Novel derivatives of arginine vasopressin antagonists

BACKGROUND OF THE INVENTION 
This invention relates to novel peptides which antagonize the antidiuretic 
and/or vasopressor action of arginine vasopressin in vivo. 
PRIOR ART STATEMENT 
Attempts to develop clinically useful synthetic antagonists of in vivo 
antidiuretic and/or vasopressor responses to arginine vasopressin, the 
antidiuretic hormone (ADH), have led to the synthesis and pharmacological 
evaluation of hundreds of analogs of the neurohypophysial peptides, 
oxytocin and vasopressin. 
Analogs of vasopressin or oxytocin which antagonize antidiuretic responses 
to ADH have been reported by Chan et al., Science, vol. 161 (1968) at 280 
and J. Pharmacol. Exp. Ther., vol. 174 (1970) at 541 vol. 196 (1976) at 
746; Dousa et al., Science, vol. 167 (1970) at 1134; Nestor et al., J. 
Med. Chem., vol. 18 (1975) at 1022 and Larsson et al., J. Med. Chem., vol. 
21 (1978) at 352, herein incorporated by reference. None of the compounds 
reported has been pharmacologically or clinically useful as an 
antidiuretic antagonist. 
The synthesis and evaluation of vasopressin analogs, incorporating 
etherified tyrosine at the 2-position, valine at the 4-position and D- or 
L-arginine at the 8-position, which antagonize the antidiuretic action of 
ADH in vivo, have been reported by Sawyer et al., Science, vol. 212 (1981) 
at 49; Manning et al., J. Med. Chem., vol. 24 (1981) at 701; and Manning 
et al., U.S. Pat. Nos. 4,367,225 and 4,399,125, herein incorporated by 
reference. 
Design of tissue-specific agonists and antagonists in the field of 
neurohypophysial peptides has been considered by Sawyer et al., Molecular 
and Cellular Endocrinology, vol. 22 (1981), 117-134; Manning et al., The 
Pituitary, Beardwell et al., eds., Butterworths, Kent, England (1981), 
265-296; Manning et al., "Peptides, Synthesis, Structure, Function," Rich 
et al., eds., Pierce Chemical Co., (1981) at 257-260 and Manning et al., 
J. Med. Chem., vol. 25 (1982) at 45 and 414. 
Modification of oxytocin, containing a D-amino acid at the 2-position has 
been disclosed by Lebl et al., Peptides, Walter de Gruyter & Co., Berlin 
(1983), at 457. Other modifications, having a penicillamine unit at the 1- 
and 6-positions of vasopressin, have been disclosed by Simek et al., ibid, 
at 461. Modification of vasopressin analogs at the 9-position, for 
example, 1-deamino[9-D-alananimide]-AVP, has been investigated by Gazis et 
al., ibid., at 465. 
It is therefore apparent that the correlation between structure of 
neurohypophysial peptides and action in vivo is not well understood and 
there is a continuing need for the development of pharmacologically and 
clinically effective antagonists of the antidiuretic action of arginine 
vasopressin. 
OBJECT OF THE INVENTION 
It is the object of the invention to provide novel antagonists of the 
antidiuretic and/or vasopressor action of ADH, which are effective in 
vivo. 
SUMMARY OF THE INVENTION 
This invention relates to novel antagonists of the antidiuretic and/or 
vasopressor action of ADH, which are compounds of the Formula I: 
##STR2## 
wherein X is (D- or L-)Tyr(R), D-Phe, D-Val, D-Ile, D-Nva, D-Nle, D-Cha, 
D-Abu, D-Thr, D-Asn, D-Gln or D-Met; Y is Val, Ile, Thr, Ala, Lys, Cha, 
Nva, Met, Nle, Orn, Ser, Asn, Gln, Phe, Tyr, Gly, Abu, or Leu; Z is (D- or 
L-)Arg, Orn or Lys; Q is Arg(NH.sub.2), Ser(NH.sub.2), (D- or 
L-)Ala(NH.sub.2), Gly, OH or NH.sub.2 and R is methyl, ethyl, propyl or 
butyl; provided that when Y is Gln or Val, R may also be H. 
This invention further relates to a method for antagonizing the in vivo 
antidiuretic and/or vasopressor response to ADH, comprising administering 
to an animal being treated an amount of a compound of Formula I, in 
admixture with a physiologically and pharmaceutically acceptable carrier, 
effective to antagonize the antidiuretic and/or vasopressor response to 
ADH. 
