WO2007038868A2

WO2007038868A2 - Novel enediyne compound and uses thereof

Info

Publication number: WO2007038868A2
Application number: PCT/CA2006/001634
Authority: WO
Inventors: Julian E. Davies; Raymond J. Anderson; Hao Wang; Koaru Warabi; Xin Hui Huang
Original assignee: University of British Columbia
Current assignee: University of British Columbia
Priority date: 2005-10-03
Filing date: 2006-10-03
Publication date: 2007-04-12
Anticipated expiration: 2008-04-03
Also published as: WO2007038868A3

Abstract

The invention relates to a novel enediyne compound, uncialamycin, including analogues thereof. The invention also relates to methods of using the compound to treat bacterial infections, to inhibit bacterial growth, and to kill bacteria. This invention may also be used to treat tumors, to prevent growth of tumor cells, to kill tumor cells, to treat cancers, to prevent growth of cancer cells, and to kill cancer cells. The invention also relates to methods of making analogues of uncialamycin. The invention also relates to compositions comprising uncialamycin and methods of administering the compositions.

Description

NOVEL ENEDIYNE COMPOUND AND USES THEREOF

Related Application

[0001] This application claims the benefit of the filing date of United

States provisional patent application No. 60/722,407 filed 3 October 2005 which is hereby incorporated by reference.

Technical Field

[0002] This application relates to a novel enediyne compound, analogues of the compound, compositions containing the compound, and uses thereof.

Background

[0003] Antibiotic resistance is an increasing problem in the treatment of bacterial infections. Hospital-acquired (nosocomial) infections are particularly difficult to treat due to bacteria acquiring resistance to commonly used drugs, often as a result of continued exposure of bacteria to multiple antibiotics. Such infections can be particularly troubling for patients who are immunocompromised or otherwise weakened from illness. As many bacteria are increasingly becoming resistant to known drugs, the need for novel antibiotics is also increasing.

[0004] There is also a demand for compounds with increased efficacy against cancers and tumors. In addition to treating bacterial infections, many antibiotics also have potent anti-tumor and anti-cancer properties.

[0005] There is a need for novel compounds with antibiotic, anti-tumor or anti-cancer properties. Summary of Invention

[0006] In one aspect, the invention relates to the enediyne compound uncialamycin having the formula:

[0007] In another aspect, the invention relates to analogues of uncialamycin having the following formula:

wherein:

X= O,

Ri, R₂, R₃, R₄, are independently H; or a one to fifteen carbon linear, branched, or cyclic, saturated, unsaturated, aromatic, or partially aromatic alkyl group optionally substituted with R₅, OR₅, OH, =O, F, Cl, Br, I, NH₂, NHR₅, N(R₅)₂, CHO, COR₅, CO₂R₅, OCOR₅, NO₂, SO₂R₅, CN, Si(R₅)₃, or with individual carbon atoms optionally replaced by O, NH, NR₅, S, SO, SO₂; or a linker to an antibody; wherein R₅ is a linear one to six carbon saturated or unsaturated alkyl group where each of the carbons may be independently substituted with OH, =O, F, Cl, Br, I, NH₂, CHO, NO₂, CN, or replaced with O, NH, or S.

[0008] In one embodiment, an analogue of uncialamycin comprises the formula

In another aspect, the invention relates to salts and prodrugs of uncialamycin and its analogues. The invention also relates to methods of making analogues of uncialamycin. [0009] In a further aspect, the invention relates to compositions comprising an effective amount of uncialamycin, or analogues, salts, or prodrugs thereof, and a pharmaceutically acceptable carrier. Such compositions include orally administrable, injectable, and topically applicable compositions. Such compositions also include antibody conjugates comprising uncialamycin, or analogues, salts, or prodrugs thereof, conjugated to an antibody through a linker. Such compositions also include other useful forms of uncialamycin, or analogues, salts, or prodrugs thereof, such as powders and solutions. In another aspect, the invention relates to methods of administering the compositions by administering effective amounts of uncialamycin.

[0010] In a further aspect, the invention relates to methods of using the compound or compositions thereof as antibiotics. The invention also relates to methods of using the compound or compositions as bacteriostatic or bactericidal agents to inhibit bacterial growth or to kill bacteria. In some aspects of the invention, the bacteria include Staphylococcus aureus, Escherichia coli, and Burkholderia cepacia complex. In another aspect of the invention, the compound or compositions can also be used against multidrug resistant bacteria.

