JPH0196189A - Novel antibiotic sf2446a2 substance, sf2446a3 substance, sf2446 b1 substance, sf2446b2 substance and sf2446b3 substance - Google Patents

Abstract

NEW MATERIAL:Antibiotic SF2446A2, A3, B1, B2 substances and salts thereof. SF2446A2 substance, color and shape: redaish brown powder. Elemental analysis (%): C 58.10, H 5.26, N 1.90. Molecular formula; C34H35NO15. Mass spectrum (FD-MS): m/Z 697 (M<+>). Melting point: 180-184 deg.C (decomposition). Color reaction: positive in 10% sulfuric acid and molybdic acid reagent. Solubility: soluble in methanol, chloroform, ethyl acetate and acetone, insoluble in water. Weakly acidic substance. USE:An antibacterial agent against Gram-positive bacteria and Microplasma. PREPARATION:A strain such as Streptomyces sp. SF2446 (FERM P-8980) belonging to the genus Streptomyces is cultivated preferably by submerged culture at 26-30 deg.C for 1-8 days.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to novel antibiotics SF244682, SF24
4683 substances, SF2446B1 substance, SF2446B
2 substance and SF2446B3 substance.

BACKGROUND OF THE INVENTION Conventional techniques and problems to be solved by the invention In the past, many antibiotics have been invented and discovered, and put into practical use in the fields of pharmaceuticals for humans, pharmaceuticals for animals, agricultural chemicals, and the like. However, there are still many medical and industrial fields that remain unsolved because no effective substances have been found.

For example, - Infectious diseases caused by common bacteria and mycoplasma are extremely troublesome diseases that can occur not only alone but also in combination with pond bacteria. There is always a need to provide material. The present inventors have focused on the above-mentioned points, and aim to solve the problems by providing a new antibiotic and establishing a method for producing the same.

Means for Solving the Problems The present inventors first cultivated a specific strain belonging to the genus Streptomyces, thereby creating a substance in the culture solution that has a strong growth-inhibiting effect against various bacteria and mycoplasma. They discovered that it was produced and accumulated, and succeeded in collecting its effective substance, naming it substance SF2446 [Patent Application 1986-311156 (filed on December 19, 1986)
1゜The present inventors found a new effective substance in the culture solution of the same strain, SF2446A2 substance, SF2446A
3 substances, SF2446B1 substance and SF244611!
Two substances were named.

In addition, a new antibiotic was obtained by treating the SF2446B1 substance or the SF244611i2 substance with an acid, and this was named the SF2446B3 substance.

These substances of the present invention have the following physical and chemical properties.

[1] SF2446A2 substance (1) Color and shape: Reddish brown powder (2) Elemental analysis value: C58, 10%, ) I 5,26
%.

N 1.90% (3) Molecular formula: C34H35NO15 (4) Mass spectrum (FD-MS): m/z 697 (M
+) (5) Melting point: 180-184℃ (decomposition) (6) Ultraviolet and visible absorption spectra (Figure 1) λ
max nm (E!ffl,) [MeOtll: 216 (648), 22Ss
h (494)-251(448), 270sh (3
70).

300sh (211), 417 (85).

[0,1811CI-MeOH]: 217 (6
10), 227sh (475),.

250 (451), 270sh (376).

300sh (211), 415 (92).

[0,IN NaOH-Me111]: 213
(1210), 240sh (535).

300sh (212), 574 (123) (7)
Infrared absorption spectrum (Figure 6) (KBram')
: 3430.2920.2820.1720°168
0.1650.1B15.1590°1560, 151
0.1460.1410°1360, 1300.126
0.1210°1160, 1110.1095.105
0°955, 940.890.870.810° (8)
1HNMR spectrum (400MHz, CDC)
l5) (Figure 11) δ (Ilpm): 1.22 (3L d) + 2
．． 32 (3L br S)+3.03 (LH, br
d), 3.14 (IH, dd).

3゜35 (IH,br d), 3.42 (3H,5
)-3,48(3H,S), 3.54(IH,dd
).

3.57 (38,s), 3.60 (38,s).

' 3.60 (IH, dc+), 3.87 (I
H, dd).

4.39 (1 skin br ddd).

4.92 (IH, d), 4.97 (IH, dd)
.

5.25 (1)1. dd), 5.96 (1)1
．． brs).

6.19 (IH, s), 6.49 (IH, br
s).

6.84 (IH, d), 8.47 (IH, s).

11.85 (ill, s), 14.26 (IH,
5(9) 13CNMR spectrum (100 Mtlz
, CDCl5) (Fig. 16) δ(ppω): 196,5! 3t 190,2
St 188.8 s.

179.5 s, 171.9 s, 162.5 s
.

160, I St 147, 3 s, 143. I 5
s142.7 St 140,5 St 133,9
s.

