CA1117045A - Antibiotic ka-6606 from saccharopolyspora - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Novel antibiotics of the following structure

Description

1117Q4~

This invention xelates to novel antibioticS~ a process for preparation of the antibiotics, and biologically pure culture for use in the process.
~ he present inventors have isolated various micro-organisms ~rom soil in search of antibiotic9 produced bythese microorganisms. Conseguently, they have succeeded in isolating an antibiotic-producing strain belonging to the genus Saccharopolyspora from the soil at Kobe, Japan. From its bacteriological properties described hereinbelow1 the strain was assumed to be a natural mutant of Saccharopolyspora hirsuta, and termed Saccharopolyspora hirsuta KC-6606.
~his K~-6606 strain was deposited as FERM-P No~ ~912 in Microorganism Research Institute, Agency of Industrial Science & ~echnology, Japan; as A~CC 20501 in American Type ~5 Culture Collection.
It has been ascertained that the antibiotics produced by the KC-6606 strain are substances not described in the literature and having an antibacterial action against Gram positive bacteria, Gram negative bacteria and acid-fast bacteria. This substance was termed "KA-6606 substance".
It has also been found that by treatment with a pharma-ceutically acceptable inorganic or organic acid, the novel antibiotic KA-6606 substance can be easily converted to an antibiotic in the form of an acid addition salt. ~urther investigation has led to the discovery that the Ka-6606 substance can be further separated into four antibiotics, KA-6606 I, KA-6606 II, KA-6606 III, and KA-6606 IV, ~nd the KA-6606 I, EA-6606 III and KA-6606 IV can be readily converted to KA-6606 II by treatment with alkalies or acids.

~17~5 It is an object of this invention therefore to provide a novel antibiotic substance KA-6606, and its acid addition salts.
Another obJect of this invention is to provide a process for preparing the KA-6606 substance.
Still another object of the invention is to pro-vide an antibiotic composition comprising the KA-6606 substance as an active ingredient.
~ further ob~ect of the invention is to provide a biologically pure culture useful for providing the KA-6606 substance.
The above and other objects of this invention along with its advantages will become more apparent from the following description.
The antibiotic KA-6606 substance of this invention can be expressed by-the following structural formula -H~ ~ - NH2 4' ~

~ 1' NH2 ~?~1 (Structure I) ~ ~2 4 ~ 3 1~17~

wherein R represents a moiety selected from the group consist-ing of hydrogen, -COCH2NH2, -COCH2NHC0~2 a 2 KA-6606 II is a compound of structure (1) wherein R is hydrogen; KA-6606 I is a compound of structure (1) wherein R is -COCH2~I2; KA-6606 III is a compound of structure (1 wherein R is -COCH2NHCONH2; and KA-6606 IV is a compound of structure (1) wherein R is -COCH2NHCHO. The KA-6606 substance of structure (1) which is produced by fermentation the KA-6606 substance-producing strain of the genus Saccharopolyspora and accumulated in the culture broth contains the four substances described above. If desired, it can be separated into these four substances or into mixtures containing two or three of these substances.
These substances are useful as antibiotics either singly or as such mixtures. Treatment of the KA-6606 I, KA-6606 III
and KA-6606 IV with ,alk,alies or acids yields KA-6606 II of structure (I) in which R is hydrogen.
The chemical structures and physical and chemical properties of the KA-6606 I, II, III and IV are described below.
KA-6606 I can be expressed by the following structural formula i~7~45 H - C- NH

H2N ~ ~ Structure (l-a) NCH

CH2~2 and haS the following molecular formula, specific rotation and melting point.
Molecular formula: C17H3505N5 Specific rotation: [~2D7 ~104 (c 1, H20) Melting point: 115 - 125C
KA 6606 II can be expressed by the following structural formula ~ 17~45 ~,.,0~

H N \ NH
\ OH~ Structure (l-b) and has the following molecular formula, specific rotation and melting point~
Molecular formula: C15H3204N4 Specific rotation: C~2D7 +13975 (c 1, H20) Melting point: 88 - 93C
KA_6606 III can be expressed by the following structural formula H - C - NH2 O~

\ ~ ~ ~ Structure (l-c) CO OCH

1~7Q4~

and has the following molecular formula, specific rotation and melting point~
Molecular formula: C18H3~06N6 Specific rotation: ~a~2D7 +103 (c 1, H20) Melting point: 145 - 152C
~-6606 IV can be expressed by the following structural formula C~H3 H~ C - NH? O

H2N \ ~2 ~ Structure (l-d) \
~NCH3--I

and has the following molecular formula, specific rotation and melting point.
Molecular formula: C18H3506N5 ~pecific rotation: [a~2D7 +101 (c 1, H20) Melting point: 145 - 140C
Known antibiotics which are most similar to the antibiotic substances KA-6606 of formula (1) are fortimicin A, B and C ~see Japanese ~aid-Open Patent Publications NosO
6487/78 (published on January 20, 1978), 29789/75 (published on March 25, 1975), 145588/75 (published on November 21, 1975), ~17Q~;~

1888~/77 (published on February 12, 1977) and 83513/77 (pub1ished on July 12, 1977), and Japanese Patent Publication No. 46311/77 (published on November 24, 1977)]. Fortimicin is an antibiotic produced by Micromonospora sp. MK-70 strain (FERM-P NoO 1560; ATCC 21819), and as disclosed in The Journal of ~ntibiotics, Vol. XXX, No. 7, pp. 552-563, has a different chemical structure from the KA-6606 substance of formula (1) given above in that the KA-6606 substance does not have a hydrox~l group at the 2-position and has a differ-ent configuration of an amino group at the l-position.
As will be described hereinbelow, the KA-6606 substance of this invention has a better antibacterial activity than fortimicin.
The novel antibiotic of the invention can be produced by fermenting the antibiotic KA-6606-producing strain of the genus Saccharopolyspora and isolating the antibiotic XA-6606 substance from the culture broth. If desired, one or more of the antibiotics KA-6606 I, KA-6606 II, KA-6606 III and KA-6606 IV can be separated from the resulting KA-6606 substance.
~he taxonomic charaterization of Saccharopolyspora hirsuta KC-6606, a typical example of the KA-6606-producing strain, is shown below. Unless otherwise specified, its properties on various culture media were observed by ordinary methods after cultivating it for 21 days at 27C.
~he colors were expressed for mature culture in accordance with the classifications of "Color Harmony Manual" (Container Corp. AmerO 1958).

