CA1041446A - Antibiotics aculeacin group - Google Patents

Abstract

Abstract of the Disclosure A novel antibiotics ative against pathogenic yeasts and fungi designated Aculeacin group antibiotics -Aculeacin-A, -B, -C, -D, -E, -F, and -G --are producted from cultured broth of Asperqillus aculeatus FERM-P 2324.

Description

~04~4~ ~:

; This invention relates to novel antibiotics Aculeacins.
More particularly, this invention relates to Aculeacin group oE antibiotics active against pathogenic yeast and fungi.
We have found thata~gus belonging to gepus Aspe.rgiilus,strain ; No. M 4214 produced unknown antibiotics active against yeas~ such as I Candida albicans and fungi such as pathogenic fungi, and desiynated as j Aculeacins.
The said Aspergillus strain M 4214 has the following ta~onomic prpperties.
.,,j : , ~ A. Macroscopic observation:
. ?,.
Czapeck agar medium:
Growth: rapid, diameter 45 ~ 60 mm. at 26C., in lO days.
Surface of colony: flat; white at an early stage of culture;
light tan t3 gc) - beaver 14 li) according ~',J!~ as formation of coniospore.
Colony partlal1y dusty yello~ish (12- gc) conidial head.
No formatlon of scxerotium.
Surrounding of colony: sli~ht arachnoid.
;Backside of colony: colorless - pearl pink ~3 ca~.
~ ~: : . .. ..
No formatlon of soluble pigment and percolate.
Growth at 37C.: slow; lO - 13 mm. in lO days.

2 Malt extract agar medium~
Growth: rapid, diameter 70 - 72 mm. at 26C., in 10 da~ys.
Surface of colony: flat; white at an early stage of culture~
rose toupe ~5 ig~ aCcording as fo~^mation ~ ~ ;
conidiospore.

Conidia: large amount o~ ~ormatlon.
Surroundlng of colony: more or less arachnoid.
Backside of colony: llght wheat (2 ea).
No formation of soluble pigment and percolate.
Growth at 37C.: 510w; 12 - 14 mm. in 10 days.

3. Potato-glucose agar medium;
Growth rate and condition: simllar to those of on malt extract agar medium.
Growth ~tt 37C.: slow; 12 - 14 mm. in 10 days.
Indicatlon o~ color is based on the lndlcatlon in "Color Harmony Manual", Ed. 1958, publlshed by Contalner Corporation of ~merica. B. Microscopic observation:
Conidial head i6 spherical at earIy stage~ ~ollowed by l~ segmented to several cylindrical. Length of conidiophore is 250 - ~;
; .~ , 1000 ~t, mainly at the range o~ 600 - 1000 y, ~ldth is 10 - 13 ~, light yellowish b~o~n and wall ls smooth and slightly thlck. Thickness is about 1.5 - 2 ~t. Vlslcle is brownt and-spherlcal or nearly spherlcal;~
. ~ ~
diameter 40 - 80~u, mainly 50 - 70 p. Segmata is single lined and size o~ 6 ~ 8 x 3 - b~ ~ is closely in line. Conidio spore ls generally . ,, ::
subspherical or spheroid~l, showing ~ariety o~ form, and size ls 3.5 ; 5~x 3 - 3.5 ~, brown colored and wall is rough. On Czapeck agar medium, light~colored rod-shaped conidio spore o~ 5 ~ 7 x 2 - 3 ~ and smooth ~-wall is ~artially formed together with the conidio spore hereinaboye!
C. C~owth condltion: ~ -G~o~th temperature: 13 ~ 40C. Growth pH: 2 ~ 9.
Optimum growth temperature and pH: 29 - 31C.
~ pH 3 - 5.
3~ ~
Accordlng to the taxonomical studies hereinbefore~ ~-thi~ strain belongs to~~spe~gillus ~ rou~ (re~e~ to Sap. ~ Agrical.
hem., 27, 806 - 809 (1953)~ and The genus ~spergill~s ~28 ~- 331 11965)~ ;~
;: : .
~ _ 2 ;
:

having black brown conidio spore. ~urther this group is differentiated ln two groups accordlng to the single or double llned segmata. This strain belongs to single llned group. At present, fungi having single lined segmata have been known as Aspergillus iaponicus and Aspergillus aculeatus, and since in the Aspergillus japonicus, conidio spore is ,:.
spherical or sub-spherical o~ 3 - 3.5 ~, and visicle is 15 - 45 ~, mainly 20 - 35,u. and in the Aspergillus aculeatus~ conidio spore is sub-spherical or spheroidal o~ 4.5 ~ 5 x 3 - 3.5 ~, and visicle is 35 - lOO~u, mainly 60 - 80~u, therefore this strain is resemble Aspergillus aculeatus in taxonomy. Further the strain Asperglllus .
aculeatus ~TCC 1034 obtained from American Type Culture Collection and this strain re~emble each other ln comparlson~ thereby this strain refered to Aspergillus aculeatus ~ 4214. This strain has been deposited in Institute for Industrial Fermentatlon and Technology, Agency of Industrial Science and Technology, Sapan, and assigned the permanent deposition number as FERM-P 2324. ~lso this strain is deposited in United States Depart~ent of Agricultu~e, Agricultural Research Service, Northern Utilization Research and De~elopment Dlvision, and added to its deposit nu~ber as NR~L.

