EP3765488A1 - Process for the manufacture of pthrp analogue - Google Patents

Process for the manufacture of pthrp analogue

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Publication number
EP3765488A1
EP3765488A1 EP19709065.7A EP19709065A EP3765488A1 EP 3765488 A1 EP3765488 A1 EP 3765488A1 EP 19709065 A EP19709065 A EP 19709065A EP 3765488 A1 EP3765488 A1 EP 3765488A1
Authority
EP
European Patent Office
Prior art keywords
leu
lys
arg
fragment
glu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19709065.7A
Other languages
German (de)
French (fr)
Inventor
Walter Cabri
Ivan DE PAOLA
Antonio Ricci
Andrea ORLANDIN
Ivan GURYANOV
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fresenius Kabi Ipsum SRL
Original Assignee
Fresenius Kabi Ipsum SRL
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Application filed by Fresenius Kabi Ipsum SRL filed Critical Fresenius Kabi Ipsum SRL
Publication of EP3765488A1 publication Critical patent/EP3765488A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis

Definitions

  • the present invention relates to a manufacturing process for an analogue of parathyroid hormone related peptide (PTHrP), abaloparatide and its related intermediates.
  • PTHrP parathyroid hormone related peptide
  • Abaloparatide is an analog of human parathyroid hormone related peptide, PTHrP (1-34), represented by Formula (I):
  • the medicinal product containing abaloparatide (marketed under the trade name TYMLOSTM), was approved by the U.S. Food and Drug Administration (FDA) in April 2017 for the treatment of postmenopausal women with osteoporosis at high risk for fracture.
  • FDA U.S. Food and Drug Administration
  • WO97/02834 describes the synthesis of human PTH (1-34) and PTHrP analogues using Solid Phase Peptide Synthesis (SPPS), which involves step-by-step addition of amino acids which are Boc-protected at the amino terminus.
  • SPPS Solid Phase Peptide Synthesis
  • This method of peptide synthesis requires repeated deprotection cycles with strong acids such as trifluoroacetic acid (TFA), for the removal of the Boc-protecting group at each addition step.
  • TFA trifluoroacetic acid
  • the process requires the use of Hydrogen Fluoride (HF) for final cleavage of the peptide from the resin.
  • Hydrogen fluoride is a highly toxic and corrosive compound and its use is not recommended in large scale production of pharmaceutical compounds. As an overall consequence, these drawbacks result in an extremely difficult and environment unfriendly process.
  • CN 106146648 discloses the synthesis of a truncated abaloparatide analogue (aa 1-33) by using a fragment-based approach consisting of the synthesis of three fragments (aa 1-15, aa 16-23 and aa 24-33) on different resins such as a chloro trltyl resin or a Rink amide resin, and condensation of these fragments using different coupling reagents.
  • One of the possible reasons for the low yield as well as the low purity of this approach is the risk of racemization of amino acid residues during the condensation of fragments in solution phase.
  • the product obtained that way needs to be purified further due to the presence of various impurities resulting in further yield loss.
  • the C-terminal carboxylic group of the fragment has to be activated prior to coupling.
  • the activation of C-terminal carboxylic groups of the fragments may however lead to both activated ester and oxazolone formation, which can be easily racemized in the basic conditions (N. L. Benoiton. Chemistry of Peptide Synthesis. Tailor and Francis Group. 2006). Therefore, the crude peptides obtained this way contain impurities corresponding to the isomerized product. Since the isomeric impurities have very similar retention times as the required target peptide, it is very difficult to separate these impurities from the desired target peptide. In summary, the activation step required for sufficient coupling of fragments often results in racemisation and hence the formation of impurities, which are extremely difficult to remove.
  • the present invention provides an improved process for the preparation of the peptide abaloparatide which is an analogue of parathyroid hormone related peptide.
  • the process of the present invention results in a product with high yield and high purity.
  • the invention relates to the preparation of abaloparatide of formula I through a fragment-based convergent synthesis.
  • the process for the preparation of abaloparatide comprises the steps of:
  • fragment A is a fragment having an achiral amino acid at the C-terminal reactive site
  • fragment B is a fragment, optionally bound to a resin at its C-terminal amino acid
  • the fragments A and B are protected fragments.
  • the invention relates to novel intermediates for the synthesis of abaloparatide wherein the intermediate may be fragment A or fragment B.
  • Fragment A is selected from
  • fragment B is selected from
  • P is a side chain protecting group or hydrogen, independently for each amino acid
  • PG is an alpha-amino protecting group or hydrogen
  • R’ is a resin or a carboxy protecting group
  • R is a resin or NH 2 .
  • peptide fragment or“fragment” describe a peptide with a partial abaloparatide amino acid sequence, that can be optionally attached to a resin at its C-terminal amino acid, and that can be protected or unprotected.
  • protected fragment or“protected peptide fragment” describe a fragment which bears either a terminal protecting group or side-chain protecting groups, or both of them.
  • terminal protecting group refers to a protecting group for the alpha- amino group of an amino acid or of a peptide, or a peptide fragment.
  • the type of the terminal protecting group used is depending on the type of resin used and on the protective groups required for side chain protection.
  • the terminal protecting group preferably is tert-butyloxycarbonyl (Boc); and wherein fragment B is used, which preferably is linked to a Rink amide resin, the terminal protecting group preferably is 9-fluorenylmethyloxycarbonyl (Fmoc).
  • side-chain protecting group is a protecting group for an amino acid side-chain functional group which is not removed when the terminal protecting group for an amino acid is removed.
  • side-chain protecting groups are included to protect side chains of amino acids which are particularly reactive or labile, to avoid side reactions and/or branching of the growing molecule.
  • acid-labile protecting groups such as tert- butyloxycarbonyl (Boc), tert-butyl (tBu), trityl (Trt), 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl (Pbf) etc.
  • the criterion for selecting side-chain protecting groups is that the protecting group should generally be stable to the reagent under the reaction conditions selected for removing the terminal protecting group at each step of the synthesis and should be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain.
  • Resin is used to describe a solid support suitable to perform peptide synthesis.
  • Resin in the present context may be selected from the group comprising Rink amide resin (RAM resin), Rink acid resin, Rink amide MB HA resin, trityl based resin, Sieber resin and Wang resin.
  • Preferred resins are the 2-chloro trityl chloride resin (CTC resin), preferably for preparation of fragment A, and the Rink amide resin, preferably for preparation of fragment B and/or preparation of abaloparatide.
  • the carboxylic group of a C-terminal amino acid can be attached to a resin.
  • it can be in the form of a primary amide (when R is NH 2 ) or it can be protected with a carboxy protecting group such as an ester. In the latter cases, the coupling of these fragments is performed in Liquid Phase Peptide Synthesis.
  • the ester is preferably an ester that is stable in basic conditions, i.e. orthogonal to the terminal protecting groups.
  • the ester is tert-butyl ester, allyl ester or benzyl ester. More preferably, the ester is tert-butyl ester, which is removable in acidic conditions.
  • the present invention in one aspect, relates to the preparation of abaloparatide, represented by formula I:
  • Abaloparatide is prepared by fragment-based convergent synthesis, comprising coupling of peptide fragments.
  • the process for the preparation of abaloparatide comprises the steps of:
  • fragment A has an achiral amino acid at the C-terminal reactive site
  • fragment B is optionally bound to a
  • the peptide fragments in step a) are protected peptide fragments.
  • the peptide fragment A and peptide fragment B can both be prepared by a fragment-based convergent approach starting from shorter peptides, both in SPPS or LPPS.
  • peptide fragment A and peptide fragment B can also be prepared using step by step SPPS by coupling of amino acids according to the required sequence to the terminal amino acid residue attached to the resin using a coupling reagent and an activating agent.
  • the resin is activated by the removal of the protecting group.
  • the activated resin is coupled with the first amino acid wherein the amino acid is protected by a terminal protecting group and optionally a side-chain protecting group. Thereafter the following steps are repeated with different amino acids until the desired fragment, which is attached to the resin, is formed: i) removal of the terminal protecting group of amino acid
  • the terminal protecting group may be removed by treatment with a base.
  • the base may be an inorganic or organic base.
  • the base is an organic base selected from the group consisting of piperidine, piperazine, DBU and diethylamine, preferably piperidine. Even more preferably, the terminal protecting group is removed with 20% piperidine in DMF.
  • the coupling of amino acids takes place in the presence of a coupling reagent.
