CA1046057A - 1-N-(.alpha.-HYDROXY-.beta.-AMINOPROPIONYL) XK-62-2 AND METHOD OF PRODUCTION THEREOF - Google Patents

Abstract

ABSTRACT: 1-N-(.alpha.-hydroxy-B-aminopropionyl) XK-62-2 is produced by chemically modifying the antibiotic XK-62-2.

Description

104~0S7 RF L~TED APPLI CATI ON~S
The present application is directed to a derivative of the antibiotic XK-62-2. The latter mentioned and the fer-mentative production thereof is described and claimed in Canadian Patent No. 1,003,772, issued January 18, 1977 and assigned to the same assignee as the present application. Applicant's assignee's Canadian Patent applications relating to semi-synthetic derivative of XK-62-2 are Serial No. 216,641 and 216,640 filed December 12, 1974, Serial No. 290,250 filed November 4, 1977 and Serial No .
218,494 filed January 23, 1975.
BACKGROUND OF THE INVENTION
The present invention relates to a derivative of the antibiotic XK-62-2 and more specifically to the derivative identified as l-N- (c~-hydroxy-~-aminopropionyl) XK-62-2 and the production thereof.
Briefly stated, as disclosed in the aforementioned Canadian Patent 1, 003,772 antibiotic XK-62-2 is produced by culturing actinomycetes such as Micromonospora sagamiensis, Micromonospora echinospora and Micromonospora purpurea by methods usually employed in the culturing of actinomycetes. More speci-fically, strains of the above-mentioned microorganisms such as Micromonospora sagemiensis ATCC 21826, ATCC 21827, ATCC 21803 and ATCC 21949 are inoculated into a li~uid medium containing a carbon source which the microorganism can utilize such as sugars, hydro-carbons, alcohols, organic acids, etc.; inorganic or organic nitrogen sources and inorganic salts and growth promoting factors and are cultured at 25 to 40 C for 2 to 12 days until substantial antibacterial activity is detected in the Culture liquor. Isolation and purification of XI~-62-2 is carried out by a combination of adsorption ~ 2 -104~QS7 1 and desorption from ion exch~nge resins and active carbon and 7 ~um~ chr~ma~o~raphy usi~ cellulos~,~;sephad~x ~, ~lumina and 3 silica gel. In this manner, XK-62-2 can be obtained in the 4 form of a salt or as 2 free base.
s XK-62-2 is a basic substance and is obtained as a 6 white power. XK-62-2 has a molecular formula of 7 C20H4lN507, and a molecular weight of 463. The substance 8 is freely soluble in water and methanol, slightly soluble in 9 ethanol and acetone and insoluble in chloroform, benzene, ethyl acetate and n-hexane.

13 In accordance with the present invention a new 14 antibacterial compound is produced by chemically modifying the antibiotic XK-62-2 having the following structural formula:
~7 6' NHCH
4' ~ 3 I') . , ' , ,~,,~ ' NH2 HO ~ N~2 'I O

23 . l"~ o,_~
HO ~--C113 ~; C1~3 27 The derivative of XK-62-2 of the present invention ~8 . exhibits a strong antibacterial activity against a variety of

2~ Gram-positive and Gram-negative bacteria and particularly

3~ has a remarkably strong antibacterial activity against those 31 bacteria that are resistant to the known aminoglycoside anti-~.L41 ~ - 3 -1046~5'7 I biotics including XK-62-2. ~ccordingly, the antibiotic of 2. the invention is useful to clean and disinfect laboratory 3 glassware and surgical instruments, and may also be used in ~ combination with various soaps for sanitation purposes and in cleaning and sanitizing hospital rooms and areas used 6 for the preparation of food. Further, the derivative is 7 expected to be effective for the treatment of various in-8 fections such as urinary tract infections and respiratory 9 infections induced by various phlogogenous bacteria, for 0 example, Staphylococcus aureus, Sarcina lutea, Escherichia coli, Pseudomonas aeruginosa and Proteus.
12 More specifically, the new derivative of XK-62-2 13 or its pharmaceutically acceptable, non-toxic acid addition

4 salts is prepared by introducing an ~-hydroxy-~-aminopropionyl - group to the amino group bonded to the carbon atom at the 6 1-position of XK-62-2.
7 . rhus, in accordance with the present invention, l-N-(a-.hydroxy-~-aminopropionyl) XK-62-2 having the formula:
1~ . - . .
4, 6' r NHCH3 22 . ~ ~ O OII
23 2 O ~ NH-c-cHcH2NH2 ~4 .; - . O
: l~o~"

27 MO ~ C1~3 2~ .
3~ is prepared by acylating XK-62-2 with an acylating agent 3l capable of introducing an ~-hydroxy-~-substituted .
. - 4 -104~iQ57 aminopropionyl group to an amino group, to prepare an inter-~ mediate compound and thereafter eliminating the substituted 3 protecting groups in a known manner. If desired, l-N-(a-~ hydroxy-~-aminopropionyl) XK-62-2 may be thereafter converted to non-toxic acid addition salts by conventional methods.
6 In a more preferred method, an enhanced yield of the 7 compound of the invention can be obtained by protecting thè
8 amino groups bonded to the carbon atoms at the 2'- and/or 9 6'-positions of XK-62-2 with a suitable protecting reagent prior to the acylation reaction for introducing an a-hydroxy-B-substituted aminopropionyl group.

l3 DESCRIPTION OF THE INVENTION
14 I. Acylation of XK-62-2 In accordance with the present invention the free 6 base of the compound, XK-62-2, is reacted with an acylating 7 agent, i.e. a compound capable of introducing an -hydroxy-~-substituted aminopropionyl group wherein at least one of l9 the hydrogen atoms of the ~-amino group is substituted by a - protecting group in a suitable solvent to prepare an inter-21 mediate compound having one of the hydrogen atoms of the amino 22 gro~p bonded to the carbon atom at the l-position of XK-62-2 23 ~ substituted by said acyl group.
2~ As the acylating agent, a-hydroxy-~-substituted aminopropionic acid and its derivatives having an acylating 26 ability may be used.
27 The reaction is carried out in one or more solvents 28 selected, depending upon the acylating agent, from the group 19 consisting of tetrahydrofuran, dimethyl formamide, dimethylaceta-3~ - mide, lower alcohols, dioxane, ethylene glycol dimethylether, 3I pyridine and water. If necessary, a clehydrating and/or condensing

