Beta-hairpin peptidomimetics as selective elastase inhibitors

Abstract

-Hairpin peptidomimetics of the general formula cyclo(-Xaa.sup.1-Xaa.sup.2-Thr.sup.3-Xaa.sup.4-Ser.sup.5-Xaa.sup.6-Xaa.sup.7-Xaa.sup.8-Xaa.sup.9-Xaa.sup.10-Xaa.sup.11-Xaa.sup.12-Xaa.sup.13-) and pharmaceutically acceptable salts thereof, with Xaa.sup.1, Xaa.sup.2, Xaa.sup.4, Xaa.sup.6, Xaa.sup.7, Xaa.sup.8, Xaa.sup.9, Xaa.sup.10, Xaa.sup.11, Xaa.sup.12 and Xaa.sup.13 being amino acid residues of certain types which are defined in the description and the claims, have elastase inhibitory properties, especially against human neutrophil elastase, and can be used for preventing infections or diseases related to such infections in healthy individuals or for slowing infections in infected patients. The compounds of the invention can further be used where cancer, or immunological diseases, or pulmonary diseases, or cardiovascular diseases, or neurodegenerative diseases, or inflammation, or diseases related to inflammation, are mediated or resulting from elastase activity. These peptidomimetics can be manufactured by a process which is based on a mixed solid- and solution phase synthetic strategy.

Claims

1. A backbone cyclized peptidic compound, built up from 13 amino acid residues, of the general formula
cyclo(-Xaa.sup.1-Xaa.sup.2-Thr.sup.3-Xaa.sup.4-Ser.sup.5-Xaa.sup.6-Xaa.sup.7-Xaa.sup.8-Xaa.sup.9-Xaa.sup.10-Xaa.sup.11-Xaa.sup.12-Xaa.sup.13-)(I), and pharmaceutically acceptable salts thereof, wherein Xaa.sup.1 is OctGly; Dab(Phe); Arg; Dab(Octanoyl); or Glu(Phe); Xaa.sup.2 is Glu; Phe; Dap(Phe); Val; or hTyr; Xaa.sup.4 is Ala; or AllylGly; Xaa.sup.6 is Ile; Xaa.sup.7 is Pro; Nglu; or Nlys; Xaa.sup.8 is Pro; Nglu; Nlys; Pip; Azt; or Oic; Xaa.sup.9 is Gln; or H-.sup.3-HGln-OH; Xaa.sup.10 is Lys; H-.sup.3-HLys-OH; or H-.sup.4-DiHLys-OH; Xaa.sup.11 is hSer; hSer(Me); Thr; alloThr; hGln; Dap; Tyr; or H-.sup.4-DiHTyr-OH; H-.sup.4-DiHThr-OH; Ser; or Asn; Xaa.sup.12 is .sup.DPro; .sup.DAla; .sup.DVal; .sup.DTyr; .sup.DLys; or .sup.DSer; and Xaa.sup.13 is Pro; .sup.DPro; Oic; Ala; Tyr; Val; Lys; H-.sup.3-HPro-OH; .sup.DGlu; or Glu; with the proviso that Xaa.sup.1 is Dab(Phe); Dab(Octanoyl); or Glu(Phe); and/or Xaa.sup.7 is Nglu; or Nlys; and/or Xaa.sup.8 is Nglu; Nlys; Pip; Azt; or Oic; and/or Xaa.sup.9 is H-.sup.3-HGln-OH; and/or Xaa.sup.10 is H-.sup.3-HLys-OH; or H-.sup.4-DiHLys-OH; and/or Xaa.sup.11 is H-.sup.4-DiHTyr-OH; or H-.sup.4-DiHThr-OH; and/or Xaa.sup.12 is .sup.DAla; .sup.DVal; .sup.DTyr; .sup.DLys; or .sup.DSer; and/or Xaa.sup.13 is .sup.DPro; Oic; H-.sup.3-HPro-OH; or .sup.DGlu; and with the further proviso that if Xaa.sup.11 is Tyr, then Xaa.sup.1 is Dab(Phe); Arg; or Glu(Phe); and/or Xaa.sup.2 is Dap(Phe).

2. A compound according to claim 1 of the formula I wherein Xaa.sup.1 is OctGly; or Dab(Phe); Xaa.sup.2 is Glu; Xaa.sup.4 is Ala; Xaa.sup.6 is Ile; Xaa.sup.7 is Pro; Nglu; or Nlys; Xaa.sup.8 is Pro; Nglu; Nlys; Pip; or Azt; Xaa.sup.9 is Gln; or H-.sup.3-HGln-OH; Xaa.sup.10 is Lys; H-.sup.3-HLys-OH; or H-.sup.4-DiHLys-OH; Xaa.sup.11 is hSer; hSer(Me); Thr; alloThr; hGln; Dap; Tyr; or H-.sup.4-DiHTyr-OH; Xaa.sup.12 is .sup.DPro; .sup.DAla; .sup.DVal; .sup.DTyr; or .sup.DLys; and Xaa.sup.13 is Pro; Oic; Ala; Tyr; or Val; with the proviso that Xaa.sup.1 is Dab(Phe); and/or Xaa.sup.7 is Nglu; or Nlys; and/or Xaa.sup.8 is Nglu; Nlys; Pip; or Azt; and/or Xaa.sup.9 is H-.sup.3-HGln-OH; and/or Xaa.sup.10 is H-.sup.3-HLys-OH; or H-.sup.4-DiHLys-OH; and/or Xaa.sup.11 is hSer; hSer(Me); alloThr; hGln; Dap or H-.sup.4-DiHTyr-OH; and/or Xaa.sup.12 is .sup.DAla; .sup.DVal; .sup.DTyr; or .sup.DLys; and/or Xaa.sup.13 is Oic; and with the further proviso that if Xaa.sup.11 is Tyr, then Xaa.sup.1 is Dab(Phe).

