GLP-1 ANALOGUES

Abstract

directed to peptide analogues of glucagon-like peptide-1, the pharmaceutically-acceptable salts thereof, to methods of using such analogues to treat mammals and to pharmaceutical compositions useful therefor comprising said analogues.

Description

BACKGROUND OF THE INVENTION

[0001]The present invention is directed to peptide analogues of glucagon-like peptide-1, the pharmaceutically-acceptable salts thereof, to methods of using such analogues to treat mammals and to pharmaceutical compositions useful therefor comprising said analogues.

[0002]Glucagon-like peptide-1 (7-36) amide (GLP-1) (SEQ ID NO: 1) is synthesized in the intestinal L-cells by tissue-specific post-translational processing of the glucagon precursor pre-proglucagon (Varndell, J. M., et al., J. Histochem Cytochem, 1985:33:1080-6) and is released into the circulation in response to a meal. The plasma concentration of GLP-1 rises from a fasting level of approximately 15 pmol/L to a peak postprandial level of 40 pmol/L. It has been demonstrated that, for a given rise in plasma glucose concentration, the increase in plasma insulin is approximately threefold greater when glucose is administered orally compared with intravenously (Kreymann, B., et al., Lancet 1987:2, 1300-4). This alimentary enhancement of insulin release, known as the incretin effect, is primarily humoral and GLP-1 is now thought to be the most potent physiological incretin in humans. In addition to the insulinotropic effect, GLP-1 suppresses glucagon secretion, delays gastric emptying (Wettergren A., et al., Dig Dis Sci 1993:38:665-73) and may enhance peripheral glucose disposal (D'Alessio, D. A. et al., J. Clin Invest 1994:93:2293-6).

[0003]In 1994, the therapeutic potential of GLP-1 was suggested following the observation that a single subcutaneous (s/c) dose of GLP-1 could completely normalize postprandial glucose levels in patients with non-insulin-dependent diabetes mellitus (NIDDM) (Gutniak, M. K., et al., Diabetes Care 1994:17:1039-44). This effect was thought to be mediated both by increased insulin release and by a reduction in glucagon secretion. Furthermore, an intravenous infusion of GLP-1 has been shown to delay postprandial gastric emptying in patients with NIDDM (Williams, B., et al., J. Clin Endo Metab 1996:81:327-32). Unlike sulfonylureas, the insulinotropic action of GLP-1 is dependent on plasma glucose concentration (Holz, G. G. 4^th, et al., Nature 1993:361:362-5). Thus, the loss of GLP-1-mediated insulin release at low plasma glucose concentration protects against severe hypoglycemia. This combination of actions gives GLP-1 unique potential therapeutic advantages over other agents currently used to treat NIDDM.

Numerous studies have shown that when given to healthy subjects, GLP-1 potently influences glycemic levels as well as insulin and glucagon concentrations (Orskov, C, Diabetologia 35:701-711, 1992; Holst, J. J., et al., Potential of GLP-1 in diabetes management in Glucagon III, Handbook of Experimental Pharmacology, Lefevbre P J, Ed. Berlin, Springer Verlag, 1996, p. 311-326), effects which are glucose dependent (Kreymann, B., et al., Lancet ii: 1300-1304, 1987; Weir, G. C., et al., Diabetes 38:338-342, 1989). Moreover, it is also effective in patients with diabetes (Gutniak, M., N. Engl J Med 226:1316-1322, 1992; Nathan, D. M., et al., Diabetes Care 15:270-276, 1992), normalizing blood glucose levels in type 2 diabetic subjects (Nauck, M. A., et al., Diagbetologia 36:741-744, 1993), and improving glycemic control in type 1 patients (Creutzfeldt, W. O., et al., Diabetes Care 19:580-586, 1996), raising the possibility of its use as a therapeutic agent.

[0004]GLP-1 is, however, metabolically unstable, having a plasma half-life (t₁/2) of only 1-2 min in vivo. Exogonously administered GLP-1 is also rapidly degraded (Deacon, C. F., et al., Diabetes 44:1126-1131, 1995). This metabolic instability limits the therapeutic potential of native GLP-1. Hence, there is a need for GLP-1 analogues that are more active or are more metabolically stable than native GLP-1.

SUMMARY OF THE INVENTION

[0005]In one aspect, the present invention is directed to a compound of formula (I),

(R²R³)-A⁷-A⁸-A⁹-A¹⁰-A¹¹-A¹²-A¹³-A¹⁴-A¹⁵-A¹⁶-A¹⁷-A¹⁸-A¹⁹-A²⁰-A²¹- -A²²-A²³-A²⁴-A²⁵-A²⁶-A²⁷-A²⁸-A²⁹-A- ³⁰-A³¹-A³²-A³³-A³⁴-A³⁵-A³⁶-A³⁷-R.s- up.1, (I)

whereinA⁷ is L-His, Ura, Paa, Pta, D-His, Tyr, 3-Pal, 4-Pal, Hppa, Tma-His, Amp or deleted, provided that when A⁷ is Ura, Paa, Pta or Hppa then R² and R³ are deleted;

A⁸ is Ala, D-Ala, Aib, Acc, N-Me-Ala, N-Me-D-Ala, Arg or N-Me-Gly;

A⁹ is Glu, N-Me-Glu, N-Me-Asp or Asp;

A¹⁰ is Gly, Acc, Ala, D-Ala, Phe or Aib;

A¹¹ is Thr or Ser;

A¹² is Phe, Acc, Aic, Aib, 3-Pal, 4-Pal, β-Nal, Cha, Trp or X¹-Phe;

A¹³ is Thr or Ser;

A¹⁴ is Ser, Thr, Ala or Aib;

A¹⁵ is Asp, Ala, D-Asp or Glu;

A¹⁶ is Val, D-Val, Acc, Aib, Leu, Ile, Tle, Nle, Abu, Ala, D-Ala, Tba or Cha;

A¹⁷ is Ser, Ala, D-Ala, Aib, Acc or Thr;

A¹⁸ is Ser, Ala, D-Ala, Aib, Acc or Thr;

A¹⁹ is Tyr, D-Tyr, Cha, Phe, 3-Pal, 4-Pal, Acc, β-Nal, Amp or X¹-Phe;

A²⁰ is Leu, Ala, Acc, Aib, Nle, Ile, Cha, Tle, Val, Phe or X¹-Phe;

A²¹ is Glu, Ala or Asp;

A²² is Gly, Acc, Ala, D-Ala, β-Ala or Aib;

A²³ is Gln, Asp, Ala, D-Ala, Aib, Acc, Asn or Glu;

A²⁴ is Ala, Aib, Val, Abu, Tle or Acc;

[0006]A²⁵ is Ala, Aib, Val, Abu, Tle, Acc, Lys, Arg, hArg, Orn, HN--CH((CH₂), --NR¹⁰R¹¹)--C(O) or HN--CH((CH₂)_e--X³)--C(O);A²⁶ is Lys, Ala, 3-Pal, 4-Pal, Arg, hArg, Orn, Amp, HN--CH((CH₂)_n--NR¹⁰R¹¹)--C(O) or HN--CH((CH₂)_e--X³)--C(O);

A²⁷ is Glu, Ala, D-Ala or Asp;

A²³ is Phe, Ala, Pal, β-Nal, X¹-Phe, Aic, Acc, Aib, Cha or Trp;

A²⁹ is Ile, Acc, Aib, Leu, Nle, Cha, Tle, Val, Abu, Ala, Tba or Phe;

[0007]A³⁰ is Ala, Aib, Acc or deleted;A³¹ is Trp, Ala, β-Nal, 3-Pal, 4-Pal, Phe, Acc, Aib, Cha, Amp or deleted;A³² is Leu, Ala, Acc, Aib, Nle, Ile, Cha, Tle, Phe, X¹-Phe, Ala or deleted;A³³ is Val, Acc, Aib, Leu, Ile, Tle, Nle, Cha, Ala, Phe, Abu, X¹-Phe, Tba, Gaba or deleted;A³⁴ is Lys, Arg, hArg, Orn, Amp, Gaba, HN--CH((CH₂), --NR¹⁰R¹¹)--C(O), HN--CH((CH₂)_e--X³)--C(O) or deleted;A³⁵ is Gly or deleted;A³⁶ is L- or D-Arg, D- or L-Lys, D- or L-hArg, D- or L-Orn, Amp, HN--CH((CH₂), --NR¹⁰R¹¹)--C(O), HN--CH((CH₂)_e--X³)--C(O) or deleted;A³⁷ is Gly or deleted; [0008]X¹ for each occurrence is independently selected from the group consisting of (C₁-C₆)alkyl, OH and halo; [0009]R¹ is OH, NH₂, (C₁-C₁₂)alkoxy, or NH--X²--CH₂-Z⁰, wherein X² is a (C₁-C₁₂)hydrocarbon moiety, and Z⁰ is H, OH, CO₂H or CONH₂; [0010]X³ is

##STR00001##

[0010]or --C(O)--NHR¹², wherein X⁴ for each occurrence is independently --C(O)--, --NH--C(O)-- or --CH₂--, and f for each occurrence is independently an integer from 1 to 29; [0011]each of R² and R³ is independently selected from the group consisting of H, (C₁-C₃0)alkyl, (C₂-C₃0)alkenyl, phenyl(C₁-C₃0)alkyl, naphthyl(C₁-C₃0)alkyl, hydroxy(C₁-C₃0)alkyl, hydroxy(C₂-C₃0)alkenyl, hydroxyphenyl(C₁-C₃0)alkyl, and hydroxynaphthyl(C₁-C₃0)alkyl; or one of R² and R³ is C(O)X⁵ in which X is (C₁-C₃0)alkyl, (C₂-C₃0)alkenyl, phenyl(C₁-C₃0)alkyl, naphthyl(C₁-C₃0)alkyl, hydroxy(C₁-C₃0)alkyl, hydroxy(C₂-C₃0)alkenyl, hydroxyphenyl(C₁-C₃0)alkyl, hydroxynaphthyl(C₁-C₃0)alkyl,

##STR00002##

[0012]where Y is H or OH, r is 0-4 and q is 0-4; [0013]n for each occurrence is independently an integer from 1-5; and [0014]R¹⁰ and R¹¹ for each occurrence is each independently H, (C₁-C₃0)alkyl, (C₁-C₃0)acyl, (C₁-C₃0)alkylsulfonyl, --C((NH)(NH₂)) or

##STR00003##

[0014]provided that when R¹⁰ is (C₁-C₃0)acyl, (C₁-C₃0)alkylsulfonyl, --C((NH)(NH₂)) or

##STR00004##

[0015]R¹¹ is H or (C₁-C₃0)alkyl; and [0016]R¹² is (C₁-C₃0)alkyl;with the proviso that:(i) at least one amino acid of a compound of formula (I) is not the same as the native sequence of hGLP-1(7-36, or -37)NH₂ (SEQ ID NOS: 1, 2) or hGLP-1(7-36, or -37)OH (SEQ ID NOS: 3, 4);(ii) a compound of formula (I) is not an analogue of hGLP-1(7-36, or -37)NH₂ (SEQ ID NOS: 1, 2) or hGLP-1(7-36, or -37)OH (SEQ ID NOS: 3, 4) wherein a single position has been substituted by Ala;(iii) a compound of formula (I) is not [Lys²⁶(N.sup.ε-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ ID NOS: 5-8), [Lys³⁴(N.sup.ε-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ ID NOS: 9-12), [Lys^26,34-bis(N.sup.ε-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ ID NOS: 13-16), [Arg²⁶, Lys³⁴(N.sup.ε-alkanoyl)]hGLP-1(8-36, or -37)-E (SEQ ID NOS: 17-20), or [Arg^26,34, Lys³⁶(N.sup.ε-alkanoyl)]hGLP-1(7-36, or -37)-E, wherein E is --OH or --NH₂ (SEQ ID NOS: 21-24);(iv) a compound of formula (I) is not Z¹-hGLP-1(7-36, or -37)-OH, Z¹-hGLP-1(7-36, or -37)-NH₂, where Z¹ is selected from the group consisting of [0017](a) [Arg²⁶] (SEQ ID NOS: 25-28), [Arg³⁴] (SEQ ID NOS: 29-32), [Arg^23,34] (SEQ ID NOS: 33-36), [Lys³⁶] (SEQ ID NOS: 37-40), [Arg²⁶, Lys³⁶] (SEQ ID NOS: 41-44), [Arg³, Lys³⁶] (SEQ ID NOS: 45-48), [D-Lys³⁶], [Arg³⁶] (SEQ ID NOS: 3,4,1,2), [D-Arg³⁶], [Arg^26,34, Lys³⁶] (SEQ ID NOS: 49-52), or [Arg^26,36, Lys³⁴] (SEQ ID NOS: 25-28); [0018](b) [Asp²¹] (SEQ ID NOS: 53-56); [0019](c) at least one of [Aib³] (SEQ ID NOS: 57-60), [D-Ala⁸] and [Asp⁹] (SEQ ID NOS: 61-64); and [0020](d) [Tyr⁷] (SEQ ID NOS: 65-68), [N-acyl-His⁷] (SEQ ID NOS: 69-72), [N-alkyl-His⁷], [N-acyl-D-His⁷] (SEQ ID NOS: 73-76) or [N-alkyl-D-His⁷];(v) a compound of formula (I) is not a combination of any two of the substitutions listed in groups (a) to (d); and(vi) a compound of formula (I) is not [N-Me-Ala⁸]hGLP-1(8-36 or -37) (SEQ ID NOS: 75, 78), [Glu¹]hGLP-1(7-36 or -37) (SEQ ID NOS: 79, 80), [Asp²¹]hGLP-1(7-36 or -37) (SEQ ID NOS: 53, 54) or [Phe³¹]hGLP-1(7-36 or -37) (SEQ ID NOS: 81, 82).

