HK1162527B

HK1162527B - Process for the production of bivalirudin

Info

Publication number: HK1162527B
Application number: HK12102674.5A
Authority: HK
Inventors: Geoffroy Sommen; Luciano Forni
Original assignee: Polypeptide Laboratories Holding (Ppl) Ab
Priority date: 2008-12-29
Filing date: 2009-12-17
Publication date: 2014-10-10

Description

Method for preparing bivalirudin

Technical Field

The present invention relates to a novel convergent synthesis process for bivalirudin, a 20-mer peptide of formula

H-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp-Phe-Glu--Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OH (I)

The invention also relates to protected peptides which are intermediates in the synthesis of bivalirudin.

Background

The proteolytic processing mediated by thrombin is critical in controlling blood coagulation. Hirudin is a potential clinical thrombin peptide inhibitor, which consists of 65 amino acids. But also shorter peptide fragments have been shown to be effective for thrombosis (life threatening condition).

US 5,196,404 discloses bivalirudin,it is one of these shorter polypeptides and is a potent thrombin inhibitor. Bivalirudin is also known as hirulog-8 (hirulog-8), BG-8967, Efludan,OrAnd has the amino acid sequence given in formula I.

WO 98/50563 describes a method for the preparation of various peptides including bivalirudin by recombinant techniques. The method comprises expressing a peptide as part of a fusion protein and then releasing the peptide from the fusion protein via an acyl acceptor.

Okayama et al chem.pharm.Bull.1996, 44, 1344-. The Wang resin requires cleavage of the peptide from the resin using concentrated trifluoroacetic acid. In a similar solid phase synthesis method for the preparation of bivalirudin, WO 91/02750 discloses the following sequential process: a single Boc-protected amino acid was attached to Boc-L-leucine-o-divinylbenzene resin, then deprotected and removed simultaneously by using HF/p-cresol/ethyl methyl sulfate, and then freeze-dried and purified. In both cases, cleavage of the peptide from the resin requires strong acid conditions which may cause total deprotection and may lead to undesired side reactions of amino acid residues, thus negatively affecting the purity of the product. In addition, side reactions are often caused in solid phase synthesis by misincorporation of amino acids, repeated linking of individual amino acids (double-hits) and/or racemization, and by-products are produced which have a structure very similar to that of the target peptide. The purification process therefore becomes laborious and leads to a reduction in yield. In particular, longer polypeptides tend to adopt an irregular conformation while still attached to a solid support, which makes it more difficult to attach additional amino acids to the growing chain. Therefore, this problem becomes more serious as the length of the peptide increases.

WO2007/033383 discloses a process for the preparation of bivalirudin based on solid phase synthesis or a combination of solid phase synthesis and solution method synthesis (mixed process). In one embodiment, the bivalirudin peptide sequence is prepared on a superacid labile resin. In another embodiment, bivalirudin is prepared by coupling a side chain protected N-terminal peptide fragment with a side chain protected C-terminal peptide fragment and subsequent deprotection with strong acidic conditions. In this case, the N-terminal fragment and the precursor of the C-terminal fragment (i.e.the peptide sequence minus leucine) are both prepared by solid phase synthesis. One disadvantage of this strategy is the massive formation of D-Tyr¹⁹-bivalirudin. This impurity is difficult to remove, thus requiring additional labor, cost, and lost yield to obtain a purified product. In addition, the amount of purified bivalirudin obtained in the example of WO2007/033383 is only in the range of grams, which indicates that this method is not suitable for large-scale preparation of bivalirudin with good purity.

Disclosure of Invention

It is an object of the present invention to provide a method for industrial scale production that overcomes the well-known disadvantages of linear, solid phase synthesis and more efficiently synthesizes bivalirudin. This object has been achieved by the method according to claim 1, the peptide fragment according to claim 19 and the use of these peptide fragments according to claim 29. Preferred embodiments constitute the subject matter of the dependent claims 2-18 and 20-28.

The present invention relates to a method that follows the convergent approach (i.e. the fragments are synthesized separately and then coupled in solution phase to constitute the target peptide). The challenge of convergent synthesis is to find suitable fragments and their order of coupling to overcome the well-known disadvantages of convergent synthesis. These disadvantages are solubility problems during coupling and separation, lower reactivity compared to solid phase synthesis and higher racemization risk of the C-terminal fragment during coupling. Bivalirudin is composed of 20 amino acidsThe residues make up so that there are a large number of possible fragments and coupling sequences. In addition, bivalirudin contains 7 amino acid residues, namely-Arg³-、-Asn⁹-、-Asp¹¹-、-Glu¹³-、-Glu¹⁴-、-Glu¹⁷-and-Glu¹⁸All of these amino acid residues have reactive side chain functionalities that require appropriate protection and deprotection. The same problem applies to the appropriate protection and deprotection of the N-and C-termini of the individual fragments, thus increasing the difficulty of finding ways to achieve the objects of the invention.

Applicants have surprisingly found that bivalirudin of formula I can be conveniently constructed by employing the [ (1+2) + (3+ {4+5}) ] strategy as described hereinafter. The numbers 1, 2 and 5 represent the three peptide fragments of formulae V, VI and X, the number 3 represents the aspartic acid derivative of formula XIII and the number 4 represents the phenylalanine derivative of formula XI. The present invention relates to a process for the preparation of bivalirudin of formula I in solution phase, comprising the following steps:

(a) reacting an optionally side-chain protected peptide of formula (V) wherein P1 is a protecting group

P1-D-Phe¹-Pro-Arg-Pro-OH(SEQ ID NO 4) (V)，

With optionally side-chain-protected peptides of formula (VI) wherein P2 is a protecting group

H-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OP2(SEQ ID NO 5)(VI)

(iii) reaction to prepare an optionally side chain protected peptide of formula (VII) wherein P1 and P2 are as defined above

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OP2(SEQ ID NO 2) (VII)，

(b) Removing P2 from the peptide prepared in step (a) to prepare an optionally side chain protected peptide of formula (II) wherein P1 is as defined above

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQ ID NO 2) (II)，

(c) Protecting a side chain of formula (X) wherein P3 is a protecting group

H-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 8)

(X)，

And phenylalanine of the formula (XI) in which P4 is a protecting group

P4-Phe¹²-OH (XI)

Reaction to prepare a side chain protected peptide of formula (XII) wherein P3 and P4 are as defined above

P4-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ IDNO 9) (XII)，

(d) Removing P4 from the peptide produced in step (c) to produce the corresponding N-terminally deprotected side chain-protected peptide of formula (XII),

(e) reacting the peptide of formula XII prepared in step (d) with side chain protected aspartic acid of formula (XIII) wherein P5 is a protecting group

P5-Asp¹¹-OH (XIII)，

To prepare side chain protected peptides of formula (XIV) wherein P3 and P5 are as defined above

P5-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 3) (XIV)，

(f) Removing P5 from the peptide prepared in step (e) to prepare a side chain protected peptide of formula (III) wherein P3 is as defined above

H-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 3) (III)，

(g) Reacting the optionally side chain protected peptide of formula II prepared in step (b) with the side chain protected peptide of formula (III) prepared in step (f) to prepare a side chain protected peptide of formula (IV) wherein P1 and P3 are as defined above

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 1)

(IV)，

(h) Removing P1, P3 and the side chain protecting groups of the peptide prepared in step (g) to prepare bivalirudin of formula (I).

The method of the present invention can synthesize bivalirudin very efficiently by convergent fragment synthesis, which can be easily applied to industrial scale production.

The C-terminal protecting groups P2 (for peptide VI) and P3 (for peptide X) may be any protecting group consistent with the orthogonality of the other protecting groups. Suitable examples are C-terminal protecting groups which can be removed by saponification, like methyl (Me) or ethyl (Et), or which can be removed by catalytic hydrogenation.

In an embodiment of the process of the present invention, in step (a), the protecting group P1 is a protecting group stable to catalytic hydrogenation reaction, and the protecting group P2 is a protecting group removable by catalytic hydrogenation reaction and orthogonal to the optional side chain protecting group; in step (c), the protecting group P3 is a protecting group removable by catalytic hydrogenation, and the protecting group P4 is a protecting group orthogonal to the side chain protecting group of the peptide of formula X and orthogonal to P3; and in step (e) the protecting group P5 is a protecting group orthogonal to the side chain protecting groups of the peptides/amino acids of formula XII and formula XIII or a protecting group orthogonal to P3, providing a solution phase process comprising the following steps

(a) (iv) optionally side chain protected peptide of formula (V) wherein P1 is a protecting group stable to catalytic hydrogenation

P1-D-Phe¹-Pro-Arg-Pro-OH(SEQ ID NO 4) (V)，

With optionally side chain-protected peptides of the formula (VI)

H-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OP2(SEQ ID NO 5) (VI)，

Wherein P2 is a protecting group removable by catalytic hydrogenation and orthogonal to the optional side chain protecting group,

to prepare optionally side chain protected peptides of formula (VII) wherein P1 and P2 are as described above

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQ ID NO 2) (II)，

(c) Subjecting a side chain-protected peptide of formula (X) wherein P3 is a protecting group removable by catalytic hydrogenation

H-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 8)

(X)，

Phenylalanine of formula (XI) wherein P4 is a protecting group orthogonal to the side chain protecting group of the peptide of formula X and to P3

P4-Phe¹²-OH (XI)

P4-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ IDNO 9) (XII)，

(d) Removing P4 from the peptide prepared in step (c) to produce the corresponding N-terminally deprotected side chain protected peptide of formula (XII),

(e) reacting the peptide of formula XII prepared in step (d) with side chain protected aspartic acid of formula (XIII)

P5-Asp¹¹-OH (XIII)，

Wherein P5 is a protecting group orthogonal to the side chain protecting groups of the peptides/amino acids of formula XII and formula XIII and to P3 to prepare peptides wherein P3 and P5 are side chain protected of formula (XIV) as defined above

P5-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 3) (XIV)，

H-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 3) (III)，

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Cly-Cly-Cly-Asn-Cly¹⁰-Asp-Phe--Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 1)

(IV)，

(h) Removing P1, P3 and side chain protecting groups of the peptide prepared in step (g) to prepare bivalirudin of formula (I)

H-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OH(SEQ ID NO 1)。

Here and hereinafter, the term "orthogonal" as a descriptive feature of the behavior of two different protecting groups should be understood to mean that one protecting group can be cleaved by some method that does not affect the other protecting group. For example, "a protecting group orthogonal to a side chain protecting group" refers to a protecting group that can be cleaved by some method that does not affect the side chain protecting group.

The advantage of this strategy is that it can be applied on an industrial scale, thus bivalirudin can be prepared with excellent purity and in kilogram quantities per batch, and does not form an image of D-Phe¹²-bivalirudin, D-Tyr¹⁹-bivalirudin or Asp⁹-undesired impurities of bivalirudin. For example, when the protected fragment Asp-Phe¹²Upon coupling with a protected peptide fragment of formula X, 5% D-Phe can be formed¹²Bivalirudin, whereas this formation can be surprisingly carried out by the process of the inventionAnd (4) inhibiting. The catalytic hydrogenation reaction as applied in the process of the present invention has the advantages of: unlike other deprotection methods, this catalytic hydrogenation reaction is a very clean reaction process that does not induce the formation of carbenium ions and therefore does not form unwanted by-products resulting from the reaction between the carbenium ions and the target peptide. For comparison, protection is achieved in the route disclosed in WO2007/033383 by using tert-butyl (tBu; e.g.tert-butyl ether or e.g.tert-butyl ester), which is a protecting group that can only be cleaved by acid hydrolysis (e.g.using hydrochloric acid or trifluoroacetic acid). The acid hydrolysis induces the formation of by-products (e.g., tert-butylated tyrosine) that are difficult to remove in the final product due to their similar physicochemical properties. Another disadvantage of acid hydrolysis is that handling large amounts of strong acids (e.g. trifluoroacetic acid) can cause production and environmental safety problems, especially on an industrial scale.

For further comparison, if a C-terminal protecting group (e.g., Et) removable by saponification is used as P2 for the peptides of formula VI and formula VII, its removal under alkaline conditions induces an asparagine residue (-Asn)⁹-) and an arginine residue (-Arg)³-) are cleaved extensively. Even if the saponification is not performed with an acid or with a base, but with a milder enzymatic method (e.g., saponification with subtilisin), it is observed that Asp is caused by cleavage of the asparagine residue⁹-the massive formation of bivalirudin.

Before, during and after the individual reaction steps of the invention, all peptide fragments as well as all coupling products may be present as such or in the form of suitable salts, depending on the physicochemical properties of the molecule and/or the reaction conditions. Suitable salts are, for example, those with Triethylamine (TEA), Dicyclohexylamine (DCHA), hydrochloric acid (HCl) and trifluoroacetic acid (TFA).

In step (h), P1, P3 and the side chain protecting groups may be removed later or simultaneously.

Preferably, in step (h), the P3 and side chain protecting groups are first removed simultaneously, followed by the removal of P1.

