CN1020853C - Pharmaceutical composition containing tricyclic compound or salt thereof - Google Patents

Pharmaceutical composition containing tricyclic compound or salt thereof Download PDF

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CN1020853C
CN1020853C CN91102788A CN91102788A CN1020853C CN 1020853 C CN1020853 C CN 1020853C CN 91102788 A CN91102788 A CN 91102788A CN 91102788 A CN91102788 A CN 91102788A CN 1020853 C CN1020853 C CN 1020853C
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milligrams
liters
hydroxyl
streptomyces
reaction
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CN1056103A (en
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奥原正国
田中洋和
后藤俊男
木野亨
畑中洋
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Fujisawa Pharmaceutical Co Ltd
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Abstract

The present invention relates to tricyclic compounds useful for the treatment and prevention of graft rejection diseases, host anti-transplant diseases caused by bone marrow transplantation, autoimmune diseases, infectious diseases, and the likeThe compounds may be represented by the following formula:
the invention also relates to a method for preparing the compounds, pharmaceutical compositions containing the compounds and their use.

Description

Tricyclic compounds, their preparation and pharmaceutical composition containing them
The present invention relates to contain the preparation of drug combination method of novel tricyclic compounds and salt thereof.
More specifically get on very well, the present invention relates to new tricyclic compound, they have such as immunosuppressive activity, antimicrobial acivity or the like pharmaceutically active, pharmaceutical composition and application that the present invention also is related to the production method of these chemical compounds and contains these chemical compounds.
Therefore, an object of the present invention is to provide new tricyclic compound, this chemical compound can be used for treatment and prevention transplant rejection, by the anti-transplant disease of host that bone marrow transplantation causes, autoimmune disease, infectious disease or the like.
Another object of the present invention provides the production method of producing this tricyclic compound with fermentation method and synthetic method.
The 3rd purpose of the present invention provides and contains the pharmaceutical composition of this tricyclic compound as active constituent.
The 4th purpose of the present invention provides the application of this tricyclic compound, is made into to be used for the treatment of and to prevent transplant rejection, by the anti-transplant disease of host that bone marrow transplantation causes, the medicament of autoimmune disease, infectious disease or the like.
About the present invention, it should be noted that, it is that new discovery first according to some new special compound proposes, these chemical compounds are FR-900506, FR-900520, FR-900523 and FR-900525 material, in more detail, pure FR-900506FR-900520, FR-900523 and FR-900525 material be up-to-date first to be subordinated to separate in the novel bacterial fermentation culture of streptomyces and to obtain.
Be the chemical constitution of explanation FR-900506, FR-900520, FR-900523 and FR-900525 material, further result of study, the present inventor has successfully measured the chemical constitution of these materials and has successfully produced tricyclic compound of the present invention.
New tricyclic compound of the present invention can be represented by following general formula:
Figure 911027882_IMG3
R wherein 1Be that hydroxyl or quilt are protected hydroxyl,
R 2Be that hydrogen, hydroxyl or quilt are protected hydroxyl,
R 3Be methyl, ethyl, propyl group or pi-allyl,
N is integer 1 or 2, and
Solid line and dotted line are represented singly-bound or two key.The present invention also comprises the salt of this general formula compound
In the above-mentioned purpose chemical compound (I), find that following four kinds of chemical compounds can be by fermenting and producing.
(1) chemical compound (I), wherein R 1And R 2Respectively be hydroxyl, R 3Be that pi-allyl, n are integers 2 and solid line and dashed signs are singly-bound, this chemical compound is called the FR-900506 material;
(2) chemical compound (I), wherein R 1And R 2Respectively be hydroxyl, R 3Be that ethyl, n are integers 2, solid line and dashed signs are singly-bound, and this chemical compound is called FR-900520 material (having another name called the WS7238A material);
(3) chemical compound (I), wherein R 1And R 2Hydroxyl by name, R 3Be that methyl, n are integers 2, solid line and dashed signs are represented singly-bound, and this chemical compound is called FR-900523 material (having another name called the WS7238B material);
(4) chemical compound (I), wherein R 1And R 2Respectively be hydroxyl, R 3Be pi-allyl, n is an integer 1, and solid line and dashed signs are singly-bound, and this chemical compound is referred to as the FR-900525 material.
About tricyclic compound of the present invention, one or more conformers or stereoisomer have been interpreted as to existing, for example optical isomer and geometric isomer, because have asymmetric carbon atom (one or more) and two key (one or more) to exist, the present invention also comprises these isomers.
According to the present invention, purpose tricyclic compound (I) can be by following method preparation
[I] fermentation method:
Belong to the streptomyces strain fermentation
Figure 911027882_IMG4
[II] synthetic method
(1) method I (introducing the protection oh group)
Figure 911027882_IMG6
(2) method 2(introduces the protection oh group)
Figure 911027882_IMG7
(3) method 3(forms two keys)
Figure 911027882_IMG9
(4) oxidation of method 4(hydroxyl ethylene group)
Figure 911027882_IMG10
(5) reduction of method 5(allyl group)
Figure 911027882_IMG11
R wherein 1, R 2, R 3, the definition of n and solid line dashed signs is all the same, R 1A and R 2A is and is protected hydroxyl, and R 2B is a leaving group.
The detailed catalogue of above-mentioned definition and preferred embodiment below will be explained in detail.
" low " this noun of Shi Yonging unless otherwise indicated, is represented 1-6 carbon atom in this manual.
Can comprise as for " being protected hydroxyl " this speech, suitable protection oh group:
The low alkylthio of low this class of alkylthiomethyl 1-() low alkyl (methylthiomethyl for example, ethylenebis dithiocarbamate methyl, propyl dithiocarbamate methyl, isopropylthio methyl, butyl sulphomethyl, isobutyl group sulphomethyl, hexyl sulphomethyl) or the like, C preferably 1-C 4Alkylthiomethyl, and methylthiomethyl most preferably;
The three replacement silicyls (for example trimethyl silyl, triethylsilyl, tributyl silicyl, tert-butyl-dimetylsilyl, three-tert-butyl silicyl or the like) that three low alkyl silicyls are such also have low alkyl-diaryl silicyl (for example methyl-diphenylmethyl silylation, ethyl-diphenylmethyl silylation, propyl group-diphenylmethyl silylation, tert-butyl-diphenylmethyl silylation or the like) and similar group.Three (C preferably wherein 1-C 4) alkyl silicyl and C 1-C 4Alkyl-diphenylmethyl silylation, and most preferably tert-butyl-dimetylsilyl and tert-butyl-diphenylmethyl silylation;
From carboxylic acid and the deutero-acyl group of sulfonic acid fatty acyl group of replacing of fatty acyl group, aroyl and aryl or the like for example.
Fatty acyl group can comprise the low-grade alkane acidyl (for example formoxyl, acetyl group, propiono, bytyry, isobutyryl, valeryl, isovaleryl, valeryl, caproyl, carboxylic acetyl group, carboxyl propionyl group, carboxylic bytyry, carboxylic caproyl or the like) that contains so one or more suitable substituent groups (as carboxyl), contain one or more suitable substituent groups (as low alkyl) ring (low) alkoxyl (low) alkanoyl (for example encircle propoxyl group acetyl group, cyclobutoxy group propiono, ring oxygen base bytyry in heptan,
Figure 911027882_IMG12
Oxygen base acetyl group, oxygen base propiono,
Figure 911027882_IMG13
Oxygen base bytyry,
Figure 911027882_IMG14
Oxygen base heptanoyl group,
Figure 911027882_IMG15
Oxygen base caproyl or the like), also comprise camphor sulfonyl or the like.
Aroyl can include the aroyl (for example benzoyl, toluyl, dimethylbenzene acyl group, naphthoyl base, nitrobenzophenone, dinitrophenyl, nitronaphthalene acyl group or the like) of one or more suitable substituent groups (as nitro), and the aryl sulfonyl (for example benzenesulfonyl, tosyl, dimethylbenzene sulfonyl, naphthalene sulfonyl base, fluorobenzene sulfonyl, chlorobenzene sulfonyl, bromobenzenesulfonyl, iodobenzene sulfonyl or the like) and the similar group of one or more suitable substituent groups (as halogen) arranged.
The fatty acyl group that replaces with aryl group can comprise have one or more suitable substituent groups virtue (low) alkanoyl (for example phenyl acetyl, phenyl propiono, phenyl bytyry, 2-trifluoromethyl-2-methoxyl group-2-phenyl acetyl, 2-ethyl-2-trifluoromethyl-2-phenyl acetyl, 2-trifluoromethyl-2-propoxyl group-2-phenyl acetyl or the like) and the similar group of (as low-alkoxy and three halos (low) alkyl).
The more preferred acyl group that had defined already can be C 1-C 4Alkanoyl (this C 1-C 4Alkanoyl can have carboxyl), ring (C 5-C 6) alkoxyl (C 1-C 4) alkanoyl (has two (C on the cycloalkanes 1-C 4) alkyl group), camphor sulfonyl, have the benzoyl of one or two nitro, have halogen benzenesulfonyl, have C 1-C 4Alkoxyl and three halo (C 1-C 4) benzene (C of alkyl 1-C 4) alkanoyl, and optimum be acetyl group, carboxyl propionyl group,
Figure 911027882_IMG16
Oxygen base acetyl group, camphor sulfonyl, benzoyl, nitro benzoyl, dinitrobenzoyl, sulphur benzenesulfonyl and 2-trifluoromethyl-2-methoxyl group-2-phenylacetyl group.
The leaving group that is fit to can be hydroxyl, acyloxy, and the acyl moiety of this acyloxy can be above-named those acyl groups and similar group.
Below explain in detail the production method of tricyclic compound of the present invention (I).
[I] fermentation method:
The bacterial strain that can produce FR-900506, FR-900520, FR-900523 and/or FR-900525 material can be fermented in Nutrient medium and prepare FR-900506, FR-900520, FR-900523 and FR-900525 material.This bacterial strain belongs to streptomyces, and for example Streptomyces tsukubaensis No.9993 and Streptomyces hygroscopicus subsp.yakushimaensis subspecies N are o.7238.
The microorganism that is used to produce FR-900506, FR-900520, FR-900523 and FR-900525 material will be described in detail below.
The bacterial strain that [A] will produce FR-900506, FR-900520 and/or FR-900525 material ferments, can produce FR-900506 of the present invention, FR-900520 and FR-900525 material in Nutrient medium, this bacterial strain belongs to streptomyces, for example Streptomyces tsukubaensis No.9993.
Microorganism
The microorganism that is used to produce FR-900506, FR-900520 and/or FR-900525 material is to belong to the streptomyces bacterial strain that can produce FR-900506, FR-900520 and/or FR-900525 material, wherein, Streptomyces tsukubaensis No.9993 is recently from Japanese Ibaraki county Toyosato-Cho, separates in the pedotheque that Tsukuba-gun gathers.
New isolated Streptomyces tsukubaensis No.9993 lyophilizing sample has been stored in the (No.1-3 of industrial science technology Room fermentation research institute, Higashi 1-Chome, yatabemachi Tsukuba-gun, Ibaraki county, Japan) storing number is FERMp-7886, store the date: on October 5th, 1984, and after press budapest treaty regulation again, change to new storage FERM BP-927 on October 19th, 1985 in same storage at.This strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number: CGMCC No.0083 on November 4th, 1985.
Should understand new FR-900506, the production of FR-900520 and/or FR-900525 material is not limited to use the peculiar microorganism of describing here, given herely only be explanation, the present invention also comprises using and anyly can produce FR-900506, FR-900520, and/or the mutant of FR-900525 material, comprise natural mutant and artificial mutation kind, the available conventional method of this artificial mutation kind produces from above-mentioned organism, for example uses X ray, ultraviolet radiation, with N-methyl-N '-nitro-N-nitrosoguanidine, 2-aminopurine processing or the like similar approach.
Streptomycete No.9993 has following form, cultivation, biology and physiological feature:
[1] morphological characteristic:
Main method (Shirling.E.B. and the D.Gottlieb:Methods for characterization of Streptomyces species that adopts by Shirling and Gottlieb narration, International Journal of Systematic Bacteriology.16,313-340,1966) carry out means of taxonomic research.
Carry out morphologic observation with optics and ultramicroscope and extract 14 days culture of growth on agar and the inorganic salt-starch agar at oatmeal agar, Fructus Hordei Germinatus-yeast in 30 ℃.Sophisticated sporophore forms Rectiflexibiles, and 10-50 or 50 was rectangle or cylindrical, 0.5-0.7 * 0.7-0.8 micron size with last spore with the electron microscope observation spore on each chain, and spore surface is slick.
[2] cultural characteristic
With Sbirling above-mentioned and Gottlieb and Waksman(Waksman, S.A.: Actinomycetal the 2nd volume; The classification, evaluation and the description that belong to and plant.The Williams and Wilkins Co.Baltimore, 1961) ten kinds of culture medium introducing are observed cultural characteristic.
In 30 ℃ of incubations 14 days, the color designation of using in this research is according to Guide standards for dyeing (by the handbook of Nippon Shikisai Kenkyusho.Tokyo publication), when growing on oatmeal agar, yeast-Fructus Hordei Germinatus extract agar and inorganic salt-starch agar, bacterium colony belongs to grey colour system.Soluble pigment produces in yeast-Fructus Hordei Germinatus extract agar, but does not produce in other culture medium.It the results are shown in table I (seeing the literary composition back).
(Becker such as Becker are adopted in the cell wall analysis, B., M.P.Lechevalier, R.E.Gordon and H.A.Lechevalier: differentiate Nocard's bacillus and streptomycete Appl.Microbiol., 12 fast with the paper chromatography of whole cell hydrolysate, 421-423,1964) and Yamaguchi(Yamaguchi, T.: the cell wall components of distinct Actinomycetal J.Bacteriol relatively on the form, 89,444-453,1965) method.The whole cell hydrolysising product analysis of bacterial strain No.9993 shows and has the LL-meso diaminopimelic acid.Therefore, be sure of that this bacterial strain should belong to the type I.
[3] biology and physiological property
The physiological property of bacterial strain No.9993 is measured according to the method for being described by Shirling and Gottlieb above-mentioned.It the results are shown in table 2.Serviceability temperature gradient incubator (by Toyo Kagaku Sangyo Co., Ltd makes) is measured growth temperature range and optimum growth temp scope on yeast-Fructus Hordei Germinatus extract agar.Growth temperature range is 18-35 ℃, and optimum temperature is 28 ℃.Blancmangeization and dispergation are positive, and melanin is produced as feminine gender.
The physiological property of table 2 bacterial strain No.9993 and Streptomyces misakiensis IFO 12891
Physiological property No.9993 IFO12891
18 ℃-35 ℃ 12 ℃-35 ℃ of the temperature ranges of growth
28 ℃ 28 ℃ of optimum temperatures
Nitrate reduction is negative
The starch hydrolysis is negative positive
Milk condensing is negative
Milk peptonizes positive weak positive
Melanin produces negative
The gelatin dissolving is positive negative
H 2S produces negative
NaCl drug-resistant capability (%)≤3% 3%＜,＜5%
Utilization of carbon source is according to Pridham and Gattlieb(Pridham, T.G. and D.Gottlieb: utilize auxiliary measuring strain J.Bacteriol., 56,107-114,1948 by some Actinomycetal carbon source) method measure.Observed growing state in 14 days later in 30 ℃ of incubations.
The utilization of carbon source of bacterial strain gathers lists in table 3.Glycerol, maltose and sodium succinate can be utilized by bacterial strain No.9993.And, the utilization of D-glucose, sucrose, D-mannose and salicin also can be observed far and away.
Table 3 bacterial strain No.9993 and streptomycete Streptomyces misakiensis IFO 12891 utilization of carbon source
Carbon source No.9993 IFO 12891
The D-glucose ±-
Sucrose ±-
Glycerol+-
The D-xylose--
D-fructose--
Lactose--
Maltose+-
Rhamnose--
Cottonseed sugar--
The D-galactose-+
L-arabinose--
The D-mannose ±-
The D-Sargassum--
Inositol--
The D-mannitol--
Inulin-+
Cellulose--
Salicin ±-
Chitin-±
Sodium citrate--
Sodium succinate+-
Sodium acetate--
Symbol :+: utilize ±: do not utilize certainly
-: do not utilize
The microscopy of bacterial strain No.9993 and cell wall composition analysis show that this bacterial strain belongs to streptomyces Waksman and Henrici1943.
So, the various streptomycetes of being introduced in the description with this bacterial strain and publication [International Journal of Systematic Bacteriology 18.69 to 189,279 to 392(1968) and 19.391 to 512(1969), and bacteriology identification handbook 8 editions (1974) such as (Bergy ' s Manual of Determinative Bacteriology) compares.
Comparative result is thought bacterial strain No.9993 and Streptomyces aburaviensis, Streptomyces(Nishimura etc.) avellaneus(Baldacci and Grein) with Streptomyces misakiensis(Nakamura) similar.Therefore with the cultural characteristic and corresponding Streptomyces aburaviensis IFO 12830 of bacterial strain No.9993, Streptomyces avellaneus IFO 13451 and Streptomyces misakiensis IFO 12891 compare.The result, bacterial strain No.9993 is the most similar to Streptomyces misakiensis IFO 12891, therefore bacterial strain No.9993 further compares with Streptomyces misakiensis IFO 12891, the results are shown in above-mentioned table 1, in 2 and 3, further result relatively, find out that bacterial strain No.9993 has any different with Streptomyces misakiensis IFO12891 on some following, therefore bacterial strain No.9993 is considered to a kind of new streptomyces species, be referred to as Streptomyces tsukubaensis sp.nov., derive from this biological soil of isolating of taking from Tsukuba-gun.
The difference of Streptomyces misakiensis IFO 12891
Bacterial strain No.9993 and Streptomyces misakiensis IFO 12891 extract agar, glucose-asparagine agar at Herba bromi japonici agar, Fructus Hordei Germinatus-yeast, and Czapek agar is different with cultural character on Rhizoma Solani tuber osi-dextrose agar.
The starch hydrolysis of bacterial strain No.9993 is negative, and the starch of Streptomyces misakiensis IFO 12891 is hydrolyzed to the positive.
