# Patent

## Title
Structural and mechanistic basis for novel compound biosynthesis using the 4-electron hexose oxidase

## Abstract

A novel pharmaceutical composition comprising a compound of formula (I) is disclosed. The novel compound can include a pharmaceutically acceptable carrier. The invention further comprises methods for making compounds of formula (I) using Dbv29, and to the use of compound of formula (I) to treat bacterial infections.

## Background
FIELD OF THE INVENTION
The invention relates to novel pharmaceutical compounds, and more particularly to novel pharmaceutical compounds useful in treating bacterial infections.
BACKGROUND OF THE INVENTION
Over the last 50 years, the number of pathogenic and commensal bacterial strains resistant to antibiotics, and the number of antibiotics to which they are resistant have increased dramatically. As a result, infections that once were readily treatable by antibiotics may no longer be responsive to such treatments. Bacteria that develop resistance to antimicrobial treatments, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistan Enterococcus (VRE), is a well recognized medical problem world-wide and has become one of the most important threats to modern health care.
For decades, vancomycin has outpaced the multidrug resistance of MRSA. It is now facing increased resistance by emerging vancomycin-intermediate S. aureus (VISA) and vancomycin-resistan Enterococcus. New antimicrobial agents and improved methods are thus needed for the treatment and prevention of infections caused by such pathogens.
SUMMARY OF THE INVENTION
The present invention relates to a novel compound, which has the formula (I),




or a pharmaceutically acceptable salt thereof,
wherein

    
    
        R1 is selected from the group consisting of aryl, alkyl, alkynyl;
        R2 is selected from the group consisting of C(O)OH, C(O)NH—R3, and CH2—NH—R4;
        
            wherein R3 is selected from the group consisting of aryl, alkyl, alkyne;
            R4 is selected from the group consisting of aryl, alkyl, alkynyl, admantyl, and C1-C10 azide.
        
        
    
    


The invention also provides a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutical acceptable carrier. Another aspect of the present invention relates to flavoenzyme, Dbv29, and the catalytic functions of Dbv29 in the preparation of the novel compound of the present invention.
Another aspect of this invention provides a method of treating a subject (e.g., humans and other mammals) with a bacterial infection. The method comprises the step of administering to a subject in need thereof an effective amount of the compound of formula (I). The compound of formula (I) can be administered by any of the accepted modes of administration including inhalation, topical, oral, rectal, implanted reservoir and parenteral (such as intravenous, intramuscular, subcutaneous, intra-articular, intra-synovial, cisternal, intrathecal, intrahepatic, intralesional and intracranial). Parenteral administration is preferred.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows the overall structure of Dbv29. FIG. 1B shows the long-loop region crossing over the binding pocket of Dbv29. FIG. 1C shows the lipid cavity of Dbv29.
FIG. 2 shows the result of a Structure-Activity Relationship study. The experimental result indicates that the compound of formula (I) with a longer side chain has a better antibacterial activity against Staphylococcus Aureus. 
FIG. 3A shows the blood bacterial counts of mice infected by E. faecalis (ATCC 51559) and treated with vancomycin, teicoplanin, compound 29 of formula (I) or saline (control) over a 3-day period. FIG. 3B shows the bacterial counts of mice on day 3 (the end of the treatment). The asterisk (*) indicates significant difference (P<0.05); data were expressed as mean±SD.



DETAILED DESCRIPTION OF THE INVENTION
Novel Compound of Formula (I)
The present invention relates to a novel compound, which has the formula (I)




or a pharmaceutically acceptable salt thereof,
wherein

    
    
        R1 is selected from the group consisting of aryl, alkyl, alkyne;
        R2 is selected from the group consisting of C(O)OH, C(O)NH—R3, and CH2—NH—R4;
        
            wherein R3 is selected from the group consisting of aryl, alkyl, alkyne;
            R4 is selected from the group consisting of aryl, alkyl, alkyne, admantyl, and C1-C10 azide.
        
        
    
    


If R4 is selected from alkyne or azide, the compound can be converted to other analogs through click chemistry for other applications such as identification of a second mode of action for broader spectrum analogs.
“Alkyl” refers to groups of from 1 to 15 carbon atoms inclusively, either straight chained, branched, cyclic or unsaturated, more preferably from 1 to 10 carbon atoms inclusively.
The term “aryl” is used herein to refer to an aromatic carbocyclic group of 6 to 14 carbon atoms inclusively having a single ring (e.g. phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Preferred aryl include phenyl and benzyl.
“Azide” refers to an anion with the formula N331.
“Alkynyl” refers to groups of from 2 to 6 carbon atoms inclusively, either straight or branched containing at least one triple bond.
The pharmaceutically acceptable salts of the compounds of formula (I) include the non-toxic salts formed from non-toxic inorganic or organic bases. For example, non-toxic salts can be formed with inorganic bases such as an alkali or alkaline earth metal hydroxide, e.g., potassium, sodium, lithium, calcium, or magnesium; and with organic bases such as an amine.
The pharmaceutically acceptable salts of the compounds of formula (I) include non-toxic salts formed from non-toxic inorganic or organic acids. Example of organic and inorganic acids are, for example, hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic, sorbic and benzoic acids.
The pharmaceutically acceptable salts can be synthesized from the compounds of formula (I) by any conventional chemical methods. In one embodiment, the salt is prepared by reacting the free acid with a requisite amount of inorganic or organic base in a suitable solvent or various combinations of solvents. In another embodiment, the salt is prepared by reacting the free base with a requisite amount of inorganic or organic acid in a suitable solvent or various combinations of solvents.
In a preferred embodiment, R1 is (CH3)2—CH—(CH2)6 and R2 is CH2—NH—CH2—C6H6 
The Pharmaceutical Composition
The present invention is also directed to a pharmaceutical composition comprising the compound of formula (I) and a pharmaceutically acceptable carrier.
A “pharmaceutically acceptable carrier” refers to a carrier that, after administration to or upon a subject, does not cause undesirable physiological effects. The carrier in a pharmaceutical composition must be “acceptable” also in the sense that is compatible with the active ingredient and, preferably, capable of stabilizing it. Suitable pharmaceutically acceptable carriers are well known in the art and vary with the desired form and mode of administration of the pharmaceutical composition. For example, they may include, but are not limited to, biocompatible vehicles, adjuvants, additives (such as pH-adjusting additives), diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface-active agents, lubricants and the like. One or more pharmaceutical carriers may be used for the delivery of a compound of formula (I).
The pharmaceutical composition can be prepared for inhalation, topical, oral, rectal, implanted reservoir and parenteral delivery (such as intravenous, intramuscular, subcutaneous, intra-articular, intra-synovial, cisternal, intrathecal, intrahepatic, intralesional and intracranial).
The pharmaceutical composition can be prepared by any method known in the art of pharmacy. Such methods include the step of bringing into association the active compound with one or more carriers. For instance, to prepare compositions suitable for injection, solutions and suspensions are sterilized and are preferably isotonic to blood. In making injectable preparations, carriers which are commonly used in this field are used, for example, water, ethyl alcohol, propylene glycol. In these instances, adequate amounts of isotonicity adjusters such as sodium chloride, glucose or glycerin can be added to make the preparations isotonic. The aqueous sterile injection solution may further comprise oxidants, buffers, and other similar additions, which are acceptable for parenteral compositions.
For instance, for oral administration in the form of a tablet or capsule, the active compound can be comminuted with a pharmaceutically acceptable carrier such as ethanol, glycerol, water and the like. Powders are prepared by comminuting the compound to a suitable fine size and mixing it with a comminuted pharmaceutical carrier such as an edible carbohydrate, for example, starch or mannitol. Flavoring, dispersing and coloring agents can also be present.
For the treatment of the eyes or other external tissues, for example, the mouth and the skin, the pharmaceutical compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active compound may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
The Flavoenzyme Dbv29
The present invention is also directed to flavoenzyme, Dbv29, and the catalytic function of the enzyme in the preparation of the novel compound of formula (I). Dbv29 is a flavin dinucleotide (FAD)-dependent primary alcohol glycopeptide hexose oxidase which carries out a 4-electron oxidation reaction. The four-electron oxidation reaction from primary alcohol to acid catalyzed by Dbv29 was disclosed in J Am Chem Soc 129:13384-13385 (2007) by Li et al, which is incorporated herein by reference. Dbv29 catalyses the last step of the N-acyl aminoglucuronyl biosynthesis. Dbv29 can be obtained from a filamentous actinomycete Nonomuraea sp. ATCC39727 and its amino acid sequence is listed in Table 1 (SEQ ID NO:1). The structure of Dbv 29 and the complex structure of Dbv29 have been deposited in the Protein Data Bank under accession numbers 2wdw and 2wdx, respectively.