1~7~45 I. Morphological propertie~
This strain forms substrate hyphae and aerial hyphae on aninorganic salts-starch agar medium. The substrate hyphae (0.5 to 0.7 ll in diameter) spread out in long branches and are tangled with each other complicately, permitting usually one,- very rarely 2 to 3, substrate spore like bodies (008 to 102 ~ in diameter) to adhere to the end of the sporangiophore (about 2.5 1l in length).
Occasionally typical nocardioform fragmentation is found in the colonyO ~he aerial hy~hae (006 to 0.9 ~ in diameter) are short, and the mature spores are observed as chains of 20 or more spores and segmented into bead-like chains of spores often separated by the length of empty hyphae. ~he spore chains are observed as loops and loose spirals. Ihe spores are oval to short cylindrical (005-0.6 ~ x 0.7-0.9 ~), and the surfaces of the spores are covered with a sheath having a hairy structure.
II. Properties on various media l. Sucrose-nitrate agar Growth: good Aerial hyphae good, powdery, shell pink (5ba) Substrate hyphae: butyrous, light tan to rose beige (3gc - 4gc) Soluble pigment pale pink with a tint of yellowish brown 20 Glycerol-nitrate agar Growth: moderate to good ~erial hyphae: good, powdery, white (a) Substrate hyphae: butyrous, light ivory (2ca) Soluble pigment pale yellow _ 9 _ 1~17Q~S

3. Glucose-asparagine agar (slant) Growth: moderate ~erial hyphae: none Substrate h~phae: butyrous to ~elatinous, buff (2fb) Soluble pigment: slightly pale yellow 4. Glycerol~aspraragine agar (ISP medium No. 5) Growth: moderate Aerial hyphae: poor, powdery, white (a) Substrate hyphae: gelatinous, light ivory (2ca) Soluble pigment: slightly pale yellow 5. Inorganic salts-starch agar (ISP medium No. 4) Growth: good Aerial hyphae: poor, powdery, white (a) Substrate hyphae: cartilaginous, light ivory to buff (2ca-2fb) Soluble pigment: none 60 ~yrosine agar (slant~ ISP medium No. 7) Growth: moderate Aerial hyphae: good, powdery, white (a) Substrate hyphae: butyrous to gelatinous, light ivory to buff (2ca-2fb) Soluble pigment: slightly pale yellow 7. Nutrient agar Growth: moderate Aerial hyphae: none Substrate hyphae: gelatinous, buff (2fb) Soluble pigment: none 80 Yeast extract-malt extract agar (ISP medium No. 2) Growth: good ~17~

A~rial hyphae: sparse, powdery, white (a) Substrate hyphae: gelatinous, bamboo (2g) Soluble pigment: pale yellow 9. Oatmeal agar (ISP medium No. 3) Growth: poor Aerial hyphae: none Substrate hyphae: colorless Soluble pigment: none 10. Peptone-yeast extract-iron agar (slant, ISP medium NoO 6) Growth: moderate Aerial hyphae: poor, powdery, white (a) Substrate hyphae: butyrous to gelatinous, light melon yellow (2ea) Soluble pigment: slightly pale yellow llo Bennett's agar Growth: good Aerial hyphae: none Sabstrate hyphae: gelatinous, light ivory (2ca) Soluble pigment: slightly pale yellow II. Physiological properties 1. Growth temperature: 18-45C

2. Liquefaction of gelatin: positive 30 Hydrolysis of starch: positive 40 Action on milk:
Coagulation: negative Peptonization: positive 5. Production of melanoid pigment:
Negative in tyrosine agar and in peptone-iron-yeast extract agar~

lli704~

60 Production of nitrite from nitrate and its accumulation: positive IV. Utilization of carbon sources Positive: D-glucose, D-fructose, raffinose, sucrose, D-mannitol, galactose, inulin, and salicinO
Negative: L-arabinose, D-xylose, inositol, I rhamnose, sorbitol, lactose, and dulcitol.
As described above, the KC-6606 strain forms substrate hyphae and aerial hyphae on various agar mediaO
Ihe substrate spore like-bodies adhere to the substrate hyphae, and the spore chains adhere to the substrate hyphaeO
~he substrate hyphae occasionally fragment into rod-shaped elements. The surfaces of the spores are covered with a sheath having hairy structure.
Analysis of its cell wall showed that it contains meso-diaminopimelic acid and arabinose and galactose as sugars. Accordingly, the cell wall of this strain is believed to be of type IVo Analysis of the fatty acids in whole cell showed that it does not contain LCN-A (lipid characteristic of nocardia).
Known strains having these characteristics have been searched through Bergey's Manual of D~terminative Bacteriology~ 8th edition (1975)o ~s a result, it has been found that the genus Micropolyspora is a similar genus, and the genus ~accharopolyspora reported by Lacey and Goodfellow (Journal of General Microbiology, Vol. 88? page 75~ 1975) _ 12 -1~17(~

can also be cited aS a similar strain. ~he strain used in this lnvention differs from strainS of the genus Micropolyspora in that it has a hairy structure in spores and the spore chains contain more than 20 sporesO But it is similar in morphology to the latter. This leads to the belief that the KC-6606 strain belongs to the genus Saccharopolyspora.
There has been no established method about the systematic cla,ssification of the genus ~accharopolyspora because only one species for one genus has been reported.
~he differenees between the KC-6606 strain and the Saeeharopolyspora hirsuta are shown in Table 1.
Table 1 __ _ __ _ _ . __ __ __ __ _ KC-6606 Saecharopolyspora ___ _ _ _ _ h rsuta _ Utilization of sugars Xylose _ Rhamnose _ Sorbitol _ +

Inositol _ +
, __. _ __ _~ ._ _ _ P~oduction and accumulation of nitrite Since the KC-6606 strain and Saeeharopolyspora hirsuta well eorrespond with each other in basic properties such as morphology~ properties on various culture media and physiological properties although differing in the utilization of earbon sourees and the formation and aecumulation of nitrite~ it would be reasonable to eonsider that these strains belong to the same speeies.

