydroxy, sulfo and amino groups, with methyl, ethyl or oxo being preferred.

X is C 1 -C 4 alkylene or C 1 -C 4 alkenylene, in which up to 2, preferably 1 carbon atoms may be replaced by oxygen, sulfur, disulfide, sulfoxy, sulfonyl or nitrogen, preferably oxygen, sulfur and the disulfide group. C 1-4 alkylene or alkenylene groups may have methyl, ethyl, oxo or amino substituents. Example of these groups: CH ₂ , ^C 2 ^H 4 ' ^C 2 ^H 2' CH (CH ₃ ) -CH ₂ , S, S-CH ₂ , CH ₂ -S, CH ₂ -SS, SS-CH ₂ , SS , S = 0, O = S = O, NH ₂ , NH-CO, CO-NH, O, O-CH ₂ , CH ₂ -O, Se;

preferred groups: CH ₂ , C ₂ H ₄ , S, S-CH ₂ , CH ₂ -S, CH ₂ -SS,

SS, NH, NH-CO, CO-NH, O, O-CH ₂ , CH ₂ -O, Se; particularly preferred groups are CH ₂ , CH ₂ -CH ₂ , S, S-CH ₂ , CH ₂ -S, SS-CH ₂ , SS, NH-CO, CO-NH, O-CH ₂ , CH ₂ -O.

Rg or Rg is an acyl group derived from a natural amino acid or a peptide having from 2 to 5 natural amino acids, wherein the first or second amino acid residues of the ring member have an aromatic side chain, i.e. tryptophan, phenylalanine, tyrosine or histidine; or C 1 -C 7 alkylaryl, wherein the carbon atoms of the C 1 -C 7 alkyl moiety may be substituted with 3 nitrogen or oxygen, with nitrogen being preferred. The inserted group may also be substituted with an oxo group;

R _a or R 4 may also be C 1 -C 4 alkyl, with methyl and ethyl being preferred, and amino, oxo and carboxyl groups.

R, R or _{R, o} is substituted with hydroxy or C1-4 alkyl hydroxy, preferably hydroxy, hydroxymethyl and hydroxyethyl; hydroxy and hydroxymethyl are more preferred, and hydroxymethyl is most preferred;

Rf, Rg ^va HY Rh is C2-5 alkyl, -C (= NH) substituted _NH2, or -NH-C (= NH) NH _2; preferably -CH ₂ -CH ₂ -CH ₂ -NH-C (= NH) NH ₂ .

R 1 or R 6 is NH ₂ ; NH ₂ may be substituted with C 1-4 alkyl or amino-C 1-4 alkyl; C 1-4 alkyl substituted with one or more, especially one or two amino groups; a five- or six-membered heterocyclic ring containing one or two nitrogen atoms; or a natural one

or an acyl group derived from 2 to 5 natural amino-peptides.

More preferred is the heterocyclic compound of formula (2), where R L is CH ₂ , C ₂ H ₄ , C ₂ H ₂ , CH (CH ₃ ) -CH ₂ , S, ch ₂ , ch ₂ -ss, ss-ch ₂ , ss, S = 0, O = S = O, NH, NH-CO, 0, o-ch ₂ , CH ₂ -O, Se; preferably CH ₂ , C ₂ H ₄ , S, CH ₂ S, CH ₂ -SS, S- S, NH, NH-CO, O, O-CH ₂ , CH ₂ -O, Se; CH _{2 is} particularly preferred , ch ₂ -

And,

-ch ₂ , ch ₂ -s, ch ₂ -ss, SS, NH-CO, och ₂ .

^R 2 is C 1-6 alkylaryl or

Particularly preferred with -CO-NH-CHC is CO-NH-CH (C 1-3 alkyl-R ₁₃ ) R ₁₃ ;

R ₂ , R ₄ , R 8 and R 8 are each independently hydrogen, C 1-4 alkyl; hydrogen, methyl or ethyl are preferred, more preferably hydrogen or methyl, and

Re; is hydroxy or hydroxy substituted

C 1 -C 4 alkyl, preferably hydroxy, methyl, hydroxyethyl, more preferably hydroxy and hydroxy hydroxy-meshene alkyl substituted with -C (= NH) NH ₂ or -NH-C (= NH) NH ₂ ; preferred is -CH ₂ -CH ₂ -CH ₂ -NH-C (= NH) NH ₂ ; the other is hydrogen or (C 1 -C 4) -alkyl, hydrogen or methyl is preferred, and hydrogen is particularly preferred.

R ₁₀ is NH ₂ ; or NH ₂ substituted with C 1 -C 4 alkyl

Substituted with NH ₂ , or 1-4

C 1 -C 4 alkyl group or -NH-CO-CH ₂ .

· · * · ♦ · * * ·· ·· · «.

-NH ₂ or -NH-CH ₂ -CH ₂ -ΝΗ ₂ optionally substituted with methyl or ethyl; NH _{2 is} preferred; NH ₂ substituted with C 1-2 alkyl; the C 1-2 alkyl group substituted at the amino group; -NH-CO-CH ₂ -NH ₂ or -NH-CO-CH (CH ₃ ) -NH ₂ ; more preferably NH ₂ ; -NH-CH ₃ ; -CH ₂ -NH ₂ ; -CH ₂ -CH ₂ -NH ₂ ; -NH-CO ₁ -CH ₂ -NH ₂ or -NH-CO-CH (CH ₃ ) -NH ₂ ; and most preferably NH ₂ ; -NH-CO-CH ₂ -NH ₂ or -NH-CO-CH (CH ₃ ) -NH ₂ .

0 ii / " \ ^R 11 is hydrogen, -COOH, -CONH ₂ , -0 - N 0 obsession -CO-R ₁₂ ; hydrogen, -COOH, -CONH _{2 are} preferred obsession the CO ' ^R 12' most preferably hydrogen, -COOH or -COOH ₂ .

R12 is an acyl group derived from a natural amino acid or a peptide consisting of 2 to 5 natural amino acids; a group derived from a natural amino acid is preferred; the residue derived from the amino acid or peptide is preferably linked via an amide bond to the central moiety.

^r13 is phenyl, hydroxyphenyl, naphthyl, indolyl, pyridyl, quinolyl, isoquinolyl or pyrrolyl, with phenyl, hydroxyphenyl or indolyl being preferred, and indolyl being particularly preferred.

Another preferred compound is represented by Formula (2) wherein

R 1 is CH ₂ , C ₂ H ₄ , S, CH ₂ -S, CH ₂ -SS, SS, NH, NH-CO, O, O-CH ₂ , H ₂ C-O, Se;

R ₂ is C 1-6 alkyl-R ₁₃ or -CO-NH-CH (C 1-3 alkyl-R ₁₃ ) R ₁₁ ;

R ₃ , R ₄ , R 8 and R 8 are independently hydrogen, methyl or ethyl;

R _{3 is} hydroxy or C 1-3 alkyl substituted by hydroxy;

one of R ₇ and R _{8 is} C 2 -C 5 alkyl substituted with -C (= NH) NH 2 or -NH-C (NH) NH 2 and the other is hydrogen, methyl or ethyl;

R ₁₀ is NH 2; ΝΗ ₇ substituted with C 1-4 alkyl; Substituted with C 1-4 alkylamino;

-NH-CH ₂ -CH ₂ -NH ₂ ;

R 1 is hydrogen, -COH, -CONH ₂ or -CO-R ₁₂ ;

an acyl group derived from the natural amino acid ^r 12 ^e 9Y,

R _{13 is} phenyl, hydroxyphenyl, naphthyl, indolyl, pyridyl, quinolyl, isoquinolyl or pyrrolyl.

Even more preferred is the group of formula (2) wherein

R 1 is CH ₂ , CH ₂ -CH ₂ , S, CH ₂ -S, CH ₂ -SS, SS, NH-CO, O, O-CH ₂ , CH ₂ O;

R ₂ is C 3 -C 6 alkyl-R ₂₃ or -CO-NH-CH (C 1 -C 3 alkyl-R ₁₃ ) R ₇ ] -;

R ₃ , R ₄ , R 8 and R 8 are each independently hydrogen, methyl or ethyl;

R ₃ is hydroxy, hydroxymethyl or hydroxyethyl;

One of R ₇ and R _{8 is} C 2 -C 5 alkyl substituted with -C (= NH) NH ₂ or -NH-C (= NH) NH ₂ ; and the other is hydrogen, methyl or ethyl;

R ₁₀ is NH ₂ ; C 1-2 alkyl

substituted NH2; C 1 -C 2 alkyl substituted with amino; -NH-CO-CH ₂ -NH ₂ , or NH-CO-CH (CH ₃ ) -NH ₂ ;

^R 11 is hydrogen, -COOH, -CONH 2, or -CO- ^R 12;

^r12 is an acyl group derived from a natural amino acid;

Is selected from the group consisting of phenyl, hydroxyphenyl and indolyl.

It is particularly preferred that a (2) wherein: ^R 1 is CH ₂ , ch ₂ -ch ₂ , s, ch ₂ -s, ch ₂ -ss, ss, nh- C 0; ^R 2 3-6 C alk alk alk alkyl-R ₂₃ or -CO-NH-CH-

_{(C1-3alkyl-R12);}

R ₃ , R ₄ , R 8 and R 8 are each independently hydrogen or methyl;

R ₃ is hydroxy, hydroxymethyl or hydroxyethyl;

One of R 7 and R ₈ is C 2 -C 5 alkyl substituted with -C (= NH) NH ₂ or -NH-C (= NH) NH ₂ ; the other is hydrogen, methyl or ethyl;

R ₁₀ is NH ₂ , -NH-CH ₃ ; -NH-CH ₂ -CH ₃ ; -CH ₂ -NH-CH ₂ -CH ₂ -NH ₂ ; -NH-CO-CH ₂ -NH ₂ or -NH-CO-CH (CH ₃ ) -NH ₂ ;

^R 11 is hydrogen, -COOH, -CONH _2, or -CO-R 1-4;

^R 12 is an acyl group of a natural amino acid;

R ₁₃ is phenyl, hydroxyphenyl or indolyl.

In a compound of formula (2) · * ··· «· *» »·

R 1 is CH ₂ , CH ₂ -CH ₂ , S, CH ₂ -S, CH ₂ -SS, SS, NH-CO, O-CH ₂ ;

R ₂ is -CO-NH-CH (C 1-3 alkyl-R ₁₃ ) R ₄₁ ;

R _{2 is} hydrogen or methyl,

R ₄ , R 8 and R 8 are hydrogen;

R8 is hydroxy, hydroxymethyl or hydroxyethyl;

One of R ₇ and Rq is -CH ₂ -CH ₂ -CH ₂ -NH-C (= NH) NH ₂ and the other is hydrogen;

R ₁₀ is NH ₂ ; -NH-CH ₃ -, -CH ₂ -NH ₂ ; -CH ₂ -CH ₂ -NH ₂ ;

-NH-CO-CH ₂ -NH ₂ or -NH-CO-CH (CH ₃ ) -NH ₂ ;

^R 11 is hydrogen, -COOH, or -CO-NH ₂ ;

And R ₁₂ is phenyl, hydroxyphenyl or indolyl.

In another compound of formula (2)

R L is CH ₂ , CH ₂ -CH ₂ , S, CH ₂ -S, CH ₂ -SS, SS, NH-CO, O-CH ₂ ;

R ₂ -CO-NH-CH (CH ₂ -R ₁₃ ) R ₁₁ ;

R _{3 is} hydrogen or methyl;

R ₄ , R 8 and R 8 are hydrogen;

R8 is hydroxy or hydroxymethyl;

One of R ₇ and R ₈ is -CH ₂ -CH ₂ -CH ₂ -NH-C (= NH) NH ₂ ; the other is hydrogen;

R ₁₀ is NH ₂ ; -NH-CO-CH ₂ -NH ₂ or -NH-CO-CH (CH ₃ ) -NH ₂ ; wherein NH ₂ is more preferred;

Rll is hydrogen, -COOH or _-CONH2;

R ₁₃ is an indolyl group.

Examples of compounds of the invention are (3), (4), · »··

(5), (6) , (7), (8) and (9) ). One another preferred ring compound of formula (2) hetero- cyclic compound wherein ^R 1 is CH ₂ , c ₂ H ₄ , c ₂ h ₂ , ch (ch ₃ ) -ch ₂ , s, ch ₂ , ch ₂ -ss, ss-ch ₂ , ss, S = O, 0 = S = 0, NH, NH-CO, 0, o-ch ₂ , CH ₂ -O, Se; CH _{2 is} preferred , c ₂ h ₄ , s, ch ₂ -s, ch ₂ -ss, s- S, NH, NH-CO, o, o-ch ₂ , ch ₂ -o, Neither ; CH _{2 is} particularly preferred , ch ₂ - -ch ₂ , s, ch ₂ -s, ss, ss -ch ₂ , NH-CO, O-CH ₂ ; ^R 2 is NH ₂ ; 1-4 alkyl-NH ₂

substituted with a group; - __ ^ NH; or -CO-R ₄₂ -NH ₂ ;

preferred is amino, C 1-4 alkyl substituted with amino,

- ^NZ 'NH, -CO-NH-CH ₂ -CO-NH ₂ -

-CO-NH-CH (CH ₃ ) -CO-NH ₂ or CO-NH-CH ₂ -CH ₂ -NH ₂ ; more preferably nh ₂ , -ch ₂ -nh ₂ , -ch ₂ -ch ₂ -nh ₂ , -co-nh-ch ₂ -co-nh ₂ , -CO-NH-CH (CH ₃ ) -CO-NH ₂ or -CO-NH-CH ₂ -CH ₂ -NH ₂ ; particularly preferably -CH ₂ -NH ₂ , -CO-NH-CH ₂ -CO-NH ₂ or -CO-NH-CH ₂ -CH ₂ -nh ₂

R ₃ , R ₄ , R 8 and R 8 are each independently hydrogen or C 1-4 alkyl; preferably hydrogen, methyl or ethyl; more preferably hydrogen or methyl, particularly preferably hydrogen;

one of R ₅ and R 8 is C 2 -C 5 alkyl substituted with -C (= NH) NH ₂ or -NH-C (= NH) NH ₂ ; preferred is -CH ₂ -CH ₂ -CH ₂ -NH-C (= NH) NH ₂ ; the other of these two is hydrogen or C 1-4 alkyl; hydrogen or methyl are preferred; hydrogen is particularly preferred.

R ₇ is hydroxy or C 1-4 alkyl substituted with hydroxy; hydroxy, hydroxymethyl and hydroxyethyl are preferred; more preferably hydroxyl and hydroxymethyl-1, especially hydroxymethyl.

R 14 is -NH- (C 1-6 -alkylaryl) or -NH-CO-CH (C 1-3 -alkylaryl) R ₁₂ , preferably -NH- (C 1-6 -alkyl) R -] - 3 or -NH-CO-CH (C 1-3 alkyl-R 1-4) R _{1: L} ; and -NH-CO-CH (C 1-3 alkyl-R 1-3) R _{1 - is} particularly preferred. ] ^R 11 is hydrogen, NH ₂ , -NH-CO (C 1-4 alkyl), or -NH-R ₁₂ , preferably hydrogen, NH ₂ , -NH-CO-CH 3, or -NHCOCH ₂ -CH ₃ or -NH-R ₁₂ and hydrogen, NH ₂ or NH-CO-CH _{3 are} particularly preferred.

^r 12 is made up from a natural amino acid or a natural amino acid peptide acyl group, wherein the number of constituent amino acids 2-5; preferred is an acyl group derived from a natural amino acid; wherein the residue derived from the amino acid or peptide is preferably linked by an amide bond to the central ring.

R 13 is phenyl, hydroxyphenyl, naphthyl, indolyl, pyridyl, quinolyl, isoquinolyl or pyrrolyl; phenyl, hydroxyphenyl or indolyl are preferred and indolyl is particularly preferred.

In another preferred compound of formula (2):

R L is CH ₂ , C ₂ H ₄ , S, CH ₂ -S, CH ₂ -SS, SS-CH ₂ , SS, NH, NH-CO, O, Se, O-CH ₂ , CH ₂ O;

• «

R ₂ is NH ₂ ; (C 1 -C 4) alkyl optionally substituted with NH ₂ ; - N ^ - NH; -CO-NH-CH ₂ -CO-NH ₂ ;

-CO-NH-CH (CH ₃ ) -CO-NH ₂ or -CO-NH-CH ₂ -NH ₂ ;

R 8, R 8 and R 8 are each independently hydrogen, methyl or ethyl;

one of R 8 and R 8 is C 2 -C 5 alkyl substituted with -C (= NH) NH ₂ or -NH-C (= NH) NH ₂ ; and the other is hydrogen, methyl or ethyl.

R ₇ is hydroxy or C 1-3 alkyl substituted with hydroxy;

R ₁₀ is -NH-C 1-6 alkyl-R ₁₃ or -NH-CO-CH (C 1-3 alkyl-R ₁₃ ) R ₁₄ ;

R _{1: 1} is hydrogen, NH ₂ , -NH-COCH ₃ , -NH-COCH ₂ -CH _3, or -NH-R ₁₂ ;

R12 is an acyl group of a natural amino acid; ^r 13 is phenyl, hydroxyphenyl, naphthyl, indolyl, pyridyl, quinolyl, isoquinolyl or pyrrolyl.

More preferred compounds of formula (2) are those wherein

R _x is CH ₂ , CH ₂ -CH ₂ , S, CH ₂ -S, CH ₂ -SS, SS-CH ₂ , SS, NH-CO, O, O-CH ₂ , CH ₂ -O;

R ₂ is NH ₂ ; C 1-4 alkyl substituted with amino; - N ^ - ^ NH; -CO-NH-CH ₂ -CO-NH ₂ ;

-CO-NH-CH (CH ₃ ) -CO-NH ₂ or -CO-NH-CH ₂ -CH ₂ -NH ₂ ;

R ₃ , R 8 and R 8 are independently hydrogen, methyl or ethyl;

One of Rg and Rg is a C2-C5 alkyl group, · ·

-C (= NH) NH ₂ or -NH-C (= NH) NH ₂ ; and the other is hydrogen, methyl or ethyl;

R ₇ is hydroxy, hydroxymethyl or hydroxyethyl;

R ₁₀ is -NH-C 3 -C 6 alkyl-R ₁₃ or -NH-CO-CH (C 1-3 alkyl-R ₄₃ ) R ₄₄ ;

R ₁ is hydrogen, NH ₂ , -NH-COCH ₃ , -NH-COCH ₂ -CH _3, or -NH-R ₄₂ ;

^r12 is an acyl group derived from a natural amino acid;

R ₁₃ is phenyl, hydroxyphenyl, naphthyl, indolyl, pyridyl, quinolyl, isoquinolyl or pyrrolyl.

A particularly preferred compound of formula (2) is that wherein

R L is CH ₂ , CH ₂ -CH ₂ , S, CH ₂ -S, CH ₂ -SS, SS-CH ₂ , SS, NH-CO, O, O-CH ₂ , CH ₂ -O;

R ₂ is NH ₂ ; -CH ₂ -NH ₂ ; -CH ₂ -CH ₂ -NH ₂ ; C 0 C _0-NH-CH2-NH2 -, -CO-NH-CH _(CH3) C 0-NH _2; or -CO-NH-CH ₂ -CH ₂ -NH ₂ ;

R ₃ , R ₄ , R 8 and R 8 are each independently hydrogen or methyl;

Rg and Rg are a hydrogen or a methyl group, the other is C2-4 alkyl group, which is -C (= NH) _NH2 or -NH-C (= NH) NH ₂ groups;

R ₇ is hydroxy, hydroxymethyl or hydroxyethyl;

R ₁₀ is -NH-C 3-6 alkyl-R ₁₃ or -NH-CO-CH (C 1-3 alkyl-R ₄₃ ) R ₄₄ ;

R 11 is hydrogen, NH ₂ , -NH-COCH ₃ or -NH-R ₁₂ ;

R12 is an acyl group derived from natural amino acids;

^r 13 is phenyl, hydroxyphenyl or indolyl.

In one of the compounds of formula (2)

R ₁ is S, CH ₂ -CH ₂ , CH ₂ -S, SS, CH ₂ -SS, NH-CO, O-CH ₂ , CH-O;

R ₂ is NH ₂ , -CH ₂ * NH ₂ , -CH ₂ -CH ₂ -NH ₂ , -CO-NH-CH ₂ -CO-NH ₂ , -CO-NH-CH (CH ₃ ) -CO-NH ₂ or -CO-NH-CH ₂ -CH ₂ -NH ₂ ;

R 8, R ₄ and R 8 are hydrogen;

One of R 8 and R 8 is hydrogen and the other is -CH ₂ -CH ₂ -CH ₂ -NH-C (= NH) NH ₂ );

R ₇ is hydroxy, hydroxymethyl or hydroxyethyl;

Rg is hydrogen or methyl;

R ₁₀ is -NH-CO-CH (C 1-3 alkyl-R ₇₃ ) R ₇₇ ;

R n is hydrogen, NH ₂ or -NH-COCH 8;

And R3 is phenyl, hydroxyphenyl or indolyl.

In another compound of formula (2)

R 8 is S, CH ₂ -S, SS, CH ₂ -SS, NH-CO;

R ₂ is -CH ₂ -NH ₂ , -CO-NH-CH ₂ -CO-NH _2, or -CO-NH-CH ₂ -CH ₂ -NH ₂ ;

R _3, R _4, Rg and R q is hydrogen;

R ₅ is -CH ₂ -CH ₂ -CH ₂ -NH-C (= NH) NH _2;

R ₇ is hydroxy or hydroxymethyl;

Rg is hydrogen or methyl;

R ₁₀ is -NH-CO-CH (CH ₂ -R ₁₃ ) R _11;

R 1-1 is NH ₂ or -NH-COCH ₃ ;

R13 is indolyl.

Examples of compounds of the invention are compounds of formulas 10 and 11.

