WO2011023680A2

WO2011023680A2 - Processes for the synthesis and purification of heterobifunctional cross-linkers

Info

Publication number: WO2011023680A2
Application number: PCT/EP2010/062303
Authority: WO
Inventors: Andrew Wood; Helen Kathryn Smith; Martin Philip Wilmshurst; Son Thanh Van
Original assignee: Cytos Biotechnology Ag
Priority date: 2009-08-24
Filing date: 2010-08-24
Publication date: 2011-03-03
Also published as: WO2011023680A3

Abstract

The present invention relates to a novel process for the production of a compound of formula (1), wherein n = 1 to 10 and m = 1 to 10, in linear two-step synthesis, wherein an N-maleimido acid NHS ester of formula (6), wherein n = 1 to 10, occurs as an intermediate. The invention further provides processes for the purification of the product and of the intermediate. In particular, the invention provides a process for the production and purification of succinimidyl-6-[β-maleimidopropionamido]hexanoate (SMPH). SMPH is a compound of formula (1), wherein m = 5 and n = 2.

Description

Processes for the Synthesis and Purification of Heterobifunctional Cross-Linkers

[0001] The present invention relates to a novel process for the production of a compound of formula (1)

, wherein n = 1 to 10 and m = 1 to 10, in linear two-step synthesis, wherein an N-maleimido acid NHS ester of formula (6)

_s wherein n = 1 to 10, occurs as an intermediate. The invention further provides processes for the purification of the product and of the intermediate. In particular, the invention provides a process for the production and purification of succinimidyl-6-[β-maleimidopropionamido]hexanoate (SMPH). SMPH is a compound of formula (1), wherein m = 5 and n = 2.

[0002] A compound of formula (1), and in particular SMPH, is a heterobifunctional cross- linking molecule. One side of the molecule comprises an NHS (N-hydroxysuccinimide) ester, which can react with primary amines at pH 7 to 9 to form amide bonds. The other side comprises a maleimide group, which can react with thiols at pH 6.5 to 7.5 to form thioether bonds. For example, SMPH has been used for coupling Cys-containing peptides in a two step reaction to exposed Lys groups of the coat protein forming a virus like particle (WO 2002/056905 A2).

[0003] The synthesis of SMPH has previously been described (WO2005/113605). However, the process given was unsuitable for the production of large (> 100 g) quantities of SMPH due to the high dilutions used (2 g / 1). There was therefore a need for the development of an improved production and purification process for producing compounds of formula (1), wherein n = 1 to 10 and m = 1 to 10, including SMPH.

[0004] One advantage of the inventive processes is that they can be performed in significantly more concentrated solution than the process known in the art, without compromising product yield and purity. It has been found by the inventors that the use of solvents with specific physico-chemical properties, and hereby in particular the choice of polar aprotic organic solvents as the first organic solvent, is highly advantageous. It has been demonstrated that kilogram quantities of SMPH with a purity at least 99.5 % and containing no unidentified impurity more than 0.1 % can be produced in accordance with the invention.

[0005] The synthetic procedure underlying the invention is illustrated by Figure 1, which shows the synthesis of SMPH in linear two-step synthesis wherein N-maleimidopropionic acid NHS ester occurs as an intermediate.

Figure Legend

Figure 1: Synthetic route for the synthesis of SMPH. In Stage 1, maleic anhydride (2) is reacted with β-alanine (3) to give a di-acid intermediate (4). This is not isolated but undergoes cyclisation upon treatment with N-hydroxysuccinimide (NHS, 5) and an activating agent to give N-maleimidopropionic acid NHS ester (6). This is subsequently coupled in the second stage of the process with 6-aminohexanoic acid (7) t o g i v e 6-[β- maleimidopropionamido]hexanoic acid (8), which upon treatment with an activating agent, re-couples with the cleaved NHS moiety to give SMPH (1).

Definitions

[0006] "polar aprotic organic solvent": The term "polar aprotic organic solvent" as used herein refers to an organic solvents which do not have an acidic hydrogen, and which comprises a polarity relative to water of greater than 0.4, wherein water is 1.0. Thus, typically and preferably, polar aprotic organic solvents neither comprises hydroxy groups nor amine groups.

[0007] "activating agent": The term "activating agent" as used herein refers to a chemical compound which is capable of activating one or more carboxyl groups within carboxylic acids or carboxylic acid derivatives for nucleophilic reactions, wherein preferably said carboxyl groups include -C(O)X groups, wherein X = OH, NH₂ or NH-R. Typically and preferably, the term "activating agent" as used herein refers to a chemical compound which is capable of activating one or more carboxyl groups within carboxylic acids for nucleophilic reactions, wherein preferably said carboxyl groups include -C(O)X groups, wherein X = OH, NH₂ or NH-R. Typically and preferably, the term "activating agent" as used herein refers to a chemical compound which is capable of activating one or more carboxyl groups within carboxylic acids or carboxylic acid derivatives for nucleophilic reactions, wherein said chemical compounds are acid chlorides, carbodiimides and uranium salts. Typically and preferably, the term "activating agent" as used herein refers to a chemical compound selected from the group consisting of acid chlorides, carbodiimides and uranium salts. Further preferably, the term "activating agent" as used herein refers to a chemical compound which is capable of activating one or more carboxyl groups within carboxylic acids or carboxylic acid derivatives for nucleophilic reactions, wherein said chemical compound is a carbodiimide. Typically and preferably, the term "activating agent" as used herein refers to a chemical compound selected from carbodiimides.

