RU2672064C2 - Antibacterial compounds against drug resistant bacteria - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to compounds useful in medicine having the structure:

.

New analogues of rifamycin, effective for the treatment of drug-resistant bacteria, especially mycobacteria, as well as a method for the preparation of such compounds, a pharmaceutical composition based on them and a method for treating bacterial infections with their use. Proposed method for the preparation of 24-desmethylrifamycin S involves the conversion of 24-desmethylrifamycin B to 24-desmethylrifamycin S in the presence of copper chloride overnight at room temperature to give 24-desmethylriphamycin S.

EFFECT: antibacterial compounds against drug-resistant bacteria have been proposed.

14 cl, 8 ex, 4 tbl, 22 dwg

Description

FIELD OF THE INVENTION

The present invention relates to the production of rifamycin analogues against drug-resistant bacteria. The present invention also relates to antibacterial compounds against drug-resistant mycobacteria. The present invention also provides a pharmaceutical composition comprising an antibacterial compound against drug-resistant bacteria, in particular mycobacteria. The present invention also provides the use of antibacterial compounds for the treatment of a disease caused by bacteria and, in particular, mycobacteria.

BACKGROUND OF THE INVENTION

Amycolatopsis mediterranei S699 (ATCC 13685) are actinobacteria that produce important antibiotic rifamycin B (Figure 1). Semi-synthetic rifamycin B derivatives are used against Mycobacterium tuberculosis and Mycobacterium leprae, pathogens of tuberculosis (TB) and leprosy, respectively. These derivatives are also used against a number of other organisms, including AIDS-associated mycobacteria. Rifamycin B and rifamycins generally belong to the ansamycin-class of antibiotics and are characterized by a naphthalene moiety pulled together by an aliphatic chain like a basket handle (Figure 1). This aromatic core gives a reddish-brown color to the rifamycin molecule. Carbon skeleton in rifamycin In bacteria bacteria A. mediterranei S699 is built from two acetate and eight propionate units and a starting unit of 3-amino-5-hydroxybenzoic acid (ANVA). Rifamycin B is a native and stable molecule and acts as a starting material for the synthesis of semisynthetic derivatives, including rifamycin S, rifamycin SV, rifampicin, rifabutin, rifapentin and rifaximin (which are in clinical use). In addition, since 1960, strain S699 has undergone a classic strain improvement program, and the successors of this strain are used to produce rifamycin B. Currently, improved industrial strains produce from about 15 g / l to 24 g / l rifamycin B.

Semi-synthetic derivatives of rifamycin B, as previously mentioned, have been developed to improve the pharmacokinetics of molecules (Figure 2). Rifampicin (3- [4-methylpiperazinylimino-methyl] rifamycin SV) was first approved for clinical use in Italy in 1968. He received the approval of the Food and Drug Administration in the United States in 1971 [Sensi, P. et al. (1983). Farmac. Ed. Sc. Vol. 14, p. 146-147]. It was the first semi-synthetic derivative of rifamycin B, it is widely used in the treatment of tuberculosis. It binds to M. tuberculosis RNA polymerase and blocks the extension of the RNA chain.

Another rifamycin B derivative is rifabutin (4-N-isobutylspiropiperidyl rifamycin S). It is sold under the name Mucobutin [Mycobutin]. It is characterized as an effective agent against several rifampicin-resistant strains and is highly active against the Mycobacterium avium complex associated with AIDS.

Rifamixin (4-deosi-3'-bromopyrido- [1 ', 2'-1,2] imidiazo [5,4-c] rifamycin SV), a derivative of rifamycin B, is practically not absorbed in the intestine when taken orally. This property makes it favorable for the treatment of local conditions in the pathogens of the gastrointestinal tract.

Rifapentin (3 {[((4-cyclopentyl-1-piperazinyl) imino] methyl} rifamycin SV) has activity against mycobacteria and a pharmacokinetic profile that provides a long-lasting effect. Sold under the name Prifith by Sanofi Aventis. It was approved for the treatment of tuberculosis by the Food and Drug administration in 1998.

A. mediterranei, a producer of rifamycin, has an interesting history. This species was isolated from a soil sample from a pine arboretum at an altitude of 200 m, about 50 m from the coast of St. Raphael, France. Due to morphological and biochemical similarities, this isolate has been classified as Streptomyces mediterranei [Margalith, P. and Beretta, G. (1960). Mycopathol. Mycol. Appl. Vol. 13, p. 321-330]. It was later classified as Amycolatopsis mediterranei [Lechevalier et al. (1986). Int. J. Sys. Bacteriol. Vol. 36, p. 29-37]. The author Lal and his colleagues developed a series of cloning vectors (pRL series) that can be used to transform and clone several strains of A. mediterranei [Lal, R., US 5985560 A (1999); Lal, R. et al. (1991). Appl. Environ. Microbiol. Vol. 57, p. 665-671; Dhingra, G. et al. (2003). Ind. Microbiol. Biotecnol. Vol. 30, p. 195-204].

It is worth noting that the synthesis of erythromycin (ery), rapamycin (rap) and rifamycin (rif) is mediated through modular polyketide synthase (multi-enzyme complexes), also known as PKS type I, which build these macrocyclic polyketides by condensation of acetate and propionate units. Type I PKSs have a collinear architecture and consist of modules, each with catalytic domains, which determine the order of substrate selection for assembly of the base chain and subsequent modification of β-ketoesters (polyketides). A type I PKS module typically consists of ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domains, which form a minimal PKS. Optionally, ketoreductase (KR), dehydratase (DH), or enoyl reductase (ER) domains (also known as reduction domains) may be present to cause modifications in the ketoester chain. The launch unit (acetyl-CoA) is loaded onto the ACP, catalyzed by the AT domain of the launch module. The extension circuit is transferred from the ACP of the previous module to the KS of the current module and is catalyzed by the KS domain. The AT domain selects the type of extension unit to add polyketide to the growing chain, KS catalyzes the condensation reaction, and the ACP binds the growing polyketide chain between sequential condensation and accepts the extension unit from AT in preparation for the next condensation reaction. The growing polyketide is modified using a reduction loop. The KR domain leads the β-keto group to the hydroxyl group. The DH domain excludes water molecules, resulting in an α-β double bond, and the ER domain converts the α-β double bond into a saturated bond. The carbon skeleton is then released from the PKS due to the hydrolytic action of the TE domain (Figure 3). While the presence of ACP, AT, and KS is important in the module, other domains, such as KR, DH, ER, are optional and may or may not be present in the module.

After the discovery of the eryPKS and rapPKS gene clusters, the entire rifPKS gene cluster, 90 kbp [kb - one thousand pairs of nucleotides], responsible for the biosynthesis of rifamycin B, was cloned from A. meditteranei S699 [August, P.R. et al. (1998). Chem Biol. Vol. 5, p. 69-71]. The biosynthetic cluster of genes is divided into four sections. Patch II (52 kb) is the largest patch and encodes modular PKS. This plot is divided into five ORFs - rifA, rifB, rifC, rifD and rifE. ORF sites include 10 modules that are collinear in accordance with their function in biosynthesis, they catalyze ten consecutive rounds of polyketide chain extension with the construction of andecatatide [undecaketide] (Figure 4). The rifA gene consists of modules 1-3, rifB from modules 4-6, rifC from module 7, rifD from module 8, and rife from modules 9-10. The order of the domains in the modules is KS-ketosynthase, AT-acyltransferase (optional recovery domains-DH-dehydratase and KR-ketoreductase) and ACP-acyl carrier protein (Figure 5). ER domain is missing from rifPKS. The rifA gene is preceded by a boot unit, which activates a launch unit to initiate the formation of a polyketide chain. There are two classes of AT domains in rifPKS. AT domains found in modules 2 and 9 catalyze the implementation of acetate extension units. The remaining eight modules have AT domains that catalyze the introduction of propionate extension units into the growing polyketide chain. Another gene, RifF, present immediately after rifE, is translationally linked to it. The role of rifF is to cyclize and simultaneously release the polyketide chain from the ACP domain of module 10 [Stratmann, A. et al. (1999). Microbiol. Vol. 145, p. 3356-3375].

The formation of a naphthalene fragment occurs between the third and fourth steps of chain extension and is not a post-PKS modification. This step was crucial in developing the swapping strategy that is implemented in this invention.

