JPH06206897A - New 16-membered ring macrolide derivative - Google Patents

Abstract

PURPOSE:To obtain a new 16-membered ring macrolide derivative for antimicrobial agents, etc., effective in Gram-positive bacteria and mycoplasma, excellent in endokinetics and capable of retaining antimicrobial activity in plasma by culturing a variant of Streptomyces mycarofaciens. CONSTITUTION:A frozen seed of Streptomyces mycarofaciens SF-837 being a variant of Streptomyces mycarofaciens which is a strain of Actinomyces is inoculated into a culture medium and subjected to shake culture at 28 deg.C for 18hr and then, the cultured mixture is centrifuged and the fungal cell is removed and transparent culture mixture is collected and the pH is adjusted to 9 and the culture mixture is extracted with ethyl acetate. This extract is concentrated under reduced pressure and the residue is purified by column chromatography to provide the objective new 16-membered ring macrolide derivative expressed by the formula [R<1> is H or the formula COR<7> (R<7> is 1-3C alkyl); R<2> and R<3> are H or (substituted)OH (with the proviso that either one of R<2> and R<3> is H); R<4> is H or the formula COR<7>; R<5> is 1-4C alkyl; R<6> is (substituted)1-10C alkyl, alkenyl, etc.).

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel 16-membered ring macrolide derivative which is effective against Gram-positive bacteria and mycoplasma, has excellent pharmacokinetics, and has sustained antibacterial activity in plasma, and a process for producing them.

[0002]

BACKGROUND OF THE INVENTION Macrolide antibiotics effective against Gram-positive bacteria, mycoplasma, chlamydia, etc. are classified as clinically important antibacterial agents because they can be administered orally and have low toxicity. Among them, commercially available 16-membered ring macrolide antibiotics are less likely to induce resistance, have less interaction with other drugs than 14-membered ring macrolides, and have less effect on the intestinal tract. Widely used around the world, mainly. Above all, it has excellent pharmacokinetics and less bitterness than natural compounds, and has less bitterness Myokamycin (MOM) (Journal of Antibiotics,
29 (5), 536 (1976), Japanese Journal of
Antibiotics, 35 (6), 1462 (1982)) is semi-synthetic 1
It is widely used clinically as a 6-membered macrolide antibiotic.

The present inventors have found that 1 is effective against various Gram-positive bacteria.
We have carried out synthetic chemical and biochemical exploratory research on 6-membered macrolide derivatives, and have conducted a biochemical reaction to specifically cleave the acyl group bonded to the 3-hydroxyl group of 16-membered macrolide by certain molds. Found and filed a patent (EP 526,9
06 (1993), US 5,219,736 (1993)). Furthermore, the present inventors
In MOM, the fact that the acyl group of the mycalose moiety is cleaved by metabolism and the antibacterial activity is clearly reduced (pharmaceutical journal, 102 (8), 781 (1982)) is fed back to drug design, and derivative research is conducted under a new idea. Was done.
That is, as a result of repeated synthetic chemical studies aimed at a 16-membered ring macrolide derivative that is stable in the living body and has a low antibacterial activity even if it is metabolized, the two hydroxyl groups in the mycarose portion of the 16-membered ring macrolide derivative are We succeeded in synthesizing a derivative with an ether bond by an alkyl group, proved that the antibacterial activity remarkably lasts in vitro , and applied for a patent (Japanese Patent Application No. 4-39013, Japanese Patent Application No. 5-43813).
9).

The correlation between the structure and various activities including the pharmacokinetics of the 16-membered ring macrolide compound, especially the 3-position in the leucomycin analog has been studied in detail (Journal of Antibiotics). Chicks, 21 (9), 532 (1968)). Among the 16-membered ring macrolide compounds, the present inventors examined the effect of the difference in the 9-position structure of the midecamycin analog on the pharmacokinetics. That is, midecamycin A whose 9-position is a hydroxyl group
₁ (medemycin / MDM) (Journal of Antibiotics, 24 (7), 452 (1971)) and midecamycin A ₃ having a carbonyl group at the 9-position (ibid, 24 (7), 476 (197).
Regarding 1)), the pharmacokinetics was observed in an animal experiment (oral administration) using mice. As a result, it was confirmed that midecamycin A ₁ , which has a hydroxyl group at the 9-position, is superior in both durability of serum concentration and recovery rate in urine to midecamycin A ₃ . That is, in order to improve the pharmacokinetics of the 16-membered ring macrolide derivative, especially the midecamycin analog, it was judged that the 9-position is preferably a hydroxyl group rather than a carbonyl group.

On the other hand, regarding the 16-membered macrolide and rosalamycin, which are not leucomycin analogues, the compound in which the carbonyl group at the 9-position is converted into a hydroxyl group and the hydroxyl group at the 3-position is acylated is compared with the original substance. It has also been confirmed that the therapeutic effect on infection in mice was improved (Japanese Patent Laid-Open No. 50-126880). Regarding the 13-position of the isotype 16-membered macrolide, it was also reported that the hydroxyl group type derivative was superior to the carbonyl type derivative in vivo activity (Meiji Seika Kenkyukai, 13 , 100 (1973)).

By the way, for the purpose of elucidating the structure-activity relationship of 16-membered ring macrolides, biosynthetic research, structural analysis, etc., a synthetic chemical approach (Journal of Organics) was used as a method for reducing the 9-position carbonyl group to a hydroxyl group.・ Chemistry, 39 (16), 2474 (1974), Journal of Antibiotics, 34 (12), 1577 (1981),
Ibid, 39 (12), 1784 (1986)) and biochemical approach (JP-A-50-126880, Journal of Antibiotics, 32 (7), 777 (1979), ibid, 33 (8), 911 (1980))
Etc. are known.