.
An ob~ect oP the present in~ention is to provide no~el :. i` antibiotics ~culeacins.

~ Another ob~ect of the present invention is to provide ;~ Aculeacin group of antibiotics actlve agalnst pathogenic fungl and ~ yeasts.

' ~ .
~' Further object of the present invention is to provlde - ~:
,~ an industrially advantageous process for the production of Aculeacins.

l~ These and other ob~ects, features and advantages of . 1 ~ :
~ the present inventlon will become more apparent to any person skllled : , J~ in the Drt upon reading the more detailed description set ~orth herein-below~ in co~nectlon with the accompanying drawings. The drawings are ~1 .
~ 3 ~
~ .
, ;, ~ L44~ :
shown as follows:
Figure 1 and 2, Ultraviolet absorptlon spectrum of ~culeacln-A in methanol.
Figures 3 and 4, Ultraviole~ absorption spectrum of Aculeacin-A ln potassium-methanol solutlon.
~ igure 5, Inrared absorptlon spectrum of Aculeacin A.
Figure 6, Ultraviolet absorption spectrum of .
Aculeacin-B in methanol.
Figure 7, Ultraviolet absorption spectru~ of Aculeacin-B
in O.Ol-N KOH~90% methanol solution.
~, Flgure 8, Ultravlolet absorption spectrum of Aculeacin-C
, in methanol.
,'~ Figuré 9, Ultravlolet absorption spectrum of Aculeacin-C
ln O.Ol-N KOH-90% methanol solution. -,-;........................................................................ . .
~, Flgure 10, Ultra~lolet absosptlon spectrum of Aculeacin-D
ln methanol.
Tigure 11, Ultraviolet absorption spectrum of Aculeacin~D
........ ......................... ........................................................... :~. :.
in O.Ol-N KOH-90% methanol solutlon.
Figure 12, Ultra~iolet absorption spectrum of Aculeacin E
``~ 20 in methanol, Flgure 13, Ultravlolet absorption spectrum o~ ~culeacin~
in O.Ol-N KO~I-90% methanol solutlon.
F~gure 14Z, Ultraviolet absorption spectrum of Aculeacin-F
Z~ ln methanol.
~ igure 155 Ultra~iolet absorption spectrum of ~culeacin-~
, . : , . : .; .
in O.Ol-N KOH-9OX methanol solutlon.
Figu~e 16, Ultraviolet abso~ptlon spectrum of Aculeacin-G '~"
in ~ethanol.
igure 17, Ult~aviolet abso~ption spectrum o~ ~culeacin-G
30 ~ in O.Ol-N KOH-~Z0% methanol solutlon.

Figu~e 18, In~rared abso~ption spectrum of ~culeacin-B.
~' ~lgure 19, Infrared absorptlon spectrum of ~culeacin-C.

~`Figure 20, Infrared absorption spectrum of Aculeacin D.
:/. ' :`
~;~Figure 21, Inrared absorption spec~rum of Aculeacin-E.

Figure 22, Infrared absorption spectrum of Aculeacin-F. ~-~
:, . .:: :.:
Flgure 23, Infrared absorption spectrum of Aculeacin-G. ~ -~
.. . .. . . .
i~ ~igure 24, NMR spectrum of Aculeacin-B.
: . ..
- Figure 25, NMR spectrum of Aculeacin-C.

~ Figure 26, NMR spectrum of Aculeacin-D.
.
10Figure 27, NMR spectrum o Aculeacin-E.

~; Figure Z8, MMR spectrum o~ ~culeacln~

Figure ?9, ~MR spectrum o~ Aculeacin-G.
.;, . . .
1 ~igure 30, Schematic chart of ~nino acid analysis of :.-~, . .
Aculeacin-B, -C, -D, -E, F and -G.
- , Figure 31, NMR spectrum of ~culeacin-S.
` Figure 32, kmino acid analysis chart of ~culeacin-A
hydrochloric acid hydrolysate.
Figure 33, Two dimensional thin layer chromatogram of ~ Aculeacin-A hydrochloric acld hydrolysate.
.'j 20Figure~34, High voltage paper electrophoresis pattern o Aculeacin-A hydrochloric acid hydrolysate.

Aculeacin g~oup antibiotics consist of Aculeacin-~, -B, C~-D,~ and -G, which~are produced by cuituring ~spergillus - /-acu}e~tu~ M 4~14 FERM-P 2324, having st~ong antiPungal actl~ity, showing : : ; the~ peak~of ultraviolet absorption at 277 ~u and conta~ning threonine as~a~ino;~acid cotponent.~l~Since the Aculeacin group oE antiblotics are peptide antibioticsj, hereinafter these group of antibiotics will be detignated ~e ~ul-acin~
According to th~s ln~entlon, aculeacins are produced by inoculating~a~stra~n o~ spergillus aculea~us M 4214 ~ERM-P 23~6 in a ~.~4~ 4~, suitable nutrient medium as the same procedure o~ antibiotic production.
The cultlvation of mlcroorganism can be carrlecl out in a number of different ways such as llquid culture or solid culture. In an industrial production, submerged aeration culture inoculated with l - 2 days cultured spore suspension of spergillus aculeatus M 4214 FERM-P 2324 , is preferable.
Nutrient media which are useEul for the production of aculeaclns may include an assimilable source of carbon such as glucose;
an assimilable source of organlc and inorganic nitrogen such as corn ; lO steep liquor, peptone and ammonlum sulfate, and the media further include salts suc4 as phosphate, magnesium or potassium.
The culturing temperature for production of aculeaclns may be changed at the range oP temperature in which the microorganism can grow and aculeacins can be produced, and prefesable at Z5 - Z8C.

