RU2130945C1 - Pth-compounds, method of their synthesis, pharmaceutical composition containing their, dna fragment and fused protein - Google Patents

Abstract

FIELD: molecular biology, genetic engineering, hormones. SUBSTANCE: invention relates to variants of parathyroid hormone (PTH) eliciting PTH-like activity and containing at least a single modification taken from a group involving:

Description

 The invention relates to variants of parathyroid hormone (PTH), a method for their preparation, pharmaceutical preparations containing these variants, as well as their use as pharmaceuticals.

 The term “PTH” as used herein refers to any genetically encoded form of parathyroid hormone, including the mature form containing 84 amino acids that are part of the PTH of certain vertebrate species, including humans, pigs, rats, bovines, chickens, and fragments thereof, as well as their analogues and derivatives exhibiting activity similar to PTH. The position of each amino acid that is part of the PTH sequence is numbered in accordance with an internationally accepted method. For continuity purposes, as well as to comply with traditional approaches, in the following description the same system of numbering of amino acids of the PTH sequence is used, regardless of the nature of the substitutions made in the molecule.

Specifically, the present invention provides a PTH compound exhibiting activity similar to PTH and containing at least one modification selected from the group including
[Leu ⁸ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTG (1-34) OH,
[Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Thr ³³ , Ala ³⁴ ] hPTG (1-34) OH,
[Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH.

[Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Gln ¹⁸ ] hPTG (1-36) OH,
[Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTG (1-34) OH,
[Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ ] hPTG (1-36) OH,
[Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTG (1-34) OH,
[Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ ] hPTH (1-36) OH and
[Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ ] hPTG (1-36) OH.

 The invention also provides a DNA fragment encoding a fusion protein having a nucleotide sequence corresponding to the N-terminal polypeptide encoded by the bacteriophage T4 gene 55, and the aforementioned compound attached to its C-terminus.

 A fusion protein is also proposed, which is an N-terminal polypeptide encoded by the T4 bacteriophage gene 55, and a compound attached to its C-terminus selected from the above group, and, if necessary, a chemically cleavable linker between them. Preferably, this linker has the amino acid sequence Asp-Pro-Pro or Asn-Gly-Pro.

 Also proposed is a method of obtaining a compound selected from the above group, which consists in cultivating a recombinant E. coli strain transformed with an expression vector containing the DNA fragment described above, inclusion particles are isolated, dissolved under acidic conditions, and they are cleaved, separated and purified. compound.

 A pharmaceutical composition is also provided that regulates calcium metabolism, containing an active ingredient and a pharmaceutically acceptable diluent or carrier, containing as an active ingredient a PTH compound selected from the above group.

The specified modification may consist in the fact that either
1. at least one radical is a group attached to the terminal amino group of the PTH compound and selected from the following: L- or D-α-amino acid, C _2-6 alkoxycarbonyl, and also optionally substituted C _1-8 alkyl A C _2-8 alkenyl, C _2-8 alkynyl, aralkyl, aralkenyl or C _3-6 cycloalkyl-C _1-4 alkyl group; and / or at least one radical is a group attached to one or more side amino groups of the PTH compound and selected from the following: C _2-6 alkoxycarbonyl, and also optionally substituted C _1-8 alkyl, C _2-8 -alkenyl, C _2-8 -alkynyl, aralkyl, aralkenyl or C _3-6 -cycloalkyl-C _1-4 -alkyl group, or
2. at least one α-amino acid residue at position 1-38 of the native PTH sequence is replaced by a residue of a natural or artificial amino acid, possibly in a protected form, the α-amino acid residues located at positions 1 and 2 of the amino-terminal part of the PTH sequence together can be replaced by a pseudopeptide,
or a combination of these modifications takes place, wherein the PTH compound lacking a L- or D-α-amino acid attached to the N-terminus, or a C _2-6 alkoxycarbonyl or possibly substituted C _1-8 alkyl, C _2-8 - an alkenyl, C _2-8 alkynyl, aralkyl, aralkenyl or C _3-6 cycloalkyl C _1-4 alkyl group is different from a PTH compound having a naturally occurring sequence of α-amino acids; or the PTH compound is different from PTH (1-34), in which
I. The α-amino acid at position 1 is Gly, D-Ser, D-Ala, or Tyr; or
II. the α-amino acid at position 2 is Ala, D-Val, Lys, Arg or Cit, and the α-amino acid at position 34 is Tyr; or the α-amino acid at position 2 is D-Val, the α-amino acid at position 34 is D-Tyr, and each of the α-amino acids at positions 8 and 18 may be Nle; or
III. the α-amino acid at position 3 and / or 6 and / or 9 is replaced by a natural or artificial amino acid; or
IV. The α-amino acid at position 23 is replaced by Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, or Thr; or
V. α-amino acid at position 25 and / or 26 and / or 27 is replaced by Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr , Trp, Tug or Val, or
VI. each of the α-amino acids at positions 8 and 18 is Nle or Met (O), and the α-amino acid at position 34 may be Tug; either each of the α-amino acids at positions 8 and 18 is Nle, the α-amino acid at position 34 is Tyr, and either the α-amino acid at position 12 is L- or D-Pro , L- or D-Ala, Aib or NMeGly, or the α-amino acid at position 23 is Phe, Leu, Nle, Val, Tyr, α-Nal or β-Nal; or
VII. the α-amino acid at position 28 is Lys, and the α-amino acid at position 30 is Leu; or
Viii. the α-amino acid at position 1 is Aib; and / or the α-amino acid at position 8 and / or 18 is Leu, Ile, Val, Phe or Trp; and / or the α-amino acid at position 11 is Ser, Lys, Phe, β-Nal, Trp or Tug, and / or the α-amino acid at position 12 is D-Leu, D-Ile, D -Nle, D-Val, D-Ser, D-Ser (Butyl), D-Abu, D-Thr, D-Nva, D-Met, D-β-Nal, D-Trp, D-Lys, D- Tyr, D-Lys (Fmoc), D-Phe or D-Asn; and / or the α-amino acid at position 13 is Leu and / or the α-amino acid at position 19 and / or at position 21 is Arg, Lys, Asn or His; and / or the α-amino acid at position 23 is 2- (1,3-dithiolan-2-yl) Trp; and / or the α-amino acid at position 25 and / or at position 26 is His; and / or the α-amino acid at position 27 is Gln or Leu; or
IX. the α-amino acid at position 8 and / or 18 is Ala or Ser; or the α-amino acid at position 8 and / or 18 is Ala, Val, Leu, Ile, Ser or Trp, and the α-amino acid at position 34 is Tyr; or the PTH compound differs from PTH (1-84) in that
I. The α-amino acid at position 1 is Tug, Val, Pro, Asp or Cys; or
II. The α-amino acid at position 2 is Ala, Glu, Leu, Ser or Arg; or
III. α-amino acids located in positions 3 and / or 6 and / or 9 are replaced by natural or artificial amino acids; or
IV. The α-amino acid at position 23 is replaced by Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser or Thr; or
V. α-amino acid at position 25 and / or 26 and / or 27 is replaced by Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; or
VI. The α-amino acid at position 8 is Met, Met (O), Ala, Val, Leu, Ile, Ser, Trp, Asn, Gln, Asp, Glu, Lys, Arg, Tyr or Gly, and the α-amino acid at position 18 is Leu; or the α-amino acid at position 8 and / or 18 is Ala, Val, Leu, Ile, Ser or Trp, and the α-amino acid at position 34 is possibly Tyr; or each of the α-amino acids located at position 8 and at position 18 is Met (O); or the α-amino acid at position 8 is Leu, and the α-amino acid at position 18 is Met (O); or
VII. The α-amino acid at position 26 is Gln;
said compound differs from Pro ⁰ PTH (1-84) or [Met ⁰ , Leu ⁸ , Leu ¹⁸ ] PTH (1-84); or the PTH compound differs from hPTG (1-36) in that the α-amino acid at position 36 is Leu;
in free form, in salt form, or in complex form.

 In the event that the PTH sequence is derived from a PTH fragment, it is a PTH fragment exhibiting activity similar to PTH and including at least the first 27 N-terminal amino acid residues of PTH, in the preferred case up to 38 N-terminal PTH amino acid residues, including from 1-34 to 1-38, for example 1-34, 1-36, 1-37 or 1-38 PTH, with at least one of the α-amino acids replaced in accordance with the present invention. In addition, one or more amino acid residues normally present in the PTH sequence may be omitted. Fragments of gPTG, especially gPTG (1-34) and gPTG (1-36), are preferred.

The C-terminus of the PTH compound can be represented by a —COOH group esterified by a COOH group, including a —COOR _a group in which R _a is a lower alkyl radical, for example, C _1-4 alkyl, —CONH ₂ or mono or a disubstituted amide radical, for example, a —CONF _b R _c radical in which one of the R _b or R _c groups is a hydrogen atom and the other is an aliphatic residue, for example, a C _1-6 alkyl group, or each of them denotes aliphatic residues or R _b and R _c together with the nitrogen atom to which they are attached, form a heterocyclic statok example pyrrolidino or piperidinyl residue.

 Hereinafter, these compounds will be referred to as "provided by the present invention compounds."

 The term "natural amino acids" means well-known amino acids. Their list, as well as their standard notation, is published [1]. These amino acids and their designations are used in the present description.

Ala, Aad (2-амино-адипиновая кислота), bAad (3-аминоадипиновая кислота), Abu (2-аминобутировая кислота), Gaba γ-аминобутировая кислота), Acp (6-аминокапроновая кислота), Dbu (2,4-диаминобутировая кислота), TMSA (триметилсилил-Ala), alle (аллоизолейцин), Nle (норлейцин), трет-Leu, Cit (цитруллин), Orn (орнитин), Dpm (2,2'-диаминопимеловая кислота), Dpr (2,3- диаминопропионовая кислота), α- или β-Nal, Cha (циклогексил-Ala), гидроксипролин, Sar (саркозин) и т.п., остатки циклических аминокислот, а также остатки N^α-алкилированных аминокислот, например, MeGly ( N^α-метил-глицин), EtGly ( N^α-этил-глицин), EtAsn ( N^α-этил-аспарагин).The following is a list of natural amino acids:
A ala alanine
D Asp Aspartic Acid
E Glu Glutamic Acid
F Phe Phenylalanine
G Gly Glycine
H His histidine
I Ile Isoleucine
K Lys Lysine
L Leu Leucine
M Met methionine
N Asn Asparagine
Q Gln Glutamine
R Arg Arginine
S Ser Serine
T thr threonine
V Val Valine
W Trp Tryptophan
Y Tyr Tyrosine
The term “artificial amino acid” residue refers to the residue of a genetically non-encoded amino acid. Artificial amino acids include, for example, the D-isomers of the above-mentioned natural α-amino acids, Aib (amino-isobutic acid), bAib (3-aminoisobutic acid), Nva (norvaline),

Ala, Aad (2-amino-adipic acid), bAad (3-aminoadipic acid), Abu (2-aminobutric acid), Gaba γ-aminobutric acid), Acp (6-aminocaproic acid), Dbu (2,4-diaminobutyric acid acid), TMSA (trimethylsilyl-Ala), alle (alloisoleucine), Nle (norleucine), tert-Leu, Cit (citrulline), Orn (ornithine), Dpm (2,2'-diaminopimelic acid), Dpr (2,3 - diaminopropionic acid), α- or β-Nal, Cha (cyclohexyl-Ala), hydroxyproline, Sar (sarcosine), etc., residues of cyclic amino acids, as well as residues of N ^α -alkylated amino acids, for example, MeGly (N ^α -methyl-glycine), EtGly (N ^α- ethyl-glycine), EtAsn (N ^α ethyl asparagine).

 Amino acids in protected form are natural or artificial amino acids having, for example, a side chain containing a heteroatom, such as O, S or N, which may be protected by an O-, S- or N-protective radical. The N-terminus of the PTH compounds provided by the present invention may also be in a protected form.

Among the N-protective radicals that can be present on the N-terminal or lateral amino groups of amino acid residues are radicals, including those listed in [2], for example, acyl radicals such as formyl, acetyl, trifluoroacetyl, methoxy succinyl, hydroxysuccinyl or benzoyl a radical optionally substituted on the phenyl ring, for example with a p-methoxycarbonyl, p-methoxy, p-nitro or p-phenylsulfonamidocarbonyl group; alkoxycarbonyl radical such as t-butyloxycarbonyl, isobutyloxycarbonyl or methoxycarbonyl radical; allyloxycarbonyl radical; trityl radical; 2,2,5,7,8-pentamethyl-chroman-6-sulfonyl radical; an arylmethoxycarbonyl radical, such as a 9-fluorenylmethoxycarbonyl or benzyloxy carbonyl radical, optionally substituted on the phenyl ring of a p-methoxy, p-nitro, o- or p-chloro, m-phenyl or 3,4-dimethyl group; an arylmethyl radical, such as a benzyl radical, optionally ring-substituted with a p-methoxy, p-nitro or p-chloro group; or an arylsulfonyl radical, such as a phenylsulfonyl radical, optionally ring-substituted with a p-methyl or p-methoxy group, or a naphthylsulfonyl radical, optionally ring-substituted, for example, with an amino or di (C _1-4 ) alkyl) amino group.

 O-protective radicals of side chains containing an oxygen atom are, for example, the radicals listed in [3]. In the case of aliphatic hydroxyl functional groups, convenient O-protecting radicals are, for example, the radicals listed in [4], including benzyl, t-butyl, methyl groups. For aromatic hydroxyl functional groups, convenient O-protecting radicals are, for example, benzyl, t-butyl, methyl, tosyl and benzyloxycarbonyl groups. In the case of carboxyl functional groups that make up the amino acid side chains, O-protecting radicals are well-known ether groups, as well as groups, for example, those listed in [5], including methyl, ethyl, t-butyl and benzyl groups. In the case of thiol functional groups that make up the amino acid side chains, S-protecting radicals are known groups, for example, those listed in [6]. Examples of such radicals are methyl, t-butyl, benzyl, p-methoxyphenylmethyl, ethylaminocarbonyl and benzyloxycarbonyl groups.

In accordance with the present invention, PTH compounds can carry at their terminal amino group at least one radical selected from the following: L- or D-α-amino acid, C _2-6 alkoxycarbonyl, and optionally substituted C _1-8 -alkyl, C _2-8 -alkenyl, C _2-8 -alkynyl, aralkyl, aralkenyl or C _3-6 -cycloalkyl-C _1-4 -alkyl group, and / or contain at least one amino group on the side a radical selected from the following: C _2-6 alkoxycarbonyl, and also optionally substituted C _1-8 alkyl, C _2-8 alkenyl a lower, C _2-8 alkynyl, aralkyl, aralkenyl or C _3-6 cycloalkyl-C _1-4 alkyl group. In the event that said group is attached to a side amino group, its location on the ε-amino group of the lysine residue is preferred. Suitable substituents of the alkyl, alkenyl, aralkyl, aralkenyl or cycloalkylalkyl radical are hydroxyl and amino, while the halogen atom and / or C _1-4 alkoxy group can also serve as substituents on the aryl part. The alkyl radical or the alkyl part of the radical can be linear or branched, as well as possibly containing O, S or N atoms. In the preferred case, any C _{1-8 is} alkyl, C _{2-8 is} alkenyl, aralkyl, aralkenyl or C _{3 The -6-} cycloalkyl-C _1-4 -alkyl group attached to the amino group of the PTH compound is unsubstituted. Examples of C _1-8 alkyl groups are C _1-6 alkyl, preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl and t-butyl groups; examples of C _2-8 alkenyl groups are C _2-4 alkenyl, preferably allyl groups, examples of C _2-8 alkenyl groups are C _2-4 alkynyl, preferably prop-2-ynyl groups, examples of aralkyl groups are phenyl or benzyl groups, an example of aralkenyl groups is a styryl group, an example of a C _3-6 cycloalkyl-C _1-4 alkyl group is a cyclohexyl methyl group. As C _2-6 alkoxycarbonyl groups, formyl or acetyl groups are preferred. Suitable D- or L-α-amino acids attached to the N-terminus include, for example, D- or LPro and Ala. If a substituent is present, an alkyl, alkynyl, alkoxycarbonyl group or a D- or L-α-amino acid attached to the N-terminus of the PTH compound and / or at least one alkyl or alkoxycarbonyl group attached to one or more side amino groups are preferred. .

