| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | May 6, 1980 |
| PATENT TITLE |
Process for manufacturing tylosin derivatives |
| PATENT ABSTRACT |
New tylosin derivatives having at least one acyl group at the 3- and 4"-positions of tylosin, and the acid addition salts thereof, which inhibit the growth of various microorganisms including drug-resistant bacterial isolants and which produce high blood levels through oral administration are produced by a biochemical reaction using the microorganisms of the genus Streptomyces which are selected for their newly-found ability to acylate at least one of the 3- and 4"-positions of macrolide antibiotics; they are recovered from the reacted mixture by conventional methods for recovering macrolide antibiotics. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | February 27, 1978 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED | The Merck Index, pp. 1260 and 1261, 9th Edition |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A process for producing acyl derivatives of tylosin having the general formula I, ##STR4## wherein R.sub.1 is H, acetyl or propionyl group and R.sub.2 is H, n-butyryl or isovaleryl group, the case where both R.sub.1 and R.sub.2 are H being excluded, and the acid addition salts thereof, from a tylosin substrate having the general formula I', ##STR5## wherein both R.sub.1 ' and R.sub.2 ' are H, or one of them is H and the other is C.sub.2 -C.sub.5 acyl group, which comprises incubating said tylosin substrate with a microbial agent obtained from a microorganism belonging to the genus Streptomyces and capable of acylating at least one hydroxyl group at the 3- and 4"-positions of said substrate in the presence of C.sub.2 -C.sub.5 acyl donor, said microbial agent being selected from a growing cell in a culture medium, a resting cell, a dried cell, a cell homogenate of said microorganism, a supernatant solution and an enzymatic preparation obtained therefrom. 2. A process, as claimed in claim 1, for producing acyl derivatives of tylosin and the acid addition salts thereof wherein the hydroxyl group at the 3-position of tylosin or the 3-position of acyl derivatives of tylosin, in which R'.sub.2 at the 4"-position is hydrogen or acyl group, as shown in formula I', is acylated. 3. A process, as claimed in claim 2, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 3-position of tylosin, shown in formula I' is acetylation. 4. A process, as claimed in claim 2, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 3-position of the derivative of tylosin, in which R'.sub.2 at the 4"-position is n-butyryl group, as shown in formula I' is acetylation. 5. A process, as claimed in claim 2, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 3-position of the derivative of tylosin in which R'.sub.2 at the 4"-position is isovaleryl group, as shown in formula I' is acetylation. 6. A process, as claimed in claim 2, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 3-position of tylosin, as shown in formula I', is propionylation. 7. A process, as claimed in claim 2, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 3-position of the derivative of tylosin in which R'.sub.2 at the 4"-position is n-butyryl group, as shown in formula I' is propionylation. 8. A process, as claimed in claim 2, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 3-position of the derivative of tylosin in which R'.sub.2 at the 4"-position is isovaleryl group, as shown in formula I' is propionylation. 9. A process, as claimed in claim 1, for producing acyl derivatives of tylosin and the acid addition salts thereof wherein the hydroxyl group at the 4"-position of tylosin or the hydroxyl group at the 4"-position of the derivative of tylosin in which R'.sub.1 at the 3-position is hydrogen or acyl group, as shown in formula I' is acylated. 10. A process, as claimed in claim 9, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 4"-position of tylosin, as shown in formula I', is n-butyrylation. 11. A process, as claimed in claim 9, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 4"-position of tylosin, as shown in formula I', is isovalerylation. 12. A process, as claimed in claim 9, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 4"-position of the derivative of tylosin in which R'.sub.1 at the 3-position is acetyl group, as shown in formula I' is n-butyrylation. 13. A process, as claimed in claim 9, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 4"-position of the derivative of tylosin in which R'.sub.1 at the 3-position is acetyl group, as shown in formula I' is isovalerylation. 14. A process, as claimed in claim 9, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 4"-position of the derivative of tylosin in which R'.sub.1 at the 3-position is propionyl group, as shown in formula I' is n-butyrylation. 