CA1137468A - Antibacterial peptide - Google Patents

Abstract

ANTIBACTERIAL PEPTIDE
Abstract of the Disclosure It has been found that dipeptides containing a 3-halo-D-alanine C-terminus are powerful antibacterials and produce a highly useful synergistic effect with anti-biotics.

Description

Detailed Description of the Invention As micro-oryanisms become resistant to known antibiotics continued effort is needed to find new com-pounds or combinations of compounds which effectively --inhibit bacteria growth.
It has now been found that a peptide of the formula R-NH-CH-COOH

wherein the shown aminoacid is in the D-configuration, X
is chlorine or fluorine, and R is the acyl moiety of an a-aminoacid in the L-configuration, wherein the a-amino group may carry a fatty acid acyl group or an aminolower-alkyl acyl group or a loweralkyl group, or the correspond-ing loweralkyl esters of said dipeptide, or nontoxic acidaddition salts thereof, are useful antibacterials7 they also represent powerful synergists for D-cycloserine and other antibiotics.
The above moiety R particulary represents the known, protein-derived aminoacids, including glycine which, of course, does not have a chiral center. The definition also includes other aminoacids where the amino group is attached to the 2- or a-poSitiOn of the acid. The amino group of substituent R may also carry an acyl group of a lower fatty acid or a loweralkyl group, primarily methyl, propyl, tert. butyl, acetyl, propionyl isobutyryl and the like. The protein-derived aminoacid may be represented by leucine, valine, norvaline, proline, serine, tyrosine, alanine, phenylalanine, threonine, methionine, glutamine, histidine, arginine, lysine and tryptophane. The new di-peptides have the unnatural sequence of an L-aminoacid (or glycine) coupled to D-haloalanine. Such a L-D sequence is usually restricted to the cell wall components of micro-organisms and its antibacterial activity is completely unexpected.
The new dipeptide can easily be synthesized by coupling the known ~-fluoro- (or chloro-)-D-alanine with ... ~
.

an active ester of a N~-protected glycine or an aminoacid in the L-configuration or a N~-alkyl homolog thereof.
Among the active esters, the hydroxysuccinimide, penta-chlorophenyl, 4-nitrophenyl, 2,4,5-trichlorophenyl, a fluorophenyl, N-hydroxyisobornyldicarboximide or similarly familiar esters of RCOO- can be used for the coupling re-action. The N ~ group and any other sensitive functional group in the aminoacid moiety represented by R above can be protected with the usual well-known groups that can subsequently be removed by a mild chemical reaction which does not affect the peptide bond formed. Among the groups frequently used as temporary protection are the carboben-zoxy (hereinafter identifed as Z) or the tert. butoxy-carbonyl for amino groups, particularly the N~- group, while benzyl or other moieties can be used to protect the hydroxy groups in serine, tyrosine or hydroxyproline or the imidazol group of histidine. Hydrogenation will re-move said benzyl group after the peptide coupling has been effected and treatment with hydrobromic acid or hydro-fluoric acid will remove other protective groups used by the skilled artisan, without cleaving the peptide bond.
The free acid can be converted into the desired alkyl ester in known fashion and/or the N~- group can be acy-lated in known manner.
In order to illustrate the preparation of the new peptides, reference is made to the following examples which, however, are not intended to limit this invention in any respect. In all examples, the optical rotations were taken at 25C. in lN HCl at the concentra-tions given.
Example 1 a) To a stirred solution of 214.2 mg. of ~-fluoro-D-alanine and 420 mg. of sodium bicarbonate in S ml. of water was added a solution of 800.8 mg. of carbobenzoxy-L-alanine-N-hydroxysuccinimide ester in 5 ml. of 1,2-dimethoxyethane. After stirring overnight at ambient temperature, the solution was concentrated to 113'7~

