WO2022090448A1 - Novel acylating reagents - Google Patents

Novel acylating reagents Download PDF

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Publication number
WO2022090448A1
WO2022090448A1 PCT/EP2021/080091 EP2021080091W WO2022090448A1 WO 2022090448 A1 WO2022090448 A1 WO 2022090448A1 EP 2021080091 W EP2021080091 W EP 2021080091W WO 2022090448 A1 WO2022090448 A1 WO 2022090448A1
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Prior art keywords
chem
acylation
compound
peptide
insulin
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PCT/EP2021/080091
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French (fr)
Inventor
Kim Birkebæk JENSEN
Jesper Hyldal MIKKELSEN
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Novo Nordisk A/S
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Publication of WO2022090448A1 publication Critical patent/WO2022090448A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C309/00—Sulfonic acids; Halides, esters, or anhydrides thereof
    • C07C309/01—Sulfonic acids
    • C07C309/28—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C309/41—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton
    • C07C309/42—Sulfonic acids having sulfo groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing singly-bound oxygen atoms bound to the carbon skeleton having the sulfo groups bound to carbon atoms of non-condensed six-membered aromatic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/22—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms
    • C07C311/29—Sulfonamides, the carbon skeleton of the acid part being further substituted by singly-bound oxygen atoms having the sulfur atom of at least one of the sulfonamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • 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
    • C07K14/605—Glucagons
    • 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
    • C07K14/62—Insulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/02—Ortho- or ortho- and peri-condensed systems
    • C07C2603/04—Ortho- or ortho- and peri-condensed systems containing three rings
    • C07C2603/06—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members
    • C07C2603/10—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings
    • C07C2603/12—Ortho- or ortho- and peri-condensed systems containing three rings containing at least one ring with less than six ring members containing five-membered rings only one five-membered ring
    • C07C2603/18—Fluorenes; Hydrogenated fluorenes

Definitions

  • the present invention relates to novel acylating reagents, their preparation, and their use in preparing Bl acylated insulin and analogues thereof.
  • Non-limiting examples of pharmaceutical peptides or proteins which have been acylated include insulin peptides.
  • acylated insulin peptides are disclosed in, e.g., WO2009/115469.
  • Methods for acylating peptides and proteins are disclosed in, e.g., WOOO/55119 and W02010/029159.
  • N-Hydroxy Succinimide is a commonly used activator for acylation of insulin.
  • Phenolic activators are disclosed in WO2018/083335, including 3,5-dichloro-2- hydroxy-N,N-dimethyl-benzenesulfonamide (3,5-DC-HBSA).
  • WO2018/083335 discloses that acylation of insulin using 3,5-DC-HBSA predominantly results in acylation at the lysine in the B29 position of the insulin analogue. The acylation reaction was run at a pH of 11.7.
  • 4-Nitrophenol is another commonly used activator for the preparation of activated esters used in peptide acylation chemistry (Metabolism 1964, 1026-1031). For instance acylation of lysine residues in peptides is described in RSC Adv. 2021, 908-914.
  • the present invention relates to a novel acylating reagent in the form of an ester of a carboxylic acid of Chem. 3:
  • the present invention relates to a novel acylating reagent of Chem. 1:
  • This compound may typically be called an activated ester, an activated phenolic ester, an activated side chain, or an acylating reagent.
  • the phenol of Chem. 2 is used to activate the side chain of Chem. 3.
  • the present invention also relates to a method of preparing the acylating reagent of the invention by reacting a compound of Chem. 3 as defined herein with a compound of Chem. 2 as defined herein or a with a compound of Chem. 4: Chem. 4: wherein Z is a suitable leaving group such as halogen.
  • the present invention also relates to a method for selectively acylating the alpha amino group of an N-terminal amino acid in a peptide or a protein, the method comprising a step of reacting the peptide or protein with the acylating reagent of the invention.
  • One such peptide or protein is human insulin or human insulin analogues.
  • the activators of the present invention leads to selective acylation at the Bl position of human insulin and human insulin analogues when the acylation reaction is run at neutral pH. To the best of our knowledge, no other activators are known which primarily results in acylation at the Bl position of insulin.
  • the invention provides novel acylating reagents.
  • the invention provides an improved acylation process whereby the selectivity for acylation at Bl of insulin or insulin analogues is increased.
  • .LM uM.
  • a waved line in a chemical formula designates a point of attachment.
  • alkyl refers to saturated, monovalent hydrocarbon radicals.
  • alkenyl as used herein, refers to monovalent hydrocarbon radicals, which contain at least one carbon-carbon double bond.
  • alkynyl refers to divalent hydrocarbon radicals, which contain at least one carbon-carbon triple bond.
  • heteroaryl refers to a radical derived from an aromatic mono- or bicyclic ring system, in which 1, 2, 3, 4 or 5 carbon atoms are replaced by heteroatoms.
  • the ring heteroatoms are generally chosen from N, O and S, wherein N includes ring nitrogen atoms which carry a hydrogen atom or a substituent as well as ring nitrogen atoms which do not carry a hydrogen atom or a substituent.
  • N includes ring nitrogen atoms which carry a hydrogen atom or a substituent as well as ring nitrogen atoms which do not carry a hydrogen atom or a substituent.
  • 'peptide' and 'protein' refer to a compound which comprises a series of amino acids interconnected by amide (or peptide) bonds.
  • 'peptidyl' refers to a radical derived from such a peptide or protein.
  • the present invention relates to novel acylating reagents, methods of their preparation, the use thereof in preparing acylated peptides and proteins, and to novel insulin derivatives.
  • the present invention relates to a compound of Chem. 1:
  • the present invention relates to an acylating reagent of Chem. la, which is a compound of Chem. 1, wherein X is N(CHs)2.
  • TSAP 5- bromo-2-hydroxy-3-(trifluromethyl)benzenesulfonic acid
  • Chem. 2a In some embodiments the present invention relates to an acylating reagent of
  • Chem. lb which is a compound of Chem. 1, wherein X is OH.
  • Chem. 2b may briefly be referred to as TSP which stands for 5- bromo-2-hydroxy-3-(trifluromethyl)benzenesulfonic acid.
  • the compound of Chem. 1 may also be referred to as an acylating reagent, an activated side chain, or an an activated ester, and it is a phenolic ester of a compound of Chem. 3 as defined herein, and an activator of Chem. 2 as defined herein.
  • the salt of Chem. 1 is an alkali metal salt or a tertiary amine salt.
  • the acylating reagent of the present invention enables introduction of a wide range of substituents or side chains at the Bl position of human insulin or human insulin analogues.
  • the organic substituent can be any organic moiety.
  • the organic substituent is alkyl, heteroalkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, each being optionally substituted.
  • the organic substituent is a peptidyl.
  • the acylating reagent of the invention is a compound of
  • Chem. 7 wherein r is an integer in the range of 8-20; wherein q is an integer in the range of 0-3; wherein p is an integer in the range of 0-3; wherein X is OH or N(CHs)2; and L is an optional linker of Chem. 8: wherein each of k, I, and m independently represents an integer in the range of 0-4; or a salt, amide, or ester thereof.
  • the Chem. 8 element may be in its L- or D-form. In some embodiments, the Chem. 8 element is in the L-form.
  • the acylating reagent of the invention is used to introduce a reactive group which can be used to introduce further chemical groups to the side chain.
  • the reactive group is an azide, which can for instance be used to couple to any terminal alkynyl compounds in a 3+2 cycloaddition in the presence of a metal catalyst (e.g copper). This is a mild efficent way for making e.g bioconjucation.
  • the acylating agent is of Chem. 1, wherein Y is N3.
  • the acylating agent is of Chem. 9: wherein X is OH or N(CHs)2.
  • the reactive group is an halide, which can for instance be used to couple to any thiol bearing compounds in an alkylation reaction. This is a mild efficent way for making e.g bioconjucation between two macro molecules or ligation to a macro molecule.
  • the acylating agent is of Chem. 1, wherein Y is a halogen. In one embodiment, Y is Cl. In another embodiment, the acylating agent is of Chem. 9a: wherein X is OH or N(CHs)2 and W is F, Cl, Br, or I. In one embodiment, W is Cl.
  • Example F of the present application acylation of desB30 human insulin is carried out at neutral pH using acylating reagents of the invention (see Table 2).
  • acylation is performed using the same side chain and reaction conditions using the prior art activators NHS, 3,5-DC-HBSA, and PNP.
  • the acylation products are determined to show the selectivity of the various acylating reagents.
  • Example G an acylation reaction using the acylating reagent of the invention is carried out in different solvents, showing that optimal reaction conditions are obtained in an aqueous solvent (see Table 3).
  • Example H it is shown that the acylating reagent of Chem. 2b is particularly suitable for Bl selective acylation for side chains with low water solubility (see Table 4).
  • Example I of the present application acylation of desB30 human insulin is carried out at neutral pH using a range of side chains activated by the activators of the invention (see Table 5).
  • Example J it is shown that Bl selective acylation is not specific to insulin analogues having a phenylalanine (Phe) in position Bl, but that Bl selective acylation is also obtained with other amino acids in the Bl position of insulin.
  • Phe phenylalanine
  • Example K demonstrates the usefulness of the acylating reagent of the invention in preparing an insulin derivative with two different side chains in position Bl and B29, respectively, without the need for extra protection/deprotection steps.
  • Example L demonstrates that the acylating reagent of the invention can also be used to attach small peptides to the Bl position of insulin.
  • the present invention also relates to a method for preparing the acylating reagent of the invention.
  • the method comprises the step of reacting a compound of Chem. 3 as defined herein with a compound of Chem. 4:
  • Chem. 4 wherein Z is a leaving group.
  • the leaving group is a halogen.
  • the leaving group is Cl or F.
  • the leaving group is Cl.
  • the method comprises the step of reacting a compound of Chem. 3 as defined herein with the phenol (also referred to as activator) of Chem. 2a: Chem. 2a:
  • the reaction takes place as a one-step esterification reaction which results in the acylating reagent of the invention of Chem. la.
  • a suitable coupling reagent for this reaction is N,N'-dicyclohexylcarbodiimide (DCC) together with dimethyl aminopyridine (DMAP).
  • the method when Y comprises chemical groups that have been protected (such as carboxylic acid groups protected with, e.g., tBu or Bn), the method also comprises a step of de-protecting the acylating reagent.
  • the acylating reagent prepared by this method is as defined in any of the acylating reagent embodiments discussed above and/or in any of the "PARTICULAR EMBODIMENTS" further below.
  • the acylating reagent of the invention can be prepared on solid support using procedures of solid phase peptide synthesis well known in the art, or in solution phase as also well known in the art. Non-limiting examples of such preparation methods are included in the Experimental part of the present application.
  • the present invention also relates to a method for acylating an N-terminal amino acid in a peptide or a protein, the method comprising a step of reacting the peptide or protein with the acylating reagent of the invention.
  • the amino group being acylated is the alfa-amino group of the N-terminal amino acid residue in the peptide or protein. In some embodiments, the amino group being acylated is the alfa-amino group of the N-terminal amino acid residue in the B-chain of human insulin or a human insulin analogue. In some embodiments, the amino group being acylated is the alfa-amino group of an N-terminal phenylalanine (Phe), serine (Ser) or glutamic acid (Glu) residue in the B-chain of human insulin or a human insulin analogue. In some embodiments, the amino group being acylated is the alfa-amino group of the N-terminal phenylalanine residue in the B-chain of human insulin or a human insulin analogue.
  • the acylating reagent for use in this method is as defined in any of the acylating reagent embodiments discussed above and/or in the "PARTICULAR EMBODIMENTS" further below.
  • the acylation method of the invention takes place under suitable conditions, which are known by the person skilled in the art.
  • the acylation reaction takes place in an aqueous reaction medium (a reaction medium that contains water). More preferably, the reaction medium is substantially free of an organic solvent. However, smaller amoumts of organic solvents are tolerated in the reaction medium.
  • the amount of organic solvent present is less than 20% (vol%).
  • the pH in the acylation reaction mixture is in the range of pH 6-8.
  • the temperature in the reaction mixture is in the range of 20°C to 50°C. The acylation method of the present invention is quite robust.
  • the acylating reagent of the invention provides great flexibility as regards the addition of the acylating reagent of the invention to the peptide or protein to be acylated. Also, or alternatively there is no need for the reaction vessel to be of any particular design. Also, or alternatively there is no need for the stirring to be optimal or optimised.
  • the acylating reagent of the invention can be added as a solution, or it can be added as a solid - without impacting the yield of the desired product. This is contrary to the known NHS- based acylation method, where the acylating reagent must be added very slowly and under rigorous control due to its hydrolytic instability.
  • the acylation method of the present invention enables acylation at the Bl position of human insulin or a human insulin analogue, i.e. acylation at the N-terminal of the B-chain of human insulin or a human insulin analogue.
  • the selectivity is for the Bl position whereas the known NHS-based acylation method and methods in WO2108/083335 are less selective for the Bl position in human insulin and analogues thereof.
  • the acylation method of the present invention comprises a further step, after the acylation reaction, of purifying the desired product of the acylation reaction. Suitable methods of purifying acylated peptides and proteins are known by the person skilled in the art.
  • the acylation method of the present invention comprises a further step, prior to the acylation reaction, of dissolving the peptide or protein to be acylated.
  • the peptide or protein is dissolved in an aqueous solution. Suitable ranges for pH, concentration of peptide or protein, and temperature are known by the person skilled in the art.
  • the acylation method of the present invention also enables the selective acylation of position Bl of human insulin or human insulin analogues with one side chain at neutral pH, followed by acylation of another position such as the B29 position with a different side chain at alkaline pH. In two subsequent steps two different side chains can be attached to an insuline analogue without the need for extra protection/deprotection steps.
  • An example of such a di-modified insulin can be seen in Example K.
  • the acylation method of the invention refers to "peptide or protein" as it is in principle applicable to any peptide or protein, whatever the size (number of amino acid residues) or other structural parameter, having an N-terminal amino acid residue.
  • peptide may not always be quite clear.
  • a peptide is sometimes defined to contain a maximum of about 50 amino acid residues
  • a polypeptide sometimes to contain a minimum of about 50 amino acid residues
  • a protein sometimes to consist of one or more peptides or polypeptides arranged in a more complex structure which may be required for biological activity.
  • insulin which consists of two peptide chains each of a length of less than 50 amino acids, coupled together via Cys-Cys bonds
  • peptide is traditionally referred to as a peptide.
  • a peptide contains up to a total of 200 amino acid residues, in one or more individual peptide chains; and a protein contains more than 200 amino acids in total, in one or more individual peptide chains.
  • Non-limiting examples of peptides for use in the method of the invention include human insulin which is a peptide of 51 amino acid residues in total (native human insulin, 30 amino acids in the B-chain and 21 amino acids in the A-chain) and analogues hereof.
  • the peptide for use in the method of the invention contains a) at least 2 amino acid residues, b) at least 5 amino acid residues, c) at least 20 amino acids; and/or d) a maximum of 150 amino acid residues.
  • the protein contains no more than 2000 amino acid residues in total.
  • the peptide or protein for use in the acylation method of the invention is a peptide.
  • the peptide or protein for use in the acylation method of the invention is a protein.
  • amino acid residues incorporated in the peptide or protein for use in the acylation method of the invention may include coded and/or non-coded amino acid residues.
  • coded amino acids refers to the 20 "natural” amino acids (see, e.g., IUPAC, table 1, section 3AA-1). Unless otherwise specified, the amino acid residue(s) in the amino acid, peptide or protein for use in the acylation method of the invention are in the L-form.
  • the peptide or protein for use in the acylation method of the invention is a pharmaceutical peptide or protein, which means that the peptide or protein has an effect, demonstrated in vitro or in vivo, which is considered at least potentially relevant for the prophylaxis or treatment of one or more diseases.
  • diseases include diabetes, obesity, and related diseases and disorders.
  • Non-limiting examples of peptides or proteins to be acylated using the method of the invention include human insulin and analogues thereof.
  • the peptide or protein to be acylated using the method of the invention is an insulin peptide.
  • insulin peptide includes human insulin and analogues thereof.
  • the human insulin A-chain has the following sequence: GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1), while the B-chain has the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO:2).
  • the insulin analogue for use in the acylation method of the invention has a maximum of 10 amino acid changes as compared to human insulin. In some embodiments the maximum of 10 amino acid changes is as compared to human proinsulin.
  • the insulin analogue comprises at least the amino acid modification desB30.
  • A14E refers to the amino acid corresponding to amino acid residue no. 14 in the A-chain of human insulin having been replaced by Glu (E);
  • B16H refers to the amino acid corresponding to amino acid residue no. 16 in the B-chain of human insulin having been replaced by His (H);
  • desB30 refers to the amino acid corresponding to amino acid residue no. 30 in the B-chain of human insulin having been deleted.
  • the insulin analogue for use in the acylation method of the present invention is desB30 human insulin (A-chain of SEQ ID NO: 1 and B-chain of SEQ ID NO:3); or a pharmaceutically acceptable salt, amide, or ester thereof.
  • the insulin peptide for use in the acylation method of the invention is an analogue of human insulin, which can be prepared by recombinant expression. Suitable recombinant expression methods are known by the person skilled in the art, see e.g. WO2009/115469 referred to above.
  • the final acylated insulin peptide produced by the acylation method of the invention (excluding acylated proinsulin, pre-proinsulin, and analogues thereof) has affinity to an insulin receptor.
  • Suitable insulin receptor affinity assays are known in the art, see e.g. Example 178 of W02009/115469. Using this assay with 0% HSA the final acylated insulin peptide produced according to the invention has an affinity of at least 0.10%.
  • sulfonic acid salt is a tertiary amine salt such as a TEA salt, or an alkali metal salt such as a K salt, a Na salt, or a Li salt.
  • L is an optional linker of Chem. 8: wherein each of k, I, and m independently represents an integer in the range of 0-4; or a salt, amide, or ester thereof.
  • a compound of Chem. 13 or a salt, amide, or ester thereof.
  • a compound of Chem. 15 or a salt, amide, or ester thereof.
  • a compound of Chem. 16 or a salt, amide, or ester thereof.
  • a compound of Chem. 40 or a salt, amide, or ester thereof.
  • a compound of Chem. 33 or a salt, amide, or ester thereof.
  • a method for acylating an N-terminal amino acid in a peptide or a protein comprising a first step of preparing an acylating reagent using an activator of
  • Chem. 2 wherein X is OH or N(CHs)2; and a second step of reacting the said acylating reagent with the peptide or protein.
  • a method for acylating an N-terminal amino acid residue in a peptide or a protein comprising a step of reacting the peptide or protein with a compound as defined in any of embodiments 1-47.
  • N-terminal amino acid residue is phenylalanine (Phe), serine (Ser) or glutamic acid (Glu).
  • the buffer is selected from Phosphate buffer, Bicine buffer (N,N-Bis(2-hydroxyethyl)glycine buffer), HEPPS buffer (3-[4-(2- Hydroxyethyl)-l-piperazinyl]propane sulfonic acid buffer), HEPES buffer (4-(2- Hydroxyethyl)-l-piperazineethanesulfonic acid buffer), and Tris buffer (2-Amino-2- (hydroxymethyl)propane-l,3-diol).
  • solvent selected from ethanol, isopropanol, N-methyl pyrrolidinone (NMP), N,N-dimethylformamide (DMF), Dimethyl sulfoxide (DMSO), tetra hydrofuran (THF), Water (H2O) and acetonitrile (CH3CN).
