KR101703137B1 - Glucose-attached aminoglycoside compounds, manufacturing method thereof and pharmaceutical compositions containing them as active ingredients - Google Patents

Abstract

상기 식에서, R₁, R₂, R₃, R₄, R₅, R₆, 및 X-Y의 정의는 명세서에서 정의한 바와 같다.The present invention relates to a sugar-added aminoglycoside compound represented by the following formula (1), which can be usefully used for the prevention or treatment of genetic diseases, an enzymatic process for producing these compounds, and a pharmaceutical composition containing the same as an active ingredient.
[Chemical Formula 1]

The definitions of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , and XY are as defined in the specification.

Description

TECHNICAL FIELD The present invention relates to aminoglycoside compounds having an amino group, a sugar group-attached amino glycoside compound, a preparation method thereof, and a pharmaceutical composition containing the same as an active ingredient.

The present invention relates to a sugar-added aminoglycoside compound which can be usefully used for the prevention or treatment of genetic diseases, an enzymatic process for producing these compounds, and a pharmaceutical composition containing the same as an active ingredient.

In the case of a number of human genetic diseases, inherited amino acid-encoding codons are replaced by three stop codons (UAA, UAG or UGA) resulting in non-sense mutation, resulting in genetic disorders. This non-sense mutation leads to premature termination of translation, resulting in fragmented inactive protein biosynthesis. To date, hundreds of the above-mentioned nonsense mutations have been known, and several of them have been known as cystic fibrosis, duchenne muscular dystrophy (DMD), ataxia telangiectasia, , Hurler's syndrome, hemophilia A, hemophilia B, and tay-sachs (Non-Patent Document 1 , 2). Although there is no effective treatment for these incurable diseases until now, gene therapy has recently been highlighted as a potential solution, but many problems remain to be solved before human application and treatment.

On the other hand, it has been shown that aminoglycosides have the activity of inducing ribosomes in vivo to read-through a nonsense mutation, thereby resulting in full-length protein that is not fragmented For the past several years, the potential of aminoglycoside as a therapeutic agent for genetic diseases has been reported (see Non-Patent Document 3-6).

Aminoglycosides are known to be broadly applicable anti-bacterial agents commonly used for the treatment of infectious diseases. The aminoglycoside-containing antimicrobials containing 2-deoxystreptamine shown in FIG. 1 are antimicrobial agents selectively acting on ribosomes of prokaryotes. Specifically, the antimicrobial agents for 2-deoxystreptamine- Site to inhibit protein translation and hinder translation fidelity (see Non-Patent Document 7-12). Among these aminoglycoside-based compounds, natural antibacterial agents such as kanamycin, tobramycin, gentamicin and related biosynthetic intermediates such as sisomicin, Amikacin, isepamicin, and netilmicin, a second-generation aminoglycoside family using a semi-synthetic process, are currently in clinical use as antibacterial agents.

On the other hand, termination of protein biosynthesis starts from the presence of termination codon in mRNA, and release factor protein is known to be involved in this process. The efficiency of termination of translation is generally very high, and the probability of incorrect insertion of the amino acid corresponding to the termination codon appears at a frequency as low as one in 10,000. It is presumed that the enhancement of the inhibitory activity of the termination of protein biosynthesis by aminoglycosides in eukaryotic cells occurs in the same mechanism as the activity of aminoglycosides which interfere with translation fidelity during protein biosynthesis. That is, the binding of aminoglycoside to the A site in the ribosome is predicted to induce a structural change in the binding, thereby stabilizing the mRNA-tRNA complex in place of the insertion of the release factor (see Non-Patent Document 13).

It was reported for the first time in 1985 that aminoglycosides inhibit early termination of translation due to nonsense mutations in mammalian cells and the potential for use as a therapeutic agent for genetic diseases has also been mentioned for the first time (see Non-Patent Document 3) . Cystic fibrosis arises from point mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The disease is the most frequent degenerative genetic disease in the Caucasians, occurring at a frequency of 1/2500 of the neonate. More than 1,000 mutants in the CFTR gene have been identified to date (see Non-Patent Document 5).

In the first study on the inhibition of CFTR non-specific mutation by aminoglycoside, gentamicin and its biosynthetic intermediates, geneticin or G-418, were activated. In experiments using bronchial epithelial cells, full-length CFTR (Refer to non-patent documents 14 and 15). Human-derived CFTR (-) transgenic mice were transfected with human-derived CFTR-G542X gene, and human-derived CFTR was expressed in gentamicin-treated experimental animals. On the other hand, In the case of tobramycin as a side, it has been reported that the non-sense read-through induction activity is weak (see Non-Patent Document 16). Above all, in a double-blind clinical trial comparing with placebo, gentamicin inhibited a nonsense mutation in the patient. In addition, gentamicin treatment resulted in nasal mucosa in 19 patients with CFTR termination mutation It was confirmed that the transverse conduction efficiency was improved (see Non-Patent Document 17). In addition, 6 types of genetic disease cell lines and experimental animal models have been conducted to report the potential of aminoglycoside as a therapeutic agent for genetic diseases (see Non-Patent Documents 18 to 23).

However, in the application of aminoglycoside as a therapeutic agent for genetic diseases, one of the main limitations is high toxicity to mammalian cells, in particular, nephrotoxicity and ototoxicity. The cause of such toxicity is presumed to be due to the combined cause of various factors such as interaction with phospholipids, inhibition of phospholipase, formation of free radicals See Patent Documents 24 and 25). Despite the sufficient selectivity for ribosomes of prokaryotic cells such as bacteria, most aminoglycosides still bind to the A site in the ribosome of eukaryotic cells. The effect of inhibiting translation in mammalian cells is also one of the major causes of the high toxicity of aminoglycosides. Another possibility associated with this toxicity is known to be binding to the 12S rRNA A-site in mitochondria (see non-patent reference 26).

A variety of studies have been conducted to develop a method to mitigate the toxicity associated with such aminoglycoside treatment. The use of antioxidants for the removal of free radicals, and the use of dextomycin to reduce the interaction between aminoglycosides and phospholipids daptomycin) and poly-L-aspartate have been reported (see Non-Patent Document 27). In addition, there have been attempts to reduce the toxicity of aminoglycoside treatment by changing the prescription schedule or the formulation treatment (see Non-Patent Documents 28 and 29).

