ADRENOMEDULLIN-ANALOGUES FOR LONG-TERM STABILIZATION AND THEIR USE

Abstract

The invention relates to stabilized Adrenomedullin derivatives and use thereof. In particular, the invention relates to novel, biologically active, stabilized Adrenomedullin (ADM) compounds. The invention further relates to the compounds for use in a method for the treatment and/or prevention of diseases, especially of cardiovascular, edematous and/or inflammatory disorders, and to medicaments comprising the compounds for treatment and/or prevention of cardiovascular, edematous and/or inflammatory disorders.

Claims

1: A compound according to formula (I) ##STR00112## a physiologically acceptable salt, a solvate or a solvate of a salt thereof, wherein X.sup.1 is selected from the group consisting of *—(CH.sub.2).sub.m1—S—S—(CH.sub.2).sub.n1—.sup.#, wherein m1 is 0-6, n1 is 0-6, with the proviso that m1+n1=0-6; *—(CH.sub.2).sub.m2—S—(CH.sub.2).sub.n2—.sup.#, wherein m2 is 0-6, n2 is 0-6, with the proviso that m2+n2=0-6; *—(CH.sub.2).sub.m3—.sup.#, wherein m3 is 1-8; *—(CH.sub.2).sub.m4—(CH.sub.2═CH.sub.2)—(CH.sub.2).sub.n3—.sup.#, wherein m4 is 0-6, n3 is 0-6, with the proviso that m4+n3=0-6; *—(CH.sub.2).sub.m5—(CH—CH)—(CH.sub.2).sub.n4—.sup.#, wherein m5 is 0-6, and n4 is 0-6, with the proviso that m5+n4=0-6; *—(CH.sub.2).sub.m6—CO—NH—(CH.sub.2).sub.n5—.sup.#, wherein m6 is 0-4, and n5 is 0-4, with the proviso that m6+n5=0-6; .sup.#—(CH.sub.2).sub.m7—CO—NH—(CH.sub.2).sub.n6—*, wherein m7 is 0-4, and n6 is 0-4, with the proviso that m7+n6=0-6; .sup.#—(CH.sub.2).sub.m8—SO—(CH.sub.2).sub.n7—*, wherein m8 is 0-4, and n7 is 0-4, with the proviso that m8+n7=0-6; .sup.#—(CH.sub.2).sub.m9—SO.sub.2—(CH.sub.2).sub.n8—*, wherein m9 is 0-4, and n8 is 0-4, with the proviso that m9+n8=0-6; *-5-6 membered heteroaryl-.sup.#; *—(CH.sub.2).sub.m10—O—(CH.sub.2).sub.n9—.sup.#, wherein m10 is 0-6, n9 is 0-6, with the proviso that m10+n9=0-6; *—(CH.sub.2).sub.m18—NH—CO—CH.sub.2—NH—CO—(CH.sub.2).sub.n—.sup.#, wherein m18 is 0-3, and n5 is 0 or 1, with the proviso that m18+n5=0-3; #—(CH.sub.2).sub.m9—NH—CO—CH.sub.2—NH—CO—(CH.sub.2).sub.n6—*, wherein m19 is 0-3, and n6 is 0 or 1, with the proviso that m19+n6=0-3; *—(CH.sub.2).sub.m20—NH—CO—CH(CH.sub.3)—NH—CO—(CH.sub.2).sub.n7—.sup.#, wherein m20 is 0-3, and n7 is 0 or 1, with the proviso that m20+n7=0-3; #—(CH.sub.2).sub.m21—NH—CO—CH(CH.sub.3)—NH—CO—(CH.sub.2).sub.n8—*, wherein m21 is 0-3, and n8 is 0 or 1, with the proviso that m21+n8=0-3; *—(CH.sub.2).sub.m22—NH—CO—CH(CH.sub.2—C(CH.sub.3).sub.2)—NH—CO—(CH.sub.2).sub.n9—.sup.#, wherein m22 is 0-3, and n9 is 0 or 1, with the proviso that m22+n9=0-3; #—(CH.sub.2).sub.m23—NH—CO—CH(CH.sub.2—C(CH.sub.3).sub.2)—NH—CO—(CH.sub.2).sub.n10—*, wherein m23 is 0-3, and n10 is 0 or 1, with the proviso that m23+n10=0-3; *—(CH.sub.2).sub.m24—NH—CO—CH(CH(CH.sub.3)C.sub.2H.sub.5)—NH—CO—(CH.sub.2).sub.n11—.sup.#, wherein m24 is 0-3, and n11 is 0 or 1, with the proviso that m24+n11=0-3; #—(CH.sub.2).sub.m25—NH—CO—CH(CH(CH.sub.3)C.sub.2H)—NH—CO—(CH.sub.2).sub.n12—*, wherein m25 is 0-3, and n12 is 0 or 1, with the proviso that m25+n12=0-3; *—(CH.sub.2).sub.m26—NH—CO—CH(CH.sub.2(C.sub.6H.sub.5))—NH—CO—(CH.sub.2).sub.n13—.sup.#, wherein m26 is 0-3, and n13 is 0 or 1, with the proviso that m26+n13=0-3; #—(CH.sub.2).sub.m27—NH—CO—CH(CH.sub.2(C.sub.6H.sub.5))—NH—CO—(CH.sub.2).sub.n14—*, wherein m27 is 0-3, and n14 is 0 or 1, with the proviso that m27+n14=0-3; —(CH.sub.2).sub.m28—NH—CO—(CH.sub.2).sub.3—NH—CO—(CH.sub.2).sub.n15—.sup.#, wherein m28 is 0 or 1, and n15 is 0 or 1, with the proviso that m28+n15=0-1; #—(CH.sub.2).sub.m29—NH—CO—(CH.sub.2).sub.3—NH—CO—(CH.sub.2).sub.n16—*, wherein m29 is 0 or 1, and n16 is 0 or 1, with the proviso that m29+n16=0-1; *—(CH.sub.2).sub.m30—NH—CO—NH—(CH.sub.2).sub.n17—.sup.#, wherein m30 is 0-5, and n17 is 0-5, with the proviso that m30+n17=0-5; .sup.#—(CH.sub.2).sub.m31—NH—CO—NH—(CH.sub.2).sub.n15—*, wherein m31 is 0-5, and n18 is 0-5, with the proviso that m31+n18=0-5; *—(CH.sub.2).sub.m32—O—CO—NH—(CH.sub.2).sub.n19—.sup.#, wherein m32 is 0-5, and n19 is 0-5, with the proviso that m32+n19=0-5; .sup.#—(CH.sub.2).sub.m33—O—CO—NH—(CH.sub.2).sub.n20—*, wherein m33 is 0-5, and n20 is 0-5, with the proviso that m33+n20=0-5; *—(CH.sub.2).sub.m34—O—CO—O—(CH.sub.2).sub.n21—.sup.#, wherein m34 is 0-5, and n21 is 0-5, with the proviso that m34+n21=0-5; *—(CH.sub.2).sub.m35—NH—CO—(CH.sub.2).sub.n22—NH—(CH.sub.2).sub.p1—, wherein m35 is 0-4, n22 is 0-4, and p1 is 0-4, with the proviso that m35+n22+p1=0-4; and *—(CH.sub.2).sub.m36—NH—CO—(CH═CH)—CO—NH—(CH.sub.2).sub.n23—.sup.#, wherein m36 is 0-2, and n23 is 0-2, with the proviso that m36+n23=0-2; wherein * and .sup.# reflect where X.sup.1 is bound within the ring structure; and X.sup.2 is absent, is hydrogen, or is an amino acid or an amino acid sequence selected from the group consisting of G.sup.14, K.sup.14, F.sup.14, SEQ ID NO:1 [Y.sup.1RQSMNNFQGLRSF.sup.14], SEQ ID NO:2 [R.sup.2QSMNNFQGLRSF.sup.14], SEQ ID NO:3 [Q.sup.3SMNNFQGLRSF.sup.14], SEQ ID NO:4 [S.sup.4MNNFQGLRSF.sup.14], SEQ ID NO:5 [M.sup.5NNFQGLRSF.sup.14], SEQ ID NO:6 [N.sup.6NFQGLRSF.sup.14], SEQ ID NO:7 [N.sup.7FQGLRSF.sup.14], SEQ ID NO:8 [F.sup.8QGLRSF.sup.14], SEQ ID NO:9 [Q.sup.9GLRSF.sup.14], SEQ ID NO:10 [G.sup.10LRSF.sup.14], SEQ ID NO:11 [L.sup.11RSF.sup.14], SEQ ID NO:12 [R.sup.12SF.sup.14], and SEQ ID NO:13 [S.sup.13F.sup.14], wherein any one of the SEQ ID NO:1 to SEQ ID NO:13 is covalently linked between F.sup.14 of said sequences by an amide bond to the N-terminal G.sup.15 of the amino acid sequence of formula (I), wherein any amino acid of X.sup.2 can optionally be replaced by a natural or unnatural amino acid; wherein A is L-Alanine; R is L-Arginine; N is L-Asparagine; D is L-Aspartic acid; Q is L-Glutamine; G is L-Glycine; H is L-Histidine; I is L-Isoleucine; L is L-Leucine; K is L-Lysine; M is L-Methionine; F is L-Phenylalanine; P is L-Proline; S is L-Serine; T is L-Threonine; Y is L-Tyrosine; V is L-Valine; X.sup.3 is absent or is a heterologous moiety which is covalently linked to the N-terminus or to a functional group of the side chain of any amino acid of X.sup.2, to the N-terminus of G.sup.15 or to Z; Z is absent or is a cleavable linker covalently bound between the N terminus of any amino acid of X.sup.2 or of G.sup.15 and X.sup.3 or between a functional group of the side chain of any amino acid of X.sup.2 and X.sup.3 wherein if X.sup.3 is absent, then Z is also absent and X.sup.2 is hydrogen or is an amino acid or amino acid sequence as defined above for X.sup.2; wherein if X.sup.3 is a heterologous moiety, then X.sup.2 is absent or is an amino acid or amino acid sequence as defined above for X.sup.2; X.sup.4 is the amino sequence *[D.sup.35 K.sup.36 D.sup.37 K.sup.38 D.sup.39 N.sup.40 V.sup.41]#, wherein at least one amino acid of said sequence can optionally be replaced by a natural or unnatural amino acid and wherein * indicates the binding site to T.sup.34 and # indicates the binding site to A.sup.42, or X.sup.4 is a moiety according to formula (A), wherein * indicates the binding site to T.sup.34 and # indicates the binding site to A.sup.42 ##STR00113## wherein X.sup.6, X.sup.7, X.sup.8, X.sup.9 and X.sup.10 are independently from another absent or an amino acid selected from L-Alanine; L-Arginine; L-Asparagine; L-Aspartic acid; L-Glutamine; L-Glycine; L-Histidine; L-Isoleucine; L-Leucine; L-Lysine; L-Methionine; L-Phenylalanine; L-Proline; L-Serine; L-Threonine; L-Tyrosine; or L-Valine, wherein k1 is 1, 2, 3 or 4, wherein k2 is 0, 1, 2, 3, 4, 5, 6, 7 or 8, wherein k3 is 1, 2, 3 or 4, X.sup.5 is the amino sequence *[R.sup.44 S.sup.45 K.sup.46 I.sup.47 S.sup.48]#, wherein the sequence can optionally comprise at least one amino acid replaced by a natural or unnatural amino acid and wherein * indicates the binding site to P.sup.43 and # indicates the binding site to P.sup.49, or X.sup.5 is a moiety according to formula (B), wherein * and .sup.# reflect where X.sup.5 is bound within the amino acid chain and wherein * indicates the binding site of X.sup.5 to P.sup.43 and # indicates the binding site to P.sup.49, ##STR00114## wherein X.sup.11 is selected from the group consisting of *—(CH.sub.2).sub.p1—S—(CH.sub.2).sub.r1—.sup.#, wherein p1 is 0-6; r1 is 0-6 with the proviso that p1+r1=0-6; *—(CH.sub.2).sub.p2—O—(CH.sub.2).sub.r2—.sup.#, wherein p2 is 0-6; r2 is 0-6 with the proviso that p1+r2=0-6; *—(CH.sub.2).sub.p3—.sup.#, wherein p3 is 1-8; *—(CH.sub.2).sub.p4—CO—NH—(CH.sub.2).sub.r4—.sup.#, wherein p4 is 0-4, and r4 is 0-4, with the proviso that p4+r4=0-6; .sup.#—(CH.sub.2).sub.p5—CO—NH—(CH.sub.2).sub.r5—*, wherein p5 is 0-4, and r5 is 0-4, with the proviso that p5+r5=0-6; wherein * and .sup.# reflect where X.sup.11 is bound within the ring structure; wherein the numbering of amino acids in formula (I) refers to the corresponding human adrenomedullin (ADM) sequence; wherein if X.sup.3 is not a di-carboxylic acid, then at least X.sup.4 is a moiety according to formula (A) as defined above and/or X.sup.5 is a moiety according to formula (B) as defined above.