The compounds of the invention are prepared in a manner known per se.

The synthesis of the compounds of the invention depends on the ring used. Crown ethers, steroids and porphyrins can be obtained commercially or isolated by standard methods. Functional groups of the compounds may be introduced by conventional methods of organic chemistry. The cyclic peptides are composed of various amino acids, and amino acid analogs or modified amino acids can be prepared by methods known per se.

Synthesis of modified amino acids is described in R.M. Williams: Synthesis of optically active α-amino acids,

Pergamon Press 1989; J.P. Greenstein and M. Winitz, Chemistry of Amino Acids, Whiley & Són, INC., 1961; R.J. Simon et al. Proc. Natl. Acad. Sci. USA (1992) 8.9, 93679371; E. Altmann et al. Helv. Chim. Acta (1991) 74, 800-806 and M. Rodrigez et al. Tetrahedron Lett. (1991), 32, 923926.

The preparation of the cyclic peptide according to the present invention can be accomplished by reacting an amide bonding fragment of a compound of formula (I) with another amide bonding fragment of a compound of formula (I). These first and second fragments are complementary to each other and are complementary to each other, so that the amide bond formed between the first and second fragments leads to the formation of compound (1). The above first and second fragments may contain free carboxyl and sulfoxy groups or reactive carboxyl or sulfoxy derivatives, and the first and second fragments above may contain free amino groups or reactive derivatives thereof, and these groups, except for the reactive group, they may be in protected form and these protecting groups may be removed and optionally salted or converted into free groups or other salts if desired. Suitable syntheses are described, for example, in M. Bodanszky, Peptide Chemistry, Springer Verlag, Berlin 1988, and E. Atherton and R.C. Sheppard, Solid Phase Peptide, Synthesis, IRL Press, Oxford, 1989.

Reactive carboxylic and sulfonic acid groups are particularly active ester and active anhydride groups, or active cyclic amides; as mentioned, reactive carboxylic acid groups may also be formed in situ.

The reactive carboxylic and sulfonic acid groups are particularly active ester and active anhydride groups or active cyclic amides; as mentioned above, reactive carboxylic acid groups may also be formed in situ, for example by using a suitable condensing agent.

The active esters of the acids are primarily those in which an unsaturated bond is attached to the bonding atom of the ester forming group, for example the type of vinyl ester such as real vinyl

esters (which can be obtained, for example, by transesterification of the ester with vinyl acetate; this is the vinyl ester method), carbamoyl vinyl esters can be obtained by treatment with isoxazolium reagent, this is the 1,2-oxazolium-Woodward method), obsession

1-lower alkoxyvinyl esters (which can be prepared by treating the corresponding acid with lower alkoxyacetylene;

), or amidino-type esters such as Ν, Ν'-disubstituted amidino esters (which can be prepared, for example, by treating the corresponding acid with the appropriate Ν, N'-disubstituted carbodiimide, such as Ν, N'-diisopropyl or N). , N'-dicyclohexylcarbodiimide; carbodiimide method) or N, N-disubstituted amidino esters (which can be prepared, for example, by treating the corresponding acid with an N, N-disubstituted cyanamide; cyanamide method). Further active esters include, for example, the corresponding aryl esters, especially phenyl esters, substituted with suitable electron withdrawing groups (these esters may be prepared, for example, by reacting a suitable acid with a suitably substituted phenol, such as 4-nitrophenyl, 4-methylsulfonyl). -phenol, 2,4,5-trichlorophenol, 2,3,4,5,6-pentachlorophenol, pentafluorophenol or 4-phenyl diazophenol, the reaction takes place in the presence of a condensing agent such as Ν, N'-dicyclohexylcarbodiimide, active aryl ester method) cyanomethyl ester (these can be prepared, for example, by treating a suitable acid with chloroacetonitrile in the presence of a base).

of appearance; cyanomethyl ester method), suitable thioesters, in particular phenylthioesters, which may be substituted, for example, by nitro (which can be prepared, for example, by treating the corresponding acid with substituted or unsubstituted thiophenol, its substituents including nitro groups and the carbide). active thiol ester method) or amino or amido esters (which can be prepared, for example, by treating the corresponding acid with an N-hydroxy amino or N-hydroxy amido compound such as N-hydroxysuccinimide, N-hydroxy). piperidine, N-hydroxyphthalimide or 1-hydroxy-1H-benzotriazole, for example, by the appropriate anhydride or carbodiimide method; active N-hydroxyester method)

Reactive anhydrides may be symmetric or may preferably be mixed anhydrides of these acids, for example anhydrides of inorganic acids such as acid halides, in particular acid chlorides (prepared by reacting the appropriate acid, including sulfonic acid, with a suitable halogenating agent such as thionyl chloride). or oxalyl chloride; acid chloride method).

Reactive anhydrides may be azides (which are prepared from an ester by treatment with a suitable hydrazide, nitric acid; azide method), anhydrides may be formed with carboxylic acid semi-derivatives such as carboxylic acid lower alkyl (semi) esters. (which can be prepared, for example, with the corresponding acid lower halo formic acid alkyl ester such as chloroformic acid lower alkyl ester, or a 1-lower alkoxycarbonyl-2-lower alkoxy-1,2-dihydroquinoline carboxylic acid); Treated with 2-ethoxy-1,2-dihydroquinoline; mixed o-alkylcarboxylic anhydride method).

Anhydrides may be formed with dihalogenated, especially dichlorinated, phosphoric acids (which may be prepared, for example, by treatment of the appropriate acid with phosphorus oxychloride;

phosphorus oxychloride method), anhydrides may also be formed from organic acids, such as mixed anhydrides of organic carboxylic acids (which may be prepared, for example, with an unsubstituted or substituted lower alkanecarboxylic acid or phenyl-lower alkanecarboxylic acid, e.g. - or by reaction with trifluoroacetic acid chloride (mixed carboxylic anhydride method). Mixed anhydrides may also be prepared from organic sulfonic acids (which may be prepared, for example, by treating the corresponding acid salt, such as its alkali metal salt, with the appropriate sulfonic acid halide, alkanesulfonic acid chloride or arylsulfonic acid chloride, such as methanesulfonic acid chloride or p-toluenesulfonic acid; sulfonic anhydride method).

The anhydrides may also be symmetric anhydrides (which may be prepared, for example, by condensation of the appropriate acid in the presence of a carbodiimide or 1-diethylaminopropane; symmetrical anhydride method).

Suitable cyclic amides are, in particular, amides of aromatic five-membered diaza-rings, such as amides of imidazoles, such as imidazole (which may be

Treated with?,? - carbonyldiimidazole; imidazole method) or pyrazole such as 3,5-dimethylpyrazole (which can be prepared, for example, by treating acid hydrazides with acetylacetone;

pyrazolide method) amides.

As mentioned, the carboxylic acid derivatives may also be formed in situ during the reactions.

For example

N, N'-disubstituted amidinoesters may be formed in situ from a mixture of a free amino group-containing additional moiety and a free carboxyl-containing peptide moiety, a suitable N, N-disubstituted carbodiimide such as N, N-diisopropyl or Ν, In the presence of N'-dicyclohexylcarbodiimide. Amino or amido esters of acids can be prepared in the presence of the amine to be acylated, wherein the acylation is carried out by reaction of the appropriate acid with an amino starting material, a Ν, N'-disubstituted carbodiimide such as N, N'-dicyclohexyl or Ν, N. in the presence of 'diisopropylcarbodiimide or in the presence of an N-hydroxyamine or N-hydroxyamide such as N-hydroxysuccinimide, preferably a suitable base such as 4-dimethylaminopyridine.

Alternatively, the process of the present invention may be carried out by reacting a fragment containing a free carboxyl group with a complementary moiety containing the amino group in a reactive form: for example, activating the amino group by reacting it with a phosphite such as diethyl chlorophosphite. , 1,1-phenylene chlorophosphite, ethyl dichlorophosphite, ethylene chlorophosphite, tetraethyl pyrophosphite, or a suitable silylating agent such as an organic halosilane such as trime. · _ 2 7 - **** ** * · · til-chlorosilane. The amino group may be activated by attachment to a halocarbonyl, such as chlorocarbonyl, or the activated form may be an isocyanate group.

Functional moieties of the reactive moieties, if not involved in the reaction, may be in protected form. These functional groups are preferably carboxyl, amino and hydroxyl groups, and may also be carbamoyl and quanidino groups.

The protecting groups, as well as the manner of their application and deprotection, are described in Protective Groups in Organic Chemistry, Plenum Press, London, New York, 1973, Methodology of Organic Chemistry Jouben-Weyl, 4th Edition, Vol. , Stuttgart, 1974, and Th. W. Greene, Protective Groups in Organic Synthesis, John Wiley and Sons, New York 1981. manuals. Protecting groups are characterized by the fact that they can be readily removed without unwanted side reactions such as solvolysis, reduction or photolysis.

Examples of hydroxy protecting groups are substituted or unsubstituted acyl groups such as halo-substituted lower alkanoyl such as

2.2-dichloroacetyl, or acyl groups of the carboxylic acid semi-esters, especially tert-butoxycarbonyl, unsubstituted or substituted benzyloxycarbonyl, such as 4-nitrobenzyloxycarbonyl or diphenylmethoxycarbonyl, or 2-halo-short carbon alkoxycarbonyl such as

2.2.2-trichloromethoxycarbonyl or formyl. Other hydroxy protecting groups are, for example, the corresponding ether-forming groups such as trityl, lower alkyl, such as tert-butyl, 2-oxa or 2-thiaaliphatic or cycloaliphatic hydrocarbons, especially 1-lower alkoxy-lower hydrocarbon groups. -alkyl or 1-lower thio-lower alkyl such as methoxymethyl, 1-methoxyethyl, 1-ethoxyethyl, methylthiomethyl, 1-methylthioethyl or 1-ethylthioethyl, or 2- oxa or 2-thiacycloalkyl, which is a 5 or 6 membered ring, for example 2-tetrahydrofuryl or 2-tetrahydropyranyl, or the corresponding thia analogs.

Further protecting groups may be unsubstituted or substituted 1-phenyl-lower alkyl, such as unsubstituted or substituted benzyl, diphenylmethyl. Substituents on the phenyl groups include, for example, halogen atoms such as chlorine, lower alkyl such as methyl groups and / or nitro groups. Further hydroxyl protecting groups may also be organic silyl or stanyl groups which preferably contain lower alkyl, preferably methyl and / or aryl groups such as phenyl, and the substituents may in particular be tri-lower alkylsilyl -, in particular trimethylsilyl, and also dimethylteryl-butylsilyl or appropriately substituted stannyl groups such as tri-n-butylstanyl.

Thiol groups may be protected analogously, for example, with trityl, acetamidomethyl or tert-butyl.

The carboxyl groups are preferably protected in an esterified form since the ester groups can be easily cleaved under mild conditions. The carboxyl thus protected29 · * ··

The esterification groups of the radicals are primarily lower alkyl, which may be branched at the 1-position or substituted at the 1-position or 2-position. Preferred esterified carboxyl groups include tert-lower alkoxycarbonyl groups such as tert-butoxycarbonyl, α-aryl lower alkoxycarbonyl groups having one or two aryl groups which may be phenyl. groups, whether or not substituted. For example, the substituents may be lower alkyl such as tertiary lower alkyl such as tert-butyl, lower alkoxy such as methoxy, hydroxy, halogen such as chlorine, nitro and / or phenyl. The groups thus formed are benzyloxycarbonyl, unsubstituted or substituted, for example, as mentioned above, 4-methoxybenzyloxycarbonyl or 4-nitrobenzyloxycarbonyl, biphenylyl-lower alkoxycarbonyl, wherein the biphenyl group is alpha position. An example is the 2- (p-biphenylyl) -2-propoxycarbonyl or diphenylmethoxycarbonyl group. The latter may also be substituted and the resulting groups are diphenylmethoxycarbonyl or di- (4-methoxyphenyl) methoxycarbonyl. The esterified carboxyl group may also be 1-lower alkoxy-lower alkoxycarbonyl such as methoxy, methoxycarbonyl, 1-methoxyethoxycarbonyl or 1-ethoxymethoxycarbonyl, 2,2-diaryl-ethoxycarbonyl. wherein the aryl group is phenyl and may be substituted. The substituent may be a nitro group such as a 4-nitrophenyl ring, and the esterified carboxyl group thus formed is 2,2-di- (4-nitrophenyl) ethoxycarbonyl, wherein the two aryl groups are, for example, phenyl. may. The aryl groups may also be fused. For example, 2- (9-fluorenyl) ethoxycarbonyl is formed. The esterified carboxyl groups may also be the following: 1-lower alkylthio-lower alkoxycarbonyl, e.g.

1-methylthiomethoxycarbonyl or 1-ethylthioethoxycarbonyl, aroylmethoxycarbonyl, in which case the aroyl may be benzoyl and may carry a substituent such as halogen such as bromine. The esterified carboxyl group may also be phenacyloxycarbonyl, 2-halo-lower alkoxycarbonyl, such as 2,2,2-trichloroethoxycarbonyl,

2-bromomethoxycarbonyl or 2-iodoethoxycarbonyl. The esterified carboxyl group may also be a 2- (trisubstituted silyl) ethoxycarbonyl group, each substituent independently being an aliphatic, araliphatic, cycloaliphatic or aromatic hydrocarbon group which may be substituted.

These substituents may be lower alkyl, lower alkoxy, aryl, halogen, and / or nitro, such as unsubstituted or appropriately substituted lower alkyl, phenyl-lower alkyl, cycloalkyl, or phenyl. The esterified carboxyl groups thus formed are 2-tri-lower alkylsilyl-ethoxycarbonyl such as 2-trimethylsilyl-ethoxycarbonyl or 2- (methyldi- (n-butyl) silyl) ethoxycarbonyl. - or 1-triarylsilyl-ethoxycarbonyl such as 2-triphenylsilylethoxycarbonyl.

Preferred protected groups are, for example, tert-lower alkoxycarbonyl such as tert-butoxycarbonyl and benzyloxycarbonyl, which may also be substituted. The esterified carboxyl groups thus formed are 4-methoxy or 4-nitrobenzyloxycarbonyl or diphenylmethoxycarbonyl and 2- (trimethylsilyl) ethoxycarbonyl.

Examples of protected amino groups include acylamino, arylmethylamino, etherified mercaptoamino, 2-acyl-lower alk-1-enylamino, silylamino or stannylamino, or the amino group in azido form. it can be.

In a suitable acylamino group, the acyl moiety may be, for example, a suitable moiety of an organic carboxylic acid, for example a C 18, especially alkane carboxylic acid, which may also be substituted, for example, with halogen, such as chlorine, lower alkoxy such as methoxy, nitro and / or phenyl. - or with a carboxylic acid half-ester group. Examples of such acyl groups include lower alkanoyl such as formyl, acetyl or propionyl, halo lower alkanoyl such as 2-haloacetyl, especially 2-chloro, 2-bromo, 2-iodo, , 2,2-trifluoro, or 2,2,2-trichloroacetyl. The acyl moiety may be a benzoyl group, with or without a substituent, which may be halogen, lower alkoxy, nitro and / or phenyl, such as benzoyl, 4-chlorobenzoyl, 4-methoxybenzoyl, or a nitrobenzoyl group. The acyl group may also be lower alkoxycarbonyl, lower alkyl branched at the 1-position, or substituted at the 1 or 2 position, such that it may be lower-alkoxycarbonyl, such as tert-butoxycarbonyl, lower alkenyl, for example, allyloxycarbonyl-α-aryl-lower alkoxycarbonyl, which may be substituted with one or two aryl groups, preferably substituted or unsubstituted phenyl. The substituents may be lower alkyl, in particular tertiary-lower alkyl such as tert-butyl. Substituents may also be lower alkoxy such as methoxy, hydroxy, halogen such as chlorine, nitro and / or phenyl, such as acyl groups which may be substituted or unsubstituted benzyloxycarbonyl, e.g. 4-nitrobenzyloxycarbonyl, biphenylyl-lower alkoxycarbonyl wherein the biphenylyl is in the α-position, for example 2- (p-biphenylyl) -2-propoxycarbonyl, or substituted diphenylmethoxycarbonyl such as benzhydryloxycarbonyl di- (4-methoxyphenyl) methoxycarbonyl, 2,2-diarylethoxycarbonyl, wherein the phenyl may be substituted. The substituent may be a nitro group so that the cyclic group is a 4-nitrophenyl group and the acyl group is a 2,2-di- (4-nitrophenyl) ethoxycarbonyl group. Here, for example, the two aryl groups may be phenyl or they may be linked together, so that the acyl group may be 9-fluorenylmethoxycarbonyl, aroylmethoxycarbonyl, where the aroyl group is preferably benzoyl. The latter may also have substituents such as halogen such as bromine. Thus, the entire group may be phenacyloxycarbonyl, 2-halo-lower alkoxycarbonyl, for example, 2,2,2-trichloroethoxycarbonyl, 2-bromoethoxycarbonyl or 2-iodoethoxycarbonyl,

-phenylsulfonyl-1-ethoxycarbonyl, nyl-phenylsulfonyl) -ethoxycarbonyl-tapped-silyl) -ethoxycarbonyl, preferably substituted as 2 - (4-methylsulfov or 2 - (tri-substituents) and the substituents are independent and may be , cycloaliphatic or aromatic hydrocarbon groups having up to 15 carbon atoms, which may also be substituted: lower alkyl, lower alkoxy-aryl, halogen, nitro, so that the groups are unsubstituted or unsubstituted. substituted lower alkyl, phenyl-lower alkyl, cycloalkyl or phenyl, and the acyl groups are formed as follows: e.g., 2-tri-lower alkylsilyl-ethoxycarbonyl, such as 2-trimethylsilyl-; ethoxycarbonyl or 2- (di-n-butylmethylsilyl) ethoxycarbonyl or 2-triarylsilyl-ethoxycarbonyl such as 2-triphenylsilyl-ethoxycarbonyl-c. soport.

Other suitable acyl groups for protecting the amino group include organic phosphoric acid, phosphonic acid or phosphinic acid such as di-lower alkyl phosphoryl such as dimethylphosphoryl, diethylphosphoryl, di-n-propylphosphoryl or diisopropylphosphoryl. , dicycloalkylphosphoryl, such as dicyclohexylphosphoryl, substituted or unsubstituted diphenylphosphoryl, such as diphenylphosphoryl, di- (phenyl-lower alkyl) phosphoryl, such as nitro-substituted or unsubstituted, e.g. or di (4-nitrobenzyl) phosphoryl, unsubstituted or substituted phenoxyphenylphosphonic acid, such as phenoxyphenylphosphonyl, di-lower alkylphosphinyl, such as diethylphosphinyl, or unsubstituted or substituted diphenyl, e.g.

In an arylmethylamino group, which may be mono-, di- or triarylmethyl, the aryl groups are unsubstituted or may also be substituted phenyl groups. These groups include, for example, benzylamino, diphenylmethylamino and especially tritylamino.

The etherified mercapto protecting group for an amino group may be primarily an arylthio or aryl-lower alkylthio group in which the aryl group is particularly phenyl and may be substituted. The substituents may be lower alkyl such as methyl or tert-butyl, lower alkoxy such as methoxy, halogen such as chlorine, and / or nitro. Suitable amino protecting groups include, for example, 4-nitrophenylthio.

An acyl group in a 2-acyl lower alk-1-en-1-yl group used as an amino protecting group may be, for example, an acyl group of a lower alkanecarboxylic acid or an acyl group of a substituted or unsubstituted benzoic acid. ......

ja. The substituents may be lower alkyl such as methyl or tert-butyl, lower alkoxy such as methoxy, halogen such as chloro and / or nitro. The protecting group may be a carboxylic acid semester ester, such as a lower carboxylic acid ester. Suitable protecting groups are in particular 1-lower alkanoylprop-1-en-2-11-, for example 1-acetylprop-1-en2-yl or 1-lower alkoxycarbonylprop-1-en-2-11 for example, 1-ecoxycarbonylprop-1-en-2-yl-

- group.

The amino group may also be protected in protonated form; Suitable anions include those of particularly strong inorganic acids, such as the corresponding anions of hydrohalides, such as chloride or bromide, or organic sulfonic acids such as p-toluenesulfonic acid.

Preferred amino protecting groups are the acyl groups of the carboxylic acid semi-esters, in particular the tert-butoxycarbonyl, allyloxycarbonyl or benzyloxycarbonyl group, with or without a substituent. Protecting groups include 4-nitrobenzyloxycarbonyl or diphenylmethoxycarbonyl, 9-fluorenylmethoxycarbonyl, or 2-halo-lower alkoxycarbonyl such as 2,2,2-trichloroethoxycarbonyl; and trityl or formyl

- group.

Unsubstituted carbamoyl groups may be protected, for example, in the form of N- (9-xanthenyl) derivatives or N- (mono-, di- or tri-arylmethyl) derivatives, where the aryl group is particularly phenyl, it may be unsubstituted. or up to five identical or different substituents. These substituents may be: preferably lower alkyl such as methyl, lower alkoxy such as methoxy. Thus, the arylmethyl protecting groups will be: 4-methoxybenzyl, 2,4,6-trimethoxybenzyl, diphenylmethyl, di- (4-methoxyphenyl) methyl, di- (4-methylphenyl) methyl and (4-methylphenyl) ([polymeric carrier] phenyl) methyl. A preferred carbamoyl protecting group is the trityl group.

Guanidino groups may be protected, for example, by protonation or nitro or by an appropriately substituted sulfonyl group. Such arylsulfonyl groups are those wherein the aryl group may be phenyl and unsubstituted or substituted. The substituents may be lower alkyl such as methyl. The substituent may also be a benzo-heterocyclic group, such as a chromanyl group, which may be substituted or unsubstituted. The substituents may be lower alkyl such as methyl and may be attached to an aromatic carbon atom. These include 4-methoxy-2,3,5- (trimethyl) phenylsulfonyl or 2,2,5,7,8-pentamethyl-6-chromanylsulfonyl.