Despite the diverse research efforts to reduce aminoglycoside toxicity, none of the results, other than the change in prescription schedule, have been improved by standardized clinical practice for genetic disease treatment. For example, gentamicin treatment below the toxic dose in clinical trials has been shown to reduce the activity of intact toxins due to aminoglycosides (see Non-Patent Document 30). On the other hand, in the case of geneticin, the best non-toxic activity was observed in in vitro test, but the fatal toxicity which appeared even at a very low concentration made it impossible to use it as a medicine (refer to Non-Patent Document 6). That is, the half-life lethal dose (LD50) of geneticin to human fibroblasts is 0.04 mg / ml, which is significantly different from the range of 2.5-5.0 mg / ml, such as gentamicin, neomycin or kanamycin 31). Thus, to date, the only aminoglycosides studied in various experimental animal models and clinical trial stages are gentamicin alone. In other words, according to a study through some cell lines, amikacin and paromomycin have been reported as substitute compounds for nonsense read-through inducer activity of gentamicin, Clinical trial data have not been reported so far (see Non-Patent Documents 32 and 33).

In the case of most non-insect intoxication activity, which has been recognized as a potential genetic disease treatment until recently, only studies on aminoglycoside products in clinical use have been conducted. In order to optimize the activity of insect intoxication by modifying the structure of the compound (See Non-Patent Document 6).

The Baasov research team at the Technion Research & Development Foundation in Israel has teamed up with a variety of new aminoglycoside compounds with pseudo-trosaccharides aminoglycosides as chemical structures, The inventors of the present invention have invented novel aminoglycosides having low cytotoxicity and selectivity for mammalian cells and have patented them as compounds usable for the treatment of genetic diseases (Refer to Patent Document 1).

The University of Tokyo and the Microbial Chemistry Research Foundation of Japan jointly patented the induction activity of arbekacin, which was developed as a semi-synthetic preparation of kanamycin natural aminoglycoside, at the laboratory level Patent Document 2).

Except for the two patents mentioned above, reports on aminoglycoside-modified compounds through organic synthesis and biosynthetic production methods that have been reported up to now are all related to the invention of novel antibacterial agents and antibiotic advantages (See Patent Documents 3 and 4).

The properties required for an effective intrinsic induction agent are those that minimize oral administration and thus the effects on intestinal bacteria. That is, it is not preferable that the antifungal activity of the Nontrivial induction drug, especially the gastrointestinal biota, which is the intestinal bacterial bacterium, is affected. From such a viewpoint, besides the above-mentioned problems of aminoglycosides, most of the commercially available aminoglycosides in clinical use have other problems in clinical application even though they have the activity of inducing insensitivity.

Recently, US biopharmaceutical company PTC Therapeutics has proposed a new method for overcoming the problems in the clinical application of aminoglycosides having the above-mentioned activity of inducing insensitivity, And found ataluren (PTC 124) as a nonaminoglycoside compound (see Patent Document 5). This compound is structurally independent of aminoglycoside, has no anti-bacterial activity, and has no toxicity. The US FDA identified PTC124 as a candidate for a new drug for cystic fibrosis and dyshiene-type muscular dystrophy, which is a rare disease, and has entered the clinical phase by fast-track, and is currently in Phase III clinical stage by 2014 (see Non-Patent Document 34 ).

As described above, all of the above-mentioned documents suggest various researches and approaches to novel aminoglycoside compounds which have a nonsense intrinsic induction activity and minimize the low activity and antibacterial activity on mammalian cells.

Accordingly, the present inventors have developed novel aminoglycoside compounds, overcoming the problems pointed out in the clinical application of existing aminoglycosides, and using them as therapeutic agents for genetic diseases.

1) Baasov, T., et al., Aminoglycosides and uses thereof in treating genetic disorders. 2011. US20130237489A1. 2) Matsuda, R., et al., Read through inducer, and therapeutic agent for nonsense-mutation-type genetic disorders. 2011. EP2535051A4. 3) Yoon, Y.J., et al., A new kanamycin compound, kanamycin-producing streptomyces species bacterium, and method of producing kanamycin. 2010. US20130096078A1. 4) Aggen, J. B., et al., Antibacterial aminoglycoside analogs. 2010. US8383596B2. 5) Almstead, N. G., et al., Crystalline forms of 3- [5- (2-fluorophenyl) - [1,2,4] oxadiazol-3-yl] -benzoic acid. 2011. US7863456B2.