1~17(~5 On the basis of the above information, the present inventors have considered the strain in guestion as a natural mutant of Saccharopolyspora~ and named it Saccharopolyspora hirsuta KC-6606 strainO The strain of Saccharopolyspora hirsuta used in this invention is susceptible to change in its characteristics, and can be mutated easily by an artificial mutating means using ultraviolet rays, X-rays, various chemicals such as nitrosoguanidines or mitomycin, etcO All such mutants which have the ability to produce ~he antibiotic KA-6606 substance can be used in this invention. In this regard, the ability of strains belonging to ~accharopolyspora hirsuta to produce antibiotics has not been reported.
According to this invention, there is provided a biologically pure culture of Sacchropolyspora hirsuta KC-6606 having characteristics identified as FERM-P No. 3912, and A~CC 20501, and alSo having the ability to produce antibiotic KA-6606 substance by fermentation in an aqueous nutrient medium containing a carbon source, a nitrogen source and minerals~
~uitable culture media for use in fermenting the KA-6606 substance-producing strain of the genus ~accharo-polyspora comprise carbon and nitrogen sources and as optional ingredients, inorganic salts (minerals), very small amounts of heavy metals, etc.
Various carbon sources can be used, and examples of preferred carbon sources are starch, glycerol, maltose, dextrin, sucrose, fructose and molasses, ~hich can be used either alone or as suitable mixtures. Hydrocarbons, organic acids and vegetable oils can also be used if the particular 1~7(~4~

strain can utilize them as a carbon source. Examples of nitrogen sources are soybean meal, yeast extract, dried yeast, peptone, meat extract, corn steep liguor, Casamino acid, Distiller's soluble, ammonium chloride, ammonium sulfate, urea and sodium nitrate, which can be used either alone or as suitable mixturesO Examples of inorganic salts include sodium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium carbonate, calcium hydroxide, cobaltic chloride, zinc sulfate, ferric chloride, and ferrous sulfate.
Inorganic substances and organic substances (e.g., amino acids) which ai-d in the growth of the strain and promote the production of KA-6606 substance can also be added to the culture medium as reguired. When an aerating cultivation method is employed, an antifoamer such as fatty acid oils, silicon oils and paraffins can also be added to the culture medium.
Cultivation may be carried out in a solid mediumO
Preferably, however, same as in the general process for producing antibiotics, a liquid cultivating method, especially a submerged cultivation method, is usedO ~he cultivation is carried out under aerobic conditions~ and the cultivation temperature is generally about 20 to about 40C, preferably about 27Co Preferably, during the cultivation, the pH of the culture medium is maintained at about 4 to about 10.
~he cultivation period is generally about 2 days to about 6 days.
~ s a result of the cultivation, the K~-6606 substance is produced and accumulated in the culture brothO

~i~7~4~i When the amo~t of the KA-6606 substance produced in the culture broth reaches a maximum, the cultivation is stopped, and the KA-6606 substance can be collected from the culture broth.
~ince the KA-6606 substance is a water-soluble basic substance soluble in water but only slightly soluble in common organic solvents, it can be separated from the culture broth by utilizing means which are customarily used in isolating and purifying water-soluble basic antibiotics.
For example, there can be used an adsorption desorption method using an ion exchange resin, active carbon, cellulose, silica gel, alumina, and a method for extracting with butanol, amyl alcohol, etc. using a higher fatty acid as an adjuvant.
For example, if the culture broth filtrate is charged into a column of a weak acidic cation exchange resin, the KA-6606 substance is adsorbed to it. ~he KA-6606 substance is then isolated by elution with a 0.1-~.0 N
alkali or acid or various salt solutionsO The resulting active eluate is lyophilized to afford a crude powder of KA-6606 substance.
~xamples of the weak acidic cation exchan~e resin used to recover the KA-6606 substance are Amberlite IRC-50, IRC-8L~ and CG-50 (Rohm ~ Haas Co.); and Diaion WK-10 and WK-20 (Mitsubishi Chemical Co., Ltd~). Alkalies that can be used for the elution are ammonium hydroxide solution, and an agueous solution of sodium hydroxide. ~xamples of the acids are formic acid, hydrochloric acid and sulfuric acid. The salt solutions may, for example, be a solution ~r~d~

