Peptoid

Abstract

The invention relates to peptoids, derivatives and analogues thereof, and to methods of chemically synthesising such compounds. The invention relates to mixed peptoids, derivatives and analogues thereof comprising lysine and arginine type monomers, which may be linear or cyclic, and to their uses in therapy, for example as antimicrobial agents, and in methods for treating microbial infections.

Claims

1. A method of preparing a peptoid, analogue or derivative thereof, comprising at least one arginine type monomer and at least one lysine type monomer, the method comprising: (i) synthesising a precursor linear peptoid, analogue or derivative thereof comprising one or more lysine type monomers protected with a first protecting group, and one or more lysine type monomers protected with a second protecting group, wherein the first and second protecting groups are orthogonal; (ii) removing the first protecting group to reveal one or more unprotected lysine type monomers; (iii) converting the one or more unprotected lysine type monomers to one or more arginine type monomers; and (iv) removing the second protecting group to obtain a peptoid, analogue or derivative thereof, comprising at least one arginine type monomer and at least one lysine type monomer.

2. The method according to claim 1, wherein the arginine type monomer comprises a monomer of Formula (I): ##STR00003## wherein x is an integer between 0 and 14.

3. The method according to claim 1, wherein the lysine type monomer comprises a monomer of Formula (II): ##STR00004## wherein y is an integer between 0 and 14.

4. The method according to claim 1, wherein the first protecting group comprises an N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) (Dde) protecting group and/or the second protecting group comprises a tert-Butyloxycarbonyl (Boc) protecting group.

5. The method according to claim 1, wherein the step of synthesising the linear precursor peptoid, analogue or derivative thereof comprises: synthesising a first monomer on a substrate to obtain a linear precursor peptoid, analogue or derivative thereof comprising one monomer; subsequently adding at least one further monomer in a step-wise fashion to obtain the linear precursor peptoid, analogue or derivative thereof containing the desired number of monomers.

6. The method according to claim 5, wherein the first and/or at least one further monomer comprise at least one monomer comprising an aromatic residue and/or at least one monomer comprising an aliphatic residue, optionally wherein the monomer comprising an aromatic residue comprises an (S)N-(1-phenylethyl) glycine (Nspe) monomer, an (R)N-(1-phenylethyl) glycine (Nrpe) monomer, an N-(phenylmethyl) glycine (Nphe) monomer, an N-(4-fluoro phenylmethyl) glycine (Npfb) monomer, an N-(3-fluoro phenylmethyl) glycine (Nmfb) monomer, an (S)N-1-(4-fluoro phenylethyl) glycine (Nsfb) monomer, an (R)N-1-(4-fluoro phenylethyl) glycine (Nrfb) monomer, an N-(3,5 difluoro phenylmethyl) glycine (Ndfb) monomer, an N-(4-chloro phenylmethyl) glycine (Npcb) monomer, an N-(4-methoxyphenylmethyl) glycine (Npmb) monomer, an N-(methylimidazole) glycine (NHis) monomer, an N-(methylindole) glycine (NTrp) monomer, an N-(4-hydroxy phenylmethyl) glycine (NTyr) monomer, an N-(4-pyridinylmethyl) glycine (NPyr) monomer, an (S)N-(1-naphthlethyl) glycine (Nsna) monomer, an (R)N-(1-naphthlethyl) glycine (Nrna) monomer, an N-(furanylmethyl) glycine (Nfur) monomer, an N-(thiofuranylmethyl) glycine (Ntfur) monomer, or an N-(diphenylmethyl) glycine (Ndpa) monomer, and the monomer comprising an aliphatic residue comprises an N-(pentyl) glycine (Namy) monomer, an N-(propyl) glycine (NNVa) monomer, an N-(isopentyl) glycine (NHLe) monomer, N-(isobutyl) glycine (NLeu) monomer, an N-(butyl) glycine (Nbut) monomer, an N-(2-carboxyethyl) glycine (NGlu) monomer, an N-(2,2,2-trifluoromethyl) glycine (Ntfe) monomer, an N-(2,2,3,3,3-pentafluoropropyl) glycine (Npfp), an N-(2,2-difluoroethyl) glycine (Ndfea) monomer, an N-(ethyl) glycine (Nea) monomer, an N-(2-thioethyl) glycine (NCys) monomer, an (S)N-(sec-butyl) glycine (Nssb) monomer, an (R)N-(sec-butyl) glycine (Nrsb) monomer, an (S)N-(1-methylbutyl) glycine (Nsmb) monomer, an (R)N-(1-methylbutvl) glycine (Nrmb) monomer, an (S)N-(1-cyclohexylethyl) glycine (Nsch) monomer, (R)N-(1-cyclohexylethyl) glycine (Nrch) monomer, an N-(1-cyclohexylmethyl) glycine (Nch) monomer, an N-(ethynylmethyl) glycine (Nem) monomer, an (S)N-(1-ethynylethyl) glycine (Nsee) monomer, or an (R)N-(1-ethynylethyl) glycine (Nree) monomer.

7.-8. (canceled)

9. The method according to claim 5, wherein the substrate comprises a resin, optionally Rink amide resin, 2-chlorotrityl chloride resin, Wang resin, 4-(1,1-dimethyl-1-hydroxypropyl) phenoxyacetyl alanyl aminomethyl polystyrene (DHPP) resin or diphenyldiazomethane (PDDM) resin.