1) Park, S. R., et al., 2-Deoxystreptamine-containing aminoglycoside antibiotics: Recent advances in the characterization and manipulation of their biosynthetic pathways. Nat. Prod. Rep., 2013. 30 (1): p. 11-20. 2) Atkinson, J. and R. Martin, Mutations to nonsense codons in human genetic diseases: implications for gene therapy by nonsense suppressor tRNAs. Nucleic Acids Res., 1994. 22 (8): p. 1327-34. 3) Burke, J. F. and A.E. Mogg, Suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside antibiotics G-418 and paromomycin. Nucleic Acids Res., 1985, 13 (17): p. 6265-72. 4) Kaufman, RJ, Correction of genetic disease by making sense of nonsense. J. Clin. Invest., 1999. 104 (4): p. 367-8. 5) Kerem, E., Pharmacologic therapy for stop mutations: how much CFTR activity is enough? Curr. Opin. Pulm. Med., 2004. 10 (6): p. 547-52. 6) Manuvakhova, M., K. Keeling, and D.M. Bedwell, Aminoglycoside antibiotics mediate context-dependent suppression of termination codons in a mammalian translation system. Rna, 2000, 6 (7): p. 1044-55. 7) Davis, B. D., Mechanism of bactericidal action of aminoglycosides. Microbiol. Rev., 1987. 51 (3): p. 341-50. 8) Jana, S. and J.K. Deb, Molecular understanding of aminoglycoside action and resistance. Appl. Microbiol. Biotechnol., 2006. 70 (2): p. 140-50. 9) Fujisawa, K., T. Hoshiya, and H. Kawaguchi, Aminoglycoside antibiotics. VII. Acute toxicity of aminoglycoside antibiotics. J. Antibiot. (Tokyo), 1974. 27 (9): p. 677-81. 10) Magnet, S. and J.S. Blanchard, Molecular insights into aminoglycoside action and resistance. Chem. Rev., 2005. 105 (2): p. 477-98. 11) Ogle, J. M., et al., Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science, 2001. 292 (5518): p. 897-902. 12) Vicens, Q. and E. Westhof, Molecular recognition of aminoglycoside antibiotics by ribosomal RNA and resistance enzymes: an analysis of x-ray crystal structures. Biopolymers, 2003. 70 (1): p. 42-57. 13) Howard, M.T., et al., Sequence specificity of aminoglycoside-induced stop condon readthrough: potential implications for treatment of Duchenne muscular dystrophy. Ann. Neurol., 2000. 48 (2): p. 164-9. 14) Bedwell, D. M., et al., Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nat. Med., 1997. 3 (11): p. 1280-4. 15) Howard, M., R.A. Frizzell, and D.M. Bedwell, Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations. Nat. Med., 1996. 2 (4): p. 467-9. 16) Du, M., et al., Aminoglycoside suppression of a premature stop mutation in a Cftr - / - mouse carrying a human CFTR-G542X transgene. J. Mol. Med., 2002. 80 (9): p. 595-604. 17) Wilschanski, M., et al., Gentamicin-induced correction of CFTR function in patients with cystic fibrosis and CFTR stop mutations. N. Engl. J. Med., 2003. 349 (15): p. 1433-41. 18) Barton-Davis, E.R., et al., Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice. J. Clin. Invest., 1999. 104 (4): p. 375-81. 19) Keeling, K.M., et al., Gentamicin-mediated suppression of Hurler syndrome stop mutations restores a low level of alpha-L-iduronidase activity and reduces lysosomal glycosaminoglycan accumulation. Hum. Mol. Genet., 2001. 10 (3): p. 291-9. 20) Sangkuhl, K., et al., Aminoglycoside-mediated rescue of a disease-causing nonsense mutation in the V2 vasopressin receptor gene in vitro and in vivo. Hum. Mol. Genet., 2004. 13 (9): p. 893-903. 21) Helip-Wooley, A., et al., Expression of CTNS alleles: subcellular localization and aminoglycoside correction in vitro. Mol. Genet. Metab., 2002. 75 (2): p. 128-33. 22) Grayson, C., et al., In vitro analysis of aminoglycoside therapy for the Arg120stop nonsense mutation in RP2 patients. J. Med. Genet., 2002. 39 (1): p. 62-7. 23) Xi, B., F. Guan, and D.S. Lawrence, Enhanced production of functional proteins from defective genes. J. Am. Chem. Soc., 2004. 126 (18): p. 5660-1. 24) Forge, A. and J. Schacht, Aminoglycoside antibiotics. Audiol. Neurootol., 2000. 5 (1): p. 3-22. 25) Nagai, J. and M. Takano, Molecular aspects of renal handling of aminoglycosides and strategies for preventing nephrotoxicity. Drug Metab. Pharmacokinet., 2004. 19 (3): p. 159-70. 26) Vicens, Q. and E. Westhof, RNA as a drug target: the case of aminoglycosides. Chembiochem, 2003. 4 (10): p. 1018-23. 27) Keeling, K.M. and D.M. Bedwell, Pharmacological suppression of premature stop mutations that cause genetic diseases. Curr. Pharmacogenomics, 2005. 3 (4): p. 259-269. 28) Kawamoto, K., et al., Antioxidant gene therapy can protect hearing and hair cells from ototoxicity. Mol. Ther., 2004. 9 (2): p. 173-81. 29) Bartal, C., et al., Pharmacokinetic dosing of aminoglycosides: a controlled trial. Am. J. Med., 2003 114 (3): p. 194-8. 30) Beauchamp, D. and G. Labrecque, Aminoglycoside nephrotoxicity: do time and frequency of administration matter? Curr. Opin. Crit. Care, 2001. 7 (6): p. 401-8. 31) Karpati, G. and H. Lochmuller, When running a stop sign may be a good thing. Ann. Neurol., 2001. 49 (6): p. 693-4. 32) Chernikov, V. G., et al., Comparison of cytotoxicity of aminoglycoside antibiotics using a panel cellular biotest system. Bull. Exp. Biol. Med., 2003. 135 (1): p. 103-5. 33) Keeling, K.M. and D.M. Bedwell, Clinically relevant aminoglycosides can suppress disease-associated premature stop mutations in the IDUA and P53 cDNAs in a mammalian translation system. J. Mol. Med., 2002. 80 (6): p. 367-76. 34) Lane, M.A. and S.J. Doe, A new era in the treatment of cystic fibrosis. Clin. Med., 2014. 14 (1): p. 76-8.

An object of the present invention is to provide a sugar-added aminoglycoside compound exhibiting low cytotoxicity and low antibacterial activity while exhibiting a nonsense intrinsic inducing activity, a process for producing the same, and a pharmaceutical composition containing the same.

In order to achieve the above object, the present invention provides a compound represented by the following formula (1), or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

In this formula,

And R ₁ is NH ₂ or OH,

R ₂ is H or OH,

R < ₃ > is H or OH,

And R ₄ is NH ₂ or OH,

R ₅ is H or CH _3,

이고,R ₆ is H, CH ₂ CH ₃ or

ego,

X-Y is CH-CH or C = C.

The present invention also relates to a method for the treatment of cystic fibrosis, duchenne muscular dystrophy (DMD), capillary vasodilator ataxia syndrome (CNS) comprising a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof a prophylactic or therapeutic agent selected from the group consisting of ataxia telangiectasia, hurler syndrome, hemophilia A, hemophilia B, and tay-sachs. A pharmaceutical composition is provided.

The present invention also relates to a method for the production of a compound of the formula (I), or a pharmaceutically acceptable salt thereof, which is useful for controlling the cystic fibrosis transmembrane conductance regulator (CFTR) or dystrophin Wherein the pharmaceutical composition has an intrinsic induction activity.

The present invention also relates to a compound represented by the formula (1), or a pharmaceutically acceptable salt thereof; And a carrier.

Also, the present invention provides a method for producing an antibiotic, comprising the steps of: 1) acid hydrolyzing aminoglycoside antibiotics containing 2-deoxystreptamine to obtain pseudodisaccharides; And 2) reacting the trisaccharide obtained in the above step 1) with a glycosidase to obtain an enzyme reaction product.

