Acylated glucagon analogues

1. A method of producing a compound having the formula:

R 1 —P 1 —P 2 —R 2

wherein

R 1 is H, C 1-4 alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R 2 is OH or NH 2 ;

P 1 is a peptide having the sequence:

His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-X24-WLE-X28-A

wherein:

X2 is selected from Aib, Ac3c, Ac4c and Ac5c;

X3 is selected from Gln and His;

X15 is selected from Asp and Glu;

X16 is selected from Glu and ψ;

X17 is selected from Arg and ψ;

X18 is selected from Ala and Arg;

X20 is selected from Lys and His;

X24 is selected from Glu and ψ; and

X28 is selected from Ser and ψ;

and P 2 is absent or is a sequence of 1-20 amino acid units independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn;

wherein the compound contains one and only one ψ,

and wherein said ψ is a residue of Lys, Arg, Orn or Cys in which the side chain is conjugated to a substituent having the formula —Z 2 Z 1 ;

—Z 1 is a fatty chain having a polar group at one end of the chain and a connection to Z 2 , —X— at the end of the chain distal from the polar group,

wherein the polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid, or a sulfonic acid group;

and —X— is a bond, —CO—, —SO—, or —SO 2 ;

—Z 2 — is a spacer of formula:

wherein:

each Y is independently —NH, —NR, —S or —O, where R is alkyl, a protecting group or forms a linkage to another part of the spacer Z 2 ;

each X is independently a bond, CO—, SO—, or SO 2 —;

with the proviso that when Y is —S, X is a bond;

each V is independently a bivalent organic moiety linking Y and X;

and n is 1-10;

the method comprising solid-phase or liquid-phase stepwise formation of peptide bonds between one or more amino acid residues to form the compound.

2. A method of producing a compound having the formula:

R 1 —P 1 —P 2 —R 2

wherein

R 1 is H, C 1-4 alkyl, acetyl, formyl, benzoyl or trifluoroacetyl;

R 2 is OH or NH 2 ;

P 1 is a peptide having the sequence:

His-X2-X3-GTFTSDYSKYL-X15-X16-X17-X18-A-X20-DFI-X24-WLE-X28-A

wherein:

X2 is selected from Aib, Ac3c, Ac4c and Ac5c;

X3 is selected from Gln and His;

X15 is selected from Asp and Glu;

X16 is selected from Glu and ψ;

X17 is selected from Arg and ψ;

X18 is selected from Ala and Arg;

X20 is selected from Lys and His;

X24 is selected from Glu and ψ; and

X28 is selected from Ser and ψ;

and P 2 is absent or is a sequence of 1-20 amino acid units independently selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Cys, Glu, Lys, Arg, Dbu, Dpr and Orn;

wherein the compound contains one and only one ψ,

and wherein said ψ is a residue of Lys, Arg, Orn or Cys in which the side chain is conjugated to a substituent having the formula —Z 2 Z 1 ;

—Z 1 is a fatty chain having a polar group at one end of the chain and a connection to Z 2 , —X— at the end of the chain distal from the polar group,

wherein the polar group comprises a carboxylic acid or a carboxylic acid bioisostere, a phosphonic acid, or a sulfonic acid group;

and —X— is a bond, —CO—, —SO—, or —SO 2 —;

—Z 2 — is a spacer of formula:

wherein:

each Y is independently —NH, —NR, —S or −0, where R is alkyl, a protecting group or forms a linkage to another part of the spacer Z 2 ;

each X is independently a bond, CO—, SO—, or SO 2 —;

with the proviso that when Y is —S, X is a bond;

each V is independently a bivalent organic moiety linking Y and X;

and n is 1-10;

the method comprising expressing a precursor peptide sequence from a nucleic acid construct that encodes the precursor peptide, recovering the expression product, and modifying the precursor peptide to yield the compound.

3. A method according to claim 2 , comprising modifying the precursor peptide to introduce the substituent at residue ψ.

4. A method according to claim 1 , wherein the compound is:

(SEQ ID NO: 56)

H-H-Ac4c-QGTFTSDYSKYLDE-K([17-carboxy-

heptadecanoyl]-isoGlu-Peg3-Peg3)-RAKDFIEWLESA-NH 2 ,

or a pharmaceutically acceptable salt thereof.

5. A method according to claim 1 , wherein the compound is:

(SEQ ID NO: 60)

H-H-Aib-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-

heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH 2 ,

or a pharmaceutically acceptable salt thereof.

6. A method according to claim 1 , wherein the compound is:

(SEQ ID NO: 61)

H-H-Ac4c-QGTFTSDYSKYLDERAAKDFI-K([17-carboxy-

heptadecanoyl]-isoGlu-GSGSGG)-WLESA-NH 2 ,

or a pharmaceutically acceptable salt thereof.

7. A method according to claim 1 , wherein the compound is:

(SEQ ID NO: 63)

H-H-Ac4c-QGTFTSDYSKYLDERAAKDFIEWLE-K([17-carboxy-

heptadecanoyl]-isoGlu-GSGSGG)-A-NH 2 ,

or a pharmaceutically acceptable salt thereof.

8. A method according to claim 1 , wherein the compound is:

(SEQ ID NO: 64)

H-H-Ac4c-QGTFTSDYSKYLDERRAKDFIEWLE-K([17-carboxy-

heptadecanoyl]-isoGlu-GSGSGG)-A-NH 2 ,

or a pharmaceutically acceptable salt thereof.