Bacterial strain No.9993 gelatin liquefaction is positive, and the gelatin liquefaction of Streptomyces misakiensis IFO 12891 is positive.
On utilization of carbon source, bacterial strain No.9993 can utilize glycerol, maltose and sodium succinate, but Streptomyces misakiensis IFO 12891 can not utilize these, and bacterial strain NO.9993 can not use D-galactose and inulin, but Streptomyces misakiensis IFO 12891 can utilize these.
FR-900506, the production of FR-900520 and FR-900525 material
The bacterial strain that belongs to streptomyces (for example Streptomyces tsukubaensis No.9993, FERM BP-927) that can produce FR-900506, FR-900520 and/or FR-900525 material is cultivated in Nutrient medium and can be produced new FR-900506 of the present invention, FR-900520 and FR-900525 material.
Generally speaking, the bacterial strain that can produce FR-900506, FR-900520 and/or FR-900525 material in the nutrient medium that contains assimilable carbon and nitrogen, preferably cultivate (for example vibration cultivate, submerged culture or the like) under the aerobic condition and can produce FR-900506, FR-900520 and/or FR-900525 material.
Carbon source is a carbohydrate preferably in Nutrient medium, for example glucose, xylose, galactose, glycerol, starch, dextrin or the like.Other be maltose, rhamnose, cottonseed sugar, arabinose, mannose, salicin, sodium succinate or the like.
Preferred nitrogenous source is a for example ammonium salt (as ammonium nitrate, ammonium sulfate, ammonium phosphate or the like) of yeast extract, peptone, gluten powder, cotton seed meal, Semen Glycines powder, corn immersion, dry yeast, Semen Tritici aestivi semina, feather powder, Semen arachidis hypogaeae powder or the like and inorganic and organonitrogen compound, carbamide, aminoacid or the like.
Though carbon and nitrogenous source be favourable in conjunction with adopting, but do not need its pure material, also share because contain the low-purity material of micro-growth factor and considerable mineral nutriment.If necessity can also add some mineral salts, for example sodium carbonate or calcium carbonate, sodium phosphate or potassium phosphate, sodium chloride or potassium chloride, sodium iodide or potassium iodide, magnesium salt, mantoquita, cobalt salt or the like in culture medium.Special when the culture medium foam is serious, if be necessary, also can add anti-foam agent, for example liquid paraffin, fatty oil, vegetable oil, mineral oil or siloxanes.
Mass production FR-900506, FR-900520 and FR-900525 material, for well, produced in small quantities then adopts the vibration in flask or the bottle to cultivate or cultivate on the surface with the aerobic condition of culture of submergence.For fear of growthing lag in FR-900506, FR-900520 and FR-900525 material production process, be grown in when carrying out in big jar, preferably use vegetative state microorganism inoculation in producing jar.Therefore at first will generate the nutrition inoculum of this microorganism and cultivate above-mentioned culture medium through inoculating with the spore of microorganism or mycelium inoculation to the culture medium of relatively small amount, the nutrition contactant that will cultivate then is aseptic to be transferred in the big jar.The culture medium that produces the nutrition inoculum be used to produce FR-900506, the culture medium of FR-900520 and FR-900525 material is identical substantially or difference arranged slightly.
Culture mix stirs and oxygenation can be finished in every way.Can provide stirring by various aerating devices or by the means such as filtrated air passage of leading to culture medium by rotation or vibration fermentation tank by propeller or similar mechanical stirring device.Filtrated air oxygenation effectively by fermenting mixture.
Fermentation is carried out about 20 ℃-40 ℃ usually, and preferably 25-35 ℃, the time is 50 hours-150 hours, and is different and different with production scale with fermentation condition.
FR-900506, the FR-900520 and/or the FR-900525 material that produce can reclaim from culture medium with reclaiming the employed conventional method of other known organism active substance.The FR-900506 that produces, FR-900520 and FR-900525 material obtain in cultivating mycelium and filtrate, and this FR-900506, FR-900520 and FR-900525 material can be from by separating culture filters or centrifugation medium obtains mycelium and the filtrate and purify, separate the method for purifying and to adopt conventional method such as concentrating under reduced pressure, lyophilization, use conventional solvent extraction, regulate pH value, with conventional resin treatment (for example anion or cation exchange resin, non-ionic adsorption resin or the like), with conventional sorbent treatment (active carbon for example, silicic acid, silica gel, cellulose, aluminium oxide or the like), crystallization, recrystallization or the like similar approach.
The physiology of FR-900506, FR-900520 and FR-900525 material and chemical characteristic
The FR-900506 of Sheng Chaning, FR-900520 and FR-900525 material have following physics and chemical property as stated above
The FR-900506 material
(1) shape and color: white powder
(2) elementary analysis:
C：64.72%H：8.78%N：1.59%
64.59% 8.74% 1.62%
(3) chromogenic reaction
Positive: cerous sulfate reaction, sulfuric acid reaction, Ehrlich reaction, Dragendorff reaction and iodine steam reaction
Negative: iron chloride reaction, ninhydrin reaction and Molish reaction
(4) dissolubility
Dissolve in: methanol, ethanol, acetone, ethyl acetate, chloroform, ether and benzene.
Be insoluble in: normal hexane, petroleum ether
Be insoluble to: water
(5) fusing point: 85-90 ℃
(6) optical rotation: [α] 23 D-73 ° of (C=0.8, CHCl 3)
(7) ultra-violet absorption spectrum: tail end absorbs
(8) infrared absorption spectroscopy:
CHCl 3
Maximum: 3,680 3,580 3,520 2,930 2,870 2830
1745 1720 1700 1645 1450 1380
1350 1330 1310 1285 1170 1135
1090 1050 1030 1000 990 960（sh）
918(centimetre -1)
(9) 13The C nuclear magnetic resoance spectrum
δ（ppm、CDCl 3）：
Figure 911027882_IMG17
39.40（t）
31.58（t） 30.79（t）
Figure 911027882_IMG18
26.46（d） 24.65（t）
Figure 911027882_IMG19
Figure 911027882_IMG20
Spectrogram is shown in Fig. 1.
(10) 1The H nuclear magnetic resoance spectrum:
Be shown in Fig. 2
(11) thin layer chromatography:
Immobile phase launches the solvent Rf value
Chloroform: methanol (10: 1, v/v), 0.58
Silica gel plate ethyl acetate 0.52
(12) physical property: neutral substance
Relevant FR-900506 material notes when measuring 13C and 1During the H nuclear magnetic resoance spectrum, this material demonstrates several to signal in various chemical shifts.
Therefore the FR-900506 material also has following characteristic
(ⅰ) in 25 ℃ and 60 ℃ of mensuration 13Each right intensity that the C nuclear magnetic resoance spectrum has disclosed various signals wherein is all different.
(ⅱ) thin layer chromatography and high pressure liquid chromatography show, the FR-900506 material for a bit, is one unimodal in high pressure liquid chromatography in thin layer chromatography.
This white powder FR-900506 material recrystallization from acetonitrile can be become crystal form, this crystal has following physics and chemical property.
(1) shape and color: colourless prism
(2) elementary analysis: C:64.30%H:8.92%N:1.77%
64.20% 8.86% 1.72%
(3) fusing point: 127 °-129 ℃
(4) optical rotation:
[α] 28 D-84.4°（C＝1.02，CHCl 3）
(5) 13The C nuclear magnetic resoance spectrum:
δ（ppm CDCl 3）：
Figure 911027882_IMG21
116.43（t）
Figure 911027882_IMG22
84.29（d）
Figure 911027882_IMG23
24.56（t）
Figure 911027882_IMG24
Figure 911027882_IMG25
Its spectrogram is shown in Fig. 3
(6) 1H nuclear magnetic resoance spectrum: be shown in Fig. 4.
Other physicochemical properties, i.e. chromogenic reaction, dissolubility, ultra-violet absorption spectrum, infrared absorption spectroscopy, the colourless prism Substance Properties of thin layer chromatography and the FR-900506 material white powder thing with this material under the same conditions is identical.
According to above-mentioned physicochemical properties and X-ray diffraction analysis, can determine that the FR-900506 structure of matter is as follows:
Figure 911027882_IMG26
17-pi-allyl-1,14-dihydroxy-12-[2-(4-hydroxyl-3-cyclohexyl methoxy hexyl)-the 1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22,3,1, O 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone
The FR-900520 material
Infrared absorption spectroscopy
νCHCl 3
Maximum: 3,680 3,575 3,520 2,940 2,875 2825
1745 1725 1700 1647 1610（sh）
1452 1380 1350 1330 1285 1170
1135 1090 1030 1005 990 980（sh）
960(sh) 913 908(sh) centimetre -1
The FR-900525 material
(1) shape and color: white powder
(2) elementary analysis:
C：65.17%，H：8.53%，N：1.76%
(3) chromogenic reaction
Positive: cerous sulfate reaction, sulfuric acid reaction, Enrlich reaction, Dragendorff reaction and iodine steam reaction
Negative: iron chloride reaction, ninhydrin reaction and Molish reaction
(4) dissolubility
Dissolve in: methanol, ethanol, acetone, ethyl acetate,
Chloroform, ether and benzene
Be insoluble in: normal hexane, petroleum ether
Be insoluble to: water
(5) fusing point: 85-89 ℃
(6) optical rotation: [α] 23 D:-88 ° of (C=1.0, CHCl 3)
(7) ultra-violet absorption spectrum: tail end absorbs
(8) infrared absorption spectroscopy
CHCl 3
ν maximum: 3,680 3,580 3,475 3,340 2,940 2880
2830 1755 1705 1635 1455 1382
1370 1330 1310 1273 1175 1135
1,093 1,050 1,020 995 970 920 867(centimetres -1)
(9) 13The C nuclear magnetic resoance spectrum:
δ（ppm CDCl 3）：
Figure 911027882_IMG27
Spectrogram is shown in Fig. 5.
(10) 1The H nuclear magnetic resoance spectrum: spectrogram is shown in Fig. 6
(11) thin layer chromatography:
Immobile phase launches the solvent Rf value
Silica gel plate ethyl acetate 0.34
(12) physical property: neutrality
Relevant FR-900525 material is worked as mensuration 13C and 1Notice during the H nuclear magnetic resoance spectrum that this material demonstrates several to signal in various chemical shifts, and when measuring thin layer chromatography and high pressure liquid chromatography, this material demonstrates single-point in thin layer chromatography, it is unimodal to demonstrate in high pressure liquid chromatography.
From above-mentioned physics and chemical property and the chemical constitution of successfully measuring the FR-900506 material, can determine that the chemical constitution of FR-500525 material is as follows:
16-pi-allyl-1,13-dihydroxy-11-[2-(4-hydroxyl-3-methoxyl group cyclohexyl-1-methylvinyl-22,24-dimethoxy-12,18,20,26-tetramethyl-10,27-two oxa-s-4-aza-tricycle-[21,3,1,0 4.8] 27 carbon-17 alkene-2,3,9, the 15-tetraketone.
[B] can with can produce bacterial strain that FR-900520 and/or FR-900523 material belong to streptomyces for example Streptomyces hygroscopicus subsp.akushimaensis No.7238 in Nutrient medium, ferment and prepare FR-900520 of the present invention and FR-900523 material.
Microorganism
The microorganism that is used to produce FR-900520 and/or FR-900523 material is to produce FR-900520 and/or the bacterial strain FR-900523 material, that belong to streptomyces, and wherein Streptomyces hygroscopicus subsp.yakushimaensis No.7238 separates to obtain recently from the pedotheque that Japanese kagoshima county yakushima gathers.
New isolated Streptomyces hygroscopicus subsp.yakushimaensis No.7238 lyophilizing sample has been stored in industrial science and (the No.1-3Higashi 1-Chome yatabemachi Tsukuba-gun of technology Room fermentation research institute, the Ibaraki county, Japan), store and number to be the FERM P-8043(storage date: on January 12nd, 1985), press the budapest treaty regulation then, on October 19th, 1985, store in same storage ground, convert new storage FERM BP-928 to.This strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on November 4th, 1985, and preserving number is CGMCE No.0082.
Should understand, produce new FR-900520 and FR-900523 material and be not limited to use special biology in this narration, that mentions here only is used for explanation.The present invention also comprises any mutant that can produce FR-900520 and/or FR-900523 material of use, comprise natural mutation and artificial mutant, this mutant can produce from mentioned microorganism by conventional method, x-ray irradiation for example, ultraviolet radiation, with N-methyl-N '-nitro-N-nitrosoguanidine, 2-aminopurine processing or the like similar approach.
Streptomyces hygroscopicus subsp.yakushimaensis No.7238 has following shape, cultivation, biology and physiology thing and levies:
[1] morphological characteristic:
Mainly adopt by Shirling and Gottlieb(Shirling, E.B.and D.Gottlieb: the method International Journal of Systematic Bacteriology of expression streptomyces strain characteristic, 16, that 313-340,1966) describes carries out means of taxonomic research.
With optical microscope and ultramicroscope, do morphologic observation to extracting the growing state of cultivating 14 days (in 30 ℃) on agar and the starch-inorganic salt agar at oatmeal agar, Fructus Hordei Germinatus-yeast, the medium length of sophisticated sporophore is with each 20 left and right sides of chain Sporulation Retinaculiaperti and spiral type.Find the suction spore ball in the gas mycelia on Herba bromi japonici agar and starch-inorganic salt agar.Spore upper surface out-of-flatness degree is between extremely short and thick stupefied and wart form.
[2] cultural characteristic:
Narrate with Shirling above-mentioned and Gottlieb, and by Waksman(Waksman, S.A. Actinomycetal, Vol.2. the classification that belongs to and plant, discriminating and description) The Williams and Wilkins Co., Baltimore, 1961) 10 kinds of culture medium of narration are observed cultural characteristics.
In 30 ℃ of incubations 14 days, the color naming of using in this research was according to Guide development criteria (by the handbook that Nippon Shikisai kenkyusho publishes, Tokyo).When at oatmeal agar, Fructus Hordei Germinatus-yeast extracts when growing on agar and the starch inorganic salt agar, and bacterium colony belongs to grey color system.Do not produce soluble pigment in test medium, the results are shown in Table 4 for it (seeing the literary composition back).
With people such as Becker [Becker, B., M.P.Lechevalier, R.E.Gordon and H.A.Lechevalier: the paper chromatographic analysis of cell wall all-hydrolytic product is differentiated Nocardia and streptomyces: Appl.Microbiol., 12 fast, 421-423,1964] and yamaguchi[yamaguchi, T.: the comparison that the cell wall of the actinomycetes that morphological properties is different is formed: J.Bacteriol, 89,444-453,1965] method is carried out the cell wall analysis.There is the LL-meso diaminopimelic acid in the analysis showed that of the cell all-hydrolytic product of No.7238 bacterial strain.Therefore, can think that the cell wall of this bacterial strain is the I type.
[3] biology and physiological property
The method of introducing according to above-mentioned Shirling and Gottlieb is measured the physiological property of No.7238 bacterial strain, the results are shown in table 5.Be determined at the temperature range and the optimum temperature of growing on yeast-Fructus Hordei Germinatus extract agar with a kind of thermograde Incubators for bacteria culture (manufacturing of Toyo kagaku Sangyo company).The temperature range of growth is 18-36 ℃, and optimum temperature is 28 ℃.Starch hydrolysis and solation effect are positive.Not having to produce with melanin is the pigment of feature.(table 5 is seen the literary composition back)
Study the utilization of carbon source according to the method [Pridham, T.G.and D.Gottlieb :] of Pridham and Gottlieb for helping some actinomycetes to do the utilization of the carbide of bacterium determination: J.Bacteriol, 56,107-114,1948.At 30 ℃, cultivate and observe growing state after 14 days.
The utilization of carbon source of this bacterial strain is listed in table 6.The No.7238 bacterial strain can utilize D-glucose, sucrose, lactose, maltose, D-trehalose, inositol, inulin and salicin.
Table 6 bacterial strain No.7238, Streptomyces antimycoticus
IFO12839Streptomyces hygroscopicus
The utilization of carbon source of Subsp.glebosus IFO13786
Carbon source IFO IFO
No.7238 12839 13786
The D-glucose+++
Sucrose+++
Glycerol-++
The D-xylose-±+
D-fructose-++
Lactose++-
Maltose+-+
Rhamnose-+-
Cottonseed sugar-++
The D-galactose-++
L-arabinose-± ±
The D-mannose-++
The D-trehalose+±+
Inositol+++
The D-mannitol-++
Inulin++-
Cellulose ±--
Salicin++-
Chitin ±--
Sodium citrate--±
Sodium succinate-++
Sodium acetate---
Symbol description :+: utilization is arranged
±: be difficult to sure utilization
-: do not utilize
The microscopic study of No.7238 bacterial strain and cell wall composition analysis show that this bacterial strain belongs to the streptomyces of Waksman and Henrici1943 discovery.
Therefore, according to disclosed explanation (International Journal of Systematic Bacteriology, 18,69 to 189,279 to 392(1968) and 19,391 to 512(1969), andBergy ' s bacteriology measures handbook 8th Edition(1974)] can compare this bacterial strain and various streptomyces strain.
Result relatively thinks that the No.7238 bacterial strain is similar to the Streptomyces antimycoticus of nineteen fifty-seven Waksman discovery and the Streptomyces hygroscopicus subsp.glebosus of people such as Ohmori discovery in 1962.Therefore, the cultural character of No.7238 bacterial strain further compares with corresponding Streptomyces antimycoticus IFO 12839 and Streptomyces hygroscopicus subsp.glebosus IFO 13786, result such as above-mentioned table 4,5 and 6.By comparative result as can be seen, several aspects below, No.7238 bacterial strain and Streptomyces antimycoticus IFO 12839 and Streptomyces hygroscopicus subsp.glebosus are distinguishing.
(ⅰ) with the difference of Streptomyces antimycoticus IFO 12839
The cultural character of No.7238 bacterial strain is different with Streptomyces antimycoticus IFO12839 on yeast-Fructus Hordei Germinatus extract agar, glucose-agedoite agar, glycerol-agedoite agar, Rhizoma Solani tuber osi-agar glucose and cruel propylhomoserin agar.
In the method for utilizing of carbon source, the No.7238 bacterial strain can utilize maltose, but bacterium Streptomyces antimycoticus IFO 12839 can not utilize maltose.And the No.7238 bacterial strain can not utilize glycerol, D-fructose, rhamnose, cottonseed sugar, D-galactose, D-mannose, mannitol and sodium succinate, but Streptomyces antimycoticus IFO12839 can utilize them.
(ⅱ) difference of Streptomyces hygroscopicus subsp.glebosus IFO 13786
