CS266156B1 - Vasopressin analogs and method of their preparation - Google Patents

Abstract

The invention falls within the field of peptide hormones and their preparation. The substances can be used as drugs, especially for substitution therapy of diabetes insipidus (thirsty dysentery). The advantage of the described substances over the previously known substances lies in a significantly higher and purer efficacy and in a higher chemical stability. Vasopressin analogues of the general formula I X-D-Tyr-Phe-X'-Asn-X"-Pro-D-Arg-Gly-NH2 (I) 1 23456 7 89 are described in which X is a butyric acid residue or a 3-mercaptopropionic acid residue X is Gln, Val or Asn X" is Cys or a ^ alpha-aminobutyric acid residue. The preparation of these substances is also addressed. The substances belong to the group of peptides.

Description

The invention relates to vasopressin analogues with a very high and very specific antidiuretic effect and a method for their preparation. These analogues are characterized by the formulae I

XD~Tyr-Phe-X'~Asn-X-Pro-D-Arg-Gly-NH ₂ 1 23456 7 8 9 in which X is a butyric acid residue or a 3-mercaptopropionic acid residue X' is Gln, Val or Asn ' X is Cys or an alpha-aminobutyric acid residue.

Highly effective and highly specific antidiuretics of the vasopressin type are used in medicine for the substitution therapy of diabetes insipidus (thirsty dysentery), nocturnal enuresis (bedwetting in children), milder forms of hemophilia including von Willebrand syndrome and in clinical diagnostics (Zaoral M.: Int. J. Peptide Protein Res. 25, 561 (1985)). The preparations used so far, primarily [1-3-mercaptopropionic acid, 8-D-arginine] vasopressin (DDAVP), (Zaoral M., Kole J.: šorm.: Collect. Czech. Chern. Commun. 32, 1 250 (1967)) are very effective and have proven themselves well in practice, as can be seen, for example, from the fact that bDAVP is ranked among the 15 most significant preparations produced so far in this century, but they still have some shortcomings. They have a low, but nevertheless quite noticeable effect on blood pressure and on the uterus. These side effects somewhat reduce their therapeutic value. The substances according to Czechoslovak AO 242 062 (Zaoral M., Bláha I., Krchňák V.) do not have the mentioned shortcomings. The substances according to AO are more advantageous than DDAVP from the point of view of medical use. Their disadvantage is only a slightly higher or lower efficacy compared to DDAVP. Considering the magnitude of the antidiuretic effect of DDAVP and the low value of its side effects, the substances according to AO 242 062 represent a significant, but still not such progress that they are clearly interesting from a practical point of view.

It is known that replacing one sulfur group of the disulfide bridge of vasopressins with a methylene group often leads to substances with increased biological effects. This can also increase the efficacy of vasopressin analogues containing an amino acid of configuration D at position 8.

It has now been found that a change in the configuration at position 2 leads to an unexpected increase in the antidiuretic effect (8 to 10-fold) and an increase in the inhibitory effects against side effects, such as effects on blood pressure and on the uterus, in these analogues, i.e. vasopressin carba analogues containing an amino acid of configuration D at position 8. These substances represent a new group of vasopressin analogues with strong agonist-antagonistic effects. Their antidiuretic effect is up to one order of magnitude higher compared to DDAVP and at the same time they are among the most potent known inhibitors of the effects on blood pressure and on the uterus. .

The essence of the present invention are vasopressin analogs according to formula I f---';

XD-Tyr-Phe-X'-Asn-X-Pro-D-Arg-Gly-NH ₂ (I)

2 3 4 5 6 7 89 in which X is a butyric acid residue or a 3-mercaptopropionic acid residue X' is Gln, Val or Asn

X is Cys or an alpha-aminobutyric acid residue.

Furthermore, the invention relates to compounds of general formula II

XD-Tyr-Phe-Gln-Asn-X-Pro-D-Arg-Gly-NH ₂ (II) where X denotes a mercaptopropionic acid residue

X is Cys or an alpha-aminobutyric acid residue of formula III

XD-Tyr-Phe-Gln-Asn-cys-Pro-D-Arg-Gly-NH ₂ , (III)

CS 266 156 Bl where X represents a butyric acid residue, and of formula IV

I ¹ ' ------------- ^- ----------1

XD-Tyr-Phe-Val-Asn-Cys-Pro-D-Arg-Gly-NH ₂ , (IV) kiln X denotes a butyric acid residue. The invention further provides a method for preparing these substances.

