METHODS OF MAKING INCRETIN ANALOGS

The disclosure relates generally to biology, chemistry and medicine, and more particularly it relates to methods of synthesizing, via hybrid liquid solid phase synthesis (HLSPS), an incretin analog having activity at one or more of the glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide- 1 (GLP-1) and glucagon (GCG) receptors.

Over the past several decades, the prevalence of diabetes has continued to rise. Type 2 diabetes (T2DM) is the most common form of diabetes, accounting for about 90% of all diabetes. T2DM is characterized by high blood glucose levels caused by insulin resistance. The current standard of care for T2DM includes dieting and exercising, as well as treating with oral glucose-lowering therapeutics and/or injectable glucose lowering therapeutics, including incretin-based therapies, such as GLP-1 receptor agonists, GIP/GLP-1 dual receptor agonists and even GIP/GLP-l/GCG (GGG) tri receptor agonists.

Inti. Patent Application Publication Nos. WO 2019/125938 and 2019/125929 generally describe incretin analogs that act as GGG tri-receptor agonists and a method of synthesizing the same via standard solid phase peptide synthesis. See also , Inti. Patent Application Publication Nos. WO 2014/049610, 2015/067716, 2016/198624, 2017/116204, 2017/153575 and 2018/100135. Likewise, Inti. Patent Application Publication Nos. WO 2013/164483 and 2016/111971 describe compounds stated to have GLP-1 and GIP activity. Moreover, Inti. Patent Application Publication No. WO 2020/023386 describes peptides having GIP and GLP1 receptor agonist activity.

There is a need, however, for alternative methods of making such incretin analogs and intermediates thereof to enable pharmaceutically elegant production with commercially desired purity. Likewise, there is a need for efficient methods and stable intermediates to provide incretin analogs efficiently, with fewer purification steps.

To address this need, the disclosure describes methods of making incretin analogs via HLSPS or native chemical ligation (NCL), where such methods use from two to four intermediate compounds to make the incretin analog.

In a first embodiment, the incretin analog can include an amino acid sequence of:

YX2QGTFTSDYSIX13LDKX17AX19X20AFIEYLLX28X29GPSSX34APPPS, where X2 is Aib, X13 is L or aMeL, X17 is any amino acid with a functional group available for conjugation, and the functional group is conjugated to a C16-C22 fatty acid, X19 is Q or A, X20 is Aib, aMeK, Q or H, X28 is E or A, X29 is G or Aib, X34 is G or Aib (SEQ ID NO:4) and the C-terminal amino acid is optionally amidated, or a pharmaceutically acceptable salt thereof. In certain instances, the incretin analog can have an amino acid sequence of: Y(Aib)QGTFTSDYSI(aMeL)LDKKAQ(Aib)AFIEYLLEGGPSSGAPPPS (SEQ ID NO:5), where the C-terminal amino acid is optionally amidated, or a pharmaceutically acceptable salt thereof.

In some instances, the C16-C22 fatty acid can be attached to the incretin analog via a linker having a structure of:

(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(gGlu)b-CO-(CH2)c-CO2H, where a can be 0, 1 or 2, b can be 1 or 2, and c can be 16 or 18.

In particular instances, the incretin analog can have the following sequence:

Y(Aib)QGTFTSDYSI(aMeL)LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(gGlu)-CO-(CH2)18-CO2H)AQ(Aib)AFIEYLLEGGPSSGAPPPS-NH2 (SEQ ID NO:6), or a pharmaceutically acceptable salt thereof, which can be depicted as having a structure of:

With regard to methods of making the incretin analog of SEQ ID NO:6 via HLSPS, the methods can include at least a step of coupling four intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7, 8, 9 and 10, or pharmaceutically acceptable salts thereof.

Alternatively, the methods can include at least a step of coupling four intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7, 11, 12 and 10, or pharmaceutically acceptable salts thereof.

Alternatively, the methods can include at least a step of coupling four intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7, 13, 14 and 10, or pharmaceutically acceptable salts thereof.

In other instances, the methods can include at least a step of coupling three intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7, 13 and 15, or pharmaceutically acceptable salts thereof.

Alternatively, the methods can include at least a step of coupling three intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS: 16, 17 and 10, or pharmaceutically acceptable salts thereof.

Alternatively, the methods can include at least a step of coupling three intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS: 18, 12 and 10, or pharmaceutically acceptable salts thereof.

Alternatively, the methods can include at least a step of coupling three intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7, 45 and 10, or pharmaceutically acceptable salts thereof.

Alternatively, the methods can include at least a step of coupling three intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7, 11 and 20, or pharmaceutically acceptable salts thereof.

In other instances, the methods can include at least a step of coupling two intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS: 19 and 15, or pharmaceutically acceptable salts thereof.

Alternatively, the methods can include at least a step of coupling two intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS: 18 and 20, or pharmaceutically acceptable salts thereof.

The methods above also can include a step of synthesizing the two to four intermediate compounds prior to the coupling step.

In the methods above, the intermediate compounds therefore can be chemically coupled or enzymatically coupled to one another to obtain the incretin analog of SEQ ID NO:6.

In the methods above, the C16-C22 fatty acid moiety and optional linker can be attached to one intermediate compound before the various intermediate compounds are coupled {i.e., acylation can occur before complete incretin analog synthesis).

Alternatively, the fatty acid moiety can be attached to the incretin analog after the various intermediate compounds have been coupled (i.e., acylation can occur after complete incretin analog synthesis). For example, the methods can include at least a step of coupling two intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:21 and 18, or pharmaceutically acceptable salts thereof, followed by coupling of a fatty acid moiety having a structure of:

(Compound 25).

Alternatively, the methods can include at least a step of coupling the following two intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:22 and 19, followed by coupling of a fatty acid moiety having a structure of:

(Compound 25).