2: The compound according to formula (I) according to claim 1, wherein X.sup.1 is selected from *—(CH.sub.2).sub.m1—S—S—(CH.sub.2).sub.n1—.sup.#, wherein m1 is 0-6, n1 is 0-6, with the proviso that m1+n1=0-6; *—(CH.sub.2).sub.m2—S—(CH.sub.2).sub.n2—.sup.#, wherein m2 is 0-6, n2 is 0-6, with the proviso that m2+n2=0-6; *—(CH.sub.2).sub.m3—.sup.#, wherein m3 is 1-8; *—(CH.sub.2).sub.m6—CO—NH—(CH.sub.2).sub.n5—.sup.#, wherein m6 is 0-4, and n5 is 0-4, with the proviso that m6+n5=0-6; .sup.#—(CH.sub.2).sub.m7—CO—NH—(CH.sub.2).sub.n6—*, wherein m7 is 0-4, and n6 is 0-4, with the proviso that m7+n6=0-6; *—(CH.sub.2).sub.m10—O—(CH.sub.2).sub.n9—.sup.#, wherein m10 is 0-6, n9 is 0-6, with the proviso that m10+n9=0-6, wherein * and .sup.# reflect where X.sup.1 is bound within the ring structure.

3: The compound according to formula (I) according to claim 1, wherein X.sup.1 is *—(CH.sub.2).sub.m1—S—S—(CH.sub.2).sub.n1—.sup.#, wherein m1 is 0-6, n1 is 0-6 with the proviso that m1+n1=0-6, and wherein * and .sup.# reflect where X.sup.1 is bound within the ring structure, or wherein X.sup.1 is *—(CH.sub.2).sub.m6—CO—NH—(CH.sub.2).sub.n5—.sup.#, wherein m6 is 0-6, n5 is 0-6 with the proviso that m6+n5=0-6, and wherein * and .sup.# reflect where X.sup.1 is bound within the ring structure.

4: The compound according to formula (I) according to claim 1, wherein X.sup.2 is absent, is hydrogen, or is an amino acid.

5: The compound according to formula (I) according claim 1, wherein X.sup.2 is G.sup.14 or K.sup.14, which is covalently linked by an amide bond to the N-terminal G.sup.15 of the compound of formula (I).

6: The compound of formula (I) according to claim 1, wherein X.sup.3 is a heterologous moiety selected from the group consisting of a polymer, a Fc, a FcRn binding ligand, albumin and an albumin-binding ligand; or a physiologically acceptable salt, a solvate or a solvate of a salt thereof, or wherein X.sup.3 is a polymer and the polymer is selected from the group consisting of linear or branched C1-C100 carboxylic acids and carboxylic di-acids, preferably C4-C30 carboxylic acids and carboxylic di-acids, optionally substituted with halo, hydroxy, alkoxy, amino, alkylamino, dialky lamino, sulfate, or phosphate, and which may be saturated, or mono- or di-unsaturated, a PEG moiety, a PPG moiety, a PAS moiety and a HES moiety; or a physiologically acceptable salt, a solvate or a solvate of a salt thereof; or wherein X.sup.3 is a carboxylic di-acid, preferably a C14-C22 carboxylic di-acid, more preferably a C14-C18 carboxylic di-acid gr derivatives thereof, or wherein X.sup.3 is a moiety according to Formula (C) ##STR00115## wherein n is 1 to 15, and wherein X.sup.1, X.sup.2, X.sup.4 and X.sup.5 and are as defined according to claim 1 and wherein # indicates the binding site to Z, wherein, if Z is absent, #indicates the binding site to X2.

7: The compound according to formula (I) according to claim 1, wherein X.sup.4 is a moiety according to formula (A) ##STR00116## wherein X.sup.6, X.sup.7, X.sup.8, X.sup.9 and X.sup.10 are independently from another absent or an amino acid selected from L-Alanine; is L-Arginine; is L-Asparagine; L-Aspartic acid; L-Glutamine; L-Glycine; L-Histidine; L-Isoleucine; L-Leucine; L-Lysine; L-Methionine; L-Phenylalanine; L-Proline; L-Serine; L-Threonine; L-Tyrosine; or V is L-Valine, wherein k1 is 1 or 2; wherein k2 is 0, 1, 2, 3, or 4; wherein k3 is 1 or 2, and .sup.# reflect where X.sup.4 is bound within the amino acid chain, wherein * indicates the binding site to T.sup.34 and # indicates the binding site to A.sup.42; or wherein X.sup.4 is a moiety according to formula (A), wherein X.sup.6 is absent or selected from the group consisting of D, N and V; wherein X.sup.7 is absent or is selected from the group consisting of D, N and V; wherein X.sup.8 is absent or is selected from the group consisting of D, N and V; wherein X.sup.9 is absent or is selected from the group consisting of D, N and; wherein X.sup.10 is absent or is selected from the group consisting of D, N and V; wherein k1 is 1 or 2; wherein k2 is 0, 1, 2, 3, or 4; wherein k3 is 1 or 2, and .sup.# reflect where X.sup.4 is bound within the amino acid chain, wherein * indicates the binding site to T.sup.34 and # indicates the binding site to A.sup.42.

8: The compound according to formula (I) according to claim 1, wherein X.sup.5 is the moiety according to formula (B), ##STR00117## wherein * and .sup.# reflect where X.sup.5 is bound within the amino acid chain and wherein * indicates the binding site of X.sup.5 to P.sup.43 and # indicates the binding site to P.sup.49, wherein X.sup.11 is selected from the group consisting of *—(CH.sub.2).sub.p1—S—(CH.sub.2).sub.r1.sup.#, wherein p1 is 0-4 and r1 is 0 or 1; .sup.#—(CH.sub.2).sub.p2—S—(CH.sub.2).sub.r2*, wherein p2 is 0-4 and r2 is 0 or 1; *—(CH.sub.2).sub.p3—.sup.#, wherein p3 is 1-4; *—(CH.sub.2).sub.p4—CO—NH—(CH.sub.2).sub.r4—.sup.#, wherein p4 is 0, 1, 2 or 3 or, and r4 is 0, 1, 2 or 3, with the proviso that p4+r4=0-4; .sup.#—(CH.sub.2).sub.p5—CO—NH—(CH.sub.2).sub.r5—*, wherein p5 is 0, 1, 2 or 3, and r5 is 0, 1, 2 or 3, with the proviso that p5+r5=0-4; wherein * and .sup.# reflect where X.sup.11 is bound within the ring structure.

9: The compound according to formula (I) according claim 1, wherein the compound is a compound according to formula (Ia) ##STR00118## wherein X.sup.1, X.sup.2, X.sup.3, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, k1, k2, and k3 are defined according to claim 1; a compound according to formula (Ib) ##STR00119## wherein X.sup.1, X.sup.2, X.sup.3 and X.sup.11 are defined according to claim 1; a compound according to formula (Ic) ##STR00120## wherein X.sup.1, X.sup.2, X.sup.3, Z, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, k1, k2, k3 and X.sup.11 are defined according to claim 1; a compound according to formula (Id) ##STR00121## wherein X.sup.3 is a di-carboxylic acid, and X.sup.1, X.sup.2, Z, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, k1, k2, k3 and X.sup.11 are defined according claim 1; a compound according to formula (Ie) according to claim 1, ##STR00122## wherein n is 1 to 30 and X.sup.1, X.sup.2, Z are defined according to claim 1, a compound according to formula (If) ##STR00123## wherein n is 1 to 30 and X.sup.1, X.sup.2, Z, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, k1, k2, k3 are defined according to claim 1; a compound according to formula (Ig) ##STR00124## wherein n is 1 to 30 and X.sup.1, X.sup.2, Z and X.sup.11 are defined according to claim 1; a compound according to formula (Ih) ##STR00125## wherein n is 1 to 30 and X.sup.1, X.sup.2, Z, X.sup.6, X.sup.7, X.sup.8, X.sup.9, X.sup.10, k1, k2, k3 and X.sup.11 are defined according to claim 1.

10: The compound of formulae (I), (Ia), (Ib), (Ic), (Id), (le), (If), (Ig) and/or (Ih) according to claim 1, wherein the compound is selected from ##STR00126## ##STR00127## ##STR00128## ##STR00129##

11: A method for the treatment and/or prevention of a cardiovascular, edematous and/or inflammatory disorder comprising administering the compound of claim 1 to a subject with the disorder.

12. A method for the treatment and/or prevention of a condition or disorder selected from heart failure, chronic heart failure, worsening heart failure, acute heart failure, acute decompensated heart failure, diastolic and systolic (congestive) heart failure, coronary heart disease, ischemic and/or hemorrhagic stroke, hypertension, pulmonary hypertension, peripheral arterial occlusive disease, pre-eclampsia, chronic obstructive pulmonary disease, asthma, acute and/or chronic pulmonary edema, allergic alveolitis and/or pneumonitis due to inhaled organic dust and particles of fungal, actinomycetic or other origin, and/or acute chemical bronchitis, acute and/or chronic chemical pulmonary edema, neurogenic pulmonary edema, acute and/or chronic pulmonary manifestations due to radiation, acute and/or chronic interstitial lung disorders, acute lung injury/acute respiratory distress syndrome (ALI/ARDS) in adult or child including newborn, ALI/ARDS secondary to pneumonia and sepsis, aspiration pneumonia and ALI/ARDS secondary to aspiration, ALI/ARDS secondary to smoke gas inhalation, transfusion-related acute lung injury (TRALI), ALI/ARDS and/or acute pulmonary insufficiency following surgery, trauma and/or burns, and/or ventilator induced lung injury (VILI), lung injury following meconium aspiration, pulmonary fibrosis, mountain sickness, chronic kidney diseases, glomerulonephritis, acute kidney injury, cardiorenal syndrome, lymphedema, inflammatory bowel disease, sepsis, septic shock, systemic inflammatory response syndrome (SIRS) of non-infectious origin, anaphylactic shock, inflammatory bowel disease, urticaria and/or edematous ocular disorders or ocular disorders associated with disturbed vascular function, including, age-related macular degeneration (AMD), diabetic retinopathy, in particular diabetic macula edema (DME), subretinal edema, and intraretinal edema comprising administering the compound of claim 1 to a subject with the condition or disorder.

13: A medicament comprising a compound of claim 1 in combination with an inert nontoxic pharmaceutically suitable excipient and/or in combination with a further active ingredient selected from the group consisting of ACE inhibitors, angiotensin receptor antagonists, beta-2 receptor agonists, phosphodiesterase (PDE) inhibitors, glucocorticoid receptor agonists, diuretics, recombinant angiotensin converting enzyme-2, acetylsalicylic acid, natriuretic peptides and derivatives thereof, and neprilysin inhibitors.

14: The medicament as claimed in claim 13 formulated for the treatment and/or prevention of cardiovascular, edematous and/or inflammatory disorders.

15: A method for the treatment and/or prophylaxis of cardiovascular, edematous and/or inflammatory disorders in humans or animals using an effective amount of the medicament of claim 13.

Description

EXPLANATION OF THE FIGURES

[1198] FIG. 1 Stability of ADM analogues in human blood plasma.

[1199] FIG. 2A 24 hour profiles of mean arterial blood pressure (MABP) recorded from telemeterd normotensive female Wistar rats after subcutaneous administration of 100 μg/kg of example 13 (compound 13) (filled squares, solid line) or vehicle (open circles, dotted line). Data points were plotted as means of averaged 30 min intervals from 11 control animals and 5 treated animals, respectively.

[1200] FIG. 2B 24 hour profiles of mean arterial blood pressure (MABP) recorded from telemeterd normotensive female Wistar rats after subcutaneous administration of 100 μg/kg of example 16 (compound 16) (filled squares, solid line) or vehicle (open circles, dotted line). Data points were plotted as means±SEM of averaged 30 min intervals from 6 control animals and 4 treated animals, respectively.

[1201] FIG. 2C 24 hour profiles of mean arterial blood pressure (MABP) recorded from telemeterd normotensive female Wistar rats after subcutaneous administration of 100 μg/kg of example 17 (compound 17) filled squares, solid line) or vehicle (open circles, dotted line). Data points were plotted as means±SEM of averaged 30 min intervals from 6 control animals and 4 treated animals, respectively.

[1202] FIG. 3A depicts the general formula (Ia).

[1203] FIG. 3B depicts the general formula (Ib).

[1204] FIG. 3C depicts the general formula (Ic).

[1205] FIG. 3D depicts the general formula (Id).

[1206] FIG. 3E depicts the general formula (Ie).

[1207] FIG. 3F depicts the general formula (If).

[1208] FIG. 3G depicts the general formula (Ig).

[1209] FIG. 3H depicts the general formula (Ihi).

