Novel fluorine-substituted vancomycin analogue as well as preparation method and application thereof

Abstract

The invention relates to a novel fluorine-substituted vancomycin analogue as well as a preparation method and application thereof. Specifically, the invention provides a fluorine-substituted adenosylmethionine analogue obtained by a chemical-enzyme catalysis method, and a novel fluorine-substituted vancomycin analogue obtained by taking the compound as a fluoroalkyl donor and combining enzyme catalysis. The obtained fluorine-substituted vancomycin analogue shows antibacterial activity which is equivalent to that of vancomycin.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and biotechnology engineering, and specifically relates to a class of novel fluorine-substituted vancomycin analogs and a preparation method and use thereof. Background Art

[0002] Vancomycin is an important glycopeptide antibiotic used to prevent and treat Gram-positive bacterial infections. Traditionally, vancomycin is known as the "last line of defense" in clinical practice and is used to treat serious infections that are ineffective against all antibiotics. However, with the abuse of antibiotics, more and more vancomycin-resistant bacteria have emerged. Introducing new pharmacophores and expanding the structure of new vancomycin analogs are important means to solve the problem of vancomycin resistance.

[0003] Fluorine is an important pharmacophore. Introducing fluorine-containing functional groups into drug molecules can usually change the lipophilicity, metabolic stability and pharmacokinetic properties of candidate drugs, and plays an irreplaceable role in the field of medicinal chemistry. Currently, more than 300 fluorine-containing drugs have been approved by the FDA, accounting for more than 20% of all drugs. Therefore, introducing fluorine-containing functional groups into vancomycin, changing the biological activity of vancomycin or changing the potential target of action, and obtaining new vancomycin analog drugs based on this is an important breakthrough.

[0004] Although the art has been trying to introduce fluorine-containing functional groups into drug molecules using various methods, for highly functionalized complex compounds such as vancomycin, no efficient and highly selective methods for introducing fluorine-containing functional groups have been successfully developed so far, nor have fluorine-substituted vancomycin analogs with advantages such as high activity or high reactivity been developed. Therefore, the art urgently needs to develop various new fluorine-substituted vancomycin analogs and their preparation methods. Summary of the invention

[0005] The object of the present invention is to provide a class of novel fluorine-substituted vancomycin analogs with high activity or reactivity.

[0006] Another object of the present invention is to provide a preparation method and use of the fluorine-substituted vancomycin analog.

[0007] Another object of the present invention is to provide a fluorine-substituted adenosylmethionine analogue used as a synthetic unit for preparing fluorine-substituted vancomycin and a preparation method thereof.

[0008] In a first aspect of the present invention, a vancomycin analog or a pharmaceutically acceptable salt thereof is provided, wherein the vancomycin analog has a structure as shown in Formula I:

[0009]

[0010] In the formula,

[0011] Rf is -(CH2) n -Rf', wherein n is an integer selected from 1, 2 or 3;

[0012] Wherein, Rf' is selected from: halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, or substituted or unsubstituted 5-8 membered heterocycloalkyl;

[0013] The above substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C3 alkyl, or C1-C3 haloalkyl.

[0014] In another preferred embodiment, n is 1.

[0015] In another preferred embodiment, the 5-8 membered heteroaryl group has 1, 2 or 3 heteroatoms selected from N, S and O.

[0016] In another preferred embodiment, the 5-8 membered heterocycloalkyl group has 1, 2 or 3 heteroatoms selected from N, S and O.

[0017] In another preferred embodiment, Rf' is selected from: halogen, substituted or unsubstituted C2-C3 alkenyl, substituted or unsubstituted C5-C6 aryl;

[0018] The substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C3 alkyl, or C1-C3 haloalkyl.

[0019] In another preferred embodiment, n is 1, and Rf' is a substituted or unsubstituted C2-C3 alkenyl group, wherein the substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C3 alkyl.

[0020] In another preferred embodiment, the halogen is selected from the group consisting of F, Cl, Br, and I.

[0021] In another preferred embodiment, the halogen is F or Cl.

[0022] In another preferred embodiment, the halogen is F.

[0023] In another preferred embodiment, the Rf is selected from the following group: 3,3-difluoroallyl, p-fluorobenzyl, p-trifluoromethylbenzyl, 3,5-difluorobenzyl.

[0024] In another preferred embodiment, the vancomycin analog is an optical isomer or a racemate. In another preferred embodiment, the vancomycin analog has a structure of Formula Ia:

[0025]

[0026] In another preferred embodiment, the vancomycin analog has a structure of Formula Ib:

[0027]

[0028] In another preferred embodiment, the vancomycin analog has a structure of Formula Ic:

[0029]

[0030] In another preferred embodiment, the vancomycin analog has a structure of Formula Id:

[0031]

[0032] In the second aspect of the present invention, there is provided a method for preparing the vancomycin analogue as described in the first aspect of the present invention, comprising the steps of:

[0033] In the presence of a methyltransferase, reacting a donor compound of formula (II) with demethylvancomycin represented by formula (IV) to produce a vancomycin analogue as described in the first aspect of the present invention;

[0034]

[0035] Wherein, the group Rf is as defined in the first aspect of the present invention.

[0036] In another preferred embodiment, the methyltransferase is the methyltransferase MtfA or a homologous enzyme thereof whose coding sequence is shown in SEQ ID NO.: 2.

[0037] In another preferred embodiment, the donor compound of formula (II) is reacted with demethylvancomycin represented by formula (IV) in the presence of methyltransferase and SAH hydrolase to produce the vancomycin analogue as described in the first aspect of the present invention.

[0038] In another preferred embodiment, the amino acid sequence of the SAH hydrolase is shown in SEQ ID NO.:3.

[0039] In another preferred embodiment, the molar ratio of the methyltransferase to the SAH hydrolase is 1:5-5:1, preferably 1:2-2:1, and more preferably 1:1.

[0040] In the third aspect of the present invention, an adenosylmethionine analogue is provided, which has a structure shown in Formula II:

[0041]

[0042] Wherein, the group Rf is as defined in the first aspect of the present invention.

