WO2014147578A2 - Composés antibactériens contre des bactéries résistantes aux médicaments - Google Patents

Composés antibactériens contre des bactéries résistantes aux médicaments Download PDF

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WO2014147578A2
WO2014147578A2 PCT/IB2014/059989 IB2014059989W WO2014147578A2 WO 2014147578 A2 WO2014147578 A2 WO 2014147578A2 IB 2014059989 W IB2014059989 W IB 2014059989W WO 2014147578 A2 WO2014147578 A2 WO 2014147578A2
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antibacterial compounds
rifamycin
present
amycolatopsis
mediterrranei
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WO2014147578A3 (fr
WO2014147578A4 (fr
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Rup LAL
Taifo Mahmud
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University of Delhi
Department of Biotechnology of Ministry of Science and Technology India
Oregon State University
State of Oregon
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University of Delhi
Department of Biotechnology of Ministry of Science and Technology India
Oregon State University
State of Oregon
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Priority to CN201480029144.8A priority Critical patent/CN105228617A/zh
Priority to RU2015144539A priority patent/RU2672064C2/ru
Publication of WO2014147578A2 publication Critical patent/WO2014147578A2/fr
Publication of WO2014147578A3 publication Critical patent/WO2014147578A3/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4162—1,2-Diazoles condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to the production of rifamycin analogsagainst drug resistant bacteria.
  • the present invention further also relates to antibacterial compounds against drag resistant mycobacteria.
  • the present invention also provides for pharmaceutical composition comprising antibacterial compound against drug resistant bacteria, particularly mycobacteria.
  • the present invention also provides for use of antibacterial compounds for the treatment of disease caused by bacteria and in particular mycobacteria.
  • Amycolatopsis mediterr mei S699 is an actinobacterium that produces an important antibioti rifamycin B ( Figure 1).
  • Semisynthetic derivatives of rifamycin B are in use against Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of tuberculosis (TB) and leprosy, respectively. These derivatives are also used against a variety of other organisms including AIDS related mycobacteria.
  • Rifamycin B and rifamycins in general belong to the ansamycm class of antibiotics and are characterized by a naphthalene moiety spanned by an aliphati chain like the handle of a basket ( Figure 1). This aromatic core imparts reddish brown colour to the rifamycin molecule.
  • the carbon skeleton in rifamycin B of A. mediterranei S699 is built from two acetate and eight propionate units and a starter unit 3-amino-5-hydroxybenzoic acid (AHBA).
  • Rifamycin B is a native and stable molecule and acts as the starting material for the synthesis of semisynthetic derivatives including rifamycin S , rifamycin SV, rifampicin, rifabutin, rifapentine and rifaximin (that are in clinical use).
  • strain S699 has been subjected to classical strain improvement program since 1960 and successors of this strain are in use for production of rifamycin B.
  • improved industrial strains produce around 15 g/i to 24 g/1 of rifamycin B.
  • Rifabutin (4-N-isobutylspiropiperidyl rifamycin S). It is marketed under the name Mycobutin. It is characterized as being effective against a few strains resistant to rifampicin and has a high activity against Mycobacterium avium complex associated with AIDS.
  • Rifamixin (4-deosy-3'-bromopyrido-[l 2'-l , 2] imidiazo [5, 4-c]-rifamycin SV), rifamycin B derivative is virtually unabsorbed in the intestine post oral administration. This property makes it favorable for treatment of local conditions within gastrointestinal pathogens.
  • Rifapentine (3 ⁇ [(4-cyclopentyl- 1 -piperazinyl) iraino] methyl ⁇ rifamycin SV) possess activity against mycobacteria and a pharmacokinetic profile which allows long lasting action. It is marketed under the name Prifith by Sanofi Aventis. It was approved for treatment of tuberculosis by Food and Drag administration in 1998.
  • A. mediterranei the producer of rifamycin, has an interesting history. It was isolated from a soil sample from a pine arboretum at an altitude of 200 m, about 50 m off the shore of St. Raphael, France. Due to morphological and biochemical similarities, this isolate was classified as Streptomyces mediterranei [Margalith, P. and Beretta, G. (1960). Mycopathol. Mycol. Appl. Vol.13, p.321 -330]. Later it was reclassified as Amycolatopsis mediterranei [Lechevalier et al. (1986). Int. J. Sys. Bacterial. ⁇ Vo ⁇ . 36, p.
  • Lai and co-workers developed a series of cloning vectors (pRL series) which can be used for transformation and cloning of several strains of A. R hinder US5985560A(1999); Lai, R.et al (1991). Appl. Environ. MicrobiolNol.51 , p.665-671; Dhingra, G.et al. ⁇ 2003). Ind.Microbiol. Biotecnol.Yol.30, p.195-204]
  • erythromycin erythromycin
  • rap rapamycin
  • rifamycin rifamycin
  • type 1 PKSs modular polyketide synthase
  • the type I PKSs have a collinear architecture and are composed of modules, each with catalytic domains that determine the order of substrate selection for basic chain assembly and subsequent modification of ⁇ -keto esters (polvketides).