1~17~45 of ammonium carbonate ~nd a solution of ammonium forma~e.
Anot;her example o~ the recovering method comprises adjusting the p~ of the culture broth filtrate to 7 to 9, contacting the filtrate with active carbon to cause the KA-6606 to adsorb to the active carbon, and eluting the substance with acidic water or hydrochloric acid-methanol.
~ he KA-6606 substance that can be isolated by the methods described above can be separated into KA-6606 I, II, III and IV by dissolving it in water or dilute ammonium hydroxide, charging it into a column of an adsorbent such as a weak acidic ion exchange resin of the type described above or a weak acidic i~ exchanger such as CM-sephadex or CM cellulose to cause the substance to be adSorbed to the adsorbent, and then eluting it with an alkaline aqueous solution such as dilute ammonium hydroxide, or an aqueous solution of ammonium carbonate or ammonium formate by a gradient method or a stepwise methodO According to this separating procedure, first se~eral components in trace amounts are eluted, and then KA-6606 IV and KA-6606 III as free baseS
are eluted in this order~ On further elution, KA-6605 I
and KA-6606 II are separated successively.
~ he resulting KA-6606 I, II, III and IV can be purified by chromatography on cellulose, silioa gel, Sephadex (e.g. LH 20), etc. For exa~ple, it can be chromatographed on a silica gel column using a mixture (1:8 3) of chloroform-methanol-17% ammonium hydroxide as an eluentO
Ihe KA-6606 I, II, III and IV that can be separated by the method described above are in the form of free bases~
and as desired, may be obtained in the form of a pure free ~ rr~de r/la~l~ _ 17 -1117~!?4~

base by causing tbem to be adsorbed to a column of a strong base anion exchange resin such as Dowex~lx2 (Dow Chemical), eluting them with deionized water, collecting acti~e fractions, and lyophilizing the collected fractions.
These KA-6606 I, II, III and IV obtained as free bases can be converted to their acid addition salts by treatment with pharmaceutically acceptable inorganic or organic acidsO Examples of such acids are inorganic acids such as hydrochloric acid, sulfuric acid and hydrobromic acid, and organic acids such as acetic acid and oxalic acid.
~he KA-6606 I, KA-6606 II~ and KA-6606 IV can be converted to KA-6606 II by treatment with alkalies or acidsO
~he conversion can be effected by treating the KA-6606 I, ,KA-6606 III and KA-6606 IV with a 0.1-4 I~ aqueous solution f an alkaline reagent such as sodium hydroxide or barium hydroxide or with a Orl~l N aqueous solution of an acidic reagent such as hydrochloric acid or sulfuric acidO
In the case of using the alkaline reagent, a strong base anion exchange resin teOgO, Amberlite IRA 400 (OH from) or Dowex lx2 (OH- from)~ may be added, and the reaction can be performed in the suspended state. ~ikewise, when the acidic reagent is used, a strong acidic cation exchange resin such as Amberlite lR 120 (H form) or Dowex 50x8 (H form) may be added, and the reaction can be performed in the suspended state. The reaction can be performed usually at about 30 to 100C for about 0.5 to

3 hoursO

Tr~de ma~l~

_ 18 --l~i7~

The physical ~nd che~ical properties of the novel antibiotics KA-6606 I, II, III and IV of structures(l-a) to (l--d) are described belowO
KA--6606 I (free ase) (1) Nature: white powder ( 2) Molecular formula: C17H~;sOsNs (~.) Elemental analysis:
C H N
Found (%): 52010 8~84 17083 Calculated (%): 52042 9006 17099

(4) Molecular weight: Z89 (mass spectrum)

(5) Melting point: 115 -- 125Co

(6) Specific rotation- (a)27 +10~ (c 17 H20)

(7) Ultraviolet absorption spectrum ht 220 ~ 360 nm, no characteristi.c absorption is shown, but only a terminal absorption existsO

(8) Infrared absorption spectrum ~he infrared absorption spectrum of a sample in pOtaSSiUm bromide tablet is as shown in ~igure lo

(9) Solubility Very readily soluble in waterO ~asily soluble in methanol, slightly soluble in ethanolO Insoluble in chloro-form, ethyl acetate, diethyl ether, hexane and petroleum etherO

(10) Color reaction Ninhydrin reaction and Rydon Smith reaction: positive;
Sakaguchi reaction, maltol reaction, ferric chloride reaction and ~ehling reaction: negative

(11) Stability 1~17~

Stable at a pH OI 7 to 8; gradually decomposed and inactivated in basicity and strong acidicity.

(12) Nuclear magnetic resonance spectrum (~D 0 ppm):
1.25 (ZH, d, C-CH~;) 7.O05 (~H, s, N-CH3) 3O07 (2H, s, COCH2N) 3040 (3H, s~ OCH3) 5.00 (lH, d, anomeric H)

(13) Mass spectrum (m/e):
389(M~), 276, 258. 248. 230. 180, 143 (lL~) Paper chromatography Rf value: 0O53 ~ilter paper: Whatman No. 1 " 15 Solvent: a lower layer of chloroform-methanol-17% ammonium hydroxide (2:1:1) (15) ~hin~layer chromatography ..
Rf value Solvent 0.56 Butanol-ethanol-chloroform-17% ammonium hydroxi,de (4:2:5:2) 0.60 Chloroform-methanol-17% ammonium hydroxide (1:8:3) OolO A lol~er la~er of chloroform-methanol-17/~ ammonium hydroxide (2:1:1) Oo76 An upper layer of chloroform-methanol-17% ammOniWD
hydroxide (2:1:1) _ _ ~LC aluminum sheet (silica gel 60 ~254 0.2 mm) (Merck) was usedO

'- - 20 -.

~117Q~

t_ve of KA 6~06 I substance.
(1) Nature: colorless crystalline powder (2) Molecular formula: C 25H4309N5 (3) Elemental analysis:
C H N
Found (%): 53051 7 7 58 12.44 Calculated (/0). 53085 7077 12.56 (4) Molecular weight: 557 (mass spectrum) (5) Melting point: 160 - 166C o (6) Specific rotation: (~)D5 +112 (c 1, H20) (7) ~uclear magnetic resonance spectrum ( ~D 0' ppm):
1.10 (3H, d, C-CH3) 1.95, 2000, 2.05, 2007 (3H, s, COCH3) 3009 (3H, s, ~CH3) 3~42 (3H, s, O-CH3) 4.11 (2H, s, COCH2N) 4.95 (lH, d. anomeric H) (8) Mass spectrum (m/e):
557(M+), 342, 314, 296, 227 KA-6606 II substance (free base) ature: white powder (2) Molecular formula: C15H3204N4 (3) El emental a.nalysis:
C H N
~ound (%): 53.97 9.51 16.55 Calculated (%): 54019 9~70 16.85 (4) Molecular weight: 332 (mass spectrum) (5) Melting point: 88 - 93Co ~17~

