DE2727048A1 - SQUARE BRACKET ON NLE HIGH 8 SQUARE BRACKET TO CERULETIDE A MODIFIED DECAPEPTIDE - Google Patents

Description

PATENTANWÄLTE ¹ [;. ~ ~.] Dlpl.-lng. P. WIRTH Dr. V. SCH M! E D-KOWAR Z IK

DlpL-lng. G. D AN N E N B E R G · Dr. P. WE IN H O LD · Dr. D. G U D E L

335024 SIEGFRICDSTHASSE

TELEPHONE: (083) _{BQQQ MQNCHEN 4Q}

Case "G 363" Wd / Sh

Parmitalia

Societa Farmaceutici Italia S.p.A.

Viale E. Bezzi, 24 1-20146 Milan / Italion

Γ si

lUlc J-Cer

ulotld, a no- identified decapeptide,

709852/0978

-1-

The present invention relates to a therapeutically useful decapeptide, its salts and to a method for the same Manufacturing.

The decapeptide according to the invention is pyroglutamyl-glutaminylaspartyl-tyrosyl-threonyl-glycyl-tryptophyl-norleucyl-aspartylphenylalaninamide, in which the phenol group of the tyrosyl residue is protected by a sulfur residue and in which all amino acids (with With the exception of glycine) have L-configuration. The invention includes also the non-toxic, pharmaceutically acceptable salts of this decapeptide with organic and inorganic bases. The inventive Decapeptide shows strong biological effectiveness and can be used in the therapy of various diseases in humans and Animals are used.

In our own US Pat. No. 3,472,832, the synthesis of a very is Similar decapeptide (Ceruletid: generic name) described and claimed. However, this decapeptide is because of the presence of a methionyl radical in the molecule associated with many disadvantages, especially in the purification of the intermediate products and the end product the synthesis and with regard to the stability of the finished dosage form.

These disadvantages also occur as a result of the slight oxidation of the mercapto group in the methionine residue to the corresponding sulfoxide by atmospheric oxygen.

The oxidized ceruletide is almost completely devoid of biological activity. To remedy this deficiency - and this is the goal of the present invention - the amino acid methionine has been substituted with n-leucine in the "ceruletide" molecule.

The new decapeptide obtained in this way is very stable to oxidizing agents and surprisingly shows in comparison with the "Ceruletid" increased biological effectiveness.

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The synthesis of the new decapeptide according to the present invention consists essentially of suitable sequentials Condensation of the protected amino acids or polypeptides. The condensation is carried out so that the decapeptide obtained has the desired sequence of ten amino acids. In the amino acids and polypeptides according to in polypeptide chemistry per se known methods are condensed, are the amino and carboxyl groups that are not involved in the formation of the peptide bond, protected by a protecting group that can be removed by acidolysis, saponification or hydrogenolysis. To protect the amino groups the following protective groups can be used: carbobenzoxy (benzyloxycarbonyl), tert-butyloxycarbonyl, trityl (triphenylmethyl), Formyl or trifluoroacetyl. To protect the carboxyl groups For example, the following protective groups can be used: methyl, ethyl, tert-butyl, benzyl or p-nitrophenyl.

The condensation between an amino group of one molecule and a carboxyl group of another molecule to form a Peptide bond can be mixed through an activated acyl derivative, such as a Anhydride, an azide, a p-nitrophenyl ester or a 2,4,5-trichlorophenyl ester and the like, or by direct condensation between a free amino group and a free carboxyl group in the presence of a condensing agent such as dicyclohexylcarbodiimide afer l-cyclohexyl-S-morpholinylcarbodiimide. the Condensation can be carried out in a solvent such as N, N-dialkylformamide, a lower aliphatic nitrile, a pyridine, tetrahydrofuran, Dimethyl formamide, acetonitrile or pyridine. The reaction temperatures are -20 C to room temperature. The reaction time is generally 6 to 120 hours.

