The present invention relates to novel urocortin-2 compounds, pharmaceutical compositions comprising the compounds, methods of using the compounds to treat disorders associated with corticotropin releasing hormone receptor-2, and intermediates and processes useful in the synthesis of the compounds.

Urocortin-2 (UCN2) is a thirty-eight amino acid endogenous peptide (SEQ ID

NO:15). It is one of three known endogenous urocortins (UCN1 and UCN3) found in mammals and is part of the corticotropin-releasing hormone (CRH; also referred to as corticotropin releasing factor) family. The CRH family exhibits many physiological functions. UCN peptides are short acting. They act through CRH receptors (CRHR) known as CRHR1 and/or CRHR2. Specifically, UCN2 selectively activates CRHR2 including known isoforms CRHR2-alpha (α) -beta (β) ) and -gamma (γ). UCN2 also has been associated with a reduction in blood pressure. European Journal of Pharmacology 469: 111-115 (2003).

Type II diabetes (T2D) is the most common form of diabetes accounting for approximately 90% of all diabetes. Over 300 million people worldwide are diagnosed with T2D. It is characterized by high blood glucose levels caused by insulin-resistance. The current standard of care for T2D includes diet and exercise as underlying adjunctive therapy along with available oral and injectable glucose lowering drugs. Nonetheless, patients with T2D still remain inadequately controlled. An alternative treatment for T2D is needed.

Chronic kidney disease (CKD) is characterized by the progressive loss of kidney function. Individuals who have CKD over time experience an increase in albuminuria, proteinuria, serum creatinine, and renal histopathological lesions. It eventually develops into end stage renal disease (ESRD) for many patients requiring either dialysis or kidney transplant. CKD may be caused by several underlying conditions including diabetes and hypertension known as diabetic nephropathy and hypertensive nephropathy, respectively. Diabetic nephropathy prevalence accounts for approximately 50% of kidney failures in the U.S. Hypertensive nephropathy prevalence accounts for nearly 25% of kidney failures in the U.S. The current standard of care for kidney diseases includes angiotensin converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs). There remains a need for an alternative treatment for CKD.

Chen et al. (Proceedings of the National Academy of Sciences (PNAS), October 31, 2006, vol. 103, NO:44, pp.16580-16585) is an article entitled“Urocortin 2 modulates glucose utilization and insulin sensitivity in skeletal muscle.” Further, in Peptides 27:

1806-1813 (2006), the authors disclose CRHR2 agonists including UCN2 analogs for the treatment of CRHR2 modulated disorders such as muscular atrophy. However, there is still a further need for novel therapeutic human UCN2 analogs that are agonists of CRHR2.

The present invention provides novel compounds that are CRHR2 agonists. The present invention also provides novel therapeutic CRHR2 agonists in the form of human UCN2 analogs which may be suitable for once weekly administration or other types of administration such as bi-monthly or monthly. The present invention also provides a novel compound that is a CRHR2 agonist for use in therapy, and in particular for use to treat T2D or CKD, or combinations thereof.

Accordingly, the present invention provides compounds which are urocortin molecules that have the amino acid sequence of Formula III:

X1IVX2SLDVPIGLLQILX3EQEKQEKEKQQATX7NAX4ILAX8V-NH2 (Formula III), wherein

X1 denotes that the I residue is unmodified or is modified at the N-terminus by either acetylation or methylation,

X2 is L or T,

X3 is L or I,

X4 is Q, R, or E,

X7 is T or E,

X8 is Q, H or R (SEQ ID NO:67), and

Formula III further comprises a modified K residue (“K*”) substituted at position 10 or at any one position between position 14 and position 30 inclusive,

K* is modified by having the epsilon amino group of the K-side chain bound to a group of the formula—X5—X6, wherein X5 is selected from the group consisting of between one to four amino acid residues, between one to four ([2-(2-Amino-ethoxy)-ethoxy]-acetyl moieties, and combinations of one to four amino acid residues and one to four ([2-(2-Amino-ethoxy)-ethoxy]-acetyl moieties, and X6 is a C14-C24 fatty acid.

The present invention provides pharmaceutical compositions comprising a compound of Formula III with the modified K residue, or a pharmaceutically acceptable salt thereof (for example, trifluoroacetate salts, acetate salts, or hydrochloride salts). In some embodiments, the terminal amino acid is amidated as a C-terminal primary amide. In further embodiments, the pharmaceutical composition may include more pharmaceutically acceptable carriers, diluents, and excipients.

As noted above, the synthetic molecules of Formula III are constructed such that the modified K residue is substituted at position 10 or at any one position between position 14 and position 30 inclusive. For example, if the modified K residue is substituted at position 10, then the G residue that normally occupies position 10 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

3 X 1 2 IVX SLDVPIK * LLQILX EQEKQEKEKQQATX 7 Rising 4 Ilakha 8 V-NH 2

wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 14, then the I residue that normally occupies position 14 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X IVX 1 2 3 SLDVPIGLLQK * LX EQEKQEKEKQQATX 7 Rising 4 Ilakha 8 V-NH 2

wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 15, then the L residue that normally occupies position 15 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQIK X3EQEKQEKEKQQATX7NAX4ILAX8V-NH2

wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 16, then the X3 residue that normally occupies position 16 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

2 X 1 IVX SLDVPIGLLQILK * EQEKQEKEKQQATX 7 Rising 4 Ilakha 8 V-NH 2

wherein K* is the modified K residue and X1, X2, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 17, then the E residue that normally occupies position 17 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3K QEKQEKEKQQATX7NAX4ILAX8V-NH2

wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 18, then the Q residue that normally occupies position 18 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EK * EKQEKEKQQATX7NAX4ILAX8V-NH2

wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 19, then the E residue that normally occupies position 19 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EQK * KQEKEKQQATX7NAX4ILAX8V-NH2

wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 20, then the K residue that normally occupies position 20 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EQEK*QEKEKQQATX7NAX4ILAX8V-NH2 wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 21, then the Q residue that normally occupies position 21 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EQEKK*EKEKQQATX7NAX4ILAX8V-NH2 wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 22, then the E residue that normally occupies position 22 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EQEKQK*KEKQQATX7NAX4ILAX8V-NH2 wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 23, then the K residue that normally occupies position 23 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EQEKQEK*EKQQATX7NAX4ILAX8V-NH2 wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 24, then the E residue that normally occupies position 24 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EQEKQEKK*KQQATX7NAX4ILAX8V-NH2 wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 25, then the K residue that normally occupies position 25 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EQEKQEKEK*QQATX7NAX4ILAX8V-NH2 wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 26, then the Q residue that normally occupies position 26 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EQEKQEKEKK*QATX7NAX4ILAX8V-NH2 wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 27, then the Q residue that normally occupies position 27 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EQEKQEKEKQK*ATX7NAX4ILAX8V-NH2 wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 28, then the A residue that normally occupies position 28 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EQEKQEKEKQQK*TX7NAX4ILAX8V-NH2 wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 29, then the T residue that normally occupies position 29 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

X1IVX2SLDVPIGLLQILX3EQEKQEKEKQQAK X7NAX4ILAX8V-NH2

wherein K* is the modified K residue and X1, X2, X3, X4, X7 and X8 have the values and features described herein.