  • a type I PKS module generally consists of ketosynthase (KS), acyl transferase (AT) and acyl carrier protein (ACP) domains which form the minimal PKS.
  • ketoreductase KR
  • dehydratase DH
  • enoyl reductase ER
  • the starter unit acetyl CoA
  • the extending chain is handed over from the ACP of the previous module to the KS of the current module and is catalyzed by the KS domain.
  • AT domain selects the type of extender unit to be added to the growing poiyketide chain
  • KS catalyses the condensation reaction
  • ACP tethers the growing poiyketide chain between successive condensation and accepts extender unit from AT in preparation for the next condensation reaction.
  • the growing poiyketide is modified by the reductive loop.
  • the KR domain reduces the ⁇ -keto group to a hydroxyl group.
  • the DH domain eliminates a molecule of water resulting in ana- ⁇ double bond and the ER domain converts the - ⁇ double bond to a saturated bond.
  • the carbon skeleton is then released from the PKS by the hydrolytic action of a TE domain (Fig tre 3). While the presence of ACP, AT and KS is essential in a module the other domains such as KR, DH, ER are optional and may or may not present in a module.
  • the ORFs comprises of 10 modules, which are coilineariy arranged in accordance with their function in biosynthesis, and catalyze ten successive rounds of poiyketide chain elongation to build an undecaketide (Figure 4).
  • the gene rifA consists of modules 1-3, ri/ of modules 4-6, rifC of module 7, rifi) of module 8 and rifE of modules 9-10.
  • the domain order in the modules is KS-ketosynthase, AT-acyl transferase (optional reductive domains- DH-dehydratase and KR-ketoreductase) and ACP-acyl carrier protein (Figure 5).
  • the domain ER is absent in rifPKS.
  • the rifA gene is proceeded by a loading unit, which activates the starter unit to initiate the poiyketide chain formation.
  • a loading unit which activates the starter unit to initiate the poiyketide chain formation.
  • AT domains There are two classes of AT domains in rifPKS.
  • the AT domains found in modules 2 and 9 catalyze the incorporation of acetate extender units.
  • the other eight modules have AT domains which catalyze the incorporation of propionate extender units into the growing polyketide chain.
  • Another gene, rift present immediately downstream to rift, is translationally coupled to it.
  • the role of RifF is to cyclise and simultaneously release polyketide chain from the ACP domain of module 10 [Strarmaiin, A.et al, ⁇ 999).MicrobiolNol. ⁇ 5, p.3356- 3375].
  • naphthalene moiety occurs between the third and fourth chain elongation step and is not a post PKS modification. This step was crucial in designing the swapping strategy followed in this invention.
  • rifamycin B The semi-synthetic derivatives of rifamycin B, such as rifampicin and rifabutin, have been widely used in the cure of tuberculosis (M. tuberculosis), leprosy ( leprae) and AIDS related mycobacterial infections.
  • Rifampicin was first introduced into the market in 1968. Since then, it has been widely used to cure tuberculosis.
  • MDR multiple drug resistant
  • the main embodiment of the present invention relates to antibacterial compounds and/or salts thereof having following chemical structure:
  • Another embodiment of the present invention relates to antibacterial compounds of the present invention which are useful against infection or a disease caused by bacteria.
  • Another embodiment of the present invention relates to antibacterial compounds of the present invention which are useful against infection or a disease caused by Mycobacterium species.
  • Yet another embodiment of the present invention relates to a method of treatment comprising administering to a patient an antibacterial compounds and/or salts thereof having following structure:
  • Yet another embodiment of the present invention relates to use of antibacterial corapounds and/or salts thereof having following chemical structure for treatment of disease or an infection.
  • Another embodiment of the present invention relates to use of a medicament comprising antibacterial compounds of the present invention for the treatment of a disease or an infection.
  • Yet another embodiment of the present invention relates to use of a medicament comprising antibacterial compounds of the present invention for the treatment of a diseases or an infection caused by bacteria.
  • Another embodiment of the present invention relates to novel strain of Amycolatopsis mediterrranei S699#34 comprising a gene rapAT2 region.
  • Yet another embodiment of the present invention relates to novel strain of Amycolatopsis mediterrranei S699#34 wherein n AT6 region is swapped with rapKTl region.