[0008] "purity": The purity of a compound of formula (1), wherein m = 1 to 10 and n = 1 to 10, and hereby in particular the purity of SMPH is preferably determined by HPLC and expressed as percentage of total peak are (% a/a). Typically and preferably, said HPLC is performed with the following parameters:

Apparatus A liquid chromatograph fitted with a variable wavelength detector

(VWD) and column heater.

Column Luna Silica (2) 5μ 250 x 4.6mm

Eluent 0.1% orthophosphoric acid (v/v) in 1 :99 (v/v) water :acetonitrile

Flow rate 1 ml/min

Monitoring Wavelength 215 nm

Injection Volume 5μl

Column Temperature 25⁰C

Run Time 60 min

Typically and preferably the purity of SMPH is determined under conditions as disclosed in

Example 3.

[0009] "impurity": The term "impurity" as used herein refers to any compound which is detected by HPLC, preferably by a HPLC which is performed with the parameters disclosed above, and most preferably by HPLC as described in Example 3, which is not a compound of formula (1), wherein m = 1 to 10 and n = 1 to 10. Typically and preferably, the term impurity refers to any compound other than SMPH.

[0010] "unidentified impurity": The term "unidentified impurity" as used herein refers to any compound other than 3-maleimidopropionic acid (NHS ester), N-hydroxysuccinimide,

SMPH, 6-[β-maleimidopropionamido]hexanoic acid, diacid (stage 1 intermediate), di-NHS ester, uncylised dimer (stage 2 impurity), and maleic anhydride.

[0011] In one aspect, the invention provides a process for the production of a compound of formula (6)

said process comprising the steps of:

(a) reacting maleic anhydride with a compound of formula (3)

NH₂-(CH₂)_n-COOH (3);

(b) contacting the reaction product of (a) with N-hydroxysuccinimide (NHS) and an activating agent to give the compound of formula (6); wherein said process is performed in a first organic solvent, wherein said first organic solvent is a polar aprotic organic solvent; and wherein n = 1 to 10.

[0012] In one embodiment, the reaction product of (a) is a di-acid intermediate. In a further embodiment the reaction product of (a), and preferably said di-acid intermediate, is not isolated.

[0013] In a further embodiment said first organic solvent exhibits a relative polarity of greater than 0.4. In a preferred embodiment, said first organic solvent does not chemically react with said compound of formula (6), wherein n = 1 to 10. Thus, in a further preferred embodiment, said first organic solvent neither comprises hydroxy groups nor amine groups.

In a further preferred embodiment said first organic solvent exhibits a relative polarity of greater than 0.4, and said first organic solvent neither comprises hydroxy groups nor amine groups.

[0014] In a preferred embodiment said first organic solvent is selected from the group consisting of: (a) acetonitrile; (b) N,N'-dimethyl formamide (DMF); and (c) dimethyl sulfoxide (DMSO); and wherein preferably said first organic solvent is acetonitrile.

[0015] In a further embodiment said activating agent is selected from the group consisting of: (a) Λ/-(3-dimethylaminopropyl)-Λ/^r'-ethylcarbodiimide hydrochloride (EDCI); (b) dicyclohexyl carbodiimide (DCC); and (c) (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP); and wherein preferably said activating agent is N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI).

[0016] In a further embodiment n = 1, 2, 3, 4, 5, 6, 7, 8, 9, orlO, wherein preferably n = 1 to

4, and wherein most preferably n = 2. Thus, in a very preferred embodiment said compound of formula (3) is β-alanine and said compound of formula (6) is N-maleimidopropionic acid

NHS ester. [0017] In a further embodiment step (a) comprises providing maleic anhydride and a compound of formula (3), wherein n = 1 to 10, and wherein preferably n = 2, in said first organic solvent, wherein the concentration of said maleic anhydride and of said compound of formula (3) in said first organic solvent together is 2 % to 20 % (w/w), preferably 5 % to 10

% (w/w), and most preferably 8 % (w/w). In a preferred embodiment the ratio of maleic anhydride and of said compound of formula (3) is 10:9 (w/w).

[0018] In a further embodiment said process is performed in the absence of atmospheric oxygen.

[0019] The reaction temperature of the process, and hereby in particular the maximum reaction temperature occurring throughout said process depends on the boiling temperature of said first organic solvent. In one embodiment the maximum reaction temperature occurring throughout said process is at most 100 ⁰C, preferably at most 80 ⁰C, and most preferably at most 70 ⁰C. In a preferred embodiment said first organic solvent is acetonitrile and the maximum reaction temperature occurring throughout said process is at most 70 ⁰C.

[0020] In one embodiment, step (a) is performed at a temperature of 20 ⁰C to 100 ⁰C, preferably of 60 ⁰C to 80 ⁰C, and most preferably of 70 ⁰C.

[0021] In a further embodiment step (b) is initiated at a temperature of 0 ⁰C to 30 ⁰C, preferably at 0 ⁰C to 10 ⁰C, and most preferably at 0 ⁰C to 5 ⁰C, wherein during step (b) the temperature is increased to 20 ⁰C to 100 ⁰C, preferably to 60 ⁰C to 80 ⁰C, and most preferably to 70 ⁰C.