15. A process, as claimed in claim 9, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 4"-position of the derivative of tylosin in which R'.sub.1 at the 3-position is propionyl group, as shown in formula I' is isovalerylation. 16. A process, as claimed in claim 1, for producing acyl derivatives of tylosin and the acid addition salts thereof wherein the hydroxyl group at the 3- and 4"-positions of tylosin, as shown in formula I', are each acylated with different acyl groups. 17. A process, as claimed in claim 16, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 3-position of tylosin, shown in formula I', is acetylation and the acylation of the hydroxyl group at the 4"-position of the same is n-butyrylation. 18. A process, as claimed in claim 16, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 3-position of tylosin, shown in formula I', is acetylation and the acylation of the hydroxyl group at the 4"-position of the same is isovalerylation. 19. A process, as claimed in claim 16, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 3-position of tylosin, shown in formula I', is propionylation and the acylation of the hydroxyl group at the 4"-position of the same is n-butyrylation. 20. A process, as claimed in claim 16, for producing an acyl derivative of tylosin and the acid addition salts thereof wherein the acylation of the hydroxyl group at the 3-position of tylosin, shown in formula I', is propionylation and the acylation of the hydroxyl group at the 4"-position of the same is isovalerylation. 21. A process, as claimed in claim 1, for producing acyl derivatives of tylosin and the acid addition salts thereof wherein a microorganism belonging to the genus Streptomyces and capable of acylating at least one of the hydroxyl groups at the 3- and 4"-positions of tylosin or the derivatives of tylosin, shown in the formula I', which is selected from the group consisting of Streptomyces thermotolerans ATCC 11416, Streptomyces fungicidicus subsp. espinomyceticus ATCC 21574, Streptomyces mycarofaciens ATCC 21454 and Streptomyces hygroscopicus ATCC 21582 is used in the presence of C.sub.2 -C.sub.5 acyl donor. 22. A process, as claimed in claim 1, for producing acyl derivatives of tylosin and the acid addition salts thereof wherein acylation is carried out in the cultivated broth of a microorganism belonging to the genus Streptomyces and capable of acylating at least one of the hydroxyl group at the 3- and 4"-positions of tylosin or the derivatives of tylosin, as shown in the formula I', in the presence of at least one acyl donor selected from the group consisting of acetyl CoA, propionyl CoA, n-butyryl CoA, isovaleryl CoA and the precursors of acyl CoAs. 23. A process, as claimed in claim 22, for producing acyl derivatives of tylosin and the acid addition salts thereof wherein a quantity of the carbon source and the nitrogen source in the medium and a sort of substrate are adjusted depending on the acyl groups used for the acylation of hydroxyl groups at the 3- and 4"-positions of tylosin or derivatives of tylosin as shown in formula I'. 24. A process, as claimed in claim 1, for producing acyl derivatives of tylosin and the acid addition salts thereof wherein a microorganism is used in the form of a cell suspension in a liquid medium selected from the group consisting of water, buffer solution and broth filtrate, in the presence of at least one acyl donor selected from the group consisting of acetyl CoA, propionyl CoA, n-butyryl CoA, isovaleryl CoA and precursors of acyl CoAs, said suspension being prepared by cultivating a microorganism belonging to the genus Streptomyces which is capable of acylating at least one hydroxyl group at the 3- and 4"-positions of tylosin or the derivatives of tylosin, as shown in formula I', collecting the cells thus produced and suspending them in said liquid medium. 25. A process, as claimed in claim 1, for producing acyl derivatives of tylosin and the acid addition salts thereof wherein the acylation is enzymatically carried out in the presence of an enzyme preparation of a mycroorganism belonging to the genus Streptomyces and capable of acylating at least one hydroxyl group at the 3- and 4"-positions of tylosin or derivatives of tylosin shown in formula I' and in the presence of at least one acyl donor selected from the group consisting of acetyl CoA, propionyl CoA, n-butyryl CoA and isovaleryl CoA. |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION (1) Field of the Invention New tylosin derivatives having at least one acyl group at the 3- and 4"-positions of tylosin, and the acid addition salts thereof, which inhibit the