(6) Specific rotation: ~)27 +13905 (c 1, E20) (7) Ultraviolet absorption spectrum At 220 to 360 nm, no specific absorption is shown, but only a terminal absorption exists.
(8) Infrared ~bsorption spectrum The infrared absorption spectrum of a sample in potassium bromide tablet is as shown in Figure 2.
(9) Solubility Very readily solub].e in water; easily soluble in methanol; slightly soluble in ethanol; insoluble in chloro-form, ethyl acetate1 diethyl ether, hexane and petroleum etherO
(10) Color reaction Ninhydrin reaction and Rydon Smlth reaction: positive;
Sak~guchi reaction, maltol reaction, ferric chloride reaction and Fehling reaction: negative (11) Stability Stable at a pH of at least 2~ gradua]ly decomposed and inactivated in strong acidityO
(12) Nuclear magnetic resonance spectrum (~D 0 ppm):
1025 (2,H, d, C-CH3) 2.70 (2H, s, N-CH3) 3~45 (~H, sl 0-CH~) 4.95 (lH, d, anomeric H) (13) Mass spectrum (m/e):
332(M ), 283, 219, 119, 143 ( 1 4) Paper chromatography Rf value: 0086 ~ilter paper: Whatman NoO 1 ~il7Q4~;

Solvent: a lower layer of chloroform-methanol-17% ammonium hydroxid~
(2:1:1) (15) Thin--layer chromatography _ Rf value Solvent ~ . l 0057 Butanol-ethanol-chloroform-17% ammonium hydroxide 0.52 Chloroform-methanol-l~/o ammonium hydroxide (1:8:~) 0.16 A lower 1a er of chloroform-methanol-17% ammonium hydroxide ~2:1:1) 0.80 An upper layer of chloroform-methanol-17% ammOnium ~ hydroxide ( 2 :1 :1 ) _ . .

Tl;C aluminum sheet (silica gel 60 :E?254 . 2 mm) (Merck Co.) was used as the plateO
Penta--N-acet~l derivative of KA 6606 II substance (1) Nature: colorless needle (2) Molecular formula: C23H4008~4oH20 (3) ~lemental analysis:
C X N
~ound (%): 52.99 7 .65 lOo 52 Calculated (%): 53.27 8.16 10.80 (4) Molecular weight: 500 (mass spectrum) (5) Melting point 139 - 141C.
(6) Specific rotation: ~tx)22 +52 (c 1, H20) (7) Nuclear magnetic resonance spectrum (~D 0' ppm):
1.09 (3H, d, C-CH3) 1.98, 1.99, 2.01,-2013 (3H, s, C0-CH3) 1~17~

~10 (ZH, s, N-CH~) 3~40 (3H, s, 0-CHz) 4.96 (lH, d, anomeric H) (8) Mass spectrum (m/e):
500(M~), Z.0~" 271. 257. 227 KA-6606 III substance (free base) (1) Nature: white powder (2) Molecular formula: C18H~606N6 (~) Elemental analysis:
C H N
~ound (%): 49.51 8.11 19.10 Calculated (%): 49.99 8.39 19.43 (4) Molecular weight: 432 (mass spectrum~
(5) Melting point: 145 - 152C.
(6) Specific rotation: ~a~D7 +103 (c 1, H20) ~7) Ultraviolet absorption spectrum At 220 to 360 nm, no specifi¢ absorption ls s~own, but only a terminal absorption exists.
(8) Infrared absorption spectrum ~he infrared absorption spectrum of a sample in potassium bromide tablet is as shown in ~igure 3.
(9) Solubility . ~ry readily solu~le in water; easaly soluble in methanol; slightly soluble~in ethanol; insoluble in chloro-form, ethyl acetate, dieth~l ether, hexane, and petroleum ether.
(10) Color reaction Ninhydrin reaction, an~ Eydon Smith reaction: positive;
Sa~a~uGhi reaction, maltol reaction, ferric chloride 13 1'7Q~

reaction, Fehling reaction: negative (ll) Stability Stable at a pH of 3 to 8; gradually decomposed and inactivated in basicity and strong acidityO
(12) Nuclear magnetic resonance spectrum (~D 0 ppm):
1021 (~H, d, C-C~) ZoO7 (~H, s, N CH~) ~040 (3H, s, 0-CH~l) 4006 (2H, s, C0-CH2-N) 4095 (lH, d, anomeric H) (13) Mass spectrum (m/e):
432(M+). 273, 219, 194, 173, l~

(14) Paper chromato~raphy:
Rf value: 0027 ~ilter paper: Whatman Mo, 1 Solvent: a lower layer of chloroform-m~thanol-17% ammonium hydroxide (2:1:1) 2.0 (15) Thin layer chromatography ___ Rf value Solvent _ 0O55 Butanol-ethanol-chloroform-17% ammonium hydroxide (4:5:2:5) 0064 Chloroform-methanol-17% ammonium hydroxide 0~06 A lower layer of chloroform-methanol-17% ammonium hydroxide (2~
0O77 An upper layer of chloroform-methanol-17% ammonium hydroxide (2:1~1) 1117~