10. The method according to claim 5, wherein the method comprises a step of cleaving the peptoid, derivative or analogue thereof from the substrate to obtain a cleaved precursor linear peptoid, analogue or derivative thereof.

11. The method according to claim 10, wherein the cleaving step is carried out subsequent to step (i) and prior to step (ii), optionally wherein the method comprises a cyclisation step comprising cyclising the precursor linear peptoid, derivative or analogue thereof to obtain a precursor cyclic peptoid, wherein the cyclisation step is carried out subsequent to the cleaving step, and prior to step (ii).

12. (canceled)

13. The method according to claim 10, wherein the cleaving step is carried out subsequent to step (iii), optionally wherein the cleaving step is carried out simultaneously to the step of removing the second protecting group.

14. (canceled)

15. A peptoid, analogue or derivative thereof, comprising at least one arginine type monomer, at least one lysine type monomer and at least one monomer comprising an aromatic residue.

16. The peptoid, analogue or derivative thereof according to claim 15, wherein the peptoid, analogue or derivative thereof comprises between 3 and 30 monomers.

17. The peptoid, analogue or derivative thereof according to claim 15, wherein the peptoid, analogue or derivative thereof comprises a linear peptoid, analogue or derivative thereof, optionally wherein the linear peptoid, analogue or derivative thereof has the structure (NLys-Nspe-Nspe).sub.2(NhArg-Nspe-Nspe).sub.2; (NhArg-Nspe-Nspe).sub.2(NLys-Nspe-Nspe).sub.2; (NLys-Nspe-Nspe)(NhArg-Nspe-Nspe)(NLys-Nspe-Nspe).sub.2; [(NhArg-Nspe-Nspe)(NLys-Nspe-Nspe)]; or [(NnArgNspeNspe)(NaeNspeNspe)].sub.2.

18. (canceled)

19. The peptoid, analogue or derivative thereof according to claim 15, wherein the peptoid, analogue or derivative thereof comprises a cyclic peptoid, analogue or derivative thereof, optionally wherein the cyclic peptoid, analogue or derivative thereof has the structure (NLys-Nphe-NhArg-Nphe-NLys-Nphe).

20. (canceled)

21. A cyclic peptoid, analogue or derivative thereof, comprising at least one arginine type monomer and at least one lysine type monomer.

22. (canceled)

23. A method of treating, ameliorating or preventing a microbial infection in a subject, the method comprising, administering to a subject in need of such treatment, a therapeutically effective amount of a peptoid, analogue or derivative thereof according to claim 15.

24. The method according to claim 23, wherein the microbial infection comprises a bacterial infection, optionally a gram positive bacterial infection or a gram negative bacterial infection and/or wherein the bacterium is from the Escherichia genus, preferably E. coli, Pseudomonas genus, preferably P. aeruginosa, Staphylococcus genus, preferably S. aureus, or Serratia genus, preferably S. marcesens.

25.-26. (canceled)

27. The method according to claim 23, wherein the microbial infection comprises a fungal infection, optionally wherein the fungus is from the Candida genus, preferably C. albicans.

28. (canceled)

29. The method according to claim 23, wherein the microbial infection comprises a parasitic infection, optionally wherein the parasite is from the Leishmania genus, preferably L. mexicana or L. donovani, the Trypanosoma genus, preferably T. brucei or T. cruzi, the Plasmodium genus, preferably P. falciparum.

30. (canceled)

31. The method according to claim 29, wherein the method is for a method of treating, ameliorating or preventing leishmaniasis, African trypanosomiasis, Chagas disease or malaria.

32.-33. (canceled)

Description

[0166] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:

[0167] FIG. 1 shows the comparative structures of an exemplary peptide and a corresponding exemplary peptoid;

[0168] FIG. 2 shows a prior art reaction sequence for synthesising guanidinylate peptoids;

[0169] FIG. 3A shows a Dde-protected NLys residue used in a method in accordance with an embodiment of the invention;

[0170] FIG. 3B shows a Boc-protected NLys residue used in a method in accordance with an embodiment of the invention;

[0171] FIG. 4 is a reaction scheme according to an embodiment of the invention showing how the Dde-protected NLys residue of FIG. 3A can be synthesised and incorporated into an extended peptoid chain;

[0172] FIG. 5 is a reaction scheme according to another embodiment of the invention showing how a linear mixed peptoid can be synthesised;

[0173] FIG. 6 is a reaction scheme according to another embodiment of the invention showing how a cyclic mixed peptoid can by synthesised;

[0174] FIG. 7 shows the structure of a first embodiment of a peptoid referred to herein as peptoid 1;

[0175] FIG. 8 shows the structure of a second embodiment of a peptoid referred to herein as peptoid 2;

[0176] FIG. 9 shows the structure of a third embodiment of a peptoid referred to herein as peptoid 3;

[0177] FIG. 10 shows the structure of a fourth embodiment of a peptoid referred to herein as peptoid 4;

[0178] FIG. 11 shows the structure of a fifth embodiment of a peptoid referred to herein as peptoid 5;