DETAILED DESCRIPTION 
Compounds of the invention, or comparison compounds of the prior art, are 
derivatives of arginine vasopressin. Amino acids are in the L-form, unless 
otherwise indicated. Each symbol, except for that of the 9-terminal 
substituent, is for the acyl (--C.dbd.O--) residue of the designated amino 
acid. For example, 9-OH represents a compound with a completed --COOH 
group of the amino acid residue at the 8-position and is, therefore, a 
desglycinamide compound. The correlation between full names and 
abbreviations is: 
AVP, arginine vasopressin; 
AVP-acid, deamido-arginine vasopressin, alternatively, vasopressinoic acid; 
desGly.sup.9 -AVP, desglycine-.sup.9 -arginine vasopressin; 
desGly(NH.sub.2).sup.9 AVP, desglycinamide.sup.9 -arginine vasopressin; 
d(CH.sub.2).sub.5 AVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic 
acid)]arginine vasopressin; 
d(CH.sub.2).sub.5 VDAVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
4-valine, 8-D-arginine]vasopressin; 
d(CH.sub.2).sub.5 Tyr(Me)VDAVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-O-methyltyrosine, 4-valine, 8-D-arginine]vasopressin; 
d(CH.sub.2).sub.5 -D-TyrVDAVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-tyrosine, 4-valine, 8-D-arginine]vasopressin; 
d(CH.sub.2).sub.5 -D-TyrVAVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-tyrosine, 4-valine]-arginine vasopressin; 
d(CH.sub.2).sub.5 -Tyr(Me)AVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-O-methyltyrosine]-arginine vasopressin; 
desGly.sup.9 d(CH.sub.2).sub.5 AVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
9-desglycine]-arginine vasopressin; 
desGly(NH.sub.2).sup.9 d(CH.sub.2).sub.5 AVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
9-desglycinamide]-arginine vasopressin; 
d(CH.sub.2).sub.5 [D-Phe.sup.2 ]VAVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-valine]-arginine vasopressin; 
desGly.sup.9 d(CH.sub.2).sub.5 [D-Phe.sup.2 ]VAVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-valine, 9-desglycine]-arginine vasopressin; 
desGly(NH.sub.2).sup.9 d(CH.sub.2).sub.5 [D-Phe.sup.2 ]VAVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-valine, 9-desglycinamide]-arginine vasopressin; 
d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4 ]AVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-isoleucine]-arginine vasopressin; 
desGly.sup.9 d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4 ]AVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-isoleucine, 9-desglycine]-arginine vasopressin; 
d(CH.sub.2).sub.5 [D-Tyr(Et).sup.2 ]VAVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-(O-ethyl)tyrosine, 4-valine]-arginine vasopressin; 
desGly.sup.9 d(CH.sub.2).sub.5 [D-Tyr(Et).sup.2 ]VAVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-(O-ethyl)tyrosine, 4-valine, 9-desglycine]-arginine vasopressin; 
d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4, Arg-NH.sub.2.sup.9 ]AVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-isoleucine, 9-argininamide]-arginine vasopressin; 
d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4, Ser-NH.sub.2.sup.9 ]AVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-isoleucine, 9-serinamide]-arginine vasopressin; 
d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4, Ala-NH.sub.2.sup.9 ]AVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-isoluecine, 9-alaninamide]-arginine vasopressin; 
d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4, D-Ala-NH.sub.2.sup.9 ]AVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-isoleucine, B 9-D-alaninamide]-arginine vasopressin; 
desGly.sup.9 d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4 ]AVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-isoleucine, 9-desglycine]-arginine vasopressin; 
d(CH.sub.2).sub.5 [Tyr(Et).sup.2 ]VAVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-(O-ethyl)tyrosine, 4-valine]-arginine vasopressin 
desGly.sup.9 d(CH.sub.2).sub.5 [Tyr(Et).sup.2 ]VAVP, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-(O-ethyl)tyrosine, 4-valine, 9-desglycine]-arginine vasopressin and 
d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4 ]AVP acid, 
[1-(.beta.-mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-isoleucine, 9-desamido]-arginine vasopressin. 
The active peptides were synthesized by solid phase synthesis, as described 
by Bankowski et al. (1978), infra; Merrifield, J. Am. Chem. Soc., vol. 85 
(1963) at 2149 and Biochemistry, vol. 3 (1964) at 1385; Manning, J. Am. 
Chem. Soc., vol. 90 (1968) at 1348; Manning et al., J. Med. Chem., vol. 19 
(1976) at 376; Lowbridge et al., J. Med. Chem., vol. 20 (1977) at 1173; 
Manning et al., J. Med. Chem., vol. 16 (1973) at 975; Kruszynski et al. 
(1980), infra; Sawyer et al., (1981), supra or Manning et al. (1981), 
supra. 
Compounds of the 9-desglycine group, that is, Q is NH.sub.2, are prepared 
as for other arginine vasopressin derivatives, except that one less cycle 
of deprotection, neutralization and coupling is employed. That is, the 
first amino acid residue, attached to resin, will be (D- or L-)Arg, rather 
than Gly, as would be the case for compounds in which Q is to be 
GlyNH.sub.2. 
Compounds in which the amino acid at the 9-position is other than 
Gly(NH.sub.2) are prepared in a similar fashion, but by attaching an amino 
acid other than glycine to the resin at the start of the synthesis. 
Desglycinamide compounds, that is Q is OH, in accordance with this 
invention are prepared as described in the examples. 
The discovery of the antidiuretic antagonists d(CH.sub.2).sub.5 
Tyr(alk)VAVP, Sawyer et al. (1981), supra, and Manning et al. (1981), 
supra, led to the synthesis of various analogs having a 
cyclopentamethylene ring structure and various substituents at the 
2-position. Other modifications at the 4-, 6-, 7- and 8-positions, have 
been proposed. 
It will also be understood that, when alkyl substituents (R) can be linear 
or branched, contemplated equivalents include all possible isomers. 