  • Another embodiment of the present invention relates to novel strain of Amycolatopsis mediterrranei S699#34 capable of producing the analogs and derivatives of rifamycin B comprising following chemical structure:
  • Another embodiment of the present invention relates a method of preparing novel strain of Amycolatopsis mediterrranei S699 comprising rapKY.2 region in n/PKS, said method comprising the steps o :
  • step (b) inserting the vector constructs of step (b) in an Amycolatopsis mediterrranei S699:
  • Another embodiment of the present invention relates to a method of preparing the desmethyrifamycin S derivative of 24-desmethyrifamycin B, said method comprising the steps of : (a) reacting 24-desmethyrifamycin B in presence of reagents selected from Copper chloride;
  • step (b) carrying the reaction of step (a) overnight at room temperature;
  • a method of preparing the 24-desmethyrifampicin derivative of 24-desmethyrifamycin B comprising the steps of;
  • step (b) adding paraformaldehyde and 1 ,3 ,5-trimethyl-hexhydro- 1 ,3,5-triazine to the mixture of step (a);
  • step (d) reacting the compound of step (c) with l-amino-4-emthyl-piperazine;
  • Another embodiment of the present invention relates to a recombinant nucleotide SEQ ID No.1.
  • Another embodiment of the present invention relates to a bacterial strains comprising nucleotide SEQ) ID NO.l .
  • Another embodiment of the present invention relates to bacterial strain/s as described in the present invention comprising nucleotide SEQ ID NO.3
  • Another embodiment of the present invention relates to bacterial strain as described in the present invention, wherein bacterial strain is capable of producing rifamycin analogues.
  • Another embodiment of the present invention relates to vector constructs comprising recombinant nucleotide sequence ID No.l .
  • Yet another embodiment of the present invention relates to vector constracts, wherein vector constracts are pAT6E and pAT6F.
  • Figure 1 Chemical structures of Rifamycin B.
  • Figure 2 Semi-Synthetic Derivatives of rifamycin B.
  • FIG. 1 Schematic representation of processive mechanism of type 1 P S.
  • Figure 5 Organization of the enzyme domain in rif PKS gene cluster.
  • Figure 6 Strategy for constructing functional cassette by swapping rif ⁇ 6 with rapNll.
  • Figure 7 Schematic representation of the strategy to showswapping rif ⁇ 6 with rapNll
  • Figure 8 Phenotypic appearance of cultures showing Single Crossover clones.
  • FIG. 9 Gel Electrophoresis profile for Single Crossover Clones.
  • Figure 11 Gel Electrophoresis profile of Double Crossover Clones.
  • Figure 12 The LC-ESI-MS profile of analog desmethylrifamycin B extracted from DCO.
  • Figure 13 NMR of rifamycin B and its analog.
  • FIG. 14 Structures of 24-desmethylrifamycm B, 24-desmethylrifamycin S and
  • Figure 15 Crystal structure of 24-desmehtylrifamycin S and drug interaction with RNA polymerase A.
  • Figure 17 NMR spectra of (a) 24-desrifamethylrifampicin; (b) chemically synthesized rifampicin and (c) commercially available rifampicin
  • Figure 18 Comparative MS/MS analysisof (a) 240desmethyirifampicin and (b) Rifampicin.
  • Figure 19 Characteristic MS fragments of rifampicin and 24-desmethylrifampicin in ESI negative ion mode.
  • Figure 20 Antibacterial assay of new rifamycin analogs against various bacterial species.
  • Figure 21 Antibacterial assay of activity of rifampicin, rifamycin S, 24-desmethyirifampicin,
  • Combinatorial approach or combinatorial biosynthesis when used in the context of the present invention refers togenetic manipulation of r PKS gene cluster by swapping of rif AT 6 domain of rijVKS gene cluster A. mediterranei S699 with rapATl domain of rapPKS gene cluster of Streptomyces hygroscopicus.
  • Vectors/Cloning vectors when used in the context of the present invention refers to development of series of vectors pAT6A, pAT6B, pAT6C, pAT6D, pAT6E, and pAT6F.
  • pAT6F is a non-replicative piasmid which was transformed into A, mediterranei S699.
  • Derivatives when used in the context of the present invention refers toderivatives obtained or prepared from 24-desmethylrifampicin B.
  • a few exemplified derivatives are 24-desmethylrifampicin,24-desmethylrifamycin S and 24-desmethylrifamycin SV.
  • Analogues when used in the context of the present invention refers toanalogs of rifamycin B, which are prepared by swappingn/AT6 domain with rap AT2 domain or contains rif AT 6 domain replaced by rapATl domain.
  • One such exemplary analog is 24- desmethylrifamycin B.
  • Mutant/ s or mutant strain/ s when used in the context of the present invention refers tostrain/s i mediterranean which rif AT6 domain is replaced with rapATl domain of the PKS-1 system and that are capable of producing analogs and derivatives of rifamycin B.
  • Amycolatopsis mediterranei #34 which produces analog such as 24-desmethylrifamycin B, etc.
  • Antibacterial compound/s when used in the context of the present invention refers to compounds which are capable of inhibiting growth of bacteria or the infection caused by bacterial or the disease caused by bacteria or disease condition caused by bacterial belonging to category of Actinobacteria or any gram negative bacteria or gram positive bacteria.