3. A compound according to claim 1 of the formula I wherein Xaa.sup.1 is OctGly; Arg; Dab(Octanoyl); or Glu(Phe); Xaa.sup.2 is Glu; Phe; Dap(Phe); Val; or hTyr; Xaa.sup.4 is Ala; or AllylGly; Xaa.sup.6 is Ile; Xaa.sup.7 is Pro; Xaa.sup.8 is Pro; or Oic; Xaa.sup.9 is Gln; Xaa.sup.10 is Lys; Xaa.sup.11 is Thr; H-.sup.4-DiHThr-OH; Tyr; Xaa.sup.12 is .sup.DPro; .sup.DVal; .sup.DTyr; .sup.DLys; or .sup.DSer; and Xaa.sup.13 is Pro; .sup.DPro; Lys; Val; Tyr; H-.sup.3-HPro-OH; .sup.DGlu; or Glu; with the proviso that Xaa.sup.1 is Dab(Octanoyl); or Glu(Phe); and/or Xaa.sup.2 is Dap(Phe); and/or Xaa.sup.8 is Oic; and/or Xaa.sup.11 is H-.sup.4-DiHThr-OH; and/or Xaa.sup.12 is .sup.DVal; .sup.DTyr; .sup.DLys; or .sup.DSer; and/or Xaa.sup.13 is .sup.DPro; H-.sup.3-HPro-OH; or .sup.DGlu; and with the further proviso that if Xaa.sup.11 is Tyr, then Xaa.sup.1 is Arg; or Glu(Phe); and/or Xaa.sup.2 is Dap(Phe).

4. A compound which is selected from Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-hSer(Me)-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Dap-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-alloThr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-hSer-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-hGln-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DPro-Oic-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Nglu-Pro-Gln-Lys-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ille-Pro-Pro-Gln-Lys-[H-.sup.4-DiHTyr-OH]-.sup.DPro-Pro-); Cyclo(-Dab(Phe)-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Tyr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Oic-Gln-Lys-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-[H-.sup.4-DiHThr-OH]-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DPro-.sup.DPro-); Cyclo(-OctGly-Phe-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-Dap(Phe)-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Tyr-.sup.DPro-Pro-); Cyclo(-Dab(Octanoyl)-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DPro-Pro-); Cyclo(-Arg-Glu-Thr-Ala-Ser-Ile-Pro-Oic-Gln-Lys-Thr-.sup.DPro-Pro-); Cyclo(-Glu(Phe)-Glu-Thr-AllylGly-Ser-Ile-Pro-Pro-Gln-Lys-Tyr-.sup.DPro-Pro-); Cyclo(-Glu(Phe)-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Tyr-.sup.DPro-Pro-); Cyclo(-Glu(Phe)-Glu-Thr-AllylGly-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DAla-Ala-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DVal-Tyr-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DVal-Lys-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DTyr-Val-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DTyr-Tyr-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DTyr-Lys-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DLys-Val-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DLys-Tyr-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DLys-Lys-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DLys-Glu-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DSer-Val-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DSer-Tyr-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DSer-Lys-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Nlys-Pro-Gln-Lys-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Nglu-Gln-Lys-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Nlys-Gln-Lys-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-[H-.sup.3-HGln-OH]-Lys-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-[H-.sup.3-HLys-OH]-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-[H-.sup.4-DiHLys-OH]-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DPro-[H-.sup.3-HPro-OH]); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DPro-.sup.DGlu-); Cyclo(-Arg-Val-Thr-Ala-Ser-Ile-Pro-Oic-Gln-Lys-Thr-.sup.DPro-.sup.DPro-); Cyclo(-Arg-hTyr-Thr-Ala-Ser-Ile-Pro-Oic-Gln-Lys-Thr-.sup.DPro-.sup.DPro-); Cyclo(-Arg-hTyr-Thr-Ala-Ser-Ile-Pro-Oic-Gln-Lys-Thr-.sup.DPro-Glu-); Cyclo(-Arg-Val-Thr-Ala-Ser-Ile-Pro-Oic-Gln-Lys-Thr-.sup.DPro-Glu-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Pip-Gln-Lys-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-Glu-Thr-Ala-Ser-Ile-Pro-Azt-Gln-Lys-Thr-.sup.DPro-Pro-); Cyclo(-OctGly-hTyr-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Ser-.sup.DPro-Pro-); Cyclo(-OctGly-hTyr-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Asn-.sup.DPro-Pro-); Cyclo(-Arg-hTyr-Thr-Ala-Ser-Ile-Pro-Pro-Gln-Lys-Thr-.sup.DPro-Glu-).

5. A method for the treatment of lung cancer; breast cancer; psoriasis; alpha 1 antitrypsin deficiency; pulmonary emphysema; cystic fibrosis; chronic obstructive pulmonary disease; idiopathic pulmonary fibrosis; bronchiectasis; pulmonary hypertension; arterial pulmonary hypertension; cardiac hypertrophy; myocarditis; acute myocardial infarction; rheumatoid arthritis; osteoarthritis; atherosclerosis; multiple sclerosis; pancreatitis; allergic rhinitis; systemic inflammatory respiratory syndrome; inflammatory dermatoses; or inflammatory bowel disease, said method comprising administering the compound according to claim 1, in free form or in pharmaceutically acceptable salt form, or a composition comprising the compound in free form or in pharmaceutically acceptable salt form, and a pharmaceutically inert carrier.