- 5 -~046QS7 I agent such as dicyclohexylcarbodiimide is added thereto. The z reactiot3 te~perature is -50 to 50C, preferable, -20 to 20C.
Usually, 0.4-2.5 moles, preferably, 0.7-1.5 moles ~ of the acylating agent is used per one mole of XK-62-2. When s an increased amount, for example, 5 moles of the acylating agent is used or where the reaction is carried out at an 7 elevated temperature of, for example, 100C, reaction may 8 proceed but the selectivity of the position to which the 9 acyl group is introduced is greatly reduced or, otherwise, o the acylating agent decomposes. Consequently, the production Il yield of the intermediate compound in the reaction mixture 12 is decreased.
13 For the protecting group of the acylating agent, 1~3 any readily eliminable protecting group usually used in peptide syntheses may be used. Such protecting group

6 and the corresponding protecting reagent which can introduce the protecting group are described in M. Bodanszky et al:
l8 Peptide Synthesis, pages 21-41 (1966) (John ~iley & Sons, I`J Inc., U.S.A.); and A. Kapoor: Journal of Pharmaceutical 2~ Sciences, Vol. 59, pages 1-27 (1970).
71 Examples of the preferred protecting groups and 27 the corresponding reagents are shown in Table 1 below.

~-3 ~s 3~
3l 1~46VS~
Tablc 1 I Protecting group ~ Protecting r~agent 3 R ~ C112-0-C- R2 ~ Cll -O-C-O-N ~ ,

7 ~ ~ R2 ~ CH2-0-C-X

8 C1~3 0 Clll3 lol H~C-C-O-C- ~13C-C-O-C-N3

9 CH3 .. . 3 , - .
O . O
, ll 12 C1~3-0-C- CH3-0-C-X

ll ll 14 C2~5-O-c- . C2H5-0-C-X

O~ , O
16 ; R3-CH2-C- R3-CH2-C-X

17 . 1O, X
22 .
23 . .
'4 : 26 - N02 29 ~ _ C2U5-0-C-~ ~`C
O
.3~ . _ .

. . - 7 -10460~i7 I In the protecting groups and protecting reagents 2 set forth in Table I above, Rl. and R2 may be the same or 3 . . different and are H, OH, NO2, Cl, Br, I, alkyl groups having 1 to-5 carbon atoms or alkoxy groups having 1 to 5 carbon 5 . atoms, R3 is H, F, Cl, Br, I or an alkyl group havin~ 1 to 5 6 carbon atoms and X is C1, Br or I.
7 . As the above-described derivatives of ~-hydroxy-~
substituted aminopropionic acid, which have the ability to acylate and are used as the acylating agent, acid halides, . . acid azide, mixed acid.anhydrides and reactive esters may be mentioned. Such derivatives are usually used in peptide : . syntheses. .Examples of these derivatives are described in 3. M. Bodanszky et al: Synthesis, page 453 (1972); and in - M. Bodanszky et al: Peptide Synthesis, pages 75-135 (1966) (John Wiley & Sons, Inc., U.S.A.).
6 . As preferred derivatives, those having a 17 structure with the hydroxy.group of the carboxy group of 18 ; a-hydroxy-B-substituted aminopropionic acid substituted 1~) by one of the following groups are appropriate:
.

~ - O-N ~ ~ - o ~ Y2 ' ~ ~ ~2 2~ . -O

; .i ~ ~ , ~ N CQ, ~r or I.
: . 26 27 . Particularly preferred derivatives are those having a t8 . structure with the OH group substituted b~
.
t~ . . O
... . ~
: - O- N
31 . . O

: - 8 -` 1046~57 1 Thus, various acylating agents are proposed.
2 Among them, N-hydroxy succinimide ester of ~-hydroxy-~-.
carbobenzoxyaminopropionic acid of the formula:
~ O OH O
~ CH2-O-C-NH-CH2-CH-C-O-N

7 iS recommended as the most preferred.
8 This acylating agent is prepared by reacting ~ a-hydroxy-~-carbobenzoxyaminopropionic acid with N-hydroxy-succinimide in the presence of a dehydrating and condensing 11 agent, for example, dicyclohexylcarbodiimide. The acylating 12 agent prepared in this manner can, of course, be isolated 13 from the reaction mixture and then reacted with XK-62-2 or 14 the reaction mixture may be directly reacted with XK-62-2 without isolating the acylating agent.
6 It is to be understood that derivatives of 17 hydroxy-~-substituted aminopropionic acid wherein the 1~ hydroxy group is substituted by other groups than that 1') disclosed above can be prepared in a known manner and are ac-ceptable for the reaction of the invention.
~t : The thus prepared intermediate compound may be 22 . isolated and purified from the reaction mixture and used 23 as the starting material for the subsequent reaction.
2~ However, it is preferred that after the completion of 2~ reaction, the reaction mixture be used as the starting 26 material for the subsequent reaction without purification.
27 The latter method is advantageous in simplifying the steps 28 ~ and enhancing the yield of recovery.
2~ If necessary, the intermediate compound may be 3~ readily isolated and purified by conventional methods, for . 31 example, column chromatography using adsorbents such as ion .
_ g -- .

104~iQ57 I exchange resins, silica gel, alumina and cellulose or thin 2 layer chromato~raphy using silica ~el, alumina and cellulose.
II. Elimination of the protecting group 4 The protecting group of the amino group contained s . in the intermediate compound prepared in the above step I
6 is thereafter eliminated to prepare l-N-(a-hydroxy-~-amino-7 propionyl) XK-62-2.
8 Elimination of the protecting group may be carried j ~) out by conventional methods. For example, where the protecting lo groups form a phthaloyl group, elimination is accomplished Il with hydrazine, where the protecting group is a carbomethoxy 12 . group or carboethoxy group, elimination is accomplished with 13 barium hydroxidei where the protecting group is a tertiary ~4 . butoxycarbonyl group, elimination is accomplished with formic acid or trifluoroacetic acid; where the protecting group is 6 . trityl group, elimination is accomplished with acetic acid 7 or trifluoroacetic acid; where the protecting group is an orthonitrophenylsulphenyl group, elimination is accomplished 1`) with acetic acid or hydrochloric acid; and where the protect-2~ ing group is a chloroacetyl group, elimination is accomplished 21 with 3-nitropyridine-2-thione ~reported by K. Undheim et al:
22 Journal of~the Chemical Society, Perkin Transactions, Part I, 23 page 829 (1973)].
~ In a preferred embodiment, the protecting group 2s of the intermediate compound is a ben7yloxycarbonyl group and 26 elimination is carried out by hydrogenolysis in the presence -7 of a metal catalyst selected from palladium, platinum, ~8. . rhodium and Raney nickel, preferably, palladium catalyst on 29 a carrier of active carbon in at least one solvent selected 3~ from the group consisting of water, tetrahydrofuran, dimethyl-1~ acetamide, dimethylformamide,lo~er alcohols, dioxane, ethylene -- 10 --.

