Antibacterial products

Abstract

The invention provides a combination of an antibacterial agent (in particular vancomycin or moenomycin) and a delivery agent, in which the delivery agent is bonded, or capable of binding, to the antibacterial agent, and in which the delivery agent is capable of binding to one or more structures on a bacterial cell membrane. The invention further provides the use of such combinations in treating or preventing bacterial infections.

Claims

1. A combination of an antibacterial agent or a derivative thereof and a delivery agent, wherein the delivery agent is a compound of formula I,
A.sup.1-D.sup.1x).sub.n  I or a pharmaceutically-acceptable salt thereof, wherein A.sup.1 represents a hydrogen atom, a terminating group or the antibacterial agent or derivative thereof; X represents a fragment comprising a group selected from —NH.sub.2, boronic acid and a boronic acid derivative; n is 2 or more; and D.sup.1 represents a dendrimer fragment according to any one of formulae A to F: ##STR00046## wherein, for the dendrimer fragments of formula A to E, the single wavy line on the left-hand side of the structure corresponds to the point of attachment of the A.sup.1 group and the other wavy lines correspond to the points of attachment of the requisite X groups; and wherein, for the dendrimer fragment of formula F, all of the wavy lines represent points of attachment of the requisite X groups, and A.sup.1 represents hydrogen and is incorporated into formula F.

2. The combination according to claim 1, wherein the antibacterial agent is a molecule or fragment that modulates the activity of a penicillin binding protein.

3. The combination according to claim 1, wherein the antibacterial agent is an inhibitor of a glycosyltransferase enzyme.

4. The combination according to claim 1, wherein the antibacterial agent is a molecule or fragment that inhibits the synthesis or repair of bacterial cell walls.

5. The combination according to claim 1, wherein the delivery agent is capable of binding to one or more structures on a bacterial cell membrane via the formation of one or more covalent bonds with said structures, or via the formation of one or more hydrogen bonds with said structures.

6. The combination according to claim 1, wherein the delivery agent comprises one or more functional groups independently selected from the list consisting of boronic acids, boronic acid derivatives, primary amines, amidines, amides, and salts thereof.

7. The combination according to claim 6, wherein the each of the one or more functional groups is independently selected from the list consisting of boronic acids, boronic acid derivatives, primary amines, amidines, guanidines, amides, ureas, and acid addition salts thereof.

8. The combination according to claim 6, wherein the delivery agent comprises at least four of said functional groups.

9. The combination according to claim 1, wherein the delivery agent is a compound of any one of formulae II to VII: ##STR00047## or a pharmaceutically-acceptable salt thereof, wherein each A independently represents a hydrogen atom, a terminating group or an antibacterial agent (or a derivative thereof); D.sup.1 to D.sup.6 each represent a dendrimer fragment according to any one of formulae A to F: ##STR00048## wherein, for the dendrimer fragments of formula A to E, the single wavy line on the left-hand side of the structure corresponds to the point of attachment of the A group and the other wavy lines correspond to the points of attachment of the groups shown in parentheses in formulae II to VII; and wherein, for the dendrimer fragment of formula F, all of the wavy lines represent points of attachment of the groups shown in parentheses in formulae II to VII, and A represents hydrogen and is incorporated into formula F; Y represents O, NH or S; each n1 is 2 or more; m, p, q and r each independently represent from 1 to 8; and R.sup.1 represents a C.sub.1-6 alkyl group; or wherein the delivery agent is a compound of formulae VIIIa, VIIIb or IX: ##STR00049## wherein each A.sup.2 independently represents an antibacterial agent (or a derivative thereof), L.sub.2 represents aliphatic linker; D.sup.7 and D.sup.8 independently represent a direct bond or a dendrimer fragment of any one of formulae A to E above and to which the boron-containing groups shown are attached, n2 is 1 or more, wherein, for compounds of formulae VIIIa, VIIIb and IX, the single wavy line on the left-hand side of each structure in formulae A to E corresponds to the point of attachment of the A.sup.2 group and the other wavy lines correspond to the points of attachment of the requisite boronic acid or boric acid-containing portions of the delivery agent; and optionally wherein D.sup.7 and D.sup.8 are attached to the boronic acid or boric acid portions of the compound of formula VIIIa, VIIIb and IX via a linker group.

10. The combination according to claim 1, wherein the delivery agent is a compound selected from the group consisting of: ##STR00050##

11. The combination according to claim 1, wherein the delivery agent is covalently bonded to the antibacterial agent.

12. The combination according to claim 1, wherein the antibacterial agent is a glycopeptide or a phosphoglycolipid molecule or fragment.

13. The combination according to claim 1, wherein the antibacterial agent is selected from the group consisting of moenomycin, moenomycin derivatives, vancomycin, vancomycin derivatives, β-lactam antibiotics and derivatives thereof.

14. The combination according to claim 13, wherein the antibacterial agent is a moenomycin A or a derivative thereof which is covalently bound to the delivery agent: (i) such that the delivery agent and any associated linker replaces part or all of the 2-amido-cyclopentane-1,3-dione portion of the moenomycin or derivative thereof; (ii) via the moenocinol portion of the moenomycin or derivative thereof; (iii) such that the delivery agent and any associated linker replaces part or all of the moenocinol portion of the moenomycin or derivative thereof; or (iv) via the 2-amino-cyclopentane-1,3-dione portion of the moenomycin or derivative thereof.