Compounds of this invention, having an action antagonistic toward the 
antidiuretic action of arginine vasopressin are generally those wherein 
the 2-substituent is an amino acid of the D-series and the 4-substituent 
is an aliphatic amino acid. When the 4-substituent is Gln and the 
2-substituent is of the L-series, it has been found that the compounds 
generally have specific antivasopressor action. 
Most of compounds of Formula I are accordingly very effective antagonists 
of the antidiuretic response to ADH. They can therefore be used in 
pharmacological studies on the contribution of ADH to a variety of 
pathological states involving water retention. It is further contemplated 
that they could be effective and specific agents for treating the syndrome 
of inappropriate secretion of ADH, that is, the Schwartz-Bartter syndrome 
or SIADH. This syndrome can complicate a number of disorders, including 
carcinomas, pulmonary diseases, intracranial diseases and head injuries, 
Bartter et. al., Am. J. Med., vol. 42 (1967) at 790. In addition, 
compounds of Formula I are effective as antagonists of the vasopressor 
response to ADH. 
Compounds of Formula I, wherein X is Tyr and Y is Gln or X is Tyr(R), Y is 
Gln and R is H, methyl, ethyl, propyl or butyl, can be used in a similar 
fashion for conditions in which antivasopressor activity is indicated. 
Surprisingly, activity of the compounds of the invention as antagonists of 
ADH or vasopressin is not lost by removal or modification of the 
9-substituent. It is known that the chemical bond between the 8- and 
9-positions is at least one point at which digestive enzymes, such as 
trypsin, attack vasopressin-type hormones. Therefore, compounds of this 
invention, if not themselves entirely stable toward digestive enzymes, 
will be useful in suggesting routes to peptide derivatives which will 
retain their activity when administered orally. 
Retention of activity following hydrolysis of the C-terminal glycinamide of 
arginine vasopressin compounds, in accordance with this invention, is 
highly unexpected, in view of tryptic digestion of AVP to give 
desglycinamide.sup.9 -AVP, which had neither the antidiuretic nor the 
vasopressor activity, characteristic of AVP. See, du Vigneaud et al., J. 
Am. Chem. Soc., vol. 75 (1953) at 4880. 
The compounds of this invention can be employed in mixtures with 
conventional excipients, i.e., physiologically and pharmaceutically 
acceptable organic or inorganic carriers suitable for parenteral or other 
application, provided that the carriers do not interact deleteriously with 
the active compounds. 
Suitable pharmaceutically acceptable carriers include, but are not limited 
to, water, salt solutions, alcohols, vegetable oils, polyethylene glycols, 
gelatine, lactose, amylose, magnesium stearate, talc, silicic acid, 
viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, 
pentaerythritol fatty acid esters, hydroxymethyl cellulose, polyvinyl 
pyrrolidone, etc. The pharmaceutical preparations can be sterilized and, 
if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, 
stabilizers, wetting agents, emulsifiers, salts for influencing the 
osmotic pressure, buffers, coloring, flavoring and/or aromatic substances, 
which do not deleteriously interact with the active compounds. 
For parenteral or intranasal application, solutions, preferably aqueous 
solutions, as well as suspensions, emulsions or implants, including 
suppositories, are particularly suitable. Ampoules are convenient unit 
dosages. 
For enteral application, particularly suitable are tablets, dragees, or 
capsules, having talc and/or a carbohydrate carrier or binder or the like, 
the carrier preferably being lactose and/or corn starch and/or potato 
starch. A syrup, elixir or the like can be used, wherein a sweetened 
vehicle is employed. Sustained release compositions can be formulated, 
including those wherein the active compound is protected with 
differentially degradable coatings, e.g., by microencapsulation, multiple 
coatings, etc. 
The compounds of Formula I are generally administered to animals, including 
but not limited to mammals, e.g, livestock, household pets, humans, 
cattle, cats and dogs. A diuretically effective daily dosage of the active 
compounds can be administered parenterally in a single dosage or as 
divided dosages throughout the day. 
Parenteral or intranasal administration is preferred. The compounds of this 
invention are particularly valuable in the treatment of humans afflicted 
with water retention of any etiology. In this regard, they can be 
administered in substantially the same manner as the known compounds 
oxytocin and vasopressin, to achieve their physiological effects. 
It will be appreciated that the actual preferred amounts of active 
compounds used will vary according to the specific compound being 
utilized, the particular compositions formulated, the mode of application, 
and the particular organisms being treated. Optimal application rates 
under/in a given set of conditions can be ascertained by those skilled in 
the art of using conventional dosage determination tests in view of the 
above guidelines.

DESCRIPTION OF PREFERRED EMBODIMENT 
Preferred antidiuretic antagonists of Formula I are those wherein: 
(a) X is D-Phe; 
(b) X is D-Tyr(R) and R is methyl, ethyl, propyl or butyl; 
(c) Y is Val, including each of (a)-(b); 
(d) Y is Ile, including each of (a)-(b); 
(e) Z is Arg, including each of (a)-(d); 
(f) Q is NH.sub.2, including each of (a)-(e); 
(f) Q is Arg(NH.sub.2), including each of (a)-(e); 
(h) Q is Ser(NH.sub.2), including each of (a)-(e); 
(i) Q is (D- or L-)Ala(NH.sub.2), including each of (a)-(e) 
(j) n is 5, including each of (a)-(i). 
Most preferred compounds are those wherein n is 5, X is D-Phe, Y is Ile, Z 
is Arg and Q is NH.sub.2 or Ala(NH.sub.2). 