[0011] In another aspect, the invention relates to methods of using the compound or compositions thereof as antitumor and anticancer agents.

[0012] Further details of the invention are described herein. Brief Description of Drawings

[0013] In drawings which illustrate various embodiments of the invention:

[0014] Figure 1 depicts HMBC correlations used to identify two major fragments A and B of uncialamycin (1).

[0015] Figure 2 is a photograph of pBR322 plasmid DNA samples incubated with uncialamycin for 15 minutes, analyzed by polyacrylamide gel electrophoresis.

[0016] Figure 3 is a photograph of pBR322 plasmid DNA samples incubated with uncialamycin for 3 hours, analyzed by polyacrylamide gel electrophoresis.

Description

[0017] Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive, sense.

[0018] In one aspect, the invention relates to a novel enediyne compound. The compound is uncialamycin and has the following structure:

(1) Uncialamycin

[0019] In another aspect, the invention relates to analogues of uncialamycin having the following formula:

wherein:

,O WRIΛi-|

<

X= O, or H

Ri, R₂, R₃, R₄, are independently H; or a one to fifteen carbon linear, branched, or cyclic, saturated, unsaturated, aromatic, or partially aromatic alkyl group optionally substituted with R₅, OR₅, OH, =O, F, Cl, Br, I, NH₂, NHR₅, N(Rs)₂, CHO, COR₅, CO₂R₅, OCOR₅, NO₂, SO₂R₅, CN, Si(R₅)₃, or with individual carbon atoms optionally replaced by O, NH, NR₅, S, SO, SO₂; or a linker to an antibody; wherein R₅ is a linear one to six carbon saturated or unsaturated alkyl group where each of the carbons may be independently substituted with OH, =O, F, Cl, Br, I, NH₂, CHO, NO₂, CN, or replaced with O, NH, or S.

[0020] In one embodiment,

and Y= . It will be appreciated by persons skilled in the art that C=X and C=Y can represent a double bond or two single bonds to the carbons bearing X and Y. When there is a double bond to X or Y, X or Y can be O. When there are two

single bonds from the carbons bearing X and Y, X can be H and Y can

[0021] In another embodiment, the analogue comprises the formula

wherein:

X= O,

,OR.

<

Y= O, or H

R₁, R₂, R₃, R₄, are independently H; or a one to seven carbon linear, branched, or cyclic, saturated, unsaturated, aromatic, or partially aromatic alkyl group optionally substituted with R₅, OR₅, OH, =O, F, Cl, Br, I, NH₂, NHR₅, N(R₅)₂, CHO, COR₅, CO₂R₅, OCOR₅, NO₂, SO₂R₅, CN, Si(R₅)₃, or with individual carbon atoms optionally replaced by O, NH, NR₅, S, SO, SO₂; or a linker to an antibody; wherein R₅ is a linear one to six carbon saturated or unsaturated alkyl group where each of the carbons may be independently substituted with OH, =O, F, Cl, Br, I, NH₂, CHO, NO₂, CN, or replaced with O, NH, or S.

[0022] In another embodiment, the analogue comprises the formula

wherein:

,OR₁

<

X= O, or H

Y= O, or H ;

Ri, R₂, R₃, R₄, are independently H; or a one to four carbon linear, branched, or cyclic, saturated, unsaturated, alkyl group optionally substituted with OR₅, OH, =O, F, Cl, Br, I, NH₂, NHR₅, N(R₅)₂; or a linker to an antibody; wherein R₅ is a linear one to three carbon alkyl group.

[0023] In more specific embodiments of the analogues, R₁, R₂, and R₄ are acetyl groups (Ac) and R₃ is H. In another embodiment, Ri and R₄ are acetyl groups (Ac), and R₂ and R₃ are H. In another embodiment, R₂ and R₄ are acetyl groups (Ac), and Ri and R₃ are H. In another embodiment, R₄ is an acetyl group (Ac), and Ri , R₂ and R₃ are H. In another embodiment, R₄ is an acyl group, and R₁, R₂ and R₃ are H. In another embodiment, R₄ is a linker to an antibody, and R₁, R₂ and R₃ are H. In another embodiment, R₄ is a linker to an antibody, Ri and R₂ are acetyl groups (Ac), and R₃ is H. In a further embodiment, R_b R₂, and R₄ are acetyl groups (Ac), and R₃ is a linker to an antibody.

