EP2283037A1 - Process - Google Patents

Process

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Publication number
EP2283037A1
EP2283037A1 EP09749882A EP09749882A EP2283037A1 EP 2283037 A1 EP2283037 A1 EP 2283037A1 EP 09749882 A EP09749882 A EP 09749882A EP 09749882 A EP09749882 A EP 09749882A EP 2283037 A1 EP2283037 A1 EP 2283037A1
Authority
EP
European Patent Office
Prior art keywords
peptide
protected
cyclic peptide
protecting group
connectable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09749882A
Other languages
German (de)
French (fr)
Inventor
Rune Severinsen
Claus Christophersen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Priority to EP09749882A priority Critical patent/EP2283037A1/en
Publication of EP2283037A1 publication Critical patent/EP2283037A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/68—Melanocyte-stimulating hormone [MSH]
    • C07K14/685—Alpha-melanotropin
    • 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
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C07K1/061—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents using protecting groups
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a process for performing a ring closure on a fully protected peptide in order to produce a protected cyclic peptide, products derived from said process and the use of said products.
  • Solid phase synthesis is a well known technique for the production of peptides.
  • SPPS solid phase peptide synthesis
  • an amino acid or peptide group is bound to a solid support resin.
  • successive amino acids or peptide groups are attached to the support- bound peptide until the peptide material of interest is formed.
  • the support-bound peptide is then typically cleaved from the support and subject to further processing and/or purification.
  • solid phase synthesis yields a mature peptide product; in other cases the peptide cleaved from the support is used in the preparation of a larger, mature peptide product.
  • Peptide modifications may be desired for a number of reasons. In particular, modifications may be made to fix or reduce the number of conformational forms the peptide may take. In other situations, peptide side chains may be modified in an attempt to increase the binding of the peptide to a specific target or, in other cases, to protect the peptide from degradation by enzymes and the like.
  • peptides comprising cyclic groups have been formed whereby bonds are created between the side chains of amino acid residues.
  • the terminals of a peptide have been linked end-on-end such that the peptide has a ring or cyclic type structure.
  • these cyclic peptides are prepared in such a way that the ring is formed by oxidation of the naturally occurring cysteine residues present in the peptide chain, thus yielding a disulfide bridged structure. This technique mimics the most common form of cyclisation found among naturally occurring peptides and proteins, but does not provide a convenient means of preparing other types of cyclic structures.
  • GB 1527252 gives an example of a process for the cyclisation of peptides containing cysteine groups.
  • a peptide containing at least two cysteine moieties is held in a solution which is substantially free of oxygen until cyclisation between the two cysteine moieties has occurred to yield a cyclic disulfide bond.
  • Further examples of processes in which cyclic peptides with disulfide bonds are formed, are given in EP 1921087.
  • end-on-end cyclic peptides In order to prepare end-on-end cyclic peptides, one technique is to employ amino acids with orthogonally protected functional groups such that some are removable selectively in the presence of others. Those skilled in the art can use these techniques to prepare peptides in solution in which the amino terminus is cyclised to the carboxyl terminus to form a ring. A naturally occurring example is the antibiotic gramicidin.
  • pairs of cysteine residues are oxidized to disulfide bonds to form one or more rings;
  • the familiar naturally-occurring cyclic peptide hormone oxytocin is an example of such a structure, such as has been prepared by O'Neil et al., Protein, 14, 509-515 (1992)); however, this example is limited to cases of disulfide forming cyclic hexapeptides.
  • US 6,008,058 discloses a method of producing terminally linked (head to tail) cyclic peptides using solid phase synthesis. The cyclic peptides of US 6,008,058 are formed whilst the peptide chain is still attached to the resin support.
  • WO 01/16162 describes a method for the preparation of a cyclic peptide wherein an aromatic group is attached to a terminal end of the peptide, said aromatic forming a link between an oxygen, thio or amine group on the peptide.
  • cyclic peptides may be prepared by forming C-backbone linkages. In this method, the linkages are formed between the carbon atoms on the peptide backbone, therefore leaving the functional groups on the amino acid side chains free for binding with a target site.
  • US 2005/0267017 discloses cyclic peptides and processes for their production wherein the cyclic linkages are formed between the carbon atoms present on the peptide backbone.
  • US 5,723,575 also discloses a process for the preparation of backbone cyclised peptides.
  • cysteine cyclisation is only appropriate where the peptide has the necessary free cysteine residues; backbone cyclisation is only appropriate where there are suitable linking sites available on the peptide.
  • ⁇ -MSH ⁇ -melanocyte-stimulating hormone
  • MC4 melanocortin 4 receptor
  • US 5,683,981 discloses a method for performing a ring closure on an ⁇ -melanocyte- stimulating hormone analogue.
  • US 5,683,981 discloses a method wherein a Lys residue and a GIu residue are condensed to form a ring structure.
  • the process for performing the ring closure comprises forming a peptide from a "key intermediate A" which comprises a number of protected amino acid residues, in particular, a protected Lys residue. This key intermediate is then coupled to a GIu residue.
  • a process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide.
  • a pharmaceutical composition comprising the cyclic peptide produced by the process of the present invention and optionally one or more pharmaceutically acceptable excipients, adjuvants, diluents or carriers.
  • a method of treating diabetes in a subject in need thereof comprising administering to the subject an effective amount of the cyclic peptide produced by the process of the present invention or a composition comprising the said cyclic peptide.
  • the process of the present invention provides an economically favourable method for producing protected cyclic peptides.
  • the process of the present invention is capable of producing cyclic peptides in greater yields than the known processes of the prior art.
  • the process of the present invention is capable of producing a cyclic peptide of a greater purity than the prior art due to a more controlled ring closure. This results in a reduction in impurities which are very similar to the desired end product and which are therefore difficult to detect by analytical methods. As a result, the process of the present invention provides a cyclic peptide with increased safety for pharmaceutical applications.
  • the process of the present invention provides a method of performing a ring closure which does not require to be attached to the support resin during cyclisation.
  • the process of the present invention does not have the disadvantages associated with performing a ring closure on a support resin.
  • a process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide.
  • a cyclic peptide may be considered to be a peptide which comprises a cyclic or ring structure which results from the linkage of two portions of a linear peptide.
  • a cyclic peptide as used herein is not considered to be a linear peptide comprising amino acids having cyclic side chains, e.g.
  • a cyclic peptide as defined herein is considered to not comprise any externally added protecting groups.
  • externally added protecting groups it is meant a protecting group which has been added during synthesis to protect a functional group on an amino acid side chain.
  • a fully protected peptide may be considered to be a peptide in which all of the amino acid side chain functional groups are protected by a suitable protecting group. It may also include a peptide which is connected to a solid phase support/ solid phase linker as this can be considered to protect the terminal amino acid to which it is attached.
  • a fully protected peptide as defined herein may be produced by a number of methods known to the person skilled in the art.
  • the fully protected peptide is produced using solid phase peptide synthesis.
  • a Sieber amide resin is used as the solid phase support resin.
  • the fully protected peptide is obtained or obtainable from the dipeptide, Fmoc-Nle-Glu-OH. In one embodiment, where the fully protected peptide is synthesised using solid phase peptide synthesis, the solid phase support or solid phase linker is acid labile.
  • the fully protected peptide is synthesised using solid phase peptide synthesis
  • the fully protected peptide is cleaved from the solid phase support or solid phase linker using a composition comprising TFA and dichloromethane.
  • the composition comprises TFA in an amount of less than 2% based on the total volume of the composition.
  • the concentration of the TFA used to cleave the fully protected peptide from the solid phase support or solid phase linker is not sufficient to remove any of the protecting groups on the fully protected peptide.
  • a protected cyclic peptide may be considered to be a peptide comprising a cyclic or ring structure which results from the linkage of two portions of a linear peptide group, wherein at least one of the amino acid side chain functional groups is protected by a suitable protecting group.
  • the definition of a protected cyclic peptide also includes those peptides comprising a cyclic group as defined above wherein substantially all, but not all, of the amino acid side chain functional groups are protected.
  • the connectable portions of the ring to be closed comprise first and second connectable portions.
  • the connectable portions of the ring to be closed comprise side chains of amino acids. In one embodiment, the connectable portions of the ring to be closed are not at the terminal ends of a peptide.
  • the amino acid side chains are those comprising at least an amine group and at least a carboxylic acid group. In one embodiment according to the process of the present invention, the amino acid side chains are side chains corresponding to Lys and GIu.
  • the protecting groups on the fully protected peptide are selected from protecting groups suitable for the protection of amino acid side chains. It is well known in the art which protecting groups may be used for the protection of amino acids. Many protecting groups have been developed for amino group protection, and fall into a number of broad classes.
  • the suitable protecting groups are selected from the group consisting of tert- butyloxycarbonyl, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, tert-butyl, trityl, benzyloxy, benzyloxycarbonyl and butyl.
  • the protecting groups suitable for removal are selected from the group consisting of but not limited to acid labile groups, Pd-labile groups and hydrazine labile groups.
  • the acid labile groups are selected from the group consisting of Mtt and OPip groups.
  • the Pd-labile groups are selected from the group consisting of Alloc and OAIyI.
  • the hydrazine labile groups are selected from the group consisting of ODmab and Dde.
  • the protecting groups of the connectable portions of the ring to be closed are selected from benzyloxy and benzyloxycarbonyl.
  • the protecting group of the first connectable portions of the ring to be closed is benzyloxy and the protecting group of the second connectable portions of the ring to be closed is benzyloxycarbonyl.
  • the abbreviations Bn, Bz and BzI are used interchangeably and are intended to mean the protecting group benzyloxy.
  • the connectable portions of the ring to be closed are protected with protecting groups which are suitable for removal by hydrogenation.
  • hydrogenation is performed using H 2 ; Pd/C.
  • the Pd/C is used in amount of at least 5% based on the theoretical weight of the fully protected peptide free of the solid phase support or solid phase linker.
  • the Pd/C is used in amount of 5 to 30% based on the theoretical weight of the fully protected peptide free of the solid phase support or solid phase linker to about 30.
  • the conditions for hydrogenation are H 2 ; Pd/C (10%).
  • hydrogenation is carried out in absolute ethanol.
  • the pH of the hydrogenation reaction should not be acidic.
  • the connectable portions of the of the ring to be closed are the side chains of amino acids, Lys and GIu, the functional group of the Lys side chain being protected with a benzyloxycarbonyl protecting group, and the functional group of the GIu side chain being protected with the benzyloxy protecting group.
  • the protecting groups may be removed by hydrogenation.
  • the connectable portions of the ring to be closed are selectively deprotected to form a selectively deprotected peptide. Accordingly, in one embodiment according to the process of the present invention, there is provided a process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting by hydrogenation, the connectable portions of the ring to be closed wherein the connectable portions consist of side chains of a Lys residue and a GIu residue; and ii) connecting the deprotected functional groups of said Lys and GIu residues to form the protected cyclic peptide.
  • the connectable portions of the ring to be closed are connected using one or more suitable connecting agents.
  • one or more suitable connecting agents are selected from the group consisting of N,N'-diisopropyl carbodiimide (DIC)/ 1-hydroxybenzotriazol (HOBt), 1- ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC)/ 1-hydroxybenzotriazol (HOBt), N,N'-diisopropylethylamine (DIPEA), benzotr ⁇ azoi-1-yi- oxy- tripyrroiidinophosphonium hexafiuorophosphate (PyBOB), O-(Benzotriazol-1-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TBTU), 2-(1 H-7-Azabenzotriazol-1-yl)-1 , 1
  • the suitable connecting agents are selected from N 1 N 1 - diisopropylethylamine (DIPEA), benzotriazol-1-yi- oxy-tripyrroiidinophosphonium hexafiuorophosphate (PyBOB) 1
  • the suitable connecting agent is O-(Benzotriazol-i-yl)-
  • step ii) of the process of the present invention is conducted in a solvent selected from THF, wet THF, Toluene, NMP/ THF, EtOAc, and similar solvents. In one embodiment, step ii) is carried out in THF.
  • the fully protected peptide may be synthesised using solid phase peptide synthesis. However, it is not necessary for the fully protected peptide to be connected to a solid phase support or a solid phase support linker when step i) of the process of the present invention is performed. Thus, in one embodiment, the fully protected peptide is not connected to a solid phase support or a solid phase support linker when it is selectively deprotected according to step i). In this regard, the fully protected peptide may be considered to be free of a solid phase support or a solid phase support linker.
  • the step of connecting the deprotected connectable portions of the ring to be closed is not particularly limited.
  • the selectively deprotected peptide is not connected to a solid phase support or a solid phase support linker during the step of connecting the connectable portions of the ring to be closed.
  • the fully protected peptide may be considered to be free of a solid phase support or a solid phase support linker.
  • process of the present invention is not particularly limited in terms of the peptides to which it is applicable. Thus, it is envisaged that any fully protected peptide may be used in the process of the present invention.
  • the fully protected peptide may be a fully protected analogue of a peptide selected from the group consisting of human insulin, a human insulin analogue, a human insulin derivative, GLP-1 , a GLP-1 analogue, a GLP-1 derivative, exendin, an exendin analogue, an exendin derivative, ⁇ -MSH, an ⁇ -MSH analogue, an ⁇ -MSH derivative, ⁇ -MSH, a ⁇ -MSH analogue, a ⁇ -MSH derivative, an MC4 receptor agonist, amylin, an amylin analogue, an amylin derivative, gastrin, a gastrin analogue, a gastrin derivative, human growth hormone, a human growth hormone analogue, a human growth hormone derivative, factor Vila, a factor Vila analogue and a factor Vila derivative, or any combination thereof.
  • the invention relates to compounds (more particularly compounds acting as melanocortin receptor agonists or antagonists) of formula I:
  • T represents tetrazol-5-yl
  • A represents a straight-chain, branched and/or cyclic C 6-2 oalkyl, C 6-2 oalkenyl or C 6-2 oalkynyl which may optionally be substituted with one or more substituents selected from halogen, hydroxy and aryl;
  • L is a bond or a chemical structure covalently linking A and P; and P represents a peptide structure comprising at least six ⁇ -amino acid residues and a ring closure.