  • Ado 8-Amino-3,6-dioxaoctanoic acid
  • DIPEA Diisopropylethylamine
  • HEPES 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid
  • HFIP Hexafluoroisopropanol
  • This section relates to methods for solid phase peptide synthesis (SPPS methods, including methods for de-protection of amino acids, methods for cleaving the peptide from resin, and for its purification), as well as methods for detecting and characterising the resulting side chains (LCMS and UPLC methods and NMR).
  • SPPS methods solid phase peptide synthesis
  • LCMS and UPLC methods and NMR X H spectra were recorded at 400 MHz on a Bruker Aeon 400 instrument.
  • 1,3-benzodioxole was used as the standard reference. Chemical shifts are reported in ppm on the 8 scale relatively to the chemical shift of the deuterated solvent.
  • the resin bound protected side chains were prepared on a 2-chlorotritylcloride resin using standard Fmoc chemistry.
  • the first Fmoc protected amino carboxylic acid (2 eq) (linker element) was dissolved in DCM and added to a DCM washed and drained resin.
  • a tertiary amine base such as DIPEA or TEA (4 Eq) was added and the resin mixture was agitated for a period of time between 12 and 17 hours at RT.
  • the resin was allowed to react with MeOH (0.79 mL/g resin) to cap free chloride sites at RT for 1 hour.
  • the resin was drained and flow washed three times with NMP or DMF (about 5.2 mL/g resin).
  • Fmoc deprotection was achieved using piperidine in NMP preferably 20% piperidine (1.05 mL/g resin) in NMP (4.15 mL/g resin), at RT for 15 to 45 min, typically 30 min, before the resin was washed thoroughly with NMP or DMF. The step was repeated until complete deprotection was obtained, typically two times or more. The resin was drained and flow washed three times or more with NMP or DMF (ca 5.2 mL/g resin) until the Chloranil test gave a negative result.
  • the resin was washed by DCM and the protected side chain was cleaved off from the resin by treatment with 1% TFA in DCM for 1-3 hours.
  • the cleavage solution was evaporated under vacuum to dryness, and the crude material was used without further purification in the activation step described in the next section.
  • Tert-butyl ester deprotection was made by following procedure; the protected activated side chain was dissolved in a mixture of TFA with 1-3% of water. The mixture was stirred at RT until the reaction was complete typically from 1 to 3 hours. Alternatively concentrated hydrochloric acid or TFA or a 1 : 1 mixture of TFA and a suitable solvent such as THF or DCM could be used instead. The product mixture was subjected to evaporation under vacuum to yield a crude oil. Precipitation of the oil in an appropriate solvent such as diethyl ether, isopropyl ether, tert-butyl methyl ether or heptane followed by filtration gave crude material.
  • an appropriate solvent such as diethyl ether, isopropyl ether, tert-butyl methyl ether or heptane followed by filtration gave crude material.
  • the activated side chains were dried under vacuum, analysed by UPLC, MS or NMR and used in the examples described in sections E, F, G and H without further purification.
  • Method 1 Solid desB30 human insulin was weighed out (28.5 mg, 5 pmol) and transferred to a 4 mL glass veil equipped with a magnetic stir bar. HEPES buffer (0.95 mL, 0.2M, pH 7.4) was added to the glass veil, giving a concentration of desB30 human insulin of 30 mg/mL. The activated side chain (1.25 equiv.) was weighed out in another glass veil and dissolved in 50 pL DMF. The side chain solution was then added to the insulin solution. The pH was adjusted to 7.4 using NaOH (0.5M) and the reaction mixture was then stirred at rt. for 24 hours. After 24 hours a small aliquot of the reaction mixture was taken out, diluted with a mixture of AcOH/HzO/MeCN (2: 1 : 1) and analysed by UPLC analysis.
  • HEPES buffer (0.95 mL, 0.2M, pH 7.4
  • the activated side chain (1.25 equiv.) was weighed out in another glass veil
  • Solid desB30 human insulin was weighed out (28.5 mg, 5 pmol) and transferred to a 4 mL glass veil equipped with a magnetic stir bar.
  • HEPES buffer (0.95 mL, 0.2M, pH 7.4) was added to the glass veil, giving a concentration of desB30 human insulin of 30 mg/mL.
  • the UPLC conditions, detector settings and mass spectrometer settings were: Gradient: Linear 95% - 0% (vol/vol) A, 0% - 95% (vol/vol) B and 5% (vol/vol) D, 4 min., 0.4 ml/min. UV detection at 214 and 280 nm. MS ionisation mode: API-ES-; Scan 50-4000 amu.
  • the UPLC conditions, detector settings and mass spectrometer settings were: Gradient: Linear 95% - 0% (vol/vol) A, 0% - 95% (vol/vol) B and 5% (vol/vol) D, 4 min., 0.4 ml/min. UV detection at 214 and 280 nm. MS ionisation mode: API-ES + ; Scan 50-4000 amu.
  • the compound was characterised by LC-MS, X H NMR.
  • the title compound was purified by flash column chromatography eluting with a gradient from 10% EtOAc in heptane to 30% EtOAc in heptane and obtained as a white solid (1.55 g, 4.44 mmol, 74%).
  • the compound was characterised by LC-MS, X H NMR.
  • the compound was prepared as described in W02010/029159.
  • Example C2 Preparation of reference compound 18-IT(lS)-l-carboxy-4-r2-r2-r2-r2-r2-r2-r2-r2- r2-(2,4-dichloro-6-sulfo-phenoxy)-2-oxo-ethoxy1ethoxy1ethylamino1-2-oxo- ethoxy1ethoxy1ethylamino1-4-oxo-butyl1amino1-18-oxo-octadecanoic acid (Chem. 11) Chem. 11:
  • the compound was prepared as described in WO2018/083335.
  • Example C3 Preparation of reference compound 22-carboxy-l-(4-nitrophenoxy)- l,10,19,24-tetraoxo-3,6,12,15-tetraoxa-9,18,23-triazahentetracontan-41-oic acid (Chem. 12)
  • Chem. 12 t-Bu protected C18-diacid-yGlu-Ado-Ado-OH (synthesized according to procedures found in section Al "methods of preparation and modifications" and procedures found in WO2018/083335) (0.5 mmol, 0.423 g), DCC (0.6 mmol, 0.124 g, 1.2 eq.) and DMAP (0.05 mmol, 0.006 g, 0.1 eq.) was dissolved in 15mL DCM, followed by addition of the prior art activator 4-Nitrophenol (PNP) (0.55 mmol, 0.077 g, l.leq). The resulting solution was stirred at RT for 18 hours. Next the DCU was removed by filtration.
  • PNP 4-Nitrophenol
  • the crude product was purified using silicagel column chromatography with a gradient eluent from DCM to 10% MeOH in DCM. After evaporation the product was obtained as a light brown oil.
  • Method Mod BU-l was used to cleave the tBu-esters for 1.5 hours. The cleavage mixture was evaporated under reduced pressure. The product was obtained as a light brown oil. (0.324 g, 0.38 mmol, 76%). Active content of material from 1H qNMR is 77% w/w The compound was characterised by LC-MS and X H NMR.
  • the activated side chain of Chem. 31 was generated in situ from N-carbobenzyloxy- glycine and 3,5-dichloro-2-hydroxy-benzenesulfonyl chloride (the prior art activator 3,5- DC-HBSA) using the general method for in situ generation of activated side chains.
  • D Synthesis of activated side chains of the invention
  • Example DI Preparation of l-(4-bromo-2-(/V,/V-dimethylsulfamoyl)-6-(trifluoromethyl)- phenoxy)-22-carboxy- 1, 10,19, 24-tetraoxo-3, 6, 12, 15-tetraoxa-9, 18,23- triazahentetracontan-41-oic acid (Chem. 13)
  • Chem. 13 t-Bu protected C18-diacid-yGlu-Ado-Ado-OH (synthesized according to procedures found in section Al "methods of preparation and modifications" and procedures found in WO2018/083335) (2 mmol, 1.692g), DCC (2.4 mmol, 0.495 g, 1.2 eq.) and DMAP (0.2 mmol, 0.024 g, 0.1 eq.) was dissolved in 15mL DCM, followed by addition of 5-Bromo-2- hydroxy-/V,/V-dimethyl-3-(trifluoromethyl)benzenesulfonamide (Chem.
  • Chem. 14 t-Bu protected C18-diacid-yGlu-Ado-Ado-OH (synthesized according to procedures found in section Al "methods of preparation and modifications" and procedures found in WO2018/083335) (1 mmol, 0.846 g) and TEA (3 mmol, 0.42 mL, 3 eq.) was dissolved in MeTHF (10 mL). 5-Bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) (1.1 mmol, 0.373 g, 1.1 eq.) was dissolved in MeTHF (5 mL) and slowly added to the mixture dropwise.
  • 2-Chlorotrityl resin (2.5 g) was transferee! to a solid phase peptide synthesis glass vessel.
  • DCM (20 mL) was added to the resin which was then stirred for 2 minutes and then drained. Then DCM (40 mL) was again added to the resin, which was then stirred for 30 minutes and then drained.
  • DIPEA 8.00 mmol, 1.4 mL
  • the resulting solution was next added to the resin followed by addition of DIPEA (8.00 mmol, 1.4 mL).
  • the resin was then stirred for 3 hours. Afterwards MeOH (2 mL) was added to the resin solution, which was then stirred for 1 hour and then drained. The resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and then drained and dried in vacuum oven at 30 °C. The loading was determined to 1.36 mmol/g resin.
  • the loaded resin (1.56 g, 1.36 mmol/g resin, 2.12 mmol) was transferred to a solid phase peptide synthesis glass vessel and treated with 20% piperidine in DMF (20 mL, 2 x 15 minutes) for fmoc deprotection. Next the resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and then drained.
  • the cleaved product was collected and concentrated under reduced pressure.
  • the product (2-[2-[2-[(2-azidoacetyl)amino]ethoxy]ethoxy]acetic aied) was used in the next step without further purification.
  • the compound was characterised by LC-MS and X H NMR.
  • the activated side chain of Chem. 16 was generated in situ from 2-(benzylthio)acetic acid and 5-bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
  • Example D5 Preparation of 4-bromo-2-(/V,/V-dimethylsulfamoyl)-6- (trifluoromethyl)phenyl((benzyloxy)carbamoyl)olvcinate (Chem. 17) Chem. 17:
  • the compound was characterised by LC-MS and X H NMR.
  • the activator of Chem. 18 was generated in situ from 2-azidoacetic acid and 5-bromo-2- hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
  • the compound was characterised by LC-MS, X H NMR.
  • the activated side chain of Chem. 19 was generated in situ from 2-(2-((4-methyl-2-oxo- 2H-chromen-7-yl)amino)-2-oxoethoxy)acetic acid and 5-bromo-2-hydroxy-3- (trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
  • Chem. 40 The activated side chain of Chem. 40 was generated in situ from chloroacetic acid and 5- bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
  • the activator of Chem. 32 was generated in situ from N-carbobenzyloxyglycine and 5- bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
  • 2-Chlorotrityl resin (2.5 g, 1.6 mmol/g) was transfered to a solid phase peptide synthesis glass vessel.
  • DCM (20 mL) was added to the resin which was then stirred for 2 minutes and then drained. Then DCM (40 mL) was again added to the resin, which was then stirred for 30 minutes and then drained.
  • DIPEA 1.4 mL, 8.00 mmol, 2.0 equiv.
  • the resulting solution was next added to the resin followed by addition of DIPEA (1.4 mL, 8.00 mmol, 2.0 equiv.). The resin was then stirred for 3 hours. Afterwards MeOH (2 mL) was added to the resin solution, which was then stirred for 1 hour and then drained. The resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and then drained and dried in vacuum oven at 30 °C. The loading was determined to 1.36 mmol/g resin.
  • the loaded resin (1.56 g, 1.36 mmol/g resin, 2.12 mmol) was transferred to a solid phase peptide synthesis glass vessel and treated with 20% piperidine in DMF (20 mL, 2 x 15 minutes) for fmoc deprotection. Next the resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and then drained.
  • Chloroacetic acid (0.57 g, 6 mmol, 3.0 equiv.) was added to a solution of Oxyma (0.853 g, 6 mmol, 3.0 equiv.) in DMF (18 mL) followed by addition of DIC (6 mmol, 0.940 mL, 3.0 equiv.) and DIPEA (2.09 mL, 12 mmol, 6.0 equiv.).
  • the resulting solution was stirred for 30 minutes and then added to the resin and stirred o/n.
  • the resin was drained, washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained.
  • the resin was treated with HFIP/DCM (1 :4, 20 mL, 2 x 30 minutes). The cleaved product was collected and concentrated under reduced pressure. The product was used in the next step without further purification.
  • the activated side chain of Chem. 33 was generated in situ from 2-(2-(2-(2- chloroacetamido)ethoxy)ethoxy)acetic acid and 5-bromo-2-hydroxy-3- (trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
  • Step 1 Preparation of (3-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5- (tert-butoxy)-5-oxopentanamido)propanamido)propanoyl)glycine
  • 2-Chlorotrityl resin (1 g, 1.06 mmol/g) was transfered to a solid phase peptide synthesis glass vessel. DCM (10 mL) was added to the resin which was then stirred for 2 minutes and then drained. Then DCM (20 mL) was again added to the resin, which was then stirred for 30 minutes and then drained.
  • the resin was treated with 20% piperidine in DMF (10 mL, 2 x 15 minutes) for fmoc deprotection.
  • the resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained.
  • the resin was treated with 20% piperidine in DMF (10 mL, 2 x 15 minutes) for fmoc deprotection.
  • the resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained.
  • the resin was treated with 20% piperidine in DMF (10 mL, 2 x 15 minutes) for fmoc deprotection.
  • the resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained.
  • the tetrapeptide was cleaved from the resin with HFIP/DCM (1:4, 20 mL, 2 x 30 minutes). The cleaved product was collected and concentrated under reduced pressure. The crude peptide were dissolved in H2O (14.5 mL), AcOH(4 mL) and MeCN (1.5mL) and purified by RP-HPLC eluting with a gradient from 60:40 to 35:65 HzO/MeCN + 0.1% trifluoroacetic acid over 40 minutes. Pure fractions were collected and lyophilized to afford the peptide as white solids. (0.6 g, 0.89 mmol, 89%).
  • acylation reactions under the alkaline reaction conditions described in WO2018/083335 were carried out for acylation of desB30 human insulin using reference activators from the prior art and two representative activators of the present invention.
  • the activated side chain are for all examples the activated side chain of Chem. 20:
  • the pH of the acylation reaction was controlled by an auto-titrator to keep a constant pH during the reaction (Titrando/Dosino®).
  • Solid desB30 human insulin was weighed out (240 mg, 43.2 pmol) and transferred to the titrate vessel and mixed with 1.00 ml water for about 15 min. The desB30 human insulin slowly dissolved. The pH was close to 9.
  • the auto-titrator vessel was connected to a cooling system and cooled at 5° C. and the pH was raised to 10.5 by adding NaOH (0.2 M, 400 pL) drop wise. The mixture was clear and colourless. 400 pL water was added manually to give a total volume of 1.80 mL.
  • the desB30 human insulin solution was titrated to pH 11.7 with NaOH (0.5 M, 185 pL). The total volume and concentration of the desB30 human insulin solution was 2.0 mL and 120 mg/mL.
  • the activated side chain (1.2 or 1.5 eq.) was dissolved in 0.5 ml NMP.
  • the activated side chain was added to the desB30 human insulin solution with a flowrate of 0.08 mL/min.
  • UPLC_method_Al The progress of the acylation was analysed by UPLC_method_Al. UPLC samples was quenched with a solution of AcOH/HzO/MeCN (2: 1: 1) before being analysed. Conversion was based on UPLC analysis.
  • acylation at alkaline pH leads predominantly to acylation at the lysine in position B29 of human insulin (Lys B29 ).
  • Some diacylated products are seen, arising from acylation of Lys B29 as well as of either GlyAl (the glycine in position 1 of the A-chain of the human insulin analogue) or of Phe B1 (the phenylalanine in position 1 of the B-chain of the human insulin analogue). This applies both for the prior art NHS activator and the prior art activator 3,5-DC-HBSA (WO2018/083335).
  • Chem. 2b SC side chain
  • SM unreacted starting material
  • the acylation at alkaline pH predominantly takes place at the Lys B29 position of the human insulin or human insulin analogue.
  • the purpose of the below example Fl is to study the use of the activators of the invention in an acylation reaction for producing a Bl-acylated insulin analogue.
  • the activated side chains of the invention used are those of Examples DI and D2, and prior art activated side chains of Examples C1-C3 are included for comparison.
  • the acylation reacion is performed at a neutral pH of 7.0-7.5.
  • the insulin analogue being acylated in this example is desB30 human insulin (A- chain of SEQ ID NO: 1 and B-chain of SEQ ID NO:3), which may be prepared, e.g., as described in W02001049742.
  • the side chain which is to be attached to the lysine at the N-terminal of the B-chain of insulin consists are identical for all acylation reactions in table 2, and the activated side chain are for all examples thus of Chem. 20:
  • Solid desB30 human insulin was weighed out (28.5 mg, 5 jimol) and transferred to a 4 mL glass veil equipped with a magnetic stir bar.
  • HEPES buffer (0.95 mL, 0.2M, pH 7.4) was added to the glass veil, giving a concentration of insulin of 30 mg/mL.
  • the activated side chain (1.25 equiv. was weighed out in another glass veil and dissolved in 50
  • the side chain solution was then added to the insulin solution.
  • the pH was adjusted to 7.4 using NaOH (0.5M) and the reaction mixture was then stirred at rt. for 24 hours. After 24 hours a small aliquot of the reaction mixture was taken out, diluted with a mixture of AcOH/H 2 O/MeCN (2: 1 : 1) and analysed by UPLC analysis.
  • the acylation reaction was performed using the activated side chains of the invention of Examples DI and D2, respectively.
  • the acylation reaction was also performed using the reference activated side chains of Examples Cl, C2 and C3; respectively. The results can be seen in table 2.
  • SM starting material
  • the side chain is identical for all the examples (see above Chem. 20).
  • different activators are used.
  • the activators of Chem. 2a and Chem. 2b, respectively, of the present invention is used.
  • the activated side chain of Chem. 10 Example Cl
  • the prior art NHS activator is used.
  • acylation at neutral pH using the activator of Chem. 2a of the present invention results in a high selectivity for acylation at the Bl position of desB30 human insulin insulin. 84% acylation at Bl is obtained, with only 8% di-acylation products and 0% of mono-acylated biproduct.
  • the insulin analogue being acylated in this example is desB30 human insulin (A-chain of SEQ ID NO: 1 and B-chain of SEQ ID NO:3), which may be prepared, e.g., as described in W02001049742.
  • the acylation reaction was performed using the activated side chain of the invention of Example DI.
  • Example Gl Preparation of desB30 insulin analogue of Chem. 21
  • Solid desB30 human insulin was weighed out (28.5 mg, 5 jimol) and transferred to a 4 mL glass veil equipped with a magnetic stir bar.
  • the solvent (0.95 mL, pH 7.4) was added to the glass veil, giving a concentration of insulin of 30 mg/mL.
  • the activated side chain (1.25 equiv. was weighed out in another glass veil and dissolved in 50 .L DMF.
  • the side chain solution was then added to the insulin solution.
  • the pH was adjusted to 7.4 using NaOH (0.5M) and the reaction mixture was then stirred at rt. for 24 hours. After 24 hours a small aliquot of the reaction mixture was taken out, diluted with a mixture of AcOH/HzO/MeCN (2: 1: 1) and analysed by UPLC analysis.