[0043] In another preferred embodiment, the adenosylmethionine analog has a structure shown in the following formula IIa, formula IIb, formula IIc, or formula IId:

[0044]

[0045] In a fourth aspect of the present invention, there is provided a method for preparing the adenosylmethionine analogue as described in the third aspect of the present invention, comprising the steps of:

[0046] In the presence of methionine adenosyltransferase or a mutant thereof, reacting the compound represented by the donor formula (III) with adenosine triphosphate (ATP) to produce the adenosylmethionine analogue as described in the third aspect of the present invention;

[0047]

[0048] Wherein, the group Rf is as defined in the first aspect of the present invention.

[0049] In another preferred embodiment, the methionine adenosyltransferase mutant is mutated from isoleucine (I) to alanine (A) at position I322 relative to the wild type.

[0050] In another preferred embodiment, the amino acid sequence of the methionine adenosyltransferase mutant is shown in SEQ ID NO.:1.

[0051] In the fifth aspect of the present invention, a pharmaceutical composition is provided, comprising: (a) the vancomycin analogue or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention; and (b) a pharmaceutically acceptable carrier.

[0052] In another preferred embodiment, the content of component (a) in the pharmaceutical composition is 0.0001-99wt%, preferably, 0.01-95wt%, more preferably, 0.1-90wt%.

[0053] In another preferred embodiment, the pharmaceutical composition further comprises other compounds having antibacterial activity.

[0054] In another preferred embodiment, the other compounds having antibacterial activity are selected from the following group: thiostrepton, chloramphenicol, lincomycin, or a combination thereof.

[0055] In the sixth aspect of the present invention, there is provided a use of the vancomycin analogue or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention, wherein the use is:

[0056] (a) for preparing an antibacterial composition;

[0057] (b) for preparing a composition for inhibiting the growth of microorganisms;

[0058] (c) for use in the preparation of a composition for treating microbial and / or bacterial infection;

[0059] (d) Used in the preparation of feed additives.

[0060] In another preferred embodiment, the composition is selected from the following group: a pharmaceutical composition, a feed composition, or a combination thereof.

[0061] In another preferred embodiment, the feed additive is an antibacterial agent.

[0062] In another preferred embodiment, the microorganisms or bacteria include Gram-positive bacteria.

[0063] In the seventh aspect of the present invention, there is provided a method for non-therapeutic inhibition of microbial growth or killing of microorganisms in vitro, comprising the steps of: using the vancomycin analogue or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention at a site requiring treatment.

[0064] In the eighth aspect of the present invention, a method for preparing a pharmaceutical composition is provided, comprising the steps of: mixing the vancomycin analogue or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention; and a pharmaceutically acceptable carrier to form a pharmaceutical composition.

[0065] In the ninth aspect of the present invention, a method for treating bacterial infection in animals is provided, comprising the steps of: administering to an animal in need of treatment the vancomycin analogue or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention or the pharmaceutical composition as described in the fifth aspect of the present invention.

[0066] In another preferred embodiment, the animals include: mammals, livestock (such as cattle, pigs, and sheep), poultry (such as chickens, ducks, and geese), and aquaculture animals.

[0067] In the tenth aspect of the present invention, a method for preparing a feed composition is provided, comprising the steps of: mixing the vancomycin analogue or a pharmaceutically acceptable salt thereof as described in the first aspect of the present invention as a feed additive with a feed raw material to form a feed composition containing the vancomycin analogue or a salt thereof.

[0068] In another preferred embodiment, the feed composition is used in livestock (such as cattle, pigs, and sheep) farming, poultry (such as chickens, ducks, and geese) farming, or aquaculture.

[0069] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] The following drawings are used to illustrate specific embodiments of the present invention, but are not used to limit the scope of the present invention.

[0071] Figure 1 Shown are SDS-PAGE gel images of hMAT2AI322A mutant, MtfA and SAHH.

[0072] Figure 2 HPLC and HR-MS results showing the production of IIa by the reaction of IIIa with ATP catalyzed by the hMAT2AI322A mutant

[0073] Figure 3 The relative activity of wild-type hMAT2A and I322A mutant in catalyzing the production of IIa is shown.

[0074] Figure 4 HPLC and HR-MS results showing the production of Ia by the reaction of IIIa with demethylvancomycin IV catalyzed by hMAT2AI322A mutant-MtfA-SAHH

[0075] Figure 5 The relative yield of SAHH on the yield of Ia production is shown.

[0076] Figure 6 The N-3,3-difluoroallyl vancomycin Ia is shown 1 H-NMR spectrum.

[0077] Figure 7 The N-3,3-difluoroallyl vancomycin Ia is shown 13 C-NMR spectrum.

[0078] Figure 8 The N-3,3-difluoroallyl vancomycin Ia is shown 19 F-NMR spectrum.

[0079] Fig. 9 The N-3,3-difluoroallyl vancomycin Ia is shown 1 H- 1 H COSY spectrum.

[0080] Fig.10 The HSQC spectrum of N-3,3-difluoroallyl vancomycin Ia is shown.

[0081] Fig.11 The HMBC spectrum of N-3,3-difluoroallyl vancomycin Ia is shown.

[0082] Fig.12The NOESY spectrum of N-3,3-difluoroallyl vancomycin Ia is shown.

[0083] Fig.13 The HR-MS / MS spectrum of N-3,3-difluoroallyl vancomycin Ia is shown.

[0084] Fig.14 hMAT2A I322A HPLC and HR-MS results of the reaction of IIIb with demethylvancomycin IV catalyzed by mutant-MtfA-SAHH to produce Ib.

[0085] Fig.15 The N-p-fluorobenzyl vancomycin Ib is shown 19 F-NMR spectrum.

[0086] Fig.16 The HR-MS / MS spectrum of N-p-fluorobenzyl vancomycin Ib is shown.

[0087] Fig.17 hMAT2A I322A HPLC and HR-MS results of the reaction of IIIc and demethylvancomycin IV catalyzed by mutant-MtfA-SAHH to produce Ic.

[0088] Fig.18 The HR-MS / MS spectrum of N-trifluoromethylbenzyl vancomycin Ic is shown.

[0089] Fig.19 hMAT2A I322A HPLC and HR-MS results of the reaction of IIId and demethylvancomycin IV catalyzed by mutant-MtfA-SAHH to produce Id.