6. A pharmaceutical composition comprising a compound or a mixture of compounds according to claim 1 of the formula I, in free form or in pharmaceutically acceptable salt form, and a pharmaceutically inert carrier.

7. A method for inhibiting activity of elastase for the treatment of infections or diseases related to said infections or cancer or immunological diseases or pulmonary diseases or cardiovascular diseases or neurodegenerative diseases or inflammation or diseases related to inflammation or immunological reactions, said method comprising administering the compound according to claim 1, in free form or in pharmaceutically acceptable salt form, or a composition comprising the compound in free form or in pharmaceutically acceptable salt form, and a pharmaceutically inert carrier.

8. A method of treating an infection or a disease or disorder associated with such an infection resulting from; or cancer mediated or resulting from; or immunological diseases mediated or resulting from; or pulmonary diseases mediated or resulting from; or cardiovascular diseases mediated or resulting from; or neurodegenerative diseases mediated or resulting from; or inflammation mediated or resulting from elastase activity; or where immunological reaction is mediated or resulting from elastase activity, comprising administering to a subject in need thereof a pharmaceutically acceptable amount of the compound according to claim 1, in free form or in pharmaceutically acceptable salt form, or a composition comprising the compound in free form or in pharmaceutically acceptable salt form, and a pharmaceutically inert carrier.

9. A process for the manufacture of a compound as defined in claim 1 of the formula I comprising the steps of (a) coupling an appropriately functionalized solid support with an appropriately N-protected derivative of that amino acid which in the desired end-product corresponds to Xaa.sup.n, wherein n is 13, 8, 7, 6, 5 or 4, 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 thus obtained; (c) coupling the product thus obtained with an appropriately N-protected derivative of that amino acid which in the desired end-product corresponds to Xaa.sup.n-1, 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 obtained in step (c); (e) effecting steps substantially corresponding to steps (c) and (d) using appropriately N-protected derivatives of amino acids which in the desired end-product are in positions n2 to 1, any functional group(s) which may be present in said N-protected amino acid derivatives being likewise appropriately protected; (f) if n is not 13, further effecting steps substantially corresponding to steps (c) and (d) using appropriately N-protected derivatives of amino acids which in the desired end-product are in positions 13 to n+1, any functional group(s) which may be present in said N-protected amino acid derivatives being likewise appropriately protected; (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 (j) 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 or into a different, pharmaceutically acceptable, salt.

10. The method according to claim 5, wherein the inflammatory bowel disease is Crohn's disease.

11. The pharmaceutical composition according to claim 6, said pharmaceutical composition being in a form suitable for inhalation, for oral, topical, transdermal, injection, buccal, transmucosal, rectal, pulmonary or inhalation administration.

12. The pharmaceutical composition according to claim 11, said pharmaceutical composition being in the form of a tablet, dragee, capsule, solution, liquid, gel, plaster, cream, ointment, syrup, slurry, suspension, powder or suppository.

Description

EXAMPLES

(1) 1. Peptide Synthesis

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

(3) 1 g (1.4 mMol) 2-chlorotritylchloride resin (1.4 mMol/g; 100-200 mesh, copoly(styrene-1% DVB) polymer matrix; Barlos et al. Tetrahedron Lett. 1989, 30, 3943-3946) was filled into a dried flask. The resin was suspended in CH.sub.2Cl.sub.2 (5 mL) and allowed to swell at room temperature under constant shaking for 30 min. A solution of 0.98 mMol (0.7 eq) of the first suitably protected amino acid residue (see below) in CH.sub.2Cl.sub.2 (5 mL) mixed with 960 l (4 eq) of diisopropylethylamine (DIEA) was added. After shaking the reaction mixture for 4 h at 25 C., the resin was filtered off and washed successively with CH.sub.2Cl.sub.2 (1), DMF (1) and CH.sub.2Cl.sub.2 (1). A solution of CH.sub.2Cl.sub.2/MeOH/DIEA (17/2/1, 10 mL) was added to the resin and the suspension was shaken for 30 min. After filtration the resin was washed in the following order with CH.sub.2Cl.sub.2 (1), DMF (1), CH.sub.2Cl.sub.2 (1), MeOH (1), CH.sub.2Cl.sub.2 (1), MeOH (1), CH.sub.2Cl.sub.2 (2), Et.sub.2O (2) and dried under vacuum for 6 hours.

(4) Loading was typically 0.6-0.7 mMol/g.

(5) The following preloaded resins were prepared:

(6) Fmoc-Ser(tBu)-O-2-chlorotrityl resin, Fmoc-Ala-O-2-chlorotrityl resin, Fmoc-Pro-O-2-chlorotrityl resin and Fmoc-Oic-O-2-chlorotrityl resin.