 Preferred PTH compounds provided by the present invention are compounds containing at least one substituted amino acid residue in one of the following positions of the PTH sequence: 1, 2, 3, 8-11, 13-19, 21, 22, 29-34, especially at positions 8-11, 16-19, 33 and / or 34. More preferred PTH compounds provided by the present invention are compounds containing more than one substituted amino acid residue, preferably more than two amino acid residues, in particular more than three residues amino acid In the best case - 5-7 replaced amino acid residues; in the preferred case, replace any combination of the above positions of the PTH sequence.

 In a number of its specific or alternative embodiments, the present invention provides the PTH compound described above, in which, in particular, the α-amino acids located at positions 1 and 2 of the N-terminus of the PTH sequence are replaced by a pseudodipeptide.

The term "pseudodipeptide" is used in the present description to denote an isosteric dipeptide in which the peptide bond between its amino acids, both natural and artificial, is replaced by any isosteric group, including -CH ₂ -NH-, -C ( halogen) = CH or —C (alkyl) = CH—.

или (б)

в которых каждый из радикалов Z и Z' независимо друг от друга представляет собой атом водорода, возможно замещенную C_1-8-алкильную, C_2-8-алкенильную, аралкильную, аралкенильную или C_3-6-циклоалкил-C_1-4-алкильную группу или защитную группу, причем не более одной групп из числа Z и Z' являются защитными группами,
Z'' обозначает атом водорода С_1-8-алкильную или защитную группу,
Y и Y' каждый независимо представляет собой возможно защищенную боковую цепь природной или искусственной α-аминокислоты,
Y_a или Y_b - один является атомом водорода, а другой - возможно защищенной боковой цепью природной или искусственной α-аминокислоты или Y_a и Y_b вместе с атомом углерода, к которому они присоединены, образуют C_3-6-циклоалкильную группу;
W представляет собой атом галогена или C_1-4-алкильную группу или
W и Y' вместе с группой

к которой они присоединены, образуют возможно замещенный ароматический циклический или гетероциклический остаток;
P₁ обозначает описанную последовательность ПТГ, в которой отсутствуют остатки аминокислот, соответствующие N-концевым положениям 1 и 2,
находящиеся в свободной форме, либо в форме соли или комплексного соединения.Examples of compounds provided by the present invention in which the α-amino acids at positions 1 and 2 are replaced by a pseudodipeptide include, for example, compounds of formula (I)
XP ₁
in which X is the residue described by formula (a)

or (b)

in which each of the radicals Z and Z 'independently of each other represents a hydrogen atom, possibly substituted C _1-8 -alkyl, C _2-8 -alkenyl, aralkyl, aralkenyl or C _3-6 -cycloalkyl-C _1-4 - an alkyl group or a protective group, and not more than one of Z and Z ′ groups are protective groups,
Z "denotes a hydrogen atom, a C _1-8 alkyl or protective group,
Y and Y 'each independently represents a possibly protected side chain of a natural or artificial α-amino acid,
Y _a or Y _b - one is a hydrogen atom and the other is a possibly protected side chain of a natural or artificial α-amino acid or Y _a and Y _b together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group;
W represents a halogen atom or a C _1-4 alkyl group or
W and Y 'with the group

to which they are attached, form a possibly substituted aromatic cyclic or heterocyclic residue;
P ₁ denotes the described PTH sequence in which there are no amino acid residues corresponding to the N-terminal positions 1 and 2,
in free form, or in the form of a salt or complex compound.

In the compounds of formula (I), P ₁ may be a PTH fragment (3-z), where z = 84 or an integer ranging from 27 to 38, preferably from 34 to 38, preferably 34, 36, 37 or 38, in particular 34 or 36. The PTH sequence shown as P ₁ may correspond to either a naturally occurring PTH sequence or a naturally occurring PTH sequence in which at least one α-amino acid residue has been replaced natural or artificial amino acid, possibly found in protected form, and / or one or more residues of α-amino acids are also removed. In addition, P ₁ may include at least one amino side group protected as described above.

 In a preferred case, the halogen atom is a fluorine or chlorine atom.

 In the compound according to formula (a), the double bond is preferably in the trans configuration (E).

As residues attached to the α-carbon atom of the α-amino acid, the radicals Y, Y ', Y _a or Y _b can be a side chain that is part of the above-mentioned natural α-amino acids, in particular, part of Gly (for example , H), Ala, Val, Ser, Leu, Ile, Phe, Trp. In addition, the radicals Y, Y ', Y _a or Y _b may be residues attached to the α-carbon atom of the aforementioned artificial α-amino acids, including Nva, Orn, Abu, Aib, Nle. In the event that the radical Y, Y ', Y _a or Y _b is the side chain of a natural or artificial amino acid containing heteroatoms such as O, S or N, these heteroatoms that make up the side chain can be protected by the above O- , S- or N-protecting groups.

 The protective groups used as radicals Z or Z 'or Z "can correspond to the above.

C _1-8 -alkyl groups used as radicals Z or Z ′ or Z ″ may correspond to the above.

In a preferred case, Z or Z ′ represents a hydrogen atom or a C _1-4 alkyl group, preferably a hydrogen atom or a methyl group.

In a preferred case, Y corresponds to a hydrogen atom or a CH ₃ group.

In the case where W is a C _1-4 alkyl group, it is preferably a CH ₃ group.

 In the event that the radicals W and Y 'together with the group to which they are attached form a possibly substituted aromatic cyclic or heterocyclic residue, it may be an aromatic 5- or 6-membered residue, possibly including 1 or 2 heteroatoms, selected from N, S and O, for example, phenyl, imidazolyl, pyridyl, oxazolyl or thiazolyl radicals. Preferred substituents are hydroxyl and methoxy, especially in the case of the phenyl ring.

 In a preferred case, Y 'corresponds to a hydrogen atom, a methyl, isopropyl or benzyl group.

In the event that Z ″ represents a C _1-8 alkyl group, it is preferably a CH ₃ -, C ₂ H ₅ - or isopropyl group.

With a _3-6 cycloalkyl group jointly formed by the radicals Y _a and Y _b , preferably is a cyclopropyl or cyclopentyl group.

 In the event that Z ″ represents a protecting group, it is preferably an acyl group, in particular an acetyl group.

аминокислота, находящаяся в положении 25 и/или в положении 26, является His; и/или α-аминокислота, находящаяся в положении 27, представляет собой Gln или Leu; либо ПТГ-соединение отличается от ПТГ(1-84), в котором
I. α-аминокислота, находящаяся в положении 1, представляет собой Tyr, Val, Pro, Asp или Cys; либо
II. α-аминокислота, находящаяся в положении 2, представляет собой Ala, Glu, Leu, Ser или Arg.In a number of its specific or alternative embodiments, the present invention provides for the PTH compound described above, in which the α-amino acid residue at position 1 and / or the α-amino acid residue at position 2 of the N-end of the PTH sequence is replaced with possibly protected the remainder of a natural or artificial amino acid, as a result of which, if the PTH compound is devoid of the L- or D-α-amino acid attached to the N-terminus, or C _2-6 -alkoxycarbonyl or possibly substituted C _1-8 -alkyl, C _2-8 alkenyl noy, C _2-8 alkynyl, aralkyl, aralkenyl or C _3-6 cycloalkyl-C _1-4 alkyl group, it differs from the PTH-compound having a sequence of naturally occurring α-amino acids; or the PTH compound is different from PTH (1-34), in which
I. The α-amino acid at position 1 is Aib, Gly, D-Ser, D-Ala, or Tyr; or
II. the α-amino acid at position 2 is Ala, D-Val, Lys, Cit or Arg, and the α-amino acid at position 34 is Tyr; or the α-amino acid at position 2 is D-Val, the α-amino acid at position 34 is D-Tyr, and each of the α-amino acids at positions 8 and 18 may be Nle; or
III. The α-amino acid at position 1 is Aib and at least one more α-amino acid residue is replaced so that the α-amino acid at position 8 and / or 18 is Leu, Ile, Val, Phe or Trp and / or the α-amino acid at position 11 is Ser, Lys, Phe, β-Nal, Trp or Tyr, and / or the α-amino acid at position 12 is D-Leu, D-Ile, D-Nle, D-Val, D-Ser, D-Ser (Butyl), D-Abu, D-Thr, D-Nva, D-Met, D-β-Nal,
D-Trp, D-Lys, D-Tyr, D-Lys (Fmoc), D-Phe or D-Asn; and / or the α-amino acid at position 13 is Leu, and / or the α-amino acid at position 19 and / or at position 21 is Arg, Lys, Asn and / or His; and / or the α-amino acid at position 23 is 2- (1,3-dithiolan-2-yl) Trp; and / or

the amino acid at position 25 and / or at position 26 is His; and / or the α-amino acid at position 27 is Gln or Leu; or the PTH compound is different from PTH (1-84), in which
I. The α-amino acid at position 1 is Tyr, Val, Pro, Asp or Cys; or
II. The α-amino acid at position 2 is Ala, Glu, Leu, Ser or Arg.

в которой каждый из радикалов Z_a и Z'_a независимо друг от друга обозначает атом водорода, C_1-6-алкильную или защитную группу, причем не более одного радикала из числа Z_a и Z'_a представляет собой защитную группу, а также
либо n=1, а
I. каждый их радикалов R₁ и R₂ является C_1-6-алкильной группой, либо
II. один из радикалов R₁ или R₂ представляет собой метильную или этильную группу, в то время как другой обозначает возможно защищенный остаток, присоединенный к α-углеродному атому природной α-аминокислоты, отличающейся от Ala, Leu, Ile или Val, либо
III. один из радикалов R₁ или R₂ является атомом водорода, метильной или этильной группой, в то время как другой представляет собой возможно защищенный остаток, присоединенный к α-углеродному атому искусственной α-аминокислоты, либо
IV. R₁ и R₂ вместе с атомом углерода, к которому они присоединены, образуют C_3-6-циклоалкильную группу, либо
V. остаток

представляет собой гетероциклический остаток, возможно сконденсированный с бензольным кольцом,
либо n = 2, 3, 4 или 5, а каждый из радикалов R₁ и R₂ обозначает атом водорода или CH₃-группу,
Y_2a представляет собой остаток возможно защищенной природной α_аминокислоты или остаток формулы (IIб),

в которой Z_b обозначает атом водорода или С_1-6-алкильную группу, и
либо m = 1, a
I. каждый их радикалов R₃ и R₄ является С_1-6-алкильной группой, либо
II. один из радикалов R₃ или R₄ представляет собой метильную или этильную группу, в то время как другой обозначает возможно защищенный остаток, присоединенный к α_углеродному атому природной α_аминокислоты, отличающейся от Ala, Leu, Ile или Val, либо
III. один из радикалов R₃ или R₄ является атомом водорода, метильной или этильной группой, в то время как другой представляет собой возможно защищенный остаток, присоединенный к α_углеродному атому искусственной α_аминокислоты,
либо n = 2, 3, 4 или 5, а каждый из радикалов R₃ и R₄ обозначает атом водорода или CH₃-группу, а P₂ представляет собой вышеописанную последовательность ПТГ, из которой удалены остатки аминокислот, в норме находящиеся в N-концевых положениях 1 и/или 2.Among the compounds provided by the present invention in which the α-amino acids in position 1 and / or in position 2 are replaced in the aforementioned manner are, for example, compounds of formula (II)
X _1a -Y _2a -P ₂ ,
in which X _1a represents a residue of a possibly protected natural α-amino acid or a residue corresponding to formula (IIa)

in which each of the radicals Z _a and Z ' _a independently represents a hydrogen atom, a C _1-6 -alkyl or protective group, and not more than one radical from among Z _a and Z' _a represents a protective group, and
either n = 1, and
I. Each of their radicals R ₁ and R ₂ is a C _1-6 alkyl group, or
II. one of the radicals R ₁ or R ₂ represents a methyl or ethyl group, while the other represents a possibly protected residue attached to the α-carbon atom of a natural α-amino acid other than Ala, Leu, Ile or Val, or
III. one of the radicals R ₁ or R ₂ is a hydrogen atom, a methyl or ethyl group, while the other represents a possibly protected residue attached to the α-carbon atom of an artificial α-amino acid, or
IV. R ₁ and R ₂ together with the carbon atom to which they are attached form a C _3-6 cycloalkyl group, or
V. balance

represents a heterocyclic residue, possibly fused to a benzene ring,
either n = 2, 3, 4 or 5, and each of the radicals R ₁ and R ₂ represents a hydrogen atom or a CH ₃ group,
Y _2a represents a residue of a possibly protected natural α_ amino acid or a residue of formula (IIb),

in which Z _b represents a hydrogen atom or a C _1-6 alkyl group, and
either m = 1, a
I. Each of their radicals R ₃ and R ₄ is a C _1-6 alkyl group, or
II. one of the radicals R ₃ or R ₄ represents a methyl or ethyl group, while the other represents a possibly protected residue attached to the α_carbon atom of a natural α_ amino acid other than Ala, Leu, Ile or Val, or
III. one of the radicals R ₃ or R ₄ is a hydrogen atom, a methyl or ethyl group, while the other represents a possibly protected residue attached to the α_carbon atom of an artificial α_ amino acid,
either n = 2, 3, 4, or 5, and each of the radicals R ₃ and R ₄ represents a hydrogen atom or a CH ₃ group, and P ₂ represents the above-described PTH sequence, from which amino acid residues normally found in N- are removed end positions 1 and / or 2.

представляет собой гетероциклический остаток, возможно сконденсированный с бензольным кольцом, он может являться производным насыщенного или ненасыщенного 5- или 6-членного гетероцикла, включающего в себя один или более гетероатомов, выбранных из числа N, О и S. Примерами такого рода гетероциклических остатков являются, в частности, 2-пиридильная, 3-морфолинильная, гексагидропиридазинильная, 2- или 3-индолильная группы.In the event that the remainder

represents a heterocyclic residue, possibly condensed with a benzene ring, it may be a derivative of a saturated or unsaturated 5- or 6-membered heterocycle, including one or more heteroatoms selected from N, O and S. Examples of such heterocyclic residues are, in particular, 2-pyridyl, 3-morpholinyl, hexahydropyridazinyl, 2- or 3-indolyl groups.

In the case where n or m> 1, the group CR ₁ R ₂ or CR ₃ R ₄ may be —CH (CH ₃ ) —CH ₂ -, a propyl, butyl or pentyl group.

A C _3-6 cycloalkyl group jointly formed by R ₁ and R ₂ is preferably a cyclopropyl or cyclopentyl group.

As residues attached to the α-carbon atom of the α-amino acid, the radicals R ₁ , R ₂ , R ₃ or R ₄ can be a side chain that is part of the above natural α-amino acids, in particular, part of Gly, Ser or Thr. One of the radicals R ₁ and R ₂ or one of the radicals R ₃ and R ₄ may denote a methyl or ethyl group, while the other may correspond to CH ₃ -, isopropyl, isobutyl, or CH ₃ —CH ₂ —CH (CH ₃ ) -group. In addition, the radicals R ₁ , R ₂ , R ₃ or R ₄ may be residues attached to the α-carbon atom of the aforementioned artificial α-amino acids, including those that are part of Abu, Gaba, Nva, Aib, TMSA or Nle. In the event that the radicals R ₁ , R ₂ , R ₃ or R ₄ are the side chains of natural or artificial amino acids, including heteroatoms, such as O, S or N, the specified heteroatom, which is part of the side chain, can be protected , for example, using the above O-, S- or N-protective groups. The protecting group used as the radical Z _a or Z ' _a may correspond to the above. The C _1-6 alkyl group serving as Z _b is preferably a methyl or ethyl group. In a preferred case, Z _b represents a hydrogen atom. The C _1-6 alkyl group serving as the radical Z _a or Z ' _a is preferably a linear or branched C _1-4 alkyl group. Preferably one of the radicals Z _a and Z _'a represents a hydrogen atom, while the other corresponds to a hydrogen atom, C _1-4 alkyl or a protective group, preferably - the hydrogen atom.