  • the antibacterial compounds used in the context of the present invention referto antibacterial compounds which are effective in inhibiting the growth of bacteria belonging to Mycohacerium species.
  • theantibacterial compounds used in the context of the present invention referto those antibacterial compounds which are effective in inhibiting any disease or or any infection or disease condition caused by bacteria belonging to Mycobac t erium species.
  • Multi-Drug Resistance/MDR when used in the context of the present invention refers to a condition enabling disease-causing bacteriato resist distinct antimicrobials or antibacterial compounds i.e. chemicals of a wide variety of structure and function targeted at eradicating the bacteria.
  • MDR in the context of the present invention refers to conditions enabling 0isQ&se-c smgAfycobacerium species to resist distinct antimicrobials or antibacterial compounds wherein the such antimicrobials or antibacterial compounds are commonly used antibiotic drags or commercially available antimicrobials or antibacterial compounds.
  • salts thereof when used in the context of the present invention refers any salt, esters, polymorphs, pure forms, isomers, mixtures of isomers, complexes and any other derivatives or analogs of 24-desmethyrifamycin B, 24-desmethyrifamycin S, 24-desmethyrifamycin SV and 24-desmethyrifampicin. More particulars salts thereof such as salt, esters, polymorphs, pure forms, isomers, mixtures of isomers, complexes and any other derivatives should at least comprise of 24-desmethyl- form or structure i.e.
  • the present invention relates to antibacterial compounds which could be effective against bacterial disease or infection. More particularly the present invention relates to the use of antibacterial compounds which can be effectively used against the infection or disease caused by bacteria group of gram positive bacteria falling under the category of Actinobacteria.
  • the antibacterial compounds of the present invention are useful against multi-drug resistant bacteria.
  • the antibacterial compounds of the present invention are useful against such as Mycobacterium species Staphylococcus speciesor strains. Bacillus species or strains, Pseudomonas species or strains and E.Coli strains
  • the present invention relates antibacterial compounds which are useful against the infection or disease caused by Mycobacterium species.
  • these antibacterial compounds are effective against Mycobacterium tuberculosis, Mycobacterium lepraeand Mycobacterium smegmatis.
  • Another aspect of the present invention provides for antibacterial compounds which are useful against multiple drug resistant strains of Mycobacteria species.
  • the mutants of mediterranei S699 were developed for production of the analog of rifamycin B, 24-desmethylrifamycin B, is also proof of concept of combinatorial biosynthesis that sets stage for the production of variety of analogs of rifamycin B by using this approach.
  • Derivatives of this analog are effective against MD strains of M. tuberculosis. More importantly this invention provides a proof of concept for the first time thai ⁇ P S gene cluster can be manipulated beyond module 4 by combinatorial approaches for the production of rifamycin B analogs. The mutant strain can now be improved for commercial use.
  • the present invention provides for combinatorial biosynthesis strategy for manipulating r PKS of A. mediterranei S699.
  • strategy devised allowed swapping the acyltransferase domain of sixth module (AT6) of r PKS(that recruits propionate) with that of acyltransferase domain of the second module of rapPKS (rap ⁇ 2 from Strepiomyces hygroscopicus (having Accession number: X86780.1 and DSM Culture Collection- DSM 41524) (which recruits acetate) onto the growing chain.
  • the swapping of these regions resulted in development and production of a produced a new analog of rifamycin B which is 24-desmethylrifamycin B.
  • the present invention also devised strategy to develop the derivatives of 24-desmethylrifamycin B,
  • the new analogs and the derivatives developed were found to effective against variety of bacterial species ( Figures 20-21). More specifically these new analogs and derivative were found to be effective against MDR strains of M. tuberculosis .
  • n PKS n PKS gene
  • the steps involved in the synthesis of rifamycin poiyketide backbone, especially na thaquionone ring the target was the rif A.T 6 region of the r /PKS gene.
  • This rif AT 6 region of the n/PKS gene cluster was swapped with rapKTl using domai -replacement strategy.
  • 24-desmethylrifamycin B and its derivatives 24-desmethylrifamycin SV, 24- desmethylrifamycin S& 24-desmethylrifampicin, were not only produced in comparable amounts but also showed stronger antibacterial activity against Staphylococcus aureus, Mycobacterium smegmatis, Bacillus subtil lis, Pseudomonas aeruginosa, Escherichia coll
  • the present invention also provides for mutant strains of A. mediterranei S699 which are capable of producing the analog/s of rifamycin B, i.e. 24-desmethylrifamycin B.
  • the present invention also provides for method developing the mutant strains of A. mediterranei S699. It has been found in the present invention that the strains of A. mediterranei S699 were found to be quite stable in the multiplication and also in the expression of the analogs of the rifamycin B. The analog production from these mutants has been carried out at a laboratory scale. The production yield was about 50 mg/L of the analog 24-desmethylrifamycin B.