In addition to the above, one can alternatively use NCL to make an incretin analog of SEQ ID NO: 6, in which the methods can include at least a step of coupling two intermediate compounds, where such compounds can have a structure selected from the following:

SEQ ID NOS:23 and 24,

SEQ ID NOS:39 and 24,

SEQ ID NOS:25 and 26,

SEQ ID NOS:40 and 26, and

SEQ ID NOS :27 and 26.

In another embodiment, the incretin analog can include an amino acid sequence of:

Y(Aib)EGT(aMeF(2F))TSD(4Pal)SI(aMeL)LD(Orn)K((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(g-Glu)-CO-(CH2)16-CO2H)AQ(Aib)EFI(D-Glu)( aMeY)LIEGGPSSGAPPPS-NH2 (SEQ ID NO:29), or a pharmaceutically acceptable salt thereof, which can be depicted as having a structure of:

.

With regard to methods of making the incretin analog of SEQ ID NO: 29 via HLSPS, the methods can include at least a step of coupling at least one of the following intermediate compounds to another intermediate compound, where such compounds have a structure as recited in SEQ ID NOS:30, 31, 32, 34, 35, 36 and/or 37, or pharmaceutically acceptable salts thereof.

Provided is a method of making an incretin analog of SEQ ID NO:29, the method comprising the step of:

coupling, via hybrid liquid solid phase synthesis, intermediate compounds selected from the groups consisting of:

a. SEQ ID NOS:7, 62, 42 and 31,

b. SEQ ID NOS:43, and 44.

In addition to the above methods, embodiments herein also include the intermidate compounds themselves ( e.g ., SEQ ID NOS:7 to 28 and 30 to 41), as well as compositions including the same.

An advantage of the analogs herein is that they can be used as effective treatments for diabetes mellitus, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), metabolic syndrome, non-alcoholic steatohepatitis (NASH) and obesity, as well as other disorders or conditions associated with modulation of GLP-1, and/or GIP, and/or Glucagon.

An advantage of the methods herein includes several process improvements such as, for example, shorter fragments initially produced via SPPS allow for generally increased purity and higher yields via HLSPS.

An advantage of the methods herein includes that efficiency of the coupling in SPPS not only is dependent on the actual residues involved in the chemical transformation but also is impacted by structure attached to the resin (i.e., solubility/aggregation issues are well known for certain sequences). With shorter fragments, more route flexibility is available for couplings of complicated amino acid residues, and an ability to redesign fragment structures to address more difficult transformations.

An advantage of the methods herein includes an improved control strategy for impurities during the synthesis, which can enable an improved final impurity profile for the crude peptide and simplify/reduce chromatography burden resulting in the cost savings.

An advantage of the methods herein includes that synthesis of shorter fragments via SPPS can allow for reduced washing cycles, for reduced volumes of reagents, and for use of greener solvent(s) leading to a reduced process mass intensity (PMI).

An advantage of the methods herein includes that with shorter fragments, risks of failure typical in linear builds of a long molecule are significantly reduced.

An advantage of the methods herein includes that a combination of liquid and solid phase synthesis is more amenable to new manufacturing platforms and introducing other innovative technologies.

An advantage of the methods herein includes flexibility in supply chain and logistics of the manufacturing process by using several independent fragments.

An advantage of the methods herein includes that use parallel manufacturing of fragments can provide reduced manufacturing cycles by parallel processing of the fragments.

An advantage of the methods herein includes that current good manufacturing practice (cGMP) convergent steps can be executed at a standard facility without a need for specialized equipment.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which the disclosure pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the incretin analog, pharmaceutical compositions and methods, the preferred methods and materials are described herein.

Moreover, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one element is present, unless the context clearly requires that there be one and only one element. The indefinite article “a” or “an” thus usually means “at least one.”

Abbreviations and Definitions

Certain abbreviations are defined as follows: “AEEA” refers to 2-[2-(2-amino-ethoxy)-ethoxy]-acetyl, “D-Glu” or “e” refers to D-Glutamic acid, “e” in an amino acid sequence refers to D-Glutamic acid, “Aib” refers to a-amino isobutyric acid, “aMeL” refers to a-methyl leucine, “aMeK” refers to a-methyl lysine, “Boc” refers to tert-butoxycarbonyl, “Bu” refers to butyl, “t-Bu” refers to tert-Butyl, “CTC” refers to

chlorotrityl chloride, “DCM” refers to dichloromethane, “DIC” refers to diisopropylcarbodiimide, “DMF” refers to dimethylformamide, “DMSO” refers to dimethyl sulfoxide, “DTT” refers to dithiothreitol, “EDTA” refers to ethylenediaminetetraacetic acid, “Fmoc” refers to fluorenylmethyloxycarbonyl chloride, “hr” refers to hour(s), “IPA” refers to isopropanol, “IPAc” refers to isopropyl acetate, “min” refers to minute(s), “Me” refers to methyl, “MTBE” refers to methyl-tert-butyl ether, “oxyma” refers to ethyl cyanohydroxyiminoacetate, “PG” refers to protecting group, “Pip” refers to piperidine, “SPPS” refers to solid phase peptide synthesis, “TFA” refers to trifluoroacetic acid, “TIPS” refers to triisopropylsilane, and “Trt” refers to trityl.

As used herein, “about” means within a statistically meaningful range of a value or values such as, for example, a stated concentration, length, molecular weight, pH, sequence identity, time frame, temperature or volume. Such a value or range can be within an order of magnitude typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The allowable variation encompassed by “about” will depend upon the particular system under study, and can be readily appreciated by one of skill in the art.