15. A pharmaceutical formulation comprising the combination of claim 1 in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

16. A method of treating a bacterial infection caused by Acinetobacter, Staphylococcus, Klebsiella, Pseudomonas, Escherichia, or Bacillus, which method comprises administration of a therapeutically effective amount of a combination as defined in claim 1 to a subject in need thereof.

17. The combination according to claim 1, wherein n is from 2 to 20.

18. The combination according to claim 9, wherein each n1 is from 2 to 20.

19. The combination according to claim 9, wherein L.sub.2 represents a C.sub.1-6 alkyl chain.

Description

FIGURES

(1) FIG. 1: The structure of moenomycin A.

(2) FIG. 2: Showing the peptide delivery of TAMRA to E. coli labelled with GFP. Confocal microscope images of GFP labelled E. coli after treatment with TAMRA labelled cell penetrating peptides. A E. coli labelled with GFP, B The same image area looking at TAMRA labelled peptides. C an overlay of images A and B.

BIOLOGICAL TESTS

(3) The effects of compounds of the invention in relation to inhibiting the growth of various microorganisms was determined using methods known to those skilled in the art, for example in vitro methods as described in J. Med. Chem. 47, 2133-2156 (2004) and in vivo methods as described in J. Med. Microbiol. 46, 208-213 (1997), the disclosures of which documents are hereby incorporated by reference.

(4) MIC Testing The bacteria were grown in 5 ml of Mueller Hinton Broth overnight at 37° C. The inhibitor was tested on its own or in conjunction with a cell penetrating peptide (CPP). When tested with a CPP they were incubated together at room temperature for 1 hour before use. Delivery agents (Compound 5, Compound 6, and 7) were used at a 1:2 ratio. CPP Linker was used at a 1:3 ratio or a 1:10 ratio where stated. All dilutions were carried out using Mueller Hinton Broth. The MIC tests were carried out across two 96 well plates unless otherwise stated. 100 μl of autoclaved Mueller Hilton Broth was added to wells 2-12 on plate 1 and 1-12 on plate 2. 200 μl of the inhibitor was added to well 1 at a concentration of 512 μg/ml. 100 μl of the inhibitor was taken from well 1 and pipetted into well 2. The mixture was pipetted up and down three times before 100 μl was taken from well 2 and added to well 3. This process was repeated until well 11 on plate 2. 100 μl was then taken up from well 11 and discarded ensuring that there was no inhibitor present in well 12 of plate 2. The pipette tips were changed between each well so that the concentration was not affected by any inhibitor on the outside of the tips. Each well was inoculated with 100 μl of bacteria that had been diluted to an OD.sub.600 nm of 0.1. This was repeated three times. The 96 well plates were then incubated for 24 hours at 37° C. The minimum inhibitory concentration (MIC) was determined to be the lowest concentration at which there was no growth visible. To determine the minimum bactericidal concentration (MBC) 10 μl of the sample was pipetted onto a Muller Hinton agar plate containing no inhibitor. This was done for each well in which there was no growth and the three wells above the MIC. These were then incubated overnight and the MBC was determined by any growth from a sample corresponding to a well that had no visible growth.

(5) Confocal Microscope XL1 blue E. coli was used for this experiment as it was transformed with pJF40 so that it produced GFP. Once the transformation had been completed, 5 ml overnights were inoculated with a single colony and grown up overnight (37° C., 180 rpm). 1 ml of bacteria was then spun down and the supernatant was removed. The bacteria pellet as then re-suspended in 0.5 ml HBSS. The labelled peptides were then added to each sample to achieve a final concentration of 10 μM of peptide. The samples were incubated at room temperature for three hours. The samples were then washed 5 times in 0.5 ml PBS by re-suspended the pellet and re-suspending in PBS. After washing 10 μl of each sample was smeared onto a microscope slide ready for viewing on the confocal. No fixation was used as the GFP bleaches quite quickly so the samples will not be able to be used for than once.

(6) Toxicity Test Methods

(7) Galleria mellonella Model

(8) This model was used to test Moenomycin A and peptides in vivo both for toxicity and antimicrobial activity. Conjugates were also tested using the same methods.

(9) Galleria mellonella were selected on the basis of being free of infection and signs of trauma, not beginning to pupate and weighing between 225 mgs and 275 mgs. All calculations were on the basis of an average weight of 250 mgs. The selected Galleria mellonella were swabbed with 70% ethanol prior to injection using a Hamilton 10 μl syringe using disposable needles. Tips were changed between solutions and between each Galleria. All injections were into the front left proleg unless otherwise stated.

(10) Preliminary Testing

(11) Ringers solution was chosen as a control, and all dilutions of bacteria, antimicrobials and peptides were carried out in this. Ringers supplemented with 20% DMSO was also used as a control to account for the DMSO used to dissolve Compound 6. Groups of 10 Galleria were used for these control groups and injected with 10 μl of the appropriate solution as previously described. These were then observed for 96 hours. The Galleria were deemed to be alive if there was movement in response to physical stimuli (touch).

