Synthetic peptide antagonists of neurokinin A, salts thereof and respective preparation processes

DESCRIPTION 
1. FIELD OF THE INVENTION 
Neurokinin A, also known as substance K or .alpha.-neurokinin, belongs like 
substance P and neurokinin B to the class of the tachykinins (nomenclature 
suggested by the IUPHAR Committee, C. Jordan and P. Oehme, Substance P: 
metabolism and biological actions, Taylor and Francis, London, 1985), 
consisting of peptides which have the C-terminal sequence 
EQU Phe-X-Gly-Leu-Met-NH.sub.2. 
For the nomenclature and the abbreviations of the amino acids, reference is 
made to the recommendations of the IU-IUB Joint Commission on 
Biochemical Nomenclature (Eur. J. Biochem. 138, 9, 1984). 
Neurokinin A was isolated in 1983 from the spinal cord of pigs (S. Kimura 
et al., Proc. Jap. Acad. Ser. B, 59, 101, 1983, and K. Kanagawa et al., 
Biochem. Biophys. Res. Com. 114, 533, 1983) and was characterized as a 
C-terminal decapeptide amide of the sequence: 
EQU H-Hys.sup.1 -Lys.sup.2 -Thr.sup.3 -Asp.sup.4 -Ser.sup.5 -Phe.sup.6 
-Val.sup.7 -Gly.sup.8 -Leu.sup.9 -Met.sup.10 -NH.sub.2. 
This sequence differs from that of substance P by the amino acid in 
position 7 and by the N-terminal sequence (positions 1-5) and differs from 
that of neurokinin B by the amino acids in positions 1-3 and 5, always in 
the N-terminal zone. The structures of substance P and neurokinin B are, 
respectively: 
Substance P: 
EQU H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH.sub.2 
Neurokinin B: 
EQU H-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH.sub.2 
Peptide antagonists of substance P and of neurokinin B are already known 
and have been described in U.S. Pat. Nos. 4,481,139 and 4,665,157 
respectively; however, no antagonists of neurokinin A are known. It is 
likewise known (D. Regoli et al., Life Sci. 40, 109, 1987) that the 
C-terminal heptapeptide fragment of neurokinin A maintains the same 
biological activity as the complete peptide. 
SUMMARY OF THE INVENTION 
The subject of the present invention is synthetic peptides of the general 
formula: 
EQU X-Asp-Y-DTrp-Val-DTrp-Z-K-NH.sub.2 I 
where: 
X=H, Arg, DArg, Lys, DLys, Thr, DThr 
Y=Tyr, Trp, DTrp, Ser, Met 
Z=Trp, DTrp 
K=Arg, Phe, DTrp, Tyr, Met 
and pharmaceutically acceptable salts thereof obtained with organic and 
inorganic acids. Such peptides are shown to possess a biological activity 
as competitive antagonists against neurokinin A, but are free of an 
agonist action. 
More particularly, the subject of the invention is a synthetic peptide 
corresponding to the following formula: 
EQU X-Asp-Y-DTrp-Val-DTrp-Z-K-NH.sub.2 
where: 
X=H, Arg, Lys, Thr 
Y=Tyr 
Z=DTrp 
K=Arg, Phe, DTrp, Tyr, Met 
as well as pharmaceutically acceptable salts thereof with organic acids and 
pharmaceutically acceptable salts thereof with inorganic acids. 
The following in particular are also part of the invention: 
an octapeptide having the following structure: 
EQU H-Arg-Asp-Tyr-DTrp-Val-DTrp-DTrp-Arg-NH.sub.2 ; 
a heptapeptide having the following structure: 
EQU H-Asp-Tyr-DTrp-Val-DTrp-DTrp-Arg-NH.sub.2 ; 
an octapeptide having the following structure: 
EQU H-Arg-Asp-Tyr-DTrp-Val-DTrp-DTrp-Phe-NH.sub.2 ; 
a heptapeptide having the following structure: 
EQU H-Asp-Tyr-DTrp-Val-DTrp-DTrp-DTrp-NH.sub.2 ; 
a heptapeptide having the following structure: 
EQU H-Asp-Trp-DTrp-Val-DTrp-DTrp-Arg-NH.sub.2. 
Synthetic peptides as defined above are competitive antagonists against 
neurokinin A (NK-2 receptor). 
A further subject of the invention is a process for obtaining the peptides 
defined above, comprising solid-phase synthesis building up the peptide 
chain from the C-terminal end towards the N-terminal end on an insoluble 
polymeric support, and subsequent detachment of the peptide from the 
polymer support by hydrolysis in anhydrous hydrofluoric acid. 