[0179] FIG. 12 is a generic structure for a peptoid in accordance with the present invention;

[0180] FIG. 13 shows monomers comprising aromatic residues which may comprise part of a peptoid of the present invention;

[0181] FIG. 14 shows monomers comprising aliphatic residues which may comprise part of a peptoid of the present invention;

[0182] FIG. 15A shows a Dde-protected Nae residue used in a method in accordance with an embodiment of the invention;

[0183] FIG. 15B shows a Boc-protected Nae residue used in a method in accordance with an embodiment of the invention;

[0184] FIG. 16 shows the structure of a sixth embodiment of a peptoid referred to herein as peptoid 6;

[0185] FIG. 17A is a graph showing the activity of peptoids 1-4 against C. albicans;

[0186] FIG. 17B is a graph showing the activity of peptoids 1-4 against S. aureus; and

[0187] FIG. 17C is a graph showing the activity of peptoids 1-4 against E. coli.

EXAMPLE 1SYNTHESIS OF LINEAR MIXED PEPTOIDS

[0188] The inventors have developed a novel method using orthogonal protecting groups to protect lysine type residues, such as NLys residues and Nae residues, to synthesise linear mixed peptoids containing lysine type resides, arginine type residues and aromatic side chains.

[0189] Materials and Methods

[0190] Procedure 1on Resin Peptoid Synthesis

[0191] Fmoc-protected Rink Amide resin (normally 100-300 mg, 0.1-0.3 mmol, typical loading between 0.6-0.8 mmol g-1) was swollen in DMF (at least 1 hour, overnight preferred, at room temperature) in a 20 mL polypropylene syringe fitted with two polyethylene frits (Crawford Scientific). The resin was deprotected with piperidine (20% in DMF v/v, 220 min) and washed with DMF (32 mL). The resin was treated with bromoacetic acid (1 ml, 0.6M in DMF) and DIC (0.18 ml, 50% v/v in DMF) for 20 minutes at room temperature at 400 rpm on a shaker platform. The resin was washed with DMF (32 mL), before the desired amine sub-monomer was added (1 ml, 0.8-2M in DMF) and allowed to react for 60 minutes at room temperature on the shaker. The resin was again washed with DMF (32 mL).

[0192] For instance, when the desired monomer was a Boc protected NLys monomer the desired amine sub-monomer was N-Boc 1,4 diaminobutane, as shown in step 1 of FIG. 4. Alternatively, when the desired monomer unit was a Boc protected Nae monomer unit the desired amine sub-monomer was N-Boc 1,2 diaminoethane.

[0193] When the desired monomer was a Dde protected NLys monomer the desired amine sub-monomer was 1,4 diaminobutane, as shown in step 3 of FIG. 4. Alternatively, when the desired monomer unit was a Dde protected Nae monomer unit the desired amine sub-monomer was 1,2 diaminoethane. In both these cases procedure 2 would then be conducted to add the Dde protecting group before procedure 1 was repeated.

[0194] When the desired monomer was Nspe the amine sub-monomer was (S)-()--methylbenzylamine.

[0195] Procedure 1 was repeated until the desired peptoid sequence had been obtained. Once the desired full length orthogonally protected peptoid had been obtained procedures 3 to 5 were undertaken sequentially.

[0196] Procedure 2Dde Protection of NLys Submonomer

[0197] Dde-OH (10 eq. wrt resin, 1M in DMF) was added to the resin and placed on the shaker at RT for 60 minutes, then the resin was washed well with DMF (32 mL).

[0198] As explained above, subsequent peptoid couplings were then made by repeating procedure 1 until the desired full length peptoid sequence was obtained.

[0199] Procedure 3Guanidinylation of the Free Amines

[0200] On resin deprotection of the Dde group was undertaken using 2% hydrazine in DMF (44 ml3 mins) and then the resin washed with DMF (32 mL). Guanidinylation of the free amines were undertaken using pyrazole-1-carboxamide (6 eq. per free amine, in the minimum amount of DMF) and DIPEA (6 eq. per free amine) on the shaker at 400 rpm, RT for 90 minutes.

[0201] Procedure 4Cleavage of Peptoid from Substrate

[0202] The resin was washed with DMF (32 mL) before cleavage. Final cleavage from resin was achieved using 95:2.5:2.5 TFA:H2O:TIPS (4 ml) for 1.5 hours and the resin removed by filtration. The cleavage cocktail was removed in vacuo, the crude product precipitated in diethyl ether (45 mL) and the precipitate retrieved by centrifuge for 15 min at 5,000 rpm. The ether phase was decanted, the crude product dissolved in a mixture of acidified H2O (0.1% TFA) and MeCN and lyophilised.

[0203] Procedure 5Purification of Crude Peptoids

[0204] Crude peptoids were dissolved into 1.5 mL (95% H2O, 5% MeCN, 0.1% TFA) and purified by preparative RP-HPLC using a Perkin Elmer 200 Series LC pump with a Perkin-Elmer 785A UV-vis detector (=250 nm) on a SB Analytical column (ODS-H Optimal), 25010 mm, 5 m; flow rate=2 mL min-1; linear gradient elution 0-50% solvent B over 60 minutes, then 50-100% B over 15 minutes (solvent A=0.1% TFA in 95% H2O, 5% MeCN, solvent B=0.1% TFA in 5% H20, 95% MeCN). Relevant fractions were collected, lyophilized and analysed by LC-MS and analytical RP-HPLC.