  • HEPES/MeCN (1: 1) and HEPES/THF (1: 1) almost no conversion was observed, and only 15% and 12% conversion to the desired Phe B1 acylated product was observed in these two reactions whereas 81% and 86% starting material was left after 24 hours.
  • the solvent has a major influence on the efficiency of the acylation reaction.
  • the reaction performs well in water (HEPES buffer), but once organic solvents are added the reaction becomes slower and the selectivity for Phe B1 acylation decreases.
  • the compound was prepared from desB30 human insulin and the activated side chain of either Chem. 17 (Example D5) using general acylation procedure method 1 or Chem. 32 (Example D9) using general acylation procedure method 2.
  • the acylation using the prior art activator 3,5-DC-HBSA is also included in table 4.
  • the prior art activator 3,5-DC-HBSA is less Bl-selective than the activators of the present invention with the formation of both diacylated products (10%), products with acylation at either Gly A1 or Lys K29 (6%).
  • acylation using lipophilic side chains using the activator of Chem. 2a results in poor conversion due to low water solubility of the activated side chains.
  • the activator of Chem. 2b can be used to increase the water solubility of the activated side chains and thus increase conversion leading to higher yields of the Bl acylated insulin product.
  • the insulin analogue being acylated in all of these examples is desB30 human insulin, which may be prepared, e.g., as described in W02001049742.
  • the compound was prepared from desB30 human insulin and activated side chain Chem.
  • the compound was prepared from desB30 human insulin and the activated side chain of
  • the compound was prepared from desB30 human insulin and the activated side chain of
  • the compound was prepared from desB30 human insulin and the activated side chain of
  • the compound was prepared from desB30 human insulin and the activated side chain of
  • the compound was prepared from desB30 human insulin and the activated side chain of Chem. 33 (Example D12) using general acylation procedure method 2.
  • table 5 also include the acylation reaction of Examples Fl and Hl, which are also included in table 2 and 4, respectively.
  • the activator of the invention can be used to introduce a number of different side chains, under aqueous conditions, on the Bl position of desB30 human insulin in highly selective manner.
  • the activator of the invention can be used to introduce a number of different side chains, under aqueous conditions, on the Bl position of desB30 human insulin in highly selective manner.
  • only minor amounts of of Gly A1 or Lys B29 acylated product as well as diacylated product are observed in these reactions. This is in stark contrast to standard methods for acylation of insulin using for instance NHS as the activator.
  • the acylation reaction was carried out using an insulin analogue with serine (Ser) and glutamic acid (Glu) in the Bl position.
  • Ser serine
  • Glu glutamic acid
  • the pKa of the N-terminal amine changes.
  • the pKa of the N-terminal amine is ⁇ 9.13, for Serine it is ⁇ 9.15 and for Glutamic acid it is 9.67. Based on these values it is expected that the serine analogue will react similar to the phenylalanine analogue.
  • Ser B1 desB30 insulin (85.0 mg, 15 pmol, 1.0 equiv.) was dissolved in 0.2M HEPES buffer (2.8 mL, pH 7.4).
  • (S)-l-(4-bromo-2-(/V,/V-dimethylsulfamoyl)-6-(trifluoromethyl)- phenoxy)-22-ca rboxy-l,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazahentetracontan- 41-oic acid (the activated side chain of Chem. 13, Example DI) (24.0 mg, 22.5 pmol, 1.5 equiv.) was dissolved in DMF (50 pL) and added to the solution.
  • the resulting mixture was stirred at rt. for 24 hours.
  • the mixture was then acidified with AcOH to pH 3 and diluted with water to a total volume of 20 mL.
  • the product was purified by RP-HPLC eluting with a gradient from 60:40 to 45:55 HzO/MeCN + 0.1% trifluoroacetic acid over 40 minutes. Pure fractions were collected and lyophilized to afford the title compound as a white solid. (49.0 mg, 7.7 pmol, 51%).
  • Glu B1 desB30 human insulin (86.8 mg, 15 pmol, 1.0 equiv.) was dissolved in 0.2M HEPES buffer (2.9 mL, pH 7.4).
  • (S)-l-(4-bromo-2-(/V,/V-dimethylsulfamoyl)-6- (trifluoromethyl)phenoxy)-22-carboxy- 1,19, 24-trioxo-3, 6, 12,15-tetraoxa-9, 18,23- triazahentetracontan-41-oic acid (the activated side chain of Chem. 13, Example DI) (24.0 mg, 22.5 pmol, 1.5 equiv.) was dissolved in DMF (50 pL) and added to the solution. The resulting mixture was stirred at rt. for 24 hours.
  • the mixture was then acidified with AcOH to pH 3 and diluted with water to a total volume of 20 mL.
  • the product was purified by RP-HPLC eluting with a gradient from 60:40 to 45:55 HzO/MeCN + 0.1% trifluoroacetic acid over 40 minutes. Pure fractions were collected and lyophilized to afford the title compound as a white solid. (43.0 mg, 6.6 pmol, 44%)
  • the Bl selective acylation using the activators of the present invention is not limited to insulin analogues having a Phe in the Bl position, but also insulin analogues having another amino acid in the Bl position.
  • the acylation method of the present invention also enables the selective acylation of position Bl of human insulin or human insulin analogues with one side chain at low pH, followed by acylation of another position such as the B29 position with a different side chain at high pH. In two subsequent steps two different side chains can be attached to an insuline analogue without the need for extra protection/deprotection steps.
  • desB30 human insulin is first acylated at position Bl at low pH, followed by acylation at position B29 at high pH.
  • Chem. 29 t-Bu protected C16-diacid-yGlu-OH (synthesized according to procedures found in WO09115469) (1 mmol, 0.528 g) and TEA (3 mmol, 0.42 mL, 3 eq.) was dissolved in MeTHF (10 mL). 3,5-dichloro-2-hydroxybenzenesulfonyl chloride (1.2 mmol, 0.314 g, 1.2 eq.) was dissolved in MeTHF (5 mL) and slowly added to the mixture dropwise. The reaction mixture was then stirred until full conversion was observed (UPLC analysis). Next the mixture was washed with 5% KHSC (3 x 5 mL).
  • the compound was characterised by LC-MS and X H NMR.
  • Solid desB30 human insulin was weighed out (228.26 mg, 0.06 jimol) and transferred to a 20 mL glass veil equipped with a magnetic stir bar.
  • HEPES buffer (7.6 mL, 0.2M, pH 7.4) was added to the glass veil, giving a concentration of insulin of 30 mg/mL.
  • Example DI (1.5 equiv.) was weighed out in another glass veil and dissolved in 100 pL DMF. The side chain solution was then added to the insulin solution. The pH was adjusted to 7.4 using NaOH (0.5M) and the reaction mixture was then stirred at rt. for 24 hours resulting in acylation of Phe B1 . Next the reaction mixture was transferred to an Amicon® Ultra - 15 centrifugal filter (3K) and the mixture was filtered on a SIGMA 4-16K refrigerated centrifuge (speed: 4000 x g) for 25 minutes. Then water was added, and the mixture filtered again. This was repeated 3 times in total.
  • desB30 human insulin is acylated at position Bl with a tetramer.
  • the examples shows an example of a ligation where a small peptide is selectively acylated onto the Phe B1 amine of desB30 human insulin. This shows that the Bl selective acylation is not limited to acylation only with small molecules.
  • Example LI Preparation of desB30 insulin analogue of Chem. 39
  • Chem. 39 DesB30 insulin (285.0 mg, 0.05 mmol, 1.0 equiv.) was dissolved in 0.2M HEPES buffer (9.5 mL, pH 7.4).

Abstract

The present invention relates to a novel acylating reagent, a method for its preparation, and a method of using it for acylating at the N-terminal of a peptide or a protein. The novel acylating reagent may be a compound which comprises 5-bromo-2-hydroxy-N,N-dimethyl-3-(trifluromethyl)benzenesulfonamide or 5-bromo-2-hydroxy-3-(trifluromethyl)benzenesulfonic acid. This novel acylating reagent enables acylation at the B1 position of insulin.

Description

DESCRIPTION
TITLE: NOVEL ACYLATING REAGENTS
The present invention relates to novel acylating reagents, their preparation, and their use in preparing Bl acylated insulin and analogues thereof.
BACKGROUND
The attachment of one or more substituents to peptides or proteins by acylation of one or more amino groups of the peptide or protein is well-known in the art. For a pharmaceutical peptide or protein this may be an efficient way of achieving a prolonged duration of action in vivo of the pharmaceutical peptide or protein.
Non-limiting examples of pharmaceutical peptides or proteins which have been acylated include insulin peptides.
Various examples of acylated insulin peptides are disclosed in, e.g., WO2009/115469. Methods for acylating peptides and proteins are disclosed in, e.g., WOOO/55119 and W02010/029159.
N-Hydroxy Succinimide (NHS) is a commonly used activator for acylation of insulin. WO2009/115469 discloses that acylation at alkaline pH (pH = 10-ll) of insulins using NHS results predominantly in acylation of the lysine residue in the B29 position of insulin. Phenolic activators are disclosed in WO2018/083335, including 3,5-dichloro-2- hydroxy-N,N-dimethyl-benzenesulfonamide (3,5-DC-HBSA). WO2018/083335 discloses that acylation of insulin using 3,5-DC-HBSA predominantly results in acylation at the lysine in the B29 position of the insulin analogue. The acylation reaction was run at a pH of 11.7.
4-Nitrophenol is another commonly used activator for the preparation of activated esters used in peptide acylation chemistry (Metabolism 1964, 1026-1031). For instance acylation of lysine residues in peptides is described in RSC Adv. 2021, 908-914.
None of the existing acylation methods present in the literature provides a single step method for selective acylation of the Bl residue of insulin (e.g. the N-terminal of the B-chain of insulin) without acylation of the alpha amine in Al (Gly). The present state of the art for high yielding Bl alpha amine acylation is a combination of steps which involves protection of the alpha amine of Al and the epsilon Lys amine followed by acylation and deprotection (Bioconjugate chem 2005, 1000-1008). The only present chemical modification which allow Bl selectivity in one step on insulin is reductive amination and so far it is limited to aromatic aldehydes (WO2012/171994). There is thus a need for an acylating reagent which will allow selective acylation at the Bl residue of insulin.
SUMMARY
The present invention relates to a novel acylating reagent in the form of an ester of a carboxylic acid of Chem. 3:
Chem. 3:
Figure imgf000003_0001
wherein Y is OR, SR, NMeC(=O)R, NHC(=O)R, halogen or N3; wherein R is an organic substituent; and a phenol (herein also referred to as an activator) of Chem. 2:
Chem. 2:
Figure imgf000003_0002
wherein X is OH or N(CHs)2.
Thus, the present invention relates to a novel acylating reagent of Chem. 1:
Chem. 1 :
Figure imgf000003_0003
wherein Y is OR, SR, NMeC(=O)R, NHC(=O)R, halogen or N3; wherein R is an organic subsitutent; and wherein X is OH or NfCHs ; or a salt thereof. This compound may typically be called an activated ester, an activated phenolic ester, an activated side chain, or an acylating reagent. The phenol of Chem. 2 is used to activate the side chain of Chem. 3.
The present invention also relates to a method of preparing the acylating reagent of the invention by reacting a compound of Chem. 3 as defined herein with a compound of Chem. 2 as defined herein or a with a compound of Chem. 4: Chem. 4:
Figure imgf000004_0001
wherein Z is a suitable leaving group such as halogen.
The present invention also relates to a method for selectively acylating the alpha amino group of an N-terminal amino acid in a peptide or a protein, the method comprising a step of reacting the peptide or protein with the acylating reagent of the invention. One such peptide or protein is human insulin or human insulin analogues.
It was surprisingly found that the activators of the present invention leads to selective acylation at the Bl position of human insulin and human insulin analogues when the acylation reaction is run at neutral pH. To the best of our knowledge, no other activators are known which primarily results in acylation at the Bl position of insulin.
In one aspect, the invention provides novel acylating reagents.
In one aspect, the invention provides an improved acylation process whereby the selectivity for acylation at Bl of insulin or insulin analogues is increased.
DESCRIPTION
In what follows, Greek letters may be represented by their symbol or the corresponding written name, for example: a = alpha; p = beta; e = epsilon; y = gamma; 6 = delta; co = omega; etc. Also, the Greek letter of ji may be represented by "u", e.g. in f l = ul, or in |.LM = uM. A waved line in a chemical formula designates a point of attachment. The term "alkyl", as used herein, refers to saturated, monovalent hydrocarbon radicals. The term "alkenyl", as used herein, refers to monovalent hydrocarbon radicals, which contain at least one carbon-carbon double bond. The term "alkynyl", as used herein, refers to divalent hydrocarbon radicals, which contain at least one carbon-carbon triple bond. The term "aryl", as used herein, refers to a radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl (= naphthalenyl). The term "heteroaryl" as used herein, refers to a radical derived from an aromatic mono- or bicyclic ring system, in which 1, 2, 3, 4 or 5 carbon atoms are replaced by heteroatoms. The ring heteroatoms are generally chosen from N, O and S, wherein N includes ring nitrogen atoms which carry a hydrogen atom or a substituent as well as ring nitrogen atoms which do not carry a hydrogen atom or a substituent. The terms 'peptide' and 'protein' refer to a compound which comprises a series of amino acids interconnected by amide (or peptide) bonds. The term 'peptidyl' as used herein refers to a radical derived from such a peptide or protein.
The present invention relates to novel acylating reagents, methods of their preparation, the use thereof in preparing acylated peptides and proteins, and to novel insulin derivatives.
Figure imgf000005_0001
The present invention relates to a compound of Chem. 1:
Chem. 1 :
Figure imgf000005_0002
wherein Y is OR, SR, NMeC(=O)R, NHC(=O)R, halogen or N3; wherein R is an organic subsitutent; and wherein X is OH or N(CHs)2; or a salt thereof.
In some embodiments the present invention relates to an acylating reagent of Chem. la, which is a compound of Chem. 1, wherein X is N(CHs)2.
Figure imgf000006_0001
wherein Y is OR, SR, NMeC(=O)R, NHC(=O)R, halogen or N3; wherein R is an organic substituent.
The activator of Chem. 2a may briefly be referred to as TSAP which stands for 5- bromo-2-hydroxy-3-(trifluromethyl)benzenesulfonic acid.
Chem. 2a:
Figure imgf000006_0002
In some embodiments the present invention relates to an acylating reagent of
Chem. lb, which is a compound of Chem. 1, wherein X is OH.
Chem. lb:
Figure imgf000006_0003
wherein Y is OR, SR, NMeC(=O)R, NHC(=O)R, halogen or N3; wherein R is an organic subsitutent.
The activator of Chem. 2b may briefly be referred to as TSP which stands for 5- bromo-2-hydroxy-3-(trifluromethyl)benzenesulfonic acid. Chem. 2b:
Figure imgf000007_0001
The compound of Chem. 1 may also be referred to as an acylating reagent, an activated side chain, or an an activated ester, and it is a phenolic ester of a compound of Chem. 3 as defined herein, and an activator of Chem. 2 as defined herein. In some embodiments, the salt of Chem. 1 is an alkali metal salt or a tertiary amine salt.
The acylating reagent of the present invention enables introduction of a wide range of substituents or side chains at the Bl position of human insulin or human insulin analogues.
In one embodiment, the acylating reagent is of Chem. 1, wherein Y is OR, SR, NMeC(=O)R, NHC(=O)R, halogen or N3; wherein R is an organic substituent.
The organic substituent can be any organic moiety. In some embodiments, the organic substituent is alkyl, heteroalkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, each being optionally substituted. In some embodiments, the organic substituent is a peptidyl. In some embodiments, when the organic susbtituent is peptidyl, Y is NMeC(=O)R or NHC(=O)R.
In some embodiments, the acylating reagent of the invention is a compound of
Chem. 7:
Figure imgf000007_0002
wherein r is an integer in the range of 8-20; wherein q is an integer in the range of 0-3; wherein p is an integer in the range of 0-3; wherein X is OH or N(CHs)2; and L is an optional linker of Chem. 8:
Figure imgf000008_0001
wherein each of k, I, and m independently represents an integer in the range of 0-4; or a salt, amide, or ester thereof.
The Chem. 8 element may be in its L- or D-form. In some embodiments, the Chem. 8 element is in the L-form.
In some embodiments, the acylating reagent of the invention is used to introduce a reactive group which can be used to introduce further chemical groups to the side chain. In one embodiment, the reactive group is an azide, which can for instance be used to couple to any terminal alkynyl compounds in a 3+2 cycloaddition in the presence of a metal catalyst (e.g copper). This is a mild efficent way for making e.g bioconjucation.
In one embodiment, the acylating agent is of Chem. 1, wherein Y is N3.
In another embodiment, the acylating agent is of Chem. 9:
Figure imgf000008_0002
wherein X is OH or N(CHs)2.
In one embodiment, the reactive group is an halide, which can for instance be used to couple to any thiol bearing compounds in an alkylation reaction. This is a mild efficent way for making e.g bioconjucation between two macro molecules or ligation to a macro molecule.
In one embodiment, the acylating agent is of Chem. 1, wherein Y is a halogen. In one embodiment, Y is Cl. In another embodiment, the acylating agent is of Chem. 9a:
Figure imgf000009_0001
wherein X is OH or N(CHs)2 and W is F, Cl, Br, or I. In one embodiment, W is Cl.
In Example F of the present application, acylation of desB30 human insulin is carried out at neutral pH using acylating reagents of the invention (see Table 2). For comparison, acylation is performed using the same side chain and reaction conditions using the prior art activators NHS, 3,5-DC-HBSA, and PNP. For these reactions, the acylation products are determined to show the selectivity of the various acylating reagents.
In Example G, an acylation reaction using the acylating reagent of the invention is carried out in different solvents, showing that optimal reaction conditions are obtained in an aqueous solvent (see Table 3).
In Example H, it is shown that the acylating reagent of Chem. 2b is particularly suitable for Bl selective acylation for side chains with low water solubility (see Table 4).
In Example I of the present application, acylation of desB30 human insulin is carried out at neutral pH using a range of side chains activated by the activators of the invention (see Table 5).
In Example J, it is shown that Bl selective acylation is not specific to insulin analogues having a phenylalanine (Phe) in position Bl, but that Bl selective acylation is also obtained with other amino acids in the Bl position of insulin.
Example K demonstrates the usefulness of the acylating reagent of the invention in preparing an insulin derivative with two different side chains in position Bl and B29, respectively, without the need for extra protection/deprotection steps.
Example L demonstrates that the acylating reagent of the invention can also be used to attach small peptides to the Bl position of insulin.
Particular embodiments of the acylating reagent of the invention are disclosed below, in the section headed "PARTICULAR EMBODIMENTS". Method of preparing the acylating reagent
The present invention also relates to a method for preparing the acylating reagent of the invention.
To prepare the acylating reagent of Chem. 1, wherein X is OH (i.e. the acylating reagent of Chem. lb), the method comprises the step of reacting a compound of Chem. 3 as defined herein with a compound of Chem. 4:
Chem. 4:
Figure imgf000010_0001
wherein Z is a leaving group. In one embodiment the leaving group is a halogen. In one embodiment the leaving group is Cl or F. In one embodiment the leaving group is Cl.
When Z is a leaving group, such as Cl, the reaction results in a carboxylic- sulfonic mixed anhydride which after a facile intramolecular acyl transfer reaction to the phenolate function results in the acylating reagent of the invention of Chem. 1, wherein X is OH (i.e. the acylating reagent of Chem. lb).