[1210] Methods

[1211] A. Synthesis of Adrenomedullin Analogues

[1212] Abbreviations

TABLE-US-00002 AA amino acid ACN acetonitrile AcOH acetic acid ADM Adrenomedullin (human) approx. approximately Boc tert-butyloxycarbonyl DCM dichloromethane Dde N-γ-(4,4-dimethy1-2,6-dioxocyclohex-1-ylidene)ethyl DIC N,N′-diisopropylcarbodiimide DIPEA N,N-diisopropyldiethylamine DMF N,N-dimethylformamide Dpr L-diaminopropionic acid EDT ethane-1,2-dithiol equiv. equivalent(s) ESI electrospray ionization (in MS) Fmoc N-[(9H-fluoren-9-ylmethoxy)carbonyl HATU O-(7-Azabenzotriazol-1-y1)-N,N,N′,N′-tetramethyluronium- hexafluorphosphat HCl hydrochloric acid HOBt 1-hydroxybenzotriazole HPLC high pressure, high performance liquid chromatography MALDI- matrix-assisted laser desorption/ionization-time ToF of flight (in MS) Mmt methoxytrityl MS mass spectrometry NaCl sodium chloride NaOH sodium hydroxyide ODD octadecanedioic acid OEG oligoethylene glycol OPp 2-phenylisopropyl Oxyma ethyl 2-cyano-2-(hydroxyimino)acetate Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl RP reversed phase (in HPLC) TA thioanisole TAM/6- 6-carboxytetramethylrhodamine TAMRA TBS tris buffered saline tBu tert-butyl TFA trifluoroacetic acid TIS triisopropylsilane Trt trityl

[1213] Nomenclature of amino acids and peptide sequences is according to:

[1214] International Union of Pure and Applied Chemistry and International Union of Biochemistry: Nomenclature and Symbolism for Amino Acids and Peptides (Recommendations 1983). In: Pure & Appl. Chem. 56, Vol. 5, 1984, p. 595-624

TABLE-US-00003 Trivial Name Symbol One-letter Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[1215] Suppliers

TABLE-US-00004 Acetic acid Grüssing ACN Biosolve Boc-Gly-OH Iris Bitotech Boc-Lys(Fmoc)-OH Iris Bitotech DCM Biosolve DIC Iris Bitotech DIPEA Roth DMF Biosolve EDT Sigma-Aldirch Fmoc-Ala-OH OPC Orpegen Fmoc-Arg(Pbf)-OH OPC Orpegen Fmoc-Asn(Trt)-OH OPC Orpegen Fmoc-Asp(OPp)-OH Merck Fmoc-Asp(tBu)-OH OPC Orpegen Fmoc-Cys(Trt)-OH OPC Orpegen Fmoc-Dpr(Mtt)-OH Iris Bitotech Fmoc-Gln(Trt)-OH OPC Orpegen Fmoc-Glu(OPp)-OH Merck Fmoc-Gly-OH OPC Orpegen Fmoc-His(Trt)-OH OPC Orpegen Fmoc-Ile-OH OPC Orpegen Fmoc-Leu-OH OPC Orpegen Fmoc-Lys(Boc)-OH OPC Orpegen Fmoc-Lys(Mmt)-OH Iris Bitotech Fmoc-Phe-OH OPC Orpegen Fmoc-Pro-OH OPC Orpegen Fmoc-Ser(tBu)-OH OPC Orpegen Fmoc-Thr(tBU)-OH OPC Orpegen Fmoc-Tyr(tBu)-OH OPC Orpegen Fmoc-Val-OH OPC Orpegen HOBt Merck NaOH Grüssing NovaSyn ® TGR R resin Novabiochem Octadecanedioic acid mono-tert-butyl ester Apollo Scientific Oxyma Iris Biotech TA Sigma-Aldric TFA Merck TIS Merck

Example/Compound List

[1216]

TABLE-US-00005 Example/ Compound Code Sequence 1 [G.sup.14, (K.sup.44, D.sup.48).sub.lac] H- ADM(14-52) GGC*RFGTC*TVQKLAHQIYQFTDKDKDNVAPK+SKID+PQGY- NH.sub.2 2 [G.sup.14, (D.sup.44, K.sup.48).sub.lac] H- ADM(l4-52) GGC*RFGTC*TVQKLAHQIYQFTDKDKDNVAPD+SKIK+PQGY- NH.sub.2 3 [G.sup.14, (K.sup.44, E.sup.48).sub.lac] H- ADM(14-52) GGC*RFGTC*TVQKLAHQIYQFTDKDKDNVAPK+SKIE+PQGY- NH.sub.2 4 [G.sup.14, (E.sup.44, K.sup.48).sub.lac] H- ADM(14-52) GGC*RFGTC*TVQKLAHQIYQFTDKDKDNVAPE+SKIK+PQGY- NH.sub.2 5 [K.sup.14(ODD)] ADM(14-52) H-K(ODD)- GC*RFGTC*TVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-NH.sub.2 6 [G.sup.14, OEG(12).sup.[35-38]] H-GGC*RFGTC*TVQKLAHQIYQFT-OEG(12)- ADM(14-52) DNVAPRSKISPQGY-NH.sub.2 7 [G.sup.14, OEG(13).sup.[35-38]] H-GGC*RFGTC*TVQKLAHQIYQFT-OEG(13)- ADM(14-52) DNVAPRSKISPQGY-NH.sub.2 8 [G.sup.14, OEG(16).sup.[35-39]] H-GGC*RFGTC*TVQKLAHQIYQFT-OEG(16)- ADM(14-52) NVAPRSKISPQGY-NH.sub.2 9 [G.sup.14, OEG(13).sup.[35-39]] H-GGC*RFGTC*TVQKLAHQIYQFTD-OEG(13)- ADM(14-52) NVAPRSKISPQGY-NH.sub.2 10 [G.sup.14, OEG(16).sup.[36-40]] H-GGC*RFGTC*TVQKLAHQIYQFTD-OEG(16)- ADM(14-52) VAPRSKISPQGY-NH.sub.2 11 [G.sup.14, OEG(19).sup.[35-40]] H-GGC*RFGTC*TVQKLAHQIYQFT-OEG(19)-VAPRSKISPQGY- ADM(14-52) NH.sub.2 12 [G.sup.14, OEG(19).sup.[36-41]] H-GGC*RFGTC*TVQKLAHQIYQFTD-OEG(19)-APRSKISPQGY- ADM(14-52) NH.sub.2 13 [K.sup.14(ODD), H-K(ODD)- (K.sup.44, D.sup.48).sub.lac] GC*RFGTC*TVQKLAHQIYQFTDKDKDNVAPK+SKID+PQGY- ADM(14-52) NH.sub.2 14 [K.sup.14(ODD), H-K(ODD)-GC*RFGTC*TVQKLAHQIYQFT-OEG(13)- OEG(13).sup.[35-38], DNVAPK+SKID+PQGY-NH.sub.2 (K.sup.44, D.sup.48).sub.lac,] ADM(14-52) 15 [K.sup.14(ODD), H-K(ODD)-G-Dpr+- (Dpr.sup.16, E.sup.21).sub.lac] RFGTE+TVQKLAHQIYQFTDKDKDNVAPRSKISPQGY-NH.sub.2 ADM(14-52) 16 [K.sup.14(ODD), H-K(ODD)-G-Dpr+.sup.1- (Dpr.sup.16, E.sup.21).sub.lac, RFGTE+.sup.1TVQKLAHQIYQFTDKDKDNVAPK+.sup.2SKID+.sup.2PQGY- (K.sup.44, D.sup.48).sub.lac] ADM(14-52) 17 [K.sup.14(ODD), H-K(ODD)-G-Dpr+.sup.1-RFGTE+.sup.1TVQKLAHQIYQFT-OEG(13)- (Dpr.sup.16, E.sup.21).sub.lac, DNVAPK+.sup.2SKID+.sup.2PQGY-NH.sub.2 OEG(13).sup.[35-38], (K.sup.44, D.sup.48).sub.lac] ADM(14-52) [1217] amino acids in brackets separated with commas ( . . . ).sub.lac indicate a lactam-bridge between the side chains of the corresponding amino acids; in sequence these lactam-bridged amino acids are marked with +; in case of two lactams, superscripted numbers reflect connected amino acids [1218] * indicates disulfide bond [1219] OEG(x).sup.[y-z] indicates that amino acids in pos y to z were replaced by a OEG-linker consisting of x atoms [1220] (ODD) indicates the attachment of octadecanedioic acid to the side chain of the corresponding amino acid [1221] Dpr is diamino propionic acid

[1222] Synthesis

[1223] All reactions and procedures were performed at room temperature if not indicated otherwise. After each coupling and deprotection step, the resins were repeatedly washed with DMF and DCM to remove excess of reagents.

[1224] General Method for Peptide Synthesis:

[1225] ADM analogues were synthesized stepwise on a NovaSyn®TGR R resin (Novabiochem) with an automated peptide synthesizer (SYRO I, MultiSynTech). The reaction vessels were loaded with 15 μmol NovaSyn®TGR R resin. Each amino acid and the reagents Oxyma and DIC were added in 8-fold molar excess (120 μmol). If not indicated otherwise, the amino acids were N-α-Fmoc-protected; the protecting groups indicated in brackets were used for side chain functionalities. All reactions were performed in DMF. Each coupling step was performed twice with a reaction time of 40 min. Cleavage of the Fmoc protecting group was achieved using 40% piperidine in DMF (v/v) for 3 min and 20% piperidine in DMF (v/v) for 10 min after each coupling step.

[1226] Lactam-Bridged Adrenomedullin-Analogues 1-4

[1227] Synthesis:

[1228] The syntheses of compounds 1-4 were performed using automated peptide synthesis as described in the general method. The amino acids used in coupling cycles 1-38 were N-α-Fmoc-protected, while Boc-Gly-OH was used as N-terminal amino acid in coupling cycle 39.

[1229] The coupling sequences were as follows:

TABLE-US-00006 AA of Coupling compound compound compound compound human Cycle 1 2 3 4 ADM 1. Tyr(tBu) 52 2. Gly 51 3. Gln(Trt) 50 4. Pro 49 5. Asp(OPp) Lys(Mmt) Glu(OPp) Lys(Mmt) 48 6. Ile 47 7. Lys(Boc) 46 8. Ser(tBu) 45 9. Lys(Mmt) Asp(OPp) Lys(Mmt) Glu(OPp) 44 10. Pro 43 11. Ala 42 12. Val 41 13. Asn(Trt) 40 14. Asp(tBu) 39 15. Lys(Boc) 38 16. Asp(tBu) 37 17. Lys(Boc) 36 18. Asp(tBu) 35 19. Thr(tBu) 34 20. Phe 33 21. Gln(Trt) 32 22. Tyr(tBu) 31 23. Ile 30 24. Gln(Trt) 29 25. His(Trt) 28 26. Ala 27 27. Leu 26 28. Lys(Boc) 25 29. Gln(Trt) 24 30. Val 23 31. Thr(tBu) 22 32. Cys(Trt) 21 33. Thr(tBu) 20 34. Gly 19 35. Phe 18 36. Arg(Pbf) 17 37. Cys(Trt) 16 38. Gly 15 39. Boc-Gly 14

[1230] For the simultaneous removal of Mmt/OPp protecting groups, the resins were treated with TFA/TIS/DCM (3:5:92, v/v/v) (15×2 min, 1 mL). Subsequently, the resins were washed with 2% DIPEA in DMF (v/v) for 10 min twice (1 mL).

[1231] Cyclization was performed using a 15-fold molar excess of HOBt and DIC in DMF as solvent for approx. 24 h.

[1232] Cleavage of the peptides from the resin and simultaneous side chain deprotection was achieved with TFA/TA/EDT (90:7:3, v/v/v) for approx. 3 h. The peptides were precipitated and washed with ice-cold diethyl ether and subsequently dried under reduced pressure.

[1233] Oxidation of the disulfide bond was achieved by solving the peptide in 10 m1 ACN/H.sub.2O/TBS (1:4:5, v/v/v), adjusting the pH to 7.6-7.8 (1 M NaOH) and subsequent shaking for 12 h. Upon completion of the oxidation, the pH was adjusted to 3-4 using 1 M HCl.

[1234] Purification of the crude peptide was performed using preparative RP-HPLC on an Aeris PEPTIDE 5 μm XB-C18 LC column (Phenomenex, 250 mm×21.2 mm, 5 m, 100 Å). A linear gradient of 10% to 60% eluent B in A over 40 min was applied (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in 15 ACN). The flow rate was 15 mL/min, UV detection was measured at λ=220 nm.

[1235] Analytics:

[1236] The identity of the peptides was confirmed via MALDI-MS (UltraflexIII, Bruker) and ESI-MS (HCT, Bruker). The purities were analyzed using analytical RP-HPLC.

