JPH05506250A - Inhibition of endothelin formation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The present invention provides a peptide capable of binding to human big-endothelin (bitET). This invention relates to compounds, their production, and pharmaceutical compositions containing them.

Endothel in (ET) is a 21-amino acid with high vasoconstrictor activity. Acidic mammalian hormones.

The presence of RN^, which encodes a large precursor of ET, in endothelial ahvi suggests that This peptide, like the precursor of many biologically relevant peptides or hormones, A 38-amino acid fragment named big-endothelin Enzymatic proteolysis of the 212 amino acid protein that forms the a et al.

Nature 332. p, 411-415J988) It shows.

The present invention provides an endothelin enzyme that is useful for the treatment of diseases that depend on endothelial action. It is a peptide that has othelin formation inhibiting activity.

The present invention can bind to human big endothelin, and has the formula ■: Ax A 4-As　l'-'<　A'x　I (wherein A and A'3 are independently S er, Gly, ^sp or ^Sn! represents the group, A and A't independently represent Vat, Pro, cry or ^1a residues. death, A, represents a ^sp or ^sn residue). Provides amino acid peptides.

The enantiomers of these peptides, which all have D-form optically active amino acid residues, are , are included within the scope of the present invention.

Salts of the peptides of the invention with pharmaceutically acceptable acids or bases are also within the scope of the invention. included.

Such acid addition salts include various inorganic or organic acids (e.g. sulfuric acid, phosphoric acid, hydrochloric acid). , Hydrogen acid, Iodide, Nitric acid, Sulfamic acid, Citric acid, Lactic acid, Pyruvate Acid, oxalic acid, maleic acid, succinic acid, tartaric acid, cinnamic acid, acetic acid, trifluoroacetic acid, amino acid derived from zozoic acid, salicylic acid, gluconic acid, ascorbic acid and related acids). can be guided. Such base addition salts include one of various inorganic or organic bases (e.g. hydroxyl Sodium hydroxide, potassium hydroxide, diethylamine, triethylamine and dihydroxide lohexylamine).

Amino acid residues are represented by a three letter code (Eur.

J. Bioebem, 138.9-37.1984>.

Seafence of formula I is a non-covalent compound of the 212 amino acid endothelial in precursor. strain to DH and Kyte and DoolitLle et al. (J, Mol.

Biol, 157.105-132.1982) The zero peptide derived from the hydropatic map of helin is usually , 0 peptides containing at least 5 amino acid residues are peptides containing 20 or less amino acids. residues, eg, up to 18 amino acid residues.

This peptide has the formula ■: X-^, -^, -^, -^, -^, 0-^, -^2-8, -^4-^5-Δ'4 −＾°, −＾゛2−＾′, −＾′1゜−＾′, −＾゛, −八戟A−＾′, W　 I [ (In the formula, A1, A4, A5.A'4 and A'3 are as defined above, and represents a hydrogen atom or a nitrogen protecting group, A and A'l are 5 independently Vat, L represents a eu, Cys or Ile residue or a valence bond, A2 and A'2 are independently Gl, ^1a or ^SO residues or valence represents a bond, A and A'a independently represent Val or a valence bond, and A and A'a independently represent Val or a valence bond; 't is independently ^rg, ^Ia-(Iy, Cys-Glu or Lys residue) represents a group or a valence bond, A- and A''g are independently ^sn-Pro , II; Iy, ^Ia represents a residue or valence bond, A9 and A'9 are independently ^sn, ^Sp, Val, Leu, Met residues or or represents a valence bond, Ate and A', 6 are independently Val-Lsu, Net-1 (is-G lu residue or valence bond, and W represents a hydroxy group or an amino group. ) is preferable.