Preferred compounds are those in which Y is Gln are those wherein Z is Arg. 
Without further elaboration, it it believed that one skilled in the art 
can, using the preceding description, utilize the present invention to its 
fullest extent. The following specific embodiments are, therefore, to be 
construed as merely illustrative and not limitative of the remainder of 
the disclosure in any way whatsoever. In the following Examples, the 
temperatures are set forth uncorrected in degrees Celsius. Unless 
otherwise indicated, all parts and percentages are by weight. 
Chloromethylated resin (Bio-Rad Bio-Beads SX-1) was esterified by the 
procedure of Gisin, Helv. Chim. Acta., vol. 56 (1973) at 1476 with Boc-Gly 
until 0.47 mmol./g. and .about.0.64 mmol/g were incorporated. Amino acid 
derivatives, including Boc-Tyr(Me) (R.sub.f (A) 0.7, R.sub.f (B) 0.8) were 
supplied by Bachem or synthesized. 
Triethylamine (TEA) and N-methylmorpholine (NMM) are distilled from 
ninhydrin. 
Acetic acid used as the HCl-acetic acid cleavage reagent was heated under 
reflux with boron triacetate and distilled from the reagent. 
Dimethylformamide (DMF) was distilled under reduced pressure immediately 
before use. Methanol was dried with magnesium methoxide and distilled. 
Other solvents and reagents were analytical grade. 
Thin layer chromatography (TLC) was done on silica gel plates (0.25 mm, 
Brinkmann Silplate) using the following solvent systems: A. 
cyclohexane-chloroform-acetic acid (2:8:1 v/v); B. propan-1-ol-ammonia 
(34%) (2:1 v/v); C. ethanol (95%)-ammonia (34%) (3:1 v/v); D. 
chloroform-methanol (7:3 v/v); E. butan-1ol-acetic acid-water (4:1:5 v/v, 
upper phase); F. butan-1-ol-acetic acid-water-pyridine (15:3:3:10 v/v). 
The applied loadings were 10-50 .mu.g. The minimum length of the 
chromatograms was 10 cm. Chloroplatinate reagent and iodine vapor were 
used for development of the chromatograms. 
Amino acid analysis of the peptides was done by the method of Spackman et 
al., Anal. Chem., vol. 30 (1958) at 1190, in which peptide samples 
weighing about 0.5 mg were hydrolyzed with constant boiling hydrochloric 
acid (400 .mu.l) in evacuated and sealed ampuoles for 18 h at 120.degree. 
C. The analyses were performed using a Beckman Automatic Amino Acid 
Analyzer, Model 121. Molar ratios were referred to Gly=1.00. Elemental 
analyses were performed by Galbraith Laboratories, Inc., Knoxville, Tenn. 
The analytical results for the elements indicated by their respective 
symbols were within .+-.0.4% of theoretical values. Optical rotations were 
measured with a Bellingham Stanley, Ltd., Model A polarimeter, type pl. 
EXAMPLE 1 
.beta.-(S-Benzylmercapto)-.beta.,.beta.-cyclopentamethylenepropionyl-Tyr(Me 
)-Phe-Gln-Asn-Cys(Bzl)-Pro-Arg(Tos)-Gly-NH.sub.2 
A. Combination of Solid Phase and Solution Methods 
Boc-Tyr(Me)-Phe-Gln-Asn-Cys(Bzl)-Pro-Arg(Tos)-Gly-NH.sub.2, prepared by the 
method of Bankowski et al., J. Med. Chem., vol. 21 (1978) at 850 (319 mg, 
0.26 mmol), was dissolved in CF.sub.3 COOH (6.5 ml) and stirred at room 
temperature for 40 mins. Cold ether (20 ml) was added to produce a 
precipitate, which was filtered and washed with ether (5.times.10 ml). The 
product was dried in vacuo over sodium hydroxide pellets. This material 
(318.5 mg) was dissolved in DMF (0.8 ml), to which was added 
N-methylmorpholine (10 l). The resulting solution had a pH of 7-8, 
measured with moist pH paper. After this neutralized solution was stirred 
at room temperature for 30 mins, a solution of p-nitrophenyl 
.beta.-(S-benzylmercapto)-.beta.,.beta.-cyclopentamethylenepropionate, 
Nestor et al., J. Med. Chem., vol. 18 (1975) at 284, (445 mg, 1.155 mmol 
in 0.4 ml of DMF) was added. The reaction mixture was stirred at room 
temperature. After 72 hours' stirring, TLC analysis using system D showed 
that the reaction mixture still contained a trace of the free octapeptide 
amide. N-Hydroxybenzotriazole monohydrate, Konig et al., Chem. Ber., vol. 
103 (1970) at 788, (39.3 mg, 0.26 mmol) was added. Coupling was complete 
within 5 hours. The precipitate was filtered, washed with cold ethyl 
acetate (4.times.10 ml) and dried in vacuo. The crude product (339 mg) was 
twice reprecipitated from DMF-methanol to give the acylpeptide amide 
(295.2 mg, 77.3%): mp 209.degree.-211.degree. C., [.alpha.].sub.D.sup.24 
=-43.6.degree. (c 0.5, DMF); R.sub.f (E) 0.45, R.sub.f (F) 0.63 Anal. 
