Note: Descriptions are shown in the official language in which they were submitted.
- 2 ~ 2 2 ~
Tht~ ention concern.~ 8~D~hom~rgiIline vn.so~re~sin
Il~ m~ny l~bor~torie~ the great ~tention ha~ be~n
~n.i.d to ~h~ preparation Or de.ri~ative~ with arginine ~t
the po~it~on ~ replaced. ~ith D-homoarginlne ~nd tyro,~ine
at the ro~it.ion 2 ~rit,h no.n~nntur~l amino ~cide.~ (Z~or~l
~t ~ Colleotion o~ CzechOCh~m.CQ~mt~. 9 31, 310, 1966;
31, ~ 56; 32, 125Q, 1967~ 35, 17169 1970; 37, 3350,
197~ 40, 905, 1975; ~19 20S~, 1976; Huguenin ~nd Boi~son-
na.~: Helv,Chim,~ct~, 46, 1669, 1963; Lindeberg et al: J~
~r~e~. ~hem,~ 15, 629, 1972S 17, 781, 1974; ~.indeberg: Int,
rtid~ Prot.Res., 7, 395, 1975; Lindeberg et al.: Int,
~T~ P ptide Prot~Re~., 8, 19~, 1976; ~odan~zky ~nd ~ind~bergs
~M~ hem~, 14, 1197, 1971). The published r~ult~ r~flect
d.i~ocintion o~ .some biologic~l effect.s (e.g.pxessoric and
ant~.dluretic) depending on the re~lacement o~ the L-nmino
~cid with the ~-~orm, a~ well ~ on the shi~t in the Fo3i-
ti~n of ~he po.~itivel.y ch~rged fl~ction gro~lp an~ on the
id ch~ln of the ~mino acid at the po~ition 8 of the pep-
tid~ ohn.i.n. Thi~ ~hirt in the ~-form of the sub~ti-tuent in-
enae~ the s,cce~ib.ility Or the peptide bond formed by
i:he ba.~ic nmino acld c~rboxyl group to try~sin clea~age
(Dim~li and ~arth: Collection of Czech~Chem.Commun., 44,
~5~ 79)~
Incre~3ing the di~tance o~ the guanidine gr~up in the
v~o~re~in (homo~rginine) line retain~ the ~ntidiuxetic
~Ct~Vit.~r at ~lmost unchanged level (Lindeberg et ~ J.Med.
C'hem., 17, 781, 1974), in dea~no ~n~logue~ w~ en¢ounter a
rel~t.iYe decre~e (~indeb~rg et ~ J.Med.Chem., 17, 781,
1974; ~ko~kova et ~ Collection of Cz~ch.Chem.Commu~.,
~6, ~50, 19~
The replacement o.~ ho~narginine with it~ form in
~e~mino annlogt~e~ (Z~oral md Rrtni}~: Collection of Czech,
Chem,Commun., 40, 905, 1975) enh~noe~ the decre~e in the
~nti~iuretic ef~ect ~y more th~.n one order (~lcopko~ et al,0
Co?.l~ction ~ C7.e~h.Chem~Co~t-~, 9 ~67 1850, 1~
~ ombinatiQn o~ replacemcnts o~ amino acid re~idues
~t the po~ition~ 2 and 8 of the 8~L-arginin~ ~a~opre~sin
peptide chain represent~ one of the mo~t potent approaches
to qu~litati~e9 a~ well a~ quRntitative alterations in the
~iological propertie~ of this type of pe~t~de tK. Jost et
al,: Handbook o~ Neurohypoph~eal Hormone Analogue~, CRC
Press, Boca Raton, U.S.A., 1987), A ~ossibility for furt-
her alter~ion~ is o~ered by the free amlno group o~ cys-
teine ~t the position 1 o~ the peptide, especially from
the ~oint of view o~ the prolonged a¢tion,
To obtain ~peci~ic~lly more e~eotiYe inhibitors o~
neurohypophy~eal hormones 9 the analogue~ o~ va~opre~in and
l~amino~asopre3~in were ~ynthe~ized with D-homoargini.ne at
the po~ition 8, which have been ~urther modified by ~ubsti-
tutions et the po3~tion 2~
Subject Or the invention cover~ new analogues of ~-D-
-homon~ginine ~Rsopres~in of the general formula
' I
R-Phe-Gln-A~n-~y~-Pro-D-~Iar-Gly~1
where ~ i3
L-O-methyltyrosine (compound of the formula I,VI)
Il-p-eth~lphenylalanine (compound of the ~ormula II,VII)
D-p_ethylphenylalanine (compound of the ~ormula III,VIII)
J,-p-methylphenglalanine (compound o~ the formula IV,IX)
