A metal complex of fmoc protected lipopeg-peptide with metal halide salts and the preparation thereof

Abstract

The present invention relates to the general field of amino acids and peptides. In particular, it relates to a solid metal complex salt of amino acids or peptides, the process for the manufacture of such solid metal complex salt and its use in the preparation of semaglutide, tirzepatide and their pharmaceutically acceptable salts.

Description

[0001] Title: A metal complex of Fmoc protected lipoPeg-peptide with metal halide salts and the preparation thereof

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the general field of amino acids and peptides. In particular, it relates to a solid metal complex salt of amino acids or peptides, the process for the manufacture of such solid metal complex salt, and its use in the preparation of semaglutide, tirzepatide and their pharmaceutically acceptable salts.

[0004] BACKGROUND OF THE INVENTION

[0005] Amino acids and peptides comprising both hydrophilic and alkylated portions in the side chain often present themselves as thick oils, gels, or waxes; this physical condition makes them very difficult to handle. Simple actions such as weighing or transferring them from one container or vessel, or the like to another, to carry out a purification, a characterization, or a further reaction, cause significant loss of material.

[0006] This is the case, for example, for compounds of Formula 1, Formula 2, Formula 3 and Formula 4:

[0007] Formula 1

[0008]

[0009] Formula 3

[0010]

[0011] Compounds of Formula 1 and Formula 3 are intermediate products in the synthesis of semaglutide, which is a human GLP-1 receptor agonist indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus and for long-term weight management in adults with obesity or overweight.

[0012] Compounds of Formula 2 and Formula 4 are intermediate products in the synthesis of tirzepatide, which is used in the same therapeutical area as semaglutide. Compound of Formula 2 differs from compound of Formula 1 since a C20 aliphatic hydrocarbon chain is present instead of the C18 one.

[0013] The use of compounds like the ones of Formula 1 - Formula 4 for preparing semaglutide and tirzepatide is well known in the art.

[0014] WO2019170895A1 describes the synthesis of GLP-1 compounds by applying an enzymatic approach to generate the target product by condensation of the corresponding two fragments catalysed by an enzyme. The approach used to generate these fragments requires the use of, for example, compound of Formula 1 to generate one of the fragments used for said semaglutide enzymatic synthesis.

[0015] A further example is provided by WO2017114191A1, where compound of Formula 3 is prepared and used as intermediate for the synthesis of semaglutide.

[0016] However, compounds of Formula 1 - Formula 4 present themselves in the form of a pale-yellow thick oil or gel, which makes them very difficult to handle.

[0017] Therefore, the general handling of these substances is not efficient and time consuming. In addition, due to the difficulties in weighing and transferring such substances and to the high probability of material loss, any operation is poorly reproducible.

[0018] OBJECT OF THE INVENTION In view of this, the first object of the present disclosure is a solid metal complex salt that does not show one or more of the above-mentioned drawbacks.

[0019] A further object of the present disclosure is to provide a process for generating such solid metal complex salt.

[0020] A further object of the present disclosure is the use of such solid metal complex salt in the synthesis of semaglutide, tirzepatide, and their pharmaceutically acceptable salts.

[0021] SUMMARY OF THE INVENTION

[0022] In a first aspect, the present disclosure relates to a solid metal complex salt obtainable by the reaction of compound of Formula I:

[0023] Rl-(AEEA)m-Al-CO-(CH2)n-COO-R2 (I) with a MX metal salt, wherein the MX metal salt is used in the range between 0.5 and 5.0 mol equivalents with respect to compound of Formula I;

[0024] M is a metal cation selected from the group of alkaline earth metal cations and transition metal cations;

[0025] X is an organic or inorganic anion;

[0026] R1 is selected from the group consisting of OH, a protected or unprotected amino acid and a peptide group consisting of 2 to 4 protected or unprotected amino acids;

[0027] R2 is H or a carboxylic protective group;

[0028] Al is a protected or unprotected amino acid or a peptide group consisting of 2 to 4 protected or unprotected amino acids;

[0029] AEEA is 2-[2-(2-aminoethoxy)ethoxy]acetyl group; m is an integer between 1 and 10; and n is an integer between 8 and 28.

[0030] The reaction of the compound of Formula I with the MX metal salt is a complexation reaction, also called complex formation reaction, which is a reaction that forms the metal complex salt.

[0031] The term "metal salt", such as the MX metal salt of the present disclosure, refers to any compound that contains metal and non-metal atoms ionically bonded together.

[0032] The term "metal complex salt", also known as coordination compounds, refers to a complex in which a central metal ion is surrounded by a certain number of ligands (atoms, ions, or molecules that donate electrons to the metal ion to form coordinate covalent bonds), where the central metal ion is typically a transition metal ion or an alkaline earth metal cation.

[0033] The term "between X and Y" is used throughout the present disclosure to define the range X to Y including the endpoints X and Y.

[0034] The term "protective group" refers to a temporarily attached group to decrease the reactivity of a functional group so that the protected functional group does not react under synthetic conditions to which the molecule is subjected in one or more subsequent steps.

[0035] The term "active pharmaceutical ingredient" refers to those substances in a pharmaceutical drug that are responsible for the beneficial health effects experienced by consumers. An example of an API is the acetaminophen contained in a pain relief tablet.

[0036] Advantageously, this solid metal complex salt does not stick to the container so it can be easily handled without losing significant amounts of such compound. Further, this solid metal complex salt can be easily isolated, for example in the form of a powder, e.g. by precipitation from a solvent with a suitable antisolvent or by removal of said solvent, for example by evaporation of said solvent. As non-limiting examples, this solid metal complex salt can thus be easily weighed, purified, characterized, transferred into another container, stored, or used as intermediate for further reactions with no significant loss of material.

[0037] In another aspect, the present disclosure relates to a process for the preparation of a solid metal complex salt by the reaction of compound of Formula I:

[0038] Rl-(AEEA)m-Al-CO-(CH2)n-COO-R2 (I) with a MX metal salt, wherein the MX metal salt is used in the range between 0.5 and 5.0 mol equivalents with respect to compound of Formula I;

[0039] M is a metal cation selected from the group of alkaline earth metal cations and transition metal cations;

[0040] X is an organic or inorganic anion;

[0041] R1 is selected from the group consisting of OH, a protected or unprotected amino acid and a peptide group consisting of 2 to 4 protected or unprotected amino acids;

[0042] R2 is H or a carboxylic protective group;

[0043] Al is a protected or unprotected amino acid or a peptide group consisting of 2 to 4 protected or unprotected amino acids;

[0044] AEEA is 2-[2-(2-aminoethoxy)ethoxy]acetyl group; m is an integer between 1 and 10; and n is an integer between 8 and 28, comprising the following steps:

[0045] (i) dispersing or solubilizing compound of Formula I in a first aliquot of a liquid medium;

[0046] (ii) treating compound of Formula I with the MX metal salt to form the metal complex salt, and

[0047] (iii) recovering the metal complex salt in a solid form.

[0048] With the wording "treating compound of Formula I with the MX metal salt to form the metal complex salt" it is meant that compound of Formula I and the MX metal salt are brought into contact to react and thus to form the metal complex salt.

[0049] The term "dispersing" means that compound of Formula I is distributed in the liquid medium in a discrete phase, consisting e.g. of particles or droplets.

[0050] Advantageously, this process transforms compound of Formula I into a solid metal complex salt which is more manageable because it is not sticking, i.e. not sticking to surfaces of the equipment used to make, store or handle it.

[0051] This process allows obtaining the solid metal complex salt with high yield and purity. In fact, any impurity soluble in the liquid medium but not able to react with the MX metal salt to form a solid, will remain in the liquid phase and thus it can be easily separated from the solid metal complex salt, e.g. by filtration.

[0052] In addition, this process requires mild conditions and involves simple and industrially viable operations.

[0053] In the present disclosure, "side chain" and "lateral chain" are used indifferently to indicate a chemical group that is attached to a core part of the molecule called the "main chain" or backbone.

[0054] In another aspect, the present disclosure relates to the use of such solid metal complex salt in the synthesis of semaglutide, tirzepatide and their pharmaceutically acceptable salts.

[0055] Advantageously, the use of such solid metal complex salt makes the synthesis of semaglutide, tirzepatide and their pharmaceutically acceptable salts very efficient, simple and industrially viable.

[0056] Also, the loss of expensive intermediates, such as the one of Formula I, is reduced. Further, cleaning operations of the apparatus of synthesis of semaglutide, tirzepatide and their pharmaceutically acceptable salts are faster and easier.

[0057] The process according to the invention allows obtaining semaglutide, tirzepatide and their pharmaceutically acceptable salts with higher yield and purity compared to processes of synthesis involving the use of the uncomplexed sticking compounds of formula I.

[0058] In another aspect, the present disclosure relates to a process for making active pharmaceutically ingredients, comprising semaglutide, tirzepatide and their pharmaceutically acceptable salts, characterized by the use of said solid metal complex salt.

[0059] BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Some features and advantages of the present invention will become apparent from the accompanying drawings, in which:

[0061] Figure 1 is a representation of a possible compound according to Formula 1 where Rl, R2, Al, and AEEA are explicated;

[0062] Figure 2 shows the HPLC-MS profile of the solid metal complex salt prepared in Example 2;

[0063] Figure 3 shows the HPLC-MS profile of the solid metal complex salt prepared in Example 4;

[0064] Figure 4 shows the HPLC-MS profiles of fragment 20-31 of semaglutide obtained by the attachment of the free lateral chain fragment (top part of the figure) and of the same fragment obtained by the attachment of the complexed lateral chain fragment (bottom), which is obtained according to the present invention, to the 21-31 fragment;

[0065] Figure 5 shows the HPLC-MS profiles of fragment 20-29 of tirzepatide obtained by the attachment of the free lateral chain fragment (top part of the figure) and of the same fragment obtained by the attachment of the complexed lateral chain fragment (bottom), which is obtained according to the present invention, to the 21- 29 fragment;

[0066] Figure 6 shows ^-NMR spectra of the compound of Formula 1 (Fl) (6 a) and the metal complex salt obtained by the reaction of compound of Formula 1 with 1 mol equivalent of MgCL (6 b) and 2 mol equivalents of MgCL (6 c). In the figure, the position of the signals attributable to the proton of the NH group of Glu (is marked with a triangle A), of both the AEEA groups (marked with a square or circle, respectively) , of a-NH group (marked with a diamond) and e-NH group of Lys (marked with a triangle on its head) are highlighted.

