WO1997007129A1 - Solution synthesis of peripheral acting analgesic opioid tetrapeptides - Google Patents

Solution synthesis of peripheral acting analgesic opioid tetrapeptides Download PDF

Info

Publication number
WO1997007129A1
WO1997007129A1 PCT/CA1996/000552 CA9600552W WO9707129A1 WO 1997007129 A1 WO1997007129 A1 WO 1997007129A1 CA 9600552 W CA9600552 W CA 9600552W WO 9707129 A1 WO9707129 A1 WO 9707129A1
Authority
WO
WIPO (PCT)
Prior art keywords
phe
process according
activating agent
tyr
group
Prior art date
Application number
PCT/CA1996/000552
Other languages
French (fr)
Inventor
Nicholas Rinaldi
Original Assignee
Biochem Pharma Inc.
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 Biochem Pharma Inc. filed Critical Biochem Pharma Inc.
Priority to AU66539/96A priority Critical patent/AU6653996A/en
Publication of WO1997007129A1 publication Critical patent/WO1997007129A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • 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 objective compound is a family of peripherally acting opioid tetrapeptides of the formula (I) Tyr-(D)R ⁇ -R 2 - R 3 -NH 2 , where R- is Ala or Arg ; R 2 is Phe or Phe(p-F); and ;R 3 is Phe or Phe(p-F) (TAPP-like peptides) having valuable pharmacological properties which enable them to be used therapeutically, in particular in me therapy and management of pain.
  • the analgesic properties of the TAPP-like peptides are manifest through the ⁇ opioid receptor subtype. Moreover, the pharmacological profile of these tetrapeptides are unique and differ markedly from standard opiates because they activate peripheral nociceptors and do not affect central nervous system centers. Unlike conventional opiates, the compounds described herein exhibit markedly attenuated side effects.
  • peptides are synthesized on a solid support or beads, which are polystyrene based, through sequential incorporation of fully-protected amino acids via a dehydrating agent as described by Stewart and Young (J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, Illinois, 1984).
  • This process is viable for research scale samples but not for pilot or commercial scale.
  • Such a process results in a yield that is relatively poor and is especially difficult to exploit industrially on account of the use of protecting agents (blocking/deblocking cycles) and of me requirement of many purification stages involving HPLC, stereochemical vulnerabiliy of the chiral centers during the synthesis and typical harsh conditions required to remove the peptide from the resin.
  • Conventional processes are expensive and require large excess of reagents and amino acids.
  • the use of a solid support (resin) limits the use of manual manufacturing equipment.
  • peripherally acting opioid tetrapeptides corresponding to formula (I): Tyr-(D)R--R 2 -R 3 -NH2 , where R- is Ala or Arg ; R 2 is Phe or Phe(p-F); and ;R 3 is Phe or Phe(p-F) and the lack of a commercial scale (i.e., yields on the order of kilogram quantities) process enabling it to be obtained with a satisfactory degree of purity, a good yield and, if possible, from fairly inexpensive commercially available starting materials and procedures, more detailed research has been carried out and has led to the discovery of a new process for their preparation.
  • a process for producing multikilo quantities of those TAPP-like peptides which overcomes the problems outlined above.
  • the process of the present invention provides a solution phase peptide synthesis process in which the yield of D-isomer of Rj is improved over the conventional processes using solid phase (eg. resin based oligopeptide building) and solution phase (employing costly blocking and deblocking agents) to increase efficiency and cost effectiveness for large scale production.
  • solid phase eg. resin based oligopeptide building
  • solution phase employing costly blocking and deblocking agents
  • the present invention provides for a synthesis of opioid analgesic tetrapeptides having the general structure represented by Formula I:
  • Ri is Ala, or Arg;
  • R 2 is Phe or Phe(p-F); and
  • R 3 is Phe or Phe(p-F); which comprises a coupling step comprising couphng (P-Tyr-(D)R ⁇ ), wherein P is an amino protecting group; with (R 2 -R3-NH 2 ) using 3-hy droxy- 1,2,3 benzotriazin- 4(3H)one (HODbht) as an activating agent, a neutralizing agent selected from the group consisting of diisopropylethylamine (DIEA) and n-ethylmorpholine (NEM); in a solvent suitable for a coupling reaction to yield to a protected intermediate of formula (II): P-Tyr-(D)R ⁇ -R 2 -R 3 -NH 2
  • DIEA diisopropylethylamine
  • NEM n-ethylmorpholine
  • the Applicant has succeeded, not without surprise, to perfect processes that minimize tlie investment costs and/or energy consumptions, and maximize the yields by adopting the most appropriate operating conditions for pre-pilot scale leading to commercial scale production.
  • the invention has also yielded an efficient process that is conserved from a microscale process to a pilot or commercial scale preparation of the desired tetrapeptide.
  • the process allows for almost racemization-free product (typically ⁇ 0.5%) on scales up to the multikilogram level.
  • the present process also removes the need for polystyrene resin as a solid phase support which is expensive on a manufacturing scale.
  • the process further circumvents the need for strong acids such as hydrofluoric acid to effect removal of side chain protecting groups.
  • the process of the present invention has important advantages, expecially as regards quality, commercial scale implementation and reproducibility.
  • the compounds of the present invention can be synthesized using conventional preparative steps and recovery methods known to those skilled in the art of organic and bio-organic synthesis, while providing new and unique combinations for the overall synthesis of each compound. Preferred synthetic routes for intermediates involved in the synthesis as well as the resulting compounds of the present invention follow.
  • the final product of this process is very similar to that obtained by costly procedures (ie. due to expensive purification steps), but it has two great advantages in that no costly protective blocking/deblocking steps are involved and only one or less purification steps are required entailing costly, time consuming chromatographic procedures.
  • the kilogram to pre-pilot scale application of this process is accordingly advantageous.
  • amino protecting group is well known in the art of peptide chemistry.
  • suitable amino protecting group can be found in Protective Groups In Organic Svnthesis. Greene T.W. and Wuts P.G., 1991, John Wiley & Sons, Inc.
  • Figure 1 shows the HPLC chromatogram of Tyr-(D)Arg-Phe-Phe-NH 2 • 2HC1 crystallized from methanol.
  • Figure 2 shows the HPLC chromatogram demonstrating separation of all the stereoisomers of the tetrapeptide Tyr-Arg-Phe-Phe.
  • Figure 3 shows chiral analysis by way of HPLC chromatography to separate the amino acids corresponding to formula (I): Tyr-(D)Arg-Phe-Phe-NH 2 following acid hydrolysis as described in Example 8. DETAILED DESCRIPTION OF THE INVENTION
  • Ri is preferably Ala, or Arg. Ri is more preferably Arg.
  • R 2 is preferably Phe or Phe(p-F).
  • R 2 is more preferably Phe.
  • R 2 is more preferably Phe(p-F).
  • R 3 is preferably Phe or Phe(p-F). R 3 is more preferably Phe. In an alternative embodiment, R 3 is more preferably Phe(p-F).
  • P is preferably selected from the group consisting of Boc (t-butyloxycarbonyl), Z (benzyloxycarbonyl), Fmoc (9-fluoromethyloxycarbonyl), Bz (benzoyl), and 3,5- dihydroxy benzoyl. P is more preferably Boc.
  • the neutrahzmg agent is preferably DIEA (diisopropylethylamine).
  • the solvent suitable for the coupling reaction is preferably dimethylformamide (DMF).