The use of rifamycin derivatives, especially rifampicin, in clinics has led to a significant reduction in TB mortality. However, a combination of poor compliance and poor medical supervision has led to the emergence of multidrug-resistant Mycobacterium tuberculosis (MDR) strains. According to a 2011 WHO report, Tuberculosis Control in the Southeast Asia Region [Tuberculosis Control in the South East Asia Region], 2.3% of MDR-TB cases among new TB cases have been reported in India. The structural complexity of rifamycin B, as mentioned earlier, limits the use of chemicals only to the C-3 and C-4 molecules. This reduces the number of permutations and combinations of altered molecules that can be generated by chemical synthesis. There is an urgent need to produce more rifamycin analogues for effective and economical treatment of MDR-TB.

Semi-synthetic rifamycin B derivatives such as rifampicin and rifabutin are widely used in the treatment of tuberculosis (M. tuberculosis), leprosy (M. leprae) and AIDS-associated mycobacterial infections. Rifampicin first entered the market in 1968. Since then, it has been widely used to treat tuberculosis. However, a combination of poor compliance and poor medical supervision led to the emergence of multidrug-resistant (MDR) strains of M. tuberculosis. Pharmaceutical companies and the medical world are now in search of cost-effective drugs that will be effective against MDR-TB. The complexity of the chemical structure of rifamycin B allows a chemical change, as mentioned above, only on the C-3 and C-4 aromatic nuclei by chemical modifications that are fully used.

Another option is combinatorial biosynthesis, which includes the permutation / removal / addition of modules / domains to an existing PKS gene cluster in rifamycin-producing organisms to create an unusual set of genes that can produce molecules of interest. Polyketide synthase gene clusters that have a collinear arrangement can be shuffled in this way and demonstrate the ability to produce erythromycin analogues. Although much work has been done in this direction on erythromycin molecules (the literature cited in the previous section), there are no reports regarding manipulations with rifPKS. Therefore, in this area there is a need to develop molecules of interest with a better and higher degree of effectiveness in the treatment of tuberculosis, as well as the development of new molecules to which TB bacteria are not resistant.

SUMMARY OF THE INVENTION

Accordingly, a basic embodiment of the present invention relates to antibacterial compounds and / or their salts having the following chemical structure:

and their salts.

Another embodiment of the present invention relates to antibacterial compounds in accordance with the present invention, which are suitable against infection or disease caused by bacteria.

Another embodiment of the present invention relates to antibacterial compounds in accordance with the present invention, which are useful against infections or diseases caused by Mycobacterium species.

Another embodiment of the present invention relates to antibacterial compounds in accordance with the present invention, which are suitable against infection or disease caused by Mycobacterium species, where the selected Mycobacterium species are Mycobacterium tuberculosis and MDR strains of M. tuberculosis.

Another embodiment of the present invention relates to a pharmaceutical composition comprising antibacterial compounds and / or their salts, having the following chemical structure:

together with their pharmaceutically acceptable carriers.

Another embodiment of the present invention relates to a method of treatment, comprising administering to a patient antibacterial compounds and / or their salts, having the following structure:

Another embodiment of the present invention relates to the use of antibacterial compounds and / or their salts having the following chemical structure for treating a disease or infection

to get the drug.

Another embodiment of the present invention relates to the use of a medicament containing the antibacterial compounds of the present invention for the treatment of a disease or infection.

Another embodiment of the present invention relates to the use of a medicament containing antibacterial compounds in accordance with the present invention, for the treatment of diseases or infections caused by bacteria.

Another embodiment of the present invention relates to a new strain of Amycolatopsis mediterranei S699 # 34, comprising a portion of the rapAT2 gene.

Another embodiment of the present invention relates to a new strain of Amycolatopsis mediterranei S699 # 34, in which the rifAT6 region is replaced by the rapAT2 region.

Another embodiment of the present invention relates to a new strain of Amycolatopsis mediterranei S699 # 34, capable of producing analogues and derivatives of rifamycin B containing the following chemical structure:

Another embodiment of the present invention relates to a method for producing a new strain of Amycolatopsis mediterranei S699 containing a rapAT2 region in rifPKS, said method comprising the following steps:

(a) isolation of the rifAT6 region of rifPKS from Amycolatopsis mediterrranei S699 and rapAT2 region of Streptomyces hygroscopicus (According to No. DSM No. 41524);

(b) obtaining vector constructs by replacing the rifAT6 region of the rifPKS rapAT2 with the region of Streptomyces hygroscopicus (According to No. DSM - 41524);

(c) inserting the vector constructs obtained in step (b) into Amycolatopsis mediterranei S699; and

(d) obtaining a new strain of Amycolatopsis mediterrranei S699 # 34 containing the rapAT2 region in rifPKS.

Another embodiment of the present invention relates to a method for producing new antibacterial compounds, said method comprising the following steps:

(a) culturing Amycolatopsis mediterranei S699 # 34 cells containing the rapAT2 region in the rifPKS region; and

(b) collecting Amycolatopsis mediterrranei S699 # 34 cells to obtain crude product fractions;

(c) purification of crude products using HPLC;

(d) preparation of antibacterial compounds 24-desmethylrifamycin B and 24-desmethylrifamycin SV.

Another embodiment of the present invention relates to a method for producing 24-desmethylrifamycin S, a derivative of 24-desmethylrifamycin B, wherein said method comprises the following steps:

(a) reacting 24-desmethylrifamycin B in the presence of copper chloride;

(b) carrying out the reaction according to step (a) overnight at room temperature;

(c) obtaining 24-desmethylrifamycin S.

A method for producing 24-desmethylrifampicin, a derivative of 24-desmethylrifamycin B, said method comprising the following steps:

(a) reacting 24-desmethylrifamycin B in the presence of dimethylformamide (DMF) and acetic acid;

(b) adding paraformaldehyde and 1,3,5-trimethyl-hexahydro-1,3,5-triazine to the mixture obtained in step (a);

(c) preparation of 3-methyl-1,3-oxazino (5,6-c) -24-desmethylrifamycin;

(d) reacting the compound obtained in step (c) with 1-amino-4-methyl-piperazine; and

(e) obtaining 24-desmethylrifampicin.

Another embodiment of the present invention relates to the recombinant nucleotide sequence ID No. one.

Another embodiment of the present invention relates to a bacterial strain comprising the nucleotide sequence of ID NO. one.

Another embodiment of the present invention relates to a bacterial strain (s) as described in the present invention, comprising the nucleotide sequence ID NO: 1.

Another embodiment of the present invention relates to a bacterial strain as described in the present invention, in which the bacterial strain is capable of producing rifamycin analogs.

Another embodiment of the present invention relates to vector constructs comprising the recombinant nucleotide sequence of ID NO: 1.

Another embodiment of the present invention relates to vector constructs in which the vector constructs are pAT6E and pAT6F.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects and advantages of the present invention will become more clear from the following detailed description with reference to the accompanying drawings, in which the same symbols represent the same details in all figures, of which:

Figure 1: Chemical structures of rifamycin B.

Figure 2: Semi-synthetic derivatives of rifamycin B.

Figure 3: Schematic representation of the translational mechanism of the PKS type 1.

Figure 4: Synthesis of a hypothetical intermediate using PKS rifamycin plot II.

Figure 5: Organization of the enzyme domain in the rifPKS gene cluster.

Figure 6: Strategy for constructing a functional cassette by replacing rifAT6 with rapAT2.

Figure 7: Schematic diagram of a strategy to demonstrate the replacement of rifAT6 with rapAT2.

Figure 8: Phenotypic crop species showing single crossover clones.

Figure 9: Gel electrophoresis profile for single crossover clones.

Figure 10: Phenotypic crop view showing dual crossover clones.

Figure 11: Gel electrophoresis profile for dual crossover clones.

Figure 12: LC-ESI-MS profile of a 24-desmethylrifamycin B analogue recovered from DCO.

Figure 13: NMR of rifamycin B and its analogue.

Figure 14: Structures of 24-desmethylrifamycin B, 24-desmethylrifamycin S and 24-desmethylrifamycin SV.

Figure 15: Crystal structure of 24-desmethylrifamycin S and drug interaction with DNA polymerase A.

Figure 16: Spectra ESI-MS [mass spectrometry and electrospray ionization] (a) rifamycin S; (b) 24-desmethylrifamycin S; (c) rifampicin; (d) 24-desmethylrifampicin.

Figure 17: NMR Spectra of (a) 24-desmethylrifampicin; (b) chemically synthesized rifampicin; and (c) commercially available rifampicin.

