16-membered cyclic macrolide derivative and its production method

The present invention relates to a 16-membered ring macrolide derivative effective against Gram-positive bacteria and a method for producing the same.

Macrolide antibiotics that are effective against Gram-positive bacteria, mycoplasma, chlamydia, etc. are classified as clinically important antibacterial agents because they can be orally administered and have low toxicity. Among them, 16-membered ring macrolide antibiotics have been studied for many derivative synthesis because they are difficult to induce resistance, have little interaction with other drugs, and have little effect on the intestinal tract. Patent Document 1 reports that rokitamycin (RKM) has been produced.

However, 16-membered ring macrolide antibiotics generally have a problem in that they have low structural stability in vivo and a short half-life in vivo. For example, in Non-Patent Document 2, 16-membered ring macrolides are used. Spiramycin, a ride antibiotic, has been reported to lose its activity by opening the aglycon by plasma esterase.

Further, regarding the 16-membered ring macrolide derivative, Patent Document 1 discloses a 2-alkyltylosin derivative in which a lower alkyl group such as a methyl group is substituted at the 2-position, but the structural stability in vivo is still still present. It wasn't enough.

As described above, none of the conventional 16-membered ring macrolide derivatives have sufficiently high structural stability in plasma, and 16-membered ring macrolide derivatives having a sufficiently extended in vivo half-life have not yet been obtained. There wasn't.<patcit num="1"><text>Japanese Unexamined Patent Publication No. 6-306094</text></patcit><nplcit num="1"><text>H.Sakakibara, et al., Acyl Derivatives of 16-Membered Macrolides, The Journal of Antibiotics, 1981, Vol.34, No.8, p.1001 ~ 1018</text></nplcit><nplcit num="2"><text>A.Inoue, et al., Biosynthesis and The Metabolic Fate of Carbon-14 Labeled Spiramycin I, The Journal of Antibiotics, 1983, Vol.36, No.4, p.442 ~ 444</text></nplcit>

<p> The present invention has been made in view of the above-mentioned problems of the prior art, and it is possible to significantly extend the in vivo half-life with sufficiently high structural stability in plasma while maintaining a high level of antibacterial activity. It is an object of the present invention to provide a 16-membered ring macrolide derivative and a method for producing the same.</p>

<p> As a result of intensive studies to achieve the above object, the present inventors have introduced at least one methyl group at the 2-position of the 16-membered ring macrolide derivative and left a carbonyl group or a hydroxyl group at the 3-position. We have found that the object is achieved and have completed the present invention.</p><p> That is, the 16-membered ring macrolide derivative of the present invention or a salt thereof is described in the following general formula (1):</p><p><chemistry num="1"><img file="JP2005298436A_D0001.tif" /></chemistry></p><p>(In the formula, R represents a hydrogen atom or a methyl group, and X represents a group represented by = CH-OH or = C = O), which is characterized by being represented by or a salt thereof.</p><p> The 16-membered ring macrolide derivative of the present invention or a salt thereof has the following general formula (2):</p><p><chemistry num="2"><img file="JP2005298436A_D0002.tif" /></chemistry></p><p>(In the formula, R represents a hydrogen atom or a methyl group), or a salt thereof is preferable.</p><p> The method for producing the 16-membered ring macrolide derivative of the present invention is described in the following general formula (3) :.</p><p><chemistry num="3"><img file="JP2005298436A_D0003.tif" /></chemistry></p><p>(In the formula, A represents an aldehyde group that may be protected, B¹Indicates hydroxyl groups that may be the same or different, and each may be protected. ) Is reduced with a reducing agent, and then a protecting group is introduced into the generated hydroxyl group at the 9-position and the acetal generated by closing the hydroxyl group at the 3-position and the aldehyde group at the 19-position. The following general formula (4):</p><p><chemistry num="4"><img file="JP2005298436A_D0004.tif" /></chemistry></p><p>(In the formula, B¹Is B in general formula (3)¹Is synonymous with B²Indicates hydroxyl groups that may be the same or different, and each may be protected. In the first step of obtaining the compound represented by), the compound represented by the general formula (4) is reacted with a base to extract the hydrogen atom at the 2-position of the lactone ring, and then the methylating agent is reacted. The following general formula (5):</p><p><chemistry num="5"><img file="JP2005298436A_D0005.tif" /></chemistry></p><p>(In the formula, B¹And B²Is B in general formula (4)¹And B²Is synonymous with, and R indicates a hydrogen atom or a methyl group. ), And the protecting group in the compound represented by the general formula (5) is removed, and then the 9-position of the lactone ring is oxidized to obtain the compound represented by the following general formula (2):</p><p><chemistry num="6"><img file="JP2005298436A_D0006.tif" /></chemistry></p><p>(In the formula, R represents a hydrogen atom or a methyl group.) The method is characterized by comprising a third step of obtaining a 16-membered ring macrolide derivative.</p><p> According to the 16-membered ring macrolide derivative of the present invention, it is not always clear why the in vivo activity is maintained at a high level, the in vivo structural stability is improved, and the in vivo half-life is significantly extended. However, the present inventors infer as follows. That is, the phenomenon that aglycon is opened by esterase in plasma and its activity is lost has not been reported with 14-membered ring macrolide antibiotics, and 14-membered ring macrolide antibiotics and 16 Comparing the chemical structures of member-ring macrolide antibiotics, 14-membered ring macrolide antibiotics have a methyl group at the 2-position, whereas 16-membered ring macrolide antibiotics have a methyl group at the 2-position. It turned out that it does not exist. Therefore, it was speculated that the 14-membered ring macrolide antibiotic has resistance to plasma esterase due to steric hindrance of the 2-position methyl group existing near the ester bond. Further, although the above-mentioned Patent Document 1 discloses a 2-alkyltylosin derivative, it is presumed that the structural stability of this compound in vivo is insufficient because the hydroxyl group at the 3-position is removed. Therefore, by introducing a methyl group at the 2-position of the 16-membered ring macrolide antibiotic and leaving a hydroxyl group or a carbonyl group at the 3-position, steric hindrance to plasma esterase acts synergistically. It is speculated that the resistance to medium esterase has improved surprisingly.</p>