аминокислоты возможно заменен таким образом, что α_аминокислота, находящаяся в положении 1, является Aib, и/или α_ аминокислота, находящаяся в положении 11, является Ser, Lys, Phe, β-Nal, Trp или Tyr, и/или α_аминокислота, находящаяся в положении 12, представляет собой D-Leu, D-Ile, D-Nle, D-Val, D-Ser, D-Ser(Butyl), D-Abu, D-Thr, D-Nva, D-Met, D-β-Nal, D-Trp, D-Lys, D-Tyr, D-Lys (Fmoc), D-Phe или D-Asn, и/или α_аминокислота, находящаяся в положении 13, является Leu и/или α_аминокислота, находящаяся в положении 19 и/или в положении 21, является Arg, Lys, Asn или His, и/или α_аминокислота, находящаяся в положении 23, представляет собой 2-(1,3-дитиолан-2-ил)Trp, и/или α_аминокислота, находящаяся в положении 25 и/или в положении 26, является His, и/или α_аминокислота, находящаяся в положении 27, представляет собой Gln или Leu; либо
III. α_аминокислота, находящаяся в положении 8 и/или 18, является Ala или Ser; либо α_аминокислота, находящаяся в положении 8 и/или 18, представляет собой Leu, а α_аминокислота, находящаяся в положении 34, является Tyr; либо ПТГ-соединение отличается от ПТГ (1-84) тем, что α_аминокислота, находящаяся в положении 8, представляет собой Met, Met(O), Ala, Val, Leu, Ile, Ser, Trp, Asn, Gln, Asp, Glu, Lys, Arg, Tyr или Gly, а α_аминокислота, находящаяся в положении 18, представляет собой Leu; либо каждая из α_ аминокислот, находящихся в положении 8 и в положении 18, представляет собой Met(O); либо α_аминокислота, находящаяся в положении 8, представляет собой Leu, а α_аминокислота, находящаяся в положении 18, представляет собой Met (О); либо ПТГ-соединение отличается от [Leu¹⁸, Tyr³⁴]гПТГ(1-84), [Leu⁸, Tyr³⁴ ] гПТГ(1-84), [Ile⁸, Leu¹⁸, Tyr³⁴]гПТГ(1-84) или [Leu⁸, Leu¹⁸, Tyr³⁴] гПТГ(1-84).In its yet another or alternative embodiment, the present invention also provides the above-described PTH compound in which the α-amino acid residue at position 8 and / or the α-amino acid residue at position 18 is replaced with a possibly protected natural or artificial amino acid residue such that in the event that the PTH compound is devoid of the L- or D-α_ amino acid attached to the N-terminus or C _2-6 alkoxycarbonyl or possibly substituted C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, aralkyl , an aralkenyl or C _3-6 cycloalkyl-C _1-4 alkyl group, it differs from the PTH compound having a naturally occurring sequence of α_ amino acids; or the PTH compound is different from PTH (1-34), in which
I. Each of the α_ amino acids located at positions 8 and 18 is Nle or Met. (O), and the α_ amino acid at position 34 is possibly Tyr; either each of the α_ amino acids at positions 8 and 18 is Nle, the α_ amino acid at position 34 is Tyr, and either the α_ amino acid at position 12 is L- or D-Pro, L- or D-Ala , Aib or NMeGly, or the α-amino acid at position 23 is Phe, Leu, Nle, Val, Tyr, α_Nal or β-Nal; either each of the α_ amino acids at positions 8 and 18 is Nle, the α_ amino acid at position 2 is D-Val, and the α-amino acid at position 34 corresponds to D-Tyr; or
II. The α_ amino acid at position 8 and / or 18 is Leu, Ile, Val, Phe or Trp, and at least one other residue

amino acids may be replaced so that the α_ amino acid at position 1 is Aib, and / or the α_ amino acid at position 11 is Ser, Lys, Phe, β-Nal, Trp or Tyr, and / or the α_ amino acid located at position 12, represents D-Leu, D-Ile, D-Nle, D-Val, D-Ser, D-Ser (Butyl), D-Abu, D-Thr, D-Nva, D-Met, D- β-Nal, D-Trp, D-Lys, D-Tyr, D-Lys (Fmoc), D-Phe or D-Asn, and / or the α-amino acid at position 13 is Leu and / or the α-amino acid located at position 19 and / or position 21 is Arg, Lys, Asn or His, and / or the α_ amino acid at position 23 is 2- (1,3-dithiolan-2-yl) Trp and / or α_ amino acid at position 25 and / or position 26 is His and / or α_ amino acid at position 27 is Gln or Leu; or
III. The α_ amino acid at position 8 and / or 18 is Ala or Ser; either the α_ amino acid at position 8 and / or 18 is Leu, and the α_ amino acid at position 34 is Tyr; or the PTH compound differs from PTH (1-84) in that the α_ amino acid at position 8 is Met, Met (O), Ala, Val, Leu, Ile, Ser, Trp, Asn, Gln, Asp, Glu , Lys, Arg, Tyr or Gly, and the α_ amino acid at position 18 is Leu; or each of the α_ amino acids located at position 8 and at position 18 is Met (O); either the α_ amino acid at position 8 is Leu, and the α_ amino acid at position 18 is Met (O); or the PTH compound differs from [Leu ¹⁸ , Tyr ³⁴ ] hPTG (1-84), [Leu ⁸ , Tyr ³⁴ ] hPTG (1-84), [Ile ⁸ , Leu ¹⁸ , Tyr ³⁴ ] hPTG (1-84) or [Leu ⁸ , Leu ¹⁸ , Tyr ³⁴ ] gPTG (1-84).

 In the event that the α-amino acid at position 8 is replaced with an artificial amino acid, it may be one of the above amino acids, including Nva, Nle or Cha. In a preferred case, it is an artificial lipophilic amino acid, preferably including a linear chain alkyl residue.

 Most preferred are Nva (norvaline) and its homologues. The artificial amino acid located at position 8 of the PTH sequence may also be α-methylated or α-ethylated natural α-amino acid.

Among the PTH compounds provided by the present invention in which the α_ amino acid residue is replaced at position 18 are, for example, PTH compounds in which the α_ amino acid residue at position 18 is replaced by a neutral amino acid residue such as Gln, Tyr, or Lys, for example [Gln ¹⁸ ] -PTH, [Lys ¹⁸ ] -PTH and [Tyr ¹⁸ ] -PTH, especially [Gln ¹⁸ or Lys ¹⁸ or Tyr ¹⁸ ] -PTH- (1-x), where x = 84 or an integer in the range from 27 to 38, preferably in the range from 34 to 38. Ala is also preferred at position 18, especially in that if the PTH sequence is a PTH fragment (1-36). In such compounds, the amino acid at position 8 may be Met or substituted with an amino acid selected from Leu, Ile, Val, Phe, Gln, Trp, Ser and Ala, or may be a residue of an artificial lipophilic amino acid, for example Nva or Nle.

 Another group of these PTH compounds includes, for example, PTH compounds in which the amino acid residue at position 8 is replaced by an artificial lipophilic amino acid residue, for example Nle or preferably Nva. In these compounds, the amino acid at position 18 may be Met or may be replaced with an amino acid selected from the group Leu, Ile, Val, Phe, Trp, Ser, Ala, Gln, Lys or Tyr, preferably Leu, Gln, Ala or Tyr.

 Another group of these PTH compounds provided by the present invention are compounds in which one or more amino acid residues in unchanged positions, for example, 1-7, 9-17 and / or 19-38, are either removed or replaced in in addition to replacing the α_ amino acid residue at position 8 or 18. In this case, the α_ amino acid at position 8 is preferably replaced with Leu, and the α_ amino acid at position 18 is replaced with Gln, Leu, Ala or Tyr.

In the case of another or alternative embodiment, the present invention provides the above-mentioned PTH compound in which at least one α_ amino acid residue included in the PTH sequence is substituted with an α_ amino acid residue located in the corresponding position of the PTH, so that in the case if the PTH compound is devoid of the L- or D-α_ amino acid attached to the N-terminus or C _2-6 alkoxycarbonyl or possibly substituted C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, aralkyl , Aralkenyl or C _3-6 cycloalkyl-C _1-4 alkyl group, it differs from the PTH-compound having a naturally occurring sequence α_aminokislot; or the PTH compound is different from PTH (1-34), in which
I. α_ amino acid at position 8 and / or 18 is Leu; and / or α_ amino acid at position 11 is Lys; and / or α_ amino acid located at position 19 and / or 21, represents Arg; and / or α_ amino acid at position 25 and / or 26 is His; and / or α_ amino acid at position 27 is Leu; or
II. the α_ amino acid at position 25 is Gln, and / or the α_ amino acid at position 26 corresponds to Asn, while the α_ amino acid at position 27 is possibly Leu; or the PTH compound is different from [Leu ⁸ , Leu ¹⁸ ] PTH (1-84).

 The term “PTGrP” refers to any genetically encoded form of PTGrP, including human, chicken, rat or mouse PTGrP. For the sake of continuity, as well as to adhere to traditional approaches, in the following description the same amino acid numbering system is applied to the PTHrP sequence starting with Ala at position 1 and including, for example, Ala at position 38.

 The specified group of compounds provided by the present invention, are hybrids between PTH and homologous sequences of PTH-rP (denoted in the present description by hybrid PTHrP molecules). The sequence of amino acids in which the substitution provided for by the present invention occurs is in the range from position 1 to position 38, preferably in region 1-36, in particular 1-34.

 In the preferred case, the PTH compounds provided by the present invention are PTH-rP hybrid molecules in which more than one α_ amino acid residue of the PTH sequence, for example at least 2, preferably 3-5, α_ amino acid residues, is replaced in accordance with the present invention .

According to the present invention, a preferred group of PTH-rP hybrid molecules includes PTH compounds in which at least one α_ amino acid residue is located at positions 8-11 of the PTH α_ amino acid sequence, i.e. Met ⁸ , His ⁹ , Asn ¹⁰ or Leu ¹¹ , is replaced by the corresponding amino acid residue that is part of the corresponding PTH-rP sequence, i.e., Leu ⁸ , His ⁹ , Asp ¹⁰ or Lys ¹¹ . Preferred is the substitution at positions 8 and / or 10, especially at positions 8 and 10. In a more preferred case, the α-amino acids located at positions 8, 10 and 11 of the indicated PTH sequence are replaced with Leu ⁸ , Asp ¹⁰ and Lys ¹¹ , respectively.

Another preferred group of PTH-rP hybrid molecules provided by the present invention includes PTH compounds in which at least one α_ amino acid residue located at positions 16-19 (the PTH α_ amino acid sequence is replaced by the corresponding α_ amino acid residue included in composition corresponding sequence of PTH-RP, i.e. to Gln ^16, Asp ^17, Leu ^18, Arg ^19. In the preferred case replace 3 or all 4 α_aminokisloty from said sequence. most preferred is provided s-PTH present invention compounds in which α_aminokislota, located at position 16 is Gln, α_aminokislota, located at position 18 is Leu, and α_aminokislota, located at position 19, corresponding to Arg.

Another preferred group of PTH-rP hybrid molecules provided by the present invention includes PTH compounds in which at least one α-amino acid residue located at positions 33-34 of the PTH α-amino acid sequence is replaced by the corresponding α- amino acids included in the corresponding sequence of PTH-RP, i.e. on Thr ³³ , Ala ³⁴ . In a preferred case, both α-amino acids are replaced.

аминокислот (8-11, 16-19, 33-34), в наиболее предпочтительном случае - комбинацию замен, выбранных из числа вышеперечисленных замен последовательностей 8-11 и 33-34.Another preferred group of PTH-rP hybrid molecules provided by the present invention are PTH compounds including any combination of the above substitutions

amino acids (8-11, 16-19, 33-34), in the most preferred case, a combination of substitutions selected from among the above substitutions of sequences 8-11 and 33-34.

 If you want to increase the value of the substitutions made, at least one more α-amino acid located in the PTH-rP hybrid molecules of the present invention at positions 1-38 can be replaced with the remainder of the natural or artificial amino acid in accordance with the above description or removed. At position 10, an α-amino acid may be selected from Gly, Gln, Glu, His, Ser, Thr, or Tyr. At position 13, there may be a D- or L-α-amino acid that is different from Arg, for example, Ala, Cys, Gln, Ile, Asn, Trp, Asp, Val, Ser, Thr, Tyr, Met, Leu or Gly: at position 16 there may be a D- or L-α-amino acid, for example, Lys, Ser, Leu, Ala, Gln or Gly; at position 17 - Ala or Ser, or preferably an amino acid whose side chain is larger than that of Ala or Ser, for example, Glu, Gln, Phe, His, Ile or Lys: at position 18 - Gln or Tyr; at position 19 - Ala, Arg, Val, Tyr, Ser, Lys, Met, His, Gly, Pro, Asn or Ile: at position 26 - Gln or Arg; and / or at position 33, a D- or L-α-amino acid, for example, Ser, Thr, Leu, Gly, Gln, Arg, Pro, Asp, Ile, Lys or Thr. In addition, PTH-rP hybrid molecules can be substituted at their N-terminus or at the amino side group as described above. If desired, the side chains of the PTH-rP hybrid molecules provided by the present invention containing S or O atoms can also be protected as described above.

Examples of preferred hybrid PTH-rP molecules provided by the present invention include [Leu ⁸ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] -PTH, [Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] -PTH, [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] -PTH, [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ ] -PTH, [Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ ] PTH, [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Gln ¹⁸ ] PTH, [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ ] PTH, [Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Thr ³³ , Ala ³⁴ ] -PTH and [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] -PTH.

 Preferred hybrid PTH-rP molecules provided by the present invention are PTH compounds (1-x '), where x' is an integer from 34 to 38, preferably 34, 36, 37 or 38, in particular gPTG (1-x ') in which one or more α-amino acids are replaced by α-amino acids located in the corresponding positions in the molecules of PTH-RP, preferably in accordance with the above description. In the event that at positions 33 and 34, substitutions are made for the corresponding sequence of α-amino acids from PTH-rP, said PTH compound is preferably a PTH fragment comprising 34 residues of α-amino acids. PTH-rP hybrid molecules containing a C-terminus are preferred.

 Preferred PTG-rP is gPTG-rP.

 Compounds provided by the present invention can be, for example, in free form, in the form of salts or in the form of their complex compounds. Salts resulting from the addition of acids can be prepared using, for example, organic acids, polymeric acids, as well as inorganic acids. Said forms of salts resulting from the addition of acids include, for example, hydrochlorides and acetates. Complex compounds are formed from compounds provided by the present invention by adding inorganic substances, for example inorganic salts or hydroxides, such as Ca and Zn salts, and / or by adding polymeric organic substances.

 The present invention also provides a method for producing compounds provided by the present invention. These compounds can be obtained stepwise or in solution, or using the method of solid-phase synthesis, or using genetic engineering.

Compounds provided by the present invention can be prepared, for example, as follows:
a) remove at least one protecting group present in the compound provided by the present invention in a protected form; or
b) two peptide fragments are connected by an amide bond, said peptide fragments being such that they obtain the desired amino acid sequence of the desired compound, after which step a) of the present method is possible; or
c) to obtain a PTH compound in which the residues of α-amino acids corresponding to amino terminal positions 1 and 2 are replaced by a pseudodipeptide in a protected or unprotected form, the pseudodipeptide is reacted with a PTH peptide in a protected or unprotected form, in which amino acid residues corresponding to positions 1 and 2 are removed, and if necessary, step a) is carried out; or
d) to obtain a PTH compound containing at least one radical selected from the following: L- or D-α-amino acid attached to the N-terminus, C _2-6 alkoxycarbonyl, and also optionally substituted C _1-8 -alkyl, C _2-8 -alkenyl, C _2-8 -alkynyl, aralkyl, aralkenyl or C _3-6 -cycloalkyl-C _1-4 -alkyl group attached to the terminal amino group and / or to the side amino group, the indicated radical is introduced into the molecule of the PTH compound, which is in a protected or unprotected form and does not contain the indicated radical, and if necessary STI carry out step a);
and isolate the compound thus obtained in free form, in the form of a salt, or in the form of a complex compound.

 Steps a), b) and c) of the present method can be carried out by analogy with well-known methods, for example, known in the field of peptide chemistry or listed in the following examples. To protect functional groups not taking part in these reactions in these reactions, protective groups suitable for use in peptides can be used if desired. The term “protecting group” may also include a polymer resin having functional groups.

 In step b), to obtain a PTH compound, in which the residues of α-amino acids corresponding to amino terminal positions 1 and 2 are replaced by a pseudodipeptide, one peptide fragment used as a starting material may contain a pseudodipeptide at its N-terminus. The specified starting material can be obtained in accordance with step c).

In step c), a pseudodipeptide used as a starting material may be a compound corresponding to the formula (IIaa) or (IIbb)
ZZ'N - CHY - CW = CH - CHY '- COOH (IIaa)
ZZ'N - CY _a Y _b - CH ₂ - NZ "- CHY '- COOH (IIbb)
or a functional derivative thereof, for example, ether, acid halide or symmetric or asymmetric anhydride.

