Title of Invention

ANALOGUES OF GLP-1

Abstract The present invention relates to 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.
Full Text 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 therefore comprising said analogues.
Glucagon-like peptide-1 (7-36) amide (GLP-1) is synthesized in the intestinal L-cells by tissue-specific post-translational processing of the glucagon precursor preproglucagon (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.t 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).
In 1994, the therapeutic potential of GLP-1 was suggested following the observation that a single subcutaneous (sic) 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 sulphonylureas, the insulinotropic action of GLP-1 is dependent on plasma glucose concentration (Holz, G.G. 4th, 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; Hoist, J.J., et al., Potential of GLP-1 in diabetes management in Glucagon III, Handbook of Experimental Pharmacology, Lefevbre PJ, Ed. Berlin, Springer Vertag, 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.
GLP-1 is, however, metabolically unstable, having a plasma half-life (t1/2) of only 1-2 min in vivo. Exogenously 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
In one aspect, the present invention is directed to a compound of formula
(I). (R2R3)-A7-A8-A9-A10-A1,-A,2-A13-Au-A15-A,6-A17-A,,-A19-A20-A21-Aa-A23-A24-A25-A29-A27-
A»-A29-A30-A31-A32-A33-A34-A3S-A3e-A37-A3,-A39-R1,
(I) wherein
A7 is L-His, Ura, Paa, Pta. Amp, Tma-His, des-amino-His, or deleted; A8 is Ala, D-Ala, Aib, Ate, N-Me-Ala, N-Me-D-Ala or N-Me-Gly; A9 is Glu, N-Me-Glu, N-Me-Asp or Asp; A10 is Gly, Ace, fl-Ala or Aib; A1'isThrorSer;
A12 is Phe, Ace, Aic, Aib, 3-Pal, 4-Pal, β-Nal, Cha, Trp or X1-Phe; A13 is Thr or Ser; A14 is Ser or Aib;

A" is Asp or Glu;
A18 is Val, Ace, Aib, Leu, lie, Tie, Nle, Abu, Ala or Cha;
A'7 is Ser or Thr;
A18isSerorThr;
A19 is Tyr, Cha, Phe, 3-Pal, 4-Pal, Ace, β-Nal or X1-Phe;
A20 is Leu, Ace, Aib, Nie, lie, Cha, Tie, Val, Phe or X1-Phe;
A21 is Glu or Asp;
A22 is Gly. Ace, fl-Ala, Glu or Aib;
A23 is Gin, Asp, Asn or Glu;
A24 is Ala, Aib, Val, Abu, Tie or Ace;
A2S is Ala, Aib, Val, Abu, Tie, Ace, Lys, Arg, hArg, Om, HN-CH((CH2)n-N(R10R11))-
C(O) or HN-CH((CH2),-X3)-C(0);
A28 is Lys, Arg, hArg, Om, HN-CH((CK2)n-N(R10R1,))-C(O) or HN-CH((CH2),-X3)-
C(O);
A27 is Glu Asp, Leu, Aib or Lys;
A28 is Phe, Pal, J3-Nal, X1-Phe, Aic, Ace, Aib, Cha or Trp;
A29 is He, Ace. Aib, Leu, Nle, Cha, Tie, Val, Abu, Ala or Phe;
A30 is Ala, Aib or Ace;
A31 is Trp, fl-Nal. 3-Pal, 4-Pal, Phe, Ace, Aib or Cha;
A32 is Leu. Ace, Aib, Nle, He, Cha, Tie, Phe, X1-Phe or Ala;
A33 is Val, Ace, Aib, Leu, He, Tie, Nle, Cha, Ala, Phe, Abu, Lys or X1-Phe;
A34 is Lys, Arg, hArg, Om, HN-CH((CH2)n-N(R10R11))-C(O) or HN-CH((CH2).-X3)-
C(O);
A38 is Gly, fl-Ala, D-Ala, Gaba, Ava, HN-(CH2)m-C(0), Aib, Acc or a D-amino acid;
A39 is L- or D-Arg, D- or L-Lys, D- or L-hArg, D- or L-Om, HN-CH((CH2)B-N(R10R11))-
C(O), HN-CH((CH2),-X3)-C A37 is Gly, /3-Ala, Gaba, Ava, Aib, Acc, Ado, Arg, Asp, Aun, Aec, HN-(CH2)m-C(0),
HN-CH((CH2)n-N(R10R,*)-C(O), a D-amino add, or deleted;
A38 is D- or L-Lys, D- or L-Arg, D- or L-hArg, D- or L-Om, HM-CH((CH((-NCR((R'1))-
C(O), HN-CH((CH2).-X3)-C(0) Ava, Ado, Aec or deleted;
A3'is D- or L-Lys, D- or L-Arg, HN-CH((CH2)n-N(R10R"))-C(O), Ava, Ado, or Aec;
X1 for each occurrence is independently selected from the group consisting of (Ct-
C8)alkyl, OH and halo;

R1 is OH, NH2, (C1-C30)alkoxy. or NH-X2-CH2-Z°, wherein X2 is a (Cr C12)hydrocarbon moiety, and Z° is H, OH, C02H or CONH2;

or -C(0)-NHR12, wherein X4 is, independently for each occurrence, -C(O)-, -NH-C(O)- or -CH2-, and wherein f is, independently for each occurrence, an integer from 1 to 29 inclusive;
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-
C10Jalkyl, hydroxy(C2-C30)alkenyl, hydroxyphenyl(C,-C10)alkyl. and

and X5 is (C1-C30)alkyl, (C2-C30)alkenyl, phenyKC-CaoJalkyl, naphthyKC-CaoJalkyl,
hydroxy(C1-C30)alkyl, hydroxy(C2-C30)alkenyl, hydroxyphenyKC-CsOalkyl or
hydroxynaphthy 1(0, -CM)alkyl;
e is, independently for each occurrence, an integer from 1 to 4 inclusive;
m is, independently for each occurrence, an integer from 5 to 24 inclusive;
n is, independently for each occurrence, an integer from 1 to 5, inclusive;
each of R'° and R11 is, independently for each occurrence, H, (C1-C30)alkyl, (C1-
C30)acyl, (C1-C30)alkylsulfonyl, -C((NH)(NH2)) or


provided that:
when A7 is Ura, Paa or Pta, then R2 and R3 are deleted;
when R10 is (C,-C30)acyl, (C1-C30)(alkylsulfonyl, -C((NH)(NH2)) or