Figure 18: Comparative MS / MS analysis of (a) 24-desmethylrifampicin and (b) rifampicin.

Figure 19: Characteristic mass spectrometric fragments of rifampicin and 24-desmethylrifampicin in the ESI [electrospray ionization] mode of negative ions.

Figure 20: Antibacterial analysis of new rifamycin analogues against various types of bacteria.

Figure 21: Antibacterial analysis of the activity of rifampicin, rifamycin S, 24-desmethylrifampicin.

Figure 22: The nucleotide sequence of ID No. one.

DETAILED DESCRIPTION

Although the invention is capable of various modifications and / or alternative methods and / or compositions, a specific embodiment of the invention is presented as an example in the figures / figures and tables and will be described in detail below. It should be understood, however, that the invention is not limited to the specific methods and / or compositions described, but rather, the invention encompasses all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the attached patent claims.

Graphs, tables, formulas and protocols are presented, as appropriate, using the usual representations in the drawings showing only those specific details that are relevant to the understanding of embodiments of the present invention, so as not to obscure the disclosure with details that would be obvious to ordinary specialists in this field, having the advantage of this description.

The following description is only illustrative embodiments and is not intended to limit the scope, applicability, or configuration of the invention in any way. Rather, the following description provides a convenient explanation for implementing illustrative embodiments of the present invention. Various changes may be made to the described embodiments with respect to the function and arrangement of the described elements without departing from the scope of the present invention.

The terms “contains”, “comprising” or any other variations thereof that are intended to cover a non-exclusive indication, for example, that one or more of the methods or compositions or systems or methods acting by definition “contains ...” are not excluded, without additional restrictions, the existence of other methods, sub-methods, compositions, sub-compositions, small or large compositions or other elements or other structures or additional methods or compositions or additional elements or additional features, or additional characteristics or additional attributes.

Definitions:

For the purposes of this invention, the following terms will have the meaning as defined here:

As used herein, the terms “combinatorial approach or combinatorial biosynthesis” as used in the context of the present invention refers to genetic manipulation of the rifPKS gene cluster by replacing the rifAT6 domain of the rifPKS gene cluster of A. mediterranei S699 with the rapAT2 domain of the rapPKS gene cluster of Streptomyces hygroscopicus gene cluster.

As used herein, the terms “vectors / cloning vectors” when used in the context of the present invention refer to the development of a series of vectors pAT6A, pAT6B, pAT6C, pAT6D, pAT6E and pAT6F. The pAT6F vector is a non-replicative plasmid that has been transformed into A. mediterranei S699.

As used herein, the terms “derivatives” as used in the context of the present invention refer to derivatives derived from or prepared from 24-desmethyl trifampicin B. Several examples of derivatives: 24-desmethyl trampicin, 24-desmethyl trifamycin S and 24-desmethyl trifamycin SV.

As used herein, the terms “analogs” as used in the context of the present invention refer to rifamycin B analogues that are obtained by replacing the rifAT6 domain with the rapAT2 domain or containing the rifAT6 domain replaced by the rapAT2 domain. One such illustrative analogue is 24-desmethylrifamycin B.

As used herein, the terms “mutant (s) or mutant strain (s)” as used in the context of the present invention refer to A. mediterraneiin strain / s, rifAT6 in whose domain is replaced by rapAT2 by the domain of the PKS-1 system and which are capable of producing analogs and derivatives rifamycin B. For example: Amycolatopsis mediterranei # 34, which produces an analogue, such as 24-desmethylrifamycin B etc.

As used here, the term “antibacterial compound (s)” as used in the context of the present invention refers to compositions that are capable of inhibiting bacterial growth or infection caused by bacteria, or a disease caused by bacteria, or a disease state caused by bacteria belonging to the category Actinobacteria or any gram-negative bacteria or gram-positive bacteria. In particular, antibacterial compounds used in the context of the present invention relate to antibacterial compounds effective in inhibiting the growth of bacteria belonging to Mycobacerium species. In addition, antibacterial compounds used in the context of the present invention relate to those antibacterial compounds that are effective in inhibiting any disease or any infection or disease state caused by bacteria belonging to Mycobacerium species.

As used here, the term "multidrug resistance / MDR" when used in the context of the present invention refers to a condition that enables pathogenic bacteria to withstand various antimicrobial or antibacterial compounds, i.e. chemicals with a wide variety of structures and functions aimed at the destruction of bacteria. In particular, MDR in the context of the present invention relates to conditions enabling pathogenic Mycobacerium species to resist various antibacterial drugs or antibacterial compounds, such antibacterial drugs or antibacterial compounds being commonly used antibiotics or commercially available antibacterial drugs or antibacterial compounds.

As used here, the term "their salts" when used in the context of the present invention refers to any salt, esters, polymorphs, pure forms, isomers, mixtures of isomers, complexes and any other derivatives or analogs of 24-desmethylrifamycin B, 24-desmethylrifamycin S, 24-desmethylrifamycin SV; and 24-desmethylrifamycin. More noteworthy salts thereof, such as salt, esters, polymorphs, pure forms, isomers, isomer mixtures, complexes and any other derivatives, should at least contain a 24-desmethyl form or structure, i.e. lose the methyl group at position 24 in analogues, derivatives, salts, esters, polymorphs, pure forms, isomers, mixtures of isomers, complexes of 24-desmethylrifamycin B, 24-desmethylrifamycin S, 24-desmethylrifamycin SV, and 24-desmethylrifamycin.

The present invention relates to antibacterial compounds that may be effective against bacterial diseases or infections. More specifically, the present invention relates to the use of antibacterial compounds that can be effectively used against infections or diseases caused by bacteria of the group of gram-positive bacteria falling under the category of Actinobacteria. The antibacterial compounds of the present invention are suitable against multidrug-resistant bacteria. The antibacterial compounds of the present invention are suitable against bacteria such as Mycobacterium species, Staphylococcus species or strains, Bacillus species or strains, Pseudomonas species or strains and Ecoli strains.

In addition, the present invention relates to antibacterial compounds that are useful against infections or diseases caused by Mycobacterium species. In particular, these antibacterial compounds are effective against Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium smegmatis. Another aspect of the present invention provides antibacterial compounds that are useful against multidrug-resistant mycobacterial strains.

Rifamycin was isolated as a related compound of the rifamycin complex [SensiP. et al. (1983). Farmac. Ed. Sc. Vol. 14, p. 146-147), and rifPKS was completely sequenced in 1998 [August, P.R. et al. (1998) Chem. Biol. Vol. 5, p. 69-79]. Since then, a number of studies have been carried out to elucidate the function of the gene cluster and structure, as well as the effects of rifamycin B. However, the unique nature of rifPKS in the production of rifamycin B has impeded the progress in obtaining rifamycin B analogues for a long time. Prior to this, it was difficult to genetically manipulate rifPKS of the bacteria A. mediterranei, since there were no productive genetic technologies or cloning vectors for A. mediterranei. Since then, one of the authors of the present invention has already developed a series of cloning vectors (PRL series) that can be used to transform and clone several strains of A. mediterranei [Lal, R., US 5985560 A (1999); Lal, R. et al. (1991). Appl. Environ. Microbiol. Vol. 57, p. 665-671; Dhingra, G. et al. (2003). Ind. Microbiol. Biotecnol. Vol. 30, p. 195-204] and the electrotransformation method developed in this way is used to manipulate the rifPKS gene cluster to produce rifamycin analogues. Manipulations with rifPKS have also been based on the modular nature of erythromycin polyketide synthases (eryPKS) and rapamycin (rapPKS), which has generated significant past interest in the development of new bioengineered polyketides. Although eryPKS and rapPKS were previously cloned, cloning and characterization of the rifamycin biosynthetic gene cluster or rifPKS in 1998 [August, P.R. et al. (1998). Chem. Biol. Vol. 5, p. 69-79] opened up the possibilities of manipulating the rifPKS gene cluster to obtain rifamycin analogues by using the same approaches that were used for eryPKS. A combinatorial approach has been used in the present invention in which genetic manipulations of a rifamycin polyketide synthase (rifPKS) A. mediterranei S699 cluster are performed to produce rifamycin B analogues.