  • the process of this invention can be carried using a further activating agent selected from the group consisting of dicyclohexylcarbodiimide (DCC); dimethylaminopropylethylcarbodiimide (EDC); and benzotriazol-l-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP).
  • DCC dicyclohexylcarbodiimide
  • EDC dimethylaminopropylethylcarbodiimide
  • BOP benzotriazol-l-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate
  • Boc-Tyr-OH is activated by esterifying the carboxylate group of Boc-Tyr-OH in nonpolar solvent such as THF. Formation of the activated succinimate ester of Boc-Tyr-OH is accomplished by dissolving Boc-tyrosine in an appropriate solvent such as THF and adding n-hydroxysuccinimide, also dissolved in the same solvent. A solution containing a dehydrating agent solution such as dicyclohexylcarbodiimide is added to effect esterification.
  • a dehydrating agent solution such as dicyclohexylcarbodiimide
  • Coupling of the (D) amino acid by activated Boc-Tyrosine-succinimide ester is accomplished by dissolution of the respective components in specified solvents (THF or H 2 O) and mixing the two solutions at room temperature, wherein the dipeptide is formed.
  • Condensation of the two dipeptidyl units D and E in a specific manner that causes mimmal racemization ( ⁇ 4 %) at the ⁇ -stereogenic center corresponding to the (D)- R* residue is accomplished by following stringent conditions including, choice of solvent, pH, neutralizing agent, choice of activating agent and rate of addition of specific activating agent solution.
  • the activating agent(s) is HODbht
  • the neutralizing agent is DIEA
  • the solvent is DMF.
  • the first activating agent is HOBhbt
  • the second activating agent can be either DCC, EPC, BOP, or an equivalent activating agent
  • the neutralizing agent can be either DIEA, or NEM
  • the solvent is DMF.
  • N terminal t-butyl carbamate protecting group is removed by acid hydrolysis in a mixture of methylene chloride and trifluoroacetic acid, followed by evaporation of the solvents.
  • CrystalUzation of the pure (> 97%) product by preferred formation of the pharmaceutically acceptible salt, such as the hydrochloride salt, is accompUshed by stirring a specified concentration of compound G in a specified solvent, such as DMF.
  • the process consists of initially preparing independently two dipeptide fragments using minimal side chain protection. Subsequently, the two fragments are joined by reacting them in a suitable solvent. It is well known to those skilled in the art that amino acids are prone to racemization. It is also known that the potential for racemization is increased in the case where an amino acid is acylated as is the case where fragment D is joined to fragment E in Scheme I. In the case cited herein racemization was observed to be as high as 44% depending on reaction conditions.
  • a solution of n-hydroxysuccinimide (343.7 g, 2.986 moles) in THF (2L) is added to an amino acid solution comprising the blocked amino acid , Boc-Tyr-OH (700 g, 2.488 moles), dissolved in THF (4.7L).
  • a solution of dicyclohexylcarbodiimide (616.1 h, 2.986 moles) in THF is added at a rate of 200 mlJminute.
  • the resulting mixture is sti ⁇ ed at room temperature for 16 - 20 hours, after which precipitated DCU is filtered off and the solution evaporated to dryness.
  • the solid material is washed on a filter with isopropanol (3 x 1 L) and ethyl ether (3 x 1 L), and the product obtained and dried under high vacuum to yield 921 grams (97.8% yield).
  • the amino acid H-(D)Arg-OH (447.69 g, 2.57 moles) is dissolved in 6.7 L H 2 O and added while stirring at room temperature to a solution of Boc-Tyr-Osu (447.69 g, 2.57 moles), previously prepared by dissolution in 1.52 L THF. After stirring the reaction mixture for 16 - 20 hours, the THF is evaporated. The product is dissolved (1.98 moles, theoretical) in a total volume equaling approximately 10L H 2 O, filtered through two Whatman fibreglass filters and one Whatman #1 paper, after which the solution is injected onto an HPLC column at a rate of 300 ml/min. The product is purified following the gradient conditions described in Table I.
  • Analytical HPLC is used to monitor the eluant, which is collected in 3 - 4 L fractions. Fractions demonstrating an HPLC purity greater than 97% by this method are combined, the acetonitrile evaporated and product lyophilized or evaporated (yield: 668.57g, 77.18% ).
  • the dipeptide, Boc-Tyr-(D)Arg-OH (760 g, 1.737 moles) is added to 15 L of a solution of CHC1 3 , H O, Methanol 1:1:2 and dissolved by sonication and mixing at 30-37 °C in a waterbath. After dissolution, 3L of DMF is added and the solution concentration by evaporation to 2.5 L. , afterwhich 4 A molecular sieves (650 mL) are added, the solution gently swirled and allowed to stand 16 - 20 hours. The solution is decanted and sieves washed with 1.9 L DMF.
  • the second dipeptide, H-Phe-Phe-NH 2 • HCl (604.18 g, 1.737 moles) is dissolved in 4.8 L DMF and one equivalent of DIEA (1.737 moles) is added.
  • the two peptide solutions are combined and HODhbt (312 g, 1.911 moles) is added, afterwhich the pH is adjusted to 7.0 - 8.0 with 250 ml DIEA, and a solution of DCC (430 g, 1.084 moles) in IL DMF is added and the reaction allowed to stir 16 - 20 hours (Note: after 30minutes of mixing, the pH is adjusted to 7.0 - 8.0 with DIEA if necessary).
  • the mixture is filtered to remove DCU, which is washed on the filter 2 x 400 mL DMF, and evaporated at 40 °C under high vacuum.
  • the almost dry mixture is dissolved in 2 L acetone and the solution aUowed to stand for 1 hour at room temperature.
  • the precipitate is filtered and washed 3 x 500 mL acetone afterwhich the total volume of the solution is adjusted by adding or evaporating acetone to a total volume of 5.1 L.
  • the Boc- terminally protected peptide (1317 g) is dissolved in a solution consisting of 55% TFA, 45% CH 2 C1 2 in a ratio equivalent to 4 mL TFA solution/gram tetrapeptide, and the solution sti ⁇ ed at room temperature for 45 minutes.
  • the reaction mixture is evaporated on a rotary evaporator at room temperature. Following the evaporation of CH C1 2 with a water aspirator pump, the remaining TFA is evaporated under high vacuum.
  • a portion of the thick residue obtained is precipitated by pouring it into 4 L of ethyl ether with stirring until stirring becomes difficult.
  • the precipitate is filtered off and washed on the filter with 2 L ethyl ether.
  • the latter precipitation step is repeated until the total amount of thick residue is precipitated.
  • the precipitates are combined and dried overnight by connecting the dessicator to an aspirator pump, foUowed by drying under high vacuum for 4 hours. (Yield: 1322.7 g)
  • the peptide (831.1 g) is dissolved in 3324 mL MeOH (ratio 4 ml MeOH/g peptide) in a water bath at 50 °C and filtered over a fritted filter. After washing the filter with 100 mL MeOH the peptide solution is transferred to a 20 L evaporating round bottom flask and gently agitated (40 RPM) at 50 °C. Small increments of acetonitrile (ratio 4.8 mL AcN/g product) are added and agitation and heat are ceased once crystals begin to form. After allowing the mixture to reach room temperature (approximately 3 hr), the flask is cooled at 4 °C for 1 hour.
  • the crystalline material is filtered on a fritted funnel and washed twice with 1.5 L of a cold solution of MeOH/AcN (1:1.2), afterwhich the crystals are dried in a dessicator connected to an aspirator pump for 2 hours and the mother liquor is evaporated and recrystaUized by repeating this entire procedure from the point of dissolution in MeOH.
  • the drying process is completed under high vacuum for a minimum of 16 hours yielding combined product totalling 707.7 g.

