Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
-- ~.os3~64
Bl~CKGROUND OF THl~ INV:ENTION
The beneficial antibacterial properties of gentamicin
Cla are known to the art. This compound, disclosed in U.S. Patent
No. 3,091,572, has a board spectrum of antibacterial activity.
Gentamicin C1a, however, is subject to inactivation by adenyltrans-
ferase. Further, although the acute toxicity (LD50) of gentamicin
Cla is generally satisfactory, it is always desirable to provide
antibiotics with further enhanced non-toxicity properties, without
sacrificing potency and broad spectrum activity.
10Recently a new antibiotic, XK-62-2, also known as
sagamicin, has been developed. This antibiotic is similar to
; gentamicin Cla with the exception that a methylamino group, rather
than an amino group, is bonded to the carbon atom at the 6' position.
Sagamicin exhibits broad antibacterial activity and is at least
eomparable to gentamicin Cla in toxicity. Sagamicin is the subject
of Canadian Patent No. 1,003,772, issued January 18, 1977. The
method for the production and the physiochemical properties of
antibiotic XK-62-2, which is used as the starting material in the
present invention, are described in detail in the aforementioned
Canadian Patent.
Briefly stated, XK-62-2, is readily produced by
culturing actinomycetes sueh as Micromonospora sagamiensis, Miero-
monospora echinospora and Micromonospora purpurea by methods usually
employed in the eulturing of actinomycetes. More specifieally,
strains of the above mentioned mieroorganisms are inoeulated into
a liquid medium eontaining a earbon source which the microorganism
can utilize such as sugars, hydrocarbons, alcohols, organic acids,
;etc.;- inorganic or organic nitrogen
. .
.~.................... .
~.
. .
,,~
., ~
,.,..
. .-~. .. -- 2 --
,
1053664
I i,sources and additionally inorganic salts and growth promoting
2 factors, and are cultured at 25-40C for 2 to 12 days. Isolation
3 jland purification of XK-62-2 is carried out by a proper combination i
~ of adsorption and desorption from ion exchange resins and active
~carbon and column chromatography using cellulose, Sephadex,
6 aluminum and silica qel. In this manner, XK-62-2 can be obtained
7 in the form of the sulfate or in the free base form.
3 ~K-62-2 is a basic substance and is obtained as a
9 white powder. XK-62-2 has a molecular formula of C20H41N507
l and a molecular weight of 463. The substance is readiiy soluble
t~ in water and methanol, slightly soluble in ethanol and acetone ;
12 ;and insoluble in chloroform, benzene, ethyl acetate and n-hexane.
l3 In accordance with this invention antibiotic derivatives
1~ having broad spectrum antibacterial activity with enhanced re-
lS sistance to inactivation by adenyltransferase andjor enhanced non-
16 toxicity as compared to sagamicin and gentamicin Cla are provided.
SU~ARY OF THE INVENTION
~19 According to the invention, there is produced a novel
20. antibiotic derivative represented by the formula:
21
' 2t
23
2~ 6 < N~p"
NNz o
,~ . - 3 ~
...... 1
'' ~o53664
I l~wherein Rl i8 a hydro~en atom or methyl group and the pharma-
2 ,ceutically acceptable non-toxic acid addition salts thereof.
3 When Rl is hydrogen, the antibiotic is represented
~ !by the formula: ¦
6' ~ NH2
~3 ~ 0 ~ ~ S'
. 12 0 ~ . 3
3 H0' ~
U NH2 . os
~s -
16 Thi$ antibiotic is referred to alternatively herein as 3n-
17 demethylgentamicin Cla, 6'-N, 3~-N-didemethyl-XK-62-2 or Compound
1~ - III.
19 When Rl is methyl, the antibiotic is represented by
the formula:
21
22 . .
23 / NHCS3
6' ~
~ 3 ~ 0 ~ ~ CU
31 , NH2
.
- _ ~ _
.
- 1053664
ThlH Antibiotic 1~ referred to alternatively herein n~ 3"-
demethylsaga~lcin, 3"-N-demethyl-XK-62-2 or Compound I.