(7) The synthesis was carried out employing a Syro-peptide synthesizer (MultiSynTech) using 24-96 reaction vessels. In each vessel 0.04 mMol of the above resin was placed and the resin was swollen in CH.sub.2Cl.sub.2 and DMF for 15 min, respectively. The following reaction cycles were programmed and carried out:

(8) TABLE-US-00003 Step Reagent Time 1 DMF, wash 5 1 min 2 20% piperidine/DMF 1 5 min, 1 15 min 3 DMF, wash 5 1 min 4 3.6 eq Fmoc amino acid, 3.6 eq HOAt/ 1 40 min DMF + 3.6 eq DIC/DMF 5 DMF, wash 1 1 min 6 3.6 eq Fmoc amino acid, 3.6 eq HOAt/ 1 40 min DMF + 3.6 eq HATU + 7.2 eq DIPEA

(9) Unless indicated otherwise, the final coupling of an amino acid was followed by Fmoc deprotection by applying steps 1-3 of the above described reaction cycle.

(10) The appropriately protected amino acid building blocks are commercially available or can be synthesized as known in the art.

(11) Attachment of Carboxylic Acids or Amino Acids to Amino Group- or Carboxylic Group-Bearing Side Chains

(12) Procedure A

(13) Attachment of Carboxylic Acids or Amino Acids to Selectively Deprotected Linear Peptides on Resin:

(14) To remove alloc-protecting groups from amino functions or allyl-protecting groups from carboxy functions present in the resin bound peptide the latter (0.04 mMol) was swollen in freshly distilled CH.sub.2Cl.sub.2 for at least 15 min followed by adding 0.2 eq tetrakis(triphenyl-phosphine)palladium(0) (10 mM) in dry CH.sub.2Cl.sub.2 and 10 eq phenylsilane. After shaking the reaction mixture for 15 min at room temperature, the resin was filtered off and a fresh solution of reagents was added to repeat the procedure. Following subsequent washing of the resin with CH.sub.2Cl.sub.2, DMF and Et.sub.2O, the resin was swollen again in CH.sub.2Cl.sub.2 and the attachment of a carboxylic acid or appropriately protected amino acid was accomplished by subsequently adding a mixture of 3.6 eq of the desired acid and 3.6 eq HOAt dissolved in DMF and 3.6 eq DIC dissolved in DMF allowing the reaction mixture to stand for 1 h disrupted only by occasionally stirring. After filtration and washing of the resin three times with DMF, the coupling was completed by repeating the procedure with a fresh solution of a mixture of 3.6 eq of the same desired acid and 3.6 eq HOAt dissolved in DMF and a mixture of 3.6 eq HATU and 7.2 eq DIPEA in DMF.

(15) In case of amino group-bearing side chains the acids used to be coupled by the above described protocol were octanoic acid or N-Boc protected phenylalanine, in case of carboxy group-bearing side chains the acid coupled by the above described protocol was phenylalanine the carboxy group being protected by tBu.

(16) Cyclization and Work Up of Backbone Cyclized Peptides

(17) Cleavage of the Fully Protected Peptide Fragment

(18) After completion of the synthesis, the resin (0.04 mMol) was suspended in 1 mL (0.13 mMol, 3.4 eq) of 1% TFA in CH.sub.2Cl.sub.2 (v/v) for 3 minutes, filtered, and the filtrate was neutralized with 1 mL (0.58 mMol, 14.6 eq) of 10% DIEA in CH.sub.2Cl.sub.2 (v/v). This procedure was repeated three times to ensure completion of the cleavage. The filtrate was evaporated to dryness and a sample of the product was fully deprotected by using a cleavage mixture containing 95% trifluoroacetic acid (TFA), 2.5% water and 2.5% triisopropylsilane (TIS) to be analyzed by reverse phase-HPLC (C.sub.18 column) and ESI-MS to monitor the efficiency of the linear peptide synthesis.

(19) Cyclization of the Linear Peptide

(20) The fully protected linear peptide (0.04 mMol) was dissolved in DMF (4 Mol/mL). Then 30.4 mg (0.08 mMol, 2 eq) of HATU, 10.9 mg (0.08 mMol, 2 eq) of HOAt and 28 l (0.16 mMol, 4 eq) DIEA were added, and the mixture was vortexed at 25 C. for 16 hours and subsequently concentrated under high vacuum. The residue was partitioned between CH.sub.2Cl.sub.2 and H.sub.2O/CH.sub.3CN (90/10: v/v). The CH.sub.2Cl.sub.2 phase was evaporated to yield the fully protected cyclic peptide.

(21) Full Deprotection of the Cyclic Peptide

(22) The cyclic peptide obtained was dissolved in 3 mL of the cleavage mixture containing 82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 5% phenol and 2.5% ethanedithiole (EDT). The mixture was allowed to stand at 25 C. for 2.5 hours and thereafter concentrated under vacuum. After precipitation of the cyclic fully deprotected peptide in diethylether (Et.sub.2O) at 0 C. the solid was washed twice with Et.sub.2O and dried.

(23) After purification of the crude products via preparative HPLC the peptides were 20 lyophilized (white powders) and analysed by the following analytical methods:

Analytical Method A for Examples 1-17, 19, 39-49

(24) Analytical HPLC retention times (RT, in minutes) were determined using a Ascentis Express C18 column, 503.0 mm, (cod. 53811-U-Supelco) with the following solvents A (H.sub.2O+0.1% TFA) and B (CH.sub.3CN+0.01% TFA) and the gradient: 0-0.05 min: 97% A, 3% B; 4.95 min: 3% A, 97% B; 5.35 min: 3% A, 97% B; 5.40 min: 97% A, 3% B. Flow rate=1.3 mL/min; UV_Vis=220 nm.