[0067] Figure 7 shows the calculated spatial arrangement of the compound of Formula 1 in dependency on the presence and amount of MgCL used.

[0068] DETAILED DESCRIPTION OF THE INVENTION

[0069] In some embodiments, the metal cation M is selected from the group consisting of Ca, Zn, Mg, Cu, Co, Ni, Ti, Zr, Fe and Ag cations. Preferably, the metal cation is selected from the group consisting of Ca2+, Zn2+, Mg2+, Cu2+, Co2+, Ni2+, Ti2+, Zr2+, Fe3+and Ag+, more preferably from the group consisting of Cu2+, Fe3+, Co2+, Mg2+, even more preferably the metal cation M is Mg2+or Cu2+, most preferably the metal cation M is Mg2+.

[0070] In some embodiments, X is a halide, preferably X is Cl1or Br-1, more preferably X is Cl-1.

[0071] In some embodiments, the MX metal salt is MgCL or CuCL, in some preferred embodiments the MX metal salt is MgCL.

[0072] In some embodiments, the MX metal salt is used in the range between 0.8 and 4.0 mol equivalents with respect to compound of formula I, preferably between 1.0 and 2.5 mol equivalents. In some preferred embodiments, the MX metal salt is MgCL and it is used in the range between 0.8 and 4.0 mol equivalents with respect to compound of formula I, preferably between 1.0 and 2.5 mol equivalents.

[0073] In the reaction the generation of the solid metal complex salt is favoured by increasing the amount of MX metal salt used with respect to compound of Formula I.

[0074] However, when the MX metal salt is used at low mol equivalents with respect to compound of formula I, wherein low refers to a range of 1.0 to 2.5, the stability of the solid metal complex salt is increased compared to the one obtained at high mol equivalents, wherein high refers to a range of 3.0 to 5.0. In particular, the undesired removal of one or more carboxylic protective groups, eventually occurring due to the formation of HCI generated during the formation of the solid metal complex salt, is reduced when the solid metal complex salt is obtained at low mol equivalents of the MX metal salt with respect to compound of Formula I.

[0075] The solid metal complex salt obtained at high mol equivalents of the MX metal salt with respect to compound of Formula I has a slightly sticking behaviour with respect to the one obtained at low mol equivalents. Further, the hygroscopicity of the solid metal complex salt obtained at low mol equivalents of the MX metal salt with respect to compound of Formula I is decreased if compared to the one obtained at high mol equivalents. Also, the solid metal complex salt obtained at low mol equivalents of the MX metal salt with respect to compound of Formula I is easier to be filtered. It is assumed that this is because it has a higher particle size.

[0076] In some embodiments, R1 is protected or unprotected Lys and / or Al is protected or unprotected Glu; in some preferred embodiments, R1 is protected or unprotected Lys and Al is protected or unprotected Glu; in some more preferred embodiments, R1 is protected Lys and Al is protected Glu.

[0077] In some other embodiments, R1 is OH and / or Al is protected or unprotected Glu; in some preferred embodiments, R1 is OH and Al is protected or unprotected Glu; in some more preferred embodiments, R1 is OH and Al is protected Glu.

[0078] In some embodiments, compound of Formula I has one or more carboxylic groups which are protected with a protective group selected independently from the group consisting of tert-Butyl (tBu), beta-3methylpent- 3-yl (Mpe), 2-Phenylisopropyl (2-Ph'Pr), and 4-(3,6,9-Trioxadecyl)oxybenzyl (TEGBz or TEGBn).

[0079] In some embodiments, Al is protected Glu and the a-carboxylic group of Glu is preferably protected with a tert-Butyl protective group.

[0080] In some embodiments, all the protective groups of all the carboxylic groups of compound of Formula I are tert-Butyl protective groups.

[0081] In some embodiments, compound of Formula I has one or more amino groups which are protected with a protective group selected independently from the group consisting of Fmoc, Boc, Smoc, and Bz.

[0082] In some embodiments, when R1 is an amino-protected amino acid, the amino-protective group of R1 is selected from the group consisting of Fmoc, Boc, Smoc, and Bz.

[0083] In some embodiments R1 is protected Lys, and the a-amino group of Lys is preferably protected with a Fmoc protective group.

[0084] In some embodiments R2 is a carboxylic protective group, preferably a tert-Butyl group.

[0085] In some preferred embodiments, Al is protected Glu and the a-carboxylic group of Glu is protected with a tert-Butyl protective group, and R1 is a protected Lys, and the a-amino group of Lys is protected with a Fmoc protective group. According to some embodiments, m is an integer between 1 and 5, preferably between 1 and 4, more preferably m is 2.

[0086] According to some embodiments, n is an integer between 10 and 25; preferably n is between 15 and 21.

[0087] According to some preferred embodiments, m is comprised between 1 and 4 and n is comprised between 15 and 21, more preferably m is 2 and n is comprised between 15 and 21.

[0088] AEEA is preferably linked to R1 through the C atom of the keto group and / or AEEA is preferably linked to Al through the N atom of the amino group and / or, when more than one AEEA is present, they are preferably linked to each other via amide bond.

[0089] When m is 2 and Al and R1 are both selected in the group consisting of a protected or unprotected amino acid or a peptide group consisting of 2 to 4 protected or unprotected amino acids, one AEEA is linked on one side to Al by an amidic link, so that the N atom of the amino group of such AEEA is covalently bonded to the C atom of the keto group of Al, and on the other side to the other AEEA by an amidic link, so that the C atom of the keto group of the first AEEA is covalently bonded N atom of the amino group of the second AEEA; the second AEEA is linked to R1 by an amidic link, so that the C atom of the keto group of the second AEEA is covalently bonded to the N atom of the amino group of Rl.

[0090] In some embodiments, where m is 2, Al is protected or unprotected Glu and Rl is protected or unprotected Lys, one AEEA is linked on one side to Glu by an amidic link, so that the N atom of the amino group of this AEEA is covalently bonded to the C atom of the keto group of Glu (8C), and on the other side to the other AEEA by an amidic link, so that the C atom of the keto group of the first AEEA is covalently bonded N atom of the amino group of the second AEEA and the second AEEA is linked to Lys by an amidic link, so that the C atom of the keto group of the second AEEA is covalently bonded to the EN atom of Lys.

[0091] The invention includes all the different embodiments that may be obtained by combining the features specifying that m is an integer between 1 and 5, preferably between 1 and 4, more preferably m is 2, and the feature specifying that n is an integer between 10 and 25, between 15 and 21. These features are also freely combinable with features of each of the embodiments disclosed for the metal cation, for the halide, for Rl, for R2, for Al, and for the different ratios of the MX metal salt and the compound of Formula I previously discussed.

[0092] The term "ratio" and "equivalent ratio" refer to the number of mol equivalents of Mx metal salt used with respect to the number of mol equivalents of compound of formula I.

[0093] In some embodiments, compound of Formula I is compound of Formula 1:

[0094] or compound of Formula 3:

[0095]

[0096] In Figure 1 compound of formula 1 is reported, where Rl, R2, Al and AEEA groups are explicated as possible examples.

[0097] In some preferred embodiments, the MX metal salt is MgCL, the MX metal salt is used in the range between 0.5 and 5.0, preferably between 1.0 and 2.5, mol equivalents with respect to compound of Formula I, Rl is protected Lys or OH, R2 is a carboxylic protective group, Al is protected Glu, m is 2 and n is 16 or 18; in some more preferred embodiments, the MX metal salt is MgCL, the MX metal salt is used in the range between 0.5 and 5.0, preferably between 1.0 and 2.5, mol equivalents with respect to compound of Formula I, R1 is Fmoc-protected Lys or OH, R2 is a tert-Butyl group, Al is a tert-Butyl-protected Glu, m is 2 and n is 16 or 18, in some most preferred embodiments, the MX metal salt is MgCL, the MX metal salt is used in the range between 1.0 and 2.5 mol equivalents with respect to compound of Formula I, R1 is Fmoc-protected Lys or OH, R2 is a tert-Butyl group, Al is a tert-Butyl-protected Glu, m is 2 and n is 16 or 18.

[0098] In a specifically preferred embodiment, the MX metal salt is MgCL, the MX metal salt is used in the range between 1.0 and 2.5 mol equivalents with respect to compound of formula I, R1 is Fmoc-protected Lys or OH, R2 is a tert-Butyl group, Al is a tert-Butyl-protected Glu, m is 2 and n is 16. The embodiment wherein R1 is Fmoc-protected Lys is even more preferred.

[0099] In a further specifically preferred embodiment, the MX metal salt is MgCL, the MX metal salt is used in the range between 1.0 and 2.5 mol equivalents with respect to compound of formula I, R1 is Fmoc-protected Lys or OH, R2 is a tert-Butyl group, Al is a tert-Butyl-protected Glu, m is 2 and n is 18. The embodiment wherein R1 is Fmoc-protected Lys is even more preferred.

[0100] Sticking characteristics of compound of Formula I, and in particular of compounds of Formula 1 to Formula 4, can reasonably be attributed to the coexistence of the hydrophilic AEEA portion and the alkylated chain portion.

[0101] Provided herein is also a process for the preparation of the solid metal complex salt described above, which comprises a step of treating compound of Formula I with the MX metal salt to form the metal complex salt, wherein the compound of Formula I is previously dispersed or solubilized in a first aliquot of a liquid medium, and a step of recovering the metal complex salt in a solid form.