<p> According to the present invention, there is provided a 16-membered ring macrolide derivative and a method for producing the same, which can maintain a high level of antibacterial activity, have sufficiently high structural stability in plasma, and can significantly extend the in vivo half-life. It becomes possible to do.</p>

Hereinafter, the present invention will be described in detail according to the preferred embodiment thereof.

First, the 16-membered ring macrolide derivative of the present invention or a salt thereof will be described. The 16-membered ring macrolide derivative of the present invention or a salt thereof is described in the following general formula (1):

<chemistry num="7"><img file="JP2005298436A_D0007.tif" /></chemistry>

(In the formula, R indicates a hydrogen atom or a methyl group, and X indicates a group represented by = CH-OH or = C = O) or a salt thereof.

As represented by the above general formula (1), the 16-membered ring macrolide derivative of the present invention has at least one methyl group bonded to the 2-position. If there is no substituent at the 2-position, that is, the 2-position is = CH₂In the case of, a phenomenon occurs in which the aglycone is ring-opened by the esterase in plasma and the activity is lost.

Further, since X in the general formula (1) is = C = O or = CH-OH, steric hindrance is improved, and the structure is synergistic with at least one methyl group substituted at the 2-position. Stability is improved. When the X is = CH-OH, the configuration of the hydroxyl group may be either the α configuration or the β configuration, but the following general formula (2):

<chemistry num="8"><img file="JP2005298436A_D0008.tif" /></chemistry>

As represented by (R represents a hydrogen atom or a methyl group in the formula), the one in which the hydroxyl group is arranged in α is particularly preferable because the antibacterial activity becomes higher.

The salt of the 16-membered ring macrolide derivative of the present invention is a salt formed by the 16-membered ring macrolide derivative represented by the general formula (1) with an acid or a base. Examples of such salts include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitrate and phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid and malein. Examples thereof include acid addition salts with organic acids such as acid, malic acid, tartaric acid, methanesulfonic acid and ethanesulfonic acid, and acidic amino acids such as aspartic acid and glutamate.

The form of use of such a 16-membered ring macrolide derivative or a salt thereof is not particularly limited, but for example, tablets, granules, tablets, granules, etc. It can be a preparation such as a pill, a capsule, a powder, a liquid, a suspension, or an injection. Further, the method of administering the preparation containing the 16-membered ring macrolide derivative of the present invention is not particularly limited, and it can be administered orally or parenterally.

Further, the dose of the 16-membered ring macrolide derivative of the present invention or a salt thereof is not particularly limited, and the dose can be appropriately increased or decreased depending on the type of disease, symptoms, age of the patient, body weight, etc. Specifically, it is preferable to administer about 200 to 1500 mg / day in several divided doses to an adult patient.

The 16-membered ring macrolide derivative of the present invention or a salt thereof can be produced by various methods, and a typical production method thereof will be described below.

First, the general formula (6): in which X in the general formula (1) is a carbonyl group (= C = O):

<chemistry num="9"><img file="JP2005298436A_D0009.tif" /></chemistry>

(In the formula, R represents a hydrogen atom or a methyl group.) A typical method for producing a 16-membered ring macrolide derivative is the following reaction formula (i) :.

<chemistry num="10"><img file="JP2005298436A_D0010.tif" /></chemistry>

(In the formula, A indicates an aldehyde group which may be protected, B indicates a hydroxyl group which may be protected, and R indicates a hydrogen atom or a methyl group.) "First manufacturing method").

That is, in the first production method, the 2-position of the 16-membered ring macrolide derivative represented by the general formula (7) in the reaction formula (i) is methylated in an inert solvent, and then the methylated 16-membered ring macrolide derivative is 16-membered. It is a method produced by removing a protecting group from a ring macrolide derivative.

Inactive organic solvents include tetrahydrofuran, diethyl ether, hexane, hexamethylphosphoramide (HMPA), N, N'-dimethylpropylene urea (DMPU), chloroform, dichloromethane, dimethyl sulfoxide, N, N'-dimethylformamide (DMF). ), Benzene, toluene or a mixed solvent thereof and the like.

Further, examples of the "optionally protected aldehyde group" represented by A in the general formula (7) include an aldehyde group or a protected aldehyde group such as acetal, thioacetal, ketal, or thioketal. Be done. Specific examples of such protected aldehyde groups include dimethyl acetal, dimethyl ketal, diethyl acetal, diethyl acetal, diethyl thioacetal, diethyl thio ketal, ethylene acetal, ethylene thio ketal, propylene acetal, propylene ketal, or Examples thereof include those to which a substituent such as a methyl group is further bonded.

Further, examples of the "hydroxyl group which may be protected" represented by B in the general formula (7) include a hydroxyl group or a hydroxyl group protected by the following protecting group. Such protecting groups include a trimethylsilyl group, a triethylsilyl group, a tri (isopropyl) silyl group, a tri (tert-butyl) silyl group, a tert-butyldimethylsilyl group, a tert-butyldiethylsilyl group, and a sexyldimethylsilyl group. Cyril-based protecting groups such as acetyl group, propionyl group, butyryl group, isobutylyl group, valeryl group, isovaleryl group and other alkanoyl-based protecting groups, 2-tetrahydrofuranyl group, 2-tetrahydropyranyl group and other protecting groups Can be mentioned.