glycol dimethyl ether, and/or pyridine, preferably, a mixture of watcr and methanol (1:1); in the presence of a small amount of hydrochloric acid, hydrobromic acid, hydriodic acid or ~ acetic acid, preferably, acetic acid, and at room temperaturc s and at atmospheric pressure.
6 The thus prepared l-N-(a-hydroxy-~-aminopropionyl) 7 XK-62-2 is isolated and purified from the reaction mixture 8 in a known manner. For example, the compound is isolated Y and purified by column chromatography using an adsorbent o such as ion exchange resins, silica gel, alumina, cellulose, Sephadex, etc., or thin layer chromatography using silica gel, alumina, cellulose, etc.
I3 ; If desired, l-N-(a-hydroxy-~-aminopropionyl) XK-1~ 62-2 prepared in accordance with the above may be converted to pharmaceutically acceptable, non-toxic acid addition 16 salts ~mono-, di-, tri-, tetra or penta salts) according to 7 conventional methods such as by interacting one molecule of 8 the compound with one to five moles of a non-toxic pharma-I'~ ceutically acceptable acid. In the present invention, non-2~ toxic acids include inorganic acids such as hydrochloric acid, 21 hydrobromic acid, hydriodic Rcid~ sulfuric acid, phosphoric 22 acid, carbonic acid, etc. and organic acids such as acetic '3 acid, fumaric acid, malic acid, citric acid, mandelic acid, '1 tartaric acid, ascorbic acid, etc.
2S Although the foregoing method accomplishes the 26 object of the invention, it has been found that by selectively 27 blocking the other free amino groups of XK-62-2, the yield of 28 the compound of the invention can be enhanced.
29 ~mong the amino groups possessed by XK-62-2, the amino ;~ group bonded to the carbon atom at the l-position is less re-3l active than those bonded to the carbon atoms at the 2'- and 1 6'-positions. As contemplated by the invention, an enhanced yield of l-N-(a-hydroxy-~-aminopropionyl) XK-62-2 can be 3 obtained by protecting the amino groups bonded to the carbon ~ atoms at the 2'- and/or 6'-positions prior to the introduction S of the -hydroxy-~-substituted aminopropionyl group to the amino 6 group bonded to the carbon atom at the l-position as follows:
7 A. Protection of the amino groups bonded to the carbon atoms at the 2'- and/or 6'-positions 9 XK-62-2 is reacted with an amino-protecting reagent in a suitable solvent to prepare at least one of: a compound 11 having a structure with the hydrogen atom of the methyl-12 - amino group bonded to the carbon atom at the 6'-position of 13 XK-62-2 protected (intermediate compound IIA); a compound 14 having a structure with at least one of the hydrogen atoms of the amino group bonded to the carbon atom at the 2'-6 position of XK-62-2 protected (intermediate compound IIB);
~7 and a compound having a structure with the hydrogen atom 18 of the methylamino group bonded to the carbon atom at the 19 6'-position and one of the hydrogen atoms of the amino group bonded to the carbon atom at the 2'-position of XK-62-2 21 protected (intermediate compc~nd IIC). Vsually, these 22 intermediate compounds are obtained as a mixture.
~ The reaction of XK-62-2 with the amino-protecting ^~ reagent is carried out under the conditions usually employed ~s in conventional methods of protecting amino groups such as 26 described above. Generally, 0.5 mole to 4.5 moles of the 27 protecting reagent is used per one mole of XK-62-2. The 28 reaction is carried out at -50C to 50C.
29 In this case, it is not desirable to use an increased 3~ amount of the protecting reagent or to carry out the reaction 31 at an elevated temperature, because, under such conditions, thc protecting group is also introduced to thc amino group ~ bonded to the carbon atom at the l-position. In order to 3 selcctively protect only those amino groups bonded to the 4 carbon atoms at the 2'- and/or 6'- positions, it is recommended to use 0.7 mole to 2.6 moles of the protecting agent per one mole 6 of XK-62-2 and to carry out the reaction at a temperature of 7 -20 to 20C.
8 The solvent for the reaction may be at least one 9 selected from the group consisting of tetrahydrofuran, dimethylacetamide, dimethylformamide, lower alcohols, dioxane, ethylene glycol dimethyl ether, pyridine and/or water.
For the amino-protecting reagent, any of the protecting 3 reagents capable of introducing readily eliminable protecting 4 groups that are usually employed in peptide syntheses may be used. Preferred protecting reagents are compounds set forth 6 in Table l above. Such protecting reagents are usuaily used 7 alone, however, the protecting reagents may, of course, be used 18 in mixture.
19 The intermediate compounds IIA, IIB and IIC of XK-62-2 can be used for the subsequent reaction as is, without '1 isolation and purification. ~owever, if desired, the inter-22 mediate compounds can be isolated and purified as described '3 ~ hereinabove.
24 B. Acylation of the amino group bonded to the carbon atom at the l-position 26 At least one of the intermediate compounds IIA, 27 IIB and IIC prepared in the above step A is reactcd with an 28 acylating agent, i.e. -hydroxy-~-substituted aminopropionic 9 acid or derivatives thereof having the ability to acylate 3`~ in a suitable solvent to prepare at least one of an inter-31 mediate compound IIIA having a structure with one of the - ]3 -1()4~iQ57 I hydrogen atoms of the amino group bonded to the carbon atom 2 at the l-position of the intermediate compound IIA substituted 3 by an ~-hydroxy-~-sutstituted aminopropionyl group; an 4 intermediate compound IIIB having a structure with one of thle hydrogen atoms of the amino group bonded to the carbon 6 atom at the l-position of the intermediate compound IIB sub-7 stituted by ~-hydroxy-~-substituted aminopropionyl group;
8 and an intermediate compound IIIC having a structure with 9 one of the hydrogen atoms of the amino group bonded to the carbon atom at the l-position of the intermediate compound 11 IIC substituted by a-hydroxy-~-substituted aminopropionyl group.
13 The acylation step and that of isolation of 14 the intermediate compounds IIIA, IIIB and IIIC can be carried out in the same manner as described in the acylation step.I
16 of XK-62-2 above, except that 0.5-l.5 moles, preferably, 0.7-17 1.2 moles of the acylating agent is used per one mole of the compound IIA, IIB and IIC. When an increased amount, for 1') example, 3 moles of the acylating agent is used or where the 2~ reaction is carried out at an elevated temperature of, for 21 example, 100C, the reaction may proceed but the selectivity of 22 the position to which an a-hydroxy-~-substituted amino-~3 propionyl group is introduced is reduced or, otherwise, the '~ acylating agent decomposes. Consequently, the production 2s yields of the intermediate compounds IIIA, IIIB and IIIC are 26 decreased.
27 C. Elimination of the protecting group ~ Elimination of the protecting groups from the thus Z'~ prepared compounds IIIA, IIIB and IIIC to prepare l-N-(-hydroxy-B-aminopropionyl) XK-62-2 is carried out in the same 31 manner as described above. Moreover, conversion of the - :l4 -resulting compounds to non-toxic acid addition salts is ~ carried out by methods well known in the art.
3 The compounds obtained by elimination of the ~ protecting group from the intermediate compounds exhibit the s same characteristics in NMR spectrum, infrared absorption 6 spectrum, melting point, specific rotation, elementary 7 analysis and MIC against various bacteria. Based on these a data, the compounds are identified as l-N-(a-hydroxy-B-g aminopropionyl) XK-62-2.
l-N-(a-hydroxy-B-aminopropionyl) XK-62-2 of the 11 present invention has an excellent antibacterial activity.
12 It is particularly notable that the compound has a strong 13 antibacterial activity against strains of Escherichia coli having R factors which show resistance to known aminoglycoside antibiotics.
6 Table 2 illustrates the antibacterial spectrum of 17 kanamycin A, gentamicin Cla, XK-62-2 and l-N-(a-hydroxy-B-8 aminopropionyl) XK-62-2 against various Gram-negative and 1q Gram-positive bacteria determined by agar-dilution method at pH 8Ø
21 From a comparison cf the minimum inhibitory con-22 centration shown in Table 2, it is apparent that the present '1 compound has a strong antibacterial activity. Characteristi-'~ cally, the compound shows a strong antibacterial activity particularly against Escherichia coli KY 8327 and 8348.

