Note: Descriptions are shown in the official language in which they were submitted.
This is a division of application Serial No.
397,464 filed March 3, 19~2.
This invention relates to novel peptides which
antagonize the antidiuretic action of arginlne vasopressin
in vivo.
~ ttempts to develop clinically useful synthetic
antagonists o~ in vivo an~idiuretic and/or vasopressor
responses to arginine vasopressln, the antidiuretic
hormone ~ADH3, have led to the synthe~l~ and
pharmacological evaluation of hundred~ of analogs of
the neurohypophysial peptide~, oxytocin and vasopressin.
~ nalogs which can effectively antagonize in vivo
vasopressor responses to ADM have been reported by
Dyckes et al., J. Med. Chem., vol. 17 (1974) at
__
250 Manning et al., J Med. _hem., vol.20 (1977) at
1228: Bankowski et al., J. Med. _hem., vol. 21 (1978)
at 850 Kruszynski et al., J. Med. Chem., vol. 23
(1980) at 364 and Lowbridge et al., J. Med. Chem.,
vol. 21 (1978) at 313.
Krus~ynski et al. reported that [l-(~-mercapto-~,
~-cyclopentamethylenepropionic acid), 2-(O-methyl)-
tyroslne]arginine vasopressin and (l-~-mercapto-~,
~-cyclopentamethylenepropionic acidl-arginine
vasopressin are potent vasopressor antagonists, which
also have very low antidluretic potency.
Manning et al. (1977) described the synthesis
of [l-dea~inopeniciilamine, 4-valine, 8-D-arginine3
va~opressin and Lowbridge et al. the ~ynthesis o
[l-(~-mercapto-~ cyclopentamethylenepropionic
acid), 4-valine, 8-D-arginine~ vasopressin. sOth of
these compounds have weak antidiuretic activity and
are potent antagonists of the vasopressor response
t~ ~VP.
i~
-~Z(~3()~7
Analogs of vasopressin or oxytocin which antagoni~e
antidiuretic responses to ~D~I have been reported by Chan
et al., Science, vol. 161 (196~) at 2~0 and J. _ha macol.
~. Ther., vol. 174 (1970) at 541 and vol. 196 (1976) at
746; Nestor et al., J. Med. Chem., vol. l~ 75) at 1022
and Larsson et al., J. Med. Chem., vol. 21 (197R) at 7~6.
None of the compounds
reported has been pharmacologically or clinically useFul
as an antidiuretic antagonist.
The synthe~is and evaluation of vasopressin analogs,
incorporating etherified tyrosine at the 2-position,
valine at the 4-position and D- or L-~rg at the 8-position,
which antagonize the anti-diuretic act;.on ~H in _~vo have
been reported by Sawyer et al., Science, vol. ~12 (1981)
at 49 and by Manning et al., J. Med. _hem., vol. 2~ (19Rl)
at 701.
Synthetic vasopressins have been disclosed in the
follow.ing U~S. Patents:
3,371,080 Boissonnas et al.
3,415,805 Siedel et al.
3,418,3U7 sOissonnas et al.
3~454,549 Boissonnas et al.
3,497,491 Zaoral
4,148,787 Mulder et al.
25 Of these references, Bolssonnas et al., '080 discloses that
2-phenylalanine-R-ornithine vasopressin has a vasoconstric-
tive action equal to that of natural vasopressins but low
antidiu~etic activity. The remaining re~erences disclose
synthetic vasopressins having high or relatively specific
3~ antidiuretic activity.
Synthetic modification~ of oxytocin are
disclosed by Mannlng in U.S. Patent~ 3,691,147
and 3,700,652.
.
'
i
~LZ()~5g~7
It is therefore apparent that there is a con-
tinuing need for the development of pharmacologically
and clinically effective antagonists of the anti-
diuretic action of arginine vasopressin.
It is an object of the invention to provide
1~ antagoni~ts to the antidiuretic action of ADH, which
are effective in vivo.
The parent apPlication relates t~ n,o~?el an-
tagonists of the antidiure,tic action of ADII, which are
compounds of the formula
1 2 3 4 5 6 7 8 9
C112-CO-Tyr(X~Phe-Val-~sn-Cy-W-Z-Gly-N~12
/CH2-C~I
CH C
~ ~I
CH -Cl S S
wherein X'is methyl, ethyl, n-propyl, isopropyl or
2~ butyl, Tyr is D- or L-; W i9 Pro or ~3-Pro and Z is L-
or D- Arg.
The parent application further relates to antagonis-ts
o~ the ADH activity of arginine vasopressin, of the formula
1 2 3 4 5 6 7 ~ 9
' CH2-CO-X-Phe-Val-~sn-Cy-Pro-%-Gly-NHz
CH -C~
CH C
~CH2-C~I '1 s
wherein X is D-Phe, D-Val, D-Leu, D-Ile, D-~rg, D-norvaline,
3~ D-norleucine, D-cyclohexylalanine, D-~-aminobutyric acid,
D-threonine or D-methionine and 7, is D- or L~rg.
:~ ~t~ S~'7
3a
The parent aPPlication also provides a Process for
the production of a compound of the formula
~ ~ Ct~2-CO-X-Phs-~tal~ n~-Cy-W-Z-Gly-N112
(CT~2 ) C . .'.
S /1
~ S ' S
whereln X is Tyr-X', D-Phe, D-Val, D-Lou, D-Il~ Arg,
-norval~ne, ~-norlauc~n~, D-cvcloh~xylalan~ne, D-~'-amlno-
butyric acid, D-threonine or D-methionine; X' is methyl,
ethyl, n-propyl, isopropyl or butyl, Tyr is D- or L-;
butyrlc ac~d, D-threon~n~ or D-~ethlorl~n~; Tyr ~ ~- or L-S
Pro or ~ -Pros Z ~ D- or L-~rg and, when X is Tyr-~',
W 1~ ~-Pro1 whlch comprl~ the st~ of:
(a3 trea~ing ~oG-~ly-reqin by ~olld phas~ ~ynthesl~
by qlx cyclo~ of deprotectlon, n~utrallzatlon and coupllng
wlth a ~lected amlno acld to pxoduce a corr~.qpond~ng pro-
t.ect~a h~ptapeptldyl re~ln of the formula
Boc-Phe-Val-~qn-Cy(~zl)-~ or ~ r~(Tos)-51Y-reslh
wl~er~in W 19 as above;
(b) tr~atlng the corr~s~ona~n~ p~ot~ct~d heptapeptldyl
resln producea in step ~a) by ~olid pha~e pe~tld~ .qynth39l~
.~n a cycle of ae~rotectlon, neutral~z~tlon and coupllng wlth
Boc-X to proauc~ a corresponalng ter~-b~toxycarbonyl-
octapeptlayl re~ln of t~ ~ormula
E~oc-X-Phe-Val-A~n-Cv tBZl) -~- (D- or L-)~rq (To~ -rQsln
(c) ammonoly21ng the corresnondln~ prot~c~e~t
octapeptldyl r~sln producea ln ~t~p (b) to a corre~pondlng
Boc octap~ptide amlde of thQ ~ormula
Boc-X-Ph~Val-A~n-Cy (B~l) -W- (D- or L-~ rg (To~ 31y-NH2 ~ .
whereln X ~g a~ above S
~ a) converltlng ~he correspona~ng!~oa-octapept:Lae
amlde produc~d ln ~tep tc) to a corresponalng ~-(S~benzyl-
marcapto)~ -cyclopentamethyleneproplonyl-o~tape~t~ a~
am~d~ of the formula
5~L~7
3b
C~2CO-X-Ph~--~al-Cy(~3zl)-W-~D- or L-)Arg(Tos)-~ly-~2
(Clt2) 5
~ _ /S-CH2Ph
by coupllng a neu-tralizod, doprote~te~ solutlon of a
corre~ponaing t~rt-butoxyc~rbonyl--octapept~de am.~da
wlth p-nltrophenyl-~-(5-benzylm~rcapto)-~,~-cyclopen-
tamethylene proplonat~, ln the pr~ence o~ ~-hy~roxy-
banzotrl~201~ monohydrate ana
~a) r~7duaing a corre~ponding ~(S-benzylmerGapto1-
cyclop~ntamethylsn~-l?rop:LonylocltapQ!?tiae am~ae
produced ln st~p (d) w~th soal~m in llquld ammonia
and oxldatively ayall~ing n r~ultlng dl3ulfhydryl compound
wlth pota~ um ~rrlcyani~e,
.
)5~7
This inven-tion further relates to a method for antagon-
izing the in vivo response to ADH, comprising administering
to an animal being reated an amount of one of -the foregoing
compounds, in admixture with a physiologically and pharma-
ceutically accep-table carrier, effective to antagonize the
antidiuretic response to ADH.