Further, as a method for methylating the 2-position of the lactone ring, a general method for alkylating the α-position adjacent to the ester can be used, and is not particularly limited, but specifically, potassium. A method of introducing a methylating agent after reacting with a base such as tert-butoxide or sodium hydride, a method of forming an enolate using silyl enolate, titanium enolate, etc., and then performing 2-position alkylation, etc. Can be mentioned. Through the methylation reaction, it is preferable to carry out the reaction in an inert organic solvent under room temperature or cooled temperature conditions (preferably 78 to 0 ° C.). When the temperature exceeds the above temperature conditions, the macrolide compound tends to decompose.

Further, the method for removing the protecting group can be a commonly used method and is not particularly limited, but specifically, a method of treating with a base such as ammonia, an inorganic acid such as hydrochloric acid or sulfuric acid, or a tri. Examples thereof include a method of treating with an organic acid such as fluoroacetic acid and difluoroacetic acid. When the protecting group is a silyl-based protecting group, a method of treating with tetrabutylammonium fluoride, hydrochloric acid, acetic acid or the like can be mentioned. When removing the protecting group, it is preferable to stir and react for 1 to 72 hours under a temperature condition of 20 to 60 ° C.

Next, the general formula (8): in which X in the general formula (1) is = CH-OH:

<chemistry num="11"><img file="JP2005298436A_D0011.tif" /></chemistry>

(In the formula, R represents a hydrogen atom or a methyl group.) A typical example of the method for producing a 16-membered ring macrolide derivative will be described. A typical method for producing such a 16-membered ring macrolide derivative is described in Reaction Scheme (ii):

<chemistry num="12"><img file="JP2005298436A_D0012.tif" /></chemistry>

(In the formula, A indicates an aldehyde group which may be protected, B indicates a hydroxyl group which may be protected, and R indicates a hydrogen atom or a methyl group.) , Called the second manufacturing method).

That is, in the second production method, the carbonyl group (oxo group) at the 3- and 9-positions of the compound represented by the general formula (9) in the reaction formula (ii) is reduced to a hydroxyl group, and then an oxidizing agent is used. This is a method for producing a 16-membered ring macrolide derivative represented by the general formula (8) by reoxidizing the 9-position and then removing the protecting group.

As a method for oxidation, a method using a chromium compound such as pyridinium chlorochromate or pyridinium dichromate, an oxidizing agent such as Dess-Martin reagent or Jones reagent, or a method such as Swern oxidation or Corey-Kim oxidation can be used. ..

Further, as the reaction solvent, dichloromethane, chloroform, acetonitrile and the like are used. Further, the "optionally protected aldehyde group" represented by A in the general formula (9) and the "optionally protected hydroxyl group" represented by B in the general formula (9) are It is the same as that described in the first manufacturing method.

Further, as a method for reducing the carbonyl group, a general method can be used and is not particularly limited, but specifically, an alcohol in the presence of a lanthanoid compound such as cerium chloride, calcium chloride, magnesium chloride or the like. Among them, a method of adding a reducing agent such as sodium borohydride for reduction can be mentioned. The reaction conditions for reduction are appropriately selected depending on the reducing agent used, but in general, the reaction is preferably carried out for 0.5 to 2 hours under a temperature condition of -20 to 25 ° C.

Further, as a method of reoxidizing the 9-position, a method of oxidizing with the oxidizing agent can be mentioned. The reaction conditions for oxidation are appropriately selected depending on the oxidizing agent used, but in general, the reaction is preferably carried out for 0.5 to 2 hours under a temperature condition of -20 to 25 ° C.

Next, a method for producing the 16-membered ring macrolide derivative of the present invention, that is, a method for producing the 16-membered ring macrolide derivative represented by the general formula (2) will be described (hereinafter, referred to as "third production method"). .). Such a method for producing a 16-membered ring macrolide derivative of the present invention is described in Reaction Scheme (iii) :.

<chemistry num="13"><img file="JP2005298436A_D0013.tif" /></chemistry>

(In the formula, A represents an aldehyde group that may be protected, B¹And B²Represent a hydroxyl group that may be protected, respectively, and R represents a methyl group or a hydrogen atom. ) Is the method represented by.

First, as the first step, the carbonyl group at the 9-position of the compound represented by the general formula (3) in the reaction formula (iii) is reduced with a reducing agent, and then the hydroxyl group at the 9-position and the hydroxyl group at the 3-position are generated. And a protecting group is introduced into the acetal generated by the closure of the aldehyde group at the 19-position to obtain the compound represented by the general formula (4).

"Aldehyde group which may be protected" represented by A in reaction formula (iii) and B¹, B²The hydroxyl group which may be protected represented by the above is the same as that described in the first production method.

The method for reducing the carbonyl group with a reducing agent is the same as the method described in the second production method. As for the blending amount of the reducing agent, it is preferable that the blending amount of the reducing agent is 3 to 6 mol with respect to 1 mol of the compound represented by the general formula (3). The intermediate obtained by purifying the compound obtained by introducing a reducing agent is hereinafter referred to as "intermediate a".