As used herein, and with reference to one or more of the GIP, GLP-1 or GCG receptors, “activity,” “activate,” “activating” and the like means a capacity of a compound, such as an incretin analog described herein, to bind to and induce a response at the receptor(s), as measured using assays known in the art, such as the in vitro assays described below.

As used herein, “amino acid with a functional group available for conjugation” means any natural or unnatural amino acid with a functional group that may be conjugated to a fatty acid by way of, for example, a linker. Examples of such functional groups include, but are not limited to, alkynyl, alkenyl, amino, azido, bromo, carboxyl, chloro, iodo and thiol groups. Examples of natural amino acids including such functional groups include Lys/K (amino), Cys/C (thiol), Glu/E (carboxyl) and Asp/D (carboxyl).

As used herein, “analog” means a compound, such as a synthetic peptide or polypeptide, that activates a target receptor and that elicits at least one in vivo or in vitro effect elicited by a native agonist for that receptor.

As used herein, “C16-C22 fatty acid” means a carboxylic acid having between 16 and 22 carbon atoms. The C16-C22 fatty acid suitable for use herein can be a saturated

monoacid or a saturated diacid. As used herein, “saturated” means the fatty acid contains no carbon-carbon double or triple bonds.

As used herein, “dual receptor activity” means an incretin analog with agonist activity at one or more of the GIP, GLP-1 and GCG receptors, especially an analog having a balanced and sufficient activity at one or more receptor to provide the benefits of agonism of that receptor while avoiding unwanted side effects associated with too much activity. Moreover, the incretin analog having dual receptor activity has extended duration of action at one or more of the GIP, GLP-1 and GCG receptors, which advantageously allows for dosing as infrequently as once-a-day, thrice-weekly, twice-weekly or once-a-week.

As used herein, “glucose-dependent insulinotropic polypeptide” or “GIP” means a peptide that plays a physiological role in glucose homeostasis by stimulating insulin secretion from pancreatic beta cells in the presence of glucose, especially human GIP (SEQ ID NO:l).

As used herein, “glucagon-like peptide-1” or “GLP-1” means a peptide that stimulates glucose-dependent insulin secretion and has been shown to prevent hyperglycemia in diabetics, especially human GLP-1 (SEQ ID NO:2).

As used herein, “glucagon” or “GCG” means peptide that helps maintain blood glucose by binding to and activating glucagon receptors on hepatocytes, causing the liver to release glucose - stored in the form of glycogen - through a process called glycogenolysis, especially human GCG (SEQ ID NO:3).

As used herein, "incretin analog" means a compound having structural similarities with, but multiple differences from, each of GIP, GLP-1 and GCG, especially human GIP (SEQ ID NO: 1), human GLP-1 (SEQ ID NO:2) and human GCG (SEQ ID NO:3). The incretin analogs described herein include amino acid sequences resulting in compounds having affinity for and activity at one or more of the GIP, GLP-1 and GCG receptors (i.e., dual agonist activity or triple agonist activity).

As used herein, “pharmaceutically acceptable buffer” means any of the standard pharmaceutical buffers known to one of skill in the art.

As used herein, “triple receptor activity” means an incretin analog with agonist activity the GIP, GLP-1 and GCG receptors, especially an analog having a balanced and sufficient activity at the receptors to provide the benefits of agonism of the receptors while avoiding unwanted side effects associated with too much activity. Moreover, the incretin analog having triple receptor activity has extended duration of action at one or more of the GIP, GLP-1 and GCG receptors, which advantageously allows for dosing as infrequently as once-a-day, thrice-weekly, twice-weekly or once-a-week.

Compositions

The structural features of the incretin analogs herein result in a compound having sufficient activity at one or more of the GIP, GLP-1 and GCG receptors to obtain the favorable effects of activity at one or more receptor (i.e., dual receptor activity or triple receptor activity), but not so much activity at any one receptor to either overwhelm the activity at the other two receptors or result in undesirable side effects when administered at a dose sufficient to result in activity at all three receptors.

The structural features of the incretin analogs herein also result in a compound having many other beneficial attributes relevant to developability as therapeutic treatments, including improving solubility of the analogs in aqueous solutions, improving chemical and physical formulation stability, extending the pharmacokinetic profile, and minimizing potential for immunogenicity.

It should be noted that the foregoing lists of structural features are exemplary, and not comprehensive, and that the combination of beneficial characteristics of exemplary analogs described herein is not the result of any modification in isolation, but is instead achieved through the novel combinations of the structural features described herein. In addition, the above-described effects of the foregoing lists of modifications are not exclusive, as many of these modifications also have other effects important to the characteristics of the compounds described herein, as described below.

The amino acid sequence of the incretin analogs herein incorporates naturally occurring amino acids, typically depicted herein using standard one or three letter codes (e.g., L/Leu = leucine), as well as a-methyl substituted residues of natural amino acids (e.g., (aMeL, aMeK, aMeY, aMeF(2F)), and certain other unnatural amino acids, such as Aib, Ornithine, 4-Pal. The structures of these amino acids are depicted below:

As noted above, the incretin analogs herein include a fatty acid moiety conjugated, for example, by way of a linker, to a natural or unnatural amino acid with a functional group available for conjugation. Such a conjugation is sometimes referred to as acylation. In certain instances, the amino acid with a functional group available for conjugation can be K, C, E and D, especially K at position 17 in SEQ ID NO:5 or SEQ ID NO:29, where the conjugation is to an e-amino group of a K side-chain. The fatty acid moiety acts as an albumin binder and provides a potential to generate long-acting compounds.

The incretin analogs herein utilize a C16-C22 fatty acid chemically conjugated to the functional group of an amino acid either by a direct bond or by a linker. The length and composition of the fatty acid impacts half-life of the incretin analog, its potency in in vivo animal models, and their solubility and stability. Conjugation to a C16-C22 saturated fatty monoacid or diacid results in an incretin analog that exhibits desirable half-life, desirable potency in in vivo animal models, and desirable solubility and stability characteristics.