. 41 ; "
~7~ The culturing period ls generally 70 - 90 hours, and ~
. ~j , . .
when the culture broth reveal;s highest potency in antibiotic production, ~ - -~the cultivation should naturally be terminated.

~; In cultured broth obtained accordingly, aculeacins are accumulated in mycelia and partially in the outside of mycella.
., .
q 20 Aculeacins Fan be assayed by cup-assay method or paper dlsc-assay method using Candida albicans or Trlchophyton asteroides as test organisms.
Aculeacins can be prePerably and effectlvely ls~olated ~ -from mycelia.
According to a preferred procedure~ the whole broth is flltered to obtain mycellum containlng aculeacins using drum-filter, ~ ilter press or centrifugal separator. The thus obtalned wet mycelia J~ is extracted wlth water mlscible organic solvent such as alcohol and acetone, ~hen ~olvent ln an extract ls distllled of~, diluted with ~ water, further the dlluent is extracted with n-butanol or ethyl acetate 6 ~
, .

\

, . .
and the extract is washed with wate~ and conce~tlated under reduced pressure to obtain oily material. The oily material can be purified by absorption or partition ch~omatography using active alumina, silica gel or the like.
' ~ The physico-chemical properties of aculeacins a~e as follows:
1) Elementary analysis: ' .. . . ... .
C% H% N%
hculeacin -~ 56.38 8.01 9.29 " -B 60.52 8.54 9.46 " -C 59.04 8.27 9.66 D57.958.02 8.83 .. . .
~' -E 57.46 8.01 9.11 54.817.59 9.04 - " -G 56.08 7.73 8.68 2) Molucular weight:

;~ Rast method: kmino acid analysis:

Ac~leacin -~ 1,021 (869) n ... .
i~ " ~B 1145r-1206 (540) n ';,~ 20 -C 1133 -1199 (540) n D 1209~V1284 (555) n -~

E 1195 ~1270 (650~ n -1227 ~1287 (565) n ~-~ ' -G 1255^~1330 (710) n J~ Calculated ~ro~ detected amount o~ threon~ne.

30;~
~ 7 ,.;~ : : . ~.:

3) Molecular ~o~mula:
Aculeacin -B: C58_61H96~104 8 15-16 11 -c C56-59H92-looN8ol6-l7 . `
-D: Csg-62Hg6olo6N8ol9-2o -E: Cs7-6lHg4-lo4N8ol9-2o -F: C56-sgHgo-ggN8o22-23 -G C5~-62H94-106N822-23

4) Melting point: -Aculeacin -A: 164 - 167 C. (fuse) " -B:148 - 151C. ( ~' " -C:164 ~ 168C. ( "
~' -D:159 - 162C. ( "
~' -E:186 - 191C. ( "
" -F:163 - 167C. ( "
" ~G:166 - 170C. ( "
Each component does not show clear meltlng point.
~,i, 5) Specific rotation: 6~7 D (C-l.0 methanol) ;:
Aculeacin ~A : -53 B : -45 ~ 20 " ~C: -47.5 `~
;~ ~' -D -46 ; reBult O~ ~3 23 (C=0.53, methanol) ~ ; ' 6) Ultraviolet absorption spectnlm:
igure 1 : ~culeacin-A in methanol (324 ga~ma/ml.).
,.,: : . ~ : .
J.~ igure~2 : Aculeacin~A in methanol (32.4 gamma~ml.).
Figure 3 : Ac~leacin-A in 0.01-N K0~-90% methanol (162 gamma/ml.-).

,~ 8 _ ,: .
~ , . .
.1 ' .

Figure 4 : Aculeac~n~A in 0.01~N KOH-90% methanol (16,2 gamma/ml.).
~igure 6 : Aculeacin-B ln methanol (40 and 400 gamma/ml.).
Figure 7 : Aculeacin-B in 0.01-N KOH-90% methanol (36 and 360 gamma/ml.).
Figure 8 : Aculeacin-C i~ methanol (40 and 400 gamma/ml.).
.
Figure 10: Aculeacin-D " ( " ).
Figure 12: ~culeac-ln-E " ( " ).
Figure 14: Aculeacin-F " ( " ).
.
Figure 16: Aculeacin-G " ( " ).
Figure 9 : Aculeacin C ln 0.01~N KOH-90% methanol (40 and 400 ga~ma/ml.).
Figure 11: Aculeacin-D
gamma/ml.).
~igure 13: Acuiea~in~E
,'' gamma/ml.,~
' ,:~; !
Figure 15: Aculeacin-F
, gamma/ml.).
Figure 17: Aculeacin-G " ( "
gamma/ml.).
: .
~, .
,, As shown in the ~igures each co~ponent has the maximum .
peak at 277 ~ and shoulders at 226 and 283 m~.
El% o~ the each component are shown in the following ' 20 1 cm ' table.
!: . in 0.01-N KOH - -in_methanol 907O methanol ~
225 m~277 m~ 283 m~ 247 ~ 295.5 ~p _ (shoul_er) (peak) (shoulder) _ I, ~ 145 15.6 13 1~8 21,9 B 144 18,5 15.6 104 21.6 (279~m~)_ (285 ~) _ (297 m~) :
C 137 18.3 15.1 100 20,0 (279 m~? _ (285 m,~) _ _ D 137 17.2 14.3 100 l9.S
~278 m~) (284 m~) . , , ~
132 16.0 13.5 120 21.2 :: : . . _ .. , ;,.
134 14.5 11 0 8 125 19.6 G _ 143 17.4 14 7 _ _ 115 23.0 . :