[0022] In a preferred embodiment step (b) comprises (i) contacting the reaction product of

(a) with N-hydroxysuccinimide (NHS); (ii) adding a first portion of said activating agent; (iii) incubating the mixture; (iv) adding a second portion of said activating agent; and (v) incubating said mixture.

[0023] In a further preferred embodiment steps (i) to (iv) are performed at a temperature of 0

⁰C to 30 ⁰C, preferably of 0 ⁰C to 10 ⁰C, and most preferably of 0 ⁰C to 5 ⁰C. In a further preferred embodiment step (v) is performed at a temperature of 20 ⁰C to 100 ⁰C, preferably of

60 ⁰C to 80 ⁰C, and most preferably of 70 ⁰C.

[0024] In a further preferred embodiment said first portion of said activating agent is about one molar equivalent of said maleic anhydride and / or said second portion of said activating agent is about one molar equivalent of said maleic anhydride.

[0025] In a preferred embodiment said first organic solvent is acetonitrile, and said activating agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI). In a further preferred embodiment said first organic solvent is acetonitrile, said activating agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI), and n = 2.

[0026] A further aspect of the invention is a process wherein a compound of formula (6) is used for the synthesis of a compound of formula (1), wherein m = 1 to 10, and n = 1 to 10. Thus, a further aspect of the invention is a process for the production of a compound of formula (1)

said process comprising the steps of:

(a) reacting a compound of formula (6)

with a compound of formula (7)

NH₂-(CH2)_m-COOH (7); and

(b) reacting the product of step (a) with an activating agent to give the compound of formula (1);

wherein said process is performed in a first organic solvent, wherein said first organic solvent is a polar aprotic organic solvent; and wherein m = 1 to 10, and n = 1 to 10.

[0027] In one embodiment said product of step (a) is a compound of formula (8)

wherein m = 1 to 10, and n = 1 to 10. In a further embodiment said product of (a), and preferably said compound of formula (8), wherein m = 1 to 10, and n = 1 to 10, is not isolated. [0028] In a further embodiment said compound of formula (6) is a compound of formula (6), wherein n = 1 to 10, and wherein said compound of formula (6) is obtainable by any one of the processes disclosed herein supra. Thus, in a further embodiment step (a) comprises providing a compound of formula (6), wherein n = 1 to 10, by any one of the processes disclosed herein supra.

[0029] In a further embodiment step (a) comprises providing a compound of formula (6), wherein n = 1 to 10, and wherein said providing comprises purifying said compound of formula (6) by any one of the processes disclosed herein infra.

[0030] In a further embodiment step (a) comprises providing a compound of formula (6), wherein n = 1 to 10, by any one of the processes disclosed herein supra, wherein said providing comprises purifying said compound of formula (6) by any one of the processes disclosed herein infra.

[0031] In a further embodiment said first organic solvent exhibits a relative polarity of greater than 0.4. In a further embodiment said first organic solvent does not chemically react with said compound of formulas (1) and (6), wherein m = 1 to 10 and / or wherein n = 1 to 10.

In a further embodiment said first organic solvent neither comprises hydroxy groups nor amine groups. In a preferred embodiment said first organic solvent exhibits a relative polarity of greater than 0.4, and neither comprises hydroxy groups nor amine groups.

[0032] In a preferred embodiment said first organic solvent is selected from the group consisting of: (a) acetonitrile; (b) N,N'-dimethyl formamide (DMF); and (c) dimethyl sulfoxide (DMSO); and wherein preferably said first organic solvent is acetonitrile.

[0033] In a further embodiment said activating agent is selected from the group consisting of: (a) Λ/-(3-dimethylaminopropyl)-Λ/^r'-ethylcarbodiimide hydrochloride (EDCI); (b) dicyclohexyl carbodiimide (DCC); and (c) (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP); and wherein preferably said activating agent is N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI).

[0034] In a further embodiment m = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and n = 1, 2, 3, 4, 5, 6, 7, 8,

9, or 10. In a preferred embodiment m = 3 to 7 and n = 1 to 4, wherein preferably m = 4 to 6 and n = 1 to 3, and wherein most preferably m = 5 and n = 2. Thus, in a very preferred embodiment said compound of formula (6) is N-maleimidopropionic acid NHS ester, said compound of formula (7) is 6-aminohexanoic acid, and said compound of formula (1) is

SMPH.

[0035] In a further embodiment step (a) comprises providing said compound of formula (6), wherein n = 1 to 10, and wherein preferably n = 2, and said compound of formula (7), wherein m = 1 to 10, and wherein preferably m = 5, in said first organic solvent, wherein the concentration of said compound of formula (6) and of said compound of formula (7) in said first organic solvent together is 2 % to 20 % (w/w), preferably 3 % to 10 % (w/w), and most preferably 7 % (w/w). In a further preferred embodiment said compound of formula (7) is provided in an amount of 0.95 to 1.1 mole equivalents of said compound of formula (6).

[0036] In a further embodiment said process is performed in the absence of atmospheric oxygen.