The compound of formula (I) according to the present invention, or a pharmaceutically acceptable salt thereof, exhibits low cytotoxicity and low antibacterial activity, and exhibits an enhanced effect of inducing no effect on gingham toxin, . ≪ / RTI >

FIG. 1 shows the results of acid hydrolysis of existing aminoglycoside antibiotics and determination of 2-deoxystreptamine (2) through the reaction of recombinant glycosaminoglycan KanM2 using pseudodisaccharides isolated and purified from the hydrolysis products as substrates -deoxystreptamine), which is a biosynthesis method for producing a sugar-added aminoglycoside compound.
FIG. 2 is an image showing the results of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel) for the separation and purification of KanM2 recombinant herbal preparations prepared through expression of kanM2 gene encoding Escherichia coli, which encodes kanamycin biosynthesis-associated glycosyltransferase (A): Result of SDS-PAGE of coenzyme KanM2 expressed in E. coli (B): Purified recombinant glycosyltransferase enzyme KanM2 (about 47 kDa), which was obtained by separation and purification of coenzyme with Talon resin SDS-PAGE results).
Fig. 3 shows the mass spectra of each of the sugar-added aminoglycoside compounds of the present invention prepared through the enzymatic reaction.
FIG. 4 is a graph showing cytotoxicity results of a sugar aminoglycoside compound of the present invention and a conventional human aminoglycoside antibiotic cell line.
Fig. 5 is a graph showing the activity of the sugar aminoglycoside compound of the present invention and gentamycin to induce nonsense intoxication.

Hereinafter, the present invention will be described in detail.

The present invention provides a compound represented by the following formula (1), or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

In this formula,

And R ₁ is NH ₂ or OH,

R ₂ is H or OH,

R < ₃ > is H or OH,

And R ₄ is NH ₂ or OH,

R ₅ is H or CH _3,

이고,R ₆ is H, CH ₂ CH ₃ or

ego,

XY is CH-CH or C = C.

Representative examples of the compound represented by the above formula (1) are as follows:

1) 3'-Dioxy-3 " -diamino-3 " -hydroxycamanamycin C,

2) 1-N-3-amino-2 (S) -hydroxypropionyl-kanamycin D,

3) 6'-Methyl-3 " -diamino-3 " -hydroxycamanamycin C,

4) 3 ', 4'-dideoxy-4', 5'-dihydro-3 "-diamino-3" -hydroxycainamycin B, and

5) 1-N-ethyl-3 ', 4'-dideoxy-4', 5'-dihydro-3 "-diamino-3" -hydroxycinnamicin B.

The compound of formula (I) according to the present invention can be used in the form of a pharmaceutically acceptable salt. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. As the free acid, inorganic acid and organic acid can be used. As the inorganic acid, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid and the like can be used. As the organic acid, citric acid, acetic acid, lactic acid, maleic acid, pumaric acid, gluconic acid, Succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, aspartic acid and the like can be used. Sulfuric acid may be preferably used.

In addition, the compound of formula (I) according to the present invention includes not only pharmaceutically acceptable salts, but also all salts, hydrates and solvates which can be prepared by conventional methods.

Further, the compound of formula (I) according to the present invention may be prepared in a crystalline form or an amorphous form, and when the compound of formula (1) is prepared in a crystalline form, it may optionally be hydrated or solvated.

The present invention also provides a pharmaceutical composition for preventing or treating a genetic disease caused by a nonsense mutation, which comprises a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof. Preferably, the genetic disease is selected from the group consisting of human degenerative genetic diseases, more preferably cystic fibrosis, Duchenne muscular dystrophy, capillary vasodilator ataxia syndrome, Hodgler's syndrome, A-type hemophilia, ≪ / RTI >

The term "prophylactic" as used herein refers to any action that inhibits or delays a genetic disease caused by a nonsense mutation by the administration of a pharmaceutical composition comprising the compound of formula 1 or a pharmaceutically acceptable salt thereof . Further, the term "treatment" used in the present invention means that the administration of the pharmaceutical composition comprising the compound represented by the above-mentioned formula (1), or a pharmaceutically acceptable salt thereof improves the symptom of the genetic disease caused by nonsense mutation It means all actions that are cured.

The compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof exhibits low antibacterial activity against Gram-negative bacterium Escherichia coli and Pseudomonas aeruginosa (Experimental Example 1) and exhibits low cytotoxicity against human cell lines 2), and exhibited an ineffective activity of intrinsic intracutaneous insensitivity (Experimental Example 3), indicating that cystic fibrosis, Duchenian type muscular dystrophy, capillary vasodilator ataxia syndrome, Hodgler's syndrome, type A hemophilia, type B hemophilia, Can be usefully used for the prevention or treatment of a genetic disease caused by a nonsense mutation.

The present invention also relates to a pharmaceutical composition comprising a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, which has an intrinsic mutagenic cystic fibrosis transmembrane conductance regulation and an activity of inducing intolerance to dystrophin .

The pharmaceutical compositions of the present invention may be formulated into oral or parenteral administration forms according to standard pharmaceutical practice. These formulations may contain, in addition to the active ingredient, an additive such as a pharmaceutically acceptable carrier, adjuvant or diluent.

When the composition of the present invention is used as a medicine, a pharmaceutical composition containing at least one compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient can be administered orally or parenterally Administration form, and the like, but are not limited thereto.

Examples of formulations for oral administration include tablets, pills, hard candles, soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs and the like, (For example, silica, talc, magnesium salt of stearic acid, calcium salt of stearic acid and / or polyethylene glycol) in the presence of a surfactant such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine . The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt The same disintegrating or boiling mixture and / or absorbing agent, coloring agent, flavoring agent, and sweetening agent.

Formulations for parenteral administration may include subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection. In this case, in order to formulate the composition for parenteral administration, the pharmaceutical composition of the present invention contains at least one compound represented by the general formula (1) or a pharmaceutically acceptable salt thereof, which is mixed with water or a stabilizer or a buffer to prepare a solution or suspension, Which can be prepared in an ampoule or vial unit dosage form. The composition may be sterilized and / or contain preservatives, stabilizers, wettable or emulsifying accelerators, adjuvants such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances and may be mixed, granulated Or a coating method.

The dose of the compound of the present invention to the human body may vary depending on the patient's age, body weight, sex, dosage form, health condition, and disease severity. When the patient is 70 kg in body weight, And may be 0.01-100 mg / day, preferably 0.01-500 mg / day. Depending on the judgment of a doctor or pharmacist, it may be administered once to several times a day at intervals of a certain period of time.