[0102] According to some embodiments, the step iii) of recovering the metal complex salt in a solid form comprises antisolvent precipitation or the removal of the liquid medium, preferably it comprises antisolvent precipitation.

[0103] Advantageously, antisolvent precipitation is a simple and flexible technique; in fact, by operating on the selection of solvent and antisolvent, their amounts, their ratio and the temperature, the precipitation can be easily optimized.

[0104] Further, antisolvent precipitation can be implemented industrially without expensive or complicated equipment.

[0105] The liquid medium can be selected from the group consisting of acetone, water, methanol, isopropyl alcohol, ethanol, dichloromethane, chloroform, ethyl acetate, methyl acetate, isopropyl acetate, acetonitrile, dimethyl carbonate, toluene, tetrahydrofuran and mixtures thereof.

[0106] In step (i) compound of Formula I can be dispersed or solubilized in the liquid medium; preferably it is solubilized in the liquid medium.

[0107] Advantageously, when in step (i) a liquid medium that is able to solubilize compound of Formula I is used, there is no loss of compound of Formula I due to its eventual sticking to the surface of the container or of the vessel.

[0108] Further, the kinetic of the formation of the metal complex salt is improved when compound of Formula I is solubilized in the liquid medium compared to when the compound of Formula I is dispersed; in other words, when compound of Formula I is solubilized in the liquid medium, the rate of obtaining the metal complex salt is increased with respect to when compound of Formula I is dispersed in the liquid medium. According to some embodiments, the liquid medium is selected from the group consisting of acetone, ethyl acetate, dimethyl carbonate, and a mixture of dichloromethane and tetrahydrofuran; preferably the mixture of dichloromethane and tetrahydrofuran is in the range 5:1- 15:1 vokvol, even more preferably in the range 8:1-10:1 vokvol.

[0109] According to some preferred embodiments, the liquid medium is acetone, ethyl acetate or dimethyl carbonate, according to some more preferred embodiments, the liquid medium is acetone.

[0110] Dimethyl carbonate is an established solvent and a green reagent which continues to attract attention due to the fact that, advantageously, it biodegrades readily in the atmosphere, and it is non-toxic.

[0111] In some embodiments, the solid metal complex salt is recovered by antisolvent precipitation and the antisolvent is selected from the group consisting of heptane, toluene, acetonitrile, ethyl acetate, cyclopentyl methyl ether, petroleum ether, diisopropyl ether, methyl tert-butyl ether, and mixtures thereof. Preferably, the antisolvent is selected from the group consisting of ethyl acetate, diisopropyl ether and mixtures thereof.

[0112] In a preferred embodiment of the process for the preparation of the solid metal complex salt as described above, wherein the solid metal complex salt is recovered by antisolvent precipitation, the liquid medium is acetone, ethyl acetate or dimethyl carbonate and the antisolvent is acetone, ethyl acetate or isopropyl ether, or a mixture of acetone and isopropyl ether, or a mixture of ethyl acetate and isopropyl ether.

[0113] In some embodiments the liquid medium and the antisolvent are the same chemical or the same mixture of chemicals.

[0114] In other words, the liquid medium dispersing or solubilizing compound of Formula I in step (i), is an antisolvent for the metal complex salt. This improves the global efficiency of the process in terms of time, reduces the use of different chemical products and makes the recycling of such liquid medium easier.

[0115] In some embodiments, step (ii) is obtained by the addition of the MX metal salt to the dispersion or the solution generated in step (i), wherein the MX metal salt is not previously dispersed or solubilized in any liquid medium; preferably, compound of Formula I is previously solubilized in a first aliquot of a liquid medium and the MX metal salt is directly added to this solution.

[0116] In alternative embodiments, step (ii) is obtained by the addition of the MX metal salt to the dispersion or the solution generated in step (i) or by the addition of the dispersion or the solution generated in step (i) to the MX metal salt, wherein the MX metal salt is previously dispersed or solubilized in a second aliquot of the liquid medium.

[0117] In some embodiments, step (ii) of treating compound of Formula I with the MX metal salt to form the metal complex salt is carried out in the presence of a buffering agent, preferably a basic buffering agent, more preferably selected from the group consisting of ammonium acetate, sodium ascorbate, sodium tetraborate, potassium tetraborate and K-Oxyma, or in the presence of a weak base, preferably said weak base being any tertiary amine, more preferably being triethylamine.

[0118] The term "basic buffering agent", also called alkaline buffering agent, refers to those substances which are able to maintain the basic pH in a solution.

[0119] Advantageously, treating compound of Formula I with the MX metal salt to form the metal complex salt in the presence of a basic buffering agent makes the metal complex salt more stable.

[0120] In some preferred embodiments, the basic buffer medium or the weak base is added in the dispersion or the solution of compound of Formula I in the first aliquot of the liquid medium of step (i). According to some embodiments, step iii) of recovering the metal complex salt in a solid form comprises antisolvent precipitation followed by filtration.

[0121] This allows for a high recovery rate of the solid metal complex salt.

[0122] According to some embodiments, step iii) of recovering the metal complex salt in a solid form comprises antisolvent precipitation, wherein the liquid medium and the antisolvent are both ethyl acetate.

[0123] In such embodiments, ethyl acetate acts as a solvent for compound of Formula I and as an antisolvent for the metal complex salt.

[0124] According to some alternative embodiments, step iii) of recovering the metal complex salt in a solid form comprises antisolvent precipitation, wherein the liquid medium is ethyl acetate and the antisolvent is a mixture of ethyl acetate and diisopropyl ether.

[0125] Also in such embodiments, ethyl acetate acts as a solvent for compound of Formula I and as an antisolvent for the metal complex salt but in these cases diisopropyl ether is added as additional antisolvent for the metal complex salt.

[0126] Advantageously, in these embodiments the addition of diisopropyl ether makes the precipitation of the solid metal complex salt faster and / or allows obtaining a solid precipitate of the metal complex salt in the form free-flowing powder, especially if the equivalent ratio between the MX metal salt and compound of Formula I is low. Further, with the addition of diisopropyl ether, yield is increased.

[0127] A powder is defined as a free-flowing powder if the particles do not stick together.

[0128] According to some other alternative embodiments, step iii) of recovering the metal complex salt in a solid form comprises antisolvent precipitation, wherein the liquid medium is dimethyl carbonate and the antisolvent is ethyl acetate and / or diisopropyl ether.

[0129] Also in this case, the use of diisopropyl ether in addition to ethyl acetate makes the precipitation of the solid metal complex salt faster and promotes the formation of the solid metal complex salt at low mol equivalents, i.e. when the MX metal salt is used in the range of 1.0 to 2.5 mol equivalents with respect to compound of formula I.

[0130] According to some preferred alternative embodiments, step iii) of recovering the metal complex salt in a solid form from a liquid medium comprises antisolvent precipitation, wherein the liquid medium is acetone and the antisolvent is acetone and / or diisopropyl ether.

[0131] The dispersion or the solubilization of compound of Formula I in a first aliquot of the liquid medium in step (i) is preferably carried out at room temperature, more preferably at temperature ranging from about 20 °C to 30 °C, even more preferably at a temperature ranging from about 20 °C to 25 °C.

[0132] The term "room temperature" refers to a temperature ranging from about 15 °C to 35 °C.

[0133] Preferably, also step (ii) is carried out at room temperature, more preferably at a temperature ranging from about 20 °C to 30 °C, even more preferably at a temperature ranging from about 20 °C to 25 °C.

[0134] In some embodiments, after step (ii), the temperature is lowered to a range between 0 °C and 15 °C, preferably from 0 °C to 10 °C or from 10 °C to 15 °C.

[0135] Advantageously, this accelerates the precipitation of the solid metal complex salt.

[0136] In some embodiments, when the liquid medium is not an antisolvent for the metal complex salt, its amount can be reduced before adding the antisolvent, for example by partial evaporation, in order to use a minor amount of antisolvent for the precipitation of the solid metal complex salt. In some alternative embodiments, precipitation of the solid metal complex salt is obtained by removal of the liquid medium, e.g., by evaporation, lyophilization, spray-drying, pervaporation membrane technology or equivalent technical methods.

[0137] This allows for recovering, at least in part, the liquid medium.

[0138] In some embodiments, the solid metal complex salt is washed after filtration.

[0139] Advantageously, this allows reducing impurities in the solid metal complex salt.

[0140] In some preferred embodiments, the solid metal complex salt is washed with diisopropyl ether after filtration.

[0141] In case ethyl acetate is used as liquid medium, the solid metal complex salt thus obtained is preferably washed one or more times with diisopropyl ether and / or with a mixture of acetonitrile and heptane.

[0142] The use of the mixture of acetonitrile and heptane allows removing the ethyl acetate that possibly remained in the solid metal complex salt.

[0143] In some embodiments, the solid metal complex salt is dried after washing, preferably under nitrogen.

[0144] In some preferred embodiments, the solid metal complex salt is dried after washing at a temperature ranging between 0 °C and 15 °C, preferably from 0 °C to 10 °C or from 10°C to 15 °C, preferably under nitrogen.

[0145] Provided is also the use of the of the solid metal complex salt according to the invention, in the synthesis of active pharmaceutical ingredients, more preferably the use of the solid metal complex salt of compounds of Formula 1 to Formula 4 in the synthesis of active pharmaceutical ingredients. Also, the use of the solid metal complex salt according to the invention, in the synthesis of semaglutide, tirzepatide and their pharmaceutically acceptable salts is provided. Most preferably, the use of the solid metal complex salt of compounds of Formula 1 to Formula 4 in the synthesis of semagutide, tirzepatide and their pharmaceutically acceptable salts is provided.

[0146] Further a process is provided to synthesize active pharmaceutical ingredients by the use of a solid metal complex salt according to the invention. More preferably, the invention comprises a process for making semaglutide, tirzepatide and their pharmaceutically acceptable salts characterized by the use of the solid metal complex salt according to the invention herein disclosed.

