Beta-hairpin peptidomimetics

Abstract

Beta-hairpin peptidomimetics of the general formula (I), cyclo(P.sup.1-p.sup.2-p.sup.3-p.sup.4-p.sup.5-p.sup.6-p.sup.7p.sup.8p.sup.9-p.sup.10-p.sup.11-p.sup.12-T.sup.1-T.sup.2], and pharmaceutically acceptable salts thereof, with P.sup.1 to P.sup.12, T.sup.1 and T.sup.2 being elements as defined in the description and the claims, have broad spectrum Gram-negative antimicrobial activity to e.g. inhibit the growth or to kill microorganisms such as Klebsiella pneumoniae and/or Acinetobacter baumannii and/or Escherichia coli. They can be used as medicaments to treat or prevent infections or as disinfectants for foodstuffs, cosmetics, medicaments or other nutrient-containing materials. These peptidomimetics can be manufactured by a process which is based on a mixed solid- and solution phase synthetic strategy.

Claims

1. A compound of the general formula (I),
cyclo[P.sup.1-P.sup.2-P.sup.3-P.sup.4-P.sup.5-P.sup.6-P.sup.7-P.sup.8-P.sup.9-P.sup.10-P.sup.11-P.sup.12-T.sup.1-T.sup.2]  (I) wherein the single elements T or P are connected in either direction from the carbonyl (C═O) point of attachment to the nitrogen (N) of the next element and wherein T.sup.1 is .sup.DPro; T.sup.2 is Pro; or Pro((3S)OH); P.sup.1 is Leu; Ile; Val; Nva; or Trp; P.sup.2 is His; Trp; or Tyr; P.sup.3 is Leu; Cha; tBuGly; Trp; Tyr; or Tyr(Me); P.sup.4 is Dab; P.sup.5 is Orn; or Lys; P.sup.6 is Dab; .sup.DDab; or Pip; P.sup.7 is Dab; P.sup.8 is Trp; P.sup.9 is Hse; or Dab; P.sup.10 is tBuGly; Ile; Val; Nva; Cha; Chg; or Trp; P.sup.11 is Ala; Val; Alb; Ser; Asn; or Tyr; and P.sup.12 is Val; Ser; or alloThr; or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1, wherein T.sup.1 is .sup.DPro; T.sup.2 is Pro; or Pro((3S)OH); P.sup.1 is Leu; Ile; Val; Nva; or Trp; P.sup.2 is His; Trp; or Tyr; P.sup.3 is Leu; Cha; tBuGly; Trp; Tyr; or Tyr(Me); P.sup.4 is Dab; P.sup.5 is Orn; or Lys; P.sup.6 is Dab; .sup.DDab; or Pip; P.sup.7 is Dab; P.sup.8 is Trp; P.sup.9 is Hse; or Dab; P.sup.10 is tBuGly; Ile; Val; Nva; Cha; Chg; or Trp; P.sup.11 is Ala; Val; Alb; Ser; or Asn; and P.sup.12 is Val; Ser; or alloThr; or a pharmaceutically acceptable salt thereof.

3. A compound according to claim 1, wherein T.sup.1 is .sup.DPro; T.sup.2 is Pro; or Pro((3S)OH); P.sup.1 is Leu; Ile; Val; Nva; or Trp; P.sup.2 is His; Trp; or Tyr; P.sup.3 is Leu; Cha; tBuGly; Trp; Tyr; or Tyr(Me); P.sup.4 and P.sup.7 are Dab; P.sup.5 is Orn; or Lys; P.sup.6 is .sup.DDab; or Pip; P.sup.8 is Trp; P.sup.9 is Hse; or Dab; P.sup.10 is tBuGly; Ile; Val; Nva; Cha; or Chg; P.sup.11 is Ala; Val; Alb; Ser; Asn; or Tyr; and P.sup.12 is Val; Ser; or alloThr; or a pharmaceutically acceptable salt thereof.