[0021]A preferred compound of the immediately foregoing compound of formula (I) is where A¹¹ is Thr; A¹³ is Thr; A¹⁴ is Ser, Aib or Ala; A¹⁷ is Ser, Ala, Aib or D-Ala; A¹⁸ is Ser, Ala, Aib or D-Ala; A²¹ is Glu or Ala; A²³ is Gln, Glu, or Ala; and A²⁷ is Glu or Ala; or a pharmaceutically acceptable salt thereof.

[0022]A preferred compound of the immediately foregoing compound of formula (I) is where A⁹ is Glu, N-Me-Glu or N-Me-Asp; A¹² is Phe, Acc or Aic; A¹⁶ is Val, D-Val, Acc, Aib, Ala, Tle or D-Ala; A¹⁹ is Tyr, 3-Pal, 4-Pal or D-Tyr; A²⁰ is Leu, Acc, Cha, Ala or Tle; A²⁴ is Ala, Aib or Acc; A²⁵ is Ala, Aib, Acc, Lys, Arg, hArg, Orn, HN--CH((CH₂), --NH--R¹⁰)--C(O); A²⁸ is Phe or Ala; A²⁹ is Ile, Acc or Tle; A³⁰ is Ala, Aib or deleted; A³¹ is Trp, Ala, 3-Pal, 4-Pal or deleted; A³² is Leu, Acc, Cha, Ala or deleted; A³³ is Val, Acc, Ala, Gaba, Tle or deleted; or a pharmaceutically acceptable salt thereof.

[0023]A preferred compound of the immediately foregoing compound of formula (I) is where A⁸ is Ala, D-Ala, Aib, A6c, A5c, N-Me-Ala, N-Me-D-Ala or N-Me-Gly; A¹⁰ is Gly, Ala, D-Ala or Phe; A¹² is Phe, A6c or A5c; A¹⁶ is Val, Ala, Tle, A6c, A5c or D-Val; A²⁰ is Leu, A6c, A5c, Cha, Ala or Tle; A²² is Gly, Aib, β-Ala, L-Ala or D-Ala; A²⁴ is Ala or Aib; A²⁹ is Ile, A6c, A5c or Tle; A³² is Leu, A6c, A5c, Cha, Ala or deleted; A³³ is Val, A6c, A5c, Ala, Gaba, Tle or deleted; or a pharmaceutically acceptable salt thereof.

[0024]A preferred compound of the immediately foregoing compound of formula (I) is where R¹ is OH or NH₂ or a pharmaceutically acceptable salt thereof.

[0025]A preferred compound of the immediately foregoing compound of formula (I) or a pharmaceutically acceptable salt thereof is where R² is H and R³ is (C₁-C₃0)alkyl, (C₂-C₃0)alkenyl, (C₁-C₃0)acyl,

##STR00005##

[0026]A most preferred compound of formula (I) is where said compound is [D-Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]-GLP-1(7-34)NH₂; [D-Ala⁸,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂; [Ala¹⁸,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 83); [Ala¹⁶,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 84); [Ala¹⁴,23,27 3-Pal^19,31]hGLP-1(7-35)-NH₂(SEQ ID NO: 85); [Ala²²,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 86); [Hppa⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 87); [Ala^23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 88); [Ala⁷,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 89); [Ala²²,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 90); [Ala¹⁵,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 91); [Ala¹⁷,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 92); [Ala^8,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 93); [Ala²¹,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 94); [Ala²²,23,26,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 95); [Ala²²,23,27,32, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 96); [Ala²²,23,26,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 97); [Ala²²,23,27,31, 3-Pal¹⁹, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 98); [Ala²²,23,27,28, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 99); [Ala²²,23,27,29, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 100); [Ala^23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 101); [Ala²⁰,22,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 102); [Ala²²,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 103); [Ala¹⁷,22,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 104); [D-Ala¹⁰, Ala²²,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂; [D-Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31]hGLP-1(7-34)-NH₂; [Ala¹⁷,23,27, 3-Pal¹⁹,26,31]hGLP-1(7-34)-NH₂ (SEQ ID NO: 105); [D-Ala⁸, Ala¹⁷, 3-Pal^19,31]hGLP-1(7-34)-NH₂; [Ala¹⁷,23,27, 3-Pal^19,31]hGLP-1(7-34)-NH₂ (SEQ ID NO: 106); [D-Ala⁸, Ala¹⁷,23,27, 3-Pal³¹, Tle²⁹]hGLP-1(7-34)-NH₂; [D-Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31, Tle¹⁶]hGLP-1(7-34)-NH₂; [D-Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP (7-34)-NH₂; [D-Ala²², Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; [Aib⁸, Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 107); [D-Ala⁸, Ala¹⁷,22,23,27, 3-Pal^19,31]hGLP-1(7-33)-NH₂; [Aib⁸, Ala¹⁷,22,23,27, 3-Pal¹⁹³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 108); [Ala¹⁷,18,23,27, 3-Pal⁹,3, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 109); [Ala¹⁷,23,27, 3-Pal^19,31, Tle³³, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 110); [Tle¹⁶, Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 111); [N-Me-D-Ala⁸, Ala¹⁷,22,23,27, 3-Pal^19,31]hGLP-1(7-33)-NH₂; [Aib⁸, Ala¹⁷,22,23,27, 3-Pal^19,31]hGLP-1(7-33)-NH₂ (SEQ ID NO: 112); [Ala¹⁷,18,22,23,27, 3-Pal^19,31, Tle^16,20, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 113); [D-Ala⁶, Ala¹⁷,18,22,23,27, 3-Pal^19,31, Tle¹⁶, Gaba³⁴]hGLP-1(7-34)-NH₂; [D-Ala^8,22 Ala¹⁷,18,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; [D-Ala^8,18, Ala¹⁷,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; [D-Ala^8,17, Ala¹⁸,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(73)-NH₂; [Ala¹⁷,18,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; or a pharmaceutically acceptable salt thereof.

[0027]Another most preferred compound of formula (I) is wherein said compound is

[Aib⁸, A6c³²]hGLP-1(7-36)NH₂ (SEQ ID NO: 114); [A6c^20,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 115); [Aib⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO: 116); [(Tma-His)⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 117); [A6c⁸]hGLP-1(8-36)-NH₂ (SEQ ID NO:118); [A6c⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 119); [A6c^16,20]hGLP-1(7-36)-NH₂ (SEQ ID NO: 120); [A6c^29,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 121); [A6c²⁰, Aib²⁴]hGLP-1(7-36)-NH₂ (SEQ ID NO: 122); [Aib²⁴, A6c^29,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 123); [A6c^16,29,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 124); [Ura⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 125); [Paa⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 126); [Pta⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 127); [N-Me-Ala⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 128); [N-Me-Ala⁸]hGLP-1(8-36)-NH₂; (SEQ ID NO.) [N-Me-D-Ala⁸]hGLP-1(7-36)-NH₂; [N-Me-D-Ala⁸]hGLP-1(8-36)-NH₂; [N-Me-Gly⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 129); [A5c⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 130); [N-Me-Glu⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO: 131); [A5c⁸, A6c^20,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 132); [Aib⁸, A6c³²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 133); [Aib^8,25]hGLP-1(7-36)-NH₂ (SEQ ID NO: 134); [Aib^8,24]hGLP-1(7-36)-NH₂ (SEQ ID NO: 135); [Aib^8,30]hGLP-1(7-36)-NH₂ (SEQ ID NO: 136); [Aib⁸, Cha²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 137); [Aib⁸, Cha³²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 138); [Aib⁸, Glu²³]hGLP-1(7-36)-NH₂ (SEQ ID NO: 139); [Aib⁸, A6c²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 140); [Aib⁸, A6c^20,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 141); [Aib^8,22]hGLP-1(7-36)-NH₂ (SEQ ID NO: 142); [Aib⁸, β-Ala²²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 143); [Aib⁸, Lys²⁵]hGLP-1(7-36)-NH₂ (SEQ ID NO: 144); [Aib⁸, A6c¹²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 145); [Aib⁸, A6c²⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO: 146); [Aib⁸, A6c³³]hGLP-1(7-36)-NH₂ (SEQ ID NO: 147); [Aib^8,14]hGLP-1(7-36)NH₂ (SEQ ID NO: 148); [Aib^8,18]hGLP-1(7-36)NH₂ (SEQ ID NO: 149); or [Aib^8,17]hGLP-1(7-36)NH₂ (SEQ ID NO: 150); or a pharmaceutically acceptable salt thereof. In another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of a compound of formula (I) as defined hereinabove or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

[0028]In still another aspect, the present invention provides a method of eliciting an agonist effect from a GLP-1 receptor in a subject in need thereof which comprises administering to said subject an effective amount of a compound of formula (I) as defined hereinabove or a pharmaceutically acceptable salt thereof.

[0029]In yet a further aspect, this invention provides a method of treating a disease selected from the group consisting of Type I diabetes, Type II diabetes, obesity, glucagonomas, secretory disorders of the airway, metabolic disorder, arthritis, osteoporosis, central nervous system disease, restenosis and neurodegenerative disease, renal failure, congestive heart failure, nephrotic syndrome, cirrhosis, pulmonary edema, hypertension, and disorders wherein the reduction of food intake is desired, in a subject in need thereof which comprises administering to said subject an effective amount of a compound of formula (I) as defined hereinabove or a pharmaceutically acceptable salt thereof. Preferred of the foregoing method is where the disease is Type I diabetes or Type II diabetes.

[0030]With the exception of the N-terminal amino acid, all abbreviations (e.g. Ala) of amino acids in this disclosure stand for the structure of --NH--CH(R)--CO--, wherein R is the side chain of an amino acid (e.g., CH₃ for Ala). For the N-terminal amino acid, the abbreviation stands for the structure of (R²R³)--N--CH(R)--CO--, wherein R is a side chain of an amino acid and R² and R³ are as defined above except in the case where A⁷ is Ura, Paa, Pta or Hppa in which case R² and R³ are not present since Ura, Paa, Pta and Hppa are considered here as des-amino amino acids. The abbreviations: β-Nal, Nle, Cha, Amp, 3-Pal, 4-Pal and Aib stand for the following α-amino acids: β-(2-naphthyl)alanine, norleucine, cyclohexylalanine, 4-amino-phenylalanine, β-(3-pyridinyl)alanine, β-(4-pyridinyl)alanine and α-aminoisobutyric acid, respectively. Other amino acid definitions are: Ura is urocanic acid; Pta is (4-pyridylthio) acetic acid; Paa is trans-3-(3-pyridyl)acrylic acid; Tma-His is N,N-tetramethylamidino-histidine; N-Me-Ala is N-methyl-alanine; N-Me-Gly is N-methyl-glycine; N-Me-Glu is N-methyl-glutamic acid; Tle is tert-butylglycine; Abu is α-aminobutyric acid; Tba is tert-butylalanine; Orn is ornithine; Aib is α-aminoisobutyric acid; β-Ala is β-alanine; Gaba is γ-aminobutyric acid; Ava is 5-aminovaleric acid; and Aic is 2-aminoindane-2-carboxylic acid.

[0031]What is meant by Acc is an amino acid selected from the group of 1-amino-1-cyclopropanecarboxylic acid (A3c); 1-amino-1-cyclobutanecarboxylic acid (A4c); 1-amino-1-cyclopentanecarboxylic acid (A5c); 1-amino-1-cyclohexanecarboxylic acid (A6c); 1-amino-1-cycloheptanecarboxylic acid (A7c); 1-amino-1-cyclooctanecarboxylic acid (A8c); and 1-amino-1-cyclononanecarboxylic acid (A9c). In the above formula, hydroxyalkyl, hydroxyphenylalkyl, and hydroxynaphthylalkyl may contain 1-4 hydroxy substituents. COX⁵ stands for --C═O.X⁵. Examples of --C═O.X⁵ include, but are not limited to, acetyl and phenylpropionyl.

[0032]What is meant by Lys(N.sup.ε-alkanoyl) is represented by the following structure:

##STR00006##

[0033]What is meant by Lys(N.sup.ε-alkylsulfonyl) is represented by the following structure:

##STR00007##

What is meant by Lys(N.sup.ε-(2-(4-alkyl-1-piperazine)-acetyl)) is represented by the following structure:

##STR00008##

What is meant by Asp(1-(4-alkyl-piperazine)) is represented by the following structure:

##STR00009##

What is meant by Asp(1-alkylamino) is represented by the following structure:

##STR00010##

The variable n in the foregoing structures is 1 to 30.

