CN1014528B - Process for producing compounds of the kind of cephalosporin - Google Patents

Abstract

The present invention relates to a compound whose structural formula is disclosed in the specification, wherein R' is HOOC-(CH2)3-CO-or HOOC-(CH2)3-CO-or H or a salt. The compound is used as an intermediate body for preparing cephem compounds, wherein some compounds can be used as antimicrobial drugs.

Description

Process for producing compounds of the kind of cephalosporin

Of the present inventionly relate to cephem compounds and production thereof.

The contriver isolates a series of microorganisms from soil and plant, find that certain micro-organisms can produce new cephem antibiotic, and the contriver is with these antibiotic called afters TAN-547, TAN-592 and TAN-591.TAN-547 has 6 components at least, i.e. TAN-547A, and B, C, D, E and F, TAN-592 have 6 component TAN-592A at least, B, C, E, and F, TAN-591 have 3 component TAN-591A, B and C at least.The contriver finds TAN-547A in addition, B or C, TAN-592A, B or C or TAN-591A are when B or C hydrolysis, get 7-formamido group cephem compounds, and with TAN-547D, E, F or TAD-592D, E or F hydrolysis then get deacetylcephalosporinC, further are converted into the amino cephem of 7-formamido--7-by enzyme reaction 7-formamido--cephem compounds.

The contriver makes further research on the basis of above-mentioned discovery, has finished the present invention.The present invention refers to: (1) following formula: compound and salt thereof

R wherein ¹Be HOOC

, HOOC-(CH ₂) ₃-CO-or be hydrogen atom

(2) preparation method of following formula: compound II and salt thereof:

This method is a wushu III compound or its salt

Contact with nutrient solution of a kind of microorganism or the material in the culturing process, this microorganism belongs to Rhodopseudomonas (pseudomonas) and can be with the HOOC-(CH of 7 of initiators ₂) ₃-CO-NH-base converts NH to ₂-Ji.

(3) preparation method of compound III and salt thereof is comprising following formula: compound IV and salt thereof

Contact with nutrient solution of a kind of microorganism or the material in the culturing process, this microorganism belongs to trigonopsis variabilis Pseudomonas (Trigonopsis) and can be with 7 of initiators

Be transformed into HOOC-CH-(CH ₂) ₃-CO-NH, and

(4) compound (IV), the production method of removing acetyl cephalo bacterium C and their salt, this method are with formula V compound or its hydrolysis

R wherein ⁴Be formamido-or hydrogen atom, R ²Two residues that are an amino acid whose residue or peptide are from Serine and L-Ala, or hydrogen atom, R ³Be-NH-C(=NH)-NH ₂Or-CH ₂NH ₂, X is L-Ala or serine residue; If X is then R of alanine residue ³Be two residues or the hydrogen atom of an amino-acid residue or L-Ala peptide, R ³Be-HN-C(=NH)-NH ₂

In following formula, the alanine residue representative

Or , the serine residue representative

Or

The formamido-representative

In patent application, the compound name shown in the formula (V) is as follows:

Antibiotic TAN-547 or TAN-547 are used to refer to one antibiotic TAN-547A sometimes, B, C, D, E or F or refer to the mixture of above-mentioned single antibiotic more than two.Antibiotic TAN-592 or TAN-592 are used to refer to one TAN-592A sometimes, B, C, D, E or F or refer to the mixture of above-mentioned single antibiotic more than at least two.Antibiotic TAN-591 or TAN-591 be sometimes in order to referring to one TAN-591A, B, or C or refer to the mixture of above-mentioned single antibiotic more than at least two.

In this application, 7-formamido--deacetylate cephalosporin is that compound IV and deacetylate cephalosporin correspondingly are called for short " 7-FA-DCPC " and DCPC in some occasion.

The salt of above-claimed cpd, therapeutical agent as medicine such as infectation of bacteria, comprise salt such as lithium salts that medicine is suitable, sodium salt, barium salt, calcium salt and magnesium salts, then comprise the above-mentioned salt of mentioning when the synthetic intermediate and such as ammonium salt, methylamine salt, diethyl amine salt, trismethylamine salt, 4-butyl ammonium and pyridinium salt.

For the above-mentioned antibiotic that obtains in nutrient solution, producing and gathering, antibiotic TAN-547,592, can produce by the method for culturing micro-organisms in substratum with 591 initial substance [this microorganism belong to dissolve Pseudomonas (Lysobacter) or vitiligoidea Pseudomonas (Xanthomonas)], corresponding TAN-547,592 and 591 can be made and gather in the crops to this initiator.

Produce TAN-547,592 or 591 microorganism example is:

Dissolve the bacterium lactan and belong to (Lysobacter lacyamgeuus) YK-90, vitiligoidea bacterium lactan belongs to (xanthomonas lactamgena) YK-280 and vitiligoidea bacterium lactan belongs to (xan Thomonas lactamgena) YK-278.These microorganisms have been stored in Japanese fermentation research institute (IFO) and Japanese foreign trade fermentation research institute of industrial technology office of the Department of Industry (FRI), preserve thing and have converted the preservation thing that this treaty of wearing admits to and be stored among the FRI.

Be storage date and the number of registration of microorganism below.

IFO FRI CGMCC

Microorganism

Registration number and registration number reach by Budapest bar registration number and reach

Registration number preservation day approximately preservation day preservation day

Lysobacte IFO14288 FERMP-7247 FECM BP-575 CGMCC 0007

Laclamgenus 1983.9.14 1983.9.19 1985.4.19

YK-90

Xanthomonas IFO 14330 FERM P-7602 FERM BP-635 CGMCC 0008

Lactamgena 1984.3.20 1984.4.28 1985.4.19

YK-280

Xanthomonas IFO 14351 FERM P-7681 FERM BP-636 CGMCC 0009

Lactamgena 1984.6.18 1984.6.25 1985.4.19

YK-278

The production process for preparing compound IV or DCPC from the compound V is under alkaline condition initial compounds to be handled, and alkaline condition comprises initial compounds is added the aqueous solution of wherein handling, and its pH value adjusted to 7 is to 11, preferably 9 to 9.7.

The described aqueous solution comprises the damping fluid that is generally used for chemical reaction, and above-mentioned damping fluid can be used as the phosphoric acid salt that is adjusted to above-mentioned pH value scope in addition that example is pointed out, borate, Citrate trianion, carbonate, hydrochloride, acetate, sodium hydroxide, glycine, veronal, borax, ammonium salt, the aqueous solution of aminomethyl propylene glycol and these analogues.

Hydrolysis reaction of the present invention is to carry out in 0 ℃ to 80 ℃ temperature range, preferably between 20 ℃ to 40 ℃, continues 1 to 72 hour, in preferably about 4 hours to 40 hours.

Any one can all can be used trigonopsis variabilis bacterium (Trigoropsis) microorganism belonging to genus that the compound IV converts the compound III to the method for preparing the compound III from the compound IV.

What be mentioned as microorganism is the microorganism that trigonopsis variabilis bacterium (Trigonopsis Valiabilis) belongs to.Used microorganism is enumerated as special case with trigonopsis variabilis bacterium (Trigonopsis Valiabilis) IFO 0671 and IFO 0755.

With the IFO number is that the microorganism of feature has been left among the Japanese IFO and lists in IFO the 7th edition culture inventory in 1984, is the storage date of microorganism at IFO below.

Microorganism stores the date

IFO on October 29th, 0,671 1954

0755 October 15 nineteen fifty-five of IFO

Those that describe in " yeast " chapters and sections in the morphological feature of trigonopsis variabilis Pseudomonas (Trigonopsis riabilis) microorganism and " means of taxonomic research " book are consistent, [this book is second version in 1970, the 1353-1937 page or leaf, editor: outstanding person, Rider (J.Ladder), northern Dutch publishing company publishes (North Holland pub.corporation)]

Produce the method for compound ii from the compound III, arbitrary Rhodopseudomonas (pseudomonas) also can all can be used the microorganism that the compound III is converted into compound ii.The microorganism that the quilt that is worth mentioning uses is Rhodopseudomonas sp.uk-2221, and this microorganism is to separate in the pedotheque collected of Japanese Fu Kuqieyema (Fukuchiyama) city Xiao brother (Hyogo) prefecture.

The feature description of bacterial strain UK-2221 microorganism is in following:

A) morphology:

Observe and find that it is bar-shaped that cell is after 5 days in 24 ℃ of cultivations on the nutrient agar slant medium, diameter is that 0.5 to 1.0 micron (μ m) length is 0.8 to 2.0 micron (μ m), and this microorganism can be observed flagellum, does not form spore, and be Gram-negative, not antiacid.

B) growth characteristic in various substratum:

24 ℃ of observations of being done after cultivating down in 1 to 14 day

1) nutrient agar plate culture: colourless garden shape bacterium colony has the surface in convex garden and complete border, does not produce the diffustivity pigment.

2) nutrient agar medium slant culture: moderate life degree, colourless bacterium colony, no diffustivity pigment produces.

3) nutritive medium is cultivated thing: bad dirt growth, form deposition, and there is not membranaceous growth.

4) gelatin nutrition jab culture: slight growth, it is active not have liquefaction.

5) reindeer moss emulsion: no reducing power, no peptone activity.

C) physiological property:

1) reduction of nitrate: (-)

2) anti-nitration reaction: (-)

3) test MR(methyl red): (-)

4) VP[lies prostrate its Si-Po Lars Kao Yier (Voges-proskauer) test: (-)

5) generation of indoles: (-)

6) generation of hydrogen sulfide (lead-acetate test): (-)

7) starch hydrolysis: (-)

8) the utilization of citric acid [the section pool (Christesen of koser ' s) (Christensen ' s) and Xi Mengsi (substratum of Simmon ' s)]: (+) [Xi Mengsi (base of Simmon ' s)-]

9) nitrogenous source utilization

I) nitric acid first: (-)

II) vitriolate of tartar: (+)

10) generation of pigment (King A and B and N.F,USP MANNITOL yeast extract paste nutrient agar): do not produce the diffustivity pigment.

King A substratum: 10 gram glycerol (glycerine), 20 gram peptones, 1.4 gram magnesium chlorides, 10 gram ammonium sulfate, 15 gram agar, 1000 milliliters of aquae destillatas, PH are 7.2.

King B substratum: 10 gram glycerine, 20 gram peptones, 1.5 gram dipotassium hydrogen phosphates, 1.5 gram sal epsom, 15 gram agar, PH is 7.2.

11) urase: (+)

12) oxydase: (+) (weak)

13) catalase: (+)

14) growth scope:

I) PH: the pH value of microorganism growth is 4.3 to 7.0, and best pH value is 5.0 to 6.0.

II) temperature: the temperature of microorganism growth is 14 to 32 ℃, and optimum temps is 18 to 26 ℃.

15) demand of oxygen: strict aerobic.

16) O-F(oxidation-fermentation) test, [Xiu Lifusen (HughLeifson) method]: do not react.

17) produce acid and gas from sugar:

Acid gas utilizes

Peptone-water peptone-water (Davis's culture)

The L-arabulose---

The D-wood sugar--+

D-glucose--+

The D-seminose--+

D-fructose--+

The D-semi-lactosi--+

Maltose---

Sucrose---

Lactose---

Trehalose---

The D-sorbyl alcohol--+

D-N.F,USP MANNITOL--+

Inositol--+

Glycerol--+

Starch---

+: the positive, ±: false positive ,-: feminine gender

18) the GC(guanine+cytosine(Cyt) that in DNA, contains):

68.3±1.5%

(Bergey ' s) family name's bacteria-measuring is learned the kind of the 8th edition description of methodology when bacterial strain UK2221 and Bergen, international system bacteriology magazine 30,225-420(1980), 32,146-149(1982) and the kind of describing on the effective inventory of document relatively, it is valid that the UK-2221 bacterial strain belongs to Rhodopseudomonas, because the UK-2221 bacterial strain is a gram negative bacillus, be that active has single polar flagellum, strict aerobic, the bacterial strain that catalase is positive does not react in the O-F test, the GC content of DNA is 68.3 ± 1.5%(guanine+cytosine(Cyt)), the inventor is with UK-2221 bacterial strain called after Rhodopseudomonas (Pseudomonas) sp.UK-2221.

Above-mentioned Rhodopseudomonas (pseudomonas) sp.UK-2221 has been stored in Japanese fermentation research institute (IFO) on August 31st, 1984, number of registration in this institute (IFO) is 14366, this microorganism once was stored in Japanese foreign trade fermentation research institute of industrial technology office of the Department of Industry (FRI) on September 7th, 1984, number of registration is FERM P-7836, stores has been converted to the stores that budapest treaty admits and has been stored among the FRI, number of registration is FERM BP-637, and be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) on April 19th, 1985, it is numbered CGMCC 0010.

Be used for trigonopsis variabilis bacterium of the present invention (Trigonopsis) and Rhodopseudomonas (pseudomonas) microorganism belonging to genus generally tends to change its proterties, and very easy-to-use artificial mutagenesis method just it is undergone mutation, for example use UV-light, the X-ray, chemical reagent is (as nitrosoguanidine, ethyl methanesulfonics etc.) no matter that a kind of above-mentioned change strain all can be used among the present invention, because they still have transfer capability described in the present invention so far." nutrient solution " of the present invention is meant the microorganism culturing product that uses in the present invention.Be used for substratum of the present invention and can be the also solid of liquid, more suitable with the former.But cultural method can be the surface cultivates also shaking culture, and the cultivation of the logical oxygen of sinking is favourable to scale operation.

In substratum, add the carbon source that Institute of Micro-biology can utilize, nitrogenous source, inorganic substance, nutritive substance or the like.As the glucose that has of carbon source, maltose, molasses, fat and oily (for example, soya-bean oil, olive wet goods) and organic acid (for example citric acid, succsinic acid, gluconic acid etc.).Organic nitrogen compound and inorganic nitrogen compound be such as soybean-cake flour, cottonseed meal, and corn steep liquor, dry yeast, yeast extract paste, meat stain cream, peptone, urea, ammonium sulfate, ammonium sulfate ammonium nitrate, ammonium chloride, ammonium phosphate all can be used as the nitrogenous source utilization.

Inorganic salt normally need during culturing micro-organisms, such as sodium-chlor, Repone K, lime carbonate, magnesiumcarbonate, potassium primary phosphate and Sodium phosphate dibasic, normal individually or appropriate combining use as inorganic salt.