The cultural character of No.7238 bacterial strain is different with the cultural character of Streptomyces hygroscopicus subsp. on yeast-Fructus Hordei Germinatus extract agar, Rhizoma Solani tuber osi-agar glucose and cruel propylhomoserin agar.
The milk peptonization of No.7238 bacterial strain is negative, but the milk peptonization of Streptomyces hygroscopicus subsp.glebosus IFO 13786 is positive.Grow under the situation that the No.7238 bacterial strain can exist at 7% NaCl, still, Streptomyces is to inhale hygroscopicus subsp.glebosusIFO13786 and can not grow under same condition.
In the application facet of carbon source, the No.7238 bacterial strain can utilize lactose, inulin and salicin, and still, Streptomyces hygroscopicus subsp.glebosusIFO13786 can not utilize them.The No.7238 bacterial strain can not utilize glycerol, D-xylose, D-fructose, cottonseed sugar, D-galactose, D-mannose, mannitol and sodium succinate, but Streptomyces hygroscopicus subsp.glebosus can utilize them.
Yet, gas on oatmeal agar and inorganic salt-starch agar is given birth in the mycelia, No.7238 forms the sporinite of suction, and its form and cultural character and Streptomyces hygroscopicus Subsp glebosus IFO13786 are similar.Therefore, think that the No.7238 bacterial strain belongs to Streptomyces hygroscopicus.Though in Streptomyces hygroscopicus subspecies, this known bacterial strain is the most similar to the No.7238 bacterial strain,, the No.7238 bacterial strain is different with Streptomyces hygroscopicus glebosus IFO13786.Can think that by the above-mentioned fact No.7238 bacterial strain is a kind of novel species of Streptomyces hygroscopicus, and called after Streptomyces hygroscopicus subsp. with separate yakushimaensis subsp.nov., this name is with to isolate soil this microorganism, that collect from yakushima relevant.
The production of FR-900520 and FR-900523 material
(for example: Streptomyces hygroscopicus subsp.yakushimaensis No.7238 belong to streptomyces by in Nutrient medium, cultivating, FERM BP-928) the bacterial strain that can produce FR-900520 and/or FR-900523 material just can be produced new FR-900520 and/or FR-900523 material.
In general, by in the nutrient medium that contains assimilable carbon and nitrogenous source, preferably (for example, vibration cultivate, submerged culture etc.) cultivated the bacterial strain that can produce FR-900520 and/or FR-900523 material and just can be produced FR-900520 and/or FR-900523 material under the condition of aerobic.
Carbon source is a saccharide preferably in Nutrient medium, as glucose, sucrose, lactose, glycerol, starch, dextrin etc.The carbon source of other that can comprise is maltose, D-trehalose, inositol, inulin, salicin etc.
Nitrogenous source is yeast extract, peptone, glutelin powder, cotton seed meal, Semen sojae atricolor powder, Semen Maydis pulp, dry yeast, wheat germ, feather powder, Semen arachidis hypogaeae powder etc. preferably, and inorganic and organic nitrogen compound, for example ammonium salt (as ammonium nitrate, ammonium sulfate, ammonium phosphate etc.), urea, aminoacid etc.
Though it is favourable that carbon source and nitrogenous source use together, do not need with pure carbon source and nitrogenous source, also be usefulness because contain the impure raw material of the mineral nutrition material of micro-growth factor and a great deal of.In the time of needs, can be added to some mineral salt in the culture medium, these mineral salts are sodium carbonate or calcium, sodium phosphate or potassium, sodium chloride or potassium, sodium iodide or potassium, magnesium salt, mantoquita, cobalt salt and similar salt.If desired, particularly when culture medium seriously foams, can add defoamer, as liquid paraffin, fatty oil, vegetable oil, mineral oil or siloxanes.
When mass production FR-900520 and FR-900523 material, under the condition of culture of buried ventilation, carry out.During produced in small quantities, with flask or bottle vibration or surface cultivation.In addition, when growing in big jar, the most handy trophosome microorganism is inoculation in producing jar, so that avoid poor growth in the process of producing FR-900520 and FR-900523 material.Therefore, at first need the culture medium taken over kind with cultivating in the spore of microorganism or the more a spot of culture medium of mycelia inoculation, produce the microbial nutrition inoculum, transfer in big jar the nutrition inoculum of being cultivated is aseptic then.The used culture medium of the culture medium that produces the nutrition inoculum and production FR-900520 and FR-900523 material is substantially the same or different.
Culture mix can be stirred and air feed with multiple mode.Can be with propeller or similarly mechanical stirring equipment, rotation or vibration fermentor, various pumping equipment or the aseptic air duct stir culture mixture by culture medium.Can make aseptic air make it air feed by fermenting mixture.
The temperature of fermenting usually is 20 ℃-40 ℃, and preferably 25-35 ℃, about 50-150 of time hour, according to the condition and the production scale of fermentation, temperature and time can change.
Therefore, the method that is generally used for reclaiming other known bioactive substances by routine can reclaim FR-900520 and/or the FR-900523 material that is generated from culture medium.FR-900520 that is generated and FR-900523 material mainly are present in the mycelium of cultivation, therefore, can from mycelium, separate and purification FR-900520 and FR-900523 material, by filtering or the centrifugalize culture fluid, method by routine is as concentrating under reduced pressure, lyophilization, solvent extraction with routine, regulate pH value, with the resin of routine (for example: sun or anion exchange resin, non-ionic adsorbent resin etc.) handle, with the adsorbent of routine (for example: active carbon, silicic acid, silica gel, cellulose, aluminium oxide etc.) handle, crystallization, recrystallization and similar method can obtain FR-900520 and FR-900523 material.
Particularly will ferment the material dissolution that contains FR-900520 and FR-900523 product that produced at suitable solvent for example in ethyl acetate, the normal hexane etc., then this solution on silicagel column with appropriate organic solvent for example ethyl acetate and normal hexane or their mixture carry out chromatographic isolation, can be separately FR-900520 and two kinds of materials of FR-900523.Two kinds of materials of FR-900520 that separates like this and FR-900523 all can further be used conventional method, for example recrystallization, once more with chromatography separation, high performance liquid chromatography and similarly method purify.
The physiology of FR-900520 and FR-900523 material and chemical characteristic
The FR-900520 material
(1) form and color
Colourless lamellar
(2) elementary analysis
C：64.81%，H：8.82%，N：1.55%
(3) chromogenic reaction
Positive: cerous sulfate reaction, sulfuric acid reaction, ehrlich reaction, road lagen doffer reaction and iodine steam reaction
Negative: iron chloride reaction, ninhydrin reaction and molisch's reaction.
(4) solubility
Soluble: methanol, ethanol, acetone, ethyl acetate, chloroform, diethyl ether and benzene.
Sl. sol.: normal hexane, petroleum ether.
Insoluble: water.
(5) fusing point:
163-165℃。
(6) specific rotatory power: [α] 23 D:-84.1 ° of (C=1.0, CHCl 3)
(7) ultra-violet absorption spectrum:
End absorption.
(8) infrared absorption spectroscopy:
ν MaximumCHCl 3:
913 908(sh 960(sh 1,452 1,380 1,350 1,330 1,285 1,170 1,135 1,090 1,030 1,005 990 980(sh 3,680 3,575 3,520 2,940 2,875 2,325 1,745 1,725 1,700 1647 1610(sh)))) (centimetre -1)
(9) 13The C nuclear magnetic resoance spectrum:
δ（ppm，CDCl 3）：
Figure 911027882_IMG29
Figure 911027882_IMG30
Its spectrogram is shown in Fig. 7.
(10) 1The H nuclear magnetic resoance spectrum:
Its spectrogram is shown in Fig. 8.
(11) thin layer chromatography:
Immobile phase launches the solvent Rf value
The silica gel plate chloroform: methanol (20: 1, V/V) 0.38
Ethyl acetate 0.51
(12) Substance Properties
Neutral substance.
About the FR-900520 material, it should be noted that, doing 13C and 1During the H nuclear magnetic resonance spectroscopy, it is paired that this material demonstrates various chemical shift signals, yet when doing the measurement of thin layer chromatography and high efficiency liquid chromatography, the FR-900520 material shows one spot at thin layer chromatography respectively, shows unimodal at high efficiency liquid chromatography.
By the chemical constitution of above-mentioned physics and chemical property and successful mensuration FR-900506 material, can determine that the FR-900520 material has following chemical constitution.
Figure 911027882_IMG31
17-ethyl-1,14-dihydroxy-12[2-(4-hydroxyl-3-cyclohexyl methoxy hexyl)-1-methyl ethylene-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle-[22.3.1.0 4.9] carbon-18-alkene-2,3,10 surplus 20, the 16-tetraketone.
The FR-900523 material
(1) form and color
Colourless needle-like.
(2) elementary analysis
C：64.57%，H：8.84%，N：1.81%
(3) chromogenic reaction
Positive: cerous sulfate reaction, sulfuric acid reaction, ehrlich reaction, De Lagen doffer reaction and iodine steam reaction
Negative: iron chloride reaction and ninhydrin reaction.
(4) solubility
Soluble: methanol, ethanol, acetone, ethyl acetate, chloroform, diethyl ether and benzene.
Sl. sol.: normal hexane and petroleum ether.
Insoluble: water.
(5) fusing point:
152-154℃。
(6) specific rotatory power: [α] 23 D:-73.0 ° of (C=0.65, CHCl 3)
(7) ultra-violet absorption spectrum:
End absorption.
(8) infrared absorption spectroscopy:
ν MaximumCHCl 3:
3670 3580 3510 2930 2875 2825 1745 1722
1700 1647 1450 1380 1350 1330 1307 1285
1170 1135 1090 1050 1030 1000 900 978
960 930 915 888 870 850(centimetres -1)
(9) 13The C nuclear magnetic resoance spectrum:
δ（ppm，CDCl 3）：
Figure 911027882_IMG32
Figure 911027882_IMG33
Its spectrogram is shown in Fig. 9.
(10) 1The H nuclear magnetic resoance spectrum:
Its spectrogram is shown in Figure 10.
(11) thin layer chromatography:
Immobile phase launches the solvent Rf value
The silica gel plate chloroform: methanol (20: 1, V/V) 0.38
Ethyl acetate 0.51
(12) Substance Properties:
Neutral.
About the FR-900523 material, it should be noted and doing 13C and 1During the H nuclear magnetic resonance spectroscopy, this material shows paired signal in various chemical shift, yet when thin layer chromatography and high efficiency liquid chromatography mensuration, the FR-900523 material shows monochromatic speckle and unimodal respectively at thin layer chromatography and high efficiency liquid chromatography.
Determine by above-mentioned physics and chemical property with to the success of the chemical constitution of FR-900506 material, can determine that the FR-900523 material has following chemical constitution:
Figure 911027882_IMG34
1,14=dihydroxy-12-[2-4-hydroxyl-3-cyclohexyl methoxy hexyl)-the 1-methyl ethylene]-23,25-dimethoxy-13,19,17,21,27-pentamethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9]-28 carbon-18-alkene-2,3,10, the 16-tetraketone
(ⅱ) synthetic method:
(1) method 1:(introduces hydroxyl protecting group)
(I is introduced hydroxyl protecting group in a) can make chemical compound (I b) at chemical compound.
The reagent of used suitable introducing hydroxyl protecting group in this reaction; can be the trial-production that resembles the such routine of the inferior vitriol of two (rudimentary) alkyl; for example: the low-carbon alkyl methyl sulfoxide (for example: dimethyl sulfoxine; the ethyl-methyl sulfoxide; the propyl group methyl sulfoxide; the isopropyl methyl sulfoxide; the butyl methyl sulfoxide; the isobutyl group methyl sulfoxide; the hexyl methyl sulfoxide; or the like); (for example: trimethylsilyl chloride resemble the so trisubstituted silyl compound of three (rudimentary) alkyl silyl halides; the triethylsilyl bromine; the tributyl silyl chloride; tert-butyldimethylsilyl chloride; or the like); low-carbon alkyl diaryl silyl halides (for example: methyl-diphenyl silyl chloride; ethyl-diphenyl silyl bromide; propyl group xylyl silyl chloride; t-butyldiphenylsilyl chlorine; or the like) and can introduce the acylation agent of above-mentioned acyl group; resemble carboxylic acid; sulphuric acid and their reactive derivative, for example: acyl halide; anhydride; active amide; active ester and analog.The example preferably of these reactive derivatives can comprise acid chloride; acid bromide RCOBr; the anhydride that contains following acid; for example the phosphoric acid of Qu Daiing (for example: dialkyl group phosphoric acid; phosphenylic acid; diphenylphosphoric acid; dibenzyl phosphoric acid; halogenated phosphoric acid; or the like) such mixed acid anhydride; dialkyl group phosphorous acid; sulfurous acid; thiosulfuric acid; sulphuric acid; alkyl carbonate (for example: methyl carbonate; ethyl carbonate; propyl carbonate; or the like); aliphatic carboxylic acid (for example: neopentanoic acid; valeric acid; isovaleric acid; the 2-ethyl-butyl; trichloroacetic acid; or the like); aromatic carboxylic acids (for example: benzoic acid; or the like); symmetric anhydride; have imido-functional group (for example: imidazoles; the 4-substituted imidazole; dimethyl pyrazole; triazole and tetrazolium) active amide of jeterocyclic chemistry thing; active ester (for example: the p-nitrophenyl ester; 2; 4-dinitrophenyl ester; the trichlorophenyl ester; the five chlorophenyl ester; the mesyl phenylester; the phenylazo phenylester; the phenyl monothioester; the p-nitrophenyl monothioester; the p-methylphenyl monothioester; the carboxymethyl monothioester; pyridyl ester; the piperidyl ester; 8-quinolyl monothioester; or have and resemble N; the N-dimethyl hydroxylamine; the 1-hydroxyl-2-(1H)-pyridone; N-hydroxy-succinamide; N-hydroxyl phthalimide; I-hydroxybenzotriazole; the ester of 1-hydroxyl-class N-hydroxy compounds such as 6-chlorobenzotriazole, and analog.
In this reaction, under the situation that two (low-carbon (LC)) alkyl sulfoxide is used as hydroxyl protecting group introducing agent, be reflected at usually under the lower alkanes acid anhydride existence resemble the acetic anhydride and carry out.
And under the situation of trisubstituted silyl compound as the introducing agent of hydroxyl protecting group, reaction is preferably in and resembles imidazoles and the so conventional condensing agent of analog thereof and carry out under existing.
Further; under the situation of acylating agent as the introducing agent of hydroxyl protecting group; reaction is preferably under following such organic or inorganic alkali existence to be carried out; as alkali metal (for example: lithium; sodium; potassium; Deng); alkaline-earth metal (for example: calcium etc.); alkali metal hydride (for example: sodium hydride etc.); alkaline earth metal hydride (for example: calcium hydride; Deng); alkali metal hydroxide (for example: sodium hydroxide; potassium hydroxide; Deng); alkali carbonate (for example: sodium carbonate; potassium carbonate; Deng); alkali metal hydrogencarbonate (for example: sodium bicarbonate; potassium bicarbonate; Deng); alkali metal alcoholates (for example: Feldalat NM; Sodium ethylate; potassium tert-butoxide; Deng); the alkali metal salt of alkanoic acid (for example: sodium acetate; Deng); trialkylamine (for example: triethylamine; Deng); pyridine compounds (for example: pyridine; lutidines; picoline; 4-N; the N-dimethyl aminopyridine, etc.); quinoline or the like.
In this reaction; during with the acylating agent of free form or its salt; reaction is preferably under the following conventional condensing agent existence to be carried out; as the carbodiimides thing (for example: N; N '-dicyclohexyl carbodiimide; N-cyclohexyl-N '-(4-lignocaine cyclohexyl)-carbodiimides; N; N '-diethyl carbodiimides; N; N '-diisopropyl carbodiimides; N-ethyl-N '-(3-dimethylamino-propyl) carbodiimides; Deng); the ketene-imine chemical compound (for example: N; N '-carbonyl two (glyoxal ethyline); 1; 5-pentylidene ketenes-N-cyclohexyl imines; diphenylethlene ketone-N-cyclohexyl imines; Deng); etherate olefinic or acetylene series (for example: ethyoxyl acetylene; β-cyclic vinyl ethylether); the sulphonic acid ester of N-hydroxybenzotriazole derivant [for example: 6-chloro-1H-benzotriazole 1-(4-chlorobenzene sulfonyloxy); Deng], and similar compound.
Reaction is carried out in to the conventional solvent that can not react ill effect usually; these solvents resemble water, acetone, dichloromethane, alcohol (for example: methanol, ethanol etc.), oxolane, pyridine, N; dinethylformamide etc.; or its mixture; and be under the situation of liquid in the introducing agent of alkali or hydroxyl protecting group, they also can be used as solvent.
Reaction temperature is not strict, and reaction can both carried out under the situation that is cooled to heat usually.
The scope of this method comprises such a case, promptly the reaction process in, chemical compound (I R a) 2The hydroxyl corresponding quilt that may convert purpose chemical compound (I b) sometimes to protect hydroxyl.
And the scope of this method also comprises such a case, and promptly when in the presence of the lower alkanes acid anhydride, two (low-carbon (LC)) alkyl sulfoxide has partial structural formula as the introducing agent of hydroxyl protecting group
Figure 911027882_IMG35
Chemical compound (I a) sometimes may be oxidized in the process of reaction, forms to have partial structural formula Chemical compound (I b), the R in two formulas wherein 2It all is hydroxyl.
(2) method 2(introduces hydroxyl protecting group)
In chemical compound (I c), introduce hydroxyl protecting group and can prepare chemical compound (I d).
Reaction is to be undertaken by the same procedure of method 1 basically, and therefore, reaction condition (for example: alkali, condensing agent, solvent, reaction temperature etc.) is exactly those conditions of method 1.
The scope of this method comprises: in the process of reaction, and the R of chemical compound (I c) 1The hydroxyl corresponding quilt that usually may be transformed into purpose chemical compound (I d) protect hydroxyl.
(3) method 3:(forms two keys)
Chemical compound (I e) can make chemical compound (I f) with alkali reaction.
Used suitable alkali can comprise alkali used in the method 1 in this reaction.
This reaction also can be in the presence of alkali be reacted with chemical compound (I e) and acylation agent and is realized the R in (I e) 2It is hydroxyl.
This reaction is being carried out can not reacting in the dysgenic conventional solvent usually, these solvents resemble water, acetone, dichloromethane, alcohol (for example: methanol, ethanol, propanol etc.), oxolane, pyridine, N, dinethylformamide etc., perhaps their mixture, in addition, at alkali is under the situation of liquid, and this alkali also can be used as solvent.