If the modified K residue is substituted at position 30, then the X7 residue that normally occupies position 30 is replaced with the modified K residue, such that these synthetic molecules would have the following formula:

3 X 1 2 IVX SLDVPIGLLQILX EQEKQEKEKQQATK declared NAX 4 8 V-NH 2

wherein K* is the modified K residue and X1, X2, X3, X4, and X8 have the values and features described herein.

As noted above, the X8 of Formula III may be Q, H, or R. However, in some of the presently preferred embodiments, the X8 group will be either an H or Q. Further preferred embodiments may have the X2 and/or the X3 of Formula III be an L residue. In yet additional preferred embodiments, the X4 of Formula III may be a Q residue and/or the X7 of Formula III is an T residue.

In other presently preferred embodiments, the X5 of Formula III may comprise between 0-2 ([2-(2-Amino-ethoxy)-ethoxy]-acetyl moieties and, more preferably, 1 or 2 amino acid residues. In other presently preferred embodiments, the X5 of Formula III may comprise two amino acid residues and two ([2-(2-Amino-ethoxy)-ethoxy]-acetyl moieties, wherein the two amino acid residues are either E or γE residues. In some embodiments, X5 comprises only amino acid residues such that there are no ([2-(2-Amino-ethoxy)-ethoxy]-acetyl moieties. In yet additional presently preferred embodiments, the X1 of Formula III will have the I residue at position 1 modified (at the N-terminus) by either acetylation or methylation.

As noted above, the amino acid sequence of Formula III is modified such that a modified K residue is substituted at position 10 or at any one position between position 14 and position 30 inclusive within the sequence. Some of the most preferred embodiments of Formula III have the modified K residue (“K*”) substituted into position 29 and have X8 be Q and X7 be T. These molecules, which are subset of Formula III, are represented below as Formula I: X1 I V X2 S L D V P I G L L Q I L X3 E Q E K Q E K E K Q Q A K* T N A X4 I L A Q V-NH2 (Formula I)

(As with the embodiments of Formula III, the embodiments of Formula I are designed such that X2 can be L or T, and X3 can be L or I, X4 can be Q, R, or E (and more preferably Q or E), and X1 can mean that the I residue at position 1 is, at its N-terminus, unmodified or is modified by either acetylation or methylation.) In some of the preferred embodiments of Formula I, the X1 will have the I residue at position 1 modified at the N-terminus by either acetylation or methylation. In the embodiments of Formula I, the modified K residue at position 29 is modified with a fatty acid side chain that is conjugated to the epsilon-amino group of the K side chain, wherein the fatty acid side chain has a formula: ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)x-CO2H where x is 16 or 18. (Stated differently, in some of the presently preferred embodiments of Formula I, the X5 and X6 groups of Formula III have the following formula: ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)x-CO2H where x is 16 or 18) (SEQ ID NO:8). Of course, as noted above, the compound and molecules of Formula I may be made into pharmaceutically acceptable salts thereof.

The present invention also provides a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof (for example, trifluoroacetate salts, acetate salts, or hydrochloride salts). In some embodiments, the terminal amino acid is amidated as a C-terminal primary amide. In further embodiments, the pharmaceutical composition may include more pharmaceutically acceptable carriers, diluents, and excipients.

In one embodiment, the compound or pharmaceutically acceptable salt of Formula I is designed such that X1 has the N-terminus of the I residue modified by acetylation; X2 is L; X3 is L; X4 is Q; the K* at position 29 is chemically modified through conjugation to the epsilon-amino group of the K-side chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)16-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:1).

In another embodiment, the compound or pharmaceutically acceptable salt of Formula I is designed such that X1 has the N-terminus of the I residue modified by acetylation; X2 is L; X3 is L; X4 is Q; the K* at position 29 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-

ethoxy]-acetyl)2-(γE)2-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:2).

In another embodiment, the compound or pharmaceutically acceptable salt of Formula I is designed such that X1 has the N-terminus of the I residue modified by methylation; X2 is L; X3 is L; X4 is Q; the K* at position 29 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)16-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:3).

In another embodiment, the compound or pharmaceutically acceptable salt of Formula I is designed such that X1 has the N-terminus of the I residue modified by methylation; X2 is L; X3 is L; X4 is Q; the K* at position 29 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:4).

In another embodiment, the compound or pharmaceutically acceptable salt of Formula I is designed such that X1 has the N-terminus of the I residue modified by methylation; X2 is T; X3 is L; X4 is E; the K* at position 29 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:5).

In another embodiment, the compound or pharmaceutically acceptable salt of Formula I is designed such that X1 has the N-terminus of the I residue modified by methylation; X2 is L; X3 is L; X4 is E; the K* at position 29 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:6).

In another embodiment, the compound or pharmaceutically acceptable salt of Formula I is designed such that X1 has the N-terminus of the I residue modified by methylation; X2 is T; X3 is I; X4 is E; the K* at position 29 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:7).

Further preferred embodiments of the present invention (which likewise fall within the scope of Formula III) may be designed in which the modified K residue (“K*”) is substituted at position 29 and X8 is Q, X7 is T. Such preferred molecules and compounds (which are subset of Formula III) can be represented as Formula II:

X1 I V X2 S L D V P I G L L Q I L X3 E Q E K Q E K E K Q Q A K* T N A X4 I L A Q V-NH2 (Formula II)

(As with the embodiments of Formula III, the embodiments of Formula II are designed such that X2 can be L or T, and X3 can be L or I, X4 can be Q, R, or E, and X1 can mean that the I residue at position 1 is, at its N-terminus, unmodified or is modified by either acetylation or methylation.) However, further preferred embodiments of Formula II may be designed in which the X1 is restricted such that the I residue (at position 1) is modified by either acetylation or methylation at the N-terminus and X4 is restricted to being either Q or E.

In the embodiments of Formula II, the modified K residue (“K*”) at position 29 is modified through conjugation to the epsilon-amino group of the K-side chain with a group of the formula–X5–X6, wherein

X5 is selected from the group consisting of:

one to four amino acids;

one to four ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) moieties; and combinations of one to four amino acids and one to four ([2-(2-Amino-ethoxy)- ethoxy]-acetyl) moieties;

X6 is a C14-C24 fatty acid (SEQ ID NO:16), or a pharmaceutically acceptable salt thereof.

