Retro-inverso analogues of C-terminal penta and hexapeptides of Substance P

This invention relates to new retro-inverso peptides and peptide derivatives in the form of analogues and the C-terminal penta and hexapeptide fragments of Substance P, which are pharmacologically active, possess prolonged action with time and are useful as vasodilators, their general formula being: ##STR1##

The undecapeptide Substance P, which is considered a neurotransmitter and a 
neuromodulator, is probably involved both in the neurotransmission of pain 
sensations and in the sensory axonal reflexes [Otsuka, M. and Takahashi, 
T., Annu. Rev. Pharmacol Toxicol, 17, 425 (1977); Henry, J. L., Brain 
Res., 114, 439 (1976); Celander, O. and Folkow, B., Acta Physiol. Scand., 
29, 359 (1953); Narumi, S. and Maki, Y., J. Neurochem., 30, 1321 (1978); 
Oehme et al., Acta biol. med. germ., 39, 469 (1980), Haeusler, G. and 
Osterwalder, R., Naunyn-Schiemedeberg's Arch. Pharmacol., 314, 111 (1980); 
Hokfelt, T. et al. in "Substance P", Von Euler, U. S. and Pernow, B., 
Editors, Raven, New York, 1977, p. 117]. 
In the central nervous system, Substance P depolarises the neurons of the 
spinal medulla and stimulates or facilitates the response of the cells 
sensitive to pain stimuli [Konishi, S. and Otsuka, M., Brain Res. 65, 397 
(1974); Henry, J. L. in "Substance P", Von Euler, U. S. and Pernow, B., 
Editors, Raven, New York, 1977, p. 231]. 
In the peripheral nervous system, Substance P, which possesses the property 
of contracting the smooth musculature and is one of the most powerful 
known vasodilators [Chernukh, A. M. et al., Exp. Biol. Med., 90, 1165 
(1980); Lembeck, F. and Zetler, G., Int. Rev. Neurobiol., 4, 159 (1962); 
Schrauwen, E. et al., Pflungers Archiv. Eur. J. Physiol. 386, 281 (1980)]. 
All these activities of Substance P are also preserved in its C-terminal 
segments, in particular in the C-terminal hexa, hepta and octapeptide 
fragments [Blumdberg, S. and Teichberg, V. I., Biochem, Byophis. Res. 
Commun. 90, 347 (1979); Bury, R. W. and Mashford, M. L., J. Med. Chem. 19, 
854 (1976); Otsuka, M. and Konishi, S. in "Substance P", Von Euler, U. S. 
and Pernow B., Editors, Raven, New York, 1977, p. 207; Rosell, S. et al. 
in Substance P, Von Euler, U. S. and Pernow B., Editors, Raven, New York, 
1977, p. 83; Yanaihara, N. et al. in Substance P, Von Euler, U. S. and 
Pernow, B., Editors, Raven, New York, 1977, p. 27]. 
Structure-function studies carried out on a series of partial sequences and 
fragments of Substance P, prepared by chemical synthesis, have shown that 
the Substance P receptor interacts best with the natural hexa and 
heptapeptide C-terminal sequences, and that the extension of the chain 
beyond the C-terminal heptapeptide produces effects of negligible 
importance. Moreover, protecting the N-terminal residue by a 
tert-butyloxycarbonyl group significantly increases the power of the 
natural C-terminal pentapeptide, whereas it produces unimportant effects 
in longer peptides. [Teichberg, V. I. and Blumdberg, S., Prog. Biochem. 
Pharmacol. 16, 84 (1980); Chorev, M. et al., in "Peptides 1980", 
Proceedings of the 16th European Peptide Symposium, Brunfeldt, K., 
Scriptor, Copenhagen, 1981, p. 451]. 
It has also been shown that Substance P and its C-terminal hexa, hepta and 
octapeptide fragments are rapidly degraded by numerous proteolytic enzymes 
with endo and aminopeptidase activity [Gullbring, B., Acta Physiol. Scand. 
6, 246 (1943); Teichberg, V. I. and Blumberg, S., Prog. Biochem. 
