Process for preparation of AMG416

Abstract

The present invention relates to a process for the preparation of AMG416. A method for preparing AMG416, or a pharmaceutically acceptable salt thereof, is provided. AMG 416 is an octa-amino acid selective peptide antagonist of the synthesized calcium-sensitive receptor. It has been developed as an intravenous treatment of secondary hyperparathyroidism (SHPT) in hemodialysis patients with chronic kidney disease-mineral and bone disorders (CKD-MBD).

Description

This application is a divisional application. The application date of the original application is April 3, 2015, the application number is 201580029560.2 (PCT / US2015 / 024347), and the name of the invention is “Method for preparing AMG416”.

[0001] CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 61 / 974,899, filed April 3, 2014, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to a sequence listing The Sequence Listing was submitted electronically via EFS as a text file created on April 3, 2015 and named "0419250488seqlist.txt" (2531 bytes), the contents of which are incorporated herein by reference in their entirety. Field of the Invention

[0003] The present disclosure relates to the field of polypeptide synthesis, and more specifically to the synthesis of AMG416 or a pharmaceutically acceptable salt thereof. Background of the Invention AMG 416 is a synthetic, eight-amino acid selective peptide antagonist of the calcium-sensing receptor. It is developed as an intravenous treatment for secondary hyperparathyroidism (SHPT) in hemodialysis patients with chronic kidney disease-mineral and bone disorder (CKD-MBD).

[0005] The hydrochloride salt of AMG416 has the chemical structure: The main chain has 7 amino acids, all of which are in D-configuration. The side chain cysteine ​​residues are in l-configuration. The molecular formula of AMG 416 (free base) is C 38 H 73 N 21 O 10 S2, and has a calculated average molecular weight of 1048.3 Da.

[0006] AMG 416 and methods for its preparation are described in International Patent Publication No. WO 2011 / 014707, which is incorporated herein by reference for any purpose. As described in International Patent Publication No. WO 2011 / 014707, AMG 416 can be assembled from the corresponding Fmoc-protected D-amino acid by solid phase synthesis. After cleavage from the resin, the material can be treated with Boc-l-Cys(NPyS)-OH to form a disulfide bond. The Boc group can then be removed with trifluoroacetic acid (TFA), and the resulting product can be purified by reverse phase high pressure liquid chromatography (HPLC) and separated by lyophilization as a TFA salt form. TFA salts can be converted to pharmaceutically acceptable salts by performing subsequent salt exchange procedures. Such procedures are well known in the art and include, for example, ion exchange techniques, optionally followed by purification of the resulting product (e.g., by reverse phase liquid chromatography or reverse osmosis).

[0007] There is a need for an efficient process for producing AMG 416 or a pharmaceutically acceptable salt thereof (eg, AMG 416 HCl), particularly a process suitable for commercial scale manufacturing. SUMMARY OF THE INVENTION In view of the above problems, one object of the present disclosure is to provide, among other things, a method for preparing AMG 416, or a pharmaceutically acceptable salt thereof.

[0009] In a first aspect, a method for preparing AMG 416 is provided, the method comprising: providing a resin-bound peptide having a structure selected from Fmoc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[resin] (SEQ ID NO:2) and Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[resin] (SEQ ID NO:3); cleaving the peptide from a solid support; and activating the side chain of the D-Cys residue of the cleaved peptide.

[0010] In one or more embodiments related to the first aspect, the cleaving and activating steps occur in the same container.

[0011] In one or more further embodiments, the resin-bound peptide is contacted with a solution comprising water, trifluoroacetic acid, triisopropylsilane, and bipyridyl disulfide.

[0012] In a second aspect, a method for preparing AMG 416 is provided, the method comprising: providing a peptide having a structure of Ac-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2 (SEQ ID NO:4); and contacting the peptide with l-Cys to produce a conjugation product.

[0013] In some embodiments related to the second aspect, the peptide is contacted with an aqueous solution comprising 1-Cys and trifluoroacetic acid.

[0014] In some further embodiments related to the second aspect, the method further comprises lyophilizing the conjugated product.

[0015] In yet further embodiments, the method of the second aspect further comprises contacting the conjugated product with an aqueous solution comprising isopropanol (IPA) and hydrochloric acid (HCl), thereby producing a precipitate comprising AMG416 HCl.

[0016] In still one or more further embodiments related to the second aspect, the method further comprises purifying the precipitate by high performance liquid chromatography (HPLC).

[0017] In yet a third aspect, a method for preparing AMG 416 is provided, the method comprising: providing a resin-bound peptide having a structure selected from the group consisting of Fmoc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[resin] (SEQ ID NO: 2) and Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[resin] (SEQ ID NO: 3); cleaving the peptide from the solid support, i.e., to provide an unsupported peptide, and activating the side chain of the D-Cys residue of the unsupported peptide to produce AMG 416 SPy intermediates (wherein SPy is 2-pyridinesulfenyl or S-Pyr), dissolving AMG 416 SPy intermediates in aqueous 0.1% TFA (trifluoroacetic acid solution), and purifying AMG 416 SPy derivatives by HPLC.

[0018] In some embodiments related to the third aspect, the method further comprises azeotropic distillation of the AMG 416 SPy intermediate, thereby causing a solvent exchange to produce a solution of AMG 416 SPy in a new solvent, for example, water and isopropanol.

[0019] In yet additional embodiments related to the third aspect, the method further comprises contacting an isopropanol-water solution of AMG416SPy, in some embodiments in the form of its trifluoroacetate salt, with an aqueous solution comprising l-Cys.

[0020] In a fourth aspect, methods are provided for preparing HD-Arg(Pbf)-OH, a suitable starting material for certain synthetic methods provided herein.

[0021] In some embodiments related to the fourth aspect, the method comprises converting Boc-D-Arg-OH to Boc-D-Arg(Pbf)-OH in the presence of NaI (sodium iodide). In some further embodiments, sodium iodide is present at a concentration of about 5% mol.

[0022] In some additional embodiments related to the fourth aspect, the method further includes converting Boc-D-Arg(Pbf)-OH to D-Arg(Pbf)-OH and crystallizing D-Arg(Pbf)-OH in an IPA / water solvent system.