124.1 d, 124. Os, 120. Os.

118.7 S, 116.7 d, 109,75t
105.3 d, 34.6 s, S3.2 d.

79.8 d, 79. IS, 77.5 d.

70.9 cl, , 68,6 d, 62,8 d.

60.8 or 5!3,9 qw 52.6 qw5
2.2 q, 38,1 t, 23.9 Qt17.
8 a (10) Color reaction: Positive for 10% sulfuric acid and molybdic acid reagents.

(11) IFH'l: Soluble in methanol, chloroform, ethyl acetate, and acetone.

Not soluble in water.

(12) Thin layer chromatography: Carrier silica gel plate (Merck & Co., Ltd.) developing solvent system
Rf value hexane-acetone (2:1) C), 2
7 Chloroform-methanol (15:1) 0.4
0(13) Distinction between basic, acidic, and neutral: Weakly acidic substances [
11] SF2446A3 substance (1) Color and shape: Reddish brown powder (2) Elemental analysis: C59.04%, H4.12%.

N 2,53% (3) Molecular formula: C26H21NO□1 (4) Mass spectrum (FD-MS): +++/z 523
(M+) (5) Melting point: >220°C (6) Ultraviolet and visible absorption spectra (Figure 2) λ1
Ilax r+m (E j style) [MeOH]: 214 (587), 228sh
(461).

258sh (321), 272sh (270).

300sh (185), 412 (48)-[0,I
N IIcI-Neo old: 216 (512), 2
30sh (400).

255sh (321), 275sh (260).

302sh (189), 410 (63).

[0,IN NaOH-MeO old: 214 (11
60), 232 (465).

300sh (193), 460sh (31).

(7) Optical external absorption spectrum (Figure 7) (KBr cm
'): 3430.3400.2940.1720°1680, 1650.1625.1590°1465,
1410.1360.1310°1265, 1220.
1160.1120°1090, 1050.1000.
960°940, 900. ! 1li75.815.
800(8)1H NMR spectrum (400Mllz,
CDCl5) (Figure 12) δ (ppm): 2.40 (3L br sL
3,34 (IHt br dL3.40 (3H,s
), 3.57 (ltl, dd) - 3,85 (3H
,s), 4.68 (IH,br d)=4.97
(IH, dd), 5.24 (IH, br s).

5.58 (2L' br), 5.95 (IL s
)+6.54 (IH, br s), 8.20 (I
H,s)'.

12.10 (IH,s)s 14.32 (1 skin 5)
(9) +3c NMR spectrum (100M) 12-
CDCl5) (Figure 17) δ (ppm): 196.3 St 190. OS
t 188,75t179.7 St 172. IS
, 162.3 s.

160, I St 149. Os, 143.3 s.

142.7 s, 140.0 s, 133.9 s
.

124.3 d, 123.9 S, 119.7 s
.

118.8 S, 116.2 d, 109.9 s
.

104.3 d= 84.63.78, 85t62.8
d, 52.6 Qt 52;3 q=38°Ot+
23,9 q (10) Color reaction: Positive for 10% sulfuric acid and molybdic acid reagents.

(11) i8MH1: Soluble in methanol, chloroform, ethyl acetate, acetone, insoluble in water.

(12) Thin layer chromatography: Carrier silica gel plate (Merck & Co., Ltd.) developing solvent system
RE value upper
2446B1 substance (1) Color and shape: Reddish brown powder (2) Elemental analysis value: C59.04%, )l 4.12
%, N 2.53% (3) Molecular formula: C34H21NO11 (4) Mass spectrum (FD-HS): I11/'Z 682 (
MH”) (5) Melting point: 188-192°C (
Decomposition) (6) Ultraviolet and visible absorption spectra (Figure 3) λmax nm (Ej!,) [MeOH]: 217 (624), 230s
h (465)=252 (461), 275sh
(341).

305sh (170), 419 (78).

[0, IN IIcI-MeO old: 218 (58
7), 227sh (461).

252 (473), 270sh (330).

300sh (191), 419 (87).

[0,IN NaOH-MeO)1]: 215
(1230), 246 (526)-3QO (195
), 589 (129) (7) Infrared absorption spectrum (Figure 8) (KBr crn'): 3450.337
0.2940.2830°1720, 1675.165
0.1620°1560, 1510.1465.140
5゜1365, 1310.1260.1240゜121
5, 1160.1130.1110°1085, 105
0.1110.980°950, 920.885.86
0.815°(8)1HNMR spectrum (400M)
1z, CDCl3) (Figure 13) 1 (ppm): 1.35 (3)I, d),
2.24 (1)1. ddd).

2.33 (38,br s)t 2,63 (ltl
,brd).