Reaction temperature is not strict, and reaction can both carried out under the situation that is cooled to heat usually.
(4) oxidation of method 4:(hydroxyl vinyl)
Can pass through oxidized compound (I g) preparation chemical compound (I h.
Used oxidant can be included in those two (low-carbon (LC)) alkyl sulfoxides that provide in the method 1 in this reaction.
This reaction is normally in the presence of the lower alkanes acid anhydride resemble the acetic anhydride, carrying out can not reacting in the dysgenic conventional solvent, these conventional solvents are as acetone, dichloromethane, ethyl acetate, oxolane, pyridine, N, dinethylformamide etc., or their mixture, and be under the situation of liquid at the lower alkanes acid anhydride, they also can be used as solvent.
Reaction temperature is not strict, and reaction can both carried out under the situation that is cooled to heat usually.
The scope of this method comprises: in the process of reaction, and the R of starting compound (I g) 1Hydroxyl may be transformed into 1-(low-carbon alkyl sulfo-on the purpose chemical compound (I h) sometimes) (low-carbon (LC)) alkoxyl.
(5) the allylic reduction of method 5:()
Can obtain chemical compound (I j) to chemical compound (I i) reduction.
In this method, can be with reducing the conventional method that pi-allyl is reduced into propyl group, as catalytic reduction or similar approach.
The appropriate catalyst of using in catalytic reduction is those conventional catalysts, as platinum catalyst (for example: platinized platinum, platinum, platinum black, colloidal state platinum, platinum oxide, platinum filament etc.), palladium catalyst (for example: palladium sponge, palladium black, Palladium monoxide, carbon carries palladium, pallamine, barium sulfate carries palladium, brium carbonate carries palladium etc.), Raney nickel (for example: reductive nickel, nickel oxide, Raney nickel etc.), Co catalysts (for example: the reduction cobalt, Raney cobalt etc.), iron catalyst (for example: reduced iron, the interior ferrum of Ruan etc.), copper catalyst (for example: go back native copper, copper in Ruan, Ullman copper etc.) and similarly catalyst.
Reduction is normally being carried out can not reacting in the dysgenic conventional solvent, and these solvents are as water, methanol, ethanol, propanol, pyridine, ethyl acetate, N, dinethylformamide, dichloromethane or their mixture.
This reductive reaction temperature is not strict, and reaction normally can both carried out under the situation that is cooled to heat.
The purpose tricyclic compound (I) that obtains according to above-mentioned synthetic method 1 to 5 can separate and purification with conventional method, for example: extraction, precipitation, fractional crystallization, recrystallization, chromatography or the like.
Chemical compound (I) and (I b) can comprise the salt that pharmaceutically can adopt resemble the alkali salt to the suitable salt of (I j), for example: alkali metal salt (for example: sodium salt, potassium salt etc.), alkali salt (for example: calcium salt, magnesium salt etc.), ammonium salt, amine salt (for example: triethylamine salt, N-benzyl-N-methylamine salt etc.) and other conventional organic salts.
It should be noted, in the reaction of above-mentioned synthetic method 1 to 5 or in the post processing of reactant mixture therein, because raw material and the asymmetric carbon atom of purpose chemical compound or the existence of two keys, isomers and/or stereoisomer may be transformed into other isomers and/or stereoisomer sometimes, and such situation is also included within the scope of the present invention.
Tricyclic compound of the present invention (I) has pharmacologically active, for example: immunosuppressive activity, antibacterial activity etc.Therefore, be used for the disease resistance for the treatment of and preventing to cause by the transplanting that resembles such organ or tissue such as heart, kidney, liver, bone marrow, skin, the anti-transplant disease of host that causes by bone marrow transplantation, autoimmune disease is as rheumatic arthritis, systemic lupus erythematosus, chronic lymphocytic thyroiditis, multiple sclerosis, myasthenia gravis, type, uveitis etc., infectious disease that is caused by morbific microorganism or the like.
Provide some examples below, with the pharmacologically active and the pharmacological test data of explanation tricyclic compound.
Test 1
Tricyclic compound (I) suppresses mixed lymphocyte reaction in vitro (MLR)
The MLR test is carried out in one group of microtitre culture dish, add 0.2 milliliter of RPMI1640 culture medium in the cave of each culture dish, add 10% hyclone, 2 mM sodium bicarbonate, penicillin (50 units per ml) and streptomycin (50 mcg/ml), contain 5 * 10 in the cave of each culture dish 5C57BL/6 cell (the H-2 that comes ture b) and with ametycin handle (at 37 ℃, handled 30 minutes, and wash three times) with the RPMI1640 culture medium with 25 mg/ml ametycins 5 * 10 5BALB/C irritation cell (H-2 d).This like cell was cultivated 68 hours at the damp environment of 5% carbon dioxide and 95% air, and is used before collecting cell at 37 ℃ 3H-thymidine (0.5 little Ci) makes it to vibrate 4 hours.Purpose compound dissolution of the present invention in ethanol, and further is diluted in the RPMI1640 culture medium, is added in the culture, the concentration that reaches at last is 0.1 mcg/ml or still less.
Gained the results are shown in table 7 to 10.Tricyclic compound of the present invention suppresses the MLR of mice.(table is seen the literary composition back)
Test 2
The antimicrobial acivity of tricyclic compound (I)
Be used in the antimicrobial acivity of the anti-various funguses of continuous agar dilution mensuration tricyclic compound (I) in the Sab.After 24 hours, minimum inhibition concentration (MIC) is represented with mcg/ml 30 ℃ of cultivations.
The table 11 of following table and 12 has been listed tricyclic compound of the present invention to true () antimicrobial acivity for example: Aspergillus fumigatus IFO5840 and Fusarium Oxysporum IFO5942.
The MIC(minimal inhibitory concentration of table 11 tricyclic compound (I) anti-aspergillus fumigatus IFO5840) value (mcg/ml)
Material MIC(mcg/ml)
FR-900506 0.025
FR-900520 0.1
FR-900523 0.3
FR-900525 0.5
The MIC value (mcg/ml) of the anti-Fusarium Oxysporum of table 12 tricyclic compound (I)
Material MIC(mcg/ml)
FR-900506 0.05
FR-900525 1
Test 3
Tricyclic compound (I) is to the influence of the allogeneic skin graft survival of Mus
The Vental of donor (Fischer) Mus is transplanted the side chest region of skin tissue transplantation to receptor (WKA) Mus.Separated wrapping at the 5th day.The situation that every day, inspection was transplanted up to repelling for extremely, is repelled to be defined as and is transplanted the necrosis more than 90% of skin tissue.
The FR-900506 substance dissolves in olive oil, and is begun continuous 14 days administered intramuscular from that day of transplanting.
As shown in table 13, with the Mus of the continuous intramuscular injection of olive oil 14 days treatment within 8 days all the skin of allografts all be ostracised, but the survival that the Mus for the treatment of with the FR-900506 material every day has obviously prolonged allogeneic skin graft.(table is seen the literary composition back)
Test 4
Tricyclic compound (I) brings out arthritic influence to the II Collagen Type VI of Mus
In cold 0.01M acetic acid, concentration is 2 mg/ml collagenolysis.This solution is with isopyknic incomplete Freund adjuvent emulsifying.Is cumulative volume several positions at 0.5 milliliter the cold emulsion back that is subcutaneously injected into female Lewis Mus and one or two position of afterbody.FR-900506 is dissolved in olive oil and oral administration.With the contrast Mus of the II Collagen Type VI immunity of same amount, only oral olive oil.Observe arthritic incidence rate.
Result of the test is listed in table 14.From the polyarthritis that begins all to have caused inflammatory on the same day with all Mus of 14 days of olive oil treatment with the immunity of II Collagen Type VI.
In the middle of the observation in three weeks, treat 14 day with the treatment of FR-900506 material every day, suppressed arthritic generation fully.
Table 14 FR-900506 material brings out the arthritic influence of Mus to the II Collagen Type VI
Dosage
The arthritic incidence rate of milligram/kg/day
Contrast (olive oil)-5/5
FR-900506 3.2 0/5
Material
Test 5
Tricyclic compound (I) is to the influence of the tentative irritated encephalomyelitis of SJL/J Mus (EAE)
Make the spinal cord homogenate by the SJL/J Mus.Take out spinal cord with inhalation, mix and homogenize with about isopyknic water at 4 ℃.Isopyknic this cold homogenate (10 mg/ml) freund complete adjuvant (CFA) emulsifying that contains 0.6 mg/ml Mycobacterium tuberculosis H37RA.
0 day and 13 days the STL/J Mus on one's body 0.2 milliliter of spinal cord CFA of double injection emulsion bring out EAE.All Mus in these tests are all carried out the clinical diagnosis evaluation of EAE and tick every day.
Do the mark of the AE order of severity according to following standard: 1 grade-tail is sagging; 2 grades-stupid gait; 3 grades-one limbs or many limbs weakness; 4 grades-paraplegia or hemiplegia.
The FR-900506 substance dissolves in olive oil, and since 0 day (immunity first day) oral administration 19 days.As table 15 expression, the FR-900506 material has prevented the development of EAE clinical symptoms significantly.
Table 15 FR-900506 material is to the influence of the tentative irritated encephalomyelitis of SJL/J mouse
Dosage was at 24 days
The number of animals that (milligram/kilogram) is ill
Contrast (olive oil)-10/10
FR-900506 32 0/5
Material
Test 6
Tricyclic compound (I) is to the influence of the local anti-transplant reaction of host of Mus (GVHR)
The great-hearted splenocyte (1 * 10 of C57BL/6 donor 7Cell) the right back foot that is subcutaneously injected into the BDF1 Mus brings out local GvHR.After 7 days Mus is killed the weighing right side (foot of injection) and a left side (not Zhu She foot) popliteal lymph node (PLN).Difference by the weight of right and left PLN is represented GvHR.
The FR-900506 material is dissolved in olive oil and began oral administration on the same day 5 days from sensitization.
The FR-900506 material prevents the ED of the anti-transplant reaction of local host 50Value is 19 milligrams/kilogram.
Test 7
The acute toxicity test of tricyclic compound (I)
At ddy Mus peritoneal injection, the acute toxicity of test FR-900506, FR-900520, FR-900523 and FR-900525 material in each case, when dosage is 100 milligrams/kilogram, is not observed dead.
Pharmaceutical composition of the present invention can use with the form of medicine preparation, for example: the form of solid, semisolid or liquid, wherein contain tricyclic compound of the present invention (I), as a kind of active component, with organic or inorganic carrier or mixed with excipients, be applicable to external, intestinal or non-intestinal, active component can be mixed with atoxic pharmaceutically adoptable carrier commonly used, makes tablet, pilule, capsule, suppository, solution, Emulsion, suspension and other any suitable forms.Operable carrier is water, glucose, lactose, arabic gum, gelatin, mannitol, gelatinized corn starch, magnesium trisilicate, Talcum, corn starch, keratin, colloidal state silicon, potato starch, urea and other carriers that is suitable in preparation, with solid, semisolid or liquid form, can use adjuvant, stabilizing agent, thickening agent and stain and spice in addition.The amount of the active purpose chemical compound that contains in the pharmaceutical composition will be enough to produce according to the state of an illness and the desired effect of treatment.
Said composition is used for human body, preferably by non-intestinal or intestinal canal administration.And the dose therapeutically effective of tricyclic compound (I) can change, the state of an illness and the age of depending on each patient that will treat, the about 0.01-1000 milligram of dosage of active component every day of usually curing the disease, be preferably the 0.1-500 milligram, 0.5-100 milligram more preferably, about 0.5 milligram, 1 milligram, 10 milligrams, 50 milligrams, 100 milligrams, 250 milligrams and 500 milligrams of usually average single doses.
Example given below illustrates purpose of the present invention.
Example 1
The separation of Streptomyces tsukubaensis No.9993
No. 9993, Streptomyces by use following dilution plate technical point from.
At Japanese ToYoSato-Cho, Tsukuba Gun, the pedotheque about the about gram that gather in the Ibaraki county adds in the sterilized test tube, and to be mixed with volume with sterilized water be 5 milliliters.This mixture mixed for 10 seconds with tubular type sonic generator (tube buzzer), continued to mix 10 minutes.Supernatant continues the sterilized water dilution with 100 times.Diluent (0.1 milliliter) coated adds vitamin B on accompanying the Czapek agar that substitutes in the culture dish 1(sucrose 30 grams, Chile saltpeter 3 grams, dipotassium hydrogen phosphate 1 gram, magnesium sulfate 0.5 gram, potassium chloride 0.5 gram, ferrous sulfate 0.01 gram, vitamin B 10.1 gram, agar 20 gram, 1000 milliliters from the beginning, pH is 7.2).After 30 ℃ of incubations 21 days, many colony lifts of growing on the plate in dilution to slant medium [yeast-malt extract broth (ISP-culture medium 2)], and 30 ℃ of cultivations 10 days.In isolating bacterium colony, can find Streptomyces tsukubaensis No.9993.
Fermentation
160 milliliters of culture medium that contain 1% glycerol, 1% soluble starch, 0.5% glucose, 0.5% cotton seed meal, 0.5% dry yeast, 0.5% corn impregnation liquid and 0.2% calcium carbonate (transferring pH is 6.5), be divided into 20 parts and pour in 20 500 milliliters of conical flasks, and 120 ℃ of sterilizations 30 minutes.Strepomyces tsukubaensis No.9993(CGMCC No.0083) the loopful slant culture be inoculated in above-mentioned 20 culture medium in 500 milliliters of conical flasks, on rotator shaker, cultivated 4 days at 30 ℃.The culture that generates is inoculated in advance in 20 minutes 200 liters fermentation tank of 120 ℃ of sterilizations, wherein contain 4.5% soluble starch, 1% corn impregnation liquid, 1% dry yeast, 0.1% calcium carbonate and 0.1%Adekanol(anti-foam agent: trade mark, maker: in 150 liters of culture medium Asahi Denka company), and under the vibration velocity of 150 liters of/minute ventilation speed and 250 rev/mins, at 30 ℃, cultivated 4 days.
Separate and purification
The culture that obtains above is by means of 5 kilograms of diatomite filtrations.The mycelium cake is with 50 liters of methanol extraction, obtain 50 liters of extracts, (trade mark, maker: Mitsubishi chemical industry company limited) the non-ionic adsorption resin post adsorbs for the methanolic extract of mycelium cake and filtrate merga pass 10 liters " Diaion HP-20 ".After washing, use methanol-eluted fractions with 30 premium on currency and 30 liters of methanol aqueous solutions.The eluent vapourisation under reduced pressure gets 2 liters of residual water.This residual water is with 2 liters of ethyl acetate extractions.Ethyl acetate extract under reduced pressure concentrates, and obtains the oily residue.The acidic silica gel of this oily residue and double weight (12 grades of silica gel specials, maker: mix this mixture ethyl acetate furnishing pasty state Fuji Devison company).After evaporating solvent, the dry powder of generation fill with normal hexane with above-mentioned 800 milliliters of identical acidic silica gel posts on, separate with chromatography.This silicagel column launches with mixture (9: 1V/V, 3 liters and 4: 1V/V, 3 liters) and 3 liters of ethyl acetate of 3 liters of normal hexane, nalka and ethyl acetate.Collection contains purpose chemical compound fraction, and under reduced pressure concentrates, and obtains the oily residue.Of the mixture dissolving of this residue, and on 500 milliliters of silica gel (maker: Merck company limited, 230-400 order) post of filling, separate with chromatography with the same solvent system with normal hexane and ethyl acetate (1: 1V/V, 30 milliliters).
Mixture eluting with normal hexane and ethyl acetate (1: 1V/V, 2 liters and 1: 2V/V, 1.5 liters).Collection contains the first purpose chemical compound fraction, and under reduced pressure concentrates, and obtains faint yellow oily thing.This grease mixes with the acidic silica gel of double weight, this mixture ethyl acetate furnishing pasty state.After evaporating solvent, the dry powder of generation is separated with chromatography on the acidic silica gel post of filling with normal hexane.And launch with normal hexane.Collection contains purpose chemical compound fraction, and under reduced pressure concentrates, and obtains the FR-900506 material crude product of 1054 milligrams of white powders.100 milligrams of this thick products separate with high pressure lipuid chromatography (HPLC).With Lichroborb SI60(trade mark, maker: Merck company) post (8 * 500 millimeters) as carrier fluid carries out eluting.This chromatography is 5 ml/min at flow velocity, and mobile phase is that dichloromethane is with diox (85: under the condition of the mixture of 15V/V, use UV-detector, locate to detect at 230 millimicrons.Collect active fractions, and evaporation, carry out this high pressure liquid chromatography once more and separate, obtain the FR-900506 material of the white powder of 14 milligrams of purification.
In addition, eluting carries out unceasingly with 1.5 liters of ethyl acetate.Collection contains the second purpose chemical compound fraction, and under reduced pressure concentrates, and obtains the FR-900525 material crude product of 30 milligrams of faint yellow oilies.
Example 2
Fermentation
Is pH that 6.5 100 milliliters of pre-culture that contain 1% glycerol, 1% corn starch, 0.5% glucose, 1% cotton seed meal, 0.5 corn impregnation liquid, 0.5% dry yeast and 0.2% calcium carbonate are poured in one 500 milliliters the conical flask, and 120 ℃ of sterilizations 30 minutes.The loopful slant culture of Streptomyces tsukubaensis No.9993 is inoculated in this culture medium, and cultivated 4 days at 30 ℃, the culture that generates is transferred in advance in the 20 liters identical pre-culture in 120 30 liters the fermentation tanks of sterilizing 30 minutes.This culture is at 30 ℃ of incubations after two days, 16 liters pre-culture is inoculated in advance and contains 4.5% soluble starch, 1% corn impregnation liquid, 1% dry yeast, 0.1% calcium carbonate and 0.1%Adekanol(anti-foam agent in 2 tons of jar of 30 minutes of 120 ℃ of sterilizations, trade mark, maker: Asahi Denka company) pH is in 1600 liters of fermentation medium of 6.8, and cultivates 4 days at 30 ℃.