In the embodiments of Formula 1 described herein, the modified K residue used in Formula I has the epsilon-amino group of the K side chain conjugated to the following group: ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)x-CO2H where x is 16 or 18. (Stated differently, in some of the presently preferred embodiments of Formula I, the X5 and X6 groups of Formula III have the following formula: ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)x-CO2H where x is 16 or 18).

Yet, as noted above, the compounds of Formula II and Formula III may use different groups for X5 and X6. For example, in some embodiments of Formula II and Formula III, X5 may be one or four E or γE amino acid residues, . Further embodiments Formula II and Formula III may may have X5 be two to four E or γE. Still further preferred embodiments Formula II and Formula III are constructed in which X5 comprises two γE amino acids. In some embodiments of Formula II and Formula III, the X5 group may comprise only amino acid residues; however, in other embodiments, the X5 group may comprise one to four amino acid residues (such as, for exmaple E or γE amino acids) used in combination with one to four ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) moieties. Specifically, in other embodiments, X5 constitutes combinations of one to four E or γE amino acids and one to four ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) moieties. Additional embodiments are designed in which X5 is combinations of two to four γE amino acids and one to four ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) moieties (such as, for example two of the ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) moieties). Other embodiments have X5 be combinations of two γE amino acids and two ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) moieties.

In one preferred embodiment of Formulas III and III, the group of the formula–X5– X6 is ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)x-CO2H, where x is 16 or 18. In other embodiments of Formulas III and III, the X5 group may comprise at least one ([2-(2-Amino-ethoxy)-ethoxy]-acetyl moiety, with or without any amino acid residues. Further preferred embodiments of Formulas III and III are constructed in which the X5 group comprises one or two ([2-(2-Amino-ethoxy)-ethoxy]-acetyl moieties. In some embodiments the X6 group is a straight chain fatty acid side chain of the formula CO-(CH2)x-CO2H, wherein x is 16, 18, or 20. Most preferable embodiments have x being either 16 or 18.

As noted above, the compounds (or pharmaceutically acceptable salts thereof) of Formulas II and III have an X6 group that is a C14 to C24 fatty acid. This C14-C24 fatty acid may be a saturated monoacid or a saturated diacid. Preferably, the fatty acid is a saturated monoacid or saturated diacid selected from the group consisting of myristic acid

(tetradecanoic acid)(C14 monoacid), tetradecanedioic acid (C14 diacid), palmitic acid

(hexadecanoic acid)(C16 monoacid), hexadecanedioic acid (C16 diacid), margaric acid (heptadecanoic acid)(C17 monoacid), heptadecanedioic acid (C17 diacid), stearic acid

(octadecanoic acid)(C18 monoacid), octadecanedioic acid (C18 diacid), nonadecylic acid (nonadecanoic acid)(C19 monoacid), nonadecanedioic acid (C19 diacid), arachadic acid (eicosanoic acid) (C20 monoacid), eicosanedioic acid (C20 diacid), heneicosylic acid

(heneicosanoic acid) (C21 monoacid), heneicosanedioic acid (C21 diacid), behenic acid (docosanoic acid) (C22 monoacid), docosanedioic acid (C22 diacid), lignoceric acid

(tetracosanoic acid)(C24 monoacid) and tetracosanedioic acid (C24 diacid). The most preferable acids are the following: myristic acid, tetradecanedioic acid, palmitic acid, hexadecanedioic acid, stearic acid, octadecanedioic acid, nonadecanedioic acid, arachadic acid, eicosanedioic acid or docosanedioic acid.

The present invention of Formula I or Formula II or Formula III provides a pharmaceutical composition comprising a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof (for example, trifluoroacetate salts, acetate salts, or hydrochloride salts among others). In other embodiments, any salt or free base suitable for human use may be made using the compounds of Formula I or Formula II or Formula III. In some preferred embodiments, a peptide acetate salt of the compounds of Formula I or Formula II or Formula III is used. In some embodiments, the C-terminal amino acid is amidated as a C-terminal primary amide. In further embodiments, the pharmaceutical composition may include more pharmaceutically acceptable carriers, diluents, and excipients.

The present invention provides a pharmaceutical composition comprising a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The present invention also provides a pharmaceutical composition comprising a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof, in combination with an additional active ingredient.

The present invention provides a method for treatment of type II diabetes in a patient comprising administering to a patient in need of such treatment an effective amount of a

compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof. The present invention also provides a method for treatment of type II diabetes in a patient comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof, wherein the administration is subcutaneous. The present invention also provides a method of treatment of type II diabetes in a patient comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof, and simultaneously or sequentially an effective amount of one or more other active ingredients. In one embodiment, the other active ingredient is a currently available oral glucose lowering drugselected from a class of drugs that is considered prior to administration the standard of care as determined by industry guidelines such as the American Diabetes Association. Examples of current standard of care include metformin, thiazolidinediones (TZDs), sulfonylureas (SUs), dipeptidyl peptidase4 (DPP-IV) inhibitors, and sodium glucose co-transporters (SGLTs). In a further embodiment of the present invention, a method of treatment of type II diabetes in a patient as defined above is combined with diet and exercise.

Furthermore, the present invention provides a method for treatment of chronic kidney disease in a patient comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof. The present invention also provides a method for treatment of chronic kidney disease in a patient comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I or Formula II or Formula III, or a

pharmaceutically acceptable salt thereof, wherein the chronic kidney disease is caused by diabetic nephropathy. The present invention also provides a method for treatment of chronic kidney disease in a patient comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I or Formula II or Formula III, or a

pharmaceutically acceptable salt thereof, wherein the chronic kidney disease is caused by hypertensive nephropathy. The present invention also provides a method for treatment of chronic kidney disease in a patient comprising administering to a patient in need of such

treatment an effective amount of a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof, wherein the administration is subcutaneous. The present invention also provides a method of treatment of chronic kidney disease in a patient comprising administering to a patient in need of such treatment an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, and simultaneously or sequentially an effective amount of one or more other active ingredients. In one embodiment, the other active ingredient is selected from currently available oral ACE inhibitors or ARBs that are considered prior to administration the standard of care as determined by industry guidelines. Examples of current standard of care are ACEs inhibitors lisinopril and captopril and ARBs losartan and irbesartan.

Moreover, the present invention provides a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof, for use in therapy. The present invention also provides a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof, for use in the treatment of type II diabetes.