a syrup under reduced pressure. The residue was dis-solved in 10 ml. of water and acidified with lN-hydro-chloric acid to precipitate 521 mg. of N-carbobenzoxy-L-alanyl- ~fluoro-D-alanine, m.p. 156-7C.
b) A solution of 2.48 g. of this protected dipeptide in 10 ml. of 32~ hydrobromic acid in acetic acid was stirred at room temperature for 30 minutes.
A gummy solid was precipitated by the addition of ether.
This material was washed with 3 portions of ether by de-cantation and crystallized from wet acetic acid, produc-ing 1.44 g. of L-alanyl-~-fluoro-D-alanine hydrobromide, m.p. 203-5C. (with decomposition); [a]D + 25.3 (c, 1.1).
Example 2 a) By repeating the process of Example l(a), but starting with 320 mg. of ~-chloro-D-alanine hydro-chloride and 588 mg. of sodium carbonate, 647 mg. of N-carbobenzoxy-L-alanyl-~-chloro-D-alanine was obtained;
m.p. 168-70C.
b) A solution of 736 mg. of this protected peptide in 101 ml. of methanol containing one equivalent of HCl was hydrogenated over 0.15 g. of 5% Pd on carbon.
The catalyst was removed by filtration after the calcu-lated amount of gaseous hydrogen had been absorbed. The catalyst was washed with methanol which was combined with the filtrate. This mixture was evaporated to dryness and the residue was placed on a 1.5 x 40 cm. column charged with a strongly basic polystyrene ion exchange resin and eluted with 0.1 molar ammonium acetate buffer of pH 7.5.
The appropriate fractions were combined to produce a solid which was crystallized from water/acetonitrile and then from water/isopropanol to give 267 mg. of L-alanyl-~-chloro-D-alanine; m.p. 196-202C. (with decomposition);
[a]D + 1 (C, 1.0).
Examples 3_- 14 In like manner, the compounds shown in Table I were made, identified by the melting point of the Na-Z-dipeptide, and the m.p. and/or optical rotation (shown as [~]D/concentration in lN HCl) of the dipeptide with the 113~

1~ XW
~ W ~ 1- 0 ~ ~ W 3 (D
t' Y G~ ~n G~ t' t' t' t' tl t' t' ,,,_~,_,,,,,II
~
O Ul ~ O
II~ IIIIIII ~
~ ~ l-- I I ~ ~ ~ ~ ~ ~ ~ ~
3 ~ ~ o ,_,_~,,,,,,,,, I I I

P~ ~

1~ ~' ~1 ~ W ~n Ul 0~ W ~ ~D ,'~.
o ~1 ~ o 1-- ~n o oo co I I I o I o I I I (D
~~ ~ ~ ~ ~
t~ o o ~ ~t O U~ WO O I'~ O
O OO D 0~ H~ W

. H
V V
1~ t~ t~ t~ t~ ~
ot~ w t~ ~I 1-- ~3 O Ul Vl O O ~ ~ ~
¦ ¦ ¦ O I I O O O D ~D ~
O O I O I O O ~
o ~ W ~D o ~h I'~ O
1-- Ul ~' ~ H~
o o S~ (D
P.

+ I + + 1-- + + I + + + +
I~ t~ ~ t~ t~ ~ O ~
~D ~n w w ~n w 1~ ~ t~ 1~ ~ _, O O O ' ~ t~
~ Ul a~ ~ ~ co ~ o ~I ~ ~n O O O O O O O O D ~ ~

~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ tl~
O O o O O 1~ 0 1~ It . . . . . . . . . . . . ~
~I t~ ~ o o o o o o o r~, 1~374~8 chemical formula of the compound. All degrees () are in Centrigrade; "d" and "s" are used to show that the compound decomposed or sintered at or before melting.
Where no optical identification is given for the N-terminal aminoacid in the above table or in the fol-lowing examples, a racemic mixture was used.
Example 15 In an ice bath, 1.07 g. of ~fluoro-D-alanine in 20 ml. of methanol was treated with 1.1 ml. of SOC12.
The mixture was stirred two days at room temperature to give a clear solution. Solvent evaporation and tritura-tion with ether gave 1.28 g. of the methyl ester of ~F-D-Ala which melts at 130C. with previous sintering above 110C.
A 630 mg. sample of this ester was treated as in Example l(a), producing 738 mg. of amorphous Z-L-Ala-3F-D-Ala-OMe.
A 620 mg. sample of the above N~ -protected dipeptide ester was hydrogenated in the presence of 0.1 -g. 5% Pd, 2 millimoles of hydrochloric acid and 100 ml.
of methanol. Evaporation of the solvent followed by ether trituration and extensive drying gave an extemely hygroscopic gum of L-Ala-~F-D-Ala-OMe~HCl; [~]D + 41 (c, 1.10).
In analogy to the above procedure, the cor-responding ethyl or butyl esters are made by replacing the above methanol with ethanol or butanol.
Example 16 a) A suspension of 512 mg. of the compound of Example 1 in 10 ml. of DMF was stirred with 1.0 ml. of acetic anhydride. After 90 minutes, the clear solution was diluted with water, evaporated to dryness and the residue ~laced on a chromatographic column containing AG-l-X2/70Ac), (an anionic exchange resin sold by the Dow Chemical Co.). Elution with 0.05 molar ammonium acetate gave 475 mg. of the ammonium salt of N~-Ac-L-Ala; [~]D ~ 21.2 (c, 1.3).
b) Acylation with N ~Z-L-Ala-ONSu followed by hydrogenation as in Example 2 afforded the tripeptide L-Ala-L-Ala-~F-D-Ala; m.p. 267-70C. (d).