(12) The kill kinetics of P. aeruginosa, S. aureus and A. baumannii were also determined by injecting a set CFU into the Galleria, the aim was to ensure that the Galleria died within 36 hours but not less than 24.

(13) To determine the appropriate antimicrobial concentration, a range was set up from 40 mg/kg to 0.5 mg/kg. Moenomycin was tested both singularly and in conjunction with either Compound 6 or 8; these two compounds were also tested separately. Groups of 2 were used for each test with control groups of 5 used to cover the whole experiment. The Galleria were injected as previously described. For toxicity testing of Moenomycin conjugates, the same procedure was followed with a concentration range up to 80 mg/kg.

(14) To determine the appropriate concentration of antimicrobial in regards to treatment of P. aeruginosa, the Galleria were first injected with 10 μl of the appropriate concentration of bacteria as previously described. They were then treated within half an hour by injection into the first right pro-leg with the compound(s) being tested.

EXAMPLES

Example 1—Purification of Moenomycin A

(15) Flavomycin (100 g) was extracted twice with methanol (400 mL) at room temperature for 16 h. The combined methanol extract was concentrated in vacuo and the residue was purified by silica gel chromatography with a gradient of 2-propanol:2 M ammonium hydroxide from 9.5:0.5 to 7:3. Moenomycin A eluted with the last gradient. The solvent was evaporated in vacuo and the obtained brown mass was lyophilized to give a brown solid. Further purification with RP-HPLC (Gemini C18) was performed with a gradient of ACN in water in 30 min to give moenomycin A as a white solid (400 mg).

(16) Calculated mass: 1581.7. Found 789.5 (M/2−H.sup.+); 1582.7 (M+H.sup.+); 1604.6 (M+Na.sup.+)

Example 2—Synthesis of Dendrimer Delivery Agents

(17) Compounds 1˜4 shown below were prepared using reported procedures from J. Mater. Chem. B 2014, 2, 2153-2167 (see Schemes 1 and 2).

(18) ##STR00015##

(19) ##STR00016##

Example 3—General Procedure for the Synthesis of Compounds 3 and 4

(20) Each solution of the crude compounds 1 (500 mg, 0.43 mmol) or 2 (175 mg, 0.08 mmol), which were dissolved in dry DCM (120 mL), was added dropwise over 2 h at 0° C. into a solution of mono-Boc-DAPMA (1.24 g, 5.08 mmol, 12 eq., dissolved in 50 mL dry DCM) employing dry reaction conditions. Immediately, the solution turned yellow due to the displacement of p-nitrophenol. A solution of DMAP (0.20 g, 1.69 mmol, 0.5 eq. per p-nitrophenyl branch) and DIPEA (0.15 mL, 1.69 mmol, 1.0 eq. per p-nitrophenyl branch) in dry DCM (30 mL) was added and the reaction mixtures were stirred at room temperature for 72 h. The solvent was then removed under reduced pressure. Purification was performed both by column chromatography (CHCl.sub.3—MeOH—NH.sub.4OH 90:9:1) and size exclusion chromatography (SEC) using a Sephadex™ LH-20 (CHCl.sub.3—MeOH 1:1). Drying under high vacuum yielded the products 11 and 12 as yellowish oils.

(21) Compound 3 was obtained as a yellowish viscous oil (1.57 g, 49%).

(22) Compound 4 was obtained as a yellowish viscous oil (0.45 g, 40%).

Example 4—General Procedure for the Synthesis of Compounds 5 and 6

(23) Compounds 5 and 6 were synthesised from compounds 3 and 4, respectively, according to the procedure below.

(24) ##STR00017##

(25) TFA (6.0 mL, excess) was slowly added to a solution of compounds 3 (0.10 g, 0.06 mmol) or 4 (0.10 g, 0.03 mmol) in DCM (6.0 mL) and stirred overnight at room temperature. The solvent was removed in vacuo and the residue washed alternately with hexane and diethyl ether. Purification was accomplished via SEC (Sephadex™ LH20, MeOH) to remove any trace amounts of impurities. Freeze drying yielded the compounds 5 and 6 as white foams.

(26) Compound 5 was obtained as a white foam (98 mg, quant.).

(27) Compound 6 was obtained as a white foam (131 mg, quant.).

Example 5—Guanidilation of PAMAM Dendrimer, 3,3′,3″,3′″-(ethane-1,2-diylbis(azanetriyl)) tetrakis(N-(2-guanidinoethyl)propanamide) (Compound 7)

(28) ##STR00018##

(29) To a solution of PAMAM dendrimer (200 mg, 0.387 mmol from a solution of 20% in MeOH) in EtOH (1.5 mL), was added 1-H-pyrazole-1-carboxamidine hydrochloride (348.3 mg, 2.322 mmol). The reaction mixture was refluxed at 86° C. and stirred overnight. After stirring, the solvent was removed in vacuo. Extraction in DCM (3×3 mL) was performed and supernatant solution was removed.

(30) Crude mass: 430 mg; crude yield: 78%

(31) Calculated mass: 684.6. Found: 685.6 (M+H.sup.+)

Example 6—Cell Penetration Studies

(32) TAMRA dye and Confocal Microscopy was used to demonstrate the principle of compound meditated delivery to Gram negative bacteria. E. coli cells were label by transforming with pJF40 which vector which harbours a gene for Green fluorescent protein (GFP) when viewed under confocal microscopy (excited at 555 nm emission at 570 nm). This was used to clearly identify bacterial cells.