TABLE 1







1
mtggtgadaa sagasstrpe lrgerclppa gpvkvtpddp rylnlklrga nsrfngepdy


 


61
ihlvgstqqv adaveetvrt gkrvavrsgg hcfedfvdnp dvkviidmsl lteiaydpsm


 


121
nafliepgnt lsevyeklyl gwnvtipggv cggvgvgghi cgggygplsr qfgsvvdyly


 


181
avevvvvnkq gkarvivatr erddphhdlw wahtgggggn fgvvtkywmr vpedvgrnpe


 


241
rllpkppatl ltstvtfdwa gmteaafsrl lrnhgewyer nsgpdspytg lwsqlmigne


 


301
vpgmgesgfm mpiqvdatrp darrlldahi eavidgvppa evpepieqrw lastpgrggr


 


361
gpasktkagy lrkrltdrqi qavyenmthm dgidygavwl igyggkvntv dpaatalpqr


 


421
dailkvnyit gwanpgneak hltwvrklya dvyaetggvp vpndvsdgay inypdsdlad


 


481
pglntsgvpw hdlyykgnhp rlrkvkaayd prnhfhhals irp









It differs from other hexose oxidoreductases in the coenzyme used and in lacking a typical Cys residue in the active site.

Two domains for the catalytic activity are identified, i.e., the F domain (residues 1-230), which binds FAD and the S domain (residues 235-523), which recognizes the substrate. See FIG. 1. The F domain can be further divided into two subdomains. The first subdomain 1 (the N-terminal subdomain (residues 1-105) consists of a central four-stranded β-sheet (B1-B4 with the strand order of 1, 2, 4, 3, wherein B1 runs antiparallel to the others), sandwiched on each side by one α-helix (H1 and H2); the second subdomain 2 (residues 113-163 and 165-230) consists of a β-sheet of five antiparallel strands (B5-B9 with the strand order of 5, 6, 9, 7, 8) that faces against five irregular α-helices (H3, H4, H6, H7 and H20). For the S domain, this domain is composed of an extended antiparallel seven-stranded β-sheet (B10-B16 with the strand order of 13, 10, 12, 11, 15, 16, 14), faced by four major α-helices in a row (H12, H10, H13, and H16). (See FIG. 1) These two domains are joined by two long-loop regions (residues 231-241 and 454-474).
A long-loop region (residues 351-362) crosses over the binding pocket. (See FIG. 1B.) The N-acyl moiety of the amino sugar is inserted further into a hydrophobic lipid cavity, which is formed by residues Phe-93, Ser-364, Tyr-395, Trp-399 and Ile-429. (See FIG. 1C.) A longer acyl side chain is favored to pack into the lipid cavity, as the longer the side chain the better the anti-bacterial activity (C10 vs C4, by >10 folds, see FIG. 2). The sugar ring anchors against the si face of the isoalloxazine ring of FAD and exposes carbon atom C5 close to the N5 nitrogen of FAD. The hydroxyl group of C4 forms hydrogen bonds with Asn-427. The hydroxyl group of C6 interacts with the side chains of Tyr-165 and Tyr-473. Most of residues that line the ligand-binding site are located in different loop regions. Several ligand-binding residues of Dbv29 were identified. Trp-399 is nearby the reducing end of the sugar, and, together with Ile-401, stands above the shoulder of the r4 sugar head and poises the head facing the isoalloxazine ring. Trp-399 was determined to be catalytically important as no product was produced by the W399A mutant, while the W399F mutant retained 15% activity (Table 2). Thus, a bulkier and hydrophobic side chain may be favored at the position. Ile-401 that stands on the other side of the sugar head, nearby the C6 carbon, is also influential as the activity was significantly reduced when this residue was mutated to smaller Ala (9%) or bulkier Trp (2%, Table 2). Thr-366 that situates opposite Trp-399 at the entry of the lipid cavity may assist the lipid chain insertion into the lipid cavity. Two mutants (T366A and T366E) showed reduced activities (11% and 5% relative activity, respectively; Table 2). Ser-364, lining at the bottom of the cavity, may force the lipid to fold in a spiral manner. The enzyme activity was reduced significantly when it was changed to bigger and charged side chains (S364K/R, Table 2).







