JP2008044877A - Antibiotic hydroxyphoslactomycin and its production method and antitumor agent and antifungal agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new phoslactomycin compound which induces the differentiation of cancer cells and/or inhibits PP2A. <P>SOLUTION: This antitumor agent contains hydroxyphoslactomycin B or its pharmaceutically acceptable salt as an active ingredient. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a novel antibiotic hydroxyphoslactomycin B, a process for producing the same, and an antitumor agent and an antifungal agent containing the antibiotic as an active ingredient.

  Phoslactomycin derivatives are conventionally known as inhibitors of protein phosphatase 2A (PP2A) (for example, Non-Patent Document 1) and are used as highly specific research reagents. Among the phoslactomycin derivatives, a method for efficiently producing phosactomycin B has been developed (for example, Non-Patent Document 2).

Journal of Biochemistry, Vol. 125, No. 5, pp. 960-965, 1999 The Journal of Biological Chemistry, Vol. 278, NO. 37, pp. 35552-35557, 2003

  However, the above-mentioned phoslactomycin derivative has a large side effect at the stage of animal experiments and has a problem in clinical application. Therefore, it has been desired to provide novel phoslactomycins that have few such disadvantages and induce differentiation of cancer cells or inhibit PP2A.

  As a substance for solving such a problem, the present inventors have found a novel antibiotic hydroxyphoslacomycin B, which is an analog of the antibiotic phoslactomycin. It was found that it has a remarkable differentiation-inducing activity, and further has an inhibitory activity against PP2A.

  The present invention has been completed based on the above findings. That is, the gist of the present invention is as follows.

(1) Hydroxyphosphomycin B represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

(2) A method for producing hydroxyphoslactomycin B, comprising culturing a hydroxyphoslactomycin B-producing bacterium belonging to the genus Streptomyces, and separating and collecting hydroxyphoslactomycin B from the culture.
(3) The production method according to (2), wherein the hydroxyphoslactomycin B-producing bacterium is Streptomyces HK-803 strain (Streptomyces sp. HK-803).
(4) Compound represented by the following formula (A):

(In the formula, R ^{1 to} R ³ each independently represent a hydroxyl-protecting group; R ⁴ represents hydrogen.)
In
Introducing a phosphate group having a protecting group into R ⁴ ;
Deprotecting the protecting groups R ^{1 to} R ³ ;
And a step of deprotecting the protecting group of the phosphate group.
(5) A compound represented by the following formula (A).

(In the formula, R ^{1 to} R ³ each independently represent a hydroxyl-protecting group; R ⁴ represents hydrogen.)
(6) An antitumor agent comprising hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof as an active ingredient.
(7) A differentiation inducer comprising hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof as an active ingredient.
(8) An antifungal agent comprising hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof as an active ingredient.
(9) Compound represented by the following formula (B):

(In the formula, R ^{5 to} R ⁷ each independently represents a hydroxyl-protecting group.)
In
A compound represented by the following formula (C), comprising a step of adding a vinyl group by an asymmetric nucleophilic addition reaction:

(In the formula, R ^{5 to} R ⁷ each independently represents a hydroxyl-protecting group.)
Manufacturing method.
(10) A compound represented by the following formula (B).

(In the formula, R ^{5 to} R ⁷ each independently represents a hydroxyl-protecting group.)

The antibiotic hydroxyphoslactomycin B of the present invention has antifungal activity, PP2A inhibitory activity and differentiation inducing activity, and therefore can be used as an antifungal agent, PP2A inhibitor and differentiation inducer. Furthermore, the hydroxyphoslactomycin B of the present invention can be used as an antitumor agent.
Moreover, according to the manufacturing method of the hydroxyphoslactomycin B of this invention, the hydroxyphoslactomycin B can be manufactured efficiently.

(1) Antibiotic hydroxyphoslactomycin B
Antibiotic hydroxyphoslactomycin B is obtained by culturing Streptomyces sp. HK-803, a soil actinomycete found in soil collected in Jizohara, Fukushima Prefecture, by the present inventor. Isolated from a new culture.

  Antibiotic hydroxyphoslacomycin B has the following structural formula and physicochemical properties.

Property: white solid molecular formula: C ₂₃ H ₃₉ O ₉ P
Specific rotation: [α] ²² _D = +75 (c 0.075, MeOH)
1H NMR data (500 MHz, CD ₃ OD): δ 0.94 (t, J = 7.5 Hz, 3 H), 1.02-1.98 (m), 2.08−2.22 (m, 1 H), 2.40−2.62 (m, 2 H), 3.60−3.83 (m, 2 H), 4.25 (t, J = 11 Hz, 1 H), 5.01 (dd, J = 6, 4 Hz, 1 H), 5.31 (t, J = 9 Hz, 1 H), 5.41 (t, J = 9 Hz, 1
H), 5.80−6.07 (m, 3 H), 6.24 (d, J = 10 Hz, 2 H), 7.08 (dd, J = 10, 5 Hz, 1 H) 13C NMR data (75 MHz, CD ₃ OD ): Δ 11.3 (-), 22.7 (+), 26.9 (+), 27.1 (+), 34.3 (+), 37.6 (-), 38.6 (+), 40.4 (+), 40.6 (-), 59.8 ( +), 64.5 (-), 78.3 (+), 78.8 (-), 82.4 (-), 121.0 (-), 123.0 (-), 124.4 (-), 126.4 (-), 134.5 (-), 138.1 ( -), 140.0 (-), 152.8 (-), 166.6 (+)

  Since the antibiotic hydroxyphoslactomycin B has PP2A inhibitory activity and differentiation-inducing activity as shown in the examples described later, hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof is a PP2A inhibitor. And a pharmaceutical composition containing a differentiation-inducing agent, a PP2A inhibitor and a differentiation-inducing agent, specifically, an antitumor agent and the like. Therefore, according to the present invention, there is provided a pharmaceutical composition comprising hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof or a combination thereof as an active ingredient.

  In addition, the antibiotic hydroxyphoslactomycin B has PP2A inhibitory activity and differentiation-inducing activity as shown in Examples described later. Accordingly, hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof is a PP2A inhibitor and differentiation inducer, and a pharmaceutical composition containing a PP2A inhibitor and differentiation inducer, specifically an antitumor agent, etc. Useful. Therefore, according to the present invention, a PP2A inhibitor or differentiation inducer consisting of hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof or a combination thereof, hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprising any or a combination thereof as an active ingredient is provided.

  Further provided by the present invention is a method comprising the step of administering a therapeutically effective amount of hydroxyphoslactomycin B to a mammal, including a human, and a method comprising the step of contacting the cell with an effective amount of hydroxyphoslactomycin B. .

  Examples of the pharmaceutically acceptable salt include mineral salts such as hydrochloride and sulfate; or organic acid salts such as p-toluenesulfonate; metal salts such as sodium salt, potassium salt and calcium salt; ammonium salt An organic ammonium salt such as methylammonium salt; and an amino acid salt such as glycine salt, but is not limited thereto.

  In the present specification, the term “antitumor agent” refers to those used for the purpose of tumor killing, tumor cell growth inhibition, tumor metastasis prevention, tumor recurrence prevention or tumor development prevention.