To prepare the acylating reagent of Chem. 1, wherein X is N(CHs)2 (i.e. the acylating reagent of Chem. la), the method comprises the step of reacting a compound of Chem. 3 as defined herein with the phenol (also referred to as activator) of Chem. 2a: Chem. 2a:
Figure imgf000010_0002
The reaction takes place as a one-step esterification reaction which results in the acylating reagent of the invention of Chem. la. One non-limiting example of a suitable coupling reagent for this reaction is N,N'-dicyclohexylcarbodiimide (DCC) together with dimethyl aminopyridine (DMAP).
In some embodiments of either of these two methods, when Y comprises chemical groups that have been protected (such as carboxylic acid groups protected with, e.g., tBu or Bn), the method also comprises a step of de-protecting the acylating reagent.
In some embodiments, the acylating reagent prepared by this method is as defined in any of the acylating reagent embodiments discussed above and/or in any of the "PARTICULAR EMBODIMENTS" further below.
The acylating reagent of the invention can be prepared on solid support using procedures of solid phase peptide synthesis well known in the art, or in solution phase as also well known in the art. Non-limiting examples of such preparation methods are included in the Experimental part of the present application.
Particular embodiments of the method of preparing the acylating reagent of the invention are disclosed below, in the section headed "PARTICULAR EMBODIMENTS".
Method of using the acylating reagent
The present invention also relates to a method for acylating an N-terminal amino acid in a peptide or a protein, the method comprising a step of reacting the peptide or protein with the acylating reagent of the invention.
More specifically, the amino group being acylated is the alfa-amino group of the N-terminal amino acid residue in the peptide or protein. In some embodiments, the amino group being acylated is the alfa-amino group of the N-terminal amino acid residue in the B-chain of human insulin or a human insulin analogue. In some embodiments, the amino group being acylated is the alfa-amino group of an N-terminal phenylalanine (Phe), serine (Ser) or glutamic acid (Glu) residue in the B-chain of human insulin or a human insulin analogue. In some embodiments, the amino group being acylated is the alfa-amino group of the N-terminal phenylalanine residue in the B-chain of human insulin or a human insulin analogue.
In some embodiments, the acylating reagent for use in this method is as defined in any of the acylating reagent embodiments discussed above and/or in the "PARTICULAR EMBODIMENTS" further below.
The acylation method of the invention takes place under suitable conditions, which are known by the person skilled in the art. Preferably, the acylation reaction takes place in an aqueous reaction medium (a reaction medium that contains water). More preferably, the reaction medium is substantially free of an organic solvent. However, smaller amoumts of organic solvents are tolerated in the reaction medium. In some embodiments, the amount of organic solvent present is less than 20% (vol%). In some embodiments, the pH in the acylation reaction mixture is in the range of pH 6-8. In some embodiments, the temperature in the reaction mixture is in the range of 20°C to 50°C. The acylation method of the present invention is quite robust. For example, it provides great flexibility as regards the addition of the acylating reagent of the invention to the peptide or protein to be acylated. Also, or alternatively there is no need for the reaction vessel to be of any particular design. Also, or alternatively there is no need for the stirring to be optimal or optimised. In the examples it is demonstrated that the acylating reagent of the invention can be added as a solution, or it can be added as a solid - without impacting the yield of the desired product. This is contrary to the known NHS- based acylation method, where the acylating reagent must be added very slowly and under rigorous control due to its hydrolytic instability.
The acylation method of the present invention enables acylation at the Bl position of human insulin or a human insulin analogue, i.e. acylation at the N-terminal of the B-chain of human insulin or a human insulin analogue. As demonstrated in Examples F and I herein, the selectivity is for the Bl position whereas the known NHS-based acylation method and methods in WO2108/083335 are less selective for the Bl position in human insulin and analogues thereof.
In some embodiments, the acylation method of the present invention comprises a further step, after the acylation reaction, of purifying the desired product of the acylation reaction. Suitable methods of purifying acylated peptides and proteins are known by the person skilled in the art.
In some embodiments, the acylation method of the present invention comprises a further step, prior to the acylation reaction, of dissolving the peptide or protein to be acylated. In some embodiments, the peptide or protein is dissolved in an aqueous solution. Suitable ranges for pH, concentration of peptide or protein, and temperature are known by the person skilled in the art.
The acylation method of the present invention also enables the selective acylation of position Bl of human insulin or human insulin analogues with one side chain at neutral pH, followed by acylation of another position such as the B29 position with a different side chain at alkaline pH. In two subsequent steps two different side chains can be attached to an insuline analogue without the need for extra protection/deprotection steps. An example of such a di-modified insulin can be seen in Example K. The acylation method of the invention refers to "peptide or protein" as it is in principle applicable to any peptide or protein, whatever the size (number of amino acid residues) or other structural parameter, having an N-terminal amino acid residue.
The distinction between peptide and protein may not always be quite clear. For example, a peptide is sometimes defined to contain a maximum of about 50 amino acid residues, a polypeptide sometimes to contain a minimum of about 50 amino acid residues, and a protein sometimes to consist of one or more peptides or polypeptides arranged in a more complex structure which may be required for biological activity. Nevertheless, insulin (which consists of two peptide chains each of a length of less than 50 amino acids, coupled together via Cys-Cys bonds) is traditionally referred to as a peptide.
For the present purpose the following definitions apply: A peptide contains up to a total of 200 amino acid residues, in one or more individual peptide chains; and a protein contains more than 200 amino acids in total, in one or more individual peptide chains.
Non-limiting examples of peptides for use in the method of the invention include human insulin which is a peptide of 51 amino acid residues in total (native human insulin, 30 amino acids in the B-chain and 21 amino acids in the A-chain) and analogues hereof.
In some embodiments, the peptide for use in the method of the invention contains a) at least 2 amino acid residues, b) at least 5 amino acid residues, c) at least 20 amino acids; and/or d) a maximum of 150 amino acid residues.
In some embodiments, the protein contains no more than 2000 amino acid residues in total.
In some embodiments, the peptide or protein for use in the acylation method of the invention is a peptide.
In some embodiments, the peptide or protein for use in the acylation method of the invention is a protein.
The amino acid residues incorporated in the peptide or protein for use in the acylation method of the invention may include coded and/or non-coded amino acid residues. The term "coded amino acids" refers to the 20 "natural" amino acids (see, e.g., IUPAC, table 1, section 3AA-1). Unless otherwise specified, the amino acid residue(s) in the amino acid, peptide or protein for use in the acylation method of the invention are in the L-form. In some embodiments, the peptide or protein for use in the acylation method of the invention is a pharmaceutical peptide or protein, which means that the peptide or protein has an effect, demonstrated in vitro or in vivo, which is considered at least potentially relevant for the prophylaxis or treatment of one or more diseases. Nonlimiting examples of diseases include diabetes, obesity, and related diseases and disorders.
Non-limiting examples of peptides or proteins to be acylated using the method of the invention include human insulin and analogues thereof.
In some embodiments, the peptide or protein to be acylated using the method of the invention is an insulin peptide. The term insulin peptide includes human insulin and analogues thereof. The human insulin A-chain has the following sequence: GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1), while the B-chain has the following sequence: FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO:2). In some embodiments, the insulin analogue for use in the acylation method of the invention has a maximum of 10 amino acid changes as compared to human insulin. In some embodiments the maximum of 10 amino acid changes is as compared to human proinsulin. In some embodiments, the insulin analogue comprises at least the amino acid modification desB30. The terminology used herein for naming insulin analogues is as usual in the art, as explained in, e.g., WO2009/115469. Thus, for example, A14E refers to the amino acid corresponding to amino acid residue no. 14 in the A-chain of human insulin having been replaced by Glu (E); B16H refers to the amino acid corresponding to amino acid residue no. 16 in the B-chain of human insulin having been replaced by His (H); and desB30 refers to the amino acid corresponding to amino acid residue no. 30 in the B-chain of human insulin having been deleted. In some embodiments, the insulin analogue for use in the acylation method of the present invention is desB30 human insulin (A-chain of SEQ ID NO: 1 and B-chain of SEQ ID NO:3); or a pharmaceutically acceptable salt, amide, or ester thereof.
In some embodiments the insulin peptide for use in the acylation method of the invention is an analogue of human insulin, which can be prepared by recombinant expression. Suitable recombinant expression methods are known by the person skilled in the art, see e.g. WO2009/115469 referred to above.
In some embodiments, the final acylated insulin peptide produced by the acylation method of the invention (excluding acylated proinsulin, pre-proinsulin, and analogues thereof) has affinity to an insulin receptor. Suitable insulin receptor affinity assays are known in the art, see e.g. Example 178 of W02009/115469. Using this assay with 0% HSA the final acylated insulin peptide produced according to the invention has an affinity of at least 0.10%.
Particular embodiments of the method of using the acylating reagent of the invention are disclosed below, in the section headed "PARTICULAR EMBODIMENTS".
PARTICULAR EMBODIMENTS
The following are particular embodiments of the invention:
1. A compound of Chem. 1:
Chem. 1 :
Figure imgf000015_0001
wherein Y is OR, SR, NMeC(=O)R, NHC(=O)R, halogen or N3; wherein R is an organic subsitutent; and wherein X is OH or N(CHs)2; or a salt thereof.
2. The compound of embodiment 1, wherein X is N(CHs)2.
3. The compound of embodiment 1, wherein X is OH.
4. The compound of embodiment 3, wherein the salt is a sulfonic acid salt.
5. The compound of embodiment 4, wherein the sulfonic acid salt is a tertiary amine salt such as a TEA salt, or an alkali metal salt such as a K salt, a Na salt, or a Li salt.
6. The compound of any one of embodiments 1-5, wherein Y is is OR.
7. The compound of any one of embodiments 1-5, wherein Y is is SR.
8. The compound of any one of embodiments 1-5, wherein Y is is NMeC(=O)R.
9. The compound of any one of embodiments 1-8, wherein the organic substituent is alkyl, heteroalkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, or peptidyl, each being optionally substituted.
10. The compound of embodiment 9, wherein the organic substituent is alkyl, heteroalkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl, or heteroarylalkyl, each being optionally substituted.
11. The compound of embodiment 9, wherein the organic substituent is peptidyl. 12. The compound of embodiment 9, wherein when the organic substituent is peptidyl, then Y is NMeC(=O)R, or NHC(=O)R.
13. The compound of any one of embodiments 1-5, wherein Y is Ns.
14. The compound of any one of embodiments 1-5, wherein the compound is of formula Chem. 9:
Figure imgf000016_0001
wherein X is OH or N(CHs)2.
15. The compound of any one of embodiments 1-5, wherein Y is a halogen.
16. The compound embodiment 15, wherein Y is Cl.
17. The compound of any one of embodiments 1-5, wherein the compound is of formula Chem. 9a:
Figure imgf000016_0002
wherein W is a halogen.
18. The compound of embodiment 17, wherein W is Cl.
19. The compound of embodiment 1, wherein the compound is a compound of Chem. 7:
Figure imgf000016_0003
wherein r is an integer in the range of 8-20; wherein q is an integer in the range of 0-3; wherein p is an integer in the range of 0-3; wherein X is OH or N(CHs)2; and
L is an optional linker of Chem. 8:
Figure imgf000017_0001
wherein each of k, I, and m independently represents an integer in the range of 0-4; or a salt, amide, or ester thereof.
20. The compound of embodiment 19, wherein r is 18.
21. The compound of any of embodiments 19-20, wherein q is 1.
22. The compound of any of embodiments 19-21, wherein p is 2.
23. The compound of any of embodiments 19-22, wherein X is N(CHs)2.
24. The compound of any of embodiments 19-23, wherein X is OH.
25. The compound of any of embodiments 19-24, wherein k is 0.
26. The compound of any of embodiments 19-25, wherein I is 0.
27. The compound of any of embodiments 19-26, wherein m is 2.
28. The compound of any of embodiments 19-27, wherein L is in its L- or D-form.
29. The compound of embodiment 23, wherein L is in its L-form.
30. A compound selected from Chem. 13, Chem. 14, Chem. 15, Chem. 16, Chem.
17, Chem. 18, Chem. 19, Chem. 40, Chem. 32, Chem. 33, and Chem. 34; or a salt, amide, or ester thereof.
31. A compound of Chem. 13; or a salt, amide, or ester thereof.
32. A compound of Chem. 14; or a salt, amide or ester thereof.
33. A compound of Chem. 15; or a salt, amide, or ester thereof.
34. A compound of Chem. 16; or a salt, amide, or ester thereof.
35. A compound of Chem. 17; or a salt, amide, or ester thereof.
36. A compound of Chem. 18; or a salt, amide, or ester thereof.
37. A compound of Chem. 19; or a salt, amide, or ester thereof.
38. A compound of Chem. 40; or a salt, amide, or ester thereof.
39. A compound of Chem. 32; or a salt, amide, or ester thereof.
40. A compound of Chem. 33; or a salt, amide, or ester thereof.
41. A compound of Chem. 34; or a salt, amide, or ester thereof.
42. The compound of any of embodiments 30-41, wherein the salt, amide, or ester thereof is a salt thereof.
43. The compound of any of embodiments 30-41, wherein the salt, amide, or ester thereof is an amide thereof. 44. The compound of any of embodiments 30-41, wherein the salt, amide, or ester thereof is an ester thereof.
45. The compound of any of embodiments 1-44 which is more selective towards acylation of the Bl position of human insulin or a human insulin analogue than a comparative compound which is identical to said compound except for the fact that Chem. 1 has been replaced by Chem. 5a:
Figure imgf000018_0001
46. The compound of any of embodiments 1-44 which is more selective towards acylation of the Bl position of human insulin or a human insulin analogue than a comparative compound which is identical to said compound except for the fact that Chem. 1 has been replaced by Chem. 6a:
Figure imgf000018_0002
47. The compound of any of embodiments 45-46, wherein the selectivity towards acylation of the Bl position of human insulin or a human insulin analogue is determined generally as described in Example F.
48. A method for preparing the compound of embodiment 1, which comprises the step of reacting a compound of Chem. 3:
Chem. 3:
Figure imgf000018_0003
wherein Y is OR, SR, NMeC(=O)R, NHC(=O)R, halogen or N3; and wherein R is an organic substituent; with a compound of Chem. 2a:
Chem. 2a:
Figure imgf000019_0001
49. The method of embodiment 48, wherein the method comprises addition of a coupling reagent.
50. The method of embodiment 49, wherein the coupling reagent is DCC and DMAP.
51. The method of any of embodiments 48-50, which is for preparing the compound of Chem. la.
52. A method for preparing the compound of embodiment 1, which comprises the step of reacting a compound of Chem. 3:
Figure imgf000019_0002
wherein Y is OR, SR, NMeC(=O)R, NHC(=O)R, halogen or N3; and wherein R is an organic substituent; with a compound of Chem. 4:
Figure imgf000019_0003
wherein Z is a leaving group.
53. The method of embodiment 52, wherein Z is Cl or F.
54. The method of embodiment 52, wherein Z is Cl.
55. The method of any of embodiments 52-54, which is for preparing the compound of
Chem. lb.
56. The method of any of embodiments 48-55, which comprises a step of purifying the desired compound.
57. A method for acylating an N-terminal amino acid in a peptide or a protein, the method comprising a first step of preparing an acylating reagent using an activator of
Chem. 2:
Figure imgf000020_0001
wherein X is OH or N(CHs)2; and a second step of reacting the said acylating reagent with the peptide or protein.
58. A method for acylating an N-terminal amino acid residue in a peptide or a protein, the method comprising a step of reacting the peptide or protein with a compound as defined in any of embodiments 1-47.
59. The method of any of embodiments 57-58, wherein the peptide or protein has two or more reactive nucleophilic functional groups.
60. The method of any of embodiments 57-59, wherein the N-terminal amino acid residue of the peptide or protein is selectively acylated.
61. The method of any of embodiments 57-60, wherein the resulting acylated peptide or protein comprises at least one reactive nucleophilic functional group which is not or only partially acylated.
62. The method of any one of embodiments 57-61, wherein the peptide or protein is human insulin or a human insulin analogue.
63. The method of any one of embodiments 57-62, wherein the N-terminal amino acid residue is phenylalanine (Phe), serine (Ser) or glutamic acid (Glu).
64. The method of embodiment 63, wherein the N-terminal amino acid residue is phenylalanine (Phe).
65. The method of embodiment 63, wherein the N-terminal amino acid residue is serine (Ser).
66. The method of embodiment 63, wherein the N-terminal amino acid residue is glutamic acid (Glu).
67. The method of any of embodiments 57-66, wherein the compound as defined in any of embodiments 1-47 is an acylating reagent.
68. The method of any of embodiments 57-67, wherein the acylation reaction takes place in a reaction mixture which is an aqueous medium.
69. The method of any of embodiments 57-67, wherein the acylation reaction takes place in an aqueous solvent.
70. The method of any of embodiments 57-67, wherein the acylation reaction takes place in an aqueous solvent substantially free of any organic solvent. 71. The method of any of embodiments 57-67, wherein the amount of organic solvent present in the reaction medium is less than 20% (vol%).
72. The method of embodiment 71, wherein the amount of organic solvent present in the reaction medium is less than 10% (vol%).
73. The method of embodiment 72, wherein the amount of organic solvent present in the reaction medium is less than 8% (vol%).
74. The method of embodiment 73, wherein the amount of organic solvent present in the reaction medium is less than 5% (vol%).
75. The method of any of embodiments 57-74, wherein the pH in the reaction mixture is in the range of pH 6-9.
76. The method of embodiment 75, wherein the pH in the reaction mixture is in the range of pH 6.5-8.5.
77. The method of embodiment 76, wherein the pH in the reaction mixture is in the range of pH 7.0-8.5.
78. The method of embodiment 77, wherein the pH in the reaction mixture is in the range of pH 7.0-8.0.
79. The method of embodiment 78, wherein the pH in the reaction mixture is in the range of pH 7.2-8.0.
80. The method of embodiment 79, wherein the pH in the reaction mixture is in the range of pH 7.2-7.8.
81. The method of embodiment 80, wherein the pH in the reaction mixture is in the range of pH 7.3-7.8.
82. The method of embodiment 81, wherein the pH in the reaction mixture is in the range of pH 7.3-7.6.
83. The method of any of embodiments 57-82, wherein the temperature in the reaction mixture is in the range of 5-60°C.
84. The method of embodiment 83, wherein the temperature in the reaction mixture is in the range of 5-50°C.
85. The method of embodiment 84, wherein the temperature in the reaction mixture is in the range of 10-40°C.
86. The method of embodiment 85, wherein the temperature in the reaction mixture is about 20°C.
87. The method of embodiment 85, wherein the temperature in the reaction mixture is RT (room temperature). 88. The method of any of embodiments 57-87, wherein the reaction mixture comprises a buffer.
89. The method of embodiment 88, wherein the buffer is selected from Phosphate buffer, Bicine buffer (N,N-Bis(2-hydroxyethyl)glycine buffer), HEPPS buffer (3-[4-(2- Hydroxyethyl)-l-piperazinyl]propane sulfonic acid buffer), HEPES buffer (4-(2- Hydroxyethyl)-l-piperazineethanesulfonic acid buffer), and Tris buffer (2-Amino-2- (hydroxymethyl)propane-l,3-diol).
90. The method of embodiment 89, wherein the buffer is HEPES buffer (4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid buffer).
91. The method of any of embodiments 57-90, wherein the reaction takes from 1-96 hours, counted from the point in time where the addition of the acylating reagent starts.