Examples of saturated C16-C22 fatty acids for use herein include, but are not limited to, palmitic acid (hexadecanoic acid) (C16 monoacid), hexadecanedioic acid (C16 diacid), margaric acid (heptadecanoic acid) (C17 monoacid), heptadecanedioic acid (C17 diacid), stearic 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), including branched and substituted derivatives thereof.

In some instances, the C16-C22 fatty acid can be a saturated C18 monoacid, a saturated C18 diacid, a saturated C19 monoacid, a saturated C19 diacid, a saturated C20 monoacid, a saturated C20 diacid, and branched and substituted derivatives thereof.

In some instances, the linker can have from one to four amino acids, an amino polyethylene glycol carboxylate, or mixtures thereof. In certain instances, the amino polyethylene glycol carboxylate has the following structure:

H-{NH-CH2-CH2-[O-CH2-CH2]m-O-(CH2)p-CO}n-OH,

where m is any integer from 1 to 12, n is any integer from 1 to 12, and p is 1 or 2.

In some instances, the linker can have one or more (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties, optionally in combination with one to four amino acids.

In instances in which the linker includes at least one amino acid, the amino acid can be one to four Glu or gGlu amino acid residues. In some instances, the linker can include one or two Glu or gGlu amino acid residues, including the D-forms thereof. For example, the linker can include either one or two gGlu amino acid residues.

Alternatively, the linker can include one to four amino acid residues (such as, for example, Glu or gGlu amino acids) used in combination with up to thirty-six (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties. Specifically, the linker can be combinations of one to four Glu or gGlu amino acids and one to four (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties. In other instances, the linker can be combinations of one or two gGlu amino acids and one or two (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

In certain instances, the incretin analog described herein includes linker and fatty acid components having the structure of the following formula:

(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)a-(gGlu)b-CO-(CH2)c-CO2H,

where a is 0, 1 or 2, b is 1 or 2, and c is 16 or 18.

In a particular instance, a is 2, b is 1, and c is 16, the structure of which is depicted below:

In another particular instance, a is 1, b is 2, and c is 18, the structure of which is depicted below:

In another particular instance, a is 0, b is 2, and c is 18, the structure of which is depicted below:

In another particular instance, a is 1, b is 1, and c is 18, the structure of which is depicted below:

In particular instances, the overall structure of the incretin analog is SEQ ID

NO:6.

In particular instances, the overall structure of the incretin analog is SEQ ID

NO:29.

The affinity of the incretin analogs herein for each of the GIP, GLP-1 and GCG receptors may be measured using techniques known in the art for measuring receptor binding levels, including, for example, those described in the examples below, and is commonly expressed as an inhibitory constant (Ki) value. The activity of the incretin analogs herein at one or more of the receptors also may be measured using techniques known in the art, including, for example, the in vitro activity assays described below, and is commonly expressed as an effective concentration 50 (ECso) value, which is the concentration of compound causing half-maximal simulation in a dose response curve.

The incretin analogs herein can be formulated as a pharmaceutical composition, which can be administered by parenteral routes ( e.g ., subcutaneous, intravenous, intraperitoneal, intramuscular or transdermal). Such pharmaceutical composition and methods of preparing the same are well known in the art. See, e.g., “Remington: The Science and Practice of Pharmacy” (Troy ed., Lippincott, Williams & Wilkins 21st ed. 2006).

The incretin analogs herein may react with any of a number of inorganic and organic acids/bases to form pharmaceutically acceptable acid/base addition salts.

Pharmaceutically acceptable salts and common techniques for preparing them are well known in the art (see, e.g. , Stahl etal., “Handbook of Pharmaceutical Salts: Properties, Selection and Use” (Wiley-VCH 2nd ed. 2011)). Pharmaceutically acceptable salts for use herein include sodium, trifluoroacetate, hydrochloride and/or acetate salts.

The disclosure also provides and therefore encompasses novel intermediate compounds and methods of synthesizing the incretin analogs herein or pharmaceutically acceptable salts thereof. The intermediate compounds and incretin analogs herein can be prepared by a variety of techniques known in the art. For example, a method using standard solid phase peptide synthesis for two or more intermediate compounds followed by HLSPS thereof is illustrated in the Examples below. The specific synthetic steps for each of the routes described may be combined in different ways to prepare the incretin analogs herein. The reagents and starting materials are readily available to one of skill in the art.

The incretin analogs herein are generally effective over a wide dosage range. For example, dosages for once-weekly administration may fall within a range of about 0.01 to about 30 mg/person/week, within a range of about 0.1 to about 10 mg/person/week or even within a range of about 0.1 to about 3 mg/person/week. Thus, the incretin analogs described herein may be dosed daily, thrice-weekly, twice-weekly or once-weekly, especially once-weekly administration.

The incretin analogs herein may be used for treating a variety of conditions, disorders, diseases or symptoms. In particular, methods are provided below for treating T2DM in an individual, where such methods include at least a step of administering to an individual in need of such treatment an effective amount of an incretin analog herein, or a pharmaceutically acceptable salt thereof.

Methods

Standard Solid Phase Peptide Synthesis of Intermediate Compounds:

The incretin analogs herein can be made via any number of standard peptide synthesis methods known in the art, especially SPPS. SPPS builds are accomplished using standard Fmoc peptide chemistry techniques employing sequential couplings with an automated peptide synthesizer. Methods of SPPS are well known in the art and need not be exhaustively described herein. See generally, “Fmoc Solid Phase Peptide Synthesis: A Practical Approach” (Chan & White ed., Oxford University Press 2000), and Merrifield (1963) J. Am. Chem. Soc.85:2149-2154.