Analytical Method B for Example 18

(25) Analytical HPLC retention times (RT, in minutes) were determined using a Ascentis Express C18 column, 503.0 mm, (cod. 53811-U-Supelco) with the following solvents A (H.sub.2O+0.1% TFA) and B (CH.sub.3CN+0.01% TFA) and the gradient: 0-0.05 min: 97% A, 3% B; 3.40 min: 33% A, 67% B; 3.45 min: 3% A, 97% B; 3.65 min: 3% A, 97% B; 3.70 min: 97% A, 3% B. Flow rate=1.3 mL/min; UV_Vis=220 nm.

Analytical Method C for Examples 20-38

(26) Analytical HPLC retention times (RT, in minutes) were determined using a Xselect CSH C18 XP column, 1003.0 mm, (cod. 186006107, Waters) with the following solvents A (H.sub.2O+0.1% TFA) and B (CH.sub.3CN+0.01% TFA) and the gradient: 0-0.05 min: 95% A, 5% B; 10.05 min: 3% A, 97% B; 12.05 min: 3% A, 97% B; 12.10 min: 95% A, 5% B. Flow rate=0.6 mL/min; UV_Vis=220 nm.

(27) Examples 1-13, 16, 20, 22, 25-33, 35-37, 43, 47, 48 are shown in Table 1. The peptides were synthesized as follows: Starting resin was Fmoc-Ser(tBu)-O-2-chlorotrityl resin, which was prepared as described above. To that resin Xaa.sup.4, finally at position 4, was grafted. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Ser.sup.5-Xaa.sup.4-Thr.sup.3-Xaa.sup.2-Xaa.sup.1-Xaa.sup.13-Xaa.sup.12-Xaa.sup.11-Xaa.sup.10-Xaa.sup.9-Xaa.sup.8-Xaa.sup.7-Xaa.sup.6.

(28) Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

(29) The HPLC-retention times and UV-purities, determined using the analytical methods as described above, are shown in Table 1.

(30) Example 14 is shown in Table 1. The peptide was synthesized as follows: Starting resin was Fmoc-Ser(tBu)-O-2-chlorotrityl resin, which was prepared as described above. To that resin Xaa.sup.4, finally at position 4, was grafted. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Ser.sup.5-Xaa.sup.4-Thr.sup.3-Dap.sup.2-Xaa.sup.1-Xaa.sup.13-Xaa.sup.12-Xaa.sup.11-Xaa.sup.10-Xaa.sup.9-Xaa.sup.8-Xaa.sup.7-Xaa.sup.6. Before the last Fmoc-deprotection procedure A was applied to attach phenylalanine to the side chain of Dap.sup.2. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

(31) The HPLC-retention times and UV-purities, determined using the analytical methods as described above, are shown in Table 1.

(32) Example 15 is shown in Table 1. The peptide was synthesized as follows: Starting resin was Fmoc-Ser(tBu)-O-2-chlorotrityl resin, which was prepared as described above. To that resin Xaa.sup.4, finally at position 4, was grafted. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Ser.sup.5-Xaa.sup.4-Thr.sup.3-Xaa.sup.2-Dab.sup.1-Xaa.sup.13-Xaa.sup.12-Xaa.sup.11-Xaa.sup.10-Xaa.sup.9-Xaa.sup.8-Xaa.sup.7-Xaa.sup.6. Before the last Fmoc-deprotection procedure A was applied to attach octanoic acid to the side chain of Dab.sup.1. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

(33) The HPLC-retention times and UV-purities, determined using the analytical methods as described above, are shown in Table 1.

(34) Examples 17-19 are shown in Table 1. The peptides were synthesized as follows: Starting resin was Fmoc-Ser(tBu)-O-2-chlorotrityl resin, which was prepared as described above. To that resin Xaa.sup.4, finally at position 4, was grafted. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Ser.sup.5-Xaa.sup.4-Thr.sup.3-Xaa.sup.2-Glu.sup.1-Xaa.sup.13-Xaa.sup.12-Xaa.sup.11-Xaa.sup.10-Xaa.sup.9-Xaa.sup.8-Xaa.sup.7-Xaa.sup.6. Before the last Fmoc-deprotection procedure A was applied to attach phenylalanine to the side chain of Glu.sup.1. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

(35) The HPLC-retention times and UV-purities, determined using the analytical methods as described above, are shown in Table 1.

(36) Examples 21, 23, 24 are shown in Table 1. The peptides were synthesized as follows: Starting resin was Fmoc-Ala-O-2-chlorotrityl resin, which was prepared as described above. To that resin Thr.sup.3, finally at position 3, was grafted. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Ala.sup.4-Thr.sup.3-Xaa.sup.2-Xaa.sup.1-Xaa.sup.13-Xaa.sup.12-Xaa.sup.11-Xaa.sup.10-Xaa.sup.9-Xaa.sup.8-Xaa.sup.7-Xaa.sup.6-Ser.sup.5. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

(37) The HPLC-retention times and UV-purities, determined using the analytical methods as described above, are shown in Table 1.

(38) Examples 34, 38, 45, 46 are shown in Table 1. The peptides were synthesized as follows: Starting resin was Fmoc-Pro-O-2-chlorotrityl resin, which was prepared as described above. To that resin Xaa.sup.12, finally at position 12, was grafted. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Pro.sup.13-Xaa.sup.12-Xaa.sup.11-Xaa.sup.10-Xaa.sup.9-Xaa.sup.8-Xaa.sup.7-Xaa.sup.6-Ser.sup.5-Xaa.sup.4-Thr.sup.3-Xaa.sup.2-Xaa.sup.1. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

(39) The HPLC-retention times and UV-purities, determined using the analytical methods as described above, are shown in Table 1.