[0023] Additional embodiments of the methods described herein will be apparent from the following description, examples, and claims. As can be understood from the foregoing and following descriptions, each feature described herein, and each combination of two or more such features, is included within the scope of the present disclosure, as long as the features included in such combination are not mutually inconsistent. In addition, any feature or combination of features may be specifically excluded from any embodiment of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The chemical structure of AMG 416 (Ac-D-Cys(l-Cys-OH)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2) (SEQ ID NO: 1) is shown.

[0025] Figure 2 The chemical structures of Rink Amide AM resin and Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-resin (SEQ ID NO: 3) are shown.

[0026] Figure 3A schematic representation of the reaction in which the SPy intermediate product (Ac-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2) (SEQ ID NO:4) is formed from the peptidyl-resin (Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-NH-resin) (SEQ ID NO:3).

[0027] Figure 4 The TFA salt of AMG 416 was prepared from the SPy intermediate (AA 1-7(SPy) ) is a schematic diagram of the reaction formed.

[0028] Figure 5 Shown is a scheme of the reaction in which the HCl salt of AMG 416 is formed from the TFA salt of AMG 416.

[0029] Figure 6 A schematic diagram of the reaction in which Boc-D-Arg(Pbf)-OH is formed from Boc-D-Arg-OH is shown.

[0030] Figure 7 A schematic diagram showing the reaction in which D-Arg(Pbf)-OH is formed from Boc-D-Arg(Pbf)-OH. DETAILED DESCRIPTION OF THE INVENTION The present disclosure will now be described more fully hereinafter. However, the disclosure may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein; rather, these embodiments are provided so that the present disclosure will be thorough and comprehensive, and its scope will be fully conveyed to those skilled in the art.

[0032] All publications, patents and patent applications cited herein, whether above or below, are incorporated herein by reference in their entirety, unless otherwise indicated. In cases where the same term is defined in both a publication, patent or patent application and the present disclosure incorporated herein by reference, the definition in the present disclosure represents the controlling definition. For publications, patents and patent applications cited for their description of a specific type of compound, peptide, chemical, etc., the portions concerning such compound, chemical, etc. are those portions of the document incorporated herein by reference.

[0033] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0034] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by context, singular terms shall include pluralities, and plural terms shall include the singular.

[0035] In general, the nomenclature used for molecular biology and protein chemistry described herein, and the techniques, are those known and commonly used in the art. The methods and techniques of the present application are generally carried out according to conventional methods known in the art and as described in each general or more specific reference cited and discussed throughout this specification, unless otherwise specified. See, for example, Laszlo, Peptide-Based Drug Design: Methods and Protocols (peptide-based drug design: methods and schemes), Humana Press (2008); Benoiton, Chemistry of Peptide Synthesis (peptide synthesis chemistry), CRC Press (2005); Ausubel et al., Current Protocols in Molecular Biology (molecular biology current scheme), Greene Publishing Associates (1992), which are incorporated herein by reference for all purposes. Purification techniques are carried out according to the manufacturer's instructions, as commonly accomplished in the art or as described herein. The terms used for analytical chemistry, synthetic organic chemistry, and drugs and pharmaceutical chemistry, and laboratory procedures and techniques are those known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0036] It is to be understood that the present disclosure is not limited to the specific methodology, protocols, reagents, etc. described herein, and that these may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure, which is defined solely by the claims.

[0037] The term "about", particularly with reference to a given amount, is meant to include a deviation of plus or minus 5%.

[0038] I General Definition The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article unless specifically stated otherwise. For example, "an element" means one element or more than one element.

[0039] The term "AMG 416", also known as etelcalcetide, formerly known as velcalcetide or KAI-4169, refers to a compound having the chemical name: N-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-arginyl-D-alanyl-D-arginylamide disulfide with L-cysteine, which has the following structural formula: Reference to AMG 416, or any compound or AMG 416 fragment, intermediate or precursor as described herein, is intended to include the neutral, uncharged form thereof, as well as pharmaceutically acceptable salts, hydrates and solvates thereof.

[0040] The terms "AMG 416 hydrochloride" and "AMG 416 HCl" are interchangeable and refer to the hydrochloride salt form of AMG 416 having the following structural formula: Typically, x has a value of 3-5 (eg, 3, 4, or 5).

[0041] "Pharmaceutically acceptable salt" refers to a salt form of a compound having at least one group suitable for salt formation, which does not cause significant adverse toxicological effects on patients. In one aspect, the term "pharmaceutically acceptable salt" may refer to a compound with one or more ionizable amine groups as provided herein, for example, AMG416, and relatively nontoxic organic or inorganic acid addition salts of AMG416 fragments, intermediates, precursors, etc. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, toluenesulfonate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate and lauryl sulfonate, etc. (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J.Pharm.Sci.66:1-19). Additional suitable pharmaceutically acceptable salt forms may be found in, for example, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zürich: Wiley-VCH / VHCA, 2002; PH Stahl and CG Wermuth, Eds.

[0042] As used herein, the terms "amino acid" and "residue" are interchangeable and, when used in the context of a peptide or polypeptide, refer to both naturally occurring and synthetic amino acids, as well as amino acid analogs, amino acid mimetics, and non-naturally occurring amino acids that are chemically similar to the naturally occurring amino acids. "Free amino acid" or "free amino group" refers to an amino acid, peptide fragment, or peptide having the -NH2 form, i.e., an unprotected amino group.

[0043] As used herein, the phrase "protecting group" or "PG" refers to one or more temporary substituents that protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include carboxylates, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, etc. See, for example, Greene, TW; Wuts, PGM Protective Groups in Organic Synthesis, Fourth Edition; Wiley: New York, 2007; Isidro-Llobet, A., et al., Amino Acid-Protecting Groups, Chem. Rev 2009, 109, 2455-2504. Reactive amino acids or peptide fragments as described herein often appropriately include one or more protecting groups on the functionality of non-subject chemical transformation targets. Exemplary protecting groups include, for example, carboxybenzyl, also known as benzyloxycarbonyl ("Cbz" or "Z"), 9-fluorenylmethyloxycarbonyl (Fmoc), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), tert-butyloxycarbonyl (Boc), trityl (Trt), methyl ester (OMe), 2-pyridinesulfinyl (SPy or S-Pyr), amide, etc. In the abbreviated structures provided herein, the NH2 at the C-terminus represents an amide protecting group (-C(O)NH2), the "H" at the N-terminus refers to the free amino group, and the designation of the protecting group in parentheses indicates that the protecting group is on the delta nitrogen of ornithine.