Furthermore, the present invention provides a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic kidney disease. The present invention provides a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof, for use in the treatment of chronic kidney disease caused by diabetic nephropathy or hypertensive nephropathy. The present invention provides the use of a compound of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of type II diabetes and/or chronic kidney disease.

The present invention also encompasses novel intermediates and processes for the synthesis of the compounds of Formula I or Formula II or Formula III.

The compounds of Formula I or Formula II or Formula III or a pharmaceutically salt thereof are particularly useful in the treatment methods of the invention.

The peptide chain of the compounds of the present invention can be synthesized using standard manual or automated solid-phase synthesis procedures. Automated peptide synthesizers are commercially available from, for example, Applied Biosystems (Foster City, CA) and Protein Technologies Inc. (Tucson, AZ). Reagents for solid-phase synthesis are readily available from commercial sources. Solid-phase synthesizers can be used according to the manufacturer’s instructions for blocking interfering groups, protecting amino acids during reaction, coupling, deprotecting, and capping of unreacted amino acids.

Typically, an N-α-carbamoyl protected amino acid and the N-terminal amino acid on the growing peptide chain attached to a resin are coupled at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidone or methylene chloride in the presence of coupling agents such as diisopropyl-carbodiimide and 1-hydroxybenzotriazole. The Nα-carbamoyl protecting group is removed from the resulting peptide resin using a reagent such as trifluoroacetic acid (TFA) or piperidine, and the coupling reaction is repeated with the next desired Nα- protected amino acid to be added to the peptide chain. Suitable amine protecting groups are well known in the art and are described, for example, in Green and Wuts,“Protecting Groups in Organic Synthesis,” John Wiley and Sons, 1991. The most commonly used examples include tBoc and fluorenylmethoxycarbonyl (Fmoc). After completion of synthesis, peptides are cleaved from the solid-phase support with simultaneous side-chain deprotection using standard treatment methods under acidic conditions.

The skilled artisan will appreciate that the peptide chain of the compounds of the invention are synthesized with a C-terminal carboxamide. For the synthesis of C-terminal amide peptides, resins incorporating Rink amide MBHA or Rink amide AM linkers are typically used with Fmoc synthesis, while MBHA resin is generally used with tBoc synthesis.

Crude peptides typically are purified using RP-HPLC on C8 or C18 columns using water- acetonitrile gradients in 0.05 to 0.1% TFA. Purity can be verified by analytical RP-HPLC. Identity of peptides can be verified by mass spectrometry. Peptides can be solubilized in aqueous buffers over a wide pH range.

As used herein, the term“AUC” means area under the curve.

As used herein, the term“average molecular weight” indicates the average of the molecular weight of the different oligomer size components with a very narrow distribution and is determined by mass spectrometry techniques.

As used herein, the term“EC50” refers to the concentration of compound that results in 50% activation of the assay endpoint, e.g., cAMP.

As used herein, the term“ED50” refers to the concentration of compound that results in a 50% response in the in vivo assay endpoint, e.g., plasma or blood glucose.

As used herein, the term“effective amount” refers to the amount or dose of compound of the invention, or a pharmaceutically acceptable salt thereof which, upon single or multiple dose administration to the patient, provides the desired effect in the patient under diagnosis or treatment for a daily administration. An effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount for a patient, a number of factors are considered by the attending diagnostician, including, but not limited to: the species of mammal; its size, age, and general health; the specific disease or disorder involved; the degree of or involvement or the severity of the disease or disorder; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.

As used herein, the term“patient” refers to a mammal, such as a mouse, guinea pig, rat, dog, cat, or human. It is understood that the preferred patient is a human.

As used herein, the term“treating” or“to treat” includes prohibiting, restraining, slowing, stopping, or reversing the progression or severity of an existing symptom or disorder.

When used herein, the term“in combination with” means administration of the synthetic molecule of the present invention either simultaneously, sequentially or in a single combined formulation with the one or more additional therapeutic agents.

Certain abbreviations are defined as follows:“ACR” refers to urine albumin/urine creatinine ratio;“amu” refers to atomic mass unit;“Boc” refers to tert-butoxycarbonyl;

“cAMP” refers to cyclic adenosine monophosphate;“DMSO” refers to dimethyl sulfoxide; “EIA/RIA” refers to enzyme immunoassay/radioimmunoassay;“hr” refers to hour;“HTRF”

refers to homogenous time-resolved fluorescent;“i.v.” refers to intravenous;“kDa” refers to kilodaltons; “LC-MS” refers to liquid chromatography-mass spectrometry;“MS” refers to ass spectrometry;“OtBu” refers to O-tert-butyl;“Pbf” refers to NG-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl;“RP-HPLC” refers to reversed-phase high performance liquid chromatography;“s.c.” refers to subcutaneous;“SEM” refers to standard error of the mean;“TFA” refers to trifluoroacetic acid; and“Trt” refers to Trityl. Standard one- letter codes are used to represent the amino acid in the compounds of Formula I. All amino acids used in the Formula I are L-amino acids. Standard three-letter codes may also be used to represent amino acids.

The compounds of the present invention utilize a C14-C24 fatty acid chemically conjugated to the epsilon-amino group of a lysine side-chain either by a direct bond or by a linker. The term“C14-C24 fatty acid” as used herein means a carboxylic acid with between 14 and 24 carbon atoms. The C14-C24 fatty acid suitable for use herein can be a saturated monoacid or a saturated diacid. By“saturated” is meant that the fatty acid contains no carbon-carbon double or triple bonds.

Examples of specific saturated C14-C24 fatty acids that are suitable for the compounds and uses thereof disclosed herein include, but are not limited to, myristic acid (tetradecanoic acid)(C14 monoacid), tetradecanedioic acid (C14 diacid), palmitic acid (hexadecanoic acid) (C16 monoacid), hexadecanedioic acid (C16 diacid), margaric acid (heptadecanoic acid)(C17 monoacid), heptadecanedioic acid (C17 diacid), stearic acid (octadecanoic acid)(C18 monoacid), octadecanedioic acid (C18 diacid), nonadecylic acid (nonadecanoic acid)(C19 monoacid), nonadecanedioic acid (C19 diacid), arachadic acid (eicosanoic acid)(C20 monoacid), eicosanedioic acid (C20 diacid), heneicosylic acid (heneicosanoic acid)(C21 monoacid), heneicosanedioic acid (C21 diacid), behenic acid (docosanoic acid)(C22 monoacid), docosanedioic acid (C22 diacid), lignoceric acid (tetracosanoic acid) (C24 monoacid), tetracosanedioic acid (C24 diacid), including branched and substituted derivatives thereof.