  • the coupling reagent may be selected from the group consisting of N,N'-diisopropylcarbodiimide (DIC), N,N’-dicyclohexylcarbodiimide (DCC), (Benzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(7-Aza-lH-benzotriazole-l- yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU), 2-(lH-benzotriazole-l-yl)- l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N-(3-di mcthylam i nopropyl )-N'- ethylcarbodiimide (EDC
  • the coupling reaction is further facilitated by the addition of an additive.
  • the additive is selected from the group consisting of l-hydroxybenzotriazole (HOBt), 2-hydroxypyridine N-oxide, N- hydroxysuccinimide, l-hydroxy-7-azabenzotriazole, endo-N-hydroxy-5-norbornene-2,3- dicarboxamide and ethyl-2-cyano-2-hydroxyimino acetate (also known as OxymaPure).
  • the reaction is carried out in the presence of ethyl-2-cyano-2-hydroxyiminoacetate.
  • step iii) possible unreacted sites after resin loading and after each coupling are blocked (“capped”) to prevent any side reactions and the formation of truncated sequences.
  • Capping is achieved by a short treatment of the peptide resin with a large excess of a highly reactive unhindered reagent, which is chosen according to the reactive site to be capped.
  • a highly reactive unhindered reagent which is chosen according to the reactive site to be capped.
  • the reagent is an acid derivative, such as an anhydride or a carboxyl chloride, in a basic medium.
  • acetic anhydride, or benzoyl chloride, and a base, for instance diisopropylethylamine, are used.
  • the reagent is an alcohol in a basic medium, such as methanol with diisopropylethylamine.
  • a basic medium such as methanol with diisopropylethylamine.
  • peptide fragments prepared as described above are optionally cleaved from the resin prior to step a) for the preparation of abaloparatide.
  • step a) coupling of a protected peptide fragment A with a protected peptide fragment B takes place to obtain protected abaloparatide or a shorter protected fragment.
  • Fragment A is selected from
  • Fragment B is selected from
  • P are a side-chain protecting groups or hydrogen, independently for each amino acid
  • PG is an alpha-amino protecting group or hydrogen
  • R’ is a resin or a carboxy protecting group
  • R is a resin or NH 2 .
  • the coupling of peptide fragments for the synthesis of abaloparatide or a shorter fragment thereof takes place in the presence of a coupling reagent.
  • the coupling reagent is selected from the list of coupling reagents reported above for step ii). More preferably, the reaction is carried out in the presence of N,N'-diisopropylcarbodiimide (DIC) .
  • DIC N,N'-diisopropylcarbodiimide
  • the coupling reaction is further facilitated by the addition of an additive.
  • the additive is selected from the list of additives reported above for step ii).
  • the reaction is carried out in the presence of ethyl-2-cyano-2-hydroxyimino acetate.
  • the coupling reaction may be carried out in the presence of a base, for instance a tertiary amine.
  • a base for instance a tertiary amine.
  • the base is selected from the group consisting of diisopropylethylamine, triethylamine, N-methylmorpholine and N-methylpiperidine; more preferably, the reaction is carried out in the presence of diisopropylethylamine.
  • the coupling of fragments takes place in the presence of a solvent selected from the group consisting of dimethylformamide, dimethylacetamide, dimethylsulfoxide, dichloromethane, chloroform, tetrahydrofuran, 2-methyl tetrahydrofuran, N-methylpyrrolidone and N- methylpyrrolidine.
  • a solvent selected from the group consisting of dimethylformamide, dimethylacetamide, dimethylsulfoxide, dichloromethane, chloroform, tetrahydrofuran, 2-methyl tetrahydrofuran, N-methylpyrrolidone and N- methylpyrrolidine.
  • the coupling is carried out in DMF.
  • the achiral amino acid at the C-terminal reactive site of fragment A may be a natural or an un natural amino acid.
  • the achiral amino acid may be selected from glycine (Gly) and 2- aminoisobutyric acid (Aib).
  • step b) abaloparatide or the shorter fragment thereof is deprotected and optionally cleaved from the resin by treatment with an acid; preferably with trifluoroacetic acid.
  • an acid preferably with trifluoroacetic acid.
  • the cleavage may be performed with a mixture TFA/water/TIPS/dodecanethiol (v/v 94/2.5/1/2.5).
  • step c) abaloparatide or the shorter fragment thereof obtained from step b) may be optionally purified by crystallization or chromatographic techniques well known in the art.
  • the coupling of peptide fragment A with fragment B, optionally bound to a resin is a promising alternative to step-by-step SPPS to produce the target peptides (i.e. abaloparatide or a shorter fragment thereof) because it reduces the formation rate of truncated sequences and unwanted isomeric forms, which are difficult to separate from the target peptide.
  • peptide fragment A is PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-OH (Fragment 1, (1-12)); and
  • R is a resin and P and PG are as defined above.
  • the coupling of Fragment 1 with Fragment 2 for the synthesis of abaloparatide takes place in the presence of a coupling reagent and an additive as described above. More preferably the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino-acetate.
  • Fragment 1 and Fragment 2 can be prepared by stepwise SPPS as explained above.
  • the fragments can be prepared by a fragment-based convergent approach starting from shorter peptides, both in SPPS or LPPS.
  • peptide Fragment 2 (13-34) is obtained by coupling of peptide Fragment 3
  • R is a resin and P and PG are as defined above.
  • the coupling of Fragment 3 with Fragment 5 for the synthesis of Fragment 2 takes place in the presence of a coupling reagent and an additive, as described above for step a). More preferably, the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino- acetate.
  • the coupling being carried out in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino-acetate; and subsequent Fmoc deblocking.
  • R is a resin and P and PG are as defined above.
  • the coupling of Fragment 4 with Fragment 5 for the synthesis of abaloparatide takes place in the presence of a coupling reagent and an additive as described above. More preferably the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino-acetate.
  • Fragment 4 and Fragment 5 can be prepared by stepwise SPPS as explained above.
  • the fragments can be prepared by a convergent approach starting from shorter peptides, both in SPPS or LPPS.
  • Fragment 4 (1-29) can be obtained by coupling of peptide Fragment 1
  • R’, P and PG are as defined above.
  • the coupling of Fragment 1 with Fragment 3’ for the synthesis of Fragment 4 takes place in the presence of a coupling reagent and an additive, as described above for step a). More preferably the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino- acetate.
  • the obtained compound is optionally deprotected and/or cleaved from the resin by treatment with an acid; preferably with trifluoroacetic acid. Subsequently, it is further reacted in a coupling reaction with another fragment or it is purified.
  • crude abaloparatide obtained by deprotection and cleavage from the resin is purified by using various methods known in the art.
  • the purification is performed by preparative HPLC.
  • a solution of the peptide is loaded into an HPLC column with a suitable solid phase, preferably Cl 8 or C8 modified silica, and a suitable aqueous mobile phase containing one or several organic solvents, preferably acetonitrile or methanol or mixtures thereof, is passed through the column. A gradient of the organic solvent is applied, if necessary.
  • the peptide with desired purity is collected and optionally lyophilized.
  • the peptide sequences of fragments A and B are partial sequences of abaloparatide, which are suitable for the coupling strategy that involves coupling via a site with an achiral amino acid at the C terminus of fragment A.
  • Step 1 Preparation of Boc-Ala-Val-Ser(tBu)-Glu(tBu)-His(Trt)-Gln(Trt)-Leu-Leu-His(Trt) ⁇ Asp(tBu)-Lys(Boc)-Gly-OH
  • Synthesis of the peptide fragment 1 was carried out by step-by-step SPPS using 2-chloro trltyl chloride resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Gly- OH and DIEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DMF were added.
  • the reaction mixture was stirred for 1 hour, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mL).
  • the loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.8 mmol/g.
  • the amino acids were pre-activated by DIC and OxymaPure (three-fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min.
  • Fmoc-Lys(Boc)- OH, Fmoc-Asp(7Bu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc- Glu(/Bu)-OH, Fmoc-Ser(7Bu)-OH, Fmoc-Val-OH and Boc-Ala-OH derivatives were used. The unreacted amino groups were capped each cycle with the mixture AC 2 0/DIPEA/DMF (2x30 min).
  • the Fmoc deprotection cycles were carried out by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml). After coupling with last Boc-protected amino acid, dry peptide resin was suspended in 4 ml of TFA/DCM 1:99 (v/v) solution and stirred for 15 min. Then the resin was filtered and the solution was neutralized by DIEA. The cleavage was repeated 3 times and the collected fractions were evaporated. The resulted oil was washed with water (3 x 1 ml) and dried in vacuo to obtain a powder that was used in the next step without further purification.