Can contain D-amino acid (for example: D-L-Ala, D-methionine(Met)) or D-α-An Jijiersuan as the substratum of cultivating trigonopsis variabilis bacterium (Trigonopsis) microorganism belonging to genus.

Under the situation of liquid culture, any static cultivation sinking is cultivated, shaking culture, and logical oxygen is cultivated and all be may be utilized, but the logical oxygen of sinking is cultivated desirable especially to scale operation.

The culture condition of trigonopsis variabilis bacterium (Trigonopsis) microorganism belonging to genus is with the state of substratum, the prescription of substratum, cultural method and changing.The preferential condition of selecting is as follows:

Temperature: about 20 to 45 ℃, preferably about 24 to 37 ℃.

The pH value of training base: about 6 to 9, preferably about 6 to 8.

Incubation time: about 24 to 144 hours, preferably about 24 to 120 hours.

The culture condition of pseudomonas microorganism belonging to genus is with the basic state of training, training based formulas, cultural method and changing.The preferential condition of selecting is as follows:

Temperature: about 20 to 45 ℃, preferably about 24 to 37 ℃

The pH value of training base: about 6 to 9, preferably about 6 to 8.

Incubation time: about 24 to 144 hours, preferably about 24 to 120 hours.

Being used for term of the present invention " material of culturing process " is that phalangeal cell or fragmented cell contain the enzyme that participates in process of the present invention, this enzyme can obtain such as filtration by above-mentioned culture being carried out any following process, centrifugal, ultrasonic fragmentation France-squeezing is handled, and osmotic pressure is handled, and freezing thawing is handled, alumina lap, N,O-Diacetylmuramidase is handled, and washing composition is handled, organic solvent processing etc.

The concentration of compound IV is about 0.5 to 20 mg/ml in compound IV and the reaction that the nutrient solution or the material in its culturing process of trigonopsis variabilis bacterium (Trigonopsis) microorganism belonging to genus contact.Preferably about 5 to 10 mg/ml, about 0.1 to 1 grams per milliliter of the quantity of cell.Preferably about 0.1 to 0.3 grams per milliliter is in wet cell.The quantity of material is calculated from the wet cell number in the culturing process.The pH value of reaction system is adjusted to about 6 to 10, preferably about 7.5 to 8.5.About 15 to 40 ℃ of reaction mixture, best 15 to 37 ℃, about 4 to 48 hours of reaction times, best 8 to 16 hours.Reaction can be used method oxygen immobilized, vibration, logical or that stir.The method of the most handy vibration, logical oxygen or stirring.

Can in reaction system, add the hydrogen peroxide enzyme inhibitors in order to obtain purpose compound III with good yield.The example of hydrogen peroxide enzyme inhibitors is such as being inorganic azide (as sodiumazide), xitix, 3-amino-1,2,3, triazole.Can be by the mensuration that colibacillary antimicrobial acivity is disappeared, or use tlc, high pressure lipuid chromatography (HPLC) is reflected at confirmation carries out or stops.After reacting completely, reaction mixture is carried out centrifugation to remove cell, purpose compound III is told from supernatant liquor and is purified.

This process can follow the cultivation of trigonopsis variabilis bacterium (Trigonopsis) microorganism to carry out together, before cultivation the compound IV is added in the substratum when this situation, and its amount is about 1 to 20 mg/ml, preferably about 2 to 10 mg/ml.Culture temperature, it is identical with the culture condition of microorganism with incubation time to train basic pH value.

In this reaction process,

Base at first converts HOOC-CO(CH to ₂) ₃-CO-NH-base, this group convert HOOC-(CH again immediately to ₂) ₃-CO-NH-promptly gets the compound III.

The concentration of compound III is about 2 to 20 mg/ml in the nutrient solution of compound III and Rhodopseudomonas (pseudomonas) microorganism belonging to genus or the contacted reaction of its culturing process material, preferably about 4 to 10 mg/ml.About 0.1 to 1 grams per milliliter of cell quantity, best 0.1 to 0.3 grams per milliliter is in wet cell.The quantity of culturing process material is calculated with wet cell quantity.The pH value of reaction system is adjusted to 6 to 10, and best 6.5 to 7.5.About 15 to 40 ℃ of the temperature of reaction mixture, best 15 to 37 ℃, about 4 hours to 66 hours of reaction times, best 16 to 54 hours.Reaction can be used mode oxygen immobilized, vibration, logical or that stir.The mode of especially vibrating, leading to oxygen or stirring is better.

This process can follow the cultivation of Rhodopseudomonas (pseudomonas) microorganism belonging to genus to carry out together, adds before cultivation in the developing medium in the compound III in this case, and its quantity is about 2 to 20 mg/ml, preferably about 4 to 10 mg/ml.Culture temperature, it is identical with the culture condition of microorganism with incubation time to train basic pH value.

In order from reaction solution, to isolate the compound IV, III, II or DCPC can be used suitable the integrating of the method that is generally used for the separating water-soluble acidic substance.Like this, the adsorption chromatography of various adsorbing agent carriers, the ion exchange chromatography of ion exchange resin, gel-filtration, anti-phase liquid chromatography, perhaps such as concentrating under reduced pressure, methods such as lyophilize can any order integrate separately or utilize repeatedly.As having of adsorbing agent carrier: activated carbon, polymeric adsorbent, anionite-exchange resin, cellulose powder etc. or have the carrier of molecular sieve effect and these all can utilize.The different eluent with the difference of kind of carrier commonly used has: water-miscible organic solvent, aqueous acetone solution, methyl alcohol, propyl alcohol, butanols, Virahol, isopropylcarbinol etc. or various acid, alkali, the aqueous solution of damping fluid or inorganic or organic salt.

More detailed situation is referring to their example, and the pH value that reaction finishes afterreaction liquid is adjusted near 6 to 7, because in question reactant is an acidic substance, reaction solution passes through Cl ^-Or ACO ^-Type carrier and absorption antibiotic.As carrier just like anionite-exchange resin (as amberlite ion exchange resin IRA-68,402, the 410(U.S. Luo Mo-Rohm of Haars Co., Ltd; Hass Co., U.S.A), ion exchange resin (the Dow Chemical Co. of The Dow Chemical Co. (US) 2030 Dow Center, Abbott Road, Midland, Michigan 48640, of road Vicks VapoRub-1(Dowex-1), U.S.A), the dianion exchange resin SA-21 C.(Mitsubishi chemical industry Mitsubishi Chemical Industries of company limited, Ltd., Japan) etc.].With sodium chloride aqueous solution or damping fluid as the eluent of adsorbed material in discussing with the wash-out active substance, in order to make the eluate desalination, eluate is neutralized to slightly acidic and uses activated carbon (the Japanese Takeda Chemical Industries of Takede Chemical Industries Ltd, Ltd., Japan) chromatography is separated, immediately with the water elution that contains alcohol, and so on.

The eluate that will contain active substance then is concentrating under reduced pressure at low temperatures, and enriched material is by DEAE or the Pharmacia Co. of QAE Sephadax(Sweden Pharmacia company, Sweden) chlorion (Cl ^-) type resin and absorption antibiotic.The antibiotic that is adsorbed is differentiated significant part with dilute sodium chloride solution classification wash-out with high pressure lipuid chromatography (HPLC), merges effective elutriant and handles with the activated carbon chromatography, desalination subsequently.Elutriant concentrates, and the concentrated solution lyophilize adds acetone in the lyophilized powder, filters and collect the precipitation that forms.

The salt of gained compound can be converted into other salt with currently known methods.For example with the salt of the gained pH value about 2 to 3 of under cooling, using the dilute hydrochloric acid regulator solution soluble in water, use such as containing sodium hydroxide, calcium hydroxide, methylamine, the pH value that the dilute alkaline soln of TBAH etc. is regulated the above-mentioned aqueous solution immediately is approximately to 7 to 8 to produce required salt, the above-mentioned aqueous solution separates with activated carbon chromatography, and behind the inorganic or organic salt of water flush away, the salt of purpose compound is promptly eluted.

Resulting 7-FA-DCPC(compound IV in the example 1 that will describe below) sodium salt is following physico-chemical property:

(1) outward appearance:

White powder.

(2) specific rotation:

(α) ²⁵ ₀+ 146.5 ° ± 30 ° (C=0.51, water)

(3) molecular weight:

The data that 438(provides according to secondary ion mass spectrometry (SIMS))

(4) ultimate analysis (%), C ₁₅H ₁₉N ₄O ₈SNaH ₂O:

The trial value calculated value

C 39.65±2.0 39.48

H 4，640±0.5 4.64

N 12.20±1.0 12.28

O 31.55

S 7.281±1.0 7.02

Na 5.2±1.0 5.04

(5) uv-absorbing (UV) spectrum:

258+ ± 2nm(209 ± 50) (referring to Fig. 1)

(6) infrared absorption (IR) spectrum (KBr sheet)

Main wave number (cm ^-1)

3430,3240,3020,1770,1680,1610,1520,1410,1375,1300,1220,1145,1070,1040,1000,800,710,540cm ^-1(referring to Fig. 2)

(7) ¹³C nucleus magnetic resonance (NMR) spectrum

(100 megacycle (MH ₂), in the deuterium):

δ179，65（S），177，22（S），171，43（S），166，30（d），162.01（S）132.76（S），122.50（S），79.47（S），66.03（d），63.75（t），57.33（d），37.28（t），32.70（t），28.22（t），23.40（t）ppm

(the S representative is unimodal; The d representative is bimodal, and t represents three peaks)

(8) high pressure liquid chromatography (HPLC) [Wal Te Lian society, the U.S. (Waters Associates, U.S.A)]:

Post: YMC-Pak A-312(Japan, mountain village chemistry laboratory (Yamamura Chemical

Laboratories，Japan）]

Moving phase: 2% methyl alcohol-0.01 mol phosphate solution (PH3.0)

Flow velocity: 2 ml/min, Rt=2.3 branch

(9) color reaction:

Positive: ninhydrin reaction

Negative: Ehrlich (Ehrlich) diazotization reaction, Ge Ruikeli Bake

Graig-Leaback) reaction, mouthful (Sakaguchi)

Reaction.

(10) solubleness:

Soluble in water

Be dissolved in methyl alcohol

Be slightly soluble in ether and ethyl acetate

Can think on the basis of above-mentioned physico-chemical property that with TAN-547 A B or the C gained compound that is hydrolyzed is the 7-FA-DCPC that represents with above-mentioned formula IV,

Resulting 7-FA-DCPC(compound IV in the example 9 that will describe below) sodium salt is following physico-chemical property:

(1) outward appearance:

White powder.

(2) specific rotation:

(α) ²⁵ _D+ 150 ° ± 30 ° (C=0.55, water)

(3) molecular weight:

The data that 438(provides according to secondary ion mass spectrometry (SIMS) analysis (SIMS) method)

(4) ultimate analysis (%), C ₁₅H ₁₉N ₄O ₈SNaH ₂O:

The trial value calculated value

C 39.8±2.0 39.48

H 4，9±0.5 4.64

N 12.3±1.0 12.28

O 31.55

S 6.9±1.0 7.02

Na 5.4±1.0 5.04

(5) uv-absorbing (UV) spectrum:

) 258 ± 2nm(200 ± 50) referring to Fig. 4

(6) infrared absorption (IR) spectrum (KBr sheet)

Main wave number (cm ^-1)

3430,3240,3020,1770,1680,1610,1520,1410,1375,1300,1220,1145,1070,1040,1000,800,710,540cm ^-1Referring to Fig. 5

(7) ¹³C nucleus magnetic resonance (NMR) spectrum

(100 megacycle (MH ₂), in the deuterium):

δ179，65（S），177，2（S），171，4（S），166，3（d），162.）（S）132.7（S），122.5（S），79.5（S），66.0（d），63.8（t），57.3（d），37.3（t），32.7（t），28.2（t），23.4（t）ppm

(the S representative is unimodal; The d representative is bimodal, and t represents three peaks)

(8) high pressure liquid chromatography (HPLC) [U.S., Wal Te Lian society, (Waters Associates, U.S.A)]:

Post: YMC-Pak A-312(Japan, mountain village chemistry laboratory (Yamamura Chemical

Laboratories，Japan）]

Moving phase: 2% methyl alcohol-0.01 mol phosphate solution (PH3.0)

Flow velocity: 2 ml/min, Rt=2.3 branch

(9) color reaction:

Positive: ninhydrin reaction

Negative: Ehrlich (Ehrlich) diazotization reaction, Ge Ruikeli Bake

Graig-Leaback) reaction, mouthful (Sakaguchi)

Reaction.

(10) solubleness:

Soluble in water

Be dissolved in methyl alcohol

Be slightly soluble in ether and ethyl acetate

Can think on the basis of above-mentioned physico-chemical property that with TAN-592 A B or C or TAN-591 A, B or the C gained compound that is hydrolyzed is the 7-FA-DCPC that represents with above-mentioned formula IV,

DCPC sodium salt resultant in the example 5 and 17 that is described below is through infrared (IR) (Fig. 3 and 6), ultraviolet (UV), ¹³C nucleus magnetic resonance (NMR), mass spectrum, ultimate analysis, high pressure liquid chromatography (HPLC) Rt(retention time) value and antimicrobial spectrum confirm consistent with the authentic sample of DCPC.

DCPC is a known compound and is the useful intermediates of producing cephalosporanic olefinic antibiotic.

The physico-chemical property of the double sodium salt of the compound III of gained is as follows in the example of mentioning below 21:

(1) outward appearance:

White powder.