  • mediterranei S699 show that there is significant difference in the NMR profiles of rifamycin B and 24-desmethyirifamycin B (Figure 13).
  • the NMR profiles show that 24-desmethylrifamycin B has quasi-molecular ions of m/z 740 and that of 24-desmethylrifamycin SV is m/z 682, which is 14 atomic mass unit less than the rifamycin B and rifamycin SV.
  • the rifamycin resistance is associated with genetic alterations in an 81 -bp region of the rpoB gene encoding the DNA-dependent RNAP P-subunit(Williams et al, 1998, Mycobacterium tubercuiosis.Antimicrob.Ageiits. Chemother., Vol.42, pages 1853-1857).
  • the present invention uses rifampicin-resistant M, tuberculosis sixains, OSDD 321 and OSDD 206, which contain S531 L mutation, and OSDD 55, which has H526T mutation in their RNAP ⁇ -subunit. The mutations were confirmed in OSDD strains.
  • the present invention finds that the drug resistant mutations disrupt hydrogen bonding in the poiyketideansa chain and a possible salt bridge (Figure 15b).
  • the loss of the methyl group in 24-desmethylrifampicin may lead to conformational changes in the ansa chain that allow for more flexibility of the compound to bind mutated RNAPs. Accordingly, it is believed thai, in addition to favourable partition coefficient, the flexibility arising from difference in gauche, syn pentane, and eclipsing interactionsof the ansa chain and the changes in the hydrogen bonding network associated with the conformational changes (Figure 15c) allow for increase binding affinities in both the wild type and drag resistant strains.
  • the present invention provides the most unexpected and unique finding in the present invention is the activity of 24- desmethylrifampicin against rifampicin sensitive and rifampicin resistant strains of M. tuberculosis, 24-desmethylrifampicin showed stronger activity' against rifampicin resistant strains of M. tuberculosis.
  • this finding of the present invention opens a complete hope for the patients suffering from Tuberculosis where the mycobacteria has become resistant to the existing rifampicin drug or related drugs.
  • the present invention demonstrate creation of novel analogs and derivatives of rifamycin B by showing for the first time that the rifPKS is amenable to domain replacement modification, leading to the formation of new rifamycin analog.
  • the present invention provides for a unique recombinant sequence ID No. l ( Figure 22) which is capable of producing analogues of rifamycin.
  • the present invention also provides for vector construct comprising the recombinant sequence ID No.l.
  • the present invention provides for novel strain of Amycolatopsis mediterranei S699 #34 carrying the nucleotide sequence ID No.1. More specifically the novel strain is having the reference number as Amycolatopsis mediterrranei S699 #34 carrying the recombinant SEQ ID No. l ( Figure 22), which provides the unique characteristic to the sixains and this unique characteristic allows the novel strains to express or produce rifamycin analogues, such as 24-desmethylrifamycin B and 24- desmethylrifamycin S .
  • the present invention also provides for pharmaceutical compositions of the antibacterial compounds and their salts thereof described in the present invention which are effective against the bacterial species, particularly mycobacteria.
  • the pharmaceutical composition of the present invention isintended for parenteral and oral administration.
  • the pharmaceutical composition described as herein in the present invention can be administered parenteraily for example, intravenously, subcutaneously, intradermaliy or intramuscularly.
  • the present invention also provides for agents which function as "pharmaceutically acceptable excipient", wherein the term “pharmaceutically acceptable excipient” means a pharmaceutically acceptable formulation carrier, solution or additive to enable the delivery, dissolution or suspension of the antibacterial compounds herein as described.
  • the pharmaceutical composition of the present invention may also contain pharmaceutically accepted auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like.
  • the pharmaceutical composition of the present invention may also contain pharmaceutically acceptable carriers, for example adjuvants, etc.
  • the pharmaceutical compositions of this invention may also be administered in any convenient form, for example tablet, capsule, injection, granule or powder form, e.g. in a sachet.
  • the main embodiment of the present invention relates to antibacterial compounds having following chemical structure:
  • Another embodiment of the present invention relates to antibacterial compounds of the present invention which are useful against infection or a disease caused by bacteria.
  • Another embodiment of the present invention relates to antibacterial compounds of the present invention which are useful against infection or a disease caused by Mycobacterium species.
  • Another embodiment of the present invention relates to antibacterial compounds of the present invention which are useful against infection or a disease caused by Mycobacterium species, where Mycobacterium species selected are Mycobacterium tuberculosis and MDR strains of tuberculosis.
  • Another embodiment of the present invention relates to a pharmaceutical composition comprising antibacterial compounds of the present invention which are useful against infection or a disease caused by bacteria.
  • Another embodiment of the present invention relates to a pharmaceutical composition comprising antibacterial compounds of the present invention which are useful against infection or a disease caused by Mycobacterium species.