[1237] Compound 1: [G.sup.14, (K.sup.44, D.sup.48).sub.lac]ADM(14-52)

##STR00093##

((4R,7S,13S,16S,19R)-19-(2-(2-aminoacetamido)acetamido)-13-benzyl-16-(3-guanidinopropyl)-7-((R)-1-hydroxyethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaazacycloicosane-4-carbonyl)-L-threonyl-ADM(23-43)-((2S,5S,8S,11S,18S)-5-(4-aminobutyl)-18-(λ.SUP.2.-azaneyl)-8-((S)-sec-butyl)-2-(hydroxymethyl)-3,6,9,13,19-pentaoxo-1,4,7,10,14-pentaazacyclononadecane-11-carbonyl)-L-prolyl-ADM(50-52)

[1238] Chemical Formula: C.sub.191H.sub.295N.sub.55O.sub.57S.sub.2

[1239] Exact Mass: 4335.13 Da

[1240] Molecular Weight: 4337.91 g/mol

[1241] Compound 1 was synthesized in a 15 μmol scale. The yield was 5.1 mg (8% f theory).

[1242] Compound 1 was analyzed via analytical RP-HPLC using a Kinetex® 5 μm C18 100 Å LC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 10% to 60% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=19.2 min, purity≥95%.

[1243] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 10% to 60% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=19.5 min, purity≥95%.

[1244] The observed mass was in accordance with the calculated mass.

[1245] ESI Ion-Trap: m/z=1085.3 [M+4H].sup.4+, 868.5 [M+5H].sup.5+, 723.9 [M+6H].sup.6+, 620.6 [M+7H].sup.7+.

[1246] MALDI-ToF: m/z=4336.1 [M+H].sup.+, 2168.5 [M+2H].sup.2+, 1446.7 [M+H].sup.3+.

[1247] Compound 2: [G.sup.14, (D.sup.44, K.sup.48).sub.lac]ADM(14-52)

##STR00094##

((4R,7S,13S,16S,19R)-19-(2-(2-aminoacetamido)acetamido)-13-benzyl-16-(3-guanidinopropyl)-7-((R)-1-hydroxyethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaazacycloicosane-4-carbonyl)-L-threonyl-ADM(23-43)-((3S,6S,9S,12S,20S)-6-(4-aminobutyl)-12-(λ.SUP.2.-azaneyl)-3-((S)-sec-butyl)-9-(hydroxymethyl)-2,5,8,11,14-pentaoxo-1,4,7,10,15-pentaazacycloicosane-20-carbonyl)-L-prolyl-ADM(50-52)

[1248] Chemical Formula: C.sub.191H.sub.295N.sub.55O.sub.57S.sub.2

[1249] Exact Mass: 4335.13 Da

[1250] Molecular Weight: 4337.91 g/mol

[1251] Compound 2 was synthesized in a 15 μmol scale. The yield was 3.9 mg (6% f theory).

[1252] Compound 2 was analyzed via analytical RP-HPLC using a Kinetex® 5 μm C.sub.18 100 Å LC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 10% to 60% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=19.3 min, purity≥95%.

[1253] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 10% to 60% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=19.6 min, purity≥95%.

[1254] The observed mass was in accordance with the calculated mass.

[1255] ESI Ion-Trap: m/z=1085.4 [M+4H].sup.4+, 868.5 [M+5H].sup.5+, 723.9 [M+6H].sup.6+, 620.6 [M+7H].sup.7+.

[1256] MALDI-ToF: m/z=4336.1 [M+H].sup.+, 2168.5 [M+2H].sup.2+, 1446.7 [M+H].sup.3+.

[1257] Compound 3: [G.sup.14, (K.sup.44, E.sup.48).sub.lac]ADM(14-52)

##STR00095##

((4R,7S,13S,16S,19R)-19-(2-(2-aminoacetamido)acetamido)-13-benzyl-16-(3-guanidinopropyl)-7-((R)-1-hydroxyethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaazacycloicosane-4-carbonyl)-L-threonyl-ADM(23-43)-((2S,5S,8S,11S,20S)-5-(4-aminobutyl)-20-(λ.SUP.2.-azaneyl)-8-((S)-sec-butyl)-2-(hydroxymethyl)-3,6,9,14,21-pentaoxo-1,4,7,10,15-pentaazacyclohenicosane-11-carbonyl)-L-prolyl-ADM(50-52)

[1258] Chemical Formula: C.sub.192H.sub.297N.sub.55O.sub.57S.sub.2

[1259] Exact Mass: 4349.15 Da

[1260] Molecular Weight: 4351.94 g/mol

[1261] Compound 3 was synthesized in a 15 μmol scale. The yield was 4.5 mg (7% f theory).

[1262] Compound 3 was analyzed via analytical RP-HPLC using a Kinetex® 5 μm C18 100 Å LC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 10% to 60% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=19.1 min, purity≥95%.

[1263] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 10% to 60% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=19.4 min, purity≥95%.

[1264] The observed mass was in accordance with the calculated mass.

[1265] ESI Ion-Trap: m/z=1088.9 [M+4H].sup.4+, 871.3 [M+5H].sup.5+, 726.2 [M+6H].sup.6+, 622.6 [M+7H].sup.7+.

[1266] MALDI-ToF: m/z=4350.1 [M+H].sup.+, 2175.5 [M+2H].sup.2+, 1451.3 [M+H].sup.3+.

[1267] Compound 4: [G.sup.14, (E.sup.44, K.sup.48).sub.lac]ADM(14-52)

##STR00096##

((4R,7S,13S,16S,19R)-19-(2-(2-aminoacetamido)acetamido)-13-benzyl-16-(3-guanidinopropyl)-7-((R)-1-hydroxyethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaazacycloicosane-4-carbonyl)-L-threonyl-ADM(23-43)-((2S,5S,8S,11S,20S)-5-(4-aminobutyl)-20-(λ.SUP.2.-azaneyl)-8-((S)-sec-butyl)-2-(hydroxymethyl)-3,6,9,17,21-pentaoxo-1,4,7,10,16-pentaazacyclohenicosane-11-carbonyl)-L-prolyl-ADM(50-52) Chemical Formula: C.SUB.192.H.SUB.297.N.SUB.55.O.SUB.57.S.SUB.2

[1268] Exact Mass: 4349.15 Da

[1269] Molecular Weight: 4351.94 g/mol

[1270] Compound 4 was synthesized in a 15 μmol scale. The yield was 6.0 mg (9% f theory).

[1271] Compound 4 was analyzed via analytical RP-HPLC using a Kinetex® 5 μm C18 100 Å LC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 10% to 60% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=19.2 min, purity≥95%.

[1272] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 10% to 60% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=19.4 min, purity≥95%.

[1273] The observed mass was in accordance with the calculated mass.

[1274] ESI Ion-Trap: m/z=1088.9 [M+4H].sup.4+, 871.3 [M+5H].sup.5+, 726.2 [M+6H].sup.6+, 622.6 [M+7H].sup.7+.

[1275] MALDI-ToF: m/z=4350.1 [M+H].sup.+, 2175.5 [M+2H].sup.2+, 1450.3 [M+H].sup.3+.

[1276] ODD-Modified Adrenomedullin Analogue 5

[1277] Synthesis:

[1278] The synthesis of compound 5 was performed using automated peptide synthesis of the sequence ADM(15-52) as described in the general method.

[1279] The coupling sequences were as follows:

TABLE-US-00007 Coupling Cycle compound 5 AA of human ADM 1. Tyr(tBu) 52 2. Gly 51 3. Gln(Trt) 50 4. Pro 49 5. Ser(tBu) 48 6. Ile 47 7. Lys(Boc) 46 8. Ser(tBu) 45 9. Arg(Pbf) 44 10. Pro 43 11. Ala 42 12. Val 41 13. Asn(Trt) 40 14. Asp(tBu) 39 15. Lys(Boc) 38 16. Asp(tBu) 37 17. Lys(Boc) 36 18. Asp(tBu) 35 19. Thr(tBu) 34 20. Phe 33 21. Gln(Trt) 32 22. Tyr(tBu) 31 23. Ile 30 24. Gln(Trt) 29 25. His(Trt) 28 26. Ala 27 27. Leu 26 28. Lys(Boc) 25 29. Gln(Trt) 24 30. Val 23 31. Thr(tBu) 22 32. Cys(Trt) 21 33. Thr(tBu) 20 34. Gly 19 35. Phe 18 36. Arg(Pbf) 17 37. Cys(Trt) 16 38. Gly 15

[1280] After automated synthesis of the sequence ADM(15-52), the N-terminal amino acid Boc-Lys(Fmoc)-OH was coupled manually with HOBt and DIC in 5-fold molar excess. The reaction was performed in DMF as solvent for 24 h.

[1281] Subsequently, the Fmoc protecting group was removed from the N-terminal amino acid using 20% piperidine in DMF (v/v) for 10 min twice.

[1282] Coupling of ODD to the free lysine side chain was achieved using a 5-fold excess (75 μmol) of octadecanedioic acid mono-tert-butyl ester, HOBt and DIC in 400 μDMF/DCM (3:1, v/v) as solvent for approx. 24 h.

[1283] Cleavage of the peptide from the resin and simultaneous side chain deprotection was achieved with TFA/TA/EDT (90:7:3, v/v/v) for approx. 3 h. The peptide was precipitated, washed with ice-cold diethyl ether and dried under reduced pressure.

[1284] Oxidation of the disulfide bond was achieved by solving the peptide in 10 m1 ACN/H.sub.2O/TBS (2:3:5, v/v/v), adjusting the pH to 7.6-7.8 (1 M NaOH) and subsequent shaking for 12 h. Upon completion of the oxidation, the pH was adjusted to 3-4 using 1 M HCl.

[1285] Purification of the crude peptide was performed using preparative RP-HPLC on an Aeris PEPTIDE 5 μm XB-C18 LC column (Phenomenex, 250 mm×21.2 mm, 5 m, 100 Å). A linear gradient of 10% to 60% eluent B in A over 40 min was applied (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN). The flow rate was 15 mL/min, UV detection was measured at λ=220 nm.

[1286] Analytics:

[1287] The identity of the peptides was confirmed via MALDI-MS (UltraflexIII, Bruker) and ESI-MS (HCT, Bruker). The purities were analyzed using analytical RP-HPLC.

[1288] Compound 5: [K.sup.14(ODD)]ADM(14-52)

##STR00097##

[1289] Chemical Formula: C.sub.212H.sub.335N.sub.58O.sub.60S.sub.2

[1290] Exact Mass: 4720.46 Da

[1291] Molecular Weight: 4723.50 g/mol

[1292] Compound 5 was synthesized in a 15 μmol scale. The yield was 3.8 mg (5% f theory).

[1293] Compound 5 was analyzed via analytical RP-HPLC using a Kinetex® 5 μm Biphenyl 100 Å LC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 20% to 70% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=14.3 min, purity≥95%.

[1294] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 20% to 70% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=17.8 min, purity≥95%.

[1295] The observed mass was in accordance with the calculated mass.

[1296] ESI Ion-Trap: m/z=1181.6 [M+4H].sup.4+, 945.5 [M+5H].sup.5+, 788.1 [M+6H].sup.6+, 675.6 [M+7H].sup.7+, 591.3 [M+8H].sup.8+.

[1297] MALDI-ToF: m/z=4721.47 [M+H].sup.+, 2361.09 [M+2H].sup.2+.

[1298] Adrenomedullin Analogues 6-12 Containing Different Length OEG-Linkers

[1299] For the synthesis of compounds 6-12, different-length oligo ethylene glycol linker blocks herein termed OEG-linker building blocks A, B, C and D (shown below) were used as replacements for four, five or six amino acids of ADM positions 35 to 41. Sometimes the term polyethylene glycole PEG is used also for very low repetition numbers, it is considered synonymous under these circumstances. The building blocks were N-α-Fmoc-protected; Here, nomenclature is defined as Fmoc-NH-OEG(x)-OH, where x describes the number of atoms in the linker upon incorporation.

[1300] Structures of Fmoc-NH-OEG(x)-OH building blocks:

##STR00098##

[1301] A: Fmoc-NH-OEG(12)-OH; CAS #: 139338-72-0; The compound was purchased from ChemPep Inc.

[1302] B: Fmoc-NH-OEG(13)-OH; CAS #: 867062-95-1; The compound was purchased from Iris Biochem.

[1303] C: Fmoc-NH-OEG(16)-OH; CAS #: 557756-85-1; The compound was purchased from Iris Biochem.

[1304] D: Fmoc-NH-OEG(19)-OH; CAS #: 882847-32-7; The compound was purchased from Iris Biochem.

[1305] Structures of —NH—OEG(x)-OH Building Blocks

##STR00099##

[1306] Synthesis:

[1307] The syntheses of compounds 6-12 were performed using automated peptide synthesis as described in the general method.