~q ~' o .S
E
~N
~ I
X ~
2 ox ~ o o ~ ~ o o o o o 3 S ~ u~ o o o o o o o o o I ~
~ O o o o o o o o o o o I
O
S I ~

7 ~
8 ~ t~l ot.,7 ~r ~ r ~J ~ ~ co ~r ~ ~ o o ~1 ~r 9 . E~ ~D u~ o o o o o o o o o ~: X o o o o o o o o ~ ,-JJ
I' ~
12 C: 0 a ~) ~J

.,1 ,~, o o ~7 ~ ~ ~ ~
t~ ,~ o o o o o o o o o 14 -' ~. -1 e O O O O O 0- 0 0 N ~-1 .J' ~

17 'al e 18 e I`J ~
r7 _I ~ ~I ~ r-~
2~) e ~ ~ D .
:~ ~ N O O ~1 ~ O 0 ~1 0 0 e .
21 U ~ L~ O O O O O O O ~ O
22 ~ X
'.1 ,~

s ~ 01 0 J~ S al c~ , a 26 1:: . ~ ~ Ir~ 0 1~ 0 O~ O O ~D 1` C~
~1 :~ 0 ~D. 0 0 ~, ~ a~ S a~ E ~ ''~
~7 o 0 ~ ,. h ~ ~ n. ) ~ O ¦~ O a~ O
0 ~ ~ V 0 ~ C C~ :~ ~ ~: ~) V 1~,) t.l V
0 :~: v E~.4 O ~ E~ C E~ v ~ Q.
C 0 ~ V ~ ~ z _1 ~O ~ ~ ¢ 0 ~ 0 ~ 0 2 8. ,~ 0 O 0 ::1 0 0 0 ~ ~ r7 ~ ~I
h O O 0 ~ 0 ~ ~ SV SV S
.9 ~ E ~ ~ 0 ~ o O .,~ -,~ ,~
U, O ~ ~ ~ ,~ C ,~ h h h ~ s ~ ~ ~, o 0 ~, a) 3ù :~ Q " v ~ E r~ ,C S S
Q~ 0 V O ,~ ~ ~ V V {~
0 v 0 h .C nJ h 0 0 0 31 P~ u) a'. ~ v~u~ ~C ~ ~ ~,.
] 6 1~46~57 1 In the above table, Escherichia coli KY 8327 and 2 KY 8348 respectively produce gentamicin adenyltransferase 3 and gentamicin acetyltransferase Type I intracellularly.
~ The former bacterium inactivates kanamycins and gentamicins by adenylation, and the latter inactivates gentamicins by 6 acetylation. It will be appreclated that the compound of the 7 present invention is not inactivated by the above-mentioned 8 anzymes and, therefore, is very effective against such bacteria.
~) Practice of certain specific embodiments of the present invention is illustrated by the following represen-11 tative examples.
1Z Example l 13 Production of 6'-N-carbobenzoxy XK-62-2, 2'-N-carbobenzoxy XK-62-2 and 2'-N, 14 6'-N-dicarbobenzoxy XK-62-2 In this example, 4.00 g (8.65 mmoles) of XK-62-2 is 6 dissolved in 92 ml of aqueous 50% dimethylformamide. To the 17 solution is added dropwise a solution of 3.23 g (12.9 mmoles) 13 of N-benzyloxycarbonyloxysuccinimide in 70 ml of dimethyl-1" formamide with stirring while maintaining the temperature at 0C to 5C. The addition is complete in 3 hours. The mix-21 ture is allowed to stand ~ at 0C to 5C overnight. By silica 2~ gel thin layer chromatography (developer : isopropanol :
23 concentrated aqueous ammonia : chloroform = 4:l:l, color reagent : ninhydrin), the presence of unreacted XK-62-2 in ~5 addition to 6'-N-carbobenzoxy XK-62-2 (Rf : 0.71), 2'-N-2h carbobenzoxy XK-62-2 (Rf : 0.62) and 2'-N, 6'-dicarbobenzoxy -7 XK-62-2 (Rf : 0.88) is confirmed.
28 Example 2 2~) Production of 2'-M, 6'-N-dicarbobenzoxy XK-62-2 .
In this example, the reaction mixture obtained in 31 Example l above is concentrated under reduced pressure. To the resulting residue are added 70 ml of water and 50 ml of 7 ethyl acetate and the resulting mixture is stirred vigorously.
~ The mixture is then allowed to stand to separate into two 4 layers (water layer and ethylacetate layer). The water layer is extracted twice with 30 ml of ethylacetate. The 6 ethylacetate layer and the ethylacetate extracts are com-7 bined, dried with anhydrous sodium sulfate and evaporated 8 to dryness. As a result 2.25 g of 2'-N, 6'-N-dicarbobenzoxy g XX-62-2 is obtained as a light yellow, amorphous solid.
~o Yield : 35.1%. The thus obtained sample may be directly ~l used as a starting material for the subsequent reaction.
12 However, if desired, the product may be further purified by 13 silica gel column chromatography (developer : iospropanol :
14 concentrated aqueous ammonia : chloroform = 4 : 1 : 1).
Analysis of purified 2'-N, 6'-N-dicarbobenzoxy 6 XK-62-2 reveals the following:
17 Melting point : 93-95C
8 Specific rotation: [alD =+81.6 (C=0.12, methanol) l~) Infrared absorption spectrum (KBr) (cm 1) [Fig. 4] : 3,800-3,000, 2,950, 1,700, 1,540, 1,456, 1,403, 1,310, 1,250, 21 1,160, 1,050, 1,010, 960, 738, 700, 605.
22 Nuclear magnetic resonance spectrum (in methanol -d4) ~ (in 2~ p.p.m. from TMS) [Fig. 1~ : 1.13 (3H, singlet), 2.62 (3H, singlet), 2~ 3.01 (3H, singlet), 5.30-4.90 (6H, broad, singlet), 7.43 (SH, singlet), 7.47 (5H, singlet) 2~ Elementary analysis:
27 calculated for C36H55N512- 2}~2 2A Found: C=58.02~; H=7.51~; N=9.70%
2~ Example 3 3~ Production of 6'-N-carbobcnzoxy X~-62-2 3l In this example, the water :layers obtaincd af~er iO46~57 I extraction with ethyl acctate in Example 2 are concentrated 2 to about 15 ml under reduced pressure. The resulting 3 concentrate is charged into a column having a diameter 4 of 2.5 cm packed with 200 ml of an ion exchange resin, S Amberlite CG-50 (ammonium form) (Product of Rohm and Haas~
6 Co., U.S.A.). The column is washed with 200 ml of water.
7 Elution is then carried out with 0.lN aqueous ammonia and 8 the eluate is taken in 10 ml portions. 6'-N-carbobenzoxy 9 XK-62-2 is eluted out in fraction Nos. 48-65. These frac-o tions are combined and concentrated to dryness under reduced ll pressure. ~s the result, 1.23 g of a colorless, amorphous 12 solid is obtained. Yield: 23.1%. The thus obtained sample 13 may be directly used as a starting material for the subse-14 quent reaction. However, if desired, the product may be further purified by the above-described ion exchange resin 6 treatment.
17 Analysis of the purified 6'-N-carboben~oxy XK-62-2 18 reveals the following:
19 Melting point; 108-110C
Specific rGtation: [a]D =+127.8 (C=0.094, methanol) Zl Infrared absorption spectrum (KBr, cm 1) ~Fig. 5] :
22 3,700-3,00~, 2,930, 1,690, 1,630, 1,596, 1,480, 1,452, t~ 1,402, 1,250, 1,143, 1,096, 1,050, 1,020, 830, 768, 750, ~ 697, 595, 550 Nuclear magnetic resonance spectrum (in methanol -d4) ~ (in 26 p.p.m. from TMS) [Fig. 2] : 1.16 (3H, singlet), 2.61 27 (3H, singlet), 3.01 (3H, singlet), 5.30-4.90 (4H, multiplet), 28 7.47 (SH, singlet) Elementary analysis:
Calculated for C28H47N509.H2 31 Found: C=54.91%; H=7.93~; N=10.90%

~ ~ - 19 -Example 4 Production of 2'-N-carbobcnzoxy XK-62-2 3 In this example, following the elution of 6'-N-t carbobenzoxy XK-62-2 in Example 3, 2'-N-carbobenzoxy XK-62-2 s is eluted out in fractions Nos. 73-97. These fractions are 6 combined and concentrated to dryness under reduced pressure 7 to obtain 1.43 g of 2'-N-carbobenzoxy XK-62-2 as a colorless, 8 amorphous solid. Yield: 26.7%. The thus obtained sample 9 may be directly used as a starting material for the subse-quent reaction. However, if desired, the product may be ll further purified by treatment with an ion exchange resin in 12 the same manner as in Example 3.
13 Analysis of the purified 2'-N-carbobenzoxy XR-62-2 tt reveals the following:
Melting point: 107-110C
6 Specific rotation: [a]D5=+87.80~ (C=0.10, water) 17 Infrared absorption spectrum (KBr) (cm ) lFig. 31 :
18 3,7~0-3,100, 2,930, 1,702, 1,530, 1,451, 1,310, 1,255, 1~ 1,141, 1,053, 1,021, 960, 735, 697, 604 Nuclear magnetic resonance spectrum (in methanol -d4) ~ (in 2t p.p.m. from TMS) [Fig. 6] : 1.13 ~3H, singlet), 2.42 (3H, 22 multiplet) ! 2.60 (3H, singlet), 5.13 (4H, broad, singlet) 2~ , Elementary analysis:
~ Calculated for C281147N59 2H2 C
Found: C=53.31%; H=8.16%; N=10.93 26 Example 5 27 - Production of N-hydroxysuccinimide ester of 28 -hydroxy-~-carbobenzoxyaminopropionic acid 29 In this example, 10 g (4.2 mmoles) of ~-hydroxy-B-3~ carbobenzoxyaminopropionic acid lThe compound is described 3l in The Carbohydrate Research, Vol. 28, pages 263-280 (1973)]