The method for introducing the protecting group is not particularly limited, but for example, a solution in which the compound (protecting agent) containing the protecting group described in the first production method is dissolved in an inert organic solvent as the intermediate a. A method of introducing a protecting group by adding to the above can be mentioned. Specifically, a method of introducing a protecting group by adding a compound such as tert-butyldimethylsilyl chloride as a protective agent and stirring and reacting is preferable. As for the blending amount of such a protective agent, it is preferable that the blending amount of the protective agent is 2 to 4 mol with respect to 1 mol of the intermediate a. The reaction conditions for introducing the protecting group are appropriately selected depending on the protective agent used, but are generally 12 to 24 hours under the temperature condition of 20 to 60 ° C in an inert organic solvent such as DMF. It is preferable to react to introduce a protecting group.

Further, a general method can be used as a method for purifying the compound represented by the general formula (4), and the method is not particularly limited. Specifically, after extracting the compound from the reaction solution, anhydrous magnesium sulfate is used. Examples thereof include a method of purifying the residue obtained by drying and concentrating using silica gel column chromatography.

Next, as the second step, the compound represented by the general formula (4) is reacted with a base in an inert organic solvent to extract the hydrogen atom at the 2-position of the lactone ring, and then the methylating agent is reacted. The compound represented by the general formula (5) is obtained.

Such an inert organic solvent is the same as that described in the first production method. The base may be any base that can be used for abstraction of hydrogen, and is not particularly limited, but bases such as butyllithium and LDA (lithium diisopropylamide) can be preferably used. In addition, examples of the methylating agent include general ones such as methyl iodide.

The reaction for extracting the hydrogen atom at the 2-position is as described above in a solution prepared by mixing a base with an inert organic solvent under a nitrogen stream, room temperature or a cooled temperature condition (preferably -78 to 0 ° C). The reaction of adding the compound represented by (4) to extract the hydrogen atom at the 2-position can be mentioned. The reaction time is appropriately selected depending on the base to be used, but it is generally preferable to carry out the reaction for 5 to 30 minutes after the introduction of the base. The amount of the base compounded is preferably 3 to 6 mol with respect to 1 mol of the compound represented by the general formula (4). The intermediate obtained by purifying the compound obtained here is hereinafter referred to as "intermediate b".

Further, as a method for reacting the methylating agent, a general method can be used, specifically, under a nitrogen stream, at room temperature, or under cold temperature conditions (preferably -78 to 0 ° C). Examples thereof include a method in which a methylating agent such as methyl iodide is added to the intermediate b in the inert organic solvent, and the mixture is stirred and reacted. In such a reaction, it is preferable to add a methylating agent and then stir for 0.5 to 2 hours to carry out the reaction. The amount of the methylating agent to be blended is preferably 3 to 6 mol with respect to 1 mol of the intermediate b.

The compound after methylation in this manner can be purified by the same method as the purification method described in the first step to obtain a compound represented by the general formula (5).

Next, as a third step, after removing the protecting group in the compound represented by the general formula (5), the 9-position of the lactone ring is oxidized to form a 16-membered ring represented by the following general formula (2). Obtain a macrolide derivative.

The method for removing the protecting group is the same as the method described in the first production method described above. The intermediate obtained by purifying the compound obtained by removing the protecting group is hereinafter referred to as "intermediate c".

Examples of the method for oxidizing the 9-position include a method in which the obtained intermediate c is dissolved in a reaction solvent to obtain a reaction solution, and then an oxidizing agent is introduced into the obtained reaction solution to cause an oxidation reaction. .. As the reaction conditions for oxidation, it is preferable to add an oxidizing agent under a temperature condition of 0 to 20 ° C. and stir for 0.5 to 2 hours for the reaction. Further, as for the blending amount of the oxidizing agent, it is preferable that the blending amount of the oxidizing agent is 1 to 2 mol with respect to 1 mol of the intermediate c. The reaction solvent and the oxidizing agent are the same as those described in the second production method.

Further, after oxidizing the 9-position of intermediate c with an oxidizing agent, a compound is extracted from this reaction solution, and the compound is purified to purify the 16-membered compound represented by the general formula (2). A ring macrolide derivative can be obtained.

The method for purifying the 16-membered ring macrolide derivative represented by the general formula (2) is not particularly limited, but the mixed solvent of chloroform and methanol (preferable compounding ratio (volume standard) is 9 Purification by pTLC using a developing solvent consisting of 1 to 2: 1) is preferable.

Hereinafter, the present invention will be described in more detail based on Examples and Comparative Examples, but the present invention is not limited to the following Examples.

<u style="single">Synthesis Examples 1-2 [Intermediate 1: 2β-methyl-3-dehydro-2', 4', 4''-tri-O-acetyldesmycosin-20-diethylacetal (in the following general formula (10), A is a diethyl acetal group, B is an acetoxyl group, R is a hydrogen atom, a methyl group at the 2-position is represented by a β configuration), and an intermediate 2: 2,2-dimethyl-3-dehydro-2', 4 ', 4''-Tri-O-acetyldesmycosin-20-diethylacetal (in the following general formula (10), A is a diethyl acetal group, B is an acetoxyl group, and R is a methyl group). Synthetic]</u> General formula (10):

<chemistry num="14"><img file="JP2005298436A_D0014.tif" /></chemistry>

The starting material 2,3-dehydro-2', 4', 4''-tri-O-acetyldesmycosin-20-diethyl acetal is a known compound (Lawrence C. Creemer, et al., 3- Keto 16-Membered Macrolides Derived from Tylosin , The Journal of Antibiotics, 2002, Vol.55, No.4, p427 ~ 436). After dissolving 1.54 g of this compound in 75 ml of tetrahydrofuran, 210.9 mg of potassium tert-butoxide and 0.2 ml of methyl iodide were added under ice-cooling, and the mixture was stirred for 2 hours. 75 ml of water was added to the reaction mixture, and this was extracted twice with 150 ml of ethyl acetate. The organic layer is dried over anhydrous magnesium sulfate and concentrated, and the obtained residue is purified by silica gel column chromatography to obtain 2β-methyl-3-dehydro-2', 4', 4''-tri-O-acetyl. Desmycosin-20-diethylacetal was obtained at 595.3 mg, and 2,2-dimethyl-3-dehydro-2', 4', 4''-triacetyldesmycosin-20-diethylacetal was obtained at 319.9 mg, respectively. Intermediate 1 and Intermediate 2 showed the following physical properties.