[0024] In one embodiment, an analogue of uncialamycin comprises the formula

[0025] In another aspect, the invention relates to salts and prodrugs of uncialamycin and its analogues. In a further aspect, the invention includes organometallic derivatives of uncialamycin and its analogues.

[0026] In a further aspect, the invention also relates to methods of making analogues of uncialamycin. Various analogues of uncialamycin can be derived from uncialamycin using any of the following schemes or any combinations thereof.

I (RCO)₂O/ pyridine

(RCO)₂O/ pyridine

(RCO)₂O/ pyridine

Scheme 2

Scheme 3

Scheme 4

As will be appreciated by persons skilled in the art, the general synthesis schemes can be combined in different ways to produce various different analogues. For example, with respect to Schemes 1 and 3, R may be methyl or a simple alkyl such as ethyl, propyl, butyl, etc. Many other R substituents are also possible. With respect to Scheme 1, R₃ may be as defined above, for example. Also, analogues with different acyl or alkyl groups can be created by using different acylation or alkylation reagents. For example, the analogue

can be created using the method of Scheme 2, and this may be followed by an acylation reaction as described in Scheme 1.

[0027] In another aspect, the invention relates to compositions comprising an effective amount of uncialamycin and pharmaceutically acceptable carriers. Such compositions include compositions comprising analogues, salts, or prodrugs of uncialamycin and pharmaceutically acceptable carriers. An effective amount of uncialamycin is an amount that is effective to treat a bacterial infection, to inhibit bacterial growth, to kill bacteria, to treat tumors, to inhibit growth of tumor cells, to kill tumor cells, to treat cancer, to inhibit cancers cells, or to kill cancer cells. Such amounts would be well within the skill of a person skilled in the art to determine. Suitable pharmaceutically acceptable carriers are also known to persons skilled in the art, including, but not limited to, suspending agents, flavorings, sweeteners, colorants, coatings, etc.

[0028] The compositions can be orally administrable, injectable, topically applied, or supplied in other suitable forms. Orally administrable compositions include compositions in the form of a tablet, a powder, a suspension, an emulsion, a capsule, a granule, a lozenge, a pill, a liquid, a syrup, or any other appropriate orally administrable form. The injectable compositions include compositions in the form of a liquid, a suspension, a solution, or any other suitable injectable form. The topical compositions include compositions in the form of a paste, an ointment, a liquid, a powder, a plaster, a suppository, an aerosol, a liniment, a lotion, an enema, an emulsion, or any other suitable topical form. Other forms of administering or otherwise providing the compositions are also contemplated, such as powder forms and solutions.

[0029] The compositions also include antibody conjugates comprising uncialamycin, or analogues, salts, or prodrugs thereof, conjugated to an antibody. The antibodies of these conjugates can be monoclonal antibodies, or any other suitable antibodies, which target bacterial antigens, tumor cell antigens, cancer cell antigens, or any other suitable antigens. Such conjugates can assist in delivering uncialamycin, or analogues, salts, or prodrugs thereof, to sites of infection or sites of tumor or cancer growth. Uncialamycin, or analogues, salts, or prodrugs thereof, can be bonded to antibodies by any bonding method known to persons skilled in the art including, but not limited to, non-covalent bonds and covalent bonds, including cleavable and non-cleavable linkers. Such bonds include, but are not limited to, peptide linkers, saccharide linkers, sulfide linkers, etc. [0030] In another aspect, the invention relates to methods of using uncialamycin or compositions thereof. Uncialamycin demonstrates inhibitory activity against both gram positive and gram negative bacteria, but does not exhibit inhibitory activity against yeasts. Accordingly, in one aspect, the invention relates to methods of using uncialamycin, or compositions thereof, as an antibiotic to treat bacterial infections in humans or other animals having a bacterial infection. In another aspect, uncialamycin, or compositions thereof, can be used as a bacteriostatic agent to inhibit bacterial growth, either in vitro or in vivo. In another aspect, uncialamycin, or compositions thereof, can be used as a bactericidal agent to kill bacteria, either in vitro or in vivo. The bacteria can comprise gram positive or gram negative bacteria, including, but not limited to, Staphylococcus aureus, Escherichia coli and Burkholderia cepacia. In particular, uncialamycin is effective against Burkholderia cepacia complex (Bcc), a group of nine species of Gram-negative non-sporulating bacilli that have emerged as serious opportunistic human pathogens in cystic fibrosis and immunocompromised patients. Because uncialamycin is a novel antibiotic, in another aspect of the invention, uncialamycin, or compositions thereof, are also useful to treat antibiotic resistant bacteria, including multidrug resistant (MDR) bacteria.