The present invention also relates to a method for producing a disaccharide, comprising the steps of: 1) acid hydrolyzing an aminoglycoside antibiotic comprising 2-deoxystretamine to obtain a disaccharide; And 2) reacting the trisaccharide obtained in the above step 1) with a glycosidase to obtain an enzyme reaction product.

In one embodiment of the present invention, the production method may further include 3) separating, purifying, or separating and purifying the enzyme reaction product obtained in the step 2) by the MPLC method.

In one aspect of the present invention, the aminoglycoside antibiotic comprising 2-deoxyestreptamine is selected from the group consisting of lividomycin, isepamicin, geneticin or G-418, sisomicin, and netilmicin, but are not limited thereto.

In one embodiment of the present invention, the disaccharide of step 1) may be isolated, purified, or separated and purified by a medium pressure liquid chromatography (MPLC) method, but is not limited thereto.

In one aspect of the present invention, preferably, the glycosyltransferase of step 2) is KanM2 or KanE, and more preferably KanM2 or KanE is expressed in E. coli.

In one aspect of the present invention, the stationary phase used in the MPLC method in the step 3) may be silica, but is not limited thereto.

In one aspect of the present invention, the mobile phase used in the MPLC method in the step 3) may be a mixed solution of chloroform: methanol: ammonium hydroxide 1: 1: 1 (v / v / v), but is not limited thereto.

In one embodiment of the present invention, the MPLC retention time in step 3) may be 8 to 14 minutes, but is not limited thereto.

Hereinafter, embodiments for explaining the present invention in more detail will be presented.

However, the following examples are provided to more easily illustrate the present invention, and the present invention is not limited by the examples.

< Example  1> Compound 1) 3'- Deoxy -3 "- Diamino -3 "- Hydroxycamnamine  Manufacture of C

(1) Through acid hydrolysis Physician's body  produce

To prepare biosynthesis of sugar - added aminoglycoside compounds through the reaction of glycosyltransferase, firstly, the disaccharide, which is an enzyme substrate, was obtained by acid hydrolysis and then separated. That is, libidomycin, an aminoglycoside antibiotic including 2-deoxyestretamine, which is currently commercially and commercially available, was purchased from Sigma-Aldrich (St. Louis, MO, USA). 100 mg of lambidomycin, an aminoglycoside antibiotic, was placed in a light-shielding glass cap tube, and 8 ml of a 1 N hydrochloric acid methanol solution (0.86 ml of concentrated hydrochloric acid + 9.14 ml of methanol) was added. After being hydrolyzed for 4 hours on a hot plate, it was confirmed that all the solution was dried, and then 8 ml of 1 N hydrochloric acid solution was added once more. During the reaction without heating for 1 hour, after confirming that all of the reactants had dissolved, the final pH of the reaction was adjusted to 7 by dropping 2 drops of 1 N sodium hydroxide methanol solution (0.6 g NaOH + methanol 15 ml). The reaction solution was centrifuged at 2000 rpm for 5 minutes, and the supernatant was dispensed into an Eppendorf tube and dried under reduced pressure using a speed bag (SpeedVac, EYELA, Tokyo, Japan).

A CombiFlash Rf MPLC System (Teledyne ISCO, Lincoln, NE, USA) was used for the separation and purification of the divalent saccharide in the acid hydrolysis reaction product. That is, in a MPLC system in which a mixture of chloroform: methanol: ammonium hydroxide 1: 1: 1 (v / v / v) was flowed to a mobile phase at a rate of 8 ml per minute to a normal-phase silica cartridge, After the dissolved reaction solution was injected, the peak detected on the chromatogram was automatically collected. Separate fractions were further concentrated under reduced pressure, and the desired disaccharides were separated by analyzing the mass spectrum with an ion trap mass spectrometer (ThermoFinnigan, San Jose, Calif., USA). Compound 6, shown in Figure 1, was hydrolyzed as a divalent sugar and was separated on a MPLC system at a retention time of 8.2 minutes. In addition, the final purified pseudo-polysaccharide compound 6 was purified to 62 mg, indicating that the acid hydrolysis efficiency was 62%, and was used as a substrate for the following enzyme reaction (see FIG. 1).

(2) In E. coli KanM2  Expression of recombinant enzyme

The kanM2 (or kanE ) gene encoding kanamycin biosynthesis secondary glycosyltransferase was expressed in Escherichia coli as follows before using the dormant saccharide obtained in (1), that is, Compound 6 as a substrate. Polymerase chain reaction (PCR) was performed using pSKC2 cosmid containing kanamycin biosynthetic gene cluster as template and kanM2 selective primer. The N-terminal primer is the sequence of SEQ ID NO: 1 below and the C-terminal primer is the sequence of SEQ ID NO: 2 below.

SEQ ID NO: 1: 5' -GG CATATG ACCGAGCCTGCCAAGGGTG-3 '( Nde I restriction site)

SEQ ID NO: 2: 5 '- CGGGGTGGGGGGA CTCGAG GAGGACCC-3' ( Xho I restriction site)

Polymerase chain reaction amplification was performed for a total of 32 cycles (initial inactivation 5 min at 98 ° C, slope at 52.8 to 69.3 ° C, followed by reaction at 72 ° C for 5 min). The PCR product was purified and ligated to pGEMR-T easy vector (pGEMR-T easy vector, Promega, Madison, Wis., USA) and then ligated to E. coli XL1 blue (XL1 blue, Stratagene, La Jolla, Lt; 0 &gt; C for 45 seconds, followed by transformation. The T-islet vector was isolated and purified from the selected transformed E. coli and the sequence thereof was confirmed. The kanM2 fragment treated with Nde I and Xho I restriction enzymes was inserted into pET-28 (a +) (Novagen, Madison, Wis., USA), a protein expression vector treated with the same restriction enzymes, BL21 (DE3) (Stratagene, La Jolla, CA, USA). At this time, 50 ppm of kanamycin antibiotics was used for selection of transformants.