The novel peptides which are the subject of the present application are 
useful for reducing or antagonizing, in animals and in humans, 
pathological effects due to an excess of neurokinin A, such as 
bronchoconstrictions or spasms in the intestines or in the urinary 
bladder. 
DETAILED DESCRIPTION OF THE INVENTION 
The peptides which are the subject of the present invention can be prepared 
by utilizing the techniques known in peptide synthesis, such as are 
described, for example, in M. Bodanszky and A. Bodanszky, The Practice of 
Peptide Synthesis, Springer-Verlag, Berlin Heidelberg 1984. By way of 
non-limiting example, the plan of the synthesis in solid phase according 
to Merrifield (R.B. Merrifield, J. Am. Chem. Soc. 85, 2149, 1963) is 
reported. When the alpha-amino function of the amino acids was protected 
by the t-butoxycarbonyl (Boc) group, 4-methylbenzhydrylamine resin (MBHA; 
amine groups: 0.4-0.6 mmol/g of resin) can be used as solid support for 
obtaining C-terminal amides; the side chains of trifunctional amino acids 
were protected by known methods described in the literature. The peptides 
were assembled by means of a semiautomatic synthesizer, using the method 
of symmetrical anhydrides; 1-2 g of resin are placed in the reactor and 
neutralized with 10% triethylamine in chloroform, and two equivalents of 
the symmetrical anhydride of the amino acid freshly prepared in solution 
in 1/1 methylene chloride/dimethylformamide are then added to the reactor. 
After a 60 minute coupling cycle and a cycle of washings of the resin with 
methylene chloride and isopropanol, the completeness of the coupling is 
verified by means of the ninhydrin test according to Kaiser et al., Anal. 
Biochem. 34, 595, 1970. The removal of the protective group from the amine 
group of the coupled amino acid is accomplished by treatment with 40% 
trifluoroacetic acid in methylene chloride and then with 5% 
diisopropylethylamine in methylene chloride (automatic protective group 
removal and neutralization cycle) in order to obtain the resin ready for 
the next coupling which is carried out by the same procedure. After the 
addition of the last amino acid (N-terminal end) and the elimination of 
the Boc group (protective group removal cycle with trifluoroacetic acid), 
the resin is removed from the reactor and dried in vacuo overnight. For 
the detachment of the peptide from the resin in the form of the C-terminal 
amide and the simultaneous removal of the protective groups from all the 
side chains of the amino acids, the resin is treated for 1 hour with 
95/5/0.5 anhydrous hydrofluoric acid/anisole/dimethyl sulfide at 0.degree. 
C. in a "Teflon" reactor. After removal of the hydrofluoric acid in an 
nitrogen stream, the resin is dried in vacuo and washed with ethyl ether, 
and the crude peptide is extracted with 50% acetic acid. The acetic acid 
solution obtained is concentrated in vacuo to a small volume and directly 
loaded on a column of steric exclusion resin for a first purification 
cycle; the fractions containing the peptide (characterized by HPLC 
analysis techniques) are combined, evaporated in vacuo and lyophilized. 
Finally, the peptide is purified by preparative reverse-phase 
high-pressure chromatographic techniques, in order to obtain a purity 
greater than 98%.

EXAMPLE 1 
Synthesis of the octapeptide having the following sequence: 
EQU H-Arg-Asp-Tyr-DTrp-Val-DTrp-DTrp-Arg-NH.sub.2 
2.0 g of MBHA resin (Novabiochem, Switzerland) carrying 0.45 
milliequivalents/g of amine groups are placed in the reactor of a Labortec 
SP 640 semiautomatic synthesizer. The resin is neutralized manually, 
carrying out two washings (5 min+15 min) with 10% triethylamine in 
chloroform (2.times.15 ml) and three washings (3.times.1 min) with 
methylene chloride (3.times.15 ml). This is followed by the coupling of 
arginine by the preformed symmetrical anhydride technique, with protection 
of the alpha-amine function by the t-butoxycarbonyl (Boc) group and of the 
guanidine function on the side chain by the p-toluene-guanidine function 
on the side chain by the p-toluene-sulfonyl group (tosyl; Tos): 1.54 g of 
Boc-Arg(Tos)-OH (3.6 milliequivalents) are dissolved in 3 ml of methylene 
chloride and 4.64 ml of an 8% weight/volume solution of 
dicyclohexylcarbodiimide in methylene chloride (equal to 1.8 
milliequivalents) are added, with continuous magnetic stirring for 15 
minutes in an ice bath, the mixture is filtered on a Gooch filter to 
remove the dicyclohexylurea formed, and the solution is added to the 
reactor, diluting with a further 5 ml of dimethylformamide and then 
initiating the automatic coupling cycle (see Table 1). At the end of the 
cycle described in the table, the Kaiser test is carried out on an aliquot 
of the resin; if the degree of incorporation of the amino acid is higher 
than 99%, the automatic removal of the protective group from the amino 
group is allowed to proceed; otherwise, the coupling is repeated. For the 
subsequent couplings, always according to the plan of Table 1, the 
following amino acids were used in the quantities indicated: Boc-DTrp 
(1.09 g for each of the three couplings), Boc-Val 0.78 
g),Boc-(O-2'-bromobenzyloxycarbonyl)-Tyr(1.78 g), cyclohexyl Boc-aspartate 
(1.12 g) and Boc-tosyl-arginine (1.54 g). 
