Search

EP-4602064-B1 - METHOD OF MANUFACTURING A PEPTIDE WITH A LYSINE DERIVATIVE

EP4602064B1EP 4602064 B1EP4602064 B1EP 4602064B1EP-4602064-B1

Inventors

  • SCHÖNLEBER, Ralph O.
  • Kümin, Michael
  • ERMERT, PHILIPP
  • HINTERMANN, TOBIAS
  • SCHWINDLING, JOACHIM
  • RÜEFLI, Pascal
  • WILLIG, Felix

Dates

Publication Date
20260513
Application Date
20231009

Claims (15)

  1. A method of manufacturing a peptide P, the method comprises a step (c) (c) condensing an alpha amino acid derivative S-am, which has one unprotected alpha amino group or one unprotected alpha imino group, with a compound of formula Pr-L wherein R L-O-1 and R L-O-2 are independently of each other a carboxylic acid protecting group, to obtain a peptide Pr-L-S, wherein R L-N-1 is an amino protecting group of formula Bsmoc with * indicating the bond to the nitrogen atom (Bsmoc).
  2. The method according to claim 1, wherein the alpha amino acid derivative S-am is a compound of formula S-I-am or S-II-am wherein R SI-1 is -(AA nx ) m -O-[resin-1], -(AA nx ) m -N-[resin-2], -O-[resin-1], -N-[resin-2], -(AA nx ) m -OR SI-1-1 , OR SI-1-1 or NH 2 , R SI-2 is H, C 1-6 alkyl or C 1-6 alkyl mono-substituted with OR SI-2-1 , SR SI-2-2 , SCH 3 , NR SI-2-3 R SI-2-4 , CO-OR SI-2-5 , CO-NR SI-2-6 R SI-2-7 , N'-R SI-2-8 -N"-R SI-2-9 -guanidino, phenyl, para-(R SI-2-10 O)-phenyl, 1-R SI-2-11 -imidazol-4-yl or 1-R SI-2-12 -indol-3-yl, -(AA nx ) m - is a substituent consisting of m condensed alpha amino acid residues AA nx with each x being an integer and x running from 1 to m, each condensed alpha amino acid residue AA nx is independently chosen and in case it possesses a side chain with a functional group, the functional group is unprotected or protected by a protecting group provided that an interfering functional group is protected, m is an integer from 1 to 30, R SI-1-1 is H or a carboxylic acid protecting group R SI-2-1 is H or a hydroxy protecting group, R SI-2-2 is a thiol protecting group, R SI-2-3 and R SI-2-4 are H, an amino protecting group or form together an amino protecting group provided that not both are H, R SI-2-5 is H or a carboxylic acid protecting group, R SI-2-6 and R SI-2-7 are H or an amide protecting group, R SI-2-8 and R SI-2-9 are H or a guanidino protecting group provided that not both are H, R SI-2-10 is a protecting group for an aromatic hydroxy group, R SI-2-11 is H or a protecting group for an imidazole nitrogen atom, R SI-2-12 is H or a protecting group for an indole nitrogen atom, R SII-1 is as defined for R SI-1 , and the peptide Pr-L-S is a compound of formula Pr-L-S-I or Pr-L-S-II wherein R L-O-1 , R L-O-2 and R L-N-1 are defined as for formula Pr-L, R SI-1 , R SI-2 and R SII-1 are defined as for formula S-I-am or S-II-am.
  3. The method according to claim 2, which comprises the step (d) (d) removing the amino protecting group R L-N-1 at the compound of formula Pr-L-S-I or Pr-L-S-II to obtain a compound of formula L-S-I-am or L-S-II-am wherein R L-O-1 , R L-O-2 , R SI-1 , R SI-2 and R SII-2 are defined as for formula Pr-L-S-I or Pr-L-S-II.
  4. The method according to claim 3, which comprises the step (e) (e) condensing the compound of formula L-S-I-am or L-S-II-am with an alpha-amino acid derivative of formula T R T-N-1 -(AA py ) q -OH (T) wherein R T-N-1 is an amino protecting group, -(AA py ) q - is a substituent consisting of q condensed alpha amino acid residues AA py with each y being an integer and y running from 1 to q, each condensed alpha amino acid residue AA py is independently chosen and in case it possesses a side chain with a functional group, the functional group is unprotected or protected by a protecting group provided that an interfering functional group is protected, q is an integer from 1 to 30, to obtain a compound of formula T-L-S-I or T-L-S-II wherein R L-O-1 , R L-O-2 , R SI-1 , R SI-2 and R SII-2 are defined as for formula L-S-I-am or L-S-II-am, R T-N-1 and -(AA py ) q - are defined as for formula T.