Pharmacol. 16, 84, (1980)]. 
The fact that Substance P and its C-terminal fragments are extremely labile 
towards enzymes makes their use problematical not only in characterisation 
studies of the Substance P receptor, but also their use in pharmacology. 
Attempts to stabilise the molecule, similar to those carried out 
successfully in the case of natural opioid peptides such as Met.sup.5 
-Encephalin and Leu.sup.5 -Encephalin (substitution of Gly with D-Ala or 
substitution of aminoacid residues with N-methylaminoacid residues) have 
led to a substantial loss of power [Blumberg, S. and Teichberg, V. I., 
Biochem. Biophys. Res. Comm. 90, 347 (1979); Sandberg, B. E. B. et al., 
Eur. J. Biochem., 114, 329 (1981)]. 
In order to adequately protect the peptide sequence against the hydrolytic 
action of peptidase, we have now discovered, according to the present 
invention, that it is extremely advantageous to apply the criterion of 
retro-inversion of suitable peptide bonds to the C-terminal penta and 
hexapeptide fragments of Substance P. 
We have therefore inverted one of the peptide bonds of the Substance P 
sequence which has proved most susceptible to the action of endopeptidase 
(the bond Phe-Gly), [Lee, C. M. et al., Eur. J. Biochem., 114, 315 (1981) 
and cited references], with the intention of making it more resistant to 
enzymatic degradation while preserving, by means of this modification, the 
three dimensional orientation of the peptide side chains, which is one of 
the indispensable requirements for maintaining the action power of the 
analogue. The inversion of a single peptide bond in the sequence requires 
the transformation of the two aminoacid residues used to form the inverted 
bond, and in particular the aminoacid residue closest to the amino end of 
the reference peptide, into a gem-diamino residue, and the transformation 
of the aminoacid residue closest to the carboxyl end into a residue of 
malonyl or 2-substituted malonyl type [Goodman, M. and Chorev, M., Acc. 
Chem. Res., 12, 1 (1979) and cited references]. 
While the incorporation of the malonyl or 2-substituted malonyl residues 
into the peptide skeleton does not present particular problems, the 
incorporation of the gem-diamino residues generally requires special and 
delicate synthesis manipulations [Goodman, M. and Chorev, M., in 
"Perspectives in Peptide Chemistry", Eberle, A., Geiger, R. and Wieland, 
T. Editors, Karger, Basel, 1980, p. 283]. 
We have considerably simplified the problem of introducing a gem-diamino 
residue into a peptide sequence by using the reagent 
I,I-bis(trifluoroacetoxy) iodobenzene as described in a copending patent 
application in the name of the same applicant. The reagent, the use of 
which is known in the direct conversion of primary carboxyl amides of 
simple structure into amines under extremely mild reaction conditions 
[Radhakrishna, A. S. et al., J. Org. Chem. 44, 1746 (1979)] is useful in 
the direct conversion of primary peptide and aminoacid amides, protected 
at the terminal NH.sub.2, into the corresponding trifluoroacetic acid 
salts of N-monoacylated gem-diamino derivatives, as described in the said 
copending patent application. We have now discovered that which 
constitutes the subject matter of the present invention, namely that it is 
possible to synthesise two new classes of peptide analogues which are 
retro-inverted at the Phe-Gly bonds of the C-terminal penta and 
hexapeptide fragments of Substance P, by employing the results obtainable 
by using I,I-bis(trifluoroacetoxy) iodobenzene, in accordance with the 
process described in the said copending patent application. 