. ~ _ .. . .. - . . , .. . .. .- - -. - - . . - , . . . . . ; , . ... ~ . ... ~ .. . , ~ .. . . . . . .

10~ 4~

7) In~rared abso~ption spectrum (KB~ t~blet);
Figure 5 : ~culeacln-~.
Figure 18 : ~culeacln-B.
Figure 19 : " -C.
Figure 20 : " -D.
Figure 21 : " -E.

Figure 22 : " -r.
Flgure 23 : ~' -G.
8) Nuclear Magnetic Resonance spect~um:
NMR spectra o~ aculeacins in deuteron dimethyl sulfoxide (D~S0-d6~ at 100 M~lz are shown in Fi~ures 24, 25, 26, 27, 28, 29 and 31 (inner standard: '~MS).
8) Color reactlon~
Shown in the ~ollowlng table.
_Color reaction ~ Aculeacins _ +: pcsitlve~ -~ negative A B C D _ ~ G
Pauli reaction + + + + + + ~ -~
Folin reaction .~ + ~ ~ ~ ~ +
HI04-benæidine reaction ~ ~ + ~ + + ~ ~ , KMnO4 decolorlzatlon reaction ~ .~ + ~ + ~ +
Ninhydrin reaction _ _ _ _ _ _ Sakaguchi reaction _ _ _ _ _ _ _ Ehlrich reactLon _ _ _ _ _ _ satin _ _ _ _ _ _ ;
. : .:
10~ Solubility:
.: .
~ Soluble: lower alrohol. ~ ~
:
~ Slight soluble: ethyl acetate and wa~er.
:
Insoluble: acetone, chlo~oform, n-hexane and pet~oleum ether, ~, ~ -10-: ' .. . .. . . . , .. , , ,, " . , 11) Aculeaclns are decomposed at an alkallne pH and due to di~ficulty soluble, aculeacins are lmmobile at electrophoresis. Aculeacins can not be transferred from butanol solutlon to ~ater at pH 2 - 9.
These may indicate the neutral nature of aculeacins.
12) Color: White crystalline powder.
13) Rf value:
Carrier: silica gel sheet (product of Eastman Rodak Co. Eastman chromagram sheet No. 6060).
Solvent: A: ethyl acetate - isopropanol - water (10 : 2 : 1).
B: chloroform - methanol (10 : 3).
C: ethyl acetate - methanol - water (20 : 4 : 1).
D: ethyl acetate - n-butanol (3 : 1).
Developer: iodine.
bioassay using Candlda alblcans (Aculeacin-A only). ~ ;
Solvent system ~ B C D

Aculeacin-A : 0.47 0.28 0.37 0.520.91 " -C : 0.46 0.87 " -D : 0.35 0.67 I! -E : 0.18 0.58 " -F : 0.15 0.38 ~-" -G : 0.13 0.32 -14) hmino acid composition:
Aculeaclns are hydrolyzed with 6-N HCl, at 110C. ~or ZO hours, and ninhydrin positlve components are analyzed by automatic a~ino acids analyzer. Results are shown in Flgure 30 ~solld line:
absorption at 570 ~p; dotted llne: absorptlon at 440 m~), following table and Flgure 32 (sol~d llne: absorptlon at 440 ~; dotted line:
absorption at 570 ~
. .

. :

T~o dimensional ~hin la~er chromatography is performed by using cellulose plate, with n-butanol~acetic acid- water ~3 : 1 : l) as primary developer and phenol-water (75 : 25) clS secondary de~eloper.
Colored by ninhydrin is shown in Figure 33, in which L-threonine and unknown five ninhydrin positive components are found.
Figure 34 is a result of paper electrophoresis using filter paper (Toyo 11ter paper No. 51, product of Toyo Roshi Co., `
Tokyo), at 2000 ~olts for 30 minutes (pH 1.8 in formic acid-acetic - , ' acid buffer). L-threonine and L histidine are used as control.
L-threonine and five unknown nlnhydrin posltive components are found.
In Flgure 33 and 34, following abbreviatlons are used.
Vlo: vlolet, ~G: yello~ish green, Y-O: orange yellow, D-Vio: dark violet, ~: yellow, Or: orange, Thr: L-threonine and His: ~-histidine.
In Figure 32, 33 and 34 are shown the result of hydrolysis of Aculeacin-A.