[0090] Fig. 20 The HR-MS / MS spectrum of N-3,5-difluorobenzyl vancomycin Id is shown. DETAILED DESCRIPTION

[0091] After extensive and in-depth research, the inventors synthesized fluorine-substituted adenosylmethionine analogs based on a chemical-enzyme catalysis method, applied them as bio-based fluoroalkylation reagents, and successfully synthesized a new type of highly active or highly reactive fluorine-substituted vancomycin analogs using methyltransferases in the vancomycin biosynthesis pathway. On this basis, the present invention was completed.

[0092] the term

[0093] Abbreviations used herein have conventional meanings in the fields of chemistry and biology.

[0094] The term "substituted" as used herein means that one or more hydrogen atoms on a specific group are replaced by a specific substituent. The specific substituent may be the substituent described above or the specific substituent appearing in the examples. Therefore, in the present invention, the substituents in the general formula may each independently be the corresponding groups in the specific compounds in the examples; that is, the present invention includes the combination of the substituents in the above general formula, and also includes the combination of some of the substituents shown in the general formula with other specific substituents appearing in the examples, as long as the desired combination of substituents is a stable or chemically feasible combination of substituents.

[0095] As used herein, the term "C1-C6 alkyl" refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like. The alkyl group may be substituted with one or more substituents, such as halogen or haloalkyl. For example, the alkyl group may be an alkyl group substituted with 1 to 4 fluorine atoms, or the alkyl group may be an alkyl group substituted with a fluoroalkyl group.

[0096] As used herein, the term "C2-C6 alkenyl" refers to a straight or branched alkenyl having 2-6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or similar groups. The alkenyl group may be substituted with one or more substituents, such as halogen or haloalkyl. For example, the alkenyl group may be an alkenyl group substituted with 1-4 fluorine atoms, or the alkenyl group may be an alkenyl group substituted with a fluoroalkyl group.

[0097] As used herein, the term "C1-C3 alkyl" refers to a straight or branched chain alkyl group having 1 to 3 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.

[0098] As used herein, the term "C2-C4 alkenyl" refers to a straight or branched alkenyl group having 2 to 4 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

[0099] As used herein, the term "cycloalkyl" refers to a non-aromatic carbocyclic group, including cyclic alkyl, alkenyl and alkynyl groups. The cycloalkyl group can be a monocyclic (e.g., cyclohexyl) or polycyclic (e.g., comprising a fused, bridged and / or spirocyclic system), wherein the carbon atoms are located inside or outside the ring system. The cycloalkyl group as a whole can have 5 to 8 ring atoms. Any suitable ring position of the cycloalkyl group can be covalently linked to the defined chemical structure. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl and homologues, isomers, etc.

[0100] As used herein, the term "heterocycloalkyl" or "heterocyclyl" refers to a saturated, partially saturated or unsaturated group (but not aromatic), having a single ring or fused rings (including bridged ring systems and spiro ring systems), having 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from nitrogen, sulfur or oxygen in the ring, in which one or more rings can be cycloalkyl, aryl or heteroaryl, as long as the point of attachment is through a non-aromatic ring. Examples of heterocyclyls include pyridyl, thienyl, piperidinyl, or similar groups.

[0101] As used herein, the term "aryl" or "aromatic ring" has the same meaning as that generally understood by those of ordinary skill in the art, and refers to a cyclic conjugated aromatic system; for example, the term "C6-C 10 The term "aryl" refers to an aromatic ring group having 6 to 10 carbon atoms and containing no heteroatoms in the ring, such as phenyl, naphthyl, etc.

[0102] As used herein, the term "heteroaryl" refers to a monocyclic or bicyclic aromatic group containing at least one (e.g., 1-3) heteroatom selected from N, O and S. Examples of heteroaryl groups include piperazinyl, morpholinyl, piperidinyl, pyrrolidinyl, thienyl, furanyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrimidinyl, and the like.

[0103] As used herein, the term "halogenated" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine or iodine atom. In addition, the term "haloalkyl" is meant to include monohaloalkyl and polyhaloalkyl. For example, the term "C1-C3 haloalkyl" is meant to include, but not limited to, trifluoromethyl, 2,2,2-trifluoroethyl, 3-bromopropyl, etc.

[0104] Unless otherwise stated, a substituted group may have a specific substituent at any substitutable position of the group, and the substituent may be the same or different at each position.

[0105] Active ingredients

[0106] As used herein, the terms "active ingredient of the present invention", "compound of the present invention" and "vancomycin derivative of the present invention" are used interchangeably and all refer to the vancomycin analogue of the first aspect of the present invention, which has the structural formula shown in Formula I below:

[0107]

[0108] In the formula, Rf is as defined in the first aspect of the present invention.

[0109] It should be understood that the term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of the present invention.

[0110] A preferred class of vancomycin analogs is fluorine-substituted vancomycin analogs, such as N-3,3-difluoroallyl vancomycin as shown in Formula Ia, N-p-fluorobenzyl vancomycin as shown in Formula Ib, N-p-trifluoromethylbenzyl vancomycin as shown in Formula Ic, and N-3,5-difluorobenzyl vancomycin as shown in Formula Id.

[0111] In another preferred embodiment, the vancomycin analog has a structure of Formula Ia:

[0112]

[0113] In another preferred embodiment, the vancomycin analog has a structure of Formula Ib:

[0114]

[0115] In another preferred embodiment, the vancomycin analog has a structure of Formula Ic:

[0116]

[0117] In another preferred embodiment, the vancomycin analog has a structure of Formula Id:

[0118]

[0119]

[0120] As used herein, the term "pharmaceutically acceptable salt" refers to a salt suitable for use as a drug formed by a compound of the present invention and an acid or base. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by a compound of the present invention and a base. Salts can be formed by cations and negatively charged groups (e.g., carboxylates) on the compounds of the present invention. Suitable cations include sodium ions, potassium ions, magnesium ions, calcium ions, and ammonium ions, such as tetramethylammonium ions. Bases suitable for forming salts include, but are not limited to, hydroxides of alkali metals and alkaline earth metals (e.g., NaOH, KOH), oxides of alkali metals and alkaline earth metals, carbonates of alkali metals and alkaline earth metals (e.g., sodium carbonate), ammonia, organic amines, and the like.