  5. The method according to claim 4, which comprises one or more further condensing cycles applied to the compound of formula T-L-S-I or T-L-S-II, each of the one or more further condensing cycles comprises a first step of removing the amino protecting group R T-N-1 of the compound of formula T-L-S-I or T-L-S-II respectively the amino protecting group of the alpha amino group of the N-terminal amino acid residue of the peptide resulting from the previous condensing cycle to obtain the related peptide with one unprotected amino group, and a second step of condensing the unprotected amino group of the related peptide obtained at the first step with an alpha amino acid derivative, which has an alpha amino group at its N-terminal amino acid residue, which is protected by an amino protecting group and which has one unprotected alpha carboxylic acid group, which is located at its C-terminal amino acid residue, to obtain a peptide resulting from the condensing cycle.
  6. The method according to any preceding claim, wherein the alpha amino acid derivative Sam is covalently linked to a resin, and which comprises a step (x) (x) cleaving the peptide obtained from the last conducted condensing step from the resin by a cleaving composition to obtain a cleaved peptide.
  7. The method according to any one of claims 2 to 6, wherein the alpha amino acid derivative S-am of step (c) is of formula S-I-am, wherein R SI-1 is a -(AA nx ) 10 -O-[resin-1], AA n1 is Phe, AA n2 is Ile, AA n3 is Ala, AA n4 is a Trp with a protected sidechain, AA n5 is Leu, AA n6 is Val, AA n7 is an Arg with a protected sidechain, AA n8 is Gly, AA n9 is an Arg with a protected sidechain and AA n10 is Gly, resin-1 is a 2-chlorotritylamidomethyl resin or a 2-chlorotrityl resin, R SI-2 is 2-(tert-butyloxycarbonyl)ethyl.
  8. The method according to any preceding claim, which comprises a step (y) (y) removing all remaining protecting groups from the peptide obtained from the last conducted condensing step to obtain a peptide free of protecting groups.
  9. The method according to any preceding claim, wherein R L-O-1 and R L-O-2 are independently from each other tert-butyl or 3-methyl-pent-3-yl.
  10. The method according to any preceding claim, wherein at step (c) the compound of formula Pr-L is activated with a condensing agent of step (c).
  11. The method according to any preceding claim, wherein at step (c) a coupling additive of step (c) is present.
  12. The method according to claims 10 and 11, wherein the condensing agent of step (c) is a carbodiimide derivative, and the coupling additive of step (c) is cyano-hydroxyimino-acetic acid ethyl ester.
  13. The method according to any preceding claim, wherein the step (c) is conducted in a solvent of step (c) and the solvent of step (c) comprises N,N-dimethylformamide.
  14. A compound of formula Pr-L wherein R L-O-1 and R L-O-2 are independently of each other a carboxylic acid protecting group, R L-N-1 is an amino protecting group of formula Bsmoc with * indicating the bond to the nitrogen atom (Bsmoc).
  15. Use of a compound of formula Pr-L as defined in claim 1 in the synthesis of a peptide P.