-: ' ' ' ' '';
,:: :. .' ' ' :

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

_ 12 ~
-:

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~L~4~ 6 ;-~ l F
~ P ~ ~ ~ " ~ ~ ~ ~
~ ~ E~ E~ E~ E~ E~ E~ E~
oo ~ ~
~ ~ l l l l ~ l l ..
....
P ~ + + + + + + +
P ~ l ~ I I I I .1 N _ , _ ~ ~ ~ 00 c~
~ t~ ~ ~ l l + + + +
00 _ _ _ _ _ ~
~ ~ + + .~ .1. + + +
_ __ _ ..
,, ~ ~ ~4 ~ + + + I I I ., _ __ _ __ ,_ ''' "
~ ~ ~ I I l l l ~ 1 _ _ I ~ ~ .
.. 0~ ;r~ ~ l ~1~ l I l l +
_ _ _ _ r~ . ~ t + + + + + + O h _ _ _ _ ~ l,q ~ P ~ ~ l l + + + +
_ _ _ ~J ~o3 :. ~, .
P ~ l ~ t l l l ~ U~
_____. _~ _ __ ~ 30 "
~ . 'C F~ I~ ~ ~ ~ C~ ~ 0 .~ ~ _ _ _ _ ~ y ~d ~:; ~rl .~.- ~ p ~ a) o o . ~ ~ : td su~ n~ ~ o o ~ ~
. ~ ~1 ~0 h O ~ .
c~ ~! _ ~ c~ +
. _ ' ' ' ' ', ', ., ~L~4~46 Aculeacins are peptlde antibiot~cs slightly soluble in ~ater in accordance with the physlco-chemlcal properties hereinabove.
kmong the antibiotics hetherto reported, having ultra-violet absorptlon maximum at 277 mu ln methanol, myrorldin (Japanese Patent Publ. 45-12276)9 athlestatln (3apanese Patent Publ. 41-12668), monilin (Takeda Institute Annual Report, 14, 8 - 10 (1955)] , oryzamycin (Japanese Patent Publ. 38-2800), saramycetin ~Antim~crobial Agen~s and Chemotherapy, 1961, 436 - 444), unamycin r3. Antibiotics, Ser. A, 13, 114 - 120 (1960)~, and vengiclde (British Patent 764,198) may resemble aculeacins. Aculeaclns are howe~er dlfferent from those antibiotlcs as follows.
Myroridin ls a basic water soluble antlbiotic which dlffer from aculeacins kthlestatin is similar to aculeacins in its physico-ehemical properties and biological property, however ~olecular formula of athlestatln -1s C32H58N5012 and ultraviolet absorption maxi~a of athlestatin are at 278 and 225 ~ showing difEerence from aculeacins.
Monilin is a basic nucleoside substance and nitrogen content in elemental analysis is higher than that of aculeacins. Oryzamycin ls an aeldic oily substance. Saramycetin is shown the aspartic acid, cystine, glycine and threonine ln hydrolysis thereof, and unamycln ls an acidlc polyene substance. Venglclde is simllar to monilin, a nucleoside s~bstance.
s illustrated hereinbefore~ aeuleaeins are new and no~el antibiotics diferent ~rom the antibiotics hetherto known in ~he prior arts.
Biological properties of aculeac~ns are as follo~s.
1. Acti~ity aga~nst bacteria: -Assay medium: glucose bouillon agar~ at 370C.? ~or 18 hours.
: :..
;~ 30 - _ 14 _ ..
' .::.' ' ' '' ~)4~L44~
Almost no actlvlty against bacte~ia such as Staphylococcus aureus, Bacillus subtills, Sarcina lutea or the lIke.
2. Acti~ity against yeasts:
Assay medium: Sabroud agar, at 3() C., for 48 hours.
Results of minimum lnhibitory concen~ration are shown in the following table.

' . '~ ' ~,, ' ,.

' ' ' ' ' ' :

;

.,.
: : .
.: . ' ' : ::

- : : ' : .. ' .

~ 15 .:

~ 446 ::

-- . o o o o o o o o ~ o ~ o o o h J\ :
: . ,' F4 o o o o o o o ~ o ~D ~ ~ O O g ' ' '' . . ...
o . . . . ... . o ~ ' ;
~D ~ ~~ ~ ~ ~ O O O U~ O O O ~
U~ ,, ", U~ "~ ,,, ~ , '. . :,, U~ o o o o o o o. ~ ~ o . /\ /\ ' ~.', U~ . , .~1 ~1 , ~ O ~ ~ O
:~ . O O O O O O O _l O ~ ~ ~ O O O ~
, /~
, ' ' ~ ~
: , . : ~ -.
~: F~ ~ ~ ~ ~ ~- ~ ~' ~ O ~C`l ~ C`~ O
. ~ , O O O O O O ~ Q O ~ D O O O
~: ': ' J~
; ~ ~ ' ~C
O O O O ~ O O ~ O', O ~1 ~ O O , 0 ~ O O O
. \f \/ \~ O ~-':
~: ' U : Ul: ~-1 h Q~ ~ ~ O u3 : h : : ~ ~ V V U~ ~, U3 F~

: ~ : ~ ~U3 U3 t~ ~r~ ~ r~
: ~: :: ~ ::1 h o~ c) ~ ~rl :1 ,LI rl r-l ~ d rd o ~d o ~ ~ ~ :~

, 3. Acti~ity against fungi:
Since aculeac~ns have no ~ungi-cidal activity, fungi are inoculated in Sabroud agar medium containing several concentrations of aculeacins, and incubated at 26C. 7 to 14 days. 80% growth inhibitory concentra~ion of aculeacins are measured by the ratio of area of giant colony and that of control without aculeacins.
I. 80Z growth inhlbitory concentration (gamma/ml.) of '' aculeacin-A.
1) dermatopathogenic fungi: ,~