The present invention rela-tes to an-tagonists of -the
antidiuretic action of ADH, the compounds being of the for-
mula
~ CIH2-CO-Tyr(h)-Phe-Val-Asn-Cy-Pro-Z-Gly-NH2
( 2)5 Cl
\ S '''
wherein Tyr- is D-Tyr and Z is L- or D- Arg. These compounds
can be used in vivo for antagonizing the an-tidiuretic res-
ponse to a vasopressor hormone in an animal being treated.
Aecordingly, the invention provides a process for the
production of a compound of the formula
~ ICH2-CO-Tyr(h)-Phe-Val-Asn-Cy-Pro-Z-Gly-NH2
( 2)5 C
~ S S
wherein Tyr is D-Tyr and Z is D- or L-Arg, which comprises the
steps of, (a) treating Boc-Gly-resin by solid phase synthe-
sis by six eyeles of deproteetion, neutralization and coupling
with a selected amino acid to produce a corresponding pro-teeted
heptapeptidyl resin of the formula
Boc-Phe-Val-Asn-Cy(Bzl)-Pro-Z(Tos)-Gly-resin,
wherein Z is as above, ~b) treating the corresponding protected
heptapeptidyl resin produeed in step (a) by solid phase peptide
synthesis in a cycle of deprotection, neutralization and coupling
with Boc-D-Tyr(H~ to produce a corresponding tert-butoxy-
carbonyloctapeptidyl resin of -the formula
Boc-D-Tyr(H)-Phe-Val-Asn-Cy(Bzl)-Pro-Z~Tos)-Gly-resin,
wherein Z is as above, (c) ammonolyzing the corresponding
-~2~ 7
4a
protected octapeptidyl resin produced in s-tep (c) to a corres-
ponding Boc-octapeptidyl amide oE the formula
soc-D-Tyr(H)-Phe-Val-Asn-Cy~szl)-Pro-Z(Tos)Gly-NH2,
(d~ converting the corresponding Boc-octapeptide amide produced
in step (c) to a corresponding ~-(S-benzylmercapto)~,~-cyclo-
pentamethylenepropionyl-octapeptide amide of the formula
f~ CH2-CO-D-Tyr(H)-Phe-Val-Asn-Cy(Bzl)-Pro-Z(Tos)-Gly-NH2
(CH2)5 IC
~-- S -CH2Ph
by coupling a neutralized, deprotected solution of a corres-
ponding tert-butoxycarbonyl-octapeptide amide with p-
nitrophenyl-~-(S-benzylmercapto)-~,~-cyclopentamethylene pro-
pionate, in the presence o N-hydroxybenzotrizaole monohydrate
and (e) reducing a corresponding ~-(S-benzylmercapto)-/~
cyclopentamethylenepropionyloctapeptide amide produced in
step (d) with sodium in liquid ammonia and oxidatively cyclizing
a resulting disulfhydryl compound with potassium ferricyanide.
In the accompanying drawings:
Fig. 1 shows urine osmolalities, as a function of time,
of rats treated intraperitoneally with an active compound;
and
Fig. 2 shows urine output, as a function of time, of
rats treated with an active compound.
Compounds provided in accordance with the invention
or that of the parent application are derivatives oE arginine
vasopressin (AVP). Amino acids are in the L- form unless
otherwise indicated. The correlation between full names and
abbreviations is:
dAVP, l~deamino-arginine vasopressin; dPAVP, [l-deamino-
penicillamine] arginine vasopressin; d(CH2)5AVP, [1-(~-
mercapto-~,~-cyclopentamethylenepropionic
5~'~
acld)~-arginine vasopressin: dvD~vP, l-deamino[~-valine,
8-D-arginine] vasopres~in; dPVD~VP, ll-deamino--
penicillamine, 4-valine, 8-D-arginine] vasopres.sin;
d(CH2~5VDAVP, [l-(~-me.rcapto-~,~-cyclopenta-
methylenepropionic acid), 4-valine, 8-D-a.rginine]
vasopressin; dTyr(Me~VP, l-deamino[2-(0-methyl)-
tyrosine] arginine vasopressin; dPTyr(Me)AVP [l-
deaminopenicillamine, 2-(0-methyl)tyro~ine] arginine
vasopressin; d(CH2)5Tyr(Me)VDAVP, [l-(~-mercapto-~,
l~ ~-cyclopentamethylenepropionlc acid), 2-0-methyltyrosine,
4-valine, 8-D-ar~inine] vasopressin; d(CH~)5Tyr(Et)VD~VP,
[l~(~-mercapto-~,~-cyclopentameth~lenepropionic acid),
2-0-ethyltyrosine, 4-valine, 8-D-arginine] vasopre~sin,
d(CH2)5Tyr(Me)VAVP, ll-(~-mercapto-~,~-cyclopenta-
methylenepropionic acid), 2-to-methyl)tyrosine~ 4-valine]
arglnine vasopress.in; d~C~I2)5Tyr(Et)V~VP, [l-(~-
mercapto-~,B-cyclopentamethylenepropionic acid),
2-0-ethyltyrosine, ~-valine] arginine va~opressin;
d~CH2)5Tyr(i-Pr)VD~VP, [l-(~-mercapto-~,~-cyclopenta-
methylenepropionic acid), 2-(0-i~opropyl)tyrosine,
4-valine, 8-D-arginine] vasopressin; d(C~I2)51yr~n-Pr~-
VD~VP, [l-(~-mercapto-~,~-cyclopentamethylenepropionic
acid), 2-(0-n-propyl)tyrosine, 4-valine, 8-~-argininel
vasopressin; d(Cli2)$Tyr(i-Pr)VAVP~ -me,rcapto-~
~-cyclopentamethylenepropionic acid), 2-(0-isopropyl)
tyro~ine, 4-valine] arginine vasopressin;
d(CH2)~Tyr(n-Pr)V~VP, [l~ mercapto-~,~ -cyclopenta-
methylenepropionic acid), 2-(0-n-propyl)tyrosine,
4-valinel arginine vasopressin; and d(CH2)5Tyr(Et)V a -
Pro AVP, ~ -mercapto-~,~ -cyclopentamethylenepropionic
acid), 2-(0-e-thyl)tyrosine, 4-valine, 7-~3,4-dehvdropro-
line)~ arginine vaso~ressin; d(C~I2)5- D-Tyr VDAVP,
-mercapto-~,~-cyclopentamethvlene- propionic acid),
2-D-tyrosine, 4-valine, 8-D-arginine vasopressin,
d(CH2)5 D-Tyr V~VP, ~ -mercapto-~,~-cvclopenta-
methylenepropionic acid), 2- n-tyrOsine, 4-valin~ -
arginine vasopressin; d(CTI2)5-D-~he vn~vP/
~f~ 7
C~ merc~to-~,~-cvclopentamethy:l~n~propionic
acid), 2-D-phenyli~ nine, 4-vi~lin~, 8-D-ar~inin~
vasopressin; d(Cll2)5 D-Phe V~VP, [l~-mercilpto-
~ cyclopentamethyleneproplonic acid), 2-D-phenylalanin~,
S 4-valine]-ar~inine v.~sopressin; d(Cll2)5 lGly21 V~VP,
[l-(~-mercapto-~,~-cyclopentamethylerlepropionic ~cid),
2-glycine, 4-vallne~-arginlne vasopre~sin ~(C1l2)5-
lD-Ala ~ V~VP, [1-(~-merCilpto-~ -cyclop~ntamethylenepro-
pionic acid), 2-D-alanLne~ ~-valine~ rginine vasopressin:
r) d (C112) 5 ~{) Val21 V.l\VP ~ -merc~pto-p ~ -cyclop~ntamc!th
lenepropionic acid), 2-D-valine, ~-v~line]-ilrclinine
vasopressin, cl(Cll2)5 [D-Leu ] V~Vl?, Il-(~-mercapto-~
cyclopentamethylenepropiot-ic acicl), 2-D-Le~lc:ine, ~-
valine~-ar~inine va~sopressin; d(CI-I~)r [~-Ile2] V~VP,
1~ [l-(~-mercapto-~,~-cyclopentamethylenepropiollic acid),
2-D-isoleucine, 4-valine]-ar~]inine vasopressill; and
d(C~l2)5 [D-Arg2] VAVP, [l-(~-mercapto-~,~cyclopentamethy-
lenepropionic acid)~ 2-D-ar~inine, ~-valine]-arginine
vasopressin.
The active peptides were synthesized by solid phase
synthesis as described b~ Bankowski et al. (1978), su~ra;
Merrifield, J. ~tm Chem. Soc., vol. 85 (1963) at 21~9 and
Biochemistry, vol. 3 (1964) at 13n5; Mannin~, J. ~m. Chern.