Abstract

This invention provides a bulk scale process for the solution synthesis of enantiomerically pure peripherally acting analgesic opioid tetrapeptides corresponding to formula (I): Tyr-(D)R1-R2-R3-NH2, where R1 is Ala or Arg; R2 is Phe or Phe(p-F); and R3 is Phe or Phe(p-F). A new and unique multi-step process is disclosed comprising the joining of two dipeptides using standard solution phase synthesis techniques, but adjusting the individual factors (e.g., solvents, activating agents, neutralizing agents etc.), to minimize racemization of the second amino acid. Tremendous cost efficiencies are achieved due to elimination of traditional sequential blocking-deblocking cycles and multiple chromatographic purification steps, which also enables these simple kilogram quantity methods to be scaled up to commercial production.

Description

Solution synthesis of peripheral acting analgesic opioid tetrapeptides
BACKGROUND OF THE INVENTION
This invention provides a method for preparing the objective compounds advantageously using a simple procedure that provides high overall yield, clinical grade purity and that uses starting material which is readily available on an industrial scale, and in a cost effective manner. The objective compound is a family of peripherally acting opioid tetrapeptides of the formula (I) Tyr-(D)Rι-R2- R3-NH2, where R- is Ala or Arg ; R2 is Phe or Phe(p-F); and ;R3 is Phe or Phe(p-F) (TAPP-like peptides) having valuable pharmacological properties which enable them to be used therapeutically, in particular in me therapy and management of pain. The analgesic properties of the TAPP-like peptides are manifest through the μ opioid receptor subtype. Moreover, the pharmacological profile of these tetrapeptides are unique and differ markedly from standard opiates because they activate peripheral nociceptors and do not affect central nervous system centers. Unlike conventional opiates, the compounds described herein exhibit markedly attenuated side effects.
Several methods are already known for the preparation of peptides, which can be synthesized by three general processes: solid phase (eg. using resins), solution phase with full orthogonal protecting groups and requiring blocking/deblocking steps, and fragment condensation utilizing minimum protecting groups. The latter is particularly frought with difficulties of solubility, low yielding coupling and high degree of racemization. The individual steps of peptide synthesis already described in the literature, however, are not particularized to this specific tetrapeptide, especially the L-D-L-L isomer thereof, and do not enable this tetrapeptide to be obtained with a satisfactory degree of purity or in a commercially acceptable yield. Furthermore, certain stages of those processes pose problems from the industrial standpoint, particularly the purification steps. Typically, peptides are synthesized on a solid support or beads, which are polystyrene based, through sequential incorporation of fully-protected amino acids via a dehydrating agent as described by Stewart and Young (J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford, Illinois, 1984). This process is viable for research scale samples but not for pilot or commercial scale. Such a process results in a yield that is relatively poor and is especially difficult to exploit industrially on account of the use of protecting agents (blocking/deblocking cycles) and of me requirement of many purification stages involving HPLC, stereochemical vulnerabiliy of the chiral centers during the synthesis and typical harsh conditions required to remove the peptide from the resin. Conventional processes are expensive and require large excess of reagents and amino acids. The use of a solid support (resin) limits the use of manual manufacturing equipment.
There is, therefore, a need for peptides acting on peripheral opioid receptors to be synthesized on a scale of sufficient size (on the pilot or commercial scale) and amenable to manufacture to allow for further toxicological evaluation and validation in clinical studies and be amenable to manufacture. There is also a need for a commercially feasible process to synthesize TAPP-like peptides, in addition to a need for a method to synthesize TAPP-like peptides on a sufficiently large scale that does not require strong acids such as hydrofluoric acid to remove protecting groups. There is also a need for a process that provides for crystalline product in a salt form that is biocompatible and whose stereogenic centers are conserved during the process.
In view of the therapeutic value of peripherally acting opioid tetrapeptides corresponding to formula (I): Tyr-(D)R--R2-R3-NH2 , where R- is Ala or Arg ; R2 is Phe or Phe(p-F); and ;R3 is Phe or Phe(p-F) and the lack of a commercial scale (i.e., yields on the order of kilogram quantities) process enabling it to be obtained with a satisfactory degree of purity, a good yield and, if possible, from fairly inexpensive commercially available starting materials and procedures, more detailed research has been carried out and has led to the discovery of a new process for their preparation. Herein we describe a process for producing multikilo quantities of those TAPP-like peptides which overcomes the problems outlined above.
The process of the present invention provides a solution phase peptide synthesis process in which the yield of D-isomer of Rj is improved over the conventional processes using solid phase (eg. resin based oligopeptide building) and solution phase (employing costly blocking and deblocking agents) to increase efficiency and cost effectiveness for large scale production.
SUMMARY OF THE INVENTION
The present invention provides for a synthesis of opioid analgesic tetrapeptides having the general structure represented by Formula I:
Tyr-(D)R1-R2.R3-NH2 Formula I wherein
Ri is Ala, or Arg; R2 is Phe or Phe(p-F); and R3 is Phe or Phe(p-F); which comprises a coupling step comprising couphng (P-Tyr-(D)Rι), wherein P is an amino protecting group; with (R2-R3-NH2) using 3-hy droxy- 1,2,3 benzotriazin- 4(3H)one (HODbht) as an activating agent, a neutralizing agent selected from the group consisting of diisopropylethylamine (DIEA) and n-ethylmorpholine (NEM); in a solvent suitable for a coupling reaction to yield to a protected intermediate of formula (II): P-Tyr-(D)Rι-R2-R3-NH2