The essence of the preparation of vasopressin analogs according to the present invention consists in preparing a linear precursor of the analog of general formula V,

X H-D~Tyr"Phe-X'-Asn-X-Pro-D-Arg/Tos/-Gly-@ (V)

COOR ' where R is tert-butyl or fluorenylmethyl (P) is a polymeric carrier

Tos denotes the residue of p-toluenesulfonic acid and the other abbreviations have the same meaning as in point 1, the protecting group R is cleaved, cyclization is performed between residues 1 and 2, the resulting cyclic precursor analogue is cleaved from the polymer support by ammonolysis, the p-toluenesulfonic residue is cleaved by the action of liquid hydrogen fluoride or by the action of sodium in liquid ammonia and the obtained product is purified.

A linear precursor of the analog according to formula V is prepared by the method of solid-phase peptide synthesis, preferably on a styrene-divinylbenzene copolymer, whereby in the fourth step of the synthesis, N-Boc-S-/tetrbutoxycarbonylpropyl/cysteine or N-Boc-S-/fluorenylmethyloxycarbonylpropyl/cysteine is not condensed to the peptide chain. After the preparation of the linear precursor according to formula V is completed, the Boc protecting group (or Boc and fluorenylmethyl protecting group) is cleaved and the ring closure is performed between the carboxyl group of S-/carboxypropionyl/cysteine and the amino group of tyrosine at position 2. The product is then cleaved from the resin by ammonolysis, the Tos residue protecting the guanidine group of D-arginine is cleaved, preferably by the action of sodium in liquid ammonia or by the action of liquid hydrogen fluoride in the presence of anisole and the obtained product is purified by gel filtration on Sephadex, continuous carrier-free electrophoresis and high-performance liquid chromatography.

The cyclization of the linear vasopressin analog of formula V is carried out in dimethylformamide.

The prepared substances were tested for antidiuretic, pressoric and uterotonic effects and for inhibition of pressoric and uterotonic effects (the tests were carried out essentially according to: Vávra I., Machová A., Krejčí I.: J. Pharmacol. Exp. Ther. 188, 241 (1974); Krejčí I., Kubková B., Vávra I.: Brit. J. Pharmacol. 30, 497 (1967); Munsick RA: Endocrinology 66, 451 (1960)). The obtained results are given in Table 1. From the data given in the table it is evident that the change of configuration in position 2 increases the antidiuretic effect of carbaanalogues 3-6 times and at the same time deepens their inhibitory effects in comparison with other analogues of this type. Furthermore, it is clear that the carba^ ^- analogue (substance I) has much higher efficacy than the corresponding carba $ analogue (substance III).

Table 1

VP AD BP UT I [But ¹ , D-Tyr ² , D-Arg ⁸ j 7.9 pA ₂ 7.2 pA ₂ 7.5 i · 1 2 4 II |But , D-Tyr , Val , D-Arg ⁸ .! III [Mpr ¹ , D-Tyr ² , Abu ⁶ , 10.0 pA ₂ 6 t8 pA ₂ 7.9 D-Arg ⁸ ] 1 to 2 pA ₂ 6.8 pA ₂ 7.8 IV DDAVP 1 0.96 5.1

CS 266 156 Bl

Abbreviations used: VP = vasopressin, AD = antidiuretic effect, BP = pressor effect, UT = uterotonic effect. The magnitude of the antidiuretic effect is expressed in relative values, with the potency of DDAVP =1, But is the butyric acid residue, Mpr is the 3-mercaptopropionic acid residue, Abu is the alpha-aminobutyric acid residue.

Substances I and II have a much higher and more specific antidiuretic effect than DDAVP. The specificity of their antidiuretic effect is not yet fully understood, since both substances are significant inhibitors of the pressor effect, and the specificity of the antidiuretic effect is determined from the ratio of the magnitude of the antidiuretic and pressor effects. Both substances appear to be clearly more advantageous for those indication areas in which DDAVP is still considered the drug of first choice. Another advantage of substances I and II over DDAVP is the increased resistance to attack by proteolytic enzymes and the elimination of chemical lability resulting from the presence of the disulfide bridge in DDAVP. The stability of substances I and II predetermines them for oral administration. The considerable efficacy of both substances in oral administration has been experimentally demonstrated.