[1308] The coupling sequences were as follows:

TABLE-US-00008 AA of comp. comp. comp. comp. comp. comp. comp. human Cycle 6 7 8 11 9 10 23 ADM 1. Tyr(tBu) 52 2. Gly 51 3. Gln(Trt) 50 4. Pro 49 5. Ser(tBu) 48 6. Ile 47 7. Lys(Boc) 46 8. Ser(tBu) 45 9. Arg(Pbf) 44 10. Pro 43 11. Ala 42 12. Val — 41 13. Asn(Trt) — — — 40 14. Asp(tBu) — — — — — 39 After automated synthesis of the C-terminal sequences, Fmoc-NH-OEG(x)-OH building block A (6), B (7, 9), C (8, 10) or D (11, 12) was coupled manually with HOBt and DIC in 5-fold molar excess. The reaction was performed in DMF as solvent for 24 h.

[1309] Subsequently, the Fmoc protecting group was removed using 20% piperidine in DMF (v/v) for 10 min twice and Fmoc-Thr(tBu)-OH (6, 7, 8, 11) or Fmoc-Asp(tBu)-OH (9, 10, 12) was coupled manually with HOBt and DIC in 5-fold molar excess. The reaction was performed in DMF as solvent for 24 h.

[1310] Elongation of the peptide chain was performed using the general method for automated peptide synthesis described above. The elongation amino acids were N-α-Fmoc-protected, while Boc-Gly-OH was used as N-terminal amino acid.

[1311] The coupling sequences were as follows:

TABLE-US-00009 AA of comp. comp. comp. comp. comp. comp. comp. human Cycle 6 7 8 11 9 10 23 ADM 1 Phe Thr(tBu) 33/34 2 Gln(Trt) Phe 32/33 3 Tyr(tBu) Gln(Trt) 31/32 4 Ile Tyr(tBu) 30/31 5 Gln(Trt) Ile 29/30 6 Hi s (Trt) Gln(Trt) 28/29 7 Ala His(Trt) 27/28 8 Leu Ala 26/27 9 Lys(Boc) Leu 25/26 10 Gln(Trt) Lys(Boc) 24/25 11 Val Gln(Trt) 23/24 12 Thr(tBu) Val 22/23 13 Cys (Trt) Thr(tBu) 21/22 14 Thr(tBu) Cys(Trt) 20/21 15 Gly Thr(tBu) 19/20 16 Phe Gly 18/19 17 Arg(Pbf) Phe 17/18 18 Cys(Trt) Arg(Pbf) 16/17 19 Gly Cys(Trt) 15/16 20 Boc-Gly Gly 14/15 21 — Boc-Gly −/14

[1312] Cleavage of the peptides from the resin and simultaneous side chain deprotection was achieved with PGP-124T, TFA/TA/EDT (90:7:3, v/v/v) for approx. 3 h. The peptides were precipitated and washed with ice-cold diethyl ether and subsequently dried under reduced pressure.

[1313] Oxidation of the disulfide bond was achieved by solving the peptide in ACN/H.sub.2O/TBS (1:4:5, v/v/v), adjusting the pH to 7.6-7.8 (1 M NaOH) and subsequent shaking for 12 h. Upon completion of the oxidation, the pH was adjusted to 3-4 using 1 M HCl.

[1314] Purification of the crude peptide was performed using preparative RP-HPLC on an Aeris PEPTIDE 5 μm XB-C18 LC column (Phenomenex, 250 mm×21.2 mm, 5 m, 100 Å). A linear gradient of 10% to 50% eluent B in A over 30 min was applied (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN). The flow rate was 15 mL/min, UV detection was measured at λ=220 nm.

[1315] Analytics:

[1316] The identity of the peptides was confirmed by MALDI-MS (UltraflexIII, Bruker) and ESI-MS (HCT, Bruker). The purities were analyzed using analytical RP-HPLC.

[1317] Compound 6: [G.sup.14, OEG(12).sup.[35-38]]ADM(14-52)

##STR00100##

[1318] Chemical Formula: C.sub.178H.sub.278N.sub.52O.sub.53S.sub.2

[1319] Exact Mass: 4056.01 Da

[1320] Molecular Weight: 4058.61 g/mol

[1321] Compound 6 was synthesized in a 15 μmol scale. The yield was 1.8 mg (3% f theory).

[1322] Compound 6 was analyzed by analytical RP-HPLC using a Kinetex® 5 μm Biphenyl 100 Å LC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 10% to 60% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=15.6 min, purity≥95%.

[1323] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 10% to 60% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=18.7 min, purity≥95%.

[1324] The observed mass was in accordance with the calculated mass.

[1325] ESI Ion-Trap: m/z=1015.4 [M+4H].sup.4+, 812.6 [M+5H].sup.5+, 677.3 [M+6H].sup.6+.

[1326] MALDI-ToF: m/z=4057.0 [M+H].sup.+, 2029.0 [M+2H].sup.2+.

[1327] Compound 7: [G.sup.14, OEG(13).sup.[35-38]]ADM(14-52)

##STR00101##

[1328] Chemical Formula: C.sub.179H.sub.280N.sub.52O.sub.53S.sub.2

[1329] Exact Mass: 4070.03 Da

[1330] Molecular Weight: 4072.64 g/mol

[1331] Compound 7 was synthesized in a 7.5 μmol scale. The yield was 6.0 mg (20% f theory).

[1332] Compound 7 was analyzed by analytical RP-HPLC using a Kinetex® 5 μm Biphenyl 100 Å LC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=21.2 min, purity≥95%.

[1333] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in 15 water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=23.4 min, purity≥95%.

[1334] The observed mass was in accordance with the calculated mass.

[1335] ESI Ion-Trap: m/z=1018.8 [M+4H].sup.4+, 815.4 [M+5H].sup.5+, 679.6 [M+6H].sup.6+, 582.6 [M+7H].sup.7+.

[1336] MALDI-ToF: m/z=4071.0 [M+H].sup.+, 2036.0 [M+2H].sup.2+.

[1337] Compound 8: [G.sup.14, OEG(16).sup.[35-39]]ADM(14-52)

##STR00102##

((4R,7S,13S,16S,19R)-19-(2-(2-aminoacetamido)acetamido)-13-benzyl-16-(3-guanidinopropyl)-7-((R)-1-hydroxyethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaazacycloicosane-4-carbonyl)-L-threonyl-ADM(23-34)-(1-(λ.SUP.2.-azaneyl)-3,6,9,12-tetraoxapentadecan-15-oyl)-L-asparagyl-ADM(41-52)

[1338] Chemical Formula: C.sub.177H.sub.279N.sub.51O.sub.51S.sub.2

[1339] Exact Mass: 3999.02 Da

[1340] Molecular Weight: 4001.61 g/mol

[1341] Compound 8 was synthesized in a 7.5 μmol scale. The yield was 5.4 mg (18% f theory).

[1342] Compound 8 was analyzed by analytical RP-HPLC using a Kinetex® 5 μm Biphenyl 100 Å LC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=21.4 min, purity≥95%.

[1343] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in 15 water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=23.5 min, purity≥95%.

[1344] The observed mass was in accordance with the calculated mass.

[1345] ESI Ion-Trap: m/z=1001.2 [M+4H].sup.4+, 801.1 [M+5H].sup.5+, 667.8 [M+6H].sup.6+, 572.5 [M+7H].sup.7+.

[1346] MALDI-ToF: m/z=4000.0 [M+H].sup.+, 2000.5 [M+2H].sup.2+, 1334.0 [M+3H].sup.3+.

[1347] Compound 9: [G.sup.14, OEG(13).sup.[36-39]]ADM(14-52)

##STR00103##

[1348] Chemical Formula: C.sub.179H.sub.280N.sub.52O.sub.53S.sub.2

[1349] Exact Mass: 4070.03 Da

[1350] Molecular Weight: 4072.64 g/mol

[1351] Compound 9 was synthesized in a 7.5 μmol scale. The yield was 2.4 mg (8% f theory).

[1352] Compound 9 was analyzed by analytical RP-HPLC using a Kinetex® 5 μm Biphenyl 100 Å LC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=21.6 min, purity≥95%.

[1353] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in 15 water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=23.7 min, purity≥95%.

[1354] The observed mass was in accordance with the calculated mass.

[1355] ESI Ion-Trap: m/z=1358.3 [M+3H].sup.3+, 1018.9 [M+4H].sup.4+, 815.4 [M+5H].sup.5+, 679.7 [M+6H].sup.6+.

[1356] MALDI-ToF: m/z=4071.0 [M+H].sup.+, 2036.0 [M+2H].sup.2+.

[1357] Compound 10: [G.sup.14, OEG(16).sup.[36-40]]ADM(14-52)

##STR00104##

[1358] Chemical Formula: C.sub.177H.sub.278N.sub.50O.sub.52S.sub.2

[1359] Exact Mass: 4000.01 Da

[1360] Molecular Weight: 4002.59 g/mol

[1361] Compound 10 was synthesized in a 7.5 μmol scale. The yield was 3.6 mg (12% f theory).

[1362] Compound 10 was analyzed by analytical RP-HPLC using a Kinetex® 5 μm Biphenyl 100 ALC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=22.2 min, purity≥95%.

[1363] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in 15 water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=24.3 min, purity≥95%.

[1364] The observed mass was in accordance with the calculated mass.

[1365] ESI Ion-Trap: m/z=1001.6 [M+4H].sup.4+, 801.3 [M+5H].sup.5+, 668.0 [M+6H].sup.6+, 572.7 [M+7H].sup.7+.

[1366] MALDI-ToF: m/z=4001.0 [M+H].sup.+, 2001.0 [M+2H].sup.2+, 1334.3 [M+3H].sup.3+.

[1367] Compound 11: [G.sup.14, OEG(19)[35-40]]ADM(14-52)

##STR00105##

[1368] Chemical Formula: C.sub.175H.sub.277N.sub.49O.sub.50S.sub.2

[1369] Exact Mass: 3929.01 Da

[1370] Molecular Weight: 3931.55 g/mol

[1371] Compound 11 was synthesized in a 7.5 μmol scale. The yield was 6.9 mg (23% f theory).

[1372] Compound 11 was analyzed by analytical RP-HPLC using a Kinetex® 5 μm Biphenyl 100 ALC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=22.1 min, purity≥95%.

[1373] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in 15 water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=24.1 min, purity≥95%.

[1374] The observed mass was in accordance with the calculated mass.

[1375] ESI Ion-Trap: m/z=983.5 [M+4H].sup.4, 787.2 [M+5H].sup.5+, 656.1 [M+6H].sup.6+, 562.5 [M+7H].sup.7+.

[1376] MALDI-ToF: m/z=3930.1 [M+H].sup.+, 1965.5 [M+2H].sup.2+.

[1377] Compound 12: [G.sup.14, OEG(19).sup.[36-.sup.41]]ADM(14-52)

##STR00106##

((4R,7S,13S,16S,19R)-19-(2-(2-aminoacetamido)acetamido)-13-benzyl-16-(3-guanidinopropyl)-7-((R)-1-hydroxyethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaazacycloicosane-4-carbonyl)-L-threonyl-ADM(23-35)-(1-(λ.SUP.2.-azaneyl)-3,6,9,12,15-pentaoxaoctadecan-18-oyl)-L-alanyl-ADM(43-52)

[1378] Chemical Formula: C.sub.174H.sub.273N.sub.49O.sub.52S.sub.2

[1379] Exact Mass: 3944.97 Da

[1380] Molecular Weight: 3947.51 g/mol

[1381] Compound 12 was synthesized in a 7.5 μmol scale. The yield was 0.7 mg (2% f theory).

[1382] Compound 12 was analyzed by analytical RP-HPLC using a Kinetex® 5 μm Biphenyl 100 ALC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=21.8 min, purity≥95%.

[1383] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 10% to 50% eluent B in A over 40 min (Eluent A=0.1% TFA in 15 water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=24.0 min, purity≥95%.

[1384] The observed mass was in accordance with the calculated mass.

[1385] ESI Ion-Trap: m/z=987.6 [M+4H].sup.4+, 790.3 [M+5H].sup.5+, 658.8 [M+6H].sup.6+, 564.8 [M+7H].sup.7+.

[1386] MALDI-ToF: m/z=3946.0 [M+H].sup.+, 1973.5 [M+2H].sup.2+.

[1387] Double Modified Adrenomedullin-Analogues 13

[1388] Synthesis:

[1389] The synthesis of compound 13 was performed using automated peptide synthesis as described in the general method.