  • Synthesis of the peptide fragment 2 was carried out by SPPS using Fmoc-protected Rink amide resin (200 mg, loading 0.65 mmol/g). After swelling of the resin in 2 ml of DMF, Fmoc group was removed by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) and the resin was washed with DMF (4x2 ml).
  • Dry peptide resin from step 3 was suspended in 4 ml of TFA/water/TIPS/dodecanethiol (v/v 94/2.5/1/2.5) and stirred for 4 h. Then the resin was filtered and washed with 2 ml of TFA. The organic solutions were collected and 15 ml of methyl /e/7-butyl ether were added. The solid residue was filtered, washed with methyl /e/7-butyl ether and dried to get crude abaloparatide with an HPLC crude purity of 81%.
  • the crude abaloparatide as obtained in step 4 was dissolved in 1 ml of a mixture ACN/water (v/v 2/8) and purified by reversed phase preparative HPLC using C18 Jupiter (Phenomenex) column and water and acetonitrile containing 0.1% of TFA (Eluents A and B, respectively). The following gradient of acetonitrile was used: 10% of Eluent B in 15 min, then from 10% to 40% of Eluent B in 60 min. The fractions containing the product with more than 99% purity were collected and lyophilized to obtain 170 mg of abaloparatide (yield 65%).
  • Synthesis of the peptide fragment 5 was carried out by SPPS using Rink amide resin (200 mg, loading 0.65 mmol/g). After swelling of the resin in 2 ml of DMF, Fmoc protective group was removed by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) and the resin was washed with DMF (4x2 ml).
  • Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu- OH, Fmoc-Lys(Boc)-OH were pre-activated by DIC and OxymaPure (three-fold excess of the reagents respect to the loading of the resin) for 3 min and coupled to the resin in 60 min.
  • the unreacted amino groups were capped each cycle with the mixture Ac 2 0/DIPEA/DCM (2x30 min).
  • the Fmoc deprotection cycles were carried out as described above in order to finally obtain the title peptide fragment (coupled to the resin).
  • Step 2 Preparation of Fmoc-Lys(Boc)-Ser(tBu)-Ile-Gln(Trt)-Asp(tBu)-Leu-Arg(Pbf)- Arg(Pbf)- Arg(Pbf)-Glu(tBu)-Leu-Leu-Glu(tBu)-Lys(Boc)-Leu-Leu-Aib-OH (3)
  • Synthesis of the peptide fragment 3 was carried out by step-by-step SPPS using 2-chloro trltyl chloride resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Aib- OH and DIEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DMF were added. The reaction mixture was stirred for 1 hour, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mF).
  • the loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.4 mmol/g.
  • the amino acids were pre-activated by DIC and OxymaPure (three-fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min.
  • Fmoc-Feu-OH, Fmoc-Fys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc- Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH protected amino acids were used. The unreacted amino groups were capped each cycle with the mixture AC 2 0/DIPEA/DCM (2x30 min).
  • the Fmoc deprotection cycles were carried out by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml). After coupling with last Fmoc- protected amino acid, dry peptide resin was suspended in 4 ml of TFA/DCM 1:99 (v/v) solution and stirred for 15 min. Then the resin was filtered and the solution was neutralized by DIPEA. The cleavage was repeated 3 times and the collected fractions were evaporated. The resulted oil was washed with water (3 x lml) and dried in vacuo (yield 82%).
  • Step 4 Purification of Abaloparatide.
  • the crude peptide was purified as described in Example 1 to obtain 175 mg of Abaloparatide (Yield 72%).
  • Synthesis of the peptide fragment 24-34 was carried out by SPPS using Rink amide resin (200 mg, loading 0.65 mmol/g). After swelling of the resin in 2 ml of DMF, Fmoc protective group was removed by 20% piperidine in DMF (2x2 ml, 5 min and 15 min) and the resin was washed with DMF (4x2 ml).
  • the unreacted amino groups were capped using Ac 2 0/DIPEA/DMF mixture (2x30 min).
  • the intermediate Fmoc deprotection cycles were carried out as described above in order to obtain side-chain protected FEKFFAibKFHTA-Rink resin.
  • Synthesis of the peptide fragment 16-23 was carried out by SPPS using CTC resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Feu-OH and DIPEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DCM were added. The reaction mixture was stirred for 2 hours, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mF). The loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.6 mmol/g.
  • the amino acids were pre-activated by DIC and OxymaPure (three fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min.
  • Fmoc-Glu(7Bu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Feu-OH, Fmoc-Asp(7Bu)-OH, Fmoc-Gln(Trt)- OH derivatives were used. In case of Arg the couplings were repeated twice.
  • Synthesis of the peptide fragment 1-15 was carried out by SPPS using CTC resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Ile-OH and DIEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DCM were added. The reaction mixture was stirred for 2 hours, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mL). The loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.5 mmol/g.
  • the amino acids were pre-activated by DIC and OxymaPure (three fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min.
  • Step 4a Condensation of fragments (16-23) + (24-34) with HBTU/HOBt/DIPEA
  • Fmoc deprotection was carried out by 20% piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml) to obtain side-chain protected QDLRRRELLEKLLAibKLHTA-Rink resin (fragment 16-34).
  • Step 4b Condensation of fragments (16-23) + (24-34) with DIC/Oxyma
  • Fmoc deprotection was carried out by 20% piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml) to obtain side- chain protected QDFRRREFFEKFFAibKFHTA-Rink resin (fragment 16-34).
  • Step 5a Condensation of fragments (1-15) + (16-34) with PyBOP/HOBt/DIPEA
  • the cleavage of the peptide was carried out with 2 ml of the mixture TFA / ethanedithiol / thiophenol / phenol / water (80:5:5:5:5 v/v/v/v/v) for 3 h.
  • the resin was filtered off, the product was precipitated in 5 ml of diethyl ether, washed twice with diethyl ether and dried to obtain crude abaloparatide with an HPFC purity of 1.58%.
  • Step 5b Condensation of fragments (1-15) + (16-34) with DIC/Oxyma.
  • the cleavage of the peptide was carried out with 2 ml of the mixture TFA / ethanedithiol / thiophenol / phenol / water (80:5:5:5:5 v/v/v/v/v) for 3 h.
  • the resin was filtered off, the product was precipitated in 5 ml of diethyl ether, washed twice with diethyl ether and dried obtain crude abaloparatide with an HPFC purity of 0.64%.

Abstract

The present invention relates to a fragment based manufacturing process for abaloparatide, which is an analogue of parathyroid hormone related peptide (PTHrP), and its related intermediates resulting in a product with high yield and high purity.

Description

PROCESS FOR THE MANUFACTURE OF PTHrP ANALOGUE
FIELD OF THE INVENTION
The present invention relates to a manufacturing process for an analogue of parathyroid hormone related peptide (PTHrP), abaloparatide and its related intermediates.
BACKGROUND OF THE INVENTION
Abaloparatide is an analog of human parathyroid hormone related peptide, PTHrP (1-34), represented by Formula (I):
1 5 10 15 20
Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg- 25 30
Glu-Leu-Leu-Glu-Lys-Leu-Leu-Aib-Lys-Leu-His-Thr-Ala-NH2 Formula (I) and is also indicated as:
AVSEHQLLHDKGKSIQDLRRRELLEKLLAibKLHTA
The medicinal product containing abaloparatide (marketed under the trade name TYMLOS™), was approved by the U.S. Food and Drug Administration (FDA) in April 2017 for the treatment of postmenopausal women with osteoporosis at high risk for fracture.
WO97/02834 describes the synthesis of human PTH (1-34) and PTHrP analogues using Solid Phase Peptide Synthesis (SPPS), which involves step-by-step addition of amino acids which are Boc-protected at the amino terminus. This method of peptide synthesis requires repeated deprotection cycles with strong acids such as trifluoroacetic acid (TFA), for the removal of the Boc-protecting group at each addition step. In addition to this, the process requires the use of Hydrogen Fluoride (HF) for final cleavage of the peptide from the resin. Hydrogen fluoride is a highly toxic and corrosive compound and its use is not recommended in large scale production of pharmaceutical compounds. As an overall consequence, these drawbacks result in an extremely difficult and environment unfriendly process.
The production of large peptides, such as abaloparatide, by SPPS is challenging, due to aggregation and a generally lower solubility of longer peptide chains. This leads to low overall yield of the final product. The chromatographic purity of crude abaloparatide prepared by SPPS is lowered by the presence of a number of impurities with similar retention times (truncated sequences), reducing the purity grade of the synthesized peptide.