The retro-inverso peptides according to the present invention are of 
general formula (I): 
##STR2## 
in which P is hydrogen, a linear or branched aliphatic alkyl group with 
1-6 carbon atoms, or a saturated or unsaturated linear or branched chain 
aliphatic acyl group such as formyl, acetyl, propionyl, n-butyryl, 
isobutyrl, n-valeryl, isovaleryl, hexanoyl, isohexanoyl, heptanoyl, 
octanoyl, crotonoyl, methacryloyl, acryloyl; or a substituted acyl group 
such as hydroxyacetyl, 2-hydroxypropionyl, 3-hydroxypropionyl, 
aminoacetyl, 4-hydroxyphenylacetyl, 4-hydroxyphenylpropionyl, 
2-aminopropionyl, 3-aminopropionyl, 0-ethylmalonyl, ethoxyformyl, 
methoxyacetyl, 3-methoxypropionyl, 3-ethoxypropionyl, chloroacetyl, 
dichloroacetyl, 2-chloropropionyl, 3-chloropropionyl, 
2,3-dichloropropionyl, bromoacetyl, 4-hydroxy-3,5-diiodophenylacetyl, 
3-oxobutyryl, 3-oxovaleryl, 4-oxovaleryl, methylthioacetyl, 
3-methylthiopropionyl, ethylthioacetyl, 3-ethylthiopropionyl, nicotinoyl, 
.gamma.-aminobutyryl, N.sup..alpha. 
-[(1-(9-adenyl)-.beta.-D-ribofuranuronosyl)], N.sup..alpha. 
-[(1-9-hypoxanthyl)-.beta.-D-ribofuranuronosyl)]; or a group such as 
benzyloxycarbonyl, tert-butyloxycarbonyl, tert-amyloxycarbonyl, 
isobornyloxycarbonyl, adamantyloxycarbonyl, chloro or nitro-substituted 
benzyloxycarbonyl; R.sup.1 is a residue of methionine, methionine 
sulphoxide, methionine sulphone, selenomethionine, leucine, norleucine, 
valine or norvaline; R.sup.2 is a residue of leucine, norleucine, valine, 
norvaline, alanine, isoleucine; R.sup.3 is hydrogen or methyl; R.sup.4 is 
the side-chain of aminoacids such as phenylalanine, tryptophan, tyrosine, 
valine, norvaline, leucine, norleucine, isoleucine, serine or derivatives, 
threonine or derivatives, histidine or derivatives, methionine, 
methionine-S-methyl, methionine sulphone, arginine or derivatives, lysine 
or derivatives, ornithine or derivatives, 2,4-diaminobutyric acid or 
derivatives, 2,3-diaminopropionic acid or derivatives, glutamic acid or 
aspartic acid or their suitable derivatives; R.sup.5 is a peptide fragment 
containing 1 or 2 amino acid residues which constitute the quintultimate 
and sextultimate residue from the carboxyamide end, and of which the 
quintultimate can be phenylalanine, tyrosine, 4-chlorophenylalanine, 
0-benzyltyrosine (or their acetyl, cyclopentyl, tert-butyloxycarbonyl or 4 
-hydroxyphenylacetyl derivatives) or glycine, and the sexultimate can be 
glutamine, pyroglutamic acid, alanine, tyrosine, lysine or derivatives, 
proline, N-formyl-proline, .beta.-alanine, N-acetyl-.beta.-alanine, 
glycine, desaminophenylalanine, desaminoglutamine, desaminoaspartic acid, 
methyldesaminoaspartic acid, or glutamic acid esters represented by 
general formula (II) 
##STR3## 
in which X is methyl, ethyl, methoxyethyl, methoxy (ethoxy).sub.n ethyl 
where N=1, 2, 3, or their tert-butyloxycarbonyl derivatives. 
In the synthesis descriptions reported hereinafter, use is made of the 
following abbreviations: Boc: tert-butyloxycarbonyl; OMe: methyl ester; 
DCC: NN'-dicyclohexylcarbodiimide; DMF: N,N-dimethylformamide; THF: 
tetrahydrofuran; NMH: N-methylmorpholine; MeOH: methanol; EtOH: ethanol; 
EtOAc: ethyl acetate; Et.sub.2 O: ethyl ether; HOBt: 
N-hydroxybenzothiazole; DCU: dicyclohexylurea; BTI: 
I.I-bis(trifluoroacetoxy) iodobenzene; 
##STR4## 
mGly=--OC--CH.sub.2 --CO--. 