According to the invention, a polymeric carrier may also be used as a protecting group, in particular a carboxyl protecting group, to which is attached a functional group to be protected, in particular a carboxyl group. This carrier is particularly suitable for so-called Merrifield peptide synthesis and is readily removable. For example, such a polymeric carrier is preferably a weakly cross-linked polystyrene resin with divinyl 3 · 4 · benzene · · · · ······························• copolymerization which contains resin bridge elements which are capable of reversibly binding the amino acid and peptide residues. In particular, the bridging elements of the aforementioned weakly crosslinked polystyrene resin are primarily methylene groups which may be substituted and bind directly to the aromatic groups of the polystyrene resin. Substituents on the methylene groups are preferably attached to the methylene groups via an ether or ester group and contain suitable functional groups to bind to the functional groups to be protected. These protecting groups are primarily carboxylic groups of the peptide fragment or amino acid. Thus protected groups, in particular protected carboxyl groups such as esterified carboxyl groups, are formed.

Such bridge elements are, for example, divalent radicals of 4-methoxybenzyl alcohols, which preferably contain a lower alkoxy group, such as methoxy group or phenyl group. The phenyl group may also be substituted, for example, in the o or p position, it may be attached to a lower alkoxy such as methoxy. In these 4-methoxybenzyl alcohols, the carbon atom of the 4-methoxy group is directly attached to the phenyl group of the polystyrene resin and the benzyl alcohol hydroxyl group esterifies the carboxyl group of the amino acid or peptide moiety.

The formation of the amide group can be carried out in a manner known per se, the reaction conditions depending on whether and how the carboxyl group involved in the reaction was activated. The reaction is usually carried out in the presence of a suitable solvent in the presence of either a diluent or a mixture thereof. and, if necessary, a condensing agent, for example, if the carboxyl group involved in the reaction is in the anhydride form, a suitable acid acceptor may be required. The reaction may be carried out under heating or cooling, e.g. -30 ° C and approx. 150 ° C, especially + 10 ° C to + 70 ° C, preferably room temperature (about + 20 ° C) to 50 ° C, in a closed reaction space and / or in an inert gas atmosphere such as nitrogen.

Conventional condensing agents include carbodiimides such as Ν, N ¹ -carbodiimide, Ν, N ¹ -diisopropylcarbodiimide, Ν, N'-dicyclohexyl or N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide. Some carbonyl compounds such as carbonyldiimidazole or 1,2-oxazolium compounds such as 2-ethyl-5-phenyl-1,2-oxazolium-3'-sulfonate and 2-ters are suitable for this purpose. butyl 5-methylisoxazolium perchlorate, a suitable acylamino compound such as 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline. The condensing agent may also be a uronium compound such as 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTV) or a phosphonium compound such as benzotriazol-1-yl. oxytris tris (dimethylamino) phosphonium hexafluorophosphate (BOP) or benzotriazol-1-yloxypyrimidinophosphonium hexafluorophosphate (PyBOP). Conventional acid scavengers are, for example, alkali metal carbonates or bicarbonates, such as sodium carbonate, potassium carbonate, sodium bicarbonate or potassium bicarbonate (optionally with a sulfate). Acid-binding agents may be bases, as is usually the case.

R Q ······

O - / --········ Spherically inhibited tri-lower alkylamines, such as N, N-diisopropyl-N-ethylamine.

The aforementioned Merrifield peptide synthesis is particularly suitable for semi-automatic and automatic synthesis of compounds of formula 2a, wherein the amino acids and / or peptide fragments and / or non-peptide moieties, in which non-reactive functional groups are usually in protected form, amide groups without attaching the resulting peptide fragments. One of the functional groups is usually the terminal carboxyl group at the peptide end, preferably having a bridge member attached to a suitable polymeric carrier as described above. In principle, this process variant can be derived in a manner analogous to conventional peptide synthesis. Care should be taken to create reaction conditions in the already constructed (peptide) moiety, which contains the polymeric carrier, when the protected functional group is liberated, usually the terminal amino group that will be present in the reaction, so that the cleaved protecting groups do not react again. in the reaction.

Carboxyl, amino, hydroxy, carboxylic acid amide, carbamoyl and / or quanidino protecting groups may be removed in a manner known per se, for example by β-elimination, solvolysis, in particular hydrolysis (in acidic or basic media), alcoholysis, acidolysis or basic processes. or by reduction, in particular by hydrogenolysis or chemical reduction, preferably stepwise or simultaneously, by enzymes.

A 2-position short-carbon procedure may also be used.

The t-lower alkoxycarbonyl or lower alkoxycarbonyl substituted with an organic silyl group or a 1-position alkoxy or lower alkylthio group, or an unsubstituted or substituted diphenylmethoxycarbonyl group can be converted, for example, into the corresponding free carboxyl group. treated with acid. Suitable nucleophilic lower alkanecarboxylic acids, which may also be halogen, such as formic acid or trifluoroacetic acid, with or without neucleophilic addition, are phenol or anisole. The unsubstituted or substituted benzyloxycarbonyl group can be liberated, for example, by hydrogenolysis, i.e., by treatment with hydrogen, in the presence of a metal hydrogenation catalyst, such as palladium. In addition, a substituted benzyloxycarbonyl group, such as 4-nitrobenzyloxycarbonyl, can also be converted by chemical reduction to a free carboxyl group, for example, by treatment with an alkali metal dithionite, e.g. as a chromium (II) salt such as chromium (II) chloride. In the presence of a hydrogen donor, the metal is capable of producing nascent hydrogen, in particular carboxylic acid, lower alkanecarboxylic acid, optionally substituted with e.g. with hydroxy groups such as acetic acid, acetic acid, glycolic acid, diphenylglycolic acid, lactic acid, mandelic acid, 4-chloro-mandelic acid, or tartaric acid or alcohol or thiol, addition of water is preferred. The 2,2-diarylethoxycarbonyl or 2- (9-fluorophenyl) ethoxycarbonyl 2- (9 · · · · · · · · · · · · · · · · · · can be cleaved (i.e., the carboxyl group is liberated). Treated with a reducing metal or metal salt as described above, it can also be 2-halo-lower alkoxycarbonyl (optionally converted from the 2-bromo-lower alkoxycarbonyl group to the corresponding 2-iodo-lower alkoxycarbonyl group) or aroylmethoxy can be converted to a carboxyl group. The aroylmethoxycarbonyl group may also be cleaved by treatment with a nucleophile, preferably a salt-forming reagent such as sodium thiophenolate or sodium iodide. The substituted 2-silylethoxycarbonyl group can also be converted to the free carboxyl group by treatment with a fluoride salt which gives the fluoride anion. These include alkali metal fluorides, such as sodium or potassium fluoride. The reaction is carried out in the presence of a macrocyclic polyether (crown ether). The reaction may also be carried out in the presence of a fluoride of an organic quaternary base. Examples include tetra-lower alkylammonium fluoride or tri-lower arylammonium fluoride such as tetraethylammonium fluoride or tetrabutylammonium fluoride, an aprotic polar solvent such as dimethylsulfoxide or N, in the presence of dimethylacetamide.

The protected amino groups may be liberated by methods known per se, but solvolysis or reduction is preferred. The 2-halo-lower alkoxycarbonylamino (preferably the 2-bromo-lower alkoxycarbonylamino group previously converted to the 2-iodo-lower alkoxycarbonylamino group) is an aroylmethoxycarbonylamino or 4-nitro-42-alkoxycarbonylamino group. The robenzyloxycarbonylamino group may be cleaved, for example, by treatment with a suitable chemical reducing agent such as zinc in the presence of a suitable carboxylic acid such as aqueous acetic acid. The aroylmethoxycarbonylamino group may be cleaved with a nucleophile, preferably a salt-forming reagent such as sodium thiophenolate, and the 4-nitrobenzyloxycarbonylamino group may be cleaved with an alkali-phen-dithionite such as sodium dithionite. Unsubstituted or substituted diphenylmethoxycarbonylamino, tert-lower alkoxycarbonylamino or 2-trisubstitutedsilylethoxycarbonylamino can be cleaved by treatment with an appropriate acid. These include the lower alkanecarboxylic acids which they may also be, for example, halogen such as fluorine. Such acids include formic acid and trifluoroacetic acid. 2,2-Diarylethoxycarbonylamino such as 2,2-di- (4-nitrophenyl) ethoxycarbonylamino, 2- (4-methylsulfonylphenylsulfonyl) ethoxycarbonylamino and 9-fluorenyl- the methoxycarbonylamino group may be cleaved by treatment with a suitable base such as an aliphatic, preferably a secondary amine such as piperidine. The amino group may be liberated from an unsubstituted or substituted benzoxycarbonylamino group, for example by hydrogenolysis, i.e. hydrogen, in the presence of a suitable hydrogenation catalyst such as palladium. The amino group may also be liberated from a triarylmethylamino or formylamino group, which may be unsubstituted or substituted, for example, by treatment with an acid, a mineral acid such as hydrochloric acid, or an organic acid such as formic acid, acetic acid, or trifluoroacetic acid.

Λ 7 ···· ♦ · with or without water. The amino group may also be liberated from an organic silylamino group, for example by hydrolysis or alcoholysis. An amino group protected with a 2-haloacetyl such as 2-chloroacetyl may be liberated, for example, by treatment with thiourea in the presence of a base, or by means of a thiolate salt such as an alkali metal foil, alcoholysis or hydrolysis. An amino group protected by a 2-substituted-silyl ethoxycarbonyl group can be converted to a free amino group by means of a fluoride salt providing a fluoride ion, as described above for the release of a similarly protected carboxyl group.

The azido-protected amino group can be converted to the free amino group, for example by reduction such as catalytic hydrogenation in the presence of a hydrogenation catalyst. Such catalysts include platinum oxide, palladium or Raney nickel. It may also be hydrogenated with zinc in the presence of an acid such as acetic acid. The catalytic hydrogenation can be carried out preferably in an inert solvent such as an alcohol such as methanol, tetrahydrofuran or water, or in a mixture of water and an organic solvent such as alcohol, dioxane at about 20 ° C to 25 ° C or under cooling or heating.

A hydroxy group protected with a suitable acyl group or a bitter silyl group or an unsubstituted or substituted 1-phenyl-lower alkyl group may be terminated in a manner analogous to a similarly protected amino group. -.

• «·· <

. . * «♦» · *

- ···· · * · ♦ * starve. The hydroxy group protected by the 2,2-dichloroacetyl group can be, for example, by basic hydrolysis, a tertiary lower alkyl group such as tert-butyl group, or a 2-oxa or 2-thiaaliphatic or cycloaliphatic hydrocarbon group. the etherified hydroxy group may be liberated by acidolysis such as treatment with a mineral acid or a strong carboxylic acid such as trifluoroacetic acid.

A 9-xanthenyl-protected carboxylic acid amide group can be liberated, for example, with hydrogen bromide and glacial acetic acid or hydrogen fluoride in the presence of anisole. A carboxylic acid group protected by a mono-, di- or triarylmethyl group can be liberated by treatment with hydrogen fluoride in the presence of anisole; a diphenylmethyl protecting group may be removed, for example, by hydrogenolysis in the presence of a palladium on carbon catalyst, and the di- (4-methoxyphenyl) methyl or 2,4,6-trimethoxybenzyl protecting group removed, for example, by treatment with trifluoroacetic acid.

Organo-sulfonyl-protected guanidino groups such as 4-methylphenylsulfonyl or 2,2,5,7,8-pentamethyl-6-chromanylsulfonyl guanidino can be liberated with the appropriate acid, TFA. The nitro-protected guanidino groups can be liberated by hydrogenolysis in the presence of palladium on carbon or by cationic reduction.

The protected functional groups, in particular the protected carboxyl groups in which the protecting group is also present as a carrier in the aforementioned Merrifield reaction, may be liberated in a manner known per se, for example as described above. A suitably esterified carboxyl group attached to the polymer support by a suitable bonding agent may be liberated according to the nature of the bonding member. For example, a carboxyl group attached via an ester group to the polymer support having an activated benzyl linker, such as a 4-methoxybenzyloxycarbonyl group in which the carbon of the methoxy group, for example, is a polystyrene resin. which is weakly bound to divinylbenzene, may be liberated in analogy to those described above for the unsubstituted or substituted benzyloxycarbonyl group, for example, by treatment with an appropriate acid such as trifluoroacetic acid.

If desired, and if we have more than one functionality, the protecting groups may be selected so that the protecting groups can be removed simultaneously, for example by acidolysis, trifluoroacetic or formic acid treatment, reduction with zinc and acetic acid, or hydrogen in the presence of a hydrogenation catalyst. -carbon catalyst.

Cyclization is generally performed after the synthesis of the linear precursor is completed, for example, by a peptide bond between the corresponding amino acids and then by the removal of any remaining protecting groups. When the cyclization is by disulfide or sulfide bonding, the protecting groups are usually removed prior to oxidative ring formation using oxygen, normal air, disodium disulfide, 1,2-diiodoethane, or an oxidizing agent. The disulfide bond formed can subsequently be modified by desulfurization, thioether bonding, and oxidation.

The present invention also provides a pharmaceutical composition comprising one or more compounds of Formula (I), which are present in a pharmaceutically effective amount, optionally containing conventional excipients, typical excipients and diluents.

The compositions of the invention may be formulated for enteral, oral, rectal and parenteral use, such as for the treatment of warm-blooded subcutaneous, intravenous or intraperitoneal routes. These formulations may contain the active ingredient alone or together with a pharmacologically acceptable carrier. The daily dose, vehicle, frequency and route of administration will depend upon the age and individual circumstances of the subject being treated, as well as the mode of treatment and the particular activity of the compound selected. An effective amount of a compound of Formula (I), which has an inhibitory effect on bone resorption, can range from about 1 µg to about 1 g, or more, in a patient weighing about 70 kg. The unit dosage forms may contain from 10 pg to 10 mg, preferably from 10 µg to 500 µg, particularly preferably from 10 µg to 100 µg.

The new pharmaceutical compositions may have an active ingredient content of 10-80%, preferably 20-60%. Dosage units for enteral or parenteral administration typically include dragees, tablets, capsules, suppositories, and ampoules. These dosage units are prepared in a manner known per se by conventional mixing, granulating, confectioning, dissolving or lyophilizing processes.

Suitable carriers include, in particular, fillers such as sugars, traditionally lactose, beet sugar, mannitol or sorbitol; cellulose formulations and / or calcium phosphates, typically tricalcium phosphate or calcium hydrogen phosphate. Conventional excipients are binders, such as starch paste, traditionally made from corn starch, wheat starch, rice starch or potato starch. The binder is gelatin, tragacanth, methylcellulose and / or polyvinylpyrrolidone. If desired, disintegrating agents such as the above starch compositions, and also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, and salts thereof such as sodium alginate may be used. Excipients are lubricants, flow control agents, alginic acid and especially lubricants, traditionally silica gel, talc, stearic acid or salts thereof, typically magnesium stearate or calcium stearate and / or polyethylene glycol. The dragee cores may be coated with a suitable enteric or insoluble coating, typically a concentrated sugar solution, which may contain gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol and / or titanium dioxide, a solution of shellac in a suitable organic solvent or solvent mixture. For enteric coatings, solutions of suitable cellulose formulations such as acetylcellulose beverage or hydroxypropylmethylcellulose beverage are used. Dyes or pigments may be added to the tablets or dragee coatings, traditionally to distinguish and identify the various dosage units.

Other oral pharmaceutical formulations include dry filled gelatin capsules and soft gelatin capsules containing glycerol or sorbitol as a plasticizer. Dry-filled capsules may contain the active ingredient in granular form, conventionally with or without the addition of a lactose filler, starch binder and / or talc or magnesium stearate lubricant and stabilizer. In soft gelatine capsules, the active ingredient may preferably be dissolved or suspended in a suitable liquid, typically fatty oil, paraffin oil or liquid polyethylene glycol, in which a stabilizer may be dissolved.

Pharmaceutical compositions for rectal administration are typically suppositories consisting of a combination of the active ingredient and a suppository base. Suitable starting materials are natural or synthetic triglycerides, paraffinic hydrocarbons, polyethylene glycols and long-chain alkanols. Gelatin capsules may also be used for rectal administration which contain a mixture of the active ingredient and the excipient. Suitable starting materials are typically liquid triglycerides, polyethylene glycol or paraffin hydrocarbons.

Parenteral compositions are preferably formulated as an aqueous solution in which non-toxic salts may be dissolved or may contain a suspension of the active ingredient. Suitable lipophilic solvents for oily injection suspensions are either fatty oils, typically sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides. Aqueous injection suspensions contain viscosity enhancing agents, traditionally sodium • ·

carboxymethylcellulose, sorbitol and / or dextran and optionally a stabilizer.

The invention also relates to the use of compounds of formula (I), preferably in the form of a pharmaceutical composition. The dosage of the active ingredient will also depend on the species of warm-blooded animal, the age, the individual condition of the patient and the mode of administration.

THE The present invention relates to a compound of formula (I), which may be used to treat the human or animal body. mazni. THE The compounds of the invention may be used in the a

bone resorption, i.e., osteoporosis, Paget's bone disease, humoral hypercalcaemia, or malignant or metastatic bone disease.

The invention is further illustrated by the following non-limiting examples.

abbreviations: Ac acetyl group Acm acetamidomethyl Boc -Butoxy-carbonyl tere. brine = saturated NaCl solution in water Bzl benzyl DCCI = dicyclohexylcarbodiimides DCE 1,2-dichloretán DCM dichloromethane DIBAH = diisobutylaluminum hydride DICD = diisopropylcarbodiimide DIPE = isopropyl ether

DIPEA = diisopropylethylamine

DMA = dimethylacetamide

DMAP = p-N-dimethylaminopyridine

DME = 1,2-dimethoxyethane

Dpr = L-diaminopropionic acid

Fmoc = 9-fluorenylmethoxycarbonyl

HOBt = 1-hydroxybenzotriazole

NArg = glycine derivative N-substituted with an arginine side chain

Pmc = 2,2,5,7,8-pentamethylchroman-6-sulfonylTBTU = 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate

TCA = trichloroacetic acid

TFA = trifluoroacetic acid

THF = tetrahydrofuran

Tra = tryptamine

Trt = trityl

MALDI = matrix-assisted laser desorption ionization mass spectrometry

Tlc = Thin Layer Chromatography on Silica Gel (Merck Silica Gel 60, F254 plates)

Marking: UV light

CMW = chloroform, methanol, water (70: 30: 5 v / v);

CM = chloroform-methanol mixture (85:15 v / v);

Z = benzyloxycarbonyl

TAB ester resin = hydroxy trialkoxybenzhydryl resin Wang resin = Fmoc-1C terminal amino acid p-benzyloxybenzyl ester polystyrene resin • ·

Unless otherwise noted, all reactions are performed at room temperature.

General procedure for solid phase synthesis of a linear precursor

Synthesis begins with a FmocC-terminal amino acid p-benzyloxybenzyl ester polystyrene resin (1% Wang Wang resin, Novabiochem, Laenfelfingen, Switzerland). A fully automatic peptide synthesis device is used which is capable of alternating removal of amino protecting groups, in this case removal of Fmoc groups, and coupling of Fmoc amino acid derivatives without isolation of peptide / resin intermediates, which are formed at each step of the synthesis. The trifunctional amino acids are introduced as appropriately protected derivatives of L and D configurations: Fmoc-Ser (But), Fmoc-Cis (Trt), Fmoc-Arg (Pmc), Boc-Dpr (Fmoc), Fmoc-Trp (Boc) or Fmoc-Trp.

Example 1

Synthesis of compound (3)

1.1. Example: Synthesis of linear precursor

Starting with 1.12 g of Fmoc-Trp-Wang resin (about 0.35 mM, Novabiochem), the following process steps are performed, the following being typical of each process:

- single wash for 0.8 minutes with isopropanol;

- pre-activation for first coupling: 1.4 mM of the partially protected Fmoc-amino acid is dissolved in a mixture of 3.08 ml of a 0.5 M HOBt-DMA solution and 0.77 ml of a 2 M DICD-DMA solution. The reaction mixture was stirred for ca. Allow to stand for 40 minutes and then use it in this form. Meanwhile, the following washes and Fmoc protecting groups were removed;

eight processes on starting resin, each lasting 1 minute, with 20% pyridine-DMA solution (removal of Fmoc protecting groups);

three washes, each lasting 0.4 minutes, with DMA (previously degassed under reduced pressure);

- single wash for 0.8 minutes with isopropanol;

five washes, each lasting 0.4 minutes, (degassed)

DMA;

- addition of a reagent, already prepared at an early stage, for coupling (see above). The reaction mixture was cooled to ca. shake for an hour. Twice 5 minutes and 15 minutes after the start of coupling, 120 μΐ DIPEA (0.7 mM) was added;

60 minutes later, removal of the coupling medium;

- two washes for 0.4 minutes with (degassed) DMA;

- single treatment 5 prig. 15 ml of a 1: 1: 8 (v / v / v) acetic anhydride: pyridine: DMA solution (to acetylate the remaining free amino groups of the growing peptide chain);

- 4 washes for 0.4 minutes each with (degassed) DMA;

- 2 washes for 0.8 minutes each with isopropanol.

This yields the following Fmoc peptide / resin intermediate:

Fmoc-Cis (Trt) -Arg (Pmc) -Ser (Butt) -D-Cis (Trt) -Trp resin from the sequential coupling of the following elements Fmoc-D-cis (Trt), Fmoc-Ser (Butt), Fmoc -Arg (Pmc) and Fmoc-Cis (Trt); wherein the resin is polystyrene esterifying carboxyl groups (1% crosslinked with methoxy-4-phenylmethoxy groups).