[1390] The coupling sequence was as follows:

TABLE-US-00010 Coupling Cycle compound 13 AA of human ADM 1 Tyr(tBu) 52 2 Gly 51 3 Gln(Trt) 50 4 Pro 49 5 Asp(OPp) 48 6 Ile 47 7 Lys(Boc) 46 8 Ser(tBu) 45 9 Lys(Mmt) 44 10 Pro 43 11 Ala 42 12 Val 41 13 Asn(Trt) 40 14 Asp(tBu) 39 15 Lys(Mmt) 38 16 Asp(tBu) 37 17 Lys(Mmt) 36 18 Asp(tBu) 35 19 Thr(tBu) 34 20 Phe 33 21 Gln(Trt) 32 22 Tyr(tBu) 31 23 Ile 30 24 Gln(Trt) 29 25 His(Trt) 28 26 Ala 27 27 Leu 26 28 Lys(Boc) 25 29 Gln(Trt) 24 30 Val 23 31 Thr(tBu) 22 32 Cys(Trt) 21 33 Thr(tBu) 20 34 Gly 19 35 Phe 18 36 Arg(Pbf) 17 37 Cys(Trt) 16 38 Gly 15

[1391] After automated synthesis, the N-terminal amino acid Boc-Lys(Fmoc)-OH was coupled manually with HOBt and DIC in 5-fold molar excess. The reaction was performed in DMF as solvent for 24 h.

[1392] Subsequently, the Fmoc protecting group was removed from the N-terminal amino acid using 20% piperidine in DMF (v/v) for 10 min twice.

[1393] Coupling of ODD to the free lysine side chain was achieved using a 5-fold excess (75 μmol) of octadecanedioic acid mono-tert-butyl ester, HOBt and DIC in 400 μDMF/DCM (3:1, v/v) as solvent for approx. 24 h.

[1394] For the simultaneous removal of Mmt/OPp protecting groups the resin was treated with TFA/TIS/DCM (3:5:92, v/v/v) (10×2 min, 1 mL). Subsequently, the resin was washed with 2.5% DIPEA in DMF (v/v) for 10 min twice (1 mL).

[1395] Cyclization was performed using a 30-fold molar excess of HOBt and DIC in DMF as solvent for approx. 24 h at T=40° C.

[1396] Cleavage of the peptides from the resin and simultaneous side chain deprotection was achieved with TFA/TA/EDT (90:7:3, v/v/v) for approx. 3 h. The peptides were precipitated and washed with ice-cold diethyl ether/n-hexane (3:1, v/v) and dried under reduced pressure.

[1397] Oxidation of the disulfide bond was achieved by solving the peptide in 20 m1 ACN/H.sub.2O/TBS (2:3:5, v/v/v), adjusting the pH to 7.6-7.8 (1 M NaOH) and subsequent shaking for 12 h. Upon completion of the oxidation, the pH was adjusted to 3-4 using 1 M HCl.

[1398] Purification of the crude peptide was performed using preparative RP-HPLC on an Aeris PEPTIDE 5 μm 20 XB-C18 LC column (Phenomenex, 250 mm×21.2 mm, 5 m, 100 Å). A linear gradient of 20% to 60% eluent B in A over 40 min was applied (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN). The flow rate was 20 mL/min, UV detection was measured at λ=220 nm.

[1399] Analytics:

[1400] The identity of the peptides was confirmed via MALDI-MS (UltraflexIII, Bruker) and ESI-Orbitrap-MS (Orbitrap Elite™, Thermo Scientific). The purities were analyzed using analytical RP-HPLC.

[1401] Compound 13: [K.sup.14(ODD), (K.sup.44, D.sup.48).sub.lac] ADM(14-52)

##STR00107##

((4R,7S,13S,16S,19R)-19-(2-((S)-2-amino-6-(17-carboxyoctadecanamido)hexanamido) acetamido)-13-benzyl-16-(3-guanidinopropyl)-7-((R)-1-hydroxyethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaazacycloicosane-4-carbonyl)-L-threonyl-ADM(23-43)-((2S,5S,8S,11S,18S)-5-(4-aminobutyl)-18-(λ.SUP.2.-azaneyl)-8-((S)-sec-butyl)-2-(hydroxymethyl)-3,6,9,13,19-pentaoxo-1,4,7,10,14-pentaazacyclononadecane-11-carbonyl)-L-prolyl-ADM(50-52)

[1402] Chemical Formula: C.sub.213H.sub.336N.sub.56O.sub.60S.sub.2

[1403] Exact Mass: 4702.44 Da

[1404] Molecular Weight: 4705.48 g/mol

[1405] Compound 13 was synthesized in a 15 μmol scale. The yield was 1.0 mg (1% f theory).

[1406] Compound 13 was analyzed via analytical RP-HPLC using a Kinetex® 5 μm C18 100 Å LC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 20% to 70% eluent B in A over 30 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=15.1 min, purity≥95%.

[1407] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 20% to 70% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=18.3 min, purity≥95%.

[1408] The observed mass was in accordance with the calculated mass.

[1409] ESI Orbitrap: m/z=1177.4 [M+4H].sup.4+, 942.1 [M+5H].sup.5+, 785.1 [M+6H].sup.6+, 673.2 [M+7H].sup.7+.

[1410] MALDI-ToF: m/z=4703.4 [M+H].sup.+, 2352.1 [M+2H].sup.2+.

[1411] Triple modified Adrenomedullin-Analogue 14

[1412] Synthesis:

[1413] The synthesis of compound 14 was performed using automated peptide synthesis as described in the general method. In coupling cycle 15, Fmoc-NH—OEG(13)-OH was used as amino acid.

[1414] The coupling sequence was as follows:

TABLE-US-00011 AA of human Coupling Cycle compound 14 ADM 1 Tyr(tBu) 52 2 Gly 51 3 Gln(Trt) 50 4 Pro 49 5 Asp(OPp) 48 6 Ile 47 7 Lys(Boc) 46 8 Ser(tBu) 45 9 Lys(Mmt) 44 10 Pro 43 11 Ala 42 12 Val 41 13 Asn(Trt) 40 14 Asp(tBu) 39 15 OEG(13) 35-38 16 Thr(tBu) 34 17 Phe 33 18 Gln(Trt) 32 19 Tyr(tBu) 31 20 Ile 30 21 Gln(Trt) 29 22 His(Trt) 28 23 Ala 27 24 Leu 26 25 Lys(Boc) 25 26 Gln(Trt) 24 27 Val 23 28 Thr(tBu) 22 29 Cys(Trt) 21 30 Thr(tBu) 20 31 Gly 19 32 Phe 18 33 Arg(Pbf) 17 34 Cys(Trt) 16 35 Gly 15

[1415] After automated synthesis, the N-terminal amino acid Boc-Lys(Fmoc)-OH was coupled manually with HOBt and DIC in 5-fold molar excess. The reaction was performed in DMF for 24 h.

[1416] Subsequently, the Fmoc protecting group was removed from the N-terminal amino acid using 20% piperidine in DMF (v/v) for 10 min twice.

[1417] Coupling of ODD to the free lysine side chain was achieved using a 5-fold excess (75 μmol) of octadecanedioic acid mono-tert-butyl ester, HOBt and DIC in 400 μDMF/DCM (3:1, v/v) as solvent for approx. 24 h.

[1418] For simultaneous removal of the Mmt/OPp protecting groups, the resin was treated with TFA/TIS/DCM (3:5:92, v/v/v) (10×2 min, 1 mL). Subsequently, the resin was washed with 2.5% DIPEA in DMF (v/v) for 10 min twice (1 mL).

[1419] Cyclization was performed using a 30-fold molar excess of HOBt and DIC in DMF for approx. 24 h at T=40° C.

[1420] Cleavage of the peptides from the resin and simultaneous side chain deprotection was achieved with TFA/TA/EDT (90:7:3, v/v/v) for approx. 3 h. The peptides were precipitated and washed with ice-cold diethyl ether/n-hexane (3:1, v/v) and dried under reduced pressure.

[1421] Oxidation of the disulfide bond was achieved by solving the peptide in 20 m1 ACN/H.sub.2O/TBS (2:3:5, v/v/v), adjusting the pH to 7.6-7.8 (1 M NaOH) and subsequent shaking for 12 h. Upon completion of the oxidation, the pH was adjusted to 3-4 using 1 M HCl.

[1422] Purification of the crude peptide was performed using preparative RP-HPLC on an Aeris PEPTIDE 5 μm XB-C18 LC column (Phenomenex, 250 mm×21.2 mm, 5 m, 100 Å). A linear gradient of 20% to 60% eluent B in A over 40 min was applied (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN). The flow rate was 20 mL/min, UV detection was measured at, =220 nm.

[1423] Analytics:

[1424] The identity of the peptides was confirmed via MALDI-MS (UltraflexIII, Bruker) and ESI-Orbitrap-MS (Orbitrap Elite™, Thermo Scientific). The purities were analyzed using analytical RP-HPLC.

[1425] Compound 14: [K.sup.14(ODD), OEG(13).sup.[35-38], (K.sup.44, D.sup.48).sub.lac]ADM(14-52)

##STR00108##

((4R,7S,13S,16S,19R)-19-(2-((S)-2-amino-6-(17-carboxyoctadecanamido)hexanamido) acetamido)-13-benzyl-16-(3-guanidinopropyl)-7-((R)-1-hydroxyethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentaazacycloicosane-4-carbonyl)-L-threonyl-ADM(23-34)-(3-(2-(2-(2-(λ.SUP.2.-azaneyl)ethoxy)ethoxy)ethoxy)propanoyl)-L-aspartyl-ADM(40-43)-((2S,5S,8S,11S,18S)-5-(4-aminobutyl)-18-(λ.SUP.2.-azaneyl)-8-((S)-sec-butyl)-2-(hydroxymethyl)-3,6,9,13,19-pentaoxo-1,4,7,10,14-pentaazacyclononadecane-11-carbonyl)-L-prolyl-ADM(50-52)

[1426] Chemical Formula: C.sub.202H.sub.319N.sub.51O.sub.56S.sub.2

[1427] Exact Mass: 4419.31 Da

[1428] Molecular Weight: 4422.20 g/mol

[1429] Compound 14 was synthesized in a 15 μmol scale. The yield was 1.7 mg (3% of theory).

[1430] Compound 14 was analyzed via analytical RP-HPLC using a Kinetex® 5 μm C18 100 Å LC column (Phenomenex, 250 mm×4.6 mm, 5 m, 100 Å) applying a linear gradient of 20% to 70% eluent B in A over 30 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=15.8 min, purity≥95%.

[1431] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 m, 90 Å) was used, applying a linear gradient of 20% to 70% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.0 mL/min; λ=220 nm). Rt=19.4 min, purity≥95%.

[1432] The observed mass was in accordance with the calculated mass.

[1433] ESI Orbitrap: m/z=1474.8 [M+3H].sup.3+, 1106.3 [M+4H].sup.4+, 885.3 [M+5H].sup.5+.

[1434] MALDI-ToF: m/z=4420.3 [M+H].sup.+, 2210.6 [M+2H].sup.2+.

[1435] N-terminal Lactam-bridged Adrenomedullin-Analogues 15-17

[1436] Synthesis:

[1437] The synthesis of compound 15-17 was performed using automated peptide synthesis as described in the general method.

[1438] The coupling sequence was as follows:

TABLE-US-00012 AA of Coupling compound compound compound human Cycle 15 16 17 ADM 1 Tyr(tBu) 52 2 Gly 51 3 Gln(Trt) 50 4 Pro 49 5 Ser(tBu) Asp(OPp) 48 6 Ile 47 7 Lys(Boc) 46 8 Ser(tBu) 45 9 Arg(Pbf) Lys(Mmt) 44 10 Pro 43 11 Ala 42 12 Val 41 13 Asn(Trt) 40 14 Asp(tBu) 39 15 Lys(Boc) OEG(13) 38 16 Asp(tBu) — 37 17 Lys(Boc) — 36 18 Asp(tBu) — 35 19 Thr(tBu) 34 20 Phe 33 21 Gln(Trt) 32 22 Tyr(tBu) 31 23 Ile 30 24 Gln(Trt) 29 25 His(Trt) 28 26 Ala 27 27 Leu 26 28 Lys(Boc) 25 29 Gln(Trt) 24 30 Val 23 31 Thr(tBu) 22 32 Cys(Trt) 21 33 Thr(tBu) 20 34 Gly 19 35 Phe 18 36 Arg(Pbf) 17 37 Cys(Trt) 16 38 Gly 15 39 Boc-Lys(Fmoc) 14

[1439] Coupling of ODD to the free lysine side chain was achieved using a 5-fold excess (75 μmol) of octadecanedioic acid mono-tert-butyl ester, HOBt and DIC in 400 μl DMF/DCM (3:1, v/v) as solvent for approx. 24 h.

[1440] The side chain protection groups OPp, Mint and Mtt were removed simultaneously by incubating the resin 15-times in TFA/TIS/DCM (2:5:93, v/v/v) for 1 min. Subsequently, the resin was neutralized two using 2.5% DIPEA/DMF (v/v) for 10 min Lactam cyclisation was achieved using 30 eq. HOBt and 30 eq. DIC in DMF, 40° C. for 12 h.