wherein P, Rj, R2, and R3 are as defined above; said process further comprising a deprotecting step which comprises deprotecting the amino function of the intermediate of formula (II) under suitable conditions to yield to a peptide of formula (I).
It will be appreciated by those skilled in the art that the compounds of formula (I) contain more than one chiral center and thus may exist in 16 different isomeric forms (enantiomeric and diastereomeric).
The Applicant has succeeded, not without surprise, to perfect processes that minimize tlie investment costs and/or energy consumptions, and maximize the yields by adopting the most appropriate operating conditions for pre-pilot scale leading to commercial scale production.
The invention has also yielded an efficient process that is conserved from a microscale process to a pilot or commercial scale preparation of the desired tetrapeptide. The process allows for almost racemization-free product (typically < 0.5%) on scales up to the multikilogram level. The present process also removes the need for polystyrene resin as a solid phase support which is expensive on a manufacturing scale. The process further circumvents the need for strong acids such as hydrofluoric acid to effect removal of side chain protecting groups.
In comparison with known procedures of peptide synthesis, the process of the present invention has important advantages, expecially as regards quality, commercial scale implementation and reproducibility.
The compounds of the present invention can be synthesized using conventional preparative steps and recovery methods known to those skilled in the art of organic and bio-organic synthesis, while providing new and unique combinations for the overall synthesis of each compound. Preferred synthetic routes for intermediates involved in the synthesis as well as the resulting compounds of the present invention follow.
The absence of by-products and racemization in die present process results in a better quality of the reaction medium and increased quality of the final product, the purity of which is typically greater than 98%. The stability, yield, purity and reproducibility which the conventional process lacks on account of the tendency of racemization are achieved in the present process, in which the absence of blocking agents also decreases cost.
The final product of this process is very similar to that obtained by costly procedures (ie. due to expensive purification steps), but it has two great advantages in that no costly protective blocking/deblocking steps are involved and only one or less purification steps are required entailing costly, time consuming chromatographic procedures. The kilogram to pre-pilot scale application of this process is accordingly advantageous.
The following common abbreviations are used throuhout the specification and in the claims:
Ala - alanine Arg - arginine Boc - t-butyloxycarbonyl
BOP - benzotriazol- l-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate Bz - benzoyl
DCC - dicyclohexylcarbodiimide DCU - dicyclohexylurea DIEA - diisopropylethylamine DMF - dimethylformamide
DPPA - diphenyphosphorylazide
EDC - dimethylaminopropylethylcarbodiimide
Fmoc - 9-fluoromethyloxycarbonyl PPA - propane phosphonic acid anhydride Phe - phenylalanine Phe(p-F) - parafluorophenylalanine TAPP - Tyr-(D)Ala-Phe-Phe-NH2 THF - tetrahydrofurane TFA - trifluoroacetic acid Tyr - tyrosine
HOBT - hydroxybenzotriazole HODbht - 3-hydroxy- 1,2,3 benzotriazin-4(3H)one HOSu - n-hydroxysuccinimide
HPLC - high performance Uquid chromatography
MeOH - methanol
NEM - n-etiiylmorpholine
Boc - t-Butoxycarbonyl
Osu - N-Hydroxysuccinimide ester
Z - benzyloxycarbonyl
The term amino protecting group is well known in the art of peptide chemistry. For example suitable amino protecting group can be found in Protective Groups In Organic Svnthesis. Greene T.W. and Wuts P.G., 1991, John Wiley & Sons, Inc.
DESCRIPTION OF TABLES AND FIGURES
Figure 1 shows the HPLC chromatogram of Tyr-(D)Arg-Phe-Phe-NH2 • 2HC1 crystallized from methanol.
Figure 2 shows the HPLC chromatogram demonstrating separation of all the stereoisomers of the tetrapeptide Tyr-Arg-Phe-Phe.
Figure 3 shows chiral analysis by way of HPLC chromatography to separate the amino acids corresponding to formula (I): Tyr-(D)Arg-Phe-Phe-NH2 following acid hydrolysis as described in Example 8. DETAILED DESCRIPTION OF THE INVENTION
Ri is preferably Ala, or Arg. Ri is more preferably Arg.
R2 is preferably Phe or Phe(p-F).
R2 is more preferably Phe.
In an alternative embodiment, R2 is more preferably Phe(p-F).
R3 is preferably Phe or Phe(p-F). R3 is more preferably Phe. In an alternative embodiment, R3 is more preferably Phe(p-F).
P is preferably selected from the group consisting of Boc (t-butyloxycarbonyl), Z (benzyloxycarbonyl), Fmoc (9-fluoromethyloxycarbonyl), Bz (benzoyl), and 3,5- dihydroxy benzoyl. P is more preferably Boc.
The neutrahzmg agent is preferably DIEA (diisopropylethylamine).
The solvent suitable for the coupling reaction is preferably dimethylformamide (DMF).
In an alternative embodiment, the process of this invention can be carried using a further activating agent selected from the group consisting of dicyclohexylcarbodiimide (DCC); dimethylaminopropylethylcarbodiimide (EDC); and benzotriazol-l-yloxy-tris(dimethylamino)-phosphonium hexafluorophosphate (BOP).
Successful preparation of these compounds is possible by way of several synthetic routes one of which is outlined in Scheme 1. SCHEME 1
Boc-Tyr-OH HOSu A
Boc-Tyr-OSu H-(D)R OH
B c
Figure imgf000010_0001
Boc-Tyr-(D)R OH H-Phe-R "92-NH '2„ HCl
Figure imgf000010_0002
Boc-Tyτ-(D)R1-Phe-R2NH2 TF:A
Figure imgf000010_0003
Tyr-(D)R1-Phθ-R2NH2 ■ TFA G
5
Tyr-(D)RrPhe-R2NH2 ■ HCl H The steps illustrated in Scheme 1 can be briefly described as follows:
Step 1:
The starting amino acid, Boc-Tyr-OH is activated by esterifying the carboxylate group of Boc-Tyr-OH in nonpolar solvent such as THF. Formation of the activated succinimate ester of Boc-Tyr-OH is accomplished by dissolving Boc-tyrosine in an appropriate solvent such as THF and adding n-hydroxysuccinimide, also dissolved in the same solvent. A solution containing a dehydrating agent solution such as dicyclohexylcarbodiimide is added to effect esterification.
Step 2:
Coupling of the (D) amino acid by activated Boc-Tyrosine-succinimide ester is accomplished by dissolution of the respective components in specified solvents (THF or H2O) and mixing the two solutions at room temperature, wherein the dipeptide is formed.
Step 3:
Condensation of the two dipeptidyl units D and E in a specific manner that causes mimmal racemization (< 4 %) at the α-stereogenic center corresponding to the (D)- R* residue is accomplished by following stringent conditions including, choice of solvent, pH, neutralizing agent, choice of activating agent and rate of addition of specific activating agent solution. In a most preferred embodiment, the activating agent(s) is HODbht, the neutralizing agent is DIEA, and the solvent is DMF. In other prefeπed embodiments the first activating agent is HOBhbt, the second activating agent can be either DCC, EPC, BOP, or an equivalent activating agent, the neutralizing agent can be either DIEA, or NEM, and the solvent is DMF. Step 4:
The N terminal t-butyl carbamate protecting group is removed by acid hydrolysis in a mixture of methylene chloride and trifluoroacetic acid, followed by evaporation of the solvents.
Step 5:
CrystalUzation of the pure (> 97%) product by preferred formation of the pharmaceutically acceptible salt, such as the hydrochloride salt, is accompUshed by stirring a specified concentration of compound G in a specified solvent, such as DMF.
The process consists of initially preparing independently two dipeptide fragments using minimal side chain protection. Subsequently, the two fragments are joined by reacting them in a suitable solvent. It is well known to those skilled in the art that amino acids are prone to racemization. It is also known that the potential for racemization is increased in the case where an amino acid is acylated as is the case where fragment D is joined to fragment E in Scheme I. In the case cited herein racemization was observed to be as high as 44% depending on reaction conditions.
The unpredictability of minimizing the racemization at step 3 can be evidenced by prior attempts to maintain the D conformation of Ri while obtaining pure product by experiments in which HOBT/DCC activating agents were used in conjunction with NEM as a neutralizing agent, yielding between 13 - 30 % racemization, but when the same activating agents were used but DIEA was substituted for the neutralizing agent, racemization dropped to 6 - 7 %. Conversely, it was observed that using HOBT/EDC as activating agents with NEM as a neutralizing agent yielded 30% racemization. When PPA was used as the activating agent and DIEA was used as the neutraUzing agent, only 3 - 4 % racemization occurred but the result yielded a very impure material. In contrast, when DPPA was the activating agent and DIEA was the neutralizing agent, 22.5 % racemization occured and a very impure material was again the result. Therefore the process described herein, was optimized to reproducibly afford product with maximum racemization equal to 1 %. In this case, the diastereomeric contaminant could be removed either by crystallization or rapid chromatography. The process described herein also allows for the conversion of the product into a biocompatible salt such as the bishydrochloride.
Moreover, when R< is Arginine, great advantages can be realized using this process due to the fact that unprotected (D)Arg-OH can be easily purchased. Conventional methods would necessitate the purchase or synthesis of (D)Arg-OH wherein the guanidine side chain would have to be protected, further necessitating a time and cost engendering de-blocking step to be incoφorated in this process. It is suprising that unprotected (D)Arg-OH could be used in this solution phase synthesis as H- (D)Arg-OH is difficult to manipulate due to solubility limitations. However, the AppUcants have succeeded with this approach by using DMF to dissolve Boc-Ty- OSu and H2O to dissolve H-(D)Arg-OH, or to use DMF to dissolve Boc-Ty-OSu and TFE to dissolve H-(D)Arg-OH .
The following examples are used to better describe the invention. These examples are for the purpose of illustration only, and are not intended to limit the invention in any manner.
EXAMPLES
The compounds of the present invention were prepared using solution phase segment condensation synthesis as outhned below wherein each individual step is generally known to persons skilled in the art. EXAMPLE 1 Preparation of Boc-Tyr-Osu
Boc-Tyr-OH + HOSu → Boc-Tyr-Osu
A solution of n-hydroxysuccinimide (343.7 g, 2.986 moles) in THF (2L) is added to an amino acid solution comprising the blocked amino acid , Boc-Tyr-OH (700 g, 2.488 moles), dissolved in THF (4.7L). To this reaction mixture, a solution of dicyclohexylcarbodiimide (616.1 h, 2.986 moles) in THF is added at a rate of 200 mlJminute. The resulting mixture is stiπed at room temperature for 16 - 20 hours, after which precipitated DCU is filtered off and the solution evaporated to dryness. The solid material is washed on a filter with isopropanol (3 x 1 L) and ethyl ether (3 x 1 L), and the product obtained and dried under high vacuum to yield 921 grams (97.8% yield).
EXAMPLE 2 Preparation and Purification of Boc-Tyr-(D)Rι-OH
Boc-Tyr-Osu + H-(D)Arg-OH ■-> Boc-Tyr-(D)Arg-OH
The amino acid H-(D)Arg-OH (447.69 g, 2.57 moles) is dissolved in 6.7 L H2O and added while stirring at room temperature to a solution of Boc-Tyr-Osu (447.69 g, 2.57 moles), previously prepared by dissolution in 1.52 L THF. After stirring the reaction mixture for 16 - 20 hours, the THF is evaporated. The product is dissolved (1.98 moles, theoretical) in a total volume equaling approximately 10L H2O, filtered through two Whatman fibreglass filters and one Whatman #1 paper, after which the solution is injected onto an HPLC column at a rate of 300 ml/min. The product is purified following the gradient conditions described in Table I. Analytical HPLC is used to monitor the eluant, which is collected in 3 - 4 L fractions. Fractions demonstrating an HPLC purity greater than 97% by this method are combined, the acetonitrile evaporated and product lyophilized or evaporated (yield: 668.57g, 77.18% ).
Figure imgf000015_0001
EXAMPLE 3 Preparation of Boc-Tyr-(D)Rι-Phe-R7-NH-
Boc-Tyr-(D)Arg-OH + H-Phe-Phe-NH2 • HCl → Boc-Tyr-(D)Arg-Phe-Phe- NH2