The antibiotic derivati.ve referred to a8 Compound
III set forth above iH prepared by reacting a compound
represented by the general formula:
r NHR,
~ O NH 2
~n. ~o~~~
HO ~/¦ o~
:, HO~ CH3
NH OH
` l
~ Rz
..
:~ whereln Rl and R2 are hydrogen atom or methyl group and when
l Rl is hydrogen, R2 i8 methyl, with an oxidizing agent.
~. ` When the compound represented by the above general
formula is Ragamicin ~XK-62-2) that is; when Rl and.R2 are
methyl, then the reaction product is a mixture of Compound I
~ (3"-N-demethyl-XK-62-2); Compound II (gentamlcin Cla) a'.so
:.` known as ~6'-N-demethyl-XK-62-2); and Compound III (6'-N-3"-
~. ~,. .
.~.. `~ N-didemethyl-XK-62-2). Compound III may be separated.and
.. ~ recovered from the mlx. If desired, Compound I may be re-
~` covered from the reaction mix and thereafter reacted with an
oxidizing agent in accordance with the present invention to
~- produce Compound III. Further, Compound II may be thereafter
recovered from the reaction mix and reacted with an oxidlzing
~, . .
. .,
-30 a8ent in accordance with the present invention to produce
i Compound III. Simllarly, Compound III may be produced by
oxidizlng Compound I or Compound II as starting
.. . .
...
:- _5_
: !1 1053664
Il, .
1 1compounds in accordance with the present invention.
2 ' The antibiotic derivative referred to as Compound I
3 !, is prepared by reacting the XK-62-2 represented by the formula:
~ r 3 -
7 ~ " , I ~H2
o ~ O
O ~
!
14 c~3
IS with an oxidizing agent. A mixture of Compounds I, II and III
u are produced. Compound I may be separated and recovered from
~t the mix.
1~1
i4 BRIEF DESCRIPTION OF DRAWINGS
Fig. l shows the infrared absorption spectrum of
21 3n-N-demethyl-XK-62-2 (Compound I);
22 Fig. 2 shows the infrared absorption spectrum of
3 6'-N-demethyl-XK-62-2 (Compound II);
2~ Fig. 3 shows the infrared absorption spectrum of
~ 6'-N, 3~-N-didemethyl-XK-62-2 ~Compound III);
26 Fig. 4 shows the nuclear magnetic responance spectrum
27 of 3N-N-demethyl-XK-62-2 ~Compound I);1
~ Fig. 5 show~ the nuclear magnetic resonance spe~trum
~ of 6'-N-demethyl-XX-62-2 (Compound II); and
~ Fig. 6 shows the nuclear magnetic resonance spectrum
of 6'-N, 3-N-didemethyl-XK-62-2 (Compound III).
-- 6 --
,,, .
.,
. .
``` i 1053664
I DESCRIPTION OF PREFERRED EMBODIMENTS
..
2 ¦ The present invention relates, inter alia, to a process
~ 1i for producing a new antibiotic derivative, Compound III, re-
-~ I' presented by the formula:
NH -
=~2 ~ 1 , 5~
O ~ C83
h~l
..
~6 and its pharmaceutically acceptable non-toxic acid addition
~9 salts. More specifically, the present invention relates to a
process for producing a new antibiotic derivative represented by
19 the above formula, by reacting XK-62-2, Compound I and Compound
II, represented by the generic formula: :
21 -~-
22 r ~HR~
C~'~,..,~ .3
~9 N~l.
,.~ ' '
7 -
s
.
:' ,, - ,
: - . . .
.~ . . ;:
. .. :................ . . -, :
1053664
.,; .
w"~..~in Rl=R2=CH3 in the case of XK62-2; Rl=C~-13 and R2-H in the
case of Compound I; and Rl=H and R2=CH3 in the case of Compound
II; with an oxidizing agent to eliminate the methyl group of the
N-methyl group bonded to the carbon atom at the 6 ' - and/or 3"-
positions of XX-62-2 or Compound I or II.