  In addition, since the antibiotic hydroxyphoslactomycin B has antifungal activity as shown in the examples described later, hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof is an antifungal agent, and It is useful as a pharmaceutical composition containing an antifungal agent. Therefore, according to the present invention, an antifungal agent comprising either hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof, or a combination thereof, any of hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof, or A pharmaceutical composition comprising a combination of the above as an active ingredient is provided.

  As the pharmaceutically acceptable salt, the same salts as described above can be used.

  Further provided by the present invention is a method comprising the step of administering a therapeutically effective amount of hydroxyphoslactomycin B to a mammal, including a human, and a method comprising the step of contacting the cell with an effective amount of hydroxyphoslactomycin B. .

  In the present specification, the term “antifungal agent” refers to those used for the purpose of sterilization, bacterial growth suppression and the like.

In the pharmaceutical composition containing hydroxyphoslactomycin B as an active ingredient, the administration method, dosage form, and dosage can be appropriately determined according to the purpose of use. For example, the dosage form of a medicament containing hydroxyphoslactomycin B as an active ingredient may be oral administration or parenteral administration. Examples of the dosage form include oral administration agents such as tablets, powders, capsules, granules, extracts, and syrups, and parenteral administration agents such as injections, drops, suppositories, and external preparations for skin. it can. These preparations can be produced by known methods using pharmaceutically acceptable additives such as excipients and binders. The dosage of the pharmaceutical composition containing hydroxyphoslactomycin B as an active ingredient varies depending on the age, weight, sensitivity, symptom level, etc. of the patient. As an adult, it is 0.1 mg to 100 mg / kg, preferably about 2 mg to 20 mg / kg per day, and 0.1 mg to 100 mg / kg for parenteral administration.
Preferably, it is about 2 mg to 20 mg / kg mg / kg. For antifungal use, the effective amount is usually 0.1 mg to 100 mg / kg, preferably about 2 mg to 20 mg / kg per day for an adult as an active ingredient, and 0.1 mg for parenteral administration. ˜100 mg / kg, preferably about 2 mg to 20 mg / kg. It is also possible to administer the above dosage once or several times a day. Further, if necessary, an amount outside the above range can be used.

  When the compound of the present invention is used as a research reagent, it can be used by dissolving in an organic solvent or a water-containing organic solvent. Examples of usable organic solvents include methanol, ethanol, water, acetone, dimethylformamide. And dimethyl sulfoxide. Examples of the dosage form include a solid agent such as powder, or a liquid agent dissolved in an organic solvent or a water-containing organic solvent. Usually, the effective use amount for exerting antitumor activity using hydroxyphoslactomycin B as a research reagent is 0.1 to 1 μg / ml in a cultured cell system. Depending on the type and purpose of use, it can be selected as appropriate. Further, if necessary, an amount outside the above range can be used.

(2) Method for Producing Antibiotic Hydroxyphosphomycin B (a) Biosynthesis of Antibiotic Hydroxyphosphomycin B As described above, the antibiotic hydroxyphoslactomycin B is the Streptomyces HK-803 strain (Streptomyces sp. HK -803) was isolated from the culture obtained. The Streptomyces HK-803 strain was deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on June 13, 2006, and its deposit number is FERM P-20933.

The mycological properties of Streptomyces HK-803 strain are as follows.
1. Morphology The morphology of the growth on inorganic salt / starch agar medium and yeast malt extract agar medium was observed with a microscope and an electron microscope. The HK-803 strain exhibited a form belonging to the genus Streptomyces. Its morphological features are as follows.
(1) Basic mycelium: Grows and branches well on agar.
(2) aerial hyphae: well grown on mediums other than glucose / asparagine agar and glycerin / asparagine agar, spore pattern is short, bent, and many dense spiral threads are formed, 20 to 50 Form a spore chain. According to observation with an electron microscope, the spore surface is smooth, elliptical or oval, and its size is 0.7-0.9 × 1.0-1.2 μm.

2. Culture properties on various media The culture properties of the cells were examined using the method of sealing and Gottliep (1996) (ISP method). In the description of the color tone, the symbols in parentheses are in accordance with the color name symbols in the 4th edition of the Descriptive Color Names Dictionary.
(1) Sucrose / nitrate agar medium Growth: Normal base mycelium color: Light wheat (2ea) to light yellow (2ic)
Aerial mycelia: Abundant aerial threads Color: Cream (2ba)
Soluble pigment: None
(2) Glucose-asparagine agar medium Growth: Bad basic mycelium color: Bright lemon color (3ea)
Aerial hyphae: Soluble pigment that does not grow: None
(3) Glycerin / asparagine agar medium Growth: Normal basic mycelium color: Bright yellow (1 1 / 2la)
Air hyphae: Slightly poor soluble pigment: Pale yellow

(4) Inorganic salt / starch agar medium Growth: Good basic mycelial color: Bright wheat (2ea) to bright yellow (2ic)
Aerial hyphae: abundant aerial hyacinth color: gray (5fe)
Soluble pigment: None
(5) Tyrosine agar medium Growth: Good basic mycelial color: Navy orange (4lc)
Aerial mycelia: Abundant aerial threads Color: White soluble pigment: None
(6) Nutrient agar growth: Normal mycelium color: Bright wheat color (2ea)
Aerial mycelia: Abundant aerial threads Color: White soluble pigment: None

(7) Yeast Extract / Malto Extract Agar Medium Growth: Normal base mycelium color: Bright yellow (3ia)
Aerial mycelia: Abundant aerial mycelia Tone: Gray (5ih) to gray brown (4ig)
Soluble pigment: yellow
(8) Oatmeal agar medium Growth: Very good basic mycelial color: Bright wheat color (2ea)
Aerial mycelia: Abundant aerial threads Color: White to gray (2fe)
Soluble pigment: None
(9) Peptone, yeast extract, iron agar medium Growth: Poor basic mycelium color: Bright wheat color (2ea)
Aerial mycelia: Abundant aerial threads Color: White soluble pigment: None

3. Physiological properties
(1) Growth temperature: 23 ° C-37 ° C
(2) Starch decomposition power: yes
(3) Coagulability of skim milk powder: negative
(4) Peptonization of skim milk powder: positive
(5) Formation of melanin-like pigment: None
(6) Nitrate reduction: positive
(7) Gelatin liquefaction power: negative

4). Utilization of carbon source Inoculate Streptomyces HK-803 strain on Prideham God Reeve medium with the following sugar or carbohydrate as the sole carbon source, incubate at 27 ° C for 20 days, and use carbon source with or without growth It was sex. ++ indicates extremely good growth, + indicates good growth and availability of the carbon source, and − indicates inability to grow and no availability of the carbon source.
L-arabinose ±
D-xylose ±
D-glucose ++
D-fructose ++
Sucrose ++
L-Inositol ++
L-Rhamnose ++
Raffinose ++
D-mannitol +
No additive −

Based on the above bacteriological properties, HK-803 strain was identified as belonging to the genus Streptomyces. Antibiotic hydroxyphoslactomycin B is produced by inoculating the above-mentioned strain into a nutrient-containing medium and culturing aerobically. The antibiotic hydroxyphoslactomycin B producing bacteria is not limited to the above strains, and any microorganism belonging to the genus Streptomyces and capable of producing the antibiotic hydroxyphoslactomycin B can be used for this production. The method for culturing microorganisms is basically the same as the method for culturing general microorganisms, but is usually preferably performed under aerobic conditions such as a shaking culture method by liquid culture and an aeration and agitation culture method. The medium used for the culture may be a medium containing a nutrient source that can be used by microorganisms belonging to the genus Streptomyces, and any of various synthetic media, semi-synthetic media, natural media, and the like can be used. As a medium composition, glucose as a carbon source, sucrose, fructose, glycerin, dextrin, starch, molasses, corn steve liquor, organic acid and the like can be used alone or in combination. As the nitrogen source, organic media such as pharmamedia, peptone, meat extract, yeast extract, soybean flour, casein, amino acid and urea, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate can be used alone or in combination.