(2) molecular weight: molecular ion peak

M/ Z 432(M+H) ⁺Secondary ion mass spectrometry SIMS)

(3) ultimate analysis (%): (40 ℃, vacuum-drying 8 hours)

The trial value calculated value

C 38.06 38.19

H 3.81 3.66

N 9.97 9.54

O 30.88

S 7.68 7.28

Na 10.44

(4) molecular formula: C ₁₄H ₁₅N ₃O ₈SNa ₂O5H ₂O

(5) ultraviolet (UV) absorption spectrum (water):

(referring to Fig. 4)

(6) the main absorption peak (cm of infrared (IR) absorption spectrum (KBr) ^-1)

3430,3230,3010,1775,1700,1690,1610,1580,1410,1305,1240,1150,1065,1045,1000,850,800,710,515 referring to Fig. 7

(7) ¹H nucleus magnetic resonance (NMR) spectrum: 100 megacycle (MH ₂), in the deuterium, δ ppm J(H ₂)

1.75-2.15 2H，m

2.15-2.55 4H，m

3.38 1H，d，J＝18

3.70 1H，d，J＝18

4.27 2H，s，

5.39 1H，s，

8.21 1H，s，

(8) high pressure liquid chromatography (HPLC) [U.S., Wal Te Lian society, (Waters Assoc, U.S.A)], model 6000 A/660/440

Post: YMC-Pack A-312,

Moving phase: 0.01 mol phosphate buffered saline buffer (PH6.3), 2 ml/min

Detect: 254nm

The Rt=3.1 branch

The physico-chemical property of the compound ii of gained is as follows in the example 22 that will mention below:

(1) outward appearance:

White powder.

(2) molecular weight: molecular ion peak

M/Z 296(M+Na) ⁺With secondary ion mass spectrometry SIMS method

(3) molecular formula: C ₉H ₁₁N ₃O ₅S(273)

(4) ultraviolet (UV) absorption spectrum

259nm（E ^1% _1cm＝365）

(5) circular dichroism (CD) spectrum:

-33300 ± 5000 Hes

+32400±5000

(6) infrared (IR) absorption spectrum: main absorption peak (cm ^-1)

3400,2980,1760,1680,1600,1510,1410,1380,1290,1240,1180,1140,1070,1040,1000,860,790,700,500 referring to Fig. 8

(7) ¹H nucleus magnetic resonance (NMR) spectrum: 400 megacycle (MH ₂), in the deuterium, δ ppm J(H ₂)

3.43 1H，d，J＝17.6

3.65 1H，d，J＝17.6

4.21 1H，d，J＝12.9

4.25 1H，d，J＝12.9

5.19 1H，s，

8.17 1H，s，

(8) high pressure liquid chromatography (HPLC) model 638-50(Japan, the Hitachi co. of Hitachi, Ltd, Japan)

Post: YMC-Pack A-312,

Moving phase: 0.01 mol phosphate buffered saline buffer (PH6.3), 2 ml/min

Detect: 254nm

The Rt=3.0 branch

To provide some references to the biological property of 7-FA-DCPC below.The 7-FA-DCPC sodium salt has shown the antimicrobial spectrum shown in the table I, and from then on table is apparent, and antibiotic 7-FA-DCPC has resisting gram-positive and negative anti-microbial activity.

Table I: the antimicrobial spectrum of antibiotic 7-FA-DCPC sodium salt

Biological subject minimal inhibitory concentration microgram (μ g)/milliliter

Intestinal bacteria N IH J JC-2 50

（Escherichiacoli）

Salmonella typhimurium IFO 12,529 25

（Salmonella typhimurium）

Streptococcus pneumoniae IFO 3,317 50

（Klebsiella pneumoniae）

Proteus vulgaris IFO 3,988 25

（proteus vulgaris）

Proteus mirabilis ATCC 21,100 12.5

（proteus mirabilis）

Serratia marcescens IFO 12,648 25

（serratia marcescens）

Alcaligenes faecalis IFO 13,111 12.5

（Alcaligenes faecalis）

Chlorine purulence bacillus IFO3080 100

（pseudomonas aeruginosa）

Streptococcus aureus FDA209p 100

（Staphylococcus aureus）

Subtilis N IH J PC I219 100

（Bacillus subtilis）

Bacillus megaterium IFO 12,108 100

（Bacillus megaterium）

Annotate: substratum nutrient agar medium (PH7.0)

The concentration of microbe inoculation: 10 ⁶The CFU(colony-forming unit)/milliliter.

In addition, the 7-FA-DCPC sodium salt is stable to various β-Nei Xiananmeis.7-FA-DCPC and DCPC show in the table II the stability of β-Nei Xiananmei.

Table 2: to the stability of β-Nei Xiananmei

The diameter of inhibition zone (using Plating)

7-FA-DCPC DCPC CMC(annotates

1）

Do not add a control group 23 30 30

Blue or green enzyme element (annotating 2)

（penicillinase） 23 30 30

Cephalosporinase (annotating 3)

（Cephalorporinase） 23 8 ＜8

Annotate:

The concentration of compound used therefor; 100 mcg/ml (μ g/ml)

Used bacterial strain: intestinal bacteria PG8

Substratum: nutrient agar (PH7.0)

Contain diaminopimelic acid (20 mg/litre)

Annotate 1:CMC rhzomorph C(cephamyeinc)

Annotate 2: belong to the plain enzyme of blue or green enzyme that (Bacillus cereus) extract (by U.S. Ka Er than chemical company (Calbio Chemical, U.S.A produces) 0.048 units per ml from bacillus cereus

Annotate 3: from cephalosporinase 0.025 units per ml of enterobacter cloacae (Entero bacter cloacae) extraction.

In addition the 7-FA-DCPC sodium salt to the mouse bacteria infection treatment of diseases effect list in the table 3

Table 3:

Infectious bacteria route of administration ED ₅₀(milligram/kilogram)

Intestinal bacteria subcutaneous about 100

0-111* subcutaneous 59.5

*: intraperitoneal infects.

After the 7-FA-DCPC sodium salt is with 1 gram/kilogram dosage mouse subcutaneous administration, do not observe the phenomena of mortality; Therefore, 7-FA-DCPC is considered to hypotoxic.These data acknowledgements, compound IV have the anti-microbial activity of resisting gram-positive and negative bacteria and are an antibiotic low to mammalian toxicity.The compound IV, wherein R is a formamido-, is stable to the bacterial strain that produces β-Nei Xiananmei, thereby can be used for treating Mammals (such as mouse, rat, rabbit, dog, human or the like) disease that causes by infectation of bacteria, adopt when the compound IV for example is used as the agent of bacterial-infection resisting treatment of diseases and be different from oral drug administration by injection approach, above-mentioned Mammals is adopted subcutaneous injection or intramuscular injection, and dosage is about 2 to 100 milligrams/kg/day, preferably about 10 to 50 milligrams/kg/day.The compound IV is made capsule and pill, and as the formulation of oral medicine, dosage is about 10 to 200 milligrams/kg/day (in the compound IV), preferably about 20 to 100 milligrams/kg/day.

In addition, compound (IV) can be used as sterilant, compound (IV) is for example made liquid preparation, the concentration that is dissolved in the compound IV in the distilled water in the said preparation is about 0.02 to 0.2 weight/volume %, make ointment and then contain 0.5 to 50 milligram, preferably every restraint agent contains 2 to 20 milligrams of compounds (IV), and above-mentioned preparation can be coated with sterilizations such as being used for above-mentioned mammiferous hand, pin, eye, ear and sterilization outward.

The compound IV also has very high value as the intermediate of a synthetic new drug.

Hydrolysis of the present invention comprises that 3 selective dissociation methods of the present invention that go up ester bonds that its result is to improve purpose compound IV yield are that superiority is arranged on industrial production.

Because the initial compounds V in the process of the present invention can be passed through microbial culture scale operation in a short time, in addition, utilization the present invention can make purpose compound (IV) mass production behind above-mentioned fermentation process, improve yield, shorten man-hour, thereby just provide one to help the compound IV is carried out industrial method.

The compound III is useful as the intermediate of producing cephem antibiotic.For example produce the The compounds of this invention II.

After the biological property of compound ii was illustrated in, the antimicrobial spectrum of compound ii sodium salt was as shown in table 4.Show visible compound ii from this and have anti-Gram-negative anti-microbial activity.

The antimicrobial spectrum of table 4 compound ii sodium salt.

The biological subject antibacterial circle diameter

(millimeter) (referring to explaining)

Intestinal bacteria CP 13 11

（Escherichia coli）

Pseudomonas aeruginosa C 141 19

（Pseudomonas aeruginosa）

Streptococcus aureus FDA209p

（Staphylococcus aureus） ＜8

Annotate: substratum: nutrient agar (PH7.0) contains diaminopimelic acid

The concentration of compound ii: 1000 micrograms (μ g)/milliliter

In addition, the sodium salt of compound (II) is stable to various β-Nei Xiananmeis.Compound ii and DCPC list in the table 5 stability of various β-Nei Xiananmeis

The stability of table 5 pair β-Nei Xiananmei

The diameter of enzyme inhibition zone (millimeter) (Plating)

Compound (II) DCPC(annotates 1) CMC(notes 2)

Do not add-control group 19 30 27.5

Penicillinase (annotating 2) 19 30 27.5

Cephalosporinase (annotating 3) 19＜8＜8

Annotate: concentration 1000 mcg/ml of compound ii

Another compound concentrations: 100 mcg/ml

Used bacterial strain: Rhodopseudomonas Pseudomonas aeruginosa (pseudomonas aeruginosa) C141

Substratum: nutrient agar (PH7.0) contains diaminopimelic acid (20 milligrams/liter)

Annotate 1:DCPC: deacetylcephalosporinC

Annotate the 2:CMC cephamycin C

Annotate 3: (U.S. Ka Er is than chemical company (Calbio Chemical, U.S.A) 0.048 units per ml for the penicillinase that extracts from bacillus cereus (Bacillus cereus)

Annotate 4: enzyme 0.025 units per ml of from enterobacter cloacae (Entero bacter cloacae), extracting.

The sodium salt of compound (II) is not found death with the dosage subcutaneous rat administration of 1 gram/kilogram, thereby compound (II) is considered to hypotoxic.These data acknowledgements, compound (II) has the anti-microbial activity of anti-gram negative bacillus, and is a hypotoxic antibiotic.

Therefore compound ii can be used for treating Mammals by bacterial infectious diseases (for example mouse, calf, horse, dog, the mankind etc.) or poultry (for example: goose, duck)

Compound (II) for example is used as the therapeutical agent that resistance to bacteria catches, then compound (II) can for example adopt and be different from oral drug administration by injection approach, above-mentioned Mammals is then adopted subcutaneous injection, or intramuscular injection, its dosage is about 5 to 200 milligrams/kg/day, is preferably 20 to 200 milligrams/kg/day.It is about 20 to 400 milligrams/kg/day (in compound ii) as its dosage of formulation of oral medicine that compound ii is made capsule and pill, preferably about 40-200 milligram/kg/day.

In addition, compound (II) can be used as sterilant.Compound (II) is for example made liquid preparation, contains the concentration that is dissolved in the compound ii in the distilled water in the said preparation and is about 0.05 to 0.4 weight/volume %, makes ointment and then contains and have an appointment 1 to 100 milligram.Preferably every restraint agent contain have an appointment 4 to 50 milligrams of compounds (II) said preparation can outside be coated with and be used for sterilization that above-mentioned mammiferous hand, pin, eye, ear etc. locate and sterilization to make anti-infective usefulness.

Compound (II) also is the intermediate that highly is worth that has of a synthetic cephem compounds.

From cephalosporin with go to learn that the acetylizad cephalosporins derivatives in 3-position is unsettled, yet has-CH the comparison of acetyl cephalo C on the 3-position ₂(this compound has-CH in the 3-position this compound of OH base (II) than acetylizad cephem compounds in the aqueous solution ₂OCOCH ₃Base) wants unexpected stable.This fact shows that this compound ii is easy to be used as intermediate in the reaction of producing cephem compounds.

Fig. 1 and Fig. 4 represent the ultraviolet absorption spectrum of the compound IV of example 1 and 9 gained.Fig. 2,3,5,6,7 and 8 expression examples, 1 gained compound IV, example 9 gained compound IV, example 5 gained DCPC, example 17 gained DCPC, example 21 gained compound III, the infrared absorption spectrum of example 22 gained compound iis.

Introduce the present invention with reference example is more detailed below.

Unless the % in substratum represents weight/volume other indicating arranged.

Reference example 1

(1) the bacterium lactan that dissolves that is grown on the nutrient agar slant medium belongs to (Lysobacter Lactamgenus) Yk-90(IFO 14288, FERM BP-575) 3 200 milliliters Alan Mei Shi of inoculation (Erlenmeyer) bottle, contain by 2% glucose in each bottle, 3% soluble starch, the 1% big bean flour of giving birth to, 1% corn steep liquor, 0.5% poly-peptone [Japanese Taihe county (Daigo Nutr it ive Chemicals of nutrient chemistry company limited, Ltd.) produce] and 0.3% sodium-chlor, 40 milliliters of the aqueous solution substratum (PH7) that the blending of 0.5% precipitated chalk is formed carry out 48 hours shaking culture to obtain inoculum in 24 ℃ on rotary shaker.

Then will be by 3% dextrin, 1.5% raw soybean face, 1.5% Semen Maydis powder, 0.2% poly-peptone, 0.1% Sulfothiorine, 4000 milliliters of the aqueous solution (PH6.5) substratum that 0.5% precipitated chalk blending is formed are assigned in 200 milliliters of erlenmeyer flasks with every part 40 ml vol, and this bottle was sterilized 20 minutes at 120 ℃ then.1 milliliter of inoculum is changed over to respectively in each the 200 milliliters erlenmeyer flasks that contain above-mentioned substratum, and the velocity of rotation with 200 rev/mins on rotary shaker was cultivated 72 hours for 24 ℃.

Regulate the pH value to 3.5 of the nutrient solution of said procedure acquisition with 7% oxalic acid, mix mutually with Harvard sieve-senior-glue (hyflo-Super-Gel) [U.S. John Mai Folan (Johns Manville) product], filter, get filtrate (16 liter), filtrate is regulated pH value to 6.8, by a pillar (1 liter) that activated carbon is housed.Wash pillar (3 liter) with water, antibiotic TAN-547 is eluted with 8% isopropylcarbinol-N/200 hydrochloric acid (8 liter).Elutriant is concentrated into 1.8 liters, and concentrated solution is by amberlite (Amberlite) CG-50(H ⁺Type, 1.4 liters) [U.S. Luo Mo, (Rohm of Haars Co., Ltd; Haas co. U.S.A.) produces] post.Water (4.5 liter) is washed post, carry out (9 liter) classification wash-out with N/100 hydrochloric acid then, collect active part, concentrate, regulate the pH value to 7.3 of concentrated solution and it is equipped with dianion exchange resin HP-20(50 to 100 order by one, 0.5 the post (pillar of Mitsubishi chemical industry company limited (Mitsubishi Chemical Industries, Ltd. Japan) produce) liter).Behind phosphoric acid buffer (PH3.5,5 liters) the flushing pillar with 0.01 molconcentration, carry out the classification wash-out with 0.01 molar phosphoric acid buffer.Collect significant part, elutriant is regulated pH value to 7.2, and by a pillar that activated carbon (100 milliliters) are housed, pillar washes (300 milliliters) with water, carries out wash-out with 8% isopropylcarbinol-N/200 hydrochloric acid (600 milliliters) with it.Elutriant concentrates, and concentrated solution is equipped with CM-dextrane gel C by one ₂₅(Na ⁺Type, 200 milliliters) pillar of [Sweden Pharmacia fine chemistry company (pharmacia Fine Chemicals co., sweden produces], use 0.02 mol sodium chloride aqueous solution (6 liter) to carry out wash-out subsequently.Each component is analyzed with high pressure liquid chromatography respectively, the corresponding TAN-A that contains, and the component of B and C collects as main component.