[1441] Cleavage of the peptide from the resin and simultaneous side chain deprotection was achieved with TFA/TA/EDT (90:7:3, v/v/v) for 3 h. The peptide was precipitated and washed with ice-cold diethyl ether.

[1442] Purification of the peptides was performed using preparative RP-HPLC on a Kinetex® 5 m Biphenyl 100A LC column (Phenomenex, 250 mm×4.6 mm, 5 μm, 100 Å). A linear gradient of 20% to 50% eluent B in 30 min was applied (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN). The flow rate was 20 mL/min, UV detection was measured at λ=220 nm.

[1443] Analytics:

[1444] The identity of the peptide was confirmed via analytical RP-HPLC and ESI-Orbitrap (Orbitrap Elite™, Thermo Fischer). The purities were analyzed using analytical RP-HPLC.

[1445] Compound 15: [K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac]ADM.sub.14-52

##STR00109##

[1446] Chemical Formula: C.sub.214H.sub.341N.sub.59O.sub.61

[1447] Exact Mass: 4713.54 Da

[1448] Molecular Weight: 4716.43 g/mol

[1449] Compound 15 was synthesized in a 15 μmol scale. The yield was 1.4 mg (1.6% f theory).

[1450] Compound 15 was analyzes via analytical RP-HPLC using a Kinetex® 5 μm Biphenyl 100 ALC column (Phenomenex, 250 mm×4.6 mm, 5 μm, 100 Å) applying a linear gradient of 20% to 70% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=16.4 min, purity≥95%.

[1451] In addition, an Aeris Peptide 3.6 μm XB-C18 100A (Phenomenex, 250 mm×4.6 mm, 3.6 μm, 100A) was used, applying a linear gradient of 20% to 70% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=17.6 min, purity≥95%.

[1452] The observed mass was in accordance with the calculated mass.

[1453] ESI Orbitrap: m/z=1179.9 [M+4H].sup.4+; m/z=944.1 [M+5H].sup.5+; m/z=786.9 [M+6H].sup.6+; m/z=674.7 [M+7H].sup.7+; m/z=590.5 [M+8H].sup.8+.

[1454] Compound 16: [K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac, (K.sup.44, D.sup.48).sub.lac]ADM.sub.14-52

##STR00110##

((3S,9S,12S,15S,21S)-15-(2-((S)-2-amino-6-(17-carboxyoctadecanamido)hexanamido)acetamido)-9-benzyl-12-(3-guanidinopropyl)-3-((R)-1-hydroxyethyl)-2,5,8,11,14,18-hexaoxo-1,4,7,10,13,17-hexaazacyclohenicosane-21-carbonyl)-L-threonyl-ADM(23-43)-((2S,5S,8S,11S,18S)-5-(4-aminobutyl)-18-(λ.SUP.2.-azaneyl)-8-((S)-sec-butyl)-2-(hydroxymethyl)-3,6,9,13,19-pentaoxo-1,4,7,10,14-pentaazacyclononadecane-11-carbonyl)-L-prolyl-ADM(50-52)

[1455] Chemical Formula: C.sub.215H.sub.339N.sub.57O.sub.61

[1456] Exact Mass: 4695.52 Da

[1457] Molecular Weight: 4698.42 g/mol

[1458] Compound 16 was synthesized in a 2×15 μmol scale. The yield was 2.1 mg (1.2% f theory).

[1459] Compound 16 was analyzes via analytical RP-HPLC using a Kinetex® 5 μm Biphenyl 100 ALC column (Phenomenex, 250 mm×4.6 mm, 5 μm, 100 Å) applying a linear gradient of 20% to 70% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=16.7 min, purity≥95%.

[1460] In addition, an Aeris Peptide 3.6 μm XB-C18 100A (Phenomenex, 250 mm×4.6 mm, 3.6 μm, 100A) was used, applying a linear gradient of 20% to 70% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=18.5 min, purity≥95%.

[1461] The observed mass was in accordance with the calculated mass.

[1462] ESI Orbitrap: m/z=1175.4 [M+4H].sup.4+; m/z=940.5 [M+5H].sup.5+; m/z=783.9 [M+6H].sup.6+; m/z=672.1 [M+7H].sup.7+.

[1463] Compound 17: [K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac, OEG(13).sup.35-38, (K.sup.44, D.sup.48).sub.lac]ADM.sub.14-52

##STR00111##

((3S,9S,12S,15S,21S)-15-(2-((S)-2-amino-6-(17-carboxyoctadecanamido)hexanamido)acetamido)-9-benzyl-12-(3-guanidinopropyl)-3-((R)-1-hydroxyethyl)-2,5,8,11,14,18-hexaoxo-1,4,7,10,13,17-hexaazacyclohenicosane-21-carbonyl)-L-threonyl-ADM(23-34)-(3-(2-(2-(2-(λ.SUP.2.-azaneyl)ethoxy)ethoxy)ethoxy)propanoyl)-L-aspartyl-ADM(40-43)-((2S,5S,8S,11S,18S)-5-(4-aminobutyl)-18-(λ.SUP.2.-azaneyl)-8-((S)-sec-butyl)-2-(hydroxymethyl)-3,6,9,13,19-pentaoxo-1,4,7,10,14-pentaazacyclononadecane-11-carbonyl)-L-prolyl-ADM(50-52)

[1464] Chemical Formula: C.sub.240H.sub.322N.sub.52O.sub.57

[1465] Exact Mass: 4412.39 Da

[1466] Molecular Weight: 4415.13 g/mol

[1467] Compound 17 was synthesized in a 2×15 μmol scale. The yield was 2.1 mg (1.2% f theory).

[1468] Compound 17 was analyzes via analytical RP-HPLC using a Kinetex® 5 μm Biphenyl 100 ALC column (Phenomenex, 250 mm×4.6 mm, 5 μm, 100 Å) applying a linear gradient of 20% to 70% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=1.55 mL/min; λ=220 nm). Rt=13.9 min, purity≥94%.

[1469] In addition, a Jupiter® 4 μm Proteo 90 Å LC column (Phenomenex, 250 mm×4.6 mm, 4 μm, 90 Å) was used, applying a linear gradient of 20% to 70% eluent B in A over 40 min (Eluent A=0.1% TFA in water; Eluent B=0.08% TFA in ACN; flow rate=0.6 mL/min; λ=220 nm). Rt=24.1 min, purity≥94%.

[1470] The observed mass was in accordance with the calculated mass.

[1471] ESI Orbitrap: m/z=1472.5 [M+3H].sup.3+; m/z=1104.6 [M+4H].sup.4+; m/z=883.9 [M+5H].sup.5+; m/z=736.7 [M+6H].sup.6+.

[1472] Synthesis of fluorescently labelled analogues for in vitro stability determination Abbreviations

TABLE-US-00013 Dde N-γ-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl DIPEA N,N-diisopropylethylamine HATU O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium- hexafluorphosphat TAM 6-Tamra

[1473] Suppliers

TABLE-US-00014 6-TAMRA EMP Biotech Dde-Lys(Fmoc)-OH Iris Bitotech DIPEA Roth HATU Merck

[1474] Fluorescently labelled versions of analogues 5 and 13-15 were synthesized by incorporation of 6-TAMRA at the N-terminus of the peptides.

TABLE-US-00015 compound analogue TAM-5 TAM-[K.sup.14(ODD)]ADM(14-52) TAM-13 TAM-[K.sup.14(ODD), (K.sup.44, D.sup.48).sub.lac]ADM(14-52) TAM-14 TAM-[K.sup.14(ODD), OEG(13).sup.[35-38], (K.sup.44, D.sup.48).sub.lac]ADM(14-52) TAM-15 TAM-[K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac]ADM(14-52) TAM-16 TAM-[K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac, (K.sup.44, D.sup.48).sub.lac]ADM(14-52) TAM-17 TAM-[K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac, OEG(13).sup.[35-38], (K.sup.44, D.sup.48).sub.lac]ADM(14-52)

[1475] The synthesis was carried out as described above. Notably, Dde-Lys(Fmoc)-OH was coupled as N-terminal amino acid (ADM pos. 14) instead of Boc-Lys(Fmoc).

[1476] Before cleavage from the resin, the Dde protecting group was removed from the N-terminal amino acid using 1 ml 3% hydrazine in DMF (v/v) (10 times, 10 min each) and coupling of 6-TAMRA (3 equiv.) was carried out with 2.5 equiv. HATU and 3 equiv. DIPEA in DMF for approximately 24h.

[1477] The identity of the peptides was confirmed by mass spectrometry with a MALDI-ToF-MS (UltraflexIII, Bruker) and an ESI-MS (HCT, Bruker). The observed masses were in accordance with the calculated masses. Purity>95% f all analogues was demonstrated by analytical RP-HPLC.

[1478] B. Assessment of Pharmacological Activity

[1479] The suitability of the compounds according to the invention for treatment of diseases can be demonstrated using the following assay systems:

[1480] (1) Test Descriptions (In Vitro)

[1481] (1a) Activity Determination of Adrenomedullin Analogues in vitro

[1482] Abbreviations

TABLE-US-00016 CLR calcitonin receptor-like receptor CRE cAMP response element DMEM Dulbecco's Modified Eagle Medium DPBS Dulbecco's Phosphate-Buffered Saline ECFP enhanced cyan fluorescent protein EYFP enhanced yellow fluorescent protein FCS fetal calf serum RAMP2 receptor activity-modifying protein 2

[1483] Suppliers

TABLE-US-00017 DMEM Lonza DPBS Lonza FCS Biochrom Ham's F-12 Fluka Metafectene ® Pro Biontex ONE Glo ™ Luciferase Assay System Promega poly-D-lysine hydrobromide Sigma-Alrich

[1484] Cell Culture HEK-293 cells (human embryonic kidney cells) were cultured in Ham's F-12/DMEM (1/1; v/v) containing 15% FCS under humidified atmosphere at 37° C. and 5% CO.sub.2 in 75 cm.sup.2 cell culture flasks.

[1485] Transient Co-Transfection of HEK293 Cells

[1486] Cells were cultured in 75 cm.sup.2 flasks to 70-80% confluency. 45 μl Metafectene® Pro was diluted in 900 ρl Ham's F-12/DMEM (1/1; v/v) and incubated for 20 min at room temperature. 9 μg plasmid containing DNA of CLR fused to EYFP and 3 μg plasmid containing DNA of RAMP2 fused to ECFP were dissolved in 900 μl Ham's F-12/DMEM (1/1; v/v). The plasmid solution was mixed with the Metafectene® Pro solution and incubated for 25 min at room temperature. Medium was removed from the cells and replaced by 6 m1 Ham's F-12/DMEM (1/1; v/v) containing 15% FCS. After addition of transfection solution the cells were incubated for 3 h under humidified atmosphere at 37° C. and 5% CO.sub.2. For the second transfection 45 μl Metafectene® Pro was diluted in 900 μl Ham's F-12/DMEM (1/1; v/v) and incubated for 20 min at room temperature. 12 μg of pGL4.29[Luc2P/CRE/Hygro] plasmid containing DNA for the luciferase reporter gene luc2P (with CRE promotor region) were dissolved in 900 μl Ham's F-12/DMEM (1/1; v/v). The plasmid solution was mixed with the Metafectene® Pro solution and incubated for 25 min at room temperature. Medium was removed from the cells and replaced by 6 m1 Ham's F-12/DMEM (1/1; v/v) containing 15% FCS. After addition of transfection solution, the cells were incubated under humidified atmosphere at 37° C. and 5% CO.sub.2 over night.

[1487] cAMP-Assay

[1488] Seeding of Transiently Transfected Cells in 96-Well-Plates.

[1489] For coating of 96-well-plates, 50 μl of a poly-D-lysine solution in DPBS (0.1 mg/ml) were pipetted in each well and incubated for 40 min. After removal of poly-D-lysine, each well was washed with 50 μl DPBS. Transiently transfected cells were detached from the cell culture flask by removal of the medium, 2-fold washing with 5 m1 DPBS and resuspending in 40 m1 Ham's F-12/DMEM (1/1; v/v) containing 15% FCS. 90,000 to 120,000 cells in 150 μl Ham's F-12/DMEM (1/1; v/v) containing 15% FCS were seeded per well and the plates were incubated under humidified atmosphere at 37° C. and 5% CO.sub.2 over night.

[1490] Cell Stimulation

[1491] For each ligand, a serial dilution with eight different concentrations was prepared using Ham's F.sup.12/DMEM (1/1; v/v). Before stimulation, the medium on the cells was replaced by 100 μl Ham's F.sup.12/DMEM (1/1; v/v) and the plates were incubated for 1 h under humidified atmosphere at 37° C. and 5% CO.sub.2. For stimulation, the medium was removed and the cells were incubated for 3 h in 80 μl of ligand-solution under humidified atmosphere at 37° C. and 5% CO.sub.2. In addition 80 μl of a 5 μM forskolin solution in Ham's F-12/DMEM (1/1; v/v) was used as a positive control and 80 μl of Ham's F-12/DMEM (1/1; v/v) as a negative control. Each concentration and the controls were tested as triplicates.