[0121] The inventors not only confirmed the structure of the compound by synthesizing a large number of fluorine-substituted vancomycin derivatives by chemical-enzymatic methods and separating and purifying them, but also confirmed through antibacterial activity experiments that the fluorine-substituted vancomycin of the present invention exhibits an antibacterial activity comparable to that of vancomycin.

[0122] In addition to being used as antibacterial active ingredients in the preparation of drugs and as feed additives for preparing feed compositions, the compounds of the present invention can also be used as intermediates for preparing other fluorine-substituted vancomycin derivatives and can be used for further chemical derivatization to produce other fluorine-substituted vancomycin analogs.

[0123] Synthetic unit: Adenosylmethionine

[0124] As used herein, the terms "adenosylmethionine", "adenosylmethionine derivatives" and "adenosylmethionine analogs" are used interchangeably and all refer to the compound described in the second aspect of the present invention, which has the structure shown in Formula II. In the present invention, the fluorinated adenosylmethionine analog is a compound that participates in the chemical-enzymatic synthesis of the fluorinated vancomycin of the present invention, and this compound is used to synthesize or prepare the fluorinated vancomycin derivatives of the present invention.

[0125] In a preferred embodiment, the adenosylmethionine analog is S-adenosine-S-3,3-difluoroallyl-L-homocysteine ​​as shown in formula IIa, S-adenosine-S-p-fluorobenzyl-L-homocysteine ​​as shown in formula IIb, S-adenosine-S-p-trifluoromethylbenzyl-L-homocysteine ​​as shown in formula IIc, or S-adenosine-S-3,5-difluorobenzyl-L-homocysteine ​​as shown in formula IId.

[0126] It should be understood that the term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of the present invention.

[0127] A preferred class of fluorine-substituted adenosylmethionine analogs has the structural formula shown in Formula II below:

[0128]

[0129] In addition to being used to prepare fluorine-substituted vancomycin analogs, the adenosylmethionine analogs described in Formula II of the present invention can also be used in conjunction with other methyltransferases to prepare other types of natural product analogs, such as other types of fluorine-substituted natural product analogs.

[0130] Preparation of Fluorine-substituted Adenosylmethionine

[0131] The present invention provides a chemical-enzymatic preparation method of an adenosylmethionine analogue represented by formula II, the method comprising the steps of:

[0132] The compound described in formula III is reacted with adenosine triphosphate (ATP) under the condition of adenosylmethionine transferase hMAT2A or its mutant to obtain the fluorine-substituted adenosylmethionine analog.

[0133]

[0134] In the formula, Rf is as defined in the first aspect of the present invention. In one embodiment, Rf is a fluorine-containing substituted functional group, such as 3,3-difluoroallyl, p-fluorobenzyl, p-trifluoromethylbenzyl, 3,5-difluorobenzyl, but not limited thereto.

[0135] In one embodiment, the amino acid sequence of the hMAT2A mutant is as shown in SEQ ID NO.: 1:

[0136] MNGQLNGFHEAFIEEGTFLFTSESVGEGHPDKICDQISDAVLDAHLQQDPDAKVACETVAKTGMILLAGEITSRAAVDYQKVVREAVKHIGYDDSSKGF DYKTCNVLVALEQQSPDIAQGVHLDRNEEDIGAGDQGLMFGYATDETEECMPLTIVLAHKLNAKLAELRRNGTLPWLRPDSKTQVTVQYMQDRGAVLPIR VHTIVISVQHDEEVCLDEMRDALKEKVIKAVVPAKYLDEDTIYHLQPSGRFVIGGPQGDAGLTGRKIIVDTYGGWGAHGGGAFSGKDYTKVDRSAAYAAR WVAKSLVKGGLCRRVLVQVSYAAGVSHPLSISIFHYGTSQKSERELLEIVKKNFDLRPGVIVRDLDLKKPIYQRTAAYGHFGRDSFPWEVPKKLKY(SEQ ID NO.:1).

[0137] The fluorine-substituted methionine analog compound represented by formula III can be prepared by conventional synthetic methods.

[0138] Preparation of active ingredients

[0139] The present invention provides a method for preparing a compound of formula I, the method comprising the steps of:

[0140] In the presence of methyltransferase MtfA, adenosylmethionine analogs are reacted with norvancomycin to obtain the vancomycin analogs of the present invention. Further, under the condition of adding SAH hydrolase SAHH, the conversion rate can be improved, and the yield of the vancomycin analogs of the present invention can be increased.

[0141]

[0142] In the formula, Rf is as defined in the first aspect of the present invention. In one embodiment, Rf is a fluorine-containing substituted functional group, such as 3,3-difluoroallyl, p-fluorobenzyl, p-trifluoromethylbenzyl, 3,5-difluorobenzyl, but not limited thereto.

[0143] In one embodiment, the amino acid sequence of the methyltransferase protein MtfA is shown in SEQ ID NO.: 2:

[0144] MGSSHHHHHHSSGLVPRGSHMSNQLERGPVRTPHADVLLASVGERGVLCDFYDEGAADTYRDLIQDADGTSEAREFATRTGPVSGPVLELAAGMGRLTFPFLDLGWEVTALELSTSVLAAFRKRLAEAPADVRDRCTLVQGDMSAFALDKRF GTVVISSGSINELDEADRRGLYASVREHLEPGGKFLLSLAMSEAAESEPLERKQELPGRSGRRYVLHVRHLPAEEIQEITIHPADETTDPFVVCTHRRRLLAPDQVVRELVRSGFDVIAQTPFASGGAGRKDMVLVEAVMPGATADAR(SEQ ID NO.:2).

[0145] In one embodiment, the amino acid sequence of the SAH hydrolase is shown in SEQ ID NO.: 3:

[0146] MGSSHHHHHHSSGLVPRGSHMKIGIIGAMEEEVTLLRDKIENRQTISLGGCEIYTGQLNGTEVALLKSGIGKVAAALGATLLLEHCKPDVIINTGSAGGLAPTLKVGDIVVSDEARYHDADVTAFGYE YGQLPGCPAGFKADDKLIAAAEACIAELNLNAVRGLIVSGDAFINGSVGLAKIRHNFPQAIAVEMEATAIAHVCHNFNVPFVVVRAISDVADQQSHLSFDEFLAVAAKQSSLMVESLVQKLAHG(SEQ ID NO.:3).