Generally, the peptide fragments obtained after each of the steps (a) to (g) will be separated before proceeding to the next step. Applicants have surprisingly found that in step (h) the isolation of the peptide obtained after the simultaneous removal of P3 and the side chain protecting groups can be dispensed with before the removal of P1, while obtaining similar yields and without negatively affecting the purity. This is surprising because normally a separation step is necessary to remove by-products which may also react in the next deprotection step and thus reduce the purity of the target peptide. Since isolation is usually accompanied by a loss of product, this finding has a positive impact on the cost and time of the overall process and generally results in higher yields of P1-deprotected peptide. Thus, in a more preferred embodiment of the process according to the invention, in step (h), the peptide obtained after simultaneous removal of P3 and the side chain protecting group is not isolated prior to removal of P1.

Any known protecting group stable to catalytic hydrogenation reactions may be used as P1. Suitable examples are tert-butoxycarbonyl (Boc), 2- (diphenyl-4-yl) prop-2-yloxycarbonyl (Bpoc), 2- (3, 5-dimethoxyphenyl) prop-2-yloxycarbonyl (Ddz), fluoren-9-ylmethoxycarbonyl (Fmoc), adamantan-1-oxycarbonyl (Adc), tert-pentyloxycarbonyl (Aoc), diphenylphosphino (Dpp), 2- (methylsulfonyl) ethoxycarbonyl (Msc) and phthaloyl (Pht). Preferably, P1 is Boc, Bpoc, Ddz, Fmoc or Msc, more preferably P1 is Boc.

Any of the well-known suitable protecting groups orthogonal to the side chain protecting groups of the fragment of formula II (for P4), the amino acid of formula XIII and the fragment of formula XIV (for P5), respectively, and to P3, can be used as protecting groups P4 and P5. Preferably, P4 and P5 are stable to catalytic hydrogenation reactions and are orthogonal to the side chain protecting groups and to P3. For example, suitable protecting groups are Boc, Bpoc, Ddz, Fmoc, Adc, Aoc, Dpp, Msc, and Pht. Preferably, P4 and/or P5 is Boc, Bpoc, Ddz, Fmoc or Msc; more preferably P4 and/or P5 is Boc.

In a preferred embodiment of the process according to the invention, at least one of P1, P4 and P5 is Boc, Bpoc, Ddz, Fmoc or Msc; preferably at least one of P1, P4 and P5 is Boc.

Any known protecting group which can be removed by catalytic hydrogenation can be used as the protecting group P3. Suitable examples are benzyl (Bzl), benzyloxymethyl (Bom), benzoyl (Pac), 4-nitrobenzyl (ONbz), 4-pyridylmethyl (Pic) and 4-sulfobenzyl. Preferably, P3 is Bzl, Bom, Pac, ONbz, Pic or 4-sulfobenzyl, with the proviso that if P4 or P5 is Boc, Bpoc or Ddz, P3 is not Bom. More preferably, P3 is Bzl. The protecting group Bom is acid sensitive, while the protecting groups Boc, Bpoc and Ddz are cleaved by acid, i.e. Bom is not orthogonal to Boc, Bpoc or Ddz. Here and below, this behavior excludes the simultaneous use of Bom and one of the protecting groups Boc, Bpoc or Ddz.

Any known protecting group which can be removed by catalytic hydrogenation-and in the case of side chain protection, is at the same time orthogonal to the side chain protecting group-can be used as protecting group P2. Suitable examples are Bzl, Bom, Pac, ONbz, Pic and 4-sulfobenzyl. Preferably, P2 is Bzl, Bom, Pac, ONbz, Pic or 4-sulfobenzyl; more preferably, P2 is Bzl.

In a preferred embodiment, at least one of P2 and P3 is Bzl, Bom, Pac, ONbz, Pic, or 4-sulfobenzyl, with the proviso that if P4 or P5 is Boc, Bpoc, or Ddz, P3 is not Bom. Preferably, at least one of P2 and P3 is Bzl.

In a more preferred embodiment, at least one of P1, P4, and P5 is Boc, Bpoc, Ddz, Fmoc, or Msc; and at least one of P2 and P3 is Bzl, Bom, Pac, ONbz, Pic, or 4-sulfobenzyl, with the proviso that if P4 or P5 is Boc, Bpoc, or Ddz, then P3 is not Bom. Preferably, at least one of P1, P4 and P5 is Boc and at least one of P2 and P3 is Bzl.

Preferably, in the method of the present invention, the side chain-protected peptides/amino acids of formulae III, IV, X and XII-XIV are protected with at least one side chain protecting group selected from Bzl, Bom, Pac, ONbz, Pic and 4-sulfobenzyl; with the proviso that if P4 or P5 is Boc, Bpoc or Ddz, none of the side chain protecting groups is Bom.

Specifically, in the methods described herein, side chain protected peptides/amino acids of formulas III, IV, X and XII-XIV are protected using at least one Bzl as a side chain protecting group.

Typically the peptides of formula V are side-chain protected at the arginine residue with a suitable side-chain protecting group such as a nitro group to avoid unwanted side reactions. In general, peptides of formula VI are side-chain protected at the asparagine residue with suitable side-chain protecting groups, in particular with Fomc at the N-terminus, to avoid unwanted side reactions. A suitable example is trityl (Trt), especially if Fomc is used for N-terminal protection.

Surprisingly, the applicants have found that for peptides of formula V and VI, side chain protection can be dispensed with, which makes it easier to assemble them and deprotect the C-terminus of their coupled product (peptide of formula VII). This finding allows to disregard the orthogonality between the C-terminal and side chain protection, thus making the route simpler. A further advantage is that the corresponding unprotected starting material is cheaper to purchase than the protected starting material, which is important especially for large-scale production.

Preferably, in the process of the invention, at least one of the optionally side chain protected peptides of formulae V and VI in step (a) is side chain unprotected. If the peptides are both side chain unprotected, the peptides of formulae VII and II obtained in steps (a) and (b) are also side chain unprotected.

More preferably, in the method of the present invention,

in step (a), the peptides of formulae V and VI are both side chain unprotected; in step (c), the peptide of formula X is protected with at least one side chain protecting group selected from Bzl, Bom, Pac, ONbz, Pic and 4-sulfobenzyl, preferably Bzl, with the proviso that if P4 and P5 are Boc, Bpoc or Ddz, none of said side chain protecting groups is Bom; and

in step (e), the aspartic acid derivative of formula XIII, preferably Bzl, is protected with a side chain protecting group selected from Bzl, Bom, Pac, ONbz, Pic and 4-sulfobenzyl, with the proviso that if P4 and P5 are Boc, Bpoc or Ddz, none of said side chain protecting groups is Bom.

Even more preferably, in the process of the invention, in step (a), the peptides of formula V and VI are both side chain unprotected; and in steps (c) and (e), at least one side chain protecting group is Bzl.

In a most preferred embodiment of the method of the invention, in step (c), the peptide of formula X is protected with five side chain protecting groups protecting the side chains of four glutamic acids and the side chain of tyrosine, thus providing a peptide of formula (Xb)

H-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-Glu(OP8)-Glu(OP9)-Tyr(P10)-Leu²⁰--OP3(SEQ ID 8) (Xb)，

Wherein P3 is a protecting group removable by catalytic hydrogenation, preferably P3 is Bzl, Bom, Pac, ONbz, Pic or 4-sulfobenzyl; more preferably P3 is Bzl; and

each of P6 to P10 is independently selected from Bzl, Bom, Pac, ONbz, Pic, and 4-sulfobenzyl; preferably each of P6 to P10 is Bzl; and in step (e) the side chain protected aspartic acid of formula XIII is

P5-Asp(OP 11)¹¹-OH，

Wherein P5 is a protecting group orthogonal to the side chain protecting groups of the peptides/amino acids of formulae XII and XIII and orthogonal to P3; preferably P5 is stable to catalytic hydrogenation reactions and is orthogonal to the side chain protecting groups and to P3; more preferably P5 is Boc, Bpoc, Ddz, Fmoc or Msc; most preferably P5 is Boc; and P11 is selected from Bzl, Bom, Pac, ONbz, Pic and 4-sulfobenzyl; preferably, P11 is Bzl.

Here and in the following, for the C-terminus and the side chain, the abbreviation "OP number" denotes the ester (after the reaction of both the side chain and the carboxylic acid at the C-terminus), while the abbreviation "P number" denotes the ether. For example, "OBzl" represents a benzyl ester (after reaction with the side chain or C-terminal carboxyl group), while the abbreviation "Bzl" represents a xylenyl ether (after reaction with a phenolic hydroxyl group such as tyrosine).

Preferably, in the process of the invention, P1, P4 and P5 are Boc; p2, P3, P6, P7, P8, P9, P10 and P11 are Bzl;

more preferably, P1, P4 and P5 are Boc, P2, P3, P6, P7, P8, P9, P10 and P11 are Bzl, and the peptides of formulae V and VI are side chain unprotected, providing a solution phase process comprising the steps of

(a) Make it

Boc-D-Phe¹-Pro-Arg-Pro-OH(SEQ ID NO 4)

And

H-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OBzl(SEQ ID NO 5)

reaction to produce

Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OBzl(SEQ ID NO 2)

(b) Removing Bzl of the peptide prepared in step (a) to prepare

Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQ ID NO 2)，

(c) Make it

H-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)-Glu(OBzl)--Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID 8)

And

Boc-Phe¹²-OH

reaction to produce

Boc-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)-Glu(OBzl)--Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID NO 9)，

(d) Removing Boc of the peptide prepared in step (c) to prepare a corresponding N-terminally deprotected peptide,

(e) contacting the peptide prepared in step (d) with

Boc-Asp(OBzl)¹¹-OH

Reaction to produce

Boc-Asp(OBzl)¹¹-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro--Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID NO 3)

(f) Removing Boc of the peptide prepared in step (e) to prepare

H-Asp(OBzl)¹¹-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID NO 3)

(g) Reacting the peptide prepared in step (b) with the peptide prepared in step (f) to prepare

Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰--Asp(OBzl)-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰-OBzl (SEQ ID NO 1), and (h) removing Boc, C-terminally protected Bzl and all side chain protected Bzl of the peptide prepared in step (g) to prepare bivalirudin of formula I.

This preferred embodiment is very simple and does not require complex protecting group strategies.

Preferably, in step (h), the C-terminal protected Bzl and all side chain protected Bzl are first removed simultaneously to give the N-terminal Boc-protected bivalirudin

Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp-Phe-Glu--Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OH (SEQ ID NO 1) and then removing Boc.

Preferably, the N-terminal Boc-protected bivalirudin obtained after simultaneous removal of the C-terminal protected Bzl and all side chain protected Bzl does not need to be isolated before removal of the Boc.

The side chain protecting groups of the C-terminal protecting groups P2, P3 and P6 to P11, in the case that they are removable by catalytic hydrogenation, can be removed by any method of catalytic hydrogenation known to those skilled in the art. The hydrogenation may be carried out by using the element hydrogen or by using a suitable hydrogen donor (e.g. formic acid, ammonium formate, 1, 3-cyclohexadiene, 1, 4-cyclohexadiene or borane adducts (e.g. tert-BuNH @)₂·BH₃) To complete). Suitable hydrogenation catalysts are, for example, noble metal-based hydrogenation catalysts, especially the metals known as platinum group metals (i.e., rhodium, ruthenium, palladium, osmium, iridium, and platinum). Conveniently, the hydrogenation catalyst is on a support such as carbon. Depending on the desired activity, the hydrogenation catalyst may be "poisoned" and in particular sulfided to reduce its activity.

Optionally, a suitable co-catalyst may be added to support the hydrogenation reaction. The cocatalyst may be a vanadium or molybdenum compound, such as vanadium (V) pentoxide₂O₅) Ammonium metavanadate (NH)₄VO₃) Or sodium molybdate (Na)₂MoO₄)。

In a preferred embodiment, the catalyst is recycled, which has a positive impact on both production costs and the environment. For the recovery of the catalyst, any treatment method suitable for recovering the catalyst may be employed.

Preferably, at least one of the steps (b) and (h) of removing is carried out in a solvent together with hydrogen and palladium on charcoal. As the solvent, any inert liquid solvent that can dissolve the reactants can be used. Solvents which may be used include halogenated aromatic hydrocarbons (e.g., chlorobenzene and trifluorotoluene); halogenated hydrocarbons (e.g., methylene chloride and ethylene dichloride); alcohols (e.g., methanol, ethanol, 2-propanol, butanol, and benzyl alcohol); halogenated alcohols (e.g., 2, 2, 2-trifluoroethanol); carboxylic acids (e.g., acetic acid); carboxylic acid esters and lactones (e.g., ethyl acetate, methyl acetate, and valerolactone); and organic solvents containing heteroatoms, such as N-methylpyrrolidone (NMP) or N, N-Dimethylformamide (DMF). The solvents can be used alone, or as a solvent mixture or as a mixture with water. Depending on the solubility of the peptide fragment, it is even possible to use only water as solvent. Thus, the removal in step (b) may be accomplished in a solvent employing only water.

Preferred solvents are selected from DMF, acetone, acetic acid, mixtures of acetone and water and mixtures of acetic acid and water.

In a preferred embodiment, said removing step (b) is performed in a solvent selected from the group consisting of DMF, acetone, water, a mixture of acetone and water; especially in DMF.

More preferably, said step (b) of removing is performed in DMF. Surprisingly, it has been found that the solvent employed in the removal step (b) has an effect on the final impurity profile of bivalirudin. It has been observed that DMF is advantageous compared with, for example, a mixture of acetone and water, so that it is possible to suppress the formation of-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰Formation of impurities following double incorporation of- (SEQ ID NO 2).