2.76 (IH, dclcl), 3.08 (IH
, ddd).

3.14 (18,ddd), 3.14 (IH,d
d).

3.23 (3H,s), 3.34 (1 skin dq)t
3.61 (3)1. s), 3.70 (1)1.
dd) -3,75 (IH9br ddd), 3.80
(3tl, s).

3.82 (3H,s)t 4,69 (IH,dd)
.

4.71 (ltl, s) - 5,86 (ltl, s
).

6.49 (IH,br s)t 6,84 (III
, d).

8.21 (IH,s)t 12.04 (IH,5)
=14.15 (IH, 5) (9) 13CNMR spectrum (100MHz,
CDCl5) (Figure 18) δ(pp+o): 197.7 St 190.3
91188,7S.

179.4 St 172. I St 162.4 s
.

160.2 St 147.1 s, 145. I 5
t142.4 S, 140.9 S, 133.7
S.

124, L d, 124. OS, 120.95t1
18.43 = 116.4 d, 109.4 S.

104.2 d, 87.1 s, 82.8 d.

79.6 d, 79.2 d, 77.95t75.
2 d, 73.3 d, 62.5 q.

61.2 Qt 52J qt 52,2 Q+26.
7 t+ 23. S q, 18,4 L+IS, Oq (10) Color reaction: Positive for 10% sulfuric acid and molybdate reagent.

(11) Monolysis: methanol, chloroform,
Soluble in ethyl acetate and acetone, insoluble in water.

(12) Thin layer chromatography: Carrier silica gel plate (Merck & Co., Ltd.) developing solvent system
Rf value hexane-acetone (2:1
) 0.43 chloroform-methanol (15:1) 0.58 (13) basic, acidic,
Neutral distinction 2 Weak acidic substance [IV] SF2446B2 Substance (1) Color and shape; Reddish brown powder (2) Elemental analysis value: C59.04%, H4.12
%, 82.05% (3) Molecular formula: C34H21NO11 (4) Mass spectrum (FD-HS): m/z 681 (M”)
(5) Melting point: 177-181℃ (decomposition) (
6) Ultraviolet and visible absorption spectra (Figure 4) λma
x nm (13 [MeOHl: 217 (617L 22Ssh
(470).

250 (455), 270sh (355).

300sh (19F), 418 (84).

[0, IN HClHe0tll : 218 (5
86), 227sh (461).

251 (464), 270sh (370).

305sh (181), 416 (85).

[0,IN NaOH-MeOIl]: 214 (
1180), 246 (511).

300 (195), 585 (128) (7) Optical external absorption spectrum (Figure 9) (KBr c+++')
:3430.2930.2330.1720°1680
, 1650.1620.1590°1560, 1515
．． 1465.1405°1365, 1310.1265
．． 1240°1215, 1165.1100.1040
゜1020, 995.950.920.885゜810
, 800 (8) 1H NMR spectrum (400 Hilz, C
DCI 3 ) (Figure 14) δ (ppm): 1.27 (3H, d), 2.
26 (IH, ddd).

2.37 (3)1. br s), 2.65 (I
H, d).

2.77 (11(,ddd), 3.08 (ill
, ddd).

3.10 (IH, dd), 3.12 (IH, dd
d).

3.25 (3fl, s), 3.50 (3H, d
q).

3.55 (3 days, s), 3.56 (3H, s)
.

3.68 (ltl, dd), 3.80 (3H,
sL4.02 (IH, ddcl), 4.86 (l
tl, 5)-5,19 (IH, dd), 6.19
(IH, 5) = 6.40 (IH, d), 6.50
(IH, br 5) s8.34 (18, s), 12.
06 (IH, 5) t14.19 (IH, 5) (9) 13CNMR Subekle (IQOt4tlz
, CDCl3) (Fig. 19) δ (ppm): 198. IS, 190,2
s, 188,751179.6 s, 172. I
S, 162,5 s.

160.2 St 147,3 s, 145. O5t
142.4 Ss 141.2 S, 133,75t
124.2 d, 124. IS, 120,9 s
.

118.4 S, 116.8 d, 109.59t
105.5 d= 87.2 s, 83. Od.

79.8 +L 78. I Ss 77.5 d.

70.8 d, 68.7460.7 Qs58.9 Q
t 52,3 q* 52,2 Q+26.7 ty
23.9 q+ IS, 5 ltl7.7 q (10) Color reaction: Positive for 10% sulfuric acid and molybdate reagent.

(11) i8 Solubility: Soluble in methanol, chloroform, ethyl acetate, acetone, insoluble in water.

(12) Thin layer chromatography: Carrier silica gel plate (Merck & Co., Ltd.) developing solvent system
Rf value hexane-acetone (2:1) 0,35
Chloroform-methanol (15:1) 0.45
(13) Distinction between basic, acidic, and neutral Weakly acidic substances [V
] SF2446B3 substance (1) Color and shape: Reddish brown powder (2) Molecular formula: C26112, NOo.