[0007]

A novel 16-membered ring macrolide derivative having two ether bonds in the mycarose moiety, which was recently synthesized by the present inventors, such as 4 "-O-depropionyl-4" -O-isoamyl. -3 "-O-methyl midecamycin A ₃ (Japanese Patent Application No. 4-39013, Japanese Patent Application No. 5-4389) is a 16-membered ring that has been launched on the market in recent years because its micarose portion is not easily attacked by esterase. Compared with the macrolide antibiotics, the highest serum concentration and urinary recovery in mouse animal experiments were clearly improved, however, its pharmacokinetics are not always completely satisfactory. As described above, the existence of the carbonyl group at the 9-position was considered to be one of the obstacles to the above, and therefore, the antibacterial activity of the present invention is stable, and even if it is metabolized, the antibacterial activity is unlikely to decrease. It is expected that a 16-membered ring macrolide derivative which is more excellent in pharmacokinetics than the derivative synthesized by the present inventors and having two ether bonds in the mycarose moiety will appear.

[0008]

[Means for Solving the Problems] The inventors of the present invention have conducted biochemical research to meet the above-mentioned expectations, and the two hydroxyl groups of the mycalose moiety, which are the 16-membered macrolide derivatives patented by the inventors of the present invention. Various derivatives in which both are ether-bonded by an alkyl group (Japanese Patent Application No. 4-39013, Japanese Patent Application No. 5-4
389) is a mutant strain of Streptomyces mycarofaciens SF-837, which is a species of actinomycete, such as SF2.
By converting microorganisms with the strain named 772, 9
Succeeded in efficiently reducing the carbonyl group at position to a hydroxyl group having a natural configuration. Moreover, these derivatives strongly inhibit the development of clinically important Gram-positive bacteria and mycoplasma, and have an extremely long-lasting antibacterial activity in rat plasma, and at the same time, high serum concentration and its sustained concentration in animal experiments using mice. The present invention has been completed by finding that it exhibits particularly excellent pharmacokinetics including sex. Note that Streptomyces mycarofaciens SF2772
Has been deposited as FERM P-13227 with the Institute of Biotechnology and Industrial Technology, Institute of Industrial Science and Technology.

The gist of the present invention lies in the following formula (I) as a novel compound.

[Chemical 2]

[In the formula, R ¹ represents a hydrogen atom or a group of the formula COR ⁷ (provided that R ⁷ represents a linear alkyl group having 1 to 3 carbon atoms).]
R ² is a hydrogen atom or an optionally substituted hydroxyl group, R ³ is a hydrogen atom or an optionally substituted hydroxyl group (however, one of R ² and R ³ is a hydrogen atom).
R ⁴ is a hydrogen atom or a group of the formula COR ⁷ (provided that R ⁷ has the same meaning as described above), R ⁵ is a linear alkyl group having 1 to 4 carbon atoms, and R ⁶ is substituted. Optionally represents a linear or branched alkyl group, alkenyl group or aralkyl group having 1 to 10 carbon atoms. ] It is related with the compound represented by these, or its pharmaceutically acceptable salt. The compound represented by the general formula (I) according to the present invention is represented by the following formula (II) prepared from the present inventors by synthetic chemical methods, for example, midecamycin A _{3 and the} like.

[0011]

[Chemical 3]

[Wherein R ¹ represents a hydrogen atom or a group of the formula COR ⁷ (provided that R ⁷ represents a linear alkyl group having 1 to 3 carbon atoms).]
R ⁵ represents a linear alkyl group having 1 to 4 carbon atoms, and R ⁶ represents an optionally substituted linear or branched alkyl group, alkenyl group or aralkyl group having 1 to 10 carbon atoms. . ] The compound represented by the above is converted into a microorganism having a function of selectively reducing the carbonyl group at the 9-position to a hydroxyl group, for example, Streptomyces mycarofaciens SF2772 strain which is one species of actinomycetes. Or, if necessary, a known selective or non-selective synthetic chemical reaction (fermentation and industry, 37 (12), 1171 (1979)) for the 9-position and / or 2'-position hydroxyl groups. It is manufactured by doing.

As a method for producing a compound represented by the formula (I) as a novel compound from a compound represented by the formula (II), a biochemical method such as microbial conversion or an enzyme produced by a living body is used. However, the synthetic chemical method including the step of protecting / deprotecting the aldehyde group at the 18-position (Journal of Organic
It can also be manufactured by Chemistry, 39 (16), 2474 (1974)). However, when the carbonyl group at the 9-position is reduced to a hydroxyl group having a natural configuration by a synthetic chemical method, the stereoselectivity of the reaction and the yield of the above-mentioned protection / deprotection step (particularly the acetalization step at the 18-position) are reduced. Considering the rate does not always give satisfactory results. Therefore, the present inventors have studied in detail the reduction of the 9-position by a biochemical method.

Regarding the biochemical method for reducing the carbonyl group at the 9-position of midecamycin A ₃ to a hydroxyl group having a natural configuration, the facts already reported by our research group (Journal of Antibiotics, 32 (7),
777 (1979)), the methodology itself for reducing the 9-position carbonyl group of a 16-membered ring macrolide compound having two alkyl groups in the mycalose moiety to the corresponding hydroxyl group by a biochemical method is easily analogized. To be done.

However, the radiation used in this method
Streptomyces mycarophassie which is one species of fungus
A mutant strain of SF-837, such as the SF2772 strain, is
Shin A ₁Producing Streptomyces mycarofaciens
Strain SF-837 strain is a non-producer strain of Midekamai related substances.
Therefore, when converting microorganisms, other than added substances and converted substances
It does not produce substances with antibacterial activity and is extremely excellent
It is a strain. Midekamai with such functions and features
Strains not producing syn-related substances are not limited to the SF2772 strain.
By treating the SF-837 strain with an artificial mutation by a known method.
In addition to the SF2772 strain, it is possible to create non-producing strains.

Incidentally, Streptomyces mycarofaciens SF-837 has been deposited as FERM P-262 at the Institute of Biotechnology, Institute of Industrial Science and Technology. Also, it has been deposited with the American Type Culture Collection as ATCC21454 and is ready for sale. In addition, the present microbial conversion method is not limited to being carried out by a midecamycin analogue non-producing strain, and can also be carried out by the same producing strain. Furthermore, the reduction of the carbonyl group at the 9-position of the 16-membered ring macrolide compound using a biochemical method can be carried out by other than a midecamycin-producing bacterium or its mutant strain, and in general, a platenomycin-producing bacterium (Journal of Antibiotics , 28 (10), 78
9 (1975)) and a maridomycin-producing bacterium (Agricultural and Biological Chemistry, 43 (6),
1331 (1979)), it is possible to use a 16-membered macrolide antibiotic-producing bacterium having a hydroxyl group at the 9-position, or a mutant strain thereof.