After removal of the Fmoc protecting group from the N-terminal cysteine residue and extensive washing with DMA and isopropanol as described above, the resin was dried in vacuo (1.45 g).

Example 1.2: Removal of resin and protecting groups

Shake 0.3 g of Cis (Trt) -Arg (Pmc) -Ser (Butt) -D-Cis (Trt) -Trp resin (about 63 μπιόΐ) twice for 5 min to remove the polymeric carrier and acid-labile protecting groups. ml of 85:15 (v / v) TFA (95%) + ethanedithiol and filtered. The residue was washed three times with 4-4 ml of DCE and also three times with 4-4 ml of TFE. The filtered solution and the washings are combined under reduced pressure for approx. It is reduced to 2 ml and the crude peptide is precipitated with 15 ml of a 1: 1 (v / v) DIPE: petroleum ether (low boiling) mixture. The precipitate was collected by filtration, washed with 5 ml of precipitated mixture and dried under reduced pressure.

For complete deprotection, the solid (64 mg) was dissolved in 4 mL of the above liquid mixture which was used to cleave the protecting groups. The solution was allowed to stand for two hours, precipitated and dried under reduced pressure.

• ·

Example 1.3: Cyclization of the Cis-Arg-Ser-D-Cis-Trp linear peptide

72.5 mg of the linear peptide were dissolved in 1100 mL of water and the pH was adjusted to 8 by addition of 25 μΐ ammonia (10% in water). Cyclization occurs during the formation of disulfide by bubbling air into the reaction mixture for 4 hours. The reaction mixture was acidified with acetic acid (5 mL) and reduced under reduced pressure to ca. 10 ml and lyophilized.

For purification, the crude peptide thus obtained was dissolved in a mixture of 4.5 ml of acetonitrile / water (1: 9 v / v) and 0.1 ml of acetic acid and chromatographed on high performance liquid chromatography (HPLC). The column volume was 20 x 250 mm, packed with Nucleosil ₇ C ₁₈ (10 nm) (Machery-Nagel, Dueren, Germany);

0.1% TFA was used as eluent in (A) 0.1% TFA in acetonitrile was used as eluent in (B). The linear gradient from 10% (B) to 50% (B) over 30 minutes, transmission rate 18 ml / min, and detection at 215 nm. The main fraction was collected with a retention time of nearly 11 minutes, concentrated under reduced pressure, and filtered through an ion exchange column ((15 mL) AG1-X8 (Bio-Rad)) treated with acetic acid to replace TFA with AcOH. The column was washed well with water and the combined fractions (40 mL) were lyophilized. The compound of formula (3) is obtained in the form of a colorless, fluffy, cotton-like precipitate.

Analysis:

HPLC: column dimensions 4.6 x 250 nm, Nucleosil 7 C - 10 g (10m) from Machery-Nagel, Dueren, Germany; 0.1% TFA was used as eluent (A) and 0.1% (CH ₃ CN) (B) Linear gradient from 10% B to 90% D over 30 min, 1 mL / min detection at 215 nm Retention time to peak

13.1 minutes.

MALDI, negative mode: 650.7 (estimated weight: 650.8).

-'- 1 H-NMR (TOCSY and ROESY experiments): water / D ₂ O (95: 5 v / v) at 25 ° C, chemical shift (ppm) relative to DSS: Cys (1) , aH 4.11, SH 3.31, 2.96; Arg, NH 8.94, aH

4.37, SH 1.84, 1.81, H 1.66, 1.60, OH 3.20, 3.20, guanido-NH 7.16, 6.61; Ser, NH 8.56, aH 4.26, SH 3.78, 3.76;

Cys (4), NH 7.71,? H 4.53, SH 3.06, 2.89, Trp, NH 7.85, aH 4.59, SH 3.38, 3.10, 2H 7, 18, 4H 7.68, 5H 7.14, 6H 7.22, 7H 7.47, NH 10.00.

Example 2

Synthesis of Compound 4:

2.1. Example: Linear peptide precursor

The linear intermediate of the compound of formula (4) is shown in Example 1.1. and 1.2. Examples are prepared in an analogous manner to Examples 1B but L-homocysteine instead of L-cysteine (Fmoc-homo-Cis (Trt); Bachem).

2.2. example

Oxidation of Homo-Cys-Arg-Ser-D-Cys-Trp

Dissolve 40 mg of the linear peptide in 600 ml of water and add 900 μΐ of 0.1 M iodine in 95% acetic acid for about 15 minutes. After one hour, 10 mg of ascorbic acid was added to reduce the excess iodine. A colorless solution is obtained which is concentrated under reduced pressure to 30 ° C at 30 ° C.

This solution was lyophilized to give a white powder (about 60 mg). This is purified by HPLC as in 1.3. Example. Compound (4) is a white cotton wool precipitate.

Analysis:

HPLC analysis was performed as described, with a single peak retention time of 13.7 minutes.

MALDI, positive mode 666.7 (calculated by weight: 666.8).

Example 3

Synthesis of compound (5)

3.1. example

Synthesis of Fmoc-NArg (Pmc):

3.1.1. example

Synthesis of N-benzyl-N-3-phthalimidopropylglycine benzyl ester N-benzylglycine benzyl ester (g, 62.6 mmol) was dissolved in dry DMF (160 mL). To this solution was added 21 g (78.2 mmol) of 3-bromopropylphthalimide and 13.4 mL (78.2 mmol) of diisopropylethylamine. The reaction mixture was stirred for 16 hours at 60 ° C with the exclusion of moisture. DMF was evaporated under reduced pressure to give an oil

The residue was chromatographed on 300 g of silica gel (70-230 mesh) using petroleum ether / ethyl acetate 95: 5 to 85:15. This gives N-benzyl-N-3-phthalimidopropylglycine benzyl ester.

TLC: R f = 0.32 (petroleum ether / ethyl acetate 4: 1) ¹ H-NMR (CDCl ₃ ; 300 MHz): 7.85 (2H); 7.71 (2H) phthalimido; 7.38-7.24 (10H) benzyl; 5.13 (2H) CH ₂ -glycine; 3.78 (2H) OCH ₂ ; 3.75 (2H); 3.39 (2H); 2.77 (2H) 3xN-CH ₂ ; 1.88 (2H) CH ₂ -propyl.

3.1.2. example

Synthesis of N-3-aminopropyl-N-benzylglycine

17.4 g (39.3 mmol) of N-benzyl-N-3-phthalimidopropylglycine benzyl ester are suspended in 100 ml of 6 N hydrochloric acid. The suspension was stirred at reflux for 6 hours. During cooling, phthalic acid is liberated from the reaction mixture. The phthalic acid was filtered off and washed with 4N hydrochloric acid. The filtered solution was narrowed to a small volume (20 mL) and concentrated several times after addition of methanol. The oily residue was dissolved in ethanol (60 mL) and excess hydrochloric acid was removed by addition of propylene oxide.

N-3-Aminopropyl-N-benzylglycine was obtained by the addition of acetone. The precipitated colorless solid was filtered off, washed with acetone and diethyl ether and dried in an oven over KOH.

TLC: _Rf = 0.44 _(CHCl3 MeOH & AeOH / H ₂ O)

MALDI: 223.9 (MH +) found for C ₁₂ H ₁₈ N ₂ O ₂ (222.29). 1.3. example

Preparation of dimethyl (2,2,5,7,8-pentamethylchroman-6-sulfonylimino) dithiocarbonate (Pmc-NC (SCH ₃ ) ₂ )

2,2,5,7,8-Pentamethylchroman-6-sulfonamide (943 mg, 3.33 mmol) was dissolved in dry DMF (3 mL). 200 μΐ of 20N sodium hydroxide (4 mmol) and 139.3 mg (1.83 mmol) of CS ₂ were added with the exclusion of oxygen. An additional 200 μl of 20N sodium hydroxide solution and 139.3 mg of CS ₂ were added in two portions after 10 and 20 minutes, respectively. Methyl iodide (415.6 μΐ, 6.66 mmol) was added to the suspension and the reaction mixture was stirred for two hours. The resulting solution was diluted with ethyl acetate (30 mL) and extracted with water (30 mL). The organic layer was dried over sodium sulfate and evaporated to dryness. The crude product was chromatographed on 20 g of silica gel (70-230 mesh) with petroleum ether / ethyl acetate 95: 5 to 85:15.

TLC: Rf = 0.44 (petroleum ether / ethyl acetate 4: 1) ^T H-NMR (CDC1 _3, 300 MHz): 2.66 (2H); 1.84 (2H) ₂ x CH ₂ chroman;

2.58 (3H); 2.55 (3H); 2.15 (3H) 3xCH ₃ chroman; 2.52 (6H)

2xSCH ₃ ; 1.34 (6H) 2 x CH ₃ chroman;

3.1.4. example

Synthesis of compound of formula 5.1

565 mg (1.914 mmol) of N-3-aminopropyl-N-benzylglycine (Example 3.1.2) are suspended in 22.5 ml of dry ethanol. Pmc-NC (SCH ₃ ) ₂ (675 mg, 1.74 mmol) (Example 3.1.3) and diisopropylethylamine (1.84 mL, 10.77 mmol) were added. The reaction • · ** · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

_ 59 - ·· * · ····· was stirred overnight at 60 ° C bath temperature. The ethanol is evaporated and the crude product is chromatographed on 20 g of silica gel (70-230 mesh) in dichloromethane / methanol 100: 1.

100: 10. This gives S-methyl-N- (2,2,5,7,8-pentamethylchroman-6-sulfonyl) -6- (3-N-benzylglycine) propylisothiourea (5.1).

TLC: R f = 0.26 (9: 1 dichloromethane / methanol)

MALDI: 563.07 (MH +) found for C ₂₈ H ₃₉ N ₃ S ₂ O ₅ (561.76) ¹ H NMR (CDCl ₃ ; 300 MHz): 8.22 (1 H) NH, ~ 7.35 (5H); 3.92 (2H);

3.45 (2H); 3.37 (2H); 2.84 (2H) 4xNCH ₂ ; 2.65 (2H); 1.88 (2H)

2xCH ₂ chroman; 2.58 (3H); 2.55 (3H); 2.17 (3H) 3xCH ₃ chroman; 2.38 (2H) SCH ₃ ; 1.91 (2H) CH ₂ -propyl; 1.33 (6H) 2 x CH ₃ chroman;

3.1.5. example

Substitution of -SCH ₃ for -NH ₂

715 mg (1.27 mmol) of S-Methyl-N- (2,2,5,7,8-pentamethylchroman-6-sulfonyl) -N- (3-N-benzylglycinyl) propyl isothiourea (17) is dissolved in 24 ml of ammonium saturated acetonitrile. The mixture was cooled in an ice bath and 208.5 mg (1.23 mmol) of silver nitrate dissolved in 6 mL of acetonitrile was added dropwise and stirred at room temperature overnight. The (AgSCH ₃ ) precipitates and is filtered. The acetonitrile was evaporated under reduced pressure and the residue was dissolved in ethyl acetate (25 mL), washed with 0.1 N HCl and brine. The organic phase is dried over sodium sulfate and evaporated again to dryness.

j uk. THE The crude product is chromatographed on 20 g of silica gel (70-230 mesh) dichloromethane / methanol 100: 2 to 100:20

N- (2,2,5,7,8-pentamethylchromane-6-sulfonyl) -6- (3-N-benzylglycine) propylguanidine.

TLC: Rf = 0.12 (9: 1 dichloromethane / methanol)

MALDI: 532.24 (MH +) found for C ₂₇ H _{3 g} N ₃ SO ₅ (530.68)

¹ H NMR (DMSO-d ⁶ / D ₂ O; 300 MHz): 7.23 (5H); 3.65 (2H); 3.12

(2 H); 3.05 (2H) 3xNCH ₂ ; 2.58 (2H); 1.75 (2H) 2xCH ₂ chroman;

2.47 (6H); 2.01 (3H) 3xCH ₃ chroman; 1.54 (2H) CH ₂ -propyl;

1.24 (6H) 2xCH ₃ chroman;

3.1.6. example

Removal of the benzyl group

630 mg (1.19 mmol) of N- (2,2,5,7,8-pentamethylchroman-6 -

sulfonyl) -7- (3-N-benzylglycinyl) propyl guanidine

(3.1.5. Example 1) is dissolved in 20 ml of dry methanol and 130 mg

hydrogenated with a palladium on carbon catalyst. After 17 hours, the theoretical amount of hydrogen is consumed and the reaction stops. The catalyst was filtered off and the filtered solution

dry simmer. The resulting foam (N- (2,2,5,7,8-pentamethyl)

-chroman-6-sulfonyl) -7- (3-glycinyl) -propyl-guanidine) was dried under high vacuum and used in the next step without purification.

TLC: _Rf (1% AcOH in MeOH) 0.38

MALDI: 441.9 (MH +) found for C ₂₀ H ₃₂ N ₄ SO ₅ (440.56).

«* *« · «44 44 44 44 44 · ♦ ♦ ♦ ♦

-61- ♦ ··· ♦♦ * ··

3.1.7. example

Introduction of the Fmoc protecting group

520 mg (1.18 mmol) of N- (2,2,5,7,8-Pentamethyl-chroman-6-sulfonyl) -N '- (3-glycinyl) -propyl-guanidine in 2.83 mL of 9% sodium carbonate solution and 2.83 ml of dimethylformamide. The reaction mixture was cooled in an ice bath and a solution of 335 mg (0.99 mmol) of succinimidyl-9-fluorenylmethyl carbonate in 2.2 ml of DMF was added in portions. The mixture is sonicated to obtain a well-distributed suspension. After stirring at room temperature for 45 minutes, the mixture was diluted with water (30 ml) and extracted with ethyl acetate (2 x 75 ml). The remaining aqueous layer was acidified to pH 2 with 4N hydrochloric acid and extracted again with 3 x 30 mL of ethyl acetate. The combined organic phases are dried over sodium sulfate and evaporated to dryness. The purified product is obtained by chromatography on 20 g of silica gel (70-230 mesh) with dichloromethane: methanol (100: 1 to 100:10). The product obtained is N- (2,2,5,7,8-pentamethylchroman-6-sulfonyl) -7- (3-N-9-fluorenalmethoxycarbonylglycinyl) -propylguanidine (Fmoc-). NARG (Pmc)). The preparation of Fmoc-NArg (Pmc) is described by Simon et al. (Proc. Natl. Acad. Sci. USA (1992) 89, 9367-9371).

TLC.-Rf = 0.52 (dichloromethane / methanol 9-1)

MALDI: 662.86 (MH +) found for C ₃₅ H ₄₂ N ₄ SO ₇ (662.80).

3.2. example

Linear peptide precursor

The linear intermediate of compound (5) is prepared according to the procedure described in 1.1.

• · · · ··· · · · ♦ * ··· ff ·· · and 1.2. Examples 1 to 4 are prepared analogously to Examples 1 to 4, but using NArg instead of Arg.

3.3. example

Oxidation of Cys-NArg-Ser-D-Cys-Trp mg mg of the linear precursor are dissolved in 1000 ml of water and the pH is adjusted to 8.0 with concentrated ammonia (about 0.5 ml). Air was bubbled through the solution for 22 hours. After addition of 15 ml of acetic acid, the solution is concentrated and lyophilized.

The crude product was purified by HPLC and converted to the acetate as described in 1.3. is described in example. The compound of formula (5) is obtained in the form of a white cotton wool powder.

Analysis:

Homogeneous analytical HPLC in Standard Tlc was performed as described with a single peak retention time of 13.1 minutes. MALDI, positive mode 652.8 (calculated 653.4)

Example 4

Synthesis of compound (6)

4.1. example

Synthesis of diaminopropionic acid (Fmoc) (Dpr (Fmoc)):

6.04 g of L-diaminopropionic acid (Fluka) (43.0 mmol) and 68.8 g of basic copper carbonate (68.8 mmol) are suspended in 144 ml of water and refluxed for 1 hour. Insoluble matter is removed by filtration and · «··« ·

was Wash with 10 ml water (70 ° C). 50 ml of DMA was added the received dark blue copper diaminopropionate solution, and a pH ca. Adjust to 9 with triethylamine (about 1 mL). 15.23 g

Fluorenal methoxycarbonyl N-hydroxysuccinimidate (Novabiochem) was dissolved in 250 mL DMA and this solution was added to the copper complex and stirred slowly for 0.5 hour. The pH was adjusted to 8-9 with triethylamine (6 mL total). The solution was allowed to stand for 1.5 hours. The mixture was reduced to 40 ml under reduced pressure at 30-40 ° C, and 950 ml of water (0-5 ° C) was slowly added with stirring. After stirring for one hour, the precipitate was filtered off, washed with water (180 ml) and dried over phosphorus pentoxide (10.3 g). After treatment with 50 ml of DIPE, the solid is filtered off and dried under reduced pressure to give 8.9 g of blue Fmoc-Dpr copper complex.

To remove copper, the complex (12.46 mmol) was dissolved in 1100 mL of ethanol / water (1: 1 v / v) and 4.0 g of ethylenediaminetetraacetic acid (EDTA) (13.71 mmol) was added. . The mixture was refluxed for 40 minutes and the solids removed by filtration. The solution was cooled to 0-5 ° C, and after 15 hours, the precipitated components were separated by filtration and dried under reduced pressure. It is then treated with 40 ml of isopropanol at 50 ° C, and the suspension is allowed to stand at 0-5 ° C for one hour and then filtered. The solid was dried under reduced pressure: 2.76 g of a white powder.

TLC CMW: main patch with a smaller accompanying component Rf ca. 0.25. The analytical use of HPLC has been described, with retention.

«« · «64 64 _ ........ time: 18.8 minutes.

4.2. example

Synthesis of Boc-α-diaminopropionic acid (Fmoc)

A suspension of 0.119 g of Drp (Fmoc) (365 mmol) and 1.3 ml of DMA (dist.) Was prepared, 50.8 μΐ (365 μπιόΐ) triethylamine and 0.087 g Boc anhydride (401 gmol) (Fluka) were added and the the mixture was stirred for 1.5 hours. Water (1 ml) was added, cooled to 0-5 ° C, then ca. Acidify to pH 4 with 120 μΐ 2N HCl. The white emulsion is extracted with 70 ml of ethyl acetate and the organic phase is washed three times with 20 ml of water each time. After drying over sodium sulfate, the solvent was evaporated under reduced pressure and the residue was lyophilized by dissolving in 1.3 ml of tert-butanol. The target compound is formed as a white powder.

Tlc CMW: Single Foot Rf ca. 0.55 1 H-NMR compatible with structure, chemical shift in ppm: tert-butyl (Boc) 1.1, CHu 4.03, CH ₂ 4.23, CH ₂ (Fmoc)

4.28, NH 6.95, 7.35, aromatic H (Fmoc) 7.4, 7.68, 7.9.

4.3. example

Synthesis of Fmoc-D-Asp (OBut) -Trp (Boc) -TAB-Amide Resin

1.1. The compound was synthesized in an analogous manner to Example 1, starting from 1.36 g of Fmoc-TAB amide resin (NOvabiochem) (0.60 mmol) and sequentially coupling the FmocTrp (Boc) and Fmoc-D-Asp (OBut) moieties. as shown in Section 1.1. example. The resulting resin was dried under reduced pressure to give 4.7 g.

4.4. example

Cleavage of Fmoc-D-Asp-Trp-amide from the TAB-amide resin and subsequent removal of the t-butyl protecting groups.

4.7 g of Fmoc-D-Asp (OBut) -Trp (Boc) -TAB-amide resin (1.79 mmol) were treated six times with 15-15 ml of 95% TFA and DCE (1: 1 v / v). .), which also contains 2% ethanedithiol. The treatment takes 5 minutes. The resin was filtered off and washed three times with 15 ml of DCE and three times with 15 ml of TFE. The combined filtered solutions were concentrated under reduced pressure at 25-30 ° C to 10 ml and stirred at 250 ml.

Pour into a mixture of DIPE and PE (1: 1 v / v). The precipitate was filtered off and washed with the mother liquor (30 mL) and dried under reduced pressure to give 0.883 g.

To completely remove the tert-butyl protecting group, the colorless precipitate was treated with 20 mL of a mixture of TFA and ethanedithiol (98: 2 v / v) for 20 minutes. The solution was diluted with ca. The reaction mixture was concentrated to 10-15 ml and the product crystallized and isolated as described above. For complete decarboxylation of the carboxy-N-indolyl intermediate, the crude product (about 0.836 g) was dissolved in 120 mL of CHgCN and DMF (1: 1 v / v) and allowed to stand for 15 hours. After concentration under reduced pressure, the product precipitated as described above and dried under reduced pressure. 0.663 g of material is obtained.

Tlc CMW: single patch Rj approx. 0.5.

Analytical HPLC as described, Retention time: 24.0 min. MALDI, positive mode, 539.9 (calculated 541.6).

Example 4.5

Coupling of Fmoc-D-Asp-Trp-Amide to TAB Ester Resin by Asp Side-chain

0.660 gFmoc-D-Asp-Trp amide (1.22 mmol) was dissolved in 2 mL DMA (dist.), 10 mL DCE and 2.10 g TAB resin (Novabiochem) (1.22 mmol) were added. with slow stirring. The mixture was cooled to 0-5 ° C and a solution of 0.503 g of DCCl (2.44 mmol) in 1 mL of DMA was added and 0.3 mL of DCE was added. A solution of 29.8 mg DMAP (0.244 mmol) in 2 mL DCE was allowed to stand for 5 min at 0-5 ° C. After 20 minutes at 0-5 ° C, 135.6 μΐ N-methylmorpholine was added. The reaction mixture was shaken for 4 hours. The resin is washed as follows: 6 times 30-30 ml DMA, 5 times 30-30 ml isopropanol. The resin is dried under reduced pressure

2.5 g (with traces of solvent).