(3) Mass spectrum (FD-MS): m/z 5
07 (H”) (4) Melting point: 187-192
℃ (5) Ultraviolet and visible absorption spectra (Figure 5) λm
ax nm (IL) [Me111]: 217 (680), 227
sh (517).

251 (464), 275sh (345).

300sh (231), 409 (87).

[0, IN IIcI-Me111]: 213
(645), 227sh (511).

251 (473), 272sh (361) = 30
0sh (241), 410 (91).

[0, IN NaOH-MeOt! ] : 215
(1220), 240sh (511).

285sh (245), 460 (47).

(6) Optical external absorption spectrum (Figure 10) (KBr a
m'): 3440, 33eo, 2950+1
72(Ltess, 1e5o, 16251159(
L1465, 1405.1360.1310°1260
, 1220.1165.1105゜1080, 1050
．． 1005.955°915, 890.815.800 (7) 1HNMR x hexle (400MHz,
CDCIz) (Figure 15) δ(pp+n)', 2.27 (IL ddd)+
2.38 (31L br s) + 2.76 (lt
l, ddd), 3. O8 (IH, ddd).

3.15 (IH, ddd), 3.23 (3H, s
).

3.84 (311, s), 4.70 (III, s
).

5.43 (2H, br), 5.94 (1) 1.5
)-6,51 (IH,br s), 8.22 (1 skin 5L12.06 (IH,s)t 14.25 (IH
, 5) (8) 1 piece CNMR 9 torr (100Mtl
z, CDCl, ) (Figure 20) δ (ppm): 197.9 St 190,3
Sl 188,85s179.8 S, 172,2
St 162,4 S.

160.3 s, 148.9 s, 145.1 s
.

142.4 St 140.9 S, 133,7 s
.

124.2 d, 124.1 s, 120.9 s
.

118.6 s, 116.4 d, 109.5 s
.

104.5 d, 87. IS, 7S, OS.

52.3 x2 q, 26.8 t.

23.9 (i, 18.5 t (9) Color reaction: Positive for 10% sulfuric acid and molybdate reagent.

(10) Solubility: methanol, chloroform,
Soluble in ethyl acetate and acetone, insoluble in water.

(11) Thin layer chromatography: Carrier silica gel plate (Merck & Co., Ltd.) developing solvent system
Rf value hexane-acetone (2:1) 0.46
Chloroform-metal-7-ol (15:1) 0.50
(12) Distinction between basic, acidic, and neutral Weakly acidic substances The chemical structural formula of the present invention substance was estimated as follows.

An example of a new antibiotic-producing bacterium used in the present invention is strain SF2446, which was newly isolated from the soil of Hyogo Prefecture.

The mycological properties of SF2446 strain are as follows.

■ Morphology The basal hyphae are elongated, well branched, and do not divide under normal conditions. Although the aerial mycelium is poorly attached, it can be attached to oatmeal agar, turch agar, glycerol/asparagine agar, sucrose/nitrate agar, etc., and spore formation is also observed. The branching of aerial hyphae is simple, and no axle branching is observed. Spore chains at the tips of aerial hyphae are wavy, loop-shaped, and hook-shaped.

Or it becomes an incomplete spiral. When observed using an electron microscope, the spores are cylindrical to elliptical and have a size of 0.5 to 1. IX
o, has a size of 6 to 1.3 μm, and one surface is wrinkled (r
It is ugose or spiny. Spores usually form chains of 50 or more. No amount of spores, motile spores, sclerotia, etc. were observed.

(2) Growth status on various media The growth status of SF2446 strain on various media is shown in Table 1. Regarding color descriptions [The standards shown in 1 are from Container Corporation of America (Co
ntainer Corporation of Ame
rica) company's [Color Competition Harmony Medium Manual (C
The one described in Color Harmony Maumal) J was used. Observations were made after culturing at 28°C for 14-21 days.

■Physiological properties (1) Growth temperature range: 20~20°C on yeast malt agar
It grows in a temperature range of 37°C and grows well at 26-30'C.

(2) Liquefaction of gelatin: Negative (3) Hydrolysis of starch: Negative (4) Reduction of nitrate: Negative (5) Peptonization of skim milk: Negative Coagulation of skim milk: Negative (6) Salt tolerance: 4% Although it grows in a salt-containing medium, 5
It will not grow above %.

(7) Production of melanin-like pigment: negative ■, carbon source availability (ISP No. 9 medium used) (
1) Use: D-glucose, glycerol (2)
Not used: D-xylose, L-arabinose, D-
Mannyl, i-inositol, raffinose, sucrose (3) 10-7 lactose, sucrose (3) whose use is suspected, cell wall composition Becker et al.'s method (Appl, Mic
As a result of analysis using LL.
It's a type.