For deprotection, a resin is swelled with DMF, and then deprotected using 20% Pip/DMF (3 x 30 min). Subsequent Fmoc deprotections use 20% Pip/DMF (1 x 5-20 min, 1 x 20-30 min) treatments, with 1 x 5-20 min, 1 x 20 min and 1 x 30 min treatment sequences being used for more difficult deprotections.

After deprotection, the resin is washed with 5 x 2 min, 10 volume DMF washes. Amino acid pre-activation uses DIC/Oxyma DMF solutions at room temperature for 30 min. Coupling of the activated amino acid to the resin-bound peptide occurs for a specified time for each individual amino acid. Solvent washing with 5 x 2 min with 10 volumes DMF is performed after each coupling.

For isolation of the final product, the resin-bound product is washed 5 x 2 min with 10 volume DCM to remove DMF. The resin is washed with 2 x 2 min 10 volume IPA to remove DCM, washed 5 x 2 min with 10 volume MTBE, and then the product is dried at 40°C under vacuum. The resin-bound product is stored cold (-20°C).

For analysis, peptide is cleaved from the resin with an acidic cocktail of TFA/H2O/TIPS/DTT in the following ratio: (0.93v/0.04v/0.03v/0.03w). The resin is swelled with DCM (4-5 vol, 3 x 30 min) and drained. Cleavage cocktail (4-5 vol) is added to the pre-swelled resin, and the suspension is stirred for 2 hr at room temperature. The solution is filtered, and then the resin is washed with a small amount of DCM and combined with the cleavage solution. The resulting solution is poured into 7-10 volumes of cold (0°C) MTBE. The suspension is aged for 30 min at 0°C, the resulting precipitate is centrifuged, and the clear solution is decanted. The residue is suspended in the same volume of MTBE, and the resulting suspension is again centrifuged and decanted. After decanting, the clear MTBE solution of the precipitated peptide is dried in vacuo at 40°C overnight.

Hybrid Liquid Solid Phase Synthesis of the Incretin Analogs:

Intermediate compounds prepared via SPPS as described above can be combined to obtain the incretin analog of SEQ ID NO:6 or 29. Methods of HLSPS are well known in the art and need not be exhaustively described herein. See generally, US Patent Application Publication No.2011/0046349; and Albericio et al. (1997) Methods Enzymol.

289:313-336, Bray et al. (2003) Nature Rev. Drug Discovery 2:587-593, Dalcol et al. (1995) J. Org. Chem.7575-60:7581, Gauthier et al. (1991) Tettrahedron Lett.32: 577-580, Schneider et al. (2005) J. Peptide Sci.11:744-753, Smith, Organic Synthesis (Academic Press 4th ed.2016), and Zhang et al. (2008) Org. Process Res. Dev.12:101-110.

Briefly, HLSPS involves independent intermediate compound synthesis and compound coupling. Applied here, one method of making the incretin analog of SEQ ID NO:6 includes at least a step of coupling the following four intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7, 8, 9 and 10.

In some instances, the fragments can be coupled in the following order: SEQ ID NO:7 to SEQ ID NO:8 to SEQ ID NO:9 to SEQ ID NO:10 (i.e., from C-terminus to N-terminus). In other instances, and with an appropriate protecting group strategy, the fragments can be coupled in a different order.

Another method of making the incretin analog of SEQ ID NO:6 includes at least a step of coupling the following four intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7, 11, 12 and 10.

In some instances, the fragments are coupled in the following order: SEQ ID NO:7 to SEQ ID NO:11 to SEQ ID NO:12 to SEQ ID NO:10 (i.e., from C-terminus to N-terminus). In other instances, and with an appropriate protecting group strategy, the fragments can be coupled in a different order.

Another method of making the incretin analog of SEQ ID NO:6 includes at least a step of coupling the following four intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7, 13, 14 and 10.

In some instances, the fragments are coupled in the following order: SEQ ID NO: 7 to SEQ ID NO: 13 to SEQ ID NO: 14 to SEQ ID NO: 10 (i.e., from C-terminus to N-terminus). In other instances, and with an appropriate protecting group strategy, the fragments can be coupled in a different order.

Alternatively, one method of making the incretin analog of SEQ ID NO: 6 includes at least a step of coupling the following three intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7 13 and 15.

In some instances, the fragments are coupled in the following order: SEQ ID NO:7 to SEQ ID NO: 13 to SEQ ID NO: 15 (i.e., from C-terminus to N-terminus). In other instances, and with an appropriate protecting group strategy, the fragments can be coupled in a different order.

Another method of making the incretin analog of SEQ ID NO: 6 includes at least a step of coupling the following three intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS: 16, 17 and 10.

In some instances, the fragments are coupled in the following order: SEQ ID NO: 16 to SEQ ID NO: 17 to SEQ ID NO: 10 (i.e., from C-terminus to N-terminus). In other instances, and with an appropriate protecting group strategy, the fragments can be coupled in a different order.

Another method of making the incretin analog of SEQ ID NO: 6 includes at least a step of coupling the following three intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS: 18, 12 and 10.

In some instances, the fragments are coupled in the following order: SEQ ID NO: 18 to SEQ ID NO: 12 to SEQ ID NO: 10 (i.e., from C-terminus to N-terminus). In other instances, and with an appropriate protecting group strategy, the fragments can be coupled in a different order.

Another method of making the incretin analog of SEQ ID NO: 6 includes at least a step of coupling the following three intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7, 45 and 10.

In some instances, the fragments are coupled in the following order: SEQ ID NO:7 to SEQ ID NO:45 to SEQ ID NO: 10 (i.e., from C-terminus to N-terminus). In other instances, and with an appropriate protecting group strategy, the fragments can be coupled in a different order.