The recombinant Escherichia coli was inoculated into the LB medium (Luria Bertani) supplemented with 1 M of sorbitol and 2.5 mM betaine at a final concentration of 1%, and cultured at 37 ° C. D-thiogalactopyranoside (IPTG, Sigma-Aldrich) at a final concentration of 0.5 mM was added to induce protein expression when growth was observed at a concentration between 0.6 and 0.8 . The recombinant E. coli strain was further cultured at 20 DEG C for 24 hours. After culturing, the culture was centrifuged at 2000 rpm for 10 minutes to collect the cells, and the cells were suspended in 50 mM sodium phosphate lysis buffer (300 nM NaCl, 10 mM imidazole, 10% glycerol, 1% Triton X-100) And sonication was carried out. After centrifugation at 12000 rpm for 30 minutes, the supernatant was collected separately, and some samples were analyzed by 12% SDS-PAGE to confirm the expression level of the recombinant kanM2 enzyme. In order to purify the expressed recombinant protein KanM2, the supernatant was diluted with a Talon-metal affinity resin (Clontech, Mountain View, Calif.) Equilibrated in 50 mM phosphate buffer (0.3 M NaCl, 20 mM imidazole) , CA, USA) and incubated at 4 ° C for one hour. After refrigerated centrifugation at 2000 rpm for 5 minutes, the resin was introduced into a disposable column and washed with phosphate buffer containing 20 mM imidazole in a volume of 10 times the resin. Finally, recombinant glycoconjugated enzyme KanM2 bound to the resin was purified with 3 ml of phosphate buffer containing 250 mM imidazole (see Fig. 2).

(3) A physician  As substrate KanM2  Biosynthesis, separation and purification of sugar aminoglycoside compounds by recombinant enzyme reaction

Separated purified divalent saccharides, ie, Compound 6, were dissolved in a reaction buffer (50 mM phosphate buffer, 10 mM magnesium chloride, 1 mg / ml bovine serum albumin) to give a concentration of 1 mM. uM and 2 mM of UDP-glucose (UDP-glucose) per nucleic acid were added, and the enzyme reaction was carried out at 37 ° C for 3 hours. After the reaction, the same amount of chloroform was added to stop the reaction. Only the supernatant was collected and lyophilized. For the separation and purification of the desired sugar-added aminoglycoside compound from the enzyme reaction solution, the above Comby Flash Rf MPLC system was used. That is, in a MPLC system in which a mixture of chloroform: methanol: ammonium hydroxide 1: 1: 1 (v / v / v) was flowed to a mobile phase at a rate of 8 ml per minute to a normal-phase silica cartridge, After the dissolved enzyme reaction product was injected, the peak detected by the chromatogram was automatically collected. Separate fractions were further concentrated under reduced pressure, and the desired sugar-added aminoglycoside compound was isolated by analyzing the mass spectrum with an ion trap mass spectrometer (LCQ ion-trap mass spectrometer, ThermoFinnigan, San Jose, CA, USA). Compound 6 used as a substrate was collected on a MPLC system at a retention time of 8.2 minutes and the desired sugar-added aminoglycoside compound, i.e., Compound 1, was detected at a retention time of 9.2 min, which is later than the substrate. The purified sugar aminoglycoside compound is 3 &apos; -dioxy-3 &quot; -diamino-3 &quot; -hydroxycainamycin C, 5 mg.

NMR samples were prepared by partially dissolving compound 1) in 200 μl of D ₂ O and then leaving the solvent in a 5 mm Shigemi advanced NMR microtube (Sigma, St. Louis, Mo.). ¹³ C NMR spectra were acquired at 298 K using a Varian INOVA 500 spectrophotometer and chemical shifts were recorded in ppm using TMS as an internal standard. All NMR data were calculated using Mnova Suite 5.3.2 software, and the ¹³ C-NMR spectra of the biosynthetic compounds were summarized in Table 1 below.

[Table 1] &lt; ¹³ &gt; C-NMR spectrum of the compounds 1 to 5

The mass spectrum of the sugar-added amino glycoside compound thus prepared is shown in Fig.

< Example  2> Compound 2) 1-N-3-Amino-2 (S) - Hydroxypropionyl - Kanamycin  Manufacturing of D

(1) Through acid hydrolysis Physician's body  produce

The same procedure as described in Example 1 was carried out except that isipamycin was used instead of libidomycin as an aminoglycoside antibiotic to obtain Compound 7 as a hydrolyzed pseudoadenoside, and the retention time collected on the MPLC system was 7.6 minutes, and it was purified to 78 mg, and the efficiency of the acid hydrolysis reaction was 78%.

(2) In E. coli KanM2  Expression of recombinant enzyme

&Lt; Example 1 >

(3) A physician As substrate KanM2  Biosynthesis, separation and purification of sugar aminoglycoside compounds by recombinant enzyme reaction

The same procedure as described in Example 1 was repeated except that Compound 7 (the retention time collected on the MPLC system was 7.6 min) was used instead of Compound 6 as a pseudo-glycoside to obtain Compound 1, 1-N-3-amino- 2 (S) -hydroxypropionyl-kanamycin D was obtained, and the retention time of Compound 2 was 8.2 minutes and finally purified to 6.1 mg.

< Example  3> Compound 3) 6'- methyl -3 "- Diamino -3 "- Hydroxycamnamine  Manufacture of C

(1) Through acid hydrolysis Physician's body  produce

The same procedure as described in Example 1 was repeated to obtain Compound 8 as a hydrolyzed pseudoadenoside, except that the geneticin was used instead of libidomycin as the aminoglycoside antibiotic. The retention time of the aliquot on the MPLC system was 8 Min, and it was purified to 65 mg, indicating that the acid hydrolysis efficiency was 65%.

(2) Expression of KanM2 recombinant enzyme in E. coli

&Lt; Example 1 >

(3) A physician As substrate KanM2  Biosynthesis, separation and purification of sugar aminoglycoside compounds by recombinant enzyme reaction

The same experiment as described in Example 1 was carried out except that Compound 8 (the retention time collected on the MPLC system was 8 minutes) was used instead of Compound 6 as a pseudo-glycoside to obtain Compound 3, 6'-methyl- Diamino-3 &quot; -hydroxycainamycin C was obtained, and the retention time of Compound 3 was 8.6 minutes and finally purified to 5.8 mg.

< Example  4> Compound 4) 3 ', 4'- Didyxy -4 ', 5'- Dihydro -3 "- Diamino -3 "- Hydroxycamnamine  Manufacture of B

(1) Through acid hydrolysis Physician's body  produce

The same procedure as described in Example 1 was carried out except that lysidomycin was used instead of libidomycin as an aminoglycoside antibiotic. As a result, the hydrolyzed pseudoadenosidal compound 9 was obtained. Min, which was purified to 72 mg, indicating that the acid hydrolysis efficiency was 72%.