Compounds corresponding to formula IIaa in which W and Y 'together with the group -C = CH-CH- to which they are attached form an optionally substituted aromatic cyclic or heterocyclic residue, as well as compounds corresponding to formula (IIbb) in which Z "" represents an alkyl or protecting group, for example an acetyl group, or the radical -CY _a Y _b represents a C _3-6 cycloalkyl group, and Z "represents a hydrogen atom, are new and are part of the present invention.

Step d) of the present method can be carried out by analogy with known methods, for example, using alkylation methods. In a preferred embodiment of the present invention, the alkylation is carried out under reduction conditions, including using the corresponding aldehyde or ketone. The reaction can be carried out, for example, in the presence of NaBH ₃ CN, preferably at an acidic pH, in particular at a pH in the range of 5 to 7. The reaction temperature may be, for example, from -20 ^° C. to 100 ^° C. It may be preferable to carry out the reaction in an inert solvent, for example, in water, alcohol, dioxane or DMF, or a mixture thereof. Using the known method, you can also attach a D - or L-α-amino acid to the N-end of the molecule.

A fragment of the peptide used as a starting material in step b) of the present method may include one or more artificial amino acid residues; in addition, it may be substituted at its N-terminal and / or amino side with a radical selected from the following: C _2-6 alkoxycarbonyl, optionally substituted C _1-8 alkyl, C _2-8 alkenyl, C _{2 -8-} alkynyl, aralkyl, aralkenyl or C _3-6 -cycloalkyl-C _1-4 -alkyl group, or may carry at its N-terminus an L- or D-α-amino acid.

 Compounds provided by the present invention or fragments thereof containing residues of natural α-amino acids can also be obtained using recombinant DNA techniques.

 According to a preferred embodiment, the present invention also provides a method for producing medium-sized polypeptides, in which a compound protein consisting of a fused N-terminal polypeptide encoded by the bacteriophage T4 gene 55 and attached to its C-terminus of the desired polypeptide is expressed in bacterial cells.

 The polypeptide encoded by gene 55 may include the complete bacteriophage T4 gp55 protein (188 amino acid residues) [7]. Alternatively, a fragment, preferably an N-terminal fragment, of the gp55 protein may be used. In particular, a gp55 protein fragment consisting of the first 112 N-terminal amino acid residues of the protein can be used. However, typically the polypeptide encoded by gene 55 includes at least the first 25 N-terminal amino acid residues of the gp55 protein.

 The mid-sized protein may have a length of from about 20 to 100, for example 150, preferably from about 20 to about 50 amino acids. Examples of medium sized proteins are calcitonins, endorphins, peptides that inhibit gastric juice enzymes, glucagon, neuropeptide Y, growth hormone release factor (GHRF), fragments of amyloid β-protein, hirudin, insulin, somatostatin, epidermal cell growth factor, nerve cell growth factor and PTH, or fragments thereof. Particularly preferred mid-sized polypeptides that can be obtained in accordance with the method provided by the present invention are provided for by the present invention compounds or fragments thereof, containing substitutions involving natural amino acids.

 In addition, the fusion protein encoded by the 55-PTH fusion genes typically contains a cleavable linker region between the polypeptide encoded by gene 55 and the desired polypeptide. In a preferred case, the cleavable linker site is capable of undergoing chemical cleavage, and more preferably consists of the amino acid sequence Asp-Pro-Pro or Asn-Gly-Pro.

 As yet another aspect of the present invention, there is provided a nucleotide sequence encoding a fusion protein consisting of an N-terminal polypeptide of bacteriophage T4 gene 55 and attached to its C-terminus of a desired mid-sized polypeptide.

 In a preferred case, the nucleotide sequence is a DNA sequence suitable for expression in bacteria. Said sequence typically contains nucleotides encoding a cleavable linker region located between sequences encoding gene 55 and the desired polypeptide.

 The present invention also provides a bacterial expression vector comprising a DNA sequence encoding the specified composite protein.

 Along with the sequence encoding the specified composite protein, the expression vector usually contains the necessary expression control sequences, including an acceptable promoter, operator and ribosome binding site, as well as other necessary regulatory sequences. In addition, the expression vector usually contains one or more selective markers.

 Any suitable promoter may be used, such as the TrpE, λP1, λPr, lac or Tac promoter. In a preferred case, the promoter is a T7 bacteriophage promoter, and the expression vector is a plasmid. For expression in E. coli, a vector constructed from plasmids R1 or Col-E1 can be used. In particular, the pET 17B expression vector, which can be obtained from Novagen, or similar expression vectors, can be used.

 As another aspect, the present invention provides bacterial host cells transformed with a sequence encoding a composite protein or the expression vector described above. Any suitable bacterial host cells may be used, with E. coli being the preferred host. In particular, E. coli strain BL21 (DE3) Lys E, as well as similar strains, can be used.

 The specified composite protein accumulates in the host cells mainly in the form of particles, which are insoluble inclusions, which facilitates the isolation of the specified composite protein from the cells. In order to isolate the desired polypeptide product from the composite protein, the protein that is part of these inclusion particles is dissolved and the desired polypeptide product is cleaved from the composite protein. Any suitable dissolution procedure may be used, including treatment with an acid, for example, at a pH of about 2-4, preferably an acid, at a pH of about 2.5; or treatment with a denaturing agent, for example, a mild denaturing agent, such as guanidine hydrochloride. In addition, it may be necessary to remove one or more undesired N-terminal amino acid residues from the product remaining after cleavage.

 Thus, as yet another aspect of the present invention, there is provided a method for producing a desired mid-sized polypeptide in bacterial host cells, the method comprising: bacterial host cells transformed as described above stimulate the expression of a composite protein in the form of inclusion particles; these particles of inclusions are isolated; dissolve the indicated inclusion particles and cleave the desired polypeptide from the specified composite protein.

 In the case of certain mid-sized polypeptides, such as PTH compounds, in order to obtain PTH compounds in active form, i.e. exhibiting activity similar to PTH, usually it is not required to carry out strictly controlled processes of denaturation and renaturation. Medium-sized polypeptide products can be prepared in active form simply by neutralizing the acidic medium used for dissolution. Dissolution and cleavage of the composite protein can be carried out in one step.

 The present invention also provides a composite protein comprising an N-terminal polypeptide encoded by the bacteriophage T4 gene 55, as well as the desired mid-sized polypeptide attached to its C-terminus.

 Based on the use of the methods and vectors provided by the present invention, it has been shown that it is possible to obtain large quantities of mid-sized polypeptides, in particular PTH products. We found that up to 50% of all proteins expressed in E. coli are the desired compound protein. In addition, the specified composite protein forms inclusion particles, which are easily separated from other substances of the host cells. Moreover, these inclusion particles often consist of at least about 90% of the desired composite protein, which significantly reduces the amount of purification required. Further, the expression of the PTH product in the form of a composite protein can significantly reduce the endogenous processing of this polypeptide in bacterial cells, which contributes to the isolation of homogeneous products.

 In a preferred case, the cleavable linker site consists of chemically cleavable amino acids attached to the N-terminus of the desired polypeptide, which allows the cleavage of the desired polypeptide from the specified composite protein using simple chemical methods. In a preferred case, the desired polypeptide does not contain these chemically cleavable groups.

 Preferred chemically cleavable linker sites contain the amino acids Asp-Pro-Pro or Asn-Gly-Pro. The Asp-Pro or Asn-Gly bonds can be chemically cleaved under acidic conditions, for example, under the action of formic acid (usually pH about 2.5) or hydroxylamine, respectively. The Pro-Pro or Gly-Pro dipeptides remaining at the N-terminus of the desired polypeptide can be further removed using an acceptable dipeptide peptidase. In particular, X-Pro dipeptide peptidase EC 3.4.14.5 L.lactis can be used [8].

 After cleavage from the fusion protein, the desired polypeptide can be purified. A purification method using high performance liquid chromatography (HPLC) can be used. Purification can be carried out before removing any undesired N-terminal amino acid residues.

 The method provided by the present invention is further illustrated in Examples 306-312, referring to the sequences given in the attached List of Sequences, in which Sequence No. 1 represents the amino acid sequence and the corresponding DNA sequence of the shortened gp55-Asp-Pro-Pro-gPTG protein (1- 38) OH cloned in plasmid pET17B, and Sequence N 3 shows the amino acid sequence and the corresponding DNA sequence of the full-length protein gp55-Asp-Pro-ProgPTH (1-38) OH cloned in plasmid pET17B.

In the examples below, all temperatures are given in ^o C. The following abbreviations are used:
Boc = tert-butyloxycarbonyl
DMF = dimethylformamide
DXM = dichloromethane
Fmoc = 9-fluorenylmethoxycarbonyl
HOBt = 1-hydroxybenzotriazole
TFA = trifluoroacetic acid
Trt = trityl = triphenylmethyl
RP-HPLC = reverse phase high resolution liquid chromatography
ct = room temperature
DNP = dinitrophenyl
Tos = tosil
DIPC = diisopropylcarbodiimide
Pmc = 2,2,5,7,8-pentamethylchroman-6-sulfonyl
IP = isopropyl
For = form
Cpe = 1-amino-cyclopentane-1-carboxylic acid
Cpp = 1-amino-cyclopropane-1-carboxylic acid
Cha = cyclohexylalanine
tBu = tert-butyl
Sequence N 11 represents the amino acid sequence of hPTG (1-34); The sequence of N 12 represents the amino acid sequence of hPTG (1-36); Sequence N 13 is the amino acid sequence of hPTG (1-38). Examples 1, 2, 5, 6, 11, 20-24, 27-30, 36, 37, 40, 41, 47, 49, 51, 53, 54, 57, 58, 61, 76-80, 82, 179 , 185, 187, 191-193, 226, 230, 237, 239, 256 and 289 are based on the sequence N 11, the replaced amino acid residues and their positions are shown in square brackets. Examples 12, 13, 18, 19, 31-35, 38, 39, 42-46, 48, 50, 52, 55, 59, 60, 62-71, 81, 83-85, 165-178, 180-184 , 186, 188-190, 194-225, 227-229, 231-236, 238, 240-255, 257-266, 268-279, 282-288, 290-300 and 302-304 based on the sequence N 12, replaced amino acid residues and their positions are shown in square brackets. Examples 3, 4, 7-10, 14-17, 25, 26, 56, 72-75, 81, 86-164, 267, 280 and 281 are based on the sequence N 13, substituted amino acid residues and their positions are shown in square brackets .

Example 1. N ^α -Isopropyl gPTG (1-34) NH ₂ .

 The specified peptide is synthesized in a stepwise manner using a polystyrene resin. To protect the alpha amino group, a Boc group is used, and the side functional groups are protected as follows: Lys (2-chlorobenzyloxycarbonyl), Ser (benzyl), Thr (benzyl), Glu (benzyl), Asp (benzyl), Met (O), His (DNP), Arg (Tos) and Trp (HCO). The remaining residues are left unprotected.

Amino-4-methylphenylmethyl-co (polystyrene-divinylbenzene = MBHA-resin) (0.7 mmol / g) is subjected to the following treatment cycle, steps (1) to (7):
(1) DXM
(2) 50% trifluoroacetic acid in DCM
(3) DXM
(4) 10% diisopropylethylamine in DMF
(5) DMF
(6) preformed symmetric anhydride (1.4 mM per 1 g of the starting resin) of the Boc amino acid in DMF
(7) DMF
Volumes of washing agents and interacting substances are from 5 to 20 ml per 1 g of the starting resin. Each step is repeated as many times as necessary to either completely enter the reaction of said resin (steps 2, 4, 6) or to completely remove the previous reagent from said resin (steps 1, 3, 5, 7). At the end of each cycle, resin samples are taken and the completion of the addition reaction is checked using a colorimetric test for the remaining amino groups using ninhydrin.

Symmetric Boc amino acid anhydrides are prepared immediately prior to use by reacting the Boc amino acid (2.8 mmol per 1 g of resin) and DCCI (1.4 mmol per 1 g of resin) in DCM containing enough DMF to completely dissolve Boc amino acids. The resulting mixture was filtered, additional DMF was added to the filtrate, which was concentrated by evaporation of volatile components at a temperature not exceeding 15 ^o C, and the resulting solution was used in step (6).

 The indicated reaction cycle (1) - (7) is repeated for each amino acid residue, thereby ensuring the synthesis of the sequence of the named compound with the exception of Boc-Gln-OH and Boc-Arg (Tos) -OH, which are added in step (6) as pre-prepared 1-hydroxybenzo-triazole esters in DMF.

 A resin with a fully synthesized peptide is treated twice with 20 ml of 5% thiophenol in DMF at rt. for 1 hour and washed with DMF, methanol and dichloromethane. The peptide resin is again subjected to steps (1) to (5), after which 0.5 ml of acetone, 84 mg of sodium cyanoborohydride and 0.2 ml of acetic acid are added in 20 ml of DMF per g of resin. After aging for 16 hours at rt said resin was washed with DMF and dichloromethane and dried.

 The peptide resin is treated with liquid HF, dimethyl sulfide, p-cresol and ethane-1,2-dithiol in accordance with the described "low-high" procedure [9]. After removal of the volatile components and washing with ethyl acetate, the resulting residue was extracted with several portions of 1% acetic acid, filtered, and the resulting filtrate was lyophilized. The resulting lyophilized product was purified using re-phase chromatography on an octadecyl silica gel column using a gradient of acetonitrile in 2% phosphoric acid or in 20 mM tetramethylammonium phosphate. The fractions containing the pure compound are combined, filtered through a weakly basic ion-exchange resin in the form of acetate, and the resulting filtrate is lyophilized to give the title compound.

 MS (Ion Spray): 4160.

Example 2. [Lys (N ^α -Isopropyl) ^26.27 ] gPTG (1-34) NH ₂ .

 The peptide is synthesized as described in Example 1, except that Lys (Fmoc) is used for inclusion at position 13, as well as Fmoc-Ser (tBu) at end position 1.

 A resin with a fully synthesized peptide is treated twice with 20 ml of 5% thiophenol in DMF at rt. for 1 hour and washed with DMF. methanol and dichloromethane. The dry resin was treated with liquid HF as described in Example 1. The resulting cleavage product (350 mg) was suspended in a mixture of methanol (5 ml), pH 5.0 phosphate buffer (5 ml), sodium cyanoborohydride (48 mg) and acetone (0.28 ml). After standing for 16 hours at rt, the resulting reaction mixture was filtered and the filtrate was evaporated. The residue was suspended in 20% piperidine in DMF for 15 minutes, diluted with 20 volumes of ether and filtered. The resulting residue was dissolved in 100 ml of water and acetic acid was added to pH 3, filtered and the resulting lyophilization filtrate was subjected. The lyophilisate is purified by reverse phase chromatography on an octadecyl silica gel column using a gradient of acetonitrile in 2% phosphoric acid. The fractions containing the pure compound are combined. Filter them through a weakly basic ion-exchange resin in the form of acetate, and the filtrate is lyophilized to give the title compound.

 MS (IS): 4205.6.

Example 3. N ^α -Isopropyl [Lys N ^α -isopropyl) ^13.26.27 ] gPTG (1-38) OH
a) Solid phase peptide synthesis.

 The peptide is synthesized in a stepwise manner using a polystyrene-based resin. The alpha amino group is protected by the Fmoc radical, and the side functional groups are protected as follows: Asp (OtBu), Glu (OtBu), His (Trt), Lys (Boc), Asn (Trt), Gln (Trt), Arg (Pmc) and Ser (tBu). The remaining amino acids are left unprotected.

Fmoc-Gly, attached via ether linkage to 4-hydroxymethyl-phenoxymethyl-co (polystyrene-1% divinylbenzene) (0.5 mM / g), is used as a starting material for stepwise solid-state synthesis consisting of repeated deprotection cycles with alpha -amino groups, washing, building (joining a new amino acid) and washing. Three to five-fold excess Fmoc amino acids are added as pre-prepared HOBt esters using DIPC. Fmoc-Arg (Pmc), Fmoc-Asn (Trt) and Fmoc-Gln (Trt) are added as asymmetric anhydrides using DIPC. Upon completion of the synthesis of the indicated peptide chain, the Fmoc-protecting group located on Ser ¹ is removed with 20% piperidine in DMF. Cleavage of the indicated peptide from the polystyrene resin, as well as the removal of all side protective groups, is carried out by treating the indicated peptide resin with a mixture of 10% ethanedithiol, thioanisole, thiocresol and water in 90% TFA for three hours at room temperature. From the combined filtrates, the product is precipitated by adding ether, filtered off and dried. The resulting product was purified by chromatography on a C-18 silica gel column using a gradient of acetonitrile in 2% H ₃ PO ₄ in water. The fractions containing the pure substance are combined, filtered through an anion exchange resin (Biorad, AG 4-X4, acetate form sieve 100-200) and lyophilized to obtain gPTG- (1-38) -OH.

 b) Alkylation.