Although in the last forty years many rifamycin derivatives have been synthesized by chemical methods, only a small group of rifamycin derivatives are currently used to treat tuberculosis. Most rifamycin derivatives, including those used in clinics, are chemically modified at the C-3 and / or C-4 positions of the naphthalene fragment. Chemical modifications of other parts of the compound have proved difficult due to the complex structure of the molecule. In addition, studies using X-ray crystallography of Thermusaquaticus RNA polymerase complexing with rifampicin showed that four free hydroxyl groups in the molecule are important for binding of RNA polymerase [Campbell, E.A. et al. (2001). Cell. Vol. 104, p. 901-912]). Therefore, modifications to these hydroxyl groups are undesirable. In connection with the foregoing, the present invention is unique in so far as it proposes a strategy for the development of new analogues and derivatives in which modifications are made in the skeleton of polyketide.

The present invention has developed A. mediterranei S699 mutants to produce a rifamycin B analog, 24-desmethylrifamycin B, which is also a proof of the concept of combinatorial biosynthesis, which gives rise to various rifamycin B analogues using this approach. Derivatives of this analogue are effective against MDR strains of M. tuberculosis bacteria. Even more importantly, this invention first confirms the concept that manipulations with the rifPKS gene cluster outside module 4 can be performed using combinatorial approaches to produce rifamycin B analogues. The mutant strain can now be improved for commercial use.

Accordingly, the present invention provides a combinatorial biosynthesis strategy for manipulating rifPKS of A. mediterranei S699. Thus, the developed strategy in accordance with the present invention allows the replacement of the acyltransferase domain of the sixth module (AT6) in rifPKS (which recruits propionate) with the same acyltransferase domain of the second rapPKS module (rapAT2 from Streptomyces hygroscopicus (under inventory number: X86780.1 and DSM collection culture-DSM 41524) (which recruits acetate) into a growing chain. The exchange of these sites has led to the development and production of a new analogue of rifamycin B, which is 24-desmethylrifamycin B. In addition, the present invention and a development strategy for derivatives of 24-desmethylrifamycin B. is also proposed. Thus, it has been found that the novel analogues and derivatives developed are effective against various types of bacteria (Figures 20-21). More specifically, these new analogues and derivatives have been found to be effective against MDR strains M. tuberculosis.

Despite the well-known fact that complex polyketide systems like rifPKS are considered to be poorly amenable to metabolic engineering by nature, either due to the inflexibility of enzymes in the forward direction, to accept modified substrates, or because of the mismatch of the architectural modular structure of modified PKS systems [Khosla , C. et al. (2009). Curr. Opin, Chem .. Biol. Vol. 13, p. 135-143], the present invention not only indicates how to explain and unravel the existing disadvantage of the rifPKS system, but also provides analogues and derivatives that are highly effective against known and / or resistant strains of mycobacteria. Thus, to solve the existing problem of the incorrigible nature of the rifPKS system, the stages of synthesis of the rifamycin polyketide skeleton, especially the naphthoquinone ring, were used; the goal was the rifAT6 region of the rifPKS gene. This rifAT6 region of the rifPKS gene cluster was replaced with rapAT2 using a domain replacement strategy. The result is a new analogue of rifamycin, i.e. 24-desmethylrifamycin B, in addition, its derivatives, namely 24-desmethylrifamycin S, 24-desmethylrifamycin SV and 24-desmethylrifamycin.

These analogs of 24-desmethylrifamycin B and its derivatives, 24-desmethylrifamycin SV, 24-desmethylrifamycin S & 24-desmethylrifamycin, were not only obtained in comparable quantities, but also showed stronger antibacterial activity against Staphylococcus aureus, Mycobacterium smegmatis, Bacillus subtillon subtas, Bacillus subtillon subtilis aeruginosa, Escherichia coli.

The present invention also provides mutant strains of A. mediterranei S699 that are capable of producing an analogue of / and rifamycin B, i.e. 24-desmethylrifamycin B. The present invention also provides a method for developing mutant strains of A. mediterranei S699. It was found in the present invention that strains of A. mediterranei S699 were quite stable when multiplied, as well as in the expression of rifamycin B analogues. An analogue of these mutants was obtained on a laboratory scale. The product yield was approximately 50 mg / L analogue 24-desmethylrifamycin B.

NMR studies of analogues obtained from mutant strains of A. mediterranei S699 show that there is a significant difference in the NMR profiles of rifamycin B and 24-desmethylrifamycin B (Figure 13). NMR profiles show that 24-desmethylrifamycin B has quasi-molecular ions with an m / z ratio of 740, and 24-desmethylrifamycin SV has an m / z ratio of 682, which is 14 atomic units less than rifamycin B and rifamycin SV.

Rifamycin resistance is associated with genetic changes in the 81-bp region of the GroV gene encoding the β subunit of DNA-dependent RNA polymerase (Williams et al. 1998. Mycobacterium tuberculosis. Antimicrob. Agents. Chemother., Vol. 42, pages 1853-1857) . The present invention uses rifampicin-resistant strains of M. tuberculosis, OSDD 321 and 206 OSDD, which contain the S531L mutation, and OSDD 55, which has the H526T mutation in the β-subunit of RNAP. Mutations have been confirmed in OSDD strains. Based on the sequence data, the present invention establishes that drug-resistant mutations disrupt hydrogen bonding in the polyketide ansa chain and in a possible salt bridge (Figure 15b). The loss of the methyl group in 24-desmethyl trifampicin, however, can lead to conformational changes in the ansa chain, which will provide higher flexibility of the compound to bind to mutated RNA polymerases. Accordingly, it is believed that, in addition to a favorable distribution coefficient, the flexibility resulting from the difference in gauche, synpentane, and the occluded interactions of the ansa chain, and changes in the hydrogen bonding network associated with conformational changes (Figure 15c) can increase the affinity binding both in wild-type strains and in drug-resistant strains. This possible conformational flexibility can also be associated with unregulated 1H and 13C NMR spectra of 24-desmethyl triampicin. In connection with the foregoing, the present invention provides the most unexpected and unique conclusion in the present invention is the activity of 24-desmethylrifampicin against rifampicin-resistant M. tuberculosis strains. 24-desmethylrifampicin showed higher activity against rifampicin-resistant M. tuberculosis strains. Thus, this finding in the present invention offers absolute hope for patients suffering from tuberculosis, where mycobacteria have become resistant to existing rifampicin drug or related drugs. These results were confirmed by computational studies of free energy perturbation (FEP), which demonstrated that RNA polymerase prefers 24-desmethylrifamycin B at 1.2 kcal / mol over rifamycin B (Table 1). This is important because this compound can now be developed as a promising step in the treatment of MDR-TB.

Thus, the present invention demonstrates the creation of new analogues and derivatives of rifamycin B, showing for the first time that rifPKS lends itself to modification of a domain substitution leading to the formation of a new rifamycin analog.

The present invention provides a unique recombinant sequence ID No. 1 (Figure 22), which is capable of producing rifamycin analogues. The present invention also provides a vector construct comprising the recombinant sequence ID No. one.

The present invention provides a new strain of Amycolatopsis mediterranei S699 # 34, carrying the nucleotide sequence ID No.l. More specifically, the new strain has an Amycolatopsis mediterranei S699 # 34 identification number carrying the recombinant sequence ID No. l (Figure 22), which provides the strains with a unique characteristic, and this unique feature allows new strains to express or produce rifamycin analogs, such as 24-desmethylrifamycin B and 24-desmethylrifamycin S.

The present invention also provides pharmaceutical compositions of the antibacterial compounds and their salts described in the present invention that are effective against bacterial species, in particular mycobacteria. The pharmaceutical composition in accordance with the present invention is intended for parenteral and oral administration. Preferably, the pharmaceutical composition described in the present invention can be administered parenterally, for example, intravenously, subcutaneously, intradermally or intramuscularly. The present invention also provides agents that act as a “pharmaceutically acceptable excipient”, the term “pharmaceutically acceptable excipient” means a carrier of a pharmaceutically acceptable composition, solution or additive to enable the delivery, dissolution or suspension of antibacterial compounds, as described herein. The pharmaceutical composition in accordance with the present invention may also contain pharmaceutically acceptable excipients, as required to approximate physiological conditions, such as pH adjustment, and buffers, tonicity agents, moisturizing agents and the like. The pharmaceutical composition in accordance with 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, in the form of a tablet, capsule, injection, in the form of granules or powder, for example, in a sachet.

The present invention will be explained below with reference to non-limiting embodiments of the invention.

Accordingly, a basic embodiment of the present invention relates to antibacterial compounds having the following chemical structure:

and their salts.

Another embodiment of the present invention relates to antibacterial compounds in accordance with the present invention, which are suitable against infection or disease caused by bacteria.