CN 106146648 discloses the synthesis of a truncated abaloparatide analogue (aa 1-33) by using a fragment-based approach consisting of the synthesis of three fragments (aa 1-15, aa 16-23 and aa 24-33) on different resins such as a chloro trltyl resin or a Rink amide resin, and condensation of these fragments using different coupling reagents.
The yield of the truncated abaloparatide (aa 1-33) obtained by condensation of these fragments followed by deprotection and cleavage from the resin as disclosed in CN 106146648 (examples 5 and 6), is only 35% and the purity of crude product is 75.2%. One of the possible reasons for the low yield as well as the low purity of this approach is the risk of racemization of amino acid residues during the condensation of fragments in solution phase. The product obtained that way needs to be purified further due to the presence of various impurities resulting in further yield loss.
In a fragment based peptide synthesis the C-terminal carboxylic group of the fragment has to be activated prior to coupling. The activation of C-terminal carboxylic groups of the fragments may however lead to both activated ester and oxazolone formation, which can be easily racemized in the basic conditions (N. L. Benoiton. Chemistry of Peptide Synthesis. Tailor and Francis Group. 2006). Therefore, the crude peptides obtained this way contain impurities corresponding to the isomerized product. Since the isomeric impurities have very similar retention times as the required target peptide, it is very difficult to separate these impurities from the desired target peptide. In summary, the activation step required for sufficient coupling of fragments often results in racemisation and hence the formation of impurities, which are extremely difficult to remove.
From the foregoing, it is apparent that the reported methods for the preparation of abaloparatide suffer from one or more of the following drawbacks: low yield, low purity, formation of side product impurities, formation of racemic impurities, use of hazardous reagents, and complicated process.
Hence, to avoid all these drawbacks, to obtain a product with higher yield, and higher purity, or with impurities that are easier to be removed, there still remains the need to provide an efficient, simple and industrially viable synthetic process which can overcome the drawbacks of the prior art and which provides abaloparatide in high yield as well as high purity. OBJECT OF THE INVENTION
It is an objective of the present invention to overcome the above-mentioned drawbacks of the prior art.
It is therefore an objective of the present invention to provide an improved process for the synthesis of abaloparatide, which provides product in high yield as well as high purity.
It is a further objective of the present invention to provide useful intermediates for the synthesis of abaloparatide.
SUMMARY OF THE INVENTION
The present invention provides an improved process for the preparation of the peptide abaloparatide which is an analogue of parathyroid hormone related peptide. The process of the present invention results in a product with high yield and high purity.
In one aspect, the invention relates to the preparation of abaloparatide of formula I through a fragment-based convergent synthesis.
Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg- Glu-Leu-Leu-Glu-Lys-Leu-Leu-Aib-Lys-Leu-His-Thr-Ala-NH2 Formula (I)
The process for the preparation of abaloparatide comprises the steps of:
a) coupling a first peptide fragment A with a second peptide fragment B, wherein
fragment A is a fragment having an achiral amino acid at the C-terminal reactive site, and fragment B is a fragment, optionally bound to a resin at its C-terminal amino acid;
b) deprotecting and optionally cleaving abaloparatide from the resin; and
c) optionally purifying abaloparatide.
During the coupling step a) preferably the fragments A and B are protected fragments.
In another aspect, the invention relates to novel intermediates for the synthesis of abaloparatide wherein the intermediate may be fragment A or fragment B.
Fragment A is selected from
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-OH (Fragment
1, (1-12)), PG-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)- Lys(P)-Leu-Leu-Aib-OH (Fragment 3, (13-29)), and
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-Lys(P)-Ser(P)- Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)-Lys(P)-Leu-Leu-Aib- OH (Fragment 4, (1-29)); and
fragment B is selected from
H-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)-Lys(P)- Leu-Leu-Aib-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 2, (13-34)),
H-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)-Lys(P)- Leu-Leu-Aib-R’ (Fragment 3’, (13-29)), and
H-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 5, (30-34)),
wherein
P is a side chain protecting group or hydrogen, independently for each amino acid,
PG is an alpha-amino protecting group or hydrogen,
R’ is a resin or a carboxy protecting group, and
R is a resin or NH2.
In a preferred embodiment, peptide fragment A is
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-OH (Fragment 1, (1-12));
peptide fragment B is
H-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)-Lys(P)- Leu-Leu-Aib-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 2, (13-34)),
wherein P, PG and R are as defined above;
and the coupling is performed in the presence of N,N’ -diisoprop ylcarbodiimide (DIC) and ethyl- 2-cyano-2-hydroxy-imino-acetate.
In another embodiment, peptide fragment A is
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-Lys(P)-Ser(P)- Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)-Lys(P)-Leu-Leu-Aib (Fragment 4, (1-29)); and
peptide fragment B is
H-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 5, (30-34)),
wherein P, PG and R are as defined above; and the coupling is performed in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino-acetate.
DETAILED DESCRIPTION OF THE INVENTION
The terms“peptide fragment” or“fragment” describe a peptide with a partial abaloparatide amino acid sequence, that can be optionally attached to a resin at its C-terminal amino acid, and that can be protected or unprotected.
The terms“protected fragment” or“protected peptide fragment” describe a fragment which bears either a terminal protecting group or side-chain protecting groups, or both of them.
The term“terminal protecting group” as used herein refers to a protecting group for the alpha- amino group of an amino acid or of a peptide, or a peptide fragment. The type of the terminal protecting group used is depending on the type of resin used and on the protective groups required for side chain protection. Herein, wherein fragment A is produced, preferably linked to a CTC resin, the terminal protecting group preferably is tert-butyloxycarbonyl (Boc); and wherein fragment B is used, which preferably is linked to a Rink amide resin, the terminal protecting group preferably is 9-fluorenylmethyloxycarbonyl (Fmoc).
As used herein,“side-chain protecting group” is a protecting group for an amino acid side-chain functional group which is not removed when the terminal protecting group for an amino acid is removed. Preferably side-chain protecting groups are included to protect side chains of amino acids which are particularly reactive or labile, to avoid side reactions and/or branching of the growing molecule. Illustrative examples include acid-labile protecting groups such as tert- butyloxycarbonyl (Boc), tert-butyl (tBu), trityl (Trt), 2,2,4,6,7-pentamethyldihydrobenzofuran-5- sulfonyl (Pbf) etc.
The criterion for selecting side-chain protecting groups is that the protecting group should generally be stable to the reagent under the reaction conditions selected for removing the terminal protecting group at each step of the synthesis and should be removable upon completion of the synthesis of the desired amino acid sequence under reaction conditions that will not alter the peptide chain.
“Resin” is used to describe a solid support suitable to perform peptide synthesis. Resin in the present context may be selected from the group comprising Rink amide resin (RAM resin), Rink acid resin, Rink amide MB HA resin, trityl based resin, Sieber resin and Wang resin. Preferred resins are the 2-chloro trityl chloride resin (CTC resin), preferably for preparation of fragment A, and the Rink amide resin, preferably for preparation of fragment B and/or preparation of abaloparatide.
The carboxylic group of a C-terminal amino acid can be attached to a resin. Alternatively, it can be in the form of a primary amide (when R is NH2) or it can be protected with a carboxy protecting group such as an ester. In the latter cases, the coupling of these fragments is performed in Liquid Phase Peptide Synthesis. The ester is preferably an ester that is stable in basic conditions, i.e. orthogonal to the terminal protecting groups. Preferably, the ester is tert-butyl ester, allyl ester or benzyl ester. More preferably, the ester is tert-butyl ester, which is removable in acidic conditions.
The present invention, in one aspect, relates to the preparation of abaloparatide, represented by formula I:
Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg- Glu-Leu-Leu-Glu-Lys-Leu-Leu-Aib-Lys-Leu-His-Thr-Ala-NPb Formula (I).
Abaloparatide is prepared by fragment-based convergent synthesis, comprising coupling of peptide fragments.
The process for the preparation of abaloparatide comprises the steps of:
a) coupling a first peptide fragment A with a second peptide fragment B, wherein
fragment A has an achiral amino acid at the C-terminal reactive site, and
fragment B is optionally bound to a
resin at its C-terminal amino acid;
b) deprotecting and optionally cleaving abaloparatide from the resin; and
c) optionally purifying abaloparatide.
Preferably the peptide fragments in step a) are protected peptide fragments.
The peptide fragment A and peptide fragment B can both be prepared by a fragment-based convergent approach starting from shorter peptides, both in SPPS or LPPS.