On the other hand, this microbial conversion method is characterized in that it does not require complicated enzyme system control such as addition of NADPH or strict adjustment of hydrogen ion concentration. The conversion using the microorganism is carried out not only in a known medium usually used for culturing actinomycetes but also in a medium having a dilute nutrient source,
A more efficient conversion and isolation of the conversion substance is realized. In addition,
Details regarding the present microbial conversion method are described in the examples described later.

When the carbonyl group at the 9-position of the compound represented by the formula (II) is reduced to a hydroxyl group by microbial conversion, when the compound represented by the formula (II) in which R ¹ represents a hydrogen atom is used as a substrate Depending on the converted strain, the 3-position hydroxyl group of the lactone ring may be acylated.

Next, using a 16-membered macrolide derivative having two alkyl groups in the mycarose moiety and a carbonyl group at the 9-position as a starting material, a method for producing a corresponding 9-position reductant will be specifically described. As described in. In order to carry out the present microorganism conversion method, first, the microorganism is seed-cultured in a liquid medium, and the 16-membered ring macrolide compound is added to the obtained seed. The macrolide compound (substrate) may be added at any time after the seed is grown, but it is preferably added 24 hours after the start of seed culture. The amount of the substrate added is usually 0.01 mg / ml to 2 mg / ml.

As the nutrient source of the medium, known ones conventionally used for culturing actinomycetes can be used. Preferably, glucose is used as the carbon source and polypeptone is used as the nitrogen source. That is, it is a feature of this method that efficient conversion and rapid isolation and purification of the conversion substance can be achieved by carrying out the present microbial conversion in a medium having a low solid content. In addition, if necessary, it is possible to add sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other inorganic salts capable of producing ions. In addition, organic substances and inorganic substances that help the growth of bacteria and promote the conversion of substrates can be added appropriately.

As the culture method, a culture method under aerobic conditions,
Particularly, the deep culture method is most suitable. Suitable temperature for culturing
The temperature is 24-32 ° C, but in most cases, the culture is performed at 28 ° C. Although the conversion efficiency of the substrate varies depending on the medium and culture conditions, the maximum accumulation of the conversion substance is usually reached in 1 to 72 hours in both shaking culture and tank culture. The culture is stopped when the accumulated amount of the conversion substance in the culture medium reaches the maximum, and the target substance is isolated and purified from the culture medium.

In order to obtain the desired conversion substance from the above culture solution, the cells are removed after culturing, and the culture solution from which solids have been removed is made alkaline, and then an organic solvent immiscible with water, such as butanol or ethyl acetate. The conversion substance is retained in the organic solvent layer by extraction with chloroform, chloroform, methylene chloride or the like. To further purify the conversion substance, chromatography using an adsorbent such as silica gel or alumina may be performed. For small-scale purification, preparative TL
It is effective to use C. By the way, the method for purifying the conversion substance is not limited to the method using the above-mentioned adsorbent, and a gel filtration method, a countercurrent distribution chromatography method, etc.
It is possible to apply a method that is usually used when purifying a natural or synthetic organic compound. Furthermore, the conversion substance obtained in the form of the free base may be converted into the corresponding salt by a conventional method using a pharmaceutically acceptable inorganic acid or organic acid. Therefore, salts thereof as well as free base converting substances are intended to be included within the scope of the present invention.

By the way, the compound represented by the formula (II) is represented by the formula (I) (wherein R ² is a hydrogen atom, R ³ is a hydroxyl group and R ⁴ is a hydrogen atom) prepared by microbial conversion. Known compounds (Fermentation and Industry, 37 (12), 1171 (1979) for selectively or non-selectively acylating the hydroxyl groups at the 9-position and / or the 2'-position for the compound or its salt. )) Or a known method of allylic rearrangement of the 9-position hydroxyl group to the 11-position or 13-position in the presence of a dilute acid (Chemical and Pharmaceutical Bretan, 18 (8), 1501 (1970), Meiji Confectionery Annual Report). , 12 , 85 (1972), Journal of Antibiotics, 35 (11), 1521 (1982)), etc., and it is possible to produce a novel useful substance based on the present invention. . As an example thereof, compound (1) (in formula (I), R ¹ is a propionyl group, R ² is a hydrogen atom, R ³ is a hydroxyl group, R ⁴ is a hydrogen atom, R ⁵ is a methyl group, R ⁶ is an isoamyl group. The 9-position hydroxyl group of the compound represented) was selectively acetylated using a known method (JP-A-48-13380) to synthesize compound (6).

The examples for obtaining the compound of the present invention and the physicochemical properties of the compound of the present invention are shown below. According to this example, a microorganism belonging to the genus Streptomyces is used.
Since the usefulness of the 9-position reduction method of the membered ring macrolide compound was shown, various methods for producing the substance by the same biochemical method can be devised based on this. On the other hand, it is also possible to devise various methods for producing the substances (1) to (6) and the like by a synthetic chemical method. Therefore, the present invention is not limited to the examples, and the compounds (1), (2) and (3) represented by the general formula (I) revealed by the present invention can be used not only in the modification means of the examples. Based on the properties of (4), (5) and (6), all methods of synthesizing, producing, extracting and purifying them by applying known means are included.