[0034]The full names for other abbreviations used herein are as follows: Boc for t-butyloxycarbonyl, HF for hydrogen fluoride, Fm for formyl, Xan for xanthyl, Bzl for benzyl, Tos for tosyl, DNP for 2,4-dinitrophenyl, DMF for dimethylformamide, DCM for dichloromethane, HBTU for 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate, DIEA for diisopropylethylamine, HOAc for acetic acid, TFA for trifluoroacetic acid, 2CIZ for 2-chlorobenzyloxycarbonyl and OcHex for Q-cyclohexyl.

[0035]A peptide of this invention is also denoted herein by another format, e.g., [A5c⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 130), with the substituted amino acids from the natural sequence placed between the set of brackets (e.g., A5c⁸ for Ala⁸ in hGLP-1). The abbreviation GLP-1 means glucagon-like peptide-1, and hGLP-1 means human glucagon-like peptide-1. The numbers between the parentheses refer to the number of amino acids present in the peptide (e.g., hGLP-1(7-36) (SEQ ID NO: 3) is amino acids 7 through 36 of the peptide sequence for human GLP-1). The sequence for hGLP-1(7-37) (SEQ ID NO: 4) is listed in Mojsov, S., Int. J. Peptide Protein Res., 40, 1992, pp. 333-342. The designation "NH₂" in hGLP-1(7-36)NH₂ (SEQ ID NO: 1) indicates that the C-terminus of the peptide is amidated. hGLP-1(7-36) (SEQ ID NO: 2) means that the C-terminus is the free acid.

DETAILED DESCRIPTION

[0036]The peptides of this invention can be prepared by standard solid phase peptide synthesis. See, e.g., Stewart, J. M., et al., Solid Phase Synthesis (Pierce Chemical Co., 2d ed. 1984). The substituents R² and R³ of the above generic formula can be attached to the free amine of the N-terminal amino acid by standard methods known in the art. For example, alkyl groups, e.g., (C₁-C₃0)alkyl, can be attached using reductive alkylation. Hydroxyalkyl groups, e.g., (C₁-C₃0)hydroxyalkyl, can also be attached using reductive alkylation wherein the free hydroxy group is protected with a t-butyl ester. Acyl groups, e.g., COE¹, may be attached by coupling the free acid, e.g., E¹COOH, to the free amine of the N-terminal amino acid by mixing the completed resin with 3 molar equivalents of both the free acid and diisopropylcarbodiimide in methylene chloride for one hour. If the free acid contains a free hydroxy group, e.g., p-hydroxyphenylpropionic acid, then the coupling should be performed with an additional 3 molar equivalents of HOBT.

[0037]When R¹ is NH--X²--CH₂--CONH₂ (i.e., Z⁰=CONH₂), the synthesis of the peptide starts with BocHN--X²--CH₂--COOH which is coupled to the MBHA resin. If R¹ is NH--X²--CH₂--COOH (i.e., Z⁰=COOH) the synthesis of the peptide starts with Boc-HN--X²--CH₂--COOH which is coupled to PAM resin.

[0038]The following describes a synthetic method for making a peptide of this invention, which method is well-known to those skilled in the art. Other methods are also known to those skilled in the art.

[0039]Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc., Louisville, Ky.) (0.9 g, 0.3 mmole) in the chloride ion form is placed in a reaction vessel of an Advanced ChemTech Peptide Synthesizer Model 200 programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 15 min each); (c) methylene chloride; (d) ethanol; (e) methylene chloride; (f) 10% diisopropylethylamine in methylene chloride.

[0040]The neutralized resin is stirred with Boc-protected amino acid which is to be the C-terminal amino acid of the desired peptide to be synthesized and diisopropylcarbodiimide (3 mmole each) in methylene chloride for 1 hour and the resulting amino acid resin is then cycled through steps (a) through (f) in the above wash program. The other amino acids (3 mmol) of the desired peptide are then coupled successively by the same procedure. The finished peptide is cleaved from the resin by mixing it with anisole (5 ml), dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) at about 0° C. and stirring it for about 45 min. Excess hydrogen fluoride is evaporated rapidly under a stream of dry nitrogen and free peptide precipitated and washed with ether. The crude peptide is then dissolved in a minimum volume of dilute acetic acid and eluted on a column (2.5×25 cm) of VYDAC® octadecylsilane silica (10 mM) and eluted with a linear gradient of 20-60% acetonitrile over about 1 h in 0.1% trifluoroacetic acid in water. Fractions are examined by thin layer chromatography and analytical high performance liquid chromatography (40-70% B at 1%/min, solution B is 80% acetonitrile/water containing 0.1% TFA) and pooled to give maximum purity rather than yield. Repeated lyophilization of the solution from water gives the product as a white, fluffy powder.

[0041]The product peptide is analyzed by HPLC. Amino acid analysis of an acid hydrolysate of the product peptide can confirm the composition of the peptide. Laser desorption MS is used to determine the molecular weight of the peptide.

[0042]The protected amino acid 1-[N-tert-butoxycarbonyl-amino]-1-cyclohexane-carboxylic acid (Boc-A6c-OH) was synthesized as follows. 19.1 g (0.133 mol) of 1-amino-1-cyclohexanecarboxylic acid (Acros Organics, Fisher Scientific, Pittsburgh, Pa.) was dissolved in 200 ml of dioxane and 100 ml of water. To it was added 67 ml of 2N NaOH. The solution was cooled in an ice-water bath. 32.0 g (0.147 mol) of di-tert-butyl-dicarbonate was added to this solution. The reaction mixture was stirred overnight at room temperature. Dioxane was then removed under reduced pressure. 200 ml of ethyl acetate was added to the remaining aqueous solution. The mixture was cooled in an ice-water bath. The pH of the aqueous layer was adjusted to about 3 by adding 4N HCl. The organic layer was separated. The aqueous layer was extracted with ethyl acetate (1×100 ml). The two organic layers were combined and washed with water (2×150 ml), dried over anhydrous MgSO₄, filtered, and concentrated to dryness under reduced pressure. The residue was recrystallized in ethyl acetate/hexanes. 9.2 g of the pure product was obtained. 29% yield.

[0043]Boc-A5c-OH was synthesized in an analogous manner to that of Boc-A6c-OH. Other protected Acc amino acids can be prepared in an analogous manner by a person of ordinary skill in the art as enabled by the teachings herein.

[0044]In the synthesis of a peptide of this invention containing A5c, A6c and/or Aib, the coupling time is about 2 hrs. for these residues and the residue immediately following them. For the synthesis of [Tma-His⁷]hGLP-1(7-36)NH₂ (SEQ ID NO: 117), HBTU (2 mmol) and DIEA (1.0 ml) in 4 ml DMF were used to react with the N-terminal free amine of the peptide-resin in the last coupling reaction; the coupling time is about 2 hours.

[0045]The full names for the abbreviations used above are as follows: Boc for t-butyloxycarbonyl, HF for hydrogen fluoride, Fm for formyl, Xan for xanthyl, Bzl for benzyl, Tos for tosyl, DNP for 2,4-dinitrophenyl, DMF for dimethylformamide, DCM for dichloromethane, HBTU for 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate, DIEA for diisopropylethylamine, HOAc for acetic acid, TFA for trifluoroacetic acid, 2CIZ for 2-chlorobenzyloxycarbonyl, 2BrZ for 2-bromobenzyloxycarbonyl and OcHex for O-cyclohexyl.

[0046]The substituents R² and R³ of the above generic formula can be attached to the free amine of the N-terminal amino acid by standard methods known in the art. For example, alkyl groups, e.g., (C₁-C₃0)alkyl, may be attached using reductive alkylation. Hydroxyalkyl groups, e.g., (C₁-C₃0)hydroxyalkyl, can also be attached using reductive alkylation wherein the free hydroxy group is protected with a t-butyl ester. Acyl groups, e.g., COX¹, can be attached by coupling the free acid, e.g., X¹COOH, to the free amine of the N-terminal amino acid by mixing the completed resin with 3 molar equivalents of both the free acid and diisopropylcarbodiimide in methylene chloride for about one hour. If the free acid contains a free hydroxy group, e.g., p-hydroxyphenylpropionic acid, then the coupling should be performed with an additional 3 molar equivalents of HOBT.

[0047]A compound of the present invention can be tested for activity as a GLP-1 binding compound according to the following procedure.

Cell Culture:

[0048]RIN 5F rat insulinoma cells (ATCC-# CRL-2058, American Type Culture Collection, Manassas, Va.), expressing the GLP-1 receptor, were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, and maintained at about 37° C. in a humidified atmosphere of 5% CO₂/95% air.

Radioligand Binding:

[0049]Membranes were prepared for radioligand binding studies by homogenization of the RIN cells in 20 ml of ice-cold 50 mM Tris-HCl with a Brinkman Polytron (Westbury, N.Y.) (setting 6, 15 sec). The homogenates were washed twice by centrifugation (39,000 g/10 min), and the final pellets were re-suspended in 50 mM Tris-HCl, containing 2.5 mM MgCl₂, 0.1 mg/ml bacitracin (Sigma Chemical, St. Louis, Mo.), and 0.1% BSA. For assay, aliquots (0.4 ml) were incubated with 0.05 nM [¹²⁵I]GLP-1(7-36) (SEQ ID NO: 151) (˜2200 Ci/mmol, New England Nuclear, Boston, Mass.), with and without 0.05 ml of unlabeled competing test peptides. After a 100 min incubation (25° C.), the bound [¹²⁵I]GLP-1(7-36) (SEQ ID NO: 151) was separated from the free by rapid filtration through GF/C filters (Brandel, Gaithersburg, Md.), which had been previously soaked in 0.5% polyethyleneimine. The filters were then washed three times with 5 ml aliquots of ice-cold 50 mM Tris-HCl, and the bound radioactivity trapped on the filters was counted by gamma spectrometry (Wallac L K B, Gaithersburg, Md.). Specific binding was defined as the total [¹²⁵I]GLP-1(7-36) (SEQ ID NO: 151) bound minus that bound in the presence of 1000 nM GLP-1(7-36) (SEQ ID NO: 3) (Bachem, Torrence, Calif.).

[0050]The peptides of this invention can be provided in the form of pharmaceutically acceptable salts. Examples of such salts include, but are not limited to, those formed with organic acids (e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids (e.g., hydrochloric acid, sulfuric acid, or phosphoric acid), and polymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of polylactic-glycolic acids). A typical method of making a salt of a peptide of the present invention is well known in the art and can be accomplished by standard methods of salt exchange. Accordingly, the TFA salt of a peptide of the present invention (the TFA salt results from the purification of the peptide by using preparative HPLC, eluting with TFA containing buffer solutions) can be converted into another salt, such as an acetate salt by dissolving the peptide in a small amount of 0.25 N acetic acid aqueous solution. The resulting solution is applied to a semi-prep HPLC column (Zorbax, 300 SB, C-8). The column is eluted with (1) 0.1N ammonium acetate aqueous solution for 0.5 hrs., (2) 0.25N acetic acid aqueous solution 0.5 hrs. and (3) a linear gradient (20% to 100% of solution B over 30 min.) at a flow rate of 4 ml/min (solution A is 0.25N acetic acid aqueous solution; solution B is 0.25N acetic acid in acetonitrile/water, 80:20). The fractions containing the peptide are collected and lyophilized to dryness.

[0051]As is well known to those skilled in the art, the known and potential uses of GLP-1 is varied and multitudinous [See, Todd, J. F., et al., Clinical Science, 1998, 95, pp. 325-329; and Todd, J. F. et al., European Journal of Clinical Investigation, 1997, 27, pp. 533-536]. Thus, the administration of the compounds of this invention for purposes of eliciting an agonist effect can have the same effects and uses as GLP-1 itself. These varied uses of GLP-1 may be summarized as follows, treatment of: Type I diabetes, Type II diabetes, obesity, glucagonomas, secretory disorders of the airway, metabolic disorder, arthritis, osteoporosis, central nervous system diseases, restenosis and neurodegenerative diseases. GLP-1 analogues of the present invention that elicit an antagonist effect from a subject can be used for treating the following: hypoglycemia and malabsorption syndrome associated with gastroectomy or small bowel resection.

[0052]Accordingly, the present invention includes within its scope pharmaceutical compositions comprising, as an active ingredient, at least one of the compounds of formula (I) in association with a pharmaceutically acceptable carrier or diluent.

[0053]The dosage of active ingredient in the compositions of this invention may be varied; however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment. In general, an effective dosage for the activities of this invention is in the range of 1×10^-7 to 200 mg/kg/day, preferably 1×10^-4 to 100 mg/kg/day, which can be administered as a single dose or divided into multiple doses.

[0054]The compounds of this invention can be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous or subcutaneous injection, or implant), nasal, vaginal, rectal, sublingual or topical routes of administration and can be formulated with pharmaceutically acceptable carriers to provide dosage forms appropriate for each route of administration.