Each aminoacid is of L form, even if not expressly specified. A 
pentapeptide derivative of general formula (I) is synthesised by the 
condensation, generally induced by DCC+HOBt, of a N-monoacetylated 
gem-diamino residue of an aminoacid or peptide residue of which the 
terminal NH.sub.2 has been conveniently protected, with a peptide fragment 
of general formula (III) 
##STR5## 
in which R.sup.3, R.sup.2 and R.sup.1 have the same meaning as heretofore. 
A hexapeptide derivative of general formula (I) is synthesised by 
condensing a suitable aminoacid derivative at the pentapeptide fragment 
synthesised as described heretofore, and having its terminal NH.sub.2 
group free, using for this purpose the known condensation methods in 
peptide synthesis as described for example in Bodansky M. and Ondetti M., 
Peptide Synthesis Interscience, New York; 1966; Finn F. M. and Hoffmann K, 
The Proteins, vol. 2, Neurath A. and Hile R. L., Editors, Academic Press, 
New York, 1976; and The Peptides, vol. 1, Gross E. amd Meilnhofer J., 
Editors, Academic Press, New York, 1979. 
After the reactions are complete, the peptides are obtained by known 
procedures in peptide isolation, such as extraction, counter-current 
distribution, precipitation, crystallisation and various types of 
chromatography. 
The presence of the products was demonstrated by reverse phase high 
pressure chromatography analysis (RP--HPLC) using the following eluent 
systems: H.sub.2 O/acetonitrile; 0.01 M NH.sub.4 H.sub.2 PO.sub.4 
/acetonitrile, 0.005 M heptanesulphonic acid, 0.01 M NH.sub.4 H.sub.2 
PO.sub.4 /acetonitrile; and chromatography analysis on a thin silica gel 
layer using the following eluent systems: n.butanolacetic acid-water 
(4:1:1); chloroform-methanol-acetic acid 85:10:5); n.butanol-isopropanol 1 
N NH.sub.4 OH-ethyl acetate (1:1:5:1) (organic phase). 
The melting points have not been corrected. 
The pharmacological activity of the retro-inverso analogues according to 
the present invention was tested by measuring the contraction of the 
isolated ileum of the guinea pig, as described by Rossel and colleagues 
(Rossel S. et al., in "Substance P", Von Euler, V. S. and Pernow B., 
Editors, Raven, New York 1977, p. 83) and by measuring the increase in the 
flow of K.sup.+ ions from testpieces of the parotidean tissue of the rat, 
as described by Rudich and Butcher [Rudich L. and Butcher F. R., Biochim 
Biophys. Acta. 444, 704 (1976)], with reference to the activity of the 
peptide 
EQU [&lt;(Glu.sup.6)]SP.sub.6-11 
The table shows the results of the pharmacological tests carried out using 
the analogue 
EQU [&lt;Glu.sup.6,gPhe.sup.8,mGly.sup.9 ]SP.sub.6-11 
TABLE 1 
__________________________________________________________________________ 
K.sup.+ release by 
Analogues of C-terminal 
Guinea pig ileum 
parotidean tissue 
Duration of action 
hexapeptides of Substance P 
contraction (%) 
(%) (in minutes) 
__________________________________________________________________________ 
[&lt;Glu.sup.6 ]SP.sub.6-11 
100 100 20 
[&lt;Glu.sup.6, gPhe.sup.8, mGly.sup.9 ]SP.sub.6-11 
15 10 No decrease 
(20 hours) 
__________________________________________________________________________ 
The subject matter and scope of the invention will be more apparent on 
reading the following example, which is merely illustrative and must in no 
way be considered as limitative of the invention.

EXAMPLE 
Synthesis of 
pyroglutamylphenylalanylgemdiaminophenylalanylmalonylleucylmethionineamide 
. Pyr-PhegPhe-mGly-Leu-Met-NH2 Synthesis of 
tert-butyloxycarbonylleucylmethionine methyl ester. Boc-Leu-Met-OMe 
1.0 equivalents of Boc-Leu are dissolved in anhydrous THF, and 1.0 
equivalents of N.M.M. and 1.1 equivalents of isobutylchloroformate are 
added to the solution, which is cooled to -15.degree. C. and maintained in 
a nitrogen atmosphere. 