  Sodium salts, potassium salts, magnesium salts, phosphates, other heavy metal salts, and the like can be added and used as necessary. When foaming is significant during culture, various known antifoaming agents such as Adecanol (registered trademark) and silicone oil can be added to the medium as appropriate, but the addition does not adversely affect the production of the target substance. Need to be. For example, it is preferably used at 0.5% or less. The pH of the medium is preferably in the optimum pH range of the microorganism to be used, usually near neutral. The temperature of the medium should be kept at a temperature at which microorganisms grow well, usually 20 to 40 ° C., particularly preferably around 27 ° C. In the case of liquid culture, the culture time is generally about 1 to 5 days, preferably about 72 hours. The target antibiotic hydroxyphoslactomycin B is produced and accumulated by the above culture. Of course, the various culture conditions described above can be appropriately changed according to the type and characteristics of the microorganism used, external conditions, and the like, and the optimum conditions are selected and adjusted from the above ranges according to each.

  Isolation of the antibiotic hydroxyphoslactomycin B produced by the culture is carried out according to the general method of collecting fermentation products when the antibiotic accumulation is maximal, It can be carried out by means of using a difference in solubility with impurities, a means of using a difference in adsorption affinity, or a means of using a difference in molecular weight. Each method can be used alone, in appropriate combination, or repeatedly. The Specifically, since it exists in the culture filtrate and the cells, the culture filtrate or the acetone extract of the cells is subjected to centrifugal liquid-liquid partition chromatography, various gel filtration chromatography, adsorption chromatography, preparative thin layer chromatography. Purified white powder of the antibiotic hydroxyphoslactomycin B can be obtained by purification in combination. An example of the method for producing the antibiotic hydroxyphoslactomycin B of the present invention will be described later as an example, but the method for producing the antibiotic hydroxyphoslactomycin B of the present invention is not limited to this example.

(B) Chemical Synthesis of Antibiotic Hydroxyphosphomycin B Hydroxyphosphomycin B can also be produced by chemical synthesis according to the method of the present invention. Hereinafter, each process in the manufacturing method of the hydroxyphoslactomycin B of this invention is demonstrated.

(First manufacturing method)
The compound represented by the following formula (A), which is a starting material in the first production method of hydroxyphoslactomycin B of the present invention [hereinafter sometimes simply referred to as compound (A)], is a novel compound, It is useful as an intermediate for the production of phoslactomycin B. Since the structure and synthesis method of the compound (A) have been clarified in this specification, the compound (A) can be understood by those skilled in the art using the specific synthesis method and the ordinary organic synthesis method shown as examples. It can be obtained by a modified method. The compound (A) can be appropriately converted into an appropriate salt form or a form provided with a protecting group for the stability during distribution, etc., and these forms are also included in the scope of the present invention.

In the formula, R ^{1 to} R ³ each independently represent a hydroxyl-protecting group; R ⁴ represents hydrogen.

The protecting groups represented by R ^{1 to} R ³ may be the same or different protecting groups. As R ^{1 to} R ³ , for example, TMS (trimethylsilyl group), TES (triethylsilyl group), TBS (t-butyldimethylsilyl group), TBDPS (t-butyldiphenylsilyl group) and the like can be used.

When introducing a protecting group into R ⁴ in relation to the stability during distribution and the previous stage of synthesis, from the viewpoint of high stereocontrollability and high efficiency of synthesis, the PMB group (4-methoxyphenyl) It is preferable to use a methyl group. That is, high selectivity in the construction of the C8 stereocenter is obtained, and a phosphate group is easily introduced. The introduction and deprotection of the protecting group can be performed based on a method used in a normal organic synthesis method.

Among the compounds (A), R ¹ and R ³ are TES groups, R ² is a TMS group, R ⁴ is hydrogen, and the physicochemical properties of the compound represented by the following structural formula are as follows.

Specific rotation: [α] ²⁸ _D +44 (c 0.063, CHCl ₃ )
UV absorption spectrum: IR (neat) 3474, 1733, 1251, 1082 cm ^-1
1 H NMR (300 MHz, CDCl ₃ ) δ 0.13 (s, 9 H), 0.57 (q, J = 8 Hz, 6 H), 0.61 (q, J = 8 Hz, 6 H), 0.80−1.80 (m , 36 H), 1.86−2.06 (m, 2 H), 2.33−2.50 (m, 2 H), 3.60−3.85 (m, 3 H), 4.88 (t, J = 9 Hz, 1 H), 4.99 ( dd, J = 6, 5 Hz, 1 H), 5.38 (t, J = 10 Hz, 1 H), 5.45 (t, J = 10 Hz, 1 H), 5.76 (dd, J = 16, 6 Hz, 1 H), 5.99 (d, J = 16 Hz, 1 H), 6.04 (d, J = 10 Hz, 1 H), 6.08 (t, J = 12 Hz, 1 H), 6.18 (t, J = 12 Hz, 1 H), 6.91 (dd, J = 10, 5 Hz, 1 H)
¹³ C NMR (75 MHz, CDCl ₃ ) δ 2.6 (-), 4.3 (+), 4.9 (+), 6.8 (-), 6.9 (-), 11.1 (-), 21.6 (+), 25.9 (+) , 26.0 (+), 33.16 (+), 33.20 (+), 36.4 (-), 39.6 (+), 39.7 (-), 40.9 (+), 58.8 (+), 66.3 (-), 72.7 (-) , 79.7 (+), 80.8 (-), 120.9 (-), 121.3
(-), 122.5 (-), 124.4 (-), 134.7 (-), 137.3 (-), 139.5 (-), 149.8 (-), 164.1 (+)

Process (1)
A phosphate group having a protecting group is introduced into R ⁴ of the compound (A). As the protecting group for the phosphate group, an allyl group, a 2-cyanoethyl group, or the like can be used. Introduction of a phosphate group having a protecting group can be carried out under the conditions used in ordinary organic synthesis techniques. For example, when introducing a phosphate group having an allyl group as a protecting group, diallyldiisopropyl phosphorimidate and 1H-tetrazole are added and reacted at 0 to 30 ° C. for 0.5 to 2 hours to have a protecting group. Phosphate groups can be introduced. The obtained compound is isolated and purified by a conventional method for isolating and purifying organic compounds as necessary.

Step (2)
The protecting groups R ^{1 to} R ³ of the resulting compound are deprotected simultaneously or separately. Deprotection can be carried out under conditions suitable for deprotection of protecting groups R ^{1 to} R ³ used in ordinary organic synthesis techniques. For example, if it is TES, TMS, etc., it can deprotect by making it react at 0-30 degreeC on acidic conditions for 10 minutes-12 hours. The obtained compound is isolated and purified by a conventional method for isolating and purifying organic compounds as necessary.