~046057 1 and 0.48 g (4.2 mmoles) of N-hydroxysuccinimide are dissolved 2 in 35 ml of ethylacetate. To the solution is added 0.86 g (4.2 mmoles) of dicyclohexylcarbodimide with stirring while ~ maintaining the temperature at 0C to 5C. The mixture is s allowed to stand at the same temperature overnight. The 6 separated dicyclohexylurea is removed by filtration. The 7 resulting filtrate is concentrated under reduced pressure O to remove ethylacetate. As the result, 1.30 g of N-hydroxy-9 succinimide ester of ~-hydroxy-~-carbobenzoxyaminopropionic o acid is obtained as a colorless, transparent oily matter.
Il Yield : 92.0~. The thus obtained product may be used as 12 is for the subsequent reaction. However, if necessary, the 13 product may be further purified by column chromatography and 1~ other well-known methods.
1s Analysis of the purified product reveals the ~6 following:
17 Infrared absorption spectrum (liquid film, cm 1): 3,700-3,100, 18 2,950, 1,816, 1,780, 1,700, 1,520, 1,320, 1,170, 1,070, 992, Nuclear magnetic resonance spectrum (in deuteriochloroform) ~
21 (in p.p.m. from TMS): 2.77 (4H, singlet), 3.67 (2H, multiplet), 27 4.64 (lH,~ multiplet), 5.11 (2H, singlet), 5.82 (lH, triplet ~ J=3.0 IIZ), 7.33 (5H, singlet) '~ Elementary analysis:
2j Calculated for C151~16N207: C=53.57%; H-4.76~; N=8.33 26 Found: C=53.42; H=4.65~; N=8.39%
27 Example 6 28 Production of l-N-(a-hydroxy-~-carbobcnzoxyamino-- propionyl)-2'-N, 6'-N-dicarbobenzoxy XK-62-2 3~ In this example, 740 mg (1.0 mmole) of 2'-N, 6'-N-~l dicarbobenzoxy XK-62-2 is dissolved in 20 ml of aqueous 50%