To contain TAN-547 A component and regulate its pH value to 7.2, it by a pillar that activated carbon (10 milliliters) are housed, after water (30 milliliters) is washed post, will be carried out wash-out with 8% isopropylcarbinol-N/200 hydrochloric acid (70 milliliters) as main component.Elutriant concentrates, and the concentrated solution lyophilize gets Powdered crude product TAN-547A dihydrochloride (61 milligrams).

The component that will contain TAN-547B and C is used with quadrat method as main component and is handled, and correspondingly obtains Powdered crude product TAN-547B dihydrochloride (144 milligrams), Powdered crude product TAN-547 C dihydrochloride (226 milligrams).

The dihydrochloride of Powdered crude product (61 milligrams) TAN-547A separates with preparation property high pressure lipuid chromatography (HPLC), and this separator column stops up with YMC-gel ODS I-15(Japan mountain village chemical test

(Yamamura Chemical Laboratories, Japan)] be carrier, and carry out the classification wash-out with 0.02 mol phosphoric acid buffer (PH3.0), various piece uses high pressure lipuid chromatography (HPLC) (HPLC) to analyze respectively, collect and only show unimodal part, regulate the pH value to 7.5 of active part with 1 normal concentration sodium hydroxide solution, again it is transferred to PH3.0 with 1 normal concentration hydrochloric acid soln again, it is passed through activated carbon column (5 milliliters), after water (25 milliliters) is washed post, with 8% isopropylcarbinol aqueous solution wash-out, elutriant concentrates, and lyophilize gets the dihydrochloride (40 milligrams) of white powder TAN-547A.The dihydrochloride of TAN-547B and C with being prepared property of method high pressure lipuid chromatography (HPLC) separate the dihydrochloride (96 milligrams) of white powder TAN-547B and the dihydrochloride (112 milligrams) of white powder TAN-547C.

(2) the bacterium lactan that dissolves that will grow on nutrient agar slant medium belongs to (Lysobacter lactamgenus) YK-90(IFO 14288, FERM BP-575) is seeded in 22 liters mouth (Sakaguchi) flask, each flask is equipped with 500 milliliters of substratum aqueous solution (PH7.0), and it consists of: glucose 2%, soluble starch 3%, raw soya bean powder 1%, corn steep liquor 1%, poly-peptone 0.5% and sodium-chlor 0.3%, and mix 0.5% precipitated chalk.Place then on the reciprocating type bottle swingging machine, cultivated 48 hours down in 24 ℃, with whole nutrient solutions of gained transfer to one 200 liters the jar in, in dress mix 0.5% antifoam agent Actcol(Japan Takede Chemical Industries Ltd and produce) above-mentioned substratum (120 liters), at (speed is 120 liters/minute) and the corn steep liquor 1% of blowing, poly-peptone 0.5% and sodium-chlor 0.3%, and mix 0.5% precipitated chalk.Place then on the reciprocating type bottle swingging machine, cultivated 48 hours down in 24 ℃, dress mixes 0.5% antifoam agent Actcol(Japan Takede Chemical Industries Ltd and produces in whole nutrient solutions of gained being transferred in one 200 liters the cylinder) above-mentioned substratum (120 liters), blow (speed is 120 liters/minute) and (150 rev/mins) condition of stirring under in 24 ℃ of cultivations 48 hours.Whole nutrient solutions of gained are transferred in one 6000 liters the cylinder again, in adorn 4000 liters of substratum aqueous solution, it consists of: dextrin 3%, raw soya bean powder 1.5%, corn matter 1.5%, poly-peptone 0.2% and Sulfothiorine 0.1%, and mix 0.5% precipitated chalk and 0.05% Actcol, blow (speed be 4000 outside/minute) and stirring (120 rev/mins) condition under in 24 ℃ of cultivations 66 hours.

The nutrient solution of said process gained (3900 liters) uses 2N hydrochloric acid to regulate PH to 6.1, adds Hyf10-Super Gel, filters washing, gets 4370 liters of filtrates.The PH of filtrate is adjusted to 7.0, and by the post of 120 liters of 50-100 purpose Dowex, 50 W Na type ion exchange resin is housed, post is used 2M sodium chloride aqueous solution (1800 liters) wash-out again after water (360 liters) is washed then.Elutriant is by activated carbon column (60 liters of charcoals).Post is used 420 liters of 8% isopropylcarbinols-N/200 hydrochloric acid wash-out again after 180 premium on currency are washed.Elutriant is concentrated into 40 liters, regulates its PH to 7.3, and by the post of 40 liters of Diaion HP-20 is housed, post is after the washing of the 0.01M of 80 liters of PH7.3 phosphoric acid buffer, with the 0.01M phosphoric acid buffer wash-out of 400 liters of PH3.5 again.

Elutriant is by activated carbon column (in adorn 10 liters of charcoals), with 30 premium on currency with the post washing after, use 70 liters of 8% isopropylcarbinols-N/200 hydrochloric acid wash-out again.Elutriant is concentrated into 2 liters, regulates PH to 7.3, and by the post of 4 liters of 50-100 order Diaion HP-20 is housed, post is after the washing of the 0.01M of 12 liters of PH7.3 phosphoric acid buffer then, and the 0.01M phosphoric acid buffer wash-out with 40 liters of PH3.5 separates again.Each wash-out part is all analyzed with HPLC, and is divided into two groups, and one group contains main ingredient TAN-547A, B, and C, and another group contains TAN-547D, E and F.Merge and contain main ingredient TAN-547D, the part of E and F, and by gac (300 milliliters) post, water is used 2100 milliliters of 8% isopropylcarbinols-N/200 hydrochloric acid wash-out after post is washed again.The post of the cross-linked glucose CM-Sephadex C25 of elutriant by 300 milliliters of Na types are housed separates with 12 liters of 0.02M sodium chloride aqueous solution wash-outs then.Each wash-out part is analyzed with HPLC, collects respectively to contain main ingredient TAN-547D, the part of E and F.

Merge the part that contains main ingredient TAN-547F, and pass through gac (50 milliliters) post, after with 150 ml waters post being washed, use 350 milliliter of 8% isopropylcarbinol-/200 hydrochloric acid wash-out again, elutriant concentrates, and enriched material gets 1.0 gram TAN-547F crude product powder through lyophilize.Handle the part that contains main ingredient TAN-547D and E respectively by same program, get 0.6 gram TAN-547E crude product powder of 0.3 gram TAN-547D crude product powder.

With 1.0 gram TAN-547F crude products preparation HPLC separation and purification, carrier is the mountain village chemistry institute production of YMC-GEL ODS30/60(Japan), separate with 2% methyl alcohol-0.02M phosphoric acid buffer (PH3.0) wash-out, each wash-out part is analyzed with HPLC, collection contains the part of main ingredient TAN-547F, regulates its PH to 7.1, and by gac (20 milliliters) post, with after the post washing, use 140 milliliters of 8% isopropylcarbinols-N/200 hydrochloric acid wash-out with 60 ml waters again.Elutriant concentrates, enriched material separates with preparation HPLC, carrier is TSK-GEL, LS-410(Japan Japan Cao Da Manufacturing Co., Ltd produces) separate with 1% methyl alcohol/0.01M phosphoric acid buffer (PH3.0) wash-out, each wash-out part is analyzed with HPLC, collection has only the part at a peak, merge and regulate PH to 7.0, use 1N hydrochloric acid re-adjustment PH to 3.0 again, and pass through gac (10 milliliters) post with 1N sodium hydroxide, post is after 60 milliliters of washings, with 140 milliliters of 8% isopropylcarbinols-N/200 hydrochloric acid wash-out, elutriant concentrates, and enriched material is all through lyophilize, get 69 milligrams of TAN-547F, the 2HCl white powder.TAN-547D and E crude product powder are also pressed with quadrat method HPLC separation and purification.Get 30 milligrams of TAN-547D HCL white powders.

It is as follows that top gained gets its physicochemical property of microbiotic TAN-547 2HCl:

（1）TAN-547A 2HCl：

1) outward appearance: white powder

2) specific optical rotation: [α] ²⁵ _D+ 71.8+20(C=0.50, water)

3) molecular weight: SIMS method, (M+H) 688

4) molecular formula: C ₂₆H ₄₁N ₉O ₁₁S2HCl(3H ₂O)

5) ultimate analysis (%):

The analytical value calculated value

C，38.29±20 C，38.33

H，6.48±1.0 H，6.06

N，15.11±1.5 N，15.47

O，27.49

S，4.12±1.0 S，3.94

Cl，8.71±1.5 Cl，8.70

* 1, sample is in decompression drying at room temperature (siccative P down ₂O ₅) 15 hours,

* 2, this is the calculated value that contains the sample of 3 mol water

6) ultra-violet absorption spectrum (UV):

260±2nm（E ^1% _1cm＝117±20）

7) garden dichroscope spectrum (CD)

-30900 ± 5000 Hes

29500±5000

(-: negative value (-) circular dichroism (cotton effect); +: on the occasion of (+) circular dichroism)

8) infrared absorption spectrum (IR): main wave number (KBr sheet):

3420，3250，3080，3000，1775，1730，1670，1510，1450，1440

1260，1165，1060，980，860，510，

9) nuclear magnetic resonance spectrum ( ¹³C-NMR): D ₂O, the signal of 100MHz (δ ppm) as follows

179.84（S），177.42（S），176.05（S），173.75（S），171.12（S），

166.40（d），162.16（S），159.62（S），134.86（s），117.40（S），

79.64（S），72.66（d），67.16（t），65.94（d），57.34（d），56.31（d）

52.03（d），43.59（t），41.23（t），37.36（t），32.80（t），29.98（t）

28.60（t），27.50（t），23.50（t），19.75（q）

(S: unimodal, d: doublet, t: triplet, q: quartet)

10) amino acid analysis: sample uses 5.5N hydrochloric acid in 110 ℃ of following hydrolysis 15 hours

L-Ala: 0.86 mol

α-An Jijiersuan: 0.94 mol

11) thin-layer chromatography (TLC): point sample membrane fiber element (production of Tokyo chemical industrial company)

The solvent system, acetonitrile: 3% ammonium sulfate (1: 1), R _f=0.52

12) HPLC (high performance liquid chromatography) (HPLC):

Post, YMC pack A312(Japan mountain village chemistry laboratory is made), mobile phase, 2% methyl alcohol/0.01M phosphoric acid buffer (PH3.0), 2 ml/min.The Rt=5.8(branch) component A, B and C(2HCl) following character be common.

13) solubleness:

Be soluble in: water, aqueous acetone solution, alcohol solution

Be slightly soluble in: dimethyl sulfoxide (DMSO), methyl alcohol, acetone vinyl acetic monomer

14) color reaction:

Positive: (hydration) triketohydrindene hydrate, Ge Ruike-Li Beike (Greig-Leaback), slope

Mouth reaction (Sakaguchi)

Negative: Ehrlich (Elrlich) Baeyer is stepped on (Barton) reaction potassium permanganate

15) stability:

Unstable in acid and alkaline aqueous solution, unstable a little in neutral aqueous solution

16) characteristic of these materials: be amphiprotic substance (dihydrochloride is neutral)

（ⅱ）TAN-547B2HCl：

1) outward appearance: white powder

2) specific optical rotation: [α] ²⁵ _D+ 54.8 ± 15(C=0.56, H ₂O)

3) molecular weight: the SIMS method, (M+H) ⁺759

4) molecular formula: C ₂₉H ₄₆N ₁₀O ₁₂S2HCl(3H ₂O)

5) ultimate analysis (%):

The analytical value calculated value

C，39.02±20 C，39.32

H，6.51±1.0 H，6.14

N，15.46±1.5 N，15.81

O，27.09

S，3.50±1.0 S，3.62

Cl，8.27±1.5 Cl，8.01

* 1, * 2, and condition and A's is identical

6) UV spectrum: Fig. 3

7) CD spectrum:

-33600 ± 5000 Hes

+30700±5000

8) IR spectrum: main wave number is: (cm ^-1)

3370，3260，3220，3080，3000，1780，1735，1675，1535，1460，1410，1260，1170，1070，880，800，530，

9) ( ¹³C-NMR) spectrum: D ₂O, (δ ppm) as follows is as follows for the signal of 100MHz

179.78（S），177.92（S），176.88（S），176.15（S），173.49（S），

170.77（S），166.41（d），162.25（S），159.59（s），134.39（S），

118.69（S），79.66（S），72.91（d），67.11（t），66.04（d），

56.95（d），56.03（d），53.14（d），51.72（d），43.61（t），41.52（t）

37.32（l），32.62（l），29.23（l），28.66（l），27.50（l），23.47（l）

19.55（g），19.51（g）

10) amino acid analysis: (condition is identical with A's)

L-Ala: 2.1 mol

α-An Jijiersuan: 1.1 mol

11) TLC:(condition and A's is identical)

R＝0.55

12) HPLC:(condition and A's is identical)

The R=6.8(branch)

（ⅱ）TAN-547C 2HCl：

1) outward appearance: white powder

2) specific optical rotation: [α] ²⁵ _D+ 25.1 ± 15(C=0.49, H ₂O)

3) molecular weight: the SIMS method, (M+H) ⁺830

4) molecular formula: C ₃₂H ₅₁N ₁₁O ₁₃S2HCl(3H ₂O)

5) ultimate analysis (%):

Analytical value ^{* 1}Calculated value ^{* 2}

C，39.61±20 C，40.17

H，6.54±1.0 H，6.22

N，15.92±1.5 N，16.10

O，26.75

S，3.41±1.0 S，3.55

Cl，6.41±1.5 Cl，7.41

* 1, * 2, and condition and A's is identical.