[0147] In some embodiments, the synthesis of semaglutide, tirzepatide and their pharmaceutically acceptable salts comprises the preparation of the solid metal complex salt of compound of formula I, preferably of compounds of Formula 1 - Formula 4, more preferably compounds of Formula 1 and Formula 2.

[0148] In fact, the solid metal complex salt of compound of Formula I, and so also compounds of Formula 1- Formula 4, can be used as intermediate product in the synthesis of semaglutide, tirzepatide and their pharmaceutically acceptable salts.

[0149] In particular, the solid metal complex salt of compound of Formula I can be used as such in coupling reactions in the synthesis of semaglutide, tirzepatide and their pharmaceutically acceptable salts: the aminoacidic or peptidic fragment of the solid metal complex salt of Formula I can directly be coupled with other amino acid or peptide fragments, without the need to have a decomplexation step in advance.

[0150] As nonlimiting example, the solid metal complex salt of compound of Formula 1 can be used as such in the coupling reaction with fragment 21-31 of semaglutide to generate fragment 20-31 of semaglutide, as well as the solid metal complex salt of compound of 2, can be directly used in the coupling reaction with fragment 21-29 of tirzepatide to generate fragment 20-29. Similarly, the solid metal complex salt of Formula 3 and Formula 4 can be activated and coupled with other amino acid or peptide fragments to generate fragments of semaglutide and tirzepatide, respectively.

[0151] EXPERIMENTAL

[0152] Example 1 - Synthesis and isolation of iron (III) solid complex (solvent: DCM / THF)

[0153] 1 g of compound of Formula 1 was solubilized at room temperature in 3.6 mL of a first solvent mixture of DCM / THF 9:1 by vol so as to have a solution of compound of Formula 1 in DCM / THF with a concentration of 280 g of compound of Formula 1 per litre of the second solvent mixture.

[0154] 0.54 g of anhydrous FeCL (3.0 mole equivalents with respect to compound of Formula 1) were solubilized at room temperature in 21.7 mL of a second solvent mixture of DCM / THF 9:1 by vol and kept under stirring for 30 min to obtain a solution of FeCL in DCM / THF.

[0155] Then, the solution of FeCL in DCM / THF and the solution of compound of Formula 1 in DCM / THF were mixed at room temperature and kept under stirring for 30 min.

[0156] Finally, 10 vol petroleum ether was used as antisolvent and added at 25±5°C °C to the solution of FeCL and compound of Formula 1 in DCM / THF to obtain the precipitation of the iron(lll) complex salt. The volume ratio of petroleum ether used as antisolvent with respect to the total amount of DCM / THF used to solubilize compound of Formula 1 and FeCL was 6:1.

[0157] The iron(lll) complex salt was then isolated by centrifugation, removal of the liquid fraction and drying; a brown solid powder was obtained; the powder was easy to be handled since the sticking behaviour was negligible.

[0158] Example 2 - Synthesis and isolation of copper (II) solid complex (solvent: DCM / THF)

[0159] 1 g of compound of Formula 1 was solubilized at room temperature in 3.6 mL of a first solvent mixture of DCM / THF 9:1 by vol so as to have a solution of compound of Formula 1 in DCM / THF with a concentration of 280 g of compound of Formula 1 per liter of the second solvent mixture.

[0160] 0.45 g of anhydrous CuCL (2.0 mole equivalents with respect to compound of Formula 1) were solubilized at room temperature in 21.7 mL of a second solvent mixture of DCM / THF 9:1 by vol and kept under stirring for 30 min to obtain a solution of CuCL in DCM / THF.

[0161] Then, the solution of CuCL in DCM / THF and the solution of compound of Formula 1 in DCM / THF were mixed at room temperature and kept under stirring for 30 min.

[0162] Finally, petroleum ether was used as antisolvent and added at 25±5 °C to the solution of CuCL and compound of Formula 1 in DCM / THF to obtain the precipitation of the copper(ll) complex salt. The volume ratio of petroleum ether used as antisolvent with respect to the total amount of DCM / THF used to solubilize compound of Formula 1 and CuCL was 6:1.

[0163] The copper(ll) complex salt thus obtained was isolated by filtration; a green solid hygroscopic powder was obtained; the powder was easily transferred and weighted without sticking to the container.

[0164] The copper(ll) complex salt was analyzed by UHPLC equipped with quaternary pump, UV detector and ESI-Q- TOF mass using as mobile phase A water +0.1% of TFA and acetonitrile as mobile phase B; the analysis proved that a slight deprotection of the tert-butoxy-protected peptide occurred during the synthesis and / or the precipitation of the magnesium(ll) complex salt. In fact, from the HPLC-MS profile of the solid metal complex salt, reported in Figure 2, about 1.5% of the tert-Butyl protecting group resulted to be removed from the peptide during the synthesis and / or the precipitation of the magnesium(ll) complex salt. In this Figure, the peaks at ca. 49 and 55 min can be attributed respectively to the compound of Formula 1 without either of the two tert-Butyl protecting groups and to the compound of Formula 1 without one of the two tert-Butyl protecting groups, while the peak at 64 min can be attributed to compound of Formula 1.

[0165] Example 3 - Synthesis and isolation of cobalt (II) solid complex (solvent: DCM / THF)

[0166] 0.33 g of anhydrous C0CI2 were solubilized at room temperature in 21.7 mL of a solvent mixture of DCM / THF 9:1 by vol and kept under stirring for 30 min to obtain a solution of C0CI2 in DCM / THF.

[0167] 1 g of compound of Formula 1 was solubilized at room temperature in 3.6 mL of a solvent mixture of DCM / THF 9:1 by vol so as to have a solution of compound of Formula 1 in DCM / THF with a concentration of 280 g of compound of Formula 1 per Liter of the solvent mixture.

[0168] Then, the solution of C0CI2 in DCM / THF and the solution of compound of Formula 1 in DCM / THF were mixed at room temperature.

[0169] Finally, MTBE was used as antisolvent and added at 10±5 °C to the solution of C0CI2 and compound of Formula 1 in DCM / THF to obtain the precipitation of the cobalt(ll) complex salt. The volume ratio of MTBE used as antisolvent with respect to the total amount of DCM / THF used to solubilize compound of Formula 1 and C0CI2 was 6:1.

[0170] The cobalt(ll) complex salt thus obtained was isolated by filtration; a blue solid hygroscopic powder was obtained; the powder was easily transferred and weighted without sticking to the container.

[0171] Also DIPE and petroleum ether were successfully used instead of MTBE as non-solvent to obtain the precipitation of the cobalt(ll) complex. Also in these cases, the volume ratio of DIPE or petroleum ether used as antisolvent with respect to the total amount of DCM / THF used to solubilize compound of Formula 1 and C0CI2 was 0.3:1 by weight.

[0172] Also in these cases, the cobalt(ll) complex salt obtained was a solid non-sticking powder.

[0173] Example 4 - Synthesis and isolation of magnesium (II) solid complex (solvent: DCM / THF)

[0174] 0.32 g of anhydrous MgCL were solubilized at room temperature in 21.7 mL of a solvent mixture of DCM / THF 9:1 by vol and kept under stirring for 30 min to obtain a solution of MgCL in DCM / THF.

[0175] 1 g of compound of Formula 1 was solubilized at room temperature in 3.6 mL of a solvent mixture of DCM / THF 9:1 by vol so as to have a solution of compound of Formula 1 in DCM / THF with a concentration of 280 g of compound of Formula 1 per Liter of the solvent mixture.

[0176] Then, the solution of MgCL in DCM / THF and the solution of compound of Formula 1 in DCM / THF were mixed at room temperature so as to have a weight ratio between MgCL and compound of Formula 1 of 0.3:1 by weight.

[0177] Finally, MTBE was used as antisolvent and added at 10±5 °C to the solution of MgCL and compound of Formula 1 in DCM / THF to obtain the precipitation of the magnesium(ll) complex salt. The volume ratio of MTBE used as antisolvent with respect to the total amount of DCM / THF used to solubilize compound of Formula 1 and MgCL was 0.3:1 by weight.

[0178] The magnesium(ll) complex salt thus obtained was isolated by filtration; it was obtained in the form of a green solid hygroscopic powder which can be handled, transferred and weighted very easily with no loosing of the material on the walls of the container.

[0179] The magnesium(ll) complex salt was analysed by UHPLC equipped with quaternary pump, UV detector and ESI-Q-TOF mass using as mobile phase A water +0.1% of TFA and acetonitrile as mobile phase B; the analysis proved that no deprotection of the tert-Butil protected peptide occurred during the synthesis and the precipitation of the magnesium(ll) complex salt. In fact, from the LC-MS profile of the solid metal complex salt, reported in Figure 3, only the peak at 64 min, attributable to compound of Formula 1, was visible.

[0180] Example 5 - Synthesis of semaglutide fragment 20-31 - Attachment of the lateral chain

[0181] 1.3 g of the magnesium(ll) complex of example 4 were used in coupling reaction with fragment 21-31 of semaglutide according to SPPS approach to obtain fragment 20-31 of semaglutide.

[0182] The solid metal complex salt in the form of powder was first solubilized in organic solvent (DMF, DCM) and then activated by adding diisopropyl carbodiimide 130 pL and 118 mg of OxymaPure® in order to generate the coupling mixture.

[0183] The coupling mixture was then added to swelled L-Glu(PG)-L-Phe-L-lle-L-Ala-L-Trp(PG)-L-Leu-L-Val-L-Arg(PG)- Gly-L-Arg(Pbf)-Gly-MBH resin (fragment 21-31, protected and attached to MBH resin) corresponding to 2 gr of starting resin with loading 0.4 mmol / gram; the reaction take place at 40°C with a reaction time of 8 hours.