4.6. example

Synthesis of the protected resin bound peptide

Boc-Dpr-Arg (Pmc) -Ser (But) -D-Asp (TAB-ester-resin) -Trp-amide

2.5 g of Fmoc-D-Asp (TAB ester resin) -Trp-amide (1.22 mmol) were subjected to solid phase synthesis in analogy to Example 1.1, successively with the following amino acid derivatives: Fmoc-Ser (Butt), Fmoc-Arg (Pmc) and Boc-Dpr (Fmoc). At the end of the synthesis, the Fmoc group on the Dpr side chain is removed (with 20% piperidine in DMA as in Example 1.1) and the resin is dried under reduced pressure: approx. 3.0 g (with traces of solvent).

4.7. example

Cleavage of the linear protected peptide from the TAB ester resin

647 μτηόΐ peptide resin (with about 3.0 g solvent traces) was shaken in ml of 99% AcOH-DCM (1: 9 v / v).

1.5 hours. The resin was filtered off and washed twice with 40-40 mL of DCE, then once with DCE / TFE (40 mL; 1: 1; v / v) and 4 times with 40-40 mL of TFE. The combined filtrate solutions were concentrated under reduced pressure at 25-30 ° C, and the oily residue was concentrated to ca. Dissolve in 100 ml (distilled) DMSO and lyophilize. To completely remove the traces of AcOH, since AcOH would interfere with the following cyclization process, the product was lyophilized twice more with 25-25 mL of DMSO: 0.704 mg (traces of solvent).

Analytical HPLC was described above with a retention time of 23.9 minutes.

MALDI, positive mode, 1091.1 (calculated 1089.3).

4.8. example

Cyclization of the protected linear peptide

Boc-Dpr-Arg (Pmc) -Ser (But) -D-Asp-Trp-amide

0.874 g HOBt (6.47 mmol) and 1.33 g DCCl (6.47 mmol) were dissolved in 162 mL DMF (degassed under reduced pressure). To this solution was added 0.704 g of linear peptide (0.647 mmol) dissolved in 162 mL of DMF with stirring. Stirring is continued for 9.5 hours and then allowed to stand for 15 hours. The reaction mixture was evaporated under reduced pressure for ca. To 5 ml, the precipitated dicyclohexylurea was filtered off and the filtered solution was added to 120 ml of DIPE. After 30 minutes at 0 ° C, the precipitate was filtered off, washed with 50 ml DIPE and dried under reduced pressure (0.649 ° C).

Analytical HPLC as described, retention time 26.8 min.

4.9. example

Cleavage of the protecting groups of the cyclic peptide

56.7 mg of the crude protected ring peptide were dissolved in 1 ml of a mixture of 95% TFA and ethanedithiol (9: 1; v / v) and allowed to react for two hours. The crude product precipitates out by adding 10 ml of a 1: 1 (v / v) mixture of DIPE and light petroleum (low boiling point). The precipitate was filtered off, washed with 3 ml of mother liquor and dried under reduced pressure. The crude product was purified by HPLC (retention time 10.0 min.) And from section 1.3. is converted to the acetate as described in Example 1b. The compound of formula (6) is obtained in the form of a white cotton wool.

Analytics:

Analytical HPLC as described, with a single peak retention time of 11.0 minutes.

MALDI, positive mode: 628.3 (calculated 630.7).

9 • ·. example

Synthesis of compound (7):

5.1. example

Boc-Cys (Acm) -Arg (Pmc) -Ser (But) -D-Cys (Trt) -Trp (Boc-Wang'-resin)

The solid phase bound protected peptide is depicted in Figure 1.1. By analogy to Example 1A, the following peptide derivatives are sequentially coupled:

Fmoc-D-Cys (Trt); Fmoc-Ser (But); Fmoc-Arg (Pmc) Boc-Cys (Acm). and 5.2. example Cyclization of peptide disulfide a resin 0.8 g, previously with isopropanol handled peptide resin (0.145 mmol) was shaken with 185 mg of iodine (0.725 mmol) 1.5 ml ethanol and 1.5 ml of chloroform made solution. 7

After 10 minutes, 10 μΐ of DIPEA was added to neutralize the hydrogen iodide. After a total reaction time of 15 minutes, the resin was filtered off and washed as follows: 5 times 10-10 ml of ethanol, 5 times 10-10 ml of chloroform and 5 times 10-10 ml of DMA. The resin was treated twice with a solution of 25 mg of ascorbic acid in 100 μΐ of water and 4.9 ml of ethanol for 1 min. This was followed by a thorough wash [3x DMA and isopropanol (10 mL)] and the resin was dried under reduced pressure to 0.8 g.

An analytical sample is cleaved from the resin according to section 1.2. Example 1A and showing that the disulfide cyclization of the peptide has occurred.

MALDI, positive mode, 652.5 (calculated: 652.7).

5.3. example

Desulfurization of the cyclic disulfide peptide on the resin

0.8 g peptide resin (0.145 mmol) was washed 12 times with 10-10 mL dry DMA. To this was added 220 μΐ of a solution of tris (diethylamino) phosphine (0.8 mmol in 5 mL of dry DMA). The mixture was allowed to stand for 48 hours with the exclusion of water and shaken slowly for 6 hours for 1 hour. filtration, and the resin was washed 8 times with 10-10 mL DMA and 5 times with 10-10 mL isopropanol, then dried under reduced pressure: 0.8 g.

5.4. example

Cleavage of peptide from resin and protecting groups:

The resolution of the above resin is shown in 1.2. Example 90 and the product was 90 mg. HPLC purification is performed as described in 1.3. Example 10 was performed analogously except that the gradient ranged from 10% to 60% over 30 minutes. The peak with a retention time of 10.5 minutes is collected, evaporated and filtered through an acetate ion exchanger as shown in 1.3. is described in example. After lyophilization, the compounds of formula (7) are obtained in the form of a white powder.

Analytical HPLC as described, Retention time 14.9 min. MALDI, positive mode, 620.6 (calculated 620.7).

• ·. example

Synthesis of compound (8)

6.1.1. example

Synthesis of 1,1-dimethylethyl-4-formyl-2,2-dimethyl-3-oxazolidinecarboxylate

1,1-Dimethylethyl 4-formyl-2,2-dimethyl-3-oxazolidinecarboxylate according to Garner et al., J. Org. Chem. (1987),

52, 2361-2364, starting from D-serine. ¹ H-NMR (CDCl ₃ ): 9.52 (s, 1H); 4.20 (m, 1H); 4.08 (m, 2H);

1.62 (s, 3H); 1.55 (s, 3H); 1.47 (s, 9H).

6.1.2. example

Introduction of a carboxyalkene group

An equimolar amount of triphenylphosphine and 3-bromopropionic acid is dissolved in toluene and refluxed for 16-20 hours. The cooled reaction mixture was filtered, washed with ether and dried in vacuo. 3.89 g (9.07 mmol) of the phosphonium salt are suspended in 50 mL of dry THF in a three neck flask under argon. The suspension was cooled to -75 ° C and n-butyllithium (9.07 mmol, 1.3 M in hexane) was added dropwise. The mixture is stirred for 0.5 hour and allowed to return to 0 ° C and then stirred for another hour. This suspension was cooled to -75 ° C and 1.22 g (5.34 mmol) of 6.1.1. The aldehyde of Example 1 was added and allowed to react for 30 minutes at 0 ° C and for another hour at room temperature. Compound (8.2) as a clear oil

Ί2.

is obtained. (P. L. Beaulieu et al., J. Org. Chem. (1991), 56),

4196-4204).

¹ H-NMR (CDCl ₃ ): 5.49 (m, 2H); 4.08 (dd, 1H); 2.67 (m, 1H); 2.45 (m, 4H); 1.59 (s, 3H); 1.52 (s, 3H), 1.47 (s, 9H).

6.1.3.példa

Methylation of the carboxyl group

To a solution of 0.91 g of compound (8.2) in methylene chloride is added 0.72 g of dicyclohexylcarbodiimide, 0.3 ml of MeOH and 0.04 g of DMAP. The reaction mixture was stirred at room temperature for 3 hours, filtered and the filtered solution was concentrated in vacuo. The residue was purified by flash chromatography (methylene chloride / methanol 10: 0.1) to give the pure product (methyl ester) as a colorless oil.

¹ H-NMR (CDCl ₃ ): 5.65 (m, 2H); 4.60 (m, 1H); 4.10 (dd, 2H); 3.70 (s, 3H); 3.25 (m, 2H); 1.59 (s, 3H); 1.52 (s, 3H); 1.47 (s, 9H).

6.1.4. example

Reduction of the ester group

To a solution of methyl ester (0.76 g) in methylene chloride was added dropwise 4.7 ml of a 1.2 molar solution of diisobutylaluminum hydride in toluene at 0 ° C. The reaction mixture was stirred at 0 ° C for 1 hour and IN hydrochloric acid (10 ml) was added dropwise. The mixture was poured into water, the organic layer was separated, dried over CaCl 2 and evaporated in vacuo. Purify by flash chromatography the alcohol compound (ethyl acetate / hexane 7: 3) to give the final product as a colorless oil. ¹ H-NMR (CDCl 3): 5.55 (m, 2H); 4.70 (m, 1H); 4.10 (dd, 2H); 3.80-3.50 (m, 4H)); 1.52 (s, 6H); 1.42 (s, 9H).

6.1.5. example

Tosylation of the alcohol group

0.680 g of the alcohol is dissolved in methylene chloride and 45 ml of triethylamine, 0.52 g of toluene-4-sulfonyl chloride and 0.04 g of dimethylaminopyridine are added at 0 ° C. The ice bath was removed immediately and the reaction mixture was stirred for 4 hours at room temperature, then poured onto ice and extracted with methylene chloride in two portions. The combined organic layers were dried over CaCl ₂ and evaporated in vacuo. The residue was purified by flash chromatography (hexane: ethyl acetate = 6: 1).

¹ H-NMR (CDCl 3): 7.8 (d, 2H); 7.35 (d, 2H); 5.55 (m, 1H); 5.40 (m, 1H); 4.53 (m, 1H); 4.18-3.90 (m, 4H); 3.60 (dd,

1H); 2.60 (dd, 1H); 2.47 (s, 3H); 1.59 (s, 3H); 1.52 (s, 3H); 1.47 (d, 9H).

Example 6.1.6

Substitution of the tosyl group with iodine

To a solution of 0.535 g of tosylate in DMF was added 0.675 g of Lil and the reaction mixture was heated at 70 ° C for 20 minutes. The reaction mixture was partitioned between water and ethyl acetate, the organic layer was separated, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (hexane / ethyl acetate 7: 1). The pure iodinated compound was obtained as a yellowish oil.

¹ H-NMR (CDCl ₃ ): 5.55 (m, 1H); 5.45 (m, 1H); 4.6 (m, 1H); 4.1 (dd, 1H); 3.70 (dd, 1H); 3.30 (m, 1H); 3.10 (m, 1H); 2.75 (m, 2H); 1.59 (s, 3H); 1.52 (s, 3H); 1.47 (s, 9H).

6.1.7. example

Synthesis of compound of formula 8.3

A solution of 1,17,1,3,3,3-hexamethyldisilazane (0.17 mL) in THF (0.5 mL) was cooled to -78 ° C and a solution of butyl lithium (0.59 mL, 1.6 M) was added. The resulting solution was stirred at -78 ° C for 10 min and then passed through 0.232 g of (2S, 4R) -3 - [(benzyloxy) carbonyl] -4-methyl-2-phenyl-1,3-oxazolin-5-one [Altmann et al., Helv. Chim. Acta (1991) 74, 800-806] in 0.8 ml of THF pre-cooled to -78 ° C. The yellowish enolate solution was stirred for 5 minutes at -78 ° C and then 0.568 g of 6.1.6. The iodinated compound of Example 1b was dissolved in 1 mL of THF. The reaction mixture was warmed to -40 ° C and stirred at this temperature for 4 hours. The reaction mixture was partitioned between saturated ammonium chloride solution and ether, the aqueous layer was separated and extracted twice with ether. The combined ether layers were dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (hexane / ether 8: 2).

H-NMR (C ₂ D ₂ Cl ₄ ; 100 ° C): : 7.36-7.10 (m, 10H) ; 6.48 (S, 1H) 5.40 (m, 1H); 5.30 (m, 1H); 4.50 (m, 1H); 4.00 (Dd, 1H) 3.60 (dd, 1H); 2.40 (m, 1H); 2.10 (m, 1H);  2.00 (M, 2 H)

1.70 (s, 3H); 1.59 (s, 3H); 1.52 (s, 3H); 1.45 (s, 9H).

1

6.1.8. example

Removal of the benzyl group

Dissolve 0.232 g of compound (8.3) in 3 ml of methanol and add to 0.5 ml of 2N aqueous LiOH. The reaction mixture was stirred for 2 hours at 45 ° C, diluted with water and extracted twice with ether. The combined ethereal extracts were dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (10: 0.4 to 10: 1 methylene chloride / methanol) to give 8.4 as a white foam.

¹ H-NMR ( CDCl ₃ ): 7, 30 (s, 5H); 6.72 (s, . 1H); 5.35 (m, 1H 5.20 (d, 2 H) ; 4.70 (m, 1H); 3, 95 (dd, 1H); 3.58 (dd, 1H 2.40 to 1.90 (M, 4H); 1.59 (s, 3H) ; 1.52 (s, 3H ); 1.48 (s, 9H

6.1.9. example

Attachment of tryptamine to the free carboxyl group

Dissolve 0.150 g of compound 8.4 in methylene chloride and add 74.5 mg DCC, 55.3 mg HOBt and 58 mg tryptamine. The reaction mixture was stirred at room temperature for 2 hours, filtered, diluted with methylene chloride and washed with brine. The organic layer was dried over calcium chloride and concentrated in vacuo. The residue was purified by flash chromatography (200: 5, methylene chloride / methanol) to give the product as a white foam.

1 H-NMR (CDCl ₃ ): 8.08 (s, 1H); 7.58 (d, 1H); 7.35 (s, 5H);

7.25-7.00 (m, 3H); 6.58 (s, 1H); 5.35 (d, 1H); 5.30 (s, 1H);

¥

5.08 (d, 2H); 4.50 (m, 1H); 3.88-3.50 (m, 3H); 2.99 (t, 2H); 2.38-1.55 (m, 4H); 1.50 (s, 6H); 1.45 (s, 3H); 1.42-1.22 (m, 2H); 1.39 (s, 9H).

6.1.10. example

Removal of the Bzl-O-CO protecting group

A 6.1.9. 0.20 g of the compound obtained in Example 1d is dissolved in 8 ml of THF and 0.030 g of 10% palladium on carbon is added under nitrogen. The reaction mixture was hydrogenated at room temperature for 3 hours. The catalyst is filtered off and the solvent is evaporated to give the free amine as a white foam.

¹ H NMR (cdci ₃ ): 8.92 (s , 1H); 7, 65 (D, 1H); 7.52 (S, IH) 7.40 (d , 1H); 7.2-7.1 ( m, 2H) ; 7.8 (s, 1H); 3.90 ( dd, IH) 3.85 (m , IH) ; 3.80 (m, 1H); 3.72 (Dd, 1H); 3, 52 (M, IH) 3.00 (t, 2 H) ; 1.90-1.40 (m, 6H ); 1.60 1 (S, 3H) ; 1.52 (s, 6H)

1.45 (s, 9H); 1.30 (s, 2H); 1.25-1.10 (m, 2H).

6.1.11. example

Protection of free amino group by Fmoc group

0.150 g of the free amine is dissolved in THF, 0.054 ml of N-ethyldiisopropylamine and 0.081 g of Fmoc-Cl are added and the reaction mixture is stirred overnight at room temperature. The reaction mixture was partitioned between methylene chloride and its salts, the organic layer was separated, dried over <RTIgt; CaCl2, </RTI> and the solvent was evaporated in vacuo. The residue was purified by flash chromatography (8: 2-9: 1 ether / hexane). The Fmoc-protected compound was obtained as a white foam.

• · ·· *

-'- 1 H-NMR ( CDC1 ₃ ): 8, 92 ( s, 1H); 7.89 (d, 2H ); 7.45-7.30 (M, 2 H) ; 7.20 (T, 1H); 7.12 (t, 1H); 7.00 (s, 1H); 6.28 (s, 1H); 5, 85 (s, 1H); 4.35 (D, 2 H); 4.15 (t, 1H); 3, 83 (m, 2H); 3.65 (M, 3H); 3, 00 (t, 2 H) ; 1.80-1.00 (m, 8H) ; I, 58 (s, 3H); 1.55 (S, 3H); 1.50 (s, 3H); 1.46 (s, 9H). 6.1. 12th example

Removal of the acylating group

0.180 g of the Fmoc-protected compound were dissolved in methanol / water (95: 5) and 2 mg of toluene-4-sulfonic acid was added. The reaction mixture was heated to 75 ° C and allowed to stand for 5 hours. The reaction mixture was concentrated in vacuo and the residue was purified by flash chromatography (200: 5 → 100: 5; ether / methanol). so we get the free alcohol.

1 H-NMR (CDCl ₃ ): 8, 92 ( S, 1H ); 7 . 89 ( d. 2 H) ; 7.45-7.30 (M, 2 H); 7.20 (t, 1H); 7.12 (t, 1H); 7.00 (s , 1H); 6.28 (s, 1H); 5.85 (s, 1H); 4.35 (D, 2 H) ; 4, 15 (T, 1H) ; 3.83 (m, 3H); 3.65 (m, 2H); 3.00 (t, 2H); 1.80-1.00 (M, 8H) ; 1.62 (s, 3H); 1.46

(s, 9H).

6.1.13. example

Oxidation of the free hydroxyl group

Dissolve 0.100 g of the free alcohol in DMF and add 0.112 g of pyridine dichromate. The reaction mixture was stirred at room temperature for 5 hours and then partitioned between ethyl acetate and its salts.

The organic layer was separated, dried over sodium sulfate and concentrated in vacuo. The residue was flash chromatographed

Purify (10: 1; methylene chloride / methanol) to give compound 8.1 in pure state.

NMR ( cdci ₃ ): 8.92 ( s, 1H ); 7.89 ( d: 2H ; 7 . 60 (D, 2 H) ; 7.45-7.30 (M, 2 H); 7.20 (T, 1H); 7.12 (T, 1H); 7.00 (S, 1H); 6.28 (s, 1H); 5.85 (s, 1H); 4.48 (m, 1H); 4.35 (D, 2 H) ; 4.15 (t, 1H); 3, 66 (m, 2H); 3, 08 (t, 2H); 1,92- 1.00 (M, 8H); 1.62

(s, 3H); 1.47 (s, 9H).

6.2. example

Coupling of compound of formula (8.1) to TAB ester resin

The compound of Formula 8.1 is prepared as described in Section 4.5. by analogy with Example 1A.

6.3. example

Synthesis of Protected Peptide Bound to Arg (Pmc) -Ser (But) - (8.1) - (TAB Ester-Resin)

In 6.2. 1.5 g (0.7 mmol) of the compound obtained in example 1.1a is subjected to solid phase synthesis. By analogy to Example 1A, the following amino acid derivatives are reacted sequentially: Fmoc-Ser (But) and Fmoc-Arg (Pmc). At the end of the synthesis, the Fmoc group is removed from the Arg and the resin is dried under reduced pressure. Approximately 1.9 g of resin are obtained (with traces of solvent).

6.4. example

Cleavage of Linear Protected Peptide from TAB Ester Resin • ·

About 1.9 g of peptide resin (approximately 0.65 mmol) were shaken in 30 mL of 99% AcOH / DCM (1: 9 v / v) for 1.5 hours. Wash with 1 ml of DCE and 1 ml of DCE-TFE (1: 1 v / v), then 4 times with 20 ml of TFE. The combined filtrate solutions are concentrated under reduced pressure at 25-30 ° C. the oily residue is dissolved in about 50 ml (distilled) DMSO and lyophilized To remove all traces of acetic acid, two additional lyophilisations are carried out, since the traces of acetic acid could interfere with the subsequent cyclization process. The final product was 0.48 g Analytical HPLC as described, retention time 26.3 min, MALDI positive mode 1027.1 (calculated 1027.3).

6.5. example

Cyclization of the protected linear peptide

0.48 g (0.467 mmol) of the linear peptide (8.4) is cyclized according to 4.9. in an analogous manner to that described in Example 1b. Analytical HPLC as described, retention time 27.2 min. MALDI, positive mode, 1009.0 (calculated 1009.2).

6.6. example

Cleavage of the protecting groups of the cyclic peptide

180 mg of the protected ring peptide were dissolved in 95% TFA / ethanedithiol (9: 1 v / v) in 2 ml and allowed to react for 2 hours. The crude peptide precipitates in 15 ml of a 1: 1 (v / v) mixture of DIPE petroleum ether (low boiling point). The precipitate was filtered off with 5 mL f

• ·

washed with mother liquor and dried under reduced pressure. The crude product was purified by HPLC with a retention time of 12.3 minutes and converted to the acetate according to section 1.3. as in the example. Compound (8) was obtained as a cotton wool powder.

Analytical HPLC as described, with a single peak retention time of 12.0 minutes.

MALDI, positive mode: 586.1 (calculated 586.7).

6.7. example

Preparation of compound (8) by other means

6.7.1. example

Opening of the oxazolidine ring in compound (8.3)

Dissolve 1.20 g of the compound of formula 8.3 (Example 6.1.7) in 95: 5 methanol: water, add 0.060 g of toluene-4-sulfonic acid and stir at 75 ° C for 7 hours. . The reaction mixture was concentrated in vacuo and the residue was purified by flash chromatography (2: 1,

ether: hexane); and so the pure product white foam shape is obtained. ¹ H-NMR (CDCl ₃ ): 7.40 ( m, 6H); 7.20 (m, 2 H) ; 6 , 90 (M, 2 H) ; 6.50 (s, 1H); 5 60 (m, 1H); 5.31 (m, 1H); 5.08 (M, 2 H) ; 4.70 (d, 1H); 4.39 (m , 1Η); 3.57 (m, 2H); 2, 60 (m, 1H); 2.22 (M, 1H); 2.02 (m, 2Η ); 1.79 (S, 3H); 1.71 ( m, 1H); 1.48 (S, 9H).

