Gipr-agonist compounds

Abstract

The present invention relates to compounds having activity against human glucose-dependent insulinotropic polypeptide (GIP) receptors. The invention also relates to compounds having a prolonged action time on the GIP receptor. Such compounds may be useful in the treatment of diabetes, including type 2 diabetes ("T2DM"). In addition, such compounds may be used to treat obesity.

Description

[0001] This application is a divisional application of PCT application PCT / US2020 / 043988, filed on July 29, 2020, with the invention name “GIPR-agonist compounds”. The date on which the PCT application entered the Chinese national phase is February 7, 2022, and the application number is 202080056086.3.

[0002] The present invention relates to compounds that are active at the human glucose-dependent insulinotropic polypeptide (GIP) receptor. The present invention also relates to compounds that have a prolonged duration of action at the GIP receptor. The compounds can be used to treat type 2 diabetes ("T2DM"). In addition, these compounds can be used to treat obesity.

[0003] Over the past few decades, the prevalence of diabetes has continued to rise. T2DM is the most common form of diabetes, accounting for approximately 90% of all diabetes. T2DM is characterized by high blood sugar levels primarily associated with insulin resistance. The current standard of care for T2DM includes diet and exercise, oral medication, and injection of hypoglycemic drugs, including incretin-based therapies, such as GLP-1 receptor agonists. There are currently a variety of GLP-1 receptor agonists available for the treatment of T2DM, but currently commercially available GLP-1 receptor agonists are typically dose-limited due to gastrointestinal side effects such as nausea and vomiting.

[0004] Subcutaneous injection is the typical route of administration for available GLP-1 receptor agonists. When treatment with oral medications and incretin-based therapies is not adequate, insulin therapy may be considered. Despite advances in today's available treatments, many T2DM patients are still unable to achieve their glycemic control goals. Uncontrolled diabetes can lead to several conditions associated with increased morbidity and mortality in patients.

[0005] Treatments that enable more patients with T2DM to achieve their glycemic treatment goals are needed.

[0006] Obesity is a complex medical disease that causes excessive accumulation of adipose tissue mass. Today, obesity is a global public health problem that is associated with adverse health outcomes and morbidity. The ideal treatment for obese patients should be to lose excess weight, improve obesity-related comorbidities, and maintain long-term weight loss. For patients with severe obesity, available obesity treatments are particularly unsatisfactory. Alternative treatment options are needed to induce therapeutic weight loss in patients in need of such treatment.

[0007] WO2016 / 111971 describes peptides believed to have GLP-1R and GIPR agonist activity. WO2013 / 164483 also discloses compounds believed to have GLP-1R and GIPR activity.

[0008] WO2018 / 181864 discloses compounds believed to have GIPR agonist activity.

[0009] There is a need for T2DM treatments that can provide effective glucose control for a large proportion of patients in need of such treatment. There is a further need for T2DM treatments that can provide effective glucose control and have a favorable side effect profile. Alternative treatment options are needed to provide therapeutic weight loss in patients in need of such treatment, such as patients with severe obesity. It is desirable to have diabetes treatment options that can be combined with insulin therapy and / or incretin therapy to provide patients with excellent glycemic results and / or a more desirable side effect profile.

[0010] Compounds with an extended duration of action at the GIP receptor are desirable to allow for less frequent dosing of the compound.

[0011] Therefore, Embodiment 1 is a compound of formula I

[0012] Z 1 X 1 X 2 BGTX 6 ISDYSIX 13 LDX 16 X 17 X 18 QX 20 X 21 X 22 VX 24 X 25 X 26 L

[0013] X 28 X 29 GPSSGAPPPSZ2 (SEQ ID NO: 4),

[0014] in:

[0015] Z1 is a modification of the N-terminal amino group, wherein the modification is selected from acetyl and absent;

[0016] X1 is selected from Y and D-Tyr;

[0017] X2 is selected from Aib, A and D-Ala;

[0018] X6 is selected from the group consisting of F, αMeF, Iva, L, αMeL and αMeF(2F);

[0019] X13 is selected from αMeL, A, L and Aib;

[0020] X16 is selected from K, E and Orn;

[0021] X17 is selected from I and K (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H;

[0022] X18 is selected from H, A and R;

[0023] X20 is selected from Aib and Q;

[0024] X21 is selected from D and E;

[0025] X22 is selected from F and αMeF;

[0026] X24 is selected from E, N, Q and D-Glu;

[0027] X25 is selected from the group consisting of Y, 4-Pal, W, and αMeY;

[0028] X26 is selected from L and K (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H;

[0029] X28 is selected from E and A;

[0030] X29 is selected from G, A, Q and T; q is selected from 16 and 18; and

[0031] Z2 is absent or is a modification of the C-terminal group, wherein the modification is an amidate;

[0032] wherein one and only one selected from X17 and X26 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO2H;

[0033] or a pharmaceutically acceptable salt thereof.

[0034] Embodiment 2 provides a compound of embodiment 1 or a pharmaceutically acceptable salt thereof, wherein Z1 is absent and X1 is Y.

[0035] Embodiment 3 provides a compound of Embodiment 1 or Embodiment 2, or a pharmaceutically acceptable salt thereof, wherein X2 is Aib.

[0036] Embodiment 4 provides a compound according to any one of Embodiments 1 to 3 or a pharmaceutically acceptable salt thereof, wherein:

[0037] X6 is selected from the group consisting of F and αMeF(2F).

[0038] Embodiment 5 provides a compound according to any one of Embodiments 1-4 or a pharmaceutically acceptable salt thereof, wherein:

[0039] X13 is selected from L and αMeL.

[0040] Embodiment 6 provides a compound according to any one of embodiments 1-5 or a pharmaceutically acceptable salt thereof, wherein X16 is K or Orn.

[0041] Embodiment 7 provides a compound of Embodiment 6 or a pharmaceutically acceptable salt thereof, wherein X16 is K.

[0042] Embodiment 8 provides a compound according to any one of embodiments 1-7 or a pharmaceutically acceptable salt thereof, wherein X18 is H.

[0043] Embodiment 9 provides a compound of any one of embodiments 1-8, or a pharmaceutically acceptable salt thereof, wherein X20 is Aib; and X22 is F.

[0044] Embodiment 10 provides a compound according to any one of embodiments 1-9 or a pharmaceutically acceptable salt thereof, wherein X21 is D.

[0045] Embodiment 11 provides a compound of any one of embodiments 1-10 or a pharmaceutically acceptable salt thereof, wherein X25 is 4-Pal or Y.

[0046] Embodiment 12 provides a compound of any one of Embodiments 1-11 or a pharmaceutically acceptable salt thereof, wherein:

[0047] X17 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H; and

[0048] X26 is L.

[0049] Embodiment 13 provides a compound according to any one of Embodiments 1-11 or a pharmaceutically acceptable salt thereof, wherein:

[0050] X17 is 1; and

[0051] X26 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO2 H.

[0052] Embodiment 14 provides a compound according to any one of embodiments 1-13, or a pharmaceutically acceptable salt thereof, wherein q is 16.

[0053] Embodiment 15 provides a compound according to any one of embodiments 1-13 or a pharmaceutically acceptable salt thereof, wherein q is 18.