[0044] As used herein, the terms "protection removing agent" or "deprotecting agent" are used interchangeably and are chemical reagents used to remove the amino protecting agent attached to the amino acid. The amino protecting agent may be well known in the art, such as, but not limited to, Fmoc and Boc.

[0045] As used herein, the terms "coupling agent", "condensing agent", "activator", and "condensation activator" are used interchangeably herein and refer to chemical reagents that promote the reaction of an amino group from one amino acid with a carboxyl group from another amino acid to form a peptide bond. Exemplary coupling agents are well known in the art and include, but are not limited to, carbodiimides such as N,N'-diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (HATU), [benzotriazol-1-yloxy (dimethylamino) methylene]-dimethylamino Tetrafluoroborate (TBTU), N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA). See, for example, El-Faham, A. and Albericio, F., "Peptide Coupling Reagents, More than a Letter Soup", Chem. Rev. 2011, 111, 6557-6602. Such compounds are readily available from commercial suppliers.

[0046] As used herein, the term "cleavage agent" refers to a chemical agent that can separate a peptide bound to a resin from the resin. Cleaving agents are well known to those of ordinary skill in the art and include acid solutions including TFA and HCl solutions.

[0047] The term "treatment" refers to successfully treating or improving any signs of an injury, pathology, or condition, including any objective or subjective parameter such as alleviating, relieving, reducing signs or symptoms, or making an injury, pathology, or condition more tolerable to the patient, slowing the rate of degeneration or decline, making the degenerative endpoint less debilitating, improving the patient's physical or mental health. The treatment or improvement of signs or symptoms can be based on objective or subjective parameters, including the results of a physical examination. For example, certain methods presented herein successfully treat SHPT in hemodialysis patients with CKD-MBD by reducing serum intact parathyroid hormone (iPTH).

[0048] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate symptoms and / or potential causes, prevent symptoms and / or their potential causes from occurring, and / or improve or remedy damage caused by or associated with a disease state (e.g., an increased PTH level). A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or to additionally prevent, hinder, delay or reverse the progression of the disease state or any other undesirable symptom associated with the disease in any way. A complete therapeutic effect does not necessarily occur by administering one dose, it may only occur after a series of doses are administered. Therefore, a therapeutically effective amount can be administered in one or more administrations.

[0049] As used herein, the terms "therapeutically effective dose" and "therapeutically effective amount" mean an amount that elicits a biological or medical response in the tissue system, animal or human being investigated by the researcher, physician or other clinician, including alleviation or amelioration of the signs or symptoms of the disease or condition being treated, i.e., an amount of velcalcetide that supports an observable level of one or more desired biological or medical responses, such as reduction of iPTH.

[0050] The terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms may also include amino acid polymers that have been modified or phosphorylated, for example, by the addition of carbohydrate residues to form glycoproteins. Peptides, polypeptides, and proteins can be produced by liquid or solid phase synthesis or by genetically engineered or recombinant cells, and include molecules having an amino acid sequence.

[0051] "Variants" of peptides or polypeptides include amino acid sequences in which one or more amino acid residues are inserted, deleted and / or substituted in an amino acid sequence relative to another polypeptide sequence. Variants include fusion proteins.

[0052] A "derivative" of a peptide or polypeptide is a peptide or polypeptide that has been chemically modified in some manner distinct from an insertion, deletion or substitution variant, e.g., via conjugation to another chemical moiety. Such modifications may include the covalent addition of a group to the amino and / or carboxyl terminus of the peptide or polypeptide, e.g., acetylation of the amino terminus of the peptide or polypeptide and / or amidation of the carboxyl terminus.

[0053] The term "amino acid" includes its normal meaning in the art. Twenty naturally occurring amino acids and their abbreviations follow conventional use. See, Immunology-A Synthesis, Second Edition, (ES Golub and DR Green, eds.), Sinauer Associates: Sunderland, Mass. (1991), which is incorporated herein by reference for any purpose. Stereoisomers (e.g., D-amino acids) of 19 conventional amino acids (except glycine), non-natural amino acids such as [α]-, [α]-disubstituted amino acids, N-alkyl amino acids and other unconventional amino acids may also be suitable components of polypeptides and are included in the phrase "amino acid". Examples of unconventional amino acids include homocysteine, ornithine, 4-hydroxyproline, [γ]-carboxyglutamate, [ε]-N,N,N-trimethyllysine, [ε]-N-acetylysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, [Σ]-N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxyl terminal direction, consistent with standard usage and convention.

[0054] As used herein, a "subject" or "patient" can be any mammal. In typical embodiments, the subject or patient is a human.

[0055] The term "qs" means adding an amount sufficient to achieve the desired function, eg, to bring a solution to a desired volume (ie, 100%).

[0056] II. Implementation Plan In one or more embodiments, AMG 416 hydrochloride is prepared via a series of process stages as follows: an exemplary stage includes solid phase peptide synthesis of a seven-member linear fragment of AMG 416 (stage I), followed by cleavage of the peptide chain from the resin with concomitant side chain deprotection and cysteine ​​activation (stage II), followed by in situ conjugation of the peptide chain with l-Cys (disulfide bond formation) to provide crude AMG 416 (stage III), followed, in some embodiments, by immediate preparative HPLC and lyophilization to provide purified AMG 416 TFA salt (stage IV). Stage IV is followed by salt exchange (TFA to HCl) by precipitation, and in some embodiments, followed by microfiltration and lyophilization to provide purified AMG 416 hydrochloride (stage V).

[0057] Solid Phase Peptide Synthesis The seven-member linear fragment of AMG 416 can be synthesized by any method known in the art, including solid phase peptide synthesis (SPPS). As used herein, the term "solid phase synthesis" or "solid phase peptide synthesis" refers to a method in which a growing peptide chain is attached to a solid support, which is well known to those of ordinary skill in the art. Solid phase synthesis generally includes the steps of: (i) covalently binding a first amino acid (whose amino group is blocked or "protected") to a solid support; (ii) removing the protecting group from the amino group using a deprotecting agent; (iii) activating the carboxyl group of a second amino acid (whose amino group is blocked) and contacting the second amino acid with the first amino acid bound to the solid support so as to obtain a dipeptide (whose amino group is blocked); (iv) repeating the peptide bond forming step, so that the peptide chain extends from the C-terminus to the N-terminus; and (v) removing the protecting group of the amino group and separating the peptide chain from the solid support using a cleavage agent to produce a peptide.