This peptide has the formula ■ or ■: X-^v-Asn-^, -^, -^, -^2-^, -Vi1-^, -^゛4-^ '=-Asp-A'1-^',. −＾′、−＾′、−＾′　A-Vil-W　n r X-Val-8゛, -＾゛6-＾°, -A',. -＾゛　1-8sp-＾′、- ^゛4-^5-Val-^, -^2-^1-^1゜-^, -■Tang■|^t-w　 W (In the formula, A represents Val, Leu or Cys residue, A'+ is Va 1 or Ile residue, A2 represents Gly or ^1a residue, A3 is Represents Ser or Gly residue, A'3 represents Asp or Asn residue , A'4 is Pro, Gly or ^Ia! g group, As is Asp or Represents an Asn residue, and A represents ^'g, ^Ia-' represents a ly or Cys residue. , A”7 represents a Glu or Lys residue, A’s is Pro, Gly or represents the ^1a residue, A represents Asn or ^sp residue, and A' represents Val , represents a Leu or HeLW group, and Ate represents a Val, Leu or Met residue. A'16 represents a His or Glu residue, and X represents a hydrogen atom or or a nitrogen protecting group, W represents a hydroxy group or an amino group) or It is more preferable to have its enantiomer which has all D-form optically active amino acid residues. preferable.

Preferred peptides are Seafence: H-＾rg-Asn-Aso-Va I-Leu-G Iy-5er- Va I -Asp-Pro-Aso-Asp-Va l |H1s-Va I -^1a-Glu-Val-OH (As bigl) and HG　y-Asn-Asn-14et-Leu-G　y-Ser-V a　I　-Asn-Pro-Asp-Asp-11e-G h　u-Net　- It has Pro-Lye-Va I-OH (8s bit2).

The peptide synthesis of the present invention can be performed by conventional solution methods or solid phase methods on polymeric supports. It can be implemented. Traditional solution synthesis methods essentially require a protected amino acid or Consists of a suitable sequential condensation of peptides. The condensation is performed so that the resulting peptide has 5 to 20 This is done to have the desired sequence of amino acid residues. Publicly recognized in peptide chemistry Amino acids and peptides fused by known methods can be blocked with suitable protecting groups. It has an amino group and a carboxy group that do not participate in the formation of a peptide bond. Hi The hydroxy functionality of the droxyamino acid (during the entire synthesis or at several steps) can be protected by a suitable protecting group or left unprotected. It can be done.

Protecting groups may be removed by hydrolysis, saponification or hydrogenolysis. Protects amino groups For example, the following protecting groups: benzyloxycarbonyl, t-butoxycarbonyl rubonyl, trityl, formyl, trifluoroacetyl, O-nitrophenyl Ruphenyl, 4-methoxybenzyloxycarbonyl, 9-fluorenylmethoxy cyclocarbonyl or 3,5-dimethoxy-α,α゛-dimethylbenzyloxycarbonyl Rubonyl may be used.

When protecting a carboxy group, for example, the following protecting groups: methyl, ethyl, t-butyl Chil, benzyl or p-nitro-benzyl can be used.

When protecting a hydroxy group, for example, the following protecting groups: acetyl, t-butoxy Cycarbonyl, benzyloxycarbonyl, 2-bromobenzyloxycarbonyl tetrahydropyranyl, t-butyl, trityl, benzyl or 2,4-di Chlorobenzyl can be used.

Amino group on one molecule and carboxyl group on the other molecule to form a peptide bond Condensation between activated acyl derivatives (e.g. mixed acid anhydrides, azides or or activated esters) or alone or through racemization inhibitors (e.g. N-hydroxysuccinimide or 1-hydroxybenzotriazole) and - In the presence of a condensing agent (e.g. dicyclohexyl lupodiimidone), the free amino acid is This can be carried out by direct condensation of the group with the free carboxyl group.

The hydrazides or substituted hydrazide derivatives of the present invention are suitable for N-protected peptides or hydrazides. amino acids and suitably substituted hydrazides (e.g. pendylcarbazate, Tylcarbazate, adamantylcarbazate, phenylhydrazine or adamantylcarbazate damantylhydrazine) or by condensation with protected peptides or amino acids hydrazide and a suitable alkylating agent (e.g. benzyl chloroformate, t-butyl Tyl chloroformate, di-t-butyl dicarbonate or adamantyl fluoroformate).

The condensation is performed in a solvent (e.g. dimethylformamide, pyridine, acetonitrile, tetra 0 reaction temperature, which can be carried out in (trahydrofuran or N-methyl-2-pyrrolidone) The temperature may be -30°C to room temperature.The reaction time is usually 1 to 120 hours. .

The synthetic scheme, protecting groups and condensing agents are chosen to avoid the risk of racemization.

D-protection reactions are carried out by methods known in polypeptide chemistry.