(33) The same cells were treated with TAMRA labelled cell penetrating peptides (sequence: PLIYLRLLRGQF (SEQ ID NO: 21); excited at 555 nm emission at 570 nm) which fluoresced under a different wavelength which was used to follow the penetration of the dye into the bacteria. Control experiment where peptides were omitted showed no fluorescence.

(34) Confocal microscope images of GFP labelled E. coli after treatment with TAMRA labelled cell penetrating peptides. A E. coli labelled with GFP, B The same image area looking at TAMRA labelled peptides. C an overlay of images A and B.

Example 7—General Procedure for the Synthesis of Compounds 8 to 19

(35) General Procedure for Peptide Synthesis

(36) Peptide syntheses was performed using standard Fmoc Solid Phase Peptide Synthesis (SPPS) protocols on Rink Amide Chemmatrix Resin, loading=0.49 mmol/g on a 0.1 mmol scale using a Biotage Initiator+Alstra fully automated microwave peptide synthesizer. All amino acid couplings were performed using 5 eq. Amino Acid with 5 eq. DIC/Oxyma in DMF as a coupling cocktail by irradiating at 70° C. for 5 min. Fmoc deprotection was performed using 20% piperidine in DMF by shaking for 3 min, followed by shaking for 10 min. 4×45 s washes were performed after each coupling cycle and 3×30 s washes were performed after each deprotection cycle.

(37) Propiolic acid was coupled using 3 eq. of acid, 2.9 eq. of HATU and 6 eq. of DIPEA in DMF and shaking for 1 h.

(38) Peptide cleavage was performed using TFA/TIS/H.sub.2O=95:2.5:2.5 (3 mL/100 mg resin). For sequences containing 1 or 2 Arg groups, the cleavage time was 2 h. For 3 or higher Arg containing peptides, cleavage time was 5 h. Peptides were precipitated using cold Et.sub.2O (−20° C.) by adding approximately 5× volume of the TFA used for cleavage and centrifuging at 7000 rpm at 0° C.

(39) Analysis and Purification of Peptides/Conjugates:

(40) All peptides/conjugates were analysed on a Thermo Scientific Dionex Ultimate 3000 RP-HPLC equipped with a Phenomenex Gemini NX C18 110 Å (150×4.6 mm) column using the following buffer systems: A: 0.1% HCOOH in milliQ water. B: ACN using a flow rate of 1 ml/min. The use of TFA was avoided as it can damage the phosphate group of Moenomycin A. The column was flushed with 100% A for 5 min prior to an injection and was flushed for 5 min with 95% B and 5% A after the run was finished.

(41) Peptides and conjugates were analysed using the following gradient: 100% A for 2 min. 0-95% B in 15 min. 95% B for 5 min. 100% A for 4 min.

(42) Peptides and conjugates were purified using the same gradient as mentioned above, on a Thermo Scientific Dionex Ultimate 3000 RP-HPLC with a flow rate of 5 mL/min using a Phenomenex Gemini NX C18 110 Å (150×10 mm) semi-prep column.

(43) LC-MS data were collected on an Agilent 1100 Series instrument with a Phenomenex Kinetex C18 100 Å column (150×4.6 mm, 5 μm at 35° C.) connected to an ESMSD type VL mass detector with a flow rate of 1.5 ml/min was used with the following solvent systems: (A): 0.1% HCOOH in H.sub.2O and (B) MeCN. The column was flushed with 100% A for 2 min, then a gradient from 0 to 100% B over 6 min was used, followed by 2 min of flushing with 100% B.

(44) The following compounds were synthesised by this method.

(45) TABLE-US-00002 Comp. Exact Mass number Peptide Derivative Chemical Formula Mass found  8 C.sub.51H.sub.99N.sub.33O.sub.9 1317.83 1453.58 [M + CF.sub.3CO.sub.2.sup.− + Na.sup.+]  9 0 C.sub.81H.sub.130N.sub.22O.sub.16 1667.00 1667.87 10 C.sub.81H.sub.130N.sub.22O.sub.16 1667.00 1667.67 12 C.sub.45H.sub.87N.sub.27O.sub.8 1133.72 1133.9  [M + H.sup.+] 13 H-P-L-I-Y-L-K-L-L-K-G-Q-F-NH.sub.2 C.sub.72H.sub.118N.sub.16O.sub.14 1430.90 1432.94 (SEQ ID NO: 6 [M + H.sup.+] 14 C.sub.75H.sub.118N.sub.20O.sub.15 1538.91 1538.90 15 C.sub.75H.sub.118N.sub.20O.sub.15 1538.91 1674.51 [M + CF.sub.3CO.sub.2.sup.− + Na.sup.+] 16 C.sub.75H.sub.118N.sub.20O.sub.15 1538.91 1674.55 [M + CF.sub.3CO.sub.2.sup.− + Na.sup.+] 17 H-P-L-I-Y-L-R-L-L-R-G-Q-F-NH.sub.2 C.sub.72H.sub.118N.sub.20O.sub.14 1486.91 1487.4  (SEQ ID NO: 21) 18 H-P-L-I-Y-L-L-L-R-R-G-Q-F-NH.sub.2 C.sub.72H.sub.118N.sub.20O.sub.14 1486.91 1487.43 (SEQ ID NO: 37) 19 C.sub.81H.sub.130N.sub.22O.sub.16 1667.00 1667.87