6) UV spectrum:

260±2nm（E ^1% _1cm）＝106±20）

7) CD spectrum:

-34700 ± 5000 Hes

+28400±5000

8) IR spectrum: main wave number (cm ^-1) (KBr sheet) be:

3350，3250，3070，3000，2950，1780，1735，1665，1530，1450

1400，1300，1250，1160，1060，790，520

9) C ¹³-NMR spectrum: D ₂O, (δ ppm) as follows is as follows for the signal of 100MHz

179.79（S），178.04（S），177.47（S），177.38（S），176.12（S），

173.47（S），171.08（S），166.32（d），162.07（s），159.52（S），

135.00（S），117.27（S），79.57（S），72.86（d），67.10（t），

65.88（d），57.28（d），55.87（d），53.07（d），52.35（d），51.62（d）

43.52（t），41.52（t），37.27（t），32.73（t），29.25（t），28.48（t）

27.36（t），23.41（t），19.45（g），19.42（g），19.25（g）

10) amino acid analysis: (condition is identical with A's)

L-Ala: 3.1 mol

α-An Jijiersuan: 1.1 mol

11) TLC:(condition and A's is identical)

R _f＝0.60

12) HPLC:(condition and A's is identical)

The R=11.7(branch)

+21000±5000

(-: negative value (-) circular dichroism; +: on the occasion of (+) circular dichroism)

8) nuclear magnetic resonance spectrum ( ¹³C-NMR): D ₂O, the signal of 100MHz (δ ppm) as follows

179.52（S），177.43（S），176.05（S），173.72（S），171.61（S），

167.89（S），159.57（S），134.40（S），118.99（s），72.64（d）

67.23（t），61.99（d），60.13（d），57.32（d），56.28（d），51.97（d）

43.53（l），41.18（l），37.58（l），32.75（l），28.94（l），28.43（l）

27.46（l），23.85（l），19.70（g），

(S: unimodal, d: double honeybee, l: triple honeybees, q: quartet)

9) infrared absorption spectrum (IR): main wave number (KBr sheet):

3420，3250，3075，2950，1765，1735，1665，1540，1450，1400

1350，1270，1160，1115，1065，1030，980，750，540

10) amino acid analysis: sample uses 5.5N hydrochloric acid in 110 ℃ of following hydrolysis 15 hours

L-Ala: about 1 mol

α-An Jijiersuan: about 1 mol

（ⅳ）TAN-547D 2HCL：

1) outward appearance: white powder

2) specific optical rotation: [α] ²⁵ _D+ 53.5 ± 10(C=0.51, H ₂O)

3) molecular weight: the SIMS method, (M+H) ⁺645

4) molecular formula: C ₂₅H ₄₀N ₈O ₁₀S HCl(3H ₂O)

5) ultimate analysis (%):

Analytical value ^{* 1}Calculated value ^{* 2}

C，40.30±2.0 C，40.84

H，6.44±1.0 H，6.44

N，15.34±1.5 N，15.24

O，28.29

S，4.39±1.0 S，4.36

Cl，4.66±1.5 Cl，4.82

* 1, sample is in decompression drying at room temperature (siccative PO) 15 hours down

* 2, this is the calculated value that contains the sample of 3 mol water

6) ultra-violet absorption spectrum (UV):

260±2nm（E ^1% _1cm＝122±20）

7) garden dichroscope spectrum (CD)

-33000 ± 5000 Hes

11) thin-layer chromatography (TLC): (Tokyo chemical industrial company produces for point sample film, Mierocrystalline cellulose

The solvent system, acetonitrile: 3% ammonium sulfate (1: 1), R _f=0.50

12) HPLC (high performance liquid chromatography) (HPLC):

Post, YMC pack A312, mobile phase, 5% methyl alcohol/0.01M

Phosphoric acid buffer (PH3.0), 2 ml/min.The Rt=4.2(branch) component D, E and F(are HCl salt) following character be common

13) solubleness:

Be soluble in: water, aqueous acetone solution, alcohol solution

Be slightly soluble in: dimethyl sulfoxide (DMSO), methyl alcohol, acetone vinyl acetic monomer

14) color reaction:

Positive: (hydration) triketohydrindene hydrate, Ge Ruike-Li Beike (Greig-Leaback), slope

Mouth reaction (Sakaguchi)

Negative: Ehrlich (Elrlich) Baeyer is stepped on (Barton) reaction potassium permanganate

15) stability:

Unstable a little in acidity and neutral aqueous solution, unstable in basic solution

16) characteristic of these materials: be amphiprotic substance (2HCl is neutral)

（ⅴ） TAN-547E2HCl：

1) outward appearance: white powder

2) specific optical rotation: [α] ²⁵ _D+ 31.1 ± 10(C=0.51, H ₂O)

3) molecular weight: SIMS method, (M+H) 716

4) molecular formula: C ₂₈H ₄₅N ₉O ₁₁S2HCl(3H ₂O)

5) ultimate analysis (%):

Analytical value ^{* 1}Calculated value ^{* 2}

C，39.86±2.0 C，39.90

H，6.28±1.0 H，6.34

N，14.64±1.5 N，14.96

O，26.58

S，3.79±1.0 S，3.80

Cl，7.83±1.5 Cl，8.41

* 1, * 2, and condition and D's is identical

6) UV spectrum:

260+2nm（E ^1% _1cm＝114±20）

7) CD spectrum:

-31000 ± 5000 Hes

+21000±5000

8) IR spectrum: main wave number (cm ^-1) as follows:

3375，3260，3220，3075，2950，1770，1735，1660，1540，1455

1400，1345，1250，1160，1115，1065，1035，980，880，815，540

9) C ¹³-NMR spectrum: D ₂O, the signal of 100MHz (δ ppm) as follows

179.48（S），177.82（S），177.36（S），176.12（S），173.46（S），

171.49（S），167.83（S），159.54（d），134.37（s），118.99（S），

72.84（d），67.19（l），61.95（d），60.10（d），57.31（d），55.96（d）

53.04（d），51.63（d），43.53（l），41.47（l），37.53（l），32.72（l）

29.14（l），28.38（l），27.43（l），23.81（l），19.44（g），

10) amino acid analysis: (condition is identical with D's)

L-Ala: about 2 mol

α-An Jijiersuan: about 1 mol

11) TLC:(condition and D's is identical)

R _f＝0.54

N，14.52±1.5 N，15.33

O，26.26

S，2.92±1.0 S，3.51

Cl，7.12±1.5 Cl，7.76

* 1, * 2, and condition and D's is identical.

6) UV spectrum:

260±2nm（E ^1% _1cm＝94±20）

7) CD spectrum:

-30000 ± 5000 Hes + 17000 ± 5000

8) IR spectrum: main wave number (cm ^-1) (KBr sheet) as follows

3360，3250，3070，3000，2950，1770，1750，1660，1535，

1455，1395，1345，1240，1160，1115，1065，1030，980，960，

870，510

9) ¹³C-NMR spectrum: D ₂O, the signal of 100MHz (δ ppm) as follows

179.39（s），177.87（s），177.26（s），176.47（s），176.15（s），

173.45（s），170.68（s），168.00（s），159.65（s），133.51（s），

121.03（s），72.90（d），67.15（t），62.04（d），52.44（d），

51.77（d），43.64（t），41.54（t），37.47（t），32.54（t），

29.30（t），28.56（t），27.41（t），23.77（t），19.55（q），

19.44（q），19.37（q）

10) amino acid analysis: (condition is identical with D's)

L-Ala: about 3 mol

α-An Jijiersuan: about 1 mol

11) TLC:(condition and D's is identical)

Rf＝0.58

12) HPLC:(condition and D's is identical)

The Rt=6.5(branch)

Measure through the HPLC method, the absolute configuration of L-Ala and α-An Jijiersuan is respectively L-and R-type.

Reference example 2

(1) will triple from Japan (Mie) Fu Ashan (Ayama) the herbarium gathered of regional Tsuge isolating vitiligoidea bacterium lactan belong to (Xanthomonas lactamgena) YK-280(IFO 14330, FERM BP-635) on agar nutrition slant medium, cultivates, be inoculated into then in one 2 liters mouth (Sakaguchi) flask, in 500 milliliters of substratum aqueous solution (PH7.0) are housed, it consists of: glucose 2%, soluble starch 3%, raw soya bean powder 1%, corn steep liquor 0.3%, poly-peptone 0.5% and sodium-chlor 0.3%, and mix 0.5% precipitated chalk.Flask is placed on the reciprocating type bottle swingging machine, cultivated 48 hours down in 24 ℃, with whole nutrient solutions of gained transfer to one 200 liters the jar in, in adorn 120 and go up and state substratum, and mix 0.05% antifoam agent Actcol, under the ventilation (120 liters/minute) and (120 rev/mins) condition of stirring, cultivated 48 hours in 24 ℃.All nutrient solutions are transferred in one 2000 liters the jar again, in adorn 1200 liters of substratum aqueous solution, it consists of (not regulating PH): dextrin 3%, raw soya bean powder 3%, poly-peptone 0.2% is also mixed 0.05% antifoam agent Ac tcol and 0.5% precipitated chalk, under ventilation (1200 liters/minute) and stirring (100 rev/mins), cultivated 66 hours in 24 ℃.

Regulate PH to 6.0 through the nutrient solution (1140 liters) that said process obtains with 2NHCl, add Hyf10-Super Gel then, filter, wash, get 1370 liters of filtrates.The PH of filtrate is adjusted to 6.3, and by 120 liters of Dowex 50W(25 liters, 50-100 order, Na are housed ⁺Type) post of ion exchange resin.Post is after water (75 liters) is washed, with 2M sodium chloride aqueous solution wash-out.Elutriant is by the post of 15 liters of gacs is housed, after washing (45 premium on currency), with 8% isopropylcarbinol-N/100 hydrochloric acid (105 liters) wash-out, the PH of elutriant is adjusted to 6.2, be concentrated into 12 liters then, the PH with concentrated solution is adjusted to 7.3 again, and by the post of 10 liters of Diaion HP-20 is housed.With the 0.01M phosphoric acid buffer washing of 20 liters of PH7.3, use the 0.01M phosphoric acid buffer wash-out of 100 liters of PH2.5 again.

Elutriant after the washing of 6 premium on currency, is used 18 liters of 8% isopropylcarbinols-N/200 hydrochloric acid wash-out by the post of 2.5 liters of gacs is housed again.Elutriant is concentrated into 1.6 liters, regulates its PH to 7.3, and by the post of 2 liters of 50-100 order Diaion HP-20 is housed, after the 0.01M phosphoric acid buffer washing with 4 liters of PH7.3, the 0.01M phosphoric acid buffer wash-out with 20 liters of PH3.0 separates again then.Each wash-out part is used the liquid chromatography(LC) analysis, and the part of gained is divided into two groups, and one group contains main ingredient TAN-592A, B, and C, and another group contains main component TAN-592D, E and F.

The part that will contain main ingredient TAN-592A, B and C merges and by the post of 500 milliliters of gacs is housed, after water (1.5 liters) is washed, with 3 liters of 8% isopropylcarbinols-N/200 hydrochloric acid wash-out.Elutriant is concentrated, and concentrated solution is by being equipped with 0.8 liter of Na ⁺The post of the CM-Sephadex C25 of type separates with 40 liters of 0.02M sodium chloride aqueous solution wash-outs, and various piece liquid chromatography (LC) analysis with the part collection that shows that TAN-59A and B are unimodal, and is collected the part that contains main ingredient TAN-592C.

Merge the part only contain TAN-592A, and by the post of 0.3 liter of gac is housed, through water (0.9 liter wash after, with 2.1 liters of 8% isopropylcarbinols-N/200 hydrochloric acid wash-out, elutriant concentrates, after lyophilize 1.5 restrain TAN-592A.2 HCl white powders.Get 2.0 gram TAN-592B HCl white powders and obtain 1.9 gram TAN-592C crude product powder from the part that only contains TAN-592B by said procedure from the part that contains main ingredient TAN-592C.

In TAN-592C crude product powder (0.7 gram) packed into 100 milliliters of Na are arranged ⁺In the post of type CM-Sephadex C-25, separate with 3 liters of 0.02M sodium chloride aqueous solution wash-outs, each wash-out part is analyzed with liquid chromatography(LC), collection contains the part of main ingredient TAN-592C, and by the post of 30 milliliters of gacs is housed, after water (100 milliliters) was washed, with 210 milliliters of 8% isopropylcarbinols-N/200HCl wash-out, elutriant concentrated.Concentrated solution HPLC [use the YMC-Pack SH-343 post of 20 millimeters X250 millimeters of φ of mountain village (yamamura) chemical company production) separate, 0.01M phosphoric acid buffer wash-out with PH3.00 separates, each wash-out is partly used liquid-phase chromatographic analysis, collects only to show unimodal part.The PH of these significant parts is adjusted to 7.3 with 1N NaOH, pulls back to PH3.0 with 1N HCL again, and by 20 milliliters of activated carbon columns are housed, after water (80 milliliters) is washed, with 200 milliliters of 8% isopropylcarbinol-water elutions.Elutriant obtains 110 milligrams of TAN-592C 2HCl-white powders after concentrated and lyophilize.

The part that contains main ingredient TAN-592D, E and F that merging obtains from said process, and by the post of 200 milliliters of gacs is housed, after water (600 milliliters) is washed, with 1.4 liters of 8% isopropylcarbinols-N/200 HCl wash-out, elutriant is after concentrating, by 300 milliliters of Na are housed ⁺The post of type CM-Sephadex c-25 separates with 15 liters of 0.02M sodium chloride aqueous solution wash-outs then, and the part that contains main ingredient TAN-592D, E and F is collected in each wash-out part liquid chromatography (LC) analysis respectively.