[0058] Suitable solid phase synthesis techniques are well known in the art and include those described in Merrifield, in Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds. 1973); Merrifield, J. Am. Chem. Soc. 85:2149 (1963); Davis et al., Biochem. Intl. 10:394-414 (1985); Stewart and Young, Solid Phase Peptide Synthesis (1969); U.S. Pat. No. 3,941,763; Finn et al., The Proteins, 3rd edition, volume 2, pp. 105-253 (1976); and Erickson et al., The Proteins, 3rd edition, volume 2, pp. 257-527 (1976). See also Houben-Weyl, Methods of Organic Chemistry. Additional Supplementary Volume to the Fourth Edition, Vol E22A, "Synthesis of Peptides and Peptidomimetics", Editor-in-Chief M. Goodman. Georg Thieme Verlag: Stuttgard and New York. 2002, pp. 685-877; Chan, WC, White, PD, "Fmoc Solid Phase Peptide Synthesis, A Practical Approach". Oxford University Press, (200), p. 9-109. Solid phase synthesis is generally the preferred technique for making individual peptides such as AMG 416, because it is generally one of the most cost-effective methods for making small peptides.

[0059] In some embodiments, the backbone linear fragments of AMG 416 are assembled using standard solid phase peptide synthesis protocols employing an Fmoc-protection strategy and, for example, Rink amide (RAM) resin (e.g., available from Sigma Aldrich) to provide a C-terminal resin-bound amide, along with peptide N-terminal acetylation. In some other embodiments, other resins and linkers (e.g., Ramage amide AM resin, also known as a tricyclic amide linker resin) may be used. In one embodiment, the assembly of the main chain linear fragment comprises the steps of: (i) mixing Fmoc-Rink amide AM resin with a deprotecting agent to obtain Rink amide AM resin; (ii) condensing Fmoc-D-Arg(Pbf)-OH with Rink amide AM resin to obtain Fmoc-D-Arg(Pbf)-Rinkamide AM resin; (iii) for each remaining amino acid residue of the main chain linear fragment of AMG 416, repeating the Fmoc deprotection in step (i) and the condensation between the amino acid and the polypeptide on the resin in step (ii), from the C-terminus to the N-terminus (for example, using Fmoc-D-Cys(Trt)-OH, Fmoc-D-Ala-OH and Fmoc-D-Arg(Pbf)-OH) to form a polypeptide resin represented by SEQ ID NO:2; and (iv) repeating the Fmoc deprotection and acetylation of the N-terminus in step (i) to form a polypeptide resin represented by SEQ ID NO:3. See Figure 2 . Fmoc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)- [Resin] (SEQ ID NO:2) Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)- [Resin] (SEQ ID NO:3)

[0060] Typically, the cleavage of the Fmoc protecting group is achieved using a deprotecting agent such as piperidine in DMF. In one embodiment, the coupling of the Fmoc-protected amino acid is carried out in a solvent such as dimethylformamide (DMF) using a suitable coupling agent such as a carbodiimide coupling agent, N, N-diisopropylcarbodiimide (DIC), optionally in the presence of an additive such as ethyl 2-cyano-2-(oxime) acetate (Oxyma) for all amino acids except cysteine. In the case of peptide chain extension with cysteine, coupling is typically carried out using N, N-diisopropylcarbodiimide (DIC) in the presence of a benzotriazole additive such as hydroxybenzotriazole (HOBT) in a solvent system such as dimethylformamide-dichloromethane (i.e., DMF, DCM, HOBt, DIC).

[0061] Acetylation of the N-terminus can be achieved by any method known in the art. In one embodiment, acetylation of the N-terminus is performed using, for example, (Ac2O) acetic anhydride in pyridine and DMF.

[0062] Cutting from resin The peptide can be separated from the support and the protecting groups can be removed from the side chains by any means known in the art. See, for example, Synthetic Peptides: A User's Guide (GA Grant, ed.), WH Freeman and Company, New York, 1992; and Chan, WC, White, PD "Fmoc Solid Phase Peptide Synthesis, A Practical Approach", Oxford University Press (2000), p. 64-66 and 105-109.

[0063] In one embodiment, the peptidyl-resin is added to a cocktial solution comprising water (e.g., deionized water (DIW)), trifluoroacetic acid (TFA), triisopropylsilane (TIPS), and dipyridyl disulfide (DPDS). This allows the peptide to be detached from the resin with concomitant side chain deprotection and cysteine ​​activation, thus preparing for in situ conjugation with l-Cys. The seven-amino acid SPy intermediate product (AA 1-7(SPy) ). See Figure 3 The sequence of the SPy intermediate product is provided in SEQ ID NO:4. Ac-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2 (SEQ ID NO:4)

[0064] In situ conjugation and preparative HPLC The SPy intermediate product can be conjugated to l-Cys by any method known in the art. In one embodiment, the conjugation of l-Cys is carried out in aqueous TFA.

[0065] The resulting AMG 416 (TFA salt) can be purified by any method known in the art. In one embodiment, AMG 416 (TFA salt) is purified by high pressure liquid chromatography (HPLC). For example, in one embodiment, the purification and concentration of AMG 416 (TFA salt) includes a reverse phase HPLC purification step and a reverse phase HPLC concentration step. See Figure 4 .

[0066] The purified and concentrated sample containing AMG 416 (TFA salt) can be lyophilized.

[0067] Salt conversion TFA salts can be converted to pharmaceutically acceptable salts, such as hydrochlorides, by any method known in the art.

[0068] In one embodiment, the lyophilized TFA salt of AMG 416 is dissolved in an aqueous solution of isopropyl alcohol (IPA). The TFA salt solution is then added to the HCl solution for salt exchange and precipitation of the HCl salt. The precipitate can then be reconstituted with water, filtered through a microfilter (e.g., a 0.2 μm filter) and lyophilized to isolate the HCl salt of AMG 416. See Figure 5 .