[0055]Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is admixed with at least one inert pharmaceutically acceptable carrier such as sucrose, lactose, or starch. Such dosage forms can also comprise, as is normal practice, additional substances other than such inert diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

[0056]Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, the elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.

[0057]Preparations according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate. Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.

[0058]Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, excipients such as coca butter or a suppository wax.

[0059]Compositions for nasal or sublingual administration are also prepared with standard excipients well known in the art.

[0060]Further, a compound of this invention can be administered in a sustained release composition such as those described in the following patents and patent applications. U.S. Pat. No. 5,672,659 teaches sustained release compositions comprising a bioactive agent and a polyester. U.S. Pat. No. 5,595,760 teaches sustained release compositions comprising a bioactive agent in a gelable form. U.S. application Ser. No. 08/929,363 filed Sep. 9, 1997, teaches polymeric sustained release compositions comprising a bioactive agent and chitosan. U.S. application Ser. No. 08/740,778 filed Nov. 1, 1996, teaches sustained release compositions comprising a bioactive agent and cyclodextrin. U.S. application Ser. No. 09/015,394 filed Jan. 29, 1998, teaches absorbable sustained release compositions of a bioactive agent. U.S. application Ser. No. 09/121,653 filed Jul. 23, 1998, teaches a process for making microparticles comprising a therapeutic agent such as a peptide in an oil-in-water process. U.S. application Ser. No. 09/131,472 filed Aug. 10, 1998, teaches complexes comprising a therapeutic agent such as a peptide and a phosphorylated polymer. U.S. application Ser. No. 09/184,413 filed Nov. 2, 1998, teaches complexes comprising a therapeutic agent such as a peptide and a polymer bearing a non-polymerizable lactone. The teachings of the foregoing patents and applications are incorporated herein by reference.

[0061]Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents and other references mentioned herein are incorporated by reference.

[0062]The following examples describe synthetic methods for making a peptide of this invention, which methods are well-known to those skilled in the art. Other methods are also known to those skilled in the art. The examples are provided for the purpose of illustration and is not meant to limit the scope of the present invention in any manner.

EXAMPLE 1

[D-Ala⁸, Ala¹⁷,22,23,27, 3-Pal^19,31, Gaba³⁴]-GLP-1(7-34)NH₂

[0063]Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc. Louisville, Ky.) (0.9 g, 0.3 mmole) in the chloride ion form was placed in a reaction vessel of an Advanced ChemTech peptide synthesizer Model 200 programmed to perform the following reaction cycle: (a) methylene chloride; (b) 33% trifluoroacetic acid in methylene chloride (2 times for 1 and 15 min each); (c) methylene chloride; (d) ethanol; (e) methylene chloride; (f) 10% diisopropylethylamine in methylene chloride.

[0064]The neutralized resin was stirred with Boc-Gaba and diisopropylcarbodiimide (3 mmole each) in methylene chloride for 1 hour and the resulting amino acid resin was then cycled through steps (a) to (f) in the above wash program. The following amino acids (3 mmole) were then coupled successively by the same procedure: Boc-Val, Boc-Leu, Boc-3-Pal, Boc-Ala, Boc-Ile, Boc-Phe, Boc-Ala, Boc-Lys(2-Cl-Z), Boc-Ala, Boc-Ala, Boc-Ala, Boc-Ala, Boc-Glu(Bzl), Boc-Leu, Boc-3-Pal, Boc-Ser(Bzl), Boc-Ala, Boc-Val, Boc-Asp(Bzl), Boc-Ser(Bzl), Boc-Thr(Bzl), Boc-Phe, Boc-Thr(Bzl), Boc-Gly, Boc-Glu(Bzl), Boc-D-Ala, Boc-His(Bom).

[0065]The resin with the completed peptide sequence was mixed with anisole (5 ml), dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) at about 0° C. and stirred for about 45 min. Excess hydrogen fluoride was evaporated rapidly under a stream of dry nitrogen and free peptide precipitated and washed with ether. The crude peptide was then dissolved in a minimum volume of dilute acetic acid and eluted on a column (2.5×25 cm) of VYDAC® octadecylsilane silica (10 mM) and eluted with a linear gradient of 20-60% acetonitrile over about 1 h in 0.1% trifluoroacetic acid in water. Fractions were examined by thin layer chromatography and analytical high performance liquid chromatography (40-70% B at 1%/min; r.t.: 14.1 min) and pooled to give maximum purity rather than yield. Repeated lyophilization of the solution from water gives the product (49.9 mg) as a white, fluffy powder.

[0066]The product was found to be homogeneous by HPLC and tlc. Amino acid analysis of an acid hydrolysate confirms the composition of the peptide. Laser desorption MS gave a MW of 2880 (Calc. M+H 2873).

EXAMPLE 2

Synthesis of Peptide Lower-Alkylamides

[0067]Peptides are assembled on O-benzyl-polystyrene resin (often referred to as Merrifield resin) using the Boc amino acid protocol described in Example 1, except that Asp and Glu amino acid carboxyl side-chains are protected with an Fm (fluorenylmethyl ester) group. Completed peptide-resins are suspended in dilute DMF solutions of an appropriate lower alkylamine (such as ethylamine, propylamine, phenethylamine, 1,2-diaminoethane, etc.) and stirred at about 60° C. (for about 18 hrs) whereupon filtration, removal of solvents under reduced pressure and trituration of cleaved peptide oil with ether gives a solid, protected alkylamide peptide. This is then subjected to HF cleavage to remove additional side chain protecting groups and HPLC purification as described in Example 1.

EXAMPLES 3-5

[0068]Examples 3-5 can be synthesized substantially according to the procedure described in Example 1 using the appropriate protected amino acids to yield the noted peptides.

Example 3: [Aib⁸, D-Ala¹⁷, Ala¹⁸,22,23,27, 3-Pal^19,31, Tle¹⁶, Gaba³⁴]-GLP-1(7-34)NH₂

Example 4: [Aib³, D-Ala¹⁷, Ala²²,23,27, 3-Pal^19,31, Tle¹⁶]-GLP-1(7-33)NH₂

Example 5: [Aib⁸ D-Ala¹⁷ Ala²²,23,27, 3-Pal^19,31, Tle^16,20]-GLP-1(7-33)NH₂

EXAMPLES 6-51

[0069]Examples 6-51 were made substantially according to the procedure described for Example 1 but using the appropriate protected amino acid to yield the noted peptide. MS were obtained by laser desorption MS (NA means not available).

Example 6: [D-Ala⁸,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂; MS=2971.0; Calc. MW=2974.4.Example 7: [Ala¹⁸,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 83); MS=2954.4; Calc. MW=2958.4.Example 8: [Ala¹⁶,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 84); MS=2943.0; Calc. MW=2946.3.Example 9: [Ala¹⁴,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 85); MS=2956.0; Calc. MW=2958.4.Example 10: [Ala²²,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 86); MS=2981.0; Calc. MW=2988.4.Example 11: [Hppa⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 87); MS=NAExample 12: [Ala¹⁵,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 88); MS=2928.0; Calc. MW=2930.4.Example 13: [Ala¹⁷,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 89); MS=2955.0; Calc. MW=2958.4.Example 14: [Ala²²,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 90); MS=2896.0; Calc. MW=2888.3.Example 15: [Ala¹⁵,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 91); MS=2852.0; Calc. MW=2844.3.Example 16: [Ala¹⁷,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 92); MS=2880.0; Calc. MW=2872.3.Example 17: [Ala¹⁸,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 93); MS=2870.0; Calc. MW=2872.3.Example 18: [Ala²¹,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 94); MS=NA.Example 19: [Ala²²,23,26,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 95); MS=2832.0; Calc. MW=2831.2.Example 20: [Ala²²,23,27,32, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 96); MS=2855.0; Calc. MW=2846.2.Example 21: [Ala²²,23,26,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 97); MS=2729.0; Calc. MW=2732.0.Example 22: [Ala²²,23,27,31, 3-Pal¹⁹, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 98); MS=2711.6; Calc. MW=2712.0.Example 23: [Ala²²,23,27,28, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 99); MS=2712.0; Calc. MW=2713.0.Example 24: [Ala²²,23,27,29, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 100); MS=2746.9; Calc. MW=2747.1.Example 25: [Ala^23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 101); MS=2777.0; Calc. MW=2,775.1.Example 26: [Ala²⁰,22,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 102); MS=2742.0; Calc. MW=2747.1.Example 27: [Ala²²,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 103); MS=2786.7; Calc. MW=2789.1.Example 28: [Ala¹⁷,22,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 104); MS=2771.0; Calc. MW=2773.1.Example 29: [D-Ala¹⁰, Ala²²,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂; MS=2802.0; Calc. MW=2803.2.Example 30: [D-Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31]hGLP-1(7-34)-NH₂; MS=2905.0; Calc. MW=2901.3.Example 31: [Ala¹⁷,23,27, 3-Pal¹⁹,26,31]hGLP-1(7-34)-NH₂ (SEQ ID NO: 105); MS=2920.0; Calc. MW=2921.3.Example 32: [D-Ala⁸, Ala¹⁷, 3-Pal^19,31]hGLP-1(7-34)-NH₂; MS=2908.0 (Na.sup.+ salt); Calc. MW=2885.3.Example 33: [Ala¹⁷,23,27, 3-Pal^19,31]hGLP-1(7-34)-NH₂ (SEQ ID NO: 106); MS=2907.0; Calc. MW=2901.3.Example 34: [D-Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31, Tle²⁹]hGLP-1(7-34)-NH₂; MS=2906.0; Calc. MW=2901.3.Example 35: [D-Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31, Tle¹⁶]hGLP-1(7-34)-NH₂; MS=2914.0; Calc. MW=2915.4.Example 36: [D-Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; MS=2856.8; Calc. MW=2858.2.Example 37: [D-Ala²², Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; MS=2871.0; Calc. MW=2872.3.Example 38: [Aib⁸, Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 107); MS=2875.0; Calc. MW=2872.3.Example 39: [D-Ala⁸, Ala¹⁷,22,23,27, 3-Pal^19,31]hGLP-1(7-33)-NH₂; MS=2786.0; Calc. MW=2787.2.Example 40: [Aib⁸ Ala¹⁷,22,23,27, 3-Pal^19,31]hGLP-1(7-33)-NH₂ (SEQ ID NO: 108); MS=2800.0; Calc. MW=2801.2.Example 41: [Ala¹⁷,18,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 109); MS=2842.5; Calc. MW=2842.2.Example 42: [Ala¹⁷,23,27, 3-Pal^19,31, Tle³³, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 110); MS=2871.0; Calc. MW=2872.3.Example 43: [Tle¹⁶, Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 111); MS=2870.0; Calc. MW=2872.3.Example 44: [N-Me-D-Ala⁸, Ala¹⁷,22,23,27, 3-Pal^19,31]hGLP-1(7-33)-NH₂; MS=2795.0; Calc. MW=2801.2.Example 45: [Aib⁸, Ala¹⁷,18,22,23,27, 3-Pal^19,31]hGLP-1(7-33)-NH₂ (SEQ ID NO: 112); MS=2784.2; Calc. MW=2785.2.Example 46: [Ala¹⁷,18,22,23,27, 3-Pal^19,31, Tle^16,20 Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 113); MS=2871.9; Calc. MW=2870.3.Example 47: [D-Ala⁸, Ala¹⁷,18,22,23,27, 3-Pal^19,31, Tle¹⁶, Gaba³⁴]hGLP-1(7-34)-NH₂; MS=2870.0; Calc. MW=2870.3.Example 48: [D-Ala^8,22, Ala¹⁷,18,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; MS=2856.3; Calc. MW=2856.3.Example 49: [D-Ala^8,18, Ala¹⁷,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; MS=NA.Example 50: [D-Ala⁸, Ala¹⁷,18,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; MS=NA.Example 51: [D-Ala⁸, Ala¹⁷,18,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; MS=2861.6; Calc. MW=2856.3.

EXAMPLE 52

[Aib⁸, A6c³²]hGLP-1(7-36)NH₂ (SEQ ID NO: 114)

[0070]The title peptide was synthesized on an Applied Biosystems (Foster City, Calif.) model 430A peptide synthesizer which was modified to do accelerated Boc-chemistry solid phase peptide synthesis. See Schnolzer, et al., Int. J. Peptide Protein Res., 40:180 (1992). 4-Methylbenzhydrylamine (MBHA) resin (Peninsula, Belmont, Calif.) with the substitution of 0.91 mmol/g was used. The Boc amino acids (Bachem, Calif., Torrance, Calif.; Nova Biochem., LaJolla, Calif.) were used with the following side chain protection: Boc-Ala-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHex)-OH, Boc-Tyr(2BrZ)-OH, Boc-His(DNP)--OH, Boc-Val-OH, Boc-Leu-OH, Boc-Gly-OH, Boc-Gln-OH, Boc-Ile-OH, Boc-Lys(2CIZ)-OH, Boc-Thr(Bzl)-OH, Boc-A6c-OH, Ser(Bzl)-OH, Boc-Phe-OH, Boc-Aib-OH, Boc-Glu(OcHex)-OH and Boc-Trp(Fm)--OH. The synthesis was carried out on a 0.20 mmol scale. The Boc groups were removed by treatment with 100% TFA for 2×1 min. Boc amino acids (2.5 mmol) were pre-activated with HBTU (2.0 mmol) and DIEA (1.0 ml) in 4 ml of DMF and were coupled without prior neutralization of the peptide-resin TFA salt. Coupling times were about 5 min except for the Boc-Aib-OH and Boc-A6c-OH residues and the following residues, Boc-Trp(Fm)-OH and Boc-His(DNP)--OH wherein the coupling times were about 2 hours.