(40) Examples 39, 40, 49 are shown in Table 1. The peptides were synthesized as follows: Starting resin was Fmoc-Pro-O-2-chlorotrityl resin, which was prepared as described above. To that resin Xaa.sup.7, finally at position 7, was grafted. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Pro.sup.8-Xaa.sup.7-Xaa.sup.6-Ser.sup.5-Xaa.sup.4-Thr.sup.3-Xaa.sup.2-Xaa.sup.1-Xaa.sup.13-Xaa.sup.12-Xaa.sup.11-Xaa.sup.10-Xaa.sup.9. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

(41) The HPLC-retention times and UV-purities, determined using the analytical methods as described above, are shown in Table 1.

(42) Examples 41, 42, 44 are shown in Table 1. The peptides were synthesized as follows: Starting resin was Fmoc-Oic-O-2-chlorotrityl resin, which was prepared as described above. To that resin Xaa.sup.7, finally at position 7, was grafted. The linear peptide was synthesized on solid support according to the procedure described above in the following sequence: Resin-Oic.sup.8-Xaa.sup.7-Xaa.sup.6-Ser.sup.5-Xaa.sup.4-Thr.sup.3-Xaa.sup.2-Xaa.sup.1-Xaa.sup.13-Xaa.sup.12-Xaa.sup.11-Xaa.sup.10-Xaa.sup.9. Following a final Fmoc deprotection as described above, the peptide was cleaved from the resin, cyclized, deprotected and purified as indicated above.

(43) The HPLC-retention times and UV-purities, determined using the analytical methods as described above, are shown in Table 1.

(44) TABLE-US-00004 TABLE 1 Examples Ex. Xaa.sup.1a) Xaa.sup.2a) Xaa.sup.3a) Xaa.sup.4a) Xaa.sup.5a) Xaa.sup.6a) Xaa.sup.7a) Xaa.sup.8a) Xaa.sup.9a) 1 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 2 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 3 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 4 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 5 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 6 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 7 OctGly Glu Thr Ala Ser Ile Nglu Pro Gln 8 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 9 Dab(Phe) Glu Thr Ala Ser Ile Pro Pro Gln 10 OctGly Glu Thr Ala Ser Ile Pro Oic Gln 11 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 12 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 13 OctGly Phe Thr Ala Ser Ile Pro Pro Gln 14 OctGly Dap(Phe) Thr Ala Ser Ile Pro Pro Gln 15 Dab(Oct).sup.c) Glu Thr Ala Ser Ile Pro Pro Gln 16 Arg Glu Thr Ala Ser Ile Pro Oic Gln 17 Glu(Phe) Glu Thr AllyGly Ser Ile Pro Pro Gln 18 Glu(Phe) Glu Thr Ala Ser Ile Pro Pro Gln 19 Glu(Phe) Glu Thr AllyGly Ser Ile Pro Pro Gln 20 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 21 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 22 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 23 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 24 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 25 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 26 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 27 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 28 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 29 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 30 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 31 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 32 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 33 OctGly Glu Thr Ala Ser Ile Nlys Pro Gln 34 OctGly Glu Thr Ala Ser Ile Pro Nglu Gln 35 OctGly Glu Thr Ala Ser Ile Pro Nlys Gln 36 OctGly Glu Thr Ala Ser Ile Pro Pro .sup.3-Gln.sup.c) 37 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 38 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 39 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 40 OctGly Glu Thr Ala Ser Ile Pro Pro Gln 41 Arg Val Thr Ala Ser Ile Pro Oic Gln 42 Arg hTyr Thr Ala Ser Ile Pro Oic Gln 43 Arg hTyr Thr Ala Ser Ile Pro Oic Gln 44 Arg Val Thr Ala Ser Ile Pro Oic Gln 45 OctGly Glu Thr Ala Ser Ile Pro Pip Gln 46 OctGly Glu Thr Ala Ser Ile Pro Azt Gln 47 OctGly hTyr Thr Ala Ser Ile Pro Pro Gln 48 OctGly hTyr Thr Ala Ser Ile Pro Pro Gln 49 Arg hTyr Thr Ala Ser Ile Pro Pro Gln Purity RT Ex. Xaa.sup.10a) Xaa.sup.11a) Xaa.sup.12a) Xaa.sup.13a) [%] MS.sup.b) [min] 1 Lys hSer(Me) .sup.DPro Pro 85 1430.8 2.53 2 Lys Dap .sup.DPro Pro 72 701.5 2.14 3 Lys alloThr .sup.DPro Pro 80 1416.8 2.35 4 Lys hSer .sup.DPro Pro 71 1416.8 2.37 5 Lys hGln .sup.DPro Pro 71 1457.8 2.29 6 Lys Thr .sup.DPro Oic 77 1470.8 2.73 7 Lys Thr .sup.DPro Pro 71 1448.8 2.41 8 Lys .sup.4-Tyr.sup.c) .sup.DPro Pro 85 1506.8 2.57 9 Lys Tyr .sup.DPro Pro 74 779.0 1.72 10 Lys Thr .sup.DPro Pro 77 1470.8 2.56 11 Lys .sup.4-Thr.sup.c) .sup.DPro Pro 70 1444.8 2.41 12 Lys Thr .sup.DPro .sup.DPro 88 1416.8 2.30 13 Lys Thr .sup.DPro Pro 88 718.0 2.64 14 Lys Tyr .sup.DPro Pro 75 1584.0 2.52 15 Lys Thr .sup.DPro Pro 71 1473.8 2.25 16 Lys Thr .sup.DPro Pro 83 1457.8 1.78 17 Lys Tyr .sup.DPro Pro 72 1612.8 2.21 18 Lys Tyr .sup.DPro Pro 85 1587.2 2.07.sup.d) 19 Lys Thr .sup.DPro Pro 77 1550.8 2.12 20 Lys Thr .sup.DAla Ala 95 1364.8 4.69.sup.e) 21 Lys Thr .sup.DVal Tyr 95 1484.7 5.17.sup.e) 22 Lys Thr .sup.DVal Lys 95 1450.0 4.26.sup.e) 23 Lys Thr .sup.DTyr Val 95 1484.6 5.17.sup.e) 24 Lys Thr .sup.DTyr Tyr 95 1548.8 4.96.sup.e) 25 Lys Thr .sup.DTyr Lys 84 1513.8 4.20.sup.e) 26 Lys Thr .sup.DLys Val 94 1450.0 4.33.sup.e) 27 Lys Thr .sup.DLys Tyr 95 1514.0 4.36.sup.e) 28 Lys Thr .sup.DLys Lys 95 1479.0 3.74.sup.e) 29 Lys Thr .sup.DLys Glu 83 1480.0 4.08.sup.e) 30 Lys Thr .sup.DSer Val 95 1408.7 4.83.sup.e) 31 Lys Thr .sup.DSer Tyr 91 1472.8 4.72.sup.e) 32 Lys Thr .sup.DSer Lys 95 1437.8 4.02.sup.e) 33 Lys Thr .sup.DPro Pro 67 724.4 4.79.sup.e) 34 Lys Thr .sup.DPro Pro 81 724.9 5.07.sup.e) 35 Lys Thr .sup.DPro Pro 88 724.4 4.69.sup.e) 36 Lys Thr .sup.DPro Pro 95 1430.7 5.19.sup.e) 37 .sup.3-Lys.sup.c) Thr .sup.DPro Pro 55 1430.7 5.03.sup.e) 38 .sup.4-Lys.sup.c) Thr .sup.DPro Pro 85 1444.7 5.13.sup.e) 39 Lys Thr .sup.DPro .sup.3-Pro.sup.c) 74 1432 2.22 40 Lys Thr .sup.DPro .sup.DGlu 95 1449.1 2.31 41 Lys Thr .sup.DPro Pro 87 1427.8 2.09 42 Lys Thr .sup.DPro Pro 92 1506.1 2.02 43 Lys Thr .sup.DPro Glu 70 1539.1 1.95 44 Lys Thr .sup.DPro Glu 70 1460.1 1.97 45 Lys Thr .sup.DPro Pro 74 1431.1 2.28 46 Lys Thr .sup.DPro Pro 80 702.2 2.49 47 Lys Ser .sup.DPro Pro 85 1451.0 2.55 48 Lys Asn .sup.DPro Pro 86 1479.0 2.46 49 Lys Thr .sup.DPro Glu 70 1484.0 1.76 .sup.a)Abbreviations of amino acid see listing above. .sup.b)MS: either [M + 1H].sup.1+ or [M + 2H].sup.2+. .sup.c).sup.4-Tyr = H-.sup.4-DiHTyr-OH; .sup.4-Thr = H-.sup.4-DiHThr-OH; Dab(Oct) = Dab(Octanoyl); .sup.3-Gln = H-.sup.3-HGln-OH; .sup.3-Lys = H-.sup.3-HLys-OH; .sup.4-Lys = H-.sup.4-DiHLys-OH; .sup.3-Pro = H-.sup.3-HPro-OH; .sup.d)Analytical method B .sup.e)Analytical method C