After two minutes, a solution prepared by dissolving 1.0 equivalents of 
HCl.Met-OMe and 1.0 equivalents of N.M.M. in DMF is added. 
During the additions, the temperature is checked to ensure that it does not 
exceed -10.degree. C. Having verified the disappearance of HCl.Met-OMe, 
the reaction is suspended by evaporating the mixture to dryness, the 
residue is taken up in EtOAc and washed with a 5% sodium bicarbonate 
solution, water, a 5% citric acid solution, and water. 
The solution of EtOAc is then dried over magnesium sulphate, and the 
product is obtained by crystallisation, by suitably adding 
30.degree.-50.degree. C. petroleum ether. M.P.=102.degree.-104.degree. C. 
[.alpha.].sub.22.sup.589 =-36.1.degree. (C=1.0 in DMF). 
Elementary analysis for C.sub.17 H.sub.32 N.sub.2 O.sub.5 S: Theoretical: 
C, 54.23%; H, 8.57%; N, 7.44%. Found: C, 54.10%; H, 8.49%; N, 7.39%. 
Chromatography analysis (thin layer chromatography and HPLC) shows no 
presence of impurities, and the .sup.1 H n.m.r. spectrum confirms the 
molecular structure. 
Synthesis of tert-butyloxycarbonylleucylmethionineamide. 
Boc-Leu-Met-NH.sub.2 
1.0 equivalents of Boc-Leu-Met-OMe are dissolved in anhydrous MeOH, and 
anhydrous ammonia is passed for 30 minutes into this solution, cooled to 
-5.degree.. 
The solution, contained in a hermetically sealed vessel, is kept overnight 
at ambient temperature, after which the product is obtained in crystalline 
form by adding a volume of water equal to about 30% of the volume of MeOH. 
M.P.=158.degree.-160.degree. C. 
[.alpha.].sub.22.sup.589 =-35.4.degree. (C=1 in DMF). 
Elementary analysis for C.sub.16 H.sub.31 N.sub.3 O.sub.4 S: Theoretical: 
C, 53.16%; H, 7.33%; N, 11.63%. Found: C, 53.03%; H, 7.23%; N, 11.50%. 
Chromatography analysis (thin layer chromatography and HPLC) shows no 
presence of impurities, and the .sup.1 H n.m.r. spectrum confirms the 
molecular structure. 
Synthesis of leucylmethionineamide hydrochloride.HCl.Leu-Met-NH.sub.2 
1.0 equivalents of Boc-Leu-Met-NH.sub.2 are dissolved in 10 ml of a 4.5 M 
solution of HCl in EtOAc. 
Having verified the disappearance of the starting substance, the reaction 
solvent is evaporated to dryness, the residue taken up with MeOH and 
crystallised by suitably adding Et.sub.2 O. 
M.P.=125.degree.-127.degree. C. 
[.alpha.].sub.22.sup.589 =10.5 (C=1.0 in H.sub.2 O). 
Elementary analysis for C.sub.11 H.sub.24 N.sub.3 O.sub.2 S.Cl.CH.sub.3 OH: 
Theoretical: C, 43.70%; H, 8.55%; N, 12.73%. Found: C, 43.55%; H, 8.42%; 
N, 12.63%. 
The .sup.1 H n.m.r. spectrum confirms the molecular structure. The product 
is pure, this having been verified by chromatography analysis (t.l.c. and 
HPLC). 
Synthesis of malonylleucylmethioneamide methylester 
(CH.sub.3)O-mGly-Leu-Met-NH.sub.2 
1.0 equivalents of methyl monomalonate are dissolved in CH.sub.2 Cl.sub.2, 
the solution is cooled to 0.degree. C., after which 1.5 equivalents of 
HOBt dissolved in DMF and 1.1 equivalents of DCC dissolved in CH.sub.2 
Cl.sub.2 are added to it. After 20 minutes, 1.0 equivalents of 
HCl.Leu-Met-NH.sub.2 are added to the cold mixture, followed by 1.1 
equivalents of N.M.M. The ice bath is removed after about one hour, and 
having verified the disappearance of the hydrochloride the reaction 
mixture is filtered, the precipitated dicyclohexylurea is washed with 
portions of THF, and the resultant solution and wash liquors evaporated to 
dryness. The residue obtained is washed with small volumes of 5% sodium 
bicarbonate, water, 5% citric acid and water, and is then dried over 
P.sub.2 O.sub.5. The product is crystallised from DMF/Et.sub.2 O. 