The dipeptide, Boc-Tyr-(D)Arg-OH (760 g, 1.737 moles) is added to 15 L of a solution of CHC13, H O, Methanol 1:1:2 and dissolved by sonication and mixing at 30-37 °C in a waterbath. After dissolution, 3L of DMF is added and the solution concentration by evaporation to 2.5 L. , afterwhich 4 A molecular sieves (650 mL) are added, the solution gently swirled and allowed to stand 16 - 20 hours. The solution is decanted and sieves washed with 1.9 L DMF.
The second dipeptide, H-Phe-Phe-NH2 • HCl (604.18 g, 1.737 moles) is dissolved in 4.8 L DMF and one equivalent of DIEA (1.737 moles) is added. The two peptide solutions are combined and HODhbt (312 g, 1.911 moles) is added, afterwhich the pH is adjusted to 7.0 - 8.0 with 250 ml DIEA, and a solution of DCC (430 g, 1.084 moles) in IL DMF is added and the reaction allowed to stir 16 - 20 hours (Note: after 30minutes of mixing, the pH is adjusted to 7.0 - 8.0 with DIEA if necessary). The mixture is filtered to remove DCU, which is washed on the filter 2 x 400 mL DMF, and evaporated at 40 °C under high vacuum. The almost dry mixture is dissolved in 2 L acetone and the solution aUowed to stand for 1 hour at room temperature. The precipitate is filtered and washed 3 x 500 mL acetone afterwhich the total volume of the solution is adjusted by adding or evaporating acetone to a total volume of 5.1 L.
The total volume of this solution is divided in 3 equal portions (1.7 L each) and each fraction is precipitated separately by transferring the peptide/acetone solution into a separatory funnel and precipitating the product at a rate of 50mIJmin into a fast stiπed EtOAc (15L). The precipitate is allowed to settle by standing for a 20 - 30 minute period and the supernatant is decanted without disturbing the precipitate. 2L of EtOAc is added to the precipitate, mixed and filtered. The powder on the filter is washed with 1 - 2 L EtOAc. The powder is transferred to a crystalUzing dish which is covered with perforated aluminum foil and dried overnight in a dessicator connected to an aspirator pump. The product is dried completely under high vacuum for 24 hours, (yield 1317.35 g)
EXAMPLE 4 TFA Deprotection of Boc-Tyr-(D)Ri-Phe-R?NH?
Boc-Tyr-(D)Arg-Phe-Phe-NH2 + TFA → Tyr-(D)Arg-Phe-Phe-NH2 • 2TFA
The Boc- terminally protected peptide (1317 g) is dissolved in a solution consisting of 55% TFA, 45% CH2C12 in a ratio equivalent to 4 mL TFA solution/gram tetrapeptide, and the solution stiπed at room temperature for 45 minutes. The reaction mixture is evaporated on a rotary evaporator at room temperature. Following the evaporation of CH C12 with a water aspirator pump, the remaining TFA is evaporated under high vacuum. A portion of the thick residue obtained is precipitated by pouring it into 4 L of ethyl ether with stirring until stirring becomes difficult. The precipitate is filtered off and washed on the filter with 2 L ethyl ether. The latter precipitation step is repeated until the total amount of thick residue is precipitated. The precipitates are combined and dried overnight by connecting the dessicator to an aspirator pump, foUowed by drying under high vacuum for 4 hours. (Yield: 1322.7 g)
EXAMPLE 5 HCl Exchange of Tyr-(D)Rι-Phe-R?-NH? » TFA
Tyr-(D)Arg-Phe-Phe-NH2 • 2TFA → Tyr-(D)Arg-Phe-Phe-NH2 • 2HC1
Deprotected tetrapeptide (1322.7 g, 1.540 moles) is dissolved in 7.94 L ethanol and a 875 mL of a 3.32 M HCl/EtOH solution is added with stirring to precipitate the peptide afterwhich the solution is stiπed for one hour at room temperature and allowed to stand at 4 °C overnight. The precipitate is filtered, washed on the filter with 2 x 2L EtOH, and dried in a dessicator under aspiration for 4 hours. The drying process is completed under high vacuum for a minimum of 16 hours. (Yield 831.1 g, 76.7 %. ).
EXAMPLE 6: Crystallization of Tyr-(D)Rι-Phe-R?NH? » HCl
The peptide (831.1 g) is dissolved in 3324 mL MeOH (ratio 4 ml MeOH/g peptide) in a water bath at 50 °C and filtered over a fritted filter. After washing the filter with 100 mL MeOH the peptide solution is transferred to a 20 L evaporating round bottom flask and gently agitated (40 RPM) at 50 °C. Small increments of acetonitrile (ratio 4.8 mL AcN/g product) are added and agitation and heat are ceased once crystals begin to form. After allowing the mixture to reach room temperature (approximately 3 hr), the flask is cooled at 4 °C for 1 hour. The crystalline material is filtered on a fritted funnel and washed twice with 1.5 L of a cold solution of MeOH/AcN (1:1.2), afterwhich the crystals are dried in a dessicator connected to an aspirator pump for 2 hours and the mother liquor is evaporated and recrystaUized by repeating this entire procedure from the point of dissolution in MeOH. The drying process is completed under high vacuum for a minimum of 16 hours yielding combined product totalling 707.7 g.
EXAMPLE 7 Final crystallization of combined product
Three batches of peptide, totalling 1870.68 g, are combined and dissolved in a solution consisting of 20% H2O/acetone (ratio approximately 3.6 peptide: 1 solvent) at 50-52 °C. Crystallization is effected with the stepwise addition of 7.8L MeOH. The flask is removed from the water bath and cooled at 4 °C overnight. The crystals are then filtered on fritted finnels and washed once with 6 L acetone followed by a wash with 6 L MeOH, transfeπed to an evaporating flask and partially dried n a dessicator connected to an aspirator pump, and then under high vacuum in a 50 °C water bath for 16 - 20 hours (yield 1606 g, 92,8%). The HPLC analysis demonstrating the purity of this product is presented in Figure 1.
EXAMPLE 8: Determination of D and L amino acids using Marfev's
Reagent
Applying the procedure of Marfey, (Carlsberg Res. Comm., 46:591-596, 1984), the relative content of each isomer in a peptide is determined. Briefly, the peptide to be analyzed is hydrolyzed (80 nmoles) using 6 N HCl at 110 °C for 24 hours, afterwhich all traces of HCl are removed by evaporation under vacuum for 20 minutes. A redrying solvent (20 ul of MeOH:H2O:TEA , 2:2:1) is added and the sample dried under vacuum for 15 minutes. After repeating the drying step once, lOOuL of Marfey's reagent (10 mg Marfey's reagent/ml acetone) is added followed by 20 uL of IM NaHCO3. The solution is mixed and heated at 30 - 40 °C for 1 hour. After coohng the solution to room temperature, 10 uL of 2 M HCl is added, the solution mixed then dried under vacuum over NaOH pellets. The resulting sample is dissolved and injected onto HPLC for analysis, the results of which are presented in Figure 3.