Merge the part that contains main ingredient TAN-592D, and, after the washing of 250 ml waters, use 560 milliliters of 8% isopropylcarbinols-N/200 HCl wash-out again by the post of 80 milliliters of gacs is housed, elutriant gets 1236 milligrams of TAN-592D crude product powder through concentrating and lyophilize.By same program, obtain 1560 milligrams of TAN-592E and 656 milligrams of TAN-592F crude product powder respectively from the part that contains main ingredient TAN-592E and F.

With the TAN-592D(1236 milligram) separate (pillar is YMC-Pack SH-343) with HPLC (high performance liquid chromatography), separate with 1% methyl alcohol-0.01M phosphoric acid buffer (PH3.0) wash-out, various piece is analyzed with liquid chromatography (LC), collect and only show unimodal part, regulate its PH to 7.3 with 1NNaOH then, and by the post of 50 milliliters of gacs is housed, after the washing of 200 ml waters, with 400 milliliter of 8% isopropylcarbinol aqueous solution wash-out, elutriant obtains 232 milligrams of TAN-592D 2HCl white powders after concentrated and lyophilize.

By as above-mentioned same program, the crude product powder of TAN-592E and F is separated with HPLC (high performance liquid chromatography), obtain 501 milligrams of TAN-592E.2HCl and 46 milligrams of TAN-592F.2HCl respectively, be white powder.

The physico-chemical property of the TAN-592.HCl of above-mentioned gained is as follows.

（ⅰ）TAN-592A.2HCl：

1) outward appearance: white powder

2) molecular weight: the SIMS method, (M+H) ⁺704

3) molecular formula: C ₂₆H ₄₁N ₉O ₁₂S2HCL(2H ₂O)

4) ultimate analysis (%):

Analytical value ^{* 1}Calculated value ^{* 2}

C，38.49±2.0 C，38.43

H，6.03±1.0 H，5.83

N，15.63±1.5 N，15.51

O，27.56

S，4.22±1.0 S，3.95

Cl，7.95±1.5 Cl，8.73

* 1, sample is in the following 60 ℃ of drying (P of decompression ₂O ₅) 8 hours.

* 2, this is the calculated value that contains the sample of 2 mol water

5) UV spectrum (UV):

260±2nm（E ^1% _1cm＝124±20）

6) garden dichroscope spectrum (CD)

-32000 ± 5000 Hes

+28000±5000

7) infrared absorption spectrum (IR): (KBr sheet) main number (cm that sprinkles ^-1) be:

3420，3080，2960，1780，1730，1670，1510，1400，1260，1170

1060，980，860，510，

8) nuclear magnetic resonance spectrum ( ¹³C-NMR): D ₂O, 100MH ₂Signal as follows

（δppm）

179.79（s），177.00（s），176.11（s），170.93（s），170.77（s），

166.42（d），162.24（s），159.63（s），134.46（s），118.40（s），

79.67（s），72.70（d），67.67（t），66.01（d），63.26（t），57.58（d）

57.03（d），56.52（t），43.61（t），41.18（t），37.35（t），32.68（t）

28.98（t），28.65（t），27.52（t），23.50（t）

(S: unimodal, d: double honeybee, t: triple honeybees, q: quartet)

9) amino acid analysis: sample uses 5.5N hydrochloric acid in 110 ℃ of following hydrolysis 15 hours.Identify Serine and α-An Jijiersuan

10) thin-layer chromatography (TLC): point sample film, Mierocrystalline cellulose (production of Tokyo chemical industrial company)

The solvent system, acetonitrile: 3% ammonium sulfate (1: 1), Rf=0.58

11) HPLC (high performance liquid chromatography) (HPLC):

Post, YMC pack A312, mobile phase, 2% methyl alcohol/0.01M phosphoric acid buffer (PH3.0), 2 ml/min.The Rt=3.7(branch) the following character of A, B, C, D, E and F is common.

12) solubleness:

Be soluble in: water, aqueous acetone solution, alcohol solution

Be slightly soluble in: dimethyl sulfoxide (DMSO), methyl alcohol, acetone vinyl acetic monomer

13) color reaction:

Positive: (hydration) triketohydrindene hydrate, Ge Ruike-Li Beike (Greig-Leaback),

Mouth reaction (Sakaguchi)

Negative: Barton reaction, potassium permanganate

（ⅱ）TAN-592B HCl：

1) outward appearance: white powder

2) molecular weight: the SIMS method, (M+H) ⁺791

3) molecular formula: C ₂₉H ₄₆N ₁₀O ₁₄S HCl(2H ₂O)

4) ultimate analysis (%):

Analytical value ^{* 1}Calculated value ^{* 2}

C，40.002±2.0 C，40.35

H，5.83±1.0 H，5.95

N，15.64±1.5 N，16.23

O，29.65

S，4.30±1.0 S，3.71

Cl，4.50±1.5 Cl，4.11

* 1, * 2, and condition and A's is identical

6) UV spectrum:

260±2nm（E ^1% _1cm＝108±20）

7) CD spectrum:

-31000 ± 5000 Hes

+29000±5000

8) IR spectrum:

3400，3080，2950，1780，1730，1660，1520，1390，1250，1170

1060，980，860，520

9) ¹³The C-NMR spectrum:

179.85（s），177.20（s），176.24（s），174.41（s），171.13（s），

166.42（d），162.23（s），159.61（s），134.55（s），117.97（s），

79.67（s），72.86（d），67.10（d），66.00（d），63.89（t），63.19（t）

59.10（d），57.40（d），57.18（d），56.30（d），43.58（t），41.46（t）

37.36（t），32.74（t），29.33（t），28.66（t），27.41（t），23.52（t）

9) amino acid analysis: (condition is identical with A's)

Serine (about 2 mol) also detects α-An Jijiersuan:

10) TLC:(condition and A's is identical)

Rf＝0.61

11) HPLC:(condition and A's is identical)

The Rt=4.2(branch)

（ⅲ）TAN-592C 2HCl：

1) outward appearance: white powder

2) molecular weight: the SIMS method, (M+H) ⁺862

3) molecular formula: C ₃₂H ₅₁N ₁₁O ₁₅S.2HCl.(4H ₂O)

4) ultimate analysis (%):

Analytical value ^{* 1}Calculated value ^{* 2}

C，38.04±2.0 C，38.17

H，6.30±1.0 H，6.11

N，14.30±1.5 N，15.30

O，30.19

S，3.18±1.0 S，3.18

Cl，7.76±1.5 Cl，7.04

* 1, condition is identical with A's.

* this sample contains 4 mol water.

5) UV spectrum:

260±2nm（E ^1% _1cm＝97+±20）

6) CD spectrum:

-28000 ± 5000 Hes

+26000±5000

7) IR spectrum

3420，3070，3000，2950，1780，1735，1660，1520，1450，1390

1250，1165，1060，860，520

8) ¹³C-NMR) spectrum:

179.52（s），178.64（s），176.16（s），175.71（s），174.49（s），

173.94（s），169.48（s），166.45（d），162.54（s），159.67（s），

132.57（s），123.59（s），79.79（s），72.33（d），66.82（t），

66.47（d），64.06（t），63.87（t），58.97（d），58.28（d），56.26（d）

56.21（d），51.93（d），43.62（t），41.46（t），37.25（t），32.33（t）

29.33（t），28.91（t），27.35（t），23.41（t），19.47（q），

9) amino acid analysis: (condition is identical with A's)

Serine (about 2 mol) also detects α-An Jijiersuan

10) TLC:(condition and A's is identical)

Rf＝0.68

11) HPLC:(condition and A's is identical)

The Rt=4.6(branch)

（ⅳ）TAN-592D 2HCl：

1) outward appearance: white powder

2) molecular weight: the SIMS method, (M+H) ⁺661

3) molecular formula: C ₂₅H ₄₀N ₈O ₁₁S2HCl(3H ₂O)

4) ultimate analysis (%):

Analytical value ^{* 1}Calculated value ^{* 2}

C，37.51±2.0 C，38.12

H，6.28±1.0 H，6.14

N，14.10±1.5 N，14.23

O，28.44

S，4.00±1.0 S，4.07

Cl，9.94±1.5 Cl，9.00

* 1 condition and A's is identical.

* 2 these samples contain 3 mol crystal water.

5) UV spectrum:

260±2nm（E ^1% _1cm＝110±20）

6) CD spectrum:

-25000 ± 5000 Hes

+22000±5000

7) IR spectrum:

3420，3075，2950，1770，1735，1670，1550，1460，1400，1260

1170，1110，1065，870，540，

8) ¹³The C-NMR spectrum:

179.36（s），176.10（s），175.86（s），170.69（s），170.18（s），

168.18（d），159.69（s），132.59（s），122.91（s），72.69（d），

67.04（t），63.21（t），62.08（d），60.24（d），57.58（d），56.57（d）

56.39（d），43.64（t），41.19（t），37.41（t），32.36（t），28.97（t）

28.71（t），27.51（t），23.71（t），

9) amino acid analysis: (condition is identical with A's)

Examine and determine out Serine and α-An Jijiersuan:

10) TLC:(condition and A's is identical)

Rf＝0.62

11) HPLC:(condition and A's is identical)

The Rt=7.9(branch)

（ⅴ）TAN-592E 2HCl：

1) outward appearance: white powder

2) molecular weight: the SIMS method, (M+H) ⁺748

3) molecular formula: C ₂₈H ₄₅N ₉O ₁₃S2HCl(H ₂O)

4) ultimate analysis (%):

Analytical value ^{* 1}Calculated value ^{* 2}

C，39.71±2.0 C，40.10

H，5.87±1.0 H，5.89

N，14.85±1.5 N，15.03

O，26.71

S，3.90±1.0 S，3.82

Cl，7.46±1.5 Cl，8.45

* 1 condition and A's is identical

* 2 these samples contain 1 mol crystal water.

5) UV spectrum:

260±2nm（E ^1% _1cm＝91+±20）

6) CD spectrum:

-24000 ± 5000 Hes

+17000±5000

7) IR spectrum:

3400，3060，2950，1760，1730，1660，1540，1460，1439，1240

1170，1110，1065，1020，870，810，500

8) ¹³The C-NMR spectrum:

179.41（s），176.44（s），176.20（s），174.23（s），171.02（s），

168.01（s），159.66（s），133.50（s），121.05（s），72.83（d），

67.11（t），63.92（t），63.12（t），62.06（d），60.21（d），58.99（d）

57.38（d），56.82（d），56.27（d），43.62（t），41.40（t），37.49（t）

32.54（t），29.23（t），28.59（t），27.40（t），23.78（t）

9) amino acid analysis: (condition is identical with A's)

Serine (about 2 mol) also detects α-An Jijiersuan

10) TLC:(condition and A's is identical)

Rf＝0.64

11) HPLC:(condition and A's is identical)

The Rt=9.6(branch)

（Ⅵ）TAN-592F 2HCl

1) outward appearance: white powder

2) molecular weight: SIMS method (M+H) ⁺819

3) molecular formula: C ₃₁H ₅₀N ₁₀O ₁₄S 2HCl(4H ₂O)

4) ultimate analysis (%):

Analytical value * ¹Calculated value * ²

C 38.00±2.0 C 38.63

H 6.87±1.0 H 6.27

N 14.35±1.5 N 14.53

O 29.88

S 3.10±1.0 S 3.33

C17.78±1.5 C17.36

* 1 condition and A's is identical.

* 2 these samples contain 4 mol water.

5) UV spectrum:

260±2nm（E ^1% _1cm＝90±20）

6) CD spectrum:

-32000 ± 5000 Hes

+20000±5000

7) IR spectrum:

3420，3070，2950，1770，1735，1660，1540，1460，1395，1340

1250，1160，1110，1065，530

8) amino acid analysis: (condition is identical with A's)

Serine (about 2 mol) also detects L-Ala and α-An Jijiersuan.

9) TLC:(condition and A's is identical), Rf=0.67

10) HPLC:(condition and A's is identical), the Rt=10.1(branch)

Above-mentioned Serine, the absolute configuration of L-Ala and α-An Jijiersuan is measured L-, L-and the D-type of being respectively through HPLC.

(2) the vitiligoidea bacterium lactan that will grow on nutrient agar slant medium belongs to

(Xanthomonas, lactamagena) YK-278(IFO 14351, FERM BP-636) culture is seeded in 15 200 milliliters the conical flask, 40 milliliters of substratum aqueous solution (PH7.0) are housed in each conical flask, and it consists of: glucose 2%, soluble starch 3%, raw soya bean powder 1%, corn steep liquor gathers peptone 0.5% and sodium-chlor 0.3%, and dopes 0.5% precipitated chalk.Conical flask places on the rotary shaker and cultivated 24 hours down in (24 ℃), and the nutrient solution of gained is as seed culture fluid.

To contain 3% dextrin, 1.5% corn steep liquor, 0.2% poly-peptone, 16 liters of substratum (PH7.0) of 0.1% Sulfothiorine and 0.5% precipitated chalk divide in 200 milliliters the conical flask of packing into, and each adorns 40 milliliters.Sterilized 20 minutes down in 120 ℃ then.1 milliliter of seed culture fluid is inoculated these be equipped with in conical flasks of substratum of sterilization, place on the rotary shaker (230 rev/mins of rotating speeds) to cultivate 90 hours down in 17 ℃.

To be adjusted to PH6.5 with 2N HCL from 16 liters of nutrient solutions that said process obtains, and add 16 premium on currency, get 32 liters of filtrates through centrifugation.

Filtrate is by being equipped with 0.5 liter of 50-100 order Na ⁺The ion exchange resin column of type Dowex-50W, after the washing of 1.5 premium on currency, with 10 liters of 2M sodium chloride aqueous solution wash-outs, elutriant is by being equipped with the post of 0.3 liter of gac, and pillar washs with 1 premium on currency, uses 2.2 liters of 8% isopropylcarbinols-N/200HCl wash-out then.Regulate the PH to 6.2 of elutriant, and be concentrated into 0.5 liter.The PH of concentrated solution is adjusted to 7.3, and by the post of 0.6 liter of Diaion HP-20 is housed, pillar is used the 0.01M phosphoric acid buffer wash-out of 6 liters of PH3.0 again after the washing of the 0.01M of 1.6 liters of PH7.3 phosphoric acid buffer.

The elutriant of three parts is respectively by being equipped with the post of 80 milliliters of gacs, pillar with 300 milliliters of washings after, with 600 8% isopropylcarbinols-N/200HCL wash-out, elutriant obtains 402 milligrams of crude product I, 760 milligrams of crude product II and 448 milligrams of crude product III respectively through lyophilize.