D-~-methylFhenylalanine (oompound of the formula V,X)
and R i~ cg~teine or ~ -mercaptopropionic acid, The sub~ect
cover~ al~o the method for the preparation of the~e com-
pound~ whi¢h is ba~ed on the solid-phase ~tep-wi~e ~ynthesi3
o~ the linenr chain ~rom hydro~ybenztriazole e~ter~ of the
amino ~cid~ proper protected with tert. butyloxycarbonyl
groups, ~ollowed by relea~ing from the resin with the ~imul-
taneous cleavage of the side-chain protecting ~roups by
mean~ of liquid hydrogen fluoride and finally, by clo8ing
th~ cycle accompJ.i~hed by the action of k~lium ferricganide
~ ~3 2 ~
and ~eFar~tion o:~ the peptide~ wlth I,- and D- i~omera
o~ ~m.ino RC~ d3 a t the po~3ition 2 o* the peptid~ ohain
by mean~ of high-pre~sure liquid chromatography (HPI.C1~
The M ntioned tyE~e3 of compounds which are able to 8Up-
~re~.~ the uterotonîc effect of the neurohypophut~e~l
ho:rmone~ may be potentially util~zed ~s the agen~ bloc-
king a premature ohildbirth provoked by endcgenic o~y-
~ocin or ~t~opre~sin.
The compounds VIII and X belong to the most effe~-
tive lnhibitors of the ~tasopre~sin aeries prep~red up
to thi3 time, See the tabl~,
N - ~ -tert.butylox~oarbonyl-~G-nitrohomoarginine
w~ ~ound to be a ~uitable D-homoarginine derivative for
the 3ynthe3is o~ all analogues, the p-methylphenyl~lQni-
ne and p-ethglphenylal~nine re~idue~ being introduced
into the ~olecule in the form of a mi~ture o~ nd D-
-amino acid~.
The ~ynthesi~ o~ oompounds of the formul~ I to X
wa~ oarried out employing the ~olid-pha~e method on benz-
hydrylamine resin~ The ~ -amino group~ were protected with
the tert,butylo~y¢arbonyl group (Boc), ~ide-¢hain group~
were blocked with the nitro group (D-homoar~inine), 4-me-
thylbenzyl (cysteine), benzylo~ycarbonyl (t~rosine) and
benzyl ( ~ -mercaptopropionic a¢id). Condensation of the
prote¢ted amino ~cid~ wa3 carried out in dimethylformami-
de (DM~) using the corre~pondin~ active hydroxybenzotria-
zole e~ters~ The prote~tive group8 ~ere clea~ed from the
~ide ohains ~lth liquid hydrogen fluoride (H~), 3imulta-
neo~ ith releQsing the peptide chain from the carrier,
Oxidation o~ SH-group~ and the ~ub~equent closing of the
~ycle were accompli~hed by the action o~ kalium ~erri-
cyanide ~olution, The compound~ were puri~ied and peptide~
with D-and L-amino aold~ at the position 2 wsre separated
by means oi HP~C. ~or the preparation of diastereo-i~ome-
rlc mixtures of the ~nalogue~ with D,l~p ~thylphenylalaline
or D,I.-p-~ethylphenglalani~e were u~ed only 1.1 equivalent~
~2~3~
of t~rtr butyloxycarbonyl-D,~p-ethu~lphenylalanine and
t~rt. butylox~carbonyl~D,L-p meth~lphenylalanine, respec-
tiV~ly, 1
The aompound~ obtai~ed were oharakterized by thinlay~r chromatograFh~ (T~C) on ~iliGa pl~-t~s (Sulifol, Ka-
~nlier, Gzecho310vnkia) u~ing the ~ollowing solvent sy~-
te~ 2-butanol 98% ~ormic acid-w~ter (10:3:8) or l-bu-
tanol-acetic ac~d-p~ridin~ ~ter (15:3:10:6)~ Electropho-
re~i~ on Whatm~nn 3 ~M p~per ~ lM-acetic ncid or in B pyridine
-acetate bu~er (pH 5,7), for 1 h at 20 Vcm 1 was al~o em-
ployed for the char~terizat~on, Amino acid analy~iq wa~
carried out on Qnaly~ers T339 or D-500 (Durrum Corp., U.S,~.).