In the phase method, a polymeric support is used. Polymers are compatible with most organic solvents. Diviny as a crosslinking agent to make polystyrene polymers completely insoluble A copolymer of rubenzene 1 to 2 wt 2 and styrene is preferred. Amino acid, chloromethylated resin, hydroxymethyl resin or benzhydride Start from the C-terminus of the peptide by coupling to the lylamine resin. a The mino and copper protecting groups are described in conventional solution methods for the preparation of the compounds of the present invention. In this case, the amine-protected amino acid is treated with a condensing agent (e.g., dicyclohexyl carbodi imide) and bound to a chloromethylated resin by a cesium salt, or or coupled to hydroxymethyl or benzhydrylamine resins. first After coupling of Removal by selecting reagents containing hydrogen chloride solution.

After removing the amino protecting group, the remaining protected amino acids can be combined as desired to obtain the desired peptide. Combine in stages in this order. Each protected amino acid is A suitable carboxy group activated 荊( For example, dicyclohexylcarbodiimide) is used in a 3-fold excess.

After completing the desired amino acid sequence, we not only cleave the peptide from the resin. , treatment with a reagent (e.g., hydrogen fluoride) that cleaves most of the remaining side chain protecting groups. The peptide is removed from the resin support by. Chloromethylation or Hydroxylation When using methyl resin, free peptide # (W = 01l) is formed. When using benzhydrylamine resin, hydrogen fluoride Treatment directly forms the free peptide amide (W=NH2). Or, Ptideamides can also be obtained from peptide acids by amitrilysis.

The compounds of the invention are of interest for inhibiting the maturation process of eadothglin. Has pharmaceutical activity.

Several proteolytic enzymes are capable of converting bigET to ET in vitro. is known (R, Tkegawa et al., BBRC167: 860-866 1990).

More specifically, α-chymotriazine is a neutral DH and human 38 It is known to cleave the residue εT precursor (brgET　1.38〉(E,G , HcMahon et al., BBRC161:406-4141989).

Inhibition of bigET cleavage uses α-chymotrypsin as a protease This was determined by "in vitro" tests.

Under our experimental conditions, during the first 5 minutes of the reaction, bigET is completely converted to big is converted to ET(1-31) and then cleaved at Trp(21) to form ET(1-21). > was formed. This second fragmentation is very slow in the presence of the peptides of the invention. Ru.

ET (1-21>, bitET (1-38) and bigET (1-31>) The fence has the following formula: %formula% α-chymotriazine (0,014zy/x1>) was added to glycine buffer (25mM> The cells were incubated with synthetic rgET (0.2 x g/xi). Enzyme/big The ET molar ratio was 11,500.

Proteolysis of bigET was followed as a function of time at 37°C, 9) 17.1 . 10ul of the initial reaction mixture was incubated at 0.45.60.100. 150.180.After 255 minutes, the RP-11P [, C column (SuperPacP epS, 4X250zz, Pharmacia).

Acetonitrile/water<0.1$ TF^) gradient solution (acetonitrile 102 l80 It was evaluated based on the yield.

Similarly, proteolysis of the substrate was performed using bigET/Peptide in the presence of peptide of formula 1. Table 1).

(a) Substrate (bigET) and inhibitor at a constant molar ratio of 1/1 (23.3 uM) It was mixed with Incubation with chymotrypsin (pH 7, 1, 37°C) was stopped with guanidine hydrochloride (414) and the mixture was transferred to RP-1 (PLC column). (see above).

(b) P/S = Product (ET>/Substrate (bitET 1-31>) corresponds to the value The standard difference in P/S value calculated as the area ratio of HPLC peak is 9z It was estimated that less than

The proteolysis of bigET was carried out at an enzyme/biirET molar ratio of 1/1000 (37°C).

p117.1>, peptide/bi at 120 and 180 min of incubation. (Table 2) as a function of gET ratio.

Table 2: (a) (a) Substrate of Asbi 2/bi ET and Uri iE′r and inhibitors at various inhibitor to substrate molar ratios, α-chymotrypsin and 12 The enzyme was incubated with guanidine for 0 or 180 min (pH 7, 1, 37°C). hydrochloride (4M) and the mixture was loaded onto a RP-IIPLC column (as described above). (see), big): Keep the T concentration constant equal to 23.3 uN.