  • R 1 represents tetrazol-5-yl or carboxy
  • R 2 represents a straight-chain, branched and/or cyclic C 6-2 oalkyl, C 6-2 oalkenyl or C 6-2 oalkynyl which may optionally be substituted with one or more substituents selected from halogen, hydroxy and aryl;
  • S 1 is absent or represents a 4-aminobutyric acid residue, GIy, ⁇ -Ala, or a glycolether-based structure according to one of the formulas llla-lllg;
  • Z 1 is absent or represents GIy, ⁇ -Ala, Ser, D-Ser, Thr, D-Thr, His, D-His, Asn, D-Asn, GIn, D-GIn, GIu, D-GIu, Asp, D-Asp, Ala, D-AIa, Pro, D-Pro, Hyp or D-Hyp;
  • Z 2 is absent or represents GIy, ⁇ -Ala, Ser, D-Ser, Thr, D-Thr, His, D-His, Asn, D-Asn, GIn, D-GIn, GIu, D-GIu, Asp, D-Asp, Ala, D-AIa, Pro, D-Pro, Hyp or D-Hyp;
  • Z 3 represents Ser, D-Ser, Thr, D-Thr, His, D-His, Asn, D-Asn, GIn, D-GIn, GIu, D-GIu, Asp, D-Asp, Ala, D-AIa, Pro, D-Pro, Hyp or D-Hyp;
  • Z 4 represents GIy, Ala, Pro, Hyp, Ser, homoSer, Thr, Tyr, GIn, Asn, 2-PyAIa, 3-PyAIa, 4- PyAIa, His, homoArg, Arg, Lys, Dab, Dap or Orn;
  • Z 5 represents GIy, Ala, Pro, Hyp, Ser, homoSer, Thr, GIn, Asn, 2-PyAIa, 3-PyAIa, 4-PyAIa, His, homoArg, Arg, Lys, Dab, Dap or Orn;
  • Z 6 represents Ala, D-AIa, VaI, D-VaI, Leu, D-Leu, lie, D-IIe, Met, D-Met, NIe or D-NIe;
  • X 1 represents GIu, Asp, Cys, homoCys, Lys, Orn, Dab or Dap;
  • X 2 represents His, Cit, Dab, Dap, CgI, Cha, VaI, lie, tBuGly, Leu, Tyr, GIu, Ala, NIe, Met,
  • (2-furyl)alanine, (3-furyl)alanine or Phe wherein one or more hydrogens on the phenyl moiety of the Phe in question may optionally and independently be substituted by a substituent selected among halogen, hydroxy, alkoxy, nitro, benzoyl, methyl, trifluoromethyl, amino and cyano
  • X 3 represents D-Phe, wherein one or more hydrogens on the phenyl moiety in D-Phe may optionally and independently be substituted by a substituent selected among halogen, hydroxy, alkoxy, nitro, methyl, trifluoromethyl and cyano;
  • X 4 represents Trp, 2-NaI, (3-benzo[b]thienyl)alanine or (S)-2,3,4,9-tetrahydro-1 H- ⁇ - carboline-3-carboxylic acid
  • X 5 represents GIu, Asp, Cys, homoCys, Lys, Orn, Dab or Dap; wherein X 1 and X 5 are joined, rendering the compound of formula Il cyclic, either via a disulfide bridge deriving from X 1 and X 5 both independently being Cys or homoCys, or via an amide bond formed between a carboxylic acid in the side-chain of X 1 and an amino group in the side-chain of X 5 , or between a carboxylic acid in the side-chain of X 5 and an amino group in the side-chain of X 1 ;
  • R 4 represents OR' or N(R') 2 , wherein each R' independently represents hydrogen or represents C 2 - 6 alkenyl or C 2 - 6 3lkynyl which may optionally be substituted with one or more amino or hydroxy; with the proviso that the compound of formula Il is not 15-carboxypentadecanoyl-Gly-Ser- Gln-His-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2 or
  • R 1 -R 2 -C( O)-R 3 -S 2 -Z 6 -c[X 1 -X 2 -X 3 -Arg-X 4 -X 5 ]R 4 [IVc] wherein R 1 represents tetrazol-5-yl or carboxy; R 2 represents a straight-chain, branched and/or cyclic C 6-2 oalkyl, C 6-2 oalkenyl or C 6-2 oalkynyl which may optionally be substituted with one or more substituents selected from halogen, hydroxyl and aryl;
  • S 2 represents a glycolether-based structure according to one of the formulas llla-lllg;
  • Z 4 represents GIy, Ala, Pro, Hyp, Ser, homoSer, Thr, Tyr, GIn, Asn, 2-PyAIa, 3-PyAIa, 4-
  • Z 5 represents GIy, Ala, Pro, Hyp, Ser, homoSer, Thr, GIn, Asn, 2-PyAIa, 3-PyAIa, 4-PyAIa,
  • Z 6 represents Ala, D-AIa, VaI, D-VaI, Leu, D-Leu, lie, D-IIe, Met, D-Met, NIe or D-NIe;
  • X 1 represents GIu, Asp, Cys, homoCys, Lys, Orn, Dab or Dap;
  • X 2 represents His, Cit, Dab, Dap, CgI, Cha, VaI, lie, tBuGly, Leu, Tyr, GIu, Ala, NIe, Met,
  • Hyp Tic, 2-PyAIa, 3-PyAIa, 4-PyAIa, (2-thienyl)alanine, 3-(thienyl)alanine, (4-thiazolyl)Ala, (2-furyl)alanine, (3-furyl)alanine or Phe, wherein one or more hydrogens on the phenyl moiety of said Phe may optionally and independently be substituted by a substituent selected among halogen, hydroxy, alkoxy, nitro, benzoyl, methyl, trifluoromethyl, amino and cyano;
  • X 3 represents D-Phe, wherein one or more hydrogens on the phenyl moiety in D-Phe may optionally and independently be substituted by a substituent selected among halogen, hydroxy, alkoxy, nitro, methyl, trifluoromethyl and cyano;
  • X 4 represents Trp, 2-NaI, (3-benzo[b]thienyl)alanine or (S)-2,3,4,9-tetrahydro-1 H- ⁇ - carboline-3-carboxylic acid;
  • X 5 represents GIu, Asp, Cys, homoCys, Lys, Orn, Dab or Dap; wherein X 1 and X 5 are joined, rendering the compound of formula IVa, IVb or IVc cyclic, either via a disulfide bridge deriving from X 1 and X 5 both independently being Cys or homoCys, or via an amide bond formed between a carboxylic acid in the side-chain of X 1 and an amino group in the side-chain of X 5 , or between a carboxylic acid in the side-chain of X 5 and an amino group in the side-chain of X 1 ;
  • R 4 represents OR' or N(R') 2 , wherein each R' independently represents hydrogen or represents C 1-6 alkyl, C 2-6 alkenyl or C 2-6 alkynyl which may optionally be substituted with one or more amino or hydroxy; with the proviso that said compound of formula IVa, IVb or IVc is not 2-[2-(15- carboxypentadecanoylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH 2 .
  • C x-y alkyl e.g. C6- 2 oalkyl
  • alkyl refers to a straight-chain, branched and/or cyclic, saturated monovalent hydrocarbon radical.
  • alkenyl refers to a straight-chain, branched and/or cyclic, monovalent hydrocarbon radical comprising at least one carbon-carbon double bond.
  • alkynyl refers to a straight-chain, branched and/or cyclic, monovalent hydrocarbon radical comprising at least one carbon-carbon triple bond, and it may optinally also comprise one or more carbon-carbon double bonds.
  • alkoxy as used herein is intended to indicate a radical of the formula -
  • aryl is intended to indicate a carbocyclic aromatic ring radical or a fused aromatic ring system radical wherein at least one of the rings is aromatic.
  • Typical aryl groups include phenyl, biphenylyl, naphthyl, and the like.
  • halogen is intended to indicate members of the 7 th main group of the periodic table of the elements, which includes fluorine, chlorine, bromine and iodine (corresponding to fluoro, chloro, bromo and iodo substituents, respectively).
  • tetrazol-5-yl is intended to indicate 1 H-tetrazol-5-yl or 2/-/-tetrazol-5-yl.
  • c[X a -...-X b ] indicates that ring closure is formed between the amino acid X a and the amino acid X b .
  • amino acids with additional amino or carboxy groups in the side chains such as Lys, Orn, Dap, GIu, Asp and others
  • At least one of Z 4 , Z 5 , X 1 , X 2 and X 5 is Dap or Dab.
  • the moiety T-A in formula I represents 10-(tetrazol-5-yl)decyl, 11-(tetrazol-5-yl)undecyl, 12-(tetrazol-5- yl)dodecyl, 13-(tetrazol-5-yl)tridecyl, 14-(tetrazol-5-yl)tetradecyl, 15-(tetrazol-5- yl)pentadecyl, 16-(tetrazol-5-yl)hexadecyl, 17-(tetrazol-5-yl)heptadecyl; 18-(tetrazol-5- yl)octadecyl or 19-(tetrazol-5-yl)nonadecyl.
  • S 1 is absent.
  • S 1 represents a structure according to formula Ilia.
  • S 1 represents a structure according to formula INb.
  • S 1 represents a structure according to formula INc.
  • S 2 represents a structure according to formula Ilia.
  • S 2 represents a structure according to formula INb.
  • the moiety R 1 -R 2 (i.e. R 1 and R 2 taken together) represents 10-(tetrazol-5-yl)decyl, 11-(tetrazol-5-yl)undecyl, 12-(tetrazol- 5-yl)dodecyl, 13-(tetrazol-5-yl)tridecyl, 14-(tetrazol-5-yl)tetradecyl, 15-(tetrazol-5- yl)pentadecyl, 16-(tetrazol-5-yl)hexadecyl, 17-(tetrazol-5-yl)heptadecyl, 18-(tetrazol-5- yl)octadecyl or 19-(tetrazol-5-yl)nonadecyl, such as 13-(tetrazol-5-yl)tridecyl, 14-(tetrazol-5-
  • R 1 -R 2 represents 10-(tetrazol-5-yl)decyl, 11-(tetrazol-5- yl)undecyl, 12-(tetrazol-5-yl)dodecyl, 13-(tetrazol-5-yl)tridecyl, 14-(tetrazol-5-yl)tetradecyl, 15-(tetrazol-5-yl)pentadecyl, 16-(tetrazol-5-yl)hexadecyl, 17-(tetrazol-5-yl)heptadecyl; 18- (tetrazol-5-yl)octadecyl or 19-(tetrazol-5-yl)nonadecyl.
  • R 1 -R 2 represents 13-(tetrazol-5-yl)tridecyl, 14-(tetrazol-5- yl)tetradecyl, 15-(tetrazol-5-yl)pentadecyl, 16-(tetrazol-5-yl)hexadecyl or 17-(tetrazol-5- yl)heptadecyl.
  • R 1 -R 2 represents 15-(tetrazol-5-yl)pentadecyl. In one embodiment hereof R 1 -R 2 represents 12-carboxydodecyl, 13-carboxytridecyl, 14-carboxytetradecyl, 15-carboxypentadecyl, 16-carboxyhexadecyl, 17-carboxyheptadecyl, 18-carboxyoctadecyl or 19-carboxynonadecyl.
  • R 1 -R 2 represents 14-carboxytetradecyl. In one embodiment hereof R 1 -R 2 represents 16-carboxyhexadecyl.
  • R 3 is absent.
  • Z 1 is absent, or Z 1 represents GIy.
  • Z 2 represents Ser, Thr, GIn, GIy or His, such as Ser or Thr.
  • Z 3 represents GIn
  • Z 4 represents Ser, homoSer, GIn, Asn, Tyr, His, Arg, homoArg, Lys, Orn, Dab or Dap.
  • Z 4 represents Ser, His, Arg or Dap.
  • Z 5 represents Ser, homoSer, Thr, Pro, Hyp, His, Lys,
  • Z 5 represents Ser, His or Dap.
  • Z 6 represents Ala, VaI, Leu, lie, Met or NIe.
  • Z 6 represents NIe.
  • X 2 represents Ser, Hyp, Cit, Dap, Asn, GIn or (4- thiazolyl)Ala.
  • X 2 represents Hyp, Dap, Cit or GIn.
  • X 2 represents Hyp.
  • X 1 is GIu
  • X 3 is D-Phe
  • X 4 is Trp
  • X 5 is Lys.
  • X 1 is Asp
  • X 3 is D-Phe
  • X 4 is Trp
  • X 5 is Lys.
  • R 4 is NH 2 .
  • R 4 is OH
  • the fully protected peptide is a fully protected alpha- melanocyte-stimulating hormone or mimic or analogue thereof. In one embodiment, the fully protected peptide has the following structure
  • the protected cyclic peptide has the following structure
  • the process of the present invention comprises, (a) providing a fully protected peptide; wherein a first connectable portion of said fully protected peptide is protected with a removable first protecting group; wherein a second connectable portion of said fully protected peptide is protected with a removable second protecting group; wherein the side chain functional groups of said fully protected peptide are protected with one or more removable third protecting group(s); wherein said removable third protecting group(s) are removable under removing conditions that are (a) different to the removing conditions for the removal of the removable first protecting group and (b) different to the removing conditions for the removal of the removable second protecting group; wherein after removal of the first protecting group and after removal of the second protecting group, the first connectable portion of said fully protected peptide is capable of being linked to the second connectable portion of said fully protected peptide thereby to form a protected cyclic peptide; (b) removing the first protecting group and removing the second protecting group to form a selectively deprotected peptide comprising said first connectable portion
  • the process according to the present invention further comprises, d) removing the one or more third removable leaving group(s) to form a cyclic peptide.
  • the connectable portions of the ring to be closed comprise first and second connectable portions which are protected with a removable protecting group.
  • the first and second connectable portions comprise a side chain of an amino acid residue.
  • first and second connectable portions comprise an amino group and a carboxylic acid group respectively.
  • first connectable portion comprises the side chain of a Lys residue and the second connectable portion comprises the side chain of a GIu residue.
  • the first and second connectable portions comprise functional groups of amino acid side chains.
  • the first removable protecting group protects a functional group on an amino acid side chain of the first connectable portion of the fully protected peptide and the second removable protecting group protects a functional group on an amino acid side chain of the second connectable portion of the fully protected peptide.
  • first and second connectable portions may be protected with any suitable removable protecting group.
  • first and second connectable portions are protected with protecting groups which are removable by hydrogenation.