[0147] In one embodiment, the norvancomycin is commercially available.

[0148] The inventors not only confirmed the structure of the compound of formula I by mass preparation and separation and purification, but also confirmed through antibacterial activity experiments that the active product fluorine-substituted vancomycin derivative of the present invention has similar antibacterial activity to vancomycin.

[0149] Pharmaceutical compositions and methods of administration

[0150] The compounds of the present invention have excellent antibacterial (antimicrobial) activity and can therefore be used as antibiotics.

[0151] The compounds of the present invention can be administered to mammals (such as humans) orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically, etc. The compounds can be administered alone or in combination with other pharmaceutically acceptable compounds. It should be noted that the compounds of the present invention can be administered in combination.

[0152] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0153] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0154] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0155] Besides these inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, and flavoring agents.

[0156] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.

[0157] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0158] Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0159] The compounds of the present invention may be administered alone or in combination with other active ingredients such as antibiotics.

[0160] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for an individual weighing 60 kg, the daily dosage is usually 1 to 1000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and individual health conditions, which are all within the skill of a skilled physician.

[0161] Feed additive and feed composition

[0162] The present invention also provides the application of the compound of the present invention in the field of feed. The compound of the present invention is very suitable as a feed additive because of its excellent antibacterial and antibacterial properties.

[0163] The present invention also provides a method for preparing a feed composition, comprising the steps of: mixing the compound of the present invention or a pharmaceutically acceptable salt thereof as a feed additive with a feed raw material to form a feed composition containing a thiostrepton analog (or a salt thereof).

[0164] The main advantages of the present invention include:

[0165] (a) Provides a class of novel fluorine-substituted vancomycin analogs having the structure of formula I and excellent activity.

[0166] (b) A chemical-enzyme catalysis method based on fluorine-substituted adenosylmethionine is provided to efficiently and highly selectively synthesize fluorine-substituted vancomycin analogs.

[0167] (c) Based on the fluorine-substituted vancomycin analogs of the present invention, it is helpful to prepare other types of fluorine-substituted vancomycin derivatives.

[0168] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0169] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only and cannot limit the content of the present invention.

[0170] Example 1

[0171] Synthesis of Fluorinated Methionine Analogues

[0172] 1. Synthesis of S-3,3-difluoroallylhomocysteine

[0173] Dissolve 10 mmol (tert-butyloxycarbonyl)-L-homocysteine ​​tert-butyl ester and 10 mmol 3-bromo-3,3-difluoropropylene in 20 mL dimethyl sulfoxide, react at room temperature for 12 hours, and then extract with ethyl acetate several times. The extracts are combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column to obtain 1.74 g of the product, with a yield of 95%.

[0174] 100 mg of the obtained S-3,3-difluoroallyl-(tert-butoxycarbonyl)-L-homocysteine ​​tert-butyl ester was dissolved in 1 ml of tetrahydrofuran, 2 mL of 6M hydrochloric acid was added, and the mixture was stirred at room temperature for 12 hours, then concentrated in vacuo to obtain a crude product, which was then washed with ethyl acetate to obtain the target product.

[0175] S-3,3-difluoroallylhomocysteine ​​was identified by NMR, and the results are as follows:

[0176] 1 H NMR (400MHz, D2O) δ4.42–4.29(m,1H),4.01(t,J=6.4Hz,1H),3.07(dm,J=8.4Hz,2H),2.62–2.49(m,2H),2.17–1.97(m,2H).

[0177] 19 F NMR (376MHz, D2O) δ-86.9 (dd, J = 41.4Hz, 1.1Hz, 1F), -89.8 (dd, J = 41.4Hz, 22.6Hz, 1F).

[0178] 13 C NMR (101MHz, D2O) δ171.9, 157.2 (t, J = 287.5Hz), 76.2 (t, J = 24.3Hz), 52.0, 29.3, 25.5, 23.2 (d, J = 6.3Hz).

[0179] HRMS(ESI):Calcd.for C7H 12 F2NO2S:212.0557(M+H) + Found:212.0563(M+H) + .

[0180] The identification results showed that S-3,3-difluoroallylhomocysteine ​​was successfully prepared.

[0181] 2. Synthesis of S-p-fluorobenzylhomocysteine

[0182] 10 mmol (tert-butyloxycarbonyl)-L-homocysteine ​​tert-butyl ester and 10 mmol 1-(bromomethyl)-4-fluorobenzene were dissolved in 20 mL dimethyl sulfoxide, reacted at room temperature for 12 hours, and then extracted with ethyl acetate several times. The extracts were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column to obtain 1.43 g of the product, with a yield of 72%.

[0183] 100 mg of the obtained S-p-fluorobenzyl-(tert-butoxycarbonyl)-L-homocysteine ​​tert-butyl ester was dissolved in 1 ml of tetrahydrofuran, 2 mL of 6M hydrochloric acid was added, and the mixture was stirred at room temperature for 12 hours, then concentrated in vacuo to obtain a crude product, and then washed with ethyl acetate to obtain the target product.

[0184] S-p-fluorobenzylhomocysteine ​​was identified by NMR, and the identification results are as follows:

[0185] 1 H NMR (400MHz, D2O) δ7.41–7.31(m,2H),7.15–7.05(m,2H),4.13(t,J=6.4Hz,1H),3.78(s,2H),2.59(t,J=7.6Hz,2H),2.25–2.05(m,2H).

[0186] 19 F NMR(376MHz,D2O)δ-116.1(m,1F).

[0187] 13 C NMR (126MHz, D2O+NaOH) δ185.4, 164.3 (d, J = 242.8Hz), 137.0 (d, J = 2.6Hz), 133.3 (d, J = 8.2Hz), 118.0 (d, J = 21.5Hz), 57.9, 37.0, 36.8, 29.6.

[0188] HRMS(ESI):Calcd.for C 11 H 15 FNO2S:244.0807(M+H) + Found:244.0802(M+H) + .

[0189] The identification results showed that S-p-fluorobenzylhomocysteine ​​was successfully prepared.