M.P.=184.degree.-185.degree. C. 
[.alpha.].sub.22.sup.589 =-33.6 (C=0.87 in DMF) 
Elementary analysis for C.sub.15 H.sub.27 N.sub.3 O.sub.5 S: Theoretical: 
C, 49.86%; H, 7.48; N, 11.63%. Found: C, 49.75%; H, 7.43%, N, 11.60% 
Chromatography analysis (t.l.c. and HPLC) shows no presence of impurities, 
and the .sup.1 H n.m.r. spectrum confirms the molecular structure. 
Synthesis of malonylleucylmethionineamide mGly-Leu-Met-NH.sub.2 
1.0 equivalents of (CH.sub.3)O-mGly-Leu-Met-NH.sub.2 are dissolved in MeOH, 
and 3 equivalents of a 3 M aqueous solution of NaOH are added to the 
solution. 
Having verified the disappearance of the starting ester, the methanol is 
diluted with water, then eliminated, and the residual solution acidified 
to pH 2 with concentrated HCl, then extracted repeatedly with EtOAc. The 
extracts are combined, dried with magnesium sulphate and evaporated to 
dryness. The product is crystallised from dioxane/30.degree.-50.degree. C. 
petroleum ether. 
M.P.=136.degree.-138.degree. C. (dec.). 
[.alpha.].sub.22.sup.589 =-38.9 (C=1.3 in DMF). 
Elementary analysis for C.sub.14 H.sub.25 N.sub.3 O.sub.5 S: Theor: C, 
48.14%; H, 7.20%; N, 12.10%. Found: C, 47.12%; H, 7.91%; N, 12.00%. 
Chromatography analysis (t.l.c. and HPLC) shows no presence of impurities, 
and the .sup.1 H n.m.r. spectrum confirms the molecular structure. 
Synthesis of tert-butyloxycarbonyl-phenylalanylphenylalanine 
methylester.Boc-Phe-Phe-Ome 
1.0 equivalents of Boc-Phe are dissolved in anhydrous THF, after which 1.0 
equivalents of N.M.M. and 1.1 equivalents of isobutylchloroformate are 
added to the solution cooled to -15.degree. C. and kept under a nitrogen 
atmosphere. After 2 minutes, a solution prepared by dissolving 1.0 
equivalents of HCl.Phe-OMe and 1.0 equivalents of N.M.M. in DMF is added. 
During the addition of the isobutylchloroformate and HCL. Phe-OMe, the 
temperature is checked to ensure that it does not exceed -10.degree. C. 
Having verified the disappearance of HCl.Phe-OMe, the reaction is suspended 
by evaporating the solvent mixture to dryness, the residue is taken up in 
EtOAc and washed with 5% sodium bicarbonate solution, water, 5% citric 
acid solution and water. The EtOAc solution is dried over magnesium 
sulphate, and the product is obtained in crystalline form by adding 
30.degree.-50.degree. petroleum ether. M.P.=121.degree.-123.degree. C. 
[.alpha.].sub.22.sup.589 =5.5.degree. (C=1.0 in acetic acid). 
Elementary analysis for C.sub.24 H.sub.30 N.sub.2 O.sub.5 : Theoretical: C, 
67.58%; H, 7.09%; N, 6.57%. Found: C, 67.49%; H, 6.99%; N, 6.78%. 
Chromatography analysis (t.l.c. and HPLC) shows no presence of impurities, 
and the .sup.1 H n.m.r. spectrum confirms the molecular structure. 
Synthesis of tert-butyloxycarbonylphenylalanylphenylalanylamide. 