~ ~r~d~lna~ k 46i~t In the same fashion, Na-propionyl, Na-glycyl, N~-valyl, Na-leucyl, and N ~ butyryl dipeptides are pre-pared.
Example 17 In analogy with Example 1, the Na-carbo-benzoxy derivative of L-a-aminobutyric acid was coupled to ~F-D-Ala, followed by the usual deprotection reaction to give L-aNH2-But-~F-D-Ala melting at 182C.(d). The mentioned intermediate showed a m.p. of 169-70C.;
[a]D + 66 (c, 0.5).
Example 18 Using 3Cl-D-Ala in the procedure of Example 17 gave a Na-protected intermediate melting at 166-8C.
The dipeptide L-aNH2-But-~Cl-D~Ala melts at 171.5-2.5C.;
~a] + 17.1 (c, 0-5)-Example 19 In a manner similar to Example l(a), Na-tert-butyloxycarbonyl-L-norvaline was coupled to ~F-D-Ala to yield the t-BOC-L-norvalyl-~F-D-Ala. It was then depro-tected as follows:
A solution of 550 mg. of the above dipeptide in 5 ml. tetrahydrofuran was added to 5 ml. 2N hydro-chloric acid. It was evaporated under reduced pressure to dryness after being stirred at room temperature for 17 hours. The residue was dissolved in 10 ml. ethanol.

2 m. of propylene oxide was added. After stirring in cold room for 24 hours, it was filtered yielding 250 mg.
L-Norval-~F-D-Ala. Recrystallization from water gave pure dipeptide; m.p. 207C (d); [a]D + 52 (c, 0.5).
Example 20 A suspension of 2.34 g. of D,L-a-amino-octanoic acid in 30 ml. of water containing 2.52 g. of NaHCO3 was stirred in an ice bath with a solution of 4.38 g. of carbobenzoxy-N-hydroxysuccinimidyl carbonate in 30 ml. of 1,2-dimethoxyethane. After 3 hours, the temperature was allowed to adjust to room temperature and stirring was continued for three days. The resulting solution was cooled in an ice bath and acidified with 11~'74~

c~ CI -7-2~ u~ to produce 2.1 g. of the desired protected amino acid; m.p. 89-92C.
The active N-hydroxysuccinimide ester of the above was made in known fashion; it melts at 90-5C. This material was coupled to ~F-D-Ala in the fashion shown in the preceding examples. The N ~pro-tected dipeptide melts at 116-22C., while the desired D,L- ~amino-octanoyl-3F-D-Ala melts at 185-92C.;
[~ D + 19 (c, 0.5).
Other compounds of the above general descrip-tion can easily be made by repeating Example l(a) but using the succinimide esters of other N ~protected amino acids. For instance, if said ester is that of isoleucine or ~aminocaproic acid, the corresponding compounds are obtained where R represents L-isoleucyl or L-~-amino-aminocaproyl. Obviously, other amino acid esters carry-ing protected additional functional groups can be em-ployed to make the dipeptides of the current invention.
Particularly, the L-threonyl-, L-tryptophyl- and L-tyrosyl-~-fluoro (or chloro)-D-alanines can be made by the above route. In all cases, the functional groups, where present, can be temporarily protected in known fashion by benzyl, carbobenzyloxy, tert. butyl or othe protective groups commonly used in the peptide art.
In order to show the pronounced synergistic activity of the new compounds with D-cycloserine, reference is made to the following in vitro tests.
In a two-fold agar dilution assay with E. coli (Juhl) and E. coli 6880 as test organisms, compounds of Examples 1 and 2 show a minimum inhibitory concentration (M.I.C.) of~800 ppm. D-cycloserine alone shows a M.I.C.
of 12.5 ppm against the former and 6.2 ppm against the latter _. coli strain. The combination of the new peptide with D-cycloserine produces the following M.I.C. test re-sults.