92. The method of embodiment 91, wherein the reaction takes from 1-72 hours, counted from the point in time where the addition of the acylating reagent starts.
93. The method of embodiment 92, wherein the reaction takes from 1-48 hours, counted from the point in time where the addition of the acylating reagent starts.
94. The method of embodiment 93, wherein the reaction takes from 12-48 hours, counted from the point in time where the addition of acylating reagent starts.
95. The method of embodiment 94, wherein the reaction takes from 12-24 hours, counted from the point in time where the addition of acylating reagent starts.
96. The method of any of embodiments 57-95, wherein the acylating reagent is added as a solution or in solid form to a solution of the peptide, or protein.
97. The method of embodiment 96, wherein the acylating reagent is added as a solution.
98. The method of embodiment 96, wherein the acylating reagent is dissolved in a solvent.
99. The method of embodiment 98, wherein the solvent is selected from ethanol, isopropanol, N-methyl pyrrolidinone (NMP), N,N-dimethylformamide (DMF), Dimethyl sulfoxide (DMSO), tetra hydrofuran (THF), Water (H2O) and acetonitrile (CH3CN).
100. The method of any of embodiments 57-99, wherein the concentration of the acylating reagent is in the range of 10-1000 mg/mL.
101. The method of embodiment 100, wherein the concentration of the acylating reagent is in the range of 10-500 mg/mL.
102. The method of embodiment 101, wherein the concentration of the acylating reagent is in the range of 10-250 mg/mL. 103. The method of embodiment 102, wherein the concentration of the acylating reagent is in the range of 20-250 mg/mL.
104. The method of embodiment 103, wherein the concentration of the acylating reagent is in the range of 30-200 mg/mL.
105. The method of any one of embodiments 57-104, wherein the acylating reagent is added in solid form.
106. The method of any of embodiments 57-105, wherein the number of moles of acylating reagent used for each mole of the peptide or protein is in the range of 1.0-7.5.
107. The method of any of embodiments 57-106, wherein the number of moles of acylating reagent used for each mole of the peptide or protein is in the range of 1.0-5.0.
108. The method of any of embodiments 57-107, wherein the number of moles of acylating reagent used for each mole of the peptide or protein is in the range of 1.0-3.0.
109. The method of any of embodiments 57-108, which comprises a step, prior to the acylation reaction step, of dissolving the peptide or protein in an aqueous solution.
110. The method of embodiment 109, wherein the peptide or protein is dissolved at a temperature of 1°C to 40°C.
111. The method of embodiment 110, wherein the peptide or protein is dissolved at a temperature of about RT (room temperature).
112. The method of any of embodiments 57-111, wherein the UPLC analysis is substantially as described in the experimental part (A2. General Methods of Detection, Analysis and Characterisation, part 3 and 4).
113. The method of embodiment 112, wherein the UPLC method is method UPLC method_3 or UPLC method_4.
114. The method of any of embodiments 57-113, wherein the desired product is a mono-acylated insulin peptide.
115. The method of embodiment 114, wherein the other products of the reaction consist of the non-acylated peptide, mono-acylated peptide and di-acylated variants thereof.
116. The method of any of embodiments 57-115, wherein the purity is at least 70%.
117. The method of any of embodiments 57-116, wherein the purity is at least 95%.
118. The method of any of embodiments 57-117, which comprises a further step of purifying the desired product of the acylation reaction.
119. The method of any of embodiments 57-118, wherein the peptide or protein is a pharmaceutical peptide or protein. 120. The method of embodiment 119, wherein the pharmaceutical peptide or protein is suitable for the treatment of diabetes or obesity.
121. The method of any of embodiments 119-120, wherein the pharmaceutical peptide or protein is selected from insulin and analogues thereof.
122. The method of embodiment 121, wherein the insulin peptide is human insulin and analogues thereof.
123. The method of any of embodiments 121-122, wherein the analogue has a maximum of 10 amino acid changes as compared to human insulin.
124. The method of any of embodiments 121-123, wherein the analogue has a maximum of 9 amino acid changes as compared to human insulin.
125. The method of any of embodiments 121-124, wherein the analogue has a maximum of 8 amino acid changes as compared to human insulin.
126. The method of any of embodiments 121-125, wherein the analogue has a maximum of 7 amino acid changes as compared to human insulin.
127. The method of any of embodiments 121-126, wherein the analogue has a maximum of 6 amino acid changes as compared to human insulin.
128. The method of any of embodiments 121-127, wherein the analogue has a maximum of 5 amino acid changes as compared to human insulin.
129. The method of any of embodiments 121-128, wherein the analogue has a maximum of 4 amino acid changes as compared to human insulin.
130. The method of any of embodiments 121-129, wherein the analogue has a maximum of 3 amino acid changes as compared to human insulin.
131. The method of any of embodiments 121-130, wherein the analogue has a maximum of 2 amino acid changes as compared to human insulin.
132. The method of any of embodiments 121-131, wherein the analogue has a maximum of 1 amino acid change as compared to human insulin.
133. The method of any of embodiments 121-132, wherein the analogue comprises the amino acid change desB30.
134. The method of any of embodiments 121-133, wherein the insulin peptide is an analogue of human insulin.
135. The method of any of embodiments 121-134, which comprises a further step of purifying the acylated insulin peptide.
136. The method of any of embodiments 121-135, wherein the acylated insulin peptide has insulin receptor affinity. 137. The method of embodiment 136, wherein the affinity is determined using the assay of Example 178 of WO2009/115469 at 0% HSA.
138. The method of embodiment 137, wherein the affinity is at least 0.10%.
EXAMPLES
This experimental part starts with a list of abbreviations, and is followed by a section including general methods for synthesising and characterising peptide analogues and derivatives of the invention. Then follows a number of examples which relate to the preparation of specific activated side chains, and their use in acylating peptide or protein analogues to produce desired derivatives thereof. The examples serve to illustrate the invention.
Abbreviations used:
The following abbreviations are used in the rest of this experimental part.
AcOH: Acetic acid
Ado: 8-Amino-3,6-dioxaoctanoic acid
Backbone: Peptide or peptide analogue (GLP-1, insulin)
CH3CN: Acetonitrile
DCC: N,N'-Dicyclohexylcarbodiimide
DCM: Dichloromethane
DCU: Dicyclohexylurea
DIC: Diisopropylcarbodiimide
DIPEA: Diisopropylethylamine
DMAP: 4-Dimethylaminopyridine
DMF: /V,/V-Dimethylformamide
DMSO: Dimethyl sulfoxide
EDC: l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
EtOAc: Ethylacetate
EtzO: Diethylether
Eq: Equivalent
Equiv: Equivalent
Fmoc: 9 H-fluoren-9-ylmethoxycarbonyl
HEPES: 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid HFIP: Hexafluoroisopropanol
H2O: Water
/-PrOH Isopropanol
KHSO4: Potassium bisulfate
LCMS Liquid chromathography mass spectrometry
MeOH Methanol
MeTHF Methyl tetra hydrofurane
MgS04: Magnesium Sulphate mL: Mililiter
MW: Molecular weight
NaOH: Sodium hydroxide
NHS: N-Hydroxysuccinimide
NMP: l-Methyl-pyrrolidin-2-one
NMR Nuclear magnetic resonance
OEG: Oligo Ethylene Glycol, alternative name to Ado, see above
OtBu: tert Butyl ester
Oxyma Pure®: Cyano-hydroxyimino-acetic acid ethyl ester
Rt: Retention time rt: Room temperature
PTFE: Polytetrafluoroethylene qNMR: Quantitative nuclear magnetic resonance
SPPS Solid phase peptide synthesis
SC: Side chain tBu: tert-Butyl
TEA: Triethylamine
TFA: Trifluoroacetic acid
THF: Tetra hydrofurane
TLC: Thin Layer Chromathography
UPLC Ultra perfomance liquid chromathography
Vol: Volume A. Materials and general methods for preparation, detection and characterisation
This section relates to methods for solid phase peptide synthesis (SPPS methods, including methods for de-protection of amino acids, methods for cleaving the peptide from resin, and for its purification), as well as methods for detecting and characterising the resulting side chains (LCMS and UPLC methods and NMR). XH spectra were recorded at 400 MHz on a Bruker Aeon 400 instrument. For qNMR 1,3-benzodioxole was used as the standard reference. Chemical shifts are reported in ppm on the 8 scale relatively to the chemical shift of the deuterated solvent. Kaiser-test (presence of free amines in SPPS) and Chloranil-test (test of piperidine in NMP) was performed according to "Fmoc solid phase peptide synthesis a practical approach" Edited by W.C. Chan and P.D. White, Oxford 2000 (2004), University Press page 61-62.
Al. Methods of preparation and modification
1, Synthesis of protected side chain
The resin bound protected side chains were prepared on a 2-chlorotritylcloride resin using standard Fmoc chemistry. The first Fmoc protected amino carboxylic acid (2 eq) (linker element) was dissolved in DCM and added to a DCM washed and drained resin. A tertiary amine base such as DIPEA or TEA (4 Eq) was added and the resin mixture was agitated for a period of time between 12 and 17 hours at RT. The resin was allowed to react with MeOH (0.79 mL/g resin) to cap free chloride sites at RT for 1 hour. The resin was drained and flow washed three times with NMP or DMF (about 5.2 mL/g resin).
Fmoc deprotection was achieved using piperidine in NMP preferably 20% piperidine (1.05 mL/g resin) in NMP (4.15 mL/g resin), at RT for 15 to 45 min, typically 30 min, before the resin was washed thoroughly with NMP or DMF. The step was repeated until complete deprotection was obtained, typically two times or more. The resin was drained and flow washed three times or more with NMP or DMF (ca 5.2 mL/g resin) until the Chloranil test gave a negative result.
Coupling of the sequential Fmoc protected amino carboxylic acids (linker) and the final mono-protected carboxylic diacid (such as 18-benzyloxy-18-oxo-octadecanoic acid) or phenoxy carboxylic diacid (such as 10-(4-benzyloxycarbonylphenoxy)decanoic acid), was achieved using conventional coupling conditions as described below. To a solution of Fmoc protected amino carboxylic acid (2-3 eq.) in a solvent like NMP or DMF and Oxyma Pure® (2-3 eq.) was added DIC (2-3 eq). The mixture was agitated for 15 to 60 min before the mixture was added to the resin. The mixture was agitated at RT for 1 to 18 hours, typically 17 hours. Alternatively, if the coupling was not completed judged by the Kaiser test, the step was repeated until a negative test was achieved.
After synthesis, the resin was washed by DCM and the protected side chain was cleaved off from the resin by treatment with 1% TFA in DCM for 1-3 hours. The cleavage solution was evaporated under vacuum to dryness, and the crude material was used without further purification in the activation step described in the next section.
2. Methods for removal of tBu protection group
Deprotection of the tBu esters was achieved using standard procedures described in the literature (Greene's Protective Group in Organic Synthesis, 4th addition, ISBN-13:978- 0471697541).
Method: Mod U l
Tert-butyl ester deprotection was made by following procedure; the protected activated side chain was dissolved in a mixture of TFA with 1-3% of water. The mixture was stirred at RT until the reaction was complete typically from 1 to 3 hours. Alternatively concentrated hydrochloric acid or TFA or a 1 : 1 mixture of TFA and a suitable solvent such as THF or DCM could be used instead. The product mixture was subjected to evaporation under vacuum to yield a crude oil. Precipitation of the oil in an appropriate solvent such as diethyl ether, isopropyl ether, tert-butyl methyl ether or heptane followed by filtration gave crude material.
The activated side chains were dried under vacuum, analysed by UPLC, MS or NMR and used in the examples described in sections E, F, G and H without further purification.
3. General method for in situ generation of activated side chains (acylating reagents) The carboxylic acid (0.24 mmol, 1.2 eq.) and TEA (0.3 mmol, 3 eq.) was dissolved in MeCN (1.5 mL). 5-Bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Chem. 4a, Example Bl; 0.2 mmol, 1 eq.) was dissolved in THF (0.5 mL) and added dropwise to the carboxylic acid solution. The resulting mixture was then stirred for 15-30 minutes at rt. This resulted in in situ generation of the activated side chain.
4. Genereral acylation procedure
Method 1: Solid desB30 human insulin was weighed out (28.5 mg, 5 pmol) and transferred to a 4 mL glass veil equipped with a magnetic stir bar. HEPES buffer (0.95 mL, 0.2M, pH 7.4) was added to the glass veil, giving a concentration of desB30 human insulin of 30 mg/mL. The activated side chain (1.25 equiv.) was weighed out in another glass veil and dissolved in 50 pL DMF. The side chain solution was then added to the insulin solution. The pH was adjusted to 7.4 using NaOH (0.5M) and the reaction mixture was then stirred at rt. for 24 hours. After 24 hours a small aliquot of the reaction mixture was taken out, diluted with a mixture of AcOH/HzO/MeCN (2: 1 : 1) and analysed by UPLC analysis.
Method 2:
Following this method, the in situ generated activated side chains are used without isolation and purification.
Solid desB30 human insulin was weighed out (28.5 mg, 5 pmol) and transferred to a 4 mL glass veil equipped with a magnetic stir bar. HEPES buffer (0.95 mL, 0.2M, pH 7.4) was added to the glass veil, giving a concentration of desB30 human insulin of 30 mg/mL.
60 |iL of mixture 1 was then added to the mixture obtained following the general method for in situ generation of activated side chains and the pH of the resulting mixture was adjusted to 7.4 using NaOH (0.5M). The reaction mixture was stirred at rt for 24 hours. After 24 hours a small aliquot of the reaction mixture was taken out, diluted with a mixture of AcOH/H2O/MeCN (2: 1 : 1) and analysed by UPLC analysis.
A2. General Methods of Detection, Analysis and Characterisation
1. LC-MS method 1
RP-analysis was performed using Waters Acquity UPLC system and Waters Xevo G2-XS Q-tof mass spectrometer fitted with Waters C18 BEH column (1.7 um, 2.1 x 150 mm, column own temperature 40 °C). Eluent A: Water. Eluent B: Acetonitrile. Eluent D: 50 mM Ammonium formate pH 9 in water. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-5 pl) onto the column which was eluted with a gradient of A and B. The UPLC conditions, detector settings and mass spectrometer settings were: Gradient: Linear 95% - 0% (vol/vol) A, 0% - 95% (vol/vol) B and 5% (vol/vol) D, 4 min., 0.4 ml/min. UV detection at 214 and 280 nm. MS ionisation mode: API-ES-; Scan 50-4000 amu.
2. LC-MS method 2
RP-analysis was performed using Waters Acquity UPLC system and Waters Xevo G2-XS Q-tof mass spectrometer fitted with Waters C4 BEH column (1.7 um, 2.1 x 150 mm, column own temperature 40 °C). Eluent A: Water. Eluent B: Acetonitrile. Eluent C: 2% FA 0.1% TFA in water. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-5 pl) onto the column which was eluted with a gradient of A and B. The UPLC conditions, detector settings and mass spectrometer settings were: Gradient: Linear 95% - 0% (vol/vol) A, 0% - 95% (vol/vol) B and 5% (vol/vol) D, 4 min., 0.4 ml/min. UV detection at 214 and 280 nm. MS ionisation mode: API-ES+; Scan 50-4000 amu.
3. UPLC method 3
RP-analysis was performed using Waters Acquity UPLC system and Acquity QDa mass detector fitted with Waters C18 BEH column (1.7 um, 2.1 x 150 mm, column own temperature 40 °C). Eluent A: 0.05% (vol/vol) TFA in water. Eluent B: 0.05% (vol/vol) TFA in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. The UPLC conditions, detector settings and mass spectrometer settings were: Gradient: Linear 5% - 60% (vol/vol) B, 16 min, 0.4 ml/min. UV detection at 214 nm. MS ionisation mode: API-ES+; Scan: 100-1250 amu.
4. UPLC method 4
RP-analysis was performed using Waters Acquity UPLC system and Acquity QDa mass detector fitted with Waters C4 BEH column (1.7 um, 2.1 x 150 mm, column own temperature 40 °C). Eluent A: 0.05% (vol/vol) TFA in water. Eluent B: 0.05% (vol/vol) TFA in acetonitrile. The analysis was performed at RT by injecting an appropriate volume of the sample (preferably 0.5-10 pl) onto the column which was eluted with a gradient of A and B. The UPLC conditions, detector settings and mass spectrometer settings were: Gradient: Linear 5% - 95% (vol/vol) B, 16 min, 0.4 ml/min. UV detection at 214 nm. MS ionisation mode: API-ES+; Scan: 100-1250 amu.
B. Synthesis of phenols
Example Bl:
Figure imgf000031_0001
of 5-bromo-2-
Figure imgf000031_0002
chloride
Chem. 4a:
Figure imgf000031_0003
4-Bromo-2-(trifluoromethyl)phenol (1 equiv.) was added in small portions to chlorosulfonic acid (6 equiv.) at 0 °C. After ended addition, the reaction mixture was allowed to reach rt and stirred until all 4-Bromo-2-(trifluoromethyl)phenol was consumed (TLC analysis). Next the reaction mixture was poured onto crushed ice leading to precipitation of the product. The precipitate was collected by filtration, washed with cold water and dried under vacuum. The product was obtained as an off white solid (2.19 g, 6.45 mmol, 39%).
The compound was characterised by LC-MS, XH NMR.
XH NMR (400 MHz, DMSO-c/6) 8 ppm 7.72 (d, 1H), 7.79 (d, 1H), 14.41 (bs, 1H).
LC-MS method 2: calculated Mass for [M+Na]+ 360.8519 Mass Found [M+Na]+ 360.8510
Example B2: Preparation of 5-bromo-2-hvdroxy-N,N-dimethyl-3-(trifluormethyl)- benzenesulfonamide (Chem. 2a) Chem. 2a:
Figure imgf000031_0004
5-Bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Example Bl; 2.04 g, 6 mmol) was added in small portions to a mixture EtOH (10 mL) and dimethylamine in water (10 mL). The reaction was stirred at rt. o/n. Next the pH was decreased to 3 using HCI (cone.). EtOAc was added and the mixture was washed with brine. The organic phase was collected, dried over MgS04, filtered and evaporated under reduced pressure. The title compound was purified by flash column chromatography eluting with a gradient from 10% EtOAc in heptane to 30% EtOAc in heptane and obtained as a white solid (1.55 g, 4.44 mmol, 74%).
The compound was characterised by LC-MS, XH NMR.
XH NMR (400 MHz, DMSO-c/6) 8 ppm 2.77 (s, 6H), 8.00 (d, 1H), 8.05 (d, 1H), 10.33 (bs, 1H).
LC-MS method 2: calculated Mass for [M+H]+ 347.9511 Mass Found [M+H]+ 347.9550
C. Synthesis of activated reference side chains
Example Cl:
Figure imgf000032_0001
of reference
Figure imgf000032_0002
18-rr(lS)-l-carboxy-4-r2-r2-r2-r2-r2- din-l-yl)oxy-2-oxo-
Figure imgf000032_0003
minol-2-oxo-
Figure imgf000032_0004
minol-4-oxo-
Figure imgf000032_0005
minol-18-oxo-octadecanoic acid
Figure imgf000032_0006
Chem. 10:
Figure imgf000032_0007
The compound was prepared as described in W02010/029159.