40 mg (9 μM) hPTG- (1-38) -OH (4458.2 g / M) was dissolved in 1.2 ml of methanol, 1.2 ml of phosphate buffer (Merck no. 9887, adjusted to pH 5.0) 45 mg (716 mmol) NaBH ₃ CN (62.84 g / M) and 0.8 ml (11 mmol) of acetone (72.52 ml / M). The reaction is monitored by RP-HPLC on a C-18 silica gel column and stopped after 15 hours at room temperature. The resulting reaction mixture was diluted with water and chromatographed on a C-18 silica gel column using a gradient of acetonitrile in 2% H ₃ PO ₄ in water. The fractions containing the pure compound are combined, filtered through an anion exchange resin (acetate form) and lyophilized to give the title compound as polyacetate, polyhydrate.

[α] $\genfrac{}{}{0ex}{}{\text{20}}{\text{D}}$ = -5.7 ^o (c = 0.317 in 95% AcOH)
MS (IS): 4626
Example 4. N ^α- Methyl [Ala ¹ ] PTH- (1-38) -OH.

The peptide chain is synthesized in the same manner as described in example 3A. At position 1, instead of Fmoc-Ser (tBu) -OH, the artificial amino acid Fmoc-N ^α -methyl-Ala-OH is attached to the peptide resin. Cleavage, deprotection and purification are carried out in the same way as in example 3A.

MS (IS) = 4455.91
Example 5. [Ala ¹ , Ala ³ , Leu ⁸ , Gln ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Phe ²³ , His ²⁵ , His ²⁶ , Leu ²⁷ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH .

 The specified peptide is obtained by analogy with the method described in example 3A. Instead of Fmoc-Gly attached via ether linkage to 4-hydroxymethyl-phenoxymethyl-co (polystyrene-divinylbenzene), a suitable Fmoc amino acid, for example Fmoc-Ala, Fmoc-Phe, Fmoc-D-Ala, Fmoc-D-Phe and etc. In addition, the side functional groups are protected as follows: Thr (tBu), Trp (Boc) and Tyr (tBu).

Reproducing the method described in examples 3 and 5, but using the corresponding starting materials, the following compounds can be obtained:
Example
6: [Leu ⁸ , Asp ¹⁰ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ ] gPTG (1-34) OH
7: [Ile ¹ ] gPTG (1-38) OH (IS-MS: 4484)
8: [Ala ¹ , Abu ² ] gPTG (1-38) OH (IS-MS: 4428)
9: [Ala ¹ , Nva ² ] gPTG (1-38) OH (IS-MS: 4442)
10: [Ala ¹ , Ile ² ] gPTG (1-38) OH (IS-MS: 4456)
11: [Ala ¹ , Ala ³ , Leu ⁸ , Gln ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , His ²⁶ , Leu ²⁷ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
12: [N-MeAla ¹ ] gPTG (1-36) OH (IS-MS: 4286)
13: [Ala ¹ , Ala ³ , Leu ⁸ , Gln ¹⁸ ] gPTG (1-36) OH (IS-MS: 4235)
14: [Thr ¹ ] gPTG (1-38) OH (IS-MS: 4472)
15: [Leu ¹ ] gPTG (1-38) OH (IS-MS: 4484)
16: [Abu ¹ ] gPTG (1-38) OH (IS-MS: 4456)
17: [Gaba ¹ ] gPTG (1-38) OH (IS-MS: 4456)
18: [Leu ⁸ , Lys ¹¹ , Gln ¹⁸ ] gPTG (1-36) OH
19: [Leu ⁸ , Gln ¹⁶ , Asp ¹⁷ , Leu ¹⁸ , Arg ¹⁹ , Arg ²² ] gPTG (1-36) OH (IS-MS: 4347)
20: [Leu ⁸ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH (IS-MS: 4007)
21: [Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH (IS-MS: 3906)
22: [Leu ⁸ , Ala ¹³ , Gln ¹⁸ , Gln ²⁶ , Phe ²⁷ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
23: Ip- [Leu ⁸ , Lys (Ip) ¹³ , Gln ¹⁸ , Lys (Ip) ^26.27 , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH (IS-MS: 4175)
24: Ip- [Leu ⁸ , Ala ¹³ , Gln ¹⁸ , Gln ²⁶ , Phe ²⁷ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
25: [Gln ¹⁶ ] gPTG (1-38) OH
26: [Ser ¹⁴ ] gPTG (1-38) OH
27: [AIa ¹ , Ala ³ , Leu ⁸ , Gln ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , His ²⁵ , His ²⁶ , Leu ²⁷ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
28: [Ala ¹ , Ala ³ , Leu ⁸ , Gln ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Gln ²⁶ , Phe ²⁷ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
29: [Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH (IS-MS: 3965)
30: [Leu ⁸ , Gln ¹⁸ , Ala ²⁹ , Glu ³⁰ , Ile ³¹ ] gPTG (1-34) OH
31: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Gln ¹⁸ ] gPTG (1-36) OH (IS-MS: 4282)
32: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ser ¹⁴ , lle ¹⁵ , Gln ¹⁶ , Asp ¹⁷ , Leu ¹⁸ , Arg ¹⁹ ] gPTG (1-36) OH
33: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Leu ¹⁸ ] gPTG (1-36) OH
34: [Leu ⁸ , Gln ¹⁶ , Asp ¹⁷ , Leu ¹⁸ , Arg ¹⁹ ] gPTG (1-36) OH
35: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁷ , Leu ¹⁸ ] gPTG (1-36) OH (IS-MS: 4252)
36: [Leu ⁸ , Gln ¹⁶ , Asp ¹⁷ , Leu ¹⁸ , Arg ¹⁹ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH.

37: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH (IS-MS: 4022)
38: [Leu ⁸ , Ala ¹⁶ , Asp ¹⁷ , Leu ¹⁸ , Ala ¹⁹ ] gPTG (1-36) OH
39: [Leu ⁸ , Asp ¹⁰ , Ala ¹⁶ , Asp ¹⁷ , Leu ¹⁸ , Ala ¹⁹ ] gPTG (1-36) OH
40: (Leu ⁸ , Gln ¹⁸ , Arg ²² , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
41: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Gln ¹⁶ , Asp ¹⁷ , Leu ¹⁸ , Arg ¹⁹ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH (IS-MS: 4077)
42: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ ] gPTG (1-36) OH (IS-MS: 4181)
43: [Leu ⁸ , Ala ¹⁶ , Asp ¹⁷ , Gln ¹⁸ , Ala ¹⁹ ] gPTG (1-36) OH (IS-MS: 4193)
44: [Leu ⁸ , Ala ¹⁶ , Ala ¹⁷ , Gln ¹⁸ , Ala ¹⁹ ] gPTG (1-36) OH (IS-MS: 4149)
45: [Leu ⁸ , Ala ¹⁷ , Gln ¹⁸ , Ala ¹⁹ ] gPTG (1-36) OH
46: [Leu ⁸ , Ala ¹⁷ , Gln ¹⁸ , Ala ¹⁹ , Arg ²² ] gPTG (1-36) OH (IS-MS: 4219)
47: [Leu ⁸ , Ala ¹⁷ , Gln ¹⁸ , Ala ¹⁹ , Arg ²² , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH (IS-MS: 3960)
48: [Leu ⁸ , Gln ¹⁸ ] gPTG (1-36) OH
49: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH (IS-MS: 3980)
50: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ ] gPTG (1-36) OH (IS-MS: 4240)
51: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH (IS-MS: 3923)
52: [Leu ¹⁸ , Asp ¹⁰ , Ala ¹⁶ , Gln ¹⁸ ] gPTG (1-36) OH (IS-MS: 4225)
53: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Thr ³³ ] gPTG (1-34) OH (IS-MS: 3999)
54: [Leu ⁸ , Gln ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , His ²⁶ , Leu ²⁷ , Thr ³³ ] gPTG (1-34) OH
55: [Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Arg ²² ] gPTG (1-36) OH
56: [Ile ¹⁵ ] gPTG (1-38) OH
57: [Leu ⁸ , Gln ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Arg ²² , His ²⁶ , Leu ²⁷ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
58: [Leu ⁸ , Gln ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , His ²⁶ , Leu ²⁷ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
59: [Leu ⁸ , Ser ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Arg ²² ] gPTG (1-36) OH
60: [Leu ⁸ , Ala ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Arg ²⁶ , Arg ²⁷ ] gPTG (1-36) OH
61: [Leu ⁸ , Gln ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , His ²⁶ , Leu ²⁷ , Arg ³³ , Ala ³⁴ ] gPTG (1-34) OH
62: [Leu ⁸ , Ala ^{16.17, 18.19} ] gPTG (1-36) OH
63: [Leu ⁸ , Ala ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Gln ²⁶ , Phe ²⁷ ] gPTG (1-34) OH (IS-MS: 4127)
64: Ip- [Leu ⁸ , Ala ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Gln ²⁶ , Phe ²⁷ ] gPTG (1-34) OH
65: Ip- [Leu ⁸ , Lys (Ip) ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Lys (Ip) ^26.27 ] gPTG (1-36) OH
66: [Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ ] gPTG (1-36) OH (IS-MS: 4166)
67: Ip- [Leu ⁸ , Lys (Ip) ¹³ , Ala ¹⁶ , Ala ¹⁷ , Ala ¹⁸ , Ala ¹⁹ , Lys (Ip) ^26.27 ] gPTG (1-36) OH
68: [Aib ³ , Gln ¹⁸ ] gPTG (1-36) OH (IS-MS: 4283)
69: Ip- [Leu ⁸ , Asp ¹⁰ , Lys (Ip) ^{11.13, 26.27,,} Gln ¹⁸ ] gPTG (1-36) OH
70: Ip- [Leu ⁸ , Ser ¹³ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Arg ²² , Lys (Ip) ^26.27 ] gPTG (1-36) OH
71: [Leu ⁸ , Tyr ¹⁸ ] gPTG (1-36) OH
72: [Ser ³³ ] gPTG (1-38) OH
73: [Thr ³³ ] gPTG (1-38) OH
74: [Leu ³³ ] gPTG (1-38) OH
75: [Gly ³³ ] gPTG (1-38) OH
76: [Leu ⁸ , His ¹⁰ , Gln ¹⁸ , Arg ²² , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
77: [Leu ⁸ , Gly ¹⁰ , Gln ¹⁸ , Arg ²² , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
78: [Leu ⁸ , Glu ¹⁰ , Gln ¹⁸ , Arg ²² , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
79: [Leu ⁸ , Thr ¹⁰ , Gln ¹⁸ , Arg ²² , Thr ³³ Ala ³⁴ ] gPTG (1-34) OH
80: [Leu ⁸ , Gln ¹⁰ , Gln ¹⁸ , Arg ²² , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
81: [Gln ³³ ] gPTG (1-38) OH
82: [Leu ⁸ , Tyr ¹⁰ , Gln ¹⁸ , Arg ²² , Thr ³³ , Ala ³⁴ ] gPTG (1-34) OH
83: [1-amino-cyclopentane-1-carboxylic acid ¹ , Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ ] gPTG (1-36) OH
84: [1-amino-cyclopentane-1-carboxylic acid ¹ , Leu ⁸ , Ala ^13.16 , Gln ¹⁸ , Ala ¹⁹ , Arg ^26.27 ] gPTG (1-36) OH
85: [1-amino-cyclopentane-1-carboxylic acid ³ , Gln ⁸ ] hPTG (1-36) OH (IS-MS: 4309)
86: [Arg ¹² ] gPTG (1-38) OH
87: [Ser ¹² ] gPTG (1-38) OH
88: [Cys ¹³ ] gPTG (1-38) OH
89: [Ile ¹³ ] gPTG (1-38) OH
90: [Asn ¹³ ] gPTG (1-38) OH
91: [Trp ¹³ ] gPTG (1-38) OH
92: [Asp ¹³ ] gPTG (1-38) OH
93: [Val ¹³ ] gPTG (1-38) OH
94: [Thr ¹³ ] gPTG (1-38) OH
95: [Ser ¹³ ] gPTG (1-38) OH
96: [Tyr ¹³ ] gPTG (1-38) OH
97: [Met ¹³ ] gPTG (1-38) OH
98: [Gln ¹³ ] gPTG (1-38) OH
99: [Leu ¹³ ] gPTG (1-38) OH
100: [Ala ¹³ ] gPTG (1-38) OH
101: [Gly ¹³ ] gPTG (1-38) OH
102: [Val ¹⁴ ] gPTG (1-38) OH
103: [Ala ¹⁴ ] gPTG (1-38) OH
104: [Lys ¹⁴ ] gPTG (1-38) OH
105: [Arg ¹⁴ ] gPTG (1-38) OH
106: [Thr ¹⁴ ] gPTG (1-38) OH
107: [Ile ¹⁴ ] gPTG (1-38) OH
108: [Tyr ¹⁴ ] gPTG (1-38) OH
109: [Tyr ¹⁵ ] gPTG (1-38) OH
110: [Arg ¹⁵ ] gPTG (1-38) OH
111: [Val ¹⁵ ] gPTG (1-38) OH
112: [Lys ¹⁶ ] gPTG (1-38) OH
113: [Ser ¹⁶ ] gPTG (1-38) OH
114: [Leu ¹⁶ ] gPTG (1-38) OH
115: [Ala ¹⁶ ] gPTG (1-38) OH
116: [Gly ¹⁶ ] gPTG (1-38) OH
117: [Ala ¹⁷ ] gPTG (1-38) OH
118: [Met ¹⁷ ] gPTG (1-38) OH
119: [Ile ¹⁷ ] gPTG (1-38) OH
120: [Ser ¹⁹ ] gPTG (1-38) OH
121: [Lys ¹⁹ ] gPTG (1-38) OH
122: [Leu ¹⁹ ] gPTG (1-38) OH
123: [Ala ¹⁹ ] gPTG (1-38) OH
124: [Tyr ¹⁹ ] gPTG (1-38) OH
125: [Met ¹⁹ ] gPTG (1-38) OH
126: [His ¹⁹ ] gPTG (1-38) OH
127: [Val ¹⁹ ] gPTG (1-38) OH
128: [G1y ¹⁹ ] gPTG (1-38) OH
129: [Pro ¹⁹ ] gPTG (1-38) OH
130: [Asp ¹⁹ ] gPTG (1-38) OH
131: [Ile ¹⁹ ] gPTG (1-38] OH
132: [Val ¹⁹ , Gln ²⁴ ] gPTG (1-38) OH
133: [Arg ¹⁹ ] gPTG (1-38) OH)
134: [Phe ²⁰ ] gPTG (1-38) OH
135: [Ala ²¹ ] gPTG (1-38) OH
136: [Gly ²¹ ] gPTG (1-38) OH
137: [Phe ²¹ ] gPTG (1-38) OH
138: [Leu ²¹ ] gPTG (1-38) OH
139: [Asn ²¹ ] gPTG (1-38) OH
140: [Gln ²¹ ] gPTG (1-38) OH
141: [Ser ²¹ ] gPTG (1-38) OH
142: [Gly ²² ] gPTG (1-38) OH
143: [Leu ²² ] gPTG (1-38) OH
144: [His ²² ] gPTG (1-38) OH
145: [Ala ²² ] gPTG (1-38) OH
146: [Ile ²² ] gPTG (1-38) OH
147: [Val ²² ] gPTG (1-38) OH
148: [Ser ²² ] gPTG (1-38) OH
149: [Arg ²² ] gPTG (1-38) OH
150: [Arg ²⁶ ] gPTG (1-38) OH
151: [Val ²⁷ ] gPTG (1-38) OH
152: [Ile ²⁷ ] gPTG (1-38) OH
153: [Leu ²⁷ ] gPTH (1-38) OH
154: [Arg ²⁷ ] gPTG (1-38) OH
155: [Ala ²⁷ ] gPTG (1-38) OH
156: [Val ²⁸ ] gPTG (1-38) OH
157: [Ile ²⁸ ] gPTG (1-38) OH
158: [Pro ³ , Thr ³³ ] gPTG (1-38) OH
159: [Arg ³³ ] gPTG (1-38) OH
160: [Pro ³³ ] gPTG (1-38) OH
161: [Asp ³³ ] gPTG (1-38) OH
162: [Ile ³³ ] gPTH (1-38) OH
163: [Lys ³³ ] gPTG (1-38) OH
164: [Ile ³¹ , Arg ³³ ] gPTG (1-38) OH
Example 165. [1-Aminocyclopentane-1-carboxylic acid ¹ ] gPTG (1-36) NH ₂ .