Co~bination E. coli (Juhl) E. coli 6880 Example Ratio M.I.C. M.I.C.

1:8 0.2:1.56 0.1:0.78 1 1:1 0.2:0.2 0.1:0.1 8:1 0.39:0-05 0-39:0-05 . . _ 1:8 0.2:1.56 0.1:0.78 2 1:1 0.78:0.78 0.78:0.78 8:1 0.62:0.78 3.1:0.39 As shown, the compounds of the current inven-tion allow the use of much lower concentrations of both compounds to get the desired antibacterial results.
The in vitro activity of the halogenated pep-tides and the halogenated peptide-antibiotic combinations provided by the present invention can be demonstrated as follows:
The halogenated peptides alone, the antibiotic alone or mixtures of the halogenated peptides and selected antibiotics are prepared in sterile concentrated aqueous solutions at the desired ratios. Serial dilutions are made to give a range of concentrations of the test sub-stances. Samples of the dilutions are mixed with an ap-propriate sterile synthetic medium in test tubes. The tubes are then inoculated with an appropriate test orga-nism and incubated at 35-37C. for 16-20 hours. Minimum inhibitory concentrations, i.e., that concentration which inhibits visible growth, are read and the frac-tional inhibitory concentration indices (F.I.C.) are calculated. The results obtained using representative - halogenated peptides and representative antibiotics are given in Table II. In all instances, E. coli (Juhl) was used as the infecting microorganism.

:1~374~8 g .
X

t~ Cl ~ C~ ~ ~ Q t~
I I I I I I P~ D I I I I I ~
~ O O Q ~ 5 O O O O O O ~ O O O O O O ~-u~ Ul Ul O
S 11 1~.
~D (D (D ~ ~D ~ (D ~D tD tD ~
`~ V V V V `~ V
CO CO CO 00 1~ ~ CO CO CO X CO CO (D ~ H
O O O O O O O O O O O O O O ~ ~ t~
o o o o o o o o o o o o o o r~
P.

1~ ~ H
a~ ~ N ~ t~ N t~ IJ. ~3 .. ... ... .,. ... ~ ~
t~ JI ~ ~ N 01 I~Jl ~1 Ul ~JI O t~
H
. ~ , .. ... ... ... ... ~,~
W W 1-- 0 1~ ~I ~ W ~ t~ ~D ~ H
0~0 O~ W~l-- ~01--W O ~ O ~ O ~ o 1--0 W CO Ul ' ~I ~ O C~ r~
.

" ,. ..... , .... .. ...... ...... ~ O
~:
. -- 1-O O O O O O O 1~ 0 0 0 0 0 0 H
.. ... ... ... ... ~5~
IJl O O 0.0 0 ~ O ~1 0 1-- 0 0 1-- ~ H
O O O W O ~ ~1 0 ~n ~ O ~ N (D ~
~P CO Co ,P X
.

1:~3'~

The _ vivo activity of the halogenated peptides and the halogenated peptide-antibiotic combinations pro-vided by the present invention can be demonstrated as follows:
Charles River mice, weighing approximately 20 g. each, are infected intraperitoneally with 10-100 times the LD of the infecting organism. At predetermined inter-vals post-infection, e.g., 1 and 5 hours, mice are dosed subcutaneously with graded doses of the halogenated peptide, antibiotic and combination thereof. The number of mice surviving each treatment for 7 days post-infec-tion is observed and the CD50 is calculated. The frac-tional inhibitory concentration (F.I.C.) for each combi-nation is calculated in the usual manner. The results using D-cycloserine as an example of the antibiotic and representative halogenated peptides are shown in Table III.
In this table, the following infecting microor-ganisms were used:
S. aureus (Smith) _. coli (Juhl) E. coli (305-101) Strep. pyrogenes (C 203).
The infecting organisms are listed by the above code; the compounds are listed by their Example number. In all in-stances, the dipeptide and D-cycloserine were tested at a ratio of 10:1. The CD50 combination column lists only the amount of peptide present; the D-cycloserine amount is 10% of the listed amount.