Soc., vol. 90 (1968) at 1348; Manning et al., J. Med. Chern.,
vol. 19 (1976) at 376; ~owbriclge et ~1., J. Me~. _hern.,
vol. 20 (1977) at 1173; Manning et al., J. Me~. _hem.,
vol. 16 (1973) at 975; Kruszynski et al. (19~0), ciu~)ra:
Sawyer et al., (1981), _u~ra or Mallnin~ et al. (19Bl)
supra.
~n Peptides containing ~3-Pro in the 7-position
were also prepared in this fashion. Incorporation
of ~ 3-Pro into peptides has been described by Felix
et al., J. Peptide Protein Re , vol. 1~ (1977) at
299 and nOtos et al., J. Med. Chem., vol. 22 (1979)
at 926.
Initlal attempt~ to design an antagonist of the
antidiuretic response to arginlne vasopressin (AVP)
47
included synth~sis of [l-deaminopenicillamine,
4-valine, 8-D-arginine] vasopressin (dPVD~VP) by
Manning et al. (1977), suJ~ra, ancl of [l-(~-mercapto-~,
~-cyclopentamethylenepropionic acid), ~-valine,
8-D-arginine] vasopressin (d(CH2)5VD~VP), Lowbridge
(1978), ~ a. The~se analog~ were designed by
replacing the two hydrogens on the ~-carbon at the
1~ position of the highly active and selective
antidiuretic peptide l-deamino~4-valine,
8-D-arginine] va~opressin (dVDAVP), Manning et al.,
J. Med. Chem., vol. 16 (1973) at 975, by two methyl
groups and a cyclopentamethylene group, respectlvely.
These substituents had previously been shown to
convert the highly potent oxytocic agonist l-deamino-
oxytocin (dOT) into potent ~ntagoni~ts of the
oxytocic response to oxytocin, specifically,
[l-deaminopenicillamine~ oxytocin (dPOT) and
ll-(p-mercapto~ cyclopentamethylenepropionic
acid)~ oxytocin (d(CH2~50T). See, ilope et al.,
~n J. siol. _hem., vol. 237 ~1962) at 1563, Schulz
et al., J. Med. Chem., vol. 9 (1966) at 647 and
__
Nestor et al., J. Med. _hem., vol. 18 (1975) at
284.
Surprisingly, neither dPVD~VP nor d(CH2)5VD~VP
was an antagonist of the antidiuretic response to
~VP although possessing 0.1 and only 0.0001 the
antidiuretic activity of dVDAVP, respectively.
Each, however, was a potent antagonist o the
vasopressor response to AVP, expressed as P~2.
3~ P~2 represents the negative logarithm to the base
10 of the average molar concentrations of ~ntagonist
which will reduce the specific biological response
to 2x units of an agonist to the level o response
to x units of the agonist. dPVDAVP and d(CH2)5VDAVP
3S had antivasopressor PA2 values of 7.82 and 7.68,
respectively.
~2~.~(.)5~7
The discovery of these two vasopressor
antagonists dPVDAVP and d(CH2)5VD~VP led to
exploration of the ef~ects of ~ dimethyl and
~ cyclopentamethylene s~lhstitutions at the 1-
position in other analocJs of AVP, partic~llarly incombination with t)-e substit-utioll of O-methyltyrosine
at the 2-position of the highly active antidiuretic
and vasopressor ag~nist l-deamino-ar~1:inine vasopressin
(dAVP) in hopes of obtaLning an antivasoE)reC;~or
1~ peptide even more potent and selective than dPVD~VP
or d(CH2)5VDAvP. See, Huguenin et al., ~lelv. Chem.
Acta., vol. 49 (1966~ at 695; Manning et al., J. Med.
Chem., vol. 19 (1976) at 842 and Law et al., J. ~m.
Chem. Soc., vol. 82 (1960) at 4579.
__ __
The discovery of the antidiuretic anta~onists
d(CH2)5Tyr(alk)VAVP, Sawyer et al., (19nl), ~su~ra,
Manning et al., (1981) ~ra, led to the synthesis of
Otller pOSitiOII two substituted analogs. ~nhanced
antl-antidiuretic poten~ies were ex}libited by the various
2~ O-alkyl D-tyrosine analogs, Mannin~ et al., in Peptides,
Struture, Function, Dan ll. ~ich and E. Gross, eds.,
Pierce Chemical Co (in press) and J. Mecl. Chem. (submitted).
The unalkylated D-tyrosine isomers of d(C~12)5VD~VP and
d(CH2)5VAVP, i.e., d(C~12)5D-Tyr-VDAVP and d(Cll2)5D-Tyr-V~VP
were also shown to be arlti-antidiuretics. ~ttempts to
further enhance anti-antidiuretic potency and selectivity
have led to the synthesis of analogs of d(C~l2)5D-Tyr2VAVP
and d(Cll2)5D-Tyr VDAVP containing other D-amino acids in
place of D-tyrosine at position two, in accordance with
3~ the present invention.
It was surprisingly founa by Bankowski et al.
(1978), supra, that of ~l-deaminopenicillamine~-
arginine vasopressin (dPAVP) an~ [l-deaminopenicillamine,
2-(O-methyl)tyrosine] arginine vasopre~sin (dPTyr(Me)-
~VP), dPAVP was less potent than~either dPVDAVP or
5~7
d(CH2)5VD~VP but dPTyr(Me)~VP had an antiva~opre~sor
P~2 of 7.96 and was the most potent antiv~sopressor
peptide then known.
The effect, on antiva~opressor potency of combining
the ~ cyclopentamethylene and 0-methyltyrosine
substitutions in d~VP was developed
[l-(~-mercapto-
~cyclopentamethylenepropionic ~cid), 2-(0-methyl)-
tyrosine]-argini~e vasopressin (d(C112)5Tyr(Me)~VP) of
the structure
1~ 1 2 3 ~t 5 fi 7 8 9
CH2-CO-Tyr(Me)-Phe-Gln-Asn-Cy-Pro-l~rg-Gly-N~12
/C112-C~ I
C~ ~ 1
C~l -Cl S
~5 ~This compouncl had ver~ hi~h antivasopresso~ potency and
very weak antidiuretic activity, as did the unmethylated
2-tyrosine derivative, d(C~12)5~VP and these compounds form
Part o~ the subiect matter of -the copending divisional
application filed of even date herewith.
The compounds of the invention of the parent applica-
tion Serial No. 397, 464 which have activity as antagonists
of the antidiuretic activity of arginine vasopressin belong
to the 4-valine-8-arginine vasopressin series and are of
Formula I
2 3 4 5 6 7 8 9
CH2-CO-Tyr(X)-Phe-Val-Asn-Cy-Pro-Z-Gly-NH2
/ 2 C ~ l
C ~ / IC
H2-CH2 S S
wherein Tyr is D- or L- and X' and Z are:
-~L2(~5~
l!)
X' %
Me D-~rg
Et D-Arg
Me ~-~r~
Et L-~rg
i-Pr D-Ar~
n-Pr D-~r~
1-Pr L-~rg
n-Pr L-~rg
~u L- or D- Arg
Compounds of Formula II are as above, wherein
Pro at the 7-position is replaced by ~3~Pro.
The compounds of Formula I are related to a
previously-reported antagonist of vasopressor
responses to ADII, Il-(~-mercapto-~,~-cyclopentamethyl-
enepropionic acid), 4-valine, 8-D-arginine] vasopressin
d(CH2)5VDAVP, Lowbridge et al. tl978). Althoug not
an antagonist of antidiuretic response~ to ~DH in _ lVO,
this analog was a competitive antagonist of the
activat~on of renal medullary adenylate cyclase by ~DH
in v tro, Butlen et al., Mol. Pharmacol., vol. 14 (1978)
at 1006. The work of Larsson et al. (1978), s~ra,
also indicated the feasibility of making O-alkyl-tyrosine
substitutions to convert thi~s type of peptide into an
2S antagonist of the antidiuretic response in _ivo.
Compounds of Formula II contain a ~ -Pro unit
thought by ~otos et al., supra, to contribute to hi~h
antidiuretic activity of certain AVP analogues.
As shown by intraperitoneal administration of
these compounds to normally-hydrated
conscious rats, (O-ethyl)-tyxosine substitution at the
2-position in compounds of Formula I is more effective
than (O-methyl)tyrosine substitution. The tO-proPyl)-
tyrosine compounds of Formula I also have impr~ssive
anti-~DH activity. Ilowever a 2-(O-Et)-tyrosine
(.}5~
11
compound of Formula II is the most effective anti-~D~I
compound evaluated to present. The 8-L-arginine
analogs are more potent than the correspondi~g
8-D-arginine analogs.
It appears that higher doses of d(Cll2)5Tyr(Et)V~VP
almost completel~ block the antidiuretic action of
endogenous ~DII. For example, the 30 pg/kg dose of
d(CH2)5Tyr(Et)VAVP raised urine flow to a mean o
27 ml/kg per hr during the second hour after injection.