The starting Compounds I and II as well as antibiotic
XX-62-2 (sagamicin) of the present invention can be readily obtained.
Antibiotic XK-62-2 is prepared according to the processes set forth
in the aforementioned Canadian Patent No~ 1,003,772. The preparation
of Compound I is described in detail hereinafter. Compound II is
gentamicin Cla, whose preparation is set forth in U.S. Patent No.
3,091,572. Compounds I and II may be obtained by reacting the
antibiotic XX-62-2 represented by the formula:
r NHCH3
--~ NH2
~0~ //
CH3
w~th an oxidizing agent.
F~l
- 8 -
, .....
1053664
Compound III i~ a novcl antiblotlc derlvative nnd
ls vAluable not only in ~lavlng broad ~pectrum antibacterial
activity per se but also in having its toxlclty reduced to
about a half of that of XK-62-2. Further, Compound III can
be converted to radloactlve XK-62-2 by lntroduclng -C14~3 to
the amlno groups bonded to the carbon atoms at the 6'- and the
3"- positions and, accordingly, it permits studies on the dis-
trlbution and the metabolic pathways of XK-62-2 in vivo.
. Compound III is generally prepared by reacting
XK-62-2, Compound I or Compound II with an oxidizing-agent in
an lnert solvent to elimlnate the methyl group of the N-methyl
group bonded to the carbon atom at the 6'- and/or 3" -
positions of XK-62-2 or Compound I or II. In accordance with
the present invention, oxidation can be carried out by any
of the conventional methods. In order to produce Compound III
in a high yield, generally, the use of Compound II is especially :~
preferred, since the methyl group of N-methyl group bonded to
the carbon atom at the 3"- positlon is more reactlve than that ~ ::
of N-methyl group bonded to the carbon atom at the 6'- position.
20 The oxidizing agent to be used in the process of the
present invention includes the conventional oxidizing agents
` and compounds having potential oxidation ability. Specifically,
`~ heavy metal salts, peroxides~ halogens, halogenic acids,
nitrogen oxides, noble metals, and the like may be used. More
specifically, the conventional oxidizing agents such as per-
', manganate, manganate, manganese dioxide, chromic anhydride,
`~ bichromate, chromate, alkyl chromate, chromyl chloride,
selenium dioxide, cobaltic salt, ceric salt, potassium ferri- :
cyanide, cupric oxide, lead oxide, mercur.ic oxide, mixtures of
.30 hydrogen peroxide with one or more of the reagents selected from
the group consisting of ferrous salt, ferric salt, selenium
dioxide, osmium tetroxide, vanadates,
_9_
,. ~ ..........
- 1053664
tungstlc acld and chromic ac~d, leAd tetraacetaee, chlorine,
bromine, iodine, hypochlorous acld, chlorlc acld, hypobromous
acld, bromic acid, perlodlc acid, nltrous oxlde, nltrogen
monoxlde, pitrogen dloxlde, etc. and noble metals such as
platlnum, nlckel, palladium, rhodlum, ruthenium, rhenlum, and
the llke may be used.
Although any of the above-mentloned oxidizing agent~
may bc used to accomplish the present lnvention, chlorlne,
bromlne, lodine, potassium ferrlcyanide, permanganate and
platlnum are preferably used and iodlne is most preferable.
As the solvent to be used ln the process of the
present lnvention, solvents whlch dissolve the reactants and
do not excessively react with the reactants may be used. For
example, water alone or in admixture with one or more solvents
selected from methanol, ethanol, tetrahydrofuran, dimethylaceta-
mlde, dlmethylformamide, dioxane and ethylene-glycol dimethyl
ether, may be usèd.