In another preferred embodiment, the removing step (h) is carried out in acetic acid or a mixture of acetic acid and water; in particular in a mixture of acetic acid and water.

The hydrogenation process of the removal steps (b) and (h) may be carried out at atmospheric or superatmospheric pressure. Typical pressures are from 1 to 100 bar. Advantageously, from 1 to 70bar is employed; in particular from 2 to 10 bar.

The hydrogenation reaction of the removal steps (b) and (h) may be carried out at low or elevated temperatures. Exemplary temperatures are-20 ℃ to 70 ℃. The preferred temperature is 0 ℃ to 60 ℃ and the more preferred temperature is 10 ℃ to 40 ℃.

The coupling steps (a), (c), (e) and (g) of the process of the invention are carried out in solution phase and under reaction conditions which are well known in the art of peptide synthesis. Coupling of the individual side chain unprotected or protected peptide fragments/amino acid derivatives can be accomplished using in situ coupling reagents such as phosphonium coupling reagents or urea coupling reagents such as benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate (BOP), benzotriazol-1-yloxy-tris (pyrrolidinyl) phosphonium hexafluorophosphate (PyBOP), O- (benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium Hexafluoroborate (HBTU), O- (6-chlorobenzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium Hexafluoroborate (HCTU), O- (6-chlorobenzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium tetrafluoroborate (TCTU), O- (7-azabenzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium Hexafluoroborate (HATU), O- (7-azabenzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium tetrafluoroborate (TATU), O- (benzotriazol-1-yl) -1, 1, 3, 3-tetramethyluronium tetrafluoroborate (TBTU), and O- [ cyano- (ethoxycarbonyl) methylamino ] -1, 1, 3, 3-tetramethyluronium tetrafluoroborate (TOTU), or carbodiimide coupling reagents such as Diisopropylcarbodiimide (DIC), Dicyclohexylcarbodiimide (DCC), and water-soluble carbodiimides (WSCDI) such as 1-ethyl-3- (3-dimethylaminopropyl) carbonbutamide (TOTU) Diimine (EDC). Other coupling methods employ previously prepared active esters (N-hydroxysuccinimide (HOSu) and p-nitrophenol (HONp) esters), previously prepared symmetrical anhydrides, asymmetric anhydrides such as N-carboxyanhydrides (NCAs), and acid chlorides (e.g., acid fluorides or acid chlorides). Preferred coupling reagents are carbodiimide coupling reagents, most preferred is DIC or EDC, suitable as EDC salts such as EDC & HCl.

The reaction mixture of the coupling steps (a), (c), (e) and (g) may advantageously comprise a base, preferably a tertiary amine base, which can deprotonate the carboxy component and neutralize the counter-ion of the amino component, thereby facilitating the in situ reaction. Suitable bases are, for example, trialkylamines, such as N, N-Diisopropylethylamine (DIPEA) or Triethylamine (TEA); n, N-dialkylanilines (e.g., N-diethylaniline); 2, 4, 6-trialkylpyridines (e.g. 2, 4, 6-trimethylpyridine); and N-alkyl morpholines (e.g., N-methyl morpholine). In particular, the reaction mixture advantageously comprises TEA or DIPEA as base.

The reaction mixture of the coupling steps (a), (c), (e) and (g) may also comprise an auxiliary affinity reagent as an additive, since the auxiliary affinity reagent has a positive effect on the inhibition of undesired side reactions. Any well-known auxiliary affinity reagent may be used. Examples of suitable auxiliary affinity reagents are 1-hydroxybenzotriazole (HOBt), N-hydroxysuccinimide (HOSu), N-hydroxy-3, 4-dihydro-4-oxo-1, 2, 3-benzotriazole (HOOBt) and 1-hydroxy-7-azabenzotriazole (HOAt). Preferably, the reaction mixture of the coupling step further comprises HOBt.

In a preferred embodiment, the coupling mixture of coupling steps (a), (c), (e) and (g) is selected from DIC/HOBt/TEA, EDC/HOBt/DIPEA and EDC/HOBt/TEA.

As solvent for the coupling steps (a), (c), (e) and (g) any inert liquid solvent can be used which can dissolve the reactants. Coupling solvents which may be used are water-soluble solvents (e.g. Dimethylsulfoxide (DMSO), dioxane, Tetrahydrofuran (THF), 1-methyl-2-pyrrolidone (NMP), N-Dimethylformamide (DMF), N-Dimethylacetamide (DMA) or any mixtures thereof); a water insoluble solvent (e.g., Dichloromethane (DCM), ethyl acetate, or any mixture thereof); and any suitable mixture between water-soluble and water-insoluble solvents (including mixtures with water). Preferred solvents are DMF or a mixture of DMF and water.

The N-terminal deprotection of steps (d), (f) and (h) may be carried out using reaction conditions well known in the art of peptide synthesis and depending on the nature of the protecting groups P1, P4 and P5. In the case where the protecting group is Boc, deprotection can suitably be accomplished using an acid (preferably trifluoroacetic acid) alone or as a mixture with an inert solvent (e.g., toluene, THF or a mixture of toluene and THF). In the case where the protecting group is Fmoc, deprotection of the N-terminus can be achieved by reaction with a base, advantageously with a secondary amine such as piperidine or diethylamine. In general, N-terminal deprotection can be carried out in a solvent, which can be any solvent that does not interfere with the reactants, such as chlorinated hydrocarbons such as dichloromethane; alkylated amides and lactams such as dimethylformamide or 1-methyl-2-pyrrolidone; aromatic hydrocarbons such as toluene; ethers such as THF or any mixture thereof. Preferably, the deprotection of the N-terminal Boc group is performed in toluene or in a mixture of phenol, toluene and THF.

The crude bivalirudin prepared in step (h) can be purified using conventional methods, such as preparative HPLC or counter-current distribution. The purification step can be repeated.

The same method can be applied to the peptide fragments obtained after steps (a) to (g).

The final bivalirudin of formula I can be isolated according to isolation methods well known in peptide chemistry (e.g. precipitation or freeze drying, also known as lyophilization).

The optionally side-chain protected peptides of formulae V and VI and the side-chain protected peptide of formula X may be prepared using conventional peptide synthesis methods such as solution phase synthesis (aka homogeneous peptide synthesis, abbreviated HPPS), Solid Phase Peptide Synthesis (SPPS), or a combination of SPPS and HPPS (aka mixed phase peptide synthesis, abbreviated MPPS) in mixed synthesis.

In one embodiment of the process of the invention, the optionally side chain protected peptide of formula V, the optionally side chain protected peptide of formula VI and the side chain protected peptide of formula X are prepared by solution phase synthesis in the aforementioned processAt least one peptide. Preferably, these peptides are represented by the corresponding dipeptides (i.e., sequence mode-D-Phe)¹-Pro-、-Arg³-Pro-、-Gly⁵-Gly-、-Gly⁷-Gly-、-Asn⁹-Gly-、-Glu¹³-Glu-、-Ile¹⁵-Pro-、-Glu¹⁷-Glu-or-Tyr¹⁹-Leu-) as a starting material. The N-terminal protecting group, C-terminal protecting group and side chain protecting group and reaction conditions may be well known to those skilled in the art, and are preferably the same as or similar to those described above.

In particular, a process for preparing an optionally side chain protected peptide of formula (V) in solution phase

P1-D-Phe¹-Pro-Arg-Pro-OH(SEQ ID NO 4) (V)，

Preferably the peptide Va with the side chain unprotected,

wherein

P1 is a protecting group, preferably P1 is a protecting group stable to catalytic hydrogenation, more preferably P1 is Boc, Bpoc, Ddz, Fmoc or Msc, most preferably P1 is Boc;

the method comprises the following steps:

(a) p12 from the removal of an optionally side chain protected dipeptide of formula (XVI)

P12-Arg-Pro⁴-OP13 (XVI)，

Preferably a dipeptide XVIa with unprotected side chains,

wherein

P12 is a protecting group, preferably P12 is a protecting group orthogonal to the side chain protecting group and orthogonal to P13, more preferably P12 is a protecting group stable to catalytic hydrogenation reactions and orthogonal to the side chain protecting group and orthogonal to P13, even more preferably P12 is Boc, Bpoc, Ddz, Fmoc or Msc, most preferably P12 is Boc; and

p13 is a protecting group such as Bzl, methyl (Me) or ethyl (Et); preferably P13 is a protecting group removable by catalytic hydrogenation and orthogonal to the side chain protecting groups, more preferably P13 is Bzl, Bom, Pac, ONbz, Pic and 4-sulfobenzyl, most preferably P13 is Bzl;

with the proviso that if P12 is Boc, Bpoc or Ddz, then P13 is not Bom; (b) reacting the N-terminally deprotected, optionally side-chain-protected dipeptide prepared in step (a), preferably the corresponding side-chain-unprotected dipeptide, with an optionally side-chain-protected dipeptide of formula (XVII)

P1-D-Phe¹-Pro-OW (XVII)，

Preferably with a dipeptide XVIIa whose side chain is unprotected,

wherein

P1 is as defined above and W is hydrogen or a preactivated group such as pentafluorophenyl

(Pfp)，

To prepare optionally side chain protected peptides of formula (XV)

P1-D-Phe¹-Pro-Arg-Pro-OP13(SEQ ID NO 4) (XV)，

Preferably the side chain unprotected peptide XVa,

wherein

P1 and P13 are as defined above, and

(c) removing P13 of the optionally side chain protected peptide of formula XV prepared in step (b), preferably the side chain unprotected peptide XVa, to prepare an optionally side chain protected peptide of formula V; preferably, peptide Va is prepared with unprotected side chains.

Also specifically, a process for the preparation of optionally side chain protected peptides of formula (VI), preferably side chain unprotected peptides VIa, in solution phase

H-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OP2(SEQ ID NO 5) (VI)，

Wherein

P2 is a protecting group, preferably P2 is a protecting group removable by catalytic hydrogenation and orthogonal to the optional side chain protecting group, more preferably P2 is Bzl, Bom, Pac, ONbz, Pic or 4-sulfobenzyl, most preferably P2 is Bzl;

the method comprises the following steps:

(a) the removal of the optionally side-chain-protected dipeptide of the formula (XVIII) is preferably P14 of the side-chain-unprotected dipeptide XVIIIa

P14-Asn-Gly¹⁰-OP2 (XVIII)，

Wherein

P14 is a protecting group, preferably P14 is a protecting group orthogonal to the side chain protecting group and orthogonal to P2, more preferably P14 is a protecting group stable to catalytic hydrogenation reactions and orthogonal to the side chain protecting group and orthogonal to P2, even more preferably P14 is Boc, Bpoc, Ddz, Fmoc or Msc, most preferably P14 is Boc; and

p2 is as defined above;

provided that if P14 is Boc, Bpoc or Ddz, then P2 is not Bom;

(b) reacting the N-terminally deprotected, optionally side-chain-protected dipeptide prepared in step (a), preferably the side-chain-unprotected dipeptide, with tetraglycine of formula (IXX)

P15-Gly⁵-Gly-Gly-Gly-OH(SEQ ID NO 10) (IXX)，

Wherein

P15 is a protecting group, preferably P15 is a protecting group orthogonal to the side chain protecting group of the peptide of formula XX, more preferably P15 is a protecting group stable to catalytic hydrogenation and orthogonal to the side chain protecting group of the peptide of formula XX, even more preferably P15 is Boc, Bpoc, Ddz, Fmoc or Msc, most preferably P15 is Boc;

to prepare optionally side chain protected peptides of formula (XX)

P15-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OP2(SEQ ID NO 5) (XX)，

Preferably the side chain unprotected peptide XXa,

wherein

P2 and P15 are as defined above,

with the proviso that if P15 is Boc, Bpoc or Ddz, then P2 is not Bom

And

(c) removing P15 of the optionally side chain protected peptide of formula XX, preferably side chain unprotected peptide XXa, prepared in step (b) to prepare an optionally side chain protected peptide of formula VI; the side chain unprotected peptide VIa is preferably prepared.