[0031] In a further aspect, the invention also relates to methods of using uncialamycin or compositions thereof to treat tumors in humans or other animals in need of treatment, to inhibit growth of tumor cells in vivo or in vitro, to kill tumor cells in vivo or in vitro, to treat cancer in humans or other animals in need of treatment, to inhibit growth of cancer cells in vivo or in vitro, or to kill cancer cells in vivo or in vitro. [0032] The invention also relates to methods of administering the compositions such that uncialamycin, or analogues, salts, or prodrugs thereof, are administered in a therapeutically effective dose. Such doses would be well within the skill of a physician or other persons skilled in the art to determine.

EXAMPLES

[0033] The following examples are intended to illustrate embodiments of the invention and are not intended to be interpreted in a limiting manner.

Isolation of Uncialamvcin

[0034] As part of a screening program aimed at discovering new antibiotics active against Bcc, it was found that crude organic extracts of cultures of a previously undescribed Streptomycete showed potent in vitro inhibition of Bcc. Bioassay guided fractionation of the crude extracts led to the identification of uncialamycin (1), a new enediyne antibiotic, as the active component. Bioactivity-guided fractionation involves thin layer chromatography of the extracts and fractions thereof and detection of the activity by overlaying a sensitive tester strain. A zone of inhibition identifies the active fraction containing the active compound.

[0035] The producing strain was extracted from the surface of the lichen Cladonia uncialis collected near Pitt River, British Columbia. Characterisation by 16S RNA sequencing showed the strain to be related, but not identical, to Streptomyces cyanogenus. Antibiotic activity of the strain was assayed by cutting plugs from solid agar cultures of the strain and placing them on lawns of tester strains of bacteria. Good inhibitory activity was detected against Gram-positive and Gram-negative bacteria (including Bcc), but not against yeasts.

[0036] Production cultures of the producing strain were grown as lawns on solid agar medium ISP4 for 14 to 21 days at room temperature. The solid agar cultures were lyophilized and extracted repeatedly with EtOAc. Concentration of the combined EtOAc extracts in vacuo gave a gummy residue that was partitioned between EtOAc and H₂O. The EtOAc soluble material was fractionated by sequential application of flash C- 18 reversed-phase chromatography (eluent: step gradient from H₂O to MeOH) and reversed-phase HPLC (column-Inertsil ODS-2; eluent: CH₃CN/H₂O 40:60) to give pure uncialamycin (1) (~ 300 μg) as a bright purple [UV(MeOH): λ_maxnm (ε) 206 (25,000), 254 (33,000), 280 (shoulder), 320 (shoulder), 539 (9,400)], optically active ([α]_D +3,300 (c 0.005, MeOH)) oil.

Chemical Characterization of Uncialamycin

[0037] Uncialamycin (1) gave a [M + Na]⁺ ion at m/z 462.0956 in the

HRESIMS appropriate for a molecular formula Of C₂₆H₁₇NO₆ (calc'd for C₂₆H₁₇NO₆Na 462.0954) requiring 19 sites of unsaturation. NMR data for uncialamycin was recorded in DMSO-^₆ at 600 MHz using a cryoprobe. The ¹³C NMR spectrum (Table 1) showed well-resolved resonances for 26 carbon atoms and the ¹H NMR spectrum contained resonances integrating for 17 protons in agreement with the HRMS data. Inspection of the HMQC data revealed that four of the protons (δ 5.39, 6.66, 10.0, and 13.2) were not attached to carbon atoms. Two major fragments A and B (Figure 1) of uncialamycin could be identified from analysis of the COSY, HMQC, and HMBC data obtained for the molecule.