[0025]

【Example】

Example 1 Compound (1) (in the general formula (I), R ¹ is propioni
Group, R ² is a hydrogen atom, R ³ is a hydroxyl group, R ⁴ is a hydrogen atom, R ⁵ is
Manufacture of a methyl group and a compound in which R ⁶ is an isoamyl group)
As the method medium, one having a composition of glucose 2.0%, polypeptone 1.0%, dipotassium hydrogen phosphate 0.05%, magnesium sulfate heptahydrate 0.05%, sodium chloride 0.3% was used, and the pH was adjusted to 7.0 before sterilization. used. The above medium
Add three 500 ml Erlenmeyer flasks containing 80 ml at 120 ° C.
After sterilizing for 0 minutes, 1.6 ml of a frozen seed of Streptomyces mycarofaciens SF2772 strain was inoculated into each Erlenmeyer flask, and cultured with shaking at 28 ° C for 24 hours. to this,
In the general formula (II), 1.2 ml of a methanol solution containing 20 mg of a compound in which R ¹ is a propionyl group, R ⁵ is a methyl group, and R ⁶ is an isoamyl group is 0.4 ml per Erlenmeyer flask.
Each of them was added and cultured with shaking at 28 ° C. for 18 hours. After completing the culture, centrifuge the culture at 3000 rpm for 10 minutes to
0 ml was obtained and solids such as bacterial cells were removed. Water on solids 120
After adding ml and stirring, centrifugation was carried out, and the obtained washing solution was combined with the above-mentioned transparent culture solution. After adjusting this to pH 9, the conversion substance was extracted twice with 300 ml of ethyl acetate, the ethyl acetate layer was dried over anhydrous sodium sulfate, and then filtered. The residue obtained by concentrating the filtrate under reduced pressure was purified by preparative TLC (developing system: chloroform-methanol (10: 1)) to obtain 9.6 mg of crude compound (1). This was purified by Sephadex LH-20 column chromatography (20 ml, methanol) to obtain 7.3 mg of compound (1). Physicochemical properties of compound (1) (1) Color and shape: Colorless solid (2) Molecular formula: C ₄₄ H ₇₅ NO ₁₄ (3) Mass spectrum (EIMS): m / z 841 (M) ⁺ (4) Specific rotation Degree: [α] _D ²⁶ -49 ° (c0.7, CH ₃ OH) (5) Melting point: 98-100 ° C (6) ¹ H NMR spectrum (400MHz, CDCl ₃ ) δ
(ppm): 2.24 (br d, 2-H), 2.76 (dd, 2-H), 5.13 (br
d, 3-H), 3.26 (br d, 4-H), 3.57 (s, 4-OCH ₃ ), 3.87 (br
d, 5-H), 1.89 (m, 8-H), 4.07 (dd, 9-H), 5.62 (dd, 10
-H), 6.68 (dd, 11-H), 6.08 (br dd, 12-H), 5.79 (ddd,
13-H), 5.03 (ddq, 15-H), 1.26 (d, 16-H ₃ ), 2.85 (br dd,
17-H), 9.63 (s, 18-H), 0.99 (d, 19-H ₃ ), 2.51 (dq, 3-
OCOC H ₂ CH ₃ ), 2.64 (dq, 3-OCOC H ₂ CH ₃ ), 1.22 (t, 3-OCOCH
₂ C H ₃ ), 4.53 (d, 1'-H), 3.22 (br dd, 2'-H), 3.48 (t,
4'-H), 3.28 (dq, 5'-H), 1.15 (d, 6'-H ₃ ), 2.62 (s, 3'-
N (CH ₃ ) ₂ ), 4.89 (d, 1 "-H), 1.57 (dd, 2" -Hax), 2.22 (d,
2 "-Heq), 1.24 (s, 3" -CH ₃ ), 2.78 (d, 4 "-H), 4.39 (dq,
5 "-H), 1.23 (d, 6" -H ₃ ), 3.25 (s, 3 "-OCH ₃ ), 3.60 (dt,
4 "-OC H ₂ CH ₂ CH (CH ₃ ) ₂ ), 3.64 (dt, 4" -OC H ₂ CH ₂ CH (CH ₃ ) ₂ ),
1.69 (m, 4 "-OCH ₂ CH ₂ C H (CH ₃ ) ₂ ), 0.89 (d, 4" -OCH ₂ CH ₂ CH
(C H ₃ ) ₂ )

Example 2Compound (2) (in the general formula (I), R ¹ is propioni
Group, R ² is a hydrogen atom, R ³ is a hydroxyl group, R ⁴ is a hydrogen atom, R ⁵ is
Methyl group, a compound in which R ⁶ is a normal butyl group)
Manufacturing method 80 ml of the medium described in Example 1 was dispensed 500
 Sterilize 3 ml Erlenmeyer flasks at 120 ℃ for 30 minutes,
Streptomyces mycarofaciens S in a flask
Inoculate 1.6 ml of F2772 strain frozen seeds at 28 ℃
It was shake-cultured for 24 hours. In addition to this, the general formula (II) smell
, R¹Is a propionyl group, R^FiveIs a methyl group, R⁶Is normal
Compound of butyl group 20 mg in methanol 1.2
 28 ml by adding 0.4 ml per Erlenmeyer flask.
The culture was carried out at 20 ° C. for 20 hours with shaking. After completing the culture, add 3000
Centrifuge at rpm for 10 minutes to obtain 200 ml of a transparent culture broth.
Etc. of solids were removed. Add 180 ml of water to the solid content and stir
Centrifugation is then performed, and the resulting wash solution is added to the transparent culture solution.
I matched it. After adjusting this to pH 9, convert the conversion substance to ethyl acetate.
Extracted twice with 380 ml of ethyl acetate and the ethyl acetate layer is washed with anhydrous sodium sulfate.
It was filtered after drying with triumnium. Obtained by concentrating the filtrate under reduced pressure
The residue obtained is used for preparative TLC (developing system: chloroform-medium).
13.5 mg of crude compound (2) after purification with ethanol (10: 1)
Got This is Sephadex LH-20 column chromatograph
Raffy (20 ml, methanol) purified, compound
(2) Obtained 9.2 mg. Physicochemical properties of compound (2) (1) Color and shape: colorless solid (2) molecular formula: C₄₃H₇₃NO₁₄ (3) Mass spectrum (EIMS): m / z 827 (M)⁺ (4) Specific rotation: [α]_D ²⁷ -50 ° (c0.9, CH₃OH) (5) Melting point: 99-101 ℃ (6)¹1 H NMR spectrum (400MHz, CDCl₃) δ
(ppm): 2.24 (br d, 2-H), 2.76 (dd, 2-H), 5.13 (br
d, 3-H), 3.25 (br d, 4-H), 3.57 (s, 4-OCH₃), 3.87 (br
 d, 5-H), 1.89 (m, 8-H), 4.07 (dd, 9-H), 5.62 (dd, 10
-H), 6.67 (dd, 11-H), 6.08 (br dd, 12-H), 5.79 (ddd,
13-H), 5.03 (ddq, 15-H), 1.26 (d, 16-H₃), 2.85 (br dd,
 17-H), 9.63 (s, 18-H), 0.98 (d, 19-H₃), 2.51 (dq, 3-
OCOCH ₂CH₃), 2.64 (dq, 3-OCOCH ₂CH₃), 1.21 (t, 3-OCOCH
₂CH ₃ ), 4.53 (d, 1'-H), 3.22 (br dd, 2'-H), 3.48 (t,
4'-H), 3.28 (dq, 5'-H), 1.15 (d, 6'-H₃), 2.62 (s, 3'-
N (CH₃)₂), 4.89 (d, 1 "-H), 1.57 (dd, 2" -Hax), 2.23 (d,
 2 "-Heq), 1.24 (s, 3" -CH₃), 2.78 (d, 4 "-H), 4.39 (dq,
5 "-H), 1.22 (d, 6" -H₃), 3.25 (s, 3 "-OCH₃), 3.57 (dt,
4 "-OCH ₂CH₂CH₂CH₃), 3.62 (dt, 4 "-OCH ₂CH₂CH₂CH₃), 1.6
0 (m, 4 "-OCH₂CH ₂ CH₂CH ₃), 1.37 (m, 4 "-OCH₂CH₂CH ₂ CH₃),
 0.91 (t, 4 "-OCH₂CH₂CH₂CH ₃ )