Separate and purification
The culture fluid that obtains above is by means of 25 kilograms of diatomite filtrations.The mycelium cake obtains 500 liters of extracts with 500 liters of acetone extractions.Mycelial acetone extract merges with 1350 liters of filtrates, and (trade mark, maker: Mitsubishi chemical industry company limited) the non-ionic adsorption resin post adsorbs by 100 liters " Diaion HP-20 ".After aqueous acetone solution washing with 300 premium on currency and 300 liter 50%, the aqueous acetone solution eluting with 75%.The eluent vapourisation under reduced pressure obtains 300 liters of residual water.This residue is with 20 liters of ethyl acetate extractions three times, ethyl acetate extract under reduced pressure concentrates, obtain the oily residue, the acidic silica gel of this oily residue and double weight (12 grades of silica gel specials, maker: mix this mixture ethyl acetate furnishing pasty state Fuji Devison company).After evaporating solvent, the dry powder of generation is separated with column chromatography on 8 liters of acidic silica gel posts same as described above of filling with normal hexane.This silicagel column launches with mixture and 30 liters of ethyl acetate of 30 liters of normal hexane, normal hexane and ethyl acetate (4: 1V/V, 30 liters).Collection contains purpose chemical compound fraction, and under reduced pressure concentrates, and obtains the oily residue.This oily residue mixes with the acidic silica gel of double weight, and this mixture ethyl acetate furnishing pasty state.After evaporating solvent, generate dry powder on 3.5 liters of acidic silica gel posts of filling with normal hexane, separate with chromatography.Silicagel column launches with 10 liters of normal hexane, normal hexane and ethyl acetate (4: 1V/V, 10 liters) mixture and 10 liters of ethyl acetate.Collection contains purpose chemical compound fraction, and under reduced pressure concentrates, and obtains faint yellow oily thing.Of the mixture dissolving of this oily residue, and on 2 liters of silica gel (maker: Merck company limited, 230-400 order) post of filling, separate with column chromatography with the same solvent system with normal hexane and ethyl acetate (1: 1V/V, 300 milliliters).Mixture and 6 liters of eluent ethyl acetates with normal hexane and ethyl acetate (1: 1V/V, 10 liters and 1: 2V/V, 6 liters).
Collection contains the first purpose chemical compound fraction, and under reduced pressure concentrates, and obtains the FR-900506 material of 34 gram white powder.This white powder is dissolved with acetonitrile, and under reduced pressure concentrate.This concentrate remains on 5 ℃ and spends the night, and obtains 22.7 gram prisms.With identical solvent recrystallize, obtain the FR-900506 material of the colourless prism of 13.6 gram purification.
In addition, collection contains the second purpose chemical compound fraction, and under reduced pressure concentrates, and obtains 314 milligrams of faint yellow powdered FR-900525 material crude products.
Example 3
Fermentation
160 milliliters of culture medium that contain 1% glycerol, 1% corn starch, 0.5% glucose, 1% cotton seed meal, 0.5% dry yeast, 0.5% corn impregnation liquid and 0.2% calcium carbonate (transferring pH is 6.5) are divided into 10 parts, pour in 10 500 milliliters the conical flask, and 120 ℃ of sterilizations 30 minutes.Strepomyces tsukubaensis No.9993 loopful slant culture is inoculated in above-mentioned each culture medium, and on rotator shaker, cultivated 4 days at 30 ℃.The culture that generates is inoculated in advance in 20 minutes 200 liters fermentation tanks of 120 ℃ of sterilizations, contains 5% soluble starch, 0.5% Semen arachidis hypogaeae powder, 0.5% dried yeast powder, 0.5% glutelin powder, 0.1% calcium carbonate and 0.1%Adekanol(anti-foam agent, trade mark, maker: in 150 liters of culture medium Asahi Denka company), in ventilation speed is that the frequency of vibration of 150 liters/minute and rotator shaker are under 250 rev/mins the vibration, cultivates 4 days at 30 ℃.
Separate and purification
The culture fluid that obtains above is by means of 5 kilograms of diatomite filtrations.The mycelium cake obtains 50 liters of extracts with 50 liters of acetone extractions.Mycelium acetone extract and 135 liters of filtrates merge, and (trade mark, maker: Mitsubishi chemical industry company limited) the non-ionic adsorption resin post adsorbs by 10 liters " Diaion HP-20 ".After the aqueous acetone solution washing with 30 premium on currency and 30 liter 50%, eluting is finished with 75% aqueous acetone solution.30 liters of eluent vapourisation under reduced pressure obtain 2 liters of residual water.This residue is with 2 liters of ethyl acetate extractions three times.Ethyl acetate extract under reduced pressure concentrates, and obtains the oily residue.The acidic silica gel of this oily residue and double weight (12 grades of silica gel specials, maker: mix this mixture ethyl acetate furnishing pasty state Fuji Devison company).After evaporating solvent, the dry powder of generation is separated with column chromatography on 800 milliliters of acidic silica gel posts same as described above of filling with normal hexane.This silicagel column launches with mixture and 3 liters of ethyl acetate of 3 liters of normal hexane, normal hexane and ethyl acetate (4: 1V/V, 3 liters).Collection contains purpose chemical compound fraction, and under reduced pressure concentrates, and obtains the oily residue.This oily residue dissolves with the mixture of normal hexane and ethyl acetate (1: 1V/V, 30 milliliters), and on 5000 milliliters of silica gel (maker: Merck company limited, 230-400 order) post of filling with the same solvent system, separates with column chromatography.Eluting is finished with mixture and 1.5 liters of ethyl acetate of normal hexane and ethyl acetate (1: 1V/V, 2 liters and 1: 2V/V, 1.5 liters).
Collection contains the first purpose chemical compound fraction, and under reduced pressure concentrates, and obtains the FR-900506 material crude product of 3 gram pale yellow powder shapes.
In addition, collection contains the second purpose chemical compound fraction, and under reduced pressure concentrates, and obtains the oily residue.This oily residue carries out chromatographic isolation again with silicagel column, obtains faint yellow oily thing.Above-mentioned oily residue mixes with the acidic silica gel of double weight, this mixture ethyl acetate furnishing pasty state.After evaporating solvent, the dry powder of generation is separated with chromatography, and is launched with normal hexane on 100 milliliters of acidic silica gel posts of filling with normal hexane.Collection contains purpose chemical compound fraction, and under reduced pressure concentrates, and obtains the FR-900525 material of 380 milligrams of light yellow powder shapes.This powder normal hexane and ethyl acetate (1: 2V/V, 5 milliliters) mixture dissolving, and at 100 milliliters of acidic silica gels (922 grades of silica gel specials of filling with dicyandiamide solution same as described above, maker: Fuji Devison company) on the post, separate with chromatography, and use and the same solvent system washing of filling acidic silica gel.Eluting is finished with ethyl acetate.Collect active fractions, and under reduced pressure concentrate, obtain the FR-900525 material of the white powder of 230 milligrams of purification.
Example 4
The separation of Streptomyces hygroscopicus subsp.Yakushimaensis No.7238
Streptomyces hygroscopicus subsp.Yakushimaensis No.7238 is to use the flat technology of following dilution isolating.
About gram pedotheque gathering at Japanese Kagoshima county yakushima adds in the sterilized test tube, and becomes 5 milliliters with the sterilized water preparation.This mixture mixed for 10 seconds with the tubular type sonic generator, and continued to mix 10 minutes.Supernatant dilutes continuously with 100 times sterilized water.0.1 on the Czapek agar of milliliter diluent coated in Petri dish, add vitamin B (1000 milliliters of sucrose 30 grams, Chile saltpeter 3 grams, dipotassium hydrogen phosphate 1 gram, magnesium sulfate 0.5 gram, potassium chloride 0.5 gram, ferrous sulfate 0.01 gram, vitamin B 0.1 gram, agar 20 grams, tap water, pH7.2).After 30 ℃ of incubations 21 days, many colony lifts of growing on the plate in dilution are supported [yeast-malt extract broth (ISP-culture medium 2)] to slant medium, and cultivated 10 days at 30 ℃.In isolating bacterium colony, can find Streptomyces hygroscopicus subsp.yakushmaensis No.7238.
Fermentation
Containing 160 milliliters of culture medium of 1% glycerol, 1% soluble starch, 0.5% glucose, 0.5% cotton seed meal, 0.5% dry yeast, 0.5% corn impregnation liquid and 0.2% calcium carbonate (transferring pH is 6.5), be divided into 20 parts, pour in 20 500 milliliters the conical flask, and 120 ℃ of sterilizations 30 minutes.Streptomyces hygroscopicus subsp.yakushmaensis No.7238(CGMCC No.0082) slant culture be inoculated on the culture medium in 20 above-mentioned 500 milliliters of conical flasks, on rotator shaker, cultivated 4 days at 30 ℃.The culture that generates is inoculated in advance and contains 4.5% glucose, 1% corn impregnation liquid, 1% dry yeast, 1% glutelin powder, 0.5% wheat germ, 0.1% calcium carbonate and 0.1%Adekanol(anti-foam agent in 200 liters of fermentation tank of 20 minutes of 120 ℃ of sterilizations, trade mark, maker: Asahi Denka company) in 150 liters of culture medium, and under the vibration velocity of 150 liters/minute ventilation speed and 250 rev/mins, cultivated 4 days at 30 ℃.
Separate and purification
The culture fluid that obtains above is by means of 5 kilograms of diatomite filtrations.The mycelium cake obtains 50 liters of extracts with 50 liters of acetone extractions.Mycelial acetone extract and 135 liters of filtrates merge, and (trade mark, maker: Mitsubishi chemical industry company limited) the non-ionic adsorption resin post adsorbs by 10 liters " Diaion HP-20 ".After washing with 30 premium on currency and 30 liters of aqueous acetone solutions, eluting is finished with acetone.The eluent vapourisation under reduced pressure obtains 2 liters of residual water.This residue aqueous solution is with 4 liters of ethyl acetate extractions.Ethyl acetate extract under reduced pressure concentrates, and obtains the oily residue.The acidic silica gel of this oily residue and double weight (12 grades of silica gel specials, maker: mix this mixture ethyl acetate furnishing pasty state Fuji Devison company).After evaporating solvent, the dry powder of generation is separated with chromatography on 800 milliliters of acidic silica gel posts same as described above of filling with normal hexane.This silicagel column launches with mixture and 3 liters of ethyl acetate of 3 liters of normal hexane, normal hexane and ethyl acetate (4: 1V/V, 3 liters).Collection contains the fraction of FR-900520 and FR-900523 material, and under reduced pressure concentrates, and obtains the oily residue.Of the mixture dissolving of this oily residue, and on 1000 milliliters of silica gel (maker: Merck company limited, 70-230 order) post of filling, separate with column chromatography with the same solvent system with nalka and ethyl acetate (1: 1V/V, 50 milliliters).With normal hexane and ethyl acetate (1: 1V/V, 3 liters and 1: 2V/V, 3 liters) mixture eluting.Collection contains purpose chemical compound fraction, and under reduced pressure concentrates, and obtains 4.5 gram pale yellow powders.This powder dissolves with 20 ml methanol, and mixes with 10 ml waters.This mixture is being used methanol and water (4: 500 milliliters of (60-200 order) " YMC " (trade marks that mixture 1V/V) is filled, maker: Yama mura chemistry institute) on the reverse phase silica gel post, separate with chromatography, and (4: mixture 1V/V) launches with the first alcohol and water.
Collection contains the fraction of FR-900520 material, and under reduced pressure concentrates, and obtains the crude product of the FR-900520 material of the light yellow powder of 1.8 grams.The a small amount of ether dissolution of this powder.After leaving standstill a night, collect the crystal of separating out by filtering, and wash, then drying under reduced pressure with ether.Recrystallize from ether obtains the colourless flaky FR-900520 material of 600 milligrams of purification.
On the reverse phase silica gel post of filling with identical dicyandiamide solution, separate with chromatography, collect the fraction that contains the FR-900523 material subsequently, and under reduced pressure concentrate, obtain the crude product of the FR-900523 material of the light yellow powder shape of 0.51 gram.This crude product dissolves with 3 milliliters of acetonitriles, and using acetonitrile, oxolane and 50 mM phosphate-buffered agent solutions (pH2.0) (3: 2: on " YMC " 70 milliliters of reverse phase silica gel posts that mixture 5V/V) is filled, carry out chromatographic isolation, (3: 2: mixture 5V/V) launched this silicagel column with acetonitrile, oxolane and 50 mM phosphate-buffered agent solutions (pH2.0), collection contains purpose chemical compound fraction, and uses ethyl acetate extraction.This extract under reduced pressure concentrates, and obtains 190 milligrams of yellowish white powder.This yellowish white powder on " YMC " reverse phase silica gel post, separates with chromatography once more, obtains 80 milligrams of white powders.The a small amount of ether dissolution of this white powder, and standing over night at room temperature obtain 56 milligrams of crystal.Use the ether recrystallize, obtain 34 milligrams of colourless acicular FR-900523 materials.
Example 5
At room temperature, 0.1 milliliter of pyridine and 0.05 milliliter of acetic anhydride adding are contained in 0.2 milliliter of dichloromethane solution of 10.4 milligrams of FR-900506 materials, this mixture stirred 5 hours.Under reduced pressure, remove solvent in the reactant mixture.Residue silica gel thin-layer chromatography (expansion solvent: ether and dichloromethane, 1: 2V/V) separate, obtain 6.0 milligrams of 12-[2-(4-acetoxy-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-17-pi-allyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4 aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum: V(CHCl 3): 3520,1728, the sharp-pointed absworption peak of 1705(), 1640,1095 centimetres -1
Example 6
At room temperature, 0.5 milliliter of pyridine and 0.3 milliliter of acetic anhydride are added in 1 milliliter of dichloromethane solution of 52.5 milligrams of FR-900506 materials, this mixture at room temperature stirred 9 hours.Under reduced pressure, remove solvent in the reactant mixture.Residue silica gel thin-layer chromatography (expansion solvent: ether and hexane, 3: 1V/V) separate, obtain 48.0 milligrams of 14-acetoxyl groups-12-[2-(4-acetoxy-3-methoxyl group cyclohexyl respectively)-the 1-methylvinyl]-17-pi-allyl-1-hydroxyl-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10,16-tetraketone and 5.4 milligrams of 12-[2-(4-acetoxy-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-17-pi-allyl-1-hydroxyl-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-14s, 28-diene-2,3,10,16-tetraketone.
First chemical compound
Infrared spectrum ν (CHCl 3): 1730, the sharp-pointed absworption peak of 1720(), 1640 centimetres -1
Second chemical compound
Infrared spectrum ν (CHCl 3): 1730,1690,1640,1627 centimetres -1
Example 7
At room temperature, the Benzenecarbonyl chloride. of 50 microlitres is added in 0.2 milliliter of dichloromethane containing 9.7 milligrams of FR-900506 materials and the 0.1 milliliter of pyridine solution, this mixture at room temperature stirred 2 hours.Under reduced pressure, remove the solvent in the reactant mixture, obtain thick grease.This grease silica gel thin-layer chromatography (expansion solvent: ether and hexane, 2: 1V/V) purify, obtain 8.0 milligrams of 17-pi-allyls-12[2-(4-benzoyloxy-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-, 14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4 aza-tricycle [22.3.1.0 4.0] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum ν (CHCl 3): 3500, the sharp-pointed absworption peak of 1735(), 1710,1640,1600 centimetres -1
Example 8
About 100 milligrams of paranitrobenzoyl chlorides adding is contained in 1 milliliter of pyridine solution of 30.5 milligrams of FR-900506 materials, and this mixture at room temperature stirred 2 hours.This reactant mixture dilutes with ethyl acetate, and by the order washing with saturated sodium bicarbonate aqueous solution, water, 1 equivalent hydrochloric acid, water, saturated sodium bicarbonate aqueous solution, water and sodium-chloride water solution, dry then.Under reduced pressure concentrate the solution that generates, residue is used chromatographic purification on silicagel column, obtain 37.7 milligrams of 17-pi-allyls-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-12-2-[4-(p-nitrophenyl formyloxy)-3-methoxyl group cyclohexyl]-the 1-methylvinyl }-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum ν (CHCl 3): 1720,1640,1610,1530-1520 centimetre -1
Example 9
According to example 8 similar methods, by 30.6 milligrams of FR-900506 materials with contain 33 milligram 3,0.5 milliliter of pyridine reaction of 5-dinitrobenzoyl chloride obtains 36.0 milligrams of 17-pi-allyls-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-12-{ 2-[4-(3,5-dinitro benzene formyloxy)-3-methoxyl group cyclohexyl]-the 1-methylvinyl }-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum ν (CHCl 3): 1730,1640,1610,1530-12520 centimetre -1
Example 10
According to example 8 similar methods, by 48 milligrams of FR-900506 materials and the 0.5 milliliter of pyridine reaction that contains 0.08 milliliter of 2-l-lid oxygen base chloroacetic chloride, obtain 50.9 milligrams of 17-pi-allyls-1,14-dihydroxy-23,25-dimethoxy-12-2-[4-(2-l-lid oxygen base acetoxyl group)-3-methoxyl group cyclohexyl]-the rare base of 1-methyl second }-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle-[22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum ν (pure) is (neat): 3520,1760, and the sharp-pointed absworption peak of 1740(), the sharp-pointed absworption peak of 1720() and, 1625 centimetres -1
Example 11