[0037] The reaction temperature of the process, and hereby in particular the maximum reaction temperature occurring throughout said process depends on the boiling temperature of said first organic solvent. In one embodiment the maximum reaction temperature occurring throughout said process is at most 100 ⁰C, preferably at most 80 ⁰C, and most preferably at most 70 ⁰C. In a preferred embodiment said first organic solvent is acetonitrile and the maximum reaction temperature occurring throughout said process is at most 70 ⁰C.

[0038] In one embodiment, step (a) is performed at a temperature of 20 ⁰C to 100 ⁰C, preferably of 60 ⁰C to 80 ⁰C, and most preferably of 70 ⁰C.

[0039] In a further embodiment step (b) comprises adding said activating agent; and incubating the mixture. In a further preferred embodiment said activating agent is added at a temperature of 0 ⁰C to 30 ⁰C, preferably at 0 ⁰C to 10 ⁰C, and most preferably at 0 ⁰C to 5 ⁰C.

In a further preferred embodiment said incubating is performed at a temperature of 0 to 40 ⁰C, preferably 10 to 40 ⁰C, and most preferably 20 ⁰C.

[0040] In a further preferred embodiment said activating agent is added in an amount of about 1.1 mole equivalents of said compound of formula (6).

[0041] In a preferred embodiment said first organic solvent is acetonitrile, and said activating agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI).

[0042] In a very preferred embodiment, said first organic solvent is acetonitrile, and m = 5 and n = 2. In a still more preferred embodiment, said first organic solvent is acetonitrile, m =

5 and n = 2, and said activating agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI).

[0043] The typical HPLC purity of SMPH produced by the process described above is 94 % to 98 % (a/a). Thus, in a further preferred embodiment said compound of formula (1) is

SMPH and the purity of said SMPH is 94 % (a/a) to 98 % (a/a), preferably the purity is at least 94 % (a/a).

[0044] A further aspect of the invention is a process for the purification of a compound of formula (6) and / or of a compound of formula (1), wherein m = 1 to 10 and / or wherein n = 1 to 10. In one embodiment said compound of formula (6) and / or said compound of formula (1) are obtainable by any one of the processes as described herein supra. The organic solvents which are used in said purification process allow to achieve high yield and purity, in particular when said compounds are produced by a process as described supra. The organic solvents used for the purification process are chosen based on their physico-chemical properties. The solubility of said compounds in the organic solvent, the water miscibility of the organic solvent and / or the chemical reactivity of the solvent towards said compounds are hereby of particular relevance.

[0045] Thus, in a further aspect the invention provides a process for the purification of a compound, wherein said compound is a compound of formula (6), wherein n = 1 to 10, or of a compound of formula (1), wherein m = 1 to 10 and n = 1 to 10, said process comprising the steps of:

(a) providing said compound, wherein preferably said compound is provided by any one of the processes disclosed herein;

(b) dissolving said compound in a second organic solvent, wherein said second organic solvent exhibits a solubility in water of less than 15 g per 100 g, wherein said compound exhibits a solubility in said second organic solvent of at least 5 g per 100 g; and

(c) precipitating said compound by replacing said second organic solvent by a third organic solvent, wherein said compound exhibits a solubility in said third organic solvent of at most 1 g per 100 g.

[0046] In one embodiment said second organic solvent and/or said third organic solvent does not chemically react with said compound. In a preferred embodiment said second organic solvent and/or said third organic solvent neither comprises hydroxy groups nor amine groups.

[0047] In a further embodiment said second organic solvent is selected from the group consisting of: (a) dichloromethane; (b) methyl tetrahydrofuran; and (c) methyl isobutyl ketone; and wherein preferably said second organic solvent is dichloromethane.

[0048] In a further embodiment said third organic solvent is selected from the group consisting of: (a) isopropylacetate; (b) t-butylmethyl ether (TBME); and (c) heptane; and wherein preferably said third organic solvent is isopropylacetate.

[0049] In a preferred embodiment said second organic solvent is dichloromethane, and said third organic solvent is isopropylacetate. [0050] In a preferred embodiment said compound is a compound of formula (6), wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a further preferred embodiment said compound is a compound of formula (6), wherein n = 1 to 4, and wherein preferably n = 1 to 3, and wherein more preferably n = 1 or 2, and wherein most preferably n = 2.

[0051] In a further preferred embodiment said compound is a compound of formula (1), wherein m = 1 to 10 and n = 1 to 10, and wherein preferably m = 3 to 7 and n = 1 to 4, and wherein further preferably m = 4 to 6 and n = 1 to 3. In a very preferred embodiment said compound is a compound of formula (1), wherein m = 5 and n = 2. Thus, in a very preferred embodiment said compound is SMPH.

[0052] Recrystallisation is removing NHS and intermediates. With respect to the synthesis of SMPH recrystallisation mainly removes NHS and 6-[β-maleimidopropionamido]hexanoic acid. The technical features disclosed in the following preferably relate to those embodiments of the invention, wherein said compound is a compound of formula (1), wherein m = 1 to 10 and n = 1 to 10, and in particular to embodiments, wherein said compound is SMPH. However, this should not imply that the disclosed process would be limited to the purification of SMPH.