[0054] Embodiment 16 provides a compound of Embodiment 1 or a pharmaceutically acceptable salt thereof, wherein:

[0055] Z1 does not exist;

[0056] X1 is Y;

[0057] X2 is selected from Aib and D-Ala; X6 is F;

[0058] X13 is selected from αMeL and L;

[0059] X16 is selected from K and Orn;

[0060] X18 is selected from H and A;

[0061] X20 is Aib;

[0062] X22 is F;

[0063] X24 is selected from E, N and D-Glu;

[0064] X25 is selected from the group consisting of Y, 4-Pal and W;

[0065] X28 is selected from E and A;

[0066] X29 is selected from G, A and Q; and q is selected from 16 and 18.

[0067] Embodiment 17 provides a compound of embodiment 16 or a pharmaceutically acceptable salt thereof, wherein:

[0068] Z1 does not exist;

[0069] X2 is Aib;

[0070] X13 is αMeL;

[0071] X18 is H;

[0072] X24 is selected from E and D-Glu;

[0073] X25 is selected from Y and 4-Pal;

[0074] X28 is E;

[0075] X29 is selected from G and A.

[0076] Embodiment 18 provides a compound of Embodiment 17 or a pharmaceutically acceptable salt thereof, wherein: X17 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H; and X26 is L.

[0077] Embodiment 19 provides a compound of Embodiment 17 or a pharmaceutically acceptable salt thereof, wherein: X17 is I; and X26 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H.

[0078] Embodiment 20 provides a compound of embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.

[0079] Embodiment 21 provides a compound of embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the compound is SEQ ID NO: 5. Embodiment 22 provides a compound of embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the compound is SEQ ID NO: 6. Embodiment 23 provides a compound of embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the compound is SEQ ID NO: 7. Embodiment 24 provides a compound of embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the compound is SEQ ID NO: 8. Embodiment 25 provides a compound of embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the compound is SEQ ID NO: 9. Embodiment 26 provides a compound of embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the compound is SEQ ID NO: 10. Embodiment 27 provides a compound of embodiment 1 or a pharmaceutically acceptable salt thereof, wherein the compound is SEQ ID NO: 11.

[0080] One embodiment provides a method for treating a condition selected from diabetes, obesity and metabolic syndrome, comprising administering an effective amount of a compound of any one of embodiments 1-27 or a pharmaceutically acceptable salt thereof to an individual in need thereof. One embodiment provides a method for treating a condition selected from T2DM, obesity and metabolic syndrome, comprising administering an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof to an individual in need thereof. One embodiment provides a method for providing therapeutic weight loss, comprising administering an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof to an individual in need thereof. One embodiment is a method for treating T2DM, comprising administering an effective amount of a compound of any one of embodiments 1-27 or a pharmaceutically acceptable salt thereof to an individual in need thereof.

[0081] One embodiment provides a compound of formula I for treatment or a pharmaceutically acceptable salt thereof. One embodiment provides a compound of any one of embodiments 1-27 for treatment or a pharmaceutically acceptable salt thereof, which is used to treat a condition selected from diabetes, obesity and metabolic syndrome. One embodiment provides a compound of formula I for treatment or a pharmaceutically acceptable salt thereof, which is used to treat a condition selected from T2DM, obesity and metabolic syndrome. In one embodiment, the condition is T2DM. In one embodiment, the condition is obesity. In one embodiment, the condition is type 1 diabetes (T1DM). In one embodiment, the condition is diabetes in a patient receiving insulin therapy. In one embodiment, the condition is metabolic syndrome.

[0082] The compound of formula I or its pharmaceutically acceptable salt can be used to treat a variety of symptoms or conditions. For example, certain embodiments provide a method for treating T2DM in a patient, comprising administering an effective amount of a compound of formula I or its pharmaceutically acceptable salt to an individual with such treatment needs. In one embodiment, a method for treating obesity in a patient, comprising administering an effective amount of a compound of formula I or its pharmaceutically acceptable salt to an individual with such treatment needs. In one embodiment, the method induces non-therapeutic weight loss to an individual, comprising administering an effective amount of a compound of formula I or its pharmaceutically acceptable salt to an individual with such treatment needs.

[0083] In certain embodiments, the present invention provides a method for treating metabolic syndrome in a patient, comprising administering an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof to an individual with such treatment needs. In one embodiment, the method is a method for treating diabetes in a patient receiving insulin therapy, comprising administering an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof to an individual with such treatment needs. Also provided herein is a compound of the present invention, which is used to treat a condition simultaneously, separately and sequentially in combination with one or more active agents, wherein the one or more active agents are selected from metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, sodium-glucose cotransporter-2 (SGLT-2) inhibitors, growth differentiation factor 15 regulators ("GDF15"), peptide tyrosine tyrosine regulators ("PYY"), modified insulin, amylin, dual amylin-calcitonin receptor agonists, and oxyntomodulin agonists ("OXM"), and the condition is selected from T2DM, obesity and metabolic syndrome. Also provided herein are compounds of the present invention for use in combination with one or more active agents simultaneously, separately and sequentially for treating a condition selected from metformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor, a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, a growth differentiation factor 15 regulator ("GDF15"), a peptide tyrosine tyrosine regulator ("PYY"), a modified insulin, an amylin receptor agonist, a dual amylin-calcitonin receptor agonist, a modified urocortin-2 (UCN-2) analog, a glucagon-like peptide-1 (GLP-1) receptor agonist, a glucagon receptor agonist, and a dual GLP-1-glucagon receptor agonist, including oxyntomodulin and its analogs, wherein the condition is selected from T2DM, obesity and metabolic syndrome. In one embodiment, a compound of the invention is provided as a fixed dose combination with one or more active agents selected from metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, SGLT-2 inhibitors, GDF15, PYY, modified insulin, amylin, dual amylin-calcitonin receptor agonists and OXM. In one embodiment, a compound of the invention is provided as a fixed dose combination with one or more active agents selected from metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, SGLT-2 inhibitors, GDF15, PYY analogs, modified insulin, amylin receptor agonists, dual amylin-calcitonin receptor agonists, modified urocortin-2 (UCN-2) analogs, glucagon-like peptide-1 (GLP-1) receptor agonists, glucagon receptor agonists and dual GLP-1-glucagon receptor agonists, including OXM and analogs thereof.In one embodiment, the compounds of the invention are provided for use in combination simultaneously, separately and sequentially with one or more active agents selected from metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, SGLT-2 inhibitors, GDF15, PYY, modified insulin, amylin, dual amylin-calcitonin receptor agonists and OXM for the treatment of a condition selected from T2DM and obesity. In one embodiment, the compound of the present invention is used for simultaneous, separate and sequential combination with one or more active agents selected from metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors, SGLT-2 inhibitors, GDF15, PYY analogs, modified insulin, amylin receptor agonists, dual amylin-calcitonin receptor agonists, modified urocortin-2 (UCN-2) analogs, glucagon-like peptide-1 (GLP-1) receptor agonists, glucagon receptor agonists and dual GLP-1 glucagon receptor agonists, including OXM and analogs. In one embodiment, the compound of the present invention is used for simultaneous, separate and sequential combination with one or more active agents selected from metformin, thiazolidinediones, sulfonylureas, dipeptidyl peptidase 4 inhibitors and SGLT-2 inhibitors for the treatment of a condition selected from T2DM and obesity.