TABLE 1 
______________________________________ 
Plan for an automatic coupling cycle 
______________________________________ 
1 Washing; methylene chloride 
1 .times. 1' 
2 Protective group removal; 40% trifluoroacetic 
1 .times. 5' 
acid in methylene chloride 
3 As above 1 .times. 15' 
4 Washing; methylene chloride 
3 .times. 1' 
5 Neutralization; 5% diisopropylethylamine 
2 .times. 1' 
in methylene chloride 
6 Washing; methylene chloride 
3 .times. 1' 
7 Coupling; anhydride of the amino acid in 
1 .times. 60' 
methylene chloride/dimethylformamide 
8 Washing; methylene chloride 
2 .times. 1' 
9 Washing; isopropanol 2 .times. 2' 
10 Washing; methylene chloride 
3 .times. 1' 
______________________________________ 
After the last coupling, the protective group removal cycle is repeated, 
the resin is taken out of the reactor and dried in vacuo over potash, 
giving 3.60 g of product. This is then followed by the detachment from the 
resin and simultaneous protective group removal from the side chains with 
anhydrous hydrofluoric acid. 2 g of the peptide-resin are placed in a 
Teflon reactor with 2 ml of anisole and 0.2 ml of dimethyl sulfide, the 
mixture is cooled to -50.degree. C. and 20 ml of anhydrous hydrofluoric 
acid are distilled in, and magnetic stirring is then maintained in an ice 
bath for 60 minutes. The hydrofluoric acid is removed by blowing with 
nitrogen, and the crude product is dried for 2 hours under suction, washed 
with ethyl ether (2.times.15 ml) and extracted in 50% acetic acid 
(3.times.15 ml) and filtered through a Gooch filter to remove the 
exhausted resin. The crude product solution thus obtained is concentrated 
to a small volume in a rotary evaporator and directly loaded on an LH 20 
column (2.5.times.100 cm) and eluted by gravity with 1/1 0.2 M acetic 
acid/acetonitrile (2 liters), collecting fractions of 10 ml. The 
peptide-containing fractions are identified by the UV plot (245 nm) of the 
effluent, combined, concentrated to a small volume in a rotary evaporator 
and lyophilized, giving 450 mg of crude product. For the final 
purification of the product by high-pressure liquid chromatography, 100 mg 
of the crude product are dissolved in 4 ml of an 8:2 aqueous solution of 
0.05% trifluoroacetic acid/acetonitrile; the clear solution thus obtained 
is loaded on an RP 18 column (1.5.times.15 cm) and eluted with a gradient 
of acetonitrile containing 0.05% trifluoroacetic acid against 0.05% 
trifluoroacetic acid from 20 to 80% in 120 minutes at a flow of 10 
ml/minute, with UV detection at 254 nm. The product comes out in about 25 
minutes; the homogeneous fractions are combined, concentrated and 
lyophilized, giving 26 mg of product. HPLC characterization: 3.9.times.150 
mm C 18 Waters Delta-Pak column, 20 to 80% gradient of acetonitrile 
against 0.05% trifluoroacetic acid in 20 minutes, flow 1 ml/minute, UV 
detection at 210 nm: RT=8.3 minutes, HPLC purity=98.0%. 
EXAMPLE 2 
Synthesis of the heptapeptide having the following sequence: 
EQU H-Asp-Tyr-DTrp-Val-DTrp-DTrp-DTrp-NH.sub.2 
1.0 g of "resin for amide peptides by the Fmoc strategy" (CH.sub.3 
O-Ph(1,4)-CH(NH-Fmoc)-Ph(1,4)-O-(CH.sub.2).sub.3 
-CONH-CH(CH.sub.3)-CONH-CH.sub.2 -Ph-polymer; Bachem, Switzerland) 
carrying 0.50 milliequivalent/g of amine groups is placed in the reactor 
of a Labortec SP 640 semiautomatic synthesizer. The hydrolysis of the 
fluorenylmethoxycarbonyl (Fmoc) group with 20% piperidine in 
dimethylformamide (DMF) is carried out automatically according to the plan 
indicated in Table 2, cycles 1 to 7. 