Another method of making the incretin analog of SEQ ID NO: 6 includes at least a step of coupling the following three intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:7, 11 and 20.

In some instances, the fragments are coupled in the following order: SEQ ID NO:7 to SEQ ID NO: 11 to SEQ ID NO:20 (i.e., from C-terminus to N-terminus). In other instances, and with an appropriate protecting group strategy, the fragments can be coupled in a different order.

Alternatively, one method of making the incretin analog of SEQ ID NO: 6 includes at least a step of coupling the following two intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS: 19 and 15.

Another method of making the incretin analog of SEQ ID NO: 6 includes at least a step of coupling the following two intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS: 18 and 20.

Alternatively, other methods of making the incretin analog of SEQ ID NO: 6 use the same disconnections as described above but instead couple all amino acid fragments of the backbone first, and then introduce the fatty acid side moiety as the last chemical transformation followed by global deprotection. Here, for example, the corresponding PG can be implemented at Lysl7, which can be selectively removed in presence of other PGs ( e.g ., Boc, tBu and/or Trt). In some instances, a method of making the incretin analog of SEQ ID NO: 6 includes at least a step of coupling the following intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:21 and 18, as well as

In some instances, a method of making the incretin analog of SEQ ID NO: 6 includes at least a step of coupling the following intermediate compounds, where such compounds have as structure as recited in SEQ ID NOS:22 and 19, as well as

Alternatively, other methods of making the incretin analog of SEQ ID NO:6 include at least of step of coupling deprotected compound intermediates ( e.g ., a thioester fragment and amide fragment) via a NCL approach. Here, for example Ala21 can be substituted with a natural enantiomer of Cys, and after completing the ligation step SEQ ID NO: 6 can be obtained by desulfurization of the Cys to deliver required Ala21 with the following intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:23 and 24.

Alternatively, the thioester (SEQ ID NO:23) may be substituted by an intermediate compound, in which the moiety of -C-P-OR Ester (CPE) at the C-terminal can serve as a masked thioester to facilitate the ligation step of compounds having a structure as recited in SEQ ID NOS:39 and 24.

In other instances, Alai 8 can be replaced with Cys and desulfurized after the native chemical ligation of the following intermediate compounds, where such compounds have a structure as recited in SEQ ID NOS:25 and 26.

Alternatively, the thioester (SEQ ID NO:25) can be substituted by an intermediate compound, in which a moiety of -Cys-Pro-OR Ester (CPE) can serve as a masked thioester to facilitate the ligation step of compounds having a structure as recited in SEQ ID NOS:40 and 26.

Alternatively, other methods of making the incretin analog of SEQ ID NO: 5 include at least of step of coupling the following intermediate compounds (e.g., a non-acylated thioester fragment and amide fragment) via a NCL approach, where such compounds have a structure as recited in SEQ ID NOS:27 and 26.

Alternatively, the thioester (SEQ ID NO:27) can be substituted by an intermediate compound, in which the moiety of -Cys-Pro-OR Ester (CPE) can serve as a masked thioester to facilitate the ligation step of compounds having a structure as recited in SEQ ID NOS:41 and 26.

In another embodiment, SEQ ID NO:29 can be synthezied by coupling SEQ ID NO:43 and SEQ ID NO:44 and then deprotect to produce SEQ ID NO:29.

In another embodiment, SEQ ID NO:48 can be synthezied by using SEQ ID NO:20 and SEQ ID NO: 18. SEQ ID NO:48 is deprotected to produce SEQ ID NO:6.

In another embodiment, SEQ ID NO:53 can be synthezied by NCL using SEQ ID NO:51 and SEQ IDNO:52.

In another embodiment, SEQ ID NO:53 can be synthezied by NCL using SEQ ID NO:52 and SEQ ID NO:54.

For effective preparation of compound intermediates of SEQ ID NOS:9, 12, 14, 15, 17, 20, 23 and 25, the following is synthesized using fatty side chain

and Fmoc-L-Lys-OH amino acid attached with fatty side chain:

.

For improved purity and efficiency of the SPPS, the following dimer, trimer and tetramer can be used for preparing SEQ ID NOS:10, 15, 20, 21, 22, 23, 25 and 27, where the structures that follow can be synthesized using amino acid building block via SPPS or liquid phase synthesis:

.

Other Methods/Uses:

The incretin analogs herein can be used in a number of therapeutic applications. For example, the incretin analogs can be used in methods of treating obesity in an

individual, where such methods include at least a step of administering to an individual in need of such treatment an effective amount of an incretin analog herein, or a pharmaceutically acceptable salt thereof.

Additionally, the incretin analogs can be used in methods of inducing non-therapeutic weight loss in an individual, where such methods include at least a step of administering to an individual in need of such treatment an effective amount of an incretin analog herein, or a pharmaceutically acceptable salt thereof.

Additionally, the incretin analogs herein can be used in methods of treating metabolic syndrome in an individual, where such methods include at least a step of administering to an individual in need of such treatment an effective amount of an incretin analog herein, or a pharmaceutically acceptable salt thereof.

Additionally, the incretin analogs herein can be used in methods of treating NASH in an individual, where such methods include at least a step of administering to an individual in need of such treatment an effective amount of an incretin analog described, or a pharmaceutically acceptable salt thereof.

Additionally, the incretin analogs herein can be used in methods of treating NAFLD in an individual, where such methods include at least a step of administering to an individual in need of such treatment an effective amount of an incretin analog herein, or a pharmaceutically acceptable salt thereof.

In these methods, effectiveness of the incretin analogs can be assessed by, for example, observing a significant reduction in blood glucose, observing a significant increase in insulin, observing a significant reduction in HbAlc and/or observing a significant reduction in body weight.