Example 8—General Procedure for the Synthesis of Peptide-Moenomycin A Conjugates Via A Ring Conjugation (Compounds 20 to 39)

(46) Peptide Synthesis:

(47) Peptide synthesis was performed using standard Fmoc Solid Phase Peptide Synthesis (SPPS) protocols on Rink Amide Chemmatrix Resin, loading=0.49 mmol/g on a 0.1 mmol scale using a Biotage Initiator+Alstra fully automated microwave peptide synthesizer. All amino acid couplings were performed using 5 eq. Amino Acid with 5 eq. DIC/Oxyma in DMF as a coupling cocktail by irradiating at 70° C. for 5 min. Fmoc deprotection was performed using 20% piperidine in DMF by irradiating at 70° C. for 3 min, followed by shaking at r.t. for 10 min. 4×45 s washes were performed after each coupling cycle and 3×30 s washes were performed after each deprotection cycle.

(48) Peptide cleavage was performed using TFA/TIS/H.sub.2O=95:2.5:2.5 (3 mL/100 mg resin). For sequences containing 1 or 2 Arg groups, the cleavage time was 2 h. For 3 or higher Arg containing peptides, cleavage time was 5 h. Peptides were precipitated using cold Et.sub.2O (−20° C.) by adding approximately 5× volume of the TFA used for cleavage and centrifuging at 7000 rpm at 0° C.

(49) Fmoc-ε-Ahx-OH and (9H-fluoren-9-yl)methyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)-carbamate and 5-Amino-2-Nitrobenzoic acid were coupled using Amino acid, HATU and DIPEA in DMF.

(50) General Procedure for Coupling Peptides to Moenomycin a (15 mg Scale):

(51) The peptides were coupled using the procedure described in Eur. J. Org. Chem. 2002, 1149-1162.

(52) Analysis and Purification of Peptides/Conjugates:

(53) All peptides/conjugates were analysed on a Thermo Scientific Dionex Ultimate 3000 RP-HPLC equipped with a Phenomenex Gemini NX C18 110 Å (150×4.6 mm) column using the following buffer systems: A: 0.1% HCOOH in milliQ water. B: ACN using a flow rate of 1 ml/min. The use of TFA was avoided as it can damage the phosphate group of Moenomycin A. The column was flushed with 100% A for 5 min prior to an injection and was flushed for 5 min with 95% B and 5% A after the run was finished.

(54) Peptides and conjugates were analysed using the following gradient: 100% A for 2 min. 0-95% B in 15 min. 95% B for 5 min. 100% A for 4 min.

(55) Peptides and conjugates were purified using the same gradient as mentioned above, on a Thermo Scientific Dionex Ultimate 3000 RP-HPLC with a flow rate of 5 mL/min using a Phenomenex Gemini NX C18 110 Å (150×10 mm) semi-prep column.

(56) LC-MS data were collected on an Agilent 1100 Series instrument with a Phenomenex Kinetex C18 100 Å column (150×4.6 mm, 5 μm at 35° C.) connected to an ESMSD type VL mass detector with a flow rate of 1.5 ml/min was used with the following solvent systems: (A): 0.1% HCOOH in H.sub.2O and (B) MeCN. The column was flushed with 100% A for 2 min, then a gradient from 0 to 100% B over 6 min was used, followed by 2 min of flushing with 100% B.

(57) Complete List of Peptides Synthesized (Peptide Nos. 1 to 17):

(58) TABLE-US-00003 Peptide Exact Mass No. Peptide-linker structure Chemical Formula Mass found  1. C.sub.37H.sub.66N.sub.20O.sub.8 918.54 919.54  2. X-R-R-R-R-R-R-R-R-NH.sub.2 C.sub.55H.sub.103N.sub.35O.sub.11 1429.86 1430 (SEQ ID NO: 1)  3. C.sub.61H.sub.114N.sub.36O.sub.12 1542.94 1543  4. 0 C.sub.67H.sub.125N.sub.37O.sub.13 1656.03 1655.8  5. C.sub.67H.sub.126N.sub.40O.sub.13 1699.04 1698.9  6. C.sub.73H.sub.138N.sub.44O.sub.14 1855.14 1855.5  7. C.sub.73H.sub.138N.sub.40O.sub.14 1799.13 1799.4  8. X-K-R-R-K-R-R-K-R-R-NH.sub.2 C.sub.61H.sub.115N.sub.33O.sub.12 1501.94 1502 (SEQ ID NO: 4)  9. C.sub.67H.sub.126N.sub.34O.sub.13 1615.02 1614.6 10. C.sub.73H.sub.137N.sub.35O.sub.14 1728.11 1728.6 11. X-K-K-K-K-K-R-NH.sub.2 C.sub.43H.sub.79N.sub.17O.sub.9 977.62 977.5 (SEQ ID NO: 14) 12. C.sub.49H.sub.90N.sub.18O.sub.10 1090.71 1090.6 13. C.sub.55H.sub.101N.sub.19O.sub.11 1203.79 1203.9 14. C.sub.66H.sub.123N.sub.25O.sub.13 1487.99 1487.8 15. C.sub.82H.sub.120N.sub.34O.sub.13 1788.98 1789.5 16. X-P-L-I-Y-L-R-L-L-R-G-Q-F-NH.sub.2 C.sub.79H.sub.122N.sub.22O.sub.17 1650.94 1651 (SEQ ID NO: 21) 17. 0 C.sub.13H.sub.19N.sub.7O.sub.4 337.15 338.1 [M + H.sup.+]

