Note: Descriptions are shown in the official language in which they were submitted.
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.
12 SUMMARY OF THE INVENTION
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
22
23 . l"~ o,_~
HO ~--C113
~; C1~3
76
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
28 CH3
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.
I2
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.
23
~-3
~s
26
27
28
29
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
13 O O
ll ll
14 C2~5-O-c- . C2H5-0-C-X
O~ , O
16 ; R3-CH2-C- R3-CH2-C-X
17 . 1O,
18 R3-CH2-C-H
X
22 .
23 . .
'4
: 26 - N02
29 ~ _ C2U5-0-C-~ ~`C
O
.3~ . _
.
31
. . - 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
6 O
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 --.
1046QS7
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
-- 11 --
1046QS7
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,
1046Q57
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 -
1046Q57
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.
26
27
28
~q
~'
31
-- 15 --
1046QS7
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 ~ ,-
0
JJ
I' ~
12 C: 0
a
~) ~J
1 3 0 C
.,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
0 0
.J' ~
16
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
- 17 -
1046Q57
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
- 18 -
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 -
1046Q57
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)]
- 20 -
~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,
19 830-500
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%
1046QS7
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
19
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
- 22 -
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)
29 XK-62-2
3~ In this example, the residue obtaincd in Example 6
3I containing l-N-(a-hydroxy-~-carbobenzoxyaminopropionyl)-2'-N,
1046057
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
- 24 -
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)
7 XK-62-2
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)
21 XK-62-2
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
2 XK-62-2
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
- 26 -
1046~S7
I is used for the subsequent reaction without purification.
2 Example 14
3 Production of l-N-(a-hydroxy-~-aminoyropionyl)
XK-62-2
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
1046057
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
I2
l4
17
18
I ~) .
2~ .
, I .
2s
~6
~7
28
3~
.~1
- 28 -
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
-29-
104~ 5'7
of the desired l-N-(~-hydroxy ~ aminopropionyl)-XK-62-2 is
obtained. Yield: 53.7ZO.
-30-