[0084] In one embodiment, a method for treating diabetes in a patient receiving insulin therapy, comprising administering an effective amount of a compound of Formula I or a pharmaceutically acceptable salt thereof to a patient in need thereof. One embodiment is a method for treating a patient who is administered insulin therapy for T1DM. One embodiment is a method for treating a patient who is administered insulin therapy for T2DM. One embodiment is administration once a week. One embodiment is subcutaneous treatment of a patient who is administered insulin therapy once a week. There is such an embodiment, wherein the insulin therapy comprises basal insulin therapy. There is such an embodiment, wherein the insulin therapy comprises mealtime insulin therapy. There is such an embodiment, wherein the insulin therapy comprises ultrafast-acting insulin therapy. Emergency infusion of insulin therapy administered with a compound of Formula I or a pharmaceutically acceptable salt thereof can promote glucagon fluctuations in patients who are receiving hypoglycemic clamps, thereby enhancing the body's natural defenses against hypoglycemia. A compound of Formula I or a pharmaceutically acceptable salt thereof may be administered once a week independently of the type of insulin used or the dose of insulin used. One embodiment is a compound of any one of Embodiments 1-27 or a pharmaceutically acceptable salt thereof, which is administered to a patient receiving insulin therapy in an effective amount, and is independent of the type of insulin used or the dose of insulin used. One embodiment is a compound of any one of embodiments 1-27 or a pharmaceutically acceptable salt thereof, which is administered to a patient receiving insulin therapy once a week in an effective amount, and is independent of the type of insulin used or the insulin dosage used. As used herein, "insulin therapy" refers to the use of approved insulin therapy to treat diabetic patients. Such insulin therapy is known to those skilled in the art and / or clinical health professionals. For example, insulin therapy may include the treatment of basal insulin. Such "insulin therapy" of basal insulin may be used in a dosing regimen with mealtime insulin and / or superfast-acting insulin. As used herein, "mealtime insulin" refers to insulin and / or modified insulin administered with meals, such as, but not limited to, insulin lispro. As used herein, "basal insulin" refers to modified insulin with a longer duration of action, such as, but not limited to, insulin glargine.

[0085] Another embodiment provides the use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a condition selected from T2DM, obesity and metabolic syndrome. One embodiment provides the use of a compound of any one of embodiments 1-27 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a condition selected from diabetes, obesity and metabolic syndrome. In one embodiment, the medicament is used to treat T2DM. In one embodiment, the medicament is used to treat obesity. In one embodiment, the medicament is used to treat diabetes in a patient receiving insulin therapy.

[0086] Another embodiment provides a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof and at least one selected from a carrier, a diluent, and an excipient. In one embodiment, a pharmaceutical composition for subcutaneous administration is provided.

[0087] As used herein, the term "treating," ...

[0088] Certain compounds of the present invention are generally effective within a wide dosage range. For example, the dosage for parenteral administration once a week falls within the range of 0.05 mg to about 60 mg / person / week.

[0089] The compound of formula I or a pharmaceutically acceptable salt thereof includes a novel amino acid sequence having affinity for the GIP receptor and has a desired effect on the receptor. GIP is a 42 amino acid peptide (SEQ ID NO: 1) that, like GLP-1, is known as an incretin that plays a physiological role in glucose homeostasis by stimulating pancreatic β cells to secrete insulin in the presence of glucose.

[0090] GLP-1 is a 36-amino acid peptide, the major biologically active fragment of which is a 30-amino acid C-terminal amidated peptide (GLP-1 7-36 )(SEQ ID NO:2).

[0091] Glucagon is a 29 amino acid peptide hormone (SEQ ID NO: 3) secreted by the α-cells of the islets of Langerhans in the pancreas that is involved in glucose homeostasis.

[0092] The compounds of the present invention provide the desired efficacy with high selectivity for the GIP receptor relative to the GLP-1R and glucagon receptors. In one embodiment, the compounds have the desired GIP receptor activity with a prolonged duration of action.

[0093] As used herein, the term "amino acid" refers to naturally occurring amino acids and non-natural amino acids. Amino acids are typically described using the standard single letter code (e.g., L = leucine) as well as α-methyl substituted natural amino acid residues (e.g., α-methylleucine or αMeL, and α-methylphenylalanine or αMeF) and certain other non-natural amino acids, such as α-aminoisobutyric acid or "Aib", "4Pal", "Orn", etc. The structures of these amino acids are shown below:

[0094]

[0095] As used herein, "Orn" refers to L-ornithine. As used herein, "4Pal" refers to 3-(4-pyridyl)-L-alanine. As used herein, "αMeF(2F)" refers to α-methyl-2-fluoro-L-phenylalanine. As used herein, "αMeY" and "αMeL" refer to α-methyl-L-tyrosine and α-methyl-L-leucine, respectively. As used herein, "e" and "D-Glu" refer to D-glutamic acid. As used herein, "D-Tyr" and "y" each refer to D-tyrosine. As used herein, "D-Ala" and "a" each refer to D-alanine. As used herein, "αMeF" refers to α-methyl-F and α-methyl-Phe. As used herein, "Iva" refers to L-isovaline.

[0096] In one embodiment, the conjugation is acylation. In one embodiment, the conjugation is to the epsilon-amino group of the K side chain. In one embodiment of the compounds of the invention, the fatty acid moiety is conjugated to K at position 17 via a linker. In one embodiment of the compounds of the invention, the fatty acid moiety is conjugated to K at position 26 via a linker.

[0097] In one embodiment, q is selected from 16 and 18. In one embodiment, q is 16. In one embodiment, q is 18.

[0098] When used herein with respect to the GIP receptor, the terms "activity," "activation," "activation," and the like refer to the ability of a compound, or a pharmaceutically acceptable salt thereof, to bind to the receptor and induce a response thereof as measured using assays known in the art, such as the in vitro assays described below.

[0099] The affinity of the compounds of Formula I or their pharmaceutically acceptable salts for the GIP receptor can be measured using techniques known in the art for measuring receptor binding levels, including, for example, those described in the Examples below, and is generally expressed as a Ki value. The activity of the compounds of the invention for the GIP receptor can also be determined using techniques well known in the art, including, for example, the following in vitro activity assays, and is generally expressed as an EC 50 The value is the concentration of compound that causes half-maximal stimulation in a dose-response curve.

[0100] In addition, data on the activity and affinity of each compound for the GLP-1 and glucagon receptors are provided to demonstrate the degree of selectivity of the compounds of the invention for the GIPR.

[0101] In one embodiment, the pharmaceutical composition of the compound of Formula I is suitable for administration by a parenteral route (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular or transdermal). Some pharmaceutical compositions and methods for their preparation are well known in the art. (See, for example, Remington: The Science and Practice of Pharmacy (DB Troy, ed., 21st ed., Lippincott, Williams & Wilkins, 2006)).

[0102] The compounds of the present invention can react with any of a variety of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and common methods for preparing them are well known in the art. (See, for example, P. Stahl et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd revised edition (Wiley-VCH, 2011)). Pharmaceutically acceptable salts of the present invention include, but are not limited to, sodium salts, trifluoroacetates, hydrochlorides, ammonium salts and acetates. In one embodiment, pharmaceutically acceptable salts are selected from sodium salts, hydrochlorides and acetates.

[0103] The present invention also encompasses novel intermediates and methods for synthesizing the compounds of the present invention or their pharmaceutically acceptable salts. The intermediates and compounds of the present invention can be prepared by various methods known in the art. In particular, the following examples describe methods using chemical synthesis. The specific synthesis steps for each of the described routes can be combined in different ways to prepare the compounds of the present invention. Reagents and raw materials are readily available to those skilled in the art.