(C.sub.73 H.sub.94 O.sub.14 N.sub.14 S.sub.3) C, H, N. 
(b) Total Synthesis on Resin 
Boc-Tyr(Me)-Phe-Gln-Asn-Cys(Bzl)-Pro-Arg(Tos)-Gly-resin (1.11 g, 0.4 mmol 
prepared from Boc-Gly-resin using solid phase methodology) was converted 
to the acylotapeptide resin (1.167 g, weight gain 57 mg, 97.6% of theory) 
in one cycle of deprotection, neutralization and coupling with 
p-nitrophenyl 
.beta.-(S-benzylmercapto)-.beta.,.beta.-cyclopentamethylenepropionate, see 
Nestor, supra. The resin was ammonolyzed, Manning, J. Am. Chem. Soc., vol. 
90 (1968) at 1348. The product was extracted with DMF. After the solvent 
was evaporated in vacuo, the residue was precipitated by addition of 
water. The crude product (410 mg) was twice reprecipitated from 
DMF-ethanol to give the acyloctapeptide (302 mg, 50.7% based upon initial 
glycine content of the resin); mp 206.degree.-208.degree. C. (decomp); 
R.sub.f (E) 0.45; R.sub.f (F) 0.63; [.alpha.].sub.D.sup.24 =-43.1.degree. 
(c 1, DMF). Anal. (C.sub.73 H.sub.94 N.sub.14 O.sub.14 S.sub.3) C, H, N. 
Amino acid analysis: Tyr, 0.79; Phe, 1.01; Glu, 1.03; Asp, 1.04; Cys(Bzl), 
0.97; Pro, 1.03; Arg, 0.99; Gly, 1.00; NH.sub.3, 2.95. 
EXAMPLE 2 
.beta.-(S-Benzylmercapto)-.beta.,.beta.-cyclopentamethylenepropionyl-Tyr(Bz 
l)-Phe-Gln-Asn-Cys(Bzl)-Pro-Arg(Tos)-Gly-HN.sub.2 
Boc-Tyr(Bzl)-Phe-Gln-Asn-Cys(Bzl)-Pro-Arg(Tos)-Gly-resin (1.46 g, 0.5 mmol) 
was converted to the acyloctapeptide resin (1.55 g, weight gain 70 mg, 
95.9% of theory) as in Example 1 by one cycle of deprotection, 
neutralization and coupling with p-nitrophenyl 
.beta.-(S-benzylmercapto)-.beta.,.beta.-cyclopentamethylenepropionate. The 
product obtained by ammonolysis of the resin was extracted with DMF. The 
solvent was evaporated in vacuo and the residue was precipitated by 
addition of water. The crude product (723 mg) was reprecipitated from 
DMF-ethanol and DMF-2% aqueous AcOH. Yield: 488 mg (62.4% based on initial 
Gly content on the resin); mp. 183.degree.-185.degree. C.; R.sub.f (E) 
0.38; R.sub.f (D) 0.41; [.alpha.].sub.D.sup.23 =-23.9.degree. (c 1, DMF). 
Anal. (C.sub.79 H.sub.98 N.sub.14 O.sub.14 S.sub.3) C, H, N. 
Amino acid analysis: Tyr, 0.97; Phe, 1.02; Glu, 1.05; Asp, 1.01; Cys(Bzl), 
0.98; Pro, 1.04; Arg, 0.98; Gly, 1.00; NH.sub.3. 
EXAMPLE 3 
[1-(.beta.-Mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-(O-methyl)tyrosine]arginine vasopressin 
(a) From Nonapeptide Amide 
A solution of the protected nonapeptide amide, prepared as in Example 1, 
(170 mg, 0.114 mmol) in 400 ml of ammonia (dried over sodium and 
redistilled) was stirred at the boiling point with sodium from a stick of 
the metal, contained in a small bore glass tube until a light blue color 
persisted in the solution for 30 sec, in accordance with du Vigneaud, J. 
Am. Chem. Soc., vol. 76 (1954) at 3115. Dry glacial acetic acid (0.4 ml) 
was added to discharge the color. The solution was evaporated. A solution 
of the residue in aqueous acetic acid (0.2%, 800 ml), was treated with 2M 
ammonium hydroxide solution to give a solution of pH 7.5. To this stirred 
solution was added gradually an excess of a solution of potassium 
ferricyanide (0.01M, 11.4 ml), Hope et al., J. Biol. Chem., vol. 237 
(1962) at 1563. The yellow solution was stirred for 90 min more and for 1 
h with anion-exchange resin (BioRad AG-3, Cl.sup.- form, 10 g damp 
weight). The suspension was filtered slowly through a bed of resin (80 g 
damp weight). The resin bed was washed with 300 ml of aqueous acetic acid 
and the combined filtrate and washings were lyophilized. The resulting 
powder (1386 mg) was desalted on a Sephadex G-15 column (110.times.2.7 cm) 
and eluted with aqueous acetic acid (50%) at a flow rate of 4 ml/h by the 
technique of Manning et al., J. Chromatog., vol. 38 (1968) at 396. The 
eluate was fractionated and monitored for absorbance at 280 nm. The 
fractions comprising the major peak were pooled and lyophilized. The 
residue (55.5 mg) was further subjected to gel filtration on a Sephadex 
G-15 column (100.times.1.5 cm) and eluted with aqueous acetic acid (0.2M) 
at a flow rate of 2.5 ml/h. The peptide was eluted in a single peak 
(absorbance 280 nm). Lyophilization of the pertinent fractions yielded the 
vasopressin analog (49 mg, 37.3%); R.sub.f (E) 0.19; R.sub.f (F) 0.30; 
[.alpha.].sub.D.sup.22 =-59.6.degree. (c 0.19, 1M AcOH). 