The essence of the invention is illustrated and substantiated by the following examples:

Melting points (m.p.) were measured on a Kofler block and are uncorrected. Optical activities were determined on an objective polarimeter. Solid-phase syntheses were performed on an automatic peptide synthesizer. Free peptides were purified by continuous carrier-free electrophoresis and/or high-performance liquid chromatography. The purity of the products was checked by silica gel thin-layer chromatography, paper electrophoresis, analytical high-performance liquid chromatography, amino acid composition analysis and elemental analysis. The biological activity of the prepared substances was tested by the methods listed on page 4. The analog synthesis program is listed in Table 2. The chemical and physicochemical properties of the analogs are listed in Table 3.

Table2 ·

Vasopressin analog synthesis program

Number Reagent Time Number step (minutes) repetition 1 55% ^TFA / _{CH2Cl2 and} 2 1 2 55% ^TFA / _{CH2Cl2 and} 30 1 3 CH ₂ ^c1 2 1 3 4 30% dioxane/Cl^C^ 1 3 5 C ^H 2 ^C 1 ₂ 1 3 6 10 _{% Et3N} / _CH2Cl2 2 1 7 ch ₂ ci ₂ 1 2 8 10 _{% Et3N} / _CH2Cl2 . 2 1 9 ^ch ₂ ci ₂ 1 6 10 Sym. anhydride/ (DMF) 30 to 120° 1 11 _{CH2C12 ​} 1 3 12 20 _% EtOH/ _CH2Cl2 • 1 3 13 ch ₂ ci ₂ 1 3

After Tyr binding, 5% anisole was added.

To a negative ninhydrin test.

CS 266 156 Bl

>1 0 O O what 0 ID Γ- rd ρ 0 σι 0 <T> 0 cn ο < . t 0 , < n rd O A xT CM N 0 Φ 0 <T> σι about '>< M X1 • · rh rh τ—4 Order d β <0 Md β M The M· ch cm x* tp V) rd > ο σ\ σ> σ> 0 σι >1 At 4 1 . · ř· X 0 rd Ο 0 0 rd O in β Ή Φ ο σι 0 ’’In » e C2 ο σ« 0 σι Ch 3 α . • κ . · ω X rM rd Ο rd O < M Md σ» co M ¹ rd CO p WHAT? rd Ο> σ\ about Ch Z άΡ 13, 13. Γθ CM xf in B 'Φ > Q CM red m Vm CM about - βΦ 0 β M φ €*> io ι© m kO 10 5.5 5.2 X. II .μ in cn >υ 0 Ρ > Ν Φ you υ ζ .21 .42 ;Z6 .99 .72 i-35 .n-acs i.5:7 L5:10 0 0 rd what about that *rt 1 ¹ · in m In m X» •red M what O M * 31 . >1 hT* About About •4 α _Λ w About < 10 £ it . 2TF 0 J. 0 CM K Λ «Ο φ 1 Ο ϋ φ M 0 N ω 0 β ω rd m O · (S's ^u o °12 ^S .3) and у 04 ΓΟ · W κ χτ υ > 0 α xf rd About CM rd CO χτ m » m ) CM · Ζ β M From M* <O Z^ CO that Λ « ·Η ítí 0 <#> Ό code IO r-4 to O rd Ch Φ m ·η •d rd ' χ^ -- 33 cm— r- X — B ΓΜ M t0 >4 0 grams X 4> 1 ch ω cT υ U · Ο ΐΛ 1 Ο řd rd Η υ ω οο ΓΟ β β χ > «J «J Ό 00 rd k£> υ +J -ο as 3 4 what in WHAT Ε Λ Λ rd ω ιι β ω CM rd •Η > υ Φ M & 0 CM -χΤ 0.01 0.60 0.01 0.53 0.01 0.60 1 h, Jan, 2. 4. ω 1 d ·. id > B > ř* U d H •2 -Q dp V* 0 1 £4 ►> tn 0 - — 0 rd CM rd ΙΠ · <0 0 ° β Md rd (ϋ ο '> rd CO 0.15 0.15 0.18 0.07 0.16 0.14 and 3MM (Strength 3.5:1 1) , 60% β C rd M tn Φ ttf >(W ·· rd 0 > d Μ Ch •r4 , rd (Μ d in tl ω 0.61 0.40 0.60 0.35 0.61 0.40 Nhatm urstv 3 (75 0 (4: 40%Me ) ) >Μ Ch cm ™ S3 ® <y mV *r ® * O Q >M 2 ’ · «Ο O O •rf I am a woman. JJ Ch Φ 8 0 1 t© W fl jj 8 0 u » w 0 P, S U 1 CM p β fl dP CO -rd 0 O 4J -. to rd cj co x ω — 3 hairs •Tyre ² •Tyr VP At for η. fie - 9 - C ran Hey Cys Vai <d 'φ I Q 1 «—* D co · ^Jd re; ra ol ol pa ro ro >ro κι μ Ed 0 CT β β Φ Qi Ch Ch Φ Λί c ¹ , VP rd < u 1 and Q r~t rof ato uta uta r s ta ta ta d >Φ ·· ( +j g X) λ o 0 0 0 Tab Ν Substance |Mpr*, D-Arg^ xT Ch rd ČĚJ * rd °°th Μ M Ch < lSj ^ř '----* Q 2 0 1 1 j « β c rt) M CM rd Of Ό Ό Ό rd Xi x 0 0 0 S υ · · w w « fl X) rd cn ο Ό 0 Md