[0104] As used herein, the term "effective amount" refers to the amount or dosage of a compound of the invention or a pharmaceutically acceptable salt thereof, which, when administered to a patient in a single or multiple administrations, provides the desired effect in the patient being diagnosed or treated. The effective amount can be determined by one skilled in the art using known techniques and by observing the results obtained under similar circumstances. In determining the effective amount for an individual, many factors are considered, including, but not limited to: the species of mammal; its size, age, and general health; the specific disease or condition involved; the degree or severity involved in the disease or disorder; the response of the individual patient; the specific compound being administered; the mode of administration; the bioavailability characteristics of the administered formulation; the dosage regimen selected; the use of concomitant medications; and other relevant circumstances.

[0105] As used herein, the term "subject in need thereof" refers to a mammal, preferably a human, having a disease or condition in need of treatment or therapy, including, for example, those listed in the above paragraphs.

[0106] As used herein, "EDTA" refers to ethylenediaminetetraacetic acid. As used herein, "DMSO" refers to dimethyl sulfoxide. As used herein, "CPM" refers to counts per minute. As used herein, "IBMX" refers to 3-isobutyl-1-methylxanthine. As used herein, "LC / MS" refers to liquid chromatography / mass spectrometry. As used herein, "HTRF" refers to homogeneous time-resolved fluorescence. As used herein, "DMF" refers to N,N-dimethylformamide. As used herein, "DCM" refers to dichloromethane. As used herein, "TFA" refers to trifluoroacetic acid. As used herein, "TFA salt" refers to trifluoroacetate. As used herein, "RP-HPLC" refers to reverse phase high performance liquid chromatography.

[0107] The present invention is further illustrated by the following examples, which should not be construed as limiting.

[0108] Peptide synthesis

[0109] Example 1

[0110] Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) 18 -CO 2 H)AQ-Aib-DFVNWLLAQGPSSGAPPPS-NH 2 (SEQ ID NO:5)

[0111] The structure of SEQ ID NO:5 is described below using the standard single letter amino acid code, with the exception of residues D-Ala2, K17, Aib20 and Ser39, where the structures of these amino acid residues have been expanded:

[0112]

[0113] The peptide backbone of Example 1 was synthesized using a Symphony multiplex peptide synthesizer (Gyros Protein Technologies. Tucson, AZ; 3.3.0.1) software version 3.3.0, using fluorenylmethoxycarbonyl (Fmoc) / tert-butyl (t-Bu) chemistry.

[0114] The resin consisted of aminomethyl polystyrene functionalized with Rink Amide linker (Polystyrene AM RAM, RAPP Polymers GmbH, H40023, 200-400 mesh) with a degree of substitution of 0.8 mmol / g. Standard side chain protecting groups were used, except for the following: Fmoc-Lys(Mtt)-OH for lysine at position 17, where Mtt is 4-methyltrityl, and Boc-Tyr(t-Bu)-OH for tyrosine at position 1. The Fmoc group was removed using 20% ​​piperidine in DMF (2 x 10 min) before each coupling step. All standard amino acid couplings were performed using equimolar ratios of Fmoc amino acid (0.3 M in DMF), diisopropylcarbodiimide (0.9 M in DCM), and Oxyma (0.9 M in DMF) at a molar excess of 9 times over the theoretical peptide loading. The couplings were allowed to proceed for 1.5 hours, except for the following: valine coupling, 3 hours; Cα-methylated amino acid coupling, 6 hours; coupling to Cα-methylated amino acids, 6-10 hours. After the peptide backbone synthesis was complete, the resin was washed extensively with DCM to remove residual DMF. The Mtt protecting group on the lysine at position 17 was selectively removed from the peptide resin using 3 treatments (3 x 20 minute treatments) of 30% hexafluoroisopropanol (Oakwood Chemicals) in DCM, and the resin was subsequently washed extensively with DCM and DMF.

[0115] The fatty acid-linker moiety was then coupled by coupling 2-[2-(2-Fmoc-amino-ethoxy)-ethoxy]-acetic acid (Fmoc-AEEA-OH, ChemPep, Inc.) and Fmoc-glutamic acid α-tert-butyl ester (Fmoc-Glu-OtBu, Ark Pharm, Inc.) following the procedure described above for step coupling and deprotection reactions. After removal of the final Fmoc protecting group, mono-OtBu-eicosandioic acid (WuXi AppTec, Shanghai, China) was coupled using a 4-fold excess of the diacid, PyBOP, and diisopropylethylamine (1:1:1 mol / mol / mol) in 1:1 DCM / DMF for 1 hour.

[0116] After the synthesis is complete, the peptide-resin is washed with DCM and then fully vacuum dried. The dried peptide-resin is treated with a cleavage mixture (10 mL TFA, 0.5 mL triisopropylsilane, 0.5 mL water and 0.5 mL 1,2-ethanedithiol) at room temperature for 2 hours. The peptide resin solution is filtered into a 50-mL conical centrifuge tube and treated with 5 times excess volume of cold ether (-20 ° C) to precipitate the crude peptide. The peptide / ether suspension is centrifuged at 3000 rcf for 1.5 min to form a solid precipitate and the supernatant is decanted. Wash with cold ether 2 times, centrifuge for 1 min each time, and then vacuum dry. The crude peptide is dissolved in 20% acetic acid / 80% water and purified by RP-HPLC with SymmetryPrep 7 μm C18 preparation column (18x 300 mm, Waters), wherein the linear gradient is 100% acetonitrile and 0.1% TFA / water buffer system (25-45% acetonitrile, 65 min). The purity of the peptide was assessed using analytical RP-HPLC with a pooling standard of >95%. The main pool purity of Example 1 compound was determined to be 96.8%. The final main product pool was then lyophilized to obtain the lyophilized peptide TFA salt. The molecular weight was determined by LC / MS (measured value: [M+3H] 3+ =1638.4; calculated value [M+3H] 3+ =1638.53; measured value MW(avg)=4912.2; calculated value MW(avg):4912.58).

[0117] Example 2

[0118] Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) 16 -CO 2 H)AQ-Aib-DFVNWLLAQGPSSGAPPPS-NH 2 (SEQ ID NO:6)

[0119] The structure of SEQ ID NO:6 is described below using the standard single letter amino acid code, with the exception of residues D-Ala2, K17, Aib20 and Ser39, where the structures of these amino acid residues have been expanded:

[0120]

[0121] The compound of SEQ ID NO: 6 was prepared substantially as in Example 1, wherein the substituted protected diacid was mono-OtBu-octadecane dioic acid (WuXi AppTec, Shanghai, China). The molecular weight was determined by LC / MS (measured value:

[0122] [M+3H] 3+ =1629.15; calculated value [M+3H] 3+ =1629.18; measured value MW(avg)=4884.45; calculated value MW(avg)=4884.53).

[0123] Example 3

[0124] Y-Aib-EGTFISDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) 18 -CO 2 H)HQ-Aib-DFVE-4-Pal-LLEAGPSSGAPPPS-NH 2 (SEQ ID NO:7)

[0125] The structure of SEQ ID NO:7 is described below using the standard single letter amino acid code, with the exception of residues Aib2, αMeL13, K17, Aib20, 4-Pal25 and Ser39, where the structures of these amino acid residues have been expanded:

[0126]

[0127] The compound of SEQ ID NO: 7 was prepared essentially as described in Example 1. The molecular weight was determined by LC / MS (measured value: [M+3H] 3+ =1662.52; calculated value [M+3H] 3+ =1662.55; measured value MW(avg)=4984.55; calculated value MW(avg)=4984.64).