TABLE 2







Relative enzymatic activities of Dbv29 mutants














Flavinylation/
Relative




Mutants
Expression
FAD binding
activitya
Rationale
Outcome





C26A
+
+
1.11
Putative point of active
Role disproven






site general base


C92A
+
+
0.95
Putative point of active
Role disproven






site general base


C151A
+
+
0.23
Putative point of active
Role disproven/






site general base/
Role proven






Putative point of






covalent attachment


C161A
+
+
0.84
Putative point of active
Role disproven






site general base


H91A
+
+
0.11
Putative point of
Role proven






covalent attachment


1401A
+
+
0.09
Potential
Role proven






ligand-binding residue


1401W
+
+
0.02
Potential
Role proven






ligand-binding residue


N44A
+
+
0.87
Potential interface
Role proven






interacting residue


R41A
+
+
0.72
Potential interface
Role proven






interacting residue


R360E
+
+
0.76
Putative point affecting
Role disproven






FAD attachment


R360L
+
+
0.23
Putative point affecting
Role disproven






FAD attachment


S364K
+
+
0.04
Potential
Role proven/






ligand-binding residue/
Role disproven






Putative point affecting






FAD attachment


S364R
+
+
0.08
Potential
Role proven/






ligand-binding residue/
Role disproven






Putative point affecting






FAD attachment


T366A
+
+
0.11
Potential
Role proven/






ligand-binding residue/
Role disproven






Putative point affecting






FAD attachment


T366E
+
+
0.05
Potential
Role proven/






ligand-binding residue/
Role disproven






Putative point affecting






FAD attachment


T366L
+
+
0.13
Potential
Role proven/






ligand-binding residue/
Role disproven






Putative point affecting






FAD attachment


W399A
+
+
—
Potential
Role proven






ligand-binding residue


W399F
+
+
0.16
Potential
Role proven






ligand-binding residue


Y135F
−
−
—
Putative point affecting
Role disproven






FAD attachment


Y165F
+
+
0.70
Putative point of active
Role proven






site general base


Y165W
+
+
0.03
Putative point of active
Role proven






site general base


Y370F
+
+
0.10
Putative point affecting
Role disproven






FAD attachment


Y403F
+
+
0.65
Putative point affecting
Role disproven






FAD attachment


Y428F
+
+
1.00
Putative point affecting
Role disproven






FAD attachment


Y453F
+
+
0.82
Putative point affecting
Role disproven






FAD attachment


Y470F
+
+
0.21
Putative point affecting
Role disproven






FAD attachment


Y473F
+
+
0.23
Putative point of active
Role proven






site general base


Y473E
+
+
—
Putative point of active
Role proven






site general base


H91A/C151A
+
+
0.05
Putative point of
Role proven






covalent attachment


R41A/N44A
+
+
0.59
Potential interface
Role proven






interacting residue


R360E/T366E
+
+
0.03
Putative point affecting
Role disproven






FAD attachment


R360L/T366A
+
+
0.03
Putative point affecting
Role disproven






FAD attachment


R360L/T366E
+
+
0.01
Putative point affecting
Role disproven






FAD attachment


Y165F/Y473F
+
−
—
Putative point of active
Role proven






site general base


Y428F/Y453F
+
+
1.08
Putative point affecting
Role disproven






FAD attachment


Y370F/Y403F
+
+
0.05
Putative point affecting
Role disproven






FAD attachment









The analyses of Dbv29 also shows that Tyr-473 and Tyr-165 function as a unique “molecular device” that governs cofactor binding, catalysis, and folding of the enzyme.
Cofactor Binding.
Even though the isoalloxazine ring of flavin is covalently linked to the side chains of His-91 and Cys-151, binding of FAD is governed by the tyrosine pair. Single mutants (H91A and C151A) retain 11% and 23% activities; respectively, relative to WT, and the double mutant (H91A/C151A) also retains 5% activity (Table 2) as well as the yellow color. On the other hand, Y165F/Y473F double mutation (but not the two single mutations) led to loss of the yellow color and total loss of catalytic activity (Table 2).
Active Site Structures and Catalysis.
Despite the crystal being grown with Teicoplanin, the density best fitted is neither Teicoplanin (substrate) nor oxo-Teicoplanin (product). It is a water-coordinated diol species because two electron densities on the C6 terminus of the amino sugar 12 (See Scheme 1) share equal weighting in sp3 configuration, thus the structure of the complex represents the transformed substrate for the second half of the oxidation. It also provides a structural basis for a proposed catalytic mechanism: in the initial stage the C6 hydroxyl group is most likely de-protonated by the hydroxyl group of Tyr-473 that is activated by the hydroxyl group of Tyr-165. This pair together drives the pro-R hydride transfer from the C6 carbon to the N5 nitrogen of isoalloxazine ring. The hydroxyl group of Tyr-165 is further facilitated by the diol-coordinated water molecule in the second oxidation stage, where a proton relay network is formed amid the tyrosine pair, the substrate diol and a water molecule.
To further support that the Y165-Y473 pair functions as a molecular device, it is necessary to show that no other di-hydroxy pairs around the active site possess similar properties. Four other tyrosine residues, Tyr-135, -370, -403 and -470, which cluster together nearby the active site, were individually mutated to Phe. These mutants (except Y135F that could not be expressed) did not lose FAD, although the enzyme activities vary (Table 2). When the distance between two hydroxyl groups is extended to 5 Å, two extra tyrosine pairs fall within this scope, which are Y403/Y370 (4.1 Å) and Y428/Y453 (2.8 Å). The double mutants, Y403F/Y370F and Y428F/Y453F, were constructed and shown to contain bicovalent FAD and retain enzyme activities (Table 2). In addition to these ‘in cis’ residue pairs, residue pairs ‘in trans’ (side chains point in opposite directions) other than tyrosine were also examined. Thr-366 that gates at the lipid cavity, along with the bulky and charged Arg-360 which sits on the same long loop (351-362) but face in opposite direction were selected. Five mutants were made, which include two single mutations R360E/T366E. The FAD was retained in all mutants, and the double mutations displayed a synergistic negative effect on overall enzyme activity (Table 2). Taken together, the results support that the tyrosine pair (Tyr-165 and -473) is unique in functioning as a molecular device.
The Processes of Producing the Compounds of Formula (I)
The compound of formula (I) is prepared using teicoplanin as a starting material. Teicoplanin is a glycopeptide antibiotic for treating serious infections caused by Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and Enterococcus faecalis. It is commercially available and can be prepared by known methods, for example, U.S. Pat. No. 7,192,743, which is incorporated herein by reference. The amount of teicoplanin used is not critical, although a concentration of about 0.1 mM to about 5 mM is preferred.
The crystal structure of the Dbv29-oxo-Tei complex indicates another important property—that the aldehyde group (its diol adduct)—is highly exposed. This can explain that, when the reaction was performed in 18O-water, both oxygens of the diol intermediate are labeled with 18O. In addition, when reactions containing Tei with/without Dbv29 were carried out in a NaBD4-containing solution, MS analyses of the Tei recovered from the reaction (a mixture of unreacted Tei and Tei obtained from reduction of the aldehyde intermediate) clearly showed M+1. This showed the possibility of trapping and modifying the aldehyde intermediate as a way to produce compounds of formula (I)).
Compounds of formula (I) can be prepared according to Scheme 1, which comprises: (a) binding teicoplanin 4 to Dbv29; and (b) incubating the mixture (a).