Physical characteristics of intermediate 1: (1) Specific rotation: [α]_D + 4.6 ° (c1.05, CHCl₃) (2) Molecular formula: C₅₀H₈₁NO₁₈(3) Mass spectrum (ESI-MS): m / z 984 [M + H]⁺(4)¹1 H NMR (500MH)_Z, CDCl₃): δ (ppm) 0.89 (17-H), 1.11 (21-H), 1.31 (2-Me) 1.80 (22-H), 1.94 (OAc), 2.03 (OAc), 2.08 (OAc), 2.30 ( 3'-NMe₂), 3.40 (2 -OMe), 3.49 (3 -OMe), 3.55 (2-H), 5.79 (13-H), 6.08 (10-H), 7.06 (11-H).

Physical characteristics of intermediate 2: (1) Specific rotation: [α]_D -31.1 ° (c1.03, CHCl₃) (2) Molecular formula: C₅₁H₈₃NO₁₈(3) Mass spectrum (ESI-MS): m / z 998 [M + H]⁺(4)¹1 H NMR (500MHz, CDCl₃): δ (ppm) 0.92 (17-H), 0.97 (18-H), 1.13 (21-H), 1.33 (2-Me)₂), 1.81 (22-H), 1.95 (OAc), 2.05 (OAc), 2.11 (OAc), 2.34 (3'-NMe)₂&gt;, 3.42 (2 -OMe), 3.51 (3 -OMe), 5.94 (13-H), 6.08 (H-10), 7.18 (11-H).

<u style="single">Example 1 [Production of Compound 1: 2-Methyl-3-dehydrodesmycosin (Compound in which R is a hydrogen atom and X is = C = O in the general formula (1))]</u> 220.8 mg of Intermediate 1 obtained as described above was dissolved in 25 ml of methanol, 0.1 ml of 28% aqueous ammonia was added thereto, and the mixture was stirred at room temperature for 72 hours. After concentrating the reaction solution, the obtained residue was dissolved in 4 ml of acetonitrile, 1 ml of 1N hydrochloric acid was added thereto, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was neutralized by adding a saturated aqueous sodium hydrogen carbonate solution, and then extracted twice with 50 ml of ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off to obtain 161.2 mg of 2-methyl-3-dehydrodesmycosin. This compound exists in an equilibrium state of keto-enol and keto-enol. Such compound 1 exhibited the following physical characteristics.

Physical characteristics of compound 1: (1) Molecular formula: C₄₀H₆₅NO₁₄(2) Mass spectrum (HRTOF-MS): m / z 782.4343 [MH]^-(Theoretical value: 782.4327 (C)₄₀H₆₅NO₁₄Calculated as)). (Four)¹1 H NMR: δ (ppm) 12.3 (enol-OH).

<u style="single">Example 2 [Compound 2: 2,2-Dimethyl-3-dehydrodesmycosin (compound in which R is a methyl group and X is represented by = C = O in the general formula (1))]</u> 262.7 mg of the above intermediate 2 was deprotected by the same method as in the method for producing Compound 1, and 96.5 mg of 2,2-dimethyl-3-dehydrodesmycosin (Compound 2) was obtained. Such compound 2 exhibited the following physical characteristics.

Physical characteristics of compound 2: (1) Specific rotation: [α]_D + 14.3 ° (c1.04, CHCl₃) (2) Molecular formula: C₄₁H₆₇NO₁₄(3) Mass spectrum (HRTOF-MS): m / z 796.4496 [MH]^-(Theoretical value: 796.4483 (C)₄₁H₆₆NO₁₄Calculated as)) (4)¹1 H NMR (500MHz, CDCl₃): δ (ppm) 0.90 (17-H), 1.07 (18-H), 1.12 (21-H), 1.29 (2-Me), 1.32 (2-Me), 1.81 (22-H), 2.49 ( 3'-NMe₂), 3.44 (2 -OMe), 3.58 (3 -OMe), 6.03 (13-H), 6.11 (10-H), 7.19 (11-H), 9.82 (20-H).

<u style="single">Synthesis Example 3 [Intermediate 3: 2β-Methyl-3-epi-9-hydro-2', 4', 4''-tri-O-acetyldesmycosin-20-diethylacetal (general formula (11) below) Synthesis of compounds in which A is a diethyl acetal group, B is an acetoxyl group, and the methyl group at the 2-position is represented by a β configuration)]</u> General formula (11):

<chemistry num="15"><img file="JP2005298436A_D0015.tif" /></chemistry>

After dissolving 261.4 mg of the intermediate 1 produced as described above in 6 ml of methanol, adding 148.4 mg of cerium (7 hydrate) chloride and stirring at room temperature for 30 minutes, sodium borohydride 0.4 under ice-cooling. g was added and the mixture was stirred for 15 minutes. 50 ml of water was added to the reaction mixture, and this was extracted twice with 50 ml of ethyl acetate. The organic layer is dried over anhydrous magnesium sulfate and concentrated, and the obtained residue is purified by silica gel column chromatography to 2β-methyl-3-epi-9-hydro-2', 4', 4''-tri. 98.7 mg of -O-acetyldesmycosin-20-diethylacetal was obtained.