[0071]At the end of the assembly of the peptide chain, the resin was treated with a solution of 20% mercaptoethanol/10% DIEA in DMF for 2×30 min to remove the DNP group on the His side chain. The N-terminal Boc group was then removed by treatment with 100% TFA for 2×2 min. After neutralization of the peptide-resin with 10% DIEA in DMF (1×1 min), the formyl group on the side of the chain of Trp was removed by treatment with a solution of 15% ethanolamine/15% water/70% DMF for 2×30 min. The partially-deprotected peptide-resin was washed with DMF and DCM and dried under reduced pressure. The final cleavage was done by stirring the peptide-resin in 10 ml of HF containing 1 ml of anisole and dithiothreitol (24 mg) at 0° C. for about 75 min. HF was removed with a flow of nitrogen. The residue was washed with ether (6×10 ml) and extracted with 4N HOAc (6×10 ml).

[0072]The peptide mixture in the aqueous extract was purified on a reverse-phase preparative high pressure liquid chromatography (HPLC) using a reverse phase VYDAC® C₁₈ column (Nest Group, Southborough, Mass.). The column was eluted with a linear gradient (20% to 50% of solution B over 105 min) at a flow rate of 10 ml/min (Solution A=water containing 0.1% TFA; Solution B=acetonitrile containing 0.1% of TFA). Fractions were collected and checked on analytical HPLC. Those containing pure product were combined and lyophilized to dryness. 92 mg of a white solid was obtained. Purity was >99% based on analytical HPLC analysis. Electro-spray mass spectrometer analysis gave the molecular weight at 3324.2 (the calculated molecular weight is 3323.7).

[0073]The synthesis of other compounds of the present invention can be carried out in the same manner as described for the synthesis of [Aib⁸, A6c³²]hGLP-1(7-36)NH₂ (SEQ ID NO: 114) in Example 52 above but using the appropriate protected amino acids depending on the desired peptide.

[0074][(N.sup.α-HEPES-His)₇]hGLP-1(7-36)NH₂ (SEQ ID NO: 152) {HEPES is (4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid)} can be synthesized as follows: After assembly of the peptide long chain on MBHA resin (0.20 mmol), the peptide-resin is treated with 100% TFA (2×2 min.) and washed with DMF and DCM. The resin is then neutralized with 10% DIEA in DMF for about 2 min. After washing with DMF and DCM, the resin is treated with 0.23 mmol of 2-chloro-1-ethanesulfonyl chloride and 0.7 mmol of DIEA in DMF for about 1 hour. The resin is washed with DMF and DCM and treated with 1.2 mmol of 2-hydroxyethylpiperazine for about 2 hours. The resin is washed with DMF and DCM and treated with different reagents ((1) 20% mercaptoethanol/10% DIEA in DMF and (2) 15% ethanolamine/15% water/70% DMF) to remove the DNP group from the His side chain and formyl group on the Trp side chain as described above before the final HF cleavage of the peptide from the resin.

[0075][(N.sup.α-HEPA-His)₇]hGLP-1(7-36)NH₂ (SEQ ID NO: 153) ([(4-(2-hydroxyethyl)-1-piperazineacetyl)-His⁷]hGLP-1(7-36)NH.s- ub.2) can be made substantially according to the procedure described immediately above for making [(N.sup.α-HEPES-His)⁷]hGLP-1(7-36)NH₂ (SEQ ID NO: 152) except that 2-bromo-acetic anhydride is used in place of 2-chloro-1-ethanesulfonyl chloride.

EXAMPLES 53-90 and 104

[0076]Examples 53-90 and 104 were made substantially according to Example 52 but using the appropriate protected amino acid.

Example 53: [A6c^20,32]hGLP-1(7-36)-NH₂ (SEQ ID NO:115); MS=3322.3; Calc. MW=3321.7.Example 54: [Aib⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 116); MS=3311.7; Calc. MW=3311.7.Example 55: [(Tma-His)⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 117); MS=3395.9; Calc. MW=3396.9.Example 56: [A6c⁸]hGLP-1(8-36)-NH₂ (SEQ ID NO: 118); MS=3214.5; Calc. MW=3214.7.Example 57: [A6c⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO:119); MS=3351.5; Calc. MW=3351.8.Example 58: [A6c^16,20]hGLP-1(7-36)-NH₂ (SEQ ID NO: 120); MS=3335.9; Calc. MW=3335.8.Example 59: [A6c^29,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 121); MS=3321.7; Calc. MW=3321.7.Example 60: [A6c²⁰, Aib²⁴]hGLP-1(7-36)-NH₂ (SEQ ID NO: 122); MS=3323.6; Calc. MW=3323.7.Example 61: [Aib²⁴, A6c^29,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 123); MS=3335.7; Calc. MW=3335.8.Example 62: [A6c^16,29,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 124); MS=3347.7; Calc. MW=3347.8.Example 63: [Ura⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 125); MS=3279.5; Calc. MW=3280.7.Example 64: [Paa⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 126); MS=3290.9; Calc. MW=3291.8.Example 65: [Pta⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 127); MS=3311.2; Calc. MW=3311.8.Example 66: [N-Me-Ala⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 128); MS=3311.4; Calc. MW=3311.7.Example 67: [N-Me-D-Ala⁸]hGLP-1(7-36)-NH₂; MS=3311.6; Calc. MW=3311.7.Example 68: [N-Me-D-Ala⁸]hGLP-1(8-36)-NH₂; MS=3174.0; Calc. MW=3174.6.Example 69: [N-Me-Gly⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 129); MS=3297.3; Calc. MW=3297.7.Example 70: [A5c⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 130); MS=3337.3; Calc. MW=3337.8.Example 71: [N-Me-Glu⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO: 131); MS=3311.4; Calc. MW=3311.7.Example 72; [A5c⁸, A6c^20,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 132); MS=3361.4; Calc. MW=3361.8.Example 73: [Aib⁸, A6c³²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 133); MS=3323.2; Calc. MW=3323.7.Example 74: [Aib^8,25]hGLP-1(7-36)-NH₂ (SEQ ID NO: 134); MS=3325.8; Calc. MW=3325.7.Example 75: [Aib^8,24]hGLP-1(7-36)-NH₂ (SEQ ID NO: 135); MS=3325.8; Calc. MW=3325.7.Example 76: [Aib^8,30]hGLP-1(7-36)-NH₂ (SEQ ID NO: 136); MS=3326.1; Calc. MW=3325.7.Example 77: [Aib⁸, Cha²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 137); MS=3351.8; Calc. MW=3351.8.Example 78: [Aib⁸, Cha³²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 138); MS=3352.0; Calc. MW=3351.8.Example 79: [Aib⁸, Glu²³]hGLP-1(7-36)-NH₂ (SEQ ID NO: 139); MS=3311.7; Calc. MW=3312.7.Example 80: [Aib⁸, A6c²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 140); MS=3323.6; Calc. MW=3323.7.Example 81: [Aib⁸, A6c^20,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 141); MS=3335.3; Calc. MW=3335.7.Example 82: [Aib⁸, Lys²⁵]hGLP-1(7-36)-NH₂ (SEQ ID NO: 142); MS=3339.8; Calc. MW=3339.8.Example 83: [Aib⁸, β-Ala²²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 143); MS=3325.6; Calc. MW=3325.8.Example 84: [Aib⁸, Lys²⁵]hGLP-1(7-36)-NH₂ (SEQ ID NO: 144); MS=3369.0; Calc. MW=3368.8.Example 85: [Aib⁸, A6c¹²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 145); MS=3289.8; Calc. MW=3289.7.Example 86: [Aib⁸, A6c²⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO:146); MS=3323.9; Calc. MW=3323.7.Example 87: [Aib⁸, A6c³]hGLP-1(7-36)-NH₂ (SEQ ID NO: 147); MS=3338.0; Calc. MW=3337.8.Example 88: [Aib^8,14]hGLP-1(7-36)NH₂ (SEQ ID NO: 148); MS=3309.8; Calc. MW=3309.7.Example 89: [Aib^8,18]hGLP-1(7-36)NH₂ (SEQ ID NO: 149); MS=3309.7; Calc. MW=3309.7.Example 90: [Aib^8,7]hGLP-1(7-36)NH₂ (SEQ ID NO; 150); MS=3309.4; Calc. MW=3309.7.Example 104: [Aib⁸, D-Arg;²⁶]hGLP-1(7-36)NH₂; MS=3310.7; Calc. MW=3311.73.

EXAMPLE 91

[Aib⁸, A5c³³]hGLP-1(7-36)NH₂ (SEQ ID NO: 154)

[0077]The title compound can be made substantially according to Example 52 using the appropriate protected amino acids.

EXAMPLE 92

[Aib⁸, A6c³², Lys³⁶(N.sup.ε-tetradecanoyl)]hGLP-1(7-36)NH₂ (SEQ ID NO: 155)

[0078]The Boc amino acids to be used are the same as those in the synthesis of [Aib⁸, A6c³²]hGLP-1(7-36)NH₂ (SEQ ID NO: 114) (Example 52) except that Fmoc-Lys(Boc)-OH is used here for the Lys³⁶(N.sup.ε-tetradecanoyl) residue. The first amino acid residue is coupled to the resin manually on a shaker. 2.5 mmol of Fmoc-Lys(Boc)-OH is dissolved in 4 ml of 0.5N HBTU in DMF. To the solution is added 1 ml of DIEA. The mixture is shaken for about 2 min. To the solution is then added 0.2 mmol of MBHA resin (substitution=0.91 mmol/g). The mixture is shaken for about 1 hr. The resin is washed with DMF and treated with 100% TFA for 2×2 min to remove the Boc protecting group. The resin is washed with DMF. Myristic acid (2.5 mmol) is pre-activated with HBTU (2.0 mmol) and DIEA (1.0 ml) in 4 ml of DMF for 2 min and is coupled to the Fmoc-Lys-resin. The coupling time is about 1 hr. The resin is washed with DMF and treated with 25% piperidine in DMF for 2×20 min to remove the Fmoc protecting group. The resin is washed with DMF and transferred to the reaction vessel of the peptide synthesizer. The remainder of the synthesis and purification procedures of the peptide are the same as those in the synthesis of [Aib⁸, A6c³²]hGLP-1(7-36)NH₂. (SEQ ID NO: 114)

[0079]The syntheses of other compounds containing Lys(N.sup.ε-alkanoyl) residue are carried out in an analogous manner as described for the synthesis of [Aib⁸, A6c³², Lys³⁶(N.sup.ε-tetradecanoyl)]hGLP-1(7-36)NH₂ SEQ ID NO: 155). Fmoc-Lys(Boc)-OH amino acid is used for the residue of Lys(N.sup.ε-alkanoyl) in the peptide, while Boc-Lys(2CIZ)-OH amino acid is used for the residue of Lys. If the Lys(N.sup.ε-alkanoyl) residue is not at the C-terminus, the peptide fragment immediately prior to the Lys(N.sup.ε-alkanoyl) residue is assembled on the resin on the peptide synthesizer first.

EXAMPLES 93-98

[0080]Examples 93-98 can be made substantially according to the procedure described for Example 92 using the appropriate amino acids.

Example 93: [Aib⁸, A6c³², Lys³⁶(N.sup.ε-tetradecanoyl)]hGLP-1(7-36)NH₂ (SEQ ID NO: 155)Example 94: [Aib⁸, Arg^26,34, A6c³², Lys³⁶ (N.sup.ε-tetradecanoyl)]hGLP-1(7-36)NH₂ (SEQ ID NO: 156)Example 95: [Aib⁸, Arg²⁸, A6c³², Lys³⁴(N.sup.ε-tetradecanoyl)]hGLP-1(7-36)NH₂ (SEQ ID NO: 157)Example 96: [Aib⁸, Lys²⁶(N.sup.ε-tetradecanoyl), A6c³², Arg³⁴]hGLP-1(7-36)NH₂ (SEQ ID NO: 158)Example 97: [Aib⁸, Lys³⁶(N.sup.ε-octanoyl)]hGLP-1(7-36)NH₂ (SEQ ID NO: 159)Example 98: [Aib⁸, A6c^20,32, Lys³⁶(N.sup.ε-octanoyl)]hGLP-1(7-36)NH₂ (SEQ ID NO: 160)

EXAMPLE 99

[Aib⁸, Arg^26,34, A6c³², Lys³⁶(N.sup.ε-tetradecanoyl)]hGLP-1(7-36)-OH (SEQ ID NO: 161)

[0081]The Boc amino acids to be used are the same as those used in the synthesis of [Aib⁸, A6c³², Lys³⁶(N.sup.ε-tetradecanoyl)]hGLP-1(7-36)NH₂ (SEQ ID NO: 162) (Example 92). Fmoc-Lys(Boc)-OH (2.5 mmol) is pre-activated with HBTU (2.0 mmol), HOBt (2.0 mmol) and DIEA (2.5 ml) in DMF (4 ml) for about 2 min. This amino acid is coupled to 235 mg of PAM resin (Chem-Impex, Wood Dale, Ill.; substitution=0.85 mmol/g) manually on a shaker. The coupling time is about 8 hrs. The remainder of the synthesis and purification procedures for making the peptide are the same as those described in Example 52.