r~C aluminum sheet (silica gel 60 F~s4 0 o 2 mm) (Merck) was used as the plateO
KA-6606 IV substance (free_base) (1) Nature: white powder (2) Molecular formula: C18H3506N5 (3) Elemental analysis:
C H
~ound (%): 51~ 39 8008 16041 Calculated (/0): 51~78 8~45 16077 (4) Molecular weight: 417 (mass spectrum) (5) Melting point: 135 - 140Co (6) Specific rotation: (c~27 +101 (c 1~ H20) (7) Ultraviolet absorption spectrum At 220 to 360 nm, no specific absorption is shown.
but only a terminal absorption existsO
(8) Infrared absorption spectrum ~he infrared absorption spectrum of a sample in pOtaSSiUm bromide tablet is as shown in Figure 40 (9) Solubility Very readily soluble in water; easily soluble in methanol; slightly soluble in ethanol; insoluble in chloro-form, ethyl acetate, diethyl ether, hexane and petroleum etherO
(lO) Color reaction ~inhydrin reattion, and Rydon Smith reaction: positive;
Sakaguchi reaction, maltol reaction, ferric chloride reaction and ~ehling reaction: negative (11 ) Stability Stable at a pH of 3 to 8; gradually deco~posed lil7~4~

and inactivated in basicity and strong acidityO
(12) Nuclear magnetic resonance spectrum (~I~ 0 ppm):
1023 (~H, d, C-CHz) Z.09 (3H, s, N-CHz) ZO40 (ZH~ s, 0-CHz) 4O18 (2H, s, C0-CH2-N) 5008 (lH, d, anomeric H) (17~) Mass spectrum (mie):
L~17(M+), Z04-, ~76, 258, 219, 17Z~ 143 (14) Paper chromatography Rf value: 0055 ~ilter paper: Whatman NoO
Solvent: a lower layer of chloroform-methanol-17% ammonium hydroxide (2:1 1)

(15) Thin~layer chromatography , _ _ Rf value Solvent . ~
0.56 Butanol-ethanol-chloroform-17% ammonium hydroxide (4:5:2:5) 0~,66 Chloroform-methanol-17% ammOnium hydroxide 0~16 . A lower layer of chloroform-methanol-17% ammonium hydroxide (2:1:1) Oo76 An upper layer of chloroform-methanol-17% ammonium hydroxide (2:1:1) _ ._ _ _ _ _ . _ TI.C aluminum sheet (silica gel 60 ~254 0.2 mm) (Merck) was used as the plateO
~he Rf values by paper chloromatography of the ~117~

novel antibiotics of KA-6606 I, II, III and IV are shown in ~able 1 below in comparison with those OI known anti-biotics. Similar data obtained by thin-1ayer chromatography are shown in '~able 2 belowO
Ta.ble Rf values of the KA-6606 substances and known antibiotics Solvent system: a lower layer of chloroform-methanol-17% ammonium hydroxide (2:1:1) :Filter paper: Whatman ~oO 1 . _.
Antib1otics Rf value _ ~ __ _ KA-~606 III 0027 Gentamicin Cl 0059 Gentamicin C2 0035 Gentamicin Cla 0012 Sagamicin 0049 Sisomicin Ool2 Verdamicin 0035 G-52 0.49 Fortimicin A 0032 ~ortimicin B 0089 Others (*1) 000 - 0005 . ~

(*1): Others represent Kanam~cin A, B And C, Paromomycin~ Neomycin A, B and C, ~utirosine A and B, 1~17~4~

Lividomycin A and B, Ribostamycin, Xylostatin, Gentamicin A and B, Tobramycin, Apramycin, Sorbicitin, antibiotic sub6tance 460, Hygromycin, or DestomycinO

~ablQ 2 Rf values of the KA~6606 substances and known antibiotics SoIvent~2~tem Solvent I: Butanol~ethanol-chloroform-17% ammonium hydroxide (4:5:2:5) Solvent II: Chloroform--methanol-17% ammonium hydr-oxide (1:8:3) Plate ILC aluminum sheet (silica gel 60 ~254 002 mm) (Merck) Rf value Antibiotics Solvent ISolvent II
_ _ KA~6606 I 0O56 0O60 KA-6606 III 0055 0.64 KA-6606 IV Oo56 0O66 Gentamicin Cl 0052 0040 Gentamicin C2 0O51 0O44 Gentamicin Cla 0O 430O34 Sagamicin 0O45 0032 Fortimicin A 0O53 0O56 Fortimicin B 0 0 0O70 .~
The antibiotics spectr~ of the novel antibiotics KA-6606 I, II 9 III and IV are shown in ~able 3O

- 29 ~

~117~

Table 3 _ _ _ KA-6606 substances . Fortimicin Amikacin ~ I I] Ill IV A
Staph aureus 209P 0.4 2 ¦ 3 6 0.8 0.8 SMITH 0.2 l ¦ 1.5 3 0.8 0.4 B. anthracis 0.2 ¦0.8 1.5 0.8 0.2 cereus 1.5 5 ¦ 6 12 6 1.5 subtilis 0.2 ¦0.8 1.5 1.5 0.4 Micrococcus luteus 0.4 'lO0 __ 6 12 Strept. faecalis 25 ~100 ~25>S0 100 100 E. coli NIHJ 1.5 50 12 25 6 3 K-12 ML 1410 3 >100 >25 50 12 3 K-12 ML 1410 R-81 12 >100 25 ~50 50 12 R-82 3 ~lO0 12 50 12 3 R-101 ) 3 '100 25 50 6 6 Prot. vulgaris OX-l9 1.5 100 6 6 6 1.5 Kleb. pneumoniae PCI-602 1.5 ~100 12 12 6 1.5 Ps. aeruginosa SHIBATA 3 ~100 25 25 12 0.8 #12 0.4 100 1.5 3 1.5 0.4 TI-13 3>100 >25~50 6 1.5 A3 3~100 25 50 12 0.2 K-ll ) 6>100 >25>50 12 1.5 3l5IV) 6>lO 1>25>50 12 25 Providencia sp.V) 1.5 ~10 ¦ 25 50 6 3 Serratia sp. 1.5 10 ¦ 6 12 3 1.5 Mycobacterium smegmatis 607 0.4>10~ ~25 12 0.8 0 8 1~7~