The synthesis scheme is chosen so that the risk of racemic

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COPY

ization is avoided; accordingly, the two pentapeptides 1 and II are coupled by the azide method. The azide pentapeptide (IA) is obtained au> Uui.t Hvdra / .id (I), which was obtained by stepwise /UL^if-iii.ui.'j U Li-'iKjation) / as described in Example 1. The l'cntapui-tid pyroglutamyl-glutaminyl-aspartyl-tyrosyl-O-acetyl-threonine azide (LA) is prepared in a solvent such as dimethylformamide with the pentapeptide glycyl-tryptophyl-norleucylaspartyl-phenylalaninamide (II), as in Example 2 described, condensed, the decapeptide pyroglutamyl-glutaniinyl-aspartyl-tyrosyl-O-acetyl-threonyl-glycyl-tryptophyl-norleucyl-aspartyl-phenylalanine amide (III) is obtained. The decapeptide (III) is treated at low temperature with an anhydrous pyridine-sulfuric anhydride complex, the decapeptide being pyroglutamyl-glutaminyl-aspartyl-tyrosyl (O-sulfate) -O-acetyl-threonyl-glycyl-tryptophyl-norleucyl-aspartyl-phenylalaninamide (IV ) is obtained in which the phenol group of the Tyrcsylrest is sulfated and the hydroxyl group of the Threonylrest is protected by an acetyl group. Mild alkaline hydrolysis of the decapaptide (IV) finally gives day decapeptide pyroglutamylglutaminyl-aspartyl-tyrosyl (O-sulfate) -threonyl-glycyl-tryptophylnorleucyl-aspartyl-phenylalaninamide (V), in which the hydroxyl group of the threonyl radical free and free of the phenyl radical is. The condensation can be represented by the following scheme in which the symbols correspond to the symbols commonly used in polypeptide chemistry.

Ac

H-Pyr-Gln-Asp-Tyr-Thr-N ₃ + H-Gly-Trp-Nle-Asp-Phe-NH ,, (IA) (II)

Ac

H-Pyr-Gln-Asp-Tyr-Thr-Gly-Trp-Nle-Asp-Phe-NH ₂ (HI)

709852/0978

COPY ^ORIGIN AL INSPECTED

- * '27270A8 " ⁴ "

Pyridine / SO, 3,

gj ^ P - »H-Pyr-Gln-Asp-Tyr Thr-Gly-Trp-Nle-Asp-PheNH ₂

SO, H ^(IV)

I.
OH _ψ H-Pyr-Gln-Asp-Tyr Thr-Gly-Trp-Nle-Asp-PheNH ₂

(V)

The following examples are intended to explain the present invention in more detail without, however, being restricted thereto target.

Example 1: Preparation of the compound (I)

Ac
I.
H-Pyr-Gln-Asp-Tyr-Thr-NH-NH ₂ .HC1 (D

8.04 ml of N-methylmorpholine and 7.01 ml of ethyl chloroformate were added successively at a temperature of -15 ° C. to a solution of 16.05 g of Boc-Thr-OH in 170 ml of anhydrous tetrahydrofuran. After stirring at this temperature for 2 minutes, 12.16 g of NH ₂ -NH-Z (Z = benzyloxycarbonyl) in 50 ml of anhydrous tetrahydrofuran were added. The reaction mixture was left with stirring for 2 hours at -10 ° C and then for 16 hours at 0 to 5 ° C. The solvent was removed in vacuo and the residue was taken up in ethyl acetate and washed successively with 1N hydrochloric acid, a 5% aqueous sodium bicarbonate solution and a saturated sodium chloride solution to neutrality. After removal of the solvent, 26.5 g of Boc-Thr-NH-NH-Z (Boc = tert-butyloxycarbonyl) (VI) were obtained as a white foam, yield 98%. The analytical sample was obtained by crystallization from diethyl ether, melting point 83 to 84 ° C. / ° tJ ^ ⁵ = -14 ° (c =] in dimethylformamide).

15 ml of 6N HCl in 15 ml of glacial acetic acid were added to a solution of 26.5 g of Boc-Thr-NH-NH-Z (VI) in 50 ml of diethyl ether. After the diethyl ether had evaporated, 150 ml of acetyl chloride were ^{added at a temperature of 0} ° C. over the course of 2 hours. The mix was

709852/0978

held at 0 C for a further 10 minutes, after which 1500 ml of diethyl ether were added. The precipitate thus formed was filtered and

Ac crystallized from methanol-isopropanol, with 21 g of H-Thr-NH-NH-Z.