  • the protecting groups of the first and second connectable portions are selected from benzyloxy, methylated benzyloxy, benzyloxycarbonyl and methylated benzyloxycarbonyl protecting groups as appropriate.
  • the protecting groups of the first and second connectable portions are selected from benzyloxy and benzyloxycarbonyl protecting groups as appropriate.
  • the fully protected peptide may be considered to be a first linear peptide.
  • the connectable portions of the ring to be closed may be considered to be first and second portions of said first linear peptide.
  • the process of the present invention comprises,
  • the one or more removable third leaving/ protecting groups are removed using a suitable deprotecting agent.
  • a suitable deprotecting agent may be selected according to the leaving/ protecting groups which are to be removed. Further, the order in which one or more of the removable third leaving/ protecting groups are removed is not particularly limited and, as a result, the deprotecting agent is not particularly limited.
  • a global protection strategy is used to remove one or more of the removable third leaving/ protecting groups.
  • the deprotecting agent used to remove the one or more removable third leaving/ protecting groups is different from the deprotecting agent used to remove the removable first and second leaving/ protecting groups.
  • the deprotecting agent used to cleave the support from the peptide may be the same or different from the protecting agent used to remove the one or more of the third removable leaving/ protecting groups.
  • trifluoro acetic acid (TFA) is used to remove one or more of the removable third leaving/ protecting groups.
  • a composition comprising
  • TFA and one or more further agents is used to remove one or more of the removable third leaving/ protecting groups.
  • the composition of TFA and one or more further agents must comprise TFA in an amount of greater than 50%.
  • the composition for removing the one or more third removable leaving/ protecting groups comprises TFA in an amount of greater than 50% and one or more scavengers based on the total volume of the composition.
  • the scavengers are selected from the group consisting of dimethoxybenzene (DMB), dithiothreitol (DTT), triisopropylsilane (TIPS) and water.
  • the composition for removing the one or more third removable leaving/ protecting groups comprises TFA in an amount of greater than 50% based on the total volume of the composition, dichloromethane, dimethoxybenzene (DMB), dithiothreitol (DTT), triisopropylsilane (TIPS) and water.
  • the concentration of each scavenger should be in the range of from about 2.5 to 3% based on the total volume of the composition.
  • the removal of the one or more removable third leaving/ protecting groups is conducted by contacting the composition as defined above with the protected cyclic peptide for about 2 hours at from about 20 0 C to about 25°C.
  • the cyclic peptide produced as a result of deprotection of the protected cyclic peptide is isolated. In one embodiment, isolation is performed by any of precipitation, reverse phase chromatography or diafiltration.
  • isolation is performed by precipitation.
  • the cyclic peptide is precipitated by transferring some or all of the cyclic peptide to a reactor containing TBME. In a specific embodiment, about half of the cyclic peptide is transferred to the reactor containing TBME, followed subsequently after a period of about 10 to 20 minutes by the remaining half of the cyclic peptide.
  • isolation is performed by neutralization followed by RP-
  • the cyclic peptide is neutralized by transferring some or all of the cyclic peptide to a reactor containing a mixture of EtOH/Water and NH 4 Ac. In a specific embodiment, about the cyclic peptide is transferred to the reactor containing EtOH/Water
  • the protected cyclic peptide is further deprotected and isolated to form a cyclic peptide.
  • the isolated cyclic peptide may also be purified by suitable means known to those in the art.
  • the cyclic peptide is derived from the protected cyclic peptide.
  • the cyclic peptide is,
  • the cyclic peptide is selected from any one of the compounds FA to FBC:
  • the cyclic peptide of the present invention is subjected to a further lyophilisation step. In one embodiment, the cyclic peptide of the present invention is subjected to a further spray drying step.
  • the ring to be closed is an internal ring.
  • An internal ring is a ring structure which is formed between either a side chain and a terminal group or a side chain and a side chain.
  • an internal ring does encompass ring structures formed as a result of links between terminal groups, i.e. end to end linkages.
  • a process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; and ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide; wherein the fully protected peptide is not connected to a solid phase support or a solid phase linker during step i).
  • a process for performing an internal ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide; and wherein the connectable portions of the ring to be closed comprise at least an amino group and a carboxylic acid group respectively and wherein the said connectable portions are protected by a protecting group removable by hydrogenation, and wherein the fully protected peptide is not connected to a solid phase support or a solid phase linker during step i).
  • a process for performing an internal ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide; and iii) deprotecting the protected cyclic peptide, wherein the connectable portions of the ring to be closed comprise at least an amino group and a carboxylic acid group respectively and wherein the said connectable portions are protected by a protecting group removable by hydrogenation and wherein the fully protected peptide is not connected to a solid phase support or a solid phase linker during step i).
  • the process of the present invention comprises,
  • a protected cyclic peptide or a cyclic peptide produced by the process as herein described.
  • the cyclic peptide may be lyophilised. In one embodiment, the cyclic peptide is suitable for the treatment of obesity.
  • composition of the present invention is suitable for the treatment of obesity.
  • a cyclic peptide as described herein for use in the treatment of obesity.
  • a pharmaceutical composition for use in the treatment of obesity in one embodiment, there is provided a pharmaceutical composition for use in the treatment of obesity.
  • a cyclic peptide or pharmaceutical composition as herein described for use in the manufacture of a medicament of the treatment of obesity.
  • the cyclic peptide or composition as described herein may be administered by any suitable means and/or route.
  • they may be delivered by parenteral administration or any one of oral, nasal, pulmonary, or transdermal administration.
  • the compositions of the present invention are encapsulated.
  • a process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide.
  • a process according to embodiment 1 comprising the steps of: (a) providing a fully protected peptide; wherein a first connectable portion of the fully protected peptide is protected with a removable first protecting group; wherein a second connectable portion of the fully protected peptide is protected with a removable second protecting group; wherein the side chain functional groups of said fully protected peptide are protected with one or more removable third protecting group(s); wherein said removable third protecting group(s) are removable under removing conditions that are (a) different to the removing conditions for the removal of the removable first protecting group and (b) different to the removing conditions for the removal of the removable second protecting group; wherein after removal of the first protecting group and after removal of the second protecting group, the first connectable portion of said fully protected peptide is capable of being linked to the second connectable portion of said fully protected peptide thereby to form a protected cyclic peptide;
  • amino acid side chains are those comprising at least an amine group and at least a carboxylic acid group.
  • amino acid side chains are side chains corresponding to Lys and GIu.
  • a process according to embodiment 9 wherein the suitable protecting groups are selected from the group consisting of tert-butyloxycarbonyl, 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl, tert-butyl, trityl, benzyloxy, benzyloxycarbonyl and butyl.
  • a process according to embodiment 9, wherein the protecting groups suitable for removal are selected from the group consisting of but not limited to acid labile groups, Pd- labile groups and hydrazine labile groups.
  • a process according to embodiment 1 1 wherein the acid labile groups are selected from the group consisting of Mtt and OPip groups.
  • a process according to embodiment 1 1 wherein the Pd-labile groups are selected from the group consisting of Alloc and OAIyI.
  • step b) as defined in claim 2
  • the first protecting group is removed before removal of the second protecting group.
  • a pharmaceutical composition comprising the cyclic peptide defined in embodiment 31 and optionally one or more pharmaceutically acceptable excipients, adjuvants, diluents or carriers.
  • a method of treating diabetes in a subject in need thereof comprising administering to the subject an effective amount of the peptide defined in embodiment 31 or the composition defined in embodiment 32.
  • a process comprising:
  • Figure 1 shows a general reaction scheme for the process of the present invention.
  • the peptides and reagents present in the scheme in Figure 1 are not limiting and only serve to show one embodiment of the present invention.
  • Figure 2 shows a general flowchart for the synthesis of cyclic peptide according to the process of the present invention.
  • the small amount of resin is drained and thoroughly washed with NMP and ethanol before it is analyzed by the colorimetric Kaiser test. If the Kaiser test is negative, the next step is the washing procedure. If the Kaiser test is positive, the coupling is not complete and DIC (0.5 eq) is added in order to form additional active ester. After 3h, a new IPC is taken out and checked by the Kaiser test. If it is positive, double coupling is necessary.
  • the resin is washed with NMP (6 x 2min x 5-10 volumes relative to the resin) to remove excess amino acid, impurities and coupling reagents.
  • the Fmoc protecting group on the N-terminal AA is removed by treatment with piperidine.
  • the resin is treated with a solution of piperidine diluted with NMP (20% Piperidine, 2 x 10 minutes x 5-10 volumes relative to the resin). After deprotection, the resin is washed again according to 1.1.2 and subsequently the peptide is ready for a new peptide coupling cycle.
  • A Fmoc protected Sieber resin weighed of for production. The resin is washed with DCM (3 x 2min x 10A volumes), with MeOH (3 x 2min x 10A volumes) and with DCM (3 x 2min x 10A volumes). A mixture of TFA diluted in DCM is prepared (2% TFA/ 1% TIPS/ DCM) and the resin is treated with the mixture (4 x 30 minutes x 10A volumes). Concentration of peptide in the cleavage filtrate should be tested by HPLC and if the last filtrate contains significant amounts of peptide the cleavage should be repeated until limited peptide is present in the cleavage filtrate.
  • pH in the filtrate is adjusted to 8-9 using DIPEA and concentrated to 2OA volumes.
  • Water (8A volumes) is added to the DCM mixture and pH in the water phase is adjusted to 8-9 using DIPEA before the phases are separated.
  • the organic phase is washed with additionally water (8A volumes) and the organic phase is concentrated (4A volumes).
  • Ethanol (14A volumes) is added and the mixture is concentrated again (4A volumes) before it is co-evaporated once again with EtOH (14A volumes to 4A volumes). The resulting yellow oil is used in the next step without further purification.
  • the Z protecting group on Lys and the BzI protecting group on GIu has to be removed.
  • the yellow oil isolated after cleavage is dissolved in absolute ethanol (10B volumes) before the mixture is transferred to the hydrogenation equipment. Pd/C (20% water, 0.5B grams) is added. After flushing with nitrogen, the mixture is placed under hydrogen atmosphere (2-3 bar) and the mixture is stirred at 50 0 C for 14 h. Removal of protecting groups should be investigated by HPLC at this stage. If IPC fragments of the protecting groups are still present Pd/C (0.1 B grams) should be added. If full deprotection has occurred, the mixture is filtrated and the filter cake is washed with absolute ethanol (5B volumes). The filtrate is concentrated to oil (maximum 2B volumes) and used in the next step without any further purification.
  • Reactants elutes at 25.5 (Reactant -Trt) and 28 minutes (Reactant).
  • the oil separated in the previous step is diluted with THF (to a total volume of 50C) and water (1 C volume).
  • DIPEA (0.75 eq compared to theoretical peptide) is added and the mixture is stirred for 5 minutes before PyBOB (0.5 eq compared to theoretical peptide) is added.
  • Addition of PyBOB and DIPEA is repeated with 30 minutes intermission (3 x 0.5 eq PyBOB, 1.5 eq overall) and the mixture is stirred 2 h overall.
  • IPC should be analyzed by HPLC in order to confirm full amide formation. If ring closure is incomplete additionally DIPEA and PyBOB is added.
  • the mixture is concentrated (maximum 3C volumes) and dissolved in DCM (15C volumes).
  • the organic phase is washed with Water (2x1 OC volumes; pH in the organic phase measured to 6-7; the separation should be allowed to separate completely over 1-4 h) before the organic phase is concentrated (2-4C volumes).
  • the oil is used in the global deprotection step without any further purification.
  • Reactants elutes at 11.9 (Reactant -Trt) and 16.8 minutes (Reactant). Products elutes at 19.5 (Product-Trt) and 25.23 (Product).
  • the peptide should be cleaved in a mixture of TFA (14D volumes; 62%), DCM (6D -
  • TIPS triisopropylsilane
  • the deprotection mixture is premixed and the protected peptide fragment is dissolved and stirred in the mixture at 20-25 0 C for 2h.
  • the volume of the mixture is reduced (to 14 volumes) over 1 h-2h under reduced pressure.
  • Half of the cleavage mixture is transferred to a reactor containing TBME (5OD volumes, 35°C-40°C) causing precipitation to occur and after 15 minutes the rest of the cleavage mixture is transferred.
  • the mixture is stirred for
  • the peptide should be cleaved in a mixture of TFA (15D volumes; 91%) and scavengers: DTT (0.5 D volumes; 3 %), water (0.5 D volumes; 3 %), and TIPS (0.5 D volumes; 3 %).
  • DTT 0.5 D volumes; 3 %)
  • TIPS 0.5 D volumes; 3 %).
  • the protected peptide fragment dissolved in DCM (4D volumes) was added to the cleavage mixture over 5-10 min and stirred in the mixture at 20-25 0 C for 2.5 h.
  • a neutralization mixture is prepared: Ammonium acetate (1.0 molar equivalent compared to TFA) in water (45 D volumes) and ethanol (10 D volumes compared to TFA).
  • pH is adjusted using NH 4 OAc or TFA to 3-3.5 and the mixture is concentrated (15D volume is removed). Consequently the mixture is

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Abstract

A process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide.

Description

PROCESS
FIELD OF THE INVENTION
The present invention relates to a process for performing a ring closure on a fully protected peptide in order to produce a protected cyclic peptide, products derived from said process and the use of said products.
BACKGROUND TO THE INVENTION
Solid phase synthesis is a well known technique for the production of peptides. In solid phase peptide synthesis (SPPS), an amino acid or peptide group is bound to a solid support resin. Then, successive amino acids or peptide groups are attached to the support- bound peptide until the peptide material of interest is formed. The support-bound peptide is then typically cleaved from the support and subject to further processing and/or purification. In some cases, solid phase synthesis yields a mature peptide product; in other cases the peptide cleaved from the support is used in the preparation of a larger, mature peptide product.
In order to synthesize a single defined peptide sequence those skilled in the art generally use the Merrifield method to "grow" peptide chains attached to solid supports. The process of synthesizing these individual peptides has been automated. Commercially available equipment can be used to synthesize peptides of one hundred or more amino acids in length. To obtain peptides of arbitrary length, the resulting peptides can be ligated with each other by using appropriate protective groups on the side chains and by employing techniques permitting the removal of the synthesized peptides from the solid supports without deprotecting them. Thus, the synthesis of individual peptides of arbitrary length is known in the art.