'74~i~

_ W
~ 1~ X
O ~~ 0 ~ U~ ~ 3 (D
u~ w u~ W ~cn W u~
c c c c c c c 8 c c c c c c 8 c c c ~D o c c c c c c c c~c c c c c c_. c c c ~ c ~
~ o ~ ~
_ 1- ~ Ul IJ.

, n N1--CO ~ ~ 1--~ O 1~ ) tl1 C~
CO CO ~ ~ O ~ ~ ~ O O ~1 ~ ~D ~ O
~ ~\ ~ . .

.... _ H

1~ o l~ ~ ~ N ~ ~ ~ O o ~1 0 ~ ~D CO ~I ~ Ul ~I IP O ~ ~I ~
. - . . . . . . (D
Ul `I ~ ~ ~ COa~ ~ w , tD~
.._ g~
i~ ~0 00 IV CO~11 ~I ~ IP ~ ~I~Jl ~ ~P IS~ ~ O 1--1--Ul O U~ W ~ E~
' . . . . . . . . . . . . . .. . P~
a~ 1 0 CO ~ I- ~ 0~ 1'-~0 q . . .. _ _ o O O O O OO O O O O O O O O O O O O O O H W
I--N ~ W ~ ~ ~ W 1-- 0 ~ ~ ~ 1~ O ~ Q- H
o o co ~ o ~~ o ~ ~n ~ co u~ 1~ U~ Ul ~ O O ~1 X ~
._ .

The compounds of the present invention can be ad-ministered intramuscularly, orally, subcutaneously or intra-venously. Sterile, liquid dosage forms can easily be pre-pared for parenteral administration by dissolving the above dipeptide in the form of a water-soluble, non-toxic salt in isotonic sodium chloride solutions containing optional buffers, stabilizers, and/or preservatives. Liquid oral dosage forms in the form of elixirs, syrups or suspensions can be made in standard fashion, also optionally containing the above additives together with coloring or flavoring agents.
Solid dosage forms for oral administration include tablets, capsules, pills and wafers. For these dosage forms, the usual solid diluents are used where required. Capsules can be filled with undiluted powdered or granulated crystals of the new compounds. For tablets, the following standard procedure may be used:
About one-half of 50 g. of cornstarch is milled together with 50 g. of the above dipeptide and 220 g. of calcium phosphate dibasic dihydrate. This blend is milled until homogenous and passed through a 40-mesh screen. The remaining portion of the cornstarch is granulated with water, heated and mixed with the above drug blend in a hot air oven at 50C and sifted through a 16-mesh screen. These granules are then mixed with 16 g. of talcum powder, 4 g. of magnesium stearate and 0.8 g. of combined coloring and fla-voring additives. The mixture is blended to homogeneity, passed through a 30-mesh screen and blended for another 15 minutes. This blend is compressed into tablets weighing approximately 350 mg. using a 9/32" standard convex punch resulting in tablets of a hardness of 7-9 with each tablet containing 50 mg. of the drug. In a similar fashion, tab-lets weighing 600 mg. containing 250 mg. of drug can be prepared, preferably in a tableting machine producing bi-sected tablets.
While the above examples are directed to the peptides per se, the acid addition salts can readily be prepared in known fashion. The nontoxic salts useful as antibacterials include primarily the hydrochloride, phos-phate, sulfate, acetate, succinate and citrate.
As will be seen from the above examples, the current dipeptides are antibacterially active in warm-S blooded animals. Against certain bacteria, the new di-peptides are powerful synergists for known antibacterials, enabling the use of the latter in quantities of only a small fraction of its curative dose. In particular, by combining the current dipeptide with a medicinally useful antibiotic in a weight ratio of 1:1 to 10:1, excellent antibacterial synergism is observed. While the demonstrated synergistic results above are based on the use of specific antibiotics, it will be under-stood that other antibiotics including penicillins other than the above carbenicillin, cephalosporins other than cephalothin, streptomycin, erythromycin, tetracyclin, etc. can be combined with the new peptides to obtain better results than with such antibiotics alone.