Physical characteristics of intermediate 3: (1) Specific rotation: [α]_D + 7.0 ° (c1.14, CHCl₃) (2) Molecular formula: C₅₀H₈₅NO₁₈(3) Mass spectrum (ESI-MS): m / z 988 [M + H]⁺(4)¹1 H NMR (500MHz, CDCl₃): δ (ppm) 0.72 (18-H), 0.88 (17-H), 0.97 (21-H), 1.36 (2-Me), 1.70 (22-H), 1.96 (OAc), 2.01 (OAc) , 2.08 (OAc), 2.30 (3'-NMe)₂), 2.65 (2-H), 3.44 (2 -OMe), 3.49 (3 -OMe), 4.11 (9-H), 5.36 (13-H), 5.67 (10-H), 6.32 ( 11-H).

<u style="single">Example 3 [Compound 3: 2β-methyl-3-epi-desmycosin (R in the general formula (1) is a hydrogen atom, X is = C-OH (hydroxyl group is β-arranged), and the methyl group at the 2-position is β. Production of compound) represented by arrangement]</u> 98.7 mg of Intermediate 3 was dissolved in 6 ml of dichloromethane, 50 mg of Dess-Martin reagent was added thereto under ice-cooling, and the mixture was stirred for 1.5 hours. 10 ml of a 0.2N aqueous sodium hydroxide solution was added to the reaction mixture, and this was extracted with 20 ml of ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and concentrated, and the obtained residue was deprotected by the same method as in Example 2 to obtain 68.7 mg of 2β-methyl-3-epi-desmycosin.

Physical characteristics of compound 3: (1) Specific rotation: [α]_D -9.2 ° (c0.95, CHCl₃) (2) Molecular formula: C₄₀H₆₇NO₁₄(3) Mass spectrum (HRTOF-MS): m / z 784.4495 [MH]^-(Theoretical value: 784.4483 (C)₄₀H₆₆NO₁₄Calculated as)) (4)¹1 H NMR (500MHz, CDCl₃): δ (ppm) 0.78 (17-H), 0.84 (18-H), 1.13 (21-H), 1.31 (2-Me), 1.73 (22-H), 2.44 (3'-NMe)₂), 2.56 (2-H), 3.41 (2 -OMe), 3.54 (3 -OMe), 5.85 (13-H), 6.16 (10-H), 7.02 (11-H), 9.67 (20- H).

<u style="single">Synthesis Example 4 [Intermediate 4: 9-hydro-9,20-di-O-tert-butyldimethylsilyl-2', 4', 4''-tri-O-acetyldesmycosin-3,20-acetal (In the general formula (4), B</u>^{<u style="single">1</u>}<u style="single">Is an acetoxyl group, B</u>^{<u style="single">2</u>}<u style="single">Is a compound represented by an O-tert-butyldimethylsilyl group)]</u> Starting material 2', 4', 4''-tri-O-acetyldesmycosin-20-diethyl acetal (in the general formula (3), A is a diethyl acetal group, B¹The compound represented by the acetoxyl group) is a known compound (Lawrence C. Creemer, et al., 3-Keto 16-Membered Macrolides Derived from Tylosin, The Journal of Antibiotics, 2002, Vol.55, No.4, p427 ~ 436). 50.1 g of this compound was dissolved in 1 L of methanol, 28.8 g of cerium (7hydrate) chloride was added thereto, and the mixture was stirred at room temperature for 30 minutes. Then, 9.7 g of sodium borohydride was added over 30 minutes under ice-cooling, and the mixture was stirred at room temperature for 10 minutes after the addition was completed. 500 ml of 1 M disodium hydrogen phosphate was added to the reaction mixture, and this was extracted twice with 500 ml of ethyl acetate. The organic layer is dried over anhydrous magnesium sulfate and concentrated, and the obtained residue is purified by silica gel column chromatography to purify 9-hydro-2', 4', 4''-tri-O-acetyldesmycosin-20. -22.4 g of diethyl acetal was obtained. Next, 22.2 g of this compound was dissolved in 300 ml of acetonitrile, 75 ml of a 1N aqueous hydrochloric acid solution was added thereto, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was neutralized with 1N aqueous sodium hydroxide solution, and this was extracted twice with 300 ml of ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and concentrated, and the obtained residue was dissolved in 250 ml of N, N-dimethylformamide. To this, 10.9 g of tert-butyldimethylsilyl chloride and 9.8 g of imidazole were added, and the mixture was stirred at 45 ° C. for 18 hours. 250 ml of water was added to the reaction mixture, and this was extracted twice with 500 ml of toluene. The organic layer is dried over anhydrous magnesium sulfate and concentrated, and the obtained residue is purified by silica gel column chromatography to 9-hydro-9,20-di-O-tert-butyldimethylsilyl-2', 4', 3.98 g of 4''-tri-O-acetyldesmycosin-3,20-acetal was obtained.

Physical characteristics of intermediate 4: (1) Specific rotation: [α]_D -8.4 ° (c1.04, CHCl₃) (2) Molecular formula: C₅₇H₁₀₁NO₁₇Si₂(3) Mass spectrum (ESI-MS): m / z 1128 [M + H]⁺(4)¹1 H NMR (500MHz, CDCl₃): δ (ppm) 0.87 (Si (CH)₃)₂C (<u style="single">CH</u>_{<u style="single">3</u>})₃), 0.88 (Si (CH)₃)₂C (<u style="single">CH</u>_{<u style="single">3</u>})₃), 0.98 (21-H), 1.73 (22-H), 1.97 (OAc), 2.04 (OAc), 2.09 (OAc), 2.33 (3'-NMe)₂), 3.46 (2 -OMe), 3.50 (3 -OMe), 3.81 (9-H), 5.36 (13-H), 5.90 (10-H), 6.18 (11-H).