[0184] PG is used in the present description to indicate a protective group. In Example 5, PG of Glu and Trp is tBU, PG of Arg is Pbf; thus, L-Glu(PG)-L-Phe-L-lle-L-Ala-L-Trp(PG)-L-Leu-L-Val-L-Arg(PG)-Gly-L-Arg(Pbf)-Gly-MBH resin is L-Glu(tBU)-L-Phe-L-lle-L-Ala-L-Trp(tBU)-L-Leu-L-Val-L-Arg(Pbf)-Gly-L-Arg(Pbf)-Gly-MBH resin.

[0185] Then, the mixture was filtered, and the resin washed with fresh organic solvent. Coupling efficiency was checked by colorimetric test (Kaiser test, TNBS test, Chloranil test) and no unreactive site were observed.

[0186] Efficiency of coupling was also checked by cleavage in the mixture of TFA / DTT / TIS / water (87.5 / 5 / 5 / 2.5 v / w / v / v) followed by HPLC analysis, as visible by Figure 4, where the HPLC profile of fragment 20-31 of semaglutide is reported. In particular, the top profile refers to uprotected fragment 20-31 obtained by coupling the lateral chain in its free form, i.e., without being complexed, with fragment 21-31, while the bottom one refers to uprotected fragment 20-31 obtained by coupling the lateral chain previously complexed by reaction with the MgCL metal salt with fragment 21-31.

[0187] As clearly visible, also in the case of the use of the complexed lateral chain, the coupling with fragment 21- 31 is successfully achieved.

[0188] Example 6 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0189] 15 g of compound of Formula 1 were solubilized at room temperature in in 75 mL of ethyl acetate to obtain a solution of compound of Formula 1 in ethyl acetate.

[0190] 1.8 g of anhydrous MgCL were added at room temperature to 75 mL of ethyl acetate and stirred for 30 minutes at 20-30 °C; then, the solution of compound of Formula 1 in ethyl acetate was added and stirred for 10 minutes at 20-30 °C, then was cooled to 0-10 °C to precipitate the magnesium(ll) complex salt.

[0191] The solid magnesium(ll) complex salt was isolated by filtration and washed two times with 30 mL of ethyl acetate, then dried first under nitrogen, then under vacuum.

[0192] Example 7 - Synthesis and isolation of magnesium (II) solid complex (solvent: acetonitrile)

[0193] 1.0 g of compound of Formula 1 was solubilized at room temperature in 32 mL of acetonitrile by stirring until obtaining a clear solution, then 320 mg of MgCL (4.0 mole equivalents with respect to compound of Formula 1) were added to the solution and the solid metal complex salt immediately started precipitating.

[0194] The mixture was stirred for 120 minutes at 20-30 °C, then cooled down to 0-10 °C and stirred for additional 120 minutes.

[0195] The solid metal complex salt was isolated by filtration, washed with 10 mL of diisopropyl ether and dried under nitrogen at 0-10 °C for 60 minutes. Yield: 1.25 g of the solid metal complex salt.

[0196] Example 8 - Synthesis and isolation of magnesium (II) solid complex (solvent: acetonitrile)

[0197] 1.0 g of compound of Formula 1 was solubilized in 32.5 mL of acetonitrile by stirring until obtaining a clear solution; then 240 mg of MgCL (3.0 mole equivalents with respect to compound of Formula 1) were added to the solution and the metal complex salt started forming.

[0198] The mixture was stirred for 30 minutes at 25 °C, then cooled down to 0-10 °C and stirred for additional 120 minutes.

[0199] 125 mL of diisopropyl ether were added to the mixture and stirred for 120 minutes at 0-10 °C.

[0200] Finally, the solid metal complex salt was isolated by filtration, washed with 5 mL of diisopropyl ether and dried under nitrogen at 0-10 °C for 120 minutes.

[0201] Yield: 1.20 g of the solid metal complex salt.

[0202] Example 9 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0203] 20 g of compound of Formula 1 were solubilized in 100 mL of ethyl acetate by stirring until obtaining a clear solution.

[0204] 2.40 g of MgCL (1.5 mole equivalents with respect to compound of Formula 1) were added to 100 mL of ethyl acetate and stirred for 30 minutes at 20-30 °C; then, the solution of compound of Formula 1 in ethyl acetate was added and solid metal complex salt immediately started precipitating; stirring was continued for 10 minutes at 20-30 °C; then the mixture was cooled down to 0-10 °C and stirred for additional 180 minutes.

[0205] Finally, the solid metal complex salt was isolated by filtration, washed with 3 aliquots of ethyl acetate (3X20 mL) and dried under nitrogen for 30 minutes and then under vacuum for 5 hours, always at a temperature comprised between 0 °C and 10 °C.

[0206] Yield: 21.00 g of the solid metal complex salt.

[0207] Example 10 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0208] 1.0 g of compound of Formula 1 was solubilized in 5 mL of ethyl acetate by stirring until obtaining a clear solution.

[0209] 320 mg of MgCL (4.0 mole equivalents with respect to compound of Formula 1) were added to the solution and the solid metal complex salt immediately started precipitating.

[0210] The mixture was stirred for 120 minutes at 20-30 °C and then cooled down to 0-10 °C and left under stirring for additional 120 minutes.

[0211] Finally, the solid metal complex salt was isolated by filtration, washed with 10 mL of ethyl acetate, and dried under nitrogen for 60 minutes and under vacuum overnight, always at a temperature comprised between 0 °C and 10 °C.

[0212] Yield: 1.30 g of the solid metal complex salt.

[0213] Example 11 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0214] 1.0 g of compound of Formula 1 was solubilized in 5 mL of ethyl acetate under stirring until obtaining a clear solution. 80 mg of MgCL (1.0 mole equivalents with respect to compound of Formula 1) were added to the solution and the metal complex salt started forming.

[0215] The mixture was stirred for 120 minutes at 20-30 °C.

[0216] Then 15 mL of diisopropyl ether were added, let under stirring overnight at 20-30 °C, cooled down to 0-10 °C and maintained under stirring for additional 120 minutes.

[0217] Finally, the solid metal complex salt was isolated by filtration, washed with 10 mL of ethyl acetate, and dried under vacuum overnight, always at a temperature comprised between 0 °C and 5 °C.

[0218] Yield: 1.00 g of the solid metal complex salt.

[0219] Example 12 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0220] 1.0 g of compound of Formula 1 was solubilized in 5 mL of ethyl acetate by stirring until obtaining a clear solution.

[0221] 88 mg of MgCL (1.1 mole equivalents with respect to compound of Formula 1) were added to 5 mL of ethyl acetate; then, the solution of compound of Formula 1 in ethyl acetate was added in 2 aliquots as follows: first 25 %vol was added at 25 °C and maintained under stirring for 30 minutes at 25 °C, then the remaining 75 %vol was added at 25 °C and maintained under stirring for additional 30 minutes at 25 °C. During the addition of the solution of compound of Formula 1 in ethyl acetate, the metal complex salt started forming.

[0222] Then 25 mL of diisopropyl ether were added, the mixture was cooled down to 0-10 °C and maintained under stirring for 120 minutes.

[0223] Finally, the solid metal complex salt was isolated by filtration, washed with 10 mL of diisopropyl ether, and dried under nitrogen for 4 hours, always at a temperature comprised between 0 °C and 10 °C.

[0224] Yield: 1.00 g of the solid metal complex salt.

[0225] Example 13 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0226] 1.0 g of compound of Formula 1 was solubilized in 5 mL of ethyl acetate by stirring until obtaining a clear solution.

[0227] 100 mg of MgCL (1.2 mole equivalents with respect to compound of Formula 1) were added to the solution and the metal complex salt started forming.

[0228] The mixture was stirred for 120 minutes at 20-30 °C.

[0229] Then 15 mL of diisopropyl ether were added, the mixture was cooled down to 0-10 °C and maintained under stirring for 120 minutes.

[0230] Finally, the solid metal complex salt was isolated by filtration, washed with 10 mL of diisopropyl ether, and dried under nitrogen for 60 minutes and under vacuum overnight, always at a temperature comprised between 0 °C and 5 °C.

[0231] Yield: 1.05 g of the solid metal complex salt.

[0232] Example 14 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0233] 1.0 g of compound of Formula 1 was solubilized in 5 mL of ethyl acetate by stirring until obtaining a clear solution. and the solid metal complex salt immediately started precipitating.

[0234] The mixture was maintained under stirring for 120 minutes at 20-30 °C; then 15 mL of diisopropyl ether were added and the mixture was cooled down to 0-10 °C and maintained under stirring for further 120 minutes.

[0235] Finally, the solid metal complex salt was isolated by filtration, washed with 10 mL of diisopropyl ether, and dried first under nitrogen for 1 hour and then under vacuum overnight, always at a temperature comprised between 0 °C and 5 °C.

[0236] Yield: 1.10 g of the solid metal complex salt.

[0237] Example 15 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0238] 1.0 g of compound of Formula 1 was solubilized in 5 mL of ethyl acetate by stirring until obtaining a clear solution.

[0239] 160 mg of MgCL (2.0 mole equivalents with respect to compound of Formula 1) were added to the solution and the solid metal complex salt immediately started precipitating.

[0240] The mixture was maintained under stirring for 120 minutes at 20-30 °C, then cooled down to 0-10 °C and maintained under stirring for further 120 minutes.

[0241] Finally, the solid metal complex salt was isolated by filtration, washed with 10 mL of diisopropyl ether, and dried under nitrogen for 60 minutes at a temperature comprised between 0 °C and 5 °C.

[0242] Yield: 1.10 g of the solid metal complex salt.

[0243] Example 16 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0244] 1.0 g of compound of Formula 1 was solubilized in 5 mL of ethyl acetate by stirring until obtaining a clear solution.

[0245] 240 mg of MgCL (3.0 mole equivalents with respect to compound of Formula 1) were added to the solution and the solid metal complex salt immediately started precipitating.

[0246] The mixture was maintained under stirring for 120 minutes at 20-30 °C, then cooled down to 0-10 °C and maintained under stirring for further 120 minutes.