I - dimethylformamide. To the solution is added dropwise a 2 . solution of 403 mg (1.2 mmole) of N-hydroxysuccinimide ester 3 of a-hydroxy-~-carbobenzoxyaminopropionic acid in 15 ml of ~ dimethylformamide with stirring while maintaining the temper-s at~lre at -5C to 0C. The addition is complete in one hour.
6 The mixture is then allowed to react overnight. By silica 7 gel thin layer chromatography (under the same conditions as 8 in Example 1), the presence of a small amount of by-products 9 and unreacted 2'-N, 6'-N-dicarbobenzoxy XK-62-2 in addition o to l-N-(-hydroxy-~-carbobenzoxyaminopropionyl)-2'-N, 6'-N-ll dicarbobenzoxy XK-62-2 (Rf : 0.95) is detected. The reaction l2 mixture is concentrated under reduced pressure to obtain a l3 slightly yellowish residue. The residue is used for the 14 subse~uent reaction without purification. If desired, the product may be purified by silica gel column chromatography 6 in the same manner as in Example 1.
I? Example 7 18 Production of l-N-(a-hydroxy-~-carbobenzoxyamino-propionyl)-2l-N-carbobenzoxy XX-62-2 In this example, 633 mg (1.0 mmole) of 2'-N-car~o-21 benzoxy XK-62-2 is dissolved in 20 ml of aqueous 50~ dimethyl-22 formamide: To the solution is added dropwise a solution of 23 403 mg (1.2 mmole) of N-hydroxysuccinimide ester of a-hydroxy-2~ ~-carbobenzoxyaminopropionic acid in 15 ml of dimethylform-amide with stirring while maintaining the temperature at -5C
26 to 0C. The addition is complete in one hour. The mixture 27 is then allowed to react overnight. By silica gel thin layer 28 chromatography (under the same conditions as in Example 1), 29 the pre5ence of a small amount of by-products and unreacted 2'-N-carbobenzoxy XK-62-2 in addition to l-N-(a-hydroxy-~-31 carbobenzoxyaminopropionyl)-2'-N-carbobenzoxy XK-62-2 104~QS7 I (~f : 0.83) is detected. The reaction mixture is concentrated under reduced pressure to obtain a slightly yellowish residue.
3 The residue is used for the subsequent reaction without ~ purification. If desired, the product may be purified ~y s silica gel column chromatography in the same manner as in 6 Example 1.
7 Example 8 s Production of l-W-~a-hydroxy-~-carbobenzoxyamino-propionyl)-6'-N-carbobenzoxy XK-62-2 o In this example 615 mg (1.0 mmole) of 6'-N-carbo-1~ benzoxy XK-62-2 is dissolved in 20 ml of aqueous 50% dimethyl-12 formamide. To the solution is added dropwise a solution of 13 403 mg (1.2 mmole) of N-hydroxysuccinimide ester of a-hydroxy-4 ~-carbobenzoxyaminopropionic acid in 15 ml of dimethylform-S amide with stirring while maintaining the temperature at -5C
l6 to 0C. The addition is complete in one hour. The mixture 17 is then allowed to react overnight. By silica gel thin layer 18 chromatography (under the same conditions as in Example 1), 1~ the presence of a small amount of by-products and unreacted 6'-N-carbobenzoxy XK-62-2 in addition to l-N-(a-hydroxy-~-2~ carbobenzoxyaminopropionyl)-6'-N-carbobenzoxy XK-62-2 (~f : 0.87) 22 is detected. The reaction mixture is concentrated under 2.1 reduced pressure to obtain a slightly yellowish residue. The 24 residue is used for the subsequent reaction without purifica-2s tion. If desired, the product may be purified by ion exchange 26 resin treatment in the same manner as in Example 3.
27 Exam le 9 p 2S Production of l-N-(a-hydroxy-~-aminopropionyl) 3~ In this example, the residue obtaincd in Example 6 3I containing l-N-(a-hydroxy-~-carbobenzoxyaminopropionyl)-2'-N, 6'-N-dicarbobenzoxy XK-62-2 as a main component is dissolved . in 20 ml of aqueous 20% methanol. To the solution is added .3 1. 0 ml of acetic acid and the mixture is subjected to hydro-~ genolysis in the presence of 120 mg of 5~ active carbon-s palladium catalyst at room temperature and atmospheric 6 pressure for 6 hours. By silica gel thin layer chromato-7 graphy (developer : isopropanol : concentrated aqueous ammonia -a chloroform = 2 : 1 : 1, color reagent : ninhydrin), the presence 9 of l-N-(a-hydroxy-f3-aminopropionyl) XK-62-2 ~Rf : 0.42) as a main component, its positional isomers and a small amount of ll XK-62-2 (due to unreacted 2'-N, 6'-N-dicarbobenzoxy XK-62-2 12 in Example 6) is confirmed. The catalyst is removed by filt-l3 ration and the filtrate is concentrated under reduced pressure.
14 The residue is then dissolved in 10 ml of water and the solu-1S tion is charged into a column (diameter : 1;5 cm) of 70 ml of 6 ion exchange resin, Amberlite CG-50 (ammonium form). The 17 column is washed with 200 ml of water and then 0.2N aqueous 18 ammonia is passed through the co umn to recover XK-62-2 1~ (58 mg). Elution is carried out with 0.4N aqueous ammonia while checking the components by thin layer chromatography.
2I The fractions'containing l-N-(a-hydroxy-f3-aminopropionyl) 22 XK-62-2 (Rf : 0.42) as the only component are combined and 23 . evaporated to dryness under reduced pressure to obtain 371 24 mg of a colorless, amorphous solid. Yield : 58.1% from 2'-N, 6'-N-dicarbobenzoxy XK-62-2.
z6 ~nalysis of the product reveals the following:
27 Melting point: 149-152C (decomposed at 160C) 2A Specific rotation: ~a]D =+91.5 (C=0.106, water) 2`~ Infrared absorption spectrum (KBr, cm 1) [~ig. 7] :