Another embodiment of the present invention relates to antibacterial compounds in accordance with the present invention, which are useful against infections or diseases caused by Mycobacterium species.

Another embodiment of the present invention relates to antibacterial compounds in accordance with the present invention, which are suitable against infection or disease caused by Mycobacterium species, where the selected Mycobacterium species are Mycobacterium tuberculosis and MDR strains of M. tuberculosis.

Another embodiment of the present invention relates to a pharmaceutical composition containing antibacterial compounds or their salts, having the following chemical structure:

together with their pharmaceutically acceptable carriers.

Another embodiment of the present invention relates to a pharmaceutical composition comprising the antibacterial compounds of the present invention that are useful against infection or disease caused by bacteria.

Another embodiment of the present invention relates to a pharmaceutical composition comprising the antibacterial compounds of the present invention that are useful against infection or disease caused by Mycobacterium spp.

Another embodiment of the present invention relates to a pharmaceutical composition comprising the antibacterial compounds of the present invention that are useful against infection or disease caused by Mycobacterium tuberculosis and MDR strains of M. tuberculosis.

Another embodiment of the present invention relates to a method of treatment, comprising administering to a patient antibacterial compounds or their salts, having the following structure:

Another embodiment of the present invention relates to the use of antibacterial compounds or their salts having the following chemical structure for treating a disease or infection.

to get the drug.

Another embodiment of the present invention relates to the use of antibacterial compounds or their salts having the following chemical structure:

to get the drug.

Another embodiment of the present invention relates to the use of a medicament containing the antibacterial compounds of the present invention for treating a disease or infection.

Another embodiment of the present invention relates to the use of a medicament containing antibacterial compounds in accordance with the present invention, for the treatment of diseases or infections caused by bacteria.

Another embodiment of the present invention relates to the use of a medicament containing the antibacterial compounds of the present invention for the treatment of diseases or infections caused by Mycobacterium species.

Another embodiment of the present invention relates to the use of a medicament containing the antibacterial compounds of the invention of claim 10 or 13 for the treatment of diseases or infections caused by Mycobacterium, Mycobacterium tuberculosis and MDR strains of M. tuberculosis.

Another embodiment of the present invention relates to a method for producing a pharmaceutical composition comprising an antibacterial compound in accordance with the present invention, together with pharmaceutically acceptable carrier (s).

Another embodiment of the present invention relates to a new strain of Amycolatopsis mediterranei S699 # 34, containing the rapAT2 gene region.

Another embodiment of the present invention relates to a new strain of Amycolatopsis mediterranei S699 # 34, where the rifAT6 region is replaced by the rapAT2 region.

Another embodiment of the present invention relates to a new strain of Amycolatopsis mediterranei S699 # 34, capable of producing new analogues and derivatives of rifamycin B, for example, as described here.

Another embodiment of the present invention relates to a new strain of Amycolatopsis mediterranei S699 # 34, capable of producing analogs and derivatives of rifamycin B, containing the following chemical structure:

Another embodiment of the present invention relates to a method for producing a new strain of Amycolatopsis mediterranei S699 # 34, comprising a rapAT2 region in rifPKS, said method comprising the steps of:

(a) isolation of the rifAT6 region of rifPKS from Amycolatopsis mediterrranei S699 and rapAT2 region of Streptomyces hygroscopicus (According to No. DSM - 41524);

(b) obtaining vector constructs by replacing the rifAT6 region of the rifPKS rapAT2 with the region of Streptomyces hygroscopicus (According to No. DSM - 41524);

(c) inserting the vector constructs obtained in step (b) into Amycolatopsis mediterrranei S699; and

(d) obtaining a new strain of Amycolatopsis mediterrranei S699 # 34 containing rapAT2 plot rifPKS.

Another embodiment of the present invention relates to a method for producing new antibacterial compounds, said method comprising the steps of:

(a) culturing Amycolatopsis mediterrranei S699 # 34 cells containing the rapAT2 region in the rifPKS region; and

(b) collecting Amycolatopsis mediterrranei S699 # 34 cells to obtain crude product fractions;

(c) purification of crude products using HPLC;

(d) preparation of antibacterial compounds 24-desmethylrifamycin B and 24-desmethylrifamycin SV.

Another embodiment of the present invention relates to a method for producing 24-desmethylrifamycin S, a derivative of 24-desmethylrifamycin B, said method comprising the steps of:

(a) reacting 24-desmethylrifamycin B in the presence of copper chloride;

(b) carrying out the reaction according to step (a) overnight at room temperature;

(c) obtaining 24-desmethylrifamycin S.

A method for producing 24-desmethylrifampicin, a derivative of 24-desmethylrifamycin B, said method comprising the steps of:

(a) reacting 24-desmethylrifamycin B in the presence of dimethylformamide (DMF) and acetic acid;

(b) adding paraformaldehyde and 1,3,5-trimethyl-hexahydro-1,3,5-triazine to the mixture obtained in step (a);

(c) preparation of 3-methyl-1,3-oxazino (5,6-c) -24-desmethylrifamycin;

(d) reacting the compound obtained in step (c) with 1-amino-4-methyl-piperazine; and

(e) obtaining 24-desmethylrifampicin.

Another embodiment of the present invention relates to the recombinant sequence ID No. one.

Another embodiment of the present invention relates to a bacterial strain (s) in accordance with the present invention containing the nucleotide sequence ID NO. one.

Another embodiment of the present invention relates to a strain of Amycolatopsis mediterranei S699 # 34, containing the nucleotide sequence of ID NO. one.

Another embodiment of the present invention relates to a bacterial strain in accordance with the present invention, wherein the bacterial strain is capable of producing rifamycin analogs.

Another embodiment of the present invention relates to a strain of Amycolatopsis mediterranei S699 # 34, in which the bacterial strain is capable of producing rifamycin analogs.

Another embodiment of the present invention relates to the recombinant nucleotide sequence ID No. 1, capable of expressing new rifamycin analogues.

Another embodiment of the present invention relates to the recombinant nucleotide sequence ID No. 1 in accordance with the present invention, the analogues being 24-desmethylrifamycin B and 24-desmethylrifamycin S.

Another embodiment of the present invention relates to vector constructs comprising the recombinant nucleotide sequence ID No. one.

Another embodiment of the present invention relates to vector constructs, the vector constructs being pAT6E and pAT6F.

Another embodiment of the present invention relates to a bacterial strain containing vector constructs as described in the present invention.

Another embodiment of the present invention relates to a strain of Amycolatopsis mediterrranei S699 # 34, containing vector constructs as described in the present invention.

Another embodiment of the present invention relates to a strain of Amycolatopsis mediterrranei S699 # 34 carrying a pAT6F vector, wherein the pAT6F vector contains the nucleotide sequence No. one.

Another embodiment of the present invention relates to a bacterial strain capable of carrying a pAT6F vector, wherein the pAT6F vector contains the nucleotide sequence No. one.

Another embodiment of the present invention relates to a microbial strain capable of carrying a pAT6F vector, wherein the pAT6F vector contains the nucleotide sequence No. one.

Another embodiment of the present invention relates to a microbial strain capable of expressing the nucleotide sequence of No. 1 and / or its products.

Another embodiment of the present invention relates to a microbial strain capable of expressing the nucleotide sequence of No. 1 and / or its products.

Although there are no reports of manipulation of the skeleton of rifamycin B using combinatorial biosynthesis, there are reports of attempts to use combinatorial biosynthesis on other polyketide synthase genes. Noticeable success has been achieved by modifying the rifamycin B molecule using a combinatorial approach, as can be seen from the description of the present invention.

The following description is only illustrative embodiments and is not intended to limit the scope, applicability, or configuration of the invention in any way. Rather, the following description provides a convenient illustration of the implementation of illustrative embodiments of the invention for various changes that can be made in the functions and arrangement of the described elements without departing from the scope of the invention.

EXAMPLES

Example 1

Genetic manipulations with A. mediterranei S699.