 The specified peptide is synthesized in a stepwise manner on a polystyrene-based resin substrate. To protect the alpha amino groups, an Fmoc group is used.

 Lateral functional groups are protected as follows: Arg (Pmc), Asn (Trt), Asp (OtBu), Gln (Trt), Glu (OtBu), His (Trt), Lys (Boc), Ser (tBu), Thr (tBu ), Trp (Boc) and Tyr (tBu). O steel amino acids are left unprotected.

0.4 mM / g of 4- (2 ', 4'-dimethoxyphenyl-Fmoc-amino-methyl) -phenoxy- (polystyrene-divinylbenzene), which can be obtained, for example, as described in [10], is subjected participating in the next cycle in which stages (1) to (5) are processing:
(1) DMF
(2) 20% piperidine in DMF
(3) DMF
(4) a mixture of HOBt, diisopropylcarbodiimide Fmoc-alanine (0.8 mm each per 1 g of the starting resin)
(5) DMF
Volumes of washing agents and interacting substances are from 5 to 20 ml per 1 g of the starting resin.

 In order to synthesize the correct amino acid sequence of the aforementioned compound on the specified resin, in the next cycle of treatments (1) - (5) Fmoc-alanine is replaced by Fmoc-valine, etc. for each subsequent cycle.

 Each step is repeated as many times as necessary to either completely enter the reaction of said resin (steps 2, 4) or to completely remove previous reagents from said resin (steps 3, 5). At the end of each cycle, resin samples are taken and the completion of the addition reaction is checked using a colorimetric test for the remaining amino groups using ninhydrin.

 At the end of the synthesis, the last cycle is carried out, consisting only of steps (1) to (3), and the obtained polypeptide resin is washed with 2-propanol, then with a mixture of methanol and methylene chloride in a volume ratio of 1: 1 and dried in a vacuum dryer.

1 g of the peptide resin is suspended in 2 ml of a mixture of trifluoroacetic acid, water and 1,2-ethanedithiol in a volume ratio of 90: 5: 5 at room temperature and washed with a small volume of TFA. The product is precipitated from the combined filtrates, adding 20 volumes of ether to them, filtered, washed with additional ether and dried. The resulting residue was dissolved in 2% acetic acid, this solution was kept at room temperature for 8 hours, and then subjected to lyophilization. The resulting lyophilisate is chromatographed on a C-18 silica gel column using a gradient of acetonitrile in 2% H ₃ PO ₄ . Fractions were tested by analytical HPLC and those that were pure were collected, filtered through an anion exchange resin (acetate) and lyophilized to give the title compound as polyacetate, polyhydrate.

 Fmoc-1-aminocyclopentane-1-carboxylic acid used in the preparation of the intermediate product of the peptide resin can be synthesized as described in [11].

MS (IS): 4312
Example 166. [1- (1-Amino-iiklopropan-1-carboxylic acid)] gPTG (1-36) NH ₂ .

 The specified peptide is obtained by a method similar to that described in example 165. Fmoc-1-aminocyclopropane-1-carboxylic acid used to obtain the intermediate product of the peptide resin can be synthesized as described in [12].

MS (IS): 4283
Reproducing the method described in example 165, but using the corresponding starting materials, the following compounds can be obtained:
Example
167: [D-Pro ¹ ] gPTG (1-36) NH ₂
168: [Nva ¹ ] gPTG (1-36) NH ₂
169: [N-Me-Ser ¹ ] gPTG (1-36) NH ₂
170: [indole-2-carboxylic acid ¹ ] gPTG (1-36) NH ₂
171: [indole-3-carboxylic acid ¹ ] gPTG (1-36) NH ₂
172: [pyridine-3-carboxylic acid ¹ ] gPTG (1-36) NH ₂
173: [pyridine-2-carboxylic acid ¹ ] gPTG (1-36) NH ₂
174: [hexahydropyridazine-3-carboxylic acid ¹ ] gPTG (1-36) NH ₂
175: [morpholine-2-carboxylic acid ¹ ] gPTG (1-36) NH ₂
176: [Pro ¹ ] gPTG (1-36) NH ₂
177: [Leu ¹ ] gPTG (1-36) NH ₂
178: [Ile ¹ ] gPTG (1-36) NH ₂
179: [Thr ³³ , Ala ³⁴ ] gPTG (1-34) NH ₂
180: [Nva ⁸ ] gPTG (1-36) NH ₂
181: [Gln ¹⁸ ] gPTG (1-36) NH ₂
182: [Tyr18] gPTG (1-36) NH ₂
183: [Lys ¹⁸ ] gPTG (1-36) NH ₂
184: [Ala ¹⁸ ] gPTG (1-36) NH ₂
185: [Phe ²³ , His ²⁵ , His ²⁶ , Leu ²⁷ ] gPTG (1-34) NH ₂
186: [Phe ²³ ] gPTG (1-34) NH ₂ (IS-MS: 4247)
187: [Phe ²³ , His ²⁵ , His ²⁶ , Leu ²⁷ , Ile ²⁸ , Ala ²⁹ , Glu ³⁰ , Ile ³¹ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) NH ₂ (IS-MS: 3934)
188: [Ala ²⁹ ] gPTG (1-36) NH ₂ (IS-MS: 4248)
189: [Ala ³⁴ ] gPTG (1-36) NH ₂ (IS-MS: 4210)
190: [Gln ²⁵ ] gPTG (1-36) NH ₂
191: [Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) NH ₂
192: [Ala ¹ , His ⁵ , Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) NH ₂
193: [Ser ¹⁴ , Ile ¹⁵ , Gln ¹⁶ , Asp ¹⁷ , Leu ¹⁸ , Arg ¹⁹ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) NH ₂
194: [D-Phe ³⁴ , D-Ala ³⁶ ] gPTG (1-36) NH ₂ [MS (IS): 4290]
195: [Ala ³ ] gPTG (1-36) NH ₂ [MS (IS): 4271]
196: [D-Ser ³ ] gPTG (1-36) NH ₂ [MS (IS): 4290]
197: [D-Glu ⁴ ] gPTG (1-36) NH ₂ [MS (IS): 4290]
198: [D-His ⁹ ] gPTG (1-36) NH ₂ [MS (IS): 4286]
199: [Ala ¹⁰ ] gPTG (1-36) NH ₂ [MS (IS): 4243]
200: [D-Asn ¹⁰ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
201: [Ala ¹² ] gPTG (1-36) NH ₂ [MS (IS): 4299]
202: [Gln ¹³ ] gPTG (1-36) NH ₂ [MS (IS): 4287]
203: [His ¹³ ] gPTG (1-36) NH ₂ [MS (IS): 4296]
204: [Leu ¹³ ] gPTG (1-36) NH ₂ [MS (IS): 4371]
205: [Ala ¹³ ] gPTG (1-36) NH ₂ [MS (IS): 4228]
206: [ALa ¹³ , Gln ²⁶ , Phe ²⁷ , D-Phe ³⁴ , D-Ala ³⁶ ] gPTG (1-36) NH ₂ [MS (IS): 4249]
207: [Ala ¹⁴ ] gPTG (1-36) NH ₂ [MS (IS): 4219]
208: [D-His ¹⁴ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
209: [D-Asn ¹⁶ ] gPTG (1-36) NH ² [MS (IS): 4284]
210: [Ala ₁₇ ] gPTG (1-36) NH ₂ [MS (IS): 4270]
211: [D-Ser ¹⁷ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
212: [Ala ¹⁹ ] gPTG (1-36) NH ₂ [MS (IS): 4228]
213: [D-Glu ¹⁹ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
214: [Ala ²¹ ] gPTG (1-36) NH ₂ [MS (IS): 4257]
215: [Ala ²² ] gPTG (1-36) NH ₂ [MS (IS): 4227]
216: [Gln ²⁶ ] gPTG (1-36) NH ₂ [MS (IS): 4287]
217: [Nle ²⁶ ] gPTG (1-36) NH ₂ [MS (IS): 4271]
218: [D-Lys ²⁶ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
219: [Nle ^8.18.27 ] gPTG (1-36) NH ₂
220: [His ²⁷ ] gPTG (1-36) NH ₂ [MS (IS): 4294]
221: [Phe ²⁷ ] gPTG (1-36) NH ₂ [MS (IS): 4304]
222: [Nle ²⁷ ] gPTG (1-36) NH ₂ [MS (IS): 4271]
223: [Asn ²⁷ ] gPTG (1-36) NH ₂ [MS (IS): 4271]
224: [Ala ²⁷ ] gPTG (1-36) NH ₂ [MS (IS): 4228]
225: [D-Gln ²⁹ ] gPTG (1-36) NH ₂ [MS (IS): 4289]
226: [D-Asp ³⁰ ] gPTG (1-34) NH ₂ [MS (IS): 4118]
227: [Ala ³⁰ ] gPTG (1-36) NH ₂ [MS (IS): 4241]
228: [D-Val ³¹ ] gPTG (1-36) NH ₂ [MS (IS): 4290]
229: [Ala ³¹ ] gPTG (1-36) NH ₂ [MS (IS): 4258]
230: [Lys ³² ] gPTG (1-34) NH ₂ CMS (IS): 3832]
231: [D-His ³² ] gPTG (1-36) NH ₂ [MS (IS): 4288]
232: [Ala ³² ] gPTG (1-36) NH ₂ [MS (IS): 4219]
233: [D-Asn ³³ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
234: [Ala ³³ ] gPTG (1-36) NH ₂ [MS (IS): 4210]
235: [NMePhe ³⁴ ] gPTG (1-36) NH ₂ [MS (IS): 4301]
236: [D-Asp ³⁰ ] gPTG (1-36) NH ₂ [MS (IS): 4290]
237: Ip- [Nle ^8.18 , Lys (Ip) ^13.26.27 , D-Asn ³³ , D-Phe ³⁴ ] gPTG (1-34) NH ₂
238: Ip- [Nle ^8.18.27 , Lys (Ip) ^13.26 ] gPTG (1-36) NH ₂
239: [Nle ^8.18 , D-Asn ³³ , D-Phe ³⁴ ] gPTG (1-34) NH ₂ [MS (IS): 4080]
240: [Lys (Ip) ¹³ ] gPTG (1-36) NH ₂ [MS (IS): 4331]
241: propargyl- [A ¹ ] gPTG (1-36) NH ₂
242: [Ala ⁰ ] gPTG (1-36) NH ₂
243: [Pro ⁰ ] gPTG (1-36) NH ₂
244: acetyl-gPTG (1-36) NH ₂
245: [HyPro ¹ ] gPTG (1-36) NH ₂
246: N-dimethyl- [Ala ¹ ] gPTG (1-36) NH ₂
247: [D-Ser ¹ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
248: [Lys (For) ¹ ] gPTG (1-36) NH ₂ [MS (IS): 4356]
249: [D-glyceric acid ¹ ] gPTG (1-36) NH ₂
250: [Asn ¹ ] gPTG (1-36) NH ₂
251: [4-aminobenzoic acid ¹ ] gPTG (1-36) NH ₂
252: [4-aminosalicylic acid ¹ ] gPTG (1-36) NH ₂
253: [TMSA ¹ ] gPTG (1-36) NH ₂ [MS (IS): 4343]
254: [Phe ¹ ] gPTG (1-36) NH ₂
255: [propargylglycine ¹ ] gPTG (1-36) NH ₂
256: [Ala ¹ , His ⁵ , Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Gln ¹⁸ , Phe ²² , Phe ²³ , His ²⁵ , His ²⁶ , Leu ²⁷ , Thr ³³ , Ala ³⁴ ] gPTG (1-34) NH ₂
257: [Abu ² ] gPTG (1-36) NH ₂
258: [D-Val ² ] gPTG (1-36) NH ₂ [MS (IS): 4288]
259: [t-Leu ² ] gPTG (1-36) NH ₂
260: [Ala ¹ ] gPTG (1-36) NH ₂
261: [D-Ile ⁵ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
262: [D-Gln ⁶ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
263: [D-Leu ⁷ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
264: [Nle ⁸ ] gPTG (1-36) NH ₂ [MS (IS): 4268]
265: [Phe ⁸ ] gPTG (1-36) NH ₂ [MS (IS): 4303]
266: [Cha ⁸ ] gPTG (1-36) NH ₂ [MS (IS): 4309]
267: [Leu ⁸ ] gPTG (1-38) NH ₂
268: [D-Leu ¹¹ ] gPTG (1-36) NH ₂ [MS (IS): 4287]
269: [Ala ¹¹ ] gPTG (1-36) NH ₂ [MS (IS): 4244]
270: [D-Lys ¹³ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
271: [D-Leu ¹⁵ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
272: [Ala ¹⁵ ] gPTG (1-36) NH ₂ [MS (IS): 4243]
273: [Ala ¹⁶ ] gPTG (1-36) NH ₂ [MS (IS): 4243]
274: [Met (O ₂ ) ¹⁸ ] gPTG (1-36) NH ₂ [MS (IS): 4320]
275: [Nle ¹⁸ ] gPTG (1-36) NH ₂ [MS (IS): 4268]
276: [D-Met ¹⁸ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
277: [Lys ²⁰ ] gPTG (1-36) NH ₂ [MS (IS): 4259]
278: [D-Arg ²⁰ ] gPTG (1-36) NH ² [MS (IS): 4288]
279: [D-Val ²¹ ] gPTG (1-36) NH ₂ GM8 (18): 4288]
280: [Trp (SO ₂ Pmc) ²³ ] gPTG (1-38) NH ₂ [MS (IS): 4723]
281: [Trp (Pmc) ²³ ] gPTG (1-38) NH ₂ [MS (IS): 4660]
282: [D-Trp ²³ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
283: [Ala ²³ ] gPTG (1-36) NH ₂ [MS (IS): 4171]
284: [D-Leu ²⁴ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
285: [Phe ²⁵ ] gPTG (1-36) NH ₂ [MS (IS): 4277]
286: [Lys ²⁵ ] gPTG (1-36) NH ₂ [MS (IS): 4258]
287: [Ala ²⁵ ] gPTG (1-36) NH ₂ [MS (IS): 4201]
288: [Ala ²⁶ ] gPTG (1-36) NH ₂ [MS (13): 4229]
289: [Lys ^26/27 (For)] gPTG (1-36) NH ₂
290: [D-Lys ²⁷ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
291: [D-Leu ²⁸ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
292: [D-Phe ³⁴ ] gPTG (1-36) NH ₂ [MS (IS): 4288]
293: [D-Val ³⁵ ] gPTG (1-36) NH ₂
294: [Ala ³⁵ ] gPTG (1-36) NH ₂
295: [Pro ³⁵ ] gPTG (1-36) NH ₂
296: [NMeVaI ³⁵ ] gPTG (1-36) NH ₂
297: [Thr ³⁵ , Ala ³⁶ ] gPTG (1-36) NH ₂
298: [D-Ala ³⁶ ] gPTG (1-36) NH ₂
299: [NMeAla ³⁶ ] gPTG (1-36) NH ₂
Example 300. (5-amino-4-fluoro) -2-isopropyl) hex-3-enoyl-hPTG (3-36) NH ₂ .

 a) Obtaining gPTG (3-36) -NH-resin.

 The specified intermediate compound is obtained in a manner analogous to that described in example 165.

 b) [(5-amino-4-fluoro-2-isopropyl) hex-3-enoyl] gPTG (3-36] -amide.

 204.6 mg of the Fmoc-hPTG (3-36) -NH resin obtained in the above step a) is treated with DMF for 5 minutes and then washed several times with isopropanol or DMF. The Fmoc protecting group is removed by treatment with a mixture of DMF and piperidine in a ratio of 8: 2 for 10 minutes. A mixture of the unprotected PTH fragment together with 38.1 mg of 4-fluoro-2-isopropyl-5-t-butoxycarbonylamino-hex-3-enoic acid, 68.7 mg of benzotriazol-1-yloxytris (pyrrolidino) phosphonium hexafluoride (PyBOP) and 26.6 mg of 4-methylmorpholine is vigorously shaken in 0.7 ml of DMF for 80 minutes. After several washes with isopropanol and DMF, the solid residue is treated with a mixture of 4.5 ml of TFA, 0.25 ml of water and 0.25 ml of ethanedithiol for 55 minutes. Then the resulting mixture was filtered and, adding diethyl ether to the remaining solution, the title compound precipitated. The specified compound is purified using preparative HPLC (Vydac column, gradient A = water, B = mixture of acetonitrile, water and phosphoric acid in a ratio of 1: 3: 0.2).