6.7.2. example

Oxidation of the hydroxyl group t 1

In 6.7.1. A solution of 0.600 g of the compound of Example 1c in 14 ml of acetone is cooled to 0 ° C and 0.90 Jones of Reagent (3.25 M CrO ₃ / 5.29 M H 2 SO 4) is added dropwise. After stirring at 0 ° C for 2 hours, isopropanol (1.3 ml) was added dropwise. The reaction mixture was filtered and the resulting solution was partitioned between ethyl acetate and its salts. The aqueous phase was extracted three times with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (10: 0.5; methylene chloride / methanol) to give the pure product as a white foam.

¹ H-NMR (CDCl 3): 7.40 ( m: 6H ); 7.20 (m, 2H); 6.85 (m, 2H); 6.54 (s, 1H); 5.55 (m, 1H); 5.33 (m, 1H); 5.08 (m, 2 H); 4.82 (m, 1H); 4.39 (m, 1H); 2, 24 (m, 1H); 2.05 (m, 2H); 1.82 (s, 3H); 1.75 (m, 1H); 1.48 (S, 9H).

6.7.3. example

Opening of the oxazolidone ring

A 6.7.2. 1.10 g of the compound of Example 1 are dissolved in methanol (ml), and LiOH (0.09) dissolved in water (1.0 ml) is added. The reaction mixture was stirred at room temperature for 30 minutes and partitioned between water and ether. The aqueous layer was separated, acidified with IN hydrochloric acid and extracted again with ether. The acidic ether extracts were combined and dried over sodium sulfate. Evaporation of the solvent afforded pure compound 8.5 as a white foam. ¹ H-NMR (CD ₃ OD): 7.30 (s, 5H); 6.12 (m, 1H); 5.10 (m, 2H);

5.5 (m, 1H); 5.10 (m, 2H); 3.80 (s, 3H); 2.30-2.10 (m, 3H);

• «· ·» * · · «♦ ♦ · · · · 4

- 82 - .........

1.60 (s, 3H); 1.42 (s, 9H); 1.28 (t, 2H).

6.7.4. example

Introduction of a 2-trimethylsilylethyl protecting group

Dissolve 0.74 g of compound 8.5 in 15 ml of methylene chloride, add 0.36 g of dicyclohexylcarbodiimide, 0.019 g of dimethylaminopyridine and 0.23 ml of 2-trimethylsilyl ethanol. After 18 hours at room temperature, the reaction mixture was filtered and the filtrate evaporated. The residue was purified by flash chromatography (1: 1, ether: hexane) to give the pure product as a colorless oil.

¹ H-NMR (CDCl ₃ ; 1: 7.36 (m , 5H) ; 6 21 (S, wide, 1H); 5.59 (i 1H); 5.20 (m, 1H); 5.10 (S, 2 H) ; 4, 20 (t, 2H); 3.75 (m, 1H) 3.70 (s, 3H); 2.00-1.90 (M, 1H); 1 60 (s, 3H); 1.40 (s, 9H 1.20 (t, 2H); 0.99 (t, 2 H) ; 0.20 3 9H)

6.7.5. example

Removal of the Z-protecting group

In Figure 6.7.4. 0.780 g of the compound of Example 1b is hydrogenated over 0.150 g of Pd / C (10%) in 40 ml of tetrahydrofuran. The catalyst was removed by filtration and the filtered solution was concentrated in vacuo. The pure product is a colorless oil.

¹ H-NMR (CDCl ₃ ): 5.02 (d, 1H); 4.21 (t, 2H); 4.10 (m, 1H); 3.80 (s, 3H); 1.90-1.10 (mn, 6H); 1.60 (s, 3H); 1.40 (s, 9H); 1.28 (t, 2H); 1.00 (t, 2H); 0.20 (s, 9H).

·· ·· ·

6.7.7. example

Condensation with Z-Ser (But) -OH

A 6.7.5. (0.400 g) was dissolved in 13 ml of tetrahydrofuran, and 0.38 ml of a solution of Z-Ser (But) -OSu (0.872 g) in triethylamine was added. The solvent was evaporated and the residue was purified by flash chromatography (10: 0.5 ether / methanol).

and a (8. 6) compound give clean material- Kent FAB MS 709 (M + H) &lt; ⁺ &gt; ^{. 1} H-NMR (DMSO;  80 ° C): 7.38- 7.29 (m, 5H) ; 5.07 (s, 2H ); 4.13 (M, 3H); 3.90 (M, 1H); 3.59 (S, 3H), 3; 47 (d, 2 H) ; 1 , 77 (m, 2 H) ; 1.60 (m, 2 H) ; 1.39 (s, 9H),  1.38 ( s, 3H), : 1.35- 1.16 (m, 4H); 1.14 (1, 9H) ; 0.95 (t, 2 H) , : 0.04 ( s, 9H).

6.7.7. example

Removal of the Z-protecting group

0.639 g of compound (8.6) is hydrogenated with Pd / C (10%) in 40 mL of tetrahydrofuran. The catalyst was removed by filtration and the filtered solution was evaporated. This gives the pure product.

1 H-NMR (CDCl ₃ ): 5.00 (d, broad, 1H); 4.21 (m, 3H); 3.50 (m,

3H); 2.20 (m, 1H); 1.80 (m, 1H); 1.65 (S, 2H); : 1.58 (s, 3H); 1.45 (S, 9H); 1.40-1.10 (m, 4H); : 1.20 (s, 9H), : 1.00 (t, 2 H) ; 0.30 (S, 9H).

6.7.8. example

Condensation with Z-Arg (Pmc) -OH

0.639 g of Z-Arg (Pmc) -OH in 15 ml of tetrahydrofuran

and 0.439 g of benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP) and 0.14 ml of triethylamine are added at 0 ° C. The reaction mixture was stirred for 5 minutes then 0.584 g of 6.7.7. The compound of Example 1b is added. The ice bath was removed and the reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was then poured into saturated NH 4 Cl solution and the aqueous layer was extracted three times with ether. The combined organic layers were washed with water and brine, dried over sodium sulfate and the solvent was evaporated. The residue was purified by flash chromatography (10: 3, methylene chloride: methanol) to give the pure product (8.7) as a foam.

FAB-MS: 1132 (M + H) ⁺ . ¹ H NMR DMSO; 120 ° C): 7,37- 7.27 (m, 5H); 5, 06 (s, 2H); 4.33 (m, 1H) ; 4.16 (t, 2H) ; 4.04 (m, 1H); 3.93 (M, 1H); 3.60 (S, 3H); 3 53 (m, 1H); 3 43 (m, 1H); 3.08 (q, 2 H) ; 2.62 (t, 2 H) ; 2.53 ( s, 3H) ; 2.52 (s, 3H ); 2.06 (s, 3H) ); 1.81 (t, 2 H) ; 1.78 to 1, 18 (m, 12H); 1.42 (s, 3H); 1.40 (S, 9H); 1.30 (S, 6H); 1 15 (s, 9H); 0 97 (t, 2 H); 0.05 (s, 9H)

6.7.9. example

Removal of the 2-trimethylsilylethyl protecting group

Dissolve 1.049 g of compound 8.7 in 18 ml of tetrahydrofuran and add 0.584 g of tetrabutylammonium fluoride. The reaction mixture was stirred at room temperature for 24 hours and then poured into saturated NH ₄ Cl solution. The aqueous layer was extracted three times with ethyl acetate. The combined organic phases are washed with water and brine, and then washed with water and brine.

The solvent was evaporated in vacuo. The residue was purified by flash chromatography (10: 0.8; methylene chloride: methanol). The pure product is obtained in the form of a white foam.

FAB-MS: 1032 (M + H) ⁺ .

1 H-NMR (DMSO; 100 ° C): 7,30- 7.25 (m, 5H) ; 5 , 06 (s, 2H); 4.33 (M, 1H); 4, 06 (M, 1H) ; 3.65 (M, 1H); 3 59 (s, 3H); 3.53 (M, 1H); 3.43 ( m. 1H); 3 08 (q, 2 H) ;  2.62 ( t, 2 H) ; : 2.53 (s, 3H); 2.52 (S, 3H) ; 2, 06 (s, 3H ); 1 80 (t, 2 H ); 1.78-1.20 (M, 12H) ; 1.42 (S, . 3H) ; 1 , 40 (s , 9H) ; 1.30 (s, 6H) ; 1.15 (s, . 9H)

6.7.10. example

Removal of the Z-protecting group

In 6.7.9. 0.697 g of the compound of Example 1b is hydrogenated with 0.136 g of Pd / C (10%) in 40 ml of methanol. The catalyst was removed by filtration, the solvent was evaporated, and the pure product was obtained as a white foam.

FAB MS: 898 (M + H) ⁺ . ¹ H NMR (CD ₃ OD): 4, 47 (M, 1H); 3.95 (m, 1H); 3.65 (M, 1H); 3.59 (S, 5H ); 3.20 (, 2 H ); 2.68 (t, 2H); 2.53 (s, 3H); 2.52 (s, 3H) / 2.10 (s, 3H) 1.90-1.20 (m, 12H); 1.82 (T, 2 H) ; 1.48 (s, 3H ); 1.42 (S, 9H ); 1.3 0 (s, 6H); 1.2 0 (s, 9H). 6.7 . 11 . example

Cyclization (ring closure)

0.341 g of 1-hydroxybenzotriazole and 0.459 g of dicyclohexylcarbodiimide are dissolved in 56 ml of dimethylformamide and 0.200 g of 6.7.10 are added dropwise over 9.5 hours. A solution of the compound of Example 1b in 56 ml of dimethylformamide. The reaction mixture was stirred at room temperature for 16 hours, then the solvent was evaporated in vacuo and the residue was purified by flash chromatography (10: 0.6, methylene chloride: methanol). The pure compound (8.8) is thus obtained in the form of a white powder.

FAB-MS: 881 (M + H) ^{+ 1} H-NMR (CD ₃ OD): 4.32 (m, 1H); 4.21 (m, 1H); 3.90

3.78 (s, 3H); 3.57 (m, 2H); 3.19 (m, 2H); 2.78 (t (S, 3H); 2.56 (s, 3H); 2.10 (s, 3H); 1.82 (t, 2H) (m, 1H);

2 H); 2.58

1.73-1.20 (m, 10H); 1.42 (s, 3H)

1.38 (s, 9H); 1.30 (s, 9H); 1.18 (m,

8H).

6.7.12. example

Cleavage of the methyl ester

Dissolve 0.087 g of compound (8.8) in 2 ml of methanol and add 12 ml of 1N NaOH. After stirring for 20 hours at 45-50 ° C, the reaction mixture was basified with water and the aqueous layer was extracted once with ether. The aqueous layer was acidified with 1% KHSO ₄ and extracted again with ethyl acetate. The combined ethyl acetate layers were washed with brine and dried over sodium sulfate. After evaporation of the solvent, the pure product is obtained in the form of a white powder.

FAB-MS: 866 (M + H) ⁺ .

¹ H NMR (cd ₃ od) : 4.32 (M, 1H) ; 4.18 (m , 1H) ; 3.90 (m, 1H); 3, 60 (m, 2H); 3.20 (M, 2 H) ; 2, 68 (Tt, 2 H) ; 2.57 (s, 3H); 2.56 (s, 3H); 2, 10 (s, 3H); 1.80 (T, 2 H) ; 1.80-1.20 (M,

• ·

10Η); 1.43 (s, 3H); 1.38 (s, 9H); 1.30 (s, 9H); 1.18 (m, 8H).

7.6.13. example

Condensation with tryptamine

A 6.7.12. A solution of 0.08 g of the compound of Example 1b in 2 ml of methylene chloride is treated with 0.021 g of dicyclohexylcarbodiimide and 0.016 g of 1-hydroxybenzotriazole. After stirring for 10 minutes at room temperature, a solution of 16.2 mg of tryptamine in 2 ml of methylene chloride was added. After stirring for 3.5 hours at room temperature, the solvent was evaporated in vacuo. The desired product (8.9) is separated by flash chromatography (methylene chloride, 10: 0.5 methylene chloride: methanol) from a mixture of the two diastereomers.

FAB-MS: 1008 (M + H) ⁺ -

^ -NMR ( cd ₃ od) : [the ( Compound 8.8 Spectrum] 7.58 (d, 1H); 7.320 (d, 1H), : 7,106.90 (m, 3H); 4.82 (m, 1H); 4.18 (m, 1H); 3.92 ( m, 1H); 3. 58 (m, 2H); 3.50 (t, 2H); 3.20 (m, 2H); 2, 98 (m, 2H ); 2.59 (t, 2H); 2.57 (s, 3H); 2.56 (S, 3H); 2, 10 (s, 3H); 1.80 (t, 2 H) ; 1.80-1.20 (m, 10H); 1.43 (S, 3H); 1.40 (s, 9H ); 1.30 (s, 9H); 1.15 (m, 8H).

7.6.14. example

Removal of protecting groups

0.035 g of compound 8.9 is dissolved in 1.6 ml of trifluoroacetic acid / water (90:10) and the reaction mixture is stirred at room temperature for 4.5 hours. The solvent was evaporated and the residue was solidified by addition of ether. Compound (9) was purified by reverse phase HPLC on a Nucleosil _C18 column; gradient: 10%

CH ₃ CN (0.09% trifluoroacetic acid) / 90% water (0.09% trifluoroacetic acid) to 90% CH ₃ CN (0.09% trifluoroacetic acid) / 10% water (0.09% trifluoroacetic acid) over 30 minutes .

_Rt = 12.00 min.

FAB-MS: 586 (M + H) &lt; ⁺ & gt ^; .

Example 7

Synthesis of 9

7.1. example

Synthesis of 3- (1,1-dimethyl) -4-methyl- (R) -2,2-dimethyl-3,4-oxazolidinecarboxylate (9.1)

Synthesis of 3- (1,1-dimethylethyl) -4-methyl- (R) -2,2-dimethyl-3,4-oxazolidinecarboxylate (9.1) from D-serine starting material according to Gardner et al. Org. Chem. (1987) 52, 2361-2364].

¹ H-NMR (CDCl ₃ ): 4.49-4.39 (m, 1H); 4.18-4.00 (m, 2H); 3.75 (s, 3H); 1.65 (d, 3H); 1.5 (d, 3H); 1.45 (s, 9H).

7.2. example

Reduction of compound of formula (9.1)

A solution of 22.3 g of compound (9.1) in 450 ml of toluene is cooled to 0 ° C and 200 ml of 1.2 M DIBAH solution (in toluene) are added dropwise. The resulting solution was stirred at 0-5 ° C for one hour and then poured into 200 mL of IN hydrochloric acid. The aqueous layer was separated and extracted with ethyl acetate (x3). The combined organic layers were dried over sodium sulfate and evaporated to dryness. Dry by flash chromatography (7: 3 hexane: ethyl acetate) to afford pure 9.2 as a colorless oil. ¹ H-NMR (CDCl ₃ ): 4.15-3.5 (m, 6H); 1.55 (s, 3H); 1.45 (s,

12H).

7.3. example

Alkylation with methyl bromoacetate

A suspension of 2.50 g of NaH in 100 ml of (abs) tetrahydrofuran is added and 10.6 g of 9.2 is added dropwise. After stirring for 1 hour, methyl bromoacetate (14 ml) was added all at once. The reaction mixture was stirred for 20 hours at room temperature. Pour into NH ₄ Cl. The aqueous layer was extracted three times with ether. The combined organic layers were dried over sodium sulfate and the solvent was evaporated. Purification by flash chromatography (3: 7, ether: petroleum ether). This gives the pure product as a colorless oil. 1 H-NMR (CDCl ₃ ): 4.2-3.9 (m, 5H); 3.72 (s, 3H); 3.68-3.30 (m, 2H); 1.55 (s, 3H); 1.52 (s, 3H); 1.9 (s, 9H).

7.4. example

Reduction of the methyl acetate group

In Figure 7.3. A solution of 6.20 g of the compound of Example 1b in 120 ml of toluene is cooled to 0 ° C and 48.0 ml of a 1.2 M solution of DIBAH in toluene are added dropwise. The resulting solution was stirred at 0-5 ° C for 1 hour and then to 150 ml of IN hydrochloric acid

It poured. The aqueous layer was separated and extracted three times with ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated in vacuo. It was dried by flash chromatography (4: 6, ethyl acetate: hexane). This gives the pure product (9.3) as a yellowish oil.

¹ H-NMR (CDCl ₃ ): 4.1-3.9 (m, 3H), 3.7-3.4 (m, 7H); 1.60 (s,

3H); 1.55 (s, 3H); 1.4 (s, 9H).

7.5. example

Reaction with toluene-4-sulfonyl chloride

6.30 g of compound 9.3 are dissolved in 100 ml of methylene chloride and 4.1 ml of triethylamine are added to 4.8 g of toluene-4-sulfonyl chloride and 0.28 g of dimethylaminopyridine at 0 ° C. The ice bath was removed immediately and the reaction mixture was stirred for 4 hours at room temperature. The reaction mixture was poured onto ice and stirred with methylene chloride in two portions. The organic extract was dried over sodium sulfate and concentrated in vacuo. The residue was dried by flash chromatography (2: 8, ethyl acetate: hexane). The pure product is a yellowish oil.

¹ H-NMR (CDCl ₃ ): 7.8 (d, 2H); 7.35 (d, 2H); 4.12 - 3.30 (m,

9H); 2.48 (s, 3H); 1.55 (s, 3H); 1.48 (s, 12H).

7.6. example

Substitution of iodine for toluene-4-sulfonyl:

The 7.5. 1.58 g of the product of example 1d is dissolved in 7.5 ml of dimethylformamide and 2.17 g of Lil are added. The reaction mixture was stirred at 70 ° C for 20 minutes. The reaction mixture was partitioned between water and ethyl acetate, the organic layer was separated, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (4: 1, methylene chloride: hexane). The purified iodine-containing product is obtained as a yellowish oil.

1 H-NMR (CDCl ₃ ): 4.1-3.2 (m, 9H); 1.56 (s, 3H); 1.54 (s, 3H); 1.48 (s, 9H).

7.7. example

Iodine Exchange to (2S, 4R) -3 - [(Benzyloxy) carbonyl] -4-methyl-2-phenyl-1,3-oxazolidin-5-tin

1.1 ml of 1,1,1,3,3,3-Hexamethyldisilazane are dissolved in 2.1 ml of tetrahydrofuran, cooled to -78 ° C and added.

3.20 ml of a 1.6 M BuLi / hexane solution. The resulting solution was stirred for 10 minutes

Stir at -78 ° C and add

1.45 g of (2S, 4R) -3 - [(benzyloxy) carbonyl] -4-methyl-2-phenyl-1,3-oxazolidin-5-one (Altmann et al., Helv. Chim. Acta (1991)). , 74, 800-806) in tetrahydrofuran (4.8 mL), cooled to -78 ° C. The yellowish enolate solution was stirred for 5 minutes at -78 ° C, and then the solution of 7.6. A solution of 3.6 g of the compound of Example 1b in 6 ml of tetrahydrofuran is added. The reaction mixture was stirred at -78 ° C for 24 hours and then partitioned between saturated ammonium chloride and ether. The aqueous layer was separated and extracted twice with ether. The combined ethereal phases were dried over sodium sulfate and concentrated in vacuo. The resulting compound (9.4) is purified by flash chromatography (3: 7, ether: hexane).

FAB-MS: 569 (M + H) @ +

¹ H-NMR ((CCl ₂ D) ₂ ; 80C) : 7.38 (s, 5H); 7.22 (s, 5H); 6.32 (s, 1H); 5.02 (d, 2H); 3 , 90 (M, 3H); 3.58 (d, 1H); 3.42 (M, 2 H); 3.28 (t, 1H); 2.52 (M, 1H) ; 2.20 (m, 1H) ; 1.72 (s, 3H);

1.53 (s, 3H); 1.46 (s, 9H); 1.43 (s, 3H).

7.8. example

Opening of oxazolidine

Dissolve 1.45 g of compound (9.4) in methanol.

nol / water (95: 5J i and a mixture of 73 mg of toluene-4 - fonic acid, and the reaction mixture was heated to 75 ° C for two hours stirred. The reaction mixture was concentrated in vacuo and a

the residue (compound 9.5) by flash chromatography

purify (3: 1, ether: hexane). ¹ H-NMR ((CCl ₂ D) ₂ ; 80 ° C): 7.38 (s, 5H) ; 7.2 8 (s, 5H), 6.37 (s 1H); 5.13 to 4.92 (m, 3H); 3.78 to 3. , 52 (m, 4H) ; 3.47 (m, 3H) 2.52 (s, (wide) , 1H); 2.28 (m, 1H) ; 2.10 (m, 1H), : 1.72 (s 3H); 1.41 (s, 9H)

7.9. example

Oxidation of the hydroxyl group

1.85 g of compound 9.5 are dissolved in 48 ml of acetone and cooled to 0 ° C. 2.82 ml are added dropwise

Jones reagent (3.25 M CrO ₃ / 5.29 M, H ₂ SO ₄ ). The reaction mixture

After stirring at 0 ° C for 2 hours, isopropanol (4 ml) was added dropwise. The reaction mixture was filtered and the resulting solution was partitioned between ethyl acetate and its salts. The aqueous layer was extracted three times with ethyl acetate. The combined organic layers were washed with brine and dried over sodium sulfate

and evaporated in vacuo. The residue was purified by flash chromatography (10: 0.75, methylene chloride: methanol).

the pure product is obtained in the form of a white foam.

FAB-MS: 542 (M + H) ⁺ .