Alternatively, the incretin analogs herein or pharmaceutically acceptable salts thereof may be used for improving bone strength in an individual in need thereof. In some instances, the individual in need thereof has hypo-ostosis or hypo-osteoidosis, or is healing from bone fracture, orthotic procedure, prosthetics implant, dental implant, and/or spinal fusion. The incretin analogs also may be used for treating other disorders such as Parkinson’s disease or Alzheimer’s disease.

EXAMPLES

The following non-limiting examples are offered for purposes of illustration, not limitation.

PEPTIDE AND POLYPEPTIDE SYNTHESIS

Example 1: Solid Phase Peptide Synthesis of Intermediate Compound 1 Intermediate Compound 1 (SEQ ID NO:7), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Sieber resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 1.

Table 1: SPPS Conditions for Example 1.

Fmoc Deprotection, Fragment Cleavage and Isolation: Fragment on Sieber resin is stirred twice with 10 V of 20% piperidine/DMF for 20-30 min, then washed six times with 10 V of DMF. The de-Fmoced fragment on Sieber resin is swelled twice using 10 V DCM for 10-20 min. A reactor with resin is cooled to about 15°C, and 20 V of 5% TFA/DCM is charged to the reactor and then stirred for 2 hr under nitrogen maintaining the temperature at about 15°C. The resin is filtered and washed with 3 x 10 V of DCM.

All the filtrates are combined together. DCM is removed from the resulting solution under reduced pressure while maintaining the internal temperature at £ 20°C to 22.5 V residual volume. MTBE (25 V) is charged to the solution, and DCM/MTBE solvents are again removed under reduced pressure while maintaining the internal temperature at £ 20°C to 22.5 V residual volume. Addition of MTBE/distillation operation is repeated until residual concentration of fragment in supernatant has not reached <0.11 wt%. Then, the resulting slurry is filtered while maintaining temperature at about 15°C. To the cake, 14 V of fresh MTBE is added, is stirred for 30 min at about 15°C, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 35°C.

Example 2: Solid Phase Peptide Synthesis of Intermediate Compound 2 Intermediate Compound 2 (SEQ ID NO:8), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Gly-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 2.

Table 2: SPPS Conditions for Example 2.

Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 10 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen at about 25°C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine, and then 5 V of DMSO is added to the filtrate. Resin treatment with 1% TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM, and stirred for 10-15 min. All the filtrates and wash are combined. The fragment solution is concentrated under vacuum to 6-10 V maintaining temperature at £ 35°C (residual DCM concentration £ 15%). DMSO solution of the fragment is added to 11-15 V of H2O over 2-6 hr period (< 1 L/min) at about 25°C. The formed slurry of precipitated fragment is stirred for 30-40 min at about 25°C and then filtered. The resulting solid is suspended in 8-12 V of H2O at about 25°C, is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40°C.

Example 3: Solid Phase Peptide Synthesis of Intermediate Compound 3 Intermediate Compound 3 (SEQ ID NO:9), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc-Ala-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 3.

Table 3: SPPS Conditions for Example 3.

Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at about 25°C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM and stirred for 10-15 min. All the filtrates and wash are combined, and the resulting mixture cooled £ 20°C. The fragment solution is concentrated under vacuum to 2-4 V maintaining temperature at £ 20°C. 5 V of ACN is added to the solution, and residual DCM removed under vacuum (residual DCM concentration £ X%) maintaining temperature at £ 20°C. An ACN solution of the fragment is added to 5 V of ice-cold H2O over 2-6 hr period (< 1 L/min) maintaining temperature at about 0°C. The resulting slurry of precipitated fragment is stirred for 30-40 min at about 0°C and then is filtered at about 0°C. The resulting solid is suspended in 3-5 V of H2O at about 25°C, is stirred 10- 15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at about 40°C.

Example 4: Solid Phase Peptide Synthesis of Intermediate Compound 4 Intermediate Compound 4 (SEQ ID NO:10), or a pharmaceutically acceptable salt thereof, can be synthesized by standard SPPS. Briefly, SPPS is conducted using Fmoc- Leu-2-CTC resin (loading factor 0.6-0.9 mmol/g) with the conditions set forth below in Table 4.

Table 4: SPPS Conditions for Example 4.

*structure for Boc-L-Tyr(t-Bu)-Aib-L-Gln(Trt)-Gly-OH is as follows:

.

Fragment Cleavage and Isolation: Fragment on CTC resin is swelled once using DCM (5 V) for 45 min. 5 V of 1% TFA/DCM is charged to the reactor, and the resulting suspension of the resin is stirred for 10-15 min under nitrogen maintaining temperature at about 25°C. The filtrate is removed and immediately neutralized by slow addition of a 1.05 equivalent amount of pyridine. Resin treatment with 1% with TFA/DCM followed by filtrate neutralization is repeated two more times. The resin is washed with 3 V of DCM, and is stirred for 10-15 min. All the filtrates and wash are combined, and the resulting mixture is cooled to £ 20°C. The fragment solution is concentrated under vacuum to 2-4 V maintaining temperature at £ 20°C. 2 V of DMSO is added to the solution, and residual DCM removed under vacuum (residual DCM concentration £5%) maintaining temperature at £ 20°C. A DMSO solution of the fragment is added to 7-9 V of ice-cold H2O over 2-6 hr (< 1 L/min), while maintaining temperature at about 0°C. The resulting slurry of precipitated fragment is stirred for 30-40 min at about 0°C and then is filtered at about 0°C. The resulting solid is suspended in 3-5 V of H2O at about 25°C, is stirred 10-15 min, and then is filtered. Washing is repeated one more time, and the resulting solid is dried at 40°C.