TABLE 2 
______________________________________ 
Plan for an automatic coupling cycle according to the 
"Fmoc" strategy 
______________________________________ 
1 Protective group removal; 20% piperidine 
1 .times. 3' 
in DMF 
2 As above 1 .times. 7' 
3 Washing; methylene chloride (DCM) 
2 .times. 1' 
4 Washing; DMF 2 .times. 1' 
5 Washing; isopropanol 2 .times. 1' 
6 Washing; DMF 2 .times. 1' 
7 Washing; DCM 2 .times. 1' 
8 Pre-equilibration; Fmoc-amino acid in 
2:1 DCM/DMF + HOBt 2' 
9 Coupling; DCC (1M in 2:1 DCM/DMF) 
90' 
10 Washing; isopropanol 1 .times. 1' 
11 Washing; DMF 1 .times. 1' 
12 Washing; DCM 1 .times. 1' 
13 Washings; repeating from 10 twice more 
______________________________________ 
This is followed by coupling of the first D-tryptophan by the active ester 
technique with hydroxybenzotriazole (HOBt) obtained "in situ" by means of 
dicyclohexylcarbodiimide (DCC). 1.5 milliequivalents (3-fold excess with 
respect to the amine groups of the resin) of Fmoc-D-tryptophan (0.639 g) 
and 2 milliequivalents of HOBt (0.310 g) are dissolved in 12 ml of 2:1 
DCM/DMF, transferred into the reactor of the apparatus and, after 
pre-equilibration for 2 minutes with stirring, 1.5 ml of 1 M DCC in 2:1 
DCM/DMF (equal to 1.5 milliequivalents) are added, thus initiating the 
automatic coupling cycle (line 9 in Table 2). At the end of the cycle 
described in the table, the Kaiser test is carried out on an aliquot of 
resin; if the degree of incorporation of the amino acid is greater than 
99%, the automatic protective group removal from the amine group is 
carried out; otherwise, the coupling is repeated. For the successive 
couplings, always according to the plan in Table 2, the following amino 
acids were used in the quantities indicated: Fmoc-DTrp (0.639 g for each 
of the 3 further couplings), Fmoc-valine (0.509 g), Fmoc-tyro-sine t-butyl 
ether (0.689 g) and Fmoc-t-butyl aspartate (0.617 g). After the last 
coupling, the protective group removal cycle is repeated, the resin is 
taken out of the reactor and dried in vacuo over potash, giving 1.650 g of 
product. This is then followed by the detachment of the peptide from the 
resin and simultaneous protective group removal from the side chains by 
trifluoroacetic acid (TFA). 1.5 g of peptide-resin are placed into a small 
flask containing 30 ml of a solution consisting of 27:1.5:1.5 
(volume/volume) TFA/ethanedithiol/p-cresol and the mixture is maintained 
for 2 hours in a water bath at 35.degree. C. with magnetic stirring. To 
precipitate the crude product, 10 volumes of ethyl ether and 5 volumes of 
petroleum ether, both cooled to 0.degree. C., are added. The mixture is 
kept overnight at -78.degree. C. and filtered over a porous funnel-shaped 
filter , giving 350 mg of crude crystalline product. For the purification 
by high-pres-sure liquid chromatography, the procedure of Example 1 is 
followed, giving 12 mg of product; characterization by HPLC analysis 
(conditions as described in Example 1): Rt=12.6 minutes, HPLC purity=98%. 