Example C2: Preparation of reference compound 18-IT(lS)-l-carboxy-4-r2-r2-r2-r2-r2- r2-(2,4-dichloro-6-sulfo-phenoxy)-2-oxo-ethoxy1ethoxy1ethylamino1-2-oxo- ethoxy1ethoxy1ethylamino1-4-oxo-butyl1amino1-18-oxo-octadecanoic acid (Chem. 11) Chem. 11:
Figure imgf000032_0008
The compound was prepared as described in WO2018/083335.
Example C3: Preparation of reference compound 22-carboxy-l-(4-nitrophenoxy)- l,10,19,24-tetraoxo-3,6,12,15-tetraoxa-9,18,23-triazahentetracontan-41-oic acid (Chem. 12)
Chem. 12:
Figure imgf000032_0009
t-Bu protected C18-diacid-yGlu-Ado-Ado-OH (synthesized according to procedures found in section Al "methods of preparation and modifications" and procedures found in WO2018/083335) (0.5 mmol, 0.423 g), DCC (0.6 mmol, 0.124 g, 1.2 eq.) and DMAP (0.05 mmol, 0.006 g, 0.1 eq.) was dissolved in 15mL DCM, followed by addition of the prior art activator 4-Nitrophenol (PNP) (0.55 mmol, 0.077 g, l.leq). The resulting solution was stirred at RT for 18 hours. Next the DCU was removed by filtration. The crude product was purified using silicagel column chromatography with a gradient eluent from DCM to 10% MeOH in DCM. After evaporation the product was obtained as a light brown oil. Method Mod BU-l was used to cleave the tBu-esters for 1.5 hours. The cleavage mixture was evaporated under reduced pressure. The product was obtained as a light brown oil. (0.324 g, 0.38 mmol, 76%). Active content of material from 1H qNMR is 77% w/w The compound was characterised by LC-MS and XH NMR.
XH NMR (400 MHz, DMSO-c/6) 8 ppm 1.22 - 1.23 (m, 24 H) 1.41 - 1.54 (m, 4 H) 1.70 - 1.82 (m, 1 H) 1.88 -2.01 (m, 1 H) 2.05 - 2.22 (m, 6 H) 2.70 (s, 6 H) 3.20 (q, 2 H) 3.28 (q, 2 H) 3.40 (t, 2 H) 3.46 (t, 2 H) 3.50 - 3.61 (m, 6 H) 3.61 - 3.75 (m, 2 H) 3.88 (s, 2 H) 4.09 - 4.19 (m, 1 H) 4.50 (s, 2 H) 7.66 (t, 1 H) 7.88 (t, 1 H) 8.03 (d, 1H) 8.27 (d, 1 H) 8.42 (d, 1 H)
LC-MS method 1: calculated Mass for [M-H]' 853.4452 Mass Found [M-H]’ 853.4547
Example C4: Preparation of 2-[2-
Figure imgf000033_0001
-3,5-dichloro- benzenesulfonic acid
Figure imgf000033_0002
Chem. 31:
Figure imgf000033_0003
The activated side chain of Chem. 31 was generated in situ from N-carbobenzyloxy- glycine and 3,5-dichloro-2-hydroxy-benzenesulfonyl chloride (the prior art activator 3,5- DC-HBSA) using the general method for in situ generation of activated side chains. D. Synthesis of activated side chains of the invention
Example DI : Preparation of l-(4-bromo-2-(/V,/V-dimethylsulfamoyl)-6-(trifluoromethyl)- phenoxy)-22-carboxy- 1, 10,19, 24-tetraoxo-3, 6, 12, 15-tetraoxa-9, 18,23- triazahentetracontan-41-oic acid (Chem. 13)
Chem. 13:
Figure imgf000034_0001
t-Bu protected C18-diacid-yGlu-Ado-Ado-OH (synthesized according to procedures found in section Al "methods of preparation and modifications" and procedures found in WO2018/083335) (2 mmol, 1.692g), DCC (2.4 mmol, 0.495 g, 1.2 eq.) and DMAP (0.2 mmol, 0.024 g, 0.1 eq.) was dissolved in 15mL DCM, followed by addition of 5-Bromo-2- hydroxy-/V,/V-dimethyl-3-(trifluoromethyl)benzenesulfonamide (Chem. 2a; the phenol of Example B2) (2.2 mmol, 0.766 g, l.leq). The resulting solution was stirred at RT for 18 hours. Next the DCU was removed by filtration. The crude product was purified using silicagel column chromatography with a gradient eluent from DCM to 10% MeOH in DCM. After evaporation the product was obtained as a light brown oil. Method Mod U l was used to cleave the tBu-esters for 1.5 hours. The cleavage mixture was evaporated under reduced pressure. The product was obtained as a light brown oil. (1.55 g, 1.5 mmol, 75%). Active content of material from 1H qNMR is 53% w/w The compound was characterised by LC-MS and XH NMR.
XH NMR (400 MHz, DMSO-c/6) 8 ppm 1.22 - 1.23 (m, 24 H) 1.41 - 1.54 (m, 4 H) 1.70 - 1.82 (m, 1 H) 1.88 -2.01 (m, 1 H) 2.05 - 2.22 (m, 6 H) 2.70 (s, 6 H) 3.20 (q, 2 H) 3.28 (q, 2 H) 3.40 (t, 2 H) 3.46 (t, 2 H) 3.50 - 3.61 (m, 6 H) 3.61 - 3.75 (m, 2 H) 3.88 (s, 2 H) 4.09 - 4.19 (m, 1 H) 4.50 (s, 2 H) 7.66 (t, 1 H) 7.88 (t, 1 H) 8.03 (d, 1H) 8.27 (d, 1 H) 8.42 (d, 1 H)
LC-MS method 1: calculated Mass for [M-H]' 1061.3621 Mass Found [M-H]’ 1061.3633 Example D2: Preparation of l-(4-bromo-2-sulfo-6-(trifluoromethyl)phenoxy)-22-carboxy- l,10,19,24-tetraoxo-3,6,12,15-tetraoxa-9,18,23-triazahentetracontan-41-oic acid (Chem. 14)
Chem. 14:
Figure imgf000035_0001
t-Bu protected C18-diacid-yGlu-Ado-Ado-OH (synthesized according to procedures found in section Al "methods of preparation and modifications" and procedures found in WO2018/083335) (1 mmol, 0.846 g) and TEA (3 mmol, 0.42 mL, 3 eq.) was dissolved in MeTHF (10 mL). 5-Bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) (1.1 mmol, 0.373 g, 1.1 eq.) was dissolved in MeTHF (5 mL) and slowly added to the mixture dropwise. The reaction mixture was then stirred until full conversion was observed (UPLC analysis). Next the mixture was washed with 5% KHSC (3 x 5 mL). The organic phase was collected, dried over MgSC , filtered and concentrated under reduced pressure. The crude product was purified by using silicagel column chromatography with a gradient eluent from DCM to 10% MeOH in DCM. After evaporation the product was obtained as a light brown oil. Method Mod U l was used to cleave the tBu-esters for 1.5 hours. The cleavage mixture was evaporated under reduced pressure. The product was obtained as a brown oil. (0.529 g, 0.51 mmol, 51%). Active content of material from 1H qNMR is 69% w/w The compound was characterised by LC-MS and XH NMR.
XH NMR (400 MHz, DMSO-c/6) 8 ppm 1.16 - 1.30 (m, 24 H) 1.41 - 1.54 (m, 4 H) 1.70 - 1.81 (m, 1 H) 1.88 -2.00 (m, 1 H) 2.05 - 2.22 (m, 6 H) 3.20 (q, 2 H) 3.28 (q, 2 H) 3.39 (t, 2 H) 3.46 (t, 2 H) 3.50 - 3.61 (m, 6 H) 3.63 - 3.72 (m, 2 H) 3.88 (s, 2 H) 4.09 - 4.19 (m, 1 H) 4.37 (s, 2 H) 7.69 (t, 1 H) 7.88 (t, 1 H) 7.99 (d, 1H) 8.03 (d, 1 H) 8.13 (d, 1 H) LC-MS method 1: calculated Mass for [M-H]' 1034.3148 Mass Found [M-H]’ 1034.3143 Example D3:
Figure imgf000036_0001
of 4-bromo-2-(/V,/V-di
Figure imgf000036_0002
-6-
Figure imgf000036_0003
2-Chlorotrityl resin (2.5 g) was transferee! to a solid phase peptide synthesis glass vessel. DCM (20 mL) was added to the resin which was then stirred for 2 minutes and then drained. Then DCM (40 mL) was again added to the resin, which was then stirred for 30 minutes and then drained. To a solution of Fmoc-8-amino-3,6-dioxaoctanoic acid (8.00 mmol, 3.10 g) in DCM (18 mL) was added DIPEA (8.00 mmol, 1.4 mL). The resulting solution was next added to the resin followed by addition of DIPEA (8.00 mmol, 1.4 mL). The resin was then stirred for 3 hours. Afterwards MeOH (2 mL) was added to the resin solution, which was then stirred for 1 hour and then drained. The resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and then drained and dried in vacuum oven at 30 °C. The loading was determined to 1.36 mmol/g resin.
The loaded resin (1.56 g, 1.36 mmol/g resin, 2.12 mmol) was transferred to a solid phase peptide synthesis glass vessel and treated with 20% piperidine in DMF (20 mL, 2 x 15 minutes) for fmoc deprotection. Next the resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and then drained.
2-Azidoacetic acid (6 mmol, 0.45 mL) was added to a solution of Oxyma (6 mmol, 0.853 g) in DMF (18 mL) followed by addition of DIC (6 mmol, 0.940 mL) and DIPEA (12 mmol, 2.09 mL). The resulting solution was stirred for 30 minutes and then added to the resin and stirred o/n. Next the resin was drained, washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained. Next the resin was treated with HFIP/DCM (1:4, 20 mL, 2 x 30 minutes). The cleaved product was collected and concentrated under reduced pressure. The product (2-[2-[2-[(2-azidoacetyl)amino]ethoxy]ethoxy]acetic aied) was used in the next step without further purification.
2-[2-[2-[(2-azidoacetyl)amino]ethoxy]ethoxy]acetic aied (2.12 mmol, 0.522 g), DCC (2.54 mmol, 0.524 g, 1.2 eq.) and DMAP (0.21 mmol, 0.026 g, 0.1 eq.) was dissolved in 15mL DCM, followed by addition of 5-Bromo-2-hydroxy-/V,/V-dimethyl-3- (trifluoromethyl)benzenesulfonamide (2.2 mmol, 0.766 g, l.leq) (Chem. 2a; the phenol of Example B2). The resulting solution was stirred at RT for 18 hours. Next the DCU was removed by filtration. The crude product was purified using silicagel column chromatography with a gradient eluent from 30% EtOAc in heptane to 50% EtOAc in heptane. The product was obtained as a yellow oil. (0.25 g, 0.42 mmol, 20%). Active content of material from 1H qNMR is 79% w/w
The compound was characterised by LC-MS and XH NMR.
XH NMR (400 MHz, DMSO-c/6) d ppm 2.74 (s, 6 H) 3.26 (q, 2 H) 3.45 (t, 2 H) 3.57 (t, 2 H) 3.68 (bs, 2 H) 3.80 (s, 2 H) 4.50 (d, 2 H) 8.15 (t, 1 H) 8.26 (s, 1 H) 8.40 (s, 1 H) LC-MS method 2: calculated Mass for [M+Na]+ 599.0189. Mass Found [M + Na]+ 599.0188
Example D4: Preparation of 2-(2-
Figure imgf000037_0001
-5-bromo-3-
Figure imgf000037_0002
benzenesulfonic acid
Figure imgf000037_0003
Chem. 16:
Figure imgf000037_0004
The activated side chain of Chem. 16 was generated in situ from 2-(benzylthio)acetic acid and 5-bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
Example D5: Preparation of 4-bromo-2-(/V,/V-dimethylsulfamoyl)-6- (trifluoromethyl)phenyl((benzyloxy)carbamoyl)olvcinate (Chem. 17) Chem. 17:
Figure imgf000037_0005
To a solution of N-benzyloxycarbonylglycine (1.0 mmol, 0.21 g, 1.0 equiv.), DCC (1.2 mmol, 0.25g, 1.2 equiv.) and DMAP (0.1 mmol, 0.01 g, 0.1 equiv.) in DCM (10 mL) was addded 5-Bromo-2-hydroxy-N,N-dimethyl-3-(trifluormethyl)-benzenesulfonamide (Chem. 2a; the phenol of Example B2) (1.1 mmol, 0.39 g, 1.1 equiv.). The mixture was stirred at rt overnight. Next, the solids were removed by filtration and the product was purifed by flash column chromathography eluting with a gradient from 30% EtOAc in heptane to 50% EtOAc in heptane. The product was isolated as a white solid (0.73 mmol, 0.38 g, 73%).
The compound was characterised by LC-MS and XH NMR.
XH NMR (400 MHz, DMSO-c/6) d ppm 2.75 (s, 6H), 4.10-4.20 (m, 2H), 5.08 (s, 2H), 7.28-7.39 (m, 5H), 8.02 (t, 1H), 8.26 (s, 1H), 8.41 (s, 1H).
LC-MS method 2: calculated Mass for [M+Na]+ 560.9913. Mass Found [M + Na]+ 560.9933
Example D6:
Figure imgf000038_0001
-5-bromo-3-
Figure imgf000038_0002
sulfonic acid
Figure imgf000038_0003
Chem. 18:
Figure imgf000038_0004
The activator of Chem. 18 was generated in situ from 2-azidoacetic acid and 5-bromo-2- hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
Example D7: Preparation of 5-bromo-2-(2-(2-((4-methyl-2-oxo-2/-/-chromen-7-yl)- amino)-2-oxoethoxy)acetoxy)-3-(trifluoromethyl)benzenesulfonic acid (Chem. 19)
Chem. 19:
Figure imgf000038_0005
Step 1 :
Preparation of: 2-(2-((4-methyl-2-oxo-2H-chromen-7-yl)amino)-2-oxoethoxy)acetic acid
Figure imgf000039_0001
7-Amino-4-methylcoumarin (1 g, 5.71 mmol, 1 equiv.) was dissolved in pyridine (40 mL). To this solution was added diglycolic anhydride (0.86 g, 7.42 mmol, 1.3 equiv.) The mixture was stirred at rt over night. Next DCM (80 mL) was added to the mixture leading to precipitation. The mixture was filtered and the precipitate was washed with DCM (200 mL) and toluene. The filtrate was then concentrated under reduced pressure. The crude product was washed with 10% KHSO4 and filtered. The solids was collected and dried under vaccuo. The product was used without further purification. The product was isolated as a brown solid (1.40 g, 4.79 mmol, 84%).
The compound was characterised by LC-MS, XH NMR.
XH NMR (400 MHz, DMSO-c/6) 8 ppm 2.40 (s, 3H), 4.24 (s 4H), 6.27 (s, 1H), 7.59 (d, 1H), 7.73 (d, 1H), 7.80 (s, 1H), 10.30 (s, 1H).
LC-MS method 1: calculated Mass for [M-H]' 290.0670 Mass Found [M-H]’ 290.0691
Step 2:
The activated side chain of Chem. 19 was generated in situ from 2-(2-((4-methyl-2-oxo- 2H-chromen-7-yl)amino)-2-oxoethoxy)acetic acid and 5-bromo-2-hydroxy-3- (trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
Example D8:
Figure imgf000039_0002
of 5-bromo-2-(2-
Figure imgf000039_0003
-3-tri
Figure imgf000039_0004
sulfonic acid
Figure imgf000039_0005
Chem. 40:
Figure imgf000039_0006
The activated side chain of Chem. 40 was generated in situ from chloroacetic acid and 5- bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
-5-bromo-3-
Figure imgf000040_0001
The activator of Chem. 32 was generated in situ from N-carbobenzyloxyglycine and 5- bromo-2-hydroxy-3-(trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
Example D12: Preparation of 5-Bromo-2-(2-(2-(2-(2-chloroacetamido)ethoxy)ethoxy)- acetoxy)-3-(trifluoromethyl)benzenesulfonic acid (Chem. 33)
Chem. 33:
Figure imgf000040_0002
2-Chlorotrityl resin (2.5 g, 1.6 mmol/g) was transfered to a solid phase peptide synthesis glass vessel. DCM (20 mL) was added to the resin which was then stirred for 2 minutes and then drained. Then DCM (40 mL) was again added to the resin, which was then stirred for 30 minutes and then drained. To a solution of Fmoc-8-amino-3,6-dioxaoctanoic acid (3.10 g, 8.00 mmol, 2.0 equiv.) in DCM (18 mL) was added DIPEA (1.4 mL, 8.00 mmol, 2.0 equiv.). The resulting solution was next added to the resin followed by addition of DIPEA (1.4 mL, 8.00 mmol, 2.0 equiv.). The resin was then stirred for 3 hours. Afterwards MeOH (2 mL) was added to the resin solution, which was then stirred for 1 hour and then drained. The resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and then drained and dried in vacuum oven at 30 °C. The loading was determined to 1.36 mmol/g resin.
The loaded resin (1.56 g, 1.36 mmol/g resin, 2.12 mmol) was transferred to a solid phase peptide synthesis glass vessel and treated with 20% piperidine in DMF (20 mL, 2 x 15 minutes) for fmoc deprotection. Next the resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and then drained.
Chloroacetic acid (0.57 g, 6 mmol, 3.0 equiv.) was added to a solution of Oxyma (0.853 g, 6 mmol, 3.0 equiv.) in DMF (18 mL) followed by addition of DIC (6 mmol, 0.940 mL, 3.0 equiv.) and DIPEA (2.09 mL, 12 mmol, 6.0 equiv.). The resulting solution was stirred for 30 minutes and then added to the resin and stirred o/n. Next the resin was drained, washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained. Next the resin was treated with HFIP/DCM (1 :4, 20 mL, 2 x 30 minutes). The cleaved product was collected and concentrated under reduced pressure. The product was used in the next step without further purification.
Step 2:
The activated side chain of Chem. 33 was generated in situ from 2-(2-(2-(2- chloroacetamido)ethoxy)ethoxy)acetic acid and 5-bromo-2-hydroxy-3- (trifluoromethyl)benzenesulfonyl chloride (Chem. 4a; the phenol of Example Bl) using the general method for in situ generation of activated side chains.
Example D14: Preparation of (S)-4-((((9/-/-fluoren-9-yl)methoxy)carbonyl)amino)-5-
-bromo-2-sulfo-6- -2-oxoethyl)amino)-3- oxopropyl)amino)-l-oxopropan-2-yl)amino)-5-oxopentanoic acid (Chem. 34)
Chem. 34:
Figure imgf000042_0001
Step 1: Preparation of (3-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5- (tert-butoxy)-5-oxopentanamido)propanamido)propanoyl)glycine
Figure imgf000042_0002
2-Chlorotrityl resin (1 g, 1.06 mmol/g) was transfered to a solid phase peptide synthesis glass vessel. DCM (10 mL) was added to the resin which was then stirred for 2 minutes and then drained. Then DCM (20 mL) was again added to the resin, which was then stirred for 30 minutes and then drained.
To a solution of Fmoc-Gly-OH (0.9 g, 3.0 mmol, 3.0 equiv.) in DCM (7 mL) was added DIPEA (0.5 mL, 3.0 mmol, 3.0 equiv.). The resulting solution was next added to the resin followed by addition of DIPEA (0.5 mL, 3.0 mmol, 3.0 equiv.). The resin was then stirred for 3 hours. Afterwards MeOH (1 mL) was added to the resin solution, which was then stirred for 1 hour and then drained. The resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained.
Next the resin was treated with 20% piperidine in DMF (10 mL, 2 x 15 minutes) for fmoc deprotection. The resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained.