Conventional genetic procedures, such as genomic DNA isolation (according to the CTAB-cetyltrimethyl bromide method), plasmid isolation (Promega DNA purification kit), and restriction endonuclease digestion were performed according to standard procedures. The pAT6F plasmid was constructed, which was transformed (using BioRadGenePulser) into A. mediterranei S699 using the previously described method [Dhingra, G. et al. (2003). J. Ind. Microbiol. Biotechnol. Vol. 13, p. 195-204], and the electroporated mixture was plated on YMG agar plates containing 4 g of yeast extract, 10 g of malt extract and 4 g of glucose per liter (pH = 7.2), which were coated with soft agar (5 g / l, DifcoTM granular agar) containing 500 μg / ml erythromycin for stability after incubation for 12-16 hours. Transformants were obtained after 5-7 days and grown in YMG medium containing 50 μg / ml erythromycin (Sigma Aldrich), with confirmation of resistance . The integration of pAT6F into these single crossover (SCO) clones was confirmed by Southern blot hybridization. In order to influence the replacement of rifAT6 with rapAT2, these single crossover clones were cultured for 3-4 rounds on a plate with YMG medium without erythromycin. Cells were plated on a plate with YMG agar under and without erythromycin pressure, and antibiotic sensitive colonies were selected. Doubled crossover (DCO) clones were further confirmed by southern blot hybridization. For Southern hybridization, genomic DNA was immobilized on a Hybond N + membrane (Amersham Biosciences). Hybridization was carried out at 65 ° C for 12 h using [α- ³² P] -labeled DNA probes. For non-radioactive methods, hybridization was performed using a DIG-labeled DNA probe at 65 ° C. Thorough washes were performed using 5 × SSC, 2 × SSC, 1 × SSC and 0.1 × SSC at 65 ° C. The resulting DCO clones contained the recombinant nucleotide sequence ID No. 1 (Figure 22). Clone A. mediterranei S699, as described and prepared, containing the recombinant nucleotide sequence ID No. 1, received the (internal) identification number of Amycolatopsis mediterranei S699 # 34.

Design of primers for PCR I [PCR] and PCR II

The flanking region of AT6, also called PCR I (41862-43533bp), located in the opposite direction from rifAT6, was amplified using oligoprimers, primers I and II (Table 1), both forward and reverse primers, respectively. The XbaI and BalI restriction sites were inserted at the 5'-ends of primer I and II, respectively, to facilitate cloning of PCR I at the SmaI site in pUC 18. These sites were subsequently used for cloning.

Similarly, PCR II (44488-45989bp), located in the forward direction from rifAT6, was amplified using a pair of primers — primer III and primer IV (Table 2). The AvrII site was inserted into primer III, and XbaI into primer IV at the end of the 5 'end for subsequent cloning in pUC 18.

Additional nucleotides inserted to generate the restriction site in all primers are marked in red. The nucleotide sequences of restriction enzyme sites formed in the direct primers PCR I and PCR II are highlighted in light blue, and in the yellow primers.

Example 2

Construction of pAT6A, pAT6B and pAT6C

PCR I and PCR II products were obtained from rifPKS on cosmid clone No. 13. Then, the polynucleotide kinase treated with the PCR I or PCR II fragment was ligated to the SmaI site of the linearized and dephosphorylated pUC18. After this, the ligation mixture was transferred to E. coli JMI0I. Plasmids containing PCR I and PCR II in pUC 18 are called pAT6A and pAT6 B, respectively. In the plasmid pAT6A, PCR I was ligated in the opposite direction to that required for subsequent cloning. In accordance with the required direction, the XbaI site in PCR I should be located next to the NdeI site of the plasmid, however, the obtained XbaI clones site was far from the NdeI site of the plasmid. To reverse the direction, PCR I was cut with EcoRI / HindIII double cleavage and inserted into pUC19, finally obtaining the plasmid, pAT6C.

Example 3

PAT6D Construction

Plasmid pMO2 [Olinyk, M. et al. (1996). Chem. Biol. Vol. 3, p. 833-839]), which has the acyltransferase of module 2 (AT2) of the biosynthetic rapamycin gene cluster from Streptomyces hygroscopicus cloned therein, cleaved with AvrII / HindIII to release the rapAT2 domain. The plasmid pAT6B was also digested with AvrII / HindIII and rapAT2 and the linearized pAT6B were ligated and the ligation mixture was transferred to E. coli JM101 and clones containing the correct insert were selected. These designs are called pAT6D.

Example 4

Construction pAT6E

PCR I was excised by cleaving pAT6C with BalI / NdeI and simultaneously pAT6D was cleaved with BalI / NdeI. Both pAT6C inserts and linearized pAT6D were ligated, and the ligation mixture was transferred to E. coli JM101. The structures thus obtained are called pAT6E.

Example 5

[Strategy development functional cassette (pAT6F - 8.3 kb) to replace (Figure 6)]: Design of the final cassette pAT6F

Plasmid pAT6E was digested with XbaI to release the PCR fragment of I-rapAT2-PCR II (approx. 4 kb) and cloned into XbaI of the digested pIJ4026 vector containing the erythromycin-resistant gene. pIJ4026 (provided by M.J. Bibb, John Innes Institute, Norwich) (Figure 6) is an E. coli plasmid but contains the ermE gene, which confers resistance to erythromycin, and is expressed only in A. mediterranei, not E. coli. The final construct contains a 3.85 fragment, consisting of fragments of PGR I, rapAT2 and PCRII in pIJ4026, called pAT6F. 3.85 kb - The fragment was named SEQ ID No. 1 (Figure 22).

Plasmid pAT6F was transformed by electroporation (7.5 kV / cm, 1000 Ohm, 25 μF) into A. mediterranei S699. Single crossover clones were selected under pressure of erythromycin in a GYM agar medium (glucose 4 g / l; malt extract 10 g / l; yeast extract 4 g / l). Single crossover (SCO) clones that did not show brown pigmentation were selected (due to the lack of rifamycin production, since the biosynthetic pathway of metabolism was blocked by the integration of pAT6F into the chromosome via homologous recombination) (Figure 7 & 8). Plasmid pTA6F contained the nucleotide sequence ID No. 1, as described in the present invention and represented in Figure 22.

For genotypic confirmation, DNA from these single crossover (S.C.O.) clones was hybridized with a plasmid (pIJ4026). Four single crossover clones (1-5, 2-2, 6-1, 6-2) showed a positive hybridization signal (Figure 9). Positive SCO clone 2-2 for double crossover selection (D.C.O.) was further cultured for 3-4 generations without erythromycin pressure. Erythromycin-sensitive dual crossover (D.C.O.) clones that were able to produce rifamycin (renewed brown pigmentation) were selected. These clones had rifAT6 replaced by rapAT2 (Figure 7). The resulting double crossover clones (Figure 10) were again checked at the genetic level for the presence of the AT2 domain instead of AT6 using Southern hybridization and DNA sequencing. Four clones showed positive signals with the rapAT2 gene (Figure 11). Double crossover clones that contained the recombinant sequence ID No. 1 were designated as Amycolatopsis mediterranei S699 # 34.

The expected rifamycin analogues produced by these DCO clones were analyzed using LC ESI-MS. Only three of these positive clones (# 3, # 34 and # 36) produced the expected analogue (Figure 12). The results were confirmed by ¹ H NMR (Figure 13). It was found that the molecular weight of the analogue is 741, which is 14 amu. (atomic mass unit) is less than the molecular weight of native rifamycin B (755).

The spores of the mutant strain were first 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 / v) YMG medium (10 × 100 ml) in 500 ml flasks. After incubation for 10 days under the same conditions, the cultures were centrifuged, the combined supernatants were acidified to pH = 3 with 1N HCl, and the metabolites were extracted with ethyl acetate (2 × 1 L). The crude extracts of rifamycin-linked compounds were chromatographed on silica gel using CHCl ₃ -MeOH / 5% NH ₄ OH (10: 1 and then 8: 1) as the mobile phase. The fractions containing the products were combined and dried using a rotary evaporator. The resulting product was further purified by HPLC [YMC-ODS-A, 250 × 10 mm, CH ₃ CN-HCOONH ₄ (0.05 M) (60:40), flow rate 2 ml / min, 254 nm]. The product was then desalted using a Sephadex LH-20 column, using MeOH as eluent, to give 24-desmethyrifamycin B (20 mg) and 24-desmethyrifamycin SV (8 mg).