The steps of protection and deprotection, whilst necessary in this form of solid phase peptide synthesis, can be considered to detract significantly from the final peptide yield. Further, it may be necessary to protect the side chains of the amino acids of the peptide in order to prevent them from participating in the coupling reaction. Thus, it may be that protecting groups which are capable of being removed under a range of conditions are utilised. This enables selective deprotection of the amino acids. Protecting groups which are removable under a range of conditions are often referred to as "orthogonal protecting groups".
In solid phase peptide synthesis, the demand of a new orthogonal protective set is significant. The established orthogonal deprotection sets are based upon the well-known Fmoc and Boc protection of amino acids. The construction of complex peptides or glycopeptides often requires a third orthogonal protecting group for side-chain amino functionalities, whose removal will not affect the protecting groups in the other orthogonal sets, or vice versa. Thus, in addition to requiring protection during the initial solid phase synthesis, functional groups and side chains may require protection if the resulting peptide is to undergo further modification once cleaved from the resin.
Peptide modifications may be desired for a number of reasons. In particular, modifications may be made to fix or reduce the number of conformational forms the peptide may take. In other situations, peptide side chains may be modified in an attempt to increase the binding of the peptide to a specific target or, in other cases, to protect the peptide from degradation by enzymes and the like.
In some instances, peptides comprising cyclic groups have been formed whereby bonds are created between the side chains of amino acid residues. In other instances, the terminals of a peptide have been linked end-on-end such that the peptide has a ring or cyclic type structure. Typically, these cyclic peptides are prepared in such a way that the ring is formed by oxidation of the naturally occurring cysteine residues present in the peptide chain, thus yielding a disulfide bridged structure. This technique mimics the most common form of cyclisation found among naturally occurring peptides and proteins, but does not provide a convenient means of preparing other types of cyclic structures. GB 1527252 gives an example of a process for the cyclisation of peptides containing cysteine groups. In GB 1527252, a peptide containing at least two cysteine moieties is held in a solution which is substantially free of oxygen until cyclisation between the two cysteine moieties has occurred to yield a cyclic disulfide bond. Further examples of processes in which cyclic peptides with disulfide bonds are formed, are given in EP 1921087.
In order to prepare end-on-end cyclic peptides, one technique is to employ amino acids with orthogonally protected functional groups such that some are removable selectively in the presence of others. Those skilled in the art can use these techniques to prepare peptides in solution in which the amino terminus is cyclised to the carboxyl terminus to form a ring. A naturally occurring example is the antibiotic gramicidin. Alternatively, pairs of cysteine residues are oxidized to disulfide bonds to form one or more rings; the familiar naturally-occurring cyclic peptide hormone oxytocin is an example of such a structure, such as has been prepared by O'Neil et al., Protein, 14, 509-515 (1992)); however, this example is limited to cases of disulfide forming cyclic hexapeptides. US 6,008,058 discloses a method of producing terminally linked (head to tail) cyclic peptides using solid phase synthesis. The cyclic peptides of US 6,008,058 are formed whilst the peptide chain is still attached to the resin support. This is taught in US 6,008,058 as being critical, as cleaving the peptide can create additional problems such as giving rise to dimeric and oligomeric structures and presenting difficulties in characterisation. In order to facilitate the formation of the terminally linked cyclic peptide, US 6,008,058 requires the presence of a multifunctional amino acid attached to the resin support. This enables the amino acid linked to the resin to also participate in cyclisation.
US 2003/0125243 also discloses a process for producing terminally linked peptides. Cyclisation is performed with 1-propoanephosphonic acid cyclic anhydride.
WO 01/16162 describes a method for the preparation of a cyclic peptide wherein an aromatic group is attached to a terminal end of the peptide, said aromatic forming a link between an oxygen, thio or amine group on the peptide.
Further cyclic peptides may be prepared by forming C-backbone linkages. In this method, the linkages are formed between the carbon atoms on the peptide backbone, therefore leaving the functional groups on the amino acid side chains free for binding with a target site. US 2005/0267017 discloses cyclic peptides and processes for their production wherein the cyclic linkages are formed between the carbon atoms present on the peptide backbone. US 5,723,575 also discloses a process for the preparation of backbone cyclised peptides.
The methods of cyclisation disclosed in the prior art have a number of disadvantages which may render them unsuitable for application in the manufacture of certain targets. For example, cysteine cyclisation is only appropriate where the peptide has the necessary free cysteine residues; backbone cyclisation is only appropriate where there are suitable linking sites available on the peptide.
One such target for which an improved process of production is desired is a cyclic analogue of α-melanocyte-stimulating hormone (α-MSH). This hormone regulates the signalling by the melanocortin 4 receptor (MC4) in hypothalamus in the central nervous system. The hormone promotes increased energy expenditure and a reduced appetite leading to weight loss.
The methods disclosed in the prior art for producing cyclic α-MSH analogues contain a number of drawbacks which impact on the purity and yield of the final product. US 5,683,981 discloses a method for performing a ring closure on an α-melanocyte- stimulating hormone analogue. In particular, US 5,683,981 discloses a method wherein a Lys residue and a GIu residue are condensed to form a ring structure. The process for performing the ring closure comprises forming a peptide from a "key intermediate A" which comprises a number of protected amino acid residues, in particular, a protected Lys residue. This key intermediate is then coupled to a GIu residue. Once the Lys and GIu residues have been attached, cyclisation is performed through condensation using BOP. Following cyclisation, amino acid coupling is continued until the desired peptide is obtained. Deprotection of the various protecing groups then occurs along with cleavage from the resin. Critically, the process of US 5,683,981 requires that the cyclisation step is conducted when the peptide is still attached to the resin. Moreover, not all of the amino acid residues present in the peptide are protected. Thus, cyclisation is conducted even though a number of the residues are unprotected.
Thus, the above process results in a relatively uncontrolled ring closure. Such an uncontrolled ring closure can result in other ring closures than the desired one. Further, the resulting impurities will be very similar to the desired final product and therefore will be difficult to remove in purification. Thus, the process of the prior art also demands a relatively high number of purification steps.
As a result, it would be desirable to provide a process for the production of a cyclic α-melanocyte-stimulating hormone analogue which does not have the drawbacks associated with the processes of the prior art.
Moreover, it would be desirable to provide a process for performing a ring closure on a peptide which does not have the drawbacks associated with the ring closure methods of the prior art.
SUMMARY OF THE INVENTION
In one aspect, there is provided a process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide.
In a second aspect there is provided a protected cyclic peptide or cyclic peptide produced by the process of the present invention.
In a third aspect, there is provided a pharmaceutical composition comprising the cyclic peptide produced by the process of the present invention and optionally one or more pharmaceutically acceptable excipients, adjuvants, diluents or carriers.
In a fourth aspect there is provided a cyclic peptide produced by the process of the present invention, or composition comprising the said cyclic peptide, for use in the treatment of obesity.
In a fifth aspect there is provided a method of treating diabetes in a subject in need thereof comprising administering to the subject an effective amount of the cyclic peptide produced by the process of the present invention or a composition comprising the said cyclic peptide.
For ease of reference, these and further aspects of the present invention are now discussed under appropriate section headings. However, the teachings under each section are not necessarily limited to each particular section.
Advantages
The process of the present invention provides an economically favourable method for producing protected cyclic peptides. The process of the present invention is capable of producing cyclic peptides in greater yields than the known processes of the prior art.
The process of the present invention is capable of producing a cyclic peptide of a greater purity than the prior art due to a more controlled ring closure. This results in a reduction in impurities which are very similar to the desired end product and which are therefore difficult to detect by analytical methods. As a result, the process of the present invention provides a cyclic peptide with increased safety for pharmaceutical applications.
The process of the present invention provides a method of performing a ring closure which does not require to be attached to the support resin during cyclisation.
Accordingly, the process of the present invention does not have the disadvantages associated with performing a ring closure on a support resin.
Abbreviations
The following abbreviations may be used herein.
DETAILED DESCRIPTION Process According to a first aspect, there is provided a process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide. A cyclic peptide may be considered to be a peptide which comprises a cyclic or ring structure which results from the linkage of two portions of a linear peptide. Thus, a cyclic peptide as used herein is not considered to be a linear peptide comprising amino acids having cyclic side chains, e.g. tryptophan, histidine etc. A cyclic peptide as defined herein is considered to not comprise any externally added protecting groups. By externally added protecting groups it is meant a protecting group which has been added during synthesis to protect a functional group on an amino acid side chain.
A fully protected peptide may be considered to be a peptide in which all of the amino acid side chain functional groups are protected by a suitable protecting group. It may also include a peptide which is connected to a solid phase support/ solid phase linker as this can be considered to protect the terminal amino acid to which it is attached.
A fully protected peptide as defined herein may be produced by a number of methods known to the person skilled in the art. In one embodiment, the fully protected peptide is produced using solid phase peptide synthesis. In one embodiment, a Sieber amide resin is used as the solid phase support resin.
In one embodiment, the fully protected peptide is obtained or obtainable from the dipeptide, Fmoc-Nle-Glu-OH. In one embodiment, where the fully protected peptide is synthesised using solid phase peptide synthesis, the solid phase support or solid phase linker is acid labile.
In one embodiment, where the fully protected peptide is synthesised using solid phase peptide synthesis, the fully protected peptide is cleaved from the solid phase support or solid phase linker using a composition comprising TFA and dichloromethane. In one embodiment, the composition comprises TFA in an amount of less than 2% based on the total volume of the composition.
In one embodiment, the concentration of the TFA used to cleave the fully protected peptide from the solid phase support or solid phase linker is not sufficient to remove any of the protecting groups on the fully protected peptide. A protected cyclic peptide may be considered to be a peptide comprising a cyclic or ring structure which results from the linkage of two portions of a linear peptide group, wherein at least one of the amino acid side chain functional groups is protected by a suitable protecting group. The definition of a protected cyclic peptide also includes those peptides comprising a cyclic group as defined above wherein substantially all, but not all, of the amino acid side chain functional groups are protected.
In one embodiment according to the process of the present invention, the connectable portions of the ring to be closed comprise first and second connectable portions.
In one embodiment according to the process of the present invention, the connectable portions of the ring to be closed comprise side chains of amino acids. In one embodiment, the connectable portions of the ring to be closed are not at the terminal ends of a peptide.
In one embodiment according to the process of the present invention, the amino acid side chains are those comprising at least an amine group and at least a carboxylic acid group. In one embodiment according to the process of the present invention, the amino acid side chains are side chains corresponding to Lys and GIu.
The protecting groups on the fully protected peptide are selected from protecting groups suitable for the protection of amino acid side chains. It is well known in the art which protecting groups may be used for the protection of amino acids. Many protecting groups have been developed for amino group protection, and fall into a number of broad classes.
A number of amino acid protecting groups are mentioned in WO 99/15510.
Accordingly, in one embodiment according to the process of the present invention, the suitable protecting groups are selected from the group consisting of tert- butyloxycarbonyl, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl, tert-butyl, trityl, benzyloxy, benzyloxycarbonyl and butyl.
In one embodiment according to the process of the present invention, the protecting groups suitable for removal are selected from the group consisting of but not limited to acid labile groups, Pd-labile groups and hydrazine labile groups.
In one embodiment, the acid labile groups are selected from the group consisting of Mtt and OPip groups.
In one embodiment according to the process of the present invention, the Pd-labile groups are selected from the group consisting of Alloc and OAIyI. In one embodiment according to the process of the present invention, the hydrazine labile groups are selected from the group consisting of ODmab and Dde.
In one embodiment according to the process of the present invention, the protecting groups of the connectable portions of the ring to be closed are selected from benzyloxy and benzyloxycarbonyl. In one embodiment according to the process of the present invention, the protecting group of the first connectable portions of the ring to be closed is benzyloxy and the protecting group of the second connectable portions of the ring to be closed is benzyloxycarbonyl.
In one embodiment according to the process of the present invention, the abbreviations Bn, Bz and BzI are used interchangeably and are intended to mean the protecting group benzyloxy.
In one embodiment according to the process of the present invention, the connectable portions of the ring to be closed are protected with protecting groups which are suitable for removal by hydrogenation. In one embodiment, hydrogenation is performed using H2; Pd/C. In one embodiment, the Pd/C is used in amount of at least 5% based on the theoretical weight of the fully protected peptide free of the solid phase support or solid phase linker. In one embodiment, the Pd/C is used in amount of 5 to 30% based on the theoretical weight of the fully protected peptide free of the solid phase support or solid phase linker to about 30. In one embodiment, the conditions for hydrogenation are H2; Pd/C (10%). In one embodiment, hydrogenation is carried out in absolute ethanol. In one embodiment, the pH of the hydrogenation reaction should not be acidic.
In one embodiment according to the process of the present invention, the connectable portions of the of the ring to be closed are the side chains of amino acids, Lys and GIu, the functional group of the Lys side chain being protected with a benzyloxycarbonyl protecting group, and the functional group of the GIu side chain being protected with the benzyloxy protecting group. In this embodiment, the protecting groups may be removed by hydrogenation.
Thus, in one embodiment, the connectable portions of the ring to be closed are selectively deprotected to form a selectively deprotected peptide. Accordingly, in one embodiment according to the process of the present invention, there is provided a process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting by hydrogenation, the connectable portions of the ring to be closed wherein the connectable portions consist of side chains of a Lys residue and a GIu residue; and ii) connecting the deprotected functional groups of said Lys and GIu residues to form the protected cyclic peptide.
In one embodiment, the connectable portions of the ring to be closed are connected using one or more suitable connecting agents. In one embodiment, one or more suitable connecting agents are selected from the group consisting of N,N'-diisopropyl carbodiimide (DIC)/ 1-hydroxybenzotriazol (HOBt), 1- ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDAC)/ 1-hydroxybenzotriazol (HOBt), N,N'-diisopropylethylamine (DIPEA), benzotrιazoi-1-yi- oxy- tripyrroiidinophosphonium hexafiuorophosphate (PyBOB), O-(Benzotriazol-1-yl)-N,N,N',N'- tetramethyluronium tetrafluoroborate (TBTU), 2-(1 H-7-Azabenzotriazol-1-yl)-1 , 1 ,3,3- tetramethyl uronium hexafiuorophosphate Methanaminium (HATU).