Tr-lchophyton asteroides 0.004 ,.....
Trichophyton ru~rum 0.006 Mlcrosporum gypseum 0.008 i1) phytopathogenic ungi:
Fusarium oxysporum ~. lini 2.0 Cercospora kikuchii 4.0 , Corynespora cassicola , 2.0 Diaporthe phaseolorum 0.01 'Sclerotium bataticola 0.08 ~scochyta soyaecola 0.08 -~
Collectotrlchum linicolum 0.4 .
Glomerella cingulata 0.06 , , ,,:
'Helmin~hosporium'~y~ 0.008 II. 80% growth inhibltory concentration of aculeacins.
-- ' Aspergillus Trichophyton fumlgatu~ asteroides (ga~ a/ml.~ (ga~ma/ml.

Aculeac~n-B 7.0 0.04 C 0.7' 0.01 ,, ,, D 0.4 0.005 E 2.0 ` 0.003 ~-:~ .
~ tl_~ 0.6 0.0~3 '"~' G 0,7 0.005 ~: . : :.:
0.5 0.004 . .
~17 ~

^

~4~ 6 4. Acute toxiclt~ on mouse: ' Aculeaclns are almost insoluble in water, soluble in 1%
sodium carboxymethyl cellulose (CMC) solution and sodium deoxycholate (DOC~. LD50 of aculeacin-A in~ection prepared by using those addittves is as follows.
~oute of administration LD50 (mg./kg,) i.p. 600 (C~C) i.m. 600 (DOC),~3460 ~CMC) --l.v. 350 (DOC) p.o. ~ 3000 (CMC) In"( )~' shows additives used.
Intra peritoneal admlnlstrat~on of aculeaclns, suspended in 0.5% carboxy methyl cellulose solution, on mice at 300 mg./kg. ls shown as follows. The ddy strain mice, male, welghed 20 g., four in one group are used.
Number of death - ::
' after 24 hours 48 hours 72 hours Aculeacin~B O 0 O

-G O O O

~' -D O O O

E O O O

F O O ~ O

G 1 ~ O

~ . :

5, Experimental chemotherapeutic effect ln mice~
Mice ~n~ected with strong pathoge~ic Candida albicans ~-were treated with acu1eacin-~, Results are illustrated as follows~
showing~effect of treatment.

:. :

4~;
Rou~e . of infectiQn , ,,,R~ute o~ admlnist~ation ,C~5~,~mg,/kg-) i.p. i.p. 2.0 ' ~' i.p. i.m. 18.0 i.p. p.o. 1~60 ~ 2 ,, i.v. i.p. 16.0 x 2 i.v. i.m. 24.0 x 2 '~oIlowing examples are only for illustratlon o the process and products of the present invention but are not to be construed as limiting. '~

Example 1.
One liter of aqueous medium consist~ng o~ glucose 2%, ~ ' polypeptone 1%, corn steep liquor 1%, KH2PO~ 0.2% and MgS0~ 0.1% were equally divided and introduced ~nto ten 500 ~l. Erlenmeyer flasks ~, (pH 6.5)~ sterilized at 120~. for 15 minutes. spergillus aculeatus -4214 FEEM-P 2324 is inoculated therein and shake cultured at 26C.
~ . .. .
for 48 hours. The cultured broth was inoculated into 180 l. of sterilized medium o the same content, hereinabove in 25p l. tank and . : . . , submerged cultured at 26,C. for 15 hou~s with agitation 250 r.p.m., ', 20 ~ aeration 180 l./min. 60 l. of the cultured broth we~e aseptlcally '~
inoculated in 2200 l. of mediuM (pH 6.5) consisting o~ saccharose 1.5~
dextrln 1,4%, polypeptone 2%, corn steep liquor 0.45%, KH2P04 0.2%, r ' ~ ;~",' ,""
MgSO4 0~.1% and anti-foamer 0.1% in 2500 l. capacity main ~ermenta~ion tank, and ermented~at 26C. ~or 90 hours at aeration of 1100 l./min. ''' ' "
and~agltatlon of 200 r.p.m. to obtain 2000 1. of fermented broth. ~ :