I) 3'-phosphotransferase I
~ phosphotransferase II
III) 2"--nucleotidyltransferase IV) 6l-acetyltransferase V) 2l~acetyltransferase The acute toxicities of the KA-6606 I, II, III
and IV of this invention determined by using mice are as followsO

KA--6606 I KA-6606 II KA--6606 III KA-6606 I~
. _ ~___.......... ~_~ ~___ __ LD 0 ivo 50-100 ~40G ?200 ~ 200 (m 3kg) _ _ ~ . _ _ _. .
scO 200-400 ?1,000 ~800 ~800 _ ~_ ~__ _ ~__ _ According to this invention, there can also be provided an antibiotic composition comprising (1) an effective amount of at least one compound selected from the group con-sisting of the KA--6606 substances of this invention and pharmaceutically acceptable acid addition salts thereof, and (2) a pharmaceutically acceptable diluent or carrierO
~he amount of the compound (1) is, for example, about OoOl to about 99O5% by weight, based on the weight of the compositionO
~he antibiotic composition of this invention m~
be in any dosage forms usually employed, but injec.ting pre-parations and capsules are especially preferredO
Preferabl~ like known water~soluble basic anti~
biotics, an injectable is prepared by filling a lyophilized powder of the antibiotic into a vial, preferably together with a stabilizer, and in use, the contents of the vial ~17~45 are dissolved in a dissolving liquid for admirlistration.
The diluent or carrier includes, for example, liquid diluents such as distilled water for injection and physiological isotonic solution, and solld carriers such as lactose, starch, white sugar, glucose, cr~stalline cellulose, calciu.m carbonate, kaolin, D-mannitol, magnesium meta-silicate aluminate, calcium sulfate, calcium phosphate and bentoniteO Addition of stabilizers such as acidic sodium bisulfite is ~lso preferredO
~he dosage of the antibiotic substance of this in~rention can be suitably selected, and is, for example, about OoOl to about 100 mg/kg/dayO
ThUs~ according to this invention, there can be provided antibiotic compositions for animals other than human, such as poultry, domesticated animals and cultivated fish, and antibiotic compositions for manO These compositions are useful as antibacterial agents having a broad antibacterial spectrum~
The KA-6606 substance of this invention is also useful as a material for producing its derivativesO
~ he following ~xamples illustrate the present in-vention in greater detailO
~xample 1 A culture medium was prepared from ~% of starch, 15% of soybean meal, 005% of corn steep liquor, 002% Of yeast extract, 0O050/o of magnesium sulfate, OoZ% of sodium chloride, 0O~% of calcium carbonate, 0c001% of cobaltic chloride hexahydrate and tap water, adjusted to a pH of 7O0 and sterilized~ ~he KC--6606 strain was inoculated in the 1~17~4~

culture medium and cultivated at 27Co for about 50 hours to form a first seed cu]tureO
Two hundred mil.liliters of the first seed culture was transferred to a 200-liter fermentor containing 100 liters of the same sterile medium as above and 1% of cotton seed oilO ~he cultivation was carried out while agitating at 2~5 ~pm at an air flow rate of 50 liters per minute at 27Co for 4 daysO
After the cultivation, sulfuric acid was added to the culture broth to adjust its pH to 200o ~hen, the culture broth was filtered with the use of Dikalyte (Dikalyte Orient CoO) as a filter aidO A dilute aqueous solution of sodium hydroxide was added to the filtrate to adjust its pH to 800 and passed through a column of a cation exchange resin, Amberlite IRC-50 (NH4+ form) (the effluent was discarded)O
After washing the resin column with deionized water, the active substance adsorbed was eluted with lN ammonium hydroxideO
'~he activity of the eluate was determined by a paper disc method using an agar plate of Bacillus subtiliso The fractions having activity were combined, and concentrated to about 50 ml under reduced pressureO The concentrate was lyophilized to afford 808 g of a crude powder of KA-6606 substanceO
Eight grams of the crude powder was dissolved in 50 ml of distilled waterO After adjusting the pH to 700, the solution w~s passed through a co].umn (~ x 150 cm) of a cation exchange resin, Amberlite CG-50 I (NH~+ form)O After wash-ing with deionized water, the active portions were obtained by gradient elution between 5 ~ of OoOl~ ammonium hydroxide and 5 ~ of lN ammonium hydroxide at a Mow rate of 150 ml~hour~