The incubation temperature ranges from about 30° C. to about 40° C., and preferably about 37° C. The incubation time ranges from about 1 hour to 10 hours, and preferably about 3 hours to 4 hours.
Compounds of formula (I) can also be prepared according to Scheme 2, comprising: (a) binding teicoplanin 4 to Dbv29; (b) adding C1-6 alkylamine to mixture (a); and (c) incubating the mixture (b).




About 5 mM to about 15 mM of C1-6 alkylamine can be used. The C1-6 alkylamine may react with the aldehyde intermediate as soon as it is formed in the first oxidation reaction, the resulting gem-hydroxyl-C1-6 alkylamine may then undergo the second oxidation reaction without leaving the active site of Dbv29. The incubation temperature ranges from about 30° C. to about 40° C., and preferably about 37° C. The incubation time ranges from about 1 hour to about 48 hours, preferably about 5 hours to about 24 hours, and more preferably about 16 hours.
Compound of formula (I) can also be prepared according to Scheme 3, comprising: (a) binding teicoplanin 4 to Dbv29; (b) adding C6-15 alkylamine, a reducing agent, and an organic solvent to mixture (a); and (c) incubating the mixture (b).




About 5 mM to about 15 mM of C6-15 alkylamine can be used in this reaction. C6-15 alkylamine maybe be too bully to enter the active site of Dbv29 so that C6-15 alkylamine reacts with the aldehyde functional group in solution to form imine that is subsequently reduced by cyanohybridoborate (Na(CN)BH3). The organic solvent used in Scheme 3 is DMSO. In one embodiment, about 10% to about 90% of DMSO is used. In a preferred embodiment, about 50% of DMSO is used.
The reducing agent used in Scheme 3 is cyanohydridoborate. In one embodiment, about 1 mM to about 20 mM of cyanohydridoborate is used. In a preferred embodiment, about 10 mM of cyanohydridoborate is used. The incubation temperature ranges from about 30° C. to about 40° C., and preferably about 37° C. The incubation time ranges from about 1 hour to about 48 hours, preferably about 5 hours to about 24 hours, and more preferably about 16 hours.
Compound of formula (I) can also be prepared according to Scheme IV, comprising: (a) binding teicoplanin 4 to Dbv21 and the mixture is incubated to form compound 5; (b) Combining compound 5 with Dbv8 and CoA derivatives, and the mixture is incubated; and (c) Combining mixture (b) with Dbv29 and O2 as the electron acceptor, and the mixture is incubated.




Dbv21 is an enzyme which catalyses the hydrolysis of acetyl from N-acetyl glucosamine moiety. The deacetylation reaction by Dbv8 and the formation of compound 5 were described by Ho et al in J Am Chem Soc 128:13694-13694 (2006), which is incorporated herein by reference. Dbv8 is an enzyme which catalyses the acylation of glucosamine moiety with various CoA derivatives. The amino acid sequences of Dbv21 and Dbv8 were reported in Chem Biol 10(6): 541-549 (2003) by Sosio et al, which are incorporated herein by reference.
CoA derivatives are selected from the group comprising acetyl CoA, propionyl CoA, butyryl CoA, hexanoyl CoA, octanoyl CoA, decanoyl CoA, lauroyl CoA, myristoyl CoA, palmitoyl CoA, crotonyl CoA, isobutyryl CoA, isovaleryl CoA, malonyl CoA, succinyl CoA, glutaryl CoA, methylmalonyl CoA, acetoacetyl CoA, benzoyl CoA, phenylacetyl CoA, biphenylacetyl CoA, and naphthoacetyl CoA. The incubation temperature for Scheme 4 ranges from about 30° C. to about 40° C., and preferably about 37° C. The incubation times for steps (a) and (b) in Scheme 4 depend on the concentration of teicoplanin. In Step (a), for each mM of teicoplanin used, the incubation time ranges from about 1 hour to about 24 hours, and preferably about 6 hours. In Step (b), for each mM of teicoplanin used, the incubation time ranges from about 2 hours to about 12 hours, and preferably about 4 hours. The incubation time for step (c) in Scheme 4 ranges from about 1 hour to about 10 hours, and preferably about 3 hours to about 4 hours.
The present invention is also directed to a method of producing compound of formula (I) according to Scheme 5, comprising: (a) binding teicoplanin 4 to Dbv21 and the mixture is incubated to form compound 5; (b) Combining compound 5 with Dbv8 and CoA derivatives, and the mixture is incubated; and (c) Combining mixture (b) with Dbv29, O2 as the electron acceptor, and alkylamine and the mixture is incubated.