[0205] All peptoids were obtained with >95% purity.

[0206] Results

[0207] NLys residues protected with Boc and Dde protecting groups are shown in FIGS. 3A and 3B respectively, and Nae residues protected with Boc and Dde protecting groups are shown in FIGS. 15A and 15B respectively.

[0208] For the synthesis of linear peptoids the procedures 1 and 2, given above, were used in combination to assemble the orthogonally protected specific peptoid sequence on a resin, as shown in FIG. 4. Using procedure 3, Dde was then easily removed using 2% hydrazine in DMF which allowed these residues to be selectively deprotected, while leaving Boc protection on other lysine type chains intact, as shown in step 1 of FIG. 5. The deprotected lysine type residues can then selectively undergo guanidinylation to introduce arginine type residues, as shown in step two of FIG. 5. Using procedure 4, the peptoids can then be cleaved from the resin, and the Boc protection group can be removed in one final step to obtain a mixed peptoid containing both lysine type residues and arginine type residues, as shown in step 3 of FIG. 5. Finally, using procedure 5, the peptoids can be purified.

[0209] Five different embodiments of linear peptoids, referred to as peptoids 1 to 4 and 6, were prepared using the above methodology. The structures of the peptoids are shown in FIGS. 7 to 10 and 16, and are:

[0210] Peptoid 1 has the structure (NLys-Nspe-Nspe).sub.2(NhArg-Nspe-Nspe).sub.2[SEQ ID No:1];

[0211] Peptoid 2 has the structure (NhArg-Nspe-Nspe).sub.2(NLys-Nspe-Nspe).sub.2[SEQ ID No:2];

[0212] Peptoid 3 has the structure (NLys-Nspe-Nspe)(NhArg-Nspe-Nspe)(NLys-Nspe-Nspe).sub.2[SEQ ID No:3];

[0213] Peptoid 4 has the structure [(NhArg-Nspe-Nspe)(NLys-Nspe-Nspe)].sub.2[SEQ ID No:4];

[0214] Peptoid 6 has the structure [(NnArgNspeNspe)(NaeNspeNspe)].sub.2[SEQ ID No:6].

[0215] All of the peptoids are 12 residue linear peptoids. Peptoids 1, 2 and 4 each contain two lysine type monomers (NLys), two arginine type monomers (NhArg) and eight aromatic residues (Nspe i.e. (S)N-1-phenylethyl), as shown in FIGS. 7, 8 and 10.

[0216] Peptoid 3 contains three lysine type monomers (NLys), one arginine type monomer (NhArg) and eight aromatic residues (Nspe i.e. (S)N-1-phenylethyl), as shown in FIG. 9.

[0217] Peptoid 6 contains two lysine type monomers (Nae) and two arginine type monomers (NnArg).

[0218] Conclusion

[0219] The inventors have found that, by using orthogonal protecting groups to protect lysine type residues, it is possible to synthesise linear peptoids containing lysine type residues, arginine type residues and aromatic side chains. This has not been possible previously.

[0220] While Nspe is used as an additional monomer in this example it will be appreciated that further monomers could be used. Examples of appropriate monomers comprising aromatic residues are shown in FIG. 13 and examples of appropriate monomers comprising aliphatic residues are shown in FIG. 14.

[0221] The orthogonal protecting groups used in this example are Boc and Dde. However, it will be appreciated that alternative protecting groups could be used.

[0222] The method devised by the inventors is a versatile method allowing the Dde-protecting group to be added and selectively deprotected in a variety of positions in the peptoid sequence, both near C and N-terminal positions and also within close proximity to each other.

EXAMPLE 2SYNTHESIS OF CYCLIC MIXED PEPTOIDS

[0223] The inventors have developed a novel method using orthogonal protecting groups to synthesise cyclic mixed peptoids comprising lysine and arginine side chains.

[0224] Materials and Methods

[0225] Procedure 1on Resin Peptoid Synthesis

[0226] 2-chlorotrityl chloride resin (0.1 mmol, typical loading 1.22 mmol g.sup.1) was swollen in dry DCM (45 mins, at room temperature) in a 20 mL polypropylene syringe fitted with two polyethylene frits. The resin was washed with dry DCM (32 mL) and loaded with bromoacetic acid (1 ml, 0.6 M in DMF) and neat DIPEA (16 eq. with respect to the resin) for 30 minutes at RT on a shaker at 400 rpm. The resin was washed with DMF (32 mL), before the desired amine sub-monomer was added (1 ml, 1.5 M in DMF) and allowed to react for 60 minutes at RT on the shaker.

[0227] For instance, when the desired monomer was a Boc protected NLys monomer the desired amine sub-monomer was N-Boc 1,4 diaminobutane, as shown in step 1 of FIG. 4.

[0228] When the desired monomer was a Dde protected NLys monomer the desired amine sub-monomer was 1,4 diaminobutane, as shown in step 3 of FIG. 4. In this case procedure 2 would then be conducted to add the Dde protecting group before procedure 1 was repeated.