Spontaneous urine flow in female rats homozygous for the
Brattleboro strain that secrete no ~D~I at all averages
32 ml/kg per hr, Sawyer, et al., Endocrinology, vol.
95 (197~) at l~0.
The importance of minor structural modificatlons
is indicated by findlngs that corresponding ~ diethyl
and~ dimethyl an~logs of d(CH~)5Tyr(Et)V~VP do not
exert detecta~le antagonistic act~vity in the
intravenous rat antidiuretic assay. The prese,nce of
the 4-valine also contributes to antagonistic
activity; Substitut~on of a 4-glutamille unit in
d(CH2)5Tyx(Et)V~VP results ~n loss of antagollistic
activity.
Compounds of the invention of the copending divisional
application having Gln in the 4-position, which antagonize
the vasopressor response to AVP, are useful in pharmacological
studies on the role of AVP in regulating blood pressure under
normal and pathophysiological conditions. Clinical applica-
tions include use as diagnostic and therapeutic antihyperten-
sive agents. For therapeutic purposes, these compounds willbe used in the same fashion as the known antihypertensive
drug Captopril, D.B. Case et al.~ "Progress in Cardiovascular
Diseases," vol. 21 ~1978) at 195.
The compounds of Formulas I and II are very effective
antagonists of the antidiuretic response to ADH.
They can therefore be used in pharmacological
'7
studies on the con-tribution of ADH to a variety of
pathological states involving water retention. I-t is
further contemplated that they could be effec-tive 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.,
10 Am. J. Med., vol. 42 (1967) at 790.
It was found that some d(CH2)5VAVP derivatives having
a D-amino acid other than tyrosine and larger than alanine
in the 2-position are more potent antagonists of the anti-
diuretic action of AVP than compounds having D- or L-
tyrosine ether units or a D-tyrosine unit at the 2- position
of d(CH2)5VAVP or d(CH2~5VDAVP.
As shown by intravenous administration of the
compounds of the parent invention to normally-hydrated
conscious rats and to hydrated rats anesthetized with
ethanol, compounds having D-Phe, D-Val, D-Leu or D-Ile
substituents a-t the 2-position have high PA2 values and
effective doses near or lower than the lowest effective
doses known heretofore.
Compounds of the parent invention having D-Phe,
D-Val, D-Leu or D-I].e at the 2-position and Arg a-t
the 8-positlon are also pure antidiuretic antagonists,
i.e., these compounds have no transient antidiuretic
agonism. Moreover, these compounds are more selective
in their activity, by virtue of high anti-ADH/antivaso-
pressor activity ratios, than known compounds.
13
The compoun~.s of this invention carl be em~loye~l inmixture with conventional excipien~:s, i.e., physioloc3ically
and pharmaceutically acceptable organlc or ino~ nic
carriers suit~lble for parenteral or ell~er~l ~pp)ic~ntion,
which do not interact deleteriously with the ~ctive
compounds.
Suitable pharmaceutically acceptable c~rr;ers
include, but are not limited tOr water, salt solutions,
alcohols, vegetable oils, polyethylene qlycols, gelatine,
lactose, amylose, magnesium stearate, talc, silicic acid,
1~ viseous paraffin, perfume oil, fatty acicl monoglycericles
and diglycerides, pentaerythritol fatty acid esters,
hydroxy-methylcellulose, polyvinyl pyrrolidone, etc.
The Pharmaceutical preparations can be sterili7ed and iE
desired mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsiEiers,
salts for influencing osmotic pressure, buffers, coloring,
flavoring and/or aromatic substances and the like whicl
do not deleteriously react with the active eompouncls.
For parenteral ox intranasal application, solutions,
2n preferably aqueous solutions, as well as suspensions,
emulsions, or implants, inctuding suppositories, are
particularly suitable. ~mpoules are convenient unit
dosages.
The compounds of this invention are generally
administered to animals, including but not limited to
mammals, e.g., livestock, household pets, humans, cattle,
cats and dogs. ~ diuretically effective daily dosage oE
the active com~ounds can be administered parenterally in
a single dosage or as divided dosages throughout the
3~ day.
L7
14
Parenteral or intrana~al administration is
preferred, the an-ti-antidiuretic com~ounds of thi~s
invention being particularly valuable itl the treatment
of humans afflicted with water retention oE any etiology.
In this regard, they can ~e 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
n amounts of active compounds used will vary according
to the specific compouncl being utilized, the
particular compositions formulated, the mode of
application, and the particular organism 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.
Preferred antidiuretic antaqonists o~ the parent
2~ i~vention are [l-(~-mercapto~ cyclopentamethylene-
propionic acid), 2-(0-ethyl)tyrosine, 4-valine]-
arginine vasopressin, most preferably the 8-L-arginine
compound. ~lso preferred is a corresponding ~3-Pro7
compound.
~ther preEerrèd compounds are those wherein X
is D-Phe, D-Val, D-Leu or D-Ile and 2 is L-~rg. The
D-Ile or D-Phe compound is most preferred.
'5~7
Without further elnboration, it i9 believed
that one skilled in the art can, u~ing the preceding
description, utili~e the present inventlon 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
ln indicated, all parts and percentages are by weight.
Chloromethylated resin (Bio~Rad*~io-13eads
SX-l) was esterified by the procedure o Gisin,
Helv. _him. ~cta, vol. 56 (1973) at 1~76 with
Boc-Gly until O.~7 mmol/g and rvO.h~4 mmol/g were
inoorporated. ~mino acid derivatives including
Boc-Tyr(Me) (Rf (A) 0.7; Rf (B) 0.8) were supplied
by Bachem Inc., or synthesi~ed.
Triethylamine (TE~) and N-methylmorpholine
(NMM) were distilled ~rom ninhydrin.
~n ~cetic acid used as the HCl-acetic acid `
cleavage reagent was heated under reflux with
boron triacetate and dtstilled from the reagent.
Dimethylformamide (DM~) was distilled under
reduced pressure immediately before use. Methanol
~S was dried with magnesium methoxide and distilled.
Other solvents and reagents were o analytical
grade.
Thin layer chromatography (TLC) done on
silica gel plates (0.25 mm, BrinkmannSilplate)
3n using the ollowing solvent systems: ~. cyclo-
hexane-chloroform-acetic acid (2:8:1 v/v);
B. propan-l-ol-ammonia (3~) (2:1 v/v); C. ethanol
* Trademarks
()S4L'~
1~
(95~)-ammonia (34%) (3:1 v/v); D. chloroform-
methanol (7:3 v~v); E. butan-l-ol-acetic acld-
water (4:1:5 v/v, upper phase1; P. butan-l-ol-
acetic acid-water-pyridine (15:3:3:10 v/v). The
applied loadings were 10-50 p~. The minimum
length of the chromatoqrams 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 me~lod of Spackman et al., ~nal.
Chem., vol. 30 (195fl) at 1190, in which peptide
samples weighing about 0.5 mg were hydrolyzed with
constant boiling hydrochloric acld (~00 ~1) in
evacuated and sealed ampules for 18 h at 120 C.
The analyses were performed using a Beckman
~utomatic Amino ~cid ~nalyzer, 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 indlcated by there respective
symbols were within ~ 0.4~ of theoretical values.
Optical rotations were measured with a Bellingham
Stanley, Ltd., Model A polarimeter, type pl.
EX~MPLE 1
~ (S-Benzylmercapto)~ -cyclopentamethylene-
~ropionyl-Tyr~Me)-Ph~e-Gln-~sn-Cys(~zl)-Pro-~rg (Tos) -
~ly-NH2.
(a) Combination of Solld Phase and Solution
Methods.
Boc-Tyr(Me)-Phe-Gln-~sn-Cys(Bzl)-Pro-~rg(Tos)-
Gly-NH2, prepared by the method of Bankowski e-t al.!
J. Med. Chem., vol. 21 (1976) at 8~2 (319 mg, 0.26 mmol),
was dissolved in TEA (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 x 10 ml). The product was dried 1~
~(3~7
17
vacuo over sodium hydroxide pellet~. Thi~ material
(318.5 mg) was dissolved in DMF (0.8 ml), to which
was added N-methylmorpholine (10 ~1). The resulting
solution had ~ pEI of 7-8, me~sured with noifit pl~
paper. ~fter this neutralized solution was stirred
at room temperature for 30 min, a solution of
p-nitrophenyl ~ (S-benzyl-mercapto)-~,~ -cyclopentamethyl-
e~epropionate, 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. ~fter 72 hours' stirring, TLC
analysis u~ing system D showed that the reaction
mixture still contained a trace of the free octapeptide
amide. N-l~ydroxybenzotriazole monohydrate, Konig et al.,
C _ . 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 x 10 ml) and dried _ vacuo. The crude
product (330 mg) was twice reprecipitated from
DMF-methanol to give the acylpeptide amide (295.2 mg,
77.3~): mp. 209-211C; [~]D2~ 3.6 (C 0.5, DMF);
Rf(E) 0.45, R~(F) 0.63 ~nal. (C73 H94Ol4S3) C, ~, N.