(59) Complete List of Conjugates Synthesized (Compounds 20 to 34):

(60) TABLE-US-00004 Exact Mass Comp. No. Conjugate description Chemical Formula Mass found 20. Conjugate with peptide 1 C.sub.106H.sub.171N.sub.26O.sub.42P 2511.18 2512.2 21. Conjugate with peptide 2 C.sub.124H.sub.208N.sub.41O.sub.45P 3022.50 3022.8 22. Conjugate with peptide 3 C.sub.130H.sub.219N.sub.42O.sub.46P 3135.58 3137.1 23. Conjugate with peptide 4 C.sub.136H.sub.230N.sub.43O.sub.47P 3248.67 3249.6 24. Conjugate with peptide 5 C.sub.136H.sub.231N.sub.46O.sub.47P 3291.68 3292.8 25. Conjugate with peptide 6 C.sub.142H.sub.243N.sub.50O.sub.48P 3447.78 3448.5 26. Conjugate with peptide 7 C.sub.136H.sub.231N.sub.40O.sub.47P 3207.67 3208.2 27. Conjugate with peptide 8 C.sub.130H.sub.220N.sub.39O.sub.46P 3094.58 3094.8 28. Conjugate with peptide 9 C.sub.142H.sub.243N.sub.46O.sub.48P 3391.77 3392.1 29. Conjugate with peptide 10 C.sub.142H.sub.242N.sub.41O.sub.48P 3320.75 3322 30. Conjugate with peptide 11 C.sub.112H.sub.184N.sub.23O.sub.43P 2570.27 2570.8 31. Conjugate with peptide 12 C.sub.118H.sub.195N.sub.24O.sub.44P 2683.35 2683.8 32. Conjugate with peptide 13 C.sub.124H.sub.206N.sub.25O.sub.45P 2796.43 2797 33. Conjugate with peptide 16 C.sub.148H.sub.227N.sub.28O.sub.51P 3243.58 3244.4 34. Conjugate with peptide 17 C.sub.82H.sub.124N.sub.13O.sub.38P 1929.79 1930

(61) General Structure of a Peptide-Moenomycin A Conjugate Via A Ring Conjugation

(62) ##STR00041##

(63) The molecular fragment shown below is present in all of peptides 1 to 17:

(64) ##STR00042##

(65) The general structure shown above for the peptide-Moenomycin A conjugates shows the nitrobenzene ring (as is present in all of peptides 1 to 17) attached to the Moenomycin skeleton.

(66) Shown below are structures of peptide conjugates that have been synthesised.