[0247] Finally, the solid metal complex salt was isolated by filtration, washed with 10 mL of diisopropyl ether, and dried under nitrogen for 1 hour at a temperature comprised between 0 °C and 10 °C.

[0248] Yield: 1.15 g of the solid metal complex salt.

[0249] Example 17 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0250] 1.0 g of compound of Formula 1 was solubilized in 5 mL of ethyl acetate by stirring until obtaining a clear solution.

[0251] 320 mg of MgCL (4.0 mole equivalents with respect to compound of Formula 1) were added and the solid metal complex salt immediately started precipitating.

[0252] The mixture was maintained under stirring for 120 minutes at 20-30 °C. then 15 mL of diisopropyl ether were added, the mixture was cooled down to 0-10 °C and maintained under stirring for further 120 minutes. Finally, the solid metal complex salt was isolated by filtration, washed with 10 mL of diisopropyl ether, dried first under nitrogen for 60 minutes and then under vacuum overnight, always at a temperature comprised between 0 °C and 10 °C.

[0253] Yield: 1.30 g of the solid metal complex salt.

[0254] Example 18 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0255] 5.0 g of compound of Formula 1 were solubilized in 50 mL of ethyl acetate by stirring until obtaining a clear solution.

[0256] 440 mg of MgCh (1.1 mole equivalents with respect to compound of Formula 1) were added to 10 mL of ethyl acetate; then, the solution of compound of Formula 1 in ethyl acetate was added in 3 aliquots as follows: first 25 %vol was added at 25 °C and maintained under stirring for 10 minutes, then other 25 %vol was added at 25 °C and maintained under stirring for 10 minutes and finally the remaining 50 %vol was added at 25 °C and maintained under stirring for additional 30 minutes. During the addition of the solution of compound of Formula 1 in ethyl acetate complex the metal salt started forming.

[0257] Then 75 mL of diisopropyl ether were added in 1 hour and the mixture was maintained under stirring for 180 minutes at 25 °C.

[0258] Finally, the solid metal complex salt was isolated by filtration, washed with 2 aliquots of diisopropyl ether (2X10 mL) and dried under nitrogen for 2 hours.

[0259] 4.75 g of the solid metal complex salt were obtained and stored at -20 °C.

[0260] Example 19 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0261] 5.0 g of compound of Formula 1 were solubilized in 50 mL of ethyl acetate by stirring until obtaining a clear solution.

[0262] 480 mg of MgCL (1.2 mole equivalents with respect to compound of Formula 1) were added to 10 mL of ethyl acetate; then, the solution of compound of Formula 1 in ethyl acetate was added in 3 aliquots as follows: first 25 %vol was added at 25 °C and maintained under stirring for 10 minutes, then other 25 %vol was added at 25 °C and maintained under stirring for 10 minutes and finally the remaining 50 %vol was added at 25 °C and maintained under stirring for additional 30 minutes. During the addition of the solution of compound of Formula 1 in ethyl acetate the metal complex salt started forming.

[0263] Then 75 mL of diisopropyl ether were added in 1 hour and the mixture was maintained under stirring for 180 minutes at 25 °C.

[0264] Finally, the solid metal complex salt was isolated by filtration, washed with 2 aliquots of diisopropyl ether (2X10 mL) and dried under nitrogen for 2 hours.

[0265] 5.10 g of the solid metal complex salt were obtained and stored at -20 °C.

[0266] Example 20 - Synthesis and isolation of magnesium (II) solid complex (solvent: dimethyl carbonate)

[0267] 5.0 g of compound of Formula 1 were solubilized in 25 mL of dimethyl carbonate by stirring until obtaining a clear solution.

[0268] 440 mg of MgCL (1.0 mole equivalents with respect to compound of Formula 1) were added to 25 mL of dimethyl carbonate; then, the solution of compound of Formula 1 in ethyl acetate was added in 3 aliquots as follows: first 25 %vol was added at 25 °C and maintained under stirring for 10 minutes, then other 25 %vol was added at 25 °C and maintained under stirring for 10 minutes and finally the remaining 50 %vol was added at 25 °C and maintained under stirring for additional 30 minutes. During the addition of the solution of compound of Formula 1 in dimethyl carbonate the metal complex salt started forming.

[0269] Then 150 mL of diisopropyl ether were added in 1,5 hour and the mixture was maintained under stirring for 180 minutes at 25 °C.

[0270] Finally, the solid metal complex salt was isolated by filtration, washed with 2 aliquots of diisopropyl ether (2X10 mL) and dried under nitrogen for 2 hours.

[0271] Example 21 - Synthesis of compound of Formula 2 - tirzepatide lateral chain

[0272] A solution of tBuOCO-(CH2)i8-CO-Glu(AEEA-AEEA)-OtBu)-OH (3.0 g, 3.43 mmol), OxymaPure® (0.73 g, 5.15 mmol) and DIC (0.531 mL, 3.43 mmol) in DCM was stirred for 2 hours. Then 0.100 mL of AcOH was added followed by Fmoc-Lys-OH (2.48 g, 5.15 mmol). The pH was adjusted to 8 with DIPEA and the reaction mixture was stirred overnight. After the completion of the reaction (HPLC control), the organic solvent was evaporated. The suspension was diluted with 50 mL of 10% NaHSC and the product was extracted twice with 30 mL of ethyl acetate. The organic phase was washed twice with 100 mL of 10% NaHSC and 100 mL of water, dried over MgSC , and evaporated. The product was purified by flash chromatography by eluting with the mixtures CHCL / methanol of 20:1, 15:1, 10:1, and 7:1 (v / v) to give 2.31 g of Fmoc-Lys(tBuOCO- (CH2)i8-CO-Glu(AEEA-AEEA)-OtBu)-OH (yield 55% and HPLC purity >95%).

[0273] Example 22 - Synthesis and isolation of magnesium (II) solid complex (solvent: ethyl acetate)

[0274] A suspension of MgCl2(0.019 g, 0.205 mmol) in 1.1 mL of ethyl acetate was stirred at room temperature and a solution of Fmoc-Lys(tBuOCO-(CH2)i8-CO-Glu(AEEA-AEEA)-OtBu)-OH (0.23 g, 0.186 mmol) in 1.1 mL of ethyl acetate was added in three equal portion every 30 minutes. Then, 3.3 mL of diisopropyl ether was added, and the reaction mixture was stirred for 4 hours. The precipitate was separated by centrifugation, washed twice with 1 mL of diisopropyl ether and dried in vacuum overnight (yield 0.24 g, purity 95%).

[0275] Example 23 - Synthesis of tirzepatide fragment 21-29

[0276] Synthesis of peptide fragment 21-29 was carried out by stepwise SPPS using 2-chlorotrityl chloride resin (250 mg, medium loading 1.6 mmol / g). After swelling of the resin in 5 mL of DCM, Fmoc-Gly-OH and DIPEA (0.8 and 3 eq, respectively, referred to the loading of the resin) in DCM were added. The reaction mixture was stirred for 1 hour and the unreacted sites of the resin were capped using 5 mL of the mixture DCM / DIPEA / MeOH (v / v / v 17 / 2 / 1) for 30 min. Then, the resin was treated with 5 mL of the solution DCM / DIPEA / Ac2O and washed with DMF (3 x 3 mL). The loading of the resin was checked by UV adsorption measurement of the solution after Fmoc deprotection and was found to be 1.24 mmol / g.

[0277] Then, Fmoc-Ala-OH, Fmoc-lle-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Val-OH, Fmoc- Phe-OH and Fmoc-Ala-OH (3 equiv respect to the loading of the resin) were preactivated by DIC (3 equiv) and OxymaPure® (3 equiv) in 5 minutes at room temperature and coupled consecutively to the resin in 60 minutes. In the case of Gin, Vai and Phe a double coupling was done. The intermediate Fmoc deprotection was carried out using 20% solution of piperidine in DMF (2 x 2.5 mL, 5 and 20 min respectively) by washing of the resin with DMF (5 x 3 mL).

[0278] Example 24 - Synthesis of tirzepatide fragment 20-29 - Attachment of the lateral chain

[0279] The introduction of the lateral chain into the peptide sequence was carried out using 1.5 equiv of Fmoc- Lys(tBuOCO-(CH2)i8-CO-Glu(AEEA-AEEA)-OtBu)-OH (in free state or complexed as described in Example 22), which was preactivated with 1.5 equiv of DIC and 1.5 equiv of OxymaPure®, and coupled in 6 hours at 40°C. At the end of the synthesis Fmoc protection was removed using 20% solution of piperidine in DMF (2 x 2.5 mL, 5 and 20 min, respectively). Then the resin was washed with DMF (3 x 3 mL) and DCM (2 x 3 mL) and dried. The efficiency of the coupling was checked by cleavage in the mixture of TFA / TIS / water (95 / 2.5 / 2.5 v / v / v). After cleavage, unprotected fragment 20-29 was obtained.

[0280] In Figure 5, the HPLC profile of unprotected fragment 20-29 of tirzepatide is reported (C18 Pore-shell column (4.6x100 mm). Eluent A, TFA / H2O 0.1% v / v; eluent B: TFA / MeCN 0.1% v / v; detection at 220 nm; gradient elution: 0 min - 5% Eluent B; 3 min - 5% Eluent B; 33 min - 95% Eluent B; 38 min - 95% Eluent B; 40 min - 5% Eluent B; 50 min - 5% Eluent B).

[0281] In particular, the top profile refers to fragment 20-29 obtained by coupling the lateral chain in its free form, i.e., without being complexed, with fragment 21-29, while the bottom one refers to unprotected fragment 20-29 obtained by coupling the lateral chain previously complexed by reaction with the MgCL metal salt with fragment 21-29.

[0282] As clearly visible, also in the case of the use of the complexed lateral chain, the coupling with fragment 20- 29 is successfully achieved.

[0283] Example 25 - Synthesis and isolation of magnesium (II) solid complex (solvent: acetone)

[0284] 20.0 g (16.71 mmol) of compound of Formula 1 were dissolved in 120 ml of acetone by stirring at 20-25 °C until a clear solution was obtained.