(s, 1H); 5.58 (s, 1H); 5.03 (m, 2H); 4.38 (s, 1H); 3.72 (m,

1H); 3.58 (m, 1H); 3.35 (m, 2H); 2.40 (m, 2H)

1.64 (s, 3H);

1.38 (s, 9H).

7:10. example

Opening of oxazolidone

In Figure 7.9. 1.65 g of the compound obtained in example 1d is dissolved in 40 ml of methanol / tetrahydrofuran (1: 1) and 0.128 g of LiOH dissolved in 0.5 ml of water are added. The reaction mixture was stirred at room temperature for 1.5 hours and then partitioned between water and ether. The aqueous layer was separated, acidified with 1 N hydrochloric acid and extracted again with ether. The acidic ether extracts were combined and dried over sodium sulfate. The solvent was evaporated to give pure product 9.6 as a white foam.

FAB-MS: 469 (M + H) ⁺ .

¹ H-NMR (CD ₃ OD): 7.16 (m, 5H); 5.10 (s, 2H); 4.30 (t, 1H); 3.78 (dd, 1H); 3.70 (s, 3H); 3.58 (dd, 1H); 3.50 (m, 2H); 2.18 (m, 2H); 1.50 (s, 3H); 1.43 (s, 9H).

7.11. example

Protection of the free carboxyl group as 2-trimethylsilylethyl ester

1.25 g of compound (9.6) are dissolved in 30 ml of methylene chloride and 0.66 g of dicyclohexylcarbodiimide, 0.032 g of dimethylaminopyridine and 0.46 ml of trimethylsilyl ethanol are added. The reaction mixture was stirred at room temperature for 4 hours, then filtered and the resulting solution was evaporated. The residue was purified by flash chromatography (75:25, hexane: ether). The pure product appears as a yellowish oil.

FAB-MS: 569 (M + H) ⁺ .

¹ H-NMR (CD ₃ OD): 7.32 (m, 5H); 5.06 ( s, 2H); 4.26 (m , 1H); 4.25 (t, 2H); 3.70 (m , 1H); 3.68 (s, 3H); 3.50 (m, 4H) ); 2.10 (T, 2 H); 1.49 (s, 3H) ); 1.44 (s, 9H); 1.00 (t, 2H); 0 , 5 (S, 9H). 7.12. example The Z-protecting group removal 7.11. example no affected product 1.08 g in 0.360 g 9 Pd / C (10 %) catalyst with the help of 20 ml of tetrahydrofuran hydrogenated. The catalyst was filtered off and filtered solution evaporated in a vacuum pure product is obtained.

FAB-MS: 435 (M + H) ⁺

¹ H-NMR ( cdci ₃ ): 5.68 (d, 1H); 4.33 (m, 1H); 4.20 (T, 2 H) ; 3.85 (m, 1H); 3.72 (s, 3H); 3.56 (m, 3H); 2.10 (m, 1H); 1.78 (M, 1H); 1.70 (s, 2H); 1.47 (s, 9H); 1.32 (s, 3H); 1.00 (T, 2 H) ; 0.50 (S, 9H).

7.13. example

Condensation protected with serine

0.780 g of the product of Example 7.12 is dissolved in 25 ml of tetrahydrofuran and 0.845 g of Z-Ser (Bu) - OSu and 0.40 ml of triethylamine are added. The reaction mixture was stirred at room temperature for 40 hours. The solvent was evaporated and the residue was purified by flash chromatography (1: 1, ether: hexane). The pure compound (9.7) is obtained in the form of a colorless oil.

FAB-MS: 712 (M + H) ⁺ .

1 H-NMR (CD ₃ OD): 7.38 (m, 5H); 5.12 (s, 2H); 5.05 (m, 1H);

4.29-4.10 (m, 4H); 3.75 (s, 3H); 3.71-3.30 (m, 5H); 2.12 (m, 2H); 1.50 (s, 3H); 1.42 (s, 9H); 1.20 ~ (s, 9H); 1.00 (t, 2H); 0.50 (s, 9H).

7.14. example

Removal of the Z-protecting group

0.855 g of compound (9.7) is hydrogenated with 0.300 g of Pd / C (10%) in 30 ml of tetrahydrofuran. The catalyst was filtered off and the solution was evaporated to give the pure product as a colorless oil.

FAB-MS: 578 (M + H) ⁺ .

1 H-NMR (CD ₃ OD): 4.25 (m, 4H); 3.75 (s, 3H); 3.71-3.30 (m,

6H); 2.12 (m, 2H); 1.52 (s, 2H); 1.49 (s, 3H); 1.44 (s, 9H); 1.21 (s, 9H); 1.00 (t, 2H); 0.50 (s, 9H).

7.15. example

Condensation protected with arginine

0.825 g of Z-Arg (Pmc) -OH is dissolved in 15 ml of tetrahydrofuran, and 0.360 g of benzotriazol-1-yloxy is added at 0 ° C.

tris (dimethylamino) phosphonium hexafluorophosphate (BOP) and 0.20 ml triethylamine. The reaction mixture is stirred for 10 minutes and then 0.75 g of the product obtained in 7.14 is added. example. The ice bath was removed and the reaction mixture was stirred at room temperature for 2.5 hours. The reaction mixture was poured into saturated NH 4 Cl solution and the aqueous layer was extracted three times with ether.

The combined organic layers were washed with water and brine, dried over sodium sulfate and the solvent was evaporated. The residue was purified by flash chromatography.

with her son (10: 0.3, ether: methanol). so pure (9.8) m.p. yellowish foam shape. FAB-MS: 1134 (M + H) ⁺ . ¹ H-NMR (CD ₃ OD): 7.32 ( m. 5H); 5 10 (s, 2H); 4.35 (M, 1H); 4.20 (m, 4H); 3.72 (s, 3H ); 3.69 (M, 2 H); 3.58 (m , 2 H) ; 3.45 (m, 2H); 3.19 (t, 2H); 2, 68 (t, 2H); 2.56 (s, 3H) ; 2.55 (S, 3H); 2.40-2.10 (m, 2H) ; 2.08 (s, 3H) ; 1.82 (t, 2 H) ; 1,68- 1.40 (m, 4H); 1.50 (s, 3H ); 1.42 (S, 9H); 1.39 (s , 6H); 1.20 (s, 9H); 1.00 (t, 2H); She, 50 (s, 9H).

7.16. example

Removal of the 2-trimethylsilylethyl group

Dissolve 0.700 g of compound 9.8 in 20 ml of tetrahydrofuran and add 0.39 g of tetrabutylammonium fluoride. The reaction mixture was stirred for 5 hours at room temperature and then poured into a saturated solution of ammonium chloride. The aqueous layer was extracted three times with ethyl acetate. The combined organic layers were washed with water and brine, and the solvent was evaporated in vacuo. The rest was flashing

purification by chromatography (10: 0.2, methylene chloride: methanol). The pure product is thus obtained as a yellowish oil.

FAB-MS: 1034 (M + H) ⁺ .

¹ H-NMR (DMSO): 8.40 (d, 1H); 7.82 (m, 1H); 7.75 (d, 1H);

7.62 (D, 1H); 7.32 (m, 5H); 6, 2 (d, 1H); 5 00 (s, 2H); 4.35 (M, 1H); 4.05 (M, 1H); 3, 79 (M, 1H); 3.66 (s, 3H); 3,42- 3.23 (M, 5H); 3.19 (M, 2 H) ; 3, 00 (M, 3H); 2.60 (t, 2H); 2.49 (S, 3H); 2.48 (s, 3H); 2.10 ( s, 3H). ; 1.82 to 1.40 (m, 4H); 1.75 (T, 2 H) ; 1.42 (s, 9H); 1.40 ( And, 3H), ; 1.22 (s, 6H); 1.10 (s, 9H).

7.17. example

Removal of the Z-protecting group

A 7.16. A solution of 0.555 g of the compound of Example 1d in 15 ml of methanol is hydrogenated with 0.110 g of Pd / C (10%). The catalyst was removed by filtration, and the filtered solution was evaporated to give the product as a white powder.

FAB-MS: 900 (M + H) ⁺ .

¹ H-NMR (CD ₃ OD): 4.35 (m , 1H); 4 , 05 (m, 1H); 3,78- 3.45 (M, 7H); 3.65 (s, 3H); 3.20 (m, 3H); 2.68 (t, 2H) ; 2.58 (s, 3H); 2.65 (s, 3H); 2.12 (m, 1H); 2, 10 (s, 3H); 1.82 (T, 2 H) ; 1.70-1.40 (m, 4H); 1.50 (s, 3H); 1.42 (s, 9H) ; 1.30 (s, 6H);

1.20 (s, 9H).

7.18. example

cyclization

To a solution of 0.207 g of 1-hydroxybenzotriazole and 0.280 g of dicyclohexylcarbodiimide in 30 ml of dimethylformamide is added dropwise over a period of 9.5 hours, the solution of 7.17. example

compound 0.122 g in 30 ml of dimethylformamide solution. The reaction mixture was stirred at room temperature 15 hours by evaporation of the solvent in vacuo, a was by flash chromatography (10: 0.3, methylene chloride: methyl

butanol) purified. so the clean (9.9) of Compounds get uk in the form of a yellowish powder FAB-MS: 882 (M + H) ⁺ . ¹ H NMR (CD ₃ OD): 4.41 (m, 1H); 4, 22 (m, IH); 4.02 (m, 1H); 3.82 (m , 1H); 3.72-3.48 (m, 4H), 3.69 (s , 3H); 3.50 (m, 1H); 3.2 (t, 2 H); 2.69 (t, 2H); 2.57 ( s, 3H); 2.56 (s, 3H); 2.02 (m, 2H) ; 1.92-1.75 (m, 2H); 1.84 (t, 2H) ; 1.65-1.58 (m, 2 H) ; 1.51 (s , 3H); 1.43 (s, 9H); 1.30 (s, 6H) ; 1.18 (s, 9H).

7.19. example

Cleavage of the methyl ester

Dissolve 0.063 g of compound (9.9) in 1.4 ml of methanol and add 0.008 g of LiOH in 0.4 ml of water.

The reaction mixture was stirred at 45-50 ° C and then diluted with water. The aqueous layer was extracted with ether once an hour. The aqueous layer was acidified with 1% KHS0 ₄ solution and extracted with ethyl acetate again. The combined ethyl acetate layers were washed with brine and dried over sodium sulfate. The solvent was evaporated to give the product as a white powder.

FAB-MS: 868 (M + H) ⁺ .

1 H-NMR (CD ₃ OD): 4.41 (m, 1H); 4.22 (m, 1H); 4.00 (m, 1H);

3.82 (m, 1H); 3.72-3.48 (m, 5H); 3.20 (t, 2H); 2.69 (t, 2H);

2.57 (s, 3H); 2.56 (s, 3H); 2.10 (s, 3H); 2.02 (m, 2H);

1.92-1.75 (m, 2H); 1.84 (t, 2H); 1.65-1.57 (m, 2H); 1.51 (s, 3H); 1.43 (s, 9H); 1.30 (s, 6H); 1.18 (s, 9H).

7.20. example

Condensation with 2-aminoethyl indole

A 7.19. A solution of 0.055 g of the product of Example 1 is dissolved in 2 ml of methylene chloride and 0.014 g of dicyclohexylcarbodiimide and 0.011 g of 1-hydroxybenzotriazole are added. After stirring for 10 minutes at room temperature, a solution of N-Boc-3- (2-aminoethyl) indole in 2 ml of methylene chloride was added. After stirring for 4 hours at room temperature, the solution was concentrated in vacuo. The residue was purified by flash chromatography

(10: 0.5, methylene chloride: methanol ). so the (9.10) formula compound two diastereomers 1: 1 ratio mixture of get uk. FAB-MS: 1110 (M + H) ⁺ . ¹ H NMR (CD ₃ OD): 7.60 (d, 1H); 7 , 49 (d, 1H) ); 7.4 0 ( and, 1H); 7.30 to 7.1 Δ (m, 2H); 4.49 ( m, 1H); 4.30 (t, 1H); 4.20 (M, 1H); 4.10 (m , 1H); 3.80-3.70 (dd, 1H ); 3.70 to 3, 50 (m, 4H) ), 3.50 (t, 2H) ; 3.20 (dd, 1H); 2.92 ( q, 2H); 2, 82-2.60 ( m. 2 H) ; 2.52 (s, 3H); 2.50 (s, 3H) ); 2.10 (s, 3H); 2.00-1.50 (M, 8H); 1.50 (d, 3H); 1.40 (s, 9H) ); 1.35 (s, 6H); 1.15 (d, 9H).

7.21. example

Deprotection and separation of diastereomers

Dissolve 0.030 g of compound (9.10) in 1 ml of a mixture of trifluoroacetic acid / ethanedithiol (85:15) and add 4.5

Stir at room temperature for 100 hours. The solvent was then evaporated in vacuo. The remaining ether solidifies. The two diastereomers were separated by HPLC using a Nucleosil reverse phase C 1-4 column. Gradient from 10% CH 2 CN (0.09% trifluoroacetic acid) / 90% H ₂ O (0.09% trifluoroacetic acid) to 20% CH 2 CN (0.09% trifluoroacetic acid) / 80% H ₂ O (O, 09% trifluoroacetic acid) over 50 minutes.

Compound R _t (9) = 30.3 min; FAB-MS: 588 (M + H) ⁺ . R _t (D) - isomer = 29.1 min ; FAB-MS: 588 (M + H) &lt; ⁺ &gt; ^. ^ -NMR (D ₂ 0) (9) compound: 7.62 (d, 1H); 7.42 (d 1H); 7.20 (t, 1H); 7.18 ( s, 1H); 7.10 '(t, 1H) ; 4, 90 (m, 1H) 4.43 (t, 1H) ; 4.18 (s, 1H); 4.16 (t, 1H); 3 , 85 (Dd, 1H) 3.67 (m, 2H) ; 3.58 (t, 2 H); 3.31 (dd, 1H); 3.19 (M, 2 H) 3.10 (m, 1H); 2.98 (t, 2H); 2.90 (t, 2H); 1.90 to 1, 57 (m, 6H)

1.42 (s, 3H).

1 H-NMR (D ₂ O) (D) -isomer: 7.63 (d, 1H); 7.42 (d, 1H); 7.20 (vol

and s, 2H); 7.10 (t, 1H); 4.90 (m, 1H); 4.50 (m, 1H); 4.10 (t, 1H); 4.05 (m, 1H) ; 3.79 (m, 2H); 3.60 (M, 1H); 3.50 (m, 4H); 3.29 (m, 1H); 3, 19 (m, 2 H); 3.00 (m, 3H) ; 1.84-1.42 (m, 6H); 1.40 (s, 3H). Example 8 Compound (10) synthesis 8.1. example Fmoc-D-Cys (Trt) - TAB-ester resin Dissolve (0.24 mmol) Fmoc-D-Cys (Trt) -t 0.4 ml (distillate)

In DMA and with slow stirring, 2 ml of DCE and 2 ml were added

101

0.40 g TAB ester resin (0.24 mmol) (Novabiochem). The mixture was cooled to 0-5 ° C and 0.1 g DCCI (0.48 mmol) was added.

In a mixture of 0.2 ml of DMA and 0.06 ml of DCE. The solution was stirred at 0-5 ° C for 5 min and a solution of 5.9 mg DMAP (0.048 mmol) in 0.4 mL DCE was added. After 20 minutes, 27.3 μΐ N-methylmorpholine was added at 0-5 ° C. The mixture was shaken for 4 hours. The resin was washed 6 times with 6-6 ml DMA and 6 times with 6-6 ml isopropanol, dried in vacuo, 0.5 g (with traces of solvent).

8.2. example

Synthesis of the protected resin bound peptide

Ac-D-Trp-Cys (Trt) -D-Ser (But) -D-Arg (Pmc) -D-Cys (Trt) - (TAB-ester-resin)

The starting material was 0.25 g of Fmoc-D-Cys (Trt) -TAB-ester resin (0.15 mmol). The following procedures are performed in each step:

- single wash for 0.8 minutes with isopropanol;

- pre-activation for first coupling: 1.4 mmol of the partially protected Fmoc amino acid is dissolved in 3.08 ml of 0.5 M HOB in DMA and 0.77 ml of 2 M DIDC in DMA is added. The reaction mixture is allowed to stand for 40 minutes and then worked up in this form.

During this time, the following washings and removal of the Fmoc group are performed:

- treatment of the resin starting material with 20% piperidine-DMA solution eight times (removal of the Fmoc protecting group) for 1 minute each;

102

- three washes for 0.4-0.4 minutes with DMA (previously degassed under reduced pressure);

- single wash for 0.8 minutes with isopropanol;

5 washes for 0.4-0.4 minutes with DMA (degassed);

- addition of the coupling reagent prepared in the meantime (above). The reaction mixture was stirred for ca. Shake for 60 minutes. After 5 and 15 minutes from the start of the reaction, 120-120 mL of DIPEA (0.7 mmol) was added;

removing the coupling reaction mixture after 60 minutes;

- two washes for 0.4 minutes with DMA (degassed);

- single treatment for 5 minutes approx. 15 ml 1: 1: 8 (v / v / v) acetic anhydride: pyridine: DMA (for acetylation of the remaining free amino groups of the growing peptide chain);

four washes for 0.4-0.4 minutes with DMA (degassed);

- two washes for 0,8-0,8 minutes with isopropanol. The following Fmoc peptide / resin intermediate is obtained:

Ac-D-Trp-Cys (trt) -D-Ser (But) -D-Arg (Pmc) -D-Cys (Trt) - (TAB ester resin) and

By sequential coupling of Fmoc-D-Arg (Pmc), Fmoc-D-Ser (But), Fmoc-Cys (Trt) and Fmoc-D-Trp.

Upon completion of the synthesis, the Fmoc group is removed and the terminal amino acid is acylated with the coupling reagent as described above. The final wash was done with isopropanol and the resin was dried under reduced pressure: 0.38 g.

8.3. example

Cleavage of the linear protected peptide from the resin

103 ···. ··:. ··. ··; . · ♦ · · · · · ··· ·· * ·

0.38 g of the resin-bound peptide in 1.5 hours beggar - 99% AcOH and DCM (1: 9, v / v). mixtures 100 ml of sulfur. The resin was filtered off and 80-80 ml DCE

Wash twice, then wash once with 80 mL of a mixture of DCE and TFE (1: 1, v / v), then four more times with 80-80 mL of TFE. The combined filtrate solutions were concentrated under reduced pressure at 25-30 ° C to give an oily residue of ca. Dissolve in 200 ml DMSO (distilled) and lyophilize. To completely remove the traces of AcOH, two additional lyophilizations were performed with 50-50 mL of DMSO, since traces of AcOH may interfere with the next cyclization reaction. After DMSO lyophilization, the product was 0.21 g of a yellow powder.

Tlc, CM. single spot Rf ca 0.4.

MALDI, positive mode, 1606.7 (calculated 1604.1).

8.4. example

Coupling of the linear protected peptide to mono-Boc-1,2-diaminoethane

0.101 g (65 µmol) of the linear protected peptide are dissolved in 0.70 ml DMA (degassed), 12.2 µΐ DIPEA (71.5 gm) and 28.8 mg TBTU (65 gm). we add. Pre-activation takes 3 minutes. 41.9 mg of mono-Boc-1,2-diaminoethane (262 μιυόΐ) are added and the mixture is allowed to stand for 16 hours. The crude product precipitated by adding 10 ml of water (0-5 ° C), filtered and washed with 3 ml of water. The resulting solid was dried over P ₂ O _{5 under} reduced pressure: 95 mg.

Tlc CM, R _f ca. 0.85.

104 • * · · • · · · · ··· ···

MALDI, positive mode, 1746.5 (calculated 1746.0).

8.5. example

Cleavage of protecting groups

To remove the polymeric carrier and the acid-labile protective anthers, 95 mg of the synthesized peptide were shaken twice with 4 ml of a 95:15 (v / v) mixture of 95% TFA and ethanedithiol and filtered. The filtrate was washed three times with 4-4 mL of DCE and three times with 4 mL of TFE. The filtered solution and the washings are evaporated under reduced pressure for approx. 2 ml and the crude peptide was precipitated with 15 ml of a 1: 1 (v / v) mixture of DIPE and petroleum ether (low boiling). The precipitate was filtered off, washed with 5 ml of mother liquor and dried under reduced pressure.

For complete deprotection, the solid was dissolved in 4 mL of the above cleavage mixture and allowed to stand for two hours. The precipitate was dried under reduced pressure. The product is a white powder of 43 ml. Analytical HPLC: retention time ca. 14.3 minutes. MALDI, positive mode: 737.3 (calculated: 738.9).

8.6. example

Cyclization of the linear peptide

43 mg (58.4 gmol) of the peptide are dissolved in 600 ml of water and the pH is adjusted to 8 by addition of ammonia (10% aqueous solution). The cyclization takes place with the formation of disulfide by bubbling air through the solution for four hours. The solution was acidified with 2.5 mL of AcOH and then kissed. · ♦ · a «·« ««

105 · «evaporate at approx. 10 ml, then lyophilize. The crude peptide is dissolved in 4.5 ml of CH ₃ CN / water for purification, 0.1 ml (1: 9,

AcOH was added and subjected to high pressure liquid chromatography (HPLC) under the following conditions: column size: 20

Machery-Nagel

250 mm, Nucleosil 7 C (d-nm) (manufactured by:

Dueren, Germany); eluted with 0.1% TFA (A)

The linear gradient from 10% B to 90% B over 30 minutes, throughput rate of 18 ml / min, and detection at 215 nm. The main fraction was ca. It was collected at 16 min retention time, concentrated under reduced pressure, filtered through an ion exchange resin (15 mL) [AG1-X8 Bio-Rad], and AcOH was added to replace the TFA with AcOH. Wash the column thoroughly with water and lyophilize the combined fractions (40 mL). The compound of formula (10) is obtained as a colorless white powder.

Analytical HPLC, single peak, ca. 15.3 minutes retention time.