At room temperature, 47 milligrams of N, N '-dicyclohexyl carbonization imines adds and contains in 10 milliliters of ethyl acetate of 51 milligrams (-)-2-trifluoromethyl-2-methoxyl group-2-phenylacetic acid.After at room temperature stirring 1.5 hours, add 25.0 milligrams of FR-900506 materials and 11 milligrams of 4-(N, the N-dimethylamino)-pyridine, then at room temperature, stirred 3.5 hours.Concentrate the solution that generates, obtain residue, this residue ether dissolution, and by the order washing with hydrochloric acid, sodium bicarbonate aqueous solution and sodium-chloride water solution.Organic layer also concentrates with dried over sodium sulfate, obtain residue, this residue is on silicagel column, with chromatography (expansion solvent: dichloromethane and ether, 10: 1V/V) separate, obtain 6.5 milligrams of 17-pi-allyls-12 2-[4-((-)-2-trifluoromethyl-2-methoxyl group-2-phenyl acetoxyl group]-3-methoxyl group cyclohexyl]-the 1-methylvinyl-1.14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10,16-tetraketone and 20.2 milligrams of 17-pi-allyl-14-{ (-)-2-trifluoromethyl-2-methoxyl group-2-phenyl acetoxyl group }-12-2-[4-((-)-2-trifluoromethyl-2-methoxyl group-2-phenyl acetoxyl group)-3-methoxyl group cyclohexyl]-the 1-methylvinyl }-1-hydroxyl-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
First chemical compound
Infrared spectrum V(is pure) (neat): 3510,1750, the sharp-pointed absworption peak of 1730(), 1710,1652,1500 centimetres -1
Second chemical compound
Infrared spectrum V(is pure) (neat): 1750,1720,1652,1500 centimetres -1
Example 12
145 milligrams of succinic anhydrides and 7 milligrams of 4-(N, N-dimethylamino) pyridine adds 7 milliliters of pyridine solutions of the stirring contain 248 milligrams of FR-900506 materials, and the mixture of generation at room temperature stirred 18 hours.This reactant mixture under reduced pressure concentrates, residue is on the silicagel column that 20 grams are filled with ethyl acetate, carry out chromatographic isolation, obtain 90 milligrams of 17-pi-allyl-12-2-[4-(3-carboxyl propionyloxy)-3-methoxyl group cyclohexyl]-the 1-methylvinyl-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum V(CHCl 3): 3500,3100-2300,1720, the sharp absworption peak of 1705(light), 1635 centimetres -1
Example 13
In 500 milligrams of 3 milliliters of pyridine solutions to 100.7 milligrams of FR-900506 materials of iodobenzene sulfonic acid chloride adding, this mixture at room temperature stirred 36 hours.This solution dilutes with ethyl acetate, and washs with saturated sodium bicarbonate aqueous solution, water and sodium-chloride water solution.The organic layer dried over sodium sulfate is filtered and is under reduced pressure concentrated.Residue is on silicagel column, with chromatography (expansion solvent: ether and hexane, 3: 1V/V) separate, obtain 61 milligrams of 17-pi-allyls-1 respectively, 14-dihydroxy-12-2-[4-(is to the iodobenzene sulfonyloxy)-3-methoxyl group cyclohexyl]-the 1-methylvinyl }-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10,16-tetraketone and 12 milligrams of 17-pi-allyl-1-hydroxyl-12-2-[4-(is to the iodobenzene sulfonyloxy)-3-methoxyl group cyclohexyl]-the 1-methylvinyl }-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-14s, 18-diene-2,3,10,16-tetraketone.
First chemical compound
Infrared spectrum V(CHCl 3): 3470,1730,1717,1692,1635,1568 centimetres -1
Second chemical compound
1H nuclear magnetic resoance spectrum δ ppm(CDCl 3):
6.15（d，J＝15Hz）
（1H），
6.25（d，J＝15Hz）
6.70（dd，J＝15Hz，10Hz）
（1H）
6.80（dd，J＝15Hz，10Hz）
7.60（2H，m），7.90（2H，m），
Example 14
According to example 13 similar methods, contain 97 milligrams of d(dextrorotation by 27 milligrams of FR-900506 materials and 0.6 milliliter) the pyridine solution reaction of sulphur acyl chloride of camphor, generate 34 milligrams of 17-pi-allyl-12-[2-(4-d(dextrorotation)-Camphora sulfonyloxy-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum V(is pure) (neat): 3500,1747, the sharp-pointed absworption peak of 1720(), the sharp-pointed absworption peak of 1710() and, 1655 centimetres -1
Example 15
118 milligrams of imidazoles and the 52.2 milligrams of tert-butyl group-diphenyl silyl chloride are added in the agitating solution of 3 milliliters of dichloromethane that contain 89.7 milligrams of FR-900506 materials.At room temperature, this mixture stirred after 2 hours, and reactant mixture is released with saturated aqueous ammonium chloride alkene, and with ether extraction three times.Dried over sodium sulfate is used in this extract water and sodium-chloride water solution washing, under reduced pressure concentrates then.Residue is on silicagel column, with column chromatography (expansion solvent: ethyl acetate and hexane, 1: 3V/V) purify, obtain 107 milligrams of 17-pi-allyls-12-[2-(4-tert-butyl group-diphenyl siloxy-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum V(is pure) (neat): 3520,1742,1705,1650 centimetres -1
Example 16
According to example 15 similar methods, by 80 milligrams of FR-900506 materials with contain 17 milligrams of tert-butyl groups-dimetylsilyl chlorine, 1 milliliter of N of (having 15 milligrams of imidazoles), the dinethylformamide reaction, generate 85 milligrams of 17-pi-allyls-12-[2-(4-tert-butyl group-dimethyl silane oxygen base-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon
-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum V(CHCl 3): 1735, the sharp-pointed absworption peak of 1720(), 1700,1640 centimetres -1
Example 17
1.5 milliliters of acetic anhydride addings are contained in 1.5 milliliters of dimethyl sulfoxide solutions of 100 milligrams of FR-900506 materials, and this mixture at room temperature stirred 14 hours.Reactant mixture dilutes with ethyl acetate, and washs with saturated sodium bicarbonate aqueous solution, water and sodium-chloride water solution.Organic layer under reduced pressure concentrates then with dried over sodium sulfate, filtration.Residue is on silicagel column, (launch solvent: ether) separate with thin layer chromatography, obtain 51 milligrams of 17-pi-allyls-1 respectively, 14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-12-[2-(4-methyl sulfo-methoxyl group-3-methoxyl group cyclohexyl)-and the 1-methylvinyl]-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-14s, 18-diene-2,3,10,16-tetraketone, 18 milligrams of 17-pi-allyl-1-hydroxyl-12-[2-(4-hydroxyl-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-14s, 18-diene-2,3,10,16 tetraketones and 10 milligrams of 17-pi-allyls-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-12-[2-(4-methyl sulfo-methoxyl group-3-methoxyl group cyclohexyl)-and the 1-methylvinyl]-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
First chemical compound
Infrared spectrum V(CHCl 3): 3470,1730,1635, the sharp-pointed absworption peak of 1630(), the sharp-pointed absworption peak of 1580() centimetre -1
Second chemical compound
Infrared spectrum V(CHCl 3): 1728,1640,1090 centimetres -1
The 3rd chemical compound
Infrared spectrum V(CHCl 3): 3480,1735,1710,1640 centimetres -1
Example 18
0.5 milliliter of acetic anhydride adding is contained 39.9 milligrams of 17-pi-allyls-12-[2-(4-tert-butyl group-dimethyl silane oxygen base-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, in 1.5 milliliters of pyridine solutions of 16-tetraketone, this mixture at room temperature stirred 6 hours.Under reduced pressure remove the solvent in the reactant mixture, obtain thick grease, this grease is on silicagel column, with thin layer chromatography (expansion solvent: ether and hexane, 1: 1V/V) purify, obtain 26.5 milligrams of 14-acetoxyl groups-17-pi-allyl-12-[2-(4-tert-butyl group-dimethyl silane oxygen base-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-1-hydroxyl-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum V(CHCl 3): 1728, the sharp-pointed absworption peak of 1715(), 1635 centimetres -1
Example 19
According to example 18 similar methods, by 10.6 milligrams of 17-pi-allyls-12-[2-(4-tert-butyl group-diphenyl siloxy-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-1,14-dihydroxy-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.O 4.9] 28 carbon-18-alkene-2,3,10,16-tetraketone and the 0.2 milliliter of pyridine reaction that contains 0.1 milligram of acetic anhydride generate 10 milligrams of 14-acetoxyl groups-17-pi-allyl-12-[2-(4-tert-butyl group-diphenyl siloxy-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-1-hydroxyl-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum V(CHCl 3): 3500,1730, the sharp-pointed absworption peak of 1720(), the sharp-pointed absworption peak of 1660(), 1640, the sharp-pointed absworption peak of 1620(), 1100 centimetres -1
Example 20
At room temperature, about 100 milligrams of potassium carbonate adding is contained 43.8 milligrams of 14-acetoxyl groups-17-pi-allyl-12-[2-(4-tert-butyl group-diphenyl siloxy-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-1-hydroxyl-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, in 1.5 milliliters of tetrahydrofuran solutions of 16-tetraketone, this mixture at room temperature stirred 3 hours.Reactant mixture dilutes with ether, and the solution of generation is used dried over sodium sulfate then with the order washing of pressing saturated aqueous ammonium chloride, water and sodium-chloride water solution.Dried solution under reduced pressure concentrates, residue is on silicagel column, with thin layer chromatography (expansion solvent: ether and hexane, 3: 2V/V) separate, obtain 30 milligrams of 17-pi-allyls-12-[2-(4-tert-butyl group-diphenyl siloxy-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-1-hydroxyl-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-14s, 18-diene-2,3,10,16-tetraketone.
Infrared spectrum V(CHCl 3): 1733, the sharp-pointed absworption peak of 1720(), 1685, the sharp-pointed absworption peak of 1640(), 1620 centimetres -1
Example 21
2 milliliters of ethyl acetate solutions that contain 50 milligrams of FR-900506 materials under atmospheric pressure and room temperature, carried out catalytic reduction reaction 20 minutes with 10 milligrams of 10%Pd-C catalyst.Filter reaction mixture, filtrate is evaporated to dried, and this residue is purified with thin layer chromatography.With chloroform and acetone (5: 1V/V) launch, obtain 50.0 milligrams of 1.14-dihydroxy-12-[2-(4-hydroxyl-3-methoxyl group cyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-17-propyl group-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
Infrared spectrum V(CHCl): 3480, the sharp-pointed absworption peak of 1735(), 1717,1700, the sharp-pointed absworption peak of 1650(), 1625 centimetres -1
Example 22
By the thick FR-900506 material of 1 gram white powder that the fermentation process similar to example 1 obtains, with 5 milliliters of acetonitriles dissolvings, use the ShimazuLC4A(trade mark, Shmazu Seisaku-sho manufacturing) high pressure lipuid chromatography (HPLC) CHPLC) separate.Use is by " YMC " S343(ODS) (trade mark, the Shimakyu company limited make) steel column (25 millimeters of internal diameters, 250 millimeters of length) of filling, flow velocity 12 ml/min.Mobile phase is the aqueous solution of 28% acetonitrile, 10% n-butyl alcohol, 0.075% phosphoric acid and 3.75 mM sodium lauryl sulphate (SDS) mixture, detects and finishes with the Hitachi UV-detector at 210 millimicrons of places.Each 100 microlitre samples that inject, high pressure liquid chromatography (HPLC) separation repeats 50 times, obtains in the post separating so that whole sample enters.Retention time is 85 to 90 minutes, collects each eluent, and with the ethyl acetate extraction of 3.6 liters of equal volume.The separating ethyl acetate layer, and, under vacuum, be concentrated to a little volume with the washing of 2 liter 1% sodium bicarbonate aqueous solution.By the sodium lauryl sulphate (SDS) that removes by filter condensing crystallizing, the corase meal that obtains dissolves with acetonitrile, and concentration is 100 mg/ml, and then carries out high pressure liquid chromatography and separate.Mobile phase is 12.5% acetonitrile, 9.75% n-butyl alcohol, 0.075% phosphoric acid and 3.75 mM sodium lauryl sulphate (SDS) mixture aqueous solutions.Pillar is an eluting under 10 ml/min at flow velocity, and retention time is 131 to 143 minutes, collects eluent, and uses isopyknic ethyl acetate extraction.Separate solvent layer, and, under vacuum, be concentrated to a little volume, by the sodium lauryl sulphate (SDS) that removes by filter condensing crystallizing with the washing of 1% sodium bicarbonate aqueous solution.
The corase meal that obtains like this dissolves with amount of ethyl acetate, (Kiesel silica gel, 230-400 order, maker: the Merck company limited), separate with column chromatography on 10 milliliters of silicagel columns.Silicagel column is with the mixture of 20 ml n-hexanes and ethyl acetate (1: 1V/V) and the mixture (1: 2V/V) wash of 60 ml n-hexanes and ethyl acetate.Eluting is finished with ethyl acetate, and fractional distillation (every part: 3 milliliters).Collect the 18-24 fraction, and under vacuum, be concentrated into driedly, obtain 24 milligrams of FR-900520 materials.
The cultural characteristic of table 1 bacterial strain No.9993 and Streptomyces misakiensis IFO12891
Cultural characteristic
Culture medium No.9993 IFO12891
Oatmeal agar G is medium
The A ash is greyish white
The R baby pink is colourless
The S nothing
It is medium that yeast-Mai Ya extracts agar G
The A light gray is greyish white
The dark red orange of R is shallow brown
The dark red orange of S does not have
Inorganic salt-starch agar G is medium
It is greyish white to light gray that A waters yellow orange
The dark orange of R is pale yellow brown
The S nothing
Culture medium No.9993 IFO12891
Glucose-asparagine G is few medium
Agar A white lime is white
R is shallow brown pale yellow brown
S does not have shallow brown
Glycerol-asparagine G is medium
Agar A is light pink greyish white to white
R is light pink pale yellow brown
S does not have shallow brown
Czapek agar G is a lot of less
A does not have greyish white
The light pink dark orange of R is to dun
The S nothing
Nutrient agar G is few
A is white, and is few white
R is colourless
The S nothing
Rhizoma Solani tuber osi-dextrose agar G is few medium
A does not have yellow ash
R is light pink brown
The S nothing
Cruel propylhomoserin agar G is medium
The A white lime is in vain to light gray
The dark orange of the dark red orange of R is to black
The S nothing
Peptone-yeast G is few
Extractum-ferrum A nothing
Agar R is colourless
The S nothing
Write a Chinese character in simplified form: G=growth A=gas is given birth to body colour
The color C=soluble pigment at the R=back side
The cultural character of table 4 bacterial strain No.7238, Streptomyces antimycoticus IFO 12839 Streptomyces hygroscopicus subsp.glebosus IFO 13786
Culture medium No.7238 IFO12839 IFO13786
Oatmeal agar G is few
A ecru brown ecru brown ecru brown
R is light yellow light yellow
The S nothing
Yeast-Mai Ya extract G is medium well medium
Agar A light gray-white Lycoperdon polymorphum Vitt Lycoperdon polymorphum Vitt
R fawn fawn darkorange
The S nothing
Inorganic salt-starch G is medium medium
Agar A ash is to the little Lycoperdon polymorphum Vitt of black Lycoperdon polymorphum Vitt
The pale yellow Lycoperdon polymorphum Vitt light orange of R light orange color color
The S nothing
Glucose-agedoite G is medium medium
Agar A light gray-white Lycoperdon polymorphum Vitt white
R light orange color light orange color light orange color
The S nothing
Glycerol-agedoite G is medium medium
Agar A white Lycoperdon polymorphum Vitt light gray
The pale yellow Lycoperdon polymorphum Vitt ecru of the pale yellow Lycoperdon polymorphum Vitt of R brown
The S nothing
Cha Baikeshi agar G is medium medium
A light gray-white light gray-white white
R fawn fawn fawn
The S nothing
Nutrition fat G is medium medium
A light gray-white light gray-white does not have
R is light yellow light yellow
The S nothing
Rhizoma Solani tuber osi-agar glucose G is medium medium
A white, few pale red brown baby pink is to white
R light orange color light orange color fawn
The S nothing
Tyrosine agar G is medium medium
A white light gray-white Lycoperdon polymorphum Vitt is to black
R fawn brown fawn
S does not have brown and does not have
Peptone-yeast extract-G is medium medium
Ferrum agar A does not have light gray-white not to be had
R is light yellow colourless
The S nothing
Abbreviation: G=growth A=gas is given birth to the color of body
The color S=soluble pigment of R=reverse side
The physiological property of table 5 bacterial strain No.7238, Streptomyces antimycoticus IFO12839 and Streptomyces hygroscopicus subsp.glebosus IFO13786
Physiological property No.7238 IFO12839 IFO13786
18 ℃-36 ℃ 18 ℃-38 ℃ 16 ℃-35 ℃ of the temperature ranges of growth
28 ℃ 28 ℃ 27 ℃ of optimum temperatures
The hydrochlorate reduction is negative negative
Starch hydrolysis is positive positive
The milk cohesion is negative negative
Blancmange turns into negative positive
It is negative positive to produce melanin
The solation effect is positive positive
Produce H 2S is negative negative
Urease activity is negative negative
NaCl drug resistance (%) 7%, 10% 7%, 10% 5%, 7%
Table 7:FR-900506 material is to the influence of MLR
FR-900506 radioactivity suppression ratio IC 50
Concentration (average count per minute (milligamma/
(millimicro grams per milliliter) ± standard error) (%) milliliter)
2.5 54±4 99.5
1.25 168±23 98.3
0.625 614±57 93.8
0.313 3880±222 60.9 0.26
0.156 5490±431 44.7
0.078 7189±365 27.6
0 9935±428
Table 8:FR-900520 material is to the influence of MLR
FR-900520 radioactivity suppression ratio IC 50
Concentration (average count per minute (milligamma/
(millimicro grams per milliliter) ± standard error) (%) milliliter)
100 175±16 99.2
10 515±55 97.8
1 2744±527 88.1 0.38
0.500 9434±1546 59.2
0.25 14987±1786 35.1
0 23106±1652 0
Table 9:FR-900523 material is to the influence of MLR
FR-900523 radioactivity suppression ratio IC 50
Concentration (average count per minute (milligamma/
(millimicro grams per milliliter) ± standard error) (%) milliliter)
100 25±12 99.9
10 156±37 99.3
1 5600±399 75.8 0.5
0.500 11624±395 49.7
0.250 17721±1083 23.3
0 23106±1052 0
Table 10:FR-900525 material is to the influence of MLR
FR-900525 radioactivity suppression ratio IC 50
Concentration (average count per minute (milligamma/
(millimicro grams per milliliter) ± standard error) (%) milliliter)
100 469±56 97.0
10 372±32 97.6
5 828±369 94.7 1.55
2.5 3564±512 77.4
1.2 10103±421 35.8
0 15741±411
Table 13:FR-900506 material is to the influence of skin allograft time-to-live
Dosage (milligram/kilogram) size of animal skin allograft survival natural law
Tester-11 7,7,7,7,7,7,8,8,8,8,8
(olive oil)
FR-900506 1 8 19，19，19，20，21，21，22，22
Material 3.2 6 22,23,23,26,27,35
10 5 56，61，82，85，89