'The bxoth we~e asRayed 22 ga~a/~l. in filtrate and 180 gammalml. of ,~, ' aculeacins in mycelia. ; ' ~ ', Cultu~ed b~oth (2000 l.) obtained in Example 1 ls :: -.
~ . : . :.: . .
~ 30 `,~ t~eated~b~ centri,fuge ~o obtaln wet mycella (130 kg.). To the wet .. ..
~ ' ': ' ` - :
~414~6 :
mycelia was added 400 1. of ~ethanol~ extracted under stirring ~or 18 hours, and 435 1. of methanol solution was obtalned by centrifuge.
Residual ~ycelia was again extracted with methanol (200 1.) to obtain 185 1. of methanol extract. Both extracts were combined (containing 265 g. of aculeacins), distllled off the methanol under reduced pressure to obtain 50 1. of concentrate. Thereto was added 50 1. of water and extracted twice with n-butanol each of 50 1. The n- butanol layer was collected (containing aculeacin 201 g.). ~fter the n-butanol layer was washed with water, the butanol was concentrated with adding small amount of water upto 22 1. 550 g. of actlve carbon was added therein -and filtered, then concentrated in vacuo to obtain 5 1. of dark brown colored viscous concentrate. The concentrate was charged on a column of actiVe alumina (10 1.). The column was washed with 20 1. of ethyl acetate, thereafter eluted with ethanol, collected each 5 1. of fractlon. Active fractions No. 3 - 13 assayed as active were combined and treated with active carbon (100 g.). After removal of carbon, filtrate was concentrated upto 500 ml. (oily substance). This oily substance was charged on a column of silicagel (15 1.) packed with ethyl acetate-methanol (10 : 1), and developed with the same solvent (40 1.) to remove fatty impurlties. Thereafter each 2 1. fractions eluted with ethyl acetate-methanol (5 : 1) were collected from fraction No. 3 to 15. The collected fractions were combined and concentrated ln vacuo to obtain yellowish powder (51 g.). The powder was dissolved in methanol (200 ml.~, and thereto was àdded ethyl acetate-n-hexane (1 : 1) (1 lit.) gradually to preclpitate the aculeacins, which was collected by filteration ànd dried in vacuo to obtain 41 g. of white powder (purity 68%).
Example 3 1.7 g. of crude aculeacins obtained in Example 2 dissolved ln 5 ml. of n-butanol was charged on silica gel column packed .

~ 20 ~4~46 with water satu~ated solvent o~ ethyl acetate~n~butanol (4 : 1). The column was eluted with the same solvent and ~ractionated in each 10 g.
fractions. Each fractions were assayed by bio-assay and thin layer chromatography to find the activi~y on No. 33 to 55 fractions. Vacuum dryness gives 629 mg. of white powder.
The powder was further purifled by dissolving n-butanol (50 ml.), washed three times with distilled water (each 10 ml.) and dried in vacuo. 577 mg. of purl~ied aculeacin A was obtalned.
Example 4.

- The fermentation in Example 1 was repeated three times to obtaln cultured broth 5.6 ~1. This broth was treated with centri-fugal separator to obtain 450 kg. oP wet mycelia. To the mycelia were added methanol (1.2 kl.), extracted with stlrring for 20 hours, and centrifuged to obtain methanol extract (1.3 kl~). Mycelia were again extracted with methanol (1 kl.). 850 1. of this extract were combined , .:
with the first extract and concentrated in vacuo upto 150 1. Thereto was added 150 1. o~ water, then extracted with n-butanol (150 1.).

- ~ Active carbon (2 kg.) was added into the separated n-butanol layer.

Decolorized solution was concentrated azeotropically to obtain dark :, . . .
~ 20 brownish viscous concentrate (6 1.). 40 1. of n-hexane were added :.
therein, collected the precipitate by centr~fuge, washed with n~

hexane to obtain dark brownish aculeaclns crude powder (847 g.).

After washing with ethyl acetate (3 1.-), ~his crude powder-was dis-; solved in methanol (l 1.), added ethyl acetate (10 1.) with stirrlng ~: .: ~ ~ : . , -, and prec~pitated Materlal was separated by centrlfuge. ~ter washlng with ethyl acetatep the powder was dried in vacuo to obtaln light brown crude aculeacins powder (369 g.).
ample 5,~
Crude aculeaclns powder obtained ~n ~xample 4 (123 g.) ~; 30~ ae~d~lssolved in n-butanol (200 ml.) and charged on a colu~n of sillca ~ . . .

gel packed ~ith mixed sol~ent o~ eth~l acetate-n~butanol-water (10 : 2 : 1), then developed with the same solvent mlxtu~e. Each 1 liter fraction was assayed by bio-assay and thln layer chromatography. Tractions No. 4 to No. 25 contalning aculeacin-B, -C and -D (fractlon I) which was eluted prior to aculeacln-~ and No. 44 to ~o. 60 containing aculeacin-E, -F and -G (fraction II) which was eluted after aculeacin-A ~ere collected and concentrated in vacuo.
~ raction I (23.4 g.) and fraction II ~S.7 g.~ were obtained as powder. Remainlng crode powder in Example 4 was treated as the same procedure to obtain the powder of fraction I (70 g.) and fraction II (17 g.). --Example 6.
Fractlon I powder (70 g.) obtained ln Example 5 was dissolved in isopropanol (100 ml.) and charged on a column of silica gel packed with mixed solution of ethyl acetate-isopropanol-water (10 : 1 : 0.5), then developed by the same solvent~ Each S00 ml. of .
fractions were checked by thin layer chromatography and collected the ractions No. 22 to 39. The comblned fractions were concentrated in ; vacuo to obtain puri~ied fraction I (14.5 g.) The powder was dissol~ed ln methanol ~15 ml.) and charged on a column of silica gel packed with chloroform-methanol (10 : 1), then developed by the same solven~. Each 20 g. fractions ; were collected.

Frac~ion No. 275 to 310 contained aculeacin-~ were .~ .
collected and con~entrated in vacûo to obtain white powder ~495 mg.).
Purified powder (447 mg.~ of aculeacln-B was obtained ~y dissolving -ln m~botanol (40 ~ , washlng twice ~lth water (10 ml.) and concentration.
.
Fractlon Mo. 311 to 340 were collected and concentrated ~`
to obtain a mlx~u~e o~ aculeacln-B and -C. (1050 mg.).