[0069] Purification of SPy intermediates In an alternative embodiment, the SPy intermediate product is purified prior to conjugation with L-Cys. In general, SPy intermediate products, particularly peptide-SPy intermediates, are considered highly unstable, i.e., they are not considered stable enough to withstand effective purification, such as by HPLC. However, in arriving at the methods provided herein, Applicants unexpectedly discovered that the peptide-SPy intermediates prepared according to the methods described herein are indeed stable enough to withstand a separate purification step. Moreover, it has been further discovered that purifying the intermediate prior to conjugation with L-Cys can in fact increase the manufacturing efficiency and reduce the manufacturing costs of the final peptide drug product.

[0070] In an exemplary embodiment, an alternative method is performed as follows. The method described below and in Example 5 can be used for the purification of SPy intermediates as provided herein, wherein the intermediate remains stable and is suitable for, for example, conjugation to a sulfhydryl-containing moiety via disulfide bond formation. For example, the peptide-SPy intermediate is dissolved in an aqueous solution containing no more than about 0.2% TFA, such as about 0.05%-0.15% TFA or about 0.1% TFA, and then directly applied to an HPLC column for chromatographic purification. Solvent exchange of the HPLC fraction containing the peptide-SPy intermediate can then be performed, for example, by azeotropic distillation. After the peptide-SPy intermediate is solvent exchanged to an appropriate solvent such as a water-isopropanol mixture, a sulfhydryl-containing moiety, such as l-Cys, is directly added to the peptide-SPy intermediate solution to cause conjugation. The resulting conjugated product, for example, in solution, can then be used for salt exchange.

[0071] A specific embodiment of the aforementioned purification method is as follows. Initially cleaving the peptide from the resin support and separating the AA 1-7(SPy) After the intermediate is produced, the intermediate is dissolved in 0.1% TFA and acetonitrile, loaded onto a stationary phase HPLC column and purified as described above. Using a 0.1% TFA solution for HPLC purification has a number of advantages when compared to using, for example, a 0.2% TFA solution. For example, 0.1% TFA damages the stationary phase less during the purification process than a higher concentration TFA solution. That is, by using this optimized TFA concentration, there is less stationary phase decomposition, resulting in a longer stationary phase life. In addition, peptide-SPy intermediates (e.g., AA 1-7(SPy) ) The product polarity is less, resulting in better retention of the intermediate on the reversed phase stationary phase. Therefore, a higher loading on the stationary phase can be achieved in each purification run. In some embodiments, the increased loading capacity increases the throughput of the manufacturing process by 1.5-2 times, or about 1.5 times.

[0072] The HPLC fraction containing the SPy intermediate product as a TFA salt was then subjected to azeotropic distillation with sufficient IPA feed to change the solvent from acetonitrile and water to a 15% water / IPA solution suitable for L-Cys conjugation and salt exchange. This method is particularly advantageous because conjugation and salt exchange can be performed in a single vessel, further improving the efficiency and feasibility of the manufacturing process.

[0073] Preparation of Fmoc-D-Arg(Pbf)-OH Starting Material AMG 416 comprises a 7 amino acid linear sequence, 4 of which are D-arginine. Disclosed herein is a method for synthesizing Fmoc-D-Arg(Pbf)-OH, an Fmoc derivative of D-arginine used in the synthesis of AMG 416. The use of high-quality Fmoc-D-Arg(Pbf)-OH starting materials can provide additional purity for crude AMG 416 from stage I to stage III, thereby increasing the purification yield in stage IV. More importantly, the use of high-quality, high-purity starting materials can provide desired and advantageous control elements to ensure the desired purity of AMG 416. A new method for preparing high-quality D-Arg(Pbf)-OH and Fmoc-D-Arg(Pbf)-OH has been developed, which produces higher yields, requires fewer unit operations, provides more robust quality control and is amenable to large-scale manufacturing. In general, the method for preparing Fmoc-D-Arg(Pbf)-OH described herein represents a significant increase in the feasibility of AMG 416 manufacturing with potential quality gains.

[0074] One of the most concise Fmoc-D-Arg(Pbf)-OH syntheses in the literature and a synthetic route that can be used to synthesize Fmoc-D-Arg(Pbf)-OH on a commercial scale is as follows. See, for example, Chinese Patent No. CN101250172B, March 2, 2012. The synthesis starts with protecting the amino group of D-Arg with di-tert-butyl dicarbonate (Boc2O), and after separation, Boc-D-Arg-OH is produced in a nearly quantitative yield. In step 2, the side chain guanidine group is protected with a Pbf group in the presence of a base such as aqueous sodium hydroxide. The product is used directly as an IPA (isopropanol) solution without separation in the next step. Step 3 includes removing the Boc protecting group under acidic conditions and isolating the corresponding product, D-Arg(Pbf)-OH, as a crystalline intermediate. Step 4 of the method includes installing an Fmoc protecting group on the amino functionality to provide Fmoc-D-Arg(Pbf)-OH as a final product. Since both the products of Step 2 and Step 4 are amorphous materials, i.e., offer limited ability to reject impurities, the crystalline intermediate D-Arg(Pbf)-OH (Step 3 product) serves as a control point to influence and ensure the purity of the final product.

[0075] Obtaining high purity products is a major challenge when relying on methods reported in the literature. Multiple recrystallizations are often required to meet purity requirements. For example, D-Arg(Pbf)-OH was recrystallized 7 times in a ternary solvent system of EtOH / EtOAc / water to bring the purity to the desired level. In addition, in step 2, 20-30% of the starting material remains unreacted even in the presence of a large excess of sodium hydroxide and PbfCl, and the overall yield (steps 1-3) is typically only less than 40%.

[0076] This paper develops and describes Figure 6 and 7 The improved method described in provides several advantages over the known methods. NaI is introduced as a catalyst for converting Boc-D-Arg-OH to Boc-D-Arg(Pbf)-OH (see Figure 6 The introduction of sodium iodide effectively significantly improves the reaction kinetics, and thus, the conversion rate of step 2 can be increased to more than 95%, and the assay yield can be increased to 90%. In addition, the total mass of impurities is also reduced.