(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, -37 or -38)NH2 or hGLP-1(7-36. -37 or -38)OH; (ii) a compound of formula (I) is not an analogue of hGLP-1 (7-36, -37 or -38)NH2 or hGLP-1 (7-36, -37 or -38)OH wherein a single position has been substituted by Ala; (Hi) a compound of formula (I) is not (Arg2834, LysM)hGLP-1(7-38)-E, (Lys((N.-alkanoyl))hGLP-1(7-36, -37 or -38)-E, (Lys34(N.-alkanoyl))hGLP-1(7-36, -37 or -38)-E, (Lys28-36-bis(N.-alkanoyl))hGLP-1(7-36, -37 or -38)-E, (Arg28, Lys34((.-alkanoyl))hGLP-1(8-36, -37 or-38)-E. (Arg28,34, Lys36((Nc-alkanoyDJhGLP-ICf-Se. -37 or -38)-E or (Arg28-34, LysM(N«-alkanoyl))hGLP-1(7-38)-E, wherein E is -OH or -NH2; (iv) a compound of formula (I) is not r-hGLP-1 (7-36, -37 or -38)-OH, Z1-hGLP-1 (7-36, -37 or -38)-NH2, wherein Z1 is selected from the group consisting of:
(a) (Arg28). (Arg34), (Arg2834). (Lys38), (Arg28, Lys38), (Arg34, Lys38), (D-Lys38),
(Arg38). (D-Arg38), (Arg2834, Lys38) or (Arg2838, Lys34);
(b) (Asp21);
(c) at least one of (Aib8), (D-Aia8) and (Asp*); and
(d) (Tyr7), (N-acyl-His7), (N-alkyl-His7), (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), (Glu19)hGLP-
1(7-36 or -37), (Asp2,)hGLP-1(7-36 or -37) or (PheJ1)hGLP-1(7-36 or -37)
or a pharmaceutically acceptable salt thereof.
A preferred group of compounds of the immediately foregoing compound is where A11 is Thr, A13 is Thr, A18 is Asp; A17 is Sen A18 is Ser or Lys; A21 is Glu; A23 is Gin or Glu; A27 is Glu, Leu, Aib or Lys; and A31 is Trp, Phe or fl-Nal; or a pharmaceutically acceptable salt thereof.
A preferred group of compounds of the immediately foregoing group of compounds is where A9 is Glu, N-Me-Glu or N-Me-Asp; A'2 is Phe, Ace, fl-Nal or Aic; A18 is Val, Ace or Aib; A19 is Tyr or fl-Nal; A20 is Leu, Ace or Cha; A24 is Ala, Aib

or Ace; A25 is Ala, Aib. Ace, Lys, Arg, hArg, Orn, HN-CH((CH2)n-N(R10R,1))-C(O) or HN-CH((CH2),-X3)-C(0); A28 is Phe or fl-Nal; A29 is lie or Ace; A30 is Ala or Aib; A32 is Leu, Ace or Cha; and A33 is Val, Lys or Ace; or a pharmaceutically acceptable salt thereof.
A preferred group of compounds of the immediately foregoing group of compounds is where A4 is Ala, D-Ala, Aib, A6c, A5c, N-Me-Ala, N-Me-D-Ala or N-Me-Gly; A10 is Gly; A12 is Phe, fl-Nal, A6c or A5c; A16 is Val, A6c or A5c; A20 is Leu, A6c, A5c or Cha; A22 is Gly, fl-Ala, Glu or Aib; A24 is Ala or Aib; A29 is lie, A6c or A5c; A32 is Leu. A6c, A5c or Cha; A33 is Val, Lys, A6c or A5c; A35 is Aib, β -Ala. Ado, A6c, A5c, D-Arg or Gly; and A37 is Gly, Aib, J3-Ala, Ado, D-Ala Ava, Asp, Aun, D-Asp, D-Arg, Aec, HN-CH((CH2)„-N(R10R1,))-C(O) or deleted; or a pharmaceutically acceptable salt thereof.
A preferred group of compounds of the immediately foregoing group of compounds is where X4 for each occurrence is -C(O)-; and R1 is OH or NH2; or a pharmaceutically acceptable salt thereof.
A preferred group of compounds of the immediately foregoing group of compounds or a pharmaceutically acceptable salt thereof is where R2 is H and R3 is (C1-C30)alkyl, (C2-C30)alkenyl, (C1-C30)acyl, (C1-C30)alkylsulfonyl,

A preferred compound of the 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; A12 is Phe, fl-Nal A6c or A5c; A18 is Val, A6c or A5c; A20 is Leu, A6c, A5c or Cha; A22 is Gly, β-Ala, Glu or Aib; A24 is Ala or Aib; A29 is lle((ASc or A5c; A32 is Leu, A6c, A5c or Cha; A33 is Val, Lys, A6c or A5c; A38 is Aib, /S-Ala, Ado, A6c, A5c D-Arg or Gly; and A37 is Gly, Aib, fl-Ala, Ado, D-Ala, Ava, Asp, Aun, D-Asp, D-Arg, Aec, HN-CH((CH2)B-N(R10R11))-C(O) or deleted; X4 for each occurrence is -C(O)-; e for each occurrence is independently 1 or 2; R1 is OH or NH2; R10 is (C1-C30)acyl. (C1-C30)(alkylsulfonyl or


pharmaceutical(( acceptable salt thereof.
A more preferred compound of formula (I) is where said compound is of the formula:
(Aib8,35)hGLP-1(7-36)NH2, ((Na-HEPES-His)7. Aib8,35)hGLP-1 (7-36)NH2, ((Na-HEPA-His)7, Aib((hGLP-l (7-36)NH2, (Aib8. fl-Ala((hGLP-l (7-36)NH2l (Aib8,35. Arg26,34, Lys38(Nε-tetradecanoyl))hGLP-1(7-36)NH2. (Aib8,35, Arg26, Lys34(Nε-tetradecanoyl))hGLP-1(7-36)NH2, (Aib83337,Arg26,34, LysM(Nε(tetradecanoyl))hGLP-1(7-38)NH2, (Aib8,35. Arg26,34, Lys38(Nε-decanoyl))hGLP-1(7-36)NH2, (Aib8,35, Arg2634, Lys38(Nε-dodecanesulfonyl))hGLP-1(7-36)NH2, (Aib8,35, Arg26,34 Lys39(Nε-(2-(4-tetradecyl-1 -piperazine)-acetyl)))hGLP-1 (7-36)NH2, (Aib835. Arg26,34, Asp3S(1-(4-tetradecyl-piperazine)))hGLP-1(7-36)NH2, (Aib8,35. Arg26,34, Asp3*(1-tetradecylamino))hGLP-1(7-36)NH2, (Aib8,35, Arg26,34, Lys38(Nε-tetradecanoyl),fl-Ala37)hGLP-1(7-37)-OH or (Aib8,35, Arg26,34, Lys*(Nε-tetradecanoyl))hGLP-1(7-36>-OH, or a pharmaceutically acceptable salt thereof.
More preferred of the immediately foregoing group of compounds is a compound of the formula: (Aib8,35)hGLP-1(7-36)NH2, (Aib8. /3-AlaM)hGLP-1(7-36)NH2, (Aib8,35. Arg28. Lys34(Nε-tetradecanoyl))hGLP-1(7-36)NH2, (Aib8,35, Arg26.34. Lys38(Nε-tetradecanoyl))hGLP-1(7-38)NH2, (Aib8,35. Arg26,34, Lys38(Nε-decanoyl))hGLP-1(7-36)NH2, or