Further non-limiting examples of compounds according to the general formula 
I included in the present invention are: 
__________________________________________________________________________ 
H--Asp--Tyr--DTrp--Val--DTrp--DTrp--Tyr--NH.sub.2 
Rt = 11.7' 
H--Asp--Tyr--DTrp--Val--DTrp--DTrp--Arg--NH.sub.2 
Rt = 9.9' 
H--Thr--Asp--Tyr--DTrp--Val--DTrp--DTrp--Arg--NH.sub.2 
Rt = 9.6' 
H--Arg--Asp--Tyr--DTrp--Val--DTrp--DTrp--Met--NH.sub.2 
Rt = 11.0' 
H--Lys--Asp--Tyr--DTrp--Val--DTrp--DTrp--Arg--NH.sub.2 
Rt = 8.8' 
H--Arg--Asp--Tyr--DTrp--Val--DTrp--DTrp--Phe--NH.sub.2 
Rt = 11.7' 
H--Arg--Asp--Ser--DTrp--Val--DTrp--DTrp--Met--NH.sub.2 
Rt = 10.5' 
H--Asp--Ser--DTrp--Val--DTrp--DTrp--Met--NH.sub.2 
Rt = 11.3' 
H--Lys--Asp--Met--DTrp--Val--DTrp--DTrp--Arg--NH.sub.2 
Rt = 8.7' 
H--Arg--Asp--Ser--DTrp--Val--DTrp--DTrp--Arg--NH.sub.2 
Rt = 8.2' 
H--Asp--Tyr--DTrp--Val--DTrp--DTrp--Met--NH.sub.2 
Rt = 12.3' 
H--Asp--Tyr--DTrp--Val--DTrp--Trp--Arg--NH.sub.2 
Rt = 9.4' 
H--Asp--Trp--DTrp--Val--DTrp--DTrp--Arg--NH.sub. 2 
Rt = 10.9' 
H--Asp--DTrp--DTrp--Val--DTrp--DTrp--Arg--NH.sub.2 
Rt = 9.4' 
__________________________________________________________________________ 
where the HPLC retention time (Rt) was determined as in Example 1. 
Biological activity 
The capacity of the peptides described in the present invention of 
interacting with the neurokinin A receptor as agonists or antagonists was 
evaluated by means of an in vitro assay using a preparation, in which the 
biological response produced by tachykinin and related peptides is 
exclusively determined by the neuro-kinin A receptor (NK-2 receptor). Such 
a preparation is the isolated vas deferens of rats, where the tachykinins 
produce a potentiation of the contractions caused by the intramural nerves 
following an electrical stimulation. The determination of the activity of 
the peptides in this preparation was carried out as described by Rovero et 
al. (Neuropeptides 10, 355, 1987). The agonistic activity of the peptides 
assayed is expressed as pD.sub.2, which represents the antilogarithm of 
the molar concentration of agonist which produces 50% of the maximum 
effect. The antagonistic activity is expressed as pA.sub.2, which 
represents the antilogarithm of the molar concentration of antagonist 
which produces a dose ratio equal to 2 between two equally effective doses 
of agonist. The pD.sub.2 were calculated according to Van Rossum (Arch. 
Int. Pharmacodyn. 143, 249, 1963), and the pA.sub. 2 were calculated from 
the analysis of the Schild plot (O. Arunlakshana and H. O. Schild, Br. J. 
Pharmacol., 14, 48, 1959). The following results were obtained: 
TABLE 3 
__________________________________________________________________________ 
Agonist or antagonist activity of the peptides described 
on the NK-2 receptor in the isolated vas deferens of rats 
Peptide pD.sub.2 
pA.sub.2 
__________________________________________________________________________ 
NKA 6.64 
n.a. 
NKA(4-10) 5.98 
n.a. 
H--Asp--Tyr--DTrp--Val--DTrp--DTrp--DTrp--NH.sub.2 
n.a. 
6.81 
H--Asp--Tyr--DTrp--Val--DTrp--DTrp--Tyr--NH.sub.2 
n.a. 
6.72 
H--Asp--Tyr--DTrp--Val--DTrp--DTrp--Arg--NH.sub.2 
n.a. 
6.60 
H--Thr--Asp--Tyr--DTrp--Val--DTrp--DTrp--Arg--NH.sub.2 
n.a. 
6.60 
H--Arg--Asp--Tyr--DTrp--Val--DTrp--DTrp--Met--NH.sub.2 
n.a. 
6.60 
H--Arg--Asp--Tyr--DTrp--Val--DTrp--DTrp--Arg--NH.sub.2 
n.a. 
6.50 
H--Lys--Asp--Tyr--DTrp--Val--DTrp--DTrp--Arg--NH.sub.2 
n.a. 
6.47 
H--Arg--Asp--Tyr--DTrp--Val--DTrp--DTrp--Phe--NH.sub.2 
n.a. 
6.24 
__________________________________________________________________________ 
The agonists produce a potentiation of the contractions caused by the nerve 
of the isolated vas deferens of rats. The antagonistic activity is 
evaluated as the capacity of the product to produce a parallel 
displacement towards the right of the concentration/response curve of 
neurokinin A after a contact time of 15 minutes. NKA denotes neurokinin A 
and NKA(4-10) is the C-terminal heptapeptide fragment of neurokinin A. 
It is intended that the method described is only an exemplification given 
solely as a practical demonstration of the invention, and this invention 
can be varied in its forms and arrangements without otherwise departing 
from the scope of the concept which is the basis of this invention.