<u style="single">Synthesis Example 5 [Intermediate 5: 9-hydro-9,20-di-O-tert-butyldimethylsilyl-2', 4', 4''-tris-O-triethylsilyldesmycosin-3,20- Acetal (in the general formula (4), B</u>^{<u style="single">1</u>}<u style="single">Is an O-triethylsilyl group, B</u>^{<u style="single">2</u>}<u style="single">Is a compound represented by an O-tert-butyldimethylsilyl group)]</u> 3.88 g of Intermediate 4 was dissolved in 250 ml of methanol, 2 ml of 28% aqueous ammonia was added thereto, and the mixture was stirred at room temperature for 72 hours. After concentrating the reaction solution, the obtained residue was dissolved in 60 ml of N, N-dimethylformamide, 3.5 ml of triethylsilyl chloride and 2.8 g of imidazole were added thereto, and the mixture was stirred at room temperature for 18 hours. 60 ml of water was added to the reaction mixture, and this was extracted twice with 120 ml of ethyl acetate. The organic layer is dried over anhydrous magnesium sulfate and concentrated, and the obtained residue is purified by silica gel column chromatography to 9-hydro-9,20-di-O-tert-butyldimethylsilyl-2', 4', 4''-Tris-O-triethylsilyldesmycosin-3,20-acetal was obtained at 3.37.

Physical characteristics of intermediate 5: (1) Specific rotation: [α]_D -17.5 ° (c1.00, CHCl₃) (2) Molecular formula: C₆₉H₁₃₇NO₁₄Si₅(3) Mass spectrum (ESI-MS): m / z 1345 [M + H]⁺(4)¹1 H NMR (500MHz, CDCl₃): δ (ppm) 0.87 (Si (CH)₃)₂C (<u style="single">CH</u>_{<u style="single">3</u>})₃), 1.18 (21-H), 1.75 (22-H), 2.42 (3'-NMe)₂), 3.47 (2 -OMe), 3.58 (3 -OMe), 5.44 (13-H), 5.84 (10-H), 6.14 (11-H).

<u style="single">Synthesis Examples 6-7 [Intermediate 6: 2β-Methyl-9-hydro-9,20-di-O-tert-butyldimethylsilyl-2', 4', 4''-Tris-O-triethylsilyl desmaiko Thin-3,20-Acetal (in the general formula (5), B</u>^{<u style="single">1</u>}<u style="single">Is an O-triethylsilyl group, B</u>^{<u style="single">2</u>}<u style="single">Is an O-tert-butyldimethylsilyl group, R is a hydrogen atom, and the methyl group at the 2-position is represented by a β configuration) and intermediate 7: 2α-methyl-9-hydro-9,20-di-O- tert-Butyldimethylsilyl-2', 4', 4''-Tris-O-triethylsilyldesmycosin-3,20-acetal (B in the general formula (5) above)</u>^{<u style="single">1</u>}<u style="single">Is an O-triethylsilyl group, B</u>^{<u style="single">2</u>}<u style="single">Is a compound in which O-tert-butyldimethylsilyl group, R is a hydrogen atom, and the methyl group at the 2-position is represented by an α configuration)]</u> A mixture of 0.35 ml of diisopropylethylamine, 2 ml of tetrahydrofuran and 2 ml of N, N'-dimethylpropinylurea was cooled to -78 ° C under a nitrogen stream, and 2 ml of a solution of intermediate 5 (675.1 mg) in tetrahydrofuran was added thereto. After 5 minutes, 0.16 ml of methyl iodide was added and stirred for another 30 minutes, then 200 ml of water was added to the reaction solution, and this was extracted twice with 200 ml of ethyl acetate. The organic layer is dried over anhydrous magnesium sulfate and concentrated, and the obtained residue is purified by silica gel column chromatography to obtain 2β-methyl-9-hydro-9,20-di-O-tert-butyldimethylsilyl-2. ', 4', 4''-Tris-O-triethylsilyldesmycosine-3,20-acetal 340.1 mg, 2α-methyl-9-hydro-9,20-di-O-tert-butyldimethylsilyl- 39.2 mg of 2', 4', 4''-tris-O-triethylsilyldesmycosin-3,20-acetal was obtained.

Physical characteristics of intermediate 6: (1) Specific rotation: [α]_D -2.1 ° (c0.84, CHCl₃) (2) Molecular formula: C₇₀H₁₃₉NO₁₄Si₅(3) Mass spectrum (ESI-MS): m / z 1359 [M + H]⁺(4)¹1 H NMR (500MHz, CDCl₃): δ (ppm) 1.26 (2-Me), 1.69 (22-H), 2.41 (3'-NMe)₂), 2.52 (2-H), 3.47 (2 -OMe), 3.58 (3 -OMe), 5.42 (13-H), 5.71 (10-H), 5.94 (11-H).

Physical characteristics of intermediate 7: (1) Specific rotation: [α]_D -63.6 ° (c0.68, CHCl₃) (2) Molecular formula: C₇₀H₁₃₉NO₁₄Si₅(3) Mass spectrum (ESI-MS): m / z 1359 [M + H]⁺(4)¹1 H NMR (500MHz, CDCl₃): δ (ppm) 1.81 (22-H), 2.42 (3'-NMe)₂), 2.46 (2-H), 3.46 (2 -OMe), 3.58 (3 -OMe), 3.82 (3-H), 5.46 (13-H), 5.98 (10-H), 6.29 (11-) H).