(Aib8'35, Arg28-34, Lys36(Nε-tetradecanoyl),β-Ala37)hGLP-1(7-37) -OH, or a pharmaceutical(( acceptable salt thereof.
Another more preferred compound of formula (I) is where said compound is of the formula:
(Aib'-38, A6c((hGLP-l (7-36)NH2;
(Aib835, Glu23)hGLP-1(7-36)NH2;
(Aib424SS)hGLP-1 (7-36)NH2;
(Aib8-39, Glu23, A6c32)hGLP-1(7-36)NH2;
(Aib8, Glu23, β-Ala35)hGLP-1(7-36)NH2;
(Aib8-35, Arg28,34)hGLP-1(7-36)NH2;
(Aib835, Arg28,34, Lys38(Nt-octanoyl))hGLP-1(7-36)NH2;
(Aib8M, Arg28,34, Lys38(N'-decanoyl))hGLP-1 (7-36)OH;
(Aib835, Lys25, Arg2834, Lys38(N,-decanoyl))hGLP-1(7-36)OH;
(Aib8, Arg28,34, S-Ala35, Lys38(Nt-Aec-decanoyl))hGLP-1(7-36)NH2;
(Aib8M,Arg28,34, Ava37, AdoM)hGLP-1(7-38)NH2;
(Aib8-35, Arg28-34. Asp37, Ava38, Ado39)hGLP-1(7-39)NH2;
(Aib835,Arg28,34, Aun37)hGLP-1 (7-37)NH2;
(Aibe-17M,)hGLP-1 (7-36)NH2;
(Aib8 .Arg2834, β-Ala35, D-Asp37, Ava38, AunM)hGLP-1 (7-39)NH2;
(Gly8,β-Ala39)hGLP-1(7-36)NH2;
(Ser8,β-Ala38)hGLP-1(7-36)NH2;
(Aib8, Glu22-23, β-Ala35)hGLP-1(7-36)NH2;
(Gly8, Aib35)hGLP-1(7-36)NH2;
(Aib8, Lys'8,β-Ala35)hGLP-1 (7-36)NH2;
(Aib8, Leu27, B-Ala35)hGLP-1(7-36)NH2;
(Aib8, Lys33,β-Ala35)hGLP-1(7-36)NH2;
(Aib8, Lys18, Leu27, β-Ala*)hGLP-1 (7-36)NH2;
(Aib8, D-Argr((hGLP-l (7-36)NH2;
(Aib8,β-Ala38, D-Arg37)hGLP-1(7-37)NH2;
(Aib8-27, /S-Ala39)hGLP-1(7-36)NH2;
(Aib8-27, β-Ala35-37, Arg38)hGLP-1(7-38)NH2;
(Aib8-27, β-Ala35-37. Arg38-38)hGLP-1(7-39)NH2;
(Aib8. Lys18-27,β-Ala35)hGLP-1(7-36)NH2;
(Aib8, Lys27, β-Ala35)hGLP-1(7-36)NH2;

(Aib8, β-Ala35, ArgM)hGLP-1(7-38)NH2;
(Aib8, Arg28'34, β-Ala3St)hGLP-1(7-36)NH2;
(Aib8. D-Arg((hGLP-l((-SSJNHj;
(Aib8,β-Ala35, Arg37)hGLP-1(7-37)NH2;
(Aib8, Phe31, β-Ala35)hGLP-1(7-36)NH2;
(Aib8-39, Phe31)hGLP-1(7-36)NH2;
(Aib8-35, Nal31)hGLP-1 (7-36)NH2;
(Aib8-38, Nal28-31)hGLP-1(7-36)NH2;
(Aib839, Arg28-34, Nal3,)hGLP-1(7-36)NH2;
(Aib835, Arg28-34, Phe31)hGLP-1(7-36)NH2;
(Aib8■*, Nal19-31)hGLP-1 (7-36)NH2;
(Aib8-35, Nal12-31)hGLP-1 (7-36)NH2;
(Aib8-35, Lys38(Nε-decanoyl))hGLP-1(7-36) NH2;
(Aib8-35, Arg34. Lys((Nε-decanoyl)hGLP-l (7-36)NH2;
(Aib8-35, Arg28-34, Lys38(Nε-dodecanoyl))hGLP-1 (7-36)NH2;
(Ai((.B-Ala35, Ser((O-decanoy((hGLPI (7-37)-NH2;
(Aib8-27, β-Ala39-37, Arg38, Lys39(N*-octanoyl))hGLP-1(7-39)NH2;
(Aib8, Arg28-34, β-Ala35, Lys3r(Nt-octanoyl))hGLP-1(7-37)NH2;
(Aib8. Arg2834. β-Ala35. Lys37(Nε-decanoyl))hGLP-1(7-37)NH2; or
(Aib8, Arg28-34, β-Ala39, Lys37(Nε-tetradecanoyl))hGLP-1 (7-37)NH2;
or a pharmaceutically acceptable salt thereof.
Another more preferred compound of formula (I) is each of the compounds that are specifically enumerated herein below in the Examples section of the present disclosure, 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 earner or diluent.
In yet 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.
In a further aspect, the present 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, 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 pharmaceutical(( acceptable salt thereof. A preferred method of the immediately foregoing method is where the disease being treated is Type I diabetes or Type II diabetes.
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 when A7 is Ura, Paa or Pta, in which case R2 and R3 are not present since Ura, Paa and Pta are considered here as des-amino amino acids. Amp, β-Nal, Nle, Chat 3-Pal, 4-Pal and Aib are the abbreviations of the following a-amino acids: 4-amino-phenylalanine, £-(2-naphthyi)aianine, norleucine, cyclohexylalanine, i3-(3-pyridinyl)alaninet fi-(4-pyridinyl)alanine and a-aminoisobutyric acid, respectively. Other amino acid definitions are: Ura is urocanic acid; Pta is (4-pyridylthio) acetic acid; Paa is frans-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; Tie is tert-butylglycine; Abu is a-aminobutyric acid; Tba is tert-butylalanine; Om is ornithine; Aib is a-aminoisobutyric acid;β-Ala is ((-alanine; Gaba is y-aminobutyric acid; Ava is 5-aminovaleric acid; Ado is 12* aminododecanoic acid, Aic is 2-aminoindane-2-carboxylic acid; Aun is 11-aminoundecanoic acid; and Aec is 4-(2-aminoethyi)-1-carboxymethyl-piperazine,
represented by the structure:
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 -

cyctooctanecarboxylic 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=OX5. Examples of -C=O.X5 include, but are not limited to, acetyl and phenylpropionyl.
What is meant by Lys(Nε-alkanoyl) is represented by the following structure:
i