[0128] Example 4

[0129] Y-Aib-EGTFISDYSI-αMeL-LD-Orn-K((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) 18 -CO 2H)HQ-Aib-DFVE-4-Pal-LLEAGPSSGAPPPS-NH 2 (SEQ ID NO:8)

[0130] The structure of SEQ ID NO:8 is described below using the standard single letter amino acid code, with the exception of residues Aib2, αMeL13, Orn16, K17, Aib20, 4-Pal25 and Ser39, where the structures of these amino acid residues have been expanded:

[0131]

[0132] The compound of SEQ ID NO: 8 was prepared essentially as described in Example 1. The molecular weight was determined by LC / MS (measured value: [M+3H] 3+ =1657.82; calculated value [M+3H] 3+ =1657.87; measured value MW(avg)=4970.46; calculated value MW(avg)=4970.62).

[0133] Example 5

[0134] Y-Aib-EGTFISDYSI-αMeL-LDKIHQ-Aib-DFVEYK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) 18 -CO 2 H)LEGGPSSGAPPPS-NH 2 (SEQ ID NO:9)

[0135] The structure of SEQ ID NO:9 is described below using the standard single letter amino acid code, with the exception of residues Aib2, αMeL13, Aib20, K26 and Ser39, where the structures of these amino acid residues have been expanded:

[0136]

[0137] The compound of SEQ ID NO: 9 was prepared essentially as described in Example 1, wherein Fmoc-Lys(Mtt)-OH was used for the lysine at position 26 instead of position 17. The molecular weight was determined by LC / MS (measured value: [M+3H] 3+ =1662.8; calculated value [M+3H] 3+ =1662.88; measured value MW(avg)=4985.4; calculated value MW(avg)=4985.63).

[0138] Example 6

[0139] Y-Aib-EGTFISDYSI-αMeL-LD-Orn-IHQ-Aib-DFVEYK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) 18 -CO 2 H)LEGGPSSGAPPPS-NH 2 (SEQ ID NO: 10)

[0140] The structure of SEQ ID NO:10 is described below using the standard single letter amino acid code, with the exception of residues Aib2, αMeL13, Orn16, Aib20, K26 and Ser39, where the structures of these amino acid residues have been expanded:

[0141]

[0142] The compound of SEQ ID NO: 10 was prepared essentially as described in Example 1, wherein Fmoc-Lys(Mtt)-OH was used for the lysine at position 26 instead of position 17. The molecular weight was determined by LC / MS (measured value: [M+3H] 3+ =1658.1; calculated value [M+3H] 3+ =1658.20; measured value MW(avg)=4971.3; calculated value MW(avg)=4971.6).

[0143] Example 7

[0144] Y-Aib-EGTFISDYSI-αMeL-LD-Orn-IHQ-Aib-EFV-(D-Glu)-YK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) 16 -CO 2 H)LEGGPSSGAPPPS-NH 2 (SEQ ID NO:11)

[0145] The structure of SEQ ID NO: 11 is described below using the standard single letter amino acid code, with the exception of residues Aib2, αMeL13, Orn16, Aib20, D-Glu24, K26 and Ser39, where the structures of these amino acid residues have been expanded:

[0146]

[0147] The compound of SEQ ID NO: 10 was prepared essentially as described in Example 1, wherein Fmoc-Lys(Mtt)-OH was used for the lysine at position 26 instead of position 17, and the protected diacid was mono-OtBu-octadecane dioic acid (WuXi AppTec, Shanghai, China). The molecular weight was determined by LC / MS (measured value: [M+3H] 3+ =1653.4; calculated value [M+3H] 3+ =1653.52; measured value MW(avg)=4957.2; calculated value MW(avg)=4957.57).

[0148] The compounds of Example 8 (SEQ ID NO: 12) through Example 122 (SEQ ID NO: 126) were prepared essentially as described in Example 1.

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161] Binding test

[0162] Glucagon (referred to as Gcg or hGcg) is a reference substance produced by Eli Lilly and Company in the United States. GLP-1(7-36)-NH 2(referred to as GLP-1 or hGLP-1) was obtained from CPC Scientific (Sunnyvale, CA, 97.2% purity, 100 μM aliquots in 100% DMSO). GIP(1-42)-NH 2 (referred to as GIP) (purity>80%, 100 μM aliquots in 100% DMSO). 125 I]-lactoperoxidase, prepared [ 125 I]-radiolabeled Gcg, GLP-1 or GIP.

[0163] Stably transfected cell lines were prepared by subcloning the receptor cDNA into the pcDNA3 expression plasmid and transfecting into human embryonic kidney (HEK) 293 (hGcgR and hGLP-1R) or Chinese hamster ovary (CHO) (hGIPR) cells, followed by selection with Geneticin (hGLP-1R and hGIPR) or Hygromycin B (hGcgR).

[0164] Two methods were used to prepare crude cell membranes.

[0165] Method 1 The frozen cell pellet was incubated on ice in 50 mM Tris HCl, pH 7.5, and Roche Complete TM Lyse in hypotonic buffer containing protease inhibitors. The cell suspension was broken up by hitting 25 times with a glass Potter-Elvehjem homogenizer with a pestle. The homogenate was centrifuged at 1100 x g for 10 minutes at 4°C. The supernatant was collected and stored on ice, while the pellet was resuspended in homogenization buffer and homogenized again as described above. The homogenate was centrifuged at 1100 x g for 10 minutes. The second supernatant was combined with the first supernatant and centrifuged at 35000 x g for 1 hour at 4°C. The resulting membrane pellet was resuspended in homogenization buffer containing about 1 to 3 mg / mL protease inhibitors, snap frozen in liquid nitrogen and stored as aliquots in a -80°C freezer until use.

[0166] Method 2 The frozen cell pellet was incubated on ice in 50 mM Tris HCl, pH 7.5, 1 mM MgCl 2 、Roche Complete TM The cells were lysed in hypotonic buffer containing EDTA-free protease inhibitors and 25 units / mL DNase I (Invitrogen). The glass Potter-Elvehjem homogenizer of pestle hits 20 to 25 times to break up the cell suspension. The homogenate is centrifuged at 1800x g for 15 minutes at 4°C. The supernatant is collected and stored on ice, and the precipitate is resuspended in homogenization buffer (without DNase I) and homogenized again as described above. The homogenate is centrifuged at 1800x g for 15 minutes. The second supernatant is merged with the first supernatant and centrifuged at 1800x g for an additional 15 minutes. The entire supernatant is then centrifuged at 25000x g for 30 minutes at 4°C. The resulting membrane precipitate is resuspended in a homogenization buffer (without DNase I) containing approximately 1 to 3mg / mL protease inhibitors, and stored in a -80°C refrigerator as aliquots until use.

[0167] Binding assay

[0168] because[ 125 I] The parent material was high in propanol, therefore, the equilibrium binding dissociation constants (K) for various receptor / radioligand interactions were determined from homogeneous competition binding assays. d ) rather than from saturation binding. K determined for receptor preparations d Values ​​are as follows: hGcgR (3.9 nM), hGLP-1R (1.2 nM), and hGIPR (0.14 nM).