Step (3)
The protecting group of the phosphate group of the obtained compound is deprotected. Deprotection can be performed under conditions suitable for deprotection of protecting groups used in ordinary organic synthesis techniques. For example, an allyl group can be deprotected by adding PdCl ₂ (PPh ₃ ) ₂ and Bu ₃ SnH and reacting at −10 to 60 ° C. for 30 minutes to 12 hours. The obtained compound is isolated and purified by a conventional method for isolating and purifying organic compounds as necessary.

(Second manufacturing method)
The compound represented by the following formula (B) as a starting material in the second production method of the present invention [hereinafter sometimes simply referred to as compound (B)] is a novel compound, and is a production of hydroxyphoslactomycin B Useful as an intermediate. Since the structure and synthesis method of the compound (B) have been clarified in the present specification, the compound (B) can be understood by those skilled in the art using the specific synthesis method and ordinary organic synthesis method shown as examples. It can be obtained by a modified method. A novel compound represented by the following formula (C) [hereinafter sometimes simply referred to as compound (C)] is produced from compound (B). Compound (C) is useful as an intermediate for the production of hydroxyphoslactomycin B. Compounds (B) and (C) can be appropriately converted into a suitable salt form or a form provided with a protecting group for the stability during distribution, etc., and these forms are also within the scope of the present invention. include.

In the formula, R ^{5 to} R ⁷ each independently represents a hydroxyl-protecting group.

In the formula, R ^{5 to} R ⁷ each independently represents a hydroxyl-protecting group.

The protecting groups represented by R ^{5 to} R ⁷ may be the same or different protecting groups. As R ^{5 to} R ⁷ , for example, TMS, TES, TBS, TBDPS, or the like can be used.

When introducing a protecting group into R ⁷ in relation to the stability during distribution and the pre-synthesis stage, PMB (4-methoxyphenylmethyl) is used from the viewpoint of high stereocontrollability and high efficiency of synthesis. Group). That is, high selectivity in the construction of the C8 stereocenter of hydroxyphoslactomycin B is obtained, and a phosphate group is easily introduced. The introduction and deprotection of the protecting group can be performed based on a method used in a normal organic synthesis method.

Among the compounds (B), the physicochemical properties of the compound represented by the following structural formula in which R ⁵ is a TBDPS group and R ⁶ is a TBS group are as follows.

Specific rotation: [α] ²⁸ _D = +15 (c 0.08, CHCl ₃ )
UV absorption spectrum: IR (neat) 3441, 1718, 1251, 1113 cm ^-1
1 H NMR (500 MHz, CDCl ₃ ) δ 0.11 (s, 3 H), 0.17 (s, 3 H), 0.92 (s, 9 H), 1.02 (s,
9 H), 1.88−2.13 (m, 2 H), 2.37 (d, J = 3 Hz, 1 H), 2.622.80 (m, 2 H), 3.80 (s,
3 H), 3.95 (t, J = 6 Hz, 2 H), 4.10 (dd, J = 10, 3 Hz, 1 H), 4.31 (d, J = 11 Hz, 1 H), 4.50 (d, J = 11 Hz, 1 H), 4.60 (dt, J = 10, 3 Hz, 1 H), 6.86 (d, J = 8.5
Hz, 2 H), 7.25 (d, J = 8.5 Hz, 2 H), 7.347.46 (m, 6 H), 7.66 (d, J = 7 Hz, 4 H) ¹³ C NMR (75 MHz, CDCl ₃ ) δ -5.0, -4.3, 18.2, 19.2, 25.9, 26.9, 40.6, 40.8, 55.3,
58.8, 59.3, 72.2, 72.9, 81.3, 85.0, 113.9, 127.8, 129.7, 129.8, 133.4, 133.5, 134.9, 135.63, 135.64, 159.5, 210.5.

Among the compounds (C), the physicochemical properties of the compound represented by the following structural formula in which R ⁵ is a TBDPS group and R ⁶ is a TBS group are as follows.

Specific rotation: [α] ²⁸ _D +16 (c 0.09, CHCl ₃ )
UV absorption spectrum: IR (neat) 3474, 3294, 1249, 1113 cm ^-1
1 H NMR (300 MHz, CDCl ₃ ) δ 0.14 (s, 3 H), 0.18 (s, 3 H), 0.92 (s, 9 H), 1.04 (s,
9 H), 1.71-1.85 (m, 2 H), 1.98−2.14 (m, 2 H), 2.41 (d, J = 2 Hz, 1 H), 3.59 (dd, J = 8, 4 Hz, 1 H ), 3.78 (s, 3 H), 3.703.92 (m, 2 H), 4.27 (s, 1 H), 4.564.70
(m, 3 H), 5.29 (dd, J = 11, 2 Hz, 1 H), 5.54 (dd, J = 17, 2 Hz, 1 H), 5.90 (dd,
J = 17, 11 Hz, 1 H), 6.83 (d, J = 9 Hz, 2 H), 7.24 (d, J = 9 Hz, 2 H), 7.337.49
(m, 6 H), 7.627.72 (m, 4 H)
¹³ C NMR (75 MHz, CDCl ₃ ) δ -4.7, -4.0, 18.3, 19.0, 26.0, 26.8, 37.2, 40.2, 55.3,
60.6, 62.0, 72.7, 73.4, 79.2, 81.3, 86.1, 113.8, 115.6, 127.8, 127.9, 129.2, 129.9, 131.2, 132.7, 132.8, 135.6, 140.3, 159.1

Process (1)
A vinyl group is added to the compound (B) by an asymmetric nucleophilic addition reaction. The asymmetric nucleophilic addition reaction can be carried out under the conditions used in ordinary organic synthesis techniques. For example, a vinyl group can be added by an asymmetric nucleophilic addition reaction by adding a nucleophilic reagent such as CH ₂ = CHMgBr and reacting at -78 to 0 ° C for 1 to 4 hours. The obtained compound is isolated and purified by a conventional method for isolating and purifying organic compounds as necessary.

  In addition, the manufacturing method of the hydroxyphoslactomycin B of this invention is demonstrated more concretely in a following example.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.

[Example 1] Biosynthesis of antibiotic hydroxyphoslactomycin B 2% glucose, 1% soluble starch, 0.1% meat extract, 0.4% dry yeast, 2.5% soy flour, 0.2% sodium chloride, diphosphate Streptomyces HK-803 strain was inoculated into 30 liters of a medium composed of 0.005% potassium, and aerated and stirred for 72 hours at 27 ° C. The whole culture filtrate was extracted with an equal volume of n-butanol. Extraction was again performed with an equal amount of n-butanol, and the n-butanol layer was concentrated under reduced pressure to obtain a crude active substance. The crude active substance was subjected to silica gel column chromatography in a solvent system consisting of chloroform, methanol and water, and the fraction containing hydroxyphoslactomycin B was concentrated and dried. After dissolving in a small amount of methanol, high performance liquid chromatography using a reverse phase column in a solvent system consisting of methanol and water was performed, and finally the fraction containing antibiotic hydroxyphoslactomycin B was concentrated to dryness under reduced pressure, As a powder, 2 mg of the antibiotic hydroxyphoslactomycin B was obtained. FIG. 1 is a chromatograph obtained by separating hydroxyphoslactomycin B by high performance liquid chromatography.