MS (IS): mass 4272
[α] $\genfrac{}{}{0ex}{}{\text{n9}}{\text{365}}$ _nm = -66.8 ^o (c = 0.25 in 95% AcOH)
Example 301. 4-Fluoro-2-isopropyl-5-tert-butoxycarbonylamino-hex-3-enoate
a) (S) -3- [1-oxo (4-Fluoro-3-hydroxy-2-isopropyl) hex-4-enyl] - 4-phenylmethyl-2-oxazolidinone.

 The specified compound is obtained by a method similar to that described in [13], using (S) -3- (1-oxoisobutyl) -4-phenylmethyl-2-oxazolidinone and 2-fluoro-but-2-enal as starting materials.

MS (FAB): MH ⁺ 350
b) (S) -3- {1-oxo [4-Fluoro-3-O- (2,2,2-trichloroethanimino) -2-isopropyl] hex-4-enyl} -4-phenylmethyl-2-oxazolidinone.

1.5 g of the compound of Example 301a) was dissolved in 6 ml of DCM. Add 0.0958 ml of 1,8-diazabicyclo (5.4.0) undec-7-ene (DBU) at 0 ^° . To the resulting solution, 0.475 ml of trichloroacetonitrile in 2 ml of DCM was added dropwise at 0 ^° . After 1 hour, the reaction mixture was evaporated, and the obtained residue was purified by chromatography on a silica gel column (elution with a mixture of hexane and diethyl ether in a 2: 1 ratio).

MS (FAB): MH ⁺ 493
c) (S) -3- [1-oxo (4-Fluoro-5-N-2,2,2-trichloroacetylamino-2-iopropyl) hex-3-enyl] -4-phenylmethyl-2-oxazolidinone.

2 g of the compound from Example 301b) was dissolved in 150 ml of o-xylene and stirred under reflux at 160 ^° C. for 3 hours. After that, o-xylene is removed and the obtained residue is purified by chromatography on a silica gel column (elution with a mixture of toluene and ethyl acetate in a ratio of 98: 2).

MS (FAB): MH ⁺ 493
d) 4-Fluoro-5-N-2,2,2-trichloroacetylamino-2-isopropyl-hex-3-enoic acid.

 The specified compound is obtained by a method similar to that described in [13], but using the compounds of Example 301c as the starting material).

MS (FAB): MH ⁺ 335
d) 5-amino-4-fluoro-2-isopropyl-hex-3-enoic acid.

 889 mg of the compound from Example 301 g) was dissolved in 20 ml of ethanol and 13.5 ml of 6N was added. sodium hydroxide solution. The resulting mixture was stirred for 20 hours at rt. After that, ethanol is removed, and the remaining aqueous fraction is acidified with 2 N. hydrochloric acid to pH 2 and extracted with n-butanol. The resulting extract was evaporated, and the residue was filtered through silica gel (eluent was a 1: 1 mixture of DCM and methanol), thereby isolating the pure title compound.

MS (FAB): MH ⁺ 190
e) 4-Fluoro-2-isopropyl-5-tert-butoxycarbonylamino-hex-3-enoic acid.

 0.56 g of the compound from Example 301d) is dissolved in 12 ml of water and 1.5 g of sodium carbonate is added. To the resulting solution was added 0.4 ml of di-tert-butyl carbonate dissolved in 12 ml of tetrahydrofuran. The mixture was stirred at room temperature for 18 hours. Then 50 ml of n-butanol and 50 ml of water are added, after which 1N is added with vigorous stirring. hydrochloric acid to pH 2. The organic fraction is evaporated, and the obtained residue is purified by chromatography on a silica gel column (elution with a mixture of hexane and acetone in a ratio of 2: 1).

MS (FAB): MH ⁺ 290
[α] _D = -61.3 ^o (1% in methanol)
Reproducing the above procedures using the appropriate starting materials, you can get the following compounds:
4-fluoro-2-methyl-5-tert-butoxycarbonylamino-hex-3-enoic acid;
4-chloro-2-methyl-5-tert-butoxycarbonylamino-hex-3-enoic acid;
4-fluoro-2-benzyl-5-tert-butoxycarbonylamino-hex-3-enoic acid;
4-chloro-2-isopropyl-5-tert-butoxycarbonylamino-hex-3-enoic acid;
4-methyl-2-isopropyl-5-tert-butoxycarbonylamino-hex-3-enoic acid
Example 302

Using the procedures described in example 300, but using the appropriate starting materials, the following compounds can be obtained:
(5-amino-4-fluoro-2-methyl) -hex-3-enoyl-gPTG (3-36) -amide
MS (IS): Mass 4245;
(5-amino-4-chloro-2-methyl) -hex-3-enoyl-gPTG (3-36) -amide
MS (IS): Mass 4260;
(5-amino-4-fluoro-2-benzyl) -hex-3-enoyl-gPTG (3-36) -amide
MS (IS): Mass 4320;
(5-amino-4-chloro-2-isopropyl) -hex-3-enoyl-gPTG (3-36) -amide
MS (IS): Mass 4289;
(5-amino-4-methyl-2-isopropyl) -hex-3-enoyl-gPTG (3-36) -amide
MS (IS): Mass 4269.

 Example 303

Using the procedures described in Example 300 above, but using the appropriate starting materials, the following compounds can be prepared:
a) (5-amino-3-aza-2-isopropyl) hexanoyl-gPTG (3-36) -amide [MS (IS): 4257] when used as starting material: (4-aza-2-t- butoxycarbonylamino-5-isopropyl) hexanoic acid;
b) (5-amino-3-aza-3-N-acetyl-2-isopropyl) hexanoyl-gPTG (3-36) -amide [MS (IS): 4299] when used as starting material: (4- aa-4-N-acetyl-2-t-butoxycarbonylamino-5-isopropyl) hexanoic acid;
c) (5-amino-3-aza-3-N-acetyl) -hexanoyl-gPTG (3-36) -amide [MS (IS): 4257] when used as starting material: (4-aza-N- acetyl-2-t-butoxycarbonylamino) hexanoic acid;
g) (5-methylamino-3-aza-2-isopropyl) -hexanoyl-gPTG (3-36) -amide [MS (IS): 4271] when used as starting material: [4-aza-2- (N t-butoxycarbonyl, N-methylamino) -5-isopropyl] hexanoic acid;
d) (5-methylamino-3-aza-3-N-methyl-2-isopropyl) hexanoyl-gPTG (3-36) -amide [MS (IS): 4285] when used as starting material: [4- aza-4-N-methyl-2- (N-t-butoxycarbonylamino, N-methylamino) -5-isopropyl] hexanoic acid;
f) (5-amino-3-aza-3-N-methyl-2-isopropyl) hexanoyl-gPTG (3-36) -amide [MS (IS): 4271] when used as starting material: (4- aa-4-N-methyl-2-t-butoxycarbonylamino-5-iopropyl) hexanoic acid;
g) (5-amino-3-aza-3-N-isopropyl) hexanoyl-gPTG (3-36) -amide [MS (IS): 4257] when used as starting material: (4-aza-4- N-isopropyl-2-t-butoxycarbonylamino-5-isopropyl) hexanoic acid;
h) [1-cyclopentane-1-amino-1-carboxylic acid (psi CH ₂ -NH) -Val ² ] -PTH (3-36) -amide using 1-cyclopentane-1-t-butoxycarbonylamino-1-carboxylic acids (psi CH ₂ —NH) —Val ² —OH.

 Example 304

Using the procedures described in Example 3a above), but using the appropriate starting materials, the compound (5-amino-3-aza-2-isopropyl) -hexanoyl- [Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ ] gPTG (3-36) -OH [MS (IS): 4135] when using (4-aza-2t-butoxycarbonylamino) -hexanoic acid as the starting material.

 Example 305. (4-Aza-4-N-Acetyl-2-tert-butoxycarbonylamino-5-isopropyl) hexanoic acid.

 a) Methyl (4-aza-2-tert-butoxycarbonylamino-5-isopropyl) - hexanoic acid.

 Boc-Ala-Val-OMe is treated with Lawesson's reagent [15]. Then, the resulting endotiopeptide is reduced in accordance with the procedure described in [16].

MS (EI): MH ⁺ 289.

 (b) Methyl (4-aza-4-N-Acetyl-2-tert-butoxycarbonylamino-5-isopropyl) hexanoate.

264 mg of the compound from Example 305a) is dissolved in 5 ml of DCM, after which 0.14 ml of triethylamine and 0.072 ml of acetyl chloride are added. The specified mixture is stirred for 24 hours at 40 ^o . After adding 50 ml of DCM, the resulting solution was washed twice with water. The organic fraction was dried and evaporated. The aforementioned compound thus obtained was purified by column chromatography (elution with a mixture of hexane and ethyl acetate in a ratio of 4: 1).

MS (EI): MH ⁺ 330
c) (4-aza-4-N-Acetyl-2-tert-butoxycarbonylamino-5-isopropyl) - hexanoic acid.

356 mg of the compound of Example 305b) was dissolved in 4 ml of a 3: 1 mixture of methanol and water and 53 mg of lithium hydroxide was added. The specified mixture was stirred at 60 ^o for two hours. Then add 25 ml of DCM, after which with vigorous stirring add 1 N. hydrochloric acid to pH 2. The organic fraction is separated, dried and evaporated, thus obtaining the above-named compound in pure form.

MS (FAB): MH ⁺ 317
By reproducing the indicated procedure, but using the appropriate starting materials, the following compounds can be obtained:
(4-aza-4-N-acetyl-2-tert-butoxycarbonylamino) hexanoic acid;
(4-aza-2-tert-butoxycarbonylamino-5-isopropyl) hexanoic acid;
(4-aza-2- (N-t-butoxycarbonyl, N-methylamino) -5-isopropyl] - hexanoic acid;
(4-aza-4-N-methyl-2-t-butoxycarbonylamino-5-isopropyl) hexanoic acid;
(4-aza-4-N-isopropyl-2-t-butoxycarbonylamino-5-isopropyl) hexanoic acid;
(4-aza-2-t-butoxycarbonylamino) hexanoic acid.

 The compounds of Examples 1-300 and 302-304 show the corresponding amino acid ratios when quantified for amino acid content.

 Unless otherwise specified, in the examples below illustrating the recombinant method provided by the present invention, the standard procedures described in [17] are used.

 Example 306. Cloning of the gene 55 of the bacteriophage T4.

Two DNA fragments, one of which contains the complete nucleotide sequence of gene 55 of the bacteriophage T4, and the other encodes a portion of the indicated gene, are amplified by polymerase chain reaction (PCR) using purified T4 DNA as a template. As primers, oligonucleotides containing recognition sites by the endonuclease NdeI or BamHI are used. PCR (25 cycles, each: 30 '' 94 ^° , 30 '' 50 ^° , 30 '' 72 ^° ) was performed using 1 ng of the template and 100 pM upstream primer (sequence No. 5, NdeI recognition site formed by nucleotides 7 -12 of the indicated sequence) and 100 pM of the downstream primer (sequence No. 6, the BamHI recognition site is formed by nucleotides 5-10 of this sequence) in 100 μl of reaction buffer containing 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl ₂ , 0.2 mM each of dATP, dTTP, dGTP, dTTP, as well as 0.1% Triton X 100, pH 9.0. Get a fragment of 0.6 TPN.

 The shortened form of gene 55 is obtained by PCR in the same way as described for the complete gene, except that another oligonucleotide is used as the primer (downstream sequence 7, the BamHI recognition site is formed by nucleotides 5-10 of this sequence ) Get a fragment of 0.35 TPN.

 Fragments of 0.6 TPN and 0.35 TPN were digested with Ndel / BamHI restriction endonucleases and cloned into the Ndel / BamHI digested pET17B expression vector (available from Novagen). A 0.6 kb DNA fragment encodes the complete gp55 protein (188 amino acid residues — see sequence No. 1), while a 0.35 kb DNA fragment encodes the 12 kD amino-terminal gp55 protein fragment (112 amino acid residues — see sequence N 3).

 Example 307. Obtaining and cloning DNA encoding hPTG (1-38) OH.

 A DNA fragment encoding a) a linker peptide corresponding to amino acid residues 110-115 of sequence N 1 and hPTG (1-38), or b) a linker peptide corresponding to amino acid residues 110-115 of sequence N 3 and hPTG (1-38), get by PCR using a cloned synthetic sequence encoding PTH as a template (1-38). As the primer located upstream, oligonucleotides encoding linker a) (sequence N 8) or encoding linker b) (sequence N 9) are used. The downstream primer is represented as sequence N 10. Each of the upstream primers includes a BamHI cloning site, and the downstream primer contains an XhoI recognition site (nucleotides 5-10 of each sequence). PCR is carried out under the same conditions as described in example 306.

 After digestion with BamHI and XhoI, a 0.23 kb PCR product was introduced into the BamHI-XhoI digest of the gp55-pER17B plasmid obtained in Example 306.

For expression of the composite proteins, the E. coli BL21 (DE3) LysE strain is transformed with the obtained plasmid constructs, after which individual bacterial colonies are propagated. The expression of the composite proteins is obtained by incubating the primary culture in a medium for cyclic growth (Bio 101) on a rotary shaker overnight at 37 ^o C. The main culture is inoculated with the specified primary culture at the rate of 1-5% of the volume of the main culture. After that, the main culture is fermented at a temperature of 37 ^o C and a pH in the range from 6.9 to 7.1 under aeration conditions with an intensity of 10 l / min and stirring at a speed of 200-400 rpm. Expression is induced by adding 1 mM IPTG at the moment when the culture reaches an OD ₅₅₀ value of approximately 1.0-5.0. After expression induction, fermentation is continued for five hours, and then the fermentation mass is collected without killing E. coli cells. The harvested cells are pelleted in a centrifuge, resuspended in a buffer sample, and examined for expression using SDS PAGE. Then, the cell pellet is frozen until the purification step. The procedures used and the host cells are fully described [18].

 Example 308. Isolation of particles of inclusions.

Frozen Precipitation E.coli. obtained in Example 307, resuspended to obtain a 25% (weight / volume) suspension in Buffer A (50 mm Tris pH 8.0; containing 2 mm DTT, 5 mm benzamidine-HCl, 1.5 mm MgCl ₂ , 1, 0 mm MnCl ₂ , 10 μg / ml DNase 1 and 2 mg / ml lysozyme), which is stirred for 1 hour at room temperature. Resuspended cells are lysed by passing them through a Manton-Golin homogenizer (2 times at 1200 bar), and the resulting lysate is stored on ice. The specified lysate was diluted 2 times with ice-cold buffer B (50 mM Tris pH 8.0; containing 2 mM DTT, 5 mM benzamidine-HCl and 4 mM EDTA) and centrifuged for 30 min at 27500 g.

The resulting supernatant was carefully removed from the centrifuge and discarded. The precipitate was resuspended in buffer B to obtain a 25% (w / v) suspension, again passed through a Manton-Golin homogenizer and centrifuged, as in the previous case. The indicated process is repeated using buffer B once and water once. The precipitates of inclusion particles thus obtained are weighed and frozen at -20 ^° C until use.

 Example 309. Dissolution and cleavage of inclusion particles containing gp55-Asp-Pro-Pro- (1-38) hPTG-OH.

Obtained in example 308, frozen inclusion particles containing gp55-Asp-Pro-Pro- (1-38) hPTG were dissolved in 10 mM HCl acid solution (analytical grade) to a final concentration of 6% (w / v). The resulting solution was incubated for 24 hours at 50 ^o C, after which the reaction was stopped by adding 1 volume of an aqueous solution of 100 mm NaOAc. The diluted supernatant is purified by centrifugation at 27500 g for 30 minutes and filtered through a glass fiber filter.

 Example 310. Dissolution and cleavage of inclusion particles containing gp55-Asnp-Gly-Pro- (1-38) gPTG-OH.