MALDI: positive mode 736.9 (calculated 736.9).

Example 9

Synthesis of compound (11)

9.1. example

Synthesis of compound of formula 11.1

106 · * · ·. , · ♦ ··· ·· «· h

9.1.1. example

Reduction of the carboxyl group of Fmoc-Cys (Trt)

Fmoc-Cys (Trt) (10 mmol) was dissolved in 10 mL DME and cooled to -15 ° C. 1.11 ml of N-methylmorpholine (10 mmol) and 1.36 ml of isobutyl chloroformate (10 mmol) are added gradually. After one minute, the precipitated N-methylmorpholine hydrochloride was filtered off, washed with DME (5 x 2 mL), and the filtered solutions and washings were combined in a large flask in an ice bath. Add 570 mg of sodium borohydride (15 mmol) in 5 mL of water at once, causing vigorous gas evolution, and then rapidly add another 250 mL of water. The resulting alcohol was extracted with diethyl ether and purified by flash chromatography (ether / petroleum ether 6: 4).

1 H-NMR (CDCl 3): 7.8-7.0 (m, 23H ); 4.84 (d, 1H); 4, 40 (d, 2 H); 4.20 (t, 1H); 3.50 (m, . 4H); 2.45 (m, 1H). 9.1.2. example Tosylation of alcohol Separated alcohol 9, 1 g, which the 9.1.1. Example 20 ml CH ₂ Dissolved in Cl ₂ , and Replies

0.65 ml of (CH 3 -CH 3) -1N, 0.710 g of toluene-4-sulfonyl chloride and 0.04 g of dimethylaminopyridine are added at 0 ° C. The ice bath was removed immediately and the reaction mixture was stirred for 4 hours at room temperature. The reaction mixture was poured onto ice and extracted with two portions of methylene chloride. The combined organic extracts were dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (ether / hexane 1: 1). so we get the right one

107. · *. ···;

2 tosylates in the form of a white foam * * · * «* · ♦ ·· <·· ♦♦ · <· * · 2 tosylate.

¹ H-NMR (CDCl ₃ ): 7.8-7.18 (m, 27H); 4.65 (d, 1H); 4.43 (m,

2 H) ; 4.15 (t, 1H); 3.91 (m, 2H); 3.52 (m, 1H); 2.42 (d, 2H);

1.58 (s, 3H).

9.1.3. example

Substitution of the alcohol group with an azido group and removal of the Fmoc protecting group

A 9.1.2. 1.5 g of the compound of Example 1 are dissolved in 20 ml of dimethylformamide and 0.160 g of sodium azide are added. The reaction mixture was stirred at 60 ° C for 3 hours and then partitioned between saturated ammonium chloride and ethyl acetate. The organic layer was separated, dried over sodium sulfate and concentrated in vacuo. The product is a yellow oil. Purify by flash chromatography (ether). The compound of formula (11.2) is obtained in pure form.

1 H-NMR (CDCl ₃ ): 7.35 (s, 15H); 3.15 (d, 2H); 2.52 (m, 1H),

2.28 (d, 2H); 1.48 (s, 2H).

IR (CH ₂ Cl ₂ ): 2106 cm ^-1 (s, N ₃ ); 1720 cm ^-1 (s, C = O).

9.1.4. example

Introduction of the Fmoc protecting group

Dissolve 1.10 g of compound 11.2 in 20 ml of methylene chloride and add 0.5 ml of N-ethyldiisopropylamine and 0.760 g of FmocCl-c at 0 ° C. The reaction mixture was warmed to room temperature and stirred for 4 hours. It is partitioned between methylene chloride and saturated ammonium chloride solution. The organic layer was separated on calcium chloride ·······················································

108 ·

Dry and evaporate the solvent in vacuo. The crude product was purified by flash chromatography (ether / hexane, 80:20). The Fmoc-protected pure product is formed as a white foam.

¹ H-NMR (CDCl ₃ ): 7.80-7.20 (m, 23H); 4.40 (d, 2H); 4.20 (t,

1H); 3.54 (m, 1H); 3.30 (m, 2H); 2.41 (m, 2H).

IR (CH ₂ Cl ₂ ): 2108 cm ^-1 (s, N ₃ ); 1730 (s, C = O).

9.1.5. example

Reduction of the azido group to the amino group

1.5 g of the Fmoc-protected compound are dissolved in 30 ml of THF, 0.735 g of triphenylphosphine and 0.54 ml of water are added. The reaction mixture was stirred for 12 hours at room temperature, poured into ice and saturated ammonium chloride and extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulfate, concentrated in vacuo,

4.40 (d,; 2.41

Adding a Boc protecting group

From compound (11.3)

Dissolve 1.5 g in ml of THF, add 0.660 g of di-terbutyl dicarbonate and add

0.45 mL of N-ethyldiisopropylamine at 0 ° C. The ice bath was removed immediately and the reaction mixture was stirred for 2 hours at room temperature. The reaction mixture is divided into saturated * · * ··> «4 · · · · wood ·

- 109 - ...........:

ammonium chloride solution and ethyl acetate, the organic layer was separated, dried over sodium sulfate and evaporated in vacuo. The residue was purified by flash chromatography (ethyl acetate / hexane, 2: 6). The product is a colorless oil.

¹ H-NMR (CDCl ₃ ): 7.80-7.20 (m, 23H); 5.22 (d, 1H); 4.45 (d,

1H); 4.35 (d, 2H); 4.18 (t, 1H); 3.58 (m, 1H); 3.10 (t, 2H);

2.40 (t, 2H); 1.40 (s, 9H).

9.1.7. example

Removal of the Fmoc protecting group

Dissolve 15 ml of dimethylamine in dimethylformamide and add 2 g of 9.1.6. Example. The reaction mixture was stirred at room temperature for 30 minutes and partitioned between saturated ammonium chloride solution and ethyl acetate. The organic layer was separated, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (ethyl acetate) to give pure 11.1.

¹ H-NMR (CD ₃ OD): 7.46-7.18 (m, 15H); 2.90 (dd, 1H); 2.82 (dd, 1H); 2.48 (m, 1H); 2.35 (dd, 1H); 2.22 (dd, 1H); 1.40 (s, 9H).

9.2. example

Fmoc-D-Arg (Pmc) - (TAB-ester-resin)

Fmoc-Arg (Pmc) -t a 8.1. In a manner analogous to that described in Example 1b, the resin is reacted.

··· «·· ··» «

- 110

9.3. example

Ac-D-Trp (Boc) -Cys (Trt) -D-Ser (But) -D-Arg (Pmc) - (TAB-ester-resin)

0.30 g (0.19 mmol) of Fmoc-D-Arg (Pmc) -TAB ester resin by solid phase synthesis as described in 8.2. by analogy with Examples 1 through 4, the following amino acid derivatives are contacted with Fmoc-D-Ser (But), Fmoc-Cys (Trt) and Fmoc-D-Trp. At the end of the synthesis, the Fmoc group is removed and the terminal amino acid is acylated as described in 8.2. capping reagent. After the final washing with isopropanol, the resin was dried under reduced pressure: 0.47 g.

9.4. example

Cleavage of the protected linear peptide from the resin

0.39 g (0.17 mmol) of the peptide-bearing resin was prepared as described in 8.3. treated as described in Example. Lyophilization from DMSO gave 0.19 g of a yellow powder.

Tlc, CM. single spot Rf approx. 0.35.

MALDI, positive mode, 1258.1 (calculated: 1258.6).

9.5. example

Coupling of the protected linear peptide to compound (11.1)

68 mg (55 µmol) of the protected linear peptide were dissolved in 0.40 ml DMA (degassed), 10.2 μΐ DIPEA (60.5 gmol) and 24.4 mg TBTU (55 pmol). and preactivate for 3 minutes. 49.3 mg of compound (11.1) are added and the mixture is allowed to stand for 16 hours. THE ·· ·

- 111 crude product precipitated with 3.0 ml of water (0-5 ° C), filtered and washed with 1.0 ml of water. The resulting solid was dried over P ₂ Og. 85 mg of material are thus obtained

Tlc CM, R _f Ca. 0.91.

MALDI, positive mode, 1688.7 (calculated 1689.2).

9.6. example

Cleavage of protecting groups

85 mg of the crude protected peptide is prepared as described in 8.5. Example 2, the complete removal of the protecting groups c. 39 mg white powder, analytical HPLC: retention time ca. 13.1 minutes.

9.7. example

Cyclization of the linear peptide

39 mg (57.3 μιπόΐ) of the pptide are oxidized according to 8.6. in 400 ml of water. The crude product was subjected to HPLC purification followed by acetate conversion under standard conditions (Example 8.6) but gradient run from 10% to 90% over 30 minutes. Retention time approx. 15 minutes. After lyophilization, 11 is obtained in the form of a white powder.

Analytical HPLC, single peak ca. With a retention time of 14.8 minutes.

MALDI: positive mode 678.9 (calculated 679.8).

Example 10

Proteolytic Stability • ··

To determine the proteolytic stability of TRSAW and its derivatives, the peptide was incubated at 37 ° C in rat serum at 200 μ-in physiologically relevant medium (0½). Aliquots of the incubated mixture (100 μ eleg)

we are defined intervals, 60 gl 20 % per- with chloric acid, stirring p and his 12,000 g centrifuge for 8 minutes. The rest was starting si material and remained in the serum decomposition products in reverse phase HPLC determined Nucleosil C ₁₈ and Nucleos il Cg autumn- lopokon UV display ? at 216 nm, flow ratio 1.0 ml / min gradient 10-90% CHgCN over 30 minutes (1. spreadsheet). First Spreadsheet Compound Half-life (minutes) TRSAW 5 (3) 22 (5) 18 (6) 22

Example 11

Effect of TRASW on trabecular bone in ovariectomized rats

Tif: RAIF (Sisseln) from Sprague-Dawley strain was performed on female rats weighing 130-150 g. The animals received ad libitum standard rat diet (8NAFAG, Grossau, Switzerland) containing 12% calcium, 0.9% phosphorus

Contains 113 and 24.8% protein. Vetanacrol was anesthetized (Veterinaria, Zurich, Switzerland) with an electric knife, dorsally ovarian exterminated (OVX-ovariectony). The animals were randomly assigned to groups of 8.

Treatment on the day of ovariectomy

TRASW or its analogs were resolved. The phosphate buffer was in saline containing 0.1% serum albumin.

test compounds were administered subcutaneously daily to the animals for 5 days per week. Non-operated and ovariectomized control animals received the appropriate amount of vehicle. The treatment lasted for 3 weeks.

The animals were sacrificed 24 hours after the last injection. Both femus were cleared of adipose tissue and trabecular bone mass was determined according to Gunness-Hey and Hock (Metab. Bone Dis. & Rel. Res. (1984), 5, 177-181). The femurs were cut in half at the mid-diaphysis with a dental saw and the proximal halves set aside. The epiphysis of the distal halves was dissected with a scalpel and the bone was broken into sagittal halves. The bone marrow was washed with water. With a dental scraper (curette), the trabecular bones were scraped from both crustaceans, combined and placed in 5% TCA. After standing at room temperature for 16 hours, the TCA extract was separated and the calcium determined by atomic adsorption spectroscopy.

Results are reported as + SEM (Table 2). Statistical analysis of the data was performed using a two-tailed Student's paired test.

114

Table 2

Tested- Dose ⁺ gg / ml potassium content of femur trabecular bone calamus [mg]

Intact control OVX control OVX + test compound TRSAW 0.2 4.06 ± 0.12 2.96 + 0.11 * 3.28 ± 0.11 TRSAW 2 3.71 ± 0.14 ^# TRSAW 20 4.45 ± 0.22 # (3) 1 5.32 ± 0.28 3.24 ± 0.12 * 3.45 ± 0.14 (3) 4 3.68 ± 0.13 # (3) 10 3.32 ± 0.13

⁺ : Daily dose Mg / kg rat #: p <0.05 vs. OVX control: p <0.05 for intact control.

Example 12

Effect on protein kinase C activity

Compounds capable of activating protein kinase C (PKC) are also likely to be active in bone anabolic actions and can effectively reverse the process of bone loss in patients with osteoporosis. [I. Jouishomme et al., Endocrinology (1992) 130, 53-60]. Therefore, PKC activity of Compound 3 was compared with that of acetyl-TRSAW, which is known to be inactive in the treatment of osteoporosis, while

TRSAW-amide is known to be active [Gagnon et al., J. Boné and

115

Mineral Slit. (1993), 8, 497-503]. Activity was measured as a percentage of the difference between membrane and cytosol localized to PKC.

12.1. example

Stimulation with test compound

Serum-free cells (osteosarcoma cells: UMR 106 line) were stimulated with the compound of interest (10 min, 30 ° C, concentrations as in Table 1).

12.2. example

Separation of cytosolic and membrane-associated PKC

The cells were washed twice with cold saline (4 ° C) and triturated with 800 μΐ buffer (A) composed of:

85.60 g of sucrose,

0, 58 g EDTA, 2.42 g Three, 1.90 EGTA

water (1000 mL) was added and the pH was adjusted to 7.5 with hydrochloric acid.

Add 10 ml of this buffer before use

20 μΐ DTT ι (1M), 20 μΐ PMSF (50 mM) -t, 40 μΐ leupeptin ( 2.5 mg / ml)

and sonicated in a centrifuge tube at low intensity for 10 minutes at 4 ° C. The cells were then centrifuged for 60 minutes at 100,000 g and the supernatant (cytosolic PKC) was separated. The pellet was suspended in 776 μΐ buffer (A); adding 25 μΐ triton (10%); the tube was shaken for 1 hour at 4 ° C and centrifuged again at 100,000 g for 60 minutes. The supernatant contains the membrane association

PKC.

Wash 3 ml of DEAE sephacel three times with 20-20 ml of buffer (B), consisting of:

2.42 g / L Tris pH 7.5

0.29 g / L EDTA

0.38 g / L EGTA.

After each wash, the resin is centrifuged. Finally, the resin is washed with buffer (C) consisting of:

40 ml (B) buffer 80 μΐ DTT (1M) 80 μΐ PMSF (50 mM) 160 μΐ leupeptin (2.5 mg / ml). 15650 μΐ (C) we give you a buffer For 333 μΐ washed resin, the cytosolic

los and membrane-associated PKC were added in separate compartments and the suspension was stirred at 4 ° C for one hour. The supernatant was separated by centrifugation and the resin was washed three times with 1 ml of buffer (B) and once with 1 ml of buffer (B) containing 20 mM NaCl.

The resin bound PKC was eluted with 300 µl NaCl (130 mM), shaken for 15 min at 4 ° C, and the supernatant was collected by centrifugation. The remaining resin bound PKC was eluted by the addition of 200 μΐ NaCl (130 mM), shaken for 15 min at 4 ° C, and the supernatant was collected by centrifugation. The two supernatants were combined.

• ·

117

12.3. example Measurement of activity buffers blind reaction Tris (0.2 M; pH 7.5) 25 μΐ 25 μΐ Mg acetate (0.5 M) 5 μΐ 5 μΐ leupeptin (0.5 mg / ml) 25 μΐ 25 μΐ radioactive ATP (100 μπι) 25 μΐ 25 μΐ CaCl ₂ (2.5 mM) 2.5 μΐ 2.5 μΐ phosphatidylserine mix - 80 μΐ Tris (0.02 M) 80 μΐ - h ₂ o 30.5 μΐ 30.5 μΐ peptide (7 mM) 7 μΐ 7 μΐ

phosphatidylserine mixture:

To 150 μΐ of phosphatidylserine μΐ diols (5 mg / ml) added 5 ml of tris (20 mM; pH 7.5) peptide:

starting material for phosphorylation (FKKSFKL-ND ₂ )

Add 50 μΐ of the cytosol and membrane fractions

200 μΐ blank (reaction time 5 minutes at 30 ° C). The reaction was quenched by the addition of 125 μΐ concentrated acetic acid. from this

Transfer 100 μΐ of the solution to a 3 x 3 cm Whatman ^R filter paper. The experiment was repeated using the reaction mixture instead of the blank. Each blank experiment was repeated twice as three times with the reaction mixture. The filter papers were counted by scintillation counting. The average value of the blank was subtracted from the average of the reaction mixtures and the amount of protein in each sample was calculated using the specific activity of radioactive ATP.

The difference between cytosolic and membrane-associated PKCs is shown in Figs

As noted in Table 1:

Table 1

Concentration of test compound in mol / l Cytosolic and membrane-associated PKC % difference TRSAW-amide Acetyl-TRSAW Compound (3) 0 0 0 0 ΙΟ ^'11 16 9 14 10 ~ ¹⁰ 22 2 31 ΙΟ ' ⁹ 27 21 48 10 " ⁸ 42 3 69

Compound (3) shows an increased activity compared to also active TRSAW-amide. Acetyl TRSAW did not exhibit typical activity as expected.

119

Claims (5)

PATENT CLAIMS C 1-4 alkyl; R 1 = hydroxy or C 1-4 alkyl substituted by hydroxy; one of R ₇ and R q is C 2 -C 5 alkyl -C (= NH) NH ₂ or -NH-C (= NH) NH ₂ PBS; and the other is hydrogen or C 1-4 alkyl; Rio ^{= _} ^ 2 ^; Substituted with ^{1 to 4} carbon atoms in the alkyl group mutated -NH ₂ ; C 1 -C 4 substituted with -NH ₂ alkyl, ; - N ^ NH; -NH-CO-CH ₂ -NH ₂ or -NH-CH ₂ -CH ₂ - -NH ₂ group, which are substituted with a methyl or ethyl group they may be tuated; 122 - Ο I, -. ^{R11 =} hydrogen, -COOH, -CONH _2, - C - or ^R12 C 0 '· ^R 12 ⁼ a group derived from a natural amino acid or a peptide composed of amino acids 2-5 natural ^e 9Y. C 1-4 alkyl; A 5- or 6-membered heterocyclic group containing one or two nitrogen atoms; or an acyl group derived from a natural amino acid or a peptide having from 2 to 5 natural amino acids. A cyclic compound of formula (I) wherein: A = ring group of 12-17 atoms, B = a bulky group attached to ring A via a carbon atom or nitrogen and containing from 1 to 6 carbon atoms, 0 to 2 nitrogen, 0 or 1 oxygen atoms in the backbone; wherein the carbon atoms may be substituted with oxo, hydroxy, sulfo, C 1-4 alkyl, morpholino, amino, carboxyl and / or a natural amino acid or a peptide composed of 2 to 5 natural amino acids; the nitrogen atoms may have a C 1-4 alkyl, a C 1-4 alkanoyl and / or a natural amino acid or a 2-5 natural amino acid peptide substituent; D = aryl (C 1 -C 4) alkyl, where the aryl may be mono- or bicyclic and may be substituted by OH, SH, NH 2 or halogen; E = NH ₂ -; C 1 -C 4 alkyl or amino (C 1 -C 4 alkyl substituted amino group; C 1 -C 4 alkyl substituted by one or more amino groups, or a peptide consisting of a natural amino acid or a 2-5 natural amino acid) an amino group substituted by an acyl group from a chain, or a five- or six-membered heterocyclic group containing one or two nitrogen atoms; F = NH or CH ₂ ; á • · ♦ ·· - 120 - X = 1-6; y = 0-4; with the proviso that the groups containing (CH ₂ ) _X -FC (= NH) NH ₂ and (CH ₂ ) y -OH and the BD group are above the plane of ring A and the group E is below the plane of ring A; or pharmaceutically acceptable salts thereof. The cyclic compound according to claim 1, wherein the distance between the binding site of the BD group and the group E on the ring A is 0.50-0.65 nm, the distance between the (CH ₂ ) y group and the location of the group E is 0, 50 to 0.62 nm, and a _{(CH2) x} group and bonding group distance BD from 0.55 to 0.75 nm. The compound of formula (Ia) according to claim 1, wherein up to 6 of the ring carbon atoms may be replaced by oxygen, sulfur or nitrogen; ring atoms up to 6 1- 4. A compound of formula (2) according to claim 1, where ^R 1 = -CH ₂ -, -C ₂ H ₄ -, -CH ₂ H ₂ -, -CH _(CH3) -ch ₂ -, -s, -ch ₂ -s-, , -ch ₂ -ss-, -ss-ch ₂ -, -SS-, s = o, o = s = o, NH-, -NH-CO-. , -O-, -Se-; -O-CH ₂ , -CH ₂ -O-; ^R 2 = C 1-6 alkylaryl or -CO- NH-CH-CH - (1-3 C 1-4 alkylaryl) R 1-4; R ₃ , R ₄ , R 8 and R _g are each independently hydrogen They may be substituted by C 4 alkyl, oxo, hydroxy, sulfo or amino; and X is C 1 -C 4 alkylene, C 1 -C 4 alkenylene, where up to 2 carbon atoms may be replaced by oxygen, sulfur, disulfide, sulfoxy, sulfonyl or nitrogen, wherein C 1 -C 4 alkylene or alkenylene the group may be substituted by methyl, ethyl, oxo or amino; R _c or R _e = C 1-4 alkyl substituted with hydroxy or hydroxy, Rf, Rg or = 2-5C alkyl radical -C (= NH) -NH _2, or may be _two groups -NH-C (= NH) -NH szubszti121 " · ♦ ·· ·· • · 9-C, -CH ₂ CH ₂ CH ₂ -NH-C (= NH) -NH _{2 is} preferred; You are Rj_ R 1 = -NH ₂ ; C 1-4 alkyl, or amino (1- (C4-C4) alkyl amino; substituted with one or more amino groups 5. A compound according to claim 4, wherein R = -CH ₂ -, -C ₂ H ₄ -, -S-, -CH ₂ -S-, -CH ₂ -SS-, -SS-, -NH-, -NH-CO-, -O-, -O-CH ₂ , -CH ₂ O-, -Se-; R _{2 is C} 1-6 alkyl-R ₄₃ or -COl-NH-CH- (C 1-3);