[Example 2] Production of antibiotic hydroxyphoslactomycin B by chemical synthesis
A solution of i-Pr ₂ NH (24.4 mL, 174.1 mmol) in THF (70 mL) was cooled to 0 ° C, and n-BuLi in hexane (66.5 mL, 2.24 M, 149 mmol) was added thereto. . After stirring for 10 minutes, the mixture was cooled to −78 ° C., and EtOAc (14.6 mL, 149 mmol) was added. After 40 minutes, a solution of “2” (10.0 mL, 124.1 mmol) in THF (10 mL) was added and stirred for 1 hour. Then, the mixture was poured into a mixed solution of saturated aqueous ammonium chloride and ethyl acetate, the organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with ethyl acetate. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product through a silica gel column gave 18.9 g of “rac-3” (yield 96%).

4A molecular sieves (9.48 g), Ti (Oi-Pr) ₄ (23.2 mL, 78.9 mmol) and CH ₂ Cl ₂
The mixture consisting of (140 mL) was cooled to −20 ° C., and L-(+)-DIPT (19.8 mL, 94.7 mmol) was added thereto. After 30 minutes, a solution of “rac-3” in CH ₂ Cl ₂ (20 mL) was added. The mixture was stirred again for 30 minutes, and a solution of t-BuOOH in CH ₂ Cl ₂ (43.6 mL, 7.26 M, 317 mmol) was added. The reaction was performed at -20 ° C for 30 hours, and added with Me ₂ S (23.2 mL, 315.9 mmol), 10% aqueous tartaric acid solution (40 mL), NaF (16.6 g, 395 mmol), and Celite (15 g). Stopped. The resulting mixture was filtered through Celite, the filtrate was concentrated under reduced pressure, and the crude product was purified on a silica gel column to obtain “(R) -3” of 22.0.
g obtained (44% yield).

“(R) -3” (13.3 g, 84.1 mmol), 4-methoxybenzyl 2,2,2-trichloroacetimidate (47.5 g, 168.1 mmol) and (+)-10-camphorsulfonic acid (586 mg, 2.52 mmol) in CH ₂ Cl ₂ (90 mL) was stirred at room temperature for 10 hours. Diluted with hexane, the resulting mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the produced “4”. This “4” was used in the next reaction as it was.

“4” was dissolved in THF (100 mL), cooled to 0 ° C., and LiAlH ₄ (2.23 g, 58.8 mmol) was added in several portions. The ice bath was removed and stirred for 1 hour. Add excess hydride to water (10.6 mL,
590 mmol) was added, and the resulting mixture was poured into 3N hydrochloric acid (98 mL, 294 mmol) and ethyl acetate. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with ethyl acetate. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product on a silica gel column gave 17.3 g of “5” (87% yield from “(R) -3”).

A solution of (COCl) ₂ (6.49 mL, 74.1 mmol) in CH ₂ Cl ₂ (180 mL) was cooled to −78 ° C., and DMSO (13.2 mL, 186 mmol) was added thereto. After 15 minutes, a solution of “5” (14.6 g, 61.8 mmol) in CH ₂ Cl ₂ (20 mL) was added. The reaction was carried out between -78 ° C and -40 ° C for 30 minutes, and then Et ₃ N (34.4 mL, 246.8 mmol) was added. The mixture was stirred at -40 ° C for 1 hour and poured into a mixture of ice-cooled saturated ammonium chloride and CH ₂ Cl ₂ . Separate the organic and aqueous layers, and separate the aqueous layer twice with CH ₂ Cl ₂
Extracted. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure gave “6” as a crude product, which was used in the next reaction as it was.

A mixture of LiCl (3.67 g, 86.6 mmol), DBU (12.5 mL, 83.6 mmol), (EtO) ₂ P (O) CH ₂ CO ₂ Et (16.1 mL, 80.4 mmol) and MeCN (85 mL) was added at 0 ° C. Then, “6” obtained in the above reaction was added. The ice bath was removed, and the mixture was stirred at room temperature for 1 hour, and saturated aqueous sodium hydrogen carbonate and ethyl acetate were added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with ethyl acetate. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product on a silica gel column gave 15.7 g of “7” (83% yield from “5”).

  A solution of “7” (15.0 g, 49.3 mmol) in THF (40 mL) was cooled to −78 ° C., and DIBAL-H in hexane (115 mL, 0.94 M, 108 mmol) was slowly added thereto. The mixture was stirred for 1 hour between -78 ° C and -60 ° C, and 3N hydrochloric acid (180 mL, 540 mmol) was added to stop the reaction. The resulting mixture was extracted twice with ethyl acetate and the combined extract was dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product with a silica gel column gave 12.4 g of the corresponding “8” (yield 96%).

A mixture of 4A molecular sieves (2.88 g), Ti (Oi-Pr) ₄ (4.24 mL, 14.4 mmol), and CH ₂ Cl ₂ (35 mL) was cooled to -20 ° C, and D-(- ) -DIPT (3.62 mL, 17.3 mmol)
Was added. After 20 minutes, a solution of “8” (12.6 g, 48.0 mmol) synthesized above in CH ₂ Cl ₂ (5 mL) was added and stirred again for 30 minutes. Next, a CH ₂ Cl ₂ solution of t-BuOOH (10.9 mL, 6.64 M, 72.4 mmol) was added. The reaction was performed at -20 ° C for 10 hours, Me ₂ S (5.29 mL, 72.0 mmol), 10%
Tartaric acid aqueous solution (7 mL) and NaF (6.05 g, 144 mmol) were added. The mixture was stirred at room temperature for 1 hour and filtered through celite. Next, 3N sodium hydroxide (28.8 mL, 86.4
mmol) was added and stirred at room temperature for 20 minutes. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted with ethyl acetate. The extracts were collected together, dried over anhydrous magnesium sulfate, dried, and concentrated under reduced pressure. The crude product was purified on a silica gel column to give 12.4 g of “9” (yield 93%).

A mixture consisting of “9” (15.6 g, 56.1 mmol), PPh ₃ (17.6 g, 67.1 mmol), baking soda (1.06 g, 12.6 mmol) and CCl ₄ (120 mL) was heated to reflux for 3 hours. Volatile material was removed under reduced pressure and hexane was added. The resulting mixture was filtered through celite, and the filtrate was concentrated under reduced pressure. The crude product was purified on a silica gel column to obtain 14.7 g of “10” (yield 88%).

  The obtained “10” (8.0 g, 27.0 mmol) in THF (40 mL) is cooled to −78 ° C., and n-BuLi in hexane (31.2 mL, 2.72 M, 84.9 mmol) is slowly added dropwise thereto. did. The mixture was stirred for 20 minutes and poured into a mixed solution of saturated aqueous ammonium chloride and ethyl acetate. After vigorous stirring for several minutes, the organic layer was separated and the aqueous layer was extracted twice with ethyl acetate. The extracts were collected together and dried over anhydrous magnesium sulfate. “11” obtained by concentration under reduced pressure was directly used in the next reaction.