~mino acid analysis: Tyr, 0.80; Phe, 1.01;
Glu, 1.04; ~sp, 1.02; Cys(Bzl), 0.98; Pro, 1.06;
Arg, 1.01; Gly, 1.00; NH3 2.91.
(b) Total Synthesis on Resin.
Boc-Tyr(Me)-Phe-Gln-~sn-Cys(Bzl~-Pro-Arg(Tos)-
Gly-resin (1.11 g, 0.4 mmol prepared from Boc-Gly-
resin using solid phase methodology) wa6 converted
to the acyloctapeptide resin (1.167 g, weight gain
57 mg, 97.6% of theory) in one cycle of deprotection,
neutralization and couplihg with p-nitrophenyl-~S-
benzylmercapto)-~,~ -cyclopentamethylenepropionate,
see Nestor ~E~a. The resin was ammonolyzed,
Manning, J. ~m. Chem. Soc., vol. 90 (1968) at
18
1348. The product was extracted with dimethyl-
formamide (DMF). After the solvent was evaporated
in vacuo, the resldue was precipitated by addition
of water. The crude product (~10 mg) was twice
reprecipitated from DMF-ethanol to give the
acyloctapeptide (302 mg, 50.7~ based upon initial
glycine content of the xesin); mp. 206-208C
(decomp); Rf (E), 0.45, Rf (F) 0.63; ~]D = -43.1
(C 1, DMF). Anal. (C73H94N14S3)
Amino acid analysis: Tyr, 0.79; Phe, 1.01;
Glu, 1.03; Asp, 1.04; Cy~(~zl), 0.97; Pro, 1.03;
Arg, 0.99; Gly 1.00; N~13, 2.g5.
EXAMPLE 2
p-(S-Benzylmercapto)-p~ -cyclopentamethylenepropionyl-
Tyr(Bzl)-PIle-Gln-~sn-Cys~B~l)-Pro-~rg(Tos)-Gly-NII2.
Boc-Tyr (~7.1) -Phe-Gln-Astl-Cys(Bzl)-Pro-Arg(Tos)Gly-
resin (1.~6 g, 0.5 mmol) was converted to the
acyloctapeptide resin (1.55 g, weight gain 70 mg,
95.9~ of theory) a~ in Example 1 by one cycle of
deprotection, neutralization and coupling with
p-nitrophenyl ~-(S-benzylmercapto)~ -cyclopenta-
methylenepropionate. The product obtained by
ammonolysis of the resin was extracted with DMF.
The solvent was evaporated in vacuo and the
residue wa~ precipitated by addition o water.
The crude product (723 mg) was reprecipitated
from D~F-ethanol and DMF-2~ aqueous AcOH. (488 mg;
62.4% based on initial Gly content on the resin);
mp. 183-185C; Rf(E) 0.38t Rf(D) 0.41; [~]D = -32~9
(C 1 DMF). ~nal.(C79H98Nl4Ol4 3
Amino acid analysis: Tyr, 0.97; Phe, 1.02;
Glu, 1.05; ~sp, 1.01; Cys(Bzl), 0.98; Pro, 1.04;
Arg, 0.98; Gly, 1.00; NH3.
t~ 5~
19
EX~MPLE 3
[1-(~ Mercapto-~ cyclopentamethylenepropionic
~cid), 2-(O-methy~)tyro3ille]argilline vasopressin.
(a) From Nonapeptide ~mide
~ 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 unt~l a light blue color persisted in the
solution for 30 sec, in accordance with duVigneaud,
J. ~m. Chem Soc., vol. 76 (1959) at 3115. Dry
glacial acetic acid ~0.4 ml) was added to discharge
the color. The solution was evaporated. ~ solution
of the residue in aqueou~ acetic acid (0.2R;
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 (O.OlM,
11.4 ml), Hope et al., J. Biol. Chem., vol. 237
(1962) at 1563. The yellow solution wa~ stirred
for 90 min more and for 1 h with anion-exchange
resin (BioRad ~G-3, Cl 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 0.2~ acetic acid
and the combined filtrate and washings were
lyophylized. The resulting powder (1386 mg) was
` desalted on a Sephadex G-15 column tllO x 2.7 cm)
and eluted with aqueou~ acetic acid (50~) at a
flow rate 4 ml/h by the technique of Manning
et al., J. Chr~matog., vol. 38 (1968) at 396.
The eluate was fractioned and monitored for
absorbance of 280 nm. The fractions comprising
the major peak were pooled and lyophylizedO The
* Trademarks
('5~7
2~
resldue (55.5 mg) was further subjected to gel
filtration on a Sephadex G~15 column (100 x 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 (~bsorbance 280 nm). Lyophilization
of the pertinent fractions yielded the vasopressin
analoq (49 mg, 37.3~) Rf(E) 0.19; ~f(F) 0.30;
~]D22_59.6 (cO.l9, lM AcOH).
~mino acid analysis: Tyr, 0.81: Phe 1.01;
Glu, 1.04; Asp, 0.9M; Pro, 1.04; ~rg, 0.95; Gly, 1.00;
NH3, 3.10. Analysis f~llowing perfo.rmic acid
oxidation p.ri.or to hydrolysis according to Moore,
J. Biol. Chem., vol. 238 (1963) at 235, qave a
.
Cys(0311)-Gly ratio of 1.03:1.00.
(b) ~rom Acyloctapeptide.
Treatment of the acyloctapeptide (160 mg,
0.107 mmol~ as described in Example 3 (a) yielded
the analog t64 mg, 51.7~), which was indistinguishable
from the foregoing preparation by TLC: 1~123D -59.1
(C 0.5, lM ~cOH).
~mino acid analysis: Tyr, 0.80; Phe, 1.02
Glu, 1.02; ~sp, 0.98; Pro, 1.03; ~rg, 0.96;
Gly, 1.00; Nl13, 3.05. ~nalysis following perormic
acid oxidat.ion prior to hydrolysis gave a Cys-
(03H1-Gly ratio of 1.02:1.00.
EXAM2LE 4
[l-(~-Mercapto-~r ~-cyclopentamethylenepropionic
acid'))arginine ~asopressin.
Treatment of the acyloctapeptide (173 mg, 0.111
mmol) as described in Example 3 (a) yielded the
analog (66 mg, S2.5%) Rf(E) 0.19, Rf(F) 0.43.
[~ D -58.7 (C 0.5, lM AcOH).
~ mino acid analysis: Tyr, 0.96; Phe, 0.98;
Glu, l.Ql; Asp, 1.01; Pro, 1..05; Gly, 1.00, NH3,
2.95. Analysis following performic acid oxidation
prior to hydrolysis gave a Cys(03H)-Gly ratio of
1.01:1.00.
('5~7
21
EX~MPLE 5
~ -Mercapto~ cyclopentamethylenepropionic
aci~), 2-(0-alkyl~tyrosine, ~-valine]-(L- a~lcl D_)-
arginine vasopressin.
Compounds of this series were prepared by
solid-phase synthesis, modified as in Manning
et al., J. Med. Ch~m., vol. 16 (1973) at 975 and
Krus~ynski et al. r J. Med. hem., vol. 23 (1980)
at 364, to obtain protected intermediates for
each analog. The procedures of Bodanszky et al.,
J. ~m. Chem. Soc., vol. 81 (1959) at 5688 and
_ __ _ _
J. Org. Chem., vol. 39 (1974) at 44~, employing
a p-nitrophenyl ester, facilitated by the use of
hydroxybenzotriazole (Konig et al., supra), were
used for the coupling of ~-(5-benzylmeecapto-~,
~-cycloperltamethylenepropionic acid in accordance
with Nestor, supra, to obtain precursor compounds.
Each precuxsor was deblocked (duVigneaud, _u~ra)
with sodi~lm in liquia ammonia. The resulting
disulfhydryl compounds were oxidatively cyclized
with potassium ferricyanide (llope et al., sl~ra).
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, Kruszynski et al., J. Med _hem., vol. 23
(1980~ at 364, and by amino acid analysis as
above.
Boc-Phe-Val-Asn-CystBzl)-Pro-D-Arg(Tos)-Gly-resin
Boc-Gly-resin (1.562 g, 1.0 mmol, of Gly) was subjected
to six cycles of deprotection, neutralization, and
coupling to yield the protected heptapeptidyl resin A
35 (2.52Z g, 1.0 mmol).
5~7
2~
soc-Phe-Val-~sn-Cys(Bzl)-Pro-~rg(Tos)-Gly-resin
The protected heptapeptidyl resin 13 (2.522 g, 1.0 mmol)
was prepared from 1.562 g (1.0 mmol) of Boc-Gly-res.in
using solid-phase methodology.