Based on the above properties, it is reasonable to consider that strain SF2446 belongs to the genus Streptomyces among actinomycetes. Therefore, the present inventors also used the SF2446 strain as TrepF Myces sp. SF2446 (Streptomyces sp.
sp, SF 2446).

Strain SF2446 has been entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology as Fiber Science and Technology Research Institute No. 8980 (FERN P-8980).

As seen in the case of actinomycetes in ponds, the SF2446 strain
Its properties change easily. For example, of SF2446 strain,
Alternatively, any mutant strain (naturally occurring or induced), transconjugant, or genetically modified strain derived from this strain that produces the antibiotic according to the present application can be used in the present invention.

In the method of the present invention, the above bacteria are cultured in a medium containing nutrients that can be used by common microorganisms. As a source of nutrients,
Glucose, starch syrup, dextrin.

Sucrose, starch, molasses, animal/vegetable oils, etc. can be used. Soy flour and wheat germ are also used as nitrogen sources.

Corn stable liquor, cottonseed meal, meat extract, peptone,
Yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used. The pond, sodium, potassium, magnesium, Kobal F as needed.

It is effective to add Kuroiso salts that can generate chlorine, phosphoric acid, sulfuric acid, and their ions. In addition, organic and inorganic substances that aid the growth of bacteria and promote the production of the antibiotics involved in the present application can be appropriately added.

The most suitable culture method is a culture method under aerobic conditions, especially a deep culture method. The appropriate temperature for culturing is 20-37°.
C, but in most cases it is cultured at around 26-30°C.

Although the production of the antibiotic related to the present application differs depending on the culture medium and culture conditions, the accumulation usually reaches its maximum within 1 to 8 days in both shaking culture and tank culture. Stop the culture when the accumulated amount of the antibiotic related to the present application in the culture solution reaches its maximum,
Isolate and purify the target substance from the culture solution.

Since the antibiotic of the present invention produced in this manner has the above-mentioned physicochemical properties, it can be extracted and purified from the culture solution according to the properties.

In particular, it is recommended that it be efficiently extracted and purified by the method described below. That is, an organic solvent that is not freely miscible with water, such as ethyl acetate, is added to the culture solution containing the active ingredient, followed by stirring and extraction. On the other hand, the solid substance is mixed with water and an organic solvent such as acetone that is freely miscible with water, and the active ingredients are extracted from the solid substance.

After distilling off the organic solvent, the active ingredient is extracted using an organic solvent that is not freely miscible with water, such as ethyl acetate. The antibiotic of the present invention is isolated by chromatography of the thus obtained oily substance using a suitable combination of carriers such as silica gel, alumina, and gel filtration agents.

Effects of the Invention The antibiotic according to the present invention has a strong antibacterial effect mainly against Durum-positive general bacteria and mycoplasma. For general bacteria, the agar plate dilution method according to the Japanese Society of Chemotherapy (
ChemoLtherapy29t 76-79.198
According to 1), for mycoplasma, liquid dilution method using PPLO broth medium (Chemotherapy 23+
Antibiotics SF2446 substance, SF2446A2 substance, SF measured according to 2569-2576, 1975)
2446A3 substance, SF2446B1 substance, SF244
The minimum inhibitory concentrations (MIC) of the 6B2 substance and SF2446B3 substance against various microorganisms are shown in Table 2.

Example 1 Starch 2.0%, glucose 1.0% as seed medium
, wheat germ 0.6%, polypeptone 0.5%, yeast extract 0.3%, soy flour 0.2%, calcium carbonate 0.1%
A medium containing the following was used. In addition, as a production medium, glucose 3.0%, wheat germ 1.5%, and soybean flour 0.5%.

Corn staple liquor 1.0%, calcium carbonate 0.
A medium containing 0.011% (cobal chloride) was used.

In addition, the pH before sterilization was adjusted to pH 7.0 in all cases.

100n+l volume triangular 7 into which 20ml of the seed medium was dispensed
Sterilize Rasph at 120°C for 30 minutes, and add Streptomyces sp. SF2446 (FERN P-8
980) was inoculated with 1-2 platinum loops of slant culture, and incubated at 28°C.
The cells were cultured with shaking for 3 days to form a type 1 culture. Next, a 500+++ l Erlenmeyer flask into which 80 n+ l of the seed medium was dispensed was sterilized at 120°C for 30 minutes, and the first type culture 4+a was
After inoculation, the cells were cultured with shaking at 28° C. for 2 days, and this was used as a second type culture. Furthermore, the 5L Erlenmeyer flask into which the seed medium IL was dispensed was sterilized at 120"C for 30 minutes, and the second seed culture 5
After inoculating 0ml, culture with shaking at 28°C for 1 day.
This was designated as type 3 culture. Furthermore, 50L jar 77 was dispensed with 25L of seed medium that had been sterilized in advance at 120°C for 30 minutes.
Mentor was inoculated with 50 seeds of the 2nd type culture and incubated with aeration at 28°C for 1 day, which was used as the 4th type culture.