In one embodiment, the suitable connecting agents are selected from N1N1- diisopropylethylamine (DIPEA), benzotriazol-1-yi- oxy-tripyrroiidinophosphonium hexafiuorophosphate (PyBOB)1 In one embodiment, the suitable connecting agent is O-(Benzotriazol-i-yl)-
N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU). In one embodiment, step ii) of the process of the present invention is conducted in a solvent selected from THF, wet THF, Toluene, NMP/ THF, EtOAc, and similar solvents. In one embodiment, step ii) is carried out in THF.
As mentioned above, the fully protected peptide may be synthesised using solid phase peptide synthesis. However, it is not necessary for the fully protected peptide to be connected to a solid phase support or a solid phase support linker when step i) of the process of the present invention is performed. Thus, in one embodiment, the fully protected peptide is not connected to a solid phase support or a solid phase support linker when it is selectively deprotected according to step i). In this regard, the fully protected peptide may be considered to be free of a solid phase support or a solid phase support linker.
Moreover, the step of connecting the deprotected connectable portions of the ring to be closed is not particularly limited. However, in one embodiment, the selectively deprotected peptide is not connected to a solid phase support or a solid phase support linker during the step of connecting the connectable portions of the ring to be closed. Again, in this regard, the fully protected peptide may be considered to be free of a solid phase support or a solid phase support linker.
Further, the process of the present invention is not particularly limited in terms of the peptides to which it is applicable. Thus, it is envisaged that any fully protected peptide may be used in the process of the present invention.
In one embodiment according to the process of the present invention, the fully protected peptide may be a fully protected analogue of a peptide selected from the group consisting of human insulin, a human insulin analogue, a human insulin derivative, GLP-1 , a GLP-1 analogue, a GLP-1 derivative, exendin, an exendin analogue, an exendin derivative, α-MSH, an α-MSH analogue, an α-MSH derivative, β-MSH, a β-MSH analogue, a β-MSH derivative, an MC4 receptor agonist, amylin, an amylin analogue, an amylin derivative, gastrin, a gastrin analogue, a gastrin derivative, human growth hormone, a human growth hormone analogue, a human growth hormone derivative, factor Vila, a factor Vila analogue and a factor Vila derivative, or any combination thereof.
In one embodiment the invention relates to compounds (more particularly compounds acting as melanocortin receptor agonists or antagonists) of formula I:
T-A-L-P [I] wherein T represents tetrazol-5-yl; A represents a straight-chain, branched and/or cyclic C6-2oalkyl, C6-2oalkenyl or C6-2oalkynyl which may optionally be substituted with one or more substituents selected from halogen, hydroxy and aryl;
L is a bond or a chemical structure covalently linking A and P; and P represents a peptide structure comprising at least six α-amino acid residues and a ring closure.
In one embodiment the invention relates to compounds having the formula II:
R1-R2-C(=O)-R3-S1-Z1-Z2-Z3-Z4-Z5-Z6-c[X1-X2-X3-Arg-X4-X5]-R4 [II] wherein
R1 represents tetrazol-5-yl or carboxy;
R2 represents a straight-chain, branched and/or cyclic C6-2oalkyl, C6-2oalkenyl or C6-2oalkynyl which may optionally be substituted with one or more substituents selected from halogen, hydroxy and aryl; R3 is absent or represents -NH-S(=O)2-(CH2)3-5-C(=O)- or a peptide fragment comprising one or two amino acid residues and containing at least one carboxy group; S1 is absent or represents a 4-aminobutyric acid residue, GIy, β-Ala, or a glycolether-based structure according to one of the formulas llla-lllg;
-HN-CH2-CH2-O-CH2-CH2-O-CH2-C(=O)- [Ilia] -[HN-CH2-CH2-O-CH2-CH2-O-CH2-C(=O)]2- [NIb]
-[HN-CH2-CH2-O-CH2-CH2-O-CH2-C(=O)]3-5- [NIc]
-[HN-CH2-CH2-O-CH2-CH2-O-CH2-CH2-NH-C(=O)-CH2-CH2-CH2-C(=O)]i-3- [llld] -[HN-CH2-CH2-O-CH2-CH2-O-CH2-CH2-NH-C(=O)-CH2-O-CH2-C(=O)]1-3- [NIe] -[HN-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)]i^- [lllfl -HN-CH2-CH2-[O-CH2-CH2]2-i2-O-CH2-C(=O)- [INg]
-HN-CH2-CH2-[O-CH2-CH2]4-i2-O-CH2-CH2-C(=O)- [NIh]
Z1 is absent or represents GIy, β-Ala, Ser, D-Ser, Thr, D-Thr, His, D-His, Asn, D-Asn, GIn, D-GIn, GIu, D-GIu, Asp, D-Asp, Ala, D-AIa, Pro, D-Pro, Hyp or D-Hyp; Z2 is absent or represents GIy, β-Ala, Ser, D-Ser, Thr, D-Thr, His, D-His, Asn, D-Asn, GIn, D-GIn, GIu, D-GIu, Asp, D-Asp, Ala, D-AIa, Pro, D-Pro, Hyp or D-Hyp;
Z3 represents Ser, D-Ser, Thr, D-Thr, His, D-His, Asn, D-Asn, GIn, D-GIn, GIu, D-GIu, Asp, D-Asp, Ala, D-AIa, Pro, D-Pro, Hyp or D-Hyp;
Z4 represents GIy, Ala, Pro, Hyp, Ser, homoSer, Thr, Tyr, GIn, Asn, 2-PyAIa, 3-PyAIa, 4- PyAIa, His, homoArg, Arg, Lys, Dab, Dap or Orn; Z5 represents GIy, Ala, Pro, Hyp, Ser, homoSer, Thr, GIn, Asn, 2-PyAIa, 3-PyAIa, 4-PyAIa, His, homoArg, Arg, Lys, Dab, Dap or Orn; Z6 represents Ala, D-AIa, VaI, D-VaI, Leu, D-Leu, lie, D-IIe, Met, D-Met, NIe or D-NIe;
X1 represents GIu, Asp, Cys, homoCys, Lys, Orn, Dab or Dap;
X2 represents His, Cit, Dab, Dap, CgI, Cha, VaI, lie, tBuGly, Leu, Tyr, GIu, Ala, NIe, Met,
Met(O), Met(O2), GIn, Gln(alkyl), Gln(aryl), Asn, Asn(alkyl), Asn(aryl), Ser, Thr, Cys, Pro, Hyp, Tic, 2-PyAIa, 3-PyAIa, 4-PyAIa, (2-thienyl)alanine, 3-(thienyl)alanine, (4-thiazolyl)Ala,
(2-furyl)alanine, (3-furyl)alanine or Phe, wherein one or more hydrogens on the phenyl moiety of the Phe in question may optionally and independently be substituted by a substituent selected among halogen, hydroxy, alkoxy, nitro, benzoyl, methyl, trifluoromethyl, amino and cyano; X3 represents D-Phe, wherein one or more hydrogens on the phenyl moiety in D-Phe may optionally and independently be substituted by a substituent selected among halogen, hydroxy, alkoxy, nitro, methyl, trifluoromethyl and cyano;
X4 represents Trp, 2-NaI, (3-benzo[b]thienyl)alanine or (S)-2,3,4,9-tetrahydro-1 H-β- carboline-3-carboxylic acid; X5 represents GIu, Asp, Cys, homoCys, Lys, Orn, Dab or Dap; wherein X1 and X5 are joined, rendering the compound of formula Il cyclic, either via a disulfide bridge deriving from X1 and X5 both independently being Cys or homoCys, or via an amide bond formed between a carboxylic acid in the side-chain of X1 and an amino group in the side-chain of X5, or between a carboxylic acid in the side-chain of X5 and an amino group in the side-chain of X1;
R4 represents OR' or N(R')2, wherein each R' independently represents hydrogen or represents C2-6alkenyl or C2-63lkynyl which may optionally be substituted with one or more amino or hydroxy; with the proviso that the compound of formula Il is not 15-carboxypentadecanoyl-Gly-Ser- Gln-His-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2 or
2-[2-(15-carboxypentadecanoylamino)ethoxy]ethoxyacetyl-Ser-Gln-Ser-Nle-c[Glu-Hyp-D-
Phe-Arg-Trp-Lys]-NH2; and pharmaceutically acceptable salts, prodrugs and solvates thereof.
In one embodiment, the peptide is a compound having one of the formulae IVa, IVb or IVc: R1-R2-C(=O)-R3-S2-Z4-Z5-Z6-c[X1-X2-X3-Arg-X4-X5]R4 [IVa]
R1-R2-C(=O)-R3-S2-Z5-Z6-c[X1-X2-X3-Arg-X4-X5]R4 [IVb]
R1-R2-C(=O)-R3-S2-Z6-c[X1-X2-X3-Arg-X4-X5]R4 [IVc] wherein R1 represents tetrazol-5-yl or carboxy; R2 represents a straight-chain, branched and/or cyclic C6-2oalkyl, C6-2oalkenyl or C6-2oalkynyl which may optionally be substituted with one or more substituents selected from halogen, hydroxyl and aryl;
R3 is absent or represents -NH-S(=O)2-(CH2)3-5-C(=O)- or a peptide fragment comprising one or two amino acid residues and containing at least one carboxy group;
S2 represents a glycolether-based structure according to one of the formulas llla-lllg;
-HN-CH2-CH2-O-CH2-CH2-O-CH2-C(=O)- [Ilia]
-[HN-CHz-CHz-O-CHz-CHz-O-CHz-C^COb- [1Mb]
-[HN-CH2-CH2-O-CH2-CH2-O-CH2-C(=O)]3-5- [NIc] -[HN-CH2-CH2-O-CH2-CH2-O-CH2-CH2-NH-C(=O)-CH2-CH2-CH2-C(=O)]i-3-[llld]
-[HN-CH2-CH2-O-CH2-CH2-O-CH2-CH2-NH-C(=O)-CH2-O-CH2-C(=O)]1-3- [NIe]
-[HN-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-O-CH2-CH2-C(=O)]1-3- [NIf]
-HN-CH2-CH2-[O-CH2-CH2]2-12-O-CH2-C(=O)- [NIg]
-HN-CH2-CH2-[O-CH2-CH2]4-12-O-CH2-CH2-C(=O)- [NIh]
Z4 represents GIy, Ala, Pro, Hyp, Ser, homoSer, Thr, Tyr, GIn, Asn, 2-PyAIa, 3-PyAIa, 4-
PyAIa, His, homoArg, Arg, Lys, Dab, Dap or Orn;
Z5 represents GIy, Ala, Pro, Hyp, Ser, homoSer, Thr, GIn, Asn, 2-PyAIa, 3-PyAIa, 4-PyAIa,
His, homoArg, Arg, Lys, Dab, Dap or Orn; Z6 represents Ala, D-AIa, VaI, D-VaI, Leu, D-Leu, lie, D-IIe, Met, D-Met, NIe or D-NIe;
X1 represents GIu, Asp, Cys, homoCys, Lys, Orn, Dab or Dap;
X2 represents His, Cit, Dab, Dap, CgI, Cha, VaI, lie, tBuGly, Leu, Tyr, GIu, Ala, NIe, Met,
Met(O), Met(O2), GIn, Gln(alkyl), Gln(aryl), Asn, Asn(alkyl), Asn(aryl), Ser, Thr, Cys, Pro,
Hyp, Tic, 2-PyAIa, 3-PyAIa, 4-PyAIa, (2-thienyl)alanine, 3-(thienyl)alanine, (4-thiazolyl)Ala, (2-furyl)alanine, (3-furyl)alanine or Phe, wherein one or more hydrogens on the phenyl moiety of said Phe may optionally and independently be substituted by a substituent selected among halogen, hydroxy, alkoxy, nitro, benzoyl, methyl, trifluoromethyl, amino and cyano;
X3 represents D-Phe, wherein one or more hydrogens on the phenyl moiety in D-Phe may optionally and independently be substituted by a substituent selected among halogen, hydroxy, alkoxy, nitro, methyl, trifluoromethyl and cyano;
X4 represents Trp, 2-NaI, (3-benzo[b]thienyl)alanine or (S)-2,3,4,9-tetrahydro-1 H-β- carboline-3-carboxylic acid;
X5 represents GIu, Asp, Cys, homoCys, Lys, Orn, Dab or Dap; wherein X1 and X5 are joined, rendering the compound of formula IVa, IVb or IVc cyclic, either via a disulfide bridge deriving from X1 and X5 both independently being Cys or homoCys, or via an amide bond formed between a carboxylic acid in the side-chain of X1 and an amino group in the side-chain of X5, or between a carboxylic acid in the side-chain of X5 and an amino group in the side-chain of X1;
R4 represents OR' or N(R')2, wherein each R' independently represents hydrogen or represents C1-6alkyl, C2-6alkenyl or C2-6alkynyl which may optionally be substituted with one or more amino or hydroxy; with the proviso that said compound of formula IVa, IVb or IVc is not 2-[2-(15- carboxypentadecanoylamino)ethoxy]ethoxyacetyl-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2.
The use of a prefix of the type "Cx-y" preceding the name of a radical, such as in Cx-yalkyl (e.g. C6-2oalkyl) is intended to indicate a radical of the designated type having from x to y carbon atoms.
The term "alkyl" as used herein refers to a straight-chain, branched and/or cyclic, saturated monovalent hydrocarbon radical. The term "alkenyl" as used herein refers to a straight-chain, branched and/or cyclic, monovalent hydrocarbon radical comprising at least one carbon-carbon double bond.
The term "alkynyl" as used herein refers to a straight-chain, branched and/or cyclic, monovalent hydrocarbon radical comprising at least one carbon-carbon triple bond, and it may optinally also comprise one or more carbon-carbon double bonds. The term "alkoxy" as used herein is intended to indicate a radical of the formula -
OR', wherein R' is alkyl as indicated above.
In the present context, the term "aryl" is intended to indicate a carbocyclic aromatic ring radical or a fused aromatic ring system radical wherein at least one of the rings is aromatic. Typical aryl groups include phenyl, biphenylyl, naphthyl, and the like. The term "halogen" is intended to indicate members of the 7th main group of the periodic table of the elements, which includes fluorine, chlorine, bromine and iodine (corresponding to fluoro, chloro, bromo and iodo substituents, respectively).