[0190] 3. Synthesis of S-trifluoromethylbenzylhomocysteine

[0191] 10 mmol (tert-butyloxycarbonyl)-L-homocysteine ​​tert-butyl ester and 10 mmol 1-(bromomethyl)-4-trifluoromethylbenzene were dissolved in 20 mL dimethyl sulfoxide, reacted at room temperature for 12 hours, and then extracted with ethyl acetate several times. The extracts were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column to obtain 2.25 g of the product, with a yield of 99%.

[0192] 100 mg of the obtained S-(p-trifluoromethyl)benzyl-(tert-butoxycarbonyl)-L-homocysteine ​​tert-butyl ester was dissolved in 1 ml of tetrahydrofuran, 2 mL of 6M hydrochloric acid was added, and the mixture was stirred at room temperature for 12 hours, then concentrated in vacuo to obtain a crude product, which was then washed with ethyl acetate to obtain the target product.

[0193] S-trifluoromethylbenzylhomocysteine ​​was identified by NMR, and the identification results are as follows:

[0194] 1 H NMR(400MHz,D2O+NaOH)δ7.43(d,J=8.0Hz,2H),7.27(d,J=8.0Hz,2H),3.59(s,2 H),3.14–3.05(m,1H),2.33–2.22(m,2H),1.77–1.64(m,1H),1.60–1.48(m,1H).

[0195] 19 F NMR(376MHz,D2O+NaOH)δ-62.3(s,3F).

[0196] 13 C NMR (126MHz, D2O+NaOH) δ185.2, 145.5, 131.9, 131.1 (q, J = 32.4Hz), 128.1 (q, J = 3.4Hz), 126.9 (q, J = 272.0Hz), 57.9, 37.2, 37.0, 29.8.

[0197] HRMS(ESI):Calcd.for C 12 H 15 F3NO2S:294.0775(M+H) ++ Found:294.0772(M+H) + .

[0198] The identification results showed that S-trifluoromethylbenzylhomocysteine ​​was successfully prepared.

[0199] 4. Synthesis of S-3,5-difluorobenzylhomocysteine

[0200] 10 mmol (tert-butyloxycarbonyl)-L-homocysteine ​​tert-butyl ester and 10 mmol 1-(bromomethyl)-3,5-difluorobenzene were dissolved in 20 mL dimethyl sulfoxide, reacted at room temperature for 12 hours, and then extracted with ethyl acetate several times. The extracts were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column to obtain 2.08 g of the product, with a yield of 99%.

[0201] 100 mg of the obtained S-3,5-difluorobenzyl-(tert-butoxycarbonyl)-L-homocysteine ​​tert-butyl ester was dissolved in 1 ml of tetrahydrofuran, 2 mL of 6M hydrochloric acid was added, and the mixture was stirred at room temperature for 12 hours, then concentrated in vacuo to obtain a crude product, which was then washed with ethyl acetate to obtain the target product.

[0202] S-3,5-difluorobenzylhomocysteine ​​was identified by NMR, and the identification results are as follows:

[0203] 1 H NMR (400MHz, D2O) δ7.03–6.96(m,2H),6.90–6.82(m,1H),4.13(t,J=6.4Hz,1H),3.79(s,2H),2.66–2.57(m,2H),2.24–2.06(m,2H).

[0204] 19 F NMR (376MHz, D2O) δ-110.7 (t, J = 8.3Hz, 2F).

[0205] 13C NMR (126MHz, D2O) δ174.4, 165.5 (dd, J = 247.0Hz, 13.6Hz), 145.0 (t, J = 9.1Hz), 114.4 (dd, J = 19.4Hz, 5.8Hz), 105.2 (t, J = 25.2Hz), 54.4, 37.0, 32.0, 28.5.

[0206] HRMS(ESI):Calcd.for C 11 H 14 F2NO2S:262.0713(M+H) + Found:262.0711(M+H) + .

[0207] The identification results showed that S-3,5-difluorobenzylhomocysteine ​​was successfully prepared.

[0208] Example 2

[0209] Synthesis of Fluorine-Substituted Adenosylmethionine Analogues

[0210] 1. Expression and purification of adenosylmethionine transferase hMAT2A mutant

[0211] The gene encoding the sequence SEQ ID NO.: 1 was constructed into the expression plasmid pET28a to obtain adenosine methyltransferase hMAT2A I322A The above expression plasmid was transformed into E. coli BL21 (DE3), and a single clone was picked and cultured overnight at 37°C in LB (containing kanamycin antibiotics). After dilution at a ratio of 1:100, the culture was expanded and the OD 600 After the pH value reached 0.6-0.8, IPTG was added at a final concentration of 0.1 mmol / L and induced at 25°C for 16 h.

[0212] The cells were collected by centrifugation, crushed by ultrahigh pressure, and the supernatant was collected by centrifugation. The supernatant was chromatographed by Ni column and tested by protein electrophoresis. The results were as follows: Figure 1 As shown. Figure 1 As a result, the target protein adenosylmethionine transferase hMAT2A was obtained I322A Mutant.

[0213] 2. Synthesis of Fluorine-Substituted Adenosylmethionine Analogues

[0214] Use the adenosylmethionine transferase hMAT2A obtained in step 1 I322A The mutant or the wild type hMAT2A is used as a catalyst, and the fluorinated methionine analogs obtained in Example 1 are reacted with ATP to obtain fluorinated adenosylmethionine analogs, as follows:

[0215] The reaction system consisted of 50 mM HEPES (pH 7.5), 50 mM KCl, 3 mM MgCl2, 1 mM fluorinated methionine analog, 1.5 mM ATP and 10 μM hMAT2A wild type or its I322A mutant.

[0216] The reaction conditions were: 30°C, reaction time 1 h.

[0217] The reaction was monitored using HPLC-MS, and the preliminary product was identified by HR-MS. The activity was further measured in conjunction with methyltransferase, and the methylated product was identified by NMR to confirm the correctness of the structure of the fluorinated adenosylmethionine analog.

[0218] When 1mM S-3,3-difluoroallyl-L-homocysteine ​​and 1.5mM ATP were catalyzed by 10μM adenosylmethionine transferase hMAT2A wild type or its I322A mutant, both fluorinated adenosylmethionine analogs IIa could be obtained. The I322A mutant was 7 times more efficient than the wild type, and the fluorinated adenosylmethionine analogs IIa could be obtained efficiently. The results of HPLC-MS detection are as follows: Figure 2 As shown. Figure 2 As a result, the target fluorine-substituted adenosylmethionine analogue was obtained.