growth of various microorganisms including drug-resistant bacterial isolants and which produce high blood levels through oral administration are produced by a biochemical reaction using the microorganisms of the genus Streptomyces which are selected for their newly-found ability to acylate the 3- and 4"-positions of 16-membered macrolide antibiotics; they are recovered from the reacted mixture by conventional methods for recovering macrolide antibiotics. The 3- and 4"-positions of 16-membered macrolide antibiotics in this specification mean the 3-position of 16-membered ring and the 4"-position of mycarose of the macrolide antibiotic, respectively. (2) Characteristics of the Invention Acylation of antibiotics is one of the practical methods for the production of new antibiotic species and their derivatives. Chemical synthesis is commonly employed for such acylation, but the acylation is liable to occur in a uniform fashion in many hydroxyl groups, whereby further reaction steps are required to obtain the desired product which is to be acylated at particular positions. Biochemical processes, on the other hand, facilitate selective acylation only at target positions due to the specificity of enzyme reactions and the yield is usually high. The present inventors have made a thorough investigation of one such biochemical conversion of macrolide antibiotics, especially of the acylation of macrolide antibiotics, and have found a number of microorganisms which are capable of specifically acylating the 3- and 4"-positions of 16-membered macrolide antibiotics. This is the first and novel finding of an enzyme reaction of the aforementioned type originating from microorganisms, and it is particularly applied to the production of new tylosin derivatives from tylosin. (3) Description of the Prior Art There has been no prior description regarding the tylosin compounds of the present invention, which are produced by the biochemical acylation of tylosin. The chemical synthesis of tylosin acetyl ester and of acetylated tylosin is described in Japanese Patent (Kokoku) Showa No. 36-22649 entitled "Method for Producing Tylosin" as described in Examples 4 and 5. However the products are obviously different from the tylosin derivatives of the present invention, for the reason presented in the Detailed Description hereinbelow. (4) Characteristics of the Process of the Invention. The acylation of antibiotics is one of the practical methods for the production of new antibiotic species and derivatives. Chemical synthesis is commonly employed for such acylation, but the acylation is liable to occur in a uniform fashion throughout the hydroxyl groups of the compound, whereby extra measures and steps are required, e.g., the proper protection of some hydroxyl groups and of other functional residues, to obtain the desired product which is acylated at one particular position. A biochemical process, on the other hand, facilitates selective acylation due to the specificity of enzyme reaction and the yield is usually high. The present inventors have made a thorough investigation of such biochemical transformation of macrolide antibiotics, and have found that a number of microorganisms are capable of specifically and simultaneously acylating the 3- and 4"-positions of 16-membered macrolide antibiotics. A particular feature of this process is that a variety of acylated derivatives can be produced by the proper combination of the two components of the acylation systems produced by one organism, namely, the substrate antibiotics and the acyl donors, which is of substantial industrial merit. There are two prior examples reported in connection with processes for the microbial acylation of 16-membered macrolide antibiotics, i.e., of spiramycin and YL-704. The process using spiramycin is presented in U.S. Pat. No. 2,943,024, entitled "Preparation of Spiramycin III", U.S. Pat. No. 2,943,025, entitled "Preparation of Spiramycin II", French Pat. No. 1,262,571, entitled "Transformation Biochimique de la Spiramycin I en Spiromycin II et III" and Japanese Patent (Kokoku) Showa No. 36-349, entitled "Process for Producing Spiramycin II, Spiramycin III and the Mixture Thereof." These patents practically deal with the same invention translated into different languages, describing processes which may be numerized as follows; 1. A spiramycin-producing organism, Streptomyces ambofaciens NRRL 2420, was cultivated in a culture medium to which spiramycin I, having a 3-hydroxyl group, was added and acylating agents, spiramcyin II, having a 3-acetyl group, and spiramycin III, having a 3-proionyl group, were produced. 