22 _ :

~4~ 6 ~ raction No. 341 to 365 were concentrated to obtain aculeacin-C powder (995 mg.). The powder was dissolved in lsopropanol (4 ml.) and charged on a column o~ silica gel packed with ethyl acetate-isopropanol-water (lO : 1 : 0,5) thereafter developed with the same solvent.
Fraction No. 46 to 80 each 20 g. contalned aculea~ln-C. The fractions were collected and concentrated in vacuo to obtaln white powder (610 mg.). Puri~ied aculeacin-C powder (522 mg.) was obtained by dissolving n-butanol (40 ml.) and washlng twice with wa~er thereo~.

Fractlon No. 531 to 625 were collected and concentrated -ln Vacuo to ohtain aculeacin-D powder (1200 mg.).
The powder was dlssolved in lsopropanol (5 ml.j and charged on a column of slllca gel packed wlth ethyl acetate-isopropanol-~=ter (10 : 1 : 0.5). Development was carrled out by the same solvent to fractionate each 20 g. Traction No. 70 to 120 which contained aculeacin-D were collected and concentrated in vacuo to obtain white ~ ;-powder (355 mg.). The powder was puri~ied by dissol~ing n-butanol (40 ml.) and washing with water. Pur~fied aculeacln~D powder (313 mg.) `
was obtalned.

Example 7~
. .
Powder (17 g.) of Eraction II obtained ln Example 5 was dissol~ed in n-butanol (40 ml.) and charged on a column o~ ethyl acetate-n-butanol-water (lO : 2 : 1). Development was carried out by ~ . .:
the eame so1vent. Each 500 ml. fraction was collected and assayed.

Traction No. 17 to 23 contained aculeacin-E and ~, and No. 2~ to 31 contained aculeacln-G.

Each fractlons were collected, treated to obtain mixed ~ ,j ~powder of aculeacin-E and -F (330 ~g.) and aculeacin~G crude power i , : ~ . :
(800 mg.}.

~:
23 , :'. ~ . " '.:',.
, 4~6 The abo~e mixture o~ Rculeac~n~E and ~F was dissolved in methanol (6 ml.), charged on a column of silica gel packed with chloroform-methanol (10 : 2) and developed with the same solvent to fractionate each 20 g. fractions. No. 135 to 175 fractions contained aculeacin-E and obtained the white powder ~175 mg.). This powder was di$solved in 20 ml. o~ n~butanol, washed twice with water (S ml.) and concentrated in vacuo to yield purified aculeacin-E powder (155 mg.).
Fraction No. 241 to 320 contained aculeacin-F. White powder (903 mg.) was obtained by concentration. Purified aculeacin-F
is yielded as white powder (872 mg.). Aculeacin-G crude powder was puriPied by dissolving in methanol (3 ml.) and chromatographed by chloroform-methanol (10 : 2) mlxture on sillca gel column. Fraction of each 20 g. No. 181 to 240 contained aculeacin-G. ~oncentration resulted white powder 408 mg. This powder was purified as the same procedure hereinabove to obtain purified aculeacin-G powder (334 mg.) .:
:

- ~
, ~ .
.~ , .
, :

:

. ~'.

Claims (16)

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

1. A process for the preparation of an antibiotic, antifungal compound selected from the group consisting of aculeacin-A, aculeacin-B, aculeacin-C, aculeacin-D, aculeacin-E, aculeacin-F and aculeacin-G which comprises innoculating a strain of Aspergillus aculeatus M4214 FERM-P2324 in a nutrient medium, terminating the cultivation when the highest potency in antibiotic production is observed and isolating the required aculeacin.

2. A process as claimed in Claim 1 wherein the required aculeacin is aculeacin-A.

3. A process as claimed in Claim 1 wherein the required aculeacin is aculeacin-B.

4. A process as claimed in Claim 1 wherein the required aculeacin is aculeacin-C.

5. A process as claimed in Claim 1 wherein the required aculeacin is aculeacin-D.

6. A process as claimed in Claim 1 wherein the required aculeacin is aculeacin-E.

7. A process as claimed in Claim 1 wherein the required aculeacin is aculeacin-F.

8. A process as claimed in Claim 1 wherein the required aculeacin is aculeacin-G.

9. Antifungal, antibiotic aculeacin compound selected from the group consisting of aculeacin-A, aculeacin-B, aculeacin-C, aculeacin-D, aculeacin-E, aculeacin-F and aculeacin-G whenever prepared by the process of Claim 1 or an obvious equivalent thereof.

10. Aculeacin-A whenever prepared by the process of Claim 2 or an obvious equivalent thereof.

11. Aculeacin-B whenever prepared by the process of Claim 3 or an obvious equivalent thereof.

12. Aculeacin-C whenever prepared by the process of Claim 4 or an obvious equivalent thereof.

13. Aculeacin-D whenever prepared by the process of Claim 5 or an obvious equivalent thereof.

14. Aculeacin-E whenever prepared by the process of Claim 6 or an obvious equivalent thereof.

15. Aculeacin-F whenever prepared by the process of Claim 7 or an obvious equivalent thereof.

16. Aculeacin-G whenever prepared by the process of Claim 8 or an obvious equivalent thereof.