CoA derivatives are selected from the group consisting of acetyl CoA, propionyl CoA, butyryl CoA, hexanoyl CoA, octanoyl CoA, decanoyl CoA, lauroyl CoA, myristoyl CoA, palmitoyl CoA, crotonyl CoA, isobutyryl CoA, isovaleryl CoA, malonyl CoA, succinyl CoA, glutaryl CoA, methylmalonyl CoA, acetoacetyl CoA, benzoyl CoA, phenylacetyl CoA, biphenylacetyl CoA, and naphthoacetyl CoA.
The alkylamine can be C1-6 alkylamine, or benzylamine. About 5 mM to about 15 mM of alkylamine can be used in this reaction. The incubation temperature ranges from about 30° C. to about 40° C., and preferably about 37° C. The incubation times for steps (a), (b) and (c) in Scheme 5 depend on the concentration of teicoplanin. In Step (a), for each mM of teicoplanin used, the incubation time ranges from about 1 hour to about 24 hours, and preferably about 6 hours. In Step (b), for each mM of teicoplanin used, the incubation time ranges from about 2 hours to about 12 hours, and preferably about 4 hours. In Step (c), for each mM of teicoplanin used, the incubation time ranges from about 1 hour to about 48 hours, preferably about 5 hours to about 24 hours, and more preferably about 16 hours.
Compounds of formula (I) can also be prepared according to Scheme 6, comprising: (a) binding teicoplanin 4 to Dbv21 and the mixture is incubated to form compound 5; (b) Combining compound 5 with Dbv8 and CoA derivatives, and the mixture is incubated; and (c) Combining mixture (b) with Dbv29, O2 as the electron acceptor, alkylamine, an organic solvents and a reducing agents, and the mixture is incubated.