Boc-Phe-Phe-NH.sub.2 
1.0 equivalents of Boc-Phe-Phe-OMe are dissolved firstly in DMF and then 
diluted with MeOH. 
Anhydrous ammonia is passed for 30 minutes through the solution cooled to 
-5.degree. C. Having interrupted the flow of ammonia, after about 1 hour, 
the solution is kept in a hermetically sealed vessel overnight at ambient 
temperature. The required product, which is obtained by adding an excess 
of water after evaporating the MeOH, is filtered, dried over P.sub.2 
O.sub.5 under vacuum, and collected. M.P.=208.degree.-210.degree. C. 
[.alpha.].sub.22.sup.589 =-24.5.degree. (C=1.0 in DMF). 
Elementary analysis for C.sub.23 H.sub.25 N.sub.3 O.sub.4 : Theoretical: C, 
67.15%, H, 7.06%; N, 10.22%. Found: C, 67.00; H, 6.99%; N, 10.15% 
Chromatography analysis (t.l.c. and HPLC) shows no presence of impurities, 
and the .sup.1 H n.m.r. spectrum confirms the molecular structure. 
Synthesis of 
tert-butyloxycarbonylalanylphenylphenylalanylgemdiaminophenylalamine 
hydrochloride. BOC-Phe-g Phe HCl 
1.0 equivalents of Boc-Phe-Phe-NH.sub.2 are suspended in a 3:2 (v/v) 
acetonitrile:water mixture, and 1.2 equivalents of BTI dissolved in 
acetonitrile are added to the solution at ambient temperature under 
vigorous stirring. 
An inert gas is bubbled through the reaction mixture in order to facilitate 
removal of the CO.sub.2 developed during the reaction. Having verified the 
disappearance of Boc-Phe-Phe-NH.sub.2, the reaction is suspended by 
evaporating to dryness 5 hours after adding the reagent, the residue is 
washed with ethyl ether, dried and dissolved in EtOH. The stoichiometric 
quantity of HCl dissolved in EtOAc is added to this solution to induce 
complete precipitation of Boc-Phe-gPhe- HCl over a period of 2 hours. The 
precipitate is filtered, washed abundantly with various portions of ethyl 
ether, dried over P.sub.2 O.sub.5 under vacuum, and collected. 
M.P.=174.degree. C. (dec.). 
[.alpha.].sub.22.sup.589 =-48.8.degree. (C=1.0 in DMF). 
Elementary analysis for C.sub.22 H.sub.30 O.sub.3 N.sub.3 Cl: Theoretical: 
C, 62.94%; H, 7.15%; N, 10.01%. Found: C, 62.39%; H, 7.12%; N, 10.27%. 
Chromatography analysis (t.l.c. and HPLC) shows no presence of impurities 
and the .sup.1 H n.m.r. spectrum confirms the molecular structure. 
Synthesis of 
tert-butyloxycarbonyl-phenylalanylgem-diaminophenylalanylmalonyl-leucylmet 
hionineamide. Boc-Phe-gPhe-mGly-Leu-Met-NH.sub.2 
1.0 equivalents of mGly-Leu-Met-NH.sub.2 are dissolved in THF, the solution 
is cooled to 0.degree. C., after which 1.5 equivalents of HOBt dissolved 
in DMF and 1.1 equivalents of DCC dissolved in THF are added to the 
solution. After 20 minutes, 1.0 equivalents of Boc-Phe-gPhe- HCl and 1.1 
equivalents of N.M.M. are added to the cold mixture. 
The ice bath is removed after about 1 hour, and the mixture is left to 
react overnight at ambient temperature. 
After filtering off the dicyclohexylurea precipitate, which is washed with 
THF, the solution and wash liquors are reduced to about 10 ml, and a white 
flaky precipitate is obtained by subsequent treatment with an excess of 
water. The precipitate is filtered, washed with numerous portions of a 5% 
citric acid solution, water, a 5% sodium bicarbonate solution and water. 
After drying over P.sub.2 O.sub.5 under vacuum, the solid residue is 
further washed with Et.sub.2 O, dried and collected. 