[0229] When the desired monomer was Nphe the amine was benzylamine.

[0230] Procedure 1 was repeated until the desired peptoid sequence had been obtained. Once the desired full length orthogonally protected peptoid had been obtained procedures 3 to 7 were undertaken sequentially.

[0231] Procedure 2Dde Protection of NLys Submonomer

[0232] Dde-OH (10 eq. wrt resin, 1M in DMF) was added to the resin and placed on the shaker at RT for 60 minutes, then the resin washed well with DMF (32 mL).

[0233] As explained above, subsequent peptoid couplings were then made by repeating procedure 1 until the desired full length peptoid sequence was obtained.

[0234] Procedure 3Cleavage of Peptoid from Substrate

[0235] Final cleavage from resin was achieved using HFIP (4 mL, 20% v/v in DCM) for 30 minutes. The resin was removed by filtration and the cleavage cocktail sparged off using a fine stream of N.sub.2. The crude product was precipitated in diethyl ether (15 mL) and the precipitate retrieved by centrifuge for 15 min at 5,000 rpm. The ether phase was decanted, the crude, protected product dissolved in a mixture of acidified H.sub.2O (0.1% TFA) and MeCN and lyophilised.

[0236] Procedure 4Cyclisation of Peptoid

[0237] The crude peptoid was cyclised in solution without further purification. The linear peptoid (100 mol) was dissolved in dry DMF (10 mL) and added dropwise to a solution of PyBOP and DIPEA (both 6 eq. with respect to the crude linear peptoid, in 10 mL DMF) over 8 hours. The reaction was allowed to proceed for a further 60 minutes at room temperature following the last addition. The DMF solvent was removed in vacuo and the crude peptoids were extracted using DCM (220 mL). The organic phases were combined, washed with water and dried over MgSO.sub.4 before filtration and solvent removal in vacuo. The resulting residue was dissolved in 50% MeCN in H.sub.2O and lyophilised.

[0238] The protected peptoids were then dissolved in 50% MeCN in H.sub.2O and purified by preparative RP-HPLC; flow rate=2 mL min.sup.1; injection made at 50% B and a linear gradient elution 50-100 solvent B over 60 minutes (solvent A=0.1% TFA in 95% H.sub.2O, 5% MeCN, solvent B=0.1% TFA in 5% H.sub.2O, 95% MeCN). Relevant fractions were collected, lyophilized and analyzed by LC-MS.

[0239] Procedure 5Guanidinylation of the Free Amines

[0240] At this stage, Dde-groups were removed using 2% hydrazine in DMF (44 ml3 mins) and then the resin washed with DMF (32 mL). Guanidinylation of the free amines were undertaken using pyrazole-1-carboxamide (6 eq. per free amine, in the minimum amount of DMF) and DIPEA (6 eq. per free amine) on the shaker at 400 rpm, RT for 90 minutes.

[0241] Procedure 6Removal of Boc Protecting Groups

[0242] The cyclic peptoids were then Boc-deprotected using 95:2.5:2.5 TFA:H.sub.2O:TIPS (4 ml) for 1.5 hours. The cleavage cocktail was removed in vacuo, the crude product precipitated in diethyl ether (45 mL) and the precipitate retrieved by centrifuge for 15 min at 5,000 rpm. The ether phase was decanted, the crude product dissolved in a mixture of acidified H.sub.2O (0.1% TFA) and MeCN and lyophilised.

[0243] Procedure 7Purification of Crude Peptoids

[0244] The peptoids were dissolved in 1.5 mL (95% H.sub.2O, 5% MeCN, 0.1% TFA) and purified by preparative RP-HPLC flow rate=2 mL min.sup.1; linear gradient elution 0-50% solvent B over 60 minutes, then 50-100% B over 15 minutes. Relevant fractions were collected, lyophilized and analyzed by LC-MS and analytical RP-HPLC.

[0245] All peptoids were obtained with >95% purity.

[0246] Results

[0247] Linear precursors were synthesised on 2-chlorotrityl chloride resin using procedures 1 and 2, given above. Again, this reaction sequence will be as shown in FIG. 4. Using procedure 3, the linear precursors were cleaved from the resin without removing either the Boc or the Dde protecting groups, as shown in step 1 of FIG. 6. Using procedure 4, a head-to-tail, solution phase cyclisation was then undertaken, as shown in step 2 of FIG. 6. Following cyclisation, the crude cyclic species was purified via RP-HPLC and then, using procedure 5, the Dde groups could be selectively deprotected and a guanidinylation reaction carried out in solution. Finally, the Boc protecting groups could also be removed, using procedure 6. These two procedures are shown as the final step in FIG. 6. Finally, using procedure 7 RP-HPLC purification was carried out to obtain peptoids with >95% purity.

[0248] One cyclic peptoid, referred to as peptoid 5, was prepared using the above methodology. The structure of the peptoid is shown in FIG. 11, and is cyclic (NLys-Nphe-NhArg-Nphe-NLys-Nphe)[SEQ ID No:5].

[0249] Peptoid 5 is a six residue cyclic peptoid containing two lysine type monomers (NLys), one arginine type monomer (NhArg), and three aromatic residues (Nphe).