Amino acid analysis: Tyr 0.81; Phe, 1.01; Glu, 1.04; Asp, 0.98; Pro, 1.04; 
Arg, 0.95; Gly, 1.00; NH.sub.3 3.10. Analysis following performic acid 
oxidation prior to hydrolysis according to Moore, J. Biol. Chem., vol. 238 
(1963) at 235, gave a Cys-(O.sub.3 H)-Gly ratio of 1.03:1.00. 
(b) From Acyloctapeptide 
Treatment of the acyloctapeptide (160 mg, 0.107 mmol) as described in 
Example 3(a) yielded the analog (64 mg, 51.7%), which was 
indistinguishable from the foregoing preparation by TLC: 
[.alpha.].sub.D.sup.23 =-59.1.degree. (c 0.5, 1M AcOH). 
Amino acid analysis: Tyr, 0.80; Phe, 1.02; Glu, 1.02; Asp, 0.98; Pro, 1.03; 
Arg, 0.96; Gly, 1.00; NH.sub.3, 3.05. Analysis following performic acid 
oxidation prior to hydrolysis gave a Cys-(O.sub.3 H)-Gly ratio of 
1.02:1.00. 
EXAMPLE 4 
[1-(.beta.-Mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-substituted, 4-substituted, 9-desglycine]-arginine vasopressin 
The compounds were made in the same manner as the compounds of Examples 
1-3, starting from Boc-Arg(Tos) resin, except that one fewer cycle of 
deprotection, neutralization and coupling was employed. Protected 
intermediates for each analog were obtained. Coupling with 
.beta.-(S-benzylmercapto)-.beta.,.beta.-cyclopentamethylenepropionate was 
done in accordance with Nestor, supra. 
Each precursor was deblocked with sodium in liquid ammonia to produce a 
sulfhydryl compound. The latter compounds were oxidatively cyclized with 
potassium ferricyanide, as in the preceding Examples. The analogs were 
desalted and purified by gel filtration on Sephadex G-15 by a two-step 
procedure using 50% acetic acid and 0.2M acetic acid, respectively, as 
eluants. The purity and identity of each analog was ascertained by 
thin-layer chromatography in three different solvent systems, BAW I 
(butan-1-ol-acetic acid, water 4:1:1 v/v), BAWP (butan-1-ol-acetic 
acid-water-pyridine 15:3:3:10 v/v) and BAW II (butan-1-ol-acetic 
acid-water 4:1:5 v/v, upper phase) with the following results: 
______________________________________ 
R.sub.f 
(BAW 
Compound (BAW I) (BAWP) II) 
______________________________________ 
desGly.sup.9 AVP 0.04 0.27 0.23 
desGly.sup.9 d(CH.sub.2).sub.5 [D-Phe.sup.2 ]VAVP 
0.39 0.59 0.39 
desGly.sup.9 d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4 ]AVP 
0.41 0.63 0.40 
desGly.sup.9 d(CH.sub.2).sub.5 [D-Tyr(Et).sup.2 ]VAVP 
0.39 0.60 0.38 
desGly.sup.9 d(CH.sub.2).sub.5 [Tyr(Et).sup.2 ]VAVP 
0.41 0.65 0.41 
______________________________________ 
EXAMPLE 5 
[1-(.beta.-Mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
9-desglycinamide]arginine vasopressin 
This compound, desGly(NH.sub.2).sup.9 d(CH.sub.2).sub.5 AVP, was obtained 
from the protected precursor, 
.beta.-(S-benzylmercapto)-.beta.,.beta.-cyclopentamethylenepropionyl-Tyr(B 
zl)Phe-Gln-Asn-Cys(Bzl)Pro-Arg(Tos)Bzl, which was synthesized in solution 
by the stepwise procedure of Bodansky et al., J. Am. Chem. Soc., vol. 81 
(1959) at 1173, starting from Boc-Arg(Tos)Bzl. Following cleavage of the 
Boc group with 1N HCl/HOAc and neutralization with triethylamine, 
successive couplings with Boc-Pro and Boc-Cys(Bzl) were performed, in the 
presence of dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBT) 
in accordance with Sheehan et al., J. Am. Chem. Soc., vol. 77 (1955) at 
1067 and Konig et al., Chem. Ber., vol. 103 (1970) at 788. 
The Asn and Gln residues were incorporated as corresponding Boc nitrophenyl 
esters. 1-Cyclohexyl-3-[2-morpholinyl-(5)-carbodiimide] (CMCD) and HOBT 
were used for coupling of Boc-Phe and Boc-Tyr(Bzl) to give the protected 
heptapeptide benzyl ester. Coupling with p-nitrophenyl 
.beta.-(S-benzylmercapto)-.beta.,.beta.-cyclopentamethylene propionate, by 
the procedure of Nestor et al., supra, yielded the required protected 
intermediate. Deblocking with sodium in liquid ammonia and purification 
were carried out as above. 