The frozen inclusion particles isolated in Example 308 containing gp55-Asp-Gly-Pro- (1-38) hPTG were dissolved in 6 M guanidine hydrochloride (containing 2 M hydroxylamine hydrochloride and adjusted to pH 9.0 by adding 4, 5 M LiOH) to give a solution with an approximate protein concentration of 5 mg / ml. By adding an additional amount of LiOH, the solution was again adjusted to pH 9 and incubated for 4 hours at 45 ^° C. The reaction was stopped by the addition of formic acid to pH 4. The solution was centrifuged and filtered as described in Example 309.

 Example 311. Preparative HPLC Cly-Pro and Pro-Pro (1-38) gPTG.

 Filtered supernatants resulting from the cleavage reaction are examined using analytical reverse phase HPLC (Orpogen HD-Gel-RP-7s-300: 4 x 150 column) and compared with the known standard (1-38) PTH (Bachem) to determine the concentration of PTH . The indicated column is titrated with 85% HPLC buffer A (90% water, 10% acetonitrile; contains 0.1 volume percent TFA) and 15% HPLC buffer B (10% water, 90% acetonitrile; contains 0.1 volume percent TFA) and elute with a gradient from 15% HPLC buffer B to 100% HPLC buffer B for 15 minutes at a flow rate of 0.75 ml / min.

 Depending on the scale of isolation, supernatants are applied either to a 1.0 x 25 cm C4 Vydac column or to a 2.2 x 25 cm C4 Vydac column. These columns are titrated with 85% HPLC buffer A and 15% HPLC buffer B at a flow rate of 4 ml / min and 10 ml / min, respectively. Columns elute with more than 80 ml gradient from 15% to 85% buffer B and more than 300 ml gradient from 15% to 55% buffer B, respectively.

 Peaks of Cly-Pro-hPTG (1-38) or Pro-Pro-hPTG (1-38) are collected manually, after which acetonitrile is removed in vacuo. In order to determine the concentration of the peptide both after cleavage and in the collected fractions, analytical HPLC was used. Solutions of the free peptide in a solvent are diluted with an equal volume of 100 mM sodium acetate, and if necessary, the pH is adjusted to 5.4. After filtration (cell size 0.2 μm), the solution was applied to a cation exchange column (either Mono S or SP-Sepharose high resolution packed in HR10 / 10 or XK16 / 10 columns, respectively), titrated with 50 mM sodium acetate pH 5, 4 (buffer B). The specified column is eluted with a gradient from 0 to 500 mM NaCl in buffer B (more than 7.5 column volumes). Fractions of 10 ml are collected; peak X-X- (1-38) hPTG is combined. The concentration of the peptide is determined using HPLC in accordance with the above description.

 This column serves both for the exchange of peptides in a more physiological buffer, and for the removal of side peptides resulting from the chemical cleavage of the compound protein itself.

 Example 312. Enzymatic removal of X-X from X-X- (1-38) hPTH-OH.

The combined peptide fractions obtained in Example 311 (concentration between 1.5-2.0 mg / ml) were adjusted to pH 8.0 by the addition of solid Tris. Purified X-prolyl dipeptidase (0.5 mg / ml in PBS pH 7.2) was added [19] in a ratio of 1: 1000 and the solution was incubated for 24 hours at 37 ^° C. The reaction was stopped by adding TFA to a concentration of 0.2% (pH 5.0), and the resulting PTH (1-38) is isolated using preparative HPLC (use the same conditions as described in Example 6). The solvent was removed and the resulting product lyophilized after removal of aliquots corresponding to the N-terminal sequence and mass spectroscopic analyzes.

Examples of yields obtained using the two approaches described above based on the use of compound proteins are as follows:
Gene 55-Asp-Pro-Pro-gPTG (1-38) OH (using the “shortened” form of gene 55)
Under conditions of partially optimized fermentation, a crude cell pellet weighing 215 g is obtained from 10 liters of fermentation mass. The percentage value of the expression of said composite protein is approximately 37%. 31 g of inclusion particles are isolated from the obtained precipitate. The protein purity (in relation to the composite protein) in these particles is equal to 61%. After dissolution, digestion, and centrifugation / filtration, approximately 620 mg of Pro-Pro-PTH was detected. This substance gives 580 mg of Pro-Pro-PTH as a result of subsequent HPLC on a C4-Vydac column. After dilution, exchange of cations for Mono S, cleavage with Chprolyl dipeptidase (at a concentration of Pro-Pro-PTH equal to 2 mg / ml), HPLC and lyophilization, 440 mg of lyophilized pure product with full biological activity are obtained. This corresponds to a 71% yield in the step from stopping the cleavage to isolating the final product.

Gene 55-Asn-Gly-Pro-gPTG (1-38) OH (using the "long" form Gene 55)
78 g of crude cell pellet, characterized by 50% expression of the indicated fusion protein, are isolated from a 20 L non-optimized fermentation mass. After lysing and centrifugation, 18 g of particles of inclusions of "90% purity" are obtained. At the completion of the acid stop step of the reaction, as well as centrifugation / filtration, approximately 470 mg of Gly-Pro-PTH is detected. After the HPLC step, 355 mg of the indicated peptide was detected on a C4-Vydac column, of which 321 mg remained after exchange of cations on SP-Sepharose. Enzymatic removal of Gly and Pro residues is carried out at a concentration of 2 mg / ml. After the final HPLC and lyophilization step, a fully biologically active gPTG (1-38) OH is obtained, which corresponds to a 52% yield from the moment the cleavage reaction is stopped.

 The compounds of examples 25, 26, 56 and 86-164 are also obtained using the method described in Examples 306-312.

 The compounds of the present invention, which are in free form or in the form of pharmaceutically acceptable salts or complex compounds, have significant pharmacological properties, as demonstrated in animal tests, and therefore these compounds are recommended for therapeutic use.

The biological activity of the compounds of the invention is shown in vitro by measuring their ability to stimulate the synthesis of cyclic AMP in rat osteosarcoma UMR 106-06 and human SaOS-2 cells according to the method of Marcus and Aurbach [20]. UMR 106 rat osteosarcoma cells are grown to obtain a continuous layer in the minimum required medium. Needle: 10% FCS in 12-well plates; human SaOS-2 cells of osteosarcoma grown in McCoy 5A medium: 10% FCS. Then, this medium was replaced with 2% FCS and 1-5 μCi / well of [3H] adenine was added. After two hours, the cells were washed twice with serum-free medium and incubated in DMEM: 1% BSA containing 1 mM 3-isobutyl-1-methyl-xanthine to prevent the action of phosphodiesterases. A test compound is added 20 minutes later. After 15 minutes, the reaction was stopped by adding glacial 5% acetic acid and cAMP was extracted. A carrier solution (0.5 ml / well) containing 5 mM unlabeled adenine, adenosine, AMP, ADP, ATP and cAMP, as well as 0.4 μCi [14C] adenosine is added to detect its utilization. [3H] cAMP is isolated using a Dowex 50W-X4 (200-400) serial sieve and chromatography on alumina, and its quantity is also calculated. The results are calculated as% of control using solvent and EC ₅₀ values determined from DRC curves. Compounds provided by the present invention stimulate the formation of cAMP in the cells of rat osteosarcoma UMR 106-06 and human SaOS-2, at a concentration of from 10 ^-11 to 10 ^-6 M.

Compounds provided by the present invention also have the ability to specifically bind to PTH receptors, including the following:
Using the lactoperoxidase method (Anawa Lab. AG, Wangen) [Tyr ³⁶ ] PTGrP (1-36), the chicken amide is iodinated to obtain a specific activity of 2200 Ci / mM. The monolayers of opossum kidney cells are washed with 200 μl of a mixture of DMEM and HAM F12 (1: 1 ratio) containing 1% BSA and incubated at 16 ^° C with [ ¹²⁵ I-Tyr ³⁶ ] xPTGrP (1-36) amide (activity 50,000 cpm per well) in the presence or absence of 1 μM [Tyr ³⁶ ] xPTGrP (1-36) amide. After incubation, the cells are washed with 0.5 ml of medium (4 ^o C), lysed with 0.5 ml of 1 N. NaOH and determine the radioactivity. Specific binding is found as the difference between total binding and non-specific binding. Competitive interaction curves are analyzed using SCTFIT, a nonlinear regression computer program [21] and present data in the form of mean pK _D values (n = 2 or 3). In this test, the compounds provided by the present invention exhibit specific binding ability, expressed as a mean pK _D ranging from 7 to 10.

 In addition, the compounds of the present invention increase plasma calcium levels after prolonged infusion of s.c., for example, as shown in male Whistar rats weighing 140-170 g and undergoing a thyroparatiroidectomy. 5 days after tyroparathyroidectomy, Alceta minipumps are implanted in the neck of rats and test compounds are infused at a rate of 0.3 to 30 μg / day / rat. After that, on the morning of the 1st, 2nd, 3rd and 6th day, blood is taken using retro-orbital puncture and plasma concentration is determined by photometry. Compounds provided by the present invention increase plasma calcium levels in said test.

 In accordance with this, the compounds provided by the present invention are recommended for the treatment of all bone diseases accompanied by progressive loss or resorption of calcium, or diseases in which it is desirable to fix calcium in the bone, for example, osteoporosis of various origins (including juvenile, menopausal, postmenopausal, post-traumatic, caused by old age, or corticosteroid therapy or inactivation), fractions, osteopathy, including acute and chronic stages associated with demine the implementation of the skeleton, softening of the bones, rarefaction of the periodontal bone or rarefaction of the bone in connection with arthritis or osteoarthritis, or for the treatment of hypoparathyroidism.

 Compounds provided by the present invention are particularly recommended for the prevention or treatment of osteoporosis of various origins.

 For all of the above uses, the recommended daily dose is in the range of about 0.003 mg to about 10 mg, preferably in the range of 0.003-3, more preferably in the range of 0.01-1 mg of the compound provided by the present invention. Compounds provided by the present invention can be used once a day or up to two times a week.

 Compounds provided by the present invention can be used in free form or in the form of a pharmaceutically acceptable salt or complex compounds. These salts and complex compounds can be obtained in the traditional way and exhibit the same degree of activity as the corresponding free compounds. The present invention also provides a pharmaceutical composition comprising a compound provided by the present invention in the form of a free base or in the form of a pharmaceutically acceptable salt or in the form of a complex compound, accompanied by a pharmaceutically acceptable diluent or carrier. These formulations can be used in a conventional manner. The compounds provided by the present invention can be applied by any conventional method, for example, parenterally, including in the form of injectable solutions and suspensions, enteral, including orally, for example, in the form of tablets or capsules, or in the form used subcutaneously through the nose or as a suppository.

In accordance with the foregoing, the present invention also covers:
a) a compound of the invention, or a pharmaceutically acceptable salt or complex thereof, for use as a pharmaceutical agent;
b) a method for improving bone formation, for example, preventing or treating all bone diseases accompanied by progressive loss or resorption of calcium, or diseases in which it is desirable to fix calcium in the bone, for example, osteoporosis of various origins (including juvenile, menopausal, post-menopausal , post-traumatic, caused by old age, or corticosteroid therapy or inactivation), fractions, osteopathy, including acute and chronic stages associated with demineralization of the skeleton, stretching bone formation, rarefaction of periodontal bone, or bone resection due to arthritis or osteoarthritis, or for the treatment of hypoparathyroidism in a patient in need of such treatment, the method comprising administering to the aforementioned patient an effective amount of a compound provided by the present invention or a pharmaceutically acceptable salt or complex compound thereof .

 c) the compound provided for by the present invention, or a pharmaceutically acceptable salt or complex compound thereof, intended for use in order to obtain a pharmaceutical composition intended for use in accordance with the above method described in paragraph b).

 In accordance with another embodiment of the present invention, the compounds provided by the present invention can be used as additional or auxiliary agents in other types of therapy, including therapy using bone resorption inhibitors, for example, in the treatment of osteoporosis, especially in therapy, based on the use of calcium, calcitonin, or an analogue or derivative thereof, including salmon, eel or human calcitonin, steroid hormones, including estrogen, a partial estrogen agonist a gene or a combination of estrogen-progestogen, bisphosphonate, vitamin D or its analogue, or any combination thereof.

 In the case where the compounds provided by the present invention are used in combination with, for example, an auxiliary agent in therapy using a bone resorption inhibitor, the dosages of the indicated compound used should certainly vary depending on the type of inhibitory medication used, including whether it is a steroid or calcitonin, on the conditions in which it should be used, on whether there is a therapeutic or prophylactic therapy, on the regimen of administration, etc.

In accordance with the above, the present invention provides another aspect:
g) a method for improving bone formation, including the prevention or treatment of calcium loss, for example, for the prevention or treatment of any of the specific conditions or diseases mentioned above in a patient in need of such treatment, the method comprising the use of an effective amount of said patient
a) provided for by the present invention compounds and
b) a secondary drug agent, wherein said secondary drug agent is an inhibitor of bone resorption, for example, as described above. Preferred are the compounds of examples 20, 29, 37, 49 and 50.

 A sequence listing is provided at the end of the text.

List of references
1. USPT.O. publication, Trademark Official Gazette, May 15, 1990, p. 33, at 46.

 2. Protective Groups in Organic Synthesis. T.W. Greene, J. Wiley & Sons, NY (1981), 219-287.

 3. The Peptides, 3 (1981), E. Gross and J. Meienhofer (Ed.).

 4. Stewart J. M., Protection of Hydroxy Groups. In "The Peptides", 3 (1981). E. Gross and J. Meienhofer (Ed.)., 169-201.

 5. Roeske R. W., Carboxy Protecting Groups, In "The Peptides", 3 (1981), E. Gross and J. Meienhofer (Ed.)., 101-135.

 6. Hiskey R. G., Sulfhydryl Group Protection, In "The Peptides", 3 (1981), E. Gross and J. Meienhofer (Ed.)., 137-167.

 7. Gram H. and Ruger R., The EMBO Journal, 1985, 4, (1): 257-264.

 8. Nardi et al., Applied and Environmental Microbiology, 1991, 57: 45-50.

 9. International Journal of Peptide and Protein Research. 1985, 26: 262-273.

 10. Tetrah. Letters, 1987, 28: 3787-3790.

 11. Valle et al., Can. J. Chem., 1988, 66: 2575-2582.

 12. Crisma et al., Int. J. Biol. Macromol., 1989, 11: 345-352.

 13. Evans et al., Org. Synth., 1990, 68.

 15. S.-O. Lawesson et al., Tetrahedron, 1981, 37: 3635.

 16. F.S. Guziec et al., THL, 1990, 23-26.

 17. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1991.

 18. Studier et al. , Use of T7 RNA Polymerase to direct Expression of Cloned Genes, Methods of Enzymology, Academic Press, 1990: 60-89.

 19. Nardi et al., App. Env. Microbiol., 1991, 57, N 1: 45-50.

 20. Endocrinology, 1969, 85: 801-810.

 21. Feyen et al., Biochem. Biophys. Res. Commun., 1992, 187: 8-13.

Claims (6)

1. The compound selected from the group including
[Leu ⁸ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH,
[Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH,
[Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH,
[Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Gln ¹⁸ ] hPTH (1-36) OH,
[Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH,
[Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ ] hPTH (1-36) OH,
[Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ , Thr ³³ , Ala ³⁴ ] hPTH (1-34) OH,
[Leu ⁸ , Asp ¹⁰ , Lys ¹¹ , Ala ¹⁶ , Gln ¹⁸ ] hPTH (1-36) OH and
[Leu ⁸ , Ala ¹⁶ , Gln ¹⁸ , Ala ¹⁹ ] hPTH (1-36) OH.  2. A DNA fragment encoding a fusion protein having a nucleotide sequence corresponding to the N-terminal polypeptide encoded by the bacteriophage T4 gene 55, and the compound according to claim 1 attached to its C-terminus.  3. A fusion protein, which is an N-terminal polypeptide encoded by the T4 bacteriophage gene 55, and the compound attached to its C-terminus according to claim 1, and, if necessary, a chemically cleavable linker between them.  4. The protein of claim 3, wherein the linker has the following amino acid sequence: Asp-Pro-Pro or Asn-Gly-Pro.  5. The method of obtaining the compound according to claim 1, which consists in culturing a recombinant E. coli strain transformed with an expression vector containing a DNA fragment according to claim 2, inclusion particles are isolated, dissolved under acidic conditions, they are cleaved, separated and the target is purified compound.  6. A pharmaceutical composition that regulates calcium metabolism, containing the active ingredient and a pharmaceutically acceptable diluent or carrier, characterized in that as the active ingredient it contains an effective amount of a compound according to claim 1.

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