Claims

1. A process for producing opioid analgesic tetrapeptides of Formula I:
Figure imgf000019_0001
Formula I wherein
Ri is Ala, or Arg; R2 is Phe or Phe(p-F); and R3 is Phe orPhe(p-F); which comprises a couphng step comprising coupUng (P-Tyr-(D)Rι), wherein P is an amino protecting group; with (R2-R3-NH2) using 3-hydroxy- 1,2,3 benzotriazin-4(3H)one (HODbht) as an activating agent, a neutralizing agent selected from the group consisting of diisopropylethylamine (DIEA) and n- ethylmorphoUne (NEM); in a solvent suitable for a coupling reaction to yield to a protected intermediate of formula (II):
P-Tyr-(D)Rι-R2-R3-NH2
(ID wherein P, R*, R2, and R3 are as defined above; said process further comprising a deprotecting step which comprises deprotecting the amino function of the inteπnediate of formula (II) under suitable conditions to yield to a peptide of formula (I).
2. A process according to claim 1 wherein Ri is Arg.
3. A process according to claim 1 wherein R2 is Phe.
4. A process according to claim 1 wherein R2 is Phe(p-F).
5. A process according to claim 1 wherein R3 is Phe.
6. A process according to claim 1 wherein R3 is Phe(p-F).
7. A process according to claim 1 wherein P is selected from the group consisting of Boc (t-butyloxycarbonyl, Z (benzyloxycarbonyl), Fmoc (9- fluoromethyloxycarbonyl), Bz (benzoyl), and 3,5-dihydroxy benzoyl.
8. A process according to claim 1 wherein P is Boc.
9. A process according to any one of claims 1 to 8 wherein the neutraUzing agent is DIE A.
10. A process according to any one of claims 1 to 8 the solvent suitable for the coupling step is dimethylformamide (DMF).
11. A process according to any one of claims 1 to 8 wherein the neutralizing agent is DIEA and the solvent suitable for the coupling step is dimethylformamide (DMF).
12. A process according to any one of claims 1 to 8 wherein in the coupling step a second activating agent is used; said second activating agent being selected from the group consisting of dicyclohexylcarbodiimide (DCC); dimethylaminopropylethylcarbodiimide (EDC); and benzotriazol- 1-yloxy- tris(dimethylamino)-phosphonium hexafluorophosphate (BOP).
13. A process according to claim 9 wherein in the coupUng step a second activating agent is used; said second activating agent being selected from the group consisting of dicyclohexylcarbodiimide (DCC); dimethylaminopropylethylcarbodiimide (EDC); and benzotriazol- 1-yloxy- tris(dimethylamino)-phosphonium hexafluorophosphate (BOP).
14. A process according to claim 10 wherein in the coupling step a second activating agent is used; said second activating agent being selected from the group consisting of dicyclohexylcarbodiimide (DCC); dimethylaminopropylethylcarbodiimide (EDC); and benzotriazol- 1-yloxy- tris(dimethylamino)-phosphonium hexafluorophosphate (BOP).
1 . A process according to claim 11 wherein in the coupling step a second activating agent is used; said second activating agent being selected from the group consisting of dicyclohexylcarbodiimide (DCC); dimethylaminopropylethylcarbodiimide (EDC); and benzotriazol- 1-yloxy- tris(dimethylamino)-phosphonium hexafluorophosphate (BOP).
16. A process according to claim 12 wherein in the coupling step a second activating agent is used; said second activating agent being selected from the group consisting of dicyclohexylcarbodiimide (DCC); dimethylaminopropylethylcarbodiimide (EDC); and benzotriazol- 1-yloxy- tris(dimethylamino)-phosphonium hexafluorophosphate (BOP).
PCT/CA1996/000552 1995-08-18 1996-08-15 Solution synthesis of peripheral acting analgesic opioid tetrapeptides WO1997007129A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU66539/96A AU6653996A (en) 1995-08-18 1996-08-15 Solution synthesis of peripheral acting analgesic opioid tetrapeptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9516994.2A GB9516994D0 (en) 1995-08-18 1995-08-18 Solution synthesis of peripheral acting analgestic opioid tetrapeptides
GB9516994.2 1995-08-18

Publications (1)

Publication Number Publication Date
WO1997007129A1 true WO1997007129A1 (en) 1997-02-27

Family

ID=10779465

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA1996/000552 WO1997007129A1 (en) 1995-08-18 1996-08-15 Solution synthesis of peripheral acting analgesic opioid tetrapeptides

Country Status (3)

Country Link
AU (1) AU6653996A (en)
GB (1) GB9516994D0 (en)
WO (1) WO1997007129A1 (en)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999047548A1 (en) * 1998-03-16 1999-09-23 Astrazeneca Ab Process for the preparation of a tetrapeptide
EP1030831A1 (en) * 1997-10-10 2000-08-30 Aventis Pharmaceuticals Products Inc. Process for the preparation of azacycloalkylalkanoyl pseudotetrapeptides
WO2001019849A1 (en) * 1999-09-15 2001-03-22 Astrazeneca Ab A process for the preparation of h-tyr-d-ala-phe(f)-phe-nh¿2?
WO2017165676A1 (en) * 2016-03-23 2017-09-28 Protagonist Therapeutics, Inc. METHODS FOR SYNTHESIZING α4β7 PEPTIDE ANTAGONISTS
CN113624898A (en) * 2021-08-23 2021-11-09 成都诺和晟泰生物科技有限公司 Purification method of chiral analgesic polypeptide drug
US11472842B2 (en) 2015-12-30 2022-10-18 Protagonist Therapeutics, Inc. Analogues of hepcidin mimetics with improved in vivo half lives
US11753443B2 (en) 2018-02-08 2023-09-12 Protagonist Therapeutics, Inc. Conjugated hepcidin mimetics
US11807674B2 (en) 2013-03-15 2023-11-07 Protagonist Therapeutics, Inc. Hepcidin analogues and uses thereof
US11840581B2 (en) 2014-05-16 2023-12-12 Protagonist Therapeutics, Inc. α4β7 thioether peptide dimer antagonists
US11884748B2 (en) 2014-07-17 2024-01-30 Protagonist Therapeutics, Inc. Oral peptide inhibitors of interleukin-23 receptor and their use to treat inflammatory bowel diseases