1~Je ~r~
~ 3 ~17(~4S

and all fractions, e~ch containing ~5 m~, were determlned by the paper di 9C method. Frac-tions containing ingredi-ents corresponding to KA 6606 I, fractions containing in-gredients corresponding to KA-6606 II, fractions containing ingredien.ts corresponding to KA-6606 III, and fractions containing ingredients corresponding to KA~6606 IV were res~
pectively collected, and lyophilized to afford 80 mg of a crude powder containing KA~606 I, 16 mg of a crude powder containing KA-6606 II, 120 mg of a crude powder containing KA--6606 III, and 794 mg of a crude powder containing KA-6606 IV, respectivelyO
~he lyophilized KA-6606 I and KA-6606 II were res~
pectively dissolved in water, and after adjusting the pH to 700, each of the solutions was passed through a column (1 x 150 cm) of an anion exchange resin, Dowex 1 x 2 (OH-form)O ~he active portion was developed with deionized water and the active fractions were combined, end lyophilized to afford 40 mg of KA-6606 I as a pure free base, and 10 mg of KA-^6606 II as a pure free baseO
Separately, 120 mg of the crude powder containing KA 6606 III substance was dissolved in water, and the solution wa8 passed through a column (2 x 60 cm) of silica gel packed with a solvent mixture of chloroform-methanol-17% ammOnium hydroxide (1:8:3) to elute the EA-6606 III with the above solventO ~he corresponding eluted fractions were conce~t-rated under reduced pressure, and lyophilized to afford 70 mg of a white powder of KA-6606 IIIo ~reatment of 794 mg of the crude powder containing EA-6606 IV in the same manner afforded 50 mg of a white powder of KA-6606 IVo ~117~45 ~ ach of the lyophilized products was dissolved in water, and a~ter adjusting the pH to 700, the solution was chromatographed on a column (1 x lO cm) of an anion exchange resin, Dowex 1 x 2 (OH form)9 and the active portion was eluted with deionized waterO ~he active fractions were collected and ]yophilized to afford 12 mg of KA-6606 III as a pure free base, and 8 mg of KA-6606 IV
as a pure free base, respectivelyO
~ ple 2 A culture medium was prepared from 2~/o of starch, 1% of soybean meal, 005/O of corn steep li~uor, 0005/O of magnesium sulfate, 0.3% of sodium chloride, 00~% of calcium carbonate and tap water, adjusted to a pH to 7007 and steri--lizedO The KC-6606 strain was inoculated, and cultivated at 27 CO for about 50 hours to form a first seed cultureO One hundred milliliters of the first seed culture was transferred to a 20-liter jar fermentor containing 10 liters of the same sterile medium as aboveO ~he cultivation was carried out while agitating at 250 rpm at an air flow rate of 10 liters per minute at 27Co for 4 daysO
In the same way as in Example 1, the culture broth was treated, and the desired products were isolated and purifiedO ~his procedure resulted in 20 mg of KA-6606 I
as a free base, 4 mg of KA-6606 II as a free base, 4 mg of KA~6606 III as a free base, and 5 mg of KA-6606 IV as a free base, all in the purified formO

~orty milligrams of the KA-6606 I obtained in ~xample:l or 2 was dissolved in 1 mol of a 4~ aqueous 7Q4~i soluti.on of sodium hydroxide, and the solution was heated for 1 hourO The reaction mixture was dissolved in 100 ml of wa-ter, neutrslized, and passed -through a column (1 x 10 cm) of ~? c~tion exchange resin, Amberlite CG- 50 (MH4+
form)0 The column wa.s washed with 100 ml of deionized water~
and eluted with 1:~ ammo:r~ium hydroxideO Fractions which had an antibacteri~.l activity and showed a positive result in a ni.nhydrin reac-tion were collected, and lyophilized to afford 23 mg of a white powder of KA-6606 II as a free base.
xamPle 4 ~orty milligrams of the KA~6606 III obtained in :E:xample 1 or 2 was dissolved in 1 ml of a 4N sodium hydroxide, and the solution was heated for 1 hourO The reaction mixture was dissolved in 100 ml of deionized water, neutralized, and then p~ssed through a column (1 x 10 cm) of a cation exchange resin, Amber].ite CG-50 (NH4 form). ~he column was washed with lOC ml of deionized water, and eluted with 1~
ammonium hydroxideD ~ractions which showed a positive result in a ni.nhydrin reaction and had an antibacterial activity were collected, and lyophilized to afford 18 m~; of a white powder of KA-6606 II as a free baseO

~orty milligrams of the KA-6606 IV obtained in Exam~?le 1 or 2 was trea1;ed in the same way as in Example As a result 9 26 mg of a white powder of KA-6606 II was ob--tained as a free base.

Claims (13)

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

1. A process for preparing an antibiotic KA-6606 of the following structure Structure (1) wherein R represents a moiety selected from the group consisting of hydrogen, -COCH2NH2, -COCH2NHCONH2 and -COCH2NHCHO; and its pharmaceutically acceptable acid addition salts, which process comprises cultivating an antibiotic KA-6606-producing strain of the genus Saccharopolyspora, isolating the anti-biotic KA-6606 from the culture broth, if required, separating from the resulting antibiotic KA-6606 at least one antibiotic selected from the group consisting of antibiotic KA-6606 I, antibiotic KA-6606 II, antibiotic KA-6606 III and antibiotic KA-6606 IV and, if required, treating the result-ing antibiotic with a pharmaceutically acceptable inorganic or organic acid to obtain an antibiotic in the form of a pharmaceutically acceptable acid addition salt.

2. The process of claim 1 wherein the antibiotic KA-6606-producing substance is Saccharopolyspora hirsuta KC-6606.

3. The process of claim 1 wherein the cultivation is performed under aerobic conditions at a temperature of about 20°C to about 40°C.

4. The process of claim 1 which comprises the further step of treating antibiotic KA-6606 I, KA-6606 III and KA-6606 IV with an alkali or acid to convert it to antibiotic KA-6606 II.

5. Antibiotic KA-6606 of Structure 1 as defined in claim 1 or a pharmaceutically acceptable acid addition salt thereof when prepared by a process according to claim 1 or an obvious chemical equivalent thereof.

6. The process of claim 1 which includes the step of separating from the product of cultivation the antibiotic KA-6606 IV, in which R is -COCH2NHCHO.

7. Antibiotic KA-6606 IV, in which R is -COCH2NHCHO, when prepared by a process according to claim 6 or an obvious chemical equivalent thereof.

8. The process of claim 1 which includes the step of separating from the product of cultivation the antibiotic KA-6606 III, in which R is -COCH2NHCONH2.

9. Antibiotic KA-6606 III, in which R is -COCH2NHCONH2, when prepared by a process according to claim 8 or an obvious chemical equivalent thereof.

10. The process of claim 1 which includes the step of separating from the product of cultivation the antibiotic KA-6606 II, in which R is hydrogen.

11. Antibiotic KA-6606 II, in which R is hydrogen, when prepared by a process according to claim 10 or an obvious chemical equivalent thereof.

12. The process of claim 1 which includes the step of separating from the product of cultivation the antibiotic KA-6606 I, in which R is -COCH2NH2.

13. Antibiotic KA-6606 I, in which R is -COCH2NH2, when prepared by a process according to claim 12 or an obvious chemical equivalent thereof.