In preferred aspects of the compounds of the present invention, the C14-C24 fatty acid is selected from the group consisting of a saturated C14 monoacid, a saturated C14 diacid, a saturated C16 monoacid, a saturated C16 diacid, a saturated C18 monoacid, a saturated C18 diacid, a saturated C19 diacid, a saturated C20 monoacid, a saturated C20 diacid, a saturated C22 diacid, and branched and substituted derivatives thereof. In more preferred aspects of the compounds of the present invention, the C14-C24 fatty acid is selected from the group consisting of myristic acid, tetradecanedioic acid, palmitic acid, hexadecanedioic acid, stearic acid, octadecanedioic acid, nonadecanedioic acid, arachadic acid, eicosanedioic acid and docosanedioic acid. Preferably, the C14-C24 fatty acid is octadecanedioic acid or

eicosanedioic acid.

The length and composition of the fatty acid impacts the half-life of the compound, the potency of the compound in in vivo animal models and also impacts the solubility and stability of the compound. Conjugation of the peptide defined herein to a C14-C24 saturated fatty monoacid or diacid results in compounds that exhibit desirable half-life, desirable potency in in vivo animal models and also possess desired solubility and stability

characteristics.

The compounds of the invention are preferably formulated as pharmaceutical compositions administered by parenteral routes (e.g., subcutaneous, intravenous,

intraperitoneal, intramuscular, or transdermal). Such pharmaceutical compositions and processes for preparing same are well known in the art. (See, e.g., Remington: The Science and Practice of Pharmacy, L.V. Allen, Editor, 22nd Edition, Pharmaceutical Press, 2012). The preferred route of administration is subcutaneous.

The compounds of the present invention may react with any of a number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. See, e.g., P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011); S.M. Berge, et al.,“Pharmaceutical Salts,” Journal of Pharmaceutical Sciences, Vol.66, NO:1, January 1977. Preferred pharmaceutically acceptable salt of the present invention are trifluoroacetate salts, acetate salts and hydrochloride salts among others.

The compounds of the present invention may be administered by a physician or self-administered using an injection. It is understood the gauge size and amount of injection volume is determined by the skilled practitioner. In one embodiment, the amount of injection volume is≤ 2ml, preferably≤1 ml. Also a further embodiment is the use of a needle gauge ^27, preferably≥29.

The compounds of Formula I or Formula II or Formula III are generally effective over a wide dosage range. For example, dosages per day normally fall within the range of about 0.01 to about 50 mg/kg of body weight. In some instances dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed with acceptable side effects, and therefore the above dosage range is not intended to limit the scope of the invention in any way.

The present invention also encompasses novel intermediates and processes useful for the synthesis of compounds of Formula I or Formula II or Formula III, or a pharmaceutically acceptable salt thereof. The intermediates and compounds of the present invention may be prepared by a variety of procedures known in the art including via both chemical synthesis and recombinant technology. In particular, the process using chemical synthesis is illustrated in the Preparation(s) and Example(s) below. The specific synthetic steps for each of the routes described may be combined in different ways to prepare compounds of Formula I, or salts thereof. The reagents and starting materials are readily available to one of ordinary skill in the art. It is understood that these Preparation(s) and Example(s) are not intended to be limiting to the scope of the invention in any way.

EXAMPLE 1

X1IVX2SLDVPIGLLQILX3EQEKQEKEKQQAK*TNAX4ILAQV-NH2

wherein the X1 at position 1 is I that is modified, at the N terminus, by acetylation; X2 is L; X3 is L; X4 is Q; and the K* at position 29 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)16-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 1). The structure of this sequence is shown below.

The structure of this sequence contains the standard single letter amino acid code with exception of residues I at position 1, and K at position 29 where the structures of these amino acid residues have been expanded.

The peptide according to SEQ ID NO: 1 of the present invention is generated by solid-phase peptide synthesis using a Fmoc/t-Bu strategy carried out on a Symphony automated peptide synthesizer (PTI Protein Technologies Inc.) starting from RAPP AM-Rink Amide resin (H40023 Polystyrene AM RAM, Rapp polymere GmbH) and with couplings using 6 equivalents of amino acid activated with diisopropylcarbodiimide (DIC) and Oxyma pure (1:1:1 molar ratio) in dimethylformamide (DMF) for 3h at 25°C.

Extended coupling for Thr30 (10h) is necessary to improve the quality of the crude peptide. A Fmoc-Lys(Alloc)-OH building block is used for K at position 29 coupling (orthogonal protecting group) to allow for site specific attachment of the fatty acid moiety later on in the synthetic process. The N-terminal residue (I at position 1) is acetylated using 10 equivalents of acetic acid with diisopropylcarbodiimide (DIC) and Oxyma pure (1:1:1 molar ratio) in dimethylformamide (DMF) for 1h at 25°C.

After finishing the elongation of the peptide-resin described above, the Alloc protecting group present in the K at position 29 is removed using catalytic amounts of Pd(PPh3)4 in the presence of PhSiH3 as a scavenger. Additional coupling/deprotection cycles using a Fmoc/t-Bu strategy to extend the K at position 29 side-chain involved Fmoc-NH-PEG2-CH2COOH (ChemPep Catalog#280102), Fmoc-Glu(OH)-OtBu (ChemPep

Catalog#100703) and HOOC-(CH2)16-COOtBu. In all couplings, 3 equivalents of the building block are used with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 4h at 25°C.

Concomitant cleavage from the resin and side chain protecting group removal are carried out in a solution containing trifluoroacetic acid (TFA): triisopropylsilane : 1,2-ethanedithiol: methanol : thioanisole 80:5:5:5:5 (v/v) for 2 h at 25°C followed by

precipitation with cold ether. Crude peptide is purified to > 99% purity (15-20% purified yield) by reversed-phase HPLC chromatography with water / acetonitrile (containing 0.1% v/v TFA) gradient on a Phenyl hexyl column (phenomenex, 5 micron, 100A), where suitable fractions are pooled and lyophilized.

In a synthesis performed essentially as described above, the purity of Example 1 was examined by analytical reversed-phase HPLC, and identity was confirmed using LC/MS (observed: M+3H+/3 =1718.8; Calculated M+3H+/3 =1720.0; observed: M+4H+/4 =1289.2; Calculated M+4H+/4 =1290.3; observed: M+5H+/5 =1031.5; Calculated M+5H+/5 =1032.4).

EXAMPLE 2

X1IVX2SLDVPIGLLQILX3EQEKQEKEKQQAK*TNAX4ILAQV-NH2

wherein the X1 is I in which the N terminus is modified via acetylation; X2 is L; X3 is L; X4 is Q; and the K* at position 29 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 2). The structure of this sequence is shown below.

The structure of this sequence contains the standard single letter amino acid code with exception of residues I at position 1 and K at position 29 where the structures of these amino acid residues have been expanded.