Alternatively, peptide fragment A and peptide fragment B can also be prepared using step by step SPPS by coupling of amino acids according to the required sequence to the terminal amino acid residue attached to the resin using a coupling reagent and an activating agent. The resin is activated by the removal of the protecting group. The activated resin is coupled with the first amino acid wherein the amino acid is protected by a terminal protecting group and optionally a side-chain protecting group. Thereafter the following steps are repeated with different amino acids until the desired fragment, which is attached to the resin, is formed: i) removal of the terminal protecting group of amino acid
ii) coupling of the next, optionally protected, amino acid (according to the required sequence) to the alpha-amino group of previous amino acid
iii) capping the unreacted groups.
In step i), the terminal protecting group may be removed by treatment with a base. The base may be an inorganic or organic base. Preferably the base is an organic base selected from the group consisting of piperidine, piperazine, DBU and diethylamine, preferably piperidine. Even more preferably, the terminal protecting group is removed with 20% piperidine in DMF.
In step ii), the coupling of amino acids takes place in the presence of a coupling reagent. The coupling reagent may be selected from the group consisting of N,N'-diisopropylcarbodiimide (DIC), N,N’-dicyclohexylcarbodiimide (DCC), (Benzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(7-Aza-lH-benzotriazole-l- yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU), 2-(lH-benzotriazole-l-yl)- l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N-(3-di mcthylam i nopropyl )-N'- ethylcarbodiimide (EDC). Preferably, the reaction is carried out in the presence of N,N'- diisopropylcarbodiimide (DIC).
The coupling reaction is further facilitated by the addition of an additive. The additive is selected from the group consisting of l-hydroxybenzotriazole (HOBt), 2-hydroxypyridine N-oxide, N- hydroxysuccinimide, l-hydroxy-7-azabenzotriazole, endo-N-hydroxy-5-norbornene-2,3- dicarboxamide and ethyl-2-cyano-2-hydroxyimino acetate (also known as OxymaPure). Preferably, the reaction is carried out in the presence of ethyl-2-cyano-2-hydroxyiminoacetate.
In step iii), possible unreacted sites after resin loading and after each coupling are blocked (“capped”) to prevent any side reactions and the formation of truncated sequences.
Capping is achieved by a short treatment of the peptide resin with a large excess of a highly reactive unhindered reagent, which is chosen according to the reactive site to be capped. For instance, when an amino group has to be capped, the reagent is an acid derivative, such as an anhydride or a carboxyl chloride, in a basic medium. Preferably, acetic anhydride, or benzoyl chloride, and a base, for instance diisopropylethylamine, are used.
As further example, when a chlorine site has to be capped, for instance after loading a trltyl based resin, the reagent is an alcohol in a basic medium, such as methanol with diisopropylethylamine. At the end of the capping step the reagents are filtered off and the resin is carefully washed before proceeding to the next alpha- amino deprotection step.
The peptide fragments prepared as described above are optionally cleaved from the resin prior to step a) for the preparation of abaloparatide.
In step a), coupling of a protected peptide fragment A with a protected peptide fragment B takes place to obtain protected abaloparatide or a shorter protected fragment.
Fragment A is selected from
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-OH (Fragment
1, (1-12)),
PG-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)- Lys(P)-Leu-Leu-Aib-OH (Fragment 3, (13-29)), and
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-Lys(P)-Ser(P)- Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)-Lys(P)-Leu-Leu-Aib- OH (Fragment 4, (1-29));
Fragment B is selected from
H-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)-Lys(P)- Leu-Leu-Aib-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 2, (13-34)),
H-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)-Lys(P)- Leu-Leu-Aib-R’ (Fragment 3’, (13-29)), and
H-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 5, (30-34));
wherein
P are a side-chain protecting groups or hydrogen, independently for each amino acid,
PG is an alpha-amino protecting group or hydrogen,
R’ is a resin or a carboxy protecting group, and
R is a resin or NH2. The coupling of peptide fragments for the synthesis of abaloparatide or a shorter fragment thereof takes place in the presence of a coupling reagent. Preferably, the coupling reagent is selected from the list of coupling reagents reported above for step ii). More preferably, the reaction is carried out in the presence of N,N'-diisopropylcarbodiimide (DIC) .
The coupling reaction is further facilitated by the addition of an additive. Preferably, the additive is selected from the list of additives reported above for step ii). Preferably, the reaction is carried out in the presence of ethyl-2-cyano-2-hydroxyimino acetate.
The coupling reaction may be carried out in the presence of a base, for instance a tertiary amine. Preferably, the base is selected from the group consisting of diisopropylethylamine, triethylamine, N-methylmorpholine and N-methylpiperidine; more preferably, the reaction is carried out in the presence of diisopropylethylamine.
The coupling of fragments takes place in the presence of a solvent selected from the group consisting of dimethylformamide, dimethylacetamide, dimethylsulfoxide, dichloromethane, chloroform, tetrahydrofuran, 2-methyl tetrahydrofuran, N-methylpyrrolidone and N- methylpyrrolidine. Preferably, the coupling is carried out in DMF.
The achiral amino acid at the C-terminal reactive site of fragment A may be a natural or an un natural amino acid. The achiral amino acid may be selected from glycine (Gly) and 2- aminoisobutyric acid (Aib).
In step b), abaloparatide or the shorter fragment thereof is deprotected and optionally cleaved from the resin by treatment with an acid; preferably with trifluoroacetic acid. For example, the cleavage may be performed with a mixture TFA/water/TIPS/dodecanethiol (v/v 94/2.5/1/2.5).
In step c), abaloparatide or the shorter fragment thereof obtained from step b) may be optionally purified by crystallization or chromatographic techniques well known in the art.
The coupling of peptide fragment A with fragment B, optionally bound to a resin, is a promising alternative to step-by-step SPPS to produce the target peptides (i.e. abaloparatide or a shorter fragment thereof) because it reduces the formation rate of truncated sequences and unwanted isomeric forms, which are difficult to separate from the target peptide.
In a preferred embodiment, peptide fragment A is PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-OH (Fragment 1, (1-12)); and
peptide fragment B is
H-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)-Lys(P)- Leu-Leu-Aib-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 2, (13-34));
wherein R is a resin and P and PG are as defined above.
Preferably, the coupling of Fragment 1 with Fragment 2 for the synthesis of abaloparatide takes place in the presence of a coupling reagent and an additive as described above. More preferably the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino-acetate.
In a more preferred embodiment, peptide fragment 1 is
Boc-Ala-Val-Ser(tBu)-Glu(tBu)-His(Trt)-Gln(Trt)-Leu-Leu-His(Trt)-Asp(tBu)-Lys(Boc)-Gly- OH (1-12));
fragment 2 is
H-Lys(Boc)-Ser(tBu)-Ile-Gln(Trt)-Asp(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Glu(tBu)-Leu- Leu-Glu(tBu)-Lys(Boc)-Leu-Leu-Aib-Lys(Boc)-Leu-His(Trt)-Thr(tBu)-Ala-Rink amide resin (13-34));
and the coupling is carried out in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino-acetate.
Fragment 1 and Fragment 2 can be prepared by stepwise SPPS as explained above. Alternatively, the fragments can be prepared by a fragment-based convergent approach starting from shorter peptides, both in SPPS or LPPS.
Preferably, peptide Fragment 2 (13-34) is obtained by coupling of peptide Fragment 3
PG-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)- Lys(P)-Leu-Leu-Aib-OH (Fragment 3, (13-29)),
with peptide Fragment 5
H-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 5, (30-34)),
wherein R is a resin and P and PG are as defined above.
Preferably, the coupling of Fragment 3 with Fragment 5 for the synthesis of Fragment 2 takes place in the presence of a coupling reagent and an additive, as described above for step a). More preferably, the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino- acetate.
More preferably, peptide fragment 2
H-Lys(Boc)-Ser(tBu)-Ile-Gln(Trt)-Asp(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Ghi(tBu)-Leu- Leu-Glu(tBu)-Lys(Boc)-Leu-Leu-Aib-Lys(Boc)-Leu-His(Trt)-Thr(tBu)-Ala-Rink amide resin (13-34))
is obtained by coupling of peptide fragment 3
Fmoc-Lys(Boc)-Ser(tBu)-Ile-Gln(Trt)-Asp(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Glu(tBu)- Leu-Leu-Glu(tBu)-Lys(Boc)-Leu-Leu-Aib-OH (13-29),
with peptide fragment 5
H-Lys(Boc)-Leu-His(Trt)-Thr(tBu)-Ala-Rink amide resin (30-34);
the coupling being carried out in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino-acetate; and subsequent Fmoc deblocking.
In another embodiment, peptide fragment A is
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-Lys(P)-Ser(P)- Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)-Lys(P)-Leu-Leu-Aib- OH (Fragment 4, (1-29)); and
peptide fragment B is
H-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 5, (30-34));
wherein R is a resin and P and PG are as defined above.