XR-62-2 and Compounds I and II have functional groups
such as hydroxyl group, amino group, and the like within the
molecule. The ob~ects of the present invention can be
accomplished avoiding the serious loss of these functional
groups by properly controlling the ~amount of the oxidizing
agent, the acidity of the reaction mixture, the temperature, the
reactlon time and the amount of the solvent in the reaction
with the above-mentioned general oxidizlng agents. When noble
metals are used as the oxidizlng agent, a good result can be
obtsined by using the noble metals lmmediately after thelr
actlvation and by carrying out the reaction ~n the presence
of air or oxygen.
,
~30 As for the amount of the oxidlzing agent, 0.5 - 15.0
moles of the oxitizlng agent is used per one mole of the stàrting
compount. However, tbe amount can be varled wlthin thls range
according to the reaction condltlons such as the type of the
' - . -10-- .
.
' , .-- . . - ', . . , ~ ~ :
1053664
nxidi~lng ag~nt, ~he reaction temperat-lre, and t~le like, ns
wlll be obvious thosc skilled in the art.
Reaction is carried out at a temperature of -20 to
100C, preferably, from 0 to 70C. The temperature can be
varied within this range according to the reaction conditions
such as the particular oxidizing agent, the amount of the
oxidizing agent, and the like, as will be obvious to those
skilled in the art.
Further, the reaction is carried out at a pH of
4.0 - 12.0 for 0.5 - 50 hours. Generally, it is preferred to
carry out the reaction at an alkaline pH.
Isolation and purification of the product from the
reaction mixture is preferably carried out as set forth below.
; After completion of the reaction, the reaction
mixture is neutralized. The neutralized reaction mixture is
contacted with a cation exchange resin as is, or is initially
concentrated under reduced preæsure and an aqueous solution of
the resulting residue is contacted with a cation exchange resin. --
The unreacted starting material and the reaction products are
~20 absorbed on the resin. Thereafter, the resin is washed with
water, and elution is carried out with 2.0N aqueous ammonia.
After the eluate is concentrated, the product is isolated and
purified by conventional methods, for example, column chromato-
graphy and thin layer chromatography using adsorbents such as
ion exchange resins, silica gel, alumina, cellulose, and the
. like.
In the preparation of Compounds I and II from XK-62-2
and isolat~on and purification thereof, the reaction conditions
described above in connection with the preparation of Compound
'30 III from XK-62-2 and Compounds I and II may be applied without
modlfications.
', In carrying out the present invention, the reaction
.~ . .
--11--
~053664
.
condition~ when iodine, which is the most preferred oxidizing
agent, is used, are explained in detail as follow~. When other
oxidizing agents such as chlorine, bromine, potasslum ferricy-
anidç, permanganate, platinum and the like, are substituted for
lodine, similar results are obtained.
Synthesis of Compound I from XK-62-2
0.7 - lO.0 moles, preferably, 2.0 - 6.0 moles of
iodine is used per one mole of XK-62-2 in order to produce
Compound I (3"-N-demethyl-XK-62-2) in a high yield by eliminating
the methyl group of N-methyl group bonded to the carbon atom at
the 3"-position of XK-62-2.
In order to maintain the pH of the reaction mixture
basic during the reaction, those material having only a remote
possibility of decomposing XK 62-2 and the demethylated product
by-reacting with them and of substantially reducing the rea-
ctivity of iodine by reacting with it, are appropriate. For
i example, hydroxides and carbonates of alkali metals and alkaline
earth meta1s, alcoholates of alkali metals, alkali metal salts
of carboxylic acid and alkaline earth metal salts of carboxylic
¦ 20 acld are compounds which satisfy these conditions. As for the
amount of the basic material, 0.5 - 6.0 moles, preferably, 2.0 -
4.0 moles of strongly basic ma_erial or 5.0 - 25.0 moles, pre-
ferably, 7.0 - 15.0 moles of weakly basic material 1s used per
one mole of the material which 1s to be demethylated. These
basic materials may be added at the start of the reaction or
adted lntermittently during the reaction without substantially
different results.
~ The above reaction is carried out generally at a
¦ temperature of -10 to 90C, preferably, at 40 to 60C ant
¦ 30 is completed ln l - 24 hours, generally within 2 - 15 hours.