Claims (2)

1, preparation contains the method for the pharmaceutical composition of logical formula I compound or its salt
Figure 911027882_IMG2
R wherein 1Be that hydroxyl or quilt are protected hydroxyl,
R 2Be that hydrogen, hydroxyl or quilt are protected hydroxyl,
R 3Be methyl, ethyl, propyl group or pi-allyl,
N be integer 1 or 2 and
Solid line and dotted line are represented singly-bound or two key,
Its condition is when n is integer 1, R 3Be propyl group or pi-allyl,
It is characterized in that:
Formula I compound or its salt and pharmaceutically acceptablely be suitable for using in external, the intestines and stomach or without conventional non-toxic carrier and/or mixed with excipients that the intestines and stomach is used is mixed with the active drug preparation that anti-organ or tissue transplant rejection disease, host are repelled graft disease or autoimmune disease.
2, by the process of claim 1 wherein that described formula I chemical compound is a 17-pi-allyl-1,14-dihydroxy-12-[2-(4-hydroxyl-3-methoxyl group cyclohexyl)-the 1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-two oxa-s-4-aza-tricycle [22.3.1.0 4.9] 28 carbon-18-alkene-2,3,10, the 16-tetraketone.
CN91102788A 1984-12-03 1985-12-03 Pharmaceutical composition containing tricyclic compound or salt thereof Expired - Lifetime CN1020853C (en)