Preferably, the coupling of Fragment 4 with Fragment 5 for the synthesis of abaloparatide takes place in the presence of a coupling reagent and an additive as described above. More preferably the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino-acetate.
Fragment 4 and Fragment 5 can be prepared by stepwise SPPS as explained above. Alternatively, the fragments can be prepared by a convergent approach starting from shorter peptides, both in SPPS or LPPS.
Fragment 4 (1-29) can be obtained by coupling of peptide Fragment 1
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-OH (Fragment 1, (1-12));
with peptide Fragment 3’ H-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)-Lys(P)- Leu-Leu-Aib-R’ (Fragment 3’, (13-29)),
wherein R’, P and PG are as defined above.
Preferably, the coupling of Fragment 1 with Fragment 3’ for the synthesis of Fragment 4 takes place in the presence of a coupling reagent and an additive, as described above for step a). More preferably the coupling takes place in the presence of DIC and ethyl-2-cyano-2-hydroxy-imino- acetate.
The coupling of fragments described above takes place in the presence of a solvent and optionally of a base, which are preferably selected in the lists as described above.
After coupling, the obtained compound is optionally deprotected and/or cleaved from the resin by treatment with an acid; preferably with trifluoroacetic acid. Subsequently, it is further reacted in a coupling reaction with another fragment or it is purified.
In a preferred aspect of present invention, crude abaloparatide obtained by deprotection and cleavage from the resin, is purified by using various methods known in the art. Preferably, the purification is performed by preparative HPLC.
To this aim, a solution of the peptide is loaded into an HPLC column with a suitable solid phase, preferably Cl 8 or C8 modified silica, and a suitable aqueous mobile phase containing one or several organic solvents, preferably acetonitrile or methanol or mixtures thereof, is passed through the column. A gradient of the organic solvent is applied, if necessary. The peptide with desired purity is collected and optionally lyophilized.
Surprisingly, it was found that the coupling of the fragments described above produces abaloparatide in greater yield and higher purity, than the coupling of any other fragment intermediates.
Throughout the specification it is preferred that the peptide sequences of fragments A and B are partial sequences of abaloparatide, which are suitable for the coupling strategy that involves coupling via a site with an achiral amino acid at the C terminus of fragment A. ABBREVIATIONS
PTH Parathyroid hormone
PTHrP Parathyroid hormone related peptide
SPPS Solid phase peptide synthesis
LPPS Liquid phase peptide synthesis
MB HA resin Methyl benzhydryl amide resin
CTC 2-chloro trltyl chloride
Aib 2-Aminoisobutyric acid
Fmoc 9-Fhiorenylmethyloxycarbonyl
Boc tert-Butyloxycarbonyl
Trt Trityl
t-Bu Tertiary butyl
Pbf 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
HPLC High performance liquid chromatography
DIE A/DIPEA Diisopropylethylamine
TFA Trifluoroacetic acid
AC20 Acetic anhydride
ACN Acetonitrile
DMF Dimethyl formamide
DCM Dichloromethane
MeOH Methanol HFIP 1,1,1 ,3,3,3-hexafluoro-2-propanol
TIPS Triisopropyl silane
DIC N,N’ -diisoprop ylcarbodiimide
DCC N,N’ -dicyclohexylcarbodiimide
EDC N-(3-dimcthylami nopropyl )-N'-cth ylcarbodiimide
HOBt 1 -Hydroxybenzotriazole
HOAt 1 -Hydroxy-7-azabenzotriazole
TBTU N,N,N',N'-Tetramethyl-0-(benzotriazol-l- yl)uronium tetrafluoroborate
PyBOP (Benzotriazol- 1 -yloxy)tripyrrolidinophosphonium hexafluorophosphate
Oxyma/OxymaPure Ethyl-2-cyano-2-hydroxyiminoacetate
HBTU 3- [Bis(dimethylamino)methyliumyl] -3H- benzotriazol- 1 -oxide hexafluorophosphate
HATU 2-(7 -Aza- lH-benzotriazole- l-yl)-l, 1,3,3- tetramethyluronium hexafluoropho sphate
EXAMPLES
Detailed experimental parameters suitable for the preparation of abaloparatide according to the present invention are provided by the following examples, which are intended to be illustrative and not limiting of all possible embodiments of the invention.
Example 1: Preparation of abaloparatide via two-fragment approach
Step 1. Preparation of Boc-Ala-Val-Ser(tBu)-Glu(tBu)-His(Trt)-Gln(Trt)-Leu-Leu-His(Trt)~ Asp(tBu)-Lys(Boc)-Gly-OH (1) Synthesis of the peptide fragment 1 was carried out by step-by-step SPPS using 2-chloro trltyl chloride resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Gly- OH and DIEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DMF were added. The reaction mixture was stirred for 1 hour, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mL). The loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.8 mmol/g. The amino acids were pre-activated by DIC and OxymaPure (three-fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min. Fmoc-Lys(Boc)- OH, Fmoc-Asp(7Bu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc- Glu(/Bu)-OH, Fmoc-Ser(7Bu)-OH, Fmoc-Val-OH and Boc-Ala-OH derivatives were used. The unreacted amino groups were capped each cycle with the mixture AC20/DIPEA/DMF (2x30 min). The Fmoc deprotection cycles were carried out by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml). After coupling with last Boc-protected amino acid, dry peptide resin was suspended in 4 ml of TFA/DCM 1:99 (v/v) solution and stirred for 15 min. Then the resin was filtered and the solution was neutralized by DIEA. The cleavage was repeated 3 times and the collected fractions were evaporated. The resulted oil was washed with water (3 x 1 ml) and dried in vacuo to obtain a powder that was used in the next step without further purification.
Step 2. Preparation of H-Lys(Boc)-Ser(tBu)-Ile-Gln(Trt)-Asp(tBu)-Leu-Arg(Pbf)- Arg(Pbf)- Arg(Pbf)-Glu(tBu)-Leu-Leu-Glu(tBu)-Lys(Boc)-Leu-Leu-Aib-Lys(Boc)-Leu- His(Trt)- Thr(tBu)-Ala-Rink amide resin (2)
Synthesis of the peptide fragment 2 was carried out by SPPS using Fmoc-protected Rink amide resin (200 mg, loading 0.65 mmol/g). After swelling of the resin in 2 ml of DMF, Fmoc group was removed by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) and the resin was washed with DMF (4x2 ml). Fmoc-Ala-OH, Fmoc-Thr(/Bu)-OH, Fmoc-His(Trt)-OH, Fmoc-Feu- OH, Fmoc-Fys(Boc)-OH, Fmoc-Aib-OH, Fmoc-Glu(/Bu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asp(7Bu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ser(7Bu)-OH, (three-fold excess respect to the loading of the resin) were pre-activated by DIC and OxymaPure (three-fold excess of the reagents respect to the loading of the resin) for 3 min and coupled to the resin in 60 min. The unreacted amino groups were capped each cycle using Ac20/DIPEA/DMF mixture (2x30 min). The Fmoc deprotection cycles were carried out as described in step 1, Example 1, in order to finally obtain protected KSIQDLRRRELLEKLLAibKLHTA-Rink amide resin, i.e. the title fragment.
Step 3. Condensation of fragments (1) + (2)
A solution of peptide fragment 1 (255 mg, 0.1 mmol), as obtained in step 1, in 5 ml of DMF, containing 1 equivalent of DIC/OxymaPure and 2 equiv of DIEA was added to 0.08 mmol of peptide fragment 2 linked to the resin. The reaction was carried on for 48 h, then the solvent was filtered and the resin washed with DMF (2x5 ml) and DCM (2x5 ml) and dried in vacuo, to obtain protected abaloparatide linked to the resin.
Step 4. Cleavage of abaloparatide from the resin
Dry peptide resin from step 3 was suspended in 4 ml of TFA/water/TIPS/dodecanethiol (v/v 94/2.5/1/2.5) and stirred for 4 h. Then the resin was filtered and washed with 2 ml of TFA. The organic solutions were collected and 15 ml of methyl /e/7-butyl ether were added. The solid residue was filtered, washed with methyl /e/7-butyl ether and dried to get crude abaloparatide with an HPLC crude purity of 81%.
Step 5. Purification of abaloparatide
The crude abaloparatide as obtained in step 4 was dissolved in 1 ml of a mixture ACN/water (v/v 2/8) and purified by reversed phase preparative HPLC using C18 Jupiter (Phenomenex) column and water and acetonitrile containing 0.1% of TFA (Eluents A and B, respectively). The following gradient of acetonitrile was used: 10% of Eluent B in 15 min, then from 10% to 40% of Eluent B in 60 min. The fractions containing the product with more than 99% purity were collected and lyophilized to obtain 170 mg of abaloparatide (yield 65%).