(67) ##STR00043## ##STR00044## ##STR00045##

Example 9—Antibacterial Efficacy Test Results

(68) TABLE-US-00005 Test material MIC MBC Organism: A. baumannii 19606 Ampicillin A 128 μg/ml Growth in all wells B 128 μg/ml Growth in all wells C 128 μg/ml Growth in all wells Moenomycin A A 4 μg/ml 8 μg/ml B 4 μg/ml 8 μg/ml C 8 μg/ml 8 μg/ml Moenomycin A + Compound 5 A 4 μg/ml 8 μg/ml B 4 μg/ml 8 μg/ml C 4 μg/ml 8 μg/ml Compound 5 A Growth in all wells Growth in all wells B Growth in all wells Growth in all wells C Growth in all wells Growth in all wells Moenomycin A + Compound 6 A 2 μg/ml 2 μg/ml B 2 μg/ml 2 μg/ml C 2 μg/ml 2 μg/ml Compound 6 A 256 μg/ml 256 μg/ml B 256 μg/ml 256 μg/ml C 256 μg/ml 256 μg/ml Moenomycin A + Compound 7 A 4 μg/ml 8 μg/ml B 4 μg/ml 8 μg/ml C 4 μg/ml 8 μg/ml Compound 7 A Growth in all wells Growth in all wells B Growth in all wells Growth in all wells C Growth in all wells Growth in all wells Moenomycin A + Compound 8  2 16 Compound 8 Growth in all wells Growth in all wells Moenomycin A + Compound 9 16 128  Compound 9 Growth in all wells Growth in all wells Moenomycin A + Compound 10 16 128  Compound 10 Growth in all wells Growth in all wells Moenomycin A + Compound 11 16 32 Compound 11 Growth in all wells Growth in all wells Moenomycin A + Compound 12  4  8 Compound 12 Growth in all wells Growth in all wells Moenomycin A + Compound 13 16 16 Compound 13 Growth in all wells Growth in all wells Moenomycin A + Compound 15  8 32 Compound 15 Growth in all wells Growth in all wells Moenomycin A + Compound 16  2 64 Compound 16 Growth in all wells Growth in all wells Moenomycin A + Compound 17  8 64 Compound 17 Growth in all wells Growth in all wells Organism: K. pneumonia 700603 Ampicillin A Growth in all wells Growth in all wells B Growth in all wells Growth in all wells C Growth in all wells Growth in all wells Moenomycin A A 64 μg/ml 128 μg/ml B 64 μg/ml 128 μg/ml C 64 μg/ml 128 μg/ml Moenomycin A + Compound 5 A 32 μg/ml 16 μg/ml B 16 μg/ml 8 μg/ml C 16 μg/ml 16 μg/ml Compound 5 A 128 μg/ml 128 μg/ml B 64 μg/ml 64 μg/ml C 64 μg/ml 64 μg/ml Moenomycin A + Compound 6 A 4 μg/ml 4 μg/ml B 4 μg/ml 4 μg/ml C 4 μg/ml 4 μg/ml Compound 6 A Growth in all wells Growth in all wells B Growth in all wells Growth in all wells C Growth in all wells Growth in all wells Moenomycin A + Compound 7 A 64 μg/ml 64 μg/ml B 32 μg/ml 32 μg/ml C 32 μg/ml 32 μg/ml Compound 7 A Growth in all wells Growth in all wells B Growth in all wells Growth in all wells C Growth in all wells Growth in all wells Moenomycin A + Compound 8  4  4 Compound 8 Growth in all wells Growth in all wells Moenomycin A + Compound 9 32 64 Compound 9 Growth in all wells Growth in all wells Moenomycin A + Compound 10 32 64 Compound 10 Growth in all wells Growth in all wells Moenomycin A + Compound 11 32 64 Compound 11 Growth in all wells Growth in all wells Moenomycin A + Compound 12  8 32 Compound 12 Growth in all wells Growth in all wells Moenomycin A + Compound 13 32 128  Compound 13 Growth in all wells Growth in all wells Moenomycin A + Compound 15 32 32 Compound 15 Growth in all wells Growth in all wells Moenomycin A + Compound 16 16 64 Compound 16 Growth in all wells Growth in all wells Moenomycin A + Compound 17  8 64 Compound 17 Growth in all wells Growth in all wells Organism: P. aeruginosa 27853 Ampicillin A Growth in all wells Growth in all wells B Growth in all wells Growth in all wells C Growth in all wells Growth in all wells Moenomycin A A 256 μg/ml Growth in all wells B 256 μg/ml Growth in all wells C 256 μg/ml Growth in all wells Moenomycin A + Compound 5 A 32 μg/ml 256 μg/ml B 32 μg/ml 256 μg/ml C 32 μg/ml 128 μg/ml Compound 5 A 256 μg/ml Growth in all wells B 256 μg/ml 256 μg/ml C 256 μg/ml 256 μg/ml Moenomycin A + Compound 6 A 4 μg/ml 16 μg/ml B 8 μg/ml 32 μg/ml C 4 μg/ml 16 μg/ml Compound 6 A 256 μg/ml 256 μg/ml B 256 μg/ml 256 μg/ml C 256 μg/ml 256 μg/ml Moenomycin A + Compound 7 A 128 μg/ml Growth in all wells B 128 μg/ml Growth in all wells C 128 μg/ml Growth in all wells Compound 7 A Growth in all wells Growth in all wells B Growth in all wells Growth in all wells C Growth in all wells Growth in all wells Moenomycin A + Compound 8 16 64 Compound 8 Growth in all wells Growth in all wells Moenomycin A + Compound 9 128  Growth in all wells Compound 9 Growth in all wells Growth in all wells Moenomycin A + Compound 10 128  Growth in all wells Compound 10 Growth in all wells Growth in all wells Moenomycin A + Compound 11 64 128  Compound 11 Growth in all wells Growth in all wells Moenomycin A + Compound 12 32 64 Compound 12 Growth in all wells Growth in all wells Moenomycin A + Compound 13 64 256  Compound 13 Growth in all wells Growth in all wells Moenomycin A + Compound 15 128  Growth in all wells Compound 15 Growth in all wells Growth in all wells Moenomycin A + Compound 16 256  Growth in all wells Compound 16 Growth in all wells Growth in all wells Moenomycin A + Compound 17 Growth in all wells Growth in all wells Compound 17 Growth in all wells Growth in all wells Organism: E. coli 25922 Ampicillin 8 μg/ml 8 μg/ml Moenomycin A 32 μg/ml 128 μg/ml Moenomycin A + Compound 5 16 μg/ml 64 μg/ml Compound 5 64 μg/ml 128 μg/ml* Moenomycin A + Compound 6 4 μg/ml 8 μg/ml Compound 6 256 μg/ml Growth in all wells Moenomycin A + Compound 7 Not tested Not tested Compound 7 Not tested Not tested Organism: B. subtilis 2410 Ampicillin 128 μg/ml 256 μg/ml Moenomycin A 256 μg/ml Growth in all wells Moenomycin A + Compound 5 8 μg/ml 64 μg/ml Compound 5 32 μg/ml 32 μg/ml Moenomycin A + Compound 6 4 μg/ml 64 μg/ml Compound 6 32 μg/ml 32 μg/ml Moenomycin A + Compound 7 Not tested Not tested Compound 7 Not tested Not tested