4. A compound according to claim 1, which is selected from cyclo(-Trp-His-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Hse-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Trp-His-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Trp-His-Cha-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Nva-Ala-Ser-.sup.DPro-Pro-); cyclo(-Trp-His-Cha-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Val-Ser-.sup.DPro-Pro-); cyclo(-Leu-His-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Leu-His-Cha-Dab-Orn-Pip-Dab-Trp-Dab-tBuGly-Alb-Ser-.sup.DPro-Pro-); cyclo(-Val-His-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Nva-His-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Trp-His-Cha-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Chg-Alb-Ser-.sup.DPro-Pro-); cyclo(-Trp-Trp-Cha-Dab-Lys-.sup.DDab-Dab-Trp-Dab-Chg-Ala-Ser-.sup.DPro-Pro-); cyclo(-Trp-Trp-Cha-Dab-Lys-.sup.DDab-Dab-Trp-Dab-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Trp-Trp-tBuGly-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Cha-Ala-Ser-.sup.DPro-Pro-); cyclo(-Trp-Trp-Leu-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Val-Ala-Val-.sup.DPro-Pro-); cyclo(-Trp-His-Cha-Dab-Orn-Pip-Dab-Trp-Dab-tBuGly-Ser-Ser-.sup.DPro-Pro((3S)OH)—); cyclo(-Trp-Trp-Cha-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ser-Ser-.sup.DPro-Pro((3S)OH)—); cyclo(-Trp-His-Tyr(Me)-Dab-Orn-.sup.DDab-Dab-Trp-Hse-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Leu-His-Cha-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ala-alloThr-.sup.DPro-Pro-); or a pharmaceutically acceptable salt thereof.

5. A compound according to claim 1, which is selected from cyclo(-Trp-His-Tyr-Dab-Orn-Dab-Dab-Trp-Dab-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Trp-His-Cha-Dab-Orn-Dab-Dab-Trp-Dab-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Ile-His-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Ile-His-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Ile-Ala-Ser-.sup.DPro-Pro-); cyclo(-Leu-His-Tyr-Dab-Orn-Dab-Dab-Trp-Dab-Chg-Ala-Ser -.sup.DPro-Pro-); cyclo(-Leu-His-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Chg-Ala-Ser-.sup.DPro-Pro-); cyclo(-Leu-His-Trp-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ala -Ser-.sup.DPro-Pro-); cyclo(-Leu-His-Tyr-Dab-Lys-.sup.DDab-Dab-Trp-Dab-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Leu-Trp-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Cha-Ala-Ser-.sup.DPro-Pro-); cyclo(-Leu-Trp-Trp-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ala-Ser-.sup.DPro-Pro-); cyclo(-Leu-Trp-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ala -Ser-.sup.DPro-Pro-); cyclo(-Leu-His-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Trp-Ala-Ser-.sup.DPro-Pro-); cyclo(-Leu-His-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ser-Ser-.sup.DPro-Pro-); cyclo(-Leu-Trp-Tyr-Dab-Lys-.sup.DDab-Dab-Trp-Dab-Cha-Ser-Ser-.sup.DPro-Pro-); cyclo(-Leu-Trp-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Cha-Ser-Ser-.sup.DPro-Pro-); cyclo(-Leu-Tyr-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Asn-Ser-.sup.DPro-Pro-); cyclo(-Leu-Trp-tBuGly-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Chg-Ala-Ser-.sup.DPro-Pro-); cyclo(-Ile-Trp-tBuGly-Dab-Lys-.sup.DDab-Dab-Trp-Dab-Cha-Ala-Ser-.sup.DPro-Pro-); cyclo(-Leu-Trp-tBuGly-Dab-Orn-.sup.DDab-Dab-Trp-Dab-Chg-Ser-Ser-.sup.DPro-Pro-); cyclo(-Leu-Trp-Cha-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Ser-Ser-.sup.DPro-Pro-); or a pharmaceutically acceptable salt thereof.

6. A compound according to claim 1, which is cyclo(-Leu-Tyr-Tyr-Dab-Orn-.sup.DDab-Dab-Trp-Dab-tBuGly-Tyr-Ser-.sup.DPro-Pro-); or a pharmaceutically acceptable salt thereof.