CoA derivatives are selected from the group consisting of acetyl CoA, propionyl CoA, butyryl CoA, hexanoyl CoA, octanoyl CoA, decanoyl CoA, lauroyl CoA, myristoyl CoA, palmitoyl CoA, crotonyl CoA, isobutyryl CoA, isovaleryl CoA, malonyl CoA, succinyl CoA, glutaryl CoA, methylmalonyl CoA, acetoacetyl CoA, benzoyl CoA, phenylacetyl CoA, biphenylacetyl CoA, and naphthoacetyl CoA.
The alkylamine in Scheme 6 is C6-15 alkylamine or benzylamine. About 5 mM to about 15 mM of alkylamine can be used in this reaction. The organic solvent used in Scheme 6 is DMSO. In one embodiment, about 10% to about 90% of DMSO is used. In a preferred embodiment, about 50% of DMSO is used.
The reducing agent used in Scheme 6 is cyanohydridoborate. In one embodiment, about 1 mM to about 20 mM of cyanohydridoborate is used. In a preferred embodiment, about 10 mM of cyanohydridoborate is used. The incubation temperature for Scheme 6 ranges from about 30° C. to about 40° C., and preferably about 37° C.
The incubation times for Steps (a) and (b) in Scheme 6 depend on the concentration of teicoplanin. In Step (a), for each mM of teicoplanin used, the incubation time ranges from about 1 hour to 24 hours, and preferably about 6 hours. In step (b), for each mM of teicoplanin used, the incubation time ranges from about 2 hours to about 12 hours, and preferably about 4 hours. The incubation time for step (c) in Scheme VI ranges from about 1 hour to about 48 hours, preferably about 5 hours to about 24 hours, and more preferably about 16 hours.
The Method of Treating a Bacterial Infection
The method of treating a bacterial infection in a subject in need of such treatment, comprises the administration of an effective amount of the compound of formula (I) and a pharmaceutically acceptable salt thereof.
The in vitro antibacterial activity of the compound of formula (I) against classically “resistant” strains is superior to vancomycin and teicoplanin. The minimum inhibitory concentration (MIC) value against VRE for compounds of formula (I) can be 8 to 32 folds lower than that of vancomycin and teicoplanin (see Table 6). In one embodiment, the method includes administering to a subject an effective amount of the compound of formula (I) against the vancomycin-susceptible enterococcus infection. In another embodiment, the method includes administering to a subject an effective amount of the active compound against the vancomycin-resistant enterococcus infection. In yet another embodiment, the method includes administering to a subject an effective amount of the active compound against the methacillin-resistant staphylococcus infection.
The term “treat” refers to administering an effective amount of active compound to a subject that has a bacterial infection, or has symptoms and signs of such an infection, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the infection, the symptoms and signs of the infection.
An “effective amount” refers to a dose of the active compound that is sufficient to eradicate or reduce the bacterial infection, which is measured by one or more of the following criteria: eradication or reduction of the bacteria by microbiological measures (such as blood culture) or serological measures (such as white blood cell count, serum C-reactive protein level), and resolution or improvement of clinical signs and symptoms (such as fever, tachycardia). Both in vivo and in vitro studies can be conducted by one skilled in the art to determine optimal administration routes and dose. The effective amount will vary depending upon several factors, including, but not limited to, the age and weight of the patient, route and frequency of administration, how advanced the disease state is, any co-morbidity, or the co-administration of other antimicrobial agents.
The term “administration” covers inhalation, topical, oral, rectal, implanted reservoir and parenteral (such as intravenous, intramuscular, subcutaneous, intra-articular, intra-synovial, cisternal, intrathecal, intrahepatic, intralesional and intracranial) delivery to a subject the active compound of the invention. Parenteral route of administration is preferred.
The composition for oral administration can be any orally acceptable dosage form including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions. The oral composition may include sustained release properties as well as rapid delivery forms.
Topical application may be formulated in carriers such as hydrophobic or hydrophilic bases to form ointments, cream, lotions, in aqueous, oleaginous or alcoholic liquids to form paints or in dry diluents to form powders.
The parenteral compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain various formulating agents. Alternatively, the active ingredient may be in powder form for reconstitution at the time of delivery with a suitable vehicle, such as sterile water.
In general, about 0.0625 mg to about 32 mg of a compound of formula (I) per kg of body weight per dose will be an effective dosage. The preferred dosage is about 0.125 mg to about 8 mg of compound of formula (I) per kg of body weight per dose, and more preferably about 0.5 mg to about 4 mg of compound of formula (I) per kg of body weight per dose. The treatment interval ranges from once per day to once per week. The dosage and treatment interval may vary somewhat depend upon the route of delivery and the condition of the patent, and determined by one skilled in the art.
The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.
EXAMPLES
Example 1
Preparation of Compounds 14, 29, 30-32, 34 and 35
Compounds 14, 29, 30-32, 34 and 35 were enzymatically synthesized using:
1. 0.5 mM of teicoplanin;
2. 10 mM of decylamine for compound 14; 10 mM of benzylamine for compound 29; 10 mM of 1-adamantanemethylamine for compound 30; 10 mM of 1-adamantaneamine for compound 31; 10 mM of 2-adamantaneamine for compound 32; 10 mM of propargylamine for compound 34; 10 mM of 5-azide pentylamine for compound 35; and
3. In the presence of 0.03 mM of Dbv29, 10 mM of Na(CN)BH3 and 50% DMSO, the mixture was incubated at 37° C. overnight.
See Table 3 for product yields of compounds 14, 29, 30-32, 34 and 35.
Example 2
Preparation of Compounds 15, 28 and 33
Compounds 15, 28 and 33 were enzymatically synthesized using:
1. 0.5 mM of teicoplanin;
2. 10 mM of hexylamine for compound 15; 10 mM of benzylamine for compound 28; and 10 mM of propargylamine for compound 33; and
3. In the presence of 0.03 mM of Dbv29, 10 mM of Na(CN)BH3 and 50% DMSO, the mixture was incubated at 37° C. overnight.
See Table 3 for product yields of compounds 15, 28 and 33.
Example 3
Preparation of Compound 16
Compound 16 was enzymatically synthesized using 0.5 mM of teicoplanin and 10 mM Butylamine, in the presence of 0.03 mM of Dbv29. The mixture was incubated at 37° C. overnight. See Table 3 for the product yield of compound 16.
Example 4
Preparation of Compound 17
Compound 17 was enzymatically synthesized using 0.5 mM of teicoplanin and 10 mM of aniline in the presence of 0.03 mM of Dbv29. The mixture was incubated at 37° C. overnight. See Table 3 for the product yield of compound 17.
Example 5
Preparation of Compound 18
Compound 18 was enzymatically synthesized using 0.5 mM of teicoplanin and 5 mM of octylamine in the presence of 0.03 mM of Dbv29, 5 mM of Na(CN)BH3 and 50% DMSO. The mixture was incubated at 37° C. overnight. See Table 3 for the product yield of Compound 18.
Example 6
Preparation of Compound 19
Compound 19 was enzymatically synthesized using 0.5 mM of Teicoplanin and 5 mM of dodecylamine in the presence of 0.03 mM of Dbv29, 5 mM of Na(CN)BH3 and 50% DMSO. The mixture was incubated at 37° C. overnight. See Table 3 for the product yield of compound 19.
Example 7
Preparation of Compounds 20, 21, 22 and 23
(a) Hydrolyzing acetyl off N-acetyl glucosamine moiety: Combined 1 mM of teicoplanin with 0.02 mM of Dbv21, and the mixture was incubated at 37° C. for 6 hours.
(b) Acylation of glucosamine moiety with various CoA derivatives: Combined mixture (a) with 0.02 mM of Dbv8 and 1 mM of CoA derivatives (e.g. Butyryl CoA for compound 20, Hexanoyl CoA for compound 21, Octanoyl CoA for compound 22 and Decanoyl CoA for compound 22), and the mixture was incubated at 37° C. for 4 hours.
(c) Oxidation: Combined mixture (b) with 0.03 mM of Dbv29 and O2 as the electron acceptor, and the mixture was incubated at 37° C. for 4 hours.
See Table 3 for product yields of compounds 20, 21, 22 and 23.
Example 8
Preparation of Compounds 24 and 25
(a) Hydrolyzing acetyl off N-acetyl glucosamine moiety: Combined 1 mM of teicoplanin with 0.02 mM of Dbv21, and the mixture was incubated at 37° C. for 6 hours.
(b) Acylation of glucosamine moiety with various CoA derivatives: Combined mixture (a) with 0.02 mM of Dbv8 and 1 mM of CoA derivatives (e.g. Hexanoyl CoA for compound 24 and Octanoyl CoA for compound 25), and the mixture was incubated at 37° C. for 4 hours.
(c) Oxidation: Combined mixture (b) with 0.03 mM of Dbv29, O2 as the electron acceptor and 5 mM of hexylamine, and the mixture was incubated at 37° C. for 24 hours.
See Table 3 for product yields of compounds 24 and 25.
Example 9
Preparation of Compound 26
(a) Hydrolyzing acetyl off N-acetyl glucosamine moiety: Combined 1 mM of teicoplanin with 0.02 mM of Dbv21, and the mixture was incubated at 37° C. for 6 hours.
(b). Acylation of glucosamine moiety with CoA derivatives: Combined mixture (a) with 0.02 mM of Dbv8 and 1 mM of Hexanoyl CoA, and the mixture was incubated at 37° C. for 4 hours.
(c) Oxidation: Combined mixture (b) with 0.03 mM of Dbv29, O2 as the electron acceptor, 5 mM of octylamine, 5 mM of Na(CN)BH3 and 50% DMSO, and the mixture was incubated at 37° C. for 24 hours.
See Table 3 for the product yield of compound 26.
Example 10
Preparation of Compound 27
(a) Hydrolyzing acetyl off N-acetyl glucosamine moiety: Combined 1 mM of teicoplanin with 0.02 mM of Dbv21, and the mixture was incubated at 37° C. for 6 hours.
(b) Acylation of glucosamine moiety with CoA derivatives: Combined mixture (a) with 0.02 mM of Dbv8 and 1 mM of Octanoyl CoA, and the mixture was incubated at 37° C. for 4 hours.
(c) Oxidation: Combined mixture (b) with 0.03 mM of Dbv29, O2 as the electron acceptor, 5 mM of dodecylamine, 5 mM of Na(CN)BH3 and 50% DMSO, and the mixture was incubated at 37° C. for 24 hours.
See Table 3 for the product yield of compound 27.
The product yields of compounds 13 to 35 by Dbv29 are summarized in Table 3. The product yields were determined by HPLC, and yield=(peak area of aminated or amidated product)/(peak areas of aminated and amidated and oxidative products)×100%.











TABLE 3





 



Coenzyme A

Product




derivative

type: amide
Yield


Compound
(Carbon No.)
Amine added
or amine
%























—
—
—
100


 






—




Amine
25


 






—




Amide
10


 






—




Amide
<10


 






—




Amide
10


 






—




Amine
25


 






—




Amine
17


 






C4
—
—
100


 






C6
—
—
100


 






C8
—
—
100


 






C10
—
—
100


 






C6




Amide
10


 






C8




Amide
10


 






C6




Amine
30


 






C8




Amine
30


 






—




Amide
<10


 






—




Amine
46


 






—




Amine
<10


 






—




Amine
<10


 






—




Amine
<10


 






—




Amide
<10


 






—




Amine
<10


 






—




Amine
25









Dbv8 was used to add various lengths of acyl side chains from corresponding CoA derivatives to the C2 amine group.