CS 266 156 Bl

Example 1 |p-Tyr ² , D-Arg ⁸ ] desamino-6-carba-vasopressin

For solid phase synthesis, 2 g of chloromethylated nickel polyol (100% benzene, 0.63 min/g) esterified with Boc-Gly by the potassium fluoride method (Horiki et al., 1999) was used.

K., Igano K., Ynouye K.: Chern. Letters 19'78, 165). On a support containing 0.30 mmol Gly/g were successively attached: Boc-D-Arg(Thos)OH, Boc-ProOH, Boc-Hcy(CH ₂ -CH ₂ COOBu ^t )OH, Boc-AsnOH, Boc-GlnOH, Boc-PheOH and Boc-D-Tyr(Z)OH by the method of symmetrical anhydrides (Hagenmaier H., Frank H.: Hoppe-Seyler's Z. Physiol. Chem. 353, 1973)). The synthesis was carried out on an automatic synthesizer according to the program given in Table 2. After the cleavage of the Boc-protecting group of tyrosine and the tert-butyl protecting group of the homocysteine derivative, Hcy (CH2~CH2 .COOBu*·) , by the action of trifluoroacetic acid, the peptide on the support was cyclized with 3 equiv. N,N'-dicyclohexylcarbodiimide in 20 ml CH2C12 ^X . After 3 days, when a negative reaction was achieved in the ninhydrin test (Kaiser E., Kolesott RL, Bossinger CD, Cook DIs Anal. Biochem. 34, 595 (1970)), the peptide on the support was washed (3x20 ml dimethylformamide, 3x20 ml CH2C1 ₂ ) and dried. 2.36 g (90%) of the substance was obtained.

The obtained peptide was cleaved from the support by treatment with a saturated solution of ammonia in methanol (30 ml, 20 C, 2 days). The support was filtered off and washed with warm dimethylformamide (50 °C, 20 ml). After concentration of the solution in vacuo, the product was precipitated with ether, filtered off and dried. 607 mg (84%) of crude peptide was obtained. The tosyl protecting group of arginine was cleaved by treatment with 10 ml of liquid HF (0 °C, 1 h) with the addition of anisole (0.3 ml) and thioanisole (0.3 ml) ^xx . After evaporation of HF, the residue was suspended in ethyl acetate (20 ml), filtered off and the filter cake was extracted with 3x20 ml of 3M acetic acid. The combined extracts were lyophilized and 470 mg (75%) of product was obtained.

^x With the same result, the cyclization can be carried out in dimethylformamide, toluene or a dimethylformamide-methylene chloride mixture (1:1).

^xx The tosyl protecting group can also be cleaved with sodium in liquid ammonia.

The resulting substance was dissolved in 9.4 ml of 20% CH ₂ COOH and subjected to separation on continuous carrier-free electrophoresis (IM, CH 2 COOH, 2600 V). The obtained fractions were detected spectrophotometrically at 280 nm, the fractions corresponding to the product were combined and lyophilized. 97.2 mg (15.5%) of a substance with satisfactory properties were obtained.