3~ 3,800-3,000, 2,930, 1,650, 1,570, 1,480, 1,385, 1,330, 1,110, J' 1,052, 1,020, 813 1046~S~
I Elementary analysis:
! Calculated for C23H46N609.H2CO3.1.5H2O: C=45.07% H=7.98~;
3 N=13.14%
4 Found: C=45.21o; ~=7.71%; N=13.32 Example 10 6 Production of l-N-(~-hydroxy-~-aminopropionyl) 8 In this example, the residue obtaincd in Example 7 , 9 containing 1-N-(a-hydroxy-~-carbobenzoxyaminopropionyl)-2'-N-0 carbobenzoxy XK-62-2 as a main component is dissolved in 20 ml ~I of aqueous 20% methanol. To the solution is added 1.0 ml of 12 acetic acid and the mixture is subjected to hydrogenolysis in 3 the presence of 110 mg of 5~ active carbon-palladium catalyst l4 at room temperature and atmospheric pressure for 6 hours.
After the completion of reaction, the react~on mixture is 6 treated in the same manner as described in Example 9 to obtain 17 397 mg of l-N-(~-hydroxy-~-aminopropionyl) XK-62-2. Yield :
18 62.1% from 2'-N-carbobenzoxy XK-62-2.
19 Example 11 2d Production of l-N-(-hydroxy-B-aminopropionyl) 22 ~n this example, the residue obtained in Example 8 .I containing l-N-(a-hydroxy-~-carbobobenzoxyaminopropionyl)-6'-N-24 carbobenzoxy XK-62-2 as a main component is dissolved in 2; 20 ml of aqueous 20% methanol. To the solution is added 26 1. 0 ml of acetic acid and the mixture is subjected to hydro-27 genolysis in the presence of 110 mg of 5~ active carbon-28 palladium catalyst at room temperature and atmospheric 29 pressure for 6 hours. After the completion of reaction, the reaction mixture is treated in the same manner as 3l ~ described in Example 9 to obtain 243 mg of l-N-(~-hydroxy-~-l~g6U~7 I aminopropionyl) XK-62-2. Yield : 38.1% from 6'-N-carbobenzoxy 3 Example 12 Production of l-N-~-hydroxy-~-aminopropionyl) XK-62-2 monosulfate _ _ 6 In this example, 6.39 g (10 mmoles) of l-N-(a-hydroxy-7 ~-aminopropionyl) XK-62-2 is dissolved in 20 ml of water. To 8 the solution is added a solution of 0.98 g (10 mmoles) of 9 sulfuric acid in 5.0 ml of water under cooling. After 30 0 minutes, cold ethanol is added to the solution until precipi-I~ tation is complete. The white precipitate is separated by 12 filtration to obtain the monosulfate of l-N-(-hydroxy-~-J3 aminopropionyl) XK-62-2.
Example 13 S Production of l-N-(-hydroxy-~-carbobenzoxyamino-propionyl) XK-62-2 7 In this example, 2.79 g (6.0 mmoles) XK-62-2 is dissolved in 50 ml of aqueous 50% ~imethylformamide. To the 1') solution is addèd dropwise a solution of 2.82 g (8.4 mmoles) 2~ of N-hydroxysuccinimide ester of -hydroxy-~-carbobenzoxy-21 2minopropionic acid in 20 ml of dimethylformamide with stirr-22 ing while maintaining the temperature at -5C to 0C. The l addition is complete in one hour. The mixture is allowed to -l react overnight. By silica gel thin layer chromatography S (developer:isopropanol : concentrated aqueous ammonia :
6 chloroform = 4 : 1 : 1, color reagent : ninhydrin), the presence ~7 of l-N-(-hydroxy-~-carbobenzoxyaminopropionyl) XK-62-2 .8 (Rf : 0.63), by-products and unreacted XK-62-2 is detected.
'9 The reaction mixture is concentrated under reduced pressure 3' to obtain a slightly yellowish residue containing l-N-(-31 hydroxy-~-carbobenzoxyarninopropionyl) YK-62-2. Thc residue 1046~S7 I is used for the subsequent reaction without purification.
2 Example 14 3 Production of l-N-(a-hydroxy-~-aminoyropionyl) In this example, the residue obtained in Example 13 6 is dissolved in 40 ml of aqueous 50% methanol. To the solu-7 tion is added 0.6 ml of acetic acid and the m~xture is 8 subject to hydrogenolysis in the presence of 250 mg of 5~ active 9 carbon-palladium catalyst at room temperature and at atmospheric 0 pressure for 6 hours. By silica gel thin layer chromatography ll (developer : isopropanol : concentrated aqueous ammonia : chloro-12 form = 2 : 1 : 1) the presence of l-N-(~-hydroxy-~-aminopro-3 pionyl) XK-62-2, its positional isomers and a small amount of ~4 XK-62-2 i5 confirmed. The catalyst is removed by filtration and the filtrate is concentrated under reduced pressure. To 16 the residue is added 15 ml of water and the solution is 17 charged into a column (diameter :2.5 cm) containing 150 ml of 18 an ion exchange resin, Amberlite CG-50 (ammonium form). The 9 column is washed with 200 ml of water and then 0.2N aqueous ammonia is passed through the column to recover XK-62-2 (116 mg).
21 ~lution is carried out with 0.4N aqueous ammonia while check-22 ing the components by thin layer chromatography. The fractions 23 containing l-N-(a-hydroxy-~-aminopropionyl) XK-62-2 (Rf : 0.42) 24 as the only component are combined and evaporated to dyrness under reduced pressure to obtain 586 mg of a colorless, amorphous 26 solid. Yield : 15.3%.
27 - Analysis of the product reveals the following:
28 Melting point: 149-152C (decomposed at 160C) 29 Specific rotation: []D =+91.5 (C=0.106, water) Infrarcd absorption spectrum (KBr, cm ): 3,800-3,000, 2,~30, 31 1,650, 1,570, 1,480, 1,385, 1,330, 1,110, 1,052, 1,020, 813 I El~mcntary analysis:
2 Calculated for C23H46N6o9 ~2co3 l sH2o 3 N=13.14%
Fo~nd: C=45.31%; H=7.54%; N=13.13%

fl l4 I ~) .