[0250] Conclusion

[0251] The synthesis of peptoid 5 shows that by using orthogonal protecting groups, it is possible to synthesise cyclic peptoids comprising lysine and arginine side chains. The inventors believe that this is the first example of a cyclic peptoid that contains an Arg type monomer in combination with a Lys type monomer.

[0252] While Nphe is used as an additional monomer in this example it will be appreciated that, as with Example 1, further monomers could be used.

[0253] As with example 1, the orthogonal protecting groups used in this example are also Boc and Dde. However, it will be appreciated that alternative protecting groups could be used.

[0254] Despite the bulky nature of the Dde group, the cyclisation reaction still occurred efficiently at room temperature and complete cyclisation was possible.

EXAMPLE 3BIOLOGICAL DATA: PLANKTONIC BACTERIA

[0255] The inventors have demonstrated that the mixed peptoids prepared as described above exhibit surprising antibacterial properties against planktonic bacteria.

[0256] Materials and Methods

[0257] Bacterial Strains

[0258] Species used in MIC assays included gram-negative Escherichia coli K12 W3110, Pseudomonas aeruginosa laboratory strain PAO2 and Serratia marcescens laboratory strain and gram-positive Staphylococcus aureus NCTC 6571 and Micrococcus luteus laboratory strain.

[0259] Overnight Culture Preparation

[0260] Bacterial cultures were prepared by streaking the bacterial strains on to agar plates with an inoculation loop and incubating overnight at 37 C. A single colony was then selected and placed in 5 mL of Iso-Sensitest broth using an inoculation loop and incubated at 37 C. with shaking overnight.

[0261] MIC Determination

[0262] MIC values were attained according to the protocol described by J. M. Andrews et al. [J. M. Andrews, J. Antimicrob. Chemother., 2001, 48, 5-16] and were conducted in 96-well microtitre plates in triplicate. 10-50 L of each overnight culture was inoculated into 1.2 mL of Iso-Sensitest broth and grown at 37 C. with shaking. An inoculum density of 10.sup.4 cfu/spot was determined by comparison with 0.5 MacFarland standard (240 M BaCl.sub.2 in 0.18 M H.sub.2SO.sub.4 aq.) and was found to relate to an A.sub.650nm of 0.07 after calibration with regular Iso-Sensitest broth. The inoculum was diluted ten-fold with Iso-Sensitest broth before use (to 10.sup.3 cfu/spot). Peptide solutions were initially dissolved in DMSO (5 mg mL.sup.1) and then diluted further with Iso-Sensitest broth to achieve a concentration range of 4 mgL.sup.1 to 512 mgL.sup.1 using 2-fold serial dilutions. Samples were vortexed between dilutions where necessary to aid dissolution. 50 L of inoculum and 50 L of peptide solution were added to each test well to achieve a final concentration range of 2 mgL.sup.1 to 256 mgL.sup.1. Separate dilutions of ampicillin and DMSO were made up in a similar manner to act as a positive antibacterial control and a +DMSO control, respectively. 50 L of inoculum and 50 L of Iso-Sensitest broth were used as a positive control and 100 L of inoculum was used as a negative control. Positive and negatives controls were conducted multiple times in parallel per plate. MIC was defined as the lowest concentration which completely inhibited bacterial growth after incubation at 37 C. for 16 h with shaking. IC.sub.50 was defined as the concentration of antibiotic which achieved a 50% inhibition of bacterial growth after incubation at 37 C. for 16 h with shaking. Quantitative data were attained as A.sub.650nm values using a BioTek Synergy H4 Hybrid Multi-Mode Microplate Reader.

[0263] Results

[0264] The anti-bacterial activity of peptoids 1 to 4 are shown in Table 1.

TABLE-US-00001 TABLE 1 Anti-bacterial properties of peptoids 1 to 4 Pep- MIC (M/mgL.sup.1) toid E. coli P. aeruginosa S. aureus S. marcescens 1 17/32 34/64 17/32 134/256 2 17/32 17/32 17/32 134/256 3 17/32 17/32 17/32 134/256 4 17/32 67/128 17/32 >134/>256

[0265] Conclusion

[0266] The mixed Arg/Lys type monomer containing peptoids (1-4) have been shown to have anti-bacterial properties against both Gram positive and Gram negative bacteria.

EXAMPLE 4BIOLOGICAL DATA: BIOFILM DATA

[0267] The inventors have demonstrated that the mixed peptoids prepared as described above exhibit surprising antibacterial and antifungal properties against bacterial and fungal biofilms.

[0268] Materials and Methods

[0269] Micro-Organism Strains and Growth Conditions

[0270] C. albicans (NCTC 3179) was subcultured aerobically on Sabouraud agar plates and propagated in yeast peptone dextrose broth. E. coli (ATCC 29522) and S. aureus (NCTC 6571) were grown on blood agar plates and propagated in brain heart infusion (BHI) broth.