  • Yet another embodiment of the present invention relates to pharmaceutical composition comprising antibacterial compounds of the present invention which are useful against infection or a disease caused by Mycobacterium tuberculosis and MDR strains of M. tuberculosis.
  • Yet another embodiment of the present invention relates to a method of treatment comprising administering to a patient an antibacterial compounds or salts thereof having following structure: 24-desmethylrifampicin
  • Yet another embodiment of the present invention relates to use of antibacterial compounds or salts thereof having following chemical structure for treatment of disease or an infection.
  • Another embodiment of the present invention relates to use of a medicament comprising antibacterial compounds of the present invention for the treatment of a disease or an infection.
  • Yet another embodiment of the present invention relates to use of a medicament comprising antibacterial compounds of the present invention for the treatment of a diseases or an infection caused by bacteria.
  • Another embodiment of the present invention relates to use of medicament comprising antibacterial compounds of the present invention for the treatment of a diseases or an infection caused by caused by Mycobacterium species.
  • Yet another embodiment of the present invention relates to use of medicament comprising antibacterial compounds of the present invention as claimed in claim 10 or 13» for the treatment of a diseases or an infection caused by Mycobacterium species, Mycobacterium tuberculosis and MDR strains of M, tuberculosis.
  • Another embodiment of the present invention relates to a method of preparing a pharmaceutical composition comprising antibacterial compounds of the present invention along with pharmaceutical acceptable carrier/s.
  • Another embodiment of the present invention relates to novel strain of Amycolatopsis mediterranei S699 #34 comprising a gene rap ⁇ 2 region.
  • Another embodiment of the present invention relates to novel strain of Amycolatopsis mediterranei S699 #34 capable of producing the analogs and derivatives of rifamycin B comprising following chemical structure:
  • Another embodiment of the present invention relates a method of preparing novel strain of Amycolatopsis mediterranei S699 #34 comprising rapKTl region in ⁇ PKS, said method comprising the steps of :
  • step (c) inserting the vector constructs of step (b) in an Amycolatopsis mediterrranei S699;
  • Another embodiraent of the present invention relates to a method of preparing the 24- desmethyrifamycin S derivative of 24-desmethyrifamycin B, said method comprising the steps of :
  • step (b) carrying the reaction of step (a) overnight at room temperature;
  • a method of preparing the 24-desmethyrifampicin derivative of 24-desmethyrifamycin B comprising the steps of :
  • step (b) adding paraformaldehyde and l,3,5-trimethyl-hexhydro-l,3,5-triazine to the mixture of step (a);
  • step (d) reacting the compound of step (c) with l-amino-4-emthyl-piperazme;
  • Another embodiment of the present invention relates to a recombinant nucleotide SEQ ID No.l .
  • Another embodiment of the present invention relates to a bacterial strain/s of the present invention comprising nucleotide SEQ ID NO. i
  • Another embodiment of the present invention relates to Amycolatopsis mediterrranei S699 #34 strain comprising nucleotide SEQ ID NO.I
  • Another embodiment of the present invention relates to bacterial strain of the present invention, wherein bacterial strain is capable of producing rifamycin analogues.
  • Another embodiraent of the present invention relates to Amycolatopsis mediterrranei S699 #34 strain, wherein bacterial strain is capable of producing rifamycin analogues.
  • Another embodiment of the present invention relates to a recombinant nucleotide SEQ ID No.l , capable of expressing novel analogues of rifamycin.
  • Another embodiment of the present invention relates to a recombinant nucleotide sequence of the present invention, wherein the analogues are 24-desmethyrifamycin B and 24-desmethyrifamycin S.
  • Another embodiment of the present invention relates to vector constructs comprising recombinant nucleotide sequence ID No.l ,
  • Yet another embodiment of the present invention relates to vector constructs, wherein vector constructs are pAT6E and pAT6F.
  • Another embodiment of the present invention relates to a bacterial strain comprising the vector constructs as described in the present invention.
  • Another embodiment of the present invention relates to Amycolatopsis mediterrranei S699#34 strain comprising vector constructs as described in the present invention.
  • Another embodiment of the present invention relates Amycolatopsis mediterrranei S699#34 strain carrying a pAT6F vector wherein the pAT6F vector comprises of a nucleotide sequence No.l.
  • Another embodiment of the present invention relates to a bacterial strain capable of carrying pAT6F vector wherein the pAT6F vector comprises of a nucleotide sequence No.l.
  • Another embodiment of the present invention relates to a microbial strain capable of carrying pAT6F vector wherein the pAT6F vector comprises of a nucleotide sequence No.l.
  • Another embodiment of the present invention relates to a microbial strain capable of expressing nucleotide sequence No.l and/or its products thereof.