Fmoc-0-Ala-OH (0.9 g, 3.0 mmol, 3.0 equiv.) and Oxyma (0.4 g, 3 mmol, 3.0 equiv.) was dissolved in DMF (8.5 mL). DIC (0.5 mL, 3.0 mmol, 3.0 equiv.) and Collidine (0.8 mL, 6 mmol, 6.0 equiv.) was added and the resulting mixture was stirred at rt. for 1 hour and then added to the resin. The resin was stirred for 3 hours then washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained.
Next the resin was treated with 20% piperidine in DMF (10 mL, 2 x 15 minutes) for fmoc deprotection. The resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained.
Fmoc-Ala-OH (0.9 g, 3.0 mmol, 3.0 equiv.) and Oxyma (0.4 g, 3 mmol, 3.0 equiv.) was dissolved in DMF (8.5 mL). DIC (0.5 mL, 3.0 mmol, 3.0 equiv.) and Collidine (0.8 mL, 6 mmol, 6.0 equiv.) was added and the resulting mixture was stirred at rt. for 1 hour and then added to the resin. The resin was stirred for 3 hours then washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained.
Next the resin was treated with 20% piperidine in DMF (10 mL, 2 x 15 minutes) for fmoc deprotection. The resin was washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained.
Fmoc-Glu(OtBu)-OH (1.3 g, 3.0 mmol, 3.0 equiv.) and Oxyma (0.4 g, 3 mmol, 3.0 equiv.) was dissolved in DMF (8.5 mL). DIC (0.5 mL, 3.0 mmol, 3.0 equiv.) and Collidine (0.8 mL, 6 mmol, 6.0 equiv.) was added and the resulting mixture was stirred at rt. for 1 hour and then added to the resin. The resin was stirred for 3 hours then washed with DCM (3 x 5 mL), DMF (3 x 5 mL) and DCM (3 x 5 mL) and drained.
The tetrapeptide was cleaved from the resin with HFIP/DCM (1:4, 20 mL, 2 x 30 minutes). The cleaved product was collected and concentrated under reduced pressure. The crude peptide were dissolved in H2O (14.5 mL), AcOH(4 mL) and MeCN (1.5mL) and purified by RP-HPLC eluting with a gradient from 60:40 to 35:65 HzO/MeCN + 0.1% trifluoroacetic acid over 40 minutes. Pure fractions were collected and lyophilized to afford the peptide as white solids. (0.6 g, 0.89 mmol, 89%).
XH NMR (400 MHz, DMSO-c/6) d ppm 1.18 (d, J = 7.2 Hz, 3H), 1.39 (s, 9H), 1.67-177 (m, 1H), 1.83-1.94 (m, 1H), 2.23 (t, J = 7.6 Hz, 2H), 2.30 (t, J = 7.6 Hz, 2H), 3.17-3.32 (m, 2H), 3.74 (d, J = 6.0 Hz, 2H), 4.01 (q, J = 6.0 Hz, 1H), 4.14-4.35 (m, 4H), 7.32 (t, J = 7.1 Hz, 2H), 7.41 (t, J = 7.1 Hz, 2H), 7.53 (d, J = 8.6 Hz, 1H), 7.72 (t, J = 6.8 Hz, 2H), 7.85-7.99 (m, 4H), 8.21 (t, J = 5.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) 6 ppm 18.4, 27.3, 27.8, 31.4, 34.9, 35.3, 40.6, 46.7, 48.2, 53.8, 65.7, 79.7, 120.1, 125.3, 127.1, 127.7, 140.7, 143.8, 156.0, 170.7, 170.9, 171.4, 171.8, 172.0. LC-MS method 1 calculated Mass for [M-H]' 623.2723. Mass Found [M-H]’ 623.2896 Step 2, activation: (5S,12S)-5-(2-carboxyethyl)-l-(9H-fluoren-9-yl)-12-methyl-3,6,10,13-tetraoxo-2-oxa- 4,7,ll,14-tetraazahexadecan-16-oic acid (0.28 g, 0.46 mmol, 1.0 equiv.) and TEA (0.19 mL, 0.14 mmol, 3.0 equiv.) was dissolved in MeTHF (15 mL). 5-Bromo-2-hydroxy-3- (trifluoromethyl)benzenesulfonyl chloride (0.17 g, 1.1 mmol, 1.1 equiv.) (Chem. 4a; the phenol of Example Bl) was dissolved in MeTHF (10 mL) and slowly added to the mixture. After ended addition, the mixture was stirred at rt for 3 hours. Afterwards, the solvent was removed under reduced pressure. Next a mixture of TFA/DCM (1: 1, 10 mL) + 1% H2O was added and the resulting mixture was stirred for 2 hours at rt. The mixture was then concentrated under reduced pressure. The crude compound was dissolved in H2O (20 mL) and purified by RP-HPLC eluting with a gradient from 65:35 to 55:45 H2O/MeCN + 0.1% trifluoroacetic acid over 40 minutes. Pure fractions were collected and lyophilized to afford the title compound as a white solid. (0.24 g, 0.28 mmol, 61%).
XH NMR (400 MHz, DMSO-c/6) 6 ppm 1.17 (d, J = 6.7 Hz, 3H), 1.68-1.81 (m, 1H), 1.83- 1.97 (m, 1H), 2.20-2.39 (m, 4H), 3.15-3.36 (m, 2H), 3.91-4.37 (m, 7H), 6.63 (bs, 2H), 7.32 (t, J = 7.4 Hz, 2H), 7.41 (t, J = 7.8 Hz, 2H), 7.55 (d, J = 7.6 Hz, 1H), 7.72 (t, J = 5.8 Hz, 2H), 7.84-8.03 (m, 5H), 8.11 (s, 1H), 8.48 (t, J = 6.4 Hz, 1H). 19F NMR (376 MHz, DMSO-d6) 6 -59.8. 13C NMR (100 MHz, DMSO-d6) 6 ppm 18.5, 27.3, 30.3, 34.8, 35.2, 40.8, 46.7, 48.2, 53.9, 65.7, 118.2, 120.1, 121.9 (q, J = 272.0 Hz), 125.4, 127.1, 127.7, 130.1 (q, J = 4.8 Hz), 135.4, 140.7, 143.8 (d, J = 3.3 Hz), 143.9, 144.1 (d, J = 2.2 Hz), 156.0, 167.7, 170.7, 171.0, 172.0, 174.0. LC-MS method 1: Calculated for [M- H]- 869.0957. Mass Found [M-H]’ 869.1063
E. Reference acylation reactions at prior art conditions (pH = 11.7)
In order to compare, acylation reactions under the alkaline reaction conditions described in WO2018/083335 were carried out for acylation of desB30 human insulin using reference activators from the prior art and two representative activators of the present invention. The activated side chain are for all examples the activated side chain of Chem. 20:
Chem. 20:
Figure imgf000044_0001
Acylation procedure at pH=11.7
The pH of the acylation reaction was controlled by an auto-titrator to keep a constant pH during the reaction (Titrando/Dosino®).
Solid desB30 human insulin was weighed out (240 mg, 43.2 pmol) and transferred to the titrate vessel and mixed with 1.00 ml water for about 15 min. The desB30 human insulin slowly dissolved. The pH was close to 9.
The auto-titrator vessel was connected to a cooling system and cooled at 5° C. and the pH was raised to 10.5 by adding NaOH (0.2 M, 400 pL) drop wise. The mixture was clear and colourless. 400 pL water was added manually to give a total volume of 1.80 mL. Just before addition of the activated side chain the desB30 human insulin solution was titrated to pH 11.7 with NaOH (0.5 M, 185 pL). The total volume and concentration of the desB30 human insulin solution was 2.0 mL and 120 mg/mL.
The activated side chain (1.2 or 1.5 eq.) was dissolved in 0.5 ml NMP. The activated side chain was added to the desB30 human insulin solution with a flowrate of 0.08 mL/min.
The progress of the acylation was analysed by UPLC_method_Al. UPLC samples was quenched with a solution of AcOH/HzO/MeCN (2: 1: 1) before being analysed. Conversion was based on UPLC analysis.
Results
In table 1, the result from acylations performed at alkaline conditions (pH=11.7) is shown. The side chain are identical for all the examples (see above Chem. 20). To activate the side chain, different activators are used. In the activated side chain of Chem. 10 (Example Cl), the prior art NHS activator is used. In the activated side chain of Chem. 11 (Example C2), the prior art activator 3,5-DC-HBSA from WO2018/083335 is used.
In the activated side chains of Chem. 13 (Example DI) and Chem. 14 (Example D2), the activators of Chem. 2a and Chem. 2b, respectively, of the present invention is used.
As described in WO2018/083335, acylation at alkaline pH leads predominantly to acylation at the lysine in position B29 of human insulin (LysB29). Some diacylated products are seen, arising from acylation of LysB29 as well as of either GlyAl (the glycine in position 1 of the A-chain of the human insulin analogue) or of PheB1 (the phenylalanine in position 1 of the B-chain of the human insulin analogue). This applies both for the prior art NHS activator and the prior art activator 3,5-DC-HBSA (WO2018/083335).
Table 1 : Acylation reactions at prior art conditions pH=11.7
Activated Activator SC Conversion (%) side chain (equiv.) SM LysB29 Di-acylation at (Example) LysB29 and either
GlyA1 or PheB1
Chem. 10 1.5 16 67 17
(Ex. Cl)
Figure imgf000046_0001
(prior art activator)
Chem. 11 1.2 0 83
Figure imgf000046_0002
(Ex. C2)
Figure imgf000046_0003
3,5-DC-HBSA
(prior art activator)
Chem. 13 FgC Br 1.2 52 40 8
(Ex. DI) Y J
HO^y
O=S=O
NMe2
Chem. 2a
Chem. 14 1.2 45 49 6
(Ex. D2)
Figure imgf000046_0004
Chem. 2b SC: side chain; SM: unreacted starting material When using the activators Chem. 2a and Chem. 2b of the present invention under alkaline conditions, the predominant site of acylation is also the lysine in position B29 of human insulin with a minor fraction of diacylated products; however, the reaction runs slowly and the conversion is thus low with 52% and 45% unreacted starting material left, respectively.
In conclusion, for both the prior art activators and the activators of the present invention, the acylation at alkaline pH predominantly takes place at the LysB29 position of the human insulin or human insulin analogue.
F. Acylation reactions at pH=7.0-7.5 using different activators and same side chain
The purpose of the below example Fl is to study the use of the activators of the invention in an acylation reaction for producing a Bl-acylated insulin analogue. The activated side chains of the invention used are those of Examples DI and D2, and prior art activated side chains of Examples C1-C3 are included for comparison.
In this example, the acylation reacion is performed at a neutral pH of 7.0-7.5.
The insulin analogue being acylated in this example is desB30 human insulin (A- chain of SEQ ID NO: 1 and B-chain of SEQ ID NO:3), which may be prepared, e.g., as described in W02001049742.
The side chain which is to be attached to the lysine at the N-terminal of the B-chain of insulin consists are identical for all acylation reactions in table 2, and the activated side chain are for all examples thus of Chem. 20:
Chem. 20:
Figure imgf000047_0001
Acylation procedure
Solid desB30 human insulin was weighed out (28.5 mg, 5 jimol) and transferred to a 4 mL glass veil equipped with a magnetic stir bar. HEPES buffer (0.95 mL, 0.2M, pH 7.4) was added to the glass veil, giving a concentration of insulin of 30 mg/mL. The activated side chain (1.25 equiv. was weighed out in another glass veil and dissolved in 50 |j.L DMF. The side chain solution was then added to the insulin solution. The pH was adjusted to 7.4 using NaOH (0.5M) and the reaction mixture was then stirred at rt. for 24 hours. After 24 hours a small aliquot of the reaction mixture was taken out, diluted with a mixture of AcOH/H2O/MeCN (2: 1 : 1) and analysed by UPLC analysis.
Example Fl :
Figure imgf000048_0001
of desB30 insulin analogue of Chem. 21
Chem. 21:
Figure imgf000048_0002
UPLC method 3: Rt 12.76
LC-MS method 2: Rt = 3.73 min m/z = 6419 = [M+ 1]+
The acylation reaction was performed using the activated side chains of the invention of Examples DI and D2, respectively. For comparison reasons, the acylation reaction was also performed using the reference activated side chains of Examples Cl, C2 and C3; respectively. The results can be seen in table 2.
Results
In contrast to the prior art conditions of Example E where the reaction was carried at alkaline pH (pH=11.7), the acylation reactions of the present example were carried out at neutral pH (pH of 7.0-7.5). Table 2: Acylation reactions at pH=7.0-7.5 using different activators and same water soluble side chain
Activated Activator Conversion (%) side chain SM Acylation PheB1 Di-acylation at
(Example) at GlyA1 PheB1 and or LysB29 either GlyA1 or
LysB29
Chem. 13 8 0 84 8
(Ex. DI)
Figure imgf000049_0001
Chem. 2a
Chem. 14 34 6 54 6
(Ex. D2)
Figure imgf000049_0002
Chem. 2b
Chem. 10 O i H 18 8 51 23
(Ex. Cl) O^Ny^O
(prior art activator)
Chem. 11 11 4 60 25
(Ex. C2)
Figure imgf000049_0003
3,5-DC-HBSA (prior art activator)
Chem. 12 87 0 13 0
(Ex. C3)
Figure imgf000049_0004
PNP
(prior art activator)
SM: starting material The side chain is identical for all the examples (see above Chem. 20). To activate the side chain, different activators are used. In the activated side chains of Chem. 13 (Example DI) and Chem. 14 (Example D2), the activators of Chem. 2a and Chem. 2b, respectively, of the present invention is used. In the activated side chain of Chem. 10 (Example Cl), the prior art NHS activator is used.
In the activated side chain of Chem. 11 (Example C2), the prior art activator 3,5-DC- HBSA from WO2018/083335 is used. In the activated side chain of Chem. 12 (Example C3), the prior art activator PNP is used.
As can be seen in table 2, acylation at neutral pH using the activator of Chem. 2a of the present invention results in a high selectivity for acylation at the Bl position of desB30 human insulin insulin. 84% acylation at Bl is obtained, with only 8% di-acylation products and 0% of mono-acylated biproduct.
Acylation at neutral pH using the activator of Chem. 2b of the present invention also results in a high selectivity for acylation at the Bl position of desB30 human insulin insulin, but the reaction results in a lower conversion to the PheB1 acylated product, and a higher amount of unreacted starting material is seen.
Acylation using the prior art activators NHS and 3,5-DC-HBSA also leads predominantly to acylation at the Bl position of insulin. However, the acylation is less selective for Bl and also results in 23% and 25%, respectively, of diacylated products, and 8% and 4%, respecitvely, of acylation at either GlyA1 or LysK29 with an overall lower conversion as compared to the activator of Chem. 2a.
In conclusion, the change to neutral pH results in higly selective Bl acylation when using the activator of Chem. 2a of the present invention.
G. Acylation reactions using different solvents
To test the solvent effect on the selectivity of the acylation reaction, the acylation reaction was carried out in different solvents. The insulin analogue being acylated in this example is desB30 human insulin (A-chain of SEQ ID NO: 1 and B-chain of SEQ ID NO:3), which may be prepared, e.g., as described in W02001049742. The acylation reaction was performed using the activated side chain of the invention of Example DI. Example Gl: Preparation of desB30 insulin analogue of Chem. 21
Chem. 21:
Figure imgf000051_0001
UPLC method 3: Rt 12.76
LC-MS method 2: Rt = 3.73 min m/z = 6419 = [M+ 1]+
Acylation procedure
Solid desB30 human insulin was weighed out (28.5 mg, 5 jimol) and transferred to a 4 mL glass veil equipped with a magnetic stir bar. The solvent (0.95 mL, pH 7.4) was added to the glass veil, giving a concentration of insulin of 30 mg/mL. The activated side chain (1.25 equiv. was weighed out in another glass veil and dissolved in 50 .L DMF. The side chain solution was then added to the insulin solution. The pH was adjusted to 7.4 using NaOH (0.5M) and the reaction mixture was then stirred at rt. for 24 hours. After 24 hours a small aliquot of the reaction mixture was taken out, diluted with a mixture of AcOH/HzO/MeCN (2: 1: 1) and analysed by UPLC analysis.
Results
Acylation of desB30 insulin at pH 7.4 using the activated sidechain of Chem. 13 (Example DI) in pure HEPES buffer resulted in 84% PheB1 acylation, along with 8% diacylated product being formed. When the solvent mixture were changed to HEPES/DMF (1: 1) only 32% PheB1 acylation was seen, along with 18% GlyA1 or LysB29 acylation and 11% diacylation. Using HEPES/EtOH (1: 1) as solvent led to 32% PheB1 acylation, 7% GlyAl or LysB29 acylation and 4% diacylation. In HEPES/MeCN (1: 1) and HEPES/THF (1: 1) almost no conversion was observed, and only 15% and 12% conversion to the desired PheB1 acylated product was observed in these two reactions whereas 81% and 86% starting material was left after 24 hours.
In conclusion, the solvent has a major influence on the efficiency of the acylation reaction. The reaction performs well in water (HEPES buffer), but once organic solvents are added the reaction becomes slower and the selectivity for PheB1 acylation decreases.
Table 3. Acylation reactions at oH=7.0-7.5 using different solvents
Activated Solvent Conversion (%) side chain SM Acylation Acylation Di-acylation of at GlyA1 at PheB1 at
Example or LysB29 PheB1 and either GlyA1 or LysB29
DI HEPES 8* 0* 84* 8*
DI HEPES/DMF (1: 1) 39 18 32 11
DI HEPES/EtOH (1 : 1) 63 7 27 4
DI HEPES/MeCN (1 : 1) 81 2 15 1
DI HEPES/THF (1 : 1) 86 2 12 0
SM: starting material; *data also included in table 2
H. Acylation with side chains of poor water solubility
Example Hl:
Figure imgf000053_0001
of desB30 insulin analogue Chem. 25
Chem. 25:
Figure imgf000053_0002
The compound was prepared from desB30 human insulin and the activated side chain of either Chem. 17 (Example D5) using general acylation procedure method 1 or Chem. 32 (Example D9) using general acylation procedure method 2.
UPLC method 3: Rt 11.08 min
LC-MS method 2: Rt = 3.11 min m/z = 5893 = [M+ 1]+
Results
To compare with an activator from the prior art, the acylation reaction was also carried out using the reference activated side chain of Chem. 31 (Example C4).
For activated side chains which are soluble in HEPES buffer (pH 7.4), the acylation reaction runs better using Chem. 2a than Chem. 2b. This can be seen from table 2, where desB30 insulin is acylated with the same side chain using activator Chem. 2a (activated side chain of Example DI) and Chem. 2b (activated side chain of Example D2), respectively.
However, for more lipophilic side chains, where the side chain activated with activator Chem. 2a is only poorly soluble in water (and HEPES buffer), the acylation reaction runs with a low conversion to the Bl acylated product. The sulfonic acid group of Chem. 2b enhances the solubility in water of the activated side chain and improves the conversion in the acylation reaction. For side chains where it is not possible to use Chem. 2a as an activator due to low solubility of the activated side chain in water, the side chains can be activated using activator Chem. 2b, resulting in the activated side chain being soluble in water. Such an example can be seen in table 4, where only 34% PheB1 acylation is observed, with 66% unreacted starting material after 24 hours. In comparison, when the activator of Chem. 2b is used, 69% acylation at PheB1 is observed, with 29% unreacted starting material after 24 hours.