24-desmethyrifamycin B: ¹ H NMR (700 MHz, D ₂ O, cryoprobe): δ 6.69 (s, 1H, H-3), 6.34 (d, 1H, J = 12 Hz, H-29), 5.98 (d , J = 11 Hz, 1H, H-17), 5.75 (dd, J = 15 Hz, 11 Hz, 1H, H-18), 5.21 (dd, J = 15 Hz, 10 Hz, 1H, H-19) 5.05 (m, 2H, H-25, H-28), 4.50 (d, J = 17 Hz, 1H, -CH ₂ -COOH), 4.42 (d, J = 17 Hz, 1H, -CH ₂ -COOH ), 3.38 (bd, J = 10 Hz, 1H, H-23), 3.22 (m, 2H, H-21 and H-27), 3.22 (s, 3H, H-37), 2.11 (s, 3H, H-36), 2.08 (s, 3H, H-14), 1.98 (m, 2H, H-20 and H-26), 1.70 (s, 3H, H-13), 1.53 (m, 2H, H- 22 and H-24), 1.28 (t, J = 12 Hz, 1H, H-24), 0.92 (d, J = 6.5 Hz, 3H, H-31), 0.85 (d, J = 7 Hz, 3H, H-32), 0.72 (d, J = 6.5 Hz, 3H, H-34). ¹³ C NMR (175 MHz, CD ₃ OD, cryoprobe): δ _C 191.7, 176.7, 174.7, 168.1, 144.7, 142.1, 141.5, 131.5, 126.5, 126.4, 119.4, 117.8, 113.7, 112.8, 112.1, 109.6, 104.4, 101.1, 80.5, 73.9, 72.4, 71.5, 68.8, 54.9, 48.8, 41.4, 39.9, 35.9, 32.2, 21.8, 20.6, 20.5, 15.4, 9.5, 9.5, 6.9. (-) - HR-ESI-TOF-MS m / z 740.2939 (calc. C ₃₈ H ₄₆ NO ₁₄ [MH] ^- : 740.2918).

24-desmethyryphamycin SV: ¹ H NMR (700 MHz, CD ₃ OD, cryoprobe): ¹ H NMR (700 MHz, CD ₃ OD): δ 6.42 (s, 1H), 6.31 (bd, J = 12 Hz, 1H) , 5.95-5.89 (m, 2H), 5.51 (s, 1H), 5.20 (bt, J = 12 Hz, 1H), 5.14 (bd, J = 9 Hz, 1H), 3.22 (s, 3H), 3.17 ( t, J = 10 Hz, 1H), 2.13 (s, 3H), 2.12-2.11 (m, 2H), 2.01 (s, 3H), 1.96 (s, 3H), 1.67 (s, 3H), 1.54-1.49 (m, 4H), 0.93 (d, 6H), 0.78 (d, J = 5 Hz, 3H). HRMS (ESI-TOF) m / z (calcd. For C ₃₆ H ₄₄ NO ₁₂ [MH] ^- : 682.2707).

Example 6

Conversion of 24-desmethylrifamycin B to 24-desmethylrifamycin S

24-desmethylrifamycin B (8 mg, 0.0107 mmol) was dissolved in MeOH-H ₂ O (10: 1, 5 ml) 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 × 5 ml). The extract was subjected to a silica gel column using CHCl ₃ -MeOH (10: 1) as an eluent to give 24-desmethylrifamycin S (6 mg).

The product was analyzed using (-) - ESI-MS (m / z = 680 [MH] -, the analysis showed the absence of a glycolate fragment and the presence of a naphthoquinone unit in the molecule) and a 1 H NMR spectrum. Interestingly, when stored in CDCl3 at -20 ° C, transparent brown-red orthorhombic crystals formed, which showed a dimeric form of coordination of 24-desmethylrifamycin S with Ca ²⁺ through C-1, C-8, C-21 and C-23 atoms oxygen (Figure 15a).

¹ H NMR (300 MHz, CD ₃ OD): 7.85 (s, 1H), 7.04 (m, 1H), 6.88 (bd, J = 11 Hz, 1H), 6.59 (m, 1H), 5.23 (dd, J = 12.6 Hz, 9.7 Hz), 4.53 (bt, J = 8 Hz, 1H), 4.22 (bd, J = 10 Hz, 1H), 3.80 (bd, J = 10 Hz, 1H), 3.48 (s, 3H) , 2.50 (s, 3H), 2.35 (s, 3H), 2.33 (s, 3H), 2.00 (s, 3H), 1.62 (m, 3H), 1.41 (d, J = 7 Hz, 3H), 1, 29 (d, J = 6.8 Hz, 3H), 0.67 (d, J = 7 Hz, 3H). HRMS (ESI-TOF) m / z 680.2730 (calcd. For C ₃₆ H ₄₂ NO ₁₂ [MH] ^- : 680.2707).

X-ray crystallography of 24-desmethylrifamycin S. Diffraction intensities were collected at 173 (2) K on a Bruker Apex CCD diffractometer using MoC radiation = 0., 1073 Å. The space group was determined based on systematic absences. Absorption adjustments were made using the SADABS program [Sheldrick, G.M. (1998). Bruker / Siemens Area Detector Absorption Correction Program (Bruker AXS, Madison. WI)]. Structures were solved using direct methods and Fourier methods and refined on F ² using full-matrix least squares methods. All non-H atoms were refined using anisotropic thermal parameters (Figure 16). Atoms H were considered in the calculated positions in the rigid group model. It was found that solvent water partially occupies a position between the molecules with a fill factor of 0.25. The H atoms in the solvent water molecule were not taken into account. The Flack parameter is 0.00 (10). The relatively high value of R _int , 0.1271, is due to the fact that the diffraction at large angles was very weak and the statistics on the intensity at large angles are insufficient. All calculations were performed using the Bruker SHELXTL software package (v. 6.10) [Sheldrick, G.M. (1998). Bruker / Siemens Area Detector Absorption Correction Program (Bruker AXS, Madison. WI)].

Example 7

Synthesis of 24-desmethylrifampicin

24-desmethylrifamycin S (5 mg, 0.0073 mmol) was dissolved in DMF (200 μl) and acetic acid (50 μl). After the mixture was stirred at 50 ° C, paraformaldehyde (3 mg) and 1,3,5-trimethyl-hexahydro-1,3,5-triazine (8 μl) were added. The reaction mixture was stirred at 50 ° C. for 2 hours until all of the starting material was converted to 3-methyl-1,3-oxazino (5,6-s) -24-desmethylrifamycin, as indicated by the blue spot on the TLC. After this, 1-amino-4-methyl-piperazine (8 μl) was added to the mixture. The reaction mixture was stirred and monitored by TLC until the blue spot disappeared and 24-desmethyl triampicin was formed. The mixture was diluted with chilled 2% acetic acid (1.5 ml) and extracted three times with CHCl ₃ (2 ml). The organic fractions were combined and concentrated to 1 ml and then washed 3 times with brine. The organic fractions were combined and dried over anhydrous sodium sulfate, then dried under a rotary evaporator. The crude fractions were chromatographed on silica gel with CHCl ₃ -MeOH at a ratio of 10: 1 & 8: 1 as eluent. The product containing fractions were combined and then purified by HPLC [CH ₃ CN - 0.05 M HCOONH ₄ (60:40)] using a column (YMC-ODS-A, internal diameter 250 × 10 mm, particle size 5 microns ) and at a flow rate of 2 ml / min. at 254 nm. The fractions containing 24-desmethylrifampicin were dried to give the title compound (2.5 mg, 0.0031 mmol) of a reddish-orange product powder.

¹ H NMR (500 MHz, CD ₃ OD): (Figure 17) HRMS (ESI-TOF) m / z 807.3829 (calcd. For C ₄₂ H ₅₅ N4O ₁₂ [MH] ^- : 807.3816).

To confirm the authenticity of 24-desmethylrifampicin, perform a comparative MS / MS analysis (Figure 18). The result showed that 24-desmethyl rifampicin (m / z 807 → 676 → 616 → 490 → 420) and rifampicin (m / z 821 → 690 → 630 → 490 → 420) have the same fragmentation patterns, although most fragments of 24-desmethyl rifampicin are 14 atomic mass units are less than the corresponding fragments from rifampicin (Figure 19). These fragmentation patterns are also consistent with the fragmentation patterns previously reported for rifampicin (Prasad, B. and Singh, S.J. (2009) Pharm. Biomed. Anal. Vol. 50, pages 475-490).

Example 8

Antibacterial analysis

The antibacterial activity of rifampicin and its analogues and derivatives, i.e. 24-desmethylrifamycin B, 24-desmethylrifamycin S, 24-desmethylrifamycin SV and 24-desmethylrifamycin are also determined using the agar diffusion method.