(b) P/S = product (ET) to substrate (bitET 1-31> ratio corresponds to ['obtained from LC area. The standard deviation in P/S is generally less than 9z. calculated.

A P/S value of 4 oz was reached in 100 minutes in the blank incubation. ^ In the presence of Sb1g1 (1/1.substrate/peptide molar ratio), the same P/S ratio has It took 150 minutes. ^In the presence of Sb1g2, these values It was not reached even after 250 minutes of treatment (Table 1). These data are bigE It represents the kinetic retardation of proteolysis of T(1-31>).

At the end of the day, a certain incubation time between bigET and sbig2 (i.e. 1 From the titration experiments (Table 2) at 20 min and 180 min), a constant P/S value is It is reached when the molar ratio of igET/^sbig2 is 1/2 or less.

The peptide of the present invention comprises an internal segment of bitET (1-38>, i.e. -13 -30) and inhibits the formation of ET(1-21>. Considering this, , the first proteolytic step that cleaves the terminal segment bigET(32-38) is irrelevant here as far as ET shadow formation is concerned.

Peptides of formula I are inhibitors of eodothelin formation and therefore can be used directly or or in cooperation with other factors, the treatment of human diseases caused by endothelin. It was briefly concluded that the method could also be applied to medical treatment.

Furthermore, these peptides can also be used to treat clinical symptoms associated with diseases such as hypertension. Can be used.

The compounds of the invention can be administered by the usual routes, such as intravenous injection or infusion, or intramuscularly. It may be administered parenterally by subcutaneous, intracavitary and intranasal administration.

The dosage depends on the age, weight and condition of the patient and the route of administration.

Therapeutic doses in humans range from 10 ng/kg to 10 my/kg 1 to 6 times a day. It is thought that the

The present invention provides as an active substance the formula ( Pharmaceutical compositions comprising compounds of I) are also provided.

The pharmaceutical composition of the present invention is manufactured according to a conventional method and administered in a pharmaceutically suitable form. given.

For example, solutions for intravenous injection or infusion may contain, for example, sterile water as a carrier. Alternatively, it may preferably be in the form of a sterile saline solution.

Suspensions or solutions for intramuscular injection contain, together with the active compound, a pharmaceutically acceptable Carriers [e.g. sterile water, olive oil, ethyl oleate, glycols (e.g. propylene glycol)] and, optionally, lidocaine hydrochloride in a suitable amount. may be included.

The following examples illustrate the invention.

UV detection operating high performance liquid chromatography (IIPLc) at -Z15nm It was carried out using a Hewlett-Paekard 1084B apparatus equipped with . Peptide, 4×250zz(1,D,) Nucleosil 300C185μ Separated on column. The following solutions: A) Adjusted to p[12,25 with triethylamine. Adjusted 0, 25N tl, Po, B)) ethylamine/CH2CH = 476 <capacity ratio) 0.258 Il, PO, adjusted to pH 12.25 was used.

This elution procedure was performed with a linear gradient of 15 to 50 B over 35 minutes at a flow rate of 1.5i+ Programmed with l/sin.

Peptides were characterized by their retention time (RT).

Em Construction method 1 price payment Yu U Wataru Wataru Tomo "Kozuer-V"" 5 Hi manifest LVal-Old 5-V Merr if 1eld method (Merrifield, JACS 85, 2149-21 54.1963). The peptide was converted into phenylacetanidomethylate. Synthesized on Les swollen fat. The α-amino protecting group on each amino acid is It was a carbonyl group (BoC).

Each binding cycle was as follows.

1. Wash the puffed fat with dichloromethane for 10 minutes.

2. Wash 3 times for 2 minutes with 5z diisopropylethylamine in DMF/CH, C1. Purify.

3. Dichloromethane washing - 1 minute x 2 4, CH, 3 equivalents of amino acid in C12 and 0.3M diisopropylcarbodiyl Combine with Mido in 60 minutes.

Same as 5.3.

6. Deprotect with 50z trifluoroacetic acid in dichloromethane for 20 minutes.

7. Dichloromethane washing - 1 minute x 6 times.

Return to 8.2.

At the completion of the binding cycle, the peptide was fluorinated with 10 g of anisole scavenger for 1 hour. Cleaved from the resin using hydrogen.

