CA1053664A - Antibiotic compounds and method of production thereof - Google Patents

Abstract

A B S T R A C T

3"-demethylgentamicin C1a is produced by selective oxidative demethylation of the 6'-and/or 3" position of XK-62-2, gentamicin C1a or 3"-N-demethyl-XK-62-2. 3"-N-demethyl XK-62-2 is produced by oxidative demethylation XK-62-2.

Description

-- ~.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 --, 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:

' 2t 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:

22 . .

6' ~
~ 3 ~ 0 ~ ~ CU

31 , NH2 .
- _ ~ _ .

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).

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., . .

``` 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.
,.~ ' ' s .
:' ,, - , : - . . .
.~ . . ;:
. .. :................ . . -, :

.,; .
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~ //

w~th an oxidizing agent.

F~l , .....

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_ ,. ~ ..........

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-- .
.

' , .-- . . - ', . . , ~ ~ :

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 .~ . .

~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 ' , db/

.~ .

~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-' db/

~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

21 .

2~

- 14 - j ' :. , 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 Staphylococcus aureus 0.0080.016<0.004<0.004 Bacillus subtilis <0.0040.008<0.004<0.004 No. 10707 Proteus vulgsris 0.0650.13 0.0330.033 Shigella sonnei 0.130.13 0.065-0.065 . ~ ` .
-~ Salmonella tyPhosa 0.0330.0650.0160.033 Kleb~iella pneumoniae 0.0160.0330.008<0.004 ~ AT~C 10031 .~ ~ .
i Escherichia coli 0.06S0.13 0.0330.016 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.

.

dbl .. ..

l jl Table 2 2 i! -3 , LD50 mg/Kg 4 j ~
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
.', "~ ' ~

, - - , . ,.. , ' ~ -. . .~.
.

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 db/

., .
:

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) ~ ¦¦ 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 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 ,1 I

~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.

, li 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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Claims (13)

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

1. Method for preparing a sagamicin(XK-62-2) derivative represented by the formula:

and pharmaceutically acceptable non-toxic acid addition salts thereof, wherein R1 is hydrogen or methyl, comprising reacting a compound represented by the formula:

wherein R1 is as defined above, R2 is a methyl group, with an oxidizing agent, and separating the desired product from the reaction mixture.

2 The process defined in claim 1, including the additional step of preparing a pharmaceutically acceptable, non-toxic acid addition salt of the product thus obtained.

3. A method of preparing 3"-demethylgentamicin C1a (6'-N,3"-N-didemethyl-XK-62-2) and pharmaceutically acceptable, non-toxic acid addition salts thereof, which comprises reacting a compound of the formula:

wherein R1 and R2 are hydrogen or methyl and when R1 is hydrogen, R2 is methyl, with an oxidizing agent, and separating the desired product from the reaction mixture.

4. The process defined in claim 3, including the step of preparing a pharmaceutically acceptable, non-toxic acid addition salt of the product thus produced.

5. The process defined in claim 4, wherein the salt is the sulfate salt.

6. A method of preparing 3"-N-demethyl-XK-62-2 and pharmaceutically acceptable, non-toxic acid addition salts thereof, which comprises reacting a compound of the formula:

with an oxidizing agent and separating the desired product from the reaction mixture.

7. The process defined in claim 6, including the step of preparing a?pharmaceutically acceptable, non-toxic acid addition salt of the product thus produced.

8. The process defined in claim 7, wherein the salt is the sulfate salt.

9. A composition of matter selected from the group con-sisting of 3"-demethylgentamicin C1a and 3"-N-demethyl-XK-62-2, said 3"-demethylgentamicin C1a and 3"-N-demethyl-XK-62-2 being represented by the formula:

Wherein : R1 is a hydrogen atom or methyl group and the pharma-ceutically acceptable non-toxic acid addition salts thereof, whenever prepared or produced by the process defined in claim 1 or 2 or by the obvious chemical equivalent.

10. 3"-demethylgentamicin C1a represented by the formula (3):

and the pharmaceutically acceptable non-toxic acid addition salts thereof, whenever prepared or produced by the process defined in claim 3 or 4 or by the obvious chemical equivalent.

11. The sulfate salt of 3"-demethylgentamicin C1a, whenever prepared or produced by the process defined in claim 5 or by the obvious chemical equivalent.

12. 3"-N-demethyl-XK-62-2 represented by the formula (1):

and the pharmaceutically acceptable non-toxic acid addition salts thereof, whenever prepared or produced by the process defined in claim 6 or 7 or by the obvious chemical equivalent.

13. The sulfate salt of 3"-N-demethyl-XK-62-2, whenever prepared or produced by the process defined in claim 8 or by the obvious chemical equivalent.