Position δ ¹W WH^mult, J(Hz)) ,

1 10.0 (d, 4.6)

2 143.6

3 110.4

4 187.0

5 134.4

6 126.1 8.23 (dd, 1.4, 7.6)

7 133.6 7.88 (ddd, 1.4, 7.6, 7.6)

8 134.9 7.94 (ddd, 1.4, 7.6, 7.6)

9 126.6 8.24 (dd, 1.4, 7.6)

10 132.2

11 182.2

12 112.7

13 154.9

14 129.9 8.51 (s)

15 135.6

16 63.5

17 63.0 5.14 (d, 3.3)

18 100.4

19 89.7

20 123.4 6.05 (dd, 0.8, 10)

21 124.0 5.97 (ddd, 1.4, 1.5, 10)

22 87.4

23 98.9

24 43.2 5.04 (dd, 1.5, 4.6)

25 76

26 63.6 4.31 (qd, 6.0, 6.0)

27 22.1 1.30 (d, 6.0)

13-OH 13.2 (brd.s)

17-OH 6.66 (brd.s)

26-OH 5.39 (d,6.0)

Table 1. C and H NMR assignments for uncialamycin (1). Data were recorded in OMSO-d₆ at 600 MHz for ¹H. [0038] A pair of olefinic resonances at δ 5.97 (H-21 ) and 6.05 (H-20), that were strongly correlated to each other in the COSY spectrum and had a coupling constant of 10 Hz, were assigned to a cis disubsituted olefin. The upfield olefinic resonance at δ 5.97 (H-21) showed strong HMBC correlations to non-protonated carbon resonances at δ 89.7 (C- 19) and 98.9 (C-23), and the downfield olefinic resonance at δ 6.05 (H-20) showed strong correlations to non-protonated carbon resonances at δ 87.4 (C-22) and 100.4 (C- 18). This suite of HMBC correlations identified an enediyne substructure in 1 (see Fragment A in Figure 1). The olefinic resonance at δ 5.97 (H-21) showed a long range COSY correlation to a methine resonance at δ 5.04 (H- 24), indicating that the carbon bearing the methine proton (C-24: δ 43.2) was attached to the C-23 alkyne carbon. A COSY correlation observed between the methine (δ 5.04, H-24) and a broad singlet at 10.0, that was not correlated to a carbon in the HMQC spectrum, and the chemical shift of the methine carbon (C-24, δ 43.2) suggested that C-24 had an NH substituent. HMBC correlations observed between the H-24 methine (δ 5.04) and the two alkyne carbon resonances at δ 87.4 (C-22) and 98.9 (C-23) confirmed the attachment of C-24 to the C-23 alkyne carbon.

[0039] A singlet methine resonance at δ 5.14 (H- 17) showed HMBC correlations to the alkyne carbon resonances at δ 89.7 (C- 19) and 100.4 (C- 18), which demonstrated that the methine carbon (C- 17: δ 63.0) was linked to the second alkyne at C-18. Both of the methine resonances at δ 5.04 (H- 24) and 5.14 (H- 17) showed HMBC correlations to a pair of deshielded resonances at δ 63.5 (C- 16) and 76.0 (C-25), assigned to non-protonated oxygen bearing carbons. This set of four HMBC correlations indicated that the two oxygenated carbons bridged the C- 17 and C-24 carbons to form a ten membered ring (C- 16 to C-25) containing the enediyne substructure. A COSY correlation between the methine resonance at δ 5.14 and a broad singlet at 6.66 (17-OH) revealed an alcohol funtionality attached to the methine carbon.

[0040] A methyl doublet at δ 1.30 (Me-27, J = 6 Hz) was correlated in the COSY spectrum to a methine at 4.31 (H-26, q, J = 6.0 Hz)) that was further correlated to a broad singlet at 5.39 (OH-26), assigned to an alcohol. The methyl resonance (δ 1.30, Me-27) showed an HMBC correlation to the carbon resonance at 76.0 (C-25), indicating that the hydroxyethyl fragment (C-26 and C-27) was the fourth subsituent on the non-protonated carbon C- 25. Both the NH-I proton (δ 10.0) and the H-17 methine (5.14) were correlated to a carbon at δ 135.6 (C- 15), and the H-24 methine (δ 5.04) was correlated to a carbon at 143.6 (C-2) in the HMBC spectrum indicating that the NH and C- 16 were vicinal substituents on an olefin or aromatic ring. A deshielded singlet at δ 8.51 showed strong HMBC correlations into carbon resonances at δ 63.5 (C-16), 143.6 (C-2), and 112.7 (C- 12) and a weak correlation into the carbon resonance at 154.9 (C- 13). This set of HMBC correlations confirmed that the NH and C-16 were attached to a benzene ring. Based on the assumption that the intense HMBC correlations were through three bonds, these correlations also indicated that the aromatic methine (δ 8.51, H-14) was ortho to C-16 (δ 63.5) and meta to the NH (C-2, δ 143.6). The weak HMBC correlation between δ 8.51 and 154.9 was attributed to a two bond coupling, placing the carbon at 154.9 (C-13) ortho to the methine carbon (C- 14) and its chemical shift required an oxygen substituent. [0041] The second fragment B of uncialamycin contained an isolated