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 dimethylforrnamide, DCM for dichloromethane, HBTU for 2-(1 H-Benzotriazol-1 -yl)-1,1,3,3-tetramethyl uranium hexafluorophosphate, DIEA for diisopropylethylamine, HOAc for acetic acid, TFA for trifluoroacetic acid, 2CIZ for 2-chlorobenzyloxycarbonyl, 2BrZ for 2-bromobenzyloxycarbonyl, OcHex for O-cyclohexyl, Fmoc for 9-fluorenylmethoxycarbonyl, HOBt for N-hydroxybenzotriazole and PAM resin for 4-hydroxymethylphenylacetamidornethyl resin.
The term "halo" encompasses fiuoro, chloro, bromo and iodo.
The term *(C1-C30)hydrocarbon moiety" encompasses alkyl, alkenyl and alkynyl, and in the case of alkenyl and alkynyl there are C2-C30-
A peptide of this invention is also denoted herein by another format, e.g.. (ASc((hGLP-l((-aejNH(( with the substituted amino acids from the natural sequence placed between the first set of parentheses (e.g., A5C8 for Ala8 in hGLP-1). The abbreviation GLP-1 means glucagon-like peptide-1; 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) is amino acids 7 through 36 of the peptide sequence for human GLP-1). The sequence for hGLP-

1(7-37) is listed in Mojsov, S., Int. J. Peptide Protein Res,. 40, 1992, pp. 333-342. The designation "NHa" in hGLP-1(7-36)NH2 indicates that the C-terminus of the peptide is amidated. hGLP-1(7-36) means that the C-terminus is the free acid. In hGLP-1(7-38). residues in positions 37 and 38 are Gly and Argt respectively.
Detailed Description
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 may 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., (C,-CM)alkylf may be attached using reductive alkylatlon. Hydroxyalkyl groups, e.g., (Cr C((hydroxyalkyl, may also be attached using reductive alkylation wherein the free hydroxy group is protected with a t-butyl ester. Acyl groups, e.g., COE\ may be attached by coupling the free acid, e.g., E'COOH, 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.
When R1 is NH-X2-CH2-CONH2, (i.e., Z((CONHa), 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., Z°=COOH) the synthesis of the peptide starts with Boc-HN-X2-CH2-COOH which is coupled to PAM resin. For this particular step, 4 molar equivalents of Boc-HN-X2-COOHV HBTU and HOBt and 10 molar equivalents of DIEA are used. The coupling time is about 8 hours.
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 HCI. The organic layer was separated. The

aqueous layer was extracted with ethyl acetate (1 x 100 ml). The two organic layers were combined and washed with water (2 x 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.
Boc-A5c-OH was synthesized in an analogous manner to that of Boc-A6c-OH. Other protected Ace amino acids can be prepared in an analogous manner by a person of ordinary skill in the art as enabled by the teachings herein.
In the synthesis of a GLP-1 analogue of this invention containing A5c, A6c and/or Aib, the coupling time is 2 hrs. for these residues and the residue immediately following them. For the synthesis of (Tma-His7)hGLP-1(7-36)NH2, HBTU (2 mmol) and DIEA (1.0 ml) in 4 ml DMF are 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.
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)hydroxyalkylt 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 add 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.
A compound of the present invention can be tested for activity as a GLP-1 binding compound according to the following procedure. Ceil Culture:
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 humidifed atmosphere of 5% C02/95% air. Radioligand Binding:
Membranes were prepared for radioligand binding studies by

homogenization of the RIN cells in 20 ml of ice-cold 50 mM Tris-HCI with a Brinkman Polytron (Westbury, NY) (setting 6, 15 sec). The homogenates were washed twice by centrifugation (39.000 g / 10 min), and the final pellets were resuspended in 50 mM Tris-HCI, containing 2.5 mM MgCI2, 0.1 mg/ml bacitracin (Sigma Chemical, St. Louis, MO)f and 0.1% BSA. For assay, aliquots (0.4 ml) were incubated with 0.05 nM (125l)GLP-1(7-36) (-2200 Ci/mmol, New England Nuclear, Boston, MA), with and without 0.05 ml of unlabeled competing test peptides. After a 100 min incubation (25 °C), the bound (t25l)GLP-1(7-36) 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-HCI, and the bound radioactivity trapped on the filters was counted by gamma spectrometry (Waliac LKB, Gaithersburg, MO). Specific binding was defined as the total (125l)GLP-1(7-36) bound minus that bound in the presence of 1000 nM GLP1(7-36) (Bachem, Torrence, CA).
The peptides of this invention can be provided in the form of
pharmaceuticaliy 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 acetifi 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 for 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.

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, neurodegenerative diseases, renal failure, congestive heart failure, nephrotic syndrome, cirrhosis, pulmonary edema, hypertension, and disorders wherein the reduction of food intake is desired. 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 gastrectomy or small bowel resection.
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.
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 1x10"' to 200 mg/kg/day, preferably 1x10* to 100 mg/kg/day, which can be administered as a single dose or divided into multiple doses.
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.
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

homogenization of the RIN cells in 20 ml of ice-cold 50 mM Tris-HCI with a Brinkman Polytron (Westbury, NY) (setting 6, 15 sec). The homogenates were washed twice by centrifugation (39,000 g / 10 min), and the final pellets were resuspended in 50 mM Tris-HCI, containing 2.5 mM MgCI2, 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 (125l)GLP-1(7-36) (-2200 Ci/mmol, New England Nuclear, Boston, MA), with and without 0.05 ml of unlabeled competing test peptides. After a 100 min incubation (25 °C), the bound (125l)GLP-1(7-36) 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-HCI, and the bound radioactivity trapped on the filters was counted by gamma spectrometry (Wallac LKB, Gaithersburg, MD). Specific binding was defined as the total (125l)GLP-1(7-36) bound minus that bound in the presence of 1000 nM GLP1(7-36) (Bachem, Torrence, CA).
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 acetifc 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) 0.1 N ammonium acetate aqueous solution for 0.5 hrs., (2) 0.25N acetic acid aqueous solution for 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.