Example 2: Preparation of abaloparatide via three-fragment approach
Step 1. Preparation of H-Lys(Boc)-Leu-His(Trt)-Thr(tBu)-Ala-Rink amide resin (5)
Synthesis of the peptide fragment 5 was carried out by SPPS using Rink amide resin (200 mg, loading 0.65 mmol/g). After swelling of the resin in 2 ml of DMF, Fmoc protective group was removed by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) and the resin was washed with DMF (4x2 ml). Fmoc-Ala-OH, Fmoc-Thr(tBu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu- OH, Fmoc-Lys(Boc)-OH (three-fold excess respect to the loading of the resin) were pre-activated by DIC and OxymaPure (three-fold excess of the reagents respect to the loading of the resin) for 3 min and coupled to the resin in 60 min. The unreacted amino groups were capped each cycle with the mixture Ac20/DIPEA/DCM (2x30 min). The Fmoc deprotection cycles were carried out as described above in order to finally obtain the title peptide fragment (coupled to the resin).
Step 2. Preparation of Fmoc-Lys(Boc)-Ser(tBu)-Ile-Gln(Trt)-Asp(tBu)-Leu-Arg(Pbf)- Arg(Pbf)- Arg(Pbf)-Glu(tBu)-Leu-Leu-Glu(tBu)-Lys(Boc)-Leu-Leu-Aib-OH (3)
Synthesis of the peptide fragment 3 was carried out by step-by-step SPPS using 2-chloro trltyl chloride resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Aib- OH and DIEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DMF were added. The reaction mixture was stirred for 1 hour, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mF). The loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.4 mmol/g. The amino acids were pre-activated by DIC and OxymaPure (three-fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min. Fmoc-Feu-OH, Fmoc-Fys(Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc- Gln(Trt)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH protected amino acids were used. The unreacted amino groups were capped each cycle with the mixture AC20/DIPEA/DCM (2x30 min). The Fmoc deprotection cycles were carried out by 20% solution of piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml). After coupling with last Fmoc- protected amino acid, dry peptide resin was suspended in 4 ml of TFA/DCM 1:99 (v/v) solution and stirred for 15 min. Then the resin was filtered and the solution was neutralized by DIPEA. The cleavage was repeated 3 times and the collected fractions were evaporated. The resulted oil was washed with water (3 x lml) and dried in vacuo (yield 82%).
Step 3. Condensation of fragments (5) and (3)
A solution of peptide fragment 3 (375 mg, 0.1 mmol) in 5 ml of DMF, containing 1 equivalent of DIC/OxymaPure and 2 equiv of DIPEA, was added to 0.08 mmol of peptide fragment 5 linked to the resin. The reaction was left to continue for 48 h, then the solvent was filtered and the resin washed with DMF (2x5 ml) and DCM (2x5 ml) and dried in vacuo to get the peptide fragment 2. Abaloparatide preparation was completed by using the same protocol as reported in example 1, step 3, to obtain crude abaloparatide with overall yield of 77% and HPFC Purity of 83%.
Step 4. Purification of Abaloparatide. The crude peptide was purified as described in Example 1 to obtain 175 mg of Abaloparatide (Yield 72%).
Example 3 (Reference): Preparation of abaloparatide via three-fragment approach according to CN106146648
Step 1. Preparation of Leu-Glu(tBu)-Lys(Boc)-Leu-Leu-Aib-Lys(Boc)-Leu-His(Trt)-Thr(tBu)- Ala-Rink resin (24-34)
Synthesis of the peptide fragment 24-34 was carried out by SPPS using Rink amide resin (200 mg, loading 0.65 mmol/g). After swelling of the resin in 2 ml of DMF, Fmoc protective group was removed by 20% piperidine in DMF (2x2 ml, 5 min and 15 min) and the resin was washed with DMF (4x2 ml). Fmoc-Ala-OH, Fmoc-Thr(/Bu)-OH, Fmoc-His(Trt)-OH, Fmoc-Feu-OH, Fmoc- Fys(Boc)-OH, Fmoc-Aib-OH, Fmoc-Feu-OH, Fmoc-Feu-OH, Fmoc-Fys(Boc)-OH, Fmoc- Glu(/Bu)-OH (three-fold excess respect to the loading of the resin) were pre-activated by DIC and OxymaPure (three-fold excess of the reagents respect to the loading of the resin) for 3 min and coupled to the resin in 60 min. The unreacted amino groups were capped using Ac20/DIPEA/DMF mixture (2x30 min). The intermediate Fmoc deprotection cycles were carried out as described above in order to obtain side-chain protected FEKFFAibKFHTA-Rink resin.
Step 2. Preparation of Fmoc-Gln(Trt)-Asp(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Glu(tBu)- Leu-OH (16-23)
Synthesis of the peptide fragment 16-23 was carried out by SPPS using CTC resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Feu-OH and DIPEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DCM were added. The reaction mixture was stirred for 2 hours, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mF). The loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.6 mmol/g. The amino acids were pre-activated by DIC and OxymaPure (three fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min. Fmoc-Glu(7Bu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Feu-OH, Fmoc-Asp(7Bu)-OH, Fmoc-Gln(Trt)- OH derivatives were used. In case of Arg the couplings were repeated twice. The unreacted amino groups were capped with the mixture Ac20/DIPEA/DMF (2x30 min). The intermediate Fmoc deprotection was carried out by 20% piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml). After completion of the synthesis dry peptide resin was suspended in 5 ml of 30% HFIP/DCM and stirred for 1 h. Then the resin was filtered and the cleavage was repeated. The collected solutions of the peptide were concentrated to 1 ml and 5 ml of diethyl ether were added. The precipitate was centrifuged, washed twice with 5 ml of diethyl ether and dried to obtain protected Fmoc-QDLRRREL-OH with HPLC purity 95%.
Step 3. Preparation of Boc-Ala-Val-Ser(tBu)-Glu(tBu)-His(Trt)-Gln(Trt)-Leu-Leu-His(Trt)- Asp(tBu)-Lys(Boc)-Gly-Lys(Boc)-Ser(tBu)-Ile-OH (1-15)
Synthesis of the peptide fragment 1-15 was carried out by SPPS using CTC resin (1.6 mmol/g). After swelling of 1.0 g of the resin using 5 ml of DCM, Fmoc-Ile-OH and DIEA (three-fold and six-fold excess, respectively, respect to the loading of the resin) in DCM were added. The reaction mixture was stirred for 2 hours, the unreacted sites of the resin were capped using a DCM/MeOH/DIPEA 17/2/1 solution (2x15 min) and the resin was washed with DMF (4x5 mL). The loading of the resin was checked by UV adsorption measurement after Fmoc deprotection and was found to be 0.5 mmol/g. The amino acids were pre-activated by DIC and OxymaPure (three fold excess of the reagents respect to the loading of the resin) before each coupling cycle during 3 min and then coupled to 200 mg of the resin in 60 min. Fmoc-Ser(7Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(7Bu)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(7Bu)-OH, Fmoc-Ser(tBu)- OH, Fmoc-Val-OH and Boc-Ala-OH derivatives were used. The unreacted amino groups were capped with the mixture AC2O/DIPEA/DMF (2x30 min). The intermediate Fmoc deprotection was carried out by 20% piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml). After completion of the synthesis dry peptide resin was suspended in 5 ml of 30% HFIP/DCM and stirred for 1 h. Then the resin was filtered and the cleavage was repeated. The collected solutions of the peptide were concentrated to 1 ml and 5 ml of diethyl ether were added. The precipitate was centrifuged, washed twice with 5 ml of diethyl ether and dried to obtain protected Boc- AV SEHQFFHDKGKSI-OH with HPFC purity 90%.
Step 4a. Condensation of fragments (16-23) + (24-34) with HBTU/HOBt/DIPEA
A solution of peptide (16-23) (780 mg, 0.325 mmol, 5 eq) in 2 ml of DMSO/DMF (1: 1 v/v), HBTU (123 mg, 0.325 mmol) and HOBt (44 mg, 0.325 mmol) were added, followed by DIPEA (84 mg, 0.65 mmol). The solution was added to peptide-resin (24-34). The reactions were continued for 4 h, the peptide-resin was filtered and washed with DMF (4x2 ml). Fmoc deprotection was carried out by 20% piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml) to obtain side-chain protected QDLRRRELLEKLLAibKLHTA-Rink resin (fragment 16-34).