(45) 2. Biological Methods

(46) 2.1 Preparation of the Peptide Samples

(47) Lyophilized peptides were weighed on a Microbalance (Mettler MT5) and dissolved in DMSO to a final concentration of 10 mM. Stock solutions were kept at +4 C., light protected. The biological assays were carried out under assay conditions having less than 1% DMSO unlike indicated otherwise.

(48) 2.2 Inhibition of Human Neutrophil Elastase

(49) The ability of the peptides of the invention to inhibit the hydrolysis activity of human neutrophil elastase (Serva Electrophoresis, Germany) using the synthetic tetrapeptidic substrate MeOSuc-AAPV-pNA (Bachem, Switzerland) was determined as follows:

(50) The above substrate (0.3 mM) and human neutrophil elastase (10 nM) were incubated at 37 C. with serial dilutions of the peptides (1% DMSO final) in assay buffer (50 mM Tris, pH 8, 300 mM NaCl, 0.01% Tween20). The release of pNA was followed by monitoring the change in absorbance at 405 nm for 30 minutes. Control assays with the same assay set-up as above, but without peptide, ran linearly. The dose-response data were fitted to the 4-parameter Hill equation providing the IC.sub.50 value using Graphpad (Prism 5).

(51) 2.3 Inhibition of Porcine Pancreatic Elastase

(52) The ability of the peptides of the invention to inhibit the hydrolysis activity of porcine pancreatic elastase (Sigma, USA) using the synthetic tripeptidic substrate MeOSuc-AAA-pNA (Bachem, Switzerland) was determined as follows:

(53) The above substrate (1 mM) and human porcine pancreatic elastase (15 nM) were incubated at 37 C. with serial dilutions of the peptides (0.5% DMSO final) in assay buffer (50 mM Tris, pH8, 100 mM NaCl, 0.01% Tween20). The release of pNA was followed by monitoring the change in absorbance at 405 nm for 30 minutes. Control assays with the same assay set-up as above, but without peptide, ran linearly. The dose-response data were fitted to the 4-parameter Hill equation providing the IC.sub.50 value using Graphpad (Prism 5).