compositions of a bioactive agent. U.S. Application No. 09/121,653 filed July 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 No. 09/131,472 filed August 10, 1998, teaches complexes comprising a therapeutic agent such as a peptide and a phosphorylated polymer. U.S. Application No. 09/184,413 filed November 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.
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.
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,
Boc-/JAIa-OH, Boc-D-Arg(Tos)-OH and Boc-D-Asp(OcHex) were purchased from Nova Biochem, San Diego, California. Boc-Aun-OH was purchased from Bachem, King of Prussia, PA. Boc~Ava-OH and Boc-Ado-OH were purchased from Chem-lmpex International, Wood Dale, IL. Boc-Nal-OH was purchased from Synthetech, Inc. Albany, OR.
(Aib8(()hGLP-1(7-36)NH2 The title peptide was synthesized on an Applied Biosystems (Foster City, CA) 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., 90:180 (1§92). 4-methylbenzhydrylamine (MBHA) resin (Peninsula, Belmont, CA) with the substitution of 0.91 mmol/g was used. The Boc amino acids (Bachem, CA, Torrance, CA; Nova Biochem., LaJoila, CA) 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-lle-OH, Boc-Lys(2CIZ)-OH, BooThr(Bzl)-OH, Boc-Ser(Bzt)-OH, Boc-Phe-OH, Boc-Aib-OH, Boc-Glu(OcHex)-OH and Boc-Trp(Fm)-OH. The

homogenization of the RIN cells in 20 ml of ice-cold 50 mM Tris-HCI with a Brinkman Polytron (Westbury, NY) (setting 6t 15 sec). The homogenates were washed twice by centrifugation (39,000 g / 10 min), and the final pellets were resuspended in 50 mM Tris-HCI, containing 2.5 mM MgCI2, 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 (125l)GLP-1(7-36) (-2200 Ci/mmol, New England Nuclear, Boston, MA), with and without 0.05 mi of unlabeled competing test peptides. After a 100 min incubation (25 °C), the bound (125l)GLP-1(7-36) 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-HCI, and the bound radioactivity trapped on the filters was counted by gamma spectrometry (Wallac LKB, Gaithersburg, MO). Specific binding was defined as the total (125l)GLP-1(7-36) bound minus that bound in the presence of 1000 nM GLP1(7-36) (Bachem, Torrence, CA).
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, methanesutfonic, toluenesulfonic, or pamoic acid), inorganic acids (e.g., hydrochloric acid, sulfuric add, 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 acetiti 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) 0.1 N ammonium acetate aqueous solution for 0.5 hrs., (2) 0.25N acetic acid aqueous solution for 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.

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 on 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.
Example 3
((Nε.HEPA-His)7lAib8'35)hGLP-1(7((36)NH2
The title compound (HEPA is (4-(2-hydroxyethyl)-1-piperazineacetyl)) can be made substantially according to the procedure described in Example 2 for making ((Na-HEPES-His)7, AibSM)hGLP-1(7-36)NH2 except that 2-bromoacetic anhydride is used in place of 2-chloro-1-ethanesulfonyl chloride.
Example 4 (Aib8, β-Ala35)hGLP-1(7-36)NH2
The title compound was synthesized substantially according to the procedure described for Example 1 using the appropriate protected amino acids. MS (ES) gave the molecular weight at 3325.7, calculated MW = 3325.8, purity = 99%, yield = 85 mg.
The synthesis of other compounds of the present invention can be accomplished in substantially the same manner as the procedure described for the synthesis of (Aib8,36)hGLP-1(7-36)NH2 in Example 1 above, but using the appropriate protected amino acids depending on the desired peptide.
Example 5 (Aib8-38, Arg2634, Lys((CNc-tetradecanoyOJhGLP-ICZ-SBJNHa
The Boc amino acids used were the same as those in the synthesis of (Aib8;M)hGLP-1(7-36)Nfl2 described in Example 1 except that Fmoc-Lys(Boc)-OH was used in this example. The first amino acid residue was coupled to the resin manually on a shaker. 2.5 mmol of Fmoc-Lys(Boc)-OH was dissolved in 4 mL of 0.5N HBTU in DMF, To the solution was added 1 mL of DIEA. The mixture was shaken for about 2 min. To the solution was then added 0.2 mmol of MBHA resin (substitution = 0.91 mmol/g). The mixture was shaken for about 1 hr. The resin was washed with DMF and treated with 100% TFA for 2x2 min to remove the Boc

protecting group. The resin was washed with DMF. Myristic acid (2.5 mmol) was pre-activated with HBTU (2.0 mmol) and DIEA (1.0 mL) in 4 mL of DMF for 2 min and was coupled to the Fmoc-Lys-resin. The coupling time was about 1 hr. The resin was washed with DMF and treated with 25% piperidine in DMF for 2x20 min to remove the Fmoc protecting group. The resin was washed with DMF and transferred to the reaction vessel of the peptide synthesizer. The following steps synthesis and purification procedures for the peptide were the same as those in the synthesis of (Aib8,35)hGLP-1(7-36)NH2 in Example 1. 43.1 mg of the title compound were obtained as a white solid. Purity was 98% based on analytical HPLC analysis. Electro-spray mass spectrometer analysis gave the molecular weight at 3577.7 in agreement with the calculated molecular weight 3578.7.
Examples 6-8 Examples 6-8 were synthesized substantially according to the procedure described for Example 5 using the appropriate protected amino acid and the appropriate acid in place of the Myristic acid used in Example 5. Example 6: (Aib8*35, Arg26, Lys34(Nε-etradecanoyl))hGLP-1(7-36)NH2; Yield = 89.6 mg; MS(ES) = 3577.2, Calculated MW = 3578.7; Purity 96%. Example 7: (Aib8,35,37, Arg26,34, LysM(Nc-tetradecanoyl))hGLP-1(7-38)NH2; Yield = 63.3 mg; MS(ES) = 3818.7; Calculated MW = 3819.5; Purity 96%. Example 8: (AibaM, Arg*34. Lys38(Nε-decanoyl))hGLP-1(7-36)NH2; Yield = 57.4 mg; MS(ES) = 3521.5; Calculated MW = 3522,7; Purity 98%; Acid = decanoic acid.
The syntheses of other compounds of the present invention containing Lys(Nc-aikanoyl) residue can be carried out in an analogous manner to the procedure described for Example 5, (Aiba,3S, Arg28-34, Lys36(N.-tetradecanoyl))hGLP-1(7-36)NH2. Fmoc-Lys(Boc)-OH amino acid is used for the residue of Lys(Nε-alkanoyl) in the peptide, while Boc-Lys(2CIZ)*OH amino acid is used for the residue of Lys. If the Lys(Nε-alkanoyl) residue is not at the C-terminus, the peptide fragment immediately prior to the Lys(Nε-alkanoyl) residue is assembled on the resin on the peptide synthesizer first. The appropriate acid corresponding to the desired alkanoyl can be purchased from Aldrich Chemical Co., Inc. Milwaukee, Wl, USA, e.g., octanoic acid, decanoic add, lauric acid and palitic acid.