7. A diastereomer or epimer of a compound of formula (I) as defined in claim 1 based on one or more chiral center(s) not explicitly specified in formula (I) or an enantiomer of a compound of formula (I).

8. A pharmaceutical composition containing a compound or a mixture of compounds according to claim 1, and at least one pharmaceutically inert carrier.

9. A pharmaceutical composition according to claim 8 in a form suitable for oral, topical, transdermal, injection, buccal, transmucosal, rectal, pulmonary or inhalation administration, especially in the form of tablets, dragees, capsules, solutions, liquids, gels, plaster, creams, ointments, syrup, slurries, suspensions, spray, nebulizer or suppositories.

10. A compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, for use as a medicament.

11. A pharmaceutical composition containing a compound or a mixture of compounds according claim 1 capable of being used as a pharmaceutically active substance having antibiotic activity.

12. A process for the preparation of a compound according to claim 1 which comprises (a) coupling an appropriately functionalized solid support with an appropriately N-protected derivative of that amino acid which in the desired end-product is in position T.sup.1 or T.sup.2 or P.sup.1 to P.sup.12 as defined above; any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (b) removing the N-protecting group from the product obtained in step (a); (c) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product is in the position of the next element (T or P), following counterclockwise or clockwise the sequence according to general formula (I) in —COOH to —NH.sub.2 orientation; any functional group which may be present in said N-protected amino acid derivative being likewise appropriately protected; (d) removing the N-protecting group from the product thus obtained; (e) repeating steps (c) and (d) until all amino acid residues have been introduced; (f) if desired, selectively deprotecting one or several protected functional group(s) present in the molecule and chemically transforming the reactive group(s) thus liberated; (g) detaching the product thus obtained from the solid support; (h) cyclizing the product cleaved from the solid support; (i) removing any protecting groups present on functional groups of any members of the chain of amino acid residues and, if desired, any protecting group(s) which may in addition be present in the molecule; (j) if desired, implementing additional chemical transformations of one or more reactive group(s) present in the molecule; and (k) if desired, converting the product thus obtained into a pharmaceutically acceptable salt or converting a pharmaceutically acceptable, or unacceptable, salt thus obtained into the corresponding free compound of formula (I) or into a different, pharmaceutically acceptable salt.

Description

EXAMPLES

(1) 1. Peptide Synthesis

(2) 1.1 General Synthetic Procedures

(3) A general method for the synthesis of the peptidomimetics of the present invention is exemplified in the following. This is to demonstrate the principal concept and does not limit or restrict the present invention in any way. A person skilled in the art is easily able to modify these procedures, especially, but not limited to, choosing a different starting position within the ring system, to still achieve the preparation of the claimed cyclic peptidomimetic compounds of the present invention.

(4) Coupling of the First Protected Amino Acid Residue to the Resin

(5) In a dried flask, 2-chlorotritylchloride resin (polystyrene, 1% crosslinked; loading: 1.4 mmol/g) was swollen in dry CH.sub.2Cl.sub.2 for 30 min (7 ml CH.sub.2Cl.sub.2 per g resin). A solution of 0.8 eq of the Fmoc-protected amino acid and 6 eq of DIPEA in dry CH.sub.2Cl.sub.2/DMF (4/1) (10 ml per g resin) was added. After shaking for 2-4 h at rt the resin was filtered off and washed successively with CH.sub.2Cl.sub.2, DMF, CH.sub.2Cl.sub.2, DMF and CH.sub.2Cl.sub.2. Then a solution of dry CH.sub.2Cl.sub.2/MeOH/DIPEA (17:2:1) was added (10 ml per g resin). After shaking for 3×30 min the resin was filtered off in a pre-weighed sinter funnel and washed successively with CH.sub.2Cl.sub.2, DMF, CH.sub.2Cl.sub.2, MeOH, CH.sub.2Cl.sub.2, MeOH, CH.sub.2Cl.sub.2 (2×) and Et.sub.2O (2×). The resin was dried under high vacuum overnight. The final mass of resin was calculated before the qualitative control.