Analytical HP~a wa~ run on the column with Sepharon IX C-
18 or Vydao Z18 TP 5, the preparative HPLC ~as carried out
on a modular ~y~tem (~n~uer) on a co~umn with Sepharon SGX-C-
-
~ t o~ abbreviation~: HP~C - high-presaure li~uid
chromatography~ MeOH m~th~nol~ T~C - thin-layer chromato-
graphy~ DMF - dimethwl ~ormnmide~ ~oc - tert.butylo~ycarbo-
n.yl group~ Mpr - ~ -mercaptopropionic acid~ Bzl - benzyl
group; T~ - tri~luoro~cetic acid,
~,~2~
The procedllre for joining the protected ~unino acid to the peptide chain being formed
on the carricr can be described as follows:
1. Cleavage of the Boc-group with 40 ml of S0% trifluoroacetic acid (TFA) in
dichloromethane containing 2% anisole, lasting 2 min, repeated again
after 30 min.
2. Rinsing with dichloromethane (3 x 40 ml, duration of each rinsing 30 sec).
3. Rinsing with 30% dioxane in dichloromethane (3 x 40 ml, each rinsing 30
sec).
4. Rinsing with dichloromethane (as in step 2).
5. Neutralization with 10% triethylarnine (40 n~) in dichloromethane (40 ml, 2
rnin cycle).
6. Rinsing with dichloromethane (2 x 40 ml, 30 sec cycle).
7. Neutralization with 40 ml of 10% triethylamine in 40 rnl dichloromethane (2
min cycle).
B. Rinsing with dichloromethane (6 x 40 ml, 30 sec cycle).
9. Additlon of the hydroxybezotriazole ester of the Boc-protected amino acid in
dichloromethane, until negative ninhydrin test is achieved (30 - 120 min).
10. Rinsing with 50% ethanol in dichloromethane (3 x 40 rnl, 30 sec cycle).
11. Rinsing with dichloromethane (3 x 15 ml, 1 rnin cycle).
Benzhydrylamine resin (4.46 g, 0.56 mmolg') was suspended in dichloromethane andafter rinsing with 5% triethylamine in dichloromethane and dimethylformamide, rnixed
with 3 molar excess of Boc-Gly-OBt. The reaction was intelrupted after 30 min and the
resin was rinsed subsequently with dimethylformamide (3 x 20 ml) and dichloromethane
(3 x 20 ml). Then, the psocedure described in Exarnple 4 was applied. Boc- aminoacids in the form of active esters (in 3 molar excess) were coupled to the amino groups
of the bound amino acid in the following sequence: Boc-D-Har(NO2)-OH, Boc-Pro-OH,
Boc-Cys(4-Me-Bzl)-OH, Boc-Asn-OH, Boc-Gln-OH and Boc-Phe-OH. A catalyst (4-
dimethylaminopyridine, 50 mg) was added to accelerate the coupling of homoarginine,
cysteine and phenylalanine.