The term "tetrazol-5-yl" is intended to indicate 1 H-tetrazol-5-yl or 2/-/-tetrazol-5-yl. In the formulas herein c[Xa-...-Xb] indicates that ring closure is formed between the amino acid Xa and the amino acid Xb.
In the present context, common rules for peptide nomenclature based on the three letter amino acid code apply, unless exceptions are specifically indicated. Briefly, the central portion of the amino acid structure is represented by the three letter code (e.g. Ala, Lys) and L-configuration is assumed, unless D-configuration is specifically indicated by "D- " followed by the three letter code (e.g. D-AIa, D-Lys). A substituent at the amino group replaces one hydrogen atom and its name is placed before the three letter code, whereas a C-terminal substituent replaces the carboxylic hydroxy group and its name appears after the three letter code. For example, "acetyl-Gly-Gly-NH2" represents CH3-C(=O)-NH-CH2-C(=O)-NH-CH2-C(=O)-NH2. Unless indicated otherwise, amino acids with additional amino or carboxy groups in the side chains (such as Lys, Orn, Dap, GIu, Asp and others) are connected to their neighboring groups by amide bonds formed at the N-2 (α-nitrogen) atom and the C-1 (C=O) carbon atom.
In one embodiment at least one of Z4, Z5, X1, X2 and X5 is Dap or Dab.
In certain embodiments of compounds of the present invention, the moiety T-A in formula I represents 10-(tetrazol-5-yl)decyl, 11-(tetrazol-5-yl)undecyl, 12-(tetrazol-5- yl)dodecyl, 13-(tetrazol-5-yl)tridecyl, 14-(tetrazol-5-yl)tetradecyl, 15-(tetrazol-5- yl)pentadecyl, 16-(tetrazol-5-yl)hexadecyl, 17-(tetrazol-5-yl)heptadecyl; 18-(tetrazol-5- yl)octadecyl or 19-(tetrazol-5-yl)nonadecyl.
In certain embodiments of compounds of the present invention, S1 is absent.
In further embodiments of compounds of the invention, S1 represents a structure according to formula Ilia.
In additional embodiments of compounds of the invention, S1 represents a structure according to formula INb.
In still further embodiments of compounds of the invention, S1 represents a structure according to formula INc. In one embodiment hereof S2 represents a structure according to formula Ilia.
In another embodiment hereof S2 represents a structure according to formula INb.
In some embodiments of compounds of the invention, the moiety R1-R2 (i.e. R1 and R2 taken together) represents 10-(tetrazol-5-yl)decyl, 11-(tetrazol-5-yl)undecyl, 12-(tetrazol- 5-yl)dodecyl, 13-(tetrazol-5-yl)tridecyl, 14-(tetrazol-5-yl)tetradecyl, 15-(tetrazol-5- yl)pentadecyl, 16-(tetrazol-5-yl)hexadecyl, 17-(tetrazol-5-yl)heptadecyl, 18-(tetrazol-5- yl)octadecyl or 19-(tetrazol-5-yl)nonadecyl, such as 13-(tetrazol-5-yl)tridecyl, 14-(tetrazol-5- yl)tetradecyl, 15-(tetrazol-5-yl)pentadecyl, 16-(tetrazol-5-yl)hexadecyl or 17-(tetrazol-5- yl)heptadecyl, e.g. 15-(tetrazol-5-yl)pentadecyl.
In one embodiment hereof R1-R2 represents 10-(tetrazol-5-yl)decyl, 11-(tetrazol-5- yl)undecyl, 12-(tetrazol-5-yl)dodecyl, 13-(tetrazol-5-yl)tridecyl, 14-(tetrazol-5-yl)tetradecyl, 15-(tetrazol-5-yl)pentadecyl, 16-(tetrazol-5-yl)hexadecyl, 17-(tetrazol-5-yl)heptadecyl; 18- (tetrazol-5-yl)octadecyl or 19-(tetrazol-5-yl)nonadecyl.
In one embodiment hereof R1-R2 represents 13-(tetrazol-5-yl)tridecyl, 14-(tetrazol-5- yl)tetradecyl, 15-(tetrazol-5-yl)pentadecyl, 16-(tetrazol-5-yl)hexadecyl or 17-(tetrazol-5- yl)heptadecyl.
In one embodiment hereof R1-R2 represents 15-(tetrazol-5-yl)pentadecyl. In one embodiment hereof R1-R2 represents 12-carboxydodecyl, 13-carboxytridecyl, 14-carboxytetradecyl, 15-carboxypentadecyl, 16-carboxyhexadecyl, 17-carboxyheptadecyl, 18-carboxyoctadecyl or 19-carboxynonadecyl.
In one embodiment hereof R1-R2 represents 14-carboxytetradecyl. In one embodiment hereof R1-R2 represents 16-carboxyhexadecyl.
In one embodiment hereof R3 is absent.
In one embodiment hereof R3 represents -NH-S(=O)2-(CH2)3-5-C(=O)-, GIu, D-GIu, Y-GIu, D-Y-GIu, Asp, D-Asp, β-Asp, D-β-Asp or Gly-γ-Glu.
In one embodiment hereof R3 represents -NH-S(=O)2-(CH2)3-C(=O)-. In one embodiment hereof R3 represents D-GIu, γ-Glu, β-Asp or Gly-γ-Glu.
In certain other embodiments of compounds of the invention, Z1 is absent, or Z1 represents GIy.
In further embodiments of compounds of the invention, Z2 represents Ser, Thr, GIn, GIy or His, such as Ser or Thr. In additional embodiments of compounds of the invention, Z3 represents GIn,
D-GIn, Asn, D-Asn, Ser or D-Ser.
In one embodiment hereof Z4 represents Ser, homoSer, GIn, Asn, Tyr, His, Arg, homoArg, Lys, Orn, Dab or Dap.
In one embodiment hereof Z4 represents Ser, His, Arg or Dap. In one embodiment hereof Z5 represents Ser, homoSer, Thr, Pro, Hyp, His, Lys,
Orn, Dab or Dap.
In one embodiment hereof Z5 represents Ser, His or Dap.
In one embodiment hereof Z6 represents Ala, VaI, Leu, lie, Met or NIe.
In one embodiment hereof Z6 represents NIe. In one embodiment hereof X2 represents Ser, Hyp, Cit, Dap, Asn, GIn or (4- thiazolyl)Ala.
In one embodiment hereof X2 represents Hyp, Dap, Cit or GIn.
In one embodiment hereof X2 represents Hyp.
In one embodiment hereof X1 is GIu, X3 is D-Phe, X4 is Trp and X5 is Lys. In one embodiment hereof X1 is Asp, X3 is D-Phe, X4 is Trp and X5 is Lys.
In one embodiment hereof R4 is NH2.
In one embodiment hereof R4 is OH.
In one embodiment, the fully protected peptide is a fully protected alpha- melanocyte-stimulating hormone or mimic or analogue thereof. In one embodiment, the fully protected peptide has the following structure
In one embodiment, the protected cyclic peptide has the following structure
In one embodiment, the process of the present invention comprises, (a) providing a fully protected peptide; wherein a first connectable portion of said fully protected peptide is protected with a removable first protecting group; wherein a second connectable portion of said fully protected peptide is protected with a removable second protecting group; wherein the side chain functional groups of said fully protected peptide are protected with one or more removable third protecting group(s); wherein said removable third protecting group(s) are removable under removing conditions that are (a) different to the removing conditions for the removal of the removable first protecting group and (b) different to the removing conditions for the removal of the removable second protecting group; wherein after removal of the first protecting group and after removal of the second protecting group, the first connectable portion of said fully protected peptide is capable of being linked to the second connectable portion of said fully protected peptide thereby to form a protected cyclic peptide; (b) removing the first protecting group and removing the second protecting group to form a selectively deprotected peptide comprising said first connectable portion and said second connectable portion;
(c) linking the first connectable portion of said selectively deprotected peptide to the second connectable portion of said selectively deprotected peptide thereby to form the protected cyclic peptide.
In one embodiment, the process according to the present invention further comprises, d) removing the one or more third removable leaving group(s) to form a cyclic peptide.
Thus, in one embodiment, the connectable portions of the ring to be closed comprise first and second connectable portions which are protected with a removable protecting group. In one embodiment, the first and second connectable portions comprise a side chain of an amino acid residue.
In one embodiment, the first and second connectable portions comprise an amino group and a carboxylic acid group respectively. In one embodiment, the first connectable portion comprises the side chain of a Lys residue and the second connectable portion comprises the side chain of a GIu residue.
In one embodiment, the first and second connectable portions comprise functional groups of amino acid side chains. In one embodiment, the first removable protecting group protects a functional group on an amino acid side chain of the first connectable portion of the fully protected peptide and the second removable protecting group protects a functional group on an amino acid side chain of the second connectable portion of the fully protected peptide.
As described above, the first and second connectable portions may be protected with any suitable removable protecting group. However, in one embodiment, the first and second connectable portions are protected with protecting groups which are removable by hydrogenation.
In one embodiment, the protecting groups of the first and second connectable portions are selected from benzyloxy, methylated benzyloxy, benzyloxycarbonyl and methylated benzyloxycarbonyl protecting groups as appropriate.
In one embodiment, the protecting groups of the first and second connectable portions are selected from benzyloxy and benzyloxycarbonyl protecting groups as appropriate.
In one embodiment, the fully protected peptide may be considered to be a first linear peptide. In this embodiment, the connectable portions of the ring to be closed may be considered to be first and second portions of said first linear peptide. Thus, in one embodiment, the process of the present invention comprises,
(a) providing a fully protected peptide; wherein a first portion of said first linear peptide is protected with a removable first leaving group; wherein a second portion of said first linear peptide is protected with a removable second leaving group; wherein the side chain functional groups of said first linear peptide are protected with one or more removable third leaving group(s); wherein said removable third leaving group(s) are removable under removing conditions that are (a) different to the removing conditions for the removal of the removable first leaving group and (b) different to the removing conditions for the removal of the removable second leaving group; wherein after removal of the first leaving group and after removal of the second leaving group, the first portion of said first linear peptide is capable of being linked to the second portion of said first linear peptide thereby to form an intermediate cyclic peptide;
(b) removing the first leaving group and removing the second leaving group to form a an intermediate cyclic peptide comprising said first portion and said second portion;
(c) linking the first portion of said intermediate cyclic peptide to the second portion of said intermediate cyclic peptide thereby to form the protected cyclic peptide; and
(d) removing the one or more third removable leaving group(s) to form a cyclic peptide.
In one embodiment, the one or more removable third leaving/ protecting groups are removed using a suitable deprotecting agent. A suitable deprotecting agent may be selected according to the leaving/ protecting groups which are to be removed. Further, the order in which one or more of the removable third leaving/ protecting groups are removed is not particularly limited and, as a result, the deprotecting agent is not particularly limited.
Accordingly, in one embodiment, a global protection strategy is used to remove one or more of the removable third leaving/ protecting groups. In one embodiment, the deprotecting agent used to remove the one or more removable third leaving/ protecting groups is different from the deprotecting agent used to remove the removable first and second leaving/ protecting groups.
Where the fully protected peptide is synthesised using a solid phase support, the deprotecting agent used to cleave the support from the peptide may be the same or different from the protecting agent used to remove the one or more of the third removable leaving/ protecting groups. In one embodiment, trifluoro acetic acid (TFA) is used to remove one or more of the removable third leaving/ protecting groups. In one embodiment, a composition comprising
TFA and one or more further agents is used to remove one or more of the removable third leaving/ protecting groups. In one embodiment, the composition of TFA and one or more further agents must comprise TFA in an amount of greater than 50%.
In one embodiment, the composition for removing the one or more third removable leaving/ protecting groups comprises TFA in an amount of greater than 50% and one or more scavengers based on the total volume of the composition. In one embodiment, the scavengers are selected from the group consisting of dimethoxybenzene (DMB), dithiothreitol (DTT), triisopropylsilane (TIPS) and water.
In one embodiment, the composition for removing the one or more third removable leaving/ protecting groups comprises TFA in an amount of greater than 50% based on the total volume of the composition, dichloromethane, dimethoxybenzene (DMB), dithiothreitol (DTT), triisopropylsilane (TIPS) and water. In one embodiment, the concentration of each scavenger should be in the range of from about 2.5 to 3% based on the total volume of the composition.
In one embodiment, the removal of the one or more removable third leaving/ protecting groups is conducted by contacting the composition as defined above with the protected cyclic peptide for about 2 hours at from about 200C to about 25°C. In one embodiment, the cyclic peptide produced as a result of deprotection of the protected cyclic peptide is isolated. In one embodiment, isolation is performed by any of precipitation, reverse phase chromatography or diafiltration.
In one embodiment, isolation is performed by precipitation. In one embodiment, the cyclic peptide is precipitated by transferring some or all of the cyclic peptide to a reactor containing TBME. In a specific embodiment, about half of the cyclic peptide is transferred to the reactor containing TBME, followed subsequently after a period of about 10 to 20 minutes by the remaining half of the cyclic peptide.
In a specific embodiment, isolation is performed by neutralization followed by RP-
HPLC. In one embodiment, the cyclic peptide is neutralized by transferring some or all of the cyclic peptide to a reactor containing a mixture of EtOH/Water and NH4Ac. In a specific embodiment, about the cyclic peptide is transferred to the reactor containing EtOH/Water
(1 :10), and NH4Ac and pH is adjusted to 2-3 before the mixture is purified on RP-HPLC.
Thus, as a result of the protected cyclic peptide being subjected to further deprotection and isolation, in one embodiment according to the process of the present invention, the protected cyclic peptide is further deprotected and isolated to form a cyclic peptide. The isolated cyclic peptide may also be purified by suitable means known to those in the art.
Thus, in one embodiment, the cyclic peptide is derived from the protected cyclic peptide.