'i '
,
-12-
db/
.~ .
-- 1053664
~vnthe61s of Compound II from XK-62-2
Compound II (gentamicin Cla or 6'-N-demethyl-XR-62-2)
ls prepared under the same conditions as ln the above-stated
synthesis of Compound I, except that 1.0 - 13.0 moles, pre-
ferably, 5.0 - 9.0 moles of iodine is used per one mole of
XK-62-2. Other oxidizing agents as previously set forth may
be substituted for iodine with similar results.
Synthesis of Compound III from XK-62-2
Compound III (6'-N 3"-N-didemethyl-XK-62-2) (3"-
demethylgentamicin Cla) is prepared under the same conditions
as in the above-stated synthesis of Compound I from XK-62-2
except that 3.0 - 15.0 moles, preferably, 7.0 - 11.0 moles of
iodine is used per one mole of XK-62-2. S$milar results are
obtained when other oxidizing agents as illustratively set
forth hereinabove are employed in place of iodine.
Synthesis of the Compound III (3"-demethylgentamicin Cla)
from ComPound I (3"-N-demethyl-XK-62-2)
Compound III is prepared under the same conditions
as in the above-stated synthesis of Compound I from XK-62-2
except that Compound I is used as the starting materl-l and that
2.0 - 15.0 moles', preferably, 6.0 - 11.0 moles of iodine is
used per one mole of Compound I. Similar results are obtained
when other aforementioned oxidizing agents are employed.
: Svnthesis of ComPound III from Compound II
Compound III is prepared under the same conditions
as in the above-stated synthesis of Compound I from XK-62-2
except that Compound II is used as the starting material and
~; that 0.7 - 10.0 moles, preferably, 2.0 - 6.0 moles of iodine
, 18 u8et per one mole of Compound II.
. Compount5 I and II obtalned from XK-62-2 can be
l~olated and recovered from the reactlon mixture in the above-
'
-13-
db/
1053664
~1
!l mentioned manner and used for the preparation of Compound III.
2 !I However, the reaction mixture can be directly used for the pre- !
3 ~ paration of the Compound III without isolation and recovery.
4 !1 Under the reaction conditions, Compounds I and II may
s be simultaneously prepared from XK-62-2. Also, under the above
6 reaction conditions, the reaction may not stop at the synthesis
7 of Compounds I and II but continues to the preparation of
~ Compound III. In these ca~es, Compound III can be recovered from
9 the reaction mixture.
It is apparent from the following tables that Compound
~1 III, XX-62-2 and Compounds I and $I exhibit high antibacterial
12 activity and that Compound III and Compound I exhibit toxicity
13 - (LD50) reduced to about a half of that of XK-62-2.
14 Table l shows the antibacterial spectrum of XK-62-2,
Compounds I and II and Compound III against various Gram-positive
16 and Gram-negative bacteria determined by the conyentional double
7 dilution method.
N
19
21 .
2~
2S
26
27
28
29
31
- 14 - j
'
:. ,
1053664
Table 1
Minlmum Inhibitory Concentratlon
(MIC mcg/mQ)
Strains XK-62-2 Compound Compound Compound
I II III
...
Pseudomonas aeruginosa 1.042.08 0.52 0.26
BMH 1
Staphylococcus aureus 0.0080.016<0.004<0.004
10ATCC 6538 P
Bacillus subtilis <0.0040.008<0.004<0.004
No. 10707
Proteus vulgsris 0.0650.13 0.0330.033
ATCC 6897
Shigella sonnei 0.130.13 0.065-0.065
ATCC 9290
. ~ ` .
-~ Salmonella tyPhosa 0.0330.0650.0160.033
AICC 9992
Kleb~iella pneumoniae 0.0160.0330.008<0.004
~ AT~C 10031
.~ ~ .
i Escherichia coli 0.06S0.13 0.0330.016
ATCC 26
20~scherichia coli 8.342.08 4.17 4.17
}~ XY 8327
Escherichia coli 1.042.08 1004 1.04
' : RY 8348
r
j' In the above table, Escherichia coli KY 8327 and
RY 8348 respectively produce an adenylating enzyme, adenyltrans-
ferase, and an acetylstlng enzyme, acetyltransferase, intra-
~i cellularly. The former bacterium inactivates kanamycins and
,
1~ gentamlcIns by adenylatlon, and the latter lnactivates gentamicins
-~ by acetylation.