Further specifically, a process for preparing a side chain protected peptide of formula (X) in a solution phase

H-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 8)

(X)，

Preferred are peptides of formula (Xb)

H-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-Glu(OP8)-Glu(OP9)-Tyr(P10)--Leu²⁰-OP3(SEQ ID 8) (Xb)，

Wherein

P3 is a protecting group, preferably P3 is a protecting group removable by catalytic hydrogenation, more preferably P3 is Bzl, Bom, Pac, ONbz, Pic or 4-sulfobenzyl, most preferably P3 is Bzl, and

each of P6 to P10 is independently selected from Bzl, Bom, Pac, ONbz, Pic and 4-sulfobenzyl, preferably each of P6 to P10 is Bzl,

the method comprises the following steps:

(a) p16 from which the side chain protected peptide of formula (VIII) was removed

P16-Glu-Glu-Ty-Leu²⁰-OP3(SEQ ID NO 6) (VIII)，

P16, preferably a peptide of formula (VIIIb)

P16-Glu(OP8)-Glu(OP9)-Tyr(P10)-Leu²⁰-OP3(SEQ ID NO 6)

(VIIIb)，

Wherein

P3 and P8 to P10 are as defined above, and

p16 is a protecting group, preferably P16 is a protecting group orthogonal to the side chain protecting group and orthogonal to P3, more preferably P16 is a protecting group stable to catalytic hydrogenation reactions and orthogonal to the side chain protecting group and orthogonal to P3, even more preferably P16 is Boc, Bpoc, Ddz, Fmoc or Msc, most preferably P16 is Boc;

provided that if P16 is Boc, Bpoc or Ddz, then P3 is not Bom;

(b) reacting the N-terminally deprotected, side-chain-protected dipeptide of formula VIII prepared in step (a), preferably the corresponding peptide VIIIb, with a side-chain-protected peptide of formula (IX)

P17-Glu-Glu-Ile¹⁵-Pro-OH(SEQ ID NO 7) (IX)，

Preferably, it is

P17-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-OH(SEQ ID NO 7) (IXb)，

Wherein

P6 and P7 are as defined above, and

p17 is a protecting group, preferably P17 is a protecting group orthogonal to the side chain protecting group, more preferably P17 is a protecting group stable to catalytic hydrogenation reactions and orthogonal to the side chain protecting group, even more preferably P17 is Boc, Bpoc, Ddz, Fmoc or Msc, most preferably P17 is Boc;

to prepare a side chain protected peptide of formula (XXI)

P17-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 8)

(XXI)，

Preferred are peptides of formula (XXIb)

P17-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-Glu(OP8)-Glu(OP9)--Tyr(P10)-Leu²⁰-OP3(SEQ ID 8) (XXIb)，

Wherein

P3, P6 to P10 and P17 are as defined above,

with the proviso that if P17 is Boc, Bpoc or Ddz, then either P3 or P6 to P10 is not Bom, and

(c) removing the side chain protected peptide of formula XXI prepared in step (b), preferably P17 of the corresponding peptide XXIb, to prepare a side chain protected peptide of formula X; preferably, the peptide Xb is prepared.

In another embodiment of the method of the invention, at least one of the peptides is selected from the group consisting of an optionally side chain protected peptide of formula V, an optionally side chain protected peptide of formula VI and a side chain protected peptide of formula X, prepared by employing a solution phase synthesis method in the aforementioned method. Therefore, any known SPPS method including known SPPS building blocks and SPPS conditions may be employed. All resins known to the person skilled in the art and which can be used for the preparation of protected peptides can be used. Here, the resin should be interpreted in a broad manner. Thus, the term "resin" is understood to mean, for example, a solid support alone or directly linked to a linker, optionally selected between the two. The resin may be insoluble or soluble. Soluble polymer polyethylene glycol (soluble PEG polymer) is an example of a soluble resin solid support, thus forming a soluble peptide-resin after assembly of the individual building blocks. Preferred resins are polystyrene based resins with trityl or bromobenzhydryl groups. Examples of trityl resins are 2-chlorotrityl chloride resin (CTC resin), trityl chloride resin, 4-methyltrityl chloride resin and 4-methoxytrityl chloride resin. Preferably, the CTC resin is used for the synthesis of the peptide fragments.

It is another object of the present invention to provide peptides useful as intermediates in the process of the invention. In particular, one of these peptides is a peptide selected from (i) optionally side chain protected peptides of formula (V)

P1-D-Phe¹-Pro-Arg-Pro-OH(SEQ ID NO 4) (V)，

Wherein P1 is a protecting group, preferably P1 is a protecting group stable to catalytic hydrogenation, more preferably P1 is Boc, Bpoc, Ddz, Fmoc or Msc, most preferably P1 is Boc; and

the peptide is optionally side-chain protected at the arginine residue with a suitable side-chain protecting group such as nitro;

(ii) optionally side chain protected peptides of formula (VI)

H-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OP2(SEQ ID NO 5) (VI)，

Wherein P2 is a protecting group, preferably P2 is a protecting group removable by catalytic hydrogenation and orthogonal to optional side chain protecting groups, more preferably P2 is Bzl, Bom, Pac, ONbz, Pic and 4-sulfobenzyl, most preferably P2 is Bzl; and

the peptide is optionally side-chain protected on an asparagine residue using a suitable side-chain protecting group such as trityl (Trt);

(iii) optionally side chain protected peptides of formula (VII)

Wherein preferred definitions of P1, P2 and optional side chain protecting groups and P1, P2 and optional side chain protecting groups are as defined above;

(iv) side chain protected peptides of formula (IIa)

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQ ID NO 2) (IIa)，

Wherein preferred definitions of P1 and side chain protecting groups and P1 and optional side chain protecting groups are as defined above,

except for Boc-D-Phe¹-Pro-Arg(Pbf)-Pro-Gly⁵-Gly-Gly-Gly-Asn(Trt)--Gly¹⁰in-OH Pbf is pentamethyldihydrobenzofuran sulfonyl and Trt is other than trityl. The peptides have been disclosed in WO2007/033383 as products of solid phase synthesis on CTC resins. In contrast, the side chain protected peptides of formula IIa according to the invention can be synthesized by different methods, i.e. by solution phase synthesis;

(v) a side chain unprotected peptide of formula (IIb)

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQ ID NO 2) (IIb)，

Wherein preferred definitions of P1 and P1 are as defined above;

(vi) side chain protected peptides of formula (X)

H-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 8)

(X)，

Wherein P3 is a protecting group, preferably P3 is a protecting group removable by catalytic hydrogenation, more preferably P3 is Bzl, Bom, Pac, ONbz, Pic or 4-sulfobenzyl, most preferably P3 is Bzl; and

(each) the side chain protecting group is at least one suitable side chain protecting group, preferably selected from Bzl, Bom, Pac, ONbz, Pic and 4-sulfobenzyl, more preferably Bzl;

(vii) side chain protected peptides of formula (XII)

P4-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 9)

(XII)，

Wherein preferred definitions of P3 and side chain protecting groups and P3 and optionally side chain protecting groups are as defined above; and

p4 is a protecting group, preferably P4 is a protecting group orthogonal to the side chain protecting group of the peptide of formula X and orthogonal to P3, more preferably P4 is Boc, Bpoc, Ddz, Fmoc or Msc, most preferably P4 is Boc, with the proviso that if P4 is Boc, Bpoc or Ddz, then P3 is not Bom;

or

P4 is hydrogen;

(viii) side chain protected peptides of formula (XIV)

P5-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQID NO 3) (XIV)，

Wherein preferred definitions of P3 and side chain protecting groups and P3 and optionally side chain protecting groups are as defined above;

and

p5 is a protecting group, preferably P5 is a protecting group orthogonal to the side chain protecting groups of peptides/amino acids of formula XII and XIII and to P3, more preferably P5 is Boc, Bpoc, Ddz, Fmoc or Msc, most preferably P5 is Boc, with the proviso that if P5 is Boc, Bpoc or Ddz, then P3 is not Bom,

except that Fmoc-Asp (OtBu) -Phe-Glu (OtBu) -Ile¹⁵-Pro--Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu²⁰In OtBu, tBu is other than tert-butyl. The peptide has been disclosed in WO2007/033383 as the product of a reaction between a precursor peptide (i.e.the peptide sequence minus Leu) and H-Leu-OtBu. The precursor peptides as disclosed in WO2007/033383 have been formed in the preceding step by solid phase synthesis on CTC resins. After reaction with H-Leu-OtBu, the disclosed peptides were not isolated before proceeding to the next step. In contrast, the side chain protected peptides of formula XIV according to the invention can be synthesized by different methods, i.e. by solution phase synthesis methods, and isolated after their synthesis;

(ix) side chain protected peptides of formula (III)

H-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQID NO 3) (III)，

Wherein preferred definitions of P3 and side chain protecting groups and P3 and optionally side chain protecting groups are as defined above, except

H-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-

Ile¹⁵-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu²⁰-OtBu. Such peptides have been disclosed in WO 2007/033383. Which is the N-terminally deprotected peptide described above. Thus, as described above, the method of its synthesis is different from the side chain protected peptide of formula III of the present invention;

and

(x) Side chain protected peptides of formula (IV)

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp-Phe--Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 1)

(IV)，

Wherein preferred definitions of P1, P3 and side chain protecting groups and P1, P3 and optionally side chain protecting groups are as defined above,

except for Boc-D-Phe¹-Pro-Arg(Pbf)-Pro-Gly⁵-Gly-Gly-Gly-Asn(Trt)--Gly¹⁰-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile¹⁵-Pro-

-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu²⁰-OtBu. Said peptide has been disclosed in WO2007/033383 as being Boc-D-Phe¹-Pro-Arg(Pbf)-Pro--Gly⁵-Gly-Gly-Gly-Asn(Trt)-Gly¹⁰-OH and H-Asp (OtBu) -Phe-Glu (OtBu) -Ile¹⁵-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)--Leu²⁰-OtBu (all of which are described above) after coupling. Thus, the synthesis of its coupled product differs from the synthesis of the side chain protected peptide of formula IV of the present invention.

In a preferred embodiment, the peptide of formula V is side chain unprotected and has the formula

Boc-D-Phe¹-Pro-Arg-Pro-OH(SEQ ID NO 4)，

Which comprises the amino acid sequence of positions 1-4 of bivalirudin.

In another preferred embodiment, the peptide of formula VI is side chain unprotected and has the formula

H-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OBzl(SEQ ID NO 5)，

Which comprises the amino acid sequence of positions 5-10 of bivalirudin.

In another preferred embodiment, the peptide of formula VII is side chain unprotected and has the formula

Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OBzl(SEQ ID NO 2)，

Which comprises the amino acid sequence of positions 1-10 of bivalirudin.

In another preferred embodiment, the side chain unprotected peptide of formula IIb is

Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQ ID NO 2)，

Which comprises the amino acid sequence of positions 1-10 of bivalirudin.

In another preferred embodiment, the side chain protected peptide of formula X is

H-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)-Glu(OBzl)--Tyr(Bzl)-Leu²⁰-OBzl (SEQ ID NO 8)，

Which comprises the amino acid sequence of positions 13-20 of bivalirudin.

In another preferred embodiment, the side chain protected peptide of formula XII is

Boc-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)-Glu(OBzl)--Tyr(Bzl)-Leu²⁰-OBzl (SEQ ID NO 9), or

H-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)-Glu(OBzl)--Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID NO 9)，

Both of which include the amino acid sequence of positions 12-20 of bivalirudin.

In another preferred embodiment, the side chain protected peptide of formula XIV is

Boc-Asp(OBzl)-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID NO 3)，

Which comprises the amino acid sequence of positions 11-20 of bivalirudin.

In another preferred embodiment, the side chain protected peptide of formula III is

H-Asp(OBzl)-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID NO 3)，

Which comprises the amino acid sequence of positions 11-20 of bivalirudin.

In another preferred embodiment, the side chain protected peptide of formula IV is

Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰--Asp(OBzl)-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID NO 1)，

Which comprises the amino acid sequence of positions 1-20 of bivalirudin.

In another aspect, the invention relates to a peptide selected from the group consisting of

(i) Optionally side chain protected peptides of formula (V)

P1-D-Phe¹-Pro-Arg-Pro-OH(SEQ ID NO 4) (V)，

Wherein P1 is a protecting group, preferably P1 is a protecting group stable to catalytic hydrogenation, more preferably P1 is Boc, Bpoc, Ddz, Fmoc or Msc; most preferably P1 is Boc; and

preferably, the peptide V is side chain unprotected and has the formula

Boc-D-Phe¹-Pro-Arg-Pro-OH(SEQ ID NO 4)；

(ii) Optionally side chain protected peptides of formula (VI)

H-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OP2(SEQ ID NO 5) (VI)，

Wherein P2 is a protecting group, preferably P2 is a protecting group removable by catalytic hydrogenation and orthogonal to the optional side chain protecting groups, more preferably P2 is Bzl, Bom, Pac, ONbz, Pic or 4-sulfobenzyl, most preferably P2 is Bzl; and

preferably, the peptide VI is side chain unprotected and has the formula

H-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OBzl(SEQ ID NO 5)；

(iii) Optionally side chain protected peptides of formula (VII)

Wherein preferred definitions of P1, P2 and side chain protecting groups and P1, P2 and optionally side chain protecting groups are as defined above;

preferably, the peptide VII is side chain unprotected and has the formula

Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OBzl(SEQ ID NO 2)；

(iv) Side chain protected peptides of formula (IIa)

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQ ID NO 2) (IIa)，

Wherein preferred definitions of P1 and side chain protecting groups and P1 and optionally side chain protecting groups are as defined above,

except for Boc-D-Phe¹-Pro-Arg(Pbf)-Pro-Gly⁵-Gly-Gly-Gly-Asn(Trt)--Gly¹⁰-OH, wherein Pbf is pentamethyldihydrobenzofuran sulfonyl and Trt is trityl. The peptides have been disclosed in WO2007/033383 as products of solid phase synthesis on CTC resins. In contrast, the side chain protected peptides of formula IIa according to the invention can be synthesized by different methods, i.e. by solution phase synthesis;

(v) a side chain unprotected peptide of formula (IIb)

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQ ID NO 2) (IIb)，

Wherein preferred definitions of P1 and P1 are as defined above;

preferably, the side chain unprotected peptide of formula IIb is

Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQ ID NO 2)；

(vi) Side chain protected peptides of formula (X)

H-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 8)