[0271] Preparation and Treatment of Single Species Biofilms

[0272] Overnight cultures of C. albicans were washed and resuspended in a modified RPMI-1640 (Sigma-Aldrich, St Louis, USA) medium to yield an inoculum of 1.010.sup.6 cells/ml. Overnight cultures of S. aureus or E. coli were washed and resuspended in brain heart infusion broth (Oxoid, Basingstoke, UK) to yield an inoculum of 5.010.sup.6 cells/ml. A total volume of 100 l of each inoculum was added to microtitre plate wells (Thermo Fisher Scientific, Roskilde, Denmark). An initial biofilm was allowed to form for 4 hours. Wells were washed three times with 200 l PBS to facilitate removal of planktonic cells and the biofilms were then treated with 100 M of peptoids 1-4 in the appropriate broth. Plates were incubated for a further 24 hours to allow biofilm maturation. After removal of planktonic cells by washing, biofilms were quantified by the crystal violet assay or by PMA-qPCR.

[0273] Biofilm quantification by crystal violet assay Washed biofilms were fixed with 100 l methanol for 10 minutes. Following removal of methanol, the wells were air dried and stained with crystal violet solution (Clin-Tech Ltd, Guildford, UK) for 20 minutes at room temperature. Excess stain was removed by washing, the plate was then air dried and bound crystal violet was re-solubilised in 16 l 33% acetic acid prior to reading at 570 nm in a microtitre plate reader (Tecan GENios, Ziirich, Switzerland).

[0274] Results

[0275] As shown in FIG. 17A, all of the peptoids 1 to 4 exhibited strong antifungal activity against the C. albicans biofilm. Furthermore, as shown in FIG. 17B, all of peptoids 1 to 4 exhibited strong anti-bacterial activity against the S. aureus biofilm, with peptoids 1 and 3 exhibiting the strongest anti-bacterial activity. Finally, as shown in FIG. 17C, all of peptoids 1 to 4 exhibited anti-bacterial activity against the E. coli biofilm, with peptoids 2 and 4 exhibiting the strongest anti-bacterial activity

[0276] Conclusion

[0277] The mixed Arg/Lys type monomer containing peptoids (1-4) have been shown to have antimicrobial properties against fungus (C. albicans), gram positive bacteria (S. aureus) and gram negative bacteria (E. coli).

EXAMPLE 5BIOLOGICAL DATA: ANTI-PARASITIC ACTIVITY

[0278] The inventors have demonstrated that the mixed peptoids prepared as described above exhibit surprising antiparasitic activity against clinically relevant parasites that cause various diseases; malaria, Chagas disease, African sleeping sickness and Leishmaniasis.

[0279] Materials and Methods

[0280] Activity against Trypanosoma brucei rhodesiense STIB900.

[0281] This stock was isolated in 1982 from a human patient in Tanzania and after several mouse passages cloned and adapted to axenic culture conditions. Minimum Essential Medium (50 l) supplemented with 25 mM HEPES, 1 g/l additional glucose, 1% MEM non-essential amino acids (100), 0.2 mM 2-mercaptoethanol, 1 mM Na-pyruvate and 15% heat inactivated horse serum was added to each well of a 96-well microtiter plate. Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 g/ml were prepared. Then 410.sup.3 bloodstream forms of T. b. rhodesiense STIB 900 in 50 l was added to each well and the plate incubated at 37 C. under a 5% CO.sub.2 atmosphere for 70 h. 10 l Alamar Blue (resazurin, 12.5 mg in 100 ml double-distilled water) was then added to each well and incubation continued for a further 2-4 h. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, Calif., USA) using an excitation wave length of 536 nm and an emission wave length of 588 nm. The IC50 values were calculated by linear regression from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, Calif., USA). Melarsoprol (Arsobal Sanofi-Aventis, received from WHO) is used as control.

[0282] Activity Against T. cruzi.

[0283] Rat skeletal myoblasts (L-6 cells) were seeded in 96-well microtitre plates at 2000 cells/well in 100 L RPMI 1640 medium with 10% FBS and 2 mM 1-glutamine. After 24 h the medium was removed and replaced by 100 l per well containing 5000 trypomastigote forms of T. cruzi Tulahuen strain C2C4 containing the -galactosidase (Lac Z) gene. After 48 h the medium was removed from the wells and replaced by 100 l fresh medium with or without a serial drug dilution of eleven 3-fold dilution steps covering a range from 100 to 0.002 g/ml. After 96 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterility. Then the substrate CPRG/Nonidet (50 l) was added to all wells. A color reaction developed within 2-6 h and could be read photometrically at 540 nm. Data were analyzed with the graphic programme Softmax Pro (Molecular Devices), which calculated IC.sub.50 values by linear regression from the sigmoidal dose inhibition curves. Benznidazole is used as control (IC50 0.5+0.2 g/ml).

[0284] Activity Against L. donovani Axenic Amastigotes

[0285] Amastigotes of L. donovani strain MHOM/ET/67/L82 were grown in axenic culture at 37 C. in SM medium.sup.24 at pH 5.4 supplemented with 10% heat-inactivated fetal bovine serum under an atmosphere of 5% CO.sub.2 in air. One hundred microlitres of culture medium with 10.sup.5 amastigotes from axenic culture with or without a serial drug dilution were seeded in 96-well microtitre plates. Serial drug dilutions of eleven 3-fold dilution steps covering a range from 90 to 0.002 g/ml were prepared. After 70 h of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. 10 l of Alamar Blue (12.5 mg resazurin dissolved in 100 ml distilled water) were then added to each well and the plates incubated for another 2 h. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, Calif., USA) using an excitation wave length of 536 nm and an emission wave length of 588 nm. Data were analyzed using the software Softmax Pro (Molecular Devices Cooperation, Sunnyvale, Calif., USA). Decrease of fluorescence (=inhibition) was expressed as percentage of the fluorescence of control cultures and plotted against the drug concentrations. From the sigmoidal inhibition curves the IC.sub.50 values were calculated.