  • Routine genetic procedures such as genomic DNA isolation (according to CTAB- Cetyltrimethyl Ammonium Bromide, method), plasmid isolation (Promega DNA purification kit), and restriction endonuclease digestion were carried out by standard techniques.
  • a plasmid pAT6F was constructed that was transformed (using BioRadGenePulser) into A, mediterranei S699 using the method described previously [Dhingra, G. et al. (2003). J. Ind. Microbiol. BiotechnolN o ⁇ . 13, p.
  • these SCOs were cultured for 3-4 rounds in YMG medium without erythromycin.
  • the cells were plated both on YMG agar plate with and without erythromycin pressure and colonies that were antibiotic sensitive were selected.
  • the double crossover (DCO) clones were further confirmed by Southern blot hybridization.
  • Southern hybridization the genomic DNA was immobilized on a Hybond N+ membrane (Amersham Biosciences). Hybridization was performed at 65°Cfor 12h [ - j2 P]-labeled DNA probes. For nonradioactive methods, hybridization was carried out using DIG-labeled DNA probe at 65°C.
  • the flanking region of AT6 also called PCR I (41862- 43533bp) located upstream of rif AT6 was amplified using oligoprimers, primers 1 and 11 (Table 1) as forward and reverse primers respectively.
  • the restriction sites Xhal and Ba were introduced at 5 ' ends of the primer 1 and II respectively in order to facilitate the cloning of PCR I in the Smal site of the pUC 18. These sites were subsequently used for cloning.
  • PCR II (44488-45989bp) located downstream of rif AT 6 was amplified using the primer pairs primer III and primer IV (Table 2). Avrll site was introduced in primer III and Xhal in primer IV at the 5 ' end for subsequent cloning in pUC 18.
  • Table 2 The sequence of the primers used to amplify PCR I and PCR II.
  • IInn oorrddeerr ttoo rreevveerrs see tthhee oorriieennttaattiioonn tthhee PPCCRR II wwaass eexxcciisseedd wwiitthh EEccooRRVV HHiinnccMMll ddoouubbllee ddiiggeessttiioonn aanndd iinnttrroodduucceedd iinn ppUUCC1199 ttoo fifinnaallllyy ggeett tthhee ppllaassmmiidd,, ppAATT66CC..
  • the plasmid pAT6E was digested with Xbal to release PGR I-rapAT2-PCR II fragment (approx. 4 kb) and cloned in Xbal digested vector pIJ4026, containing erythromycin resistance gene.
  • pIJ4026 (provided by M. J. Bibb, John Innes Institute, Norwich) ( Figure6) is an is. coli plasmid but contains the ermE gene that confers resistance to erythromycin and is expressed only in A. mediterranei but not in E. coli.
  • the final construct comprises of 3.85 fragment consisting of PGR I, rapAT2 and PCRII fragments in pIJ4026 was named pAT6F, The 3.85 kb fragment designated as SEQ ID NO.l ( Figure 22).
  • This plasmid pAT6F was transformed by electroporation (7.5 V/Cm, 1000 ⁇ , 25 ⁇ ) into A. mediterranei S699.
  • the single cross over clones were selected under erythromycin pressure in GYM agar media (Glucose 4g/l; Malt Extract l Og/I; Yeast Extract 4g/l).
  • the single cross over (SCO) clones which did not show brown pigmentation (due to absence of rifamycin production as the biosynthetic pathway was blocked by the integration of pAT6F into the chromosome through homologous recombination were selected ( Figure 7 & 8).
  • the plasmid pTA6F contained the nucleotide sequence ID No. I as described in the present invention and represented in Figure 22.
  • Spores of the mutant strain were initially grown on a shaker in YMG medium for 3 days at 30°C and 200 rpm.
  • the seed culture was then used to inoculate (10%, v/vj YMG medium (10 x 100 mL) in 500 raL flasks. After incubation for 10 days under the same conditions, the cultures were centrifuged, the pooled supematants acidified to pH 3 with IN HC1, and the metabolites extracted with ethyl acetate (2 x 1 L).
  • 24-desmethylrifamycin B (8 mg, 0.0107 mmol) was dissolved in MeOH-H 2 0 (10: 1 , 5mL) containing CuCl 2 (0.1 mM). The reaction mixture was stirred at RT overnight to convert 24-desmethylrifamycin B to 24-desmethylrifamycin S. The mixture was acidified to pH 3 and the product was extracted with ethyl acetate (3 x 5mL). The extract was subjected to silica gel column using CHCl 3 -MeOH (10: 1) as eluent to give 24-desmethylrifamycin S (6 mg).