The peptide according to SEQ ID NO: 2 of the present invention is synthesized similarly as described above in Example 1. HOOC-(CH2)18-COOtBu is incorporated using 3 equivalents of the building block with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 4h at 25°C.

In a synthesis performed essentially as described above, the purity of Example 2 was examined by analytical reversed-phase HPLC, and identity was confirmed using LC/MS (observed: M+3H+/3 =1728.2; Calculated M+3H+/3 =1729.4; observed: M+4H+/4 =1296.3; Calculated M+4H+/4 =1297.3; observed: M+5H+/5 =1037.4; Calculated M+5H+/5 =1038.0).

EXAMPLE 3

X1IVX2SLDVPIGLLQILX3EQEKQEKEKQQAK*TNAX4ILAQV-NH2

wherein the X1 is I in which the N terminus is modified via methylation; X2 is L; X3 is L; X4 is Q; and the K* at position 29 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2- CO-(CH2)16-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 3). The structure of this sequence is shown below.

The structure of this sequence contains the standard single letter amino acid code with exception of residues N-methyl isoleucine at position 1 and K at position 29, where the structures of these amino acid residues have been expanded.

The compound according to SEQ ID NO: 3 of the present invention is synthesized similarly as described above for Example 1. The N-terminal residue (N-methyl isoleucine at position 1) is incorporated as Boc-NMeIle-OH using 6 equivalents of the building block with PyBOP (6 equiv) and DIEA (12 equiv) in DMF-DCM (1:1, v/v) for 15h at 25°C.

In a synthesis performed essentially as described above, the purity of Example 3 was examined by analytical reversed-phase HPLC, and identity was confirmed using LC/MS (observed: M+3H+/3 =1709.6; Calculated M+3H+/3 =1710.7; observed: M+4H+/4 =1282.2; Calculated M+4H+/4 =1283.3; observed: M+5H+/5 =1025.8; Calculated M+5H+/5 =1026.8).

EXAMPLE 4

X1IVX2SLDVPIGLLQILX3EQEKQEKEKQQAK*TNAX4ILAQV-NH2

wherein X1 is I in which the N terminus is modified via methylation; X2 is L; X3 is L; X4 is Q; and the K* at position 29 is chemically modified through conjugation to the epsilon- amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 4). The structure of this sequence is shown below.

The structure of this sequence contains the standard single letter amino acid code with exception of residues N-methyl Isoleucine at position 1 and K at position 29, where the structures of these amino acid residues have been expanded.

The compound according to SEQ ID NO: 4 of the present invention is synthesized similarly as described above for Example 1. The N-terminal residue (N-methyl Isoleucine at position 1) is incorporated as Boc-NMeIle-OH using 6 equivalents of the building block with PyBOP (6 equiv) and DIEA (12 equiv) in DMF-DCM (1:1, v/v) for 15h at 25°C. HOOC-(CH2)18-COOtBu is incorporated using 3 equivalents of the building block with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 4h at 25°C.

In a synthesis performed essentially as described above, the purity of Example 4 was examined by analytical reversed-phase HPLC, and identity was confirmed using LC/MS (observed: M+3H+/3 =1719.4; Calculated M+3H+/3 =1720.1; observed: M+4H+/4 =1289.8; Calculated M+4H+/4 =1290.3; observed: M+5H+/5 =1031.8; Calculated M+5H+/5 =1032.4).

EXAMPLE 5

X1IVX2SLDVPIGLLQILX3EQEKQEKEKQQAK TNAX4ILAQV-NH2

wherein X1 is I in which the N terminus is modified via methylation; X2 is T; X3 is L; X4 is E; and the K* at position 29 is chemically modified through conjugation to the epsilon- amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO- (CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 5). The structure of this sequence is shown below.

The structure of this sequence contains the standard single letter amino acid code with exception of residues N-methyl Isoleucine at position 1, and K at position 29 where the structures of these amino acid residues have been expanded.

The compound according to SEQ ID NO: 5 of the present invention is synthesized similarly as described above for Example 4.

In a synthesis performed essentially as described above, the purity of Example 5 was examined by analytical reversed-phase HPLC, and identity was confirmed using LC/MS (observed: M+3H+/3 =1715.7; Calculated M+3H+/3 =1716.4; observed: M+4H+/4 =1287.0; Calculated M+4H+/4 =1287.5; observed: M+5H+/5 =1029.7; Calculated M+5H+/5 =1030.2).

EXAMPLE 6

X1IVX2SLDVPIGLLQILX3EQEKQEKEKQQAK TNAX4ILAQV-NH2

wherein X1 is I in which the N terminus is modified via methylation; X2 is L; X3 is L; X4 is E; and the K* at position 29 is chemically modified through conjugation to the epsilon- amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO- (CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 6). The structure of this sequence is shown below.

The structure of this sequence 6 contains the standard single letter amino acid code with exception of residues N-Methyl Isoleucine at position 1 and K at position 29 where the structures of these amino acid residues have been expanded.

The compound according to SEQ ID NO: 6 of the present invention is synthesized similarly as described above for Example 4.

In a synthesis performed essentially as described above, the purity of Example 6 was examined by analytical reversed-phase HPLC, and identity was confirmed using LC/MS (observed: M+3H+/3 =1719.7; Calculated M+3H+/3 =1720.4; observed: M+4H+/4 =1289.8; Calculated M+4H+/4 =1290.5; observed: M+5H+/5 =1032.2; Calculated M+5H+/5 =1032.6).

EXAMPLE 7

X1IVX2SLDVPIGLLQILX3EQEKQEKEKQQAK*TNAX4ILAQV-NH2

wherein X1 is I in which the N terminus is modified via methylation ; X2 is T; X3 is I; X4 is E; and the K* at position 29 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)18-CO2H; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO: 7). The structure of this sequence is shown below.

The structure of this sequence contains the standard single letter amino acid code with exception of residues N-methyl Isoleucine at position 1 and K at position 29, where the structures of these amino acid residues have been expanded.

The compound according to SEQ ID NO: 7 of the present invention is synthesized similarly as described above for Example 4.

In a synthesis performed essentially as described above, the purity of Example 7 was examined by analytical reversed-phase HPLC, and identity was confirmed using LC/MS (observed: M+3H+/3 =1715.6; Calculated M+3H+/3 =1716.4; observed: M+4H+/4 =1286.8; Calculated M+4H+/4 =1287.5; observed: M+5H+/5 =1029.8; Calculated M+5H+/5 =1030.2).

EXAMPLE 8

The following compounds of the present invention are synthesized similarly as described above for Example 4. The structures shown below contains the standard single

letter amino acid code with exception of residues N-methylated I at position 1 and K at position 29 where the structures of these amino acid residues have been expanded.