  The obtained mixed solution consisting of “11”, TBSCl (4.88 g, 32.4 mmol), imidazole (3.67 g, 53.9 mmol) and DMF (50 mL) was stirred at room temperature for 1 hour. Saturated aqueous sodium bicarbonate and hexane were added and stirred vigorously for several minutes. The organic layer was separated, and the aqueous layer was extracted twice with hexane. The extracts were collected together and dried over anhydrous magnesium sulfate. After concentration under reduced pressure, the resulting crude product was purified by silica gel column to give 9.22 g of “12” (91% yield from “10”).

A solution of “12” (12.1 g, 32.3 mmol) and 2,6-lutidine (5.64 mL, 48.4 mmol) in MeOH (70 mL) was cooled to −78 ° C., and ozone-containing oxygen gas was added to it. Infused for 2 hours. After removing excess ozone by blowing in argon, Me ₂ S (7.12 mL, 97.0 mmol) was added, the refrigerant was removed, and the mixture was stirred for 1 hour. Thereafter, the solution was concentrated under reduced pressure to obtain “13”. This compound was used for the next reaction through a short silica gel column.

n-BuLi in hexane solution in THF (50 mL) solution containing i-Pr ₂ NH (11.3 mL, 80.6 mmol)
(30.0 mL, 2.26 M, 67.8 mmol) was added. After stirring at 0 ° C. for 10 minutes, the mixture was cooled to −78 ° C., and EtOAc (6.64 mL, 67.8 mmol) was added. After 1 hour, a solution of “13” in THF (10 mL) was added and stirred for another 30 minutes. After completion of the reaction, the mixture was poured into an ice-cooled mixed solution of saturated aqueous ammonium chloride and ethyl acetate. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with ethyl acetate. The extracts were collected together and dried over anhydrous magnesium sulfate. After concentration under reduced pressure, the obtained “14” was used in the next reaction as it was.

The obtained “14” was dissolved in THF (60 mL), cooled to 0 ° C., and LiAlH ₄ (1.84 g, 48.5 mmol) was slowly added. The mixture was stirred for 30 minutes, and the excess hydride was treated with water (4.36 mL, 242 mmol). In addition, NaF (10.2 g, 243 mmol) was also added. After stirring for 30 minutes, the mixture was filtered through Celite. The filtrate was concentrated under reduced pressure and purified by silica gel column to give 8.42 g of “15” (64% yield from “12”).

  A mixed solution consisting of “15” (5.35 g, 13.2 mmol), TBDPSCl (4.06 mL, 15.8 mmol), imidazole (1.79 g, 26.3 mmol) and DMF (35 mL) was stirred at room temperature for 1 hour. Saturated aqueous sodium hydrogen carbonate and ether were added for dilution, and the mixture was stirred vigorously for several minutes. The organic layer was separated, and the aqueous layer was extracted twice with ether. The extracts were collected together and dried over anhydrous magnesium sulfate. After concentration under reduced pressure, the resulting crude product was passed through a short silica gel column to obtain “16”. This compound was used in the next reaction as it was.

DMSO (2.80 mL, 39.4 mmol) was added to a solution of (COCl) ₂ (1.50 mL, 17.1 mmol) in CH ₂ Cl ₂ (55 mL) cooled to −78 ° C. After 5 minutes, a solution of “16” in CH ₂ Cl ₂ (5 mL) was added. Stir for 30 minutes between -78 ° C and -50 ° C, then add Et ₃ N (9.17 mL, 65.8 mmol). Stir for another 30 minutes and add saturated aqueous sodium bicarbonate. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with CH ₂ Cl ₂ . The extracts were collected together and dried over anhydrous magnesium sulfate. After concentration under reduced pressure, the crude product was passed through a short silica gel column to give “17”. This product was used in the next reaction as it was.

“17” was dissolved in THF (50 mL), cooled to −78 ° C., and a THF solution of CH ₂ = CHMgBr (29.2 mL, 0.90 M, 26.3 mmol) was slowly added dropwise over 10 minutes. After 1 hour, this solution was poured into an ice-cooled mixed solution of saturated aqueous ammonium chloride and ethyl acetate. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with ethyl acetate. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentrate under reduced pressure and purify the crude product with a silica gel column.
(Yield 87% from “15”).

A CH ₂ Cl ₂ (20 mL) solution containing “18” (5.58 g, 10.2 mmol) and 2,6-lutidine (2.37 mL, 20.4 mmol) was ice-cooled, and TESOTf (2.53 mL, 11.2 mmol) was added thereto. ) Was slowly added dropwise. The mixture was stirred at the same temperature for 30 minutes, and saturated aqueous sodium hydrogen carbonate was added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with CH ₂ Cl ₂ . The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product on a silica gel column gave 6.13 g of “19”
(Yield 91%).

A mixed solution of MeOH and i-PrOH (1: 1, 100 mL) containing “19” (13.5 g, 17.2 mmol) and 2,6-lutidine (4.0 mL, 34.3 mmol) was cooled to -78 ° C. Oxygen gas containing ozone was blown into this for 5 hours. After removing excess ozone by blowing argon, Me ₂ S (3.8
mL, 51.74 mmol) was added, the refrigerant was removed, and the mixture was stirred for 1 hour. Thereafter, the solution was concentrated under reduced pressure to obtain “20”.

60% NaH (1.37 g, 34.3 mmol) in mineral oil was washed three times with hexane to remove the mineral oil. THF (80 mL) was added thereto, and after cooling to 0 ° C., (EtO) ₂ P (O) CH ₂ CO ₂ Et (7.21 mL, 36.0 mmol) was added. After 20 minutes, “20” THF (20 mL) was slowly added dropwise. After removing the ice bath and stirring overnight, it was poured into a saturated aqueous ammonium chloride solution. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with hexane. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product on a silica gel column gave 7.14 g of “21” (48% yield from “19”).

“21” (7.14 g, 8.31 mmol) and “22” (2.35 g, 9.95 mmol) were dissolved in benzene (40 mL), and t-BuNH ₂ (8.68 mL, 83.1 mmol), Pd (PPh ₃ ) ₄ (0.481 g, 0.416 mmol)
And CuI (0.238 g, 1.25 mmol) were added. The mixture was stirred overnight at room temperature and protected from light, and the reaction was stopped by adding saturated aqueous ammonium chloride and hexane. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with hexane. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product on a silica gel column gave 7.0 g of “23” (yield 87%
).

A solution of “23” (4.32 g, 4.46 mmol) in THF (30 mL) was cooled to −70 ° C., and DIBAL-H in hexane (11.9 mL, 0.94 M, 11.2 mmol) was slowly added dropwise thereto. -78 ℃ ~
The mixture was stirred at -60 ° C for 30 minutes and water (1.0 mL, 56 mmol) was added to stop the reaction. After raising the temperature to 0 ° C, NaF (2.34 g, 56 mmol) was added. The mixture was stirred vigorously for 30 minutes and filtered through celite. Concentration of the filtrate under reduced pressure and purification on a silica gel column gave 4.01 g of the corresponding “24” (yield 97%).

Et ₃ N (9.49 mL, 68.1 mmol) and DMSO (14.4 mL, 204.1 mmol) were added to a solution of “24” (6.30 g, 6.81 mmol) in CH ₂ Cl ₂ (40 mL), and cooled to 0 ° C. , SO ₃ • Py (3.25 g, 20.4 mmol) was added little by little. After removing the ice bath and stirring for 30 minutes, saturated aqueous sodium bicarbonate and hexane were added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with hexane. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product on a silica gel column gave 6.10 g of “25” (yield 97%).