h
The biologi.oal ~ctivity of the peptide~ wa~ te~ted
on rats namely: the uterotonic activity (agon~ stlc and
Qntagonistic) according to the Holton'~ method (Holton,J.:
~rit,J.Pharmacol., 3, 328, 1960) modified by Munsick(En-
docrinology, 66S 451, 1960~, the in}~bitory activitg being
expre~sed as PA2 (Eggen et al.: J~Gen.Phy~iol,, 56, ~50,
1970), the pre~oric activity was determlned on de~pinali-
zed male rats ac¢ording to Krejci et al.(Brit.J.Pharm,~he-
mother., 30, 497, 1967) and the antidiuretic activity
according to Burn (Burn et al.: Blol.Stand,O~ford Univ.
Press, London, 1950) with ~deaminol, D-arginine8Jva~o-
pre~in (dD~VP) u~ed a~ the ~t~ndard. Summary of the bio-
logical activities is pre~ented in ~able.
Further, e~amples describing the preparation of com-
pounds I to X.are pre~ented~
2 ~ ~
Example 1
Preparation of [ 1 -mercaptopropionic acid, 2-O-methyltyrosirle R-I)-
homoar~inine]vasopressin (compound o the formula VI),
Boc-Tyr(Me)OH and Mpr(Bzl)OH were coupled to the resin with the bound
heptapetide (0.91 g, 0.33 mmol). The resin with the bound nonapeptide (1 g ) wassubn~itted to the action of liquid hydrogen fluoride (10 ml, 60 min, 0C) in the presence
of anisole ~l.S rnl). The hydrogen fluoride was then removed with nitrogen at 0C (in
the course of 30 min). The mixture of the free nonapetide and the resin was shaken
wi~h ether, filtered off and rinsed with ethylacetate. The free peptide was then dissolved
in 20% acetic acid (100 ml) at 40C, diluted with water and the solution was
Iyophiliæd. The Iyophilized product was dissolved in water (300 ml) and the pH of the
solution was adjusted to 7Ø Kalium ferricyanide (0.01 moll-' was added to the solution
until the colour remained yellow. When the oxidation was terrninated (after 30 min),
the pH was adjusted to 4.5 with acetic acid. The solution was applied to a column with
Amberlite CG-SOI and, after rinsing with 0.25% acetic acid ~150 ml), the product was
eluted with 50% acetic acid (60 rnl), Iyophilized (47 mg) and purified by means of
HPLC to afford 9 mg of the peptide.
Amino acid analysis: Phe 0.87, Cys - 0.91, Asp - 1.01, Glu - 0.98, Pro - 0.96, Gly -
1.00, Tyr(Me) - 0.96, Har - 0.92.
Example 2
Preparation of [1-mercaptopropionic acid, 2-p-ethyl-D,L-phenylalanine-8-D-
homoarginine]vasopressin (compounds of the formulae VII and VIII),
Boc-L,D-p-ethylphenylalanine (1.1 equivalent) was coupled to the resin with the
bound heptapeptide (1.35 g, 0.5 rnmol) in the course of 24 h. Then, during 30 min,
further 1 equivalent was added in the presence of dimethylaminopyridine. After the
coupling of Mpr(Bzl)OH to the resin according to the scheme presented,the nonapepdde
bound to the resin (1.4 g) was submitted to the acdon of liquid hydrogen fluoride (10
ml, 60 min, 0C) in the presence of anisole (l.S ml). The hydrogen fluoride was
removed with nitrogen at 0C (in the course of 30 min). The released nonapeptidetogether with the resin was mixed with ether, filtered off and rinsed with ethylace~ate.