X1IVLSLDVPIGLLQILLEQEKQEKEKQQAK*TNAQILAQV-NH2

wherein X1 has the N-terminus of the I residue modified by methylation;

wherein the K* at position 29 has been chemically modified with the following fatty acid side chain:

-γE-([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)18-COOH; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:9). X1IVLSLDVPIGLLQILLEQEKQEKEKQQAK*TNAQILAQV-NH2

wherein X1 has the N-terminus of the I residue modified by methylation;

wherein the K* at position 29 has been chemically modified with the following fatty acid side chain:

-γE-([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2–(γE)2-CO-(CH2)16-COOH;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:10).

X1IVLSLDVPIGLLQILLEQEKQEKEKQQAK*TNAQILAQV-NH2

wherein X1 has the N-terminus of the I residue modified by methylation;

wherein the K* at position 29 has been chemically modified with the following fatty acid side chain:

-γE-γE-γE-γE-CO-(CH2)18-COOH;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:11).

X1IVLSLDVPIGLLQILLEQEKQEKEKQQAK*TNAQILAQV-NH2

wherein X1 has the N-terminus of the I residue modified by methylation;

wherein the K* at position 29 has been chemically modified with the following fatty acid side chain:

-γE-γE-([2-(2-Amino-ethoxy)-ethoxy]-acetyl)-γE-γE-CO-(CH2)18-COOH; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:12).

X1IVLSLDVPIGLLQILLEQEKQEKEKQQAK*TNAQILAQV-NH2

wherein X1 has the N-terminus of the I residue modified by methylation;

wherein the K* at position 29 has been chemically modified with the following fatty acid side chain:

-γE-γE-([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-γE-γE-CO-(CH2)18-COOH; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:13).

X1IVLSLDVPIGLLQILLEQEKQEKEKQQAK*TNAQILAQV-NH2

wherein X1 has the N-terminus of the I residue modified by methylation;

wherein the K* at position 29 has been chemically modified with the following fatty acid side chain:

-γE-([2-(2-Amino-ethoxy)-ethoxy]-acetyl)-γE-γE-CO-(CH2)18-COOH;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:14).

EXAMPLE 9

The following compounds of the present invention are synthesized similarly as described above for Example 4. The structures shown below contains the standard single letter amino acid codes. All of the following compounds or synthetic molecules fall within the scope of Formula III. The purity of these compounds was tested by analytical reversed-phase HPLC, and identity was confirmed using LC/MS, in the manner outlined herein.

IVLSLDVPIGLLQK*LLEQEKQEKEKQQATTNARILARV-NH2

wherein the K* at position 14 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:21).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 14).

IVLSLDVPIGLLQIK*LEQEKQEKEKQQATTNARILARV-NH2

wherein the K* at position 15 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:22).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 15).

IVLSLDVPIGLLQILLK*QEKQEKEKQQATTNARILARV-NH2

wherein the K* at position 17 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:23).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 17).

IVLSLDVPIGLLQILLEQK*KQEKEKQQATTNARILARV-NH2

wherein the K* at position 19 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:24^).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 19).

IVLSLDVPIGLLQILLEQEK*QEKEKQQATTNARILARV-NH2

wherein the K* at position 20 has been chemically such that the epsilon-amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:25).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 20).

IVLSLDVPIGLLQILLEQEKK*EKEKQQATTNARILARV-NH2

wherein the K* at position 21 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:26).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 21).

IVLSLDVPIGLLQILLEQEKQK*KEKQQATTNARILARV-NH2

wherein the K* at position 22 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:27).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 22).

IVLSLDVPIGLLQILLEQEKQEK*EKQQATTNARILARV-NH2

wherein the K* at position 23 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:28).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 23).

IVLSLDVPIGLLQILLEQEKQEKK*KQQATTNARILARV-NH2

wherein the K* at position 24 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:29).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 24).

IVLSLDVPIGLLQILLEQEKQEKEK*QQATTNARILARV-NH2

wherein the K* at position 25 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:30).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 25).

IVLSLDVPIGLLQILLEQEKQEKEK * QQATTNARILARV-NH2

wherein the K* at position 25 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-γE- CO-(CH2)14-CH3 group; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:31).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is two γE residues and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 25).

IVLSLDVPIGLLQILLEQEKQEKEK * QQATTNARILARV-NH2

wherein the K* at position 25 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -([2-(2-Amino-ethoxy)-ethoxy]- acetyl)-γE- CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:32).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is a combination of one γE residue and one ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) group and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 25). IVLSLDVPIGLLQILLEQEKQEKEKK*QATTNARILARV-NH2

wherein the K* at position 26 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:33).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 26).

IVLSLDVPIGLLQILLEQEKQEKEKK * QATTNARILARV-NH2

wherein the K* at position 26 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-γE- CO-(CH2)14-CH3 group; and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:34).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is two γE residues and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 26).

IVLSLDVPIGLLQILLEQEKQEKEKQK * ATTNARILARV-NH2

wherein the K* at position 27 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:35).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 27).

IVLSLDVPIGLLQILLEQEKQEKEKQQK * TTNARILARV-NH2

wherein the K* at position 28 has been chemically modified such that the epsilon- amino group of the K-side chain is bonded with a -γE-CO-(CH2)14-CH3 group;

and the C-terminal amino acid is amidated as a C-terminal primary amide (SEQ ID NO:36).

This sequence falls within the scope of Formula III (in that, in this particular embodiment, X1 is an unmodified I residue, X2 is L, X3 is L, X4 is R, X7 is T, X8 is R, X5 is one γE residue and X6 is a C16 mono fatty acid and the K* residue has replaced the original amino acid at position 28).

CLAIM:

1. A compound of the Formula:

wherein X1 denotes that the I residue is modified by either acetylation or methylation at e N-terminus;

wherein X2 is L or T;

wherein X3 is L or I;

wherein X4 is Q or E; and

wherein a modified K residue (“K*”) at position 29 is modified through conjugation to the silon-amino group of the K-side chain with a group of the formula–X5–X6, wherein

X5 is selected from the group consisting of:

one to four more amino acids;

one to four ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) moieties; and combinations of one to four amino acids and one to four ([2-(2-Amino-ethoxy)- ethoxy]-acetyl) moieties;

X6 is a C14-C24 fatty acid (SEQ ID NO:16);

or a pharmaceutically acceptable salt thereof.

2. The compound or salt of claim 1, wherein X5 is selected from the group consisting :

one to four E or γE amino acids;

one to four ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) moieties; and combinations of one to four E or γE amino acids and one to four ([2-(2-Amino- ethoxy)-ethoxy]-acetyl) moieties.