HCl (VII), m.p. 120 ° C (dec.), JV] ^ ⁰ = + 10 ° (c = 1 in dimethylformamide), yield 84%.

Ac

15.19 g of H-Thr-NH-NH-Z.HCl (VII) were replaced by ethoxyformic anhydride with 7.52 g of Boc-Tyr-OH, as described for the preparation of the compound (VI), condensed. The raw synthetic material was crystallized from diethyl ether, whereby

Ac

15.19 g of boc-Tyr-Thr-NH-NH-Z (VIII), melting point 133 to 134 ° C., Lj _n = -1.6 (c = 1 in dimethylformamide), were obtained, yield

⁷⁸ * • Ac

12.8 g of Boc-Tyr-Thr-NH-NH-Z (VIII) were added to 130 ml of 1.33N

Hydrogen chloride dissolved in acetic acid. After 2 5 minutes at room temperature the solution was evaporated to dryness in vacuo and the residue was triturated with diethyl ether. The solid thus obtained was filtered and crystallized from anhydrous ethanol diethyl ether

Ac

siert, whereby in quantitative yield 11.4 g of H-Tyr-Thr-NH-NH-Z.HCl (IX), melting point 150 to 160 ° C, fo (Jp ⁰ = + 29 ° (c = 1 in AcOH) obtained, yield 95%.

Ac

11.4 g of H-Tyr-Thr-NH-NH-Z.HCl (ΐχ) were replaced by ethoxy ant

OBzI

Acid anhydride with 7.28 g of Boc-Asp-OH (BzI = benzyl) (prepared as in J.Am.Chem. Soc. 81, p. 620 (1965)) condensed. To Crystallization from dimethylformamide-ethyl acetate gave 15.66 g

OBzI Ac ο Γ 720

Boc-Asp-Tyr-Thr-NH-NH-Z (X) obtained, m.p. 185 to 186 C, / * J _n = -15.7 (c = 1 in dimethylformamide), yield 90%.

OBzI Ac By acidolysis of 12.2 g of Boc-Asp-Tyr-Thr-NH-NH-Z (X) with

a solution of 1.33N hydrogen chloride in acetic acid were Hg

OBzI Ac
Obtained H-Äsp-Tyr-Thr-NH-NH-Z.HCl (XI). After crystallization from

Methanol diethyl ether melted the compound at 173 to 176 ° C,

709852/0978

OM _n ^ +16.3 (c = 1 in AcOH 95%), yield 98%. ^JD OBzI Ac

11 g of H-Äsp-Tyr-Thr-NH-NH-Z.HCl (XI) were replaced by ethoxyformic anhydride condensed with 3.79 g of Boc-Gln-OH, after

OEzI

Crystallization from methanol-ethyl acetate 11.6 g Boc-Gln-Asp-Tyr-

Thr-NH-NH-Z (XII), m.p. 205 to 266 ° Ο, ^^ ° = -19.9 ° (c = 1 in dimethylformamide) were obtained, yield 83%. By acidolysis

OBzI Ac

of 11.58 g of Boc-Gln-Asp-Tyr-Thr-NH-NH-Z (XII) with a solution of 1.33N hydrogen chloride in acetic acid were 10.76 g (quantita-

9BzI Ac
tive yield) H-Gln-Asp-Tyr-Thr-NH-NH-Z.HCl (XIII) obtained, melting point 120 to 140 ° C. After crystallization from anhydrous ethanol-diethyl ether, the product melted at 125 to 135 ° C (decomp.), -8.7 (c = 1 in dimethylformamide).

OBzI Ac

9.25 g of H-Gln-Äsp-Tyr-Thr-NH-NH-Z.HCl (XIII) were treated with ethoxyformic anhydride with 2.89 g of Z-Pyr-OH (prepared according to Liebigs Ann. 640, p. 1 ⁴ ^ (1961)) condensed, whereby according to crystalline

OBzI Ac

I I

cation from dimethylformamide-methanol 8.55 g of Z-Pyr-Gln-Asp-Tyr-Thr-NH-NH-Z (XIV) were obtained, m.p. 216 to 220 ° C, [«J ^ ⁰ = -24 ° ( c = 1 in dimethylformamide). Yield 74%.

OBzI Ac

8.55 g Z-Pyr-Gln-AESP-Tyr-Thr-NH-NH-Z (XIV) was dissolved in 150 ml of dimethylformamide containing 1.1 equivalents of hydrochloric acid at room temperature in the presence of van 10% Palladium on carbon 5 Hydrogenated for hours. The catalyst was filtered off and washed thoroughly with dimethylformamide. The filtrate was then evaporated to dryness in vacuo. The solid residue turned off

Ac crystallized from absolute ethanol, with 4.75 g of Pyr-Gln-Asp-Tyr-fhr-

.HCl (I), m.p. 189 to 191 ° C, / «/ p ⁰ = -12 ⁽

(c = 1 in dimethylformamide), yield 80%.

Example 2: Preparation of the compound (II)

H-Gly-Trp-Nle-Asp-Phe-NH ₂ (II)

OBzI To a solution of 20 g of Boc-Asp-OH in 120 ml of anhydrous

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Tetrahydrofuran was added successively 6.8 ml of N-methylmorpholine and 5.9 ml of ethyl chloroformate at a temperature of -15 ° C. After stirring at this temperature for 2 minutes, 12.4 g of Phe-NH ₂ .HC1 in 100 ml of anhydrous dimethylformamide containing 6.8 ml of N-methylmorpholine were added.

The reaction mixture was allowed to stand with stirring for 2 hours at -10 ° C and then for 16 hours at 0 to 5 ° C. That Solvent was removed in vacuo and the residue off

OBzI

Ethyl acetate-petroleum ether crystallized, with 20.5 g of Boc-Asp-Phe-NH (XV), melting point 138 ° C., pJ _D = -28.3 ° (c = 1 in dimethylformamide), yield 70%.

OBzI

20.2 g of Boc-Asp-Phe-NH ₂ (XV) were dissolved in 200 ml of 1.33N hydrogen chloride in acetic acid. After 25 minutes at room temperature, the solution was evaporated in vacuo and the residue was triturated with diethyl ether. The solid product thus obtained was filtered and crystallized from ethyl acetate-petroleum ether, 17.3 g of OB:
Asj

foil ²⁰ = + 11.9 ° (c = 1.2 in MeOH), yield 99%. ^LJü OBzI

14.77 g of H-Asp-PheNH ₂ .HCl (XVI) were replaced by ethoxy ant

acid anhydride, as described for compound (XV), condensed with 8.4 g of Boc-Nle-OH, with 19.2 g of protected tripeptide

OBzI _{r 72O}

Boc-Nle-Asp-PheNH ₂ (XVII), m.p. 156 ° C / ° <J _D = -30 ° (c = 1.3 in MeOH), yield 91%.

By acidolysis of 19.2 g of Boc-Nle-Asp-PheNH (XVII) with

1.33N hydrogen chloride in acetic acid, as for the compound (XVI)

OBzI
described, 16.6 g H-Nle-AESP-Phe-NH ₂ .HC1 (XVIII), mp. 224-225 ⁰ CPJ ²⁰ = -2.4 ° (c = 1.1 in MeOH), yield 97%.

16.5 g of H-NIe Asp-Phe-NH ₂ .HCl (XVIII) were replaced by ethoxy

formic anhydride with 10.76 g of Z-Trp-OH, as for the preparation of the compound (XVJ described, condensed, 24.7 g of the

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9
H-Asp-PheNH ₂ .HCl (XVI), m.p. 183 to 184 C,

^ o. 2727CK8 " ⁸ "

OBzI
I.
protected tetrapeptide Z-Trp-Nle-Asp-PheNH ₂ (XIX) were obtained, melting point 220 to 222 ° C, £ * J £ ° = -33.7 ° (c = 1.2 in dimethylformamide), yield 97% . OBzl

I.
24.7 g of Z-Trp-Nle-Asp-PheNH ₂ (XIX) were in 200 ml of a

Mixture of dimethylformamide and acetic acid (1: 1 V / V) dissolved and reduced in a stream of hydrogen in the presence of 10% palladium on carbon at room temperature. After 1 equivalent of hydrochloric acid had been added, the catalyst was filtered off, washed with dimethylformamide and the solvent was evaporated off in vacuo, giving 15.75 g of H-Trp-Nle-Asp-PheNH ₂ .HCl (XX), m.p.

⁰ = -21.1 ° (c = 1 in MeOH), yield 83%. To a solution of the tetrapeptide H-Trp-Nle-Asp-PheNH ₂ .HCl (XX) in 100 ml of dimethylformamide, 12.7 g of Boc-Gly-OCP (OCP = 2,4,5-trichlorophenyl ester) and 2.86 ml of N-methylmorpholine were added at 0 ° C. After stirring for 60 hours at 0 to 5 ° C., the solvent was removed in vacuo and the residue was triturated with diethyl ether, filtered and crystallized first from dimethylformamide-ethyl acetate and then from methanol, with 8.55 g of Boc-Gly-Trp-Nle -Asp-Phe-NH ₂ (XXI), m.p. 218 ° C, = -18.3 ° (c = 1 in dimethylformamide), yield 45%.

By acidolysis of 8.55 g of the protected pentapeptide Boc-Gly-Trp-Nle-Asp-Phe-NH ₂ (XXI) in 170 ml of formic acid for 4 hours at room temperature and in the presence of 0.85 ml of 2-mercaptoethanol, 5, 98 g of H-Gly-Trp-Nle-Asp-Phe-NH ₂ (II) obtained, m.p. 212 ° C, / * J ^ ⁰ = -35 ° (c = 0.44 in dimethylformamide-HMPA 1: 1) , Yield 81%.

Example 3: Preparation of the compound (III)

Ac
H-Pyr-Gln-Asp-Tyr-Thr-Gly-Trp-Nle-Asp-PheNH ₂ (III J

709852/0978

Ac To a solution of 1.095 g of Pyr-Gln-Asp-Tyr-Thr-NH-NH-.HCl (I)

in 10 ml of anhydrous dimethylformamide were successively 1.22 ml 1.84N hydrogen chloride in anhydrous tetrahydrofuran and 0.176 ml n-Butyl nitrite added at a temperature of -25 ° C. After stirring at this temperature for 15 minutes were 0.58 ml of N-methylmorpholine and a solution of 0.953 g of H-Gly-Trp-Nle-Asp-Phe-NH- (II) in 20 ml of a mixture of dimethylformamide and hexamethylphosphoramide (1: 1 V / V) containing 0.165 ml of N-methylmorpholine added. The reaction mixture was kept at -10 ° C for 3 days and at 5 ° C for 2 days. After evaporation of the volatile solvents the product was precipitated in vacuo by dilution with a cold aqueous citric acid solution. The raw material yielded after two Crystallizations from dimethylformamide-methanol 0.780 g of the deca-

Ac
peptides Pyr-Gln-Asp-Tyr-Thr-Gly-Trp-Nle-Asp-Phe-NH ₂ (III), m.p. 225 ° C, = -15 ° (c = 1 in dimethylformamide), yield 40%.

Example 4: Preparation of the compound (IV)

SO, H Ac

f ³ I

Pyr-Gln-Asp-Tyr Thr-Gly-Trp-Nle-Asp-Phe-NI ^ (IV)

0.320 g of the compound (III), dissolved in 10 ml of anhydrous dimethylformamide, were added at -10 ° C to 5 g of a pyridine-sulfuric anhydride complex, suspended in 25 ml of anhydrous pyridine, added. The mixture was allowed to stand at room temperature for 18 hours left and then with an IM aqueous potassium bicarbonate solution cooled down. After evaporation to dryness in vacuo, the residue was triturated with dimethylformamide and filtered to remove the insoluble remove inorganic salts. The filtrate was again evaporated to dryness in vacuo, leaving 0.320 g of the crude sulfated decapeptide of formula (IV) were obtained.