Step 4b. Condensation of fragments (16-23) + (24-34) with DIC/Oxyma
A solution of peptide (16-23) (780 mg, 0.325 mmol, 5 eq) in 2 ml of DMSO/DMF (1: 1 v/v) DIC (41 mg, 0.325 mmol) and OxymaPure (46 mg, 0.325 mmol) was stirred for 3 min and added to peptide-resin (24-34). The reactions were continued for 4 h, the peptide-resin was filtered and washed with DMF (4x2 ml). Fmoc deprotection was carried out by 20% piperidine in DMF (2x2 ml, 5 min and 15 min) with following washing of the resin with DMF (4x2 ml) to obtain side- chain protected QDFRRREFFEKFFAibKFHTA-Rink resin (fragment 16-34).
Step 5a. Condensation of fragments (1-15) + (16-34) with PyBOP/HOBt/DIPEA
To a solution of peptide (1-15) (940 mg, 0.325 mmol, 5 eq) in 2 ml of DMSO/DMF (1: 1 v/v), PyBOP (170 mg, 0.325 mmol) and HOBt (44 mg, 0.325 mmol) were added, followed by DIPEA (84 mg, 0.65 mmol). The solution was added to peptide-resin obtained in step 4a. The reactions were continued for 4 h, the peptide-resin was filtered and washed with DMF (3x2 ml), DCM (3x2 ml) and dried. The cleavage of the peptide was carried out with 2 ml of the mixture TFA / ethanedithiol / thiophenol / phenol / water (80:5:5:5:5 v/v/v/v/v) for 3 h. The resin was filtered off, the product was precipitated in 5 ml of diethyl ether, washed twice with diethyl ether and dried to obtain crude abaloparatide with an HPFC purity of 1.58%.
Step 5b. Condensation of fragments (1-15) + (16-34) with DIC/Oxyma.
To a solution of peptide (1-15) (940 mg, 0.325 mmol, 5 eq) in 2 ml of DMSO/DMF (1: 1 v/v), DIC (41 mg, 0.325 mmol) and Oxyma (46 mg, 0.325 mmol) were added. The solution was stirred for 3 min and added to peptide-resin obtained in step 4b. The reactions were continued for 4 h, the peptide-resin was filtered and washed with DMF (3x2 ml), DCM (3x2 ml) and dried. The cleavage of the peptide was carried out with 2 ml of the mixture TFA / ethanedithiol / thiophenol / phenol / water (80:5:5:5:5 v/v/v/v/v) for 3 h. The resin was filtered off, the product was precipitated in 5 ml of diethyl ether, washed twice with diethyl ether and dried obtain crude abaloparatide with an HPFC purity of 0.64%.

Claims

1. A process for the preparation of abaloparatide of formula I
1 5 10 15 20
Ala-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg- 25 30
Glu-Leu-Leu-Glu-Lys-Leu-Leu-Aib-Lys-Leu-His-Thr-Ala-NFb (I) comprising the steps of:
a) coupling a first peptide fragment A with a second peptide fragment B, wherein
fragment A has an achiral amino acid at the C-terminal reactive site, and
fragment B is optionally bound to a resin at its C-terminal amino acid;
b) deprotecting and optionally cleaving abaloparatide from the resin; and
optionally purifying abaloparatide.
2. The process according to claim 1, wherein
fragment A is selected from the group of fragments consisting of
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-OH
(Fragment 1, (1-12)),
PG-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Ghi(P)- Lys(P)-Leu-Leu-Aib-OH (Fragment 3, (13-29)), and
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-Lys(P)-
Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Ghi(P)-Lys(P)-Leu-
Leu-Aib-OH (Fragment 4, (1-29)); and wherein
fragment B is selected from the group of fragments consisting of
H-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)- Lys(P)-Leu-Leu-Aib-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 2, (13-34)),
H-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)- Lys(P)-Leu-Leu-Aib-R’ (Fragment 3’, (13-29)), and
H-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 5, (30-34)),
wherein
P is a side chain protecting group or hydrogen, independently for each amino acid,
PG is an alpha-amino protecting group or hydrogen, R’ is a resin or a carboxy protecting group, and
R is a resin or NH2.
3. The process according to claim 2, wherein
fragment A is
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-OH
(Fragment 1, (1-12));
peptide fragment B is
H-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Ghi(P)- Lys(P)-Leu-Leu-Aib-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 2, (13-34)).
4. The process according to claim 2, wherein
fragment A is
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-Lys(P)- Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Ghi(P)-Lys(P)-Leu- Leu-Aib (Fragment 4, (1-29)); and
peptide fragment B is
H-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 5, (30-34)).
5. The process according to claim 3, wherein
Fragment 2 (13-34) is obtained by coupling of
PG-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Ghi(P)-
Lys(P)-Leu-Leu-Aib-OH (Fragment 3, (13-29)),
with
H-Lys(P)-Leu-His(P)-Thr(P)-Ala-R (Fragment 5, (30-34)).
6. The process according to claim 4, wherein
Fragment 4 (1-29) is obtained by coupling of
PG-Ala-Val-Ser(P)-Glu(P)-His(P)-Gln(P)-Leu-Leu-His(P)-Asp(P)-Lys(P)-Gly-OH
(Fragment 1, (1-12));
with
H-Lys(P)-Ser(P)-Ile-Gln(P)-Asp(P)-Leu-Arg(P)-Arg(P)-Arg(P)-Glu(P)-Leu-Leu-Glu(P)- Lys(P)-Leu-Leu-Aib-R’ (Fragment 3’, (13-29)).
7. The process according to any one of the previous claims, wherein the coupling is carried out in the presence of a coupling reagent selected from the group consisting of N,N’- diisopropylcarbodiimide (DIC), N,N’-dicyclohexylcarbodiimide (DCC), (Benzotriazol-l- yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(7-Aza-lH-benzotriazole- l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HATU), 2-(lH-benzotriazole-l-yl)- l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) and ethyl-dimethylaminopropyl carbodiimide (EDC), preferably in the presence of N,N’-diisopropylcarbodiimide (DIC).
8. The process according to any one of the previous claims, wherein the coupling is carried out in the presence of an activating agent selected from the group consisting of 1- hydroxybenzotriazole (HOBt), 2-hydroxypyridine N-oxide, N-hydroxysuccinimide, 1- hydroxy-7-azabenzotriazole (HOAt), endo-N-hydroxy-5-norbomene-2, 3-dicarboxamide and ethyl-2-cyano-2-hydroxyimino acetate, preferably ethyl-2-cyano-2-hydroxyimino acetate.
9. The process according to any one of the previous claims, wherein the coupling is carried out in the presence of N,N’ -diisopropylcarbodiimide and ethyl-2-cyano-2-hydroxyimino acetate.
10. The process according to any one of the previous claims, wherein the side chain protecting group P is a protecting group selected from the group consisting of tert-butyloxycarbonyl (Boc), tert-butyl (tBu), trityl (Trt) and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) group, and wherein the alpha-amino protecting group PG is selected between Fmoc and Boc group.
11. The process according to claim 3, wherein
fragment A is
Boc-Ala-Val-Ser(tBu)-Glu(tBu)-His(Trt)-Gln(Trt)-Leu-Leu-His(Trt)-Asp(tBu)-Lys(Boc)- Gly-OH (1-12));
fragment B is
H-Lys(Boc)-Ser(tBu)-Ile-Gln(Trt)-Asp(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Glu(tBu)- Leu-Leu-Glu(tBu)-Lys(Boc)-Leu-Leu-Aib-Lys(Boc)-Leu-His(Trt)-Thr(tBu)-Ala-Rink resin (13-34)); and the coupling is carried out in the presence of N,N’ -diisopropylcarbodiimide and ethyl-2-cyano-2-hydroxyimino acetate.
12. The process according to claim 11, wherein fragment B is obtained by coupling of
Fmoc- Lys(Boc)-Ser(tBu)-Ile-Gln(Trt)-Asp(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-Ghi(tBu)-
Leu-Leu-Glu(tBu)-Lys(Boc)-Leu-Leu-Aib-OH (13-29),
with
H-Lys(Boc)-Leu-His(Trt)-Thr(tBu)-Ala-Rink resin (30-34);
the coupling is carried out in the presence of N,N’-diisopropylcarbodiimide and ethyl-2-cyano-
2-hydroxyimino acetate;
and subsequent Fmoc deblocking.
EP19709065.7A 2018-03-12 2019-03-12 Process for the manufacture of pthrp analogue Withdrawn EP3765488A1 (en)

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