¹H spin system comprised of four contiguous aromatic protons (δ 8.23, dd, J = 1.4, 7.6 Hz H-6; 7.88, ddd, 1.4, 7.6, 7.6 Hz H-7; 7.94, ddd, J = 1.4, 7.6, 7.6 Hz H-8; 8.24, dd, J = 1.4, 7.6 Hz H-9). HMBC correlations observed between the proton resonance at δ 8.23 (H-6) and a carbon resonance at 187.0 (C-4) and between the proton resonance at 8.24 (H-8) and a carbon resonance at 182.2 (C-11) suggested that the other two subsituents on the benzene ring were quinone carbonyls. Fragments A and B shown in Figure 1 accounted for all of the carbon, hydrogen, and nitrogen atoms in the molecular formula of uncialamycin (1), but contained one extra oxygen atom. In order to complete the quinone and satisfy the remaining aromatic valences in Fragment A, the two carbonyl carbons of fragment B (C-4 and C-I l) had to be attached to the two substituted aromatic carbons (C-3 and C- 12) of fragment A. Finally, it was apparent that the two oxygentated carbons C- 16 and C-25 had to be bridged by an epoxide to account for the number of oxygen atoms and sites of unsaturation required by the molecular formula of 1. This implied that the C- 13 oxygen substituent had to be part of a phenol functionality that would engage in intramolecular hydrogen bonding with the C-I l carbonyl consistent with the observed OH chemical shift of δ 13.2.

[0042] A ROESY correlation between δ 5.14 (H- 17) and 4.31 (H-26) showed that C-26 and C- 17 were cis oriented about the C-16/C-25 epoxide and also defined the relative stereochemistry of H- 17 as shown. Molecular models revealed that due to steric and bond angle strain the C- 17 to C-23 enediyne containing bridge could only reasonably be cis fused to the piperidine ring. Uncialamycin (1) shares structural features with dynemicin A (2) and deoxydynemicin A (3) isolated from Micromonospora chersina. The H-24 resonance in uncialamycin (1) has a chemical shift of δ 5.04 and a 4.6 Hz coupling to the NH-I proton, which is nearly identical to the chemical shift (δ 5.05) and coupling (J = 4.3 Hz) of the corresponding methine proton (H-2) in dynemicin A (2), in agreement with the relative stereochemical assigment at C-24 in 1. Comparison of the additional NMR assigments reported for dynemicin A (2) and its triacetate derivative provided further strong support for the assigned structure of uncialamycin

(I)-

Structures of Dynemicin A (2) and Deoxy dynemicin A (3)

Biological Activity of Uncialamycin

[0043] Uncialamycin (1) shows potent in vitro antibacterial activity against Staphylococcus aureus (Minimal Inhibitory Concentration (MIC) 0.0000064 μg/mL), Escherichia coli (MIC 0.002 μg/mL) and Burkholderia cepacia (MIC 0.001 μg/mL). B. cepacia K56-2, C2822, CEP559, C3430, and CEP1016 were sensitive to uncialamycin at low concentrations. Candida albicans and Sacchromyces cerevisiae were not sensitive at relatively high concentrations of uncialamycin. [0044] MICs were determined as follows. Successive two-fold dilutions of uncialamycin (100 ng/ml) were made in LB medium. Tubes were inoculated with 5 ul of a 10-fold dilution of an overnight culture of the test strain. Visible growth was checked after overnight incubation at 37⁰C. The lowest concentration which inhibited the growth was designated the MIC.

[0045] All enediynes identified to date characteristically act on duplex

DNA and cause single and double stranded breaks due to the action of benzenoid diradicals formed as a result of Bergmann rearrangement of the antibiotic molecule within the minor groove of the target DNA. The identification of uncialamycin as an enediyne led to an examination of its activity as a DNA damaging agent. Initial studies indicate that uncialmycin interacts with plasmid DNA leading to extensive degradation.