A portion of the product (8 mg) was analyzed by gradient elution (15% to 40% aqueous methanol containing 0.1% trifluoroacetic acid) on a Separon Si-C-18 column (5x2.5 cm). A completely uniform product was obtained chromatographically.

Example 2

I* 2 81

D-Tyr , D-Arg desamino-l-carba-vasopressin

The synthesis was carried out by the procedure used in example 1, with the only difference that Boc-Cys (CH ₂ -CH ₂ -CH ₂ -COOBu* ^: ) OH was used as the fourth amino acid. After the addition of Boc-AsnOH, the resin was divided in half. The obtained intermediates were used for the synthesis of other vasopressin analogues (example 3). Cyclization was carried out with 3eq. N,N'-dicyclohexylcarbodiimide in dichloromethane (3 days). After ammonolysis, the tosyl group of arginine was cleaved off with cap.

HF as in the previous case and also the purification procedure was analogous. The product characteristics are given in Table 3.

Example 3

[ό-Tyr ² , Val ⁴ , D-Arg ⁸ ]desamino-l-carba-vasopressin

CS 266 156 Bl

The synthesis used part of the resin obtained during the preparation of the previous compound (Example 2) and instead of glutamine, valine was condensed. The condensation, cyclization, cleavage and purification procedures were analogous (a gradient up to 50% methanol was used during HPLC purification). The properties of the obtained peptide are given in Table 3.

The substances which are the subject of this invention can be used for therapeutic purposes in <>1> I. i: 11 <·<-I i, l ^[ . tei é are indicated in the introductory part, namely in the form suitable for intranasal, sublingual, subcutaneous, intravenous and oral application. They can be used in aqueous solutions, in emulsions with vegetable oils, in liposomes, further adsorbed, absorbed, encapsulated and covalently bound to/in polymeric carriers. Due to their high chemical and especially enzymatic stability they are very suitable for oral use.

Table 3

List of abbreviations "

Abbreviation Full name

Hcy homocysteine

Boc tert-butyloxycarbonyl residue

Tos p-toluenesulfonyl residue

TFA trifluoroacetic acid

VP vasopressin

AD antidiuretic effect

BP pressor effect

UT uterotonic effect

But the rest of the butyric acid.

Mpr residue of 3-mercaptopropionic acid'

Abn alpha-aminobutyric acid residue

Claims (4)

OBJECT OF THE INVENTION Vasopressin analogues of general formula I XD-Tyr-Phe-X'-Asn-X-Pro-D-Arg-Gly-NH ₂ (I) 1 2 3 4 5 6 7 89 in which X is a butyric acid residue or a 3-mercaptopropionic acid residue X 'is Gln, Val or Asn X is Cys or an alpha-aminobutyric acid residue 2. XD-Tyr-Phe-Gln-Asn-X * '- Pro-D-Arg-Gly-NH ₂ (ID where X denotes a 3-mercaptopropionic acid residue X is Cys or an alpha-aminobutyric acid residue 3. xD-Tyr-Phe-Gln-Asn-Cys-Pro-D-Arg-Gly-NH ₂ (III) where X is a butyric acid residue 4. XD-Tyr-Phe-Val-Asn-Cys-Pro-D-Arg-Gly-NH ₂ (IV) where X is a butyric acid residue CS 266 156 Bl Process for the preparation of vasopressin analogues of the general formula I according to item 1, characterized in that a linear precursor of the analogue of the general formula V is prepared by the solid phase method F... X H-U-Tyr-Phe-X'-Asn-X'-Pro-D-Arg (Tos) -Gly--P; (V) roOR 'wherein R is a target. Butyl or fluorenylmethyl Qpj is a polymeric carrier Tos denotes the p-toluenesulfonic acid residue and the other symbols have the same meaning as in 1, the Boc group Tyr and the group R are cleaved by the action of trifluoroacetic acid, the cyclisation between residues 1 and 2 is carried out by treatment with Ν, Ν'-dicyclohexylcarbodiimide in dichloromethane, dimethylformamide, toluene or a mixture of dimethylformamide and dichloromethane, the cyclic analog precursor is cleaved from the polymeric support by ammonolysis, the p-toluenesulfonyl residue is cleaved by liquid hydrogen fluoride or sodium in liquid ammonia and the product obtained is purified by continuous supportless electrophoresis or high performance liquid chromatography.