In one embodiment, the cyclic peptide is,
In one embodiment, the cyclic peptide is selected from any one of the compounds FA to FBC:
16-(Tetrazol-5-yl)hexadecanoyl-Gly-Thr-Gln-His-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Tφ-Lys]- NH2
^-(Tetrazol-δ-yOhexadecanoyl-Gly-Thr-Gln-Dap-Ser-Nle-ctGlu-Hyp-D-Phe-Arg-Trp-Lys]- NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Thr-Gln-Dap-Ser- Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Ser-Nle- c[Glu-Dap-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Dap-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
^(i θ-CTetrazol-S-yOhexadecanoylsulfamoyObutanoyl-Gly-Ser-D-Gln-His-Dap-Nle-ctGlu- Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(2-{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]- ethoxy}acetyl-Nle-c[Glu-Dap-D-Phe-Arg-Trp-Lys]-NH2
(2-{2-[4-(16-(Tetrazol-5-yl)hexadecanoylsulfamoyl)butanoylamino]ethoxy}ethoxy)acetyl-His- Nle-c[Glu-Dap-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(2-{2-[2-(15-Carboxypentadecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}- acetyl-Pro-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(2-{2-[2-(15-Carboxypentadecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}- acetyl-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
the compound: the compound: the compound: the compound: the compound: the compound:
(2-{2-[2-(2-{2-[(R)-4-Carboxy-2-(16-(1 H-tetrazol-5-yl)hexadecanoylamino)butanoylamino]- ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl-Ser-Ser-Nle-c[Glu-Hyp-D-Phe-Arg-Trp- LyS]-NH2
the compound:
the compound:
{2-[2-(15-(Carboxy)pentadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Dap-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
the compound:
the compound:
the compound:
15-Carboxypentadecanoyl-Gly-Ser-Ser-Tyr-Thr-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(15-Carboxypentadecanoylamino)ethoxy]ethoxy}acetyl-Ser-Tyr-Hyp-Nle-c[Glu-Hyp-D- Phe-Arg-Trp-Lys]-NH2
{2-[2-(15-Carboxypentadecanoylamino)ethoxy]ethoxy}acetyl-Asn-Asn-Pro-Nle-c[Glu-Hyp- D-Phe-Arg-Trp-Lys]-NH2
(2-{2-[(R)-4-Carboxy-2-(16-(tetrazol-5-yl)hexadecanoylamino)butanoylamino]ethoxy}- ethoxy)acetyl-Gly-Ser-Gln-His-Dap-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(2-{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]- ethoxy}acetyl-Ser-Gln-His-Dap-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
(2-{2-[4-(16-(Tetrazol-5-yl)hexadecanoylsulfamoyl)butanoylamino]ethoxy}ethoxy)acetyl- Arg-Dap-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-Dap-Ser- Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
(2-{2-[4-(16-(Tetrazol-5-yl)hexadecanoylsulfamoyl)butanoylamino]ethoxy}ethoxy)acetyl-His- Dap-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(2-{2-[2-(16-(Tetrazol-5- yl)hexadecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl-Dap-Nle-c[Glu- Hyp-D-Phe-Arg-Trp-Lys]-NH2
(2-{2-[4-(16-(Tetrazol-5-yl)hexadecanoylsulfamoyl)butanoylamino]ethoxy}ethoxy)acetyl-Gly- Ser-Gln-His-Dap-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
^(i θ-CTetrazol-S-yOhexadecanoylsulfamoyObutanoyl-Gly-Ser-Gln-His-Dap-Nle-ctGlu-Hyp- D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-D-Ser-His-His- Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-OH
(2-[2-{(2-[2-{16-(Tetrazol-5-yl)hexadecanoylamino}ethoxy]ethoxy)acetylamino}ethoxy]- ethoxy)acetyl-Gly-Ser-Gln-His-Dap-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
(2-[2-{(2-[2-{(2-[2-{(2-[2-{16-(Tetrazol-5-yl)hexadecanoylamino}ethoxy]ethoxy)acetylamino}- ethoxy]ethoxy)acetylamino}ethoxy]ethoxy)acetylamino}ethoxy]ethoxy)acetyl-Gly-Ser-Gln- His-Dap-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
); {2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-His-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Ser-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
^(I S-CarboxypentadecanoylsulfamoyObutanoyl-Gly-Ser-Gln-His-Dap-Nle-ctGlu-Hyp-D- Phe-Arg-Trp-Lys]-NH2
(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butanoylamino]ethoxy}ethoxy)- acetyl-Gly-Ser-Gln-His-Dap-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
[2-(2-{(S)-4-Carboxy-4-[2-(17-carboxyheptadecanoylamino)acetylamino]butanoylamino}- ethoxy)ethoxy]acetyl-Gly-Ser-Gln-His-Dap-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
(2-{2-[(S)-3-Carboxy-3-(17-carboxyheptadecanoylamino)propanoylamino]ethoxy}ethoxy)- acetyl-Gly-Ser-Gln-His-Dap-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Thr-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Dab-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His- homoSer-Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Orn-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Lys-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Arg-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-2-PyAla- Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
{2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-4-PyAla- Nle-c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2
In one embodiment, the cyclic peptide of the present invention is subjected to a further lyophilisation step. In one embodiment, the cyclic peptide of the present invention is subjected to a further spray drying step.
In one embodiment according to the process of the present invention, the ring to be closed is an internal ring. An internal ring is a ring structure which is formed between either a side chain and a terminal group or a side chain and a side chain. Thus, an internal ring does encompass ring structures formed as a result of links between terminal groups, i.e. end to end linkages.
In one embodiment according to the process of the present invention, there is provided a process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; and ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide; wherein the fully protected peptide is not connected to a solid phase support or a solid phase linker during step i).
In one embodiment according to the process of the present invention, there is provided a process for performing an internal ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide; and wherein the connectable portions of the ring to be closed comprise at least an amino group and a carboxylic acid group respectively and wherein the said connectable portions are protected by a protecting group removable by hydrogenation, and wherein the fully protected peptide is not connected to a solid phase support or a solid phase linker during step i).
In one embodiment according to the process of the present invention, there is provided a process for performing an internal ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide; and iii) deprotecting the protected cyclic peptide, wherein the connectable portions of the ring to be closed comprise at least an amino group and a carboxylic acid group respectively and wherein the said connectable portions are protected by a protecting group removable by hydrogenation and wherein the fully protected peptide is not connected to a solid phase support or a solid phase linker during step i).
In one embodiment, the process of the present invention comprises,
(a) providing a fully protected peptide; wherein a first connectable portion of said fully protected peptide is protected with a removable first protecting group; wherein a second connectable portion of said fully protected peptide is protected with a removable second protecting group; wherein the side chain functional groups of said fully protected peptide are protected with one or more removable third protecting group(s); wherein said removable third protecting group(s) are removable under removing conditions that are (a) different to the removing conditions for the removal of the removable first protecting group and (b) different to the removing conditions for the removal of the removable second protecting group; wherein after removal of the first protecting group and after removal of the second protecting group, the first connectable portion of said fully protected peptide is capable of being linked to the second connectable portion of said fully protected peptide thereby to form a protected cyclic peptide;
(b) removing the first protecting group and removing the second protecting group to form a selectively deprotected peptide comprising said first connectable portion and said second connectable portion;
(c) linking the first connectable portion of said selectively deprotected peptide to the second connectable portion of said selectively deprotected peptide thereby to form the protected cyclic peptide, wherein the first and second connectable portions are connected to define an internal ring, wherein the step of removing the first and second protecting groups to form a selectively deprotected peptide is performed when the fully protected peptide is not connected to a solid phase support or a solid phase linker.
A general reaction scheme for the process of the present invention is outlined in Figure 1. The peptides and reagents present in the scheme in Figure 1 are not limiting and only serve to show one embodiment of the present invention. Peptide
According to a second aspect of the present invention, there is provided a protected cyclic peptide or a cyclic peptide produced by the process as herein described.
In one embodiment, the cyclic peptide may be lyophilised. In one embodiment, the cyclic peptide is suitable for the treatment of obesity.
Composition
According to a third aspect of the present invention, there is provided a pharmaceutical composition comprising the cyclic peptide as herein described. In one embodiment, the composition of the present invention is suitable for the treatment of obesity.
Applications
In a further aspect there is provided a cyclic peptide as described herein for use in the treatment of obesity.
In one embodiment, there is provided a pharmaceutical composition for use in the treatment of obesity.
In one embodiment, there is provided a cyclic peptide or pharmaceutical composition as herein described for use in the manufacture of a medicament of the treatment of obesity.
The cyclic peptide or composition as described herein may be administered by any suitable means and/or route. For example, they may be delivered by parenteral administration or any one of oral, nasal, pulmonary, or transdermal administration. In one embodiment, the compositions of the present invention are encapsulated.
The present invention also contemplates the following embodiments:
1. A process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide.
2. A process according to embodiment 1 comprising the steps of: (a) providing a fully protected peptide; wherein a first connectable portion of the fully protected peptide is protected with a removable first protecting group; wherein a second connectable portion of the fully protected peptide is protected with a removable second protecting group; wherein the side chain functional groups of said fully protected peptide are protected with one or more removable third protecting group(s); wherein said removable third protecting group(s) are removable under removing conditions that are (a) different to the removing conditions for the removal of the removable first protecting group and (b) different to the removing conditions for the removal of the removable second protecting group; wherein after removal of the first protecting group and after removal of the second protecting group, the first connectable portion of said fully protected peptide is capable of being linked to the second connectable portion of said fully protected peptide thereby to form a protected cyclic peptide;
(b) removing the first protecting group and removing the second protecting group to form a selectively deprotected peptide comprising said first connectable portion and said second connectable portion;
(c) connecting the first connectable portion of said selectively deprotected peptide to the second connectable portion of said selectively deprotected peptide thereby to form the protected cyclic peptide.
3. A process according to embodiments 1 or 2 wherein the protected cyclic peptide is further subjected to deprotection to form a cyclic peptide.
4. A process according to any one of embodiments 1 to 3 wherein the ring closure is an internal ring closure.
5. A process according to any one of embodiments 1 to 4 wherein the removal of the protecting groups from the connectable portions of the fully protected peptide is performed when the fully protected peptide is free from a solid phase support or a solid phase linker.
6. A process according to any one of embodiments 1 to 5 wherein the connectable portions comprise side chains of amino acids.
7. A process according to embodiment 6 wherein the amino acid side chains are those comprising at least an amine group and at least a carboxylic acid group. 8. A process according to embodiment 7 wherein the amino acid side chains are side chains corresponding to Lys and GIu.
9. A process according to any one of embodiments 1 to 8 wherein the protecting groups are selected from protecting groups suitable for the protection of functional groups of amino acid side chains.
10. A process according to embodiment 9 wherein the suitable protecting groups are selected from the group consisting of tert-butyloxycarbonyl, 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl, tert-butyl, trityl, benzyloxy, benzyloxycarbonyl and butyl.
11. A process according to embodiment 9, wherein the protecting groups suitable for removal are selected from the group consisting of but not limited to acid labile groups, Pd- labile groups and hydrazine labile groups.
12. A process according to embodiment 1 1 , wherein the acid labile groups are selected from the group consisting of Mtt and OPip groups.
13. A process according to embodiment 1 1 , wherein the Pd-labile groups are selected from the group consisting of Alloc and OAIyI.
14. A process according to embodiment 1 1 , wherein the hydrazine labile groups are selected from the group consisting of ODmab and Dde.
15. A process according to any one of embodiments 1 to 14 wherein the connectable portions of the fully protected peptides are protected with protecting groups removable by hydrogenation.
16. A process according to embodiment 1 1 wherein the hydrogenation is carried out using H2; Pd/C (10%).
17. A process according to any one of embodiments 1 to 16 wherein the protecting groups on the connectable portions are selected from benzyloxy and benzyloxycarbonyl. 18. A process according to any one of embodiments 1 to 17 wherein the protecting groups are selected from the group consisting of tert-butyloxycarbonyl, 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl, tert-butyl, trityl, benzyloxy, benzyloxycarbonyl and butyl, Mtt, OPip, Alloc, OAIyI, ODmab, Dde, benzyloxy, benzyloxycarbonyl
19. A process according to any one of embodiments 1 to 17 wherein the fully protected peptide is formed by solid phase peptide synthesis.
20. A process according to embodiment 2 and any embodiment dependent thereon wherein in step b) as defined in claim 2, the first protecting group is removed before removal of the second protecting group.
21. A process according to any one of embodiments 2 to 20 wherein the removable third protecting group(s) are the same.
22. A process according to any one of embodiments 1 to 21 wherein the fully protected peptide is a mimic or analogue of an alpha-melanocyte-stimulating hormone.
23. A process according to any one of embodiments 1 to 22 wherein the fully protected peptide is
24. A process according to any one of embodiments 1 to 23 wherein the protected cyclic peptide is
25. A process according to any one of embodiments 3 to 24 wherein the cyclic peptide is {2-[2-(16-(Tetrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Dap-Nle- c[Glu-Hyp-D-Phe-Arg-Trp-Lys]-NH2.
26. A process according to embodiment 25 wherein the cyclic peptide is lyophilised.
27. A process according to any one of embodiments 3 to 26 wherein said cyclic peptide is for use in medicine.
28. A process according to any one of embodiments 3 to 27 wherein said cyclic peptide is for use in the treatment of obesity.
29. A process according to any one of embodiments 3 to 28 wherein said cyclic peptide is for use in the preparation of a medicament for the treatment of obesity.
30. A protected cyclic peptide produced by the process of any one of embodiments 1 to 29.
31. A cyclic peptide produced by the process of any one of embodiments 3 to 29.
32. A pharmaceutical composition comprising the cyclic peptide defined in embodiment 31 and optionally one or more pharmaceutically acceptable excipients, adjuvants, diluents or carriers.
33. A cyclic peptide or pharmaceutical composition as defined in embodiment 31 or 32 for use in the treatment of obesity. 34. A method of treating diabetes in a subject in need thereof comprising administering to the subject an effective amount of the peptide defined in embodiment 31 or the composition defined in embodiment 32.
35. A process comprising:
(i) preparing a cyclic peptide by the process according to any one of embodiments 3 to 29; and
(ii) making a medicament using the cyclic peptide.
36. A process according to embodiment 35 wherein said medicament is for the treatment of obesity.
37. A process as substantially defined herein with reference to the examples.
38. A protected cyclic peptide as substantially defined herein with reference to the examples.
39. A cyclic peptide as substantially defined herein with reference to the examples.
40. A method of treatment as substantially defined herein with reference to the examples.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 (A,B) shows a general reaction scheme for the process of the present invention. The peptides and reagents present in the scheme in Figure 1 are not limiting and only serve to show one embodiment of the present invention.