Example 9 (Aib8-35, Arg26-34, Lys((Nc-dodecanesulfony()hGLP-l(7-36)NH2
The Boc amino acids to be used in this synthesis are the same as those used in the synthesis of Example 5. 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 2x2 min to remove the Boc protecting group. The resin is washed with DMF and to it is added 0.25 mmol of 1-dodecanesulfonyl chloride in 4 mL of DMF and 1 mL of DIEA. The mixture is shaken for about 2 hrs. The resin is washed with DMF and treated with 25% piperidine in DMF for 2 x 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 synthesis of the rest of the peptide and purification procedures are the same as those described in Example 1.
The syntheses of other compounds of the present invention containing Lys(Nc-alkylsulfonyl) residue can be carried out in an analogous manner to the procedure described in Example 9. Fmoc-Lys(Boc)-OH amino acid is used for the residue of Lys(Nc-alkylsulfonyl) in the peptide, while Boc*Lys(2ClZ)-OH amino acid is used for the residue of Lys. If the Lys(Nε-alkylsulfonyi) residue is not at the C-terminus, the peptide fragment immediately prior to the Lys(Nt-alkylsulfonyl) residue is assembled on the resin on the peptide synthesizer first. The appropriate akylsulfonyl chloride can be obtained from Lancaster Synthesis Inc., Windham, NH, USA, e.g., 1-octanesulfonyl chloride, 1-decanesulfonyl chloride, 1-dodecanesulfonyl chloride, 1-hexadecanesulfonyl chloride and 1-octadecylsulfonyl chloride.
(Aib8,35, Arg2834, Lys36(Nε-(2-(4-tetradecyl-1-piperazine)-acetyl)))hGLP-1(7-36)NH2 The Boc amino acids to be used for this example are the same as those used in the synthesis of Example 5. 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 (substitution =

0.91 mmol/g) resin. The mixture is shaken for about 1 hr. The resin is washed with DMF and treated with 100% TFA for 2x2 min to remove the Boc protecting group. The resin is washed with DMF. The 2-bromoacetic acid (2.5 mmol) is pre-activated with HBTU (2.0 mmol) and DIEA (1 mL) in 4 mL of DMF for about 2 min and is added to the resin. The mixture is shaken for about 10 min and washed with DMF. The resin is then treated with 1.2 mmol of piperazine in 4 mL of DMF for about 2 hrs. The resin is washed with DMF and treated with 2 mmol of 1-iodotetradecane for about 4 hrs. After washing with DMF, the resin is treated with 3 mmol of acetic anhydride and 1 mL of DIEA in 4 mL of DMF for about 0.5 hr. The resin is washed with DMF and treated with 25% piperidine in DMF for 2x20 min. The resin is washed with DMF and transferred to the reaction vessel of the peptide synthesizer to continue the synthesis. The remaining synthesis and purification procedures for the peptide are the same as the procedures described for Example 1.
The syntheses of other compounds of the present invention containing Lys(Nc-(2-(4-a!kyl-1-piperazine)-acetyl)) residue are carried out in an analogous manner as the procedure described for the synthesis of Example 10. Fmoc-Lys(Boc)-OH amino acid is used for the residue of Lys(N«-(2-(4-alkyM-piperazine)-acetyl)) in the peptide, while Boc-Lys(2CIZ)-OH amino acid is used for the residue of Lys. The corresponding iodoalkane is used for the residue of Lys(Ne-(2-(4-alkyl-1-piperazine)-acetyl)) during the alkylation step. If the Lys(N«-(2-(4-alkyl-1-piperazine)-acetyl)) residue is not at the C-terminus, the peptide fragment immediately prior to the Lys(Nε-(2-(4-alkyl-1-piperazine)-acetyl)) residue is assembled oh the resin on the peptide synthesizer first.

The Boc amino acids to be used in this example are the same as the amino acids used in synthesis of Example 5 except Fmoc-Asp(0-tBu)-OH is used at position 36. The first £mino acid residue is coupled to the resin manually on a shaker. 2.5 mmol of Fmoc-Asp(0-tBu)-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 (substitution = 0.91 mmol/g) resin. The mixture is shaken for about 1 hr. The resin is washed with DMF and treated with 100% TFA for 2x15 min to remove the tBu protecting group. The resin is washed with DMF and is treated with HBTU (0.6 mmol) and DIEA (tmL) in 4 mL

of DMF for about 15 min. 0.6 mmol of piperazine is added to the reaction mixture and the mixture is shaken for about 1 hr. The resin is washed with DMF and treated with 3 mmol of 1-iodotetradecane for about 4 hrs. After washing with DMF, the resin is treated with 3 mmol of acetic anhydride and 1 mL of DIEA in 4 mL of DMF for about 0.5 hr. The resin is washed with DMF and treated with 25% piperidine in DMF for 2x20 min to remove the Fmoc protecting group. The resin is washed with DMF and transferred to the reaction vessel of the peptide synthesizer to continue the synthesis. The remaining synthesis and purification procedures for the peptide are the same as those for the synthesis of Example 1.
The syntheses of other compounds of the present invention comprising Asp(H4-alkylpiperazine)) or Glu(1-(4-alkylpiperazine)) residue are carried out in an analogous manner as the procedure described for the synthesis of Example 11. Fmoc-Asp(OtBu)-OH or Fmoc-Glu(0-tBu)-OH amino acid is used for the residue of Asp(1-(4-alkylpiperazine)) or Glu(1-(4-alkylpiperazine» in the peptide, while Boc-Asp(OcHex)-OH or Boc-Glu(OcHex)-OH amino acid is used for the residue of Asp or Glu. The corresponding iodoalkane is used for the residue of Lys(Nt-(2-(4-alkyl-1-piperazine)-acetyl)) during the alteration step. If the Asp(1-(4-alkylpiperazine)) or Glu(1-(4-alkylpiperazine)) residue is not at the C-terminus, the peptide fragment immediately prior to the Asp(1-(4-alkylpiperazine)) or GIu(1-(4-alkylpiperazine)) residue is assembled on the resin on the peptide synthesizer first
Example12
(Aib8,35,Arg26,34,Asp36(1-tetradecylamino))hglp-1(7-36)NH2
(The Boc amino acids to be used for this example are the same as those used in Example 5. The first amino acid residue is coupled to the resin manually on a shaker, 2.5 mmol of Fmoc-Asp(0-tBu)-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 (substitution = 0.91 mmol/g) resin. The mixture is Shaken for about 1 hr. The resin is washed with DMF and treated with 100% TFA for 2x15 min to remove the t-Bu protecting group. The resin is washed with DMF and is treated with HBTU (0.6 mmol) and DIEA (1mL) in 4 mL of DMF for about 15 min. 0.6 mmol of 1-tetradecaneamine is added to the reaction mixture and the mixture is shaken for about 1 hr. The resin is washed with DMF and treated with 25% piperidine in DMF for 2x20 min to remove the Fmoc protecting group. The resin is washed with DMF and transferred to the reaction