[0077] The ethyl esters of D-Orn(Pbf)-OH and D-Arg(Pbf)-OH are the main impurities formed in Step 2 and Step 3, respectively. Figure 7 ), and is difficult to remove by crystallization, thus facilitating the need for multiple recrystallizations. The improved process described herein further comprises using isopropyl acetate (IPAc), a more sterically hindered ester compared to the more commonly used EtOAc, as a solvent for step 3, wherein Boc-D-Arg(Pbf)-OH is converted to D-Arg(Pbf)-OH. The use of IPAc significantly slows down the side reaction, transesterification, catalyzed by the strong acid HCl. As a result, the content of the corresponding impurity, the ester of D-Arg(Pbf)-OH, is reduced to less than about 0.5% (relative to greater than 1.0% in the process using EtOAc). In addition, isopropyl alcohol (IPA) / water was found to be a more powerful solvent system that allows removal of all process impurities and also minimizes product losses in the crystallization step. In a pilot run conducted at a laboratory scale (20 g), the purity of D-Arg(Pbf)-OH (step 3 intermediate) was increased to greater than 99.7% with only 2 crystallizations, and the overall yield (steps 1-3) was about 70%. One embodiment of this improved process is described in Example 6 below.

[0078] The skilled person will readily appreciate that the disclosure also extends to variants and derivatives of AMG 416. For example, in one embodiment, the methods provided herein may also be used with N-acetyl-D-cysteinyl-D-alanyl-D-arginyl-D-arginyl-D-alanyl-D-arginyl-D-alanyl-D-arginylamide and the disulfide of D-cysteine. In another embodiment, the disclosed formulation may also be used with N-acetyl-l-cysteinyl-l-alanyl-l-arginyl-l-arginyl-l-arginyl-l-alanyl-l-arginylamide and the disulfide of D-cysteine ​​and / or N-acetyl-l-cysteinyl-l-alanyl-l-arginyl-l-arginyl-l-arginyl-l-alanyl-l-arginylamide and the disulfide of l-cysteine. In another embodiment, the disclosed conception may also be used with the disulfide of N-acetyl-D-cysteinyl-D-arginyl-D-alanyl-D-arginyl-D-arginyl-D-alanyl-D-arginyl amide and l-cysteine, and / or the disulfide of N-acetyl-D-cysteinyl-D-arginyl-D-alanyl-D-arginyl-D-arginyl-D-alanyl-D-arginyl amide and D-cysteine. In addition, the present method may be used to prepare one or more compounds provided in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9 and / or Table 10 of International Patent Publication No. WO 2011 / 014707. In further embodiments, the methods described herein may also be used to prepare the compounds described in International Patent Publication No. WO 2011 / 014707. Example

[0079] The following examples, including experiments performed and results obtained, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims.

[0080] Example 1A Synthesis of linear backbone fragments The synthesis of the main chain linear fragment Figure 2 Rink Amide AM resin (Fmoc 2,4-dimethoxy-4'(carboxymethyloxy)-benzhydrylamine attached to aminomethyl resin) (1 kg) was added to DMF (5.8 L / kg) and the solution was stirred at 22°C. The resin was filtered and slurry washed. The sample was tested for residual Fmoc (UV test) and residual piperidine (pH). Kaiser and / or TNBS color test was performed to ensure that Fmoc deprotection had been performed.

[0081] The first six amino acid derivative couplings followed the same pre-activation, coupling and washing procedures. 1.6 eq of protected Fmoc amino acid was added to DMF at 22 ° C, 8.9 L / kg. Then Oxyma (2.45 eq) was added. The solution was cooled to 21 ° C and DIC (2.13 eq) was added to allow the reaction to proceed. The pre-activated solution was combined with the resin and allowed to react. DIC (1.07 eq) was added and the reaction was allowed to proceed at 22 ° C. The incomplete coupling of the sample was checked using Kaiser and / or TNBS color test. The material was washed, followed by Fmoc deprotection and further washing. The sample was checked for residual Fmoc (UV inspection) and residual piperidine (pH). Kaiser and / or TNBS color test was performed to ensure that Fmoc deprotection had occurred.

[0082] To a 1:1.7 DMF:DCM solution, 12 L / kg, was added Fmoc-D-Cys(Trt)-OH 1.6 eq followed by HOBt.H2O (2.45 eq). The solution was cooled to 20°C, DIC (2.13 eq) was added and the reaction was allowed to proceed. The preactivated solution was combined with the resin and the reaction was allowed to proceed at 22°C. DIC 1.07 eq was added to the SPPS reaction with a DMF / DCM ratio of about 1:1. The reaction was allowed to proceed at 22°C. The samples were checked for incomplete coupling using Kaiser and / or TNBS colorimetric tests.

[0083] The material was washed, followed by Fmoc deprotection and further washing. The samples were checked for residual Fmoc (UV inspection) and residual piperidine (pH). Kaiser and / or TNBS colorimetric tests were performed to ensure that Fmoc deprotection had occurred.

[0084] DMF (0 L / kg), acetic anhydride (1.06 L / kg) and pyridine (1.06 L / kg) were added to the solution and the solution was stirred for pre-activation. The pre-activated solution was combined with the resin and stirred at 22°C. The material was filtered and washed. The samples were checked for incomplete capping using Kaiser and / or TNBS color test. The material was slurried. The resin was dried without stirring under nitrogen. A sample of the dry resin was taken and checked for LOD and residual solvent. See Figure 2 .

[0085] Example 1B Synthesis of linear backbone fragments The main chain linear fragment of AMG 416 was synthesized in Figure 2 The peptide chain was built from the C-terminus to the N-terminus on Rink AM amide resin, with one amino acid per cycle. Each cycle consisted of two reaction steps: 1) cleavage of Fmoc from the N-terminus; 2) coupling of the next Fmoc-protected amino acid or final acetylation.

[0086] The beginning of SPPS: Rink AM amide resin (1.0 mole) was transferred into the SPPS reactor and washed with N,N'-dimethylformamide (DMF).

[0087] Fmoc cleavage: The resin from the previous step was suspended in a 20% piperidine / DMF solution for at least 10 min. The completion of the Fmoc cleavage was monitored by ultraviolet (UV) absorption measurements. After the Fmoc cleavage was complete, the resin was washed alternately with DMF and isopropanol (IPA) until a neutral pH was reached.

[0088] Fmoc-amino acid coupling: After the Fmoc cleavage step, the coupling reaction was carried out by mixing the resin with a solution of an Fmoc-protected amino acid derivative (>1.2 moles) and an activator (>1.8 moles) (N,N'-diisopropylcarbodiimide (DIC) and ethyl(oxime)-cyanoacetate (Oxyma)) in DMF. For the coupling of Fmoc-D-Cys(Trt)-OH, DIC and 1-hydroxybenzotriazole hydrate (HOBt) were used as activators, and a mixture of DMF and dichloromethane (DCM) was used as the reaction solvent. The reaction mixture was stirred at room temperature overnight. Kaiser and TNBS tests were performed to monitor the completion of the coupling. Negative results in both Kaiser and TNBS tests were required before the process proceeded to the next cycle. After each coupling or capping step, the resin was washed alternately with DMF and IPA.