(X)，

the side chain protecting group(s) is/are preferably selected from Bzl, Bom, Pac, ONbz, Pic and 4-sulfobenzyl, more preferably Bzl;

preferably, the side chain protected peptide of formula X is

H-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)-Glu(OBzl)--Tyr(Bzl)-Leu²⁰-OBzl (SEQ ID NO 8)；

(vii) Side chain protected peptides of formula (XII)

P4-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ IDNO 9) (XII)，

p4 is a protecting group, preferably P4 is a protecting group orthogonal to the side chain protecting group of the peptide of formula X and to P3, more preferably P4 is Boc, Bpoc, Ddz, Fmoc or Msc, most preferably P4 is Boc,

provided that if P4 is Boc, Bpoc or Ddz, then P3 is not Bom;

or

P4 is hydrogen;

preferably, the side chain protected peptide of formula XII is

H-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)-Glu(OBzl)--Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID NO 9)；

(viii) Side chain protected peptides of formula (XIV)

P5-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQID NO 3) (XIV)，

and

except that Fmoc-Asp (OtBu) -Phe-Glu (OtBu) -Ile¹⁵-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu²⁰In OtBu, tBu is other than tert-butyl. The peptide has been disclosed in WO2007/033383 as the product of the reaction between the precursor peptide (i.e.the peptide sequence minus Leu) and H-Leu-OtBu. The precursor peptide as disclosed in WO2007/033383 has been formed in the preceding step by solid phase synthesis on CTC resin. After reaction with H-Leu-OtBu, the disclosed peptides were not isolated before proceeding to the next step. After reaction with H-Leu-OtBu, the disclosed peptides were not isolated before proceeding to the next step. In contrast, the side chain protected peptides of formula XIV according to the invention can be synthesized by different methods, i.e. by solution phase synthesis methods and isolated after their formation; preferably, the side chain protected peptide of formula XIV is

Boc-Asp(OBzl)-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID NO 3)；

(ix) Side chain protected peptides of formula (III)

H-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQID NO 3)

Wherein preferred definitions of P3 and side chain protecting groups and P3 and optionally side chain protecting groups are as defined above,

except that H-Asp (OtBu) -Phe-Glu (OtBu) -Ile¹⁵-Pro--Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu²⁰-OtBu. Such peptides have been disclosed in WO 2007/033383. It is the N-terminally deprotected peptide described above. Thus, as described above, the method of its synthesis is different from the side chain protected peptide of formula III of the present invention;

preferably, the side chain protected peptide of formula III is

H-Asp(OBzl)-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID NO 3)；

And

(x) Side chain protected peptides of formula (IV)

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO 1) (IV)，

except for Boc-D-Phe¹-Pro-Arg(Pbf)-Pro-Gly⁵-Gly-Gly-Gly-Asn(Trt)--Gly¹⁰-Asp(OtBu)-Phe-Glu(OtBu)-Glu(OtBu)-Ile¹⁵-Pro--Glu(OtBu)-Glu(OtBu)-Tyr(tBu)-Leu²⁰-OtBu. Said peptide has been disclosed in WO2007/033383 as being Boc-D-Phe¹-Pro-Arg(Pbf)-Pro--Gly⁵-Gly-Gly-Gly-Asn(Trt)-Gly¹⁰-OH and H-Asp (OtBu) -Phe-Glu (OtBu) -Ile¹⁵-Pro-Glu(OtBu)-Glu(OtBu)-Tyr(tBu)--Leu²⁰-OtBu (all of which are described above) after coupling. Thus, the synthesis of its coupled product differs from the synthesis of the side chain protected peptide of formula IV of the present invention;

preferably, the side chain protected peptide of formula IV is

Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰--Asp(OBzl)-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰-OBzl(SEQ ID NO 1)；

Use as an intermediate in the synthesis of bivalirudin of formula (I)

H-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OH(SEQ ID NO 1) (I)。

The invention also relates to a process for the preparation of bivalirudin of formula I, comprising the following steps

(a) Reacting a peptide of formula (IIc) wherein P1 is a protecting group

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQ ID NO 2) (IIc)，

With a peptide of formula (IIIc) wherein each of P3 and P6 to P11 is Bzl

H-Asp(OP11)-Phe-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-Glu(OP8)-Glu(OP9)-Tyr(P10)-Leu²⁰-OP3(SEQ ID NO 3) (IIIc)，

To prepare peptides of formula (IVc) wherein P1, P3 and P6 to P11 are as defined above

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰--Asp(OP11)-Phe-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-Glu(OP8)--Glu(OP9)-Tyr(P10)-Leu²⁰-OP3(SEQ ID NO 1) (IVc)；

(b) Removing the side chain and C-terminal protecting groups P3 and P6 to P11 of the peptide prepared in step (a); and

(c) removing the N-terminal protecting group P1 of the peptide prepared in step (b) to prepare bivalirudin of formula I.

The method can efficiently synthesize bivalirudin by a convergent fragment synthesis method, and is easy to be applied to industrial production. Moreover, the route for preparing bivalirudin is very simple and does not require the adoption of complex protecting group strategies. In addition, various building blocks (fragments) can be selected to avoid or reduce racemization during assembly.

Before, during and after the individual reactions of the invention, all fragments and all coupling products may be present as such or in the form of suitable salts, depending on the physicochemical properties of the molecule and/or the reaction conditions. Suitable counterions are, for example, in the form of salts with Triethylamine (TEA), Dicyclohexylamine (DCHA), hydrochloric acid (HCl) and trifluoroacetic acid (TFA).

As the protecting group P1, any protecting group stable to catalytic hydrogenation such as t-butoxycarbonyl (Boc), fluoren-9-ylmethoxycarbonyl (Fmoc), 2- (3, 5-dimethoxyphenyl) propan-2-yloxycarbonyl (Ddz), adamantyl-1-yloxycarbonyl (Adc) (adamantyl-1-oxocarbonyl), t-pentyloxycarbonyl (Aoc), diphenylphosphino (Dpp), 2- (methylsulfonyl) ethoxycarbonyl (Msc) and phthaloyl (Pht) may be used. Preferably, the protecting group P1 is Boc, Fmoc or Ddz.

Preferably, the protecting groups P3 and P6 to P11 are benzyl (Bzl). Here and hereinafter, the abbreviation "OBzl" stands for benzyl ester (after reaction with the side chain or C-terminal carboxyl group), while the abbreviation "Bzl" stands for ditolyl ether (after reaction with the phenolic hydroxyl group of, for example, tyrosine).

Steps (a) to (c) can be carried out using standard reaction conditions well known in the art of peptide synthesis.

Coupling and deprotection steps (a), (b) and (c) are preferably carried out in solution.

For the coupling step (a), DMF is preferably used as solvent. The first deprotection step (b) is preferably carried out in acetic acid and/or water, while the second deprotection step (c) is preferably carried out in toluene.

In a preferred embodiment, the coupling step (a) is accomplished using a combination of HOBt, EDC. HCl and TEA.

In a preferred embodiment, the first deprotection step (b) is carried out using hydrogen and palladium on charcoal.

In a preferred embodiment, the second deprotection step (c) is performed using TFA.

The crude product obtained after step (c) can be purified by conventional methods, such as preparative HPLC, counter-current distribution or equivalent methods. If the intermediates obtained after steps (a) and (b) are to be purified, the same procedure can be used for purification.

Protected peptide fragments IIc and IIIc may be prepared using conventional peptide synthesis methods, such as solution phase synthesis (HPPS) or solid phase synthesis (SPPS). In the case of SPPS, all resins known to the person skilled in the art and which can be used for the preparation of protected peptides can be used. In this context, the resin should be interpreted in a broad manner. Thus, the term "resin" is understood to mean, for example, a solid support alone or directly linked to a linking molecule, optionally with a choice between the two. The resin may be insoluble or soluble. The soluble polymer polyethylene glycol is an example of a soluble resin solid carrier. Preferred resins are polystyrene based resins with trityl or bromobenzhydryl groups. Examples of trityl resins are 2-chlorotrityl chloride resin (CTC resin), trityl chloride resin, 4-methyltrityl chloride resin and 4-methoxytrityl chloride resin. Preferably, the CTC resin is used for the synthesis of fragments comprising free carboxyl functions.

In a preferred embodiment, the protected peptide fragments IIc and IIIc are prepared using solution phase synthesis.

It is another object of the present invention to provide peptides useful as intermediates in the process of the invention. Specifically, one of these peptides is a protected peptide of formula IVc, wherein P1 is a protecting group; preferably P1 is Boc or H; and P3 and P6 to P11 are Bzl.

Another peptide particularly useful as an intermediate in the process of the invention is an N-terminally protected peptide of formula IIc, wherein P1 is a protecting group, preferably P1 is Boc.

In another aspect, the invention also relates to a process for preparing an N-terminally protected peptide of formula (IIc) wherein P1 is Boc

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQ ID NO 2) (IIc)，

It includes:

(a) removing the C-terminal protecting group of the peptide of formula (Vc)

P1-D-Phe¹-Pro-Arg-Pro-OP13(SEQ ID NO 4) (Vc)，

Wherein P1 is Boc and P13 is a protecting group such as Bzl, methyl (Me) or ethyl (Et); preferably P13 is Bzl;

to obtain an N-terminally protected, C-terminally unprotected peptide of formula Vc wherein P13 is H;

(b) removing the N-terminal protecting group of the peptide of formula (VIc) wherein P15 is Boc

P15-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OBzl(SEQ ID NO 5)

(VIc)，

Obtaining a C-terminally protected, N-terminally unprotected peptide of formula VIc wherein P15 is H;

(c) reacting the peptide of formula Vc wherein P13 is H prepared in step (a) with the peptide of formula VIc wherein P15 is H prepared in step (b) to give a peptide of formula (VIIc) wherein P1 is Boc

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OBzl(SEQ ID NO 2) (VIIc)；

And

(d) removing the C-terminal protecting group of the peptide prepared in step (C) to obtain an N-terminally protected peptide of formula IIc.

Steps (a) to (d) may be carried out using standard reaction conditions well known in the art of peptide synthesis.

Coupling and deprotection steps (a), (b), (c) and (d) are preferably carried out in solution.

For the coupling step (c), DMF is preferably used as solvent. The first deprotection step (a) is preferably carried out in acetone, while the second deprotection step (b) is preferably carried out in toluene and/or THF. The third deprotection step (d) is preferably carried out in a solvent selected from DMF, acetone, water, a mixture of acetone and water.

In a preferred embodiment, the first deprotection step (a) is carried out using hydrogen and palladium on charcoal.

In a preferred embodiment, the second deprotection step (b) is performed using TFA.

In a preferred embodiment, the coupling step (c) is accomplished using a combination of HOBt, DIC and TEA.

In a preferred embodiment, the third deprotection step (d) is carried out using hydrogen and palladium on charcoal.

The crude product obtained after step (d) can be purified by conventional methods, such as preparative HPLC, counter-current distribution or equivalent methods. If the intermediates obtained after steps (a), (b) and (c) need to be purified, the same procedure can be used for purification.

The protected peptide fragments Vc and VIc may be prepared using conventional peptide synthesis methods, such as solution phase synthesis (HPPS) or solid phase synthesis (SPPS). In the case of SPPS, all resins known to the person skilled in the art and which can be used for the preparation of protected peptides can be used. In this context, the resin should be interpreted in a broad manner. Thus, the term "resin" is understood to mean, for example, a solid support alone or directly linked to a linking molecule, optionally with a choice between the two. The resin may be insoluble or soluble. The soluble polymer polyethylene glycol is an example of a soluble resin solid support, thus creating a soluble peptide-resin conjugate. Preferred resins are polystyrene based resins with trityl or bromobenzhydryl groups. Examples of trityl resins are 2-chlorotrityl chloride resin (CTC resin), trityl chloride resin, 4-methyltrityl chloride resin and 4-methoxytrityl chloride resin. Preferably, the CTC resin is used for the synthesis of fragments comprising free carboxyl functions.

In a preferred embodiment, the protected peptide fragments Vc and VIc are prepared using solution phase synthesis.

It is another object of the present invention to provide protected peptides useful as intermediates in the processes described herein for preparing N-terminally protected peptides of formula IIc. Specifically, one of these peptides is a C-terminally and N-terminally protected peptide of formula Vc, wherein P13 is a protecting group, preferably Bzl; another of these peptides is an N-terminally protected peptide of formula Vc with a free C-terminus.

Another peptide particularly useful as an intermediate in the process of the invention is a C-terminally protected peptide of formula VIc, wherein P15 is Boc; or P15 is H.

Another peptide particularly useful as an intermediate in a process for preparing bivalirudin is a protected peptide of formula IIIc, wherein P3 and P6 to P11 are Bzl.