(6) The following preloaded resins were prepared: Fmoc-Dab(Boc)-2-chlorotrityl resin, Fmoc-Pro-2-chlorotrityl resin, Fmoc-.sup.DPro-2-chlorotrityl resin, Fmoc-alloThr-2-chlorotrityl resin, and Fmoc-Tyr(Me)-2-chlorotrityl resin.

(7) Synthesis of the Fully Protected Peptide Fragment

(8) The synthesis was carried out on a Syro-peptide synthesizer (MultiSynTech GmbH) using 24 to 96 reaction vessels. In each vessel were placed approximately 80 mg of the above resin (weight of the resin before loading). The following reaction cycles were programmed and carried out:

(9) TABLE-US-00001 Step Reagent Time 1 CH.sub.2Cl.sub.2, wash and swell (manual) 1 × 3 min 2 DMF, wash and swell  2 × 30 min 3 20% piperidine/DMF 1 × 5 min and 1 × 15 min 4 DMF, wash 5 × 1 min 5 3.5 eq. Fmoc amino acid/DMF +  1 × 60 min 3.5 eq. PyBOP + 7 eq. DIPEA 6 3.5 eq. Fmoc amino acid/DMF +  1 × 60 min 3.5 eq. HATU or PyBOP or HCTU + 7 eq. DIPEA 7 DMF, wash 5 × 1 min 8 20% piperidine/DMF 1 × 5 min and 1 × 15 min 9 DMF, wash 5 × 1 min 10 CH.sub.2Cl.sub.2, wash (at the end of the 3 × 1 min synthesis)

(10) Steps 5 to 9 are repeated to add each amino-acid residue.

(11) After the synthesis of the fully protected peptide fragment had been terminated, the cleavage, cyclization and work up procedures, as described herein below, were used for the preparation of the final compounds.

(12) Cleavage, Backbone Cyclization and Deprotection

(13) After assembly of the linear peptide, the resin was suspended in 1 ml of 1% TFA in CH.sub.2Cl.sub.2 (v/v; 0.14 mmol) for 3 minutes and filtered, and the filtrate was neutralized with 1 ml of 20% DIPEA in CH.sub.2Cl.sub.2 (v/v; 1.15 mmol). This procedure was repeated four times to ensure completion of the cleavage. The resin was washed three times with 1 ml of CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2 layers containing product were evaporated to dryness.

(14) The fully protected linear peptide was solubilised in 8 ml of dry DMF. Then 2 eq. of HATU and 2. eq. of HOAt in dry DMF (1-2 ml) and 4 eq. of DIPEA in dry DMF (1-2 ml) were added to the peptide, followed by stirring for ca. 16 h. The volatiles were removed by evaporation. The crude cyclic peptide was dissolved in 7 ml of CH.sub.2Cl.sub.2 and washed three times with 4.5 ml 10% acetonitrile in water (v/v). The CH.sub.2Cl.sub.2 layer was then evaporated to dryness.

(15) To fully deprotect the peptide, 7 ml of cleavage cocktail TFA/DODT/thioanisol/H.sub.2O (87.5:2.5:5:5) were added, and the mixture was kept for 2.5-4 h at room temperature until the reaction was completed. The reaction mixture was evaporated close to dryness and the peptide precipitated with 7 ml of cold Et.sub.2O. The precipitate was washed 3 times with 4 ml of cold Et.sub.2O.

(16) Purification Procedure (Preparative Reverse Phase LC-MS)

(17) Compounds were purified by reverse phase chromatography using a Phenomenex Gemini NX-C18 column, 30×100 mm, 5 μm (Cat No. 00D-4435-U0-AX) or a Waters XBridge C18 OBD column, 30×100 mm, 5 μm (Cat No. 186002982). Mobile phases used were: A: 0.1% TFA in Water/Acetonitrile 95/5 v/v B: 0.1% TFA in Acetonitrile

(18) Gradient slopes in the preparative runs were adapted each time based on analytical LC-MS analysis of the crude product. As an example, a typical run (purification of Ex. 11) was executed using the Phenomenex column with a flow rate of 35 ml/min running a gradient from 0-1 min 0% B, at 1.1 min 25% B to a final of 8 min 45% B (retention time: 5.96 min in this case). Detection: MS and UV @ 220 nm Fractions collected were evaporated using a Genevac HT4 evaporator or a Büchi system.