The preparation method of compounds 13 to 35 are summarized in Table 4.











TABLE 4





Example






No.
R1
R2
Scheme
Enzyme(s) used







13
(CH3)2CH(CH2)6
C(O)OH
I
Dbv 29


14
(CH3)2CH(CH2)6
C(O)NH(CH2)9CH3
III
Dbv 29


15
(CH3)2CH(CH2)6
C(O)NH(CH2)5CH3
II
Dbv 29


16
(CH3)2CH(CH2)6
C(O)NH(CH2)3CH3
II
Dbv 29


17
(CH3)2CH(CH2)6
C(O)NH—C6H6
II
Dbv 29


18
(CH3)2CH(CH2)6
CH2NH(CH2)7CH3
III
Dbv 29


19
(CH3)2CH(CH2)6
CH2NH(CH2)11CH3
III
Dbv 29


20
CH3(CH2)2
C(O)OH
IV
Dbv 21, Dbv 8






and Dbv 29


21
CH3(CH2)4
C(O)OH
IV
Dbv 21, Dbv 8






and Dbv 29


22
CH3(CH2)6
C(O)OH
IV
Dbv 21, Dbv 8






and Dbv 29


23
CH3(CH2)8
C(O)OH
IV
Dbv 21, Dbv 8






and Dbv 29


24
CH3(CH2)4
C(O)NH(CH2)5CH3
V
Dbv 21, Dbv 8






and Dbv 29


25
CH3(CH2)6
C(O)NH(CH2)5CH3
V
Dbv 21, Dbv 8






and Dbv 29


26
CH3(CH2)4
CH2NH(CH2)7CH3
VI
Dbv 21, Dbv 8






and Dbv 29


27
CH3(CH2)6
CH2NH(CH2)7CH3
VI
Dbv 21, Dbv 8






and Dbv 29


28
(CH3)2CH(CH2)6
C(O)NH—CH2—C6H6
II
Dbv 29


29
(CH3)2CH(CH2)6
CH2—NH—CH2—C6H6
III
Dbv 29


30
(CH3)2CH(CH2)6
CH2−—NH—CH2-
III
Dbv 29




admantyl


31
(CH3)2CH(CH2)6
CH2−—NH-admantyl
III
Dbv 29


32
(CH3)2CH(CH2)6
CH2−—NH-admantyl
III
Dbv 29


33
(CH3)2CH(CH2)6
C(O)NH—CH2—
II
Dbv 29




HC≡CH


34
(CH3)2CH(CH2)6
CH2—NH—CH2—
III
Dbv 29




HC≡CH


35
(CH3)2CH(CH2)6
CH2—NH—(CH2)5—N3
III
Dbv 29









Physiochemical properties of compounds 13 to 35 are summarized in Table 5.












TABLE 5





Example

Molecular
Exact
MS
LC


No.
Molecular formula
Weight
Mass
(m/z)
traces




















13
C88H94Cl2N9O34
1892.63
1890.53

21.2


14
C98H117Cl2N10O32
2017.93
2015.72
2019.19
22.11


15
C94H107Cl2N10O33
1975.81
1973.64
1975.46
21.41


16
C92H104Cl2N10O33
1947.76
1946.61
1949.41
20.62


17
C94H99Cl2N10O33
1967.75
1965.58
1969.23


18
C96H114Cl2N10O32
1989.88
1988.70
1991.50
20.75


19
C100H121Cl2N10O32
2045.98
2043.75
2047.11


20
C82H83Cl2N9O34
1809.48
1807.44
1810.26
14.56


21
C84H87Cl2N9O34
1837.54
1835.47
1837.88
16.1


22
C86H91Cl2N9O34
1865.59
1863.50
1866.29
17.75


23
C88H95Cl2N9O34
1893.64
1891.54
1894.38
19.4


24
C90H99Cl2N10O33
1919.7
1917.58
1921.13


25
C92H103Cl2N10O33
1947.76
1945.61
1948.22


26
C92H105Cl2N10O32
1933.77
1931.63
1935.35


27
C94H109Cl2N10O32
1961.83
1959.66
1963.52


28
C95H101Cl2N10O33
1981.77
1979.59
1983.14
21.19


29
C95H103Cl2N10O32
1967.79
1965.61
1969.32
19.3


30
C99H113Cl2N10O32
2025.91
2023.69
1013.67


31
C98H111Cl2N10O32
2011.88
2009.67
1006.56


32
C98H111Cl2N10O32
2011.88
2009.67
1006.55


33
C91H98Cl2N10O33
1930.7
1928.57
1931.29


34
C91H100Cl2N10O32
1916.7
1914.59
1917.42


35
C93H107Cl2N13O32
1989.81
1987.65
1990.25









Example 11
In Vitro Anti-Bacterial Test
The antibacterial activity of the compounds of formula (I) can be demonstrated in vitro. The results of the antibacterial testing of vancomycin, teicoplanin and compounds 5-10, 13-15 and 29, using the Minimum Inhibition Concentrations (MICs) Test, are set forth in Table 6, which shows the MICs of vancomycin 1, teicoplanin 4, compounds 5-10, 13-15 and 29 against the testing bacterial strains (Enterococcus Faecalis and Staphylococcus Aureus).









TABLE 6









Strains















E. faecalis
E. faecalis
E. faecalis
E. faecalis
E. faecalis
S. Aureus
S. Aureus


Compound
ATCC 29302
ATCC 33186
ATCC 51299
ATCC 51559
ATCC 700221
ATCC 29213
ATCC 700699

















1
1
4
>64
>32
>64
1
16


(vancomycin)


4
0.125
0.25
0.5
>32
>32
0.5
8


(Teicoplanin)


5
8
>32
>32
>32
>32
16
>32


6
8
>32
>32
>32
>32
16
>32


7
8
>32
>32
>32
>32
8
>32


8
2
>32
>32
>32
>32
4
32


9
0.125
0.25
0.25
>32
>32
0.5
8


10 
>64
>64
>64
>64
>64


13 
0.125
0.25
0.25
>32
>32
0.5
8


(Oxo-Tei)


14 
0.5
0.5
0.5
1
4
4
16


15 
0.0625
0.0625
0.125
8
32
2
16


29 
0.125
0.25
0.5
0.25
2





1. The concentration unit is ug/ml. MIC was determined as given concentrations wherein there were no growths for given testing strains in duplicate.