Analyze through HPLC, show that the crude product II contains TAN-592A, B and C, the crude product III contains TAN-592D, E and F.

By as (1) described method with purifying crude, 18 milligrams of TAN-592A(HCL), 39 milligrams of B, 35 milligrams of C, 12 milligrams of D, 15 milligrams of E and 24 milligrams of F.

Crude product I (400 milligrams) is dissolved in 100 ml waters, and solution is by being equipped with 50 milliliters of Na ⁺The post of type CM-Sephadex C-25, the sodium chloride aqueous solution wash-out with 1.5 liters of 0.02M separates then.The part that contains main ingredient TAN-591A, B and C is collected in each wash-out part liquid chromatography (LC) analysis respectively.

The part that will contain main ingredient TAN-591A, B and C is the post (every post is adorned 10 milliliters) by gac is housed respectively, every post with 30 milliliters of washings after, use 70 milliliter of 8% isopropylcarbinol aqueous solution wash-out respectively, elutriant concentrates and separates (post: YMC Pack SH-343,20 millimeters X250 millimeters of φ) 0.01M phosphoric acid buffer wash-out separation with PH4.5 with HPLC (high performance liquid chromatography).The liquid chromatography (LC) analysis of each wash-out part is collected and is shown unimodal part, with 1N NaOH the PH of these parts is adjusted to 7.3, pulls back to PH3.0 with 1N HCL again, and respectively by the post of 5 milliliters of gacs is housed.Pillar is after 20 milliliters of washings, and with 50 milliliter of 8% isopropylcarbinol aqueous solution wash-out, elutriant obtains 3 milligrams of TAN-591A, 18 milligrams of B and 22 milligrams of C hydrochlorides respectively after concentrated and lyophilize, be white powder.

The physico-chemical property of the microbiotic TAN-591HCL of above-mentioned gained is as follows:

（ⅰ）TAN-591A 2HCl

1) outward appearance: white powder

2) molecular weight: SIMS method (M+H) ⁺676

3) molecular formula: C ₂₆H ₄₁N ₇O ₁₂S2HCl(2H ₂O)

4) ultimate analysis (%):

Analytical value * ¹Calculated value * ²

C 40.38±2.0 C 39.79

H 6.53±1.0 H 6.04

N 12.81±1.5 N 12.50

O 28.54

S 4.07±1.0 S 4.09

C18.40±1.0 C19.04

* 1, sample is under reduced pressure in 60 ℃ of drying (P ₂O ₅) 8 hours.

* 2, this is the calculated value that contains the sample of 2 mol water.

5) ultraviolet (UV) spectrum:

260±2nm（E ^1% _1cm＝118±20）

6) garden dichroscope spectrum (CD):

-34000 ± 5000 Hes

+27000±5000

7) infrared spectra (IR): (KBr sheet) main number (cm that sprinkles ^-1) be

3420，3250，3080，2950，1780，1735，1675，1515，1410，1360

1280，1160，1060，980，860，520

8) nuclear magnetic resonance spectrum ( ¹³C-NMR): D ₂O, the signal of 100MHz (δ ppm) as follows

179.74（s），177.19（s），176.16（s），170.90（s），170.66（s），

166.42（d），162.12（s），134.89（s），117.77（s），79.70（s），

72.71（d），67.12（t），66.02（d），63.22（t），57.58（d），57.35（d）

56.67（d），42.22（t），41.21（t），37.36（t），32.77（t），31.28（t）

29.30（t），28.62（t），25.08（t），23.50（t）

(S: unimodal, d: doublet, t: triplet, g: quartet)

9) amino acid analysis: sample uses 5.5N HCL in 110 ℃ of following hydrolysis 15 hours.

Detect Serine and α-An Jijiersuan.

10) thin-layer chromatography (TLC): point sample film, Mierocrystalline cellulose (production of Tokyo chemical industrial company)

The solvent system, acetonitrile 3%; 3% ammonium sulfate (1: 1), Rf=0.45

11) HPLC (high performance liquid chromatography) (HPLC): post, YMC pack A312,

Mobile phase, 5% methyl alcohol/0.01M phosphoric acid buffer (PH3.0), 2 ml/min

The Rt=2.4(branch)

The following character of the component of A, B and C is common

12) solubleness:

Be soluble in: water, aqueous acetone solution, alcohol solution

Be slightly soluble in: dimethyl sulfoxide (DMSO), methyl alcohol, acetone, vinyl acetic monomer

13) color reaction: the positive: (hydration) triketohydrindene hydrate, Greig-Leaback reaction

Negative: Barton reaction, potassium permanganate mouth (Sakaguchi)

Reaction

（ⅱ）TAN-591 B 2HCl

1) outward appearance: white powder

2) molecular weight: SIMS method (M+H) ⁺763

3) molecular formula: C ₂₉H ₄₆N ₈O ₁₄S2HCl(2H ₂O)

4) ultimate analysis (%):

Analytical value * 1 calculated value * 2

C 39.34±2.0 C 39.95

H 6.02±1.0 H 6.01

N 12.52±1.5 N 12.85

O 29.38

S 4.40±1.0 S 3.68

C17.57±1.5 C18.13

* 1, * 2, and condition is identical with A's.

5) UV spectrum:

260±2nm（E ^1% _1cm＝124±20）

6) CD spectrum:

-39000 ± 5000 Hes

+29000±5000

7) IR spectrum:

3400，3270，3080，2970，1780，1735，1670，1530，1410，1260

1160，1060，980，875，520

8) ¹³The C-NMR spectrum:

179.69（s），177.19（s），176.21（s），174.14（s），171.04（s），

170.89（d），166.38（d），162.07（s），134.95（s），117.55（s）

79.68（s），72.84（d），67.09（t），66.00（d），63.90（t），63.10（t）

59.00（d），57.41（d），57.36（d），56.37（d），45.25（t），41.41（t）

37.35（t），32.77（t），31.47（t），29.20（t），28.60（t），24.94（t）

23.49（t），

9) amino acid analysis: (condition is identical with A's)

Serine (about 2 mol) also detects α-An Jijiersuan.

10) TLC:(condition and A's is identical)

Rf＝0.47

11) HPLC:(condition and A's is identical)

The Rt=2.8(branch)

（ⅲ）TAN-591C.3HCl

1) outward appearance: white powder

2) molecular weight: SIMS method (M+H) ⁺834

3) molecular formula: C ₃₂H ₅₁N ₉O ₁₅S3HCl(4H ₂O)

4) ultimate analysis (%):

Analytical value * ¹Calculated value * ²

C 36.74±2.0 C 37.85

H 6.31±1.0 H 6.16

N 11.74±1.5 N 12.42

O 29.94

S 3.48±1.0 S 3.16

CL11.86±1.5 CL10.48

* 1 condition and A's is identical.

* 2 these samples contain 4 mol water.

5) UV spectrum:

260±2nm（E ^1% _1cm＝110±20）

6) CD spectrum:

-57000 ± 5000 Hes

+39000±5000

7) IR spectrum:

3440，3270，3080，2950，1780，1740，1675，1530，1410，1250，1150，1060，960，800，540

8) amino acid analysis: (condition is identical with A's)

Serine (about 2 mol), and detect L-Ala and α-An Jijiersuan.

9) TLC:(condition and A's is identical)

Rf＝0.51

10) HPLC:(condition and A's is identical)

The Rt=3.3(branch)

Above-mentioned Serine, the absolute configuration of L-Ala and α-An Jijiersuan determines to be respectively L-, L-and D-type through the HPLC analysis.

Embodiment 1

With the TAN-547A(1.0 gram) be dissolved in 200 milliliters of 0.02M Sodium phosphate dibasic aqueous solution, solution is regulated PH to 9.4 with the 2N aqueous sodium hydroxide solution, stirs 33 hours under room temperature then.Added 2N sodium hydroxide every 5 hours during this, so that the PH of solution remains between the 9.0-9.4.Adding 100 ml waters are finished in reaction, and its PH to 7.0 of re-adjustment is then by being equipped with 100 milliliters of CL ^-The production of type QAE-Sephadex A-25(Sweden Pharmacia Fine Chemical Works) post, the 0.02M phosphoric acid buffer wash-out with PH7.0 separates subsequently.Each several part is analyzed with HPLC, collects to show unimodal part, and regulates its PH to 7.0.The solution of these parts is merged, and by the post of 50 milliliters of gacs is housed.Pillar is after water (150 milliliters) is washed, and with 300 milliliter of 8% isopropylcarbinol aqueous solution wash-out, elutriant gets 253 milligrams of 7-FA-DCPC sodium salt white powders through concentrating and lyophilize.

Embodiment 2

8.5 milligrams of TAN-547B are dissolved in 8.5 milliliters of 0.02M disodium phosphate solns, and regulate PH to 9.4 with 1N sodium hydroxide.Solution stirred under room temperature 24 hours, added 0.1 sodium hydroxide therebetween so that its PH remains between the 9.0-9.4.Reaction solution is analyzed with HPLC.Can obtain 1.3 milligrams of 7-FA-DCPC sodium salts thus.Its physico-chemical property shows that the compound of it and example 1 gained is identical.

Embodiment 3

7.0 milligrams of TAN-547C are dissolved in 7 milliliters of 0.02M disodium phosphate solns, and solution is regulated PH to 9.4 with 0.1N sodium hydroxide.And under room temperature, stirred 24 hours, add 0.1 sodium hydroxide therebetween PH is remained between the 9.0-9.4.Reaction solution is analyzed with HPLC.Can obtain 1.0 milligrams of 7-FA-DCPC sodium salts thus.Its physico-chemical property shows that it is identical with example 1 resulting compound.

Embodiment 4

In 2 liters of TAN-547A(9 gram), the B(8 gram) and the C(1.5 gram) add 7.2 gram Sodium phosphate dibasics in the aqueous solution of mixture, solution is regulated PH to 9.4 with 2N sodium hydroxide, and stirring 24 hours under room temperature.Adding 4 premium on currency are finished in reaction, regulate its PH to 7.0, then by 20 liters of CL are housed ^-After the post of type QAE-Sephadex A-25, pillar are washed with 3 premium on currency, use the 0.02M phosphoric acid buffer wash-out of 8 liters of PH7.0 again.Merge water lotion and damping fluid, and by the post of 1 liter of powdered carbon is housed, post is used 3 liter of 8% isopropylcarbinol wash-out after washing with 4 premium on currency again.Elutriant concentrates, and concentrated solution is by being equipped with 0.5 liter of 50-100 purpose Cl ^-The ion exchange resin column of type Dowex/x2.After post is washed with 1 premium on currency, use 2.5 liters of 0.1M sodium chloride aqueous solution wash-outs again.This elutriant and the above-mentioned elutriant that obtains from QAE-Sephadex A-25 (8 liters) merge, and by the post of 0.7 liter of powdered carbon is housed.Post is after 2 premium on currency are washed, with 2.8 liter of 8% isobutanol solution wash-out.Elutriant gets 3.0 gram 7-FA-DCPC sodium salt powder after concentrated and lyophilize.Its physico-chemical property shows that the compound that it and example 1 obtain is identical.

Embodiment 5

90 milligrams of TAN-547D are dissolved in 20 milliliters of 0.01M Sodium phosphate dibasic aqueous solution, and solution is regulated PH to 9.4 with the 1N aqueous sodium hydroxide solution, and stirs 33 hours under room temperature.Added 0.1N sodium hydroxide every 5 hours during this, so that its PH remains between the 9.0-9.4.Adding 20 ml waters are finished in reaction, the PH to 7.0 of regulator solution, and by 15 milliliters of CL are housed ^-The post of type QAE-Sephadex A-25, the 0.02M phosphoric acid buffer wash-out with PH7.0 separates subsequently.Each wash-out part is analyzed with HPLC, and collect to show unimodal wash-out part, and regulate its PH to 7.0, and by the post of 10 milliliters of gacs is housed.Post is used 70 milliliter of 8% isopropylcarbinol wash-out again after 40 milliliters of washings.Elutriant obtains 18 milligrams of DCPC sodium salts through concentrating and lyophilize, is white powder.

Embodiment 6

7.0 milligrams of TAN-547E are dissolved in 3.8 milliliters of 0.02M disodium phosphate solns, and solution is regulated PH to 9.4 with 0.2N sodium hydroxide.And under room temperature, stirred 24 hours, add 0.1 sodium hydroxide therebetween to keep its PH between 9.0-9.4.Reaction solution liquid chromatography (LC) analysis can obtain 1.4 milligrams DCPC sodium salt thus.Its physico-chemical property shows, it and example 5 resulting compounds are identical.

Embodiment 7

3.5 milligrams of TAN-547F are dissolved in 3.8 milliliters of disodium phosphate solns, and solution is regulated PH to 9.4 with 0.2N sodium hydroxide, and stirs 32 hours under room temperature, adds 0.1 sodium hydroxide therebetween so that its PH remains between the 9.0-9.5.Reaction solution liquid chromatography (LC) analysis can obtain 0.64 milligram DCPC sodium salt thus.Its physico-chemical property shows, it and example 5 resulting compounds are identical.

Embodiment 8

With 4.3 milligrams of TAN-547D, 3.6 milligram TAN-547E and 2.7 milligrams of TAN-547F are dissolved in 10 milliliters of 0.2M disodium phosphate solns, solution is regulated PH to 9.4 with 0.1N sodium hydroxide, and under room temperature, stirred 25 hours, add during this 0.1N sodium hydroxide so that its PH remain between the 9.0-9.5.Reaction solution is analyzed with HPLC, can obtain 1.7 milligrams of DCPC sodium salts thus.Its physico-chemical property shows, it and example 5 resulting compounds are identical.

Embodiment 9

0.5 gram TAN-592A is dissolved in 100 milliliters of 0.02M Sodium phosphate dibasic aqueous solution, solution is regulated PH to 9.5 with the 2N aqueous sodium hydroxide solution, and under room temperature, stirred 30 hours, keep the PH of reaction solution between 9.4-9.6, to react complete 50 ml waters that add therebetween, regulate its PH to 7.0, and by 50 milliliters of CL are housed ^-The post of type QAE-Sephadex A-25, the 0.02M phosphoric acid buffer wash-out with PH7.0 separates subsequently.Each wash-out part is analyzed with HPLC, collects to show unimodal part, and regulates its PH to 7.0, and by the post of 25 milliliters of gacs is housed, post is after 75 milliliters of washings, with 150 milliliter of 8% isopropylcarbinol wash-out then.Elutriant obtains 112 milligrams of 7-FA-DCPC sodium salts through concentrating and lyophilize, is white powder.