3. The compound or salt of claim 2, wherein X5 is a combination of one to four E or E amino acids and one to four ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) moieties.

4. The compound or salt of claim 3, wherein X5 is a combination of two to four γE mino acids and one to four ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) moieties.

5. The compound or salt of claim 3, wherein X5 is a combination of two γE amino ids and two ([2-(2-Amino-ethoxy)-ethoxy]-acetyl) moieties.

6. The compound or salt of any one of claims 1 to 5, wherein X6 is a straight chain tty acid of the formula CO-(CH2)x-CO2H, wherein x is 16, 18, or 20.

7. The compound or salt of any one of claims 1 to 6, wherein group of the formula –X5–X6 is ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)x-CO2H where x is 16 18.

8. The compound or salt according to any one of claims 1 to 7 wherein the C-terminal mino acid is amidated as a C-terminal primary amide.

9. The compound or salt according to any one of claims 1 to 8 wherein:

X1 denotes that the I residue is modified by acetylation at the N-terminus;

X2 is L;

X3 is L;

X4 is Q; and

the group of the formula–X5–X6 is ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-CH2)x-CO2H where x is 16 or 18.

10. The compound or salt according to claim 9 wherein x is 18.

11. The compound or salt according to claim 9 wherein x is 16.

12. The compound or salt according to any one of claims 1 to 8 wherein:

X1 denotes that the I residue is modified by methylation at the N-terminus;

X2 is L;

X3 is L;

X4 is Q; and

the group of the formula–X5–X6 is ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)18-CO2H.

13. The compound or salt according to any one of claims 1 to 8 wherein:

X1 denotes that the I residue is modified by methylation at the N-terminus;

X2 is L;

X3 is L;

X4 is Q; and

the group of the formula–X5–X6 is ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)16-CO2H.

14. The compound or salt according to any one of claims 1 to 8 wherein:

X1 denotes that the I residue is modified by methylation at the N-terminus;

X2 is T;

X3 is L;

X4 is E; and

the group of the formula–X5–X6 is ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO- CH2)18-CO2H.

15. The compound or salt according to any one of claims 1 to 8 wherein:

X1 denotes that the I residue is modified by methylation at the N-terminus;

X2 is L;

X3 is L;

X4 is E; and

the group of the formula–X5–X6 is ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO- CH2)18-CO2H.

16. The compound or salt according to any one of claims 1 to 8 wherein: X1 denotes that the I residue is modified by methylation at the N-terminus;

X2 is T;

X3 is I;

X4 is E; and

the group of the formula–X5–X6 is ([2-(2-Amino-ethoxy)-ethoxy]-acetyl)2-(γE)2-CO-(CH2)18-CO2H.

17. A compound comprising:

the amino acid sequence

IVX2SLDVPIGLLQILX3EQEKQEKEKQQATX7NAX4ILAX8V-NH2 (SEQ ID NO:67) wherein X2 is L or T;

wherein X3 is L or I;

wherein X4 is Q, R, or E;

wherein X7 is T or E; and

wherein X8 is Q, H or R,

wherein a modified K residue (“K*”) is substituted for the amino acid residue at position 10 or at any amino acid residue between position 14 and position 30 inclusive, wherein K* is modified by having the epsilon amino group of the K-side chain bound to a group of the formula—X5—X6, wherein

X5 is selected from the group consisting of:

between one to four amino acid residues;

between one to four ([2-(2-Amino-ethoxy)-ethoxy]-acetyl moieties; and combinations of one to four amino acid residues and one to four ([2-(2-Amino- hoxy)-ethoxy]-acetyl moieties;

X6 is a C14-C24 fatty acid;

or a pharmaceutically acceptable salt thereof.

18. A compound or salt according to claim 17, wherein X8 is either an H or Q residue.

19. A compound or salt according to claim 17, wherein X5 comprises 1 or 2 amino id residues and 1 or 2 ([2-(2-Amino-ethoxy)-ethoxy]-acetyl moieties.

20. A compound or salt according to claim 19, wherein X5 comprises two amino acid sidues and two ([2-(2-Amino-ethoxy)-ethoxy]-acetyl moieties, wherein the two amino acid sidues are either E or γE residues.

21. A compound or salt according to claim 17, wherein X1 is modified at the N-rminus by either acetylation or methylation.

22. A compound or salt according to claim 17, wherein X2 is an L residue.

23. A compound or salt according to claim 17, wherein X3 is an L residue.

24. A compound or salt according to claim 17, wherein X4 is a Q residue.

25. A compound or salt according to claim 17, wherein X7 is an T residue.

26. A compound or salt according to claim 17, wherein X5 comprises between one to ur amino acid residues such that there are no ([2-(2-Amino-ethoxy)-ethoxy]-acetyl moieties in e synthetic molecule.

27. A compound or salt according to claim 17 wherein the I residue at position 1 is odified at the N-terminus by one of acetylation or methylation

28. A pharmaceutical composition comprising a compound or salt according to any one claims 1 to 27 and one or more pharmaceutically acceptable carriers, diluents, and excipients.

29. A method for treating type II diabetes in a patient comprising administering to a tient in need of such treatment an effective amount of a compound or salt according to any one claims 1 to 28.

30. A method for treating chronic kidney disease in a patient comprising administering a patient in need of such treatment an effective amount of a compound or salt according to any ne of claims 1 to 28.

31. The method according to claim 30 wherein the chronic kidney disease is caused by abetic nephropathy.

32. The method according to claim 30 wherein the chronic kidney disease is caused by ypertensive nephropathy.

33. The methods according to any one of claims 30 to 32, wherein the administration the compound or salt to the patient in need of such treatment is subcutaneous.

34. A compound or salt according to any one of claims 1 to 28 for use in therapy.

35. A compound or salt according to any one of claims 1 to 28 for use in the treatment type II diabetes.

36. A compound or salt according to any one of claims 1 to 28 for use in the treatment chronic kidney disease.

37. A compound of the Formula:

X1 I V X2 S L D V P I G L L Q I L X3 E Q E K Q E K E K Q Q A K T N A X4 I L A Q V- NH2

wherein X1 denotes that the I residue is modified by either acetylation or methylation at e N-terminus;

wherein X2 is L or T;

wherein X3 is L or I;

wherein X4 is Q or E (SEQ ID NO:18).

38. A method for treating type II diabetes in a cat comprising administering to a cat in ed of such treatment an effective amount of a compound or salt according to any one of SEQ. D NOS.1, 2, 3, 5, 6, and 7.

39. A method for treating chronic kidney disease in a cat comprising administering to cat in need of such treatment an effective amount of a compound or salt according to any one of EQ. ID NOS.1, 2, 3, 5, 6, and 7.