[0046] To analyze the DNA degradation activity of uncialamycin, supercoiled, covalently closed pBR322 DNA was incubated with uncialamycin. The total volume of each reaction was 20 ul, containing 400ng DNA in 2OmM Tris buffer (pH 7.5), with and without uncialamycin, in the presence and absence of ImM NADPH or O.lmM dithiothreitol (DTT), at 37⁰C for 15 minutes or 3 hours. Samples from the reactions were loaded into wells of a 0.9% agarose gel (0.1 ug/ml ethidium bromide), and electrophoresis was employed to separate the DNA fragments (see Figures 2 and 3).

[0047] In Figure 2, the reaction samples were incubated for 15 minutes. Lane 1 = control (no drug), lane 2 = control (no drug) + DTT, lane 3 - 20 μM drug + DTT, lane 4 = 10 μM drug + DTT, lanes 5 and 6 = size standards. Lanes 3 and 4 show degradation of DNA compared to control lanes 1 and 2. In Figure 3, the reaction samples were incubated for 3 hours. Lane 1 = control (no drug) + DTT, Lane 2 = control (no drug) +NADPH, Lane 3 = 20 μM drug, Lane 4 = 20 μM drug +NADPH, Lane 5 = 20 μM drug + DTT, lane 6 = 10 μM drug + DTT, lanes 7 and 8 contain size standard markers. Lanes 4, 5, and 6 show degradation of DNA.

[0048] It is known that the triacetate of dynemicin A is more potent than the natural product, dynemicin A. As discussed above, uncialamycin shares structural features with dynemicin A and the present invention encompasses triacetate and trimester analogues of uncialamycin as shown in Schemes 1 and 4 which may exhibit enhanced biological activity.

[0049] As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof.

REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A compound having the formula:

and salts and prodrugs thereof.

2. An analogue of the compound according to claim 1 comprising the formula

wherein:

X= O,

OR₂

<

Y= O, or H ;

Ri, R₂, R₃, R₄, are independently H; or a one to fifteen carbon linear, branched, or cyclic, saturated, unsaturated, aromatic, or partially aromatic alkyl group optionally substituted with R₅, OR₅, OH, =O, F, Cl, Br, I, NH₂, NHR₅, N(R₅)₂, CHO, COR₅, CO₂R₅, OCOR₅, NO₂, SO₂R₅, CN, Si(R₅)₃, or with individual carbon atoms optionally replaced by O, NH, NR₅, S, SO, SO₂; or a linker to an antibody; wherein R₅ is a linear one to six carbon saturated or unsaturated alkyl group where each of the carbons may be independently substituted with OH, =O, F, Cl, Br, I, NH₂, CHO, NO₂, CN, or replaced with O, NH, or S; and salts and prodrugs of the analogue.

3. An analogue according to claim 2, wherein

4. An analogue of the compound according to claim 1 comprising the formula

wherein:

,OR,

<

Y= O, or H

Ri, R₂, R₃, R₄, are independently H; or a one to seven carbon linear, branched, or cyclic, saturated, unsaturated, aromatic, or partially aromatic alkyl group optionally substituted with R₅, OR₅, OH, =O, F, Cl, Br, I, NH₂, NHR₅, N(R₅)₂, CHO, COR₅, CO₂R₅, OCOR₅, NO₂, SO₂R₅, CN, Si(R₅)₃, or with individual carbon atoms optionally replaced by O, NH, NR₅, S, SO, SO₂; or a linker to an antibody; wherein R₅ is a linear one to six carbon saturated or unsaturated alkyl group where each of the carbons may be independently substituted with OH, =O, F, Cl, Br, I, NH₂, CHO, NO₂, CN, or replaced with O, NH, or S; and salts and prodrugs of the analogue.

5. An analogue of the compound according to claim 1 comprising the formula

wherein:

R₁, R₂, R₃, R₄, are independently H; or a one to four carbon linear, branched, or cyclic, saturated, unsaturated, alkyl group optionally substituted with OR₅, OH, =O, F, Cl, Br, I, NH₂, NHR₅, N(R₅)₂; or a linker to an antibody; wherein R₅ is a linear one to three carbon alkyl group; and salts and prodrugs of the analogue.