Bu ₂ BOTf CH ₂ Cl in ice-cooled (R) -N-butyryl-4-benzyl-2-oxazolidinone (“26”) (2.53 g, 10.2 mmol) in CH ₂ Cl ₂ (40 mL) ₂ solutions (9.91 mL, 1.0 M, 9.91 mmol) and (i-Pr) ₂ NEt (2.53 mL, 14.5 mmol) were added, stirred for 30 minutes, cooled to -78 ° C, and “25” ( 6.10 g, 6.61 mmol) in CH ₂ Cl ₂ (10 mL) was added dropwise over 10 minutes. The mixture was stirred at -78 ° C for 10 minutes and at room temperature for 3.5 hours, and saturated aqueous ammonium chloride solution was added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with CH ₂ Cl ₂ . The extracts were collected together and dried over anhydrous magnesium sulfate. After concentration under reduced pressure, the crude product was purified by silica gel column to give “27”.

A CH ₂ Cl ₂ (5 mL) solution containing “27” and pyridine (7.96 mL, 99 mmol) was ice-cooled, and TESCl (2.21 mL, 13.2 mmol) was added dropwise thereto. The mixture was stirred at the same temperature for 30 minutes, and the reaction was stopped by adding saturated aqueous sodium hydrogen carbonate and hexane. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with hexane. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product through a silica gel column gave 8.10 g of the corresponding “28” (95% yield from “25”).

N-BuLi in hexane in ice-cooled EtSH (2.36 mL, 31.5 mmol) in THF (20 mL)
(6.95 mL, 2.72 M, 18.9 mmol) was added to prepare an EtSLi / THF solution. On the other hand, a solution of “28” (8.10 g, 6.30 mmol) in THF (20 mL) was prepared, and this was ice-cooled, and then EtSLi / THF solution was added. Stir for 1.5 hours and add saturated aqueous ammonium chloride. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with hexane. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product through a silica gel column gave 6.80 g of “29” (yield 90%).

  A solution of “29” (2.77 g, 2.37 mmol) in toluene (20 mL) was cooled to −78 ° C., and DIBAL-H in hexane (3.27 mL, 0.94 M, 3.07 mmol) was slowly added thereto. Stir at -78 ° C for 30 min, add a few drops of ethyl acetate, and raise the temperature to 0 ° C. Water (0.24 mL, 13 mmol) and NaF (550 mg, 13 mmol) were added, and the mixture was vigorously stirred for 30 minutes, and then filtered through Celite. The filtrate was concentrated under reduced pressure, and “30” was directly used in the next reaction.

A solution of Bu ₄ NOH in MeOH (4.15 mL, 1.0 M, 4.15 mmol) was added to a solution of (PhO) ₂ P (O) CH ₂ CO ₂ Et (1.37 g, 4.27 mmol) in THF (20 mL). After 20 minutes, the mixture was cooled to -78 ° C and a THF (5 mL) solution of the aldehyde synthesized above was added dropwise. After that, it was transferred to a -18 ° C refrigerant, stirred at that temperature for 6 hours, and saturated ammonium chloride aqueous solution and hexane were added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with hexane. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product on a silica gel column gave 2.40 g of “31” (86% yield from “29”).

  “31” (4.86 g, 4.12 mmol) was dissolved in a mixed solvent of THF (10 mL) / MeOH (30 mL), and PPTS (31 mg, 0.12 mmol) was added thereto. The mixture was stirred overnight at room temperature, and saturated aqueous sodium hydrogen carbonate and ethyl acetate were added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with ethyl acetate. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product on a silica gel column gave 3.78 g of “32” (yield 86%).

Ti (Oi-Pr) ₄ (0.21 mL, 0.71 mmol) was added to a solution of “32” (3.78 g, 3.54 mmol) in benzene (30 mL) and heated at 80 ° C. for 20 minutes. After returning to room temperature, several drops of water were added to treat Ti (Oi-Pr) ₄ and then diluted with ethyl acetate. The resulting mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure and purified on a silica gel column to give 3.40 g of “33” (yield 94%).

A THF solution of “33” (3.10 g, 3.04 mmol) was placed in an ice bath, and a THF solution of Bu ₄ NF (12.2 mL, 1.0 M, 12.2 mmol) was added dropwise. The mixture was stirred at 0 ° C for 20 minutes, then returned to room temperature over 30 minutes, and saturated aqueous ammonium chloride and ethyl acetate were added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with ethyl acetate. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product through a silica gel column gave 1.48 g of “34” (88% yield).

Dibromoethane (1.35 mL, 15.7 mmol) was added dropwise into a mixture of zinc powder (24.1 g, 369 mmol) and EtOH (50 mL) heated to 80 ° C. Gas evolved immediately, but dibromoethane (1.40 mL, 16.2 mmol) was added again after the evolution stopped. The mixture was stirred at 80 ° C for 15 minutes, and then cooled to 50 ° C. A THF (35 mL) solution containing LiBr (8.27 g, 95.2 mmol) and CuBr (5.52 g, 38.5 mmol) was added thereto. A solution of “34” (1.33 g, 2.41 mmol) in EtOH (5 mL) was added to the zinc powder thus activated. The mixture was again heated to 80 ° C, reacted for 2 hours, returned to room temperature, and diluted with ethyl acetate. The mixture was filtered through Celite, the filtrate was concentrated under reduced pressure, and saturated aqueous ammonium chloride and ethyl acetate were added to the residue. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with ethyl acetate. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product on a silica gel column gave “35” as 1.10
g obtained (yield 83%).

CH ₂ Cl ₂ (2 with “35” (910 mg, 1.64 mmol) and pyridine (3.95 mL, 49.2 mmol)
mL) The solution was ice-cooled, and TESCl (1.10 mL, 6.57 mmol) was added dropwise thereto. The mixture was stirred at the same temperature for 30 minutes, and saturated aqueous sodium hydrogen carbonate and ether were added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted with ether twice. The extracts were collected together, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, and the crude product was purified on a silica gel column to give 1.10 g of the corresponding “36” (yield)
85%).

A CH ₂ Cl ₂ (2 mL) solution containing “36” (1.10 g, 1.40 mmol) and 2,6-lutidine (4.91 mL, 42.2 mmol) was ice-cooled, and TMSOTf (1.00 mL, 5.67 mmol) was added to this. ) Was added dropwise. The mixture was stirred at the same temperature for 20 minutes, and saturated aqueous sodium hydrogen carbonate and ether were added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted with ether twice. The extracts were collected together and dried over anhydrous magnesium sulfate. After concentration under reduced pressure and passing through a short silica gel column, 1.04 g of “37” was obtained (yield 86%).

A solution of “37” (280 mg, 0.327 mmol) in CH ₂ Cl ₂ / H ₂ O (19: 1, 5 mL) was ice-cooled, and DDQ (89 mg, 0.39 mmol) was added thereto. The mixture was stirred at the same temperature for 30 minutes, and saturated aqueous sodium hydrogen carbonate was added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with CH ₂ Cl ₂ . The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product through a silica gel column gave 172 mg of “38” (yield 87%).