2. The cells cultivated free of spiramycin and the acylating agents were suspended in a reaction medium, to which was added spiramycin I and the acylating agents. After incubation, spiramcyin II and III were produced. 3. In the fermentational production of spiramycins with the aforementioned organism, the addition of the acylating agents enhanced the production ratio of spiramycin II or III. The process according to the present invention is clearly distinguishable in principle from the prior art process for microbially acylating spiramycin for the following reasons. 1. The prior art process uses an organism which is a direct producer of spiramycin, and the process is closely linked to antibiotic fermentation. The four favorable strains used in the present invention are non-producers of the desired antibiotic species, and this indicates that the process of the present invention is an enzymatic process in principle. 2. The substrate antibiotic used in the prior art process is limited to spiramycin I which is a direct product of the organism of the prior art process, while the process of the present invention can employ most of the 16-membered macrolide antibiotics, indicating the non-specific nature of the reaction in terms of substrate specificity. 3. The prior art process can effect the conversion of only the 3-position of spiramycin I, while the process of the present invention can, if intended, carry cut the simultaneous conversion of the 3- and 4"-positions with one organism. A much wider variety of products can be obtained with the proper combination of the substrate, the acyl donor and the reaction conditions. Also of significance is the simplicity of the process in converting the two functional positions simultaneously with one cell system. With regard to YL-704, which is a family of leucomycins, Japanese Patent (Kokoku) Shown No. 49-13992 "Process for Producing an Antibiotic YL-704A.sub.1 " is presented. This patent indicates that an organism selected from the group consisting of Strectomyces eurocidicus NIHJ-267, Streptomyces albireticuli IFC-12737, Streptomyces kitasatcensis NRRL-2486 and Streptomyces sp. MCRL-0737 is cultivated in a medium containing "DHP compound", 4"-deacyl YL-704A.sub.1, or the DHF compound and L-leucine and after its cultivation, YL-704A.sub.1, which has an isovaleryl group at 4"-position of the DHP compound, is separated. The processes of the present invention are also obviously different from that of the last mentioned patent, relating to YL-704A.sub.1 production, in respects like those described in the case of the prior art spiramycin process, namely with respect to the organisms employed, the enzymatic nature of the processes of the present invention using enzyme system having no strict substrate specificity, the variety of products to be produced by the processes of the present invention and the fact that acylation at the 3- and 4"- positions can be effected simultaneously with one type of strain, all of which are of substantial industrial utility. OBJECTS OF THE INVENTION Accordingly, it is an object of this invention to provide a new compound having infection-control activities. Another object of this invention is to provide a new pharmaceutical, a veterinary drug and a feed-additive composition and the methods for their use. Still another object of this invention is to provide a new process for the acylation of at least one hydroxyl group at the 3- and 4"-positions of 16-membered macrolide antibiotics. SUMMARY OF THE INVENTION 1. The present invention provided a new compound of the formula: ##STR1## wherein R.sub.1 is hydrogen, acetyl or propionyl and R.sub.2 is hydrogen, n-butyryl or isovaleryl, the case where both R.sub.1 and R.sub.2 are hydrogen being excluded, and the nontoxic, pharmaceutically acceptable acid addition salts thereof, the new compounds being produced by acylating biochemically at least one of the 3- and 4"-hydroxyl groups of tylosin with the organism of the genus Streptomyces capable of exerting such acylation. The tylosin derivatives thus produced are recovered from the reacted mixture by conventional methods for recovering macrolide antibiotics. The present invention thus provides new tylosin derivatives which (a) inhibit the growth of various micoorganisms including drug-resistant bacterial isolants, and (b) produce high blood levels through oral and enteric administration. 2. Other embodiments of the present invention provide a pharmaceutical, a veterinary drug, and a feed-additive composition for administration to humans and animals comprising such a compound in an amount sufficient to control infectious diseases caused by microorganisms. 3. Still other embodiments of the present invention provide a method for chemotherapeutically controlling infectious diseases caused by microorganisms in humans and animals by the administration of such a compound to the humans and animals in a dosage sufficient to control the infections. 