[0053] In a further embodiment said process further comprises recrystallising said compound, wherein said recrystallising comprises the steps of:

(a) drying the precipitated compound, wherein preferably said drying is performed at a temperature of 20 ⁰C to 60⁰C, preferably at 25 ⁰C to 35 ⁰C, most preferably at 30 ⁰C

(b) dissolving the dried compound in a fourth organic solvent, wherein said compound exhibits a solubility in said fourth organic solvent of at least 5 g per 100 g;

(c) precipitating said compound by the addition of a fifth organic solvent, wherein said compound exhibits a solubility in said fifth organic solvent of at most 1 g per 100 g.

[0054] In one embodiment said fourth and/or said fifth organic solvent do not chemically react with said compound. In a preferred embodiment said fourth and/or said fifth organic solvent neither comprise hydroxy groups nor amine groups.

[0055] In a preferred embodiment said fourth organic solvent is acetonitrile or dichloromethane, preferably acetonitrile.

[0056] In a further preferred embodiment said fifth organic solvent is t-butylmethyl ether

(TBME). [0057] In a very preferred embodiment said fourth organic solvent is acetonitrile, and said fifth organic solvent is t-butylmethyl ether (TBME).

[0058] In a further embodiment said dissolving is performed at a temperature of 20 ⁰C to 70

⁰C, preferably 60 ⁰C.

[0059] In a further embodiment the dried compound is dissolved in said fourth organic solvent to a concentration of 15 % (w/w) to 35 % (w/w), preferably 25 % (w/w).

[0060] In a further embodiment during the addition of said fifth organic solvent the temperature of the mixture is maintained about constant, and preferably the temperature of the mixture is maintained at about the same temperature as during said dissolving step.

[0061] In a further embodiment said fifth organic solvent is added to a concentration of 5 %

(w/w) to 15 % (w/w).

[0062] In a further embodiment said precipitating is continued at a temperature of 0 ⁰C to 30

⁰C, preferably 0 ⁰C to 10 ⁰C, and most preferably at 5 ⁰C.

[0063] The typical purity as determined by HPLC of recrystallised SMPH was found to be more than 98 %. Thus, in a preferred embodiment said compound is SMPH and the purity of said SMPH after recrystallisation is at least 98 % (a/a).

[0064] Further purification to remove unidentified polar impurities can be carried out by passing a solution of said compound and hereby in particular of SMPH through silica.

[0065] Thus, in a further embodiment said process comprises the step of further purifying said compound wherein said purifying comprises: (a) dissolving said compound in acetonitrile; (b) contacting the solution with silica; (c) passing said solution through a bed of silica; and (d) precipitating said compound from the eluate obtained in step (c).

[0066] In a preferred embodiment said compound is dissolved in acetonitrile to a concentration of 2 % (w/w) to 10 % (w/w), preferably 5 % (w/w).

[0067] In a further preferred embodiment said dissolving of said compound in acetonitrile is performed at a temperature of 20 ⁰C to 25 ⁰C.

[0068] In a further preferred embodiment said silica comprises a particle size of 2 μm to 25 μm and / or wherein said silica comprises a pore volume of about 0.75 cm³ / g.

[0069] In a further preferred embodiment said contacting said solution with silica comprises suspending said silica in said solution. In a further preferred embodiment said solution is contacted with 1 to 4, preferably 2, weight equivalents of silica as compared to said compound.

[0070] In a further preferred embodiment, said solution is passed through a bed of silica comprising 4 to 8, preferably 6, weight equivalents of silica as compared to said compound. [0071] In a further preferred embodiment step (c) of said process further comprises the step of washing said bed of silica with acetonitrile. In a further preferred embodiment said washing is performed with 5 to 100 times, preferably with 10 to 40 times the volume of acetonitrile as compared to the volume of acetonitrile used for dissolving said compound.

[0072] In a further preferred embodiment said precipitating is achieved by replacing acetonitrile with said fifth organic solvent, wherein preferably said fifth organic solvent is t- butylmethyl ether (TBME).

[0073] In a further preferred embodiment said process further comprising the step of drying the precipitated compound at a temperature of 20 ⁰C to 60 ⁰C, preferably of 25 ⁰C to 35 ⁰C.

[0074] It has been demonstrated that passing SMPH through silica as described below allows to achieve a HPLC purity of at least 99.5 % (a/a) with no more than 0.1 % (a/a) unidentifiable impurities. Thus in a preferred embodiment said compound is SMPH and the purity of said SMPH after further purification is at least 99.5 % (a/a), wherein preferably said SMPH does not comprise more than 0.1 % (a/a) unidentifiable impurities.

[0075] A further aspect of the invention is a process for the purification of a compound of formula (1), said process comprising: (a) dissolving a compound of formula (1) in acetonitrile; (b) contacting the solution with silica; (c) passing said solution through a bed of silica; and (d) precipitating said a compound of formula (1) from the eluate obtained in step (c); wherein m = 1 to 10 and n = 1 to 10, and wherein preferably m = 3 to 7 and n = 1 to 4, and wherein further preferably m = 4 to 6 and n = 1 to 3. In a very preferred embodiment said compound of formula (1) is SMPH.

[0076] In a preferred embodiment said formula (1), preferably said SMPH, is dissolved in acetonitrile to a concentration of 2 % (w/w) to 10 % (w/w), preferably 5 % (w/w). In a further preferred embodiment said dissolving of said compound in acetonitrile is performed at a temperature of 20 ⁰C to 25 ⁰C.