Example 9—Antibacterial Efficacy Test Results for Conjugated Delivery Agents

(69) TABLE-US-00006 K. pnumoniae A. baumannii B. subtilis ATCC ATCC P. aeruginosa Compound 168 700603 19606 ATCC 27853 number MIC MBC MIC MBC MIC MBC MIC MBC Compound 20 32 GAW 64 — 32 64 GAW GAW Compound 21 — — 128 GAW 8 256 128 GAW Compound 22 16 32 64 — 32 64 256 GAW Compound 23 2 64 8 256 64 128 16 256 Compound 24 2 64 GAW GAW 32 64 256 GAW Compound 25 1 64 GAW 256 64 128 256 256 Compound 26 4 GAW 16 64 8 32 128 256 Compound 27 — — GAW GAW 4 64 128 GAW Compound 28 8 64 GAW GAW 64 128 256 GAW Compound 29 — — 256 GAW 16 64 128 GAW Compound 30 — — 64 64 4 32 64 128 Compound 31 0.5 64 64 128 1 — 64 64 Compound 32 — — 64 GAW 2 8 128 GAW Compound 33 — — GAW GAW 64 GAW GAW GAW Compound 34 — — 64 64 4 256 256 GAW S. Aureus ATCC 25922 Compound number MIC MBC Compound 34 0.25 —

Example 10—Antibacterial Efficacy Test Results for Peptide Delivery Agents

(70) TABLE-US-00007 B. subtilis K. pnumoniae A. baumannii P. aeruginosa Peptide/anti-bacterial 168 ATCC 700603 ATCC 19606 ATCC 27853 agent MIC MBC MIC MBC MIC MBC MIC MBC Peptide 4 128 GAW GAW GAW GAW GAW GAW GAW Peptide 4 + Moenomycin A 1 GAW 16 32 2 4 64 256 Peptide 5 128 GAW GAW GAW GAW GAW GAW GAW Peptide 5 + Moenomycin A 0.25 8 32 GAW 1 4 32 256 Peptide 6 128 256 GAW GAW GAW GAW GAW GAW Peptide 6 + Moenomycin A 0.25 8 16 GAW 1 8 32 GAW Peptide 9 GAW GAW GAW GAW GAW GAW GAW GAW Peptide 9 + Moenomycin A 0.5 GAW 16 16 2 16  32 256 Peptide 15 GAW GAW GAW GAW GAW GAW GAW GAW Peptide 15 + 0.25 16   8 16 1 4 32  64 Moenomycin A S. Aureus Peptide/anti-bacterial ATCC 25922 agent MIC MBC Peptide 17 256 — Peptide 17 + Moenomycin A 0.25 — “GAW” = growth in all wells

Example 11—Toxicity Data for Unbound Delivery Agents

(71) Wax Moth Galleria mellonella

(72) In vivo toxicity tests were conducted for Compounds 1 to 19 using wax moth Galleria mellonella. No toxicity was observed below 40 mg/kg (24-96 hr, 100% survival).

(73) Mammalian Cytotoxicity

(74) Moenomycin A—no toxicity was observed up to 10 mg/mL.

(75) Compound 8—no toxicity was observed up to 110 μg/mL.

(76) Compound 6 did not affect the cell viability in the tumor cell line 786-0 in initial in vitro testing. Compound 6 was toxic in HeLa cells at 20 μg/ml.

Example 12—Toxicity Data for Covalent Conjugates of Moenomycin A Wax Moth Galleria mellonella

(77) Compounds 26, 29, 32 were tested—no toxicity was observed below 80 mg/kg (24 hr, 100% survival).

(78) Mammalian Cytotoxicity

(79) In Vitro Testing in HeLa cells showed no toxicity up to the concentrations mentioned in the table below.

(80) TABLE-US-00008 Compound number 100% survival in μM 100% survival in μg/mL 29 10 33.23 26 2 6.42 32 10 27.98

(81) Abbreviations

(82) ACN=Acetonitrile

(83) Boc=tert-butyloxycarbonyl

(84) DCM=dichloromethane

(85) DIBAL=diisobutylaluminium hydride

(86) DIPEA=N,N-diisopropylethylamine

(87) DMAP=4-dimethylaminopyridine

(88) DMF=N,N-dimethylformamide

(89) eq.=equivalents

(90) Et=ethyl

(91) h=hour(s)

(92) HCl=hydrochloric acid

(93) HPLC=high performance liquid chromatography

(94) IR=infra red (in relation to spectroscopy)

(95) MBC=minimum bactericidal concentration

(96) mcpba=meta-chloroperoxybenzoic acid

(97) Me=methyl

(98) MIC=minimum inhibitory concentration

(99) min.=minute(s)

(100) MS=mass spectrometry

(101) PAMAM=polyamidoamine

(102) RP=reverse phase

(103) rt/RT=room temperature

(104) THF=tetrahydrofuran

(105) TFA=trifluoroacetic acid

(106) Prefixes n-, s-, t- and tert- have their usual meanings: normal, secondary, iso, and tertiary.