  • 24-desmethylrifamycin S (5mg, 0.0073 ramol) was dissolved in DMF (200 ⁇ ) and acetic acid (50 ⁇ ). After stirring the mixture at 50°C, paraformaldehyde (3mg) and l,3,5-trimethyl-hexahydro-l,3,5- triazine (8 ⁇ iL) were added. The reaction was stirred at 50°C for 2h until all starting material was converted to 3-methyi-l,3-oxazino(5,6-c)-24-desmethylrifarnycin, indicated by a blue spot on TLC, Subsequently, l-amino-4-methyl-piperazine (8 ⁇ ) was added to the mixture.
  • Antibacterial activity of rifampicin and its analogues and derivatives i.e. 24-desmethyrifamycin B, 24-desmethyrifamycin S, 24-desmethyrifamycin SV and 24-desmethyriampicin also determined by agar diffusion assay.
  • Mycobacterium smegmatis, Mycobacterium smegmatis, Bacillus subtilis, Staphylococcus aureus and Escherichia coli were streaked on nutrient agar and incubated overnight at 37 °C. Colonies were transferred to nutrient broth and incubated at 37 °C for 24 h. The growth of the cultures was measured to a proper density at 600 nm(BioRad, SmartSpec 300). Inoculum (ImL) was mixed thoroughly with warm nutrient agar (24 mL) and poured into petri dishes. The agar plates were allowed to solidify and dry for 30 min before assay.
  • MDR strains were procured from Open Source Drug Discover (OSDD; www.osdd.net) and the drug testing was performed at Prernas Biotech, Haryana, India, according to WHO guidelines.
  • the drug sensitivity tests were carried out against tworifampicin-sensitive and three-resistant strains of M. tuberculosis:OSDO 209 & H37Rv and OSDD 55, OSDD 206 & OSDD 321 , respectively (Table 3).
  • Table 3 Comparitive data of the drag Sensitivity assays on various Mycobacterium tuberculosis (resistant and sensitive) strains (procured from OSDD) against commercially available rifampicin and the novel compound 24-desmethylrifamycin S & 24-desmethylrifampicin.
  • Rifampicin (commercially available from HiMedia), 24-desmethylrifampicin and 24- desmethylrifamycin Swere tested against the above mentioned pathogenic strains. The tests were done at various concentrations (0.01 - SO ⁇ xg/rnL) of drugs using BacT/ALERT MB System[Crump, J. A. et ah (2011). J. Clin. Microbiol. Vol. 49, p. 3054-3057]. The results revealed that 24-desmethylrifamycin S &24-desmemylrifampicin showed strong antibacterial activity against both rifamycin-sensitive and - resistant strains of M, tuberculosis.
  • Drug sensitivity assays were done by Premas Biotech, Haryana, India, using various concentrations of drugs (0.0 i - i Lig/ml).
  • the drug testing was done using MB BacT/ALERT SystemSystem[Crump, J. A. et al. (201 1 ). J. Clin. Microbiol. Vol. 49, p. 3054-3057], which is a mycobacterial detection system that utilizes a colorimetric sensor and reflectance detector to determine the level of carbon dioxide within the bottle.
  • C0 2 With the growth of microorganism, there is production of C0 2 resulting in color change of the sensor (at the bottom of the bottle).
  • concentration of CO ? increases there is change in color from green to yellow.
  • the bottle contains a media and MB/BacT Reconstitution fluid, which promotes the growth of mycobacteria.
  • the sample is inoculated into MP BacT/ALERT bottle.
  • the testing is performed with two controls: Direct growth control (DGC) and Proportionate growth control (PGC).
  • DGC involves O. imL seed culture into MP BacT/ALERT bottle along with 0.5ml reconstitution fluid.
  • PGC involves 0.5 mL of DGC into MP BacT/ALERT bottle along with 0.5ml reconstitution fluid.
  • Test Bottle involves 0.5 mL seed culture into MP BacT/ALERT bottle along with 0.5 mL reconstitution fluid as well as antibiotic. The test is considered as complete when PGC bottle flags positive.
  • gacgtatccg gcggcgagct C3.CCQciC3.Q3. 2280 gtgaccgacc accgcatccg gccgtacacc ccacgattcc agcaacccga acagagccac 2340 ctgcaaagcg aacagggccg gctgggcata cccggtctca ttcacatcga gatcgggcac 2400 atccagcaga tcccacacct gctgatggat ccgcgcgaag acggggaacg cggcggccag 2460 ttcctcaccc 3, " t SLC C 3.C) C 3.C gctgcgaccc tggccagccg gctgggcata cccggtctca 2520 ttcacatcga gatcgggcac atccag

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Abstract

L'invention concerne la production d'analogues de rifamycine contre des bactéries résistantes aux médicaments. L'invention concerne également des composés antibactériens contre des mycobactéries résistantes aux médicaments. L'invention concerne également une composition pharmaceutique comprenant un composé antibactérien contre des bactéries résistantes aux médicaments, notamment des mycobactéries. L'invention concerne également l'utilisation de composés antibactériens pour le traitement d'une maladie causée par des bactéries, et notamment des mycobactéries.
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