In another aspect, the invention also relates to a method for preparing protected peptides of formula (IIIc) wherein P3 and P6 to P11 are Bzl

H-Asp(OP11)-Phe-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-Glu(OP8)--Glu(OP9)-Tyr(P10)-Leu²⁰-OP3(SEQ ID NO 3) (IIIc)，

It includes:

(a) removing the N-terminal protecting group of the peptide of formula (VIIIc)

P16-Glu(OP8)-Glu(OP9)-Tyr(P10)-Leu²⁰-OP3(SEQ ID NO 6)

(VIIIc)，

Wherein P3 and P8 to P10 are Bzl and P16 is Boc,

to obtain a C-terminally protected, N-terminally unprotected peptide of the formula VIIIc in which P16 is H;

(b) reacting the peptide of formula VIIIc prepared in step (a) wherein P16 is H and P3 and P8 to P10 are Bzl with the peptide of formula (IXc) wherein P17 is Boc and P6 and P7 are Bzl

P17-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-OH(SEQ ID NO 7)

(IXc)，

To obtain a peptide of formula (Xc) wherein P17 is Boc and P3 and P6 to P10 are Bzl

P17-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-Glu(OP8)-Glu(OP9)--Tyr(P10)-Leu²⁰-OP3(SEQ ID NO 8) (Xc)；

(c) Removing the N-terminal protecting group of the peptide of formula Xc prepared in step (b), wherein P3 and P6 to P10 are Bzl; and P17 is a Boc, and,

to obtain a C-terminally protected, N-terminally unprotected peptide of the formula Xc in which P3 and P6 to P10 are Bzl and P17 is H;

(d) reacting the peptide of formula Xc prepared in step (c) wherein P17 is H and P3 and P6 to P10 are Bzl with a protected amino acid of formula (XI)

Boc-Phe¹²-OH (XI)，

To obtain a peptide of formula (XIic) wherein P3 and P6 to P10 are Bzl and P4 is Boc

P4-Phe-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-Glu(OP8)-Glu(OP9)--Tyr(P10)-Leu²⁰-OP3(SEQ ID NO 9) (XIIc)，

(e) Removing the N-terminal protecting group of the peptide of formula XIic prepared in step (d), wherein P3 and P6 to P10 are Bzl and P4 is Boc,

to obtain a C-terminally protected, N-terminally unprotected peptide of the formula XIIc in which P3 and P6 to P10 are Bzl and P4 is H;

(f) contacting the peptide of formula XIIc prepared in step (e) wherein P4 is H and P3 and P6 to P10 are Bzl with a protected amino acid of formula (XIIIC) wherein P11 is Bzl

Boc-Asp(OP11)¹¹-OH (XIIIc)，

Reacting to obtain a peptide of formula (XIVc) wherein P3 and P6 to P11 are Bzl

Boc-Asp(OP11)-Phe-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-Glu(OP8)--Glu(OP9)-Tyr(P10)-Leu²⁰-OP3(SEQ ID NO 3)；

And

(g) removing the N-terminal protecting group of the peptide prepared in step (f) to obtain a C-terminally protected peptide of formula IIIc.

Steps (a) to (g) are carried out using standard reaction conditions well known in the art of peptide synthesis.

Coupling and deprotection steps (a) to (g) are preferably carried out in solution.

For coupling steps (b), (d) and (f), preferably DMF is used as solvent. The deprotection steps (a), (c), (e) and (g) are preferably carried out in a mixture of toluene and THF as solvent.

In a preferred embodiment, coupling steps (b), (d) and (f) are accomplished using a combination of HOBt, EDC & HCl, while step (b) uses base TEA and steps (d) and (f) use DIPEA, respectively. In another preferred embodiment, the deprotection steps (a), (c), (e) and (g) are carried out using TFA and phenol.

The crude product obtained after step (g) can be purified by conventional methods, such as preparative HPLC, counter-current distribution or equivalent methods. If the intermediates obtained after steps (a) to (f) are to be purified, the purification can be carried out in the same way.

The protected peptide fragments VIIIc, IXc, XI and XIIIc can be prepared using conventional peptide synthesis methods, such as solution phase synthesis (HPPS) or solid phase synthesis (SPPS). In the case of SPPS, all resins known to the person skilled in the art and which can be used for the preparation of protected peptides can be used. In this context, the resin should be interpreted in a broad manner. Thus, the term "resin" is understood to mean, for example, a solid support alone or directly linked to a linking molecule, optionally with a choice between the two. The resin may be insoluble or soluble. The soluble polymer polyethylene glycol is an example of a soluble resin solid carrier. Preferred resins are polystyrene based resins with trityl or bromobenzhydryl groups. Examples of trityl resins are 2-chlorotrityl chloride resin (CTC resin), trityl chloride resin, 4-methyltrityl chloride resin and 4-methoxytrityl chloride resin. Preferably, the CTC resin is used for the synthesis of fragments comprising free carboxyl functions.

In a preferred embodiment, the protected peptide fragments VIIIc, IXc, XI and XIIIc are prepared using solution phase synthesis.

It is another object of the invention to provide protected peptides which are useful as intermediates in the process of the invention for the preparation of C-terminally protected peptides of formula IIIc. Specifically, one of these peptides is a protected peptide of formula XIVc, where P3 and P6 through P11 are Bzl.

Another peptide that may be particularly useful as an intermediate in the process of the invention is a side chain protected peptide of formula XIIc, wherein P3 and P6 to P10 are Bzl, and P4 is a Boc protecting group, or P4 is H.

Another peptide that may be particularly useful as an intermediate in the process of the invention is a protected peptide of formula Xc, wherein P3 and P6 to P10 are Bzl, and P17 is a Boc protecting group, or P17 is H.

Another peptide that may be particularly useful as an intermediate in the process of the invention is a protected peptide of formula IXc wherein P6 and P7 are Bzl and preferably P17 is Boc.

Another peptide that may be particularly useful as an intermediate in the process of the invention is a protected peptide of formula VIIIc, wherein P3 and P8 through P10 are Bzl, and P16 is a Boc protecting group, or P16 is H.

In another aspect, the present invention also relates to the use of any of the above peptides as intermediates in the synthesis of bivalirudin.

Detailed Description

The following non-limiting examples will describe in detail representative embodiments of the invention.

Abbreviations:

example 1: preparation of H-Asn-Gly ¹⁰ -OBzl·TFA

Boc-Asn-Gly-OBzl (90.20 kg; Hexagon Labs Inc., USA) was added to a mixture of TFA (90L), toluene (388L) and THF (45L) at 20 ℃. To the resulting mixture was added TFA (198L) slowly at ≦ 22 ℃. The reaction was allowed to complete at 20 ℃. Completion of the cleavage was monitored by HPLC.

Then, THF was added slowly and the reaction mixture was evaporated in vacuo. A triple azeotropic distillation was performed using a mixture of toluene and THF. Obtaining H-Asn-Gly¹⁰-OBzl · TFA as oily residue, diluted with ethyl acetate. The resulting solution was used directly in the next chemical step (see example 3). Yield: 100 percent. Purity (HPLC): 99.3 percent.

Example 2: preparation of Boc-Gly ⁵ -Gly-Gly-Gly-OH·TEA(SEQ ID NO 10)

To Boc-Gly at 20 deg.C⁵-Gly-OEt (SEQ ID NO 10) [98.11 kg; bonora et al, Gazzetta Chimica Italiana 1980, 110, 503-]TEA (73.1L) was added slowly to the suspension in a mixture of acetone (78.44L) and treated water (491L). The reaction was allowed to complete at 20 ℃. Completion of the saponification reaction was monitored by HPLC.

The solution was evaporated in vacuo and the volume of the residue was adjusted to 456L with treated water. Boc-Gly⁵The solution of-Gly-Gly-Gly-OH. TEA (SEQ ID NO 10) was used directly in the next chemical step (see example 3). Yield: 100 percent. Purity (HPLC): 99.6 percent.

Example 3: preparation of Boc-Gly ⁵ -Gly-Gly-Gly-Asn-Gly ¹⁰ -OBzl(SEQ ID NO 5)

Adjusting H-Asn-Gly with TEA at 0 deg.C¹⁰-OBzl. TFA (573L, see example 1) to pH 6-6.5 in ethyl acetate. Boc-Gly as prepared in example 2⁵A solution of-Gly-Gly-Gly-OH (SEQ ID NO 10) was cooled to 0 ℃ and added to the above solution, followed by HOBt (28.52kg) and EDC & HCl (69.81 kg). The pH was adjusted to 6-6.5 with TEA (121L) at 0 ℃. The reaction mixture was allowed to warm to room temperature.

After completion of the coupling reaction (after about 10 h; as indicated by HPLC), NaCl was added to the reaction mixture. The resulting suspension was cooled and filtered to give a solid residue, which was washed several times with an aqueous NaCl solution and then cooled. The resulting solid was dried in vacuo to yield 130.15kg (100%) of Boc-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰OBzl (SEQ ID NO 5) with a purity of 97.9% (HPLC).

Example 4: preparation of H-Gly ⁵ -Gly-Gly-Gly-Asn-Gly ¹⁰ -OBzl·TFA (SEQ ID NO 5)

Boc-Gly was added slowly to a mixture of TFA (131L), toluene (525L) and THF (105L) at ≤ 22 deg.C⁵-Gly-Gly-Gly-Asn-Gly¹⁰OBzl (SEQ ID NO 5) (131.20kg, see example 3). To the resulting reaction mixture was added slowly TFA (289L) at ≦ 22 ℃. The reactionThe reaction was complete (monitored by HPLC) at 20 ℃ over about 1.5 h.

The resulting reaction mixture was concentrated in vacuo and the resulting residue was poured into diisopropyl ether. The resulting suspension was filtered to give a solid, which was washed with diisopropyl ether several times and then dried in vacuo to give 130.99kg of H-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰OBzl. TFA (SEQ ID NO 5) with a purity of 97.9% (HPLC).

Example 5: preparation of H-Arg-Pro-OBzl.2HCl

To a suspension of Boc-Arg-Pro-OBzl & HCl (95.00 kg; Hexagon LabsInc., USA) in acetic acid (1910L) and THF (19L) was slowly added a solution of hydrochloric acid (1M) in acetic acid (380L) at ≤ 22 deg.C. Completion of the cleavage reaction was monitored by HPLC (after about 1 hour).

The resulting reaction mixture was concentrated in vacuo. First with acetic acid and then with DMF. This gave H-Arg-Pro-OBzl.2HCl as an oily residue, which was diluted with DMF to give a volume of about 380L. The resulting solution was used directly in the next chemical step (see example 7). Yield: 100 percent. Purity (HPLC): 96 percent.

Example 6: preparation ofBoc-D-Phe-Pro-OPfp

To a suspension of Boc-D-Phe-Pro-OH (33.33 kg; Bachem AG, Switzerland) in ethyl acetate (183L) was added a solution of HOPfp (17.78kg) in ethyl acetate (10L) at ≤ 24 ℃. To the resulting reaction mixture was slowly added a solution of DCC (21.44kg) in ethyl acetate (83.3L) at-6 ℃. The resulting mixture was then allowed to warm to room temperature. The completion of the coupling reaction was monitored by HPLC (about 2 h).

The DCU salt was removed by filtration and washed with ethyl acetate. The resulting filtrate was evaporated in vacuo until the residual volume reached about 80L. Multiple azeotropic distillations are carried out with toluene. The resulting oily residue was precipitated in petroleum ether. The resulting solid was filtered, washed with petroleum ether and dried in vacuo to give 26.3kg (54%) of Boc-D-Phe-Pro-OPfp with a purity of 99.3% (HPLC).

Example 7: preparation of Boc-D-Phe ¹ -Pro-Arg-Pro-OBzl·HCl(SEQ ID NO 4)

Dilution of H-Arg with DMF (360L) at 20 deg.C³-Pro-OBzl.2HCl (103.00 kg; 561L solution; see example 5). DMF (82L) was evaporated in vacuo at below 55 ℃. Then to H-Arg at 20 DEG³Boc-D-Phe was added to a solution of-Pro-OBzl.2HCl¹Pro-OPfp (121.54 kg; see example 6). The pH of the resulting mixture was adjusted to 6.5 at 0 ℃ using TEA (58L). The reaction was allowed to complete at 0 ℃ for about 20h as indicated by HPLC monitoring.

The solid portion of the resulting suspension was filtered off and washed with DMF. The resulting filtrate was concentrated in vacuo until a residual volume of about 385L was obtained. A mixture of deionized water, NaCl, and ethyl acetate was added. The phases are separated and successively treated with NaHCO₃Aqueous solution, Na₂CO₃Aqueous solution, HCl solution in brine and finally the organic phase obtained is washed with brine. The organic phase was concentrated in vacuo to give an oily residue, dried by azeotropic distillation with toluene and precipitated in diisopropyl ether at 20 ℃. The resulting suspension was filtered to give a solid, which was washed several times with diisopropyl ether and dried under vacuum to give 106kg of Boc-D-Phe¹-Pro-Arg-Pro-OBzl & HCl (SEQ ID NO 4) with a purity of 96% (HPLC).

Example 8: preparation of Boc-D-Phe ¹ -Pro-Arg-Pro-OH(SEQ ID NO 4)

Boc-D-Phe adjustment with a mixture of TFA/THF (50/50, V/V, 0.03L) at 20 deg.C¹-Pro-Arg-Pro-OBzl HCl (SEQ ID NO 4) (33.58kg, see example 7) in acetone (67L) to a pH of 4. To a suspension of palladium on charcoal (3.36kg) in acetone (17L) was added the resulting mixture. At 20 ℃ at 3The hydrogenation is carried out at a hydrogen pressure of bar for at least 2 h. The completion of the hydrogenation reaction was monitored by HPLC.