A 300L jar fermenter containing 20OL of production medium previously sterilized at 120°C for 30 minutes was inoculated with 25L of the above-mentioned type 4 culture, and aerated (20L/20L) at 28°C for 5 days.
minutes), stirring (initial 100r, 130r after 100rp time)
p+n) was cultured. After the culture was completed, diatomaceous earth was added as a filter aid and filtered.

158 L of 70% acetone water was added to the solid content containing bacterial cells obtained after filtration, stirred for 1 hour, and filtered again to obtain 158 L of bacterial cell extract containing active ingredients. Furthermore, the bacterial cell extract was concentrated under reduced pressure to remove acetone, and the aqueous layer (54 L) was adjusted to pH 7, and 54 L of ethyl acetate was added thereto and stirred for 1 hour to extract the active ingredients. This grinding process was repeated twice, and anhydrous sodium sulfate was added to the obtained ethyl acetate extract to dry it, and the extract was concentrated under reduced pressure to about 2 L.

The concentrated solution was left at 5°C for one week, and the precipitated crystals were filtered out.
After washing with cold methanol and then hexane and drying, 8
．． 59 g of SF2446 material was obtained. The filtrate and washings were combined and concentrated under reduced pressure to obtain a reddish-brown oily substance.

This oily substance was placed on the top of a 2.4 L silica gel column (Wacodel C 300, manufactured by Wako Pure Chemical Industries, Ltd.) that had been filled with chloroform in advance, and washed with 5 L of chloroform.

The active fractions were developed with chloroform-methanol (10:1) and concentrated under reduced pressure to obtain an oily substance.

This operation was repeated twice, the resulting oily substance was placed on top of a similar 2.4 L silica gel column, and chloroform I
After washing with L, chloroform-methanol (50:1),
When expanded with a 200m17 lux cut.

SF2446 for 7 Lacs, 9 N3-6 (Fr, I)
The active area mainly containing B1 substances is fractions 7 to 14 (
Fr, II) with SF2446 substance and SF2446
The active area mainly containing 83 substances is 7 lactones 15 to 18.
The active area mainly containing SF2446B2 substance in (Fr, m) is the active region containing SF2446B2 substance in 7-lactation 19-28 (Fr, IV) and 7-lactation 29-40 (Fr, V).
An active area containing mainly A2 substance was obtained. Fr, I was concentrated to dryness under reduced pressure to obtain 650 n+g of an oily substance. This oily substance was placed on top of a 120 ml silica gel column previously filled with hexane-acetone (2:1) and developed with the same solvent system to obtain a parent substance containing mainly SF2446B1 substance. This parent substance was further purified by preparative silica gel TLC (developing system: hexane-acetone 1:1) to obtain 80.5 mg of SF2446B1 substance. Fr.

(2) was concentrated to dryness under reduced pressure to obtain 10.51 g of the parent substance. This parent substance was mixed with chloroform-methanol (1:
1) What if you perform recrystallization using a mixed solution? SF of 1.828
2446 substances were obtained. The filtrate) was concentrated under reduced pressure, and the obtained parent substance was purified using Cef7dex LH-20 (750 ml,
Column chromatography (manufactured by Pharmacia) was performed, and the active fraction was concentrated to dryness under reduced pressure. This process was repeated twice to obtain 570 mg of SF2446 substance and 303 mg of parent substance containing mainly SF2446^3 substance. This parent substance was purified using a silica gel column (30 g*Developing system: chloroform-methanol 30:1) and then using a preparative silica gel column TLC (7 system: chloroform-methanol 15:1), resulting in 23
．． 7 mg of SF2446A3 material was obtained. Fr,
III was concentrated to dryness under reduced pressure to obtain the parent material of 772H. This parent substance is chloroform-methanol (30:
Column chromatography was performed on silica gel (80 g) using 1) as a developing solvent, and the obtained active fraction was concentrated to dryness under reduced pressure to obtain 123 mg of the parent substance. When this parent substance was further purified by preparative silica gel TLC (developing system: toluene-ethyl acetate 1:2°, then chloroform-methanol 15:1), the result was +78.81111?
SF2446B2 material was obtained. Fr.