[0089] Final acetylation: After the final Fmoc deprotection, the N-terminal amino group of the peptide was acetylated using acetic anhydride and pyridine in DMF. Kaiser and TNBS tests were performed to verify the completion of acetylation. If acetylation was incomplete, the same acetylation procedure was repeated until negative results were obtained from both Kaiser and TNBS tests.

[0090] Finally, the protected peptide backbone of the drug substance on the resin (AMG 416-resin) was isolated by filtration, washed with DMF, IPA and acetonitrile (ACN) and dried under reduced pressure.

[0091] Example 2A Cleavage of backbone linear fragments from the resin A mixed solution was prepared by combining DIW (0.16 L / kg), TFA (5.64 L / kg), TIPS (0.46 eq) and DPDS (6.41 eq) at room temperature and then cooling the solution to 0±2°C. The peptide on the resin was added to the mixed solution at 0±2°C, the solution was heated to 25°C and the reaction was allowed to proceed. The resin was removed by filtration and washed. The solution was maintained at -10°C and a 6.8:1 solution of IPE:MeCN (24.5 L / kg) was added at -10°C over time to control temperature and precipitation. The reaction was allowed to proceed and the AMG416SPy intermediate product was filtered and washed at -5°C. The SPy intermediate product was dried under full vacuum at 20°C. See Figure 3 .

[0092] Example 2B Cleavage of backbone linear fragments from the resin The cleavage solution was prepared in a reactor by mixing TFA, H2O and triisopropylsilane (TIPS) in a ratio of about 96.9:2.6:0.5 (v / v / v). DPDS (>1.2 moles) was added to the cleavage solution as an activation reagent for the thiol group of D-cysteine. AMG 416-resin (1.0 mole) was added to the reactor and the reaction mixture was stirred at room temperature for >1 h. The resin was removed by filtration. The filtrate and washing solution were transferred to another container and cooled. A cold anti-solvent mixture of diisopropyl ether (IPE) and ACN was then added to the solution to precipitate AMG 416-SPy. The suspension was filtered through a filter dryer, and the AMG 416-SPy filter cake was subsequently washed with ACN and IPE and dried under reduced pressure on the filter dryer at approximately 20°C.

[0093] Example 3 In situ conjugation of L-Cys / preparative HPLC AMG 416 SPy intermediate (1.0 mole) was added to 0.2% TFA solution. L-cysteine ​​(>1.1 moles) was added to the solution and the reaction was allowed to proceed at room temperature for at least 15 min.

[0094] Purification of crude AMG 416 was performed by preparative chromatography using a C18 silica gel stationary phase using ACN / H2O as mobile phase and TFA as modifier. The crude AMG 416 solution from stage III was loaded onto the column and a linear gradient method was used for the purification step. Elution was monitored by UV absorbance at 230 nm. After each load, the column was flushed with 80% ACN / water (v / v) until a stable UV baseline was reached. The fractions were stored at 5°C, sampled, and then the fractions with the desired purity (determined by HPLC) were pooled. The combined pool from the purification run was concentrated by performing a concentration run using the same HPLC column. The fractions were stored at 5°C. The fractions with the desired purity (determined by HPLC) were lyophilized to isolate the AMG 416 TFA salt. See Figure 4 .

[0095] Example 4 Salt conversion AMG 416 TFA salt was added to 10 L / kg of 15% water / IPA (v / v) solution at 10°C until complete dissolution was observed. This solution was added to 12M aqueous HCl 0.27 L / kg and IPA 49.4 L / kg solution by subsurface addition over 3 hours, resulting in direct precipitation of AMG 416 4.5 HCl salt. The batch was aged for 3 hours and sampled for analysis.

[0096] The material was filtered and slurried with 10 L / kg of 96 wt% IPA. The filter cake was then reslurried in 10 L / kg of 96% wt% IPA for 4 hours. The material was filtered and further slurried with 96 wt% IPA 10 L / kg and then IPA 10 L / kg. The material was dried at 25°C under full vacuum. The dried filter cake was dissolved in 8 L / kg of water and the batch was concentrated by distillation to remove residual IPA and to the desired concentration. The solution temperature was maintained below 25°C throughout the distillation. See Figure 5 .

[0097] Example 5 Purification of SPy intermediates and generation of AMG 416HCl An alternative method for preparing AMG 416HCl salt is described here. As described in Example 2 above, after cutting from the resin, precipitation and filtration, the SPy intermediate product is dried at 20°C under complete vacuum. The precipitate is then dissolved in 0.1% TFA aqueous solution and loaded onto a C-18 column for HPLC purification. The column is run at <60bar, and the solution temperature is 15-25°C from beginning to end. The eluent is 0.1% TFA / acetonitrile and 0.1% TFA / water. The fractions are stored at 5°C, sampled, and then the fractions are pooled. The combined pool from the two runs is diluted and the same HPLC column is used for concentration / purification runs to reduce the total volume and remove additional impurities. The fractions are stored at 5°C.

[0098] The fraction containing the AMG 416 SPy intermediate was subjected to azeotropic distillation to change the solvent from 0.1% TFA to 15% water / IPA solution, with IPA added as needed. L-cysteine ​​1.15eq was then added to the resulting AMG 416 SPy intermediate in IPA solution and the reaction was allowed to proceed at room temperature for conjugation to occur and the AMG 416 TFA salt to form, as described above in Example 4. The AMG 416 TFA solution was added to 0.27 L / kg of 12M aqueous HCl and 49.4 L / kg of IPA solution by subsurface addition over 3 hours, resulting in the direct precipitation of the AMG 416 4.5 HCl salt. The batch was aged for 3 hours and sampled for analysis.

[0099] The material was filtered and slurried with 96 wt% IPA 10 L / kg. The filter cake was then reslurried in 10 L / kg 96% wt% IPA for 4 hours. The material was filtered and further slurried with 10 L / kg 96 wt% IPA and then IPA 10 L / kg. The material was dried at 25°C under full vacuum. The dried filter cake was dissolved in 8 L / kg of water and the batch was concentrated by distillation to remove residual IPA and to the desired concentration. The solution temperature was maintained below 25°C throughout the distillation.