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2359817A1 (en) * 1976-07-27 1978-02-24 Reckitt & Colmann Prod Ltd Peptide cpds. contg. tyrosine, glycine, phenylalanine and d-amino acid - local anaesthetics, smooth muscle relaxants, adrenergic neutron blockers, beta-stimulants etc.
FR2465713A1 (en) * 1979-09-20 1981-03-27 Erba Farmitalia NOVEL BIOLOGICALLY ACTIVE PEPTIDES AND THEIR USE AS MEDICAMENTS
WO1995022557A1 (en) * 1994-02-21 1995-08-24 Astra Aktiebolag Novel opioid peptides for the treatment of pain and use thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2359817A1 (en) * 1976-07-27 1978-02-24 Reckitt & Colmann Prod Ltd Peptide cpds. contg. tyrosine, glycine, phenylalanine and d-amino acid - local anaesthetics, smooth muscle relaxants, adrenergic neutron blockers, beta-stimulants etc.
FR2465713A1 (en) * 1979-09-20 1981-03-27 Erba Farmitalia NOVEL BIOLOGICALLY ACTIVE PEPTIDES AND THEIR USE AS MEDICAMENTS
WO1995022557A1 (en) * 1994-02-21 1995-08-24 Astra Aktiebolag Novel opioid peptides for the treatment of pain and use thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MISICKA, ALEKSANDRA ET AL: "Structure-activity relationships of analogs of highly potent opioid peptide, biphalin", REGUL. PEPT. (1994), (SUPPL. 1), S131-S132 CODEN: REPPDY;ISSN: 0167-0115, XP000611981 *
P.W. SCHILLER ET AL.: "Dermorphin Analogues an Increased Positive Net Charge in Their Message Domain Display Extremely High mu Opioid Receptor Selectivity", JOURNAL OF MEDICINAL CHEMISTRY, vol. 32, no. 3, March 1989 (1989-03-01), WASHINGTON US, pages 698 - 703, XP002021558 *
SCHILLER, P. W. ET AL: "Two new families of opioid peptide analogs displaying extraordinary.mu.-receptor selectivity and preference for either peripheral or central sites", ADV. BIOSCI. (OXFORD) (1989), 75(PROG. OPIOID RES.), 85-8 CODEN: AVBIB9;ISSN: 0065-3446, XP000612379 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1030831A1 (en) * 1997-10-10 2000-08-30 Aventis Pharmaceuticals Products Inc. Process for the preparation of azacycloalkylalkanoyl pseudotetrapeptides
EP1030831A4 (en) * 1997-10-10 2003-01-02 Aventis Pharm Prod Inc Process for the preparation of azacycloalkylalkanoyl pseudotetrapeptides
WO1999047548A1 (en) * 1998-03-16 1999-09-23 Astrazeneca Ab Process for the preparation of a tetrapeptide
WO2001019849A1 (en) * 1999-09-15 2001-03-22 Astrazeneca Ab A process for the preparation of h-tyr-d-ala-phe(f)-phe-nh¿2?
US11807674B2 (en) 2013-03-15 2023-11-07 Protagonist Therapeutics, Inc. Hepcidin analogues and uses thereof
US11840581B2 (en) 2014-05-16 2023-12-12 Protagonist Therapeutics, Inc. α4β7 thioether peptide dimer antagonists
US11884748B2 (en) 2014-07-17 2024-01-30 Protagonist Therapeutics, Inc. Oral peptide inhibitors of interleukin-23 receptor and their use to treat inflammatory bowel diseases
US11472842B2 (en) 2015-12-30 2022-10-18 Protagonist Therapeutics, Inc. Analogues of hepcidin mimetics with improved in vivo half lives
WO2017165676A1 (en) * 2016-03-23 2017-09-28 Protagonist Therapeutics, Inc. METHODS FOR SYNTHESIZING α4β7 PEPTIDE ANTAGONISTS
US10407468B2 (en) 2016-03-23 2019-09-10 Protagonist Therapeutics, Inc. Methods for synthesizing α4β7 peptide antagonists
US11753443B2 (en) 2018-02-08 2023-09-12 Protagonist Therapeutics, Inc. Conjugated hepcidin mimetics
CN113624898B (en) * 2021-08-23 2023-08-25 成都诺和晟泰生物科技有限公司 Purification method of chiral analgesic polypeptide medicine
CN113624898A (en) * 2021-08-23 2021-11-09 成都诺和晟泰生物科技有限公司 Purification method of chiral analgesic polypeptide drug

Also Published As

Publication number Publication date
AU6653996A (en) 1997-03-12
GB9516994D0 (en) 1995-10-18

Similar Documents

Publication Publication Date Title
AU2010241171B2 (en) Method for the manufacture of degarelix
JP3249178B2 (en) Solid phase synthesis of peptides containing a C-terminal azaamino acid amide
Dudash Jr et al. Comparative study of selected coupling reagents in dipeptide synthesis
USH1312H (en) Method for the preparation of gyk-dtpa
US20190055190A1 (en) Peptide nucleic acid (pna) monomers with an orthogonally protected ester moiety and novel intermediates and methods related thereto
WO1997007129A1 (en) Solution synthesis of peripheral acting analgesic opioid tetrapeptides
US5166394A (en) Coupling reagent for peptide synthesis
US5391711A (en) Biotinylating reagent and purification process for synthesized peptide using thereof
CS235072B2 (en) Method of l-tyrosyl-d-alanyl-glycyl-l-phenylalanylamide&#39;s new derivatives production
GB2127830A (en) Amides of tripeptides and tetrapeptides
Funakoshi et al. Affinity purification method using a reversible biotinylating reagent for peptides synthesized by the solid-phase technique
US4491541A (en) Peptides
CS197268B2 (en) Process for preparing l-pyroglutamyl-l-histidyl-3-alkylprolinamides
US5770692A (en) Carbamoylation of amino groups in peptides via N-aryloxycarbonyl intermediates
US4301066A (en) Preparation of (D-Trp 6)-LH-RH via the heptapeptide H-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH2
HU192206B (en) Process for preparing novel peptides
US4369137A (en) N-Protected pentapeptides useful as intermediates in the preparation of thymopoietin pentapeptide
Okada et al. Amino acids and peptides. Part 45. Development of a new N π-protecting group of histidine, N π-(1-adamantyloxymethyl) histidine, and its evaluation for peptide synthesis
WO2021026800A1 (en) Method for synthesizing degarelix acetate
KR100336139B1 (en) Novel peptide active materials and methods for their preparation
Ohno et al. Partial enzymic deprotection in the synthesis of a protected octapeptide bearing a free terminal carboxyl group
EP0311392B1 (en) Solution synthesis of an octapeptide
US3247178A (en) Synthesis of peptides containing alpha, omega-diamino acids protected by phthalyl and t-butyloxycarbonyl groups
KR20200078999A (en) Process for the Preparation of Tripeptide
CN106699839B (en) Process for synthesizing Diapin (Gly-Gly-leu) by chemical method

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA