Claims
- 1. A chemical compound having the formula: ##STR17## in which: n is 0 or 1;
- P is a single bond or a D or L isomer of Pro, Pro, Ala, MeAla, Arg, Lys, iUrg, MeArg, MeLys or MeHArg;
- A is a single bond, Gly, or a D or L isomer of Arg, Lys, Orn HArg, MeArg, MeLys, MeOrn, MeHArg or Gln
- D is a single bond, Gly, or a D or L isomer of Argm Lys, HArg, Me Arg, MeLys, MeHArg, Gln or Orn;
- B is OH, NH.sub.2, NHAlk or NH (CH.sub.2).sub.m NHR;
- Z is Phe, Phe(4'-Alk), Tyr(Alk), Ile or Tyr;
- X is a D or L isomer of Phe, Phe(4'-Alk), Val, Nva, Leu, Ile, Pba, Nle, Cha, Abu, Met, Chg, Tyr, Trp or Tyr(Alk); and
- Y is Val, Ile, Abu, Ala, Chg, Gln, Lys, Cha, Nle, Thr, Phe, Leu or Gly;
- m is 2 to 6;
- R is H or C(=NH)NH2 and
- * is to indicate D isomer, L isomer or a D,L mixture.
- 2. The compounds of claim 1 in which X is a D isomer.
- 3. The compounds of claim 1 in which X is a D-Tyr(Alk) and Y is Val.
- 4. The compounds of claim 1 in which X is D-Tyr(Alk), Y is Val, n is 1 and P is a single bond.
- 5. The compounds of claim 1 in which X is D-Tyr(Et), Y is Val, n is 1 and B is NH.sub.2.
- 6. The compound of claim 1 being [1,6-(6,6-cyclopentamethylene-2-amino-D-suberic acid)-2-(O-ethyl-D-tyrosine)-4-valine-8-arginine-9-desglycine]vasopressin.
- 7. The compound of claim 1 being [1,6-(6,6-cyclopentamethylene-2-amino-L-suberic acid)-2-(O-ethyl-D-tyrosine)-4-valine-7-desproline-8-arginine-9-desglycine]vasopressin.
- 8. The compound of claim 1 being [1,6-(6,6-cyclopentamethylene-2-amino-D-suberic acid)-2-(O-ethyl-D-tyrosine)-4-valine-7-desproline-8-arginine-9-argininamide]vasopressin.
- 9. The compound of claim 1 being [1,6-(6,6-cyclopentamethylene-2-amino-D-suberic acid)-2-(O-ethyl-D-tyrosine)-4-valine-7-D-proline-9-desglycine]vasopressin.
- 10. The compound of claim 1 being [1,6-(6,6-cyclopentamethylene-2-amino-L-suberic acid)-2-(O-ethyl-D-rteiaubw)-4-valine-7-arginine-8-D-arginine-9-desglycine]vasopressin.
- 11. The compound of claim 1 being [1.6-(6,6-cyclopentamethylene-2-amino-D-suberic acid)-2-O-ethyl-D-tyrosine)-4-valine-7-arginine-8-D-arginine-9-desglycine]vasopressin.
- 12. A pharmaceutical composition comprising a pharmaceutical carrier and, dispersed therein, an vasopressin antagonist effective but nontoxic quantity of a compound of claim 1.
- 13. The composition of claim 12 in which said compound is [1,6-(6,6-cyclopentamethylene-2-amino-D-suberic acid)-2-(O-ethyl-D-tyrosine)-4-valine-8-arginine-9-desglycine]vasopressin.
- 14. The composition of claim 12 in which said compound is [1,6-(6,6-cyclopentamethylene-2-amino-L-suberic acid)-2-(O-ethyl-D-tyrosine)-4-valine-8-arginine-9-desglycine]vasopressin.
- 15. The method of inducing a vasopressin antagonist effect in a patient in need of such an effect comprising administering parenterally or intranasally to said patient a nontoxic, effective therefor quantity of a compound of claim 1.
- 16. The method of claim 15 in which the onist effect is manifested by a water diuresis.
- 17. The method of claim 15 in which the compound is [1,6-(6,6-cyclopentamethylene-2-amino-D suberic acid)-2-(O-ethyl-D-tyrosine)-4-valine-8-arginine-9desglycine]vasopressin.
BACKGROUND OF THE INVENTION
This application is a continuation-in-part of U.S. Ser. No. 819,336, filed Jan. 16, 1986, now abandoned.
This invention relates to new vasopressin compounds whose structures are distinguished by having a 1,6-dicarba bridge which is combined with a spirocycloalkyl substituent at the 1-position and to pharmaceutical compositions of these compounds and their use as vasopressin antagonists. The compounds have potent in vivo and in vitro vasopressin antagonist activity.
Examples of the dithia or vasopressin antagonist art are U.S. Pat. Nos. 4,469,679, 4,481,193, 4,481,194 and 4,491,577.
Carba analogues of oxytocin and vasopressin, which have agonist activity, have been repeatedly reported in the prior art, such as in U.S. Pat. Nos. 3,980,631, 4,285,858, 4,482,486, 4,237,119 as well as in F. Fahrenholz et al., Biochem. Biophys. Res. Com. 122, 974 (1984) and J. Biol. Chem. 258 14861 (1983). Dicarba or 1,6-amino-suberic acid analogs of lysine-and arginine-vasopressin and oxytocin have been reported to have variable agonist activity. These prior art structures have no substituent on the 1,6-suberic acid and are agonists, i.e. they have the same biological activity as do vasopressin (VSP) or oxytocin (OXT).
We have now found that, when the 1-unit and the dithia connection of certain vasopressin structures is replaced by a 6,6-spiroalkylenesuberic acid, the resulting compounds have potent, even enhanced antagonist activity, especially aquaretic activity.
In the description herein and in the claims, the nomenclature common in the art of peptide and vasopressin chemistry is used. When no configuration is noted, the amino acid unit is in the L, or naturally occurring, form.
Certain of the peptide art designations used herein are the following: Pas, 6,6-cyclopentamethylene-2-aminosuberic acid; Tas, 6,6-cyclotetramethylene-2-aminosuberic acid; Pas(Bzl), .omega.-benzyl ester of Pas; Abu, .alpha.-amino-n-butyric acid; Cad, cadaverine; Chg, cyclo hexylglycine; Cha, cyclohexylalanine; Thr, threonine; Pba, .alpha.-aminophenylbutyric acid; Gln, glutamic acid amide or glutamine; Pro, proline, .DELTA.Pro, .DELTA..sup.3 -proline; Gly, glycine; Tyr, tyrosine; Tyr(Alk), lower alkyl ether of Tyr; Phe, phenylalanine; Phe(4'-Alk), 4'-alkylphenylalanine; MeAla, N-methylalanine; Val, valine; Ile, isoleucine; Nle, norleucine; Leu, leucine; Ala, alanine; Lys, lysine; Arg, arginine; HArg, homoarginine; MeArg, N-methylarginine; MeHArq, N-methylhomoarqinine; MeLys, N-methyllysine; Met, methionine; Asn, asparagine; Put(G), 1-amino-4-quanidinobutane (--NH(CH.sub.2).sub.4 NHC(NH)NH.sub.2); Glu, glutamic acid; Orn, ornithine; Asp, aspartic acid, MeOrn, N-methylornithine; Tos, tosylate; BHA, benzhydrylamine; DMAP, 4-dimethylaminopyridine; DIEA, diisopropylethylamine; Trp, tryptophan; HF, hydrogen fluoride; 4-MeBzl, 4-methylbenzyl; TFA, trifluoroacetic acid; DCC, dicyclohexylcarbodiimide; Boc, t-butyloxycarbonyl; Z, benzyloxycarbonyl; VSP, vasopressin; HBT, hydroxybenzotriazole. In the definitions such as MeArg above, Me denotes a methyl located on the amido nitrogen of the peptide unit concerned. The designation, Pas.sup.1,6, is used to denote the cyclized peptide as described hereafter. "Alk" represents a lower alkyl of 1-4 carbons. For example, these may be optionally attached to the oxygen substituent of a tyrosine unit at position 2 of the peptide of formula I, to the N-terminal nitrogen of the tail, or to the 4'-position of a Phe unit at position 2 or 3 of the peptide of formula I. Such alkyl substituents include methyl, ethyl, n-propyl, isopropyl or butyl. Ethyl is preferred. When the term "vasopressin" is used, it means L-arginine vasopressin (AVP) unless otherwise modified.
The dicarba-vasopressin compounds of this invention are illustrated by the following structural formula: ##STR1## in which: n is 0 or 1;
Subgeneric groups of compounds of this invention comprise compounds of formula I in which the P--A--D--B tail is Pro--Arg--NH.sub.2, Arg--Gly--NH.sub.2, Pro--Arg--Gly--NH.sub.2, Arg--DArg--NH.sub.2, DArg--DArg--NH.sub.2, Put(G), NMeArg--Arg--NH.sub.2, Pro--Arg--NH(CH.sub.2).sub.2 NH.sub.2, Arg--NH.sub.2, and Arg--Gly--Arg--NH.sub.2. In formula I, n is preferably 1 and X is a D-isomer unit.
Also included in this invention are addition salts, complexes or prodrugs, such as esters of the compounds of this invention when B is OH, especially the nontoxic, pharmaceutically acceptable acid addition salts. The acid addition salts are prepared in standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic, ethane disulfonic or methanesulfonic acids. Certain end pro ducts of formula I such as the Arg.sup.7 --Arg.sup.8 compounds have two strong basic groups in their structures, therefore, their acid addition salt derivatives are easily prepared. The ester derivatives of the acid forms of the end products, such as the methyl, ethyl or benzyl esters, are prepared as known to the art.
The end products (I) of this invention are prepared by cyclization of the corresponding novel linear peptide (II): ##STR2## in which X, Z, Y, P, A, D, B, n and * are as defined above with any chemically reactive centers protected as described below. Also included are any addition salt forms of compounds of Formula II as noted above for the end compounds of formula I. It should be emphasized that the linear intermediate structure II can be cyclized only in the form of the free base of X. No cyclization occurs when the salt form is present but the salt forms are important derivatives for isolation and characterization of the intermediates.
The cyclization involves the formation of an amide bond between the .alpha.-amino group of the X unit at position 2 and the free .omega.-carboxy group of the 6,6-substituted 2-amino suberic acid unit at position 6 of structure II. Any chemical method of amide formation is employed such as reaction using dicyclohexylcarbodiimide plus hydroxybenzotriazole or 4-dimethylaminopyridine, reaction using diphenylphosphoryl azide (DPPA) and base, 1-(3 -dimethylaminopropyl)-3-ethylcarbodiimide (EDC) as the methiodide or hydrochloride; the azide derivative of the carboxylic acid; a mixed anhydride such as the benzoyl, pivaloyl or isovaleroyl containing anhydride with the suberic acid; ethoxyacetylene or N-ethyl-5-phenyl-isoxazolium-3'-sulfonate. All these general synthetic methods are described in Peptide Synthesis, M. Bodanszky, John Wiley, 1976, page 191 and Table 3, pages 116-121. These coupling reactions are usually run in either aqueous or organic solvents until reaction is complete. Among the preferred solvents are dimethylformamide, dimethylacetamide, methylene chloride, acetonitrile or combinations thereof. The reactions are usually carried out at room temperature and in dilute solution to minimize polymerization.
Diphenylphosphoryl azide (DPPA) is a particularly useful cyclization agent. The linear peptide (II) is treated with an excess of DPPA in dried dimethylformamide and triethylamine at room temperature until thin layer monitoring demonstrates completion of the reaction. The desired product is isolated by evaporation and purified by gel filtration and high performance liquid chromatography.
The resin supported peptide chain of the linear peptides (II) is usually built up, stepwise, proceeding from the B unit and working toward the X unit through the novel Pas or Tas unit at position 6 of the linear product. Each unit is properly protected as known in the peptide art and as described below. The sequence of step-reactions is conveniently carried out in a Beckman 990B peptide synthesizer or its equivalent without isolation of each intermediate peptide. The details of the procedure are in the working examples presented hereinafter.
The various amino acids (AA), which are consecutively added to the resin-supported chain, are protected as known to the art. For example, the Boc protecting group is used for an amino group, especially at the .alpha.-position; an optionally substituted benzyl for the carboxy group of the Pas or Tas unit; tosyl for the Arg, HArg or MeArg unit; and an optionally substituted carbobenzyloxy (Z) for the Tyr, Orn or Lys units. The protective groups are, most conveniently, those which are not easily removed by using mild acid treatment, such as for removing the Boc group. Rather one should use HF, sodium-liquid ammonia or, for benzyl or carbobenzyloxy groups, catalytic hydrogenation.
Often, the uncyclized, resin-supported polypeptide of Formula II is synthesized in a number of synthetic cycles to offer good supplies of oligopeptide intermediates to vary the chain as described above.
The assembled, resin-supported peptide is treated with an excess of anhydrous hydrogen fluoride with an appropriate scavenger compound, such as anisole, to give the linear peptide intermediate of formula II in good yield.
The compounds of formula I are also prepared by reacting the cyclized Pas.sup.6 carboxylic acids or derivative thereof with a protected form of PADB in the terminal acid ester or amide form and likewise the cyclized Pro.sup.7 with protected ADB, cyclized Arg.sup.7 with protected DB or cyclized Arg.sup.8 with B, in each case employing reaction conditions and intermediates of standard peptide methods of synthesis. Such starting material acids, such as those of formula I in which P is an arginine-like unit as defined above and A is hydroxy, are prepared as described above by either a resin-supported or a solution reaction sequence.
The key to the synthesis of the compounds of formula I and the discovery of their biological activity was the availability of the 6,6-spirocycloalkylene-2-aminosuberic acid which must be available for use in the peptide synthesis of the intermediates of formula II discussed above. 2-Amino and 2,7-diaminosuberic acids had been prepared earlier using a Kolbe electrolysis as the key step of the synthesis, R. Nutt et al., J. Org. Chem. 45 3078 (1980). The presence of the bulky spirocycloalkylene substituent at the 6,6-position of the desired 2-aminosuberic acid made the preparation of these intermediates and the cyclization of the linear peptides of formula II unpredictable prior to the present invention
The synthetic preparation of 6,6-spirocycloalkylene-2-aminosuberic acids as well as the corresponding 7,7-substituted azelaic and 5,5-substituted pimelic acids involves (1) the insertion of a terminally unsaturated hydrocarbon chain at the spiro carbon of a cycloalkane carboxylate, (2) homologating the carboxylate, (3) functionalizing the unsaturated center of the side chain and (4) preparation of the amino acid therefrom. Details of the preparation are presented in Example 1.
The spirocycloalkylene-2-aminoalkandioic acid intermediates are represented by the following formula (III): ##STR3## in which: m is 2-4;
Carboxy protecting groups, which are useful at R or R.sup.1 of formula III, are well known in the peptide art, Peptide Synthesis, loc. cit. 49-57. Representative groups are lower alkyl of 1-8 carbons, benzyls, benzhydryls, phenyls all which form ester derivatives but which are easily removed by methods known to the art. Preferably, selective removal methods must be available after the linear peptide of formula II has been prepared.
Amino protective groups, which are useful in formula III at R.sup.2 are also well known, Peptide Synthesis, loc. cit. 18-48. Particularly useful are carbobenzoxy, t-butyloxycarbonyl, p-toluene sulfonyl, trifluoroacetyl, arylsulfenyl or formyl groups.
Also included in formula III are the optically active isomers. These are separated from isomeric mixtures into optically pure D- and L-isomeric form by fractional crystallization of salts with chiral bases. They can also be obtained using the novel Kolbe method of synthesis described below.
Another aspect of the invention is the chiral synthesis of these 6,6-spirocycloalkylene-2-aminoalkanedioic acids. It was unexpectedly found that the bulky cycloalkylene group did not prevent formation of the desired compounds by the Kolbe electrolysis procedure. For example, 1,1-cyclohexanediacetic acid monobenzylester was electrolyzed under Kolbe conditions with N-.alpha.-Boc-L-glutamic acid .alpha.-benzyl ester to give the pure L-isomer benzyl 2-L-Boc-amino-5-(1-carbobenzoxymethylcyclohexyl)pentanoate which is dibenzyl ester of 6,6-cyclopentamethylene-2-L-Boc-aminosuberic acid. The synthetic procedure to prepare these optically pure intermediates of formula III permits the preparation of the optically pure isomers of Formula I and Formula II compounds at the * position. Details of this procedure which gives optically pure products are given in Example 17.
The end compounds (Formula I) of the invention have vasopressin antagonist activity. Vasopressin is known to contribute to the anti diuretic mechanism of action within the kidney. When the action of these compounds antagonizes that of the natural anti-diuretic hormone (ADH), the body excretes water due to an increased permeability of the terminal portions of the renal tubule. The mechanism of action is at the vasopressin receptors (V.sub.2 -receptors) located on the plasma membrane of certain renal epithelial cells. The most notable pharmacodynamic effect of the ADH antagonists of the invention is that of a water diuretic rather than of a natriuretic such as hydrochlorothiazide.
Any patient suffering from the syndrome of inappropriate antidiuretic hormone secretion (SIADH) or from an undesirable edematous condition is a target for the claimed compounds. Examples of clinical conditions indicated for the compounds of this invention include hypertension, hepatic cirrhosis, hyponatremia, congestive heart failure or a component of any traumatic condition resulting from serious injury or disease.
The second group of vasopressin receptor sites are the vascular pressor sites (V.sub.1 -receptors) within the cardiovascular system itself. These are also somewhat antagonized by the compounds of this invention thereby inducing antipressor or hypotensive activity.
The compounds of this invention are also oxytocine antagonists and as such are useful to prevent premature labor and in the treatment of primary dysmenorrhea.
The compounds of this invention, therefore, are used especially to induce anti-hypertensive or diuretic activity in patients in need of such treatment. This comprises the administration internally, parenterally, buccally or by insufflation, of a nontoxic but effective quantity of the chosen compound, preferably dispersed in a pharmaceutical carrier. Dosage units of the active ingredient are selected from the range of 0.01 to 10 mg/kg, preferably 0.1 to 1 mg/kg, of base based on a 70 kg patient. The dosage units are administered to the human or animal patient from 1 to 5 times daily.
The novel pharmaceutical compositions of this invention, which contains an active antagonist ingredient of formula I, comprises a dosage unit which is dissolved or suspended in a standard liquid carrier, such as isotonic saline, and is contained in an ampoule or a multiple dose vial suitable for a parenteral injection such as for intravenous, subcutaneous or intramuscular administration. A composition for insufflation may be similar but is usually administered in a metered dose applicator or inhaler. Pulverized powder compositions may, also, be used along with oily preparation, gels, buffers for isotonic preparations, buccal losenges, trans-dermal patches and emulsions or aerosols.
Antagonistic activity toward the natural anti-diuretic hormone (anti ADH activity) is determined, in vitro, in the medullary tissue of hog or human kidneys and, in vivo, in the hydropenic rat. The in vitro assay procedures for vasopressin stimulated adenylate cyclase activation or vasopressin binding activity are described by F. Stassen et al., J. Pharmacology and Experimental Therapeutics, 223, 50-54 (1982).
In the test procedure for assay of adenylate cyclase activity, the amount of .sup.32 P/cAMP formed in the absence of medullary membrane is determined (blank). The blank value is subtracted from all experimental data. The compound is tested for its effect on basal adenylate cyclase activity and/or on vasopressin stimulated activity. Each determination is carried out in triplicate. The Ka value is derived from a Lineweaver-Burke plot. Rel. V.sub.max =(V.sub.max drug/V.sub.max vasopressin) x 100. K.sub.i =I/[Ka'/Ka)-1] where I is the concentration of the antagonist and Ka' and Ka are the concentrations of vasopressin required to give half maximal activity of adenylate cyclase in the presence and absence of antagonist, respectively.
In the test procedure for binding assays, the amount of .sup.3 H-vasopressin bound in the absence and in the presence of an excess of vasopressin (7.3.times.10-.sup.6 M) is measured in triplicate. These values represent total and non specific binding, respectively. The K.sub.B of a compound is derived from the equation for competitive inhibition K.sub.B =IC.sub.50 /(1+L/K.sub.D), where IC.sub.50 is the concentration required for 50% inhibition of .sup.3 H-vasopressin (K.sub.D =3.6.times.10.sup.-9 M; 1 SD=0.4.times.10 .sup.-9 M). This is the average K.sub.D value determined on 3 preparations of hog kidney membranes.
The assay for anti-ADH activity in vivo is the hydropenic rat protocol described below:
Food and water are removed from male rats approximately 18 hours prior to testing. Animals are housed 4 per metabolism cage. At 0 hour, the test compound is administered intraperitoneally to the test group and an equivalent volume of vehicle is administered to both control groups (fasted and non-fasted). Urine volume and osmolality are measured every hour for 4 hours. Test values are recorded as ml of urine excreted (cumulative), mEg/rat electrolyte excreted, mg/rat urea excreted, and osmolality in milli-Osmoles/kg H.sub.2 O. A tolerance test is used to determine significance. ED.sub.300 is defined as the dose of compound (.mu.g/kg) required to lower urine osmolality to 300 m-Osmoles/kg.
The stereochemistry of the PAS unit in compounds C and D is assigned based on the biological activities of the two compounds as well as by amino acid analysis using chiral gas chromatographic analysis with the optically active PAS obtained from the Kolbe method as the standard. The representative data of Table I demonstrate that the Pas.sup.1,6 compounds possess equivalent or substantially greater biological activity than their respective dithia compounds of the prior art.
US Referenced Citations (8)
Non-Patent Literature Citations (6)
| Entry |
| S. Hase et al., Journal of American Chemical Society, pp. 3591-3600, 5/17/1972. |
| Fahrenholz et al., Journal of Biological Chemistry, pp. 14861-14867, 12/25/1983. |
| Fahrenholz et al., Biochemical and Biophysical Research Communications, vol. 122, No. 3, pp. 974-982, Aug. 16, 1984. |
| Jost et al., Collection Czechoslov. Chem. Commun. Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences, Prague 6, Jun. 5, 1970. |
| Sawyer et al., Molecular and Cellular Endocrinology, pp. 117-134, 1981. |
| Nutt et al., J. Org. Chem., pp. 3078-3080, 1980. |
Continuation in Parts (1)
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Number |
Date |
Country |
| Parent |
819336 |
Jan 1986 |
|
Patent Grant
4760052