(54) 2.4 Inhibition of Human Proteinase 3

(55) The inactivation of human proteinase 3 (Elastin Products Company, USA) by the peptides of the invention using synthetic tripeptidic substrate Boc-Ala-Ala-Nva-SBzl (Elastin Products Company, USA) was determined as follows:

(56) The above substrate (1 mM), 4,4-dithiodipyridine (250 M) and human proteinase 3 (10 nM) were incubated at 37 C. with serial dilutions of the peptides (0.5% DMSO final) in assay buffer (50 mM Tris, pH7.4, 150 mM NaCl, 0.01% Tween20). The reaction process was followed by monitoring the change in absorbance at 340 nm for 30 minutes. Control assays with the same assay set-up as above, but without peptide, ran linearly. The dose-response data were fitted to the 4-parameter Hill equation providing the IC.sub.50 value using Graphpad (Prism 5).

(57) 3. Results

(58) The results of the experiments described under 2.2-2.4, above, are indicated in Table 2 herein below.

(59) TABLE-US-00005 TABLE 2 Human Porcine neutrophil pancreatic Human elastase hNE elastase PPE proteinase 3 hPr3 (hNE) IC.sub.50 (PPE) IC.sub.50 (hPr3) IC.sub.50 hNE/PPE hNE/hPr3 Ex. IC.sub.50 [nM] SD [nM] IC.sub.50 [M] SD [M] IC.sub.50 [M] SD [M] selectivity selectivity 1 4.8 1.2 0.62 0.10 0.72 0.44 129 150 2 5.5 0.7 1.20 0.44 3.54 1.07 218 644 3 6.8 1.1 1.42 0.03 1.01 0.41 209 149 4 8.6 2.8 1.35 0.01 1.02 0.13 157 119 5 9.9 2.1 1.59 0.33 2.35 0.80 161 237 6 12 4.9 3.85 0.63 4.46 3.07 321 372 7 13.3 3.7 3.92 0.88 4.06 1.27 295 305 8 19.8 9.6 1.81 0.08 3.33 0.47 91 168 9 17.3 1.0 3.72 0.34 12.3 3.5 215 711 10 7.3 0.4 36.2 1.8 >100 n.d. 4959 >13699 11 11.5 2.9 48.5 6.3 21.1 12.5 4217 1835 12 15.2 5.7 >100 n.d. >100 n.d. >6579 >6579 13 10.1 1.0 21.4 0.6 >100 n.d. 2119 >9901 14 15 9.5 5.5 1.1 26 0.7 367 1733 15 19.1 3.2 92.8 n.d. 45.2 19.9 4859 2366 16 15 8.1 >100 n.d. >100 n.d. >6667 >6667 17 15 1.3 31.2 0.1 60.2 13.3 2080 4013 18 15.9 5.4 74.1 12 46.2 23.2 4660 2906 19 33 5.7 >100 n.d. >100 n.d. >3030 >3030 20 13.3 9.3 28.3 0.1 8.9 5.4 2128 669 21 12.9 7.9 10.6 0.2 13.0 1.9 822 1008 22 12.4 11.6 60.1 4.5 70.4 23.5 4847 5677 23 6.0 3.7 10.3 2.1 10.3 3.3 1717 1717 24 11.6 3.9 12.9 0.3 7.7 4.7 1112 664 25 6.4 1.9 45.0 0.7 41.8 21.9 7031 6531 26 10.6 8.3 46.8 8.1 5.0 0.2 4415 472 27 10.1 n.d. 44.1 4.0 39.6 n.d. 4366 3921 28 12.0 4.6 >100 n.d. >100 n.d. >8333 >8333 29 11.8 7.9 12.8 1.6 62.6 3.7 1085 5305 30 12.0 10.1 51.2 11.2 14.3 10.1 4267 1192 31 22.9 1.0 52.1 7.1 32.8 19.4 2275 1432 32 22.3 8.5 90.0 4.1 >100 n.d. 4036 >4484 33 7.0 4.0 8.3 1.1 6.1 1.8 1186 871 34 30.3 15.1 15.5 0.3 5.2 1.7 512 172 35 7.7 7.3 1.6 0.8 1.5 0.4 208 195 36 16.2 6.4 11.1 0.9 5.9 0.9 685 364 37 41.5 32.5 8.8 0.5 15.4 2.1 212 371 38 17.2 10.1 9.2 0.6 9.2 3.1 535 535 39 2.9 0.1 87.9 13.6 71.5 26.0 30310 24655 40 1.3 0.2 >100 n.d. >100 n.d. >76923 >76923 41 7.9 2.8 >100 n.d. >100 n.d. >12658 >12658 42 4.8 3.1 >100 n.d. >100 n.d. >20833 >20833 43 2.7 1.9 >100 n.d. >100 n.d. >237037 >237037 44 9.6 4.9 >100 n.d. >100 n.d. >10417 >10417 45 7.1 1.9 2.3 0.5 0.9 0.4 324 127 46 7.1 5.5 1.9 0.1 2.2 0.1 268 310 47 1.4 0.2 0.5 0.3 1.0 0.2 357 714 48 2.7 1.0 0.4 0.3 1.1 0.7 148 407 49 15.3 6.4 38.1 1.6 9.6 7.1 2490 627 n.d. = not determined