[0286] Activity Against P. falciparum.

[0287] In vitro activity against erythrocytic stages of P. falciparum was determined using a 3H-hypoxanthine incorporation assay using the drug sensitive NF54 strain (Schipol airport) or the chloroquine and pyrimethamine resistant K1 strain that originate from Thailand and the standard drug chloroquine (Sigma C6628). Compounds were dissolved in DMSO at 10 mg/ml and added to parasite cultures incubated in RPMI 1640 medium without hypoxanthine, supplemented with HEPES (5.94 g/l), NaHCO.sub.3 (2.1 g/1), neomycin (100 U/ml), Albumax (5 g/l) and washed human red cells A.sup.+ at 2.5% haematocrit (0.3% parasitaemia). Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 g/ml were prepared. The 96-well plates were incubated in a humidified atmosphere at 37 C.; 4% CO.sub.2, 3% O.sub.2, 93% N.sub.2. After 48 h 50 l of 3H-hypoxanthine (=0.5 Ci) was added to each well of the plate. The plates were incubated for a further 24 h under the same conditions. The plates were then harvested with a Betaplate cell harvester (Wallac, Zurich, Switzerland), and the red blood cells transferred onto a glass fibre filter then washed with distilled water. The dried filters were inserted into a plastic foil with 10 ml of scintillation fluid, and counted in a Betaplate liquid scintillation counter (Wallac, Zurich, Switzerland). IC.sub.50 values were calculated from sigmoidal inhibition curves by linear regression using Microsoft Excel. Chloroquine and artemisinin are used as control.

[0288] Cell Culture of Leishmania Mexicana M379 Promastigotes and Amastigotes

[0289] Leishmania mexicana (M379) promastigote parasites were maintained at 26 C. in Schneider's Insect medium (Sigma-Aldrich) supplemented with heat-inactivated foetal bovine sera (FBS, 15%; Biosera Ltd). Cells were counted using a Neubauer Improved Haemocytometer. Promastigotes were transformed into axenic amastigotes by a pH and temperature shift as previously described. A culture of recently transformed (three days) promastigotes in the late log phase was transferred into Schneider's Insect medium supplemented with 20% heat-inactivated FBS (pH 5.5) at 5105 parasites/mL. After 6 days, the parasites were in the metacyclic stage and used for transformation to amastigote-like forms by transfer in the same medium at 32 C. at 5105 parasites/mL. After additional 5-7 days, the parasites should be in the amastigote stage and be ready for cytotoxicity studies and infections.

[0290] Cytotoxicity Assays with L. Mexicana M379 Promastigotes and Amastigotes

[0291] Cytotoxicity analyses were performed in 96-well plates (Costar, Fisher Scientific) using Alamar Blue (Invitrogen) for cell viability detection as previously described.

[0292] Promastigote and amastigote L. mexicana were pre-incubated with the compounds in triplicate (5 mM stock solutions in DMSO; Amphotericin B was used as a positive control; untreated parasites with DMSO as a negative control) in 50 l of the corresponding media at 4106 mL-1 for 1 hour. Afterwards, 40 l were removed from each well before the addition of 90 l of the corresponding media, followed by incubation for 24 hours at 4105 mL-1. Then, 10 l Alamar Blue solution (Invitrogen) was added to each well for an incubation of 4 hours prior to assessing cell viability using a fluorescent plate reader (Biotek; Ex 560 nm/Em 600 nm). To investigate the effects of serum on the efficacy of the peptoids, the assay described above was modified using serum-free medium for the pre-incubation time. For these assays, the parasites were washed three times in serum-free medium before adding them to the compound solutions. All of the experiments described above were carried out on a minimum of two separate occasions to ensure a robust data set was collected.

[0293] Results

[0294] The anti-parasitic activity of peptoids 1, 2, 4 and 6 against biofilm are shown in Table 2.

TABLE-US-00002 TABLE 2 Anti-parasitic activities of peptoids 1, 2, 4 and 6. IC50 (M) Pep- L. T. brucei T. L. P. toid mexicana rhodesiense cruzi donovani falciparum 1 37 6.73 12.35 11.14 1.50 2 34 4 37 16.70 21.50 19.95 2.72 6 6.44 6.58 9.51 0.99

[0295] Peptoids 1, 2 and 4 were all found to be active against L. mexicana, which causes cutaneous leishmaniasis. Additionally, peptoids 1, 4 and 6 were all found to be active against T. brucei rhodesiense, which causes causes African sleeping sickness, T. cruzi which causes Chagas disease, L. donovani which causes visceral leishmaniasis, and P. falciparum, which causes malaria.

[0296] Conclusion

[0297] Peptoids 1, 2, 4 and 6 have been shown to have anti-parasitic activities against various protozoan parasites.