CLAIMS

The invention claimed is:

1. A method of making an incretin analog of SEQ ID NO:6, the method comprising the step of:

coupling, via hybrid liquid solid phase synthesis, four intermediate compounds selected from the groups consisting of:

a. SEQ ID NOS:7, 8, 9 and 10,

b. SEQ ID NOS:7, 11, 12, and 10, and

c. SEQ IDNOS:7, 13, 14 and 10.

2. A method of making an incretin analog of SEQ ID NO:6, the method comprising the step of:

coupling, via hybrid liquid solid phase synthesis, three intermediate compounds selected from the groups consisting of:

a. SEQ IDNOS:7, 13 and 15,

b. SEQ ID NOS: 16, 17 and 10,

c. SEQ ID NOS: 18, 12 and 10, and

d. SEQ IDNOS:7, 45 and 10.

3. A method of making an incretin analog of SEQ ID NO:6, the method comprising the step of:

coupling, via hybrid liquid solid phase synthesis, two intermediate compounds selected from the groups consisting of:

a. SEQ ID NOS: 15 and 19 and

b. SEQ ID NOS: 18 and 20.

4. An intermediate compound comprising:

SEQ ID NO: 7 or a pharmaceutically acceptable salt thereof.

5. An intermediate compound comprising:

SEQ ID NO: 8 or a pharmaceutically acceptable salt thereof.

6. An intermediate compound comprising:

SEQ ID NO: 9 or a pharmaceutically acceptable salt thereof.

7. An intermediate compound comprising:

SEQ ID NO: 10 or a pharmaceutically acceptable salt thereof.

8. An intermediate compound comprising:

SEQ ID NO: 11 or a pharmaceutically acceptable salt thereof.

9. An intermediate compound comprising:

SEQ ID NO: 12 or a pharmaceutically acceptable salt thereof.

10. An intermediate compound comprising:

SEQ ID NO: 13 or a pharmaceutically acceptable salt thereof.

11. An intermediate compound comprising

SEQ ID NO: 14 or a pharmaceutically acceptable salt thereof.

12. An intermediate compound comprising:

SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.

13. An intermediate compound comprising:

SEQ ID NO: 16 or a pharmaceutically acceptable salt thereof.

14. An intermediate compound comprising:

SEQ ID NO: 17 or a pharmaceutically acceptable salt thereof.

15. An intermediate compound comprising:

SEQ ID NO: 18 or a pharmaceutically acceptable salt thereof.

16. An intermediate compound comprising:

SEQ ID NO: 19 or a pharmaceutically acceptable salt thereof.

17. An intermediate compound comprising:

SEQ ID NO:20 or a pharmaceutically acceptable salt thereof.

18. An intermediate compound comprising:

SEQ ID NO:21 or a pharmaceutically acceptable salt thereof.

19. An intermediate compound comprising:

SEQ ID NO:22 or a pharmaceutically acceptable salt

20. An intermediate compound comprising:

SEQ ID NO:23 or a pharmaceutically acceptable salt thereof.

21. An intermediate compound comprising:

SEQ ID NO:24 or a pharmaceutically acceptable salt thereof.

22. An intermediate compound comprising:

SEQ ID NO:25 or a pharmaceutically acceptable salt thereof.

23. An intermediate compound comprising:

SEQ ID NO:26 or a pharmaceutically acceptable salt thereof.

24. An intermediate compound comprising:

SEQ ID NO:27 or a pharmaceutically acceptable salt thereof.

25. An intermediate compound comprising:

SEQ ID NO:28 or a pharmaceutically acceptable salt thereof.

26. An intermediate compound comprising:

SEQ ID NO: 38 or a pharmaceutically acceptable salt thereof.

27. An intermediate compound comprising:

SEQ ID NO:39 or a pharmaceutically acceptable salt thereof.

28. An intermediate compound comprising:

SEQ ID NO:40 or a pharmaceutically acceptable salt thereof.

29. An intermediate compound comprising Boc-Y(Aib)EGT(aMeF(2F))TSD(4Pal)SI(aMeL)L (SEQ ID NO:30), or a pharmaceutically acceptable salt thereof.

30. An intermediate compound comprising:

SEQ ID NO:31 or a pharmaceutically acceptable salt thereof.

31. An intermediate compound comprising Q(Aib)EFI(D-Glu)( aMeY)LIEG (SEQ ID NO:32), or a pharmaceutically acceptable salt thereof.

32. An intermediate compound comprising GPSSGAPPPS (SEQ ID NO:33), or a pharmaceutically acceptable salt thereof.

33. An intermediate compound comprising Boc-Y(Aib)EGT(aMeF(2F))TS (SEQ ID NO:34), or a pharmaceutically acceptable salt thereof.

34. An intermediate compound comprising Q(Aib)EFI(D-Glu)( aMeY)LIEGGPSSGAPPPS-NH2 (SEQ ID NO:35), or a pharmaceutically acceptable salt thereof.

35. An intermediate compound comprising EFI(D-Glu)( aMeY)LIEGGPSSGAPPPS-NH2 (SEQ ID NO:36), or a pharmaceutically acceptable salt thereof.

36. An intermediate compound comprising CQ(Aib)EFI(D-Glu)( aMeY)LIEGGPSSGAPPPS-NH2 (SEQ ID NO:37), or a pharmaceutically acceptable salt thereof.

37. An intermediate compound selected from the group consisting of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45. SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID

NO:53, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, or a pharmaceutically acceptable salt thereof.

38. A method of making an incretin analog of SEQ ID NO:29, the method comprising the step of:

coupling, via hybrid liquid solid phase synthesis, intermediate compounds selected from the groups consisting of:

a. SEQ ID NOS:7, 62, 42 and 31,

b. SEQ ID NOS:43, and 44.