When ■ is concentrated to dryness under reduced pressure, it is 5.071? (7) SF24
46A2 material was obtained. Similarly, Fr and V were concentrated to dryness under reduced pressure to obtain 5.72 g of the parent substance. This parent substance was subjected to column chromatography on silica gel (300 g, H amount system: chloroform-methanol 50:1), then silica sol (300 g, developing system: hexane-acetone).
2:1) column chromatography, and further Sephadex LH-20 (developing system: chloroform-methanol 1:
1) Perform column chromatography to remove 1.13H S
F2446A2 material was obtained.

Example 2 Concentrated hydrochloric acid (0.2 ml) was added to methanol solution (2.2 ml) of SF2446B1 substance (15.2+ng) and left at 40°C for 1 hour.

Thereafter, the solvent was distilled off under reduced pressure, and the residual aroma was analyzed by preparative silica gel TLC (developing system: chloroform-methanol 15:
1), 5.4 mg of SF2446B3
Substance obtained.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet and visible absorption spectra of SF2446A2 substance (2'Oμg/+1), where the solid line (-) is in methanol solution and the broken line (----) is in 0.1N hydrochloric acid. In the methanol solution, the - dotted chain line (---) indicates 0.
Shown in 1N sodium hydroxide-methanol solution. Figure 2 shows the ultraviolet and visible absorption levels of SF2446A3 substance (20Hg7xfl), where the solid line (-) is in methanol solution and the broken line < --------- is 0.1
In normal hydrochloric acid-methanol solution, - dotted line (---) is 0
．． Shown in 1N sodium hydroxide-methanol solution. Figure 3 shows the ultraviolet and visible absorption spectra of SF2446B1 substance (20Hg/xl), where the solid line (-) is in methanol solution and the broken line (-------) is in 0.1N hydrochloric acid-methanol solution. In the middle, the -dotted chain line (---) is 0.1
Shown in normal sodium hydroxide-methanol solution. Figure 4 shows the ultraviolet and visible absorption spectra of SF2446B2 substance (20 μg) (Substance 2446B2 (20 μg)), where the solid line (-) is in methanol solution and the broken line (-----) is in 0.1N hydrochloric acid-methanol solution. Inside, the dashed line (---) indicates the 0.1N sodium hydroxide-methanol solution. Figure 5 shows the ultraviolet and visible absorption spectra of SF2446B3 substance (20μg/1Ip), where the solid line (-) is in methanol solution and the broken line (----) is in 0.1N hydrochloric acid. - In the methanol solution, - the dashed line (---) is 0.
Shown in 1N sodium hydroxide-methanol solution. FIG. 6 shows the infrared absorption spectrum of SF2446A2 substance in potassium bromide tablets. FIG. 7 shows the infrared absorption spectrum of SF2446^3 substance in potassium bromide tablets. FIG. 8 shows the infrared absorption spectrum of SF2446B1 substance in potassium bromide tablets. FIG. 9 shows the infrared absorption spectrum of SF2446B2 substance in potassium bromide tablets. FIG. 10 shows the infrared absorption spectrum of SF2446B3 substance in potassium bromide tablets. Figure 11 shows SF244682 in deuterated chloroform solution;
400MHz1HNM measured using TMS as a reference material
The R spectrum is shown. Figure 12 shows SF2446A3 in deuterated chloroform solution,
400MHz1HNM measured using TMS as a reference material
The R spectrum is shown. Figure 13 shows SF2446B1 in deuterated chloroform solution,
400MHz1HNM measured using TMS as a reference material
The R spectrum is shown. Figure 14 shows SF2446B2 in deuterated chloroform solution,
400MHz1HNM measured using TMS as a reference material
The R spectrum is shown. Figure 15 shows SF2446B3 in deuterated chloroform solution,
400MHz1HNM measured using TMS as a reference material
The R spectrum is shown. Figure 16 shows SF2446A2 in deuterated chloroform solution,
100MHz”CNM measured using TMS as a reference material
The R spectrum is shown. Figure 17 shows SF2446A3 in deuterated chloroform solution,
100MHz”CNM measured using TMS as a reference material
R specklere is shown. Figure 18 shows SF2446B1 in deuterated chloroform solution,
100MHz”CNM measured using TMS as a reference material
R also indicates a vector. Figure 19 shows SF2446B2 in deuterated chloroform solution,
100MHz13CN measured using TMS as a reference material
An MR spectrum is shown. Figure 20 shows SF2446B3 in deuterated chloroform solution,
100MHz13CN measured using TMS as a reference material
An MR spectrum is shown.