[0219]

[0220] Example 3

[0221] Synthesis of fluorine-substituted vancomycin analogs

[0222] 1. Expression and purification of methyltransferase MtfA

[0223] The gene with the coding sequence of SEQ ID NO.: 2 was constructed into the expression plasmid pET28a to obtain the expression plasmid of the methyltransferase in the biosynthesis pathway of vancomycin. The above expression plasmid was transformed into E. coli BL21 (DE3), and a single clone was picked and cultured in LB (containing kanamycin antibiotics) at 37°C overnight, and then diluted at a ratio of 1:100 and expanded. 600 After the pH value reached 0.6-0.8, IPTG was added at a final concentration of 0.1 mmol / L and induced at 25°C for 16 h.

[0224] The cells were collected by centrifugation, crushed by ultrahigh pressure, and the supernatant was collected by centrifugation. The supernatant was chromatographed by Ni column and tested by protein electrophoresis. The results were as follows: Figure 1 As shown. Figure 1 As a result, the target protein methyltransferase MtfA was obtained.

[0225] 2. Expression and purification of SAH hydrolase SAHH

[0226] The gene with the coding sequence of SEQ ID NO.: 3 was constructed into the expression plasmid pET28a to obtain the expression plasmid of SAH hydrolase SAHH. The above expression plasmid was transformed into E. coli BL21 (DE3), and a single clone was picked and cultured in LB (containing kanamycin antibiotics) at 37°C overnight, and then diluted at a ratio of 1:100 and expanded. 600 After the pH value reached 0.6-0.8, IPTG was added at a final concentration of 0.1 mmol / L and induced at 25°C for 16 h.

[0227] The cells were collected by centrifugation, crushed by ultrahigh pressure, and the supernatant was collected by centrifugation. The supernatant was chromatographed by Ni column and tested by protein electrophoresis. The results were as follows: Figure 1 As shown. Figure 1 As a result, the target protein SAH hydrolase SAHH was obtained.

[0228] 2. Synthesis of fluorine-substituted vancomycin analogs

[0229] Using the methyltransferase MtfA obtained in step 1 as a catalyst, the fluorinated adenosylmethionine analogs prepared in situ in Example 2 are reacted with demethylvancomycin to obtain fluorinated vancomycin analogs, as follows:

[0230] The reaction system was 50 mM HEPES (pH 7.5), 50 mM KCl, 3 mM MgCl2, 1 mM fluoro-substituted methionine analog, 1.5 mM ATP, 0.5 mM demethylvancomycin, 20 μM hMAT2A I322A mutants and 20 μM methyltransferase MtfA.

[0231] Using the methyltransferase MtfA and SAH hydrolase SAHH obtained in step 1 and step 2 as catalysts, the fluorinated adenosylmethionine analogues prepared in situ in Example 2 are reacted with demethylvancomycin, respectively, to improve the reaction efficiency and the yield of the fluorinated vancomycin analogues obtained, as follows:

[0232] The reaction system was 50 mM HEPES (pH 7.5), 50 mM KCl, 3 mM MgCl2, 1 mM fluoro-substituted methionine analog, 1.5 mM ATP, 0.5 mM demethylvancomycin, 20 μM hMAT2A I322A mutants, 20 μM SAH hydrolase and 20 μM methyltransferase MtfA.

[0233] The reaction conditions are: 30°C, reaction time 4 to 8 hours.

[0234] HPLC and HPLC-MS were used to monitor the corresponding conversion. After the reverse conversion was complete, the reaction solution was crudely separated using an MCI gel column (chromatographic column CHP20 / P120, Mitsubishi Chemical Corporation, Japan) and further semi-preparatively separated using HPLC (chromatographic column: Waters BEH C18 column) to obtain pure fluorine-substituted vancomycin analogs.

[0235] Preparation of N-3,3-difluoroallyl vancomycin

[0236] 16 mg of norvancomycin was mixed with S-3,3-difluoroallyl-L-homocysteine ​​in the presence of adenosylmethionine transferase hMAT2A I322A Under the catalysis of mutant, methyltransferase MtfA and SAH hydrolase SAHH, fluorinated vancomycin analog Ia was obtained with a conversion rate of more than 99% and an isolation yield of 84%. HPLC-MS detection, the results are as follows Figure 3 shown.

[0237]

[0238] N-3,3-Difluoroallyl vancomycin:

[0239] HRMS (m / z) [M+2H] 2+ [C 68 H 77 Cl2F2N9O 24 ] 2+ calcd:755.7213,found:755.7206( Figure 4 ). HR-MS / MS results are as follows Fig.13 .

[0240] 19 F NMR (470MHz, DMSO): -89.3 (m), -87.7 (d, J = 45.1Hz). ( Figure 8 )

[0241] Figure 6 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 and Fig.12 N-3,3-difluoroallyl vancomycin 1 H(500MHz,DMSO), 13 C(500MHz,DMSO), 19 F(500MHz,DMSO), 1 H-1 HCOSY (500 MHz, DMSO), HSQC (500 MHz, DMSO), HMBC (500 MHz, DMSO) and NOESY (500 MHz, DMSO) spectra.

[0242] The identification results show that the inventors have successfully prepared N-3,3-difluoroallyl vancomycin.

[0243] Preparation of N-p-fluorobenzyl vancomycin

[0244] Combination of norvancomycin with S-p-fluorobenzyl-L-homocysteine ​​in the presence of adenosylmethionine transferase hMAT2A I322A The mutant, methyltransferase MtfA and SAH hydrolase SAHH catalyzed to obtain fluorine-substituted vancomycin analog Ib with a conversion rate of more than 66%. HPLC-MS detection, the results are as follows Fig.14 shown.

[0245]

[0246] N-p-Fluorobenzyl vancomycin:

[0247] HRMS (m / z) [M+2H] 2+ [C 72 H 80 Cl2FN9O 24 ] 2+ calcd:771.7338,found:771.7293( Fig.14 ). HR-MS / MS results are as follows Fig.16 .

[0248] 19 F NMR (470MHz, DMSO): -116.4 (s) ( Fig.15 ).