(2) Expression of KanM2 recombinant enzyme in E. coli

&Lt; Example 1 >

(3) A physician As substrate KanM2  Biosynthesis, separation and purification of sugar aminoglycoside compounds by recombinant enzyme reaction

The same experiment as described in Example 1 was carried out except that Compound 9 (the retention time collected on the MPLC system was 13 minutes) was used instead of Compound 6 as a pseudo-glycoside to obtain Compound 4, 3 ', 4'- -4 ', 5'-dihydro-3 "-diamino-3" -hydroxycainamycin B, and the retention time of Compound 4 was 13.8 minutes and finally purified to 4.2 mg.

< Example  5> Compound 5) 1-N-ethyl-3 ', 4'- Didyxy -4 ', 5'- Dihydro -3 &quot; -diamino-3 &quot; - Hydroxycamnamine  Manufacture of B

(1) Through acid hydrolysis Physician's body  produce

The same procedure as described in Example 1 was carried out except that nethylamycin was used instead of libidomycin as an aminoglycoside antibiotic. The hydrolyzed pseudoadenosidal compound 10 was obtained, and the retention time of the aliquot on the MPLC system was 7.9 Min, and purified to 66 mg, indicating that the acid hydrolysis efficiency was 66%.

(2) Expression of KanM2 recombinant enzyme in E. coli

&Lt; Example 1 >

(3) A physician As substrate KanM2  Biosynthesis, separation and purification of sugar aminoglycoside compounds by recombinant enzyme reaction

The same procedure as described in Example 1 was repeated except that Compound 10 was used instead of Compound 6 as a pseudo-glycoside (the retention time collected on the MPLC system was 7.9 minutes) , 4'-dideoxy-4 ', 5'-dihydro-3 "-diamino-3" -hydroxycainamycin B, and the retention time of Compound 5 was 9 minutes and finally purified to 5.8 mg.

< Experimental Example  1> Example  Comparison of antibiotic activity of compounds and existing antibiotics

(ATCC (American Type Culture Collection) (Manassas, VA, USA) and an antibiotic resistant strain bank (CCARM, Seoul, Korea) to examine the antibacterial activities of the compounds 1 to 5 obtained in Examples 1 to 5 Antibacterial activity was tested by securing two type strains and two clinical isolates of Escherichia coli and Pseudomonas aeruginosa belonging to gram - negative bacteria.

Specifically, four test bacterial strains were cultured and maintained in a Mueller-Hinton broth (Difco, BD Biosciences, San Jose, Calif., USA) at 28 ° C. Subsequently, the commercial aminoglycoside antibiotics before acid hydrolysis and the five compounds obtained in the examples were treated in the medium. Antibacterial activity against test strains was measured using a microdilution method recommended by the Clinical and Laboratory Standard Institute (CLSI). Serial dilutions of each treatment were diluted to 0.25 ~ 128 ㎍ / ml with distilled water, and distilled water was used as a negative control. The growth of the bacteria was monitored at 600 nm using a Labsystem Bioscreen C reader and compared to conventional antibiotics diluted in a broth medium to inhibit the growth of the test microorganism and of the compound of Example The minimum inhibitory concentration (MIC) of antibiotics was determined as the lowest concentration. All analyzes were performed at least three times and the results are shown in Table 2 below.

[Table 2] Anti-bacterial activity of Gram-negative bacteria of the compound of Example and the aminoglycoside antibiotics

As shown in Table 2, the antibacterial activities of two species of E. coli susceptible to kanamycin and two species of P. aeruginosa resistant to kanamycin showed that the five compounds of the present invention had lower antimicrobial activity than the conventional commercial antibiotics , Indicating that aminoglycoside may overcome the effects of gastrointestinal bioter, one of the problems in the clinical application of genetic diseases as a therapeutic agent for genetic diseases.

< Experimental Example  2> Example  Comparison of cytotoxicity of compounds and existing antibiotics to human cell lines

In order to test toxicity against renal cytotoxicity, which is another problem in clinical application of 5 compounds of the present invention, the normal kidney cell line HEK293, the kidney proximal tubular cell line LLC-PK1 and the kidney epithelial cell line A498 And its cytotoxicity was confirmed by MTT method. Gentamycin, an aminoglycoside antibiotic, and amikacin, a related semisynthetic antibiotic, were compared at the same concentration as the control group. First, three kinds of human kidney cell lines for assay, that is, HEK293 normal kidney cell line, LLC-PK1 kidney proximal tubular cell line, and kidney epithelial cell line A498 were activated. Each cell line was pre-cultured in DE medium, centrifuged and dispensed into T-flask for 5 days. On the third day of culturing, each of the compound of the present invention was treated so as to have a final level of 0.1 to 2.5 ppm, followed by further culturing for 2 days, and if necessary, treated cell lines were separated and sampled at intervals of one day. Gentamicin at the same concentration, which was reported to be toxic as a control with each treatment, was also treated in the same manner and then subjected to a color reaction with final MTT to measure the individual LD50 for each cell line. The results are shown in FIG. 4 .

As shown in Fig. 4, in the case of HEK293, the compounds 4 and 5 having chemical structures similar to those of gentamicin exhibited similar cytotoxicity to the LD50 values of gentamicin-treated plants, whereas compounds 1, 2 and 3 Showed low cytotoxicity. Similar results were also obtained in the renal proximal tubular cell line, LLC-PK1. In particular, the treatments of compound 1 and 3 showed cytotoxicity twice as low as those of the control gentamycin treatment. Finally, in the cytotoxicity test results of kidney epithelial cell line, the compound 2 treatment showed higher cytotoxicity than the control, whereas the compound 1 and compound 3 treatments also showed reduced cytotoxicity more than twice The present inventors have shown that aminoglycoside can be used as a compound capable of overcoming renal toxicity which is another problem in clinical application as a therapeutic agent for genetic diseases.