[0077] In a further preferred embodiment said silica comprises a particle size of 2 μm to 25 μm and / or wherein said silica comprises a pore volume of about 0.75 cm³ / g. In a further preferred embodiment said contacting said solution with silica comprises suspending said silica in said solution.

[0078] In a further preferred embodiment said solution is contacted with 1 to 4, preferably 2, weight equivalents of silica as compared to said compound. [0079] In a further preferred embodiment, said solution is passed through a bed of silica comprising 4 to 8, preferably 6, weight equivalents of silica as compared to said compound of formula (1), preferably as compared to said SMPH.

[0080] In a further preferred embodiment step (c) of said process further comprises the step of washing said bed of silica with acetonitrile. In a further preferred embodiment said washing is performed with 5 to 100 times, preferably with 10 to 40 times the volume of acetonitrile as compared to the volume of acetonitrile used for dissolving said compound of formula (1), preferably SMPH.

[0081] In a further preferred embodiment said precipitating is achieved by replacing acetonitrile with said fifth organic solvent, wherein preferably said fifth organic solvent is t- butylmethyl ether (TBME).

[0082] In a further preferred embodiment said process further comprising the step of drying the precipitated compound of formula (1), preferably SMPH, at a temperature of 20 ⁰C to 60

⁰C, preferably of 25 ⁰C to 35 ⁰C.

EXAMPLE 1

N-Maleimidopropionic acid NHS ester

[0083] Maleic anhydride, β-alanine (1 mol eq.), and acetonitrile (ACN, 25 volumes) were charged to a vessel. The slurry was stirred under nitrogen and heated to 70 ⁰C. The temperature was maintained at 70 ⁰C for a further 5 to 8 hours. After cooling to 5 ⁰C, NHS (1 mol eq.) was charged, followed by Λ/-(3-dimethylaminopropyl)-Λf'-ethylcarbodiimide hydrochloride (EDCI, 1 mol eq.). The temperature was maintained at 0 - 5 ⁰C for 1 hour before adding a further charge of EDCI (1 mol eq.). The mixture was heated to 70 ⁰C and maintained at this temperature for 7 hours. The reaction mixture was cooled to 20⁰C and concentrated under vacuum at a maximum temperature of 45°C until the rate of solvent distillation was negligible. Dichloromethane (DCM, 40 volumes) was added to the residue, which was stirred until a solution was formed. This solution was washed with aqueous ammonium chloride (12 % w/w, 25 wt eq.) then with aqueous sodium chloride (24 % w/w, 25 wt eq.). The organic solution was dried by stirring with magnesium sulphate (1 wt eq.) at ambient temperature for 1 to 2 hours. The inorganics were filtered off under vacuum and the filter cake was washed with DCM (4 volumes). The filtrates were concentrated under vacuum, whilst gradually replacing DCM with isopropylacetate (IPAC). The residual slurry was cooled and stirred at ambient temperature for 1 hour before filtering under vacuum. The product filter cake was washed with IPAC (4 volumes) and then dried to constant mass under vacuum with slow rotation at 40 ⁰C to yield the desired compound as an off white solid. The typical yield is 60 to 80 % of the maximum theoretical yield.

[0084] The product was characerised by NMR as follows: ¹H NMR (400MHz, d6-DMSO): δ 2.80 (4H, br s), δ 3.05 (2H, t, J ~ 7Hz), δ 3.75 (2H, t J ~ 7Hz), δ 7.05 (2H, s).

EXAMPLE 2

Succinimidyl-6- [β-maleimidopropionamido] hexanoate (SMPH)

[0085] N-Maleimidopropionic acid (NHS ester), 6-aminohexanoic acid (1.04 mol eq.) and ACN (20 volumes) were charged to a vessel. The slurry was stirred and heated under nitrogen to 70 ⁰C. This temperature was maintained for 5 to 8 hours. After cooling to 20 to 25 ⁰C, the mixture was filtered through GF/F paper to remove fine insoluble particles. The filtrates were charged back to the vessel and cooled to 0 to 5 ⁰C. EDCI (1.1 mol eq.) was charged and the reaction mixture was then heated to 21 ⁰C. This temperature was maintained for 10 to 16 hours.

[0086] The reaction mixture was discharged and concentrated under vacuum at a maximum temperature of 30 ⁰C until the rate of solvent distillation was negligible. DCM (15 volumes) was added to the residue, which was stirred until a solution is formed.

[0087] This solution was washed with aqueous ammonium chloride (12 % w/w, 10 wt eq.) then with aqueous sodium chloride (24 % w/w, 10 wt eq.). It was then dried by stirring with magnesium sulphate (1 wt eq.) at ambient temperature for 1 to 2 hours. The inorganics were filtered off under vacuum and the filter cake was washed with DCM (2 volumes). The filtrates were concentrated under vacuum at a maximum temperature of 30 ⁰C, whilst gradually replacing DCM with IPAC. The residual slurry was cooled and stirred at ambient temperature for 1 hour before filtering under vacuum. The product filter cake was washed with IPAC (2 volumes) and then dried to constant mass under vacuum with slow rotation at 30 ⁰C to yield crude SMPH as a yellow solid. The typical yield is 60 to 80 % of the maximum theoretical yield.