Table 4: Acylation reactions at pH=7.0-7.5 for side chains with low water solubility
Activated side Activator Conversion (%) chain SM Acylation PheB1 Di-acylation
(Example) at GlyA1 at or LysB29 PheB1 and either GlyA1 or LysB29
Chem. 17 66 0 34 0
(Ex. D5)
Figure imgf000054_0001
Chem. 2a
Chem. 32 F3C^^^Br 29 0 69 2
(Ex. D9) Il
Ho y o=s=o
OH
Chem. 2b
Figure imgf000054_0003
Chem. 31 ® 56 10
(Ex. C4)
Figure imgf000054_0002
3,5-DC-HBSA
(prior art activator)
SM: starting material Although a high selectivity for acylation at PheB1 is obtained for both the activator of Chem. 2a and Chem. 2b, a higher conversion is seen with activator Chem. 2b. This is due to solubility issues in water using Chem. 2a.
For comparison, the acylation using the prior art activator 3,5-DC-HBSA is also included in table 4. As can be seen from the results, the prior art activator 3,5-DC-HBSA is less Bl-selective than the activators of the present invention with the formation of both diacylated products (10%), products with acylation at either GlyA1 or LysK29 (6%).
In conclusion, acylation using lipophilic side chains using the activator of Chem. 2a results in poor conversion due to low water solubility of the activated side chains. Instead, the activator of Chem. 2b can be used to increase the water solubility of the activated side chains and thus increase conversion leading to higher yields of the Bl acylated insulin product.
I. Acylation reactions at pH=7.0-7.5 using an activator of the invention and various side chains
The below examples illustrate the use of the activators of the invention in acylation reactions of insulin with high selectivity for the Bl position.
The insulin analogue being acylated in all of these examples is desB30 human insulin, which may be prepared, e.g., as described in W02001049742.
Example II: Preparation of desB30 insulin analogue of Chem. 23
Chem. 23:
Figure imgf000055_0001
The compound was prepared from desB30 human insulin and activated side chain Chem.
15 (Example D3) using general acylation procedure method 1.
UPLC method 3: Rt 10.52 min
LC-MS method 2: Rt = 2.92 min m/z = 5932 = [M+ 1]+ Example 12: Preparation of desB30 insulin analogue of Chem. 24
Chem. 24:
Figure imgf000056_0001
The compound was prepared from desB30 human insulin and the activated side chain of
Chem. 16 (Example D4) using general acylation procedure method 2.
UPLC method 3: Rt 11.26 min
LC-MS method 2: Rt = 3.14 min m/z = 5867= [M+ 1]+
Example 13: Preparation of desB30 insulin analogue of Chem. 26
Chem. 26:
Figure imgf000056_0002
The compound was prepared from desB30 human insulin and the activated side chain of
Chem. 18 (Example D6) using general acylation procedure method 2.
UPLC method 3: Rt 10.68 min
LC-MS method 2: Rt = 2.94 min m/z = 5786 = [M + 1]+ Example 14: Preparation of desB30 insulin analogue of Chem. l
Chem. 27:
Figure imgf000057_0001
The compound was prepared from desB30 human insulin and the activated side chain of
Chem. 19 (Example D7) using general acylation procedure method 2.
UPLC method 3: Rt 10.95 min
LC-MS method 2: Rt = 3.07 min m/z = 5975 = [M + 1]+
Example 15: Preparation of desB30 insulin analogue of Chem. 28
Chem. 28:
Figure imgf000057_0002
The compound was prepared from desB30 human insulin and the activated side chain of
Chem. 40 (Example D8) using general acylation procedure method 2.
UPLC method 3: Rt 11.11 min
LC-MS method 2: Rt = 2.92 min m/z = 5779.75 = [M+ 1] Example 16: Preparation of desB30 insulin analogue of Chem. 35
Chem. 35:
Figure imgf000058_0001
The compound was prepared from desB30 human insulin and the activated side chain of Chem. 33 (Example D12) using general acylation procedure method 2.
UPLC method 3: Rt 11.00 min
LC-MS method 2: Rt = 3.0 min m/z = 5928.6 = [M+ 1]+
Results The above acylation reactions are included in table 5. For completeness, table 5 also include the acylation reaction of Examples Fl and Hl, which are also included in table 2 and 4, respectively.
As can be seen from table 5, the activator of the invention can be used to introduce a number of different side chains, under aqueous conditions, on the Bl position of desB30 human insulin in highly selective manner. Thus, only minor amounts of of GlyA1 or LysB29 acylated product as well as diacylated product are observed in these reactions. This is in stark contrast to standard methods for acylation of insulin using for instance NHS as the activator.
Table 5: Acylation reactions at pH=7.0-7.5 using the activator of the invention and various side chains
Acylated Activated Activator Conversion (%) insulin side chain SM Acylation Acylation Di-
(Example) (Example) at GlyA1 or at PheB1 acylation
LysK29 at
PheB1 and either GlyA1 or LysK29
Chem. 21 Chem. 13 Chem. 2a 8* 0* 84* 8*
(Ex. Fl) (Ex. DI)
Chem. 23 Chem. 15 Chem. 2a 9 0 79 12
(Ex. Il) (Ex. D3)
Chem. 24 Chem. 16 Chem. 2b 44 2 52 2
(Ex. 12) (Ex. D4)
Chem. 25 Chem. 32 Chem. 2b 29* 0* 69* 2*
(Ex. Hl) (Ex. D9)
Chem. 26 Chem. 18 Chem. 2b 42 6 47 5
(Ex. 13) (Ex. D6)
Chem. 27 Chem. 19 Chem. 2b 22 3 68 6
(Ex. 14) (Ex. D7)
Chem. 28 Chem. 40 Chem. 2b 20 4 58 18
(Ex. 15) (Ex. D8)
Chem. 35 Chem. 33 Chem. 2b 43 9 40 8
(Ex. 16) (Ex. D12)
SM: starting material; *data also presented in table 2; #data also presented in table 4. J. Acylation of Insulin analogues with different amino acids in Bl position
To investigate whether the Bl selective acylation using the activators of the invention is specific to insulin analogues having a phenylalanine (Phe) in position Bl, the acylation reaction was carried out using an insulin analogue with serine (Ser) and glutamic acid (Glu) in the Bl position. When changing the N-terminal amino acid, the pKa of the N-terminal amine changes. For phenylalanine the pKa of the N-terminal amine is ~ 9.13, for Serine it is ~ 9.15 and for Glutamic acid it is 9.67. Based on these values it is expected that the serine analogue will react similar to the phenylalanine analogue. For the Glutamic acid insulin analogue, a higher amount of the N-terminal amines will be protonated at neutral pH (due to the higher pKa value of the N-terminal amine) and therefore be a little less reactive than the two others.
Example JI : Acylation of SerB1 desB30 human insulin
Chem. 36:
Figure imgf000060_0001
SerB1 desB30 insulin (85.0 mg, 15 pmol, 1.0 equiv.) was dissolved in 0.2M HEPES buffer (2.8 mL, pH 7.4). (S)-l-(4-bromo-2-(/V,/V-dimethylsulfamoyl)-6-(trifluoromethyl)- phenoxy)-22-ca rboxy-l,19,24-trioxo-3,6,12,15-tetraoxa-9,18,23-triazahentetracontan- 41-oic acid (the activated side chain of Chem. 13, Example DI) (24.0 mg, 22.5 pmol, 1.5 equiv.) was dissolved in DMF (50 pL) and added to the solution. The resulting mixture was stirred at rt. for 24 hours. The mixture was then acidified with AcOH to pH 3 and diluted with water to a total volume of 20 mL. The product was purified by RP-HPLC eluting with a gradient from 60:40 to 45:55 HzO/MeCN + 0.1% trifluoroacetic acid over 40 minutes. Pure fractions were collected and lyophilized to afford the title compound as a white solid. (49.0 mg, 7.7 pmol, 51%).
UPLC method 4: Rt 6.94 min
LC-MS method 2: Rt = 3.63 min
Calc. : 6356.97 m/z; found: m/3: 2120.26; m/4: 1590.46; m/5: 1272.58; m/6: 1060.65
Table 6. Acylation of SerB1 desB30 human insulin.
Conversion (%)
SM Acylation at GlyA1 or Acylation at SerB1 Di-acylation at
LysK29 SerB1 and either GlyA1 or LysK29
18 1 78 2
SM: starting material As can be seen from table 6, the acylation of SerB1 desB30 human insulin results in a highly selective acylation of the SerB1 amine at position Bl.
Example J2: Acylation of GluB1 desB30 human insulin:
Chem. 37:
Figure imgf000061_0001
GluB1 desB30 human insulin (86.8 mg, 15 pmol, 1.0 equiv.) was dissolved in 0.2M HEPES buffer (2.9 mL, pH 7.4). (S)-l-(4-bromo-2-(/V,/V-dimethylsulfamoyl)-6- (trifluoromethyl)phenoxy)-22-carboxy- 1,19, 24-trioxo-3, 6, 12,15-tetraoxa-9, 18,23- triazahentetracontan-41-oic acid (the activated side chain of Chem. 13, Example DI) (24.0 mg, 22.5 pmol, 1.5 equiv.) was dissolved in DMF (50 pL) and added to the solution. The resulting mixture was stirred at rt. for 24 hours.
The mixture was then acidified with AcOH to pH 3 and diluted with water to a total volume of 20 mL. The product was purified by RP-HPLC eluting with a gradient from 60:40 to 45:55 HzO/MeCN + 0.1% trifluoroacetic acid over 40 minutes. Pure fractions were collected and lyophilized to afford the title compound as a white solid. (43.0 mg, 6.6 pmol, 44%)
UPLC method 4: Rt 6.89 min
LC-MS method 2: Rt = 3.63 min
Calc. : 6529.03 m/z; found: m/3: 2177.35; m/4: 1633.26; m/5: 1306.81; m/6: 1089.17
Table 7. Acylation of GluB1 desB30 human insulin.
Conversion (%)
SM Acylation at GlyA1 or Acylation at GluB1 Di-acylation at
LysK29 GluB1 and either GlyA1 or LysK29
34 1 63 2
SM: starting material As can be seen from table 7, the acylation of GluB1 desB30 human insulin results in a highly selective acylation of the GluB1 amine at position Bl.
From examples JI and J2 it can thus be seen that the Bl selective acylation using the activators of the present invention is not limited to insulin analogues having a Phe in the Bl position, but also insulin analogues having another amino acid in the Bl position.
K. Preparation of a di-modifed insulin analogue
The acylation method of the present invention also enables the selective acylation of position Bl of human insulin or human insulin analogues with one side chain at low pH, followed by acylation of another position such as the B29 position with a different side chain at high pH. In two subsequent steps two different side chains can be attached to an insuline analogue without the need for extra protection/deprotection steps.
In the below example, desB30 human insulin is first acylated at position Bl at low pH, followed by acylation at position B29 at high pH.
Example KI: Preparation of desB30 insulin analogue of Chem. 38
Chem. 38:
Figure imgf000062_0001
Step 1: Preparation of 16-[[l-carboxy-4-(2,4-dichloro-6-sulfo-phenoxy)-4-oxo- butyl ]amino ]-16-oxo-hexadecanoic acid
Chem. 29:
Figure imgf000063_0001
t-Bu protected C16-diacid-yGlu-OH (synthesized according to procedures found in WO09115469) (1 mmol, 0.528 g) and TEA (3 mmol, 0.42 mL, 3 eq.) was dissolved in MeTHF (10 mL). 3,5-dichloro-2-hydroxybenzenesulfonyl chloride (1.2 mmol, 0.314 g, 1.2 eq.) was dissolved in MeTHF (5 mL) and slowly added to the mixture dropwise. The reaction mixture was then stirred until full conversion was observed (UPLC analysis). Next the mixture was washed with 5% KHSC (3 x 5 mL). The organic phase was collected, dried over MgSC , filtered and concentrated under reduced pressure. The crude product was purified by using silicagel column chromatography with a gradient eluent from DCM to 5% MeOH in DCM. After evaporation the product was obtained as a colorless oil. Method Mod U l was used to cleave the tBu-esters for 1.5 hours. The cleavage mixture was evaporated under reduced pressure. The product was obtained as a brown oil. (0.557 g, 0.870 mmol, 87%). Active content of material from 1H qNMR is 73% w/w
The compound was characterised by LC-MS and XH NMR.
XH NMR (400 MHz, DMSO-c/6) 8 ppm 1.23 (s, 21 H), 1.44 - 1.55 (m, 4 H), 1.90 - 2.05 (m, 1 H), 2.06 -2.16 (t, 3 H), 2.16 - 2.22 (t, 2 H), 2.56 - 267 (m, 2 H), 4.27 (bs, 1 H), 7.65 (s, 1 H), 7.74 (s, 1 H), 8.15 (d, 1 H).
LC-MS method 1: calculated Mass for [M-H]' 638.1599 Mass Found [M-H]’ 638.1649
Step 2, Acylation procedure:
Solid desB30 human insulin was weighed out (228.26 mg, 0.06 jimol) and transferred to a 20 mL glass veil equipped with a magnetic stir bar. HEPES buffer (7.6 mL, 0.2M, pH 7.4) was added to the glass veil, giving a concentration of insulin of 30 mg/mL. l-(4- bromo-2-(/V,/V-dimethylsulfamoyl)-6-(trifluoromethyl)phenoxy)-22-carboxy- 1,10, 19,24- tetraoxo-3,6,12,15-tetraoxa-9,18,23-triazahentetracontan-41-oic acid (activated sidechain of Chem. 13, Example DI) (1.5 equiv.) was weighed out in another glass veil and dissolved in 100 pL DMF. The side chain solution was then added to the insulin solution. The pH was adjusted to 7.4 using NaOH (0.5M) and the reaction mixture was then stirred at rt. for 24 hours resulting in acylation of PheB1. Next the reaction mixture was transferred to an Amicon® Ultra - 15 centrifugal filter (3K) and the mixture was filtered on a SIGMA 4-16K refrigerated centrifuge (speed: 4000 x g) for 25 minutes. Then water was added, and the mixture filtered again. This was repeated 3 times in total. The mixture was then transferred to 20 mL glass veil and diluted with a NazCCh buffer (0.2M, pH 10.8) to a give a concentration of the peptide of 60 mg/mL. To this solution was then added 16-[[l-carboxy-4-(2,4-dichloro-6-sulfo-phenoxy)-4-oxo-butyl]amino]-16-oxo- hexadecanoic acid (Chem. 29, prepared in step 1) (1.6 equiv.) in DMF (200 pL) and the resulting mixture was stirred at rt. for 3 hours resulting in acylation of LysB29. Next the mixture was diluted with water and the pH was decreased to 3 using AcOH. Purification of the product was performed on a GILSON HPLC system using a Gemini C18 30 x 250 mm column (45-65% MeCN in MilliQ (0.1 vol.% TFA) over 40 min, 20 mL/min. (152.8 mg, 0.022 mmol, 56%).
UPLC method 3: Rt 9.40 min
LC-MS method 2: Rt = 4.52 min
Calc. : 6813.25 m/z; found: m/3: 2272.71; m/4: 1704.31; m/5: 1364.04
L. Ligation of insulin with a tetrapeptide
In this example, desB30 human insulin is acylated at position Bl with a tetramer. The examples shows an example of a ligation where a small peptide is selectively acylated onto the PheB1 amine of desB30 human insulin. This shows that the Bl selective acylation is not limited to acylation only with small molecules.
Example LI: Preparation of desB30 insulin analogue of Chem. 39 Chem. 39:
Figure imgf000064_0001
DesB30 insulin (285.0 mg, 0.05 mmol, 1.0 equiv.) was dissolved in 0.2M HEPES buffer (9.5 mL, pH 7.4). (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((3-(((S)-l-((2- (4-bromo-2-sulfo-6-(trifluoromethyl)phenoxy)-2-oxoethyl)amino)-l-oxopropan-2- yl)amino)-3-oxopropyl)amino)-5-oxopentanoic acid (65.4 mg, 0.075 mmol, 1.5 equiv.) (activated side chain of Chem. 34, Example D14) was dissolved in DMF (200 pL) and added to the insulin solution. The resulting mixture was then stirred at rt for 24 hours. The mixture was then diluted with Water/ AcOH (50 mL, 1: 1) and the peptide was purified by RP-HPLC eluting with a gradient from 70:30 to 45:55 HzO/MeCN + 0.1% trifluoroacetic acid over 45 minutes. Pure fractions were collected and lyophilized to afford the title compound as a white solid. (81.3 mg, 0.013 mmol, 26%). UPLC method 4: Rt 6.51 min LC-MS method 2: Rt = 3.32 min.
Calc. : 6251.79 m/z; found: m/3: 2085.27; m/4: 1564.20; m/5: 1251.56; m/6: 1043.14
While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

65 CLAIMS
1. A compound of Chem. 1:
Chem. 1 :
Figure imgf000066_0001
wherein Y is OR, SR, NMeC(=O)R, NHC(=O)R, halogen or Ns; wherein R is an organic subsitutent; and wherein X is OH or N(CHs)2; or a salt thereof.
2. The compound according to claim 1, wherein X is N(CHs)2.
3. The compound according to claim 1, wherein X is OH.
4. The compound according to any one of claims 1 to 3, wherein the organic substituent is alkyl, heteroalkyl, alkenylalkyl, alkynylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, or peptidyl, each being optionally substituted.
5. The compound according to any one of claims 1 to 4, wherein Y is OR.
6. The compound according to any one of claims 1 to 5, wherein the compound is of Chem. 7:
Figure imgf000066_0002
wherein r is an integer in the range of 8-20; wherein q is an integer in the range of 0-3; wherein p is an integer in the range of 0-3; wherein X is OH or N(CHs)2; and 66
L is an optional linker of Chem. 8:
Figure imgf000067_0001
wherein each of k, I, and m independently represents an integer in the range of 0-4; or a salt, amide, or ester thereof.
7. The compound according to claim 6, wherein r is 16; wherein q is 1; wherein p is
1; wherein k is 0, I is 0, and m is 2.
8. The compound according to any one of claims 1 to 5, wherein the compound is of Chem. 9:
Figure imgf000067_0002
wherein X is OH or N(CHs)2.
9. The compound according to any one of claims 1 to 5, wherein the compound is of Chem. 9a:
Figure imgf000067_0003
wherein W is halogen, and wherein X is OH or N(CHs)2.
10. A compound according to claim 1 selected from the following:
Chem. 13:
Figure imgf000067_0004
67
Chem. 14:
Figure imgf000068_0001
68
Chem. 19:
Figure imgf000069_0001
69 or a pharmaceutically acceptable salt, amide, or ester thereof.
11. A method for acylating an N-terminal amino acid in a peptide or a protein, the method comprising a first step of preparing an acylating reagent using an activator of Chem. 2:
Figure imgf000070_0001
wherein X is OH or N(CHs)2; and a second step of reacting the said acylating reagent with the peptide or protein.
12. The method of claim 11, wherein the pH is 6-8 and the solvent is an aqueous solvent.
13. The method of claim 11, wherein the peptide or protein is human insulin or a human insulin analogue.
14. The method of claim 11, which comprises a further step of purifying the desired product of the acylation reaction.
15. The method of any of claims 11-12, wherein the product of the acylation reaction is selected from the following compounds:
Chem. 21; Chem. 23, Chem. 24; Chem. 25; Chem. 26; Chem. 27; Chem. 28; Chem. 35; Chem. 36; Chem. 37; Chem. 38; and Chem. 39; or a pharmaceutically acceptable salt, amide, or ester thereof.
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