(19) Alternatively for larger amounts the following LC-purification system was used: Column: Waters XBridge C18 OBD column, 50×250 mm, 10 μm (Cat No. 186003900) Mobile phase A: 0.1% TFA in Water Mobile phase B: Acetonitrile Flow rate: 150 ml/min Detection: UV @ 220 nm

(20) After lyophilisation the products were obtained typically as white to off-white powders and analysed by HPLC-ESI-MS methods as described below. Analytical data after preparative HPLC purification are shown in Table 1.

(21) 1.2 Analytical Methods

(22) Analytical Method A:

(23) Analytical HPLC retention times (RT, in minutes) were determined using an Ascentis Express C18 column, 50×2.1 mm, 2.7 μm, with the following solvents A (H.sub.2O+0.1% TFA) and B (CH.sub.3CN+0.085% TFA) and the gradient: 0-0.05 min: 97% A, 3% B; 3.3 min: 15% A, 85% B; 3.32 min: 3% A, 97% B; 3.32-3.55 min: 3% A, 97% B; 3.57-3.7 min: 97% A, 3% B. Flow rate=1.6 ml/min at 55° C.

(24) Analytical Method B:

(25) Analytical HPLC retention times (RT, in minutes) were determined using an Ascentis Express C18 column, 50×3.0 mm, 2.7 μm, with the following solvents A (H.sub.2O+0.1% TFA) and B (CH.sub.3CN+0.085% TFA) and the gradient: 0-0.05 min: 97% A, 3% B; 4.95 min: 3% A, 97% B; 4.95-5.35 min: 3% A, 97% B; 5.37-5.4 min: 97% A, 3% B. Flow rate=1.3 ml/min at 55° C.

(26) Analytical Method C:

(27) Analytical HPLC retention times (RT, in minutes) were determined using an Gemini NX C18 column, 50×2.0 mm, 3.0 μm, with the following solvents A (H.sub.2O+0.1% TFA) and B (CH.sub.3CN+0.085% TFA) and the gradient: 0-0.1 min: 97% A, 3% B; 2.7 min: 3% A, 97% B; 2.7-3.0 min: 3% A, 97% B; 3.05-3.3 min: 97% A, 3% B. Flow rate=0.8 ml/min at 45° C.

(28) 1.3 Synthesis of Peptide Sequences

(29) Examples 1-13, 18-38 are shown in Table 1.

(30) The peptides were synthesized according the general method starting with the amino acid L-prolin, which was grafted to the resin (Fmoc-Pro-2-chlorotrityl resin). The linear peptides were synthesized on the solid support according to the procedure described above in the following sequence: Resin-Pro-T.sup.1-P.sup.12-P.sup.11-P.sup.10-P.sup.9-P.sup.8-P.sup.7-P.sup.6-P.sup.5-P.sup.4-P.sup.3-P.sup.2-P.sup.1. The products were cleaved from the resin, cyclized, deprotected, and finally purified by preparative reverse phase LC-MS as described above.

(31) After lyophilisation the products were obtained as white to off-white powders and characterised by HPLC-MS. For analytical data, see Ex. 1-13, 18-38 in Table 1.

(32) Example 14 is shown in Table 1.

(33) The peptide was synthesized according the general method starting with the amino acid D-proline, which was grafted to the resin (Fmoc-.sup.DPro-2-chlorotrityl resin). The linear peptide was synthesized on the solid support according to the procedure described above in the following sequence: Resin-.sup.DPro-Ser-Ser-tBuGly-Dab-Trp-Dab-Pip-Orn-Dab-Cha-His-Trp-Pro((3S)OH). The product was cleaved from the resin, cyclized, deprotected and finally purified by preparative reverse phase LC-MS as described above.

(34) After lyophilisation the product was obtained as a white to off-white powder and characterised by HPLC-MS. For analytical data, see Ex. 14 in Table 1.

(35) Example 15 is shown in Table 1.