2. E. faecalis (ATCC 29302) is a standard strain; E. faecalis (ATCC 33186) is an antibiotics sensitive strain; E. faecalis (ATCC 51299) is a low level VRE; E. faecalis (ATCC 51559) is a multidrug-resistant strain (VRE, ampicillin, ciprofloxacin, gentamicin, rifampicin, teicoplanin and vancomycin); E. faecalis (ATCC 700221) is a strain resistant to vancomycin; S. Aureus (ATCC 29213) is a methacillin-sensitive strain; and S. Aureus (ATCC 700699) is a strain resistant to methacillin, and reduced vancomycin susceptibility through thickening of the cell wall.






Compared to vancomycn and teicoplanin, the compounds of formula (I) showed significantly enhanced bactericidal activities against the five tested Enterococcus faecalis strains. In addition, it appears that compound 15 with a shorter chain is more effective against antibiotics-sensitive strains (e.g. ATCC 33186), whereas the longer-chain compound 14 is more effective against drug-resistant strains (e.g., ATCC 51559 and 70021). Strikingly, compound 29, tailored with benzylamine, is equally effective against the sensitive and resistant strains but the dosage is much lower compare to that of vancomycin and tecoplanin. Compound 29 complements the inadequacy of vancomycin against the Vancomycin-Resistant Enterococcus. 
Compounds 9 and 13 are also effective in treating Staphylococcus Aureus and MRSA infections.
Example 13
In vivo Animal Study
12 ICR female mice with an average body weight of 27 g to 30 g were used in this experiment. Mice had free access to water and food at all time. Sepsis was induced in mice by intravenous (i.v.) injection of 1.3×105 cfu/mouse of E. faecalis (ATCC 51559) on day 0. The mice were randomized into the following four treatment groups at the start of the experiment:

    
    
        (a) Vancomycin (n=3);
        (b) Teicoplanin (n=3);
        (c) Compound 29 (n=3); and
        (d) Saline (control).
    
    


Each mouse received 10 mg/kg/dose of the medication by intravenous route, twice a day from day 1 to day 3, for a total of 6 doses. Mice were anesthetized and blood was taken from the orbital sinus on day 1, day 2, and day 3. The blood sample was diluted with PBS, plated on Brain Heart infusion agar (BHI agar; Difico, Detroit, Mich., USA) and cultured for colony formation unit.
The results of the in vivo study are shown in FIGS. 3A and 3B. Data were expressed as mean±SD and the statistical analysis was performed using SigmaStat®. Differences were considered to be significant if the P value was <0.05.
FIG. 3A shows the whole blood bacterial counts of mice infected by E. faecalis (ATCC 51559) and treated with vancomycin, teicoplanin, compound 29 or saline for three days. The bacterial counts for the control mice increased steadily over the 3-day period, while the bacterial counts in mice treated with vancomycin, teicoplanin and compound 29 were suppressed, but to different extents. These results show compound 29 is more efficacious than vancomycin and teicoplanin in treating E. faecalis infection. FIG. 3B shows the bacterial counts on day 3 (the end of the treatment). In general, these three drugs all show positive effects against the infection, but only compound 29 displays significant effect. The asterisk (*) indicates significant difference (P<0.05).

## Claims

1. A compound of formula (I)



or a pharmaceutically acceptable salt thereof,
R1 is alkyl;
R2 is CH2—NH—R4; and
R4 is selected from the group consisting of aryl, alkyl, alkynyl, adamantyl, and C1-C10 azide.

2. The compound of claim 1, wherein R1 is C1-C15 alkyl.

3. The compound of claim 1, wherein R1 is (CH3)2CH(CH2)6; CH3(CH2)2; CH3(CH2)4; CH3(CH2)6 or CH3 (CH2)8.

4. The compound of claim 1, wherein R4 is benzyl, (CH2)7 CH3, (CH2)11CH3, adamantyl, adamantyl-CH2, CH2—HC≡CH or (CH2)5N3.

5. The compound of claim 1, wherein R1 is (CH3)2CH(CH2)6 and R2 is CH2—NH—CH2—C6H6.

6. A pharmaceutical composition comprising a compound of formula (I)



R1 is alkyl;
R2 is CH2—NH—R4; and
R4 is selected from the group consisting of aryl, alkyl, alkynyl, adamantyl, and C1-C10 azide; and
a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein R1 is C1-C15 alkyl.

8. A method of making a compound of formula (I) of claim 1, comprising the steps of:
(a) binding teicoplanin to Dbv29;
(b) adding C6-15 alkylamine, a reducing agent, and an organic solvent to the mixture in step (a); and
(c) incubating the mixture in step (b).

9. The method according to claim 8, wherein about 5 mM to about 15 mM of C6-15 alkylamine is used.

10. The method according to claim 8, wherein the organic solvent is about 10% to about 90% of DMSO.

11. The method according to claim 8, wherein the reducing agent is about 1 mM to about 20 mM of cyanohydridoborate.

12. The method according to claim 8, wherein the incubation temperature is about 30° C. to about 40° C.

13. The method according to claim 8, wherein the incubation time is about 1 hour to about 48 hours.

14. A method for treating bacterial infection in a subject, comprising the step of:
administering to the subject an effective amount of a compound of formula (I) to treat said disease;




wherein
R1 is alkyl;
R2 is NH—R4;
and
R4 is selected from the group consisting of aryl, alkyl, alkynyl, adamantyl, and C1-C10azide.

15. The method according to claim 14, wherein the effective amount is about 0.0625 to about 32 mg of compound of formula (I) per kg of body weight per dose.

16. The method according to claim 14, wherein the compound of formula (I) is administered once per day to once per week.

17. The method according to claim 14, wherein said administering is by parenteral route.

18. The method according to claim 14, wherein the bacterial infection is a vancomycin-resistant enterococcus infection.

19. The pharmaceutical composition of claim 6, wherein R1 is one of the following: (CH3)2CH(CH2)6; CH3(CH2)2; CH3(CH2)4; CH3(CH2)6 or CH3(CH2)8.

20. The pharmaceutical composition of claim 6, wherein R4 is one of the following: benzyl, (CH2)7 CH3, (CH2)11CH3, adamantyl, adamantyl-CH2, CH2—HC≡CH and (CH2)5N3.

21. The pharmaceutical composition of claim 6, wherein R1 is (CH3)2CH(CH2)6 and R2 is CH2—NH—CH2—C6H6.