M.P.=242.degree.-243.degree. C. 
[.alpha.].sub.22.sup.589 =-12.33 (C=10.7 in DMF). 
Elementary analysis for C.sub.36 H.sub.52 N.sub.6 O.sub.7 S: Theoretical: 
C, 60.67%; H, 7.30%; N, 11.80%. Found: C, 60.60%; H, 7.09%; N, 11.69%. 
Analysis of aminoacids: Theoretical: Phe, 1.00; Leu, 1.00; Met, 1.00. 
Found: Phe, 1.03; Leu, 1.00; Met, 0.87. 
Chromatography analysis (t.l.c. and HPLC) shows no presence of impurities, 
and the .sup.1 H n.m.r. spectrum confirms the molecular structure. 
Synthesis of 
phenylalanyl-gemdiaminophenylalanylmalonylleucylmethionineamide hydrochlor 
ide. HCl.Phe-gPhe-mGly-Leu-Met-NH.sub.2 
1.0 equivalents of Boc-Phe-gPhe-mGly-Leu-Met-NH.sub.2 are dissolved in 15 
ml of a 4.5 M solution of HCl in EtOAc. Having verified the disappearance 
of the starting substance, the reaction solvent is evaporated to dryness, 
the residue taken up in DMF and crystallised by adding a suitable quantity 
of Et.sub.2 O. 
M.P.=236.degree.-238.degree. C. 
[.alpha.].sub.22.sup.589 =-10.2 (C=1.0 in DMF). 
Elementary analysis for C.sub.31 H.sub.45 N.sub.6 O.sub.5 SCl Theoretical: 
C, 57.37%; H, 6.94%; N, 12.95%. Found: C, 57.30%; H, 6.80%; N, 12.88%. 
Chromatography analysis (t.l.c. and HPLC) shows no presence of impurities, 
and the .sup.1 H n.m.r. spectrum confirms the molecular structure. 
Synthesis of 
pyroglutamylphenylalanylgemdiaminophenylalanylmalonylleucylmethionineamide 
. Pyr-Phe-gPhe-mGly-Leu-Met-NH.sub.2 
1.0 equivalents of pyroglutamic acid are dissolved in DMF and the solution 
is cooled to 0.degree. C., after which 1.5 equivalents of HOBc dissolved 
in DMF and 1.1 equivalents of DCC dissolved in THF are added. After 20 
minutes, 1.0 equivalents of HCl.Phe-gPhe-mGly-Leu-Met-NH.sub.2 and 1.1 
equivalents of N.M.M. are added. The ice bath is removed after about 1 
hour, and the mixture is left to react overnight at ambient temperature. 
Having verified the disappearance of HCl.Phe-gPhe-mGly-Leu-Met-NH.sub.2, 
the solution is filtered, and the dicyclohexylurea precipitate is washed 
with THF. The resultant solution and the wash liquors are reduced to a 
small volume, and a flaky precipitate is obtained by treatment with an 
excess of water. The required product is isolated by reverse phase high 
pressure preparative liquid chromatography, the stationary phase 
consisting of Lichroprep 25.40 .mu.m (Merck), and using H.sub.2 O/CH.sub.3 
Cn 20% as eluent. The product is recovered by lyophilisation after 
evaporating the acetonitrile. M.P.=261.degree.-265.degree. C. 
[.alpha.].sub.22.sup.546 =-10.0 (C=0.5 in DMF). 
Elementary analysis for C.sub.36 H.sub.49 N.sub.7 O.sub.7 S: Theoretical: 
C, 59.75%; H, 6.77%; N, 13.55%. Found: C, 59.67%; H, 6.69%; N, 13.49%. 
Analysis of aminoacids: Theoretical: Gln, 1.00; Phe, 1.00; Leu, 1.00; Met, 
1.00. Found: Gln, 1.04; Phe, 1.00; Leu, 1.00; Met, 0.93. 
Chromatography analysis (t.l.c. and HPLC) shows no presence of impurities, 
and the .sup.1 H n.m.r. spectrum confirms the molecular structure.