[1492] Luminescence Measurement

[1493] After 3 h of stimulation, the solutions were removed and the cells were washed with 50 μl of Ham's F-12/DMEM (1/1; v/v) perwell. After 10 m incubation in 30 μl of Ham's F-12/DMEM (1/1; v/v) at room temperature, 30 μl of luciferase-solution (ONE-Glo™ Luciferase Assay System) was added and the luminescence was directly measured using an Infinite M200 (Tecan).

[1494] Data Analysis

[1495] Data analysis of the luminescence measurement was carried out with GraphPad Prism 5. Therefore, the measured luminescence values of each plate were first corrected on the base of the respective average of forskolin stimulation. Afterwards they were normalized to [G14]ADM(14-52), which was used as standard peptide in every assay. After correction and normalization, data was analyzed using non-linear regression giving dose-response curves for each tested ligand.

[1496] Representative EC.sub.50 values for the embodiment examples are given in the following Table 1 below.

TABLE-US-00018 AM.sub.1R activity data EC.sub.50 E.sub.max ± SEM Example Example [nM] [%] n Reference [G.sup.14]ADM(14-52) 3.5 99 ± 1 38 1 [G.sup.14, (K.sup.44, D.sup.48).sub.lac]ADM(14-52) 3.9 102 ± 3 2 2 [G.sup.14, (D.sup.44, K.sup.48).sub.lac]ADM(14-52) 4.8 91 ± 1 2 3 [G.sup.14, (K.sup.44, E.sup.48).sub.lac]ADM(14-52) 16 93 ± 2 2 4 [G.sup.14, (E.sup.44, K.sup.48).sub.lac]ADM(14-52) 5 [K.sup.14(ODD)]ADM(14-52) 17 105 ± 5 2 6 [G.sup.14, OEG(12).sup.[35-38]]ADM(14-52) 21 95 ± 7 2 7 [G.sup.14, OEG(13).sup.[35-38]]ADM(14-52) 13 103 ± 4 6 8 [G.sup.14, OEG(16).sup.[35-39]]ADM(14-52) 16 95 ± 3 3 9 [G.sup.14, OEG(13).sup.[36-39]]ADM(14-52) 49 87 ± 4 3 10 [G.sup.14, OEG(16).sup.[36-40]]ADM(14-52) 81 77 ± 3 3 11 [G.sup.14, OEG(19).sup.[35-40]]ADM(14-52) 39 94 ± 2 2 12 [G.sup.14, OEG(19).sup.[36-41]]ADM(14-52) 431 79 ± 5 3 13 [K.sup.14(ODD), (K.sup.44, D.sup.48).sub.lac]ADM(14-52) 2.6 94 ± 7 2 14 [K.sup.14(ODD), OEG(13).sup.[35-38],(K.sup.44, D.sup.48).sub.lac] ADM(14-52) 6.6 92 ± 5 2 15 [K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac]ADM(14-52) 3.4 89 ± 2 2 16 [K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac, (K.sup.44, D.sup.48).sub.lac]ADM(14-52) 1.4 100 ± 5 3 17 [K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac, OEG(13).sup.[35-38], 5.0 77 ± 3 3 (K.sup.44, D.sup.48).sub.lac]ADM(14-52)

[1497] (1b) Stability Determination of Adrenomedullin Analogues in Human Blood Plasma Suppliers

TABLE-US-00019 ACN Biosolve Ethanol (absolute for molecular biology) ITW Reagents Human blood plasma Haema

[1498] The stability of the peptides was investigated using fluorescently labeled analogues, which were prepared as described above.

[1499] The TAMRA-labeled analogues were dissolved in 1.5 m1 of human blood plasma to a concentration of 10.sup.−5 M and incubated at 37° C. under constant shaking. Samples of 150 μl were taken at different time points and precipitated with 300 μl Ethanol/ACN (1:1) for at least 1 h at −20° C. After centrifugation for 30 s at 12000 rpm, the supernatant was collected and incubated at −20° C. for at least 3 h. Subsequently, it was transferred into Costar® Spin-X® Centrifuge Tube Filters (0.22 μm) and centrifuged for 1 h at 12000 rpm. The samples were analyzed by RP-HPLC using a VariTide RPC column (Agilent Technologies, 250 mm×4.6 mm, 6 m, 200 Å) with linear gradients of 0.1% TFA in water and 0.08% TFA in ACN; fluorescence measurement (λ.sub.ex=525 nm; λ.sub.em=572 nm) was used for the detection of the analogues and their N-terminal fragments. The percentage of intact peptide was determined by peak integration. The values of peaks containing additional cleavage fragments were corrected by comparing intensities of cleavage fragments and intact peptide using MALDI-MS analysis (UltraflexIII, Bruker). The stability of the peptides was calculated with GraphPad Prism 5 (GraphPad Software) using a two phase exponential decay function for the determination of slow-decay phase half-lives (ln(2)/K.sub.slow; K.sub.slow: rate constant of slow part of exponential decay).

[1500] The stability of ADM analogues in human blood plasma are shown in Table 2 and FIG. 1.

TABLE-US-00020 TABLE 2 stability in human blood plasma compound analogue t.sub.1/2 (slow) TAM-control TAM-[G.sup.14]ADM(14-52) 12 h TAM-5 TAM-[K.sup.14(ODD)]ADM(14-52) 149 h TAM-13 TAM-[K.sup.14(ODD), (K.sup.44, D.sup.48).sub.lac]ADM(14-52) >>144 h TAM-14 TAM-[K.sup.14(ODD), OEG(13).sup.[35-38], (K.sup.44, D.sup.48).sub.lac,]ADM(14-52) >>144 h TAM-15 TAM-[K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac]ADM(14-52) >>144 h TAM-16 TAM-[K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac, (K.sup.44, D.sup.48).sub.lac]ADM(14-52) >>144 h TAM-17 TAM-[K.sup.14(ODD), (Dpr.sup.16, E.sup.21).sub.lac, OEG(13).sup.[35-38](K.sup.44, D.sup.48).sub.lac]ADM(14-52) >>144 h

[1501] 1c) Tests on a Recombinant Adrenomedullin-Receptor Reporter Cell

[1502] The activity of the compounds according to the invention is quantified with the aid of a recombinant Chinese hamster ovary (CHO) cell line that carries the human adrenomedullin-receptor. Activation of the receptor by ligands can be measured by aequorin luminescence. Construction of the cell line and measurement procedure has been described in detail [Wunder F., Rebmann A., Geerts A, and Kalthof B., Mol Pharmacol, 73, 1235-1243 (2008)]. In brief: Cells are seeded on opaque 384-well microtiter plates at a density of 4000 cells/well and are grown for 24 h. After removal of culture medium, cells are loaded for 3 h with 0.6 μg/ml coelenterazine in Ca.sup.2+-free Tyrode solution (130 mM sodium chloride, 5 mM potassium chloride, 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 1 mM magnesium chloride, and 4.8 mM sodium hydrogen carbonate, pH 7.4) supplemented with 0.2 mM 3-Isobutyl-1-methylxanthine (IBMX) in a cell culture incubator. Compounds are added for 6 min in calcium.sup.2+-free Tyrode solution containing 0.1% bovine serum albumin. Immediately before adding calcium.sup.2+ to a final concentration of 3 mM measurement of the aequorin luminescence is started by use of a suitable luminometer. Luminescence is measured for 60 s. In a typical experiment compounds are tested in a concentration range of 1×10.sup.−13 to 3×10.sup.−6 M.

[1503] 1d) Transcellular Electrical Resistance Assays in Endothelial Cells

[1504] The activity of the compounds according to the invention is characterized in in vitro-permeability assays in human umbilical venous cells (HUVEC, Lonza). By use of an xCELLigence® apparatus (ACEA Biosciences, Inc.; San Diego, Calif.) changes of transendothelial electrical resistance (TEER) over an endothelial monolayer are continuously measured by use of a small gold electrodes on which the cells have been seeded. HUVEC are grown on the 96-well sensor electrode plates (OMNI Life Science, 2801035) to confluent monolayers and hyperpermeability can be induced by inflammatory stimuli such as Thrombin, TNF-α, IL-1β, VEGF, Histamine and hydrogen peroxide which all have been demonstrated to cause break down of endothelial cell contacts and reduction of TEER. Test compounds are added before or after addition of thrombin. In a typical experiment compounds are tested in a concentration range of 1×10.sup.−10 to 1×10.sup.−6 M.

[1505] 1c) In Vitro-Permeability Assays in Endothelial Cells

[1506] In another in vitro model of endothelial hyperpermeability the activity of compounds according to the invention is examined with respect to modulation of macromolecular permeability. Human umbilical vein endothelial cells (HUVECS) are grown to confluency on fibronectin-coated Transwell® filter membranes (24-well plates, 6.5 mm-inserts with 0.4 μM polycarbonate membrane; Costar #3413) which separate an upper from a lower tissue culture chamber with endothelial cells growing on the bottom of the upper chamber. The medium of the upper chamber is supplemented with 250 μg/ml of 40 kDa FITC-Dextran (Invitrogen, D1844). Hyperpermeability of the monolayer is induced by addition of thrombin. Medium samples are collected from the lower chamber every 30 min and relative fluorescence as a parameter for changes of macromolecular permeability over time is measured in a suitable fluorimeter. Thrombin challenge typically induces a significant increase of FITC-dextran transition across the endothelial monolayers. In a typical experiment compounds are tested in a concentration range of 1×10.sup.−10 to 1×10.sup.−6 M.

[1507] (2) Test Descriptions (In Vivo)

[1508] 2a) Measurement of Blood Pressure and Heart Rate in Telemetered, Normotensive Wistar Rats

[1509] The cardiovascular effects induced by compounds according to the invention are investigated in freely moving conscious female Wistar rats (body weight>200 g) by radiotelemetric measurement of blood pressure and heart rate. Briefly, the telemetric system (DSI Data Science International, MN, USA) is composed on 3 basic elements: implantable transmitters (PhysioTel HD-S10), receivers (PhisioTel RPC-1 with PhisioTel MX2 Data Exchange Matrix) and a computer-based acquisition software (Dataquest™ A.R.T for Windows). Rats are instrumented with pressure implants for chronic use at least 14 days prior to the experiments. During catheter implantation rats are anesthetized with pentobabital (Nembutal, Sanofi: 50 mg/kg i.p.). After shaving the abdominal skin, a midline abdominal incision is made, and the fluid-filled sensor catheter is inserted upstream into the exposed descending aorta between the iliac bifurcation and the renal arteries. The catheter is tied several times at the stopper. The tip of the telemetric catheter is located just caudal to the renal arteries and secured by tissue adhesive. The transmitter body is affixed to the inner peritoneal wall before closure of abdomen. A two-layer closure of the abdominal incision is used, with individual suturing of the peritoneum and the muscle wall followed by closure of the outer skin. For postsurgical protection against infections and pain a single dosage of an antibiotic (Oxytetracyclin® 10%, 60 mg/kg s.c., 0.06 m1/100 g body weight, Beta-Pharma GmbH & Co, Germany) and analgesic were injected (Rimadyl®, 4 mg/kg s.c., Pfizer, Germany). The hardware configuration is equipped for 24 animals. Each rat cage is positioned on top of an individual receiver platform. After activation of the implanted transmitters, an on-line data acquisition system, samples data and converts telemetric pressure signals to mm Hg. A barometric pressure reference allows for relation of absolute pressure (relative to vacuum) to ambient atmospheric pressure. Data acquisition software is predefined to sample hemodynamic data for 10-s intervals every 5 minutes and are displayed as average values every 30 minutes. Data collection to file is started 2 hours before administration of test compounds and finished after completion of 24-h cycles. In a typical experiment test compounds are administered as bolus either subcutaneously or intravenously at doses of 1 to 1000 μg/kg body weight (as referred to the peptide component).

[1510] In this test single dose application of substances according to the present invention induce long lasting blood pressure reduction at doses of <500 μg/kg body weight [FIGS. 2A, 2B, 2C].

ADRENOMEDULLIN-ANALOGUES FOR LONG-TERM STABILIZATION AND THEIR USE

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A61P9/00

HUMAN NECESSITIES

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A61K45/06

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A61P9/12

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A61K47/64

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A61K47/545

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A61K31/616

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C07K14/575

CHEMISTRY; METALLURGY

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A61K47/542

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A61K38/00

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A61K47/64

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A61K31/616

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A61K45/06

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A61K47/54

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A61P9/12

HUMAN NECESSITIES

Abstract

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