Applications Claiming Priority (9)

Application Number Priority Date Filing Date Title
JP30455/84 1984-12-03
JP8430455/84 1984-12-03
GB848430455A GB8430455D0 (en) 1984-12-03 1984-12-03 Fr-900506 substance
JP02869/85 1985-02-05
JP8502869/85 1985-02-05
JP8508420/85 1985-04-01
GB858508420A GB8508420D0 (en) 1985-04-01 1985-04-01 Fr-900506 & fr-900525 substances
JP08420/85 1985-04-01
CN85109492A CN1013687B (en) 1984-12-03 1985-12-03 Tricycle compounds, a process for their production and a pharmaceutical composition containing the same

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Cited By (1)

* Cited by examiner, † Cited by third party
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CN100338043C (en) * 2002-02-13 2007-09-19 特瓦药厂私人有限公司 Method for extracting a macrolide from biomatter

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Publication number Priority date Publication date Assignee Title
CN112121173B (en) * 2020-08-22 2022-05-06 浙江大学 Fatty acid modified immunosuppressant MMF and FK506 nano preparation and preparation method and application thereof

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100338043C (en) * 2002-02-13 2007-09-19 特瓦药厂私人有限公司 Method for extracting a macrolide from biomatter

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CN1056103A (en) 1991-11-13

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