0.91 mmol/g) resin. The mixture is shaken for about 1 hr. The resin is washed with DMF and treated with 100% TFA for 2x2 min to remove the Boc protecting group. The resin is washed with DMF. The 2-bromoacetic acid (2.5 mmol) is pre-activated with HBTU (2.0 mmol) and DIEA (1 mL) in 4 mL of DMF for about 2 min and is added to the resin. The mixture is shaken for about 10 min and washed with DMF. The resin is then treated with 1.2 mmol of piperazine in 4 mL of DMF for about 2 hrs. The resin is washed with DMF and treated with 2 mmol of 1-iodotetradecane for about 4 hrs. After washing with DMF, the resin is treated with 3 mmol of acetic anhydride and 1 mL of DIEA in 4 mL of DMF for about 0.5 hr. The resin is washed with DMF and treated with 25% piperidine in DMF for 2x20 min. The resin is washed with DMF and transferred to the reaction vessel of the peptide synthesizer to continue the synthesis. The remaining synthesis and purification procedures for the peptide are the same as the procedures described for Example 1.
The syntheses of other compounds of the present invention containing Lys(Nε-(2-(4-alkyl-1-piperazine)-acetyl)) residue are carried out in an analogous manner as the procedure described for the synthesis of Example 10. Fmoc-Lys(Boc)-OH amino acid is used for the residue of Lys(Nε-(2-(4-alkyM-piperazine)-acetyl)) in the peptide, while Boc-Lys(2CIZ)-OH amino acid is used for the residue of Lys. The corresponding iodoalkane is used for the residue of Lys(Ne-(2-(4-alkyl-1 -piperazine)-acetyl)) during the alkylation step. If the Lys(Ne-(2-(4-alkyt-1 -piperazine)-acetyl)) residue is not at the C-termtnus, the peptide fragment immediately prior to the Lys(Nε-(2-(4-alkyi-1-piperazine)~acetyl)) residue is assembled oh the resin on the peptide synthesizer first.
Example 11 (Aib8,35, Arg28,34, Asp36(1-(4-tetradecyl-piperazine))hGLP-((7-36)NH2 The Boc amino acids to be used in this example are the same as the amino acids used in synthesis of Example 5 except Fmoc-Asp(0-tBu)-OH is used at position 36. The first amino acid residue is coupled to the resin manually on a shaker. 2.5 mmol of Fmoc-Asp(0-tBu)-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 (substitution = 0.91 mmol/g) resin. The mixture is shaken for about 1 hr. The resin is washed with DMF and treated with 100% TFA for 2x15 min to remove the tBu protecting group. The resin is washed with DMF and is treated with HBTU (0.6 mmol) and DIEA (1mL) in 4 mL

The syntheses of other analogs of hGLP-1(7-36)-OH, hGLP-1(7-37)-OH and hGLP-1(7-38)-OH of the instant invention which contain Lys(Nt-alkanoyl) residue can be carried out in an analogous manner according to the procedure described for the synthesis of Example 14. Fmoc-Lys(Boc)-OH amino acid is used for the residue of Lys(Nε-alkanoyl) in the peptide, while Boc-Lys(2CIZ)-OH amino acid is used for the residue of Lys.

A mixture of MBHA resin (0.2mmol, substitution-0.91mmol/g), Fmoc-Aec-OH (0.40gf 0.829 mmol), HBTU (1.5 mL @ 0.5M in DMF) and DIEA (0.5mL) in a reaction vessel was shaken on a shaker for 4h at room temperature. The resin was then washed with DMF and treated with 25% piperidine in DMF for 2X20min. The resin was washed with DMF and DCM and transferred to the reaction vessel of the peptide synthesizer to continue the assembly of the rest of the peptide according the procedure described for Example 1. The purification procedure was also the same as the one described in Example 1. Electro-spry mass spectrometer analysis gave the molecular weight at 3494.8 in agreement with the calculated molecular weight 3494.99. Purity 93%; Yield 79.1mg.

Example 367 was synthesized substantially according to the procedure described for Example 366. MS(ES)=3551.7, calculated MW*3552.04; Purity 97%; Yield 97.4mg.

A mixture of MBHA resin (0.2mmoi, substitution((O((lmmol/g), Fmoc-Aec-OH (0.289g, 0.6 mmol), HBTU (1.12 mL @ 0.5M in DMF) and DIEA (0.4mL) in a reaction vessel was shaken on a shaker for 2h at room temperature. The resin was then washed with DMF and treated with 30% piperidine in DMF for 2X15min. The resin was washed with DMF. To the reaction vessel were added Fmoc-Aec-OH (0.289g, 0.6 mmol), HBTU (1.12 mL @ 0.5M in DMF) and DIEA (0.4mL). The mixture was shaken at room temperature for 2h. The resin was washed with DMF

and treated with 30% piperidine in DMF for 2X15min. The resin was washed with DMF and DCM and transferred to the reaction vessel of the peptide synthesizer to continue the assembly of the rest of the peptide according the procedure described for Example 1. The purification procedure was also the same as the one described in Example 1. Electro-spry mass spectrometer analysis gave the molecular weight at 3663.9 in agreement with the calculated molecular weight 3664.26. Purity 100%; Yield 75.3mg.

A mixture of MBHA resin (0.2mmol, substitution=0.91mmol/g), Boc-Lys(Fmoc)-OH (1.17g. 2.5mmol)( HBTU (4 mL @ 0.5M in DMF) and DIEA (1mL) in a reaction vessel was shaken on a shaker at room temperature for 10min. The resin was washed with DMF and treated with 25% piperidine in DMF for 2X15min. The resin was washed with DMF. To the reaction vessel were added Fmoc-Aec-OH (0.289gf 0.6 mmol), HBTU (1.12 mL @ 0.5M in DMF) and DIEA (0.4mL). The mixture was shaken at room temperature for 10min. The resin was washed with DMF and treated with 30% piperidine in DMF for 2X15min. The resin was washed with DMF and treated with a mixture of decanoic acid (431 mg, 2.5 mmol), HBTU (4 mL @ 0.5M in DMF) and DIEA (1mL) for 10 min. The resin was washed with DMF and treated with 100% TFA for 2X2 min. The resin was washed with DMF and DCM and transferred to the reaction vessel of the peptide synthesizer to continue the assembly of the rest of the peptide according the procedure described for Example 1. The purification procedure was also the same as the one described in Example 1. Electro-spry mass spectrometer analysis gave the molecular weight at 3677.0 in agreement with the calculated molecular weight 3677.25. Purity 97.6%;Yie»d 44.8mg.
