(36) The peptide was synthesized according the general method starting with the amino acid (S)-2-amino-4-(tert-butoxycarbonylamino)butanoic acid, which was grafted to the resin (Fmoc-Dab(Boc)-2-chlorotrityl resin). The linear peptide was synthesized on the solid support according to the procedure described above in the following sequence: Resin-Dab-Cha-Trp-Trp-Pro((3S)OH)-.sup.DPro-Ser-Ser-tBuGly-Dab-Trp-Dab-.sup.DDab-Orn. The product was cleaved from the resin, cyclized, deprotected and finally purified by preparative reverse phase LC-MS as described above.

(37) After lyophilisation the product was obtained as a white to off-white powder and characterised by HPLC-MS. For analytical data, see Ex. 15 in Table 1.

(38) Example 16 is shown in Table 1.

(39) The peptide was synthesized according the general method starting with the amino acid (S)-2-amino-3-(4-methoxyphenyl)propanoic acid, which was grafted to the resin (Fmoc-Tyr(Me)-2-chlorotrityl resin). The linear peptide was synthesized on the solid support according to the procedure described above in the following sequence: Resin-Tyr(Me)-His-Trp-Pro-.sup.DPro-Ser-Ala-tBuGly-Hse-Trp-Dab-.sup.DDab-Orn-Dab. The product was cleaved from the resin, cyclized, deprotected and finally purified by preparative reverse phase LC-MS as described above.

(40) After lyophilisation the product was obtained as a white to off-white powder and characterised by HPLC-MS. For analytical data, see Ex. 16 in Table 1.

(41) Example 17 is shown in Table 1.

(42) The peptide was synthesized according the general method starting with the amino acid (2S,3S)-2-amino-3-hydroxybutanoic acid, which was grafted to the resin (Fmoc-alloThr-2-chlorotrityl resin). The linear peptide was synthesized on the solid support according to the procedure described above in the following sequence: Resin-alloThr-Ala-tBuGly-Dab-Trp-Dab-.sup.DDab-Orn-Dab-Cha-His-Leu-.sup.DPro-Pro. The product was cleaved from the resin, cyclized, deprotected and finally purified by preparative reverse phase LC-MS as described above.

(43) After lyophilisation the product was obtained as a white to off-white powder and characterised by HPLC-MS. For analytical data, see Ex. 17 in Table 1.