< Experimental Example  3> Example  Comparing the induction activity of the compound with the conventional antibiotics

In vitro and in vivo NRI assays were carried out on 5 compounds of the Examples to confirm intrinsic induction activity. First, a dystrophin containing a nonsense mutation codon and a gene encoding CFTR were expressed in E. coli, respectively, and E. coli containing the expressed protein was sonicated to obtain a cell-free extract. Five compounds were reacted at 30 ° C for three hours in the final sections of 2.4, 3.6 and 4.8 ppm, respectively, and the proteins were precipitated with 0.1% trichrolo acetic acid (TCA). Then, the precipitated protein was dissolved again in tris buffer (pH 7.4), separated on HPLC / ESI-MS, and the peak appeared on the chromatogram was deconvoluted with an algorithm program to determine the protein size Respectively. The peak intensities of the cleaved and full length proteins were measured to determine the intrinsic activity of each compound.

On the other hand, an in vitro NRI assay, which is an early stage of the preclinical study, was conducted on cell lines isolated from degenerative genetic disease patients, ie, cystic fibrosis patients. This is a method that can provide more specific and clinically close data compared to the in vitro NRI assay described above. First, activated cystic fibrosis genetic disease cell lines were activated. That is, the cells were pre-cultured in DE medium, centrifuged and cultured in T-flask for 5 days. On the third day of culture, each of the five compounds was treated to have three sections of final 2.4, 3.6 and 4.8 ppm, followed by further culture for 2 days. Gentamicin, the activity of which was reported as a control with each treatment, was compared and compared. The treated and control samples were sonicated and centrifuged to obtain the supernatant. Protein was precipitated by adding 0.1% trichloroacetic acid to the supernatant of each treatment, and proteins precipitated by high-speed refrigeration centrifugation were collected and dissolved once in Tris buffer (pH 7.4). The dissolved protein was separated on HPLC / ESI-MS, and the peak shown on the chromatogram was deconvoluted by an algorithm program to confirm its protein size. The peak intensities of the cleaved protein and the full-length protein were measured, and the activity of inducing nonsense intact toxicity of each compound was determined and shown in FIG.

As shown in FIG. 5, in some compounds, the same inactivation activity (4.8 ug / ml) in comparison with the gentamycin control group was found to be comparable or improved in in vitro inducing activity of intrinsic trait. That is, the compound 4 and the compound 5 treatments showed lower induction activity than that of the control gentamycin, the compounds 1 and 2 showed no induction activity similar to that of the control, and the compound 3, Activity. On the other hand, similar results were obtained as the in vitro activity test results when comparing the induction activity of intact insulin in vitro. That is, the treatment with Compound 4 and Compound 5 treated at the levels of 2.4, 3.6 and 4.8 ug / ml showed lower induction activity than that of the control gentamycin, Compound 2 showed no induction activity similar to that of the control, 1 and compound 3 showed an enhanced activity of intact allergen inducing activity compared with the control group. From the above results, it was confirmed that the compound of the Example was clinically applicable as a therapeutic agent for genetic diseases.

Five compounds of the Examples (Compound 1 to Compound 5) were formulated as follows.

< Formulation example  1> Purification (direct pressurization)

After Compound 1 (5.0 mg) was sieved, 14.1 mg of lactose, 0.8 mg of crospovidone (USNF) and 0.1 mg of magnesium stearate were mixed and pressed to prepare tablets.

< Formulation example  2> Purification (wet assembly)

Compound 1 (5.0 mg) was sieved, and 16.0 mg of lactose and 4.0 mg of starch were mixed. After 800.3 mg of polysorbate was dissolved in pure water, an appropriate amount of this solution was added to make it finely-ground. After drying, the granules were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The fine particles were pressed to prepare tablets.

< Formulation example  3> Powder and Capsule

Compound 1 (5.0 mg) was sieved and then mixed with 14.8 mg of lactose, 10.0 mg of polyvinylpyrrolidone and 0.2 mg of magnesium stearate. The mixture was filtered through a hard No. 5 gelatin capsules.

< Formulation example  4> injection

180 mg of mannitol, 26 mg of Na _{2 H 2} PO _{4 12 H 2} O and 2974 mg of distilled water were added to 100 mg of the active ingredient to prepare an injection.

The sugar aminoglycoside compound exhibiting the intrinsic intrinsic induction activity of the present invention, a method for producing the same, and a pharmaceutical composition containing the same, overcome the problems in development of a conventional aminoglycoside genetic disease drug, Can be usefully used.

<110> Industry-University Cooperation Foundation Sunmoon University <120> Glucose-attached aminoglycoside compounds, manufacturing method          and pharmaceutical compositions containing them active          ingredients <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 27 <212> DNA <213> Escherichia coli <400> 1 ggcatatgac cgagcctgcc aagggtg 27 <210> 2 <211> 27 <212> DNA <213> Escherichia coli <400> 2 cggggtgggg ggactcgagg aggaccc 27

Claims (10)

1) 3'-Dioxy-3 &quot; -diamino-3 &quot; -hydroxycamanamycin C,
2) 1-N-3-amino-2 (S) -hydroxypropionyl-kanamycin D,
3) 6'-Methyl-3 &quot; -diamino-3 &quot; -hydroxycamanamycin C,
4) 3 ', 4'-dideoxy-4', 5'-dihydro-3 "-diamino-3" -hydroxycainamycin B, and
5) a compound selected from the group consisting of 1-N-ethyl-3 ', 4'-dideoxy-4', 5'-dihydro-3 "-diamino-3" -hydroxycainamycin B, A pharmaceutically acceptable salt. delete The present invention relates to the use of a compound of claim 1 for the treatment of cystic fibrosis, duchenne muscular dystrophy (DMD), ataxia telangiectasia, hurler syndrome, hemophilia A), hemophilia B (hemophilia B), and tay-sachs. delete delete 1) acid hydrolyzing aminoglycoside antibiotics containing 2-deoxystreptamine to obtain pseudodisaccharides; And
2) obtaining the enzyme reaction product by reacting the trisaccharide obtained in the above step 1) with the glycosidase
Lt; RTI ID = 0.0 &gt; 1. &Lt; / RTI &gt; The method according to claim 6,
3) separating, purifying, or separating and purifying the enzyme reaction product obtained in step 2) by medium pressure liquid chromatography (MPLC)
&Lt; / RTI &gt; The method according to claim 6,
Wherein the enzyme of step 2) is KanM2 or KanE. 9. The method of claim 8,
Wherein said KanM2 or KanE is expressed in Escherichia coli. 8. The method of claim 7,
Wherein the MPLC retention time in step 3) is 8 to 14 minutes.

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