A solution of “38” (131 mg, 0.217 mmol) in CH ₂ Cl ₂ (3 mL) was ice-cooled, and 1H-tetrazole (30 mg, 0.43 mmol) and diallyldiisopropyl phosphorimidate (75 mg, 0.31) were added to the solution. mmol) was added. Remove the ice bath, stir for 30 minutes, put it in the ice bath again, and 35% H ₂ O ₂ (0.15 mL,
0.84 mmol) was added dropwise. After stirring for 2 hours, saturated aqueous sodium hydrogen carbonate and CH ₂ Cl ₂ were added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with ethyl acetate. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product on a silica gel column gave 121 mg of “39” (yield 73%).

AcCl (0.0053 mL, 0.075 mmol) was added to MeOH (0.20 mL) to prepare a HCl / MeOH solution. This solution was added to an ice-cooled solution of “39” (95 mg, 0.12 mmol) in CH ₂ Cl ₂ / THF / MeOH (5: 5: 1, 4.4 mL). The mixture was stirred for 20 minutes while maintaining 0 ° C, and saturated aqueous sodium hydrogen carbonate and ethyl acetate were added. The organic layer and the aqueous layer were separated, and the aqueous layer was extracted twice with ethyl acetate. The extracts were collected together and dried over anhydrous magnesium sulfate. Concentration under reduced pressure and purification of the crude product through a silica gel column gave 68 mg of “40” (yield 92%).

A CH ₂ Cl ₂ (0.4 mL) solution containing “40” (21 mg, 0.035 mmol) and water (0.019 mL, 1.06 mmol) was ice-cooled, and PdCl ₂ (PPh ₃ ) ₂ (1.3 mg, 0.0018 mmol) and Bu ₃ SnH (0.024 mL, 0.089 mmol) were added. Stir for 1 hour at this temperature and add water and hexane to precipitate a white solid. The white and hexane layers were removed, and the white solid was purified by silica gel column to give 14 mg of hydroxyphoslactomycin B “1” (yield 77%).

  This synthesis scheme is shown below.

[Example 3] PP2A inhibitory activity test of antibiotic hydroxyphoslactomycin B 150 μM in reaction buffer (20 mM HEPES, 0.5 mM MgCl ₂ , 10 mM NaCl, 1 mM dithiothreitol, [pH 7.5]) Add synthetic peptide (KR-pT-IRR; Sigma) and 1% evaluation sample solution (DMSO solution) to a total volume of 100 μl, add 0.01 unit of purified PP2A (Upstate), and then in an incubator at 30 ° C. Let stand for 30 minutes. After the reaction, add 100 μl of malachite green solution (0.034% malachite green in 1N HCl aqueous solution, 4.2% ammonium molybdate, 0.01% Tween 20) and let it stand at room temperature for 15 minutes to fully color and release. The amount of phosphoric acid was measured using the absorbance at 600 nm wavelength as an index. The inhibition rate was calculated by setting a sample containing only the synthetic peptide and the buffer as a blank and adding a purified enzyme thereto as 100% phosphatase activity. The results are shown in Table 1.

[Example 4] Differentiation-inducing activity test of antibiotic hydroxyphoslactomycin B Human promyelocytic leukemia cell line HL-60 was 10% heat-inactivated fetal bovine serum (JRH Bioscience) in RMPI1640 medium (Sigma). And 50 units / ml penicillin and 50 μg / ml streptomycin (Sigma) were used as a medium and cultured at 37 ° C. in a humidified atmosphere containing 5% carbon dioxide.

In 96-well multiwell plates aliquoted 5 × 10 ^3/100 μl of HL-60 cells min, a 1% rating sample solution to stand (50% 2-propanol solution) was added. This was incubated for 96 hours at 37 ° C in a humidified atmosphere containing 5% carbon dioxide, and then 100 µl of NBT reagent (1 mg / ml nitroblue tetrazolium, 0.6 mg / ml phorbol butyl ester) was added. Incubated for 2 hours. Differentiated HL-60 cells, that is, cells stained purple with the NBT reagent, were measured by microscopic observation, and when purple staining was seen in 10% or more of the total number of cells, differentiation induction was Judged that there was.

[Example 5] Antifungal activity test of hydroxyphoslactomycin B Aspergillus fumigatus was mixed with PS agar medium (potato dextrose (Difco) 2.4%, yeast extract (Difco) 0.1%, agar (Japanese) Inoculated on the photopure drug) 1.3%) and allowed to stand at 30 ° C. for about 1 week to 10 days to sufficiently grow spores. The spores were suspended in sterilized water and prepared to 10 ⁵ cells / ml.

  Hydroxyphoslactomycin B diluted stepwise with 50% 2-propanol was added to the PS agar medium, and 100 μl each was poured into a 96-well well to solidify. On the agar medium, 10 μl of the spore suspension prepared above was added, cultured overnight at 37 ° C., and the growth was observed with the naked eye, and antifungal against Aspergillus fumigatus of hydroxyphoslactomycin B was observed. Activity was measured. As a result, the minimum growth inhibitory concentration was 10 μg / ml.

Since the hydroxyphoslactomycin B exhibits antifungal activity, PP2A inhibitory activity and differentiation-inducing activity, the composition containing hydroxyphoslactomycin B is useful as an antifungal agent and an anticancer agent.

FIG. 1 shows a chromatograph when hydroxyphoslactomycin B is separated from a culture by high performance liquid chromatography. Using a reverse-phase ODS column, fractionation was carried out with a solvent system composed of acetonitrile, water, and acetic acid with a gradient of acetonitrile of 17% to 60%.

Claims (10)

Hydroxyphosphomycin B represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

  A method for producing hydroxyphoslactomycin B, comprising culturing a hydroxyphoslactomycin B-producing bacterium belonging to the genus Streptomyces, and separating and collecting hydroxyphoslactomycin B from the culture.   The method according to claim 2, wherein the hydroxyphoslactomycin B-producing bacterium is Streptomyces HK-803 strain (Streptomyces sp. HK-803). Compound represented by the following formula (A):

(In the formula, R ^{1 to} R ³ each independently represent a hydroxyl-protecting group; R ⁴ represents hydrogen.)
In
Introducing a phosphate group having a protecting group into R ⁴ ;
Deprotecting the protecting groups R ^{1 to} R ³ ;
And a step of deprotecting the protecting group of the phosphate group. A compound represented by the following formula (A).

(In the formula, R ^{1 to} R ³ each independently represent a hydroxyl-protecting group; R ⁴ represents hydrogen.)   An antitumor agent comprising hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof as an active ingredient.   A differentiation inducer comprising hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof as an active ingredient.   An antifungal agent comprising hydroxyphoslactomycin B or a pharmaceutically acceptable salt thereof as an active ingredient. Compound represented by the following formula (B):

(In the formula, R ^{5 to} R ⁷ each independently represents a hydroxyl-protecting group.)
In
A compound represented by the following formula (C), comprising a step of adding a vinyl group by an asymmetric nucleophilic addition reaction:

(In the formula, R ^{5 to} R ⁷ each independently represents a hydroxyl-protecting group.)
Manufacturing method. A compound represented by the following formula (B).

(In the formula, R ^{5 to} R ⁷ each independently represents a hydroxyl-protecting group.)

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