[0082]The syntheses of other analogs of hGLP-1(7-36)-OH (SEQ ID NO: 3) and hGLP-1(7-37)-OH, (SEQ ID NO: 4) which contain Lys(N.sup.ε-alkanoyl) residue, are carried out in an analogous manner as described for the synthesis of [Aib⁸, Arg^26,34, A6c³², Lys³⁶(N.sup.ε-tetradecanoyl)]hGLP-1(7-36)-OH (SEQ ID NO: 161). Fmoc-Lys(Boc)-OH amino acid is used for the residue of Lys(N.sup.ε-alkanoyl) in the peptide, while Boc-Lys(2CIZ)-OH amino acid is used for the residue of Lys.

EXAMPLES 100-103

[0083]Examples 100-103 can be made substantially according to the procedure described for Example 99 using the appropriate amino acids.

Example 100: [Aib⁸, Arg²⁶, A6c³², Lys³⁴(NF-tetradecanoyl)]hGLP-1(7-36)-OH (SEQ ID NO: 162)Example 101: [Aib⁸, Lys²⁶(N.sup.ε-tetradecanoyl), A6c³², Arg³⁴]hGLP-1(7-36)-OH (SEQ ID NO: 163)Example 102: [Aib⁸, Arg^26,34-A6c³², Lys³⁶(N.sup.ε-tetradecanoyl)]hGLP-1(7-37)-OH (SEQ ID NO: 164)Example 103: [Aib⁸, Arg²⁶, A6c³², Lys³⁴(N.sup.ε-tetradecanoyl)]hGLP-1(7-37)-OH (SEQ ID NO: 165)

Sequence CWU 1

165130PRTArtificial SequenceDescription of Artificial Sequence Mutagen 1His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30231PRTArtificial SequenceDescription of Artificial Sequence Mutagen 2His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30330PRTHomo sapiens 3His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30431PRTHomo sapiens 4His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30530PRTArtificial SequenceDescription of Artificial Sequence Mutagen 5His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30631PRTArtificial SequenceDescription of Artificial Sequence Mutagen 6His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30730PRTArtificial SequenceDescription of Artificial Sequence Mutagen 7His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30831PRTArtificial SequenceDescription of Artificial Sequence Mutagen 8His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30930PRTArtificial SequenceDescription of Artificial Sequence Mutagen 9His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg 20 25 301031PRTArtificial SequenceDescription of Artificial Sequence Mutagen 10His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25 301130PRTArtificial SequenceDescription of Artificial Sequence Mutagen 11His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg 20 25 301231PRTArtificial SequenceDescription of Artificial Sequence Mutagen 12His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25 301330PRTArtificial SequenceDescription of Artificial Sequence Mutagen 13His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg 20 25 301431PRTArtificial SequenceDescription of Artificial Sequence Mutagen 14His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25 301530PRTArtificial SequenceDescription of Artificial Sequence Mutagen 15His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg 20 25 301631PRTArtificial SequenceDescription of Artificial Sequence Mutagen 16His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25 301729PRTArtificial SequenceDescription of Artificial Sequence Mutagen 17Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10 15Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg 20 251830PRTArtificial SequenceDescription of Artificial Sequence Mutagen 18Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10 15Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25 301929PRTArtificial SequenceDescription of Artificial Sequence Mutagen 19Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10 15Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg 20 252030PRTArtificial SequenceDescription of Artificial Sequence Mutagen 20Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10 15Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Xaa Gly Arg Gly 20 25 302130PRTArtificial SequenceDescription of Artificial Sequence Mutagen 21His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Xaa 20 25 302231PRTArtificial SequenceDescription of Artificial Sequence Mutagen 22His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Xaa Gly 20 25 302330PRTArtificial SequenceDescription of Artificial Sequence Mutagen 23His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Xaa 20 25 302431PRTArtificial SequenceDescription of Artificial Sequence Mutagen 24His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Xaa Gly 20 25 302530PRTHomo sapiens 25His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 302631PRTHomo sapiens 26His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 302730PRTArtificial SequenceDescription of Artificial Sequence Mutagen 27His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 302831PRTArtificial SequenceDescription of Artificial Sequence Mutagen 28His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 302930PRTHomo sapiens 29His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg 20 25 303031PRTHomo sapiens 30His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25 303130PRTArtificial SequenceDescription of Artificial Sequence Mutagen 31His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg 20 25 303231PRTArtificial SequenceDescription of Artificial Sequence Mutagen 32His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25 303330PRTHomo sapiens 33His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg 20 25 303431PRTHomo sapiens 34His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25 303530PRTArtificial SequenceDescription of Artificial Sequence Mutagen 35His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg 20 25 303631PRTArtificial SequenceDescription of Artificial Sequence Mutagen 36His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25 303730PRTHomo sapiens 37His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Lys 20 25 303831PRTHomo sapiens 38His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Lys Gly 20 25 303930PRTArtificial SequenceDescription of Artificial Sequence Mutagen 39His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Lys 20 25 304031PRTArtificial SequenceDescription of Artificial Sequence Mutagen 40His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Lys Gly 20 25 304130PRTHomo sapiens 41His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Lys 20 25 304231PRTHomo sapiens 42His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Lys Gly 20 25 304330PRTArtificial SequenceDescription of Artificial Sequence Mutagen 43His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Lys 20 25 304431PRTArtificial SequenceDescription of Artificial Sequence Mutagen 44His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Lys Gly Lys Gly 20 25 304530PRTHomo sapiens 45His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Lys 20 25 304631PRTHomo sapiens 46His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Lys Gly 20 25 304730PRTArtificial SequenceDescription of Artificial Sequence Mutagen 47His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Lys 20 25 304831PRTArtificial SequenceDescription of Artificial Sequence Mutagen 48His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Lys Gly 20 25 304930PRTHomo sapiens 49His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Lys 20 25 305031PRTHomo sapiens 50His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Lys Gly 20 25 305130PRTArtificial SequenceDescription of Artificial Sequence Mutagen 51His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Lys 20 25 305231PRTArtificial SequenceDescription of Artificial Sequence Mutagen 52His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Leu Val Arg Gly Lys Gly 20 25 305330PRTHomo sapiens 53His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 305431PRTHomo sapiens 54His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 305530PRTArtificial SequenceDescription of Artificial Sequence Mutagen 55His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 305631PRTArtificial SequenceDescription of Artificial Sequence Mutagen 56His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Asp Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 305730PRTArtificial SequenceDescription of Artificial Sequence Mutagen 57His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 305831PRTArtificial SequenceDescription of Artificial Sequence Mutagen 58His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 305930PRTArtificial SequenceDescription of Artificial Sequence Mutagen 59His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 306031PRTArtificial SequenceDescription of Artificial Sequence Mutagen 60His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 306130PRTHomo sapiens 61His Ala Asp Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 306231PRTHomo sapiens 62His Ala Asp Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 306330PRTArtificial SequenceDescription of Artificial Sequence Mutagen 63His Ala Asp Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg

20 25 306431PRTArtificial SequenceDescription of Artificial Sequence Mutagen 64His Ala Asp Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 306530PRTHomo sapiens 65Tyr Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 306631PRTHomo sapiens 66Tyr Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 306730PRTArtificial SequenceDescription of Artificial Sequence Mutagen 67Tyr Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 306831PRTArtificial SequenceDescription of Artificial Sequence Mutagen 68Tyr Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 306930PRTArtificial SequenceDescription of Artificial Sequence Mutagen 69Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 307031PRTArtificial SequenceDescription of Artificial Sequence Mutagen 70Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 307130PRTArtificial SequenceDescription of Artificial Sequence Mutagen 71Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 307231PRTArtificial SequenceDescription of Artificial Sequence Mutagen 72Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 307330PRTArtificial SequenceDescription of Artificial Sequence Mutagen 73Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 307431PRTArtificial SequenceDescription of Artificial Sequence Mutagen 74Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 307530PRTArtificial SequenceDescription of Artificial Sequence Mutagen 75Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 307631PRTArtificial SequenceDescription of Artificial Sequence Mutagen 76Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 307729PRTArtificial SequenceDescription of Artificial Sequence Mutagen 77Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10 15Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 257830PRTArtificial SequenceDescription of Artificial Sequence Mutagen 78Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10 15Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 307930PRTHomo sapiens 79His Ala Glu Gly Thr Phe Thr Ser Glu Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 308031PRTHomo sapiens 80His Ala Glu Gly Thr Phe Thr Ser Glu Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 308130PRTHomo sapiens 81His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Arg 20 25 308231PRTHomo sapiens 82His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Phe Leu Val Lys Gly Arg Gly 20 25 308329PRTArtificial SequenceDescription of Artificial Sequence Mutagen 83His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ala Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Lys Gly 20 258429PRTArtificial SequenceDescription of Artificial Sequence Mutagen 84His Ala Glu Gly Thr Phe Thr Ser Asp Ala Ser Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Lys Gly 20 258529PRTArtificial SequenceDescription of Artificial Sequence Mutagen 85His Ala Glu Gly Thr Phe Thr Ala Asp Val Ser Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Lys Gly 20 258629PRTArtificial SequenceDescription of Artificial Sequence Mutagen 86His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Lys Gly 20 258730PRTArtificial SequenceDescription of Artificial Sequence Mutagen 87Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 308829PRTArtificial SequenceDescription of Artificial Sequence Mutagen 88His Ala Glu Gly Thr Phe Thr Ser Ala Val Ser Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Lys Gly 20 258929PRTArtificial SequenceDescription of Artificial Sequence Mutagen 89His Ala Glu Gly Thr Phe Thr Ser Asp Val Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Lys Gly 20 259028PRTArtificial SequenceDescription of Artificial Sequence Mutagen 90His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Xaa 20 259128PRTArtificial SequenceDescription of Artificial Sequence Mutagen 91His Ala Glu Gly Thr Phe Thr Ser Ala Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Xaa 20 259228PRTArtificial SequenceDescription of Artificial Sequence Mutagen 92His Ala Glu Gly Thr Phe Thr Ser Asp Val Ala Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Xaa 20 259328PRTArtificial SequenceDescription of Artificial Sequence Mutagen 93His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ala Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Xaa 20 259428PRTArtificial SequenceDescription of Artificial Sequence Mutagen 94His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Ala Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Xaa 20 259528PRTArtificial SequenceDescription of Artificial Sequence Mutagen 95His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Ala Ala Phe Ile Ala Xaa Leu Val Xaa 20 259628PRTArtificial SequenceDescription of Artificial Sequence Mutagen 96His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Ala Val Xaa 20 259727PRTArtificial SequenceDescription of Artificial Sequence Mutagen 97His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Ala Ala Phe Ile Ala Xaa Leu Xaa 20 259827PRTArtificial SequenceDescription of Artificial Sequence Mutagen 98His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Ala Leu Xaa 20 259927PRTArtificial SequenceDescription of Artificial Sequence Mutagen 99His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Ala Ile Ala Xaa Leu Xaa 20 2510027PRTArtificial SequenceDescription of Artificial Sequence Mutagen 100His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ala Ala Xaa Leu Xaa 20 2510127PRTArtificial SequenceDescription of Artificial Sequence Mutagen 101His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Xaa 20 2510227PRTArtificial SequenceDescription of Artificial Sequence Mutagen 102His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Ala Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Xaa 20 2510327PRTArtificial SequenceDescription of Artificial Sequence Mutagen 103His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Xaa 20 2510427PRTArtificial SequenceDescription of Artificial Sequence Mutagen 104His Ala Glu Gly Thr Phe Thr Ser Asp Val Ala Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Xaa 20 2510528PRTArtificial SequenceDescription of Artificial Sequence Mutagen 105His Ala Glu Gly Thr Phe Thr Ser Asp Val Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Xaa Ala Phe Ile Ala Xaa Leu Val Lys 20 2510628PRTArtificial SequenceDescription of Artificial Sequence Mutagen 106His Ala Glu Gly Thr Phe Thr Ser Asp Val Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Lys 20 2510728PRTArtificial SequenceDescription of Artificial Sequence Mutagen 107His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Xaa 20 2510827PRTArtificial SequenceDescription of Artificial Sequence Mutagen 108His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ala Ser Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val 20 2510928PRTArtificial SequenceDescription of Artificial Sequence Mutagen 109His Ala Glu Gly Thr Phe Thr Ser Asp Val Ala Ala Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Xaa 20 2511028PRTArtificial SequenceDescription of Artificial Sequence Mutagen 110His Ala Glu Gly Thr Phe Thr Ser Asp Val Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Xaa Xaa 20 2511128PRTArtificial SequenceDescription of Artificial Sequence Mutagen 111His Ala Glu Gly Thr Phe Thr Ser Asp Xaa Ala Ser Xaa Leu Glu Gly 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Xaa 20 2511227PRTArtificial SequenceDescription of Artificial Sequence Mutagen 112His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ala Ala Xaa Leu Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val 20 2511328PRTArtificial SequenceDescription of Artificial Sequence Mutagen 113His Ala Glu Gly Thr Phe Thr Ser Asp Xaa Ala Ala Xaa Xaa Glu Ala 1 5 10 15Ala Ala Ala Lys Ala Phe Ile Ala Xaa Leu Val Xaa 20 2511430PRTArtificial SequenceDescription of Artificial Sequence Mutagen 114His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Arg 20 25 3011530PRTArtificial SequenceDescription of Artificial Sequence Mutagen 115His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Arg 20 25 3011630PRTArtificial SequenceDescription of Artificial Sequence Mutagen 116His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3011730PRTArtificial SequenceDescription of Artificial Sequence Mutagen 117Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3011829PRTArtificial SequenceDescription of Artificial Sequence Mutagen 118Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln 1 5 10 15Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 2511930PRTArtificial SequenceDescription of Artificial Sequence Mutagen 119His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3012030PRTArtificial SequenceDescription of Artificial Sequence Mutagen 120His Ala Glu Gly Thr Phe Thr Ser Asp Xaa Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3012130PRTArtificial SequenceDescription of Artificial Sequence Mutagen 121His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Xaa Ala Trp Xaa Val Lys Gly Arg 20 25 3012230PRTArtificial SequenceDescription of Artificial Sequence Mutagen 122His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln Xaa Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3012330PRTArtificial SequenceDescription of Artificial Sequence Mutagen 123His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Xaa Ala Lys Glu Phe Xaa Ala Trp Xaa Val Lys Gly Arg 20 25 3012430PRTArtificial SequenceDescription of Artificial Sequence Mutagen 124His Ala Glu Gly Thr Phe Thr Ser Asp Xaa Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Xaa Ala Trp Xaa Val Lys Gly Arg 20 25 3012530PRTArtificial SequenceDescription of Artificial Sequence Mutagen 125Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3012630PRTArtificial