[0169] [ 125 I]-glucagon binding

[0170] Human Gcg receptor binding assays were performed using wheat germ agglutinin (WGA) beads (Perkin Elmer) in scintillation counting proximity assay (SPA) mode. The binding buffer contained 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 0.1% (w / v) bacitracin (Research Products), 0.003% (w / v) polyoxyethylene sorbitan monolaurate and Roche Complete EDTA-free TM Protease inhibitors. Peptides and Gcg were thawed and serially diluted 3-fold in 100% DMSO (10-point concentration response curve). Subsequently, 5 μL of the serially diluted compound or DMSO was transferred into 3632 clear bottom assay plates containing 45 μL of assay binding buffer or unlabeled Gcg control (non-specific binding or NSB, final concentration 1 μM). Subsequently, 50 μL of [ 125I]-Gcg (final concentration 0.15 nM), 50 μL human GcgR membrane (1.5 μg / well) and 50 μL WGA SPA beads (80 to 150 μg / well). The plate was sealed and mixed on a plate shaker (setting 6) for 1 minute and then incubated at room temperature for 12 hours after incubation / sedimentation time using a PerkinElmer Trilux Scintillation counter readings. Final assay concentrations typically ranged from 1150 nM to 0.058 nM for the peptides tested in the response curves and from 1000 nM to 0.05 nM for the control Gcg.

[0171] [ 125 I]-GLP-1 binding

[0172] Human GLP-1 receptor binding assay was performed with WGA beads using SPA mode. Binding buffer contained 25 mM HEPES, pH 7.4, 2.5 mM CaCl 2 , 1 mM MgCl 2 , 0.1% (w / v) bacitracin, 0.003% (w / v) -20 and Roche Complete EDTA-free TM Protease inhibitors. Peptides and GLP-1 were thawed and serially diluted 3-fold in 100% DMSO (10 point concentration response curve). Subsequently, 5 μL of the serially diluted compound or DMSO was transferred into 3632 clear bottom assay plates containing 45 μL of assay binding buffer or unlabeled GLP-1 control (non-specific binding or NSB, final concentration 0.25 μM). Subsequently, 50 μL of [ 125 I]-GLP-1 (final concentration 0.15 nM), 50 μL human GLP-1R membrane (0.5 μg / well) and 50 μL WGA SPA beads (100 to 150 μg / well). The plate was sealed and mixed on a plate shaker (setting 6) for 1 minute and after 5 to 12 hours incubation / sedimentation time at room temperature with a PerkinElmer Trilux Scintillation counter readings. The final assay concentration range for the peptides tested in the response curves was generally 1150 nM to 0.058 nM and for the control GLP-1 was 250 nM to 0.013 nM.

[0173] [ 125 I]-GIP binding

[0174] Human GIP receptor binding assay was performed with WGA beads using SPA mode. The binding buffer contained 25 mM HEPES, pH 7.4, 2.5 mM CaCl 2 , 1 mM MgCl 2, 0.1% (w / v) bacitracin, 0.003% (w / v) -20 and Roche Complete EDTA-free TM Protease inhibitors. Peptides and GIP were thawed and serially diluted 3-fold in 100% DMSO (10 point concentration response curve). Subsequently, 5 μL of the serially diluted compound or DMSO was transferred into 3632 clear bottom assay plates containing 45 μL of assay binding buffer or unlabeled GIP control (non-specific binding or NSB, final concentration 0.25 μM). Subsequently, 50 μL of [ 125 I]-GIP (final concentration 0.075-0.15 nM), 50 μL human GIPR membrane (3 μg / well) and 50 μL WGA SPA beads (100 to 150 μg / well). The plate was sealed and mixed on a plate shaker (setting 6) for 1 minute and after 2.5 to 12 hours incubation / sedimentation time at room temperature with a PerkinElmer Trilux Scintillation counter readings. The final assay concentration range for the peptides tested in the response curves was typically 1150 to 0.058 nM or 115 nM to 0.0058 nM, while the control GIP was 250 nM to 0.013 nM.

[0175] Binding assay data analysis

[0176] The raw CPM data of the concentration curves of peptide, Gcg, GLP-1 or GIP were converted to percent inhibition by subtracting nonspecific binding (binding in the presence of excess unlabeled Gcg, GLP-1 or GIP, respectively) from each CPM value and dividing by the total binding signal also corrected for subtracting nonspecific binding. Four parameters (curve maximum, curve minimum, IC 50 The data were analyzed using the nonlinear regression routine (Genedata Screener, version 12.0.4, Genedata AG, Basel, Switzerland). i =IC 50 / (1+D / K d ), from IC 50 Absolute value calculation of affinity constant (K i ), where D = the concentration of radioligand used in the experiment, IC 50 is the concentration causing 50% inhibition of binding, K d is the equilibrium binding dissociation constant of the radioligand (described above). K iValues ​​are reported as geometric means, with errors expressed as standard errors of the mean (SEM), and n equal to the number of independent replicates (measured in experiments performed on different days). The geometric mean is calculated as follows:

[0177] Geometric mean = 10 (Log Ki值的算术平均值)

[0178] n = y / x means that only a subset of replicates (y) out of the total number of replicates (x) was used to represent the mean. SEM was calculated only when y = 2 or more. Means are expressed as geometric means and standard error of the mean (SEM) with the number of replicates (n) shown in parentheses.

[0179] Table 1. In vitro binding affinities (Ki) of the indicated example and comparative molecules to human GcgR, GIPR and GLP-1R in the presence of 0.1% bacitracin.

[0180]

[0181]

[0182]

[0183]

[0184] As shown in Table 1, the present examples are very potent binders to human GIPR, and have lower affinity for GLP-1R and GcgR.

[0185] cAMP pharmacological functional assay in the presence of 0.1% casein

[0186] A panel of cAMP assays was performed in HEK293 cells expressing human GLP-1 receptor (GLP-1R), glucose-dependent insulinotropic peptide (GIPR), or glucagon receptor (GcgR). Each cell line overexpressing the receptor (20 μl) was treated with the test peptide in DMEM (Gibco catalog number 31053) supplemented with 0.1% casein (Sigma catalog number C4765), 250 μM IBMX, 1X GlutaMAXTM (Gibco catalog number 35050), and 20 mM HEPES (HyClone catalog number SH30237.01) in a 20 μl assay volume. After 60 minutes of incubation at room temperature, the resulting intracellular cAMP increase was quantitatively determined using the CisBio cAMP Dynamic 2HTRF assay kit (62AM4PEJ). Lysis buffer containing cAMP-d2 conjugate (20 μl) and antibody anti-cAMP-Eu3+-cryptate (20 μl) was then added to determine cAMP levels. After incubation at room temperature for 60 minutes, the HTRF signal was detected using an Envision 2104 plate reader (PerkinElmer). Fluorescence emission at 620 nm and 665 nm was measured and the ratio between 620 nm and 665 nm was calculated, which was then converted to nM cAMP per well using a cAMP standard curve. The dose-response curves of the compounds were plotted as percentage stimulation normalized to the minimum (buffer only) and maximum (maximum concentration of each control ligand) and analyzed using a four-parameter nonlinear regression fit with a variable slope (Genedata Screener13). EC50 is the concentration of compound that causes half the maximum stimulation in the dose-response curve. Relative EC was derived by nonlinear regression analysis using the maximum response percentage relative to the concentration of the added peptide, based on a four-parameter logistic equation fit. 50 value.