[0088] Crude SMPH was added to ACN (3 volumes) and the slurry heated to 60 ⁰C to completely solubilise the solid. t-Butyl methyl ether (TBME, 9 volumes) was slowly added, whilst maintaining temperature. Insoluble material was filtered off and the filtrates cooled to

5 ⁰C to crystallise. SMPH was filtered off as an off white solid. Typical recovery was 60 to

80 %.

[0089] Recrystallised SMPH was stirred with ACN (20 volumes) and silica (2 wt eq.) at 20 to 25 ⁰C for 2 to 4 hours before charging to a bed of silica (4 wt eq.). The bed was pulled dry and more ACN (20 volumes) was charged to the silica bed to wash SMPH through. The filtrates were concentrated under vacuum at a maximum of 30 ⁰C whilst gradually replacing the ACN with TBME. The resulting precipitate was filtered off under vacuum. The white solid was dried to constant mass under vacuum with slow rotation at 30 ⁰C to yield high purity SMPH. Typical recovery was 60 to 80 %.

[0090] The end product was characerised by NMR as follows: IH NMR (400MHz, d6-

DMSO): δ 1.30 (4H, m), δ 1.60 (2H, m), δ 2.30 (2H, t, J ~ 7Hz), δ 2.65 (2H, t, J ~ 7Hz), δ

2.80 (4H, t, J ~ 7Hz), δ 3.0 (2H, m), δ 3.60 (2H, t, J ~ 7Hz), δ 7.0 (2H, s); LCMS: m/z (ES),

380 (M + H)+

EXAMPLE 3

Determination of the Purity of SMPH by HPLC

[0091] A solution of the test substance was dissolved in acetonitrile to a final concentration of 1 mg/ml. HPLC was performed under the following conditions:

Apparatus A liquid chromatograph fitted with a variable wavelength detector (VWD) and column heater.

Column Luna Silica (2) 5 μ 250 x 4.6 mm

Eluent 0.1 % orthophosphoric acid (v/v) in 1 : 99 (v/v) water : acetonitrile

Flow rate 1 ml/min

Monitoring Wavelength 215 nm

Injection Volume 5 μl

Column Temperature 25 ⁰C

Run Time 60 min [0092] The following compounds were identified by NMR and their retention times were determined:

Compound Approx. retention time (min)

3-maleimidopropionic acid (NHS ester) - 3.5

N-hydroxysuccinimide - 4 2

SMPH _^ 5 9

6- [ β-maleimidopropionamido ] hexano ic

acid ~ ⁶-⁶

diacid (stage 1 intermediate) _3 9

di-NHS ester (stage 1 impurity) _3 9

uncylised dimer (stage 2 impurity) _7 5

maleic anhydride _3 5

[0093] The purity of SMPH was determined as the percentage of the peak area of SMPH in the total peak area of the chromatogram (% a/a). Purified SMPH of Examples 2 showed a purity of at least 99.5 % (a/a), meaning that 0.5 % of the peak area were assigned to other compounds, which are generally referred to as "impurities". Identified compounds other than SMPH contributed 0.4 % (a/a). Unidentified compounds contributed 0.1 % (a/a).

Claims

1. A process for the production of a compound of formula (6)

said process comprising the steps of:

(a) reacting maleic anhydride with a compound of formula (3)

NH₂-(CH₂)_n-COOH (3);

(b) contacting the reaction product of (a) with N-hydroxysuccinimide (NHS) and an activating agent to give the compound of formula (6); wherein said process is performed in a first organic solvent, wherein said first organic solvent is a polar aprotic organic solvent; and

wherein n = 1 to 10.

2. The process of the preceding claim, wherein said first organic solvent exhibits a relative polarity of greater than 0.4, wherein preferably said first organic solvent neither comprises hydroxy groups nor amine groups.

3. The process of any one of the preceding claims, wherein said first organic solvent is selected from the group consisting of:

(a) acetonitrile;

(b) N₅N' -dimethyl formamide (DMF); and

(c) dimethyl sulfoxide (DMSO);

and wherein preferably said first organic solvent is acetonitrile.

4. The process of any one of the preceding claims, wherein said activating agent is selected from the group consisting of:

(a) Λ/-(3-dimethylaminopropyl)-Λf'-ethylcarbodiimide hydrochloride (EDCI);

(b) dicyclohexyl carbodiimide (DCC); and

(c) (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP); and wherein preferably said activating agent is N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDCI).

5. The process of any one of the preceding claims, wherein n = 1 to 4, and wherein preferably n = 2.

6. The process of any one of the preceding claims, wherein step (a) comprises providing maleic anhydride and a compound of formula (3), wherein n = 1 to 10, and wherein preferably n = 2, in said first organic solvent, wherein the concentration of said maleic anhydride and of said compound of formula (3) in said first organic solvent together is 2 % to 20 % (w/w), preferably 5 % to 10 % (w/w), and most preferably 8 % (w/w).

7. A pro cess for the pro duction o f a compound o f formula ( 1 )

said process comprising the steps of:

(a) reacting a compound of formula (6)

with a compound of formula (7)

NH₂-(CH2)_m-COOH (7); and

wherein said process is performed in a first organic solvent, wherein said first organic solvent is a polar aprotic organic solvent; and

wherein m = 1 to 10, and n = 1 to 10.

8. The process of claim 7, wherein said first organic solvent exhibits a relative polarity of greater than 0.4, and wherein preferably said first organic solvent neither comprises hydroxy groups nor amine groups.