Embodiment 10

0.5 gram TAN-592B is dissolved in 100 milliliters of 0.02M Sodium phosphate dibasic aqueous solution, and solution is regulated PH to 9.4 with the 2N aqueous sodium hydroxide solution, and stirs 32 hours under room temperature, keeps its PH therebetween between 9.2-9.8.Adding 50 ml waters are finished in reaction, regulate PH to 7.0, and by 50 milliliters of CL are housed ^-The post of type QAE-Sephadex A-25, the 0.02M phosphoric acid buffer wash-out with PH7.0 separates subsequently.Each wash-out part is analyzed with HPLC, collects to show unimodal part, merges and regulates its PH to 7.0, and by the post of 25 milliliters of gacs is housed, post is after 75 milliliters of washings, with 150 milliliter of 8% isopropylcarbinol wash-out then.Elutriant obtains 56 milligrams of 7-FA-DCPC sodium salts after concentrated and lyophilize, be white powder.

Embodiment 11

8.0 milligrams of TAN-592C are dissolved in 8.0 milliliters of 0.02M disodium phosphate solns, and solution is regulated PH to 9.4 with 0.1N sodium hydroxide, and stirs 20 hours under room temperature, add therebetween 0.1N sodium hydroxide so that its PH remain between the 9.2-9.7.Reaction solution is analyzed with HPLC, can obtain 1.1 milligrams of 7-FA-DCPC thus.Its physico-chemical property shows, it and example 9 resulting compounds are identical.

Embodiment 12

In TAN-592A(10.2 gram), the B(8.2 gram) and C(1.6 restrain) in the aqueous solution (2 liters) of mixture, add 7.2 gram Sodium phosphate dibasics, solution is regulated PH to 9.4 with 2N sodium hydroxide, as for stirring 31 hours under the room temperature, keeps its PH between 9.0～9.4 with 2N sodium hydroxide therebetween.Reaction is finished adding 4 premium on currency and is regulated its PH to 6.7, and by 1 liter of Na is housed ⁺The post of type Dowex 50 WX2, post are after 1 premium on currency is washed, and washing lotion and elutriant merge, and by the post of 1.5 liters of powdered carbons is housed, post is after 4 premium on currency are washed, with 7.5 liter of 8% isopropylcarbinol wash-out.Elutriant concentrates, and concentrated solution is by being equipped with 1 liter of Cl ^-The post of type QAE-Sephadex A-25 separates with 0.02M sodium chloride aqueous solution wash-out subsequently.Contain antibiotic part (4 liters) by the post of 1.5 liters of powdered carbons is housed, post is after 4.5 premium on currency are washed, with 4.5 liter of 8% isopropylcarbinol wash-out.Elutriant obtains the 7-FA-DCPC sodium salt powder of 4.1 grams through concentrating and lyophilize.Its physico-chemical property shows that the mixture that it and example 9 obtain is identical.

Embodiment 13

10 gram TAN-591A are dissolved in 200 milliliters of 0.02M Sodium phosphate dibasic aqueous solution, and solution is regulated PH to 9.4 with the 2N aqueous sodium hydroxide solution, and stirs 33 hours under room temperature, keeps its PH therebetween between 9.0～9.4.Reaction is finished adding 100 ml waters and is regulated its PH to 7.0 solution by 100 milliliters of Cl are housed ^-The post of type QAE Sephadex A-25, the 0.02M phosphoric acid buffer wash-out with PH7.0 separates subsequently.Each wash-out is partly analyzed with the high-speed liquid chromatography, collects to show unimodal part, merges and regulates its PH to 7.0.And by the post of 50 milliliters of gacs is housed, post is after 150 milliliters of washings, with 300 milliliter of 8% isopropylcarbinol wash-out.Elutriant obtains 122 milligrams of 7-FA-DCPC sodium salts through concentrating and lyophilize, is white powder.

Embodiment 14

9.2 milligrams of TAN-591B are dissolved in 9.2 milliliters of disodium phosphate solns, and solution is regulated PH to 9.4 with 0.1N sodium hydroxide, as for stirring 20 hours under the room temperature, keeps its PH between 9.2～9.7 with 0.1N sodium hydroxide therebetween.Reaction soln is analyzed with the high-speed liquid chromatography, can get 1.5 milligrams of its physico-chemical properties of 7-FA-DCPC thus and show that the compound that it and example 9 obtain is identical.

Embodiment 15

7.8 milligrams of TAN-591C are dissolved in 7.8 milliliters of 0.02M use 120 milliliter of 8% isopropylcarbinol wash-out again.Elutriant concentrates, and enriched material obtains 140 milligrams of 7-DCPC sodium salt powder through lyophilize.Its physico-chemical property shows that the compound that it and example 9 obtain is identical.

Embodiment 16

In 50 milliliters of TAN-591A(400 milligrams), the B(112 milligram) and the C(53 milligram) in the aqueous solution of mixture, add 180 milligrams of Sodium phosphate dibasics, solution is regulated PH to 9.4 with 2N sodium hydroxide, keeps its PH between 9.0～9.4 with 2N sodium hydroxide therebetween.Adding 100 ml waters are finished in reaction, regulate PH to 7.0, and by 25 milliliters of Na are housed ⁺The post of type Dowex 50 WX2 washs with 25 ml waters subsequently.Water lotion and elutriant merge, and by the post of 25 milliliters of powdered carbons is housed, post is used 75 milliliter of 8% isopropylcarbinol wash-out again after 100 milliliters of washings.Elutriant concentrates, and concentrated solution is by being equipped with 25 milliliters of Cl ^-The post of type QAE-SephadexA-25 separates with 350 milliliters of 0.02M sodium chloride aqueous solution wash-outs subsequently.To contain antibiotic part (100 milliliters) by the post of 40 milliliters of powdered carbons is housed, post is after 120 milliliters of washings, and voltinism matter shows that the compound that it and example 17 obtain is identical.

Embodiment 17

100 milligrams of TAN-592D are dissolved in 20 milliliters of 0.01M disodium phosphate solns, and solution is regulated PH to 9.4 with 1N sodium hydroxide, and stirs 30 hours under room temperature, keeps its PH therebetween between 9.0～9.4.Adding 20 ml waters are finished in reaction, regulate its PH to 7.0, and by 25 milliliters of Cl are housed ^-The post of type QAE-Sephadex A-25, the 0.02M phosphoric acid buffer wash-out with PH7.0 separates subsequently.Each wash-out part is analyzed with HPLC, collects to show unimodal part, and merging is also regulated PH to 7.0.And by the post of 10 milliliters of gacs is housed, post is after 40 milliliters of washings, with 70 milliliter of 8% isopropylcarbinol wash-out.Elutriant obtains 21 milligrams of DCPC sodium salts through concentrating and lyophilize, is white powder.

Embodiment 18

9.2 milligrams of TAN-592E are dissolved in 9.2 milliliters of 0.02M disodium phosphate solns, and solution is adjusted to PH9.4 with 0.2N sodium hydroxide, and stirs 20 hours under room temperature, adds 0.1N sodium hydroxide therebetween to keep PH between 9.2～9.7.Reaction solution liquid chromatography (LC) analysis can get 2.4 milligrams of DCPC sodium salts thus.Its physico-chemical property shows that the compound that it and example 17 obtain is identical.

Embodiment 19

8.0 milligrams of TAN-592F are dissolved in 16 milliliters of 0.02M disodium phosphate solns, and solution is regulated PH to 9.4 with 0.2N sodium hydroxide, and stirs 20 hours under room temperature, adds 0.1N sodium hydroxide therebetween to keep its PH between 9.2～9.7.Reaction solution liquid chromatography (LC) analysis can get 2.0 milligrams of DCPC sodium salts thus.Its physico-chemical property shows that the compound that it and example 17 obtain is identical.

Embodiment 20

With 3 milligrams of TAN-592D, 3 milligrams of TAN-592E and 3 milligrams of TAN592 C are dissolved in 10 milliliters of 0.02M disodium phosphate solns, solution is regulated PH to 9.4 with 0.1N sodium hydroxide, and stirs 25 hours under room temperature, adds 0.1N sodium hydroxide therebetween to keep its PH between 9.0～9.5.Reaction solution high-speed liquid chromatography analysis can get 1.5 milligrams of DCPC sodium salts thus.In its thing disodium phosphate soln, solution is regulated PH to 9.4 with 0.1N sodium hydroxide, and stirs 20 hours under room temperature, keeps its PH between 9.2～9.7 with 0.1N sodium hydroxide therebetween.Reaction solution is analyzed with the high-speed liquid chromatography, can obtain 0.82 milligram of 7-FA-DCPC sodium salt thus.Its physico-chemical property shows that the compound that it and example 9 obtain is identical.

Embodiment 21

IFO 0755 is seeded in 200 ml flasks that 40 milliliters of seed culture mediums are housed with trigonopsis variabilis bacterium (Trigonopsis variabilis).Every liter of seed culture medium (PH6.0) contains: 20 gram glucose, 4 gram KH ₂PO ₄1 gram Mg SO ₄7H ₂O, 0.5 gram CaCl ₂, 0.1 gram H ₃PO ₄, 40 milligrams of (NH ₄) ₆Mo ₇O ₂₄4H ₂O, 40 milligrams of MnSO ₄4H ₂O, 40 milligrams of ZnSO ₄7H ₂O, 45 milligrams of CuSO ₄5H ₂O, 25 milligrams of FeSO ₄7H ₂O, 4 milligrams of thiamine hydrochlorides, 20 microgram vitamin Hs, 4 gram DL-α-Bing Ansuans.Nutrient solution was in 28 ℃ of following joltings 2 days.

1 milliliter of nutrient solution is transferred in the main medium (except that the DL-α-Bing Ansuan replaces with DL-methionine, other component is identical), cell is separated with supercentrifuge, behind distilled water wash, be scattered in 40 milliliters of 1M tetra-sodium damping fluids (PH8.0) that include 10 millimole sodiumazide.The aqueous solution that in suspension, adds 4 milliliters 440 milligrams compounds (IV).In mixture impouring 200 ml flasks, reacted 16 hours down in 28 ℃.With the cell elimination, get 91 milliliters of supernatant liquors with high speed centrifugation.

Supernatant liquor adds 500 ml waters, regulates PH to 7.0, then by 200 milliliters of Cl ^-The post of type QAE Sephadex A-25 with 0.05M phosphoric acid buffer (PH7.0) wash-out, merges significant part, and by the post of 200 milliliters of gacs is housed, post earlier with 1000 milliliter of 8% isopropylcarbinol aqueous solution, is used 400 milliliter of 8% isopropylcarbinol aqueous solution-N/100 ammoniacal liquor wash-out again after washing.Elutriant concentrates, and concentrated solution is by being equipped with 200 milliliters of Cl ^-The post of type QAE Sephadex A-25, the pillar 0.03M phosphoric acid buffer wash-out of PH7.0.Each wash-out part is analyzed with HPLC, collects to show unimodal part, and by the post of 300 milliliters of gacs is housed.Pillar earlier with 900 milliliter of 8% isopropylcarbinol aqueous solution, is used 500 milliliters of 8% isopropylcarbinols-N/100 ammoniacal liquor wash-out subsequently again after 900 milliliters of washings.Merge wash-out, concentrate and lyophilize, get the double sodium salt of 360 milligrams of compounds (III).

Embodiment 22

With Rhodopseudomonas (Pseudomonas) SP.UK-2221(IFO14366 of-loopful, FERM BP-637) be inoculated in 200 milliliters of substratum that are contained in 1 liter of conical flask.Substratum contains 1% peptone, 0.5% meat medicinal extract, and 0.1% yeast extract, 0.05% pentanedioic acid and 0.5% sodium-chlor, its PH are 10.0.Culture was in 30 ℃ of following joltings 7 days.

Centrifugation goes out cell, and it is dispersed in (PH7.0) in the 0.1M potassium phosphate buffer, and being made into concentration is 500 mg/ml.This suspension (20 milliliters) and 60 ml concns are that the 0.1M potassium phosphate buffer of the compound (III) (obtaining from example 21) of 15 mg/ml mixes, and place 48 hours down in 37 ℃.Reaction mixture is removed cell through centrifuging, and the PH of gained supernatant liquor is adjusted to 7.2, and by the post of 5 milliliters of gacs is housed, pillar is used 10 ml waters earlier after 10 milliliters of washings, use 40 milliliter of 8% isopropylcarbinol wash-out more then.Elutriant concentrates down in decompression, and concentrated solution (1 milliliter) is by being equipped with 5 milliliters of Cl ^-The post of type QAE-Sephadex A-25, pillar is used earlier 25 ml waters, and 0.05M sodium chloride aqueous solution wash-out is used after washing with 25 milliliters of 0.02M sodium chloride aqueous solutions again in the back, collects a for per 5 milliliters.Each wash-out part is analyzed with HPLC, collects the unimodal part of demonstration required compound, gets 20 milliliters of significant parts.Its PH is adjusted to 6.9, and by activated carbon column (5 milliliters) column chromatography.Pillar is after 25 milliliters of washings, and is first with 25 milliliter of 8% isopropylcarbinol, back with 25 milliliters of 8% isopropylcarbinols-N/100 ammoniacal liquor wash-out.Elutriant concentrates down in decompression, and lyophilize gets 8.5 milligrams of free compounds (II), is white powder.

Fig. 1

Optical density VS wave number

Fig. 2

Transparence VS wave number

Fig. 3

Transparence VS wave number

Fig. 4

Optical density VS wave number

Fig. 5～Fig. 8

Transparence VS wave number

Claims (1)

1, the method for the compound or its salt that the production structure formula is following:

It comprises cultivates compound or its salt shown in the following formula with the nutrient solution of a kind of microorganism or the processed products of nutrient solution:

This microorganism is Rhodopseudomonas Pseudomonas sp.UK-2221 (CGMCC 0010), and it can be with the HOOC-(CH on the initial compounds 7-position ₂) ₃-CO-NH-group changes NH into ₂-group.

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