Purity of the compound was determined as above, with the following R.sub.f 
values: 
BAW I: 0.07 
BAWP: 0.18 
BAW II: 023 
EXAMPLE 6 
[1-(.beta.-Mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-valine, 9-desglycineamide]-arginine vasopressin 
The compound, desGly(NH.sub.2).sup.9 d(CH.sub.2).sub.5 [D-Phe.sup.2 ]VAVP, 
was obtained from the protected precursor, 
.beta.-S-(benzylmercapto)-.beta.,.beta.-cyclopentamethylenepropionyl-D-Phe 
-Phe-Val-Asn-Cys(Bzl)-Pro-Arg(Tos)Bzl, which was synthesized in solution, 
starting from Boc-Phe-Val-Asn-Cys(Bzl)-Pro-Arg(Tos)-Bzl, prepared by 
custom synthesis by Alpha Biomedicals, Inc., San Carlos, Calif., except 
that cleavage of the Boc groups was done by trifluoroacetic acid, 
Boc-D-Phe was used instead of Boc-Tyr(Bzl) and coupling of 
.beta.-(S-benzylmercapto)-.beta.,.beta.-cyclopentamethylenepropionic acid 
was mediated by DCC and HOBT to give the required protected intermediate. 
The product was characterized by TLC with the following R.sub.f values: 
BAW I: 0.40 
BAWP: 0.46 
BAW II: 0.39 
EXAMPLE 7 
[1-(.beta.-Mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-isoleucine, 9-desamido]-arginine vasopressin 
A compound of the formula d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4 ]AVP 
acid was obtained from a protected precursor, 
.beta.-S-(benzylmercapto)-.beta.,.beta.-cyclopentamethylenepropionyl-D-Phe 
-Phe-Ile-Asn-Cys(Bzl)-Pro-Arg(Tos)-Gly, which was synthesized by the solid 
phase method, starting from Boc-resin as in the foregoing examples, except 
that the partially protected precursor was cleaved from the resin by 
acidolysis with hydrogen bromide in trifluoroacetic acid containing 
anisole by the method of Walter et al., J. Med. Chem., vol. 19 (1976) at 
376. After evaporation of the trifluoroacetic acid, the product was 
obtained by precipitation from an acetic acid solution with diethyl ether. 
The intermediate was deblocked with sodium in liquid ammonia, reoxidized, 
lyophilized and purified as in the foregoing examples. The product was 
characterized by TLC, with the following values: 
BAW I: 0.15 
BAWP: 0.39 
BAW II: 0.29 
EXAMPLE 8 
Antagonism to the vasopressor response was estimated in accordance with 
Dyckes et al., J. Med. Chem., vol. 17 (1974) at 969. The values are 
expressed as pA.sub.2 values, defined as in Schild et al., Br. J. 
Pharmacol., vol. 2 (1947) at 189. 
Activity as antidiuretic agonists was determined by intravenous injection 
of the compounds being evaluated in ethanol-anesthesized water-loaded rats 
in accordance with Sawyer, Endocrinology, vol. 63 (1958) at 694. 
Antagonism of the antidiuretic response to subsequent injections of 
arginine vasopressin was tested by the method of Sawyer et al., Science, 
vol. 212 (1981) at 49. 
Antagonistic potencies were determined and expressed as "effective doses" 
and pA.sub.2 values. The "effective dose" is defined as the dose (in 
nanomoles per kilogram) that reduces the response seen from 2x units of 
agonist injected 20 min after the dose of antagonist to the response with 
1x units of agonist. Estimated in vivo "pA.sub.2 " values represent the 
negative logarithms of the effective doses, divided by the estimated 
volume of distribution (67 ml/kg). Results are given in Table 1. 
EXAMPLE 9 
[1-(.beta.-Mercapto-.beta.,.beta.-cyclopentamethylenepropionic acid), 
2-D-phenylalanine, 4-isoleucine, 9-substituted]-arginine vasopressin 
(a) Synthesis 
Compounds of this series were prepared as in the foregoing examples, except 
that an amino acid, other than glycine, was attached to the resin in the 
first cycle of the synthesis. The compounds obtained were of the formula 
d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4, Q.sup.9 ]AVP and were 
characterized by TLC as in the foregoing examples. The following results 
were obtained: 
______________________________________ 
R.sub.f 
Q (BAW I) (BAWP) (BAW II) 
______________________________________ 
Arg(NH.sub.2) 
0.11 0.37 0.27 
Ser(NH.sub.2) 
0.39 0.59 0.35 
Ala(NH.sub.2) 
0.41 0.64 0.37 
D-Ala(NH.sub.2) 
0.42 0.64 0.37 
NH.sub.2 0.45 0.64 0.41 
______________________________________ 
(b) Evaluation 
The compounds were evaluated as in Example 6 to determine effective doses 
as antagonists of the antidiuretic action of arginine vasopressin. The 
following results were obtained: 
______________________________________ 
Effective dose (nmoles/kg) 
Q Anti-ADH AntiVP 
______________________________________ 
Arg(NH.sub.2) 0.67 .+-. 0.15 
0.55 .+-. 0.09 
Ser(NH.sub.2) 0.55 .+-. 0.09 
0.54 .+-. 0.09 
NH.sub.2 0.66 .+-. 0.17 
0.96 .+-. 0.09 
Ala(NH.sub.2) 0.31 .+-. 0.07 
4.8 .+-. 1.2 
D-Ala(NH.sub.2) 2.9 .+-. 0.4 
1.93 .+-. 0.23 
______________________________________ 
These results show that the indicated modifications at the 9-position 
produce compounds which antagonize the antidiuretic action of AVP. 