Table 2 shows the acute toxlcity (LD50) of XR-62-2,
Compo~nte I and II and Compound III in mice.
.
-15-
dbl
.. ..
053664
l jl Table 2
2 i! -
3 , LD50 mg/Kg
4 j
XR-62-2 93
~
6 l Compound I 200
Compound II 88 ¦ -
, Compound III 138
. g
~lo Compound III and Compound I, if desired, can be con-
1l verted to pharmaceutically acceptable non-toxic acid addition
12 salts ~amine salts). In the present invention, non-toxic acids
1~ include inorganic acids such as hydrochloric acid, hydrobromic
~. .. -
u acid, hydriodic acid, sulfuric acid, phosphoric acid, carbonic -
acid, and the like and organic acids such as acetic acid, fumaric
6 acid, malic acid, citric acid, mandelic acid, tartaric acid,
~17 ascorbic acid, and the li~e. The methods for producing the
above-mentioned acid addition salts are convention and well known
3l9 to the art.
~20 Certain specific embodiments of the invention are
~2l illustrated by the following representative examples.
Example 1
~U On this example, 2.9 g (6.3 mmoles) of XK-62-2 and 9.4 g
~2~ ~69.1 mmoles) of sodium acetate trihydrate are dissolved in 145 ml -
125 of aqucous 50% dimethylformamide. To the solution is added, 7.3 g
~28.8 mmoles) of iodine and the mixture is allowed to react
~n at 55C with stirring overnight. Then the reaction mixture is
?~ passed through a column packed with 150 ml of Amberlite~product
¦ of Rohm ~ Haas Co.) IRC-SO ~H form). After the column is washed
with 600 ml of water for complete desalting and decoloring,
1 , ~ .
~l 2.ON aqueous ammonia is passed through the colamn. About 250 ml
-. :
~ 16 - I
.', "~ ' ~
, - - , . ,.. , ' ~ -. . .~.
.
1053664
of fractlon~ which develop color by the add~tion of nlnhydrin
are combined and concentrated under reduced pressure to obtain
2.60 g of a ~lightly yellowish residue. The thus obtained
residue is subjected to column chromatography using 130 g of
sllica gel and a solvent of isopropanol:chloroform:concent~ated
aqueous ammonia (4:1:1). The eluate is taken in 13 ml port~o~
and fraction Nos. 55 - 68 are combined and concentrated to
dryness under reduced pressure to recover 450 mg of unreacted
XK-62-2. Then, fraction Nos. 75 - 83 are combined and con-
centrated to dryness under reduced pressure to obtai~ 70 mg
of 6'-N-demethy-l-XK-62-2 (Compound II, gentamicin Cla) having
the following characteristics:
Melting point: 113-117C
Spec~fic rotation: [a]24 + 171.1 (c=0.98, water)
Infrared absorption spectrum (KBr, cm 1) (Fig. 2):
3700-3000, 2940, 1630, 1575, 1480, 1380, 1340,
1286, 1146, 1108, 1052, 1021, 957, 820
Nuclear magnetic resonance spectrum (in D20) ~ (in
p.p.m. from DSS) (Fig. 5): 1.20 (3H, s), 2.53 (3H,
8), 5.13 (lH, d, J~4.0 Hz), 5.23 (lH, d, J~4.0 Hz)
~` Elementary analysis:
Calculated for ClgH3gN57-H2; C = 48-81%; H - 8-84%;
N - 14.98Z
Found: C ~ 49.36Z; H - 8.65X; N = 14.77%
Fraction nos. 93 - 135 are concentrated under
reducet pres~ure to obtain 1.38 g of 3"-N-demethyl-XK-62-2
` (Compound I).