(44) 1.4 Sequence Data

(45) TABLE-US-00002 TABLE 1 Examples (Ex.) Pu- Analyt. RT rity Ex. P.sup.1 a) P.sup.2 a) P.sup.3 a) P.sup.4 a) P.sup.5 a) P.sup.6 a) P.sup.7 a) P.sup.8 a) P.sup.9 a) P.sup.10 a) P.sup.11 a) p.sup.12 a) T.sup.1 a) T.sup.2 a) Meth. MS .sup.b) [min] [%] 1 Trp His Tyr Dab Orn .sup.DDab Dab Trp Hse tBuGly Ala Ser .sup.DPro Pro B 827.3 1.81 87 2 Trp His Tyr Dab Orn .sup.DDab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro B 827.0 1.82 88 3 Trp His Cha Dab Orn .sup.DDab Dab Trp Dab Nva Ala Ser .sup.DPro Pro B 815.2 1.93 93 4 Trp His Cha Dab Orn .sup.DDab Dab Trp Dab tBuGly Val Ser .sup.DPro Pro A 558.0 1.33 82 5 Leu His Tyr Dab Orn .sup.DDab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro B 790.5 1.80 87 6 Leu His Cha Dab Orn Pip Dab Trp Dab tBuGly Alb Ser .sup.DPro Pro B 827.4 1.98 85 7 Val His Tyr Dab Orn .sup.DDab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro B 522.6 1.73 95 8 Nva His Tyr Dab Orn .sup.DDab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro B 783.5 1.72 89 9 Trp His Cha Dab Orn .sup.DDab Dab Trp Dab Chg Alb Ser .sup.DPro Pro A 576.4 1.33 68 10 Trp Trp Cha Dab Lys .sup.DDab Dab Trp Dab Chg Ala Ser .sup.DPro Pro C 578.1 1.60 94 11 Trp Trp Cha Dab Lys .sup.DDab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro C 569.4 1.57 99 12 Trp Trp tBuGly Dab Orn .sup.DDab Dab Trp Dab Cha Ala Ser .sup.DPro Pro C 847.0 1.57 92 13 Trp Trp Leu Dab Orn .sup.DDab Dab Trp Dab Val Ala Val .sup.DPro Pro A 550.6 1.53 88 14 Trp His Cha Dab Orn Pip Dab Trp Dab tBuGly Ser Ser .sup.DPro Pro B 851.0 1.90 90 ((3S)OH) 15 Trp Trp Cha Dab Orn .sup.DDab Dab Trp Dab tBuGly Ser Ser .sup.DPro Pro C 575.5 1.51 98 ((3S)OH) 16 Trp His Tyr(Me) Dab Orn .sup.DDab Dab Trp Hse tBuGly Ala Ser .sup.DPro Pro B 557.6 1.89 83 17 Leu His Cha Dab Orn .sup.DDab Dab Trp Dab tBuGly Ala alloThr .sup.DPro Pro A 528.9 1.35 89 18 Trp His Tyr Dab Orn Dab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro A 827.4 1.14 90 19 Trp His Cha Dab Orn Dab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro A 822.4 1.29 82 20 Ile His Tyr Dab Orn .sup.DDab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro B 790.8 1.8 85 21 Ile His Tyr Dab Orn .sup.DDab Dab Trp Dab Ile Ala Ser .sup.DPro Pro B 790.7 1.79 92 22 Leu His Tyr Dab Orn Dab Dab Trp Dab Chg Ala Ser .sup.DPro Pro B 803.7 1.79 90 23 Leu His Tyr Dab Orn .sup.DDab Dab Trp Dab Chg Ala Ser .sup.DPro Pro A 803.8 1.23 91 24 Leu His Trp Dab Orn .sup.DDab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro A 802 1.25 89 25 Leu His Tyr Dab Lys .sup.DDab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro A 797.9 1.17 87 26 Leu Trp Tyr Dab Orn .sup.DDab Dab Trp Dab Cha Ala Ser .sup.DPro Pro A 835.3 1.33 95 27 Leu Trp Trp Dab Orn .sup.DDab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro A 826.7 1.31 94 28 Leu Trp Tyr Dab Orn .sup.DDab Dab Trp Dab tBuGly Ala Ser .sup.DPro Pro A 815.3 1.2 95 29 Leu His Tyr Dab Orn .sup.DDab Dab Trp Dab Trp Ala Ser .sup.DPro Pro A 827 1.15 87 30 Leu His Tyr Dab Orn .sup.DDab Dab Trp Dab tBuGly Ser Ser .sup.DPro Pro A 798.5 1.18 82 31 Leu Trp Tyr Dab Lys .sup.DDab Dab Trp Dab Cha Ser Ser .sup.DPro Pro A 850.2 1.3 94 32 Leu Trp Tyr Dab Orn .sup.DDab Dab Trp Dab Cha Ser Ser .sup.DPro Pro A 843.2 1.32 94 33 Leu Tyr Tyr Dab Orn .sup.DDab Dab Trp Dab tBuGly Asn Ser .sup.DPro Pro A 825.3 1.21 88 34 Leu Trp tBuGly Dab Orn .sup.DDab Dab Trp Dab Chg Ala Ser .sup.DPro Pro A 803.2 1.26 94 35 Ile Trp tBuGly Dab Lys .sup.DDab Dab Trp Dab Cha Ala Ser .sup.DPro Pro A 817.3 1.32 90 36 Leu Trp tBuGly Dab Orn .sup.DDab Dab Trp Dab Chg Ser Ser .sup.DPro Pro A 811.3 1.27 95 37 Leu Trp Cha Dab Orn .sup.DDab Dab Trp Dab tBuGly Ser Ser .sup.DPro Pro A 818.3 1.35 89 38 Leu Tyr Tyr Dab Orn .sup.DDab Dab Trp Dab tBuGly Tyr Ser .sup.DPro Pro A 849.8 1.26 87 .sup.a) Abbreviations of amino acid see listing above .sup.b) MS: either [M + 2H].sup.2+ or [M + 3H].sup.3+.
2. Biological Methods
2.1. Preparation of the Peptides

(46) Lyophilized peptides were weighed on a Microbalance (Mettler MT5) and dissolved in sterile water to a final concentration of 1 mg/ml. Stock solutions were kept at +4° C., light protected.