SequenceDescription of Artificial Sequence Mutagen 126Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3012730PRTArtificial SequenceDescription of Artificial Sequence Mutagen 127Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3012830PRTArtificial SequenceDescription of Artificial Sequence Mutagen 128His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3012930PRTArtificial SequenceDescription of Artificial Sequence Mutagen 129His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3013030PRTArtificial SequenceDescription of Artificial Sequence Mutagen 130His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3013130PRTArtificial SequenceDescription of Artificial Sequence Mutagen 131His Ala Xaa Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3013230PRTArtificial SequenceDescription of Artificial Sequence Mutagen 132His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Arg 20 25 3013330PRTArtificial SequenceDescription of Artificial Sequence Mutagen 133His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Arg 20 25 3013430PRTArtificial SequenceDescription of Artificial Sequence Mutagen 134His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Xaa Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3013530PRTArtificial SequenceDescription of Artificial Sequence Mutagen 135His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Xaa Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3013630PRTArtificial SequenceDescription of Artificial Sequence Mutagen 136His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Xaa Trp Leu Val Lys Gly Arg 20 25 3013730PRTArtificial SequenceDescription of Artificial Sequence Mutagen 137His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3013830PRTArtificial SequenceDescription of Artificial Sequence Mutagen 138His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Arg 20 25 3013930PRTArtificial SequenceDescription of Artificial Sequence Mutagen 139His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Glu Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3014030PRTArtificial SequenceDescription of Artificial Sequence Mutagen 140His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3014130PRTArtificial SequenceDescription of Artificial Sequence Mutagen 141His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Arg 20 25 3014230PRTArtificial SequenceDescription of Artificial Sequence Mutagen 142His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3014330PRTArtificial SequenceDescription of Artificial Sequence Mutagen 143His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3014430PRTArtificial SequenceDescription of Artificial Sequence Mutagen 144His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Lys Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3014530PRTArtificial SequenceDescription of Artificial Sequence Mutagen 145His Xaa Glu Gly Thr Xaa Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3014630PRTArtificial SequenceDescription of Artificial Sequence Mutagen 146His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Xaa Ala Trp Leu Val Lys Gly Arg 20 25 3014730PRTArtificial SequenceDescription of Artificial Sequence Mutagen 147His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Xaa Lys Gly Arg 20 25 3014830PRTArtificial SequenceDescription of Artificial Sequence Mutagen 148His Xaa Glu Gly Thr Phe Thr Xaa Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3014930PRTArtificial SequenceDescription of Artificial Sequence Mutagen 149His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Xaa Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3015030PRTArtificial SequenceDescription of Artificial Sequence Mutagen 150His Xaa Glu Gly Thr Phe Thr Ser Asp Val Xaa Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3015130PRTArtificial SequenceDescription of Artificial Sequence Mutagen 151His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3015230PRTArtificial SequenceDescription of Artificial Sequence Mutagen 152Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3015330PRTArtificial SequenceDescription of Artificial Sequence Mutagen 153Xaa Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 3015430PRTArtificial SequenceDescription of Artificial Sequence Mutagen 154His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Xaa Lys Gly Arg 20 25 3015530PRTArtificial SequenceDescription of Artificial Sequence Mutagen 155His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Xaa 20 25 3015630PRTArtificial SequenceDescription of Artificial Sequence Mutagen 156His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Xaa Val Arg Gly Xaa 20 25 3015730PRTArtificial SequenceDescription of Artificial Sequence Mutagen 157His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Xaa Val Xaa Gly Arg 20 25 3015830PRTArtificial SequenceDescription of Artificial Sequence Mutagen 158His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Xaa Val Arg Gly Arg 20 25 3015930PRTArtificial SequenceDescription of Artificial Sequence Mutagen 159His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Xaa 20 25 3016030PRTArtificial SequenceDescription of Artificial Sequence Mutagen 160His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Xaa Glu Gly 1 5 10 15Gln Ala Ala Lys Glu Phe Ile Ala Trp Xaa Val Lys Gly Xaa 20 25 3016130PRTArtificial SequenceDescription of Artificial Sequence Mutagen 161His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Xaa Val Arg Gly Xaa 20 25 3016230PRTArtificial SequenceDescription of Artificial Sequence Mutagen 162His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Xaa Val Xaa Gly Arg 20 25 3016330PRTArtificial SequenceDescription of Artificial Sequence Mutagen 163His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Xaa Glu Phe Ile Ala Trp Xaa Val Arg Gly Arg 20 25 3016431PRTArtificial SequenceDescription of Artificial Sequence Mutagen 164His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Xaa Val Arg Gly Xaa Gly 20 25 3016531PRTArtificial SequenceDescription of Artificial Sequence Mutagen 165His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15Gln Ala Ala Arg Glu Phe Ile Ala Trp Xaa Val Xaa Gly Arg Gly 20 25 30

Claims

1. A compound of formula (I),(R²R³)-A⁷-A⁸-A⁹-A¹⁰-A¹¹-A¹²-A- ¹³-A¹⁴-A¹⁵-A¹⁶-A¹⁷-A¹⁸-A¹⁹-A²⁰-A.s- up.21-A²²-A²³-A²⁴-A²⁵-A²⁶-A²⁷-A²⁸-A.sup- .29-A³⁰-A³¹-A³²-A³³-A³⁴-A³⁵-A³⁶-A³- 7-R¹, (I)whereinA⁷ is L-His, Ura, Paa, Pta, D-His, Tyr, 3-Pal, 4-Pal, Hppa, Tma-His, Amp or deleted, provided that when A⁷ is Ura, Paa, Pta or Hppa then R² and R³ are deleted;A⁸ is Ala, D-Ala, Aib, Acc, N-Me-Ala, N-Me-D-Ala, Arg or N-Me-Gly;A⁹ is Glu, N-Me-Glu, N-Me-Asp or Asp;A¹⁰ is Gly, Acc, Ala, D-Ala, Phe or Aib;A¹¹ is Thr or Ser;A¹² is Phe, Acc, Aic, Aib, 3-Pal, 4-Pal, β-Nal, Cha, Trp or X¹-Phe;A¹³ is Thr or Ser;A¹⁴ is Ser, Thr, Ala or Aib;A¹⁵ is Asp, Ala, D-Asp or Glu;A¹⁶ is Val, D-Val, Acc, Aib, Leu, Ile, Tle, Nle, Abu, Ala, D-Ala, Tba or Cha;A¹⁷ is Ser, Ala, D-Ala, Aib, Acc or Thr;A¹³ is Ser, Ala, D-Ala, Aib, Acc or Thr;A¹⁹ is Tyr, D-Tyr, Cha, Phe, 3-Pal, 4-Pal, Acc, β-Nal, Amp or X¹-Phe;A²⁰ is Leu, Ala, Acc, Aib, Nle, Ile, Cha, Tle, Val, Phe or X¹-Phe;A²¹ is Glu, Ala or Asp;A²² is Gly, Acc, Ala, D-Ala, β-Ala or Aib;A²³ is Gln, Asp, Ala, D-Ala, Aib, Acc, Asn or Glu;A²⁴ is Ala, Aib, Val, Abu, Tle or Acc;A²⁵ is Ala, Aib, Val, Abu, Tle, Acc, Lys, Arg, hArg, Orn, HN--CH((CH₂)_n--NR¹⁰R¹¹)--C(O) or HN--CH((CH₂)_e--X³)--C(O);A²⁶ is Lys, Ala, 3-Pal, 4-Pal, Arg, hArg, Orn, Amp, HN--CH((CH₂)_n--NR¹⁰R¹¹)--C(O) or HN--CH((CH₂)_e--X³)--C(O);A²⁷ is Glu, Ala, D-Ala or Asp;A²⁸ is Phe, Ala, Pal, β-Nal, X¹-Phe, Aic, Acc, Aib, Cha or Trp;A²⁹ is Ile, Acc, Aib, Leu, Nle, Cha, Tle, Val, Abu, Ala, Tba or Phe;A³⁰ is Ala, Aib, Acc or deleted;A³¹ is Trp, Ala, β-Nal, 3-Pal, 4-Pal, Phe, Acc, Aib, Cha, Amp or deleted;A³² is Leu, Ala, Acc, Aib, Nle, Ile, Cha, Tle, Phe, X¹-Phe, Ala or deleted;A³³ is Val, Acc, Aib, Leu, Ile, Tle, Nle, Cha, Ala, Phe, Abu, X¹-Phe, Tba, Gaba or deleted;A³⁴ is Lys, Arg, hArg, Orn, Amp, Gaba, HN--CH((CH₂), --NR¹⁰R¹¹)--C(O), HN--CH((CH₂)_e--X³)--C(O) or deleted;A³⁵ is Gly or deleted;A³⁶ is L- or D-Arg, D- or L-Lys, D- or L-hArg, D- or L-Orn, Amp, HN--CH((CH₂), --NR¹⁰R¹¹)--C(O), HN--CH((CH₂)_e--X³)--C(O) or deleted;A³⁷ is Gly or deleted;X¹ for each occurrence is independently selected from the group consisting of (C₁-C₆)alkyl, OH and halo;R¹ is OH, NH₂, (C₁-C₁₂)alkoxy, or NH--X²--CH₂-Z⁰, wherein X² is a (C₁-C₁₂)hydrocarbon moiety, and Z⁰ is H, OH, CO₂H or CONH₂;X³ is ##STR00011## or --C(O)--NHR¹², wherein X⁴ for each occurrence is independently --C(O)--, --NH--C(O)-- or --CH₂--, and f for each occurrence is independently an integer from 1 to 29;each of R² and R³ is independently selected from the group consisting of H, (C₁-C₃0)alkyl, (C₂-C₃0)alkenyl, phenyl(C₁-C₃0)alkyl, naphthyl(C₁-C₃0)alkyl, hydroxy(C₁-C₃0)alkyl, hydroxy(C₂-C₃0)alkenyl, hydroxyphenyl(C₁-C₃0)alkyl, and hydroxynaphthyl(C₁-C₃0)alkyl; or one of R² and R³ is C(O)X⁵ in which X⁵ is (C₁-C₃0)alkyl, (C₂-C₃0)alkenyl, phenyl(C₁-C₃0)alkyl, naphthyl(C₁-C₃0)alkyl, hydroxy(C₁-C₃0)alkyl, hydroxy(C₂-C₃0)alkenyl, hydroxyphenyl(C₁-C₃0)alkyl, hydroxynaphthyl(C₁-C₃0)alkyl, ##STR00012## where Y is H or OH, r is 0 to 4 and q is 0 to 4;e for each occurrence is independently an integer from 1 to 4;n for each occurrence is independently an integer from 1-5; andR¹⁰ and R¹¹ for each occurrence is each independently H, (C₁-C₃0)alkyl, (C₁-C₃0)acyl, (C₁-C₃0)alkylsulfonyl, --C((NH)(NH₂)) or ##STR00013## provided that when R¹⁰ is (C₁-C₃0)acyl, (C₁-C₃0)alkylsulfonyl, --C((NH)(NH₂)) or ##STR00014## R¹¹ is H or (C₁-C₃0)alkyl; andR¹² is (C₁-C₃0)alkyl;with the proviso that:(i) at least one amino acid of a compound of formula (I) is not the same as the native sequence of hGLP-1 (7-36, or -37)NH₂ (SEQ ID NOS: 1, 2) or hGLP-1 (7-36, or -37)OH (SEQ ID NOS: 3, 4);(ii) a compound of formula (I) is not an analogue of hGLP-1(7-36, or -37)NH₂ (SEQ ID NOS: 1,2) or hGLP-1(7-36, or -37)OH (SEQ ID NOS: 3, 4) wherein a single position has been substituted by Ala;(iii) a compound of formula (I) is not [Lys²⁶(N.sup.ε-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ ID NOS: 5-8), [Lys³⁴(N.sup.ε-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ ID NOS: 9-12), [Lys^26,34-bis(N.sup.ε-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ ID NOS: 13-16), [Arg²⁶, Lys³⁴(NE-alkanoyl)]hGLP-1(8-36, or -37)-E (SEQ ID NOS: 17-20), or [Arg^26,34, Lys³⁶(N.sup.ε-alkanoyl)]hGLP-1(7-36, or -37)-E, wherein E is --OH or --NH₂ (SEQ ID NOS: 21-24);(iv) a compound of formula (I) is not Z¹-hGLP-1(7-36, or -37)-OH, Z¹-hGLP-1(7-36, or -37)-NH₂, where Z¹ is selected from the group consisting of(a) [Arg²⁶] (SEQ ID NOS: 25-28), [Arg³⁴] (SEQ ID NOS: 29-32), [Arg^26,34] (SEQ ID NOS: 33-36), [Lys³⁶] (SEQ ID NOS: 37-40), [Arg²⁶, Lys³⁶] (SEQ ID NOS: 41-44), [Arg³⁴, Lys³⁶] (SEQ ID NOS: 45-46), [D-Lys³⁶], [Arg³⁶] (SEQ ID NOS: 37-40), [D-Arg³⁶], [Arg^26,34, Lys³⁶] (SEQ ID NOS: 49-52), or [Arg^26,36, Lys³⁴] (SEQ ID NOS: 25-28);(b) [Asp²¹] (SEQ ID NOS: 53-56);(c) at least one of [Aib⁸] (SEQ ID NOS: 57-60), [D-Ala⁸] and [Asp⁹] (SEQ ID NOS: 61-64); and(d) [Tyr⁷] (SEQ ID NOS: 65-68), [N-acyl-His⁷] (SEQ ID NOS: 69-72), [N-alkyl-His⁷] (SEQ ID NOS: 73-76), [N-acyl-D-His⁷] or [N-alkyl-D-His⁷];(v) a compound of formula (I) is not a combination of any two of the substitutions listed in groups (a) to (d); and(vi) a compound of formula (I) is not [N-Me-Ala⁸]hGLP-1(8-36 or -37) (SEQ ID NOS: 77, 78), [Glu¹⁵]hGLP-1(7-36 or -37) (SEQ ID NOS: 79, 80), [Asp²¹]hGLP-1(7-36 or -37) (SEQ ID NOS: 53, 54) or [Phe³¹]hGLP-1(7-36 or -37) (SEQ ID NOS: 81, 82).