TABLE 1 
__________________________________________________________________________ 
Agonistic Activities 
units/mg Anti-ADH Anti-VP 
Compound ADH VP ED pA.sub.2 
ED pA.sub.2 
__________________________________________________________________________ 
AVP 330 .+-. 23 
382 .+-. 5 
-- -- -- -- 
AVP-acid.sup.a,b 4.7 .+-. 0.6 
0.03 -- -- -- -- 
desGly.sup.9 AVP 164 .+-. 4 
0.05.sup.+ 
-- -- 68 .+-. 16 
6.09 .+-. 0.10 
desGly.sup.9 (NH.sub.2)AVP.sup.c 
5.6 .+-. 1.1 
0.02.sup.+ 
-- -- -- -- 
d(CH.sub.2).sub.5 AVP 
0.03 .+-. 0.01 
-- -- -- 0.56 .+-. 0.11 
8.16 .+-. 0.09 
desGly.sup.9 -d(CH.sub.2).sub.5 AVP 
0.003.sup.+ 
-- -- -- 0.27 .+-. 0.04 
8.40 .+-. 0.06 
desGly(NH.sub.2).sup.9 --d(CH.sub.2).sub.5 AVP 
0.04 .+-. 0.01 
-- -- -- 0.73 .+-. 0.07 
7.88 .+-. 0.06 
d(CH.sub.2).sub.5 [D-Phe.sup.2 ]VAVP 
weak.sup.+ 
-- 0.67 .+-. 0.13 
8.06 .+-. 0.09 
0.58 .+-. 0.04 
8.06 .+-. 0.03 
desGly.sup.9 -d(CH.sub.2).sub.5 [D-Phe.sup.2 ]VAVP 
-- -- 0.58 .+-. 0.11 
8.09 .+-. 0.08 
0.47 .+-. 0.04 
8.15 .+-. 0.03 
desGly(NH.sub.2).sup.9 --d(CH.sub.2).sub.5 -- 
-- -- 1.30 .+-. 0.35 
7.75 .+-. 0.11(4) 
0.80 .+-. 0.08 
7.93 .+-. 0.05(4) 
[D-Phe.sup.2 ]VAVP 
d(CH.sub.2).sub.5 [Tyr(Et)].sup.2 VAVP 
0.03.sup.+ 
-- 1.9 .+-. 0.2 
7.57 .+-. 0.06 
0.49 .+-. 0.11 
8.16 .+-. 0.09 
desGly-d(CH.sub.2).sub.5 [Tyr(Et).sup.2 ]VAVP 
-- -- 1.0 .+-. 0.2 
7.89 .+-. 0.09 
0.45 .+-. 0.02 
8.18 .+-. 0.02 
d(CH.sub.2).sub.5 [D-Tyr(Et).sup.2 ]VAVP 
weak.sup.+ 
-- 1.1 .+-. 0.2 
7.81 .+-. 0.07 
0.45 .+-. 0.11 
8.22 .+-. 0.12 
desGly-d(CH.sub.2).sub.5 [D-Tyr(Et).sup.2 ]VAVP 
-- -- 1.8 .+-. 0.3 
7.58 .+-. 0.07 
0.45 .+-. 0.04 
8.17 + 0.04 
d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4 ]AVP 
-- -- 0.46 .+-. 0.07 
8.24 .+-. 0.08 
0.99 .+-. 0.12 
7.86 .+-. 0.05 
desGly.sup.9 -(CH.sub.2).sub.5 - 
-- -- 0.66 .+-. 0.17 
8.05 .+-. 0.09 
1.0 .+-. 0.1 
7.84 .+-. 0.03 
[D-Phe.sup.2, Ile.sup.4 ]AVP 
d(CH.sub.2).sub.5 [D-Phe.sup.2, Ile.sup.4 ]AVP acid 
-- -- 6.42 .+-. 2.06 
7.11 .+-. 0.11 
7.95 .+-. 1.28 
6.94 .+-. 0.08 
__________________________________________________________________________ 
.sup.a AVPacid was purchased from Bachem, Inc. 
.sup.b This compound was originally reported as being an ADH antagonist i 
vitro and in vivo, Dousa et al., supra. 
These results show it is an antidiuretic agonist in vivo. 
.sup.c Originally obtained by tryptic cleavage of AVP, du Vigneaud et al. 
J. Am. Chem. Soc., vol. 75 (1953) at 4880. 
.sup.+ Compounds showed weak partial agonistic activities in these assays 
in a way not clearly related to dose. 
The preceding examples can be repeated with similar success by substituting 
the generically or specifically described reactants and/or operating 
conditions of this invention for those used in the preceding Examples. 
From the foregoing description, one skilled in the art can easily ascertain 
the essential characteristics of this invention and, without departing 
from the spirit and scope thereof, can make various changes and 
modifications of the invention to adapt it to various usages and 
conditions.