[0187] Using homogeneous time-resolved fluorescence, experiments were performed to determine the intrinsic potency of the example and control molecules in the presence of casein (instead of serum albumin) as a non-specific blocking agent that does not interact with the fatty acid moieties of the molecules being analyzed.

[0188] Intracellular cAMP levels were determined by extrapolation using the standard curve. Compound dose response curves were plotted as percent stimulation normalized to minimum (buffer only) and maximum (maximum concentration of each control ligand) and analyzed using a four-parameter nonlinear regression fit with variable slope (Genedata Screener 13). EC 50 is the concentration of compound that causes half-maximal stimulation in the dose-response curve. Each relative EC50 value used for geometric mean calculations was determined based on curve fitting.

[0189] Compound concentration response curves were plotted as percent stimulation normalized to minimum (buffer only) and maximum (maximum concentration of each control ligand) values ​​and analyzed using a four-parameter nonlinear regression fit with variable slope (GenedataScreener 13). 50 EC is the concentration of compound that causes half-maximal stimulation in a dose-response curve. EC is calculated as 50 Summary Statistics: Geometric Mean:

[0190] GM=10(log 10 Converted EC 50 arithmetic mean of the values).

[0191] The standard error of the mean is reported as follows:

[0192] SEM = geometric mean x (log 10 Converted EC 50 standard deviation of the values / square root of the number of runs) x log e 10.

[0193] Logarithmic transformation leads to EC 50 Values ​​decrease on a multiple scale rather than an arithmetic scale.

[0194] Each time the test was performed, the peptides to be tested and the natural ligands GIP, GLP-1 and glucagon were run, with buffer only as a baseline (minimum value), and the maximum concentration of each of the GIP, GLP-1 and glucagon standards was used as the maximum value for the calculation. For illustration, as shown in Example 1, the peptides were tested in 8 runs of the hGIPR cAMP assay. For the avoidance of doubt, the hGIP amide, hGLP-1 amide and glucagon EC50 examples in Table 2 are from the geometric mean of a series of 18 assay values, and these values ​​vary from day to day compared to zero buffer. Therefore, each example uses the geometric mean of these values ​​to normalize the experimental run results of the example.

[0195] Table 2. Functional activation of hGcgR, hGIPR and hGLP-1R in the presence of 0.1% casein.

[0196]

[0197]

[0198]

[0199]

[0200]

[0201] As shown by the data in Table 2, the Example compounds of the present invention are very effective in stimulating cAMP from human GIPR in the presence of 0.1% casein.

[0202] In vivo studies

[0203] Pharmacokinetics in male cynomolgus monkeys

[0204] The pharmacokinetics of selected examples were evaluated following a single subcutaneous administration of 50 nmol / kg to male cynomolgus monkeys. Blood samples were collected over 504 hours and the resulting individual plasma concentrations were used to calculate pharmacokinetic parameters. 3 The concentration of EDTA was determined using a qualified LC / MS method that measured the intact mass of the compound. Each peptide and the analog as internal standard were extracted from 100% cynomolgus monkey plasma using protein precipitation. A combined instrument was used for LC / MS detection. The average pharmacokinetic parameters are shown in Table 3.

[0205] Table 3. Mean pharmacokinetic parameters of peptides following a single subcutaneous administration of 50 nmol / kg to male cynomolgus monkeys.

[0206]

[0207] Abbreviation: T 1 / 2 = half-life, Tmax = time to reach maximum concentration, Cmax / dose = maximum plasma concentration divided by dose, AUC Inf / dose=AUC Inf Divided by the dose, CL / F = clearance / bioavailability. Note: Data are average values, where n = 2 / group,

[0208] As can be seen in Table 3, the results for the example peptides tested in this study were consistent with the extended pharmacokinetic profile.

[0209] Pharmacokinetics after subcutaneous administration in male Sprague Dawley rats

[0210] The pharmacokinetics of selected embodiments were evaluated after a single subcutaneous (SC) administration of 100 nmol / kg to male Sprague Dawley rats. Blood samples were collected within 168 hours after SC administration. Pharmacokinetic parameters were calculated using individual plasma concentrations. A qualified LC / MS method measuring the intact mass of the embodiments was used to determine plasma (K3 EDTA) concentrations. Each peptide and analog as an internal standard were extracted from 100% rat plasma using protein precipitation. A combined instrument was used for LC / MS detection. The average pharmacokinetic parameters of the embodiments are shown in Table 4.

[0211] Table 4. Mean (+ / - SD) pharmacokinetic parameters of peptides following a single subcutaneous administration of 100 nmol / kg to male Sprague Dawley rats.

[0212]

[0213] Abbreviation: T 1 / 2 = half-life, Tmax = time to reach maximum concentration, Cmax / dose = maximum plasma concentration divided by dose, AUC Inf / dose=AUC Inf Divided by dose, CL / F = Clearance / Bioavailability. Note: Data are mean values ​​where n=3 / group, except for Example 3 where data are from n=1 animal.

[0214] As can be seen in Table 4, the results of this study using these example peptides are consistent with the extended pharmacokinetic profile.

[0215] In vivo effects on insulin secretion in male Wistar rats

[0216] Male Wistar rats (Envigo, Indianapolis, IN) (280-320 grams) with femoral artery and femoral vein catheters were housed individually in polycarbonate cages with filter tops. Rats maintained a 12:12h light-dark cycle (6:00 A.M. lights on) at 21°C and received food and deionized water ad libitum. Rats were randomly grouped according to body weight and given 1.5 mL / kg subcutaneously (sc) at a dose of 0.3, 1.0, 3, 10, 30, and 100 nmol / kg 16 hours before glucose administration, then fasted. Animals were weighed and anesthetized with sodium pentobarbital by intraperitoneal (ip) (65 mg / kg, 30 mg / mL). At time 0, blood samples were collected in EDTA tubes, after which glucose (0.5 mg / kg, 5 mL / kg) was administered intravenously (iv). Blood samples were collected at 2, 4, 6, 10, 20, and 30 min after intravenous administration of glucose to determine glucose and insulin levels. Blood glucose levels were determined using a clinical chemistry analyzer. Plasma insulin was determined using an electrochemiluminescence assay (Meso Scale, Gaithersburg, MD). Glucose and insulin AUCs were compared to vehicle controls, with n=5 animals per group. Results are shown (SEM) (N).

[0217] Table 5: Effects of Example Compounds on Insulin Secretion During an Intravenous Glucose Tolerance Test.

[0218]

[0219] The data presented in Table 5 demonstrate a dose-dependent increase in insulin secretion.

[0220] Table 6: ivGTT insulin secretion shown by the following data:

[0221]

[0222] The data presented in Table 6 demonstrate a dose-dependent increase in insulin secretion.

[0223] Evaluation of the immunogenicity of the compounds of Examples 1, 2, 3, 5 and 6

[0224] The purpose of this study was to determine the relative potential of Example Compounds 1, 2, 3, 5 and 6 for clinical immunogenicity.