2~ .

, I .
2s ~6 ~7 3~
.~1 l04~as, _ mple 15 Production of l-N-(~-hydroxy-~-aminopropionyl)-XK-62-2 In this example, 926 mg (2.0 mmols) of XK-62-2 is dissolved in 100 ml of aqueous 50% N,N-dimethylformamide.
To the solution is added dropwise a solution of 1.18 g (5.4 m mols of N-ethoxycarbonylphthalimide in 10 ml of N~N-dimethylformamide with stirring while maintaining the temperature at 20 to 25C. The addition is complete in 15 minutes. The mixture is allowed to react overnight.
m e reaction mixture contains 2'-N-phthaloyl-XK-62-2 as a main component and is used for the subsequent reaction without isolation and purification.
To the reaction mixture is added dropwise a solution of 1.05 g (3.2 mmols) of N-hydroxysuccinimide ester of q-hydroxy-~ ~ phthaloylpropionic acid in 10 ml of N~N-dimethylformamide while maintaining the temperature at 20 to 25 C. The addition is complete in 15 minutes. The mixture is allowed to react overnight. As a result, the reaction mixture containing 2'-N-phthaloyl-l-N-(qthydroxy--N-phthaloylpropionyl)-XK-62-2 as a main component is obtained.
To the reaction mixture is then added dropwise a solution of 12.9 g of aqueous 80% hydra~ine in 70 ml of methanol while maintaining the temperature at 10 to 15 C. The addition is complete in 30 minutes. The mixture is allowed to react overnight to eliminate the phthaloyl group. The reaction mixture is concentrated under reduced pressure and the resulting residue is dissolved in 20 ml of water.

By column chromatography using Amberlite CG-50 (ammonium form) under the same conditions as in Example 14, 685.5 mg 104~ 5'7 of the desired l-N-(~-hydroxy ~ aminopropionyl)-XK-62-2 is obtained. Yield: 53.7ZO.

Claims (13)

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

1. A process for producing a compound of the formula:

and non-toxic pharmaceutically acceptable acid addition salts thereof, which comprises acylating a compound of the formula:

with from 0.4 to 2.5 moles of an acylating agent, per mole of starting compound, capable of introducing an .alpha.-hydroxy-.beta.-sub-??ituted aminopropionyl group wherein said .beta.-amino group is substituted by a protecting group selected from the group con-sisting of:

wherein R1 and R2 may be the same or different and are H, OH, NO2, Cl, Br, I, alkyl groups having 1 to 5 carbon atoms or alkoxy groups having 1 to 5 carbon atoms, and R is H, F, Cl, Br, I or an alkyl group having 1 to 5 carbon atoms; in a solvent at from -50° to 50°C
to introduce said .alpha.-hydroxy-.beta.-substituted aminopropionyl group to the carbon atom at the 1-position of said starting compound; and thereafter eliminating said protecting group.

2. A process according to claim 1 wherein said acylating agent is selected from .alpha.-hydroxy-.beta.-substituted aminopropionic acid and its derivatives having an acylating ability.

3. A process according to claim 3 wherein said acylating agent is:

4. A process according to claim 1 wherein said reaction temperature is -20° to 20° C and 0.7 to 1.5 moles of acylating agent is used per mole of starting compound.

5. A process according to claim 1 wherein said protecting group is a benzylcarbonyl group and elimination is carried out ?y hydrogenolysis.

6. A process according to claim 2 wherein said reaction solvent is selected from the group consisting of tetrahydrofuran, dimethyl formamide, dimethylacetamide, lower alcohols, dioxane, ethylene glycol dimethylether, pyridine and water.

7. A process for producing a compound of the formula:

and non-toxic pharmaceutically acceptable acid addition salts thereof, which comprises reacting a compound of the formula:

with from 0.5 moles to 4.5 moles of an amino protecting reagent per one mole of starting compound at from -50°C to 50°C, said amino protecting reagent being selected from the group consisting of:

wherein R1 and R2 may be the same or different and are H, OH, NO2, Cl, Br, I, alkyl groups having 1 to 5 carbon atoms, alkoxy groups having 1 to 5 carbon atoms and X is Cl, Br or I; in a solvent to produce a mixture of intermediate compounds wherein at least one of the amino groups bonded to the carbon atoms at the 2' position and 6' position of the starting compound is protected by a protecting group; acylating said compounds with from 0.5 to 1.5 moles an acylating agent, per mole of said intermediate compound, capable of introducing an .alpha.-hydroxy-.beta.-substituted amino-propionyl group wherein said .beta.-amino group is substituted by a protecting group selected from the group consisting of:

wherein R1 and R2 may be the same or different and are H, OH, NO2, Cl, Br, I, alkyl groups having 1 to 5 carbon atoms or alkoxy groups having 1 to 5 carbon atoms and R3 is H, F, Cl, Br, I or an alkyl group having 1 to 5 carbon atoms in a solvent at -50°C to 50°C to introduce said .alpha.-hydroxy-.beta.-substituted amino-propionyl group to the carbon atom at the 1-position of said starting compound; and thereafter eliminating said protecting groups.

8. A process according to claim 7 wherein said acylating agent is selected from .alpha.-hydroxy-.beta.-substituted aminopropionic acid and its derivatives having an acylating ability.

9. A process according to claim 8 wherein said acylating agent is:

10. A process according to claim 7 wherein said protecting group is a benzylcarbonyl group and elimination is carried out by hydrogenolysis.

11. A process according to claim 7 wherein said solvent for said amino protecting reaction is selected from the group consisting of tetrahydrofuran, dimethylacetamide, dimethylfor-mamide, lower alcohols, dioxane, ethylene glycol dimethylether, pyridine and water.

12. A process according to claim 7 wherein said solvent for said acylating step is selected from the group consisting of tetrahydrofuran, dimethylformamide, dimethylacetamide, lower alcohols, dioxane, ethylene glycol dimethylether, pyridine and water.

13. A compound having the formula:
or a non-toxic pharmaceutically acceptable acid addition salt thereof, whenever prepared or produced by the process defined in claim 1, 3 or 7 or by the obvious chemical equivalent.