[0100] Example 6 Synthesis of HD-Arg(Pbf)-OH The synthesis of the backbone linear fragment as described in Example 1 requires the use of Fmoc-D-Arg(Pbf)-OH for the addition of D-arginine subunits, which account for 4 of the 7 residues in the linear fragment. An improved and more efficient method for synthesizing Fmoc-D-Arg(Pbf)-OH is described below.

[0101] The synthesis begins with protection of the amino group of D-Arg by di-tert-butyl dicarbonate (Boc2O), yielding Boc-D-Arg-OH in nearly quantitative yield after isolation by crystallization under standard procedures reported in the literature. The arginine side chain guanidinium group is protected with a Pbf group by addition of a 10 / 1 acetone / THF solution of Pbf-Cl (1.3 eq) in the presence of aqueous NaOH (4.3 eq) / NaI (5% mol) as base for 1 h (0-5°C), yielding Boc-D-Arg(Pbf)-OH (85-90% assay yield). See Figure 6 The Boc-D-Arg(Pbf)-OH / IPAc solution was treated with 4.8 eq of concentrated HCl at 20°C for about 6 h. After the reaction, the organic layer was discarded and the crude product was separated from the aqueous layer by adjusting the pH to 5 using NaOH, after which a white suspension was observed. Figure 7 . When the supernatant has a concentration of about 3 mg / ml at 20°C, the supernatant is filtered at room temperature, the resulting filter cake is washed with water and dried under vacuum. The overall assay yield after this step is about 80-85%. The purity of HD-Arg(Pbf)-OH is increased to >98.5% by the first recrystallization from 3 / 1 water / IPA (v / v). When the supernatant has a concentration of about 7 mg / mL, it is filtered and dried under vacuum. After this step, the overall assay yield is about 75%. (About 10% product loss is typically observed in this step). A second recrystallization from 4 / 1 water / IPA (v / v) is performed. Filtration is performed when the supernatant concentration is about 3.7 mg / mL. The second recrystallization increases the purity of HD-Arg(Pbf)-OH to about 99.84 area percent purity (LCAP) by HPLC, and impurities do not exceed 0.2 LCAP. The typical yield of this step is about 93%, with a product loss of about 5%. HD-Arg(Pbf)-OH is then reacted with FmocOSu following standard protocols and the product Fmoc-D-Arg(Pbf)-OH is isolated using standard procedures from the literature.

Claims

1. Method for preparing AMG 416 shown in SEQ ID NO: 1: The method comprises: providing a resin-bound peptide having a structure selected from the group consisting of Fmoc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[resin] (SEQ ID NO:2) and Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[resin] (SEQ ID NO:3); cleaving the peptide from the solid support; and Activates the side chain of D-Cys residues.

2. The method of claim 1, wherein said cleaving and said activating occur in the same container.

3. The method of claim 1, wherein the resin-bound peptide is contacted with a solution comprising water, trifluoroacetic acid, triisopropylsilane, and bipyridyl disulfide.

4. The method of claim 1, wherein D-Arg(Pbf)-OH is synthesized by converting Boc-D-Arg-OH into Boc-D-Arg(Pbf)-OH by mixing Boc-D-Arg-OH with PbfCl, NaOH and NaI.

5. The method of claim 4, further comprising converting Boc-D-Arg(Pbf)-OH to D-Arg(Pbf)-OH by mixing Boc-D-Arg(Pbf)-OH with HCl and IPAc.

6. The method of any one of claims 1 to 5, wherein after activation, a peptide of SEQ ID NO: 4 is provided: Ac-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2 (SEQ ID NO: 4).

7. The method of claim 6, wherein a peptide having the structure of Ac-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2 (SEQ ID NO: 4) is contacted with L-Cys.

8. A method for preparing AMG 416, comprising: Providing a resin-bound peptide selected from the following structures: Fmoc-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[resin] (SEQ ID NO:2) and Ac-D-Cys(Trt)-D-Ala-D-Arg(Pbf)-D-Arg(Pbf)-D-Arg(Pbf)-D-Ala-D-Arg(Pbf)-[resin] (SEQ ID NO:3); cleaving the peptide from the solid support; and Activates the side chain of D-Cys residues.

9. The method of claim 8, wherein said cleaving and said activating occur in the same container.

10. The method of claim 8, wherein the resin-bound peptide is contacted with a solution comprising water, trifluoroacetic acid, triisopropylsilane, and bipyridyl disulfide.

11. A method for preparing AMG 416, comprising: Providing a peptide having the structure of Ac-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2 (SEQ ID NO:4); and The peptide is contacted with L-Cys to produce a conjugated product.

12. The method of claim 11, wherein the peptide is contacted with an aqueous solution comprising L-Cys and trifluoroacetic acid.

13. The method of claim 11, further comprising lyophilizing the conjugated product.

14. The method of claim 11, further comprising contacting the conjugated product with an aqueous solution comprising IPA and HCl, thereby producing a precipitate comprising AMG 416HCl (SEQ ID NO: 1).

15. The method of claim 14, further comprising purifying the precipitate by HPLC.

16. A method for preparing AMG 416, comprising: providing a peptide having the structure of Ac-D-Cys(SPy)-D-Ala-D-Arg-D-Arg-D-Arg-D-Ala-D-Arg-NH2 (SEQ ID NO:4) in a TFA solution; Purify the peptide by HPLC; Solvent exchange by azeotropic distillation; and The peptide is contacted with L-Cys to produce a conjugated product.

17. The method of claim 16, wherein the peptide is contacted with an aqueous solution of L-Cys and water in IPA.

18. The method of claim 17, further comprising contacting the conjugated product with an aqueous solution comprising IPA and HCl, thereby producing a precipitate comprising AMG 416 HCl (SEQ ID NO: 1).

19. A method for synthesizing D-Arg(Pbf)-OH, comprising: The Boc-D-Arg-OH was converted to Boc-D-Arg(Pbf)-OH by mixing Boc-D-Arg-OH with PbfCl, NaOH and NaI.

20. The method of claim 19, further comprising: The Boc-D-Arg(Pbf)-OH was converted to D-Arg(Pbf)-OH by mixing the Boc-D-Arg(Pbf)-OH with HCl and IPAc.

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