Boc-Tyr(Me)-Phe-Val-~sn-Cys(Bzl)-Pro-D-~rg(Tos)-Gly-resin
A single cycle oL solid-phase pept.ide synthesis with
Boc-Tyr(Me) as the carboxy component converted
heptapeptidyl r~sin ~ (1.261 g, 0.5 mmol) to the
corresponding tert-butyloxycarbonyloctapepti~yl resin
C (1.35 g, 0.5 mmol).
Boc-Tyr(Et)-Phe-Val-~sn-Cys~Bzl)-Pro-D-~rg(Tos-Gly-resin
The heptapeptidyl resin ~ (1.261 g, 0.5 mmol) yielded
the tert-butyloxycarbonyloctapeptidyl resirl D (1.357 g,
0.5 mmol) in one cycle of solid-phase peptide synthesis
with Boc-Tyr(Et) as the carboxy component.
Boc-Tyr(Me)-PIle-Val-~sn-Cys(Bzl)-Pro-~rg(Tos)-Gly-resin
The heptapeptidyl resin B (1.261 gr 0.5 mmo.U was converted
to protected octapeptidyl resin E (1.35 g, 0.5 mmol) in
one cycle of deprotection, neutralization and coupling
with Boc-Tyr(Me).
~ (S-Benzylmercapto)~ ~cyclopentamethylenepropiony-Tyr(F.t)-
Phe-Val-~sn-Cys(Bzl)-Pro ~rg(Tos)-Gly-resin
The heptapeptidyl resin B (1.261 g, 0.5 mmol) was
converted to the ~cyloctapeptide resin tl.43 g, 0.5 mmol)
in two cycles of solid pha~e peptide synthesis using as
the carboxy component, respectively: Boc-Tyr(Et) and
p-nitrophenyl ~-(S-~enzylmercapto)-~, ~ -cyclopentamethylen-
propionate.
~oc-Tyr(Me)-Phe-Val-Asn-Cys(Bzl)-Pro-D-~r~ (Tos) -Gly-NH~
The protected oc-tapeptide resin C (1.35 g, 0.5 mmol)
was ammonolyæed and the product extracted with warm DMF.
The product was precipitated by addition of water. The
crude product was reprecipitated from DMF-ethanol-ethyl
ether to give the pure product as a white powder (0.581 g,
88.52% based on initial Gly content o~ the resin) mp.
239-240C; [~]D = -14.9 (C = 1, DMF); Rf(~), 0.54,
R (D) 0.73; ~na~- (CS3H85Nl3Ol4S2)
~2~`~()S~7
23
Amino acid anal~sis: Tyr, 1.02; Phe, 0.98;
Val, 1.02; ~sp, 1.00; Cy~(Bzl), 0.98; ~ro, 1.01;
Arg, 0.97; Gl~, 1.00; N~13, 2.1.
Boc-Tyr(Et)-Phe-Val-~sn-Cys~Bzl)-Pro-D-Arg(Tos~Gly-NI-I2
Treatment of the protected octapeptide resin D (1.357 g,
O.5 mmol) a~ above yielded the Boc-octap~ptideamide
(0.535 g, 80.69~ based on initial Gly content of the
resinj mp. 211-213 C; 1O~D = -16.4 (C = 1, DMF);
R (E) 0 61 R (D), 0.83: Anal. (C6~H87N13Ot~ 2
Amino acid analysis: Tyr, 0.99; Phe, 1.00; Val,
1.01 ~sp, 1.02; cys(nzl), O.9R, Pro, 1.00 Arg, 0.98;
Gly, 1.00; N~13, 213.
~oc-Tyr(Me)-Phe-Val-~sn-Cys(nzl)-Pro-Arg(To~tGly-NH2
Treatment of the protected octapeptide re~sin F (1.35 g,
lS 0.5 mmol) as a~ove gave the correspondin~
Boc-octapeptideam.ide (0.S97 g, 90.96~ basecl on initial
Gly content Or the resin). mp. 216-217 C (c]ecomp. );
1~]D = -34.82 (C = 1, DMF); Rf(E), 0.S4, Rf(D), 0.73;
Anal. (C63~85N13014S2)
Amino ~cid analysis: Tyr, 0.99; Phe, 1.00; Val,
1.02; Asp, 1.01; Cys(B~l), 0.98; Pro, 1.01, ~rg, 0.98;
Gly, 1.00; Nl13, 2.09.
~-(S-Benzylmercapto-~, ~ -cyclopentamethylenepropionyl-
Tyr(Et)-Phe-Val-Asn-Cys(Bzl)-Pro-Arq('l'os)-Gly-NH2
The protected acyloctapeptide resin (1.43 gr O.S mmol)
was ammonoly~ed and the product extracted w.ith warm
DM~. The product was precipltate~ by addition of water.
The crude product was reprecipitated from DMF-ethanol-
ethyl ether to give the pure product. (0.490 g,
~6.S4~ based on initial Gly content of the resin).
mp. 211-213 C; ['~D 4 = -39.8 (C =1, DMF); Rf(E),
0-59, Rf(D), 0.75; Anal. (C74H97Nl3Ol3S3) C, H, N-
Amino acid analysis: Tyr, 0.99; Phe, 1.01; Val,
~ ; Asp, 1.01; Cys(B~l), 0.99; Pro, 1.02; Arg, 0.98;
Gly, 1.00; NH3, 2.07.
24
~-(S~Ben7.ylmercapto)~ cycl~pentamethylenepropionyl-
Tyr(Me)-phe-val-~sn-cys(Rzl)-pro-D-~r~(Tos)Gly-NJl2
The tert-butyloxycarbonyloctapeptide amide prepared
above (0 270 gr 0.206 mmol) wa~ dissolved in TF~
(3 ml) and allowed to stand at room temperature for
20 min. Cold ethex was added. The precipitated material
was filtexed and washed with eth~r (5 x 10 ml). The
p~oduct was dried in vacuo over sodium hydroxide pellets.
This material (250 mg) was dissolved in DMF (0.8 ml)
to which solution N-methylmorpholine was added to give
a ~olution of pH 7-Q (moist p~l paper). l`h~ neutralized
solution was stirred at room temperature or 20 min.
solution of p-nitrophenyl ~-(S-ben7ylmercapto-~,
~-cyclopentamethylenepropionate (0.135 g, 0.37 mmol)
and N-hydroxybenzotri~%ole monohyclrate (57 mq, 0.37 mmol)
in DMF (1.0 ml) w~s nddcd. The reaction mixture was
stirred at room temperature overnight and TLC (system
E) showed that the reaction was complete. Methanol
(80 ml) and ether (20 ml) were added with vigorou~
mixing. The precipitated material was filtered, washed
with a mixture of methanol-ether (8:2), and clried ln
acuo. ~he crude product (270 mg) was reprecipitated
orm DMF-methanol to give the acyl pepticle amide (263 mg,
75.2~); Mp. 220-221 C: ~)D = -25.7 (C - 1, DMF);
Rf(E), 0.55, R~D), 0.83: ~nal. (C73H95Nl3Ol3S3) C, H, N.
~mino ~cid analys.is: Tyr, 0.98; Phe, 1.01; Val, 1.02
Asp, 1.02; Cy~(Bzl), 0.97; Pro, 1.03; ~rg, 1.0; Gly, 1.00;
NH3, 2.06.
~-(S-Benzylmercapto-~ cyclopentamethylenepropionyl-Tyr(Et)-
Phe-Val-~sn-Cys(~zl)-Pro-D-~rg(Tos)-Gly-NII2
l'he tert-butyloxycarbonyloctapeptide amicle (0.398 9,
O.3 mmol) was deprotected and coupled with p-nitrophenyl
~-(S-benzylmercapto)-~, ~-cyclopentamethylenepropionate
(0.232 g, 0.6 mmol) as described above to give the
acyloctapeptide amicle ~0.361 g, 81.67~) mp. 222-22~" C;
1~]D = -22.8D (C = 0.5, DMF); R~(E), 0.5, Rf(D), 0.83;
~nal. (C7~ll97Nl3Ol3s3)
~ mino acid analysis: Tyr, 1.0; Phe, 1.02; Val,
1.03; ~sp, 1.02; Cys(B~.l), 0.98; Pro, 1.03; ~rg, 0.99;
Gly, 1.00; Nl13, 2.11.
~ (S-~en~.ylmercapto)~ cyclopentamethylenepropionyl-
Tyr(Me)-Phe-Val-~sn-Cys(Bzl)-Pro-~rg(l'os)-Gly-NII2
Tert-8utyloxycarbonyloctapeptide amide (0.~94 g, 0.3 mmol)
was cleprotected and coupled with p-nitrophenyl ~-(S-
benzylmercapto)-~ cyclopentamethylenepropionate
(0.232 q, 0.6 mmol) as above to produce the
acyloctapeptide amide (0.38~ g, 8~.65~); mp. 211-214 C;
~D 1 = -39.2 (C = 1, DMF); Rf(E), 0.~7, ~f(D), O.R5;
Anal- (C73~lgsNl30l3 3
~ mino acid analysis: Tyr, 0.99; Phe, 1.02; Val,
1.03; ~sp, 1.01; Cys(l~.l), 0.99; Pro, 1.02; ~rc~, 0.9~;
Gly, 1.00; N~3, 2.04.