Melting point: 105 - 115C
,
Speclfic rotation: la]28 + 148.5 (c~0.097, water)
Infraret absorption spectrum (KBr, cm 1) (Flg. 1):
3800 - 3000, 2940, 1640, 1570, 1465, 1379, 1330,
.
1284, 1142, 1110, 1050, 1020, 995, 865, 810
-17-
db/
., .
:
053664
I!
~ Nuclear magnetic resonance spectrum (in methanol -d4)
2 , ~ (in p.p.m. from TMS) ~Fig. 4): 1.16 (3H, s), 2.43 (3~1, s)
!; 5.06 (lH, d, J=3.9 Hz), 5.20 (lH, d, Js3.8 Hz)
~ : Elementary analysis:
Calculated for ClgH39N5O7.H20: C - 48.81~; H = 8.84%;
6 N = 14.98~
7 Found: C - 49.36%: H = 8.65%; N = 14.77%
8 Based on the foregoing it i8 therefore believed that
9 the structure of Compound I is repre~ented by formula:
:~
u . - ~ NHCH3
12 1;~< ,
C113'ii'
19 ~0 0~1 . I
N}~2
2I Fraction Nos. 151 - 179 are concentrated to dryness
22 under reduced pressure to o~tain 310 m~ of 6'-N, 3n-N-didemethyl-
23 XK-62-2 (Compound III).
2~ Melting point: 130 - 140C
Specific rot~tion: [329 + 97.0o (c=0.10, water)
26 Infrared absorption spectrum (KBr, cm 1) (Fig. 3):
27 3700-3000, 2950, 1630, 1570, 1480, 1380, 1333, ~.290,
1~.49, 1113, 1052, 1025, 958
~ Nuclear magnetic resonance spectrum (in D20) ~ (in p.p.m.
from DSS) (~ig. 6): 1.18 (3~1, s), 5.12 ~1l, d, J=4.01 Hz3,
31 5.29 (lil, d, J=3.9 Hz)
-- 18 -- -
11 1053664
~ ¦¦ Elementary analysis:
2 Calculated for C18H37N507.H2o
3 N = 15.44%
~ ll Found: C = 47.54% H = 8.39%; N = 15.23%
Based on the foregoing it is therefore believed that
6 , the structure of Compound III is represented by formula:
~: ;
9 ~ 6' ~ N~2
H2
3 ~ ~ 2
6 ' ~ii2 ~CH3 "`'
17 N 2 ~
9 Example 2
In this example, 463 mg (1.0 mmole) of XK-62-2 is
21 dissolved in 25 ml of water and to the solution is added 929 mg
22 (5.9 mmoles) of potassium permanganate. The mixture is allowed
23 to react at room temperature overnight. Then the reaction
2~ mixture is passed through the column packed with 30 ml of Amberlite
2s IRC-50 (H form). After the column is washed with 120 ml of
26 water, 2.ON aqueous ammonia is passed through the column. About
27 80 ml of fractions which develop color by the addition of ninhydrin
28 are combined and conccntrated under reduced pressure to obtain
29 426 my of a slightly yellowish residue. The thus obtained
residue i6 subjected to colun~n chromatography using silica gel
31 in the same manner as in Example 1. As the result, 110 mg of
,,
I1 105;~6~4
I !~unreacted XX-62-2 is recovered and subsequently, 15 mg of
2 ,ICompound Il, 135 mg of Compound I and 70 mg of Compound III
3 jl are obtained.