Figure 2 shows a general flowchart for the synthesis of cyclic peptide according to the process of the present invention. Examples
The invention will now be described with reference to the following non-limiting examples.
1.1 General peptide coupling cycle 1.1.1 Solid phase Peptide coupling
The Fmoc protected Amino Acid (Fmoc-AA-OH, 2 eq) was dissolved in N- methylpyyrolidone (NMP) (5-10 volumes relative to the resin). N-hydroxybenzotriazol (HOBt) (2 eq) and N, N'-diisopropylcarbodiimide (DIC) (2 eq) is added and the mixture is stirred for 15 minutes before it is added to the drained resin. The reaction mixture is stirred 0.5 h and DIPEA (0.5 eq) is added. After 3-6h a small in process control (IPC) is taken out from the reaction mixture. The small amount of resin is drained and thoroughly washed with NMP and ethanol before it is analyzed by the colorimetric Kaiser test. If the Kaiser test is negative, the next step is the washing procedure. If the Kaiser test is positive, the coupling is not complete and DIC (0.5 eq) is added in order to form additional active ester. After 3h, a new IPC is taken out and checked by the Kaiser test. If it is positive, double coupling is necessary.
1.1.2 Wash of resin
After the coupling is completed the resin is washed with NMP (6 x 2min x 5-10 volumes relative to the resin) to remove excess amino acid, impurities and coupling reagents.
1.1.3 Fmoc deprotection
The Fmoc protecting group on the N-terminal AA is removed by treatment with piperidine. The resin is treated with a solution of piperidine diluted with NMP (20% Piperidine, 2 x 10 minutes x 5-10 volumes relative to the resin). After deprotection, the resin is washed again according to 1.1.2 and subsequently the peptide is ready for a new peptide coupling cycle.
1.2 Cleavage from resin
A=Fmoc protected Sieber resin weighed of for production. The resin is washed with DCM (3 x 2min x 10A volumes), with MeOH (3 x 2min x 10A volumes) and with DCM (3 x 2min x 10A volumes). A mixture of TFA diluted in DCM is prepared (2% TFA/ 1% TIPS/ DCM) and the resin is treated with the mixture (4 x 30 minutes x 10A volumes). Concentration of peptide in the cleavage filtrate should be tested by HPLC and if the last filtrate contains significant amounts of peptide the cleavage should be repeated until limited peptide is present in the cleavage filtrate. Immediately after cleavage, pH in the filtrate is adjusted to 8-9 using DIPEA and concentrated to 2OA volumes. Water (8A volumes) is added to the DCM mixture and pH in the water phase is adjusted to 8-9 using DIPEA before the phases are separated. Subsequently the organic phase is washed with additionally water (8A volumes) and the organic phase is concentrated (4A volumes). Ethanol (14A volumes) is added and the mixture is concentrated again (4A volumes) before it is co-evaporated once again with EtOH (14A volumes to 4A volumes). The resulting yellow oil is used in the next step without further purification.
1.2.1 HPLC Method Column: Jupiter 4μ Proteonomics 90
Method: Eluent A: 50% MeCN, 0.1 % H3PO4
Eluent B: 90% MeCN, 0,1 % H3PO4
Time Eluent A Eluent B Flow
0 min 100 % 0 % 1 mL/min
5 min 80 % 20 % 1 mL/min
20 min 40 % 60 % 1 mL/min
25 min 0 % 100 % 1 mL/min
30 min 0 % 100 % 1 mL/min
31 min 100 % 0 % 1 mL/min
35 min 100 % 0 % 1 mL/min
Chromatogram:
Products elutes at 25.5 (Product -Trt) and 28 minutes (Product).
1.3 Lys and GIu deprotection 1.3.1 Procedure B=Theoretical weight of protected peptide cleaved from resin.
In order to perform ring closure between side chains on GIu and Lys, the Z protecting group on Lys and the BzI protecting group on GIu has to be removed.
The yellow oil isolated after cleavage is dissolved in absolute ethanol (10B volumes) before the mixture is transferred to the hydrogenation equipment. Pd/C (20% water, 0.5B grams) is added. After flushing with nitrogen, the mixture is placed under hydrogen atmosphere (2-3 bar) and the mixture is stirred at 500C for 14 h. Removal of protecting groups should be investigated by HPLC at this stage. If IPC fragments of the protecting groups are still present Pd/C (0.1 B grams) should be added. If full deprotection has occurred, the mixture is filtrated and the filter cake is washed with absolute ethanol (5B volumes). The filtrate is concentrated to oil (maximum 2B volumes) and used in the next step without any further purification.
1.3.2 HPLC method
Column: Jupiter 4μ Proteonomics 90
Method: Eluent A: 50% MeCN, 0.1 % H3PO4 Eluent B: 90% MeCN, 0,1 % H3PO4
Time Eluent A Eluent B Flow
0 min 100 % 0 % 1 mL/min
5 min 80 % 20 % 1 mL/min
20 min 40 % 60 % 1 mL/min
25 min 0 % 100 % 1 mL/min
30 min 0 % 100 % 1 mL/min
31 min 100 % 0 % 1 mL/min
35 min 100 % 0 % 1 mL/min
Chromatogram:
Reactants elutes at 25.5 (Reactant -Trt) and 28 minutes (Reactant).
Products elutes at 11.9 (Product-Trt) and 16.8 (Product).
Toluene 13.6
Product + Z elututes at 26.7
Product + BzI elutes at 20.1
1.4 Ring closure 1.4.1 Procedure C=Theoretical weight of partially protected peptide
The oil separated in the previous step is diluted with THF (to a total volume of 50C) and water (1 C volume). DIPEA (0.75 eq compared to theoretical peptide) is added and the mixture is stirred for 5 minutes before PyBOB (0.5 eq compared to theoretical peptide) is added. Addition of PyBOB and DIPEA is repeated with 30 minutes intermission (3 x 0.5 eq PyBOB, 1.5 eq overall) and the mixture is stirred 2 h overall. IPC should be analyzed by HPLC in order to confirm full amide formation. If ring closure is incomplete additionally DIPEA and PyBOB is added. The mixture is concentrated (maximum 3C volumes) and dissolved in DCM (15C volumes). The organic phase is washed with Water (2x1 OC volumes; pH in the organic phase measured to 6-7; the separation should be allowed to separate completely over 1-4 h) before the organic phase is concentrated (2-4C volumes). The oil is used in the global deprotection step without any further purification.
1.4.2 HPLC method
Column: Jupiter 4μ Proteonomics 90
Method: Eluent A: 50% MeCN, 0.1 % H3PO4
Eluent B: 90% MeCN, 0,1 % H3PO4
Time Eluent A Eluent B Flow
0 min 100 % 0 % 1 mL/min
5 min 80 % 20 % 1 mL/min
20 min 40 % 60 % 1 mL/min
25 min 0 % 100 % 1 mL/min
30 min 0 % 100 % 1 mL/min
31 min 100 % 0 % 1 mL/min
35 min 100 % 0 % 1 mL/min
Chromatogram:
Reactants elutes at 11.9 (Reactant -Trt) and 16.8 minutes (Reactant). Products elutes at 19.5 (Product-Trt) and 25.23 (Product).
1.5 Global deprotection 1.5.1 Procedure I
D=Theoretical weight of protected ring closed peptide.
The peptide should be cleaved in a mixture of TFA (14D volumes; 62%), DCM (6D -
Volume of concentrated peptide from 7.4; 26%), and scavengers Dimethoxy benzene
(DMB) (0.63D volumes; 2.8 %), DTT (0.63D volumes; 2.8 %), water (0.63D volumes; 2.8
%), and triisopropylsilane (TIPS) (0.63D volumes; 2.8 %).
The deprotection mixture is premixed and the protected peptide fragment is dissolved and stirred in the mixture at 20-250C for 2h. The volume of the mixture is reduced (to 14 volumes) over 1 h-2h under reduced pressure. Half of the cleavage mixture is transferred to a reactor containing TBME (5OD volumes, 35°C-40°C) causing precipitation to occur and after 15 minutes the rest of the cleavage mixture is transferred. The mixture is stirred for
30 min and filtrated under nitrogen atmosphere at 300C- 35°C (the filter cake has a tendency to crack). When the solvent reaches the top of the filter cake, the solid is washed with TBME (3x5D volumes).
1.5.2 Procedure Il D=Theoretical weight of protected ring closed peptide.
The peptide should be cleaved in a mixture of TFA (15D volumes; 91%) and scavengers: DTT (0.5 D volumes; 3 %), water (0.5 D volumes; 3 %), and TIPS (0.5 D volumes; 3 %). The protected peptide fragment dissolved in DCM (4D volumes) was added to the cleavage mixture over 5-10 min and stirred in the mixture at 20-250C for 2.5 h. A neutralization mixture is prepared: Ammonium acetate (1.0 molar equivalent compared to TFA) in water (45 D volumes) and ethanol (10 D volumes compared to TFA). The neutralization mixture is cooled to 15°C and the cleavage mixture was slowly added (Tmax=25°C) under constant stirring. After complete addition pH is adjusted using NH4OAc or TFA to 3-3.5 and the mixture is concentrated (15D volume is removed). Consequently the mixture is cooled to 10-150C for 30 minutes and filtrated. The filtrate is collected and transferred to the purification facility.
1.5.3 HPLC Method
Column: Vydac Protein and peptide, C18
Method: Eluent A: 10% MeCN, 0.1 % H3PO4
Eluent B: 90% MeCN, 0,1 % H3PO4
Time Eluent A Eluent B Flow
0 min 100 % 0 % 1 mL/min
30 min 0 % 100 % 1 mL/min
35 min 0 % 100 % 1 mL/min
36 min 100 % 0 % 1 mL/min
45 min 100 % 0 % 1 mL/min
Chromatogram:
Product elutes at 13.1 ; Product + tButyl elutes at 17.4
2.1 Formation of the starting material 16-(Tetrazol-5-yl) hexadecaneoic acid
The synthetic procedure of the starting material 16-(Tetrazol-5-yl) hexadecaneoic acid is summarized below. Synthesis of 16-(Tetrazol-5-yl) hexadecaneoic acid.
An overview of the process of the present invention is given in Table 1 (Figure 2).
All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law). All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.
The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents. This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

Claims

1. A process for performing a ring closure on a fully protected peptide to form a protected cyclic peptide comprising the steps of: i) selectively deprotecting the connectable portions of the ring to be closed; ii) connecting the deprotected connectable portions of step i) to form the protected cyclic peptide, wherein the removal of the protecting groups from the connectable portions of the fully protected peptide is performed when the fully protected peptide is free from a solid phase support or a solid phase linker.
2. A process according to claim 1 comprising the steps of:
(a) providing a fully protected peptide; wherein a first connectable portion of the fully protected peptide is protected with a removable first protecting group; wherein a second connectable portion of the fully protected peptide is protected with a removable second protecting group; wherein the side chain functional groups of said fully protected peptide are protected with one or more removable third protecting group(s); wherein said removable third protecting group(s) are removable under removing conditions that are (a) different to the removing conditions for the removal of the removable first protecting group and (b) different to the removing conditions for the removal of the removable second protecting group; wherein after removal of the first protecting group and after removal of the second protecting group, the first connectable portion of said fully protected peptide is capable of being linked to the second connectable portion of said fully protected peptide thereby to form a protected cyclic peptide;
(b) removing the first protecting group and removing the second protecting group to form a selectively deprotected peptide comprising said first connectable portion and said second connectable portion; (c) connecting the first connectable portion of said selectively deprotected peptide to the second connectable portion of said selectively deprotected peptide thereby to form the protected cyclic peptide.
3. A process according to claims 1 or 2 wherein the protected cyclic peptide is further subjected to deprotection to form a cyclic peptide.
4. A process according to any one of claims 1 to 3 wherein the ring closure is an internal ring closure.
5. A process according to any one of claims 1 to 4, wherein during cyclisation the performing of ring closure does not require attachment to the support resin.
6. A process according to any one of claims 1 to 5 wherein the connectable portions comprise side chains of amino acids.
7. A process according to any one of embodiments 1 to 6 wherein the fully protected peptide is a mimic or analogue of an alpha-melanocyte-stimulating hormone.
8. A process according to any one of claims 1 to 7 wherein the cyclic peptide is {2-[2-(16- (T etrazol-5-yl)hexadecanoylamino)ethoxy]ethoxy}acetyl-Gly-Ser-Gln-His-Da p-Nle-c[Glu- Hyp-D-Phe-Arg-Trp-Lys]-NH2.
9. A process according to any one of embodiments 1 to 8 wherein the protecting groups on the connectable portions are selected from benzyloxy and benzyloxycarbonyl.
10. A protected cyclic peptide produced by the process of any one of claims 1 to 9.
11. A cyclic peptide produced by the process of any one of claims 1 to 10.
12. A pharmaceutical composition comprising the cyclic peptide defined in claim 1 1 and optionally one or more pharmaceutically acceptable excipients, adjuvants, diluents or carriers.
13. A cyclic peptide or pharmaceutical composition as defined in claim 1 1 or 12 for use in the treatment of obesity.
14. A method of treating diabetes in a subject in need thereof comprising administering to the subject an effective amount of the peptide defined in claim 1 1 or the composition defined in claim 12.
15. A process comprising: (i) preparing a cyclic peptide by the process according to any one of claims 1 to 9; and (ii) making a medicament using the cyclic peptide.
EP09749882A 2008-05-22 2009-05-20 Process Withdrawn EP2283037A1 (en)

Priority Applications (1)

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EP09749882A EP2283037A1 (en) 2008-05-22 2009-05-20 Process

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US20030125243A1 (en) * 2000-07-20 2003-07-03 Jun Liu Synthesis of cyclic peptides
CA2523408A1 (en) * 2003-05-09 2004-11-18 Novo Nordisk A\S Peptides for use in treating obesity
ES2564167T3 (en) * 2004-07-08 2016-03-18 Novo Nordisk A/S Conjugates of long-acting polypeptides containing a tetrazole fraction
WO2008087190A2 (en) * 2007-01-18 2008-07-24 Novo Nordisk A/S Use of peptides in combination with surgical intervention for the treatment of obesity

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