The free peptide was then dissolved in 20% acetic acid (100 ml) at 40C, diluted with
water and the solution was Iyophilized. The Iyophiliæd product was dissolved in water
(500 ml) and the pH of the solution was adjusted to 7.0 with ammonium hydroxide.Kalium ferricy<~lide (V.01 moll~' was added, until the colour of the solution remained
yellow. When the oxidation was terminated (after 30 rnin), the pH was adjusted to 4.5
with acetic acid. The solution was applied to a column with Amberlite CG-50I and,
after rinsing with 0.25% acetic acid (150 ml), the product was eluted with 50% acetic
acid (60 ml). Lyophilisation of the eluate afforded 41 mg of the product.
Separation of the racemate of [l-mercaptopropionic acid, 2-p-ethyl-D,L-phenylalanine-8-
I)-homoarginine]vasop}essin:
a The preparative HPLC was canied out using a modular settup (Knauer settupcomprising Knauer HPLC Programmer 50, Knauer HPLC Pump 364 and Knauer
detector with variable wave1ength setting) on a column filled with Sepharon SG-X-C-18
(10 um, 250 x 16 mm). The peptide was injected in the amount of 5 mg and eluted
with a concentration gradient of methanol (MeOH) - start 50%, gradient 1% min~' (the
retention time for the L-form: 10.05 min, for the D-form: 11.97 min). Yield of the L-
derivative was 10 mg, that of the D-derivative X mg.
Amino acid analysis: L-form: EtPhe - 0.80, Phe - 1.03, Cys - 0.93, Asp - 0.95, Glu -
1.12, Pro - 0.98, Gly - 1.00, Har - 0.87;
.D-form: tPhe - 0.85, Phe - 1.00, Cys - 0.93, Asp - 0.93, Glu - 1.16, Pro - 0.97, Gly -
1.03, Har- 0.87.
Example 3
Preparation of [1-mercaptopropionic acid, 2-p-methyl-D,L-phenylalanine-8-D-
homoarginine]vasopressin (compounds of the formulae I~: and X).
Boc-L,D-p-MePhe-OH and Mpr(Bzl)OH were coupled to the resin with the
bound heptapeptide (1.35 g, 0.5 mmol) according to the procedure described in Example
7. Peptides were released from the resin and isolated as described in Example 7. Yield:
50 mg of the peptide.
Separation of the racemate ll-mercaptopropionic acid, 2-p-methyl-D,L-phenylalanine-8-
D-homoarginine]vasopressin;
Separation of the rnixture was carried out as in Example 2. Under the conditionsstated, the mobility of the L-p-methylphenylalanine isomer is higher (retention eime
C~, ~ 2 2
13.57 - 13.73 min) in comparison to that of the D- isomer (retentioll time 15.72 - 15.75
Itlill). Yield of the L-derivative: 4.2 mg, of the D-derivative: 5.6 mg.
Amillo acid analysis: L-rorm: MePhe - 0,90, Phe - 1.03, Cys - 1.05, Asp - ().97, Glu -
1.13, Pro - 0.99, Gly - 1.05, Har - 0.87;
D-form: MePhe - 0.87, Phe - 1.00, Cys - 1.06, Asp - 0.95, Glu - 1.10, Pro - 1.00,
~31y - 1.06, Har - 0.87.