[0285] 1.67 mg (17.55 mmol) of anhydrous MgCL (1.05 mole equivalents with respect to compound of Formula 1) were added to the solution of compound of Formula 1 in acetone and maintained under stirring for 60 minutes at 20-25 °C under nitrogen atmosphere. After the addition of MgCL to the solution of compound of Formula 1 in acetone, metal complex salt in the form of a gel started forming.

[0286] Then 360 ml of diisopropyl ether were added at 20-25 °C. The mixture was cooled to 10-15 °C and maintained under stirring for 3 - 4 hours at 10-15 °C under nitrogen atmosphere.

[0287] Finally, the solid metal complex salt was isolated by filtration, washed with diisopropyl ether (40 ml) and dried for 15 hours at 15-20 °C.

[0288] 19.6 g of the solid metal complex salt were obtained and stored at -20 °C.

[0289] Example 26 - Synthesis and isolation of magnesium (II) solid complex (solvent: acetone, buffering agent: ammonium acetate)

[0290] 5.0 g (4.18 mmol) of compound of Formula 1 were solubilized in 30 ml of acetone by stirring at 20-25 °C until a clear solution was obtained.

[0291] 100 mg (1.29 mmol) of ammonium acetate ammonium acetate (0.3 mole equivalents with respect to compound of Formula 1) were added to the solution of compound of Formula 1 in acetone and maintained under stirring for 20 minutes at 20-25 °C under nitrogen atmosphere. 440 mg (4.60 mmol) of anhydrous MgCL (1.10 mole equivalents with respect to compound of Formula 1) were added and maintained under stirring for 75 minutes at 20-25 °C under nitrogen atmosphere. After the addition of MgCL to the solution of compound of Formula 1 in acetone metal complex salt in the form of a gel started forming.

[0292] Then 90 ml of diisopropyl ether were added at 20-25 °C. The mixture was cooled to 10-15 °C, maintained under stirring for 2.5 hours at 10-15 °C under nitrogen atmosphere, and dried for 12 hours at 10-15 °C.

[0293] 5.5 g of the solid metal complex salt were obtained and stored at -20 °C.

[0294] Example 27 - Synthesis and isolation of magnesium (II) solid complex (solvent: acetone, buffering agent: sodium ascorbate) 1.0 g (0.836 mmol) of compound of Formula 1 were solubilized in 6 ml of acetone by stirring at 20-25 °C until obtaining a clear solution.

[0295] 80 mg (0.418 mmol) of sodium ascorbate (0.5 mole equivalents with respect to compound of Formula 1) were added to the solution of compound of Formula 1 in acetone under nitrogen atmosphere and maintained under stirring for 20 minutes at 20-25 °C under nitrogen atmosphere. 80 mg of anhydrous MgCL (1.05 mole equivalents with respect to compound of Formula 1) were added and maintained under stirring for 75 minutes at 20-25 °C under nitrogen atmosphere. After the addition of MgCL to the solution of compound of Formula 1 in acetone metal complex salt in the form of a gel started forming.

[0296] Then 18 ml of diisopropyl ether were added at 20-25 °C. The mixture was cooled to 10-15 °C and maintained under stirring for 3 - 4 hrs at 10-15 °C under nitrogen atmosphere.

[0297] Finally, the solid metal complex salt was isolated by filtration, washed with diisopropyl ether (2 ml) and dried for 15 hours at 15-20 °C.

[0298] 1.0 g of the solid metal complex salt was obtained and stored at -20 °C.

[0299] Example 28 - Synthesis and isolation of magnesium (II) solid complex (solvent: acetone, weak base: triethylamine)

[0300] 2.0 g (1.67 mmol) of compound of Formula 1 were solubilized in 12 ml of acetone by stirring at 20-25 °C until a clear solution was obtained.

[0301] 170 mg (1.67 mmol) of triethylamine (1.0 mole equivalents with respect to compound of Formula 1) were added to the solution of compound of Formula 1 in acetone and maintained under stirring for 10 minutes at 20-25 °C under nitrogen atmosphere. 167 mg (1.755 mmol) of anhydrous MgCL (1.05 mole equivalents with respect to compound of Formula 1) were added and maintained under stirring for 60 minutes at 20-25 °C under nitrogen atmosphere. After the addition of MgCL to the solution of compound of Formula 1 in acetone metal complex salt in the form of a gel started forming.

[0302] Then 36 ml of diisopropyl ether were added at 20-25 °C. The mixture was cooled to 10-15 °C and maintained under stirring for 3 - 4 hours at 10-15 °C under nitrogen atmosphere.

[0303] Finally, the solid metal complex salt was isolated by filtration, washed with diisopropyl ether (4 ml) and dried for 15 hours at 15-20 °C.

[0304] 2.1 g of the solid metal complex salt were obtained and stored at -20 °C.

[0305] Example 29 - Synthesis and isolation of magnesium (II) solid complex (solvent: acetone, weak base: triethylamine)

[0306] 2.0 g (1.67 mmol) of compound of Formula 1 were solubilized in 12 ml of acetone by stirring at 20-25 °C until a clear solution was obtained.

[0307] 190 mg (1.839 mmol) of triethylamine (1.1 mole equivalents with respect to compound of Formula 1) were added to the solution of compound of Formula 1 in acetone and maintained under stirring for 10 minutes at 20-25 °C under nitrogen atmosphere. 167 mg (1.755 mmol) of anhydrous MgCL (1.05 mole equivalents with respect to compound of Formula 1) were added and maintained under stirring for 60 minutes at 20-25 °C under nitrogen atmosphere. After the addition of MgCL to the solution of compound of Formula 1 in acetone metal complex salt in the form of a gel started forming.

[0308] Then 36 ml of diisopropyl ether were added at 20-25 °C. The mixture was cooled to 10-15 °C and maintained under stirring for 3 - 4 hours at 10-15 °C under nitrogen atmosphere. Finally, the solid metal complex salt was isolated by filtration, washed with diisopropyl ether (4 ml) and dried for 15 hours at 15-20 °C.

[0309] 2.1 g of the solid metal complex salt were obtained and stored at -20 °C.

[0310] Example 30 - Synthesis and isolation of magnesium (II) solid complex (solvent: acetone, weak base: triethylamine)

[0311] 5.2 g (4.35 mmol) of compound of Formula 1 were solubilized in 31.2 ml of acetone by stirring at 20-25 °C until obtaining a clear solution.

[0312] 90 mg (0.869 mmol) of triethylamine (0.2 mole equivalents with respect to compound of Formula 1) were added to the solution of compound of Formula 1 in acetone and maintained under stirring for 10 minutes at 20-25 °C under nitrogen atmosphere. 430 mg of anhydrous MgCL (1.05 mole equivalents with respect to compound of Formula 1) were added and maintained under stirring for 60 minutes at 20-25 °C under nitrogen atmosphere. After the addition of MgCL to the solution of compound of Formula 1 in acetone metal complex salt in the form of a gel started forming.

[0313] Then 93.5 ml of diisopropyl ether were added at 20-25 °C. The mixture was cooled down to 10-15 °C and maintained under stirring for 3 - 4 hrs at 10-15 °C under nitrogen atmosphere.

[0314] Finally, the solid metal complex salt was isolated by filtration, washed with diisopropyl ether (10 ml) and dried for 15 hours at 15-20 °C.

[0315] 5.2 g of the solid metal complex salt were obtained and stored at -20 °C.

[0316] Example 31 - Synthesis and isolation of magnesium (II) solid complex (solvent: acetone, buffering agent: K- Oxyma)

[0317] 5.0 g (4.18 mmol) of compound of Formula 1 were solubilized in 30 ml of acetone by stirring at 20-25 °C until obtaining a clear solution.

[0318] 791 mg (4.39 mmol) of K-Oxyma (1.05 mole equivalents with respect to compound of Formula 1) were added to the solution of compound of Formula 1 in acetone and maintained under stirring for 20 minutes at 20-25 °C under nitrogen atmosphere. 420 mg (4.39 mmol) of anhydrous MgCL (1.05 mole equivalents with respect to compound of Formula 1) were added and maintained under stirring for 75 minutes at 20-25 °C under nitrogen atmosphere. After the addition of MgCL to the solution of compound of Formula 1 in acetone metal complex salt in the form of a gel started forming.

[0319] Then 90 ml of diisopropyl ether were added at 20-25 °C. The mixture was cooled to 10-15 °C and maintained under stirring for 2.5 hrs at 10-15 °C under nitrogen atmosphere, and dried for 12 hours at 10-15 °C under vacuum (<10 Mbar)

[0320] 5.6 g of the metal complex salt were obtained and stored at -20 °C.

[0321] Example 32 - Synthesis and isolation of magnesium (II) solid complex (solvent: acetone, buffering agent: sodium tetraborate)

[0322] 5.0 g (4.18 mmol) of compound of Formula 1 were solubilized in 30 ml of acetone by stirring at 20-25 °C until obtaining a clear solution.

[0323] 883 mg (4.39 mmol) of sodium tetraborate (1.05 mole equivalents with respect to compound of Formula 1) were added to the solution of compound of Formula 1 in acetone and maintained under stirring for 20 minutes at 20-25 °C under nitrogen atmosphere. 420 mg (4.39 mmol) of anhydrous MgCL (1.05 mole equivalents with respect to compound of Formula 1) were added and maintained under stirring for 75 minutes at 20-25 °C under nitrogen atmosphere. After the addition of MgCL to the solution of compound of Formula 1 in acetone metal complex salt in the form of a gel started forming.

[0324] Then 90 ml of diisopropyl ether were added at 20-25 °C. The mixture was cooled to 10-15 °C and maintained under stirring for 2.5 hrs at 10-15 °C under nitrogen atmosphere, and dried for 12 hours at 10-15 °C under vacuum.

[0325] 5.8 g of the metal complex salt were obtained and stored at -20 °C.