[0249] The identification results show that the inventors have successfully prepared N-3,3-difluoroallyl vancomycin.

[0250] Preparation of N-trifluoromethylbenzyl vancomycin

[0251] Combination of norvancomycin with S-trifluoromethylbenzyl-L-homocysteine ​​in the presence of adenosylmethionine transferase hMAT2A I322A The mutant, methyltransferase MtfA and SAH hydrolase SAHH catalyzed the fluorinated vancomycin analog Ic with a conversion rate of more than 44%. HPLC-MS detection showed that the results were as follows Fig.17 shown.

[0252]

[0253] N-trifluoromethylbenzyl vancomycin:

[0254] HRMS (m / z) [M+2H] 2+ [C 73 H 80 Cl2F3N9O 24 ] 2+ calcd:796.7322,found:796.7252( Fig.17 ). HR-MS / MS results are as follows Fig.18 .

[0255] The identification results show that the inventors have successfully prepared N-trifluoromethylbenzyl vancomycin Ic.

[0256] Preparation of N-3,5-difluorobenzyl vancomycin

[0257] Combination of norvancomycin with S-3,5-difluorobenzyl-L-homocysteine ​​in the presence of adenosylmethionine transferase hMAT2A I322A The mutant, methyltransferase MtfA and SAH hydrolase SAHH catalyzed the fluorinated vancomycin analog Id with a conversion rate of more than 78%. HPLC-MS detection showed that Fig.19 shown.

[0258]

[0259] N-3,5-difluorobenzyl vancomycin:

[0260] HRMS (m / z) [M+2H] 2+ [C 72 H 79 Cl2F2N9O 24 ] 2+ calcd:780.7291,found:780.7227( Fig.19 ). HR-MS / MS results are as follows Fig. 20 .

[0261] The identification results showed that N-3,5-difluorobenzyl vancomycin Id was successfully prepared.

[0262] Example 4

[0263] Determination of Antibacterial Activity of Fluorosubstituted Vancomycin Derivatives

[0264] The antibacterial activity of fluorinated vancomycin derivatives was determined as follows: In a 96-well plate, fluorinated vancomycin dissolved in dimethyl sulfoxide was added to the culture medium to 4 μg / mL and diluted to 0.000125 μg / mL. The test bacteria were added to the culture medium to 10 7-10 8 cfu / mL (according to the 0.5 McFarland standard) and cultured overnight. The minimum concentration at which the test bacteria cannot grow is the minimum inhibitory concentration (MIC).

[0265] The minimum inhibitory concentration (MIC) test results are as follows:

[0266] Table 1

[0267] MRSA MSSA VRE Vancomycin (1μg / mL) (1μg / mL) (64μg / mL) N-3,3-difluoroallyl vancomycin (8μg / mL) (4μg / mL) (128μg / mL)

[0268] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A vancomycin analog or a pharmaceutically acceptable salt thereof, characterized in that: The vancomycin analog has a structure shown in Formula I: In the formula, Rf is -(CH2) n -Rf', wherein n is an integer selected from 1, 2 or 3; Wherein, Rf' is selected from: halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, or substituted or unsubstituted 5-8 membered heterocycloalkyl; The above substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C3 alkyl, or C1-C3 haloalkyl.

2. The vancomycin analog or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Wherein Rf' is selected from: halogen, substituted or unsubstituted C2-C3 alkenyl, substituted or unsubstituted C5-C6 aryl; The above substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C3 alkyl, or C1-C3 haloalkyl.

3. The vancomycin analog or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: The Rf is selected from the group consisting of 3,3-difluoroallyl, p-fluorobenzyl, p-trifluoromethylbenzyl, and 3,5-difluorobenzyl.

4. A method for preparing the vancomycin analogue according to claim 1, comprising the steps of: In the presence of a methyltransferase, reacting the donor compound of formula (II) with the demethylvancomycin of formula (IV) to produce the vancomycin analogue as claimed in claim 1; In the formula, Rf is -(CH2) n -Rf', wherein n is an integer selected from 1, 2 or 3; in, Rf' is selected from the group consisting of halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, or substituted or unsubstituted 5-8 membered heterocycloalkyl; The above substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C3 alkyl, or C1-C3 haloalkyl.

5. The method according to claim 4, characterized in that The method comprises the steps of: reacting a donor compound of formula (II) with demethylvancomycin represented by formula (IV) in the presence of methyltransferase and SAH hydrolase, thereby producing the vancomycin analogue as claimed in claim 1.

6. An adenosylmethionine analogue, characterized in that: The adenosylmethionine analog has a structure shown in Formula II: In the formula, Rf is -(CH2) n -Rf', wherein n is an integer selected from 1, 2 or 3; Wherein, Rf' is selected from: halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, or substituted or unsubstituted 5-8 membered heterocycloalkyl; The above substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C3 alkyl, or C1-C3 haloalkyl.

7. The adenosylmethionine analogue according to claim 6, characterized in that The adenosylmethionine analogue has a structure shown in the following formula IIa, formula IIb, formula IIc, or formula IId:

8. A method for preparing the adenosylmethionine analogue as claimed in claim 6, comprising the steps of: In the presence of methionine adenosyltransferase or a mutant thereof, reacting the compound represented by the donor formula (III) with adenosine triphosphate (ATP) to produce the adenosylmethionine analogue as claimed in claim 6; In the formula, Rf is -(CH2) n -Rf', wherein n is an integer selected from 1, 2 or 3; in, Rf' is selected from the group consisting of halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C5-C8 aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted C3-C8 cycloalkyl, or substituted or unsubstituted 5-8 membered heterocycloalkyl; The above substitution refers to substitution by one or more substituents selected from the group consisting of halogen, C1-C3 alkyl, or C1-C3 haloalkyl.

9. A pharmaceutical composition comprising: (a) the vancomycin analogue or a pharmaceutically acceptable salt thereof according to claim 1; and (b) a pharmaceutically acceptable carrier.

10. A use of the vancomycin analogue or a pharmaceutically acceptable salt thereof as claimed in claim 1, wherein the use is: (a) for preparing an antibacterial composition; (b) for preparing a composition for inhibiting the growth of microorganisms; (c) for use in the preparation of a composition for treating microbial and / or bacterial infection; (d) Used in the preparation of feed additives.

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