The reaction mixture was filtered through a cellulose filter. The resulting filter cake was washed several times with acetone. The combined filtrates were concentrated in vacuo. The oily residue obtained was dried by azeotropic distillation with a mixture of acetone and toluene. The resulting oily residue was diluted with ethyl acetate and poured into diisopropyl ether. The resulting suspension was filtered and the resulting precipitate was washed several times with diisopropyl ether and dried under vacuum to give 31.4kg of Boc-D-Phe¹-Pro-Arg-Pro-OH (SEQ ID NO 4) with a purity of 99.4% (HPLC).

Example 9: preparation of Boc-D-Phe ¹ -Pro-Arg-Pro-Gly ⁵ -Gly-Gly-Gly-Asn- -Gly ¹⁰ -OBzl(SEQ ID NO 2)

Regulating H-Gly with TEA (9L) at a temperature of less than or equal to 22 DEG C⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OBzl. TFA (SEQ ID NO 5) (25.20kg, see example 4) to pH 6.5-7 in DMF (504L) and deionized water (25L). Slowly adding Boc-D-Phe at a temperature of less than or equal to 22 DEG C¹-Pro-Arg-Pro-OH (SEQ ID NO 4) (25.46kg, see example 8) and HOBt (1.02 kg). DIC (9.5L) was slowly added to the resulting reaction mixture at ≤ 12 deg.C. The pH of the resulting mixture was adjusted to 7-7.5 using TEA (0.3L) at ≤ 12 deg.C. The reaction was carried out at 10 ℃ for 10 days until completion as indicated by HPLC and TLC monitoring.

The resulting reaction mixture was concentrated in vacuo to give an oily residue which precipitated into a mixture of ethyl acetate and diisopropyl ether. The supernatant was removed several times and replaced with the same volume of diisopropyl ether. The mixture was filtered to give a solid, which was washed three times with diisopropyl ether and dried under vacuum to give 30.50kg (76%) of Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OBzl(SEQ IDNO 2)，The purity was 83.4% (HPLC).

Example 10: preparation of Boc-D-Phe ¹ -Pro-Arg-Pro-Gly ⁵ -Gly-Gly-Gly-Asn- -Gly ¹⁰ -OH(SEQ ID NO 2)

Reacting Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰A solution of OBzl (SEQ ID NO 2) (23.43kg net peptide weight, see example 9) in a mixture of acetone (43L) and deionized water (9L) was added to a suspension of palladium on charcoal (0.75kg) in acetone (3L). The hydrogenation was carried out at 20 ℃ under a hydrogen pressure of about 3bar for 11 h. The completion of the hydrogenation reaction was monitored by HPLC.

Deionized water (36L) was added to the resulting reaction mixture, and the reaction mixture was filtered through a cellulose filter cartridge. The resulting filter cake was washed several times with a 2: 8 mixture of deionized water and acetone. The combined filtrates were concentrated in vacuo. Drying by azeotropic distillation of the resulting residue with a mixture of DMF and toluene gave 21.5kg of Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH (SEQ ID NO 2) with a purity of 80% (HPLC).

Example 11: preparation of Boc-Tyr (Bzl) -Leu ²⁰ -OBzl

H-Leu-OBzl p-toluenesulfonate (22.2 kg; Bachem AG, Switzerland) was suspended in ethyl acetate (108L) and TEA (ca. 7.1L) was added at room temperature. Boc-Tyr (Bzl) -OH (20 kg; Senn Chemicals, Switzerland) and HOBt (7.3kg) were added as solids. To the resulting mixture was added a solution of DCC (12.2kg) in DMF (40L) at-6 ℃. Completion of the reaction was monitored by TLC and HPLC.

The resulting DCU was removed by filtration. Sequentially adopting KHSO₄Aqueous solution and common saltMixture of water, KHSO₄Aqueous solution, NaHCO₃The resulting filtrate was washed with an aqueous solution and brine. The resulting organic phase was evaporated in vacuo. The residue obtained is dissolved in ethyl acetate and then precipitated in petroleum ether. The solid was filtered, washed with petroleum ether and dried in vacuo. The product is obtained again after vacuum concentration of the mother liquor solution and precipitation in petroleum ether. The solid was filtered, washed with petroleum ether and dried in vacuo. The resulting product was mixed twice to obtain 25.9kg (84%) of Boc-Tyr (Bzl) -Leu²⁰OBzl, 97% purity (HPLC).

Example 12: preparation of H-Tyr (Bzl) -Leu ²⁰ -OBzl·TFA

To Boc-Tyr (Bzl) -Leu at 20 deg.C²⁰To a mixture of-OBzl (25.9kg, see example 11), phenol (1.3kg), toluene (104L) and THF (21L) was added TFA (83L) slowly. The reaction was complete at 20 ℃. Completion of the cleavage reaction was monitored by HPLC.

The resulting reaction mixture was evaporated in vacuo. Azeotropic distillation was performed using a mixture of toluene and THF. Ethyl acetate and petroleum ether were then added to the residue. The solid was filtered, washed with a mixture of ethyl acetate and petroleum ether, then with petroleum ether and finally dried under vacuum to yield 23.7kg (89%) of H-Tyr (Bzl) -Leu²⁰OBzl. TFA, 98% pure (HPLC).

Example 13: preparation of Boc-Glu (OBzl) -Tyr (Bzl) -Leu ²⁰ -OBzl (SEQ ID NO 6)

To a solution of H-Tyr (Bzl) -Leu-OBzl. TFA (23.7kg, see example 12) in DMF (80L) was added Boc-Glu (OBzl) -OSu (42.90kg, Senn Chemicals, Switzerland). The pH of the resulting reaction mixture was slowly adjusted to 7-7.5 with DIPEA at 0 ℃. The completion of the coupling reaction was monitored by HPLC.

The resulting reaction mixture was evaporated in vacuo andthe oily residue was poured into treated water. The solid was filtered, washed with treated water, reslurried in a mixture of acetonitrile and treated water, and finally dried under vacuum to give 40.3kg of Boc-Glu (OBzl) -Tyr (Bzl) -Leu²⁰OBzl (SEQ ID NO 6) with a purity of 92% (HPLC).

Example 14: preparation of H-Glu (OBzl) -Tyr (Bzl) -Leu ²⁰ - OBzl·TFA(SEQ ID NO 6)

To a mixture of TFA (117L), phenol (5.87kg), toluene (470L) and THF (94L) was slowly added Boc-Glu (OBzl) -Tyr (Bzl) -Leu at 15 ℃²⁰OBzl (SEQ ID NO 6) (117.45kg, see example 13). Additional TFA (282L) was added slowly to the resulting mixture at ≦ 17 deg.C. The reaction was completed at 15 ℃. Completion of the cleavage reaction was monitored by HPLC (3 h).

The resulting reaction mixture was evaporated in vacuo at below 35 ℃. Residual TFA was removed by azeotropic distillation with toluene/THF and then with toluene. To the resulting oily residue was added MTBE (825L). Then, petroleum ether was added to the resulting suspension. After cooling, the solid obtained is filtered and washed several times with petroleum ether. After resuspension in petroleum ether and filtration, the resulting solid was washed with petroleum ether. This operation is repeated again. The solid was then dried under vacuum to yield 114.86kg (96%) of H-Glu (OBzl) -Tyr (Bzl) -Leu²⁰-OBzl. TFA (SEQ ID NO 6) with a purity of 96% (HPLC).

Example 15: preparation of Boc-Glu (OBzl) -Ile ¹⁵ -Pro- -OH·DCHA(SEQ ID NO 7)

To a solution of H-Ile-Pro-OH & TFA (36kg, Bachem AG, Switzerland) in DMF (433L) was slowly added Boc-Glu (OBzl) -OSu (62kg, Senn Chemicals, Switzerland). The pH was adjusted to 7-7.5 with DIPEA at 0 ℃. The completion of the reaction was monitored by HPLC.

The resulting reaction mixture was evaporated under vacuum and the resulting oily residue was diluted with ethyl acetate. With KHSO₄The resulting mixture was washed with an aqueous solution and brine. The resulting organic phase was evaporated in vacuo and the oily residue was dried by azeotropic distillation with toluene. The resulting oily residue was diluted with toluene and the pH adjusted to 7-7.5 with DCHA. Petroleum ether is then added to the resulting mixture. The resulting solid was then filtered, washed with petroleum ether and dried under vacuum to give 102kg of Boc-Glu (OBzl) -Ile¹⁵Pro-OH DCHA (SEQ ID NO 7) with a purity of 89% (HPLC).

Example 16: preparation of Boc-Glu (OBzl) -Ile ¹⁵ -Pro-Glu(OBzl)- -Glu(OBzl)-Tyr(Bzl)-Leu ²⁰ -OBzl(SEQ ID NO 8)

At the temperature of less than or equal to 24 ℃ to H-Glu (OBzl) -Tyr (Bzl) -Leu²⁰Boc-Glu (OBzl) -Ile (OBzl) -TFA (SEQ ID NO 6) (110.39kg, net peptide weight, see example 14) in DMF (451L) was added slowly to the solution¹⁵Pro- -OH DCHA (SEQ ID NO 7) (98.88kg, see example 15) and HOBt (13.82 kg). EDC. HCl (22.99kg) was added in small portions to the resulting mixture at-6 ℃. The pH of the resulting reaction mixture was adjusted to 6.5-7 with TEA (12L) at-6 ℃. Completion of the coupling reaction was monitored by HPLC at 10 ℃.

The salt is removed by filtration. The resulting filtrate was evaporated in vacuo. The oily residue obtained (470L) is in NaHCO₃Precipitating in the solution. The solid was filtered and washed with treated water several times. After resuspending in treated water, acetonitrile was added. The resulting suspension is then cooled. The solid was filtered, washed with water and dried under vacuum to give 160.34kg (88%) of Boc-Glu (OBzl) -Ile¹⁵-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)--Leu²⁰OBzl (SEQ ID NO 8) with a purity of 90% (HPLC).

Example 17: preparation of H-Glu (OBzl) -Ile ¹⁵ -Pro-Glu(OBzl)- -Glu(OBzl)-Tyr(Bzl)-Leu ²⁰ -OBzl·TFA(SEQ ID NO 8)

Adding Boc-Glu (OBzl) -Ile to a mixture of TFA (72L), toluene (289L) and THF (6L) at ≤ 15 deg.C¹⁵-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)--Leu²⁰OBzl (SEQ ID NO 8) (72.24kg, see example 16). To the resulting mixture was added slowly additional TFA (159L) at ≦ 22 ℃. The reaction was completed (monitored by HPLC) at 20 ℃ for 2.5 h. The reaction mixture was evaporated in vacuo. Residual TFA was removed by multiple azeotropic distillations with a mixture of toluene and THF. The oily residue was diluted with toluene and poured into diisopropyl ether. The solid was filtered and washed several times with diisopropyl ether. After resuspension in a mixture of acetonitrile and diisopropyl ether, the solid is filtered, washed several times with diisopropyl ether and finally dried in vacuo to yield 61.7kg (87%) of H-Glu (OBzl) -Ile¹⁵-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)--Leu²⁰OBzl. TFA (SEQ ID NO 8) with a purity of 88.9% (HPLC).

Example 18: preparation of Boc-Phe-Glu (OBzl) -Ile ¹⁵ -Pro- -Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu ²⁰ -OBzl(SEQ ID NO 9)

At the temperature of less than or equal to 24 ℃ to H-Glu (OBzl) -Ile¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰A solution of-OBzl & TFA (SEQ ID NO 8) (61.63kg, see example 17) in DMF (264L) was added Boc-Phe-OH (10.03 kg; Senn Chemicals AG, Switzerland) and HOBt (4.95kg) slowly. EDC & HCl (8.53kg) was added slowly at-5 ℃. The pH of the resulting reaction mixture was gradually adjusted to 6.5-7 with DIPEA (47.5L) at-5 ℃. The coupling reaction is carried out at 10 DEG CIt should take 23h until the reaction is complete (monitored by HPLC).

The resulting reaction mixture was evaporated in vacuo. The oily residue obtained is taken up in NaHCO₃Suspending in a solution. The solid was filtered, washed several times with treated water and dried under vacuum to give 67.64kg (95%) of Boc-Phe-Glu (OBzl) -Ile15-Pro-Glu (OBzl) -Tyr (Bzl) -Leu²⁰OBzl (SEQ ID NO 9) with a purity of 87.9% (HPLC).

Example 19: preparation of H-Phe-Glu (OBzl) -Ile ¹⁵ -Pro- -Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu ²⁰ -OBzl·TFA(SEQ ID NO 9)

Boc-Phe-Glu (OBzl) -Ile (OBzl) -was added in small portions to a mixture of TFA (68L), phenol (3.38kg), toluene (271L) and THF (54L) at 15 deg.C¹⁵-Pro--Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu²⁰OBzl (SEQ ID NO 9) (67.66kg, see example 18). To the resulting mixture was added slowly additional TFA (149L) at 15 ℃. Completion of the cleavage reaction was monitored by HPLC (4.3 h).

The resulting reaction mixture was evaporated in vacuo. Residual TFA was removed by multiple azeotropic distillations with a mixture of toluene and THF. The oily residue was diluted with toluene and poured into diisopropyl ether. The solid was filtered, washed several times with diisopropyl ether and dried under vacuum to give 67.25kg (99%) of H-Phe-Glu (OBzl) -Ile¹⁵-Pro--Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu²⁰OBzl. TFA (SEQ ID NO 9) with a purity of 86.6% (HPLC).