[0326] Example 33 - Synthesis and isolation of magnesium (II) solid complex (solvent: acetone, buffering agent: potassium tetraborate)

[0327] 5.0 g (4.18 mmol) of compound of Formula 1 were solubilized in 30 ml of acetone by stirring at 20-25 °C until obtaining a clear solution.

[0328] 1025 mg (4.39 mmol) of potassium tetraborate (1.05 mole equivalents with respect to compound of Formula 1) were added to the solution of compound of Formula 1 in acetone and maintained under stirring for 20 minutes at 20-25 °C under nitrogen atmosphere. 420 mg (4.39 mmol) of anhydrous MgCL (1.05 mole equivalents with respect to compound of Formula 1) were added and maintained under stirring for 75 minutes at 20-25 °C under nitrogen atmosphere. After the addition of MgCL to the solution of compound of Formula 1 in acetone metal complex salt in the form of a gel started forming.

[0329] Then 90 ml of diisopropyl ether were added at 20-25 °C. The mixture was cooled to 10-15 °C and maintained under stirring for 2.5 hrs at 10-15 °C under nitrogen atmosphere, and dried for 12 hours at 10-15 °C under vacuum.

[0330] 5.9 g of the metal complex salt were obtained and stored at -20 °C.

[0331] Example 34 - Characterization of the metal complex salt

[0332] The structure of the metal complex salts was investigated by using ID and 2D ^-NMR experiments in CDCh.

[0333] ^-NMR is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the structure of its molecules. Proton NMR spectra of most organic compounds are characterized by chemical shifts in the range +14 to -4 ppm and by spin-spin coupling between protons. The integration curve for each proton reflects the abundance of the individual protons.

[0334] Three different samples were analyzed, compound of Formula 1 (Fl) and metal complex salt obtained by the reaction of compound of Formula 1 with 1 mol equivalent of MgCL (Cl) and 2 mol equivalents of MgCL (C2), respectively.

[0335] The ^-NMR spectra of the samples are reported in Figure 6; in particular, the ^-NMR spectrum of Fl is reported in Figure 6 a), the one of Cl in Fig. 6 b) and the one of C2 in Fig. 6 c). In the figure, the position of the signals attributable to hydrogen of NH group of Glu, of both the AEEA groups, of a-NH group and E-NH group of Lys are highlighted.

[0336] The experiments outlined a noticeable change in the position of the ^-NMR signals of amide protons with the addition of the magnesium chloride, thus suggesting that coordination likely occurs between the nitrogen atom of the amine (NH) groups and the metal cation. Further, this also excludes the formation of a metal salt and proves that the structure obtained by the reaction of compound of Formula 1 with MgCL is a metal complex salt. You would not expect a shift in the protons if a salt would have formed, and the amount of the metal cation would change.

[0337] Here, the resulting change of position clearly is dependent on the amount of the metal salt added. To investigate more deeply this dependency, several bi-dimensional studies were performed on the samples. The analysis of two-dimensional nuclear Overhauser effect (NOESY) spectra in the absence and in the presence of MgCL by using CYANA program indicated a significant increase in the number of intra- and interresidual nuclear Overhauser effects (NOEs) in C2 compared to Fl and Cl (Table 1). CYANA (combined assignment and dynamics algorithm for ^-NMR applications) is a program for automated structure calculation of biological macromolecules on the basis of conformational constraints from ^-NMR.

[0338] Table 1. Analysis of NOESY spectra of Fl, Cl and C2

[0339] This trend is further confirmed by the results of the Chemical Shift Perturbation (CSP) analysis, confirming that the protons 14, 16 and 17 of the AEEA region and the tert-butyl group of glutamic acid appeared to be the most sensible to the addition of the salt. This chemical shift perturbation was used to calculate the theoretical spatial arrangement of compound of Formula I. According to this analysis, compound of Formula 1 has a different spatial arrangement in dependency of the presence and amount of MgCL used, as reported in Figure 7 for compound of Formula 1 (Fig. 7 a), compound of Formula 1 after reaction with 1 mol eq (Fig. 7 b) and 2 mol equivalents (Fig. 7 c) of MgCL, respectively.

[0340] Abbreviations

[0341] AC2O Acetic anhydride

[0342] AEEA 2-[2-(2-aminoethoxy)ethoxy]acetyl group

[0343] Boc tert-buthoxycarbonyl

[0344] Bz benzyl

[0345] CSP Chemical Shift Perturbation

[0346] CDCI3 deuterated chloroform

[0347] DCM dichloromethane

[0348] DIC N,N'-Diisopropylcarbodiimide

[0349] DIPE Diphenyl ether

[0350] DIPEA N,N-Diisopropylethylamine

[0351] DMF Dimethylformamide

[0352] DTT 1,4-Dithiothreitol Fmoc 9-Fluorenylmethoxycarbonyl

[0353] HPLC High Performance Liquid Chromatography

[0354] K-Oxyma 2-Cyano-2-(hydroxyimino)acetic acid ethyl ester potassium salt

[0355] MeCN acetonitrile

[0356] MeOH methanol min minutes

[0357] Mpe beta-3methylpent-3-yl

[0358] MS Mass Spectroscopy

[0359] MTBE methyl tertial butyl ether

[0360] ^-NMR proton nuclear magnetic resonance

[0361] NOE nuclear Overhauser effect

[0362] NOESY 1H two-dimensional nuclear Overhauser effect

[0363] Pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl

[0364] 2-Ph'Pr 2-Phenylisopropyl

[0365] Smoc 2,7-disulfo-9-fluorenylmethoxycarbonyl

[0366] SPPS Solid Phase Peptide Synthesis tBU tert-Butyl

[0367] TEGBn, TEGBz 4-(3,6,9-Trioxadecyl)oxybenzyl

[0368] TFA trifluoroacetic acid

[0369] TIS triisopropylsilane

[0370] THF tetrahydrofuran

[0371] Trt Trityl

Claims

Claims1. Solid metal complex salt obtainable by the reaction of compound of Formula I: Rl-(AEEA)m-Al-CO-(CH2)n-COO-R2 (I) with a MX metal salt, wherein the MX metal salt is used in the range between 0.5 and 5.0 mol equivalents with respect to compound of Formula I;M is a metal cation selected from the group of alkaline earth metal cations and transition metal cations; X is an organic or inorganic anion;R1 is selected from the group consisting of OH, a protected or unprotected amino acid and a peptide group consisting of 2 to 4 protected or unprotected amino acids;R2 is H or a carboxylic protective group;Al is a protected or unprotected amino acid or a peptide group consisting of 2 to 4 protected or unprotected amino acids;AEEA is 2-[2-(2-aminoethoxy)ethoxy]acetyl group; m is an integer between 1 and 10; and n is an integer between 8 and 28.

2. The solid metal complex salt according to claim 1, wherein M is selected from the group consisting of Ca, Zn, Mg, Cu, Co, Ni, Ti, Zr, Fe and Ag cations.

3. The solid metal complex salt according to claim 1 or claim 2, wherein X is a halide, preferably X is Cf or Br4. The solid metal complex salt according to any of claims 1 to 3, wherein the MX metal salt is used in the range between 0.8 and 4.0 mol equivalents, preferably between 1.0 and 2.5 mol equivalents, with respect to compound of Formula I.

5. The solid metal complex salt according to any of claims 1 to 4, wherein R1 is protected or unprotected Lys and / or Al is protected or unprotected Glu.

6. The solid metal complex salt according to any of claims 1 to 5, wherein m is an integer between 1 and 5.

7. The solid metal complex salt according to any of claims 1 to 6, wherein n is an integer between 10 and 25.

8. The solid metal complex salt according to any of claims 1 to 7, wherein compound of Formula I is compound of Formula 1:or compound of formula 3:A process for the preparation of a solid metal complex salt according to any of claims 1 to 8 comprising the following steps: i dispersing or solubilizing compound of Formula I in a first aliquot of a liquid medium; ii treating compound of Formula I with the MX metal salt to form the metal complex salt, and iii recovering the metal complex salt in a solid form. 0 The process for the preparation of the solid metal complex salt according to claim 9, wherein step (iii) comprises antisolvent precipitation or removal of the liquid medium, preferably step (iii) comprises antisolvent precipitation.11.The process for the preparation of the solid metal complex salt according to claim 9 or claim 10, wherein the liquid medium is selected from the group consisting of water, methanol, isopropyl alcohol, ethanol, dichloromethane, chloroform, ethyl acetate, methyl acetate, isopropyl acetate, acetonitrile, dimethyl carbonate, toluene, tetrahydrofuran, acetone and mixtures thereof.12.The process for the preparation of a solid metal complex salt according to any of claims from 9 to 11, wherein the solid metal complex salt is recovered by antisolvent precipitation and the antisolvent is selected from the group consisting of heptane, toluene, acetonitrile, ethyl acetate, cyclopentyl methyl ether, petroleum ether, diisopropyl ether, methyl tert-butyl ether, acetone and mixtures thereof.13.The process for the preparation of the solid metal complex salt according to any of claims 9 to 12, wherein the MX metal salt is previously solubilized or dispersed in a second aliquot of the liquid medium before treating the compound of Formula I with the MX metal salt.14.The process for the preparation of the solid metal complex salt according to any of claims 10 to 13, wherein the solid metal complex salt is recovered by antisolvent precipitation, the liquid medium is acetone, ethyl acetate or dimethyl carbonate and the antisolvent is acetone, ethyl acetate or isopropyl ether, or a mixture of acetone and isopropyl ether, or a mixture of ethyl acetate and isopropyl ether.

15. Use of the solid metal complex salt according to any of claims 1 to 8, in the synthesis of semaglutide, tirzepatide and their pharmaceutically acceptable salts.

16. Process for making active pharmaceutically ingredients, comprising semaglutide, tirzepatide and their pharmaceutically acceptable salts, characterized by the use of the solid metal complex salt according to any of claims 1 to 8.

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