Example 5: Preparation of the decapeptide (V)

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SO ₃ H

Pyr-Gln-Asp-Tyr Thr-Gly-Trp-Nle-Asp-Phe-NH ₂ (V)

0.320 g of the raw material (IV) obtained in Example 4 was dissolved in 10 ml of water. 2.5 equivalents of 1 N sodium hydroxide was added and the solution was kept at room temperature for 3 hours. After neutralizing the alkaline solution with solid CO ₂ , the mixture was evaporated to dryness in vacuo. The crude residue obtained in this way was taken up in dimethylformamide and filtered from inorganic salts. The yellow filtrate was again evaporated to dryness in vacuo and the residue was dissolved in a little water and treated at a temperature of 0 C with Dowex ^ SOW (H ⁺ ) (brand name) with stirring for 5 minutes. The mixture was filtered and the filtrate was extracted three times with n-butanol. The combined organic extracts were washed with a little water and made alkaline with diethylamine. The solvent was evaporated in vacuo and the concentrated _Λ

Diethyl ether solution was added, 0.320 g of crude Pyr-Gln-Asp-Tyr-Thr-Gly-Trp-Nle-Asp-Phe-NH- (V) being precipitated as the diethyl anunonium salt. The product was then purified by dissolving it in 2 ml of dimethylformamide and pouring the resulting solution on a small pot (27 χ 1.5 cm) Amberlite ^ XA-D2 (brand name) using water and then a mixture of water and methanol was eluted as the eluent with increasing amount of methanol up to a concentration of 50%. 0.150 g of end product were obtained, £ <J = -23 ° (c = 1 in dimethylformamide). Biological activities
JNIe ⁸ J-Ceruletide

The biological activities of the ^ NIe J ceruletide were determined in comparison with ceruletide. Ceruletide is a decapeptide developed by Erspamer et al. (Nature 212 , 2θ4, 1966) in the skin

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from Australian frogs (HyIa caerulea) and which shows physiological activities similar to those of cholecystokinin-pancreozymin (G.Bertaccini et al., Br.J. Pharmacol. 3 ±, 291, 1968; G.Bertaccini et al, ibidem .37 . » ¹⁸ S, 1969).

1) The effect of [Nie J-Ceruletide on gallbladder motility was examined in situ in guinea pigs by intravenous injection. If the strength of the ceruletide is set equal to 100, the relative strength of the [Nie ⁸ J-ceruletide is found to be 130 to 160.

2) The effect via the pancreatic secretion was determined in anesthetized rats, as described by GJDockray (J.Physiol. 225 , 679, 1972). Two 10 minute fractions of the pancreatic juice were collected before and seven fractions after intravenous administration of the peptides. The ratio of dose to volume and protein output within 70 minutes after the treatment are in

shown in the table.

If one sets the strength of the ceruletide equal to 100, the strength of the [kle J-ceruletide appears to be about 120 to 130.

Based on the above results, [Nie J-Ceruletide advantageous as a diagnostic agent for the same clinical indications as ceruletide in both biliary and pancreatic diseases be used. «

Tabel -

Effect of intravenous injection of [_Nle J-ceruletide and ceruletide on pancreatic secretion in rats Dose volume output protein output

ng / kg IV

, ul / 70 min

mg / 70 min

25th

50

100

200

59 67 79 96

Ceruletid 57 66 70 93

[Never J-ceruletide

11 18 28

Ceruletide

16 21

mean value of 6 to 7 rats

709852/0978 ORIGINAL INSPECTED

Claims (4)

Patent claims: The decapeptide pyroglutamyl-glutaminyl-aspartyl-tyrosylthreonyl-glycyl-tryptophyl-norleucyl-aspartyl-phenylalaninamide, in which the phenol group of the tyrosyl residue is free or protected by a sulfuric acid residue and the hydroxyl group of the threonyl residue is free or protected by an acetyl radical, and its non-toxic pharmaceutically acceptable salts with an organic or inorganic base. 2. The decapeptide of the formula Ac I H-Pyr-Gln-Asp-Tyr-Thr-Gly-Trp-Nle-Asp-Phe-NH ₂ , wherein Ac represents an acetyl radical. 3. The decapeptide of the formula SO ₀ H Ac I ³ I. H-Pyr-Gln-Asp-Tyr Thr-Gly-Trp-Nle-Asp-Phe-NH-, wherein Ac represents an acetyl radical. 4. The decapeptide of the formula SO H H-Pyr-Gln-Asp-Tyr Thr-Gly-Trp-Nle-Asp-Phe-NH ₂ and its non-toxic pharmaceutically acceptable salts from the group of sodium, potassium, magnesium, calcium, diethylamine and dicyclohexylamine salts. 709852/0978