) при концентрации 1 мг/мл и высушили при комнатной температуре. Диски поместили на чашки с инокулированным агаром и инкубировали при 37°C в течение 24 ч. Для получения контрастного фона зоны ингибирования по всей верхности чашек добавили 0,25% проявляющийся краситель МТТ (2 мл).Mycobacterium smegmatis, Bacillus subtilis, Staphylococcus aureus and Escherichia coli were streaked onto nutrient agar and incubated overnight at 37 ° C. The colonies were transferred to a nutrient broth and incubated at 37 ° C for 24 hours. The growth of the cultures was measured to the proper density at 600 nm (BioRad, SmartSpec 300). The inoculum (1 ml) was thoroughly mixed with warm nutrient agar (24 ml) and poured into Petri dishes. The agar plates were left to harden and dry for 30 minutes before analysis. Whatman sterile discs were saturated with rifampicin and its analogues (20

) at a concentration of 1 mg / ml and dried at room temperature. Disks were placed on inoculated agar plates and incubated at 37 ° C. for 24 hours. 0.25% MTT stain (2 ml) was added to obtain a contrast background inhibition zone over the entire surface of the plates.

Antibacterial assays were performed with 24-desmethylrifamycin S and 24-desmethylrifamycin SV for gram-positive and gram-negative bacteria such as Mycobacterium smegmatis, Bacillus subtilis, Staphylococcus aureus and Escherichia coli (Figure 20). The results showed that the new compounds (24-desmethylrifamycin S and 24-desmethylrifamycin SV) obtained from the new analogue of 24-desmethylrifamycin B were more active than rifamycin B against M. smegmatis. Modeling studies were performed that suggested that 24-desmethylrifampicin binds to M. tuberculosis rifamycin-resistant RNA polymerase better than rifampicin.

Similarly, an antibacterial assay was performed using 24-desmethylrifampicin, 24-desmethylrifamycin S and commercially available rifampicin and rifamycin S against Mycobacterium smegmatis and Staphylococcus aureus. The results showed that 24-desmethylrifamycin S and 24-desmethylrifamycin are active against M. smegmatis and S. aureus, compared with rifamycin S and rifampicin, respectively (Figure 21).

Based on the above results, an additional study was conducted on the effect of analogues and derivatives in accordance with the present invention in order to study multidrug-resistant M. tuberculosis strains. To do this, MDR strains were purchased from Open Source Drug Discovery (OSDD; www.osdd.net) and tested for drugs at Premas Biotech, Haryana, India, according to WHO guidelines. Drug sensitivity tests were performed on two rifampicin-sensitive and three rifampicin-resistant M. Tuberculosis strains: OSDD 209 & H37Rv and OSDD 55, OSDD 206 & OSDD 321, respectively (Table 3).

Rifampicin (commercially available from HiMedia), 24-desmethylrifampicin and 24-desmethylrifamycin S were tested against the above pathogenic strains. Tests were performed at various concentrations (0.01-50 μg / ml) of drugs using the BacT / ALERT MB system [Crump, J.A. et al. (2011). J. Clin. Microbiol. Vol. 49, p. 3054-3057]. The results showed that 24-desmethylrifamycin S & 24-desmethylrifampicin showed strong antibacterial activity against both rifamycin-sensitive and rifamycin-resistant M. tuberculosis strains.

Drug sensitivity analyzes were performed at Premas Biotech, Haryana, India, using various drug concentrations (0.01-1 μg / ml). Drug testing was performed using the BacT / ALERT MB system [Crump, JA et al. (2011). J. Clin. Microbiol. Vol. 49, p. 3054-3057], which is a mycobacterium detection system in which a colorimetric sensor and a reflection sensor are used to determine the level of carbon dioxide inside the bottle. With the growth of microorganisms, CO _{2 is} produced, resulting in a color change of the sensor (at the bottom of the bottle). As the concentration of CO ₂ increases, the color changes from green to yellow. The bottle contains the medium and MV / VasT reducing fluid, which promotes the growth of mycobacteria. A sample is inoculated into an MP BacT / ALERT vial. Testing is carried out with two controls: direct growth control (DGC) and proportional growth control (PGC). DGC provides 0.1 ml inoculum in a MP BacT / ALERT vial along with 0.5 ml of reducing fluid. PGC provides 0.5 ml of DGC in a MP BacT / ALERT vial along with 0.5 ml of reducing fluid. The test vial provides 0.5 ml inoculum culture in an MP BacT / ALERT vial along with 0.5 ml of recovery fluid, as well as an antibiotic. The test is considered complete when the PGC vial signals a positive reading.

Free energy disturbance studies

Since analogues of 24-desmethylrifamycin showed comparable or higher activity than rifampicin against both rifampicin-sensitive and rifampicin-resistant M. tuberculosis, the authors decided to conduct free-energy perturbation (FEP) studies using wild-type RNAP [Postma, JP M. et al. (1982). Faraday Symp. Chem. Soc. Vol. 17, p. 55-67; Singh, U.C. et al. (1987) J. Am. Chem. Soc. Vol. 109, p. 1607-1614] (Table 4). Although calculations of FEP with rifampicin-resistant RNAP are more desirable, unfortunately, the crystal structure of mutant holo-RNAP has yet to be identified. Rifamycin B and 24-desmethylrifamycin B were selected in the study based on their potentially low computational costs, while for rifampicin derivatives they are slightly higher. In addition, it was generally accepted that C-3 or C-4 side chains do not significantly alter drug binding, but significantly change transport properties.

FEP calculations were performed using Gromacs 4.5 [Pronk, S. et al. (2013). Bioinformatics. Vol. 29, p. 845-854] with 21 parallel windows from a previously formed structure, and the Bennett acceptance ratio was used to tabulate the free binding energy. FEP results showed that 24-desmethylrifamycin B has a similar affinity for wild-type RNA polymerase with rifamycin B, but has a lower affinity for water than the latter compound. Such a significant decrease in the solvation affinity of 24-desmethylrifamycin B entails a higher distribution coefficient for protein than rifamycin B, which leads to an increase in the relative binding affinity of 24-desmethylrifamycin B for RNA polymerase by 1.2 kcal / mol (Table 4) .

Claims (25)

1. Antibacterial compounds having the following chemical structure:

. 2. The antibacterial compounds according to claim 1, characterized in that the antibacterial compounds are suitable against bacterial infection. 3. Antibacterial compounds according to paragraphs. 1, 2, characterized in that the antibacterial compounds are suitable against infection caused by species of the genus Mycobacterium. 4. Antibacterial compounds according to paragraphs. 1-3, characterized in that the selected species of the genus Mycobacterium are Mycobacterium tuberculosis and strains of M. Tuberculosis with multidrug resistance. 5. An antibacterial pharmaceutical composition comprising an antibacterial compound selected from 24-desmethylrifamycin S, 24-desmethylrifamycin SV and 24-desmethylrifampicin

together with their pharmaceutically acceptable carriers. 6. The pharmaceutical composition according to claim 5, characterized in that the antibacterial compounds are suitable against bacterial infection. 7. The pharmaceutical composition according to paragraphs. 5, 6, characterized in that the antibacterial compounds are suitable against infection caused by species of the genus Mycobacterium. 8. The pharmaceutical composition according to paragraphs. 5, 6, characterized in that the antibacterial compounds are suitable against infection caused by Mycobacterium tuberculosis and M. tuberculosis strains with multidrug resistance. 9. A method for treating bacterial infections, comprising administering to a patient antibacterial compounds selected from 24-desmethylrifamycin S, 24-desmethylrifamycin SV and 24-desmethylrifampicin

. 10. The use of antibacterial compounds selected from 24-desmethylrifamycin S, 24-desmethylrifamycin SV and 24-desmethylrifamycin

, to treat a bacterial infection. 11. The use of antibacterial compounds selected from 24-desmethylrifamycin S, 24-desmethylrifamycin SV and 24-desmethylrifamycin

, to obtain a medicine for the treatment of bacterial infections. 12. The use of antibacterial compounds according to claim 11 for the manufacture of a medicament for the treatment of infections caused by species of the genus Mycobacterium. 13. The use of the antibacterial compounds of claim 11 for the manufacture of a medicament for the treatment of an infection caused by species of the genus Mycobacterium, Mycobacterium tuberculosis and M. tuberculosis strains with multidrug resistance. 14. The method of producing 24-desmethylrifamycin S according to claim 1, wherein said method comprises the following steps: (a) converting 24-desmethylrifamycin B to 24-desmethylrifamycin S in the presence of copper chloride; (b) carrying out the reaction according to step (a) overnight at room temperature; and (c) obtaining 24-desmethylrifamycin S.

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