[0225] method:

[0226] CD4+T cell assay: The CD4+T cell assay was used to compare the potency of Example Compounds 1, 2, 3, 5 and 6 in inducing an immune response in vivo according to methods known in the art (see, e.g., Jones et al. (2004) J. Interferon Cytokine Res. 24:560-572; and Jones et al. (2005) J. Thromb. Haemost. 3:991-1000), where evaluation of clinically tested monoclonal antibodies and peptides showed a certain degree of correlation between T cell proliferation observed in vitro and clinical immunogenicity. Protein therapeutics that induce less than 30% positive responses in the CD4+T cell proliferation assay are associated with a low risk of immunogenicity. Briefly, to assess the propensity for clinical immunogenic responses to the compounds of Examples 1, 2, 3, 5, and 6, CD8+ T cell-depleted peripheral blood mononuclear cells (PBMCs) were prepared from a group of 10 healthy donors with different human leukocyte antigen (HLA) class II haplotypes and labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen). Each donor was tested in triplicate with 2.0 mL of culture medium control, keyhole limpet hemocyanin (KLH; 0.33 μM), and compounds of Examples 1, 2, 3, 5, and 6 (0.33 μM). The cultures were incubated at 37° C., 5% CO2 for 7 days. On day 7, samples were analyzed by flow cytometry using a BD LSR II Fortessa (Becton Dickinson; Franklin Lakes, NJ) equipped with a high-throughput sampler (HTS). Data were analyzed using FlowJo, LLC / TreeStar software (FlowJo, LLC / TreeStar; Ashland, OR).

[0227] Results and discussion

[0228] All donors generated positive T cell responses to KLH (100%). The frequency and magnitude of CD4+ T cell responses to the example compounds are analyzed as shown in Table 7.

[0229] Table 7: CD4+ T cell responses of example compounds and positive control (KLH).

[0230]

[0231] Cell division index ("CDI"): The ratio of dividing CD4+ T cells to the total number of CD4+ T cells in stimulated samples compared to unstimulated samples.

[0232] These data show that the frequency of positive CD4+ T cell responses (CDI>2.5) was low for the tested Example compounds, and the magnitude of the response was low (CDI<3) for one positive donor from the Example 2 group. Therefore, based on this assay, these compounds have a low risk of immunogenicity.

[0233] Amino acid sequence

[0234] SEQ ID NO:1

[0235] GIP 1-42 (people)

[0236]

[0237] SEQ ID NO:2

[0238] GLP-1(7-36)amide(human)

[0239]

[0240] SEQ ID NO:3

[0241] Glucagon (human)

[0242]

[0243] SEQ ID NO:4

[0244] Z 1 X 1 X 2 EGTX 6 ISDYSIX 13 LDX 16 X 17 X 18 QX 20 X 21 X 22 VX 24 X 25X 26 LX 28 X 29 GPSSGAPPPSZ 2 。

Claims

1. A compound of the following formula: Z1XX2BGTXSDYSIX13LDX16X17X18QX20X21X22VX24X25X26L X28X29GPSSGAPPPSZ2 (SEQ ID NO:4) Wherein: Z1 is a modification of the N-terminal amino group, and the modification is selected from acetyl and absent; X1 is selected from Y and D-Tyr; X2 is selected from Aib, A and D-Ala; X6 is selected from F, αMeF, Iva, L, αMeL and αMeF(2F); X13 is selected from αMeL, A, L and Aib; X16 is selected from K, E and Orn; X17 is selected from I and K (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H; X18 is selected from H, A and R; X20 is selected from Aib and Q; X21 is selected from D and E; X22 is selected from F and αMeF; X24 is selected from E, N, Q and D-Glu; X25 is selected from Y, 4-Pal, W and αMeY; X26 is selected from L and K (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H; X28 is selected from E and A; X29 is selected from G, A, Q and T; q is selected from 16 and 18; and Z2 is absent or is a modification of the C-terminal group, and the modification is an amidate; wherein one and only one selected from X17 and X26 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein X1 is Y and Z1 is absent.

3. The compound according to claim 1 or claim 2 or a pharmaceutically acceptable salt thereof, wherein X2 is Aib.

4. The compound according to any one of claims 1-3 or a pharmaceutically acceptable salt thereof, wherein X6 is selected from F and αMeF(2F).

5. The compound according to any one of claims 1-4 or a pharmaceutically acceptable salt thereof, wherein X13 is selected from L and αMeL.

6. The compound according to any one of claims 1-5 or a pharmaceutically acceptable salt thereof, wherein X16 is K or Orn.

7. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein X16 is K.

8. The compound according to any one of claims 1-7 or a pharmaceutically acceptable salt thereof, wherein X18 is H.

9. The compound according to any one of claims 1-8 or a pharmaceutically acceptable salt thereof, wherein X20 is Aib; and X22 is F.

10. The compound according to any one of claims 1-9 or a pharmaceutically acceptable salt thereof, wherein X21 is D.

11. The compound according to any one of claims 1-10 or a pharmaceutically acceptable salt thereof, wherein X25 is 4-Pal or Y.

12. The compound according to any one of claims 1-10 or a pharmaceutically acceptable salt thereof, Wherein: X17 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H; and X26 is L.

13. The compound according to any one of claims 1-10 or a pharmaceutically acceptable salt thereof, wherein X17 is I; and X26 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H.

14. The compound according to any one of claims 1-12 or a pharmaceutically acceptable salt thereof, wherein q is 16.

15. The compound according to any one of claims 1-12 or a pharmaceutically acceptable salt thereof, wherein q is 18.

16. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, Wherein: X1 is Y, and Z1 is absent; X2 is selected from Aib and D-Ala; X6 is F; X13 is selected from αMeL and L; X16 is selected from K and Orn; X18 is selected from H and A; X20 is Aib; X22 is F; X24 is selected from E, N, and D-Glu; X25 is selected from Y, 4-Pal, and W; X28 is selected from E and A; X29 is selected from G, A, and Q; q is selected from 16 and 18.

17. The compound according to claim 16 or a pharmaceutically acceptable salt thereof, wherein: X2 is Aib; X13 is αMeL; X18 is H; X24 is selected from E and D-Glu; X25 is selected from Y and 4-Pal; X28 is E; X29 is selected from G and A.

18. The compound according to claim 17 or a pharmaceutically acceptable salt thereof, wherein: X17 is K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H; and X26 is L.

19. The compound according to claim 17 or a pharmaceutically acceptable salt thereof, wherein: X17 is I; and X26 is K (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γ-Glu)-CO-(CH 2 ) q -CO 2 H.

20. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:

11.

21. The compound according to claim 20 or a pharmaceutically acceptable salt thereof, wherein the compound is SEQ ID NO:

7.

22. The compound according to claim 20 or a pharmaceutically acceptable salt thereof, wherein the compound is SEQ ID NO:

8.

23. The compound according to claim 20 or a pharmaceutically acceptable salt thereof, wherein the compound is SEQ ID NO:

10.

24. A method for treating a disorder selected from diabetes, obesity, and metabolic syndrome, comprising administering to a patient in need thereof an effective amount of the compound according to any one of claims 1-22 or a pharmaceutically acceptable salt thereof.

25. The method according to claim 24, wherein the disorder is obesity.

26. The method according to claim 24, wherein the disorder is type 2 diabetes.

27. A method for treating diabetes in a patient receiving insulin therapy, comprising administering to a patient in need thereof an effective amount of the compound according to any one of claims 1-22 or a pharmaceutically acceptable salt thereof.

28. A pharmaceutical composition comprising the compound according to any one of claims 1-22 or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent, or excipient.

29. The compound according to any one of claims 1-22 or a pharmaceutically acceptable salt thereof for treating a disorder selected from diabetes, obesity, and metabolic syndrome.

30. The compound according to claim 29 or a pharmaceutically acceptable salt thereof, wherein the disorder is type 2 diabetes.

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