6. An analogue according to claim 2, wherein Ri, R₂, and R₄ are acetyl groups (Ac) and R₃ is H.

7. An analogue according to claim 2, wherein R₁ and R₄ are acetyl groups (Ac), and R₂ and R₃ are H.

8. An analogue according to claim 1, wherein R₂ and R₄ are acetyl groups (Ac), and Ri and R₃ are H.

9. An analogue according to claim 1 , wherein R₄ is an acetyl group (Ac), and Ri, R₂ and R₃ are H.

10. An analogue according to claim 1, wherein R₄ is an acyl group, and Ri, R₂ and R₃ are H.

1 LAn analogue according to claim 1, wherein R₄ is a linker to an antibody, and R₁, R₂ and R₃ are H.

12.An analogue according to claim 1, wherein R₄ is a linker to an antibody, R₁ and R₂ are acetyl groups (Ac), and R₃ is H.

13. An analogue according to claim 1, wherein R₁, R₂, and R₄ are acetyl groups (Ac), and R₃ is a linker to an antibody.

14. A composition comprising an effective amount of a compound according to claim 1 and a pharmaceutically acceptable carrier.

15. A composition comprising an effective amount of a compound according to claim 2 and a pharmaceutically acceptable carrier.

16. An antibody conjugate comprising an effective amount of a compound according to claim 1 conjugated to an antibody.

17. An antibody conjugate comprising an effective amount of a compound according to claim 2 conjugated to an antibody.

18. An antibody conjugate according to claim 16, wherein the antibody binds to bacterial antigens, tumor cell antigens, or cancer cell antigens.

19. An antibody conjugate according to claim 17, wherein the antibody binds to bacterial antigens, tumor cell antigens, or cancer cell antigens.

20. A method of using a compound according to claim 1 as an antibiotic to treat bacterial infections in humans or other animals, to inhibit bacterial growth, or to kill bacteria.

21. A method of using a compound according to claim 2 as an antibiotic to treat bacterial infections in humans or other animals, to inhibit bacterial growth, or to kill bacteria.

22. A method according to claim 20, wherein the bacteria are selected from the group consisting of Staphylococcus aureus, Escherichia coli, and Burkholderia cepacia complex.

23. A method according to claim 21, wherein the bacteria are selected from the group consisting of Staphylococcus aureus, Escherichia coli, and Burkholderia cepacia complex.

24. A method according to claim 22, wherein the bacteria comprises Burkholderia cepacia complex.

25. A method according to claim 23, wherein the bacteria comprises Burkholderia cepacia complex.

26. A method of treating tumors, inhibiting growth of tumor cells, killing tumor cells, treating cancer, inhibiting growth of cancer cells, and killing cancer cells comprising administering an effective amount of a compound according to claim 1 to a patient in need of treatment.

27. A method of treating tumors, inhibiting growth of tumor cells, killing tumor cells, treating cancer, inhibiting growth of cancer cells, and killing cancer cells comprising administering an effective amount of a compound according to claim 2 to a patient in need of treatment.

28. A method of administering a compound according to claim 1, wherein the compound is administered in a therapeutically effective dose.

29. A method of administering a compound according to claim 2, wherein the compound is administered in a therapeutically effective dose.

30. A method of administering a composition according to claim 14, wherein the compound is administered in a therapeutically effective dose.

31. A method of administering a composition according to claim 15, wherein the compound is administered in a therapeutically effective dose.

32. A compound selected from the group consisting of compounds represented by the following formula and salts and prodrugs thereof:

wherein:

X= O, or

R₁, R₂, R₃, R₄, are independently H; or a one to fifteen carbon linear, branched, or cyclic, saturated, unsaturated, aromatic, or partially aromatic alkyl group optionally substituted with R₅, OR₅, OH, =O, F, Cl, Br, I, NH₂, NHR₅, N(R₅)₂, CHO, COR₅, CO₂R₅, OCOR₅, NO₂, SO₂R₅, CN, Si(R₅)₃; or with individual carbon atoms optionally replaced by O, NH, NR₅, S, SO, SO₂; or a linker to an antibody; wherein R₅ is a linear one to six carbon saturated or unsaturated alkyl group where each of the carbons may be independently substituted with OH, =O, F, Cl, Br, I, NH₂, CHO, NO₂, CN, or replaced with O, NH, or S.