WE CLAIM:

wherein
A7 is L-His, Ura, Paa, Pta, Amp, Tma-His, des-amino-His, or deleted;
A8 is Ala, D-Ala, Aib, Acc, N-Me-Ala, N-Me-D-Ala or N-Me-Gly;
A9 is Glu, N-Me-Glu, N-Me-Asp or Asp;
A10 is Gly, Ace, β-Ala or Aib;
A11 is Thr or Ser;
A12 is Phe, Ace, Aic, Aib, 3-Pal, 4-Pal, B-Nal, Cha, Trp or X'-Phe;
A13 is Thr or Ser;
A14 is Ser or Aib;
A15 is Asp or Glu;
A16 is Val, Acc, Aib, Leu, Ile, Tle, Nle, Abu, Ala or Cha;
A17 is Ser or Thr;
A18 is Ser or Thr;
A19 is Tyr, Cha, Phe, 3-Pal, 4-Pal, Acc, B-Nal or X'-Phe;
A20 is Leu, Ace, Aib, Nle, Ile, Cha, Tle, Val, Phe or X'-Phe;
A21 is Glu or Asp;
A22 is Gly, Acc, B-Ala, Glu or Aib;

A23 is Gln, Asp, 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-N(R10R11))-C(O)
or HN-CH((CH2)e-X3)-C(0);
A26 is Lys, Arg, hArg, Orn, HN-CH((CH2)n-N(R10R11))-C(O) or HN-CH((CH2)e-X3)-C(0);
A is Glu Asp, Leu, Aib or Lys;
A28 is Phe, Pal, B-Nal, X'-Phe, Aic, Acc, Aib, Cha or Trp;
A29 is Ile, Acc, Aib, Leu, Nle, Cha, Tle, Val, Abu, Ala or Phe;


OH and halo;
R1 is OH, NH2, (C,-C3o)alkoxy, or NH-X2-CH2-Z°, wherein X2 is a (C1-C12)hydrocarbon
moiety, and Z° is H, OH, C02H or CONH2;

*
or -C(0)-NHR12, wherein X4 is, independently for each occurrence, -C(O)-, -NH-C(O)- or -CH2-, and wherein f is, independently for each occurrence, an integer from 1 to 29 inclusive; each of R and R 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,



or NH2; r is 0 to 4; q is 0 to 4; and 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 or hydroxynaphthyl(C1-C30)alkyl;
e is, independently for each occurrence, an integer from 1 to 4 inclusive;
m is, independently for each occurrence, an integer from 5 to 24 inclusive;
n is, independently for each occurrence, an integer from 1 to 5, inclusive;
each of R10 and R11 is, independently for each occurrence, H, (C1-C30)alkyl, (C1-C30)acyl,

(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, -37 or -38)NH2 or hGLP-l(7-36, -37 or -38)OH; (ii) a compound of formula (I) is not an analogue of hGLP-l(7-36, -37 or -38)NH2 or hGLP-1(7-36, -37 or -38)OH wherein a single position has been substituted by Ala;



5. The compound as claimed in claim 4 or a pharmaceutically acceptable salt thereof, wherein X4 for each occurrence is -C(O)-; e for each occurrence is independently 1 or 2; and R1isOHorNH2.

6. The compound as claimed in claim 5 or a pharmaceutically acceptable salt thereof,
wherein R2 is H and R3 is (C1-C30)alkyl, (C2-C30)alkenyl, (C1-C30)acyl, (C1-C30)alkylsulfonyl,

7. The compound as claimed in claim 5 or a pharmaceutically acceptable salt thereof,
wherein R10 is (C1-C30)acyl, (C1-C30)alkylsulfonyl or

8. The compound as claimed in claim 7 or a pharmaceutically acceptable salt thereof,
wherein R10 is (C4-C20)acyl, (C4-C20)alkylsulfonyl or



11. A pharmaceutical composition comprising an effective amount of the compound as claimed in claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.






























14. 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.
15. 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, 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.
16. A method according to claim 15, wherein said disease is Type I diabetes or Type II
diabetes.
17. Use of a compound as claimed in claim 1, in the preparation of a medicament for the treatment of disease.
18. Use as claimed in claim 17, in which the disease is selected from the group consisting of Type I diabetes, Type II diabetes, obesity, glucaganomas, secretory disorders of the airway, metabolic disorder, arthritis, osteoporosis, central nervous system disease, restenosis and neurodegenerative disease.


Documents:

3870-chenp-2007 english translation 29-06-2011.pdf

3870-chenp-2007 form-13-1 29-06-2011.pdf

3870-chenp-2007 form-13-2 29-06-2011.pdf

3870-chenp-2007 form-13-3 29-06-2011.pdf

3870-chenp-2007 power of attorney 29-06-2011.pdf

3870-CHENP-2007 AMENDED CLAIMS 20-09-2011.pdf

3870-CHENP-2007 AMENDED PAGES OF SPECIFICATION 20-09-2011.pdf

3870-chenp-2007 correspondence others 29-06-2011.pdf

3870-CHENP-2007 CORRESPONDENCE OTHERS 09-02-2011.pdf

3870-CHENP-2007 CORRESPONDENCE OTHERS 28-12-2011.pdf

3870-CHENP-2007 EXAMINATION REPORT REPLY RECEIVED 20-09-2011.pdf

3870-CHENP-2007 FORM-1 06-01-2012.pdf

3870-CHENP-2007 FORM-2 06-01-2012.pdf

3870-CHENP-2007 FORM-3 20-09-2011.pdf

3870-CHENP-2007 OTHER PATENT DOCUMENT 20-09-2011.pdf

3870-CHENP-2007 OTHER PATENT DOCUMENT 28-12-2011.pdf

3870-CHENP-2007 POWER OF ATTORNEY 20-09-2011.pdf

3870-CHENP-2007 CORRESPONDENCE OTHERS 04-10-2011.pdf

3870-CHENP-2007 CORRESPONDENCE OTHERS 06-01-2012.pdf

3870-CHENP-2007 POWER OF ATTORNEY 04-10-2011.pdf

3870-chenp-2007-abstract.pdf

3870-chenp-2007-claims.pdf

3870-chenp-2007-correspondnece-others.pdf

3870-chenp-2007-description(complete).pdf

3870-chenp-2007-form 1.pdf

3870-chenp-2007-form 26.pdf

3870-chenp-2007-form 3.pdf

3870-chenp-2007-form 5.pdf


Patent Number 250923
Indian Patent Application Number 3870/CHENP/2007
PG Journal Number 06/2012
Publication Date 10-Feb-2012
Grant Date 07-Feb-2012
Date of Filing 05-Sep-2007
Name of Patentee IPSEN PHARMA S.A.S
Applicant Address 65 QUAI GEORGE GORSE,BOULOGNE-BILLANCOURT
Inventors:
# Inventor's Name Inventor's Address
1 ZHENG XIN DONG 40 ANGELICA DRIVE FRAMINGTON MASSACHUSETTS 01701
PCT International Classification Number A61K 38/00
PCT International Application Number PCT/EP99/09660
PCT International Filing date 1999-12-07
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/111,255 1998-12-07 U.S.A.
2 09/206601 1998-12-07 U.S.A.