[l-(~-Mercapto~ -c~clopentamethylenepropionic acid),
2-(0-ethyl)tyrosine, ~-valine]-arqinine vasopressin
solution of protected acylocta-peptide amide (1~0 mg, 0.095
mmol) in 400 ml of ammonia (dried and xedistilled from
sodium) was st.trred and treated 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 s. Dry glacial acetic acid
(0.4 ml) was added to discharge the color. The solution
was evaporated by passing N2 through the flask. ~fter
5 min, the residue was dissolved in aqueous acetic
acid (10~, 50 ml) to wh~ch was added 800 ml of water.
The solution was treated with 2M ammonium hydroxide
solution to give a solut~on of pH-6.5. ~n excess of
a solution of potassium ferricyanide (O.OlM, 16 ml)
was added gradually with stirring. The yellow solution
was stirred for 10 min more and for 10 min with anion
exchange resin (~io-Rad ~G-3, Cl form, 10 g damp
weight). The suspension was slowly filterecl through
a bed of resin ~50 9 damp weight). ~fter washing the
bed with aqueous acetic acid (0.2~, 200 ml), the combinecl
~t.~(~5~7
2~
filtrate and wa~hings were lyophyli~ed. The resulting
powder (1.63 g) wa~ desalted on a Sephadex G-15 column
1110 x 2.7 cm) by elution with aqueous acetic acid (50~)
at a flow rate 5 ml/h. The eluate was fraction~ted and
monitored or absorb~nce o~ 280 nm. The fractions
comprising the major peak were pooled an~l lyophylized.
The residue (28 mg) wa~ subjected to gel filtration on a
Sephadex G-1~ column (100 x 1.5 cm). Product was eluted
with aqueous acetlc acid ~0.2M) at a flow rate of ~ ml/h.
The peptide was eluted in a slngle peak (absorbancr2
280 nm). Lyophyli7.ation of the pertinent fractlons
yielded the vasopressin analog (2~ mg, 20.6~). TL,C
Rf(E), 0.31, Rf(F), 0.62, [~]D = -65.1 (C = 0.2, lM
I\cO~I ) .
Amino acid analysis: Tyr, 1.00; Phe, 1.01; Val,
1.01, Asp, ].01; Pro, l.Ol;~rg, 1.00; Gly, 1.00; N~13,
1.97. ~nalysis ollowing performic acid oxidation
prior to hydrolysis gave a Cys (03H)-Gly ratio of 1.01:
1 .00.
[~ -Mercapto-p~ -cyclopentamethylene~ropionic ~cid],
2-(0-methyl)tyroslne, 4-valine, 8-1)-arginille] vasopressin
The peptide intermediate (168 mg, 0.115 mniol) was reduced
~y sodium in liquid ammonia, reoxidize~1, deionized, and
purified as above to give 49.5 mg o product (35.5~)
Rf(E), 0.30, Rf(F), 0.61; [~]D = -~6.~1 (C - 0.~, lM
~cOH).
Amino acid analysis: Tyr, 0.98: Phe, 1.01; Val,
0.98; ~sp, 0.99; Pro, 1.03; ~rg, 0.98; Gly, 1.00; NH3,
12.1. ~nalysis following performic acid oxidation prior
to hydrolysis gave a Cys(03H)-Gly ratio 1.03:1.00.
[l-(~-Mercapto-~, ~-cyclopentamethylenepxopionic acid),
2-(0-ethyl)tyrosine, 4 valine, 8-D-arginine] vasopressin
~he yield of analog from 167 mg (0.113 mmol) of inter-
mediate was 29 mg (20.9~). Rf(E), 0.29, Rf(F), 0.57,
[~JD = -41.1 (C = 0.3, lM ~cOH).
Q5~7
27
~ mino acid analysis: Tyr, 0.98; Phe, 1.01; Val,
1.03; ~sp, n.g9; Pro, 1.03: ~rg, 1.02 Gly, 1.00 N1~3,
1.98. ~nalysis following performic ac.id oxidation
prior to hydrolysis gave a Cys ~O3l~)-Gly ratio 1.01:
1.00.
~ -Mercapto-~,~ -cyclopentamethylenepropionic acid~,
2-(O-methyl)tyrosine, ~-valine]-arqinine vasopressin
Treatmen~ of the acyl octapeptide (174 mg, 0.119 mmol)
as above yielded the 51.5 mg of prod~ct (35.6~).
RftE), 0.28, Rf(F), 0.60; [~]D = -66.3 (C = 0.4, lM
~cO~I ) .
~mino acicl analysis: Tyr, 0.99; Phe, 1.01; Val,
1.02; ~sp, 1.01; Pro, 1.00; ~rg, 1.01; Gly, 1.00; N~13,
2.11. ~nalysis followinc3 perform.ic acid oxiclation
prior to hydrolysis g~ve a Cys(O3ll)-~ly ratio ].. 03:1.00.
EX~MPLE 6
[l-(~-Mercapto~ -cyclopentamethylenepropionic acid),
2-(O-ethyl)tyrosLle, 4-valine, 7-(3,~-dellydroproline)~
arginine vas~pressin
The compound was prepared as i.n Example 5, using
~3-proline instead of proline.
EX~MPLE 7
~ ercapto-~,~-cyclopentamethvlenepropionic acid),
2-substituted, ~-valine, 7-proline~ -(L- and D-)
arginine vasopressin
Compounds of this series ~ere preparec1 as in Example
5. Purity was determined by TLC assay on silica gel in
~two solvent systems: E. butanol/acetic acicl/water
(B~W) (4:1:5) or F. butanol/acetic acicl/water/pyridine
3~ (15:3:3:10). Results were:
()5~
X Z l~f ~ l~f ~;~
__ _ _. _
D-Tyr L-~rg O.:L 7 O. r~o
D-~he L-~rg 0.1 7 0 r~
Gly L-~rg0.15 0.~
D-~la L-~rg O.l6 0.~9
D-Val L-~rg 0.17 0.49
D-Leu L-~rg 0.17 0.53
~-lle L-~rg0.17 0.51
D-~rg L-~rg 0.0~ 0.30
D-Phe ~-~r~ O.l~i 0.51
~X~MPLE 8
Antagonism to the vasopressor response was
estima~ed in accordance with Dyckes et al., J. Med. Chem.,
vol. 17 ~1974) at 969~ The values are expressed as P~2
values, defined by Schild et al.~ Br. J. Pllarmacol.,
vol. 2 (1~7) ~t 1~9.
~ ctivity as antidiuretic agoni.sts was determined
by intravenous injection of compounds being evaluated
into ethanol-anestlles.ized water-loadecl rats in accordance
2~ witll Sawyer et al., Endocrinology, vol~ 63 (1958) at 694.
~ ntagonistic potencies were determined and expressed
as "effective doses" and as P~2 values. The "efectlve
dose" is deined as the dose (in nanomoles per kilo~ram)
that reduces the response seen from 2x units o agonist
injected 20 min after the dose of antagonist to the
response with lx units of agonist. Estimated in _ lVO
"P~2~ values represent the negative loga~ithms o~ the
effective doses divided by the estimated volume of
distribution ~67 mL/kg). Results are given in Table r.
~a'~0s~7
2
,~ o ~ I_
I )U~ o o o o
h tJl~) o ~-1 ~ o o o
+1 ~ +~+~ o
t I ~
d' Or~ 1 0 In ~
o ~ ~ o c~ ~ o
~n
~n
,1 ~Io~r~ o o o o
l~J
~ ~ o o o r~ o o o
~n ~
~d n~ +1 +1 ~r
V O ~ ~ O co ~0 C C~
~ ~ . . . . . t,-~ .
rS r~ r~
~I h h
rlr~1 ~ t~) b'
a~ ~I h h
w ~ ,~ a n
C~ ~ ,
a) a)
r~ h h $-1 h ~I h h h
~E~ E~ E-l E~ t~ t~ t-~ E~
Il)
Cr ~ U t~ r~l rJ r~
r~
~ I
r~ r,~
r a~
r~
a
a) t-~
,~ ~n, > ~ p ~ h ;~
.a Q, ~ ~ rJ ~ r ) ~ t I r
~ ~ ~ r-~ ~' 'r~, -~ t ~ r
t~ , r~ rt3 ~r~ d ~U ~d
~r~ 5~7
3~
Results displayed in Table I show that eompounds of
this invention, particularly [l-(~-mere~pto-
~cyelopentamethylenepropionie ae.id), 8-nrginine] vasopressin
and ~ mercapto-~,~ -eyclopentamethylellepropionic acid),
2-(0-methyl)tyrosine, 8-argin.ine] vasopr~ssin antagonize
tne vasopressor response to argi.nine va~sopressin
and also exhibit a marked reduetion in alltid.iuretie
activity.