<u style="single">Example 4: [Compound 4: Preparation of 2β-methyldesmycosin (compound in which R is a hydrogen atom and the methyl group at the 2-position is represented by a β configuration in the above general formula (2))]</u> 107.3 mg of Intermediate 6 was dissolved in 5 ml of acetonitrile, to which 5 ml of water was added. To this, 50 μl of difluoroacetic acid was added, and the mixture was stirred at 45 ° C. for 72 hours. A saturated sodium hydrogen carbonate solution was added to the reaction mixture for neutralization, and this was extracted twice with 50 ml of ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and concentrated. The obtained residue was dissolved in 3 ml of dichloromethane, 25 mg of Dess-Martin reagent was added thereto under ice-cooling, and the mixture was stirred for 1.5 hours. 10 ml of a 0.2N aqueous sodium hydroxide solution was added to the reaction mixture, and this was extracted with 20 ml of ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and concentrated, and the obtained residue was purified by pTLC using chloroform-methanol 4: 1 as a developing solvent to obtain 25.9 mg of 2β-methyldesmycosin.

Physical characteristics of compound 4: (1) Specific rotation: [α]_D -21.3 ° (c1.15, CHCl₃) (2) Molecular formula: C₄₀H₆₇NO₁₄(3) Mass spectrum (HRTOF-MS): m / z 784.4477 [MH]^-(Theoretical value: 784.4483 (C)₄₀H₆₆NO₁₄Calculated as) (4)¹1 H NMR (500MHz, CDCl₃): δ (ppm) 0.86 (17-H), 0.95 (18-H), 1.13 (21-H), 1.21 (2-Me), 1.72 (22-H), 2.28 (2-H), 2.51 ( 3'-NMe₂), 3.41 (2 -OMe), 3.54 (3 -OMe), 5.82 (13-H), 6.15 (10-H), 7.11 (11-H), 9.63 (20-H).

<u style="single">Example 5 [Production of Compound 5: 2α-methyldesmycosin (compound in which R is a hydrogen atom and the methyl group at the 2-position is represented by an α configuration in the above general formula (2))]</u> 53.5 mg of Intermediate 7 was treated in the same manner as in Example 4 to obtain 18.9 mg of 2α-methyldesmycosin.

Physical characteristics of compound 5: (1) Specific rotation: [α]_D + 3.8 ° (c0.43, CHCl₃) (2) Molecular formula: C₄₀H₆₇NO₁₄(3) Mass spectrum (HRTOF-MS): m / z 786.4659 [M + H]⁺(Theoretical value: 786.4640 (C)₄₀H₆₈NO₁₄Calculated as) (4)¹1 H NMR (500MHz, acetone-d₆): δ (ppm) 0.94 (17-H), 0.99 (2-Me), 1.01 (18-H), 1.88 (22-H), 2.48 (2-H), 2.48 (2'-NMe)₂), 3.45 (2 -OMe), 3.52 (3 -OMe), 5.88 (13-H), 6.40 (10-H), 7.23 (11-H), 9.69 (20-H).

<u style="single">(Evaluation of the compound obtained in the example)</u> &lt;Test Example 1&gt; For compounds 1 to 5 and desmycosin and rokitamycin obtained in Examples 1 to 5, the minimum inhibitory concentration (MIC) for the test bacteria was measured by a trace liquid dilution method according to the NCCLS standard method. As the measurement medium, Cation-adjusted Mueller-Hintonbroth (2% LHB-added CAMHB) was used for S. pneumoniae, and Haemophilus test medium (HTM) was used for H. influenzae. Final inoculation count of 10⁴The cells were CFU / well and aerobically cultured at 35 ° C for 20 to 24 hours. Using the growth of bacteria in the drug-free medium of each plate as a control, the minimum drug concentration at which no growth of bacteria was observed was used as the MIC. The results are shown in Table 1. The unit of the numerical value in the table is (μg / ml).

<tables num="1"><img file="JP2005298436A_D0016.tif" /></tables>

Thus, compounds 1 to 5 had antibacterial activity against clinically important Gram-positive bacteria, and compound 4 had antibacterial activity equivalent to that of desmycosin. In addition, Compound 5 showed particularly strong antibacterial activity against macrolide-resistant bacteria.

&lt;Test Example 2&gt; Compound 4 was evaluated for resistance to plasma esterase as follows. That is, after incubating compound 4 in rat plasma at 37 ° C. for 1 hour, the abundance ratio of the lactone ring-opened product was evaluated as the abundance ratio with the unchanged product. Desmycosin was used as a comparative substance. The ring-opening body abundance ratio was calculated by the following formula. Ring-opened body abundance ratio (%) = (lactone ring-opened body amount / unchanged body residual amount) × 100 The result is that the ring-opened body abundance ratio of desmycosin is 16.6%, and the ring-opened body abundance ratio of Compound 4 is 1. It was less than%. As described above, since compound 4 was stably present in rat plasma for 1 hour, it was confirmed that the methyl group at the 2-position and the hydroxyl group at the 3-position synergistically acquired resistance to plasma esterase.

As described above, according to the present invention, a 16-membered ring macrolide derivative capable of significantly extending the in vivo half-life with sufficiently high structural stability in plasma while maintaining a high level of antibacterial activity. And its manufacturing method can be provided.

Therefore, the 16-membered ring macrolide derivative of the present invention is useful as an antibacterial agent, particularly as an antibiotic, etc., because it sufficiently exerts antibacterial activity and prolongs the half-life in vivo.