Description

The present invention relates to a method of manufacturing a peptide, which contains a lysine residue with a fatty acid conjugation motif covalently linked to its epsilon nitrogen atom, the method comprising a condensing of an alpha amino acid derivative with a lysine derivative, which possesses the fatty acid conjugation motif covalently linked to its epsilon nitrogen atom and which possesses a specific amino protecting group, namely 1,1-dioxobenzo[b]thiophen-2-ylmethyloxycarbonyl, at its alpha nitrogen atom. It relates further to the lysine derivative and a method of manufacturing the lysine derivative. It relates also to a lysine precursor, which possesses a part of the fatty acid conjugation motif covalently linked to its epsilon nitrogen atom and which possesses the specific amino protecting group at its alpha nitrogen atom, and a method of manufacturing the lysine precursor. It relates additionally to a method of manufacturing a lysine intermediate, which possesses the specific amino protecting group at its alpha nitrogen atom. Semaglutide (CAS-No. 910463-68-2) is an active pharmaceutical ingredient and is known as a glucagon-like peptide-1 receptor agonist. Semaglutide, when it is written in the three-letter-code of peptides, has the following formula: Accordingly, Semaglutide has a linear 31-mer peptide backbone and carries at the epsilon nitrogen atom of its lysine20 a fatty acid side-chain, the epsilon nitrogen atom of lysine20 is substituted with HO-CO-(CH2)16-CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxy]acetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl]. WO 2006-097537 A2 discloses in its example 4 the peptide Semaglutide. Semaglutide is synthesized by an acylation of the eplison-nitrogen atom of lysine20 at an already complete linear peptide backbone. Accordingly, the fatty acid side chain at lysine20 is introduced after the complete linear peptide backbone of Semaglutide has been synthesized. CN 104356224 A discloses a synthesis of Semaglutide, which employs as a building block a Fmoc-protected lysine derivative, which carries already the fatty acid side chain. Accordingly, lysine20 carrying already a fatty acid side chain is introduced during the synthesis of the linear peptide backbone of Semaglutide. The employed Fmoc-protected lysine derivative (CAS-No. 1662688-20-1) is depicted below CN 113461801 A discloses at its example 1 a solid phase peptide synthesis of the aforementioned Fmoc-protected lysine derivative (CAS-No. 1662688-20-1) via a solid-phase conjugated Alloc-protected lysine derivative (CAS-No. 2721349-46-6), which is depicted below CN 115677827 A discloses at its example 4 a synthesis of Boc-Lys(tert-BuO-CO-(CH2)16-CO-gamma-Glu-2-[2-(2-aminoethoxy)ethoxyacetyl]-2-[2-(2-aminoethoxy)ethoxyacetyl])-Ala-OAII (CAS-No. 2921565-74-2) via a Boc-protected lysine derivative (CAS-No. 2921565-73-1), which is depicted below WO 96-25394 discloses 2-(4-nitrophenylsulfonyl)ethoxycarbonyl respectively Nsc-group as an amino-protecting group in solid phase peptide chemistry and states on its page 18 that Nsc-group is perfectly resistant to the action of acidic reagents usually used for the cleavage of protective groups of tert-butyl type. Protein and Peptide Letters (1997), vol. 4, No. 5, p. 307-312 discloses a comparative study of Fmoc- and Nsc-groups in automated solid phase peptide synthesis. It is concluded that comparative synthesis of three test peptides result in similarly looking HPLC-UV chromatograms. On page 312, a retention time of Nsc-Phe-OH with 16 minutes versus a retention of Fmoc-Phe-OH with 24 minutes is stated for an analytical HPLC with a C18 column and 0.1 % trifluoroacetic acid in a water / acetonitrile gradient. A decomposition seems not to occur at Nsc-Phe-OH in view of the statement of one retention time. Tetrahedron Letters (1994), vol. 35, no. 42, p. 7821-7824 discloses on its page 7822 the preparation of Nsc-Lys(Boc)-OH via its trimethylsilyl derivative. A yield of 83% is reported for a homogeneous product after recrystallization. During the work-up, an exposure to an aqueous 5% NaHCO3 solution at an extraction has taken place. Organic Letters (2001), vol. 3, no. 5, p. 781-783 discloses solid phase peptide synthesis with alpha-azide-protected amino acids and states at its table 2 that alpha-azido acids outperform comparative Fmoc-amino acid at several tested peptide sequences. Journal American Chemical Society (1997), vol. 119, p. 9915-9916 discloses Bsmoc group, which is an abbreviation for 1,1-dioxobenzo[b]thiophene-2-ylmethyloxycarbonyl, as an amino-protecting group in peptide synthesis. Bulletin of Korean Chemical Society (1998), vol. 19, no. 6, p. 696-698 discloses 2-(phenylsulfonyl)ethoxycarbonyl respectively Psc group for the orthogonal protection of the epsilon amino group of lysine in conjunction with the Boc group, i.e. Boc-Lys(Psc)-OH, and its usage in liquid phase peptide synthesis. RU 2196144 C1 discloses at its example 11 a peptide Psc-D-Phe-Cys(Bzm)-Phe-D-Trp(For)-Lys(P