EX~MPLE 9
3~ ~l-(B-Mereapto~ ,~-eyelopentamethylenepl-opioni.e
aeid), 2-(0-alkyl)tyrosine, 4-vali.ne, ~-(],- anc.l ~-)
arginine] vasopressin compounds, eva].l.~ated as in
Example 8I were weak antidiuretic agonists. They
eaused an initial submaximal inhib.il:ion of urine 10w
lasting about 10 min, followecl by a per.iocl of
inhibition of respons.es to ADII lastitlg 1 to 3 h,
depending on the dose. This inhibition was
reversible, that is, eould be overcome by raising the
dose of ADIT. Repeated assays permitted estimation
2~ of the "effeetive dose" of eaeh analog, whieh is de-
fined as the dose whieh reduees the ~ntidiuretie
response to 2x units of ~DII injeeted 20 min after
the dose of antagonist to eqùal the response to lx
units injeeted prior to the antagollist. 'I'he
estimated effeetive doses for these analoc3s
/in nmoles/kg) and antivasopressor activity are given
in Table ~I.
Whereas [l-(~-mereapto-~ eyclopentamethylenepro-
ptonie acid), 2-(0-alkyl)tyrosine, ~-valine, ~-(.T,- and D-)
3n arginine] vaso~ressin compouncls were weak ~nt:id;-lretic
a~onists, cnusincl an i.nltial submax;.m~l in~llbit.ion of
urine flow lasting about 10 min, followed by a period
of inhibit.ion of responses to ADH lastinc3 1 to 3 h, the
preferred compounds of this invention, indicated by
asterisks in the table below, had no antidluretic agonistic
aetivity.
~;J
:: .
Table II
~nti-~rtidiuretic ~ntivaso~ressor
ED PA2 ED pA2
C~?~und n~ol~s/K~ r.r~les/Xg
d(CH2)5 D-Tyr VAVP 2.2 +0.27.51+0.08(4) 0.29T0.098.41+0.11(4)
d(CH2)5 ~-P~e ~iA~ 0.67+0.13*8.07+0.09(8)0.58+0.048.05+0.03(4)
d(CH2)5[Gly ~'tr~VP agonist - a~onist
d(CH2)5~D-~la ]VA'IP agonist - 177 T31 5.79+0.08(4)
d(CH2~5[~-Val jVAVP 2.3 +0.3 * 7.48+0.06(4) 27 T3 6.41+0.05(4)
d(CE2)5[~-reu ]V~VP 1.2 +0.3 * 7.79T0.12(4) 26 '5 6.45+0.09(4) ~ ~
d(CH2)5~D-Ile2~VA~rP 0.70+Q.0 * 7.38+0.05(4) 8.2 T1.4 6.94iO.08(5) Y
d(CH2)5[D-~rg jV~v~ ~39 ~5.9 rV 260 ~v 5.4 Gn
d(CH2)5 D-P~e V~A~ 6.9 +1.37,01fO.10~9) 0.73 7.93 T 0.07(4)
d(CH2)5Tyr(:'e)~,~AVP 15 +3 6.68T0.11(~) 0.28+0.05 3.4~ 0.07(~)
d(CH2)5~yr(~t)VD~.~YrP 5.7 +0.5 '.lC 0.08(4) n.34 0.0 8.31-0-~5~3)
. .
.
.~
s"
Table II(Continued)
Anti-a~ti~iuretic Antivas~pressor
Co~o~nd E ?A2 _ 2
d(C~2)5Tyr(i-Pr)VDAVP 8.5 0.C7 6.88-0.07(4) 0.28TO.Q7 8.41T0.08 (8)
d(cH2)5Tyr(n-pr)vDAvp 14~2 6.67+0.05(4) 1.1~0.2 7.86~^.10 (8)
d(cH2)5Tyr(L~l2)vAvp 3.1'0.4 7.35+0.06(4) 0.29+0.06 8.32T0.08(4) w
d(C;~2)5Tyr(Et)VAV~ 1.9~0.2 7.57T0.06(4) 0.49+0.11 8.16+0.09(4) N
d(Cri2)5Tyr(i-Pr)Vr.VP 3.6+0.9 7.32+G.06(6) 0.31+0.06 8.36+0.09(4)
d(CH2)5Tyr(~-2r)VAVP 3.5-0.06 7.29TO.07(4) 0.40~0.04 8.22+0.04(4) 67
d(CH2)~-D--yr(~e)VAVP 1.2-0.3 7,77+Q.07(6) 0.23fo.04 8.48+Q.08(4)
d(CF.2)5-D--yr( r t)VAVP 1.1~0.2 7.81+0.07(5) 0.45f0.11 8.22 0.12(4)
d(C~2)5~TYr~Et)~V~
~3 Pro7 A~7P 1. 5TO . 3
~2Q(~4 :7
33
The preEerred compounds of this invention are ~lso
more select.ive than prior art antidiurctic ant~gonists
with respect to antivasopressor potencies, as shown by
the ratios of antivasopressor/anti-antidiuretic effective
doses:
, ~nt.ivasopressor
Compounds ED s ~n~i-.tn~i~liur~tic
d(Cil2)5 D-Tyr(Et) V~vP 0.41
d(CI~ ) D-r~ V~VP 0. n7
d(Cll2)5[D-Val2]V~VP 12
d(Cll2)5 [D-Leu ~ V~VP 22
d(CI12)5[D~ ]V~Vp 12
F.X~MPLE 1~
(a) ~ntagonism to ~ -mercapto-~,~-cyclopenta-
methylenepropionic acic~), 2-(0-alkyl)tyrosine, ~-vaJille]-
(L- and D-)-ar~ine vasopressin compounds to en~o~enous
ADI~ was shown by injection of the compound
intraperitoneally .into conscious rats. Urine was
collected for 4 h after the injections. l`he
data in the Table below are the means ~ SE of
2~ results on groups of 4-6 rat~. *P <0.05 and
**P ~0.005 are given for d.iferences between the
means for rats receiving antagonists and the
means for reponses of the same rats injected with
solvent only. The mean control urine volume
.rate for ~olvent-injected r~ts was 0.9 + 0.1 ml/kg
per hr and the mean osmolality was 15~4 + 85
mOsm/kgll20 (n=32~.
54~7
Dose Ur~ne volume Osmolality
ug/kg ml/kg per hr m~sm/kgll2o
d(Cll2)5~yr(Me)100 1.5 + 0.313~1 ~ 428
V~7~VP
300 ~.2 ~ 0.4* 961 ~ 20~*
d(CH2)5Tyr(Et)- 30 2.8 ~ 0.~** 6~0 -t 47
VD~VP
100 9.5 ~ *23~ ~ 25**
d(Cll2)5Tyr(Me)- 10 1.1 + 0.5 1303 ~ 190
1 ~Vl\VP
3.4 ~ 0.9~ 51~ ~ 105~
d(CIi2)5T~r(~t)- 10 7.~ + 1.0~ 3lfi + 38~*
. Vir\VP
13.~ + 2.5~ 194 ~ 17*~
tb) Responses by intact female rats, weighincJ
20~ to ~ .J, to llltl-~t?e~lto~ je~ )t
d(C112)5Tyr(Et)V~VI' were determined in ~ block design
experiment in which each rat received solvent and
both doses of the ~DII antagon.ist. Injections were
?~ given at l~ast two days apart. The rats were on
water ad lib. Injections we.re m~de at 11 ~.m.,
~fter which spontaneously vo.icled urine was collected
hourly Eor four hours.
In ~ig. 1 i.s shown os~ol.~li.ty Or tllt. ~lrinc~
~s a ~uncti.on Or t.imc. tJrine osmol~]i.tics for
the control (solvcnt-injecttcl) were .lvcr.lrlcrl ovcr
2 hour l~criods owin(l to infrc(lucl)cy ol l.ll~in-ltiOIl.
In lir~. 2 i~ shown tllc urinc ollt.l~uL ns a function
of time.
3~ In l)oth lirlurcs, vcrtic.~l ].inc~s tllrou~ ?oints
indic.~tc Sl's.
5 4 7
EXAMPLE 11
Compounds wherein Tyr at the 2-position is of
the D-series and is unetherified (X' is H) are pre-
pared as in Examples 1-5. The compound in which Z-is
L-Arg is very active as an antagonist of the anti-
diuretic activity of arginine vasopressin.
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 chan~es and
modifications of the invention to adapt it to various
usages and conditions