Example 3Compound (3) (in the general formula (I), R ¹ is propioni
Group, R ² is a hydrogen atom, R ³ is a hydroxyl group, R ⁴ is a hydrogen atom, R ⁵ is
Methyl group, compound in which R ⁶ is represented by hexyl group) 80 ml of the medium described in Example 1 was dispensed 500
 Sterilize 3 ml Erlenmeyer flasks at 120 ℃ for 30 minutes,
Streptomyces mycarofaciens S in a flask
Inoculate 1.6 ml of F2772 strain frozen seeds at 28 ℃
It was shake-cultured for 24 hours. In addition to this, the general formula (II) smell
, R¹Is a propionyl group, R^FiveIs a methyl group, R⁶Is hexyl
Compound of the group represented by 20 mg in methanol solution 1.2 ml
Add 0.4 ml per Erlenmeyer flask to 28 ℃
The cells were shake-cultured for 19 hours. After culturing, add 3000 rp of culture
Centrifuge at m for 10 minutes to obtain 190 ml of transparent culture solution
Solids were removed. Add 160 ml of water to the solid content and stir
Centrifuge and combine the wash solution with the transparent culture solution.
I let it. After adjusting this to pH 9, the conversion substance was converted to ethyl acetate.
Extract twice with 350 ml and add ethyl acetate layer to anhydrous sodium sulfate.
It was filtered off after drying on a glass. Obtained by concentrating the filtrate under reduced pressure
TLC for preparative residue (development system: chloroform-methano
(10: 1)) to give 12.2 mg of crude compound (3).
It was Sephadex LH-20 column chromatograph
(20 ml, methanol) and then the compound (3)
8.3 mg was obtained. Physicochemical properties of compound (3) (1) Color and shape: colorless solid (2) molecular formula: C₄₅H₇₇NO₁₄ (3) Mass spectrum (EIMS): m / z 855 (M)⁺ (4) Specific rotation: [α]_D ^{twenty four} -50 ° (c0.8, CH₃OH) (5) Melting point: 96-102 ℃ (6)¹1 H NMR spectrum (400MHz, CDCl₃) δ
(ppm): 2.24 (br d, 2-H), 2.76 (dd, 2-H), 5.13 (br
d, 3-H), 3.25 (br d, 4-H), 3.57 (s, 4-OCH₃), 3.87 (br
 d, 5-H), 1.89 (m, 8-H), 4.07 (dd, 9-H), 5.62 (dd, 10
-H), 6.68 (dd, 11-H), 6.08 (br dd, 12-H), 5.79 (ddd,
13-H), 5.03 (ddq, 15-H), 1.26 (d, 16-H₃), 2.85 (br dd,
 17-H), 9.63 (s, 18-H), 0.99 (d, 19-H₃), 2.51 (dq, 3-
OCOCH ₂CH₃), 2.64 (dq, 3-OCOCH ₂CH₃), 1.22 (t, 3-OCOCH
₂CH ₃ ), 4.53 (d, 1'-H), 3.22 (br dd, 2'-H), 3.48 (t,
4'-H), 3.28 (dq, 5'-H), 1.15 (d, 6'-H₃), 2.63 (s, 3'-
N (CH₃)₂), 4.89 (d, 1 "-H), 1.57 (dd, 2" -Hax), 2.23 (d,
 2 "-Heq), 1.24 (s, 3" -CH₃), 2.78 (d, 4 "-H), 4.39 (dq,
5 "-H), 1.23 (d, 6" -H₃), 3.25 (s, 3 "-OCH₃), 3.55 (dt,
4 "-OCH ₂CH₂(CH₂)₃CH₃), 3.61 (dt, 4 "-OCH ₂CH₂(CH₂)₃C
H₃), 1.61 (m, 4 "-OCH₂CH ₂ (CH₂)₃CH₃), 0.88 (t, 4 "-OCH₂
CH₂(CH₂)₃CH ₃ )

Example 4 Compound (4) (in the general formula (I), R ¹ is propioni
Group, R ² is a hydrogen atom, R ³ is a hydroxyl group, R ⁴ is a hydrogen atom, R ⁵ is
Method for producing a compound in which a methyl group and R ⁶ are allyl groups) 80 ml of the medium described in Example 1 was dispensed 500
Sterilize 3 ml Erlenmeyer flasks at 120 ° C for 30 minutes, then add Streptomyces mycarofaciens S to each Erlenmeyer flask.
Inoculate 1.6 ml of F2772 strain frozen seeds at 28 ℃
It was shake-cultured for 24 hours. In this, in the general formula (II), R ¹ is a propionyl group, R ⁵ is a methyl group, R ⁶ is a compound represented by an allyl group 20 mg methanol solution 1.2 ml,
Add 0.4 ml / Erlenmeyer flask at 28 ℃
The culture was shaken for an hour. After the culture is completed, the culture solution is set at 3000 rpm for 1
Centrifugation was carried out for 0 minutes to obtain 180 ml of a transparent culture solution, and solids such as bacterial cells were removed. 120 ml of water was added to the solid content, and the mixture was stirred and then centrifuged, and the obtained washing liquid was combined with the transparent culture medium. After adjusting this to pH 9, the conversion material was converted to ethyl acetate 300
It was extracted twice with ml, the ethyl acetate layer was dried over anhydrous sodium sulfate and then filtered. The residue obtained by concentrating the filtrate under reduced pressure was used for preparative TLC (developing system: chloroform-methanol).
(10: 1)) to obtain 9.8 mg of crude compound (4).
This was purified by Sephadex LH-20 column chromatography (20 ml, methanol) to give compound (4) 6.4 m
got g. Physicochemical properties of compound (4) (1) Color and shape: colorless solid (2) Molecular formula: C ₄₂ H ₆₉ NO ₁₄ (3) Mass spectrum (EIMS): m / z 811 (M) ⁺ (4) Specific rotation Degree: [α] _D ²⁶ -55 ° (c0.6, CH ₃ OH) (5) Melting point: 101-106 ° C (6) ¹ H NMR spectrum (400MHz, CDCl ₃ ) δ
(ppm): 2.24 (br d, 2-H), 2.76 (dd, 2-H), 5.13 (br
d, 3-H), 3.26 (br d, 4-H), 3.57 (s, 4-OCH ₃ ), 3.87 (br
d, 5-H), 1.89 (m, 8-H), 4.07 (dd, 9-H), 5.62 (dd, 10
-H), 6.68 (dd, 11-H), 6.08 (br dd, 12-H), 5.79 (ddd,
13-H), 5.03 (ddq, 15-H), 1.26 (d, 16-H ₃ ), 2.84 (br dd,
17-H), 9.63 (s, 18-H), 0.99 (d, 19-H ₃ ), 2.51 (dq, 3-
OCOC H ₂ CH ₃ ), 2.64 (dq, 3-OCOC H ₂ CH ₃ ), 1.22 (t, 3-OCOCH
₂ C H ₃ ), 4.54 (d, 1'-H), 3.50 (t, 4'-H), 3.28 (dq, 5'-
H), 1.16 (d, 6' -H 3), 2.65 (s, 3'-N (CH 3) 2), 4.91 (d,
1 "-H), 1.57 (dd, 2" -Hax), 2.24 (d, 2 "-Heq), 1.24 (s,
3 "-CH ₃ ), 2.87 (d, 4" -H), 4.40 (dq, 5 "-H), 1.23 (d, 6"
-H ₃ ), 3.25 (s, 3 "-OCH ₃ ), 4.11 (br dd, 4" -OC H ₂ CH = C
H ₂ ), 4.19 (br, dd, 4 "-OC H ₂ CH = CH ₂ ), 5.95 (ddt, 4" -OCH
₂ C H = CH ₂ ), 5.17 (br d, 4 "-OCH ₂ CH = C H ₂ ), 5.23 (br d, 4"
-OCH ₂ CH = C H ₂ )

Example 5 Compound (5) (in the general formula (I), R ¹ is propioni
Group, R ² is a hydrogen atom, R ³ is a hydroxyl group, R ⁴ is a hydrogen atom, R ⁵ is
Method for producing compound in which methyl group and R ⁶ are represented by benzyl group) 100 ml of the medium described in this Example 1 was dispensed 50
Sterilize two 0 ml Erlenmeyer flasks at 120 ° C for 30 minutes, then add Streptomyces mycarofaciens to each Erlenmeyer flask.
Inoculate each with 2.0 ml of frozen seeds of SF2772 strain, 28 ℃
The cells were shake-cultured for 24 hours. In the formula (II), R ¹ is a propionyl group, R ⁵ is a methyl group, and R ⁶ is a benzyl group.
Add 0.5 ml per Erlenmeyer flask to 28 ℃
The cells were shake-cultured for 20 hours. After culturing, add 3000 rp of culture
Centrifugation was carried out at m for 10 minutes to obtain 160 ml of a transparent culture solution, and solids such as bacterial cells were removed. Water (160 ml) was added to the solid content, and the mixture was stirred and then centrifuged, and the obtained washing solution was combined with the transparent culture solution. After adjusting this to pH 9, the conversion substance was converted to ethyl acetate.
It was extracted twice with 320 ml, the ethyl acetate layer was dried over anhydrous sodium sulfate and then filtered. The residue obtained by concentrating the filtrate under reduced pressure was purified by preparative TLC (developing system: chloroform-methanol-concentrated aqueous ammonia (100: 10: 1)) to obtain 10.8 mg of a crude compound (5). This was purified by Sephadex LH-20 column chromatography (20 ml, methanol) to obtain 7.9 mg of compound (5). Physicochemical properties of compound (5) (1) Color and shape: colorless solid (2) Molecular formula: C ₄₆ H ₇₁ NO ₁₄ (3) Mass spectrum (SIMS): m / z 862 (M + H) ⁺ (4) Specific rotation: [α] _D ¹⁵ -52 ° (c0.8, CH ₃ OH) (5) Melting point: 112-116 ° C (6) ¹ H NMR spectrum (400MHz, CDCl ₃ ) δ
(ppm): 2.24 (br d, 2-H), 2.76 (dd, 2-H), 5.13 (br
d, 3-H), 3.25 (br d, 4-H), 3.57 (s, 4-OCH ₃ ), 3.87 (br
d, 5-H), 1.89 (m, 8-H), 4.07 (dd, 9-H), 5.62 (dd, 10
-H), 6.68 (dd, 11-H), 6.08 (br dd, 12-H), 5.79 (ddd,
13-H), 5.03 (ddq, 15-H), 1.26 (d, 16-H ₃ ), 2.31 (br dd,
17-H), 2.84 (br dd, 17-H), 9.63 (s, 18-H), 0.98 (d,
19-H ₃ ), 2.51 (dq, 3-OCOC H ₂ CH ₃ ), 2.64 (dq, 3-OCOC H ₂ CH
₃ ), 1.21 (t, 3-OCOCH ₂ C H ₃ ), 4.54 (d, 1'-H), 3.49 (t,
4'-H), 3.28 (dq, 5'-H), 1.15 (d, 6'-H ₃ ), 2.62 (s, 3'-
N (CH ₃ ) ₂ ), 4.90 (d, 1 "-H), 1.57 (dd, 2" -Hax), 2.22 (d,
2 "-Heq), 1.15 (s, 3" -CH ₃ ), 3.00 (d, 4 "-H), 4.45 (br d
q, 5 "-H), 1.23 (d, 6" -H 3), 3.25 (s, 3 "-OCH 3), 4.62
(d, 4 "-OC H ₂ C ₆ H ₅ ), 4.70 (d, 4" -OC H ₂ C ₆ H ₅ ), 7.3-7.4 (m,
4 "-OCH ₂ C ₆ H ₅ )

Example 6Compound (6) (in the general formula (I), R ¹ is propioni
Group, R ² is hydrogen atom, R ³ is acetoxy group, R ⁴ is hydrogen source
A compound in which R ⁵ is a methyl group and R ⁶ is an isoamyl group
Thing) To 13.0 mg of compound (1), add 0.64 ml of anhydrous toluene and dissolve.
Dissolve 5.6 μl of anhydrous pyridine and 4.8 μl of acetyl chloride.
After sequentially adding, the mixture was stirred at room temperature for 45 minutes. Vinegar in the reaction mixture
Add 3.2 ml of ethyl acidate and 8.0 μl of triethylamine and extract.
And wash the ethyl acetate layer twice with 3.2 ml of water.
After drying over sodium sulfate, it was filtered. Reduce the pressure of the filtrate
The residue obtained by concentration is used for preparative TLC (development system: chloro
Form-methanol (10: 1)) to give compound (6)
10.7 mg was obtained. Physicochemical properties of compound (6) (1) Color and shape: Colorless solid (2) Molecular formula: C₄₆H₇₇NO₁₅ (3) Mass spectrum (SIMS): m / z 884 (M + H)⁺ (4) Specific rotation: [α]_D ¹³ -56 ° (c1.0, CH₃OH) (5) Melting point: 104-108 ℃ (6)¹1 H NMR spectrum (400MHz, CDCl₃) δ
(ppm): 2.25 (br d, 2-H), 2.74 (dd, 2-H), 5.11 (br
d, 3-H), 3.24 (br d, 4-H), 3.57 (s, 4-OCH₃), 3.93 (br
 d, 5-H), 2.01 (m, 8-H), 5.08 (dd, 9-H), 2.02 (s, 9-O
COCH₃), 5.57 (dd, 10-H), 6.74 (dd, 11-H), 6.09 (br dd,
 12-H), 5.88 (ddd, 13-H), 2.17 (dt, 14-H), 4.98 (ddq,
 15-H), 1.26 (d, 16-H₃), 2.83 (br dd, 17-H), 9.64 (s,
 18-H), 0.95 (d, 19-H₃), 2.51 (dq, 3-OCOCH ₂CH₃), 2.6
7 (dq, 3-OCOCH ₂CH₃), 1.21 (t, 3-OCOCH₂CH ₃ ), 4.51 (d,
1'-H), 3.15 (dd, 2'-H), 2.39 (t, 3'-H), 3.46 (t, 4'-
H), 3.27 (dq, 5'-H), 1.14 (d, 6'-H₃), 2.56 (s, 3'-N (C
H₃)₂), 4.88 (d, 1 "-H), 1.56 (dd, 2" -Hax), 2.22 (d, 2 "
-Heq), 1.24 (s, 3 "-CH₃), 2.78 (d, 4 "-H), 4.42 (dq, 5"
-H), 1.23 (d, 6 "-H₃), 3.25 (s, 3 "-OCH₃), 3.59 (dt, 4 "
-OCH ₂CH₂CH (CH₃)₂), 3.64 (dt, 4 "-OCH ₂CH₂CH (CH₃)₂),
1.51 (m, 4 "-OCH₂CH ₂ CH (CH₃)₂), 1.69 (m, 4 "-OCH₂CH₂CH
(CH₃)₂), 0.89 (d, 4 "-OCH₂CH₂CH (CH ₃ ) ₂ )

[0031]

The compounds (1), (2), (3) and (5) represented by the general formula (I) obtained in the present invention are strong against clinically important Gram-positive bacteria and mycoplasma. Has antibacterial activity. In particular, the compounds (1) and (2) retain excellent antibacterial activity equivalent to or higher than that of MDM as shown in Table 1. Further, the compound (1) shows a practically effective anti-mycoplasma activity. The compound (6) also has an antibacterial activity which is approximately comparable to that of the compound (1).

The compounds (1), (2) and (3) represented by the general formula (I) obtained in the present invention have the property that the antibacterial activity lasts extremely long in rat plasma. . This stability is directly related to the fact that the hydroxyl groups of the mycalose moiety in the compound obtained in the present invention are not ester-bonded by an acyl group, but all hydroxyl groups are ether-bonded by an alkyl group.
FIG. 1 shows the relative antibacterial activity against M. luteus when compounds (1), (2), (3) and MOM were incubated in thawed rat plasma at 37 ° C. for 24 hours. 0 hours antibacterial activity in
Specific activity when 100%) is shown. When experimented with rat plasma, compounds (1), (2) and (3), which have two ether bonds in the mycarose moiety and a hydroxyl group at the 9-position, are less likely to be metabolized and released to the neutral sugar moiety. Since the hydroxyl group of OH does not appear, the degree of decrease in antibacterial activity in plasma is M
Clearly less than OM. This fact directly affects the excellent pharmacokinetics of the compound of the present invention described below. By the way, when MDM was tested under the same conditions, it showed almost no antibacterial activity after 24 hours. By the way, MOM
It has been reported that the pattern of metabolism of the mycarose moiety of erythromycin is generally similar in human and rat (Pharmaceutical Journal, 102 (8), 781 (1982)). Therefore, it is easily suggested that the compounds (1), (2) and (3) of the present invention have a long lasting strong antibacterial activity even in human blood.

Next, the compound (1) of the present invention was subjected to a pharmacokinetics test using mice. That is, 200 mg / kg of the compound (1) was orally administered to mice to obtain M. l as a test strain .
Serum concentration was measured by a bioassay method using uteus . As a result, the maximum serum concentration of compound (1) was 11.
3 μg / ml, MOM 6.2 μg / ml, MDM 5.2 μ
g / ml, Rokitamycin (RKM) (Journal of
Antibiotics, 34 (8), 1001 (1981)) 2.9 μg / ml
, Which is comparable to the typical new macrolide, clarithromycin (CAM). This maximum serum concentration in mice is the highest value in the 16-membered ring macrolide compound in which the 9-position is a free hydroxyl group, as far as the present inventors know, and has been pointed out in the past.
The problem of "low serum concentration", which is the weak point of 6-membered macrolide antibiotics, was fundamentally solved. Compound (1)
When a similar animal test was carried out on the compound (6) in which the 9-position hydroxyl group of (3) was acetylated, the maximum serum concentration (about 2 hours after administration) exceeded that of compound (1), and 6 hours after administration. However, its high serum concentration of 80% or more, which is the maximum value, continues, and it is attracting attention as a macrolide derivative that exhibits new pharmacokinetics.

Then, 200 mg / kg of the compound (1) was orally administered to the mice, and the recovery rate in urine within 24 hours after the administration was measured by the same bioassay method. As a result, compound (1)
The recovery rate in urine is 20%, which is slightly less than that of CAM, but compared with MOM, MDM or RKM, which has a recovery rate of less than 2%, the urinary recovery rate of the 16-membered macrolide derivative is outstanding. showed that. That is, extremely high stability (ability to retain antibacterial activity) of the compound (1) in the living body of mice was shown.

As described above, the compound (1) of the present invention not only sustains the antibacterial activity in rat plasma, but also exhibits particularly excellent pharmacokinetics in mouse animal experiments. Further, the compound (6) also showed an excellent duration of serum concentration in mouse animal experiments. These include that the side chain of the mycarose moiety is an alkyl group instead of an acyl group, and that the 9-position of the lactone ring is a hydroxyl group (or an acylated hydroxyl group) instead of a carbonyl group.
It depends largely on two structural reasons. It is easily inferred that the compound (2) and other related compounds will also show extremely excellent pharmacokinetics in the same manner as the compound (1).

[Table 1]

[Brief description of drawings]

FIG. 1 shows the relative antibacterial activity of Compounds (1), (2), (3) and MOM against M. luteus in thawed rat plasma after 24 hours (0 hour was defined as 100% for each compound). (Specific activity over time) is shown.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Tsuneo Ishizuka 760, Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Seika Co., Ltd., Pharmaceutical Research Laboratory (72) Nobue Kikuchi 760, Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Seika Co., Ltd., Pharmaceutical Research Laboratory (72) Inventor, Aiko Miyata, 760, Shimooka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Seika Co., Ltd., Pharmaceutical Research Laboratory (72) Inventor, Minako Araaki, Kohoku-ku, Yokohama-shi, Kanagawa 760 Okamachi Meiji Confectionery Co., Ltd., Pharmaceutical Research Laboratory (72) Inventor Osamu Hara 760 Shishioka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Meiji Confectionery Co., Ltd. Chemical Research Laboratory (72) Inventor Seiji Shibahara Yokohama, Kanagawa Prefecture Meiji Seika Co., Ltd. Pharmaceutical Research Institute, 760 Shimooka-cho, Kohoku-ku

Claims (7)

[Claims] 1. The following formula (I): [In the formula, R ¹ represents a hydrogen atom or a group of the formula COR ⁷ (provided that R ⁷ represents a linear alkyl group having 1 to 3 carbon atoms), and R ² represents a hydrogen atom or an optionally substituted hydroxyl group. R ³ represents a hydrogen atom or an optionally substituted hydroxyl group (provided that either one of R ² and R ³ represents a hydrogen atom), and R ⁴ represents a hydrogen atom or a group of the formula COR ⁷ (provided that R ⁷ Has the same meaning as above.)
R ⁵ represents a linear alkyl group having 1 to 4 carbon atoms, and R ⁶ represents an optionally substituted linear or branched alkyl group, alkenyl group or aralkyl group having 1 to 10 carbon atoms. ]
A compound represented by: or a pharmaceutically acceptable salt thereof. 2. In the formula (I) of claim 1, R ¹ is a propionyl group, R ² is a hydrogen atom, R ³ is a hydroxyl group, R ⁴ is a hydrogen atom, R ⁵ is a methyl group, and R ⁶ is an isoamyl group. A represented compound, or a pharmaceutically acceptable salt thereof. 3. In the formula (I) of claim 1, R ¹ is a propionyl group, R ² is a hydrogen atom, R ³ is a hydroxyl group, R ⁴ is a hydrogen atom, R ⁵ is a methyl group, and R ⁶ is a normal butyl group. A compound represented by: or a pharmaceutically acceptable salt thereof. 4. In the formula (I) of claim 1, R ¹ is a propionyl group, R ² is a hydrogen atom, R ³ is a hydroxyl group, R ⁴ is a hydrogen atom, R ⁵ is a methyl group, and R ⁶ is a hexyl group. The compound represented,
Or a pharmaceutically acceptable salt thereof. 5. In the formula (I) of claim 1, R ¹ is a propionyl group, R ² is a hydrogen atom, R ³ is a hydroxyl group, R ⁴ is a hydrogen atom, R ⁵ is a methyl group, and R ⁶ is an allyl group. A represented compound, or a pharmaceutically acceptable salt thereof. 6. In the formula (I) of claim 1, R ¹ is a propionyl group, R ² is a hydrogen atom, R ³ is a hydroxyl group, R ⁴ is a hydrogen atom, R ⁵ is a methyl group, and R ⁶ is a benzyl group. The compound represented,
Or a pharmaceutically acceptable salt thereof. 7. The formula (I) according to claim 1, wherein R ¹ is a propionyl group, R ² is a hydrogen atom, R ³ is an acetoxy group, R ⁴ is a hydrogen atom, R ⁵ is a methyl group, and R ⁶ is an isoamyl group. A compound represented by: or a pharmaceutically acceptable salt thereof.