The peptide was washed with ether, dried, dissolved in 151 acetic acid and lyophilized. Ta. RT=27.9 minutes.

Yoshi λ engineering The following peptides were produced by operating as described in Example 1.

a) H-Gly-^5n-kn-Net-I, eu-GIy-Ser-VaI -Asn-Pro-Asp-Asp-11e-GIu-Ne soot|Pro-Lys -Val　-OH (Asbig2) RT=20.7 minutes b) H-^1a-^5o-Asr+-Leu-Vat-^1a-5er-Val -Aso-GIy-Asp-Asp-VaI-flis-keu-Pro-G lu-Val -0HRT=27.8 minutes c) H-Cys-Gly-5er-Val-Asn-Pro-Asp-As Iu-Net-Pro-Lys-Vat-Og on p-11e- RT=21.3 minutes d) H-Cys-Gly-5er-Vat-^5n-Pro-8sp-85 g to 0RR = 15.9 minutes e) FIIoc-5er-Vil-Asn-Pro-Asp-Asp-G ly-Lys-0■RT=18. powder □ ! car (wherein A and A' are independently 5er-Gly, Asp or ^sn residues represents, A4 and A°i independently represent Vat, Pro, Gly or ^1a residues, A, represents an Asp or ^sn residue) human big- It can bind to endotheI. 20 or fewer amino acids pharmaceutically acceptable The peptide, which also provides a salt, is an inhibitor of endotbelin formation and Therefore, we believe that it can be applied to the treatment of human diseases caused by endothelin. I found it.

Claims (9)

[Claims] 1. capable of binding to human big endothelin and having the formula I: A3-A4-A 5-A'4-A'3I (wherein A3 and A'3 are independently Ser, Gly, Asp or Asn residues represents, A4 and A'4 independently represent Val, Pro, Gly or Ala residues, A5 represents an Asp or Asn residue). a peptide of amino acids, or its enantiomers in which each amino acid is in the D form, or A pharmaceutically acceptable salt thereof. 2. Formula II: X-A6-A7-A8-A9-A10-A1-A2-A3-A4-A5-A'4 -A'3-A'2-A'1-A'10-A'9-A'8-A'7-A'6-WI I (wherein A3, A4, A5, A'4 and A'3 are as defined in claim 1) , X represents a hydrogen atom or a nitrogen protecting group, A1 and A'1 are independently Val , represents a Leu, Cys or He residue or a valence bond, A2 and A'2 are independently Gly, Ala or Asp residues or valence represents a bond, A6 and A'6 independently represent Val or a valence bond, and A7 and A'7 , independently Arg, Ala, Cly, Cys, Glu or Lys residues or , represents a valence bond, and A8 and A'8 independently represent Asn, Pro, Gly, A represents a la residue or valence bond, A9 and A'9 are independently Asn, Asp, Val, Leu, Met residues or represents a valence bond, A10 and A'10 are independently Val, Leu, Met, His, Glu residues or represents a valence bond, and W represents a hydroxy group or an amino group) 2. The peptide according to claim 1, wherein each amino acid residue is in the D form. thiomers, or pharmaceutically acceptable salts thereof. 3. formula: [There is an array] or a pharmaceutically acceptable salt thereof. 4. formula: [There is an array] or a pharmaceutically acceptable salt thereof. 5. The method may include single amino acids and/or preformed pairs of two or more amino acids. The peptides are condensed in the desired order and then, if desired, the terminal carboxy group is converted to an amide. and/or optionally convert the peptide into a pharmaceutically acceptable form. of the peptide according to any one of claims 1 to 4, comprising converting it into a salt of Manufacturing method. 6. Claims 1 to 4, as active ingredient, with a pharmaceutically acceptable diluent or carrier. A medicament comprising the peptide according to any one of the above or a pharmaceutically acceptable salt thereof composition. 7. The method according to claims 1 to 4 for use in a treatment method for the treatment of the human or animal body. A peptide according to any one of the preceding paragraphs or a pharmaceutically acceptable salt thereof. 8. according to claim 7 for use as an inhibitor of endothelin formation. peptide or salt. 9. Peptide or salt according to claim 7 or 8 for use in the treatment of hypertension. .