2. A compound according to claim 1 or a pharmaceutically acceptable salt thereof wherein A¹¹ is Thr; A¹³ is Thr; A¹⁴ is Ser, Aib or Ala; A¹⁷ is Ser, Ala, Aib or D-Ala; A¹⁸ is Ser, Ala, Aib or D-Ala; A²¹ is Glu or Ala; A²³ is Gln, Glu, or Ala; and A²⁷ is Glu or Ala.

3. A compound according to claim 2 or a pharmaceutically acceptable salt thereof wherein A⁹ is Glu, N-Me-Glu or N-Me-Asp; A¹² is Phe, Acc or Aic; A¹⁶ is Val, D-Val, Acc, Aib, Ala, Tle or D-Ala; A¹⁹ is Tyr, 3-Pal, 4-Pal or D-Tyr; A²⁰ is Leu, Acc, Cha, Ala or Tle; A²⁴ is Ala, Aib or Acc; A²⁵ is Ala, Aib, Acc, Lys, Arg, hArg, Orn, HN--CH((CH₂), --NH--R¹⁰)--C(O); A²⁸ is Phe or Ala; A²⁹ is Ile, Acc or Tle; A³⁰ is Ala, Aib or deleted; A³¹ is Trp, Ala, 3-Pal, 4-Pal or deleted; A³² is Leu, Acc, Cha, Ala or deleted; A³³ is Val, Acc, Ala, Gaba, Tle or deleted.

4. A compound according to claim 3 or a pharmaceutically acceptable salt thereof wherein A⁸ is Ala, D-Ala, Aib, A6c, A5c, N-Me-Ala, N-Me-D-Ala or N-Me-Gly; A¹⁰ is Gly, Ala, D-Ala or Phe; A¹² is Phe, A6c or A5c; A¹⁶ is Val, Ala, Tle, A6c, A5c or D-Val; A²⁰ is Leu, A6c, A5c, Cha, Ala or Tle; A²² is Gly, Aib, β-Ala, L-Ala or D-Ala; A²⁴ is Ala or Aib; A²⁹ is Ile, A6c, A5c or Tle; A³² is Leu, A6c, A5c, Cha, Ala or deleted; A³³ is Val, A6c, A5c, Ala, Gaba, Tle or deleted.

5. A compound according to claim 4 or a pharmaceutically acceptable salt thereof wherein R¹ is OH or NH.sub.2.

6. A compound according to claim 5 or a pharmaceutically acceptable salt thereof wherein R² is H and R³ is (C₁-C₃0)alkyl, (C₂-C₃0)alkenyl, (C₁-C₃0)acyl, ##STR00015##

7. A compound according to claim 1 wherein said compound is[D-Ala⁸, Ala¹⁷,22,23,27, 3-Pal^19,31, Gaba³⁴]-GLP-1(7-34)NH₂;[D-Ala⁸,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂;[Ala¹⁸,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 83);[Ala¹⁶,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 84);[Ala¹⁴,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 85);[Ala²²,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 86);[Hppa⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 87);[Ala¹⁵,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 88);[Ala¹⁷,23,27, 3-Pal^19,31]hGLP-1(7-35)-NH₂ (SEQ ID NO: 89);[Ala²²,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 90);[Ala¹⁵,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 91);[Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 92);[Ala¹⁸,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 93);[Ala²¹,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 94);[Ala²²,23,26,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 95);[Ala²²,23,27,32, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 96);[Ala²²,23,26,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 97);[Ala²²,23,27,31, 3-Pal¹⁹, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 98);[Ala²²,23,27,28, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 99);[Ala²²,23,27,29, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 100);[Ala^23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 101);[Ala²⁰,22,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 102);[Ala²²,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 103);[Ala¹⁷,22,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 104);[D-Ala¹⁰, Ala²²,23,27, 3-Pal^19,31, Gaba³³]hGLP-1(7-33)-NH₂;[D-Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31]hGLP-1(7-34)-NH₂;[Ala¹⁷,23,27, 3-Pal¹⁹,26,31]hGLP-1(7-34)-NH₂ (SEQ ID NO: 105);[D-Ala⁸, Ala¹⁷, 3-Pal^19,31]hGLP-1(7-34)-NH₂;[Ala¹⁷,23,27, 3-Pal^19,31]hGLP-1(7-34)-NH₂ (SEQ ID NO: 106);[D-Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31, Tle²⁹]hGLP-1(7-34)-NH₂;[D-Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31, Tle¹⁶]hGLP-1(7-34)-NH₂;[D Ala⁸, Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂;[D-Ala²², Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂;[Aib⁸, Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 107);[D-Ala⁸, Ala¹⁷,22,23,27, 3-Pal^19,31]hGLP-1(7-33)-NH₂;[Aib⁸, Ala¹⁷,22,23,27, 3-Pal^19,31]hGLP-1(7-33)-NH₂ (SEQ ID NO: 108);[Ala¹⁷,18,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 109);[Ala¹⁷,23,27, 3-Pal^19,31, Tle³³, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 110);[Tle¹⁶, Ala¹⁷,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 111);[N-Me-D-Ala⁸, Ala¹⁷,22,23,27, 3-Pal^19,31]hGLP-1(7-33)-NH₂;[Aib⁸, Ala¹⁷,18,23,27, 3-Pal^19,31]hGLP-1(7-33)-NH₂ (SEQ ID NO: 112);[Ala¹⁷,18,22,23,27, 3-Pal^19,31, Tle^16,20, Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 113);[D-Ala⁸, Ala¹⁷,18,22,23,27, 3-Pal^19,31, Tle¹⁶, Gaba³⁴]hGLP-1(7-34)-NH₂;[D-Ala^8,22, Ala¹⁷,18,23,27,3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂;[D-Ala^8,18, Ala¹⁷,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂;[D-Ala^8,17, Ala¹⁸,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; or[D-Ala⁸, Ala¹⁷,18,22,23,27, 3-Pal^19,31, Gaba³⁴]hGLP-1(7-34)-NH₂; or a pharmaceutically acceptable salt thereof.

8. A compound according to claim 1 wherein said compound is[Aib⁸, A6c³²]hGLP-1(7-36)NH₂ (SEQ ID NO: 114);[A6c^20,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 115);[Aib⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 116);[(Tma-His)⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 117);[A6c⁸]hGLP-1(8-36)-NH₂ (SEQ ID NO: 118);[A6c⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 119);[A6c^16,20]hGLP-1(7-36)-NH₂ (SEQ ID NO: 120);[A6c^29,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 121);[A6c²⁰, Aib²⁴]hGLP-1(7-36)-NH₂ (SEQ ID NO: 122);[Aib²⁴, A6c^29,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 123);[A6c^16,29,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 124);[Ura⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 125);[Paa⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 126);[Pta⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 127);[N-Me-Ala⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 128);[N-Me-Ala⁸]hGLP-1(8-36)-NH₂ (SEQ ID NO: ______);[N-Me-D-Ala⁸]hGLP-1(7-36)-NH₂;[N-Me-D-Ala⁸]hGLP-1(- 8-36)-NH₂;[N-Me-Gly⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 129);[A5c⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 130);[N-Me-Glu⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO: 131);[A5c⁸, A6c^20,32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 132);[Aib⁸, A6c³²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 133);[Aib^8,25]hGLP-1(7-36)-NH₂ (SEQ ID NO: 134);[Aib⁸²⁴]hGLP-1(7-36)-NH₂ (SEQ ID NO: 135);[Aib.sup.8.30]hGLP-1(7-36)-NH₂ (SEQ ID NO: 136);[Aib⁸, Cha²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 137);[Aib⁸, Cha³²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 138);[Aib⁸, Glu²³]hGLP-1(7-36)-NH₂ (SEQ ID NO: 139);[Aib⁸, A6c²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 140);[Aib⁸, A6c.sup.20.32]hGLP-1(7-36)-NH₂ (SEQ ID NO: 141);[Aib^8,22]hGLP-1(7-36)-NH₂ (SEQ ID NO: 142);[Aib⁸, β-Ala²²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 143);[Aib⁸, Lys²⁵]hGLP-1(7-36)-NH₂ (SEQ ID NO: 144);[Aib⁸, A6c¹²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 145);[Aib⁸, A6c²⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO: 146);[Aib⁸, A6c³³]hGLP-1(7-36)-NH₂ (SEQ ID NO: 147);[Aib^8,14]hGLP-1(7-36)NH₂ (SEQ ID NO: 148);[Aib^8,18]hGLP-1(7-36)NH₂ (SEQ ID NO: 149);[Aib^8,17]hGLP-1(7-36)NH₂ (SEQ ID NO: 150); or[Aib⁸, D-Arg;²⁶]hGLP-1(7-36)NH₂; or a pharmaceutically acceptable salt thereof.

9. A pharmaceutical composition comprising an effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

10. A method of eliciting an agonist effect from a GLP-1 receptor in a subject in need thereof which comprises administering to said subject an effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof.

11. A method of treating a disease selected from the group consisting of Type I diabetes, Type II diabetes, obesity, glucagonomas, secretory disorders of the airway, metabolic disorder, arthritis, osteoporosis, central nervous system disease, restenosis, neurodegenerative disease, renal failure, congestive heart failure, nephrotic syndrome, cirrhosis, pulmonary edema, and hypertension, in a subject in need thereof which comprises administering to said subject an effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof.

12. A method according to claim 11 wherein said disease is Type I diabetes or Type II diabetes.

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