Claims (1)

[Claims] 1. Novel antibiotic SF2446A2 having the following properties
, A3, B1, B2 substances and their salts; (a) SF2446A2 substance (1) Color and shape: reddish brown powder (2) Elemental analysis values: C58.10%, H5.26%, N
1.90% (3) Molecular formula: C_3_4H_3_5NO_1_5 (4) Mass spectrum (FD-MS):_m_/_z6
97 (M^+) (5) Melting point: 180-184°C (decomposed) (6) Ultraviolet and visible absorption spectra: Figure 1 (7) Infrared absorption spectrum: Figure 6 (8)^1H NMR spectrum ( 400MHz, CDC
l_3): Figure 11 (9)^1^3CNMR spectrum (100MHz, C
DCl_3): Figure 16 (10) Color reaction: Positive for 10% sulfuric acid and molybdate reagent. (11) Solubility: Soluble in methanol, chloroform, ethyl acetate, acetone, insoluble in water. (12) Thin layer chromatography: Support silica gel plate (manufactured by Merck & Co.) ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (13) Distinction between basic, acidic, and neutral: Weakly acidic substances (b)
SF2446A3 substance (1) Color and shape: Reddish brown powder (2) Elemental analysis: C59.04%, H4.12%, N
2.53% (3) Molecular formula: C_2_6H_2_1NO_1_1 (4) Mass spectrum (FD-MS):_m_/_z5
23 (M^+) (5) Melting point: >220°C (6) Ultraviolet and visible absorption spectra: Figure 2 (7) Infrared absorption spectrum: Figure 7 (8)^1H NMR spectrum (400MHz, CDC
l_3): Figure 12 (9)^1^3CNMR spectrum (100MHz, C
DCl_3): Figure 17 (10) Color reaction: Positive for 10% sulfuric acid and molybdate reagent. (11) Solubility: Soluble in methanol, chloroform, ethyl acetate, acetone, insoluble in water. (12) Thin layer chromatography: Carrier silica gel plate (manufactured by Merck & Co.) ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (13) Distinction between basic, acidic, and neutral: Weakly acidic substances (c)
SF2446B1 Substance (1) Color and shape: Reddish brown powder (2) Elemental analysis: C59.72%, H5.35%, N
2.21% (3) Molecular formula: C_3_4H_3_5NO_1_4 (4) Mass spectrum (FD-MS):_m_/_z6
82 (MH^+) (5) Melting point: 1SS-192°C (decomposition) (6) Ultraviolet and visible absorption spectra: Figure 3 (7) Infrared absorption spectrum: Figure 8 (8)^1H NMR spectrum ( 400MHz, CDC
l_3): Figure 13 (9)^1^3CNMR spectrum (100MHz, C
DCl_3): Figure 18 (10) Color reaction: Positive for 10% sulfuric acid and molybdate reagent. (11) Solubility: Soluble in methanol, chloroform, ethyl acetate, acetone, insoluble in water. (12) Thin layer chromatography: Carrier silica gel plate (manufactured by Merck & Co.) ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (13) Distinction between basic, acidic, and neutral: Weakly acidic substances (d)
SF2446B2 substance (1) Color and shape: Reddish brown powder (2) Elemental analysis: C59.16%, H5.18%, N
2.05% (3) Molecular formula: C_3_4H_3_5NO_1_4 (4) Mass spectrum (FD-MS):_m_/_z6
81 (M^+) (5) Melting point: 177-181°C (decomposed) (6) Ultraviolet and visible absorption spectra: Figure 4 (7) Infrared absorption spectrum: Figure 9 (8)^1H NMR spectrum ( 400MHz, CDC
l_3): Figure 14 (9)^1^3CNMR spectrum (100MHz, C
DCl_3): Figure 19 (10) Color reaction: Positive for 10% sulfuric acid and molybdate reagent. (11) Solubility: Soluble in methanol, chloroform, ethyl acetate, acetone, insoluble in water. (12) Thin layer chromatography: Carrier silica gel plate (manufactured by Merck & Co.) ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (13) Distinction between basic, acidic, and neutral: Weakly acidic substances (e)
New antibiotic SF2446B3 substance (1) Color and shape: Reddish brown powder (2) Molecular formula: C_2_6H_2_1NO_1_0 (3) Mass spectrum (FD-MS):_m_/_z5
07 (M^+) (4) Melting point: 1S7-192°C (5) Ultraviolet and visible absorption spectra: Figure 5 (6) Infrared absorption spectrum: Figure 10 (7) ^1H NMR spectrum (400MHz, CDC
l_3): Figure 15 (8)^1^3CNMR spectrum (100MHz, C
DCl_3): Figure 20 (9) Color reaction: Positive for 10% sulfuric acid and molybdate reagent. (10) Solubility: Soluble in methanol, chloroform, ethyl acetate, acetone, insoluble in water. (11) Thin layer chromatography: Carrier silica gel plate (manufactured by Merck) ▲ Contains mathematical formulas, chemical formulas, tables, etc. ▼ (12) Distinction between basic, acidic, and neutral: Weakly acidic substances