Example 3
,
j In this example, 449 mg (1.0 mmole) of 3n-N-demethyl-
6 lXK_62_2 (Compound I) and 2.04 g (15.0 mmoles) of sodium acetate
7 Itrihydrate are dissolved in 30 ml of aqueous 50% dioxane. To the
9 solution is added 2.04 g (8.0 mmoles) of iodine and the mixture
9 iS allowed to react at 60C with stirring overnight. Then the
jreaction mixture is passed through a column packed with 30 ml of
11 Amberlite IRC-S0 (H+ form). After the column is washed with
12 150 ml of water for complete desalting and decoloring, 2.0N
3 aqueous ammonia is pa~sed through the column. About 50 ml
~ of fractions which develop color by the addition of ninhydrin are
1S combined and concentrated under reduced pressure to obtain 420 mg
~6 of a slightly yellowish residue. The thus obtained residue is
17 subjected to column chromatography using 20 g of silica gel and a
18 solvent of isopropanol:chloroform:concentrated aqueous ammonia
19 ~4:1:1). The eluate is taken in 13 ml portions and fraction Nos.
37 - 49 are combined and concentrated to dryness under reduced
n pressure whereby, 150 mg of unreacted 3"-N-demethyl-XK-62-2
22 (Compound I) is recovered. Then fraction Nos. 61 - 76 are com-
3 bined and concentrated to dryness under reduced pressure to
2~ obtain 170 mg of 6'-N,3"-N-didemethyl-XK-62-2 (Compound III).
Elementary analysis:
26 Calculated for C18H37N57-H2
27 N ~ 15.44%
2~ Found: C ~ 47.41S; H = 8.45S: N = 15.72%
29 Example 4
~ In this example, 449 mg (1.0 mmole) of 6'-N-d~methyl-
31 X~-62-2 (Compound II) and 2.04 g (15.0 mmoles) of sodium acetate
- 20 -
,1 I
1053664
~ril)ydrate are dis601ved ln 40 ml of aqueous 50% dimethyl-
formamide. To the ~olution i8 added 761 mg (3.0 mmoles) of
iodirle and tl~e înix~ure i~ allowed to react at 50C with stlrring
overnight. Then the reac~ion mi~ture i8 passed through a column
packed with 30 ml of Amberlite IRC-50 (H+ form). After ~he
column is washed with 130 ml of water for complete desalting
and decoloring, 2.0N aqueous ammonia i9 passed through the
column. About 60 ml of fractions which develop color by the
addition of ninhydrin are combined and concentrated under
reduced pressure to obtain 430 mg of a slightly yellowish
residue. The thus obtained residue is subjected to column
chromatography using 20 g of silica gel and a solvent of
lsopropanol:chloroform:concentrated aqueous ammonia (4:1:1).
The eluate is taken in 13 ml portions and fractions Nos.
33 - 42 are combined and concentrated to dryness under reduced
pressure whereby, 89 mg of unreacted 6'-N-demethyl-XK-622
(Compound II) is recovered. Then fraction Nos. 63 - 85 are
combined and concentrated to dryness under reduced pressure
to obtain 295 mg of 6'-N, 3"-N-didemethyl-XK-62-2 (Compound
III).
Elementary analysis:
Calculated for CloH97N507.H20: C = 47.67~;
H ~ 8.67%; N ~ 15.44%
~ Found: C ~ 47.79%; H ~ 8.82~; N = 15.61
Example 5
In this example, 454 mg (l.Ommoles) of 6' -N, 3"-N- -
tldemethyl-xK-62-2~compoundIII) is dissolved in 4 ml of water.
To~the solution is atded a solution of 98 mg (1.0 mmole) of
sulfuric acid in l ml of water under cooling conditions. After
30 30 minutes, cold ethanol is added to the solution untll precipi-
tatlon is complete. By filtering the mixture containing the
preclpltated whlte solid, monosulfate of Compound III is obtained.
,
11
li
1053664
Example 6
i,
2ln this example, 4.67 9 (10.0 mmoles) of 3~ -demethyl-
X~-62-2 ~Compound 1) is dissolved in 20 ml of water. To the
~ solution is added a solution of 980 mg (10.0 mmoles) of sulfuric
; r acid in S ml of water under cooling conditions. After thirty
minute~, cold ethanol is added to the~mixture until the precipi-
tation is complete. The resulting mixture containing the deposited
~ white solid i~ subjected to filtration to obtain the monosulfate
9 of Compound 1.
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23
24
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29
31
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