Example 4
Preparation of ~2-O-methyltyrosine, 8-D-homoarginine]vasopressin (compound of the
formula 1)
Boc-Tyr(Me)OH and Boc-Cys(4-Me-Bzl)OH were coupled to the resin with the
bound heptapeptide (1.7 g, 0.48 mmol) according to the general scheme,
'Ihe resin with the coupled nonapeptide (1.8 g ) was submitted to the action of liquid
hydrogen fluoride (20 ml, 60 min, 0C) in the presence of anisole (1.0 ml) and 1,2-
ethandithiole (1 ml). The hydrogen fluoride was removed with nitrogcn at oC ~in the
course of 30 min). The released nonapeptide together with the resin was mixed with
ether, ~lltered off and rinsed with ethylacetate. The free peptide was extracted with 50%
acetic acid, acetic acid and water and finally, Iyophilized (yield 0.69 g). The Iyophilized
product was dissolved in water (700 ml~ and the pH of the solution was adjusted to 7.0
with sodium hydroxide (0.1 moll'~,Kalium ferricyanide (0.01 moll') was added, until
the colour of the solution remained yellow. The pH of the solution was maintained at
7.2 during the oxidation (20 min) by means of sodium hydroxide solution (0.1 moll'),
lhell it was adjusted to 4.5 with acetic acid. The solution was applied to a column with
Amberlite CG-SOI and, after rinsing with 0.25% acetic acid, the product was eluted with
50% acetic acid (60 ml), Iyophilized (353 mg) and purified on a column Vydac TPS by
met!lanol elution (linear gradient 20% - 40% MeOH - 0.05% trifluoroacetic acid)
Lyophilisation yielded 160 mg of the product.
Elemental analysis: for C~,,H67NI30,2S~ x 3 TFA x 2 H~O calculated: 43.26% C, 5.18%
H, 14.01% N; found: 42.92% C, 4.92% H, 14.31% N.
AMino acid analysis: Asp - 1.00, Glu - 1.00, Pro - 0.75, Gly - 0.99, Cys - 1.87,Tyr(Me) - 1.1, Phe - 1.07, Har - 1.05.
~(~
2 ~ 2 s~
Exntnyle 5
~reparation of [2-p-ethyl-L-pllenylalanine, 8-D-homoarginine]Y~Isopressin (compound of
the fonnula 11)
Thc resin with the bound heptapeptide (1.35 g, 0.5 mmol - Fxamplc4 ) was
submitted to the reaclion with 1.1 equivalent of Boc-L,D-
PIle(p-Et)-OH for 24 h, with further 1 equivalent in the presence of
dimethylaminopyridine for 30 min, and finally, with Boc-Cys(4-Me-Bzl~OH. After
removing the Boc-protec~ive gtOUp, ~he resin was mixed with hydrogen fluoride (15 ml,
6O min, 0C) in the presence of anisole (2 ml). The released nonapeptide was together
wi~h the resin (after removing hydrogen fluoride with nitrogen) rnixed with ether,
filtered off, rinsed with ethylacetate and the free peptide was extracted subsequently
with acetic acid, 50% acetic acid and water and finally, neutralised to the pH value Gf
7.0 with sodium hydroxide (0.1 moll-'). Kalium ferricyanide (0.01 moll-') was added,
until the colour of the solution remained yellow. The pH of the solution was maintained
nt 7.2 during tlle oxidation (20 rnin) by means of sodium hydroxide solution (0.1 moll 3 )
then it was adjusted to 4~5 with acetic acid~ The soluti~on was applied to a column
with Amberlite CG-50l and, after rinsing with 0.25% acetic acid, the product was eluted
with 50% acetic acid, Iyophili~ed (185 mg) and purifled by means of HPLC on a
-column with Sepharon SGX C-18 by a linear gradi,ent MeOH (20 - 70%) - 0.1% TFA.'rhe first peak characterized by a mobilitylf k'- 7.4~ ~e~)~f) - 0.~ A
colresponds to the analogue with p-ethyl-L-phenylalanine at the position 2.
For C49H,,N,sO"Sl x 4 TFA x 2 H1O (1602.5) calculated: 42.79% C, 4.97% H, 13.11%N; found: 42~85% C, 5~05% H, 13~37% N.
~mino acid analysis: Asp - 0~99, Glu - 1~00, Pro - 1.12, Gly - 1.01, Cys (determined
as cys~cic ncid) - 2.12, 4-Et-Phc - 0.78, Phc - 1.02, H~r - 0.80.