Claims (21)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for preparing a peptide of the formula:
wherein the shown amino acid is in the D-configuration, X
is a halogen selected from the group consisting of: chlorine and fluorine and R is the acyl moiety of an .alpha.-aminoacid in the L-configuration; said process comprising: coupling an alanine selected from the group consisiting of: .beta.-chloro-and .beta.-fluoro-D-alanine with a reactant selected from the group consisting of: an active ester of an N,.alpha.-protected glycine, an amino acid in the L-configuration and an N,.alpha.-alkyl homolog thereof.

2. A process as defined in claim 1, wherein the .alpha.-amino group of said acyl moiety carries an additional moiety selected from the group consisting of: a fatty acid acyl group, an aminoloweralkyl acyl group, a loweralkyl group and a corresponding loweralkyl ester of the said peptide.

3. A process as defined in claim 2, wherein any N,.alpha.-amino group of said L-configuration amino acid carries a protecting group; and removing said protecting group.

4. A process as defined in claim 3, wherein the carhoxy group of said alanine is esterified with a loweralkyl alcohol.

5. A process as defined in claim 1, wherein X is fluorine and R is the acyl moiety of L-serine.

6. A process as defined in claim 1, wherein said peptide has the formula:

wherein R' is selected from the group consisting of: hydrogen and an alkyl group of 1 to 8 carbon atoms.

7. A process as defined in claim 6, wherein X is fluorine and R' is methyl.

8. A process as defined in claim 6, wherein X is fluorine and R' is isobutyl.

9. A process as defined in claim 6, wherein X is fluorine and R' is isopropyl.

10. A process as defined in claim 6, wherein X is chlorine and R' is methyl.

11. A process as defined in claim 6, wherein X is chlorine and R' is isobutyl.

12. A peptide of the formula:
wherein the shown amino acid is in the D-configuration, X
is a halogen seleeted from the group consisting of: chlorine and fluorine and R is the acyl moiety of an .alpha.-aminoacid in the L-configuration; or a pharmaceutically acceptable acid addition salt thereof; when prepared by the process defined in claim 1, or an obvious chemical equivalent thereof.

13. A peptide of the formula:
wherein the shown amino acid is in the D-configuration, X
is a halogen selected from the group consisting of: chlorine and fluorine and R is the acyl moiety of an .alpha.-aminoacid in the L-configuration, and wherein the .alpha.-amino group of said acyl moiety carries an additional moiety selected from the group consisting of: a fatty acid acyl group, an aminoloweralkyl acyl group, a loweralkyl group and a corresponding lower-alkyl ester of the said peptide; or a pharmaceutically acceptable acid addition salt thereof; when prepared by the process defined in claim 2, or an obvious chemical equivalent thereof.

14. A peptide of the formula:
wherein the shown amino acid is in the D-configuration, X
is a halogen selected from the group consisting of: chlorine and fluorine, R is the acyl moiety of an .alpha.-aminoacid in the L-configuration and Y is loweralkyl; or a pharmaceutically acceptable acid addition salt thereof; when prepared by the process defined in claim 4, or an obvious chemical equivalent thereof.

15. A peptide of the formula:
wherein the shown amino acid is in the D-configuration and Rs is the acyl moietv of L-serine; or a pharmaceutically acceptable acid addition salt thereof; when prepared by the process defined in claim 5, or an obvious chemical equivalent thereof.

16. A peptide of the formula:

wherein X is a halogen selected from the group consisting of:
chlorine and fluorine and R' is selected from the group consisting of: hydrogen and an alkyl group of 1 to 8 carbon atoms; or a pharmaceutically acceptable acid addition salt thereof; when prepared by the process defined in claim 6, or an obvious chemical equivalent thereof.

17. A pepticle of the formula:
or a pharmaceutically acceptable acid addition salt thereof;
when prepared by the process defined in claim 7, or an obvious chemical equivalent thereof.

18. A peptide of the formula:

or a pharmaceutically acceptable acid addition salt thereof;
when prepared by the process defined in claim 8, or an obvious chemical equivalent thereof.

19. A peptide of the formula:
or a pharmaceutically acceptable acid addition salt thereof;
when prepared by the process defined in claim 9, or an obvious chemical equivalent thereof.

20. A peptide of the formula:

or a pharmaceutically acceptable acid addition salt thereof;
when prepared by the process defined in claim 10, or an obvious chemical equivalent thereof.

21. A peptide of the formula:

or a pharmaceutically acceptable acid addition salt thereof;
when prepared by the process defined in claim 11, or an obvious chemical equivalent thereof.