9. The process of any one of claims 7 to 8, wherein said first organic solvent is selected from the group consisting of:

(a) acetonitrile;

(b) N₅N' -dimethyl formamide (DMF); and

(c) dimethyl sulfoxide (DMSO);

and wherein preferably said first organic solvent is acetonitrile.

10. The process of any one of claims 7 to 9, wherein said activating agent is selected from the group consisting of:

(a) Λ/-(3-dimethylaminopropyl)-Λf'-ethylcarbodiimide hydrochloride (EDCI);

(b) dicyclohexyl carbodiimide (DCC); and

(c) (benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate (PyBOP);

and wherein preferably said activating agent is N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDCI).

11. The process of any one of claims 7 to 10, wherein m = 3 to 7 and n = 1 to 4, and wherein preferably m = 5 and n = 2.

12. The process of any one of claims 7 to 11, wherein step (a) comprises providing said compound of formula (6) and said compound of formula (7) in said first organic solvent, wherein the concentration of said compound of formula (6) and of said compound of formula (7) in said first organic solvent together is 2 % to 20 % (w/w), preferably 3 % to 10 % (w/w), and most preferably 7 % (w/w).

13. The process of any one of claims 7 to 12, wherein m = 5 and n = 2, wherein said activating agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI); and wherein said first organic solvent is acetonitrile.

14. The process of any one of claims 7 to 13, wherein said compound of formula (6) is obtainable the process of any one of claims 1 to 6 and/or by the process of any one of claims 15 to 19.

15. A process for the purification of a compound, wherein said compound is a compound of formula (6), wherein n = 1 to 10, or of a compound of formula (1), wherein m = 1 to 10 and n = 1 to 10, said process comprising the steps of:

(a) providing a compound of formula (6) by the process of any one of claims 1 to 6; or providing a compound of formula (1) by the process of any one of claims 7 to 14;

16. The process of claim 15, wherein said second organic solvent and/or said third organic solvent neither comprises hydroxy groups nor amine groups.

17. The process of any one of claims 15 or 16, wherein said second organic solvent is selected from the group consisting of:

(a) dichloromethane;

(b) methyl tetrahydrofuran; and

(c) methyl iso butyl ketone;

and wherein preferably said second organic solvent is dichloromethane.

18. The process of any one of claims 15 to 17, wherein said third organic solvent is selected from the group consisting of:

(a) isopropylacetate;

(b) t-butylmethyl ether (TBME); and

(c) heptane;

and wherein preferably said third organic solvent is isopropylacetate.

19. The process of any one of claims 15 to 18, wherein said second organic solvent is dichloromethane, and wherein said third organic solvent is isopropylacetate.

20. The process of any one of claims 15 to 19 wherein said compound is a compound of formula (1), wherein m = 1 to 10 and n = 1 to 10, and wherein preferably m = 3 to 7 and n = 1 to 4.

21. The process of any one of claims 15 to 19, wherein said compound is a compound of formula (1), and wherein m = 5 and n = 2.

22. The process of any one of claims 20 or 21, further comprising recrystallising said compound, wherein said recrystallising comprises the steps of:

(a) drying the precipitated compound;

(c) precipitating said compound by the addition of a fifth organic solvent, wherein said compound exhibits a solubility in said fifth organic solvent of at most 1 g per 100 g;

wherein preferably said fourth and said fifth organic solvent neither comprise hydroxy groups nor amine groups.

23. The process of claim 22, wherein said fourth organic solvent is acetonitrile or dichloromethane, preferably acetonitrile.

24. The process of any one of claims 22 or 23 wherein said fifth organic solvent is t- butylmethyl ether (TBME).

25. The process of any one of claims 22 to 24, wherein said fourth organic solvent is acetonitrile, and wherein said fifth organic solvent is t-butylmethyl ether (TBME).

26. The process of any one of claims 22 to 25, wherein the dried compound is dissolved in said fourth organic solvent to a concentration of 15 % (w/w) to 35 % (w/w), preferably

25 % (w/w).

27. The process of any one of claims 22 to 26, wherein said fifth organic solvent is added to a concentration of 5 % (w/w) to 15 % (w/w).

28. The process of any one of claims 22 to 27, wherein said process comprises the step of further purifying said compound wherein said purifying comprises:

(a) dissolving said compound in acetonitrile;

(b) contacting the solution with silica;

(c) passing said solution through a bed of silica; and

(c) precipitating said compound from the eluate obtained in step (c).

29. The process of claim 28, wherein said compound is dissolved in acetonitrile to a concentration of 2 % (w/w) to 10 % (w/w), preferably 5 % (w/w).

30. The process of any one of claims 28 to 29, wherein said silica comprises a particle size of 2 μm to 25 μm and / or wherein said silica comprises a pore volume of about 0.75 cm³ / g.

31. The process of any one of claims 28 to 30, wherein said precipitating is achieved by replacing acetonitrile with said fifth organic solvent, wherein preferably said fifth organic solvent is t-butylmethyl ether (TBME).

32. The process of any one of claims 28 to 32, wherein the purified compound comprises a purity of at least 99.5 %, and wherein preferably said purified compound does not contain more than 0.1 % unidentifiable impurities as determined by HPLC.