Example 20: preparation of Boc-Asp (OBzl) -Phe-Glu (OBzl) -Ile ¹⁵ - -Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu ²⁰ -OBzl(SEQ ID NO 3)

At 20 deg.C or below to H-Phe-Glu (OBzl) -Ile¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰-OBzl. TFA (SEQ ID NO 9) (60.46kg net peptide weight, see example 19) in DMF (370L) Boc-Asp (OBzl)¹¹OH (20.22kg) (Senn Chemicals AG, Switzerland) and HOBt (8.42 kg). EDC. HCl (13.38kg) was slowly added to the resulting reaction mixture at-5 ℃. The pH of the resulting reaction mixture was gradually adjusted to 6.5-7 with DIPEA (27L) at-5 ℃. The coupling reaction was then allowed to react at 10 ℃ for 35h until the reaction was complete (monitored by HPLC).

The resulting reaction mixture was then evaporated in vacuo. The oily residue obtained was slowly added to NaHCO₃In aqueous solution. The resulting suspension was filtered and resuspended in deionized water. After filtration and washing with deionized water several times and once with acetonitrile and deionized water, the resulting solid was dried under vacuum to give 64kg (96%) of Boc-Asp (OBzl) -Phe-Glu (OBzl) -Ile¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰OBzl (SEQ ID NO 3) with a purity of 81.4% (HPLC).

Example 21: preparation of H-Asp (OBzl) -Phe-Glu (OBzl) -Glu- -Ile ¹⁵ -Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu ²⁰ -OBzl·TFA (SEQ ID NO 3)

Boc-Asp (OBzl) -Phe-Glu (OBzl) -Ile (Ile) was added in small portions to a mixture of TFA (64L), phenol (3.2kg), toluene (265L) and THF (51L) at a temperature below 15 deg.C¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰OBzl (SEQ ID NO 3) (64.00kg, see example 20). Additional TFA (141L) was added slowly to the resulting reaction mixture at ≦ 15 deg.C. The cleavage reaction was carried out at 15 ℃ for 4.8h (monitored by HPLC).

The resulting reaction mixture was concentrated in vacuo. At 20 ℃ the mixture is heatedThe oily residue obtained is precipitated by addition to diisopropyl ether. The resulting suspension was filtered, resuspended in diisopropyl ether and filtered again. The resulting solid was washed with diisopropyl ether and dried in vacuo to give 61.4kg (95%) of H-Asp (OBzl) -Phe-Glu (OBzl) -Ile (net peptide weight)¹⁵-Pro-Glu(OBzl)--Glu(OBzl)-Tyr(Bzl)-Leu²⁰OBzl. TFA (SEQ ID NO 3) with a purity of 77.6% (HPLC).

Example 22: preparation of Boc-D-Phe ¹ -Pro-Arg-Pro-Gly ⁵ -Gly-Gly-Gly- Asn-Gly ¹⁰ -Asp(OBzl)-Phe-Glu(OBzl)-Glu(OBzl)-Ile ¹⁵ -Pro-Glu(OBzl)- -Glu(OBzl)-Tyr(Bzl)-Leu ²⁰ -OBzl(SEQ ID NO 1)

To Boc-D-Phe at ≤ 24 deg.C¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH (SEQ ID NO 2) (65.47kg, see example 10) in a solution of DMF (345L) H-Asp (OBzl) -Phe-Glu (OBzl) -Ile was added in small portions¹⁵-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)-Leu²⁰-OBzl. TFA (SEQ ID NO 3) (104.40kg, see example 21) and HOBt (8.42 kg). EDC. HCl (12.46kg) was added slowly at-5 ℃. The pH of the resulting reaction mixture was gradually adjusted to 6.5-7 with TEA (16.5L) at-5 ℃. The coupling reaction was carried out at-5 ℃ for 22h until the reaction was complete (monitored by HPLC).

The resulting reaction mixture was slowly diluted with deionized water. The resulting suspension was filtered, washed with deionized water, and dried under vacuum to yield 72kg (50%) of Boc-D-¹Phe-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp(OBzl)--Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)-Glu(OBzl)-Tyr(Bzl)--Leu²⁰-OBzl(SEQ ID NO 1) The purity was 81.4% (HPLC).

Example 23: preparation of H-D-Phe ¹ -Pro-Arg-Pro-Gly ⁵ -Gly-Gly-Gly- -Asn-Gly ¹⁰ -Asp-Phe-Glu-Glu-Ile ¹⁵ -Pro-Glu-Glu-Tyr-Leu ²⁰ -OH·2TFA (bivalirudin) (SEQ ID NO 1)

Reacting Boc-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp(OBzl)-Phe-Glu(OBzl)-Glu(OBzl)-Ile¹⁵-Pro-Glu(OBzl)-Glu(OBzl)--Tyr(Bzl)-Leu²⁰A suspension of OBzl (SEQ ID NO 1) (15.00kg, see example 22) in acetic acid (53L) was added to a mixture of palladium on charcoal (1.13kg), deionized water (6L) and acetic acid (5L). The hydrogenation reaction was carried out at < 37 ℃ under a hydrogen pressure of 3bar (monitored by HPLC).

After completion of the reaction (5h), the resulting reaction mixture was cooled and diluted with deionized water and acetic acid. The reaction mixture was filtered through a cellulose cartridge and the cellulose cartridge was eluted multiple times with a mixture of acetic acid and treated water. The resulting filtrate was concentrated in vacuo. The water content of the residue obtained was determined by Karl Fischer titration (water content < 5.0%).

Toluene was then added to the oily residue, followed by TFA. The completion of the cleavage reaction (after one hour) was monitored by HPLC.

The resulting reaction mixture was concentrated in vacuo. The oily residue obtained is precipitated by addition to diisopropyl ether. The resulting suspension was filtered to give a solid, which was washed several times with diisopropyl ether and then dried in vacuo to give 8.41kg (78% by weight of neat peptide) of crude H-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰--Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-H.2TFA (bivalirudin) (SEQ ID NO 1), the purity of the crude bivalirudin was 65% (HPLC). The crude product is detected by HPLC to contain no D-Phe¹²Bivalirudin, i.e. no racemization at position 12.

The crude peptide was purified by preparative HPLC on a C18 reverse phase stationary phase. The crude peptide was purified by elution with an ammonium acetate/water/acetonitrile gradient in the first step, and then with a trifluoroacetic acid/water/acetonitrile gradient in the second step. The eluted fractions containing pure product were concentrated and lyophilized to give 5.89kg (70% based on bivalirudin content in crude peptide) of H-D-Phe as a white powder¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp-Phe-Glu--Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-H.2TFA (bivalirudin) (SEQ ID NO 1) with a purity of 99% (HPLC). No D-Phe could be detected¹²-bivalirudin and D-Tyr¹⁹Bivalirudin, and no more than 0.2% of each other impurities detected (HPLC).

Claims

1. Process for the preparation of bivalirudin of formula (I) in solution phase

H-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp-Phe--Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OH(SEQ ID NO1)（I），

Which comprises the following steps:

(a) optionally side chain protected peptides of formula (V) wherein P1 is a protecting group stable to catalytic hydrogenation

P1-D-Phe¹-Pro-Arg-Pro-OH(SEQ ID NO4)（V），

With optionally side chain-protected peptides of the formula (VI)

H-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OP2(SEQ ID NO5)（VI），

to prepare optionally side chain protected peptides of formula (VII) wherein P1 and P2 are as defined above

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OP2(SEQID NO2)（VII），

(b) Removing P2 from the peptide prepared in step (a) to prepare an optionally side chain protected peptide of formula (II) wherein P1 is as described above

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-OH(SEQID NO2)（II），

H-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO8)（X），

With phenylalanine of the formula (XI)

P4-Phe¹²-OH（XI），

Wherein P4 is a protecting group orthogonal to the side chain protecting group of the peptide of formula X and orthogonal to P3,

to prepare side chain protected peptides of formula (XII) wherein P3 and P4 are as defined above

P4-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO9)（XII），

P5-Asp¹¹-OH（XIII），

Wherein P5 is a protecting group orthogonal to the side chain protecting groups of the peptides/amino acids of formulae XII and XIII and to P3,

to prepare the side chain protected peptide P5-Asp-Phe-Glu-Glu-Ile wherein P3 and P5 are of formula (XIV) as defined above¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ IDNO3)（XIV），

H-Asp-Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ IDNO3)（III）

(g) Contacting the optionally side chain protected peptide of formula II prepared in step (b) with the peptide prepared in step (b)

(f) The side chain protected peptide of formula (III) prepared in (1) to prepare a side chain protected peptide of formula (IV) wherein P1 and P3 are as defined above

P1-D-Phe¹-Pro-Arg-Pro-Gly⁵-Gly-Gly-Gly-Asn-Gly¹⁰-Asp--Phe-Glu-Glu-Ile¹⁵-Pro-Glu-Glu-Tyr-Leu²⁰-OP3(SEQ ID NO1)（IV），

(h) Removing P1, P3 and the side chain protecting groups of the peptide prepared in step (g) to prepare bivalirudin of formula I.

2. The process of claim 1, wherein at least one of P1, P4, and P5 is tert-butoxycarbonyl, 2- (diphenyl-4-yl) propan-2-yloxycarbonyl, 2- (3, 5-dimethoxyphenyl) propan-2-yloxycarbonyl, fluoren-9-ylmethoxycarbonyl, or 2- (methylsulfonyl) ethoxycarbonyl.

3. The method of claim 2, wherein at least one of P1, P4, and P5 is Boc.

4. The process according to any one of claims 1 to 3, wherein at least one of P2 and P3 is benzyl, benzyloxymethyl, benzoyl, 4-nitrobenzyl, 4-pyridylmethyl, or 4-sulfobenzyl,

with the proviso that if P4 or P5 is Boc, Bpoc or Ddz, P3 is not Bom.

5. The method of claim 4, wherein at least one of P2 and P3 is Bzl.

6. The method according to claim 1, wherein the side chain-protected peptides/amino acids of formulae III, IV, X and XII-XIV are protected with at least one side chain protecting group selected from benzyl, benzyloxymethyl, benzoyl, 4-nitrobenzyl, 4-pyridylmethyl and 4-sulfobenzyl;

with the proviso that if P4 or P5 is Boc, Bpoc or Ddz, none of said

The side chain protecting group is Bom.

7. The method of claim 6, wherein the side chain protected peptides/amino acids of formulas III, IV, X and XII-XIV are protected with at least one Bzl.

8. The method of claim 1, wherein at least one of the optionally side chain protected peptides of formulae V and VI in step (a) is side chain unprotected.

9. The method according to claim 1, wherein the peptide of formula X in step (c) is a peptide of formula (Xb) below

H-Glu(OP6)-Glu(OP7)-Ile¹⁵-Pro-Glu(OP8)-Glu(OP9)--Tyr(P10)-Leu²⁰-OP3(SEQ ID8)（Xb），

Wherein

P3 is a protecting group removable by catalytic hydrogenation; and

each of P6 to P10 is independently selected from Bzl, Bom, Pac, ONbz, Pic, and 4-sulfobenzyl;

and in step (e) the side chain protected aspartic acid of formula XIII is

P5-Asp(OP11)¹¹-OH，

Wherein

P5 is a protecting group orthogonal to the side chain protecting groups of the peptides/amino acids of formulae XII and XIII and orthogonal to P3; and

p11 is selected from Bzl, Bom, Pac, ONbz, Pic and 4-sulfobenzyl.

10. The method of claim 9, wherein P3 is Bzl, Bom, Pac, ONbz, Pic, or 4-sulfobenzyl; each of P6 to P10 is independently Bzl, Bom, Pac, ONbz, Pic, or 4-sulfobenzyl; p5 is Boc, Bpoc, Ddz, Fmoc or Msc; and P11 is Bzl, Bom, Pac, ONbz, Pic or 4-sulfobenzyl.

11. The method of claim 10, wherein P3 is Bzl, each of P6-P10 is Bzl, P5 is Boc, and P11 is Bzl.

12. The method of claim 9, wherein P1, P4, and P5 are Boc; and P2, P3, P6, P7, P8, P9, P10 and P11 are Bzl.

13. The method of claim 1, wherein in step (h), P3 and the side chain protecting group are removed first simultaneously, followed by removal of P1.

14. The method of claim 13, wherein the peptide obtained after simultaneous removal of P3 and side chain protecting groups does not require isolation prior to removal of P1.

15. The method of claim 1, wherein at least one of the removing steps (b) and (h) is performed with hydrogen and palladium on charcoal in a solvent.

16. The process of claim 15, wherein the solvent is selected from the group consisting of N, N-dimethylformamide, acetone, a mixture of acetone and water, acetic acid, and a mixture of acetic acid and water.

17. The method of claim 1, wherein at least one selected from the group consisting of the optionally side chain protected peptide of formula V, the optionally side chain protected peptide of formula VI and the side chain protected peptide of formula X is prepared by employing a solution phase synthesis method in the foregoing methods.

18. The method of claim 1, wherein at least one selected from the group consisting of the optionally side chain protected peptide of formula V, the optionally side chain protected peptide of formula VI and the side chain protected peptide of formula X is prepared by employing a solid phase synthesis method in the foregoing methods.