4. The present invention further provides a process for producing 16-membered macrolide antibiotic compounds having at least one acyl group at the 3- and 4"-position, e.g., the tylosin derivatives having the above formula, which comprises culturing the organisms of the genus Streptomyces possessing the necessary acylation activity in a medium conventionally employed for culturing the organisms of this genus, performing such acylation reaction in a combination of the enzymatic sources, e.g., growing and non-growing cells of the cultured organism or the enzymatic preparations therefrom, the antibiotic substrate of 16-membered macrolide antibiotics having at least one hydroxyl group at the 3- and 4"-positions, e.g. tylosin, and the acyl-group donor of C.sub.2 -C.sub.5 acyl compounds, e.g., acyl CoAs and their metabolic precursors, and recovering the acylated products from the reaction mixture by means of the conventionally employed method for recovering macrolide antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS FIGS. 1 and 2 show the ultraviolet absorption spectra of 4"-n-butyryltylosin and 3-acetyl-4"-isovaleryltylosin, respectively. FIGS. 3,4,5,6,7,8,9 and 10 show the infrared absorption spectra of 3-acetyltylosin, 3-acetyl-4"-n-butyryltylosin, 3-acetyl-4"-isovaleryltylosin, 3-propionyltylosin, 3-propionyl-4"-n-butyryltylosin, 3-propionyl-4"-isovaleryltylosin, 4"-n-butyryltylosin and 4"-isovaleryltylosin, respectively. FIGS. 11,12,13,14,15,16,17 and 18 are the NMR spectra (CDCl.sub.3) of the aformentioned, respective compounds presented in the same order as the infrared absorption spectra. FIGS. 19,20 and 21 are the chemical ionization mass spectra for 3-acetyltylosin, 3-propionyltylosin and 3-acetyl-4"-isovaleryltylosin, respectively. FIGS. 22 and 23 are C.sup.13 -NMR spectra (25.2 MHz) for 3-acetyltylosin and 3-acetyl-4"-isovaleryltylosin FIG. 24 is a thin layer chromatogram of the tylosin derivatives of this invention. DETAILED DESCRIPTION OF THE INVENTION This invention relates to new derivatives of macrolide antibiotics and their production. More particularly, it relates to new acylated derivatives of tylosin and to processes for the preparation thereof by the biochemical acylation of tylosin or its derivatives. In further detail, it relates to new derivatives of tylosin as shown in the following general formula I: ##STR2## wherein R.sub.1 is hydrogen, an acetyl group or a propionyl group and R.sub.2 is hydrogen, an n-butyryl group or an isovaleryl group, the case where both R.sub.1 and R.sub.2 are hydrogen being excluded, which are produced by acylating tylosin or its derivatives of the formula I': ##STR3## wherein R.sub.1 ' and R.sub.2 ' are both hydrogen or wherein either R.sub.1 ' or R.sub.2 ' is hydrogen and the other is an acyl group with a 2-5 carbon number, at least one of the hydroxyl at the 3- and 4"-position by the use of growing or resting cells, cell preparations or enzyme preparations of the microorganisms belonging to the genus Streptomyces which are capable of producing the enzyme for this acylation. The acylation of antibiotics is one of the practical methods for the production of new antibiotic species and derivatives. Chemical processes are commonly employed in such acylation. However, the acylation is liable to occur in a uniform fashion, making necessary further steps, e.g., selective protection of functional residues, to obtain the desired product which is acylated at particular positions. Biochemical processes, on the other hand, cause selective acylation only at a target position due to the specificity of enzymatic reactions and the yield is usually high. The present inventors have made a thorough investigation of one such biochemical reaction, especially of the acylation of macrolide antibiotics, and have found that a number of microorganisms can specifically acylate both the 3-position and 4"-position of 16-membered macrolide antibiotics. The finding by the present inventors of the existence of this type of enzyme derived from microorganisms and enzymatic reactions is novel and the first of its kind. These reactions were studied further to establish biochemical conversion of macrolide antibiotics industrially and was particularly applied to the production of new tylosin derivatives from tylosin. The newly developed tylosin derivatives include 3-acetyltylosin, 3-acetyl-4"-n-butyryltylosin, 3-acetyl-4"-isovaleryltylosin, 3-propionyltylosin, 3-propionyl-4"-n-butyryltylosin, 3-propionyl-4"-isovaleryltylosin, 4"-n-butyryltylosin and 4"-isovaleryltylosin. |
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