Yield 18 mg~
Example 6
Preparation of ~2-p-ethyl-D-phcnylalanine, 8-D-homoarginine]vasopressin (compound of
lhe formula 111)
The compound WAS prepared accorsling to the procedure described in Example 5,
in a mixture with compound 1l, from which it has been separated by rneans of HPLC
on a column with Sepharon SGX C-18 as described in Example 4. The compound III
~ ,4~ s~ ?J ~
WAS cluted in the second peak, after the linear gradient MeOH - 0.05% TF~ (20 -
7() %) ha(l been applied and is characteriz~d by the following parameters: k - 3,60,MeOH
- 0,OS5~ TF~ (6: 4), k = 19,57 MeOH - 0,05,'~ TFA (5: 5)O
For C49~7,N,~O"Sl x 4 I~A (1566.4) calculatcd: 43.71% C, 4.83% H, 13.41% N; found:
43.74% C, 5.16% H, 13.52% N.
Amino acid analysis: Asp - 1.00, ~lu - 0.98, Pro - 1.05, Gly - 1.12, Cys (detennined
as cysteic acid) - 2.02, 4-Et-Phe - 0.68, Phe - 1.05, Har^ 0.8~.
Example 7
Preparation of [2-p-methyl-L-phenylalanlne, 8-D-homoarginine]vasopressin (compound of
the forrmula IV)
The resin with the bound heptapeptide (3.4 g, 0.96 mmol ) ` was
treated according to the scheme described.. With 1.1 equivalent of Boc-
L,D-Phe(p-Me)-OH for 18 h and with further 0.5 equivalent for 4 h. Then the arnino
acid Boc-Cys(4-Me-Bzl)-OH was nttached. The resin with the coupled protected peptide
was treated then according to the procedure described in Exarnple 5~ Elution with 50%
acetic acid from the column with Amberlite CG-50I affor~ed 614 mg of the crude
product which was Iyophili~ed and purified funher by means of HPLC on a co1urnn
Vydac 218 TPS by the elution with a linear concentration gradient MeOH (25 - 60%) -
0.()5% TFA. [2-p-rnethyl-L-pllenylalanine, 8-D-homoarginine]vasopressin was elutcd in
the first pcak (in the yield of 40 mg), the characteristics of which wcre as follows: k's
3.18, MeOH - 0.05% TFA (55:45).
I~or C~,H6,N"O,2S~ x 3 TFA ~c 2 H20 (1474.4) calculated: 43.99% C, 5.20% H, 14.25%
N; found: 44.19% C, 4.96% H, 14.41% N.
~mino acid analysis: Asp - 1.08, Glu - 1.01, Pro - 0.87, Gly - 1.08, Cys (detcrmined
as cysleic acid) - 2.04, 4-Me-Phe - 0.70, Phe - 0.92, Har - 1.01.
Exatnple 8
E'reparation of l2-p-methyl-D-phenylalanine, 8-D-homoarginine]vasopressin (compound of
tlle formula V)
Ttle compound was prepared according to the procedure described in the
Example 7 in a rnixture with compound IV and separated from it by HPLC on a
:
12
2~2g~
column Vydac Z18 TPS by elution with a linear concsntration ~radient MeOH (25 -
60%) - 0.05% TFA. [2-p-methyl-D-phenylalanine, 8-D-homoarginine]vasopressin wns
eluted in the second peak (16.2 mg), the characteristics of which were as follows: k'=
5.18, MeOH - 0.05% TFA (55:45).
For C4"H~,,N"O,2S2x3 ,5TFAx 1 H20 (1522.4) calculated: 43.39% C, 5.00% H, 13.80%N, found: 43.24% C, 4.78% H, 14.13% N.
Amino acid analysis: Asp - û.89, C31u - 1.02, P~ - 1.00, Gly - 1.05, Cys (detern~ined
as cysteic acid) - 2.03, 4-Me-Phe - 0.88, Phe - 1.00, Har - 1.08.
~22~
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