(47) 2.2. Antimicrobial Activity of the Peptides

(48) The selective antimicrobial activities of the peptides were determined in 96-well plates (Greiner, polystyrene) by the standard NCCLS broth microdilution method (National Committee for Clinical Laboratory Standards 1993. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 3rd ed. Approved standard M7-A6; National Committee for Clinical laboratory standards, Wayne, Pa.) with slight modifications. Inocula of the microorganisms were diluted into Mueller-Hinton II (MH, cation adjusted) broth+0.02% BSA and compared with a 0.5 McFarland standard to give appr. 10.sup.6 colony forming units (CFU)/ml. Aliquots (50 μl) of inoculate were added to 50 μl of MH broth+0.02% BSA containing the peptide in serial two-fold dilutions. The following microorganisms were used to determine antibiotic selectivity of the peptides: Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883 and Acinetobacter baumannii DSM 30008. Antimicrobial activities of the peptides were expressed as the minimal inhibitory concentration (MIC) in μg/ml at which no visible growth was observed after 18-20 hours of incubation at 35° C.

(49) 2.3. Hemolysis

(50) The peptides were tested for their hemolytic activity against human red blood cells (hRBC). Fresh hRBC were washed three times with phosphate buffered saline (PBS) and centrifuged for 10 min at 2000×g. Compounds (100 μg/ml) were incubated with 20% hRBC (v/v) for 1 h at 37° C. and shaking at 300 rpm. The final erythrocyte concentration was approximately 0.9×10.sup.9 cells/ml. A value of 0% and 100% cell lyses, respectively, was determined by incubation of hRBC in the presence of PBS containing 0.001% acetic acid and 2.5% Triton X-100 in H.sub.2O, respectively. The samples were centrifuged, the supernatants were 8-fold diluted in PBS buffer and the optical densities (OD) were measured at 540 nm. The 100% lyses value (OD.sub.540H.sub.2O) gave an OD.sub.540 of approximately 0.5-1.0.

(51) Percent hemolysis was calculated as follows: (OD.sub.540peptide/OD.sub.540H.sub.2O)×100%.

(52) The results of the experiments described in 2.2-2.3 are indicated in Table 2 herein below.

(53) TABLE-US-00003 TABLE 2 Minimal inhibitory concentrations (MIC) in Mueller-Hinton broth II and hemolysis Escherichia Klebsiella Acinetobacter coli pneumoniae baumannii Hemolysis ATCC 25922 ATCC 13883 DSM 30008 at MIC MIC MIC 100 μg/ml Ex. [μg/ml] [μg/ml] [μg/ml] [%] 1 0.5 1 2 1 2 0.25 1 1 1 3 0.5 1 2 3 4 0.5 1 2 5 5 0.5 2 2 0 6 0.5 1 1 3 7 0.5 1 1 1 8 1 4 2 1 9 1 0.5 1 n.d. 10 2 1 4 1 11 1 1 2 1 12 1 0.5 2 3 13 2 1 2 2 14 0.25 0.5 1 4 15 1 1 4 0 16 0.5 2 n.d. 1 17 1 2 4 5 18 2 2 2 0 19 2 1 2 0 20 2 1 2 0 21 1 2 2 1 22 2 2 4 0 23 2 2 4 3 24 2 1 4 1 25 2 2 2 0 26 1 1 2 1 27 2 1 4 0 28 2 2 4 1 29 2 2 4 0 30 1 1 2 1 31 1 1 2 1 32 1 0.5 2 3 33 1 1 2 5 34 2 2 4 1 35 1 1 4 1 36 2 1 4 1 37 2 2 4 1 38 2 2 2 0 n.d.: not determined